key: cord-260099-mthwab4p authors: li, yi; hao, bingtao; kuai, xuezhang; xing, guichun; yang, juntao; chen, jie; tang, li; zhang, lingqiang; he, fuchu title: c-type lectin lsectin interacts with dc-signr and is involved in hepatitis c virus binding date: 2009-02-21 journal: mol cell biochem doi: 10.1007/s11010-009-0056-y sha: doc_id: 260099 cord_uid: mthwab4p hepatitis c virus (hcv) is a major cause of liver disease. however, the detailed mechanism underlying hepatocyte infection with hcv is not yet completely understood. we previously identified a novel c-type lectin—lsectin predominantly expressed on liver sinusoidal endothelial cells. here we demonstrate that lsectin can interact with two hcv receptors, dc-signr and cd81, through its central ectodomain. furthermore, cells expressing lsectin specifically can be attached by the naturally occurring hcv in the sera of infected individuals. this binding was found to be mediated by the hcv e2 glycoprotein and could be efficiently inhibited by egta but not by mannan treatment. the present study suggests that lsectin interaction with dc-signr might contribute to hcv binding to liver sinusoidal endothelial cells. electronic supplementary material: the online version of this article (doi:10.1007/s11010-009-0056-y) contains supplementary material, which is available to authorized users. hcv, a positive single-stranded rna virus in the flaviviridae family, is the major causative agent of non-a, non-b hepatitis in humans and has infected *170 million people worldwide [1] [2] [3] . the hcv structural protein includes two envelope glycoproteins e1 and e2; these glycoproteins form a heterodimer and mediate viral binding and entry into host cells [4, 5] . to infect a cell, hcv must first attach itself to the cell surface by interaction with specific cell surface receptors, and consequently induce conformational changes in the glycoproteins e1 and e2 and fusion of the viral and cellular membranes [2, 4, 5] . by using surrogate models such as the truncated soluble e2 glycoprotein and hcv pseudoparticles, several potential hcv receptors have been identified, including the human tetraspanin cd81, scavenger receptor class b type 1/2 (sr-b1/2), and low density lipoprotein receptor (ldl-r) [5] [6] [7] [8] . since liver is the major target organ of hcv infection, the expression pattern of hcv receptors has been suggested to be liver specific or dominant within the liver. in addition to cause an infection, hcv can use multiple attachment factors and receptors in parallel or in succession [4] . dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign; also termed cd209) and dc-sign related (dc-signr; also known as l-sign and cd209l) are two members of the c-type lectin family; these bind to viral glycoproteins such as e2 in hcv [9] [10] [11] [12] . electronic supplementary material the online version of this article (doi:10.1007/s11010-009-0056-y) contains supplementary material, which is available to authorized users. recently, we have identified a novel c-type lectin predominantly expressed on liver sinusoidal endothelial cells (lsec) and denominated it as liver sinusoidal endothelial cell c-type lectin (lsectin) [13] . lsectin shares 32% and 31% amino acid sequence identities with dc-signr and dc-sign, respectively, and has been shown to bind in vitro with mannose, glcnac, and fucose. interestingly, lsectin can be colocalized with dc-signr on the lsecs [13] . recent studies showed that lsectin could bind to ebola virus surface glycoprotein, filovirus glycoproteins, and the spike protein of sars coronavirus [14] [15] [16] . in addition, lsectin was also demonstrated to be expressed in human peripheral blood and thymic dendritic cells isolated ex vivo. lsectin expression confers ebola virus-binding capacity to leukemic cells, participates in antigen uptake and internalization, and therefore it functions as a pathogen receptor [17] . however, the potential role of lsectin in hcv binding to lsecs was not fully understood. in this study, we present evidence that lsectin could interact with dc-signr and cd81, the two previously identified hcv receptors. we further showed that lsectin could bind to the hcv e2 glycoprotein in a calciumdependent manner. these results might contribute to the understandings of the complicated mechanism of hcv infection. cell lines and antibodies cho cells stably expressing the fc-lsectin fusion protein were cultured in dmem and 10% fetal bovine serum (hyclone) supplemented with 400 lg/ml geneticin (g418; gibco) and 100 units/ml penicillin/streptomycin. lsectin and dc-signr were subcloned into the a site of the pires expression vector. lsectin, dc-signr and empty vectors (for mock cells) were individually transfected into nih3t3 cells, and 800 lg/ml g418 was added after 48 h. clones that stably expressed lsectin or dc-signr were screened, and the expression was confirmed by western blotting and fluorescence-activated cell sorter (facs) analysis, using anti-lsectin mouse monoclonal antibody we previously raised [18] and anti-dc-signr antibody (igg2b; r&d systems). the anti-lsectin monoclonal antibody could specifically recognize lsectin and its specificity was further shown in supplementary fig. 1 . full-length dc-signr and cd81 were cloned into the pcdna3.1 vector (invitrogen). coimmunoprecipitation was carried out as described previously [19] . expression and purification of hcv e2 protein the hcv e2 (384-664 aa) fused to human igg1-fc was generated. cho clones with stable fc-e2 were obtained. the fc-e2 proteins were purified by affinity chromatography with the rprotein aff column (amersham biosciences). the amount of e2 antigen was quantified by elisa using anti-fc monoclonal antibody (sigma). hcv e2 was also cloned into the psectag2/hygro vector for producing myc-tagged e2. soluble e2 binding and inhibition nih3t3 cells (2 9 10 5 ) with stable lsectin or dc-signr expression were seeded in six-well plates. the cells were then harvested with the cell dissociation solution (sigma) and washed three times with an facs buffer. after adding 20 lg/ml mannan or 5 mm egta, the cells were incubated with the mock antigen or soluble e2 glycoprotein in the facs buffer for 1 h at 4°c. after washing three times, the adherence of e2 was quantified by facs using anti-fc or anti-myc antibody. for the soluble lsectin protein inhibition experiment, hek293t cells were transfected with pflag-cmv-lsectin. the cells were preincubated with the soluble lsectin protein before addition of e2. fc protein was used as negative control and data was normalized by the total input protein. pseudotype particles production and cell infection plasmid containing hcv e1e2 was constructed with 5 0 -gct aag ctt gga tgg ccg acc tca tgg ggt ac-3 0 (hind iii) and 5 0 -cga gaa ttc cgc ctc cgc ttg gga tat-3 0 (ecor i) using hcv genome-containing pbrtm-hcv1-3011 vector (infectious molecular clone h77 (genbank accession no. af009606) as the template. the pcr product was digested and cloned into pcdna3.1 vector. to generate the pseudotype viral particles, the envelope-deficient pnl4-3.luc.r-e-plasmid and the hcv e1e2 plasmid were cotransfected into hek293t cells. the culture supernatant was harvested, sterile filtered, divided into aliquots, and stored at -80°c. the bound pseudotype particles were measured using elisa. stable lsectin or dc-signr nih3t3 cells (2 9 10 5 ) were seeded 24 h before infection in 12-well plates. after washing three times with serum-free dmem, 200 ll of the pseudotype retroviral particles in medium were added to the cells followed by incubation for 3-5 h at 37°c under gentle agitation every 15 min. subsequently, 300 ll of dmem was added, and the luciferase activity in cell lysates was determined at 48 h after infection (assay kit was from promega). the hcv virus-positive and negative donors' sera (all hiv negative) were obtained from the general hospital of pla, and informed consent was obtained from each subject. the cells were blocked with 10% heat-inactivated goat serum for 20 min at 37°c. after washing, the sera were diluted in an adhesion buffer and were allowed to incubate with the cells for 1 h at 37°c; the unbound viruses were removed. the hcv rna copies bound to the cells were measured by quantitative pcr using the hcv kit (shenzhen pg bio-tech). for the inhibition assays, mannan (20 lg/ml) or egta (5 mm) was preincubated for 20 min at room temperature or the hcv virions were preincubated with the soluble lsectin protein for 20 min at 4°c. we have previously demonstrated that similar to its close paralogue dc-signr, lsectin could form homo-tetramer [13] . dc-signr and cd81 are known hcv receptors expressed on lsecs. this knowledge prompted us to investigate whether lsectin could interact with dc-signr or cd81. first we detected the physical interactions of lsectin with dc-signr using co-immunoprecipitation assay (co-ip) in the hek293t cells co-transfected with lsectin and dc-signr expressing plasmids. as shown in fig. 1a , ectopic expressed lsectin could be coimmunoprecipitated with myc-tagged dc-signr in hek293t cells. the reciprocal co-ip assays also indicated that dc-signr could be coimmunoprecipitated with lsectin (fig. 1b) . then the soluble lsectin adhesion assay was performed to confirm the interaction. the soluble lsectin protein consisting of only the central ectodomain (aa 55-162) was incubated with the hek293t cells transfected with dc-signr or with vector-mock cells. their associations were assessed by facs assays with anti-lsectin antibody. about a half of the dc-signr-hek293t cells were bound to the soluble fc-lsectin but not control-fc protein, whereas only 6% of the mock cells were adhered to lsectin (fig. 1c) . since soluble lsectin contains only the ectodomain, this result implicated that the ectodomain of lsectin was sufficient for its interaction with dc-signr. lsectin protein contains an n-terminal transmembrane domain (tm), a potential encocytic motif (aa 6-9), a central ectodomain (aa 55-162) including a coiled-coil region (aa 96-136), and a c-terminal carbohydrate to further investigate whether the ectodomain of lsectin was required for and whether other regions were involved in the interaction with dc-signr, a series of lsectin truncates or point mutants were constructed and used in the co-ip assays. the expressions of these mutants in cells were adjusted to a comparable level and the lysates in same amount were used as input for immunoprecipitation. the 5% inputs and ip products were run on sds-page gel and detected by western blotting. as shown in fig. 1d to extend our conclusion, we further tested the interaction of lsectin with another hcv receptor cd81. co-ip assays and facs analysis both showed that lsectin could also interact with cd81 (fig. 2a-c) . due to the fact that hek293t expresses high level of endogenous cd81, we also detected the interaction between flag-lsectin with endogenous cd81 (fig. 2b) . adhesion assay result implicated that ectodomain of lsectin was sufficient for its interaction with cd81, although the affinity with cd81 seems to be weaker than that with dc-signr (compare fig. 2c with fig. 1c) . since lsectin could interact with dc-signr and cd81, which are the well-defined hcv receptors, we asked whether lsectin could interact with hcv by binding hcv e2 glycoprotein similar to the manner of dc-signr and cd81. nih3t3 cells stably expressing lsectin or dc-signr were generated and the expressions of these proteins on the cell surface were roughly comparable (supplementary fig. 1a) . then the cells were incubated with purified soluble fc-tagged hcv e2 fusion protein and washed and binding was assessed by flow cytometry. the soluble fctagged e2 but not control-fc protein significantly bound the nih3t3-lsectin or nih3t3-dc-signr expressing cells but not the mock cells (fig. 3a) . to further confirm the binding of e2 with lsectin, hek293t cells were transiently transfected with pflag-cmv-lsectin and incubated with e2. the soluble e2 glycoprotein was found to bind efficiently with the hek293t-lsectin cells in a dose-dependent manner but not with the empty vector ( fig. 3b and supplementary fig. 1b) . pre-incubation with the soluble lsectin protein resulted in a dramatic decrease in the affinity of hcv e2 to the hek293t-lsectin cells (fig. 3c) . the binding of myctagged e2 to the hek293t-lsectin cells was also inhibited by the soluble his-tagged lsectin protein (fig. 3d) . previous studies have shown that egta, chelators of calcium ion, and mannan could block the binding of the virus to dc-sign and dc-signr [9] [10] [11] [12] . lsectin can bind to carbohydrates such as mannose, glcnac, and fucose [13] ; this prompted us to investigate whether the interaction between the e2 glycoprotein and lsectin was influenced by these agents. the binding of e2 with nih3t3-lsectin stable transfectants was efficiently inhibited by egta and less (fig. 3e) ; the latter was a little different from that in the case of dc-signr. taken together, these results indicated that the soluble hcv e2 protein could bind to lsectin in the cell surface. to evaluate whether the hcv glycoprotein binds to lsectin in the context of a viral particle, we tested the binding of the hiv pseudotype particles bearing the hcv e1 and e2 chimeras to the nih3t3-lsectin cells. the affinity of the hcv pseudotypes with lsectin was approximately fivefolds that of the control pseudotype particles alone (fig. 4a, middle) . as a positive control, the binding of hcv pseudotypes to dc-signr-expressing cells was eightfolds than the control pseudotype particles (right). interestingly, lsectin adherence could be blocked by egta but not by mannan whereas dc-signr adherence could be blocked by both (fig. 4a) , indicating their specificity in carbohydrate binding. previous studies showed that dc-sign and dc-signr augment infection of various viruses such as hiv, hcv, ebola virus, cmv in trans-infection form, suggesting that they capture viruses and then transfer them to nearby other target cells rather than mediate virus entry into the cells where they are expressed [reviewed in ref. 9 ]. similar to dc-signr, lsectin is expressed on liver sinusoidal endothelial cells, which are not the host cells in hcv infection. so we speculated whether lsectin could capture viruses and had any effects on hcv infection in a manner similar to that of dc-sign and dc-signr. nih3t3 transfectants and hep3b hepatocellular carcinoma cells (positive control) were infected with the hcv pseudotype particles and the luciferase activities were measured 48 h post-infection. hep3b cells are highly permissive to hcv pseudovirus entry. by contrast, the infection of the nih3t3-mock, nih3t3-dc-signr, and nih3t3-lsectin cells resulted in only low levels of luciferase activities, indicating that neither dc-signr nor lsectin facilitated hcv infection of nih3t3 cells (fig. 4b) . to detect and confirm the binding of the hcv virions present in the hcv-positive sera to lsectin, we performed the virus-binding assay with patient serum. nih3t3-lsectin cells were bound to the hcv virions in all the three high-titer hcv-positive sera (above 10 5 copies), and the binding levels of lsectin ranged from 10 to 30-folds that of the control cells (fig. 4c) . the results suggested that lsectin could interact with hcv virions in the hcv-positive sera. to investigate the binding specificity, we performed the inhibition experiments. the binding effects of nih3t3-lsectin cells to hcv virions could be blocked by either soluble lsectin or egta, indicating the binding specificity and the ca 2? -dependence (fig. 4c) . consistently with the conclusion of the soluble hcv-e2 binding assay (fig. 3) , lsectin binding to hcv virions could not be inhibited by mannan (fig. 4c) , suggesting the similarity but not identity of lsectin with dc-signr in the hcv adhesion. our present study suggests that lsectin, a recently identified c-type lectin, could interact with dc-signr and cd81 and was involved in hcv binding. the coimmunoprecipitation assays and the soluble lsectin adherence assays showed the interaction of lsectin with dc-signr and cd81. moreover, liver sinusoidal endothelial cells (lsecs) serve as an active barrier between circulating blood and hepatocytes. during hcv infection in the liver, lsecs capture the circulating hcv particles, transmit the captured particles to the endothelial cells, and present them to the entry-permissive hepatocytes. the adherence of cd81-expressing cells to soluble lsectin also implicated a potential interaction between cd81 on hepatocytes and lsectin on lsecs, which may be involved in the transmission of the hcv virions to hepatocytes. this possibility should be further investigated in the future. mapping analysis showed that the central ectodomain of lsectin was both sufficient and necessary for interaction with dc-signr and cd81. deletion of the coiled-coil nih3t3 cells were incubated with the sera from 3 hcv-infected patients for 2 h, washed three times, and then lysed. the hcv rna was extracted and measured using real-time qpcr assay. the y-axis represents the log-fold increases above nih3t3 cell binding in the graph region which might mediate the homo-tetramerization or hetero-oligomerization completely abolished the binding of lsectin to dc-signr (fig. 1d) [15] . l-sign showed high affinity to high mannose oligosaccharides, but lsectin bound with high selectivity to glycoproteins terminating in glcnacb1-2man. we noted that mannan, a mannose polymer obtained from yeast cell wall, could not significantly block the binding of the e2 glycoprotein and hcv pseudotype particles with lsectin (fig. 3e, 4a and c) , while mannan could compete for binding to l-sign. these results suggested that the presence of various types of incompletely matured glycans may facilitate the interaction of enveloped viruses with multiple cell surface receptors. in addition, we observed similar affinities of lsectin and dc-signr to hcv-e2 in binding assays with soluble e2 proteins (fig. 3e ) but different affinities in the assays with pseudotype particles (fig. 4a) . this might be caused by different conformations of e2 proteins in different contexts or different length of ectodomains of lsectin and dc-signr. in this aspect, ectodomain length of type 2 c-lectins has been demonstrated critical for pathogen recognition [10, 11, 17] , and lsectin ectodomain is shorter than dc-signr ectodomain [13, 16] . gramberg et al. previously reported that they could not detect the binding of hcv pseudotype particles to lsectin expressed cells [14] , which is inconsistent with our results. this inconsistency may be from the different affinity of lsectin to the variants of e2 protein from different hcv isolates. hcv can be classified into six major genotypes and further subdivided into at least 70 subtypes, which exhibit different phenotypic properties. it was reported that a soluble form of e2 protein had a significant genotypespecific difference in cd81 binding assay. in our present study, hcv e1e2 glycoprotein constructs were amplified using the infectious molecular clone h77 as the template. another possibility is the different sensitivity in detecting the p24 antigen recovery with the different titer of hcv pseudotype particles because the affinity of lsectin with hcv pseudotype particles was only half of dc-signr. in summary, the present study suggests that lsectin interaction with dc-signr might contribute to hcv binding to liver sinusoidal endothelial cells. analysis of a successful immune response against hepatitis c virus hepatitis c virus infection analysis of successful immune responses in persons infected with hepatitis c virus a cdna fragment of hepatitis c virus isolated from an implicated donor of posttransfusion non-a, non-b hepatitis in japan cell entry of hepatitis c virus binding of hepatitis c virus to cd81 the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus the lowdensity lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis c virus the role of dc-sign and dc-signr in hiv and siv attachment, infection, and transmission l-sign (cd209l) and dc-sign (cd209) mediate transinfection of liver cells by hepatitis c virus l-sign (cd 209l) is a liver-specific capture receptor for hepatitis c virus dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances transinfection of t cells characterization of a novel c-type lectin-like gene, lsectin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus a novel mechanism for lsectin binding to ebola virus surface glycoprotein through truncated glycans interactions of lsectin and dc-sign/dc-signr with viral ligands: differential ph dependence, internalization and virion binding the dc-sign-related lectin lsectin mediates antigen capture and pathogen binding by human myeloid cells preparation and characterization of monoclonal antibody against human lsectin targeting of ww domains linker of hect-type ubiquitin ligase smurf1 for activation by ckip-1 acknowledgments we are grateful to dr. robert w. doms for kindly providing the pcdna3/dc-signr plasmid. this work was partially supported by grants from the chinese national basic research programs (2006cb910802), the chinese national natural science foundation projects (30621063, 30730050, 30772030) , the chinese national high-tech program (2006aa02z165), and the beijing science and technology nova program (2007a063). key: cord-265588-1tcaleeo authors: yousaf, tahir; rafique, shazia; wahid, fazli; rehman, sidra; nazir, abdul; rafique, javeria; aslam, kashif; shabir, ghulam; shah, shahid masood title: phytochemical profiling and antiviral activity of ajuga bracteosa, ajuga parviflora, berberis lycium and citrus lemon against hepatitis c virus date: 2018-03-20 journal: microb pathog doi: 10.1016/j.micpath.2018.03.030 sha: doc_id: 265588 cord_uid: 1tcaleeo hepatitis c is a serious health issue and cause liver disorders in millions of people. available therapeutic agents require long term administration with numerous side effects. therefore, there is a dire need to find alternative treatment options for this disease. since ancient times, medicinal plants are widely used to cure various diseases with no or less harmful effects. therefore, this study was designed to find out phytochemicals and investigate antiviral activity of methanol extract of ajuga bracteosa, ajuga parviflora, berberis lycium and citrus lemon against hepatitis c virus (hcv infection). phytochemical analysis of the plant extract was performed using various chemical tests. toxicity of the plant extract was determined against using trypan blue exclusion method. antiviral activity of the selected plant extract was find out against hcv infected hepg2 cells. for this purpose, hepg2 cells were seeded with hcv positive and negative serum and nontoxic doses of plant extract for 24 and 48 h. after this rna was extracted and viral load was determined using real-time pcr. phytochemical analysis showed the presence of flavonoids and phenols in all plant extracts while amino acids, alkaloids and tannins were present in b. lycium and saponins were detected in c. lemon. toxicity assay showed that all plant extracts were nontoxic at maximum concentration of 200 μg/ml except b. lycium, which showed mild toxicity at 40 μg/ml and were extremely toxic at 60 μg/ml and above doses. real-time pcr quantitation result revealed that after 24 h treatments a. parviflora showed highest antiviral activity, followed by a. bracteosa, while b. lycium extract had low (35%) and c. lemon has no antiviral effects. the 48 h treatments showed an increase antiviral activity by a. bracteosa followed by a. parviflora and b. lycium while c. lemon showed negative effect. our results depicted that mentioned plants might be used as an alternative therapeutic regime or in combination with existing treatments against hcv. plants the primitive source of food are utilized for centuries to cure diseases by the human being in different forms. medicinal plants are valuable source of compounds that are inclusive part of drug administrated presently. natural product plays vital role in the cure of bacterial and tumour diseases. but their role in case of viral disease is still limited with a few examples such as in coronavirus, coxsackievirus, dengue virus, enterovirus, herpes simplex, measles virus respiratory syncytial virus, influenza, human immunodeficiency, hepatitis b and c [1] . the main advantage of using natural molecules from plant extracts is their low cost, with no need of chemical synthesis. this mode of production might lead to less expensive treatments, available for populations of low-income countries. ajuga bracteosa (a. bracteosa) and ajuga parviflora (a. parviflora) belongs to ajuga genus and lamiaceae family. a. bracteosa is native to the hilly areas of pakistan, china, india and malaysia. in pakistan, it is found in the northern hilly areas locally known as trakha boti (bitter herb). it has many ethnopharmacological uses as its leaves been used to make herbal medicine to cure diabetes, malaria and digestion related problems [2] . moreover, leaves extract has been traditionally used to remove toxicity from the blood. the root extract has also been used for the curing of digestion related issues [3] . a. parviflora is found in pakistan, kashmir, and afghanistan within the low temperature and hilly https [4] . it was reported that this plant has been traditionally used to cure fever, asthma, hcv, jaundice [5] , arthritis, cancer and wounds [6] . this plant also has been used in the treatment of eye irritation, poisoning from insect attack, stomach pain and liver tissue damage [7] . berberis lycium (b. lycium) naturally grow in all regions of himalayas and also found in temperate and semi temperate regions of pakistan, nepal, bangladesh, afghanistan and india. in pakistan, they were found in the area which lies between 900 and 2900 m like azad kashmir, khyber pakhtunkhwa, punjab and baluchistan. it is used to cure disease like liver disorders, abdominal pain, skin diseases, cough and ophthalmic. citrus lemon (c. lemon) comes under the family rutaceae and grown all over the world. it has been used against cancer, to reduce the cytotoxicity of low density lipoprotein and to prevent edema of the legs. hepatitis c is a liver inflammatory disorder caused by hepatitis c virus (hcv). hcv is a tiny (55-65 nm in size) virus having an envelope, a positive ssrna genome and belongs to the flaviviridae family. hcv is present in chronic form in around 130-150 million peoples around the world and are responsible of 33% death alone as a member of hepatitis group [8] . in pakistan, hcv sero-prevalence among the general adult population is 6.8%, while active hcv infection was found in approximately 6% of the population [9] . currently, the most common treatment for hcv is the combination of ribavirin and pegylated interferonα. this treatment is given for 24-48 weeks depending on viral genotype and races [10] . the long term uses of this treatment produces severe side effects including transient virologic response, hemolytic anemia and teratogenic effects [11, 12] . hence, it is needed to find new and alternative ways of treatment for hcv with lower side effects and higher efficacy. the purpose of the study was to find out more effective alternative therapeutic option with reduced or no side effects. an antiviral regimen which could be less expensive and easily available to all, especially to the people who cannot access the current hcv treatment aim of our basic study. the results demonstrated that 3 out of 4 selected plants had a notable anti-hcv activity. these plants can be subjected to further bioassay guided isolation of anti-hcv compounds which may ultimately leads to an alternative hcv treatment. all plants were selected based on their medicinal uses by the local community for the treatment of viral and other infectious diseases. three medicinal plants, a. bracteosa, a. parviflora and b. lycium were collected from dir lower malakand division, khyber pakhtunkhwa, pakistan, while c. lemon was collected from ajman, united arab emirates (table 1 ). plants were identified by dr. abdul nazir, plant taxonomist at department of environmental sciences, comsats institute of information technology abbottabad, pakistan and the voucher specimen was placed in islamabad herbarium at quaid-i-azam university, islamabad. the plants were washed with tap water and dried at room temperature in the shade. at 8-12% moisture level, the plant materials were grounded and weighted. then, extraction was performed using previously reported method with some modifications [13] . briefly, 100 gm of each plant material was soaked in 750 ml of 70% methanol for 10 days. the mixture was filtered using a mousseline cloth in order to remove large waste particles of the plant, followed by filtration with whatman filter paper 42. the extract was concentrated using a rotatory evaporator at 50°c. the dried samples were weighted and stored. stock were prepared by dissolving 20 mg of extract in 1 ml of dimethyl sulfoxide and kept at -20°c for further use. phytochemical analysis of the plant extract was done as per standard protocols [14, 15] . in short, mayer's test was performed for the detection of alkaloids. in which the 3 ml of extracts were separately dissolved in 70% hcl and filtered. then, mayer's reagent was added to the filtrates and appearance of yellow color precipitate indicated the presence of alkaloids. fehling's test was used for the detection of carbohydrates. in this, 0.5 g extracts were dissolved in 5 ml of distilled water and filtered. then, the filtrates were hydrolyzed with 70% hcl, neutralized with alkali and at last heated with fehling's a and b solutions. the presence of reducing sugars was indicated by the formation of red precipitate. for the detection of saponins, foam test was applied in which 0.5 gm of extract was dissolved in 2 ml of distilled water and well shaken. the production of foam, and its persistence up to 10 min indicated the presence of saponins. phenols were detected with ferric chloride test in which 2 ml of extracts were treated with 2-3 drops of 10% ferric chloride solution. formation of bluish black color indicated the presence of phenols. tannins were detected with gelatin test. it was performed by adding 1% gelatin solution containing sodium chloride to the extract. the presence of tannins was indicated by the formation of white precipitate. for the detection of flavonoids (lead acetate test), 2 ml of extracts were treated with 2-3 drops of lead acetate solution. formation of yellow color precipitate indicated the presence of flavonoids. ninhydrin test was used for the detection of amino acids. ninhydrin solution was added to the extract and boiled for a few min. the formation of blue color indicated the presence of amino acids. fourteen chronically infected patients (diagnosed based on serum hcv rna by pcr) with hcv 3a were selected based on consent without any age and gender discrimination ( table 2 ). sera were taken from patients at the genome center of center for applied molecular biology, lahore, pakistan and stored at -80°c. only those patients were selected which were negative for hepatitis b surface antigen. hepg2 cells were purchased from american type culture collection (usa). these cells were maintained at 37°c in a 95% air and 5% co 2 humidified atmosphere. cells were cultured in complete dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs), 1% antibiotics (combination of penicillin & streptomycin), and sodium bicarbonate (0.0037 gm/ml). when reach to 70-80% confluency, subcultring were performed and plates were prepared. the hepg2 cells were split and plated for antiviral analysis of plant extracts. for this purpose, cells were grown at a density of 3.1 × 10 5 cells/well in 12-wells culture plates. briefly, the 70-80% confluent plates media were aspired and washed with 5-8 ml of 1× phosphate buffered saline (pbs) followed by the addition of trypsin edta (invitrogen life technologies, carlsbad, usa) to dislodge the cells. then, 2-3 ml of complete medium (dmem+10% fbs + antibiotics) was added to the flask to neutralize the trypsin edta. the cells were then shifted with medium to 15 ml falcon tube, centrifuges, counted and seeded at the required density and dislodged the cells into a single cell suspension (10-20 times). then cells were incubated at 37°c, 5% co 2 for 24 h before treatments. the toxicity assay was performed using trypan blue exclusion method. all plant extracts were subjected to toxicity analysis. for this purpose, hepg2 cells were cultured in 24 well plates at a density of 8 × 10 4 cells/well. the first 3 wells were taken as control while the other wells were treated with different concentration of plant extracts ranging from 40 μg/ml to 200 μg/ml. after 24 h, media was aspired and wells were washed with 1× pbs solution and tripsinized with trypsin edta to dislodge the cells. after 2 min, complete culture media was added to stop further trypsinization. cells were suspended into a single cell suspension through generous pipetting. ratios of 1:1 (10 μl each) of trypan blue and suspended cells were mixed well. after this, 10 μl of the mixture was poured onto hemocytometer and the viable and dead cells were counted on the basis unstained and stained cells, respectively. initially, percent viability was found out using below formula in the next step, viability of the control cells was considered as 100% and all other treatments were compared with control. hepg2 cells were seeded in complete media at density of 3 × 10 5 cells per well in 24 wells plate. the plates were incubated at 37°c for 24 h. five wells of the 1st row was seeded with 100 μl of hcv positive serum of known viral load, in which the 5th well was kept as positive control (in which only virus added; no plant extract added) and the 6th was kept as negative control (in which no virus & no plant extract added). another plate was kept as negative control (no virus, no drug). each row was treated with separate plant extract, a. bracteosa (i st row), a. parviflora (2 nd row), b. lycium (3rd row) and c. lemon (4th row) at a concentration of 200 μg/ml except b. lycium, which was 20 μg/ml. after 24 h incubation, total rna was extracted using mache-rey-nagel nucleic acid extraction kit (macherey-nagel gmbh & co. germany) according to the manufacturer's instructions. hcv rna quantifications were determined by real-time pcr smart cycler ii system (cepheid sunnyvale, usa) using the sacace hcv quantitative analysis kit (sacace biotechnol-ogies caserta, italy) according to the manufacturer's instructions. all experiments were conducted in triplicate to check the reproducibility of the results. all statistical comparisons were made by means of student's t-test and p˂ 0.05 were considered significant. phytochemical analysis of the methanol plant extracts of a. bracteosa, a. parviflora, b. lycium and c. lemon revealed the presence of phytoconstituents such as alkaloids, carbohydrates, amino acids, flavonoids, saponins, tannins, and phenols as shown in table 3 . flavonoids and phenols were detected in all of the above mentioned four tested extracts. alkaloids and tannins were only present in b. lycium, whereas saponins were only detected in c. lemon. carbohydrate and amino acid were detected in all except c. lemon and b. lycium in which it was not detected, respectively. toxic extracts and compounds cannot be used for biological activities. so, it was important to find out the toxic effects of four medicinal plants used in the current study before evaluating for anti-hcv activities. results have been summarized in fig. 1 . it was observed that a. bracteosa, a. parviflora, and c. lemon exhibited no significant cytotoxic effects on cell proliferation of liver cells up to concentration of 200 μg/ ml. it is worth mentioning that b. lycium methanol extract was significantly toxic even at a dose of 40 μg/ml and were extremely toxic at higher doses. hcv serum can be used to infect human hepatocyte cultures as reported by rehman et al. [10] . antiviral activities of methanol extract of four medicinal plants were tested against hcv and results has been summarized in fig. 2 for 24 h and fig. 3 for 48 h treatments. results table 3 the phytochemical constituents of the a. bracteosa, a. parviflora, b. lycium and c. lemon. chemical a. bracteosa a. parviflora b. lycium c. lemon amino acid + + -+ 4 flavonoids tannins --+ -7 phenols + + + + fig. 1 . cellular toxicity of plant extract via trypan blue exclusion method. a. bracteosa, a. parviflora, and c. lemon exhibited non-significant cytotoxic effects. showed that after 24 h viral count dramatically decreased in case of a. parviflora (70%) treated sample followed by a. bracteosa (60%) and b. lycium (35%), while c. lemon had shown negative antiviral activity and viral count increased by 2.7 times as compared to untreated control. similarly, real-time pcr results showed that after 48 h the viral count was decreased up to a very low titer in case of a. bracteosa (75%) treated sample, followed by a. parviflora (65%) and b. lycium (20%), while c. lemon still had negative effects having viral count of about 1.6 times as compared to control. a large variety of traditional medicinal herbs and plants have been reported to exhibit antiviral activities against different viruses [16, 17] . as hcv infect liver, so the idea was to check pakistani medicinal plants having traditional uses in the treatment of various viral, infectious diseases and liver disorders for the anti-hcv activities. methanol extract from the leaves of a. bracteosa, a. parviflora, roots of b. lycium and from the pulp of c. lemon were checked for their anti-hcv activity. it is always important to know the phytoconstituents of extracts under investigation. therefore, plants were subjected to phytochemical analysis. the plants under study were also previously analyzed by researchers for phytoconstituents. they had reported that a. bracteosa methanol extract is a promising bioactive extract due to presence of polyphenols and phytoecdysteroids. kayani et al. [18] . the results in current study agreed as phenols were detected (table 3) . similarly, previous study reported the presence of aromatic compounds, carbohydrates, glycosides, tannins, alkaloids, polyphenols, quinines and dions, aminophenols, steroids/sterols, flavonoids and terpenoids in a. parviflora methanol extract. in the present study flavonoids, carbohydrates, amino acids and phenols were detected, however alkaloids were not detected ( table 3 ). the reason for absence of alkaloids could be the difference in season of collection or age of the collected plants in the present and previous study. likewise, the presence of flavonoids, alkaloids, tannins, carbohydrates was reported in b. lycium [19] . all these constituents were also present in the currently investigated extract (table 3 ). in a previous study, the phytochemical analysis of citrus juice concentrates revealed the presence of flavonoids and saponins which are detected in the current study [20] . non-toxic plant extracts can be used for biological activities; therefore, it was important to find out non-toxic doses of all plant extracts. extracts of a bracteosa, a. parviflora, c. lemon were found nontoxic at the maximum concentration (200 μg/ml) while the b. lycium was observed toxic even at very low dose of 40 μg/ml therefore its concentration was kept at a minimum dose (20 μg/ml). in past, anti hcv activity has not been reported for a. bracteosa, a. parviflora extracts but another specie belonging to the same family lamiaceae has been reported to possess antiviral activity. ma et al. [21] found that the plant of a. decumbens poses potent antiviral activity against respiratory syncytial virus (rsv) with an ic 50 value of 131.6 μg/ ml. plant extracts (a. bracteosa, a. parviflora, b. lycium) investigated in current study showed significant activity against hcv at nontoxic concentration, while the c. lemon had negative effects in antiviral activity. antiviral efficacy of tested could be explained based on presence or absence of chemical constituents of plant extracts. from these results, it was concluded that the medicinal plants specially a. bracteosa and a. parviflora as well as b. lycium have a significant ability to combat with the hcv virus and reduce/stop the growth of the virus. these medicinal plants can be used as an alternative treatment option for hcv infection after further pre-clinical and clinical studies. in future, bioassay guided isolation can be performed to isolate bioactive anti-hcv compound(s) from these plants. consent were taken from all participants. this study was approved by research ethics committee, of comsats institute of information technology, abbottabad. all author agrees to publish the data. the authors declare that they have no competing interests. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. antiviral natural products and herbal medicines quantitative ethnobotanical survey of medicinal flora thriving in malakand pass hills phytotherapy of hypertension and diabetes in oriental morocco ethnobotanical studies of some plants of chagharzai valley, district buner, pakistan a new phthalic acid ester from ajuga bracteosa evaluation of the wound healing effect of some jordanian traditional medicinal plants formulated in pluronic f127 using mice (mus musculus) plants used as remedies antirheumatic and antineuralgic in the traditional medicine of lebanon global health sector strategy on viral hepatitis hepatitis c virus prevalence and genotype distribution in pakistan: comprehensive review of recent data antiviral activity of acacia nilotica against hepatitis c virus in liver infected cells management of adverse effects of peg-ifn and ribavirin therapy for hepatitis c ribavirin: current role in the optimal clinical management of chronic hepatitis c intestinal and vascular smooth muscle relaxant effect of viscum album explains its medicinal use in hyperactive gut disorders and hypertension phytochemical screening and extraction: a review qualitative and quantitative phytochemical analysis in four pteridophytes antiviral activities of indonesian medicinal plants in the east java region against hepatitis c virus therapeutic potential of taraxacum officinale against hcv ns5b polymerase: in-vitro and in silico study evaluation of ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities phytochemical and pharmacological screening of anti-inflammatory activity of berberis lycium root extract phytochemical, antimicrobial, and antioxidant activities of different citrus juice concentrates, food science & nutrition antiviral chinese medicinal herbs against respiratory syncytial virus we gratefully acknowledge pro. dr. idrees khan, director camb for their support and guidance. we are also thankful all participants whose provided serum for the current study. key: cord-261287-l4649du3 authors: puoti, massimo; zanini, barbara; quinzan, gian paolo; ravasio, laura; paraninfo, giuseppe; santantonio, teresa; rollo, adriano; artioli, stefania; maggiolo, franco; zaltron, serena; master hiv/hcv co-infection study group; raise, enzo; mignani, ermenegildo; resta, francesco; verucchi, gabriella; pastore, giuseppe; suter, fredy; carosi, giampiero title: a randomized, controlled trial of triple antiviral therapy as initial treatment of chronic hepatitis c in hiv-infected patients() date: 2004-05-06 journal: j hepatol doi: 10.1016/j.jhep.2004.04.016 sha: doc_id: 261287 cord_uid: l4649du3 background/aims: interferon and ribavirin combination therapy for chronic hepatitis c induces a low response rate in human immunodeficiency virus (hiv) infected patients. to assess the impact of intensification of interferon administration and of the addition of amantadine on the efficacy and safety of standard anti-hepatitis c virus (hcv) treatment in hiv-infected patients. methods: multicentre, prospective, open-label, randomized, phase iii clinical trial. eighty co-infected patients were randomized to receive ribavirin 800–1000 mg/day in combination with, group a: interferon alpha2a 3 miu thrice weekly; group b: ifnα2a 3 miu daily, plus amantadine 200 mg/day; treatment duration was 24–48 weeks according to hcv genotype. results: forty-one patients were randomized in group a and 39 in group b. intention-to-treat analysis showed a sustained virological response, defined as hcv-rna negativization, 6 months after stopping treatment in 22% of patients from group a and 13% from group b (p>0.05). the lack of a 2-log drop in hcv-rna levels after 12 weeks of treatment showed a 100% predictive value of lack of sustained response. conclusions: amantadine addition and interferon intensification do not improve the low efficacy of combination of interferon alfa plus ribavirin in hiv/hcv co-infected patients. patients with no early virologic response did not have any probability of sustained response. prevalence of hepatitis c virus (hcv) infection among anti-human immunodeficiency virus (hiv) seropositive patients with a history of intravenous drug use (idu) or transfusion is greater than 80%. the extensive use of highly active antiretroviral therapy (haart) has dramatically changed the prognosis of hiv infection, prolonging and improving life of anti-hiv seropositives [1] . on the other hand, mortality and morbidity for liver disease have increased significantly [2] . consequently, treatment of chronic hepatitis c in co-infected patients has become mandatory. interferon in combination with ribavirin was the gold standard for treatment of chronic hepatitis c in hiv-uninfected patients, inducing a 40% rate of sustained response [3] . however, a cumulative sustained virological response (svr) was observed in only 22% (95% confidence interval (ci), 14 -30%) of 111 patients enrolled in four pilot uncontrolled studies aiming to assess the efficacy and tolerability of ribavirin plus interferon alfa1 administered thrice weekly in hiv/hcv co-infected patients [4 -7] . so there is an urgent need for new and more effective treatment schedules for hepatitis c in hiv co-infected patients. among patients treated with interferon alfa three times weekly an intermittent increase of hcv viral load is observed on treatmentfree days [8] . daily administration of interferon could maintain a sustained antiviral effect on hcv; this schedule is expected to increase the efficacy of interferon alfa 2. amantadine (1-aminoamantadan) is a tricyclic amine with antiviral activity against toga-, myxo-, arena-, flavi-and coronavirus [9] . a comparative study recently demonstrated a statistically significant advantage of the addition of amantadine to interferon and ribavirin combination in hiv-uninfected patients with chronic hepatitis c and nonresponders to a previous cycle of interferon monotherapy [10] . thus addition of amantadine and intensification of interferon schedule with daily administration could be expected to increase the efficacy of standard combination treatment with ribavirin and interferon administered thrice weekly. in order to test this hypothesis we designed a multicentre, randomized controlled trial to compare the efficacy and safety of a new treatment schedule for chronic hepatitis c including ribavirin, amantadine and daily interferon alfa administration with the standard combination treatment. from april 2000 to october 2001, 80 hiv/hcv co-infected patients were consecutively enrolled in a multicentre, prospective, open-label, randomized, phase iii clinical trial. the study was conducted by the master hiv/hcv co-infection study group. eligibility criteria included: age between 18 and 60 years; alanine aminotransferase (alt) levels above the upper limit of normal (uln) 6 months before enrolment in the study; detectable plasma hcvrna by qualitative test (polymerase chain reaction (pcr) with amplicor w roche diagnostic system, hoffman-la roche, basel, switzerland); proven hivab seropositivity by elisa confirmed by western blot; stable hiv disease with cd4 cell count persistently over 300/ml during the last 8 months; anti-retroviral treatment (art) started at least 3 months before enrolment and demonstrated to be effective or no need for art; exclusion of hepatocellular carcinoma by imaging and alphafetoprotein level lower than 100 ng/ml; willingness not to consume alcohol during the treatment period. exclusion criteria were: reactivity for hepatitis b surface antigen (hbsag), neutropenia (fewer than 1500 neutrophils per ml), anaemia (less than 12 g/dl of haemoglobin in women and less than 13 g/dl in men), thrombocytopenia (fewer than 90,000 platelets per ml), decompensated liver disease, serum creatinine level more than 1.5 times the uln, poorly controlled psychiatric disease, alcohol or drug dependence in the year prior to enrolment; substantial coexisting medical conditions except hiv co-infection, previous treatment with ifn alfa or ribavirin or amantadine; systemic anti-neoplastic or immunomodulatory treatment in the preceding 6 months; current/present hiv-related opportunistic infection or malignancy classified as aids defining events (according to 1993 cdc aids surveillance case definitions); concomitant medication with rifampicin and/or rifabutin and/or isoniazid and/or pyrazinamide and/or gancyclovir; evidence of excessive alcohol consumption (.40 g in males and 20 g in females) and/or illegal substance abuse within the last 6 months; coexisting causes of liver disease; any additional contraindication to any of the drugs used in the study. additional exclusion criteria were pregnancy or lactation, and refusal to practise effective contraception during treatment and follow-up. this randomized controlled clinical trial was conducted at 15 italian centres from april 2000 to december 2002. patients were randomly assigned at a 1:1 ratio (with a block size of five) to receive: group a: interferon alpha 2a (ifna) 3 million units (miu) subcutaneously (sc) three times per week plus daily per oral3 (po) ribavirin (copegus w , roche, 200 mg tablets) or group b: ifna 2a 3 miu sc daily plus ribavirin (copegus w , roche, 200 mg tablets) and amantadine (mantadan w , boheringer ingelheim, 100 mg tablets) po 100 mg every 12 h. ribavirin was given orally with food at a dose of 800 mg/day (two tablets bid every 12 h) for patients ,75 kg of body weight and 1000 mg/day (two tablets in the morning and three in the evening after 12 h) for patients .75 kg of body weight. treatment duration differed according to hcv genotype: 24 weeks for hcv genotype 2 or 3, and 48 weeks for hcv genotype 1 or 4, only if hcvrna was negative at week 24 according to international guidelines [11] . randomization was centralized in the coordinating centre and stratified according to hcv genotype (genotype 1 or 4 vs. 2 or 3). genotype was performed by reverse hybridization assay (inno lipa hcv ii; innogenetics, ghent, belgium). patients were followed up for a treatment-free period of 24 weeks after cessation of therapy. the institutional review boards of the participating centres approved the protocol and all patients provided written informed consent. the study was conducted according to the declaration of helsinki, the applicable regulatory requirements and the ich/cpmp guidelines 'good clinical practice'. clinical examination, laboratory testing (including lactic acid and bicarbonate) and haematological count, including cd4 cell count, were performed monthly; plasma hivrna was monitored every 3 months using commercially available tests (quantiplex hiv-1 rna v.3.0 assay chiron corporation emeryville california, usa); hcvrna was measured at time 0 and after 1, 2, 4 and 12 weeks by a commercially available secondgeneration rt-pcr test (amplicor monitor version 2.0, roche diagnostic system, pleasanton, ca) according to the manufacturer's instructions. presence of hcvrna in plasma was determined at weeks 4, 12 and 24, at the end of treatment and 24 weeks after cessation of therapy using a commercially available second-generation rt-pcr test (amplicor hcv 2.0; roche diagnostic systems, pleasanton, ca) with a low end detection limit of 50 iu/ml according to the manufacturer's instructions. primary measures of efficacy were end-of-treatment response (eotr) and svr, respectively, defined as hcvrna levels below 50 iu/ml at the end of treatment, and 24 weeks after treatment. measures of safety were: any change in cd4 cell count, hivrna level, rate of withdrawal for adverse events (ae) or drop-out (do), rate of withdrawal from art or switch of anti-retrovirals. in difficult-to-treat patients (such as 'non-responders' to interferon monotherapy), triple therapy has been proven to increase by at least three times the sustained viral response rate obtained with standard interferon and ribavirin combination [10] . in order to establish that the svr in the triple therapy arm is at least three times higher than the 18% sustained response rate observed in hiv-co-infected patients treated with interferon and ribavirin in pilot studies [4] [5] [6] [7] , it was calculated that at least 64 patients should have been enrolled. a confidence probability of 80% and a significance level of 0.05 were used. intention to treat (itt) and per protocol (pp) analyses were performed. categorical variables were compared using fisher's exact test; distribution of continuous variables was compared using the t-test, mann-whitney two-sample statistic test or wilcoxon ranksum test. relationship between patients' baseline characteristics and svr was examined by univariate logistic regression analysis. to assess the independence of these factors, a multivariate logistic regression analysis was performed with backward selection ðp . 0:2þ: all p values reported are two-sided, and p was considered significant when ,0.05. statistical analysis was performed using stata software, version 7.0 (stata corporation, college station, tx, usa). eighty patients were enrolled in the study; 41 were randomly assigned to group a and 39 to group b; their baseline characteristics are shown in table 1 . no difference was found in demographics or clinical and immunovirological characteristics between the two groups. sixty patients (75%) were taking art, all for more than 3 months according to inclusion criteria, when they started anti-hcv treatment. the rates of hcvrna clearance in both groups are shown in table 2 . itt analysis showed 32.5% eotr, 31.7% in group a and 33.3% in group b. at the end of 6 months, follow-up response rates had decreased by about half: svr was observed in 17.5, 22% in group a and 12.9% in group b. differences in absolute hcvrna levels or hcvrna change-over baseline between the two groups at any time were not statistically significant. table 3 shows the distribution of some baseline characteristics in patients with and without svr; viroimmunologic and art characteristics were also analyzed, but the tests did not reveal a relationship with svr; genotype 2 or 3 and gamma glutamyl transferase (ggt) baseline level less than 1.5 times the upper limit of the normal range were more frequent in patients with svr (p , 0:05 fisher's exact test); univariate logistic regression showed their role as predictive factors of svr, with an odds ratio of 6 (1.2 -28.9 ci 95%) and 13.5 (1.6 -111.8 ci 95%), respectively. aspartate aminotransferase (ast) and neutrophil baseline levels were, respectively, lower and higher in patients who obtained an svr (p , 0:05 with mann -whitney test). by performing a multivariate logistic regression we found that genotype 2 or 3 and ggt baseline value were independent predictive factors for svr (table 4 ). by week 12, 32 of the 68 patients (47%) still on active treatment had a virologic response defined as a 2-log decrease from baseline hcvrna levels or no detectable serum hcvrna (fig. 1) . of those with a 12-week virologic response 14 (44%) subsequently had an svr, 5 dropped out (2 because of an adverse event and 3 spontaneously stopped treatment), 5 did have sustained hcvrna negativization, 2 showed a breakthrough after reducing the dose of ribavirin, and 6 patients relapsed after stopping treatment. eighteen out of 32 tested hcvrna negative at 12 weeks. the sustained response rate in this group (9/18, 50%) did not differ significantly from that observed in subjects with detectable hcvrna at 12 weeks (5/14; 36%). by contrast, of the 36 patients who did not have a 12-week virologic response 20 dropped out (8 because of ae and 12 prematurely stopped treatment), and 14 did not show hcvrna negativization; only two patients showed an etr, but none showed an svr. twenty-five out of 80 patients (31.3%) stopped treatment prematurely: 10 patients (18.8%) withdrew due to ae and 15 independently stopped therapy: distribution of ae and do was similar in the two treatment groups. pp analysis showed 32.1% of svr in group a and 14.8% in group b: there was not a significant difference between the two treatment arms. the treatment schedule was modified for more than showed an increase in hivrna level at any time during treatment and only 4 of them needed a switch of art due to loss of efficacy. no statistically significant difference was found in cd4 and hivrna levels between the two treatment groups at any time. eleven patients were undergoing treatment with didanosine, 39 with stavudine, and six with both of them; lactic acidosis was not observed in any patients. the main finding of this study was the very poor rate of svr to combination of standard interferon and ribavirin observed in both treatment arms, with and without amantadine and interferon dose intensification. cumulatively 17.5% of treated patients and 23.6% of those who completed the treatment course cleared hcvrna. this response rate is significantly lower than the 40% svr rate reported in trials performed on hiv seronegatives [12] the high withdrawal rate (31%), the large number of subjects requiring adjustment of the treatment schedule (31%) and the low response rate observed in those who completed the treatment schedule suggest that the tolerability and efficacy of interferon alfa and ribavirin in combination are reduced in hiv-co-infected subjects. the proportion of patients who needed interferon dose reduction was significantly higher in group b. we did not find a statistically significant difference in the rates of eotr and svr between the two treatment arms. therefore, the combination of interferon alfa schedule intensification and amantadine addition neither increase the efficacy nor improve the tolerability of combination therapy in co-infected hiv/hcv patients. clinical trials with pegylated interferon formulations [13, 14] in combination with ribavirin are ongoing in hiv/hcv-co-infected patients; preliminary results suggest that increased interferon levels induced by pegylated interferons could improve response rates in hiv/hcv-co-infected patients [15, 16] . amantadine addition did not improve the efficacy or tolerability of interferon alpha and ribavirin in chronic hcv infection. however, given the low power of this study, additional studies are needed before this drug is discarded from the therapeutic armamentarium for hiv/hcv co-infection. univariate logistic regression analysis of factors predictive of svr did not confirm that of age, sex, degree of fibrosis or baseline hcvrna levels, reported in hiv seronegative patients, but this was probably due to the small size of the sample under study [17] . the results of our study show that half of the patients with svr had in the past reached a cd4 cell count of less than 250/ml; such a low nadir of cd4 count in the years preceding treatment did not preclude achievement of a svr. these findings, together with the absence of an association between fibrosis score and cd4 counts and svr, suggest that progression of both hcv and hiv infections does not decrease the response rate to interferon and ribavirin combination in patients without severe immune depletion. we can therefore hypothesize that in patients without a high probability of response and early stage of liver fibrosis a watchful waiting strategy would not reduce the potential efficacy of anti-hcv treatment. this hypothesis needs to be confirmed by larger studies or by a meta-analysis of multiple pilot studies. hcv genotype, early hcvrna clearance and ggt levels were significant predictors of svr in multivariate analysis. the association between hcv genotypes 2 and 3 and a higher rate of svr is well known and has been confirmed in anti-hiv seropositives by pilot studies [4 -7] . all but two responders were infected by hcv genotype 2 or 3 and the svr rate in patients infected by genotype 1 or 4 was 5%. so, taking into account the side effects of treatment, these data suggest a very low cost effectiveness of treatment with standard interferon and ribavirin in hiv seropositives infected by hcv genotype 1 or 4. the association between earlier hcv clearance and svr suggests that suppression of hcv replication in the early phase of treatment is necessary but not sufficient to induce an svr. high serum ggt in chronic hepatitis c patients was frequently associated with more severe hepatic fibrosis or cirrhosis, or with steatosis, and may in part account for poor response to interferon therapy [18 -21] . ggt alteration in hiv seropositive patients is frequent and possibly correlated with one or more of the following factors: hepatic steatosis and/or lipidic dismetabolism, drug-related hepatic toxicity (especially in patients undergoing art), excessive alcoholic consumption, hcv-related hepatic damage (especially genotype 3) and immuno-mediated biliary duct damage. although all the included patients denied alcohol abuse, we cannot rule out that high ggt levels were due to undeclared excessive alcohol consumption. body mass index was not significantly associated with treatment response. only one of the svrs showed high ggt at baseline; in this patient ggt normalized the third month after hcvrna negativization. evaluation of art toxicity, careful assessment of alcohol intake and correction of alcohol abuse, diagnosis and treatment of altered lipid metabolism are factors to evaluate before starting treatment for chronic hepatitis c in patients co-infected with hiv. in this study the lack of a 2-log drop in hcvrna levels after 12 weeks of treatment showed a 100% negative predictive value of svr. although this data should be interpreted with caution, given the high proportion of dos, this result reinforces/supports the observation of a nearly absolute negative predictive value of the lack of a 12-week virologic response in hcv non-infected patients treated with pegylated interferons [13, 14] . as we enter an era in which hcv treatment is going to be pursued aggressively in hiv/hcv-co-infected persons, more and more emphasis must be placed on identifying at an early stage subjects who will not benefit from an expensive and poorly tolerated therapy. if these results are confirmed by larger studies, anti-hcv treatment could be stopped after 12 weeks, without decreasing the expected rate of svr, in hiv-infected patients who did not show at least a 2-log drop in hcvrna level. in conclusion, intensification of interferon alpha schedule and amantadine addition do not appear to improve the limited efficacy of standard combination therapy including interferon thrice weekly plus ribavirin for the treatment of chronic hepatitis c in hiv-co-infected patients. the best candidates for anti-hcv treatment are patients infected by hcv genotype 2 or 3 with normal ggt levels. the lack of a 2-log drop in hcvrna level after 12 weeks of treatment seems to be highly predictive of the poor efficacy of anti-hcv treatment. paolo and ospedale umberto i venezia-mestre) a.poggio, v. mondino (divisione di malattie infettive, ospedale di verbania), m. tinelli, mc. cerri (divisione di malattie infettive ospedale di lodi). mortality for liver disease in patients with hiv infection: a cohort study mortality due to chronic viral liver disease among patients with human immunodeficiency virus randomised trial of interferon a2b plu ribavirin for 48 weeks or for 24 weeks vs. interferon a2b plus placebo for 48 weeks for treatment of chronic hepatitis c virus chronic hepatitis c in hiv infection: feasibility and sustained efficacy of therapy with interferon alfa-2b and ribavirin long term efficacy of combination therapy with interferon alfa 2b and ribavirin for severe chronic hepatitis c in hiv infected patients interferon and ribavirin combination therapy in chronic hepatitis c in human immunodeficiency virus co-infected patients with congenital coagulation disorders pilot study of interferon alpha high-dose induction therapy in combination with ribavirin for chronic hepatitis c in hivco-infected patients the effects of a high dose, short course of interferon on hepatitis c a controlled trial of amantadine and rimantadine in the prophylaxis of influenza a infection triple antiviral therapy as a new option for patients with interferon non-responsive chronic hepatitis c international consensus conference on hepatitis c. consensus statement introduction to therapy of hepatitis c peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial multicenter, randomized trial comparing pegylated interferon alpha-2b (peg-ifn) plus ribavirin (rbv) vs. peg-ifn for treatment of hiv/hcv co-infected patients anrs hc02-ribavic: a randomised controlled trial of pegylatedinterferona-2b plus ribavirin vs interferona-2b plus ribavirin as primary treatment of chronic hepatitis c in hiv co-infected patients is an 'à la carte' combination interferon alfa-2b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis c? retreatment with interferon plus ribavirin of chronic hepatitis c non-responders to interferon monotherapy: a metaanalysis of individual patient data gamma-glutamyl transpeptidase as a response predictor when using alpha-interferon to treat hepatitis c treatment of chronic sporadic-type non-a, non-b hepatitis with lymphoblastoid interferon: gamma gt levels predictive for response steatosis accelerates the progression of liver damage of chronic hepatitis c patients and correlates with specific hcv genotype and visceral obesity the authors wish to thank monica bertoletti and angela braga for their invaluable help and technical assistance in preparing the manuscript. key: cord-001834-6xf4o3oy authors: sung, pil soo; shin, eui-cheol; yoon, seung kew title: interferon response in hepatitis c virus (hcv) infection: lessons from cell culture systems of hcv infection date: 2015-10-07 journal: int j mol sci doi: 10.3390/ijms161023683 sha: doc_id: 1834 cord_uid: 6xf4o3oy hepatitis c virus (hcv) is a positive-stranded rna virus that infects approximately 130–170 million people worldwide. in 2005, the first hcv infection system in cell culture was established using clone jfh-1, which was isolated from a japanese patient with fulminant hcv infection. jfh-1 replicates efficiently in hepatoma cells and infectious virion particles are released into the culture supernatant. the development of cell culture-derived hcv (hcvcc) systems has allowed us to understand how hosts respond to hcv infection and how hcv evades host responses. although the mechanisms underlying the different outcomes of hcv infection are not fully understood, innate immune responses seem to have a critical impact on the outcome of hcv infection, as demonstrated by the prognostic value of ifn-λ gene polymorphisms among patients with chronic hcv infection. herein, we review recent research on interferon response in hcv infection, particularly studies using hcvcc infection systems. hepatitis c virus (hcv) is a positive-stranded rna virus in the family flaviviridae, and it is estimated 130-170 million people are infected with hcv worldwide [1] . acute hcv infection is spontaneously cured in 20%-30% of patients, but the majority of infected patients fail to clear the virus and develop chronic persistent infection [2] [3] [4] . in addition to a combination regimen of pegylated interferon (ifn)-α and ribavirin, direct acting antiviral drugs (daas) against hcv have been developed, and a high rate of sustained virological response (svr) has been achieved by using these antiviral drugs [5] . however, the high cost of these drugs results in limited access in developing nations where the disease burden is high; therefore, there is still a need for the development of a prophylactic vaccine. until now, the pathogenesis of hcv infection has not been clearly elucidated yet. importantly, the detailed mechanism of innate immune activation by hcv and its implications for viral persistence and treatment response have not been clearly explained. therefore, understanding hcv-host interactions and immune responses are important novel therapeutics with a higher barrier to viral resistance can be developed [6] . however, there is no established small animal model for the study of the entire life cycle of hcv infection and immunopathogenesis [7] . severe combined immunodeficiency mice grafted with human hepatocytes are the only small animals that can be infected with hcv, although they cannot exert adaptive immune responses [8] . recently, genetically-humanized mouse models are being developed to recapitulate the entire life cycle of hcv [9, 10] , but these models have restricted replication of hcv, limiting their utility. as an experimental tool, development of cell culture-derived hcv (hcvcc) systems has dramatically facilitated hcv research over the last 10 years. here, we review recent advances in the research on innate immune response in hcv infection and focus primarily on interferon response of host cells. it was not until 2005, more than 15 years after the discovery of hcv [11] , that the first efficient cell culture model of hcv became available. the identification of a clinical isolate (genotype 2a) that replicates efficiently in huh-7 hepatoma cells [12] made the first cell culture system possible. this isolate was obtained from a japanese patient with fulminant hcv infection and was called jfh-1 [13] [14] [15] . viral particles produced by the transfection of huh-7 cells with in vitro transcribed jfh-1 rna could infect naïve cells in cell culture and the liver of chimpanzees in vivo [14] . the hcv virion particles derived from the cell culture system were named "hcvcc" [13] . until now, only jfh-1 spontaneously replicates in huh-7 cells without adaptive mutations and releases infectious virus particles [14, 15] . after the discovery of jfh-1-based hcvcc system, other hcv cell culture systems with various genotypes were established. for genotype 2 cell culture systems, j6cc (genotype 2a) [16] and j8cc/dh8cc/dh10cc (genotype 2b) [16, 17] were developed. they replicated and propagated efficiently in huh-7.5 cells, although they had adaptive mutations to facilitate their replication [16, 17] . the first genotype 1a strain, h77-s, replicated and released infectious particles in huh-7 cells and immortalized human hepatocytes, although the amount of released virus was lower than jfh-1 [18, 19] . the con1 (genotype 1b) cell culture system was also reported, but a very low level of replication has also limited its utility [20] . recently, a new cell culture system of genotype 1a was developed. the tn genome with eight mutations (tncc) [21] and h77c recombinant harboring 19 mutations (h77ccc) replicated and spread efficiently in huh-7.5 cells [22] . recently, a cell culture system for infectious genotype 3a was also established by introducing adaptive mutations into the s310 strain [23] . hcvcc system has some limitations that should be considered. the most important limitation is the restricted availability of genotypes established in cell culture models. currently, hcvcc systems for genotypes 4, 5, and 6 are unavailable. for genotypes 1 and 2, only specific patient clones have been propagated in cell culture systems. it should be noted, however, that a new host factor, sec14l2 was recently reported to enable replication of non-adapted hcv in hepatoma cells [24] . new cell culture system utilizing sec14l2-expressing hepatoma cells may overcome the limited availability of hcvcc system. another limitation of the current hcvcc system is the non-polarized nature of huh-7-based cells [25, 26] . hepatocytes are highly polarized in the liver and cell-to-cell transmission takes an important part in the spread of hcv, but the current hcvcc system does not reflect the viral spread occurring in the infected liver. in addition, huh-7 cells are not fully differentiated [27] and, thus, have a defect in activation of the innate immune response by hcvcc infection [28] . in primary human hepatocytes (phhs), replication and virus production by hcvcc infection have been reported [27] , but it is difficult to obtain phhs for experimental use. immortalized human hepatocyte was reported to support hcv genome replication, virus assembly, and robust ifn response against the virus [19, [29] [30] [31] and, thus, can be used as an alternative. differentiated hepatocyte-like cells (dhcs) induced from pluripotent stem cells have also been used for hcvcc infection [32] [33] [34] . dhcs were found to mount an efficient innate immune response after hcvcc infection, including the production of chemokines and type iii ifns [33] . recently, dhcs from adipose tissue-derived human mesenchymal stem cells (at-hmscs) were used for hcvcc infection [35] , and the entry and replication of hcvcc were found to occur efficiently in dhcs from at-hmscs. hcvcc infection systems provide a unique opportunity to study innate immune responses to hcv infection. here, we focus mainly on recent advances in the study of interferon response in hcv infection. in hcv-infected cells, viral rna is sensed by retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein 5 (mda-5) in the cytoplasm and toll-like receptor 3 (tlr3) in the endosome, which leads to downstream signaling that results in the induction of type iii and i ifns and other inflammatory cytokines [28, [36] [37] [38] [39] . among these receptors, a role of mda-5 in hcv sensing has remained controversial for several years, and it was recently proven that mda-5 also participates in hcv sensing in the cytoplasm using hcvcc infection systems [28, 36, 40] . intracellular signals from rig-i, mda-5, and tlr3 are transmitted via mitochondrial antiviral signaling protein (mavs) and toll/il-1 receptor domain-containing adaptor inducing ifn- (trif), respectively, which leads to the interferon regulatory factor-3 (irf-3)-dependent induction of ifns and nf-κb activation in hcv-infected cells [38, 39] . similar to other viruses, hcv uses several mechanisms to interfere with the induction of ifns, particularly ns3/4a protease. ns3/4a cleaves mavs, which leads to the impairment of signaling and ifn production in response to hcv rna [41] . mavs cleavage by ns3/4a has also been confirmed in hcv-infected liver tissue [42] . ns5a also contributes to immune evasion from the host. ifn-γ expression is inhibited in ns5a-transgenic mice after adenoviral challenge, meaning that ns5a plays an important role toward establishment of chronic hcv infection [43] . despite hcv interference with the induction of ifns, ifns are endogenously produced by hcv-infected cells [28, 29, [44] [45] [46] . both genotype 2a [28, 29, [44] [45] [46] and genotype 1a [29] were reported to activate intracellular interferon signaling pathways. ifns are currently classified into three major classes: type i, type ii, and type iii. among them, type i and iii ifns are considered innate immune response ifns. among type i ifns, there are 13 ifn-αs, in addition to ifn-β, ifn-ω, ifn-ε, and ifn-κ [47] . ifn-λs (ifn-1 or il-29; -2 or il-28a; and -3 or il-28b) are a new family of ifns that have been designated as type iii ifns. since the discovery of ifn-λs in 2003, their functions have been considered to overlap with type i ifns because signaling via the ifn- receptor is similar to that via the ifn-α/β receptor. after binding to their receptors, type i and iii ifns initiate a signaling cascade through the janus kinase (jak)-signal transducer and activator of transcription (stat) pathways. the cellular actions are then mediated by the induction of interferon-stimulated genes (isgs) that have antiviral and/or immunomodulatory activity [48, 49] . recently, it was demonstrated that ifn-s are major ifns produced by hcv-infected cells [28, [44] [45] [46] . ifn-s activate the same jak-stat pathway as type i ifns [48] [49] [50] , thereby inducing a similar set of isgs. although the exact source of ifn-s in hcv-infected liver remains to be clarified, it seems that the production of ifn-s by hcv-infected hepatocytes results in the expression of isgs, presumably through autocrine and/or paracrine signaling via the ifn-λ receptor [28, [44] [45] [46] . although hcv interferes with the induction of ifns, continuous isg up-regulation in hcv-infected liver has been demonstrated in chimpanzee models [51, 52] and hcv-infected patients [53, 54] . interestingly, hcv rna and isg mrna are detected simultaneously in hepatocytes from patients with chronic hcv infection [55] . this finding suggests that hcv infection potently stimulates the production of endogenous ifns, which leads to isg up-regulation in infected liver [55] , and that hcv survives under the isg up-regulation perhaps due to the protein kinase r (pkr)-mediated suppression of isg protein translation [56, 57] . as a rapid response to type iii and i ifns, ifn stimulated gene factor 3 (isgf3), which consists of tyrosine-phosphorylated stat1 (py-stat1), tyrosine-phosphorylated stat2 (py-stat2), and irf9, mediates the induction of numerous isgs, including stat1, stat2, and irf9 themselves [47] . recently, it was demonstrated that prolonged induction of a set of isgs is mediated by unphosphorylated isgf3 (u-isgf3), which is composed of unphosphorylated stat1 (u-stat1), unphosphorylated stat2 (u-stat2), and irf9 [58, 59] . the u-isgf3 level is increased by sustained exposure to ifns, and u-isgf3 leads to enhanced expression of a set of isgs (u-isgf3-downstream isgs, u-isgs) [58] . in other words, there appear to be two phases of isg expression following type iii or i ifn stimulation. the initial rapid response is driven by the classical phosphorylated form of isgf3, which is followed by a second, more prolonged response driven by u-isgf3 [58] . in line with this report, we recently demonstrated that endogenous production of type iii and i ifns by hcv infection increases the levels of u-isgf3, composed of u-stat1, u-stat2, and irf9 proteins [60] . using hcvcc infection systems with immune-competent liver cells such as phhs and tlr3-transfected huh7 cells, we demonstrated that u-isgf3 induces the expression of u-isgs [60] . as mentioned above, isgs induced by endogenous type iii or i ifns are up-regulated in hcv-infected liver [53, 54, [60] [61] [62] . representative isgs that are maintained at high levels of expression include isg15, ifi27, ifi44, mx1, and oas-1 [53, 54, [60] [61] [62] . these isgs are regulated not only by isgf3 but also by u-isgf3, and they are mainly antiviral [58, 60] . we recently found that increased levels of u-stat1, u-stat2, and irf9 are able to inhibit hcv rna replication without exogenous ifn treatment [60] . this finding suggests that the sustained expression of isgs by u-isgf3 has antiviral activity against hcv in the infected liver but is insufficient to clear the virus. previously, it was demonstrated that patients with high levels of isgs in their liver at baseline respond poorly to combined therapy with pegylated ifn- and ribavirin [53, 54, [60] [61] [62] [63] [64] . moreover, it has been shown that increased isg expression at baseline is a stronger predictor of a poor response to pegylated ifn-/ribavirin therapy than is the il28b genotype [53] . some reports have emphasized usp18 as a critical factor conferring unresponsiveness to exogenous ifn-α treatment by suppressing intracellular signaling [65] [66] [67] [68] . however, the mechanism underlying the increase and maintenance of usp18 protein levels in hcv-infected liver has not been clearly elucidated. recently, we found that prolonged exposure to ifn- up-regulates u-isgf3 and u-isgs, including isg15, and that isg15 causes the refractoriness to exogenous ifn- treatment by stabilizing usp18 protein [60] . in 2013, the gene ifnl4 was first described [69] . ifnl4 expression is influenced by a germline dinucleotide frameshift variant located in exon 1 of ifnl4 [69] . the ifnl4-∆g allele generates the full-length ifn-λ4 protein, whereas the ifnl4-tt allele does not create ifn-λ4 due to a premature stop [69] . the ifnl4-∆g allele is associated with a poor response to pegylated ifn-α/ribavirin therapy [69, 70] , and a recent study concluded that the ifnl4-∆g/tt genotype is the primary polymorphism underlying poor treatment response in hcv-infected patients [71] . another study showed that ifnl4-∆g genotype is associated with high levels of isgs and that hepatic levels of isg15 in chronic hepatitis c are strongly associated with ifn-λ4 expression, suggesting that ifn-λ4 contributes to induction of isgs in hcv-infected liver [72] . forced expression of ifnl4 gene up-regulates isgs in phhs and hepg2 cells [69, 73] , and has antiviral effects against hcv [74] . recombinant ifn-λ4 protein activates the jak-stat pathway through binding to the ifn-λ receptor [75] , evokes similar gene expression pattern to ifn-λ3 [76] . future study using hcvcc infection systems will explain the mechanism of ifn-λ4 induction in hcv-infected cells and the effects of endogenous ifn-λ4 on both isg induction and the response to exogenous ifn-α treatment. after hcv infection, the expression of some genes is down-regulated, and the expression of those genes tends to be further down-regulated in the liver of non-responders to ifn treatment [61, 77] . one of the genes down-regulated in hcv-infected liver is dual specificity phosphatase 1 (dusp1), a mitogen-activated protein kinase phosphatase (mkp) that de-phosphorylates mitogen-activated protein kinases (mapks) [78] . we demonstrated that silencing dusp1 expression inhibits hcv replication in hcvcc-infected cells and hcv replicon cells by up-regulating antiviral isgs [77] . dusp1 silencing enhances the nuclear translocation of stat1 and causes the induction of isgs [77] . although the detailed mechanism of dusp1 down-regulation in hcv-infected liver remains to be elucidated, this serves as an example of how hosts regulate the expression of isgs and restrict viral infection while bypassing endogenous ifn production. in virus-infected cells, viral peptides are processed and loaded onto major histocompatibility complex (mhc) class i molecules and presented to viral peptide-specific cd8 + cytotoxic t cells [79] . recently, we demonstrated using an hcvcc system that ifn-induced up-regulation of mhc class i molecules is attenuated by hcv infection [57] . hcv rna activates pkr, which phosphorylates the translation initiation factor eif2α to block the translation of proteins, including isgs [56] and mhc class i [57] . the attenuated expression of mhc class i by hcv infection causes a reduction of the effector functions of hcv-specific cd8 + t cells [57] . before our study, several studies had investigated the effect of hcv proteins on mhc class i expression with conflicting results. the expression of mhc class i was not affected by overexpression of hcv proteins in one study [80] , whereas another study showedup-regulation of mhc class i expression by the hcv core [81] . by using an hcvcc model, we were able to evaluate the effect of the whole life cycle of hcv infection on mhc class i expression, and we demonstrated the attenuation of ifn-induced mhc class i expression by hcvcc infection. the isolation of the jfh-1 clone and the establishment of hcvcc infection systems made it possible to perform various studies on host-virus interactions and innate immune responses against hcv infection. now is the era of daas, and hcvcc systems have greatly contributed to the advent of the daa era. however, much remains to be resolved. above all, the precise mechanism of interferon response and its paradoxical contribution to viral persistence should be elucidated. novel cell culture models that closely mimic host responses with various clinical strains are the prerequisite for understanding the pathogenesis of hcv infection and clarifying the mechanism of viral persistence. epidemiology and natural history of hcv infection hepatitis c virus-induced hepatocellular carcinoma hepatitis c virus and hepatocarcinogenesis cd8(+) t-cell responses in acute hepatitis c virus infection current and future therapies for hepatitis c virus infection antiviral resistance and direct-acting antiviral agents for hcv animal models for the study of hepatitis c virus infection and related liver disease hepatitis c virus replication in mice with chimeric human livers completion of the entire hepatitis c virus life cycle in genetically humanized mice murine models of hepatitis c: what can we look forward to? isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome efficient replication of the genotype 2a hepatitis c virus subgenomic replicon complete replication of hepatitis c virus in cell culture production of infectious hepatitis c virus in tissue culture from a cloned viral genome robust hepatitis c virus infection in vitro robust full-length hepatitis c virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains highly efficient infectious cell culture of three hepatitis c virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5a, and polymerase inhibitors production of infectious genotype 1a hepatitis c virus (hutchinson strain) in cultured human hepatoma cells generation of infectious hepatitis c virus in immortalized human hepatocytes production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations highly efficient full-length hepatitis c virus genotype 1 (strain tn) infectious culture system efficient infectious cell culture systems of the hepatitis c virus (hcv) prototype strains hcv-1 and h77 development of hepatitis c virus genotype 3a cell culture system sec14l2 enables pan-genotype hcv replication in cell culture the hcv life cycle: in vitro tissue culture systems and therapeutic targets hepatitis c virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum production of infectious hepatitis c virus in primary cultures of human adult hepatocytes hepg2 cells mount an effective antiviral interferon-lambda based innate immune response to hepatitis c virus infection hepatitis c virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes hepatitis c virus genotype 1a growth and induction of autophagy hepatitis c virus infection impairs irf-7 translocation and alpha interferon synthesis in immortalized human hepatocytes human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis c virus modeling hepatitis c virus infection using human induced pluripotent stem cells productive hepatitis c virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation microrna-27a modulates hcv infection in differentiated hepatocyte-like cells from adipose tissue-derived mesenchymal stem cells mda5 plays a critical role in interferon response during hepatitis c virus infection control of temporal activation of hepatitis c virus-induced interferon response by domain 2 of nonstructural protein 5a regulation of hepatic innate immunity by hepatitis c virus immune responses to hcv and other hepatitis viruses eftud2 is a novel innate immune regulator restricting hepatitis c virus infection through the rig-i/mda5 pathway cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system inhibition of intrahepatic gamma interferon production by hepatitis c virus nonstructural protein 5a in transgenic mice hepatitis c virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures il-29 is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection hcv infection induces a unique hepatic innate immune response associated with robust production of type iii interferons interferons at age 50: past, current and future impact on biomedicine ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il28b inhibits hepatitis c virus replication through the jak-stat pathway interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes genomic analysis of the host response to hepatitis c virus infection stealth and cunning: hepatitis b and hepatitis c viruses interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c interferon signaling and treatment outcome in chronic hepatitis c simultaneous detection of hepatitis c virus and interferon stimulated gene expression in infected human liver hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation hepatitis c virus attenuates interferon-induced major histocompatibility complex class i expression and decreases cd8+ t cell effector functions ifnβ-dependent increases in stat1, stat2, and irf9 mediate resistance to viruses and dna damage unphosphorylated stat1 prolongs the expression of interferon-induced immune regulatory genes roles of unphosphorylated isgf3 in hcv infection and interferon responsiveness hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection cell-type specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis c infection hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response hepatic isg expression is associated with genetic variation in interleukin 28b and the outcome of ifn therapy for chronic hepatitis c protein isgylation modulates the jak-stat signaling pathway silencing of usp18 potentiates the antiviral activity of interferon against hepatitis c virus infection interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp18/ubp43 interferon-β and interferon-λ signaling is not affected by interferon-induced refractoriness to interferon-α in vivo a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus ifn-λ4: the paradoxical new member of the interferon lambda family comparison of functional variants in ifnl4 and ifnl3 for association with hcv clearance hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis c: a phenotype-genotype correlation study prokunina-olsson, l. expression of interferon λ 4 is associated with reduced proliferation and increased cell death in human hepatic cells interferon-lambda4 is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden interferon λ 4 signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses transcriptome analysis reveals a classical interferon signature induced by ifnlambda4 in human primary cells suppression of dual specificity phosphatase i expression inhibits hepatitis c virus replication dual-specificity phosphatases: critical regulators with diverse cellular targets mhc class i antigen presentation: learning from viral evasion strategies expression of hepatitis c virus proteins does not interfere with major histocompatibility complex class i processing and presentation in vitro upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p53 and tap1 impairs natural killer cell cytotoxicity the authors declare no conflict of interest. key: cord-284904-qw1ig2v4 authors: mizui, tomokazu; yamashina, shunhei; tanida, isei; takei, yoshiyuki; ueno, takashi; sakamoto, naoya; ikejima, kenichi; kitamura, tsuneo; enomoto, nobuyuki; sakai, tatsuo; kominami, eiki; watanabe, sumio title: inhibition of hepatitis c virus replication by chloroquine targeting virus-associated autophagy date: 2009-09-17 journal: j gastroenterol doi: 10.1007/s00535-009-0132-9 sha: doc_id: 284904 cord_uid: qw1ig2v4 background: autophagy has been reported to play a pivotal role on the replication of various rna viruses. in this study, we investigated the role of autophagy on hepatitis c virus (hcv) rna replication and demonstrated anti-hcv effects of an autophagic proteolysis inhibitor, chloroquine. methods: induction of autophagy was evaluated following the transfection of hcv replicon to huh-7 cells. next, we investigated the replication of hcv subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. the effect on hcv replication was analyzed after transfection with sirna of atg5, atg7 and light-chain (lc)-3 to replicon cells. the antiviral effect of chloroquine and/or interferon-α (ifnα) was evaluated. results: the transfection of hcv replicon increased the number of autophagosomes to about twofold over untransfected cells. pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of hcv replicon. silencing of autophagy-related genes by sirna transfection significantly blunted the replication of hcv replicon. treatment of replicon cells with chloroquine suppressed the replication of the hcv replicon in a dose-dependent manner. furthermore, combination treatment of chloroquine to ifnα enhanced the antiviral effect of ifnα and prevented re-propagation of hcv replicon. protein kinase r was activated in cells treated with ifnα but not with chloroquine. incubation with chloroquine decreased degradation of long-lived protein leucine. conclusion: the results of this study suggest that the replication of hcv replicon utilizes machinery involving cellular autophagic proteolysis. the therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis c. the genome of hcv, a member of the family flaviviridae, consists of a positive-sense single-stranded rna. peginterferon/ribavirin combination therapy, which is the most effective therapy against hcv infection, is effective in around 50% for genotype 1 and 80% for genotypes 2 and 3 [1] [2] [3] , however, many people cannot tolerate the serious side effects and are resistant to peg-interferon/ribavirin combination therapy. difficulties in eradicating hcv are attributable to the limited number of treatment options against hcv [4, 5] . therefore, the search for novel therapeutic agents remains a strong aspiration. autophagy is an evolutionarily conserved cellular pathway in which the cytoplasm and organelles are engulfed within double-membraned vesicles, known as autophagosomes. while cellular autophagy is thought to be in preparation for the turnover and recycling of cellular constituents [6] [7] [8] , this process has been proposed as a mechanism of virus replication complex formation in positive-stranded rna viruses including poliovirus, equine arteritis virus and coronavirus [9] [10] [11] [12] . in these viruses, the replication complexes consist of double membrane vesicles in the cytoplasm, suggestive of an autophagosome origin [9, 12] . recently, it was reported that transfection of hcv replicon induced autophagy [11] . additionally, sir et al. [13] demonstrated that the suppression of autophagy inhibited the replication of hcv. these findings suggested that the autophagy plays a pivotal role in hcv replication. chloroquine, which is widely used for the treatment of malaria, is a well-established inhibitor of autophagic proteolysis which acts by inhibiting acidification of lysosomes and endosomes [14] . it has been reported that chloroquine exerts direct antiviral effects on several rna viruses including coronaviruses, flaviviruses and human immunodeficiency virus (hiv) [8, [15] [16] [17] . moreover, clinical studies have demonstrated the safety, tolerability, and efficacy of chloroquine in the antiviral treatment of hiv infection [18, 19] . here, we have demonstrated that autophagic proteolysis plays a pivotal role on hcv replication, moreover, the inhibition of autophagic proteolytic pathways can constitute an effective new therapeutic target against hcv. cell culture and treatment huh-7 cells were stably transfected with hcv replicon expressing chimeric protein of firefly luciferase and neomycin phosphotransferase [20, 21] . they were cultured in dulbecco's modified essential medium (dmem) (sigma, st. louis, mo) supplemented with 10% foetal bovine serum (fbs) at 37°c under 5% co 2 . to maintain cell lines carrying the hcv replicon, g418 (wako, osaka, japan) was added to the medium at a final concentration 500 lg/ml. luciferase activities were quantified to evaluate the replication of hcv replicon by a luminometer (lumat lb9507; berthold, germany) using a bright-glo luciferase assay system (promega, madison, wi). assays were performed in triplicate, and the results were expressed as mean ± sd as percents of controls. the viability of cells was assessed by wst-1 assay. cells were cultured in 96-well plates at 5 9 10 3 /well for 24 h, and then treated with 3-methyladenine [22] (10 mm), mixture of e64d (1 lg/ml) and pepstatin a [23] (1 lg/ml), and chloroquine (10 -6 -10 -3 m) for 18 h. cell proliferation reagent wst-1 (roche, swiss) was added to each well, and the cells were incubated for another 1 h at 37°c. the absorbance was measured against a background control by microplates reader (spectra max 340pc, molecular devices, sunnyvale, ca) at 450 nm. the reference wavelength was 650 nm. inhibition of autophagy and replication of hcv replicon cells were treated with 3-methyladenine (10 mm) or mixture of e64d (1 lg/ml) and pepstatin a (1 lg/ml), chloroquine (10 -7 -10 -3 m), interferon (ifn)a (100 u/ml) for 18 h, the levels of replication of hcv replicon were assessed by luciferase assay. moreover, cells were cultured with chloroquine (10 -5 m) and/or ifna (100 u/ml) for 7 days, then continued to incubate without drugs for another 21 days. replication levels of hcv replicon were determined by luciferase assay at 7th and 21st days from cessation of drugs. naïve huh7 cells, huh7/rep-feo cells, and huh7/rep-feo treated with ifna for 14 days were seeded on 30 mm dishes and incubated for 48 h. in addition, huh7/rep-feo cells were treated with chloroquine (10 -5 m) for 18 h. cells were prefixed with 2% glutaraldehyde, post-fixed with 1% osmic acid, dehydrated in graded ethanol, embedded in resin, and cut into sections on an ultramicrotome. the cells were analyzed by a transmission electron microscope (hitachi h7100, japan). the number of autolysosomes in 100 lm 2 of cytoplasm was counted by using transmission electron microscopy. small interfering rna knockdown of atg5, 7, lc-3 a combination of four chemically synthesized sirna duplex molecules targeted to the human atg5, 7, lc-3a, lc-3b mrna sequence (dharmacon, lafayette, co) was transiently transfected (final concentration 50 nm) into huh7/rep-feo cells using a transfection reagent (dharmacon, lafayette, co). sirna targeted to enhanced green fluorescence protein was used as a control. forty-eight hours after transfection, levels of hcv replication were analyzed by luciferase assay. twenty-five micrograms of total cell lysates were subjected to sds/page on a 10% gradient gel and electrophoretically transferred onto polyvinylidene fluoride membranes. after blocking with 5% non-fat dry milk in tris-buffered saline, membranes were incubated with primary rabbit monoclonal antibody against phospho-protein kinase r (p-pkr) (cell signaling technology, danvers, ma) or light-chain 3 (lc3), followed by a secondary horseradish peroxidase (hrp)-conjugated anti-rabbit igg antibody (cell signaling technology, danvers, ma). subsequently, specific bands were visualized using ecl detection kit (amersham pharmacia biotech, midland, on, canada). long lived protein is mainly degraded by autophagy [24] . cells were incubated with williams' e/10% fbs containing 0.5 lci/ml [ 14 c]leucine for 24 h to label long-lived proteins. cells were washed with williams' e/10% fbs containing 10 -5 m of unlabeled leucine and incubated with the medium for 2 h to allow degradation of short-lived proteins and minimize the incorporation of labeled leucine. the cells were then washed with phosphate-buffered saline (pbs) and incubated at 37°c with williams' e/10% fbs in the presence or absence of chloroquine (10 -5 m). after 4 h, aliquots of the medium were taken and a one-tenth volume of 100% trichloroacetic acid was added to each aliquot. the mixtures were centrifuged at 12,000g for 5 min, and the acid-soluble radioactivity was determined using a liquid scintillation counter. at the end of the experiment, the cultures were washed twice with pbs, and 1 ml of cold trichloroacetic acid was added to fix the cell proteins. the fixed cell monolayers were washed with trichloroacetic acid and dissolved in 1 ml of 1 n naoh at 37°c. radioactivity in an aliquot of 1 n naoh was determined by liquid scintillation counting. the percentage of protein degradation was calculated according to published procedures [25] . differences were compared using anova. basically p values less than 0.05 were considered as statistically significant. we counted numbers of autophagosome and autolysosome in cells transduced with hcv replicon rep-feo by using electron microscopy. double membrane vesicles with the morphology of autophagosomes were identified at 2.3 vacuoles/cells in naïve huh-7 cells, while transfection of hcv replicon increased the number of vacuoles to about fourfold over untransfected huh-7 cells (fig. 1a, b) . subsequent treatment of the cells with ifna (100 u/ml) for 14 days to eliminate hcv replicon substantially decreased the autophagolysosome in cytoplasm of huh7/rep-feo cells (fig. 1a, b) . these observations suggested that hcv replicon induces formation of autophagosomes. to clarify the role of autophagy on the replication of hcv, huh7/ rep-feo cells were treated with 3-methyladenine (10 mm) or a mixture of e64d (10 lg/ml) and pepstatin a (10 lg/ml) which inhibited autophagic protein degradation. replication level of hcv replicon in cells was increased to about twofold after 18 h in control media, however incubation with 3-methyladenine completely blunted increases in replication of hcv replicon. treatment with 3-methyladenine decreased the number of autophagosomes to about 19% of huh7/rep-feo cells. furthermore co-incubation with e64d and pepstatin a decreased replication of hcv replicon to about 66% of control (fig. 2a) . next, wst-1 assay was performed to check the cytotoxicity of these drugs. treatment with 3-methyladenine or a mixture of fig. 2 inhibition of autophagy suppressed replication of hcv replicon. a cells were treated with 3-methyladenine (3-ma) (10 mm) or a mixture of e64d (1 lg/ml) and pepstatin a (pep) (1 lg/ml) for 18 h, the levels of replication of hcv replicon were assessed by luciferase assay. b cells were treated with 3-methyladenine (10 mm), mixture of e64d (1 lg/ml) and pepstatin a (1 lg/ml) for 18 h. cell proliferation reagent wst-1 was added to each well, and the cells were incubated for 1 more hour at 37°c. the absorbance was measured against a background control by microplates reader at 450 nm. the reference wavelength was 650 nm. c a combination of four chemically synthesized sirna duplex molecules targeted to the human atg5, 7, lc-3a, lc-3b mrna sequence was transiently transfected into huh7/rep-feo cells using a transfection reagent. sirna targeted to enhanced green fluorescence protein was used as the control. forty-eight hours after transfection, levels of hcv replication were analyzed by luciferase assay e64d and pepstatin a did not affect cell viability (fig. 2b) . to clarify the role of autophagy induction in the replication of hcv, we suppressed the induction of autophagy by silencing autophagy-related genes (atg5, atg7, lc-3a and lc-3b) by sirna transfection. silencing of autophagy-related genes reduced the replication of hcv replicon to about 70% of control (fig. 2c) . transfection with sirna of autophagy related genes decreased the number of autophagosomes to about 30% of control. these results indicated that autophagy plays a pivotal role in replication of hcv. chloroquine inhibits the replication of hcv replicon next, we evaluated the anti-hcv effect of chloroquine, which is a lysosomotropic agent that raises intralysosomal ph and impairs autophagic protein degradation. to assess the effects of chloroquine on the intracellular replication of the hcv replicon, huh7/rep-feo cells were cultured with various concentrations of chloroquine in the medium. the replication of the hcv replicon was increased to about twofold within 18 h in the control media, however, which was suppressed by chloroquine in a dose-dependent manner (fig. 3a) . next, cytotoxicity of chloroquine was analysed by wst-1 assays. huh7/rep-feo cells treated with chloroquine showed no significant effect on cell viability in doses of lower than 10 -5 m (fig. 3b) . however, incubation with 10 -4 m of chloroquine reduced the cell viability to 25% of control. on the basis of the toxicity curve, the ic 50 of the drug was calculated to be 3.6 9 10 -5 m (fig. 3c) . the average ec 50 of chloroquine was calculated as 2.2 9 10 -7 m (fig. 3c) . the replication of hcv replicon was suppressed to nearly 40% of control at 10 -5 m of chloroquine, which did not affect cell viability. these data indicated that chloroquine efficiently inhibited the replication of hcv replicon in the absence of toxic effect to cells at the concentration of 10 -5 m. accordingly, we used 10 -5 m of chloroquine for the following study. next, we conducted the following assay to determine the synergistic inhibitory effect of chloroquine to ifna on hcv replication. treatment with chloroquine for 18 h . c calculation of ec 50 and ic 50 . concentration of chloroquine inhibiting 50% of the replication of hcv replicon is showed as ec 50 . ic 50 is the concentration of chloroquine which inhibits 50% of the cell proliferation of huh-7 rep/feo cells resulted in a significant decrease of hcv replicon to about 40% of control. on the other hand, incubation with ifna for 18 h inhibited the replication of hcv replicon to the levels about 15% of controls as expected. however, coincubation with 100u/ml of ifna and 10 -5 m of chloroquine further decreased hcv replication significantly (fig. 4a) . to determine whether long-term chloroquine treatment inhibits post-treatment re-propagation of hcv replicon, we followed up luciferase activity of the cells at the 7th and 21st days after 7 days of treatment with chloroquine and/or ifna (fig. 4b) . in hcv replicon cells treated by chloroquine, luciferase activities recovered to 53 and 88% on 7 and 21 days after cessation of treatment. in cells that were treated by ifna, luciferase activity maintained background level for 7 days post-treatment. however, it reappeared in 21 days. in sharp contrast, co-incubation with ifna and chloroquine for 7 days suppressed hcv replication for the extensive period up to 21 days, even in the absence of these drugs (fig. 4b) . anti-hcv effect of chloroquine independent of ifn signaling pathway ifn-inducible double-stranded rna-activated protein kinase r (pkr) plays a key antiviral role against hepatitis c virus [26, 27] . to elucidate the mechanisms of the inhibitory effect of chloroquine on hcv replication, phosphorylated pkr (p-pkr) was evaluated by western blotting analysis. p-pkr was detectable in cells treated with ifna after 24 h; this increase in p-pkr expression peaked at 24 h after ifna treatment and was reduced at 48 h (fig. 5a) . in contrast, p-pkr was not observed in cells treated with chloroquine at any time point. it is reported that chloroquine disrupts lysosomal function, preventing effective autophagic protein degradation, leading to the accumulation of ineffective autophagosomes [28] . therefore, we investigated if chloroquine led to the accumulation of autolysosomes as a result of suppression of proteolysis. we performed electron microscopic investigation to evaluate quantities of autophagosomes and autolysosomes. ultrastructural analysis identified 0.94 ± 0.1 vacuoles/100 lm 2 of autolysosomes in control cells; however, treatment with chloroquine increased the number of autolysosomes dramatically to about 13-fold over control (fig. 5b) . furthermore, the molecular form of lc3 protein of the cells, which is a component of autophagosomes, was examined by western blot analysis to ensure that chloroquine treatment leads to the accumulation of autophagosomes and autolysosomes. as shown in fig. 5c , immunopositive protein bands for lc3-i and lc3-ii forms were clearly evident in control cells. after chloroquine treatment, lc3-ii expression increased at 4 h (fig. 5c) to about threefold over control without enhancing lc3-i expression, and at 8 h (fig. 5c ) lc3-ii expression was further enhanced. finally, we evaluated turnover of the long-lived protein leucine, which was mainly degraded by autophagy. huh7/rep-feo cells were labeled with [ 14 c]leucine for 24 h, and degradation of [ 14 c]leucine in cells treated with or without chloroquine was measured. chloroquine treatment decreased degradation of leucin to 76% of control, indicating that chloroquine blunts degradation of proteins via an autophagic b assessment of re-propagation of hcv replicon after long term treatment of chloroquine and/or ifna. huh-7 rep/feo was incubated with chloroquine (10 -5 m) and/or ifna (100 u/ml) for 7 days, then drugs were removed from the medium and incubation continued for another 21 days. luciferase assay was performed at the 7th and 21st days from cessation of drugs. values are shown as percentages of the control cells [*p \ 0.05 vs. ifna treatment group (day 28) by anova] pathway (fig. 6) . these results demonstrate that chloroquine-induced the accumulation of autolysosomes was due to disruption of autophagic proteolysis. previous reports have disclosed that autophagy plays a pivotal role on the replication of several rna viruses [10] [11] [12] . our present results demonstrate that autophagy is induced by transfection of hcv replicon and is reduced by deletion of replicon due to ifna (fig. 1a, b) . these results suggest that autophagy is induced in the presence of hcv replication in its host cells. however, the role of autophagy in the pathogenesis of hcv is largely unclear. we found that the inhibition of autophagosome formation and autophagic proteolysis blunt the replication of genotype 1b subgenomic hcv replicon (fig. 2a, c) . sir et al. [13] reported that inhibition of autophagy also reduced the replication of the jfh1-based full length genotype 2a genome. therefore, the utilization of autophagy on viral replication is shown by hcv strains across different genotypes. on the other hand, not only a silencing of autophagic gene but also pharmacological inhibition of autophagic proteolysis possesses anti-hcv effects (fig. 2a, c) . however, treatment with both chloroquine and the mixture of e64d and pepstatin induced the accumulation of autophagosomes in cytoplasm. therefore, it is likely that hcv does not utilize the double membrane structure as the localization of the viral replication formation. these results support the hypothesis that protein degradation due to autophagy is important for hcv replication. chloroquine is a well-known inhibitor of autophagic protein degradation and is often used as an anti-malarial agent. moreover, the anti-viral effect of chloroquine on other rna viruses has been already reported in clinical trials [15, 16] . in our results, chloroquine inhibits the intracellular replication of an hcv replicon in a dosedependent manner (fig. 3a) . this antiviral effect of chloroquine was clearly not due to cytotoxic effects (fig. 3b) . moreover, chloroquine possesses a synergistic effect with ifna on hcv replication (fig. 4a) . although ifna possesses strong anti-hcv effects, re-propagation of hcv replicon was observed after 3 weeks following 7 days of treatment with ifna. interestingly, co-incubation with ifna and chloroquine for 7 days prevented repropagation of hcv replicon (fig. 4b) . chloroquine is a lysosomal weak base that is known to affect acid vesicles leading to dysfunction of several proteins [29] . it was demonstrated that disruption of lysosomal function impairs maturation of viruses through inhibiting the low-ph dependent proteases in trans-golgi vesicles in hiv and the sars coronavirus infection in vitro [15, 29] . however, little is understood about the mechanism of its antiviral effect. in previous reports, various drugs which possess inhibitory effects on the replication of hcv and have a synergistic action with ifna have been proposed as new therapeutic agents to treat hcv. some of them have proved to exhibit their anti-hcv effects through augmentation of ifn-induced antiviral gene responses [30, 31] . however, the anti-hcv effect of chloroquine was not associated with activation of one of ifn receptors signaling molecule pkr (fig. 5a) . our results showed chloroquine induced the accumulation of ineffective autophagosomes in cytoplasm of huh7/rep-feo cells (fig. 5b) and inhibited the degradation of long-lived protein leucine (fig. 6) . these findings imply that chloroquine effectively impairs the function of autophagy in our experiment. these results indicated that chloroquine is a new anti-hcv agent that targets the autophagic proteolysis. previous reports have shown that chloroquine possesses anti-viral effects on various rna viruses. its beststudied effects are those against hiv replication, which are being tested in clinical trials [17, 18] . hcv coinfection is common in hiv-positive patients in usa and europe [32, 33] . since hiv infection accelerates the progression of hcv-related liver disease, treatment of hcv is generally recommended. however, co-infected patients have a greater risk of antiretroviral therapy-associated hepatotoxicity than patients with hiv only [34] . moreover, treatment with ribavirin is believed to increase the risk of anemia in patients taking the hiv drug zidovudine [35] . a clinical study designed for hiv patients showed the safety and efficacy of chloroquine used for long terms up to 48 weeks [36] . therefore, the combination therapy of interferon and chloroquine is, possibly, a hopeful therapy for hcv-hiv co-infected patients. since chloroquine is known as one of the inexpensive drugs, therefore, chloroquine might provide a new effective, safe and economical therapeutic option for patients with hcv. in conclusion, autophagic proteolysis might be a new therapeutic target on the replication of hcv. drug resistance in antiviral treatment for infections with hepatitis b and c viruses sustained virological response to interferonalpha is associated with improved outcome in hcv-related cirrhosis: a retrospective study peginterferon alfa-2a in patients with chronic hepatitis c and cirrhosis poor response to pegylated interferon and ribavirin in older women infected with hepatitis c virus of genotype 1b in high viral load efficacy of ribavirin plus interferon-a in patients aged 60 years with chronic hepatitis c the molecular mechanism of autophagy autophagy as a regulated pathway of cellular degradation autophagy in the eukaryotic cell coronavirus replication complex formation utilizes components of cellular autophagy acidotropic amines inhibit proteolytic processing of flavivirus prm protein formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response effect of weak bases on the intralysosomal ph in mouse peritoneal macrophages chloroquine is a potent inhibitor of sars coronavirus infection and spread anti-hiv effects of chloroquine: mechanisms of inhibition and spectrum of activity effects of chloroquine on viral infections: an old drug against today's diseases? comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1 hydroxyurea and didanosine as initial therapy for hiv-infected patients with low viral load: safety, efficacy and resistance profile after 144 weeks synergistic inhibition of intracellular hepatitis c virus replication by combination of ribavirin and interferonalpha inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas 3-methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes autolysosomal membrane-associated betaine homocysteine methyltransferase. limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy autophagy as a regulated pathway of cellular degradation protein degradation in 3t3 cells and tumorigenic transformed 3t3 cells pkr-a protein kinase regulated by double-stranded rna evidence that hepatitis c virus resistance to interferon is mediated through repression of the pkr protein kinase by the nonstructural 5a protein comparison of different autophagic vacuoles with regard to ultrastracture, enzymatic composition, and degradation capacity-formation of crinosomes chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion new therapies for chronic hepatitis c virus infection combination of a hepatitis c virus ns3-ns4a protease inhibitor and alpha interferon synergistically inhibits viral rna replication and facilitates viral rna clearance in replicon cells hepatitis c in the hiv (human immunodeficiency virus) atlanta v.a. (veterans affairs medical center) cohort study (havacs): the effect of coinfection on survival weinbreck p. seroprevalence of hbv, hcv and hdv hepatitis markers in 500 patients infected with the human immunodeficiency virus therapeutic issues in hiv/hcvcoinfected patients zidovudine use but not weight-based ribavirin dosing impacts anaemia during hcv treatment in hiv-infected persons hydroxychloroquine, hydroxycarbamide, and didanosine as economic treatment for hiv-1 key: cord-006436-61mgtbj4 authors: yoshiba, makoto; sekiyama, kazuhiko; iwamura, yukari; sugata, famio title: development of reliable artificial liver support (als)-plasma exchange in combination with hemodiafiltration using high-performance membranes date: 1993 journal: dig dis sci doi: 10.1007/bf01316501 sha: doc_id: 6436 cord_uid: 61mgtbj4 a new artificial liver support system (alss) consisting of plasma exchange (pe) in combination with hemodiafiltration (hdf) using high-performance membranes of polymethyl metacrylate (pmma) and cellulose triacetate (cta) was developed to efficiently remove middle molecules from plasma and treat fulminant hepatic failure (fhf) complicated, by the onset of hepatic coma. twenty-seven patients with fhf due to viral hepatitis, two with type a (ha), nine with type b (hb), and 16 with type non-a, non-b (nanb) underwent therapy with this new alss over the last five years. three patients, with an exacerbation of chronic hb and 15/16 with type nanb hepatitis were treated with interferon (ifn) also. of these, 25 patients (92.6%), regained consciousness and 15 (55.6%) [1/2 (50%) with type a, 6/9 (66.7%) with type b and 8/16 (50%) with type nanb hepatitis] survived including four patients who survived with intensive, care and plasma exchange alone, 19/31 (61.3%) patients survived. because of its biocompatibility, both survivors and nonsurvivors could be sustained with the alss without complications for long periods (19.3 days for the survivors and 32.4 days for nonsurvivors). with this alss the ability to sustain life for such prolonged periods allows hepatic regeneration to occur and result in patient survival. it is anticipated that this new alss will not only be of value in cases of fulminant hepatic failure but that it may also play a role in sustaining life for those, awaiting liver transplantation. tients include exchange transfusion (et) (1), plasma exchange (pe) (2) , charcoal hemoperfusion (hp) (3), charcoal plasma peffusion (pp) (4) , and hemodiafiltration (hdf) using a polyacrylonitrile (pan) membrane (5) . the effect of et on survival of fhf patients was not demonstrated by a controlled trial (6) . although charcoal hp was at one time claimed to have achieved a high survival rate (10/22) in an open study (3) , its effectiveness could not be confirmed by a subsequent large scale controlled trial (7) . the survival rate with pan-hdf has been reported at 21.5% in one report (5) , and as being 33% in another (8) . due to the relatively poor therapeutic effectiveness of these alss, liver transplantation currently is advocated as the best treatment for fhf (9, 10) . unfortunately, not all patients with fhf can be treated with liver transplantation because compatible donor livers are not readily obtainable (11) . worse yet, liver transplant patients, particularly those with viral liver diseases, frequently experience recurrent viral liver disease, which can on occasion present as fhf (12) . the liver plays a central role in the metabolism of various materials, including carbohydrates, proteins, amino acids, lipids, vitamins, and hormones. from a clinical viewpoint, however, two functions are particularly important. these are: (1) the production of plasma proteins, especially coagulation factors; and (2) the metabolism of various molecules having a potential for central nervous system (cns) toxicity. loss of these two functions is responsible for the two major clinical problems associated with fhf: bleeding and coma. thus restoration of these two hepatic functions is a prerequisite for an effective alss. the poor effect of all existing alss can be attributed to a failure to either completely or at least partially restore these two functions. in japan pe is currently the most frequently used method of liver support in cases of fhf because of the technical advances in the practice of pe and the readily available supply of fresh frozen plasma (ffp) provided by the japanese red cross. pe serves the two critically important hepatic functions required of an effective alss by supplying plasma components that are synthesized by the liver and removing toxic materials that are normally metabolized by the liver. experience has shown that although pe is effective in restoring plasma components, it has only a limited value in removing putative neurotoxic metabolites from a large body pool (13) . as a result, most patients with severe fhf die of coma despite aggressive pe. with the aim of developing a more reliable alss, we have combined pe with a method capable of efficient and safe removal of putative neurotoxic substances thought to be responsible for hepatic coma. we have developed hdf using a recently developed high-performance membrane with large pores, which allows proteins to be filtered for the effective removal of middle-sized molecules (mms), which have been thought to be responsible for hepatic coma (14) . preliminary use of this new alss has shown it to be effective in reversing grade v hepatic coma in a patient with fulminant liver failure (15) . in the present communication, the experience in a substantially larger number of patients with viral-induced fhf treated with alss is reported. the subjects in the present study were 27 consecutive patients with fhf including six with late-onset hepatic failure (lohf), with coma occurring 8-24 weeks after the onset of hepatitis (16) . all were treated at showa university fujigaoka hospital in the last five years. in each case their coma had not resolved with standard intensive care. virological studies. the methods utilized for the determination of various hepatitis markers were as follows: igm ha-ab, hbsag , and igm hbc ab were determined by ria; anti-hcv (c-100-3) was measured using a hcv-ab elisa kit (ortho diagnostic systems); hbv dna polymerase (dna-p) was assayed; hbv dna was detected using the polymerase chain reaction (pcr), using primers capable of amplifying nucleotide (nt) sequences 1653-1972; hcv rna was assayed by a twostage pcr using two pairs of oligonucleotides from the 5'-noncoding region of hcv as primers (17); anti-core for hcv was determined by elisa using two core peptides (cp9 and cp10) (18, 19) . in order to avoid false positive results for the various hcv markers due to contamination by blood donor plasma, all virus markers were assayed prior to the start of the plasma exchange (pe) therapy. type a hepatitis was diagnosed by the identification of igm ha-ab. acute type b hepatitis was diagnosed by the presence of a positive hbsag and/or igm hbc-ab. two patients were negative for hbsag but had igm hb c and were diagnosed as having type b hepatitis. both were positive for hbv dna by pcr. an acute exacerbation of hb in an hbv carrier was diagnosed by a history of known hbsag positivity for more than six months, the presence of hbv dna and hbv dna-p, and a markedly elevated igg hb c level. type c hepatitis was diagnosed by the presence of either a positive anti-c-100, hcv rna or hcv anti-core result. morphological and histological diagnosis. all patients underwent ultrasonographic (us) and computer tomographic (ct) examinations for determination of liver volume. us was performed on admission and at least once a week thereafter. ct scanning was performed on admission and at least twice a week thereafter. laparoscopy was performed in 12 of the 15 patients. liver biopsy was obtained in 11 of the 12 patients who underwent laparoscopic examinations. three serial biopsies (5, 4 & 3) were obtained in three patients. four patients had two biopsies. an autopsy was obtained in 10 of the 12 patients that died. alss. the high-performance membranes used were a polymethyl metacrylate membrane (pmma, bk series: toray medical inc., tokyo) and a cellulose triacetate membrane (cta, nipro co., tokyo), which were originally developed for the removal of e~2-microglobulin (mol wt 118.00) (20) . according to the manufacturer, the pmma and the cta membranes had an inulin (mol wt 5200) permeability three times greater than the pan membrane used by others. before its clinical use, the biocompatibility of the pmma membrane was assessed in dogs (21) . the biocompatibility of the cta membrane was assessed directly with its clinical use. adverse effects, including hemolysis, significant platelet loss, and dic attributable to either the pmma hdf or cta hdf, were not observed. blood access was established with a double-lumen catheter inserted into a central vein. two patients who required continuous alss for more than three months had a surgical a-v fistula created using the dorsalis pedis artery. a 10-mg bolus of nafamostat mesilate, a proteinase inhibitor (futhan, torii pharmaceutical co., tokyo) was added to the alss circuit and was delivered at the rate of 20 mg/hr in the course of the alss treatment just prior to the initiation of pe. pe was performed using a membrane separation method marketed as plasmaflo (asahi medical co., tokyo). ffp was supplied by the japanese red cross kawasaki blood center (kawasaki city, kanagawa prefecture). the amount of ffp used during pe was adjusted to keep the patient's pt level above 30% and averaged 3.5 liters. blood, the plasma of which was exchanged as ffp, was drawn into the circuit of the pmma or cta hdf. hdf was performed using a commercially available hdf apparatus (tr 701: toray medical inc., tokyo). filtration was performed at a flow rate of 4-6 liters/hr using a bicarbonate buffer, ph 7.4, having a potassium concentration of 4.0 meq/liter. the volume of the substitution fluid was adjusted over a range of 6-30 liters, depending on the patient's response. dialysis was performed concomitantly at a flow rate of 500 ml/min using a conventional acetate buffer. the circuit diagrams for the alss are shown in figure 1 . all three patients with an acute exacerbation of hbv and 11 of the 15 patients with nanb fhf were treated with ifn-13 daily at a dose of 300 mu for 3-60 days. two patients were diagnosed as having type a hepatitis. six patients had acute type b hepatitis. three patients had an acute exacerbation of their chronic hb carrier state. all three developed fhf following repeated intensive chemotherapy for non-hodgkin's lymphoma in spite of being negative for hbeag and having completely normal liver function tests before the start of their chemotherapy. the remaining 15 patients were diagnosed as having non-a, non-b hepatitis because of negative tests for all hav and hbv markers together with negative tests for other viruses known to cause hepatitis and negative results when tested for the presence of various autoantibodies. three nanb patients were positive for all three hcv markers assessed. four nanb patients were positive for two of the hcv markers utilized and six were positive for only one hcv marker. thus, 13 of 16 nanb patients were diagnosed as having type c hepatitis. both patients with type a hepatitis were positive for one or another hcv marker. this high rate of superinfection in type b hepatitis is explained by the fact that all six had a history of prior blood transfusion. thus, 21/27 (77.8%) had evidence for recent or past hcv infection. the cause of the fhf in the hav group was assessed to the hav because the igm ha level was elevated throughout the course of the liver failure. the cause of the fhf appeared to be hbv in four patients because their liver failure resolved after the cessation of identifiable hbv replication. in one patient, however, liver failure was protracted after the loss of hbsag. hcv infection may have played a role in the hepatic failure in this particular case. fifty-five out of 27 patients (92.6%) regained consciousness, and 15 patients (one patient with type a, six patients with type b, and eight patients with type nanb) survived. twelve patients (one patient with type a, three patients type b, and eight patients type nanb) died. the survival rate was 1/2 (50%) for type a, 6/9 (67%) for hepatitis b, and 8/16 (50%) for hepatitis nanb. another two hb patients with fh, one with hepatitis a + c and one with fatty liver of pregnancy survived with pe alone. overall, 19 of 31 patients (61.3%) with fhf treated at this institution survived. 6 ---6. t liters) (p < 0.01) for those who died. thus hdf was utilized more intensively in the latter cases but to no avail. the alss was initiated at grade i coma in two, at grade ii coma in six, and at grade iii coma in seven of the 15 surviving patients. it was started at grade ii coma in 10, at grade iii coma in one, and at grade iv coma in one of the 12 patients who died. no significant difference in coma grade between survivors and nonsurvivors was evident at the time the alss was started. the 27 patients included in this study could be divided into three groups (group i: survivors; group ii: patients who died of either a complication or interruption of the alss, and group iii: patients who died of coma despite alss). several parameters reflecting residual hepatic function in each of these three groups were compared including minimum prothrombin time (pt), maximum total bilirubin (tbil), ratio of direct bilirubin to total bilirubin (dbil/tbil) and minimum bun level. the minimum pt level was 22.6 ___ 11.3%, 16.3 ___ 14.7%, and 14.0 __+_ 12.3 min in groups i, ii, and iii, respectively. because of the wide variability in each group, these values did not differ between groups. the maximum yoshiba et al tbil level was 17.7 ___ 9.2, 26.2 ___ 3.4 and 24.8 mg/dl, respectively, for groups i-iii; again no group differences were evident. the minimum dbil/tbil was 0.58 ---0.12, 0.49 ___ 0.17, and 0.19 ---0.14, respectively, for groups i-iii. this parameter statistically distinguished group i from group iii (p < 0.01) and group ii from group iii (p < 0.02). the minimum bun level was 10.3 __. 3.9, 10.3 _+ 6.6 and 2.7 __-3.9 mg/dl for groups i-iii, respectively. this value distinguished group i from group iii (p < 0.001) and group ii from group iii (p < 0.01). the hepatic volume measured by abdominal ct was 507-1427 ml (average 895 __-412 ml) for 14 of the 15 patients in group i. liver weight at autopsy ranged from 450 to 1040 g (average 688 ---218 g) in four of the five patients in group ii who died. the liver volume of the one patient who was not autopsied was 356 ml by ct scan. liver weight at autopsy was 210-790 g (average 495 _+ 227 g) in six of the seven patients in group iii who died. the liver volume of the one patient who was not autopsied in this group was 585 ml by ct at the time of death. the complications of hepatic failure experienced in this series, in addition to coma, included pulmonary aspergillosis, sepsis, adult respiratory distress syndrome (ards), and bleeding from esophageal varices. each of these caused a patient's death. nonvariceal gastrointestinal bleeding occurred in one surviving and six nonsurviving patients. renal failure occurred in one surviving patient and two patients who died. brain edema detected by ct was noted in two surviving patients and in three patients who died. in each case, it occurred during grade iv or v coma. fungal and bacterial infection and ards occurred in seven surviving patients and in one patient who died. in the following case report, the course of alss treatment of a 30-year-old female who had the lowest (worst case) dbil/tbil, bun, and liver weight values in this series is described. the patient maintained consciousness while ingesting a 60-g protein 1600-cal diet with the use of alss requiring 51 liters of ffp and 30 liters of replacement fluid. her illness had begun with nausea and dark urine on february 17, 1989 on admission, physical examination revealed profound jaundice, anemia, and grade ii coma. liver dullness was only 6 cm to percussion. the results of her laboratory tests on admission demonstrated an abnormally low direct bilirubin level compared with the level of the total bilirubin and a very low bun level. her pt was low at 29.3% despite two runs of pe on each of the preceding two days. fischer's ratio (plasma val + leu + ile/try + phe) was low at 0.57 (normal range 3.0-4.0), and her plasma gin was high at 2414 nmol/ml (normal range 478.3-658.3 nmol/ml). after three additional runs of pe using 4.8 liters of ffp and cta hdf using 30 liters of fluid, she regained consciousness. although her pt transiently rose to around 60% after pe, it regularly fell to levels of 20-25% just prior to the start of the next pe. any reduction in the amount of substitution fluid to a value less than 24 liters invariably induced coma. her dbil/tbil ratio fell to its lowest value of 0.05 on the 36th hospital day (dbil 1.0 mg/dl and tbil 20.1 mg/dl). her bun level fell to its lowest value 0.6 mg/dl on the 14th hospital day (figure 2) . because consciousness could be maintained by daily pe using 4.8 liters of ffp and cta hdf utilizing 30 liters of the substitution fluid, oral nutrition was started with 300 cal of carbohydrate beginning on the 6th hospital day. protein was added to the diet on the 10th hospital day with an increase in the caloric intake to 800 cal. she was finally placed on a 60-g protein, 1600-cal diet without any signs of encephalopathy. moreover, she occasionally enjoyed overnight stays at home between alss sessions which lasted about 10 hr. she regularly experienced severe hypoglycemic episodes in the early morning hours. serial abdominal ct obtained every other week showed progressive shrinkage of her liver (figure 3) . the calculated fischer's ratio fell as low as 0.22 and the plasma gin level often exceeded 10,000 nmol/ml after the initiation of the oral diet. after the 100th hospital day she began to lapse into coma again despite continuation of the alss, and ultimately she died of deepening coma on the ll5th hospital day. at autopsy, her liver weight was only 210 g. histologic examination revealed only a few residual liver parenchymal cells forming pseudo-bile ductules around the portal areas ( figure 4 ). a new alss system consisting of pe and hdf using high-performance membranes [polymethyl metacrylate (pmma) and cellulose triacetate (cta)] was devised to enhance the removal of the middle-sized molecules thought to be responsible for hepatic coma. before its use clinically, the biocompatibility of the pmma membrane was as-sessed using dogs with experimental fhf, because previous experience with pp using uncoated charcoal had shown that, although charcoal pp is effective in relieving coma in patients with mild liver failure, it causes severe bleeding due to thrombocytopenia. subsequent experimental studies using dogs with experimental f h f disclosed that the charcoal pp enhanced coagulation and fibrinolysis resulting in severe dic (22) . activation of factor xii on the surface of uncoated activated charcoal may be responsible for this untoward phenomenon. unlike charcoal, the pmma membrane used in this system is biocompatible and both the pmma and the cta membrane produced no adverse clinical effects. the effectiveness of this new alss is demonstrated by the observation that 25/27 (92.6%) patients with fhf treated with it completely recovered consciousness from grade i-v coma. six of these 23 patients lapsed into coma again as hepatic regeneration failed to occur. nonetheless, this high rate of transient as well as complete recovery from hepatic coma is in marked contrast with the 50% value reported for charcoal hp (3), 17/41 (43.6%) (5), and 9/24 (37.5%) (8) for the pan hdf. moreover, it is considerably better than the 14/26 (53.8%) recovery reported by us using charcoal pp (4). the four patients who failed to respond to the alss and the six patients who relapsed into coma all had either markedly atrophied livers on admission or experienced progressive hepatic atrophy despite alss. this point is documented by the average liver weight of seven autopsied cases, which was 460 + 170 g. the lowest mean serum dbil/tbil ratio that reflects hepatic bilirubin conjugation capacity was 0.19 ---0.14 for the seven patients who died. their mean lowest bun level, which reflects overall urea cycle capacity of the liver, was markedly reduced at 2.7 +__ 3.9 mg/dl. moreover the extremely high plasma gin level, the end product of an alternative ammonia detoxifying pathway, further documents the severity of the urea cycle dysfunction in these seven cases. it is noteworthy that even the most severe case, in which liver weight was only 210 g at autopsy and in which occurred the lowest dbil/tbil ratio and bun levels at 0.05 and 0.6 mg/dl, respectively, could be maintained conscious while ingesting a 60-g protein 1600-cal diet while on the alss and requiring 4.8 liters of ffp and 30 liters of substitution fluid. most importantly, the effectiveness of this alss was demonstrated by the high rate (15/27, 55.6%) of survival among patients who received the therapy. this survival rate has to be compared with that reported for et (0/8, 0%) (6), for charcoal hp started at grade iv coma (10/22, 45.5%) (3), for pan hdf in two reports (9/42, 22.0% (5) and 8/24, 33.3% (8)), and for charcoal pp in our own hands (5/26, 19.2%) (4). in the present study the alss was instituted at a coma grade of less than iii in 26 of the 27 patients. the early application of alss may have contributed to the high survival rate (23) . it needs to be recalled, however, that the major determinant of prognosis in cases of fhf is the etiology of fhf. thus, with charcoal hp while 54.7% and 63.6% of patients in grade iii coma due to acetaminophen overdose or hepatitis a and b survive, only 12.5% and 33.3% of patients with nanb and halotheme hepatitis, respectively, survive (24) . virological study of the 27 patients in this study disclosed that two had type a, nine had type b, and 16 had nanb hepatitis. evidence for hcv infection was present in 13 of 16 patients with nanb, both of the type a hepatitis cases, and six of nine with hepatitis b also had evidence of prior hcv infection. thus, the hcv infection rate in this series was 21/27 (77.8%). the high rate of coinfection in the cases with hepatitis a was unexpected. the high prevalence of hcv in cases of hepatitis b is explained by the fact that all of them had received blood transfusion before the onset of their fhf. currently it is not known how hcv infection influences the clinical course of either hepatitis a or b associated fh. the two cases with hepatitis a in the present series experienced unusually protracted liver failure. one of these, although manifesting the clinical features of acute hepatitis at the onset, ultimately died of bleeding of an esophageal varix with a hepatic histology consistent with rapidly progressive cirrhosis. another patient with hepatitis b failed to recover despite clearance of hbsag. the liver histology at autopsy showed active hepatitis probably due to coinfection with hcv. despite the high prevalence of hcv coinfection, the survival rate in hepatitis a, b, and nanb in this series was 1/2 (50%), 6/9 (67%), and 8/16 (50%), respectively. this very high survival rate in cases of nanb is remarkable and may have been due to the use of the alss. alss appears to sustain the patient free of complications sufficiently long until the liver is able to regenerate and regain adequate function to sustain life. an average duration of alss use of 19.3 days for survivors and 32.4 days for nonsurvivors stands in marked contrast to the 3, 4, and 8.6 days for et, charcoal, and pan hdf (6, 7, 8) reported by others in similar types of cases. the high survival rate in the cases with nanb and acute exacerbation of hb carriers may be attributable to the use of ifn. it is generally believed that the hb virus is eliminated at the onset of fhf as a result of the host immune response (25, 26) . in nanb hepatitis, however, replication of the responsible virus persists, as shown by the findings of hcv rna in seven of the 14 nanb patients tested in this study. persistent viral replication in such cases presum-ably allows continued liver cell necrosis to occur and renders fhf caused by hcv intractable to current clinical management. ifn may play a role in related hcv and may help to induce viral clearance. the only treatment that may have helped the seven patients who died in this series that was not used is liver transplantation. unfortunately this therapy is not available in japan currently. treatment of hepatic coma by exchange blood transfusion plasmapheresis in hepatic coma charcoal hemoperfusion in the treatment of fulminant hepatic failure new artificial liver support system (plasma perfusion detoxification) for hepatic coma treatment of encephalopathy during fulminant hepatic failure by hemodialysis with high permeability membrane controlled trial of exchangetransfusion therapy in fulminant hepatitis controlled trials of charcoal hemoperfusion and prognostic factors in fulminant hepatic failure treatment of fulminant hepatic failure by polyacrilonitrile membrane hemodialysis theft dhv: liver transplantation fulminant hepatic failure and orthotopic liver transplantation orthotopic liver transplantation for acute and subacute hepatic failure in adults liver transplantation in patients with b viral hepatitis and delta infection plasmapheresis in acute liver failure. in plasmapheresis therapeutic applications and new techniques, y nose significance of middle molecules in the pathogenesis of hepatic encephalopathy hemodiafiltration treatment of deep hepatic coma by protein passing membrane: case report late onset hepatic failure: clinical, serological and histological features detection of hepatitis c virus rna by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region enzyme-linked immunosorbent assay for antibodies against the capsid protein of hepatitis c virus with a synthetic oligopeptide antibodies against synthetic oligopeptides deduced from the putative core gene for the diagnosis of hepatitis c virus infection evaluation of beta2-microglobulin removal with high-performance hemodiafiltration biocompatibility of polymethyl metacrylate (pmma) membrane---effect on blood coagulation and the fibrinolysis system aggravation of coagulation abnormality by charcoal plasma perfusion in dogs with experimental acute liver failure earlier charcoal haemoperfusion in fulminant hepatic failure early indicators of prognosis in fulminant hepatic failure multiplication of hepatitis b virus in fulminant hepatitis b hepatitis b virus dna in fulminant hepatitis b authors are greatly indebted to staffs of the emergency center and the dialysis center of showa university fujigaoka hospital for their dedication to the treatment of fhf patients. professor van thiel's advice and discussion in preparing this manuscript is greatly acknowledged.this study is partly supported by a grant-in-aid for scientific research of the japanese ministry of education, science and culture, and the ministry of health and welfare. key: cord-264713-38dlh3wg authors: vernet, guy title: molecular diagnostics in virology date: 2004-08-20 journal: j clin virol doi: 10.1016/j.jcv.2004.06.003 sha: doc_id: 264713 cord_uid: 38dlh3wg molecular biology has significantly improved diagnosis in the field of clinical virology. virus discovery and rapid implementation of diagnostic tests for newly discovered viruses has strongly beneficiated from the development of molecular techniques. viral load and antiviral resistance or subtyping assays are now part of the biological monitoring of patients chronically infected by human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv) and cmv. it will be important to add to this panel assays for other viruses of the herpesviridae family. qualitative assays for the detection of blood-borne viruses have increased safety of blood donation and organ transplantation. screening of other blood-borne viruses (parvovirus b19, hav), multiplexing of detection and test automation to improve practicability and reduce costs will be the next steps. a major evolution in the near future will be the generalization of nat for the diagnosis of viral etiology in patients, mostly with respiratory, cns or gastro-intestinal diseases. major technical improvements have been made to avoid obstacles that still limit this generalization, i.e. genetic variability of viruses, multiplex detection, contamination risk. commercial offers already exist but menus must be extended to limit the validation and documentation work associated with home-brew assays. real-time amplification has allowed the development of new nat platforms but automation and integration of all steps of the reaction are still required to reduce hands-on-time, time-to-result and costs, and to increase throughput. molecular biology has revolutionized all domains of viruses diagnosis including the rapid identification of emerging or re-emerging viruses, viral safety of blood products or organ transplants and viral disease management. one of the major driving forces for the introduction of molecular techniques in virology has been the absence of easy and performing multiplication techniques similar to those developed for bacteriology. the most striking illustration of the power of molecular techniques concerns blood transmitted viruses-human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) for which spectacular progresses in the detection and treatment of viral diseases have been made following the introduction of qualitative and quantitative nucleic acid tests (nat). the recent discovery of a new human coronavirus responsible for severe acute respiratory syndrome (sars) epidemic is another example. it is obvious that nat will encourage the development of antiviral drugs which, compared to antibiotics, has been delayed partly because performing efficiency assessment techniques were lacking. during the last 15 years, many new human pathogens have been discovered among which eight were viruses with various pathogenicity. molecular techniques have played a central role in their discovery (table 1) . the construction of cdna libraries by cloning techniques has been used to identify hcv and hepatitis e virus. representational difference analysis (rda) was successful in identifying human herpes virus 8 (hhv-8), and the hepatic viruses gbv-c and ttv. rda allows the detection of viral sequences by comparing whole nucleic acids sequences in cells from humans or animals before and after infection. reverse-trancription polymerase chain reaction (rt-pcr) with random or degenerate primers has been used to identify human metapneumovirus (hmpv), the virus sen and the coronavirus associated to sars (sars-cov). molecular techniques alone have allowed the characterization of very important human pathogens like hhv-8, responsible of kaposi sarcoma or hcv which induce acute or chronic hepatitis, cirrhosis and hepatocarcinoma. however, other viruses detected in a similar way are still waiting for the demonstration of their clinical importance. this illustrates the need to verify koch's postulate and the importance of keeping laboratory competencies for classical virology-tissue culture, electron microscopy and animal experiments-which plays a major role in virus discovery, together with epidemiological studies. large epidemiological studies are also required to assess the clinical interest of new pathogens. molecular diagnostics have been very rapidly implemented in clinical virology laboratories following the discovery of hmpv (van den hoogen et al., 2001; peiris et al., 2003; boivin et al., 2003) and sars-cov (ksiazek et al., 2003; drosten et al., 2003; anderson, 2003) . a prospective study on the prevalence of hmpv could be initiated as early as during the 2000-2001 winter, although the virus has been discovered in 2000 only. there has been only a few weeks lag between sequencing sars-cov and availability of the first commercial nat. nat are more and more used to exclude blood donations from patients infected by viruses (allain, 2003) . hcv and hiv testing has been implemented as part of routine screening in blood banks in 14 and 7 european countries respectively. in france, two commercial offers -procleix (gen-probe, inc. usa) and nuclisens extractor (biomerieux, france) associated with cobas ampliscreen (roche diagnostics, switzerland)-are used to screen donations for hiv and hcv infections. this allowed the reduction of residual risk from 1 in 400,000 to 1 in 2.5 millions for hiv and 1 in 760,000 to 1 in 5 millions for hcv (assal et al., 2003) . however, there is room from improvement as few contaminated blood units are still missed due to the lack of sensitivity induced by pooling strategies. cost constraints must also be considered if further improvements are to be considered. cost effectiveness of hiv and hcv nat addition to serology testing is already very low: in usa it has been calculated that the cost of each saved life is 4.7-11.2 millions us$ per year (jackson et al., 2003) . hepatitis b virus screening using molecular biology should also be included as even the most recent antigen assays (hbsag) assays miss infected blood units. as an example, single-sample hbv testing would allow the detection of 35-50 additional contaminated units among 10 7 units tested (biswas et al., 2003) . monovalent or trivalent assays (hiv, hbv, hcv) are proposed by roche diagnostics (ampli nat) and gen-probe (procleix ultrio) but blood units should not be pooled to provide sufficient sensitivity. procleix ultrio detects single seroconversions 20 days earlier than abbott prism-hbsag but only 13 days in pools of 8 and 11.5 days in pools of 24 (cambié, 2002) . alternatively, an ultracentrifugation step could be introduced following pooling to obtain a higher sensitivity compared to current antigen assays (roth et al., 2002) . screening for west-nile virus contamination has been implemented in us blood banks. from late june to mid-september 2003, approximately 2.5 million donations were screened. twelve hundred eighty-five (0.05%) were initially reactive for wnv by using nucleic acid-amplification tests and 601 (0.02% of the total donations) are considered presumptive viremic donations (i.e. a donation that is repeatedly reactive by the primary and/or alternate nat assay or a primary nat assay with a very high signal) (cdc, 2003) .other viruses (parvovirus b19, hepatitis a virus) are also transmitted by blood donation and may be part of the screening in a near future. however, nat may not always be the best diagnosis approach and antigen tests or antibody tests may be efficient and less expensive alternatives. automation and integration of nat is necessary to reduce costs and quarantine delays for blood units supply. in this respect, the recent approval by us food and drug administration (fda) of the tigris molecular diagnostic system (gen-probe, san diego, usa) is a major breakthrough as it has been designed to process 500 samples in 8 h. integration and time-to-result are also very important parameters to be considered to insure viral safety of transplant organs, especially lung, heart and liver. it is very important to determine the status of transplants regarding infection by hiv, hbv, hcv and viruses from the herpesviridae family. nat have significantly improved identification of viruses as etiologic agents of human diseases affecting various organs, especially respiratory and gastro-enteric tracts and central nervous system (cns). as a consequence, rapid antiviral treatments can be initiated and considerably reduce morbidity and mortality as, for example, in the case of herpes encephalitis. the increasing number of available antiviral drugs will even accentuate the need for positive viral identification. similarly, unnecessary antibiotic treatments can be avoided or reduced and hospitalisation durations shorten. treatments of chronic viral diseases are very efficiently monitored by viral load assays. however there are still obstacles that prevent a wider dissemination of molecular assays. the extreme genetic variability of some viruses, especially rna viruses (which rna-polymerases have no proofreading activity), often makes their diagnosis difficult. the most striking examples are found in the norovirus family which contains viruses responsible for the vast majority of gastro-intestinal epidemics in adults. hiv diagnosis is also quite difficult to achieve due to its high genetic variability. gardner et al. (2003) have deduced from sequence alignments that a real-time taqman assay should contain not less than nine primer and probe sets to detect with the same sensitivity all hiv strains in a geographically representative panel. to reduce the impact of variability on amplification and detection efficiency, one can use primers and probes with 2 o-methyl bases, degenerate bases or "universal" bases, such as inosine or nebularine. touchdown pcr protocols, in which the annealing temperature slightly decreases during the successive amplification cycles to bracket the melting temperature tm of the reaction, provides sensitivity even when primers have mismatches with target sequences of divergent species in a viral family. finally, degenerate primers or probes, with mixtures of the bases found in sequence databases among various species, may be useful to detect all species of a viral family. it is often desirable to provide the capability for panel detection, i.e. to detect several viruses that can be responsible for a disease. for example, coyle et al. have described at the winter meeting of european society for clinical virology (copenhagen, january 2004) a molecular viral respiratory strip for the detection of 12 common respiratory viruses. whenever possible, consensus primers able to detect all viruses from a family or genus must be used. there are several examples of such consensus primers for enterovirus (kammerer et al., 1994) , flavivirus (scaramozzino et al., 2001) or herpesviridae (tenorio et al., 1993) . however, their ability to amplify all viruses with the same efficiency must be carefully evaluated. if such an approach is not possible, two different possibilities exist for panel detection: multiplex detection in single tubes or parallel detection in individual tubes. mixing primers in a single amplification tube to achieve multiplex detection of viruses usually results in decreased sensitivity of assays compared to single tests. for example, we have observed, using a dna-microarray assay (see below), that the analytical sensitivity of multiplex rt-pcr detection of six viruses, i.e. influenza a, influenza b, rsv a/b, parainfluenza 1, 2 and 3 is reduced by a factor of <1-2 logs compared to single detections, depending on the virus. nevertheless, this multiplex assay was able to identify correctly 21/22 infections in respiratory specimen (one rsv b infection was misidentified as rsv a; unpublished data). the formation of primer dimers is generally considered as the major cause of sensitivity loss but careful optimisation of all parameters of amplification including primer, enzymes, nucleotides and salts concentrations as well as protocol conditions are required to obtain expected performances. realtime assays (see below) that monitor signal apparition during the amplification step are also limited in their capacity to realize multiplex detection by the number of available wavelengths in existing equipments which currently allows the detection of three viruses only. instead of mixing several pairs of primers in a single tube, nucleic acids purified from the original clinical specimen can be distributed into several tubes for independent amplification and detection. major drawbacks of this approach are the reduction of sensitivity because of lower amounts of nucleic acid available for each individual amplification, higher hands-on times required to manipulate all the different tubes, difficulty to automate the distribution of small volumes of purified nucleic acids without introducing cross-contaminations and higher costs due to the need for enzymes in each tube. internal controls (ic) are important components to monitor each step of the assay from extraction of nucleic acids to detection. because inhibitors of the amplification reaction, which are frequent in some specimen types, will also impact its amplification, the presence of an ic is a strong validation in case of negative result. niesters (2002) has described an original approach for internal control of nat: the use of viral universal controls that can be added to each specimen and be amplified with specific primers, preferably in a multiplex format with primers for the virus to detect. seal herpes virus 1 and phocine distemper virus can be used to control nat for dna and rna viruses respectively. of course, animal viruses which can not infect humans are required. other strategies for ic involve synthetic materials, i.e. plasmids or transcripts which contain sequences able to bind the test primers and a specific probe. clinical virology laboratories have high expectations in term of automation and integration of molecular assays. a major bottleneck in the workflow of these laboratories is at the level of sample preparation. table 2 shows automated systems for nucleic acid purification that are currently commercialised. most of them use the nucleic acid binding properties of silica (boom et al., 1990) . as many as 96 samples can be handled in a single run on the biorobot 9604 (qiagen gmbh, germany). time-to-result and, more important, hands-on-time tend to decrease, with the most recent equipments requiring no longer than 20 min of technician time for more than 24 samples. new labelling technologies that do not need solid-phase separation have allowed the development of real-time molecular assays in which the detection of amplicons is done as soon as they appear during amplification. the most simple real-time detection chemistry uses the sybr green dye which specifically binds during double-stranded dna generated during pcr. several probe technologies (fig. 1 ) have also been designed for real-time assays like taqman ( fig. 1a ) or molecular beacons (fig. 1b) in which a quencher molecule is removed from the vicinity of the fluorescent marker upon binding to rna or dna generated during amplification cycles. in the fret technology (fig. 1c) , two probes, one with a fluorescence donor and one with a fluorescence acceptor molecule are designed to bind adjacent sequences of the amplified material to generate signal (mackay et al., 2002) . real-time techniques have been designed for pcr or nasba amplification and have several advantages which facilitate automation and reduce time-toresult (30-120 min) and hands-on-times. they are performed in closed devices which do not need to be opened to transfer amplified material for end-point detection, thus reducing the risk of laboratory or samples cross-contamination. the use of real-time platforms makes the general organisation of molecular biology laboratories easier by reducing the constraints on activities segregation in different rooms to control contaminations. table 3 shows existing real-time automates. the next generation of nat platforms will integrate sample preparation, amplification and detection in a single test device genexpert (cepheid, usa) is the first fully integrated system which allows the detection of bacillus anthracis or group b streptococcus in approximately 45 min directly from clinical specimen. several viral assays based on real-time nasba on this platform will soon be available from biomerieux. several ivd companies offer commercial nat for the diagnosis of viral diseases. besides technical difficulties, another obstacle to the development of molecular assays is the importance of resources needed to optimise, produce and validate home-brew assays and to build up documentation required for qualification of techniques and laboratories. new european community regulations will even increase this need. it is the role of in vitro diagnostic (ivd) companies to provide reagents which are the results of careful optimisation and are produced according to high quality manufacturing procedures. they have clinical and regulatory affairs departments that conduct validation studies and assemble documentation required to get approval of the reagents. however, even for ivd companies, the conception and validation process is time-consuming and timeto-market may be long. this is especially a problem when a diagnostic tool is urgently needed in case of emergence of a new virus. one possibility to reduce time-to-market is to release "research use only" (ruo) assays or assays that have the "ce analytical" approval in europe or the status of "analyte specific reagent" (asr) in the usa. in this case, commercial products that have excellent analytical sensitivity and are manufactured according to quality standards of the ivd industry can be used by clinical virology laboratories, which have the responsibility to validate their use as diagnostic tools and obtain authorization to use them. infections by hiv and hepatitis b and c viruses are usually well diagnosed using serology. only diagnosis of early primo-infections may benefit from nat. platforms like amplicor amplicor from roche diagnostics or easyq from biomerieux that are usually used for viral load measurement during therapy (see below) are also suitable for the early detection of hiv or hcv infections. many other infections and especially acute infections for which igm appear only several days after onset of symptoms cannot be efficiently diagnosed using serology assays. forty to sixty percent of community acquired pneumonia that require hospitalisation 2 have no known aetiology despite intensive investigation and this percentage is even higher when less severe lower respiratory tract infections (lrti) are considered (l. kaiser, personal communication). in a recent study, henrickson et al. (2004) , have shown, using multiplex rt-pcr that 40% of children hospitalised for lrti are infected with the seven most common respiratory viruses. similarly, a recent survey of encephalitis leading to hospitalisation in the usa from 1988 to 1997 has revealed that nearly 60% had no aetiology (khetsuriani et al., 2002) . absence of specific antiviral treatments for most viruses, which limits prescription of biological tests and weak performances of diagnostic tests based on viral culture or serology are major explanations of this situation. nat, which have been implemented by many large european hospitals, significantly improve viral diagnosis. however, there are several obstacles to the generalization of molecular diagnostics in smaller, decentralized laboratories. a major obstacle is the fact that, except for hiv, hbv, hcv and cmv, virology nat are most often home-brew assays which sometimes suffer from bad performances and poor batch to batch consistency. however, quality of nat is the percentage of false-positive results which reflects laboratory or cross-contaminations and used to be high, dropped to 5.5% (wallace et al., 2003) . table 5 shows some products currently commercialised by major companies for viruses other than hiv, hbv and hcv, although the list may not be exhaustive. most of them are asr or ruo kits although some are ec marked. the majority of these reagents have been designed to run on realtime platforms. results shown by liolios et al. in 2001 illustrate the interest of multiplex detection of respiratory viruses by nat. one hundred forty-three clinical specimen were tested using the hexaplex assay from prodesse inc. (usa) which detects six viruses in a single tube using pcr and detection with microplate capture and peroxidase-labelled probes. 9 samples were detected with the prodesse assay only and not with immunofluorescence or viral culture (table 6) . table 6 multiplex nat for the detection of respiratory viruses (hexaplex, prodesse inc.) dna-microarrays or dna-chips are very powerful detection tools that can be combined with amplification techniques to detect viruses or virus variants (reviewed in clewley, 2004) . wang et al. (2002) have described a microarray spotted with 70-mer oligonucleotides which represent the five most conserved sequences (more than 20/70 bases are conserved among all representative sequences in a virus sequence alignment) of each virus of interest. the chip contains 1600 different probes. following pcr amplification of genetic material in the clinical specimen using random primers, hybridisation onto the microarray was able to identify respiratory viruses in the enterovirus, rhinovirus, paramyxovirus, adenovirus and herpesvirus families. however, this technique has not yet been extensively validated for routine diagnosis in a clinical virology laboratory. we have developed assays combining rt-pcr and dna-microarrays for the detection of viruses. the arrays are manufactured using the photolithography in situ synthesis technology (affymetrix, usa) and contain 20-mer oligonucleotides. sequence signatures are identified using extensive sequence databases because they are conserved in all viruses of a genus or family or in all subtypes or isolates of a virus and are not found in other viruses. for each base of the signature, probes perfectly matching the target and probes with a mismatch at the interrogated position are present on the array. if polymorphisms are present in or near the signature, variant probes may also be present. consensus primers have been designed for enterovirus, flavivirus, herpesviridae, parainfluenza and influenza virus, rsv and adenovirus. they can be combined in multiplex detection pcr or rt-pcr assays for the diagnosis of viral respiratory or cns infections. following amplification, dna is labelled using a newly developed diazomethyl chemistry (laayoun et al., 2003) . a complete line of instruments (sample preparation, thermocycler, hybridisation station and laser scanner) is available to perform the assay. as described above, a respiratory assay designed to detect six major viruses (parainfluenza 1, 2 and 3, rsv a/b, influenza a and b) in a single specimen has demonstrated high clinical sensitivity in preliminary evaluation. fig. 2 illustrates the discriminatory capacity of this technology for cns viruses. assays for the identification of human papilloma virus (hpv) are commercialised by several companies. the detection of a highly pathogenic hpv type has a very high predictive value for cervical carcinoma. however, the number of hpv types that are more or less closely associated to cervical cancer is high. dna-microarrays may thus be appropriate for the multiplex detection of all these types. such an assay has been described by and is distributed by biomedlab co. (south-korea). it is based on a consensus amplification and on 30-mer probes that are able to detect and discriminate 22 hpv types and has shown an association of 92.5-97.5% between hpv positivity and lesions of different severity or carcinoma whereas hpv infection was only found in 35.1% of cases when cytology was normal. viral load platforms are available from several ivd companies (versant from bayer diagnostics; cobas ampliprep/ amplicor from roche diagnostics, minimag/nuclisens easyq from biomerieux). significant progress have been made in the ability of most hiv assays to detect all subtypes of hiv-1 but no commercial assay exist for hiv-2. the analytical sensitivity of hbv viral load assays should be increased to reach the same performances as those of hiv assays, especially in the case of infections by variants with low replication competencies. for efficient treatment monitoring of immunosuppressed patients, for example in the case of organ transplantation, viral load assays should also be developed for epstein-barr virus, varicella-zooster virus and hhv6. genotyping tests are now commercially available and are part of the biological follow-up of treated patients although home-brew assays are still used in most laboratories (korn et al., 2003) . hiv and hbv resistance assays as well as hcv subtyping assays are generally based on the sequencing technology (trugene hiv and hbv from bayer healthcare; vi-roseq hiv-1 from celera diagnostics; 5 genotyping hcv kit from bayer healthcare). hybridisation techniques, such as lipa assays (innogenetics, belgium) are also used. however, the number of probes that can be spotted on nitrocellulose strips is limited and only few polymorphisms can be detected which is convenient for hcv subtyping but does not for resistance tests which are based on the detection of a high number of mutations. in addition, hiv and hbv have highly variable genomes and naturally occurring polymorphisms that are present in the vicinity of resistance mutations may affect the binding efficiency of probes. dna-microarrays are an alternative for resistance or typing reagents for viruses or bacteria . we have developed an assay based on rt-pcr and detection with fig. 2 . biomerieux dna-microarray for the detection of neurotropic viruses. following nucleic acid purification, amplification is performed by pcr using a single touchdown protocol in three tubes, one for herpesviridae (one primer pair for hhv 1, 2, 3, 5 and 6), one for enteroviruses (one primer pair for all serotypes) and one for flaviviruses (one primer pair for all viruses). amplification products are combined and labelled using diazomethyl chemistry and hybridised on a dna-microaaray which contains 20-mer oligonucleotides. two or four probes are used for the detection of each base of sequence signatures determined for each virus. a total of 20,000 probes are synthesised on this dna-microarray which has been designed for the simultaneous detection of viruses from the herpesviridae family and from the flavivirus, enterovirus, paramyxovirus, poliomavirus, bunyavirus and orthopoxvirus genus. a: amplicons generated resolved using the bioanalyzer (agilent technologies). b: image of the dna-microarray obtained with a confocal laser reader. c: resolution capability of the array. closely related viruses hybridise with very different efficiency on heterologous probes. high density probe arrays, designed to detect 204 antiretroviral resistance mutations simultaneously in gag cleavage sites, protease, reverse transcriptase, integrase and gp41. this assay has been tested on a panel of 99 hiv-1 patients on a total of 4465 relevant codons in comparison with the classic sequence-based method. key resistance mutations were correctly identified in 95 and 92% of codons in protease and reverse transcriptase, respectively (gonzalez et al., 2004) . we have also developed a similar assay for the detection of polymorphisms in the complete hbv genome: 78 antiviral resistance mutations in pol gene, 146 vaccine, diagnostic or immunotherapy mutation in s gene and 209 mutations in basic core promoter, pre-core, core, x, pre-s1 and pre-s2 regions that may have an impact on disease evolution or treatment efficiency. this assay is currently under evaluation in the frame of hepbvar a european collaborative group for the study of emerging variants of hepatitis b virus. in the coming years, more and more laboratories will offer to clinicians viral diagnosis based on nucleic acid tests. many biological and instrumentation problems that have slowed the generalization of molecular assays have been resolved but several others remain and need to be addressed. major im-provements are expected in the integration and automation of nat diagnostic platforms to reduce hands-on-time, time-toresult and costs and to increase throughput. ivd companies have engaged in development programs to provide clinical virologists with equipments and application menus adapted to their diagnosis needs. technical constraints and recent improvements of molecular assays transfusion risks of yesterday and of today correlation of cervical carcinoma and precancerous lesions with human papillomavirus (hpv) genotypes detected with the hpv dna chip microarray method a novel coronavirus associated with severe acute respiratory syndrome application de la biologie moléculaireà la sécurité virale transfusionnelle: le dépistage génomique viral comparative sensitivity of hbv nats and hbsag assays for detection of acute hbv infection human metapneumovirus infections in hospitalized children rapid and simple method for purification of nucleic acids update: detection of west nile virus in blood donations-united states a role for arrays in clinical virology: fact or fiction identification of a novel coronavirus in patients with severe acute respiratory syndrome limitations of taq-man pcr for detecting divergent viral pathogens illustrated by hepatitis a, b, c, and e viruses and human immunodeficiency virus detection of hiv-1 antiretroviral resistance mutations with highdensity dna probe arrays national disease burden of respiratory viruses detected in children by polymerase chain reaction the cost-effectiveness of nat for hiv, hcv, and hbv in whole-blood donations nested pcr for specific detection and rapid identification of human picornaviruses burden of encephalitisassociated hospitalizations in the united states quality control trial for human immunodeficiency virus type 1 drug resistance testing using clinical samples reveals problems with detecting minority species and interpretation of test results characterization of a novel coronavirus associated with severe acute respiratory syndrome aryldiazomethanes for universal labeling of nucleic acids and analysis on dna chips comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens real-time pcr in virology clinical virology in real-time children with respiratory disease associated with metapneumovirus in hong kong nat for hbv and anti-hbc testing increase blood safety comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns5 gene sequences a newly discovered human pneumovirus isolated from young children with respiratory tract disease species differentiation and antibiotic susceptibility testing with dna microarrays linkage between the journal and quality control molecular diagnostics (qcmd) microarray-based detection and genotyping of viral pathogens key: cord-270498-hh6h50t2 authors: tseng, chin-kai; hsu, sung-po; lin, chun-kuang; wu, yu-hsuan; lee, jin-ching; young, kung-chia title: celastrol inhibits hepatitis c virus replication by upregulating heme oxygenase-1 via the jnk mapk/nrf2 pathway in human hepatoma cells date: 2017-09-19 journal: antiviral res doi: 10.1016/j.antiviral.2017.09.010 sha: doc_id: 270498 cord_uid: hh6h50t2 background and purpose: celastrol, a quinone methide triterpene isolated from the root extracts of tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase-1 (ho-1) to achieve disease prevention and control. ho-1 induction was recently shown to result in anti-hcv activity by inducing type i interferon and inhibiting hepatitis c virus (hcv) ns3/4a protease activity. the aim of the present study is to evaluate the anti-hcv activity of celastrol and characterize its mechanism of inhibition. methods: the anti-hcv activity of celastrol was evaluated using the hcv subgenomic replicon and hcvcc infection systems. the anti-hcv mechanism of celastrol targeting ho-1 expression was clarified using specific inhibitors against several signaling pathways. the transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. the synergistic effect of celastrol and a numbers of clinically used anti-hcv drugs was determined via a drug combination assay. results: celastrol inhibited hcv replication in both the hcv subgenomic and hcvcc infection systems with ec50 values of 0.37 ± 0.022 and 0.43 ± 0.019 μm, respectively. celastrol-induced heme oxygenase 1 (ho-1) expression promoted antiviral interferon responses and inhibition of ns3/4a protease activity, thereby blocking hcv replication. these antiviral effects were abrogated by treatment with the ho-1-specific inhibitor snmp or silencing of ho-1 expression by transfection of shrna, which indicates that ho-1 induction contributes to the anti-hcv activity of celastrol. jnk mitogen-activated protein kinase and nuclear factor erythroid 2-related factor 2 (nrf2) were confirmed to be involved in the inductive effect of celastrol on ho-1 expression. celastrol exhibited synergistic effects in combination with interferon-alpha, the ns5a inhibitor daclatasvir, and the ns5b inhibitor sofosbuvir. conclusion: celastrol can serve as a potential supplement for blocking hcv replication. targeting the jnk/nrf2/ho-1 axis presents a promising strategy against hcv infection. hepatitis c virus (hcv) is an enveloped single-stranded positive-sense rna virus belonging to the hepacivirus genus of the flaviviridae family (brass et al., 2006) . the genome of the virus includes 9600 base pairs encoding 4 structural proteins (c, e1, e2 and p7) and 6 nonstructural proteins (ns2, ns3, ns4a, ns4b, ns5a and ns5b) (brass et al., 2006) . hcv infection is a risk factor of chronic liver diseases, including cirrhosis, fibrosis and hepatocellular carcinoma. hcv-infected patients have been estimated to number up to 200 million worldwide (alter, 2007) . in the past, the standard of care (soc) for hcv infection is treatment with a combination of pegylated interferon (peg-ifn-a) and ribavirin (rbv); however, the efficacy of this treatment is only 40%e50% in patients infected with genotype 1 hcv (ghany et al., 2009) . as well, this soc therapy is associated with several adverse effects, such as anemia, headache, fatigue and depression (schoggins et al., 2011) . in 2011, telaprevir and boceprevir, the first daas were approved by the us food and drug administration (fda) and found to exhibited higher rates of sustained virologic responses (svrs) in patients infected with genotype 1 hcv when combined with peg-ifn-a, which provided a new soc for the treatment of chronic hcv infection (kiser et al., 2012) . despite the increasing efficacy of the treatment modes developed for hcv infection, however, undesirable side effects continue to be observed during therapy (kiser et al., 2012) . the fda recently approved the two-agent combo harvoni and the fouragent combo viekira pak for distribution; these treatments could achieve svrs up to 95% in patients infected with genotype 1 hcv (koretz, 2014) . unfortunately, while these antiviral agents achieve higher rates of svr while avoiding the adverse effects often induced by ifn (koretz, 2014) , they also exist the risk to reactivate the hepatitis b virus (hbv) in treating patients with hcv and hbv co-infection (yeh et al., 2017) . moreover, these two drugs relatively more expensive than other drugs. thus, development of a more safety and cost-effective substitute for treatment is an important endeavor. celastrol is a quinone methide triterpene isolated from tripterygium wilfordii, a medicinal plant used to treat a range of illnesses including inflammation, swelling, fever, sores, and pain in india, japan, china, korea, and other asian countries (ju et al., 2015; lee, j.y. et al., 2015; youn et al., 2014) . celastrol is a meal supplement that is widely used in herbal medicine because of its diverse biological activities, which include anti-inflammation, anti-cancer, hepatoprotection, and anti-microbial properties (ju et al., 2015; lee et al., 2015) . several reports have demonstrated that celastrol exhibits inhibitory effects on human immunodeficiency virus (youn et al., 2014) , dengue virus (yu et al., 2017a) , and other severe acute respiratory syndrome-associated coronaviruses (ryu et al., 2010) . celastrol can induce heme oxygenase-1 (ho-1) gene expression via nuclear factor erythroid 2-related factor 2 (nrf2) activation to prevent circulatory failure prevention and protect against myocardial ischemia (der sarkissian et al., 2014) . ho-1 induction was recently shown to result in anti-hcv activity by inducing type i interferon and inhibiting hcv ns3/4a protease activity (lehmann et al., 2010; zhu et al., 2010) . ho-1 is an inducible and rate-limiting enzyme that degrades heme to produce biliverdin, carbon monoxide (co) and ferrous iron (fe3þ) via the heme catabolic pathway (maines, 2005) . the enzyme and its metabolites exhibit protective effects against oxidative stress-induced tissue damage upon exposure to multiple stimuli such as viral and bacterial products including lipopolysaccharides, cytokines, oncogenes, mitogens, and various growth factors (farombi and surh, 2006; paine et al., 2010) . upon hcv infection, down-regulation of ho-1 expression has been observed in hcv-infected patients and hcv core protein expression although the biological role of ho-1 suppression and the detailed mechanism of hcv against ho-1 expression were unclear (abdalla et al., 2004) . ho-1 is a detoxifying enzyme that is mainly regulated by nrf2 (shan et al., 2006) . activation of ho-1 expression occurs when nrf2 binds to the antioxidant response element (are) of the ho-1 promoter region. by contrast, bach1 suppresses ho-1 expression by competing with the are binding site (shan et al., 2006) . under normal conditions, nrf2 is sequestered in the cytoplasm by kelchlike ech-associated protein (keap1)-mediated proteasome degradation (shan et al., 2006) . upregulating nrf2/are-dependent ho-1 expression to achieve anti-inflammatory and anti-oxidative stress functions is mediated by several host factors, including the mitogene-activated protein kinase (mapk) molecules p38 mapk, extracellular signal-regulated kinase 1 and 2 (erk1/2), and c-jun nterminal kinase (jnk) (paine et al., 2010) . in this study, our data revealed that celastrol significantly inhibits hcv replication, and that the anti-hcv effect of celastrol was attenuated by the ho-1 specific inhibitor tin mesoporphyrin (snmp) or ho-1 gene expression silencing. celastrol-mediated ho-1 induction contributed to the anti-hcv action through inducing antiviral ifn response and inhibiting hcv ns3 protease activity. moreover, the jnk/nrf2/ are axis was the critical pathway involved in celastrol-mediated ho-1 induction against hcv replication. combinations of celastrol and ifn-a, sofosbuvir or daclatasvir were tested to determine their ability to enhance of anti-hcv activity. human hepatoma (huh-7), ava5 (huh-7 cells containing hcv genotype 1b subgenomic rna replicon cells) (blight et al., 2000) , and huh7.5/j6/jfhemcviresrlucneo (huh-7 cells harboring hcv genotype 2a subgenomic rna and renilla luciferase reporter gene) obtained from apath llc (st. louis, mo) were maintained in dulbecco's modified eagle's medium containing 10% fetal bovine serum, 1% non-essential amino acids, and 1% antibioticeantimycotic in a 5% co 2 atmosphere at 37 c. huh-7.5 cells stably expressing the pef/jfh1-rz/n plasmid were grown to produce cell culture-derived infectious hcv particles (hcvcc), and the conditional medium was collected to harvest viral particles according to a protocol described previously (kato et al., 2007) . celastrol (pubchemcid:122724) was purchased from fusol-material co., ltd (tainan, taiwan). ifn-a-2a (roferon © -a) was purchased from roche ltd. an ho-1 specific inhibitor [tin mesoporphyrin (snmp)], and mapk-specific inhibitors (sp600125, sb203580, and pd98059) were purchased from sigma (st. louis, mo). daclatasvir and sofosbuvir was purchased from shanghai haoyuan chemexpress co., ltd. the final concentration of dmso in all reactions was maintained at 0.1%. ava5 cells were seeded in 96-well plates at a density of 5 â 10 4 cells per well and treated with celastrol at the indicated concentrations. after 3 days incubation, the cell viability was determined by the celltiter 96 aq ueous one solution cell proliferation assay (promega, madison, wi, usa) according to the manufacturer's instructions. pho-1-luc (hill-kapturczak et al., 2003) and p3xare-luc (liao et al., 2008) vectors were used to measure the transcriptional activity of ho-1 and nrf2, respectively. pisre-luc, a reporter vector containing firefly luciferase under the control of an ifn-stimulated response element (isre), was used to measure ifn response-dependent transcriptional activity (stratagene, agilent technologies, ca, usa). all cloned dna fragments were verified by dna sequencing. western blotting was performed as described previously (lee et al., 2014) . in brief, 20 mg of cell lysates was analyzed by sds-page, and then transferred to a pvdf membrane. the membranes were probed with anti-hcv ns5b (1:5000; abcam, cambridge, ma, usa), anti-ho-1 (1:3000; abcam), anti-nrf2 (1:3000; genetex, ca, usa), anti-phospho-erk1/2 (1:1000; cell signaling technology, inc. danvers, ma, usa), anti-phospho-p38 (1:1000; cell signaling), anti-phospho-jnk (1:1000; cell signaling), anti-erk1/2 (1:1000; cell signaling), anti-p38 (1:1000; cell signaling), or anti-jnk (1:1000; cell signaling)antibody. a loading control was determined using anti-gapdh antibody (1:10000; genetex). rna isolation and quantitative real-time rt-pcr (qrt-pcr) were performed as described previously (lee et al., 2014) . relative mrna levels were determined by normalization against the cellular endogenous glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene. the primers used in the study are listed in table 1 . to evaluate the transcriptional regulation of ho-1, nrf2, or ifn response by celastrol, the ava5 cells were seed on the 24-wells plates at a density of 4 â 10 5 cells per well. after 24 h incubation, the 0.5 mg of promoter-driven firefly luciferase plasmids, including pho-1-luc, p3xare-luc, or pisre-luc, were respectively transfected into pre-seeded ava5 cells using t-pro™ transfection reagent (ji-feng biotechnology co., ltd. taiwan), according to the manufacturer's instructions. the transfected cells were then treated with celastrol at various concentrations for 3 days. cell lysates were prepared for the luciferase activity assay and western blotting using the bright-glo luciferase assay system (promega, madison, wi, usa). the cell-based ns3/4a activity assay was performed as described previously (lee et al., 2014) . in brief, the huh-7 cells were seed on the 24-wells plates at a density of 4 â 10 5 cells per well. after 24 h incubation, the cells were co-transfected with the ns3/ 4a protease reporter vector peg(ded4ab)seap and ns3/4a expression vector pns3/4a, followed by celastrol treatment with or without snmp or 3 days. the 0.6 mm telaprevir treatment server as the positive control. supernatants were harvested for the seap activity assay using the phospha-light assay kit. each transfection mixture contained 0.1 mg of the firefly luciferase expression vector (pfluc) as a transfection control for normalization against seap activity. cells were seeded in 24-well plates at a density of 4 â 10 4 cells/ well and treated with various concentrations of celastrol for 3 days. the cell culture medium was harvested to measure ifn-a concentration using a human ifn-a elisa kit (uscnk life science inc., usa) according to the manufacturer's protocol. absorbance was detected at 450 nm using an epoch microplate spectrophotometer. ava5 cells were seeded in a 10-cm dish at a density of 1.4 â 10 6 cells per 10-cm dish, and treated with increasing concentrations of celastrol for 3 days. the cells were then collected for preparation of a nuclear fraction as described previously (lee et al., 2014) . ava5 cells were treated with serially diluted celastrol (0, 0.05, 0.1, or 0.2 mm) in combination with serially diluted ifn-a (7.5, 15, 30, or 60 u/ml), telaprevir (0.075, 0.15, 0.3, and 0.6 mm) sofosbuvir (10, 20, 40, or 80 nm), or daclatasvir (0.5, 1, and 2 pm). after 3 days of incubation, hcv rna levels were determined by qrt-pcr. multiple drug combination data were analyzed using calcusyn2™ software (biosoft, cambridge, uk) which compares single and multiple drug dose effects and determines the combination index (ci) value. the effect of a multiple drug combination is presented as antagonism (ci > 1), additivity (ci ¼ 1), or synergism (ci < 1). data were presented as the mean ± standard deviation of at least three independent experiments. statistical significance was analyzed using student's t-test. a significant difference was considered as *p < 0.05. celastrol is a quinone methide triterpene (fig. 1a) possessing anti-denv activity (lee et al., 2015) . to discover whether celastrol exhibits anti-hcv activity, we first treated hcv subgenomic replicon ava5 cells with celastrol at increasing concentrations for 3 days. western blotting assay and qrt-pcr analysis were performed to determine hcv protein and rna levels under celastrol treatment, respectively. the cytotoxic effect of celastrol on ava5 cells was also tested using the mts assay. the results indicated that celastrol dose-dependently reduced hcv protein synthesis (fig. 1b) and rna replication with a 50% effective concentration (ec 50 ) of 0.37 ± 0.022 mm (fig. 1c ) without cytotoxicity at effective antiviral concentrations (50% cytotoxicity concentration: cc 50 ¼ 4.83 ± 0.32 mm) (fig. 1d ). based on the results collected from hcv replicon, the selectivity index of celastrol against hcv replication approximates 13. the jfh1 replicon and hcvcc infectious assay was performed to confirm the anti-hcv activity of celastrol. here, huh7.5/j6/jfhemcviresrlucneo replicon cells and jfh1infected huh-7 cells were treated with increasing concentrations of celastrol for 3 days. as shown in fig. 1e and f, qrt-pcr analysis revealed that celastrol dose-dependently reduced hcv rna levels and fully inhibited hcv replication at a concentration of 0.7 mm. celastrol was recently shown to induce on ho-1 gene expression for antiviral activity (youn et al., 2014) . we first examined whether celastrol could induce ho-1 expression by determining ho-1 promoter activity, as well as ho-1 rna and protein levels, in ava5 cells in the presence of celastrol. ava5 cells were transfected pho-1-luc containing a firefly luciferase gene driven by the ho-1 promoter. then, the transfected-cells were treated with celastrol at increasing concentrations for 3 days and subjected to luciferase activity assay. the results indicated that celastrol significantly induced the ho-1 promoter activity in a concentrationdependent manner ( fig. 2a) . as expected, the ho-1 rna and protein levels were also dose-dependently induced by celastrol ( fig. 2b and c). to investigate whether celastrol-induced ho-1 expression is involved in the anti-hcv activity of celastrol, ava5 cells were cotreated with a fixed concentration of celastrol and increasing concentrations ho-1 specific inhibitor snmp for 3 days. western blotting assay was preformed to determine the restorative effect of snmp on hcv protein synthesis upon celastrol treatment. as shown in fig. 2d , celastrol inhibited hcv protein synthesis compared with non-celastrol treated cells (lanes 1 and 2) by contrast, snmp treatment dose-dependently attenuated the suppressive effect of celastrol on hcv protein synthesis (lanes 3e5). as expect, the ho-1 specific shrna mediated ho-1 gene silencing also can attenuate the suppressive effect of celastrol on hcv protein synthesis (fig. 2e) . these results reveal that celastrol inhibited hcv replication is correlated with ho-1 induction. the results of qrt-pcr analysis indicated that ifn-a-5 and ifn-a-17 rna levels were gradually induced by celastrol (fig. 3a) . by contrast, the inductive effect of celastrol on ifn-a-5 and ifn-a-17 rna levels was significantly attenuated by ho-1 inhibitor snmp in a concentration-dependent manner (fig. 3b) . we next measured ifn-a protein secretion levels in the culture medium after celastrol treatment. as expected, elisa results indicated that ifn-a protein secretion levels were gradually induced by celastrol after 3 days of treatment (fig. 3c ). an antiviral effect on cells may be attributed to the interaction of ifn-a and cell surface ifn-a receptors, leading to the activation of isre and upregulation of downstream antiviral genes (yu et al., 2017b) . to evaluate whether celastrol-induced ifna could activate downstream antiviral genes, isre activity and the expression of three critical ifn-mediated antiviral genes, including 2 0 -5 0 -oligoadenylate synthetase 1e3 (oas1e3), were measured. ava5 cells were transfected with isre-driven firefly luciferase reporter plasmid followed by treatment with celastrol for 3 days. luciferase assay results indicated that the isre promoter activity was increased by approximately 2.8e6.5-fold by celastrol (fig. 4a ). oas1e3 gene expression levels were significantly upregulated approximately 1.8 ± 0.04, 3.9 ± 0.06, and 4.7 ± 0.09-fold by celastrol, respectively (fig. 4b) . another proposed anti-hcv action of ho-1 induction is inhibition of ns3/4a protease activity (zhu et al., 2010) . therefore, we performed a cell-based hcv protease assay to examine the ability of celastrol to target hcv ns3/4a protease activity. huh-7 cells were co-transfected with peg(de4ab)seap reporter and hcv pns3/4 protease expression plasmids, followed by celastrol treatment for 3 days. as shown in fig. 5a , hcv ns3/4a protease activity decreased by approximately 1.2e4-fold in comparison with that of the control by celastrol. by contrast, the inhibitory effect of celastrol on hcv ns3/4a protease activity was dose-dependently attenuated by ho-1 inhibitor snmp (fig. 5b) . these data indicate that targeting hcv ns3/4a protease is an alternative anti-hcv action of celastrol. nrf2 functions as an important upstream regulator in the mediation of ho-1 expression by binding to the are response element (farombi and surh, 2006) . to investigate whether celastrol-induced ho-1 induction is mediated by nrf2 activation, we first investigated nrf2 nuclear translocation. ava5 cells were treated with increasing concentrations of celastrol for 3 days, after which the total cells lysate and nuclear fraction were harvested and subjected to western blotting assay. as shown in fig. 6a , total nrf2 protein levels were not affected by celastrol (upper panel). by contrast, nuclear nrf2 protein levels significantly accumulated upon celastrol treatment at a concentration of 0.5 mm, which is the effective dosage against hcv replication. we then examined the nrf2-mediated are activation caused by celastrol. here, ava5 cells were transfected with are-driven firefly luciferase reporter plasmid. the transfected-cells were treated with increasing concentrations of celastrol for 3 days. as expected, are-driven firefly luciferase activity was elevated by celastrol in a concentrationdependent manner (fig. 6b ). considering these results, the nrf2-are-ho-1 axis can be concluded to be strongly associated with the anti-hcv activity of celastrol. activation of the mapk signaling cascade, which includes p38, erk1/2, and jnk, has been reported to be involved in ho-1 induction resulting in anti-hcv activity (huang et al., 2006 whether mapks are involved in anti-hcv effect of celastrol, ava5 cells were treated with 0.5 mm celastrol for 0e120 min. the phosphorylation levels of p38, erk1/2, and jnk were then measured by western blotting with phospho-specific antibodies. ava5 cells were incubated with celastrol for 3 days, and the total cell lysate and cellular nuclear fraction were harvested. total nrf2 (t nrf2) and nuclear nrf2 (ne nrf2) levels were analyzed by western blotting using anti-nrf2 antibody. lamin b1 served as the internal control for the nuclear fraction, and gapdh served as internal control for the total cell lysate. (b) celastrol increases nrf2-mediated are transactivation. ava5 cells were transfected with 0.5 mg of p2xare-luc followed by celastrol treatment for 3 days, and then harvest to analyze their luciferase activity. here, luciferase activity was used to represent isre activity. error bars denote the mean ± sd of three independent experiments. *p < 0.05. as shown in fig. 7a , the jnk phosphorylation levels were elevated by celastrol in a time-dependent manner compared with that at the time point of 0 min. by contrast, celastrol showed no significant effect on erk/1/2 or p38 phosphorylation at any time point. to clarify the role of jnk in the ho-1 induction of celastrol, we used specific inhibitors against erk1/2 (pd98059), jnk (sp600125), and p38 (sb203580), to measure ho-1 rna expression. as shown in fig. 7b , ho-1 rna expression levels were induced by celastrol compared with non-celastrol treated cells and the jnk inhibitor sp600125 significantly reduced the ho-1 inductive effect of celastrol. by contrast, the erk1/2 inhibitor pd98059 and p38 inhibitor sb203580 showed no significant effect on celastrol-induced ho-1 induction. these results suggest that the anti-hcv effect of celastrol is associated with jnk-mediated ho-1 induction in ava5 cells. 3.7. celastrol synergistically or additively inhibits hcv replication when combined with ifn-a, sofosbuvir, daclatasvir or telaprevir to determine whether celastrol can enhance the anti-hcv activity of several of clinically used anti-hcv drugs, such as ifn-a, the ns3/4a inhibitor telaprevir, the ns5b inhibitor sofosbuvir (lam et al., 2012) and the ns5a inhibitor daclatasvir (lee et al., 2011a) . ava5 cells were co-treated with each drug and celastrol at various concentration ratios for 3 days. the synergistic effect of each combination was evaluated by calcusyn 2.1 as described by chou and talalay (1984) . the ci values for the ec 50 , ec 75 , and ec 90 of ifn-a ranged from 1.01 to 0.86, those of telaprevir ranged from 1.03 to 0.97, sofosbuvir ranged from 0.31 to 0.63 and daclatasvir is ranging from 0.4 to 0.46 (table 1) . no significant cytotoxicity was observed in any combination treatment, as assessed by a colorimetric mts assay (data not shown). these findings reveal that celastrol may serve as a dietary supplement for enhancing the therapeutic effect of clinically used anti-hcv drugs. the type i ifn system presents important innate immunity to effectively block virus replication (jones et al., 2005; morrison et al., 2013) . hcv infection has been reported to inhibit antiviral ifn responses that facilitate virus replication by promoting the hcv ns3/ 4a protease-mediated cleavage of mitochondrial antiviralsignaling protein (mavs)/tir-domain-containing adapterinducing interferon-b (trif) (gokhale et al., 2014) . in the present study, we found that a natural product celastrol could effectively inhibit hcv ns3/4a protease activity and enhance ifn-mediated antiviral gene expression through ho-1 induction (figs. 3e5) . the ho-1 metabolite biliverdin has been proven to be a blocker of hcv ns3/4a protease activity. therefore, the regulatory effect of celastrol on the mitochondria-mediated inf signaling pathway against virus replication presents promising prospects for future investigations. our data revealed that nrf-2-mediated ho-1 induction contributed to the anti-hcv activity of celastrol based on the accumulation of nuclear nrf-2 and enhancement of nrf-2 binding activity on the are response element (fig. 6) . given that the activation of nrf2 nuclear translocation is regulated by keap1dependent ubiquitination and bach1, a competitor of nrf2 for binding to are in the ho-1 promoter region (maines, 2005) , future studies should be performed to determine whether celastrol alters the expression levels of keap-1 or bach1 for regulating ho-1 induction. knowledge in this area will help provide alternative targets for screening anti-hcv agents. we further found that the ho-1 inductive effect of the celastrol was also associated with jnk activity (fig. 7) . however, several kinases are involved in jnk activation, including mitogen-activated protein kinase kinase 4 (mkk4), mkk7 and mixed-lineage kinases (mlks) (wasserman et al., 2010) . future studies should examine the effect of celastrol on the kinases involved in jnk activation comprehensively describe the relationship between celastrol and ho-1 induction. several studies have indicated that celastrol exhibits anti-inflammatory activity by inhibiting of nuclear factor-kb (nf-kb) and downstream cycloxygenase-2 (cox-2) (ding et al., 2013; joshi et al., 2016) . on the basis of earlier findings on a promising tactic against hcv infection via down-regulation of nf-kb-mediated cox-2 expression lee et al., 2011b) , we propose that the inhibitory effect of celastrol on nf-kb-mediated cox-2 expression may, at least in part contribute to its anti-hcv activity. hence, more work is necessary to elucidate additional signaling pathways involved in the anti-hcv activity of celastrol. drug combination therapy is considered to be a promising approach to increase therapeutic efficacy and decrease drug resistance in comparison with mono-drug therapy (hajj et al., 2015) . the two-agent combo harvoni and the four-agent combo viekira pak, which could achieve the svrs of up to 95% in patients infected with genotype 1 hcv. however, the low fidelity of hcv polymerase during viral replication may lead to the emergence of drug resistance, which poses a major challenge in treating hcv infection (lee et al., 2014) . targeting host factors could be an alternative strategy to eliminate drug resistance in hcv therapeutic regimens because the mutation rate of the host genome is lower than that of the rna virus genome (liao et al., 2008) . in this study, we showed that celastrol can be considered as a suitable candidate for hcv therapy to minimize the risk of drug resistance by targeting host ho-1 signaling pathway and synergistically inhibiting hcv replication in combination with clinically used drugs against different viral targets (table 1) . further studies are warranted to clarify the potential clinical relevance of our findings. in summary, the data indicated that celastrol efficiently inhibited hcv replication via the induction of the jnk/nrf2/ho-1 axis, which may represent a therapeutic target for the future development and discovery of anti-hcv drugs. celastrol exhibited synergetic effects on anti-hcv activity in combination with ifn, sofosbuvir or daclatasvir. these results reveal that celastrol may serve as a dietary supplement for enhancing therapeutic effects of the anti-hcv drugs currently available. down-regulation of heme oxygenase-1 by hepatitis c virus infection in vivo and by the in vitro expression of hepatitis c core protein epidemiology of hepatitis c virus infection efficient initiation of hcv rna replication in cell culture aqueous extract of the edible gracilaria tenuistipitata inhibits hepatitis c viral replication via cyclooxygenase-2 suppression and reduces virus-induced inflammation quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors celastrol protects ischaemic myocardium through a heat shock response with up-regulation of haeme oxygenase-1 celastrol, an inhibitor of heat shock protein 90beta potently suppresses the expression of matrix metalloproteinases, inducible nitric oxide synthase and cyclooxygenase-2 in primary human osteoarthritic chondrocytes american association for the study of liver diseases, diagnosis, management, and treatment of hepatitis c: an update hepatitis c virus. strategies to evade antiviral responses combination of acamprosate and baclofen as a promising therapeutic approach for parkinson's disease an internal enhancer regulates heme-and cadmium-mediated induction of human heme oxygenase-1 mechanistic link between the anti-hcv effect of interferon gamma and control of viral replication by a ras-mapk signaling cascade dengue virus inhibits alpha interferon signaling by reducing stat2 expression celastrol modulates inflammation through inhibition of the catalytic activity of mediators of arachidonic acid pathway: secretory phospholipase a 2 group iia, 5-lipoxygenase and cyclooxygenase-2 celastrol ameliorates cytokine toxicity and pro-inflammatory immune responses by suppressing nf-kb activation in rinm5f beta cells production of infectious hepatitis c virus of various genotypes in cell cultures review and management of drug interactions with boceprevir and telaprevir acp journal club: review: telaprevir, boceprevir, simeprevir, or sofosbuvir improves response in hcv type 1 genotype and subtype profiling of psi-7977 as a nucleotide inhibitor of hepatitis c virus. antimicrob the hepatitis c virus ns5a inhibitor (bms-790052) alters the subcellular localization of the ns5a non-structural viral protein anti-hepatitis c virus activity of acacia confusa extract via suppressing cyclooxygenase-2 andrographolide exerts anti-hepatitis c virus activity by up-regulating haeme oxygenase-1 via the p38 mapk/nrf2 pathway in human hepatoma cells celastrol blocks binding of lipopolysaccharides to a toll-like receptor4/myeloid differentiation factor2 complex in a thiol-dependent manner cinnamaldehyde inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion molecules in endothelial cells by suppressing nf-kappab activation: effects upon ikappab and nrf2 the heme oxygenase system: update dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential regulation of hepatitis c virus replication and gene expression by the mapk-erk pathway sars-cov 3clpro inhibitory effects of quinone-methide triterpenes from tripterygium regelii a diverse range of gene products are effectors of the type i interferon antiviral response role of bach1 and nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin a novel c-jun n-terminal kinase (jnk)-binding protein wdr62 is recruited to stress granules and mediates a nonclassical jnk activation oxidative stress induces anti-hepatitis c virus status via the activation of extracellular signal-regulated kinase reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals celastrol ameliorates hiv-1 tat-induced inflammatory responses via nf-kappab and ap-1 inhibition and heme oxygenase-1 induction in astrocytes celastrol inhibits dengue virus replication via up-regulating type i interferon and downstream interferon-stimulated responses schisandrin a inhibits dengue viral replication via upregulating antiviral interferon responses through stat signaling pathway biliverdin inhibits hepatitis c virus nonstructural 3/4a protease activity: mechanism for the antiviral effects of heme oxygenase we are grateful to dr. charles rice (rockefeller university and aapth, lcc, usa) for kindly supporting con1b replicon plasmid, human hepatoma cell; huh-7 and hcv subgenomic replicon containing cell line; ava5, huh7.5/j6/jfhemcviresrlucneo hcv replicon cells and dr. t. wakita (national institute of infectious diseases, japan) for providing the jfh1 plasmid. key: cord-000473-jpow6iw1 authors: astrovskaya, irina; tork, bassam; mangul, serghei; westbrooks, kelly; măndoiu, ion; balfe, peter; zelikovsky, alex title: inferring viral quasispecies spectra from 454 pyrosequencing reads date: 2011-07-28 journal: bmc bioinformatics doi: 10.1186/1471-2105-12-s6-s1 sha: doc_id: 473 cord_uid: jpow6iw1 background: rna viruses infecting a host usually exist as a set of closely related sequences, referred to as quasispecies. the genomic diversity of viral quasispecies is a subject of great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. high-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software was originally designed for single genome assembly and cannot be used to simultaneously assemble and estimate the abundance of multiple closely related quasispecies sequences. results: in this paper, we introduce a new viral spectrum assembler (vispa) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art shorah tool on both simulated and real 454 pyrosequencing shotgun reads from hcv and hiv quasispecies. experimental results show that vispa outperforms shorah on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. while shorah has a significant advantage over vispa on reads simulated with sequencing errors due to its advanced error correction algorithm, vispa is better at assembling the simulated reads after they have been corrected by shorah. vispa also outperforms shorah on real 454 reads. indeed, 7 most frequent sequences reconstructed by vispa from a real hcv dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and sanger sequencing. in contrast, only one of the sequences reconstructed by shorah is viable. on a real hiv dataset, shorah correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas vispa correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. vispa source code is available at http://alla.cs.gsu.edu/~software/vispa/vispa.html. conclusions: vispa enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. we are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations. results: in this paper, we introduce a new viral spectrum assembler (vispa) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art shorah tool on both simulated and real 454 pyrosequencing shotgun reads from hcv and hiv quasispecies. experimental results show that vispa outperforms shorah on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. while shorah has a significant advantage over vispa on reads simulated with sequencing errors due to its advanced error correction algorithm, vispa is better at assembling the simulated reads after they have been corrected by shorah. vispa also outperforms shorah on real 454 reads. indeed, 7 most frequent sequences reconstructed by vispa from a real hcv dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and sanger sequencing. in contrast, only one of the sequences reconstructed by shorah is viable. on a real hiv dataset, shorah correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas vispa correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. vispa source code is available at http://alla.cs.gsu.edu/~software/vispa/vispa.html. conclusions: vispa enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. we are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations. many viruses (including sars, influenza, hbv, hcv, and hiv) encode their genome in rna rather than dna. unlike dna viruses, rna viruses lack the ability to detect and repair mistakes during replication [1] and, as a result, their mutation rate can be as high as 1 mutation per each 1,000-100,000 bases copied per replication cycle [2] . many of the mutations are well tolerated and passed down to descendants, producing a family of co-existing related variants of the original viral genome referred to as quasispecies, a concept that originally described a mutation-selection balance [3] [4] [5] [6] [7] . the diversity of viral sequences in an infected individual can cause the failure of vaccines and virus resistance to existing drug therapies [8] . therefore, there is a great interest in reconstructing genomic diversity of viral quasispecies. knowing sequences of the most virulent variants can help to design effective drugs [9, 10] and vaccines [11, 12] targeting particular viral variants in vivo. briefly, the 454 pyrosequencing system shears the source genetic material into fragments of approximately 300-800 bases. millions of single-stranded fragments are sequenced by synthesizing their complementary strands. repeatedly, nucleotide reagents are flown over the fragments, one nucleotide (a, c, t, or g) at a time. light is emitted at a fragment location when the flown nucleotide base complements the first unpaired base of the fragment [13, 14] . multiple identical nucleotides may be incorporated in a single cycle, in which case the light intensity corresponds to the number of incorporated bases. however, since the number of incorporated bases (referred to as a homopolymer length) cannot be estimated accurately for long homopolymers, it results in a relatively high percentage of insertion and deletion sequencing errors (which respectively represent 65%-75% and 20%-30% of all sequencing errors [15, 16] ). the software provided by instrument manufacturers were originally designed to assemble all reads into a single genome sequence, and cannot be used for reconstructing quasispecies sequences. thus, in this paper we address the following problem: given a collection of 454 pyrosequencing reads generated from a viral sample, reconstruct the quasispecies spectrum, i.e., the set of sequences and the relative frequency of each sequence in the sample population. a major challenge in solving the qsr problem is that the quasispecies sequences are only slightly different from each other. the amount and distribution along the genome of differences between quasispecies varies significantly between virus species, as different species have different mutation rates and genomic architectures. in particular, due to the lower mutation rate and longer conserved regions, hcv quasispecies are harder to reconstruct than quasispecies of hbv and hiv. additionally, the qsr problem is made difficult by the limited read length and relatively high error rate of high throughput sequencing data generated by current technologies. the qsr problem is related to several well-studied problems: de novo genome assembly [17] [18] [19] , haplotype assembly [20, 21] , population phasing [22] and metagenomics [23] . as noted above, de novo assembly methods are designed to reconstruct a single genome sequence, and are not well-suited for reconstructing a large number of closely related quasispecies sequences. haplotype assembly does seek to reconstruct two closely related haplotype sequences, but existing methods do not easily extend to the reconstruction of a large (and a priori unknown) number of sequences. computational methods developed for population phasing deal with large numbers of haplotypes, but rely on the availability of genotype data that conflates information about pairs of haplotypes. metagenomic samples do consist of sequencing reads generated from the genomes of a large number of species. however, differences between the genomes of these species are considerably larger than those between viral quasispecies. furthermore, existing tools for metagenomic data analysis focus on species identification, as reconstruction of complete genomic sequences would require much higher sequencing depth than that typically provided by current metagenomic datasets. in contrast, achieving high sequencing depth for viral samples is very inexpensive, owing to the short length of viral genomes. mapping based approaches to qsr are naturally preferred to de novo assembly since reference genomes are available (or easy to obtain) for viruses of interest, and viral genomes do not contain repeats. thus, it is not surprising that such approaches were adopted in the two pioneering works on the qsr problem [24, 25] . eriksson et al. [24] proposed a multi-step approach consisting of sequencing error correction via clustering, haplotype reconstruction via chain decomposition, and haplotype frequency estimation via expectation-maximization, with validation on hiv data. in westbrooks et al. [25] , the focus is on haplotype reconstruction via transitive reduction, overlap probability estimation and network flows, with application to simulated error-free hcv data. recently, the qsr software tool shorah was developed [26] and applied to hiv data [27] . another combinatorial method for qsr was also developed and applied to hiv and hbv data in [28] , with results similar to those of shorah. our contributions in this paper are as follows: • a novel qsr tool called viral spectrum assembler (vispa) taking into account sequencing errors at multiple steps, • comparison of vispa with shorah on hcv synthetic data both with and without sequencing errors, and • statistical and experimental validation of the two methods on real 454 pyrosequencing reads from hcv and hiv samples. our method for inferring the quasispecies spectrum of a virus sample from 454 pyrosequencing reads consists of the following steps (see fig. 1 ): • constructing the consensus virus genome sequence for the given sample and aligning the reads onto this consensus, • preprocessing aligned reads to correct sequencing errors, • constructing a transitively reduced read graph with vertices representing reads and edges representing overlaps between them, • selecting paths in the read graph that correspond to the most probable quasispecies sequences, and assembling candidate sequences for selected paths by weighted consensus of reads, and • estimating candidate sequence frequencies by em below we describe each step separately. we assume that a reference genome sequence of the particular virus strain is available (e.g., from ncbi [29] ). since viral genomes do not have sizable repeats and the quasispecies sequences are usually close enough to the reference sequence, the majority of reads can typically be uniquely aligned onto the reference genome. however, a significant number of reads may remain unaligned due to differences between the reference genome and sequences in the viral sample. in order to recover as many of these reads as possible, we iteratively construct a consensus genome sequence from aligned reads. in particular, we first align 454 pyrosequencing reads to the reference sequence using the segemehl software [30] . then we extend the reference sequence with a placeholder i for each nucleotide inserted by at least one uniquely aligned read. similarly, we add a placeholder d to the read sequence for each reference nucleotide missing from the aligned read. then we perform sequential multiple alignment of the previously aligned reads against this extended reference sequence. finally, the consensus genome sequence is obtained by (1) replacing each nucleotide in the extended reference with the nucleotide or placeholder in the majority of the aligned reads and (2) removing all i and d placeholders, respectively corresponding to rare insertions and to deletions found in a majority of reads. reads may contain a small portion of unidentified nucleotides denoted by n'swe treat n as a special allele value matching any of nucleotides a, c, t, g, as well as placeholders i, and d. iteratively, we replace the reference with the consensus and try to align the reads, for which we could not find any acceptable alignment previously. our experiments on a dataset consisting of approximately 31,000 454 pyrosequencing reads generated from a 5.2kb-long hcv fragment (see data description in results and discussions) show that 85% of reads are uniquely aligned onto the reference sequence and an additional 9% of the reads are aligned onto the final consensus sequence. reads that cannot be aligned onto the final consensus are removed from the further consideration. since aligned reads contain insertions and deletions, we use placeholders i and d to simplify position referencing among the reads. all placeholders are treated as additional allele values but they are removed from the final assembled sequences. first, we substitute each deletion in the aligned reads with placeholder d. deletion supported by a single read is replaced either with the allele value, which is present in all other reads overlapping this position, or with n, signifying an unknown value, otherwise. next, we fill with placeholder i each gap in a read corresponding to the insertions in the other reads. all insertions supported by a single read are removed from consideration. we begin with the definition of the read graph, introduced in [25] and independently in [24] , and then describe the adjustments that need to be made to read graph construction and edge weights to account for sequencing errors as well as the high mutation rate between quasispecies. the read graph g = (v, e) is a directed graph with vertices corresponding to reads aligned with the consensus sequence. for a read u, we denote by b(u), respectively e(u), the genomic coordinate at which the first, respectively the last, base of u gets aligned. a directed edge (u, v) connects read u to read v if a suffix of u overlaps with a prefix of v and they coincide across the overlap. two auxiliary vertices -a source s and a sink t are added such that s has edges into all reads with zero indegree and t has edges from all reads with zero outdegree. then each st-path corresponds to a possible candidate quasispecies sequence. the read graph is transitively reduced, i.e., each edge e = (u, v) is removed if there is a uv-path not including edge e. note that certain reads can be completely contained inside other reads. let a superread refer to a read that is not contained in any other read and let the rest of the reads be called subreads. subreads are not used in the construction of the read graph, but are taken into account in the final assembly of candidate sequences and frequency estimation. since the number of different st-paths is exponential, we wish to generate a set of paths that have high probability to correspond to real quasispecies sequences. in order to estimate path probability, we independently estimate for each edge e the probability p(e) that it connects two reads from the same quasispecies, and then multiply estimated probabilities for all edges on the path. under the assumption of independence between edges, if we assign to each edge e a cost equal tolog (p(e)) = log(1/p(e)), then the minimum-cost st-path will have the maximum probability to represent a quasispecies sequence. for reads without errors, [25] estimated the probability that two reads u and v connected by edge (u, v) belong to the same quasispecies as is the overhang between reads u and v [25] , n = #reads, q = #quasispecies, and l = #starting positions. thus, in this case the cost of an edge with overhang δ can be approximated by δ ∝ log(1/p δ ). to account for sequencing errors, we adjust the construction of the read graph to allow for mismatches. we use three parameters: (1) n = #mismatches allowed between a read and a superread, (2) m = #mismatches allowed in the overlap between two adjacent reads, and (3) t = #mismatches expected between a read and a random quasispecies. the probability that two reads u and v with j mismatches within an overlap of length o = e(u) b(v) belong to the same quasispecies can be estimated as: where ε is the estimated 454 sequencing error rate. as in the case of error-free reads, defining the edge costs as ensures that stpaths with low cost correspond to most likely quasispecies sequences. to generate a set of high-probability (low-cost) paths that are rich enough to explain observed reads, we compute for each vertex in the read graph the minimum cost st-path passing through it. finding these paths is computationally fast. indeed, we only need to compute two shortest-paths trees in g, one outgoing from s and one incoming into t; the shortest st-path passing through a vertex v is the concatenation of the shortest s v-and vt-paths. preliminary simulation experiments (see additional file 1) show that better candidate sets are generated when edge costs c defined by (1) and (2) are replaced by e c . in fact, if we use even faster dependency on c then we obtain better candidate sets. the fastest growing cost effectively changes the shortest path into so called maxbandwidth path, i.e., paths that minimizes maximum edge cost for the entire path and for each subpath. so, vispa generates candidate paths using this strategy. when no mismatches are allowed in the construction of the read graph, finding the candidate sequence corresponding to a st-path is trivial, since by definition adjacent superreads coincide across their overlap. when mismatches are allowed, we first assemble a consensus sequence from superreads used by the st-path. it may be not the best choice, especially when the coverage with superreads is low. hence, we replace each initial candidate sequence with a weighted consensus sequence obtained using both superreads and subreads of the path, as described below. for each read r, we compute the probability that it belongs to a particular initial candidate sequence s as: where l and l denote the lengths of the read and initial candidate sequence, respectively, k is the number of mismatches between the read and the initial candidate sequence s, and t/l is the estimated mutation rate. then final candidate sequence is computed as the weighted consensus over all reads, where the weight of a read is the probability that it belongs to the sequence. note that, unlike the case without mismatches, the same candidate sequence can be obtained from different candidate st-paths, so we remove duplicates at the end of this step. we assume that reads r with observed frequencies where generated from a quasispecies population q as follows. first, a quasispecies sequence q q is randomly chosen accordingly to its unknown frequency f q . a read starting position is generated from the uniform distribution and then a read r is produced from quasispecies q with j sequencing errors. the probability of this event is calculated as h q r j l lj j , ( ) where l is the read length and ε is the sequencing error rate. in our simulation studies we use the following read data sets. in order to perform cross-validation on the assembly method, we simulate reads data from 1739-bp long fragment from the e1e2 region of 44 hcv sequences [32] when sequence frequencies are generated according to some specific distribution. in our simulation experiments, we use geometric distribution (i-th sequence is constant factor more frequent than the (i + 1)-th sequence) to create sample quasispecies populations with different number of randomly selected above-mentioned quasispecies sequences. we first simulate reads without sequencing errors: the length of a read follows normal distribution with a particular mean value and variance 400, and a starting position follows the uniform distribution. this simplified model of reads generation has two parameters: number of the reads that varies from 20k up to 100k and the average read length that varies from 200bp up to 600bp. additionally, we simulate 454 pyrosequencing reads from 10 quasispecies sequences (following geometric distribution of frequencies) out of 44 hcv sequences [32] using flowsim [33] . we generated 30k reads with average length 350bp. the data set data1 has been received from hcv research group in institute of biomedical research, at university of birmingham. data1 contains 30,927 reads obtained from the 5.2kb-long fragment of hcv-1a genome (which is more than a half of the entire hcv genome). the average (aligned) read length average is 292bp but it significantly varies as well as the depth of position coverage (see additional file 1 for details). the depth of reads coverage variability is due to a strong bias in the sequence start points, reflecting the secondary structure of the template dna or rna used to generate the initial pcr products. as a result, shorter reads are produced by gc-rich sequences. data1 is available upon request from the authors. the hiv dataset [27] contains 55,611 reads from mixture of 10 different 1.5kb-long region of hiv-1 quasispecies, including pol protease and part of the pol reverse transcriptase. the aligned reads length varies from 35bp to 584bp with average about 345bp (see additional file 1 for details). in contrast to [27] , we do not filter out reads with low-quality scores. in all our experimental validations, we compare the proposed algorithm vispa with the state-of-the-art tool shorah as well as with vispa on shorah-corrected reads (shorahreads + vispa). we say the quasispecies sequence is captured if one of the candidate sequences exactly matches it. we measure the quality of assembling by portion of the real quasispecies sequences being captured by candidate sequences (sensitivity = + ) and its portion among candihere, we see advantage of vispa over shorah. following [24] , we measure the prediction quality of frequency distribution with kullback-leibler divergence, or relative entropy. given two probability distributions, relative entropy measures the "distance" between them, or, in the other words, the quality of approximation of one probability distribution by the other distribution. formally, the relative entropy between true distribution p and approximation distribution q is given by the formula: where summation is over all reconstructed original sequences i = {i | p(i) > 0, q(i) > 0} , i. e., over all original sequences that have a match (exact or with at most k mismatches) among assembled sequences. the relative entropy is decreasing with increasing of the average read length. it is expected since sensitivity is increasing with increasing of the average read length and em predicts underlying distribution more accurately. vispa algorithm considerably outperforms shorah (see fig. 2 (right)). however, shorah has a significant advantage over vispa on a read data simulated by flowsim both in prediction power and in robustness of results (see table 1 ). indeed, shorah correctly infers 3 out of 10 real quasispecies sequences whereas vispa reconstructs only 1 sequence. additionally, 10 most frequent assemblies inferred by shorah are more robust with repeating up to 45% of times on 10%-reduced data versus 1% of times for vispa's assemblies. this advantage can be explained by superior read correction in shorah. if vispa is used on shorah-corrected reads, the results drastically improves: 5 quasispecies sequences are inferred and exactly 95% of times are repeated on reduced data, confirming that vispa is better in assembling sequences (see table 1 ). experimental validation on 454 pyrosequencing reads from hcv samples we first discuss the choice of parameters of the read graph and candidate sequence assembly from stpaths. then we give statistical validation for obtained 10 most frequent quasispecies sequences. we infer quasispecies spectrum based on the read graphs constructed with various numbers n and m (numbers of mismatches allowed for superreads and overlaps corresponding to edges). we sort the estimated frequencies in descending order and count the number of sequences which cumulative frequency is 85%, 90%, and 95%. fig. 3 reports these numbers as a percent of the total number of candidate sequences. there is an obvious drop in percentage for all three categories if we allow up to n = 6 mismatches to cluster reads and up to m = 15 mismatches to create edges. in this case, the constructed read graph has no isolated vertices. to refine assembled candidate sequences, we use all reads and parameter t varying from 80bp till 350bp, or, in the other words, mutation rate varying from 1.75% up to 8% per sequence (which is in the range observed in [34] ). out of 3207 max-bandwidth paths, we obtain as much as 938 distinct sequences (t = 80) and as low as 755 sequences (t = 350) for different values of t [80; 350]. the neighbor-joining tree for the most frequent 10 candidate sequences obtained by vispa and shorah (see fig. 4 ) reminds a neighbor-joining tree for hcv quasispecies evolution. additionally, the most frequent candidate sequence found by vispa is 99% identical to one of the actual orfs obtained by cloning the quasispecies. the quasispecies sequence is considered found if one of candidate sequences matches it exactly (k = 0) or with at most k (1 or 9) mismatches. all methods are run 100 times on 10% -reduced data. for the i-th (i = 1, .., 10) most frequent sequence assembled on the whole data, we record its reproducibility, i.e., percentage of runs when there is a match (exact or with at most k mismatches) among 10 most frequent sequences found on reduced data. "reproducibility: max" and "reproducibility: average" report respectively maximum and average of those percentages." figure 3 percentage of candidate sequences which cumulative frequency is 85%, 90%, and 95%. the values on x-axis corresponds to the number of allowed mismatches during read graph construction. n_m means that up to n mismatches are allowed in superreads and up to m mismatches are allowed in edges. viral sequences containing internal stop codons are not viable since the entire hcv genome consists of a single coding region for a large polyprotein. so the number of reconstructed viable sequences can serve as an accuracy measure for quasispecies assembly. out of 10 most frequent sequences reconstructed by vispa, only 3 are not viable while shorah is able to reconstruct only one viable sequence. this sequence has 99.94% similarity with the vispa's fourth most frequent assemblies. both methods returned similar frequency estimations for this sequence: 0.017% (shorah) and 0.019% (vispa). both shorah and vispa (n = 6, m = 15) are run on eight 2.66ghz-cpus with 8m cache. they take around 40 minutes to assemble sequences and estimate their frequencies. smaller value of n increases vispa's runtime since its bottleneck (candidate sequences assembling) is proportional to the number of reads times number of paths. indeed, smaller value of n results in larger number of superreads in built read graph, thus, in larger set of candidate paths. for example, vispa runs 90 minutes for n = 2, m = 2. the plot on fig. 5 shows validation results for 10 most frequent quasispecies sequences with respect to em estimations assembled on data1 by shorah and vispa (n = 6, m = 15, and t = 120). repeatedly, 100 times we have deleted randomly chosen 10% of reads and run both methods on each reduced read instance to reconstruct quasispecies spectrum. the plot reports the percentage of runs when each of 10 most frequent sequences assembled on data1 are reproduced among the 10 most frequent quasispecies figure 4 the neighbor-joining phylogenetic tree for 10 most frequent hcv quasispecies variants on a 5,205bp-long fragment obtained by vispa and shorah. sequences are labeled with software name and its rank among 10 most frequent assembled sequences. percentage of runs when the i-th most frequent sequence is reproduced among 10 most frequent quasispecies assembled on the 10%-reduced set of reads. the i-th point at x-axis corresponds to the i-th most frequent sequence assembled on the 100% of reads. no data are shown for the sequences that are reproduced less than 5% of runs. inferred on the reduced instances with no mismatches (k = 0), or with k = 1, 2, 5 mismatches. for example, for k = 0 shorah repeatedly (35% of times) reconstructs only the third most frequent sequence while vispa reconstructs 7 sequences in at least 15% times, and the most frequent sequence is reconstructed 40% times. this plot shows that the found sequences are pretty much reproducible for vispa. in order to compare vispa and shorah, we run both of the methods on hiv dataset, used in the first experiment in [27] . as said above, we do not preprocess reads with respect to its 454 quality score, and it can explain poorer performance of shorah. indeed, shorah correctly infers only 2 quasispecies sequences with at most 4 mismatches: one assembly has 3 mismatches with real quasispecies sequence, and the other has 4 mismatches. vispa correctly reconstructs 5 quasispecies with at most 2 mismatches (3 of them among 10 most frequent assemblies): two sequences are inferred without any mismatches (one is among 10 most frequent assemblies), one assembly has 1 mismatch with real quasispecies sequence (and it is among 10 most frequent assemblies), and the rest sequences have 2 mismatches (one is among 10 most frequent assemblies). the assemblies correspond to a viable protein sequences. if vispa is applied to shorah-corrected reads, it can successfully infer three real quasispecies without any mismatches. in this paper, we have proposed and implemented vispa, a novel software tool for quasispecies spectrum reconstruction from high-throughput sequencing reads. the vispa assembler takes into account sequencing errors at multiple steps, including mapping-based read preprocessing, path selection based on maximum bandwidth, and candidate sequence assembly using probability-weighted consensus techniques. sequencing errors are also taken into account in vispa's em-based estimation of quasispecies sequence frequencies. we have validated our method on simulated error-free reads, flowsim-simulated reads with sequencing errors, and real 454 pyrosequencing reads from hcv and hiv samples. we are currently exploring extensions of vispa to paired-end reads; the main difficulty is selection of pair-aware candidate paths. we also foresee application of vispa's techniques to the analysis of high-throughput sequencing data from microbial communities [23] and ecological samples of eukaryote populations [35] . the vispa source code is available at http://alla.cs.gsu. edu/~software/vispa/vispa.html. additional file 1: supplementary materials. the file contains derivation of edge cost formula (2) and em algorithm, example of read graph construction and analysis of 454 pyrosequencing data. rna virus quasispecies: significance for viral disease and epidemiology mutation rates among rna viruses rna virus mutations and fitness for survival the quasispecies (extremely heterogeneous) nature of viral rna genome populations: biological relevance -a review the molecular quasi-species hepatitis c virus (hcv) circulates as a population of different but closely related genomes: quasispecies nature of hcv genome distribution rapid evolution of rna viruses rna virus populations as quasispecies. current topics in microbiology and immunology computational methods for the design of effective therapies against drug resistant hiv strains hiv-1 subtype b protease and reverse transcriptase amino acid covariation the rational design of an aids vaccine diversity considerations in hiv-1 vaccine selection pyrosequencing: an accurate detection platform for single nucleotide polymorphisms genome sequencing in microfabricated high-density picolitre reactors pyrobayes: an improved base caller for snp discovery in pyrosequences quality scores and snp detection in sequencing-by-synthesis systems short read fragment assembly of bacterial genomes building fragment assembly string graphs whole-genome sequencing and assembly with high-throughput, short-read technologies hapcut: an efficient and accurate algorithm for the haplotype assembly problem algorithmic strategies for the single nucleotide polymorphism haplotype assembly problem 2snp: scalable phasing based on 2-snp haplotypes environmental genome shotgun sequencing of the sargasso sea beerenwinkel n: viral population estimation using pyrosequencing hcv quasispecies assembly using network flows deep sequencing of a genetically heterogeneous sample: local haplotype reconstruction and read error correction error correction of nextgeneration sequencing data and reliable estimation of hiv quasispecies combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing fast mapping of short sequences with mismatches, insertions and deletions using index structures maximum likelihood from incomplete data via the em algorithm (with discussions) hepatitis c virus continuously escapes from neutralizing antibody and t-cell responses during chronic infection in vivo characteristics of 454 pyrosequencing data-enabling realistic simulation with flowsim the quasispecies nature and biological implications of the hepatitis c virus. infection robust haplotype reconstruction of eukaryotic read data with hapler inferring viral quasispecies spectra from 454 pyrosequencing reads authors contributions ia designed algorithms, developed software, performed analysis and experiments, wrote the paper. bt performed analysis and experiments. sm contributed to developing software. kw designed algorithms and developed software. im contributed to designing the algorithms and writing the paper. pb supplied the hcv data and contributed to performing the analysis. az designed the algorithms, wrote the paper and supervised the project. all authors have read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-264326-teahway7 authors: eleftheriou, phaedra; amanatidou, dionysia; petrou, anthi; geronikaki, athina title: in silico evaluation of the effectivity of approved protease inhibitors against the main protease of the novel sars-cov-2 virus date: 2020-05-29 journal: molecules doi: 10.3390/molecules25112529 sha: doc_id: 264326 cord_uid: teahway7 the coronavirus disease, covid-19, caused by the novel coronavirus sars-cov-2, which first emerged in wuhan, china and was made known to the world in december 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to april 16th, 2020. it has 79.5% sequence identity with sars-cov-1 and the same strategy for host cell invasion through the ace-2 surface protein. since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. the 3d structure of the sars-cov-2 main protease was compared with the 3d structures of seven proteases, which are drug targets, and docking analysis to the sars-cov-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. increased 3d structural similarity between the sars-cov-2 main protease, the hcv protease and α-thrombin was found. according to docking analysis the most promising results were found for hcv protease, dpp-4, α-thrombin and coagulation factor xa known inhibitors, with several of them exhibiting estimated free binding energy lower than −8.00 kcal/mol and better prediction results than reference compounds. since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. dpp-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe sars-cov-2 induced pneumonia. the coronavirus sars-cov-2, responsible for the pandemic which started in wuhan, china, during the latter part of 2019, has spread to 213 countries, areas and territories according to the who [1, 2] by april 16th 2020 and continues to spread, infecting approximately 84,500 and causing the death of approximately 8000 people daily. although young people usually develop mild symptoms, it can cause severe lower respiratory disease affecting mostly the elderly and individuals with other co-morbidities such as cardiovascular problems, pre-existing respiratory disease, diabetes, hypertension or cancer [1] . sars-cov-2 belongs to the βcov genera of the coronavirinae subfamily of the order of nidovirals [3] . compared to sars and mers-coronavirus strains that caused the two most recent epidemics, in 2002-2003 and 2012, respectively-it mostly resembles sars-cov-1 (2002-03), with 79.5% sequence identity and the same mechanism for host cell entrance, via the angiotensin-converting enzyme-2 (ace-2) surface protein of the cell [4] . as far as cleavage recognition motifs are concerned, the sars-cov-2 main protease cleaves the viral polyprotein at no less than 11 sites, recognizing the sequence leu-gln ↓ ser/ala/gly [22] . the hiv-1 protease recognizes nine cleavage sites in its polyprotein substrate, with sequence differences that keep the feature of cleavage, most commonly, between phe and pro. alternatively, it cleaves between amino acids with hydrophobic or aromatic side chains (tyr/met/leu/phe ↓ pro/phe/tyr/ala/leu/met) [23] . the hcv protease cleaves its polyprotein substrate at four sites, recognizing a glu/asp-glu/asp-xxx-cys ↓ ser general motif [24] . the human organism protease dpp-4 preferentially cleaves at x ↓ pro or x ↓ ala sites [25] , whereas thrombin preferentially cleaves at the gly/ala/val/ile/pro-arg ↓ ser/ala/gly/thr/(note/d)-x(note/d)-arg/(note) motif [27] . the coagulation factor xa cleaves after arg, recognizing a motif val/pro/ile-gln/asp/glu-phe/gly-arg ↓ ser-leu [26] . renin recognizes the sequence ile-his-pro-phe-his-leu↓ [29] , while angiotensin-converting enzyme (ace) recognizes the angiotensin i sequence asp-arg-val-tyr-ile-his-pro-phe ↓ his-leu [28] . similarities between proteases at the catalytic amino acids of the active site and in the nature of amino acids of the substrate may be an indication for efficient inhibition of the proteases by the same inhibitors. however, the nature of surrounding amino acids of the active site and the 3d structure of the active center are also of great importance. in the present study, the 3d structure of the sars-cov-2 main protease was compared with the 3d structures of proteases which are drug targets with approved inhibitors. in addition, a number of approved inhibitors of the above proteases were evaluated by docking analysis for their ability to inhibit the sars-cov-2 main protease, interacting with the active site of the enzyme. since the 3d structure of the active site of the enzyme is crucial for catalytic activity, we proceeded to a comparison of the sars-cov-2 main protease, mpro, with the hiv-1 protease, the hcv protease (ns3 protein) and the human proteases dpp-4, thrombin, factor xa, renin and ace, which constitute known drug targets with approved inhibitors. significant 3d similarity was found between the sars-cov-2 and the hcv protease (p = 1.34 × 10 −4 ) as well as between the sars-cov-2 protease and α-thrombin (p = 7.42 × 10 −4 ). interestingly, the protein parts with increased structural alignment included the catalytic area of the enzyme. the lowest similarity was observed in the case of angiotensin-converting enzyme and coagulation factor xa (figures 1 and 2) . the structural similarity between the sars-cov-2 protease and some of the selected proteases, in combination with the existence of the same amino acids at certain positions of the substrate cleavage site, such as ser at the p1' position of the recognition sequence of the hcv protease and thrombin are promising features in the effort to identify effective sars-cov-2 protease inhibitors among the approved drugs of the selected proteases. to investigate this possibility further, we proceeded to docking analysis of randomly selected approved or on-trial inhibitors of the selected proteases (table 1 ). an exception from the random selection was made for the hiv-1 inhibitors where lopinavir and ritonavir were selected because they have already been proposed for the treatment of sars-cov-2. the rest of the drugs that were selected are the anti-hcv drugs telaprevir and boceprevir, the dpp-4 inhibitor sitagliptin, the renin inhibitor alisliren, the ace inhibitor captopril, the thrombin direct inhibitors argatroban and dabigatran and the coagulation factor xa inhibitor rivaroxaban. docking analysis was performed using the sars-cov-2 structure 6lu7. the results were promising for most of the compounds, with the exception of the ace and renin inhibitors, captopril and aliskiren, for which the calculated free binding energy was −5.17 and −4.66 kcal mol −1 , respectively ( table 2 ). according to our previous experience and to the literature, a free binding energy higher than −5.5 kcal mol −1 is an indication that the compound is inactive [32, 33] . the best results were obtained for the hcv protease inhibitors telaprevir and boceprevir, with free binding energies of −10.05 and −9.16 kcal mol −1 , respectively, followed by the thrombin inhibitor argatropan and the dpp-4 inhibitor, sitagliptin, with free binding energies of −9.03 and −8.80 kcal mol −1 , respectively. the hiv-1 protease inhibitors ritonavir and lopinavir showed a little lower predicted activity, with free binding energies of −8.96 and −8.65 kcal mol −1 , respectively. docking analysis to the whole enzyme indicated that the enzyme active site is the second preferred binding site of lopinavir, ritonavir and boceprevir. according to this observation, the expected inhibitory action of these compounds is to be lower than that predicted based on their binding energy to the active site [32] . rivaroxamban and dabigatran are also expected to be active, with calculated free binding energies of −7.97 and −6.57 kcal mol −1 , respectively. since the evaluation of the randomly selected hcv protease, dpp-4, α-thrombin and coagulation factor xa inhibitors showed promising results, we proceeded to the evaluation of a more extended sample of approved drugs of these categories, including a number of drugs currently in phase iii clinical trial (table 3, table 4 ). for better estimation of the probable inhibitory effect of the selected compounds, a second protein structure of the sars-cov-2 protease was also used (pdb id: 6m2n). the whole enzyme, including both protease subunits, was selected for the process. for comparison reasons, the estimated binding energy of the compounds to their initial targets were also calculated. for the same reasons, for some of the most promising sars-cov-2 candidate inhibitors, the estimated binding energy to the hiv-1 4rvj protease structure was calculated. docking analysis of both sars-cov-2 protease structures gave similar results. interestingly, 20 out of the 24 compounds which were selected for evaluation exhibited lower estimated binding energy than the initial inhibitor of the 6m2n structure (−7.45 kcal mol −1 ) [34] and of the n3 inhibitor (−7.41 kcal mol −1 ) [35] . docking of the compounds to the target proteases for which they were designed showed a generally higher binding probability (lower estimated binding energy) for the initial targets compared to the sars-cov-2 protease but the most active compounds showed comparable results for the two targets. for example, faldaprevir exhibited an estimated binding energy of −11.27 kcal mol −1 for the sars-cov-2 protease and −11.15 kcal mol −1 for the hcv protease, while teneligliptin showed −9.87 kcal mol −1 for the new protease and −9.16 kcal mol −1 for its initial target. table 3 . estimated binding energies of approved or under-trial iii protease inhibitors to the structure of the sars-cov-2 main protease structures (pdb code: 6lu7 and pdb code: 6m2n), to their initial protease target (hcv protease structure 2wf8, dpp-4 structure 2ffw, a-thrombin structure 1dwe, factor xa structure 4bti) and to the hiv-1 protease structure 4rvj. according to the estimated free binding energies, all evaluated drugs could act as sars-cov-2 protease inhibitors. among the hcv protease inhibitors, faldaprevir exhibited the lowest free binding energy. teneligliptin and gemigliptin are the most promising drugs among the dpp-4 inhibitors. in the group of anti-coagulants, inogartan and argatroban of the first randomly selected compounds were the most promising among a-thrombin inhibitors and edoxaban among factor xa inhibitors. docking analysis of the whole enzyme indicated that the enzyme active site is the first preferred binding site for all the hcv protease and dpp-4 inhibitors presented in table 3 but the third preferred site for inogatran and the second for edoxaban. consequently, the expected inhibitory action of inogatran and edoxaban are to be lower than that predicted based on their relative binding energy to the active site. in conclusion, faldaprevir, teneligliptin and gemigliptin are the compounds expected to exhibit the highest activity against the sars-cov-2 main protease. the sars-cov-2 protease preferentially cleaves its substrate after gln which follows leu and before a ser or ala or gly amino acid (leu-gln ↓ ser/ala/gly). the gln amino acid, at the p1 position of the substrate, is an amino acid with a relatively long side chain leading to a polar amide group with hydrogen donor/acceptor possibility. as shown by studies of complexes of the mpro enzyme with substrate analogues [34] , gln is placed in vicinity to cys145, at the subsite s1 of the active center, surrounded by the side chains of the amino acids asn142, glu166, his163, and his172 and the main chains of phe140 and leu141. the p2 amino acid, leu, has a relatively long, hydrophobic but not bulky or aromatic side chain. the side chain of leu is inserted at the s2 subsite, which consists of the side chains of the amino acids his41, met49, tyr54 and met165 placed in vicinity to asp187. the p4 amino acid of the peptide interacts with the amino acids met165, leu167, phe185, and gln192 and the main chain of gln189. in the case of the substrate analogue n3, in addition to polar or hydrophobic interactions, a hydrogen bond (hb) is formed between the his163 side chain and o of the lactam ring which serves as a mimetic of the gln amino acid of the natural substrate [34] . a second hb is formed between the h of the closest to the lactam ring nh group of the backbone of the ligand and the co of the main chain of his164 of the enzyme, while the nh group of the backbone of leu amino acid of the ligand interacts with the co of the backbone of gln189 of the enzyme. the lactam ring as a mimetic of the gln amino acid of the substrate is also present at the structure of the a-ketomide inhibitors designed by zang et al. [22] . in this case, the h of the nh group of the lactam ring forms two hydrogen bonds with the co of the main chain of phe140 and the carboxylate of glu166, while the co oxygen of the lactam moiety forms a hydrogen bond with the imidazole of his163. in the most active compound of this series, the p2 leu of the natural substrate has been replaced by a cyclopropane, while the pyridon ring at the p3 position participates in complex stabilization through hydrogen bonds with the o of the side chain of gln189 and the nh of the main chain of glu166. the two above substrates constitute peptidomimetics with peptide bonds between amino acid mimetic moieties. in both cases, the catalytic amino acid cys145 forms a covalent bond with the ligand. most of the approved drugs proposed as sars-cov-2 protease inhibitors according to in silico studies are not peptidomimetics. according to docking analysis, various groups may be placed at the s1 subsite such as the 7-methoxy-8-methyl-quinoline moiety of simeprevir [15] or the oxadiazole group of raltegravir, which adopts a curved form within the binding pocket [13] . interaction with thr24, thr25 and ser46 also seems to play an important role in complex stabilization of many compounds including raltegravir and ribavirin [12] . most inhibitors used in this study do not contain the classic peptide bonds but share with peptide substrates the characteristic of many polar, hd/a groups. in gemigliptin, the tetrahydropyridine ring and the 2-amino-4-oxobutyl bridge are placed at the s1 subsite between glu166 and cys145. a hydrogen bond between the co group of the backbone of glu166 and the nh 2 group of the 2-amino-4-oxobutyl bridge contributes to complex stabilization, while cys145 participates in hydrophobic interactions with carbons of the bicyclic moiety ( figure 3 ). the trifluoromethyl pyrimidine moiety interacts with his41 of the s2 subsite which stabilizes the complex by halogen bond formation with the f atoms and pi interactions with the pyrimidine ring. halogen bonds are also formed between the f atoms of the fluoropiperidinone ring and amino acids val186, arg188 and thr190. in linagliptin, the n of the piperidine ring participates in hydrogen bond interactions with the o of the co group of the main chain of glu166 of the s1 subsite, while the nh 2 substituent of the ring forms hb with pro168. the his41 of the s2 subsite participates in complex stabilization by polar interaction with the n atom of the methylquinazoline and by hydrophobic and pi interactions with the carbons of this moiety, while met49 also forms hydrophobic interactions. additional stabilization is achieved by the interaction of met165 with the dihydropurine moiety ( figure 3 ). in faldaprevir, the cyclopentyl carbamate moiety participates in hydrogen bond formation with the asn142 of the s1 subsite, while hydrogen bonds are also formed between the 2-ethenylcyclopropane-1-carboxylic acid group and the amino acids thr25 and ser46 (figure 4 ). in the case of melagatran, a curved conformation is adopted. the catalytic amino acid, cys145, forms hydrophobic interactions with cyclohexan, while asn144 of the s1 subsite strongly contributes to complex stabilization via polar interaction with the n atom of the aminoacetyl acid group. at the s2 subsite, the nh group of the side chain of his41 participates in hydrogen interaction with the o of the methyl carbamoyl bridge, while met49 is involved in hydrophobic interactions with the phenyl ring of the molecule. in addition, the oh group of the nearby ser46 participates in hydrogen bond formation with the nh 2 of the benzimidamide group ( figure 5) . in betrixaban, the chloropyridin moiety is placed at the s1 subsite, interacting via halogen bonds with the amino acids glu166, phe140 and leu141 and via hydrophobic interactions with cys145. in addition, a hydrogen bond is formed between cys145 and the n of the amide bridge connecting the chloropyridin-2yl and the methoxybenzyl rings of the molecule. his41 of the s2 subsite forms pi interactions with the carbons of the methoxyphenyl ring. the n,n-dimethylbenzimidamide moiety, placed in the area of the s4 subsite, strongly contributes to complex stabilization via the hydrophobic interaction between met165 and the phenyl ring as well as by hydrogen bond formation between the amino acids arg188, thr190 and gln192 and the n of the n,n-dimethylbenzimidamide moiety ( figure 5 ). in general, there is a great variety between the groups placed at the s1 or s2 subsites of the enzyme, as concluded by our results and the literature [12, 13, 15] . most of the amino acids involved in complex stabilization of the natural substrate and the peptide analogues [22, 34] participate in interactions with the studied compounds as well. however, the substantial interaction with his163 in peptidomimetic complexes is not observed in the interaction pattern of the most potent compounds. different structures may offer the possibility of interaction with the crucial amino acids of the sars-cov-2 active site contributing to complex stability. however, the right distances between polar, hd/a, hydrophobic/aromatic moieties constitute important features of each structure. according to docking analysis, all active molecules are long enough to interact simultaneously with amino acids surrounding glu166, cys145 and amino acids in the vicinity of thr25 and his41 and contain several hydrophobic and aromatic moieties as well as hydrogen bond donor/acceptor groups capable of hydrogen bond formation and polar interactions. the enzyme amino acids involved in hydrogen bond formation are glu166, pro168, gln189, arg188, thr24, thr25, thr26, his41, cys44, ser46, asn142, gly143, cys145, his164, thr190, and glu192 (figures 3-7) . apart from the polar and hydrophobic interactions, at least two hydrogen bonds are formed in the case of the most active compounds, while interactions with phe140 were also involved in complex stabilization of argatroban. the most promising compounds of all categories contain multiple hydrophobic moieties as well as polar/hydrogen donor/acceptor moieties. the most active molecules of the categories of gliptines and previrs are relatively long molecules and adopt linear conformations. hydrophobic moieties at distances of approximately 9 å, 11 å, 14 å and 16 å are present in all potent compounds. met165, his41, cys44, cys145, leu167 of the mpro active site are mainly involved in hydrophobic and pi interactions, with a maximum distance between them of approximately 18 å (figures 3-7) . polar or hydrogen donor/acceptor moieties are present at multiple distances, with a distance of 8 å being common in active compounds. glu166 and his 41, at a distance of approximately 13 å, participate in hydrogen bond and polar interactions of the most potent gliptins (figures 3 and 6) , while ser46, thr25 and asn142, at a distance of approximately 12 å, participate in hydrogen bond formation of faldaprevir. in the sars-cov-2 protease, amino acids involved in hydrophobic and polar interactions are distributed in all the active sites, although pi interactions are mostly observed at the region of his41. distances of approximately 5 å, 9 å, 11 å and 16 å are observed between amino acids participating in polar and hydrophobic interactions. amino acids with hydrophobic or polar/hydrogen donor acceptor properties exist at similar distances at the active sites of the initial target of these compounds, dpp-4 and the hcv protease, respectively. for example, in the case of the gliptins' initial target dpp-4, a great number of tyrosine amino acids are present at the active site. as shown in the case of teneligliptin, figure 6 , the amino acids tyr586, phe355, tyr667, tyr667, ty548, and tyr632 are involved in hydrophobic and pi interactions with distances among them varying between 8 å and 19 å. the amino acids arg123, glu203, glu204, ser631, tyr633 and asn711 are involved in polar interactions and hydrogen bond formation. all hydrophobic interactions originate from amino acids oriented at the same moiety of the active site, while the amino acids participating in polar interaction are placed in the other moiety. distances, between 11 and 18 å are observed between amino acids of the active site participating in polar and hydrophobic interactions. the length of the molecules, the great number of polar and hydrophobic groups, the flexibility of the molecules and the appropriate distances between characteristic groups seem to favor binding of gliptins at the mpro active site. a higher binding energy was estimated for the smaller molecule alogliptin (figure 7) . in the case of thrombin inhibitors, a number of hydrogen bonds and hydrophobic interactions mainly stabilize the complex. his41, ser46 and met49 participate in hydrogen bond formation between melagatran and mpro enzyme, while leu27, his41, met49 and cys145 are involved in hydrophobic interactions. although these interactions lead to a substantially low estimated binding energy of the compound, the significantly higher hydrogen bonds, pi bonds, polar and hydrophobic interactions may be responsible for the much better prediction results for binding to the initial protease target. ala189, ala190, gly219, cys191, cys220, trp215, his 57 and leu99 seem to be involved in binding to thrombin. the evaluated compounds were approved drugs, currently on the market with the exception of four compounds which were under phase iii trial (table 4 ). there are several compounds among the most promising ones, which are characterized as well-tolerated or having no side effects, such as danoprevir and sovaprevir of the hcv protease inhibitors, teneligliptin and gemigliptin but also trelagliptin, evogliptin and gosogliptin among the dpp-4 inhibitors, inogatran and melagatran of α-thrombin inhibitors. the dpp-4 inhibitors developed for the treatment of diabetes type ii as well as the α-thrombin and factor xa inhibitors, used as anti-coagulants, may not be appropriate for patients who do not suffer such problems. however, they may be worth consideration for patients already being treated for type ii diabetes or with anti-coagulants. moreover, higher platelet count and increased coagulation features are observed in covid-19 patients with severe pneumonia compared to patients with non sars-cov-2-induced pneumonia. furthermore, according to recent results, anti-coagulant treatment by heparin administration reduced mortality of covid-19 patients with severe pneumonia and markedly elevated d-dimers [35] . in general, the results can be considered as promising for some of the evaluated drugs and could be useful for scientists working in the field. in vitro evaluation is also within our future targets if the recombinant sars-cov-2 protease becomes available in the market. the 3d alignment was performed between the 3d structure of the sars-cov docking analysis was carried out on a molecular docking server using autodock [38, 39] , as previously described [32] . the lamarckian genetic algorithm (lga) and the solis and wets local search method [40] were used for the docking simulation. during docking, all rotatable torsions were released. autodock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der waals and the electrostatic terms, respectively-100 different runs that were set to terminate after a maximum of 2,500,000 energy evaluations were performed in each docking experiment. the population size was set to 150. a translational step of 0.2 å, and quaternion and torsion steps of 5 were applied during the search. docking analysis of the hcv protease and the hiv-1 protease presented in table 3 was performed using autodock4.2. via ligandscout. among the values calculated by the program is the estimated free binding energy, which is an indicator of the probability of the compounds to form a stable complex with the selected enzyme target and consequently the probability to be effective enzyme inhibitors. the protein structures chosen from the protein data bank and the docking center and docking box chosen for each docking analysis were as follows: for docking analysis of the sars-cov-2 main protease enzyme, the structure 6lu7 of the enzyme in a complex with the competitive inhibitor n3 for active compounds, the docking was repeated, extending the docking box to cover the whole enzyme, to ensure that the compound does not preferentially bind to other sites of the enzyme instead of the active site [32] . for better estimation of the probable inhibitory effect of the selected compounds, a second protein structure of the sars-cov-2 protease complexed with the inhibitor 5,6,7-trihydroxy-2-phenyl-4h-chromen-4-one was also used (pdb id: 6m2n). in this structure, both subunits were included in the pdb file and the whole enzyme constructed by the two identical subunits was used. the use of the two subunit form was preferred as it could enable the evaluation of a probable interference of the second subunit in binding of the inhibitors, since glu166 which commonly participates in complex stabilization interacts with ser1 of the other protein chain. the docking center was set at x = −61.73, y = −35.18, z = 23.26 and the docking box was set at x = 20, y = 20, z = 20. for comparison reasons, the estimated binding energy of the compounds to their initial targets was also calculated. the structures chosen were the hcv structure 2wf8 in complex with asunaprevir (center: for some of the most promising sars-cov-2 candidate inhibitors, the estimated binding energy to the hiv-1 protease 4rvj structure (crystallized in complex with amprenavir) was evaluated (center: x = −1.935, y = 4.006, z = 48.913, box x = 40, y = 40, z = 30). in all cases, during enzyme preparation, the inhibitor of the initial enzyme complex was abstracted, and the ph was set at 7.0 for ligand preparation. for the evaluation of the docking method, the initial ligand was docked to the enzyme and the position was compared with its initial position at the complex. examples of the similarity of the two positions are shown at figure 8 . stomach ache, abnormal results of blood tests that measure liver function, anemia [57] otamixaban -ended at phase iii sanofi figure 8 . docking of the initial ligand (5,6,7-trihydroxy-2-phenyl-4h-chromen-4-one) of the 6m2n complex to the sars-cov-2 protease structure 6m2n and of the initial ligand (sitagliptin) to the 4ffw structure of dpp-4. the position of the docked ligand (green) is very close to that of the initial ligand (magenta). the 3d structure alignment of the sars-cov-2 main protease with the hiv-1 and hcv viral proteases as well as with the human proteases dpp-4, thrombin, coagulation factor xa, renin and ace showed greater similarity with the hcv protease and with thrombin. this finding, in combination with the fact that these two proteases preferentially cleave before ser like sars-cov-2 protease, enhances the possibility of finding effective sars-cov-2 inhibitors among the approved anti-hcv protease and anti-thrombin inhibitors. docking analysis of 34 approved or phase iii trial protease inhibitors to the sars-cov-2 protease revealed several drugs that may act as sars-cov-2 protease inhibitors, within the classes of the hcv protease, dpp-4, α-thrombin and coagulation factor xa inhibitors. twenty five of them exhibited estimated binding energy lower than the reference inhibitor n3, and twenty one lower than −8.00 kcal mol −1 . several of the most promising compounds are approved drugs characterized as well tolerated in subjects. compared to lopinavir and ritonavir, most of the evaluated compounds showed better results, as characterized by lower predicted free binding energy and high preference for binding to the active site compared to other sites of the enzyme. these promising in silico results encourage further evaluation for in vitro and in vivo anti-viral activity. the dpp-4 inhibitors developed for the treatment of type ii diabetes, as well as α-thrombin and factor xa inhibitors used as anti-coagulants, may not be appropriate for patients who do not suffer such problems but may be useful for patients who need such treatment. it is worth mentioning that diabetic patients belong to the high-risk group for this virus, while anti-coagulant therapy has been proposed for covid-19 patients with severe pneumonia. the authors declare no conflict of interest. rna-dependent rna polymerase dpp-4 dipeptidyl peptidase-4 covid-19) outbreak situation covid-19 coronavirus pandemic. available online genomic characterization of the 2019 novel human pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin therapeutic options for the 2019 novel coronavirus (2019-ncov) coronaviruses-drug discovery and therapeutic options hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro apeiron biologics to trial coronavirus drug candidate in china rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds potential inhibitors against 2019-ncov coronavirus m protease from clinically approved medicines in silico screening of natural compounds against covid-19 by targeting mpro and ace2 using molecular docking virtual screening and repurposing of fda approved drugs against covid-19 main protease targeting sars-cov-2: a systematic drug repurposing approach to identify promising inhibitors against 3c-like proteinase and 20-o-ribose methyltransferase ul-haq, z. identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach molecular investigation of sars-cov-2 proteins and their interactions with antiviral drugs putative inhibitors of sars-cov-2 main protease from a library of marine natural products: a virtual screening and molecular modeling study an investigation into the identification of potential inhibitors of sars-cov-2 main protease using molecular docking study potential inhibitors of coronavirus 3-chymotrypsin-like protease (3cl pro ): an in silico screening of alkaloids and terpenoids from african medicinal plants potential anti-sars-cov-2 drug candidates identified through virtual screening of the chembl database for compounds that target the main coronavirus protease. febs open bio 2020 emerging principles in protease-based drug discovery the use of stems in the selection of international nonproprietary names (inn) for pharmaceutical substances; world health organization crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors comparative studies on retroviral proteases: substrate specificity new details of hcv ns3/4a proteinase functionality revealed by a high-throughput cleavage assay dipeptidyl peptidase iv inhibition for the treatment of type 2 diabetes: potential importance of selectivity over dipeptidyl peptidases 8 and 9 factor xa active site substrate specificity with substrate phage display and computational molecular modeling the extended cleavage specificity of human thrombin molecular recognition and regulation of human angiotensin-i converting enzyme (ace) activity by natural inhibitory peptides the his-pro-phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin direct thrombin inhibitors an ensemble view of thrombin allostery docking analysis targeted to the whole enzyme: an application to the prediction of inhibition of ptp1b by thiomorpholine and thiazolyl derivatives prediction of enzyme inhibition and mode of inhibitory action based on calculation of distances between hydrogen bond donor/acceptor groups of the molecule and docking analysis: an application on the discovery of novel effective ptp1b inhibitors structure of mpro from covid-19 virus and discovery of its inhibitors difference of coagulation features between severe pneumonia induced by sars-cov2 and non-sars-cov2 mmdb and vast+: tracking structural similarities between macromolecular complexes application of the pm6 semi-empirical method to modeling proteins enhances docking accuracy of auto-dock automated docking using a lamarckian genetic algorithm and an empirical binding free energy function minimization by random search techniques a phase 3 study in combination with bms-790052 and bms-650032 in japanese hepatitis c virus (hcv) patients. available online daclatasvir plus asunaprevir for chronic hcv genotype 1b infection pharmacokinetics, safety, and tolerability of faldaprevir in patients with renal impairment management of adverse events during the treatment of chronic hepatitis c infection patients with hcv genotype 1 infection: safety, antiviral activity, resistance, and pharmacokinetics fda drug safety communication: fda adds warnings about heart failure risk to labels of type 2 diabetes medicines containing saxagliptin and alogliptin multiple-dose pharmacokinetics and pharmacodynamics of evogliptin (da-1229), a novel dipeptidyl peptidase iv inhibitor, in healthy volunteers addition of dipeptidyl peptidase-4 inhibitors to sulphonylureas and risk of hypoglycaemia: systematic review and meta-analysis inhibitors for type 2 diabetes: drug safety communication-may cause severe joint pain sitagliptin phosphate monograph for professionals available online this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord-000008-3dgjv0x1 authors: vali, bahareh; yue, feng yun; jones, r. brad; sheth, prameet m.; kaul, rupert; betts, michael r.; wong, david; kovacs, colin; loutfy, mona; common, andrew; halpenny, roberta; ostrowski, mario a. title: hiv-specific t-cells accumulate in the liver in hcv/hiv co-infection date: 2008-10-20 journal: plos one doi: 10.1371/journal.pone.0003454 sha: doc_id: 8 cord_uid: 3dgjv0x1 background and aims: hepatitis c virus (hcv)-related liver disease progresses more rapidly in individuals co-infected with human immunodeficiency virus-1 (hiv), although the underlying immunologic mechanisms are unknown. we examined whether hiv-specific t-cells are identified in the liver of hcv/hiv co-infected individuals and promote liver inflammation through bystander immune responses. methods: ex-vivo intra-hepatic lymphocytes from hcv mono-infected and hcv/hiv co-infected individuals were assessed for immune responses to hiv and hcv antigens by polychromatic flow cytometry. results: hcv/hiv liver biopsies had similar frequencies of lymphocytes but lower percentages of cd4(+) t-cells compared to hcv biopsies. in co-infection, intra-hepatic hiv-specific cd8(+) and cd4(+) t-cells producing ifn-γ and tnf-α were detected and were comparable in frequency to those that were hcv-specific. in co-infected individuals, viral-specific cd8(+) t-cells produced more of the fibrogenic cytokine, tnf-α. in both monoand co-infected individuals, intra-hepatic hcv-specific t-cells were poorly functional compared to hiv-specific t-cells. in co-infection, haart was not associated with a reconstitution of intra-hepatic cd4(+) t-cells and was associated with reduction in both hiv and hcv-specific intra-hepatic cytokine responses. conclusion: the accumulation of functional hiv-specific t-cells in the liver during hcv/hiv co-infection may represent a bystander role for hiv in inducing faster progression of liver disease. approximately 25% of human immunodeficiency virus-1 (hiv) infected individuals are also infected with hepatitis c virus (hcv) [1] . hiv adversely affects each stage of the natural history of hcv infection. fewer individuals recover spontaneously from hcv infection when also infected with hiv [2] . among those with persistent hcv infection, hiv co-infection is associated with higher hcv viremia and more rapid progression to cirrhosis and hepatocellular carcinoma [3] . a recent meta-analysis showed that hiv co-infection increased the risk of histological hepatic cirrhosis by two-fold and clinically decompensated liver disease by six-fold [4] . in addition, hcv co-infection is associated with increased incidence of haart (highly active antiretroviral therapy) related liver injury [5] . the mechanisms for hepatic damage in hcv/hiv co-infection are poorly defined. although intra-hepatic t-cell immune responses are necessary for hcv clearance, they have also been shown to play a central role in mediating hepatocellular injury by direct cytotoxicity or indirectly by releasing cytokines. in this regard, ifn-c has been shown to be anti-fibrogenic, whereas, tnf-a activates hepatic stellate cells, which induce fibrosis, and likely contributes to progression to cirrhosis [6, 7] . potent and broad cd4 + and cd8 + t-cell immunity are important for virologic control in both hcv and hiv viral infections. ex-vivo hcv-specific cd8 + t-cell responses in peripheral blood mono-nuclear cells (pbmcs) from mono-infected individuals are generally weak [8] . although, peripheral hcvspecific cd4 + and cd8 + t-cell responses are somewhat weaker in hcv/hiv co-infected individuals [9] , similar frequencies of intra-hepatic hcv-specific responses appear to be obtained in hcv versus hcv/hiv co-infection [10, 11] . however, ex-vivo hiv-specific cd8 + t-cell responses in pbmcs from hiv monoinfected individuals are about one log higher than ex-vivo hcvspecific responses in hcv mono-infection. in addition, impairment in cellular immune responses to hcv compared to hiv has been shown in hcv/hiv co-infection [12] . hiv-specific cd8 + t-cells are easily detectable in blood of untreated hiv infected individuals [13] . such high frequencies of hiv-specific t-cells circulating in peripheral blood led us to question whether these cells could also migrate to the liver in hcv/hiv co-infection and through bystander responses add to the inflammation induced by hcv-specific t-cells. hcv mono-infected and hcv/hiv co-infected individuals who required liver biopsies for work up of liver disease were recruited for the study (see results and table 1 ). all study participants provided informed, written consent and the study protocol was approved by the research ethics board at the university of toronto and st. michael's hospital. both blood and liver biopsy samples were received from each participant. liver biopsy samples were washed in rpmi-1640 to remove contaminating blood lymphocytes, manually homogenized with a plastic plunger, and treated with dnase (0.002%, sigma) and collagenase iv (0.02%, sigma) for 30 minutes, stirring at 37uc. the digested cell suspension was filtered through a 70 mm strainer, washed and re-suspended in r-10 medium (10% fetal calf serum). in order to identify candidate epitope-specific responses to be detected in ex-vivo liver samples, we first mapped antigen-specific t-cell responses in blood against the entire hiv-1 clade-b and hcv-1a proteome using the matrix approach by ifn-c eli-spot assay as described previously [14] . mapped peptides were then pooled to evaluate hepatic responses. in order to address the possibility that differing epitopes were only targeted in the liver, we also used four peptide pools that previously were shown to target a majority of responses. these pools spanned hiv-gag and hcv-ns3, hcv-ns4 and hcv-core protein (2 mg/peptide/ml, from national institute of health reagent program). of the hcv pools, the pool that gave the strongest elispot response in pbmcs was used for hepatic cell stimulation (see below). all the extracted cells from each liver biopsy were split in three wells and stimulated on the same day as pbmcs. 1610 6 pbmcs and liver-isolated cells were stimulated with either dmso, hiv or hcv peptide pools as described previously [14] . hiv pools consisted of peptides that were screened by the matrix approach in that individual plus the hiv-gag pool. likewise, hcv pools consisted of mapped peptides plus an hcv pool that gave the strongest response in pbmcs. cd107a antibody (pe-cy5, bd pharmingen) was added at the time of stimulation. the following antibodies were used for staining: cd8-pe texas-red (beckman coulter), cd4-pacific blue (e-bioscience), cd3-apccy7, ifnc-fitc, tnfa-pecy7, il2-apc, mip-1b-pe (bd bioscience), pd-1 fitc (biolegend) and dead cell stain aqua (invitrogen, molecular probes). cells were analyzed on a multi-color facsaria flow cytometer (bd biosciences). for blood samples between 500,000 to 1,000,000 total events and for liver biopsy samples between 50,000 to 200,000 total events were collected. data analysis was performed using flowjo version 8.6 (treestar inc., san carlos, ca). polychromatic flowjo data were analyzed with pestle software, and pie-chart graphs were generated using spice software (obtained from m. roederer, national institutes of health, bethesda md). multi-parameter analysis of hiv-gag: 77-85 (slyntvatl: sl9) specific cd8 + t-cells was conducted in both blood and liver of co-infected individuals initially identified with a positive elispot response to the 15-mer hiv peptide including sl9 epitope, using the corresponding tetramer (itag mhc class-i tetramer, beckman coulter). tetramer staining was performed prior to peptide stimulation, at room temperature for 20 minutes. tetramer stained cells were then washed and stimulated with 10 mg/ml of sl9 peptide followed by ics staining as mentioned above. additional hiv, hcv and cmv-specific pentamer staining (pro5 mhc class i pentamers -proimmune) was conducted followed by pd-1 staining. data were analyzed by performing two-tailed non-parametric mann-whitney test using graphpad prism version 4.00. p-values#0.05 were considered significant. three groups of individuals were studied as depicted in table 1 ; hcv mono-infected (n = 6), hcv/hiv co-infected who were not receiving haart (n = 8) and hcv/hiv co-infected who were receiving haart for greater than one year at the time of evaluation (n = 12). all individuals never received prior treatment for hcv and underwent liver biopsies for staging and evaluation for pegylated-interferon/ribavirin treatment. hcv/hiv co-infected individuals had higher hcv viral loads. on average, cd4 t-cell counts of hiv infected individuals were .400/ml in both groups. of note, the mean hepatic fibrosis scores were higher in the haart treated and mono-infected groups in this cohort, indicating that individuals in these groups had more advanced disease at the time of biopsy in this study. although similar frequencies of intra-hepatic lymphocytes were obtained in dual versus mono-infection, haart-treated individuals showed a trend towards greater percentages of lymphocytes in their biopsies ( figure 1a ). the percentage of intra-hepatic cd4 + t-cells was significantly reduced in dual infection [31.6%613.8 for hcv vs 6.5%62.9, for hcv/hiv therapy naïve, p,0.01], and was not associated with any improvement in haart-treated individuals, as previously shown in the gut [15] (figure 1b) . however, compared to hcv mono-infected individuals the percentage of intra-hepatic cd8 + t-cells was higher in both co-infected groups [33.8%65.5% for hcv vs 67.3%615.5% for hcv/hiv therapy naïve vs 59.5615.1% for hcv/hiv on haart, p,0.01] (figure 1b ). to determine the presence of intra-hepatic viral specific t-cell responses, liver isolated cells were stimulated with hcv and hiv peptide pools. summary data of viral specific responses are depicted in figure 2 . in response to stimulation with hiv peptide pool, untreated co-infected individuals showed significantly higher frequencies of intra-hepatic cd4 + t-cells producing ifn-c, compared to hcv mono-infected [0.1660.05% vs 0.0260.01%, p,0.05], and haart-treated co-infected individuals [0.1660.05% vs 0.0360.05%, p,0.05] (figure 2a ). untreated co-infected individuals showed a trend towards lower frequencies of intra-hepatic ifn-c producing cd4 + t-cells in response to hcv peptides. surprisingly, haart-treated co-infected individuals had significantly reduced hcv-specific ifn-c producing cd4 + t-cells when compared to untreated co-infected individuals [0.0260.01% vs 0.4660.11%, respectively, p,0.01] (figure 2a). therapy naïve co-infected subjects had greater ifn-c producing cd8 + t-cells in response to hiv peptides compared to hcv mono-infected individuals [1.3960.37% vs 0.0260.0%, p,0.05], and haart was associated with a significant reduction in the frequencies of these cells [1.3960.37% vs 0.3060.26%, p,0.05] (figure 2b). although there was a trend for enhanced intra-hepatic cd8 + t-cells producing ifn-c in response to hcv peptides in therapy-naive co-infection compared to hcv mono-infection, this was not found to be statistically significant. haart on the other hand, was associated with a significant reduction in hcv-specific, intra-hepatic cd8 + t-cells producing ifn-c [1.360.37% vs 0.0360.01%, p,0.05] (figure 2b). similarly, co-infected individuals had significantly greater intrahepatic tnf-a expressing cd4 + t-cells after hiv peptide stimulation compared to hcv mono-infected [0.260.05 vs 0.0260.01, p,0.01], although haart had no significant effect on their frequencies (figure 2c ). hcv mono-infected individuals showed significantly higher frequencies of hcv-specific tnf-a producing cd4 + t-cells compared to haart-treated co-infected individuals [0.9160.25% for hcv vs 0.2360.20 for hcv/hiv on haart, p,0.01] (figure 2c), but did not show significant differences with the untreated co-infected group. the therapy-naïve co-infected group showed significantly higher frequencies of intra-hepatic tnf-a producing cd8 + tcells in response to both hiv-1 and hcv antigens. both types of to study the functional profile of virus-specific t-cells in hcv/ hiv co-infection, simultaneous expression of 5 distinct cd8 + t-cell markers were analyzed in 3 individuals within each cohort using a previously developed multicolor flow cytometry method [16] . expression levels of degranulation marker cd107a and cytokines ifn-c, tnf-a and il-2, as well as the chemokine mip-1b were simultaneously measured in response to hcv or hiv peptides in both blood and liver of each individual. figure 3 depicts a representative multi-parameter analysis of cd8 + t-cell responses in liver and blood of a therapy-naïve, co-infected subject in response to hiv and hcv peptide pools. these data indicate that both hcv and hiv specific cd8 + t-cells expressing one or more functions are detectable in the liver and blood. compared to blood, the frequency of hiv-specific t-cells producing cd107a and il-2 was shown to be significantly higher in the liver of therapy-naïve, co-infected individuals. consistent with previously reported data [10] , hcv-specific responses were compartmentalized to the liver and stronger than peripheral hcv-specific responses (figure 3c ). recognition of functional ctl, specific for the hla-a0201-restricted hiv-sl9 epitope in hcv/hiv co-infected liver to determine if t-cells specific for an hiv immuno-dominant epitope are present in hcv/hiv co-infected liver, we quantified cd8 + t-cells specific for hla-a*0201-restricted slyntvatl (sl9) epitope in the liver of individuals with positive sl9 responses in their blood. we identified 3 co-infected hla-a*0201 individuals, among them one showed a response to the sl9 epitope of hiv-gag antigen. figure 4 shows the multi-parameter analysis of tetramer positive cd8 + t-cells in blood and liver of this therapy-naïve, co-infected individual. the tetramer cytokine response pattern was shown to be different in the liver compared to blood of the same individual, with diminished intra-hepatic tetramer-specific ifn-c responses and an increase in both cd107a and tnf-a responses, with the majority of sl9 tetramer positive cells expressing these two markers. we also included cmv as a non-hepatotropic control virus in our liver analysis. using a pool of hla-a*0201-and hla-a*2402-restricted, cmv-specific pentamers, we did not detect any cmv-specific cd8 + t-cells in hcv/hiv co-infected liver, although we could readily detect them in blood (figure 4c ). using the panel of markers tnf-a, ifn-c, mip-1b, il-2, and cd107a, we characterized the ability of cd8 + t-cells to simultaneously exert these 'functions' in response to both hcv and hiv peptides. figure 5 depicts a representative functional profile of virus specific cd8 + t-cells in blood and liver during hcv/hiv co-infection (fig. 5a) and hcv mono-infection (fig. 5b) . analysis of co-infected subjects demonstrates a very limited functional hierarchy of hcv-specific t cells in the blood, with majority of t-cells producing one function. hiv-specific tcells from blood had a more expanded functional hierarchy. in accordance with previous reports on hiv mono-infected individuals [16] , cells expressing all 5 functions were absent in the blood of co-infected subjects, mainly due to lack of il-2 production. in co-infection, intra-hepatic cd8 + t-cells responding to hcv peptides were within the single-responding and 2+ populations. intra-hepatic cd8 + t-cell responses to hiv peptides produced a larger spectrum of responses. cd107a responding cells were represented in nearly all of the different hiv-specific populations in the liver of co-infected individuals. as expected, hcv-specific responses in the blood of hcv mono-infected subjects were mainly single functional. the profile of hcv-specific responses in the liver of hcv mono-infected individuals showed the appearance of a very small population of 4+ responding cells in the liver. we and others have considered a cutoff point of more than 2 simultaneously expressed markers to demonstrate poly-functional characteristic of responding t-cells [16, 17] . figure 5c shows a comparison between average frequency of intra-hepatic viralspecific responses within the pool of cd8 + t-cell populations expressing 2 markers or less, and those within the pool of populations expressing more than 2 markers simultaneously. for both hcv and hiv-specific cd8 + t-cells the majority of responses had two or less functions. however, intra-hepatic hiv-specific responses demonstrated more poly-functionality, compared to hcv-specific responses either within co-infected or mono-infected individuals [0.0560.01 vs 0.00760.00, p,0.05; hiv-specific responses vs hcv-specific responses in hcv/hiv co-infected group]; [0.0560.01 vs 0.0160.00, p,0.05; hivspecific responses in hcv/hiv co-infected group vs hcvspecific responses in hcv mono-infected group]. in summary, although viral-specific t-cells, simultaneously expressing all 5 measured markers were rarely found in the liver, intra-hepatic hiv-specific t-cells showed greater functional capacity when compared to those being hcv-specific. based on the recently highlighted role of pd-1 contributing to the dysfunction of t-cells in chronic viral infections, we also determined whether hiv and hcv-specific intra-hepatic t-cells differ in the degree of pd-1 expression. in an hcv/hiv coinfected liver, we found that 100% of intra-hepatic hcv-specific cd8 + t-cells were pd-1 positive, compared to 48.8% of those cells that were hiv-specific (figure 5d ). this is the first study to demonstrate the presence of hivspecific t-cells within the liver of hcv/hiv co-infected individuals. the finding of hiv-specific t-cells within liver of co-infected individuals may not altogether be surprising, given the high frequencies of hiv-specific cd8 + t-cells found in the peripheral blood in untreated hiv infection. nevertheless, it is surprising to find functional t-cells of such viral specificities to be accumulating in liver. in contrast, we could not detect cmvspecific t-cells in co-infected liver despite their abundance in blood indicating that different viruses target t-cells to the liver. recent studies have demonstrated that systemic viral infections may recruit viral-specific t-cells to the liver. the significance of non-hepatotropic viral-specific t-cells that are found in liver is unclear. it has been postulated that the liver can non-selectively trap activated t-cells during any infection, and thus act as a 'sink' or 'graveyard' [18] , however it is unclear whether these cells are rendered anergic while traveling in the liver or contribute to inflammation and damage as a result of bystander activation. of note, is that hepatitis has been observed in measles [19] , sars [20] and in 20% of individuals with acute hiv infection [21] . polakos et. al. [22] found that some individuals infected with influenza-a develop transaminitis and showed in a murine influenza model that influenza-specific cd8 + t-cells migrate to figure 3 . polychromatic facs analysis of viral-specific t cells in hcv/hiv co-infection. shown are representative facs data of the hiv and hcv specific multi-parameter cd8 + t-cell responses from (a) liver and (b) blood of subject om 405, a therapy-naïve hcv/hiv co-infected individual, after in vitro stimulation using pool of hiv and hcv peptides. initial gating on forward scatter area (fsc-a) versus height (fsc-h) was used to remove doublets. the events were further gated on forward scatter (fsc) versus the dead cell marker to remove dead cells. lymphocytes were gated on the remaining live cells on a fsc versus ssc plot. gates on cd3 + /cd8 + cells were then generated. all responses are gated on a cd3 + /cd8 + population and presented against tnf-a on the x-axis. figure (c) shows a comparison of the frequency of hiv and hcv-specific cd8 + t-cells in the liver and blood of therapy-naïve, co-infected individuals. all intra-hepatic hcv-specific responses are significantly stronger than peripheral hcv-specific responses. doi:10.1371/journal.pone.0003454.g003 non-hepatotropic viruses such as hiv, cmv and ebv, in general do not induce chronic hepatitis, thus, it is possible that the coexistence of hepatotropic viruses may alter the hepatic environment to allow recruitment of activated t-cells non-specifically. this could be due to an up-regulation of integrins such as icam-1 and vcam-1 in hepatic sinusoids as previously shown during hcv infection [23] that could enhance t-cell recruitment. in this regard, spangenberg et. al. [24] demonstrated the presence of influenza-specific t-cells in about 50% of liver biopsies from hcv mono-infected individuals. there are several lines of evidence demonstrating that the liver efficiently clears many foreign pathogens, including rna viruses. it is shown that liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys [25] . there is also evidence for the detection of hiv rna in the liver of hiv infected individuals [26] . these findings support the identification of hiv-specific t-cells in the liver. in hcv/hiv co-infection, it is possible that intra-hepatic hcv-specific cd4 + t-cells become infected with hiv and recruit hiv-specific immune responses to this site. evidence for these potential mechanisms will need further analysis on liver biopsies of co-infected individuals. our analysis of liver biopsies from hcv/hiv co-infected individuals not only demonstrate that hiv-specific t-cells producing ifn-c and tnf-a are detected in the liver, but also exhibit comparable frequencies of responses to those that are hcv-specific. this observation may explain the added contribution of hiv-specific immune responses to the ongoing intrahepatic damage induced by hcv-specific t-cell responses that are inefficient in clearing the virus. therapy naïve co-infected individuals demonstrated a higher frequency of intra-hepatic cd8 + t-cells that produce tnf-a in response to both hcv and hiv antigen stimulation compared to hcv mono-infected individuals. in addition, we identified cd8 + t-cells specific for an immunodominant hiv epitope in co-infected liver, demonstrating high frequency of tnf-a expression. intra-hepatic tnf-a has been previously associated with liver fibrosis, and the accumulation of cells expressing this marker may explain in part the faster rate of liver disease progression found in hcv/hiv coinfection. further comparisons of tnf-a responses between immunodominant hcv and other hiv epitopes in a larger cohort of individuals are warranted. contrary to our expectation, viral-specific, intra-hepatic levels of ifn-c were also higher in the therapy-naïve co-infected group, which would be against the expected protective role of ifn-c. however, we interpret this observation as a potential effect of the fibrogenic tnf-a to mask ifn-c protection. on the other hand, viral-specific t-cells are composed of several major populations with unique functional patterns. therefore, measurement of only one or two t-cell functions may not provide a comprehensive picture of the quality of t-cell responses. recent lines of evidence demonstrate the importance of the qualitative rather than quantitative characteristics of cd8 + t-cell responses to efficient viral control [13, 27] . the significance of tcell populations simultaneously representing 5 different functions has been discussed as a hierarchical functional model in viral infections such as cmv and ebv which are effectively controlled by respective cd8 + t-cells [16] . hcv-specific cd8 + t-cells were not poly-functional which is consistent with the notion that although hcv-specific t-cells are found in hepatic tissue, their loss of poly-functionality may be associated with inefficient control of hcv replication. hiv-specific t-cells in the liver of co-infected individuals however, simultaneously could express 4 and 5 of the measured markers. recently, t-cell exhaustion has been related to signaling pathways through pd-1 [28, 29] . our analysis of pd-1 levels of antigen-specific cd8 + t-cells from co-infected liver demonstrates higher expression of pd-1 on hcv-specific t-cells, compared to those specific for hiv, supporting the notion that the former are less functional. the observed poly-functionality of intra-hepatic hiv-specific t-cells, should have little effect on hcv replication but would further enhance the cytokine milieu induced from bystander activation, and contribute to liver damage during co-infection with hcv. in this regard, we found that the degranulation marker cd107a dominates the hiv-specific cd8 + t-cell responses in the liver, with the majority of the responding cells expressing cd107a, a surrogate marker for the cytotoxic function of cd8 + t-cells. activated hiv-specific cd8 + t-cells with the potential to degranulate could induce bystander damage. in addition, the release of chemokines such as mip-1b by the same cells could also attract further lymphocytes without hcv specificity to the liver. bystander function of these non-specific t-cells could expand the tissue damage triggered by hcv infection and ultimately activate fibrogenesis. we found that the frequency of cd4 + t-cells within livers of coinfected individuals was reduced compared to hcv monoinfection. surprisingly, haart did not appear to reconstitute the cd4 + t-cell population within liver. despite this defect of cd4 + t-cell help, comparable frequencies of hcv-specific-cd8 + t-cells were found in co-infected livers. haart-treated biopsies showed further reduced frequencies of hcv-specific responses. these data support previous findings that show haart induces cd4 + t-cell recovery but not any restoration of hcv-specific tcell responses peripherally [30] . further investigation is needed to clarify the role of cd4 + t-cell help in affecting the frequencies of hcv-specific cd8 + t-cells in hcv/hiv-1 co-infection. haart was also associated with a reduction in frequencies of hiv-specific t-cell responses within liver, indicating that removing the hiv antigenic load may also reduce the opportunity for such cells to accumulate within hepatic tissue. here, we propose a novel mechanism for enhanced hcvrelated liver disease progression in hiv co-infection; that of bystander activation and induced inflammation from hiv-specific t-cells accumulated in the liver. our data however are limited in the cross-sectional nature of our cohort, the low number of analyzed liver biopsies and the narrow range of cd4 + t-cell counts among the studied individuals. we should also acknowledge that ex-vivo functional t-cell capacity may not exactly reflect the situation in-vivo. further studies, particularly, those which are prospective are warranted in order to understand the role that hiv-specific t-cells play in contributing to fibrosis and in particular how haart modulates these responses. frequency of cd8 + t-cells specific for hcv, hiv and cmv in hcv/hiv co-infected liver (om 385). liver isolated mononuclear cells were stained with pools of hla-a*0201 and hla-a*2402-restricted pentamers (pro5 mhc class i pentamers, proimmune), followed by staining for cell surface markers cd3 and cd8. the following pentamers were used for each group: hcv pentamers: ns3-cingvcwtv and ns3-klvalginav; hiv pentamers: pol-ilkepvhgv and gag p24-tlnawvkvv; cmv pentamers: pp65-nlvpmvatv and pp65-qydpvaalf. no cd8 + t-cells specific for cmv were detected in this co-infected liver sample. similar findings were found in another individual (data not shown). doi:10.1371/journal.pone.0003454.g004 background is shown to become extremely low when examining combinations of functions, nearly reaching 0 events for multiple functions simultaneously. this permits a very low threshold for detection of positive responses from multiple combinations. consequently, for multi-parameter analysis, the results were thresholded based on a minimum criterion of positivity, as calculated by spice software and presented as the 90 th percentile of negative values for each analysis. each pie chart generated by spice software, represents the hierarchy of responses to either hcv or hiv antigen stimulation. for simplicity, responses are grouped by number of functions and matched to the colored bars, with black bars representing the percentage of responding cells to hiv peptides and gray bars representing the percentage of responses to hcv peptide stimulation. in all pie charts, color red represents the 5+ responding population and the colors blue, green, turquoise, and yellow representing the 4+, 3+, 2+, and 1+ populations respectively. color-coded arcs represent the dominant marker within each pie slice, with color blue representing tnf-a, red for cd107-a, green for ifn-c and black for mip-1b. although il-2 is included in the presentation and demonstrated by bar graphs, the software would not allow for arc colors for more than 4 responses. as a result there is no arc representative for il-2. figure (c) represents the average frequency of intra-hepatic viral specific responses within the pool of cd8 + t-cell populations simultaneously expressing 2 functions or less, compared with those within the pool of populations expressing more than 2 functions simultaneously; as analyzed in 3 subjects within each cohort of hcv mono and hcv/hiv co-infected individuals. the cutoff point of simultaneous expression of more than 2 measured markers is considered to show cd8 + t-cell poly-functionality. effect of human immunodeficiency virus on hepatitis c virus infection among injecting drug users impaired hepatitis c virus-specific t cell responses and recurrent hepatitis c virus in hiv coinfection retrospective analysis of the impact of hiv infection and alcohol use on chronic hepatitis c in a large cohort of drug users influence of human immunodeficiency virus infection on the course of hepatitis c virus infection: a meta-analysis hepatotoxicity associated with antiretroviral therapy in adults infected with human immunodeficiency virus and the role of hepatitis c or b virus infection pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis the role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride impaired effector function of hepatitis c virus-specific cd8+ t cells in chronic hepatitis c virus infection hiv longterm non-progressors maintain brisk cd8 t cell responses to other viral antigens cd8+ cell responses to hepatitis c virus (hcv) in the liver of persons with hcv-hiv coinfection versus hcv monoinfection comparison of hcv-specific intrahepatic cd4+ t cells in hiv/hcv versus hcv human immunodeficiency virus type 1-hepatitis c virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses analysis of total human immunodeficiency virus (hiv)-specific cd4(+) and cd8(+) t-cell responses: relationship to viral load in untreated hiv infection hiv-specific cd8+ lymphocytes in semen are not associated with reduced hiv shedding severe cd4+ t-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy hiv nonprogressors preferentially maintain highly functional hiv-specific cd8+ t cells polyfunctional analysis of human t cell responses: importance in vaccine immunogenicity and natural infection the liver as a site of t-cell apoptosis: graveyard, or killing field? measles associated hepatobiliary disease: an overview clinical significance of hepatic derangement in severe acute respiratory syndrome acute human immunodeficiency virus type 1 infection kupffer cell-dependent hepatitis occurs during influenza infection inflammatory markers in chronic hepatitis c intrahepatic cd8+ t-cell failure during chronic hepatitis c virus infection the liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys identification and quantitation of hiv-1 in the liver of patients with maintenance of large numbers of virus-specific cd8+ t cells in hivinfected progressors and long-term nonprogressors pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcvspecific cd8 exhaustion pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression differences in hcv-specific t cell responses between chronic hcv infection and hiv/hcv co-infection we are grateful to the patients who contributed their time and effort to this study. we would also like to thank ms. dionne white for her expertise and assistance in flow cytometry and dr. george makedonas for spice and pestle software training. we specially thank dr. mario roederer for providing us with the mentioned software. key: cord-000638-ss1435el authors: beq, stephanie; rozlan, sandra; pelletier, sandy; willems, bernard; bruneau, julie; lelievre, jean-daniel; levy, yves; shoukry, naglaa h.; cheynier, rémi title: altered thymic function during interferon therapy in hcv-infected patients date: 2012-04-16 journal: plos one doi: 10.1371/journal.pone.0034326 sha: doc_id: 638 cord_uid: ss1435el interferon alpha (ifnα) therapy, despite good efficacy in curing hcv infection, leads to major side effects, in particular inducement of a strong peripheral t-cell lymphocytopenia. we here analyze the early consequences of ifnα therapy on both thymic function and peripheral t-cell homeostasis in patients in the acute or chronic phase of hcv-infection as well as in hiv/hcv co-infected patients. the evolution of t-cell subsets and t-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of t-cell receptor excision circles (trec) and estimation of intrathymic precursor t-cell proliferation during the first four months following the initiation of ifnα therapy. beginning with the first month of therapy, a profound lymphocytopenia was observed for all t-cell subsets, including naïve t-cells and recent thymic emigrants (rte), associated with inhibition of intrathymic precursor t-cell proliferation. interleukin (il)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. this was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble cd127. decrease in il-7 plasma concentration under ifnα therapy correlated with the decline in hcv viral load, thymic activity and rte concentration in blood. these data demonstrate that ifnα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs t-cell homeostasis. such a side effect might be detrimental for the continuation of ifnα therapy and may lead to an increased level of infectious risk, in particular in hiv/hcv co-infected patients. altogether, this study suggests the therapeutic potential of il-7 in the maintenance of peripheral t-cell homeostasis in ifnα-treated patients. the hepatitis c virus (hcv) causes persistent infection in approximately two thirds of cases leading to chronic liver disease, liver failure, and, eventually, hepatocellular carcinoma in a substantial proportion of infected individuals. the most common therapy for chronic hepatitis c consists of pegylated interferon-a (ifna) and ribavirin administration which results in viral clearance in 43-46% (genotype 1) to 80%, (genotype 3) of treated patients [1] . interferon will continue to be a major component of new direct acting antivirals for treatment of hcv [2] . ifna is produced in large amounts during the acute phase of many viral infections [3, 4, 5, 6] . direct activation of interferonstimulated genes enhances naïve t-cell survival through increased bcl-2 and reduced bax activation [7] and contributes to clonal expansion of antigen-specific t-cells [8] . recent data suggest that early therapeutic intervention with pegylated ifna rescues polyfunctional memory t-cells expressing high levels of the il-7 receptor alpha chain (cd127) and bcl-2, allowing a higher rate of sustained viral response [9] . however, despite good efficacy, ifna-based therapies lead to sustained anemia, thrombocytopenia, neutropenia and lymphocytopenia [10, 11, 12, 13, 14] . moreover, pegylated ifna therapy enhances the risk of infection in older hcv-infected patients and hiv-infected individuals, independently from neutropenia [15, 16, 17] . the mechanisms of action of ifna include inhibition of different hematopoietic growth factors [18, 19] , possibly affecting lymphoid differentiation at an early stage [20] , and modifications in cell homing [12, 21, 22] . the mechanisms involved in ifna therapy-associated leukocyte depletion remain poorly understood. others and we have documented a strong reduction in the ability of hiv-infected patients to sustain thymic production as a direct consequence of a drop in intrathymic precursor t-cell proliferation [23, 24, 25] . similar thymic impact was also seen during early siv-infection in the rhesus macaque model [26] . the capacity of the thymus to produce recent thymic emigrants (rtes) is, in large part, dependent on thymocyte proliferation [27] . indeed, extensive thymocyte proliferation occurs between t-cell receptor beta (tcrb) and alpha (tcra) chain rearrangements. the extent of this proliferation directly correlates with thymic export [28] . the extent of cell proliferation in the thymus can be measured in patients through estimating, in peripheral blood cells, the ratio (sj/btrec ratio) between the frequency of signal joint t-cell receptor excision circles (sjtrec), produced during the excision of the tcrd locus prior to tcra chain rearrangement, and that of dbjbtrec t-cell receptor excision circles (trecs) produced during tcrbd to tcrbj rearrangement [29] . these by-products of tcr rearrangement processes are generated by the circularization of the chromosomal dna excised during tcr rearrangements respectively occurring at the dn3 (dbjbtrec) and dp (sjtrec) stages of differentiation. due to the fact that trecs do not replicate during mitosis, increased proliferation between dn3 and dp leads to the reduction of dbjbtrec frequency in rtes as compared to sjtrec frequency and consequently to an increase of the sj/btrec ratio [23] . the correlation between initial plasma ifna levels and the speed of thymic dysfunction observed during hiv primary infection suggested that ifna, produced as part of the innate immune response to infection, participates in the impairment of thymopoiesis. however, no direct evidence of the relationship between ifna production and thymic dysfunction was provided by these studies. in contrast, arizcorreta and colleagues showed that ifna and ribavirin therapy induces a substantial reduction of circulating sjtrecs, in hiv/hcv co-infected patients, accompanied by sustained naïve cd4 + t-cell defect, suggesting thymic dysfunction [10] . similarly, in the siv-infected rhesus macaque model, we showed that ifna therapy induced a strong decrease of circulating rte numbers as defined either by sjtrec frequency and numbers or by cd31 hi expression on naïve t-cells [30] . interestingly, in these animals, recombinant interleukin (il)-7 therapy more than abrogated the deleterious effects of ifna therapy [30] . il-7 is a key cytokine implicated at various levels of thymocytes differentiation. it allows cell survival during the rearrangement processes, and is implicated in the extensive thymocyte proliferation, in particular in intermediate single positive (isp) and early dp cells [31, 32, 33, 34] . this proliferation participates in the development of naïve t-cell diversity [35] . while up regulated by ifna [36, 37] , the cyclin-dependent kinase inhibitor p27/kip1 is negatively regulated by il-7 [38] , allowing isp and early dp thymocytes to proliferate. moreover, ifna also inhibit il-7 dependent proliferation through down modulation of the common c chain, that participates, together with cd127 to the il-7 receptor [39] . we here investigated the early impact of ifna therapy on thymic function and naïve t-cell homeostasis in both hcv-infected and hiv/hcv co-infected patients who started ifna therapy. we first evaluated the evolution of naïve t-cell subsets in three groups of hcv infected individuals: 1) acute hcv infection (n = 8), defined as ,6 months post estimated date of infection; 2) chronic hcv infection (n = 8), defined as .6 months post estimated date of infection; and 3) hiv/hcv co-infected individuals (n = 10). in all groups, patients were enrolled at the beginning of ifna therapy and were followed for a total of 4 months. while, for any group of patient's, naïve cd4+ and cd8+ t-cell counts were not significantly different from healthy individuals (figure 1a), as early as one month following treatment initiation, naïve cd4+ t-cell counts were significantly reduced in chronically hcv-infected patients (39%, 58%, 46% and 35% decrease at m1, m2, m3 and m4 respectively; p#0.025; figure 1b , top central panel). a similar trend was also observed in the cd8 compartment (40%, 39%, 33% and 33% decrease; figure 1b , bottom central panel). a comparable effect was also observed in most co-infected patients (mean cell count declines were 19%, 32%, 52% and 43% at m1, m2, m3 and m4 in the cd4+ t-cell compartment and 9%, 21%, 41% and 42% in cd8+ t-cell subset; p#0.05 by m2-m3; figure 1b , right panels). in contrast, naïve t-cell counts were only barely affected in acutely-hcv infected patients under ifna therapy ( figure 1b , left panels). similarly, central memory cd4+ t-cells (cd45 ra-ccr7+; tcm) demonstrated 38% and 28% decrease in hcv and hiv/hcv patients respectively (59% and 60% in cd8+ tcm) while effector memory (cd45ra2 ccr7-; tem) cd4+ t-cell counts declined by 45% and 10% in the same groups (61% and 65% in cd8+ tem) ( figure s1 ). within cd4+ naïve t-cells, rtes can be identified by their higher expression of the platelet endothelial cell adhesion molecule-1 (pcam-1 or cd31) [40] . while the number of rtes was similar in hcv-infected patients at study entry and healthy individuals ( these data demonstrate that, as early as one month following treatment initiation, ifna induces stronger alterations of naïve tcell subsets, and more specifically in the rte compartment than in any other t-cell subset, suggesting a specific effect on thymopoiesis. we thus analyzed the evolution of intrathymic precursor t-cell proliferation, peripheral t-cell cycling, il-7 plasma concentration and il-7 receptor alpha chain (cd127) expression, different factors affecting naive t-cell homeostasis. despite differences between the 3 groups at study entry, rte cycling rate, as estimated through measurement of ki-67 expression, did not change significantly during the follow-up period ( figure 3a ). these data demonstrate that the observed changes in sjtrec frequencies were not a consequence of variations of rte proliferation during ifna therapy but more probably due to reduced thymic production. we thus estimated thymic output through quantification of the sj/btrec ratio in all groups of patients ( figure 3b ). the sj/ btrec ratio estimates the extent of thymocyte proliferation between tcrb rearrangement and the excision of the t-cell receptor delta (tcrd) locus [23] . this parameter directly reflects the extent of thymic production and, contrarily to sjtrec values, is independent from peripheral rte proliferation or survival capacity [28] . the sj/btrec ratio was already low in hivinfected patients (p,0.005 as compared to healthy control donors; figure 3b bottom left panel) and did not evolve further under ifna therapy in co-infected patients ( figure 3b , bottom right panel). in contrast, acutely hcv-infected patients demonstrated higher than normal sj/btrec ratio at baseline (p,0.05 as compared to aged matched healthy controls), showed a significant reduction in sj/btrec ratio at m1 (p = 0.014) and m2 (p = 0.001; figure 3b , top panel). finally, a similar decline in the sj/btrec ratio was observed during ifna therapy in chronically hcv-infected patients (p,0.02 at m1, m2 and m3; figure 3b , central panel). precursor t-cell proliferation in the thymus is, at least in part, dependent upon il-7. we thus quantified plasma il-7 concentration in all groups of patients. at study entry, hcv-and hiv/ hcv-infected patients presented with elevated plasma il-7 (median = 10.3 pg/ml, range (6.7-12.9) in acutely hcv-infected patients; 8.3 pg/ml (6.3-10.5) in chronic hcv-infected patients and 7.15 pg/ml (4.3-13.5) in co-infected subjects), as compared to that observed in healthy control individuals (p,0.001 for any patients' group; figure 4a) . surprisingly, while lymphocytopenia established, il-7 plasma concentrations significantly decreased in both groups of hcv-infected patients (30, 54, 18 and 29% decrease at m1 to m4 in acute infection, p,0.05; 25, 46, 26 and 16% decrease at m1 to m4 in chronic infection, p,0.05; figure 4b left and central panels). in contrast, il-7 plasma levels did not significantly evolve in co-infected individuals during the first month of ifna therapy ( figure 4b right panel) . only patients with the highest il-7 plasma levels showed a reduction in the concentration of this cytokine. decreased plasma il-7 concentrations could be a consequence of reduced il-7 production, increased consumption by t-cells or sequestration by soluble il-7 receptor (scd127). in both hcvinfected and hiv/hcv co-infected patients, neither scd127 considering the variations in all the parameters we used to evaluate thymic function, we then sought to evaluate the impact of changes in il-7 plasma levels on de novo production from the thymus and on the number of both sjtrec and circulating cd4+ rtes. in a majority of patients, il-7 plasma level, sj/btrec ratio, sjtrec/ml and blood rte concentration fluctuated in parallel ( figure s2 ). variation of il-7 plasma concentration (dil-7) during the first month of therapy correlated with variations in naïve t-cell counts (cd4+ + cd8+; dnaïve t-cell counts) and rte cd4+ t-cell counts (drte t-cell counts) in both hcv (r = 0.521, p = 0.039 and r = 0.595, p = 0.025; figure 5a and 5b, left panels) and, to a lesser extent, hiv/hcv co-infected patients (r = 0.636, p = 0.048 and r = 0.539, p = 0.108; figure 5a and 5b, right panels). moreover, in hcv-infected patients, dil-7 also correlated with variations in intrathymic precursor t-cell proliferation (dsj/btrec ratio; r = 0.601, p = 0.020; figure 5c ). variations in plasma il-7 levels also correlated with changes in the proportions (d%ki-67+ in cd4+rtes; r = 0.806, p = 0.0002; figure 5d , left panel) and numbers (dki-67+rtes; r = 0.706, p = 0.002; figure 5e , left panel) of cycling rtes in acute and chronic hcv infected patients and with d%ki-67+rte counts in co-infected patients (r = 0.709, p = 0.022; figure 5e , right panel). overall, il-7 concentration was associated with reduced thymopoiesis and rte proliferation, lower consequently leading to limited circulating rte and naïve t-cell counts. these data strongly suggest that changes in il-7 plasma levels during ifna therapy directly impact the homeostasis of rtes. we herein demonstrated that ifna-based therapy leads to major lymphocytopenia in naïve t-cell compartments, in particular in the rte subset. several mechanisms could be implicated in the establishment of such a lymphocytopenia [41] . among these, enhanced apoptosis [42, 43] , cell sequestration in lymphoid or non-lymphoid organs [12, 21, 22] and regulation of peripheral t-cell homeostasis [20] . in our study, no major change in cell survival (bcl-2 expression) or t-cell activation (cd25 and cd69 expression) was observed during the follow-up period (data not shown). moreover, we did not observe any significant modification in ki-67 expression in any t-cell subset during the first month of therapy (data not shown and figure 3 ). finally, ifna-induced t-cell homing, although rapid and massive, is only a transient process [22] suggesting that this mechanism marginally contributes to the observed long lasting lymphocytopenia. interestingly, both sjtrec quantification (sjtrec/ml) and intrathymic precursor t-cell proliferation (sj/btrec ratio) were affected very early on after initiation of therapy ( figures 2b and 3b ). while sjtrec frequency and concentration in peripheral blood can be affected by modifications of parameters that impact on peripheral t-cell homeostasis (cycling, survival/apoptosis, homing), the sj/btrec ratio is a marker of the intrathymic proliferation history of rtes. indeed, this parameter is generated by cell proliferation that occurs between tcrb chain rearrangement and the excision of tcrd locus. further cell cycling after tcra chain rearrangement does not modify the sj/btrec ratio as both type of trecs are similarly diluted upon cell proliferation. accordingly, while exported to the periphery, the sj/btrec ratio of mature t-cells cannot be modified. therefore, while the observed decrease in sjtrec concentration (figure 2) can be a consequence of modifications of circulating t-cell homeostasis, the decline of the sj/btrec ratio observed during the first months of ifna therapy (figure 3) defines changes in thymocyte proliferation, thus in thymic output [28] . acutely infected patients demonstrated a higher sj/btrec ratio at baseline than patients in the chronic phase. however, this group was younger (median = 31.5 (26-47)) versus median = 53.5 (37-61)) than the chronic group (p,0.01; data not shown) and demonstrated normal sj/btrec ratio for their age. similar evolution of thymic function and circulating t-cell subsets were observed in both groups of hcv-infected patients, irrespective of the development stage of hcv pathology. the lack of effect of ifna therapy in hiv/hcv co-infected patients might be due to the fact that, as expected for chronically hiv-infected individuals, these patients already had a low thymic function at study entry. the impairment of thymopoiesis in hcv-infected patients under ifna therapy is reminiscent of that observed during the acute phase of hiv-1 infection [23] which suggested that long term production of ifna, as part of the anti-hiv innate immune response, may play a role in the observed thymic defect. the correlation between decline in il-7 plasma levels under ifna therapy and both thymic dysfunction and reduced t-cell counts, in particular in the naïve and rte compartments ( figures 5a and 5b) , confirms this hypothesis. finally, in a recent study, we showed that ifna treatment leads to decreased sjtrec frequency as well as reduced naïve t-cell and rte counts in siv-infected rhesus macaques [30] . such an effect was accompanied by a 30-40% decrease in il-7 plasma levels in these animals and could be counteracted by injection of recombinant simian il-7 [30] . one could expect that such an effect of type i ifns is not restricted to hiv-infection as many viral infections induce ifna responses and cause transient lymphocytopenia in the infected hosts [3, 4, 5, 6] . moreover, the ifna-induced reduction of thymic function and its probable consequences on naïve t-cell diversity may contribute to the higher infectious risk associated with ifna therapy, in particular observed in older patients [15, 16, 44] . there are multiple sources for circulating il-7 during viral infections including lymphoid organs, epithelial cells and recently the liver was identified as a major source of il-7. moreover, increased plasma il-7 levels can also be observed during viral infection in non-lymphopenic individuals ( [33] and unpublished data), suggesting a role in the development of immune responses. indeed, this cytokine participates to t-cell homing in various lymphoid and non-lymphoid tissues through stimulation of local chemokine productions [45] . increased il-7 plasma levels in lymphopenic individuals is likely due to reduced consumption [46] yet augmented production to counteract lymphopenia cannot be excluded [33] . the recent identification of the liver as an il-7 producing tissue upon tlr stimulation [47] makes it tempting to speculate that hcv-infection can also, through tlr activation, stimulate il-7 production by the liver. indeed, non-lymphopenic hcv-infected patients demonstrate similar il-7 plasma levels than lymphopenic hiv-infected individuals [33, 48] suggesting that most of the il-7 production in untreated hcv-infected patients was not linked to circulating t-cell counts. the reduction of il-7 plasma levels while lymphocytopenia establishes under ifna therapy, the absence of a correlation between il-7 plasma levels and cd127 expression and the concomitance of decreases in il-7 plasma levels and hcv viral load under therapy suggest that viremia might be driving il-7 production before initiation of therapy. our data suggest that, before initiation of ifna therapy, actively replicating hcv leads to the overproduction of il-7. subsequent reduction of il-7 production upon initiation of therapy probably reflects the elimination of il-7 producing hcv-infected hepatocytes. this sudden reduction of il-7 plasma levels may lead to diminished thymopoiesis. the fact that il-7 plasma levels did not reach normal levels when hcv became undetectable may suggest that, after the initial decline that follows the drop in viremia, il-7 plasma levels were regulated, as in hivinfected patients [33] and in ifna-treated siv-infected rhesus macaques [30] , as a consequence of lymphocytopenia through either reduced consumption or increased production in lymphoid organs [49] . future studies with a longer follow-up period, in particular after the end of ifna therapy and recovery from lymphocytopenia are required to further elucidate this point. we herein demonstrated that a substantial reduction in thymic export was observed in hcv-infected patients, during the first months of ifna therapy. this effect directly paralleled ifnainduced lymphocytopenia and decreased il-7 plasma levels, initially high in hcv-infected patients. these data suggest that il-7 production by the liver, a consequence of active hcv replication, was reduced while patients controlled hcv viremia. restricted il-7 plasma levels might, in association with the antiproliferative effect of ifna, limit t-cell production in the thymus. our study highlights the therapeutic potential of il-7 as a complement to the standard ifna based treatment to help hcvinfected patients to sustain normal circulating t-cell counts, and restore the diversity of the peripheral t-cell repertoire through its central thymopoietic effect. restoring the breadth and intensity of t-cell control over the hcv virus might be immediately beneficial for the hiv/hcv co-infected population and offer new promising avenues for chronic hcv in the context of massive drop of hcv viral load after short term treatment with new antiviral compounds that will continue to be administered in combination with ifna [50] . sixteen hcv-infected patients (c-1 to c-16) and ten hiv/ hcv co-infected patients (i-1 to i-10) naïve to ifna therapy were enrolled in this study. a summary of the virological and immunological status of patients at baseline is shown in table 1. all the hiv/hcv co-infected patients but one were under haart with undetectable viremia (,40 hiv copies/ml). chronically infected patients (c-9 to c-16 and i-1 to i-10) initiated pegylated ifna/ribavirin treatment (ifna-2a: pegasys, 180 mg weekly, ribavirin: copegus, 800 mg to 1000 mg daily) and were followed over a 4 months period. patients included in the acute phase of hcv infection (c-1 to c-8) were treated with pegylated ifna (ifna-2a: pegasys, roche, 180 mg weekly) [51, 52] . blood samples were taken monthly on edta. two milliliters of total blood were 2-fold diluted in fcs/20%dmso frozen at 280uc and conserved in liquid nitrogen. these total blood samples were subsequently used for flow cytometry analyses. plasma was separated from the remaining eight milliliters and mononuclear cells were purified on ficoll hypaque (eurobio, courtaboeuf, france) and frozen for further analyses. patients from the hcv mono-infection group were followed at the centre de recherche du chum, hôpital saint luc, montreal, qc, canada and its collaborators as previously described [9, 53] . patients from the hiv-hcv groups were followed at the hôpital henri mondor, creteil, france. clinical protocols conformed to figure 5 . variations in il-7 plasma levels correlate with evolution of rte production. correlations. between variations in il-7 plasma levels (dil-7) and either variations in (a) total (cd4 + + cd8 + ) naïve t-cell counts (dnaïve t-cell counts), (b) rte defined as cd31 hi naïve cd4 + t-cells (drte cd4 counts), (c) the sj/btrec ratio (dsj/btrec ratio), (d) the frequency of ki-67 + cells in the rte cd4 + t-cell subset (d%ki-67 + in cd4 + rtes) or (e) the number of circulating ki-67 + cd4 + rtes (dki-67 + rte counts) between study entry and month 1 of therapy were calculated for acutely (black symbols) and chronically (white symbols) hcv-infected patients (left panels) and hiv/hcv co-infected patients (right panels). correlation coefficients (spearman's r) and the associated probabilities (p) are shown. doi:10.1371/journal.pone.0034326.g005 ethical guidelines of the authors' institutions and the us department of health and human services' human experimentation guidelines. this study was approved by both the ethical committee of centre hospitalier de l'université de montreal (chum) and the ethical committee of hôpital henri mondor, créteil, france. samples were obtained with the written subjects' informed consent. immunophenotyping and flow cytometry analysis facs analyses were performed on cryopreserved samples. after thawing blood cells were incubated for 15 minutes at 4uc with conjugated monoclonal antibodies (mabs). for intracellular labeling, cells were permeabilized with the cytofix/cytoperm kit (becton dickinson) before incubation with specific mabs according to the manufacturer's instructions. samples were then washed, fixed in 2% paraformaldehyde phosphate-buffered saline (pbs/pfa 2%) and acquired using a cyan cytofluorometer (dako) and analyzed with flowjo 8.7 software. the monoclonal antibodies used in this study were: cd3-pacific blue (pb) (clone ucht-1; dako, trappes, france), cd4-peridin chlorophyll protein-cyanine 5.5 (percp-cy5.5) (clone l200; bd, le-pont-de-claix, france), cd45ra-phycoerythrin (pe) (clone hi100; bd), ccr7-allophycocyanin (apc) (clone 150503; r&d systems europe, lille, france); cd8-phycoerythrin-cyanine 7 (pe-cy7) (rpa-t8; bd), cd31-biotin (clone wm59; abdserotec, düsseldorf, germany); ki-67-fluorescein isothiocyanate (fitc) (clone mib-1; dako), bcl-2-fitc (clone 124; dako) and strepatavidin-pe-texas-red (bd). il-7 was quantified in the plasma using the il-7 quantikine hs kit according to the manufacturer's instructions (r&d systems europe). plasma soluble-cd127 quantification soluble plasma il-7 receptor (scd127) quantification was performed as previously described [54] . parallel quantification of the sjtrec and the 13 djbtrecs, together with cd3c gene (used as a housekeeping gene) was performed for each sample using lightcyclertm technology (roche diagnostics) with a technique adapted from [29] . intrathymic precursor t-cell proliferation was evaluated through calculation of the sj/btrec ratio as described [23] . hcv rna quantification was performed using an in-house quantitative real-time reverse transcription-pcr assay as previously described [9] , cobas amplicor hcv monitor test tm , version 2.0 (sensitivity 600 iu/ml)), qualitative cobas ampli-prep/cobas amplicor hcv test tm , version 2.0 (sensitivity 50 iu/ml) or abbott realtime hcv assay tm (sensitivity 12 iu/ ml). statistical analyses (spearmans rank correlations and wilcoxon matched -paired signed-rank tests) were performed using the stata/ic 10.0 (stata corporation, college station, tx u.s.a.). due to the exploratory nature of the study there was no correction for multiple comparisons, and calculated p values are reported herein. mechanism of action of interferon and ribavirin in treatment of hepatitis c a new standard of care for the treatment of chronic hcv infection selective lymphocyte depletion during the early stage of the immune response to foot-and-mouth disease virus infection in swine quantitation of t lymphocyte subsets helps to distinguish dengue hemorrhagic fever from classic dengue fever during the acute febrile stage effects of severe acute respiratory syndrome (sars) coronavirus infection on peripheral blood lymphocytes and their subsets extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients a dual role of ifnalpha in the balance between proliferation and death of human cd4+ t lymphocytes during primary response type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection early interferon therapy for hepatitis c virus infection rescues polyfunctional, long-lived cd8+ memory t cells t cell receptor excision circles (trecs), cd4+, cd8+, and their cd45ro+, and cd45ra+, subpopulations in hepatitis c virus (hcv)-hivco-infected patients during treatment with interferon alpha plus ribavirin: analysis in a population on effective antiretroviral therapy efficacy and safety of combination therapy with interferon-alpha2b and ribavirin for chronic hepatitis c in hiv-infected patients efficacy and safety of alpha-interferon treatment for chronic hepatitis c in hivinfected patients. hiv-hepatitis spanish study group future trends in managing hepatitis c treatment of hepatitis c and anemia in human immunodeficiency virus-infected patients use of pegylated interferons is associated with an increased incidence of infections during combination treatment of chronic hepatitis c: a side effect of pegylation? incidence of neutropenia and infections during combination treatment of chronic hepatitis c with pegylated interferon alfa-2a or alfa-2b plus ribavirin opportunistic infections and cd4 lymphocytopenia with interferon treatment in hiv-1 infected patients regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (cfu-gemm, cfu-mk, bfu-e, and cfu-gm) from patients with myelofibrosis with myeloid metaplasia impairment of t and b cell development by treatment with a type i interferon cd4+ t-lymphocytopenia in hiv-infected patients receiving interferon therapy for chronic hepatitis c. hiv-hepatitis spanish study group type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia hiv infection rapidly induces and maintains a substantial suppression of thymocyte proliferation changes in thymic function with age and during the treatment of hiv infection slow disease progression and robust therapy-mediated cd4+ t-cell recovery are associated with efficient thymopoiesis during hiv-1 infection il-7 induces immunological improvement in siv-infected rhesus macaques under antiviral therapy t cell homeostasis: thymus regeneration and peripheral t cell restoration in mice with a reduced fraction of competent precursors the magnitude of thymic output is genetically determined through controlled intrathymic precursor t cell proliferation estimating thymic function through quantification of t-cell receptor excision circles interleukin-7 treatment counteracts ifn-{alpha} therapy-induced lymphopenia and stimulates siv-specific cytotoxic t lymphocyte responses in siv-infected rhesus macaques interleukin-7 promotes survival and cell cycle progression of t-cell acute lymphoblastic leukemia cells by down-regulating the cyclin-dependent kinase inhibitor p27(kip1) interleukin-7 mediates the homeostasis of naive and memory cd8 t cells in vivo increased production of il-7 accompanies hiv-1-mediated t-cell depletion: implications for t-cell homeostasis the role of interleukin-7 in early t-cell development a direct estimate of the human alphabeta t cell receptor diversity how cells respond to interferons augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27kip1 in human hepatocellular carcinoma cells down-regulation of p27kip1 expression is required for development and function of t cells endogenous interferon-alpha production by differentiating human monocytes regulates expression and function of the il-2/il-4 receptor gamma chain two subsets of naive t helper cells with distinct t cell receptor excision circle content in human adult peripheral blood tlymphocyte populations in hepatitis c and hiv co-infected patients treated with interferon-alfa-2a and ribavirin interferon alpha augments activation-induced t cell death by upregulation of fas (cd95/apo-1) and fas ligand expression ifn-alpha suppresses activation of nuclear transcription factors nf-kappa b and activator protein 1 and potentiates tnf-induced apoptosis rapid decline of cd4+ cells after ifn alpha treatment in hiv-1 infection injection of glycosylated recombinant simian il-7 provokes rapid and massive t-cell homing in rhesus macaques interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of cd4+ t cells hepatic interleukin-7 expression regulates t cell responses loss of il-7 receptor alpha-chain (cd127) expression in acute hcv infection associated with viral persistence interleukin-7 receptor expression: intelligent design hepatitis c virus (hcv) genotype 1 subtype identification in new hcv drug development and future clinical practice determinants of antiviral treatment initiation in a hepatitis c-infected population benefiting from universal health care coverage response rates to pegylated interferon and ribavirin in hcv/hiv coinfection: a research synthesis rare birds in north america: acute hepatitis c cohorts a validated assay to measure soluble il-7 receptor shows minimal impact of il-7 treatment the authors acknowledge the subjects who participated in this study. we also thank c. chesnel for clinical and logistical support. key: cord-011026-iapgkz0p authors: el-bitar, alaa m. h.; sarhan, moustafa; abdel-rahman, mohamed a.; quintero-hernandez, veronica; aoki-utsubo, chie; moustafa, mohsen a.; possani, lourival d.; hotta, hak title: smp76, a scorpine-like peptide isolated from the venom of the scorpion scorpio maurus palmatus, with a potent antiviral activity against hepatitis c virus and dengue virus date: 2019-07-06 journal: int j pept res ther doi: 10.1007/s10989-019-09888-2 sha: doc_id: 11026 cord_uid: iapgkz0p growing global viral infections have been a serious public health problem in recent years. this current situation emphasizes the importance of developing more therapeutic antiviral compounds. hepatitis c virus (hcv) and dengue virus (denv) belong to the flaviviridae family and are an increasing global health threat. our previous study reported that the crude venom of scorpio maurus palmatus possessed anti-hcv and anti-denv activities in vitro. we report here the characterization of a natural antiviral peptide (scorpion-like peptide smp76) that prevents hcv and denv infection. smp76 was purified from s. m. palmatus venom and contains 76 amino acids with six residues of cysteine. smp76 antiviral activity was evaluated using a cell culture technique utilizing huh7it-1, vero/slam, hcv (jfh1, genotype 2a) and denv (trinidad 1751, type 2). a potential antiviral activity of smp76 was detected in culture cells with an approximate ic(50) of 0.01 μg/ml. moreover, smp76 prevents hcv infection and suppresses secondary infection, by inactivating extra-cellular infectious particles without affecting viral replication. interestingly, smp76 is neither toxic nor hemolytic in vitro at a concentration 1000-fold higher than that required for antiviral activity. conclusively, this report highlights novel anti-hcv and anti-denv activities of smp76, which may lay the foundation for developing a new therapeutic intervention against these flaviviruses. hepatitis c virus (hcv) is a single-stranded rna viruses that belongs to family flaviviridae (mohammed et al. 2013; supanee et al. 2014) . around 150 million people worldwide are chronically infected with hcv and the annual mortality from hcv-related liver diseases reach up to 700,000 individual (ministry of health and 2015; world health organization 2016; jefferies et al. 2018 ). in the past decade, interferon-based therapy was the gold standard for hcv treatment with a sustained virological response (svr) rate hovering around 50%. the recent approval of oral direct-acting antivirals (daas), like hcv ns3 protease inhibitors, ns5a inhibitors and ns5b rna-dependent rna polymerase inhibitors, for clinical use improved the svr rates to more than 90% (pawlotsky 2016; falade-nwulia et al. 2017 ). nevertheless, cirrhosis patients remain at risk for severe complications. in addition, treatment with daas is not affordable for many patients and they are still not readily available around the globe. therefore, uncovering novel hcv inhibitors is still a clinical priority. dengue virus (denv) is another single-stranded rna flaviviridae virus that is transmitted by mosquitoes causing dengue fever (rodenhuis-zybert et al. 2010) . denv is currently endemic in more than 100 countries with the highest prevalence in south-east asia, africa and the americas (mackenzie et al. 2004; malavige et al. 2004; deen et al. 2006; bhatt et al. 2013) . each year, there are around 390 million denv infections are recorded worldwide and among them 50 to 100 million patients are presented with the clinical manifestations of dengue fever (bhatt et al. 2013) . dengue fever leads to 20,000 annual deaths, mainly in young children (rui-feng et al. 2008) . to date, four denv serotypes, denv-1, denv-2, denv-3 and denv-4, have been identified and infections with one serotype does not offer protection from infection with the remaining three serotypes (weaver and vasilakis 2009; messina et al. 2014; mustafa et al. 2015) . a major impediment to the development of vaccines is that vaccines should have a tetravalent effect, i.e. sufficient protective immune responses against all four denv serotypes. owing to the absence of specific treatments against denv and the limitations of the available vaccine (dengvaxia® or cyd-tdv), the global burden of denv infection is becoming enormous (behnam et al. 2016) . therefore, the development of new antiviral compounds against denv infections is urgently needed. scorpion venom is a rich source for drug discovery and prototyping ghosh et al. 2019) . scorpion venoms are highly complex mixture of nucleotides, enzymes, mucoproteins, biogenic amines, nucleotides, salts, as well as peptides and proteins (omran 2003; rodriguez de la vega and possani 2005; ozkan et al. 2006a, b, c; feng et al. 2008; kanoo and deshpande 2008; ortiz et al. 2015) . antimicrobial peptides (amps), isolated from several venomous animals, exhibit a wide range of antibacterial and antiviral activity with direct or indirect microbicide activity (hv et al. 2006; ortiz et al. 2015) . several studies demonstrated an antiviral effect for certain scorpion venom peptides (carballar-lejarazu et al. 2008; el-bitar et al. 2015; ortiz et al. 2015) . in this study, we report the molecular and functional characterization of a new antiviral peptide (smp76), a scorpion-like peptide derived from an egyptian scorpion's venom, s. m. palmatus. our findings will broaden the currently known antiviral peptides and open a new avenue for the development of novel hcv and denv therapies. adult s. m. palmatus scorpions were collected from the western coastal mediterranean desert (alexandria governorate, egypt) and were housed individually in clear plastic containers. scorpions were fed small insects and were given water. crude venom was extracted using electrical stimulation (20 v) and the milked venom was collected and centrifuged for 20 min at 13,000 rpm/4 °c as detailed previously (abdel-rahman et al. 2013) . clear supernatants were pooled, freeze-dried and stored at − 20 °c until use. venom samples were dissolved in bi-distilled water and the total protein concentration was determined by bca protein assay kit (pierce biotechnology, rockford, il, usa) according to the standard protocols. the human hepatoma-derived cell line, huh7it-1, was cultured in dulbecco's modified eagle's medium (dmem; wako, osaka, japan) supplemented with non-essential amino acids (invitrogen, carlsbad, ca, usa), fetal bovine serum (biowest, nuaille, france), streptomycin (100 μg/ml) and penicillin (100 iu/ml) (invitrogen) in a 5% co 2 incubator at 37 °c (aoki et al. 2014 ). huh7it-1 cells were infected with cell culture-adapted hcv (jfh1 strain of genotype 2a) and supernatants were collected at day 3 post-infection (wakita et al. 2005; yu et al. 2010 ). next, supernatants were, concentrated by 100 k amicon centrifugal filters and used for antiviral screening. denv type 2 (trinidad 1751 strain) (hotta et al. 1983; hotta and homma 1994) was infected into vero/slam cells (ono et al. 2001) . following an hour of virus adsorption, the virus-infected cells were cultured with dmem medium containing 10% fetal bovine serum at 37 °c in 5% co 2 . supernatants were collected at 3 to 5 days post-infection and stored at − 80 °c. measles virus (k52 strain) was inoculated to vero/slam cells and the culture supernatants was collected from the virus-infected cells as described previously (otaki et al. 2006 ). the cytotoxicity of smp76 was estimated using wst-1 assay as described previously with a some modification (deng et al. 2008) . briefly, huh7it-1 cells seeded in 96-well plate (2.5 × 10 4 cells/well) were treated with serial dilutions of smp76 (0.1 to 10 µg/ml) or medium (control) for 48 h at 37 °c in 5% co 2 . then, supernatants were discarded and replaced with fresh dmem medium containing 10 μl of wst-1 reagent (roche, mannheim, germany) and incubated for 4 h. the number of viable cells was quantified by using a microplate reader at 450 and 630 nm. for each dilution, the percentage of viable cells were compared to the control sample and used to calculate the 50% cytotoxic concentrations (cc 50 ) values according to the following formula: hemolytic activity of smp76 was performed as previously described (evans et al. 2013 ). briefly, a total of 10 μl of smp76 peptide was mixed with 190 μl of diluted human red blood cells (rbcs) to achieve a final dilution 1/20 of the original venom peptide per well. alternatively, the rbcs were incubated with 200 μl of 0.5% triton x-100 or pbs to serve as both positive and negative controls, respectively. after an hour incubation period at 37 °c, the plate was centrifuged for 5 min at 500×g and 100 μl of supernatant was transferred to a clear 96-well plate. the released hemoglobin was measured on a microplate reader at 400:541 nm. the percentage of hemolysis was calculated relative to the positive control (0.5% triton x100). the hemolysis concentration (hc 50 ) value was defined as the peptide concentration that can lyse 50% of the rbcs. huh7it-1 cells were grown on coverslips (13-mm in diameter; 1.9 × 10 5 cells/well) 1 day before viral infection. different concentrations of the venom fractions were mixed with hcv at multiplicity of infection (moi: 1) for 2 h at 37 °c. then, the virus/venom fraction mixture was inoculated in huh7it-1 cells for 2 h at 37 °c. medium-treated virus and cells were used as controls. the percentage of inhibition for absorbance of sample/absorbance of control × 100. virus infectivity was compared to the control samples and the 50% inhibitory concentrations (ic 50 ) were calculated. hcv infectivity was determined as described previously (deng et al. 2008) . in brief, huh7it-1 cells, grown on glass coverslips, were incubated with tenfold serially diluted virus samples for 2 h; then, the cells were washed with free medium and cultured for another 24 h. following fixation and permeabilization, huh7it-1 cells were incubated for 1 h with the serum of hcv-infected patients, followed by fitc-conjugated goat anti-human igg (medical & biological laboratories co., ltd., nagoya, japan). finally, the cells were counterstained by hoechst 33342 (molecular probes, eugene, or, usa) and mounted using vectashield h-1000 reagent (vector laboratories, inc. burlingame, ca, usa). hcv antigen positive cells were counted under a fluorescence microscope (bz-9000, keyence, osaka, japan). for dengue virus infectivity, serially diluted venom fractions and smp76 were mixed with fixed amount of denv and incubated for 2 h at 37 °c. the virus/venom fractions mixture was inoculated for 2 h at 37 °c on vero/slam cells. the cells were washed twice after the virus inoculation and incubated with a fresh medium for 24 h. the infected cells were incubated with mouse monoclonal antibody against dengue virus followed\alexa fluor a488 goat anti-mouse igg (life technologies). to determine the infectivity of measles virus, serially diluted smp76 was mixed separately with fixed amount of measles virus and incubated for 2 h at 37 °c. virus/venom fraction mixture was inoculated to vero/slam cells for 2 h at 37 °c and the cells were washed twice then, incubated with fresh medium for 24 h. the plaques (virus-induced syncytia) forming on the infected monolayer cells were counted. the smp76 venom peptide was mixed with a fixed amount of hcv jfh1 for 2 h at 37 °c. next, the virus/smp76 mixture was inoculated to huh7it-1 cells and incubated for 2 h at 37 °c. the cells were washed and cultured without smp76 for 24 h. finally, the cells were subjected to an indirect immunofluorescence assay as previously described (el-bitar et al. 2015) . huh7it-1 cells were lysed in sds sample buffer and equal amounts of protein were separated on a sds-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (pvdf) (millipore, bedford, ma, usa). the pvdf membrane was blocked by 5% skim milk and probed with anti-hcv ns3 antibody and anti-gapdh antibody (millipore). followed by horseradish peroxidaseconjugated goat anti-mouse immunoglobulin (invitrogen) as a secondary antibody and visualized using the enhanced chemiluminescence detection system (ecl; ge healthcare, buckinghamshire, uk). the amounts of hcv rna in the infected cells were determines as described previously (el-bitar et al. 2015) . rna was extracted by rna cell miniprep system reliaprep (promega, madison, wi, usa). the cdna was transcribed from one µg total rna using a goscript reverse transcription system (promega) with oligo(dt) primers. quantitative real-time pcr was performed using sybr premix ex taq (takara, kyoto, japan) in a microamp 96-well reaction plate. pcr was conducted on a abi prism 7500 fast system (applied biosystems, foster city, ca, usa) with specific primers used to amplify the ns5a region of the hcv genome 5′-aga cgt att gag gtc cat gc-3′ (sense) and 5′-ccg cag cga cgg tgc tga tag-3′ (antisense). the expression of gapdh mrna was also measured as a housekeeping gene using the 5′-gcc atc aat gac ccc ttc att-3′ (sense) and 5′ tct cgc tcc tgg aag atg g-3′primers. chromatographic separation of s. m. palmatus venom was conducted using reverse phase high performance liquid chromatography (rp-hplc; waters, milford, massachusetts, united states) (abdel-rahman et al. 2013) .a total of 4 mg scorpion venom was reconstituted in 200 ml 0.05% trifluoroacetic acid (tfa) and fractionated by a c18 rp-hplc column (250 × 10 mm, 5 µm; vydac, california, united states). a gradient of buffer a (0.12% tfa in milliq water) and sixty percent buffer b (0.10% tfa in acetonitrile) were used to separate scorpion venom in 1 h (1 ml/min flow rate). individual venom fractions were collected manually according to the peak's absorbance (at 230 nm). all collected fractions were dried using a rotary evaporator (savant speed vac sc210a, minnesota, united states). the active fraction eluted at retention time 36.4 min was further characterized using mass spectrometry and amino acids sequencing. in addition, recombinant and synthetic smp76 derivatives (n-terminal 32 aa and c-terminal 44 aa) were prepared as described below. the average molecular mass of native smp76 peptide (8398 da), the recombinant fusion protein thioredoxine-smp76 (22123 da) and a recombinant c-terminal of smp76 (4775.57 da) were determined using esi-ms, esi lcq fleet spectrometer (thermo scientific, ca, usa). the sequence of native smp76 (approximately 250 pmol) was determined using edman degradation (protein sequencer ppsq-31a, shimadzu scientific biotech, maryland, united states). synthetic n, c-terminal and fulllength smp76 peptides were manufactured by genscript japan inc. six oligonucleotides were designed to cover the c-terminal region of smp76 (44 aa) (supplementary table 1 ). the oligonucleotides bhek-dir1 and scsmp-lw5 included the bamhi and xhoi restriction sites, respectively. subsequently, this enabled the cloning into the pet22b-thio-ek expression vector as detailed previously (jiménez-vargas et al. 2017; vargas-jaimes et al. 2017) . pcr assembly of the c-terminal peptides was carried out using vent dna polymerase (new england biolabs, ma, united states). the final concentration of external primers bhek-dir1 and scsmp-lw5 was 0.2 pmol/μl while the concentration of internal oligonucleotides was 0.02 pmol/μl. in order to express the fusion protein thioredoxine-c-terminal, pet22b-thio-c-terminal plasmid was transformed into e. coli bl21 (de3) using electroporation. the pellet was harvested and the fusion protein was purified using the ni-nta agarose resin columns (qiagen) as previously described (vargas-jaimes et al. 2017) . hplc purification was further performed using a c18 rp-hplc column (250 × 10 mm, 5 µm; vydac, california, united states). the purified fusion protein thioredoxine-c-terminal was digested with enterokinase (new england biolabs) in 200 mm tris hcl (ph 8.0), 500 mm nacl, 20 mm cacl 2 for 16 h at 25 °c. then, the pure recombinant c-terminal of smp76 was finally isolated and purified by hplc as described above. data are presented as mean ± standard error of mean (sem). the difference between data sets was determined by student's two-tailed t test. a p value of < 0.05 was considered to be statistically significant. since a whole s. m. palmatus soluble venom strongly inhibited hcv infectivity in vitro and displayed anti-hcv activity in cell culture (el-bitar et al. 2015) , the crude venom was fractionated to identify active molecule(s) with anti-hcv activity. accordingly, 74 fractions from four milligrams of the venom were separated by hplc analytical method ( fig. 1a; table 1 ). subsequently, the anti-hcv activity of fractions obtained from the venom of s. m. palmatus were tested against jfh1 strain of genotype 2a. based on protein concentrations, 30 fractions were tested for anti-hcv activity. the other fractions showed very low protein concentrations and, therefore, it was not possible to check their antiviral activity (table 1) . each fraction was incubated separately with fixed amount of hcv for 2 h at 37 °c. then, huh7it-1 cells were infected with the virus/venom-fraction mixture (fig. 2a) and virus infectivity was measured by infectious center assay. the anti-hcv activity started to appear from the retention time (rt) 35.5 until 43.8 min. the fraction at rt 36.4 min showed the most potent anti-hcv activity with ic 50 being 0.01 μg/ml ( fig. 2b ; table 1 ). in order to identify the bioactive compound(s) present in rt 36.4, lc-ms-esi analysis was performed. interestingly, the data of mass spectrometry showed that the active fraction contains a unique peptide with molecular mass of 8398 da (fig. 1b) . to go further into the characterization of the active peptide, the amino acid sequence was determined (fig. 1c) . the sequence of this peptide contains 76 amino acids (gwinekkmqqkidekigkniiggmakavihk-maknefqcvanvdtlgnckkhcakttgekgych-gtkckcgielsy). the obtained sequence belongs to the scorpion venom antimicrobial peptides and matched with the scorpine-like peptide smp76, which was identified in the scorpion venom gland of s. m. palmatus using transcriptomic analysis (abdel-rahman et al. 2013) . the amino acid sequence of smp76 was confirmed until the amino acid number 40 by edman degradation method and the molecular mass was confirmed by mass spectrometry resulting in 8398 da (see "materials and methods"). the cytotoxic activity of smp76 against huh7it-1 cells was tested using the wst-1 assay, and hemolytic activity was examined on human red blood cells. the cc 50 and the hc 50 were calculated. as shown in table 2 , cc 50 of smp76 against huh7it-1 cells and hc 50 were > 10 μg/ml. these results indicate that this peptide has no cytotoxic or hemolytic effects up to 10 μg/ml with selectivity index (si) > 1000. since smp76 displayed a significant inhibitory effect at the early stage of hcv infection, we examined whether the smp76 peptide can also inhibit hcv ns3 protein production and hcv rna replication in the cells. virus at multiplicity of infection of 2 pfu/cell was inoculated to the huh7it-1 cells for 3 ~ 4 h at 37 °c. after virus adsorption, the cells were cultured with media supplemented with 0.1 μg/ml of smp76 for 44 h at 37 °c (fig. 3a) . the cells were harvested and subjected to immunoblot and rt-qpcr analyses. the results showed that the post-treatment of hcv rna replication was not significantly inhibited (fig. 3b) or hcv ns3 protein synthesis in the cells (fig. 3c) . the above results suggest that the smp76 directly affects hcv particles and/or host cells in the culture medium to inhibit the viral infection and does not have an antiviral effect in the cells. we previously showed that the crude venom of s. m. palmatus inhibits denv (el-bitar et al. 2015) . therefore, anti-denv activity of the selected 30-fractions obtained from the crude venom of s. m. palmatus was tested. each fraction was incubated separately with fixed amount of denv for 2 h at 37 °c. after that, the virus/venom fractions mixtures were used to infect vero/slam cells and virus infectivity was measured by infectious center assay. interestingly, the results were consistent with the data of anti-hcv activity obtained in this study (table 1) . also, the fraction identified at 36.4 min which contains smp76 showed the potent anti-denv activity with ic 50 being 0.01 μg/ml (table 1 and fig. 4b ). to determine whether the antiviral activity of smp76 peptide (previously described) was specific to hcv and denv, a schematic of infection assay. b amounts of hcv infectious particles. the data represents mean ± sem of two independent experiments. § below the detection limit; ‡ ≤ 0.07%; # < 0.5% we tested its possible effects on another enveloped virus such as measles virus (otaki et al. 2006) . in this investigation, the virus was incubated with smp76 (10-0.01 μg/ml) for 2 h. then, vero/slam cells were infected with the virus/smp76 mixture (fig. 4a) and virus infectivity was measured using an infectious center or plaque assay. the results revealed that while smp76 peptide showed strong activity against denv with ic 50 10 ng/ml, it induced weak inhibition on measles virus at 10 μg/ml (fig. 4b ). in an attempt to identify the active domain of smp76, the antiviral activity (anti-hcv and anti-denv) of synthetic n-terminal (32 aa) and c-terminal (44aa without disulfide bonds) were tested. although, the purified native smp76 showed strong antiviral activity with ic 50 of 10 ng/ml, there was no antiviral activity for both synthetic terminals (table 3) . moreover, antiviral activity of recombinant c-terminal (44 aa) was examined. also, no antiviral activity for recombinant c-terminal was detected (table 3) . these results indicate that the full-length of smp76 may be required for its activity against hcv and denv. the above mentioned results imply that the full-length of smp76 may be required for its activity against hcv and denv. the full-length smp76 peptide (76 aa) was synthesized but without disulfide bonds. the antiviral activity of the synthesized smp76 peptide was examined against hcv and denv. these results showed no antiviral activity for the synthetic full-length peptide without disulfide bonds against hcv and denv (table 3) . scorpine is firstly isolated from the venom of pandinus imperator. the structure of scorpine is a hybrid between a cecropin and a defensin. the sequence of scorpine carboxyl terminal region is similar to that of β-ktx family, with cysteine-stabilized α/β fold, and three disulfide bridges. on the other hand, its amino-terminal region is identical to the cecropin family peptides (conde et al. 2000) . scorpine has also amino acid sequences similar to amps and k + channel blocking peptides (luna-ramirez et al. 2015) . scorpine homologs were thereafter identified from the venom of various scorpions such as opistophthalmus carinatus (zhu and tytgat 2004) , heterometrus laoticus (uawonggul et al. 2007) , h. gertschi ), s. m. palmatus (abdel-rahman et al. 2013 ), genus vaejovis (quintero-hernandez et al. 2015 and urodacus yaschenkoi (luna-ramirez et al. 2015) . importantly, all these peptides possess anti-malaria as well as antimicrobial activities (conde et al. 2000; carballar-lejarazu et al. 2008 ) and act also as potassium channel blockers ). the present data clearly showed that smp76 inhibits the ability of hcv virus to infect the host cells. indeed, our previous study demonstrated that the crude venom of s. m. palmatus venom prevents hcv infection with direct virocidal activity (el-bitar et al. 2015) . on the other hand, we cannot rule out the possibility that, smp76 peptide might has an independent effect on the receptor complexes of host cell components or interacts with components that inactivate viral entry. this possibility will be further investigated by the incubation of smp76 with cells in a free-virus condition prior to hcv infection. however, it worth to mention that the incubation of s. m. palmatus crude venom with cells prior to the hcv infection of cells did not impair the viral infectivity (el-bitar et al. 2015) . thus, the possible effect of smp76 on the host cell to abrogate hcv infection is unlikely. in the present study, smp76 prevents the early stages of life cycle of hcv and denv most probably through interacting with viral particles. the viral particle can be neutralized by targeting the envelope of the hcv or host factors related to the mature viral particle (zeisel et al. 2013) . notably, it has been reported in various studies that the structure of biological membranes could be altered by amps (zasloff 2002; harrison et al. 2016) . currently, the new approach for hcv infection treatment probably based on the combination of several drugs (pereira and jacobson 2009; sarrazin and zeuzem 2010; zeisel et al. 2011; qian et al. 2016 ). therefore, the use of smp76 with anti-hcv drugs for treatment of hcv infection may have synergistic effect. however, further experiments are needed to check this possibility. the present study reported distinctive data on the ability of smp76 peptide to protect cellular systems from attack of denv and neutralize viral infection. currently dengue virus considered as one of the most important arthropod born viral disease worldwide (botta et al. 2018) . despite the global efforts, there is no antiviral therapy against denv infections clinically approved and only symptomatic treatment and hospital supportive care setting are available for infected people (behnam et al. 2016) . it was shown that recombinantly expressed scorpine (rscrp) inhibited denv-2 replication in c6/36 mosquito cells. also, it was suggested that the development of transgenic mosquitoes that overexpress and correctly secrete rscrp and could eventually break the dengue fever transmission (carballar-lejarazu et al. 2008 ). on the other hand, smp76, as an infection inhibitor, has some advantages compared to antiviral drugs that target the viral replication stages inside the target cell. smp76 can inhibit denv infection before viral entry and is, therefore, helpful for treatment of denv viraemia. the prospective pharmaceutical potential of smp76 cannot be neglected, especially considering its potent antiviral activity. however, smp76 isolation from natural sources is ineffectual and time-consuming. synthetic smp76 without disulfide bonds showed no antiviral activities against hcv and denv. one possibility is that the synthetic smp76 peptide without disulfide bonds did not have the properly folded structure necessary for activity. recently, recombinant scorpine with antimalarial and antibacterial activities was produced by different fusion technology using small ubiquitin-related modifier (sumo) (zhang et al. 2014) and maltose binding protein (mbp) (zhang et al. 2016 ). these methods have improved efficiency and reduced the cost of producing scorpine and can contribute to the future production of active recombinant smp76. interestingly, the recombinant smp76 was shown to inhibit denv and zikv infections in cultured cell lines and primary mouse macrophages. however, rsmp76 did not inactivate the viral particles directly but suppressed the established viral infection by upregulating the expression of ifn-β (ji et al. 2018 ). this mechanism is significantly different from the virucidal effect of native smp76 peptides. the exact mechanism by which smp76 exerts its antiviral activity against hcv and denv to inhibit infecting their target cells need further studies. in vivo studies should also assess the future role of smp76 in managing hcv and denv infections. venom proteomic and venomous glands transcriptomic analysis of the egyptian scorpion scorpio maurus palmatus (arachnida: scorpionidae) isolation and identification of substances with anti-hepatitis c virus activities from kalanchoe pinnata the medicinal chemistry of dengue virus the global distribution and burden of dengue drug repurposing approaches to fight dengue virus infection and related diseases recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom the who dengue classification and case definitions: time for a reassessment hepatitis c virus infection induces apoptosis through a bax-triggered, mitochondrionmediated, caspase 3-dependent pathway wide phylogenetic distribution of scorpine and long-chain beta-ktx-like peptides in scorpion venoms: identification of "orphan" components virocidal activity of egyptian scorpion venoms against hepatitis c virus ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs oral direct-acting agent therapy for hepatitis c virus infection: a systematic review isolation and characterization of a hyaluronidase from the venom of chinese red scorpion buthus martensi scorpion venomtoxins that aid in drug development: a review phospholipid dependent mechanism of smp24, an α-helical antimicrobial peptide from scorpion venom lectin-mediated enhancement of dengue virus infection in a mouse macrophage cell line mk1 enhancement of dengue virus type 2 replication in mouse macrophage cultures by bacterial cell walls, peptidoglycans, and a polymer of peptidoglycan subunits peptide antimicrobial agents update on global epidemiology of viral hepatitis and preventive strategies world the scorpion venom peptide smp76 inhibits viral infection by regulating type-i interferon response design and expression of recombinant toxins from mexican scorpions of the genus centruroides for production of antivenoms involvement of phospholipase a2 pathway for the indian red scorpion venom-induced augmentation of cardiopulmonary reflexes elicited by phenyldiguanide whole transcriptome of the venom gland from urodacus yaschenkoi scorpion emerging flaviviruses: the spread and resurgence of japanese encephalitis, west nile and dengue viruses dengue viral infections global spread of dengue virus types: mapping the 70 year history international (2015) egypt health issues survey 2015. ministry of health and population/egypt and icf international inhibition of dengue virus entry into target cells using synthetic antiviral peptides discovery of fifth serotype of dengue virus (denv-5): a new public health dilemma in dengue control cytotoxic and apoptotic effects of scorpion leiurus quinquestriatus venom on 293t and c2c12 eukaryotic cell lines measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (cdw150) but not cd46 as a cellular receptor scorpion venom components as potential candidates for drug development inhibition of measles virus and subacute sclerosing panencephalitis virus by rna interference optimization of antiscorpion venom production oliver, 1807) scorpionism in the saniliurfa region in turkey study of the relationship between androctonus crassicauda (oliver, 1807; scorpiones, buthidae) venom toxicity and telson size, weight and storing condition hepatitis c virus resistance to direct-acting antiviral drugs in interferon-free regimens new and experimental therapies for hcv entry inhibitors: new advances in hcv treatment emerg transcriptome analysis of scorpion species belonging to the vaejovis genus dengue virus life cycle: viral and host factors modulating infectivity overview of scorpion toxins specific for na+ channels and related peptides: biodiversity, structure function relationships and evolution esistance to direct antiviral agents in patients with hepatitis c virus infection transcriptome analysis of the venom gland of the mexican scorpion hadrurus gertschi (arachnida: scorpiones) comparison of dengue virus and hcv: from impact on global health to their rna-dependent rna polymerases purification and characterization of heteroscorpine-1 (hs-1) toxin from heterometrus laoticus scorpion venom quintero-hernández v (2017) recombinant expression of intrepicalcin from the scorpion vaejovis intrepidus and its effect on skeletal ryanodine receptors production of infectious hepatitis c virus in tissue culture from a cloned viral genome molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease guidelines for the screening, care and treatment of persons with chronic hepatitis, c infection. world health organization development of a simple system for screening anti-hepatitis c virus drugs utilizing mutants capable of vigorous replication antimicrobial peptides of multicellular organisms hepatitis c virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies host-targeting agents for prevention and treatment of chronic hepatitis c-perspectives and challenges high-level expression, purification, and characterization of bifunctional scfv-9r fusion protein production and characterization of scorpine by mbp fusion technology in escherichia coli the scorpine family of defensins: gene structure, alternative polyadenylation and fold recognition the authors are grateful to dr. lin deng and dr. ming chen, division of microbiology, kobe university graduate school of medicine, japan, for their assistance in virological analyses. the authors also acknowledge dr. fernando zamudio, m. sc. leonel vargas jaimes and m. sc. maría teresa romero gutiérrez for their assistance in the proteomics work done in this work. we also grateful key: cord-013244-d6saaiu9 authors: eijsink, job f. h.; al khayat, mohamed n. m. t.; boersma, cornelis; ter horst, peter g. j.; wilschut, jan c.; postma, maarten j. title: cost-effectiveness of hepatitis c virus screening, and subsequent monitoring or treatment among pregnant women in the netherlands date: 2020-10-16 journal: eur j health econ doi: 10.1007/s10198-020-01236-2 sha: doc_id: 13244 cord_uid: d6saaiu9 background: the prevalence of diagnosed chronic hepatitis c virus (hcv) infection among pregnant women in the netherlands is 0.26%, yet many cases remain undiagnosed. hcv screening and treatment of pregnant hcv carriers could reduce the burden of disease and limit vertical transmission from mother to child. we assessed the impact of hcv screening and subsequent treatment with new direct-acting antivirals (daas) among pregnant women in the netherlands. methods: an hcv natural history markov transition state model was developed, to evaluate the public-health and economic impact of hcv screening and treatment. besides all 179,000 pregnant women in the netherlands (cohort 1), we modelled 3 further cohorts: all 79,000 first-time pregnant women (cohort 2), 33,000 pregnant migrant women (cohort 3) and 16,000 first-time pregnant migrant women (cohort 4). each cohort was analyzed in various scenarios: i no intervention, i.e., the current practice, ii screen-and-treat, i.e., the most extensive approach involving treatment of all individuals found hcv-positive, and iii screen-and-treat/monitor, i.e., a strategy involving treatment of symptomatic (f1–f4) patients and follow-up of asymptomatic (f0) hcv carriers with subsequent treatment only at progression. results: for all cohorts, comparison between scenarios (ii) and (i) resulted in icers between €9,306 and €10,173 per qaly gained and 5 year budget impacts varying between €6,283,830 and €19,220,405. for all cohorts, comparison between scenarios (iii) and (i) resulted in icers between €1,739 and €2,749 per qaly gained and budget impacts varying between €1,468,670 and €5,607,556. for all cohorts, the icers (scenario iii versus ii) involved in delayed treatment of asymptomatic (f0) hcv carriers varied between €56,607 and €56,892, well above the willingness-to-pay (wtp) threshold of €20,000 per qaly gained and even above a threshold of €50,000 per qaly gained. conclusion: universal screening for hcv among all pregnant women in the netherlands is cost-effective. however, it would be reasonable to consider smaller risk groups in view of the budget impact of the intervention. electronic supplementary material: the online version of this article (10.1007/s10198-020-01236-2) contains supplementary material, which is available to authorized users. hepatitis c is a serious disease caused by infection with hepatitis c virus (hcv). worldwide 80-130 million people are chronically infected with hcv [1, 2] . exposure to the virus results in 80% of cases in a chronic infection [3] . approximately 20% of chronically infected patients develop serious hcv-related liver disease after onset of the infection [4] . currently, hepatitis c affects 8% of pregnant women globally [5] . hcv may be transmitted vertically, mostly perinatally, from mother-to-child [6] [7] [8] . with the development of new drug therapies which are highly effective and well tolerated, there is a potential for these drugs to be used by pregnant patients with hepatitis c [9] . hcv screening of pregnant women potentially contributes to the goal of the world health organization (who) to achieve 90% diagnosis of hcv and 80% treatment by 2030 worldwide through scaling-up screening strategies and prevention of hcv transmission [10] . two major developments have contributed to the demand for hcv screening of specific risk groups. the first and most important development is the improved hcv treatment with direct-acting antivirals (daas) [11] . more than 90% of chronically infected hcv patients are cured through daa treatment compared to only 50% with previous treatments [12] [13] [14] . the second development is the increase of hepatocellular cancer (hcc) incidence, hcv infection being the leading cause of hcc in western countries [15] . screening and daa treatment of risk groups could prevent reinfection, new infections, hcc and vertical transmission from mother-to-child. the health council in the netherlands has recommended to investigate the cost-effectiveness of screening of pregnant women for hcv with subsequent daa treatment [16] [17] [18] . prevalence of diagnosed chronic hcv infection among women in the dutch population is 0.26% (95% confidence interval (ci): 0.15-0.46%), which is similar to the prevalence in the general population in europe [17] . firstgeneration non-western migrants are more likely to be hcvpositive (0.7-2.3%) than western women (0.1-0.4%) [18] . notably, these immigrants represent 5.9% of the total dutch female population [19] . in industrialized countries, hcv is the most common cause of chronic liver disease among children and perinatal transmission is the leading cause of infection [20] . the current best estimate of vertical transmission risk is between 4.5 and 7.1% [21] . treatment with daas during pregnancy is not yet recommended, and lactation during treatment is contra-indicated, because of a lack of information on potential toxicity [22] . however, it is conceivable that in the near future daa treatment of hcv-infected women during pregnancy becomes available, not only to limit disease progression in the patient, but also to prevent vertical transmission of the virus to the child. the aim of this study is to estimate the public-health and economic impact of hcv screening and treatment among pregnant women from a public-health perspective [23] . in particular, we estimated the health gains, cost-effectiveness and the budget impact of implementing such a programme. the results of our study can be used to reach a rational decision as to whether hcv screening and potential treatment of pregnant women should be implemented in the netherlands and elsewhere [24] . a screening model linked to hcv-disease states within a markov model was used to evaluate the cost-effectiveness (ce) of hcv screening of pregnant women, with initial treatment during pregnancy, compared to current practice (no screening and no intervention) from a health-care payer perspective in the netherlands. our ce analysis includes health benefits for pregnant women and their children, and the corresponding budget impact. the costs and effects of hcv screening and various modalities of subsequent treatment versus current practice were calculated for four cohorts of pregnant women and were expressed in terms of incremental cost-effectiveness ratio (icer), as further elaborated below. we used a deterministic, hcv natural history, closed-cohort markov model, as presented in fig. 1 . the model includes annual cycles and a life-time horizon of 70 years, representing the approximate period from the age at which a woman can become pregnant until her death. hcv carriers were classified in metavir scores f0-f4. f0 is a (fully) healthy, but hcv-infected, state. f1-f3 represent mild to severe stages of liver fibrosis. f4 represents liver cirrhosis. in the model, patients with metavir score f4 may develop hepatocellular cancer (hcc), decompensated cirrhosis (dcc) and, subsequently, patients with dcc can progress to liver transplantation (lt). lt-patients move to the follow-up state (post-lt). post-lt patients are described as patients during the first 12 months after their liver transplantation. after 1 year, they move to the follow-up state post-lt + until their death. without screening, hcv-infected patients generally develop symptoms in a late stage of infection [18, 25] . implementation of screening will result in detection of increased numbers of asymptomatic patients [26] and, later on, fewer patients with fibrosis or cirrhosis relative to the current situation without screening. in this study, we assumed that testing a cohort comprising of all pregnant women is a 'one-time' screening for each women (independent of the number of pregnancies), rather than having repeat testing in their potential subsequent pregnancies. in the model, we used a conservative sustained virologic response (svr) of 95% for patients with metavir scores of f0, f1, f2 and f3, and 90% for f4 patients [11] . we only included treatment regimens for 12 weeks, independent on the metavir scores and in accordance with the dutch hcv-guidelines [27, 28] . it was assumed that if patients were not cured, they proceed to the next lower health state. vertical transmissions are included in the model as potentially prevented hcv infections, after screening of the mothers and subsequent daa treatment. the probabilities to move from one health state to another are given in table s1 of the appendix. the first hcv screening step represents a serologic antibody test to determine the presence of a current or past hcv infection. the second test is a reverse-transcription polymerase-chain reaction (rt-pcr) viral rna test to confirm the serologic test, and to determine whether the hcv infection had been cleared spontaneously. the rt-pcr test has a sensitivity between 61.0% and 81.8% and a specificity between 97.5 and 99.7% [29] . the third test concerned a fibroscan examination, which is a quantitative analysis technique to support the diagnostics of liver fibrosis in patients and to determine the metavir score (f0-f4). individuals are screened first for anti-hcv antibodies and, if found positive, are subsequently screened for hcv rna. outpatient visit consultation costs were included for each test. for individuals who are rna-positive, we incorporated fibroscan costs for disease staging. the annual costs for daa treatment were assumed at their list price levels in the netherlands [30] . weighted average treatment costs for daa were estimated at €34,000 based on actual use of daa medication (sofosbuvir, ledipasvir/ sofosbuvir, grazoprevir/elbasvir, velpatasvir/sofosbuvir, daclatasvir, ombitasvir/paritaprevir/ritonavir) for a 12-week treatment period in 2018 [31] . we did not consider other treatments for hcv infection, such as protease inhibitors, ribavirin or peg-interferons. for the budget-impact analysis, we included total medical costs in the first 5 years, costs of hcv treatment, screening costs and follow-up costs with possible hcv-related diseases. the pregnant women included for hcv screening in this study are between 20 and 45 years of age, with an average hcc hepatocellular cancer, dcc decompensated cirhossis, lt liver transplantation. lrd: liver-related death. *in case of treatment failure, patients will be in the same metavir state after the treatment age of 29 [19, 32] . we excluded women with recurrent hcv infection, women with hiv infection and injecting drug users [33] . in this study, we considered four different cohorts of pregnant women. the characteristics of the four cohorts were obtained from statistics netherlands (cbs); we took the average size of the years 2000 to 2017. details of the cohorts, including size, hcv prevalence and vertical transmission estimates [21] , are as follows: quality of life depends on the state of health and the age of the pregnant woman. in the model, all hcv health states were assigned a particular utility, ranging from 0 to 1. utility 0 reflects death and utility 1 reflects full health without any complaints. the utility of hcv-positive, but asymptomatic, patients (f0) was reduced with 0.02 [34, 35] , because of reasons of anxiety and worries and among these individuals. utilities after successful treatment were assumed to increase by 0.05 [36] . the utilities are presented in s1 of the appendix. quality-adjusted life years (qalys) were calculated as the product of remaining life years of the patient in a particular health state after the intervention (screening and monitoring or treatment) and the quality of life after the intervention [36] . we investigated three scenarios with different comparisons between the scenarios. scenario i, the no-intervention scenario, reflects the current practice of absence of screening. scenario ii, the screen-and-treat scenario, reflects the most extensive approach with daa treatment of all individuals found hcv-positive after screening. finally, scenario iii, the screen-and-treat/monitor scenario, reflects the approach in which, after screening, the f0 patients are not treated but actively monitored (and, if indicated, treated later on). we specifically considered this third scenario to avoid delayed overtreatment. indeed, 20% of asymptomatic hcv-infected individuals spontaneously clear the virus and, in addition, approximately 80% of chronically infected patients will never develop hcv-related liver disease [37] . obviously, one does not know a priori which patients will develop chronic infection and symptoms of disease. therefore, we chose to periodically monitor these patients. we assumed that just monitoring asymptomatic hcv carriers instead of treatment would contribute to lower treatment costs and result in higher patient value. three comparisons between the different scenarios were performed: • scenario ii versus scenario i, reflecting screening and treatment of all hcv-positive patients versus the current practice of no intervention. • scenario iii versus scenario i, reflecting treatment of symptomatic (f1-f4) patients and monitoring of asymptomatic (f0) hcv carriers versus the current practice. • scenario iii versus scenario ii, focusing specifically on the additional costs and health gains due to immediate treatment of all f0 hcv carriers versus just monitoring these asymptomatic individuals until some of them progress to disease. as avoidance of mother-to-child transmission of hcv is one of the most important reasons for hcv screening and treatment of pregnant women, vertical transmissions are explicitly taken into account in the model. specifically, we included the effects of vertical transmission on the healthcare costs, treatment costs and qalys for the (unborn) children. we express the cost-effectiveness of the different scenarios described above in terms of incremental cost-effectiveness ratio (icer), using the following formula: in which c represents the costs and e the quality-adjusted life years (qalys); subscript 1 represents the case where the intervention has been applied and subscript 0 represents the case where the intervention has not been applied. therefore, the icer represents the costs per quality-adjusted life year (qaly) gained. in the netherlands, icers are considered against an informal willingness-to-pay (wtp) threshold of €20,000 per qaly gained [38] . notably, we also considered a wtp-threshold of €50,000 per qaly gained, reflecting the burden of disease [39] . the budget-impact analysis gives a perspective on total future hcv-related costs. for the budget-impact analysis, we included direct medical costs, costs of hcv treatment and costs of screening, in the first 5 years, 10 years and 15 years of implementation of screening according to the budget impact guidelines [40] . the total costs were discounted with an annual rate of 4%, the qalys were discounted with 1.5%, according to dutch guidelines [41] . price levels in the year 2018 were applied. a one-way sensitivity analysis was performed to estimate the effect of variation in specific parameters on the icer and to determine which parameter has the most pronounced effect on the icer. the parameters were varied between minus 10% and plus 10% of the base-case parameter value. the prevalence was varied in the range of the 95% ci of the hcv prevalence of 0.26% (0.15-0.46%). a probabilistic sensitivity analysis (psa) was performed to assess the uncertainty around the different input parameters and the effect on the cer. here, input parameters are considered as random quantities based on the underlying parameter distributions. for every simulation (5000 in total), the parameters were sampled from the parameter space of 95% ci. if the 95% ci was unknown for a specific parameter, we varied the parameter between minus 10% and plus 10%, following a triangle distribution. all variables and ranges are represented in table s2 of the appendix. we first determined the health benefits involved in implementation of hcv screening and daa treatment among pregnant women in the netherlands. in all four cohorts, we found significant reductions in liver disease after 2-3 decades, specifically a reduction of 30% in dcc, of 37% in hcc, of 27% in liver transplantation (lt) and of 34% in liver-related death (lrd). we also found significant reductions in vertical hcv transmissions. since each cohort consists of a different number of pregnant women with a specific hcv prevalence, the absolute number of avoided vertical transmission varied between the different cohorts. specifically, in the cohort of all pregnant women, we found 27 avoided cases of vertical transmission, in the cohort of first-time pregnant women 18 avoided cases, in the cohort of pregnant migrants 14 avoided cases, and in the cohort of first-time pregnant migrants 10 avoided cases. we subsequently determined the cost-effectiveness and budget impact of hcv screening and treatment among the four cohorts of pregnant women following the different scenarios and comparisons. table 1 presents an overview of the results. for each of the cohorts, the table shows the values of the icer for comparisons between the two respective intervention scenarios and the scenario; no intervention (current practice), table 1 also presents the icers for scenario screen-and-treat versus screen-and-treat/monitor and in table 2 the total 5 years, 10 years and 15 years budget impact (bi) of the different interventions. below, we further elaborate on the results obtained for each of the cohorts. the (table 2) . limiting the intervention to the cohort of first-time pregnant migrants further improved cost-effectiveness results, with the most favorable outcomes for the screen-and-treat/ monitor scenario. specifically, comparison in this group between the screen-and-treat and no intervention scenarios yielded 664 qalys gained at incremental costs of €6,179,374, resulting in an icer of €9,306 per qaly gained. the total bi over 5 years of this screening scenario we conducted an additional comparison ( as indicated above, hcv screening and treatment of pregnant women prevents significant numbers of vertical transmission cases. yet, the effects of vertical transmission on the icers of the screen-and-treat and the screen-and-treat/ monitor scenarios remain limited. this is primarily due to the relatively low rate of vertical transmission of 4.5-7.1% [21] . with inclusion of vertical transmission in the markov model, the icers for the four different cohorts range between €9306 and €10,173, and without inclusion of vertical transmission in the model, the icers range between €8780 and €9703. we performed both a one-way sensitivity analysis and a probabilistic sensitivity analysis (psa), to assess the effect of parameter uncertainty on the cost-effectiveness outcomes. the effect of the cohort size on the icer outcomes was found to be minimal. here, we present the result on the univariate sensitivity analysis for the screen-and-treat/monitor versus no intervention scenario in the cohort of first-time pregnant migrants. this is the scenario with the most favorable cost-effectiveness. the one-way sensitivity-analysis for this scenario in this fig. 2 one-way sensitivity analysis for the comparison between the screen-and-treat/monitor and no intervention scenarios among first-time pregnant migrants. the diagram shows the change in the icer when each parameter is increased or reduced with 10% cohort shows that the cost-effectiveness outcome is most sensitive to variation in the prevalence of hcv (fig. 2 ). for the screen-and-treat versus no intervention scenario in the same cohort, the cost-effectiveness outcome was most sensitive to variation in medication price (fig. 3 ). for the screen-and-treat versus screen-and-treat/monitor scenario, monitoring disutility is most sensitive to variation (fig. 4) . the results of the ceac are presented in figs. 5 and 6. these results indicate that, among the four cohorts investigated, the icers for both the screen-and-treat versus no intervention and screen-and-treat/monitor versus no intervention scenarios remain well below the informal dutch wtp-threshold of €20,000 per qaly gained. overall, the results of the psa showed limited variation around the mean cost-effectiveness estimate upon varying the model inputs independently, underlining the robustness of the model. finally, fig. 7 shows the respective cost-effectiveness acceptability curve based on varying the wtp-threshold. these results indicate that among the four cohorts investigated, the icers for screen-and-treat versus screenand-treat/monitor is not below the informal dutch wtpthreshold of €20,000 per qaly gained. our study demonstrates that, after screening of pregnant women, identification of hcv patients at early metavir stages and implementation of daa treatment would prevent one out of three liver-related diseases caused by hcv on the long term. in addition, depending on the specific screening/ treatment strategy, the size and the hcv prevalence of the cohorts, hcv screening and treatment results in prevention of 10-27 vertical transmissions in the netherlands. our present study demonstrates that hcv screening of pregnant women and subsequent immediate treatment of all hcv-positive individuals with daas is a cost-effective intervention in the netherlands. this applies not only to the cohorts of non-western migrant women in the netherlands with a relatively high hcv prevalence, but also to the cohorts of all pregnant dutch women in which on average the hcv prevalence is lower. indeed, in all four different cohorts studied, the icers of the screen-and-treat versus no intervention scenario were similar, varying between €9306 and €10,173 per qaly gained, and thus remained well below the wtp-threshold of €20,000 per qaly gained. still considerably lower icers were obtained for the screen-and-treat/monitor scenario in which only the symptomatic f1-4 patients are treated and the asymptomatic f0 hcv carriers are just monitored until some of them progress fig. 3 one-way sensitivity analysis for the comparison between the screen-and-treat and no intervention scenarios among first-time pregnant migrants. the diagram shows the change in the icer when each parameter is increased or reduced with 10% fig. 4 one-way sensitivity analysis for the comparison between the screen-and-treat and screen-and-treat/monitor scenarios among first-time pregnant migrants. the diagram shows the change in the icer when each parameter is increased or reduced with 10% cost-effectiveness acceptability curve (ceac) for the comparison between the screen-and-treat and screen-and-treat/monitor scenarios among the four cohorts of pregnant women to disease. indeed, for this scenario the icers among the different cohorts varied between only €1739 and €2749 per qaly gained, the most cost-effective result being obtained for the cohort of first-time pregnant migrant women. while, as indicated above, the icer of hcv screening and treatment (or monitoring of f0 and treatment of f1-4 patients), remained below the dutch wtp-threshold of € 20,000, the budget impact of these interventions was substantially different between the four cohorts. clearly, the budget impact is directly proportional to the size of the cohort, and thus was much higher for the cohorts of all pregnant dutch women, as opposed to the migrant women. for the screen-and-treat scenario, the budget impact varied between €6,283,830 and €19,220,405 in the migrant cohort and all pregnant women, respectively. also, the extent of treatment strongly affects the budget impact. for example, in the cohort of all pregnant women, the budget impact of the screen-and-treat/monitor scenario was, with €5,607,556, much lower than the €19,220,405 of the screen-and-treat scenario. likewise, in the cohort of migrant women, the budget impact varied substantially between these two scenarios, ranging from €1,734,575 and €9,323,994. the above results illustrate that implementation of a strategy of active monitoring of f0 patients, rather than immediate treatment of these asymptomatic individuals, represents an effective way of reducing the costs of hcv screening and treatment. the reason is that approximately 20% of hcvinfected individuals spontaneously clear the virus, while furthermore 80% of those who do become chronic hcv carriers, will never develop hcv-related liver disease [42] . clearly, postponing treatment of f0 patients saves potentially unnecessary costs. accordingly, restriction of treatment to f1-4 patients represents the most cost-effective scenario and thus contributes to optimization of value for hcv patients. this is also illustrated by the comparison of our scenarios ii and iii, resulting in an icer above €50,000 per qaly gained in all cohorts studied, which directly demonstrates that treatment of f0 patients is not cost-effective. a 66% daa discount, in the comparison of screen-and-treat versus screen-and-treat/monitor all hcv-infected pregnant women, would be cost-effective at a threshold of €20,000 per qaly gained, in different cohorts of pregnant women. 66% discount, is comparable with the discount rate from biologicals versus biosmilars in the netherlands, therefore in the future screen-and-treat could also be a cost-effective scenario compared to screen and monitoring [43] . while just monitoring of asymptomatic chronic hcv carriers does reduce costs, it does not prevent spread of the virus through vertical transmission from mother-to-child. monitoring of f0 carriers does not prevent hcv infection in subsequent pregnancies either; our model did not take further transmission of hcv and spreading of infection into account in untreated women. in this respect, our model can be considered to reflect a conservative estimate of cost-effectiveness. inclusion of transmission effects beyond the child would further enhance the cost-effectiveness profile. however, these effects do not outweigh the benefits of restricting treatment to f1-4 patients. we therefore conclude that monitoring of f0 hcv-positive patients instead of immediate treatment prevents significant costs and thus results in the most favorable cost-effectiveness with a substantially lower budget impact [44] . in this study, we focused on screening of pregnant women and subsequent treatment of hcv-positive individuals with daas. however, currently, hcv treatment with daas is contraindicated for pregnant women, because of a lack of studies regarding direct teratogenic effects and pharmacological effects later in life of the offspring. consequently, under the present circumstances, hcv-positive mothers can only be treated after childbirth and thus only children from subsequent pregnancies would be protected. according to bernstein et al., universal hcv screening and treatment with daas during pregnancy is on the horizon [45] . clearly, these interventions should be urgently evaluated for safety and implemented if appropriate [46] . several studies regarding daa treatment of hcv infection during pregnancy are ongoing. for example, the results of a phase i study in magee women's hospital in pittsburgh are expected to be presented in 2020 [47] . in the future, we anticipate a development for hcv screening and treatment similar to that in the case of hiv/aids, where hiv-positive women are treated with combination antiretroviral therapy (cart) to prevent mother-to-child transmission of the virus [33, 48] . perinatal transmission is the primary hcv transmission route among children responsible for 70-90% of cases. many children often remain untested and potentially hcv undiagnosed. therefore, next to the direct benefit of treatment for the women in curing their infection and preventing serious liver-related diseases, benefits for the child exist in avoiding hcv with possible extrahepatic effects of hcv infection in childhood and significant reductions in both physical and psychosocial health as well as in cognitive functions. the outcome of our study that hcv screening and treatment of pregnant women in the netherlands is a cost-effective intervention against the informal dutch wtp-threshold of €20,000 per qaly gained, is in apparent disagreement with the findings of urbanus et al. [18] in 2013. these authors estimated that only if costs per treatment were to decline to €3750 (a reduction in price of €31,000), screening of all pregnant women would be cost-effective. however, the results of urbanus et al. [18] were obtained before the introduction of the highly effective daas in 2015. now, it appears that screening and daa treatment, of hcv-positive individuals would be a cost-effective intervention. nonetheless, as discussed above, screening of the entire population of pregnant women is not necessarily preferred, because of the large budget impact of the intervention and the low hcv prevalence in the total dutch population. kracht et al. have proposed "micro-elimination" of hcv by screening and treatment of various pre-defined hcv risk groups [49] . these authors concluded, in agreement with our results, that hcv screening of risk groups is the most pragmatic and efficient approach. our study could be helpful with decisions on the implementation of hcv screening programmes in europe. the estimated fraction of hcv cases that remain undiagnosed in the general or proxy populations in europe ranges between 20% in denmark to 91.2% in greece [50] . razavi et al. estimated the overall proportion of undiagnosed hcv cases in the eu at 64% [1] . daa treatment of hcv in pregnancy is not (yet) in clinical guidelines, our model is hypothetical currently in that respect. the main difference between, for example, the assld/idsa-guidelines and our model is that we assumed that pregnant women are treated with daas after hcv diagnosis during pregnancy. a simplified treatment algorithm [51] , for treatment-naive patients without cirrhosis, possibly would reduce the costs in the model, which could also improve the performance of treatment, corresponding with favorable to cost-effectiveness [52] . this study reflects a single cohort model in the netherlands, with effects on children for that specific cohort. our current analysis does not include future pregnancies in the very same cohorts. in the future, the total amounts of screened and treated pregnant women will be lower and preferably result from the standard prenatal screening for infectious diseases, which means higher numbers to screen to identify patients, but also less patients to be treated with relatively expensive treatments. our study demonstrates that screening and monitoring or treatment of smaller subgroups of pregnant women is highly cost-effective approach and has a comparatively low budget impact in the netherlands. on the other hand, in other countries with a higher hcv prevalence, screening of all pregnant women could be a more cost-effective option [6, 53] . our study indicates that universal hcv screening of pregnant women in the netherlands is cost-effective, independent of the specific cohort involved. however, the budget impact is substantially different between subgroups, and is largely determined by the cohort size and by the extent of treatment of hcv-positive individuals. screening and subsequent monitoring of f0 patients and treatment of f1-f4 patients with the daas appeared to be the most cost-effective approach. hcv screening and treatment of pregnant women results in a substantial reduction of hcv-related liver diseases and deaths. it also prevents vertical transmission of the virus from mother to child. from a public-health and health-economic perspective, it would be reasonable to consider smaller risk groups of first-time pregnant or/and non-western pregnant women for an active hcv screening programme in the netherlands, and possibly elsewhere. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. the estimated future disease burden of hepatitis c virus in the netherlands with different treatment paradigms natural history of chronic hepatitis c the effects of female sex, viral genotype, and il28b genotype on spontaneous clearance of acute hepatitis c virus infection hepatitis c virus treatment: is it possible to cure all hepatitis c virus patients? effects of mode of delivery and infant feeding on the risk of mother-to-child transmission of hepatitis c virus: european paediatric hepatitis c virus network the management of hcv infected pregnant women and their children european paediatric hcv network clinical course and management of acute and chronic viral hepatitis during pregnancy european convention on human rights hcv vertical transmission in pregnancy: new horizons in the era of daas hepatitis elimination by 2030: progress and challenges systematic review: current concepts and challenges for the direct-acting antiviral era in hepatitis c cirrhosis peginterferon alfa-2b plus ribavirin compared with interferonalfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection sterfte aan chronische hepatitis b en c in nederland hepatitis c virus and hepatocellular carcinoma: a narrative review screening van risicogroepen op hepatitis b en c (screening of risk groups for hepatitis b and c )| advies | gezondheidsraad hepatitis c in the general population of various ethnic origins living in the netherlands: should non-western migrants be screened? is adding hcv screening to the antenatal national screening program in amsterdam, the netherlands, cost-effective? a review of hepatitis c virus (hcv) vertical transmission: risks of transmission to infants born to mothers with and without hcv viraemia or human immunodeficiency virus infection the prevalence of hepatitis b and c in an antenatal population of various ethnic origins perinatal transmission of hepatitis c virus infection cost-effectiveness of universal hepatitis c virus screening of pregnant women in the united states hepatitis c in pregnancy: screening, treatment, and management hepatitis c virus prevalence in the netherlands: migrants account for most infections health strategy on hcv in the netherlands longterm treatment outcomes of patients infected with hepatitis c virus: a systematic review and meta-analysis of the survival benefit of achieving a sustained virological response hcv richtsnoer 12 weeks treatment daas systematic review of the accuracy of antibody tests used to screen asymptomatic adults for hepatitis c infection. c open ank?infot ype=g&label =00-totaa l&tabel =b_01-basis &geg=vs_ gebr&item=j05ap ank?zoekt erm=sofos buvir %2fvel patas vir%2fvox ilapr evir&toedi ening svorm =table tten en capsules the use of highly active antiretroviral therapy (haart) in patients with advanced hiv infection: impact on medical, palliative care, and quality of life outcomes guideline for conducting economic evaluations in healthcare utiliteitsmeting bij de klinische besluitvorming systematic review: health-state utilities in liver disease: a systematic review natural history of hepatitis c the nice cost-effectiveness threshold: what it is and what that means quantification of the potential impact of cost-effectiveness thresholds on dutch drug expenditures using retrospective analysis principles of good practice for budget impact analysis: report of the ispor task force on good research practices -budget impact analysis. value heal richtlijn voor het uitvoeren van economische evaluaties in de gezondheidzorg (protocol for the execution of economic evaluation in healthcare cost effectiveness of screening for hepatitis c virus in asymptomatic, average-risk adults identifying key benefits in european off-patent biologics and biosimilar markets: it is not only about price! hepatitis c virus prevalence and level of intervention required to achieve the who targets for elimination in the european union by 2030: a modelling study hepatitis c in pregnancy in the era of direct-acting antiviral treatment: potential benefits of universal screening and antepartum therapy considering direct-acting antivirals to cure hepatitis c virus during pregnancy: is this the last treatment frontier? ther study of hepatitis c treatment during pregnancy -clinical-trials.gov n.d treatment of hepatitis c during pregnancy-weighing the risks and benefits in contrast to hiv strategies for achieving viral hepatitis c micro-elimination in the netherlands hepatitis b and c testing strategies in healthcare and community settings in the eu/ eea: a systematic review a simplified algorithm for the management of hepatitis c infection liver diseases in pregnancy: liver transplantation in pregnancy key: cord-000833-m6abyuvx authors: sekiguchi, satoshi; kimura, kiminori; chiyo, tomoko; ohtsuki, takahiro; tobita, yoshimi; tokunaga, yuko; yasui, fumihiko; tsukiyama-kohara, kyoko; wakita, takaji; tanaka, toshiyuki; miyasaka, masayuki; mizuno, kyosuke; hayashi, yukiko; hishima, tsunekazu; matsushima, kouji; kohara, michinori title: immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis c virus suppresses viral protein levels in mouse liver date: 2012-12-17 journal: plos one doi: 10.1371/journal.pone.0051656 sha: doc_id: 833 cord_uid: m6abyuvx chronic hepatitis c, which is caused by infection with the hepatitis c virus (hcv), is a global health problem. using a mouse model of hepatitis c, we examined the therapeutic effects of a recombinant vaccinia virus (rvv) that encodes an hcv protein. we generated immunocompetent mice that each expressed multiple hcv proteins via a cre/loxp switching system and established several distinct attenuated rvv strains. the hcv core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis c-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. immunization with one rvv strain (rvv-n25), which encoded nonstructural hcv proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis c within 7 days after injection. furthermore, hcv protein levels in liver tissue also decreased in a cd4 and cd8 t-cell-dependent manner. consistent with these results, we showed that rvv-n25 immunization induced a robust cd8 t-cell immune response that was specific to the hcv nonstructural protein 2. we also demonstrated that the onset of chronic hepatitis in cn2-29((+/−))/mxcre((+/−)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) tnf-α and (interleukin) il-6. thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of hcv proteins because these mice had not developed immune tolerance to the hcv antigen. in addition, we propose that rvv-n25 could be developed as an effective therapeutic vaccine. hepatitis c virus (hcv) is a major public health problem; approximately 170 million people are infected with hcv worldwide [1] . hcv causes persistent infections that can lead to chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (hcc) [2] . antiviral drugs are not highly effective in individuals with a chronic infection; furthermore, an effective vaccine against hvc has not been developed. a convenient animal model of hcv infection will greatly facilitate the development of an effective hcv vaccine. transgenic mice that express hcv proteins have been generated to study hcv expression [3, 4] ; however, in each of these cases, the relevant transgenes is expressed during embryonic development; therefore, the transgenic mice become immunotolerant to the transgenic products, and consequently, the adult mice are not useful for investigations of the pathogenesis of chronic hepatitis c. to address this problem, we developed a system that can drive conditional expression of an hcv transgene; our system involves the cre/loxp system and a recombinant adenovirus capable of expressing cre recombinase [5, 6] . concerns have been expressed that an adenovirus and transient expression of hcv proteins could induce immune responses [5] and, therefore, obscure any evidence of the effect of the host immune responses on chronic liver pathology. therefore, here, we used a cre/loxp switching system to generate an immunocompetent mouse model of hcv protein expression; with this system, we could study the host immune responses against hcv proteins. folgori et al. (2006) reported effective vaccination of chimpanzees with an adenoviral vector and plasmid dna encoding the hcv nonstructural region. this technique protected the liver tissues from acute hepatitis, which results when whole animals are challenged with virus [7] . however, this vaccine has not yet been shown to be effective against chronic hcv infection. here, we aimed to address how hcv expression causes chronic liver diseases and to provide new options for hcv vaccine development. using lc16m8, a highly attenuated strain of vaccinia virus (vv), we generated three recombinant vaccinia viruses (rvvs) that each encoded one of three different hcv proteins and found that one recombinant virus (rvv-n25), which encoded nonstructural hcv proteins, resolved pathological chronic hepatitis c symptoms in the liver. we also found that immunization with rvv-n25 suppressed hcv core protein levels in the livers of transgenic mice; moreover, this suppression was mediated by cd4 and cd8 t cells, as has been previously reported [8] . to produce adult mice that express an hcv transgene, we bred cn2-29 transgenic mice, which carry an hcv transgene, [5, 6, 9] with mx1-cre transgenic mice [10] , which express cre recombinase in response to interferon (ifn)-a or a chemical inducer of ifn-a, poly(i:c) ( figure 1a ). following poly(i:c) injection, the hcv transgene was rearranged, and hcv sequences were expressed in the livers of f1 progeny (cn2-29 (+/2) /mxcre (+/2) mice) within 7 days after poly(i:c) injection ( figure 1b) . to evaluate the characteristic features of these cn2-29 (+/2) / mxcre (+/2) mice, we analyzed serum alanine aminotransferase (alt) and liver hcv core protein levels after poly(i:c) injection. as illustrated in figure 1c , serum alt levels increased and reached a peak at 24 h after the first poly(i:c) injection; this elevation appeared to be a direct result of the poly(i:c) treatment, which causes liver injury [11] . after this peak, serum alt levels dropped continuously until day 4, and then alt levels began to increase, as did hcv core protein levels. thereafter, the hcv core protein was expressed consistently for at least 600 days. histological analysis showed hcv core protein expression in most hepatocytes of the transgenic mice; these mice showed evidence of lymphocytic infiltration that was caused by the hcv core proteins ( figure 1d and e). these observations, in addition to the modified histology activity index (hai) scores, indicated that expression of hcv proteins caused chronic hepatitis in the cn2-29 (+/2) /mxcre (+/2) mice because a weak, though persistent, immune response followed an initial bout of acute hepatitis ( figure s1 ). moreover, we observed a number of other pathological changes in these mice -including swelling of hepatocytes, abnormal architecture of liver-cell cords, abnormal accumulation of glycogen, steatosis, fibrosis, and hcc (figures 1e and f, table s1 ). steatosis was mild in the younger mice (day 21) and became increasingly severe over time (days 120 and 180; figure s2 ). importantly, none of the pathological changes were observed in the cn2-29 (+/2) /mxcre (2/2) mice after poly(i:c) injection ( figure 1f ). to determine whether activation of the host immune response caused the reduction with hcv protein levels in the livers of cn2-29 (+/2) /mxcre (+/2) mice, we used a highly attenuated vv strain, lc16m8, to generate three rvvs [12] . each rvv encoded a different hcv protein; rvv-cn2 encoded mainly structural proteins, rvv-n25 encoded nonstructural proteins, and rvv-cn5 encoded the entire hcv protein region (figure 2a ). because rvvs can express a variety of proteins and induce strong and long-term immunity, they have been evaluated as potential prophylactic vaccines [13] . we used western blots to confirm that each hcv protein was expressed in cell lines. each of seven proteins -the core, e1, e2, ns3-4a, ns4b, ns5a, and ns5b -was recognized and labeled by a separate cognate antibody directed ( figure s3 ). to induce effective immune responses against hcv proteins in transgenic mice, we injected an rvv-hcv (rvv-cn2, rvv-cn5, or rvv-n25) or lc16m8 (as the control) intradermally into cn2-29 (+/2) / mxcre (+/2) mice 90 days after poly(i:c) injection ( figure 2b ). analysis of liver sections 7 days after immunization with rvv-n25 revealed dramatic improvement in a variety of pathological findings associated with chronic hepatitis -including piecemeal necrosis, hepatocyte swelling, abnormal architecture of liver-cell cords, abnormal accumulation of glycogen, and steatosis ( figures 2c-e) . collectively, these results demonstrated that only the rvv-n25 treatment resulted in histological changes indicative of improvement in the chronic hepatitis suffered by the transgenic mice. to determine whether rvv-n25 treatment induced the same effect in other strains of hcv transgenic mice, we analyzed rzcn5-15 (+/2) /mxcre (+/2) mice, which express all hcv proteins; in these mice, chronic hepatitis was resolved within 28 days of immunization with rvv-n25. taken together, these findings indicated that rvv-n25 had a dramatic therapeutic effect on both types of hcv transgenic mice ( figure s4 ). to assess in detail the effects of rvv-hcv immunization on hcv protein clearance from the livers of cn2-29 (+/2) /mxcre (+/ 2) mice, we monitored the levels of hcv core protein in liver samples via elisa. we found that within 28 days after immunization the hcv core protein levels were significantly lower in livers of rvv-n25-treated mice than in those of control mice ( figure 3a ). immunohistochemical analysis indicated that, within 28 days after immunization, levels of hcv core protein were substantially lower in the livers of cn2-29 (+/2) /mxcre (+/2) mice than in those of control mice ( figure 3b ). importantly, neither resolution of chronic hepatitis nor reduction in the hcv protein levels was observed in the mice treated with lc16m8, rvv-cn2, or rvv-cn5. these results indicated that hcv nonstructural proteins might be important for effects of therapeutic vaccines. in contrast, rvv-cn5 which encoded hcv structural and non-structural proteins did not show any significant effects. these results indicated that hcv structural proteins might have inhibited the therapeutic effects of the non-structural proteins. therefore, it may be important to exclude the hcv structural proteins (aa 1-541) as antigenic proteins when developing therapeutic vaccines against chronic hepatitis c. in addition, we measured serum alt levels in cn2-29 (+/2) / mxcre (+/2) mice from all four treatment groups 28 days after rvv-hcv immunization. serum alt levels were not significant(+/2) and the cremediated activation of the transgene unit. r6cn2 hcv cdna was cloned downstream of the cag promoter, neomycin-resistant gene (neo), and poly a (pa) signal flanked by two loxp sequences. this cdna contains the core, e1, e2, and ns2 regions. (b) cre-mediated genomic dna recombination. after poly(i:c) injection, genomic dna was extracted from liver tissues and analyzed by quantitative rtd-pcr for cre-mediated transgenic recombination. the transgene was almost fully recombined in transgenic mouse livers 7 days after the injection. in all cases, n = 3 mice per group. (c) hcv core protein expression was sustained for at least 600 days after poly(i:c) injection. (d) immunohistochemical analysis revealed that most hepatocytes expressed the hcv core protein within 6 days after injection. (e) liver sections from cn2-29 (+/2) /mxcre (+/2) mice after the poly(i:c) injection. infiltrating lymphocytes (arrows) were observed on days 6 and 180; hepatocellular carcinoma (hcc) was observed on day 360. in contrast, these pathological changes were not observed in cn2-29 (+/2) /mxcre (2/2) mice after the injection. the inset image shows abnormal mitosis in a tumor cell. (f) hepatocyte swelling and abnormal architecture of liver-cell cords (silver staining), as well as abnormal glycogen accumulation (pas staining) were observed on day 90 in cn2-29 (+/2) /mxcre (+/2) mice. we observed steatosis (oil-red-o staining) on day 180 and, subsequently, fibrosis (azan staining) on day 480. the scale bars indicate 50 mm. doi:10.1371/journal.pone.0051656.g001 ly different in the rvv-n25-treated mice and control mice ( figure 3c ); this finding indicated that rvv-n25 treatment did not cause liver injury and that the antiviral effect was independent of hepatocyte destruction. we hypothesized that the reduction in the levels of hcv core protein in rvv-hcv-treated mice was not caused by cytolytic elimination of hepatocytes that expressed hcv proteins. to investigate this hypothesis, we conducted an rtd-pcr analysis of genomic dna from liver samples of cn2-29 (+/2) /mxcre (+/2) mice. the recombined transgene was similar in rvv-n25-treated and control mice 28 days after immunization ( figure 3d ). we also measured the expression of hcv mrna in lc16m8-treated cn2-29 (+/2) /mxcre (+/2) mice with that in rvv-n25-treated cn2-29 (+/2) /mxcre (+/2) mice 28 days after immunization; the hcv mrna levels did not differ between rvv-n25-treated cn2-29 (+/2) /mxcre (+/2) and control mice ( figure 3e ). these results indicated that rvv-n25-induced suppression of hcv core protein expression could be controlled at a posttranscriptional level. viral clearance is usually associated with cd4 and cd8 t-cell activity that is regulated by cytolytic or noncytolytic antiviral mechanism [14] . to determine whether cd4 or cd8 t-cell activity was required for the reduction in hcv core protein levels in the livers of transgenic mice, we analyzed the core protein levels in cn2-29 (+/2) /mxcre (+/2) mice immunized with rvv-n25 in the absence of cd4 or cd8 t cells ( figure 4a ). as expected, the mice lacking cd4 or cd8 t cells failed to show a reduction in hcv core protein levels ( figure 4b ). however, in mice lacking either cd4 or cd8 t-cells, the pathological changes associated with chronic hepatitis were resolved following rvv-n25 immunization, and the steatosis score of rvv-n25-treated mice was significantly lower than that of control mice (figures 4c-e) . these results indicated that cd4 and cd8 t cells were not responsible for the rvv-n25-induced amelioration of histological findings and that other inflammatory cell types may play an as-yet-unidentified role in the resolution of the pathological changes in these mice. because we found that hcv protein reduction in the liver required cd8 t cells, we tested whether hcv-specific cd8 t cells were present in splenocytes 28 days after immunization. to determine the functional reactivity of hcv-specific cd8 + t cells, we performed a cd107a mobilization assay and intracellular ifnc staining. cn2-29 transgenic mice expressed the hcv structural protein and the ns2 region. however, rvv-n25 comprised only isolated splenocytes from immunized mice were co-cultured with el-4cn2 or el-4ns2 cell lines for 2 weeks and analyzed. cytolytic cell activation can be measured using cd107a, a marker of degranulation [15] . the ratio of cd8 + cd107a + cells to all cd8 t cells significantly increased in rvv-n25-treated splenocytes after co-culture with el-4cn2 or el-4ns2 (p,0.05), whereas splenocytes that had been treated with any other rvv were not detected ( figure 5a, b and c) . these results indicated that rvv-n25 treatment increased the frequency of hcv ns2specific activated cd8 t cells. consistent with these results, the ratio of cd8 + ifn-c + cells to all cd8 t cells for rvv-n25-treated mice was also significantly higher than that for mice treated with any other rvv (p,0.05). taken together, these findings indicated that rvv-n25 induced an effective cd8 t-cell immune response and that ns2 is an important epitope for cd8 t cells. to determine whether rvv-n25 treatment affected inflammatory cytokine production, we measured serum levels of inflammatory cytokines after rvv immunization. the serum levels of these inflammatory cytokines increased in the cn2-29 (+/2) / mxcre (+/2) mice ( figure 6a, figure s5 ). immunization with rvv-n25 affected serum levels of inflammatory cytokines in cn2-29 (+/ 2) /mxcre (+/2) mice and caused a return to the cytokine levels observed in wild-type untreated mice ( figure 6a ). in wild-type mice, the cytokine levels remained unchanged after immunization ( figure 6a ). these results indicated that inflammatory cytokines were responsible for liver pathogenesis in the transgenic mice. to test the hypothesis that inflammatory cytokines were responsible for liver pathogenesis in cn2-29 (+/2) /mxcre (+/2) mice, we administered transgenic mouse serum intravenously into nontransgenic mice. we observed the development of chronic hepatitis in the nontransgenic mice within 7 days after the serum transfer ( figures 6b and c) . this finding was consistent with the 28 days after immunization with lc16m8 or rvv-n25. the scale bars indicate 100 mm (c) and 50 mm (d). (e) histological evaluation of steatosis in liver samples from cd4-depleted or cd8-depleted cn2-29 (+/2) /mxcre (+/2) mice 28 days after immunization with lc16m8 or rvv-n25. significant relationships are indicated by a p-value. doi:10.1371/journal.pone.0051656.g004 hypothesis that inflammatory mediators played a key role in inducing hepatitis. furthermore, to investigate whether tnf-a and il-6 played particularly critical roles in the pathogenesis of chronic hepatitis in the transgenic mice, we neutralized tnf-a and blocked the il-6 receptor in the livers of these mice. as expected, chronic hepatitis did not develop in these mice. (figure 6d and e) . next, to determine which cell population(s) produced tnf-a, il-6, or both during continuous hcv expression in cn2-29 (+/2) / mxcre (+/2) mice, we isolated intrahepatic lymphocytes (ihls) and labeled the macrophages (the f4/80 + cells) with anti-tnf-a and anti-il-6 antibodies using an intracellular cytokine detection method. macrophages in cn2-29 (+/2) /mxcre (2/2) mice produced small amounts of tnf-a and il-6, while those in cn2-29 (+/2) /mxcre (+/2) mice produced much larger amounts of these cytokines ( figure 6f ). finally, we evaluated whether rvv-n25 treatment affected the number of macrophages, cytokine production by macrophages, or both; specifically, we isolated ihls from cn2-29 (+/2) /mxcre (+/2) mice 7 days after immunization with rvv-n25 or with lc16m8. the percentage of macrophages (cd11b + f4/80 + ) among ihls and il-6 production from these macrophages were significantly lower in rvv-n25-treated mice than in control mice ( figure 6g ). though the percentage of tnf-a-producing macrophages was not significantly different in rvv-n25-treated and control mice (p = 0.099), rvv-n25 treatment appeared to suppress these macrophages. these results demonstrated that rvv-n25 had a suppressive effect on activated macrophages, and they indicated that this suppression ameliorated the histological indicators of chronic hepatitis. various hcv transgenic mouse models have been developed and used to examine immune response to hcv expression and the effects of pathogenic hcv protein on hepatocytes [4, 16, 17] . however, these transgenic mice develop tolerance to the hcv protein; therefore, examining immune response to hcv protein has been difficult. to overcome the problem of immune tolerance in mouse models of hcv expression, we developed an hcv model in mice that relies on conditional expression of the core, e1, e2, and ns2 proteins and the cre/loxp switching system [5, 6] ; we showed that the injection of an ad-cre vector enhanced the frequency of hcv-specific activated cd8 t cells in the liver of these mice and caused liver injury. however, the ad-cre adenovirus vector alone causes acute hepatitis in wild-type mice. nevertheless, the transgenic model was useful for evaluating interactions between the host immune system and viral protein (serum alt level over 2,000 iu/l) [5] ; hcv core protein levels were reduced and expression of this protein was transient (about 2 weeks). therefore, this ad-cre-dependent model cannot be used to effectively investigate immune responses to chronic hcv hepatitis. here, we used poly (i:c)-induced expression of cre recombinase to generate hcv transgenic mice in order to study the effect of hcv protein and confirmed that these mice developed chronic active hepatitis-including steatosis, lipid deposition, and hepatocellular carcinoma. these pathological findings in the transgenic mice were very similar to those in humans with chronic hepatitis c; therefore, this mouse model of hcv may be useful for analyzing the immune response to chronic hepatitis. however, experimental results obtained with this mouse model may not directly translate to clinical findings from patients with hcv infection because the expression of hcv proteins was not liver specific in these mice. furthermore, poly(i:c) injection can activate innate immune responses and, consequently, might induce temporary liver injury [18] . additionally, poly(i:c) injection has an adjuvant effect; specifically, it stimulates tlr3 signaling [19] . to evaluate whether poly(i:c) injection caused hepatitis in cn2-29 (+/2) /mxcre (2/2) mice, we examined serum alt levels and liver histology following poly(i:c) injection. we found that, following poly(i:c) injection, serum alt levels in cn2-29 (+/2) / mxcre (2/2) mice increased, reached a peak one day after injection, declined from day 1 to day 6, and were not elevated thereafter; this time-course indicated that poly(i:c) injection alone did not induced continuous liver injury ( figure s6 ). based on these findings, we believe that the effects of poly(i:c) injection in these mice did not confound our analysis of chronic hepatitis. immunization with rvv-n25 suppressed hcv protein levels in the liver, and this suppression was associated with ameliorated pathological chronic hepatitis findings (see figure 3 ). importantly, rvv-n25 treatment did not cause liver injury based on the serum alt levels; therefore, this treatment was unlikely to have cytopathic effects on infected hepatocytes. these findings provided strong evidence that rvv-n25 treatment effectively halted the progression of chronic hepatitis. immunization with plasmid dna or with recombinant vaccinia virus can effectively induce cellular and humoral immune responses and exert a protective effect against challenge with hcv infection [20, 21] . however, findings from these previous studies revealed hcv immunization of both uninfected, naïve animals and immune-tolerant animals induced a hcv-specific immune response. in the model describe here; the animals were immune competent for hcv; therefore, our findings provided further important evidence that rvv-n25 was effective in the treatment of chronic hepatitis. in addition, we demonstrated that rvv-n25 treatment in the absence of cd4 and cd8 t cells had no effect on hcv clearance. this important observation indicated that rvv-n25-induced hcv clearance was mediated by cd4 and cd8 t cells. many studies have shown that spontaneous viral clearance during acute hcv infection is characterized by a vigorous, broadly reactive cd4 and cd8 t-cell response. [8, 22] hcv clearance and hepatocellular cytotoxicity are both mediated by cd8 antigenspecific (cytotoxic t lymphocyte) ctls [23] . consistent with these observations, rvv-n25 treatment effectively induced the accumulation of ns2-specific cd8 t cells, which express high levels of figure 6 . immunization with rvv-n25 suppresses serum inflammatory cytokine levels. (a) daily cytokine levels in the serum of cn2-29 (+/ 2) /mxcre (+/2) mice during the week following immunization with lc16m8, rvv-cn2, rvv-n25, or rvv-cn5. values represent means 6 sd (n = 3) and reflect the concentrations relative to those measured on day 0. the broken lines indicate the baseline data from wild-type mice. in all cases, n = 6 mice per group. (b) liver sections from cn2-29 (+/2) /mxcre (+/2) and cn2-29 (+/2) /mxcre (2/2) mice. (c) histology activity index (hai) scores of liver samples taken from cn2-29 (+/2) /mxcre (+/2), or cn2-29 (+/2) /mxcre (2/2) mice. (d) liver sections from cn2-29 (+/2) /mxcre (+/2) mice in which tnf-a was neutralized and the il-6 receptor was blocked. the scale bars indicate 50 mm. (e) hai scores of liver samples taken from cn2-29 (+/2) /mxcre (+/2) in which tnf-a was neutralized and the il-6 receptor was blocked. tg and non-tg indicate cn2-29 (+/2) /mxcre (+/2) and cn2-29 (+/2) /mxcre (2/2) , respectively. (f) macrophages were the main producers of tnf-a and il-6 in cn2-29 (+/2) /mxcre (+/2) mice following poly(i:c) injection. (g) immunization with rvv-n25 reduced the number of macrophages in liver samples from cn2-29 (+/2) /mxcre (+/2) mice and suppressed tnf-a and il-6 production from macrophages ( figure 6g ). significant relationships are indicated by a p-value. doi:10.1371/journal.pone.0051656.g006 cd107a and ifn-cin the spleen. notably, even with rvv-n25 immunization, the frequency of activated cd8 t cells was very low, and a minimum of 2-weeks incubation was required to distinguish the difference between rvv treatments. even if a small population of specific cd8+ t cells played a relevant role in the reduction of core protein, it is difficult to assert that the only ns2specific cd8+ t cells were important to this reduction. however, based on the results presented in figure 4b , we are able to conclude that at least cd8+ and/or cd4+ t cells were important to the reduction in hcv core protein. therefore, to elucidate the mechanism of hcv protein clearance, further investigation of not only the other t cell epitopes but also other immunocompetent cells is required. interestingly, rvv-n25 treatment-but not the rvv-cn2 or rvv-cn5 treatment-efficiently induced a hcv-specific activated cd8 t cells response; this difference in efficacy could have one or more possible causes. the hcv structural proteins (core, e1, and e2 proteins) in the rvv-cn construct may cause the difference; saito et al. reported that injection with plasmid constructs encoding the core protein induced a specific ctl response in balb/c mice [24] . reportedly, ctl activity against core or envelope protein is completely absent from transgenic mice immunized with a plasmid encoding the hcv structural proteins, but core-specific ctl activity is present in transgenic mice that were immunized with a plasmid encoding the hcv core [21] . in contrast, when recombinant vaccinia virus expressing different regions of the hcv polyprotein were injected into balb/c mice, only the hcv core protein markedly suppressed vaccinia-specific ctl responses [25] . thus, the hcv core protein may have an immunomodulatory function [26] . based on these reports and our results, we hypothesize that the causes underlying the effectiveness of rvv-n25 treatment were as follows: 1) this rvv construct included the core and envelope proteins and 2) the core protein had an immune-suppressive effect on ctl induction. therefore, we suggest that exclusion of the core and envelope antigen as immunogen is one important factor in hcv vaccine design. interestingly, immunization with rvv-n25 rapidly suppressed the inflammatory response; however, immunization with either of the other rvvs did not (see figure 6a ). this result indicated that rvv-n25 may modulate inflammation via innate immunity, as well as via acquired immunity. reportedly, toll-like receptor (tlr)-dependent recognition pathways play a role in the recognition of poxviruses [27] . tlr2 and tlr9 have also been implicated in the recognition of the vaccinia virus [28, 29] . these findings indicate that tlr on dendritic cells may modulate the immunosuppressive effect of rvv-n25 in our model of hcv infection; however, further examination of this hypothesis is required. the finding that pathological symptoms in the hcv transgenic mice were completely blocked by intravenous injection of tnf-a and il-6 neutralizing antibodies indicated that the progression of chronic hepatitis depended on inflammatory cytokines in serum, rather than the hcv protein levels in hepatocytes. lymphocytes, macrophages, hepatocytes, and adipocytes each produce tnf-a and il-6 [30, 31] , and hcv-infected patients have elevated levels of tnf-a and il-6 [32, 33] . both cytokines also contribute to the maintenance of hepatosteatosis in mice fed a high-fat diet [34] , and production of tnf-a and il-6 is elevated in obese mice due to the low grade inflammatory response that is caused by lipid accumulation [35] . these findings indicate that both cytokines are responsible for hcv-triggered hepatosteatosis, and anti-cytokine neutralization is a potential treatment for chronic hepatitis if antiviral therapy is not successful. the reduction of macrophages in number might be due to the induction of apoptosis by vaccinia virus in vitro infection as previous reported [36] . to understand the mechanisms responsible for the reduction of the number of macrophage, we performed another experiment to confirm whether the macrophages were infected with vaccinia virus inoculation. however, based on pcr analyses; vaccinia virus dna was not present in liver tissue that contained macrophages ( figure s7 ). furthermore, apoptosis of macrophages was not detected in liver samples (data not shown). based on these results, it is unlikely that the reduction in the number of macrophages was due to apoptosis induced by vaccinia virus infection. although rvv-n25 reduced the number of macrophage, precise mechanism is still unknown. further examination to elucidate the mechanism is required. in conclusion, our findings demonstrated that rvv-n25 is a promising candidate for an hcv vaccine therapy. additionally, the findings of this study indicate that rvv-n25 immunization can be used for prevention of hcv infection and as an antiviral therapy against ongoing hcv infection. r6cn2 hcv cdna (nt 294-3435) [37] and full genomic hcv cdna (nt 1-9611) [38, 39] were cloned from a blood sample taken from a patient (#r6) with chronic active hepatitis (text s1). the infectious titer of this blood sample has been previously reported [40] . r6cn2hcv and r6cn5hcv transgenic mice were bred with mx1-cre transgenic mice (purchased from jackson laboratory) to produce r6cn2hcv-mxcre and r6cn5hcv-mxcre transgenic mice, which were designated cn2-29 (+/2) /mxcre (+/2) and rzcn5-15 (+/2) /mxcre (+/2) mice, respectively. cre expression in the livers of these mice was induced by intraperitoneal injection of polyinosinic acid-polycytidylic acid [poly(i:c)] (ge healthcare uk ltd., buckinghamshire, england); 300 ml of a poly(i:c) solution (1 mg/ml in phosphate-buffered saline [pbs]) was injected three times at 48-h intervals. all animal care and experimental procedures were performed according to the guidelines established by the tokyo metropolitan institute of medical science subcommittee on laboratory animal care. tissue samples were fixed in 4% paraformaldehyde in pbs, embedded in paraffin, sectioned (4-mm thickness), and stained with hematoxylin and eosin (h&e). staining with periodic acid-schiff stain, azan stain, silver, or oil-red-o was also performed to visualize glycogen degeneration, fibrillization, reticular fiber degeneration, or lipid degeneration, respectively. for immunohistochemical staining, unfixed frozen liver sections were fixed in 4% paraformaldehyde for 10 min and then incubated with blocking buffer (1% bovine serum albumin in pbs) for 30 min at room temperature. subsequently, the sections were incubated with biotinylated mouse anti-hcv core mono-clonal antibody (5e3) for 2 h at room temperature. after being washed with pbs, the sections were incubated with streptavidin-alexa fluor 488 (invitrogen). the nuclei were stained with 4',6diamidino-2-phenylindole (dapi). fluorescence was observed using a confocal laser microscope (laser scanning microscope 510, carl zeiss). the pbr322-based plasmid vector pbmsf7c contained the ati/p7.5 hybrid promoter within the hemagglutinin gene region of the vaccinia virus, which was reconstructed from the psfj1-10 plasmid and pbm vector [41, 42] . separate full-length cdnas encoding either the hcv structural protein, nonstructural protein, or all hcv proteins were cloned from hcv r6 strain (genotype 1b) rna by rt-pcr. each cdna was inserted into a separate pbmsf7c vector downstream of the pbmsf7c ati/p7.5 hybrid promoter; the final designation of each recombinant plasmid was pbmsf7c-cn2, pbmsf7c-n25, or pbmsf-cn5 (figure 2 ). they were then transfected into primary rabbit kidney cells infected with lc16m8 (multiplicity of infection = 10). the viruscell mixture was harvested 24 h after the initial transfection by scrapping; the mixture was then frozen at 280uc until use. the hemagglutinin-negative recombinant viruses were cloned as previously described [42] and named rvv-cn2, rvv-n25, or rvv-cn5. insertion of the hcv protein genes into the lc16m8 genome was confirmed by direct pcr, and expression of each protein from the recombinant viruses was confirmed by western blot analysis. the titers of rvv-cn2, rvv-n25, and rvv-cn5 were determined using a standard plaque assay and rk13 cells. data are shown as mean 6 sd. data were analyzed using the nonparametric mann-whitney or kruskal-wallis tests or an-ova as appropriate; graphpad prism 5 for macintosh (graph-pad) was used for all analyses. p values ,0.05 were considered statistically significant. table s1 incidence of hepatocellular carcinoma in male and female transgenic mice at 360, 480, and 600 days after poly(i:c) injection. text s1 supporting information including material and methods, and references. (docx) hepatitis c virus infection epidemiology of hepatitis c in the west transgenic expression of hepatitis c virus structural proteins in the mouse the core protein of hepatitis c virus induces hepatocellular carcinoma in transgenic mice possible role of cytotoxic t cells in acute liver injury in hepatitis c virus cdna transgenic mice mediated by cre/loxp system efficient conditional transgene expression in hepatitis c virus cdna transgenic mice mediated by the cre/loxp system a t-cell hcv vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees hepatitis b virus immunopathology inhibition of cytochrome c release in fas-mediated signaling pathway in transgenic mice induced to express hepatitis c viral proteins inducible gene targeting in mice distinct poly(i-c) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes characteristics of an attenuated vaccinia virus strain, lc16m0, and its recombinant virus vaccines evidence for protection against chronic hepatitis c virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus viral clearance without destruction of infected cells during acute hbv infection a novel flow cytometric assay for evaluating cell-mediated cytotoxicity hepatitis c virus core and e2 protein expression in transgenic mice steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis c virus immunoprivileged status of the liver is controlled by toll-like receptor 3 signaling ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer immunization with hepatitis c virus-like particles results in control of hepatitis c virus infection in chimpanzees genetic immunization of wild-type and hepatitis c virus transgenic mice reveals a hierarchy of cellular immune response and tolerance induction against hepatitis c virus structural proteins the liver as a lymphoid organ unscrambling hepatitis c virus-host interactions plasmid dna-based immunization for hepatitis c virus structural proteins: immune responses in mice suppression of host immune response by the core protein of hepatitis c virus: possible implications for hepatitis c virus persistence flying under the radar: the immunobiology of hepatitis c a46r and a52r from vaccinia virus are antagonists of host il-1 and toll-like receptor signaling innate immunity against vaccinia virus is mediated by tlr2 and requires tlr-independent production of ifnbeta survival of lethal poxvirus infection in mice depends on tlr9, and therapeutic vaccination provides protection hepatitis c virus infection: molecular pathways to metabolic syndrome gut, inflammation and osteoporosis: basic and clinical concepts serum interleukin 6 concentrations in chronic hepatitis c patients before and after interferon-alpha treatment tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis c dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing il-6 and tnf expression inflammatory mechanisms in obesity vaccinia virus induces apoptosis of infected macrophages isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome activation of the cki-cdk-rb-e2f pathway in full genome hepatitis c virusexpressing cells hepatitis c virus impairs p53 via persistent overexpression of 3beta-hydroxysterol delta24-reductase correlation between the infectivity of hepatitis c virus in vivo and its infectivity in vitro prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov sars-cov spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus we thank dr. fukashi murai for supporting this study. we also thank dr. keiji tanaka for providing the mxcre mice, dr. shigeo koyasu for providing the gk1.5 (anti-cd4) and 53-6.72 (anti-cd8) monoclonal antibodies, and dr takashi tokuhisa for helpful discussions. key: cord-011182-nbtfb39r authors: dehghani, behzad; hashempour, tayebeh; hasanshahi, zahra; moayedi, javad title: bioinformatics analysis of domain 1 of hcv-core protein: iran date: 2019-04-20 journal: int j pept res ther doi: 10.1007/s10989-019-09838-y sha: doc_id: 11182 cord_uid: nbtfb39r hepatitis c virus (hcv) infection is a serious global health problem and a cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (hcc). bioinformatics software has been an effective tool to study the hcv genome as well as core domains. our research was based on employing several bioinformatics software applications to find important mutations in domain 1 of core protein in iranian hcv infected samples from 2006 to 2017, and an investigation of general properties, b-cell and t-cell epitopes, modification sites, and structure of domain 1. domain 1 sequences of 188 hcv samples isolated from 2006 to 2017, iran, were retrieved from ncbi gene bank. using several tools, all sequences were analyzed for determination of mutations, physicochemical analysis, b-cell epitopes prediction, t-cell and ctl epitopes prediction, post modification, secondary and tertiary structure prediction. our analysis determined several mutations in some special positions (70, 90, 91, and 110) that are associated with hcc and hepatocarcinogenesis, efficacy of triple therapy and sustained virological response, and interaction between core and ccr6. several b-cell, t-cell, and ctl epitopes were recognized. secondary and tertiary structures were mapped fordomain1 and core proteins. our study, as a first report, offered inclusive data about frequent mutation in hcv-core gene domain 1 in iranian sequences that can provide helpful analysis on structure and function of domain 1 of the core gene. hcv infection is a serious global health problem and causes chronic hepatitis, liver cirrhosis, and hcc (akuta et al. 2007; ajorloo et al. 2015; alborzi et al. 2017 alborzi et al. , 2015 moayedi et al. 2018; hashempoor et al. 2018) . it is estimated that 160 million people are infected worldwide (lavanchy 2011) . the prevalence rate of hcv infection is from 0.2 up to 40% in different countries, and this prevalence in iran is 0.16% (sefidi et al. 2013) . lack of an effective vaccine and therapeutic choices has leaded to the rapid growth of hcv infection (lauer and walker 2001) . hcv has six large different genotypes (1-6) and 70 distinct subtypes (a, b, c, etc.) globally (martro et al. 2011) . genotyping analysis showed 30-33% difference in each genotype, and in subtypes around 20-25% (sefidi et al. 2013) . according to the current investigations in iran, the predominant hcv subtype is 1a, followed by 3a and 1b (sefidi et al. 2013) . hcv is a positive-strand rna virus encoding three structural components, the core protein, and two e1 and e2 envelope glycoproteins (ajorloo et al. 2015) . the core protein has many confirmed roles: core binds rna and dna and has an important function in rna packing. it has been determined that hcv-core is a nucleic acid chaperone similar to retroviral nucleocapsid (nc) proteins in act acting to rearrange hcv 3′utr, resulting in rna dimerization in vitro (caval et al. 2011; cristofari gl; ivanyi-nagy et al. 2004; steinmann et al. 2008) . hcv-core is a highly basic protein that forms the viral nc and has interactions with cellular proteins and signal transduction pathways. as a result of hcv-core and host cell interactions, core may have a function in persistent infection and the pathogenesis of hcv liver disease (polyak et al. 2006; hashempour et al. 2015) . core protein consisted of three predicted domains: aa1-aa117 domain 1 (domain d1), aa117-aa177 domain 2 (domain d2), and 177-191 domain 3 (domain d3) (strosberg et al. 2010) . domain 1 contains frequent positively charged amino acids, and is involved in rna binding, promotes dimerization of the viral rna, and has a significant role in nc formation and core envelopment by endosomal membranes (ivanyi-nagy et al. 2006) . several identified mutations in domain 1 are involved in the development of hcc and hepatocarcinogenesis, the efficacy of triple therapy, and interaction between core and cxcl6 (akuta et al. 2007 (akuta et al. , 2010 (akuta et al. , 2011 fishman et al. 2009; ogata et al. 2002; takahashi et al. 2001; idrees and ashfaq 2013) . humoral and cellular immune responses against hcv infections are inefficient and there is no convincing explanation to understand hcv immune pathogenesis (gremion and cerny 2005) . however, hcv-specific igm and igg together were detected in acute infection. there are some outstanding proofs supporting a role for abs in control of hcv infection and especially in reinfection (cashman et al. 2014 ). some researchers have described a rise in anti-hcv humeral immune after immunization with core protein (aghasadeghi et al. 2006) . other researchers have claimed that in acute hcv infection the titer of antibodies is very low, and delay in neutralizing antibody production is responsible for ineffective ability to prevent hcv infection (netski et al. 2005) . other sources have introduced a large diversity of epitopes in hcv proteins as a possible way to escape the humoral response (pavio and lai 2003) . cellular immune responses have an important role in the clearance of hcv infection. patients with cellular immune dysfunction like human immunodeficiency virus (hiv) have rapid hcv progression. some researchers have claimed that this system promotes liver injury by cytolysis activity of infected cells. in spite of cellular immune responses, hcv often evades recognition and has the ability to persist (ward et al. 2002) . several studies have described a number of pathways for hcv to escape from cell responses, including impaired oligo-/mono-specific or no virus-specific cd4 + and cd8 + , mutation of epitopes, weakness of proliferative capacity, and cytotoxicity and ability to secrete tnf-α and ifn-γ by cd8 + t cells (neumann-haefelin et al. 2005) . in addition, regulatory t cells (tregs) induced by hcv infection have a significant role in the impaired activity of cellular immune response hashempoor et al. 2010) . bioinformatics tools are efficient means to study viruses and different parts of the hcv genome like core domains (idrees and ashfaq 2013; moattari et al. 2015; dehghani et al. 2017; nezafat et al. 2018; atapour et al. 2018; sarvari et al. 2014; behzad dehghani and zahra hasanshahi 2019) . bioinformatics tools are efficient means to study viruses and different parts of the hcv genome like core domains. many programs have been developed to analyze function, structures, and modification of core protein, providing a large amount of information about core domains and important mutation sites (akuta et al. 2007 (akuta et al. , 2010 (akuta et al. , 2011 . current data are useful for prediction of hcv disease development and treatment response. in this study, we employed several bioinformatics tools to find important mutations in domain 1 of the core protein, general properties of b-cell and t-cell epitopes, modification sites, and structure of domain 1 in iranian hcv infected samples from 2006 to 2017. domain 1 sequences of 188 iranian hcv samples and reference sequences of hcv genotypes that were registered in ncbi gene bank (http://www.ncbi.nlm.nih.gov/) from 2006 to 2017 were downloaded. homology among sequences was determined using multiple sequence alignment available in clc-sequence viewer software under the following parameters: gap open cost, 10; gap extension cost, 1.0; and very accurate progressive alignment algorithm. also, phylogenetic trees were analyzed through clustal x software, version 1.81, by neighbor-joining times to confirm the reliability of phylogenetic trees. the accession numbers of all sequences are displayed in table 1 . by considering previous studies, several significant mutations that are involved in: 1-hepatocellular carcinoma, 2-viral response to triple therapy, and 3-the interaction between core and cxcl6, were determined. all sequences were compared to find mentioned mutations (akuta et al. 2007; fishman et al. 2009; ogata et al. 2002) . general properties of domain 1 (genotype 1a) were determined by employing "expasy'sprotparam" (http://expas y.org/tools /protp aram.html) and protscale at (http://web. expas y.org/prots cale/) (gasteiger et al. 2005 ). chou and fasman, karplus and schulz, kolaskar & tongaonkar, emini, parker, and bepipred methods at http:// www.immun eepit ope.org (http://tools .immun eepit ope.org/ tools /bcell /iedb_input ) were run for prediction of b-cell epitopes positions (chou and fasman 2009; karplus and 1 3 schulz 1985; emini et al. 1985; parker et al. 1986; larsen et al. 2006 ). on hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface and turns features by bcepred (http://www.imtec h.res.in/ragha va/bcepr ed) b-cell epitopes prediction were performed (saha and raghava 2004) . abcpred software (http://www.imtec h.res.in/ragha va/ abcpr ed/) predicted 16 meric b-cell epitopes (saha and raghava 2006a, b) . propred-i (http://www.imtec h.res.in/ragha va/propr ed1/) (singh and raghava 2003) was employed for mhc class-i binding peptide prediction and proposed (http://www. imtec h.res.in/ragha va/propr ed/) was used for mhc class-ii binding peptide prediction. programs were worked at a 4% default threshold by the proteasome and immunoproteasome filters on at 5% threshold (singh and raghava 2001) . mhc class i and ii predictions were determined using the immune epitope database (iedb) (http://tools .immun eepit ope.org/main/). for prediction of ctl epitopes, "ctlpred" and ann methods were used (48). probability of antigenicity was expected by vaxijen software at http://www.ddg-pharm fac.net (doytchinova and flower 2007) . ige epitopes and allergic properties were estimated at http://www.imtec h.res.in/ragha va/algpr ed/index .html by using algpred (saha and raghava 2006) . serine, threonine, and tyrosine phosphorylation sites prediction was done using disphos (http://www.dabi.templ e.edu/disph os/pred.html) (iakoucheva et al. 2004 ) and net-phos (http://www.cbs.dtu.dk/servi ces/netph os/) (blom et al. 1999) . kinase specific phosphorylation sites were determined by netphosk (http://www.cbs.dtu.dk/servi ces/ netph osk/) (blom et al. 2004) . netnglyc (http://www.cbs. dtu.dk/servi ces/netng lyc/) (gupta and brunak 2002) and glycoep (http://www.imtec h.res.in/ragha va/glyco ep/submi t.html) were employed for n-glycosylation sites prediction (chauhan et al. 2013 ). to predict secondary and tertiary structures of core and domain 1 of genotype 1a, sopma at (http://npsa-pbil. ibcp.fr/cgi-bin/npsa_autom at.pl?page=npsa_sopma .html) (geourjon and deleage 1995) , i-tasser at (http://zhang lab.ccmb.med.umich .edu/i-tasse r) (roy et al. 2010) , phyre 2 server at (http://www.sbg.bio.ic.ac.uk/~phyre the signal peptide was predicted by "signal-blast", and "signalp 4.1 server". all data were collected anonymously in accordance with legal requirements regarding data protection and medical confidentiality. approval from the faculty human research ethics committee (shiraz university of medical sciences) was obtained before the commencement of the study. by considering all submitted sequences in ncbi genbank we could not find any sequences related to 2017. phylogenetic tree for all sequences was shown in fig. 1 . all 2006 sequences were placed in a cluster at the bottom of the tree, and a sequence of 2012 has a high similarity to kf218585.1 (2014) . the majority of sequences were closer to 1a and 3a than other reference sequences. all important mutation positions were listed in table 2 ; the majority of mutations happened in 2013 and 2016 samples. no mutation was detected in12, 23, 25, 39, 45 positions. protparam results for domain 1 are listed in table 3 . because of the high percentage of basic amino acids, domain 1 is a highly basic peptide (theoretical pi: 12). the instability index, an estimate of the stability of a protein in a test tube, showed that domain 1 is an unstable peptide. aliphatic index, a positive factor for the increase of thermostability of proteins, indicated that this peptide is a thermostable peptide. gravy is a hydropath city index and increasing phylogenetic tree based on domain1 sequences and by using neighbor joining method. the phylogenetic tree was constructed by the nj method. the numbers at the forks show the numbers of occurrences of the repetitive groups to the right out of 100 bootstrap samples. all used reference sequences were showed after accession numbers (1a, 1b, and etc.). sequences were categorized in five major clusters positive score indicates a greater hydrophobicity, so this peptide is a hydrophilic peptide. hydropathicity analysis by kyte j. and doolittle r.f. method showed that the major part of the peptide had a negative score; the maximum hydropath city score was on aa34 (valin) and the minimum hydropath city score was on aa 14 (asparagine). amino acids flexibility predicted by bhaskaran r ponnuswamy p.k method indicated that the maximum flexibility was around amino acid 58 (proline) and the minimum was around aa 95 (glycine). transmembrane (tm) tendency calculated by zhao, g., london e. method, showed that the major part of peptide had a negative score, and the maximum transmembrane tendency was on aa34 (valine) and the minimum was on aa10 (lysine). peptide polarity predicted by grantham r method showed that the maximum polarity was on amino acid 12 and 13 (lysine and arginine) and the minimum polarity was on aa 34 (valine). http://www.immun eepit ope.org online software: chou and fasman beta-turn prediction, which is based on the rationale for predicting turns to predict antibody epitopes, showed one high score region, 99-112. emini surface accessibility prediction, which is based on surface accessibility scale, showed two high score positions (4-20 and 49-61). karplus and schulz flexibility scale was used for b-cell prediction; this method is based on mobility of protein segments on the basis of the known temperature b factors of the a-carbons of 31 proteins of known structure. results demonstrated two positions with the highest score (50-60 and 5-12). kolaskar & tongaonkar antigenicity method is based on physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes to predict antigenic determinants on protein. results showed five positions of 20-39, 43-49, 63-69, 78-85, and 93-101. parker hydrophilicity prediction method is based on peptide retention times during high-performance liquid chromatography (hplc) on a reversed-phase column. by this method, two regions were found: 9-16, 51-57. linear b-cell epitopes were determined using bepipred. this method is based on a combination of a hidden markov model and a propensity scale method. three regions (1-25, 51-84, and 102-116) were founded by bepipred analysis. for a combination of all physicochemical properties (hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface, and turns) for linear b-cell epitope prediction based on physicochemical properties on a non-redundant dataset: using bcepred online software, three regions (4-22, 47-59, and 109-115) with the highest combined score were found. five 16 meric conserved regions (78, 49, 1, 64 and 102) were found by abcpred prediction server (table 4 ). according to a predefined cutoff of vaxijen program, domain 1 was confirmed as a probable antigen (model: virus and threshold: 0.4). the prediction of allergenic proteins by mapping of ige epitope, svm, and hybrid methods showed that domain 1 was not an allergen protein. regarding t-cell responses against hcv, previous researches found some hosts' human leukocyte antigen (hla) alleles associations with hcv infection in iranian patients. we found several epitopes of hla's shown in table 5 . some studies found both cd4 helper and cd8 ctl responses against hcv infection. ctlpred found several epitopes for ctl (table 6 ). prediction of serine, threonine, and tyrosine phosphorylation sites by disphos showed one position (116) in domain1. by "netphos" software we found 10 phosphorylation sites (fig. 2) , 6 sites for serine (53, 56, 99, 103, 106, and 116) 3 sites for threonine (15, 49, and 52) and one site for tyrosine (86). netphosk results determined four phosphorylation sites, three threonine amino acids (3, 15, and 49) for protein kinase c and one serine (116) for protein kinase a. no glycosylation site was found by netnglyc and glycoep. secondary structure prediction for core and domain1 by using sompa software was summarized in table 7 and fig. 3 . sompa showed there was no alpha helix structure in domain1 and the major part of it was the random coil. but the combination of (ps) 2 -v2 and phyre 2 showed there was an alpha helix structure in 8-15 region (figs. 4, 5) . all programs displayed extended strand in the 29-36 region. 3d structures were determined by all three online software but only structures predicted by i-tasser were fig. 2 phosphorylation sites prediction for domain 1 using "netphos" online software. green lines indicate 6 sites for serine, blues lines show 3 sites for threonine, and one purple line shows tyrosine. all sites with scores above the threshold of 0.5 were considered as phosphorylation sites reliable. final structures (figs. 6, 7) were validated by qmean. qmeanscore and z-score for calculated for core were 0.242 and − 5.43. the scores were not satisfactory but at least provided an overview of the core protein structure. qmeanscore and z-score for domain 1 were 0.61 and − 1.24 confirming the quality and reliability of the predicted structure. ramachandran plot was assessed by rampage, and percentages of the favoured region, allowed region, and outlier region for core were 63.0%, 27.5%, and 9.5% respectively . 3 secondary structure prediction using sompa. red region is extended strand, blue is the alpha helix, green is beta turn, and purple is the random coil. the majority of core structure belongs to random coil fig. 4 secondary structure prediction using phyre2. the result of this tool shows that the majority of the core structure (40%) is alpha helix which is indicated with green helix, also the confidence keys of the predicted structure for these regions are high (fig. 8) . rampage results for domain1 showed 61.7% of residues in favored region and 24.3 in allowed region (fig. 9 ). figure 10 showed the showed t cell and b-cell epitopes on the surface of the core protein. both online tools "signal-blast" and "signalp 4.1 server" were not able to predict any signal peptide for core protein. the results of "peptidecutter" prediction were summarized in table 8 . the prediction was done for all the predicted epitopes. according to the results, the antigenic epitopes that had the lower number of cleavage positions for enzymes were more potential for b cell or t cell epitopes. although emerging bacterial viral diseases have caused great catastrophes in human history which can affect from a small and localized group to millions of people across continents, several vaccine and therapies have been introduced to control them (dehghani et al. 2014 (dehghani et al. , 2013 . fishman et al. (2009) using multivariable logistic regression models ,found 10 hcv-core gene polymorphisms extensively associated with increased hcc risk (36g/c, 209a, 271c/u, 309a/c, 384u, 408u, 435a/c, 465u, 481a, 546a/c) and one significantly linked with decreased hcc risk (78u). mentioned mutations related to change in domain1 amino acid sequence: n11s/t, k12silent/n, a25v, g69s, q70r, m91l, and l102p. all current amino acid changes decreased hcc risk except a25v (78u) (fishman et al. 2009 ). in selected sequences, we found that amino acid 11 was t in all sequences except one sequence in 2013 (p) and in 1a (n). positions 12(k) did not show any change; amino acid 25 was p and amino acid 69 was r in all sequences. (l)). in amino acid 102 we did not find any change but in one of the reference sequences, we found one change (5a (g to s)). akuta et al. (2007) employed pcr for detecting substitutions of aa 70 and aa 91 in hcv-core gene of genotype 1b by using the mutation-specific primer as an important predictor of hepatocarcinogenesis. for wild samples, aa 70 was arginine (r) and aa 91 was leucine (l) but for mutant aa 70 was glutamine(q)/histidine (h) and aa 91 was methionine (m) (akuta et al. 2007) . also , furui et al. (2011) identified aa 70 and aa 91 substitutions among japanese volunteer blood donors (furui et al. 2011) . in terms of aa 70 substitutions, we recognized 2 glutamine substitutions in 2006 sequences and 3 glutamine, 2 histidines, one proline in 2013 sequences; also 4 glutamine substitutions in 2014 sequences, and 25 glutamine, and 1 fig. 8 ramachandran plot was used to visualize energetically allowed regions for backbone dihedral angles ψ against ϕ of amino acid residues in modeled protein structure (lcc model) for tertiary structure of core protein by rampage; the majority of amino acids residues were in favored region (119 amino acids) and allowed region (52 amino acids) 1 3 histidine in 2016. we found several methionine residues in aa 91 in , and 2016 . ogata et al. (2002 compared sequences of the core protein of subtype 1b hcv strains obtained from patients with and without hcc and found some amino acid mutation sites (ogata et al. 2002) . k23q, q70r, and t110m substitutions were found by ogata et al. (2002) . in comparison with our results, in all sequences, aa 23 was k, in the majority of sequences aa 70 was r and in 34 sequences it was q. in aa 110 we did not find any methionine (ogata et al. 2002) . akuta et al. (2010) confirmed the role of gln70 (or his70) in the efficacy of triple therapy and sustained a virological response, the patient with both genotype non-tt and gln70 (his70) had the worst sustained virological response. also, akuta et al. (2011) by following up twenty-six patients determined the role of gln70 (his70) substitution in the development of hcc they suggested detection of aa substitutions in the core region before antiviral therapy (akuta et al. 2010 (akuta et al. , 2011 . alestig et al. (2011) showed substitution in aa 70 of the core was related to treatment response, but that was less important than il28b polymorphism. in our study, 37 sequences had gln70 (or his70) substitution (alestig et al. 2011) . tokita et al. (2000) approved the role of hcv-core region (thr49pro) to reduce the fluorescence enzyme immunoassay fig. 9 ramachandran plot was used to visualize energetically allowed regions for backbone dihedral angles ψ against ϕ of amino acid residues in modeled protein structure (lcc model) for tertiary structure of domain1 by rampage; majority of amino acids residues were in favored region (71 amino acids) and allowed region (28 amino acids) (feia) sensitivity. we found 2 sequences in 2013 and one in 2016 with t to p substitutions (tokita et al. 2000) . findings of horie et al. (1999) indicated that alteration from glycine to serine at core codon 45 was dominant in noncancerous liver portions rather than in cancerous liver portions and sera from hcc patients (horie et al. 1999) . our results did not show any glycine to serine mutation in aa 45 and in all sequences, it was glycine. idrees and ashfaq (2013) by using molecular docking software, reported interactions of amino acid residues arginine 149, arginine 39, arginine 74, and arginine 78 in hcvcore protein and leu44, ala71, ser76 and pro97 in cxcl6. this finding clues to understanding hcv pathogenesis. any change in these positions can relate to hcv infection and hcc. our results showed no alteration in aa 39 and 74, and in aa 78 just one sequence was glutamine to arginine (idrees and ashfaq 2013) . using a combination of predicted b-cell antibody epitopes by all methods on the immune epitope website and also considering bcepred and abcpred prediction ,we could define three major epitopes (4-20, 50-60, and 100-112) for domain 1. ferroni et al. (1993) by using the algorithm of jameson and wolf identified four epitopes in hcv-core protein (7-21, 31-45, 49-63, and 99-113) . harase et al. (1995) analyzed the response to hcv-core protein in mice and found a major b cell epitope (21-40). pirisi et al. (1995) analyzed sera from 97 hcv infected patients and found three 15-mer peptides as antigens in an enzyme immunoassay. they concluded that anti-r15p (50-64, rktsersqprgrrqp) as a potent antigen might help to identify a subgroup at higher risk to develop hcc (pirisi et al. 1995) . also, a study on hcv positive blood donor by lechmann et al. (1996) determined a region (aa 1-24) of domain1 that bound the antibodies from the sera of all patients and showed a great potential for detection of hcv infection by using serological b-cell responses tests. comparison of our results with previous studies revealed that all predicted epitopes in our research have a good potential for future studies of the immune response against hcv infection, and are useful for recognition of all kinds of hcv infections. gededzha et al. (2014) used several bioinformatics tools to predict hla class i and hla class ii in hcv genotype 5a. they found three t-cell epitopes of ns3, ns4b, and ns5b. some hla class ii alleles were found in iranian patients by samimi-rad et al. (2015) ; drb1*0301, dqa1*0501, dqb1*0201, drb1*1101, and dqb1*0301 were demonstrated in patients with hcv clearance, and drb1*0701, dqa1*0201, dqb1*0602, drb1*0301, drb1*11, and dqb1*0201 occurred more frequently in chronic patients (samimi-rad et al. 2015) . khorrami et al. (2015) found a relationship between hla-g, il-10, and response to combined therapy in hcv positive patients. they concluded that hla-g, and il-10 have a significant role in response to therapy with ifn-α2α and ribavirin. also, hla-a01 and hla-b38 were determined as important alleles associated with peg-ifn plus ribavirin therapy in egyptian patients by farag et al. (2013) . pourhassan et al. (2014) found several hla alleles associated with hcv in iranian patients (2011) (2012) (2013) . a2, a3, b35, b38, bw4, cw4and cw7 were the most frequent alleles found by this group. fig. 10 a: the position of the b-cell epitopes (yellow region) on core tertiary structure and b: the t-cell epitopes (yellow region) on core 3d structure table 8 the results of predicted cleavage positions for 12 common proteases: b-cell, t-cell, and ctl predicted epitopes positions caspase chymotrypsin clostripain elastase pepsin proteinase k asp-n staphylococcal peptidase i thrombin proline trypsin b-cell epitopes 78-93 3(8,9,16) 5(4, 7, 9,15, 16) 6(4, 6, 8, 9, 12, 16) 1 (11) 1(12) 49-64 5 (2, 7, 11, 13, 14) 3(1, 4, 6) 1 (5) 1 (6) 6 (2, 3, 7, 11, 13, 14) 1_16 7 9 16) 12 (2, 3, 6, 7, 8, 9, 16, 18, 20, 21, 24, 26,) hla-cw*0401 78-93 3(8, 9 ,16) 5(4, 7, 9, 15, 16) 6(4, 6, 8, 9, 12, 16) 1(11) 1(12) hla-cw*0702 1(4) 4(6 7 9 10) in accordance with the above-mentioned studies, we collected hla alleles associated with hcv infection, and by employing in-silico analysis we established numerous t-cell epitopes for domain1 that can be helpful for future studies to design effective vaccine against hcv genotype 1a, and can provide benefit data for better understanding the role of domain1 in immune response. many researchers have proved broader ctl responses to hcv infection and the usefulness of ctl epitopes mapping to develop therapeutic interventions or vaccines (sabet et al. 2014; saeedi et al. 2014; jazayeri and carman 2005; arashkia et al. 2011) . in our research, we utilized reliable software to predict ctl epitopes and extracted data that can be useful for vaccine development studies. by considering results of phosphorylation sites prediction by 3 programs, we concluded that 3 sites (15, 49, and 116) were the main phosphorylation sites in domain 1. amino acid 116 is a serine that located in arg-arg-arg-ser-arg region; this region was similar to the usual target sequence for protein kinase a [arg-arg-x-ser/thr-x]. two threonine amino acid residues (15, 49) were calculated as protein kinase c target sites where this kinase acts through the phosphorylation of hydroxyl groups of amino acid residue. previous studies indicated that core protein is phosphorylated by pka and pkc. core phosphorylation regulates the suppressive activity of hcv-core protein on hbv gene replication and expression. also, it has been shown that phosphorylation in core relates to the nuclear localization of the core protein. they demonstrated three serine residues (ser-53, ser-116, and ser-99) as the potential phosphorylated sites in core protein, that were similar to netphos software results in our research (yassin 2001; shih et al. 1995; lu and ou 2002) . secondary structure prediction indicated that the majority of domain1 was the random coil, and all b-cell epitopes and important mutations placed on random coil structure. tertiary structures were designed by three significant and reliable online programs, but just one of them provided a reliable and high-quality protein structure model for domain1. the quality and reliability of models were confirmed by qmean and rampage software. by examining all the predictions core epitopes with and without signal peptide, we found out that there is no difference between these two different strategies of analysis (pene et al. 2009; targett-adams et al. 2008; ma et al. 2007; okamoto et al. 2008; oehler et al. 2012) . based on previous studies core protein has a c terminal signal peptide and because the focus of our study was on domain 1, it was expected that the deletion of this region could not affect the epitope perdition results. digestion analysis to predict possible proteases was shown that each epitope can be digested by at least 5 the position of each epitope, the number of cleave sites in each epitope as well as the position of the cleave sites in each epitope are mentioned in the table selected proteases which can have a significant effect on the reduction of the half-life of epitopes. finally our investigations in this research provided comprehensive data about frequent mutations in domain1, and as a first report can be useful for future study about significant mutations and their role in therapeutic pathway and response to antiviral therapy for iranian patients. also, identification of domain1 properties provides practical information for domain1 cloning and more researches. evaluation of a native preparation of hcv core protein (2-122) for potential applications in immunization, diagnosis and mab production detection of specific antibodies to hcv-arf/core + 1 protein in cirrhotic and non-cirrhotic patients with hepatitis c: a possible association with progressive fibrosis amino acid substitutions in the hepatitis c virus core region are the important predictor of hepatocarcinogenesis amino acid substitution in hepatitis c virus core region and genetic variation near the interleukin 28b gene predict viral response to telaprevir with peginterferon and ribavirin amino acid substitutions in hepatitis c virus core region predict hepatocarcinogenesis following eradication of hcv rna by antiviral therapy insights into the role of hcv plus-/minus strand rna, ifn-γ and il-29 in relapse outcome in patients infected with hcv role of serum level and genetic variation of il-28b in interferon responsiveness and advanced liver disease in chronic hepatitis c patients core mutations, il28b polymorphisms and response to peginterferon/ribavirin treatment in swedish patients with hepatitis c virus genotype 1 infection immunoinformatics modeling, construction of dna plasmids carrying ctl epitopes of hepatitis c virus and their preliminary immunological analysis designing a fusion protein vaccine against hcv: an in silico approach qmean: a comprehensive scoring function for model quality assessment prediction of ctl epitopes using qm, svm and ann techniques sequence and structurebased prediction of eukaryotic protein phosphorylation sites1 prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence the humoral immune response to hcv: understanding is key to vaccine development packaging of hcv-rna into lentiviral vector in silico platform for prediction of n-, o-and c-glycosites in eukaryotic protein sequences protein structure prediction server amino acid sequence the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand rna in vitro immunogenicity of salmonella enterica serovar enteritidis virulence protein, invh, and cross-reactivity of its antisera with salmonella strains immunoprotectivity of salmonella enterica serovar enteritidis virulence protein, invh, against salmonella typhi functional and structural characterization of ebola virus glycoprotein (1976-2015)-an in silico study vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide human leukocyte antigen class i alleles can predict response to pegylated interferon/ribavirin therapy in chronic hepatitis c egyptian patients identification of four epitopes in hepatitis c virus core protein mutations in the hepatitis c virus core gene are associated with advanced liver disease and hepatocellular carcinoma prevalence of amino acid mutation in hepatitis c virus core region among japanese volunteer blood donors protein identification and analysis tools on the expasy server prediction of t-cell epitopes of hepatitis c virus genotype 5a sopma: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments hepatitis c virus and the immune system: a concise review prediction of glycosylation across the human proteome and the correlation to protein function immune response to hepatitis c virus core protein in mice expansion of cd4+ cd25+ foxp3+ regulatory t cells in chronic hepatitis c virus infection a decline in anti-core + 1 antibody titer occurs in successful treatment of patients infected with hepatitis c virus f protein increases cd4+ cd25+ t cell population in patients with chronic hepatitis c mutations of the core gene sequence of hepatitis c virus isolated from liver tissues with hepatocellular carcinoma the importance of intrinsic disorder for protein phosphorylation hcv infection and ns-3 serine protease inhibitors analysis of hepatitis c virus rna dimerization and core-rna interactions virus escape ctl or b cell epitopes? prediction of chain flexibility in proteins protein structure prediction on the web: a case study using the phyre server the relationship between hla-g and viral loads in non-responder hcv-infected patients after combined therapy with ifn-α2α and ribavirin improved method for predicting linear b-cell epitopes hepatitis c virus infection evolving epidemiology of hepatitis c virus t-and b-cell responses to different hepatitis c virus antigens in patients with chronic hepatitis c infection and in healthy anti-hepatitis c virus-positive blood donors without viremi phosphorylation of hepatitis c virus core protein by protein kinase a and protein kinase c characterization of the cleavage of signal peptide at the c-terminus of hepatitis c virus core protein by signal peptide peptidase hepatitis c virus sequences from different patients confirm the existence and transmissibility of subtype 2q, a rare subtype circulating in the metropolitan area of silico functional and structural characterization of h1n1 influenza a viruses hemagglutinin comparison of il-28b favorable genotype frequency between healthy and patients infected with hcv humoral immune response in acute hepatitis c virus infection t cell response in hepatitis c virus infection exploring dengue proteome to design an effective epitope-based vaccine against dengue virus au-sabetian, soudabeh structural analysis of hepatitis c virus core-e1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase comparative sequence analysis of the core protein and its frameshift product, the f protein, of hepatitis c virus subtype 1b strains obtained from patients with and without hepatocellular carcinoma intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis c virus core protein and viral propagation new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites the hepatitis c virus persistence: how to evade the immune system? sequential processing of hepatitis c virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment reactivity to b cell epitopes within hepatitis c virus core protein and hepatocellular carcinoma assemble and interact: pleiotropic functions of the hcv core protein. hepatitis c viruses: genomes and molecular biology the first report in azeri patients i-tasser: a unified platform for automated protein structure and function prediction immunogenicity of multi-epitope dna and peptide vaccine candidates based on core, e2, ns3 and ns5b hcv epitopes in balb/c mice enhanced immune responses of a hepatitis c virus core dna vaccine by co-inoculating interleukin-12 expressing vector in mice bcepr(ed): prediction of continuous b-cell epitopes in antigenic sequences using physico-chemical properties prediction of continuous b-cell epitopes in an antigen using recurrent neural network algpred: prediction of allergenic proteins and mapping of ige epitopes association of hla class ii alleles with hepatitis c virus clearance and persistence in thalassemia patients from iran comparative proteomics of sera from hcc patients with different origins distribution of hepatitis c virus genotypes in iranian chronic infected patients modulation of the trans-suppression activity of hepatitis c virus core protein by phosphorylation propred: prediction of hla-dr binding sites propred1: prediction of promiscuous mhc class-i binding sites efficient trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles core as a novel viral target for hepatitis c drugs hepatitis c virus (hcv) genotype 1b sequences from fifteen patients with hepatocellular carcinoma: the 'progression score'revisited maturation of hepatitis c virus core protein by signal peptide peptidase is required for virus production hepatitis c virus core mutations reduce the sensitivity of a fluorescence enzyme immunoassay cellular immune responses against hepatitis c virus: the evidence base unraveling the mystery of liver diseases in egypt the authors would like to acknowledge shiraz publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-000786-ofpcgxce authors: chua, brendon y.; johnson, douglas; tan, amabel; earnest-silveira, linda; sekiya, toshiki; chin, ruth; torresi, joseph; jackson, david c. title: hepatitis c vlps delivered to dendritic cells by a tlr2 targeting lipopeptide results in enhanced antibody and cell-mediated responses date: 2012-10-16 journal: plos one doi: 10.1371/journal.pone.0047492 sha: doc_id: 786 cord_uid: ofpcgxce although many studies provide strong evidence supporting the development of hcv virus-like particle (vlp)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. in this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the tlr2 agonist pam(2)cys (e(8)pam(2)cys) to enhance the immunogenicity of vlps containing the hcv structural proteins (core, e1 and e2) of genotype 1a. while co-formulation of this lipopeptide with vlps only resulted in marginal improvements in dendritic cell (dc) uptake, its ability to concomitantly induce dc maturation at very small doses is a feature not observed using vlps alone or in the presence of an aluminium hydroxide-based adjuvant (alum). dramatically improved vlp and e2-specific antibody responses were observed in vlp+e(8)pam(2)cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted vlps was required to match the antibody titres obtained with a single dose of vlps formulated with this lipopeptide. this result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of vlp+e(8)pam(2)cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of vlps by huh7 cells. moreover, vaccination of hla-a2 transgenic mice with this formulation also induced better vlp-specific ifn-γ-mediated responses compared to non-adjuvanted vlps but comparable levels to that achieved when coadministered with complete freund’s adjuvant. these results suggest overall that the immunogenicity of hcv vlps can be significantly improved by the addition of this novel adjuvant by targeting their delivery to dcs and could therefore constitute a viable vaccine strategy for the treatment of hcv. hepatitis c virus (hcv) infection affects an estimated 200 million individuals worldwide and contributes to significant morbidity and mortality rates associated with liver cirrhosis and hepatocellular carcinoma. approximately 80% of infected individuals do not clear the virus following acute infection and will develop chronic infection that can lead to end-stage liver disease and complications. although treatment options using a combination of pegylated interferon-a and ribavirin are available, sustained clearance of the virus is only achieved in approximately 40% of individuals infected with hcv genotype 1 and 60-70% of those who are infected with genotypes 2 or 3 [1] . recent advances in the treatment of hcv using directly acting antiviral agents (daas) such as boceprevir and telaprevir have improved svr rates in both treatment naïve and experienced patients (reviewed in [2] ). however, treatment can be prolonged, expensive and also associated with substantial side effects. the development of an effective vaccine that can significantly reduce the number of new infections and improve sustained virological response rates could therefore be a useful adjunct to current therapeutic approaches and reduce the impact of infection on global health care systems. whilst the immune correlates mediating the clearance of virus are still not entirely clear or defined, there is substantial evidence demonstrating that the development of a broad multifunctional t cell response against an array of key viral proteins such as core, e1, ns3, ns4 and ns5 during acute hcv infection is associated with disease resolution [3, 4] and may also provide a level of protection against reinfection [5] . it is also becoming increasingly apparent that such responses alone are not enough [6] and that neutralising antibodies also play an integral role in conferring protection [7, 8] and facilitating viral clearance by mechanisms including antibodydependent cellular cytotoxic mechanisms [9] . an effective hcv vaccine will need to induce antibody and cell-mediated responses and also provide cross protection against different viral genotypes and quasispecies. neutralising antibodies induced against conserved, conformational epitopes in the viral envelope e1 and e2 glycoproteins [10] [11] [12] , notably antigenic region 3 (ar3) [13] of e2, including the critical neutralisation contact residues contained within domain i of e2 [14] and amino acids 313-327 of e1 [15] , can be broadly cross-neutralising. the fact that these antibodies neutralise different hcv genotypes highlights the importance of including epitopes from both envelope proteins for a vaccine strategy to be effective. virus-like particles (vlps) possess features which make them ideal vehicles for the delivery of viral antigens to the immune system; (i) antibody epitopes are presented in the native conformation for induction of potentially neutralising antibodies (ii) multiple t cell, cd4 + and cd8 + , epitopes are packaged in vlps (iii) vlps lack regulatory proteins as well as genetic material that could pose a risk of reversion or mutation (iv) encouraging results have been obtained using insect cell-derived recombinant vlps expressing hcv antigens which induce virus-specific humoral and cellular responses [16] [17] [18] (v) hcv vlps appear to possess properties favourable for dendritic cell uptake [19] and (vi) they exhibit superior immunogenicity and antigenicity over recombinant protein and dna-based vaccine approaches [16, 17] . an important consideration in the manufacture of hcv-based vlps is the cell-type used for their manufacture. for example, it has been shown that vaccination with recombinant hcv envelope proteins expressed in mammalian cells, but not in yeast or insect cells, protect chimpanzees from primary infection by an homologous hcv isolate [20] . similarly, rosa et al have demonstrated that mammalian cell-derived recombinant envelope proteins bind to human cells with higher affinity than those produced in yeast or insect cells and appear to be antigenically and functionally similar to the viral proteins produced in an infected host cell [21] . more recently, vaccination of macaques using vlps in a prime-boost regime has been reported to induce broadly neutralising antibody responses against different hcv genotypes [22] . although all of these studies provide encouraging results supporting the development of hcv vlp-based vaccines, the fact that heterologous viral vectors and/or unrealistic dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. in this study we evaluate the immunogenicity of mammalian cell-derived vlps containing structural proteins (core, e1 and e2) of hcv genotype 1a when delivered directly to dendritic cells (dcs) using a toll-like receptor 2 (tlr2) targeting lipopeptide. this lipopeptide contains the tlr2 agonist dipalmitoyl-sglyceryl-cysteine (pam 2 cys) and associates electrostatically with protein antigens significantly improving their ability to induce both humoral and cell-mediated responses [23] . the ability of lipopeptide-vlp complexes to facilitate dc uptake, induce antibody capable of inhibiting vlp entry into target cells and to elicit cell-mediated antigen-specific responses were each determined. hcv virus-like particles were constructed using a recombinant adenovirus containing encoding the hcv structural proteins (core, e1 and e2) of hcv 77h, genotype 1a. briefly, the core/e1 genes were amplified from pbrtm_hcv 1-3011 plasmid containing the genome of hcv h77 genotype 1a (a gift from prof c rice). the core/e1 genes were amplified from pbrtm_hcv 1-3011 plasmid containing the genome of hcv h77 genotype 1a (a gift from prof c rice). the forward core/e1 primer (59gcctctagagccaccatgcatcaccatcaccat-cacacaagcacgaatcctaaactcaaagaaaaacc 39) was designed to introduce an xbai enzyme restriction site followed by a kozak sequence, a start codon and a his(6) tag at amino terminal end of the core protein. the reverse primer of core/e1 (59 ggcttaagcccggtgacgtgggtttcc gcgtcgac 39) was designed to amplify from the sequence downstream of the region corresponding to e1/e2 cleavage site (amino acids 383/384) and to introduce an afl ii restriction site at the 39 end. next, the e2 genome was amplified using a forward primer (59cgactagt-gaaacccacgtcaccgggggaagtgccggccgc 39) that also introduced a spei site at the 59 end. the reverse e2 primer (59cggatatctcatcac gcctccgcttgggatatgagtaa-catcatcc 39) was designed to introduce a double stop codon and an ecorv restriction site at the 39 end of the amplicon. the core/e1 and e2 amplicons were cloned into pgemeasy (promega) and subsequently subcloned and ligated to produce a construct which was verified by dna sequencing. this construct was subsequently subcloned into padtrack-cmv (provided by b vogelstein, howard hughes medical centre, baltimore), digested with pmei and transformed into adeasier-1 cells by electroporation (bio-rad gene pulser) as previously described [24] . high titres of recombinant adenovirions encoding the hcv proteins (radhcv-ce1e2) were produced in 293t cells by serial passaging and the equivalent multiplicity of infection (moi) was determined as described previously [24] . to produce hcv vlps, huh7 cells were infected with radhcv-ce1e2 at a moi of 1. at 72 hours post-infection, cells were collected and disrupted using a dounce homogeniser and centrifuged at 17,000 g for 5 min. the supernatant was further centrifuged through a 30% sucrose cushion (containing 20 mm tris ph 7.4 and 150 mm nacl) at 178,000 g for 4 hours at 4uc. the resulting pellet was resuspended in 50 mm tris ph 7.and 100 mm nacl and purified through a 33% caesium chloride gradient by ultracentrifugation at 14uc at 143,000 g) for 72 hours. twelve 1 ml gradients were recovered and dialysed against sterile pbs at 4uc overnight. fluorescent labelling of vlps was achieved by adding vlps (6 mg/ml) to 2 mg/ml of fluorescein isothiocyanate (fitc; sigma aldrich) in 50 ml of dmso. the suspension was vortexed vigorously and incubated overnight at 4uc before dialysis against pbs the next day. all vlp preparations were stored in aliquots at 270uc until use. huh7 and 293t cells were grown in dulbecco's modified eagle's medium (dmem; invitrogen usa) supplemented with 10% fetal calf serum (fcs) and streptomycin 50 mg/ml at 37 c in 5% co 2 . the syntheses of the branched anionic peptide construct containing eight n-terminal glutamic acid residues (e 8 ) using traditional fmoc chemistry has been described previously [23] . briefly, synthesis was carried out manually using peg-s ram solid support (rapp polymere, tübingen, germany; substitution factor 0.27 mmol/g). fmoc-lysine(mtt)-oh (novabiochem, läufelfingen, switzerland) was first coupled to the support and the fmoc protecting group present on the a-amino group then removed and fmoc-lysine(fmoc)-oh was then coupled to the exposed n-terminal amino group. subsequent de-protection and acylation of another two rounds of fmoc-lysine(fmoc)-oh yielded eight branch points to which glutamic acid residues were coupled. the primary amino groups of the glutamic acid residues were then acetylated using a 20-fold excess of acetic anhydride and a 40-fold excess of diisopropylethylamine (dipea; sigma, australia) to generate e 8 which has an overall charge of 28. lipidation of e 8 was then carried out by removing the mtt protective group present on the e-amino group of the c-terminal lysine followed by acylation of the exposed e-amino group with two serially added serine residues. the pam 2 cys lipid moiety was then coupled according to zeng et al [25] to generate e 8 (pam 2 cys) ( figure 1a ). following assembly, lipopeptides were cleaved from the solid phase support and all side-chain protecting groups removed with 88% tfa, 5% phenol, 2% tips, 5% water for 3 hours at room temperature. lipopeptides were analysed by reversed phase highpressure liquid chromatography (rp-hplc) using a vydac c4 column (4.66300 mm) installed in a waters hplc system. the chromatogram was developed at a flow rate of 1 ml/min using 0.1% tfa in h 2 o and 0.1% tfa in acetonitrile as the limit solvent. lipopeptides were purified if necessary. all products presented as a single major peak on analytical rp-hplc and had the expected mass when analysed using an agilent series 1100 ion trap mass spectrometer. a line of murine balb/c-derived dcs (d1 cells) was prepared and propagated according to the method described by chua et al [26] . after a minimum of 21 days in culture, cells were stained for class ii mhc using fitc conjugated anti-ia/ie antibody (clone m5/114.15.2; becton dickinson, usa) and pe-conjugated cd11c (clone 2g9; becton dickinson, usa) prior to use. cells were verified to be cd11c + mhc class ii + by flow cytometry using a facscaliber (becton dickinson, usa). d1 cells (2610 5 ) were seeded onto a petri dish in 1 ml of fresh d1 media [26] and incubated at 37uc and 5% co 2 in the presence or absence of 5 mg fitc-labelled vlps alone or with vlps mixed with e 8 pam 2 cys (0.2 pmole/ml). cells were harvested 24 hours later and washed with facs wash (1% fcs/5 mm edta in pbs) before fixation in 1% paraformaldehyde in pbs. to examine cellular association of vlps, cell fluorescence was analysed by flow cytometry (facscaliber, becton dickinson, usa). for examination of intracellular uptake of vlps, extracellular fluorescence was quenched by addition of an equal volume of 0.1 m citrate buffer (ph 4.0) containing 250 mg/ml trypan blue (merck, damstadt, germany) prior to analysis [27] . data were analysed using flowjo software (tree star, san carlos, ca). to assess the degree of dc maturation resulting from exposure to different adjuvants, cells were exposed to varying concentrations of aluminium hydroxide gel, alhydrogel (sigma aldrich, missouri, usa), e 8 pam 2 cys or lipopolysaccharide (5 mg/ml) as a positive control (lps; sigma aldrich, milwaukee, usa). in some experiments, cells were incubated with vlps alone (5 mg/ml) or in the presence of e 8 pam 2 cys (0.032 mg/ml). after 16 hours, cells were harvested, washed and analysed for expression of surface class ii mhc antigen. all experimental procedures involving animals were approved by the university of melbourne's animal ethics committee under the aec numbers 0707207 and 061061. hla-a2k b transgenic mice (hhd mice) were obtained from the queensland institute for medical research and bred in the animal house facility of the department of microbiology and immunology at the university of melbourne under specific pathogen free conditions. these mice do not express h-2d b but instead express the chimeric monochain of the a1 & a2 domains of hla-a2.1 and the a3 cytoplasmic and transmembrane domains of h-2d b linked at its n-terminus to the c terminus of human b2 microglobulin [28] . female hhd mice (3 per group) were inoculated subcutaneously on each side of the base of tail (50 ml per dose) with vlps (30 mg) either alone, emulsified in an equal volume of complete freund's adjuvant (cfa) or with an equal amount of e 8 pam 2 cys on days 0 and 14. spleens were removed 28 days after the second dose and splenocytes restimulated in vitro at a concentration of 3610 6 cells/ml in rf-10 medium consisting of rpmi 1640 medium (gibco, usa) supplemented with 10% fetal calf serum (csl, parkville, australia) 7.5 mm hepes, 2 mm l-glutamine, 76 mm 2-mercaptoethanol, 150 u/ml penicillin, 150 mg/ml streptomycin, 150 mm non-essential amino acids and 10 u/ml of recombinant il-2 (roche, indianapolis, usa) at 37uc in an atmosphere of 5% co 2 . restimulation was carried out in the presence of 10 mg of vlps or 10 mm of an irrelevant hcvderived hla-a2-restricted epitope (ns5b2594-2602) that is not contained in the vlp construct. cells were harvested 5 days later, washed and serial dilutions commencing at 5610 5 cells/ml performed in polyvinylidene fluoride (pvdf) membrane-lined 96-well plates (millipore, ireland) previously coated with 5 mg/ml anti-ifn-c capture antibody (clone r4-6a2-bd pharmingen, san diego, usa). cells were then cultured in the presence of 3.75610 5 irradiated autologous vlp-pulsed (10 mg) splenocytes for 40 hours at 37uc and 5% co 2 . after washing with pbst (pbs containing 0.05% tween 20), biotinylated anti-ifn-c detection antibody (clone xmg1.2; becton dickinson, usa) was added and incubated for 2 hours at room temperature in a humidified atmosphere. plates were then washed and streptavidin-alkaline phosphatase (becton dickinson, usa) added and incubated for a further 2 hours. spots representative of ifn-c-producing cells were developed by the addition of 100 ml of 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 2-amino-2-methyl-1-propanol buffer (sigma-aldrich, usa) for 30 minutes. individual spots were counted using an aid ispot elispot reader (gmbh, strassberg, germany). analysis of variance in all experiments and all p values in this study were conducted and obtained using one-way anova nonparametric statistical analysis and tukey's post-hoc range tests performed with prism 5 (graphpad software, la jolla, california usa). flat bottom 96-well polyvinyl plates were coated with either purified hcv vlps (10 mg/ml) or recombinant e2 protein (5 mg/ ml) in pbsn 3 overnight at 4uc. prior to coating plates with hcv vlps, wells were pre-incubated with galanthus nivalis lectin (2 mg/ml; sigma aldrich australia) in carbonate buffer (15 mm naco 3 , 35 mm nahco 3 , 0.21 mm nacl) for 30 minutes at room temperature following removal of antigen, 100 ml of bsa (10 mg/ml) in pbs was added and plates incubated for 1 hour at room temperature before washing four times with pbst (pbs containing v/v 0.05% tween-20 [sigma aldrich, milwaukee, usa]). serial dilutions of sera obtained from immunised mice were added to wells and incubated in a humidified atmosphere overnight. after washing, bound antibody was detected using horseradish peroxidase-conjugated rabbit antimouse igg antibodies (dako, glostrup, denmark) in conjunction with enzyme substrate (0.2 mm 2,2_-azino-bis 3-ethylbenzthiazoline-sulfonic acid in 50 mm citric acid containing 0.004% hydrogen peroxide). the reaction was stopped by addition of 50 ml of 0.05 m naf. titers of antibody are expressed as the reciprocal of the highest dilution of serum required to achieve an optical density of 0.2. for the detection of specific antibody secreting cells by elispot, pvdf membrane-lined 96-well plates (mabtech, nacka strand, sweden) were coated with 100 ml of pbs containing vlps (10 mg/ml), recombinant e2 (10 mg/ml) or anti igg antibody (10 mg/ml) overnight at 4uc. plates were washed 5 times with pbs and blocked for 2 hours using rpmi 1640 medium (gibco, usa) supplemented with 20% bsa (sigma, australia). wells were emptied before 5610 5 splenocytes in 200 ml of rf-10 medium was added and incubated for 36 hours at 37uc in an atmosphere of 5% co 2 . spot forming units representative of specific antibody-producing cells were developed as previously described for the detection of ifn-c-secreting cells except that biotinylated anti-igg antibody and streptavidin-conjugated horseradish peroxidase (both from mabtech, nacka strand, sweden) were used as detecting reagents. in order to determine any inhibition of cell entry by vlps using antibodies present in sera of vaccinated animals, huh7 cells were enhancement of hcv-vlp immunogenicity plos one | www.plosone.org first incubated with pbs (10% fcs) for 15 min at 4uc to reduce non-specific binding of antibodies subsequently added. cells were washed twice, resuspended in pbs (0.1% fcs) and incubated with fitc-labelled vlps at 4uc for 1 hr. serial dilutions of sera from vaccinated or non-vaccinated mice were then added and incubated for a further 1 hour at 37uc. for each reaction, 5610 5 huh7 cells and 200 ng of fitc-labelled vlps were used in a total volume of 500 ml. at the end of this incubation period, cells were washed with pbs (0.1% fcs) and then fixed in bd cytofix (becton dickinson, usa). inhibition of vlp entry was determined by flow cytometry and analysed using weasel 2.0 software (walter and eliza hall institute, melbourne, australia). sera from vaccinated mice that demonstrated a decrease in specific cellular binding of 50% or more compared to sera from naïve mice were considered to contain neutralising antibodies [18] . hcv genotype 1a vlps were produced by transducing a human hepatocyte-derived cell line with recombinant adenovirus containing encoding the hcv structural proteins (core, e1 and e2) of genotype 1a to produce particles that harbour antigenic resemblance to virions produced in an infected host cell. because of the essential role that dcs play in the induction of both humoral and cell-mediated responses, we first examined the ability of a spleen-derived dc line (d1 cells) to take up fluorescein isothiocyanate-labelled hcv vlps (fitc-vlps). flow cytometric analysis revealed that dcs incubated with fitc-vlps exhibited higher whole cell fluorescence intensities compared to untreated dcs indicating the presence of cell-associated vlps ( figure 1b) . exposure of dcs to vlps pre-mixed with the lipopeptide e 8 pam 2 cys also resulted in higher levels of cell fluorescence compared to untreated dcs. the percentage of fluorescenated cells in these cultures was similar to cultures that contained fitc-vlps alone ( figure 1c) . to determine the magnitude of vlp cell uptake, intracellular fluorescence was measured by quenching extracellular fluorescence after exposure of cells to trypan blue prior to flow cytometric analysis [27] . although the resulting fluorescence intensities of dcs incubated with fitc-vlps were now lower following this treatment, the levels were still notably higher than untreated cells confirming the presence of intracellular fitc-vlps ( figure 1d ). equivalent fluorescence cell intensities were also observed in dcs that were incubated with fitc-vlps pre-mixed with e 8 pam 2 cys. however, a higher percentage of fluorescenated cells were detected in those cultures compared to those exposed to fitc-vlps alone ( figure 1e ) indicating that an increase in uptake of these constructs is facilitated using the lipopeptide. to investigate the ability of hcv vlps and e 8 pam 2 cys to cause activation of dcs, we measured the expression of surface mhc class ii molecules following incubation with the various antigens. the results (figure 2a) indicate that untreated dcs contained two populations of cells which were mhc class ii low and mhc class ii high , the latter comprising ,24% of the population analysed. while the distribution of these populations was not affected by exposure to hcv vlps alone, incubation with hcv vlps mixed with e 8 pam 2 cys caused a dramatic shift in the distribution of mhc class ii expressing cells such that 83% of cells were mhc class ii high . the upregulation of mhc class ii expression on these cells were comparable to those cultured in the presence of lps which is a potent dc maturation stimulus. further dosing experiments showed that increasing the concentrations of hcv vlps to 20 mg/ml, did not induce dc activation because the percentage of mhc class ii high dcs in cultures containing escalating doses of vlps remained similar to those containing untreated dcs ( figure 2b ). in contrast, exposure to as little as 0.1 nmoles of e 8 pam 2 cys was sufficient at inducing a greater than two-fold increase in activation of dc compared to untreated cells ( figure 2c) and was similar to the levels of activation observed with lps. no dc activation was observed in the presence of alhydrogel ( figure 2d ). the presence of e 8 pam 2 cys in hcv vlp-containing formulations however, not only promotes uptake of hcv vlps by dcs but also considerably increases the level of dc activation. to determine if the dc activating properties of vlp formulations containing e 8 pam 2 cys translate to an improvement in hcv vlp immunogenicity, balb/c mice were inoculated with vlps alone or with vlps mixed with e 8 pam 2 cys. hcv vlp-specific antibody titres in sera obtained after one, two or three doses of each formulation were then determined by elisa. administration of vlps alone in saline was able to elicit detectable titres of specific antibody that were marginally increased after each dose of antigen ( figure 3a ). in animals that received hcv vlps mixed with e 8 pam 2 cys, however, antibody levels were significantly higher, in some cases by up to ten-fold more than those from animals that received the same dose of hcv vlps alone. in fact the titre of specific antibody induced following administration of 3 doses of hcv vlps alone was achieved using a single dose only of hcv vlp mixed with e 8 pam 2 cys. when compared to animals that were inoculated with hcv vlps formulated with alhydrogel, an adjuvant widely used to induce antibody responses to both human and veterinary vaccines [29] , lower antibody titres were observed in these animals than in those that received the vlp-lipopeptide formulation. in examining levels of e2 specific antibodies elicited by vaccination, higher titres were once again demonstrated in animals that received 3 doses of vlps mixed with e 8 pam 2 cys compared to those that were inoculated with vlps alone or with alhydrogel ( figure 3b ). the hierarchical pattern of antibody responses induced by e 8 pam 2 cys and alhydrogel was also confirmed by the numbers of specific antibody secreting cells that were detected in the spleens of vaccinated mice. once again significantly higher numbers of cells secreting both hcv vlp ( figure 4a ) or e2-specific antibodies ( figure 4b ) were detected in animals that received hcv vlps mixed with e 8 pam 2 cys than those that were inoculated with hcv vlps alone or vlps formulated with alhydrogel. to assess the neutralising activity of antibodies induced by vaccination, we first set out to investigate if vlp entry into human hepatocyte cell line huh7 could be inhibited. pre-incubation of fitc-labelled vlps (vlp-fitc) with pbs or naïve serum resulted in minimal inhibition of vlp entry ( figure 5a ). however, the presence of an antibody against cd81, a cell surface molecule implicated in hcv entry into hepatocytes [30] , was able to prevent vlp entry into these cells by .90% confirming that these vlps also utilise this molecule to facilitate cell entry. we next analysed the ability of sera obtained from mice inoculated with vlps to inhibit the binding and entry of vlps into huh 7 cells ( figure 5b ). neutralisation of binding of vlps to huh7 cells was significantly greater in sera obtained from mice inoculated with vlps in e 8 pam 2 cys (,50%) compared to sera obtained from mice inoculated with vlps administered in alhydrogel (,30%) or in saline (,20%). the ability of vlp formulations containing e 8 pam 2 cys to induce a cell-meditated immune response was examined by inoculating transgenic mice expressing the mhc class i (hla-a2) allele but not endogenous h-2d b molecules [28] . control transgenic animals were inoculated with vlps alone or vlps emulsified with an equal amount of complete freund's adjuvant (cfa). splenocytes from vaccinated animals were obtained 28 days post-inoculation and re-stimulated with antigen in vitro. the results ( figure 6 ) of an elispot assay carried out revealed significantly higher numbers of hcv vlp-specific ifn-c producing cells in the spleens of mice inoculated with vlps in the presence of e 8 pam 2 cys or vlps emulsified in cfa than in those that received vlps alone. the development of novel, effective anti-viral vaccine strategies in recent times has seen a notable shift away from the use of traditional formulations which utilize whole inactivated or live attenuated viruses towards approaches based on recombinant subunit protein antigens which are more easily characterised and defined. vlps offer features that make them a useful platform for delivering viral antigens in a single vaccine construct which not only minimises the risks that may be associated with preparations containing or requiring the use of a replicating pathogen but will also closely resemble native viral antigens from which they are derived. the most convincing demonstration of vlps efficacy is the quadrivalent vlp-based vaccine gardasil which prevents persistent infection and associated disease caused by human papillomavirus [31] . other studies of vlp-based vaccination strategies have also shown promising results and led to phase i testing against a number of disease indications including seasonal and pandemic influenza, hepatitis b, malaria and hiv (reviewed in [32] ). depending on the type of virus used to manufacture a vlp construct, studies have shown that protective responses induced by vlps can be elicited without co-administration of adjuvant [33] [34] [35] . in many cases, however, the induction of useful immune responses may require multiple doses [36, 37] , a regime that may be impractical to implement in the field or involve a viral vector to provide an initial priming dose followed by a boost using vlps [22, 38] . of relevance to the present study, the use of adjuvants to enhance vlp immunogenicity has been shown to induce strong antibody responses using dose-sparing amounts of hiv [39] or norwalk virus-derived vlps [40] and also elicits cell-mediated were incubated with vlps (5 mg) alone or formulated with e 8 pam 2 cys (0.01 nmoles/ml) or alhydrogel (5 mg) in a total volume of 1 ml. for comparative purposes within all experiments, cells were also either left untreated, exposed to lps (5 mg/ml) or to similar amounts of each adjuvant alone. cell surface mhc class ii expression was determined after 16 hours using a peconjugated anti-ia/ie antibody. cells expressing low levels of mhc class ii molecules were deemed to be immature whilst those expressing high levels were considered to be mature. shown are representative histograms depicting cell surface mhc class ii expression from one of three experiments conducted separately. mhc class ii high expressing cells are shaded in grey. for dosing experiments, cells were also incubated with increasing amounts of (b) vlps, (c) e 8 pam 2 cys, (d) alhydrogel or vlps (5 mg) formulated with increasing amounts of (e) e 8 pam 2 cys, or (f) alhydrogel. doi:10.1371/journal.pone.0047492.g002 responses that culminate in improved protection against lethal influenza viral [41, 42] and tumorigenic challenge [43] . our previous studies have shown peptide epitope and proteinbased antigens can be made far more immunogenic when covalently attached to pam 2 cys in order to target their delivery via tlr2 to dendritic cells (dcs) [44] . this results in the induction of robust antibody and cd8 + t cell-mediated immune responses and has been shown for multiple indications [44] [45] [46] [47] . each of these vaccine candidates demonstrated the ability of this simple lipid structure to dramatically enhance the immunogenicity of antigens that are otherwise immunologically inert. nevertheless, the approach introduces complexities into the vaccine manufacturing process due to the requirement for covalent attachment of pam 2 cys to an antigen. the use of the anionic lipopeptide e 8 pam 2 cys overcomes many of the technical complexities related to this process, especially the use of covalent chemistries, by making use of electrostatic association with antigen [23] . the ability of pam2cys to dramatically enhance the immunogenicity of hcv vlps was demonstrated in the improved overall antibody responses that we observed. not only are greater antibody titres induced following vaccination with vlps formulated with e 8 pam 2 cys compared to the use of vlps alone or when co-administered with alhydrogel but up to 3 doses of nonadjuvanted or traditionally adjuvanted antigen were required to match the titres obtained with a single dose using lipopeptide. most importantly in the context of hcv the trend translates to improved e2-specific antibody responses and the use of lower doses of vlps to achieve this while maintaining efficacy has major advantages by providing cost benefits to vaccine manufacturers. our studies examining the interaction of vlps with dcs indicate that while improvements in vlp uptake mediated by this lipopeptide is minimal, its ability to concomitantly induce the maturation of dcs at very small doses is a feature not observed using vlps alone or vlps administered in the presence of alum. our previous work also demonstrated that association of antigen with charged lipopeptide facilitates trafficking of antigen to lymph cys. supernatants were clarified by centrifugation, incubated with huh7 cells (5610 5 ) in a total volume of (500 ml) for 1 hour. cells were then harvested and cellular fluorescence levels analysed by flow cytometry. all bar graphs represent the percentage reduction in vlp entry relative to baseline levels obtained using serum from naïve mice. doi:10.1371/journal.pone.0047492.g005 figure 6 . cell-mediated responses elicited by vaccination. hla-a2k b transgenic mice (n = 3 per group) were inoculated (100 ml) subcutaneously at the base of the tail on days 0 and 14 with 30 mg of vlps alone, emulsified with an equal amount of complete freund's adjuvant (cfa) or pre-mixed with 30 mg of e 8 pam 2 cys in saline. splenocytes were obtained 28 days later and restimulated for 7 days in the presence of 10 mg vlps or an irrelevant hcv-derived hla-a2restricted epitope not part of the vlp construct. the frequency of peptide-specific t cells producing ifn-c was determined in an elispot assay. each bar represents the average number of ifn-c producing t cells and standard deviation in each group after subtracting nonspecific responses from corresponding samples stimulated with the irrelevant peptide. doi:10.1371/journal.pone.0047492.g006 enhancement of hcv-vlp immunogenicity plos one | www.plosone.org nodes draining from the vaccination site [23] and together with the results presented in this study provide an explanation for the dose-sparing neutralising antibody responses that we observe. coadministration of vlps using charged lipopeptide has the added benefit of eliciting vlp-specific cell-mediated responses in hla-a2 transgenic mice, a fact that may also be attributed to dc targeting and activation. given the results of the work described in this study, we conclude that the use of this branched anionic lipopeptide together with vlps containing hcv antigens in order to provide a broad spectrum of conformational epitopes can provide benefits in terms of inducing improved levels of neutralising antibody titres and eliciting cell-mediated responses. this strategy could therefore constitute a valuable addition to the armamentarium of current vlp-based vaccine developments against hcv. peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial antiviral strategies in hepatitis c virus infection full-breadth analysis of cd8+ t-cell responses in acute hepatitis c virus infection and early therapy differential antigenic hierarchy associated with spontaneous recovery from hepatitis c virus infection: implications for vaccine design hepatitis c virus reinfection in injection drug users cd4+ immune escape and subsequent t-cell failure following chimpanzee immunization against hepatitis c virus rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis c spontaneous clearance of chronic hepatitis c virus infection is associated with appearance of neutralizing antibodies and reversal of t-cell exhaustion serum antibodies against the hepatitis c virus e2 protein mediate antibodydependent cellular cytotoxicity (adcc) exploiting information inherent in binding sites of virus-specific antibodies: design of an hcv vaccine candidate cross-reactive with multiple genotypes characterization of the hepatitis c virus e2 epitope defined by the broadly neutralizing monoclonal antibody ap33 induction of neutralizing antibody responses to hepatitis c virus with synthetic peptide constructs incorporating both antibody and t-helper epitopes broadly neutralizing antibodies protect against hepatitis c virus quasispecies challenge the disulfide bonds in glycoprotein e2 of hepatitis c virus reveal the tertiary organization of the molecule isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis c virus hepatitis c virus-like particles induce virus-specific humoral and cellular immune responses in mice immunization with hepatitis c virus-like particles protects mice from recombinant hepatitis c virus-vaccinia infection inhibition of hepatitis c virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis c uptake and presentation of hepatitis c virus-like particles by human dendritic cells vaccination of chimpanzees against infection by the hepatitis c virus a quantitative test to estimate neutralizing antibodies to the hepatitis c virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells a prime-boost strategy using virus-like particles pseudotyped for hcv proteins triggers broadly neutralizing antibodies in macaques soluble proteins induce strong cd8+ t cell and antibody responses through electrostatic association with simple cationic or anionic lipopeptides that target tlr2 modulation of mapk pathways and cell cycle by replicating hepatitis b virus: factors contributing to hepatocarcinogenesis synthesis of a new template with a built-in adjuvant and its use in constructing peptide vaccine candidates through polyoxime chemistry comparison of lipopeptidebased immunocontraceptive vaccines containing different lipid groups dendritic cell acquisition of epitope cargo mediated by simple cationic peptide structures h-2 class i knockout, hla-a2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies aluminium compounds for use in vaccines binding of hepatitis c virus to cd81 safety, immunogenicity, and efficacy of quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine in women aged 24-45 years: a randomised, double-blind trial developments in virus-like particle-based vaccines for infectious diseases and cancer chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix 1 efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov a microbial platform for rapid and low-cost virus-like particle and capsomere vaccines vaccine potential of nipah virus-like particles vaccination with multimeric l2 fusion protein and l1 vlp or capsomeres to broaden protection against hpv infection effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice dna-vlp prime-boost intra-nasal immunization induces cellular and humoral anti-hiv-1 systemic and mucosal immunity with cross-clade neutralizing activity effects of adjuvants on igg subclasses elicited by virus-like particles an intranasally delivered toll-like receptor 7 agonist elicits robust systemic and mucosal responses to norwalk virus-like particles adjuvants that stimulate tlr3 or nlpr3 pathways enhance the efficiency of influenza virus-like particle vaccines in aged mice intranasal immunization with influenza vlps incorporating membrane-anchored flagellin induces strong heterosubtypic protection murine polyomavirus virus-like particles carrying full-length human psa protect balb/c mice from outgrowth of a psa expressing tumor a totally synthetic vaccine of generic structure that targets toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic t cell responses protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of l2 intranasal lipopeptide primes lung-resident memory cd8+ t cells for long-term pulmonary protection against influenza a selfadjuvanting multiepitope immunogen that induces a broadly cross-reactive antibody to hepatitis c virus key: cord-017948-fqhl1qb4 authors: hu, yuan title: molecular techniques for blood and blood product screening date: 2012-04-05 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-1-4614-3970-7_28 sha: doc_id: 17948 cord_uid: fqhl1qb4 the food and drug administration (fda) is responsible for ensuring the safety of the more than 15 million units of blood and blood components donated each year in the united states. “blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries,” said david a. kessler, md, former fda commissioner [1]. screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. the united states has the safest blood supply in the world [1] and the fda is striving to keep it safe by decreasing the risk of infectious disease transmission. the regulatory agency is continuously updating its requirements and standards for collecting and processing blood. as mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases. in the united states, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (table 28.1). the field of clinical microbiology and virology are now focusing on molecular technology. currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. it is time for all blood safety procedures to include molecular detection techniques. this approach can significantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. this chapter reviews the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety. the food and drug administration (fda) is responsible for ensuring the safety of the more than 15 million units of blood and blood components donated each year in the united states. "blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries," said david a. kessler, md, former fda commissioner [ 1 ] . screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. the united states has the safest blood supply in the world [ 1 ] and the fda is striving to keep it safe by decreasing the risk of infectious disease transmission. the regulatory agency is continuously updating its requirements and standards for collecting and processing blood. as mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases. in the united states, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (table 28 .1 ). the fi eld of clinical microbiology and virology are now focusing on molecular technology. currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. it is time for all blood safety procedures to include molecular detection techniques. y. hu (*) u.s. food and drug administration, northeast regional laboratory , 158-15 liberty avenue , jamaica , ny 11433 , usa e-mail: yuan.hu@fda.hhs.gov no of fi cial support or endorsement of this article by the food and drug administration is intended or should be inferred. this approach can signi fi cantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. this chapter reviews the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety. direct detection of viral antigens and virus speci fi c antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. there are some limitations. for direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts suf fi ciently detectable in the body by an antibodymediated assay. for indirect virus detection by virus speci fi c antibodies (e.g., an immuno fl uorescence assay or enzyme immunoassay (eia), etc.), there is a problem in that shortly after infection by a pathogenic virus, there is a window period in which antibody generation is insuf fi cient for detection [ 2 ] . to reduce this window period of low detection, direct nucleic acid tests are needed. through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. the nucleic acid tests can also provide evidence for genetic variation in viruses. molecular methods include the use of nucleic acid probes as well as ampli fi cation-based and dna sequence-based techniques. an increasing number of molecular diagnostic methods are now available commercially. in comparison to classical methods, molecular biological methods are superior in terms of rapidness, speci fi city, and sensitivity. the current nucleic acid detection methods in the fi eld may be grouped into two major classes: amplifying techniques such as pcr and nonamplifying techniques such as southern blot hybridization. amplifying techniques are more sensitive than nonamplifying techniques. there are two different types of amplifying methods [ 3 ] , target ampli fi cation methods and signal ampli fi cation methods. target amplifying techniques include pcr, nucleic acid sequence-based ampli fi cation (nasba) [ 4, 5 ] , self-sustaining sequence ampli fi cation (3sr), transcription-based ampli fi cation (tas), transcription-mediated ampli fi cation (tma), strand displacement ampli fi cation (sda), and ligase chain reaction (lcr). signal ampli fi cation methods include branched dna (bdna) signal ampli fi cation [ 6 ] , cleavage-based signal ampli fi cation (cycling probe technologies and invader assay), qß replicase, hybrid capture, cycling probe technologies (cpt), and rolling-circle ampli fi cation (rca) [ 7 ] . to further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing. southern blotting [ 8 ] was named after edward m. southern who developed this procedure at edinburgh university in the 1970s. this technique is used to detect speci fi c sequences within mixtures of dna, which is size-fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. after blocking of nonspeci fi c binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the speci fi c dna sequence of interest. should speci fi c dna be present on the blot, it will combine with the labeled probe and be detectable. by coelectrophoresing dna fragments of known molecular weight, the size(s) of the hybridizing band(s) can then be determined. southern blotting hybridization technology is one of the major tools that have already helped clinical staffs worldwide interpret genomic information. other competing methodologies include in situ hybridization and solution hybridization. important clinical examples of the use of this technology are dna fi ngerprinting and the ability to detect dna gene rearrangements. in 1983, dr. kary mullis at cetus corporation conceived of polymerase chain reaction [ 9 ] . there is not a single technique that has had a greater impact on the practice of molecular biology than pcr. with this technique, we can detect infectious diseases agents at an extremely low level. it is based on the ability of sense and antisense dna primers to hybridize to a dna of interest. following extension from the primers on the dna template by dna polymerase, the reaction is heat-denatured and allowed to anneal with the primers once again. another round of extension leads to a multiplicative increase in dna products. therefore, a minute amount of dna can be ef fi ciently ampli fi ed in an exponential fashion to result in larger amounts of dna that are more easily manipulated. by including critical controls, the technique can be made quantitative. the current level of the sensitivity and detection limit is as low as 10-50 copies per ml blood in hiv testing [ 1, 10, 11 ] . important clinical examples of the use of pcr are detection of hiv and hcv [12] [13] [14] . pcr techniques have evolved into different branches. some of them are now widely in use for virus detection in clinical diagnostics. these are real-time pcr by taqman (roche), light cycler (roche) and smart cycler (cepheid), and in situ pcr, nested-pcr, nested-real time pcr [ 15 ] , broad-range pcr, multiplex pcr, rt-pcr, arbitrarily primer pcr, long pcr, and quantitative pcr. real-time sequence technology will be coming soon for more detailed detection. in the past, identi fi cation of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (rflp). with real-time sequences technology, we will be able to detect a virus early as well as to obtain the viral sequence. microarrays were developed at stanford university by schena and coworkers in the early 1990s [ 16 ] . for medical applications, a microarray analysis offers a very accurate screening technology. it allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. it promises to be a fast and accurate diagnostic tool in the fi eld of clinical microbiology and virology. applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. compared with existing technologies, it allows for a wider variety of speci fi c tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. with the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identi fi cation of many types of pathogenic contaminants. in the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. it can provide vast information about the identity of bloodborne pathogens as well as their gene expression pro fi les [ 17 ] . to ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents. after donation, each unit of donated blood undergoes a series of tests for bloodborne agents such as human immunode fi ciency virus (hiv)-1, hiv-2, hepatitis b virus (hbv), hepatitis c virus (hcv), human t-cell lymphotropic virus (htlv)-1 and htlv-ii, west nile virus (wnv), and treponema pallidum , the agent of syphilis. all of the above tests are referred to as screening tests, and are designed to detect as many infectious agents as possible. because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. in order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more speci fi c tests called con fi rmatory tests. thus, con fi rmatory tests help determine whether a donor is truly infected. if any one of these tests fails, affected blood products are considered unsuitable for transfusion [ 18 ] . nucleic acid testing (nat) employs testing technology that directly detects the genomes of viruses. because nat detects a virus's genetic material instead of waiting for the body's response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [ 19 ] , thus further improving blood safety. nat will become the gold standard because of greater sensitivity compared to antibody tests. since 1999, nat has been approved by the fda and used to detect hiv-1 and hcv; this technology currently is under investigation for detecting other infectious disease agents. we know that for many viral infections, viral rna appears very early in the infection, in 1-2 weeks, but the antibody does not appear until 10-12 weeks, e.g., hiv and hcv [ 20 ] . in order to virtually prevent infection by all the transfusion associated viruses, we need to detect the viruses in their window period, and a nat or gene-based testing method is needed. nat also provides an opportunity for the viral, e.g., hiv or hcv, infected donor to seek early treatment. on the other hand, nat is not only a sensitive method, but also a rapid method which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical. the hbv is a highly infectious and often nonsymptomatic virus that is transmitted primarily through blood and blood-derived fl uids and is a leading cause of liver infection worldwide. the world health organization (who) estimates that two billion people worldwide have been infected with hbv and 350,000,000 people are chronically infected. chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1,000,000 deaths each year. each year up to 200,000 people become newly infected in the united states alone. since the beginning of screening for hbv in 1969, the rate of infection through blood transfusions has greatly decreased. however, as of 2000, hbv is still transmitted through blood transfusions in 1 out of 137,000 units of blood. one reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. serologic tests for hbv include hepatitis b surface antigen (hbsag) and hepatitis b core antibody (hbcab). hbv, which mainly infects the liver, has an inner core and an outer envelope (the surface). the hbsag test detects the outer envelope, identifying an individual infected with the hbv. this virus can cause in fl ammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. fortunately, most patients recover completely and test negative for hbsag within a few months after the illness. a small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. chronically infected people can develop severe liver disease as time passes, and need to be followed carefully by an experienced physician. to reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hbsag by the most sensitive and speci fi c assays. blood donations that are found to be reactive in the hbsag test are automatically con fi rmed by the hbsag con fi rmatory assay. if the specimen is neutralizable in the con fi rmatory test, the specimen is considered positive for hbsag. hbsag testing of donated blood has begun in 1975 (table 28.1 ) . currently, all blood donors are screened for hbsag, but occasional transmission of hbv still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). detection of early hbv infection of blood donors is still a major problem of blood transfusion. the current third-generation licensed hbsag tests (mostly radioimmunoassay and enzyme immunoassays) can not detect hbv in the window period for hbv infection. this is a strong motivation for introducing molecular detection techniques to the fi eld. there are some commercially available test methods for detecting hbv dna in the market now, such as chiron's quantiplex hbv dna [ 21 ] , digene's hybrid capture, abbott's hbv dna assay, and roche's amplicor hbv monitor. using these commercial hybridization or pcr-based assays, hbv dna can be detected 1-3 weeks before the appearance of hbsag [ 22 ] . some chronically infected patients who have lost their hbsag remain hbv dna positive, but are disquali fi ed as potential blood donors. molecular detection of hbv dna is more sensitive than current methods employed for hbsag screening. determination of antibodies to the hepatitis b core antigen (anti-hbc) (total) is also used to monitor the progress of the hepatitis b viral infection. determination of anti-hbc (igm) is employed to distinguish an acute hepatitis b infection from a chronic infection. the anti-hbc test developed in 1987 detects an antibody to the hbv that is produced during and after infection. if an individual has a positive anti-hbc test, but the hbsag test is negative, it may mean that the person once had hepatitis b, but has recovered from the infection. of the individuals with a positive test for anti-hbc, many have not been exposed to the hbv; thus, there is a frequent problem of false positives. although the individual may be permanently deferred from donating blood, it is unlikely that the person's health will be negatively affected. (note: this antibody is not produced following vaccination against hepatitis b). the hcv is a member of the flaviviridae family of viruses, which are associated with both human and animal diseases. hepatitis caused by hcv is the most common chronic bloodborne infection in the united states. over four million americans are believed to be infected. hcv can also be transmitted through blood transfusion. hcv causes in fl ammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver in fl ammation, cirrhosis, cancer, and death. eventually, up to 20% of people with hcv may develop cirrhosis of the liver or other severe liver diseases. as in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. because of the risk of serious illness, people with hcv need to be followed closely by a physician with experience evaluating this infection. since the fi rst cloning of fulllength hcv cdna in 1989, signi fi cant progress has been made in characterizing its molecular biology [ 11 ] . but, the natural history of hcv infection is still largely unclear and current treatment options for patients are limited. there is no vaccine for hcv, and the only available treatment, a combination of alpha interferon and ribavirin, is ef fi cacious in only a minority of patients [ 23 ] . the life cycle of the hcv is poorly understood due to the lack of an ef fi cient cell culture system [ 24 ] . there is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hcv. we have recently established a cell culture system for the replication of hcv by using human t and b leukemia cell lines [ 25 ] . this model should represent a valuable tool for the detailed study of the initial steps of the hcv replication cycle and for the evaluation of antiviral molecules. currently, appropriate vaccine strategies for hcv have not been developed. early detection and prevention of hcv infection are most important for blood safety. it is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the hcv genome because of a high mutation rate in this genomic region. however, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of hcv. in 1990, the fi rst speci fi c test for hcv, the major cause of "non-a, non-b" hepatitis was introduced. now, a third generation elisa kit is available to detect antibodies to hcv and screening blood for hcv antibodies is recommended. these assays are based on detection of serum antibody to various hcv antigens because these antibodies are nearly universally present in patients who are chronically infected with hcv [ 26 ] . the hcv screening tests are known to have signi fi cant limitations and positive samples should be further tested by hcv con fi rmatory tests. guidelines provided by the cdc recommend that hcv antibody screening test positive samples should be con fi rmed with serologic or nucleic acid supplemental testing. hcv con fi rmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. in this way, the response to individual antigens can be recognized, and some false-positive elisa results can be eliminated (e.g., riba, chiron hcv 3.0, and pcr assay) (e.g., roche cobas amplicor hcv test, version 2.0). laboratories can choose to perform this testing on all positive specimens or based on screening test positive (signal to cutoff) ratios. the positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened. hcv antibodies are not generally detectable for at least 6 weeks and may not appear for several months. acute hcv infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. high sensitivity detection of hcv during the window period is a longterm technical challenge in the fi eld. tests for hcv rna genome detection based on the pcr or other highly sensitive rna detection systems have been used for the diagnosis of acute hepatitis [ 26 ] . sensitive detection of hcv rna based on rt-pcr or other nucleic acid ampli fi cation techniques can be readily accomplished with kits that are now available commercially. for example, in 1999 the fda approved roche's amplicor hiv-1 monitor ultra sensitive quantitative assay. it can measure hiv levels at as few as 50 copies/ml and another commercial kit, the lcx hiv rna quantitative assay from abbott laboratories, also has a detection limit at 50 copies/ml. some studies even showed a sensitivity limit at 1 copy [ 27 ] . in fact, a qualitative assay should be much more sensitive than a quantitative assay for hiv/hcv screening. a sensitive qualitative hcv molecular detection assay will possibly interdict and virtually prevent all transfusion-associated hiv/hcv. the current sensitivity standard for clinical diagnostics is 100 copies/ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies/ml. hiv-1 and/or hiv-2 virus cause acquired immunode fi ciency syndrome, or aids. the test is designed to detect antibodies directed against antigens of the hiv-1 or hiv-2 viruses. hiv-1 is much more common in the united states, whereas hiv-2 is prevalent in western africa. donors are tested for both viruses because both are transmitted by infected blood, and a few cases of hiv-2 have been identi fi ed in us residents. in1985, the fi rst blood-screening eia test to detect hiv was licensed and quickly implemented by blood banks to protect the blood supply. in 1992, testing of donor blood for both hiv-1 and hiv-2 antibodies (anti-hiv-1 and anti-hiv-2) was implemented. in 1996, hiv p24 antigen testing of donated blood was mandated. now, the p24 antigen testing is going to be compared with a pcr-based test for their ability to detect hiv in the window period. htlv retroviruses are endemic in japan and the caribbean but relatively uncommon in the united states. they cause adult t-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. the infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. in rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. htlv-ii infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. disease associations with htlv-ii have been hard to con fi rm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections, or rare cases of neurological disease. in 1989, human-t-lymphotropic-virus-antibody testing of donated blood was begun. blood is now routinely screened for antibodies to htlv-i/ii. these test screens for antibodies directed against epitopes of the htlv-i/ii viruses. several commercial assays based on the enzyme-linked immunosorbent assay (elisa) or particle agglutination formats are used for screening of htlv antibodies, followed by con fi rmatory assays using western blotting. in some infected individuals, the serologic response to htlv infection is very low. these problems have been solved by the application of pcr ampli fi cation of speci fi c sequences in the virus genome. pcr can be used to detect htlv-i/ii proviruses and is now the method of choice for detection of htlv dna directly from blood and many other tissues. commercial pcr kits for htlv are available [ 28 ] . the wnv is a single-stranded rna virus of the flaviviridae family and is the most recent emerging infectious disease threat to public health and, potentially, to the safety of our blood supply. in 2002, wnv was identi fi ed as transfusion transmissible. it is transmitted by mosquitoes to birds and other animals through a mosquito bite. the virus can infect people, horses, many types of birds, and some other animals. wnv was shown in 2002 to be transmissible by blood [ 29 ] , with an estimated mean risk of 2/10,000-5/10,000 in outbreak regions in the united states. the most common symptoms of transfusion-transmitted cases of wnv were fever and headache. detection of wnv includes either a measurement of wnv antibodies or of wnv nucleic acid (detecting genetic material from the virus itself). there are two types of wnv antibody testing: igm and igg. in most individuals, igm antibodies will be present within 8 days after the initial exposure to wnv, followed by igg production several weeks later. but, the antibodies tested to detect wnv are not expedient for donor blood screening. nat involves amplifying and measuring the wnv's genetic material to detect the presence of the virus in blood or tissue. wnv nat will be negative in the blood once clinical illness has occurred. in this situation, both nat and igm antibody testing may be needed. nucleic acid tests to screen blood for wnv are commercially available and in current use. but, the viral yield for wnv infection is much lower than other viruses. consequently, a more sensitive wnv nat system for donor blood screening will be required, which could further reduce the risks of transfusion transmitted wnv. serum samples from all blood units should be subjected to either the venereal disease research laboratory (vdrl) test or a treponemal test, such as the t. pallidum haemagglutination (tpha) test before transfusion. any unit found positive should be discarded as per standard safety procedures. this test is done to detect evidence of infection with the spirochete that causes syphilis. blood centers began testing for this shortly after world war ii, when syphilis rates in the general population were much higher. the risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors, and because the spirochete is fragile and unlikely to survive blood storage conditions. sensitivity and speci fi city of serologic tests vary depending on the type of test performed and the stage of the disease. if the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. syphilis serological tests for donors have less clinical signi fi cance. a nucleic acid test for accurately detecting syphilis is needed. it can be used to determine whether a blood donor is currently or has recently been infected with the spirochete. in recent years, numerous infectious agents found worldwide have been identi fi ed as potential threats to the blood supply and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. even if the hepatitis virus test is negative for all known a-e hepatitis agents, there are some unidenti fi ed hepatitis viruses, called non a-e hepatitis viruses that can still be transmitted by blood transfusion. in the future, advances in nat may allow rapid discovery of the unknown hepatitis viruses. hepatitis delta virus (hdv) is a small rna virus that can infect only individuals who have hbv; worldwide more than 15 million people are coinfected [ 30 ] . hdv is clinically important because it generally makes hbv infections more damaging to the liver. increased understanding of the molecular virology of hdv will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. pcr and real-time pcr methods are available for hdv rna detection [ 31 ] . tt virus (ttv) [ 32 ] , named for the patient from whom it was fi rst isolated with non-a-e and g posttransfusion hepatitis in japan in 1997, is a newly discovered transfusion transmitted, single-stranded and circular dna virus [ 33 ] . ttv is nonenveloped and its entire sequence of ~3.9 kb has been determined. it is also often interpreted as a transfusion-transmitted virus [ 32 ] . at least 16 genotypes have been identi fi ed, and ttv is now found all over the world. ttv infection was sought by detection of ttv dna in serum by polymerase chain reaction using primers generated from a conserved region of the ttv genome, e.g., the utr region [ 34 ] . donor blood and blood product can be screened for ttv dna by using pcr or real-time pcr. the signi fi cance of positive fi ndings is still unclear, because high level ttv carriers in healthy populations are currently found [ 35, 36 ] . whether ttv actually causes hepatitis remains to be determined. cytomegalovirus (cmv) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. donor blood is not routinely tested for cmv, and the prevalence of cmv antibody ranges from 50 to 80 % of the population. but, blood contaminated with cmv can cause problems in neonates or immunocompromised patients. it also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis and colitis. potential problems in selected patient populations can be prevented by transfusing cmv negative blood or frozen, deglycerolized red blood cells. serologic tests for antibody to cmv are useful for determining whether a patient had cmv infection in the past, a determination of great clinical importance for organ and blood donors, and in the pretransplant evaluation of prospective transplant recipients [ 37 ] . commercial nat kits are available for cmv [ 3 ] , and these include the amplicor pcr cmv monitor test and hybrid capture system cmv dna test. sensitive screening tests for malaria are neither commercially available nor of fi cially approved yet. the most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria. donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. because there are no practical laboratory tests available to test donor blood, donors traveling to high risk malaria areas are excluded from donating blood for 6 months. however, there is a need to develop suitable screening tests, especially for use in an endemic area. a number of clinical research approaches have been developed for the extraction, ampli fi cation and detection of malaria parasite dna from blood products [ 37 ] . variant creutzfeldt-jakob disease (vcjd-a rare but fatal brain infection) [ 38 ] was fi rst described in 1996 in the united kingdom. vcjd is strongly linked with exposure to the bovine spongiform encephalopathy (bse) agent. bse is a transmissible spongiform encephalopathy (tse) affecting cattle and was fi rst reported in the uk in 1986. it has different clinical and pathologic characteristics from classic vcjd. each disease also has a particular genetic pro fi le of the prion protein gene. in recent years, questions have been raised concerning the potential risk of vcjd disease for recipients of plasma-derived clotting factors, including united states licensed factor eight (pdfviii), factor nine (pdfix), and other plasma-derived products such as immune globulins and albumin. in the past 10 years, there have been some reported cases of probable vcjd transmission by red blood cell transfusions in the united kingdom. prion infections are associated with long and clinically silent incubations. the number of asymptomatic individuals with vcjd prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. in order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vcjd prion infection. recently research papers have shown that sensitivity detection methods are available for vcjd prion [ 39 ] . however, commercial detection kits are not yet available. the dengue virus (denv) is a member of the virus family flaviviridae and is transmitted to people through the bite of an infected mosquito. the dengue virus has been shown to have four subtypes. these subtypes are different strains of dengue virus that have 60-80 % homology between each other. dengue has emerged as a worldwide problem only since the 1950s. with more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. according to cdc, as many as 100 million people are infected yearly. dengue is caused by any one of four related viruses transmitted by mosquitoes. there are not yet any vaccines to prevent denv infection, and the most effective protective measure is to avoid mosquito bites. there have been healthcare-related transmissions, including transmission by blood products [ 40 ] . dengue infection has a viremic phase that lasts 4-8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [ 40 ] . there are currently no tests for direct detection of dengue virus, but there are however, commercial elisa tests to detect antibodies of the dengue virus in blood samples from patients. recently, research papers have shown that pcr detection methods are available for any dengue virus strain [ 41 ] . babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis. babesiosis is a malaria-like parasitic disease, and there are over 100 species of babesia identi fi ed. in the united states, babesia microti is the agent most commonly reported to cause human infection. clinical confusion between human babesiosis and malaria is often reported in literature [ 42 ] . babesia infection can also be acquired by blood transfusion. in fact, there have been many cases of transfusioninduced babesiosis documented [ 43 ] . risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. current standards issued by the american association of blood banks (aabb) require the inde fi nite deferral of a blood donor with a history of babesiosis. [ 44 ] there is a need to develop methods for identi fi cation b. microti in order to reduce the risk of transmission of babesiosis by transfusion. diagnosis depends upon fi nding parasites on blood fi lm examination which can be detected 2-4 weeks after a tick bite. hamster inoculation and serology have also been used for diagnosis. the indirect fluorescent antibody test (ifat) is available for b. microti and is the most useful serological test for early diagnosis. also, the pcr screen tests for babesiosis are technically available in the fi eld [ 45 ] . chagas disease is named after the brazilian physician carlos chagas, who discovered the disease in 1909. chagas disease is spread mainly by blood-sucking insects infected with trypanosoma cruzi . chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. national screening of the blood supply was instituted in early 2007 by fda, and more than 1,000 donors with t. cruzi infection have been identi fi ed within the past 3 years of testing. "screening for t. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of chagas disease," says karen midthun, m.d., acting director of the fda's center for biologics evaluation and research. currently, serological elisa tests are available for diagnose chronic chagas disease. pcr test is not a tool for diagnosis of chronic chagas disease in clinical practice yet, although some research results have showed that pcr is a very sensitive parasitological test for chagas' disease in active transmission regions [ 46 ] . more studies are needed for the development of this molecular method. coronavirus is an rna virus known to be associated with respiratory disease. severe acute respiratory syndrome (sars) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from asia in 2003. there has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with sars is present in the blood of people who are sick, and it is possible that the virus could be present in blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit sars through a blood donation. to help determine whether or not an individual might be infected with sars, a blood collection facility will ask a potential donor orally or in writing about any travel to a sars-affected country or a history of sars or possible exposure to sars. enzyme-linked immunoassays for detection of speci fi c igg and igm antibodies and rt-pcr for detection of sars coronavirus speci fi c rna in the sars patients has been developed. rapid, sensitive, and speci fi c identi fi cation of sars and other novel coronaviruses by molecular methods will be very important in the future. based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non a-e, g, and ttv posttransfusion hepatitis. it is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. in recent years, numerous new infectious agents found worldwide have been identi fi ed through time-consuming procedures. by the time a new virus, such as hcv, hiv and sars, is found, many people are infected and there could be a large number of fatalities. there is an urgent need to develop methods for rapid identi fi cation and characterization of previously unknown pathogenic viruses. the most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (rda), and broad-range polymerase chain reaction (br-pcr). but they are all time-consuming approaches. the new unrecognized and uncharacterized viral agents can be rapid identi fi ed by some of the new molecular approaches, e.g., subtraction hybridization [ 47 ] and dna microarray. ensuring the safety and ef fi cacy of blood and blood products is a critical regulatory challenge. the high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. it will be achieved by introducing more updated nucleic acid tests to the fi eld of blood safety. nat is a method of testing blood that is more sensitive and speci fi c than conventional tests that require the presence of antibodies to trigger a positive test result. also, nat works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. this improved sensitivity should enable us to signi fi cantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. we are to protect the blood supply from not only known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents. the nat methods are more sensitive and speci fi c compared with non-nat. in the future, nat technology, such as pcr, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety. progress in blood supply safety emerging infectious disease issues in blood safety white tj (eds) molecular microbiology: diagnostic principles and practice evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents detection of piscine nodaviruses by real-time nucleic acid sequence based ampli fi cation (nasba) molecular-based methods for quantifying hiv viral load molecular diagnostics of infectious diseases dna, 50 years of the double helix: from the concept of molecular hybridization to microarrays in vitro nucleic acid ampli fi cation techniques multicenter evaluation of the performance characteristics of the nuclisens hiv-1 qt assay used for quantitation of human immunode fi ciency virus type 1 rna serological and molecular biology screening techniques for hcv infection clinical evaluation of an hiv-1 and hcv assay and demonstration of signi fi cant reduction of the hcv detection window before seroconversion hiv-1 monitor ultrasensitive quantitative assay. roche, nutley 14. lcx hiv rna quantitative assay from nested real-time pcr for hepatitis a detection introduction to microarray analysis development and validation of a diagnostic dna microarray to detect quinolone-resistant escherichia coli among clinical isolates testing requirements for communicable disease agents detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-ampli fi cation testing immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infection control practices advisory committee (hicpac) assessment of hepatitis b virus dna stability in serum by the chiron quantiplex branched-dna assay section two: speci fi c virus families recent advances in prevention and treatment of hepatitis c virus infections the scienti fi c challenge of hepatitis c hirsh fi eld i (2003) detection of extrahepatic hcv replication by a novel highly sensitive single tube nested-pcr section two: speci fi c virus families new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunode fi ciency virus type 1 rna in plasma human t-cell leukemia virus types 1 and 2 (chap. 58) estimated risk of transmission of the west nile virus through blood transfusion in the us hepatitis delta virus quanti fi cation of hepatitis delta virus rna in serum by consensus real-time pcr indicates different patterns of virological response to interferon therapy in chronically infected patients ttv-a virus searching for a disease a novel unenveloped dna virus (tt virus) associated with acute and chronic non-a to g hepatitis role of transfusion-transmitted virus in acute viral hepatitis and fulminant hepatic failure of unknown etiology a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology tt virus section two: speci fi c virus families emerging infectious disease agents and their potential threat to transfusion safety detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay threat of dengue to blood safety in dengue-endemic countries pcr detection of nearly any dengue virus strain using a highly sensitive primer cocktail do babesiosis and malaria share a common disease process? babesia infection through blood transfusions: reports received by the us food and drug administration transfusion-transmitted babesia spp.: bull's-eye on babesia microti detection of babesia species from infected dog blood by polymerase chain reaction pcr-based diagnosis for chagas' disease in bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis rapid approach to identify an unrecognized viral agent complete list of donor screening assays for infectious agents and hiv diagnostic assays key: cord-009263-w8bmmgtj authors: colpitts, che c.; tsai, pei-ling; zeisel, mirjam b. title: hepatitis c virus entry: an intriguingly complex and highly regulated process date: 2020-03-18 journal: int j mol sci doi: 10.3390/ijms21062091 sha: doc_id: 9263 cord_uid: w8bmmgtj hepatitis c virus (hcv) is a major cause of chronic hepatitis and liver disease worldwide. its tissue and species tropism are largely defined by the viral entry process that is required for subsequent productive viral infection and establishment of chronic infection. this review provides an overview of the viral and host factors involved in hcv entry into hepatocytes, summarizes our understanding of the molecular mechanisms governing this process and highlights the therapeutic potential of host-targeting entry inhibitors. recent estimates from the world health organization (who) indicate that approximately 71 million individuals are infected by the hepatitis c virus (hcv) worldwide [1] . there is no vaccine to prevent hcv infection. following infection, the majority of individuals will develop chronic hepatitis c that may subsequently lead to liver cirrhosis and cancer. although chronic hepatitis c can now be cured using direct-acting antivirals (daas), the majority of individuals with chronic hepatitis c remain undiagnosed and untreated. furthermore, a successful antiviral treatment does not prevent reinfection of patients with risk behaviors. the who has recently launched a global program to achieve hcv elimination and so far, only a minority of countries have implemented measures aiming at the elimination of hcv infection as a public health threat within the next decade(s) [2] . while increasing the number of diagnosed/treated cases and reducing risky behavior in defined populations will contribute to micro-elimination of hcv, global eradication of hcv remains challenging and will likely require a protective vaccine [3, 4] . hcv was discovered in 1989 and subsequently classified in the genus hepacivirus of the flaviviridae family of viruses [5] . this highly variable rna virus is further classified into six major genotypes that have distinct geographical distributions. the hcv genome encodes a polyprotein that is subsequently processed into three viral structural proteins that form the viral particle and seven non-structural proteins that are essential for viral replication. the structural proteins comprise the envelope glycoproteins e1 and e2 as well as the capsid protein core. the core protein and the viral rna form the nucleocapsid that is surrounded by a lipid envelope decorated with the e1 and e2 glycoproteins, which drive viral entry. in chronically infected patients, hcv particles circulate as "lipo-viro particles" (lvps), i.e., virions associated with low-density to very low-density lipoprotein (ldl, vldl) components including apolipoproteins b (apob) and e (apoe) [6-10]. by shielding the virus from neutralizing antibodies targeting the hcv envelope glycoproteins, the association of hcv with ldl/vldl components may contribute to viral evasion of host immune defenses. lvps appear to be dynamic structures and their composition is influenced by factors affecting lipid metabolism [11] . electron microscopy observation of viral particles recently showed the long-suspected ultrastructure of hcv [12] . in line with the results from mass spectrometry analyses of viral particles [13, 14] , electron microscopy confirmed that hcv particles are comprised of both viral and host factors [12, 15] . the hcv protease ns3 has also been found associated with hcv particles in proteomic studies [14] . viral entry is the first step of the viral life cycle and a major target for neutralizing antibodies preventing productive infection. researchers have aimed to identify the hcv receptor(s) and understand the hcv entry process for more than 20 years. increasing knowledge about the viral life cycle coupled with technological advances have enabled the development of ever more sophisticated model systems, allowing the discovery of key host factors essential for hcv entry, including those responsible for hcv tissue and species tropism (reviewed in [16, 17] ). deciphering their essential roles and interplay in hcv entry has led to the identification of targets for entry inhibitors and has provided clues for rational vaccine design (reviewed in [18, 19] ). this review provides an overview of the viral and host factors involved in hcv entry into hepatocytes and summarizes our current understanding of the molecular mechanisms governing this process. the interaction of hcv with hepatocytes leading to viral entry is largely dependent on the interaction of host lipoprotein components and viral envelope glycoproteins with host factors expressed at the hepatocyte surface. within the past two decades, researchers have identified an abundance of host factors involved in the processes leading from viral attachment to the hepatocyte to receptor-mediated endocytosis of the viral particle and endosomal fusion using various approaches (reviewed in [16, 17, 20] ). cluster of differentiation 81 (cd81), scavenger receptor class b type i (sr-bi), claudin-1 (cldn1) and occludin (ocln) are the four main host factors mediating hcv entry. indeed, expression of one or several of these host factors can confer cell susceptibility to infection by hcv [21] [22] [23] . while none of those factors individually confers tissue tropism to hcv, cd81 and ocln are responsible for the human species-specific tropism of hcv [22, 24, 25] . in addition to these four essential entry factors, additional host factors play a role in hcv attachment (attachment/binding factors) and internalization/fusion (co-factors). hcv can infect hepatocytes by two distinct routes, i.e., via cell-free virus entry or through cell-to-cell transmission. summarized below are the host factors and sequence of events leading from initial viral attachment to release of the hcv genome in the cytosol of hepatocytes for the cell-free virus entry pathway ( figure 1 ). hcv cell-to-cell transmission is described in section 5. schematic representation of the cell-free hepatitis c virus (hcv) entry pathway. this cartoon summarizes the host factors and sequence of events leading from initial viral attachment of lipo-viro particles (lvps) to hcv internalization and release of the viral genome in the cytosol of hepatocytes. the initial binding step primarily involving the lipoprotein component of lvps likely is a rather unspecific event, which results in the concentration of the virus at the basolateral membrane of hepatocytes and exposure of viral envelope glycoprotein domains that enable the virus to specifically interact with sr-bi, cd81, and cldn1 (post-binding). the formation of an hcv coreceptor complex is essential for subsequent viral internalization via clathrin-mediated and dynamindependent endocytosis. this process is highly regulated by various kinases. endocytotic vesicles ultimately mature into acidic endosomes, thus promoting low ph-dependent hcv fusion. hcv infection occurs via the parenteral route and hcv reaches the liver with the bloodstream. liver sinusoidal cells may then capture circulating lvps and facilitate viral infection of neighboring hepatocytes [26] [27] [28] [29] . the liver is the major organ of lipid homeostasis, and hepatocytes express several lipoprotein receptors at their surface. initial attachment of lvps to hepatocyte basolateral membranes likely involves virus-associated lipoprotein components (particularly apoe [9,13,30-35]), and virus envelope glycoproteins, which interact with highly sulfated heparan sulfate proteoglycans (hspg) [36] [37] [38] (particularly syndecans [39] [40] [41] ), ldl receptor (ldlr) [42] [43] [44] [45] and sr-bi [46] [47] [48] [49] [50] [51] on the cell surface ( figure 1 ). interestingly, in addition to their role in viral attachment, these host factors have been shown to also contribute to later steps of the viral life cycle, such as post-binding steps [52, 53] , internalization [54] or replication [55] . recently, tim-1/human hepatitis a virus cellular receptor 1 (havcr1)/cd365, a phosphatidylserine receptor that serves as a host factor for various flaviviridae viruses has been identified as an additional factor contributing to hcv attachment via interaction with phosphatidylserine exposed on the hcv envelope [56] . it has been suggested that hcv-tim-1 interaction may stabilize/enhance viral attachment and promote subsequent interaction with the main entry factors [57] . given the importance of virus-associated lipoprotein-derived components for the interaction with basolateral hepatocyte membranes, various lipoproteins have been shown to modulate these processes: e.g., high density lipoprotein (hdl) increases hcv figure 1 . schematic representation of the cell-free hepatitis c virus (hcv) entry pathway. this cartoon summarizes the host factors and sequence of events leading from initial viral attachment of lipo-viro particles (lvps) to hcv internalization and release of the viral genome in the cytosol of hepatocytes. the initial binding step primarily involving the lipoprotein component of lvps likely is a rather unspecific event, which results in the concentration of the virus at the basolateral membrane of hepatocytes and exposure of viral envelope glycoprotein domains that enable the virus to specifically interact with sr-bi, cd81, and cldn1 (post-binding). the formation of an hcv co-receptor complex is essential for subsequent viral internalization via clathrin-mediated and dynamin-dependent endocytosis. this process is highly regulated by various kinases. endocytotic vesicles ultimately mature into acidic endosomes, thus promoting low ph-dependent hcv fusion. hcv infection occurs via the parenteral route and hcv reaches the liver with the bloodstream. liver sinusoidal cells may then capture circulating lvps and facilitate viral infection of neighboring hepatocytes [26] [27] [28] [29] . the liver is the major organ of lipid homeostasis, and hepatocytes express several lipoprotein receptors at their surface. initial attachment of lvps to hepatocyte basolateral membranes likely involves virus-associated lipoprotein components (particularly apoe [9,13,30-35]), and virus envelope glycoproteins, which interact with highly sulfated heparan sulfate proteoglycans (hspg) [36] [37] [38] (particularly syndecans [39] [40] [41] ), ldl receptor (ldlr) [42] [43] [44] [45] and sr-bi [46] [47] [48] [49] [50] [51] on the cell surface ( figure 1 ). interestingly, in addition to their role in viral attachment, these host factors have been shown to also contribute to later steps of the viral life cycle, such as post-binding steps [52, 53] , internalization [54] or replication [55] . recently, tim-1/human hepatitis a virus cellular receptor 1 (havcr1)/cd365, a phosphatidylserine receptor that serves as a host factor for various flaviviridae viruses has been identified as an additional factor contributing to hcv attachment via interaction with phosphatidylserine exposed on the hcv envelope [56] . it has been suggested that hcv-tim-1 interaction may stabilize/enhance viral attachment and promote subsequent interaction with the main entry factors [57] . given the importance of virus-associated lipoprotein-derived components for the interaction with basolateral hepatocyte membranes, various lipoproteins have been shown to modulate these processes: e.g., high density lipoprotein (hdl) increases hcv pseudoparticle (hcvpp) entry and cell culture-derived hcv (hcvcc) infection while oxidized hdl/ldl inhibit hcvcc infection [58] [59] [60] . lipoprotein lipase, which has been reported to function as a bridge between virus-associated lipoproteins and hspg, can also modulate hcv infection [61] [62] [63] . furthermore, a recent study reported that long-chain fatty acyl-coenzyme a can inhibit hcv attachment by targeting virus-associated lipoproteins [64] . the initial attachment step primarily involving the lipoprotein component of lvps likely is a rather unspecific event, which serves to concentrate virions at the basolateral membrane of hepatocytes. it also leads to exposure of viral envelope glycoprotein domains that enable the virus to specifically interact with sr-bi, cd81, and cldn1 ( figure 1 ). these three cell surface factors act as viral receptors and contribute to viral entry in a temporally-regulated manner [21, 37, 52, 65, 66] . indeed, they not only directly bind the viral envelope glycoproteins [46, 67, 68] but also interact with each other [69] [70] [71] [72] [73] , thereby contributing to the formation of a hcv co-receptor complex that is essential for subsequent viral internalization. interestingly, several additional host factors that associate with these entry factors have been shown to also contribute to hcv entry (figure 1 ), through direct mechanisms or through indirect regulatory mechanisms (see section 4). hcv e2 is first thought to bind to the extracellular loop of sr-bi [46] . the lipid transfer activities of sr-bi [50] may facilitate exposure of binding sites on hcv e2, allowing for the transfer of the viral particle to cd81, which has key roles in subsequent entry steps. sr-bi itself also has a role in post-binding entry steps [53] . expression levels of sr-bi have been shown to define virus internalization rates, demonstrating the key role of sr-bi in hcv internalization [74, 75] . interestingly, while a splice variant of sr-bi (i.e., sr-bii) has also been demonstrated to promote hcvcc infection, its overexpression did not affect internalization rates, suggesting that sr-bi trafficking plays a role in hcv internalization [74] . high sr-bi surface expression may facilitate the assembly of protein complexes between cd81, sr-bi, and other hcv entry factors to drive internalization [75] . although initial engagement with sr-bi likely primes e2 for subsequent interactions with cd81, it was recently shown that a small fraction of virions are able to achieve entry in the absence of sr-bi [76] . cd81 is critical for post-binding steps of hcv entry, through its interactions with the hcv e2 glycoprotein [67] . binding to cd81 activates signaling pathways that promote virion internalization, including activation of receptor tyrosine kinases, such as the epidermal growth factor receptor (egfr) [77] , and rho and ras gtpases [71, 78] . activation of egfr leads to hras activation [71] . this has been reported to promote actin rearrangements, thus inducing lateral diffusion of cd81 to promote interaction with cldn1, a protein that is expressed both on the hepatocyte basolateral membrane and at tight junctions. the formation of the cd81-cldn1 co-receptor complex is a pre-requisite for hcv entry [69, 70] . the role of egfr in regulating the cd81-cldn1 association is essential for this process [77] . however, egfr has also been proposed to help recruit clathrin-coated vesicles to aid in hcv internalization [72] . consistently, egfr ligands have been shown to enhance the kinetics of hcv entry by inducing the endocytosis of egfr-cd81 complexes [79] . the kinase mknk1 has also been suggested to contribute to hcv entry downstream of egfr [80] , although the mechanisms remain to be elucidated. through these activities, egfr has a key role in regulating the hcv entry process. the association of cd81 with the tight junction protein cldn1 drives hcv internalization [21] . cldn1 is comprised of four transmembrane domains, with two extracellular loops (el1 and el2). residues within the small highly conserved el1 of cldn1 are key for viral entry [21, 81] , and genetic evidence has suggested a direct interaction between the hcv e1 glycoprotein and cldn1 [82] . furthermore, it has also been reported that e1-e2 complexes can interact cldn1 el1, whereas soluble e2 did not [68] . interestingly, other claudins appear to be capable of mediating hcv entry, in a genotype-dependent manner [83] . cldn6 and cldn9 are functional as hcv entry factors for some genotypes [84, 85] , and cldn12 was recently implicated in hcv entry as well [86] [87] [88] . ocln is another tight junction protein that facilitates hcv uptake at a post-binding step [22] , although the mechanisms remain less clear. like cldn1, ocln has four transmembrane domains, with two large extracellular loops (el1 and el2). ocln el2 has been shown to be essential in mediating hcv entry, possibly through its interactions with the endocytosis-promoting gtpase dynamin ii [89] . while ocln does not appear to interact directly with hcv particles, ocln acts at a similar step as cldn1 to enable hcv entry. multiple lines of evidence suggest that ocln is critical for a late, post-binding entry step [90, 91] . alternative entry routes have also been suggested. for example, it has been reported that the vldl receptor that mediates lipoprotein uptake into hepatocytes might enable hcv to enter hepatocytes in vivo in a cd81-independent manner [92] . since hepatoma cell lines used to study hcv entry do not express vldlr under classical culture conditions [92] , the role of this host factor has not yet been widely studied in vitro. while most of the hcv entry factors described herein were identified in the context of classical hepatoma cell lines, the recent development of more sophisticated systems has allowed the validation of these factors under conditions that more closely mimic the in vivo hepatic environment. for example, single particle imaging of polarized hepatoma organoids [72] recently showed that hcv localizes with sr-bi, cd81 and egfr at the basolateral membrane before actin-dependent trafficking to tight junctions, which for the most part fits nicely with the model proposed from studies in hepatoma cell lines. several other factors have been shown to contribute to hcv internalization, although the specific mechanisms still remain unclear. the abl tyrosine kinase was recently identified as a host factor for hcv entry, acting during clathrin-mediated endocytosis [93] . similarly, the transferrin receptor 1 (tfr1) plays a role in hcv particle uptake [94] , although the mechanisms and significance are still poorly understood. niemann-pick c1-like 1 (npc1l1) cholesterol absorption receptor was also identified as an hcv entry factor and likely contributes to hcv entry through its roles as a cholesterol receptor [95] . ultimately, interactions with these entry factors promote hcv internalization via clathrin-mediated and dynamin-dependent endocytosis [89, 96, 97] (figure 1) , although alternative endocytotic pathways may also play a role [98] . endocytotic vesicles ultimately mature into acidic endosomes, thus promoting low ph-dependent hcv fusion. low endosomal ph is critical to drive conformational rearrangement of the glycoproteins and exposes the fusion peptide. however, interactions of viral glycoproteins with cd81 are also thought to prime the viral particle for fusion by inducing conformational rearrangements in hcv e1 and e2 [99] . the hcv e1 and e2 glycoproteins form a noncovalent heterodimer that mediates fusion. three regions on the e1 and e2 glycoproteins (at positions 270 to 284, 416 to 430, and 600 to 620 on the hcv polyprotein) have been identified to play a role in the membrane fusion process [100] , but the fusion mechanism of hcv remains poorly defined. although hcv was expected to have a class ii fusion protein like other flaviviridae, the hcv fusion machinery does not resemble any other known fusion protein, suggesting hcv fusion to be a unique process compared to other fusion mechanisms. despite harboring a central immunoglobulin (ig)-fold domain common among class ii fusion proteins, the e2 crystal structure revealed a compact globular fold that is inconsistent with the highly-extended class ii fusion fold [101, 102] . given these disparities with known fusion proteins, it has been proposed that although e2 may mediate hcv entry through interactions with cellular factors, e1 is the hcv fusion protein. indeed, e1 forms trimers, a feature that is typical for fusion proteins, although e1 trimer formation was dependent on the co-expression of e2 [103] . furthermore, several studies have identified a hydrophobic region in e1 (csalyvgdlc) that may represent a putative fusion peptide [104] [105] [106] . an e1 deletion mutant (lacking residues 268-292) was defective in its ability to form fusion pores and thus rendered hcv pseudoparticles non-infectious [107] . another recent study identified a central hydrophobic region in e1 that controls requirements for low ph-dependent fusion [108] . however, the n-terminal domain of e1 does not possess the expected class ii fusion protein fold [109] and e1 is likely too small to connect viral and cellular membranes [110] . therefore, hcv fusion is most likely mediated by both e1 and e2, depending on intra-and intermolecular interactions to drive conformational rearrangements of the heterodimer required for fusion. consistent with this model, interactions between e1 and e2 are critical for entry [68, 111] and recent coevolution analysis revealed that e1 and e2 refold interdependently during fusion [112] . in a chaperone-like role, e2 likely supports the fusion properties of e1. the molecular details underlying precisely how the e1-e2 heterodimer mediates membrane fusion still remain to be elucidated. in contrast to the host entry factors that act as receptors/co-receptors, i.e., via direct interaction with the hcv envelope glycoproteins, others contribute to the regulation of viral entry without directly interacting with viral components, despite being essential for the viral entry process. cd81 and its interacting partners play key roles in regulating entry. cellular factors that regulate the localization and activity of tight junction proteins also affect hcv entry. several cd81-associated factors have been described [71, 73] that indirectly regulate hcv entry. as a tetraspanin cd81 forms large complexes with other membrane proteins and different proteomic approaches identified several cd81-associated factors that have roles in hcv entry, including hras, integrin î²1 (itgb1), ras-related protein rap2b, calpain-5 (capn5) and ubiquitin ligase casitas b-lineage lymphoma proto-oncogene b (cblb) [71, 73] . hras, through promotion of actin rearrangements, may promote the lateral diffusion of cd81 and its interaction with cldn1 [78] . additional cd81 binding partners involved in hcv entry include the serum response factor binding protein 1 (srfbp1), which is recruited to cd81 during hcv uptake [113] and is thought to act at a late post-binding entry step. collectively, these data suggest that complex tetraspanin networks may provide platforms for hcv entry and contribute to regulating this process. this is in line with studies having shown that in contrast to hepatocytes, other cell types which are not susceptible to hcv express distinct cd81 partners that restrict hcv entry [114] [115] [116] . interestingly, tetraspanin assemblies are emerging as key platforms regulating the entry of unrelated viruses [117] [118] [119] . localization of cldn1 at the cell surface to facilitate contact with cd81 is critical for hcv entry, and several factors that affect the localization of cldn1 are important in the hcv entry process (figure 1 ). cell surface localization of cldn1 (regulated by vesicular transport proteins such as sec24c) is associated with enhanced hcv entry [120] . tumor-associated calcium signal transducer 2 (tacstd2) interacts with cldn1 and ocln, and regulates their localization through protein kinase c (pkc)-mediated phosphorylation [121] . a recent study showed that serotonin 2a receptor (5-ht 2a r) controls cldn1 localization through protein kinase a (pka)-mediated phosphorylation [122] . serotonin receptor 6 (5-ht6) antagonists were similarly shown to mediate cldn1 localization in a pka-dependent (yet 5-ht6-independent) manner [123] . these findings are consistent with a pioneering study that demonstrated the importance of pka for cell surface localization of cldn1 [124] . e-cadherin is also an important regulator of the cell-surface localization and distribution of cldn1 and ocln [125] . the mechanisms described above are thought to apply to cell-free infection of hepatocytes by lvps distributed to the liver via the bloodstream. following this initial hepatocyte infection, hcv is thought to disseminate within the liver using different mechanisms, including cell-free infection following release of newly synthesized viral particles from infected hepatocytes as described above. however, cell-to-cell transmission from an infected hepatocyte to adjacent hepatocytes is critical for viral persistence in the liver [126] . in contrast to cell-free virus entry, hcv cell-to-cell transmission is resistant to the majority of neutralizing antibodies [126, 127] . however, this process can be targeted by a variety of entry inhibitors [53, 77, 128] . indeed, numerous host entry factors involved in cell-free hcv entry appear to be similarly involved in hcv cell-to-cell transmission. however, since this process has been less extensively studied than cell-free virus entry, the relative contribution of viral and host factors as well as their spatio-temporal interplay remains less characterized. several studies using various approaches have shown that cd81, sr-bi, cldn1, ocln, egfr, ephrin receptor a2 (epha2), npc1l1 and ldlr likely contribute to hcv cell-to-cell transmission [41, 77, 95, 126, 127] . as for cell-free entry, sr-bi-independent hcv cell-to-cell transmission has been reported [129] . studies using apoe-silenced donor cells demonstrated that apoe also plays an important role in this process [130] [131] [132] while apoe expressed by the recipient cells does not appear to be relevant for hcv cell-to-cell transmission [131, 132] . since apoe is required for a late step in the morphogenesis of viral particles and their infectivity [130, 133, 134] , this suggests that mature enveloped viral particles are transferred between adjacent hepatocytes. this is in line with data from a recent reporter-based live-cell visualization study using mutant viruses showing that hcv structural genes and p7 gene are essential for functional hcv cell-to-cell transmission [132] . despite these numerous similarities between cell-free entry and cell-to-cell transmission of hcv, differences in the molecular mechanisms underlying these distinct viral entry pathways have been reported. indeed, in contrast to cell-free hcv entry that appears to require cd81 (unless hypoxic culture conditions are used [92] ), cd81-independent cell-to-cell transmission has been described [135, 136] . furthermore, while apoe/vldl containing serum has been shown to inhibit cell-free hcv infection, it did not interfere with hcv cell-to-cell transmission [41] . this indicates that although viral and host factors involved in both hcv entry pathways are the same overall, subtle differences in virus-host interactions may exist between both entry routes. whether these in vitro observations have consequences for dissemination of hcv in vivo remains to be determined. notably, hcv rna containing exosomes have been reported [137] [138] [139] [140] [141] . however, whether and to what extent these exosomes transmit replication competent hcv genomes, proteins and/or virions remains a matter of debate [132, 140, 142] . until the approval of daa therapy in 2014, viral clearance rates using pegylated interferon î± and ribavirin (then the standard-of-care for chronic hepatitis c) were only~50%. therefore, researchers were actively investigating alternative antiviral strategies against hcv, including the development of entry inhibitors. as an essential prerequisite for productive viral infection, hcv entry is an attractive antiviral target, with several advantages. inhibiting viral entry prevents subsequent steps of the viral life cycle and limits viral dissemination. due to their mechanism of action, entry inhibitors represent an interesting strategy to prevent graft infection in hepatitis c patients undergoing liver transplantation and may also be valuable in the setting of transplantation of organs from hcv positive donors. entry inhibitors also protect cells from virus-induced modifications and may limit the emergence of resistant variants during viral replication. of note, entry inhibitors may synergize with daas as they act through complementary mechanisms of action [128, 143, 144] . entry inhibitors could therefore prove useful in combination therapy regimens. the numerous host factors involved in the hcv entry process offer several possible targets for antiviral intervention. indeed, several such entry factors-including monoclonal antibodies (mabs) and small molecule inhibitors-have been evaluated as antiviral targets (reviewed in [18, 20, 145] ) and some have reached preclinical development (table 1) . their clinical efficacy remains to be demonstrated. table 1 . host factors involved in hcv entry that have been suggested as antiviral targets. only host factors for which host-targeting entry inhibitors, e.g., monoclonal antibodies (mabs) or small molecule inhibitors have at least reached in vivo preclinical development are listed. a comprehensive list of host factors and their role(s) in hcv entry is provided in [17] . scavenger receptor bi (sr-bi) attachment, postbinding, cell-to-cell transmission anti-sr-bi mabs itx5061 [46] [47] [48] [49] [50] [51] [52] [53] 128, [146] [147] [148] cd81 postbinding, endocytosis, signaling, cell-cell transmission anti-cd81 mabs [37, 47, 67, 78, 96, [149] [150] [151] [152] claudin-1 (cldn1) postbinding, endocytosis, cell-cell transmission anti-cldn1 mabs [21, 66, 69, 70, 96, 143, 144, 153, 154] occludin (ocln) postbinding, endocytosis, cell-cell transmission anti-ocln mabs [22, 89, 90, [155] [156] [157] [158] [159] epidermal growth factor receptor (egfr) postbinding, endocytosis, signaling, cell-cell transmission anti-egfr mabs erlotinib [77, 79] niemann-pick c1-like 1 (npc1l1) postbinding, fusion, cell-cell transmission ezetimide [95] 5-ht 2a r endocytosis, fusion phenoxybenzamine [122] given its importance for public health and the limited approaches to manage hcv-infected patients prior to 2014, the field has developed highly relevant model systems and tools to study this virus. as a result of these efforts over the years, hcv entry is now a well-characterized process involving a tremendous array of host factors. deciphering the essential role played by host factors in viral entry has led to the development of host-targeting entry inhibitors (reviewed in [18, 20, 145] ), a class of antivirals that not only prevent hcv infection, but may also in some cases clear established hcv infection [154] . increasing knowledge about the viral and host determinants involved in virus-host interactions leading to viral entry also provided valuable information for the understanding of viral escape from neutralizing antibodies and the design of a protective vaccine (reviewed in [160, 161] , which is a challenge that still remains to be addressed for the global eradication of hcv [3, 4] . furthermore, what has been learned from the study of hcv entry may contribute to understanding the entry pathways of other, less well-characterized, viruses. funding: m.b.z. is supported by inserm, the university of lyon, the impulsion program of the idexlyon (anr-16-idex-0005-funding from the state managed by the french national research agency as part of the investments for the future program), and comitã© dã©partemental du rhã´ne de la ligue contre le cancer. c.c.c. is supported by a research initiation grant from queen's university. the authors declare no conflict of interest. inserm, the university of strasbourg and aldevron/genovac have filed patent applications on monoclonal antibodies targeting host factors and kinases inhibiting hcv infection as antiviral targets. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. proteomics of hcv virions reveals an essential role for the nucleoporin nup98 in virus morphogenesis ultrastructural analysis of hepatitis c virus particles hepatitis c virus entry hepatitis c virus entry: protein interactions and fusion determinants governing productive hepatocyte invasion. cold spring harb host-targeting agents to prevent and cure hepatitis c virus infection hepatitis c virus (hcv)-apolipoprotein interactions and immune evasion and their impact on hcv vaccine design hepatitis c virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies claudin-1 is a hepatitis c virus co-receptor required for a late step in entry human occludin is a hepatitis c virus entry factor required for infection of mouse cells reconstitution of the entire hepatitis c virus life cycle in non-hepatic cells completion of the entire hepatitis c virus life cycle in genetically humanized mice mice expressing minimally humanized cd81 and occludin genes support hepatitis c virus uptake in vivo l-sign (cd 209l) is a liver-specific capture receptor for hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dc-signr dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e2 altmeyer, r. c-type lectins l-sign and dc-sign capture and transmit infectious hepatitis c virus pseudotype particles characterization of the heparin binding sites in human apolipoprotein e the interaction of natural hepatitis c virus with human scavenger receptor sr-bi/cla1 is mediated by apob-containing lipoproteins infectivity of hepatitis c virus is influenced by association with apolipoprotein e isoforms human apolipoprotein e peptides inhibit hepatitis c virus entry by blocking virus binding hepatitis c virus attachment mediated by apolipoprotein e binding to cell surface heparan sulfate apolipoprotein e mediates attachment of clinical hepatitis c virus to hepatocytes by binding to cell surface heparan sulfate proteoglycan receptors viral and cellular determinants of the hepatitis c virus envelope-heparan sulfate interaction characterization of the early steps of hepatitis c virus infection by using luciferase reporter viruses the roles of cd81 and glycosaminoglycans in the adsorption and uptake of infectious hcv particles syndecan-1 serves as the major receptor for attachment of hepatitis c virus to the surfaces of hepatocytes syndecan 4 is involved in mediating hcv entry through interaction with lipoviral particle-associated apolipoprotein e attachment and postattachment receptors important for hepatitis c virus infection and cell-to-cell transmission hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor low density lipoprotein receptor as a candidate receptor for hepatitis c virus characterization of hepatitis c virus (hcv) and hcv e2 interactions with cd81 and the low-density lipoprotein receptor the low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis c virus the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus cell entry of hepatitis c virus requires a set of co-receptors that include the cd81 tetraspanin and the sr-b1 scavenger receptor scavenger receptor class b type i and hepatitis c virus infection of primary tupaia hepatocytes high-avidity monoclonal antibodies against the human scavenger class b type i receptor efficiently block hepatitis c virus infection in the presence of high-density lipoprotein receptor complementation and mutagenesis reveal sr-bi as an essential hcv entry factor and functionally imply its intra-and extra-cellular domains characterization of hepatitis c virus particle subpopulations reveals multiple usage of the scavenger receptor bi for entry steps scavenger receptor bi is a key host factor for hepatitis c virus infection required for an entry step closely linked to cd81 the postbinding activity of scavenger receptor class b type i mediates initiation of hepatitis c virus infection and viral dissemination hepatitis c virus infection propagates through interactions between syndecan-1 and cd81 and impacts the hepatocyte glycocalyx role of low-density lipoprotein receptor in the hepatitis c virus life cycle tim-1 promotes hepatitis c virus cell attachment and infection determinants in the ig variable domain of human havcr1 (tim-1) are required to enhance hepatitis c virus entry an interplay between hypervariable region 1 of the hepatitis c virus e2 glycoprotein, the scavenger receptor bi, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies high density lipoproteins facilitate hepatitis c virus entry through the scavenger receptor class b type i oxidized low-density lipoprotein inhibits hepatitis c virus cell entry in human hepatoma cells lipoprotein lipase mediates hepatitis c virus (hcv) cell entry and inhibits hcv infection lipoprotein lipase inhibits hepatitis c virus (hcv) infection by blocking virus cell entry lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis c virus (hcv) through their catalytic activities on hcv-associated lipoproteins long-chain fatty acyl-coenzyme a suppresses hepatitis c virus infection by targeting virion-bound lipoproteins different domains of cd81 mediate distinct stages of hepatitis c virus pseudoparticle entry inhibition of hepatitis c virus infection by anti-claudin-1 antibodies is mediated by neutralization of e2-cd81-claudin-1 associations binding of hepatitis c virus to cd81 critical interaction between e1 and e2 glycoproteins determines binding and fusion properties of hepatitis c virus during cell entry cd81 and claudin 1 coreceptor association: role in hepatitis c virus entry claudin association with cd81 defines hepatitis c virus entry hras signal transduction promotes hepatitis c virus cell entry by triggering assembly of the host tetraspanin receptor complex single particle imaging of polarized hepatoma organoids upon hepatitis c virus infection reveals an ordered and sequential entry process hepatitis c virus enters liver cells using the cd81 receptor complex proteins calpain-5 and cblb scavenger receptor bi and bii expression levels modulate hepatitis c virus infectivity hepatoma cell density promotes claudin-1 and scavenger receptor bi expression and hepatitis c virus internalization building a mechanistic mathematical model of hepatitis c virus entry egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy cd81 is a central regulator of cellular events required for hepatitis c virus infection of human hepatocytes hepatitis c virus induces epidermal growth factor receptor activation via cd81 binding for viral internalization and entry contrasting roles of mitogen-activated protein kinases in cellular entry and replication of hepatitis c virus: mknk1 facilitates cell entry residues in a highly conserved claudin-1 motif are required for hepatitis c virus entry and mediate the formation of cell-cell contacts selection of a hepatitis c virus with altered entry factor requirements reveals a genetic interaction between the e1 glycoprotein and claudins isolate-dependent use of claudins for cell entry by hepatitis c virus claudin-6 and claudin-9 function as additional coreceptors for hepatitis c virus the tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis c virus a genome-wide genetic screen for host factors required for hepatitis c virus propagation genetic dissection of flaviviridae host factors through genome-scale crispr screens copii cargo claudin-12 promotes hepatitis c virus entry the second extracellular loop dictates occludin-mediated hcv entry temporal analysis of hepatitis c virus cell entry with occludin directed blocking antibodies the tight junction-associated protein occludin is required for a postbinding step in hepatitis c virus entry and infection hepatitis c virus utilizes vldlr as a novel entry pathway abl tyrosine kinase regulates hepatitis c virus entry. front identification of transferrin receptor 1 as a hepatitis c virus entry factor identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor hepatitis c virus induces cd81 and claudin-1 endocytosis hepatitis c virus entry depends on clathrin-mediated endocytosis alternative endocytosis pathway for productive entry of hepatitis c virus hepatitis c virus is primed by cd81 protein for low ph-dependent fusion characterization of fusion determinants points to the involvement of three discrete regions of both e1 and e2 glycoproteins in the membrane fusion process of hepatitis c virus hepatitis c virus envelope glycoprotein e1 forms trimers at the surface of the virion proteomics computational analyses suggest that hepatitis c virus e1 and pestivirus e2 envelope glycoproteins are truncated class ii fusion proteins flunarizine prevents hepatitis c virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within e1 functional analysis of hepatitis c virus (hcv) envelope protein e1 using a trans-complementation system reveals a dual role of a putative fusion peptide of e1 in both hcv entry and morphogenesis the deletion of residues 268-292 of e1 impairs the ability of hcv envelope proteins to induce pore formation a central hydrophobic e1 region controls the ph range of hepatitis c virus membrane fusion and susceptibility to fusion inhibitors unexpected structure for the n-terminal domain of hepatitis c virus envelope glycoprotein e1 a novel membrane fusion protein family in flaviviridae? identification of interactions in the e1e2 heterodimer of hepatitis c virus important for cell entry a protein coevolution method uncovers critical features of the hepatitis c virus fusion mechanism quantitative proteomics identifies serum response factor binding protein 1 as a host factor for hepatitis c virus entry the cd81 partner ewi-2wint inhibits hepatitis c virus entry interacting regions of cd81 and two of its partners, ewi-2 and ewi-2wint, and their effect on hepatitis c virus infection ewi-2wint promotes cd81 clustering that abrogates hepatitis c virus entry the tetraspanin cd9 facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases tetraspanins: architects of viral entry and exit platforms sec24c-dependent transport of claudin-1 regulates hepatitis c virus entry infection with hepatitis c virus depends on tacstd2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma identification of serotonin 2a receptor as a novel hcv entry factor by a chemical biology strategy identification of piperazinylbenzenesulfonamides as new inhibitors of claudin-1 trafficking and hepatitis c virus entry protein kinase a-dependent step(s) in hepatitis c virus entry and infectivity hepatitis c virus depends on e-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies neutralizing antibody-resistant hepatitis c virus cell-to-cell transmission synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis c different requirements for scavenger receptor class b type i in hepatitis c virus cell-free versus cell-to-cell transmission apolipoprotein e codetermines tissue tropism of hepatitis c virus and is crucial for viral cell-to-cell transmission by contributing to a postenvelopment step of assembly apolipoprotein e, but not apolipoprotein b, is essential for efficient cell-to-cell transmission of hepatitis c virus visualizing the essential role of complete virion assembly machinery in efficient hepatitis c virus cell-to-cell transmission by a viral infection-activated split-intein-mediated reporter system apolipoprotein e likely contributes to a maturation step of infectious hepatitis c virus particles and interacts with viral envelope glycoproteins the association of hepatitis c virus glycoproteins with apolipoproteins e and b early in assembly is conserved in lipoviral particles cd81 is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells real-time imaging of hepatitis c virus infection using a fluorescent cell-based reporter system short-range exosomal transfer of viral rna from infected cells to plasmacytoid dendritic cells triggers innate immunity exosome-mediated transmission of hepatitis c virus between human hepatoma huh7.5 cells exosomes from hepatitis c infected patients transmit hcv infection and contain replication competent viral rna in complex with ago2-mir122-hsp90 virion-independent transfer of replication-competent hepatitis c virus rna between permissive cells knockdown of autophagy inhibits infectious hepatitis c virus release by the exosomal pathway look who's talking-the crosstalk between oxidative stress and autophagy supports exosomal-dependent release of hcv particles hepatitis c virus cell-cell transmission and resistance to direct-acting antiviral agents humanisation of a claudin-1-specific monoclonal antibody for clinical prevention and cure of hcv infection without escape host-targeting agents for prevention and treatment of chronic hepatitis c-perspectives and challenges role of scavenger receptor class b type i in hepatitis c virus entry: kinetics and molecular determinants small molecule scavenger receptor bi antagonists are potent hcv entry inhibitors novel human sr-bi antibodies prevent infection and dissemination of hcv in vitro and in humanized mice cd81 is required for hepatitis c virus glycoprotein-mediated viral infection diverse cd81 proteins support hepatitis c virus infection serum-derived hepatitis c virus infection of primary human hepatocytes is tetraspanin cd81 dependent a novel monoclonal anti-cd81 antibody produced by genetic immunization efficiently inhibits hepatitis c virus cell-cell transmission monoclonal anti-claudin 1 antibodies prevent hepatitis c virus infection of primary human hepatocytes clearance of persistent hepatitis c virus infection in humanized mice using a claudin-1-targeting monoclonal antibody species-specific regions of occludin required by hepatitis c virus for cell entry monoclonal antibodies against occludin completely prevented hepatitis c virus infection in a mouse model a novel occludin-targeting monoclonal antibody prevents hepatitis c virus infection in vitro characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin human-rat chimeric anti-occludin monoclonal antibodies inhibit hepatitis c virus infection designing a b cell-based vaccine against a highly variable hepatitis c virus hepatitis c virus vaccine: challenges and prospects. vaccines (basel) 2020, 8, 90 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-015941-4fz79wzf authors: hu, yuan title: molecular techniques for blood and blood product screening date: 2018-11-10 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-3-319-95111-9_2 sha: doc_id: 15941 cord_uid: 4fz79wzf blood product safety is a high priority for manufacturing industries, hospitals, and regulatory agencies. an important step in ensuring safety is the screening of donated blood for infectious diseases. molecular technologies for screening infectious diseases have improved remarkably over the years. molecular biological assay significantly reduced the risk of transfusion-transmitted infections. unlike previous methods, molecular technologies for screening infectious diseases are specific, efficient, easy to use, and economical. a new era in molecular biology is coming to the field of blood safety. direct detection of viral antigens and virus-specific antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. there are some limitations. for direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts sufficiently detectable in the body by an antibody-mediated assay. for indirect virus detection by virus-specific antibodies [e.g., an immunofluorescence assay or enzyme immunoassay (eia), etc.], there is a problem in that shortly after infection by a pathogenic virus, and there is a window period in which antibody generation is insufficient for detection [4] . to reduce this window period of low detection, direct nucleic acid tests are needed. through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. the nucleic acid tests can also provide evidence for genetic variation in viruses. molecular methods include the use of nucleic acid probes as well as amplification-based and dna sequence-based techniques. an increasing number of molecular diagnostic methods are now available commercially [2] . in comparison to classical methods, molecular biological methods are superior in terms of rapidness, specificity, and sensitivity. the current nucleic acid detection methods in the field may be grouped into two major classes: amplifying techniques such as pcr and non-amplifying techniques such as southern blot hybridization. amplifying techniques are more sensitive than non-amplifying techniques. there are two different types of amplifying methods [5] , target amplification methods and signal amplification methods. target amplifying techniques include pcr, nucleic acid sequence-based amplification (nasba) [6, 7] , self-sustaining sequence amplification (3sr), transcription-based amplification (tas), transcription-mediated amplification (tma), strand displacement amplification (sda), and ligase chain reaction (lcr). signal amplification methods include branched dna signal amplification (bdna) [8] , cleavage-based signal amplification (cycling probe technologies and invader assay), qß replicase, hybrid capture, cycling probe technologies (cpt), and rolling-circle amplification (rca) [9] . to further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing [2] [3] [4] [5] . southern blotting [10] was named after edward m. southern who developed this procedure at edinburgh university in the 1970s. this technique is used to detect specific sequences within mixtures of dna, which is size fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. after blocking of non-specific binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the specific dna sequence of interest. should specific dna be present on the blot, it will combine with the labeled probe and be detectable. in 1983, dr. kary mullis at cetus corporation conceived of polymerase chain reaction [11] . there is not a single technique that has had a greater impact on the practice of molecular biology than pcr. with this technique, we can detect infectious disease agents at an extremely low level. it is based on the ability of sense and antisense dna primers to hybridize to a dna of interest. following extension from the primers on the dna template by dna polymerase, the reaction is heat denatured and allowed to anneal with the primers once again. another round of extension leads to a multiplicative increase in dna products. therefore, a minute amount of dna can be efficiently amplified in an exponential fashion to result in larger amounts of dna that are more easily manipulated. by including critical controls, the technique can be made quantitative. the current level of the sensitivity and detection limit is as low as 10-50 copies per ml blood in hiv testing [1, 12, 13] . important clinical examples of the use of pcr are detection of hiv and hcv [14] [15] [16] . pcr techniques have evolved into different branches. some of them are now widely in use for virus detection in clinical diagnostics. these are real-time pcr by taqman (roche), lightcycler (roche), smartcycler (cepheid), in situ pcr, nested pcr, nested real-time pcr [17] , broad-range pcr, multiplex pcr, rt-pcr, arbitrarily primer pcr, long pcr, and quantitative pcr. real-time sequence technology will be coming soon for more detailed detection. in the past, identification of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (rflp). with real-time sequence technology, we will be able to detect a virus early as well as to obtain the viral sequence. microarrays were developed at stanford university by schena and co-workers in the early 1990s [18] . for medical applications, a microarray analysis offers a very accurate screening technology. it allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. it promises to be a fast and accurate diagnostic tool in the field of clinical microbiology and virology. applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. compared with existing technologies, it allows for a wider variety of specific tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. with the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identification of many types of pathogenic contaminants. in the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. it can provide vast information about the identity of blood-borne pathogens as well as their gene expression profiles [19] . to ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents [20] . all of the above tests are referred to as screening tests and are designed to detect as many infectious agents as possible. because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. in order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more specific tests called confirmatory tests. thus, confirmatory tests help determine whether a donor is truly infected. if any one of these tests fails, affected blood products are considered unsuitable for transfusion [20] . nucleic acid testing (nat) employs testing technology that directly detects the genomes of viruses. because nat detects a virus's genetic material instead of waiting for the body's response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [21] , thus further improving blood safety. nucleic acid testing is becoming the gold standard because of greater sensitivity compared to antibody tests [2] . since 1999, nat has been approved by the fda and used to detect hiv-1 and hcv; this technology currently is under investigation for detecting other infectious disease agents. we know that for many viral infections, viral rna appears very early in the infection, in 1 to 2 weeks, but the antibody doesn't appear until 10-12 weeks, e.g., hiv and hcv [21] . in order to virtually prevent infection by all the transfusion-associated viruses, we need to detect the viruses in their window period, and a nat or gene-based testing method is needed. nat also provides an opportunity for the viral, e.g., hiv or hcv, infected donor to seek early treatment. on the other hand, nat is not only a sensitive method but also a rapid method, which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical. the hepatitis b virus (hbv) is a highly infectious and often non-symptomatic virus that is transmitted primarily through blood and blood-derived fluids and is a leading cause of liver infection worldwide [22] . the world health organization (who) estimates that 2 billion people worldwide have been infected with hbv and 350,000,000 people are chronically infected. chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1000,000 deaths each year. each year up to 200,000 people become newly infected in the united states alone. since screening for hbv began in 1969, the rate of infection through blood transfusions has greatly decreased. however, as of 2000, hbv is still transmitted through blood transfusions in 1 out of 137,000 units of blood. one reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. serologic tests for hepatitis b virus include hepatitis b surface antigen (hbsag) and hepatitis b core antibody (hbcab). hbv, which mainly infects the liver, has an inner core and an outer envelope (the surface). the hbsag test detects the outer envelope, identifying an individual infected with the hepatitis b virus. this virus can cause inflammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. fortunately, most patients recover completely and test negative for hbsag within a few months after the illness. a small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. chronically infected people can develop severe liver disease as time passes and need to be followed carefully by an experienced physician. to reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hepatitis b surface antigen by the most sensitive and specific assays. blood donations that are found to be reactive in the hbsag test are automatically confirmed by the hbsag confirmatory assay. if the specimen is neutralizable in the confirmatory test, the specimen is considered positive for hbsag. hepatitis b surface antigen testing of donated blood has begun in 1975 (table 1) . currently, all blood donors are screened for hbsag, but occasional transmission of hbv still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). detection of early hbv infection of blood donors is still a major problem of blood transfusion. the current third-generation licensed hbsag tests (mostly radioimmunoassay and enzyme immunoassays) cannot detect hbv in the window period for hbv infection. this is a strong motivation for introducing molecular detection techniques to the field [23] . there are some commercially available test methods for detecting hbv dna in the market now, such as chiron's quantiplex hbv dna [24] , digene's hybrid capture, abbott's hbv dna assay, and roche's amplicor hbv monitor. using these commercial hybridization or pcr-based assays, hbv dna can be detected 1-3 weeks before the appearance of hbsag [25] . some chronically infected patients who have lost their hbsag remain hbv dna positive but are disqualified as potential blood donors. molecular detection of hbv dna is more sensitive than current methods employed for hbsag screening [22] [23] [24] [25] . determination of anti-hbc (total) is also used to monitor the progress of the hepatitis b viral infection. determination of anti-hbc (igm) is employed to distinguish an acute hepatitis b infection from a chronic infection. the anti-hbc test developed in 1987 detects an antibody to the hepatitis b virus that is produced during and after infection. if an individual has a positive anti-hbc test, but the hbsag test is negative, it may mean that the person once had hepatitis b but has recovered from the infection. of the individuals with a positive test for anti-hbc, many have not been exposed to the hepatitis b virus; thus, there is a frequent problem of false positives. although the individual may be permanently deferred from donating blood, it is unlikely that the person's health will be negatively affected. (note: this antibody is not produced following vaccination against hepatitis b [26] . the hepatitis c virus (hcv) is a member of the flaviviridae family of viruses, which are associated with both human and animal diseases [27] . hepatitis caused by hcv is the most common chronic blood-borne infection in the united states. over 4 million americans are believed to be infected. hcv can also be transmitted through blood transfusion. hcv causes inflammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver inflammation, cirrhosis, cancer, and death. eventually, up to 20% of people with hcv may develop cirrhosis of the liver or other severe liver diseases. as in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. because of the risk of serious illness, people with hcv need to be followed closely by a physician with experience evaluating this infection. since the full-length hcv cdna was first cloned in 1989, significant progress has been made in characterizing its molecular biology [13] . but, the natural history of hcv infection is still evolving, and current treatment options for patients are either limited or expensive [27] . there is no vaccine for hcv, and the current treatment includes a combination of alpha interferon and ribavirin as well as the combination of the nucleotide polymerase inhibitor sofosbuvir and the ns5a inhibitor velpatasvir [28] . although the former is efficacious in only a minority of patients [29] , the latter has been shown to be effective in a broad range of patients [28] . the life cycle of the hcv continues to be poorly understood due to the lack of an efficient cell culture system [30] . there is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hepatitis c virus. we recently established a cell culture system for the replication of hcv by using human t and b leukemia cell lines [31] . this model should represent a valuable tool for the detailed study of the initial steps of the hcv replication cycle and for the evaluation of evolving antiviral molecules. currently, appropriate vaccine strategies for hcv have not been developed. early detection and prevention of hcv infection are most important for blood safety. it is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the hcv genome because of a high mutation rate in this genomic region. however, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of hcv. in 1990, the first specific test for hepatitis c virus, the major cause of "non-a, non-b" hepatitis, was introduced. now, a third-generation elisa kit is available to detect antibodies to hcv, and screening blood for hcv antibodies is recommended. these assays are based on detection of serum antibody to various hcv antigens because these antibodies are nearly universally present in patients who are chronically infected with hcv [32] . the hcv screening tests are known to have significant limitations, and positive samples should be further tested by hcv confirmatory tests. guidelines provided by the cdc recommend that hcv antibody screening testpositive samples should be confirmed with serologic or nucleic acid supplemental testing. hcv confirmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. in this way, the response to individual antigens can be recognized, and some false-positive elisa results can be eliminated (e.g., riba, chiron hcv 3.0, and pcr assay) (e.g., roche cobas amplicor hcv test, version 2.0). laboratories can choose to perform this testing on all positive specimens or based on screening test-positive (signal to cutoff) ratios. the positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened. hcv antibodies are not generally detectable for at least 6 weeks and may not appear for several months. acute hcv infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. high-sensitivity detection of hcv during the window period is a long-term technical challenge in the field. tests for hcv rna genome detection based on the pcr or other highly sensitive rna detection systems have been used for the diagnosis of acute hepatitis [26] . sensitive detection of hcv rna based on rt-pcr or other nucleic acid amplification techniques can be readily accomplished with kits that are now available commercially. for example, in 1999 the fdaapproved roche's amplicor hiv-1 monitor ultrasensitive quantitative assay. it can measure hiv levels at as few as 50 copies/ml, and another commercial kit, the lcx hiv rna quantitative assay from abbott laboratories, also has a detection limit at 50 copies/ml. some studies even showed a sensitivity limit at one copy [33] . in fact, a qualitative assay should be much more sensitive than a quantitative assay for hiv/ hcv screening. a sensitive qualitative hcv molecular detection assay will possibly interdict and virtually prevent all transfusion-associated hiv/hcv. the current sensitivity standard for clinical diagnostics is 100 copies per ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies per ml. hiv-1 and/or hiv-2 virus cause acquired immunodeficiency syndrome or aids. the test is designed to detect antibodies directed against antigens of the hiv-1 or hiv-2 viruses. hiv-1 is much more common in the united states, whereas hiv-2 is prevalent in western africa. donors are tested for both viruses because both are transmitted by infected blood, and a few cases of hiv-2 have been identified in us residents. in 1985, the first blood screening eia test to detect hiv was licensed and quickly implemented by blood banks to protect the blood supply. in 1992, testing of donor blood for both hiv-1 and hiv-2 antibodies (anti-hiv-1 and anti-hiv-2) was implemented. in 1996, hiv p24 antigen testing of donated blood was mandated. now, the p24 antigen testing is going to be compared with a pcr-based test for their ability to detect hiv in the window period. htlv retroviruses are endemic in japan and the caribbean but relatively uncommon in the united states [34] . they cause adult t-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. the infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. in rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. htlv-ii infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. disease associations with htlv-ii have been hard to confirm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections or rare cases of neurological disease. in 1989, human t-lymphotropic virus antibody testing of donated blood was begun. blood is now routinely screened for antibodies to htlv-i/ii. these test screens for antibodies directed against epitopes of the htlv-i and htlv-ii viruses. several commercial assays based on the enzyme-linked immunosorbent assay (elisa) or particle agglutination formats are used for screening of htlv antibodies, followed by confirmatory assays using western blotting. in some infected individuals, the serologic response to htlv infection is very low. these problems have been solved by the application of pcr amplification of specific sequences in the virus genome. pcr can be used to detect htlv-i/ii proviruses and is now the method of choice for detection of htlv dna directly from blood and many other tissues. commercial pcr kits for htlv are available [34] . the west nile virus (wnv) is a single-stranded rna virus of the flaviviridae family and is one of the most recent emerging infectious disease threats to public health and, potentially, to the safety of our blood supply [35] [36] [37] . in 2002, wnv was identified as transfusion transmissible. it is transmitted by mosquitoes to birds and other animals through a mosquito bite. the virus can infect people, horses, many types of birds, and some other animals. wnv was shown in 2002 to be transmissible by blood [35] , with an estimated mean risk of 2/10,000-5/10,000 in outbreak regions in the united states. the most common symptoms of transfusion-transmitted cases of wnv were fever and headache. detection of wnv includes either a measurement of wnv antibodies or of wnv nucleic acid (detecting genetic material from the virus itself). there are two types of wnv antibody testing: igm and igg. in most individuals, igm antibodies will be present within 8 days after the initial exposure to wnv, followed by igg production several weeks later. but, the antibodies tested to detect wnv are not expedient for donor blood screening. nucleic acid testing involves amplifying and measuring the west nile virus's genetic material to detect the presence of the virus in the blood or tissue. wnv nat will be negative in the blood once clinical illness has occurred. in this situation, both nat and igm antibody testing may be needed. nucleic acid tests to screen blood for wnv are commercially available and in current use. but, the viral yield for wnv infection is much lower than other viruses. consequently, a more sensitive wnv nat system for donor blood screening will be required, which could further reduce the risks of transfusiontransmitted wnv. serum samples from all blood units should be subjected to either the vdrl (venereal disease research laboratory) test or a treponemal test, such as the treponema pallidum hemagglutination (tpha) test before transfusion. any unit found positive should be discarded as per standard safety procedures. this test is done to detect evidence of infection with the spirochete that causes syphilis. blood centers began testing for this shortly after world war ii, when syphilis rates in the general population were much higher. the risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors and because the spirochete is fragile and unlikely to survive blood storage conditions. sensitivity and specificity of serologic tests vary depending on the type of test performed and the stage of the disease. if the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. syphilis serological tests for donors have less clinical significance. a nucleic acid test for accurately detecting syphilis is needed. it can be used to determine whether a blood donor is currently or has recently been infected with the spirochete. in recent years, numerous infectious agents found worldwide have been identified as potential threats to the blood supply, and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. even if the hepatitis virus test is negative for all known a-e hepatitis agents, there are some unidentified hepatitis viruses, called non-a-e hepatitis viruses that can still be transmitted by blood transfusion. in the future, advances in nat may allow rapid discovery of the unknown hepatitis viruses. hepatitis delta virus (hdv) is a small rna virus that can infect only individuals who have hbv; worldwide more than 15 million people are coinfected [38] . hdv is clinically important because it generally makes hbv infections more damaging to the liver. increased understanding of the molecular virology of hdv will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. pcr and real-time pcr methods are available for hdv rna detection [39] . tt virus (ttv) [40] , named for the patient from whom it was first isolated with non-a-e and g posttransfusion hepatitis in japan in 1997, is a newly discovered transfusion-transmitted, single-stranded and circular dna virus [41] . ttv is nonenveloped, and its entire sequence of ~3.9 kb has been determined. it is also often interpreted as a transfusion-transmitted virus [42] . at least 16 genotypes have been identified, and ttv is now found all over the world. ttv infection was sought by detection of ttv dna in serum by polymerase chain reaction using primers generated from a conserved region of the ttv genome, e.g., the utr region [42] . donor blood and blood product can be screened for ttv dna by using pcr or real-time pcr. the significance of positive findings is still unclear, because high-level ttv carriers in healthy populations are currently found [42] [43] [44] . whether ttv actually causes hepatitis remains to be determined. cytomegalovirus (cmv) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. donor blood is not routinely tested for cmv, and the prevalence of cmv antibody ranges from 50 to 80% of the population. but, blood contaminated with cmv can cause problems in neonates or immunocompromised patients. it also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis, and colitis. potential problems in selected patient populations can be prevented by transfusing cmv negative blood or frozen, deglycerolized red blood cells. serologic tests for antibody to cmv are useful for determining whether a patient had cmv infection in the past, a determination of great clinical importance for organ and blood donors and in the pretransplant evaluation of prospective transplant recipients [45] . commercial nat kits are available for cmv [5] , and these include the amplicor pcr cmv monitor test and hybrid capture system cmv dna test. chagas disease is caused by the blood-borne parasite, trypanosoma cruzi, which is transmitted to humans through insects. in the united states, chagas disease is considered one of the neglected parasitic infections, a group of five parasitic diseases that have been targeted by cdc for public health action [46] . commercial anti-t. cruzi assay kits are available for the qualitative detection of antibodies, trypanosoma cruzi (t. cruzi), the causative agent of chagas disease in human serum and plasma specimens by abbott diagnostics [47] . sensitive screening tests for malaria are neither commercially available nor officially approved yet. the most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria [48] . donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. because there are no practical laboratory tests available to test donor blood, donors traveling to high-risk malaria areas are excluded from donating blood for 6 months. however, there is a need to develop suitable screening tests, especially for use in an endemic area. a number of clinical research approaches have been developed for the extraction, amplification, and detection of malaria parasite dna from blood products [49] . variant creutzfeldt-jakob disease [50] (vcjd, a rare but fatal brain infection) was first described in 1996 in the united kingdom. vcjd is strongly linked with exposure to the bovine spongiform encephalopathy (bse) agent. bse is a transmissible spongiform encephalopathy (tse) affecting cattle and was first reported in the united kingdom in 1986. it has different clinical and pathologic characteristics from classic cjd. each disease also has a particular genetic profile of the prion protein gene [51] . in recent years, questions have been raised concerning the potential risk of variant creutzfeldt-jakob disease for recipients of plasma-derived clotting factors, including the united states licensed factor viii (pdfviii), factor ix (pdfix), and other plasma-derived products such as immune globulins and albumin. in the past 10 years, there have been some reported cases of probable variant creutzfeldt-jakob disease (vcjd) transmission by red blood cell transfusions in the united kingdom [52] . prion infections are associated with long and clinically silent incubations [50, 51] . the number of asymptomatic individuals with vcjd prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. in order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vcjd prion infection [52] . recently research papers have shown that sensitivity detection methods are available for vcjd prion [53] [54] [55] . however, commercial detection kits are not yet available. the dengue virus (denv) is a member of the virus family flaviviridae and is transmitted to people through the bite of an infected mosquito [56] . the dengue virus has been shown to have four subtypes. these subtypes are different strains of dengue virus that have 60-80% homology between each other. dengue has emerged as a worldwide problem only since the 1950s. with more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. according to cdc, as many as 100 million people are infected yearly [57] . dengue is caused by any one of the four related viruses transmitted by mosquitoes. there are not yet any vaccines to prevent denv infection, and the most effective protective measure is to avoid mosquito bites. there have been healthcare-related transmissions, including transmission by blood products [58] . dengue infection has a viremic phase that lasts 4-8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [58] . there are currently no tests for direct detection of dengue virus, but there are, however, commercial elisa tests to detect antibodies of the dengue virus in blood samples from patients [59] . recently, research papers have shown that pcr detection methods are available for any dengue virus strain [57, 60] . babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis [61] . babesiosis is a malaria-like parasitic disease [62] , and there are over 100 species of babesia identified [63] . in the united states, babesia microti is the agent most commonly reported to cause human infection. clinical confusion between human babesiosis and malaria is often reported in literature [62] . babesia infection can also be acquired by blood transfusion [64, 65] . in fact, there have been many cases of transfusion-induced babesiosis documented [64, 65] . risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. current standards issued by the american association of blood banks (aabb) require the indefinite deferral of a blood donor with a history of babesiosis [65] . there is a need to develop methods for identification babesia microti in order to reduce the risk of transmission of babesiosis by transfusion. diagnosis depends upon finding parasites on blood film examination which can be detected 2-4 weeks after a tick bite. hamster inoculation and serology have also been used for diagnosis. the indirect fluorescent antibody test (ifat) is available for b. microti and is the most useful serological test for early diagnosis [66] . also, the pcr screen tests for babesiosis are technically available in the field [67] . chagas disease is named after the brazilian physician carlos chagas, who discovered the disease in 1909 [68] . chagas disease is spread mainly by blood-sucking insects infected with trypanosoma cruzi. chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. national screening of the blood supply [69] was instituted in early 2007 by fda, and more than 1000 donors with t. cruzi infection have been identified within the past 3 years of testing. "screening for t. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of chagas disease," says karen midthun, m.d., acting director of the fda's center for biologics evaluation and research. currently, serological elisa tests are available to diagnose chronic chagas disease [70] . pcr test is not a tool for diagnosis of chronic chagas disease in clinical practice yet, although some research results have showed that pcr is a very sensitive parasitological test for chagas disease in active transmission regions [71] . more studies are needed for the development of this molecular method. coronavirus is an rna virus known to be associated with respiratory disease. severe acute respiratory syndrome (sars) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from asia in 2003. there has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with sars is present in the blood of people who are sick, and it is possible that the virus could be present in the blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit sars through a blood donation. to help determine whether or not an individual might be infected with sars, a blood collection facility will ask a potential donor orally or in writing about any travel to a sars-affected country or a history of sars or possible exposure to sars. enzyme-linked immunoassays for detection of specific igg and igm antibodies and rt-pcr for detection of sars coronavirus-specific rna in the sars patients have been developed. rapid, sensitive, and specific identification of sars and other novel coronaviruses by molecular methods will be very important in the future. ebola virus disease (evd) is a rare and deadly disease caused by infection with one of the ebolavirus species [72] . the recent outbreak in 2014-2015 is the largest ebola outbreak since the ebola virus was first discovered in 1976, first in yambuku, democratic republic of congo, and then in nzara, south sudan. the virus family filoviridae includes three genera: cuevavirus, marburgvirus, and ebolavirus. there are five species that have been identified: zaire, bundibugyo, sudan, reston, and taï forest. the first three, bundibugyo ebolavirus, zaire ebolavirus, and sudan ebolavirus, have been associated with large outbreaks in africa. the virus responsible for causing the 2014 west african outbreak belongs to the zaire species (who) [72] . samples from patients are an extreme biohazard risk. currently, a number of approaches have been developed and are available for diagnoses of ebola virus disease: (1) antibody-capture enzyme-linked immunosorbent assay (elisa), (2) antigen-capture detection tests, (3) serum neutralization test, (4) reverse transcriptase polymerase chain reaction (rt-pcr) assay, (5) electron microscopy, and (6) virus isolation by cell culture tests. the who and fda are working to help expedite the development and availability of medical products -such as treatments, vaccines, diagnostic tests, and personal protective equipment -with the potential to help bring the ebola epidemic in west africa under control as quickly as possible (fda) [73] . based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non-a-e, g, and ttv posttransfusion hepatitis. it is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. in recent years, numerous new infectious agents found worldwide have been identified through time-consuming procedures. by the time a new virus, such as hcv, hiv, and sars, is found, many people are infected, and there could be a large number of fatalities. there is an urgent need to develop methods for rapid identification and characterization of previously unknown pathogenic viruses. the most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (rda), and broad-range polymerase chain reaction (br-pcr). but they are all time-consuming approaches. the new unrecognized and uncharacterized viral agents can be rapidly identified by some of the new molecular approaches, e.g., subtraction hybridization [74] and dna microarray. ensuring the safety and efficacy of blood and blood products is a critical regulatory challenge. the high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. it will be achieved by introducing more updated nucleic acid tests to the field of blood safety [2, 3] . nucleic acid testing is a method of testing blood that is more sensitive and specific than conventional tests that require the presence of antibodies to trigger a positive test result. also, nat works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. this improved sensitivity should enable us to significantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. we are not only to protect the blood supply from known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents [4, 36, 37, 75] . the nat methods are more sensitive and specific compared with non-nat. in the future, nat technology, such as pcr, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety. progress in blood supply safety advances in testing technology to ensure transfusion safety -nat and beyond the safety of the blood supply -time to raise the bar emerging infectious disease issues in blood safety in vitro nucleic acid amplification techniques evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (nasba) molecular-based methods for quantifying hiv viral load molecular diagnostics of infectious diseases 50 years of the double helix: from the concept of molecular hybridization to microarrays in vitro nucleic acid amplification techniques. diagnostic molecular microbiology principles and applications. mayo foundation multicenter evaluation of the performance characteristics of the nuclisens hiv-1 qt assay used for quantitation of human immunodeficiency virus type 1 rna serological and molecular biology screening techniques for hcv infection clinical evaluation of an hiv-1 and hcv assay and demonstration of significant reduction of the hcv detection window before seroconversion roche amplicor hiv-1 monitor ultrasensitive quantitative assay quantitative assay from abbott laboratories nested real-time pcr for hepatitis a detection introduction to microarray analysis. microarray analysis development and validation of a diagnostic dna microarray to detect quinolone-resistant escherichia coli among clinical isolates food & drug administration. testing requirements for communicable disease agents detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-amplification testing update on hepatitis b virus infection challenges in hepatitis b detection among blood donors assessment of hepatitis b virus dna stability in serum by the chiron quantiplex branched-dna assay hepatitis b virus. section two: specific virus families immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infection control practices advisory committee (hicpac) hepatitis c virus: virology, diagnosis and treatment sofosbuvir and velpatasvir for hcb genotype 1, 2, 4, 5, and 6 infection recent advances in prevention and treatment of hepatitis c virus infections the scientific challenge of hepatitis c detection of extrahepatic hcv replication by a novel highly sensitive single tube nested-pcr chapter 34: hepatitis c viruses. section two: specific virus families new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunodeficiency virus type 1 rna in plasma chapter 58: human t-cell leukemia virus types 1 and 2. section two: specific virus families estimated risk of transmission of the west nile virus through blood transfusion in the us transfusion-transmitted emerging infectious diseases: 30 years of challenges and progress the potential treat to blood transfusion safety of emerging infectious disease agents hepatitis delta virus. the lancet, early online publication quantification of hepatitis delta virus rna in serum by consensus real-time pcr indicates different patterns of virological response to interferon therapy in chronically infected patients ttv-a virus searching for a disease a novel unenveloped dna virus (tt virus) associated with acute and chronic non-a to g hepatitis role of transfusion-transmitted virus in acute viral hepatitis and fulminant hepatic failure of unknown etiology a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology chapter 77: cytomegalovirus. section two: specific virus families evaluation of a prototype trypanosoma cruzi antibody assay with recombinant antigens on a fully automated chemiluminescence analyzer for blood donor screening transfusion-transmitted malaria in countries where malaria is endemic: a review of the literature from sub-saharan africa detection and species identification of malaria parasites by isothermal thda amplification directly from human blood without sample preparation 18 years of research and surveillance prions: beyond a single protein crerutzfeldt-jakob disease and blood transfusion: updated results of the uk transfusion medicine epidemiology review study detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay preclinical detection of variant cjd and bse prions in blood quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus threat of dengue to blood safety in dengueendemic countries comparison of a commercial igm capture elisa with dengue antigen focus reduction microneutralization test and the centers for disease control dengue igm capture-elisa pcr detection of nearly any dengue virus strain using a highly sensitive primer cocktail do babesiosis and malaria share a common disease process? babesia: a world emerging babesia infection through blood transfusions: reports received by the us food and drug administration transfusion-transmitted babesia spp.: bull's-eye on babesia microti continuous in vivo culture and indirect fluorescent antibody test for zoonotic protozoa of babesia microti detection of babesia species from infected dog blood by polymerase chain reaction silver spring, m.d. 20993. complete list of donor screening assays for infectious agents and hiv diagnostic assays elisa versus pcr for diagnosis of chronic chagas disease: systematic review and meta-analysis pcr-based diagnosis for chagas' disease in bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis virus disease updated ebola response updates from fda rapid approach to identify an unrecognized viral agent emerging infectious disease agents and their potential threat to transfusion safety key: cord-002410-2zi5iv2t authors: bruening, janina; weigel, bettina; gerold, gisa title: the role of type iii interferons in hepatitis c virus infection and therapy date: 2017-02-01 journal: j immunol res doi: 10.1155/2017/7232361 sha: doc_id: 2410 cord_uid: 2zi5iv2t the human interferon (ifn) response is a key innate immune mechanism to fight virus infection. ifns are host-encoded secreted proteins, which induce ifn-stimulated genes (isgs) with antiviral properties. among the three classes of ifns, type iii ifns, also called ifn lambdas (ifnls), are an essential component of the innate immune response to hepatitis c virus (hcv). in particular, human polymorphisms in ifnl gene loci correlate with hepatitis c disease progression and with treatment response. to date, the underlying mechanisms remain mostly elusive; however it seems clear that viral infection of the liver induces ifnl responses. as ifnl receptors show a more restricted tissue expression than receptors for other classes of ifns, ifnl treatment has reduced side effects compared to the classical type i ifn treatment. in hcv therapy, however, ifnl will likely not play an important role as highly effective direct acting antivirals (daa) exist. here, we will review our current knowledge on ifnl gene expression, protein properties, signaling, isg induction, and its implications on hcv infection and treatment. finally, we will discuss the lessons learnt from the hcv and ifnl field for virus infections beyond hepatitis c. interferons (ifn) are innate cytokines, which interfere with virus infections. while type i ifns were discovered in the 1950s, it was not until 2003 that the first type iii ifns, namely, ifn lambda 1 (ifnl1), lambda 2 (ifnl2), and ifn lambda 3 (ifnl3), were described [1, 2] . the most recent member of the type iii ifns, ifn lambda 4 (ifnl4), was discovered even ten years later [3, 4] . all four ifnls are encoded on chromosome 9 in the 19q13.13 region. infls share their open reading frame structure with the interleukin-10 (il-10) family of cytokines comprising five exons and four introns [5] [6] [7] . therefore, they are also termed il-29 (ifnl1), il-28a (ifnl2), and il-28b (ifnl3). ifnl1 through ifnl3 have a high degree of sequence similarity with 72% to 96% amino acid conservation with ifnl2 and ifnl3 being most closely related. these findings suggest a common ancestor gene for all ifnls [3] . ifnl4 expression is the consequence of a frameshift mutation and this gene product shares 27% to 29% sequence similarity with the other three ifnls (table 1 and figure 1 ). ifnl1-3 proteins are roughly 22 kda in size, while ifnl4 is slightly smaller with 20 kda. they share an alpha helical bundle structure with type i and type ii ifn family members. significant differences occur in the side chains of ifnl1, ifnl2, and ifnl3 and amino acid differences at the receptor binding site likely contribute to the differences in ifnl responses as detailed below. the expression of ifnl genes is tightly controlled and expression profiles of ifnl subtypes are ligand and tissue specific [8] . typically, rna virus infection and the concomitant exposure of cells to foreign rna in cytoplasmic or endosomal compartments lead to ifnl induction. in particular, sindbis virus, dengue virus, vesicular stomatitis virus, encephalomyocarditis virus [1, 2] , respiratory syncytial virus [9, 10] , influenza virus, sendai virus [11, 12] , and hepatitis c virus (hcv) [13] [14] [15] were shown to induce ifnls in vitro and in vivo. in addition to rna viruses, dna viruses including cytomegalovirus and herpes simplex virus can induce ifnls [16, 17] . while almost any cell type can express ifnls, the most prominent producers b ifnl4_human ifnl1_human ifnl2_human ifnl3_human ifnl4_human ifnl1_human ifnl2_human ifnl3_human ifnl4_human ifnl1_human ifnl2_human ifnl3_human ifnl4_human ifnl1_human ifnl2_human ifnl3_human ----maaawtvvlvtlvlglavagpvptsk---ptttgkgchigrfkslspqelasfkka ------------------------------------------------------------mkldmtgdctpvlvlmaavltvtgavpvarlhgalpdargchiaqfkslspqelqafkra ----mtgdcmpvlvlmaavltvtgavpvarlrgalpdargchiaqfkslspqelqafkra rdaleeslklknwscsspvfp-gnwdlrllqvrerpvaleaelaltlkvleaa--agpal kdaleeslllkdcrchsrlfp-rtwdlrqlqvrerpmaleaelaltlkvleatadtdpal kdaleeslllkdckcrsrlfp-rtwdlrqlqvrerpvaleaelaltlkvleatadtdpal : : : : : : . . [37] of ifnl proteins (ids: q8iu54, q8izj0, q8izi9, and k9m1u5). exons are indicated by the black and white boxes below the sequences. positions of helices are indicated by the lines above the sequences. identical amino acids are marked by an asterisk ( * ); conserved amino acids by a colon (:); and semiconserved amino acids by a period (.). of these antiviral cytokines are myeloid and plasmacytoid dendritic cells [8, [18] [19] [20] [21] . tissues with strong ifnl induction upon virus infection are the lung and the liver with a strong contribution of airway epithelial cells and hepatocytes [15, [22] [23] [24] [25] [26] [27] [28] [29] . limited data is available on the expression kinetics of ifnls in different cell types. it seems, however, that ifnl expression onset and duration differ for the four subtypes. for instance, primary human hepatocytes (phh) carrying the single nucleotide polymorphism (snp) responsible for ifnl4 expression show an early and short ifnl4 expression (2 to 6 h after stimulation), while ifnl3 was detectable from 2 to 24 h after stimulation with a synthetic poly i:c rna ligand [4] . differences in positive or negative feedback mechanisms may explain the varying expression kinetics for ifnl subtypes. ifnl1 through ifnl3 are typically induced simultaneously and this is reflected by common transcription factors and binding sites in the promoter regions. activator protein 1, ifn response factor 3 (irf3), irf7, and nuclear factor kappa beta (nf-kb) are thought to bind to the promoter of all infl genes [11, 12, [30] [31] [32] [33] [34] [35] [36] . additionally, med23 seems to be a transcriptional coactivator [17] . taken together, ifnls are induced upon sensing of virus infection in particular after lung and liver infection. the receptor for all four ifnls is composed of two subunits, the alpha-subunit ifnlr1 encoded on chromosome 1 and the beta-subunit il10rb encoded on chromosome 21 [40] [41] [42] [43] [44] . the former is specific for the ifnl receptor (ifnlr), while the latter is shared with the type ii cytokine receptors for il-10, il-22, and il-26 [45] . restricted expression of the ifnlr1 subunit leads to a tissue specific response to ifnls. in particular tissues with high epithelial cell content like intestine, liver, and lung express ifnlr1 and respond to ifnls [46] . apart from primary human hepatocytes, hepatocellular carcinoma cell lines including huh-7 and hepg2 cells respond to ifnl [14, 47] . in addition to the full length ifnlr1, a secreted form lacking exon vi has been described and may function as a decoy receptor dampening ifnl responses [2, 48, 49] . the ifnl ligand-receptor interface is comprised of helix a, loop ab, and helix f for ifnl and the n-terminal domain as well as the interdomain hinge region for the ifnlr. van der waals and hydrophobic forces determine the ligandreceptor interaction [40] . amino acids critical for receptor binding differ between ifnl subtypes and this might lead to different ligand binding affinities as well as differences in the stability of the ligand-receptor complex [4, 50] . additionally, mutations in the ifnl3 and ifnl1 receptor binding sites journal of immunology research 3 have been described with the ifnl4 generating frameshift mutation being the best described [40, 41, 50] . the impact of these genetic variants is discussed in detail below. taken together all four ifnl proteins share the same cell surface receptor, which is primarily expressed in intestine, lung, and liver tissue. signaling in response to ifnls is initiated by dimerization of the two ifnlr subunits. initial binding of ifnls to ifnlr1 induces the recruitment of il-10rb, leading to the activation of the receptor associated kinases janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2). cross-tyrosine phosphorylation of the ifnlr subsequently recruits signal transducer and activator of transcriptions (stat) 1 and 2 to the receptor platform. phosphorylated stat1 and stat2 form a heterotrimer together with ifn regulatory factor 9 (irf9). this trimeric complex, called ifnstimulated gene factor 3 (isgf3), translocates to the nucleus where it binds to the ifn regulated response element (isre) to drive ifn-stimulated gene (isg) expression [51] . antiviral effects of ifnls are largely shared with type i ifns. however, differences in receptor tissue expression and the kinetics of stat pathway induction exist between the two ifn classes [52] [53] [54] . in huh-7 cells, ifnl induces a slower and more sustained isg response [55, 56] . among the hundreds of isgs induced by ifnls and type i ifns are isg15, myxovirus (influenza virus) resistance 1 (mx1), 2 -5 -oligoadenylate synthetase 1-3 (oas-1-3), and protein kinase r (pkr). isgs interfere with different stages of viral life cycles as reviewed in [57] . the anti-inflammatory isgs usp18 and suppressor of cytokine signaling 1-3 (socs1-3), however, are specifically induced by ifnls and not by type i ifns [58] . both proteins interfere with stat signaling and therefore lead to desensitization to type i ifns and ifnls [59] [60] [61] . ifnl4 additionally induces expression of rantes and fos genes in hepatoma cells [4] . these genes are hallmarks of hcv-induced liver damage. interestingly and in contrast to type i infs, ifnls are themselves isgs as ifn stimulation of hepatoma cells induces their expression [11] . although ifnl2 and ifnl3 have high sequence homology, they differ in their antiviral activity with ifnl3 displaying the strongest antiviral activity in a hepg2 challenge experiment with encephalomyocarditis virus [62] . this finding is in line with a strong isg (mx1 and irf9) induction by ifnl3 in hepatocytes [55] . ifnl4, in turn, displays antiviral activities which are comparable to ifnl3 as shown in reporter cells expressing the ifnlr and a luciferase gene under the control of the ifi6 promoter [3] . in conclusion, ifnls signal through the jak1/stat pathway for isg induction and the set of isgs largely overlaps with that induced by type i ifns. to the genus hepacivirus in the flaviviridae family. hcv is an enveloped virus with a single-stranded, positive-orientated rna genome of 9.6 kbp length. according to genome sequence diversity hcv can be classified into seven genotypes and multiple subtypes [63] . the liver tropic virus enters hepatocytes in a multistep process involving several host cell proteins (as reviewed, e.g., in [64] ). after ph-dependent fusion of the viral membrane with the endosomal membrane, the viral genome is released into the cytoplasm. there the positive-orientated rna genome is directly translated into a single polyprotein, which is cleaved by viral and cellular proteases into 10 structural and nonstructural (ns) proteins. replication and virus assembly occurs in endoplasmic reticulum-(er-) associated membranous structures, called the membranous web (mw) (as reviewed, e.g., in [65] ). hcv assembly, maturation, budding, and release occur in close contact with the cellular very low density lipoprotein synthesis pathway. nascent hcv particles are released from the cells via the secretory pathway into the bloodstream or directly infect bystander cells (as reviewed, e.g., in [64] ). a schematic overview of the hcv life cycle is depicted in figure 2 . worldwide 92-149 million people, representing approximately 2% of the world's population, are chronically infected with hcv [66] , one of the causative agents of viral hepatitis. hcv is a blood borne virus and transmission occurs parenterally, mainly by reusing injection material, insufficient sterilization of medical tools, or by transfusion of unscreened blood or blood products. as screening of blood products is a standard procedure nowadays in most countries, people who inject drugs have the highest risk of contracting hepatitis c. in fact, over 60% of injecting drug users are positive for hcvantibodies [67] . naturally hcv infects only humans, but chimpanzees can be experimentally infected. in both cases, hcv targets the liver, in particular hepatocytes. the narrow host range is determined by the presence or absence of certain host cell factors; proteins critically needed for hcv entry, like the cell surface receptors scavenger receptor bi (srbi), cd81, claudin-1 (cldn1), and occludin (ocln) (as reviewed, e.g., in [68] ) or molecules needed for viral replication, like microrna 122, are expressed in hepatocytes. on the other hand, proteins suppressing hcv infection, like ewi-2wint, are absent in hepatocytes [69] . after acute infection, which is mainly asymptomatic, hcv establishes a lifelong, persistent intrahepatic infection in approximately 80% of the patients. development of chronic hepatitis c (chc) leads to progressing liver fibrosis and eventually cirrhosis (15-30% of chc patients), which can cause liver failure or the development of hepatocellular carcinoma (2-4% of chc induced cirrhosis patients per year) [70] . consequently, hcv causes app. 700,000 deaths per year [70] . chc was classically treated with recombinant peg-ifnalpha in combination with ribavirin (rbv). the treatment duration was long (24-48 weeks) and wearing, with peg-ifn-alpha being administered 3 times a week, severe side effects occurring frequently, and still only approximately half of the patients being cured. since 2014 hcv therapy improved drastically, as several direct acting antivirals targeting hcv ns3/4a protease, ns5a, or ns5b rna-polymerase were approved. these inhibitors, either alone or in combination with rbv, now heal over 90% of patients treated. direct acting antivirals (daas) are more effective than peg-ifn-alpha in eliminating hcv, but also treatment duration is shorter (minimum of 8 weeks), they can be administered orally, and adverse events are fewer. despite advances in drug development, a vaccine against hcv is still not available. for more details we refer the reader elsewhere, for example [71, 72] . the innate immune response serves as the first line of defense against infections; pathogen associated molecular patterns (pamps) are recognized by extra-or intracellular pattern recognition receptors (prrs), which triggers signaling cascades leading to the production of cytokines including interferons. the innate immune response to hcv is summarized in figure 3 . in hcv infected cells, double-stranded (ds) rna replication intermediates are generated and recognized as pamps by the cytosolic rna sensor retinoic acid-induced gene i (rig-i) [73] and melanoma differentiation-associated gene 5 (mda5) [74, 75] , both belonging to the family of rig-i like receptors (rlrs). sensing of hcv by rig-i or mda5 then leads to the oligomerization of the adaptor protein mitochondria antiviral signal (mavs; also called cardif, visa, ips-1) into large signaling complexes [76] . besides the cytosol, hcv dsrna can also be present in extracellular, er, or endosomal compartments. extracellular dsrna, maybe released from dying cells, can be taken up into uninfected neighboring cells by class a scavenger receptors [77] . after endocytosis, dsrna is brought to the endosome, where it is bound by toll-like receptor 3 (tlr3). alternatively, tlr3 might engage hcv in autophagic vesicles, as hcv replicating cells display an enhanced amount of them [78] . recognition of dsrna by tlr3 activates tir domain-containing adapter-inducing ifn-(trif; also called ticam-1) signaling. mavs and trif then trigger signaling cascades leading to the activation of different cytosolic kinases (i b kinases (ikk) and tank-binding kinase 1 (tbk1)), which in turn induce activation of the key transcription factors nf-b and irf3 [79, 80] . upon activation, these proteins translocate to the nucleus where they bind to promoter elements in type i and type iii ifn genes. by this, inflammatory cytokine and ifn expression is initiated. binding of secreted ifns to their receptors in an autocrine or paracrine manner leads to the activation of the jak/stat pathway, as depicted in figure 3 . ultimately, this triggers the expression of hundreds of ifn-stimulated genes isgs, which generate an antiviral state limiting hcv replication. during chc, the innate immune response can only control hcv replication but not completely eliminate the virus. this is partially due to viral mechanisms counteracting the immune response: briefly, hcv ns3/4a serine protease has been shown to inhibit ifr3 phosphorylation [81] by cleaving and inactivating the rig-i adaptor protein mavs [82] [83] [84] and the tlr3 adaptor protein trif [85] . interestingly, recently discovered members of the genus hepacivirus infecting nonhuman mammals carry an ns3/4a enzyme capable of cleaving not only their cognate host's mavs, but also human mavs [86] . this suggests that all yet studied hepaciviruses can antagonize the human antiviral innate immune response and that there is no barrier to zoonotic transmission at the level of innate immune interference. humans chronically infected with hcv display increased ifnl expression. specifically, dolganiuc et al. showed that ifnl serum levels are higher in chc patients than in hcv-negative with liver inflammation [87] . the authors observed elevated expression of the ifnlr in liver biopsies from chc patients. studying liver biopsies from infected patients also helped to confirm that there is an association between ifnl and isg expression levels; namely, that ifnl expression leads to elevated isg induction [88, 89] . in particular, a correlation between the activity of the ifnl4 protein and isg induction has been discovered. surprisingly, high ifnl4 and isg levels negatively impacted the outcome of hcv infection and treatment (see section 3.2) [90] . the host immune response to acute hcv infection has been studied in experimentally infected chimpanzees and in genetically humanized mice. in the livers of chimpanzees a strong host response can be detected, including the induction of type iii ifn transcription and isg expression [88, 91] . especially ifnl1 mrna and protein levels are elevated, correlating with isg expression and viremia. however, there is no link between type iii ifn expression in the liver or peripheral organs of infected chimpanzees and the outcome of the acute infection [91] . consistently, in immunocompetent transgenic mice expressing the human hcv entry factors, hcv infection results in upregulation of several isgs [92] . this is consistent with the observation that mouse-liver derived cells produce type i and iii ifns when transfected with hcv subgenomic rna and this leads to abrogation of hcv replication [93] . of note, current mouse models do not allow chronic hcv infection and since the ban on chimpanzee experimentation there are no immunocompetent animal models to study chc. recent efforts on establishing alternative nonhuman primate models for hepatitis c [94, 95] and on using rodent hepaciviruses as surrogate infectious agents to study chc, might resolve this hurdle in the future [96, 97] . hepatocytes [88, 91] . type iii ifns and isgs are similarly inducted upon hcv infection of primary human fetal liver cells [98, 99] . here the magnitude of induction differs from donor to donor but correlates with virus replication. to study different aspects of the hcv life cycle, hepatoma cell lines are frequently used. similar to hcv infection of primary cells, also the hepatoma cell line hepg2 induces ifnl transcription upon infection [74, 88] . interestingly, israelow et al. showed that ifnl induction attenuates hcv replication and that the ifnl response in hepg2-hfl cells stably replicating a hcv subgenome is blunted, probably due to mavs cleavage by hcv ns3/4a [100] . with regard to ifnl4, hong et al. found that endogenous ifnl4 transcription is only poorly induced upon stimulation with hcv in different hepatoma cell lines and phh. also no or reduced levels of secreted ifnl4 as compared to ifnl3 are detectable upon hcv infection [3] . the partial retention of ifnl4 in the cytoplasm, as observed in hepg2 cells and phh in a different study, might explain this observation [101] . the clear correlation between ifnl induction and hcv attenuation observed in hepatoma cell lines does not reflect observations in chc patients for several reasons. first, the complexity of the liver with contributions of kupffer cells, liver sinusoidal endothelial cells, stellate cells, and infiltrating additional immune cells (reviewed in [102] ) is obviously not mimicked by simple cell culture models. second, transformed cell lines do not necessarily resemble primary hepatocytes. in fact, most hepatoma cell lines that can be infected with hcv do not mount a strong innate immune response [73] . nevertheless hepatoma cell lines are regularly used to study the effect of exogenously added ifnl on hcv infection as they typically express all components of the ifnlr pathway. ifnl stimulation reduces levels of subgenomic or full length genomic hcv (+)rna in huh-7 cells in a dose dependentmanner [49, 103, 104] . these results were confirmed in several other hepatoma cell lines, including the huh-7 derived lunet hcd81 cells expressing a firefly luciferase gene or hepg2 cells expressing microrna122 and cd81 [3] . hepatoma cell lines and phh have also been used to study how the ifnl subtypes differ in their ability to limit viral infections; ifnl3 and ifnl4 induce the same set of isgs in phh [105] and the two subtypes have the same antiviral activity against hcv in an overexpression setup in hepatoma cells. in summary, expression of specific ifnl subtypes is induced in phh and some hepatoma cell lines upon infection with hcv, resulting in limiting virus production. however, the majority of hepatoma cell lines do not elicit a strong immune response and ifnl expression. novel model systems including stem cell derived hepatocytes [106] [107] [108] and tissue engineering systems [109] might in the future allow to more faithfully mimic host responses to hepatotropic virus infection. after establishment of peg-ifn-alpha and rbv as the standard of care treatment for hepatitis c [110] , it became clear that patients of african ancestry had significantly lower cure rates than those of european ancestry during ifn-alpha/rbv treatment. in 2009, two genome-wide association studies discovered ifnl gene polymorphisms as the underlying genetic basis for the different ifn-alpha/rbv treatment responses as well as for different spontaneous clearance rates [111, 112] . this work spurred further investigations on ifnl gene snps and their role during hcv infection and treatment. three major snps near the ifnl3 and ifnl4 genes correlate with hcv treatment response and are in high linkage disequilibrium [113, 114] . these polymorphisms are rs12979860(c/t) located 3 kb upstream of the ifnl3 gene [111, 112] , rs8099917 (t/g) located between the ifnl2 and ifnl3 genes [42, 115] , and rs368234815(tt/î�g) (originally named ss469415590), which creates a frameshift upstream of the ifnl3 gene leading to generation of the new ifnl4 gene product [4, 38, 116] . for all three snps the first allelic variant is associated with a higher probability of sustained virological response to ifn-alpha/rbv treatment. the location of these three snps on chromosome 19 is schematically depicted in figure 4 . treatment response dependency on ifnl polymorphisms was demonstrated for several hcv genotypes and in chronic patients with genotype 4 the ifnl snps are the strongest predictors for response known to date [117] . in addition to the three above described snps, six additional snps in the ifnl locus have been described to strongly associate with sustained virological response after ifn-alpha/rbv treatment (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, and rs7248668) [115] . how the ifnl snps mechanistically influence treatment outcome is mostly unclear. initially, it was suspected that the snps alter the transcriptional regulation of ifnl3 as they are located upstream of the ifnl3 coding sequence, where they could influence transcription factor binding and dna methylation. however, while some studies detect a correlation of protective ifnl snps and higher ifnl expression levels, other fail to do so (see [39] for detailed description). mechanistically, the rs8099917 snp has been suggested to influence ifnl3 mrna stability with the favorable allele being more stable [118] . for the rs368234815(tt/î�g) snp the functional impact is best described [4] . the î�g variant results in the expression of ifnl4, which is a pseudogene in tt carriers. ifnl4 expression is associated with increased isg levels and this in turn worsens treatment outcome. while this might seem counterintuitive, it is in line with observations that patients with increased pretreatment isg levels in the liver respond more poorly to ifn-alpha/rbv therapy [119] [120] [121] . thus, it seems at least in the case of ifnl4 that it has an adverse effect during hepatitis c by desensitizing the liver to ifn-alpha/rbv treatment. this has been confirmed in an independent study on the ifnl4 coding snp rs117648444 [90] , which results in a less active ifnl4 variant and consequently in improved treatment response. while one might question the value of these genetic markers in the age of ifnfree daa treatment with high cure rates, it should be noted that ifnl locus snps are also predictive for daa treatment outcome and moreover influence the daa response kinetics [122] [123] [124] . genetic markers might therefore allow prediction of treatment duration and consequently reduce costs and exposure time to daas. human polymorphisms in the ifnl locus responsible for improved response to ifn-alpha/rbv treatment are also associated with better spontaneous clearance of hcv. allele frequency of the rs12979860 snp differs between individuals with european or african ancestry with the favorable rs12979860(c) variant predominating in the former population. this finding correlates with better clearance of hcv in european ancestry individuals. the rs368234815(tt/î�g) snp similarly predicts hcv clearance rates. however, it is a better predictor than the rs12979860 snp in african-americans, while the predictive value of both snps is similar in european-americans [4, 125] . causative for this difference is the degree of linkage disequilibrium between both snps in the two populations [116] . the third snp with strong predictive value (rs8099917) for the outcome of hcv infection also shows a higher frequency of the protective allele (t) in individuals of european and asian ancestry as compared to individuals of african ancestry. in summary, there is a clear link between ifnl gene snps and hcv treatment outcome as well as spontaneous clearance. notably, except for the snp resulting in ifnl4 expression, the mechanisms causing the association remain elusive and the associated snps may not be the true causal variants. nonetheless, the predictive value of the ifnl snps extends beyond hepatitis c as genetic associations with nonalcoholic fatty liver disease, allergy, and infection with cytomegalovirus, human t-lymphotropic virus, hepatitis b virus, hiv, and herpes simplex virus have been suggested (reviewed in [126] ). the discovery of ifnl polymorphisms in the context of hcv infection may therefore importantly contribute to the understanding of other hepatic and extrahepatic diseases. before the rise of daas targeting hcv, ifnls were considered an attractive alternative to ifnalpha/rbv treatment for several reasons. the antiviral profile of ifnl resembles the one of ifn-alpha, as both interferons signal via isgf3. this holds true in primary human hepatocytes [105] as well as in hepatoma cell lines [49] . however the kinetics and magnitude of isg induction differ between ifnalpha and ifnl [55, 103, 127] . another difference between the two ifn types is their tissue specificity caused by the divergent expression pattern of their receptors; in contrast to the ifnl receptor complex, which shows a restricted tissue expression, the ifn-alpha receptor is expressed ubiquitously. thus compared to ifnl, ifn-alpha acts more systemic, causing more adverse effects, which are often limiting treatment options and compliance. the overlapping response of ifn-alpha and ifnl signaling on the one hand and the tissue specificity of the ifnlr on the other hand made ifnl promise that the replacement of ifn-alpha by ifnl would yield the same therapy outcome with fewer side effects. indeed, clinical studies revealed an improved safety and tolerability profile for peg-ifnl1a compared to peg-ifnalpha [128, 129] . when used in combination with rbv and the daa daclatasvir, a 24-week peg-ifnl1a based treatment does not only show less adverse events, but also leads to a higher sustained virological response than treatment with a peg-ifn-alpha based regime; 12 weeks after treatment no hcv rna is detectable in the blood of 88% of patients in the peg-ifnl1a group compared to only 70.5% of patients in the peg-ifnl1a group [130] . in a different clinical study peg-ifnl1a or peg-ifn-alpha2a were given together with rbv and telaprevir, but here no noninferiority of peg-ifnl1a regarding safety, tolerability, and efficacy was observed [131] . ifnl3 has the highest activity among the ifnl types and therefore might be more suitable as therapeutic agent than ifnl1 [55] . nevertheless, only ifnl1 has been evaluated in clinical trials so far, probably due to the fact that recombinant ifnl3 is difficult to produce. recently ifnl3 analogs, which allow high yield production and are comparable to ifn-alpha2a in their ability to inhibit hcv replication in huh-7.5.1 cells, have been designed [132] . however, the licensing of effective daa paved the way for an ifn-free therapy of chc, which is becoming the standard of care nowadays. thus most likely ifnl will not be needed as therapeutic agent for hepatitis c in the future. while the development of hepatitis c drugs is fortunately an unprecedented success story [133] , we still lack specific drugs for other hepatotropic viruses. for instance, hepatitis e virus is sensitive to ifnl in in vitro models [134] and currently treatment options for hepatitis e are limited. consequently, the mechanistic insights on the interplay of ifnl and hcv might spur important future work on the role and possible therapeutic application of ifnl during infection with other viruses infecting ifnlr expressing tissues, in particular the liver and the lung. ifn-s mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28r interferon lambda 4 signals via the ifn receptor to regulate antiviral activity against hcv and coronaviruses a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus interferon-lambda: a new addition to an old family the family of il-10-related cytokines and their receptors: related, but to what extent? the role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand-and cell-dependent suppression of the induction of alpha, beta, and lambda interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages toll-like receptor expression and induction of type i and type iii interferons in primary airway epithelial cells lambda interferon (ifn-), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo viral infection and toll-like receptor agonists induce a differential expression of type i and interferons in humans plasmacytoid and monocyte-derived dendritic cells interferonlambda serum levels in hepatitis c comparative analysis of the lambda-interferons il-28a and il-29 regarding their transcriptome and their antiviral properties against hepatitis c virus interferon type i gene expression in chronic hepatitis c the impact of the interferon-lambda family on the innate and adaptive immune response to viral infections a systematic analysis of host factors reveals a med23-interferon-regulatory axis against herpes simplex virus type 1 replication maturing dendritic cells are an important source of il-29 and il-20 that may cooperatively increase the innate immunity of keratinocytes type iii ifns are produced by and stimulate human plasmacytoid dendritic cells mouse cd8 + dcs and human bdca3+ dcs are major producers of ifn-in response to poly ic il-4 enhances ifn-1 (il-29) production by plasmacytoid dcs via monocyte secretion of il-1ra lambda interferon is the predominant interferon induced by influenza a virus infection in vivo respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells myd88 is required for protection from lethal infection with a mouseadapted sars-cov hcv infection selectively impairs type i but not type iii ifn signaling lambda interferon inhibits human immunodeficiency virus type 1 infection of macrophages an important role for type iii interferon (ifn-/il-28) in tlr-induced antiviral activity alpha/beta interferon (ifn-/ )-independent induction of ifn-1 (interleukin-29) in response to hantaan virus infection viral evasion and subversion of pattern-recognition receptor signalling virus infection induces the assembly of coordinately activated transcription factors on the ifnenhancer in vivo viral infections activate types i and iii interferon genes through a common mechanism ifn regulatory factor family members differentially regulate the expression of type iii ifn (ifn-) genes the role of transposable elements in the regulation of ifn-1 gene expression the role of differential expression of human interferon-a genes in antiviral immunity type i inteferon gene induction by the interferon regulatory factor family of transcription factors transcriptional regulation of ifn-genes in hepatitis c virus-infected hepatocytes via irf-3â�¢irf-7â�¢nf-b complex fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega ifn-4: the paradoxical new member of the interferon lambda family interferon lambda: opportunities, risks, and uncertainties in the fight against hcv crystal structure of human interferon-1 in complex with its high-affinity receptor interferon-r1 il28ra polymorphism (rs10903035) is associated with insulin resistance in hiv/hcv-coinfected patients il28b is associated with response to chronic hepatitis c interferon-and ribavirin therapy interferon-is functionally an interferon journal of immunology research but structurally related to the interleukin-10 family comparative genomic analysis of the interferon/interleukin-10 receptor gene cluster the expanded family of class ii cytokines that share the il-10 receptor-2 (il-10r2) chain ifnlambda (ifn-) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo interferon-lambda (ifn-) induces signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes purification, crystallization and preliminary crystallographic studies of the complex of interferon-1 with its receptor interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes the structure of human interferon lambda and what it has taught us transcriptional regulation by stat1 and stat2 in the interferon jak-stat pathway interferon-inducible antiviral effectors interferon lambdas: the next cytokine storm the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities dynamic expression profiling of type i and type iii interferonstimulated hepatocytes reveals a stable hierarchy of gene expression differential effects of type i and ii interferons on myeloid cells and resistance to intracellular bacterial infections interferonstimulated genes: a complex web of host defenses new regulators of nf-b in inflammation combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread socs proteins, cytokine signalling and immune regulation usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon response human interferon-3 is a potent member of the type iii interferon family expanded classification of hepatitis c virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource the ins and outs of hepatitis c virus entry and assembly hepatitis c virus rna replication and assembly: living on the fat of the land global epidemiology and genotype distribution of the hepatitis c virus infection global epidemiology of hepatitis b and hepatitis c in people who inject drugs: results of systematic reviews the impact of hepatitis c virus entry on viral tropism the cd81 partner ewi-2wint inhibits hepatitis c virus entry guidelines for the screening, care and treatment of persons with chronic hepatitis c infection recent advances in understanding hepatitis c emerging therapies for the treatment of hepatitis c regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i hepg2 cells mount an effective antiviral interferon-lambda based innate immune response to hepatitis c virus infection hepatitis c virus replicative doublestranded rna is a potent interferon inducer that triggers interferon production through mda5 mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response class a scavenger receptor 1 (msr1) restricts hepatitis c virus replication by mediating toll-like receptor 3 recognition of viral rnas produced in neighboring cells the autophagy machinery is required to initiate hepatitis c virus replication ikke and tbki are essential components of the irf3 signalling pathway identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-b and irf3 regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatitis c virus ns3-4a inhibits the peroxisomal mavs-dependent antiviral signalling response immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif hepacivirus ns3/4a proteases interfere with mavs signaling in both their cognate animal hosts and humans: implications for zoonotic transmission type iii interferons, il-28 and il-29, are increased in chronic hcv infection and induce myeloid dendritic cell-mediated foxp3+ regulatory t cells hcv infection induces a unique hepatic innate immune response associated with robust production of type iii interferons ifn-receptor 1 expression is induced in chronic hepatitis c and correlates with the ifn-3 genotype and with nonresponsiveness to ifntherapies reduced ifn 4 activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes il-29 is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection completion of the entire hepatitis c virus life cycle in genetically humanized mice control of hepatitis c virus replication in mouse liver-derived cells by mavs-dependent production of type i and type iii interferons hepatitis c virus infects rhesus macaque hepatocytes and simianized mice hepatic cells derived from induced pluripotent stem cells of pigtail macaques support hepatitis c virus infection identification of rodent homologs of hepatitis c virus and pegiviruses evidence for novel hepaciviruses in rodents hepatitis c virus induces interferon-and interferon-stimulated genes in primary liver cultures interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness interferon lambda 4 expression is suppressed by the host during viral infection expression of interferon lambda 4 is associated with reduced proliferation and increased cell death in human hepatic cells living in the liver: hepatic infections interferons and inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics lambda interferon inhibits hepatitis b and c virus replication transcriptome analysis reveals a classical interferon signature induced by ifn 4 in human primary cells engrafted human stem cell-derived hepatocytes establish an infectious hcv murine model productive hepatitis c virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation modeling hepatitis c virus infection using human induced pluripotent stem cells new methods in tissue engineering: improved models for viral infection 25 years of interferon-based treatment of chronic hepatitis c: an epoch coming to an end genetic variation in il28b and spontaneous clearance of hepatitis c virus genetic variation in il28b predicts hepatitis c treatment-induced viral clearance genetic variation in il28b is associated with chronic hepatitis c and treatment failure: a genome-wide association study genome-wide association study of spontaneous resolution of hepatitis c virus infection: data from multiple cohorts genome-wide association of il28b with response to pegylated interferonand ribavirin therapy for chronic hepatitis c il28b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction il28b polymorphisms predict response to therapy among chronic hepatitis c patients with hcv genotype 4 the favorable ifnl3 genotype escapes mrna decay mediated by au-rich elements and hepatitis c virus-induced micrornas interferon signaling and treatment outcome in chronic hepatitis c liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis c hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection il28b in the era of direct-acting antivirals for hepatitis c faldaprevir and deleobuvir for hcv genotype 1 infection ifnl4-î�g genotype is associated with slower viral clearance in hepatitis c, genotype-1 patients treated with sofosbuvir and ribavirin association of the ifnl4-î�g allele with impaired spontaneous clearance of hepatitis c virus gene-disease association with human ifnl locus polymorphisms extends beyond hepatitis c virus infections kinetic differences in the induction of interferon stimulated genes by interferon-and interleukin 28b are altered by infection with hepatitis c virus phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis c virus infection a randomized phase 2b study of peginterferon lambda-1a for the treatment of chronic hcv infection peginterferon lambda-1a/ribavirin with daclatasvir or peginterferon alfa-2a/ribavirin with telaprevir for chronic hepatitis c genotype 1b a randomized study of peginterferon lambda-1a compared to peginterferon alfa-2a in combination with ribavirin and telaprevir in patients with genotype-1 chronic hepatitis c design and evaluation of novel interferon lambda analogs with enhanced antiviral activity and improved drug attributes bringing the hepatitis c virus to life antiviral activities of different interferon types and subtypes against hepatitis e virus replication the authors have no competing interests. key: cord-006129-5rog0s98 authors: hemida, maged gomaa; ye, xin; thair, simone; yang, decheng title: exploiting the therapeutic potential of micrornas in viral diseases: expectations and limitations date: 2012-08-16 journal: mol diagn ther doi: 10.1007/bf03256383 sha: doc_id: 6129 cord_uid: 5rog0s98 new therapeutic approaches are urgently needed for serious diseases, including cancer, cardiovascular diseases, viral infections, and others. a recent direction in drug development is the utilization of nucleic acidbased therapeutic molecules, such as antisense oligonucleotides, ribozymes, short interfering rna (sirna), and microrna (mirna). mirnas are endogenous, short, non-coding rna molecules. some viruses encode their own mirnas, which play pivotal roles in viral replication and immune evasion strategies. conversely, viruses that do not encode mirnas may manipulate host cell mirnas for the benefits of their replication. mirnas have therefore become attractive tools for the study of viral pathogenesis. lately, novel therapeutic strategies based on mirna technology for the treatment of viral diseases have been progressing rapidly. although this new generation of molecular therapy is promising, there are still several challenges to face, such as targeting delivery to specific tissues, avoiding off-target effects of mirnas, reducing the toxicity of the drugs, and overcoming mutations and drug resistance. in this article, we review the current knowledge of the role and therapeutic potential of mirnas in viral diseases, and discuss the limitations of these therapies, as well as strategies to overcome them to provide safe and effective clinical applications of these new therapeutics. therapeutic strategies based on mirna technology for the treatment of viral diseases have been progressing rapidly. although this new generation of molecular therapy is promising, there are still several challenges to face, such as targeting delivery to specific tissues, avoiding off-target effects of mirnas, reducing the toxicity of the drugs, and overcoming mutations and drug resistance. in this article, we review the current knowledge of the role and therapeutic potential of mirnas in viral diseases, and discuss the limitations of these therapies, as well as strategies to overcome them to provide safe and effective clinical applications of these new therapeutics. rna interference (rnai) is a system in living cells that regulates the activation and silencing of gene expression. rnai governs the regulation of host cell genes, mainly through two types of rna molecules: small interfering rna (sirna) and microrna (mirna). [1] sirnas are a class of double-stranded rna molecules, 20-25 nucleotides in length, which play different roles in cellular biology. experimental application or targeting of sirnas in various cell types and animal models has shown promise for the potential treatment of diseases induced by viruses such as hepatitis c, influenza, and hiv. [2] [3] [4] the suppression of viral gene expression by sirnas makes them very attractive for antiviral therapy, and some sirnas are already being used in clinical trials. [5] however, sirnas still have a long way to go before being brought to market, because of their potential side effects. one of the major concerns in using sirnas as molecular therapeutics is their induction of a strong immune response. mirnas, which were discovered in 1993, [6] are a group of non-coding, single-stranded rna molecules, ranging in size from 19 to 25 nucleotides. [7] it is now believed that mirnas compose one percent of the total human genome. mirnas are widely expressed in various species, including viruses. the first virally encoded mirna was discovered in the epstein barr virus (ebv) genome. [8] now, there are more than 141 identified mirnas encoded by 15 viruses, with more expected to be identified in the near future as a result of the improvement in online prediction and validation tools. [9] cellular mirnas in animals seem to be conserved, while virally encoded mirnas are highly variable even within the same group of viruses. [10] this may be due to the high frequency of viral mutations relative to eukaryotes. mirnas play an important role in regulation of almost one-third of all known human mrnas. [11] most mirnas have a specific tissue expression profile. their unique expression pattern may explain their roles in different biologic activities, such as cellular differentiation, environment adaptation, oncogenesis, and host-pathogen interaction. [12] the therapeutic potential of mirnas was first realized with the discovery that downregulation of mir-15 and mir-16 is associated with development of b-cell leukemia. [13] shortly after that, the potential for treatment of several other cancers was realized. [14] scientists have aimed to control the expression level of key genes via manipulation of cellular or viral mirnas to treat disease. these approaches include anti-mirna oligonucleotides (amos), peptide nucleic acids, and mirna sponges. [15] initial experiments have obtained promising results in controlling various viral infections. [16] [17] [18] in addition, it has been found that restoring or over-expressing certain mirnas may also be beneficial for reverse pathologic conditions, especially in cancer treatments. [19] the goal of this article is to review the recent progress in the understanding of the roles of mirnas in viral diseases, and to discuss the potential of these molecules in serving as a therapeutic target or as a useful therapeutic tool. we also specifically highlight the major obstacles faced by mirna technology in both therapeutics and vaccine strategies. viruses are obligate intracellular parasites. they lack the essential machinery required for their replication. thus, viruses adopt several clever strategies to ensure the success of their replication in a suitable host, one of which is manipulation of the host mirnas to modify the cellular environment for their own benefit. [20] some dna viruses are capable of encoding their own mirnas to modulate both the viral and cellular protein expression in order to provide a favorable environment for viral replication. [12] answering back, certain host mirnas alter the cell gene expression to defend the cells against the viral infection by interfering with viral proteins or other cellular factors as a type of immune response against these particular viruses. [21] therefore, the relationship between viruses and mirnas is complicated, to say the least. since mirnas play essential roles in viral infections, they are considered to be promising therapeutic targets in infectious diseases. their endogenous nature, small size, and flexible function make mirnas very good candidates, as they may trigger lower immunogenic responses and have fewer side effects than sirnas. [12, 14] in view of the current data regarding the roles of different viral and cellular mirnas in various viral replication cycles, we believe that manipulation of these mirnas will have a promising therapeutic role in infectious diseases. [12, 14, [22] [23] [24] [25] currently, there are more than 700 mirnas encoded by the human genome alone. [12] we will discuss the roles and therapeutic potential of cellular as well as viral mirnas (if any) in the pathogenesis and treatment of different viral diseases. human polyomaviruses (hpyvs) are a group of oncogenic, circular, non-enveloped, double-stranded dna (dsdna) viruses. [26] five polyomaviruses have been found to infect humans. of particular interest are two strains of these viruses, named (according to the initials of the first affected patients) bk virus (bkv) and james canyon virus (jcv). [27] the reservoir species for human infection is the rhesus macaque. humans have also acquired simian virus 40 (sv40) infection from contaminated poliovirus vaccines, and recent studies have reported horizontal transmission between people. [28] hpyvs induce a wide range of tumors affecting almost all body organs (including the brain, bones, colon, pancreas, stomach, and urogenital tract), as well as lymphomas and leukemia. [28, 29] the viral genome of sv40 encodes five proteins; two large t antigen (lt), one small t antigen (st-ag), and three that encode the capsid proteins (vp1, vp2 and vp3). hpyvs are able to encode viral mirnas for their own benefit. both bkv and jcv encode the same mirna, named mir-j1. it is upregulated in the brain in progressive multifocal leukoencephalopathy syndrome, suggesting a major role in this particular disease. [30] sv40 encodes mirnas called v-mirnas during the late stages of infection. [31] they are complementary to the viral mrnas produced at the early stage of viral infection. [31] these v-mirnas slow down the expression of the viral t-antigen genes and lower the level of interferon (ifn)-g produced by cytotoxic t lymphocytes, thus reducing the influx of inflammatory cells and facilitating evasion of the immune response. [31] human papillomaviruses (hpvs) are also oncogenic viruses. [32] they are usually associated with different forms of both benign and malignant tumors, especially those affecting the skin and the genital tract. [32] these viruses are usually classified, on the basis of their virulence, into either low-or high-pathogenic variants. [26] only a few hpv strains can produce mirnas during their replication. [33] for example, hpv-31 encodes viral mirnas at a very early stage of the infection, but these mirnas are usually degraded once latent infection takes place. [34] in another independent study, both hpv-31 and hpv-18 were found to encode viral mirnas, but they are not involved in cell transformation or cancer development. [33] it is well known that host mir-34a is involved in inhibition of abnormal cell growth in tumors. [35] mir-34a inhibits cell cycle progression at the g2 phase and subsequently induces apoptosis. [36] studies have reported success using the tumor suppressor complex (mrna-cellular mirna-34a), which targets downstream genes of tumor protein p53 (tp53). mir-34a is usually downregulated during hpv infection in primary keratinocytes. [36] thus, restoration of the normal expression level of this mirna is a potential strategy for therapeutic intervention. [36] adenoviruses are a group of non-enveloped dsdna viruses. over 50 serotypes have been identified in different clinical diseases, such as respiratory, gastrointestinal, urogenital, and eye diseases. [36] adenovirus usually encodes several small noncoding rna molecules called virus-associated rnas, such as va1 and va2. [37] they facilitate immune evasion by inhibiting dsrna-induced protein kinase r (pkr), which blocks ifn-a activity. [38] one study reported that adenovirus va1-rna interferes with the biogenesis of host mirnas and the function of sirna shrna (short hairpin rna), through inhibition of the nuclear transport of the pre-mirnas and the shrnas and direct inhibitory action of dicer. [37] usually, a small part of the va1 rna is subjected to processing by dicer, and this results in generation of mirna. use of anti-mirna antisense inhibitors (2 0 -o-methyl amo) to downregulate this mirna resulted in inhibition of virus production. [39] herpesviruses are a group of enveloped dsdna viruses, classified into three subfamilies (a, b, and g). they are characterized by induction of latent infections in their target hosts. [40] these virus-encoded mirnas play important roles in the establishment of latent infection, as well as the pathogenesis of virally induced diseases. according to the most recent studies, herpesviruses utilize their encoded mirnas in a wide range of biologic functions, such as inhibition of apoptosis, immune evasion, control of cellular proliferation, and regulation of viral replication. [41] [42] [43] in the following section, we will discuss herpesvirus-encoded mirnas. one of the most important genes encoded by herpes simplex virus (hsv) is called the latency-associated transcript (lat). [44] this gene does not encode proteins but may be involved in the production of mirnas or in cell survival after viral infection. [44] there has been debate around the origin of mir-lat as to whether it is a virus-encoded or cell-encoded mirna. [41] this mirna is believed to act by downregulating transforming growth factor (tgf)-b and smad3. tgf-b plays an important role in cell proliferation and induction of apoptosis. smad3 is a signaling pathway mediator, which is triggered by the action of tgf-b. [41] hsv-1 mir-lat-icp34.5 has been recently discovered during hsv-1 infection. [45] according to bioinformatic analysis, the hsv-1 genome encodes 24 mirnas, eight of which were found to be conserved between both hsv1 and hsv-2, and thus are believed to be functional. [46] six of these mirnas are upregulated in the trigeminal ganglia of mice infected with hsv-1. these mirnas are encoded by lat (table i) . [46] in addition, quantitative reverse transcription pcr showed that both mir-1 and mir-6 were highly expressed in vero cells infected with hsv-1 -as high as 1200 and 300 copies, respectively -whereas the other four mirnas showed downregulation, with only 40 copies per infected cell. [55] the mirna expression profile during hsv-1 infection revealed that several mirnas among the eight candidates mentioned earlier were upregulated. those mirnas were believed to play major roles in induction of the latent phase of viral infection. this assumption was based on comparison between the mirna expression profiles of the latent and active infections. for example, mir-h2 was found to be upregulated in latent infection to a level of 63 000 copies cell, compared with less than 40 copies cell during active infection. [56] furthermore, mirna-2 inhibits the production of the infected cell polypeptide (icp)-0 protein, which is responsible for triggering the active phase of hsv infection. another upregulated mirna, mir-6, is responsible for downregulation of icp4, which is responsible for the increased expression level of many hsv genes during the active phase of hsv-1 infection. [55, 56] human cytomegalovirus (hcmv) is a herpesviruses affecting humans, and can result in acute or latent infections. [57] the form of infection largely depends on the immune status of the affected host. [57] it can be fatal in immune-compromised patients, such as those with aids or recent organ transplants. [57] it may also be responsible for birth defects and congenital abnormalities in pregnant women. [21] as with other herpesviruses, there is evidence supporting the presence of mirnas that modulate viral pathogenesis in different tissues. [21] specifically, hcmv has been recently reported to encode 15 mirnas. [21] the most commonly expressed three mirnas during the active phase of hcmv infection are mir-ul23-5p, mir-ul23-3p, and mir-us24, [48] which target different cellular proteins, such as transcription factors (hfn3 and tgif2), receptors (cd206 receptors for interleukin-18), and other proteins involved in signal transduction pathways, such as rab2l. [48] hcmv is able to induce the latent phase of infection by a cunning immune-evasion strategy through the action of mir-ul112, which targets several cellular proteins such as major histocompatibility complex (mhc) class i associated proteins, especially the mhc class i-related chain b (micb). [58, 59] mir-ul112-1 also regulates early viral protein expression, such as immediate early protein (ie)-72. ie72 is a key mediator in the shift from the latent to the active phase of viral infection, because it suppresses ie1. [58, 59] thus, if the hcmv infection is synchronized with overexpression of the mir-ul112, the ie1 protein expression level is greatly reduced, which mediates the latent phase of infection. [43] it is also thought that mir-ul112-1 targets another viral protein called ul114. [58] downregulation of ul114 protein, using mir-ul112-1, results in inhibition of viral dna replication and subsequently triggers the latent phase of infection, making the virus able to evade the host immune system. [34] exploitation of this mechanism is being considered as a potential therapeutic strategy. [21] ebv is another oncogenic virus affecting humans. it is usually associated with induction of latent infection in more than 95% of affected patients. [60] in most cases, benign tumors develop; in some cases, however, malignant tumors may also develop, such as hodgkin's lymphoma, t-cell lymphoma, nasopharyngeal carcinoma, and gastric tumor. [61] during the active phase of ebv infection, more than 100 genes are usually expressed, whereas only 11 of them are expressed during latent infection. [21] it has been recently reported that ebv encodes more than 20 mirnas. [33] these mirnas are divided into two groups: one group is encoded from the intronic regions of a gene called bart, which is expressed at high levels in epithelial cells, but at lower levels in b cells, in the latent phase of infection; the other group is encoded from the untranslated region of a gene called bhrf1, a viral bcl2 homolog that prevents apoptosis. [8] although the functions of most ebv-encoded mirnas have not been completely identified, it has been found that mirna-bart2 targets balf5 mrna, a viral dna polymerase, and mirna-bart2 induces cleavage in the 3 0 untranslated region (utr) of balf5, resulting in inhibition of the lytic viral infection ( figure 1 and table i) . [63] according to bioinformatic prediction data, ebv mirna-bart5 is believed to target puma, a modulator of apoptosis in the bcl2 protein group regulated by tp53. [64] in some cases, puma can also induce apoptosis through the tp53-independent pathway. therefore, targeting puma with ebv mir-bart5 results in suppression of its action in apoptosis. [64] other ebv-encoded mirnas target the ifng-inducible chemokine cxcl11. [65] without cxcl11, ebv is able to evade the host immune response and subsequently enhance ebv replication. [65] the same group performed bioinformatic analysis and showed that cxcl11 contains a target sequence for mir-bhrf1-3 at its 3 0 utr sequence. targeting cxcl11 using this viral encoded mirna will have an immunomodulatory effect on the viral-induced tumor. [65] therefore, it is believed that targeting bhrf1-3 could be a good therapeutic approach for viral ebv-induced tumors. [65] kshv is one of the gamma-herpes viruses groups and is usually associated with kaposi's sarcoma infection, from which it acquired its name. [47] like other herpesviruses, kshv usually induces latent infections. [47] kshv encodes several mi-rna candidates from the genomic region spanning 4 kilobases between orf12 and orf71. [66] kshv-encoded mirnas target important genes involved in cell proliferation, modulation of the host immune system, apoptosis, and angiogenesis. for example, mir-k5 targets the mrna of the bcl2 (prosurvival gene) interacting protein called bclaf1. [62] this leads to reactivation of the kshv lytic infection. [62] thus kshvencoded mirnas play an important role in the virus host interactions, and silencing of those mirnas using different approaches, particularly the amo, is therefore a promising therapeutic strategy against such virus infection (figure 1). [62] marek's disease virus (mdv) is one of the alpha herpesviruses, characterized by rapid production of t-cell lymphomas in chickens. [67] the viral genome encodes several mirnas, which are mainly encoded from the oncogenes, especially meq and lat. [68] the mirnas of this virus are highly expressed in mdv-transformed cells, suggesting an essential role in mdv oncogenesis. however, their target genes have not been well identified. [68, 69] it is known that several host cell mirnassuch as mir-17, mir-92, mir-21, and let-7i -are involved in the cancer development induced by mdv in chickens. [68] they are most often upregulated in an msb-1 (mdv-transformed cd4+ t-cell line derived from a spleen lymphoma induced by the bc-1 strain of mdv-1) library after mdv infection. [69] this makes for an ideal model for the study of mirnas. comparative analysis of mirna expression profiles during mdv infection, using microarray analysis, may enable identification of specific mirnas involved in neoplastic transformation when compared with non-infected cells. this may become a useful diagnostic approach through examination of mirna signatures profiles. [70] this is supported by a recent report showing an association between downregulation of the host cellular mir-155 expression and upregulation of mdvencoded mirnas (m2-5p, m12-3p, m5-3p, m4-5p, m3-5p, m11-5p, m2-3p, m4-3p, and m3-3p), a potential marker for mdv-induced tumor formation. [69] subsequent identification of the putative target of these mirnas will lead to a better understanding of the molecular mechanism of mdv-induced tumorigeneses. [69] the human immunodeficiency virus (hiv) genome encodes several important genes, such as nef, vef, tat, and vpu. [71] bioinformatic analysis suggests that the hiv genome encodes five pre-mirnas, which are processed into ten mature mi-rnas, but their definite functions are still not well identified. [72] the hiv nef gene is located at the 3 0 end of the hiv genome and is highly expressed in early viral infection. [73] it has been shown to downregulate the expression levels of cd4, cd28, and mhc class i molecules. [73] hiv nef encodes mir-n367, which has a unique function at both the transcriptional and translational levels, rather than at the post-transcriptional level. [51, 74] the mechanism of action of mir-n367 is believed to be suppression of hiv promoter activity; however, further studies are required to explain the exact mechanism of such an action. [51] several studies reported that downregulation of the nef gene results in inhibition of hiv replication. [51, 74] this may lead to production of low-pathogenic strains of hiv or may favor latent hiv infection. [74] targeting of these viral mirnas using different approaches, especially amo treatment, could potentially have a therapeutic effect on hiv. [ hybridizing with corresponding targets of host genes (blue sequences). the viral mirna sequence and the 3 0 untranslated region (utr) sequence of the targeted host and viral genes were obtained from the referenced articles or from the national center for biotechnology information (ncbi) website and then submitted to the rnahybrid software program (http://bibiserv.techfak. uni-bielefeld.de/rnahybrid/). the predicted secondary structure for the hybridization between viral mirnas and their targets are generated by the rnahybrid program. (a) mir-k5 encoded by kaposi's sarcoma-associated herpesvirus (kshv), targeting host gene bclaf1 (bcl2-associated transcription factor 1). [62] (b) mir-bart2 encoded by ebv, targeting the viral dna polymerase gene balf5. [63] (c) mir-ul112 encoded by herpesviruses, targeting host gene micb (mhc class i-related chain b). [59] mfe = minimum free energy. currently, there are more than 170 million people affected by hepatitis c virus (hcv) infection worldwide. [75] drug resistance is one of the major hindrances in treating such viral infection. [76] hcv induces different forms of tumors in humans and is one of the major causes of liver diseases all over the world, resulting in hepatocellular carcinoma and, finally, complete liver failure. [77] it has been recently reported that host cellular mirna-122 has two recognition sites in the 5 0 utr of the hcv genome, resulting in upregulation of hcv infection. [78] further investigation showed that interaction between mirna-122 and the viral genome causes accumulation of viral rna in the liver tissues (table i) . furthermore, the level of viral rna in the liver tissues is usually controlled by mir-122 binding sites. [78] interestingly, hcv infection also modulates cellular mirna expression profiles. following hcv infection, three mirnas (mir-122, mir-100, and mir-10a) are upregulated, while other two mirnas (mir-198 and mir-145) are downregulated. [78] ura et al. [52] found that cyclin g1 acts as a putative target for mir-122. use of a primate model targeting mir-122 with specific antagonists resulted in a reduction in the level of hcv replication in the affected livers, demonstrating the promise of this strategy. [79] in addition, a recent study demonstrated the therapeutic potential of silencing mir-122 in chronic hcv viral infection in primate models, whereby chimpanzees that were positive for hcv infection were treated with a specific lna-modified oligonucleotide (spc3649). [79] these lna-oligomers targeted the complement sequence of mir-122 and resulted in a decrease in the duration of the viremia following acute hcv infection. there were no reports of any side effects or any viral resistance observed after the treatment. [79] this approach provided long-lasting effects in the hcv-infected animals, as well as great improvement in the liver pathology. [79] more clinical trials are needed to further confirm the promising results of this new molecular therapeutic approach. [78] hepatitis b virus (hbv) belongs to the genus orthohepadnavirus in the family of hepadnaviridae. hbv infection progresses into cirrhosis and hepatocellular carcinoma in most cases. [80] it is believed that hbv encodes mirnas that regulate their own gene expression. [81] according to bioinformatic predictions, hbv encodes only one mirna. however, several studies have failed to identify any cellular genes regulated by this virus-encoded mirna, implying alternative gene expression mechanisms. [81, 82] according to the mirna expression profiles of several patients suffering from cirrhosis due to hbv infection, the host hsa-mirna-615-3p is usually upregulated. in recent clinical studies, ura et al. [52] studied the role of different mirnas in the pathogenesis of both hbv and hcv in the context of development of hepatocellular carcinoma in infected liver tissues. [52] in this study, the differential expression levels of 188 mirnas from 12 hbv and 18 hcv patients were tested using qrt-pcr. according to this study, 19 mirna candidates were highly expressed in both hbv and hcv infections, and 31 mirnas served as markers for the severity of liver damage. it is known that hbv infection triggers pathways associated with dna damage, recombination, and signal transduction pathways -whereas hcv infection usually triggers an immune response, antigen presentation, cell cycle progression, proteosome activation, and lipid metabolism. therefore, the overall conclusion was that certain mirnas may act as important mediators in the pathogenesis of both hbv and hcv infections. [52] these studies have paved the way to a new era in molecular antiviral therapy through modulation of the expression levels of those key mirnas. there is now a new antiviral therapy for controlling hbv infection, using artificial mi-rnas. this approach has revealed a dramatic reduction in hbv protein expression levels and a remarkable reduction in viral dna replication in vitro. [83] [84] [85] severe acute respiratory syndrome coronavirus (sars-cov) is a single-stranded rna virus, which belongs to the family coronaviridae. although several trials have been performed to treat this pathogen using conventional drugs such as ribavirin, antibiotics, anti-inflammatory steroids, and different kinds of immune stimulators, these approaches still lack viral specificity. sars-cov infection in bronchioalveolar stem cells (bascs) is a prime example of how mirna modulates the virus-host interaction. sars-cov is unable to replicate in well differentiated cells, so it has to control basc cellular differentiation in order to establish a successful viral infection. [86] this virus usually hijacks cellular mirnas such as mir-17 * , mir-574-5p, and mir-214 for the benefits of its replication and immune evasion. the nucleocapsid and spike glycoproteins downregulate the expression levels of mir-223 and mir-98, respectively. this action enables the virus to hinder basc cellular differentiation and the production of inflammatory chemokines, creating an environment that is optimal for virus replication. restoration of the levels of mir-223 and mir-98 poses a potential novel approach in treating sars-cov infection. [86] the influenza a outbreak of 2009 provided a warning about the urgent need for new alternative molecular therapeutic approaches for both the treatment and prophylaxis of such viral infections. molecular therapy using mirna technology may offer a new therapeutic approach to cope with the continuous changes in virus strains every year. recent bioinformatics tools have paved the way for the discovery of new mirnas and their target sequences for the design of nucleic acid-based therapeutics. for example, there are two human-encoded mirnas that have potential binding sites within both the viral polymerase (pb2) and hemagglutinin (ha) genes (mir-507 and mir-136, respectively) [table i]. [22] the target sequences of these two mirnas are highly conserved among different influenza virus strains. the ha protein is involved in the attachment of the virus to its receptors, and the pb2 protein is an essential component in the ribonucleoprotein complex, needed for rna transcription and replication. the presence of human mirnas that target conserved regions of influenza rna suggests that the human genome has evolved to use this as a defense mechanism against infection. this supports the argument that targeting viral genes with mirnas may be an effective strategy. this may also suggest that it is a futile attempt, since we have the mirnas and yet still succumb to influenza infections. a recent study has been conducted to determine mi-rna expression profiles after avian influenza virus (aiv) infection in chickens. this study showed changes in the cellular mirna profile in response to the aiv infection, suggesting that the mi-rnas play a role in the host-pathogen interaction during aiv infection. [87] specifically, there were alterations in the mirna profiles of mir-146, which had been previously reported to play a role in immune-related signal pathways in mammals. [87] one recent study utilized mirna technology in the development of influenza virus vaccines, whereby a new influenza a virus vaccine was developed using mirna-based gene silencing. the method involves introducing an mirna sequence of non-avian origin, known as a mirna-responsive element (mre), into the viral nucleoprotein gene, resulting in construction of new reassortant h1n1 and h5n1 viruses. with this strategy, the degree of the viral attenuation is controlled by the expression level of mir-124, which targets the introduced mre sequence. this novel strategy offered a very good vaccine that was species specific, offering a high level of protection. [4] the nascent viruses were attenuated for mice, while they still propagated well in embryonated chicken eggs and were able to generate high levels of neutralizing antibodies in animals. this novel approach for influenza vaccine development may be used in combination with the currently available vaccine in order to increase both the safety and the efficacy of influenza virus vaccines in the near future. [4] coxsackievirus, especially coxsackievirus b3 (cvb3), is the most common pathogen of human myocarditis. anti-cvb3 drug development has been recently focused on a nucleic acidbased strategy. our laboratory first reported the successful inhibition (>92%) of cvb3 replication in hela cells by transfection of sirnas targeting viral protease 2a rna. we also found that the antiviral effect was disrupted by mutations in the central strand region, and mismatch was tolerated near the 3 0 end but not near the 5 0 end of the sirna; [88] furthermore, the sirna effect was mediated by the antisense strand to the viral genome, rather than the sense strand complimentary to the viral negative-strand of the replicating intermediate. this finding was further conformed by another report. [89] when applied systemically to mice, sirna targeting 2a had a significant protective effect if applied 6 and 14 hours after infection, including reduced viral replication and tissue injury, as well as an increased survival rate. [90] recently, we tested a packaging rna (prna) vector (a component of the bacterial phage nano-motor) for targeted delivery of sirnas. through conjugation of a folate ligand to the prna vector, we specifically delivered the sirnas targeting cvb3 2a to hela cells and hl-1 cardiomyocytes that expressed folate receptors. [91] in addition to the transfection of mature sirnas, overexpression of shrnas was also effective against cvb3 3d rna polymerase and structural protein vp1, both in cells and in mice, where viral pancreatitis was significantly reduced. [92] schubert et al. [89] used the sidex double expression vector to simultaneously transfect two sirna sequences targeting the cvb3 3d rna polymerase sequence in a green fluorescent protein (gfp) reporter construct. this double expression of both sirnas successfully suppressed reporter expression despite the intentional introduction of an artificial point mutation (simulating an escape mutation or a mirna target) that caused a mismatch with one of the two sirnas. as we have discussed above, there have been some promising results supporting the development of mirnas for the treatment of several viral infections, and some of these mirna-based drugs have reached the clinical trial stage. despite this great progress, their clinical applications are still hampered by several challenges. in the following section, we briefly discuss the current obstacles or limitations facing mirnas-based antiviral therapy. one of the major limitations for the use of mirna-based antiviral therapy is the production of transgene-specific immunity. [93] delivery of mirnas using viral vectors usually results in the development of immune response against the viral vector. basically, the delivery vector will stimulate an innate immune response in the forms of cytotoxic t-lymphocytes, humoral neutralizing antibody against their viral capsid proteins, and cytokine-mediated inflammatory responses in vivo. [93, 94] direct correlation between the immune response to the adenovirus capsid protein and the concentration of the viral vector has been reported; this interaction is usually associated with undesirable side effects in the host, especially if the construct moves from the target tissue into the blood circulation. [95] the targeted delivery of sirna, mirna, and other nucleic acid-based therapies is another major concern of using these molecular therapeutic approaches. in contrast to the great progress in local administration of both sirna-and mirna-based therapies in the eyes, lung, and vagina, systemic delivery to target organs such as the liver, heart, and intestine is still undergoing optimization. [96, 97] it is interesting to note that some studies have shown success in administering sirnas via the intracerebral route; however, the risk is that foreign nucleic acid may be delivered to the central nervous system. [93] several laboratory techniques -such as real-time pcr, microarray analysis, luminex bead arrays, northern blotting, in situ hybridization, formalin fixation, and paraffin embedding -are currently in use in mirna detection and quantification. however, all of these techniques still require further optimization. [98] [99] [100] once they are optimized, a clear choice for sensitivity and specificity will emerge, and this approach will allow early and sensitive detection of mirna expression in different disease syndromes. this will have a great impact on the early tracking of serious viral diseases. [101] the off-target effects of sirnas were one of the major concerns in earlier studies using both sirna and shrna technologies in gene therapy. as a new generation of molecular gene therapy, mirnas would be expected to have a high degree of specificity for their targets. however, since mirna action is based on imperfect base pairing with the target sequence in most circumstances, the specificity will be lower than that of sirna. this prediction has been confirmed by recent clinical trials, followed up by microarray analysis, which revealed possible off-target effects of mirnas. [102] another follow-up study by birmingham et al., [103] using the combination of bioinformatics and microarray analysis, found that using either the sirna or the mirna could result in off-target silencing. in addition, in vivo studies have revealed that one mirna may target several genes at the same time, and the targets are not clearly identified. this suggests diverse modes of action of a given single mirna. on the other hand, one gene may be regulated by several mirnas, [104] indicating that the mode of action is more complicated than expected. since drug therapies must precisely target the virus in question and nothing else, a large undertaking is needed to gather all possible information regarding all targets of each mirna that is being considered for drug development. [104, 105] although the currently used viral vectors in mirna delivery are non-pathogenic, there is always the possibility of mutations within those viral vectors. these mutations may not only result in abnormal gene expression of the viral mirna construct but may also cause possible insertion of vectors into the human genome, increasing the risk of cancer. [23] moreover, the targeted viruses (especially the rna viruses) are prone to mutation, which may drive drug resistance. there are currently two possible approaches to conquer these issues: one is the targeting of cellular factors that are essential for virus replication or use of more than one mirna for the same target gene; the other possible solution is the targeting of several conserved regions of the viral genome by different sirnas or mirnas. viruses are among the most common causes of human diseases. because of the unique biologic properties of viruses, there is no effective and specific antiviral therapy available so far. several vaccines and antiviral drugs have shown a limited degree of efficacy for prophylaxis and treatment of some viral infections. however, high mutation rates enable viral diseases to emerge and re-emerge frequently. thus, new strategies for drug and vaccine development must be devised to fight the threat of viral diseases to human health. recent advances in the understanding of mirna structure, function, and particularly their association with the molecular pathogenesis of a variety of complex diseases, have served as a theoretical basis for drug development. on the one hand, as key factors for viral replication and latency, mirnas are ideal targets for inhibition. in this regard, construction of mrnas that contain multiple tandem binding sites of a given mirna may be useful to produce decoys or 'mirna sponges' to inhibit the function of a specific mirna. in addition, chemically synthesized antisense rna oligomers ('antagomirs') targeting a mirna of interest could be also be a promising approach to inhibit mirna activity. on the other hand, mirna expression vectors can be used to overexpress specific mirnas to achieve a long-term effect of reversing the imbalance of mirna expression caused by infection. further, introduction of pre-mirna mimetics for transient replacement is another option for investigation. in summary, although there are many limitations at present, we believe that with the rapid progress in mirna research, these small molecules will become an invaluable target and a useful tool for basic research and drug development. it is expected that an mirna-based antiviral therapy will become available for clinical application in the near future. duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells hiv may produce inhibitory microrna (mirnas) that block production of cd28, cd4 and some interleukins modulation of hepatitis c virus rna abundance by a liver-specific microrna microrna-mediated species-specific attenuation of influenza a virus future prospect of rna interference for cancer therapies the c. elegans heterochronic gene lin-4 encodes small rnas with antisense complementarity to lin-14 the microrna registry identification of virus-encoded micrornas cellular versus viral microrna in hostvirus interaction conserved seed pairing often flanked by adenosines, indicates that thousands of human genes are microrna targets micrornas: genomics, biogenesis, mechanism, and function viruses and micrornas frequent deletions and downregulation of micro-rna genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia micrornas fine-tune oncolytic viruses microrna sponges: competitive inhibitors of small rnas in mammalian cells lna-mediated micrornas silencing in non-human primates inhibition of microrna with antisense oligonucleotides emerging role of micrornas in disease pathogenesis and strategies for therapeutic modulation current perspectives in micrornas (mirna) host-virus interaction: a new role for micrornas silencing viral microrna as a novel antiviral therapy? host-virus interaction: a new role for micrornas the promises and pitfalls of rna-interference-based therapeutics viral and cellular micrornas as determinants of viral pathogenesis and immunity microrna-122 stimulates translation of hepatitis c virus rna papillomaviruses and cancer: from basic studies to clinical applications human polyomavirus bkv and renal disease the role of polyomaviruses in human disease human polyomaviruses: molecular mechanisms for transformation and their association with cancer evolutionarily conserved function of a viral microrna sv40-encoded micrornas regulate viral gene expression and reduce susceptibility to cytotoxic t cell the oncogenic potential of human papillomaviruses: a review on the role of host genetics and environmental cofactors human papillomavirus genotype 31 does not express detectable microrna levels during latent or productive virus replication identification of micrornas of the herpesvirus family microrna-34a suppresses invasion through downregulation of notch1 and jagged1 in cervical carcinoma and choriocarcinoma cells identification of differentially expressed mirnas in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis anti-interferon activity of adenovirus-2-encoded vai and vaii rnas in translation in cultured human cells suppression of rna interference by adenovirus-associated rna is processed to functional interfering rnas involved in virus production the family herpesviridae: a brief introduction anti-apoptic function of a microrna encoded by hsv-1 latency-associated transcripts host immune system gene targeting by a viral mirna a human cytomegalovirus-encoded mi-crorna regulates expression of multiple viral genes involved in replication virology: micro mystery the replication of herpes simplex viruses prediction and identification of herpes simplex virus-1 encoded micrornas transcriptional origin of kaposi's sarcoma associated herpesvirus micrornas human cytomegalovirus expresses novel micrornas during productive viral infection marek's disease virus encodes micrornas that map to meq and the latency associated transcripts merkel cell polyomavirus encodes a microrna with the ability to auto-regulate viral gene expression regulation of human immunodeficiency virus 1 transcription by nef microrna differential expression between hepatitis b and hepatitis c leading disease progression to hepatocellular carcinoma a cellular microrna mediates antiviral defense in human cells human papillomaviruses modulate expression of microrna 203 upon epithelial differentiation to control levels of p63 proteins micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas herpes simplex virus type 1 icp4 promotes transcription preinitiation complex formation by enhancing the binding of tfiid to dna severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review identification and function of human cytomegalovirus micrornas suppression of immediate early viral gene expression by herpesvirus encoded micrornas: implications for latency latent epstein-barr virus (ebv) infection and cytomegalovirus (cmv) infection in synovial tissue of autoimmune chronic arthritis determined by rna-and dna-in situ hybridization epstein-barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions tandem array-based expression screens identify host mrna targets of virus-encoded micrornas epstein barr virus encoded microrna mir-bart2 down-regulates the viral dna polymerase balf5 the nuclear function of p53 is required for pumamediated apoptosis induced by dna damage ebv micrornas in primary lymphomas and targeting of cxcl-11 by ebv-mir-bhrf1-3 micrornas of kaposi's sarcoma-associated herpes virus marek's disease: a model for herpesvirus oncology analysis of the expression profiles of marek's disease virus-encoded micrornas by real-time quantitative pcr differential expression of micrornas in marek's disease virus-transformed t-lymphoma cell lines micrornas accurately identify cancer tissue origin are viral-encoded micrornas mediating latent hiv-1 infection? hiv-1 encoded candidate micro-rnas and their cellular targets hiv-1 nef suppression by virally encoded microrna challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection mechanisms of drug resistance to current and future antiviral therapies for hepatitis c virus infection occurrence of hcc in asymptomatic hcv-related chronic hepatitis therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection mir-122 regulation of lipid metabolism revealed by in vivo antisense targeting association between hepatitis b virus and pancreatic cancer hbv-encoded microrna candidate and its target targeted deletion of dicer in the heart leads to dilated cardiomyopathy and heart failure expressed anti-hbv primary micro-rna shuttles inhibit viral replication efficiently in vitro and in vivo inhibition of hepatitis b virus gene expression and replication by artificial microrna micrornaome of splenic macrophages in hypersplenism due to portal hypertension in hepatitis b virus-related cirrhosis an animal model of sars produced by infection of macaca mulatta with sars coronavirus oncogenic hpv infection interrupts the expression of tumor-suppressive mir-34a through viral oncoprotein e6 inhibition of coxsackievirus b3 replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand maintaining inhibition: si-rna double expression vectors against coxsackieviral rnas targeting 2a protease by rna interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice targeted delivery of anti-coxsackievirus sirnas using ligand-conjugated packaging rnas expression of short hairpin rnas against the coxsackievirus b3 exerts potential antiviral effects in cos-7 cells and in mice prospects and obstacles to using small interfering rnas as small molecule drugs progress and prospects: immune responses to viral vectors lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons inhibition of respiratory viruses by nasally administered sirna an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection identification of micrornas and other small regulatory rnas using cdna library sequencing mustering the micromanagers analysis of microrna expression by in situ hybridization with rna oligonucleotide probes transcription and processing of human microrna precursors expression profiling reveals off-target gene regulation by rnai a protocol for designing sirna with high functionality and specificity a pattern-based method for the identification of microrna binding sites and their corresponding heteroduplexes serum response factor regulates a muscle-specific microrna that targets hand2 during cadiogenesis correspondence: professor decheng yang, 166-1081 burrard street, the heart and lung institute, st paul's hospital this work was supported by grants from the canadian institutes of health research and the heart and stroke foundation of bc and yukon. dr. maged gomaa hemida is a recipient of the cihr-impact postdoctoral training fellowship. xin ye is supported by a ugf award from the university of british columbia.the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord-017506-t86v3zw3 authors: knox, tamsin a.; jerger, logan; tang, alice m. title: alcohol, hiv/aids, and liver disease date: 2012-04-27 journal: alcohol, nutrition, and health consequences doi: 10.1007/978-1-62703-047-2_23 sha: doc_id: 17506 cord_uid: t86v3zw3 globally, there are over 33 million persons living with hiv/aids resulting in 1.8 million deaths annually. while the rate of hiv transmission is slowing, it is estimated that 2.6 million new infections occur yearly [1]. in the united states, there are approximately 1.2 million living with hiv/aids, with 50,000 new hiv infections and 17,000 deaths from the disease annually [2]. for those who can obtain effective antiretroviral therapy (art), hiv/aids has become a chronic disease with life expectancies over 30 years [3]. research in the last 10 years has revealed the importance of alcohol in the hiv/aids epidemic. alcohol use, in moderate or hazardous amounts, has been associated with increased acquisition of hiv infection, progression of hiv infection, deleterious effects on hiv treatment, and acceleration in the comorbidities of hiv infection [4–9]. yet alcohol remains the “forgotten drug” of the hiv/aids epidemic [10]. globally, there are over 33 million persons living with hiv/aids resulting in 1.8 million deaths annually. while the rate of hiv transmission is slowing, it is estimated that 2.6 million new infections occur yearly [ 1 ] . in the united states, there are approximately 1.2 million living with hiv/aids, with 50,000 new hiv infections and 17,000 deaths from the disease annually [ 2 ] . for those who can obtain effective antiretroviral therapy (art), hiv/aids has become a chronic disease with life expectancies over 30 years [ 3 ] . research in the last 10 years has revealed the importance of alcohol in the hiv/ aids epidemic. alcohol use, in moderate or hazardous amounts, has been associated with increased acquisition of hiv infection, progression of hiv infection, deleterious effects on hiv treatment, and acceleration in the comorbidities of hiv infection [4] [5] [6] [7] [8] [9] . yet alcohol remains the "forgotten drug" of the hiv/aids epidemic [ 10 ] . alcohol has a complex relationship with hiv acquisition. risky sexual behaviors, among heterosexuals or among men who have sex with men (msm), that promote hiv transmission are increased in the setting of alcohol these include increased frequency of sexual encounters with new or anonymous partners and reduced condom use [ 11, 12 ] . attention to the locations and clientele where alcohol is served [ 13 ] has led to the development of an "ecological epidemiology" of the interplay of multiple risk factors around hiv transmission [ 14 ] . once infected with hiv, alcohol use is associated with progression of hiv infection from asymptomatic infection, to symptomatic aids with declining immune function measured by low cd4 t-cell counts (<200 cells/mm [ 3 ] ) in the blood, to death from wasting or an opportunistic infection. again, the relationship between alcohol use and progression of hiv infection is multifaceted. hazardous drinking has been associated with delayed testing and treatment for hiv infection [ 12, 15, 16 ] , poor adherence to art therapy [ 6, 17 ] , and increased hiv viral replication and shedding [18] [19] [20] . simian immunode fi ciency virus (siv) infection in monkey models has con fi rmed fi ndings that regular intake of alcohol leads to more rapid progression of disease, weight loss, and death [21] [22] [23] [24] . alcohol use also complicates the care of persons with hiv infection. not only is adherence to art decreased, but drug interactions between alcohol and speci fi c art medications may increase the toxicity of therapy [ 25 ] . hiv infection has numerous comorbidities including coexisting infections such as chronic viral hepatitis or tuberculosis as well as progressive organ dysfunction involving the liver, cardiovascular system, neurological dysfunction, or pulmonary disease. concurrent alcohol use may have a deleterious effect on any of these conditions [26] [27] [28] [29] [30] . thus, the management of alcohol misuse is central to control and treatment of hiv/aids. this chapter summarizes recent research on the effects of alcohol on hiv infection. epidemiologic studies of alcohol use in hiv infection inconsistently de fi ne alcohol intake and problem drinking. many studies categorize alcohol intake as "none," "moderate" drinking (ranging from any alcohol intake to daily intake over the period studied), and "hazardous" drinking (including regular daily intake or binge drinking and may or may not include a diagnosed alcohol disorder). in addition, the studies screening for alcohol disorders use different criteria including the cage questions, audit questionnaire, self-reported drinking, or a physician's report of an alcohol disorder [ 31 ] . thus, varying methodology and study population selection will greatly in fl uence the results from studies of alcohol use in hiv. prevalence of alcohol use among populations with hiv. there are wide apparent differences in rates of alcohol use and hazardous alcohol use due to the populations surveyed, the de fi nitions of "problem" alcohol use even in the same cohort, and the methods used to determine alcohol intake. in general, the prevalence of alcohol use disorders is several fold higher among populations with hiv infection compared to the general us population. some of the highest prevalence rates from problem drinking are among us veterans and homeless veterans [ 6, 58 ] . among the veterans administration (va) population, hazardous drinking patterns are found more frequently in african-americans (26%) than in whites (18%, p < 0.001) [ 59 ] . cook et al. determined that the prevalence of moderate and hazardous drinking among women with hiv infection was also higher than in the general us population [ 42, 56, 57 ] . other characteristics were associated with hazardous drinking patterns such as lower education, unemployment, nonwhite race, depression, and drug use. in both this cohort and in a va cohort, hazardous alcohol use was associated with hepatitis c virus (hcv) infection [ 42, 60 ] . among veterans with hcv infection, 35% were hazardous drinkers compared with 12% hazardous drinkers among matched controls without hcv infection [ 60 ] . the increased alcohol use among idu and the high correlation of idu and hcv infection likely explain this fi nding [ 46, 61 ] . alcohol intake appears to decline over time in persons with hiv infection as it does in noninfected persons with medical illness [ 62 ] . lefevre et al. examined alcohol intake in a group of 111 hivpositive patients of a university hospital clinic, mostly msm. in surveys repeated every 6 months for a mean follow-up of 30 months, the frequency of drinking decreased from 6.4 to 3.9 drinks/week ( p < 0.001) [ 63 ] . in the swiss hiv cohort study, lower alcohol use was found in those who had been on art for longer periods of time [ 64 ] . cook analyzed data from the women's interagency hiv study (wihs) on 2,770 hiv-positive women followed for 11 years [ 42 ] . there was a slight, approximately 5%, decrease in hazardous drinking over time but no change in the overall amount of drinking, possibly as some switched categories from hazardous to nonhazardous drinking. however, there was a signi fi cant decrease in alcohol consumption among women who were coinfected with hepatitis c and hiv from 31% with hazardous drinking patterns in 1995 to 10% in 2006. alcohol has been implicated in accelerating the progression of hiv disease through a number of mechanisms. persons drinking alcohol heavily delay testing for hiv and have less connection with and retention in the health-care system [ 12, 15, 16 ] , delaying the initiation of art. thus, heavy alcohol use predisposes persons to late presentation in the course of infection, with high hiv viral loads, low cd4 counts, and opportunistic infections, and promotes continued spread of hiv [ 45, 65 ] . one of the central ways alcohol intake adversely affects hiv disease is by decreasing adherence to art. adherence to art is key to suppression of hiv replication, prevention of developing drug resistance, and long-term survival [ 66 ] . this has been well documented among all subgroups with hiv infection [ 6, 64, 65, [67] [68] [69] [70] [71] . while there are few studies of adherence in developing countries, one study from india con fi rms the association of alcohol use and risk of nonadherence or discontinuation of art medications [ 72 ] . convincingly, there is a dose-response relationship between alcohol intake and adherence, with higher amounts of alcohol or more hazardous drinking being associated with poorer measures of adherence. samet et al. found that the amount of alcohol consumption was the strongest predictor of adherence with highest levels of adherence being found in those abstinent from alcohol compared to moderate use or at-risk use [ 70 ] . chander et al., studying nearly 2,000 hivinfected persons receiving care at johns hopkins hospital in baltimore, maryland, found that adherence was 22% lower in moderate alcohol users and 54% lower in hazardous alcohol users compared to no alcohol use. adherence was further decreased by 68% with concurrent drug use [ 65 ] . there may be several reasons for lower adherence in persons who use alcohol. drinking pattern affects the likelihood of noncompliance. braithwaite et al., studying 2,700 members of the veterans administration aging cohort study (vacs), found that abstainers missed art on 2% of days. nonbinge drinkers missed medication on 4% of drinking days and post-drinking days but only on 2% of nondrinking days. binge drinkers, in contrast, missed art on 11% of drinking days, 5.5% of postdrinking days, and 4% on nondrinking days [ 6 ] . therefore, while medication adherence was lower on drinking days for binge and non-binge drinkers, missing medications was increased twofold among binge drinkers on days they were either not drinking or post-drinking. this suggests that nonadherence was also due to factors not directly related to alcohol but related to characteristics common among binge drinkers [ 6 ] . sankar et al. studied beliefs about alcohol and art medication interactions in a group of african-american patients treated for hiv [ 71 ] . over three quarters of those surveyed felt that "alcohol and art do not mix"; one-third attributed this to alcohol making art ineffective and another third felt that alcohol made art more toxic. in this study, participants reported purposely skipping art doses when they drank, with light drinkers skipping 64% of the times when they drank and moderate drinkers 55% of the times. however, heavy drinkers skipped art only 29% of the time when they drank and reported that they felt no ill effects from drinking and taking art [ 71 ] . thus, medication adherence is determined by amount of alcohol intake, drinking pattern (binge or non-binge drinking), and beliefs about the safety of alcohol combined with art. issues of medication adherence and alcohol are further discussed in chap. 18 and in a meta-analysis by hendershot [ 17 ] . alcoholics have increased susceptibility to bacterial infections including tuberculosis, pneumonia, and sepsis [ 73 ] . in vitro studies have shown that alcohol impacts several areas of immune function, acting largely as an immunosuppressant. alcohol decreases t-cell proliferation reducing cd4, cd8, and natural killer (nk) cell numbers [ 7 ] and reduces cd8 cell responses to bacteria [ 74 ] . cell-mediated immune responses are decreased [ 75 ] , and myeloid dendritic cells, which are involved in antigen presentation to the immune system, are decreased in number and function with chronic alcohol ingestion [ 76, 77 ] . alcohol increases expression of pro-in fl ammatory cytokines such as tnf-alpha [ 78 ] which may enhance immune dysfunction. experiments by bagasra et al. on human peripheral blood mononuclear cells (pbmc) have shown that cells from healthy persons who are infected in vitro with hiv-1 have higher levels of hiv replication when harvested after alcohol consumption [ 19 ] . enhanced hiv replication was associated with a concurrent inhibition of cd8 cells by alcohol [ 18 ] . siv infection, a macaque model for hiv, has produced evidence of the effect of alcohol on immune function and hiv replication. in rhesus macaques inoculated with siv infection, siv replication was 31-to 85-fold higher in monkeys with chronic alcohol ingestion compared to controls [ 21 ] . siv replication persisted in the central nervous system of alcohol-fed monkeys but was undetectable in control monkeys. poonia et al. proposed that the mechanism of alcohol's effect on siv replication is through its effect on intestinal lymphocytes since the small intestine is one of the most lymphocyterich organs. alcohol-fed monkeys had lower numbers of cd8 cells (before and after siv infection) and higher numbers of cd4 cells in the small intestine after siv infection. they suggested that the 1-2 log 10 increase in siv replication in alcohol-fed monkeys occurs because of the increase in number of cd4 cells susceptible to siv infection in the small intestine and reduction in cd8 cells which may control siv replication [ 22 ] . chronic alcohol ingestion also altered the course of hiv infection with alcohol-fed monkeys having lower cd4 cell counts, lower caloric intake, higher tnf-alpha expression, and a more rapid progression to end-stage siv disease (mean 374 days compared to 900 days in controls) [ 23, 24 ] . alcohol use has been shown to affect hiv progression and survival. in the pre-haart era, alcohol use was not associated with progression to aids [79] [80] [81] . however, two well-controlled, longitudinal studies since the introduction of combination art have shown that alcohol is associated with hiv disease progression. samet et al. studied alcohol use in 595 participants in the macs cohort over 7 years [ 82 ] . heavy alcohol use was associated with a lower mean cd4 cell count (by ~50 cells/ml) but not a decline in cd4 percentage or hiv viral load when adjusted for adherence. baum et al. studied 231 hiv-positive persons followed for 2.5 years [ 5 ] . frequent alcohol users of ³ 2 drinks/day were almost 3 times more likely to develop a cd4 count £ 200 cells/ml, which is an aids-de fi ning event. this effect was particularly marked in alcohol users not on art whose risk of developing a cd4 count £ 200 cells/ml was nearly 8 times nondrinkers. in this study, alcohol use was associated with higher hiv viral load in those on art but not in those without art. these results suggest that the effect of alcohol on hiv viral load is mediated through adherence. however, the effect of alcohol in lowering absolute cd4 count rather than percentage could be in fl uenced by the splenomegaly and secondary lymphopenia seen with alcoholism and chronic viral hepatitis [ 83 ] . moderate to heavy alcohol use has also been associated with increased hiv viral shedding in the female genital tract after controlling for plasma viral load [ 20 ] suggesting that alcohol may affect hiv transmission by physiological as well as behavioral risk factors. the vacs study has provided models for estimating the effect of alcohol on survival in hiv infection. using data on art adherence in the vacs cohort, braithwaite et al. developed a model simulating survival based on levels of alcohol consumption (nondrinkers, hazardous drinkers consuming ³ 5 drinks on drinking days, and nonhazardous drinkers) [ 84 ] . the model predicted decreased survival by >1 year in nonhazardous drinkers drinking at least once a week, 3.3 years in nonhazardous drinkers drinking daily, and up to 6.4 years in hazardous drinkers drinking daily. however, the vacs index, subsequently developed to predict decreases in life expectancy based on hiv and non-hiv characteristics, does not include a separate variable for alcohol or drug abuse beyond adjusting for severity of liver disease and coexisting hcv infection [ 85 ] . in addition, a longitudinal study of changes in physical function with age in the same cohort did not show an effect of alcohol [ 86 ] . further longitudinal studies in this cohort and others should de fi ne the impact of alcohol use on survival in hiv. persons with hiv infection are particularly vulnerable to the effects of alcohol. the detrimental effects of alcohol on the immune system have been covered above, and the effects of alcohol on general health and nutrition are covered in other chapters in this book. persons with hiv infection are at risk of poor nutritional status, and even a 3% weight loss has been associated with increased mortality [87] [88] [89] [90] . thus, further changes in nutritional status due to alcohol use, particularly lower body weight or micronutrient de fi ciencies, would exacerbate the nutritional effects of hiv [ 91, 92 ] . approximately one-third of persons with hiv infection are coinfected with hcv, and approximately 10% have evidence of chronic hepatitis b virus (hbv) infection [ 93 ] . the prevalence of hcv coinfection increases to almost 90% in those who acquired hiv from idu. persons with coinfection with chronic hepatitis have accelerated liver fi brosis leading to cirrhosis [ 9, 94 ] . in a study of liver histology of idu who had acquired hcv infection, those with concurrent hiv infection developed cirrhosis in a mean of 6.9 years after infection compared to 23.2 years among hcv mono-infected persons ( p < 0.001) [ 95 ] . persons with coinfection also have an increased risk of death from end-stage liver disease [96] [97] [98] [99] . they are also at higher risk for drug-induced hepatotoxicity from art [ 100, 101 ] which may be related to altered cytochrome metabolism with progressive liver disease [ 102 ] . other metabolic abnormalities are more common in coinfected persons including hyperglycemia, diabetes, and bacterial translocation from the small intestine to the portal system, predisposing coinfected chronic in fl ammation and progressive liver disease [103] [104] [105] . hazardous alcohol use is increased in some populations with coinfection, particularly idus [ 64, 106 ] . alcohol use further exacerbates the effect of coinfection on liver disease. alcohol use of > 50 g/ day is associated with increased hcv replication [ 107, 108 ] and progressive liver fi brosis assessed by serum markers [ 109 ] , transient elastometry [ 110 ] or by liver biopsy [ 9, [111] [112] [113] . death from endstage liver disease is also more common in coinfected persons who use alcohol [ 29, 30, 114, 115 ] . the incidence of, as well as deaths related to, hepatocellular carcinoma is also increased in those with coinfection who drink alcohol [ 30, 116 ] . only one study did not fi nd an association of alcohol use and an hcv-related severe event (including decompensated cirrhosis, hepatocellular carcinoma, or death) [ 117 ] , but in this cohort, only 10% consumed >30 g of alcohol daily. alcohol use also contributes to metabolic abnormalities in coinfected persons. it is associated with higher rates of liver steatosis [ 110 ] and drug-induced liver disease [ 25, 118 ] . the association of alcohol use with hepatocellular carcinoma is also discussed in chap. 32. the adverse effects of alcohol in coinfection argue strongly for intervention. hazardous alcohol use is a common reason for coinfected persons not receiving treatment for hcv infection, where treatment rates may be as low as 7% [ 106, [119] [120] [121] [122] . fortunately, alcohol use seems to decrease with interventions after hcv diagnosis in some populations [ 123, 124 ] . treatment of chronic viral hepatitis whether due to hcv or hbv infection slows the progression of liver fi brosis [ 125, 126 ] and reduces the incidence of drug-induced liver disease [ 127 ] . treatment outcomes with pegylated interferon and ribavirin [ 128, 129 ] and with the new protease inhibitors for hcv infection should continue to improve as more coinfected persons are being enrolled in treatment [ 130 ] . persons with hiv infection have an increased risk of cardiovascular disease, particularly accelerated atherosclerosis and myocardial infarction [131] [132] [133] . cardiovascular disease is likely due to a combination of additional risk factors found in hiv infection [ 26 ] including (1) chronic in fl ammation from hiv viral replication and subsequent immunode fi ciency [ 134 ] , (2) the effect of chronic in fl ammation on serum lipid levels [ 133 ] , (3) the metabolic effects of certain classes of antiretroviral medications [ 131, 133 ] , (4) increased prevalence of insulin resistance [ 135 ] , and (5) increased translocation of bacteria across the small intestine into the bloodstream as a result of immunode fi ciency [ 136 ] . persons with hiv infection have been shown to have more rapid progression of atherosclerosis measured by intermediate markers such as carotid intima-media thickness, and this has correlated with mortality [ 134, 137, 138 ] . alcohol use further increases the risk of cardiovascular disease in hiv infection. freiberg et al., studying the vacs cohort, found that the risk of cardiovascular disease was increased (or 1.55, 95% ci 1.07-2.23) in hiv-infected men with alcohol abuse or dependence, when controlled for cardiac risk factors, art use, and cd4 count [ 8 ] . furthermore, hcv infection may have an independent effect in increasing the risk of cardiovascular disease (or 4.7, 95% ci 1.7-12.7) although alcohol use does not seem to affect this relationship [ 139 ] . chapters 24 and 25 explore further the relationship of alcohol and cardiovascular disease. alcohol and hiv infection are both risk factors for pulmonary diseases. alcoholics have increased prevalence of oropharyngeal colonization by pathogenic bacteria and an increased risk of aspiration [ 140 ] . in addition, they have impaired pulmonary immune function leading to a higher incidence of pneumonia [ 27, 140 ] . studies have shown that alcohol use is a risk factor for the development of pneumonia in the absence of hiv, as well as more severe, multilobar pneumonia and more virulent pathogens including candida , gram-negative bacteria, and staphylococcus aureus infections. this, in turn, leads to longer hospitalizations and increased mortality related to alcohol use [ 141 ] . the risk for adult respiratory distress syndrome (ards), which has a mortality of 40-60% [ 142 ] , is increased three-to fourfold in those with heavy alcohol intake [ 143, 144 ] . similarly, persons with hiv infection are at an increased risk of community-acquired pneumonias, including unusual pathogens such as pseudomonas aeruginosa [ 145 ] . hiv infection is also associated with pulmonary opportunistic infections, such as pneumocystis [ 146 ] . while both alcohol use and hiv infection have an increased risk of pneumonia and tuberculosis, there are no studies to date that demonstrate the interaction of these risk factors for acute pulmonary disease [ 27 ] . there is suggestive literature that depletions in zinc levels or pulmonary glutathione stores may mediate impaired host defense [ 27 ] . chronic lung disease in alcoholics is largely related to associated tobacco use [ 27 ] . however, persons with hiv infection have an increased risk of emphysema, lung cancer, and pulmonary hypertension, independent of smoking, and this is particularly evident in those with poorly controlled hiv infection [ 147 ] . the adverse effects of alcohol use on hiv are evident, and interventions to mitigate alcohol use among hiv-infected individuals are needed. to date, clinical studies and a few randomized controlled trials (rcts) assessing the effectiveness of interventions have shown mixed results. in this section, we will brie fl y review the types of interventions that have been evaluated and discuss results from a few published trials. interested readers can refer to recent review articles for more complete reviews of the literature [ 13, 148, 149 ] . many types of alcohol interventions have been tested among hazardous alcohol users with and without hiv infection. these include brief interventions as well as more intensive behavioral, social network, and medication interventions. brief interventions, also referred to as brief motivational interviews, are typically a single session discussing the patients' alcohol use. studies employing this type of intervention often involve exploration of the pros and cons of a patient's alcohol use, self-assessment of the patient's alcohol consumption severity, and a more formal assessment of the patient's alcohol consumption as compared to the general population [ 150 ] . more extensive behavioral interventions have also been investigated, including cognitive-behavioral therapy, motivation enhancement, or 12-step programs. each of these behavioral interventions is directly aimed at investigating personal motivation behind alcohol consumption and developing personal behavior modi fi cation strategies [ 151 ] . these interventions typically require multiple sessions. in addition to individualized plans and programs for those with increased alcohol consumption, social network and structural interventions which target larger populations and communities have also been evaluated. social network interventions have most commonly focused on employing in fl uential community leaders to change speci fi c behaviors or promote health-conscious decisions. these studies, often referred to as popular opinion leader (pol) or peer-based model interventions, may be particularly effective in communities that are dif fi cult for outside researchers to impact [ 152 ] . alternatively, structural interventions, which may include political and legal action, may also be effective in altering individuals' behavior and environment. lastly, medications, such as disul fi ram, naltrexone, and acamprosate, have been shown to decrease alcohol consumption via physiologic effects, including decreasing cravings or causing adverse reactions when alcohol is consumed [ 149 ] . in addition to the type of alcohol intervention, there are several other factors to consider when evaluating results from clinical trials of alcohol interventions. the fi rst is the setting in which the interventions are conducted. interventions have been conducted in various settings including primary care clinics [ 153, 154 ] , hospital inpatient settings [ 155 ] , emergent care settings [ 156 ] , and social settings or drinking venues (places where alcohol is served) [ 13 ] . a second important factor to consider is the population being targeted, which may vary depending on severity of alcohol use (dependent vs. nondependent drinkers), geographic region, and cultural practices around drinking. a third factor to consider is the outcome that is being targeted. for example, previous trials have examined the effects of alcohol interventions on decreasing alcohol consumption, improving adherence to antiretroviral medication and/or reducing sexual risk behaviors. the combination of the type of intervention, the setting in which the intervention is implemented, the population that is being targeted, and the expected outcomes of the trial will all contribute to the success or failure of an intervention. the published literature on rcts of alcohol interventions among populations affected by hiv re fl ects the various combinations of factors described above. for example, one study targeting msm in the usa with alcohol use disorders combined two types of interventions (motivational interviewing and peer-group education/support strategies) and examined the effects on reducing at-risk drinking and sexual risk behaviors [ 157 ] . in this study, individuals receiving the combined intervention reported signi fi cantly lower number of days of drinking and number of heavy drinking days per 30-day period compared to control participants. another study tested the effects of a brief theorybased behavioral hiv-alcohol risk-reduction intervention on sexual risk behaviors in men and women recruited from informal drinking establishments in a suburban township of capetown, south africa [ 50 ] . the authors reported signi fi cant reductions in unprotected intercourse, increased use of condoms, and less use of alcohol before sex in the intervention group compared to controls, with the largest impacts among lighter drinkers. these two studies illustrate the success of individual counseling interventions for reducing risk behaviors around alcohol consumption among persons at risk for or living with hiv. other interventions among individuals with hiv who consume alcohol have targeted the outcome of antiretroviral medication adherence. in two speci fi c studies [ 151, 158 ] , motivational interviews and cognitive-behavioral skills training were not effective in improving long-term medication adherence. given the importance of adherence to art to controlling hiv infection, more research is needed to develop novel interventions targeting this outcome. interventions directed at alcohol-serving establishments have had mixed results. studies have focused on popular opinion leader (pol) models, in which community-de fi ned opinion leaders are identi fi ed and trained to help shift social norms and behaviors toward safer sexual practices [ 152 ] . this type of intervention in gay bars in several us cities signi fi cantly reduced episodes of risky sexual behavior compared to control bars [ 152, 159, 160 ] ; however, when this intervention was adapted for testing in several international settings, the fi ndings were negative in that comparable reductions in risky sexual behaviors and incidence of sexually transmitted infections were seen in both intervention and control communities [ 161 ] . another study testing the effects of a peer-based intervention on reducing episodes of unprotected sex with non-wife partners in beer halls in zimbabwe found no difference compared to controls [ 162 ] . in summary, interventions involving varied counseling approaches directed at decreasing alcohol consumption and/or risky sexual behavior appear promising in speci fi c settings. other areas of investigation, such as interventions aimed at improving art adherence among alcohol users or use of medications for alcohol dependence (such as naltrexone) in hiv-infected populations, need further research. more intervention studies will help to generalize fi ndings across different contexts and help to improve health outcomes and minimize the effects of alcohol on persons living with hiv. in this chapter, we have examined the prevalence of hazardous alcohol use in hiv which is much higher than found in the general us population. alcohol use and frequenting venues where alcohol is consumed has been shown to be an important risk factor for the acquisition of hiv infection. understanding the complex interrelationships between individual characteristics and venues should improve our approach to prevention [ 12, 14 ] . the effect of alcohol on adherence to art is well documented. there are also good laboratory models, particularly with siv infection in macaques, to show that chronic alcohol use accelerates the progression of disease. finally, alcohol use has deleterious effects on health, particularly related to progression of liver disease in persons with hiv/hcv coinfection. health-care providers may underestimate the extent of hazardous drinking among their hiv patients. a study in the va population showed that the sensitivity for health-care providers' ability to diagnose hazardous drinking was only 22% [ 163 ] . thus far, trials of interventions to reduce hazardous drinking in populations affected by hiv have shown mixed results. the underdiagnosis of hazardous alcohol use and lack of proven, effective treatment strategies raise the question of whether there is any "safe" level of alcohol intake in hiv. justice et al. examined the relationship of medical illness related to alcohol use in veterans with hiv infection [ 164 ] . for diseases associated with alcohol use (hcv infection, hypertension, diabetes, chronic obstructive lung disease, and certain infections), there was a linear relationship between alcohol intake category (none, moderate, hazardous) and the disease. this suggests that there may be no "safe" level of alcohol intake for hiv-infected persons [ 165 ] . more aggressive screening and treatment of alcohol-related disorders is clearly warranted to prevent hiv transmission and to improve treatment and outcomes of persons with hiv infection [ 84 ] . racial and sex disparities in life expectancy losses among hivinfected persons in the united states: impact of risk behavior, late initiation, and early discontinuation of antiretroviral therapy med care (alcohol in hiv infection: insights from the veterans aging cohort study and the veterans affairs national health information system) alcohol use accelerates hiv disease progression a temporal and dose-response association between alcohol consumption and medication adherence among veterans in care alcohol's role in hiv transmission and disease progression the association between alcohol consumption and prevalent cardiovascular diseases among hiv-infected and hiv-uninfected men incidence and predictors of severe liver fi brosis in human immunode fi ciency virus-infected patients with chronic hepatitis c: a european collaborative study alcohol: the forgotten drug in hiv/aids causal links between binge drinking patterns, unsafe sex and hiv in south africa: its time to intervene integrating hiv/aids and alcohol research social and structural hiv prevention in alcohol-serving establishments: review of international interventions across populations hiv risk and the alcohol environment: advancing an ecological epidemiology for hiv/aids alcohol and hiv disease progression: weighing the evidence health services utilization for people with hiv infection: comparison of a population targeted for outreach with the us population in care alcohol use and antiretroviral adherence: review and meta-analysis increased human immunode fi ciency virus type 1 replication in human peripheral blood mononuclear cells induced by ethanol: potential immunopathogenic mechanisms alcohol intake increases human immunode fi ciency virus type 1 replication in human peripheral blood mononuclear cells alcohol consumption and hiv-1 vaginal rna shedding among women increased viral replication in simian immunode fi ciency virus/ simian-hiv-infected macaques with self-administering model of chronic alcohol consumption chronic alcohol consumption results in higher simian immunode fi ciency virus replication in mucosally inoculated rhesus macaques chronic binge ethanol consumption accelerates progression of simian immunode fi ciency virus disease chronic alcohol accentuates nutritional, metabolic, and immune alterations during asymptomatic simian immunode fi ciency virus infection risk factors for severe hepatic injury after introduction of highly active antiretroviral therapy focus on the heart: alcohol consumption, hiv infection, and cardiovascular disease modeling hiv and alcohol's effects: focus on the lung focus on the brain: hiv infection and alcoholism prevalence factors associated with signi fi cant liver fi brosis assessed by transient elastometry in hiv/hepatitis c virus-coinfected, patients liver-related deaths in, h. i. v. infected patients between in the french, germivic joint study group network detecting alcohol problems in hiv-infected patients: use of the cage questionnaire the association between hiv infection and alcohol use: a systematic review and meta-analysis of african studies alcohol as a correlate of unprotected sexual behavior among people living with hiv/aids: review and meta-analysis early alcohol initiation and subsequent sexual and alcohol risk behaviors among urban youths alcohol use, partner type, and risky sexual behavior among college students: fi ndings from an event-level study hiv and sexually transmitted diseases: lethal synergy alcohol and sexual hiv risk behavior among problem drinking men who have sex with men: an event level analysis of timeline followback data alcohol use and risk of hiv infection among men who have sex with men alcohol use and sexual risk behavior among human immunode fi ciency virus-positive persons depressive symptoms, utilization of mental health care, substance use and sexual risk among young men who have sex with men in explore: implications for age-speci fi c interventions risk factors for hiv infection among men who have sex with men longitudinal trends in hazardous alcohol consumption among women with human immunode fi ciency virus infection alcohol consumption, art usage and high-risk sex among women infected with hiv hiv, alcohol, and noninjection drug use alcohol abuse and stage of hiv disease in intravenous drug abusers the impact of illicit drug use and harmful drinking on quality of life among injection drug users at high risk for hepatitis c infection alcohol use and sexual risks for hiv/aids in sub-saharan africa: systematic review of empirical fi ndings multiple recent sexual partnerships and alcohol use among sexually transmitted infection clinic patients socioeconomic status and risk of hiv infection in an urban population in kenya randomized trial of a community-based alcohol-related hiv riskreduction intervention for men and women in cape town south africa hiv rates and risk behaviors are low in the general population of men in southern india but high in alcohol venues: results from 2 probability surveys male alcohol use and unprotected sex with non-regular partners: evidence from wine shops in chennai women and substance use in india and bangladesh the impact of hiv and high-risk behaviours on the wives of married men who have sex with men and injection drug users: implications for hiv prevention estimation of hiv-1 incidence among fi ve focal populations in dehong, yunnan: a hard hit area along a major drug traf fi cking route the 12-month prevalence and trends in dsm-iv alcohol abuse and dependence: united states binge drinking among us adults associations between alcohol use and homelessness with healthcare utilization among human immunode fi ciency virusiinfected veterans alcohol problems and health care services use in human immunode fi ciency virus (hiv)-infected and hiv-uninfected veterans co-morbid medical and psychiatric illness and substance abuse in hcv-infected and uninfected veterans high prevalence of alcohol use among hepatitis c virus antibody positive injection drug users in three us cities reduction in alcohol consumption and health status alcohol consumption among hiv-infected patients self-reported alcohol consumption and its association with adherence and outcome of antiretroviral therapy in the swiss hiv cohort study hazardous alcohol use: a risk factor for non-adherence and lack of suppression in hiv infection adherence to protease inhibitor therapy and outcomes in patients with hiv infection gender differences in factors associated with adherence to antiretroviral therapy problem drinking and medication adherence among persons with hiv infection alcohol use and incarceration adversely affect hiv-1 rna suppression among injection drug users starting antiretroviral therapy alcohol consumption and antiretroviral adherence among hiviinfected persons with alcohol problems sero-positive african americans' beliefs about alcohol and their impact on anti-retroviral adherence access, adherence, quality and impact of arv provision to current and ex-injecting drug users in manipur (india): an initial assessment alcohol abuse, alcoholism, and damage to the immune system-a review chronic ethanol induces inhibition of antigen-speci fi c cd8+ but not cd4+ immunodominant t cell responses following listeria monocytogenes inoculation consequences of alcohol consumption on host defence alcohol exposure impairs myeloid dendritic cell function in rhesus macaques alcohol suppresses the granulopoietic response to pulmonary streptococcus pneumoniae infection with enhancement of stat3 signaling regulation of macrophage activation in alcoholic liver disease no evidence for a role of alcohol or other psychoactive drugs in accelerating immunode fi ciency in hiv-1-positive individuals. a report from the multicenter aids cohort study cofactors of progression to acquired immunode fi ciency syndrome in a cohort of male sexual contacts of men with human immunode fi ciency virus disease determinants of hiv disease progression among homosexual men registered in the tricontinental seroconverter study alcohol consumption and hiv disease progression the impact of cirrhosis on cd4+ t cell counts in hiv-seronegative patients estimating the impact of alcohol consumption on survival for hiv + individuals towards a combined prognostic index for survival in hiv infection: the role of 'non-hiv' biomarkers association of age and comorbidity with physical function in hivinfected and uninfected patients: results from the veterans aging cohort study malnutrition in a population of hiv-positive and hiv-negative drug users living in chennai, south india. drug alcohol depend weight loss and survival in hivpositive patients in the era of highly active antiretroviral therapy increasing risk of 5% or greater unintentional weight loss in a cohort of hiv-infected patients weight loss and body-composition changes in men and women infected with hiv in fl uence of chronic alcohol abuse on body weight and energy metabolism: is excess ethanol consumption a risk factor for obesity or malnutrition? inadequate dietary intake and altered nutrition status in early hiv-1 infection hepatitis c virus infection as an opportunistic disease in persons infected with human immunode fi ciency virus in fl uence of human immunode fi ciency virus infection on the course of hepatitis c virus infection: a meta-analysis human immunode fi ciency virus infection modi fi es the natural history of chronic parenterally-acquired hepatitis c with an unusually rapid progression to cirrhosis increasing mortality due to end-stage liver disease in patients with human immunode fi ciency virus infection rates and risk factors of liver fi brosis progression in patients with chronic hepatitis c natural history of liver fi brosis progression in patients with chronic hepatitis c. the obsvirc, metavir, clinivir, and dosvirc groups little evidence that hepatitis, c. virus leads to a higher risk of mortality in the absence of cirrhosis excess alcohol intake: the swiss hepatitis c cohort, study. j viral hepatitis hepatotoxicity associated with antiretroviral therapy in adults infected with human immunode fi ciency virus and the role of hepatitis c or b virus infection highly active antiretroviral therapy-induced liver injury ritonavir greatly impairs cyp3a activity in hiv infection with chronic viral hepatitis hepatitis c virus antibody-positive patients with hiv infection have a high risk of insulin resistance: a cross-sectional study the effect of haart and hcv infection on the development of hyperglycemia among hiv-infected persons human immunode fi ciency virus-related microbial translocation and progression of hepatitis c relative prevalence of comorbidities and treatment contraindications in hiv-mono-infected and hiv/hcv-co-infected veterans effect of alcohol use and highly active antiretroviral therapy on plasma levels of hepatitis c virus (hcv) in patients coinfected with hiv and hcv an overview of hiv and chronic viral hepatitis co-infection hazardous drinking is associated with an elevated aspartate aminotransferase to platelet ratio index in an urban hiv-infected clinical cohort risk factors for advanced liver fi brosis in hiv-infected individuals: role of antiretroviral drugs and insulin resistance liver fi brosis progression in human immunode fi ciency virus and hepatitis c virus coinfected patients. the multivirc group factors affecting liver fi brosis in human immunode fi ciency virusand hepatitis c virus-coinfected patients: impact of protease inhibitor therapy a comparison of fi brosis progression in chronic liver diseases the in fl uence of human immunode fi ciency virus coinfection on chronic hepatitis c in injection drug users: a long-term retrospective cohort study mortality related to chronic hepatitis b and chronic hepatitis c in france: evidence for the role of hiv coinfection and alcohol consumption hepatocellular carcinoma and nonhodgkin's lymphoma: the roles of hiv, hepatitis c infection, and alcohol abuse the french national prospective cohort of patients co-infected with hiv and hcv antiretroviral drugs and liver injury rates and predictors of hepatitis c virus treatment in hcv-hiv-coinfected subjects rates of hcv treatment eligibility among hcv-monoinfected and hcv/ hiv-coinfected patients in tertiary care referral centers barriers to treatment of hepatitis c in hiv/hcv coinfected adults in brazil barriers to treatment of hepatitis c in hiv/hcvcoinfected adults with alcohol problems medical care and alcohol use after testing hepatitis c antibody positive at std clinic and hiv test site screening programs awareness of hepatitis c diagnosis is associated with less alcohol use among persons co-infected with hiv liver fi brosis changes in hiv-hbv-coinfected patients: clinical, biochemical and histological effect of long-term tenofovir disoproxil fumarate use slower fi brosis progression in hiv/hcv-coinfected patients with successful hiv suppression using antiretroviral therapy managing symptomatic drug-induced liver injury in hiv-hepatitis c virus-coinfected patients: a role for interferon peginterferon alfa-2a plus ribavirin versus interferon alfa-2a plus ribavirin for chronic hepatitis c in hiv-coinfected persons peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection in hiv-infected patients care of hepatitis c virus infection in human immunode fi ciency virusinfected patients: modi fi cations in three consecutive large surveys between trends in rates of myocardial infarction among patients with hiv epidemiological evidence for cardiovascular disease in hiv-infected patients and relationship to highly active antiretroviral therapy cardiovascular disease risk factors in hiv patients-association with antiretroviral therapy. results from the dad study markers of atherosclerosis and in fl ammation and mortality in patients with hiv infection combination antiretroviral therapy and the risk of myocardial infarction microbial translocation is a cause of systemic immune activation in chronic hiv infection framingham risk score and early markers of atherosclerosis in a cohort of adults infected with hiv progression of atherosclerosis as assessed by carotid intima-media thickness in patients with hiv infection the association between hepatitis c infection and prevalent cardiovascular disease among hiv-infected individuals alcohol, immunosuppression, and the lung high alcohol intake as a risk and prognostic factor for community-acquired pneumonia incidence and outcomes of acute lung injury chronic alcohol abuse is associated with an increased incidence of acute respiratory distress syndrome and severity of multiple organ dysfunction in patients with septic shock the alcoholic lung: epidemiology, pathophysiology, and potential therapies bacterial pneumonia in hospitalized patients with hiv infection: the pulmonary complications, icu support, and prognostic factors of hospitalized patients with hiv (pip) study hiv-associated opportunistic pneumonias hiv infection and risk for incident pulmonary diseases in the combination antiretroviral therapy era effectiveness of hiv prevention for youth in sub-saharan africa: systematic review and meta-analysis of randomized and nonrandomized trials interventions targeting hiv-infected risky drinkers: drops in the bottle brief alcohol intervention and alcohol assessment do not in fl uence alcohol use in injured patients treated in the emergency department: a randomized controlled clinical trial motivational interviewing and cognitive-behavioral intervention to improve hiv medication adherence among hazardous drinkers: a randomized controlled trial randomised, controlled, community-level hiv-prevention intervention for sexual-risk behaviour among homosexual men in us cities screening in brief intervention trials targeting excessive drinkers in general practice: systematic review and meta-analysis effectiveness of brief alcohol interventions in primary care populations effectiveness of opportunistic brief interventions for problem drinking in a general hospital setting: systematic review preventive care in the emergency department: screening and brief intervention for alcohol problems in the emergency department: a systematic review reducing sexual risk behaviors and alcohol use among hiv-positive men who have sex with men: a randomized clinical trial a randomized controlled trial to enhance antiretroviral therapy adherence in patients with a history of alcohol problems community aids/hiv risk reduction: the effects of endorsements by popular people in three cities hiv prevention with male prostitutes and patrons of hustler bars: replication of an hiv preventive intervention results of the nimh collaborative hiv/sexually transmitted disease prevention trial of a community popular opinion leader intervention evaluation of a peer network-based sexual risk reduction intervention for men in beer halls in zimbabwe: results from a randomized controlled trial veterans aging cohort 3-site s. how harmful is hazardous alcohol use and abuse in hiv infection: do health care providers know who is at risk? med care (alcohol in hiv infection: insights from the veterans aging cohort study and the veterans affairs national health information system) role of alcohol in determining human immunode fi ciency virus (hiv)-relevant outcomes: a conceptual model to guide the implementation of evidence-based interventions into practice. med care (alcohol in hiv infection: insights from the veterans aging cohort study and the veterans affairs national health information system) the prevalence of alcohol consumption and heavy drinking among people with hiv in the united states: results from the hiv cost and services utilization study longitudinal assessment of the effects of drug and alcohol abuse on hiv-1 treatment outcomes in an urban clinic determinants of heterogeneous adherence to hiv-antiretroviral therapies in the multicenter aids cohort study key: cord-010273-0c56x9f5 authors: simmonds, peter title: virology of hepatitis c virus date: 2001-10-10 journal: clin ther doi: 10.1016/s0149-2918(96)80193-7 sha: doc_id: 10273 cord_uid: 0c56x9f5 hepatitis c virus (hcv) has been identified as the main causative agent of post-transfusion non-a, non-b hepatitis. through recently developed diagnostic assays, routine serologic screening of blood donors has prevented most cases of post-transfusion hepatitis. the purpose of this paper is to comprehensively review current information regarding the virology of hcv. recent findings on the genome organization, its relationship to other viruses, the replication of hcv ribonucleic acid, hcv translation, and hcv polyprotein expression and processing are discussed. also reviewed are virus assembly and release, the variability of hcv and its classification into genotypes, the geographic distribution of hcv genotypes, and the biologic differences between hcv genotypes. the assays used in hcv genotyping are discussed in terms of reliability and consistency of results, and the molecular epidemiology of hcv infection is reviewed. these approaches to hcv epidemiology will prove valuable in documenting the spread of hcv in different risk groups, evaluating alternative (nonparenteral) routes of transmission, and in understanding more about the origins and evolution of hcv. hepatitis c virus (hcv) has been identified as the main causative agent of posttransfusion non-a, non-b hepatitis. 1,2 the identification of hcv led to the development of diagnostic assays for infection, based either on detection of antibody to recombinant polypeptides expressed from cloned hcv sequences or direct detection of virus ribonucleic acid (rna) sequences by polymerase chain reaction (pcr) using primers complimentary to the hcv genome. routine serologic screening of blood donors now prevents most or all cases of posttransfusion hepatitis. assays 0149-2918/96/$ 3.50 for antibody also are important diagnostic tools and have been used to investigate the prevalence of hcv in different risk groups, such as intravenous drug users, patients with hemophilia, and other recipients of blood products, and to conduct epidemiologic studies of hcv transmission. the complete genomic sequence of hcv has been determined for several isolates, revealing both its overall genome organization and its relationship to other rna viruses. deducing possible methods of replication by analogy with related viruses is possible, although such studies currently are hampered by the absence of a satisfactory in vitro culture method for hcv. as a consequence, most conventional virologic studies are difficult and artificial. hcv contains a positive-sense rna genome approximately 9400 bases in length. in overall genome organization and presumed method of replication, it is most similar to members of the family flaviviridae, particularly in coding for a single polyprotein that is then cleaved into a series of presumed structural and nonstructural proteins (figure 1 ). 3 the roles for these different proteins have been inferred by comparison with related viruses and by in vitro expression of cloned hcv sequences in prokaryotic and eukaryotic systems. these artificial systems allowed the investigation of protein expression, cleavage, and posttranslational modifications. there are numerous positive-stranded rna virus families whose coding capacity is contained within a single open read-ing frame (orf) as is found in hcv, and with which it may be usefully compared (table i) . among human viruses, these include both picomaviridae (eg, poliovirus, coxsackievirus a and b, and hepatitis a virus) and flaviviridae (eg, dengue fever and yellow fever virus). the genomes of those viruses have a similar organization with structural proteins at the 5' end and nonstructural proteins at the 3' end. however, virus families differ in genome size, the number of proteins produced, the mechanism by which the polyprotein is cleaved, and the detailed mechanism of genome replication. for example, the genome of the picornaviridae is shorter than that of hcv (approximately 7200 to 8400 bases), contains four nucleocapsid proteins (compared with the single protein of hcv), is nonenveloped (and therefore contains no homologues of the two hcv-encoded hcv glycoproteins e 1 and e2), and uses exclusively virus-encoded proteases to cleave its polyprotein. this is different from both hcv and the flaviviridae, in which cleavage of the structural proteins is thought to be carried out by the host cell--derived signalase. members of the flaviviridae have many features in common with hcv. they have a similar genome size (yellow fever virus has 10,862 bases 4 compared with 9379 for hcv 3) and package a viral-encoded glycoprotein into the virus envelope (el). the homologue of e2 in flaviviruses (a membrane-bound glycoprotein called ns 1; "ns" stands for nonstructural) is expressed only on the infected cell surface. like hcv, the polyprotein is cleaved by a combination of viral and host cell proteases. although there is no close sequence similarity between hcv and other known viruses, at least two regions with conserved amino acid residues provide another fundamental aspect of genome organization that differs between the flavivirus and picornavirus families is the structure of the 5' and 3' untranslated regions (utrs). these parts of the genome are involved in hcv replication and initiation of translation by cellular ribosomes of the virus-encoded polyprotein. pestiviruses and hcv show evidence for a highly structured 5'utr and 3'utr, in which internal base-pairing produces a complex set of stem-loop structures that are thought to interact with various host cell and virus proteins during replication. 7,8 in particular, studies have shown that for the picornaviridae and, more recently, for hcv and pestiviruses, 7,9a° such structures are involved in internal initiation of translation, in which binding to the host cell ribosome directs translation to an internal methionine (aug) codon. this contrasts strongly with translation of flavivirus genomes, which act much like cellular messenger rna in which ribosomal binding initially occurs to the capped 5' end of the rna, followed by scanning of the sequence in the 5' to 3' direction with translation commencing from the first aug codon. structurally, hcv is also more similar to the pestiviruses than the flaviviruses, with an exceptionally low buoyant density in sucrose (1.08 to 1.11 g/cm3), 11 similar to that reported for pestiviruses and attributable in both cases to heavily gly-cosylated external membrane glycoproteins in the virus envelope. by contrast, flavivirus envelope glycoproteins contain few sites for n-linked glycosylation, and the virion itself is relatively dense (1.2 g/cm3) . the arrangement and number of cleavage sites of the hcv polyprotein are more similar to pestiviruses, particularly in the further cleavage of both ns4 and ns5 proteins into two subunits, in both cases with ns5b corresponding to the rna polymerase. recently, two distinct rna viruses have been discovered in new world primate tamarins (sanguinis). this monkey species had previously been shown to harbor an infectious agent causing chronic hepatitis originally derived from inoculation with plasma from a surgeon (gb) in whom chronic hepatitis of unknown etiology had developed. 12 parts of the genome of the two viruses (provisionally called gbv-a and gbv-b) show measurable sequence similarity to certain regions of hcv. for example, a 200-amino-acid sequence of part of ns3 of gbv-a and gbv-b shows 47% and 55% sequence similarity with the homologous region in hcv (positions 1298 to 1497 in the hcv polyprotein) 3 and 43.5% sequence similarity to each other. similarly, in ns5, the region around the active site of the rna polymerase (including the gdd motif and positions 2662 to 2761 in hcv) shows 36% and 41% sequence similarities and 43% between gbv-a and gbv-b. 12 in these nonstructural regions, these similarity values are greater than those between hcv and pestiviruses or flaviviruses, although little homology can be found on comparison of the regions of the genomeencoding structural proteins (ie, the core and envelope), nor with the normally highly conserved 5'utr. the degree of relatedness between hcv and other positive-stranded rna viruses can be more formally analyzed by phylogenetic analysis of highly conserved parts of the genome, such as the ns5 region (and homologues in other viruses) encoding the rna-dependent rna polymerase, which invariably contains the canonical gdd motif necessary for the enzymatic activity of the protein. comparisons of a 100-amino-acid sequence surrounding this motif indicate a close relationship between hcv and gbv-a and gbv-b, an intermediate degree of relatedness with the pestivirus bovine viral diarrhea virus, and a much more distant relatedness to fiaviviruses ( figure 2 ). 6 '13 remarkably, a series of plant viruses that are structurally distinct from each of the mammalian virus groups, and with different genome organizations, have rna-dependent rna polymerase amino acid sequences that are perhaps more similar to those of hcv than are the flaviviruses. hcv replication has been studied using a variety of experimental techniques. however, little progress has been made toward the development of a practical hcv culture. hcv does not produce obvious cytopathology, and the amount of hcv released from cells infected in vitro often is low. 14-18 this might be because the cells used for culture are not representative of those infected in vivo, or because productive infection requires a combination of cytokines and growth factors that might be present in the liver but which cannot be recreated in cell culture. the observation that low levels of hcv replication might be detected in lymphocyte 14,18 and hepatocyte cell lines 16, 17 indicates that either the tropism of hcv for different cell types may be greater than first imagined or that the virus replication detected so far does not represent the full replicative cycle of hcv that occurs in vivo. transfection of full-length dna sequences of the hcv genome might be expected to initiate the full replicative cycle of hcv, as it does when similar experiments are done in picornavirus sequences. however, only a low level of expression of virus proteins was observed when a complete hcv sequence was transfected into a transformed hepatocyte (hepatoma) cell line (huh7). 19 despite this, there was evidence of replication of the hcv genome and the production of low concentrations of progeny virus particles. such models provide an important experimental system for future investigations of hcv replication. in common with other positive-strand rna viruses, hcv is presumed to replicate its rna genome through the production of a replication intermediate (ie, an rna copy of the complete genome) and is synthesized by the activity of a virally encoded rna-dependent rna polymerase. the minus-strand copy would then be used to generate positive-stranded copies. because templates can be reused, several minus-strand copies can be synthesized from the infecting positive strand, and each of these transcripts can be used several times to produce positive-strand progeny sequences. in this way, a single input sequence may be amplified several thousandfold. although initiation of transcription is well understood for some positive-strand rna viruses (such as the picornaviridae), no information currently is available on how rna synthesis of hcv or other flakoonin 6 for sources of non-hcv sequences. gbv-a, gbv-b, and hcv (genotypes la, lb, 2a, 2b, and 3a shown) were aligned using the program clustal, and phylogenetic analysis was done using the programs protdist (pam matrix), neighbor, and drawtree in the phylip package. 13 viviruses is primed. hcv lacks homopolymeric tracts (such as poly [u] in the picornaviruses) at the 5' end of the genome, whereas the 3' end is variable, containing either poly(u) or poly(a) tracts, or possibly neither, as now appears to be the case with the related pestiviruses. furthermore, there appears to be no homologue of the vpg protein of picornaviridae. for these reasons, it is likely that the mechanism of transcription initiation for hcv is different. using a strand-specific pcr method, antisense hcv rna sequences have been detected in the liver of hcv-infected patients, confirming the presumed method of replication of hcv via a replication intermediate. 2° such assays provide a valuable technique for detecting hcv replication, as both a sensitive method of monitoring hcv replication in virus culture experiments and a way of investigating the range of cell types and distribution of hcv infection in hcv-infected patients. in particular, the possibility of replication at extrahepatic sites has been proposed on the basis of such assays; these studies have been reviewed by lau et al. 21 the 5'utr is thought to play a significant role in initiating and regulating translation of the large orf of hcv. this region is approximately 341 to 344 bases long, and a combination of computer analysis, nuclease mapping experiments, and studies of covariance has led to a proposed secondary structure model for this part of the genome ( figure 3 ). 22 using the same methods, researchers have predicted a remarkably similar structure for pestiviruses, s despite the virtual absence of nucleotide sequence similarities with hcv, indicating the importance of the overall structure of this region in interactions with viral and cellular proteins or other rna sequences. direct evidence for internal initiation of translation has been obtained from in vitro translation of reporter genes downstream from the 5'utr sequence placed in mono-or dicistronic vectors. 9'23'24 the nonpaired tip of the stem-loop structure 3 is partially complimentary to the 18s subunit of ribosomal rna and may, therefore, be the site of binding during internal initiation. 25 the internal ribosomal entry site activity of 5'utr is consistent with the hypothesis that translation is initiated from the aug methionine codon at position 341.22 there is no evidence for translation from any of the variable number of aug triplets upstream from position 341, although production of the small proteins from these upstream potential orfs may play some role in regulating expression of the large orf. 26 in the absence of a cell culture system for hcv, most information available on the expression and processing of hcv proteins has been obtained from transfection experiments with cloned dna sequences corresponding to the different proteins, and more recently by direct observations of the cellular distributions and properties of hcv proteins detected in liver or plasma in vivo. transfection of prokaryotic or eukaryotic cells with dna copies of different parts of the hcv genome under the control of artificial promoters allows expression of the encoded proteins, and provides a useful technique for studying their synthesis, biochemical properties, and table ii ). expression of this part of the genome in cells 27-29 or reticulocyte-lysate-containing microsomal membranes 3°,31 leads to the synthesis of a polyprotein and its cleavage into a series of proteins. the protein identified as the capsid protein on the basis of comparisons with related viruses is expressed as a protein of approximate size 21 to 22 kd. the assignment of this protein as the nucleocapsid protein is supported by the presence of regions within the protein containing numerous basic (positively charged) amino acids that may have rna-binding properties associated with the encapsidation of hcv rna during virus assembly. binding of core protein to ribosomal rna has recently been reported. 31 using similar techniques, expression of the putative envelope proteins of hcv (el and e2) leads to the synthesis in mammalian cells of two heterogeneous proteins with sizes ranging from 31 to 35 kd and 68 to 72 kd, respectively. 27-31 cleavage between the capsid protein and el, e1 and e2, and e2 and ns2 depends on .~o the addition of microsomal membranes, implying that the host cell signalase has a role in these processing steps. the sizes of e1 and e2 are greater than could be explained by their amino acid sequences alone and support biochemical evidence for extensive glycosylation of both proteins after translation. both e1 and e2 have a large number of potential n-linked glycosylation sites, although the details of which sites are used, the extent to which the glycoprotein moieties are modified, and whether there is also o-linked glycosylation await further biochemical analysis. two cleavage sites between e2 and ns2 (both microsome dependent) have recently been identified, 32 leading to the production of e2 proteins differing in size by 80 amino acid residues. 33 evidence for intermolecular associations between e1 and e2 has been obtained through immunoprecipitation experiments, in which antibody to e1 or e2 could precipitate both proteins under nondenaturing conditions. 28,29.34 the nature or significance of this association is unclear, although current evidence suggests that the association is predominantly noncovalent 34 and does not occur simply through hydrophobic interactions between the membrane anchors of the two proteins. 33,35 recently, monoclonal antibodies to either e1 or e2 were shown to coprecipitate ns2 and ns3, 35 and there also is evidence for associations between e2, ns2, and ns4b. 33 in vitro translation of the rest of the genome leads to the production of proteins of sizes 23, 70 to 72, 4, 27, 56 to 58, and 66 kd, corresponding to ns2, ns3, ns4a, ns4b, ns5a, and ns5b, respectively ( figure 1 ; table ii ). proteolytic cleavage pathways that generate the nonstructural proteins are mediated by ns2 and ns336 and have been extensively studied by several groups, as they represent possible targets of antiviral treatment. ns3 is a serine protease that catalyzes cleavage reactions between ns3/ns4a, ns4a/ns4b, ns4b/ns5a, and ns5a/ ns5b. 36~° ns2a is a metalloproteinase that cleaves the ns2/ns3 junction. 36.41 the ns3/ns4a cleavage reaction mediated by ns3 and the ns2/ns3 cleavage mediated by ns2 occur in cis, whereas other reactions can occur through intermolecular associations between ns3 and the rest of the polyprotein. accounts of the complex sequence of events and the interactions between nonstructural proteins involved in cleavage reactions differ in detail depending on the experimental methods used. however, cleavage may be a sequential process modulated by the activities of other proteins, such as ns4a. ns2 protease activity is zinc dependent and contains an active site dependent on residues in ns3. therefore, after the cis cleavage of the ns2/ns3 junction, the protease is inactivated and will not act in trans on other substrates. this cleavage reaction has been shown to be essential for activating ns3 protease, and that natural variation in the efficiency of the reaction may modulate the pathogenicity of hcv in vivo. 42 when released, ns3 cleaves other sites with varying efficiencies. the active site of ns3 has been mapped by deletion experiments to lie at the amino terminus of the protein (residues 1409 to 1215). 43 the substrate specificity of the serine protease activity has been defined by sequence comparisons and mutagenesis experiments 44-47 and generally conforms to the consensus sequence d/e----c/t$s/a in the target protein. there is some evidence for a less stringent requirement for spe-cific amino acids around the cis cleavage site (ns3/ns4a) than for those cleaved in trans. 46 several investigators have described the requirement for other protein cofactors for the activity of ns3. in particular, it appears that binding of ns4a to ns348,49 is necessary at least for the cleavage of ns4b/ns5a and may modulate the activity of ns3 in other ways. although there is now some information on the proteolytic cleavage steps used to process the hcv polyprotein, the difficulty associated with in vitro culture of hcv and production of infectious molecular clones of hcv so far has prevented a more detailed understanding of the sites of hcv replication in cells and the processes of virus assembly and release from the cell. future research should reveal the nature of the interaction between the capsid protein and virus rna and how this is packaged into the assembled provirion, the posttranslation modifications to the envelope proteins and where these occur in the cell, and the sites of budding of hcv through cellular membranes. to understand replication more fully, we must also identify the mechanism of priming of rna synthesis from the ends of the genome, the nature of the primers, or whether circularization is necessary for transcription. because a cell culture system to investigate differences in neutralization and cytopathic properties of hcv is not available, nucleotide sequence comparisons and typing assays developed from se-quence data have become the principal techniques for characterizing different variants of hcv. this type of analysis is fairly easy to perform, especially since virus sequences can be amplified by pcr directly from clinical specimens. in common with other rna viruses, variants of hcv show considerable sequence variability, many differing considerably from the prototype hcv (hcv-pt). 3 differences of up to 29% have been found between the complete genomic sequences of the most extremely divergent variants analyzed to date, 5° comparable to those observed between serotypes of other human positive-strand rna viruses such as poliovirus, coxsackievirus, and coronaviruses. sequence variability is evenly distributed throughout all virus genes (table 111 )9 -57 apart from the highly conserved nucleotide (and amino acid) sequence of the core (nucleocapsid) protein and 5'utr and the greater variability of the envelope gene (table iii) . nucleotide sequence comparison of complete genomes or subgenomic fragments between variants has shown that variants of hcv obtained from japan are substantially different from the hcv-pt variant obtained in the united states. 3 comparison of the complete genome sequence of hcv-j 53 and hcv-bk 51 from japan showed 92% sequence similarity to each other but only 79% with hcv-pt. at that time, the former variants were classified as the "japanese" type (or type ii), while those from the united states (hcv-pt and hcv-h) were classified as type i. comparisons of subgenomic regions of hcv, such as el, 58 core, 59,60 and ns5, 61 sources of sequences: la = hcv-h52; lb = hcv-j53; lc = hc-j954; 2a = hc-j655; 2b = hc-j85°; 3a = nzli56; 3b = tr. 57 in all comparisons, the 5'ncr is the most conserved subgenomic region (maximum 9% nucleotide sequence divergence), whereas highly variable regions are found in parts of the genome encoding el and ns2 (35% to 44% nucleotide sequence and 34% to 45% amino acid sequence differences). provide evidence of at least six major groupings of hcv sequences, each of which contains a series of more closely related clusters of sequences ( figure 4 ). 62 the current widely used nomenclature for hcv variants reflects this hierarchy of sequence relationships between different isolates. based on previous suggestions, 63,64 the major branches in the phylogenetic tree are referred to as "types," while "subtypes" correspond to the more closely related sequences within most of the major groups ( figure 4) . although ns5 sequences are analyzed in figure 4 , equivalent sequence relationships exist in other parts of the genome. the types have been numbered 1 to 6 and the subtypes a, b, and c, in both cases in order of discovery. therefore, the sequence cloned by chiron 3 is assigned type la, hcv-j and hcv-bk are type lb, hc-j6 is type 2a, and hc-j8 is type 2b. this nomenclature closely follows the schemes originally described by enomoto (type 3a) on the basis of phylogenetic analysis of sequences in the ns5, ns3, core, and 5'utr noncoding regions. this approach avoids the inconsistencies of earlier systems and should be easier to extend when new genotypes are discovered. some genotypes of hcv (types la, 2a, and 2b) show a broad worldwide distribution, whereas others, such as types 5a r simmonds and 6a, are found only in specific geographic regions. blood donors and patients with chronic hepatitis from countries in western europe and the united states frequently are infected with genotypes la, lb, 2a, 2b, and 3a, although the relative frequencies of each may vary. 58,61,65-76 there is a trend for more frequent infection with type lb in southern and eastern europe. in many european countries, genotype distributions vary with the age of the patients, reflecting rapid changes in genotype distribution with time within a single geographic area. a striking geographic change in genotype distribution is apparent between southeast europe and turkey (both mainly type lb) and several countries in the middle east and parts of north and central africa where other genotypes predominate. for example, a high frequency of hcv infection is found in egypt (20% to 30%), 77-79 of which almost all corresponds to type 4a. 8°,81 hcv type 4 also is the principal genotype in countries such as yemen, kuwait, iraq, and saudi arabia in the middle east 6° and in zaire, burundi, and gabon in central africa. 58, 69, 71 hcv genotype 5a is frequently found among patients with non-a, non-b hepatitis and blood donors in south africa 58,61,7°,82 but is found only rarely in europe and elsewhere. 58, 72 in japan, taiwan, and some parts of china, genotypes lb, 2a, and 2b are the most frequently found. 63,83-90 infection with type l a in japan appears to be confined to patients with hemophilia who received commercial (us-produced) blood products, such as factor viii and xi clotting concentrates. 63,91 the geographic distribution of type 3 varies; it is only rarely found in japan 92 and is also infrequent in taiwan, hong kong, and macau. 93 how-ever, this genotype is found with increasing frequency in countries to the west, frequently occurring in singapore and accounting for most hepatitis infections in thailand. 93,94 in a small sample, it was the only genotype found in bangladesh and eastern india. 6° as with type 4 in africa, there is now evidence of considerable sequence diversity within the type 3 genotype, with at least 11 different subtypes of type 3 identified in nepal, 95 india, and bangladesh. 6° a genotype with a highly restricted geographic range is type 6a. this type was originally found in hong kong 69,8° and was shown to be a new major genotype by sequence comparisons in the ns5 and e1 regions. 58,61 approximately one third of anti-hcv-positive blood donors in hong kong are infected with this genotype, as are an equivalent proportion in neighboring macau sl and vietnam. 96 a series of novel genotypes has been found in vietnam 96 and thailand6°; these genotypes are distinct from types 1 to 6 classified to date but are more closely related to type 6 than to other genotypes, 96 consistent with their overlapping geographic range with type 6 in southeast asia. numerous investigations are being conducted into possible differences in the course of disease associated with different hcv genotypes, such as the rate of development of cirrhosis and hepatocellular carcinoma, and whether certain genotypes are more or less likely to respond to interferon treatment. a large number of clinical investigations have documented severe and progressive liver disease in patients infected with each of the well-char-acterized genotypes (types la, lb, 2a, 2b, 3a, and 4a), so there is little evidence thus far of variants of hcv that are completely nonpathogenic. however, possible variation in the rate of disease progression, differences between genotypes in routes and frequency of person-to-person transmission, or differences in the probability of achieving a sustained response to antiviral treatment would indicate the potential usefulness of identifying the infecting genotype in certain clinical situations. several clinical studies have catalogued a variety of factors (including genotype) that correlate with the severity of liver disease and show predictive value for response to antiviral treatment. factors that frequently have been shown to influence response to interferon treatment include age and duration of infection, presence of cirrhosis before treatment, genotype, and pretreatment level of circulating viral rna in plasma. 97 a consistent finding reported by several different groups that used a variety of typing assays has been the greatly increased rate of long-term response found when treating patients infected with genotypes 2a, 2b, and 3a compared with type lb. 74 '85'98-106 for example, chemello et al m2 found that long-term (>12 months) normalization of alanine aminotransferase levels was achieved in only 29% of patients infected with type 1 variants, compared with 52% of those infected with type 2 and 74% of those infected with type 3. in a study by tsubota et al, m6 infection with type lb, the presence of cirrhosis, and a high pretreatment virus load were each independently associated with a reduced chance of response (relative risks of 16, 5, and 4, respectively) . the mechanism by which different genotypes differ in response to treatment remains obscure. for treatments such as in-terferon, we do not know whether the effect of the drug is directly antiviral or whether the inhibition of virus replication is secondary to increased expression of major histocompatibility complex class i antigens on the surface of hepatocytes and greater cytotoxic t-cell activity against virus-infected cells. elucidating the mechanism of action of interferon and whether there are virologic differences between genotypes in sensitivity to antiviral agents awaits a cellculture model for hcv infection. although determination of the nucleotide sequences is the most reliable method of identifying different genotypes of hcv, this method is not practical for large clinical studies. many of the published methods for "genotyping" are based on amplification of viral sequences in clinical specimens, either by using type-specific primers that selectively amplify different genotypes, by analyzing the pcr product by hybridization with genotype-specific probes, or by using restriction fragment length polymorphisms (rflp). the assays have different strengths and weaknesses. for example, methods based on amplification and analysis of 5'ncr sequences have advantages of sensitivity, because this region is highly conserved and can be more frequently amplified from hcv-infected patients than other parts of the genome. however, few nucleotide differences are found between different genotypes. although reliably differentiating six major genotypes by using rflp or by type-specific probes is possible, it is not always possible to reliably identify virus subtypes. types 2a and 2b consistently differ at position -124, allowing them to be differentiated by the restriction enzyme scrf167 orby probes 10 to 13 in the inno-lipa (innogenetics, zwijnaarde, belgium). 71 however, sequences of type 2c are indistinguishable from some of those of type 2a. similarly, some of the novel subtypes of type 1 often show sequences identical to those of type la or lb, 54,60 and a small proportion of type la variants are identical to type lb and vice versa. 22 typing methods based on coding regions, such as core and ns5, can reliably identify subtypes as well as major genotypes because the degree of sequence divergence is much greater (table iii) . however, amplifying sequences in coding regions of the genome generally is difficult because sequence variability in the primer-binding sites may reduce the effectiveness of sequence amplification by pcr. nevertheless, the variation is exploited in a genotyping assay that uses type-specific primers complimentary to variable regions in the core gene. currently, this assay can identify and differentiate types la, lb, 2a, 2b, and 3a, 1°7a°8 although the method is technically complicated to perform reliably 1°9 and may be difficult to extend to the great range of hcv genotypes now described. serologic typing methods have advantages over pcr-based methods in terms of the speed and simplicity of sample preparation and the use of simple equipment found in any diagnostic virology laboratory. by careful optimization of reagents, such assays may show high sensitivity and reproducibility. for example, type-specific antibody to ns4 peptides can be detected in approximately 95% of patients with non-a, non-b hepatitis. h° furthermore, the assays can be readily extended to detect new genotypes. one ns4based assay can reliably identify type-spe-cific antibody to six major genotypes, n° although the antigenic similarity between subtypes currently precludes the separate identification of types la and lb and 2a and 2b using the ns4 peptides alone. in contrast to the highly restricted sequence diversity of the 5'ncr and adjacent core region, the two putative envelope genes are highly divergent between different variants of hcv (table iii) 111-114 and show a three-to-four-times higher rate of sequence change with time in persistently infected patients, ll5 because these proteins are likely to lie on the outside of the virus, they would be the principal targets of the humoral immune response to hcv elicited on infection. changes in the e1 and e2 genes may alter the antigenicity of the virus to allow "immune escape" from neutralizing antibodies, 112 therefore accounting for both the high degree of envelope sequence variability and the observed persistent nature of hcv infection. supporting this model is the observation that much of the variability in the e1 and e2 genes is concentrated in discrete "hypervariable" regions, 112-114 possibly reflecting the pressures on the virus to evade immune recognition at specific sites where hcv may be neutralized. experimental evidence supporting this theory includes the observations that variants of hcv with changes in the e1 and e2 genes are antigenically distinct, and, in many cases, the in vivo appearance of variants with different sequences in the hypervariable region is followed by development of antibodies that specifically recognize the new variants. 1~2,116-h8 in one report, 119 persistent hcv infection developed in a patient with deficient anti-body responses (agammaglobulinemia), but without the development of sequence variability in e2 consistent with the role of antibody-driving variation in immunocompetent persons. on the other hand, envelope sequences obtained sequentially from persistently infected patients sometimes show no significant change, 12°-122 whereas in others, variants coexist with antibodies that recognize the corresponding hypervariable region peptides, u6 cytotoxic t-cell responses also may play a protective role in hcv infection, 123,124 as they do in other virus infections for which they are more important in virus clearance than antibody response to infection. although circumstantial evidence supports the theory of immune escape, additional studies are needed to confinn this as a plausible model of virus persistence. many of the current uncertainties may be resolved when a satisfactory in vitro neutralization assay is developed for hcv that enables the effect of amino acid changes in the envelope gene to be directly investigated. additional information also is needed on the relative importance of humoral and cell-mediated immunity to hcv and to determine which is more important in virus clearance and protection from reinfection. persistent infection with hcv entails continuous replication of hcv over years or decades of infection in hcv carders. the large number of replication cycles, combined with the relatively error-prone rna-dependent rna polymerase, leads to measurable sequence drift of hcv over time. for example, over an 8-year interval of persistent infection in a chim-panzee, the rate of sequence change for the genome as a whole was 0.144% per site per year, 115 similar to the rate calculated for sequence change in the 5' half of the genome over 3 years observed in a human carrier (0.192%) 125 and in a crosssectional study. 126 using this "molecular clock," it is possible in principle to calculate times of divergence between hcv variants and therefore to establish their degree of epidemiologic relatedness. for example, the finding of relatively few sequence differences between variants infecting two individuals would provide evidence of recent hcv transmission between them. sequence comparisons in variable regions, such as e2 and ns5, of the hcv genome have been used to document transmission between persons, either from mother to child, 127 within families, 128 by iatrogenic routes, 129-132 or by sexual contact. [133] [134] [135] in these studies, the possibility of transmission by different risk behaviors was assessed by measuring the degree of relatedness of hcv recovered from implicated persons. phylogenetic analysis of nucleotide sequences provides a more formal method of investigating relationships between sequences. phylogenetic trees produced by such methods indicate the degree of relatedness between sequences, while the branching order of the different lineages shows the most likely evolutionary history of the sampled population. for example, clustering of hcv sequences into a single phylogenetic group among recipients of an hcv-contaminated blood product (anti-d immunoglobulin) was still apparent 17 years after infection ( figure 5 ). these approaches to hcv epidemiology will prove valuable in documenting the spread of hcv in different risk groups, evaluating alternative (nonparenteral) routes of transanti-d ig recipients pt figure 5 . phylogenetic relationships between sequences from the ns5 region of patients exposed to an implicated batch of anti-d immunoglobulin (ig) in 1977 (o) and those of epidemiologically unrelated type lb variants from japan (j), the united states (u), and europe (e). b250 = ns5 sequence of hepatitis c virus recovered from batch b250 of anti-d ig; donor ---sequence of variant infecting suspected donor to plasma pool used to manufacture batch b250. phylogenetic analysis was done on a segment (222 base pairs; positions 7975 to 8196) of the ns5 gene that was amplified, sequenced, and analyzed as previously described. 61 sequence distances were calculated using the program dnaml in a data set containing the prototype hepatitis c virus (type la) as an outgroup. sequences were obtained from published sources. 61'76 mission, and understanding more about the origins and evolution of hcv. this paper attempts to review a rapidly expanding area of research. it is hoped that a combination of basic science and clinical studies may eventually lead to a greater understanding of the ways in which hcv infection may be prevented or cured by the use of antiviral vaccines. the information provided here will clearly form the basis of many of these developments. of the hepatitis c virus core protein. j virol. 1994; 68:3631-3641. 32 isolation of a cdna derived from a bloodborne non-a, non-b hepatitis genome. science an assay for circulating antibodies to a major etiologic virus of human non-a, non-b hepatitis genetic organization and diversity of the hepatitis c virus nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution hepatitis c virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups the phylogeny of rna-dependent rna polymerases of positivestrand rna viruses internal ribosome entry site within hepatitis c virus rna secondary structure of the 5' nontranslated region of hepatitis c virus and pestivirus genomic rnas a conserved helical element is essential for internal initiation of translation of hepatitis c virus rna pestivirus translation initiation occurs by internal ribosome entry extraordinarily low density of hepatitis c virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction identification of two flavivirus-like genomes in the gb hepatitis agent phylip inference package version 3.5. seattle, wash: department of genetics evidence for in vitro replication of hepatitis c virus genome in a human t-cell line correlation between the infectivity of hepatitis c virus in vivo and its infectivity in vitro susceptibility of human liver cell cultures to hepatitis c virus infection multicycle infection of hepatitis c virus in cell culture and inhibition by alpha and beta interferons susceptibility of human t-lymphotropic virus type i infected cell line mt-2 to hepatitis c virus infection transfection of a differentiated human hepatoma cell line (huh7) with in vitro-transcribed hepatitis c virus (hcv) rna and establishment of a long-term culture persistently infected with hcv demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis c virus using strand-specific rt/pcr. virology in situ detection of hepatitis c virus: a critical appraisal variation of the hepatitis c virus 5'-non coding region: implications for secondary structure, virus detection and typing translation of human hepatitis c virus rna in cultured cells is mediated by an internal ribosome-binding mechanism complete 5' noncoding region is necessary for the efficient internal initiation of hepatitis c virus rna unusual folding regions and ribosome landing pad within hepatitis c virus and pestivirus rnas end-dependent translation initiation of hepatitis c viral rna and the presence of putative positive and negative translational control elements within the 5' untranslated region expression, identification and subcellular localization of the proteins encoded by the hepatitis c viral genome characterization of hepatitis c virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses expression and identification of hepatitis c virus polyprotein cleavage products gene mapping of the putative structural region of the hepatitis c virus genome by in vitro processing analysis a second hepatitis c virus-encoded proteinase hepatitis c virus ns3 serine proteinase: transcleavage requirements and processing kinetics identification of the domain required for trans-cleavage activity of hepatitis c viral serine proteinase substrate requirements of hepatitis c virus serine proteinase for intermolecular polypeptide cleavage in escherichia coli specificity of the hepatitis c virus ns3 serine protease: effects of substitutions at the 3/4a, 4a/4b, 4b/5a, and 5a/5b cleavage sites on polyprotein processing substrate determinants for cleavage in cis and in trans by the hepatitis c virus ns3 proteinase nucleotide sequence of hepatitis c virus (type 3b) isolated from a japanese patient with chronic hepatitis c at least 12 genotypes of hepatitis c virus predicted by sequence analysis of the putative e1 gene of isolates collected worldwide sequence analysis of the core gene of 14 hepatitis c virus genotypes investigation of the pattern of hepatitis c virus sequence diversity in different geographical regions: implications for virus classification classification of hepatitis c virus into six major genotypes and a series of subtypes by phylogenetic analysis of the ns-5 region a proposed system for the nomenclature of hepatitis c viral genotypes there are two major types of hepatitis c virus in japan analysis of a new hepatitis c virus type and its phylogenetic relationship to existing variants serological responses to infection with three different types of hepatitis c virus two french genotypes of hepatitis c virus: homology of the predominant genotype with the prototype american strain detection of three types of hepatitis c virus in blood donors: investigation of type-specific differences in serological reactivity and rate of alanine aminotransferase abnormalities identification of hepatitis c viruses with a nonconserved sequence of the 5' untranslated region sequence analysis of the 5' noncoding region of hepatitis c virus at least five related, but distinct, hepatitis c viral genotypes exist typing of hepatitis c virus isolates and new subtypes using a line probe assay sequence analysis of the 5' untranslated region in isolates of at least four genotypes of hepatitis c virus in the netherlands use of the 5' non-coding region for genotyping hepatitis c virus genotypes of hepatitis c virus in italian patients with chronic hepatitis c heterogeneity of hepatitis c virus genotypes in france genotypic analysis of hepatitis c virus in american patients hepatitis c virus infection in egyptian volunteer blood donors in riyadh risk factors associated with a high seroprevalence of hepatitis c virus infection in egyptian blood donors high hcv prevalence in egyptian blood donors sequence variability in the 5' non coding region of hepatitis c virus: identification of a new virus type and restrictions on sequence diversity geographical distribution of hepatitis c virus genotypes in blood donors: an international collaborative survey new genotype of hepatitis c virus in south-africa typing of hepatitis c virus (hcv) genomes by restriction fragment length polymorphisms distribution of plural hcv types in japan clinical backgrounds of the patients having different types of hepatitis c virus genomes genomic typing of hepatitis c viruses present in china hcv genotypes in china hcv genotypes in different countries differences in the hepatitis c virus genotypes in different countries prevalence, genotypes, and an isolate (hc-c2) of hepatitis c virus in chinese patients with liver disease imported hepatitis c virus genotypes in japanese hemophiliacs genotypic subtyping of hepatitis c virus survey of major genotypes and subtypes of hepatitis c virus using restriction fragment length polymorphism of sequences amplified from the 5' non-coding region a new type of hepatitis c virus in patients in thailand hepatitis c virus variants from nepal with novel genotypes and their classification into the third major group hepatitis c virus variants from vietnam are classifiable into the seventh, eighth, and ninth major genetic groups prediction of response to interferon treatment of chronic hepatitis c hcv genotypes in chronic hepatitis c and response to interferon detection of hepatitis c virus by polymerase chain reaction and response to interferon-alpha therapy: relationship to genotypes of hepatitis c virus factors useful in predicting the response to interferon therapy in chronic hepatitis c hepatitis c virus genotypes--an investigation of type-specific differences in geographic origin and disease simmonds p. hepatitis c serotype and response to interferon therapy prediction of interferon effect in chronic hepatitis c by both quantification and genotyping of hcv-rna genotypes and titers of hepatitis c virus for predicting response to interferon in patients with chronic hepatitis c antiviral effect of lymphoblastoid interferon-alpha on hepatitis c virus in patients with chronic hepatitis type c factors predictive of response to interferon-alpha therapy in hepatitis c virus infection typing hepatitis c virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources characterization of the genomic sequence of type v (or 3a) hepatitis c virus isolates and pcr primers for specific detection application of six hepatitis c virus genotyping systems to sera from chronic hepatitis c patients in the united states use of ns-4 peptides to identify typespecific antibody to hepatitis c virus genotypes 1, 2, 3, 4, 5 and 6 characterization of hypervariable regions in the putative envelope protein of hepatitis c virus evidence for immune selection of hepatitis c virus (hcv) putative envelope glycoprotein variants: potential role in chronic hcv infections marked sequence diversity in the putative envelope proteins of hepatitis c viruses hypervariable regions in the putative glycoprotein of hepatitis c virus genetic drift of hepatitis-c virus during an 8.2-year infection in a chimpanzee--variability and stability humoral immune response to hypervariable region-1 of the putative envelope glycoprotein (gp70) of hepatitis c virus hypervariable 5'-terminus of hepatitis c virus e2/ns1 encodes antigenically distinct variants a structurally flexible and antigenically variable n-terminal domain of the hepatitis c virus e2/ns1 protein--implication for an escape from antibody hypervariable region of hepatitis c virus envelope glycoprotein (e2 ns1) in an agammaglobulinemic patient the degree of variability in the amino terminal region of the e2/ns 1 protein of hepatitis c virus correlates with responsiveness to interferon therapy in viraemic patients sequence variation in the large envelope glycoprotein (e2/ns 1) of hepatitis c virus during chronic infection dynamics of genome change in the e2/ns1 region of hepatitis c virus in vivo intrahepatic cytotoxic t lymphocytes specific for hepatitis-c virus in persons with chronic hepatitis hepatitis c virus (hcv)-specific cytotoxic t lymphocytes recognize epitopes in the core and envelope proteins of hcv nucleotide sequence and mutation rate of the h strain of hepatitis c virus analysis of genomic variability of hepatitis c virus a unique, predominant hepatitis c virus variant found in an infant born to a mother with multiple variants risk of hepatitis c virus infections through household contact with chronic carriers--analysis of nucleotide sequences comparison of hepatitis c virus strains obtained from hemodialysis patients hepatitis c viral markers in patients who received blood that was positive for hepatitis c virus core antibody, with genetic evidence of hepatitis c virus transmission hepatitis c transmission in a hemodialysis unit: molecular evidence for spread of virus among patients not sharing equipment confl,~mation of hepatitis c virus transmission through needlestick accidents by molecular evolutionary analysis heterosexual transmission of hepatitis c virus analysis of nucleotide sequences of hepatitis c virus isolates from husband-wife pairs acute hepatitis c infection after sexual exposure key: cord-281389-sht0yx4a authors: tal, michal caspi; torrez dulgeroff, laughing bear; myers, lara; cham, lamin b.; mayer-barber, katrin d.; bohrer, andrea c.; castro, ehydel; yiu, ying ying; lopez angel, cesar; pham, ed; carmody, aaron b.; messer, ronald j.; gars, eric; kortmann, jens; markovic, maxim; hasenkrug, michaela; peterson, karin e.; winkler, clayton w.; woods, tyson a.; hansen, paige; galloway, sarah; wagh, dhananjay; fram, benjamin j.; nguyen, thai; corey, daniel; kalluru, raja sab; banaei, niaz; rajadas, jayakumar; monack, denise m.; ahmed, aijaz; sahoo, debashis; davis, mark m.; glenn, jeffrey s.; adomati, tom; lang, karl s.; weissman, irving l.; hasenkrug, kim j. title: upregulation of cd47 is a host checkpoint response to pathogen recognition date: 2020-06-23 journal: mbio doi: 10.1128/mbio.01293-20 sha: doc_id: 281389 cord_uid: sht0yx4a it is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. we now show that the very early innate response also has an immunosuppressive component. infected cells upregulate the cd47 “don’t eat me” signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (apc) functions. a cd47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but cd47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that encode no mimic. cd47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (prrs). furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis c patients, upregulated cd47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. indeed, results from antibody-mediated cd47 blockade experiments as well as cd47 knockout mice revealed an immunosuppressive role for cd47 during infections with lymphocytic choriomeningitis virus and mycobacterium tuberculosis. since cd47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify cd47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents. adaptive immune responses. indeed, results from antibody-mediated cd47 blockade experiments as well as cd47 knockout mice revealed an immunosuppressive role for cd47 during infections with lymphocytic choriomeningitis virus and mycobacterium tuberculosis. since cd47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify cd47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents. importance immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (prrs). prr binding sets off a cascade of events that activates immune responses. we now show that, in addition to activating immune responses, prr signaling also initiates an immunosuppressive response, probably to limit inflammation. the importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the cd47 molecule, can lead to enhanced immune responses to any pathogen that triggers prr signaling. since most or all pathogens trigger prrs, cd47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. such immunotherapy could be done without a requirement for knowing the hla type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile. keywords cd47, host response, immune checkpoint, innate immunity, pathogen recognition receptors t he earliest immune responses to invasion by pathogenic microorganisms begin with the sensing of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) such as toll-like receptors (tlrs). ligation of prrs initiates signal transduction pathways that ultimately lead to the activation of broad innate and highly specific adaptive immune responses. discoveries in recent years have demonstrated that the induction of adaptive immune responses involves not only activation mechanisms but also inhibitory mechanisms or "checkpoints," which regulate immune function at a cellular level to prevent immunopathological damage by overactivated effector responses (1) . antibody (ab)-mediated blockade of checkpoint molecules such as ctla4 and pd-1 is being used therapeutically to enhance anticancer immune responses (2, 3) , and blockade of cd47 is now in clinical trials to activate macrophagemediated phagocytosis of cancer cells (4) (5) (6) (7) , which upregulate cd47 expression as an immune evasion mechanism (8) (9) (10) (11) . cd47 is an abundantly expressed transmembrane cell surface glycoprotein that can act as a receptor for thrombospondins, form complexes with integrins, and bind to the inhibitory receptor signal-regulatory protein alpha (sirp␣) (12) (13) (14) . cd47 binding to sirp␣ has emerged as an important innate immune checkpoint by regulating immune cell clearance and inflammatory signaling (6) . cd47 engagement of sirp␣ results in the phosphorylation of cytoplasmic itim motifs by inhibitory protein tyrosine phosphatases, shp-1 and shp-2 (15) . given the well-established role of cd47 in cancer cell immune evasion, we investigated whether cd47 expression is modified in other disease contexts, specifically infectious diseases. the cd47-sirp␣ axis has immunomodulatory functions that impact phagocytosis, chemokine and cytokine responses, innate and adaptive immune cell homeostasis and activation, t cell killing, and b cell antibody production (15) (16) (17) (18) . viruses have evolved mechanisms to evade host defenses (19) and take advantage of inhibitory signaling pathways for selective advantage. of interest, poxviruses, which devote many genes toward immune suppression and evasion, encode a cd47 mimic (20) . the cd47 mimic of myxomavirus, m128l, can be deleted with no effect on in vitro replication, but the deletion mutant loses pathogenicity in vivo. this loss of pathogenicity was associated with increased monocyte/macrophage activation (20) . the present study examines cd47 expression in the context of infectious agents that encode no cd47 mimic. both mouse and human cells showed a significant upregulation of cd47 upon infection with various pathogens. the results indicated that stimulation of either cytosolic or endosomal pprs resulted in cd47 upregulation. in addition, inflammatory cytokines present in the serum of hepatitis c virus (hcv)-infected patients could also induce cd47 upregulation, even in the context of no virus. in addition to viruses, clinically relevant bacteria such as mycobacterium tuberculosis induce the upregulation of cd47 that limits host resistance. our results indicate that cd47 upregulation is a very early innate checkpoint response and that immunological inhibitory mechanisms are activated not only at the effector phase of immune responses but also already at the induction phase of prr sensing. thus, cd47 is a promising target for checkpoint therapies against a wide range of infectious diseases. to examine the role of cd47 expression during the innate response to infection, we investigated whether hematopoietic cells upregulated cd47 expression in several unrelated infection models during the first days after infection. we began by analyzing cd47 expression on cells from mice inoculated with friend virus (fv), a naturally occurring retroviral infection in mice (21) . fv primarily infects erythroid progenitor cells in the spleen but can also infect immune cells (22) . cd47 was significantly upregulated on several hematopoietic cell lineages from mouse spleens at 3 days postinfection (dpi) compared to cells from naive mice (fig. 1a) . cd47 expression was also analyzed at 2 dpi in mice infected with lymphocytic choriomeningitis virus (lcmv). compared to naive controls, all of the spleen cell types analyzed showed significantly increased cell surface expression of cd47 (fig. 1b) . a significant upregulation of cd47 expression was also observed in response to lcmv at 3 dpi in a previous report (23) . infections with la crosse arbovirus were also analyzed at 2 dpi, and we also observed significantly upregulated cd47 expression in hematopoietic spleen cells compared to naive controls (fig. 1c) . cd47 expression is upregulated on human cells in response to in vitro infection. examination of a publicly available gene expression data set (gene expression omnibus [geo] accession number gse147507) from severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection of a549 human lung cells also showed a significant upregulation of cd47 compared to mock-infected controls (fig. 1d ). to determine whether bacterial infection would also upregulate cd47 expression, human peripheral blood mononuclear cells (pbmcs) were examined 24 and 48 h after infection with borrelia burgdorferi in vitro. multiple pbmc subsets showed significantly upregulated cd47 expression in response to borrelia burgdorferi infection compared to naive cells (fig. 1e) . we also investigated cd47 expression on human pbmcs infected in vitro with mcherry-expressing strains of salmonella enterica serovar typhi. wild-type salmonella typhi (ty2 wt) and salmonella typhi δflic (ty2 δflic), a mutant strain that lacks flagella, were examined. infections were done by centrifugation to compensate for the lack of motility of the flagellum mutant. compared to naive cells, mcherry-positive b cells significantly upregulated cd47 expression when infected with wild-type salmonella typhi for 24 h (fig. 1f ). in contrast, cd47 expression was not significantly upregulated in b cells infected with the mutant strain lacking flagella, salmonella typhi δflic (fig. 1f) . the reduced upregulation of cd47 expression by salmonella typhi δflic compared to wild-type salmonella typhi suggested that the sensing of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) might play a role in cd47 upregulation, as flagellin is a potent pamp recognized by extracellular tlr5 (24) and the nlr family of apoptosis-inhibitory proteins (naips) (25) . overall, the combined in vivo and in vitro results from multiple pathogen infections in both human and mouse cells indicated that the upregulation of cd47 was a conserved host response possibly related to host sensing mechanisms. cd47 is upregulated in response to host recognition of pathogens. to determine whether cd47 upregulation was initiated by prr stimulation, we specifically stimulated prrs using small-molecule agonists rather than infectious agents. cd47 upregulation on human dendritic cells (dcs) and monocytes was tested in vitro using pbmcs stimulated with either muramyl dipeptide (mdp) to activate the bacterial peptidoglycan prr, nucleotide-binding oligomerization domain-containing protein 2 (nod2), or cl264 to activate the single-stranded rna (ssrna) endosomal prr, tlr7. flow cytometry was used to identify cell subsets and measure cd47 expression at 24 h poststimulation. both human dcs and monocytes responded to either type of prr stimulation with a significant upregulation of cd47 surface expression ( fig. 2a) . we also tested tlr stimulation using a dual tlr7/8 agonist, r848. since this dual agonist, which also has in vivo activity (26) , produced dose-dependent upregulation of cd47 on human pbmcs (fig. 2b) , we proceeded to test prr stimulation in an in vivo mouse model. daily intraperitoneal injections of r848 into mice led to strongly upregulated cd47 expression on both macrophages and dcs isolated from spleens on day 3 (fig. 2c) . together, these data demonstrated that cd47 was upregulated on both human and mouse immune cells via pathogen-sensing mechanisms. furthermore, tlr7 stimulation via endosomal uptake indicated that cd47 could be upregulated not only tal et al. ® by infected cells, as would be reflected by cytosolic nod2 stimulation, but also by surveilling immune cells. to examine cd47 expression in human viral infection, we first compared transcriptional levels of cd47 from publicly available microarray data (geo accession number gse38597) from healthy and hepatitis c virus (hcv) patient liver biopsy specimens. the analysis revealed significantly higher expression levels of cd47 in the liver biopsy specimens from acutely infected hcv patients than for healthy controls (fig. 3a) . we then used cytometry by time of flight (cytof) to examine cd47 expression on pbmcs during hcv infection in the context of sofosbuvir (sof) therapy (27) , comparing healthy controls to hcvinfected patients prior to treatment, midway through treatment, and at 6 months posttreatment. compared to healthy controls, monocytes and dcs from hcv patients demonstrated a sustained upregulation of cd47 at all treatment time points, including the 6-month posttreatment time point ( fig. 3b (b) cd47 mfi on human total pbmcs from 4 separate donors stimulated with titrated concentrations of r848 from 0.1 g/ml to 10 g/ml, as indicated, for 48 h. statistics were done by a paired two-way t test with bonferroni correction. (c) mice (n ϭ 5/group) were injected intraperitoneally with 1 mg/kg r848 for 3 days, and on day 3, splenocytes were isolated and macrophages and dcs were analyzed for cd47 mfi. statistics were done by an unpaired two-way t test. (ns, p ͼ 0.05; *, p ͻ 0.05; **, p ͻ 0.01; ***, p ͻ 0.001). error bars represent sem. upregulation of cd47 is a host checkpoint response ® differences between pre-, mid-, and posttreatment cd47 levels in either monocytes or dcs, and all of these time points were significantly different from those for healthy controls ( fig. 3b and c). conventional dendritic cells (cdcs) are classified into cdc1s and cdc2s, which can be distinguished in part through the specific expression of the cd47 receptor, sirp␣, on cdc2s (28) . when we compared cd47 expression levels within sirp␣ lo and sirp␣ hi dcs, we observed that cd47 expression was highest on sirp␣ hi dcs (fig. 3d ). there was no correlation between viral titers and cd47 expression on either monocytes or dc subsets in this patient cohort (see fig. s1a in the supplemental material). between healthy controls and pretreatment hcv patients, there was significant cd47 upregulation in sirp␣ lo dcs but not in sirp␣ hi dcs (fig. 3d ). however, compared to healthy controls, the proportion of sirp␣ hi dcs was significantly increased at both pretreatment and midtreatment, which could also contribute to higher cd47 expression levels in dcs (fig. s1b) . thus, hcv infections were associated with the increased expression of both cd47 and its receptor, sirp␣, on antigen-presenting cells (apcs). hcv patient plasma-induced cd47 expression ex vivo. to determine whether inflammatory factors present in hcv patient plasma could affect cd47 upregulation, we derived dcs from healthy donor monocytes with plasma added from either patients in the hcv patient cohort or healthy controls. we found that monocyte-derived dcs (mdcs) cultured in the presence of hcv patient plasma significantly upregulated cd47 compared to plasma from healthy donors (fig. 4a ). luminex analysis of patient plasma was used to characterize the inflammatory milieu over the course of hcv infection and treatment. plasma isolated from patients at the pretreatment time point contained both virus and inflammatory cytokines, indicating that cd47 upregulation could have been due to infection of the dcs. however, plasma isolated from patients at the midtreatment and posttreatment time points contained no detectable virus (data not shown) but still had increased levels of inflammatory cytokines, including tumor necrosis factor alpha (tnf-␣), cxcl10, and interferon alpha (ifn-␣), compared to healthy controls (fig. 4b to d). cd47 expression was increased under all hcv patient plasma conditions compared to healthy controls despite undetectable virus at the midtreatment and posttreatment time points. the differences between the pre-, mid-, and posttreatment time points within hcv patients were not statistically significant. these results suggested that cytokines in the inflammatory milieu of hcv patient plasma could upregulate cd47 expression. to confirm the ability of cytokines to upregulate cd47 surface expression, we performed in vitro stimulations of human pbmcs isolated from healthy donors using tnf-␣, cxcl10, and ifn-␣, as single treatments and in combinations. at 72 h poststimulation, the only single cytokine that induced significant cd47 upregulation was ifn-␣ (fig. 4e) . combinations of these inflammatory cytokines enhanced the upregulation of cd47 surface expression, with the triple combination of tnf-␣, cxcl10, and ifn-␣ having the strongest effect (fig. 4e) . these experiments demonstrated that in addition to pathogen recognition, host immune cells could also upregulate cd47 surface expression in response to the inflammatory milieu induced by the pathogen. to determine the effects of cd47 blockade during a live viral infection, c57bl/6 mice were injected with anti-cd47 (blocking) antibody daily beginning 2 days prior to infection with lcmv. to ascertain whether both anti-cd47-treated and mock-treated animals were equivalently infected, lcmv titers in the spleens and liver were determined at 3 dpi. the analyses showed no significant differences in viral titers at this time point regardless of treatment (fig. 5a) . anti-cd47 injections were continued through 5 dpi, and plasma virus levels were then measured at 8 and 12 dpi. significantly reduced viremia levels were observed in anti-cd47-treated mice compared to mock-treated mice at both time points (fig. 5b ). since it is known that host clearance of lcmv is highly dependent on cd8 ϩ t cell responses (29, 30) , it was of interest to determine if cd47 blockade affected those responses. total cd8 ϩ t cell levels were determined tal et al. ® from blood samples of lcmv-infected mice at 8, 10, and 12 dpi in the presence or absence of cd47 blockade. at both 8 and 10 dpi, there were significantly increased cd8 ϩ t cell numbers responding to lcmv infection in the treated mice compared to the isotype-matched antibody control-treated mice (fig. 5c) . genetic inactivation of cd47 expression prolongs survival from mycobacterium tuberculosis infection in vivo. we next investigated the role of cd47 during mycobacterium tuberculosis infection in vitro and in vivo. first, human macrophages derived from healthy pbmc samples were used for in vitro infection with m. tuberculosis to examine cd47 expression levels. cd47 expression levels are shown for four donors, and compare naive macrophages from cultures that were not infected to macrophages from cultures that were infected with phrodo-labeled m. tuberculosis. in comparison to naive macrophages, cd47 was not upregulated on m. tuberculosis-infected (phrodopositive [phrodo ϩ ]) macrophages (fig. 5d) . interestingly, m. tuberculosis-uninfected (phrodo-negative [phrodo ϫ ]) macrophages from the m. tuberculosis-infected cultures downregulated cd47 expression in comparison to naive macrophages, an observation for which we do not yet have an explanation (fig. 5d ). the lack of cd47 upregulation by m. tuberculosis-infected macrophages was supported by metasignature analysis upregulation of cd47 is a host checkpoint response ® comparing microarrays for gene expression signatures specific to hcv and tuberculosis (tb) (fig. s2) . the disease-specific gene expression signature for hcv consistently showed an upregulation of cd47, whereas the disease-specific gene expression signature for tb showed downregulation. for an in vivo assessment of a possible functional role for cd47 in host resistance to m. tuberculosis infection, cd47 knockout (ko) mice were infected via the aerosol route with a low dose (100 to 200 cfu) of m. tuberculosis strain h37rv. cd47-deficient mice displayed significantly increased host resistance to m. tuberculosis infection, with significantly longer survival (humane endpoints), compared to wild-type c57bl/6 mice (fig. 5e) . furthermore, spleens and lungs from the cd47-deficient mice at humane endpoints yielded significantly lower mycobacterial cfu (fig. 5f ). these results indicated that cd47 expression can exert significant suppressive effects on immune responses to infectious pathogens in vivo. previous results demonstrating that malignant cells upregulate cd47 expression to evade cellular clearance (9, 31) led us to hypothesize that pathogens might also induce cd47 surface expression as an immune evasion mechanism. in fact, poxviruses encode a cd47 mimic that acts as a potent virulence factor (20) . curiously, we repeatedly observed an upregulation of cd47 surface expression across diverse infections with both viruses and bacteria, none of which are known to encode a cd47 mimic or have any cd47 sequence homology. flagella are potent inducers of tlrs, so the finding that salmonella typhi δflic mutants lacking flagella were poor at inducing cd47 suggested a possible connection to host sensing via prr signaling (fig. 1f) . indeed, direct stimulation of prrs with synthetic ligands such as mdp, cl264, and r848 induced cd47 upregulation in vitro and in vivo (fig. 2) . since sirp␣-mediated recognition of cd47 on infected cells by macrophages and dcs transduces an inhibitory signal that attenuates phagocytosis and downstream antigen presentation functions, it was counterintuitive that cd47 upregulation would be due to a host response. why would the host dampen its immune response to infection? we have shown that in vivo, cd47 blockade improved the control of lcmv infection (fig. 5a) , increased cd8 ϩ t cell responses (fig. 5c) , and, in a previous report, increased the expression of costimulatory molecules on apcs as well (23), thus confirming that cd47 induces immunosuppressive signals. similarly, mice with genetic inactivation of cd47 had reduced bacterial loads and longer survival times when infected with m. tuberculosis ( fig. 5e and f) . we conclude that, like coinhibitory molecules such as pd-1 that dampen adaptive immune responses, cd47 upregulation acts as an intrinsic governor of the innate immune response to prevent overactivation that can lead to immunopathology. thus, the initial immune response to infections is attenuated until proinflammatory signals overcome anti-inflammatory signals. cd47 was previously identified by microarray analysis as an interferon-stimulated gene (isg), upregulated as part of a coordinated program of host defense mechanisms upon ifn-␣ stimulation (32) . in addition, tnf-␣ was demonstrated to induce the upregulation of cd47 on vascular smooth muscle cells in vitro (33) . in breast cancer, tnf-␣-mediated cd47 upregulation is transcriptionally controlled by nf-b through an nf-b motif within an enhancer of the cd47 gene (34) . these reports are consistent with our findings showing that the inflammatory milieu in patient blood during hcv infection is sufficient to upregulate cd47 surface expression (fig. 4e) . while our study focused on tnf-␣, cxcl10, and ifn-␣, redundant mechanisms of cd47 upregulation by additional inflammatory mediators are possible and should be examined in depth in future experiments. in the experimental models utilized, it is difficult to distinguish the relative contributions of cytokine-induced cd47 upregulation from those of direct pathogen-induced cd47 upregulation where both are present and either is sufficient. the results clearly indicate that cd47 surface expression can be upregulated either in a cell-intrinsic manner via stimulation of prrs or by surveilling immune cells in response to extrinsic signaling by inflammatory cytokines. it may also be possible that signaling via cis interactions within a cell could occur in cells such as macrophages, which express both cd47 and sirp␣. of the infectious agents that we analyzed, m. tuberculosis infection was unique in failing to induce cd47 expression upon infection. of interest, uninfected cells from m. tuberculosis-infected cultures downregulated cd47 expression. this may not be too surprising because the life cycle of m. tuberculosis is dependent on phagocytosis by alveolar macrophages, and m. tuberculosis is known to induce strong inflammatory responses via the induction of cytokines and chemokines. further research will be required to identify the mechanisms involved in these unique responses to m. tuberculosis, but despite its failure to upregulate cd47, improved survival from m. tuberculosis infection was observed in mice genetically deficient in cd47 compared to wildtype mice (fig. 5e ). better recoveries from malaria parasites (35, 36) and escherichia coli infections (37) have also been shown in cd47 ko mice. in addition, cd47 ko mice have improved influenza virus vaccine responses (16) . however, from an evolutionary standpoint, the upregulation of cd47 expression in response to pathogens must result in a competitive advantage for the host. as an example, cd47 ko mice show poorly controlled inflammatory responses to and increased morbidity and mortality from candida albicans infection (38) . the contrasting results from various infections in cd47 ko mice illustrate how tightly balanced the immune system has evolved to be and the care that must be taken when immune interventions are undertaken. that said, the results from cd47 ko mice, in which the entire immune system has developed in the absence of a critical immunomodulatory molecule, might produce different results than the same infection in an immune-replete mouse treated with anti-cd47 antibodies during the infectious process. indeed, while we found that treating wild-type mice with anti-cd47 before lcmv strain we (lcmv-we) infection improved recovery, it has been reported that cd47 ko mice have decreased resistance to lcmv clone-13 infections (39) . additional experiments will be required to determine which experimental factors may account for the differences in these outcomes. the accelerated cd8 ϩ t cell responses and clearance of lcmv infections following prophylactic blockade of cd47 were most likely due to enhanced apc function evidenced by the increased expression of costimulatory cd86 on dendritic cells observed at 3 dpi (23). however, it is also possible that there were direct effects on cd8 ϩ t cells since it was previously shown that activated cd8 ϩ t cells express sirp␣, the receptor for cd47 (17) . it was shown that only activated effector cells and not naive cd8 ϩ t cells express sirp␣, and any direct effects on cd8 ϩ t cells would occur only after expansion and development of effector functions. furthermore, in contrast to the negative signal delivered to cells of the monocytic lineage via sirp␣ ligation to cd47, evidence suggested that cd47-sirp␣ signaling in cd8 ϩ t cells delivered a positive signal associated with improved cytolytic killing of infected target cells in vivo. the cd8 ϩ t cell responses measured in this prophylactic anti-cd47 study were total cd8 ϩ t cell responses rather than tetramer-stained cells known to be virus specific. thus, the expansion of bystander cd8 ϩ t cells by anti-cd47 was not excluded. however, when anti-cd47 was used in a therapeutic setting against lcmv infections, the predominant cd8 ϩ t cell expansions were virus specific, and the mechanism of protection was dependent on dcs and cd8 ϩ t cells (23) . the current results demonstrate that cd47 plays a prominent role in modulating inflammatory responses to infections. while these findings open new possibilities for therapeutic intervention against pathogenic agents, it is important to note that the context of the host response to specific types of infections will determine whether cd47 blockade would be protective or detrimental. there may be circumstances where host responses need boosting, and cd47 represents a novel target for host-directed therapies in such cases. possibilities include viruses such as sars-cov-2, human immunodeficiency virus, human papillomavirus, cytomegalovirus, epstein-barr virus, varicella-zoster virus, and ebola virus, etc. there is also a potential application for treating infections with bacteria, including m. tuberculosis and multidrug-resistant bacterial strains that might otherwise be untreatable. although not addressed in this study, other infectious agents, such as fungi or parasites, that elicit prr responses might also be tractable to anti-cd47 therapy. a key factor is that infected cells also express damage-associated molecular patterns (damps), which act as "eat me" signals that are being masked by the cd47 "don't eat me" signal (11, 40) . therefore, releasing the inhibition of phagocytosis of these cells would need to be weighed cautiously with the extent of infection and the replaceability of the infected cell types. . virus stocks were passaged no more than 3 times in vero cells. for analysis of cd47 expression in mice during lacv infection, 21-day-old c57bl/6 (jackson laboratories) male or female mice were inoculated intraperitoneally with a 10 5 -pfu dose of virus diluted into a 200-l volume of sterile pbs. mice of the same strain, age, and sex inoculated with an equivalent volume of a vero cell culture supernatant in pbs were used as controls. at 2 dpi, whole blood and spleen were isolated from mice and processed for flow cytometry as described above. lcmv viral titers were detected by plaque-forming assays on mc57 fibroblasts (obtained from by the ontario cancer institute, canada). organs were dissociated, and plasma was diluted in dulbecco's modified eagle medium (dmem) containing 2% fetal calf serum (fcs), titrated 1:3 over 12 steps, and incubated on mc57 cells. after 4 h of incubation at 37°c, a methylcellulose overlay was added, and the cells were incubated for 48 h, followed by staining of lcmv plaques using an anti-lcmv-np antibody (clone vl4). c57bl/6 mice were infected with 2 ϫ 10 6 pfu of lcmv strain we and treated with anti-cd47 via daily intraperitoneal injections of 100 g of anti-cd47 (clone 410, catalog number be0283; bioxcell) or an isotype control (rat igg2a isotype) (bioxcell) from day ϫ2 to day 6 postinfection. to analyze infected spleen cells, splenocytes were isolated by tissue homogenization through a 100-m filter, and red blood cells were removed using ack lysis buffer (0.15 m nh 4 cl, 10 mm khco 3 , 0.1 m edta). the gating strategy for spleen cell subset analyses was the same as the one described previously (23) . all antibodies were from bd biosciences, biolegend, or ebioscience/thermo fisher scientific, including brilliant violet 605-anti-cd11b (clone m1/70), phycoerythrin (pe)-cf594-anti-cd19 (clone id3), pe-cy7-anti-cd11c (clone hl3), pe-cy7-anti-ter119 (clone ter-119), alexa fluor 647-anti-cd47 (clone miap301), and fluorescein isothiocyanate (fitc)-anti-major histocompatibility complex class ii (mhcii) (i-a/i-e) (catalog number fab6118f); lymphocyte populations were initially gated on single live cells on the basis of forward scatter (fsc) versus side scatter (ssc). mouse dcs were defined as cd11c ϩ cd11b ϫ , and macrophages were defined as cd11c ϫ cd11b ϩ . human dcs were also gated on the basis of high mhcii expression levels. the cd11c ϩ subset contained a minor population of cd11bintermediate cells that could have been inflammatory macrophages. the multiparameter data were collected with an lsrii instrument (bd biosciences) and analyzed using flowjo software. bacterial strains. bacterial strains included a b31 borrelia burgdorferi clone (gcb726) with the cp9 plasmid replaced by a cp9-based ptm61 construct containing green fluorescent protein (gfp) (42) . salmonella enterica serovar typhi ty2 mcherry mutant strains were generated via red recombination and included the salmonella enterica serovar typhi ty2 mcherry δfla (flic::kan) mutant (25) . m. tuberculosis h37rv δlysa and δpancd, used for the in vitro studies, were provided by william r. jacobs, jr. (43) . m. tuberculosis strain h37rv was used for the in vivo studies. affymetrix array profiles of liver biopsy specimens. affymetrix arrays were obtained as cel files, mas5 normalized using the "affy" package in bioconductor, mapped to ncbi entrez gene identifiers using a custom chip definition file (brainarray version 19 [http://brainarray.mbni.med.umich.edu/ brainarray/]), and converted to hugo gene symbols (44) . a publicly available gene expression data set from sars-cov-2 infection of a549 cells (accession number gse147507) (n ϭ 10) was downloaded from the gene expression omnibus (geo). independent biological replicates of transformed lung alveolar (a549) cells were mock infected (uninfected [un]) (n ϭ 13) or infected (inf) (n ϭ 6) with sars-cov-2 (usa-wa1/2020). cdna libraries were sequenced from each sample using the illumina nextseq 500 platform. raw sequencing reads were aligned to the human genome (hg19) using the rna-seq alignment app (v2.0.1) on basespace (illumina, ca). gene expression values were summarized using counts per million (cpm) and converted to a log 2 scale using the formulas log 2 (cpm) if the cpm were ͼ1 and cpm ϫ 1 if the cpm were ͻ1. a standard t test was performed using the python scipy.stats.ttest_ind package (version 0.19.0) with welch's two-sample t test (unpaired, unequal variance [equal_varϭfalse], and unequal sample size) parameters. the results were independently validated with r statistical software (r version 3.6.1, 5 july 2019). cd47 was significantly upregulated (p ϭ 0.00332) in sars-cov-2-infected samples. (45) . hcv sofosbuvir cohort. pbmcs, plasma, and serum were studied in 14 hcv-infected patients previous to direct-acting antiviral therapy (sofosbuvir [sof] and simeprevir [sim] ; sof and ribavirin [rbv]; and sof, rbv, and pegylated interferon [peg]) before treatment, during treatment, and after treatment. ten patients underwent at least one previous treatment with interferon, and the other four were treatment naive. thirteen patients experienced a sustained virologic response (svr) after 12 weeks of therapy. pbmcs, plasma, and serum were collected from noninfected patients as a control (table 1) . one patient relapsed. patients provided written informed consent for research testing under protocols by the stanford university institutional review board. phospho-cytof sample processing and staining. cryopreserved pbmcs stored at ϫ180°c were thawed in warm rpmi medium supplemented with 10% fbs, benzonase, and a penicillin-streptomycin mixture (complete rpmi medium). cells were transferred into serum-free rpmi medium containing 2 mm edta and benzonase, incubated with cisplatin for 1 min, and immediately quenched with 4 volumes of complete rpmi medium. next, 1 million cells per sample were transferred into complete rpmi medium and rested for 30 min at 37°c. following this rest period, cells were fixed in pbs with 2% paraformaldehyde (pfa) at room temperature for 10 min. cells were then washed twice with cyfacs buffer and barcoded as previously described (46) . following barcoding, samples were combined for surface marker staining, performed at room temperature for 1 h. subsequently, cells were washed and permeabilized in methanol (meoh) at ϫ80°c overnight. the next day, cells were washed and incubated with the intracellular cytokine cocktail at room temperature for 1 h. dna staining was performed for 20 min with iridium (191/193) in pbs with 2% pfa at room temperature. finally, cells were washed twice with cyfacs buffer and then twice with milliq water before data acquisition on the cytof2 instrument. data were debarcoded and manually analyzed on cytobank (www.cytobank.org/). monocyte-derived dc and macrophage cultures. healthy donor leukocyte reduction system cones were provided by the stanford blood center. pbmcs were isolated by a 1.077-g/ml ficoll gradient using sep-mate tubes. monocytes were selected for by plastic adherence after 20 min of incubation at 37°c with 5% co 2 in rpmi medium plus 10% human serum (gemini). selected monocytes were then cultured for 72 h in rpmi medium supplemented with 1% serum, 10 ng/ml interleukin-4 (il-4), and 800 iu/ml granulocyte-macrophage colony-stimulating factor (gm-csf); the concentration of gm-csf was increased to 1,600 iu/ml for the final 24 h. immature dcs were matured by replacing culture medium with rpmi medium supplemented with 1% healthy donor or hcv patient plasma, 10 ng/ml il-4, 800 iu/ml gm-csf, 10 mg/ml lipopolysaccharide (lps), and 100 iu/ml ifn-␥. for macrophage derivation, monocytes were cultured in rpmi medium supplemented with 10% human serum for 7 days. human and murine luminex assays. the human samples were analyzed at the human immune monitoring center at stanford university. human 63-plex or mouse 38-plex kits were purchased from ebioscience/affymetrix and used according to the manufacturer's recommendations, with modifications as described below. briefly, beads were added to a 96-well plate and washed in a biotek elx405 washer. samples were added to the plate containing mixed antibody-linked beads and incubated at room temperature for 1 h, followed by incubation overnight at 4°c with shaking. cold and room-temperature incubation steps were performed on an orbital shaker at 500 to 600 rpm. following incubation overnight, plates were again washed in a biotek elx405 washer, and a biotinylated detection antibody was then added for 75 min at room temperature, with shaking. the plate was washed as described above, and streptavidin-pe was added. after incubation for 30 min at room temperature, another wash was performed as described above, and reading buffer was added to the wells. each sample was measured in duplicate. plates were read using a luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. custom assay control beads by radix biosolutions were added to all wells. in vitro stimulations and infections of human pbmcs and macrophages. healthy donor leukocyte reduction system cones were provided by the stanford blood center. human pbmcs were isolated by a 1.077-g/ml ficoll gradient using sep-mate tubes. isolated pbmcs were cultured in rpmi medium supplemented with 10% fbs and 100 u/ml penicillin-streptomycin at a concentration of 1 ϫ 10 6 cells/ml. to activate prrs, cells were stimulated with either 1 g/ml cl264-rhodamine (invivogen), 1 g/ml muramyl dipeptide (invivogen), or r848 at concentrations of 0.1 g/ml, 1 g/ml, and 10 g/ml or left unstimulated. cells were collected at 48 h poststimulation prior to flow cytometry. pbmcs were stimulated with single treatments or combination treatments of 10 ng/ml tnf-␣, 100 ng/ml cxcl10, and 100 ng/ml ifn-␣. cells were then analyzed for cd47 expression 72 h after cytokine stimulation. in vitro bacterial infections of pbmcs were performed at a multiplicity of infection (moi) of 10 for salmonella enterica serovar typhi strains and at an moi of 40 for borrelia burgdorferi, for 24 and 48 h, respectively. salmonella enterica serovar typhi strains were spun onto the cells to compensate for the motility differences. indeed, the strain of salmonella enterica serovar typhi infected a lower percentage of cells, but infected versus uninfected cells were differentiated for the analyses. for m. tuberculosis in vitro infection of macrophages, m. tuberculosis was stained for 1 h in pbs with a 1:20,000 dilution of phrodo (essen biosciences) at 37°c to fluorescently label infected macrophages. macrophages were plated into 96-well u-bottom plates. fluorescently labeled m. tuberculosis bacteria were used to infect macrophages at an moi of 1:10 for 24 h. for antibodies for flow cytometry, anti-cd11c (clone 3.9), anti-hla-dr (clone l243), anti-cd11b (clone m1/70), anti-cd14 (clone m5e2), anti-cd16 (clone 3g8), and anti-sirp (clone se5a5) were purchased from biolegend, except for allophycocyanin (apc)-anti-cd47 (clone b6h12; ebioscience), which was purchased from invitrogen. cells were analyzed with 4=,6-diamidino-2phenylindole (dapi) for dead-cell exclusion and then gated on single cells using fsc-a (forward scatter all human in vitro experiments were repeated in at least two independent experiments with a minimum of 4 biological replicates. in vivo and in vitro stimulation of mouse cells. female c57bl/6 rrid:imsr_jax:000664 (wt) mice were bred at the stanford university stem cell institute barrier facility (stanford, ca) and used at between 8 and 12 weeks of age at the beginning of the experiments. for r848 in vivo analysis, 10 naive mice were injected intraperitoneally with either 1 mg/kg of body weight of r848 or pbs, all at a volume of 0.1 ml, for three consecutive days. on day 3 after the first treatment, splenocytes were isolated. spleens were dissociated by collagenase treatment in the presence of dnase i and mechanical dissociation to obtain a single-cell suspension of splenocytes. red blood cells were removed using ack lysis buffer (gibco), and the remaining splenocytes were seeded at a density of 1 ϫ 10 6 splenocytes/well in a 96-well u-bottom low-adherence plate. cells were then stained for the macrophage and dc markers cd11b, mhcii, and cd11c, as well as sirp␣ and cd47, with dapi for live/dead exclusion. for in vitro stimulation, splenocytes were isolated from naive mice as described above and then stimulated with either 1 g/ml cl264-rhodamine (invivogen) overnight or 1 g/ml poly(i·c)-rhodamine (invivogen) complexed with lipofectamine 2000 for 1 h or left unstimulated. cells were collected at 24 h poststimulation for analysis by flow cytometry. antibodies for flow cytometry were purchased from biolegend or bd biosciences. m. tuberculosis infections. m. tuberculosis infections were done in c57bl/6 mice or cd47 ko rrid:imsr_jax:003173 (cd47 ko) mice that were bred at niaid facilities. for infections with m. tuberculosis h37rv (100 to 200 cfu), 8-to 12-week-old male and female mice were placed in a whole-body inhalation system (glas-col, terre haute, in) and exposed to aerosolized m. tuberculosis. delivery doses were set by measuring lung cfu at 2 to 24 h postexposure from three to five control mice through mechanical homogenization using precellys evolution (precellys, atkinson, nh). lung homogenates were then serially diluted in pbs-tween 20 and cultured on middlebrook 7h11 agar plates supplemented with oleic acid-albumin-dextrose-catalase (difco, detroit, mi), and cfu were counted 21 days later. cell isolation from m. tuberculosis-infected lung tissue and flow cytometry. lungs were digested and dissociated using gentle magnetically activated cell sorting (macs) and lung cell isolation buffer (miltenyi biotec). the digested lung was passed through a 100-mm cell strainer, and an aliquot was removed for the determination of cfu. cells were washed and purified with 37% percoll. cells for sorting were washed, counted, and subsequently surface stained in a biosafety level 3 (bsl3) containment area under sterile conditions. the following cell populations were sorted to 90 to 97% purity and plated for cfu counts: cd45.1 ϩ (wt) or cd45.1 ϫ (il1r12/2) cd11b ϩ , cd11b ϩ gr1 high (neutrophils), and cd11b ϩ gr1 low (myeloid). abs against i-ab (clone m5/114.15.2), ly6g (clone 1a8), cd11c (clones hl3 and n418), cd45.1 (clone a20), cd45.2 (clone 104), t cell receptor ␤ (tcr␤) (clone h57-597), nk1.1 (clone pk136), cd11b (clone m1/70), cd45 (clone 30-f11), and gr-1 (clones rb6 and 8c5) and live/dead fixable cell stains were obtained from ebioscience/thermo fisher scientific, biolegend, or bd pharmingen. samples were acquired on a 350 symphony flow cytometer or sorted on a facsaria instrument (bd biosciences, san jose, ca) and analyzed using flowjo software. supplemental material is available online only. was supported by grant f30dk099017 and the stanford medical scientist training program. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. k.j.h. and i.l.w. are listed as inventors on u.s. patent 2019/0092873 a1 cd47, targeted therapies for the treatment of infectious disease. i.l.w. is a cofounder, director, and stockholder in forty seven inc., a public company that was involved in cd47-based immunotherapy of cancer during this study but was acquired by gilead. at the time of this submission, i.l.w. has no formal relationship with gilead. restoring function in exhausted cd8 t cells during chronic viral infection immune checkpoints and their inhibition in cancer and infectious diseases combination anti-ctla-4 plus anti-pd-1 checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies cd47 blockade by hu5f9-g4 and rituximab in non-hodgkin's lymphoma first-in-human, first-in-class phase i trial of the anti-cd47 antibody hu5f9-g4 in patients with advanced cancers the macrophage 'do not eat me' signal, cd47, is a clinically validated cancer immunotherapy target therapeutic targeting of the macrophage immune checkpoint cd47 in myeloid malignancies the cd47-signal regulatory protein alpha (sirpa) interaction is a therapeutic target for human solid tumors cd47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis dysregulated gene expression networks in human acute myelogenous leukemia stem cells calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by cd47 integrin-associated protein is a receptor for the c-terminal domain of thrombospondin integrin-associated protein is a ligand for the p84 neural adhesion molecule integrin-associated protein (cd47) and its ligands the interaction between signal regulatory protein alpha (sirp␣) and cd47: structure, function, and therapeutic target cd47 plays a role as a negative regulator in inducing protective immune responses to vaccination against influenza virus a functional subset of cd8ϩ t cells during chronic exhaustion is defined by sirp␣ expression functional cd47/signal regulatory protein alpha (sirp(alpha)) interaction is required for optimal human t-and natural killer-(nk) cell homeostasis in vivo recent advances in understanding viral evasion of type i interferon myxoma virus m128l is expressed as a cell surface cd47-like virulence factor that contributes to the downregulation of macrophage activation in vivo friend retrovirus studies reveal complex interactions between intrinsic, innate and adaptive immunity suppression of acute anti-friend virus cd8 ϩ t-cell responses by coinfection with lactate dehydrogenase-elevating virus immunotherapeutic blockade of cd47 inhibitory signaling enhances innate and adaptive immune responses to viral infection toll-like receptors stimulate human neutrophil function cutting edge: inflammasome activation in primary human macrophages is dependent on flagellin human tlr7 or tlr8 independently confer responsiveness to the antiviral compound r-848 sofosbuvir: a novel treatment option for chronic hepatitis c infection dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny immune response against lymphocytic choriomeningitis virus infection in mice without cd8 expression lymphocytic choriomeningitis virus cd47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells functional classification of interferon-stimulated genes identified using microarrays cd47-blocking antibodies restore phagocytosis and prevent atherosclerosis a cd47-associated super-enhancer links pro-inflammatory signalling to cd47 upregulation in breast cancer cd47 regulates the phagocytic clearance and replication of the plasmodium yoelii malaria parasite cd47-sirp␣ interactions regulate macrophage uptake of plasmodium falciparum-infected erythrocytes and clearance of malaria in vivo cd47 deficiency protects mice from lipopolysaccharide-induced acute lung injury and escherichia coli pneumonia cd47 promotes protective innate and adaptive immunity in a mouse model of disseminated candidiasis cd47 expression in natural killer cells regulates homeostasis and modulates immune response to lymphocytic choriomeningitis virus macrophages eat cancer cells using their own calreticulin as a guide: roles of tlr and btk enhancement of spleen focus formation and virus replication in friend virus-infected mice real-time high resolution 3d imaging of the lyme disease spirochete adhering to and escaping from the vasculature of a living host long-term protection against tuberculosis following vaccination with a severely attenuated double lysine and pantothenate auxotroph of mycobacterium tuberculosis robust enumeration of cell subsets from tissue expression profiles sars-cov-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems platinum-conjugated antibodies for application in mass cytometry we thank members of the weissman, hasenkrug, davis, and glenn laboratories, especially da yoon no and nathaniel fernhoff, for helpful advice, discussions, and reagents. we specifically acknowledge aaron newman for assistance and guidance in the analysis of microarray data. we thank susan matlock brewer for assistance and guidance with salmonella experiments. we thank yael rosenberg-hasson and the staff of the stanford human immune monitoring center for their technical expertise and for running the mouse and human luminex assays. we also acknowledge the laboratory of benjamin r. tenoever and daniel blanco-melo for the rapid sharing of critical sars-cov-2 data at the onset of this pandemic (geo accession number gse147507).research key: cord-013176-6ckuya1w authors: ninfali, paolino; antonelli, antonella; magnani, mauro; scarpa, emanuele salvatore title: antiviral properties of flavonoids and delivery strategies date: 2020-08-21 journal: nutrients doi: 10.3390/nu12092534 sha: doc_id: 13176 cord_uid: 6ckuya1w this review summarizes the latest advancements in phytochemicals as functional antiviral agents. we focused on flavonoids, like apigenin, vitexin, quercetin, rutin and naringenin, which have shown a wide range of biological effects including antiviral activities. the molecular mechanisms of their antiviral effects mainly consist in the inhibition of viral neuraminidase, proteases and dna/rna polymerases, as well as in the modification of various viral proteins. mixtures of different flavonoids or combination of flavonoids with antiviral synthetic drugs provide an enhancement of their antiviral effects. recent strategies in drug delivery significantly contribute to overcoming the low bioavailability of flavonoids. frequent viral infections worldwide have led to the need for new effective antiviral agents, which can be identified among the various phytochemicals. in this light, screening the antiviral activities of a cocktail of flavonoids would be advantageous in order to prevent viral infections and improve current antiviral therapies. in the past two decades, studies conducted in our laboratory focused on the antioxidants present in vegetable foods and on their capacity to reduce the adverse physiological effects of the oxygen free radicals [1, 2] . the research was also aimed at the technologies of food transformation in order to preserve, as much as possible, the antioxidants in the final products [3] . antioxidants are mainly represented in nature by the liposoluble vitamins e and a, β-carotene, hydro-soluble vitamin c and a wide range of amphipathic molecules, broadly termed phenolic compounds [1] . these compounds are divided into several sub-classes, including phenolic acids and analogues, stilbenes, flavonoids and their analogues. flavonoids possess important health protective effects, including anti-inflammatory, anticancer and antiviral properties [4] [5] [6] [7] [8] [9] [10] . there are in nature more than 6000 flavonoids, which have been structurally identified and divided in classes: flavones (e.g., apigenin), flavanols (e.g., quercetin), flavins (e.g., epigallocatechin-3-gallate), isoflavones (e.g., genistein) and anthocyanidins (e.g., cyanidin). flavonoids occur in their free or conjugated form or are often esterified with one or two sugars with o-glycosidic or c-glycosidic bonds [11] . in the past decade we purified and studied the biological effects of a group of the flavonoid c-glycosides derived from apigenin, namely vitexin, vitexin-2-o-rhamnoside and vitexin-2-o-xyloside ( figure 1 ). the interest in the antiviral activity of natural flavonoids has increased in the last decade because of the frequency of viral infections, particularly influenza infections, which affect several million patients annually [12] . while vaccination is the primary strategy for influenza prevention, there are scenarios for which vaccination is not possible and antiviral molecules represent an important sanitary presidium. synthetic antiviral drugs often show limited efficacy and serious adverse effects [13] , whereas herbal extracts, known for their antiviral properties with no or mild side effects, may be a viable alternative for treating various viral diseases [14] . viruses consist of nucleic acid (dna or rna) enclosed in a protein structure, called capsid, which can be surrounded by a lipid membrane named the envelope. the infective unit of the virus is called the virion and this parasite can replicate only inside host cells, hijacking the molecular machinery and controlling dna replication, rna transcription and the protein translation processes. viruses attack the host cells through adsorption to receptors specific for each type of target cells, penetrating through the cell membrane, then the genetic material of the virus is liberated to replicate its own genome, produce new viral proteins and obtain new virions [15] . in 2017, the antiviral effects of various phytochemicals were reviewed by kapoor et al. [15] , taking into consideration many categories of compounds, ranging from flavonoids to saponins and lignans. in the same year, another group published a review focused specifically on the antiviral effects of the six classes of flavonoids [16] . in 2020, an interesting review regarding the methods for delivery of phytochemicals to increase their bioavailability in human tissues has been published [17] . in this review, we first summarize flavonoids along with their class and plant sources, with particular attention to apigenin, vitexin and its derivatives (table 1) . we then discuss the antiviral action mechanisms of flavonoids, their combinations with conventional antiviral drugs in multitarget cocktails, and the delivery strategies used to increase their bioavailability and antiviral efficacy. the interest in the antiviral activity of natural flavonoids has increased in the last decade because of the frequency of viral infections, particularly influenza infections, which affect several million patients annually [12] . while vaccination is the primary strategy for influenza prevention, there are scenarios for which vaccination is not possible and antiviral molecules represent an important sanitary presidium. synthetic antiviral drugs often show limited efficacy and serious adverse effects [13] , whereas herbal extracts, known for their antiviral properties with no or mild side effects, may be a viable alternative for treating various viral diseases [14] . viruses consist of nucleic acid (dna or rna) enclosed in a protein structure, called capsid, which can be surrounded by a lipid membrane named the envelope. the infective unit of the virus is called the virion and this parasite can replicate only inside host cells, hijacking the molecular machinery and controlling dna replication, rna transcription and the protein translation processes. viruses attack the host cells through adsorption to receptors specific for each type of target cells, penetrating through the cell membrane, then the genetic material of the virus is liberated to replicate its own genome, produce new viral proteins and obtain new virions [15] . in 2017, the antiviral effects of various phytochemicals were reviewed by kapoor et al. [15] , taking into consideration many categories of compounds, ranging from flavonoids to saponins and lignans. in the same year, another group published a review focused specifically on the antiviral effects of the six classes of flavonoids [16] . in 2020, an interesting review regarding the methods for delivery of phytochemicals to increase their bioavailability in human tissues has been published [17] . in this review, we first summarize flavonoids along with their class and plant sources, with particular attention to apigenin, vitexin and its derivatives (table 1) . we then discuss the antiviral action mechanisms of flavonoids, their combinations with conventional antiviral drugs in multi-target cocktails, and the delivery strategies used to increase their bioavailability and antiviral efficacy. the most common flow chart for studies regarding the antiviral activity of phytochemicals is focused on the immediate testing of the whole natural extract, by dividing the polar from the non-polar constituents. after that, fractions with remarkable activity in in vitro antiviral assays, such as cytophatic effect, neutralization assay, hemagglutination, viral enzyme inhibition, or virion number reduction assay, are further fractionated through chromatographic techniques in order to purify the active phytochemicals. effective compounds are then used on virus-infected animals or in human clinical trials in order to assess their effective antiviral properties [15] . the complete procedure, when interfaced with chemical libraries, represents the basis for high throughput screening assays [15] . the parameter used for assessing antiviral efficiency of drugs and phytochemicals is represented by 50% inhibitory concentrations (ic 50 ) or by 50% effective concentration (ec 50 ). table 2 shows the main flavonoids studied for antiviral activity along with their investigated mechanisms of action. in the first section, we point out the studies focused on apigenin, vitexin and their derivatives, which were found active against many viruses like human hepatitis c virus (hcv), herpes simplex virus-1 (hsv-1), human hepatitis a and b viruses (hav; hbv), rhesus rotavirus (rrv) and influenza virus. the natural extract of eclipta alba (asteraceae) was shown to be able to inhibit the hcv replicase in a cell culture system, which resulted in reduced hcv rna titer and translation level of viral proteins [19] . through bioassay-based fractionations of the natural extract, the authors identified two flavonoid compounds: apigenin and luteolin, which, tested individually, exhibited a dose-dependent inhibition of hcv replicase in vitro [19] . quercetin, extracted from embelia ribes (mirsinaceae), exhibited antiviral effects against hcv, exerted through activity inhibition of the viral protease non-structural protein 3 (ns3), leading to a decrease in hcv replication [36] . furthermore, the flavanol quercetagetin was found to inhibit hcv rna-dependent rna polymerase (rdrp) through inhibition of rna binding to the viral polymerase, a mechanism associated with broad genotypic activity against several hcv strains and a high barrier to resistance mechanisms [39] . luteolin and quercetin showed anti-hcv effects in hepatoma huh-7 cells transfected with non-structural protein 5b (ns5b) cloned gene vector, that codifies for the ns5b polymerase of hcv virus [40] . naringenin and quercetin possess antiviral activity against hcv, but naringenin showed stronger inhibition of virion assembly, whereas quercetin inhibited viral translation by blocking non-structural protein 5a (ns5a) and internal ribosome entry site (ires)-mediated translation, as well as heat shock protein 70 (hsp70) induction [41] . bioinformatics tools were also used to study the interaction of various phytochemicals with viral proteins that possess pivotal roles in the production of new virions and in the infection of the host cells. this approach may be a useful premise for deeper investigation regarding flavonoids which have provided interesting evidence of interactions with viral proteins. for instance, naringenin, diosmetin, apigenin and luteolin were all able to bind to the ns5b protein of hcv with higher affinity when compared with the antiviral drug sofosbuvir, inhibiting the activity of this viral enzyme [25] . epigallocatechin-3-gallate (egcg), the principal tea derived catechin, efficiently inhibited cell culture-derived hbv entry into hepatocellular cell lines, independent of the hbv genotypes, through a mechanism that involves the clathrin-dependent endocytosis of the hbv receptor sodium taurocholate co-transporting polypeptide (ntcp) from the plasma membrane, followed by its protein degradation [26] . the extract obtained from erythrina speciosa (fabaceae) exerted antiviral effects against hav-h10 viruses mainly due to vitexin which, isolated from the extract, exhibited an antiviral activity against this virus with ec 50 = 41.6 ± 7.6 µm [42] . the authors showed that the flavone vitexin can interact with the binding pocket of hav 3c proteinase, inhibiting this enzyme [42] . hcv virions inhibition of virion assembly vitexin, extracted from the plant flos trollii, (caryophyllaceae), exerted its anti-h1n1 influenza virus effects through partially down-regulating toll-like receptor 3 (tlr3) and toll-like receptor 7 (tlr7) pathways and up-regulating toll-like receptor 4 (tlr4) molecular pathway [38] . tlrs are transmembrane glycoproteins, which are privileged targets of several viruses and can be activated by endogenous molecules in the context of inflammation. tlr activation produces pro-inflammatory cytokines through nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) signaling pathway or through interferon regulatory factor 3 (irf3) and interferon regulatory factor 7 (irf7). some viruses enter the host cells by binding tlr3, but after their entrance the viruses are able to inhibit cytokine production, thus impairing the immune response. phytochemicals able to decrease the binding between tlrs and virus particles can slow the infective process. interestingly, virus infection can lead to an induction of the inflammation process, characterized by excessive production of nitric oxide (no), interleukin-1 (il-1), interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α). it was shown that various flavonoids enhance the production of interferon-β (ifn-β) in order to counteract the viral infections [38] . baicalin, baicalein, wogonin, chrysin and oroxylin a, isolated from scutellaria baicalensis, showed anti-h1n1 activities, with ic 50 values of 7.4, 7.5, 2.1, 7.7 and 12.8 µm, respectively, and were all more potent than the conventional antiviral drug oseltamivir phosphate, which had an ic 50 of 45.6 µm [22] . these flavonoids increased the transcriptional activity of nuclear factor erythroid 2-related factor 2 (nrf2), the master regulator of the antioxidant responses, whose activation is related to the antiviral effects of scutellaria baicalensis [22] . the natural extract of tetrastigma hemsleyanum (vitaceae) contains many flavonoids, including vitexin, vitexin-2-o-rhamnoside, isorhamnetin, rutin, kaempferol, astragalin, quercitrin, quercetin and iso-quercetin, which were shown to be able to exert anti-influenza virus activity, with different efficiency, through the reduction of the number of plaques induced by the influenza virus in infected madin-darby canine kidney (mdck) cells [21] . similarly, the flavonoid quercetin-3-rhamnoside, extracted from houttuynia cordata (saururaceae), exerted anti-influenza a/ws/33 activity reducing virus-mediated cytopathic effects, directly interacting with virus particles [37] . furthermore, the same authors showed that quercetin-3-rhamnoside exerted anti-influenza virus activity in mice, when used at 6.25 mg/kg/day for six days after influenza virus infection [45] . flavonoids sanggenon o, egcg and chamaejasmin were all potentially able to inhibit dengue virus replication by blocking the asn-130 glycosylation site of the viral protein non structural protein 1 (ns1) [23] . baicalin, a flavonoid derived from scutellaria baicalensis (lamiaceae), exhibited viricidal activity against dengue virus-2 (denv-2) extracellular virions with ic 50 = 19.6 ± 0.2 µm, exerted an anti-adsorption effect with ic 50 = 40.5 ± 0.4 µm and also inhibited virus replication with ic 50 = 30.2 ± 0.2 µm [44] . studies of the antiviral effects of the flavonoids fisetin, quercetagetin, and pinocembrin showed that fisetin can inhibit the replication molecular machineries of dengue virus and enterovirus a71 [35] . furthermore, other antiviral mechanisms of the pinocembrin consist in targeting the molecular machinery used by the zika virus to replicate its own genome inside the host cells [33] . this flavanone acts on the post-entry processes of zika virus replication cycle through the inhibition of both viral rna production and synthesis of envelope proteins [33] . interestingly, the plant moringa oleifera lam (moringaceae) provides a rich and rare combination of several phytochemicals, including the flavonoids quercetin and kaempferol, and its leaves extract can be applied as a prophylactic or therapeutic anti-hsv-1 medicine [29] . the extract obtained from moringa oleifera lam remarkably reduced the plaque formation induced by wt hsv, thymidine kinase-deficient hsv and phosphonoacetate-resistant hsv strains [29] . furthermore, the extract obtained from erythrina speciosa possessed antiviral properties against the hsv-1 virus, mainly due to vitexin which exhibited an antiviral activity with ec 50 = 80.9 ± 6.2 µm, exerted through the interaction of this flavone with the binding pocket of hsv-1 thymidine kinase [42] . vitexin and luteolin from aspalathus linearis (fabaceae) showed antiviral activity against rrv with ic 50 of 129 µm and 116 µm , respectively; interestingly, apigenin-7-o-glucoside from melissa officinalis (labiateae) inhibited viral growth, with an ic 50 of 150 µm, through the reduction of the number of rrv-induced plaques in infected ma104 cells [20] . tangeretin and nobiletin, two polymethoxyflavones extracted from citrus reticulate "chachi", possess anti-rsv properties. tangeretin exhibited a dose-dependent inhibition of rsv-induced plaque formation on hep-2 cells, through inhibition of rsv entry into host cells and viral replication. furthermore, tangeretin decreased the levels of rsv phosphoprotein (p protein), which is associated with the viral genome to form the holo-nucleocapsid. the extent of the antiviral effect of this phytochemical was comparable to the conventional antiviral drug ribavirin [32] . the knowledge of the molecular mechanisms of virus infection and phytochemical antiviral actions is fundamental in planning an effective therapeutic approach. the main mechanisms involved in the antiviral effects of phytochemicals are focused on the targeting of viral enzyme activities. many natural molecules target the catalytic activity of the influenza virus neuraminidase, also called sialidase. this enzyme is a glycoside hydrolase that cleaves the glycosidic linkages of sialic acids ( figure 2 ). various phytochemicals inhibit the activity of viral sialidases, hampering the release of new virions from the infected cells and preventing new infections [47] . another enzyme with a pivotal role in influenza a virus infection is rdrp, which is composed of three subunits: polymerase acidic subunit (pa), protein binding 1 subunit (pb1) and protein binding 2 subunit (pb2). the enzyme synthesizes viral mrnas using short capped primers derived from host cellular mrnas, which are cut by the viral endonuclease. the n-terminal domain of the pa subunit contains a typical endonuclease active site and harbors the rna/dna endonuclease activity, which is essential for viral growth [48] . the knowledge of the molecular mechanisms of virus infection and phytochemical antiviral actions is fundamental in planning an effective therapeutic approach. the main mechanisms involved in the antiviral effects of phytochemicals are focused on the targeting of viral enzyme activities. many natural molecules target the catalytic activity of the influenza virus neuraminidase, also called sialidase. this enzyme is a glycoside hydrolase that cleaves the glycosidic linkages of sialic acids (figure 2 ). various phytochemicals inhibit the activity of viral sialidases, hampering the release of new virions from the infected cells and preventing new infections [47] . another enzyme with a pivotal role in influenza a virus infection is rdrp, which is composed of three subunits: polymerase acidic subunit (pa), protein binding 1 subunit (pb1) and protein binding 2 subunit (pb2). the enzyme synthesizes viral mrnas using short capped primers derived from host cellular mrnas, which are cut by the viral endonuclease. the n-terminal domain of the pa subunit contains a typical endonuclease active site and harbors the rna/dna endonuclease activity, which is essential for viral growth [48] . enzymes involved in the hcv virus replication, like the protease ns3 and the polymerase ns5b can also be efficiently inhibited or modulated by phytochemicals ( figure 3 ). ns3 is a hcv nonstructural protein, which acts as a serine protease. its n-terminal domain can interact with the viral non structural protein 2 (ns2), while the c-terminal domain acts both as helicase and nucleoside triphosphatase. enzymes involved in the hcv virus replication, like the protease ns3 and the polymerase ns5b can also be efficiently inhibited or modulated by phytochemicals ( figure 3 ). ns3 is a hcv nonstructural protein, which acts as a serine protease. its n-terminal domain can interact with the viral non structural protein 2 (ns2), while the c-terminal domain acts both as helicase and nucleoside triphosphatase. the ns5b protein is rdrp with a pivotal role in replicating hcv's viral rna by using the viral ssrna+ as a template to catalyze the polymerization of ribo-nucleoside triphosphates during replication of the viral genome. interestingly, quercetin, apigenin and luteolin effectively inhibit ns5b polymerase activity [33] . when phytochemicals have been combined among them or with synthetic antiviral drugs, synergistic therapeutic effects were often evidenced. overall, when synergy occurred, it was often justified by the fact that the different molecules target different steps in the the ns5b protein is rdrp with a pivotal role in replicating hcv's viral rna by using the viral ssrna+ as a template to catalyze the polymerization of ribo-nucleoside triphosphates during replication of the viral genome. interestingly, quercetin, apigenin and luteolin effectively inhibit ns5b polymerase activity [33] . when phytochemicals have been combined among them or with synthetic antiviral drugs, synergistic therapeutic effects were often evidenced. overall, when synergy occurred, it was often justified by the fact that the different molecules target different steps in the molecular mechanisms of viral infection, and the final antiviral effect results therefore potentiated. naringenin is a flavanone, which exhibits anti-hcv activity by blocking the assembly of hcv particles [41] . the phytochemical quercetin exerted anti-hcv activity by reducing internal ribosome entry site-(ires-)mediated translation and also inhibiting the viral non-structural protein ns5a as well as the protease ns3 [41] . when naringenin and quercetin were used together they suppressed hcv rna replication and inhibited viral replication to a higher extent when compared with the phytochemicals used individually, thus demonstrating a synergistic effect [41] . ladanein, a flavone isolated from marrubium peregrinum l. (lamiaceae), exploited antiviral effects through the inhibition of the post-attachment entry step of hcv virions, with an ic 50 of 2.5 µm. ladanein, in combination with cyclosporine, showed a remarkable synergistic antiviral effect against various hcv genotypes [30] . the natural extracts from nymphaea alba l. (nymphaeaceae), containing iso-quercetin, hyperoside, quercetin, reynoutrin, apigenin and isokaempferide, showed anti-hcv activity, suppressing hcv ns3 gene expression in the transfected huh-7 cell line and inhibiting the viral genotype 1a replication. furthermore, the combination of nymphaea alba l. extract with the conventional drug boceprevir displayed synergistic effects for inhibition of hcv replication in a docking bioinformatics model [28] . an antiviral role of phytochemicals was also linked to the receptors recognized by viruses for their endocytosis, such as the membrane receptor ntcp. this protein has a mass of 56 kda and is involved in the transport of bile salt molecules, steroid hormones, thyroid hormones and xenobiotics. ntcp is required for the entry in hepatocytes of both hbv and human hepatitis d virus (hdv). in fact, the virus-receptor interaction leads to the clathrin-dependent endocytosis of hbv virions, which can replicate inside the host cells [26] . egcg, used at the dose of 50 µm, induced clathrin-dependent endocytosis of ntcp from the plasma membrane, leading to its degradation and inhibiting the entry of hbv virus into immortalized human primary hepatocytes (figure 3 ). two hiv enzymes address pivotal roles in virus replication and virion production: hiv reverse transcriptase and the homodimer of hiv protease (figure 4 ). hiv reverse transcriptase is an rna-dependent dna polymerase (rddp) and builds ssdna based on an rna template in its polymerase active site; the enzyme also cleaves the original rna template into pieces through its nuclease active site and finally it polymerizes a second dna strand to form the final dsdna, which is integrated in the host cell genome. interestingly, it was shown that egcg suppressed hiv-1 infection in hela-cd4-ltr-β-gal cells, with a ec 50 of 1.6 µm, by acting as a non-nucleoside hiv reverse transcriptase inhibitor [43] . furthermore, it was demonstrated that the flavonoids myricetin-3-rhamnoside and myricetin-3-(6-rhamnosylgalactoside) possessed antiviral activity in vitro against hiv with ec 50 of 120 µm and 45 µm, respectively [31] . this antiviral effect was exerted through the inhibition of hiv reverse transcriptase with ic 50 of 10.6 µm for myricetin-3-rhamnoside and of 13.8 µm for myricetin-3-(6-rhamnosylgalactoside) [31] . hiv protease is a retroviral aspartyl protease, which cleaves newly synthesized viral polyproteins (namely gag-pol) at nine cleavage sites to create the mature protein components of the virion. mature hiv-1 protease exists as a 22 kda homodimer, each one containing an asp25 amino acid, which acts as the catalytic residues are able to hydrolyze peptide bonds on the gag-pol polyproteins into fully functional viral proteins, like reverse transcriptase and integrase. kehinde et al. (2019) [27] showed that the phytochemicals kaempferol-7-o-glucoside and egcg were able to interact with hiv-1 protease, showing pronounced structural evidence as potential hiv-1 protease inhibitors ( figure 4) . interestingly, phytochemicals can also be used to reduce the extrusion of antiviral drugs from infected cells. in fact, apigenin, fisetin and luteolin were able to slow down the elimination of the antiviral drugs atazanavir, lopinavir, darunavir and saquinavir, which target the viral protease of hiv-1 [27] . dependent dna polymerase (rddp) and builds ssdna based on an rna template in its polymerase active site; the enzyme also cleaves the original rna template into pieces through its nuclease active site and finally it polymerizes a second dna strand to form the final dsdna, which is integrated in the host cell genome. interestingly, it was shown that egcg suppressed hiv-1 infection in hela-cd4-ltr-β-gal cells, with a ec50 of 1.6 µm, by acting as a non-nucleoside hiv reverse transcriptase inhibitor [43] . furthermore, it was demonstrated that the flavonoids myricetin-3-rhamnoside and myricetin-3-(6-rhamnosylgalactoside) possessed antiviral activity in vitro against hiv with ec50 of 120 µm and 45 µm, respectively [31] . this antiviral effect was exerted through the inhibition of hiv reverse transcriptase with ic50 of 10.6 µm for myricetin-3-rhamnoside and of 13.8 µm for myricetin-3-(6-rhamnosylgalactoside) [31] . hiv protease is a retroviral aspartyl protease, which cleaves newly synthesized viral polyproteins (namely gag-pol) at nine cleavage sites to create the mature protein components of the antiviral activity of flavonoids may be also due to the modulation of host cell enzymes used by the virus to take advantage for infection, such as the enzymes with a pivotal role in the production of radical oxygen species (ros), utilized to increase the number of virions. regarding hsv-1, which persists in the host in sensory neurons in latency, the enzyme nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox) family is a useful source of ros, which can be used to reactivate the viral infection under oxidative stress conditions [52, 53] . interestingly, delphinidin-3-rutinoside obtained from extracts of solanum melongena l. (solanaceae), inhibited hsv-1 replication through the reduction of nox4 protein levels, when added for 24 h after viral adsorption on vero cells [24] . the extract obtained from veronica persica poir (plantaginaceae) possessed a dose-dependent inhibitory activity against the herpes simplex virus strains hsv-1 and hsv-2 and a prominent synergistic activity in combination with acyclovir in anti-hsv therapy, exerted through a reduction of the percentage of plaque numbers of both hsv-1 and hsv-2 in the infected cero cells [54] . the natural extract of disticella elongata (bignoniaceae) exhibited antiviral effects against denv-2 virus and this effect was mainly due to pectolinarin and acacetin-7-o-rutinoside [18] . when the two flavonoids pectolinarin and acacetin-7-o-rutinoside were used in combination, the antiviral effect was eight times more potent against denv-2 virus (with ec 50 = 18.3 ± 2.6 µm) than the flavonoid pectolinarin used alone (with ec 50 = 138.8 ± 6.1 µm). the selectivity index of the combination (si = 45) was remarkably higher than the si of the isolated pectolinarin (si = 4.6), indicating that the phytochemical mixture specifically inhibited viral growth, with negligible effects on the vitality of the cells infected by denv-2 virus. the ethanol extract obtained from the leaves of disticella elongata showed antioxidant activities; therefore, it could detoxify cell damaging free radicals present in denv-2 viral infections [18] . the low water solubility and low bioavailability of natural flavonoids represent the major limit for their use in the nutraceutical sector. many delivery strategies have been used by researchers for increasing flavonoid bioavailability following oral consumption [16] , including: micelles, nanoparticles, microspheres, crystals, dendrimers, the self-micro-emulsifying drug delivery system (smdds) and the self-nanoemulsifying drug delivery systems (snedds), as recently reviewed [17, 55] . for instance, it was shown that the catechin and egcg-loaded chitosan nanoparticles led to a higher rate of intestinal absorption of the two phytochemicals [56] . interestingly, in chitosan particles the flavonoids maintain their antioxidant activity and can exploit their antioxidant effects in the blood stream [57] . the loading of myricetin into a cationic polymeric nanoparticle carrier with a cationic corona and hydrophobic core was investigated in order to improve the clinical relevance of this natural flavonoid by increasing its solubility [58] . smdds has been used to overcome the problem of low bioavailability of hydrophobic molecules as, in the intestinal lumen, the oil-in-water microemulsions containing phytochemicals may be efficiently formed with a consequent increase of the intestinal absorption of the flavonoid [59] . interestingly, puerarin, an isoflavone isolated from the root of pueraria lobata, exhibited 2.6 fold higher bioavailability when prepared using the smdds technique [34] . furthermore, the synthesis of silver nanoparticles linked with phytochemicals and their use for antimicrobial and antiviral treatments have been described, highlighting the various molecular mechanisms that lead to the phytochemical-mediated inhibition of viral replication [60] . poly (d,l-lactide) (pla) nanoparticles and polymeric micelles contributed to a more sustainable and efficient release of flavonoids characterized by a poor bioavailability, like quercetin and apigenin [61, 62] . quercetin was successfully encapsulated on the most uniformly distributed type of pla-4 nanoparticle, synthesized using lonicera japonica leaf extract, showing that these nanoparticles allowed a slow release of quercetin [63] . this nanoparticle approach paves the way for encapsulating drugs, small molecules, nutraceuticals and other bioactive ingredients to obtain safer cellular uptake, improved biodistribution, specific targeted delivery and enhancement of the therapeutic antiviral efficacy of encapsulated drugs and phytochemicals [64] . the increase in antiviral efficacy and bioavailability of flavonoids may be attained through their encapsulation into red blood cells (rbcs), as has occurred for other type of antiviral drugs and molecules, such as fludarabine (figure 4 ) [65] , vincristine and vinblastine [66, 67] . the idea of using rbcs as delivery system for flavonoids takes its advantage from the favorable characteristics of these cells. they have a long life-span of about 120 days and have a widespread circulation throughout the body, and hence can be used as drug reservoirs, enabling them to facilitate sustained drug release. moreover, rbcs protect encapsulated drugs and molecules from degradation; they are completely biodegradable and show no or only minor immunogenic responses. interestingly, the rbcs were used to determine cellular antioxidant activity of some flavonoids, specifically vitexin, vitexin-2-o-xyloside and vitexin-2-o-rhamnoside, with the aim of predicting their bioavailability [68] . moreover, it was demonstrated that flavonoids could have beneficial effects in preventing oxidative damage of erythrocyte membrane [69, 70] . some authors have also evidenced the possibility that human rbcs play a pivotal role in the distribution and bioavailability of circulating flavonoids such as quercetin [71] . furthermore, it was shown that drug-loaded rbcs can be modified in order to increase their macrophage-mediated phagocytosis by inducing band 3 clustering and opsonization through the complementary and autologous iggs [72] . in future perspective, this approach could be considered in order to possibly improve the antiviral activity of some flavonoids, like baicalin, that was able, like fludarabine [65] , to act against hiv-1 chronic infection of human monocytes and macrophages, inhibiting the fusion of hiv virus envelope proteins with these cells [73] . although polymeric nanoparticles, liposomes, dendrimers, micelles and inorganic nanoparticles have been widely accepted as drug delivery systems, they show toxicity and a short lifetime, thus making them relatively disadvantageous when compared with natural cell carriers, such as rbcs. for this reason, some authors in recent years have tried to mimic the erythrocyte cell membrane to produce biocompatible nanocarriers in order to decrease their toxicity and to prolong their survival in blood circulation [74] [75] [76] . rbcs, which are biodegradable and non-immunogenic, can be used as a valuable carrier system with a lifespan that is remarkably prolonged and controllable when compared to synthetic carriers. several approaches have been developed to load peptides, drugs and molecules into rbcs or to attach these agents onto rbcs' outer membrane surface by either chemical or physical methods [77] and the possibility of loading drugs into autologous rbcs prior to their transfusion into patients has been studied in small animal models and primates, as well as in clinical studies of human patients [78] [79] [80] [81] . the topic of phytochemical encapsulation in rbcs remains open for further studies, but we believe that flavonoid derivatives and nanoparticles able to bind flavonoids could be successfully considered for this application in the near future. diet and life-style play important roles in the defense against the attacks of viruses. the relationship between diet and the immune system involves the microbiota, i.e., the ecological community of commensals and potentially pathogenic microbes and symbionts that live in the gut [82] . the diet modulates the microbiota composition, leading to an increase or a decrease in immune defenses [82] . the mediterranean diet (and in general diets rich in fruits, vegetables and herbs) maintains gut microbiota homeostasis and provides protection against microbes and viruses [83] . the cross-talk between microbiota and immunity is supported by the aryl hydrocarbon receptor (ahr), which is ubiquitous, but mainly present in the cytoplasm of immune cells [84] . it was demonstrated that ahr binds different ligands, namely metabolites, pollutants and pathogenic molecules, and after this interaction it translocates into the nucleus, where it induces specific transcription programs and modulates the defensive functions of both t and b cells, dendritic cells and monocytes [84] . interestingly, it was shown that flavonoids can bind ahr [84] . on this basis, people eating vegetables, all of which contain flavonoids to a different extent, would strengthen their immune system [83] . this strengthening is a general effect that occurs with many nutrients, but there are more specific antiviral effects attributable to the flavonoids treated in this review. flavonoids, like apigenin, vitexin, quercetin, rutin and naringenin, show a wide range of antiviral effects (table 2) , because they are able to target different pathways of viral infections. it is therefore possible to increase the chances of blocking viral replication using mixtures of flavonoids with synergistic antiviral effects, because of the pleiotropic effects of their combination. an important question that arises is focused on the reasonable concentration range of flavonoids that should be used for an effective antiviral therapy, which is hard to be reached by diet alone. recent experiments performed in in vivo studies demonstrated the protective efficacy of various flavonoids, tested in the range 10-50 mg/kg body weight per day, in newborn mice challenged with a lethal dose of the enterovirus 71 [85] . interestingly, apigenin (50 mg/kg), luteolin (10 mg/kg) and quercetin (10 mg/kg) conferred survival protection of 88.9%, 91.7% and 50.1%, respectively, from the lethal enterovirus 71 infection; moreover, isorhamnetin provided the highest survival protection of 100% at a dose of 10 mg/kg. the authors hypothesized that the differences in concentrations are due to different times of absorption and renal clearance of these flavonoids [85] . the flavanol isorhamnetin was studied also by dayem et al. [86] , who showed that oral administration of this flavonoid at 1 mg/kg/day for five days in mice infected with the influenza a virus significantly decreased lung virus titer by two-fold, increased the survival rate (which ranged from 70% to 80%) and decreased mice body weight loss by 25%. these authors hypothesized that the methyl group of the b ring of isorhamnetin may contribute to its strong antiviral potency against influenza a virus [86] . guo et al. [87] focused their in vivo studies on the flavone wogonin, showing that, in human hbv-transgenic mice, this flavonoid administered once a day at 7, 14 and 28 mg/kg reduced plasma hbsag level and immunohistological staining of the liver confirming the hbsag reduction exerted by wogonin [87] . the potentiality of flavonoid bioactivity in vivo depends on the extent of their absorption after ingestion and their ability for distribution in various target tissues. in this light, liu et al. [88] developed a quercetin-loaded cationic nanostructure lipid carrier (q-cnlc), which increased the in vivo bioavailability of this flavonoid. this quercetin-nanostructure complex benefits from its strong interactions with negatively charged intestinal mucosa, which could increase its residence in the gastrointestinal tract. moreover, the authors showed an entrapment efficiency of quercetin of 89.3% and a slower release of this flavonoid from the q-clnc, when compared with the release profile of a simple quercetin solution, indicating that q-clnc exhibited a sustained and controlled release of this flavanol, in order to maintain its effective therapeutic concentration [88] . in addition, the same authors performed in vivo tissue distribution studies in c57bl/6j mice, comparing treatment with 25 mg/kg of orally administered quercetin solution with the administration of 25 mg/kg of q-cnlc, showing that the relative quercetin uptake from q-cnlc was 1.57 fold higher in lungs, 1.51 fold higher in liver and 1.68 fold higher in kidneys. on the contrary, the relative quercetin uptake from q-cnlc was lower in spleen, heart and brain, when compared with the quercetin solution [88] . these results indicate that the most suitable delivery strategy should be chosen in order to target a specific organ with a particular flavonoid-nanostructure complex for future clinical applications. furthermore, the safety of the used phytochemical should also be considered, as has already been done for hydroxytyrosol, which is quickly absorbed and eliminated by the kidneys in either free or conjugated forms. hydroxytyrosol has been considered safe at 50 mg/kg/day by the european food safety authority (efsa) panel [89] . in this light, we think that, for a 70 kg person, a flavonoid range between 0.5-3.0 g/day should be proposed. these values are close to the daily polyphenol intake in humans, calculated in various diet surveys [90] , such as the supplementation en vitamines et mineraux antioxydants (su.vi.max) study, which ranked the polyphenol intake at 1.19 ± 0.51 g/day [91] . accordingly, in another dietary intervention trial aimed at improving cognition in older adults, a total of 1.04 g/day of flavonoids was applied [92] . indeed, based on these results, we think that 0.5-3.0 g/day of flavonoids could be a reasonable concentration range in order to start preliminary experiments, focused on assessing the in vivo antiviral effects of flavonoids. concerning the combination of flavonoids in an antiviral cocktail, each phytochemical may be used initially at a concentration of about 0.5 g/day with the aim of reaching an intake of 3 g/day of antiviral flavonoids. in the case of antiviral synergistic effects [5, 6] or increase of the absorption of one specific flavonoid exerted by other phytochemicals [93] , the individual flavonoid concentrations can be modulated, according to the extent of the effect. it should also be emphasized that a significant antiviral effect is linked to the type of flavonoid mixture, the delivery system used, the pharmacokinetic pattern, the number of targets involved and the number of required daily doses. once these aspects have been defined, the more suitable regimen of administration consists of starting with the lowest effective concentration for a fixed time and proceeding with increasing doses of the antiviral flavonoids. monitoring the markers of antiviral efficacy and any side effects should also be considered in order to evaluate the risks-benefits pattern. overall, our review shows that many flavonoids exhibit antiviral activity and could offer a promising alternative for prevention of and therapy for viral infections. flavonoids are present in many vegetables and the first protection for the immune system resides in the ability to seek foods rich in bioactive nutrients. education programs for a healthy diet should be implemented during the outbreaks of viral infections [94] . in fact, a diet rich in vegetables activates the ahr in the gut for maintenance of microbiota homeostasis, which in turn regulates the immune system. in the critical periods of viral infections, oral dietary supplementation with nutraceutical preparations based on combinations of flavonoids can be useful in order to inhibit different steps of the viral infective cycle. molecular mechanisms underlying the antiviral effects of flavonoids, herein described, mainly focus on the inhibition of viral enzyme activities: neuraminidases, dna/rna polymerases and proteases. therefore, a cocktail of flavonoids, selected for their efficacy in the inhibition of different viral enzymes, could be associated with elevated immune response and offer a promising option for antiviral therapies. this option acquires great importance considering that the viral genome frequently mutates, due to the lack of proof-reading activity of most of the viral polymerases. these mutations could hamper the efficacy of antiviral synthetic drugs. on the contrary, antiviral flavonoids, as well as the combination of synthetic antiviral drugs with flavonoids, would enhance therapeutic strategies by targeting the multiple signaling pathways involved in the viral infections [95] . the active concentration of the flavonoids should be investigated, considering the pharmacokinetic studies available in the literature and the synergistic effects of the specific flavonoid combinations [15] [16] [17] . the scarce intestinal absorption and bioavailability of flavonoids, when given through food or in pills, may be enhanced by the use of new drug delivery strategies [96] . in fact, since flavonoids have some drawbacks after oral administration such as low stability, bioavailability and bio-efficacy, researchers are developing biocompatible nanomaterial synthesis as novel delivery systems (including nanospheres, nano-capsules, micro and nano-emulsions, micelles, solid lipid nanoparticles and capsules), for overcoming the delivery challenges of flavonoids in the biomedical sector. phytochemical-nanomaterial complexes can represent innovative drug delivery strategies (alongside those already known) for new antiviral therapies against the seven baltimore virus classes [97] . interestingly, three patents regarding the antiviral effects of flavonoids (us 7,998,937; ep1245230; us 6,399,654) have been already assigned to the korea research institute of bioscience and biotechnology and advanced life sciences inc. [35] . however, an important step that must be achieved is to obtain the authorization of novel food including flavonoids with antiviral properties, following regulation eu 2015/2283 [98] . nowadays, this authorization has been given only to hydroxytyrosol [89] and few other nutrients. if the antiviral efficacy and safety of flavonoids and their mixtures can be clearly demonstrated in vivo, it would be possible to obtain european food safety authority (efsa) authorization of novel foods, in order to provide new natural tools for preventing and facing outbreaks of viral infections. indeed, when flavonoids are administered through nano-sized delivery systems, they show increased stability and bioavailability with an enhanced and prolonged activity. however, the in vivo behavior and the antiviral actions of these nano-delivery systems are still under experimental evaluation. interferon regulatory factor 3 irf7 interferon polyphenols and antioxidant capacity of vegetables under fresh and frozen conditions antioxidant capacity of extra-virgin olive oils antioxidant capacity of vegetables, spices and dressings relevant to nutrition effect of flavonoids on human health: old subjects but new challenges vitexin 2-o-xyloside, raphasatin and (-) eepigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells betalains increase vitexin-2-o-xyloside cytotoxicity in caco-2 cancer cells antiproliferative activity of vitexin-2-o-xyloside and avenanthramides on caco-2 and hepg2 cancer cells occurs through apoptosis induction and reduction of pro-survival mechanisms natural and synthetic avenathramides activate caspases 2,8,3 and downregulate htert, mdr1 and cox-2 genes in caco-2 and hep3b cancer cells a combination of moringin and avenanthramide 2f inhibits the proliferation of hep3b liver cancer cells inducing intrinsic and extrinsic apoptosis flavonoids from beta vulgaris cicla and betalains from beta vulgais rubra: antioxidant, anticancer, antiinflammatory activities-a review characterization and biological activity of the main flaonoids from swiss chard (beta vulgaris subspecies cycla) oseltamivir resistance-disabling our influenza defenses twenty years of research into medicinal plants: results and perspectives combination of western medicine and chinese traditional patent medicine in treating a family case of covid-19 in wuhan. front antiviral phytochemicals: an overview flavonoids: promising natural compounds against viral infections antiviral effects of phytochemicals from medicinal plants: applications and drug delivery strategies antiviral activity of disticella elongata (vahl) urb. (bignoniaceae), a potentially useful source of anti-dengue drugs from the state of minas gerais identification and evaluation of anti hepatitis c virus phytochemicals from eclipta alba an evaluation of the inhibitory effects against rotavirus infection of edible plant extracts qualitative and quantitative analysis for the chemical constituents of tetrastigma hemsleyanum diels et gilg using ultra-high performance liquid chromatography/hybrid quadrupole-orbitrap mass spectrometry and preliminary screening for anti-influenza virus components anti-h1n1 virus, cytotoxic and nrf2 activation activities of chemical constituents from scutellaria baicalensis molecular docking based screening of plant flavonoids as dengue ns1 inhibitors a polyphenol rich extract from solanum melongena l. dr2 peel exhibits antioxidant properties and anti-herpes simplex virus type 1 pharmacoinformatics approach for investigation of alternative potential hepatitis c virus nonstructural protein 5b inhibitors epigallocatechin-3-gallate inhibits entry of hepatitis b virus into hepatocytes the pharmacokinetic properties of hiv-1 protease inhibitors: a computational perspective on herbal phytochemicals anti-hepatitis c virus activity and synergistic effect of nymphaea alba extracts and bioactive constituents in liver infected cells moringa oleifera: a food plant with multiple medicinal uses a plant-derived flavonoid inhibits entry of all hcv genotypes into human hepatocytes the role of the glycosyl moiety of myricetin derivatives in anti-hiv-1 activity in vitro antiviral activity of polymethoxylated flavones from guangchenpi, the edible and medicinal pericarps of citrus reticulata "chachi antiviral activity of pinocembrin against zika virus replication characterization and evaluation of self-microemulsifying sustained-release pellet formulation of puerarin for oral delivery phytochemicals as antiviral agents: recent updates suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns3 protease activity inhibitory effects of quercetin 3-rhamnoside on influenza a virus replication anti-influenza a virus mechanism of three representative compounds from flos trollii via tlrs signaling pathways inhibition of rna binding to hepatitis c virus rna-dependent rna polymerase: a new mechanism for antiviral intervention therapeutic potential of taraxacum officinale against hcv ns5b polymerase: in-vitro and in silico study divergent antiviral effects of bioflavonoids on the hepatitis c virus life cycle breaking down the barriers to a natural antiviral agent: antiviral activity and molecular docking of erythrina speciosa extract, fractions, and the major compound epigallocatechin gallate inhibits the hiv reverse transcription step baicalin, a metabolite of baicalein with antiviral activity against dengue virus quercetin-3-rhamnoside exerts antiinfluenza a virus activity in mice virology blog: about viruses and viral disease inhibition of sialidase activity as a therapeutic approach anti-influenza activity of marchantins, macrocyclic bisbibenxyls contained in liverworts a sequence data resource for hepatitis b virus corriere nazionale: i 5 virus che spaventano la scienza infectious agents and neurodegeneration influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by nox4-derived ros antiviral activity of veronica persica poir. on herpes virus infection nanoparticulate delivery systems for antiviral drugs chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (+)-catechin and (-)-epigallocatechin gallate orac of chitosan and its derivatives. food hydrocoll electrostatic interactions enable nanoparticle delivery of the flavonoid myricetin development of self-microemulsifying drug delivery system (smedds) for oral bioavailability enhancement of simvastatin in beagle dogs phytochemical-assisted synthetic approaches for silver nanoparticles antimicrobial applications: a review development of biodegradble nanoparticles for delivery of quercetin preparation and in vitro evaluation of apigenin-loaded polymeric micelles plant extract synthesized pla nanoparticles for controlled and sustained release of quercetin: a green approach domínguez-perles, r. nanoparticles and controlled delivery for bioactive compounds: outlining challenges for new "smart-foods" for health drug-loaded red blood cell-mediated clearance of hiv-1 macrophage reservoir by selective inhibition of stat1 expression the terpene-indole alkaloids loaded erythrocytes as a drug carrier: design and assessment study of desorbtion and exemption of terpeno-indole alkaloids of vinkristin and vinblastin from erythrocitary cell carriers the cellular antioxidant activity in red blood cells (caa-rbc): a new approach to bioavailability and synergy of phytochemicals and botanical extracts protective effect of flavonoids against red blood cell hemolysis by free radicals inhibition of free radical initiated peroxidation of human erythrocyte ghosts by flavonols and their glycosides human red blood cell sas a natural flavonoid reservoir drug delivery through phagocytosis of red blood cells flavonoids as antiviral agents for enterovirus a71 (ev-a71) red blood cell membrane-camouflaged nanoparticles: a novel drug delivery system for antitumor application red blood cell-mimicking synthetic biomaterial particles using mechanobiological mimicry of red blood cells to extend circulation times of hydrogel microparticles engineering erythrocytes for the modulation of drugs' and contrasting agents' pharmacokinetics and biodistribution red blood cell-based delivery of drugs and nanomaterials for therapeutic and diagnostic applications delivery of drugs bound to erythrocytes: new avenues for an old intravascular carrier observation of erythrocyte dynamics in the retinal capillaries and choriocapillaris using icg-loaded erythrocyte ghost cells erythrocytes as carriers for drug delivery in blood transfusion and beyond the potential impact of gut microbiota on your health: current status and future challenges. asian pac effect of fruit and vegetable consumption on immune function in older people: a randomized controlled trial polyphenols and tryptophan metabolites activate the aryl hydrocarbon receptor in an in vitro model of colonic fermentation antiviral efficacy of flavonoids against enterovirus 71 infection in vitro and in newborn mice antiviral effect of methylated flavonol isorhamnetin against influenza anti-hepatitis b virus activity of wogonin in vitro and in vivo characterization and biodistribution in vivo of quercetin-loaded cationic nanostructured lipid carriers ec regulation 258/97. safety of hydroxytyrosol as a novel food pursuant to regulation (ec) no 258/97 systematic review on polyphenol intake and health outcomes: is there sufficient evidence to define a health-promoting polyphenol-rich dietary pattern? nutrients dietary intake of 337 polyphenols in french adults enhancing dentate gyrus function with dietary flavanols improves cognition in older adults enhancement of flavonoid ability to cross the blood-brain barrier of rats by co-administration with α-tocopherol the food systems in the era of the coronavirus (covid-19) pandemic crisis. foods viral mutation rates flavonoids loaded in nanocarriers: an opportunity to increase oral bioavailability and bioefficacy nanomaterials designed for antiviral drug delivery transport across biological barriers of the european parliament and of the council and repealing regulation (ec) no 258/97 of the european parliament and of the council and commission regulation (ec) no 1852 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors want to thank university of urbino carlo bo for financial support. the authors declare no conflict of interest. the funders had no role in the writing of the manuscript or in the decision to publish this manuscript. key: cord-003993-3bozjfv7 authors: cagliani, rachele; forni, diego; sironi, manuela title: mode and tempo of human hepatitis virus evolution date: 2019-10-25 journal: comput struct biotechnol j doi: 10.1016/j.csbj.2019.09.007 sha: doc_id: 3993 cord_uid: 3bozjfv7 human viral hepatitis, a major cause of morbidity and mortality worldwide, is caused by highly diverse viruses with different genetic, ecological, and pathogenetic features. technological advances that allow throughput sequencing of viral genomes, as well as the development of computational tools to analyze such genome data, have largely expanded our knowledge on the host range and evolutionary history of human hepatitis viruses. thus, with the exclusion of hepatitis d virus, close or distant relatives of these human pathogens were identified in a number of domestic and wild mammals. also, sequences of human viral strains isolated from different geographic locations and over different time-spans have allowed the application of phylogeographic and molecular dating approaches to large viral phylogenies. in this review, we summarize the most recent insights into our understanding of the evolutionary events and ecological contexts that determined the origin and spread of human hepatitis viruses. human hepatitis viruses are extremely diverse and consequently belong to different viral families (table 1 ). in recent years, the availability of high-throughput technologies has revealed that relatives of human hepatitis viruses can be found in a wide variety of animals. this finding, as well as the increasing availability of the genome sequences of human-infecting viruses sampled across different geographic areas, has largely expanded our knowledge about the genetic diversity and evolutionary origin of these human pathogens. in this review, we thus focus on the latest insights into the possible events and ecological contexts that determined the origin and spread of human hepatitis viruses. a short presentation of the most widely used approaches to estimate the ages of viral lineages is also provided to contextualize recent research efforts on these viruses. molecular dating analyses using virus genetic data can be particularly informative due to the rapid rate of evolution of many viral species. by converting genetic differences among sequences into time units, molecular dating provides information on the timing of viral spread or emergence. most molecular dating approaches are based on maximum-likelihood or bayesian phylogenetic frameworks [5, 6] , and they usually exploit two strategies: molecular clock calibration using the sampling dates of the viral sequences (tip dating) and/or calibration using information on some internal nodes of the phylogeny. tip dating is well-suited to study relatively recent events (e.g., epidemics or intra-host evolution), but requires that a temporal signal is present in the dataset (i.e., that the sequences have accumulated a measurable amount of change between sampling times) [6] . calibration using internal nodes can in principle allow to dig deeper into the past, but requires some a priori knowledge about the virus evolutionary history (e.g., host-virus co-evolution, paleontological information). the widespread use of molecular dating has however revealed that the relationship between genetic divergence and time is complex, as evolutionary rates tend to vary with the time frame of measurement. in particular, high evolutionary rates are observed in the short term, whereas low rates are inferred in long time span studies [7] [8] [9] . this pattern was observed for many viral lineages and is sometimes referred to as the time-dependent rate phenomenon (tdrp) [10] . failure to account for the tdrp can potentially lead to erroneous molecular dating results [10, 11] . the tdrp reflects mutation rate in very short timescales and substitution rate in very long timescales, during which transient deleterious mutations are removed by the action of natural selection, leading to lower rate estimates [12] . another factor most likely contributing to the tdrp is the saturation of nucleotide substitutions, which is extremely rapid in viral genomes, especially when the polymerase is error-prone [12] . thus, recent analyses indicated that, for all baltimore classification groups, viral evolutionary rates tend to decrease continuously with the timescale of measurement [10, 11] . because the rate of decay is consistent with a power law relationship between substitution rate and sampling timescale, a model using a simple regression was at first proposed to estimate the tdrp effect on viral phylogenies [10] . very recently, this approach was implemented in a bayesian statistical framework, in which evolutionary rates can vary among different time epochs [13] . before the introduction of such an approach, effective attempts to correct for the tdrp were performed by the use of nucleotide substitution models that allow site-and branch-specific variation in selective pressure (selectioninformed models). these models, which were applied to analyze the ancient evolution of some viral lineages, at least partially correct for the effects of both purifying selection and substitution saturation in branch length estimation [14] [15] [16] . hav is mainly transmitted via the faecal-oral route through exposure to contaminated food or water, or through direct contact with infected people. hav is a single-strand, positive rna virus with a genome of approximately 7.5 kb in length ( table 1 ). the hav genome contains a single orf flanked by a relatively long 5 0 utr and a 3 0 utr. the 5 0 utr harbors an internal ribosome entry site that directs the cap-independent translation of hav proteins. the orf encodes a polyprotein processed in 11 mature proteins: 5 structural proteins involved in capsid formation (vp4, vp2, vp3, vp1, and px, deriving from p1 segment) and 6 nonstructural proteins with a role in rna genome amplification (2b, 2c, 3a, 3b, 3cpro, and 3dpol, deriving from p2 and p3 segments) [4, 17] . based on genomic structure, hav belongs to the family picornaviridae within the genus hepatovirus. nevertheless, many characteristics distinguish hav (and hepatoviruses in general) from other picornaviridae family members. some peculiar features include the primary tropism for hepatocytes, the ability to shed as nonenveloped virus in feces and as enveloped particles in blood, as well as some genomic features such as low g/c ratio, low cpg levels, and strong codon bias [18, 19] . hav was identified as the etiologic agent of hepatitis a by feinstone and colleagues [20] in 1973. unlike hbv and hcv, which establish chronic infections in humans, hav infection is usually acute and generates lifelong immunity. this condition is able to determine the disappearance of the virus in small and isolated populations [21, 22] and did not probably favor its maintenance in early human communities. it is thus legitimate to wonder how hav survived and evolved during early human history, a question that remains presently unanswered. for a long time, it was thought that hepatoviruses were restricted to humans and non-human primates (nhps), with genetically distinct variants classified as six main different genotypes [23] : three isolated from humans (hav, genotype i-iii) and subclassified in 6 subgenotypes (ia, ib, iia, iib, iiia, iiib) and three of simian origin (shav, genotype iv-vi). however, despite genetic heterogeneity, hav viruses belong to a single common serotype. in recent years, the advent of new sequencing approaches has led to an exponential increase in the identification of new viral speacute cies, including highly diverse non-primate hepatoviruses. several hav-related viruses were identified in different mammalian orders. in particular, a number of hav-like viruses were recovered in placental mammals, mainly in bats and rodents, but also in tree shrews, hedgehogs, seals and chinese woodchucks [24] [25] [26] . recently, de oliveira carneiro and colleagues [27] identified a novel hav-related virus in didelphis aurita, a brazilian common opossum, further extending the host range of mammal-infecting hepatoviruses. moreover, viruses related to mammalian hepatoviruses were detected in reptiles, amphibians, and fish [28] . these advances allowed new insights into the evolutionary history of hav. the phylogenetic relationships among hepatovirus that infect small mammals only partially reflects those among their hosts, suggesting multiple, non-recent cross-species and cross-order host switches during hepatovirus evolution [24] . this observation is supported by recombination events observed in hepatoviruses that have been identified in genetically and geographically distant hosts [29] . these cross-species transmission events also involved the opossum hepatovirus, which most likely originated from an ancestral host switch from rodents into marsupials [27] . conversely, hepatovirus phylogenies suggest no host switch involving a primate donor. this evidence, the absence of recombination events between havs and non-primate hepatoviruses, as well as the observation that primate hepatoviruses form, regardless of the genomic region considered, a monophyletic group in the topology of hepatovirus phylogenies, support the hypothesis that humans and nhp have acquired hepatoviruses from other animal reservoirs relatively recently [24, 29] . however, if and when this hypothetical host-jump occurred into primates remains to be clarified. phylogenetic and ancestral state reconstruction suggested a likely cricetid rodent origin for primate havs and marsupial hepatoviruses, whereas a laurasiatherian host origin was proposed for all mammalian hepatoviruses [24, 27] (fig. 1) . in this scenario, the evolutionary history of hepatoviruses is evocative of that of hantaviruses, as the origin of mammalian hantaviruses is traced back to bats and insectivores [30] . thus, the supposed origin of hepatoviruses in insectivorous laurasiatherian mammals, as well as the preservation of some structural and functional characteristics similar to present-day insect picorna-like viruses (dicistroviridae) [31] led drexler and colleagues to hypothesize a more ancient evolutionary origin of havs, with an ancestry in a primordial insectborne virus [24] . in conclusion, hav emergence in humans likely represents a relatively recent evolutionary event, probably of zoonotic origin. nonetheless, the ancestor of human hepatoviruses has yet to be identified. the characterization of other hepatoviruses in primates, and mammals in general, will be instrumental to the identification of the hav ancestors and will clarify the evolutionary history of hepatoviruses. hbv was the first human hepatitis virus to be isolated and identified in 1970 [32] . hbv transmission varies depending on the prevalence of infection. in areas with a low prevalence (<2%), the most common mode of transmission is through infected blood or high-risk behaviors (e.g. unprotected sex or injecting drug use). in high-and intermediate-prevalence areas, hbv is commonly spread through perinatal and horizontal (especially among children) routes [33] . hbv belongs to the hepadnaviridae family, which comprises two genera: orthohepadnavirus (mammal-infecting viruses) and avihepadnavirus (bird-infecting viruses) ( fig. 2a) . its genome, a partially double-stranded circular dna of about 3.2 kb (table 1) , is composed of four overlapping frameshifted open reading frames (orfs) [34] . viral replication is carried out by a reverse transcriptase with no proofreading ability and considerable variability exists among strains. thus, at least nine genotypes (a-i) plus a tentative one (j), with a heterogeneous global distribution, have been described to date [35] [36] [37] [38] [39] (fig. 2b) . despite its worldwide diffusion and the accumulation of detailed knowledge on the associated pathologies, the origin and evolutionary history of hbv are still debated [34, 40] . hepadnaviruses were detected in several mammals, including nhps, rodents and bats, birds, fish, and reptiles [41, 42] (fig. 2a) . recently, lauber et al. discovered a family of fish viruses with genomic features similar to those of hbv, dating the origin of the hepdnaviridae family to at least 300 million years ago [43] ; this finding, together with the discovery of endogenous hepadnavirus elements integrated in the genome of birds and reptiles [44] [45] [46] [47] , suggests a long and complex relationship between this viral family and its hosts. concerning hbv, different theories were proposed to explain its origin, but all of them have some sort of limitation. hbv was initially thought to have emerged quite recently in the new world from genotypes f/h infecting amerindians [48, 49] ( fig. 2a and b). however, the discovery of an ancient strain in a 16th century asian mummy, as well as the worldwide diffusion of hepadnaviruses in nhps, questioned this hypothesis [50] (fig. 2a ). an alternative theory suggests that hbv followed the out-of-africa migration of modern humans, which occurred approximately 60,000 years ago [51] [52] [53] . in particular, paraskevis et al. found a good correspondence between the demographic histories of hbv and those of human populations [53] . these authors also showed that the substantial divergence of the f and h genotypes ( fig. 2a) , a major evidence in favor of the new world origin hypothesis [49] , was probably due to positive selection acting on those branches [54] . nonetheless, the extensive application of ancient dna sequencing revealed a more complex scenario. in fact, two european neolithic hbv genomes did not cluster with any extant human strain in the phylogenetic tree, but did cluster with nhp viruses [55] (fig. 2a ). other authors [9] , who sequenced 12 ancient hbv genomes of different ages (800-4500 years old), obtained a similar result, with the ancient strains clustering with known modern genotypes or forming new clades ( fig. 2a) . this implies that some hbv lineages of the past went extinct ( fig. 2a) . moreover, muhlemann and coworkers showed that the geographic distribution of ancient samples does not match the modern genotype distribution [9] . they thus suggested that early evolutionary scenarios can be concealed and overwritten by more recent migratory events [9] . a third hypothesis for the origin of hbv posits that hepadnaviruses co-speciated with their primate hosts in the new world and in the old world. thus, multiple zoonotic transmissions would have originated hbv genotypes found in humans [56] . this scenario is supported by the diffusion of hepadnaviruses in diverse primate species and by the inferred divergence time of the orthohepadnavirus and avihepadnavirus genera, that is very similar to that of their host classes [56] . however, the recent identification of a novel hepadnavirus in capuchin monkeys confirmed that new world monkeys are infected by viruses that are very distantly related to hbv ( fig. 2a) , indicating that they do not represent the direct ancestors of genotypes h and f [41] . instead, evolutionary analyses with human and nhp viral strains placed the origin of hbv ancestors in hominoid old world primates, preceding the formation of the human lineage [41] . in summary, although considerable progress was achieved in recent years, a high level of uncertainty concerning the ultimate origin of hbv still exists. the particular organization of the viral genome (i.e. overlapping reading frames in a short genome) limits the variability of most of nucleotide positions (i.e. to avoid the introduction of nonsynonymous mutations) and results in a relatively slow mutation rate. this characteristic, along with the action of natural selection on particular genotypes [54] , as well as the adaptation to different human populations [34] , contributes to hbv variability and complicates inferences about its origin. finally, different studies [9, 50, 57] have shown that, as for other viruses (see section 2), hbv substitution rates are affected by viral sampling time frames. indeed, the evolutionary rates generated using information from ancient hbv genomes were shown to fit well with the tdrp regression line calculated for baltimore group vi and vii viruses (i.e., reverse-transcribing viruses) [11] . crucially, these results indicate that, whereas tip calibration approaches have demonstrated to be useful in the reconstruction of recent epidemiological events [58] [59] [60] , limiting analysis to extant strains for the reconstruction of ancient hbv evolution can be misleading [9] and that approaches that correct for the tdrp should be envisaged. hcv is an enveloped virus belonging to the flaviviridae family (genus hepacivirus). in analogy to other members of the family, hcv has a 9.6 kb positive-stranded linear rna genome. the virus encodes a single polyprotein that is processed by cellular and viral proteases to yield at least 10 mature products. hcv was identified in 1989 by houghton and colleagues as a cause of non-a and non-b hepatitis [61] . if left untreated, hcv can persist lifelong in humans, often resulting in cirrhosis and hepatocellular carcinoma. presently, the hcv worldwide seroprevalence is estimated to be $1% [1] , with about 71 million persons living with chronic infection [1] . the hcv epidemic apparently started recently, in the 1930s-1940s, as a consequence of practices that determined parenteral or percutaneous exposure (e.g., blood transfusion, vaccination campaigns, and intravenous drug injection) [62] [63] [64] . for instance, one of the most affected countries is egypt, where the virus was most likely disseminated through nationwide vaccination programs or contaminated blood-derived products [65] . in fact, sexual or vertical transmission of hcv are relatively rare, and the overwhelming majority of infections occur via the parenteral/percutaneous route. thus, due to historical reasons and to the transmission pattern, a small number of so-called ''epidemic" hcv subtypes (1a, 1b, 3a, and 2a) account for most infections worldwide [64, 66] . hcv is, however, genetically heterogeneous and the epidemic subtypes represent a minor fraction of viral diversity. eight major hcv genotypes (hcv-1 to -8) have been described, and these are further divided into at least 90 subtypes (https://talk.ictvonline. org/ictv_wikis/flaviviridae/w/sg_flavi/56/hcv-classification) (fig. 3a) . several of these genotypes and subtypes were identified and classified only recently [67, 68] , suggesting that a considerable proportion of hcv diversity remains undescribed. moreover, a number of natural inter-genotype recombinants were reported [24, 27] was generated using phyml [136] . hepatovirus host silhouettes are colored according to taxonomic order. the human hav subgenotypes are also reported. (https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/38/ table-4-recombinant-rf-hcv-genomes). hcv genotypes display antigenic variability and viral genetic diversity is geographically structured: in sub-saharan africa and south-east asia highly divergent subtypes of the same genotype dominate transmissions across contiguous areas (fig. 3a) . these subtypes are referred to as ''endemic" [62, 64] and their presence is consistent with a long-standing association of hcv with populations living in these regions. because parenteral exposure became common only in the relatively recent past, several hypotheses were formulated to account for the maintenance of endemic hcv transmission. some authors proposed that traditional practices such as circumcision, tattooing, piercing or acupuncture facilitated and maintained hcv infection among human populations [64, 69] . others indicated that sexually transmitted infections (stis) that disrupt mucosal integrity are responsible for increased sexual hcv transmission [70] . this was indeed shown to be the case in modern high-risk populations [71] and stis have probably been common throughout human history [72] . an alternative scenario is that the bite of arthropods, especially those taking large blood [138] ). graphical representation of genotype (gt) diversity. the number of distinct recognized subtypes is represented on the y axis. circle size is proportional to the average pairwise distance between subtypes. genotype 7 is marked with an asterisk as only two subtypes are known. (b) phylogenetic tree of the rdrp domain of known hepaciviruses. the tree was obtained using raxml with 1000 bootstrap replicates [139] . nodes with support equal to or higher than 0.7 are marked with a black dot. (c) hypothetical viral phylogenies that illustrate the effect of viral lineage extinction on the evolutionary inference about the origin of hcv and ehv/chv. in the left panel, a horse-to human transmission event is hypothesized, with the following extinction of the transmitted ehv lineage. in the right panel, a reverse zoonosis introduces a hcv-like virus in horse populations; the following extinction of several ehv lineages accounts for the low genetic diversity of extant strains. meals, can mechanically transmit hcv, possibly from other animal reservoirs such as horses [73, 74] . hcv infection is in fact restricted to our species but, thanks to extensive field work, a number of hepaciviruses have been described in domestic and wild mammals, as well as in reptiles and fish [28, [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] (fig. 3b ). at present, the largest diversity of hepacivirus species seems to be hosted by rodents and bats (fig. 3b) . instead, as previously noted [75] , the lowest genetic diversity is observed for hepaciviruses that infect cattle and horses (fig. 3b) , suggesting that husbandry practices may have resulted in the artificial selection of specific viral strains or facilitated recent viral transmission from some other animal source (e.g., commensal rodents). non-human primates also host hepaciviruses, but these are highly divergent from hcv. overall, the phylogenetic relationships among hepaciviruses poorly mirror those among their hosts (fig. 3b ), suggesting several crossspecies and cross-order host switches during viral evolution. up to now, the closest relatives of hcv were identified in horses/donkeys (equine hepacivirus, ehv) and dogs (canine hepacivirus, chv) (fig. 3b) . because chv is less genetically diverse than ehv, the canine virus possibility originated as a recent cross-species transmission from horses [85] , hinting at the ability of hepacivirus to shift among genetically distant hosts. despite these advances, the events that led to the origin of hcv are still unknown. taking as a fact the relatedness of the human virus to ehv/chv, possible scenarios include that: i) hcv originated from a cross-species transmission of ehv, ii) that ehv was transmitted to horses by humans infected with hcv, which leaves the question on hcv origin open; iii) that hcv and ehv originated from the cross-species transmissions of the same (or similar) virus, with subsequent host adaptation and divergence. if this were the case, multiple crossspecies transmission events may have originated distinct hcv genotypes [85] . teasing apart these possibilities clearly requires understanding of the timing and circumstances of hcv (and ehv) evolution. up to date, no archaeological sample carrying traces of hcv or ehv has been described and the oldest hcv sequence dates to 1953 [86] (1979 for ehv [82] ). thus, molecular dating efforts have relied on extant sequences, with the difficulties associated with the tdrp. studies that did not account for the tdrp provided estimates of the time to the most recent common ancestor (tmrca) of hcv genotypes in a range between $200 and 1000 years ago [63, 64, 76, 87, 88] ; the origin of the horse virus was dated around 1800 ce [85] . a study that separately accounted for the rate of synonymous and nonsynonymous substitutions estimated hcv to have originated at least 2000 years ago [89] . recently, a method based on an a selection-informed model was used to estimate the divergence time of hcv genotypes and the origin of extant ehv/chv strains [70] . this approach, provided estimates of $3000 years ago for the tmrca of extant hcv genotypes (with a low-bound estimate of $5000 years before present) and of $800 years ago for ehv/chv [70] . if these dates are taken to provide at least an indication of the real evolutionary scenario, the possibility that hcv was transmitted to humans by horses infected with ehv can be excluded, an observation in line with the low diversity of ehv/chv [82] . the origin of ehv/chv as reverse zoonosis (i.e., the transmission of a human virus to animals) seems also unlikely, as in this case horse viruses should cluster within hcv diversity, unless the hcv lineage that originated ehv went extinct in the last 800 years. indeed, as the hbv story exemplifies, viral lineage extinction can occur and was previously documented for other human pathogens such as parvovirus b19 [90] , and variola virus [91] . as anticipated above, breeding practices may facilitate this process in the case of animal viruses. we know, for instance, that a minimum of two horse lineages went extinct during the domestication process and that horse genetic diversity has largely been shaped by events that occurred in the last few centuries [92] . it is thus possible that human-mediated selection on the host also resulted in the artificial selection of viral lineages. this would explain the relatively recent origin of extant ehv strains and their low diversity. if viral lineage extinction did occur, the time frames of ehv and hcv evolution would be underestimated and the scenario of hcv originating as a zoonosis from horses (or ehv as a reverse zoonosis) may still hold (fig. 3c) . of course, the alternative possibility that ehv and hcv were transmitted independently to their present-day hosts by a third unknown reservoir is also in line with data on extant diversity and, if the transmission to horses occurred recently, does not require to postulate viral lineage extinction. thus, a number of open questions remain on the origin of hcv. hopefully, technological advances that allow sequencing of trace genetic material from ancient samples will provide information on viral strains hosted by humans and horses back in the past. at the same time, the extensive application of metagenomic approaches to different animal hosts across diverse geographic regions will expand our knowledge on hepacivirus diversity and eventually uncover the direct ancestor of hcv (if it still exists). indeed, the possibility that the different hcv genotypes derived from independent cross-species transmission events [85] would imply that viruses related to hcv are relatively common in the wild, thus giving good chances to be recovered in large field surveys. hdv is a defective virus incapable of autonomous propagation [93] . its genome, a self complementary circular rna of $1700 nucleotides, encodes a single protein (the hdv-encoded delta antigen) ( table 1) . hdv requires hbv surface proteins, that are complexed with the delta antigen, to form transmissible virions [94] . thus, hdv is usually considered a satellite of hbv, although recent data have shown that other enveloped viruses can promote hdv propagation, at least in vitro (e.g. hcv, dengue virus, vesicular stomatitis virus) [95] . genetic heterogeneity among strains is quite high for hdv, which is thus classified in eight different genotypes (from 1 to 8), although a three major genogroup classification was recently proposed (group 1 for previous genotype 1, group 2 for genotypes 2 and genotypes from 4 to 8, and group 3 for previous genotype 3) [96] hdv genetic diversity is highest in africa, suggesting that the defective virus emerged in and spread from this continent [40, 97] . the evolutionary origin of hdv is nonetheless unknown. hdv-like circular rnas were only recently described in birds, reptiles, amphibians, fish and insects [98] [99] [100] . however, these hdvlike elements were not found to be associated with hepadnavirus infection, reinforcing the idea the hdv is not necessarily only transmitted in conjunction with hbv-related viruses, at least in these animals [100] . current evidence suggests that hdv evolved from the human cellular transcriptome [101] . the first indications that a virus was responsible for waterborne, epidemic hepatitis came from studies of asian outbreaks in the 1950-70s and, in analogy to hcv, the agent was referred to as ''epidemic non-a, non-b hepatitis" [102] [103] [104] . hepatitis e virus was eventually isolated and sequenced in the early 1990s [105, 106] . since then, a number of hev strains responsible for human infection were identified. hev is a positive-strand rna virus belonging to the hepeviridae family (table 1) . in common with all other members of this viral family, the hev genome comprises three partially overlapping open reading frames (orfs): orf1 and orf2 encode a non-structural [136] . the piscihepevirus branch is in red, orthohepevirus branches are in blue. the enlargement shows phylogenetic relationships for viruses belonging to the orthohepevirus a species, with representative hosts. (b) geographic distribution of anthropotropic (hev-1 and hev-2) and enzootic (hev-3-hev-4) hev strains. genotypes were assigned to countries irrespective of their prevalence. thus, even if a single case was reported in a given country, the genotype was recorded as present. cases that could be clearly ascribed to migration/travels were excluded. data derive from forni et al. [121] , with updates from [110, [140] [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] . (c) time-scaled phylogenetic tree of a non-recombing orf1 region [121] . branch lengths represent evolutionary time. the time-frames of historical events mentioned in the text are reported. the rabbit and camel silhouettes mark the split of the rabbit-infecting and camel/dromedary-infecting genotypes. the pig silhouette marks the human-restricted/enzootic genotype split. polyprotein and the viral capsid, respectively, whereas orf3 codes for a small phosphoprotein. a fourth orf (orf4), overlapping the helicase domain in orf1, was recently described but seems to be specific for some hev genotypes (hev genotype 1, hev-1) [107] . members of the hepeviridae family are currently classified into two genera, orthohepevirus and piscihepevirus [108] (https://talk. ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rnaviruses/w/hepeviridae). the piscihepevirus genus includes only one species (piscihepevirus a) with one member (cutthroat trout virus), whereas the orthohepevirus genus is divided into four species of viruses infecting mammals and birds (orthohepevirus a-d) [108] (fig. 4a) . this classification is, however, likely to change in the near future following the identification of novel hepeviruses in fish other than trout and in amphibians [28] (fig. 4a) . human-infecting hev strains are genetically heterogeneous and display distinct epidemiologic patterns, but all belong the orthohepevirus a species. orthohepeviruses a account for a minority of the overall diversity of hepeviruses that infect vertebrates and their closest relative is a presently unclassified virus detected in a swedish moose (fig. 4a ) [109] . field surveys revealed a high prevalence of hev in moose populations from sweden and other baltic regions [110, 111] . in general, ungulates represent major orthohepeviruses a reservoirs. at present, eight orthohepevirus a genotypes are recognized (hev-1 to -8) (fig. 4a ). hev-1 and hev-2 infect only humans and cause waterborne outbreaks mainly in tropical and subtropical regions (fig. 4b) . conversely, genotypes 3 and 4 account for the majority of hepatitis e human cases in industrialized countries. hev-3 and hev-4 also infect several other domestic (mainly pigs) and wild (e.g., ungulates and small carnivores) animals, their transmission to humans being usually zoonotic [2] (fig. 4a) . phylogenetic analyses showed that hev-3 and hev-4 sequences derived from human cases are interspersed within those isolated from swine, indicating that pig-infecting hev-3 and hev-4 can easily cross the species barrier and infect humans [112] . notably, though, evolutionary rates are higher for genotypes 3 and 4 than for the human-specific genotypes, suggesting cyclical adaptation to different mammalian hosts [113] . a distinct hev-3 clade, mainly detected in rabbits (hev-3ra), can also cause human hepatitis e [114] [115] [116] [117] (fig. 4a) . the remaining genotypes hev-5/hev-6 and hev-7/hev-8 have been detected in boars and camels, respectively [2] (fig. 4a) . however, they are also thought to have zoonotic potential, as hev-5 and hev-7 can infect cynomolgus monkeys [118, 119] and hev-7 was detected in a patient who consumed camel meat and milk [120] . thus, viruses belonging to all hev genotypes seem to be transmissible to humans. conversely, experimental infection with hev-1 and hev-2 indicated that these viruses have a host range restricted to primates [2] . hev genotypes are therefore usually referred to as enzootic (hev-3 and ã�4) or human-restricted/anthropotropic (hev-1 and -2). although several human hepatitis e cases have a zoonotic origin and orthohepeviruses a are found in diverse mammals, recent data indicated that one or more reverse zoonoses led to the emergence and radiation of hev genotypes [121] . in fact, character state reconstruction on a large phylogeny revealed that humans were the most likely hosts of the ancestor of extant orthohepeviruses a [121] . this notion is in line with the observation that most, if not all, hev genotypes can infect our species, whereas other animals are differentially susceptible to distinct hev genotypes. moreover, increasing evidence suggests that reverse zoonotic events (also known as zooanthroponoses) are all but rare, and examples include other rna viruses such as rotaviruses, enteroviruses, and human influenza viruses [122] [123] [124] [125] . for both swine influenza a viruses and swine vesicular disease virus onward transmission in pigs is well documented [123, 125] and is facilitated by intensive husbandry practices. molecular dating using a selection-informed method inferred that the ancestor of extant orthohepeviruses a existed $6800 to $3200 years ago, most likely in east asia [121] . these inferences well correlate with historical circumstances that may have favored hev emergence and host range expansion. in this period, sedentary agriculture promoted the appearance of large human settlements in several asian regions and pig husbandry practices started to intensify in east asia [126] [127] [128] [129] [130] (fig. 4c) . crowded living conditions and poor sanitation possibly favored the emergence and spread of the waterborne, human-specific hev strains. the close contact between humans and pigs most likely promoted hev zooanthroponotic transmission and emergence of the enzootic strains (fig. 4c) . additional reverse zoonotic transmissions may have also originated the camel-infecting and rabbit-infecting strains. in fact, the estimated timing of hev-7/8 emergence (4055 bce to 1192 bce) [121] encompasses the time of domestication of bactrian and dromedary camels [131] [132] [133] (fig. 4c) . as for hev-3ra, it was estimated to have diverged from hev-3 around 600 ce, in europe [121] . this time frame corresponds to the middle ages, when historical evidence indicates that rabbits were kept in warrens and bred for meat [134] (fig. 4c ). of course, these estimates do not necessarily imply that camels and rabbits acquired hev from humans, as the domestication process may have exposed these animals to various viral sources, including other domestic (e.g., pigs) and peridomestic mammals. these scenarios provide a credible framework for orthohepevirus a origin, as well as for the diversification of hev genotypes, and selection-informed methods should at least partially correct for the tdrp, as they explicitly model purifying selection [14] [15] [16] . nonetheless, the above-mentioned data on hbv [9] suggest caution in the inference of time and location of ancestral events based on extant viral diversity. also, pig infection with hev-3 and hev-4 is generally asymptomatic [135] , possibly indicating a long-standing host-virus association that might even predate swine husbandry development. it should also be noted that the ultimate origin of orthohepevirus a remains unknown. humans may have acquired hev through cross-species transmission from other animals. however, known orthohepeviruses that infect mammals and birds are distantly related to orthohepevirus a, indicating that none of them represents the source of human-infecting hev. likewise, the origin and evolutionary relationship between the moose virus and human-infecting orthohepeviruses remain unclear. by allowing the large-scale identification of novel viral species, as well as the sequencing of viral genomes from archaeological samples, technological advances have largely expanded our knowledge on the evolution and origin of human hepatitis viruses. these insights have been paralleled by the development of computational tools and theoretical frameworks to analyze and mine viral sequence data (e.g., the above-mentioned recognition of the tdrp and the development of approaches to correct for it). the overall picture emerging from these studies clearly indicates that, with the possible exception of hdv, viruses related to human hepatitis viruses infect several other mammalian and non mammalian vertebrates. the specific events that originated these human pathogens remain, however, largely unknown. for hcv and hev, the evolutionary history of the human viruses is likely intertwined with that of related viruses that infect domestic animals. conversely, in the case of hav and hbv, the closest relatives of the human viruses are found in nhps. in general, these observations indicate that a deeper understanding of the evolutionary dynamics of human viruses has a relevance not only to improve our ability to treat and prevent present infections, but also to gain insight into the ecological contexts that may fuel the emergence of novel human pathogens, with particular reference to zoonotic ones. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. world health organization (who) zoonotic hepatitis e virus: classification, animal reservoirs and transmission routes hepatitis a: old and new type a viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention bayesian molecular dating: opening up the black box inferences from tip-calibrated phylogenies: a review and a practical guide bayesian estimates of the evolutionary rate and age of hepatitis b virus enigmatic origin of hepatitis b virus: an ancient travelling companion or a recent encounter ancient hepatitis b viruses from the bronze age to the medieval period time-dependent rate phenomenon in viruses prisoners of war -host adaptation and its constraints on virus evolution time-dependent estimates of molecular evolutionary rates: evidence and causes bayesian inference of evolutionary histories under time-dependent substitution rates a case for the ancient origin of coronaviruses purifying selection can obscure the ancient age of viral lineages evolutionary origins of human herpes simplex viruses 1 and 2 hepatitis a virus genome organization and replication strategy a pathogenic picornavirus acquires an envelope by hijacking cellular membranes fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis a virus capsid hepatitis a: detection by immune electron microscopy of a viruslike antigen associated with acute illness hepatitis ain greenland: importance of specific antibody testing in epidemiologic surveillance prevalence of antibody to hepatitis a and hepatitis b viruses in selected populations of the south pacific genetic variability and molecular evolution of hepatitis a virus evolutionary origins of hepatitis a virus in small mammals discovery of a novel hepatovirus (phopivirus of seals) related to human hepatitis a virus a novel hepatovirus identified in wild woodchuck marmota himalayana a novel marsupial hepatitis a virus corroborates complex evolutionary patterns shaping the genus hepatovirus the evolutionary history of vertebrate rna viruses evolutionary origins of enteric hepatitis viruses phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents hepatitis a virus and the origins of picornaviruses virus-like particles in serum of patients with australia-antigen-associated hepatitis hepatitis b virus epidemiology hepatitis b virus: the challenge of an ancient virus with multiple faces and a remarkable replication strategy genotypes and genetic variability of hepatitis b virus heterogeneous recombination among hepatitis b virus genotypes a genetic variant of hepatitis b virus divergent from known human and ape genotypes isolated from a japanese patient and provisionally assigned to new genotype possible origins and evolution of the hepatitis b virus (hbv) the global hepatitis b virus genotype distribution approximated from available genotyping data origins and evolution of hepatitis b virus and hepatitis d virus a novel hepatitis b virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses bat hepadnaviruses and the origins of primate hepatitis b viruses deciphering the origin and evolution of hepatitis b viruses by means of a family of nonenveloped fish viruses endogenous hepadnaviruses, bornaviruses and circoviruses in snakes early mesozoic coexistence of amniotes and hepadnaviridae endogenous hepadnaviruses in the genome of the budgerigar (melopsittacus undulatus) and the evolution of avian hepadnaviruses the first full-length endogenous hepadnaviruses: identification and analysis hepatitis b virus has a recent new world evolutionary origin reconstructing the origins of human hepatitis viruses tracing hepatitis b virus to the 16th century in a korean mummy complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis b virus, four of which represent two new genotypes subtypes, genotypes and molecular epidemiology of the hepatitis b virus as reflected by sequence variability of the s-gene dating the origin and dispersal of hepatitis b virus infection in humans and primates dating the origin of hepatitis b virus reveals higher substitution rate and adaptation on the branch leading to f/h genotypes neolithic and medieval virus genomes reveal complex evolution of hepatitis detection of hepatitis b virus infection in wild-born chimpanzees phylogenetic relationships with human and other primate genotypes the paradox of hbv evolution as revealed from a 16th century mummy epidemic history and evolutionary dynamics of hepatitis b virus infection in two remote communities in rural nigeria molecular epidemiology of hepatitis b virus in misiones molecular diversity in irregular or refugee immigrant patients with hbvgenotype-e infection living in the metropolitan area of naples isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome phylogeny and molecular evolution of the hepatitis c virus the epidemic behavior of the hepatitis c virus the origin of hepatitis c virus genetic epidemiology of hepatitis c virus throughout egypt global distribution and prevalence of hepatitis c virus genotypes identification of a novel hepatitis c virus genotype from punjab, india: expanding classification of hepatitis c virus into 8 genotypes identification of 19 novel hepatitis c virus subtypes-further expanding hcv classification global epidemiology of hepatitis c virus infection evolutionary analysis provides insight into the origin and adaptation of hcv sexually acquired hepatitis c virus infection: a review infectious diseased and human population history investigating the endemic transmission of the hepatitis c virus exploring the possibility of arthropod transmission of hcv highly divergent hepaciviruses from african cattle characterization of a canine homolog of hepatitis c virus identification of rodent homologs of hepatitis c virus and pegiviruses surveying the global virome: identification and characterization of hcv-related animal hepaciviruses evidence for novel hepaciviruses in rodents bats are a major natural reservoir for hepaciviruses and pegiviruses serology-enabled discovery of genetically diverse hepaciviruses in a new host differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys identification of a novel hepacivirus in domestic cattle from germany detection of non-primate hepaciviruses in uk dogs hepacivirus cross-species transmission and the origins of the hepatitis c virus evolutionary analysis of hepatitis c virus gene sequences from 1953 origin of hepatitis c virus genotype 3 in africa as estimated through an evolutionary analysis of the fulllength genomes of nine subtypes, including the newly sequenced 3d and 3e full-length genomes of 16 hepatitis c virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes the origin of hepatitis c virus genotypes ancient human parvovirus b19 in eurasia reveals its long-term association with humans 17(th) century variola virus reveals the recent history of smallpox tracking five millennia of horse management with extensive ancient genome time series transmission of the hepatitis b virus-associated delta antigen to chimpanzees hepatitis delta virus enveloped viruses distinct from hbv induce dissemination of hepatitis d virus in vivo a comprehensive bioinformatic analysis of hepatitis d virus full-length genomes genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains a divergent hepatitis d-like agent in birds identification of a novel deltavirus in boa constrictors novel hepatitis d-like agents in vertebrates and invertebrates hepatitis delta virus: a fascinating and neglected pathogen study of an epidemic of non-a, non-b hepatitis. possibility of another human hepatitis virus distinct from post-transfusion non-a, non-b type infectious hepatitis in delhi (1955-56): a critical studyepidemiology. 1957 epidemic and endemic hepatitis in india: evidence for a non-a, non-b hepatitis virus aetiology isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis e virus international committee on taxonomy of viruses hepeviridae study group novel hepatitis e like virus found in swedish moose prevalence and phylogenetic analysis of hepatitis e virus in pigs, wild boars, roe deer, red deer and moose in lithuania high prevalence of hepatitis e virus in swedish moose -a phylogenetic characterization and comparison of the virus from different regions close similarity between sequences of hepatitis e virus recovered from humans and swine genotypespecific evolution of hepatitis e virus rabbit hepatitis e virus infections in humans rabbit hev in immunosuppressed patients with hepatitis e acquired in switzerland transmission of hepatitis e virus from rabbits to cynomolgus macaques hepatitis e virus strains in rabbits and evidence of a closely related strain in humans genotype 5 hepatitis e virus produced by a reverse genetics system has the potential for zoonotic infection production of infectious dromedary camel hepatitis e virus by a reverse genetic system: potential for zoonotic infection chronic infection with camelid hepatitis e virus in a liver transplant recipient who regularly consumes camel meat and milk origin and dispersal of hepatitis e virus reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldom-documented human biological threats to animals reverse zoonosis of influenza to swine: new perspectives on the human-animal interface a systematic review of evidence that enteroviruses may be zoonotic evolutionary histories of coxsackievirus b5 and swine vesicular disease virus reconstructed by phylodynamic and sequence variation analyses bar-yosef of the neolithic demographic transition and its consequences rapid, global demographic expansions after the origins of agriculture changes in regional settlement patterns and the development of complex societies in southeastern shandong social complexification and pig (sus scrofa) husbandry in ancient china: a combined geometric morphometric and isotopic approach agricultural origins and the isotopic identity of domestication in northern china the prehistory of the silk road the history of the camel bone dating project the appearance of the domestic camel in southeast arabia rabbits and the specious origins of domestication recent knowledge on hepatitis e virus in suidae reservoirs and transmission routes to human estimating maximum likelihood phylogenies with phyml distribution of hbv genotypes in latin america global prevalence and genotype distribution of hepatitis c virus infection in 2015: a modelling study raxml version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies complete genome sequence of a hepatitis e virus genotype 1e strain from an outbreak in nigeria hepatitis e in italy: 5 years of national epidemiological, virological and environmental surveillance hepatitis e virus genotypes and subgenotypes causing acute hepatitis quantification and genetic diversity of hepatitis e virus in wild boar (sus scrofa) hunted for domestic consumption in central italy dakovic rode o. genetic diversity of hepatitis e virus (hev) strains derived from humans, swine and wild boars in croatia from a case of incidental infection of hepatitis e virus (hev) genotype 1 in a domestic pig hepatitis e virus genotype 1 and hepatitis a virus dual infection in pediatric patients with a low socioeconomic status from mexico epidemiology and genotype 3 subtype dynamics of hepatitis e virus in belgium first detection of hepatitis e virus genotype 3 as a common infectious agent in patients with chronic liver damage in mexico a new hepatitis e virus genotype 2 strain identified from an outbreak in nigeria hepatitis e virus seroprevalence and correlates of anti-hev igg antibodies in the rakai district hepatitis e virus infection in different groups of estonian patients and people who inject drugs the worldwide burden of viral hepatitis in terms of death and disability is enormous. in 2015, viral hepatitis caused 1.34 million deaths, the majority of which imputable to the effects of chronic hbv (hepatitis b virus) and hcv (hepatitis c virus) infection [1] . an estimated 5% of hbv-infected persons are also co-infected with hdv (hepatitis delta virus), which worsens the clinical outcome compared to hbv monoinfection [1] . less than 5% of overall hepatitis mortality is caused by hav (hepatitis a virus) and hev (hepatitis e virus), that are usually associated with acute, self-limiting disease [1] . however, the case fatality rate of hev is particularly high in specific groups, notably pregnant women and elderly individuals [2] . although rare, infection with hav can also cause acute liver failure and death, and the risk increases with age [3] . despite the existence of a safe and effective vaccine, hav remains a common cause of acute viral hepatitis in many regions of the world [4] . key: cord-017764-h1w9gbxk authors: meanwell, nicholas a.; belema, makonen title: the discovery and development of daclatasvir: an inhibitor of the hepatitis c virus ns5a replication complex date: 2018-06-08 journal: hcv: the journey from discovery to a cure doi: 10.1007/7355_2018_47 sha: doc_id: 17764 cord_uid: h1w9gbxk the discovery of the hepatitis c virus (hcv) ns5a replication inhibitor daclatasvir (1) had its origins in a phenotypic screening lead. however, during the optimization campaign, it was observed that some members of the chemotype underwent a radical dimerization under the assay conditions. this redirected the effort to focus on palindromic molecules, a species subsequently shown to complement the dimeric nature of the ns5a protein. the most challenging aspect of the discovery program was extending antiviral activity to encompass gt-1a virus which was accomplished only after the development of extensive structure-activity relationships. in clinical trials, oral administration of daclatasvir (1) produced a profound effect on viral load with onset that was more rapid than had been seen previously with either ns3 protease or ns5b polymerase inhibitors. a groundbreaking clinical trial that combined daclatasvir (1) with the protease inhibitor asunaprevir (52) established that a chronic hcv infection could be cured with small molecule therapy in the absence of immune stimulation, setting the stage for approval of this regimen for the treatment of gt-1b-infected subjects by the japanese health authorities on july 4, 2014. the discovery of the hepatitis c virus (hcv) nonstructural 5a (ns5a) replication complex inhibitor daclatasvir (1) began with the development of a genotype 1b (gt-1b) replicon that was implemented as a phenotypic screen using a design that conferred a stringent triaging of hit molecules [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . this screening campaign was part of a broader strategy that pursued the identification of mechanistically orthogonal inhibitors of hcv that could be used in combination therapy, an approach that anticipated clinical use of drug cocktails to minimize the selection of resistant viruses. more specifically, the replicon screen comprised of simultaneously assessing the antiviral activity of test molecules toward a sub-genomic hcv gt-1b construct and a bovine viral diarrhea virus (bvdv) replicon, both replicating in the same human liver huh-7 cell line background and seeded in the same well of a 96-well plate [6] . hcv inhibition was determined indirectly using a fluorescence resonance energy transfer (fret)-based assay that assessed ns3 protease activity toward a synthetic substrate incorporating both a fluorescence donor [(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (edans)] in the amino terminus and a fluorescence acceptor [4-((4-(dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester (dabcyl)] at the carboxyl terminus. the bvdv replicon incorporated a firefly luciferase reporter gene that provided an orthogonal readout of replication activity based on the emission of light, reflecting the amount of enzyme present after adding a substrate. the third piece of information harvested from the assay, which was also the first experimental data obtained, was alamarblue cell viability staining which assessed mitochondrial function, providing an indication of the cytotoxicity of test compounds. this assay configuration was used to interrogate a representative selection of the bristol-myers squibb proprietary compound collection and identified the iminothiazolidinone 2 as hit that met the criteria of exhibiting activity toward the hcv replicon at concentrations at least tenfold lower than either inhibitory activity toward the bvdv replicon or cytotoxicity [12] . the specific details of the antiviral profile of 2 in the screening assays and toward a panel of viruses are summarized in table 1 . compound 2 had an interesting background since it had its origin in a prospective library that had been prepared as part of a campaign to embellish the bristol-myers squibb proprietary compound collection. a notable structural feature of 2 is that it had been designed to include a phenyl substituent at c-5, unusual since c-5 benzylidene derivatives are far more prevalent in the literature, a function of convenient access by way of a knoevenagel condensation reaction between a c-5 unsubstituted iminothiazolidinone and an aldehyde [13, 14] . the data accumulated for 2 that are compiled in table 1 revealed a profile of significant and selective inhibitory potency toward the gt-1b hcv replicon with an ec 50 value of 570 nm, a finding that encouraged further study of the chemotype [15] . adding to the intrigue with 2 as a lead inhibitor was the generation and validation of resistant mutants arising in the replicon in response to selective pressure that mapped to the amino terminus of the ns5a protein, specifically a tyr98his and a leu31val/gln54leu combination [12] . at the time of this discovery, little was known about the specific functions of the ns5a protein in viral replication although, perhaps not surprisingly for such a small virus, it was known to be an essential protein [16] [17] [18] [19] [20] . the z-configuration of the 2-arylimino moiety of 2 was assigned based on prior studies which indicated that this topology minimized steric effects. however, attempts to separate the individual stereoisomers at c-5 were thwarted by facile racemization of the benzylic center after chiral chromatographic separation [15, 21] . seminal insights into structure-activity relationships (sars) were gleaned from a survey that explored variation of the three major peripheral elements of the moleculethe furanylmethyl substituent, the arylimino moiety, and the amino acid amide. changes to both the furanylmethyl and arylimino moieties in the context of the cbz-alanine amide were found to influence potency, with compounds incorporating polar heterocycles being the more potent, while more lipophilic substituents generally exhibited poorer replicon inhibitory activity. overall, a good dynamic range of potency was observed with the structural variations sampled, with ec 50 values ranging from 10 to 2.5 μm. however, more profound and precise effects on hcv gt-1b inhibitory potency were associated with changes to the cbz-alanine amide element, with the key findings summarized in fig. 1 . the unnatural alanine analogue 3 was 170-fold less potent, while the glycine homologue 4 was eightfold weaker than 2. the strong dependence of antiviral activity on the absolute configuration of the amino acid element was reproduced in the proline derivatives 5 and 6, both of which were fivefold more potent than their alanine congeners [15] . replacing the cbz element with a dihydrocinnamate moiety resulted in an erosion of potency in both series, as exemplified by 7 and 9. however, a significant increase in potency was observed with the phenylacetamide moiety found in 8 and 10, with both compounds expressing single digit nanomolar ec 50 values in the replicon and confirming the inherent advantage offered by proline. only weak antiviral effects were observed with the many other amino acids and additional structural variations that were explored at this site of the pharmacophore, with some representative examples of those sampled compiled in fig. 2 [15] . while these collective sars appeared to be coherent and were readily interpretable, as highlighted by the sensitivity of potency to changes to the amino acid element, as exploration of 2 and its analogues continued, it became apparent that the iminothiazolidinone chemotype was unstable under some conditions [15, 22] . the first indication of a problem was the observation that a sample of 2 degraded upon standing as a solution in dmso for several days, with the thiohydantoin 15 (r 3 ¼ nhco 2 ch 2 ph) and thiourea 17 (r 3 ¼ nhco 2 ch 2 ph) determined to be two of the degradation products after a preparative experiment (scheme 1). in the replicon, 15 exhibited a modest inhibitory activity, ec 50 while 17 was essentially inactive. compounding the intrigue, incubating the more potent 8 in replicon media until degradation was complete and then assessing the antiviral activity of the preparation in the gt-1b replicon demonstrated that the hcv inhibitory activity and potency were fully preserved [15, 22] . a more detailed analysis of the degradation products from 8 guided by high-performance liquid chromatography (hplc) biogram analysis, in which fractions taken from the chromatography column are concentrated and assessed for replicon inhibition, led to the identification of two potent constituents present in just trace amounts in the cell media [22, 23] . a preparative experiment allowed isolation of a sufficient quantity of each component to allow for a more detailed characterization. 1 h-nmr and mass spectrum analyses indicated that the two compounds were dimers with an isomeric relationship and assigned as 18 based on the absence of the c-5 hydrogen atom of the iminothiazolidinone ring in the 1 h-nmr spectrum and connectivity that was confirmed after the analysis of the 13 c-nmr spectrum. although neither the exact stereo-composition of the benzylic centers of 18 nor the precise relationship between these two compounds was determined, it is of note that the isomer that was less mobile on a reversed phase column converted to the more mobile isomer upon heating in cd 3 cn at 50 c, an observation made while conducting an nmr experiment as part of the structure determination process [22] . with the acquisition of these data, the mechanistically holistic picture of the degradation process that is presented in scheme 1 was proposed. abstraction of the c-5 hydrogen atom of 8 by oxygen, which is a diradical in the ground state, would lead to the c-5 radical 11 that is stabilized in a classic captodative fashion by the adjacent c¼o moiety, the sulfur atom, and the phenyl substituent [24, 25] . combination of 11 with o 2 and hydrogen atom acquisition would lead to the hydroperoxide species 12 which in dmso would be reduced to alcohol 13, an unstable ring system that would be expected to undergo ring opening to give 14. reclosure of 14 would then afford the thiohydantoin 15, which had been isolated in the original dmso degradation experiment, while hydrolytic decomposition of 14 would afford acid 16 and the thiourea 17, the latter of which had also been isolated. however, in cell culture media, the stability of radical 11 is presumably such that dimerization can occur, a process that may well be facilitated by aggregative association of 8 and/or 11 in the aqueous medium. the replicon inhibitory potency of the dimers 18a and 18b was striking, with the less mobile isomer eluting from the reversed phase chromatography column exhibiting an ec 50 value of 600 pm in the gt-1b replicon, while the thermodynamically more stable and chromatographically more mobile isomer was 70-fold weaker, ec 50 ¼ 43 nm [15, 22] . with the elucidation of the structures of 18a and 18b, consideration was given to the concept that the ns5a-inhibiting pharmacophore represented by these dimers may be the embedded symmetrical bibenzyl element. this notion was based on an appreciation of the precise sars surrounding the amino acid moiety in contrast to the relatively more nebulous effects associated with structural variation to the iminothiazolidinone substituents. under this concept, the iminothiazolidinone ring system of 2 was suggested to act as a scaffolding element by which a bivalent ns5a inhibitor pharmacophore was convened through a radical-mediated dimerization process [15, 22] . this hypothesis was readily tested in the context of the proline-based chemotype, with 19 demonstrating an ec 50 value of 30 nm and confirming the pharmacophore proposal. more interestingly, the unsaturated synthetic precursor 20 32 n. a. meanwell and m. belema was almost 350-fold more potent than 19, ec 50 ¼ 0.086 nm, providing further insight into the topography of the ns5a-inhibiting pharmacophore. the discovery of the structurally simpler symmetrical pharmacophore represented by 19 and 20 turned out to be prescient since x-ray crystallographic structure data for ns5a constructs that were published several years after this discovery revealed a dimeric, c2-symmetric protein complex [26] [27] [28] . of note, this structure was determined using a protein construct lacking the membrane-associating amino terminus but, nevertheless, containing some of the key elements of the anticipated binding site for ns5a inhibitors based on the location of resistance mutations. the availability of a structure of the amino terminus of ns5a, acquired by nmr methodology, facilitated the construction of models of ns5a that, when combined with the resistance mutation data, suggested that these compounds bound across the ns5a dimer interface at a site residing between the membrane and the core of the protein. dimerization of the ns5a protein in cells was subsequently confirmed as was association of ns5a with rna, a prediction from the x-ray data based on the preponderance of basic amino acids lining the inner surface of the u-shaped dimeric form of ns5a in the solid state [29] [30] [31] [32] . while the antiviral activity of 20 was attractive, there were concerns around elements embedded within the structure, with the olefin observed to be configurationally unstable in some analogues, while the potential for release of one or both aniline moieties after the action of an amidase or protease in vivo raised the specter of toxicity. however, a considerably more challenging problem arose when 20 was evaluated in a newly developed gt-1a replicon where the compound was found to be poorly active, with an ec 50 value that was in excess of 10 μm. the gt-1a strain of hcv is clinically relevant, with prevalence that varies across the globe; consequently, securing activity toward this genotype was considered to be a critical objective. enhancing gt-1a inhibitory activity became the immediate focus of further study, and the approach adopted, while primarily directed toward the peripheral elements, was broad-based in prosecution. modifications to all facets of the molecule were explored, some of which were also directed toward simultaneously addressing the structural liabilities highlighted above. however, as described below, introducing and retaining gt-1a coverage while optimizing adme properties would end up posing a considerable challenge. while deeper insight into sars for gt-1b inhibition emerged from the initial phases of this effort, only a small number of compounds were identified that exhibited modest but reproducible gt-1a inhibition. among these were the oxazole 21 and the substituted proline derivative 22, both of which were inhibitory in the gt-1a replicon with ec 50 values of less than 1 μm [33] . however, attempts to optimize these molecules were unproductive, in each case leading to sar cul-de-sacs. as the studies progressed, the 2-ethyl-substituted benzamide derivative 23 and its unsaturated homologue 24 were discovered to exhibit weak inhibition of gt-1a and gt-1b replicons, with modeling studies suggesting that the effect of substitution was not a function of modulating the conformation between the phenyl ring and proline carbonyl [34] . the pyridine derivative 25 further advanced the sar associated with this cap element but, more importantly, formed the basis of the discovery of the isoquinoline derivative 26, in which the ethyl substituent was incorporated into a fused ring, as the first compound to exhibit potent and balanced antiviral activity toward gt-1a and gt-1b replicons [34] . the promise of this compound was further underscored when it was screened in replicons or hybrid replicons representing genotypes 2a, 3a, and 5a where the ec 50 values ranged from 2.2 to 14 nm. further examination of the sars revealed that gt-1a inhibitory activity was much more sensitive to the nature and the substitution pattern of the isoquinoline ring than gt-1b [34] . for example, 27, the parental analogue of 26, exhibited gt-1a and gt-1b ec 50 values that were 20-and 3-fold weaker than that of 26, respectively. in addition, the methoxy-substituted derivative 28 and its chloro-substituted analogue 29 retained potent gt-1b inhibition, but their gt-1a ec 50 values were >10 μm. however, a more fruitful avenue of investigation was found when the effects of deannulating the isoquinoline ring were probed [35] . the α-keto amide 30 preserved the gt-1a inhibition exhibited by 26, while the derived secondary alcohols 31 and 32 demonstrated that planarity at this site was not a specific requirement. the tertiary alcohol homologues 33 and 34 added further to the sars, with the (s,s)-analogue 34 the more potent isomer, particularly toward the gt-1b replicon where the ec 50 value was 6 pm. another encouraging observation was made with 35 which, although poorly active in the gt-1a replicon, demonstrated 64% bioavailability following oral dosing to rats, indicating that securing systemic exposure after oral delivery of these symmetrical stilbene derivatives was feasible. replacing the stilbene element with an alkyne, which resolved a cis-trans isomerization issue observed with some analogues, added further to the understanding of the topography of the pharmacophore. additional probing of the amino acid terminal region using this alkyne-based scaffold identified potent arylglycine-based analogues for which the gt-1a ec 50 values for some compounds, including 36-38, were single digit nm [35] . notably, the change in stereochemical preference in evolving the chemotype from the mandelamide analogues 33 and 34 to the phenylglycine analogues 36 and 37 further highlighted the relatively intricacy and sensitivity of the gt-1a inhibition sars that were being uncovered during this phase of the project. equally intriguing was the accumulating evidence indicating that the gt-1b virus was highly tolerant of many structural changes, an observation that could not readily be explained based on the differences in amino acid composition at the putative binding site of the compounds. a concurrent effort examined scaffold modifications directed toward the identification of a less problematic replacement for the anilide moiety that would decrease or eliminate the potential for aniline release in vivo which led to the emergence of two noteworthy sar findings. firstly, the replacement of the anilide moieties of 36 and 38 with a benzimidazole, a design intended to preserve both the h-bond donor and acceptor properties of the anilide, resulted in a 4-to 30-fold reduction in potency toward the gt-1a replicon, as exemplified by 39 and 40 [36] . secondly, a mix and match sar exercise accentuated the sensitivity of gt-1a inhibitory potency to topological parameters, exemplified by the 70-fold difference in gt-1a inhibitory potency between regioisomers 41 and 42. these sar findings were attributed to the altered topology of the peripheral pharmacophoric elements with respect to the core, a shortcoming that was ultimately addressed by the biphenyl derivatives 43 and 44 which recapitulate the linearity the discovery and development of daclatasvir: an inhibitor of the. . . associated with the core alkynes in 36-40. in 43 and 44, a motif that was arrived at only after considerable experimentation, deannelation of the benzimidazole ring provided a structural arrangement that compensated for the reduced length of the core of the pharmacophore relative to 39 and 40. the success of this design strategy is readily apparent since both 43 and 44 are exquisitely potent hcv antiviral agents with balanced gt-1a and gt-1b inhibition, with ec 50 values ranging from 7 to 42 pm [4] . however, the oral bioavailability and systemic exposure of both 43 and 44 in the rat were poor, a result attributed, in part, to the high molecular weight (747 and 807 da, respectively) and structural composition. this notion was reinforced by pk studies with the smaller (mw ¼ 713) and unsymmetrical tetrahydrofuran 45 which divests of a h-bond donor. although the oral bioavailability of 45 was low in rodents (f in mouse ¼ 17%, f in rat ¼ 6.8%), its exposure in the dog was much improved, with bioavailability measured at 45%. in an effort to reduce the molecular weight of the carbamate 44, the two aromatic rings of the phenyl glycine moiety were curtailed to isopropyl substituents affording the d-valine derivative 46. however, this structural modification resulted in a significant reduction in potency toward both hcv genotypes, with the gt-1a inhibition particularly sensitive, eroding by 44,000-fold. this sar observation took some time to understand and was resolved only after further study of the chemotype, which revealed that the preferred absolute configuration of the alkyl-glycine caps was the opposite of that of the aryl-glycine caps. the initial observation in this direction was made when the tetrahydrofuran ring of 45 was replaced with l-alanine to provide 47, which restored potency to sub-nanomolar levels. in an observation that proved to be pivotal, the symmetrical alanine derivative 48 performed similarly, and further optimization of the amino acid appendage led to the discovery of the bis-l-valine derivative 1, an exercise that also included assessing the potential of unsymmetrical derivatives. the decision to advance 1 into ind-enabling toxicology evaluation was taken after a careful comparison with 49, an analogue with two changes to the periphery that demonstrated similar antiviral properties to 1 (table 2 ) but a different pk profile ( table 3 ). the decision to select 1 for development rather 49 was based on the observation of a twofold accumulation of the latter compound in the plasma, livers, and hearts of mice after 4 days of daily drug administration which occurred at all of the dose levels (15, 50, and 100 mg/kg) examined. the antiviral profiles of 1 and 49 toward wild-type and hybrid replicons representing all of the genotypes and subtypes that were available at the time are summarized in table 2 [1] [2] [3] [4] [37] [38] [39] [40] . adding to confidence in the potential of 1 as it negotiated the development path toward clinical evaluation was the potent inhibition observed in a newly established gt-2a jfh-replicating virus assay. the ec 50 value of 28 pm in this assay exhibited a good correlation with the inhibitory potency toward the gt-2a jfh replicon [1] [2] [3] [4] . the pharmacokinetic parameters of 1 and 49 in rat, dog, and cynomolgus monkey are compiled in table 3 and demonstrate good systemic exposure following oral administration, with the drug concentration measured at 24 h post-dose well in excess of the ec 50 values for the gt-1a and gt-1b replicons and the proteinbinding-adjusted ec 90 value of 383 pm determined for the gt-1a replicon. more importantly, target organ exposure was also demonstrated, with liver levels of 103 nm measured in the rat 24 h following a 5 mg/kg dose of 1, a concentration that was fivefold higher than that in plasma. the favorable absorption properties of 1 have been attributed, in part, to the formation of an intramolecular h-bond between the carbamate c¼o and imidazole n-h moieties that enhances lipophilicity and reduces the apparent psa of the molecule based on a chromatographic analysis and which is supported by modeling studies [41] . despite its high potency and broad genotype inhibitory activity, the precise mode of inhibition of hcv replication by 1 remains as enigmatic as does the biochemical function of the ns5a protein [16, 18, [42] [43] [44] [45] [46] [47] [48] . hcv ns5a has no known enzymatic activity but is a critical element in the assembly and function of the replication complex on intracellular membranes and also in virion assembly [16, [42] [43] [44] [45] [46] [47] [48] . hcv ns5a is a 447-residue phosphoprotein that is comprised of three functional domains and an amphipathic helix at the amino terminus that associates with but does not traverse to biological membranes. domain 1 contains a zn 2+ binding motif and several serine residues that are sites of basal phosphorylation and hyperphosphorylation. the phosphorylation state of ns5a may modulate its function in virus replication and assembly with the hyperphosphorylated form, which can be produced by the action of the host cell lipid kinase, phosphatidylinositol 4-kinase, involved in the assembly of virions. domain 2 has been shown to bind to the ns5b rna-dependent rna polymerase and has been associated with the sensitivity of the virus to interferon therapy although that function is controversial. while domain latter effect has been postulated to explain the rapid decline in viremia observed in hcv-infected patients administered clinically effective doses of 1 (vide infra) [3, 51] . in addition to associating with all of the viral nonstructural proteins, hcv ns5a has also been shown to bind to an extensive repertoire of host cell proteins that exceeds 130 entities [52] [53] [54] [55] [56] [57] . as a consequence, the ns5a protein is typically viewed as a master regulator of virus replication and virion production, orchestrating both viral proteins and the host cell environment to ensure the successful production and release of progeny virus [42] [43] [44] [45] [46] [47] [48] . an association of ns5a inhibitors with the viral protein was originally demonstrated by studies with the biotin-labeled derivative 50 which is an effective inhibitor of gt-1b replication [3] . the antiviral activity of 50 is highly sensitive to the absolute configuration of the proline moiety since the (r,r)-isomer 51 is inactive, while inhibition is substantially reduced by the tyr93his resistance mutation that arises in response to virus passaging in the presence of 1. this sar profile is strictly analogous to that established for the stilbene chemotype, and 50 was thus viewed as a useful tool molecule with which to probe drug-target binding interactions. in an initial experiment, gt-1b replicon lysate was incubated with 50, and the mixture passed over streptavidin immobilized on beads; however, this experimental protocol failed to pull down any viral protein. in contrast, incubating replicon cells with 50 for 18 h before lysing and passing the lysate over streptavidin beads identified only ns5a as a binding partner, while a control experiment with inactive diastereomer 50 failed to isolate any virial proteins. these results indicate that 50 binds to hcv ns5a and that binding is dependent on the absolute configuration of the proline element, an observation concordant with the sars developed in the stilbene-based series. while the experiments conducted with 50 indicate that the binding of inhibitors to the ns5a is choreographically somewhat complex, the binding of inhibitors of ns5a to both domain 1 and the full-length viral protein was subsequently demonstrated in a series of independent biochemical experiments [58, 59] . these studies have suggested that the binding of inhibitors to ns5a interferes with the association of viral rna with the protein, with the binding of compounds competed out by other ns5a inhibitors and demonstrating diminished affinity for the tyr93his mutant protein [58, 59] . however, profiling of inhibitors in cell-based assays has indicated that disruption of rna binding to ns5a does not appear to occur and that the introduction of key resistant mutations leads to only a modest reduction in the the discovery and development of daclatasvir: an inhibitor of the. . . binding of inhibitors [60, 61] . studies with 50 in resistant gt-1b replicons indicated that while the tyr93his-resistant mutation reduced inhibitory potency by 220-fold, an estimate of the amount of ns5a protein pulled down by the chemical probe, as determined by an analysis of western blots, suggested similar levels of protein-drug association for the resistant and wild-type strains [60, 61] . adding further to the complexity of the biochemistry was the observation that in a protein pull-down experiment, an attempt to outcompete the biotinylated tool compound 50 with a non-biotinylated analogue in a gt-1b tyr93his-containing mutant (ec 50 ¼ 290 nm for stated analogue vs >7 μm for 50) not only failed but, at low concentration, appeared to have incrementally enhanced the amount of ns5a pulled down. from these data, it was inferred that the development of resistance to 1 does not lead to exclusion of binding to the ns5a protein, as is often observed with other classes of antiviral agents. rather, these observations suggested a scenario in which hcv ns5a develops resistance by accommodating rather than expelling inhibitors, with the mutations presumably allowing restoration of protein function in the presence of the bound inhibitor. consistent with this suggestion, several of the resistant mutations incorporate smaller or more flexible amino acid side chains, exemplified by tyr93his, tyr93cys, leu31met, and leu31val, which may restore conformational flexibility compromised by the binding of inhibitors. these observations stimulated an experiment designed to evaluate the effect of combining 1 with structurally related compounds on the function of hcv ns5a incorporating resistance mutations. two possible outcomes were contemplated: in the first scenario, a molecule would simply compete with bound 1 and the observed effect would be one of silence. however, the alternative scenario speculated on the potential of a second molecule to act in conjunction with 1 to restore inhibition by binding to an adjacent site on the ns5a molecule. a screen of compounds selected from the library of hcv ns5a inhibitors assessed in the presence of 1 using the tyr93asn gt-1aresistant replicon, followed by sar optimization, identified syn-395 (52) as a molecule capable of restoring the sensitivity of resistant virus to the inhibitory effects of 1. for example, in a typical experiment, 1 exhibited ec 50 values of 33 pm and 339 nm toward the wild type and tyr93asn mutant replicons, respectively, whereas 52 was poorly active in both replicons, with ec 50 values of 214 and 215 nm, respectively. however, the ec 50 of 1 toward the tyr93asn mutant replicon improved to 0.13 nm when titrated in the presence of a suboptimal concentration (40 nm) of 52. this result represented a 2,600-fold increase in the sensitivity of the tyr93asn replicon to 1 in the presence of 52. the synergistic relationship between 1 and 52 was confirmed in a reciprocal experiment where 52 was titrated in the presence of suboptimal amount of 1 affording a similar outcome [60, 61] . this has presented a significant challenge to developing a more coherent and detailed understanding of the mechanism of action of hcv ns5a inhibitors and the modeling of drug-target interactions of these potent antiviral agents and the allosteric modulators [60] [61] [62] [63] [64] [65] [66] [67] . while the bivalent nature of ns5a inhibitors, including the allosteric modulators represented by 52, complements the dimeric form of the protein observed in solid-state structures of elements of domain i, the binding interfaces between the proteins vary [26-28, 60, 61] . one interpretation of this observation anticipates an oligomeric form of hcv ns5a in cells that can bind to the viral rna and protect it from chemical and enzymatic degradation while providing a platform for rna presentation to the polymerase and translocation to the developing virion [68] [69] [70] . however, the biochemical pharmacological effects of ns5a inhibitors are multifaceted and complex and include altering the subcellular distribution of ns5a, modulating the phosphorylation state of the protein, interfering with the formation of the membranous factories where virus replication occurs, and blocking the transfer of the viral genome to assembly proteins, leading to a clustering of hcv proteins at endoplasmic reticulum membranes [71] [72] [73] [74] [75] [76] . the phase i clinical trial with 1 comprised of a randomized, double-blind, placebocontrolled, single ascending dose study involving administration of 1, 10, 25, 50, 100, and 200 mg of the drug to normal healthy volunteers (nhvs) [3] . a doseproportional increase in plasma exposure was observed over the dosing range, and the concentration of 1 in plasma 24 h after dosing exceeded the protein-bindingadjusted ec 90 values of 49 pm (0.04 ng/ml) and 383 pm (0.28 ng/ml) recorded for the gt-1b and gt-1a replicons, respectively [3] . compound 1 was quickly absorbed and exposure extended beyond 24 h, with plasma drug concentration maintained above the less sensitive gt-1a protein-binding-adjusted ec 90 value of 383 pm at 72 h for all but the 1 mg dose, predicting the potential for once-daily dosing [51] . the absolute oral bioavailability of 1 in humans is 67%, and the compound is~99% bound to plasma proteins [77] [78] [79] [80] . in this trial, 1 was safe and well tolerated at all of the administered doses with no clinically significant adverse effects observed, a profile that set the stage for a proof-of-concept study in hcv gt-1infected subjects. doses of 1, 10, and 100 mg were administered in a randomized, double-blind, placebo-controlled, single ascending dose format similar to that used for the nhv study, and plasma hcv rna levels were monitored until 172 h postdose. the results of this study are compiled in table 4 with mean plasma hcv rna declining by 1.8 log 10 iu/ml 24 h following the 1 mg dose, while the 10 and 100 mg doses provided increased efficacy, with viral load declines of 3.2 and 3.3 log 10 iu/ml, respectively, measured at 24 h. the mean maximal viral load reduction in the 100 mg dose cohort was 3.6 log 10 iu/ml with hcv rna measured at 35 iu/ml in one of the gt-1b-infected subjects, while plasma rna in another was below the the discovery and development of daclatasvir: an inhibitor of the. . . 41 lower limit of quantification (25 iu/ml) at 144 h post-dose. the decline in plasma viral rna concentration following administration of the 10 and 100 mg doses of 1 was both rapid and profound in nature, with a mean reduction of 1.95 log 10 iu/ml measured at 6 h post-dose for nine of the patients [49, 51] . the steep decline in viral load was subsequently explained after the development of a multiscale model of viral kinetics that took into account the effects of 1 on both viral replication and virus assembly and secretion, with the latter being the source of an immediate effect on virion production. the mean effectiveness of 1 on virus rna production and virion assembly/secretion was estimated to be 99 and 99.8%, respectively, and yielded an estimate of plasma hcv half-life of 45 min, significantly shorter than the 2.7 h halflife estimated from an analysis of viral kinetics during treatment with older, interferon-based therapies [49] . the profile of 1 was further explored in a double-blind, placebo-controlled multiple ascending dose study in which the drug was administered for 14 consecutive days to gt-1-infected subjects at doses of 1, 10, 30, 60, and 100 mg once daily and 30 mg twice daily [51] . each panel comprised of five patients randomized such that four received drug and one was administered a placebo control, with drug pk parameters determined on days 1 and 14. median peak plasma concentrations of 1 occurred 1-2 h after dosing, and the pk profile was supportive of once-daily dosing with a mean terminal half-life of 12-15 h and steady state achieved after 3-4 days of drug administration. the mean maximal reduction in hcv rna levels in plasma are compiled in fig. 3 with 30 and 60 mg qd cohorts comprised solely of gt-1a-infected subjects. in the other dosing cohorts, those infected with gt-1b virus exhibited a greater response compared to those infected with gt-1a virus. however, most patients experienced viral rebound on or before day 7 of therapy with viral rna levels below the mean maximal decline except in the 30 mg bid cohort (fig. 4) . rebound was typically more rapid in the gt-1a-infected subjects which can be explained by a lower genetic barrier to resistance in this subtype [81] [82] [83] . in hcv gt-1a, only a single base pair change in the viral rna is typically required to code for an alternative amino acid, while gt-1b frequently requires two base pair changes for coherent coding [81] [82] [83] . population sequencing indicated the appearance of mutations at met28, gln30, leu31, and tyr93 all of which had been identified as resistance mutation hotspots in response to selective pressure by 1 in replicon studies in vitro [84, 85] . while the results of this trial further confirmed the potential of hcv ns5a as a therapeutic target, the rapid emergence of resistance to 1 anticipated that optimal clinical application would be as part of combination therapy [79, [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] . the combination of 1 as add-on therapy to the extant standard of care, pegylated ifnα and ribavirin (53) , was explored clinically in patients infected with hcv genotypes 1-4 and a subset of patients who were co-infected with hiv-1. the results indicated that sustained virologic responses could be achieved with shorter 24-week durations of therapy with a burden of side effects comparable to that of pegylated ifnα and 53 [92] [93] [94] [95] [96] . however, it was the opportunity availed by the contemporaneous development of the hcv ns3 protease inhibitor asunaprevir (54) that allowed the pursuit of a parallel clinical program that had a significant impact on the course of the clinical development of hcv therapeutic agents [97] [98] [99] [100] [101] [102] . in a small open-label clinical trial conducted in hcv gt-1-infected subjects who had no evidence of cirrhosis and who had previously failed to respond to peg-ifn/53 therapy (referred to as null responders), a combination of 1 (60 mg qd po) and 54 (600 mg bid po) with peg-interferon α2a (180 μg per week subcutaneously) and 53 (1,000 or 1,200 mg qd po, depending on body weight) administered for 24 weeks was compared with a regimen comprised of only the two direct-acting antiviral agents (daas) [99, 102] . all of the ten patients receiving the quadruple drug regimen had undetectable levels of hcv rna in plasma measured at 12 weeks following the last dose (svr 12 ), while nine also achieved svr 24 . one patient in this group had detectable hcv rna in plasma at week 24, but this was not quantifiable, and viral rna was not detected in plasma 5 weeks later [99, 102] . of the 11 patients receiving only the dual daa combination, five had undetectable levels of hcv rna in plasma at the end of therapy, and four maintained this status at weeks 4, 12, and 24 after the last drug dose. this cohort was comprised of nine subjects infected with gt-1a and two infected with gt-1b, with both gt-1b-infected patients achieving svr 24 , while six patients infected with gt-1a virus experienced virological breakthrough while on therapy and the remaining patient infected with gt-1a virus relapsed after completing drug therapy. this study, which was conducted in a very challenging patient population, provided the first indication that a chronic hcv infection could be cured solely by treatment with daas in the absence of the immune stimulation provided by peg-interferon α2a and 53 [100] . the successful treatment of hcv gt-1b infections with 1 and 54 redirected the clinical development plan for this dual combination to japan where gt-1b is the 44 n. a. meanwell and m. belema most prevalent, accounting for approximately 70% of the estimated two million infections at the time [103, 104] . the combination of 1 and 54 has been studied extensively in gt-1b-infected japanese patients, leading to approval of the drug combination by the japanese pharmaceutical and medical devices agency (pmda) on july 4, 2014 [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] . the marketing authorization of 1 as daklinza™ and 54 as sunvepra ® in japan represented the first approval of a combination of daas for the treatment of hcv infection although the combination of sofosbuvir (55) and 53 had been approved by the fda in december of 2013 [107] . the phase iii japanese clinical trials of 1 (60 mg qd) and 54 (100 mg bid) in gt-1b-infected subjects that subtended marketing approval evaluated 24 weeks of therapy and were associated with svr 12 rates of 81% in non-responders and 87% in those intolerant of or ineligible for pegylated interferon therapy. in a multinational study conducted in a broad gt-1b patient population, the svr 12 rates were 90% in those naïve to therapy and 82% in those intolerant of or ineligible for interferon-based therapies. the pre-existence of the tyr93his polymorphism in the hcv ns5a gene was a predictor of lower clinical efficacy, with the svr rates declining to 45 from 95% in those patients that harbored this mutation at baseline, which has a 15% prevalence in the japanese patient population. broadening the utility of 1 and 54 to treat hcv gt-1a infections required the addition of a third agent, the allosteric rna-dependent rna polymerase inhibitor beclabuvir (56) which was developed as a fixed-dose combination comprising of 30 mg of 1, 200 mg of 54, and 75 mg of 56 administered as a bid regimen for 12 weeks [120] [121] [122] [123] [124] [125] [126] . in the unity 1 international study which was conducted in 415 non-cirrhotic patients with hcv gt-1 infection, 91% of the patients achieved svr 12 . svr 12 rates of 92% were observed in treatment-naive patients and 89% in treatment-experienced patients, while virologic failure occurred in 8% of the patients. in the unity-2 phase iii study conducted in the united states, canada, france, and australia in 202 gt-1-infected patients with compensated cirrhosis, the svr 12 rates were 93% for patients in the treatment-naive group and 87% for those in the treatment-experienced group. svr 12 rates were improved to 98% for patients in the treatment-naive group and 93% for those in the treatment-experienced group when 53 was included in the regimen. in a phase iii trial (unity 3) conducted in 217 japanese patients infected with gt-1 hcv, svr 12 rates of 95% were achieved in both treatment-naive (n ¼ 152) and interferon-experienced (n ¼ 65) cohorts after 12 weeks of therapy. the svr 12 rates were similar across the patient subgroups evaluated that included patients with cirrhosis and those aged 65 years. these studies contributed to the approval of the fixed-dose combination of 1, 54, and 56 for marketing in japan on december 20, 2016. a number of clinical studies have demonstrated that co-dosing of 1 with the nucleoside-based ns5b rna-dependent rna polymerase inhibitor 55, with and without 53, achieves a high cure rate across hcv genotypes and patient population groups, including in those with comorbidities such as hiv-1 infection [127] [128] [129] . in a compassionate use program that reflected a real-world experience, including some subjects with advanced liver disease that would have been excluded from phase iii studies, the combination of 1 and 55 (with and without 53) demonstrated a high efficacy [130] . in addition, long-term follow-up studies have demonstrated a 99% durability for the svr 12 associated with various regimens that include 1 [131] . daclatasvir (1) has been approved in more than 60 countries for use in combination with 54, 56, or other hcv inhibitors, including 55 [132] . a combination of all four of these agents has been evaluated in gt-1-infected patients as a drug intensification approach to shortening the duration of therapy to 4 or 6 weeks [133] . however, while the majority (96%) of patients experienced undetectable levels of hcv rna at the end of therapy, relapse occurred in 77% of those treated for 4 weeks and 43% of those subject to 6 weeks of treatment with quadruple therapy [133] . the ns5a replication complex inhibitor class of hcv inhibitor has become established as a critical component of all of approved pan-genotypic daa combination therapies [132] . the discovery of 1, the prototype ns5a inhibitor that is the founding member of the class, was identified only after considerable optimization of a lead discovered by phenotypic screening, a powerful approach to drug discovery that has proven to be well-suited as a means of identifying mechanistically novel antiviral agents [134] [135] [136] [137] [138] . conflict of interest the authors are employees of bristol-myers squibb and own company stock. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. discovery of daclatasvir, a pan-genotypic hepatitis c virus ns5a replication complex inhibitor with potent clinical effect case history: the discovery of the first hepatitis c virus ns5a replication complex inhibitor daclatasvir (daklinza™) chemical genetics strategy identifies an hcv ns5a inhibitor with a potent clinical effect hepatitis c virus ns5a replication complex inhibitors: the discovery of daclatasvir the discovery and development of hepatitis c virus ns5a replication complex inhibitors development of a cell-based high-throughput specificity screen using a hepatitis c virusbovine viral diarrhea virus dual replicon assay replication of subgenomic hepatitis c virus rnas in a hepatoma cell line hepatitis c virus replicons: potential role for drug development the hepatitis c virus replicon system: from basic research to clinical application evolution of cell culture systems for hcv on the history of hepatitis c virus cell culture systems identification of hepatitis c virus ns5a inhibitors 4-thiazolidinone -a biologically active scaffold convenient synthesis of 5-arylidene-2-imino-4-thiazolidinone derivatives using microwave irradiation inhibitors of hcv ns5a: from iminothiazolidinones to symmetrical stilbenes hepatitis c virus ns5a: tales of a promiscuous protein the non-structural 5a protein of hepatitis c virus hepatitis c virus ns5a: enigmatic but still promiscuous 10 years on! biochemical and immunologic properties of the nonstructural proteins of the hepatitis c virus: implications for development of antiviral agents and vaccines mutations that permit efficient replication of hepatitis c virus rna in huh-7 cells prevent productive replication in chimpanzees regioselective synthesis of 3-(heteroaryl)-iminothiazolindin-4-ones the discovery and development of daclatasvir: an inhibitor of the discovery of potent hepatitis c virus ns5a inhibitors with dimeric structures hplc biogram analysis: a powerful tool used for hit confirmation in early drug discovery the captodative effect advances in radical-trapping antioxidant chemistry in the twentyfirst century: a kinetics and mechanisms perspective structure of the zinc-binding domain of an essential component of the hepatitis c virus replicase crystal structure of a novel dimeric form of ns5a domain i protein from hepatitis c virus the crystal structure of ns5a domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric hcv inhibitors correlation between ns5a dimerization and hcv replication hepatitis c virus nonstructural protein 5a (ns5a) is an rna-binding protein hepatitis c virus nonstructural protein 5a: biochemical characterization of a novel structural class of rna-binding proteins all three domains of the hepatitis c virus nonstructural ns5a protein contribute to rna binding hcv ns5a replication complex inhibitors. part 3: discovery of potent analogs with distinct core topologies hcv ns5a replication complex inhibitors. part 4: optimization for genotype 1a replicon inhibitory activity hcv ns5a replication complex inhibitors. part 5: discovery of potent and pan-genotypic hcv ns5a replication complex inhibitors hepatitis c virus ns5a replication complex inhibitors. part 6: the discovery of a novel and highly potent biarylimidazole chemotype with inhibitory activity toward genotype 1a and 1b replicons resistance analysis of the hepatitis c virus ns5a inhibitor bms-790052 in an in vitro replicon system vitro activity of bms-790052 on hepatitis c virus genotype 4 ns5a in vitro activity of daclatasvir on hepatitis c virus genotype 3 ns5a comparison of daclatasvir resistance barriers on ns5a from hepatitis c virus genotypes 1 to 6: implications for cross-genotype activity the discovery of potent nonstructural protein 5a (ns5a) inhibitors with a unique resistance profile -part 2 hepatitis c virus ns5a protein -a master regulator ed) hepatitis c viruses ns5a -from obscurity to new target for hcv therapy targeting the ns5a protein of hcv: an emerging option small molecule inhibitors of the hepatitis c virus-encoded ns5a protein ns5a: a new target for antiviral drugs in the treatment of hepatitis c virus infection hcv ns5a replication complex inhibitors modeling shows that the ns5a inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis c virus half-life kinetic analyses reveal potent and early blockade of hepatitis c virus assembly by ns5a inhibitors multiple ascending dose study of bms-790052, a nonstructural protein 5a replication complex inhibitor, in patients infected with hepatitis c virus genotype 1 interaction between hepatitis c virus and host cell factors hcvpro: hepatitis c virus protein interaction database understanding the biological context of ns5a-host interactions in hcv infection: a network-based approach affinity capture and identification of host cell factors associated with hepatitis c virus (+) strand subgenomic rna the discovery and development of daclatasvir: an inhibitor of the identification and comparative analysis of hepatitis c virus-host cell protein interactions host cell kinases and the hepatitis c virus lifecycle potent hepatitis c inhibitors bind directly to ns5a and reduce its affinity for rna direct binding of ledipasvir to hcv ns5a: mechanism of resistance to an hcv antiviral agent resensitizing daclatasvir-resistance hepatitis c variants by allosteric modulation of ns5a synergistic activity of combined ns5a inhibitors asymmetric binding to ns5a by daclatasvir (bms-790052) and analogs suggests two novel modes of hcv inhibition resistance patterns associated with hcv ns5a inhibitors provide limited insight into drug binding a refined model of the hcv ns5a protein bound to daclatasvir explains drug-resistant mutations and activity against divergent genotypes a comprehensive computational analysis for the binding modes of hepatitis c virus ns5a inhibitors: the question of symmetry overall structural model of ns5a protein from hepatitis c virus and modulation by mutations conferring resistance of virus replication to cyclosporin a mechanisms of hepatitis c viral resistance to direct acting antivirals from structure to function: new insights into hepatitis c virus rna replication inhibition and avoidance of mrna degradation by rna viruses attacked from all sides: rna decay in antiviral defense small molecules targeting hepatitis c virus-encoded ns5a cause subcellular redistribution of their target: insights into compound modes of action the effect of ns5a inhibitors on ns5a phosphorylation, polyprotein processing and localization daclatasvir-like inhibitors of ns5a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication daclatasvir prevents hepatitis c virus infectivity by blocking transfer of the viral genome to assembly sites the hepatitis c virus ns5a inhibitor (bms-790052) alters the subcellular localization of the ns5a non-structural viral protein cyclophilin and ns5a inhibitors, but not other anti-hepatitis c virus (hcv) agents, preclude hcv-mediated formation of double-membrane-vesicle viral factories practical and efficient strategy for evaluating oral absolute bioavailability with an intravenous microdose of a stable isotopically-labeled drug using a selected reaction monitoring mass spectrometry assay daclatasvir: a review of its use in adult patients with chronic hepatitis c virus infection daclatasvir: a review of preclinical and clinical pharmacokinetics daclatasvir: a review in chronic hepatitis c contribution of a mutational bias in hepatitis c virus replication to the genetic barrier in the development of drug resistance antiviral resistance and the future landscape of hepatitis c virus infection therapy importance of hcv genotype 1 subtypes for drug resistance and response to therapy genotypic and phenotypic analysis of variants resistant to hepatitis c virus nonstructural protein 5a replication complex inhibitor bms-790052 in humans: in vitro and in vivo correlations hepatitis c virus rna elimination and development of resistance in replicon cells treated with bms-790052 hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-α therapy new kinetic models for the hepatitis c virus modeling hcv kinetics under therapy using pk and pd information treatment of hepatitis c virus infection with interferon and small molecule direct antivirals: viral kinetics and modeling quantifying the diversification of hepatitis c virus (hcv) during primary infection: estimates of the in vivo mutation rate the discovery and development of daclatasvir: an inhibitor of the mathematical modeling of hcv infection: what can it teach us in the era of direct-acting antiviral agents? daclatasvir plus peginterferon alfa and ribavirin for treatment-naive chronic hepatitis c genotype 1 or 4 infection: a randomised study daclatasvir plus peginterferon and ribavirin is noninferior to peginterferon and ribavirin alone, and reduces the duration of treatment for hcv genotype 2 or 3 infection efficacy and safety of daclatasvir plus pegylated-interferon alfa 2a and ribavirin in previously untreated hcv subjects coinfected with hiv and hcv genotype-1: a phase iii, open-label study a randomized trial of daclatasvir with peginterferon alfa-2b and ribavirin for hcv genotype 1 infection daclatasvir combined with peginterferon alfa-2a and ribavirin in japanese patients infected with hepatitis c genotype 1 the discovery of asunaprevir (bms-650032), an orally efficacious ns3 protease inhibitor for the treatment of hepatitis c virus infection preclinical profile and characterization of the hepatitis c virus ns3 protease inhibitor asunaprevir (bms-650032) preliminary study of two antiviral agents for hepatitis c genotype 1 a watershed moment in the treatment of hepatitis c resistance analysis of the hepatitis c virus ns3 protease inhibitor asunaprevir randomized trial of daclatasvir and asunaprevir 52 n. a. meanwell and m. belema with or without pegifn/rbv for hepatitis c virus genotype 1 null responders changes in hepatitis c virus genotype distribution in japan changing trends in hepatitis c infection over the past 50 years in japan dual therapy with the nonstructural protein 5a inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis c virus genotype 1b-infected null responders effectiveness and safety of daclatasvir plus asunaprevir for hepatitis c virus genotype 1b: systematic review and meta-analysis daclatasvir + asunaprevir: first global approval characterization of virologic escape in hepatitis c virus genotype 1b patients treated with the direct acting antivirals daclatasvir and asunaprevir dual oral therapy with daclatasvir and asunaprevir for patients with hcv genotype 1b infection and limited treatment options daclatasvir plus asunaprevir for chronic hcv genotype 1b infection high sustained virologic response to daclatasvir plus asunaprevir in elderly and cirrhotic patients with hepatitis c virus genotype 1b without baseline ns5a polymorphisms daclatasvir plus asunaprevir treatment for real-world hcv genotype 1-infected patients in japan randomized comparison of daclatasvir+asunaprevir versus telaprevir+peginterferon/ ribavirin in japanese hepatitis c virus patients favorable efficacy of daclatasvir plus asunaprevir in treatment of elderly japanese patients infected with hcv genotype 1b aged 70 and older efficacy and tolerability of an ifn-free regimen with dcv/asv for elderly patients infected with hcv genotype 1b the efficacy and safety of dual oral therapy with daclatasvir and asunaprevir for genotype 1b in japanese real-life settings the practical management of chronic hepatitis c infection in japan -dual therapy of daclatasvir + asunaprevir the discovery and development of daclatasvir: an inhibitor of the daclatasvir for the treatment of hepatitis c virus infection efficacy and safety of daclatasvir in hepatitis c: an overview discovery and preclinical characterization of the cyclopropylindolobenzazepine bms-791325, a potent allosteric inhibitor of the hepatitis c virus ns5b polymerase unity-1 study group (2013) fixed-dose combination therapy with daclatasvir, asunaprevir, and beclabuvir for noncirrhotic patients with hcv genotype 1 infection daclatasvir in combination with asunaprevir and beclabuvir for hepatitis c virus genotype 1 infection with compensated cirrhosis a randomized trial of daclatasvir in combination with asunaprevir and beclabuvir in patients with chronic hepatitis c virus genotype 4 infection daclatasvir/asunaprevir/ beclabuvir fixed-dose combination in japanese patients with hcv genotype 1 infection daclatasvir/asunaprevir/beclabuvir, all-oral, fixed-dose combination for patients with chronic hepatitis c virus genotype 1 beclabuvir in combination with asunaprevir and daclatasvir for hepatitis c virus genotype 1 infection: a systematic review and meta-analysis daclatasvir with sofosbuvir and ribavirin for hepatitis c virus infection with advanced cirrhosis or post-liver transplantation recurrence daclatasvir plus sofosbuvir for hcv in patients coinfected with hiv-1 all-oral 12-week treatment with daclatasvir plus sofosbuvir in patients with hepatitis c virus genotype 3 infection: ally-3 phase iii study daclatasvir plus sofobuvir, with and without ribavirin, achieved high sustained virological response rates in patients with hcv infection and advanced liver disease in a real-world cohort long-term follow-up of clinical trial patients treated for chronic hcv infection with daclatasvir-based regimens current therapy for chronic hepatitis c: the role of direct-acting antivirals short-duration treatment for chronic hepatitis c virus with daclatasvir, asunaprevir, beclabuvir and sofosbuvir (fourward study) daclatasvir: the first of a new class of drugs targeted against hepatitis c virus ns5a how were new medicines discovered? impact of highthroughput screening on biomedical research cell-based assays to identify inhibitors of viral disease anti-infectives: can cellular screening deliver? the discovery and development of daclatasvir: an inhibitor of the key: cord-018785-tcr5xlf8 authors: nambiar, puja; silibovsky, randi; belden, katherine a. title: infection in kidney transplantation date: 2018-06-27 journal: contemporary kidney transplantation doi: 10.1007/978-3-319-19617-6_22 sha: doc_id: 18785 cord_uid: tcr5xlf8 infection is an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. pretransplant screening, immunizations, and optimal antibacterial and antiviral prophylaxis can help to reduce the impact of infection. awareness of the approach to infection in the transplant recipient including diagnostic and management strategies is essential to optimizing outcomes. a total of 17,600 kidney transplants were performed in the united states in 2013. as the incidence of acute rejection has declined, the probability of graft and patient survival continues to improve (usrds 2015) . infection, however, remains an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007 ). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009 ). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. the kidney transplant recipient's net state of immune suppression and epidemiologic exposures determine the risk for infection at a given time. a traditional timeline has been used to predict patterns of infection after organ transplantation. this timeline has been altered in recent years with changes in immunosuppressive therapy and the routine use of antibacterial and antiviral prophylaxis. treatment for acute rejection and coinfection with viruses such as cytomegalovirus (cmv) and epstein-barr virus (ebv) may also alter predictable patterns of infection (fishman 2007) . the basic concepts of the traditional timeline, however, are still used to establish a differential diagnosis for infection at varied intervals posttransplantation (fig. 1) . within the first month, infections are noted to include those related to surgical complications, nosocomial exposures, and donor-derived pathogens. multidrug-resistant organisms including methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococcus (vre), and carbapenem-resistant enterobacteriaceae (cre) are important considerations, as is clostridium difficile. urinary tract infections are common within the first 6 months. opportunistic infections are more likely to occur 1-6 months after transplantation, reflecting the greater impact of immune suppression during this time. reactivation of latent pathogens such as polyoma virus bk, hepatitis c virus (hcv), and mycobacterium tuberculosis may also occur. prophylaxis for pneumocystis jiroveci, herpes viruses including cmv, and hepatitis b virus (hbv) makes these infections less common during this time period. beyond 6 months, the degree of immune suppression for most patients decreases. risk remains, however, for community-acquired infection, environmental exposures, recurrent infection, and the late presentation of viral infection, in particular cmv, once prophylaxis has been discontinued (fishman 2007; karuthu and blumberg 2012) . interventions can be undertaken to reduce the impact of infection after kidney transplantation. pretransplant screening of donors and recipients for infection that can be transmitted with organ donation or reactivated in an immune suppressed recipient is essential for optimizing transplant outcomes. guidelines for pretransplant screening are available from the american society for transplantation (fischer et al. 2013) , kidney disease: improving global outcomes (kdigo 2009) and the us public health service (seem et al. 2013) . recommended screening tests for donors and recipients are listed in table 1 . screening of living donors is performed prior to transplantation with varied timing. if there is a hiv(þ) consider if hiv is well controlled hcv: anti-hcv and hcv nat hcv(þ) hcv(à) reject, may be a consideration in the future hcv(à) hcv(þ) consider, hcv(þ) candidates should have a liver biopsy, improved outcomes if hcv is treated pretransplant hcv(þ) hcv(þ) consider (as for dà/rþ) hbv: hbsag, hbsab and hbcab (igm/igg); hbv nat (center dependent) sag(à), cab(à) sag(à), cab(þ), sab(þ/à) accept, vaccinate sab(à) candidates consider, with prophylaxis posttransplant sag(à), cab(þ) sag(à), cab (þ/à), sab (þ/à) accept if donor is cigm(à) and vaccinate sab(à) candidates, offer prophylaxis posttransplant if sab(à) or lost; reject if donor is cigm(þ) sag(þ), cab(þ) sag(à), cab (þ/à), sab (þ/à) reject cmv igg cmv(þ) or (à) cmv(þ) accept; will need posttransplant prophylaxis or preemptive therapy cmv(þ) cmv(à) accept; high risk for cmv infection, will need posttransplant prophylaxis ebv igg ebv(þ) or (à) ebv(þ) accept ebv(þ) ebv(à) accept; at risk for primary ebv and ptld, monitor posttransplant hsv 1/2 igg hsv(þ) hsv(þ) or (à) (fischer et al. 2013) . deceased donor screening, in contrast, is under time constraints and is usually performed within hours of transplantation in coordination with organ procurement organizations. infection with hiv, hbv, and hcv may not be detected in the early stages of infection. many transplant centers now perform more sensitive rapid molecular testing on potential organ donors including nucleic acid amplification (nat) testing for hiv, hbv, and hcv. a comprehensive medical and social history on potential organ donors is required in order to identify risk factors for blood borne pathogens. in efforts to expand the pool of available organs, recipients may consent to receipt of a kidney from a nat negative donor who is deemed "high risk" for blood borne infection based on identified risk factors. recipients of such organs are monitored posttransplantation with testing for hiv, hbv, and hcv between 1 and 3 months and for hbvagain at 12 months (fischer et al. 2013; seem et al. 2013; kovacs et al. 2014; len et al. 2014 ). use of hcv-and hbv-positive organs can be considered in respective positive recipients. furthermore, in 2013 the hiv organ policy equity act lifted a long-standing ban on allowing hivpositive organs to be donated to hiv-positive recipients (mgbako et al. 2013; muller et al. 2015) . donors who have active bacterial infection at the time of kidney procurement may transmit infection to the recipient. screening for bacterial infection in kidney donors includes assessing for urinary tract infection and bacteremia. urine and blood culture data are reviewed. if a kidney donor is known to have a urinary tract or systemic infection with a virulent organism such as staphylococcus aureus, pseudomonas aeruginosa, or candida species, the organ recipient is usually treated with a 10-14 day course of targeted antimicrobial therapy since these bacteria can compromise vascular and urinary anastomoses leading to mycotic aneurysms, anastomotic, and organ failure (fischer et al. 2013 ). allograft contamination can occur during organ procurement or processing. interpretation of organ preservation fluid cultures is challenging. the risk of transmission of infection to the organ recipient from contaminated preservation fluid, however, is low (fischer et al. 2013; len et al. 2014 ). candidates for kidney transplantation should have their vaccine status reviewed and updated in accordance with recommendations issued by the advisory committee on immunization practices with the centers for disease control and prevention (cdc 2012). while vaccinations in end stage renal disease patients may be less effective and durable than in healthy patients, a better response can be anticipated prior to transplantation than after (janus et al. 2008; kausz and pahari 2004) . special consideration should be given to vaccination for pneumococcus, influenza, and hbv. two pneumococcal vaccines are currently licensed for use in the united states: the 13-valent pneumococcal conjugate vaccine (pcv 13, prevnar 13) and the 23-valent-pneumococcal-polysaccharide vaccine (ppsv 23, pneumovax 23). current guidelines recommend that unvaccinated patients with chronic renal failure receive pcv 13 followed at least 8 weeks later by ppsv 23 (kobayashi et al. 2015) . a second dose of ppsv 23 is recommended 5 years after the first dose (cdc 2012). influenza vaccination should be administered annually. there are a number of influenza vaccine formulations available. live attenuated influenza vaccination (flumist) is not recommended in chronic kidney disease patients. an inactivated vaccine option should be used (cdc 2012) . a high dose inactivated influenza vaccine is now available and was shown to induce a higher antibody response than traditional vaccines in adults over the age of 65 (diaz-granados et al. 2014) . the use of this vaccine in transplant candidates and recipients is currently under investigation. transplant candidates not immune to hbv should receive high dose hbv vaccination (40 micrograms antigen per dose) due to decreased response rates with standard dosing (huprikar et al. 2015) . human cytomegalovirus-human herpes 5 (cmv), a member of the family herpesviridae, is an opportunistic pathogen occurring in 20-60% of solid organ transplant recipients (brennan 2001) . cmv is a cause of significant morbidity and mortality in this population (mwintshi and brennan 2007) . the incidence of cmv in the renal transplant population is estimated to be between 8% and 32% (patel and paya 1997) . renal transplant patients are at lower risk for primary cmv compared with other organ transplant recipients owing to a lower burden of latent virus in renal allograft tissue. the risk factors for development of cmv disease include donor seropositivity/recipient seronegativity(dþ/rà), use of induction immunosuppression (antilymphocyte antibodies), donor age >60 years, simultaneous kidney-pancreas transplantation, treatment for acute rejection, impaired transplant function, and concurrent infection from other viruses (like ebv and hhv-6 and 7) (de keyzer et al. 2011) . cmv-seronegative recipients of cmv-seropositive donors (d + /r à ) are at the highest risk, whereas d + /r + or d à /r + transplantations are considered to be moderate risk with d à /r à being lowest risk, with an incidence of cmv disease <5% (de keyzer et al. 2011) . immunosuppressive drugs also influence the incidence and severity of cmv disease. for instance, cyclosporine increases the risk of cmv disease, whereas use of sirolimus seems to have a protective effect (san juan et al. 2008) the use of antilymphocyte antibody (antithymocyte globulin or muromonab-cd3) is associated with a two to fivefold increase in the rate of cmv, but basiliximab and daclizumab do not seem to increase its incidence (de keyzer et al. 2011) . cmv infection may occur in solid organ transplantation recipients as primary infection when a cmv seronegative individual receives cells latently infected with cmv from a seropositive donor, donor-derived reinfection, or reactivation of latent recipient infection (patel and paya 1997) . the following definitions are commonly used in the transplant literature to differentiate cmv infection from cmv disease. cmv infection is evidence of cmv replication regardless of symptoms, and cmv disease is evidence of cmv infection with symptoms, such as viral syndrome, leukopenia, thrombocytopenia, or invasive tissue disease (e.g., pneumonitis, hepatitis, retinitis, gastrointestinal disease) (humar and snydman 2009) . cmv disease and even asymptomatic cmv infection have been shown to be independent risk factors for reduced graft survival and overall mortality beyond 100 days posttransplantation . infection with cmv has been implicated in acute allograft dysfunction and chronic allograft nephropathy (mclaughlin et al. 2002; tong et al. 2002) . cmv disease is also associated with posttransplant lymphoproliferative disorder (ptld), posttransplant diabetes mellitus, and transplant artery stenosis (pouria et al. 1998; hjelmesaeth et al. 2004; manez et al. 1997) . cmv infection can occur as acute infection between the first and 6 months following transplant, when immunosuppression is at its maximum or as delayed infection from reactivation of latent virus after antiviral prophylaxis has completed, later in the first year. given the significant effect of cmv on patient outcomes, prevention plays an important role. serologic screening for cmv should be performed on both donor and recipient prior to transplant to categorize high risk patients. several cmv vaccine candidates are under investigation although none are currently available. universal prophylaxis involves giving antivirals to those recipients at risk posttransplant before the onset of infection, whereas in preemptive therapy patients are monitored at regular intervals and started on antivirals when there is early evidence of replication prior to onset of clinical disease. chemoprophylaxis in high risk patients (dþ/rà) has shown to reduce the incidence of cmv disease by 60% and has decreased cmv associated mortality and opportunistic infection (hodson et al. 2005) . preemptive therapy in high risk patients based on cmv viral load monitoring has not shown reduction in acute rejection or all-cause mortality (strippoli et al. 2006) . a randomized controlled trial by kliem et al. in 2008 comparing oral ganciclovir chemoprophylaxis with viral load monitoring revealed improved graft survival in those who received ganciclovir chemoprophylaxis (kliem et al. 2008) . a recent cochrane review from 2013 concluded that the efficacy of preemptive therapy compared with prophylaxis to prevent cmv disease remains unclear due to significant heterogeneity between studies and that additional head-tohead studies are required to determine the relative benefits and harms of preemptive therapy and prophylaxis to prevent cmv disease in solid organ transplant recipients (owers et al. 2013) . standard prophylactic guidelines recommend therapy in dþ/rà, dþ/rþ, and dà/rþ using oral ganciclovir or valganciclovir for a minimum of 3 months posttransplant and 1-3 months after treatment of rejection with antilymphocyte therapy (humar and snydman 2009; kotton et al. 2013 ). valganciclovir has replaced ganciclovir because of better bioavailability, lower pill burden, and reduced availability of oral ganciclovir (paya et al. 2004 ). the optimal length of prophylaxis is unknown, but recent trials have shown that 6 months of prophylaxis is more effective in decreasing the incidence of cmv disease in dþ/ rà kidney transplant recipients (humar et al. 2010; doyle et al. 2006) . current guidelines recommend dosing valganciclovir at 900 mg daily (adjusted for renal dysfunction) if tolerated in dþ/ rà recipients. some centers have successfully treated patients with half of this dose (450 mg daily) with less drug toxicity. however, dþ/rà recipients may be at higher risk of breakthrough infection and the development of resistance with this lower dosing strategy (kotton et al. 2013) . the diagnosis of cmv disease can be made by several techniques including cmv antigenemia assay, nucleic acid testing (nat), serology, antibody testing, viral culture, and histopathology. nat is generally more sensitive than antibody testing or culture. higher values by nat are suggestive of cmv disease and weekly viremia testing can be used to monitor response to therapy. the interlaboratory variability of nat is expected to be reduced with the recent establishment of international standards, intended to be used in the standardization of nucleic acid amplification technique (nat)-based assays for hcmv (karuthu and blumberg 2012) . patients with gastrointestinal and neurologic cmv disease often fail to exhibit cmv viremia and histopathology is necessary to establish diagnosis in these instances. treatment of active cmv disease requires a combination of immunomodulation, antiviral therapy with or without adjuvant therapy and if possible, reduction of immunosuppression (kotton et al. 2013; green et al. 2004 ). the mainstay of therapy is intravenous ganciclovir. the victor trial (valcyte in cmv disease treatment of solid organ recipients) demonstrated oral valganciclovir was not inferior to intravenous ganciclovir in mild to moderate cmv disease in solid organ transplant recipients (asberg et al. 2009 ). the current guidelines recommend renally adjusted intravenous ganciclovir 5 mg/kg twice daily or oral valganciclovir, 900 mg twice daily for mild cmv disease (kotton et al. 2013) . in severe cmv disease, intravenous ganciclovir is preferred with reduction of immunosupression despite the increased risk of rejection (de keyzer et al. 2011) . the use of adjuvant therapy with cmv-specific hyperimmune globulin or standard intravenous immunoglobulin may be considered in individuals with hypogammaglobulinemia, severe systemic infection, or in failure to respond to standard therapy (humar et al. 2010) . cmv resistance to ganciclovir has been noted in renal transplant recipients due to mutations in ul 97, the gene responsible for the first phosphorylation step in ganciclovir activation and ul 54, the gene responsible for dna polymerase (limaye et al. 2000) . cmv resistance should be considered when patients have worsening disease or persistent, unchanged viremia at 2 weeks of therapy and in such cases, genotype testing for mutations of the genes encoding ul 97 and ul 54 should be performed (weikert and blumberg 2008). treatment options for drug resistant cmv include the use of high dose ganciclovir, foscarnet, and cidofovir; however, no clinical trial data are available regarding optimal therapy options for resistant cmv. the use of novel agents including leflunomide and artesunate has been attempted as salvage therapy with varying success. several new antiviral treatment options are currently under investigation including maribavir and brincidofovir (an oral prodrug of cidofovir with less nephrotoxicity) for use in the treatment of drug resistant cmv (limaye et al. 2000) . epstein barr virus -human herpesvirus 4 (ebv) is a ubiquitous gamma herpes virus that remains latent in lymphocytes following primary infection. it is responsible for posttransplant lymphoproliferative disorder (ptld) which increases morbidity and mortality in the transplant population. approximately 62-79% of ptld cases have been associated with ebv (karuthu and blumberg 2012) . ptld most commonly occurs in the first year posttransplant (cockfield et al. 1993) . the risk factors for ptld include ebv naïve recipients who receive ebv seropositive organs, active primary ebv infection, younger recipient, coinfection by cmv and other viruses, prior splenectomy, second transplant, acute or chronic graft versus host disease, immunosuppressive drug regimen (okt3 or polyclonal antilymphocyte antibody), and the type of organ transplanted. kidney transplant recipients are at lower risk compared with other types of transplants and have an incidence of approximately 1-3% (gulley and tang 2010; allen et al. 2009; taylor et al. 2005) . the majority of symptomatic ebv infections in renal transplant recipients are primary infection likely related to transmission of donor virus. ebv disease can be asymptomatic or presents with a nonspecific febrile syndrome, lymphadenopathy, hepatosplenomegaly, atypicalþ lymphocytosis, hematologic disorders including anemia, leukopenia, thrombocytopenia, and organ-specific diseases like gastroenteritis, hepatitis, or pneumonitis (allen et al. 2009 ). ptld typically follows primary infection and frequently presents as a rapidly enlarging mass in the grafted organ, lymph nodes, bone marrow, or extranodal sites (manez et al. 1997) . ptld is divided into four major histopathologic subtypes as per the world health organization (who): early lesions, polymorphic ptld, monomorphic ptld, and classical hodgkin lymphoma type ptld. definitive diagnosis of ptld requires histopathologic confirmation by tissue excision biopsy with immunologic cell typing, cytogenetics, immunoglobulin gene rearrangements, and ebvspecific staining (allen et al. 2009 ). staging is performed by histologic types (monoclonal versus polyclonal, t cell versus b cell) and location (allograft, other organs, metastasis) (weikert and blumberg 2008) . clinical management of ptld typically involves reduction of immunosuppression which can lead to remission in 23-86% of the patients (weikert and blumberg 2008). antiviral therapy with acyclovir or ganciclovir is controversial and no evidence supports its efficacy (taylor et al. 2005) . rituximab (monoclonal antibody to cd20) is commonly used for treatment of ptld in recipients who failed reduction of immunosuppression alone (allen et al. 2009 ). in isolated graft ptld, surgical resection is an option (weikert and blumberg 2008) . in patients that fail the above strategies, ifn and ivig have been used with varying success and cytotoxic chemotherapy with radiation remains salvage therapy (green et al. 2004 ). there is no standardized therapy to prevent ptld. kdigo guidelines recommend monitoring ebv viral load in high risk renal transplant patients within the first week after transplant, then at least monthly for 3-6 months and then every 3 months for the rest of the first posttransplant year. additional viral load monitoring is recommended after treatment for acute rejection in high risk groups (children, ebv dþ/rà). outcomes with ptld in renal transplant patients vary according to the site involved. patients with isolated graft involvement have a 5-year survival of 68% compared with those patients with ptld extending beyond the allograft whose survival varied between 36% and 38% (weikert and blumberg 2008). human herpesvirus 1herpes simplex virus types 1 and 2 (hsv)and human herpesvirus 3varicella zoster virus (vzv)are alpha herpes viruses. hsv 1 has a seroprevalence of 60% in the adult population, while hsv 2 has a seroprevalence of 15% and vzv rates can be as high as 90% (green et al. 2004 ). the incidence of hsv disease in renal transplant recipients is approximately 53% and vzv 4-12% (patel and paya 1997) . hsv may cause primary infection following which the virus remains latent in the sensory nerve ganglia or more commonly causes reactivation infection. hsv may be seen as early as in the first posttransplant month in the absence of prophylaxis. hsv infection usually presents with oral or genital mucocutaneous lesions, occasionally pneumonitis, tracheobronchitis, esophagitis, hepatitis, encephalitis, or disseminated infection (green et al. 2004) . vzv causes localized dermatomal or multidermatomal or disseminated zoster with or without visceral involvement (pneumonitis, hepatitis, pancreatitis, encephalitis). pretransplant screening for prior vzv infection should be performed, and naïve patients should be vaccinated with live attenuated varicella vaccine before transplant whenever possible in order to avoid primary vzv infection posttransplantation (fehr et al. 2002) . since vzv is a live vaccine, it should not be given if transplant is expected within 4-6 weeks in order to avoid active shedding of virus at the time of transplant. posttransplant prophylaxis is recommended with acyclovir, valacyclovir, or ganciclovir (in those who need cmv prophylaxis) for approximately 1-3 months posttransplant in order to avoid hsv and vzv reactivation (green et al. 2004) . diagnosis of hsv and vzv infection can be made with pcr or direct fluorescence antibody for hsv from vesicular lesions, csf, or visceral tissue samples. serologies are rarely helpful in active infection owing to high seroprevalence. kdigo guidelines recommend that renal transplant recipients who develop less severe hsv or vzv infections can be treated with an appropriate oral antiviral agent (e.g., acyclovir, valacyclovir, or famciclovir), and those with systemic infection should be treated with intravenous acyclovir and a reduction in immunosuppressive medication and subsequently switched to an appropriate oral antiviral agent (green et al. 2004 ). the use of foscarnet, cidofovir, or topical trifluridine may be considered in patients with acyclovir resistant virus with careful monitoring of renal functions (kotton and fishman 2005; tan and goh 2006) . human herpesvirus 6 and human herpesvirus 7 (hhv 6 and hhv 7) are ubiquitous with high seroprevalence in adults. these viruses are common causes of fever in children and remain latent in lymphocytes following primary infection. hhv 6 uses the cd46 molecule as its receptor but may also infect other cell types, such as monocytes, and epithelial and endothelial cells. hhv 7 uses the cd4 molecule as its receptor and is more strictly lymphotropic. infection occurs as a result of reactivation in the first 4 weeks following transplant often in recipients not on cmv prophylaxis (singh and carrigan 1996) . clinical manifestations include fever, rash, hepatitis, interstitial pneumonitis, encephalitis, leukopenia, and myelosuppression. owing to its immunomodulatory effects, it is hypothesized that hhv 7 may act as a cofactor for hhv 6 and cmv reactivation, while both hhv 6 and hhv 7 may act as cofactors in the pathogenesis of cmv disease and acute rejection (kidd et al. 2000; chapenko et al. 2000; dockell and paya 2001) . the diagnosis of hhv 6 and hhv 7 is made by tissue immunohistochemistry or nat testing of peripheral blood lymphocytes. treatment includes reduction in immunosuppression and ganciclovir, but cidofovir and foscarnet have also been utilized (green et al. 2004; kotton and fishman 2005; dockell and paya 2001) . hhv 8 is associated with primary effusion lymphoma, kaposi's sarcoma, and multicentric castleman's disease. infection can be acquired as primary through the allograft or through reactivation of latent virus (diociaiuti et al. 2000; regamy et al. 1998 ). hhv 8 causes kaposi's sarcoma, the most common presentation in renal transplant recipients, through upregulation of vascular endothelial growth factor (vegf) receptor f1 k1/kdr in endothelial cells (stallone et al. 2005) . treatment includes reduction in immunosuppression and cytotoxic chemotherapy. sirolimus, an immunosuppressive drug used in renal transplant patients is thought to inhibit not only the production of vegf but also dampens its effect on endothelial cells (stallone et al. 2005 ). bk polyomavirus (bkv) and jc polyomavirus (jcv) belong to the family polyomaviridae. bkv is responsible for causing polyomavirus associated nephropathy (pvan) in 95% of cases and jcv in less than 5% of the cases. pvan occurs in 1-10% of patients with renal transplantation and causes renal allograft loss in 10-80% of cases dadhania et al. 2008) . the risk factors for bkv associated pvan include the use of potent immunosuppressive regimens, caucasian race, older age, diabetes mellitus, cadaveric renal transplant, and combined kidney and pancreas transplant trofe et al. 2003) . bkv is known to cause interstitial nephritis, ureteral stenosis, and ureteral stricture of the allograft kidney most commonly occurring within the first 3-4 months after renal transplant patients when immunosuppression is at its highest (randhawa and brennan 2006) . jcv less commonly causes pvan and is more frequently associated with progressive multifocal leukoencephalopathy (pml), a demyelinating disorder of the white matter presenting as neurologic impairment and dementia (phillips et al. 2004) . diagnosis of bkv includes the use of viral load assays (blood, urine), detection of viral cytopathic effect (decoy cells), nat, bkv-specific antibody, or histopathology (hariharan 2006) . kdigo guidelines recommend screening all renal transplant recipients for bkv with quantitative plasma nat at least monthly for the first 3-6 months after transplantation, then every 3 months until the end of the first posttransplant year, whenever there is an unexplained rise in serum creatinine, and after treatment for acute rejection. the guidelines suggest reducing immunosuppressive medications when bkv plasma nat is persistently greater than 10,000 copies/ ml (107 copies/l) (kdigo 2009). sustained high bk viremia in spite of reduction in immunosuppression may need additional antiviral therapy, although data regarding optimal treatment options are unknown. there are limited data regarding the effectiveness of leflunomide and/or cidofovir or the use of fluoroquinolones or ivig for treatment of bkv infection (randhawa and brennan 2006; josephson et al. 2006) . to date there is no effective treatment for pml. patients with allograft loss due to pvan have undergone successful retransplantation (hariharan 2006) . patients with chronic renal failure, in particular those receiving hemodialysis, are at increased risk for contracting hepatitis b virus (hbv). the prevalence of hepatitis b surface antigen (hbsag)positive patients has declined because of hbv vaccination, strict segregation of hbsag-positive patients in dialysis units, improved screening of blood products, and the use of erythropoiesis stimulating agents (karuthu and blumberg 2012) . approximately 2-10% of patients with a history of hbv prior to transplant will reactivate posttransplant (weikert and blumberg 2008) . serial monitoring of hbv dna every 3-6 months is required after transplantation as liver enzyme levels do not reflect infection status and elevated viral loads suggest resistance to therapy (levitsky et al. 2013) . in a meta-analysis conducted by fabrizi and his colleagues, hbsag seropositivity was an independent risk factor for allograft loss and posttransplant death (fabrizi et al. 2005) . the treatment options currently approved for chronic hbv include: ifn alpha, pegylated ifn, lamivudine, entecavir, telbivudine, tenofovir, and adefovir (fabrizi et al. 2005; chan et al. 2002; chang et al. 2010) . kdigo recommends that interferon treatment generally be avoided because of the high associated incidence of rejection. tenofovir or entecavir are preferable to lamivudine, to minimize the development of drug resistance, unless medication cost requires that lamivudine be used. during therapy with antivirals, hbv dna and alt levels should be measured every 3 months to monitor efficacy and to detect drug resistance. all hbsagpositive renal transplant recipients should receive prophylaxis with tenofovir, entecavir, or lamivudine. hbsag-positive patients with cirrhosis should be screened for hepatocellular carcinoma every 12 months with liver ultrasound and alpha feto-protein. patients who are negative for hbsag and have hbsab titer <10 miu/ml should receive booster vaccination to raise the titer to >100 miu/ ml (kdigo 2009). hepatitis c virus (hcv) infection has been increasingly recognized in end stage renal disease patients (esrd). donor-derived hcv may uncommonly occur after transplantation. screening of patients with esrd and testing renal transplant patients for newly acquired hcv should include nat (levitsky et al. 2013) . hcv-positive donors can be considered for hcv-positive recipients and possibly will be considered for hcv-negative recipients in the future given improved treatment options for cure of hcv that could be administered post transplant. hcv-infected renal transplant recipients have decreased survival and increased complication rates. posttransplant complications include glomerulonephritis (gn), posttransplant diabetes mellitus, and accelerated progression to cirrhosis with fibrosing cholestatic hepatitis (morales et al. 2010) . liver biopsy within 6-12 months of transplantation and subsequent biopsies are required for evaluation of liver disease posttransplant as 20-51% of patients may have normal liver enzyme levels with abnormal histologic features (ashry ahmed gheith 2011). hcv-infected recipients should be tested for proteinuria every 3-6 months, and patients with new onset proteinuria should undergo allograft biopsy (kdigo 2009) . the effect of immunosuppression on the progression of hcv-related liver injury and the management of immunosuppression in the hcvinfected renal transplant recipient remain uncertain. thus, it is preferable to treat hcv in transplant candidates prior to transplantation given the potential for improved outcomes with successful hcv treatment and the complications associated with treatment posttransplant. patients with a sustained virologic response to pretransplant treatment have a reduced risk for hcv recurrence and decreased posttransplant gn (domınguez-gil and morales 2009). options for treatment include interferon/ peginterferon alone or in combination with ribavirin. the risk of toxicity with the addition of ribavirin has limited the use of combination therapy in chronic kidney disease (ckd) patients. the availability of direct acting hcv protease and polymerase inhibitors has sparked new enthusiasm for treating hcv-infected ckd patients and studies are ongoing evaluating the use of these agents in ckd. if treatment cannot be given prior to transplant, kdigo recommends monotherapy with standard interferon for hcv-infected renal transplant recipients in whom the benefits of antiviral treatment clearly outweigh the risks (kdigo 2009). the use of direct acting hcv antivirals posttransplantation can also be considered and will likely be preferred in the future given improved tolerance and efficacy with these agents with an understanding that drug interactions with calcineurin inhibitors may occur.a study looking at 20 hcv-positive kidney transplant recipients (60% treated pre-transplant with interferon unsuccessfully) treated with direct acting antivirals posttransplant found that 100% cleared the virus and had a sustained virologic response at 12 weeks. the most common agents used were sofosbuvir and simeprevir (sawinski et al. 2016 ). human immunodeficiency virus (hiv) belongs to the family of retroviridae. with the introduction of antiretroviral therapy (art) in the mid-1990s, the incidence of hiv related deaths has been reduced. renal diseases related to hiv infection include hiv associated nephropathy (hivan), immune complex diseases, and thrombotic microangiopathy (frassetto et al. 2009 ). a total of 10% of patients with hiv develop hivan and it remains an important complication of hiv infection, progressing rapidly to end stage renal disease (esrd) (shahinian et al. 2000) . a large prospective clinical trial examining outcomes among 150 hiv + kidney transplant recipients reported 3-year patient and graft survival rates of 88.2% and 73.7%, respectively, which were similar to survival rates among a cohort of unmatched elderly (>65 years) hivnegative (hiv à ) kidney recipients (stock et al. 2010 ). the candidates for transplantation include those with well-controlled hiv infection with undetectable viral loads, cd4 >200 cells per microliter, and absence of untreatable infections or malignancies (blumberg et al. 2009 ). the most significant complications in this patient population posttransplant include increased rejection rates (up to 25%), managing drug interactions between art and immunosuppressive therapy and complications related to cardiovascular risk factors and hepatitis coinfection (blumberg et al. 2009 ). the choice of art should be based on susceptibility results and if possible, the use of protease inhibitors should be avoided owing to significant drug interactions with this class of art. with regards to immunosuppressive therapy, the use of thymoglobulin may result in prolonged depression of cd4 counts, whereas monocloncal anti-il2 receptor antibodies, such as basiliximab/daclizumab, have been shown to increase cd4 cell counts (ciuffreda et al. 2007; carter et al. 2006) . the risks of antilymphocyte therapy should be balanced with the risks of rejection in hiv-infected recipients. of note, hiv-positive donors can now be considered in hivpositive recipients. the various respiratory viruses that cause infection affecting the renal transplant patient population include adenovirus, respiratory syncytial virus (rsv), influenza, parainfluenza, human metapneumovirus, rhinovirus, and coronavirus (green et al. 2004) . clinical manifestations include upper respiratory tract infection, bronchitis, and pneumonia. in addition to respiratory illness, adenovirus is known to cause gastroenteritis, hemorrhagic cystitis, pancreatitis, meningoencephalitis, necrotizing hepatitis, and nephritis/renal dysfunction in renal transplant recipients (pham et al. 2003; alsaad et al. 2007) . infection with these viruses may also be associated with rejection (weikert and blumberg 2008) . prevention involves hand hygiene and the use of droplet precautions for those suspected of having infection. influenza vaccination is recommended prior to transplant and yearly following transplant. treatment of respiratory viral infection involves supportive care and antiviral medications. influenza can be treated with oseltamivir or zanamavir. ribavirin is approved for the treatment of rsv. adenovirus infection is treated with reduction of immunosuppression with consideration of cidofovir (ison 2006) . emerging viral pathogens include newly recognized viruses or previously known viruses that are either increasing or threatening to increase in incidence. some of the emerging viruses causing infections in renal transplant population include human t-cell leukemia virus type 1 (htlv-1), hepatitis e virus (hev), measles virus, rabies virus, lymphocytic choriomeningitis virus (lcmv), dengue virus (denv), west nile virus, and zika virus. case reports of adult t-cell leukemia (atl) following renal transplantation in htlv-1-positive patients have been documented, though in a case series of renal transplant recipients with long-term follow-up, no cases of atl or htlv-1-associated myelopathy (ham) developed (nakamura et al. 2005; tanabe et al. 1998) . hev may induce kidney injury with significant reduction in glomerular filtration rate. glomerular injuries such as membranoproliferative glomerulonephritis have been described in kidney transplant patients with acute and chronic hev infections (kamar et al. 2012) . the incidence of measles in transplant recipients is unclear. cases of subacute measles encephalitis (sme) have developed in renal transplant recipients. the clinical course is one of deteriorating mental status and treatment refractory seizures (waggoner and deresinski 2013) . worldwide, vector-borne viral disease is increasing in incidence and can be transmitted with blood products and organ transplantation. fatal cases of dengue have been reported within the first month following renal transplant (waggoner and deresinski 2013) . west nile virus has also been reported in transplant recipients with a high incidence of neuroinvasive disease and poor outcomes. zika virus is also now a concern. cases of donor-derived rabies in the sot population have been reported. patients typically developed encephalitis between 1 and 2 months posttransplant, and all symptomatic reported patients died (srinivasan et al. 2005) . cases of lcmv causing severe disease in organ transplant patients have been documented. the 4 clusters of lcmv infection occurred in the united states and involved kidney, liver, and lung transplants; symptoms included fever, abdominal pain, nausea, diarrhea, and altered mental status (srinivasan et al. 2005; barry et al. 2008; fischer et al. 2006) . two renal transplant recipients survived lcmv infection. ribavirin has been employed in some cases, though the benefits remain unclear (waggoner and deresinski 2013) . data regarding the incidence, screening and treatment options of the above-mentioned emerging viruses are limited. given the risk of donor-derived viral transmission, organs should not be accepted from donors with unexplained febrile or neurologic illness. in unclear cases, the risk of donor-derived infection should be balanced with the benefit of the transplant. bacterial infections after renal transplantation can be due to surgical complications at the time of transplantation, nosocomial infection, immunosuppression, or community-acquired infection. donorderived bacterial infections from the transplanted kidney or blood stream can occur as well. about 47% of kidney transplant recipients develop bacterial infections (patel and paya 1997) . occurring any time posttransplantation, urinary tract infections account for the overwhelming majority of these infections and are the most common bacterial infections prolonging or leading to re-hospitalization (wyner 1994) . enterococci, staphylococci, enteric gram-negative organisms, and p. aeruginosa are the most common bacteria isolated (wyner 1994) . bacterial pneumonia, postoperative wound infections, and bacteremia or sepsis, although less common, also prolong or lead to rehospitalizations after transplantation (karuthu and blumberg 2012) . common bacterial pathogens for these infections are gram-negative organisms, including multidrug resistant bacteria; gram positive organisms, including methicillin-resistant staphylococcus aureus (mrsa), and vancomycin-resistant entercococci (vre), as well as organisms more typically seen in immunocompromised patients such as listeria. months after the operation, bacterial pathogens include streptococcus species, mycoplasma, legionella, listeria, salmonella, and nocardia. trimethoprim-sulfamethoxazole (tmp-smx) prophylaxis has been shown to reduce the incidence of some of these infections. increased antimicrobial resistance, urgency of treatment, drug interactions, and toxicities, as well as the risk for clostridium difficile colitis all contribute to the complex decision making required for antimicrobial management. risk factors for urinary tract infection after transplantation are a prolonged period of hemodialysis before transplant, prolonged bladder catheterization, female sex, deceased donor transplant, kidney-pancreas transplant with bladder drainage, uretero-vesical stents, and an increased immunosuppressed state (karuthu and blumberg 2012; lapchik et al. 1992) . prophylaxis to lower the risk of infection after transplant with trimethoprim-sulfamethoxazole is routine (karuthu and blumberg 2012) . controversy regarding the exact dosing and duration of prophylaxis exists. typically it is given at a dose of 160 mg trimethoprim and 800 mg of sulfamethoxazole daily for 6-12 months (kdigo 2009 ). trimethoprim-sulfamethoxazole reduces the risk of uti and bacteremia (karuthu and blumberg 2012; patel and paya 1997) . symptoms of uti include frequency, urgency, and dysuria as well as nausea and vague abdominal complaints. some patients are asymptomatic. escherichia coli is the most common pathogen and an increasing number of pathogens are multidrug resistant. sensitivity testing is required. treatment of asymptomatic bacteriuria in the renal transplant recipient is controversial and is not routinely recommended (coussement and abramowicz 2013) . although not well studied, since utis in renal transplant patients are complicated, 7-14 days of antibiotics is a typical duration. removal of stents and catheters as well as drainage of abscesses are frequently required to prevent relapse and for cure. surgical wound infections, occurring at a rate of 3-4%, usually present within the first 4 weeks after transplant (ramos et al. 2008) . obesity, urine leaks, re-operation through the original incision, diabetes, high creatinine levels in plasma, and prolonged bladder catheterization are risk factors for wound infections (humar et al. 2001; khoury and brennan 2005) . improved organ procurement, preservation, and surgical techniques along with preoperative antibiotics all reduce the risk of subsequent postoperative wound infection. bacterial organisms causing these types of infections may be nosocomial and multidrug-resistant making antibiotic treatment difficult due to limited options or toxicities. source control with good wound care is critical in the management of these types of infections. although pneumonia is the most common bacterial infection in all solid organ transplant recipients, its incidence is lowest in those who have received a kidney (khoury and brennan 2005) . occurring early in the posttransplant period, cmv infection and rejection treatment with antilymphocyte preparations increase the pneumonia risk. hospital-acquired pneumonia due to resistant pathogens, such as mrsa, and extended spectrum beta lactamase (esbl) or carbapenemresistant (cre) gram-negative organisms are increasing in incidence and sometimes require nephrotoxic agents for treatment. communityacquired pneumonia can occur any time after transplantation and the incidence of communityacquired pneumonia specifically due to streptococcus pneumoniae can be lowered with vaccination. bacteremia and sepsis are most commonly due to a urinary source, followed by lung, wound, and abdomen (khoury and brennan 2005) . intravenous catheters also play a role. diabetes mellitus and posttransplant dialysis increase the incidence of sepsis which decreases the survival rate in these patients (abbott et al. 2001) . prompt treatment with broad spectrum antibiotics followed by rapid de-escalation to pathogen-specific therapy based on sensitivities is required. removal of foreign bodies such as intravenous catheters and stents is also necessary for cure. nocardia is a rare infection seen in the renal transplant recipient occurring in less than 4% of patients (wilson et al. 1989 ). trimethoprim-sulfamethoxazole prophylaxis used after transplant to prevent pneumocystis jiroveci pneumonia (pcp) likely prevents nocardia infection as well. nocardia asteroides is the most common species and causes pulmonary infections, including cavitary lesions and pleural effusions (patel and paya 1997) . other common sites of infection, due to dissemination, are central nervous system (cns) and cutaneous. all patients with nocardia should be evaluated for cns disease. allograft rejection, high-dose prednisone, azathioprine, instead of cyclosporine based immunosuppression, and neutropenia are risk factors for this infection (patel and paya 1997) . diagnosis is made by the identification of branching and beading rods on gram and modified acid fast staining and cultures of infected sites. antimicrobial susceptibility testing should be performed on all isolates. high dose trimethoprim-sulfamethoxazole sometimes in combination with amikacin is the treatment of choice, but allergic reactions and other side effects sometimes limit their use. alternatives include imipenem, minocycline, and ceftriaxone, but choices should be based on susceptibilities and site of infection (spelman 2016) . nocardia infections can relapse and prolonged therapy up to a year is recommended followed by chronic suppressive therapy (spelman 2016; arduino et al. 1993 ). listeria monocytogenes is a bacterial organism that is transmitted most commonly during summer and early fall to humans via the gastrointestinal tract from contaminated dairy products, raw vegetables, and meat. although more common during the first 2 months after transplantation, infection may occur at any point, and risk is increased with rejection therapy (patel and paya 1997) . infections involving the central nervous system, such as meningitis and meningoencephalitis, are most common and present with headaches, fever, meningismus, altered mental status, and possibly focal neurologic deficits including cranial nerve palsies and seizures (patel and paya 1997) . cerebrospinal fluid examination typically reveals a pleocytosis, mostly polymorphonuclear leukocytes, decreased glucose, and elevated protein, but as the name implies, a mononuclear predominance may occur instead. gram staining has a low sensitivity and may be negative or reveal gram positive bacilli which may be confused with diphtheroids. other sites of infection include bacteremia, pneumonia, endophthalmitis, and septic arthritis. while trimethoprim-sulfamethoxazole, used for p. carinii prophylaxis, may also prevent infection with listeria, the treatment of choice is intravenous ampicillin and gentamicin for up to 8 weeks in those with cns infections to prevent relapses. gentamicin is usually continued for a shorter duration, about 2 weeks if kidney function is stable. (gelfand 2016) . trimethoprim-sulfamethoxazole is an alternative treatment for those who are allergic to penicillin. decreasing immunosuppressive agents is sometimes, but not always necessary. legionella infections in renal transplant recipients most commonly occur early in the posttransplantation period, but can be seen any time, especially during episodes of rejection. legionella pneumophila is the most common species to infect humans, and although more commonly community-acquired, nosocomial transmission occurs (patel and paya 1997) . most infections are pulmonary including pneumonia, and abscess with cavitation. symptoms are typical of lung infections but also may include headache and diarrhea. a legionella urinary antigen test and culture of lower respiratory secretions on selective media are used for diagnosis. empiric treatment for legionella is appropriate while waiting for results. quinolone antibiotics, such as levofloxacin, are preferred over macrolides in renal transplant patients because of drug interactions between macrolides and immunosuppressive medications. initially given intravenously, quinolone antibiotics can be quickly deescalated to oral treatment when the patient has defervesced. renal transplant patients, especially those who are severely ill at presentation, should receive 21 days of treatment (yu 2016) . along with pcp and listeria, as noted above, prophylaxis with trimethoprim-sulfamethoxazole may also prevent legionella infection. immunosuppression increases the risk of developing mycobacterium tuberculosis (tb) disease. although the majority of tuberculosis infections in renal transplant recipients occur in the first 18 months, tb can occur any time after transplantation (khoury and brennan 2005) . its overall incidence is lower in the united states when compared to the rest of the world, and foreign-born recipients are at greatest risk. having a high index of suspicion is important in renal transplant patients because presentation can be atypical and pretransplant screening with tuberculin skin tests or ifn-gamma release assays are unreliable in chronic kidney disease patients due to anergy. extra-pulmonary sites of infection and disseminated disease occur in about a third of cases (karuthu and blumberg 2012). laryngeal, meningeal, skeletal, cutaneous, intestinal, and renal infections are examples of extra-pulmonary disease. fevers are common, but sweats and weight loss may be absent (patel and paya 1997) . screening prior to transplant should include a history regarding prior exposures, and treatment for tb, as well as a chest x-ray and urine afb culture. prophylaxis with isoniazid or rifampin should be offered to patients prior to transplantation with a history of inadequately treated tb, an abnormal chest x-ray suggestive of prior tb exposure, a positive ppd or ifn gamma assay, contact with someone with active tb, or a kidney from a ppd-positive donor in order to minimize reactivation disease after transplantation (khoury and brennan 2005) . patients receiving treatment for latent tb may undergo renal transplantation and complete their defined course afterwards with special attention to potential drug interactions and toxicities (karuthu and blumberg 2012) . diagnosis of tb after renal transplantation often requires a biopsy of the infected site with stains for acid fast bacilli and cultures for sensitivity testing. treatment of active disease after transplantation requires multiple drugs and should follow the american thoracic society, center for disease control, and infectious disease society of america guidelines (mmwr 2003) . special attention to drug toxicities and interactions with immunosuppressive agents is required. rifampin, in particular, decreases cyclosporine levels and increases the risk for rejection. fungal infections in kidney transplant recipients occur less frequently than in other solid organ transplant recipients. most present within the first 6 months after transplantation (hagerty et al. 2003 ) and can represent primary, reactivated, or donor-derived infection. those associated with geographic and environmental exposures include histoplasmosis, coccidioidomycosis, blastomycosis, and paracoccidioidomycosis. others are considered opportunistic and include infections such as candida, aspergillus, and cryptococcus (karuthu and blumberg 2012) . broad spectrum antibiotics, corticosteroids, diabetes mellitus, rejection therapy, cmv infection, and duration of pretransplant dialysis are risk factors (khoury and brennan 2005) . esophageal candidiasis, urogenital candidiasis, and pneumonia are the three most common sites of fungal infections in these patients (abbott et al. 2001) . clinical presentation may be nonspecific and diagnosis difficult due to testing limitations. positive cultures may represent colonization rather than infection with pathogens such as candida and aspergillus. cultures, antigen assays, serum galactomannan assays, and radiography may be helpful, but are not always diagnostic. subsequently, biopsy with pathology and cultures is considered the gold standard for diagnosing fungal infections (karuthu and blumberg 2012) . drug interactions and toxicities as well as immune reconstitution, due to lowering of immunosuppressive medications, further complicate the management of fungal disease in these patients and require expert advice (karuthu and blumberg 2012) . pneumocystis jiroveci (formerly pneumocystis carinii (pcp), protozoa) is a pathogen currently considered a fungus based on nucleic acid and biochemical analysis. presenting as pneumonia with interstitial infiltrates on chest x-ray within the first year after transplantation in those not receiving prophylaxis, mortality may be high. nonproductive cough and shortness of breath with rapid progression to hypoxia is a classic presentation. diagnosis is based on silver staining of deep respiratory specimens from induced sputum, bronchoalveolar lavage, or transbronchial biopsy (martin and fishman 2013) . the treatment of choice is high dose trimethoprim-sulfamethoxazole for 21 days with corticosteroids in hypoxic patients (partial pressure of oxygen of <70 mmhg on room air) tapered over 14 days. atovaquone or clindamycin plus pyrimethamine are alternative agents (martin and fishman 2013) . trimethoprim-sulfamethoxazole prophylaxis for 6-12 months after transplantation is highly effective in preventing this infection and should be administered to all renal transplant patients if tolerated. frequently used alternatives for prophylaxis in allergic patients include dapsone (if glucose-6 phosphate dehydrogenase levels are normal) and atovaquone. infection remains an important concern in patients undergoing kidney transplantation. attention to pretransplant screening of the potential organ donor and recipient is essential to optimizing transplant outcomes. advances in the management of transplant-related infections include the increasing use of rapid molecular diagnostic testing as well as improvements in the approach to prophylaxis and treatment. ongoing challenges include the need for prolonged immunosuppression to prevent organ rejection, drug-drug interactions, and the management of resistant and emerging pathogens. continued awareness of the risks, timing, and presentation of infection posttransplant and strategies to reduce its impact will contribute further to progress in the field of kidney transplantation. hospitalizations for bacterial septicemia after renal transplantation in the united states epstein-barr virus and posttransplant lymphoproliferative disorder in solid organ transplant recipients late-onset acute haemorrhagic necrotizing granulomatous adenovirus tubulointerstitial nephritis in a renal allograft nocardiosis in renal transplant recipients undergoing immunosuppression with cyclosporine long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients dilemma of hcv infection in renal transplant recipients lymphocytic choriomeningitis virus transmitted through solid organ transplantation -massachusetts solid organ transplantation in the hiv-infected patient cytomegalovirus in renal transplantation thymoglobulin-associated cd4þ t-cell depletion and infection risk in hiv-infected renal transplant recipients hiv transmitted from a living organ donor recommended adult immunization schedule-united states preemptive lamivudine therapy based on hbv dna level in hbsag-positive kidney allograft recipients entecavir treatment for up to 5 years in patients with hepatitis b e antigen-positive chronic hepatitis b co-infection of two β-herpesviruses (cmvand hhv-7) as an increased risk factor for "cmv disease" in patients undergoing renal transplantation effects of immunosuppressive drugs on hiv infection: implications for solid-organ transplantation post-transplant lymphoproliferative disorder in renal allograft recipients: clinical experience and risk factor analysis in a single center should we treat asymptomatic bacteriuria after renal transplantation? epidemiology of bk virus in renal allograft recipients: independent risk factors for bk virus replication human cytomegalovirus and kidney transplantation: a clinician's update risk factors for hospitalization for bacterial or viral infection in renal transplant recipients-an analysis of usrds data efficacy of high-dose versus standard-dose influenza vaccine in older adults hhv 8 in renal transplant recipients human herpesvirus-6 and-7 in transplantation transplantation in the patient with hepatitis c 24-week oral ganciclovir prophylaxis in kidney recipients is associated with reduced symptomatic cytomegalo-virus disease compared to a 12-week course polyomavirus disease in renal transplantation: review of pathological findings and diagnostic methods hbsag seropositive status and survival after renal transplantation: meta-analysis of observational studies disseminated varicella infection in adult renal allograft recipients: four cases and a review of the literature transmission of lymphocytic choriomeningitis virus by organ transplantation ast infectious diseases community of practice (2013) screening of donor and recipient in solid organ transplantation infection in solid organ transplant recipients renal transplantation in patients with hiv clinical manifestations and diagnosis of listeria monocytogenes infection guidelines for the prevention and management of infectious complications of solid organ transplantation using epstein-barr viral load assays to diagnose, monitor, and prevent posttransplant lymphoproliferative disorder fungal infections in solid organ transplant patients bk virus nephritis after renal transplantation polyoma-virus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations asymptomatic cytomegalovirus infection is associated with increased risk of new-onset diabetes mellitus and impaired insulin release after renal transplantation antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients ast infectious diseases community of practice: cytomegalovirus in solid organ transplant recipients are wound complications after a kidney transplant more common with modern immunosuppression the efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients solid organ transplantation from hepatitis b virus-positive donors: consensus guidelines for recipient management adenovirus infections in transplant recipients vaccination and chronic kidney disease polyomavirus-associated nephropathy: update on antiviral strategies hepatitis e virus and the kidney in solid-organ-transplant patients common infections in kidney transplant recipients the value of vaccination in chronic kidney disease kdigo clinical practice guideline for the care of kidney transplant recipients infectious complications in kidney transplant recipients: review of the literature prospective study of human betaherpesviruses after renal transplantation. association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection improvement in long-term renal graft survival due to cmv prophylaxis with oral ganciclovir: results of a randomized clinical trial intervals between pcv13 and ppsv23 vaccines: recommendations of the advisory committee on immunization practices (acip) viral infection in the renal transplant recipient transplantation society international cmv consensus group: international consensus guidelines on the management of cytomegalovirus in solid organ transplantation selecting suitable solid organ transplant donors: reducing the risk of donor-transmitted infections risk factors for nosocomial urinary tract and postoperative wound infections in renal transplant patients: a matched-pair case-control study recommendations for screening of donor and recipient prior to solid organ transplantation and to minimize transmission of donor-derived infections infectious diseases community of practice (2013) viral hepatitis in solid organ transplant recipients emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants posttransplant lymphoproliferative disease in primary epstein-barr virus infection after liver transplantation: the role of cytomegalovirus disease american society of transplantation and american society of transplant surgeons, pneumocystis pneumonia in solid organ transplantation cytomegalovirus seromismatching increases the risk of acute renal allograft rejection allowing hiv-positive organ donation: ethical, legal and operational considerations renal transplantation in patients with hepatitis c virus antibody. a long national experience hiv-positive-to-hiv-positive kidney transplantation-results at 3-5 years prevention and management of cytomegalovirus infection in solid-organ transplantation prognosis of htlv-i-positive renal transplant recipients pre-emptive treatment for cytomegalovirus viraemia to prevent cytomegalovirus disease in solid organ transplant recipients infections in solid-organ transplant patients valganciclovir solid organ transplant study group: efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cyto-megalovirus disease in solid organ transplant recipients fatal disseminated adenovirus infections in immunocompromised patients polyoma nephropathy and progressive multifocal leukoencephalopathy in a renal transplant recipient cmv infection is associated with transplant renal artery stenosis incisional surgical site infection in kidney transplantation bk virus in transplant recipients: an overview and update transmission of human herpesvirus 8 infection from renal-transplant donors to recipients impact of early cytomegalovirus infection and disease on longterm recipient and kidney graft survival resitra network of the spanish study group of infection in transplantation (2008) impact of current transplantation management on the development of cytomegalovirus disease after renal transplantation successful treatment of hepatitis c in renal transplant recipients with direct-acting antiviral agents phs guideline for reducing human immunodeficiency virus, hepatitis b virus, and hepatitis c virus transmission through organ transplantation prevalence of hiv-associated nephropathy in autopsies of hiv-infected patients human herpesvirus-6 in transplantation: an emerging pathogen rates of first infection following kidney transplant in the united states up to date transmission of rabies virus from an organ donor to four transplant recipients sirolimus for kaposi's sarcoma in renal transplant recipients outcomes of kidney transplantation in hiv-infected recipients preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients viral infections affecting the skin in organ transplant recipients: epidemiology and current management strategies long-term results in human t-cell leukemia virus type 1-positive renal transplant recipients post-transplant lymphoproliferative disorders (ptld) after solid organ transplantation the association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation polyomavirus in kidney and kidney-pancreas transplant recipients epidemiology of kidney disease in the unites states. national institutes of health, national institute of diabetes and digestive and kidney diseases in: safdar a (ed) principles and practice of transplant infectious diseases nocardial infections in renal transplant recipients the evaluation and management of urinary tract infections in recipients of solid organ transplants treatment and prevention of legionella infection key: cord-016343-wc3i54fc authors: frese, michael; dazert, eva title: interferon-induced effector proteins and hepatitis c virus replication date: 2008 journal: hepatitis c virus disease doi: 10.1007/978-0-387-71376-2_6 sha: doc_id: 16343 cord_uid: wc3i54fc hepatitis c virus (hcv) is a small, enveloped rna virus that is often capable of establishing a persistent infection, which may lead to chronic liver disease, cirrhosis, hepatocellular carcinoma, and eventually death. for more than 20 years, hepatitis c patients have been treated with interferon-alpha (ifn-α). current treatment usually consists of polyethylene glycol-conjugated ifn-α that is combined with ribavirin, but even the most advanced ifn-based therapies are still ineffective in eliminating the virus from a large proportion of individuals. therefore, a better understanding of the ifn-induced innate immune response is urgently needed. by using selectable self-replicating rnas (replicons) and, more recently, recombinant full-length genomes, many groups have tried to elucidate the mechanism(s) by which ifns inhibit hcv replication. this chapter attempts to summarize the current state of knowledge in this interesting field of hcv research. interferons (ifns) are a diverse class of cytokines with key functions in the innate immune response to viruses (reviewed in pestka et al., 2004; goodbourn et al., 2000; samuel, 2001) . three types of ifns can be distinguished that have partially overlapping biological properties. type i ifns are secreted by most virus-infected cells and by a highly specialized leukocyte population, termed natural ifn-producing cells or plasmacytoid dendritic cells (colonna et al., 2002) . the human genome contains many type i ifn genes encoding 12 ifn-α subtypes, ifn-β, ifn-ε, ifn-κ, and ifn-ω. the reason why the human genome encodes so many ifn-α subtypes is not known but has been speculated that different subtypes elicit a slightly different antiviral response. furthermore, it is tempting to speculate that the most recent multiplication of ifn-α genes is a consequence of an ongoing arms race between viruses that encode soluble ifn receptors and the innate immune defense system. type i ifn genes differ from all other ifn genes by the fact that they lack introns. recently, three distantly related cytokines have been identified that share sequence similarities with type i ifns and the interleukin-10 (il-10) family. accordingly, these cytokines have been named ifn-λ1, ifn-λ2, and ifn-λ3 (kotenko et al., 2003) or il-29, il-28a, and il-28b, respectively (sheppard et al., 2003) . although it seems that many biological properties of this most recently discovered group of ifn-like cytokines resemble those of type i ifns, they are referred to as type iii ifns. in contrast to types i and iii ifns, of which numerous genes have been identified, the human genome contains only one type ii ifn gene. the gene product, ifn-γ, is only expressed in specialized immune cells such as activated t lymphocytes and natural killer (nk) cells. all types of ifns bind to highly specific cell surface receptors that trigger the phosphorylation and nuclear translocation of a family of latent transcription factors, known as signal transducers and activators of transcription (stats). type i ifns bind to the ifn-α receptor (ifnar), which leads to the formation of the ifn-stimulated gene factor-3 (isgf-3), a heterotrimer consisting of stat1, stat2, and ifn-response factor-9 (irf-9/p48). isgf-3 activates gene transcription via the ifn-stimulated response element (isre). type iii ifns bind to a different receptor complex consisting of the ifnlr1/il-28rα subunit and the il-10β subunit (donnelly et al., 2004) but nevertheless trigger a signaling cascade that is very similar to that of type i ifns (doyle et al., 2006) . a slightly different signaling pathway has been described for ifn-γ. the type ii ifn binds to the ifn-γ receptor (ifngr) which leads to the phosphorytation of the gamma activation factor (gaf), a phosphorylated stat1 homodimer, is translocated to the nucleus, where it enhances gene expression by binding to the gamma activation site (gas). beside these wellestablished signaling pathways, alternative pathways have been described, but their contribution to the antiviral activity of ifn remains to be further elucidated (pestka et al., 2004) . types i and iii ifns are believed to execute their antiviral activities through the induction of proteins that accumulate inside an infected host cell. these effector proteins may interfere with distinct steps in viral replication or trigger the degradation of viral rnas. by contrast, ifn-γ predominantly induces the expression of proteins with systemic functions, such as those involved in antigen processing and presentation. in addition, ifn-γ induces the expression and release of chemokines that activate and orchestrate the adaptive immune response (e.g., . however, at least in some virus infections, ifn-γ may also contribute to the establishment of an antiviral state by the induction of proteins with direct antiviral activities (reviewed in guidotti & chisari, 2001) . of note, all ifns that have been tested so far inhibit hepatitis c virus (hcv) rna replication in cultured cells, although differences have been noted in respect to the ic 50 and the kinetics of inhibition. hcv is a member of the genus hepacivirus that belongs to the family flaviviridae (van regenmortel et al., 2000) . hcv isolates can be grouped into at least 6 genotypes that differ in their nucleotide sequence by 31% to 34%. furthermore, within a given genotype, subtypes can be defined that differ in their nucleotide sequence by 20% to 23%. different genotypes show a remarkable degree of heterogeneity with respect to antiviral treatment (mchutchison & fried, 2003) . for example, only 50% of patients infected with genotype 1 mount a sustained antiviral response, whereas 80% to 90% of those infected with genotype 2 and genotype 3 viruses do so. this is of immediate medical significance, and numerous attempts have been made to identify the viral factor(s) that determine the outcome of current ifn therapies. the underlying molecular mechanisms are, however, still controversial. hcv has a ∼9.6-kb single-stranded rna genome of positive polarity (reviewed in bartenschlager et al., 2004) . the genome encodes a large polyprotein that is coand post-transcriptionally cleaved by cellular and viral proteinases into 10 proteins (core, e1, e2, p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b). the production of an additional protein (f) by ribosomal frame shifting has been reported, but its function remains to be defined. the coding sequence is flanked by nontranslated regions (ntrs) that are important for rna translation (5 ntr) and replication (5 and 3 ntr). both ntrs are highly structured and contain numerous stem loops, most notably in the internal ribosome entry site (ires) of the 5 ntr (honda et al., 1999) but also in other regions such as the x-tail sequence of the 3 ntr (blight & rice, 1997) . furthermore, ntr sequences have been shown to interact with complementary coding sequences, which further increases the amount of intramolecular base pairing (kim et al., 2003; friebe et al., 2005) . figure 1 shows the organization of the hcv genome and depicts some of its key structural elements. the figure also summarizes major protein functions, including those that counteract the innate immune response. hcv replication takes place in the cytoplasm of persistently infected hepatocytes, the principal host cells of the virus. detailed information on the mode of rna replication is not available for hcv, but by analogy to other flaviviruses (westaway et al., 2002) , it has been proposed that the incoming positive-stranded rna genome is used as a template for the synthesis of a negative-stranded rna molecule that remains base-paired with its template. the resulting double-stranded rna (also called "replicative form") is then transcribed multiple times, which results in the generation of numerous full-length, positive-stranded progeny rnas that may used for replication, translation, or packaging into newly formed virus particles (for details, see bartenschlager et al., 2004) . double-stranded rna that is formed by intramolecular base pairing between complementary sequences of positive-stranded hcv rnas or during viral replication should alert the double-stranded rna detection system of the host cell. this would the virus genome contains a large open reading frame (orf) that encodes all major viral proteins and an alternative orf that encodes the frame shift protein (f), which has an unknown function. the structural proteins c, e1, e2, and p7 are liberated from the polyprotein by cellular signal peptidases, and all other cleavages are performed by viral proteases. rna secondary structures are drawn according to blight & rice (1997) , honda et al. (1999) , you et al. (2004), and friebe et al. (2005) . black dots indicate the position of the start and the stop codon of the large orf. the minimum regions in the 5 and 3 nontranslated regions (ntrs) required for replication and initiation of translation are encircled with dotted lines. rdrp, rna-dependent rna polymerase; ires, internal ribosome entry site; 5bsl3.2, stem loop 3.2 within the coding sequence of ns5b normally result in the production of type i ifns and the subsequent expression of ifn-induced effector proteins, which in turn would establish an antiviral state in the ifn-producing cell itself and in neighboring cells. hcv-infected hepatocytes, however, seem not to produce much ifn, as liver biopsy samples of most chronic hepatitis c patients lack detectable amounts of type i ifn mrnas (mihm et al., 2004) . this finding is in line with the observation that cultured human hepatoma cells produce only small amounts of ifns in response to viral infections or other stimuli such as poly(inosine[i])-poly(cytosine[c]), suggesting that human hepatocytes are generally rather poor ifn producers (keskinen et al., 1999) . however, primary chimpanzee and tamarin hepatocytes were found to be highly responsive to poly(i)-poly(c), which raises the question of why the liver of chronic hepatitis c patients does not contain type i ifns (lanford et al., 2003) . this enigma has recently been solved by foy and co-workers, who reported that the hcv ns3/4a protease interferes with the ability of cells to sense double-stranded rna (foy et al., 2003) . ns3/4a-mediated cleavage of the adaptor protein trif reduces its abundance and inhibits poly(i)-poly(c)-activated signaling through the toll-like receptor 3 pathway before its bifurcation to irf-3 and nuclear factor-κb (nfκb)-mediated gene activation pathways (li et al., 2005) . furthermore, ns3/4a cleaves the adapter protein mavs/ips-1/visa/cardif, which interrupts the signaling between the doublestranded rna binding protein rig-i and kinases that phosphorylate the ifn regulatory factors irf-3 and irf-7 (meylan et al., 2005) . this act of sabotage efficiently prevents the nuclear import of the latent transcription factors irf-3 and irf-7, a crucial step in the activation of type i ifn gene transcription (reviewed by hiscott et al., 2006) . taken together, these findings suggest that hcv-infected hepatocytes are prevented from producing the amount of type i ifn that is needed to assist virus clearance. nevertheless, type i ifn-induced mrnas/proteins are readily detectable in liver biopsies of hepatitis c patients, even in liver samples that do not contain detectable amounts of type i ifn mrnas (mihm et al., 2004) . this begs the question as to where the ifn that is not locally produced comes from. according to mihm and co-workers, natural ifn-producing cells or plasmacytoid dendritic cells may represent an important extrahepatic source of ifn in hepatitis c patients (mihm et al., 2004) . however, natural ifn-producing cells have the propensity to migrate to secondary lymphoid organs rather than to sites of inflammation (penna et al., 2002) . as a consequence, the expression of type i ifn-induced proteins may never reach levels required to eliminate the virus from already infected cells and/or to prevent the infection of new host cells. all currently licensed hcv therapies rely on the antiviral activity of type i ifn (mostly polyethylene glycol-conjugated ifn-α2) that is given alone or in combination with ribavirin. the administration of recombinant ifn bypasses the block of ifn production in hcv-infected host cells and dramatically increases the expression of type i ifn-induced proteins throughout the body. this leads in most cases to a rapid decline of hcv rna levels (first-phase response), which is believed to reflect an inhibition of virus replication. later on, hcv rna levels decline more gradually (second-phase response) as the liver is cleared of virus-infected cells (neumann et al., 1998; layden & layden, 2002) . although ifn-α initially reduces the viral load in almost all patients, a sustained response (as defined by the loss of detectable hcv rna during therapy and its continued absence for at least 6 months after the treatment has been ended) is not experienced by all patients. especially those patients who suffer from an infection with genotype 1b viruses often fail to eradicate the virus (manns et al., 2001; fried et al., 2002) . the correlation between therapy success and the infecting genotype suggests the involvement of viral factors, but the underlying molecular mechanisms are not yet understood. with the development of hcv replicons (lohmann et al., 1999) , it became possible to analyze the role of individual cytokines in the innate immune response against hcv. because most patients respond, at least initially, to a treatment with ifn-α, it was not unexpected that this ifn and other type i ifns also block rna replication of different hcv genotypes in human hepatoma cells (blight et al., 2000; frese et al., 2001; guo et al., 2001; cheney et al., 2002; larkin et al., 2003; okuse et al., 2005; windisch et al., 2005; miyamoto et al., 2006) and in cells of nonhepatic origin, e.g., hela cells (guo et al., 2003) and 293 cells (ali et al., 2004) . similar results were obtained by using type iii ifns (robek et al., 2005; marcello et al., 2006) and ifn-γ (cheney et al., 2002; frese et al., 2002) but not other antivirally active cytokines such as tnf-α (frese et al., 2003) . the idea that ifn-γ enforces the critical first line of defense in the hcv-infected liver was further elaborated by li and co-workers, who demonstrated in a co-culture experiment that nk cells block hcv replication in huh-7 cells through the secretion of ifn-γ (li et al., 2004) . clinical data are limited and it is still controversial discussed whether hepatitis c patients benefit from ifn-γ administrations. nevertheless, it is interesting to note that types i and ii ifns inhibit hcv rna replication in huh-7 cells in a highly synergistic manner (larkin et al., 2003; okuse et al., 2005) . given the power of combination therapies in the treatment of other persistent virus infections, it might be rewarding to elucidate the mechanism(s) responsible for the observed synergistic antiviral effects of different ifn types. for example, do ifn-α and ifn-γ enhance the expression level of one or more effector proteins in a synergistic manner as suggested by tan et al. (2005) , or do they induce the expression of different, ifn type-specific effector proteins that interfere with more than one step of the hcv life cycle as suggested by windisch et al. (2005) ? the answers to these questions may help physicians to predict the outcome of ifn therapies and lead to the improvement of ifn-based therapies. several attempts have been made to analyze systematically the ifn-induced changes in the gene expression of hcv host cells. in one approach, liver biopsy samples were taken from expetimentally infected chimpanzees and the gene expression profile was monitored by using cdna microarrays. the results revealed that the infection of the liver rapidly leads to the upregulation of numerous genes including those encoding well-known ifn-induced effector proteins such as the chimpanzee homologue of mxa (bigger et al., 2001; su et al., 2002) . in both studies, the expression of mxa and that of other type i ifn-induced proteins correlated with the magnitude and duration of the infection. however, transient and sustained viral clearances were rather associated with the production of ifn-γ and the subsequent expression of type ii ifn-induced genes, suggesting a biphasic course of the innate immune response and a crucial role for ifn-γ in virus clearance. in another approach, cdna microarrays were used to analyze ifn-induced changes in the gene expression profile of cultured human cells containing hcv replicons hayashi et al., 2005) . even if these and similar studies did not lead to the identification of the effector proteins that inhibit hcv replication in ifn-stimulated cells, they will guide present and future investigations by suggesting potential candidate genes. the contribution of some of the most prominent ifn-induced effector proteins (figure 2 ) to the ifn-induced inhibition of hcv replication is discussed in the following paragraphs. antiviral pathways that may contribute to the establishment of the so-called antiviral state in ifn-stimulated human cells. from left to right: the mxa gtpase inhibits viral replication by missorting and trapping of viral components into large membrane-associated complexes (the role of gtp hydrolysis in this process is not fully understood). three different ifn-induced oligoadenylate synthetases (oas1, oas2, and oas3) are encoded by the human genome. binding to double-stranded rna (dsrna) leads to hetero-and/or homo-oligomerization and subsequently to the production of oligoadenylates with a 2 ,5 -phosphodiester bond linkage. these 2-5a oligonucleotides activate the latent endoribo-nuclease rnase l, which leads to the degradation of viral and cellular rnas (in some cell types, the expression of rnase l is also regulated by ifns). the p150 isoform of the adenosine deaminase adar1 binds to double-stranded rna and catalyzes the conversion of adenosine to inosine (a to i). such editing may occur selectivity at one or a few positions, or more frequently, at a large number of sites. editing of viral rnas may change the coding sequence, activate an i-specific rnase, and/or destroy rna secondary structures by disrupting adenosine/uracil base pairings. the double-stranded rna-activated protein kinase pkr may block viral protein translation by the phosphorylation and thereby inactivation of the eukaryotic initiation factor eif2a. furthermore, pkr may activate intracellular signaling pathways that contribute to the establishment of a robust antiviral response. the inducible nitric oxide synthetase nos2 produces large amounts of nitric oxide (no), which is implicated in a variety of immune functions such as the activation of macrophages mx proteins belong to the superfamily of dynamin-like large gtpases and their expression is tightly regulated by type i and type iii ifns (holzinger et al, 2007; reviewed in haller & kochs, 2002; haller et al, 2007) . of the two human mx proteins, mxa and mxb, only mxa has demonstrable antiviral activity. mxa is a cytoplasmic protein with a size of ∼78 kda that has been shown to inhibit the replication of a broad variety of rna viruses. cell culture experiments demonstrate that mxa inhibits orthomyxoviruses, bunyaviruses, rhabdoviruses, birnaviruses, reoviruses, and togaviruses (mundt 2007 reviewed in haller et al., 1999 . in some cases, viral replication is almost completely blocked by mxa. for example, stably transfected vero cells that constitutively express mxa produce up to 1,000,000-fold lower virus titers than control cells that did not express any mx proteins . the antiviral effect of mxa has also been analyzed in vivo by using transgenic mice that constitutively express the human mxa protein but lack functional mouse mx proteins and mice that constitutively express mxa but cannot mount a proper ifn-induced antiviral response due to a disruption in the gene for the β subunit of the ifn type i receptor. in both cases, mxa-expressing animals were found to be completely resistant to thogoto virus, a tick-borne orthomyxovirus hefti et al., 1999) . furthermore, mxa-expressing animals exhibited an enhanced resistance against . . . influenza a virus (family orthomyxoviridae), vesicular stomatitis virus (vsv; family rhabdoviridae), lacrosse virus (family bunyaviridae), and semliki forest virus (family togaviridae) hefti et al., 1999) . other reports suggest that mxa has an even wider antiviral activity, but the supporting data are less convincing. the modus operandi of mxa is not completely understood, but accumulating data indicate that cytoplasmic mx proteins missort and immobilize viral components. in cells that had been infected with lacrosse virus, mxa binds and translocates the viral nucleocapsid protein into membrane-associated perinuclear complexes reichelt et al., 2004) . a similar phenomenon was observed in cells that had been infected with thogoto virus. in this case, however, mxa inhibited the nuclear transport of incoming viral nucleocapsids , thereby preventing primary transcription and leading to an early and very efficient block of virus replication. in healthy individuals, mxa expression is below the detection limit, but expression levels increase dramatically during many viral infections and as a consequence of ifn-α treatment (roers et al., 1994; chieux et al., 1998) . mxa mrna quantification in peripheral blood mononuclear cells has even been used to monitor the bioavailability of administered type i ifns in hepatitis c patients (gilli et al., 2002; jorns et al., 2006) . not surprisingly, elevated mxa expression levels have also been found in the liver of chronic hepatitis c patients, indicating an ongoing struggle between the innate immune system and hcv (macquillan et al., 2002 (macquillan et al., , 2003 patzwahl et al., 2001) . a genetic study from japan addressing a single nucleotide polymorphism at position -88 in the promoter sequence of the mxa gene revealed that a thymidine (t) in that position favors a sustained response of hepatitis c patients to treatment with ifn-α, whereas a guanosine (g) is more frequently found among nonresponders (hijikata et al., 2000) . interestingly, a t at that position increases the homology of the first isre in the mxa promoter to the isre consensus sequence (hijikata et al., 2000) . furthermore, experiments with reporter constructs suggest that the t allele has a higher transcriptional activity than the g allele when stimulated with ifn-α (hijikata et al., 2001) . a similar association between the g/t single nucleotide polymorphism at position -88 of the mxa gene and the response of hepatitis c patients to ifn-α therapy was found in a european study, in which the t genotype was also found to be associated with the ability to clear hcv naturally without the help of recombinant ifn (knapp et al., 2003) . since mxa has the ability to efficiently inhibit a variety of different rna viruses, mxa was the first ifn-induced effector protein to be analyzed for its antiviral activity in the hcv replicon system (frese et al., 2001) . however, no evidence was found for an involvement of mxa in the ifn-induced inhibition of hcv rna replication. the constitutive expression of mxa did not inhibit subgenomic hcv replicons, and the expression of a dominant-negative mutant of mxa did not restore hcv rna replication during ifn-α treatment (frese et al., 2001) . these earlier observations are in line with the more recent finding that ifn-α inhibits hcv rna replication in huh-7 cells and huh6 cells with a similar ic 50 (3 to 5 iu/ml and 5 to 10 iu/ml, respectively), although the former produce nearly 75-fold more mxa mrnas than the latter (windisch et al., 2005) . taken together, the data indicate that ifn-α inhibits hcv rna replication by mxa-independent pathways. most recently, it has been noted that brefeldin a, a golgi apparatus disrupting agent, renders the replication of kunjin virus susceptible to mxa (hoemen et al., 2007) . since kunjin virus and hcv are both flaviviruses that use host cell-derived membranes to establish replication factories, it is tempting to speculate whether a disruption of the membranous web in hcv-infected cells would expose hcv rna-protein complexes to antivirally active proteins such as mxa. the oas/rnase l pathway (also known as ifn-inducible 2-5a response) requires two types of enzymes, an oligoadenylate synthetase and a ribonuclease (reviewed in samuel, 2001) . the human genome contains four gene loci that encode ifn-induced oligoadenylate synthetases (oas1, oas2, and oas3) and an oas-like protein. this and alternative splicing leads to the expression of numerous isoforms with sizes ranging from 40 to 100 kda (rebouillat & hovanessian, 1999) . oas protein expression is enhanced in response to most, if not all, ifns, but the magnitude of induction can vary dramatically with different ifns and the type of the producing cell. newly produced oas proteins are believed to be inactive, but binding to double-stranded rna leads to their oligomerization and starts the production of oligoadenylates with a 2 ,5 -phosphodiester bond linkage (2-5a oligonucleotides). these oligonucleotides bind to and activate the latent ribonuclease rnase l, a process that is associated with the formation of stable rnase l homodimers. once activated, rnase l can degrade single-stranded rnas of viral and cellular origin. cleaving of target rnas occurs preferentially on the 3 side of uracil-adenosine (ua) and uu dinucleotides (floyd-smith et al., 1981; wreschner et al., 1981) . the cleavage of mrna and rrna may trigger a general protein shut-off in virusinfected cells, thereby limiting virus replication and spread. different oas proteins are associated with different cellular compartments, vary with respect to the amount of double-stranded rna needed for activation, and produce 2-5a oligonucleotides of different sizes (samuel, 2001) . it is therefore tempting to speculate that the diversity of oas proteins and isoforms evolved to fight a rather wide spectrum of dna and rna viruses including poxviruses, reoviruses, and picornaviruses. members of the family picornaviridae seem to be especially sensitive to the oas/rnase l pathway. for example, overexpression of the 40-kda form of the human oas1 protein confers resistance to mengovirus but not vsv (chebath et al., 1987) , and the constitutive expression of the 69-kda form of the oas2 protein inhibited the replication of encephalomyocarditis virus but not that of vsv, sendai virus, and a reovirus (ghosh et al., 2000) . in this context it is interesting to note that one of the oas gene loci has recently been identified to confer increased resistance to the west nile virus (family flaviviridae) in laboratory mice (perelygin et al., 2002) and that the transcript of the oas1b allele in susceptible mice contains a premature stop codon, which results in a truncated protein (mashimo et al., 2002) . experiments with congenic mice and cells derived from those mice revealed that expression of the full-length oas1 protein limited virus production in vivo and in cell culture. surprisingly, however, rnase l activity was highest in susceptible cells, and downregulation of rnase l activity in resistant cells did not restore virus titers to levels observed in susceptible cells (scherbik et al., 2006) . as with many other ifn-induced proteins, oas protein expression is slightly upregulated in hepatitis c patients (macquillan et al., 2003) and further enhanced in response to the administration of recombinant ifn-α (murashima et al., 2000) . rather indirect evidence that the oas/rnase l pathway may indeed target hcv replication/translation was recently provided by taguchi and co-workers, who reported that the n-terminal portion of ns5a (amino acids 1 to 148), which lacks the so-called pkr-binding domain, binds to oas proteins and there by counteract the antiviral activity of ifn-α (taguchi et al., 2004) . furthermore, it has been demonstrated that purified recombinant rnase l and that from hela cell extracts efficiently cleaves hcv rna in vitro (han et al., 2004) . however, further investigations are needed to determine whether rnase l also cleaves hcv rnas in infected hcv host cells and to what extent an oas-induced block of hcv protein translation contributes to the ifn-induced inhibition of hcv rna replication. adar1 forms together with adar2 and the less extensively studied adar3 protein a small family of constitutively expressed adenosine deaminases that act on rna (reviewed in valente & nishikura, 2005; toth et al., 2006) . adar1 and adar2 bind highly structured rnas and catalyze the hydrolytic c6 deamination of adenosine, a reaction that converts adenosine to inosine (a to i editing). adarmediated editing may occur selectively at one or a few positions or, more frequently, at a larger number of sites (hyperediting or hypermutation). a to i exchanges may have severe consequences: (1) editing of coding sequences may lead to amino acid exchanges because i is recognized as g by the translational machinery (of note, a to i editing does not create stop codons); (2) editing of noncoding regions may affect rna splicing, stability, or translational efficiency (e.g., by disrupting au base pairs); (3) editing may regulate gene silencing (e.g., by disrupting au base pairs); and (4) hyperedited rna may be recognized and cleaved by an i-specific rnase (scadden & smith, 1997 , 2001 . a prominent example of a cellular rna that is edited by adar proteins is the mrna of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor subunit glur-2. adar2 edits a codon in exon 11, which results in an amino acid exchange that changes the ca 2+ permeability of the receptor. this highly specific editing event has far-reaching consequences. adar2 knockout mice are prone to seizures and die young. the impaired phenotype appears to result entirely from a single underedited position in the glur-2 mrna, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically (higuchi et al., 2000) . likewise, genetic targeting of the adar1 locus revealed an essential requirement for this adar protein in the embryogenesis of mice (wang et al., 2000 (wang et al., , 2004 hartner et al., 2004) , and it has been suggested that its expression protects against stress-induced apoptosis (wang et al., 2004) . a closer look at the adar1 gene locus revealed that protein expression is controlled by three promoters and alternative splicing (reviewed in toth et al., 2006) . two constitutively active promoters drive the expression of a ∼110-kda protein (p110), whereas an ifn-regulated promoter with an isre controls the expression of a larger isoform (p150) that is expressed in response to inflammation or ifn treatment (patterson & samuel, 1995; george & samuel, 1999; yang et al., 2003a yang et al., , 2003b . both isoforms contain multiple nuclear localization signals, but only the ifn-induced p150 isoform has a nuclear export signal. accordingly, p110 is a nuclear protein and p150 has been detected in both nuclear and cytoplasmic compartments. hypermutation of viral rnas has been observed for several rna viruses, including measles virus, parainfluenza virus 3 (both family paramyxoviridae), and vsv (o'hara et al., 1984; cattaneo et al., 1988; murphy et al., 1991) . it has been speculated that subacute sclerosing panencephalitis (sspe), a fatal necropathic response in patients with a persistent measles virus infection of the brain, is associated with extensive editing of the matrix protein mrna. this prevents virion assembly and release because these steps in the viral life cycle require a functional matrix protein. other transcripts, however, are less frequently edited, which is thought to result in a persistent virus replication (cattaneo et al., 1989; baczko et al., 1993) . thus, an incomplete adar-mediated innate immune response might contribute to the pathology of sspe. interestingly, certain viruses abuse adar proteins to control important checkpoints in replication and particle formation. a well-known example is the hepatitis d virus (hdv), a subviral human pathogen that depends on hepatitis b virus as a helper virus (reviewed in casey, 2006) . hdv has a small, circular rna genome that encodes only a single protein, the hepatitis delta antigen (hdag). without editing, a 195-amino acid version of hdag is made that is essential for virus replication (kuo et al., 1989) . later on, in the viral life cycle a highly specific a to i editing event changes a uag amber stop codon to an uig tryptophan codon, and a 214-amino acid hdag-l is produced that mediates genome packaging (chang et al., 1991) . hcv rnas may also be subject to adar-mediated modifications, but in this case, editing seems to be less specific and to inhibit virus replication. it has recently been reported that the silencing of adar1 expression in hcv replicon cells increases the amount of hcv rna about 40-fold . moreover, taylor and co-workers noted that ifn-α increases the frequency of a to g mutations in subgenomic replicon rnas and that the transfection of adar-specific sirnas rescues hcv rna replication in the presence of moderate ifn-α concentrations. based on these findings, taylor et al. concluded that ifn-α inhibits hcv replication through adar-mediated hyperediting of viral rna. in our laboratory, we have used specific antibodies to determine the intracellular localization of p150 anddespite its nuclear export signal -, we observed that p150 accumulates predominantly in the nucleus of ifn-treated huh-7 cells. in the presence of subgenomic or full-length hcv rnas, however, we observed that p150 localizes to distinct cytoplasmic structures (e. dazert, r. bartenschlager, and m. frese, unpublished results) . we also analyzed the antiviral effect of constitutively expressed p150 on hcv replication and found that the overexpression of p150 in huh-7 cells did not block hcv rna replication. taken together, our findings support the idea of taylor et al. that p150 interacts with hcv rnas, but we argue that p150 does that only in the context off other ifn-induced proteins. further studies are under way to fully elucidate the role of p150 in the ifn-induced inhibition of hcv replication. another prominent protein of the innate immune defense that has long been suspected of interfering with hcv replication is the double-stranded rna-activated protein kinase pkr. this serine/threonine kinase is constitutively expressed and has multiple functions in the control of host cell transcription and translation (reviewed in garcia et al., 2006) . upon stimulation with ifns, most cells respond by increasing the expression of pkr. ifn-induced pkr accumulates in the cytoplasm and was found in association with ribosomes (thomis et al., 1992) . pkr may exert its antiviral activity through different pathways (reviewed in toth et al., 2006) . first, pkr is able to control the cellular translation machinery through phosphorylation of the α subunit of the eukaryotic translation initiation factor eif-2α, which would affect the production of both host and virus proteins. second, pkr-mediated phosphorylation is implicated in several signaling pathways that contribute to the establishment of a robust antiviral response. for example, pkr has been shown to activate the latent transcription factor nfκb, which may lead to the enhanced expression of proinflammatory genes (gil et al., 2004) . in addition, pkr may activate other kinases such as the p38 mitogen-activated protein (map) kinase, which further intensifies and diversifies the innate immune response (goh et al., 2000) . the concept that pkr-mediated phosphorylation events play an important role in the innate immune response against viral infections is largely based on the fact that many rna and dna viruses try to inhibit pkr by (1) overexpressing small rnas that bind to but do not activate pkr, (2) producing eif-2α decoys, and (3) enhancing pkr degradation (reviewed in langland et al., 2006) . if pkr is a key player in ifn-induced antiviral defense, genetically targeted knockout mice that lack functional pkr proteins should be extremely sensitive to viral infections. two lines of pkr −/− mice have been generated in which the coding sequences of either the n-terminal or the c-terminal part of the protein have been disrupted (yang et al., 1995; abraham et al., 1999, respectively) . pkr −/− mice are indeed more susceptible to certain virus infections than wild-type animals (balachandran et al., 2000; stojdl et al., 2000; carr et al., 2006; samuel et al., 2006) , but at least in some cases, this seems to depend on the mouse strain used, and other experimental conditions (murphy et al., 2003) . additional experiments have been conducted by using mefs from pkr −/− mice, but a direct antiviral activity of pkr (e.g., the inhibition of virus multiplication by blocking protein translation) is still controversial. interestingly, priming of pkr −/− mice with poly(i)-poly(c) or ifns before the virus challenge points to a rather indirect mode of pkr action, such as the enhancement of double-stranded rna-induced signaling events (yang et al., 1995) . however, it should be noted that most of these experiments have been performed with viruses that encode pkr inhibitors. it would be interesting to re-evaluate the phenotype of pkr −/− mice with genetically modified viruses that cannot express functional pkr inhibitors. another problem in the characterization of pkr −/− mice is the presence of related kinases that also phosphorylate eif-2α (toth et al., 2006) . even if these kinases differ from pkr in their response to double-stranded rna and/or other physiological stress signals, they may partially substitute for the lack of pkr in pkr −/− mice, thereby making it difficult to quantify the contribution of pkr to the innate immune response (as exemplified in smith et al., 2005) . two hcv proteins have been described as interacting with the kinase. by analyzing hcv sequences from japanese hepatitis c patients, mutations within a discrete region of ns5a, the so-called ifn sensitivity determining region (isdr), were proposed to confer resistance to ifn-α (enomoto et al., 1995 (enomoto et al., , 1996 . since the original reports by enomoto and co-workers, numerous studies have been conducted in japan as well as in other countries to determine the predictive value of ns5a sequences in the outcome of ifn-based therapies, but the existence of an isdr is still controversial (reviewed in tan & katze, 2001; reinvestigated by pascu et al., 2004; brillet et al., 2007) . whether or not an isdr really exists, the description of such a sequence put ns5a in the focus of hcv research. the subsequent finding that mutations in the isdr affect the ability of ns5a to bind to and inhibit pkr (gale et al., 1998) led to the hypothesis that pkr blocks hcv replication and that ns5a is able to counteract the antiviral activity of pkr. however, experiments with hcv replicons provided no further evidence for an involvement of ns5a in ifn resistance. on the contrary, point mutations within the isdr or a deletion of 47 amino acids encompassing the entire isdr enhanced viral replication without affecting the ifn sensitivity of hcv replicons (blight et al., 2000; guo et al., 2001) . these findings were extended by a. kaul and r. bartenschlager, who analyzed the function of ns5a by using two subgenomic genotype 1b replicons that differ only in the ns5a coding sequence. in one replicon, the isdr was identical to that of ifn-susceptible strains, whereas the isdr sequence of the other replicon contained mutations that have been suspected to confer pkr binding and ifn resistance (gale et al., 1998) . despite these differences, both replicons were found to be equally sensitive to ifn-α (unpublished results) . this result argues against the hypothesis that ns5a counteracts an ifn-induced and pkr-mediated block of viral protein translation. however, the result does not contradict the idea that ns5a inhibits other activities of pkr (e.g., a pkr-mediated priming of intracellular signaling pathways). of note, several reports suggest that ns5a may sabotage the innate immune response through pkr-independent activities (discussed in macdonald & harris, 2004) . for example, it has been reported that ns5a increases the production of interleukin (il)-8, thereby attenuating the antiviral properties of ifns (polyak et al., 2001a (polyak et al., , 2001b . it would be interesting to study the immunomodulatory activities of ns5a in an immunocompetent small animal model, which might finally put an end to the discussion about the role of pkr in hcv pathology. a second hcv protein has been reported to interact with pkr. it was found that e2 binds to pkr through its pkr-eif2α homology domain (pephd) (taylor et al., 1999 (taylor et al., , 2001 pavio et al., 2002) . however, the significance of this observation has been questioned because an increasing number of clinical studies demonstrate that the pephd is a highly conserved region with no conspicuous mutations accumulating during ifn-α therapy (reviewed in tan & katze, 2001) . furthermore, e2 expression does not increase the resistance of hcv genotype 1b replicons toward ifns. a genomic replicon that encodes an e2 protein with the pephd sequence of a resistant hcv isolate had a similar degree of susceptibility as a subgenomic replicon lacking e2 (frese et al., 2002; a. kaul and r. bartenschlager, unpublished results) . the adenovirus-associated rna i (va i ), a small, highly structured rna that binds to pkr and but does not trigger its dimerization and activation, has recently been found to stimulate hcv rna replication in the replicon system . it was also reported that recombinant va i rna efficiently rescues hcv rna replication in the presence of as much as 500 iu/ml of ifn-α . since va i rnas may also bind to other proteins of the innate immune response such as adar1, more research is needed to define the role of pkr in limiting hcv protein translation. a more direct approach to the question of pkr interference with hcv rna replication/translation has recently been undertaken by using rna silencing. a. kaul and r. bartenschlager transfected cells containing subgenomic hcv replicons with sirnas that target pkr mrnas for degradation and subsequently treated the cells with different concentrations of ifn-α. in no case did they observe that a downregulation of pkr expression levels results in a restoration of hcv replication in the presence of ifn (unpublished results). with the establishment of a new generation of hcv replicons that contain the consensus sequence from a japanese genotype 2a isolate and replicate efficiently without the need for adaptive mutations, it became possible to study hcv rna replication in a variety of new host cells including those of nonhepatic and nonhuman origin (kato et al., 2005; uprichard et al., 2006) . most recently, genotype 2a replicons were employed by chang and co-workers, who set out to analyze the antiviral effect of type i ifns on hcv replication in mefs from pkr −/− mice and congenic wild-type mice. interestingly, ifn-α as well as ifn-β inhibited hcv rna replication in pkr −/− mefs as efficiently as in pkr +/+ mefs (chang et al., 2006) , suggesting that pkr-mediated translational control plays only a minor role in the ifn-induced inhibition of hcv rna replication. the inducible nitric oxide (no) synthetase, originally named inos but also abbreviated as nos2, belongs to a small family of no-producing enzymes. in unstimulated cells, nos2 is virtually absent, but expression levels increase rapidly in response to pro-inflammatory cytokines, especially ifn-γ. the expression of nos2 results in a long-lasting production of no (karupiah et al., 2000) . the no free radical has been recognized for its strong antimicrobial activity against various protozoa, bacteria, and viruses. for example, the replication of a coxsackievirus is suppressed by no through inactivation of the viral cysteine protease by s-nitrosylation (saura et al., 1999) . furthermore, no production is essential for the t cell-mediated noncytopathic inhibition of hepatitis b virus replication in virus-transgenic mice (guidotti et al., 2000) . other viruses that have been reported as sensitive to no include severe acute respiratory coronavirus (akerstrom et al., 2005) , respiratory syncytial virus (stark et al., 2005) , mouse hepatitis virus (pope et al., 1998) , and herpes simplex virus type 1 (adler et al., 1997) . however, the use of no by the infected host as an antimicrobial substance is a double-edged sword. no-induced oxidative stress may cause severe cellular and organ dysfunction. influenza a virus-infected wild-type mice, for example, suffer from an excessive production of no in the lungs, which often leads to respiratory failure and death, whereas knockout mice that cannot express functional nos2 survive the infection with little evidence of pneumonitis (akaike et al., 1996; karupiah et al., 1998) . in the liver of most hcv-infected individuals, nos2 is easily detectable (mihm et al., 1997; majano et al., 1998; schweyer et al., 2000) . the enhanced expression of nos2 in the liver of hepatitis c patients has largely been attributed to ifn-γ that is released by resident and infiltrating immune cells. in addition, it has been speculated that the hcv replication itself may stimulate the expression of nos2 in infected hepatocytes (machida et al., 2004) . based on genetic studies, it has been suggested that the production of no is involved in hcv clearance, as certain nos2 haplotypes were more frequently found among hcv-infected individuals who spontaneously cleared the infection and in ifn-treated hepatitis c patients who could mount a sustained antiviral response (yee et al., 2004) . this hypothesis, however, lacks supporting evidence from cell culture experiments. the treatment of huh-7 cells with the no donor (z)-1-[2-(aminoethyl)-n-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (deta nonoate) or the arginase inhibitor ng-hydroxy-l-arginine (noha) did not result in an inhibition of hcv rna replication (frese et al., 2002) . furthermore, the nos inhibitor l-n6-(1-iminoethyl)lysine (l-nil) did not even partially restore hcv replication in the presence of ifn-γ (frese et al., 2002) . one should, however, not overinterpret these results. the in vivo production of no may have more complex consequences than those that could be investigated in cell culture. no may act as a messenger rather than as an effector molecule, or no may induce dna damage and apoptosis (jaiswal et al., 2000) . thus, further studies are needed to fully elucidate the role of no in hepatitis c pathology. so far, only a few further ifn-induced effector proteins have been investigated with respect to their potential to inhibit hcv rna replication, most notably indoleamine 2,3-dioxygenase (ido). the ido-mediated depletion of tryptophan is well known as a defense mechanism against certain intracellular parasites (carlin et al., 1989) . rather recently, this pathway has also been recognized as an ifn-γ-induced antiviral defense mechanism against herpesviruses (bodaghi et al., 1999) and poxviruses (terajima & leporati, 2005) . if ido inhibits hcv replication as well, inhibition of the effector protein or addition of tryptophan to the cell culture medium should restore viral protein synthesis. however, the ido inhibitor α-methyl-dl-tryptophan did not restore the replication of subgenomic hcv replicons in the presence of ifn-γ (frese et al., 2002) . likewise, increased concentrations of l-tryptophan could not rescue the viral protein synthesis (frese et al., 2002) , suggesting that the depletion of tryptophan is not-or is not the only-mechanism by which ifn-γ inhibits the replication of hcv rnas. beside the induction of direct antiviral activities in infected host cells, ifns may enhance and direct the activities of nk cells and t cells. such indirect effects are usually ascribed to ifn-γ, but shin and co-workers noted that type i ifns also stimulate the generation of immunoproteasomes in the liver of hepatitis c patients (shin et al., 2006) . it would be interesting to determine the extent to which these indirect effects contribute to the antiviral activity of type i ifns. characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded rna-dependent protein kinase suppression of herpes simplex virus type 1 (hsv-1)-induced pneumonia in mice by inhibition of inducible nitric oxide synthase (inos, nos2) pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus hepatitis c virus subgenomic replicons in the human embryonic kidney 293 cell line essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection novel insights into hepatitis c virus replication and persistence clonal expansion of hypermutated measles virus in an sspe brain dna microarray analysis of chimpanzee liver during acute resolving hepatitis c virus infection secondary structure determination of the conserved 98-base sequence at the 3 terminus of hepatitis c virus genome rna efficient initiation of hcv rna replication in cell culture role of ifn-induced indoleamine 2,3 dioxygenase and inducible nitric oxide synthase in the replication of human cytomegalovirus in retinal pigment epithelial cells the nonstructural 5a protein of hepatitis c virus genotype 1b does not contain an interferon sensitivitydetermining region interferons and indoleamine 2,3-dioxygenase: role in antimicrobial and antitumor effects the lack of rnadependent protein kinase enhances susceptibility of mice to genital herpes simplex virus type 2 infection rna editing in hepatitis delta virus biased hypermutation and other genetic changes in defective measles viruses in human brain infections mutated and hypermutated genes of persistent measles viruses which caused lethal human brain diseases the large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus replication of hepatitis c virus (hcv) rna in mouse embryonic fibroblasts: protein kinase r (pkr)-dependent and pkr-independent mechanisms for controlling hcv rna replication and mediating interferon activities constitutive expression of (2 -5 ) oligo a synthetase confers resistance to picornavirus infection comparative analysis of anti-hepatitis c virus activity and gene expression mediated by alpha, beta, and gamma interferons the mxa protein levels in whole blood lysates of patients with various viral infections interferon-producing cells: on the front line in immune responses against pathogens the expanded family of class ii cytokines that share the il-10 receptor-2 (il-10r2) chain interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes comparison of full-length sequences of interferon-sensitive and resistant hepatitis c virus 1b. sensitivity to interferon is conferred by amino acid substitutions in the ns5a region mutations in the nonstructural protein 5a gene and response to interferon in patients with chronic hepatitis c virus 1b infection interferon action: rna cleavage pattern of a (2 -5 )oligoadenylate-dependent endonuclease regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease human mxa protein inhibits tick-borne thogoto virus but not dhori virus interferon-alpha inhibits hepatitis c virus subgenomic rna replication by an mxa-independent pathway interferon-gamma inhibits replication of subgenomic and genomic hepatitis c virus rnas hepatitis c virus rna replication is resistant to tumour necrosis factor-alpha kissing-loop interaction in the 3 end of the hepatitis c virus genome essential for rna replication peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection control of pkr protein kinase by hepatitis c virus nonstructural 5a protein: molecular mechanisms of kinase regulation impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action human rna-specific adenosine deaminase adar1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible cell growth regulatory and antiviral effects of the p69 isozyme of 2-5 (a) synthetase traf family proteins link pkr with nf-kappa b activation evaluation of ifn-alpha bioavailability by mxa mrna in hcv patients the protein kinase pkr is required for p38 mapk activation and the innate immune response to bacterial endotoxin interferons: cell signalling, immune modulation, antiviral response and virus countermeasures noncytolytic control of viral infections by the innate and adaptive immune response nitric oxide inhibits hepatitis b virus replication in the livers of transgenic mice effect of alpha interferon on the hepatitis c virus replicon cytopathic and noncytopathic interferon responses in cells expressing hepatitis c virus subgenomic replicons mx proteins: mediators of innate resistance to rna viruses interferon-induced mx proteins in antiviral host defense interferon-induced mx proteins: dynamin-like gtpases with antiviral activity sensitivity of hepatitis c virus rna to the antiviral enzyme ribonuclease l is determined by a subset of efficient cleavage sites liver disintegration in the mouse embryo caused by deficiency in the rna-editing enzyme adar1 the transcriptome of hcv replicon expressing cell lines in the presence of alpha interferon human mxa protein protects mice lacking a functional alpha/beta interferon system against la crosse virus and other lethal viral infections point mutation in an ampa receptor gene rescues lethality in mice deficient in the rna-editing enzyme adar2 identification of a single nucleotide polymorphism in the mxa gene promoter (g/t at nt -88) correlated with the response of hepatitis c patients to interferon genetic polymorphism of the mxa gene promoter and interferon responsiveness of hepatitis c patients: revisited by analyzing two snp sites (-123 and -88) in vivo and in vitro mastercard: a priceless link to innate immunity west nile virusinduced cytoplasmic membrane structures provide partial protection aginst the interferoninduced antiviral mxa protein induction of mxa gene expression by influenza a virus requires type i or type ii interferon signaling a phylogenetically conserved stem-loop structure at the 5 border of the internal ribosome entry site of hepatitis c virus is required for cap-independent viral translation inflammatory cytokines induce dna damage and inhibit dna repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism rapid and simple detection of ifn-neutralizing antibodies in chronic hepatitis c non-responsive to ifn-alpha rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice nadph oxidase, nramp1 and nitric oxide in the host microbiol response nonhepatic cell lines hela and 293 support efficient replication of the hepatitis c virus genotype 2a subgenomic replicon impaired antiviral response in human hepatoma cells long-range rna-rna interaction between the 5 nontranslated region and the core-coding sequences of hepatitis c virus modulates the ires-dependent translation polymorphisms in interferon-induced genes and the outcome of hepatitis c virus infection: roles of mxa, oas-1 and pkr interferon-induced human mxa gtpase blocks nuclear import of thogoto virus nucleocapsids antivirally active mxa protein sequesters la crosse virus nucleocapsid protein into perinuclear complexes ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex initiation of replication of the human hepatitis delta virus genome from cloned dna: role of delta antigen antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis c virus subgenomic replicons inhibition of pkr by rna and dna viruses synergistic antiviral activity of human interferon combinations in the hepatitis c virus replicon system viral kinetics of hepatitis c: new insights and remaining limitations immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif natural killer cells inhibit hepatitis c virus expression replication of subgenomic hepatitis c virus rnas in a hepatoma cell line hepatitis c virus ns5a: tales of a promiscuous protein hepatitis c virus infection activates the immunologic (type ii) isoform of nitric oxide synthase and thereby enhances dna damage and mutations of cellular genes intrahepatic mxa and pkr protein expression in chronic hepatitis c virus infection upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis c virus infection inducible nitric oxide synthase expression in chronic viral hepatitis. evidence for a virus-induced gene upregulation peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial interferons alpha and lambda inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics a nonsense mutation in the gene encoding 2 -5 -oligoadenylate synthetase/l1 isoform is associated with west nile virus susceptibility in laboratory mice current therapy for hepatitis c: pegylated interferon and ribavirin cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatic expression of inducible nitric oxide synthase transcripts in chronic hepatitis c virus infection: relation to hepatic viral load and liver injury interferon type i gene expression in chronic hepatitis c comparison between subgenomic replicons of hepatitis c virus genotypes 2a (jfh-1) and 1b (con1 nk5.1) human mxa confers resistance to double-standed rna viruses of two virus families effect of interferon treatment on serum 2 ,5 -oligoadenylate synthetase levels in hepatitis c-infected patients numerous transitions in human parainfluenza virus 3 rna recovered from persistently infected cells herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions enhancement of antiviral activity against hepatitis c virus in vitro by interferon combination therapy sustained virological response in hepatitis c virus type 1b infected patients is predicted by the number of mutations within the ns5a-isdr: a meta-analysis focused on geographical differences expression and regulation by interferon of a double-stranded-rna-specific adenosine deaminase from human cells: evidence for two forms of the deaminase enhanced expression of interferonregulated genes in the liver of patients with chronic hepatitis c virus infection: detection by suppression-subtractive hybridization detection of a novel unglycosylated form of hepatitis c virus e2 envelope protein that is located in the cytosol and interacts with pkr enhanced virus resistance of transgenic mice expressing the human mxa protein differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells positional cloning of the murine flavivirus resistance gene interferons, interferon-like cytokines, and their receptors hepatitis c virus nonstructural 5a protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response elevated levels of interleukin-8 in serum are associated with hepatitis c virus infection and resistance to interferon therapy resistance to murine hepatitis virus strain 3 is dependent on production of nitric oxide the human 2 ,5 -oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties missorting of lacrosse virus nucleocapsid protein by the interferon-induced mxa gtpase involves smooth er membranes lambda interferon inhibits hepatitis b and c virus replication mxa gene expression after live virus vaccination: a sensitive marker for endogenous type i interferon antiviral actions of interferons pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons an antiviral mechanism of nitric oxide: inhibition of a viral protease a ribonuclease specific for inosine-containing rna: a potential role in antiviral defense specific cleavage of hyper-edited dsrnas rnase l plays a role in the antiviral response to west nile virus liver infiltrating t lymphocytes express interferon-gamma and inducible nitric oxide synthase in chronic hepatitis c virus infection il-28, il-29, and their class ii cytokine receptor il-28r virus-induced type i ifn stimulates generation of immunoproteasomes at the site of infection involvement of the interferon-regulated antiviral proteins pkr and rnase l in reovirus-induced shutoff of cellular translation immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection the murine double-stranded rna-dependent protein kinase pkr is required for resistance to vesicular stomatitis virus genomic analysis of the host response to hepatitis c virus infection hepatitis c virus ns5a protein interacts with 2 ,5 -oligoadenylate synthetase and inhibits antiviral activity of ifn in an ifn sensitivity-determining region-independent manner global transcriptional profiling demonstrates the combination of type i and type ii interferon enhances antiviral and immune responses at clinically relevant doses how hepatitis c virus counteracts the interferon response: the jury is still out on ns5a inhibition of the interferoninducible protein kinase pkr by hcv e2 protein hepatitis c virus envelope protein e2 does not inhibit pkr by simple competition with autophosphorylation sites in the rna-binding domain new antiviral pathway that mediates hepatitis c virus replicon interferon sensitivity through adar1 role of indoleamine 2,3-dioxygenase in antiviral activity of interferon-gamma against vaccinia virus mechanism of interferon action: cdna structure, expression, and regulation of the interferon-induced, rna-dependent p1/eif-2 alpha protein kinase from human cells interferon action and the double-stranded rna-dependent enzymes adar1 adenosine deaminase and pkr protein kinase replication of a hepatitis c virus replicon clone in mouse cells adar gene family and a-to-i rna editing: diverse roles in posttranscriptional gene regulation the viith report of the international committee on taxonomy of viruses requirement of the rna editing deaminase adar1 gene for embryonic erythropoiesis stress-induced apoptosis associated with null mutation of adar1 rna editing deaminase gene replication and gene function in kunjin virus dissecting the interferon-induced inhibition of hepatitis c virus replication by using a novel host cell line interferon action -sequence specificity of the ppp(a2 p)na-dependent ribonuclease widespread inosine-containing mrna in lymphocytes regulated by adar1 in response to inflammation intracellular localization of differentially regulated rna-specific adenosine deaminase isoforms in inflammation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase inducible nitric oxide synthase gene (nos2a) haplotypes and the outcome of hepatitis c virus infection a cis-acting replication element in the sequence encoding the ns5b rna-dependent rna polymerase is required for hepatitis c virus rna replication gene expression associated with interferon alfa antiviral activity in an hcv replicon cell line we are indebted to kerry mills, sandra thomas, ali zaid, friedemann weber, brett lidbury and ralf bartenschlager for helpful suggestions and careful reading of the manuscript; and artur kaul and r. bartenschlager for the communication of unpublished results. key: cord-005617-bxbogskm authors: conway, anna; fernàndez-lópez, laura; reyes-urueña, juliana; casabona, jordi title: hepatitis c screening in community-based voluntary counselling and testing services in europe: an observational study from the cobatest network 2014–2018 date: 2019-11-20 journal: j community health doi: 10.1007/s10900-019-00780-0 sha: doc_id: 5617 cord_uid: bxbogskm the cobatest network links community-based voluntary counselling and testing (cbvct) services in the european region and collects testing data using standardised data collection tools. this study aims to describe the population being screened for anti-hcv antibodies in the cobatest network and identify risk factors associated with a reactive hcv screening test result in the period 2014–2018. clients aged > 16 screened for hcv in the period 2014–2018 at one of the network’s cbvct services were included in the study. in the 5 year period, 7426 clients were screened for hcv in 22 centres in 10 countries and anti-hcv antibodies were detected in 113 (1.5%). the majority of people screened were aged 25–44, men who have sex with men (msm), not hiv+ , not reporting a history of injecting drug use or sex work. detection of anti-hcv antibodies was associated with being hiv + msm (aor 9.1, 95% ci 3.8; 21.8 compared to hiv-clients) and being a person who injects drugs (pwid, aor 28.1, 95% ci 17.6; 45.0, compared to people with no history of injecting drug use). this study demonstrates that hiv-msm with no history of injection drug use are using cbvct services for hcv screening, but reactive screening test is associated with being hiv+ or pwid. the integration of hcv screening into the cbvct service model may widen access to testing for populations that may otherwise not be tested. electronic supplementary material: the online version of this article (10.1007/s10900-019-00780-0) contains supplementary material, which is available to authorized users. in 2017, 31,273 cases of hepatitis c virus (hcv) were reported to the ecdc in 29 eu/eea member states, corresponding to a crude rate of 7.3 cases per 100,000 population [1] . these figures are likely to be an underestimate as hepatitis infection often shows no symptoms. only 26.0% of the cases in 2017 included data on the mode of transmission, the most common of which was injecting drug use accounting for 44.0% of those cases with complete information on transmission status [1] . who in 2016 recommended a dramatic scale-up of hcv testing and linkage to care to achieve elimination of hcv by 2030 [2] . hcv screening and treatment interventions carry a double public health benefit: reducing both morbidity and incidence through a treatment-as-prevention effect [3] . a recent scoping review of studies investigating barriers to hcv testing, found that low self-perceived risk of acquiring hcv, perceived stigma and fear of a positive result were reported as barriers to hcv screening the cobatest study group members are listed in acknowledgement section. the online version of this article (https ://doi.org/10.1007/s1090 0-019-00780 -0) contains supplementary material, which is available to authorized users. and testing. there are also barriers to providers wishing to offer hcv screening, including time constraints, lack of specific knowledge about hcv and discomfort in asking about risk behaviours [4] . peer counselling offered in community-based voluntary counselling and testing (cbvct) services can help men who have sex with men (msm) overcome additional barriers to testing such as homophobia and internalised homonegativity [5] . the who recommends that cbvct services form part of a country's national testing strategy, including testing by trained non-medical professionals. many cbvct services, recognising the needs of their clients, have moved beyond hiv screening and now offer screening for hcv and syphilis [6] . who guidelines state that rapid diagnostic tests have acceptable sensitivity and specificity compared to laboratory-based testing and can be successful at increasing testing uptake and reducing loss to follow-up (world health organization (who) [7] ). once the rapid diagnostic test performed in cbvct services detects anti-hcv, a hcv rna nucleic acid test has to be performed to establish active hcv infection and ensure linkage to care. the cobatest network links organisations across europe who offer cbvct services and promotes testing, early diagnosis and linkage to care in at-risk populations. services are heterogenous, variously targeting the general population or mix of key populations; (msm), people who inject drugs (pwid), migrants or sex workers (sw). clients enter cbvct services seeking an anonymous and confidential hiv, hepatitis c or syphilis screening. not all services offer testing for all diseases. the hcv screening tests detect antibodies to hcv in human serum, plasma or whole blood (giving result: reactive/unreactive). clients with a reactive specimen then require referral for an rna test to identify current hcv infection (giving result: active hcv infection/no active hcv infection). rna testing is not part of the standard offer for cbvct services but they can report to the cobatest data collection tool if they have the result of the rna test performed at another site. the cobatest network began in 2013 with the goal of homogenizing the monitoring and evaluation of hiv testing activities at the community level [8, 9] and later expanded to syphilis and hcv screening. the standardised data collection tools collect information on sociodemographic characteristics, reasons for testing, previous hiv/ hcv/syphilis screening testing, risk behaviours, hiv/ hcv/syphilis screening test results, linkage to care. since 2014 this data has been collected and centralised in a single database. in 2018, 45 organisations in 20 countries in the who european region submitted data on their testing activity. this study is the first pan-european observational study using data from cbvct services which share common data collection tools to investigate screening for hcv in a range of populations. using cobatest data, this study aims to understand who is using cbvct services for hcv screening and what factors determine a reactive test. this study describes hcv screening activity in cbvct services, describes the populations being screened, describes the proportion of reactive hcv screening tests and identifies risk factors associated with a reactive hcv screening test result in the cobatest network in the years 2014-2018. this study is based on disaggregated data collected from members of the cobatest network which offered hcv screening for a minimum of 6 months during the period of study (january 1st 2014-december 31st 2018). counsellors complete pre-test counselling and collect information on the client using the cobatest standardised data collection tools. cobatest network members can submit their data in one of three ways: using the online cobatest tool; in disaggregated format according to data file specifications; in aggregated format with the cobatest indicators already calculated. for this study, all disaggregated data submitted before the censuring date of 28 february 2019 was included. cbvct services use a unique client identification code to anonymise the client data and allow the identification of repeat testers. in the case that one person was screened multiple times for hcv over the study period, only the most recent hcv screening test was included in the study. those with no reported hcv screening test result or aged under 16 were removed from the analysis. the flowchart of inclusion criteria is presented in fig. 1 . the same inclusion criteria were applied to people tested for hiv, not hcv, in the cobatest network and a comparison between the two groups is presented in online annex 1. firstly, we described characteristics and activity in cbvct services which offer hcv screening. to do this we used an existing database created as part of a 2017 study into the quality of the cobatest network's data [10] . in the scope of that study, all the cbvct services that were partners of the cobatest network in 2017 were invited to complete the online survey which was a piloted, structured ad hoc instrument hosted by the survey monkey website. cbvct services that did not send the information in 2017 were contacted for this study via email to report their services' characteristics. we refer to each cbvct service by its number which is randomly assigned when a service joins the cobatest network. according to cbvct service, we report testing setting, target population, which hcv screening tests were offered (rapid oral test, rapid blood test, conventional test) and number of people screened for hcv. secondly, the clients screened for hcv and clients with a reactive screening test were described by socio-demographic characteristics (gender [men, women, transgender], age group [16-24, 25-44, 45-64, > 65], migrants [defined as been born in a country different to the country where the cbvct service is placed], region of origin), key populations (msm, pwid, sw) and epidemiological variables (hiv status, hcv screening test results and rna test results). differences between groups were assessed using the pearson's chi 2 test with a p value of < 0.05 considered statistically significant. finally, the univariate logistic regression analysis was performed and the odds ratios for a reactive hcv screening test were presented with their 95% confidence intervals and p values. clients with missing information on key populations were considered in the non-risk category (see below for sensitivity analysis). the multivariate logistic regression model was decided using a forwardstepwise method, using the significant variables from the univariate analysis. in the case that two variables that were both significant in the univariate analysis showed colinearity, the one likely to be a bigger contributor to hcv incidence according to the literature was selected for the forward-stepwise method. for example, if the variables hiv + msm and msm were both significantly associated with reactive hcv test, msm was discarded when selecting variables for the multivariate model. there were high proportions of missing data in some variables included in the logistic regression model. a sensitivity analysis was performed to understand the impact of recoding missing data to the non-risk category in the logistic regression model. the results from the analysis of two different logistic remove clients aged <16 n=59 regression models were compared; the first (the model used for this study) considered all missing and "don't know" responses in the non-risk category and the second eliminated all missing and "don't know" answers from the analysis. data analysis was performed using statase 14.1. the description of cobatest members in table 1 shows the heterogeneity of their services; testing is variously offered on-site, in outreach or in venues using rapid oral test, rapid blood test or conventional laboratory tests. the majority of centres target the general population or msm. the services are not representative, neither at the national nor european level, of cbvct services offering hcv screening. in table 2 describes the testers; the majority aged ≥ 25 and < 45, msm, not hiv+ , not reporting a history of injecting drug use or sex work. of the 75 persons reporting to be hiv+ , 68 were msm (not displayed). the proportion of reactive hcv screening test was higher in transgender people compared to men and women (not statistically significant), higher in people living with hiv (plhiv) than others, higher in sex workers (sw) than non-sex workers and higher in pwid than those not reporting history of injecting drug use. missing data is displayed as a percentage of each variable. more than 80% of information was complete for each of the following variables: gender, migrant, msm. other variables were less complete: age group (77.6% complete), sex worker (64.2% complete) result of last hiv test (51.9% complete) ( table 2) . table 3 presents the results of the univariate and multivariate logistic regression analysis. a reactive hcv screening test result was associated with being aged ≥ 25 and < 45, msw, pwid; a migrant or a plhiv. for the multivariate model, age was excluded given the large proportion of missing data. the final multivariate model included hiv + msm, pwid and migrant status, finding each to be independently associated with a reactive hcv screening test. the results of the sensitivity analysis are presented in online annex 1. the factors associated with a reactive hcv screening test remained the same in both models and the strength of the association differed only slightly indicating that considering the missing responses in the non-risk category did not greatly impact the results of the study. this study demonstrates that hiv-msm with no history of injection drug use are using cbvct services for hcv screening, but a reactive screening test is associated with being hiv+ or pwid. cbvct services in the european region are increasingly integrating hcv screening into their service model to ensure testing is more widely available for populations that may otherwise not be tested. the use of standardised data collection tools in the cobatest network allows for hcv screening data to be pooled and analysed in a timely and robust manner. the study identifies client characteristics associated with a reactive hcv screening test which could assist cbvct services in targeting testing. the number of cbvct services in the cobatest network offering hcv testing and the number of tests performed has increased every year since 2014 but the large numbers of hiv tests carried out over the study period shows that this is still services' main testing activity (see fig. 1 ). although this sample is not representative of all cbvct services in europe or cbvct services in each country, it may reflect the increasing number of cbvct services offering hcv screening. the rising number of tests performed in the network comes with a decrease in proportion of reactive hcv tests, reflecting services increasingly offering screening to populations at low risk of hcv. this could have implications for the cost-effectiveness of cbvct services and requires more investigation to understand how services fund their hcv screening programme and to understand if cbvct services have the knowledge to identify people at higher risk of hcv. services offered by cobatest members are heterogeneous but the majority of clients being screened for hcv are msm, as has been seen in previous studies on hiv screening [8] . we found a high proportion of reactive hcv tests in pwid, in line with the lower end of the estimated range from the ecdc (7-95.4%) [11] . the low number of pwid tested reflects the fact that pwid are not the target population for the majority of the cbvct services in this study. pwid could benefit from improved access to services offering hcv screening which are tailored to their needs. peer counselling for this group has been suggested as a way to destigmatise testing [12] . in this study, being msm was not associated with a reactive hcv screening test but being hiv + msm was. hiv + msm account for a small fraction of all people tested but show a high proportion of reactive tests, reflecting studies that find the hcv burden in msm is concentrated in hiv + msm [13] . the cobatest questionnaire does not collect enough information to identify other subpopulations of msm at higher risk of hcv such as preexposure prophylaxis (prep) users. a study in france in 2016-early 2017 found the incidence of primary hcv in hiv + msm similar to that of hiv-msm who use prep and recommends both groups should be targeted for hcv screening (cotte et al. [14] . ecdc public health guidance for hcv testing recommends that msm, trans people and sw be tested for hcv every 6-12 months depending on ongoing risk (sexualised drug use, prep or pep use, hiv infection, history of rectal bacterial sti), that pwid be tested up to every 6 months and migrants be tested once with re-testing based on individual risk assessment [15] . to assess clients' ongoing risk, from the beginning of 2019 the cobatest data collection tool will collect information on prep use and chemsex. there are several limitations to this study. the study includes services in 10 countries but the results are not representative of all cbvct activity in these countries, nor in the european region, and therefore cannot be generalised to the region. we do not have complete information on how centres select who to screen for hcv, and why over 84,000 clients were screened for hiv and not hcv. there was a high proportion of missing data for some variables but, according to the sensitivity analysis, the estimated effect of this was minimal. there are issues with the data quality and documentation at service level which have been identified in a recent study [10] and have not all yet been resolved. cbvct services offer anonymous and confidential screening services, and no on-site rna test, thus it is unsurprising that there is a high percentage of missing information on rna tests. in order to understand if cbvct services are diagnosing active hcv infections, the reporting of rna tests and results should be improved. this could be achieved by collaborating with local infectious disease clinics or laboratories to share rna test results with the consent of the client. further investigation is needed to understand if cbvct services have functional linkage to care pathways for clients with a reactive hcv screening test. this study shows that the cbvct service model can be used to screen for hcv and that services are detecting possible active hcv infection. cbvct services that historically focussed on hiv testing are now expanding to offer screening for hcv and other diseases. this could boost testing coverage, especially for key populations, which is an essential element of any european country's strategy to eliminate hcv. countries should incorporate the cbvct service model as part of a national strategy to increase hcv diagnosis and linkage to care in order to reduce the burden of disease. governments can support cbvct services by facilitating circuits that allow fast referral to an rna test for clients who have had a reactive hcv screening and ensure linkage to care for those with an active infection. annual epidemiological report for 2017-hepatitis c combating hepatitis b and c to reach elimination by scaling up prevention and treatment towards the elimination of hepatitis c: a global mathematical model barriers to and facilitators of hepatitis c virus screening and testing: a scoping review internalised homonegativity predicts hiv-associated risk behavior in european men who have sex with men in a 38-country cross-sectional study: some public health implications of homophobia cobatest network: community-based voluntary counselling and testing in europe who guidelines on hepatitis b and c testing the cobatest network: a platform to perform monitoring and evaluation of hiv community-based testing practices in europe and conduct operational research guidelines for data collection for monitoring and evaluation of community based voluntary counselling and testing (cbvct) for hiv in the cobatest network assessing the quality of routine hiv testing data in the community setting 'cobatest network hepatitis b and c epidemiology in selected population groups in the eu/eea the berlin hepatitis c manifesto: access to prevention, testing, treatment and care for people who use drugs behavioural, not biological, factors drive the hcv epidemic among hiv-positive msm: hcv and hiv modelling analysis including hcv treatment-as-prevention impact hepatitis c virus incidence in hiv-infected and in preexposure prophylaxis (prep)-using men having sex with men public health guidance on hiv, hepatitis b and c testing in the eu/ eea-an integrated approach publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest there are no conflict of interest for any author. key: cord-007305-pkjfnhro authors: iosub, silvia; gromisch, donald s. title: leukonychia partialis in kawasaki disease date: 1984-10-17 journal: j infect dis doi: 10.1093/infdis/150.4.617-a sha: doc_id: 7305 cord_uid: pkjfnhro nan legend. different treatment courses for travellers highly suspected of being infected with borrelia recurrentis. no t some of the travellers in groups iii and iv had minor signs of tbrf (headache and low grade fever). colleagues -some areas in the northern part of the israel desert (negev) are known to be endemic for borrelia recurrentis infection [1] (tick-borne relapsing fever). four groups of subjects, aged 18-21 years, had an overnight stay in a few caves along a small valley in this area. the groups camped separately overnight in the same caves within an interval of four weeks. after an overnight stay in the caves, 50% of the individuals in each group found ticks or had signs of tick bites on their bodies. most actually noted the ticks biting them. fifteen (52%) of 29 subjects in groups i and ii had signs of bites. four to five days later, 12 of them developed fever (>39 c), rigors, headache, anorexia, nausea, vomiting, and generalized muscular pain. the clinical diagnosis of tick-borne relapsing fever was confirmed by dark-field microscopy of fresh blood samples (12 patients) or serologically (proteus oxk agglutination test) in the three patients in whom spirochete was not identified in the blood. tetracycline (tevacycline; teva ltd, jerusalem) [2, 3] was administered in a dose of 2.0 g/day for seven days and all of the patients recovered. following this experience, 13(46%) of 28 individuals (groups iii and iv) were given tetracycline (1.0 g/day) please address requests for reprints to dr. eithan galun, department of medicine a, hadassah university hospital, ein kerem, jerusalem 91120, israel for three to five days, 24-48 hr after evidence of tick bites. only two of these 13 subjects developed fever « 38c) for one day and three had headache for about two days. none of them developed overt symptoms and signs of tick-borne relapsing fever as observed in the subjects from groups i and ii. it would appear that a 'short-term, low-dose course of tetracycline may have prevented the appearance of the disease following a tick bite. ) . examination of the patients' nails showed that most fingernails and some toenails were abnormally white proximally, with a distinct sharp 2-4 mm transverse band of normal pink nail distally. the free edge of the nail was of normal white color and the nails were of normal texture. the color changes persisted for four to seven days. leukonychia (white nails) has been divided into four types: total, partial, striate, and punctate. all but the punctate type may be hereditary (autosomal dominant) or acquired. our patients had nail abnormalities typical for leukonychia partialis of the acquired type, since color changes had not been noted before the present illness and were transient. this type of leukonychia has been associated with tuberculosis, arteriosclerosis, nephritis, hodgkin's disease, chilblains, metastatic carcinoma, anemia, and hepatic cirrhosis [1, 2] . most researchers agree that it is caused by abnormal keratinization [2] [3] [4] . some [2] postu-correspondence late that altered vascular patterns of the nailbed or intravascular changes (anemia and hypoproteinemia) may also lead to increased whiteness of the nail. since our patients were not anemic or hypoproteinemic, we suggest that vasculitis was the cause of leukonychia partialis. although the heart and coronary arteries are mainly involved in kawasaki disease, other small and medium-sized arteries may exhibit intimal thickening, round cell infiltration, and, rarely, fibrinoid necrosis [5] . according to kawasaki et al. [6] an arteritis similar to that seen in infantile periarteritis nodosa and accompanied by coronary thrombosis and aneurysm was found in 13 autopsy cases. we would welcome correspondence from others who have seen nail color abnormalities in kawasaki disease. relapsingfever in children in the northern negev borrelia recurrentis infection: singledose antibiotic regimens and management of the jarisch-herxheimer reaction single-dose doxycyclinetreatment of louse-borne relapsingfever and epidemictyphus plorde 11. penicillin vs. tetracycline in the treatment of louse-borne relapsing fever phagocytosis of borreliarecurrentis by blood polymorphonculear leukocytesis enhanced by antibiotic treatment diseases of the skin acute febrile mucocutaneous lymph node syndrome a new infantile acute mucocutaneous lymph node syndrome (mlns) prevailing in japan no.4· october 1984 © 1984 by the university of chicago. all rights reserved colleagues -we examined paired sera from 62 infants with acute non bacterial gastroenteritis and from 50 age-matched controls (admitted to the hospital for nondiarrheal diseases) for antibody to human coronavirus (hcv) oc43 and neonatal-calf diarrhea coronavirus (ncdcy). antibody response was observed with greater frequency in patients (27.4010) than in controls (2.0%). it was characterized by the presence of neutralizing and often hal antibody to hcv oc43, but not to the antigenically related ncdcv [1] . these serological data suggested indirectly the existence of a human enteric coronavirus (hecy) antigenically related to hcv oc43, and prompted us to examine both infants and young children with acute gas-this work was supported by grant grt c6/i81/124 from the world health organization.please address requests for reprints to professor goiuseppe gerna, virus laboratory, institute of infectious diseases, university of pavia, 27100 pavia, italy. troenteritis and age-matched controls for detection of antibody to coronavirus in sera and coronavirus-like particles in stools. coronavirus-like particles were detected by electron microscopy in 34 (16.3%) of 208 patients and in 3 (1.6%) of 182 controls tested (p < .01), subsequently, we purified hecvs from stools of two patients (va24 and va 35) by sucrose density gradient centrifugation. antisera from mice and guinea pigs immunized with purified virus were examined by conventional immune electron microscopy (lem) [2] for reactivity to hcv oc43, ncdcv, and the two hecv strains. results showed a two-way crossreactivity between hcv oc43 and the two hecv strains. the antigenic relatedness to hcv oc43, as well as the typical morphology (figure), suggest that the coronavirus-like particles detected were actually hecvs. furthermore, the buoyant density of hecv-24 and hecv-35 in sucrose was approximately 1.20 glml, a value in agreement with those reported for human and animal coronavirus. convalescent-phase sera from all the patients positive for excretion of coronavirus-like particles in stools, and seronegative for previous hcv oc43 infections, reacted by iem with hecy-24 and hecv-35 and, to a lesser extent, with hcy oc43. acute-phase sera from the same patients were poorly reactive or nonreactive by iem. conversely, key: cord-000174-mgj9zfft authors: slavenburg, serena; heijdra, yvonne f.; drenth, joost p. h. title: pneumonitis as a consequence of (peg)interferon-ribavirin combination therapy for hepatitis c: a review of the literature date: 2009-04-28 journal: dig dis sci doi: 10.1007/s10620-009-0797-1 sha: doc_id: 174 cord_uid: mgj9zfft combination of peginterferon and ribavirin is the current therapy for chronic hepatitis c infection (hcv). interstitial pneumonitis is a rare side-effect of hcv therapy and is an important cause of dose reduction or discontinuation, impairing success of antiviral therapy. we performed a review of the literature in order to present diagnostic modalities and possible treatments for pneumonitis and to offer guidelines. we searched for cases where pneumonitis as a side-effect of hcv treatment was documented. first we performed a literature search via pubmed and web of science interface and second we searched three drug toxicity databases. we systematically analyzed all case reports with respect to clinical manifestations, type of treatment, and outcome. a literature search revealed 19 articles, containing 25 case descriptions, while we traced 33 cases from the drug toxicity databases. pneumonitis presented with any of the combination of fever, dyspnea, and cough and can arise with any type of (conventional or pegylated) interferon. mortality secondary to pneumonitis was seen in 7% of cases, exclusively with peginterferon α-2b. in most cases therapy was discontinued and steroids were started. interferon-induced pneumonitis during hcv treatment is a severe complication and should be recognized in order to prevent further pulmonary damage and/or death. hepatitis c (hcv) persists in up to 85% of patients and may result in liver cirrhosis and hepatocellular carcinoma [1] . the combination of peginterferon and ribavirin is the mainstay of treatment. eradication of the virus can be achieved in up to 90% of chronic hcv patients, depending on a number of host-and virus-related factors [1, 2] . successful treatment results in resolution of hepatic necroinflammation and regression of fibrosis. though potentially successful, peginterferon and ribavirin are known to cause various side-effects in hcv patients. most individuals suffer from some side-effects such as flu-like symptoms, myalgia, fatigue, gastrointestinal disturbances, psychiatric disorders, and hematological abnormalities such as anemia and leukopenia [3] [4] [5] . these adverse effects are relatively common and can be managed with supportive care. there are also less wellknown side-effects of antiviral treatment that may hamper successful eradication of the virus. knowledge of the extensive gamut of side-effects of combination therapy is critical for the adequate management of side-effects. pulmonary toxicity in patients undergoing hcv combination treatment is rare, and may include interstitial pneumonitis, sarcoidosis, pleuritis, bronchiolitis obliterans organizing pneumonia (boop), and exacerbation of asthma [6, 7] . pneumonitis occurs only rarely as a side-effect of hcv combination therapy and can arise at any stage of the treatment. most commonly it presents with cough, which is difficult to differentiate from the ubiquitous cough that may occurs as a common side-effect of combination treatment. interferon is the agent that is thought to be associated with pneumonitis in these patients. this paper aims to review the salient issues of pneumonitis in the setting of hcv combination treatment and aims to offer guidelines for diagnosis and treatment of pneumonitis. a 51-year-old male was seen in august 2005 because of hcv infection. he had been using intravenous (i.v.) drugs until 1985 and his medical history revealed multiple total hip replacements and revisions because of hip necrosis attributed to i.v. drug-related blood-borne infections. liver function tests were abnormal: alanine aminotransferase (alat) 237 iu/l (normal \45 iu/l), aspartate aminotransferase (asat) 235 iu/l (normal \50 iu/l), c-glutamyl transpeptidase 241 iu/l (normal \55 iu/l), and bilirubin 19 lmol/l (normal \10 lmol/l). the patient was infected with genotype 3 hcv virus with high viral load ([5 9 10 5 iu/ml). a liver biopsy was performed and showed moderate necroinflammation with portoportal fibrosis (metavir; grade a2, score f2). in view of this advanced stage we offered the patient a 24-week treatment for hcv consisting of peginterferon a-2b (pegintron ò ; 150 lg s.c. weekly) in combination with ribavirin (rebetol ò ; 400 mg twice daily). four weeks after starting treatment he consulted us for a dry cough. chest x-ray at that time showed no abnormalities. although dry, nonproductive cough is usually caused by ribavirin, it typically clears after stopping the drug. the dry cough, however, persisted after end of treatment, being the reason to consult a chest physician. physical examination revealed bibasal crepitations. a new chest radiograph showed bilaterally a diffuse, interstitial pattern ( fig. 1a) and chest high-resolution computed tomography (hrct) demonstrated bilateral ground-glass opacities in central and upper zones (fig. 1b) . pulmonary function tests indicated a restriction and a diminished diffusing capacity. maximal incremental cycle ergometry showed decreased breathing reserve (0 l/min), high breathing frequency (65/ min), and exercise-induced hypoxemia caused by an oxygen uptake problem, compatible with interstitial pulmonary disease. results from bronchoalveolar lavage supported a diagnosis of drug-induced interstitial pneumonitis (cell count 1.68 9 10 9 /l, 73% lymphocytes, cd4/cd8 ratio 0.5). the results of cultures and stains of the bronchoalveolar lavage specimens were negative for bacteria, fungi, acid-fast bacteria, cytomegalovirus, herpes simplex virus, and malignant cells. there were no signs of infection (blood culture was negative). as after 6 weeks the cough persisted, steroids up to 40 mg daily were started, which resulted in slow amelioration of symptoms, normalization of pulmonary function tests, and disappearance of the ground-glass effect on hrct. one year after end of treatment the patient is well, without evidence of recurrence of hepatitis c. first we performed a literature search for articles on pneumonitis as a side-effect of hcv treatment in order to obtain a comprehensive overview of this particular side-effect. we took advantage of the pubmed and web of science interface (http://www.ncbi.nlm.entrez and http://apps.isiknowledge. com) and searched with the following keywords: interferon, interstitial, pneumonitis, and hepatitis c, for the period 1990-2008. articles written in english, french or german were included in the analysis. furthermore, we searched the three drug toxicity databases. we included the netherlands pharmacovigilance center (www.lareb.nl) database and the fig. 1 a chest x-ray with diffuse, bilateral interstitial pattern. b high-resolution computer tomography image showing bilateral ground-glass opacities in central zone drug-induced lung diseases database (www.pneumotox. com), and lastly we performed an exhaustive search of the database of the world health organization (who) via the uppsala monitoring center (http://www.who-umc.org). we used as keywords interferon and pneumonitis. the who database is a collection of data about adverse drug reactions from around the world, especially from countries that are members of the who, and the generation of signals of drugs which might possibly have problematic side-effects. inclusion criteria were cases that developed interstitial pneumonitis in the setting of hcv treatment, written in english, french or german. exclusion criteria were other pulmonary diseases, e.g., sarcoidosis, exacerbation of asthma, boop, acute respiratory distress syndrome (ards), infectious pneumonia, liver transplanted patients, hiv co-infected patients, and interferon beta treatment. patients who developed interstitial pneumonitis during interferon therapy with a disease other then hepatitis c were also excluded. the references of the traced articles were scrutinized for additional articles. initial analysis yielded a total of 291 articles. the abstracts of this set of articles were scrutinized for cases that developed (interstitial) pneumonitis in the setting of hcv treatment. subsequently data were retrieved with special attention to the following items: demographics (ethnicity, gender, and age), dosage and type of (peg)interferon, concomitant use of ribavirin (dosage), symptoms, interstitial pneumonitis, onset, symptoms, and outcome on follow-up. we retrieved 19 articles, which contained detailed clinical descriptions of 25 cases of interstitial pneumonitis during or after hcv treatment. our own case was added to the analysis. articles were published in the time frame 1988-2008. a total of 25 cases, 12 (48%) males and 13 (52%) females, with mean age of 55.2 years, developed an interstitial pneumonitis. ethnicity was mentioned in seven cases, being caucasian (five) and asian (two). tables 1 and 2 present the demographic characteristics of all cases. fourteen patients were treated with conventional interferon, while 11 patients developed pneumonitis during or after treatment with peginterferon and ribavirin. interferon a-2b had been used in eight patients (62%), while interferon a-2a and lymphoblastoid interferon (6 mu q.d. 9 10 days, then 6 mu t.i.w.) was used in one single case. interferon type was not specified in three cases. dosages regimes varied with type of interferon and are presented in table 2 . onset of symptoms of interstitial pneumonitis ranged from 20 days to 23 weeks of therapy. symptoms included cough, dyspnea, and fever. peginterferon a-2b was used in eight patients (67%), while peginterferon a-2a was used in four patients (33%). consensus interferon (infergen, 9 lg q.d.) was given in a single case. dosage regimes varied from 100 to 180 lg/week, depending on type of peginterferon, and from 400 to 1,200 mg/day for ribavirin. in two cases amantadine was added to the treatment regimen. onset of symptoms of interstitial pneumonitis ranged from 2 to 12 weeks of therapy. in all interferon cases therapy was discontinued, and five of these cases resolved without treatment [8] [9] [10] . eight patients needed to be treated with various steroids. interstitial pneumonitis in a 72-year-old female patient was treated with 30 mg/day prednisolone, and she was then maintained on a regimen of intermittent pulse therapy with 100 mg/day; a 56-year-old male patient was started with 2 g/day methylprednisolone for 3 days and 40 mg/day prednisolone for 2 days [10] . prednisolone (60 mg/day) and 100 mg/day azathioprine were given after a relapse of interstitial pneumonitis that was initially treated with 30 mg/day prednisolone [11] . in one case of therapy with methylprednisolone pulse therapy, 1 g/day i.v. for 3 days was given, followed by 40 mg/day oral prednisolone twice over 2 weeks; the other cases in that article received 50 mg/day oral prednisone [12] . therapy for one case was not specified [13] . in eight cases combination therapy was discontinued and steroid therapy was initiated. while dosage and length of steroid treatment was highly variable, most authors started at a relatively high dosage. intravenous therapy with 180 mg/day methylprednisolone was started in one case [14] , while other authors started with 125 mg/day prednisolone [15, 16] . others started patients on oral steroid therapy (40 mg) [6, 17, this study], while two authors initiated inhalation steroids [18, 19] . interstitial lung disease resolved in one case with oral antibiotics, given under presumptive diagnosis of community-acquired pneumonia. she recovered without sequel [7] . in four combination-therapy cases the dosage of steroid therapy was not defined [20] [21] [22] [23] . we identified 60 pneumonitis cases in association with (peg)interferon a from the who database. in 33 cases the indication of (peg)interferon (and ribavirin) combination treatment was hcv. there were 16 (48%) males and 15 (45%) females in this cohort, with mean age of 53 years. we failed to retrieve cases that met the inclusion criteria from the netherlands pharmacovigilance center and drug-induced lung diseases databases. interferon monotherapy was used in three (9%) patients. interferon a-2b in combination with ribavirin was used in eight patients (24%), while interferon a-2a in combination with ribavirin was used in a single case. peginterferon a-2b in combination with ribavirin had been used in 6 patients (18%), while in 13 patients (39%) combination therapy with peginterferon a-2a was used. in two cases type of interferon and ribavirin was not described. dosages regimes varied from 80 to 180 lg/week, depending on type of peginterferon, in combination with ribavirin (800-1,200 mg/day). onset of symptoms of interstitial pneumonitis ranged from 23 days to 10 months of therapy. four patients recovered and one patient died after drug-induced pneumonitis; outcomes of the remaining patients were not described. there was no description on treatment regimen for pneumonitis. collectively, the cohort consisted of 25 patients from literature cohort and 33 patients from the drug toxicity database. four patients (7%) from these two cohorts died following development of interstitial pneumonitis. all patients had been treated with peginterferon a-2b. death in one case was due to multiple organ failure [14] , in another case due to cerebral edema [15] , and in one case because of development of acute cholestatic hepatitis [16] . the cause of death of one case from the who database was not described. interstitial pneumonitis occurs only rarely as a side-effect of hcv combination treatment and often leads to discontinuation of therapy, which has great implications for patients. in this article we presented our case of pneumonitis during combination therapy and performed a review in order to generate guidelines to manage symptoms and treatment. given the paucity of reports with interstitial pneumonitis after ribavirin monotherapy, we suspect that interferon is the culprit. the most common presenting symptom of pneumonitis is any combination of dry cough, dyspnea, fever, and fine inspiratory crackles noted on examination. hemoptysis, wheezing, and signs of consolidation are rare. onset of pneumonitis can be at any stage of hcv treatment, supporting the idiosyncratic nature of this side-effect. chest radiographs usually show bilateral patchy infiltrates or opacifications, and thin-section ct scans show bilateral patchy consolidation as well as ground-glass attenuation [24] . in most cases, symptoms of pneumonitis are reversible after cessation of treatment with (peg)interferon and ribavirin, again in support of drug-induced interstitial pneumonitis. there is no consensus regarding treatment of interstitial pneumonitis induced by (peg)interferon and ribavirin. upon review of the literature, three options are possible: first, to stop combination treatment of hcv and wait until the disease resolves, which was done in a limited number of cases [7] [8] [9] [10] ; second, to give steroids, although dosage and route of administration regimes vary widely [6, 10-18, this study]; and thirdly, in therapy-resistant or relapsing cases, adding azathioprine to steroids may be beneficial in order to resolve the interstitial pneumonitis [11] . the relatively high mortality rate in our series suggests that a more aggressive approach is warranted, and favors the early administration of steroids. one remarkable finding is that mortality occurred exclusively in patients with peginterferon-induced pneumonitis in comparison with conventional interferon (7% versus 0%). the reason for this is unclear, but (peg)interferon has been associated with other pulmonary toxicity such as sarcoidosis, pleuritis, boop, and exacerbation of asthma. patients who died in the combination-therapy group were relatively young and had no relevant pulmonary or other severe diseases in their medical history. the doses of peginterferon and ribavirin varied but ranged within limits offered by treatment guidelines [25] . the mortality, only observed in patients treated with peginterferon and ribavirin combination therapy, raises the issue of whether the peg molecule increases the severity of pneumonitis once it arises. interferon toxicity is generally dose and duration dependent [26] , which leads us to speculate that pulmonary toxicity may occur more severely with long-acting peginterferon; however, we saw no effect of dosage on the occurrence of pneumonitis. patients died due to different causes (e.g., hypoxia-induced cerebral edema, acute cholestatic hepatitis, and multi-organ failure), all were induced by complications after initially interstitial pneumonitis. one alternative explanation for the increased mortality might be that all patients with peginterferon were also treated with ribavirin, although ribavirin per se is not associated with pulmonary toxicity. our data suggest no significant difference between interferon a-2a or 2b, which suggest it is not due to the interferon molecule. in most interferon monotherapy cases of interstitial pneumonitis in hcv treatment in japan the use of sho-saiko-to, a herbal medicine, led to pneumonitis. sho-saiko-to has been approved by the japanese ministry of health and welfare and has often been administered in chronic viral liver disease [27] . the mechanism of this side-effect, probably caused by interferon, remains unclear and several pathophysiological mechanisms have been proposed, centering on the known immunomodulatory activity of interferon [28] . interferon has direct antiviral and immunomodulatory effect, including cytokine reduction, increased natural killer cell function, and enhanced cellular expression of major histocompatibility class 1 antigens [28] [29] [30] . it is plausible that interferon triggers a lung-specific immune-mediated response resulting in interstitial pneumonitis, similar to other autoimmune diseases. failure to recognize interferon-associated pulmonary toxicity may result in persistence of pulmonary damage. mortality was seen only in patients treated with pegylated interferon combination therapy. therefore, clinicians should be aware of development of interstitial pneumonitis in chronic hepatitis c patients who develop pulmonary symptoms such as cough or dyspnea. the threshold for obtaining chest x-ray or pulmonary hrct scan in these patients should be low. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. peginterferon and ribavirin for chronic hepatitis c hepatitis c virus infection seizures associated with low-dose alpha-interferon side effects of therapy of hepatitis c and their management a survey of adverse events in 11, 241 patients with chronic viral hepatitis treated with alfa interferon significant pulmonary toxicity associated with interferon and ribavirin therapy for hepatitis c spectrum of pulmonary toxicity associated with use of interferon therapy for hepatitis c. case report and review of literature side effects of high-dose interferon therapy for chronic hepatitis c interstitial pneumonitis and interferon-alfa pneumonitis associated with natural and recombinant interferon alfa therapy for chronic hepatitis c a patient with chronic hepatitis c who simultaneously developed interstitial pneumonia, hemolytic anemia and cholestatic liver dysfunction after alphainterferon administration pneumonitis during interferon and/or herbal drug therapy in patients with chronic active hepatitis induction of interstitial pneumonitis during interferon treatment for chronic hepatitis c pegylated interferon and ribavirin-induced interstitial pneumonitis with ards severe interstitial pneumonitis secondary to pegylated interferon alpha-2b and ribavirin treatment of hepatitis c infection interstitial pneumonitis associated with pegylated interferon alpha-2b therapy for chronic hepatitis c. case report consensus interferon induced interstitial pneumonitis in a patient with hcv pneumonia recurrence during chronic hepatitis c treatment interstitial pneumonitis during combination therapy with interferon-a and ribavirin in a patient with chronic hepatitis c interstitial pneumonitis after combination therapy with pegylated interferon alpha-2b and ribavirin for chronic hepatitis c pulmonary toxicity by pegylated interferon alpha-2a in a patient with chronic hepatitis c induced interstitial pneumonitis: role of pegylated interferon alpha 2b interstitial pneumonitis due to pegylated interferon alfa-2b and ribavirin nonspecific interstitial pneumonia with fibrosis: radiographic and ct findings in seven patients diagnosis, management, and treatment of hepatitis c sarcoidosis after use of interferon for chronic hepatitis c: report of a case and review of the literature report on drug side effect effector mechanisms of immune responses a perspective on the clinical effectiveness and tolerance of interferon alpha new insights into the mechanisms of interferon alfa: an immunoregulatory and anti-inflammatory cytokine acknowledgments the authors are indebted to the national pharmacovigilance centers that contributed data for this study. the opinions and conclusions, however, are not necessarily those of the various centers, nor of the who. key: cord-006061-z9r5htm9 authors: hu, bo; li, shanshan; zhang, zhanfeng; xie, shenggao; hu, yuqian; huang, xianzhang; zheng, yi title: hcv ns4b targets scribble for proteasome-mediated degradation to facilitate cell transformation date: 2016-06-17 journal: tumour biol doi: 10.1007/s13277-016-5100-4 sha: doc_id: 6061 cord_uid: z9r5htm9 hepatitis c virus (hcv) nonstructural protein 4b (ns4b) is a multi-transmembrane protein, but little is known about how ns4b contributes to hcv replication and tumorigenesis. its c-terminal domain (ctd) has been shown to associate with intracellular membrane, and we have previously shown that ns4b ctd contains a class i pdz-binding motif (pbm). here, we demonstrated that ns4b pbm interacts with the pdz-containing tumor suppressor protein, scribble, using immunofluorescence and co-immunoprecipitation assays, and this interaction requires at least three contiguous pdz domains of scribble. in addition, ns4b pbm specifically induced scribble degradation by activating the proteasome-ubiquitin pathway. similar scribble degradation was also observed in hcv-infected cells, suggesting ns4b could work in the context of hcv. finally, ns4b pbm mutants showed reduced colony formation capacity compared with its wild-type counterpart, indicating that ns4b pbm plays important roles in ns4b-mediated cell transformation. altogether, we provide a mechanism by which ns4b induces cell transformation through its pbm, which specifically interacts with the pdz domains of scribble and targets scribble for degradation. hepatitis c virus (hcv) is an enveloped positive singlestranded rna virus that causes hepatitis c and hcv-related liver diseases including hepatocellular carcinoma (hcc) [1] . up until now, there is no effective hcv vaccine available and hepatitis c is becoming a serious global health problem that affects 170 to 200 million people worldwide. the current standard of care is the combined use of nucleoside analog ribavirin and pegylated interferon-α; however, the efficacy of this treatment is limited and depends largely on the viral genotypes [2] [3] [4] . the hcv genome encodes a polyprotein of about 3010 amino acids, which is cleaved into functional proteins including the mature structural capsid protein (c), envelope proteins (e1 and e2), ion channel (p7), and six nonstructural proteins (ns2-ns5b) [5] . although the precise mechanism underlying hcv replication process remains largely unclear, most of the hcv nonstructural (ns) proteins are known to be involved in membranous web (mw) and double-membrane vesicles (dmvs) formed by protein-protein and protein-lipid interactions, which is the site of the viral rna synthesis [6] [7] [8] . one of the ns proteins that are indispensable for viral replication is hcv ns4b, which is a hydrophobic protein highly conserved in flaviviridae family [9] . ns4b expression causes alteration of the endoplasmic reticulum (er) membrane and formation of mw, which could provide a platform for hcv viral replication [8, 10] . our previous work has shown that ns4b expression activates unfolded protein response (upr), er overload response, and nf-κb pathway in human hepatic cells, which could contribute to hcv replication and pathogenesis [11] [12] [13] . hcv ns4b is a 27-kda protein consisting of an nterminal domain (aa 1 to 69), c-terminal domain (aa 191 to 261), and a central transmembrane domain (aa 70 to 190). topology structure studies of ns4b showed that its nterminal domain comprises two amphipathic α-helices (ah1 and ah2) with several functional properties. its central domain consists of four transmembrane domains (tms) and a nucleotide-binding walker a motif, mediating hcv viral replication and replication-related focus formation [9] . the cterminal domain has been suggested to be the major membrane-binding domain, but recent studies showed that it is involved in protein-protein and protein-rna interactions [14, 15] . in addition, the predicted helices and motifs of the cterminal domain are involved in hcv replication [16] . we have previously analyzed the interaction motifs within ns4b using basic elm software and found that its cterminus contains a pdz-binding motif (pbm) [9] , which could bind cellular pdz-containing proteins such as the membrane protein, scribble. scribble is a pdz-containing protein and has been well known for its roles in cellular polarity [17] and apoptosis [18] . the functions of scribble have been shown to be modulated by viral proteins [19] . human tlymphotropic virus type 1 (htlv-1) tax protein affects the localization of scribble protein and inhibits its activity to attenuate t cell receptor (tcr)-induced activation of nuclear factor of activated t cells (nfat). tick-borne encephalitis virus (tbev) ns5 protein interacts with scribble to inhibit interferon-mediated jak-stat signaling. in addition, highrisk human papillomavirus (hpv) e6 protein binds scribble protein to cause its degradation through proteasome-mediated proteolysis [19] . hcv ns4b has been reported to transform nih3t3 cells, but little is known about the underlying mechanism [14] . here, we showed that ns4b pbm is a key motif that enables ns4b to interact with scribble protein, and at least three pdz domains of scribble are required for this interaction. furthermore, this interaction leads to scribble degradation through the proteasome-ubiquitin pathway. we also observed this proteasome-dependent scribble degradation in hcv-infected huh7.5.1 cells, suggesting that ns4b has some roles for hcv-mediated scribble degradation. in addition, ns4b pbm confers the ability of ns4b to transform cells. taken together, our study revealed a novel mechanism by which ns4b contributes to hcv tumorigenesis by targeting tumor suppressor protein, scribble, for degradation. 293t, hepg2, huh7.5.1, and hela cells were maintained in dulbecco's modified eagle medium (dmem) with 10 % fetal bovine serum (fbs) and cultured at 37°c in a 5 % co 2 incubator. the replicon con-1 was kindly provided by prof. ralf bartenschlager (university of heidelberg, germany). to construct ns4b pbm mutants, the last four amino acids of ns4b were deleted or site-direct-mutated as alanine (aaaa) with appropriate sets of primers. the full-length cdna of ns4b gene was designed as ns4bwt, the ns4b with last four amino acids deleted as ns4bd, and the ns4b with last four amino acids mutated as ns4bm. these ns4b sequences were inserted into pegfpc1 and pflag-cmv2 plasmids (invitrogen, usa) to construct pegfpc1-ns4b, pegfpc1-ns4bd, and pegfpc1-ns4bm and pflag-cmv2-ns4b, pflag-cmv2-ns4bd, and pflag-cmv2-ns4bm, respectively. the plasmid pcdna/flag-scribble (kindly provided by prof. rice, baylor college of medicine, usa) was used as template to amplify all the fragments of scribble and cloned into indicated mammalian expression vectors. all the constructs were verified by dna sequencing. the plasmids were transfected into cells at approximately 80 % confluence by lipofectamine 2000 (invitrogen, karlsruhe, usa) in 150-μl dmem medium without 10 % fbs, according to manufacturers' instructions. at 24 h post-transfection, cells were washed with phosphate-buffered saline (pbs) and fixed in 4 % formaldehyde for 15 min at room temperature. cells were permeabilized in 0.1 % triton x-100 in pbs, blocked with 3 % fbs in pbs, and then incubated with primary antibody overnight at 4°c followed by incubation with secondary antibody conjugated to red fluorochromes. the cell nuclei were stained with 1 μg/ml dihydrochloride (beyotime, china). cover slips were mounted upside down on slides with suitable volume of prolong gold antifade reagent (invitrogen, usa). the cells were examined using an olympus ix81 fluorescence microscope equipped with the appropriate filter sets and olympus fluoview ver.2.0a viewer software. the antibodies used were anti-scribble (santa cruz biotechnology, usa), anti-goat tritc labeled igg antibody (kpl, usa). at 48 h post-transfection, 293t cells were washed in ice-cold pbs and lysed on ice with lysis buffer (20 mm tris-hcl ph 7.5, 150 mm nacl, 1 mm edta, 1 mm egta, 1 % triton x-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm navo 4 , 1 μg/ml leupeptin, 1 mm pmsf). cell-free lysate was mixed with anti-flag agarose (abmart, china), washed three times with lysis buffer, and boiled in 40 μl of 1× sds-page loading buffer. proteins were separated on sds-page gel and transferred into pvdf membrane. membrane was blocked with 5 % nonfat milk and incubated with primary antibodies overnight at 4°c and secondary antibodies (kpl company) in 2.5 % nonfat milk at room temperature for 2 h. proteins were visualized by supersignal ® west pico chemiluminescent substrate (thermo scientific). plasmid pjfh1 that contains the full-length hcv genotype 2a jfh1 strain cdna was kindly provided by professor wakita (national institute of infectious diseases, tokyo, japan). the infectious jfh1 hcv 2a virus was generated, and virus titers were quantified by rt-pcr as previously described [20] . huh7.5.1 was infected by jfh1 at tcid50 of 100. cells were harvested at the indicated time, and scribble expression was analyzed by qrt-pcr and western blot. at 24 h post-transfection, trypsinized cells were suspended in dmem medium and subsequently 0.3 % low-melting-point agarose overlaid onto a solidified layer of dmem medium and 0.6 % agarose in 20-mm plates (1000 cells/plate). two weeks later, the colonies were stained with 0.005 % crystal violet (sigma, usa). the colony-forming units (cfu) with more than 100 cells were counted under a light microscope. we have previously reported that hcv ns4b contains a pbm domain within its c-terminal domain, which enables it to interact with pdz domain-containing proteins [9] . as scribble protein contains four pdz domains and interacts with the pbm of some viral proteins [19] , we hypothesized that hcv ns4b pbm domain could interact with the pdz domain of scribble. to test this hypothesis, we co-transfected pegfp-ns4b and pflag-cmv2-scribble expression vectors into 293t cells and analyzed their co-localization by immunofluorescence. hcv ns4b localized throughout the cytoplasm and has a perinuclear staining (fig. 1a, second panel) , consistent with previous reports about ns4b localization [21, 22] . most importantly, we found that ns4b co-localized with scribble (fig. 1a , second and third panels), indicating an interaction between these two proteins. we also observed the co-localization between ns4b and scribble in other cells lines including hela (fig. 1a , fourth panel) and hepg2 cells (fig. 1a, fifth panel) , indicating that hcv ns4b interacts with scribble protein irrespective of cell lines. to identify which ns4b domain(s) interacts with scribble, we constructed vectors that express egfp-fused ns4b nterminal domain (egfp-ns4bn), central domain (egfp-ns4bcore), and c-terminal domain (egfp-ns4bc) and transfected them into 293t cells. we found that ns4b nterminal domain distributes around the nucleus but not nearby the membrane (fig. 1b, first panel) . ns4b central domain displays small punctate or dot-like structures throughout the cells (fig. 1b, second panel) . however, neither ns4b n-terminal domain nor central domain co-localized with scribble. only ns4b c-terminal domain showed colocalization with scribble (fig. 1b, third panel) , indicating that ns4b c-terminal domain is required for ns4b-scribble interaction. as pbm resides in ns4b c-terminal domain, to further explore whether ns4b pbm mediates the ns4b-scribble interaction, we mutated ns4b pbm either by deleting all pbm residues (egfp-ns4bd) or changing all pbm residues to alanine (egfp-ns4bm) and transfected these two mutants into 293t cells. we then analyzed the interaction between ns4b and endogenous scribble protein by immunofluorescence assay. as shown in fig. 1b , egfp-ns4bd and egfp-ns4bm proteins were expressed throughout the cytoplasm, almost the same as egfp-ns4b, indicating that ns4b pbm domain does not affect its cellular localization. however, there is no overlay area of endogenous scribble with either egfp-ns4bd or egfp-ns4bm (fig. 1b, fourth and fifth panels) , indicating that ns4b pbm domain is required for ns4b-scribble interaction. we further analyzed ns4b-scribble interaction by coimmunoprecipitation (co-ip). we transfected 293t or hepg2 cells with plasmids expressing flag-tagged ns4b (flag-ns4b) or its pbm mutants (flag-ns4bd or flag-ns4bm). ns4b was immunoprecipitated with anti-flag resin. as expected, scribble protein was precipitated with wild-type ns4b but not ns4b pbm mutants (fig. 2 , compare lane 6 to lanes 7 and 8), confirming that ns4b pbm is required for ns4b to interact with scribble. the pdz domains of scribble are required for its interaction with ns4b pbm scribble consists of three primary domains: leucine-rich repeats (lrrs), lap-specific domain (lapsd), and four contiguous pdz domains (fig. 3a) . both lrrs and pdz domains are protein-protein interaction modules. to determine whether hcv ns4b interacts with pdz domains of scribble, we transfected the plasmid expressing myc-tagged four scribble pdz domains into 293t cells and examined its interaction with ns4b by co-ip assay. as shown in fig. 3b , ns4b binds the four pdz fragment of scribble, indicating that the other two domains, lrr and lapsd, are not required for ns4b-scribble interaction. to identify which pdz domains are required for this interaction, we constructed a series of plasmids expressing myc-tagged scribble pdz domains and examined interactions with ns4b by co-ip assay. when pdz1, pdz2, pdz3, or pdz4 was expressed alone, they were not able to interact with ns4b ( fig. 3c-f ). expression of two tandem pdz domains (pdz1-2, pdz2-3, pdz3-4) of scribble also failed to interact with ns4b ( fig. 3g-i) . only scribble that contains the first three (pdz1-3) or the last three pdz domains (pdz2-4) interacts with ns4b (fig. 3j, k) , indicating that the interaction between hcv ns4b pbm and scribble involves at least three contiguous pdz domains of scribble. as hcv ns4b interacts with scribble, we next investigated the effects of ns4b on scribble. viral proteins have been shown to regulate scribble either by altering its localization or triggering scribble degradation [19] . from fig. 1a , we found that ns4b does not affect the localization of scribble. hence, we explored whether ns4b affects the protein levels of scribble. we transfected different amounts of ns4b expression plasmids into 293t and hepg2 cells and then examined the protein levels of scribble. increased expression of ns4b significantly reduced scribble protein levels in both 293t (fig. 4a) and hepg2 cells (fig. 4b) . in ns4b-transfected 293t and hepg2 cells, ns4b expression was higher at 48-h post-transfection compared with 24-h post-transfection; however, scribble protein levels were lower at 48-h post-transfection compared with 24-h post-transfection, revealing a negative correlation between ns4b expression and scribble protein level. we also examined the impact of ns4b on the other two pdz-containing proteins, lethal giant larvae2 (lgl2) and protease-activated receptor-3 (par3). our data showed that their intracellular protein levels were not altered by ns4b expression (fig. 4b -d, f; the par3 protein data was not shown). these results indicate that ns4b specifically induced scribble degradation. to understand how hcv ns4b triggered scribble degradation, we examined the involvement of the proteasomeubiquitin pathway. first, we examined the effects of ns4b on the protein levels of ubiquitin-specific peptidase 14 (usp14), a cytoplasmic protein that belongs to the ubiquitin-specific processing family of deubiquitinating enzymes [23, 24] . as shown in fig. 5a , expression of hcv ns4b led to reduced usp14 protein levels in both 293t cells and hepg2 cells especially at 48-h post-transfection, indicating that hcv ns4b activates the proteasome-ubiquitin pathway. to examine whether the proteasome pathway is involved in the scribble protein degradation, we performed a series of ubiquitination assays. cells were transfected with plasmids expressing full-length flag-tagged ns4b and then treated cells with the proteasome inhibitor, mg132. our data showed that ns4b induced higher ubiquitination of scribble than empty plasmids in both 293t cells and hepg2 cells (fig. 5b) , suggesting that ns4b induced scribble degradation via the proteasomeubiquitin pathway. we also transfected ns4b pbm mutants (flag-ns4bd and flag-ns4bm) into 293t and hepg2 cells and found that the protein levels of scribble were not significantly reduced by these two ns4b mutants when compared with the fulllength ns4b (fig. 5c) . as a control, the expression of endogenous β-actin and exogenous gfp was not altered (fig. 5c) , indicating that the specific degradation of scribble requires its interaction with ns4b pbm. to analyze whether ns4b triggers scribble degradation in the context of hcv, we investigated the effect of hcv jfh1 infection on scribble expression. huh7.5.1 cells were infected with hcv jfh1, and the expression of scribble was analyzed by qrt-pcr and western blotting. our results showed that scribble mrna was not significantly changed by hcv infection (fig. 6a) . fig. 4 hcv ns4b induced scribble degradation. a, b 293t (a) and hepg2 (b) cells were transfected with 1, 3, and 5 μg pcdna3.1-ns4b expression plasmid. twenty-four hours posttransfection, cell lysates were prepared and analyzed by western blot with indicated antibodies. pcdna3.1(−) plasmid was used as a negative control. c, d 293t (c) and hepg2 (d) cells were transfected with 1 μg of pcdna3.1-ns4b expression plasmid. twenty-four and 48 h post-transfection, the protein levels of ns4b, scribble, and lgl2 were analyzed by western blot. e, f relative protein levels of scribble (e) and lgl2 (f) were quantitated with standard deviation (sd). gapdh levels were used to normalize these two proteins. *p < 0.05; **p < 0.01 however, scribble protein was decreased in hcvinfected huh7.5.1 cells compared with mock-infected cells (fig. 6b) , indicating that hcv infection resulted in scribble degradation. altogether, these results implied that ns4b works in the context of hcv to trigger scribble degradation. as scribble is a tumor suppressor protein and hcv ns4b pbm targets scribble for degradation, it is highly possible that ns4b pbm may promote cell transformation by targeting huh7.5.1 cells were infected by hcv jfh1 and harvested at day 3 and day 5. a total mrna was extracted, and rt-pcr was performed using primers for scribble and β-actin. data were presented as mean ± se (n = 3, p>0.05). b cells lysates were subjected to western blot analysis using antibodies against hcv core protein, scribble, and βactin scribble. to test this hypothesis, we transfected hepg2 cells with ns4b-expressing plasmids. our results showed that ns4b induced more colonies (increased anchorageindependent growth) than empty vector-transfected cells (fig. 7) , consistent with its reported roles in transformation [14] . moreover, transfection of full-length ns4b significantly yielded more colonies than its pbm mutants, ns4bd or ns4bm (fig. 7) . these data suggest that ns4b promotes cell transformation primarily by its pbm domain-mediated scribble degradation. cell-derived membrane is the key platform for viral replication and assembly. the interaction between hcv proteins and host cellular proteins plays important roles in hcv life cycle and pathogenesis [25] . hcv ns4b is a highly hydrophobic protein and considered to be the least characterized hcv protein [26] . the major function of ns4b is to induce intracellular host membrane alteration and provide a platform for virus replication [8, 9, 27] . hcv ns4b has also been shown to cause cell transformation [14] . however, it remains to be determined about the underlying mechanism. in this study, we provided a plausible mechanism that ns4b induces cell transformation by degrading the tumor-suppressor protein, scribble, through the interaction between ns4b pbm and scribble pdz domains. our study provides the first evidence that hcv ns4b interacts with the tumor suppressor protein scribble via its pbm. several virus proteins have been shown to contain pbm that can bind pdz domain(s). these proteins include human adenovirus e4-orf1, hepatitis b virus (hbv) core protein, influenza a virus ns1 protein, hpv e6 protein, htlv1 tax protein, tbev ns5 protein, dengue virus (dv) ns5 protein, rabies g protein, sars e protein, rhpv e7 protein, and crpv le6 and se6 proteins (reviewed in [19] ). among these proteins, influenza a virus ns1 protein, hpv e6 protein, htlv1 tax protein, and tbev ns5 protein have been shown to interact with scribble. here, we provide evidence that hcv ns4b is a novel pbm-containing protein that interacts with scribble. the pdz domain is a protein-protein interaction module that is widespread throughout evolution. many cellular pdz proteins are targets for viral proteins including dlg1, dlg4, magl-1, magl-2, magl-3, scribble, pdlim2, mupp-1, patj, zo-1, zo-2, tip-1, tip-2/gipc, erbin, pro-il-16, ß1-syntrophin, pdzd8, ptpn4, and pals1 [19] . in this study, we found that hcv ns4b specifically interacts with scribble. moreover, we found that at least three contiguous pdz domains of scribble are required for this interaction (fig. 3 ). this phenomenon is quite different from the other reported virus proteins (hpv e6, htlv-1 tax, and tbev ns5) that target the cellular pdz protein [19] . for example, only pdz domains 4 and 5 of patj are required to bind hpv e6 [19] . it is highly likely that the interaction between hcv ns4b and scribble requires the three contiguous scribble pdz domains to form a favorable conformation and cooperative binding among these pdz domains. different viruses employ various strategies to modulate scribble to facilitate their replication and/or pathogenesis. some viral proteins alter scribble localization. the influenza a virus ns1 and htlv-1 tax proteins interact with scribble and result in mislocalization of scribble into cytoplasmic puncta [19] . the influenza a virus ns1-mediated relocalization of scribble was thought to inhibit the proapoptotic function of scribble. however, we did not observe any location change of scribble in ns4b expression cells (fig. 1a) . other viral proteins regulate scribble protein level. the hpv e6 binds scribble and results in its degradation values are means ± sd (n = 3). *p < 0.01,**p < 0.05 through the proteasome pathway [28] [29] [30] . similar to hpv e6, we found that hcv ns4b specifically induced scribble protein degradation with little to no effects on the other two pdzcontaining proteins, lgl2 and par3. this is the first report regarding the role of hcv ns proteins in regulating scribble functions by affecting its protein levels. moreover, hcv ns4b pbm induced scribble degradation through the ubiquitin-proteasome pathway, indicating that hcv ns4b utilized the same strategy with hpv e6 in regulating scribble. the tbev ns5 protein directly interacts with scribble and causes the inhibition of the jak/stat pathway by an unclear mechanism [19] . it is unknown whether hcv ns4b affects the jak/stat pathway. the ns4b pbm-scribble pdz interaction is important for colony formation of transfected cells, indicating that ns4b pbm contributes to hcv pathogenesis and cellular transformation by inducing scribble degradation. interestingly, colony formation assay also showed that in the cells expressing the ns4b pbm mutants, more colonies were formed compared to empty vector-transfected cells, suggesting that there are additional unknown mechanisms whereby ns4b promotes transformed cell growth in addition to pbm. as ns4b nucleotidebinding motif (nbm) has been reported to induce tumor transformation [14] , it is highly likely that both nbm and pbm confer ns4b the potential to promote tumor transformation. taken together, we identified an important motif, pbm, within ns4b and characterized its functions in facilitating tumor cell transformation through binding the pdz domains of tumor suppressor, scribble, and inducing scribble degradation via the proteasome-ubiquitin pathway. our findings reveal a novel mechanism by which ns4b contributes to hcv pathogenesis and tumorigenesis. the outcome of acute hepatitis c predicted by the evolution of the viral quasispecies vaccination for hepatitis c virus: closing in on an evasive target understanding the hepatitis c virus life cycle paves the way for highly effective therapies chronic hepatitis c: future treatment unravelling hepatitis c virus replication from genome to function protein-protein interactions between hepatitis c virus nonstructural proteins identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons hepatitis c virus rna replication and assembly: living on the fat of the land interaction networks of hepatitis c virus ns4b: implications for antiviral therapy expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex hepatitis c virus ns4b induces unfolded protein response and endoplasmic reticulum overload responsedependent nf-kappa b activation hepatitis c virus non-structural protein ns4b can modulate an unfolded protein response the roles of endoplasmic reticulum overload response induced by hcv and ns4b protein in human hepatocyte viability and virus replication the nucleotide binding motif of hepatitis c virus ns4b can mediate cellular transformation and tumor formation without ha-ras co-transfection the hepatitis c virus ns4b protein can trans-complement viral rna replication and modulates production of infectious virus the future of hcv therapy: ns4b as an antiviral target polarity complex proteins deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma emerging theme: cellular pdz proteins as common targets of pathogenic viruses complete replication of hepatitis c virus in cell culture the hepatitis c virus nonstructural protein 4b is an integral endoplasmic reticulum membrane protein topology of the membrane-associated hepatitis c virus protein ns4b enhancement of proteasome activity by a small-molecule inhibitor of usp14 trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna hepatitis c virus ns4b carboxy terminal domain is a membrane binding domain the ins and outs of hepatitis c virus entry and assembly hpv e6 degradation of p53 and pdz containing substrates in an e6ap null background viral oncoprotein-induced mislocalization of select pdz proteins disrupts tight junctions and causes polarity defects in epithelial cells patj, a tight junction-associated pdz protein, is a novel degradation target of high-risk human papillomavirus e6 and the alternatively spliced isoform 18 e6 acknowledgments this work was supported by scientific research funding for returned scholars of ministry of education (no. 2012940) and by natural science foundation of guangdong province (no. 2014a030313726). we thank prof. ralf bartenschlager, university of heidelberg, germany, for providing the subreplicon con-1 and prof. andrew rice for providing the plasmid pcdna/flag-scribble. conflicts of interest none key: cord-026112-58sa5z03 authors: dehghani-dehej, farzaneh; hosseini, zinat; mortazkar, poupak; khanaliha, khadijeh; esghaei, maryam; fakhim, atousa; bokharaei-salim, farah title: prevalence of hcv and/or hbv coinfection in iranian hiv-infected patients date: 2020-04-24 journal: nan doi: 10.2217/fvl-2019-0066 sha: doc_id: 26112 cord_uid: 58sa5z03 aim: hiv-infected patients risk coinfection with hbv and hcv. this study aimed to investigate molecular epidemiology of hbv and hcv coinfection in iranian hiv-infected individuals. materials & methods: in this cross-sectional study, serological markers of hbv and hcv infection (hepatitis b surface antigen [hbsag], hepatitis b e-antigen [hbeag], hepatitis b e-antibody [hbeab] and hepatitis b core antibody [hbcab]) and anti-hcv antibodies [anti-hcv abs] were tested in 198 iranian hiv-infected patients. from plasma, hbv viral load was determined using cobas taqman 48, and hcv-rna was detected by reverse transcriptase-nested pcr. results: 85 out of 198 (42.9%) patients were anti-hcv ab positive and 42/198 (21.2%) had detectable hcv-rna. eight (4.0%) had traceable hbv-dna. all these patients were infected by hbv genotype d. 55 (27.8%) were hbcab positive. nine (4.4%) were hbsag and anti-hcv ab positive. conclusion: none were hiv-rna/hcv-rna/hbv-dna positive, 21.2% were hiv-rna/hcv-rna positive and 4.0% were hiv-rna/hbv-dna positive. therefore, studies on diagnosing these infections in hiv-infected individuals may be valuable. epidemiology of the infection [11, 12] . hepatitis c has a global impact in terms of mortality and morbidity with over 70 million people infected all around the world [13] . according to studies in iran, the prevalence of hcv infection is nearly 0.5% (1.0% in men and 0.1% in women) [14, 15] . for hcv infection and the liver damage associated with it, the leading cause of mortality and morbidity is among hiv-infected patients. according to available evidence, hiv/hcv-coinfected patients are at higher risk for liver cirrhosis and hepatocellular carcinoma (hcc) [16, 17] . given that the transmission routes of hiv, hbv and hcv viruses are common, these infections can occur simultaneously. worldwide, nearly 40 million people are living with hiv, about 2.6 million people are infected with hbv and about 2.8 million people are hcv-infected [2] . hiv infection intensifies natural history of hbv infection, which can lead to an increase in rates of hbv persistence, relapse of hbv (resurgence of hepatitis b surface antigen [hbsag] , hepatitis b e-antigen [hbeag] or hbv-dna) and considerable clinical disease. previous studies of the hbv/hiv coinfection have shown that hiv leads to a lack of protective immunity against hbv, increased risk of cirrhosis and hcc and liver-related mortality [18, 19] . the effect of antiretroviral therapies (arts) on the natural history of hbv-related disease have been different, in some studies, it leads to recovery from hbv infection and in other studies, with relapse of hepatitis b [18, 20] . the death rate in hiv-positive patients decreased after taking combination arts, but only in those with hiv/hbv or hiv/hcv coinfection. the mortality rate is high due to liver damage. hiv/hbv-coinfected people have a higher rate of progression to liver fibrosis, cirrhosis, hcc, less clearance of hbsag and occult hbv infections (obi) are more frequent in these patients [13] . therefore, it seems that screening for hbv infection in the hiv-infected individuals should be done. testing hbsag, hbeag and ab and determining hbv viral load are an essential part of hbv infection assessment in hiv/hbv-coinfected patients. obviously, determination of cd4 counts and hiv viral load are necessary along with the antiretroviral drug-resistant response [21] . according to evidence, hcv/hiv-coinfected patients are at higher risk for cirrhosis and hcc [22] . hiv infection exacerbates natural history of hcv infection. hcv-rna loads in these patients are higher and clearance of hepatitis c viremia after acute infection in hiv-positive patients are less, and liver diseases in these patients show more progress than patients with hiv infection alone [20] . it is known that hbv and hcv infections have been associated with various clinical manifestations in people with hiv infection including impaired immune response during arts, and also increased susceptibility to artsrelated liver toxicity [23] . therefore, prior to the administration of art, patients should be tested for the presence of these infections. the aim for this study is to investigate the prevalence of hcv and/or hbv coinfection in iranian hiv-infected individuals. from september 2015 to june 2018, 198 consecutive iranian hiv-positive individuals who were referred to hospitals affiliated with iran university of medical sciences (iums), tehran, iran, were entered to this study. the research was approved by the iums' ethical committee, and all of the studied population were informed about this survey, and a written informed consent was obtained from all the subjects and also from parents of hiv-infected children in this cross-sectional study. collection of the specimens 5 ml of the patient's blood was taken from each participant into an edta-containing vacutainer tube. after separation of the plasma by centrifugation (5 min at 3000 rpm), plasma was stored at -80 • c until analysis. plasma specimens from ten individuals who were infected with hcv, and ten subjects who were infected with hbv were used as positive controls, and also plasma samples from ten healthy blood donors were used as negative controls for the experiments. serologic tests by enzyme immunoassay serological markers of hbv and hcv infection such as hbsag, hbeag, hepatitis b e-antibody (hbeab), hepatitis b core antibody (hbcab) and anti-hepatitis c virus antibodies (anti-hcv) abs were tested by the commercial enzyme immunoassay kits (dia. pro, milano, italy), according to the manufacturer's protocols. hbv viral load was assessed in 500 μl of the studied subjects plasma specimens using the high pure dna extraction kit and cobas taqman 48 kit (roche diagnostics, ca, usa) according to the manufacturer's procedure [24] . this test is a real-time pcr assay that is based on dual-labeled hybridization probe that targets two regions (precore and core) of hbv. the detection limit of the cobas taqman 48 kit is 6 to >1 × 10 8 iu/ml [25] . the hbv genotyping was determined in hbv-dna-positive specimens using the inno-lipa™ hbv kit (innogenetics, ghent, belgium) according to the manufacturer's protocols [26] . hcv detection by reverse transcriptase-nested pcr method & hcv genotyping with restriction fragment length polymorphism assay to detect genomic hcv-rna in the plasma samples of studied subjects, the viral rna was isolated from 140 μl of plasma using the qiaamp viral rna isolation kit (qiagen gmbh, hilden, germany) based on the manufacturer's procedure. the quantity and quality of the extracted rna was evaluated using the nanodrop™ spectrophotometer (thermo fisher scientific, wilmington, nc, usa) [27] . the hcv-rna was detected in extracted rna of plasma samples by the reverse transcriptase-nested pcr (rt-nested pcr) assay using two sets of primers for the 5non-translated region (5 -ntr) of hcv, as previously described in detail [28, 29] . the amplified pcr products of subjects' samples, negative and positive control specimens, and 100 bp dna size marker were electrophoresed on a 2.2% gel agarose and then stained with syber green and visualized by a uv transilluminator. the genotyping of hcv was determined in hcv-positive samples with restriction fragment length polymorphism (rflp) assay, based on a protocol that previously described in detail [28, 29] . the statistical analysis was performed using spss software version 20 (spss inc., il, usa). the kolmogorov-smirnov test was conducted to determine the quantitative variables' normality. the analysis of continuous variables was done using kruskal-wallis and one-way analysis of variance (anova) tests. the statistical differences between the two groups were evaluated by fisher's exact test and chi-square test when appropriate. p-values <0.05 were considered statistically significant. from september 2015 to june 2018, a total of 198 hiv-infected individuals (anti-hiv abs and hiv-rna positive) were enrolled in this cross-sectional study. the mean age of subjects was 35.3 ± 13.5 years (a range of 1-68 years old). out of 198 studied individuals, 126 (63.6%) were male. complete information of the demographic, laboratory and epidemiological characteristics were presented in table 1 . a significant association was observed between the sex of the participants and alanine aminotransferase (alt), aspartate aminotransferase (ast) level, anti-hcv abs, hcv-rna (p < 0.001) in plasma samples, and also in epidemiological parameters such as history of having unprotected sex, history of imprisonment, injection drug users (idus), idu sexual partners, history of tattooing, history of needle stick (p < 0.001) and history of transfusion (p = 0.034) ( table 1) . a significant relationship was observed between coinfection with hcv or hbv in hiv-infected patients and cd4 + t-cell count (p = 0.044), in other words, cd4 + t-cell count was very low in patients with these coinfections. a strong association was observed between the sex of the participants and level of education (p = 0.042) and marital status (p < 0.001) ( table 2) . 85 (42.9%) of studied subjects were positive for anti-hcv abs in plasma samples; and 42 (21.2%) had detectable hcv-rna in the plasma ( table 1 ). the hcv genotyping was performed using rflp assay for hcv-rna-positive specimens, and the results of hcv genotyping are presented in table 3 . eight (4.0%) of the studied cases had detectable hbv-dna in the plasma samples ( table 1 ). the hbv genotyping was carried out for these samples by the inno-lipa hbv kit. all these patients were infected by hbv genotype d. 55 (27.8%) of the participants were hbcab positive, and a strong relationship was observed between the sex of the studied participants and anti-hbcab in plasma specimens (p < 0.001) ( table 1) . this survey demonstrated that none of the iranian hiv-infected individuals were hiv-rna/hcv-rna/hbv-dna positive simultaneously, 21.2% were hiv-rna/hcv-rna positive and 4.0% were hiv-rna/hbv-dna positive. no significant association was observed between the hiv viral load in coinfected patients and monoinfected patients (p = 0.054). nine (4.4%) of the studied hiv-infected patients were hbsag and anti-hcv ab positive. all the information about demographic and laboratory parameters of these patients are presented in table 4 . despite the existence of successful prevention and treatment methods, the simultaneous infection of hiv, hbv and hcv is still a worldwide health issue. with the use of antiretroviral medicines and longer life expectancy in 10 hiv-infected patients, the complications of this chronic disease and its intersection with other viral infections are more evident [30] . in iran, the prevalence of hiv and other blood-related viral infections, such as hcv is relatively low in the general population [31] . the present survey was conducted on 198 individuals who were infected with hiv to investigate the molecular epidemiology of hcv/hbv coinfection in these individuals. this study showed that none of the hiv-infected people were hiv-rna/hcv-rna/hbv-dna positive simultaneously, 4.0% were hiv-rna/hbv-dna positive and 21.2% were hiv-rna/hcv-rna positive. hcv infection is more common in people infected with hiv than in hiv-negative individuals [32] . the rate of hcv/hiv coinfection is different around the world and is heavily dependent on geographical location, socioeconomic conditions of that particular location and high-risk groups [32] . nearly 37 million people are infected with hiv so far and about 70 million people all over the world are infected with hcv [2, 13] . approximately 2,278,400 people have hiv/hcv coinfection in the world, and about 62% of them are people that have been infected through injecting drugs [32] . the present study showed that approximately 81 (40.9%) of the patients are those who have injected drugs and about 46 (23.2%) of them are those who had idu sexual partner (that includes only women) ( table 1 ). the epidemiologic parameters such as injection drug abuse, needle sharing, tattooing, history of imprisonment and history of having unprotected sex revealed higher prevalence of hcv-coinfection in these patients compared with monoinfected cases. in the coinfected patients, the level of liver enzymes alt/ast was significantly high. while the first way of hiv transmission in iran is from sharing injection needles among idus [33] , hiv transmission cases in idus has begun to decline from 2005, a trend that has continued so far, and today hiv transmission through unprotected sexual contact is increasing (36.8%) [34, 35] . in this study the number of idus was 81 (40.9%). since the transmission of hcv by sexual contact is a rare phenomenon, the number of people coinfected with hcv/hiv is expected to decrease in the future [3, 36] . in the current study, 75 (37.9%) of the hiv-positive patients had a history of unprotected sex and probably the hiv virus in these patients transferred through unprotected sex. perhaps this is the reason for the decrease in the number of patients with hcv/hiv coinfection (21.2%) compared with previous reports [37, 38] . of course, in recent years, the increase in the transmission of hcv in males that have sexual intercourse with other males has been seen due to high-risk sexual behaviors. in addition, there have been cases of hcv spontaneous clearance in people who inject drugs (pwid) after art [39, 40] . the hcv genotyping on the plasma specimens showed a prevalence for subtypes, 1a (40.5%), 1b (16.7%) and 3a (33.3%). in four samples, the divergence genotype detected a mixed infection of two subtypes (1a/3a-1ab/3a) ( [41] [42] [43] . according to various reports from around the world, it seems that more research is required in this field with a wider population. like hcv infection, all hiv-infected patients should be screened for hbv infection. cirrhosis, hcc and hepatotoxicity after arts are the effects of hbv/hiv coinfection [44] . hbv vaccination should be done in all hiv individuals with hbv-negative laboratory tests. similar to hcv, hcc occurs in hbv/hiv-coinfected patients without cirrhosis [45] . hbv viral load is higher in hiv/hbv-coinfected patients than hbv-monoinfected individuals [46] . the present study found that 55 (27.8%) of these patients have anti-hbcab and 25 (12.6%) of them have hbsag. in some people, antibodies of the hbv core can be detected without anti-hbsag and hbeag [47] . our study confirms previous reports that male subjects are at high risk of developing hbv infection. typical methods for the transmission of both hbv and hiv are the sexual pathway and injection drug abuse [48] , while transmission of hcv by sexual pathway is unusual and given that in recent years the pathway for hiv transmission has changed, the prevalence of hcv is changing, but a significant change in the prevalence of hbv is unlikely to occur [49] . in a study on blood donors with an nat test in tehran, the incidence and residual risk for hiv was lower than those in developed countries, whereas hbv and hcv was higher compared to developed countries. in the iranian population, hiv infection is lower than the other countries, and screening tests are effective for blood donors. in the case of hcv, an increased incidence of hcv infection has been observed in the iranian society and in blood donors in recent years; this may be due to the highest number of idus in iran compared to other middle eastern countries [50] . incidence and high residual risk in iran indicates the nature of the endemic hbv virus. due to the launch of hbv vaccination in iran in 1993, we have to wait for the effects of the vaccination among the iranian population. based on that study, blood donors should have a more accurate technique similar to the accurate nat-screening techniques [51] . in patients with coinfection of hiv with hcv and/or chronic hbv, progressive liver fibrosis, cirrhosis and hcc can occur and coinfection of hiv with hbv and/or hcv can affect the management of hiv infection and complicate it [44, 52] . therefore, it is best to identify infection of hepatitis as quickly as possible. the result of this study revealed that none of the participants were hiv-rna/hcv-rna/hbv-dna positive simultaneously. to the best of our knowledge, the current survey is the first research that has analyzed the presence of the molecular epidemiology of hcv/hbv coinfection in iranian hiv-infected individuals; therefore, the results of this study cannot be compared with the result of other iranian research. there have been reports of coinfection with hbv and hcv in hiv-positive people, for example, 0.5% in singapore [53] , 0.62% in germany [54] and 1.7% in serbia [55] . it seems that further research focusing on this issue is needed. although there are studies that indicate seroprevalence of hiv/hcv/hbv coinfection in iran. for example, bakhti et al. found that 8% of hiv-infected individuals are coinfected with hcv/hbv (hbsag and anti-hcv ab positive), and in a meta-analysis, bagheri amiri et al. reported that coinfection of hiv/hbv/hiv was close to zero in the general population, street children and healthcare workers, while it peaked to 1.25% in pwid [56] . this study revealed that in iranian hiv-infected individuals, 4.4% of the individuals are seropositive for hbv/hcv (hbsag and anti-hcv ab positive). according to a previous study, prevalence of cirrhosis in hiv/hbv/hcv triple-infected patients was higher than hiv/hbv-or hiv/hcv-coinfected individuals [57] . therefore, prevention programs for hiv/hbv/hcv coinfection are in need of development. this study reveals that there is a high prevalence of hcv infection (21.2%) in hiv-infected individuals (hiv-rna/hcv-rna positive), as well as 4% of these people infected with hbv (hiv-rna/hbv-dna positive). also, the result of this survey highlighted that none of the hiv-infected subjects were hcv-rna/hbv-dna positive simultaneously. therefore, it seems that in hiv-positive patients, in addition to routine diagnosis of all the authors of this article are very grateful to those who volunteered to participate in this research. the current research was funded by research deputy of iran university of medical sciences (iums), tehran, iran with grant number 8921215087. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. ethical approval for this research was obtained from the local ethics committee of iran university of medical sciences (iums), tehran, iran, that is accordance with helsinki declaration (ethical code: ir.iums.fmd.rec 1396. 8921215087). all of the volunteers participating in this study were informed about this research prior to their enrollment. in addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. hiv diagnosis and treatment through advanced technologies world health organization global health observatory current diagnostic methods for hiv interference of apoptosis by hepatitis b virus characterization of hepatitis b virus with complex structural variations hiv-hepatitis b virus coinfection: epidemiology, pathogenesis, and treatment global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity hepatitis b: screening, awareness, and the need to treat hepatitis b virus infection in the general population of iran: an updated systematic review and meta-analysis hepatitis c virus (hcv) genotyping by annealing reverse transcription-pcr products with genotype-specific capture probes identification of a novel hepatitis c virus genotype from punjab, india: expanding classification of hepatitis c virus into 8 genotypes molecular epidemiology of hepatitis c virus in cambodia during 2016-2017 hepatitis b, hepatitis c, and mortality among hiv-positive individuals seroprevalence of hepatitis c virus: the first population-based study from iran the impact of ifn-γ gene polymorphisms on spontaneous clearance of hcv infection in fars province, southern of iran the presence of autoantibodies to cytoplasmic rod and ring particles in the serum of patients with chronic hepatitis c virus infection hepatocellular carcinoma after sustained virological response with interferon-free regimens in hiv/hepatitis c virus-coinfected patients hiv-1, hepatitis b virus, and risk of liver-related mortality in the multicenter cohort study (macs) hepatitis b and long-term hiv outcomes in co-infected haart recipients survival in patients with hiv infection and viral hepatitis b or c: a cohort study management of hiv and hepatitis virus coinfection human immunodeficiency virus/hepatitis c virus (hcv) co-infected patients with cirrhosis are no longer at higher risk for hepatocellular carcinoma or end-stage liver disease as compared to hcv mono-infected patients liver fibrosis and hepatitis b coinfection among art naive hiv-infected patients at a tertiary level hospital in northwestern tanzania: a cross-sectional study detection of hepatitis b virus covalently closed circular dna in the plasma of iranian hbeag-negative patients with chronic hepatitis b detection of hbv genome in the plasma and peripheral blood mononuclear cells of iranian hbsag negative patients with hiv infection: occult hbv infection distribution of hepatitis b virus genotypes in azerbaijani patients with chronic hepatitis b infection prevalence of jc polyomavirus large t antigen sequences among iranian patients with central nervous system tumors hepatitis c genotypes in finland determined by rflp occult hepatitis c virus infection in iranian patients with cryptogenic liver disease trends in underlying causes of death in people with hiv from co-infection of human immunodeficiency virus, hepatitis c and hepatitis b virus among injection drug users in drop in centers prevalence and burden of hcv co-infection in people living with hiv: a global systematic review and meta-analysis trend of hiv/aids prevalence and related interventions administered in prisons of iran − 13years' experience antiretroviral drug resistance mutations in naïve islamic republic of iran aids progress report hiv-1 genetic diversity and transmitted drug resistance frequency among iranian treatment-naive, sexually infected individuals epidemiological distribution and genotype characterization of the hepatitis c virus among hiv patients in kashan high prevalence of hcv coinfection in hiv-infected individuals in shiraz, islamic republic of iran sexually transmitted hepatitis c infection: the new epidemic in msm? occasional spontaneous clearance of chronic hepatitis c virus in hiv-infected individuals hiv and hcv coinfection: prevalence, associated factors and genotype characterization in the midwest region of brazil prevalence of hepatitis c virus (hcv) coinfection in hiv infected individuals in south india and characterization of hcv genotypes detection and analysis of hepatitis c virus in hiv-infected patients in the guangxi province of china long-term liver diseases after initiation of antiretroviral therapy in hiv-infected patients with and without hbv or hcv coinfection risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus dna level hiv-hepatitis b virus co-infection: epidemiology, pathogenesis and treatment isolated antibody to hepatitis b core antigen in human immunodeficiency virus type-1− infected individuals hepatitis c, hepatitis b, and human immunodeficiency virus infections among non-intravenous drug-using patients attending clinics for sexually transmitted diseases epidemiology of hepatocellular carcinoma: new trends prevalence and trend of hepatitis c virus infection among blood donors in iran: a systematic review and meta-analysis incidence and residual risk of hiv, hbv and hcv infections among blood donors in tehran hepatic decompensation in antiretroviral-treated patients co-infected with hiv and hepatitis c virus compared with hepatitis c virus-monoinfected patients: a cohort study factors associated with hepatitis b and c co-infection among hiv-infected patients in singapore daclatasvir plus sofosbuvir, with or without ribavirin, in real-world patients with hiv-hcv coinfection and advanced liver disease comparison of demographic, epidemiological, immunological, and clinical characteristics of patients with hiv mono-infection versus patients co-infected with hcv or/and hbv: a serbian cohort study hbv and hcv coinfection prevalence in iran-a systematic review and meta-analysis hepatic decompensation in patients with hiv/hepatitis b virus (hbv)/hepatitis c virus (hcv) triple infection versus hiv/hcv coinfection and the effect of anti-hbv nucleos(t)ide therapy key: cord-003407-f5v3hhr8 authors: hung, ting-chun; jassey, alagie; lin, chien-ju; liu, ching-hsuan; lin, chun-ching; yen, ming-hong; lin, liang-tzung title: methanolic extract of rhizoma coptidis inhibits the early viral entry steps of hepatitis c virus infection date: 2018-11-27 journal: viruses doi: 10.3390/v10120669 sha: doc_id: 3407 cord_uid: f5v3hhr8 hepatitis c virus (hcv) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis c-associated end-stage liver diseases. despite improvement of hcv treatment using the novel direct-acting antivirals (daas) targeting viral replication, there is a lack of prophylactic measures for protection against hcv infection. identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against hcv. herein, we investigated the anti-hcv activity of the methanolic extract from rhizoma coptidis (rc), a widely used traditional chinese medicine documented by the who and experimentally reported to possess several pharmacological functions including antiviral effects. using the cell culture-derived hcv system, we demonstrated that rc dose-dependently inhibited hcv infection of huh-7.5 cells at non-cytotoxic concentrations. in particular, rc blocked hcv attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to hcv. moreover, rc robustly suppressed hcv pseudoparticles infection of huh-7.5 cells and impeded infection by several hcv genotypes. collectively, our results identified rc as a potent antagonist to hcv entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-hcv agent. hepatitis c virus (hcv) is an important liver pathogen belonging to the flaviviridae family with an enveloped positive single-stranded rna genome. hcv has seven genotypes (genotype 1~7) and a genome size of about 9.6 kb which encodes a polyprotein that is approximately 3000 amino acids long. the polyprotein upon translation is processed by viral and host proteases to yield 10 matured protein including structural proteins, core, e1, e2, and p7 ion channel, as well as non-structural proteins ns2, ns3, ns4a, ns4b, ns5a, and ns5b [1] . hcv entry into the host hepatocytes is mediated by interaction basal media for all viral infection analyses consisted of dulbecco's modified eagle's medium (gibco-invitrogen, carlsbad, ca, usa) containing 2% fetal bovine serum. rhizoma coptidis roots from coptis chinensis franch (id#kew-2736105 from the plant list [18] ) were obtained from local pharmacy store (kaohsiung, taiwan) and authenticated by dr. ming-hong yen using anatomical methods as well as by hplc analysis through comparison to known molecular standards as previously described [10, 19] . a voucher specimen was deposited at the kaohsiung medical university herbarium (ctm-rcc03). for methanol extraction [20] , the roots were washed, dried, and homogenized before extraction with 100 % methanol, followed by concentration in vacuo. the methanolic rc stock was dissolved in dimethyl sulfoxide (dmso; sigma, st. louis, mo, usa) prior to use. huh-7.5 cells seeded at 1 × 10 4 cells/well in 96-well plates overnight were treated with increasing concentrations of rc for 5 days. the cells were then washed twice with phosphate buffered saline (pbs) before xtt cell viability analysis as previously described [21] . for examining antiviral activity, huh-7.5 cells (1 × 10 4 cells/well in 96-well plates) were concurrently treated with the virus (moi = 0.01) and the test drug at various concentrations before incubation at 37 • c for 3 days. the anti-hcv activity was determined by measuring the luciferase reporter signals using the biolux™ gaussia luciferase assay kit (new england biolabs; pickering, on, canada) and a luminometer (promega; madison, wi, usa) as previously reported [22] . data were expressed as percent (%) hcv infectivity from test treatments relative to medium control (virus only). ifn-α (sigma) served as positive control. the time-of-drug-addition assay which provides information on the target of test agents in the viral life cycle consisted of pre-treatment, co-addition treatment, and post-infection treatment, and was performed as previously described [22] . for all analyses, huh-7.5 cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well overnight and infection with hcvcc was carried out at moi = 0.01. luciferase activity for all conditions was determined as described earlier following 72 h of incubation at 37 • c. the synchronized infection analysis to determine the effect of test agents on early viral entry was performed as previously described [22] . to examine viral inactivation, the test drug was incubated with the cell-free virus particles prior to diluting the virus-drug mixture to a subtherapeutic concentration and infecting the host cells (final hcvcc moi = 0.01). to investigate the influence on viral attachment, pre-chilled huh-7.5 cells were challenged with the hcvcc (moi = 0.01) in the presence/absence of the test agent at 4 • c, which allows binding of the viral particles to the host cells while precluding entry. to test for impact on viral entry/fusion, huh-7.5 cells were pre-bound with hcvcc (moi = 0.01) at 4 • c and then shifted to 37 • c incubation in the presence/absence of the test drug. for all the above analyses, huh-7.5 cells were seeded in 96-well plates (1 × 10 4 cells/well) and luciferase activity for all conditions was determined as described earlier following 72 h of incubation at 37 • c. the enzyme-linked immunosorbent assay (elisa)-based binding assay was performed as previously described [23] . briefly, pre-chilled huh-7.5 cells were challenged with hcvcc in the presence/absence of test drug at 4 • c for 3 h before washing with pbs and fixing the cells with 4 % paraformaldehyde. cell-bound virus was detected using primary anti-hcv e2 antibody (1:20000; austral biological, san ramon, ca, usa) and secondary goat anti-mouse igg conjugated with horseradish peroxidase antibody (1:36000, invitrogen), followed by assessment with tmb (3,3',5,5'-tetramethylbenzidine) two-component microwell peroxidase substrate kit (kpl; gaithersburg, md, usa) and absorbance reading at 450 nm with a elx800 microplate reader (instrument, inc. ; winooski, vt, usa). retroviral pseudoparticles bearing hcv glycoproteins e1/e2 were produced following a previously described method [24] with some modifications. a pcdna3.1 plasmid vector (invitrogen) containing complete hcv core and e1/e2 glycoproteins of the hc-j6ch strain (genotype 2a; nc_009823) was co-transfected in conjunction with the pnl.luc.env − r + construct (env-defective retroviral backbone encoding firefly luciferase; kindly provided by dr. éric a. cohen) into 293t cells using omnifect™ (transomic technologies inc.; huntsville, al, usa). supernatant containing the viral pseudoparticles was harvested and filtered (0.45 µm) before being concentrated using 25% (v/v) polyethylene glycol and stored at −80 • c before use. for the infectivity experiment, the hcv pseudoparticles (hcvpp) were used to inoculate huh-7.5 cell monolayers in 12-well plates in the presence or absence of the test agent for 2 h at 37 • c. the cells are then washed with pbs and further incubated in basal media for 72 h before being harvested and assessed for reporter luciferase activity using the firefly luciferase assay kit (promega) and a luminometer. viral infectivity was calculated as percent (%) relative light units (rlu) compared to control (virus only). antiviral activity against recombinant hcv carrying glycoproteins from genotype 2b (j8/jfh1), 3a (s52/jfh1), and 7a (qc69/jfh1) viruses (kindly provided by dr. jens bukh) [25, 26] was performed using a previously reported method [23] with some modifications. huh-7.5 cells (1 × 10 4 cells/well in 96-well plates) were challenged with the viruses at moi = 0.01 in the presence/absence of the test agent. after 3 days of further incubation, detection of viral infectivity was performed by immunofluorescence staining of hcv foci using primary mouse anti-core clone b2 antibody (1:200; anogen; mississauga, on, canada), which cross-reacts different hcv core antigenic determinants, and a secondary alexa fluor 488 goat anti-mouse igg (h+l) (1:400; invitrogen) [27] . hcv-positive foci were quantitated and results were plotted against the dmso control [28] . statistical analysis was conducted using one-way analysis of variance (anova) followed by tukey's multiple comparison test or unpaired t test. a p value of less than 0.05 (p < 0.05) was considered to be statistically significant. all data are expressed as means ± standard error of the means (sem) from three independent experiments. given the numerous pharmacological properties of rc, we hypothesized that the medicinal herb could potentially possess antiviral activities against hcv. to explore this possibility, huh-7.5 cells were infected with gaussia luciferase reporter-tagged hcvcc in the presence of different concentrations of the methanolic extract of rc and the luciferase activity was subsequently assessed to determine viral infectivity. a cytotoxicity assay was simultaneously performed on the cells with the same drug concentrations. our results showed that rc could inhibit hcv infection dose-dependently and up to 50 µg/ml without inducing significant cytotoxicity ( figure 1a ). the 50 % cytotoxic concentration (cc 50 ), the 50 % effective concentration (ec 50 ), and the selective index (si = cc 50 /ec 50 ) were determined to be 168.9 ± 1.06 µg/ml, 20.07 ± 1.08 µg/ml, and 8.42, respectively ( figure 1b ). since the antiviral efficacy of rc appeared to be comparable between 20 and 50 µg/ml, a concentration of 20 µg/ml was chosen for the remainder of our experiments. given the numerous pharmacological properties of rc, we hypothesized that the medicinal herb could potentially possess antiviral activities against hcv. to explore this possibility, huh-7.5 cells were infected with gaussia luciferase reporter-tagged hcvcc in the presence of different concentrations of the methanolic extract of rc and the luciferase activity was subsequently assessed to determine viral infectivity. a cytotoxicity assay was simultaneously performed on the cells with the same drug concentrations. our results showed that rc could inhibit hcv infection dosedependently and up to 50 μg/ml without inducing significant cytotoxicity ( figure 1a ). the 50 % cytotoxic concentration (cc50), the 50 % effective concentration (ec50), and the selective index (si = cc50/ec50) were determined to be 168.9  1.06 μg/ml, 20.07 ± 1.08 μg/ml, and 8.42, respectively ( figure 1b ). since the antiviral efficacy of rc appeared to be comparable between 20 and 50 μg/ml, a concentration of 20 μg/ml was chosen for the remainder of our experiments. in order to narrow down the window of antiviral activity from rc, we performed a time-ofdrug-addition assay wherein the drug was either added to cells 24 h before hcvcc infection ("pretreatment"), concurrently added at the time of viral infection ("co-addition"), or added after infection ("post-infection") and incubated for 3 days before measuring the luciferase reporter activity. results indicated no significant difference in hcv infectivity in the presence or absence of rc during pretreatment (figure 2) , suggesting that the drug does not appear to modulate the host cells before hcv infection. in contrast, rc significantly inhibited hcv infectivity when simultaneously added during the viral challenge as indicated by the substantial drop in the luciferase signal (figure 2) . only a moderate decrease in viral infectivity was observed when the drug was added after the establishment of viral infection. in contrast, ifn-α, which served as positive control, effectively inhibited the hcvcc infection in all 3 types of treatment. thus, rc's anti-hcv activity appeared in order to narrow down the window of antiviral activity from rc, we performed a time-of-drug-addition assay wherein the drug was either added to cells 24 h before hcvcc infection ("pre-treatment"), concurrently added at the time of viral infection ("co-addition"), or added after infection ("post-infection") and incubated for 3 days before measuring the luciferase reporter activity. results indicated no significant difference in hcv infectivity in the presence or absence of rc during pretreatment (figure 2) , suggesting that the drug does not appear to modulate the host cells before hcv infection. in contrast, rc significantly inhibited hcv infectivity when simultaneously added during the viral challenge as indicated by the substantial drop in the luciferase signal (figure 2) . only a moderate decrease in viral infectivity was observed when the drug was added after the establishment of viral infection. in contrast, ifn-α, which served as positive control, effectively inhibited the hcvcc infection in all 3 types of treatment. thus, rc's anti-hcv activity appeared strongest when concurrently present on the host cell with the viral particles, suggesting that its inhibitory effect mainly targeted the early phase of the hcv infection, including viral entry. viruses 2018, 10, x for peer review 6 of 12 strongest when concurrently present on the host cell with the viral particles, suggesting that its inhibitory effect mainly targeted the early phase of the hcv infection, including viral entry. to further characterize the mechanism(s) underlying rc's anti-hcv effect, which was strongest when rc was simultaneously present with the virus on the host cell surface, we performed a synchronized infection assay on early viral entry. to test whether rc could inactivate the cell-free viral particles, the drug was pre-incubated with the hcvcc for 3 h before dilution and infecting huh-7.5 cells. the drug-virus mixture was then diluted 20x to yield a non-effective concentration which was subsequently added to the cells and incubated for 3 days. luciferase readings following 3 days of incubation showed no significant difference between samples treated with or without rc, suggesting that the drug does not impact the free viral particles ( figure 3a ). to determine the effect of rc on viral attachment, we specifically added rc during hcv cell binding at 4 °c, which allows for virus binding but precludes viral internalization, and then tested the reporter readout at the end of the incubation. results demonstrated a significant decrease in the luciferase reporter activity, which indicated that rc interfered with the attachment of the virus to the host cells ( figure 3a ). to ascertain whether rc influenced the post-attachment viral entry/fusion step, huh-7.5 cells were prebound with hcv at 4 °c before shifting the temperature to 37 °c in the presence of rc. similar to the effect observed on viral attachment, our data revealed a significant decrease in hcv infectivity at the end of the incubation, suggesting that rc equally blocked hcv entry/fusion ( figure 3a ). to further characterize the mechanism(s) underlying rc's anti-hcv effect, which was strongest when rc was simultaneously present with the virus on the host cell surface, we performed a synchronized infection assay on early viral entry. to test whether rc could inactivate the cell-free viral particles, the drug was pre-incubated with the hcvcc for 3 h before dilution and infecting huh-7.5 cells. the drug-virus mixture was then diluted 20x to yield a non-effective concentration which was subsequently added to the cells and incubated for 3 days. luciferase readings following 3 days of incubation showed no significant difference between samples treated with or without rc, suggesting that the drug does not impact the free viral particles ( figure 3a ). to determine the effect of rc on viral attachment, we specifically added rc during hcv cell binding at 4 • c, which allows for virus binding but precludes viral internalization, and then tested the reporter readout at the end of the incubation. results demonstrated a significant decrease in the luciferase reporter activity, which indicated that rc interfered with the attachment of the virus to the host cells ( figure 3a ). to ascertain whether rc influenced the post-attachment viral entry/fusion step, huh-7.5 cells were pre-bound with hcv at 4 • c before shifting the temperature to 37 • c in the presence of rc. similar to the effect observed on viral attachment, our data revealed a significant decrease in hcv infectivity at the end of the incubation, suggesting that rc equally blocked hcv entry/fusion ( figure 3a) . to directly validate our finding that rc inhibits hcv attachment to the host cells, we employed an elisa-based binding assay. huh-7.5 cells were inoculated with hcvcc in the presence of rc at 4 • c for 3 h, after which the cells were washed, fixed, and subjected to colorimetric analysis by detecting cell surface-bound hcv particles using anti-hcv e2 antibody. as depicted in figure 3b , rc treatment dose-dependently decreased viral attachment as demonstrated by the sharp decline in the absorbance signal, which is in agreement with the above results. hcv entry steps are mediated by viral glycoproteins [2] . based on the inhibitory effects observed from rc against hcv entry steps, and to validate its antiviral potency against hcv entry, we further examined the impact of rc treatment on infection by retroviral pseudoparticles bearing hcv glycoproteins e1/e2 (hcvpp). specifically, luciferase-tagged hcvpp were used to infect huh-7.5 cells in the presence or absence of rc, before further incubating the cells and assessing luciferase reporter activity as a readout of viral infectivity. as demonstrated in figure 3c , rc robustly suppressed hcvpp infection of the huh-7.5 hepatoma cells compared to the dmso control. this result therefore confirms and validates the antiviral capacity of rc against the viral entry stage of hcv. hcv infection of hepatocytes is a well-orchestrated process involving the engagement of several host factors including cd81, sr-bi, cldn-1, and ocln. in addition, apoe has also been reported to play a role in mediating hcv entry [5] . given that rc treatment blocked hcv infection mainly by targeting viral entry, we next asked whether the drug could modulate the expression of key host cells entry factors. to this end, huh-7.5 cells were pre-treated with rc for 24 h before harvesting the cell lysates for western blot analysis. as depicted in figure 3d , pre-treatment of huh-7.5 cells with rc did not alter the expression of cd81, sr-bi, cldn-1, and ocln. similarly, the expression of apoe was not affected by rc treatment ( figure 3d) . apob, which participates in hcv virion release was included for comparison and was neither affected by the drug treatment. these results therefore suggested that rc did not inhibit hcv infection via influencing the host cells and is consistent with our data from the pre-treatment analysis (figure 2 ). since rc appeared to primarily target hcv entry, we finally sought to examine whether the medicinal herb also possesses a pan-genotypic activity against hcv. for this purpose, huh-7.5 cells were seeded and infected with recombinant hcvcc expressing glycoproteins from genotypes 2b (j8/jfh1), 3a (s52/jfh1), and 7a (qc69/jfh1) in the presence of the rc before further incubation and analysis of hcv infectivity by immunofluorescence staining. results showed a significant decrease in viral infection across all the tested hcv genotypes when rc was present, suggesting that it may possess a pan-genotypic activity against hcv (figure 4) . continuous identification of novel antivirals with various modes of action is important given that development of drug resistance is commonplace especially with viruses that exhibit genetic variability, including hcv. in this study, we demonstrated for the first time that the methanolic extract of rc robustly inhibited hcv infection. specifically, rc mainly targeted the hcv early viral entry steps such as attachment and entry/fusion to the host cells. interestingly, rc also inhibited hcv infection of several other genotypes. our results therefore identified rc as a promising antagonist with pan-genotypic function against hcv entry, which could be useful for developing hcv prophylaxis. currently no approved therapeutic treatment exists for targeting hcv entry, and patients with chronic hepatitis c are at risk for end-stage liver diseases such as cirrhosis and liver cancer, which necessitate liver transplantation. importantly, donor livers inadvertently become re-infected almost immediately after transplantation in hepatitis c patients [29] . given that the daas in current use cannot prevent liver graft re-infection and have the propensity to select for drug-resistant mutants, combining entry inhibitors with the daas would be expected to broaden the treatment strategies against hepatitis c especially in the liver transplant setting. interestingly, previous studies have demonstrated that combining daas and virus entry inhibitors can produce a synergistic effect to improve drug efficacy [30] . our discovery that rc can antagonize hcv entry makes it an ideal candidate for use in liver transplant scenarios and to test in combination with the daas for better therapeutic efficacy. natural resources such as plant materials serve as excellent starting point for antiviral discovery [31] . rc's anti-hcv bioactivity identifies the medicinal herb as an important antiviral source for the treatment of hepatitis c. various medicinal plant extracts have been shown to possess anti-hcv properties and thereafter served as source for further identifying small molecule inhibitors. examples of those that specifically target hcv entry include silibinin and silymarin from silybum marianum [32, 33] , gallic acid found in limonium sinense [22] , loliolide and the butenolide (4r,6s)-2dihydromenisdaurilide derived from phyllanthus urinaria [28, 34] , ladanein isolated from marrubium peregrinum l. (lamiaceae) [35] , and bupleurum kaoi and its terpenoid saikosaponin b2 [23] . while the molecular constituents in rc contributing to its anti-hcv activity remain to be identified, the alkaloids are potential candidate bioactives which are known to be the major components of rc and are typically present in the alcoholic extracts of the herb [11] . examples include berberine, coptisine, palmatine, epiberberine, columbamine, jatrorrhizine, and groenlandicine, among other major compounds [11] . whether a single compound or a combination of them contributes to rc's antiviral actions against hcv remains to be explored. further in-depth analysis would be required to fully elucidate the bioactive ingredients responsible for rc's anti-hcv effect. our results indicated that rc specifically blocked hcv attachment and entry/fusion into the host cells without significantly influencing the cell-free viral particles or modulating key host cell continuous identification of novel antivirals with various modes of action is important given that development of drug resistance is commonplace especially with viruses that exhibit genetic variability, including hcv. in this study, we demonstrated for the first time that the methanolic extract of rc robustly inhibited hcv infection. specifically, rc mainly targeted the hcv early viral entry steps such as attachment and entry/fusion to the host cells. interestingly, rc also inhibited hcv infection of several other genotypes. our results therefore identified rc as a promising antagonist with pan-genotypic function against hcv entry, which could be useful for developing hcv prophylaxis. currently no approved therapeutic treatment exists for targeting hcv entry, and patients with chronic hepatitis c are at risk for end-stage liver diseases such as cirrhosis and liver cancer, which necessitate liver transplantation. importantly, donor livers inadvertently become re-infected almost immediately after transplantation in hepatitis c patients [29] . given that the daas in current use cannot prevent liver graft re-infection and have the propensity to select for drug-resistant mutants, combining entry inhibitors with the daas would be expected to broaden the treatment strategies against hepatitis c especially in the liver transplant setting. interestingly, previous studies have demonstrated that combining daas and virus entry inhibitors can produce a synergistic effect to improve drug efficacy [30] . our discovery that rc can antagonize hcv entry makes it an ideal candidate for use in liver transplant scenarios and to test in combination with the daas for better therapeutic efficacy. natural resources such as plant materials serve as excellent starting point for antiviral discovery [31] . rc's anti-hcv bioactivity identifies the medicinal herb as an important antiviral source for the treatment of hepatitis c. various medicinal plant extracts have been shown to possess anti-hcv properties and thereafter served as source for further identifying small molecule inhibitors. examples of those that specifically target hcv entry include silibinin and silymarin from silybum marianum [32, 33] , gallic acid found in limonium sinense [22] , loliolide and the butenolide (4r,6s)-2-dihydromenisdaurilide derived from phyllanthus urinaria [28, 34] , ladanein isolated from marrubium peregrinum l. (lamiaceae) [35] , and bupleurum kaoi and its terpenoid saikosaponin b2 [23] . while the molecular constituents in rc contributing to its anti-hcv activity remain to be identified, the alkaloids are potential candidate bioactives which are known to be the major components of rc and are typically present in the alcoholic extracts of the herb [11] . examples include berberine, coptisine, palmatine, epiberberine, columbamine, jatrorrhizine, and groenlandicine, among other major compounds [11] . whether a single compound or a combination of them contributes to rc's antiviral actions against hcv remains to be explored. further in-depth analysis would be required to fully elucidate the bioactive ingredients responsible for rc's anti-hcv effect. our results indicated that rc specifically blocked hcv attachment and entry/fusion into the host cells without significantly influencing the cell-free viral particles or modulating key host cell entry factors to hcv. the ability of rc to inhibit hcvpp infection suggests that its mechanistic target(s) may involve the hcv glycoproteins-mediated interactions with the host cell. a potential mechanism is the transient interaction(s) with the hcv glycoproteins that are inadequate to inactivate free viral particles but sufficiently effective to hinder contact with cell surface receptors/co-receptors. additional mechanisms of action could also include concentration-dependent reversible conformational alterations in receptors/co-receptors or viral particles' structure to block viral entry. we also noted a moderate effect from rc in the post-infection stage (figure 2) , albeit less pronounced compared to its impact in inhibiting hcv entry steps. this effect was not entirely due to inhibition of entry in subsequent de novo infection cycles, since treatment of subgenomic hcv replicon cells with rc also showed a moderate decrease in viral rna in a preliminary experiment ( figure s1 ). it is possible that rc may possess multiple mechanisms against hcv infection, which would necessitate further studies for clarification. nonetheless, our results clearly demonstrated that rc most potently inhibited hcv entry. therefore, we suggest that rc could be further explored for prophylactic/therapeutic management of hepatitis c. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/10/12/669/ s1, figure s1 : effect of rc treatment on hcv subgenomic replicon cells. replication of hepatitis c virus the mechanism of hcv entry into host cells strategies to preclude hepatitis c virus entry roles of lipoproteins and apolipoproteins in particle formation of hepatitis c virus neglected but important role of apolipoprotein e exchange in hepatitis c virus infection antiviral treatment of hepatitis c drug interactions with new hepatitis c oral drugs ledipasvir/sofosbuvir (harvoni): improving options for hepatitis c virus infection hepatitis c virus treatment in the real world: optimising treatment and access to therapies coptidis rhizoma and its main bioactive components: recent advances in chemical investigation, quality evaluation and pharmacological activity english version; ministry of health and welfare anti-inflammatory and antimicrobial effects of heat-clearing chinese herbs: a current review anti-herpes simplex virus effects of berberine from coptidis rhizoma, a major component of a chinese herbal medicine in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex coptidis rhizoma extract inhibits replication of respiratory syncytial virus in vitro and in vivo by inducing antiviral state broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry the plant list-version 1.1 characterization of protoberberine alkaloids in coptidis rhizoma (huanglian) by hplc with esi-ms/ms application of analytical and preparative high-speed counter-current chromatography for separation of alkaloids from coptis chinensis franch hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread limonium sinense and gallic acid suppress hepatitis c virus infection by blocking early viral entry saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry studying hcv cell entry with hcv pseudoparticles (hcvpp) robust hepatitis c genotype 3a cell culture releasing adapted intergenotypic 3a/2a (s52/jfh1) viruses development and characterization of hepatitis c virus genotype 1-7 cell culture systems: role of cd81 and scavenger receptor class b type i and effect of antiviral drugs highly bioavailable silibinin nanoparticles inhibit hcv infection activity-based and fraction-guided analysis of phyllanthus urinaria identifies loliolide as a potent inhibitor of hepatitis c virus entry hepatitis c and liver transplantation: enhancing outcomes and should patients be retransplanted synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis c antiviral natural products and herbal medicines multiple effects of silymarin on the hepatitis c virus lifecycle silibinin inhibits hepatitis c virus entry into hepatocytes by hindering clathrin-dependent trafficking a plant-derived flavonoid inhibits entry of all hcv genotypes into human hepatocytes the authors would like to thank charles m. rice, jens bukh, and éric a. cohen for reagents, christopher d. richardson for experimental support for the antiviral assay against the various hcv genotypes, and shun-pang chang and chueh-yao chung for technical assistance. the authors declare no conflicts of interest. key: cord-024003-698d15gk authors: loutfy, samah a; elberry, mostafa h; farroh, khaled yehia; mohamed, hossam taha; mohamed, aya a; mohamed, elchaimaa b; faraag, ahmed hassan ibrahim; mousa, shaker a title: antiviral activity of chitosan nanoparticles encapsulating curcumin against hepatitis c virus genotype 4a in human hepatoma cell lines date: 2020-04-22 journal: int j nanomedicine doi: 10.2147/ijn.s241702 sha: doc_id: 24003 cord_uid: 698d15gk purpose: current direct-acting antiviral agents for treatment of hepatitis c virus genotype 4a (hcv-4a) have been reported to cause adverse effects, and therefore less toxic antivirals are needed. this study investigated the role of curcumin chitosan (cucs) nanocomposite as a potential anti-hcv-4a agent in human hepatoma cells huh7. methods: docking of curcumin and cucs nanocomposite and binding energy calculations were carried out. chitosan nanoparticles (csnps) and cucs nanocomposite were prepared with an ionic gelation method and characterized with tem, zeta size and potential, and hplc to calculate encapsulation efficiency. cytotoxicity studies were performed on huh7 cells using mtt assay and confirmed with cellular and molecular assays. anti-hcv-4a activity was determined using real-time pcr and western blot. results: the strength of binding interactions between protein ligand complexes gave scores with ns3 protease, ns5a polymerase, and ns5b polymerase of -124.91, -159.02, and -129.16, for curcumin respectively, and -68.51, -54.52, and -157.63 for cucs nanocomposite, respectively. cucs nanocomposite was prepared at sizes 29–39.5 nm and charges of 33 mv. hplc detected 4% of curcumin encapsulated into csnps. ic50 was 8 µg/ml for curcumin and 25 µg/ml for the nanocomposite on huh7 but was 25.8 µg/ml and 34 µg/ml on wish cells. csnps had no cytotoxic effect on tested cell lines. apoptotic genes’ expression revealed the caspase-dependent pathway mechanism. csnps and cucs nanocomposite demonstrated 100% inhibition of viral entry and replication, which was confirmed with hcv core protein expression. conclusion: cucs nanocomposite inhibited hcv-4a entry and replication compared to curcumin alone, suggesting its potential role as an effective therapeutic agent. hepatitis c virus (hcv) currently infects over 71 million individuals around the world. its persistence causes infected individuals to enter a chronic phase that then leads to several different outcomes of liver disease. 1 hcv therapies are becoming highly efficient and well tolerated due to improved understanding of hcv biology and to the discovery of antiviral targeting of essential viral functions. 1, 2 the ns5b polymerase, ns5a and ns3 proteases are considered crucial targets for the development of direct-acting antiviral drugs. 3 the current treatment of hcv genotype 4a (hcv-4a) includes sofosbuvir, ledipasvir, and ombitasvir. the response rate has reached more than 90%. 4 despite the promise of such therapy, the suboptimal efficacy and adverse effects like anemia, rash, bilirubin, nausea, pruritus, and photosensitivity 5 as well as the increasing number of patients with advanced liver disease and no therapeutic options emphasize a major need for improved therapeutic regimens. in addition, the high mutation rate of viral rna and development of mutant resistant strains has slowed development of innovative anti-hcv drugs. therefore, natural compounds extracted from plants have attracted the attention of researchers for their therapeutic activities. 6 a substantial variety of natural compounds has demonstrated antiviral activity, including against hcv, 7 or hepatoprotective effect from natural compounds like naringenin, epigallocatechin-3-gallate (egcg), silymarin, and caffeine. 8, 9 turmeric curcumin, known as curcuma longa, is a natural plant with a significant therapeutic role that harbors no side effects. 10 curcumin's activity as an antiviral emerged recently, and it exhibits a significantly high inhibition against several viruses, like hepatitis b virus, 11 hiv, influenza viruses, and human herpes viruses. 12 several reports demonstrated curcumin's activity against hcv; it can obstruct viral attachment and fusion to hepatocytes and prevent cell to cell transmission due to loss of the membrane's integrity. 10, 13 however, curcumin's positive effect is hampered by its lower solubility, bioavailability, and low cellular uptake, but the progress in nanotechnology has enabled formulation of nanoparticles, and polymeric nanoparticles can be used to encapsulate curcumin. this could resolve curcumin's limitations by enhancing its sustained release to target diseased cells, improve its bioavailability, prevent it from degradation or metabolism, and increase its therapeutic potential. 14 polymeric nanoparticles are biocompatible and biodegradable and have been developed as drug delivery vectors and have desirable properties like better encapsulation of compounds to protect and deliver them efficiently with high pharmacokinetics and endocytosis efficiency. 15 chitosan is derived from chitin, which is a natural biopolymer and is the second most abundant natural polysaccharide. 16 it is found primarily in marine animals' exoskeleton, ie, lobsters, shrimps and crabs. chitosan is immunogenic, and its antitumor, anti-inflammatory, antibacterial, antifungal, and antimicrobial activities have been observed.17, 18 it has been reported that preparation of chitosan as nanoparticles revealed no toxicity at lower concentration (30 µg/ ml) but showed anticancer activity at higher concentrations (>100 µg/ml) against different cancer cell lines, which supports its application as a drug delivery system. 17, 19 previously, chitosan nanoparticles (csnps) have been conjugated with antiretroviral agents like saquinavir, a protease inhibitor, to improve anti-hiv therapy; cell targeting efficiency increased by 92% compared to the soluble drug control. 20 accordingly, to increase the antiviral activity of curcumin, in the present study we conjugated chitosan and curcumin as a nanocomposite and evaluated it for antiviral activity against entry and replication of hcv-4 in hepatoblastoma cells and performed in silico studies. in silico docking was performed using molegro virtual docker (mvd) 2013.6.0.0 (clc bio-qiagen company, aarhus, denmark). 21 the 3d structure of cucs nanocomposite was predicted using schrödinger's materials science suite 2.6 ( figure 1 ). curcumin ligand was retrieved from pubchem bioassay. homology models of ns3 protease and ns5a polymerase were built using the prime program of the schrödinger software suite. blast search was performed to identify a template protein structure. 22 loops refinement and verification was performed using the protein refinement program of schrödinger software. [23] [24] [25] the binding sites of ns3 protease, ns5a polymerase, and ns5b polymerase were detected using mvd cavities prediction. all docking calculations were carried out using the grid-based moldock score (grid) function with 0.30 å grid resolution. the csnps were prepared using an ionotropic gelation method of chitosan with tpp anions where positive charges of the amino groups of chitosan were slightly modified to be crosslinked with the tpp negative charges as we presented previously. 19 cucs nanocomposite was prepared by dissolving 320 mg of curcumin in 80 ml ethanol and incubated with 500 mg csnps suspended in 40 ml of millipore water at acidic ph from 4.0 to 5.0 with some modifications. 17, 26 finally, the curcumin was bound to csnps and dried using vacuum. high-resolution tem was used to determine the size, shape, and size distribution of the csnps and cucs nanocomposite by using a drop from diluted nanoparticles solution in order to form a monolayer through deposition on an amorphous carbon-coated copper grid and left to evaporate at room temperature. the particle and size distribution measurements were identified with a jem-2100 transmission electron microscope (jeol, tokyo, japan) that accelerates voltage at 200 kv, and the imaging was performed using the same microscope. zeta potential was performed to measure the electrical charges of the particles in nano colloids using a nano zs apparatus (malvern instruments, malvern, uk), through electrophoretic light scattering of the nanoparticles in an aqueous solution and using smoluchowski equation to calculate the zeta potentials at 25ºc. 27 the measurement was completed during 6 minutes and the migration voltage was fixed at 25 v. the ftir was carried out for csnps, curcumin, and cucs nanocomposite to show at which bonds the curcumin and chitosan were formed. ftir was used with the following specifications: spectral range 400-4500 cm −1 , number of scans 16, spectral resolution 4 cm −1 . the software was opus (bruker (germany), vertex 70, ramii). nanoparticles from the nanocomposite were weighed and acidified at ph 3.0; curcumin was extracted from the compound using a mixture of ethyl acetate and isopropanol (9:1 v/v) by shaking for 6 minutes. the mixture was centrifuged at 3864 × g for 20 minutes, and the upper layer of ethyl acetate was discarded and the extraction step was repeated. the final extraction solution was evaporated with vacuum to be completely dry, and its residue was dissolved using 100 μl ethanol. an aliquot of the dissolved solution was used for curcumin quantification via hplc by calculating the peak area of absorbance of the samples at wavelength 428 nm and comparing it to the standard peak area of curcumin. finally, the mobile phase was prepared by mixing 1% citric acid ph 3.0/acetonitrile (45:55, v/v), and the flow rate was 1 ml/min. the release of curcumin from its encapsulation in csnps was performed at ph 7.4, and the nanoparticles were redispersed in pbs. the total volume of the solution was divided into 5 tubes at 37°c under orbital shaking. subsequently, curcumin in nanoparticles was centrifuged at 966 × g for 10 minutes, where the sediment was extracted in methanol and quantified using spectrophotometry. finally, the release of curcumin from csnps was quantified according to the following equation: huh7 cells, derived from human hepatoma cells, were received as a gift from the lab of radiobiology and experimental radiooncology, uke, hamburg, germany and were used and maintained at virology and immunology unit, cancer biology dept., national cancer institute, cairo university. the wish cell line, human amniotic cells, was used as a model for normal cells. the cells were maintained as a monolayer in a 25 cm 2 flask with approximately 6 ml dmem supplemented with 10% fbs, 2% penicillin, and 2 mg/ml streptomycin. the cells were incubated under standard conditions of 37ºc, 5% co 2 , and 95% humidity. cellular toxicity of the tested materials (curcumin, csnps and cucs nanocomposite) was investigated against huh7 cells using 2-fold dilutions starting from 100 to 6.25 µg/ml, according to our previously published protocol. 19 the mtt formazan product was identified via measuring the absorbance using an enzyme linked immunosorbent assay (elisa) plate reader (model elx800, biotek instruments, inc., winooski, vt, usa), and positive and negative controls were run in the plate. the viability of cells (%) in relation to the control wells with untreated cells was calculated using the following equation: where a test is the absorbance of the test sample and a control is the absorbance of the control sample. the results were the average of three wells, and 100% viability was determined from the negative control (untreated cells). the cytotoxic effect of the ic50 concentration of cucs nanocomposite was followed microscopically with phase contrast microscopy (100x magnifications) after treatment of huh7 and wish cells after 24 hours. compusyn software (version 1.0, combosyn, inc., paramus, nj, usa) was used to predict and simulate the combination index (ci) and fraction effect (fa) of the cucs nanocomposite to estimate its activity and the amount of drug released into cells. 28 simulated cucs nanocomposite was used to investigate its cellular response using a response additivity model (linear interaction effect). such a model can demonstrate the positive effect of a drug combination (chitosan and curcumin) that might occur when the combination effect (e ab ) is greater than the expected additive effect given by the sum of the individual effects (e a +e b ). therefore, we investigated such possible favorable outcomes for better efficacy of therapeutic application and the minimal development of drug toxicity to provide a selective synergistic effect against the target. accordingly, this can be achieved by using a minimal set of mathematical and pharmacological concepts, according to an equation where the ci was calculated as follows: where e a refers to curcumin, e b refers to csnps and e ab refers to the nanocomposite. the resulting ci theorem offers a quantitative definition for additive effect (ci = 1), synergism (ci < 1), and antagonism (ci > 1) in drug combinations. such analysis was also performed to confirm the toxic effect of curcumin when combined with csnps, which also may indicate its possible role in the antiviral mechanism. the in vitro cellular uptake of the particles was carried out using tem, and the cells were first counted using a hemocytometer. then, the cells (3 × 10 6 /75 cm 2 flask) were treated with 100 µm of cucs nanocomposite for 24 hours. the cells were then washed with pbs buffer, fixed with 2% glutaraldehyde for 2 hours, then washed twice with pbs and finally fixed in 1% oso 4 for 1 hour. following agarose (1.5%) enrobing, spurr's resin embedding, and ultrathin (50 nm) sectioning, the samples were stained with 2% aqueous uranyl acetate and 25 mg/ml lead citrate and imaged with a jeol 100s microscope, 29 but cell control was tested using a field emission scanning electron microscope equipped with a scanning transmission electron (stem) detector (quattro s, thermo fisher scientific). flow cytometry was performed to identify the nature of cell death that was induced after treatment of huh7 with ic50 concentration of cucs nanocomposite. this was done by trypsinization of cells and centrifugation at 314 × g for 5 minutes. then, 100 µl of cell suspension was added to 100 µl of dna prep lpr (<0.1% potassium cyanide, <0.1% nan 3 , nonionic detergents, saline, and stabilizers) to obtain final cell concentration of 3-5 x10 6 cells/ml. afterward, 2 ml of dna prep stain was added to the cell suspension solution and incubated at room temperature for 30 minutes. finally, acquisition was done on a flow cytometer (beckman coulter, epicsxl, brea, ca, usa) of 10,000 events to calculate the percentages of cells occupying the different phases of the cell cycle. 30 quantification of mrna expression of apoptotic genes; caspase 3 and bcl-2 two 25 cm 2 flasks were seeded with 3 × 10 5 huh7cells and were left for 24 hours under optimum culture conditions to have a confluent sheet of cells, and then cells were treated with the estimated ic50 concentration of curcumin and cucs nanocomposite. afterwards, cells were harvested and total rna extraction was performed using nucleic acid extraction kit nucleospin ® (macherey-nagel gmbh, duren, germany) according to the manufacturer's instructions. the rna extract was used in real-time pcr assay using sensifast™ sybr ® hi-rox one-step kit (bioline, london, uk) with a total reaction volume of 25 µl and primers specific for caspase 3, bcl-2 and the housekeeping gene β-actin. the reaction consisted of three stages: stage 1 at 45°c for 10 minutes, stage 2 at 95°c for 2 minutes, stage 3 repeated for 35 cycles that consisted of 95°c for 5 seconds, then 60°c for 10 seconds, and 72°c for 5 seconds. finally, calculation of the relative quantification of the cycle threshold (ct) was conducted according to the reaction of each target gene. the relative quantification (rq) of each gene was quantified according to the calculation of δδct as the rq of each gene is calculated by taking its 2 −δδct according to the following equation: six well tissue culture plates were seeded with 3 × 10 4 of huh7 cells and left for 24 hours under optimum culture conditions to have a confluent sheet of cells, and then they were treated with the estimated ic50 concentration of the cucs nanocomposite and paclitaxel as a standard chemotherapeutic agent to allow its comparison with nanocomposite. afterwards, the cells were harvested and their total proteins were extracted to measure the levels of apoptotic proteins caspase 3, caspase 8, and p53, as well as levels of the antiapoptotic marker bcl-2. then, the isolated proteins' levels were measured using human bcl-2 platinum elisa assay kit according to the manufacturers' instructions (ebioscience, madison, wi, usa). the standard curve for each protein was drawn according to each kit and the reaction products were measured at 450 nm using a microplate reader (tecan group ltd., seestrasse, männedorf, switzerland). six well tissue culture plates were seeded with 500,000 cells/ well; huh7cells were treated with nontoxic doses of curcumin, csnps, and cucs nanocomposite at concentrations of 15 μg/ml, 100 μg/ml, and 20 μg/ml, respectively. this was performed according to a previously published protocol. 32 a 500 µl aliquot of the positive serum for hcv-4a was inoculated and kept for 90 minutes for adsorption. dmem (10%) was added to a final volume of 3 ml and the incubation was continued for 24 hours. infected cells were treated with either curcumin, chitosan, or cucs nanocomposite. untreated infected cells and cell control were treated with media only. the plates were incubated for 24 hours, and viral rna was extracted prior to real-time pcr assay according to the previously published protocol. 32 the six well plates were again used as previously described in the antiviral assay, then the infected and treated huh7 cells were subjected to cell harvesting with a cell scraper, and the protein content of the cells was extracted using lysis buffer, then the protein content was measured with a bradford protein assay kit. subsequently, 20 µg of protein concentration of each sample was loaded into 10% sds-page with an equal volume of 2x laemmli sample buffer (4% sds, 10% β-mercaptoethanol, 2% glycerol, 0.004% bromophenol blue, and 0.125 m tris hcl). afterward, the gel was transferred to a polyvinylidene fluoride (pvdf) membrane and then blocked with trisbuffered saline with tween 20 buffer and 3% bovine serum albumin (bsa) for 1 hour. the membrane was incubated overnight at 4°c with 1:1000 dilution of the primary antibody (anti-hcv core 1b antibody, ab2740, thermo scientific, boston, ma, usa) and then incubated for 1 hour at room temperature with horseradish peroxidase hrp-conjugated secondary antibody goat anti-rabbit igg. finally, the membrane was washed and bands were visualized with the aid of chemiluminescent substrate, and signals were detected using a ccd camera-based imager. ultimately, band images of the target proteins were analyzed using chemidoc mp imager in relation to β-actin as a positive control. 33 the workflow of methods performed in the current research is presented graphically in scheme 1. the in silico study showed that the binding affinity of curcumin to ns3 protease and ns5a polymerase was higher than that of cucs nanocomposite chitosan. however, cucs nanocomposite showed good binding affinity toward ns5b polymerase as compared to curcumin. curcumin showed a higher docking score toward ns3 protease and ns5a polymerase with −124.91 and −159.02 moldock score, respectively ( table 1) . binding of curcumin with hcv-4a target proteins (ns3, ns5a and ns5b) showed that it could form h-bonds at the cavity of ns3 protease ( figure 1a and b and table 1 ) and ns5a polymerase ( figure 1c and d and table 1) , where it forms 2 h-bonds and 5 h-bonds with ns3 protease and ns5a polymerase inside the protein cavity, respectively. however, cucs nanocomposite formed 9 h-bonds with ns5b polymerase ( figure 1e and f and table 1 cucs nanocomposite was subjected to several characterization procedures to study encapsulation efficiency. the tem results showed that the size ranged from 29 to 39.5 nm (figure 2a) with charges of 33 mv as measured with the zeta analyzer ( figure 2b ). the ftir spectra of chitosan, curcumin, and cucs nanocomposite are shown in figure 2c . characteristic bands at 1511 cm −1 and 1062 cm −1 were attributed to the stretching vibrations of the c=c vibrations, and c-o-c stretching modes, respectively, in curcumin. for csnps, the characteristic band at 3450 cm −1 was assigned to stretching vibration mode of n-h overlapped with o-h stretching vibration mode, the band at 2880 cm −1 was due to c-h stretching vibration mode, the band at 1653 cm −1 was assigned to the c=o, and the band at 1428 cm −1 was assigned to bending vibration of n-h stretching vibration. in comparison with curcumin, a different spectrum was observed for cucs nanocomposite as new bands appeared at 1070 cm −1 and the n-h group of csnps disappeared ( table 2 ). it can be assumed that the ammonium groups of chitosan were linked with oh groups of curcumin to form the nanocomposite. results from hplc indicate that 42 mg curcumin was encapsulated in the csnps, giving 4% of curcumin encapsulated inside csnps ( figure 2d and e) . the in vitro drug release experiment showed that initially, a gradual increase with the exponential release rate of curcumin was observed for the first 2 hours with an increment rate of 30 ( figure 2f ). this large increase is due to the curcumin molecules that deposited on the surface of cs nanocapsules. afterward, there was a relatively constant release of curcumin for the next 4 hours and there was a linear plateau in release that was figure s1 shows the behavior profile of curcumin-encapsulated into csnps and the equation used for the calculation. from the release profile of curcumin, it is clear that about 45% of curcumin was released from the nanoparticles at time 0, and that afterward the amount of curcumin released from the nanoparticles was about 84% after the first 2 hours. finally, there was no further release obtained for the other 4 hours. the cytotoxic effect of various concentrations of curcumin, chitosan, and cucs nanocomposite showed that the ic50s of curcumin and cucs nanocomposite were detected at concentrations of 8 and 25 µg/ml, respectively, on huh7 after 48 hours of cell exposure, and csnps were nontoxic at more than 100 µg/ml on huh7 or wish ( figure 3 ). regarding cytotoxic effects of the same materials on wish, our results revealed that the ic50s of curcumin and cucs nanocomposite were detected at concentrations of 25.8 and 34 µg/ml, respectively, after the same time of cell exposure. the light microscopy results showed aggressive changes at the concentration of the ic50 for both curcumin and cucs nanocomposite without any changes in the morphology of cells treated with csnps at 100 µg/ml (figure 4 ). the cucs nanocomposite showed a good therapeutic effect against human liver cancer cells of fraction effect 75 that can kill 75% for the simulated drug release amount upon combining 48 µg/ml of curcumin and 193 µg/ml chitosan. but upon increasing these concentrations, the effect would be antagonistic according to compusyn software (table 3, supplementary tables s1 and s2 ). tem images showed binding and internalization of csnps into huh7 cells. aggregation of csnps to form nanoparticle clusters on the cell membrane and its dispersion in the cytoplasm was evident ( figure 5a-c) . the cucs nanocomposite was further investigated with flow cytometry of cell cycle and dna contents of huh7 cells treated with the ic50 concentration. results showed cell arrest in the preg1 phase, preventing cells from entry into s and g2/m phases of the cell cycle ( figure 6a and b and table 4 ), whereas the untreated control cells showed the expected cell cycle pattern for continuously growing cells. the mrna expressions of caspase 3 gene increased posttreatment with ic50 concentrations of curcumin, and it was doubled after treatment of cells with cucs nanocomposite. however, the expression of bcl-2 decreased post-treatment with both materials ( table 5 , supplementary figure s2 ). treatment of huh7 cells with the ic50 concentrations of cucs nanocomposite was associated with an increase in the activity of caspase 3 protein from 9.7 µg/ml to 12 µg/ml, and it markedly decreased the protein level of anti-apoptotic bcl-2 from 2.8 µg/ml to 0.2 µg/ml. moreover, a marked increase in the protein levels of caspase 8 and p53 is shown in figure 7a . likewise, the effect of paclitaxel as a standard anticancer drug on antiviral activities of curcumin, csnps and cucs nanocomposite were tested against the viral entry in infected huh7 cells from positive hcv-4a patients through mixing of equal volumes of the nontoxic concentrations of the investigated materials and viral titer; in this assay one viral titer was involved (10 5 iu/ml). as a result, the tested compounds showed high antiviral activities against entry of hcv-4a into huh7 cells by almost 100% reduction in viral titer, and curcumin inhibited viral entry by almost 95%. (table 6 ). the hcv core protein expression level post-treatment of hcv infected huh7 cells with curcumin, csnps, and cucs nanocomposite against viral replication and entry was quantified in relation to β-actin as a control ( table 7 , figure 8 ). the expression post-treatment against viral replication showed a decrease in cells treated with the three compounds and specifically for that treated with nanocomposite with a range 0.9 to 5.6 ratio to the positive infected huh7 cells. regarding expression post-treatment with curcumin, csnps, and nanocomposite against viral entry, there was a decrease in the hcv core protein expression, especially in those treated with nanocomposite with a 1.05 to 5.6 ratio to the positive infected huh7 cells. recently, the advent of new direct-acting antiviral drug therapy for hcv-4a has greatly improved sustained virology response, although there is the possibility of serious adverse effects and development of resistant mutant strains. therefore, discovery of more effective and less toxic antiviral agents is still needed. computational screening of large chemical libraries using molecular docking and the establishment of hcv culture model has led to numerous studies and the discovery of many anti-hcv agents. accordingly, we started working on csnps, 19 and the in silico results demonstrated high binding affinity towards hcv-4a target proteins (ns3 protease, ns5b polymerase, and ns5a helicase) and this encouraged us to continue our research on csnps as a drug delivery system for antiviral agents, hoping to increase therapeutic efficacy and decrease therapeutic dose. in the current research, curcumin was encapsulated into csnps and evaluated for its antiviral activity against hcv-4a first with in silico analysis and subsequently with in vitro studies, with the hope to discover new candidates for use as hcv-4a inhibitors. our in silico analysis gave promising results by showing an increase in the affinity of our designed nanocomposite against hcv-4a ns5b compared to curcumin alone, suggesting its potential role against viral replication, first with in silico analysis and subsequently with in vitro studies, to discover new candidates for use as hcv-4a inhibitors. our in silico analysis gave promising results by showing an increase in the affinity of our designed nanocomposite against hcv-4a ns5b compared to curcumin alone, suggesting its potential role against viral replication. here we found that curcumin conjugated to chitosan via the oh group of curcumin and the ammonia group of csnps crosslinked with tpp, which resulted in small sizes (29.6-39.5nm) 34 compared to results previously published by das and colleagues who used sodium alginate as a crosslinker instead of tpp salt, which led to chitosan with a bigger size (100±20 nm). 35 different fundamental and clinical investigations illustrate curcumin's restricted adequacy because of its poor dissolvability, rapid metabolism, low bioavailability, and pharmacokinetics. in addition, its physicochemical properties are unstable in several conditions like neutral ph or alkaline ph ≥7.0, and ambient temperature. 36 therefore, we conjugated curcumin into csnps to improve its properties, control its release, and enhance its antiviral activity. our preparation was performed at acidic ph with certain modifications, 26 using ascorbic acid as solvent for csnps instead of acetic acid, where positive charges of amino groups on chitosan can interact with negative charges on oh of curcumin through electrostatic interactions, leading to formation of cucs nanocomposite. this binding property was found to be much less at neutral or alkaline ph due to the higher tendency of csnps to aggregate because of neutralization of the amino groups on csnps. 17 our hplc experiment demonstrated 4% entrapment of curcumin into csnps after 6 minutes of polymeric digestion; this time needs to be lengthened in the future preparations. drug release profile showed that 50% of loaded curcumin was released during the first 11 minutes-the release time known as "burst release" meaning the amount of curcumin deposited on the outer surface of the csnps-and 34% of curcumin was released at 120 minutes. this means that the total release of curcumin (84%) was obtained after 2 hours. afterward, a relatively constant release of curcumin was observed for the next 4 hours (ie 240 minutes). this is supported by results of akhtar et al who reported pharmacokinetic analysis for chitosan bound to curcumin in parasite plasmodium yoelii infected mice. 17 they found that the curcumin concentration detected in the blood was significantly higher in mice fed with curcumin loaded onto csnps compared to blood from mice administered an equimolar concentration of curcumin alone. results showed that administration of csnps orally ensured a sustained release of curcumin over 8 hours post-feeding, whereas, in the case of curcumin alone, the levels declined significantly after 3 hours and were not detectable beyond 6 hours. in a study by samrot et al, curcumin-loaded csnps were synthesized from crab shell for drug delivery using two chelators (tpp and bacl 2 ) in preparation of csnps. 37 they observed an initial drug burst from the biopolymer that then become steady, and the csnps chelated with bacl 2 led to sustained drug release for more than 70 minutes. regarding csnps chelated with tpp, the samples had a stable drug release profile; drug release increased from the 20 th to the 60 th minute and then gradually decreased until the 100th minute, followed by a stable release. this indicates that chitosan synthesized using tpp showed a slow release of curcumin for 180 minutes. such an observation might explain why the cytotoxic effect of our synthesized nanocomposite was lower than that of curcumin alone (25 µg/ml versus 8 µg/ml for curcumin). the literature shows that the cytotoxic effect of a nanoformulation of curcumin is equal to or higher than that of free curcumin. 38, 39 this does not match our results where the cytotoxic effect of cucs was lower than that of free curcumin. this might be due to i) the lower % of the entrapment efficiency, ii) the nature of the nanocarrier, which is different in the current study than in previous studies, and iii) the antioxidant properties of csnps as previously reported by nair et al. 40 further extensive investigations using simulated compusyn software (linear interaction effect) were conducted to investigate possible additivity between curcumin and csnps, which might predict the synergistic effect of cucs nanocomposite as antiviral activity. upon using multiple inhibitors of different types and mechanisms of inhibition on enzyme kinetic models, several hundred equations have been derived that can be reduced to general equation by comparing theoretical biological activities in the absence and presence of inhibitors. this mathematical work leads to the ci. the obtained data are unique to each laboratory because data vary according to instrument, reagents, and type of cells grown in each lab. 41 the nature of cell death induced by the formulated cucs nanocomposite was analyzed with flow cytometry, and results showed that huh7 treated with an ic50 concentration of nanocomposite induced cell cycle arrest at the preg1 phase in comparison to the untreated cells, which hindered their entrance into the s and g2/m phases. this might be due to the small amount of dna content and admission into programmed cell death, as previously illustrated by kumar et al. 42 the results of molecular apoptotic gene expression level of cells treated with an ic50 concentration of curcumin and its composite showed high expression levels in caspase 3 and lowered expression of bcl2 in huh7 cells treated with curcumin and its nanocomposite. this indicates that the apoptosis was induced via a caspasedependent pathway. such cytotoxic effect could be due to the production of reactive oxygen species in response to the toxic dose of curcumin, resulting in dna breakage, which in turn causes activation of p53 that can recruit other molecules like bax, bak. this, in turn, causes pores in the mitochondria membrane, disrupting its membrane, leading to the release of cytochrome c, which stimulates a cascade of death compounds and ultimately cell death. 43 finally, protein expression levels of caspase 3, caspase 8, p53, and bcl2 were detected using elisa assay. cells treated with paclitaxel, a standard anticancer drug served as a control. our results matched that of mrna transcriptional level, which showed an increase in the expression levels of caspase 3, caspase 8, and p53 proteins and a decrease in the expression levels of bcl2 protein compared to untreated huh7. these results confirmed the designated roles of the nanocomposite in mediation in the apoptotic process through cell death receptors and induction of apoptosis through activation of intrinsic and extrinsic apoptotic pathways. 44, 45 concerning assessment of the antiviral activity of csnps, curcumin, and its nanocomposite, curcumin demonstrated inhibition of hcv-4a entry by 94.5%, which is in line with results reported by kusuma et al, 46 where curcumin was confirmed to block the hcv entry pathway into its targeted hepatoma cells with no effect on viral assembly or virion release. moreover, our data match those of anggakusuma et al who reported that curcumin nanoformulation increased its antiviral activity with no toxic effect; they demonstrated that the antiviral mechanism occurred by affecting virion membrane fluidity without disrupting virion integrity. 10 in the present study, the same observations were obtained upon analyzing antiviral activity against replication of hcv-4a; these results confirm the in silico analysis and demonstrate the dual roles of csnps and its composite to inhibit both viral entry and replication. these results were confirmed with hcv core protein expression using western blot assay, which demonstrated a remarkable decrease in the expression level after the treatment of cells with csnps, curcumin, and nanocomposite. nevertheless, the nanocomposite exhibited the lowest decrease in the hcv core protein expression, which indicates that the nanocomposite has the highest antiviral activity whether against viral entry or replication. further investigations are still required to study the anti-hcv activity of nanocomposite in the replicating system, which is underway in our laboratory. in conclusion, the current study obtained a clear view on the potency of csnps in synergizing the antiviral activity of curcumin on binding as a nanocomposite form. additionally, the prepared nanocomposite demonstrated a powerful multitarget antiviral agent against hcv-4a entry and replication, and this was proved on the molecular and protein levels. however, it is mandatory to confirm the novelty of the nanocomposite as a potential alternative therapy against hcv-4a infection through analysis of pharmacokinetics and pharmacodynamics as well as further analysis on the hcv-4a replicating system to obtain the therapeutic index and ultimately an in vivo study. the research has been approved by the national cancer institute-cairo university, irb irb00004025 organization no. iorg0003381. the international journal of nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. this journal is indexed on pubmed central, medline, cas, scisearch ® , current contents ® /clinical medicine, journal citation reports/science edition, embase, scopus and the elsevier bibliographic databases. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. submit your manuscript here: https://www.dovepress.com/international-journal-of-nanomedicine-journal world health organization. hepatitis c hepatitis c in egypt -past, present, and future hcv ns3/4a protease inhibitors and the road to effective direct-acting antiviral therapies. hepatitis c virus ii hcv genotype 4, 5 and 6: distribution of viral subtypes and sustained virologic response rates in clinical trials of approved direct-acting antiviral regimens defining the possibilities: is short duration treatment of chronic hepatitis c genotype 1 with sofosbuvir-containing regimens likely to be as effective as current regimens? plant-derived antivirals against hepatitis c virus infection hepatitis c virus and natural compounds: a new antiviral approach? viruses naringenin prevents obesity, hepatic steatosis, and glucose intolerance in male mice independent of fibroblast growth factor 21 coffee and caffeine are associated with decreased risk of advanced hepatic fibrosis among patients with hepatitis c turmeric curcumin inhibits entry of all hepatitis c virus genotypes into human liver cells curcumin inhibits hepatitis b virus infection by down-regulating cccdna-bound histone acetylation a review on antibacterial, antiviral, and antifungal activity of curcumin curcumin against hepatitis c virus infection: spicing up antiviral therapies with 'nutraceuticals'? curcumin-hybrid nanoparticles in drug delivery system (review) bacterial components as naturally inspired nano-carriers for drug/gene delivery and immunization: set the bugs to work? preparative methods of phosphorylated chitin and chitosan-an overview oral delivery of curcumin bound to chitosan nanoparticles cured plasmodium yoelii infected mice preparation of 5-fluorouracilloaded chitosan nanoparticles and study of the sustained release in vitro and in vivo synthesis, characterization and cytotoxic evaluation of chitosan nanoparticles: in vitro liver cancer model evaluation of chitosan nanoformulations as potent anti-hiv therapeutic systems moldock: a new technique for highaccuracy molecular docking new york: schrödinger, llc l2-solutions for nonlinear schrödinger equations and nonlinear groups a comparative study of available software for high-accuracy homology modeling: from sequence alignments to structural models structural and biochemical basis for the difference in the helicase activity of two different constructs of sars-cov helicase inventors; arab republic of egypt, ministry of scientific research. a method for preparation of anti hcv compounds against genotype 4a ca2+ adsorption to lipid membranes and the effect of cholesterol in their composition cytotoxicity, biocompatibility and cellular response of carbon dots-plasmonic based nano-hybrids for bioimaging multiplexed screening of cellular uptake of gold nanoparticles using laser desorption/ionization mass spectrometry dna measurement and cell cycle analysis by flow cytometry quantitative analysis of the mrna expression levels of bcl2 and bax genes in human osteoarthritis and normal articular cartilage: an investigation into their differential expression characterization of chronic hcv infection-induced apoptosis effects of hepatitis c virus core protein and nonstructural protein 4b on the wnt/β-catenin pathway curcumin-loaded chitosan tripolyphosphate nanoparticles as a safe, natural and effective antibiotic inhibits the infection of staphylococcus aureus and pseudomonas aeruginosa in vivo encapsulation of curcumin in alginatechitosan-pluronic composite nanoparticles for delivery to cancer cells an in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts synthesis of curcumin loaded polymeric nanoparticles from crab shell derived chitosan for drug delivery galactosylated chitosan-polycaprolactone nanoparticles for hepatocyte-targeted delivery of curcumin a comparison between the cytotoxic effects of pure curcumin and curcumin-loaded plga-peg nanoparticles on the mcf-7 human breast cancer cell line an evaluation of curcuminencapsulated chitosan nanoparticles for transdermal delivery generalized equations for the analysis of inhibitions of michaelis-menten and higher-order kinetic systems with two or more mutually exclusive and nonexclusive inhibitors synthesis of substituted phenanthrene-9-benzimidazole conjugates: cytotoxicity evaluation and apoptosis inducing studies apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies analysis of drug combinations: current methodological landscape decreased expression level of apoptosisrelated genes and/or proteins in skeletal muscles, but not in hearts, of growth hormone receptor knockout mice turmeric curcumin inhibits entry of all hepatitis c virus genotypes into human liver cells this project was supported financially by the science and technology development fund (stdf), egypt, grant no.15108. we would like to thank national cancer institute, cairo university, for supporting us with equipment, reagents, and consumables. our sincere gratitude goes to the lab of radiobiology and experimental radiooncology, uke, hamburg, germany, for supplying huh7 and wish cell lines as a gift to complete our research. we also thank ahmed mamdouh osman for his technical assistance. the authors declare no conflicts of interest in this work. key: cord-255781-55zrmgxq authors: bergman, scott j.; ferguson, mckenzie c.; santanello, cathy title: interferons as therapeutic agents for infectious diseases date: 2011-12-31 journal: infectious disease clinics of north america doi: 10.1016/j.idc.2011.07.008 sha: doc_id: 255781 cord_uid: 55zrmgxq this article explains the rationale for development of interferons as therapeutic agents, and describes commercial products available today. it also provides a summary of studies that have been performed with interferons for use as exogenous biological response modifiers in viral infections. overall, the best data exist for treatment of viral hepatitis b and c, for which interferons are a cornerstone of therapy. although infections with human papillomavirus and common cold viruses sometimes respond favorably to interferons, their outcomes are far from ideal. finally, the role of interferons as vaccine adjuvants is still being explored but could be promising. replication in vitro at concentrations as low as pg/ml-the development of ifns as clinically useful drugs has been largely disappointing. this fact can be attributed partly to their short half-life in vivo and their extensive side effects. in fact, many symptoms of viral infections such as influenza can be blamed on endogenous ifn release. the adverse effects prevalent at therapeutic doses include fever, myalgia, and headache, dubbed "flulike symptoms," along with bone marrow suppression leading to leukocytopenia and thrombocytopenia, plus central nervous system manifestations including depression. 1 ifns have been studied for the treatment or prevention of herpes zoster, herpes simplex, and cytomegalovirus infections, but the successful development of acyclovir and ganciclovir gave clinicians safer and more effective alternatives for dealing with these viruses. 5, 6 ifns can also be used in the treatment of multiple sclerosis and certain cancers, but this article reviews the therapeutic applications of ifns for infectious diseases, focusing on viral infections. ifns are not absorbed orally because of their large amino acid sequence, which is susceptible to the proteolytic enzymes in the digestive tract. however, ifn-a is readily absorbed after both intramuscular and subcutaneous injection. 7 this rapid absorption combined with a short half-life means that frequent injections are needed to maintain adequate concentrations in the body. both commercially available ifn-a products in the united states have now been chemically attached to polyethylene glycol (peg) to enhance their half-life and make once-weekly dosing possible. this coupling not only makes administration easier, but also reduces side effects by having a predictably lower peak concentration of the exogenous cytokine. both pegylated inf-a2a (pegasys) and ifn-a2b (peg-intron) are obtained from escherichia coli by recombinant methods. these agents consist of naturally occurring small proteins with molecular weights of 15,000 to 27,600 da. 3 each is considered a first-line option for the treatment of chronic hepatitis c virus (hcv) infection in combination with ribavirin. more details on this use and others are described later in this article. along with the list of additional indications approved by the food and drug administration shown in table 1 , ifn-a was shown to be an effective treatment for the symptoms of an aggressive case of chronic active epstein-barr virus, but did not eliminate infection entirely. 8 therefore, additional studies would need to be performed before recommendation for this use. human leukocyte derived ifn-an3 (alferon n) injection contains a spectrum of a ifns, and is only approved for the treatment of refractory or recurring condylomata acuminata in adult patients. a low-dose oral version is in development for use in the treatment and prevention of influenza. 9 both versions have been studied against human immunodeficiency virus (hiv)-1 infection, but with little success. 10,11 ifn alfacon-1 (infergen) is considered the synthetic "consensus interferon" because it contains a nonnatural sequence of ifn-a amino acids all chosen for the highest activity against viral hepatitis. to date, no pegylated formulation of this product has been brought to market. all the a ifns include a black-box warning in their prescribing information about how their use .may cause or aggravate fatal or life-threatening neuropsychiatric, autoimmune, ischemic and infectious disorders. patients should be monitored closely with periodic clinical and laboratory evaluations. therapy should be withdrawn in patients with persistently severe or worsening signs and symptoms related to side effects. in many, but not all cases, these resolve after stopping therapy. 12, 13 ifn-b1a (avonex or rebif) and ifn-b1b (betaseron) are recombinant proteins with 166 and 165 amino acids, respectively. these b ifns have antiviral and immunomodulatory properties too, but their use at this time is limited to treatment of multiple sclerosis, not infections. ifn-g1b (actimmune) injection is used regularly for the prevention of infections in patients with chronic granulomatous disease along with antibacterials and antifungals. 14 its mechanism of action for this purpose is not entirely known, but long-term studies show a definite benefit. 15 ifn-g can also be used as a salvage therapy for mycobacterial infections, but is not routinely used for treatment of this or other infections. 16 topical imiquimod 5% (aldara) and 3.75% (zyclara) creams do not have inherent antiviral activity alone, but instead induce ifn-a, ifn-b, and ifn-g plus tumor necrosis factor (tnf)-a through toll-like receptors (tlrs). local application to external genital and perianal warts results in an immunomodulatory response that stimulates cytokines, which have antiviral action and cause a reduction in both viral load and wart size. 3 chronic infection with hepatitis b virus (hbv) and hcv affects over 400 million people worldwide. [17] [18] [19] chronic viral hepatitis is a leading cause of cirrhosis, liver transplantation, and hepatocellular carcinoma. with the development of a vaccination series for hepatitis b in the mid-1980s, along with increased public education and awareness, acute infection rates of both hbv and hcv in the united states have declined steadily. 19 hbv is a double-stranded dna virus whereas hcv is a single-stranded rna virus, both of which are capable of significant morbidity and mortality in chronic infection. the exact mechanisms of hepatic injury from hbv and hcv infection are not completely understood. because asymptomatic carriers with normal liver transaminases exist, it is likely multiple immune-mediated mechanisms result in hepatocyte damage as opposed to the virus itself being directly cytotoxic. following acute viral infection, the innate immune response initiates formation of nk cells, followed by virus-specific cd4 1 t cells and cd8 1 cytotoxic t lymphocytes. nk cells stimulate production of ifn-a/b and promote cellular clearance of viral proteins through disruption of the replication process. following successful clearance, either spontaneously or by treatment with ifn, peripheral cytotoxic t lymphocytes and cd4 1 t-cell response persists. 20 chronic infection is likely a result of failed innate and adaptive immunity. specifically, chronic infection with hcv has been associated with impaired t-cell and nk-cell response. [21] [22] [23] [24] genetic factors also likely influence progression of disease and predisposition to adverse effects. 25 although an abundance of research has investigated the immune response in relation to chronic viral hepatitis, many areas of uncertainty still exist. standard ifn-a, the first approved ifn for viral hepatitis, lacked several desirable pharmacokinetic properties. the addition of peg created an ifn that has a slower rate of absorption, reduced elimination, and a longer half-life, necessitating less frequent dosing and fewer adverse effects. furthermore, the peg moiety results in reduced immunogenicity and sterically hinders the antigenic binding site. 26, 27 although pegylated ifn has replaced standard ifn-a in treatment of chronic hbv and hcv, as many as 40% to 50% of patients still fail to respond to treatment. successful response depends on many factors including but not limited to viral genotype, viral load, and degree of liver fibrosis. 25 chronic hepatitis b and c are treated similarly with peginterferon (pegifn); however, only pegifn-a2a is fda-approved in the united states for treatment of hbv. both pegifn products are administered as subcutaneous injections once weekly for durations up to 48 weeks, dependent on viral genotype and early viral response for treatment of hcv. pegifn-a2b is dosed based on body weight (1.5 mg/kg once weekly) whereas pegifn-a2a is a fixed dose (180 mg/wk). ribavirin is used in combination with pegifn for treatment of hcv. the exact mechanism of action of ribavirin as an adjunctive antiviral agent in hcv is not completely understood. 12, 13 some studies have proposed ribavirin to act as an ifn-stimulated gene inducer to improve second-phase viral decline. 28 protease inhibitors (boceprevir and telaprevir) are recently approved adjunctive oral agents for the treatment of chronic hcv with pegifn and ribavirin. to date, all studies of protease inhibitors have been conducted in patients with hcv genotype 1, and have shown an increase in sustained virologic response (svr) rates particularly for patients previously unresponsive to ifn therapy. [29] [30] [31] [32] [33] [34] [35] the use of ifn for the treatment of chronic hbv and hcv has represented a mainstay of treatment for several decades. the specific mechanisms behind the antiviral effects of ifn for hepatitis are complex. ifn-stimulated genes are induced by ifn and disrupt viral replication. hundreds of ifn-stimulated genes are thought to exist. viperin, isg20 and protein kinase r (pkr) are just a few of the most commonly cited. it is also highly possible that ifn-stimulated genes work synergistically to produce antiviral activities. 36, 37 a lack of pkr can lead to an environment conducive to hcv replication, though it may not be a good predictor of exogenous ifn response. the study of ifn-stimulated genes and their role in determining who responds to ifn therapy has been evaluated in several studies. 25, 36, 38 additional studies of ifnstimulated gene expression are needed to clarify which are directly involved in successful viral response, in what capacity they affect response, and whether pharmacotherapy directed at induction of ifn-stimulated genes can help improve treatment response. chronic hbv infection can be successfully treated with ifn monotherapy. 39 loss of viral dna and antibody formation are successful outcomes associated with ifn treatment. the mechanism of ifn antiviral activity varies depending on hepatitis be antigen (hbeag)-positive or hbeag-negative disease. in hbeag-positive patients, an immune response is stimulated by ifn whereas in hbeag-negative disease, ifn acts directly as an antiviral. 5 hbeag-negative disease tends to be more difficult to treat, and is associated with a longer duration of disease and a higher likelihood of complications such as cirrhosis. 20 several oral nonnucleoside reverse transcriptase inhibitors are also available for treatment of hbv (entecavir, tenofovir, adefovir, lamivudine, and telbivudine). although ifn is still considered a first-line alternative and provides the advantage of defined treatment duration rather than potentially lifelong administration, these oral agents are often used in therapy because of their ease of use and reduced number of side effects associated with treatment. the ability of hcv to evade the host immune response has produced a complex rna virus capable of lingering infection, ultimately resulting in opportunities for increased risk of transmission and complications from advanced liver disease. much of the research regarding the use of ifn for chronic viral hepatitis has focused on use in hcv. following treatment with ifn, a decline in hcv rna occurs over several phases. a rapid inhibition of rna production within the first 1 to 2 days of treatment is followed by a second, slower phase associated with clearance of infected cells. 23, 40, 41 the interferons for infectious disease immune response to endogenous ifn produced by innate immunity and that administered exogenously can differ in terms of antiviral activities based on the phase of viral decline. studies have shown that response to ifn-based treatment for hcv may be affected by differences in ifn signaling and induction. it is likely that hcv has mechanisms to avoid recognition by the innate immune response, and as such inhibits the ability of hcv-infected cells to generate ifn. 25, 38, 42 early studies conducted in nonresponders to current therapy showed wide genetic diversity, with many showing no common traits to predict nonresponse to ifn therapy. 25, 38, 43 however, in 2009 several major studies were published associating a singlenucleotide polymorphism (snp) just upstream from interleukin-28b gene (il28b) with ifn response in patients with hcv genotype 1. [44] [45] [46] [47] [48] additional evidence points to the fact that the il28b polymorphism is also linked to spontaneous clearance of hcv. 48, 49 the il28b variant encodes for ifn-l3, a type iii ifn belonging to the interleukin (il)-10 superfamily, which function in a manner similar to type i ifns, resulting in ifn-stimulated gene induction. [49] [50] [51] the genome-wide association study conducted by ge and colleagues 45 evaluated more than 1600 treatment-naïve hcv genotype 1 patients, the majority of whom originated from the ideal study. results from logistic regression showed that the il28b polymorphism was a stronger predictor of svr than baseline viral load, ethnicity, or degree of fibrosis. further research in this area is needed to clearly identify a future role for genotype testing and further clarify whether it may influence response to therapy in other hcv genotypes. a multicenter, randomized, controlled study by mangia and colleagues 52 analyzed 268 caucasian patients with hcv genotype 2 (n 5 213) and 3 (n 5 55). out of 61% of patients who achieved rapid virologic response (rvr), il28b genotype was not associated with svr, whereas in those patients who did not achieve rvr a significant difference in svr was noted based on il28b genotype. at this time genotype testing for il28b is not routinely recommended for all hcv patients planning to undergo treatment, but it may be in the future. if done, it should not be used as the only factor when choosing a treatment strategy. 49 the complexity of viral defense mechanisms and subsequent effect on the host response has led not only to development of chronic infections but also to a lack of a viable vaccine. hcv viral polymerase lacks a proofreading capability, creating a more diverse target for vaccine development. 18 additional challenges include the lack of a suitable animal model to mimic a human environment and medium for viral growth. 18 one of the major limitations to ifn therapy is adverse effects. malaise, gastrointestinal effects, neuropsychiatric effects, neutropenia, and anemia can all limit the effectiveness of treatment by necessitating dosage reductions or treatment discontinuation. for newer ifn therapies to be successful, they must induce an antiviral response while at the same time limiting adverse effects. albinterferon is a new ifn therapy currently in development for the treatment of chronic hcv. this product is a combination of ifn-a2b fused to recombinant human albumin. one of the advantages with this product is that it only requires once or twice monthly dosing. 53 not much is known at this time about the immunomodulating effects of albinterferon in hcv. it has been shown to have similar svr and adverse event rates to traditional pegifn when used in combination with ribavirin. [54] [55] [56] research into ifn-l as an agent to treat hcv has also been initiated. it is hypothesized that l ifns may be associated with less adverse effects than ifn-a because ifn-l receptors are primarily found in hepatocytes. 49, 57 specifically, research into new investigational pharmacotherapy in the form of pegylated il-29 (ifn-l1) in patients with hcv genotype 1 who relapsed following traditional treatment with peg-ifn-a and ribavirin appears promising. 50, 57 both ifn-l1 and ifn-l3 share a common receptor and have a similar sequence identity. 57 a 4-week, open-label study conducted in 56 patients with chronic hcv genotype 1 was designed to assess pegifn-l1 in combination with ribavirin. 57 it was a dose escalation study conducted in 3 parts. parts 1 and 2 evaluated patients who relapsed following treatment with ifn-a, and part 3 included treatment-naïve patients. in part 1, pegifn-l monotherapy (1.5 mg/kg or 3 mg/kg) was administered subcutaneously every 2 weeks or weekly. in parts 2 and 3, a range of pegifn-l dosages (0.5 mg/kg, 0.75 mg/kg, 1.5 mg/kg, or 2.25 mg/kg) were administered weekly in combination with ribavirin twice daily (1000 mg if weight <75 kg and 1200 mg if weight !75 kg). the primary outcomes were safety and tolerability. pharmacokinetics and viral load reduction were evaluated as secondary end points. commonly reported adverse effects with pegifn-l included fatigue (29%), nausea (12%), myalgia (11%), and headache (9%). most adverse events were mild or moderate in severity. four patients (7%) experienced treatment-related toxicity and required doses to be withheld. one patient experienced grade 3 thrombocytopenic purpura and another patient had elevated alanine aminotransferase, aspartate aminotransferase, and bilirubin levels. both events were considered to be related to treatment with pegifn-l. aminotransferase elevations occurred most often in patients who received high-dose (3 mg/kg) pegifn-l monotherapy. no clinically relevant decreases in absolute neutrophil count occurred. also, hemoglobin values remained consistent with known effects in patients who received ribavirin therapy. viral activity decreased in the majority of patients who relapsed with previous treatment, with 23 of 24 patients achieving at least a greater than 2-log reduction in hcv rna. six of 7 treatment-naïve patients achieved a similar reduction in viral load and 2 achieved undetectable hcv rna levels. kinetic data showed a linear relationship between dose and exposure independent of body weight, which may prompt future research to evaluate a fixed dose of pegifn-l. 57 larger, longer, controlled, and blinded studies of ifn-l as a viable treatment option in hcv are needed to define its place in therapy and benefits over existing ifn therapy. studies in other hcv genotypes are also needed. in addition, with the advent of protease inhibitors, more research will be necessary to evaluate how direct antivirals and il28b genotyping interact in guiding treatment decisions. adjunctive therapy with agents that induce or restore ifn-stimulated gene expression has recently been evaluated in patients with hcv. s-adenosylmethionine (same) given orally was evaluated in an open-label study in 24 patients with chronic hcv, genotype 1 who were considered nonresponders to previous ifn and ribavirin treatment. 41 same was administered at a dose of 800 mg twice daily in combination with pegifn-a2a (180 mg/kg weekly) and weight-based ribavirin (1000 mg if weight <75 kg and 1200 mg if weight !75 kg). the primary outcome was change in firstphase and second-phase viral decline. treatment response and ifn-stimulated gene expression were also evaluated after up to 72 weeks of treatment. results showed significant improvement in second-phase viral decline assessed at 2 weeks. svr was also evaluated; however, this study was not powered to detect differences in virologic response rates. furthermore, at the time of publication not all patients had reached 24 weeks post treatment, so the full effects on svr were not fully known. the addition of same showed greater induction of ifn-stimulated genes, including viperin, myxovirus resistance protein, and isg15, compared with control. adverse effects noted with same were mild and mostly related to gastrointestinal upset, likely as a result of lactose in the tablet preparation. 41 additional research is aimed at investigating structure-activity relationships, and preliminary pharmacokinetic studies on oral ifn inducers that act on tlrs in the treatment of hcv. 58 upper respiratory tract infection in the form of "the common cold" can be caused by a variety of viruses including rhinovirus, coronavirus, influenza, parainfluenza, respiratory syncytial virus, adenovirus, coxsackie, and echovirus families among others. 59 symptoms may include rhinorrhea, nasal obstruction, cough, fever, and sore throat. the disease is usually mild and self-limited, but several trials have addressed treatment or prevention of the common cold with therapeutic agents. ifns were once one of the most popular prospects for this purpose, but the minor benefit that was derived from them was counteracted by the adverse effects inflicted. 60 an early double-blind trial with ifn-a2b intranasal drops did demonstrate that with use for several days before experimentally induced rhinovirus infection, common cold symptoms were significantly fewer in study participants compared with placebo-drop users. 61 administration of the drops 4 times daily was superior to a higher dose given once daily at preventing infection. short-term use was well tolerated, but obviously it is not realistic for everyone to use intranasal drops 4 times daily throughout the entire cold season. in an attempt to prevent natural infection during the period of increased acute respiratory tract virus activity, a twice-daily nasal spray was studied in volunteers over 28 days. 62 there was a significant decrease in the number of rhinovirus infections noted, but not in any other types of viral respiratory tract infections including parainfluenza. adverse events with the ifn formulation were common in this placebocontrolled trial. during the first week alone, 20% of participants receiving ifn spray reported nosebleeds. this number increased to 41% by the end of the study. providing ifn prophylaxis for family members of those infected with common cold viruses is a more targeted approach to therapy. several studies have addressed the usefulness of ifn nasal sprays in this scenario. seven days of use did significantly reduce rhinovirus infections in 2 different trials when compared with placebo for both individuals (7.9% vs 15.5%) and their families (3.3% vs 33.3%, both p<.05), but not in 2 other studies when lower doses were given for a shorter 5-day course. [63] [64] [65] [66] overall, the intranasal dose of ifn needed to protect against upper respiratory tract infection appears to cause significant unwanted effects. 67 infection with coronavirus and respiratory syncytial virus has also been an object of investigation for ifn-a2b nasal sprays, but with little success. 68, 69 a study of intranasal human lymphoblastoid ifn-an1 (wellferon) suggested lower prophylactic activity for influenza than it did for rhinovirus. 70 because results of prophylactic trials with ifns for common cold viruses were not favorable, use in the treatment of infection seemed a logical application for this biological response modifier. although some benefit was originally seen with twice-daily ifn-a2b intranasal drops for treatment of experimentally induced rhinovirus, 71 no advantage was clear when an intranasal spray was used once daily for 5 days to treat natural infection. 72 increased rates of blood in the mucus were again noted for participants receiving the intervention, and the ifn group experienced more secondary complications requiring prescription of antibiotics. the investigators concluded that intranasal ifn was ineffective for treating the common cold and was associated with clinically significant side effects. similar trials with ifn-b-serine and ifn-g formulations, although initially positive, have shown equally disappointing clinical results. [73] [74] [75] [76] even though the prospects of further study on ifns for upper respiratory tract infection appear limited, one modern trial did demonstrate an added benefit of intranasal ifn-a2b in combination with an antihistamine (chlorpheniramine) and nonsteroidal anti-inflammatory drug (ibuprofen) at reducing common cold symptoms, showing that at least one group is still interested in studying the topic. 77 investigators have also recently begun research on an alternative therapeutic approach for rhinovirus infections using the ifn and tnf-a inducer, imiquimod. application of this intranasal cream in primates has shown promising results in terms of enhancing cytokine response, but human trials have not yet been published. 78 human papillomaviruses (hpvs) are now known to be the cause of cervical cancer and are also responsible for genital warts. hpvs are nonenveloped, double-stranded dna viruses that invade mucosal and epithelial tissues during sexual contact with an infected partner. it is estimated that more than 50% of the sexually active american population has been or will be infected with hpv at one point in their lives. 79 when hyperproliferation of infected cells occurs, this can lead to genital warts or cancer of the cervix, vagina, vulva, and penis, among others. there are more than 100 different types of hpv and approximately 40 of them infect genital mucosa. fifteen carcinogenic types of hpv have been identified, but 2 of them are associated with 70% of cervical cancers. 80 two vaccines have recently been introduced that prevent infection with these most common high-risk types of hpv, 16 and 18. 81 one of these vaccines can also induce protection against the most prevalent hpv types that have a low risk of malignancy, but instead cause genital warts: hpv-6 and hpv-11. hpv has the ability to persist in stratified epithelia for decades because of mechanisms that avoid immune eradication. ifn plays a large role in this cycle. 82 ifns are normally secreted by keratinocytes, but hpv reduces their expression. introduction of low-level ifn can actually increase early gene transcription and hpv replication, which may explain why use of the agent therapeutically has had mixed results. 83 overall outcomes have been positive more often for cases of genital warts than reduction of hpv lesions associated with cancers. a study comparing the in vitro activity of ifn-a2b and ifn-an3 on oncogenic hpv-16, hpv-18, and hpv-31b demonstrated that increasing concentrations did not always correlate with a stepwise inhibition of hpv replication. 84 meanwhile, a meta-analysis recently analyzed locally used and systemic ifn for genital warts. 85 seven randomized studies of ifn intralesional injection or topical gel met criteria for inclusion, and overall there was a benefit in complete response rates over placebo (44.4% vs 16.15%, relative risk 2.68, 95% confidence interval 1.79-4.02). however, there was no difference in outcomes for trials comparing systemic ifns with placebo. in comparison, clearance of genital and perianal warts occurs in 50% of patients with the topical ifn inducer imiquimod, usually after 8 to 10 weeks of use. 3 the 5% imiquimod cream (aldara) should be applied to affected areas 3 times a week for up to 16 weeks, whereas the newer 3.75% cream (zyclara) can be applied once daily for as little as 8 weeks to treat external genital warts caused by hpv. systemic ifn therapy may be useful when hpv affects areas of the body other than the anogenital region. 86 successful treatment with systemic pegifn-a and a topical retinoid has been reported for mucosal carcinomas from epidermodysplasia verruciformis, a genetic abnormality leading to persistent and widespread hpv infection of the skin. 87 recurrent respiratory hpv infection has also been effectively treated with ifn-a (12 of 18 patients), although it had no effect on viral load or replication. 88 a 20-year follow-up of patients treated with ifn-a for recurrent respiratory papillomatosis confirmed better response rates for hpv-6 than hpv-11, which had a higher likelihood of malignant transformation. 89 for recurrent conjunctival papilloma, topical plus systemic or intralesional ifn has been effective with partial excision. 90, 91 the rapid resolution of significant hpv-associated warts on the hand, foot, and face has also occurred in an hiv-infected patient on antiretrovirals while being treated for hepatitis c with pegifn-a2b and ribavirin. 92 case reports of treatment with the topical ifn inducer, imiquimod, have shown promise for its use in focal epithelial hyperplasia (heck disease), a rare disorder caused by specific types of hpv (13, 14, 32, and 55) affecting oral mucosa primarily in children. 93 in addition, imiquimod 5% cream has been used successfully in the treatment of plantar warts, a smoother, flatter manifestation of hpv-1, hpv-2, and hpv-4 on the foot. 94 of interest, the oral h2-antagonist cimetidine, along with reducing stomach acid, also induces production of ifn-g and il-2, which eliminates viral warts in some patients. 95 in the future the improved application of more effective topical ifns may become a reality, 96 which could provide a valuable treatment for hpv infections without the systemic side effects of current injectable formulations. adjuvants (adjuvare, latin for "to help") are substances that augment the immunogenicity of an antigen when mixed with the antigen for use in a vaccine. adjuvants (1) stimulate granuloma (which is a macrophage-rich mass), (2) enhance costimulatory signals, (3) stimulate nonspecific lymphocyte production, (4) prolong the antigen concentration in a site for lymphocyte exposure, 97 and (5) induce cytokines. 2, 98 research in vaccine development has shown that one of the most promising uses of ifns is as an adjuvant with specific antigens in prophylactic vaccines. 99 toporovski and colleagues 99 provide a current review of the use of ifn-a, ifn-b, ifn-g, and ifn-l in vaccine studies that focus primarily on murine, avian, porcine, and nonhuman species. regardless of the species, the use of ifns as adjuvants seems to improve the efficacy and safety of most vaccines while providing the immunomodulatory effect of stimulating the t-helper 1 response. in humans, ifn-a, predominantly produced by plasmacytoid dendritic cells, plays a large role in the body's immune response against viruses. 100 it induces plasma cell differentiation from b cells causing an increase in the serum level of influenzaspecific immunoglobulins, and channels antigen-presenting cells (apcs) to the site of infection. 101 most research on ifn-a adjuvant activity and its subsequent use in approved vaccines seems to indicate that it is a potent adjuvant. 99 when mixed with the influenza vaccine and injected intramuscularly, it is a highly effective adjuvant. 100 oromucosal administration of recombinant ifn-a, like that of natural oromucosal ifn production, has been shown to provide immunity against viral infection and tumor cell growth. 102 nonresponders low responders to a previous vaccine showed an improved immunoglobulin response with a recombinant ifn-a and hbv vaccine. 103 although research is also focused on the other classes of ifns as adjuvants, thus far they have not yielded results as promising as that of ifn-a. the use of ifn-b has yielded mixed results; ifn-g has been used primarily in dna vaccines; and even less is known about the use of ifn-l in vaccines. 99 nevertheless, the use of ifns as adjuvants shows great promise in augmenting vaccine efficiency, and should continue to be a top priority in the development of vaccines. ifns have been tested repeatedly against infectious diseases, but injections are used mostly for the treatment of viral hepatitis c and prevention of infections in patients with chronic granulomatous disease clinically. intralesional ifn and topical inducers are effective in reducing the manifestations of genital warts, but they do not eliminate cancer-causing hpv from the body. ifn has not proved to be consistently effective for treatment of respiratory tract infections from the common cold or influenza viruses, and prophylactic use is not currently feasible. the severity and quantity of adverse effects from systemic ifn therapy make it unattractive for many uses. several infections, including herpes simplex, herpes zoster, cytomegalovirus, and even viral hepatitis b have other effective pharmacologic treatments. ifn has been successfully used as a vaccine adjuvant, and further research may allow for its additional use for this application in the future. mim's medical microbiology goodman & gilman's the pharmacological basis of therapeutics role of endogenous biological response modifiers in pathogenesis of infectious diseases interferons at age 50: past, current and future impact on biomedicine immunoglobulins, vaccines or interferon for preventing cytomegalovirus disease in solid organ transplant recipients interferon: mechanisms of action and clinical value interferon-alpha therapy for chronic active epstein-barr virus infection: potential effect on the development of t-lymphoproliferative disease fda authorizes alferon ldo clinical study for treatment and prevention of influenza a multicenter, randomized, controlled trial of three preparations of low-dose oral alpha-interferon in hivinfected patients with cd41 counts between 50 and 350 cells/mm(3). division of aids treatment research initiative (datri) 022 study group vitro activity of interferon-alpha n3 (alferon n) against hiv-1. interscience conference on antimicrobial agents and chemotherapy nj: pegasysò (peg-interferon alfa-2a) nj: peg-intronò (peginterferon alfa-2b) a controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. the international chronic granulomatous disease cooperative study group long-term interferon-gamma therapy for patients with chronic granulomatous disease biological response modifiers as adjunct treatment for refractory localized mycobacterium avium complex infections fact sheet: world health organization viral cancers: world health organization surveillance for acute viral hepatitis-united states antiviral drugs (other than antiretrovirals) cytoskeleton rearrangement induced by tetraspanin engagement modulates the activation of t and nk cells natural killer cells: primary target for hepatitis c virus immune evasion strategies? interferon-alpha-induced trail on natural killer cells is associated with control of hepatitis c virus infection impaired effector function of hepatitis c virus-specific cd81 t cells in chronic hepatitis c virus infection association of genetic polymorphisms with interferon-induced haematologic adverse effects in chronic hepatitis c patients reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation pegylated interferons: what role will they play in the treatment of chronic hepatitis c? ribavirin improves early responses to peginterferon through improved interferon signaling boceprevir for previously treated chronic hcv genotype 1 infection boceprevir, an ns3 protease inhibitor of hcv antiviral effects and safety of telaprevir, peginterferon alfa-2a, and ribavirin for 28 days in hepatitis c patients antiviral activity of telaprevir (vx-950) and peginterferon alfa-2a in patients with hepatitis c sch 503034, a novel hepatitis c virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders telaprevir with peginterferon and ribavirin for chronic hcv genotype 1 infection telaprevir and peginterferon with or without ribavirin for chronic hcv infection the interferon inducible gene: viperin identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus the induction of type i interferon production in hepatitis c-infected patients a treatment algorithm for the management of chronic hepatitis b virus infection in the united states: 2008 update effect of ribavirin on hepatitis c viral kinetics in patients treated with pegylated interferon s-adenosyl methionine improves early viral responses and interferon-stimulated gene induction in hepatitis c nonresponders the interferon inducing pathways and the hepatitis c virus genetic variability of hepatitis c virus before and after combined therapy of interferon plus ribavirin a new era of hepatitis c therapy begins genetic variation in il28b predicts hepatitis c treatment-induced viral clearance il28b is associated with response to chronic hepatitis c interferon-alpha and ribavirin therapy genome-wide association of il28b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c genetic variation in il28b and spontaneous clearance of hepatitis c virus hepatitis c pharmacogenetics: state of the art in 2010 pharmacogenetics of hepatitis c therapy snipping away at hepatitis c. hepatology an il28b polymorphism determines treatment response of hepatitis c virus genotype 2 or 3 patients who do not achieve a rapid virologic response albinterferon alfa-2b dosed every two or four weeks in interferon-naive patients with genotype 1 chronic hepatitis c safety and antiviral activity of albinterferon alfa-2b dosed every four weeks in genotype 2/3 chronic hepatitis c patients albinterferon alfa-2b was not inferior to pegylated interferon-alpha in a randomized trial of patients with chronic hepatitis c virus genotype 2 or 3 albinterferon alfa-2b was not inferior to pegylated interferon-alpha in a randomized trial of patients with chronic hepatitis c virus genotype 1 phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis c virus infection design and optimisation of orally active tlr7 agonists for the treatment of hepatitis c virus infection the common cold alpha 2-interferon for the common cold intranasal interferon alpha 2 for prevention of rhinovirus infection and illness intranasal interferon-alpha 2b for seasonal prophylaxis of respiratory infection prophylactic efficacy of intranasal alpha 2-interferon against rhinovirus infections in the family setting prevention of natural colds by contact prophylaxis with intranasal alpha 2-interferon ineffectiveness of postexposure prophylaxis of rhinovirus infection with low-dose intranasal alpha 2b interferon in families intranasal interferon (rifn-alpha a, ro 22-8181) for contact prophylaxis against common cold: a randomized, doubleblind and placebo-controlled field study tolerance of one-month intranasal interferon prevention of experimental coronavirus colds with intranasal alpha-2b interferon the efficacy of intranasal interferon alpha-2a in respiratory syncytial virus infection in volunteers wellferon") prophylaxis against rhinovirus and influenza virus in volunteers intranasal interferon-alpha 2 treatment of experimental rhinoviral colds intranasal recombinant alfa-2b interferon treatment of naturally occurring common colds tolerance and efficacy of intranasal administration of recombinant beta serine interferon in healthy adults ineffectiveness of recombinant interferon-beta serine nasal drops for prophylaxis of natural colds interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers recombinant human interferongamma as prophylaxis against rhinovirus colds in volunteers combined antiviral-antimediator treatment for the common cold immune responses induced by intranasal imiquimod and implications for therapeutics in rhinovirus infections genital hpv infection-fact sheet epidemiologic classification of human papillomavirus types associated with cervical cancer update on human papillomavirus vaccines: life saver or controversy magnet? clin microbiol newslett human papillomaviruses and the interferon response interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (irf)-1 efficacy of two commercial preparations of interferon-alpha on human papillomavirus replication interferon for the treatment of genital warts: a systematic review successful treatment of disseminated human papillomavirus infection with pegylated interferon and ribavirin epidermodysplasia verruciformis with multiple mucosal carcinomas treated with pegylated interferon alfa and acitretin human papillomavirus, viral load and proliferation rate in recurrent respiratory papillomatosis in response to alpha interferon treatment use of interferon-alpha in recurrent respiratory papillomatosis: 20-year follow-up topical alpha-interferon in recurrent conjunctival papilloma resolution of recurrent conjunctival papilloma after topical and intralesional interferon alpha2b with partial excision in a child treatment of human papillomavirus with peg-interferon alfa-2b and ribavirin treatment of focal epithelial hyperplasia with topical imiquimod: report of three cases plantar warts treated with an immune response modifier: a report of two cases cimetidine treatment for viral warts enhances il-2 and ifn-gamma expression but not il-18 expression in lesional skin topical delivery of interferon alpha in human volunteers and treatment of patients with human papillomavirus infections new york: w.h. freeman and co advances in vaccine adjuvants interferons as potential adjuvants in prophylactic vaccines plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin 6 adjuvant activity of interferon alpha: mechanism(s) of action oromucosal interferon therapy: marked antiviral and antitumor activity randomized comparative trial of interferon-alpha versus placebo in hepatitis b vaccine non-responders and hyporesponders key: cord-003158-mhlqnj52 authors: wang, qi; hagedorn, curt; liu, shuanghu title: adapted hcv jfh1 variant is capable of accommodating a large foreign gene insert and allows lower level hcv replication and viral production date: 2018-07-13 journal: int j biol sci doi: 10.7150/ijbs.27411 sha: doc_id: 3158 cord_uid: mhlqnj52 infectious hcv carrying reporter genes have further applications in understanding the hcv life cycle including replication, viral assembly and release. in this study, a full-length 3039bp lacz gene was inserted into the derivative of jfh1-am120 to develop an additional reporter virus. the results showed that the recombinant reporter virus jfh1-am120-lacz can replicate and produce lower titers of infectious virus. however, insertion of the lacz gene in the c-terminal region of the ns5a in hcv jfh1-am120-lacz decreased viral replication and dramatically impaired the production of infectious viral particles. moreover, the jfh1-am120-lacz reporter virus lost the lacz gene after serial passage. nevertheless, the jfh1-am120-lacz reporter virus displayed the entire life cycle of hcv, from replication to production of infectious virus, in huh7.5 cells. this study demonstrates that the ns5a region of hcv jfh1-am120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site. hepatitis c virus (hcv) is a member of the flaviviridae family and is a single-stranded positive-sense rna. it infects approximately 3% of the worldwide population (1, 2) . hcv can cause acute and chronic hepatitis, and lead to fibrosis, cirrhosis and hepatocellular carcinoma (3) . although much progress has been made in developing highly effective hcv antivirals, an effective vaccine has yet to be developed (4, 5) . an internal ribosome entry site (ires) of hcv drives rna translation to produce a polyprotein of approximately 3,000 amino acids (aa) that encodes both structural and nonstructural proteins (6) (7) (8) . the development of hcv replicon systems advanced the understanding of hcv replication, viral protein processing and viral-host cell interactions (9, 10 ). an establishment of an infectious hcv cell culture system using a genotype 2a isolate (jfh1 strain) of hcv and huh-7 cells was a major achievement (11) (12) (13) . in this system, infectious hcv particles are secreted in an envelope glycoproteindependent manner and enable a variety of questions to be answered regarding hcv biology and cell infection. however, virus titers of jfh1 released from infected cells are relatively low and limit some applications of this system. studies have identified adaptive or compensatory mutations that enhance infectious virus particle production from either wild-type jfh1 or intergenotypic chimeras (13) (14) (15) (16) (17) (18) (19) . non-structural protein 5a (ns5a) of hcv is a phosphoprotein, which has 1398 nt and a calculated molecular mass of 49 kda. it migrates as 56-and 58-kda species in sds-polyacrylamide gels and is ivyspring international publisher organized into three domains: i (aa28-213), ii (aa250-342), and iii (aa356-447). domain i coordinate a single zinc atom, and domains ii and iii are less well characterized but are important for rna replication and/or virion assembly (20) (21) (22) (23) . previous studies have demonstrated that the hcv jfh1 ns5a c-terminal is a flexible region which is capable of accommodating foreign gene inserts (such as egfp, 720bp and rennilla luciferase [rluc] , 930bp) and still permit hcv replication and viral production (18, 19, (24) (25) (26) (27) (28) (29) . vesicular stomatitis virus (vsv) is negative-stranded rna virus that has been used as a gene expression vector. it can accommodate a gene insert as large as 5955 bases (30) . one of the more common reporter genes, the e. coli lacz gene, is 3039 bp in length and encodes the protein β-galactosidase (β-gal). the expression of lacz can be identified by staining with x-gal substrate to produce a blue color as a measure of enzyme activity (31) (32) (33) . human hepatoma cells (huh7) can express β-galactosidase using adenoviral vectors carrying the lacz gene (34) . it is unclear whether the hcv jfh1 ns5a c-terminal is capable of accommodating a large foreign gene such as lacz, and still permit hcv replication and production of infectious virus. in a previous study we demonstrated that there was no viral production after insertion of egfp (720bp) and rennilla luciferase (rluc; 930bp) into the c-terminus of ns5a of wild type jfh1 (18) . however, a higher titer of hcv reporter virus was produced after inserting egfp and rluc into ns5a c-terminus of an adaptively mutated jfh1 strain designated jfh1-am120 (18) . in this study, we used the jfh1-am120 as a vector to explore if infectious reporter virus would be produced following insertion of lacz gene that was three time larger than rluc, into the ns5a c-terminus. the result showed that the ns5a region of hcv jfh1-am120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site. human hepatoma cells, huh7.5, were generously provided by charles rice (35) (rockefeller university) and maintained in dulbecco's modified eagle's medium (dmem) (invitrogen) supplemented with 100 u/ml of penicillin, 100 µg/ml of streptomycin, nonessential amino acids, and 10% fetal bovine serum (fbs) (invitrogen) at 37˚c in 5% co 2 . all experiments described in this study were performed using these cells. the monoclonal antibody to the ns5a protein (9e10) was a gift from charles rice (13) . goat anti-mouse conjugated with horseradish peroxidase (hrp) (sigma), goat anti-mouse igg conjugated with alexa fluor 594 (invitrogen), x-gal (genlantis, cat#a10300k), mammalian β-gal activity assay kit (thermofisher, #75707), renilla luciferase assay system (promega) were obtained commercially. plasmids constructs were based on jfh-am120 that has adaptive mutations previous reported (18) and jfh-am120 based on the consensus sequence of hcv pjfh1, which was kindly provided by dr. wakita (11) . jfh-am120-egfp and jfh-am120-rluc had been described previously (18) . the egfp and renilla luciferace (rluc) genes were inserted in frame into a unique rsrii site located in the amino acid 398 codon of jfh1-am120. jfh-am120-lacz was also constructed by inserting the lacz gene (3039bp) into the rsrii site of the ns5a c-terminal region of jfh-am120. the lacz fragment was amplified from vector psv-β-galactosidase control (promega, e1081) using the primers lacz-foward (5'-tctcggtc cgatcgatcccgtcgttttaca-3'), lacz-reverse (5'-cttacggaccgcctttttgacaccagaccaa ct-3'). the new construct was sequenced and the correct full-length clone, jfh-am120-lacz, was used for the subsequent experiments. to generate full-length genomic rna, pjfh1-am120-lacz and controls of pjfh1-am120, pjfh1-am120-egfp, pjfh1-am120-rluc and were linearized at the 3' end of the hcv cdna with xbai. the linearized plasmid dna was purified and used as a template for t7 in vitro transcription (megascript; ambion, austin, tx). in vitro transcribed rnas were transfected into cells by electroporation as described previously (29, 36) . briefly, trypsinized cells were washed twice and resuspended with serum-free opti-mem (invitrogen) at the concentration of 1x10 7 cells per ml. ten micrograms of rna were mixed with 0.4 ml of the cells in a 4-mm cuvette. a bio-rad gene pulser system was used to deliver a single pulse at 270v and 960 µf and the cells were plated in t75 costar flasks (corning). transfected cells were cultured in complete dmem for the indicated times. cells were passaged every three days and the presence of hcv in the corresponding supernatants was determined by immunofluorescence assays (ifa) for the ns5a protein or x-gal staining. cells infected by hcv or hcv reporter virus were washed with pbs, fixed with 4% paraformaldehyde, and permeabilized with 0.2% triton x-100. fixed cells were blocked with 1% bovine serum albumin and 1% normal goat serum in pbs. ns5a protein was detected in cells with an ns5a-specific monoclonal antibody and visualized with a secondary goat anti-mouse antibody conjugated with alexa fluor 594 (invitrogen, 1: 1,000 dilutions). cover slips were mounted onto slides with dapi (vector labs) and immunostaining visualized by fluorescence microscopy (nikon e400). the titer of infectious hcv was determined by the number of cell foci staining for ns5a as previously described in detail (12) . briefly, cell supernatants were serially diluted 10-fold in complete dmem. the supernatant was used to infect 5 × 10 4 naive huh 7.5 cells per well in 24-well plates. the inocula were incubated with cells for three hours at 37 o c and then supplemented with fresh complete dmem. the level of hcv infection was determined three days postinfection by immunofluorescence staining for hcv ns5a and ns5a-rluc, or x-gal staining for ns5a-lacz and directly visualized the egfp for ns5a-egfp. the viral titer is expressed as focus-forming units per milliliter of supernatant (ffu/ml). x-gal positive cells were identified using an x-gal staining assay kit (genlantis, cat#a10300k) and visualized by bright field microscopy (nikon) following the manufacturer's instructions. briefly, 5 × 10 4 naive huh 7.5 cells per well were seeded in 24-well plates and were transfected with rna of jfh-am120-lacz or infected with the supernatant collected from transfected cells of rna of jfh-am120-lacz or controls. 72 hours post-transfection or infection, cells were fixed with formaldehydeglutaraldehyde buffer for 15 minutes and stained the cells with x-gal staining solution for three hours. blue stained cells were identified by bright field microscopy (nikon e400). the viral titer of jfh-am120-lacz was expressed as focus-forming units per milliliter of supernatant (ffu/ml). colocalization of x-gal pictures and immunofluorescence images were processed by photoshop cc software. the β-galactosidase activity was measured using a mammalian β-galactosidase assay kit (thermofisher, #75707) and microplate reader (bio-tek) following the manufacturer's instructions. briefly, huh7.5 cells were transfected with rna of jfh-am120-lacz and controls of jfh-am120, jfh-am120-egfp and jfh-am120-rluc and the cells were incubated for 72 hours. then cells were washed once, 100μl of β-galactosidase assay reagent was added to each well, and cells were incubated for 30 minutes at 37°c. reactions were stopped by adding 100 μl of β-galactosidase assay stop solution to each well and absorbance at 405nm was measured. the hcv-transfected huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mm tris-hcl, ph 7.5, 150 mm sodium chloride, 1% nonidet p40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (roche). the total protein for each sample was measured with a standard protein assay (bio-rad). twenty-five micrograms of total protein for each sample was analyzed by 10% sds-page and transferred to nitrocellulose membranes. the membranes were blocked by incubating them with 5% skim milk. hcv proteins were detected with monoclonal antibodies specific to ns5a, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin g (bio-rad) and a chemiluminescence substrate (pierce). β-actin was used as a control and was detected with an anti-β-actin monoclonal antibody (sigma). total rna was extracted with trizol (invitrogen) and hcv rna was measured by qpcr analysis as described previously (37) . the relative quantity of hcv rna in control and hcv samples was calculated with the comparative ct (cycling threshold) method. a reference gene (β-actin) was used as the control. huh7.5 cells were transfected with the rna of jfh1-am120-lacz and controls of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc and the cells were passaged at every three days for a total of 15 days. the total rna in the huh7.5 cells were extracted with trizol reagent (invitrogen). the rna was reverse transcribed using superscript iii reverse transcriptase (invitrogen) and random primers. the resulting cdna was used as a template for subsequent pcr with platinum® pfx dna polymerase (invitrogen) and the following primers: jfh-6815-for, 5'-ttaattcctatgctgtcgggtcc cagct-3'; jfh-7765-rev, 5'-gtgcgttgtacagta caccttgttatgg-3'. the pcr amplicons (950bp for ns5a wild type) were analyzed by 1% agarose gel electrophoresis and sequenced using standard methods. the mean and standard deviation of the mean for data were determined and the results for different experimental conditions were compared using the students t -test. the p value<0.05 was considered to be significant. a previous study (14) demonstrated hcv jfh1 wild type is capable of producing infectious virus but the titer is very low. it also cannot bear the foreign genes of egfp and rluc to produce viable reporter viruses (18) . therefore, we used the adaptively mutated jfh1 strain, jfh1-am120, that produces higher titers of infectious virus as a vector for these studies (18) . on this strain, egfp and renilla luciferace (rluc) genes were inserted in frame into a unique rsrii site located in the amino acid 398 codon of ns5a and reporter viruses of jfh1-am120-egfp and jfh-am120-rluc were produced in huh7.5 (18) . the jfh1-am120-lacz was constructed by in frame inserting full lacz gene fragment (3039bp) to the same rsrii site of jfh-am120 ns5a c-terminal region and the lacz fragment was amplified from vector psv-β-galactosidase control (promega, e1081) using primers lacz-f(5'-tctcggtccgatagatcc cgtcgttttaca-3') and lacz-r(5'-cttacggacc gcctttttgacaccagaccaact-3'). the new clone was sequenced and the correct full-length clone was chosen for the subsequent experiments. all the constructs used in this study are shown in fig. 1 . in this study the lacz gene (3039 bp) was inserted into the ns5a c-terminal region of jfh1-am120, designated jfh1-am120-lacz. jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc were used as controls. rnas were transcribed from plasmids of jfh1-am120, jfh1-am120-egfp, jfh1-am120-rluc and jfh1-am120-lacz and were transfected to huh7.5 cells to determine whether cells transfected with the rnas from each plasmid expressed hcv proteins and hcv rna. three days after rna transfection of cells, wild-type ns5a (58kd) and the ns5a-egfp (84kd), ns5a-rluc (92kd) and ns5a-lacz (169kd) fusion protein with the predicted molecular mass were detected in cell lysate by western blotting (fig. 2a) . however, the band of ns5a-lacz protein was obviously weaker than ns5a and other fusion proteins. the hcv rnas were also quantified by qpcr. the results showed that hcv rna amounts (fig. 2b ) correlated with changes in protein levels by western blotting. these results indicated that the replication level of jfh1-am120-lacz was significantly decreased after insertion of lacz gene into the ns5a c-terminal of jfh1-am120. however, jfh1-am120-lacz can still replicate in huh7.5 cells even though the lacz gene is 3039bp in length. fig. 1 . schematic representation of hcv constructs used in this study. jfh1-am120, jfh1-am120-rluc and jfh1-am120-egfp were generated in our previous study (18) . egfp and renilla luciferace (rluc) genes were inserted in frame into a unique rsrii site located in the amino acid 398 codon of ns5a of jfh1-am120. jfh-am120-lacz was generated by in frame insertion of full lacz gene, which was amplified from vector psv-β-galactosidase control (promega, e1081) to the same rsrii site. the detailed protocol was described in material and method. jfh-am120-lacz and jfh-am120, jfh-am120-egfp and jfh-am120-rluc, the total rna was extracted from cells and qpcr analysis of hcv rna was performed. experiments were performed three times and the data presented as the mean ± sd (* p< 0.05, * * p< 0.01). the lacz gene encodes β-galactosidase (β-gal) and the protein levels of this gene can be determined via staining with x-gal substrate and measuring the activity of the β-galactosidase enzyme. the protein levels and enzyme activity of β-galactosidase should be detected in jfh1-am120-lacz infected huh7.5 cells. to verify this statement, the rna of jfh1-am120-lacz was transfected into huh7.5 cells. rnas of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc were used as controls. three days post transfection, β-galactosidase activity was measured using a mammalian β-galactosidase assay kit (thermofisher). the results showed only the sample of jfh1-am120-lacz had detectable β-galactosidase activity. all other transfected rnas were negative (fig. 3a ). using the same method, additional cells were transfected with jfh-am120-lacz rna or control rnas and fixed with paraformaldehyde, stained using an x-gal staining assay kit (genlantis) and visualized the cell with blue color under bright field microscope. blue cells were only seen in cells transfected with jfh1-am120-lacz rna (fig.3b) and there was not any on the other three transfected rnas (data not shown). these results confirmed the jfh1-am120-lacz could replicate in huh7.5 cells. to examine the concurrent expression of hcv ns5a and the β-galactosidase fusion protein in huh7.5 cells after transfection of jfh1-am120-lacz rna, the colocalization of the ns5a protein and β-galactosidase protein were determined by staining with ns5a immunofluorescence assays and x-gal three days after transfection. cells were fixed with 4% paraformaldehyde and immunostained with anti-ns5a antibody (red) and nuclei counterstained with dapi (blue). images were taken by immunofluorescence microscopy (200x). the coverslip was then washed and x-gal staining was performed, followed by visualizing and imaging by bright field microscopy (200x). colocalization assay was performed with photoshop cc software (fig.3c) . images showed that ns5a positive cells (red) were also with the blue color of x-gal staining. this result provided evidence that fusion protein of ns5a and β-galactosidase can be co-expressed in huh7.5 cells after transfection of jfh1-am120-lacz rna. to determine if transfection of huh 7.5 cells with jfh1-am120-lacz rna resulted in production of infectious viral particles, we inoculated naïve huh 7.5 cells with supernatants collected at 6 th days post-transfection with jfh1-am120-lacz rna. supernatants collected from the cells transfected with rnas from jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc were used as controls. at three days post-infection, the cells infected with the supernatant of jfh-am120-lacz were stained by x-gal and blue color cells were detected, while all of the others were negative. egfp positive cells were only found in jfh-am120-egfp infected cells. jfh1-am120 and jfh1-am120-rluc infected cells expressed ns5a and ns5a-rluc, as determined by the immunofluorescence assay (ifa). the viral titers are expressed as focus-forming units per milliliter of supernatant (ffu/ml). the titer of jfh1-am120, jfh1-am120-egfp, jfh1-am120-rluc and jfh1-am120-lacz were 5.5x10 5 ffu/ml, 4.5x10 4 ffu/ml, 4.2x10 4 ffu/ml and 0.98x10 2 ffu/ml, respectively. the viral titers of jfh1-am120-egfp and jfh1-am120-rluc were about 10 times lower than jfh-am120, however, the titer of jfh1-am120-lacz was dramatically lower (more than 5000 times) compared to jfh-am120 (fig.4) . these results demonstrated that the insertion of lacz in ns5a c-terminal region of jfh1-am120 significantly impaired the production of infectious viral particles. this result provided direct evidence that long exogenous genes can influence the capability of infectious hcv virus production at difference levels with a gene length dependent manner. fig. 3 . analysis of ns5a-lacz activity followed rna transfection of jfh1-am120-lacz. (a) huh7.5 cells were transfected with the rna of jfh1-am120-lacz and controls of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc as described in methods. three days post-transfection, the absorption peaks of β-galactosidase activity were measured using a bio-tec plate reader at 405 nm. the values were relative to jfh1-am120-lacz. experiments were performed three times and the data presented as the mean ± sd. (b) three days post-transfection of huh7.5 cells in 24-well plates with rna of jfh1-am120-lacz, cells were fixed with 4% paraformaldehyde followed x-gal staining. cover slips were visualized and images were taken (100x) by bright field microscopy. experiments were performed two times and representative results are shown. (c) co-localization of ns5a and β-galactosidase. three days post-transfection of huh7.5 cells in 24-well plates with rna of jfh1-am120-lacz, cells were fixed with 4% paraformaldehyde and were immunostained with anti-ns5a antibody (red) and nuclei were counterstained using dapi (blue). images were taken by immunofluorescence microscopy (200x). then the coverslip was washed and were processed with x-gal staining followed visualizing and imaging by bright field microscopy (200x). colocalization assay was performed by photoshop cc software. experiments were performed three times and representative results are shown. fig. 4 . infectivity assay of virus particles followed rnas transfection of jfh1-am120-lacz and controls. the rna of jfh1-am120-lacz and controls of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc were electroporated into huh-7.5 and the infectivity titers in the cultured supernatants at the 6th day were measured (described in material and method). the viral titer is expressed as focus-forming units per ml of supernatant (ffu/ml) as determined by the average number of ns5a-positive foci detected by immunofluorescence for ns5a (mock, jfh-am120 and jfh-am120-rluc), or directly visualized egfp positive cells(jfh1-am120-egfp) and detected blue color cells after x-gal staining (jfh1-am120-lacz). assays were performed three times and the data are presented as mean ± standard deviation. (** p< 0.01, *** p< 0.001) fig. 5 . kinetics assay of the jfh1-am120-lacz hcv reporter after multiple passages. the cells transfected with the rna of jfh1-am120-lacz were passaged at every three days for a total of 15 days. supernatants collected were designated p1 to p5. the double titrations were carried out by x-gal staining for β-galactosidase and immunofluoresence staining for ns5a protein (see materials and methods). experiments were performed three times and the data presented as the mean ± sd. our data show that jfh1-am120-lacz can replicate and produce a low titer of jfh1-am120-lacz hcv reporter virus in huh7.5 cells. however, the genetic stability of a reporter virus is essential for its further application. to explore the durability of the jfh1-am120-lacz reporter in huh7.5 cells subculture, a kinetics assay was performed by passaging the cells every three days for a total of 15 days. supernatants were collected and designated p1 to p5. double titrations were carried out by x-gal staining for β-galactosidase and immunofluorescence staining for ns5a protein (see materials and methods). the titer measured by these two methods was different. the titer after ns5a antibody staining was increased from p1 to p5. the titer after x-gal staining was increase from p1 to p2 then decreased from p2 to p3 and reached zero at p4 and p5 (fig.5) . the discrepancy of these result indicated the lacz gene may be deleted and one strain lost lacz gene became a dominant and produced a higher titer virus. to confirm these results, western blotting was carried out for cells transfected with jfh1-am120-lacz rna and control rnas at p1 and p5. the results show no band of ns5a-lacz (163kd) in p5 cells and the ns5a fragment reverting to the size of wild-type ns5a (56 and 58kd) (fig. 6a ) compared to the much larger ns5a-lacz fusion protein in p1 cells ( fig.2a) . ns5a-egfp and ns5a-rluc did not change after the same number of passages ( fig.2a & 6a) . to verify these results, rt-pcr was performed on p1 and p5 cells and agarose gel analysis showed the predicted size pcr products ( fig. 6b and 6c ). the size of the ns5a-wt, ns5a-egfp and ns5a-rluc pcr products did not change at p1 and p5. however, the size of ns5a-lacz pcr product was reverted to the same size of the ns5a-wt product at p5. this pcr product was sequenced and only six nucletitides of the lacz gene remained, while the other nucleotides of the lacz gene were absent, providing direct evidence for the loss of the lacz gene with multiple passaging of cells. on the fig. 2a and 6b , a weak band of similar size to wild-type ns5a were seen in the lacz lane. this suggests that the deletion of the lacz gene may occur early in the first passage. these results indicated the jfh1-am120-lacz reporter virus was unstable after progressive cell culture. some rna viruses can tolerate the insertion of relatively large exogenous genes without disrupting replication and the production of functional recombinant viruses (30, 38) . vesicular stomatitis virus (vsv), has been used as a gene expression vector and can accommodate the insertion of a foreign gene as large as 5955 bp (30) . previous reports have shown that the hcv adapted jfh-1 strain can tolerate the insertion of exogenous genes less than 1000 bp, such as egfp (720bp) and rluc(930bp), into the c terminus of ns5a without disrupting viral replication and the production of infectious virions (18, 19, 25, 26, 28) . a reporter gene as large as the lacz gene (3039bp), has not been successfully inserted into the hcv genome with production of infectious virions. our results demonstrate that the lacz reporter gene can be inserted into the ns5a c-terminus of hcv jfh1-am120 and will express the predicted ns5a-lacz fusion protein, which can be detected by western blotting three days after rna transfection of cells. however, compared to the control vectors of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc, there was an obvious decrease in the expression of the ns5a fusion protein ( fig. 2a) . β-galactosidase was also detected in these cells by several measures (fig.3 ) and the supernatants of these cells produced infectious virus of jfh1-am120-lacz (fig.4) . however, the infectious virus titer was much lower, 0.98 x10 2 ffu/ml compared to 5.5x10 5 ffu/ml of parent jfh1-am120 virus. in addition, the titer of infectious hcv jfh1-am120-lacz progressively decreased with serial passaging of cells, with the eventual loss of x-gal positive cells (fig.5) . this observation is consistent with western blot and ns5a pcr analysis indicating the loss of the lacz reporter gene (fig.6) . the reason for the loss of the inserted lacz reporter gene is unclear. there may be three possible mechanisms on it. first, the hcv genome rna might have the capability of repairing itself and removing exogenous genes. second, the hcv ns5a gene has a limit to it's carrying capacity for exogenous genes, and lacz is too large for stable expression. third, the combined effects of viral rna repair, expression stability and replication might result in lacz depletion in some strains, which could become the dominant strains during subsequent replication process, and this is consistent with the basic principle of natural adaptive mutation selection. 6 . stability assay of jfh1-am120-lacz and controls followed the rna transfection. (a) huh7.5 cells were transfected with the rna of jfh1-am120-lacz and controls of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc and the cells were passaged at every three days for a total of 15 days. the cells of passage 1 ( fig.2a) and passage 5 were lysed for western blotting using anti-ns5a and anti-β-actin bodies as described in methods.the experiment was performed twice and representative example are shown. (b & c) detection of hcv rna. huh7.5 cells were transfected with the rna of jfh1-am120-lacz and controls of jfh1-am120, jfh1-am120-egfp and jfh1-am120-rluc and the cells were passaged at every three days for a total of 15 days. the total rna was extracted and rt-pcr was performed described as in methods. the pcr products were analyzed by 1% agarose gel electrophoresis. experiments were performed two times and a representative experiment is shown (b showed passage 1 st result and c showed passage 5 th result). although hcv-egfp and hcv-rluc reporter viruses we have described previously were quite stabile with repeated passages, resulted in relatively high titers of infectious virions and have been used in a number of studies (19, (39) (40) (41) , it is still unclear the capacity of hcv to accommodate large foreign genes. the production of infectious jfh1-am120-lacz virions reported here demonstrate that much larger gene inserts can be engineered into the c terminus of ns5a of jfh1-am120 for other applications and approaches. however, the instability of this reporter virus and the loss of lacz gene are likely to limit its use. nevertheless, the fact that a reporter gene up to 3039bp in size can be inserted into the c terminus of ns5a indicates that additional studies can be performed to guide further engineering of jfh1-am120 in the research of hcv. global burden of hepatitis c: considerations for healthcare providers in the united states clinical practice. chronic hepatitis c infection hepatitis c virus to hepatocellular carcinoma sofosbuvir and velpatasvir for hcv genotype 1, 2, 4, 5, and 6 infection oral direct-acting agent therapy for hepatitis c virus infection: a systematic review turning hepatitis c into a real virus structural biology of hepatitis c virus overview of hepatitis c virus genome structure, polyprotein processing, and protein properties efficient initiation of hcv rna replication in cell culture replication of subgenomic hepatitis c virus rnas in a hepatoma cell line production of infectious hepatitis c virus in tissue culture from a cloned viral genome robust hepatitis c virus infection in vitro complete replication of hepatitis c virus in cell culture cell culture adaptation of hepatitis c virus and in vivo viability of an adapted variant persistent hepatitis c virus infection in vitro: coevolution of virus and host robust production of infectious viral particles in huh-7 cells by introducing mutations in hepatitis c virus structural proteins advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis c virus a cell culture adapted hcv jfh1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses direct visualization of hepatitis c virus-infected huh7.5 cells with a high titre of infectious chimeric jfh1-egfp reporter virus in three-dimensional matrigel cell cultures the effects of ns5a inhibitors on ns5a phosphorylation, polyprotein processing and localization identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns5a protein the ns5a protein of hepatitis c virus is a zinc metalloprotein structure of the zinc-binding domain of an essential component of the hepatitis c virus replicase insertion of green fluorescent protein into nonstructural protein 5a allows direct visualization of functional hepatitis c virus replication complexes compensatory mutations in ns3 and ns5a proteins enhance the virus production capability of hepatitis c reporter virus monitoring the antiviral effect of alpha interferon on individual cells insertion and deletion analyses identify regions of non-structural protein 5a of hepatitis c virus that are dispensable for viral genome replication analysis of hepatitis c virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes measuring antiviral activity of benzimidazole molecules that alter ires rna structure with an infectious hepatitis c virus chimera expressing renilla luciferase genetically modified vsv(nj) vector is capable of accommodating a large foreign gene insert and allows high level gene expression expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells characterization of the nuclear localization signal and subcellular distribution of hepatitis c virus nonstructural protein ns5a augmentation of antitumor effects of p53 gene therapy by combination with hdac inhibitor constitutive activation of nf-kappab in human hepatocellular carcinoma: evidence of a cytoprotective role highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication enhancement of hepatitis c virus rna replication by cell culture-adaptive mutations rna-sequencing analysis of 5' capped rnas identifies many new differentially expressed genes in acute hepatitis c virus infection coronaviruses as vectors: stability of foreign gene expression interferon-alpha inducible protein 6 impairs egfr activation by cd81 and inhibits hepatitis c virus infection interferon lambda 4 expression is suppressed by the host during viral infection activation of perk-nrf2 oncogenic signaling promotes mdm2-mediated rb degradation in persistently infected hcv culture we thank dr. t. wakita for providing the plasmid containing the hcv jfh1 plasmid. we also thank dr. c.m. rice for providing huh7.5 cells and the anti-ns5a monoclonal antibody. this work was supported in part by nih grant ca176130 to c.h.h. the authors have declared that no competing interest exists. key: cord-264936-3posyr5n authors: mohammadzadeh, sara; khabiri, alireza; roohvand, farzin; memarnejadian, arash; salmanian, ali hatef; ajdary, soheila; ehsani, parastoo title: enhanced-transient expression of hepatitis c virus core protein in nicotiana tabacum, a protein with potential clinical applications date: 2014-11-24 journal: hepat mon doi: 10.5812/hepatmon.20524 sha: doc_id: 264936 cord_uid: 3posyr5n background: hepatitis c virus (hcv) is major cause of liver cirrhosis in humans. hcv capsid (core) protein (hcvcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. although invention of transgenic (stable) tobacco plants expressing hcvcp with proper antigenic properties was recently reported, no data for “transient-expression” that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for hcvcp. objectives: the purpose of this study was to design a highly codon-optimized hcvcp gene for construction of an efficient transient-plant expression system for production of hcvcp with proper antigenic properties in a regional tobacco plant (iranian jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. materials and methods: a codon-optimized gene encoding the kozak sequence, 6xhis-tag, hcvcp (1-122) and kdel peptide in tandem (from nto c-terminal) was designed and inserted into potato virus-x (pvx) and classic pbi121 binary vectors in separate cloning reactions. the resulted recombinant plasmids were transferred into agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. the effect of gene silencing suppressor p19 protein derived from tomato bushy stunt virus on the expression yield of hcvcp by each construct was also evaluated by co-infiltration in separate groups. the expressed hcvcp was evaluated by dot and western blotting and elisa assays. results: the codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced gc content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of “ggtaag” splice site in native hcvcp. blotting assays via specific antibodies confirmed the expression of the 15 kda hcvcp. the expression level of hcvcp was enhanced by 4-5 times in p19 co-agroinfiltrated plants with better outcomes for pvx, compared to pbi121 vector (0.022% versus 0.019% of the total soluble protein). the plant-derived hcvcp (phcvcp) could properly identify the hcvcp antibody in hcv-infected human sera compared to escherichia coli-derived hcvcp (ehcvcp), indicating its potential for diagnostic/immunization applications. conclusions: by employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of hcvcp in tobacco with proper antigenic properties could be possible. hepatitis c virus (hcv), the major cause of blood-borne chronic hepatitis with potential progression to cirrhosis, has infected around 170 million people globally (1) . due to heterogeneity of hcv proteins, their high mutation rates and complex pathogenesis, no approved vaccine for human application against this viral infection is available to date (2, 3) . recognition of conserved epitopes in hcv proteins and advancements in the formulation of vaccines in novel modalities, however, have led to the placement of a few hcv vaccines in the pipeline of human clinical trials in recent years (4, 5) . hcv holds a single-stranded positive-sense rna genome which encodes for three structural (core protein and envelope proteins e1 and e2) and six nonstructural proteins (4) . among the hcv proteins, core (hcvcp) is the most conserved hcv antigen, and hence, a good candidate to be considered for hcv vaccine formulations (4) (5) (6) . accordingly, application of even isolated hcvcp-ctl epitopes (8-10 amino acids) in the context of synthetic multi-epitope hcv vaccines has also been reported (7) (8) (9) . besides, anticore antibodies are the first to be raised after the onset of infection, a property that has provided an important diagnostic value for this protein and has located it among antibody-capturing antigens in commercially available serological assays for hcv diagnosis (10) . in addition, nucleotide sequence of hcvcp is supposed to encode for other alternated frame-shift proteins (arfps) with important pathogenic roles in chronic hcv infection and cirrhosis (4, 11) . therefore, to fulfill different diagnostic, research and therapeutic demands, production of hcvcp in various expression systems has been addressed (4) (5) . however, since the c-terminal hydrophobic region of hcvcp exerts immune-suppressive effects (4, 12) , its first 120 n-terminal residues, the hydrophilic region (hcvcp n-120), which contains both nuclear localization signals (nls) (13) and most of the conserved b and t cell epitopes, is usually employed for different diagnostic (10, 14) and vaccine applications (15) (16) (17) . plants have an extensive potential to provide safe and inexpensive sources of biopharmaceutical and vaccination proteins (18) . currently, several plant-derived viral proteins such as hepatitis b surface antigen (19) and norwalk virus capsid protein (20) are in vaccination clinical trials. recently, invention of transgenic tobacco plants expressing hcvcp stably at t0 and t1 generations (21) or directing the expression of its 143-amino acid nterminal in tobacco chloroplasts (22) were reported. although these prior reports proved the proper antigenic structure of the plant-derived hcvcp for diagnostic purposes and indicated its full functionality to react with poly/monoclonal anti-core antibodies and hcv-infected human sera, however, both studies addressed only the transgenic (stable) tobacco plant generation for hcvcp. however, transient expression in plants, compared with transgenic plant generation, is currently the method of choice due to several advantages like the simplicity and feasibility of rapid protein expression, omitting tissue culture and regeneration costs and ease of large scale industrial plans (23) (24) (25) . an important example of transient expression in plants is the production of seasonal influenza vaccines in tobacco (26) . the success in efficient plant transient expression, however, depends on several factors such as: codon-optimization of the heterologous gene according to the plant-codon adaptation index (27, 28) , selection of the proper expression vector (29, 30) , and efficient inhibition of the gene silencing phenomenon which suppresses the expression of foreign genes in plants (31) . while at present knowledge and techniques for the abovementioned parameters are being improved, providing novel data on transient expression process for the regionally-adapted plant hosts in general, and overcoming the plant-based gene silencing phenomenon in specific, is of high importance. in this study, we aimed to: i) construct an efficient transient tobacco expression system for hcvcp n-121 by designing a highly codon-optimized gene and employment of the iranian jafarabadi-tobacco plant cultivar which is a high biomass producer ii) evaluate the expression level of hcvcp for a potato virus x-based vector (pvx) (32) com-pared to a classic pbi121 binary plant vector in co-agroinfiltration with p19 gene silencing suppressor plasmid and iii) assess the antigenicity of this tobacco-derived hcvcp for potential clinical (diagnostic and vaccine formulation) applications. the pivex2.4a core plasmid encoding hcvcp n-121, used as both the source of the sequence for construction of plant-based expression vectors and for the expression of n-terminally 6xhis-tagged hcvcp protein in bl21-ai strain of escherichia coli by arabinose induction (as positive control) was previously described (33, 34) . several modifications for optimized (plant) codon-usage of hcvcp nucleotide sequence were considered, including: i) codon optimization according to the codon adaptation index of nuclear-encoded genes of tobacco (35) , ii) removal of (plant) mrna destabilizing sequences from the native hcvcp coding sequence, iii) addition of kozak (gccac-catggc) sequence (36) and hexahistidine (6xhis)-tag for nickel affinity purification at the 5′ site, iv) addition of nucleotides encoding endoplasmic reticulum retrieval signal (kdel) at the 3′ end (37) , v) addition of bamhi and saci restriction sites at both ends of the gene for directional cloning into the same sites of the plant expression binary vector pbi121 ( figure 1 ). the plant-optimized hcvcp gene for recombinant expression in tobacco (also termed tr-hcvcp) was synthesized and delivered as a clone in puc57 plasmid by shinegene molecular biotech inc. (shanghai, china). as shown in figure 3 a, the synthetic gene was subcloned into bamhi and saci sites of the pbi121 vector (38) under the control of camv 35s promoter and upstream of the nopaline synthase transcriptional terminator (nos-ter). in this study, the pvx-gw vector (32) , which was kindly gifted by dr. cristiano lacorte (embrapa recursos geneticos e biotecnologia, brasília, brazil), was used as the pvx-based viral vector. for cloning of the tr-hcvcp sequence into pvx-gw vector, the synthetic gene was pcr amplified using a forward primer, f-kozak-vx: 5΄-taatatcgatctcgagccaccatggctcatcacc-3΄ and a reverse primer, r-core-vx: 5΄-taatgtcgacggatcct-cagagttcgtccttctttc-3΄ harboring clai and sali restriction sites (underlined sequences). pcr amplification was performed with 25 cycles at 94˚c for 30 seconds, 58˚c for 30 seconds, and 72˚c for 90 seconds and 72˚c for 15 minutes. the 439-bp amplicon was subsequently digested by clai and sali enzymes and cloned into the same sites of pvx-gw vector under the control of duplicated pvx coat protein subgenomic promoter (cpp) (figure 3 b ). all the recombinant constructs were confirmed by restriction and sequencing analyses using f-kozak-vx and r-core-vx specific primers. all the cloning and molecular procedures were according to the standard protocols (39). the changes to the original sequence are shown by lower case letters. location of the kozak sequence, 6 xhis-tag, nucleotides encoding kdel and restriction sites for bamhi and saci are indicated. atg and tga denote the start and termination codons, respectively. a. tumefactions strain lb4404 was transformed with pvxcore and pbi-core vectors ( figure 3 a and 3b) in separate reactions via the standard freeze-thaw protocol (40) . to this end, the hcvcp-coding plant constructs, pbi-core and pvx-core were introduced into agrobacterium lb4404 (in the case of pbi-core) and into agrobacterium lb4404; the latter had already harbored the helper plasmid psoup (41) (in the case of pvx-core), which is essential for replication of the pvx-gw-based plasmids in agrobacterium species, respectively. subsequently, transformed agrobacterium cells were selected on plates containing 100 µg/ ml rifampicin (rif) and 50 µg/ml kanamycin (kan) and incubated for 72 hours at 28°c. the transformed colonies were confirmed by gene-specific colony pcr using f-kozak-vx and r-core-vx-specific primers, as described above. all the chemicals, culture media and antibiotics were purchased from sigma (us) and merck (germany) companies. to inhibit the silencing of foreign gene expression in tobacco, the constructed expression vectors were coinfiltrated with agrobacterium strain lb4404, harboring the p19 silencing-suppressor gene (42) from tomato bushy stunt virus (tbsv) (genbank accession number: m21958), which was previously cloned into pcambia 1304 vector (cambia co., australia) in our lab (p19-pcambia 1304 vector). the agroinfiltration protocol was according to kapila et al. study (43) . in brief, the agrobacterium cultures were first adjusted to od 600 of 1.0 with ms medium supplemented with 2% (w/v) sucrose, 10 mm mes ph = 5.5, 10 mm mgcl 2 , and induced for an additional 3 hours with 400 µm acetosyringone. the agrobacterium suspensions were subsequently mixed in equal ratios (half for p19 plasmid and half for each of the expression constructs, either pvx-core or pbi-core) and the n. tabacum (cultivar jafarabadi; kindly provided by dr. tohidfar, agricultural biotechnology research institute of iran) leaves were immersed in the bacterial suspension while a vacuum of 0.5 mbar was applied for two minutes. the infiltrated leaves were placed adaxial side down on wet whatman paper in glass containers and subsequently incubated at 25°c with a 16-hour photoperiod for five days. the infiltrated tobacco leaves were ground into a fine powder in liquid nitrogen, using mortar and pestle. subsequently, 0.5-ml extraction buffer (50 mm nah 2 po 4 , ph = 8, 300 mm nacl, 0.1% tween 20, 10 mm beta-mercaptoethanol, 1 mm pmsf, and 5 mm edta) was added to each gram of powdered leaf tissue. the total soluble protein (tsp) was removed from cell debris by centrifugation of the leaf extracts for 20 minutes at 4°c (14000 rpm). the e. coli-derived hcvcp (ehcvcp) and plant-derived hcvcp (phcvcp) were purified through application of nickel-nitrilotriacetic acid (ni-nta) chromatography, as previously described (15, 34) . protein concentrations were determined by bradford assay (44) using bovine serum albumin (bsa) as standard. for dot blot analysis of proteins, equal concentration of tsp (5 µg-25 µg) from plant extracts, 5 µg from ehcv as positive control and 25 µg of non-agroinfiltrated plant extract were directly dotted on a nitrocellulose membrane. the membrane was dried at room temperature, blocked with 5% (w/v) skim milk in pbs-tween (1/1000 v/v), and after three washing steps incubated with anti-c/n terminal 5xhis antibody (qiagen) 1:10000 in tbs-t for two hours at room temperature. following the washing steps, the membrane was incubated with 1:8000 diluted horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (sigma, usa) for one hour. finally, 3-3'diaminobenzidine (dab, sigma, usa) was used for color development. for sds-page and western blotting analyses, purified hcv core from e. coli and plant and plant crude extracts of the recombinant hcv core protein expressed by p19 coagroinfiltrated pbi121 and pvx vectors were loaded onto a 12% sds-polyacrylamide gel; by the end of electrophoresis, the protein bands were either stained with coomassie brilliant blue (bio-rad) or transferred to the pvdf membrane. subsequently, the corresponding protein bands on the membrane were identified by biotinylated anticore polyclonal antibody (abcam, uk) (1:1000 dilution) and streptavidin hrp conjugate (sigma, usa) (1:4000 dilution), respectively, and were detected by tmb as substrate. elisa was performed using the streptavidin-biotin peroxidase complex (abc) assay. briefly, wells were coated overnight at room temperature with 100 µl of 5 µg/ml mouse monoclonal antibody against 21-40 amino acids of hcv core antigen (abcam, uk) and blocked with 1% bovine serum albumin (bsa) for two hours at 37˚c. after several washing steps, 50 μg of tsp from each sample or ehcvcp (for standard curve, serial dilutions in the range of 0.1-1 µg/ml) were added to the coated wells in duplicates and incubated for one hour at 37°c. following the washing steps, biotinylated anti-core polyclonal antibodies (1:1000 dilution, abcam, uk) were immersed into the wells and incubation continued at 37˚c for one hour followed by washing steps and another similar incubation with streptavidin hrp conjugate (1:4000; sigma). finally, by adding tmb and color development, the absorbance at 450 nm was measured. to detect the immuno-reactivity (antigenicity) of tobacco-derived hcvcp, an indirect elisa for detection of anticore antibodies in human sera was developed as previously described (15) . in brief, purified phcvcp was coated in elisa plates (100 µl of 10 µg/ml). the pooled human sera (1/20 dilution) from five hcv seropositive patients and one serum negative (obtained from tehran blood transfusion center) were incubated with the plate for two hours at room temperature. hrp-labeled anti-human igg (1:5000 dilution, sigma, usa) was used for detection of bound antibodies and tmb was used for color development, as described above. prism 5.0 software (graphpad, usa) was used for data handling and statistical analysis was carried out using mann-whitney non-parametric and anova tests. statistical significance was set at p ≤ 0.05. figure 1 represents the nucleotide changes applied in the basal sequence of hcvcp to improve its codon utilization for efficient expression in plants. other elements (kozak, kedel, his-tag and restriction sites) were also included in hcvcp basal sequence in tr-hcvcp ( figure 1 ). in addition, to remove the plant mrna destabilizing sequence "ggtaag" (nucleotides 358-363) which is present in native hcvcp sequence, it was modified ( figure 1 ). as shown in figure 2 a, the corresponding nucleotide alterations increased the codon adaptation index (cai) value from 0.65 to 0.85 and reduced the gc content from 62.62 to 51.05 (figure 2 b) . results of dna sequencing (not shown) indicated that the kozak (gccaccatggc) sequence (36) , harboring the start codon (atg) at 5', kdel nucleotides at 3′ end, the designed restriction sites and all nucleotide modifications, was properly located in the tr-hcvcp sequence (figure 1 figure 3 represents the schematic of the constructed pbi-core (figure 3 a) and pvx-core (figure 3 b ) plant expression vectors harboring the tr-hcvcp, located under the control of camv 35s promoter (in pbi-core) and cp promoter (in pvx-core), upstream of the nos-ter transcriptional terminator. the sequencing results (not shown) confirmed the presence of the cloned tr-hcvcp gene in pbi-core and pvx-core expression vectors with no alterations in the nucleotides. the resulting plasmids were transferred into a. tumefaciens lba4404 target hosts (in the case of pvx-core, a. tumefaciens lba4404 harboring the psoup vector). the transformed agrobacteria were screened for recombinant constructs using core-specific primers (f-kozak-vx and r-core-vx) and the colony pcr assay indicated the expected 439-bp fragments (figure 3 c) . one positive clone from each construct was subsequently used for agroinfiltration of n. tabacum leaves by p19 silencing-suppressor gene procedure as outlined in 3.3. to detect and analyze phcvcp, the transformed tobacco leave extracts were evaluated by dot and western blot assays ( figure 4 ). as shown in figure 4 a, dot blot analysis showed that hcv core was present in extracts of the tobacco leaves, transformed by each of the vectors (pbi-core and pvx-core), compared to negative controls with the same amount of loaded tsp. although dot blot is a qualitative assay rather than a quantitative one, our result tentatively implied a higher level of expression for pvx-core compared with that of pbi-core construct, while the same amounts of tsp were applied (figure 4 a) . coomassie-stained sds-page showed that nonspecifically-bound plant proteins were eluted from ni-nta affinity chromatography besides a band with an electrophoretic mobility of 15 kda (figure 4 b) . the recent band was visible on a coomassie-stained sds-page gel (figure 4 b) and was also detected using anticore polyclonal antibodies in a western blot assay. there was no degradation of phcvcp and the protein was not glycosylated because the size of the produced protein was as predicted by the protein sequence (https://www.genscript. com/ssl-bin/site2/peptide_calculation.cgi). as shown in figure 4 c, the results of western blot analysis indicated that phcvcp from pvx-core and pbi-core expressed a protein of approximately 15 kda, while the negative control lacked the core protein and ehcvcp showed a higher molecular weight (~21 kda, which is due to addition of vector-derived amino acids in pivex2.4a plasmid) and a few other lower-molecular weight protein bands which were presumably the results of ribosomal release and uncompleted translation, as previously reported (15) . it should be noted that dot blotting in this study was performed based on prior studies that used this procedure in complementation of western blotting (7, 8) . indeed, in western blotting, the protein will be completely denatured (by boiling in sds-page loading buffer which contains 2-me and sds) and therefore some information that might be related to the native/soluble structure of the protein might be lost (e.g. interaction of protein with a conformational antibody), while dot blot assay was used as a preliminary and fast test to show if the protein was expressed. the expression levels of phcvcp by each of the vectors (pbi-core as a classical plant binary vector and pvx-core as a viral-based vector) in the presence or absence of p-19 co-agroinfiltration were compared by elisa. sandwich elisa is the assay of choice and is usual in quantification of antigen proteins (when an exact and precise standard curve is prepared and a specific antibody against the antigen is used) and a number of diagnostic elisa kits work on this basis (45, 46) . in this context and to perform an exact and precise quantitative elisa, we used a specific monoclonal antibody against the core antigen (at the bottom of elisa plates) and polyclonal anticore antibodies (at the top of the phcvcp) in a sandwich elisa-based protocol and also prepared a precise standard curve by applying serial dilution (0.1-1 µg/ml; data not shown) of purified ehcvcp to calculate the expression levels of phcvcp. they were expressed in form of the percentage of tsp, which was determined using a bradford protein assay. the leaves transformed by pvx (vector without hcvcp) and co-agroinfiltrated with p19 constructs were used as negative control (figure 5 a) . the results indicated that the expression of pbi-core and pvx-core was around 0.004% of tsp in the absence of p19 co-agroinfiltration, whereas in its presence, the expression level of pbi-core and pvx-core increased to 0.019% (210 ng/g) and 0.022% (240 ng/g) of tsp fresh leaf weight, respectively, which was almost five times improvement for phcvcp expression (figure 5 a) . the diagnosis potency of phcvcp for detection of hcvinfected human sera was also analyzed by elisa. as shown in figure 5 b, purified phcvcp could detect hcv-infected sera in a comparable way to that of the ehcvcp. a). dot blotting; at control column: row 1: negative control (25 μg tsp of untransformed tobacco leaves); row 2: 5 μg positive control (ehcvcp). at pvx column: negative control (tobacco leaves transformed by pvx vector alone; ie, without tr-hcvcp). pvx-core and pbi-core columns: the extracts from the pvx-core and pbi-core p19 co-agroinfiltrated leaves, respectively. rows 2 and 1 in these two last columns correspond to 5 µg and 25 µg of tsp, respectively. b) coomassie-stained 12% sds-page gel, loaded with; lane 1: 5 μg concentrated plant-purified hcvcp (from the pvx-core expression system). lane 2: 5 μg of purified ehcvcp. lane 3: crude protein extract from agroinfiltrated leaves with pvx-core (20 µg). lane 4: untransformed leaves (20 µg). c) western blotting; lane 1: positive control (700 ng purified ehcvcp). lane 2: purified phcvcp (700 ng from the pvx-core expression system). lanes 3 and 4: the extracts from p19 co-agroinfiltrated pvxcore and pbi-core leaves, respectively (50 µg of plant tsp was applied in each lane). lane 5: negative control (50 µg of plant tsp of untransformed tobacco leaves). lane m: prestained protein ladder (fermentas). hcvcp denote to hcv core protein, ehcvcp and phcvcp dnote to e.coli-derived and plant-derived hcvcp, respectively. in western blot and sds-page figures, the location of hcvcp under the 20 kda molecular weight range is shown by arrows. the reason for multiple bands in the case of ehcvcp is explained in the corresponding result section of the text. the results of sds-page showed that ni-nta pull-downs of plant extract contained endogenous nonspecific plant proteins besides phcvcp. according to elisa data, the concentration of phcvcp was 1/20 of the column-purified protein. therefore, although 1 µg (100 µl of 10 μg/ml coated) was coated, only a small amount reacted (less than 50 ng) (figure 5 b) . however, none of these endogenous nonspecific plant proteins reacted with anti-hcvcp in western blotting of the purified protein fraction. the primary objective of the present study was to provide an efficient transient tobacco system for expression of hcvcp in a regionally-adapted tobacco host (the iranian jafarabadi-tobacco plant cultivar). while other tobacco plant such as australian nicotiana species, n. benthamiana, is a well-known and hyper-susceptible host for plant-derived recombinant protein expression (47) , to our knowledge there was no prior report available on expression in jafarabadi tobacco cultivar (which is one of the currently available tobacco cultivar in iran). to address this concern, the first strategy that should be considered would be the optimization of the gene sequence for efficient codon usage by the plant (27) . in agreement, it is recently shown that the plant codonoptimized bpv-1 li gene product increased significantly in comparison to the unmodified counterpart (28) . as shown in figure 2 a, our codon optimization approach increased the cai value from 0.65 to 0.85, which was in favor of codon utilization for n. tabacum (35) . although increasing the cai value is usually inevitably accompanied by increase of a + t percentage (which enhances mrna instability and decreases the efficiency of translation) (48, 49) , our codon optimization approach that was based on the codon usage of nuclear-encoded genes of tobacco could keep the a + t percentage at 49%, while reducing the gc content from 62.62 to 51.05 (figure 2 b) , almost the perfect range desired for gc content (50%). to ameliorate the expression level, insertion of the kozak sequence (gccaccatggc) (36) at the translation start site (atg) was also considered ( figure 1 ). however, since the two base pairs immediately following the atg start codon were "c" and "a" and were not compatible with the optimized kozak sequence (underlined nucleotides), consistent with the approach undertaken by amani et al. (50) , we had to insert the gct sequence that codes for alanine (a nonpolar amino acid). moreover, to improve the stability of phcvcp, kdel-encoded bases were also considered at the 3' site of the tr-hcvcp sequence ( figure 1 ). employing this strategy for expression of human epidermal growth factor in tobacco resulted in a 10 4 -fold higher yield of protein expression (51) . finally, in our codon optimization strategy, the possible deleterious splicing motif in plants "ggtaag", resulting in rna degradation and gene silencing of the gene (52) , was removed from the native hcvcp-coding sequence (nucleotides 358-363). in our study, two kinds of plant vectors were employed: a classic, nonreplicative, binary vector (pbi121) and a pvxbased viral replicative vector (29, 30) . our results indicated that the pvx-based vector provided higher expression levels of phcvcp compared to that of pbi121 (figure 4 a and 5a). our result was in complete agreement with the results of a recent report comparing the expression levels by tmv and pvx replicative viral vectors which was three times of that of pbi121 vector (53) . a) the expression level of pbi-core and pvx-core were assessed by elisa and compared in the presence or absence of co-agroinfiltration by gene silencing suppressor p19 construct. leaf control denotes the tobacco leaves transformed by pvx vector alone (ie, without tr-hcvcp). b) result of elisa assay for confirmation of plant-derived hcvcp, using hcv-positive human serum. negative control corresponds to hcv negative human sera. bsa was also used as another negative control. phcvcp (corresponding to 100 µl of 10 µg/ml of phcvcp from pvx-core-transformed tobacco leaves) and ehcvcp denote plant-and e. coli-derived hcvcp, respectively. statistical analysis was performed by one-way analysis of variance (anova), using the bonferroni's multiple comparison test. for obtaining the highest expression levels, it is important to prevent the silencing mechanisms that limit foreign gene expressions in plants. it is shown that coexpression of viral silencing suppressor proteins eliminates the effects of post-transcriptional gene silencing (ptgs) in a transient expression system (42) . application of p19-mediated co-agroinfiltration has already shown to improve recombinant expression in plants by several folds (42) . our results indicated that the presence of p19 increased the expression levels of phcvcp by five times in both pbi121 and pvx expression constructs (figure 5 a) . our data were consistent with recent reports on enhancement of the expression levels of sars-cov n (54), antibodies (53) and gfp (55) proteins in the presence of p19 by 3-5 and even 100 folds, respectively. the 10 times differences in the transient expression levels of human growth hormone peptide by pbi121 in n. tabacum was 0.002% of tsp (56) to that of our study (0.02% of tsp for phcvcp). the enhanced expression levels in our study compared to this prior report should be a direct result of the codon optimization and gene silencing suppression strategies undertaken for tr-hcvcp in our gene modification approaches. finally, the results of elisa in our study ( figure 5 ) demonstrated that the transient expression of phcvcp in tobacco leaves could provide the proper protein for diagnostic purposes and indicated its full functionality to react with poly/monoclonal anticore antibodies and hcv infected human sera compared to that of ehcvcp (14) . in addition, the proper conformation of phcvcp to interact with specific monoclonal antibody against 21-40 amino acids of the core antigen (coated at the bottom of the elisa plates) and the polyclonal anticore antibodies (at the top of the phcvcp in elisa), supported the presence of various interactive epitopes on phcvcp. the monoclonal antibody used in this study was against amino acids 21-40 on hcv core antigen. this region carries two important immunological epitopes which are conserved between hcv strains: 1a, 1b, and 2 (57): moreover, the amino acids of 23-44 are highly promiscuous cd4+ t cell epitopes and the 35-44 amino acids are hcvspecific hla-a2-restricted ctl epitopes. they have been shown to be effective immunogens in the vaccine phase of two clinical trials while are present on the hcv core particle or in multi-epitope complexes (58) . therefore, the primary results have proposed that the plant-produced phcvcp might have the promising structure to be used as a vaccine candidate too. however, these primary results should be confirmed through animal and immunization studies. although these ending results were consistent with the prior two reports on appropriate antigenicity of tobaccoderived hcvcp, both of those previous studies addressed only transgenic (stable) tobacco-plant generations for hcvcp (21, 22) . however, transient expression in plants is currently the method of choice due to the simplicity and feasibility of rapid protein expression, omitting tissue culture and regeneration costs, and ease of large scale industrial plans growth (23) (24) (25) . while transgenic (stable) plant generation needs longer periods of transgenic production and more deleterious effects on gene silencing (21) which is not encountered in transient-expression approach (59) . taken together, the present study was the first (to our best of knowledge) to address construction of a transient tobacco expression system for hcvcp. for highest possible expression efficiency, the hcvcp dna sequence was codon optimized (based on the cai for nuclear-encoded genes of tobacco), destabilizing of the ggtaag sequence in the native hcvcp coding sequence was altered and kozak and kdel sequences were included at the 5' and 3' ends, respectively. two types of plant expression vectors (pvx-based and classic pbi121 binary) for hcvcp were constructed and their levels of protein expression in the presence and absence of p19 co-agroinfiltration were compared. the results indicated the significant effect of our approach in enhancement of the expression levels for phcvcp. to our knowledge, all the above mentioned reports were first of their kinds for hcvcp and provided valuable information for plant-based expression studies on this antigen. the preliminary results (with five hcv positive sera) showed that plant-hcvcp has the capability to capture anti-hcvcp antibodies for potential clinical applications which should be further evaluated and confirmed by statistically acceptable numbers of positive sample sera. the next steps might be standardization of phcvcp for application in diagnostic elisa through application of positive sera with determined viral loads as well as precisely predetermined standard positive sera together with high number of optimization studies with different concentrations of antigen/antibody in elisa with precise statistical analyses and evaluation of the phcvcp for immunization studies. trends of obesity in iranian adults from 1990s to late 2000s; a systematic review and meta-analysis virology of hepatitis c virus infection hepatitis c virus vaccines--progress and perspectives advances in hepatitis c virus vaccines, part one: advances in basic knowledge for hepatitis c virus vaccine design advances in hepatitis c virus vaccines, part two: advances in hepatitis c virus vaccine formulations and modalities enhancement of immune responses by co-delivery of ccl19/mip-3beta chemokine plasmid with hcv core dna/protein immunization polytope dna vaccine development against hepatitis c virus: a streamlined approach from in silico design to in vitro and primary in vivo analyses in balb/c mice construction of hcv-polytope vaccine candidates harbouring immune-enhancer sequences and primary evaluation of their immunogenicity in balb/c mice fusion of hbsag and prime/ boosting augment th1 and ctl responses to hcv polytope dna vaccine the role of core antigen detection in management of hepatitis c: a critical review expression and characterization of escherichia coli derived hepatitis c virus arfp/f protein the wild-type hepatitis c virus core inhibits initiation of antigen-specific t-and b-cell immune responses in balb/c mice identification of a functional, crm-1-dependent nuclear export signal in hepatitis c virus core protein collaborative team of the international pasteur network . the impact of hcv diversity on diagnosis tools for hcv infection hcv core protein immunization with montanide/cpg elicits strong th1/th2 and long-lived ctl responses hcv core protein-expressing dna vaccine induces a strong class i-binding peptide dth response in mice immunization of mice by bcg formulated hcv core protein elicited higher th1-oriented responses compared to pluronic-f127 copolymer expression of shigella flexneri ipab gene in tobacco overview of expression of hepatitis b surface antigen in transgenic plants bioprocessing of plant-derived virus-like particles of norwalk virus capsid protein under current good manufacture practice regulations expression of an hcv core antigen coding gene in tobacco (n. tabacum l.) a hepatitis c virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera expression of hiv-1 antigens in plants as potential subunit vaccines transient expression systems for plant-derived biopharmaceuticals a plant-produced influenza subunit vaccine protects ferrets against virus challenge. influenza other respir viruses a plant-based system for rapid production of influenza vaccine antigens. influenza other respir viruses modification of the coding sequence enhances plant expression of insect control protein genes in planta production of a candidate vaccine against bovine papillomavirus type 1 magnifection--a new platform for expressing recombinant vaccines in plants plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins advances in plant molecular farming potatovirus x and tobacco mosaic virus-based vectors compatible with the gateway cloning system fcgamma receptor-like activity of hepatitis c virus core protein initiation of hepatitis c virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein the codon adaptation index--a measure of directional synonymous codon usage bias, and its potential applications the scanning model for translation: an update a c-terminal signal prevents secretion of luminal er proteins complete sequence of the binary vector pbi121 and its application in cloning t-dna insertion from transgenic plants molecular cloning storage of competent cells for agrobacterium transformation pgreen: a versatile and flexible binary ti vector for agrobacterium-mediated plant transformation an enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus an agrobacterium-mediated transient gene expression system for intact leaves a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding production and secretion of human interleukin-18 in transgenic tobacco cell suspension culture generation of biologically active multi-sialylated recombinant human epofc in plants nicotiana benthamiana: its history and future as a model for plant-pathogen interactions effects of codon modification on human bmp2 gene expression in tobacco plants effect of codon optimization and subcellular targeting on toxoplasma gondii antigen sag1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice in silico analysis of chimeric espa, eae and tir fragments of escherichia coli o157:h7 for oral immunogenic applications expression of active human epidermal growth factor (hegf) in tobacco plants by integrative and non-integrative systems prediction of splice sites in plant pre-mrna from sequence properties plant-made trastuzumab (herceptin) inhibits her2/neu+ cell proliferation and retards tumor growth boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice a new viral vector exploiting rna polymerase i-mediated transcription transient expression of human growth hormone in potato (solanum tuberosum), tobacco (nicotiana tobacum) and lettuce (lactuca sativa) leaves by agroinfiltration progress in the development of preventive and therapeutic vaccines for hepatitis c virus immunogenicity and safety of different injection routes and schedules of ic41, a hepatitis c virus (hcv) peptide vaccine. vaccine transgene expression variability (position effect) of cat and gus reporter genes driven by linked divergent t-dna promoters it is with affection and appreciation that dr. a. jafari from the molecular biology department of pasteur institute of iran is acknowledged for her supports and help concerning this project the present study was in part to fulfill the thesis of mrs. sara mohammadzadeh in ph.d. s. m. the ph.d. candidate; she was responsible for most experimental works and prepared the predraft of the manuscript. alireza khabiri helped with elisa assays. farzin roohvand: advisor; helped with expression of ehcvcp, diagnosis potency of phcvcp by elisa and editing of the manuscript. arash memarnejadian: co-advisor; soheila ajdary and ali hatef salmanian: co-supervisors; parastoo ehsani: supervisor; design of the experimental works and editing the manuscript. this work was supported by pasteur institute of iran, tehran, iran. key: cord-001421-6t5puo6p authors: marfà, santiago; crespo, gonzalo; reichenbach, vedrana; forns, xavier; casals, gregori; morales-ruiz, manuel; navasa, miquel; jiménez, wladimiro title: lack of a 5.9 kda peptide c-terminal fragment of fibrinogen α chain precedes fibrosis progression in patients with liver disease date: 2014-10-02 journal: plos one doi: 10.1371/journal.pone.0109254 sha: doc_id: 1421 cord_uid: 6t5puo6p early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. this study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, hcv-positive patients who underwent liver transplantation (lt). we analyzed 93 lt patients with hcv recurrence, 41 non-lt patients with liver disease showing a fibrosis stage f≥1 and 9 patients without hcv recurrence who received antiviral treatment before lt, as control group. blood obtained from 16 healthy subjects was also analyzed. serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by seldi-tof-ms. characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. marked differences were observed between the serum proteome profile of lt patients with early fibrosis recurrence and non-recurrent lt patients. a robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent lt patients. however, the same peak was barely detected in recurrent lt patients. similar results were found when comparing samples of healthy subjects with those of non-lt fibrotic patients, indicating that our findings were not related to either lt or hcv infection. using tandem mass-spectrometry, we identified the protein peak as a c-terminal fragment of the fibrinogen α chain. cell culture experiments demonstrated that tgf-β reduces α-fibrinogen mrna expression and 5905 m/z peak intensity in hepg2 cells, suggesting that tgf-β activity regulates the circulating levels of this protein fragment. in conclusion, we identified a 5.9 kda c-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease. early detection of fibrosis progression and the development of portal hypertension is of major relevance in the prognosis and treatment of patients with chronic liver disease [1] . indeed, early recognition of subjects prone to develop these alterations may allow prompt initiation of therapeutic interventions. therefore, identification of noninvasive biomarkers related to the activation of the fibrogenic process is of major relevance, particularly in those subjects with sustained liver injury [2] . however, despite the numerous attempts to uncover such molecules, this objective has resulted elusive. this is likely related to the natural history of liver disease. with the exception of fulminant hepatic failure, liver disease is an insidious process in which clinical detection and symptoms of hepatic decompensation may occur weeks, months or many years after the onset of injury, and healing may occur without clinical detection [3] . however, in particular clinical circumstances, i.e. patients infected with the hepatitis c virus (hcv), submitted to liver transplantation (lt), it is possible to expect recurrence of hepatic fibrosis and portal hypertension to occur within a short period of time [4] . thus, these patients constitute a population particularly suitable to identify noninvasive markers of early fibrogenesis. the current investigation took advantage of the faster development of hepatic fibrosis in hcv-positive lt patients. serum samples were collected shortly after lt and high-throughput proteomic techniques were used to ascertain whether the proteomic pattern of these samples differs from the proteomic pattern expression obtained from serum samples of non-infected lt patients. ultimately, the investigation was aimed to identify early circulating serum biomarkers of active fibrogenesis in patients with liver disease. one hundred and nineteen patients admitted to the liver unit to undergo a liver biopsy from june 2001 to january 2006 were prospectively considered for this study. exclusion criteria were presence of ascites, chronic kidney failure in hemodyalisis and moderate or severe acute graft rejection during the first three months, biliary complications or antiviral treatment during the first year after lt in the case of lt recipients. in addition 16 healthy volunteers were also included in the study. the design of the study was two folded: first we assessed whether the serum proteomic profile of recurrent hcv-lt patients differs from that of non-recurrent hcv-lt patients. the serum proteomic profile and routine liver and renal function tests were initially analyzed in a training set of 10 hcv-rna recurrent lt patients 6 months post lt that showed a fibrosis stage f$1 at 1 year after lt. paired hepatic venous pressure gradient (hvpg) determination was also available in 7 of these patients. the control group consisted in 9 patients without hcv-rna recurrence, who underwent antiviral treatment before lt and achieve sustained virological response. in addition, serum samples were also collected from 41 non-lt patients with advanced liver disease. the hcv or hepatitis b virus (hbv) was present in 8 and 3 of these patients, respectively, whereas the etiology of liver disease was other than viral in the remaining (9 nonalcoholic steatohepatitis, nash; 10 alcoholic liver disease, ald; 4 autoimmune hepatitis, ah; and 7 cryptogenic). thereafter, the results were validated in a test set of 83 hcv recurrent lt patients. serum samples were also collected 6 months post-transplantation and the proteomic profile was evaluated along with liver and renal function tests. hvpg measurement in 53 of these patients was also performed. percutaneous and transjugular liver biopsies and hvpg measurements were performed as we have previously described [5] . fibrosis stage was scored using the scheuer classification: no fibrosis (f0), minimal portal fibrosis (f1), periportal fibrosis (f2), fibrosis beyond the portal tract making septums (f3) and cirrhosis (f4) [6] . see (data s1). protein profiling was performed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms) using the eight-spot format cm10 (weak cationic exchange) proteinchip arrays (bio-rad). in a preliminary study performed to set up the experimental conditions, 2 pooled serum samples from the 9 patients without hcv-rna recurrence and the 10 patients included in the training set were loaded onto three different types of protein chip arrays: h50 (that binds proteins through reverse phase or hydrophobic interactions), cm10 (negatively charged surface that acts as a weak cation-exchanger) and imac-30 (immobilized metal affinity capture surface preactivated with copper). the resulting spectra from each pool were compared and the cm10 array showed the highest number of peaks detected and the highest total signal intensity compared to h50 and imac-30; therefore only the cm10 array was used in the subsequent studies. prior to sample loading, spots were equilibrated two times with 200 ml of cm binding/washing buffer (0.1 m sodium acetate, ph 4). each sample was loaded in duplicate randomly in order to minimize any systematic error. forty microliters of fractionated serum sample was incubated in 60 ml of cm binding buffer for 30 minutes on a shaker at room temperature. afterwards, arrays were washed three times with 200 ml cm washing buffer for 5 minutes at room temperature. unbound serum proteins were removed by washing twice with deionized water. thereafter, arrays were air-dried and 1 ml of energy-absorbing matrix (saturated sinapinic acid in an aqueous solution containing 50% acetonitrile and 0.5% tfa) was added twice to each spot. the surface was allowed to air dry between each application. the array was read by using the proteinchip pbs ii reader (biorad). each spot was read at low (2500 nj), medium (3000 nj) and high (3500 nj) energy laser intensities. the mass-to-charge ratio (m/z) was set from 1.000 to 25.000 m/z for the low-energy laser intensity, between 2.500 and 200.000 m/z for the medium-energy laser intensity and from 5.000 to 200.000 for the high-energy laser intensity. all spectra were calibrated using two external calibration standards (all-in-one peptide standard and all-in-one protein standard, biorad). a peak resolution was optimized within 5.000 m/z, 12.000 m/z or 19.000 m/z according to low, medium or high energy laser intensity, respectively. all data were processed with the proteinchip data manager client 4.1 software (bio-rad). to minimize the possible random error and spectral outliers, all the raw data was normalized by the average total ion current across the group and all spectra differing by twice the standard deviation or more from the mean were deleted. furthermore, the baseline was also corrected by adjusting the parameter to 30 times the expected peak width. for the peak selection, several parameters were selected for the identification of peak clusters. thus, only peaks with a signal to noise equal or greater than 5; with a valley depth superior than three; found in a minimum of 20% of all spectra and with an m/z error below the 0.3% for the low-energy laser intensity spectra and below 2% for the medium-and high-energy laser intensity spectra, were considered. subsequently, all peak clusters detected were verified manually. relabeling, removal or addition of peaks was performed when necessary. to test the quality of the assay, pooled normal sera from two individuals was assessed. five protein peaks randomly selected over the course of the study were used to calculate the coefficient of variance (cv) as described [7] . we then determined the reproducibility of the seldi spectra, both within and between arrays (intra-assay and inter-assay, respectively). the intra-assay (spot to spot) cv was 11.95% for peak intensity and 0.02% for mass accuracy. the inter-assay (chip to chip) cv was 21.96% for peak intensity and 0.03% for mass accuracy. see (data s2). hepg2 cells were obtained from american type culture collection (atcc, manassas, va, usa). this immortalized, stable cell line can be repeatedly frozen, thawed and propagated. hepg2 cells were seeded (2610 6 cells/well) in vented t-75 flasks and grown to confluence in dulbecco's modified eagle medium (dmem), supplemented with 50 u/ml penicillin, 50 mg/ml streptomycin and 10% of fetal calf serum (fcs). thereafter, cells were switched to 1% fcs and incubated (37uc) under normoxic (21% o 2 , 5% co 2 ) or hypoxic conditions (5% o 2 , 5% co 2 ) in a controlled o 2 water-jacketed co 2 incubator (forma scientific series ii, 3131, marietta, oh) or treated with tnf-a (10 ng/ml, sigma, st louis, mo), lipopolysaccharide (lps, 10 ng/ml, the same day of the liver biopsy, 20 ml of blood were obtained in a fasting status. serum was stored at 280uc, and serum albumin, aspartate aminotransferase (ast), alanine transaminase (alt), bilirrubin and blood urea nitrogen (bun) were measured with the advia 2400 instrument (siemens healthcare diagnostics, tarrytown, ny, usa). amino-terminal propeptide of type iii procollagen (piiinp), hyaluronic acid (ha), and tissue inhibitor of matrix metalloproteinase type-1 (timp-1) were measured in all patients by a ce-marked random-access automated clinical immunochemistry analyzer that performs magnetic separation enzyme immunoassay tests (advia centaur, siemens healthcare diagnostics, tarrytown, ny, usa). the enhanced liver fibrosis (elf) score was calculated using the algorithm recommended in the ce-marked assay [elf = 2.278 + 0.851 ln(ha) + 0.751 ln(piiinp) + 0.394 ln(timp-1)] blood tests. statistical analysis of the results was performed by the non parametric mann-whitney u test and the kruskal-wallis test with the dunn post hoc test as appropriated. quantitative data were analyzed using graphpad prism 5 (graphpad software, inc. san diego, ca). we obtained written informed consent from all patients included in the study and the investigation was approved by the investigation and ethics committee of the hospital clinic of barcelona following the ethical guidelines of the 1975 declaration of helsinki. the principal demographic values of patients included in the definition group are shown in table 1 . as per the selection criteria, most recurrent hcv patients showed higher fibrosis and elf scores, elevated hvpg measures and greater ast and alt values than non recurrent hcv subjects. figure 1 depicts a portion of the spectra of all the samples investigated in this training group, ranging between 3000 and 11000 daltons; the mass to charge ratio analyzed. the expression pattern of the spectrograms obtained from non recurrent hcv patients clearly differed from those of recurrent hcv subjects. six statistically different peaks, identified in the figure as peptides a, b, c, d, and e, were detected. as shown in table 2 the signal intensity of four of these peaks (a, b, c, and f) was markedly higher in non recurrent than in recurrent patients whereas an inverse situation was observed in the remaining two peaks (d and e). the most remarkable difference was detected on analyzing peptide a (5905 m/z), since it was fully evident in all samples obtained from non recurrent hcv patients but almost suppressed in the serum of recurrent hcv individuals. an intriguing question arising from the above described results was to elucidate whether these findings result from the particular characteristics of hcv transplanted patients rather than to a differential feature characterizing early fibrogenic processes. thus, the serum proteomic spectrum was next analyzed in healthy individuals, in non-lt fibrotic hcv infected patients and in non hcv fibrotic patients. the clinical and demographic characteristics of these subjects are shown in table 3. fibrosis scores and liver function tests of the two groups of fibrotic patients were similar to those obtained in hcv lt patients. the mass spectrograms of all individuals included in this portion of the investigation are shown in figure 2 . the upper, middle and lower parts correspond to healthy subjects, fibrotic non hcv subjects and fibrotic hcv patients, respectively. since in the previous experiments the most striking differences were observed with peptide a, in this and the subsequent experiments we focused on the peak with a mass/charge ratio of 5905 da. all serum samples analyzed from healthy subjects showed a spectrogram compatible with the presence of this peptide whereas pathological serum samples, either from fibrotic non lt hcv patients or fibrogenic non hcv subjects showed the absence of this peptide or very low intensity peaks. these results indicate that neither hcv nor lt account for the suppressed expression of the a peptide in serum samples of fibrotic patients. to further confirm that peptide a behaves as an early serum biomarker of fibrosis, mass spectrometric analysis was performed in a test group of serum samples obtained from 83 hcv recurrent patients 6 months after lt. hvpg was assessed in 53 of these patients and the average value was of 5.560.8 mm hg. all the serum samples showed a quite similar expression pattern and coincidences included both the different peptide fragments detected and the signal intensity of these fragments (data s4). the most relevant finding was, however, that the mass spectrum corresponding to peptide a showed a very low peak intensity in all samples obtained from hcv recurrent patients. in fact, in 53 out of the 83 patients the peak intensity at m/z 5905 was under the background levels of 10 ma and in the remaining samples, intensities ranged between 11.5 and 81.4 ma ( figure 3) . thus, these results confirm the findings obtained in the training group in a larger group of subjects. to isolate the protein of interest and to determine candidate protein identity, serum samples from two healthy subjects containing high seldi intensity were pooled and separated by tricine-sds-page (figure 4b). the band at 5.9 kda was excised trypsinized and analyzed by lc-ms/ms. as shown in figure 4c , two peptide sequences were identified, which matched with the human fibrinogen a c-chain at 3.23% coverage, suggesting that suppression of the fibrinogen a c-chain 5.9 kda fragment is an early surrogate indicative of active fibrogenesis in patients with liver disease. to unveil potential mechanisms governing the release of the 5.9 kda peptide c-terminal fragment of the fribrinogen a chain in the serum of patients under an early fibrogenic process, hepg2 cells were treated with well known proinflammatory stimuli (tnfa, lps and il-1b) or profibrogenic substances (aii, et-1, apelin, fibronectin and tgf-b) for 6 hours. with the exception of tgfb, none of these substances induced significant changes in the expression of human fibrinogen chains messenger. however, evaluation of the extension and aggressiveness of the fibrogenic process in the injured liver is of major relevance for the diagnosis, prognosis and treatment of patients with hepatic disease [8] . the methods currently available to assess liver fibrosis include the serological determination of several parameters related to liver function and hepatic remodeling, imaging techniques, such as fibroscan or arfi and the use of invasive procedures such as hvpg measurement or liver biopsy, the latter still being the most widely accepted gold standard method for assessing liver fibrosis [9] . the specific limitations of each of these methods have been extensively discussed previously [10] . the risk of complications and low sensitivity for mild or moderate fibrosis are among the most remarkable limitation for invasive and non invasive methods, respectively [11] . recently, a liver fibrosis score, namely elf, which combines the serum concentrations of substances related to collagen metabolism (piiinp) and tissue remodeling (timp-1 and ha), has progressively been incorporated among the most common diagnostic tools to evaluate liver fibrosis. however, whereas this technique was found to be highly accurate in patients with advanced fibrosis (f3-f4 stage) [12] [13] [14] it appeared to be less efficacious in the diagnosis of mild or moderate fibrosis (f1-f2 stage) [15, 16] . early detection of active fibrogenic activity, therefore, still remains an open challenge in liver disease. fibrosis progression evolves over long periods of time, with this representing one of the most relevant difficulties to identify specific early biomarkers of fibrosis. in the current investigation this issue was overcome by assessing the proteomic profile of hcv-positive lt recipients in a training set of serum samples. blood samples were obtained at 6 months after lt and a liver biopsy was performed 1 year after surgery to define fibrosis stage. it is well known that fibrosis progression is accelerated in recurrent hepatitis c, with 15% to 47% of lt recipients developing fibrosis/cirrhosis within the first 3 years post transplantation [17] . therefore, rapid fibrosis progression is a major characteristic of this group of patients and for this reason they are particularly suitable to uncover serum tags of hepatic fibrosis. seldi-tof-ms technology or protein chip profiling combines mass spectrometry with a surface enhanced biochip which allows uniform and reproducible binding and desorption of biomarkers [18] . seldi-tof-ms also incorporates sample prefractionation. this markedly decreases the complexity of protein rich fluids, such as serum, allowing comparison of peak intensity between samples using large sample sets [19] . in the current study, serum proteins were fractionated by anion exchange chromatography based on their isoelectric points using a ph gradient. the resulting fraction was bound to a weak cation exchange surface to create an array of protein chip spots. this surface was selected according to its higher accuracy and reproducibility yields. using this technology we were able to simultaneously detect relative protein expression levels over a range of molecular masses of 2 to 180 kda, although the 2-20 kda range appeared to be the most sensitive. by means of this profiling system, we found at least 6 serum biomarkers that were differentially increased or decreased in recurrent hcv patients. among them, a protein of 5.9 kda (protein a) was fully suppressed in the serum of all the hcv patients included in the training set. in contrast, readily detectable levels of this protein were detected in all non-recurrent hcv patients. we assessed whether lt and/or hcv infection account for the different expression patterns of peak a in serum samples of nontransplanted hcv positive and hcv negative subjects with fibrosis. the demographic and biochemical characteristics of patients with fibrosis included in the training set of samples were quite similar to those displayed by the fibrotic patients of this protocol with the exception of hepatic enzymes which, as expected, were higher in lt recurrent hcv patients than in fibrotic non-transplant patients. both, the proteomic profile of the hcv positive samples and the proteomic profile of fibrotic patients non-infected sera, showed no or very low intensity peaks at the 5.9 kda spectra. this markedly differed from the proteomic analysis of the serum samples of healthy subjects included in this set of experiments because all displayed consistent amounts of protein a. interestingly, different etiologies (nash, ald, hbv, ah, cryptogenic) accounted for liver fibrosis in negative hcv patients, further emphasizing the close relationship between the lack of the 5.9 kda protein and the fibrogenic process. next, the spectral data obtained in the test set were applied for validation purposes. all serum samples included in the test set showed an intensity m/z 5905 peak well below the values found in both healthy subjects and non recurrent hcv patients. indeed, in most of these samples the a peak was not detected (figure 3). overall, our results showing markedly decreased expression of the m/z 5905 in the spectral profile of all samples from patients with fibrosis further strengthen the highly sensitive diagnostic performance of this peak. a major limitation of seldi-tof-ms technology is related to the unfeasibility of directly identifying the protein of interest. in fact, for the majority of protein identifications it is necessary to achieve the enrichment of the specific peak by chromatography procedures and purification by sds gel electrophoresis with subsequent triptic digest. in our investigation, amino acid sequencing of the trypsin digest of the 5.9 kda protein revealed it to be a fragment of the fibrinogen a c-chain. human fibrinogen is a circulating 340 kda glycoprotein which has been shown to be of hepatic origin in vivo. moreover, inflammatory stimuli may induce in vitro secretion of this glycoprotein in non hepatic cells including epithelial cells, granulosa cells, cervical carcinoma cells and trophoblasts [20] . however, current evidence strongly suggests that the largest site of human plasma fibrinogen is the hepatocytes [21] . it is comprised of two symmetric half molecules bound by a disulphide knot, each consisting in one set of three different polypeptide termed aa, bb and c. each of these polypeptides is encoded by a separate gene located on chromosome four. the predominant aa of circulating fibrinogen contains 610 aa (63.5 kda), the bb chain 461 aa (56 kda) and the c chain is heterogeneous, but the most abundant form consists of 411 aa (48 kda). the protein shows extensive post translational modification including phosphorylation, sulphation, glycosylation and hydroxylation. the fibrinogen a c-domain of the human fibrinogen is the c-terminal two-thirds of the aa chain that extends from the coiled oil portion of each half of the dimeric fibrinogen molecule [22, 23] . the a c-fragments are released into circulation as natural by-products of fibrinolytic systemic activation and are therefore, found in the systemic circulation in healthy individuals [24] . our results showing almost suppressed expression of the 5.9 kda fragment of the a c-chain of fibrinogen in patients undergoing a fibrogenic process are in agreement with those previously reported by nomura f et al in heavy drinkers [25] . furthermore, these authors showed that serum levels of this fragment were recovered when alcohol intake has ceased for more than 3 months and they also extended their findings to hcv infected patients [26] . later, this fragment was described as having diagnostic value in patients with acute respiratory syndrome [27] , breast cancer [28] and pancreatic adenocarcinoma [29] . the regulation of total human fibrinogen by a number of proinflammatory agents has been previously investigated using the hepg2 hepatocellular carcinoma cell line [30] . this in vitro model faithfully recapitulates fibrinogen expression including a, b and c fibrinogen [31] and has been used to study fibrinogen production and regulation in vitro [32] . accordingly we subsequently assessed the potential regulatory role of several candidate mediators on a-fibrinogen expression in hepg2 cells. a number of proinflammatory/profibrogenic agents that have previously been involved in the pathogenesis of the fibroproliferative processes [33] [34] [35] were tested. among them, only tgf-b showed significant regulatory activity on a-fibrinogen mrna expression and decreased 5.9 kda fibrinogen ac-fragment intensity. of note was, however, that the fold change in the fibrinogen ac-fragment induced by tgf-b in hepg2 cells was makedly lower than that observed in samples from fibrotic patients. the marked differences between the in vivo and in vitro experimental conditions likely account for this discordance. for instance, hepg2 is a human derived carcinoma cell line that shows altered abundance of tgfb receptors [36, 37] which in turn could result in some sort of resistance to this cytokine. on the other hand it is well known that regulation of acute-phase proteins is mediated by a combination of cytokines thus raising the possibility that additional factors involved in inflammatory processes also regulate the expression of the 5.9 kda fragment of fibrinogen [38] . our results are in line with past studies in which tgf-b inhibited the induction of fibrinogen produced by il-6 and decreased the synthesis of fibrinogen in hepg2 and hepb cells [38] , respectively. these latter experiments also showed a parallel diminution in afibrinogen mrna levels. this phenomenon seems to be mediated by post-transcriptional mechanisms since tgf-b did not modify fibrinogen gene transcription, suggesting that the effect of this cytokine in liver cells is regulated at the level of mrna stability [39] . overall, all these results indicate that tgf-b may regulate the synthesis of a-fibrinogen at the postranscriptional level. in summary, the current investigation took advantage of the faster development of hepatic fibrosis in hcv-positive lt patients to identify early circulating serum biomarkers of active fibrogenesis in patients with liver disease. using high throughput seldi-tof-ms technology we unveiled a differential protein pattern profile between early fibrosis recurrence and non recurrent lt patients. six protein peaks displaying statistically significant different intensities were observed within a range of 1000 to 25000 m/z. the peak located at 5905 m/z showed the most remarkable difference, since it was fully detected in non-recurrent lt patients but was almost suppressed in recurrent lt patients. similar results were found when comparing samples of healthy subjects with those of non lt fibrotic patients both hcv positive and negative, indicating that our findings were not related to either lt or hcv infection. identification of this protein peak showed more than a 99% coincidence with a c-terminal fragment of the fibrinogen a chain. moreover, cell culture experiments demonstrated that tgf-b downregulates a-fibrinogen mrna expression and decreases the peak intensity of the m/z 5.9 kda protein in hepg2 cells. in conclusion, we identified a 5.9 kda c-terminal fragment of the fibrinogen a chain as a serum biomarker of early fibrogenic processes in patients with liver disease. since tgf-b inhibited a-fibrinogen mrna expression in hepg2 cells it is temptative to speculate that the activation of this cytokine in the early phases of liver injury could be responsible for the impairment in the circulating levels of the fibrinogen a c-chain fragment in patients with active hepatic fibrogenesis. data s1 materials and methods corresponding to the serum fractionation procedure. fibrosis and cirrhosis reversibility: clinical features and implications biomarkers of hepatic fibrosis, fibrogenesis and genetic pre-disposition pending between fiction and reality pathogenesis of liver fibrosis liver fibrosis hepatic venous pressure gradient identifies patients at risk of severe hepatitis c recurrence after liver transplantation the nomenclature of chronic hepatitis: time for a change proteomic profiling of cholangiocarcinoma: diagnostic potential of seldi-tof ms in malignant bile duct stricture evaluation of liver fibrosis: a concise review biopsy and non-invasive methods for the diagnosis of liver fibrosis: does it take two to tango? non invasive markers of liver fibrosis noninvasive assessment of liver fibrosis serum markers detect the presence of liver fibrosis: a cohort study the enhanced liver fibrosis (elf) score: normal values, influence factors and proposed cut-off values assessment of liver fibrosis before and after antiviral therapy by different serum markers panels in patients with chronic hepatitis c noninvasive assessment of liver fibrosis arfi, fibroscanß, elf and their combinations in the assessment of liver fibrosis: a prospective study progression of liver fibrosis in post-transplant hepatitis c: mechanisms, assessment and treatment evaluation of serum protein profiling by surface-enhanced laser desorption/ionization timeof-flight mass spectrometry for the detection of prostate cancer: i. assessment of platform reproducibility clinical proteomics: searching for better tumour markers with seldi-tof mass spectrometry fibrinogen and fibrin human plasma fibrinogen is synthesized in the liver the structure and biological features of fibrinogen and fibrin comparative structural and functional features of the human fibrinogen alpha c-domain and the isolated alpha c fragment. characterization using monoclonal antibodies to defined coohterminal a alpha chain regions identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry serum fibrinogen a c-chain 5.9 kda fragment as a biomarker for early detection in hepatic fibrosis related to hepatitis c virus serum proteomic fingerprints of adult patients with severe acute respiratory syndrome serum proteomic analysis identifies a highly sensitive and specific discriminatory pattern in stage 1 breast cancer serum diagnosis of pancreatic adenocarcinoma using surface-enhanced laser desorption and ionization mass spectrometry human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis b surface antigen recombinant human fibrinogen and sulfation of the gamma' chain effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines hypoxia and proinflammatory factors upregulate apelin receptor expression in human stellate cells and hepatocytes bacterial lipopolyshaccaride inhibits cb2 receptor expression in human monocytic cells apelin mediates the induction of profibrogenic genes in human hepatic stellate cells transforming growth factor-b and response to anticancer therapies in human liver and gastric tumors in vitro and in vivo differential inhibition of the tgf-b signaling pathway in hcc cells using the small molecule inhibitor ly2157299 and the d10 monoclonal antibody against tgf-b receptor type ii transforming growth factor beta 1 regulates production of acute-phase proteins transformin growth factor-b1 induced smad signaling, cell cycle arrest and apoptosis in hepatoma cells the authors are indebted to drs. f. elortza and i. iloro for their collaboration in the identification of the 5.9 kda protein peak. key: cord-253768-y35m3vh1 authors: springer, sandra a; barocas, joshua a; wurcel, alysse; nijhawan, ank; thakarar, kinna; lynfield, ruth; hurley, hermione; snowden, jessica; thornton, alice; del rio, carlos title: federal and state action needed to end the infectious complications of illicit drug use in the united states: idsa and hivma’s advocacy agenda date: 2020-10-01 journal: j infect dis doi: 10.1093/infdis/jiz673 sha: doc_id: 253768 cord_uid: y35m3vh1 in response to the opioid crisis, idsa and hivma established a working group to drive an evidenceand human rights-based response to illicit drug use and associated infectious diseases. infectious diseases and hiv physicians have an opportunity to intervene, addressing both conditions. idsa and hivma have developed a policy agenda highlighting evidence-based practices that need further dissemination. this paper reviews (1) programs most relevant to infectious diseases in the 2018 support act; (2) opportunities offered by the “end the hiv epidemic” initiative; and (3) policy changes necessary to affect the trajectory of the opioid epidemic and associated infections. issues addressed include leveraging harm reduction tools and improving integrated prevention and treatment services for the infectious diseases and substance use disorder care continuum. by strengthening collaborations between infectious diseases and addiction specialists, including increasing training in substance use disorder treatment among infectious diseases and addiction specialists, we can decrease morbidity and mortality associated with these overlapping epidemics. the epidemiology of the us opioid epidemic continues to evolve and presents new challenges. in recent years, the epidemic has shifted from prescription opioid pills to injection of illicitly produced opioids, including heroin and fentanyl, with concomitant increasing injection of stimulants including cocaine and methamphetamine [1] [2] [3] . as a result, the incidence of injection drug use (idu)-related infections such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), and invasive bacterial and fungal infections, including staphylococcus aureus bacteremia, endocarditis, and skin and soft tissue infections, is rising [2, [4] [5] [6] [7] [8] [9] [10] [11] . injection of fentanyl or heroin alone and in combination with stimulants have led to new hiv outbreaks among people who use drugs throughout the country [4, [10] [11] [12] . in addition to hiv, both acute hcv and hbv infection incidence has mirrored the rise in injection opioids [5, 13] and hospitalizations for injection opioid-related endocarditis have increased more than 12-fold in recent years [6, 8] . at the state of the union address in february 2019, president trump called for a plan to end hiv as an epidemic in the united states. this plan seeks to reduce new infections by 75% in the next 5 years and by 90% in the next 10 years. even amid the opioid epidemic, such ambitious goals can be achieved if policy changes occur and adequate resources are provided. thus, more than ever, addressing the hiv epidemic as well as hcv and other idu-related infections also requires a focus on the opioid and co-occurring stimulant epidemics. doing so will improve patients' outcomes and reduce the public health risk of infectious disease transmission. nevertheless, a number of barriers to care in people who use drugs need to be addressed to end the opioid and hiv epidemics in the united states as well as reduce the other infectious disease health outcomes. to address these barriers we recommend expanding medicaid, expanding access to harm reduction services, improving treatment and surveillance to enhance the continuum of care, and treating opioid and other substance use disorders (sud), including through lowbarrier hospital and community-based treatment, as well as in the criminal justice setting. the authors of this paper are members of a working group created by the infectious diseases society of america (idsa) and the hiv medicine association (hivma) in 2017 to enhance their efforts to educate and advocate on the urgent need to better prevent and treat serious infections linked to the opioid and stimulant epidemics and underlying sud. the working group developed a policy agenda reflecting issues raised by infectious diseases and hiv physicians and health care professionals working at the intersection of infectious diseases and opioid use disorder (oud) and other sud epidemics more broadly. in this paper, we outline practice and policy suggestions that are likely to positively impact the oud, stimulant epidemics, and infectious diseases epidemics, and that have been reviewed and approved by the idsa and hivma board of directors as a call to action for infectious diseases and hiv practitioners. medications for treatment of opioid use disorder (mouds, which is now the preferred term to medication-assisted therapy) are recognized as the most effective treatments for oud [14] . there are 3 food and drug administrationapproved mouds-methadone, buprenorphine, and extended-release naltrexone (xr-ntx). methadone is a full opioid agonist and buprenorphine is a partial opioid agonist, while xr-ntx is an opioid antagonist. all are successful in treating oud and in decreasing mortality. all reduce illicit opioid use, opioid craving, overdose, and hiv and hcv transmission [14, 15] ; and buprenorphine and xr-ntx also improve hiv viral suppression in people living with hiv, the gold standard of care in treatment of hiv that is associated with reduced mortality and reduced transmission [16, 17] . of the 3 mouds, access to methadone and buprenorphine are limited by regulations. prescribing requires special training outside postgraduate programs and either a waiver from the drug enforcement agency in the case of buprenorphine or treatment in a federally certified opioid treatment program in the case of methadone. unfortunately, many clinical settings lack physicians trained in oud treatment. only about 5% of the nation's physicians have waivers to prescribe buprenorphine and most substance use treatment programs do not have opioid treatment programs, which makes methadone treatment challenging to obtain [18] . therefore, the prevailing care for these patients typically consists of withdrawal management or detoxification and referral to outpatient resources for follow-up treatment. this asks patients with severe oud to tolerate withdrawal symptoms, risking premature exit from hospital, and relapse to opioid use after failure to connect with oud treatment referrals. such inadequate care results in prolonged hospitalizations due to concern about relapse and nonadherence if patients leave the hospital, readmissions after oud relapse, and, if concomitant infection is present, lack of antibiotic adherence and reinfection. ultimately, this cycle leads to poor clinical outcomes, high health care costs, and excess deaths. infectious disease specialists are at the frontlines in many hospitals treating infectious diseases in people who use drugs and have an opportunity to screen and treat co-occurring suds. in 2018, congress passed legislation offering opportunities to heighten the response to the opioid epidemic and its infectious diseases complications. on 24 october 2018, president trump signed into law the substance use disorder prevention that promotes opioid recovery and treatment (support act). this bill includes a range of prevention, care, workforce, and public health provisions to strengthen the response to the opioid epidemic (table 1 ) [19] . the bill was passed with strong bipartisan support from congressional members recognizing that the status quo was woefully inadequate to respond to the opioid epidemic. idsa and hivma supported the support act, including provisions that improved medicaid and medicare coverage of sud treatment and services, and that increased the patient cap for which physicians could prescribe moud. priority issues for idsa and hivma were provisions authorizing funding for the centers for disease control and prevention (cdc) to eliminate opioid related infections through improved surveillance and prevention for infections linked to idu and funding for the health resources and services administration to build workforce capacity through a new substance use treatment provider loan forgiveness program, offering up to $250 000 in loan repayment over 6 years for providers working in substance use treatment facilities [20] . both programs depend on congress to appropriate funding. five million dollars was appropriated for fiscal year 2019 for the cdc eliminate opioid related infections funding provision. the fiscal year 2020 appropriations bills were signed into law on december 20, 2019 and included $10 million for the cdc's eliminate opioid related infections program and $12 million for the new substance use disorder loan forgiveness program [21] . other legislative proposals supported by idsa and hivma that have been introduced in the 116th congress include: the medicaid re-entry act that would allow medicaid coverage for inmates during the 30-day period preceding release from a public institution [22] ; the comprehensive addiction resources emergency act modeled after the highly successful ryan white hiv/aids program and that would provide funding to states to support comprehensive prevention, care, and treatment programs [23] ; and the mainstreaming addiction treatment act that would eliminate the requirement for clinicians to obtain a waiver to prescribe buprenorphine [24] . as outlined in this paper, urgent policy action is needed to reduce illness and death due to our nation's substance use epidemics. in addition to state and jurisdictional bans or restrictions on syringe services programs (ssp), funding remains a significant barrier to expanding access to ssp services [25] . increased state and federal funding are needed to expand ssp and other harm reduction services, including access to moud and infectious diseases treatment services in order to decrease hcv, hiv, idu-related infections, and vaccine-preventable diseases, and improve oud-related outcomes [26, 27] . studies have demonstrated that incorporation of ssps combined with moud is associated with a decrease in hcv and hiv acquisition risk by 76% and 34%, respectively [28] . ssps also can facilitate vaccine uptake for hepatitis a virus (hav), hbv, influenza, and invasive pneumococcal disease, which disproportionately impact people who inject drugs or experience unstable housing or homelessness [29, 30] . ssps can offer a safe space without stigma for individuals with suds to also receive counseling regarding safe sexual practice, safe injection practice, and provision of hiv preexposure prophylaxis (prep) and contraception. ssps that also provide treatment for oud can facilitate linkage to care for effective evidence-based treatments [31] . furthermore, persons with suds may be reluctant to seek nonemergent care for skin and soft tissue infections and postpone medical evaluation until the need is more urgent. providing care for skin and soft tissue infections in a supported setting may reduce progression to serious infections and reduce complications like wound botulism or partial drainage of abscesses. given the increased incidence of idu-associated infections and overdose deaths [1, 2] , there is a need to provide ongoing support for and increased access to ssps. the hiv outbreak in scott county, indiana, in addition to other emerging hbv and hcv epidemics, has highlighted the need to expand ssps, particularly in nonurban areas [32] . although federal funds to support ssps was an important step, additional federal funding and flexibility are needed to fully cover services and costs associated with these programs, including purchasing of sterile syringes and to support delivery of moud at ssps [33] . in jurisdictions where ssps are prohibited or sparse, cities and states should be incentivized to modify their laws and to encourage uptake by local jurisdictions. in some states, there is a limit on the number or location of such programs, or ssps may only be allowed during certain circumstances (ie, public health emergencies) [34] . such limitations should be eliminated given the documented need for these programs and their potential to reduce infectious diseases [34, 35] . additionally, drug paraphernalia laws, which prohibit possession of syringes, pose barriers to ssp expansion and effectiveness [36] . state and local governments should be encouraged to employ innovative programming, including mobile delivery and contracting with communitybased organizations. additionally, states should be incentivized to eliminate 1-for-1 syringe exchange (ie, exchanging 1 used syringe for 1 sterile syringe) because they create barriers to individuals who inject drugs having an adequate supply of sterile 2. incentivize states to give more authority to local governments to establish ssps and to eliminate barriers to sterile syringes, such as one-for-one needle exchange requirements. 3. allow jurisdictions that have approved overdose prevention sites or supervised injection facilities to implement and evaluate the intervention in the united states. 4. urge all states to expand medicaid. 5. fund demonstration projects and pilot studies to identify effective care models for comanagement of infectious diseases and sud. 6. increase funding for national and regional warmlines and peer-to-peer mentoring, programs for prescribers of mouds, and for cotreatment of related infections. 7. eliminate the buprenorphine waiver, remove patient caps, and offer grant funding for case management and other support services to clinics and practices that prescribe mouds. 8. increase funding and reimbursement for telehealth and other low-barrier access care delivery models. 9. support implementation of universal hcv testing. 10 . develop a national surveillance system to report and track idu-related infections to predict and respond to emerging epidemics. 11. integrate moud and counseling services during incarceration. 12. integrate screening for oud and treatment with moud into jails and prisons. 13. expand access to harm reduction during and after incarceration. 14. allow states to initiate medicaid coverage 30 days prior to release from criminal justice settings to facilitate care initiation and coordination during the transition to the community. syringes. secondary exchange, or the distribution of sterile syringes from 1 person to a social network, is often necessary due to distance and transportation barriers. in addition to ssps, other harm reduction services are needed to address the expanding epidemics. overdose prevention sites (also known as supervised injection facilities or safe injection sites) are facilities in which persons can inject drugs in a safe, clean environment under medical supervision. overdose prevention sites enable rapid, life-saving intervention in the case of drug overdose and can also provide injection equipment and referrals to care for sud and other health care services. overdose prevention sites have existed for many years in europe, australia, and canada. studies of overdose prevention sites in vancouver and sydney have found an increase in withdrawal management or detoxification service referrals and a decrease in drug overdose rates, syringe sharing, public injections, and publicly discarded syringes [37] [38] [39] . several us municipalities have advocated for overdose prevention sites, but political opposition has so far impeded implementation. a recent modeling study in seattle estimated that an overdose prevention site would yield cost savings through prevention of overdose deaths, enrollment in mouds, prevention of emergency medical services deployments, and emergency department visits and hospitalizations [40] . although concerns have been raised about violation of federal and state drug laws, overdose prevention sites have been legally established successfully in areas outside of the united states. review of the processes and experience could facilitate implementation in us jurisdictions that have approved overdose prevention sites. jurisdictions that have approved overdose prevention sites should be allowed to implement and evaluate the intervention in the united states. significant work needs to be done to improve the care continuum for people with infectious diseases and co-occurring sud. the first step needs to be ensuring that everyone has access to health care. federal support for the medicaid expansion must continue and the 14 states that have not expanded medicaid should be incentivized to do so [19] . recent studies have shown that expansion of health care services, mostly via medicaid expansion, increased utilization of moud [3, 41, 42] . expanding access to health coverage is necessary to prevent and treat the infection, underlying sud, and improve overall mortality and quality of life as evidenced by studies finding an association between enrollment in an affordable care act qualified health plan and improved outcomes for people with hiv [43] . as a next step, treatment programs that integrate substance use care and treatment for infectious complications in order to improve outcomes are needed. treatment of both the sud and associated infections (eg, hiv, hcv) can be cost-effective and is associated with improved infection and sud outcomes [44] . previous studies have shown that patients with either hiv or hcv who receive mouds have improved viral suppression (hiv) [16, 17] , achieve sustained virologic response/ cure (hcv) [45, 46] , and have increased retention in care [47] . however, significant gaps remain in understanding the role substance use treatment plays in caring for people with other idurelated infections, such as endocarditis, deep tissue abscesses, skin and soft tissue infections, and bone and joint infections. one innovative care model combined outpatient parenteral therapy with buprenorphine treatment and showed similar clinical and drug use outcomes to completing inpatient therapy and resulted in reduced hospital length of stay by 24 days [48] . studies are needed to evaluate novel approaches to antimicrobial treatment for idu-associated infections such as the role of long-acting glycopeptides. increased funding is necessary for other demonstration projects and pilot studies to identify effective care models for comanagement of infectious diseases and oud as well as other suds. additionally, we need to expand the network of providers prescribing moud. most infectious diseases and hiv physicians receive little to no formal training in the management of oud and other suds. training to identify and treat oud and other suds should be increased in medical schools, nursing schools, physician assistant schools, residency programs, and within hospitals. while all infectious diseases and hiv physicians should become familiar with harm reduction principles and be able to counsel patients regarding safe injection practices, we need broader national support for physicians table 2 to comanage oud, suds, and co-occurring infectious diseases. lack of confidence has been identified as a major barrier preventing some physicians from integrating buprenorphine into their practice for the treatment of oud [49] . warmlines, such as the one run by the clinical consultation center at the university of california san francisco, and videoconferencingbased learning communities such as project echo, are excellent resources to provide support on a number of clinical aspects of disease management (table 2 ) [44] . increasing funding for national and regional warmlines, telehealth-based learning communities, peer-to-peer mentoring programs, and other technical assistance programs such as the opioid response network will help decrease barriers to providing substance use treatment. the opioid response network is a network of experienced clinicians that is funded by the substance abuse mental health and services administration to provide technical assistance to improve access to substance use treatment. in addition, a reorganization of the buprenorphine prescribing system is needed. in order to increase the number of providers who prescribe moud and improve patient access, we recommend eliminating the buprenorphine waiver requirement, removing the patient caps, and dedicating grant funding for case management and other support services to clinics that prescribe mouds. increased funding and reimbursement are also needed for low-barrier care delivery models such as telehealth. these innovative programs, which have already begun to be tested in infectious diseases/oud comanagement [50] , have the potential to increase medication uptake and improve outcomes by increasing access to treatment where people reside. in addition, multidisciplinary team meetings, including surgeons, sud specialists, inpatient internal medicine clinicians, nurses, social work, and case management, are being piloted in several hospitals across the country in order to make informed and collaborative decisions on complex patients, such as those with recurrent endocarditis following valve repair. evaluation of the impact of these types of collaborative efforts, both on patient outcomes and workplace satisfaction, can help inform best practice for all hospitals. we also need to address the requirements of particularly highrisk patients, including pregnant women and infants born to mothers with oud, and persons experiencing homelessness who may be unable to access traditional care. oud among pregnant women has increased significantly and there is an urgent need to build capacity to manage oud among pregnant women [51] . infants born to mothers with oud during pregnancy are at increased risk for hiv, hbv, and hcv. screening for hiv, hbv, and hcv is recommended for all pregnant women [52] and has been successfully integrated into most prenatal screening paradigms, allowing for perinatal management that decreases the risk of infant infection. in september 2006, the cdc recommended screening all sexually active persons 13-65 years old for hiv at least once, but this has not occurred and needs emphasis in order to end the epidemic. in august 2019, the us preventive services task force issued a draft recommendation for universal hcv screening [53] . given that overall incidence of hcv is increasing alongside the opioid epidemic [54] , strategies including provider education and increased resources are needed to ensure universal hcv testing is performed, particularly among women of child-bearing age and in prenatal care to prevent infant infection [55] [56] [57] . persons experiencing homelessness and unstable housing are similarly at increased risk for infections associated with sud. this is, in part, due to the high prevalence of concomitant untreated mental illness and sud among these individuals and sanitation issues [58, 59] . it is also due to our inability to implement effective management strategies for sud and infections in this vulnerable population. in addition to ensuring persons who experience homelessness receive treatment of their infectious diseases and sud through low-barrier and street-based medicine programs, expanding access to stable housing would also improve short-and long-term outcomes and should be part of a comprehensive strategy [60, 61] . finally, to monitor progress of these interventions, we need a standardized mechanism for reporting idu-related infections. other than for hiv and, in some states, for hcv infection, there is no national database of idu-related infections for surveillance, prevention activities, and program evaluation. this makes it difficult-if not impossible-to identify, predict, and prevent new infectious disease epidemics related to substance use in the united states. in addition, the majority of federal funding has been directed towards opioid overdose treatment and hiv resultant from idu, but not toward the bacterial and fungal infection complications, partly due to lack of integrated surveillance systems for serious idurelated infections, such as endocarditis. developing national surveillance systems to track and predict new epidemics before they happen and increasing national institutes of health funding for research into other infectious diseases related to the worsening sud epidemics in this country are urgently needed. over half of the criminal justice-involved population (cjip) have oud or sud, with a 10-fold higher prevalence than found in the general adult population [62] . as such, employing and enforcing evidence-based treatment guidelines that address the overlap of sud and infectious diseases in the criminal justice system has the potential to improve morbidity and mortality substantially. key components include evaluating new entrants for sud and idu-related infections, integrating moud and counseling services during incarceration, providing both appropriate medical care during incarceration and harm reduction during and after incarceration, care coordination, seamless referral to outpatient care for sud and chronic infections, and uninterrupted insurance coverage for cjip. intertwined with national increases in sud and incarceration rates, there have been substantial increases in hiv and hcv in cjip, as well as outbreaks of hav and hbv [63, 64] . inequities that exist in health care access in the community are amplified by criminal justice involvement, leading to premature deaths [65] . mortality rates are high following release, primarily driven by untreated oud leading to fatal overdose, progression of hiv, and hbv/hcv-induced liver disease [66] [67] [68] . additionally, overall infectious disease testing rates and rates of vaccination against hav and hbv are low [69, 70] . integration of infectious disease management with treatment for oud in cjip is an endorsed strategy for reducing these health inequities that will likely lead to improved infectious disease outcomes and facilitates linkage to care postrelease [16, 17, 71] . despite the evidence, however, few incarcerated settings offer moud. sud screening in jails and prisons with linkage to substance use treatment also decreases postrelease mortality [72, 73] and increases postrelease hiv viral suppression [16, 17] . clearly, prevention and treatment for oud and associated infections in this population can improve both individual outcomes and public health, especially when initiated during incarceration. time spent in prison or jail provides a reachable moment-an opportunity to engage a vulnerable population. screening for oud and treatment with moud need to be integrated into jails and prisons to improve substance use and infectious diseases outcomes. in addition to testing and treatment, access to harm reduction tools to prevent infection needs to be prioritized in the cjip. harm reduction tools like condoms and clean needles are not routinely available in prisons or jails despite several research studies demonstrating the need for such tools, and the consequences of not providing them [74, 75] . increasing awareness and availability of prep in jails and prisons-continued from the community, initiating while detained, or initiated before release-need to be urgently deployed, especially in communities deemed to be at high risk of hiv outbreak [76] . as evidenced by previous successful implementation of intensified harm reduction, expansion can be implemented in jails, effectively containing outbreaks [77] . substance use treatment coupled with uninterrupted health insurance is needed to improve outcomes among persons who are released from jail and prison. as a case study, expansion of hcv treatment during incarceration is feasible, cost-effective, and the best option to move closer to national hcv elimination [78, 79] . hcv diagnosis in jails with linkage postrelease is a feasible alternative if hcv treatment costs are deemed prohibitive [80] . a major barrier in hcv linkage to care postrelease is that 90% of states have policies that withdraw enrollment in insurance programs when people are incarcerated, outsourcing health care to medical corporations hired by criminal justice administrators [81] . prior to release, there are often attempts to reestablish health insurance, but this is complex because of uncertainty around the date of release and place the person will live. the process of re-entry is a vulnerable time for people who are incarcerated, with high mortality related to drug use but also associated with suboptimal postrelease management of chronic conditions like liver disease [82] . increased flexibility of medicaid, allowing initiation of insurance before release and sustained coverage prior to conviction, would improve health care transitions into and out of correction settings. finally, additional research funding is needed to develop and evaluate strategies to manage non-hiv/hcv-related infections secondary to idu, such as skin and soft tissue infections or infective endocarditis. despite increasing frequency of endocarditis in people with oud/sud [7] , and high rates of history of incarceration in people with skin and soft-tissue infections [7] , there are limited data on the epidemiology of disseminated bacterial and fungal infections in cjip. since the time this manuscript was accepted in december of 2019 the covid-19 pandemic has changed the world and has made the implementation of many of these recommendations even more urgent. we are at a pivotal moment in the opioid epidemic in the united states. as we desperately attempt to decrease the staggering number of overdose deaths, we must also grapple more broadly with idu in general, which is causing increases in hiv, hcv, and other idu-related infections. as a result, we as infectious disease specialists need a paradigm shift in our clinical approach, and we need broad and aggressive policy changes to support that shift. throughout history, infectious diseases clinicians have risen to the challenge. in 1998, dr jonathan mann said in an address, "when the history of aids and the global response is written, our most precious contribution may well be that, at a time of plague, we did not flee, we did not hide, we did not separate ourselves. " this time is no different-it is our epidemic too. disclaimer. the funders were not involved in the research design, analysis, or interpretation of the data or the decision to publish the manuscript. financial support. this work was supported by the national institute on drug abuse, national institutes of health (grant number k02 da032322 for career development to s. a. s.). supplement sponsorship. this supplement is sponsored by the centers for disease control and prevention. potential conflict of interests. s. a. s. has provided scientific consultation to alkermes inc. j. a. b. reports providing waiver training courses for providers to become buprenorphine prescribers through the providers clinical support system. a. w. is a consultant to gensler architectural firm. a. n. reports grants from gilead focus program outside the submitted work. r. l. reports royalties for a book on infectious disease surveillance donated to minnesota department of health. all other authors report no potential conflicts. increases in drug and opioid-involved overdose deaths-united states hospitalizations related to opioid abuse/dependence and associated serious infections increased sharply nonopioid overdose death rates rose almost as fast as those involving opioids community outbreak of hiv infection linked to injection drug use of oxymorphone-indiana increases in acute hepatitis c virus infection related to a growing opioid epidemic and associated injection drug use, united states trends in drug use-associated infective endocarditis and heart valve surgery increasing infectious endocarditis admissions among young people who inject drugs hospitalizations for endocarditis and associated health care costs among persons with diagnosed drug dependence-north carolina invasive methicillinresistant staphylococcus aureus infections among persons who inject drugs-six sites notes from the field: hiv diagnoses among persons who inject drugs-northeastern massachusetts outbreak of human immunodeficiency virus infection among heterosexual persons who are living homeless and inject drugs notes from the field: hiv infection investigation in a rural area-west virginia increases in acute hepatitis b virus infections-kentucky tip 63: medications for opioid use disorder-executive summary oral substitution treatment of injecting opioid users for prevention of hiv infection retention on buprenorphine is associated with high levels of maximal viral suppression among hiv-infected opioid dependent released prisoners extended-release naltrexone improves viral suppression among incarcerated persons living with hiv with opioid use disorders transitioning to the community: results of a double-blind, placebo-controlled randomized trial opioid treatment programs and related federal regulations education, defense, state, foreign operations, and energy and water development appropriations act, 2020. passed by the us house of representatives hr 1865 -further consolidated appropriations act hr 2569 comprehensive addiction resources emergency act of 2019 hr 2482 mainstreaming addiction treatment act of 2019 national academies of sciences, engineering, and medicine. integrating responses at the intersection of opioid use disorder and infectious disease epidemics: proceedings of a workshop integrating treatment at the intersection of opioid use disorder and infectious disease epidemics in medical settings: a call for action after a national academies of sciences, engineering, and medicine workshop needle and syringe programmes and opioid substitution therapy for preventing hcv transmission among people who inject drugs: findings from a cochrane review and meta-analysis homelessness and hepatitis a opioid analgesic use and risk for invasive pneumococcal diseases optimising health and safety of people who inject drugs during transition from acute to outpatient care: narrative review with clinical checklist reduction of injectionrelated risk behaviors after emergency implementation of a syringe services program during an hiv outbreak the policy surveillance program. a lawatlas project. syringe service program laws interventions to prevent hiv and hepatitis c in people who inject drugs: a review of reviews to assess evidence of effectiveness state syringe and drug possession laws potentially influencing safe syringe disposal by injection drug users impact of a medically supervised safer injection facility on community drug use patterns: a before and after study attendance at supervised injecting facilities and use of detoxification services medically supervised injecting centres the projected costs and benefits of a supervised injection the role of health insurance on treatment for opioid use disorders: evidence from the affordable care act medicaid expansion the affordable care act in the heart of the opioid crisis: evidence from west virginia evidence from a multistate cohort: enrollment in affordable care act qualified health plans' association with viral suppression addiction medicine consultations reduce readmission rates for patients with serious infections from opioid use disorder intensive models of hepatitis c care for people who inject drugs receiving opioid agonist therapy: a randomized controlled trial low hepatitis reinfection following direct acting antiviral therapy among people who inject drugs on opioid agonist therapy treatment of medical, psychiatric, and substance-use comorbidities in people infected with hiv who use drugs outpatient parenteral antimicrobial therapy plus buprenorphine for opioid use disorder and severe injection-related infections prescribing practices of rural physicians waivered to prescribe buprenorphine integrated, co-located, telemedicine-based treatment approaches for hepatitis c virus management in opioid use disorder patients on methadone state strategies to address opioid use disorder among pregnant and postpartum women and infants prenatally exposed to substances, including infants with neonatal abstinence syndrome american college of obstetricians and gynecologists. routine tests during pregnancy us preventive services task force. draft recommendation statement hepatitis c virus infection in adolescents and adults: screening increases in acute hepatitis c virus infection related to a growing opioid epidemic and associated injection drug use, united states hepatitis c virus infection among reproductive-aged women and children in the united states reducing risk for mother-to-infant transmission of hepatitis c virus: a systematic review for the u.s. preventive services task force hepatitis c screening in mothers and infants exposed to opioids the opioid epidemic in veterans who were homeless or unstably housed experience and outcomes of hepatitis c treatment in a cohort of homeless and marginally housed adults hepatitis c treatment outcomes among homelessexperienced individuals at a community health centre in boston adherence to protease inhibitors, hiv-1 viral load, and development of drug resistance in an indigent population drug use, dependence, and abuse among state prisoners and jail inmates prevalence of infection with hepatitis b and c viruses and co-infection with hiv in three jails: a case for viral hepatitis prevention in jails in the united states incarceration, drug use, and infectious diseases: a syndemic still not addressed hivpositive and in jail: race, risk factors, and prior access to care risks of drug-related death, suicide, and homicide during the immediate postrelease period among people released from new york city jails mortality after prison release: opioid overdose and other causes of death, risk factors, and time trends from substance use disorders, psychiatric disorders, and mortality after release from prison: a nationwide longitudinal cohort study preventive healthcare for underserved women: results of a prison survey hepatitis b vaccination practices in state and federal prisons linkage to hepatitis c care after incarceration in jail: a prospective, single arm clinical trial postincarceration fatal overdoses after implementing medications for addiction treatment in a statewide correctional system the impact of opioid substitution therapy on mortality post-release from prison: retrospective data linkage study the furthest left behind: the urgent need to scale up harm reduction in prisons a spray bottle and a lollipop stick": an examination of policy prohibiting sterile injecting equipment in prison and effects on young men with injecting drug use histories the path to implementation of hiv pre-exposure prophylaxis for people involved in criminal justice systems the global state of harm reduction in prisons cost-effectiveness and budgetary impact of hcv testing, treatment and linkage to care in u.s. prisons prevention of hepatitis c by screening and treatment in u.s. prisons a budget impact analysis of newly available hepatitis c therapeutics and the financial burden on a state correctional system filling the gap: the importance of medicaid continuity for former inmates public health implications for adequate transitional care for hivinfected prisoners: five essential components key: cord-258234-qn8xp4v9 authors: striker, rob; mehle, andrew title: inhibitors of peptidyl proline isomerases as antivirals in hepatitis c and other viruses date: 2014-11-06 journal: plos pathog doi: 10.1371/journal.ppat.1004428 sha: doc_id: 258234 cord_uid: qn8xp4v9 nan viruses have small genomes with limited coding capacity. a common strategy by which viral genomes maximize their coding capacity is to express multifunctional proteins that promiscuously interact with various cellular partners to perform an array of essential functions. these interactions often involve flexible, and in some cases intrinsically disordered, viral domains or entire proteins that assume distinct conformations only upon binding cellular partners (see review, [1] ). viral coding capacity is further enhanced by relying on host factors and protein folding machinery to access different conformations and functions. these disordered peptide regions can be computationally recognized by features such as glycine, serine, and proline residues in contexts that are not conducive to b-strands or a-helices (reviewed by [2] ). bioinformatic analysis was used to predict the rigidity of proteins encoded by nearly 3,500 genomes from archaea, bacteria, eukaryotes, and viruses. this analysis suggests that almost all genomes with greater than 50% of their encoded residues in a predicted disordered state are viral genomes [3] . thus, disordered proteins are enriched in the viral proteome and are common features to a large number of viruses. flexible viral proteins and/or domains interact with the cellular folding machinery, including proline isomerases. while proline is traditionally thought of as being a rigid amino acid that can ''kink'' the polypeptide chain, prolines can slowly rotate between two energetically similar configurations, cis or trans. this rotation is only fast enough to be physiologically relevant when facilitated by proline isomerases (rotamases) such as mammalian cyclophilins [4] . at least four structurally distinct classes of cellular proline isomerases exist in bacteria and eukaryotes, and some viruses encode their own proline isomerase [4, 5] . identification of the host isomerases exploited by viruses and the viral proteins that require them to perform essential viral functions for replication in culture, or more importantly, in animals, presents an obvious antiviral strategy. whether or not inhibition of host proline isomerases could be an antiviral strategy for hepatitis c virus (hcv) was a subject of debate for several years. cyclophilins are the most-studied proline isomerases and likely the least discriminating in terms of substrate choice. cyclophilins were discovered as a target of the immunosuppressive drug cyclosporine. cyclosporine, as well as immunosuppressant tacrolimus, both inhibit the adaptive immune response by inhibiting distinct classes of peptidyl proline isomerases; cyclosporine inhibits cyclophilins while tacrolimus inhibits fk506-binding proteins (fkbps) [4] . a decade ago two groups showed that self-replicating hcv rnas (replicons) are dependent upon hcv nonstructural proteins and are inhibited by cyclosporine, but not tacrolimus [6, 7] . this led to the proposal that hcv replication requires cyclophilins, but not fkbps. this conflicted with observations by many clinical scientists. from 1983 until around 1998, when tacrolimus began supplanting cyclosporine, hcv-positive transplant patients received cyclosporine to prevent organ rejection, but this treatment did not simultaneously cure their hcv infection. then and now, hcv is not only the most common reason for a liver transplant but also a common reason for needing a second liver transplant because immunosuppression (or the associated higher viral load that may arise from increased replication in the transplanted liver) clearly accelerated hcv-mediated disease in the liver graft. thus, defining a role for proline isomerases during hcv infection in patients was confounded by the fact that any antiviral benefit from cyclosporine must overwhelm its immunosuppressive effect. replicon data provided critical evidence for the role of cyclophilins during hcv infection in culture, yet cyclosporine and tacrolimus appeared to be ''equally bad'' for hcv-infected solid organ transplant patients; thus, controversy persisted [8] . there is now no longer a question that cyclophilin a plays a crucial role during hcv replication and cyclophilin inhibitors possess potent anti-hcv activity. using an innovative mousehuman chimera model, it was shown that hcv replicates to significantly lower levels in cyclophilin a-deficient animals than in mice with cyclophilin a [9] . several cyclophilin inhibitors that retain anti-hcv activity but do not have the immunosuppressive properties of cyclosporine and tacrolimus have also been studied. these compounds maintain their antiviral activity, and at least one has reached late phase iii trials for hcv [10] . whether there is a role for cyclophilin inhibitors in future cures of hcv remains unclear. multiple inhibitors that target viral enzymes and promote viral clearance in a high percentage of patients are being adopted [10] . still, cyclophilin inhibitors may ultimately prove clinically useful for viral infections that resist new treatment regimes or useful when used in combination with existing therapies. hcv encodes only ten proteins, including structural and nonstructural (ns) proteins. cyclophilin inhibitors that reduce viral replication also block interactions between cyclophilin a and ns5a, suggesting that this association is important during the viral life cycle and might be the relevant target of the antiviral activity of cyclosporine [11] [12] [13] . an interaction between cyclophilin b and the viral polymerase ns5b was also reported [14] . selection of subgenomic replicons for cyclosporine resistance created mutations in both ns5a and ns5b [15] . mutating specific ns5b residues in isolation conferred low level or no resistance to cyclosporine [16] , whereas mutations in ns5a, the most prolinerich hcv protein, conferred the highest levels of resistance [15] . these data suggest that ns5a is the most important substrate of cyclophilins for hcv replication. additional work by multiple groups showed that cyclophilin a is the major, if not only, cyclophilin to play a role in the hcv life cycle [16, 17] . ns5a is approximately 54 kd with three distinct domains separated by low-complexity sequences (figure 1 ). approximately 10% of the amino acids in ns5a are prolines. in addition, ns5a has regions of high disorder, multiple reversible posttranslational modifications, and helical tendencies [18] . ns5a displays significant sequence variability across viral genotypes, yet it typically contains approximately 8 pro-pro motifs. it is not clear if these conserved diprolines are biologically relevant cyclophilin ainteracting sites. mapping studies have implicated the residues in domain 2 as critical for the effects of cyclophilin a and cyclophilin inhibitors during viral replication ( figure 1a , 1b) [11] [12] [13] . nuclear magnetic resonance (nmr) studies indicate that much of domain 2 is disordered [18] . additionally, nmr studies show that several prolines in domain 3 occupy both cis and trans configurations and may thus be substrates for isomerases [18] . domain 3 from genotype 2 strains also contains a proline-rich insert ( figure 1b) . but, despite the presence of the proline-rich insert at least one genotype 2 strain has less cyclophilin dependence [19] , suggesting that specific proline context, rather than the number or percentage of proline residues may determine the importance of cyclophilin and whether cyclophilin inhibitors are an applicable antiviral strategy. within ns5a domain 2, most evidence implicates a single proline (p319) and the tryptophan, aspartate, and tyrosine residues surrounding it in a warpdyn motif as being especially significant [11, 13, 20] . the warpdyn motif itself is bracketed by additional proline residues (p[a/i]warpdynp). mutations conferring resistance to cyclophilin inhibitors map to the warpdyn motif, e.g., r318w and d320e [13] . the d320e mutation had little to no effect on the binding of ns5a to cyclophilin a. however, even though this is a conservative change, the d320e mutation appears to alter the local protein conformation. nmr spectra of a 20-amino-acid peptide that includes the prolines bracketing the warpdyn motif showed that the isomerization state of p319 exists in equilibrium with approximately 75% in the trans conformations. conversely, spectra collected on peptide containing the resistance mutation d320e revealed that approximately 70% of p319 was now in the cis conformation. thus, mutations that confer resistance to cyclophilin inhibitors shift the cis:trans ratio of configurations in the motif, reducing dependence on the isomerase activity of cyclophilins [13] . cyclophilin a has at least low-level affinity for multiple other stretches of domain 2, including two tripeptide alanine-hydrophobic residue-proline motifs [20] that surround the warpdyn. additional mutations adjacent to the warpdyn motif arise in patients treated with cyclosporine. an atypical proline (p328, which is the consensus amino acid in only 5% of genotype 1 strains) downstream of the motif was detected in one patient prior to treatment that mutated to serine following exposure to cyclosporine [21] . the ns5a p328 variant possessed enhanced susceptibility to cyclosporine in replicon experiments that was lost upon mutation to serine, suggesting in at least this patient a figure 1 . hcv ns5a is a protein that is rich in both proline residues and disorder and that associates with cyclophilin a via domain 2. a) crystallographic model of domain 1 of ns5a (residues 37-213, pdb 1zh1), which has a well-defined structure. interestingly, a similar, but alternate structure for domain 1 with a completely different dimer interface (pdb 3fqm) has also been solved and it is currently unknown whether a single conformation or both best represent the intracellular state. b) linear representation of ns5a. current evidence suggests the entire carboxy terminus is disordered, but it has traditionally been studied as seperate domains termed domain 2 and 3 [11] . red bars represent diprolines. plot of ns5a disorder prediction from iupred (iupred.enzim.hu/). doi:10.1371/journal.ppat.1004428.g001 concentration of cyclosporine was achieved in vivo that had an antiviral effect [21] . these data identify critical regions in ns5a that recruit and utilize cyclophilin a to participate in viral genome replication. despite the depth of information regarding ns5a domain 2, our basic understanding of which viral prolines in hcv or other viruses require isomerases is limited. this is in part because only one other example has been investigated extensively-the association between cyclophilin a and capsid (ca) from the hiv gag protein [22] . that interaction was captured in crystal structures, revealing that cyclophilin a binds a relatively flexible loop between structured parts of the viral ca ( figure 2 ) [23] . it is certainly premature to draw general conclusions about viralcyclophilin interactions when only two have been characterized to any depth, but some noteworthy similarities and differences can be made. the hiv ca loop contains a single glycine-proline motif, with the proline (p90) existing in both trans and cis conformations in different structures and in solution [22, 24] . the lack of structural rigidity for this loop is exemplified by the multiple conformations detected in solution structures, the higher b-factor for this region of the protein in crystal structure without cyclophilin, or disorder and the lack of structural information in some models (figure 2 ). the glycine-proline motif of ca has no obvious resemblance to the cyclophilin a interaction site in hcv ns5a. however, these regions share several common properties: the flexible cyclophilin-interacting loop in ca is also bracketed by prolines, just as the pawarpdynp motif in hcv; residues 86, 91, and 96 that surround the glycine-proline in ca influence viral susceptibility to cyclophillin inhibitors similar to mutations surrounding ns5a p319 [25] ; and the critical glycine-proline is amino-terminal to regions of ca that are actually more proline rich and predicted by bioinformatics analysis to be disordered, analogous to the positioning of the pawarpdynp motif in hcv ns5a. the interaction of hiv ca with cyclophilin a leads to packaging of cyclophilin a into the viral particle. yet, cyclophilin a appears to function primarily in capsid uncoating [25] , rather than particle assembly, and does not have a role in hiv genome replication as it does for hcv. while limited, this comparison provides common themes that may facilitate the identification of other viral proteins that rely on host proline isomerases for function and may thus be susceptible to intervention by blocking isomerization. the hiv gag protein also contains proline stretches termed proline-rich motifs (prms) [26] , but they do not appear to be critical targets for cyclophilin a. while some proline content favors disorder, consecutive prolines impart rigidity. prms generally have proline content surpassing 30%, and neither viral nor human prms have yet been described interacting with cyclophilins specifically. new viral threats emerge much faster than rationally designed antivirals. the number of clinically useful antivirals remains limited, so a drug that works on multiple viruses would be welcomed. identifying viruses and viral proteins that depend on host proline isomerases is an appealing strategy. for example, at least some lethal coronaviruses are suppressed by cyclophilin inhibitors [27] . unfortunately, merely identifying a proline-rich viral protein is not sufficient to predict interactions with isomerase or sensitivity to isomerase inhibitors. creative, systematic approaches are needed to determine which viral proteins contain prolines that are substrates for isomerases and access multiple conformations to perform critical viral functions. identifying host factors that regulate viral infection sequences and topology: intrinsic disorder in the evolving universe of protein structure orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the mimivirus specific inhibition of hepatitis c virus replication by cyclosporin a cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes the natural history of recurrent hepatitis c and what influences this completion of the entire hepatitis c virus life cycle in genetically humanized mice hepatitis c ns5a protein: two drug targets within the same protein with different mechanisms of resistance cyclosporine inhibits a direct interaction between cyclophilins and hepatitis c ns5a hcv resistance to cyclosporin a does not correlate with a resistance of the ns5a-cyclophilin a interaction to cyclophilin inhibitors deb025 (alisporivir) inhibits hepatitis c virus replication by preventing a cyclophilin a induced cis-trans isomerisation in domain ii of ns5a cyclophilin b is a functional regulator of hepatitis c virus rna polymerase sensitivity of hepatitis c virus to cyclosporine a depends on nonstructural proteins ns5a and ns5b cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics hepatitis c virus proteins: from structure to function suppression of viral rna binding and the assembly of infectious hepatitis c virus particles in vitro by cyclophilin inhibitors a conserved tandem cyclophilinbinding site in hepatitis c virus nonstructural protein 5a regulates alisporivir susceptibility phenotypic analysis of ns5a variant from liver transplant patient with increased cyclosporine susceptibility structural insights into the catalytic mechanism of cyclophilin a crystal structure of human cyclophilin a bound to the amino-terminal domain of hiv-1 capsid structure of the amino-terminal core domain of the hiv-1 capsid protein correlation of naturally occurring hiv-1 resistance to deb025 with capsid amino acid polymorphisms proline-rich regions and motifs in trafficking: from escrt interaction to viral exploitation cyclophilins as modulators of viral replication we sincerely apologize to the many authors whose work was not cited because of space limitations. key: cord-280330-ibvbowl0 authors: lin, liang-tzung; chen, ting-ying; lin, song-chow; chung, chueh-yao; lin, ta-chen; wang, guey-horng; anderson, robert; lin, chun-ching; richardson, christopher d title: broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry date: 2013-08-07 journal: bmc microbiol doi: 10.1186/1471-2180-13-187 sha: doc_id: 280330 cord_uid: ibvbowl0 background: we previously identified two hydrolyzable tannins, chebulagic acid (chla) and punicalagin (pug) that blocked herpes simplex virus type 1 (hsv-1) entry and spread. these compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (gags). based on this property, we evaluated their antiviral efficacy against several different viruses known to employ gags for host cell entry. results: extensive analysis of the tannins’ mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. chla and pug were effective in abrogating infection by human cytomegalovirus (hcmv), hepatitis c virus (hcv), dengue virus (denv), measles virus (mv), and respiratory syncytial virus (rsv), at μm concentrations and in dose-dependent manners without significant cytotoxicity. moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. specifically, the tannins blocked all these steps of infection for hcmv, hcv, and mv, but had little effect on the post-fusion spread of denv and rsv, which could suggest intriguing differences in the roles of gag-interactions for these viruses. conclusions: chla and pug may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell gags for entry. further studies testing the efficacy of these tannins in vivo against certain viruses are justified. viral infections are responsible for causing a significant number of human diseases, epidemic outbreaks, morbidity, and mortality. while vaccine efforts have proven successful for preventing and eradicating some viral infections, many viruses cannot be targeted by immunization, including dengue virus (denv), human cytomegalovirus (hcmv), hepatitis c virus (hcv), human immunodeficiency virus (hiv), and respiratory syncytial virus (rsv) [1] [2] [3] [4] [5] . alternative means of control include the use of antiviral drugs; however, there are currently few licensed and efficacious drugs available for prophylactic and therapeutic antiviral treatments. global public health is therefore under constant threat of emerging and re-emerging viral infections, particularly those that do not currently have effective vaccines or have the potential to develop drug-resistant mutations [6] . furthermore, due to increased global travel, trade, and rapid urbanization, increased numbers of viral pathogens are being introduced or reintroduced into areas where they are not normally indigenous [7] . this is reflected by the recent emergence of viral outbreaks caused by severe acute respiratory syndrome (sars) virus, influenza virus (h1n1 and h5n1), denv, west nile virus (wnv), and measles virus (mv) [7] [8] [9] . in addition, the potential for outbreaks due to the intentional or accidental release of virus has also raised serious concerns. thus, efforts in developing antiviral therapies are required to safeguard the public against viral pathogens. most antiviral therapies target defined steps in the viral life cycle, or more specifically, a particular viral protein. examples include nucleoside analogues that inhibit herpes simplex virus (hsv) replication [10] , protease inhibitors directed against the hcv ns3 protease [11] , and neuraminidase inhibitors that block the release of influenza virus particles from infected cells [12] . however, the use of these antivirals is inevitably associated with the potential risk of selecting for drug-resistant viruses, which can pose a significant problem in the clinical management of these viral infections [10, 12, 13] . a combination cocktail of several inhibitors is often necessary to reduce the risk of generating drug resistant mutants. this is best exemplified by highly active antiretroviral therapy (haart) for treating hiv infections [14] . however, experience with combination therapies is still limited, and the potential of producing viral escape mutants cannot be ruled out. an alternative, albeit less specific antiviral therapy is interferon (ifn) which, however, is only effective against a limited number of viral pathogens [15] . moreover, because ifn treatment is prohibitively expensive and burdened with adverse side-effects, the therapy often results in low patient compliance [16, 17] . these characteristics make ifn impractical for widespread use in clinical settings. in view of these shortcomings, there is a clearly a need to develop novel and cost-effective antiviral therapeutics, particularly those that harbor broad-spectrum bioactivities, which can be employed to control and limit the spread of viral infections when immunization and standard therapies are unavailable. glycosaminoglycans (gags) are negatively charged linear polysaccharides that are typically sulfated and include chondroitin sulfate (cs) and heparan sulfate (hs). they represent a repertoire of complex natural glycans that are localized within extracellular matrices and on cell surfaces, and exhibit heterogeneous structures that allow them to bind to a wide range of protein partners such as adhesion molecules, chemokines, cytokines, growth factors, and matrix proteins [18] . thus, gags play important roles in many biologic processes, which have profound physiological consequences that include cell signaling, inflammation, angiogenesis, and coagulation [18, 19] . many viruses employ gags as primary entry factors that facilitate the infection of the host cell. these include denv, hcmv, hcv, hiv, hsv, mv, rsv, and others [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . interactions of viral glycoproteins with gags are usually thought to increase the frequency of initial attachment of viral particles to the target cell surface. they, in turn, enable subsequent higher affinity binding with virusspecific entry receptors that promote virus entry. the importance of gags in facilitating viral infections has been demonstrated by using soluble heparin or gag-deficient cell lines to block the entry of several viruses [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . in our previous study, we identified chebulagic acid (chla) and punicalagin (pug) (figure 1 ), two hydrolyzable tannins isolated from terminalia chebula retz., (t. chebula) as inhibitors of hsv type 1 (hsv-1) entry and spread [33] . we demonstrated that the two structurally-related compounds mediated their antiviral activities by targeting hsv-1 viral glycoproteins that interact with cell surface gags. taking note of the fact that many viruses employ gags to initially bind to the host cell, and based on evidence that chla and pug may act as gag-competitors, we explored the antiviralpotential of these two tannins against a number of viruses known to interact with gags. viral models included denv, hcmv, hcv, mv, and rsv ( table 1 ). many of the diseases associated with these viruses lack preventative vaccines and/or drug treatment options [1] [2] [3] [4] 13, [34] [35] [36] . indeed, both chla and pug efficiently inhibited entry and spread of these viruses to varying degrees. we suggest that chla and pug have potential as novel cost-effective and broad-spectrum antivirals for controlling emerging/recurring infections by viruses that engage host cell surface gags. dulbecco's modified eagle's medium (dmem) and alpha minimal essential medium (amem) were purchased from gibco-invitrogen (carlsbad, ca, usa). fetal bovine serum (fbs), penicillin g, streptomycin, and amphotericin b were purchased from chemicon (billerica, ma, usa). heparin, dimethylsulfoxide (dmso), and in vitro toxicology assay kit (xtt based) were purchased from sigma (st. louis, mo, usa). vero (african green monkey kidney cells, atcc ccl-81), hel (human embryonic lung fibroblast, atcc ccl , and a549 (human lung carcinoma, atcc ccl-185) cells were obtained from the american type culture collection (atcc; rockville, md, usa) and cultured in dmem supplemented with 10% fbs, 200 u/ml penicillin g, 200 μg/ml streptomycin, and 0.5 μg/ml amphotericin b. huh-7.5 (human hepatocarcinoma huh-7 cell derivative; provided by dr. charles m. rice, the rockefeller university, new york, ny, usa) and hep-2 (human epithelial cells derived from a larynx carcinoma; provided by r. anderson) cells were cultured in the same medium condition as just described. cho-slam or chinese hamster ovary cells expressing human signaling lymphocyte activation molecule, the receptor for wild-type measles, were generated as previously reported and cultured in amem supplemented with 10% fbs and 800 μg/ml of g418 [37, 38] . hcmv (ad169 strain; provided by dr. karen l. mossman, mcmaster university, hamilton, on, canada), wild-type human adenovirus type-5 (adv-5), and vsv-gfp (vesicular stomatitis virus with green fluorescent protein tag) have been described elsewhere and viral titers and antiviral assays were determined by standard plaque assay using methanol fixation followed by crystal violet (sigma) [33, 39, 40] . cell-culture derived hcv particles were produced by electroporation of huh-7.5 cells using the jc1flag2(p7-nsgluc2a) construct (genotype 2a; kindly provided by dr. charles m. rice), which harbors a gaussia luciferase reporter that allows detection of virus infectivity, as previously described [41] . hcv viral titer and antiviral assays were determined by immunofluorescence staining of tcid 50 using anti-ns5a 9e10 antibody (gift from dr. charles m. rice) and luciferase assays. denv-2 (dengue virus type 2; strain 16681) and rsv (serogroup a, long strain; atcc vr-26) were propagated in vero and hep-2 cells, respectively [42, 43] . viral titers and antiviral assays for denv-2 and rsv were determined by immunohistochemical staining plaque assay using anti-flavivirus group antibody (1:1,000; millipore, billerica, ma, usa), anti-rsv fusion protein antibody (1:5,000; millipore), and goat anti-mouse igg (h + l) alkaline phosphatase (ap) conjugate (invitrogen; denv-2, 1:5,000; rsv, 1:10,000), followed by development with vector black ap substrate kit (vector laboratories; burlingame, c, usa) based on previously reported method [42] . mv-egfp (recombinant ichinose-b 323 wild-type measles virus isolate, ic323) expressing enhanced green fluorescent protein was originally obtained from dr. roberto cattaneo (mayo clinic, rochester, mn, usa) and propagated in marmoset b lymphoblastoid cells (b95a) [44] ; viral titer and antiviral assays were determined by tcid 50 on cho-slam cells. the basal medium containing 2% fbs with antibiotics was used for all virus infection experiments. virus concentrations are expressed as plaque forming units (pfu) per well or multiplicity of infection (moi). chla and pug ( figure 1 ) were isolated and purified as previously described, with their structures confirmed by high-performance liquid chromatographic method coupled with uv detection and electrospray ionization mass spectrometry (hplc-uv/esi-m), and their purities checked by hplc with photodiode array detection (hplc-pda) [33] . both compounds were dissolved in dmso and the final concentration of dmso was equal to/or below 1% for the experiments. heparin served as control and was dissolved in sterile double-distilled water. for all assays, unless otherwise specified, test compound concentrations used were as follows based on antiviral dose response deter cells (1 × 10 4 per well of 96-well plate) were treated with the test compounds for 3 days. treatment effects on cell viability (%) and the 50% cytotoxic concentration (cc 50 ) values of the test compounds were determined based on the xtt (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-phenylamino)-carbonyl]-2h-tetrazolium hydroxide) assay as previously reported [33] . the respective cell lines and relative viral dose used, as well as the incubation periods for test compound treatment and for viral cytopathic effects to take place, are indicated in table 2 and figure 2a for each specific virus. for assessing the antiviral activities of the tannins on the panel of viruses, hel (1 × 10 5 cells/well), vero (2 × 10 5 cells/well), hep-2 (1.5 × 10 5 cells/well), and a549 (2 -3 × adv-5 adenoviridae dsdna -+/-10 5 cells/well) cells were seeded in 12-well plates and cotreated with the respective viral inoculum ( figure 2a ) and increasing concentration of test compounds for 1 -2 h. the inoculum and drug mixtures were removed from the wells that were subsequently washed with pbs twice and then overlaid with 2% fbs medium containing either methylcellulose (sigma; hcmv: 0.6%; denv-2: 0.75%; rsv and vsv: 1%) or seaplaque agarose (lonza, basel, switzerland; adv-5: 1%). after further incubation for 24 h -10 days depending on the specific virus, wells containing adv-5, hcmv, and vsv infections were analyzed by standard plaque assays, and wells containing denv-2 and rsv infections were analyzed by immunohistochemical staining as described above. viral infection (%) and the 50% effective concentration (ec 50 ) of test compounds against different viral infections were calculated as previously described [33] . for evaluating the antiviral activities of the tannins on mv-egfp infection, cho-slam cells (2 × 10 4 cells/ well) were seeded in 96-well plates and viral inoculum and increasing concentration of the test compounds were co-added onto the cell monolayer for 1.5 h. the inoculum and drug mixtures were then removed and the wells were washed with pbs twice before overlay with amem containing 2% fbs. after further incubation for 24 h, the plates were then scanned by the typhoon 9410 variable mode imager (amersham biosciences; baie d'urfe, quebec, canada) and the egfp expression was analyzed by imagequant tl software (amersham biosciences). viral inhibition (%) and the ec 50 for each compound based on viral egfp expression were determined as previously reported [33] . for analyzing antiviral activities of the tannins on hcv infection, huh-7.5 cells (1 × 10 4 cells/well) were seeded in 96-well plates and the cell monolayer was cochallenged with the viral inoculum and increasing concentration of the test compounds for 3 h. the inoculum and drug mixtures were removed from the wells, followed by washing with pbs twice and overlaying with dmem containing 2% fbs. after further incubation for 72 h, the supernatant was collected and then assayed for luciferase activity using the biolux™ gaussia luciferase assay kit (new england biolabs; pickering, on, canada) and a luminometer (promega; madison, wi, usa). hcv infectivity was expressed as log 10 of relative light units (rlu) for determining viral inhibition (%) and the ec 50 of the drugs against hcv infection was calculated using graphpad prism 5 software (san diego, ca, usa). all values were plotted against the dmso control treatment of virus infection. viral inactivation assays were performed as previously described [33] and the incubation periods and viral dose used are listed in figure 3a . different viruses were mixed with the test compounds and incubated at 37°c ( figure 3a , long-term). the drug-virus mixtures were subsequently diluted (50 -100 fold) to "sub-therapeutic" (ineffective) concentrations with low serum medium and then inoculated on to the respective host cells seeded in multiwell plates. the dilution to sub-therapeutic concentration prevents effective interaction between the drugs and the host cell surface. for comparison, viruses were also mixed with test compounds and immediately diluted (no incubation period) to sub-therapeutic concentration prior to infection ( figure 3a , short-term). following incubation for viral absorption, the diluted inocula were removed and the wells were washed with pbs twice before applying the overlay medium. the plates were further incubated before being subjected to assessment by plaque assays, egfp expression analysis, or luciferase assay as described above. analyses of drug effect on viral attachment were performed based on host cell infection (method 1) or virusspecific cellular enzyme-linked immunosorbent assay (elisa; method 2) as previously described [33] . experiments were all carried out at 4°c which allows for virus binding but precludes entry which occurs most efficiently at 37°c. in method 1 ( figure 4a ), different cell types were prechilled at 4°c for 1 h and then co-treated with dose of respective viruses and test compounds at 4°c for the indicated times. the inocula and drugs were removed and the cell monolayers were washed with ice-cold pbs twice before applying the overlay medium. after further incubation at 37°c, plaque assays, egfp expression analysis, or luciferase assay were performed as described above to assess host cell infection. in method 2 ( figure 5a ), different cell types (2 × 10 4 cells/well) were seeded in 96-well plates and grown overnight. the cell monolayers were pre-chilled at 4°c for 1 h and then co-treated with the respective viruses (hcmv, moi = 5; hcv, moi = 0.1; denv-2, moi = 5; mv, moi = 1; rsv, moi = 5) and various concentrations of test compounds at 4°c for an additional 2 h. following the virus binding period, the inocula and drugs were removed and the cell monolayers were washed with icecold pbs before fixation with pre-chilled 4% paraformaldehyde (pfa) in pbs for 1 h on ice. at that point, the wells were blocked with 5% bovine serum albumin (bsa) at 4°c overnight to prevent any non-specific binding. bound viruses on the cellular surfaces were then detected by elisa assay whereby wells were incubated with the following respective mouse monoclonal primary antibodies (diluted in pbs containing 5% bsa) at 37°c for 1 h before washing with pbst (0.1% tween 20 in pbs) three times: anti-hcmv gb antibody (1:10,000; thermo pierce, rockford, il, usa), anti-hcv e2 antibody (1:20,000; austral biologicals, san ramon, ca, usa), anti-flavivirus group antibody (1:5,000) for denv-2, anti-measles hemagglutinin antibody (1:5,000; millipore), and anti-rsv fusion protein antibody (1:15,000). samples were then subjected to incubation at 37°c for 1 h with goat anti-mouse igg conjugated with horseradish peroxidase (hrp; invitrogen), diluted the viral penetration assay was performed as previously reported [33] and the incubation periods and viral dose used are indicated in figure 4a . monolayers of different cell types were pre-chilled at 4°c for 1 h and then infected for the indicated times with the respective viruses at 4°c to allow virus binding but not entry. the inocula were removed and the wells were washed with ice-cold pbs twice before treating with the test compounds for the indicated times at 37°c. this shift to 37°c facilitates viral penetration and therefore allows assessment of drug effect on viral internalization. the drugs were afterwards removed and non-internalized extracellular viruses were detached by either citrate buffer (50 mm sodium citrate, 4 mm kcl, ph 3.0) or pbs washes. the wells were then further washed with pbs twice prior to covering the cell monolayers with overlay medium. after additional incubation at 37°c, plaque assays, egfp expression analysis, or luciferase assay were performed as described above. for examining drug effects post viral entry, cell monolayers were infected with respective viruses at 37°c with the viral dose and incubation times as specified in figure 6a . following the absorption period, the inocula were removed and extracellular viruses were detached by citrate buffer or pbs washes as just described before treating with the test compounds mixed in the overlay medium at 37°c for the indicated times. plaque assay, egfp expression assessment, or luciferase assay were performed as described above for analysis. for hcmv, the infection was titered by standard plaque assay on newly seeded hel cells. alpha interferon (ifn-α) from human leukocytes (1,000 u/ml; sigma) was included as control for hcv. viral cell-to-cell spread assay was performed as previously described [33, 45] with some modifications and the viral dose and incubation periods are indicated in figure 7a . briefly, different cell types were infected with the respective viruses and extracellular viruses were removed by citrate buffer or pbs washes as specified earlier. the wells were then covered with overlay medium containing either methylcellulose (denv-2: 0.75%; rsv: 1%), seaplaque agarose (lonza; mv: 1%), or in the case of hcmv with 0.1% of neutralizing gamunex antibodies (purified clinical human iggs; provided by dr. andrew c. issekutz, dalhousie university, halifax, ns, canada) [33] . the overlay medium helps limit viral secondary infection, thus allowing monitoring of cell-to-cell spread of virus in the presence or absence of the drugs. the plates were incubated until initial plaque formation, to which the test compounds were then added into the overlay medium and monitored in subsequent incubation for analysis of viral plaque size by immunofluorescence assay. the fusion inhibitory peptide (fip, z-d-phe-l-phe-gly-oh, 200 μm; sigma) also served as control for mv [46] . the examination of hcv spread is based on previously described protocol with some modifications [47] . huh-7.5 cells were electroporated with hcv jc1flag2 (p7-nsgluc2a) rna (10 μg) as described above to establish random productive infections in the cell population, and then mixed with naïve cells at a ratio of 1:1 before seeding in 12-well plates. assembled hcv particles (within 24 -48 h post-transfection) would transmit to neighboring cells that do not harbor viral rna during viral spread and form localized foci in ensuing incubation period [48] . medium was changed 24 h postelectroporation with an overlay medium containing the test drugs or control and 0.5% methylcellulose, and the plates were further incubated for 5 days before analysis of hcv-positive foci through immunostaining. the s29 cell line (provided by dr. rodney s. russell, memorial university of newfoundland, st. john's, nl, canada), which is a huh-7 derivative deficient in the hcv receptor cd81, does not allow cell-to-cell transmission of hcv infection and was included as control [49] . for immunofluorescence analysis of viral plaque size due to spread, the overlay media were removed and the wells were fixed with ice-cold methanol before blocking with 3% bsa. samples were then treated at 37°c for 1 h with the respective mouse monoclonal primary antibodies diluted in pbs containing 3% bsa: anti-hcmv gb antibody (1:1,000) , anti-ns5a 9e10 antibody for hcv (1:25,000), anti-flavivirus group antibody (1:400) for denv-2, and anti-rsv fusion protein antibody (1:1,000). after incubation, the wells were washed with pbs three times before applying alexa fluor 488 goat anti-mouse igg (h + l) antibody (invitrogen), diluted at 1:1,000 (hcmv and rsv) or 1:400 (denv-2 and hcv) in pbs containing 3% bsa. following incubation at 37°c for 1 h, the samples were washed with pbs three times prior to visualization by fluorescence microscopy. the fluorescence expression of mv-egfp could be readily detected without addition of antibodies. photomicrographs were taken at × 100 magnification (leica microsystems; wetzlar, germany) and viral plaque sizes were then analyzed with metamorph software (molecular devices; sunnyvale, ca, usa). in the case of hcv, cellular nuclei were stained with hoechst dye (sigma) prior to visualization and the number of cells in the viruspositive foci was determined. for all virus tested, a total of five random virus-positive plaques were evaluated for each treatment group per independent experiment. comparison was made between viral plaques stained prior to drug addition and those at the endpoint of the experiment, and the data were plotted as "fold change of plaque area". broad-spectrum antiviral effects of chla and pug chla and pug were evaluated for their antiviral effects against a panel of enveloped viruses whose entry involves cellular surface gags (table 1) . vesicular stomatitis virus (vsv) and adenovirus type 5 (adv-5) were included for comparison. the 50% indices of cytotoxicity (cc 50 ) and effective antiviral concentrations (ec 50 ), as well as the selective index (si = cc 50 /ec 50 ), were determined for each virus infection host cell system and are listed in table 2 . as shown in figure 2 , chla and pug displayed broad-spectrum antiviral effects in a dosedependent manner. both compounds exhibited significant inhibitory effect on enveloped viruses known to engage gags for infection, including hcmv, hcv, denv-2, mv, and rsv, with their ec 50 < 35 μm and si > 10 ( table 2 ). both tannins were especially effective against rsv with their ec 50 values being < 1 μm. the two compounds, however, displayed only limited efficacy (si < 10) against infections by vsv and adv-5. this is consistent with the fact that these viruses have previously been shown not to require gags for entry. vsv can infect gag-deficient cells [22, 23, 50] , whereas hs-mediated entry is only important for adv-5 in the absence of its primary receptor car (coxsackievirus and adenovirus receptor) and can only be inhibited to a maximum of 50% by soluble heparin [51, 52] . for the remainder of the studies, we focused on the effects of the tannins against hcmv, hcv, denv-2, mv, and rsv. chla and pug were previously observed to inactivate hsv-1 particles and prevent their interaction with the host cell surface [33] . we examined whether the tannins could also inactivate the different enveloped viruses and prevent subsequent infection. these natural products were pre-incubated with the viruses and then diluted to sub-therapeutic concentrations prior to infecting the respective host cell. results indicated that both chla and pug were able to interact with hcmv, hcv, denv-2, mv, and rsv virions. their effects were irreversible and abrogated subsequent infections (figure 3) . a 60 -80% block against the paramyxoviruses mv and rsv was observed, whereas near 100% inhibition was achieved against hcmv, hcv, and denv-2. the data suggest that chla and pug can directly inactivate these free virus particles and neutralize their infectivity. in further characterizing the antiviral mechanism(s) involved, we explored the effect of chla and pug against hcmv, hcv, denv-2, mv, and rsv attachment to the host cell surface and upon subsequent membrane fusion. the temperature change between 4°c (permitting virus binding but not entry) and 37°c (facilitating virus entry/ penetration) allows examination of the drug effect on each specific event [53] . both tannin compounds effectively prevented attachment of the investigated viruses as shown by readouts of inhibition of infection (method 1; figure 4 ) and by elisa-based binding assays using virus-specific antibodies to detect bound virus on the cell monolayer (method 2; figure 5 ). the inhibition of virus attachment by chla and pug were similar against hcmv, hcv, denv-2, and rsv, and ranged from 90 -100% (figure 4 ). against mv, pug appeared to be more effective than chla, and inhibition of entry varied between 50 -80%. the compounds' ability to abolish binding of the above viruses was confirmed by the decrease of virions detected on cell surfaces. this occurred in a dose-dependent manner with increasing concentrations of the tannins ( figure 5) . to see whether the chla and pug retained their activity during the virus penetration phase, the test viruses were allowed to bind to the cell surface at 4°c and then allowed to penetrate the target cell membrane by a temperature shift to 37°c in the presence or absence of the tannins. chla and pug were again observed to impair virus entry by these viruses, resulting in 50 -90% protection of the host cell from infection from the virus being examined (figure 4 ). heparin exhibited similar inhibitory effects as the tannins against attachment of the test viruses, but was less potent in inhibiting cell penetration by hcmv, hcv, and mv (< 40% inhibition on average). therefore, chla and pug are able to abrogate host cell binding and penetration by hcmv, hcv, denv-2, mv, and rsv during the cell entry process. we next determined the antiviral activity of the two hydrolyzable tannins in controlling spread of established infections. target cell monolayers were infected with the respective test virus, and then incubated with or without the compounds. as shown in figure 6 , both chla and pug effectively inhibited hcmv, hcv, and mv infections (80 -100% protection), but were ineffective against the growth of denv-2 and rsv (< 25%). to further validate the tannins' effect on virus cell-to-cell transmission, we examined the effects of the drugs on viral plaque size. the change in the area of the plaques was measured using either viral immunofluorescence or egfp-tagged reporter viruses. neutralizing antibodies, methylcellulose or agarose were included in the overlay medium to prevent secondary infection of uninfected cells throughout the monolayer, ensuring that viral spread occurs via intercellular junctions between neighboring infected and virus-free populations. the data indicated that viral plaques from hcmv, hcv, and mv infections were restricted by chla and pug to near initial size, whereas plaques due to denv-2 and rsv infections were unaffected and expanded further (figure 7 and additional file 1: figure s1 , additional file 2: figure s2 , additional file 3: figure s3 , additional file 4: figure s4 and additional file 5: figure s5 ). these results are in agreement with the data obtained following post-entry drug treatment in figure 6 , where hcmv, hcv, and mv, but not denv-2 and rsv, were shown to be sensitive to the tannins' antiviral effects. thus, it appears that the two tannins are effective in limiting post-infection spread of hcmv, hcv, and mv, but are inefficient in preventing cell-to-cell transmission of denv-2 and rsv. heparin, on the other hand, displayed limited effect against the spread of the viruses post-entry ( figures 6 and 7) . the window of antiviral activity from chla, pug, and heparin at different stages of viral entry and spread are summarized in table 3 . the inhibition of virus-host cell entry is an effective antiviral control strategy. based on the way a virus infects a host cell through interactions between viral glycoproteins and cellular membrane molecules, countermeasures against this process have been developed. for example, protective antibodies elicited by vaccines bind to viral particles and prevent infection [54] . another strategy consists of using monoclonal antibodies or small molecules to bind host cell receptors and block virus interactions. examples include an antibody directed against the hcv receptor claudin 1, and another is the antagonist maraviroc, which interacts with the hiv coreceptor ccr5 [14, 55] . another hiv inhibitor called enfuvirtide blocks gp41-mediated membrane fusion during virus entry. amantidine blocks influenza m2 ion channel activity during entry and viral assembly [14, 56] . on the other hand, non-specific approaches directed against the virus can influence membrane fluidity (lipid bilayer intercalator lj001), membrane fusion (rigid amphipathic fusion inhibitors, rafis) [57, 58] , or neutralize surface charge (cationic amphipathic sterol, squalamine) [59] . these are effective against a wide range of enveloped viruses. similarly, we recently considered gag receptors as targets for potential antiviral therapy. two natural molecules of the hydrolyzable tannin class, chla and pug, possess gag-competing properties [33] . in this study, both compounds displayed significant in vitro antiviral activity against a variety of viruses, suggesting that blocking interaction with gags is a feasible way to prevent infection by some viruses. our finding adds to the list of molecular strategies that are being developed to prevent and limit viral infections. we previously showed that chla and pug exerted their antiviral effects against hsv-1 by binding viral glycoproteins that interact with cell surface gags [33] . in the current study, these compounds were demonstrated to be effective against infection by other viruses, including hcmv, hcv, denv-2, mv, and rsv, whose entry is known to be sensitive to neutralization by heparin (table 3 ). similar to hsv-1 [33] , the tannins are hypothesized to bind to viral glycoproteins on these viruses and the cell surfaces of infected cells, blocking virus attachment, entry, and cell to cell spread. the two tannins may target more than one step of infection, including attachment, membrane fusion, and cell-to-cell fusion. many viral glycoproteins have multiple roles including binding to host cell surface gags, interaction with higher affinity receptors, and mediating membrane fusion [25, 33, [60] [61] [62] [63] [64] . since chla and pug could not block the spread of denv-2 and rsv, this might reflect situations where the inhibitors interact with specific sites on the viral glycoproteins involved with attachment, membrane fusion, or cell spread, but not all these functions. conformational changes in the viral glycoproteins could result from binding to the tannins and interactions with the cellular microenvironment may vary for the different viruses. for example, heparin was observed to be relatively ineffective against post-entry spread for all viruses examined. this could be due to the fact that the molecular size of heparin limits its accessibility to viral glycoproteins in the intercellular junctions [33] . in addition, the tannins displayed differential efficacies against the viruses examined (table 2 ). it is interesting that chla and pug appeared to be particularly selective against rsv, which could be due to higher affinity of the compounds against the rsv glycoproteins. detailed structure-activity relationship (sar) studies coupled with the analysis of individual viral glycoproteins would be necessary to clarify these issues. in addition, the use of genetically altered virus lacking certain glycoproteins, for example the deltag rsv with deleted glycoprotein g [31] , could further help clarify the tannins' antiviral mechanism. although vaccines represent the preferred method for protection against viruses, they have limited use against individuals who are already infected with a virus. vaccines are also associated with problems of supply, cost of development, coverage and deployment, and efficacy against newly emerging and rapidly mutating viruses [65] . while some antiviral therapies have proven successful, treatment of many pathogenic viral infections have yet to be developed or approved. these include several of the infectious agents investigated in this study. the clinical value of current antiviral drugs is also frequently compromised by development of drug resistant variants causing recurrence of viral infections. broad-spectrum antivirals may offer some relief in the treatment of these infections. although many viruses use gags to initiate infection, therapies exploiting this interaction have yet to be developed. heparin, which is also a type of gag, is known to block the interaction of viral glycoproteins with gags in cell culture studies. however, it is not clinically useful in vivo for frequent/long-term administration due to side effects related to its anticoagulation activity [66] . conversely, while the chla and pug are structurally different from heparin, they also target the gag-interacting properties of viruses and possess a much higher potency. in vivo toxicological and metabolic studies of these tannins have been explored with both showing minimal toxicity [67, 68] . furthermore, the two compounds could be massproduced by chemical synthesis or extracted from t. chebula, which is widespread throughout southeast asia, making them attractive, cost-effective drug candidates [69] [70] [71] [72] . therefore, development of broad-spectrum antivirals using chla and pug or their structure as lead compounds could be useful. they could help control viruses such as hcv, denv, mv, and rsv, especially in epidemic areas and resource-poor countries where active vaccine or commercial programs are unavailable. potential applications include formulations of the tannins as topical creams, gels, aerosol inhalers, or incorporating these compounds in materials, such as wipes, surgical masks, and protective gloves. in conclusion, we have demonstrated that chla and pug have the ability to function as broad-spectrum antivirals in vitro. they effectively prevented infections by viruses utilizing gag-assisted entry, and included hcmv, hcv, denv, mv, and rsv. these natural molecules could serve as new therapeutic agents and help limit infections by viruses for which vaccines or fda-licensed drugs do not yet exist. future clinical applications and studies investigating their efficacy in vivo against specific viruses should be explored. additional file 1: figure s1 . examination of chla and pug treatment on hcmv cell-to-cell spread. hel cell monolayers were inoculated and infected with hcmv for 2 h, washed with pbs to remove excess surface bound virus, and covered with an overlay medium to prevent secondary infection. initial virus plaques were allowed to form in the subsequent infections and chla, pug, heparin, dmso control were added to the overlay medium for an additional incubation time before analysis of viral plaque size by immune fluorescence microscopy at 5 days post-infection as described in methods. representative virus plaques/foci are shown after three independent experiments were performed. scale bar indicates 100 μm. additional file 2: figure s2 . examination of chla and pug treatment on hcv cell-to-cell spread. huh-7.5 cells were electroporated with fulllength hcv replicon rna and covered with an overlay medium to prevent secondary infection. initial virus plaques were allowed to form in the subsequent infections and chla, pug, heparin, and dmso control were added to the overlay medium for an additional incubation time before analysis of viral plaque size by immune fluorescence microscopy at 7 days post-electroporation as described in methods. representative virus plaques/foci are shown after three independent experiments were performed. scale bar indicates 100 μm. additional file 3: figure s3 . examination of chla and pug treatment on denv-2 cell-to-cell spread. vero cells were inoculated and infected with denv-2 for 1.5 h, washed with citrate buffer to remove excess surface bound virus, and covered with an overlay medium to prevent secondary infection. initial virus plaques were allowed to form in the subsequent infections and chla, pug, heparin, and dmso control were added to the overlay medium for an additional incubation time before analysis of viral plaque size by immune fluorescence microscopy at 6 days post-infection as described in methods. representative virus plaques/foci are shown after three independent experiments were performed. scale bar indicates 100 μm. additional file 4: figure s4 . examination of chla and pug treatment on mv-egfp cell-to-cell spread. cho-slam cells were inoculated and infected with mv-egfp for 1.5 h, washed with citrate buffer to remove excess surface bound virus, and covered with an overlay medium to prevent secondary infection. initial virus plaques were allowed to form in the subsequent infections and chla, pug, heparin, fip, and dmso control were added to the overlay medium for an additional incubation time before analysis of viral plaque size by egfp fluorescence microscopy at 48 h post-infection as described in methods. representative virus plaques/foci are shown after three independent experiments were performed. scale bar indicates 100 μm. additional file 5: figure s5 . examination of chla and pug treatment on rsv cell-to-cell spread. hep-2 cells were inoculated and infected with rsv for 1.5 h, washed with citrate buffer to remove excess surface bound virus, and covered with an overlay medium to prevent secondary infection. initial virus plaques were allowed to form in the subsequent infections and chla, pug, heparin, and dmso control were added to the overlay medium for an additional incubation time before analysis of viral plaque size by immune fluorescence microscopy at 48 h postinfection as described in methods. representative virus plaques/foci are shown after three independent experiments were performed. scale bar indicates 100 μm. immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms progress in the development of preventive and therapeutic vaccines for hepatitis c virus update on the current status of cytomegalovirus vaccines respiratory syncytial virus prevention and therapy: past, present, and future hiv vaccines: progress to date zoonotic viral diseases and the frontier of early diagnosis, control and prevention the global burden of bacterial and viral zoonotic infections the socio-ecology of zoonotic infections measles vaccination in humanitarian emergencies: a review of recent practice oral and perioral herpes simplex virus type 1 (hsv-1) infection: review of its management directly acting antivirals against hepatitis c virus advances in the structure-based design of the influenza a neuraminidase inhibitors treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus recent advances in antiretroviral drugs interferons as therapeutic agents for infectious diseases human interferons alpha, beta and omega management of adverse effects of peg-ifn and ribavirin therapy for hepatitis c the structure of glycosaminoglycans and their interactions with proteins heparan sulphate proteoglycans fine-tune mammalian physiology cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis c virus adsorption cellular binding of hepatitis c virus envelope glycoprotein e2 requires cell surface heparan sulfate sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans the fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate heparin-like glycosaminoglycans prevent the infection of measles virus in slam-negative cell lines initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes viral and cellular determinants of the hepatitis c virus envelope-heparan sulfate interaction characterization of the early steps of hepatitis c virus infection by using luciferase reporter viruses envelope-dependent, cyclophilin-independent effects of glycosaminoglycans on human immunodeficiency virus type 1 attachment and infection contribution of the respiratory syncytial virus g glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread important advances in the field of antidengue virus research the search for new therapies for human cytomegalovirus infections measles control-can measles virus inhibitors make a difference? measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (cdw150) but not cd46 as a cellular receptor the interaction between measles virus and its receptor. slam'. dissertation: university of toronto human cytomegalovirus glycoprotein b is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress modified apoptotic molecule (bid) reduces hepatitis c virus infection in mice with chimeric human livers cell culture-produced hepatitis c virus does not infect peripheral blood mononuclear cells dramatic caspase-dependent apoptosis in antibody-enhanced dengue virus infection of human mast cells murine host responses to respiratory syncytial virus (rsv) following intranasal administration of a protollin-adjuvanted, epitope-enhanced recombinant g protein vaccine measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed sulfated derivatives of escherichia coli k5 capsular polysaccharide are potent inhibitors of human cytomegalovirus specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the n-termini of the f1 or ha2 viral polypeptides identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor complete replication of hepatitis c virus in cell culture advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis c virus sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions molecular umbrellas: a novel class of candidate topical microbicides to prevent human immunodeficiency virus and herpes simplex virus infections vaccines: correlates of vaccine-induced immunity monoclonal anti-claudin 1 antibodies prevent hepatitis c virus infection of primary human hepatocytes drugs in development for influenza a broad-spectrum antiviral targeting entry of enveloped viruses rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential viral entry mechanisms: the increasing diversity of paramyxovirus entry structural basis of viral invasion: lessons from paramyxovirus f molecular mechanisms involved in the early steps of flavivirus cell entry virus entry and innate immune activation hepatitis c virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies vaccines: past, present and future heparin induced thrombocytopenia: diagnosis and management repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for 37 days is not toxic anti-hyperglycemic effect of chebulagic acid from the fruits of terminalia chebula retz structural features and biological properties of ellagitannins in some plant families of the order myrtales tannins and related compounds from combretaceae plants tannins and related compounds. cii. structures of terchebulin, an ellagitannin having a novel tetraphenylcarboxylic acid (terchebulic acid) moiety, and biogenetically related tannins from terminalia chebula retz synthesis of ellagitannin natural products submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to thank drs. andrew c. issekutz, charles m. rice, karen l. mossman, and rodney s. russell for reagents, and dr. michael g. the authors declare that they have no competing interests. key: cord-273555-i1d4quos authors: stättermayer, a. f.; ferenci, p. title: letter: does the ifnl4 gene discovery really provide a causal role for the il28b haplotype blocks? authors' reply date: 2014-02-04 journal: aliment pharmacol ther doi: 10.1111/apt.12626 sha: doc_id: 273555 cord_uid: i1d4quos nan while ifnl4 variant was strongly associated with a sustained virological response in both hcv genotypes 1 and 4, and thus confirmed previous findings by prokunina-olsson et al. 2 and bibert et al., 3 the study confirms a strong linkage between ss469415590 and rs12979860 variants (q = 0.988) compared with the modest correlation between rs8099917 and ifnl4 (q = 0.598). these findings therefore do not support an additional clinical benefit of ifnl4 in the prediction of a response to peg/ rbv in caucasian patients. recently, the finding that recombinant ifnl4 protein exerts a potent antiviral activity against both hcv and human coronaviruses 4 works against the assumption that the same protein does inhibit clearance of hcv, i.e. negatively interfering with hcv infection, while being endowed with a strong anti-hcv activity. this suggests the existence of a complex relationship between ifnl4 and hcv in humans, similar to that recently described for ifn-1 and lymphocytic choriomeningitis virus in mice, 5 where chronic ifn-i signalling due to persistent infection drives immunosuppression and disease progression. although validation studies in large cohorts of patients, like the one investigated by st€ attermayer et al., 1 may help understand the clinical utility of ifnl4, these studies have so far failed to demonstrate any causal rela-tionship between ifnl4 protein and nonresponse to interferon therapy in chronic hcv patients. among other approaches, the assessment of the missense variants previously described in the ifnl4 coding region, 2 may help unravel this issue. letter: does the ifnl4 gene discovery really provide a causal role for the il28b haplotype blocks? authors' reply sirs, we thank dr galmozzi and dr lampertico for their comment on our recently published paper on the effect of the dinucleotide frameshift variant in ss469415590 in the interferon (ifn)-k4 gene on interferon/ribavirin treatment and its relationship with the two commonly used single nucleotide polymorphisms (snp) in il28b (rs12979860, rs8099917). 1, 2 we agree that our study does not provide insights on the causal relationship between ifnl4 and treatment response in patients with chronic hepatitis c virus (hcv) infection. nevertheless, our study was designed to investigate the clinical usefulness of the different snps in il28b and ifnl4 in a large cohort of caucasian patients infected with chronic hcv. hamming et al. demonstrated that ifnl4 encodes an active type iii interferon with potent anti-viral activity against both hcv and coronaviruses. 3 galmozzi and lampertico therefore conclude that this strong anti-viral activity works against the assumption of an inhibition of the ifnl4 protein of treatment-induced hcv clearance. we would like to remind them that up-regulation of intrahepatic interferon-stimulated genes (isg) is associated with treatment failure in patients with chronic hcv, and levels of isg are differently distributed according to different il28b genotypes. 4 furthermore, prokunina-olsson and her collaborators demonstrated that ifnl4 induces isg expression in hepg2 hepatoma cells. 5 thus, high baseline isg levels might be associated with poor response to exogenous ifn, by exhausting the ifn response pathways. 6 finally, we agree that the causal role of ifnl4 genotypes for viral clearance is conflicting, and further studies need to be performed to elucidate the molecular mechanistic relationship. the authors' declarations of personal and financial interests are unchanged from those in the original article. 2 sirs, bager et al. 1 reported a randomised controlled trial comparing oral (100 mg twice daily ferrous sulphate for 3 months) or intravenous (1000 mg ferric carboxymaltose, single-dose infusion) iron supplementation and placebo for anaemia after nonvariceal upper gastrointestinal (gi) bleeding. peptic ulcers were the most common sources of bleeding (70%). patients in the placebo group prematurely discontinued the study medication because 25% of patients were still anaemic at the end of the 13-week follow-up period and, therefore, required rescue therapy. the study shows that either oral or intravenous were significantly more effective than placebo to overcome anaemia, and iron stores were more effectively replenished with intravenous iron when compared with oral. on reading the abstract, one may tend to think the results were foreseeable, but this study deserves a higher consideration. the first important conclusion we can draw is that no patient should be discharged after upper gi bleeding without iron supplementation whenever anaemia is present. although logical, this protocol is not applied in routine daily practice, and had been seldom addressed in the literature. intravenous iron did not lead to better results at week 1, 4 or 13 when compared with oral supplementation, even though only 56% had complete adherence to oral therapy. at the very least, these findings seem counterintuitive. whether the efficacy of oral iron was related to higher intestinal absorption because of few patients on proton pump inhibitor therapy (17%), even though peptic ulcer was the most common cause of gi bleeding, remains unknown. in addition, oral iron is notably cheaper. this study highlights the importance of iron supplementation after post-bleeding anaemia, with a clear cost-effective advantage for oral administration, but with similar results for the intravenous route in case of oral iron intolerance. the results are especially relevant given that restrictive transfusion strategies for upper gi bleeding (transfusion when haemoglobin level <7 g/dl) have been associated with better outcomes for nonvariceal upper gi bleeding 2 . in this context, most patients will be expected to be anaemic at discharge, and this study sheds light on the optimal short-term clinical management regarding iron supplementation. declaration of personal and funding interests: none. polymorphisms of interferon-k4 and il28b -effects on treatment response to interferon/ribavirin in patients with chronic hepatitis c a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus il28b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction interferon lambda 4 signals via the ifnk receptor to regulate antiviral activity against hcv and coronaviruses blockade of chronic type i interferon signaling to control persistent lcmv infection letter: does the ifnl4 gene discovery really provide a causal role for the il28b haplotype blocks polymorphism of interferon-k and il28b -effects on treatment response to interferon/ribavirin in patients with chronic hepatitis c interferon lambda 4 signals via the ifnk receptor to regulate antiviral activity against hcv and coronaviruses hepatic isg expression is associated with genetic variation in interleukin 28b and the outcome of ifn therapy for chronic hepatitis c a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus il28 variation affects expression of interferon stimulated genes and peg-interferon and ribavirin therapy letter: the irony of oral iron -not an underdog for post-gastrointestinal bleeding anaemia acknowledgement declaration of personal and funding interests: none. key: cord-007236-8hiymqyb authors: sun, ji-min; kim, seong-jun; kim, geon-woo; rhee, jin-kyu; kim, nam doo; jung, heeyong; jeun, jungae; lee, seung-hoon; han, seung hyun; shin, chul soo; oh, jong-won title: inhibition of hepatitis c virus replication by monascus pigment derivatives that interfere with viral rna polymerase activity and the mevalonate biosynthesis pathway date: 2011-11-09 journal: j antimicrob chemother doi: 10.1093/jac/dkr432 sha: doc_id: 7236 cord_uid: 8hiymqyb objectives: hepatitis c virus (hcv) infection causes chronic liver disease and is a major public health problem worldwide. the aim of this study was to evaluate the potential of monascus pigment derivatives, which were derived from a microbial secondary metabolite synthesized from polyketides by monascus spp., as hcv antiviral agents. methods: we performed an in vitro rna-dependent rna polymerase (rdrp) assay to screen for hcv rdrp inhibitors. the anti-hcv activity of rdrp inhibitors in hcv-replicating cells was evaluated by quantification of the rna viral genome. molecular docking analysis was performed to predict the binding sites of the selected rdrp inhibitors. results: we have identified a monascus pigment and its derivatives as inhibitors of the hcv ns5b rdrp. a group of monascus orange pigment (mop) amino acid derivatives, in which the reactive oxygen moiety was changed to amino acids, significantly inhibited hcv replication. further, combination of the mop derivatives (phe, val or leu conjugates) with interferon (ifn)-α inhibited hcv replication more than ifn-α treatment alone. lastly, molecular docking studies indicate the inhibitors may bind to a thumb subdomain allosteric site of ns5b. the antiviral activity of the mop derivatives was related to a modulation of the mevalonate pathway, since the mevalonate-induced increase in hcv replication was suppressed by the mop compounds. conclusions: our results identify amino acid derivatives of mop as potential anti-hcv agents and suggest that their combination with ifn-α might offer an alternative strategy for the control of hcv replication. hepatitis c virus (hcv) infects 170 million people worldwide, and is often associated with chronic hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. 1 currently, pegylated interferon (ifn) and the nucleoside analogue ribavirin are used as the standard therapy to treat chronic hcv infection. however, ifn-a alone or in combination with ribavirin often leads to a range of side effects, and the sustained virological response rate after combination therapy of pegylated ifn-a and ribavirin is ,50%, particularly for those infected with hcv genotype 1 or 4. 2 therefore, there is an urgent need for the development of alternative anti-hcv agents, especially for patients who do not respond to ifn-a therapy. hcv is an enveloped rna virus with a positive-sense single-strand rna genome of 9.6 kb. the viral genome consists of one long open reading frame (orf) flanked by untranslated regions (utrs) at both the 5 ′ and 3 ′ ends of the genome. the orf encodes a single polyprotein of 3010 amino acids that is proteolytically processed by cellular and viral proteases into ≥10 polypeptides corresponding to the viral structural and non-structural (ns) proteins. the 65 kda hcv ns5b protein has rna-dependent rna polymerase (rdrp) activity and is a key player in hcv rna replication. 3 rdrp activity is not present in mammalian cells, offering the opportunity to identify selective inhibitors of the hcv rdrp. the crystal structure of hcv ns5b resembles a right hand shape with finger, thumb and palm domains similar to other polymerases. 4 over the last decade, researchers have begun developing nucleoside and non-nucleoside analogue inhibitors, which target the active site in the palm subdomain and the allosteric sites in the thumb subdomain of ns5b. 5 microbial secondary metabolites have a variety of biological properties that make them useful as antibiotics, anticancer drugs and antiviral drugs, and in other applications. 6 monascusfermented products, first mentioned in a monograph of chinese medicine in 1590, are produced by monascus species, and historically have been used to treat indigestion, muscle bruises and dysentery. 7 monacolin k, also known as lovastatin, is the major secondary metabolite produced by monascus spp. and a potent inhibitor of 3 ′ -hydroxy-3-methylglutaryl-coenzyme a (hmg-coa) reductase. 8, 9 monascus pigments are also secondary metabolites synthesized from polyketides by monascus spp. 10 there are six major monascus pigments, including the yellow pigments monascin and ankaflavin, the orange pigments monascorubin (c 23 h 26 o 5 ) and rubropunctatin (c 21 h 22 o 5 ), and the red pigments monascropunctamine and rubropunctamine. 11 monascus pigments have been used as food additives and traditional medicines in east asian countries, including china, korea, japan and taiwan. 12 further, the pigments have many useful biological activities, such as antimicrobial, 13 tumour suppressive and immunosuppressive activities, 14 as well as hypolipidaemic activities; 8 however, their mechanisms of action have not been well defined. here, we report that monascus pigment derivatives have anti-hcv activity. these compounds were identified from a screen of microbial secondary metabolites for hcv ns5b rdrp inhibitors. we demonstrated that a group of monascus orange pigment (mop) derivatives effectively inhibited ns5b rdrp activity and interfered with the mevalonate synthesis pathway, thereby suppressing hcv replication in cells harbouring an hcv genotype 1b subgenomic replicon and in cells infected with genotype 2a hcv. the huh7 human hepatoma cell line was grown in dulbecco's modified eagle's medium (dmem; biowhittaker, walkersville, ma, usa) with supplements, as described previously. 15 the huh7-derived cell line r-1, which supports stable, autonomous replication of a genotype 1b hcv subgenomic replicon, was maintained in dmem with 1 mg/ml g418, as described previously. 15 monascus pigments and reagents mop amino acid derivatives (aads) were produced using monascus sp. kccm 10093 and purified from thin layer chromatography, as described previously. 11, 16 the purity of the mop aads was evaluated by hplc, as described previously. 11 the purified mop aad compounds were stored as a 10 mm stock solution in dmso at 2208c and diluted in serum-free medium for use such that the final dmso concentration did not exceed 0.05%. ifn-a was purchased from sigma-aldrich (i-4276, st louis, mo, usa). infectious hcv rna of genotype 2a hcv jfh1 17 was prepared by in vitro transcription using the megascript t7 kit (ambion, austin, tx, usa) and electroporated into huh7 cells, as described previously. 18 huh7 cells were infected with jfh1 virus at a multiplicity of infection of 0.3, as described previously. 18 for evaluation of antiviral activity, the indicated doses of ifn-a and/or mop aads were added to dmem containing 5% fetal bovine serum. after 3 days, cells were harvested and the relative hcv genomic rna levels were assessed by quantitative reverse transcription real-time pcr (qrt-pcr). the half-maximal inhibitory concentration (ic 50 ) value was determined by fitting the data to a three-parametric sigmoidal function using sigmaplot software (version 10.0; systat software inc., richmond, ca, usa). the cytotoxicity of mop derivatives was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) reagent, as described previously. 19 briefly, huh7 cells grown on a 96-well plate to 70% confluence were incubated for 72 h with mop derivatives at various concentrations in complete dmem. formazan formation was measured by reading the optical absorbance at 570 nm on a microplate reader (fluostar optima; bmg labtech gmbh, offenburg, germany). recombinant hcv ns5b protein with an n-terminal hexahistidine tag was expressed in escherichia coli and purified, as described previously. 15 in vitro rna polymerase activity assays were performed in a streptavidincoated flashplate (perkinelmer life and analytical science, waltham, ma, usa), as described previously. 18 in brief, the reaction was performed in a 25 ml total volume mixture containing 50 mm tris-hcl (ph 7.5), 50 mm nacl, 5 mm mgcl 2 , 1 mm dtt, 20 u of rnase inhibitor (promega, madison, wi, usa), 6 mm utp, 1 mg of poly(a) rna, 10 pmol of biotinlyated oligo(u) 12 , 5 mci [a-32 p]utp (3000 ci/mmol; amersham pharmacia biotech, piscataway, nj, usa) and 3.75 pmol of purified ns5b. the reaction mixture was incubated at 328c for 2 h. to stop the reaction, 25 ml of 100 mm edta was added. after 30 min incubation at room temperature, the reaction mixture was removed and the plates were washed with phosphate-buffered saline. the captured, labelled rna products were measured in a perkinelmer topcount scintillation counter. assays were performed using the hmg-coa reductase assay kit (sigma-aldrich), according to the manufacturer's protocol. the enzyme activity based on nadph oxidation was determined by measuring the absorbance at 340 nm for 10 min (378c) using a spectrophotometer (fluostar optima; bmg labtech gmbh). the reaction was performed in the presence of each derivative of mop (10 mm) or with pravastatin (1 mm; sigma-aldrich). total cholesterol was extracted from the cells as described previously. 20 the cholesterol content was determined with the amplexred cholesterol assay kit (invitrogen, carlsbad, ca, usa). mevalonate was prepared by the hydrolysis of mevalonolactone (sigma-aldrich) with koh, as previously described. 21 viral rna genome quantification total rna was extracted from r-1 cells or hcv-infected cells by using the trizol ls reagent (invitrogen). the hcv rna level in each sample was quantified by qrt-pcr using a primer pair and the taqman probe targeting a region within the hcv 5 ′ -utr, as described previously. 18 sun et al. western blot analysis r-1 cells or hcv-infected huh7 cells were resuspended in lysis buffer containing 25 mm tris-hcl (ph 7.5), 150 mm nacl, 1% triton x-100 and an edta-free protease inhibitor cocktail (roche diagnostics gmbh, mannheim, germany). cell lysates containing equal amounts of proteins were resolved by sds-page, transferred to nitrocellulose membranes and immunoblotted with anti-ns5a (virogen, watertown, ma, usa) or anti-ns5b 18 antibodies, as described previously. 22 to demonstrate equal loading of cell lysates, a-tubulin was measured by western blot analysis using an anti-a-tubulin antibody (oncogene research products, cambridge, ma, usa). blots were developed using the enhanced chemiluminescence western blot system (ge healthcare life sciences, piscataway, nj, usa), as described previously. 22 quantitative analyses of western blots were performed using the scion image software (scion co., frederick, md, usa). crystal structures of hcv ns5b protein (protein data bank codes 1nhu, 1nhv, 2gir, 2brk, 2qe2 and 3fqk) were used for molecular docking simulation analyses. the ns5b structures were kept rigid, whereas the torsional bonds in mop aads were set free to flexible docking. affinity grids on the binding pocket were constructed using autogrid4 23 with a grid spacing of 0.375 å . each grid map consisted of 40×40×40 grid points. autodock4 23 was used to find the binding positions for mop aads on the hcv ns5b by molecular docking simulation. molecular docking was performed by a global genetic algorithm combined with local minimization, lamarckian genetic algorithm to explore the compound conformational space. after each docking job with 100 trials, the final docked conformations were clustered using a tolerance of 1 å root-mean-square deviation. all other parameters were set to default values. the docking conformation properly oriented towards the hcv ns5b inhibitor binding pocket was selected and the free energy of binding was estimated. the molecular graphics for the inhibitor binding pocket and refined docking model for the selected mop aads were generated using the pymol software package. 24 the hcv ns5b rdrp is an essential enzyme for hcv replication and is therefore a promising antiviral drug target for blocking viral genome replication. 3, 5 we screened an in-house microbial secondary metabolite library and identified the mop secondary metabolite (figure 1a ) as an inhibitor of hcv rdrp activity. to screen the library, we performed an in vitro rdrp assay using recombinant ns5b. the in vitro rdrp assay was carried out in a flashplate coated with streptavidin using poly(a) rna/biotinylated oligo(u) 12 as a substrate in the presence of 10 mm of each library compound. as shown in figure 1 (b), the natural mop inhibited ns5b activity by 28% at 10 mm concentration. in an attempt to improve the inhibitory potency, we produced various aads of mop in which the aromatic ring oxygen of mop was changed to different amino acids (figure 1a) . these derivatives were tested for rdrp inhibitory activity. among the resulting mop aads, we found that derivatives containing asn, lys, phe, gly, ala, val, leu or ile side chains had greater inhibitory potency (up to 45%) than the parent mop. however, the activity was still 50-fold lower than that of benzothiadiazinylquinone (42% inhibition at 200 nm), a potent hcv inhibitor designed to target the palm site of the ns5b polymerase. 25 anti-hcv activity of monascus pigment derivatives 51 jac considering the novel potential of these microbial secondary metabolites as anti-hcv agents, we proceeded to characterize their structure-function relationships. we tested the hcv inhibitory effect of the above-described mop aads in r-1 cells that harboured an hcv genotype 1b subgenomic replicon. this replicon consists of the hcv internal ribosome entry site (ires), which directs expression of a g418 selectable neo r marker, and an encephalomyocarditis virus ires, which directs the expression of the hcv ns proteins (ns3 to ns5b) required for hcv replication (figure 2a, top panel) . r-1 cells were treated with 10 mm of each mop aad for 48 h and then the remaining steady-state level of ns5b protein was determined by western blot analysis. as shown in figure 2 (a), among the selected group of mop aads that inhibited ns5b rdrp activity in vitro, lys, phe, val, leu and ile derivatives also inhibited hcv replication in huh7 cells, which harbour a hcv subgenomic replicon rna. treatment of cells with the positive control, ifn-a (100 iu/ml), or the mop aad both reduced the ns5b protein levels to different degrees. ifn-a reduced ns5b by 75% while the reductions for the aads were: lys 30%; phe 39%; val 38%; leu 45%; and ile 35%. in the conditions of this assay, none of the mop compounds significantly affected huh7 cell proliferation (at most ,10% at 10 mm concentration), as assessed by a colorimetric mtt assay (data not shown). notably, in contrast to the in vitro rdrp assay results, the parental mop compound did not significantly reduce hcv protein expression levels in the huh r-1 cells, which might be due to its lower affinity to the rdrp or inability to pass through the plasma membrane. we next confirmed the anti-hcv activity of the five selected mop derivatives (lys, phe, val, leu and ile derivatives) by measuring hcv subgenomic rna levels by real-time qrt -pcr. the hcv rna levels were decreased by 38% -53% by treatment with 10 mm of each of the mop derivatives (figure 2b) . we finally selected mop phe, val and leu derivatives to further characterize the mechanism of hcv inhibition in hcv-infected cells. we used the hcv infection system established with the genotype 2a hcv clone jfh1, which yields infectious hcv virus from huh7 or huh7derived cell lines. 17 because combination therapy with pegylated ifn-a and ribavirin is the current standard therapy for the treatment of hcv infection, 26 we also sought to evaluate the potential of the mop aads to act in combination with ifn-a. hcv-infected huh7 cells were treated with 10 mm of each mop aad alone or in combination with ifn-a (100 iu/ml) for 72 h and the hcv genome level was analysed by real-time qrt -pcr. as shown in figure 3 rna when combined with ifn-a and inhibited hcv replication in a dose-dependent manner, with an ic 50 value of 8.9 mm (figure 3b ). we also monitored viral ns5a and ns5b protein expression by western blot analysis. as shown in the figure 3 (b) inset, the mop-leu derivative reduced ns5a and ns5b expression in a dose-dependent manner. lastly, the compound displayed no significant cytotoxicity at concentrations up to 50 mm (,20% decrease of cell viability at 50 mm), as assessed by the mtt cell viability assay (figure 3c ). together, these results demonstrate that mop aads had antiviral activity against both genotype 2a hcv (from the huh7 infection model) and genotype 1b hcv (from the subgenomic replicon system in figure 2 ), and suggest their potential as novel hcv antiviral drugs when used in combination with ifn-a. to understand the molecular mechanism of rdrp inhibition by the selected mop aads, we performed molecular docking of the inhibitors to the crystal structures of hcv ns5b -inhibitor complexes. 27, 28 mop and its aads (the phe, ile and leu derivatives; figure 4a ) were successfully docked to the thumb subdomain of ns5b, which is 35 å away from the polymerase active site in the palm domain ( figure 4b ). as shown in figure 4 hydrogen bond interaction site defined by amino acids arg501 and lys533. the propenyl moiety of mop aads is bound to the surface of one of the hydrophobic pockets defined by trp528/ leu497/tyr477. the predicted binding mode of this moiety is similar to that of the ethylfuran moiety of the reference compound. the hexanoyl group of the mop aads, like the 4-fluorophenyl ring of the reference compound, is buried into a narrow deep hydrophobic pocket defined by leu489, ile482 and met423. lastly, two hydrogen bonds between oxygen atoms of a carboxylate group on the inhibitors and arg501 and lys533 side chains on ns5b were also observed, as with the carboxylic acid moiety of the reference compound. the location of this inhibitorbinding site suggests that the binding of these inhibitors could interfere with a conformational change essential for hcv ns5b polymerase activity. 29 interference with mevalonate pathway by the mop aads lipid metabolism and cholesterol synthesis play a critical role in hcv replication. because the hmg-coa reductase, which catalyses the conversion of hmg-coa to mevalonate, is the ratelimiting step in cholesterol biosynthesis, various inhibitors of this enzyme were previously tested for anti-hcv activity. 30 in particular, lovastatin, one of the monascus-produced secondary metabolites, was shown to effectively suppress hcv replication by inhibiting the interaction between hcv ns5a and the host protein fbl2. 31, 32 we also observed that lovastatin treatment (10 mm) for 48 h reduced viral genome abundance by 70% in hcv-replicating r-1 cells (data not shown), confirming previous reports. 30, 31, 33 currently, it is not known whether mop, like lovastatin, also interferes with cholesterol biosynthesis. to assess the possibility that mop aads may inhibit hcv replication by inhibiting cholesterol biosynthesis, we first performed in vitro hmg-coa reductase assays in hcv replication-supporting huh7 cells. as shown in figure 5 (b), mop derivatives (the phe, val and leu derivatives) did not have any observable hmg-coa reductase inhibitory activity, even at a high concentration (10 mm), while pravastatin, a hmg-coa reductase inhibitor, significantly decreased the enzyme activity at a concentration of 1 mm. in the mevalonate pathway, acetyl-coa is converted into hmg-coa, mevalonate, farnesyl diphosphate, squalene and cholesterol (figure 5a ). 34 the production of geranylgeranyl lipids and cholesterol through the mevalonate pathway is important for hcv rna replication. 31 consistent with this, we found that mevalonate supplementation to hcv-replicating cells increased hcv subgenomic rna levels (figure 5c ). this result prompted us to further investigate whether the mop aads block hcv replication by inhibiting the cholesterol biosynthetic pathway downstream of mevalonate. to test this possibility, we examined whether the increase in hcv replication by the addition of mevalonate can be suppressed by the mop aads. huh7 r-1 cells, which harbour an hcv subgenomic replicon, were incubated with 10 mm mevalonate in the absence or presence of 10 mm of the phe, val or leu mop aads. after 2 days, the cells were harvested and hcv subgenomic rna levels analysed by real-time qrt-pcr. as shown in figure 5 (c), the hcv rna level increased by 37% with the addition of mevalonate; however, the mevalonate-induced increase was completely blocked by mop aad treatment. moreover, the increase in intracellular cholesterol caused by mevalonate supplementation was also inhibited by mop aad treatment and the cholesterol levels were reduced to the level of mock-treated cells (figure 5d ). together, these results suggest that in addition to a direct inhibition of ns5b rdrp activity, these mop aad compounds also inhibit hcv replication by interfering with the cholesterol biosynthetic pathway downstream of the hmg-coa reductase step. microbial secondary metabolites have been shown to be effective as anticancer or antimicrobial agents, and for the treatment of metabolic diseases such as hypercholesterolaemia. these natural products are attractive for developing antiviral drugs to treat or control viral infectious diseases, but have previously been overlooked in antiviral research. in this study, we screened an in-house library of microbial secondary metabolites and found a series of mop aads that have anti-hcv activity. we have identified two novel activities of these mop aads: inhibition of hcv rdrp activity; and interference with the mevalonate pathway. both of these inhibitory activities were found to contribute to the suppression of hcv replication. currently, four different allosteric binding sites for hcv ns5b non-nucleoside inhibitors (nnis) have been identified: two sites in the thumb domain; and two sites in the palm domain. 35 the mop leu, ile and phe aads were predicted to bind primarily to the thumb subdomain site ii, namely the nni site ii, 35 å away from the active site of the ns5b protein. the mop aads failed to dock to the other three nni sites and did not show any common binding patterns shared with previously reported inhibitors known to bind to nni-binding sites i, iii and iv (data not shown). the pharmacophore of hcv ns5b nni binding to thumb subdomain site ii is composed of the hydrogen-bond interaction sites (arg501 and lys533) and a hydrophobic cavity, which appear to be occupied by a carboxylic acid residue and prophenyl residue of mop aads, respectively. among those two predicted interacting parts of the mop aads, the carboxylic acid residue seems to be more critical for ns5b inhibition, since the mop lacking this carboxylic acid residue showed comparably lower ns5b inhibitory activities and no detectable anti-hcv activity in hcv-replicating cells. further, molecular docking simulations showed that the parental mop compound did not make hydrogen bonds with arg501 and lys533. the nnis binding to this site, like the inhibitors binding to the nni-binding site i on the upper section of the hcv ns5b thumb domain, are likely to prevent the polymerase from adopting the closed conformation required for productive polymerization during elongation. 29 genotype-dependent antiviral activity, which is due to the polymorphism of ns5b, is a major obstacle for the development of nnis against hcv rdrp. indeed, except for hcv796, an inhibitor that binds to nni site iv on the palm domain, the activity of nnis against non-genotype 1 hcv is limited. 36, 37 the mop aads selected in the present study demonstrated similar antiviral potency against hcv genotype 1b-derived subgenomic replicon cells and genotype 2a hcv-infected cells, suggesting the binding mode of these inhibitors was not limited to the genotype 1 ns5b polymerase. however, further studies are needed to determine whether these mop derivatives provide a novel scaffold binding to the thumb subdomain allosteric sites of ns5b from other genotypes as well. in addition to genotype diversity, the emergence of resistant mutants is another obstacle for the development of nnis. for instance, treatment of patients with nni site ii inhibitors, such as filibuvir and vch759, in clinical trials resulted in the selection of resistant mutants with mutations at leu419, met423 and ile482 of ns5b. 35, 38, 39 our molecular docking simulation predicted that leu489/ile482/ met423 is also involved in the binding of the selected mop aads to the site ii hydrophobic pockets. thus, it will be of interest to investigate whether the mop aads show cross-resistance with other nni site ii inhibitors. if so, mop aads would be additional combination choices for nni cocktail therapy. several lines of evidence suggest that cellular lipid and cholesterol metabolism plays either a direct or indirect role in the hcv life cycle. in particular, cholesterol biosynthesis was proposed as an integral part of hcv rna replication, which occurs on lipid rafts. 31, 40 in addition, hcv entry through the low-density lipoprotein receptor and viral assembly occurring on the surface of lipid droplets are also linked to cholesterol biosynthesis. 41, 42 accordingly, various inhibitors of hmg-coa reductase have been shown to suppress hcv replication. 33, 43 our results suggest that the anti-hcv activity of mop aads (the phe, val and leu derivatives) might also be partially due to their ability to interfere with the mevalonate biosynthetic pathway. the key enzyme in the mevalonate -cholesterol pathway, hmg-coa reductase, was not inhibited by the mop derivatives. however, the increased hcv rna levels induced by mevalonate supplementation were inhibited by the mop aads. it was previously suggested that geranylgeranylation of the host factor fbl2 is required for targeting of hcv ns5a to the intracellular membranous structure to form the rna replicase complex. 43 this previous study also demonstrated that the addition of cholesterol failed to rescue hcv rna replication in the presence of lovastatin. 43 thus, it is likely that the mop aads prevented hcv replication in part by blocking the generation of geranylgeranyl or its precursors, since the hcv ns5a -fbl2 interaction required for hcv replication depends on geranylgeranylation of fbl2. 43 of note, the increased intracellular cholesterol level in the mevalonate-treated cells decreased upon treatment with the mop aads. therefore, mop aads appear to be capable of inhibiting the step(s) involved in converting mevalonate to cholesterol or the enzyme converting farnesyl diphosphate to geranylgeranyl diphosphate. in summary, the results presented here demonstrate that mop aads can effectively inhibit hcv replication. a double-hit strategy, including inhibition of hcv rdrp activity and interference with the mevalonate synthetic pathway, to inhibit hcv amplification may provide the basis for successful antiviral therapy using the mop aads derived from this microbial secondary metabolite. the selected aads of mop potentiated the antiviral activity of ifn-a, suggesting that combination therapy with ifn-a or other drugs may offer an alternative strategy for controlling hcv replication. hepatitis c and liver transplantation pegylated interferons for the treatment of chronic hepatitis c: pharmacological and clinical differences between peginterferon-a-2a and peginterferon-a-2b from structure to function: new insights into hepatitis c virus rna replication crystal structure of the rna-dependent rna polymerase of hepatitis c virus progress towards improving antiviral therapy for hepatitis c with hepatitis c virus polymerase inhibitors. part i: nucleoside analogues microbial drug discovery: 80 years of progress production and isolation of an antibiotic from monascus purpureus and its relationship to pigment production in vivo hypolipidemic effects and safety of low dosage monascus powder in a hamster model of hyperlipidemia chemistry, biochemistry, and pharmacology of hmg-coa reductase inhibitors the effect of different nitrogen sources on pigment production and sporulation of monascus species in submerged, shaken culture color characteristics of monascus pigments derived by fermentation with various amino acids monascus rice products antimicrobial activities of amino acid derivatives of monascus pigments azaphilones inhibit tumor promotion by 12-o-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mice protein kinase c-related kinase 2 regulates hepatitis c virus rna polymerase function by phosphorylation effect of monascus pigment derivatives on the electrophoretic mobility of bacteria, and the cell adsorption and antibacterial activities of pigments production of infectious hepatitis c virus in tissue culture from a cloned viral genome suppression of hepatitis c virus replication by protein kinase c-related kinase 2 inhibitors that block phosphorylation of viral rna polymerase interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses sars coronavirus replication vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-c-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis interaction of hepatitis c virus core protein with hsp60 triggers the production of reactive oxygen species and enhances tnf-a-mediated apoptosis automated docking using a lamarckian genetic algorithm and empirical binding free energy function the pymol molecular graphics system a highly efficient, asymmetric synthesis of benzothiadiazine-substituted tetramic acids: potent hepatitis c virus rna-dependent rna polymerase specifically targeted antiviral therapy for hepatitis c virus non-nucleoside analogue inhibitors bind to an allosteric site on hcv ns5b polymerase. crystal structures and mechanism of inhibition structure-based design of a novel thiazolone scaffold as hcv ns5b polymerase allosteric inhibitors crystal structures of the rna-dependent rna polymerase genotype 2a of hepatitis c virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors different anti-hcv profiles of statins and their potential for combination therapy with interferon reliance of host cholesterol metabolic pathways for the life cycle of hepatitis c virus identification of fbl2 as a geranylgeranylated cellular protein required for hepatitis c virus rna replication hepatitis c virus rna replication is regulated by host geranylgeranylation and fatty acids regulation of the mevalonate pathway new ns5b polymerase inhibitors for hepatitis c development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis c virus polymerase inhibitors binding-site identification and genotypic profiling of hepatitis c virus polymerase inhibitors evaluation of vch-759 monotherapy in hepatitis c infection selection and characterization of replicon variants dually resistant to thumb-and palm-binding nonnucleoside polymerase inhibitors of the hepatitis c virus characterization of the hepatitis c virus rna replication complex associated with lipid rafts the lipid droplet is an important organelle for hepatitis c virus production hepatitis c virus and host cell lipids: an intimate connection disruption of hepatitis c virus rna replication through inhibition of host protein geranylgeranylation we thank drs christoph seeger and takaji wakita for providing pzs2 and pjfh1 plasmids, respectively. none to declare. key: cord-002369-shk4n8f6 authors: fahmy, ahmed m.; labonté, patrick title: the autophagy elongation complex (atg5-12/16l1) positively regulates hcv replication and is required for wild-type membranous web formation date: 2017-01-09 journal: sci rep doi: 10.1038/srep40351 sha: doc_id: 2369 cord_uid: shk4n8f6 hepatitis c virus (hcv) infection induces intracellular membrane rearrangements, thus forming a membranous web (mw) in which hcv replication and assembly occur. the hcv-induced mw is primarily composed of double membrane vesicles (dmvs) transfused by multi-membrane vesicles. the autophagy machinery has been proposed to participate in the formation of such vesicles. however, no clear evidence has been found linking autophagy to the formation of these dmvs. in this study, we evaluated the role of the autophagy elongation complex (atg5-12/16l1) in hcv replication and mw formation. using a dominant negative form of atg12 and an sirna approach, we demonstrated that the atg5-12 conjugate, but not lc3-ii formation, is crucial for efficient viral replication. furthermore, purification of hcv mw revealed the presence of atg5-12 and atg16l1 along with hcv nonstructural proteins. interestingly, lc3 was not recruited along with the elongation complex to the site of viral replication. finally, inhibition of the elongation complex, but not lc3, greatly impaired the formation of the wild-type mw phenotype. to our knowledge, this study provides the first evidence of the involvement of autophagy proteins in the formation of wild-type mws. hepatitis c virus (hcv) infection is a leading cause of liver diseases, including cirrhosis and hepatocellular carcinoma. hcv, a member of the flaviviridae family, is a hepacivirus with a positive-strand rna genome 1 . the virus replicates exclusively in the cytoplasm of the host cell. after cell entry, the 9.6 kb hcv genome is released and translated at the rough endoplasmic reticulum (rer) into a single polyprotein. this translated polyprotein is then proteolytically processed by cellular and viral proteases into 10 distinct proteins consisting of structural (core, e1, and e2) and nonstructural (p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b) proteins 2 . the expression of hcv proteins results in the induction of a major rearrangement of host cell membranes, thus leading to the formation of a complex membranous compartment termed the membranous web (mw), which favors viral rna replication and assembly 3, 4 . this massive remodeling of the host cell membrane network is associated with all positive-strand rna viruses and is typically characterized by the generation of either convoluted membranes or double membrane vesicles (dmvs) [5] [6] [7] [8] . importantly, the hcv-induced mw is primarily composed of dmvs thus suggesting that autophagy plays a role in the construction of the hcv replication scaffold 7,9 . macroautophagy, referred to hereafter as autophagy, is a catabolic pathway that degrades proteins and organelles, thereby maintaining cell homeostasis and directing cell fate. during cellular stress such as amino acid starvation, autophagy is triggered, thereby forming an organelle called the autophagosome. the de novo formation of the autophagosome begins by initiation of the growth of a double-membraned phagophore that, by closing, sequesters cytoplasmic contents. the autophagosome then fuses with the lysosome, thus allowing the degradation of the intra-autophagosomal cargo by the action of lysosomal enzymes and the release of free amino acids and other products. this process is orchestrated by more than 30 autophagy-related gene (atg) proteins and other autophagy-linked proteins 10 . hcv does not perturb formation of the atg5-12/16l1 complex. atg5 forms a conjugate with atg12, but the monomeric forms of these two proteins have been shown to be nearly undetectable under normal conditions 35 . we first tested whether this conjugation occurs in hcv-infected cells. the assessment of the atg5 protein by western blotting showed that monomeric atg5 (32 kda) was undetectable in both infected and uninfected cells. atg5 was detected only in the atg5-12-conjugated form (55 kda) (fig. 1a) . infection with hcv jfh1 strain was confirmed by detecting hcv ns3 protein using anti-ns3 antibody (fig. 1a) . the difficulty in detecting unconjugated atg5 suggested that the majority of the atg5 is readily conjugated to atg12 in huh7 cells, as has previously been reported for other cell types 36 . in addition, hcv infection did not inhibit this conjugation. furthermore, hcv infection induced lc3-ii accumulation (fig. 1a) . this result confirms the capability of hcv to modulate autophagy, as has previously been reported by several groups 9, 18, 19 . after atg5 is conjugated to atg12, it forms a multimeric complex by associating with atg16l1. to test whether this complex forms in hcv-infected cells, we overexpressed atg12-flag in infected and uninfected cells. using co-immunoprecipitation with an anti-flag monoclonal antibody, we detected the atg5-12/16l1 complex under both conditions (fig. 1b) . whereas atg5-12 associates spontaneously with atg16l1 37,38 , our results indicated that hcv infection did not disturb the formation of the atg5-12/16l1 complex. to test whether the decoupling of the atg5-12 conjugate influences hcv replication, we overexpressed the dominant-negative form of atg12 (atg12dn) in hcv-infected huh7 cells. this mutated form of the atg12 protein lacks the c-terminal glycine, which is crucial for conjugation with atg5 39 . interestingly, the overexpression of this conjugation-defective mutant had an adverse effect on hcv lifecycle, as indicated by a decrease in the ns3 protein (fig. 1c) , as well as the viral rna level (fig. 1d ), as compared with the levels in mock-treated cells. the specificity of the effect of atg12dn overexpression was assessed by trans-complementation with wild-type atg12. as expected, trans-complementation with atg12 restored the normal level of replication (fig. 1c,d) . altogether, these results suggest that the atg5-12 conjugate, rather than the monomeric form of the atg5 and atg12 proteins, acts as an hcv proviral factor. the atg5-12 conjugate is involved in the hcv lifecycle at a post-translational step. the conjugation of atg12 to lysine 130 of atg5 is mediated by atg7, an e1-like enzyme, thus allowing the formation of the atg5-12/16l1 complex. a fraction of the atg5-12/16l1 complex localizes to the isolation membrane, where it facilitates lc3 lipidation. another role of atg7, along with atg3, an e2-like enzyme, is to activate lc3 after its processing at the c-terminus by atg4b, thus allowing its conjugation to the amino group of phosphatidylethanolamine (pe) and formation of the membrane-associated lc3-ii, which assists in the expansion and closure of the autophagosome 40, 41 . therefore, silencing of atg7 allows the inhibition of lc3-ii and atg5-12 conjugation (fig. 2b) . thus, by silencing lc3, atg7 or atg12, we were able to analyze the independent contributions of the two autophagy conjugation systems in the hcv lifecycle. for this purpose, we first determined the efficiency of the selected sirna to knock down their respective targets ( fig. 2a ,b,c). we then analyzed the effects of the silencing of atg12, lc3 or atg7 on hcv entry, viral rna translation/replication and virion maturation and secretion. as shown in fig. 2d , huh7 infection by hcvpp was not altered in cells treated with sirna against lc3, atg7 or atg12, thus suggesting that neither lc3 nor the atg5-12 conjugate is involved in viral entry. previously, dreux and colleagues have reported that hcv rna translation is affected by inhibition of lc3 conjugation using sirna against atg4b. in that report, the authors followed the luciferase activity expressed from a replication-defective subgenomic replicon rluc/sgr harboring an inactivation mutation (gdd to gnd) at the active site of the hcv polymerase ns5b (rluc/sgr-gnd) 31 . using a similar approach, we evaluated the effects of lc3, atg7 and atg12 silencing on viral rna translation and/or replication. we used full-length hcv jfh1 rna with a firefly luciferase reporter containing the gnd mutation in the active site of ns5b (jfh1/fluc-gnd) and then analyzed the fluc activity expressed from the hcv internal ribosomal entry site (ires). a significant decrease in luciferase activity (> 80%) was observed in jfh1/fluc-gnd-transfected cells pretreated with silc3 ( fig. 2e) . silencing of atg7, which inhibits lc3-ii formation as well as atg5-12 conjugation, decreased viral translation by 50%. this effect probably occurred through the inhibition of lc3-ii conjugation, because silencing of atg12 expression was much less efficient than lc3 silencing at inhibiting viral rna translation (fig. 2e ). in contrast, silencing of atg12 severely affected the luciferase activity of a replication competent jfh1/fluc wild-type virus. the effect on replication was not due to the toxicity of sirna treatment (fig. s1 ), thus suggesting that the atg5-12 conjugate is involved in an hcv lifecycle step(s) beyond entry and rna translation (fig. 2e ), whereas lc3 expression and/or conjugation is primarily important for viral translation, as previously suggested 31 . assessed by western blotting in mock (ui) or huh7 cells infected at more than 90% with jfh1 using specific anti-atg5 antibody. hcv infection and lc3 lipidation were detected using anti-ns3 and anti-lc3 antibodies, respectively. β -actin represents loading control. (b) uninfected or jfh1-infected huh7 cells at more than 90% were transiently transfected with a plasmid encoding for flag-atg12 protein. two days later, cells were lysed and atg12 was immunoprecipitated using anti-flag antibody or igg as a control followed by western blot analysis using anti-flag and anti-atg16l1. (c) huh7 cells were infected with jfh1 (> 90% infected) before being transfected with control (mock), flag-atg12, gfp-atg5, flag-atg12dn or flag-atg12 and flag-atg12dn encoding plasmids. cell lysates were analyzed for hcv ns3, atg5 and atg12 protein expressions at 72 h post-transfection by western blotting. (d) jfh1-infected cells at more than 90% were transfected with control plasmid (mock), flag-atg12dn or flag-atg12 and flag-atg12dn. two days later, intracellular hcv rna was quantified by rt-qpcr. data were collected from three independent experiments (n = 3). (**p = 0.005, ns, none-significant. statistical analysis was performed by using one-way anova). scientific reports | 7:40351 | doi: 10.1038/srep40351 the atg5-12 conjugate positively regulates hcv rna replication in an lc3-independent manner. to determine whether lc3 or the atg5-12 conjugate modulates hcv rna replication, we analyzed the effects of silencing these autophagy genes on viral rna replication in huh7 cells stably expressing the jfh1 subgenomic replicon (sgr). using these specific cells, which are capable of only intracellular hcv rna replication and lacked the capacity to produce infectious viral particles, allowed us to study hcv rna replication independently of viral entry and egress. again, silencing of atg12 but not lc3 efficiently inhibited rna replication, thus supporting the role of the atg5-12 conjugate in viral replication and ruling out the possibility that lc3 participates in viral replication (fig. 3a,b) . because silencing of lc3 expression led to a clear inhibition of hcv rna translation after electroporation of the viral rna but did not significantly affect replication in jfh1-sgr cells, we sought to compare the effect of sirna treatment before and after infection with hcvcc jfh1 (fig. 3c) . the results clearly demonstrated that silc3 inhibited hcv only when it was transfected before infection, whereas siatg7 was effective when it was transfected before or after infection. these results suggest that lc3 is important early in infection and primarily for initial hcv rna translation, as has previously been reported 31 . finally, we evaluated the effects of sirna treatment on intracellular and extracellular hcv infectious particle production in jfh1-infected cells (fig. 3d) . these results suggested that hcv maturation and secretion was not significantly affected by sirna treatment. although silencing atg12 led to a significant decrease in hcv particle formation, this effect was attributed to a severe reduction in viral replication. collectively, atg12 silencing and to a lesser extent atg7 but not lc3, impaired viral replication. atg5-12 and atg16l1 are associated with purified mw extract. in a previous study from our lab, we have shown that atg5 interacts with the hcv polymerase (ns5b) and co-localizes with the mw associated protein ns4b 34 . the major limitation in our ability to investigate the composition of the mw has recently been resolved by dr. ralf bartenschlager's group, which has developed a method to purify the hcv mw by using hcv replicon cells harboring an ha-tag ns4b (ns4b-ha) 42 . this method allowed us to evaluate the presence of the autophagy elongation complex proteins in purified mw extract. through this protocol, after membrane enrichment from a discontinuous sucrose gradient via ultracentrifugation, we pooled fractions that were rich in viral nonstructural proteins (fraction 3 to 7) but mostly devoid of soluble proteins (gapdh or lc3i, fractions 8-10) (fig. 4a) . the mw vesicles were then pulled down from pooled fractions by using a specific antibody against the ha-tag of the hcv ns4b protein. subsequently, the mw-enriched extract was used for either western blot analysis or vesicle visualization by transmission electron microscopy (tem). as expected, hcv ns4b ha , ns3 and ns5a were readily detectable in the purified extract from ns4b ha replicon cells but not that from control untagged ns4b replicon cells (fig. 4b) . the autophagy elongation complex proteins (atg5-12 and atg16l1) were also detected in the purified mw from ns4b ha replicon cells, but not in the control extract, thus indicating that the elongation complex is indeed present at the hcv replication site. in contrast, we were unable to detect lc3ii in the purified mw (fig. 4b) , thereby suggesting that lc3 is not recruited with the autophagy elongation complex to the mw. we then examined the morphology of purified membranes and compared them with er membranes purified from a cell line expressing ha-tagged calnexin, as previously described 42 . the results showed that almost 90% of the ns4b ha purified membranes were spherical vesicles as compared with clnxn ha purified material, in which the majority of membranes were composed of partially collapsed large membranes (fig. 5a,b) . our results are in agreement with those of paul and colleagues, who have demonstrated that most of the purified er membranes are composed of elongated structures, as opposed to the spherical vesicles found in mw extracts 42 . finally, the specificity of the pull-down using ha-beads was confirmed by using extracts from untagged sgr cells (fig. 5c) . altogether, these results indicate that at least a fraction of the autophagy elongation complex is localized in the virus-induced mw compartments. silencing of atg12 or atg7, but not lc3, alters the phenotype of the mw. to evaluate the putative role of host cell proteins in mw formation, reiss and colleagues have established a t7-polymerase-based specific antibodies were used to detect ns3, ns4b ha , ns5a, lc3 and gapdh as indicated on the left. fractions containing membrane-associated proteins (boxed in red) were pooled for affinity capture immunoprecipitation. the density of the different fractions is shown in the lower panel. tcl, total cell lysate. (b) ha-specific affinitycaptured protein content from pooled fraction in (a) was analyzed by western blotting to detect viral ns3, ns4b, ns5a and autophagy elongation complex atg5-12/16l1 by using specific antibodies. pooled fractions from sgr cell lysate was used to demonstrate pull-down specificity. hcv rna synthesis system in which continuous production of hcv polyproteins persists even when hcv replication is abrogated 43 . this system is particularly useful to evaluate the formation of the mw while targeting host cell proteins in the absence of hcv rna replication. with this system, it has been shown that alterations in mw formation result in a clustered phenotype of hcv nonstructural proteins 43, 44 . thus, using the same system (obtained from dr. volker lohmann), we analyzed mw formation indirectly by monitoring viral protein localization after treatment with silc3, siatg7 and siatg12. under normal conditions, the ns3 and ns5a cellular distribution appeared as small punctate structures that appeared to be membrane associated (fig. 6a,b) . treatment with silc3 did not alter the cellular distribution of the viral proteins, as observed by confocal microscopy ( fig. 6a-c) . strikingly, silencing of atg7 or atg12 resulted in the formation of larger protein clusters in most of the transfected cells ( fig. 6a-c) . this effect was not due to decreased hcv protein expression level (fig. 6d) , thus suggesting that the atg5-12 conjugate, but not lc3, is required to obtain a wild-type mw phenotype. silencing of atg12 or atg7, but not lc3, modifies mw morphology. next, using tem, we analyzed mw morphology after treatment with silc3, siatg7 or siatg12. expression of ptm-ns3-5b in cells treated with sictl induced heterogeneous membrane alterations composed of dmvs of an average size of 200 nm interspersed by multi-membrane vesicles (mmvs) that were distributed throughout the cytoplasm (fig. 7a,b) and that were not seen in negative cells (fig. 7e) . silencing of atg7 resulted in more homogenous dmvs with a markedly decreased average size (90 nm) and led to the disappearance of mmvs (fig. 7a,b,d) . silencing of atg12 led to a similar effect but with a much lower abundance of dmvs (fig. 7c) . however, silencing of lc3 had no effect on the dmv size (average diameter 200 nm) (fig. 7b) or on the vesicle types in which both dmvs and mmvs coexist (fig. 7c,d) . thus, a similar morphology of membrane alterations was observed in sictl-treated cells (fig. 7a,b) . these results strongly suggest that the atg5-12 conjugate is crucial for the formation of a typical hcv-induced mw architecture. in the present study, we demonstrated the requirement of the atg5-12/16l1 complex for the completion of the hcv lifecycle. hcv infection does not hamper atg12 conjugation to atg5 or the formation of the multimeric complex atg5-12/16l1 (fig. 1a,b) . in contrast, the conjugation of atg12 to atg5 is crucial for the hcv lifecycle. more specifically, our study suggests a role of the atg5-12/16l1 complex in hcv genome replication and the formation of the mw. the involvement of the autophagy elongation complex in the hcv replication step was investigated by using sirna targeting of atg7, atg12 or lc3. because the silencing of atg7 is known to inhibit the conjugation of both lc3 and atg5, we were able to address the importance of these two conjugation systems in hcv replication. indeed, the atg5-12 conjugate acted as a proviral factor at a step beyond entry and rna translation but before virion maturation and secretion, as depicted in figs 2 and 3. we also observed that silencing of lc3 interfered with hcv rna translation after electroporation of replication-defective replicon (fig. 2e) . these results are consistent with those of dreux and colleagues, who have found a defect in viral rna translation after silencing of beclin-1 or atg4b, thus leading to inhibition of lc3-ii formation 31 . silencing of atg12 had little effect on replication-deficient virus but was detrimental to the replication of the jfh1/fluc virus, thus indicating that its primary target is beyond the translation step (fig. 2e) . this result was further confirmed in cells stably expressing the jfh1 subgenomic replicon, in which silencing of atg7 or atg12, but not lc3, significantly inhibited hcv replication (fig. 3a,b) . silencing of lc3 impeded hcv only when performed before infection, thus suggesting that the atg5-12 conjugate, but not lc3, is important in viral replication after the establishment of infection (fig. 3c ). in addition, the co-purification of the elongation complex proteins with the mw suggested that the atg5-ns5b interaction previously described by our laboratory 34 might actually participate in targeting of the elongation complex to the mw and/or in supplying of autophagic isolation membranes for the formation of the virus-induced vesicles. in canonical autophagy, the atg5-12/16l1 complex is recruited to the isolation membrane prior to lc3 and is released just before the completion of autophagosomes. the absence of lc3 in the purified mw suggests that hcv either hijacks atg5-12/16l1-positive lc3-negative isolation membranes or initiates the de novo formation of the isolation membrane at the mw rather than utilizing lc3-positive autophagosomes for the formation of dmvs within the mw (fig. 4) . interestingly, the recruitment of the elongation complex to the mw was not accompanied by lc3 lipidation or its relocation at that site. recently, it has been demonstrated that the atg5-12/16l1 complex has a membrane-tethering activity that is independent of lc3 45, 46 . this finding highlights the possibility that in hcv infected cells the major role of the elongation complex is to tether vesicles during mw formation. concomitantly, it has been reported that some atg proteins, including atg16l1, can traffic in lc3-free vesicle-like structures to the site where they probably act to generate de novo isolation membranes 47 . this finding also raises the possibility that hcv may recruit similar structures that aid in the formation of the mw. recently, reiss and colleagues have developed a system to evaluate the importance of host factors in membranous web formation 43 . using this system, we demonstrated that atg7 as well as atg12 expression, but not lc3, are important to obtain a wild-type mw phenotype, as observed using confocal microscopy (fig. 6) . furthermore, the morphology of the hcv-induced vesicles was severely altered after silencing of atg7 or atg12, but not lc3. notably, knocking down atg12 decreased the size and the number of dmvs, whereas silencing of atg7 mainly affected their size (fig. 7) . at the moment, it remains unknown whether the altered mw is hcv-replication competent. however, the importance of the atg5-12 conjugate in hcv rna replication suggests that the autophagy elongation complex inhibits hcv replication through destabilization of the viral replication factories present within the mw. in summary, recruitment of the autophagy elongation complex to the mw, which is normally involved in dmv formation, promotes viral replication and maintains proper formation of the wild type mw. cell culture and reagents. huh7 and huh7-lunet cells stably expressing calnexin or ns4b-ha replicon were obtained from dr ralf bartenschlager. huh7-lunet cells stably expressing the t7 polymerase (huh7-lunet-t7) was obtained from dr. volker lohmann, and the huh7.5 cell line was obtained from dr. charles rice. all huh7-derived cell lines were cultured in dulbecco's modified eagle's medium (dmem; gibco) supplemented with 10% v/v fetal bovine serum (fbs) (multicell), 100 u/ml penicillin, 100 μ g/ml streptomycin, and 2 mm l-glutamine (gibco) at 37 °c, 5% co 2 , in a humidified incubator. cell lines harboring the wild-type replicon or ns4b ha were maintained in medium supplemented with g418 (gibco) at a final concentration of 500 μ g/ml. huh7-lunet-t7 and huh7-lunet cells stably expressing calnexin were cultured in the presence of 10 μ g/ml blasticidin (in vivo gen). plasmids and antibodies. the hatg5 and hatg16l1 sequences were cloned into the pegfp-c1 plasmid (clontech), thus forming pgfp-atg5 and pgfp-atg16l1, respectively. the flag-tagged atg12 (patg12) and its dominant-negative derivative patg12δ g140 (atg12dn) constructs were kindly provided by dr. adi kimchi 39 . the ptm vector for the expression of hcv nonstructural proteins ns3 to 5b (ptm-ns3-5b) was kindly provided by dr. volker lohmann. rabbit polyclonal anti-lc3, rabbit polyclonal anti-atg5, mouse monoclonal anti-flag, and mouse monoclonal anti-β -actin antibodies were purchased from sigma aldrich. rabbit polyclonal anti-atg12 and anti-atg7 were purchased from cell signaling. rabbit polyclonal anti-atg16l1 antibody was purchased from mbl. mouse monoclonal anti-ha was purchased from roche. mouse monoclonal anti-ns3 and anti-ns5a antibodies were purchased from biofront. rabbit polyclonal anti-ns3 and ns5a were obtained from dr. olivier nicolas. rabbit polyclonal anti-ns4b and anti-ns5b antibodies were kindly provided by drs. kouacou konan and takaji wakita, respectively. mouse monoclonal anti-gapdh was purchased from santa cruz. the cell culture-derived hcv (hcvcc) jfh1 virus was generated in huh7 cells by transfection of in vitro-transcribed full-length jfh1 rna (megascript, ambion). viral stocks were produced by infection of huh7 cells at a multiplicity of infection (moi) of 0.01, as described previously 48 . a replicative bicistronic jfh1-based full-genome construct expressing firefly luciferase (pjfh1/fluc) and a clone with a mutation in the viral polymerase (gdd-to-gnd) (pjfh1/fluc-gnd) were generated as previously described 49 . to reach 90% infected cells, huh7 cells were infected at an moi of 0.01, passaged for 7 days and then analyzed by immunofluorescence using an anti-ns5a antibody. western blot analysis. cells were lysed in 300 μ l of lysis buffer [25 mm tris-hcl, 150 mm nacl, 1 mm edta, 1% np40, complete protease inhibitor (roche)]. the lysates were normalized for total protein content using the bca protein assay kit (pierce). the proteins were then resolved by sds-page, transferred to polyvinylidene fluoride (pvdf) membranes (bio-rad), blocked for 30 min at room temperature (rt) with pbs-5% milk, and then incubated overnight at 4 °c with primary antibody in pbs-2% bsa. after being washed with 0.4% tween 20 in pbs (pbst), the membranes were incubated for 1 h at rt with a goat-anti-rabbit or goat-anti-mouse igg conjugated to horseradish peroxidase in pbs-5% milk. protein bands were visualized with either the clarity western ecl (bio-rad) or femto chemiluminescence substrates (pierce). purification of hcv-induced mw. hcv-remodeled membrane purification was performed using a method adopted from a previously described protocol 42 . briefly, 7.5 × 10 7 huh7-lunet cells harboring either wild-type or ha-tagged ns4b replicons or control cells stably overexpressing canx ha were washed, scraped and then resuspended in 500 μ l of hypotonic buffer and incubated on ice for 30 min. the cells were lysed with 50 strokes with a dounce homogenizer. the lysates were centrifuged at 800× g for 10 min at 4 °c. supernatants were collected and layered on top of a discontinuous sucrose gradient (70% to 30%) and centrifuged at 130,000× g for 4 h at 4 °c using an sw60i rotor (beckman coulter). ten fractions were collected from the bottom (300 μ l each) and analyzed for protein content. for ha affinity capture, fractions 3 to 7 were pooled, and then an equal amount of protein contained in pooled fractions was equilibrated to 150 mm nacl. incubation with ha-agarose beads (sigma-aldrich) was performed as previously described 42 . membrane visualization by transmission electron microscopy. to examine purified membranes, 50 μ l of eluted material was centrifuged at 10 p.s.i. on a copper grid for 5 min at rt in an airfuge (beckman). structures were negatively stained using 2% aqueous uranyl acetate for 30 sec and examined with an h-7100 (hitachi) transmission electron microscope. quantification of hcv rna by rt-qpcr. isolated rna was reverse transcribed with m-mlv (invitrogen). the generated cdna was used for qpcr using taqman probes, as previously described 50 . results were analyzed using the comparative δ ct method. small interfering rna (sirna) transfection. huh7 cells were reverse transfected in a 24-well plate with sirna (25 nm final concentration) using lipofectamine rnaimax reagent (life technologies) according to the manufacturer's protocol. huh7 cells were transfected with sirna to gfp, lc3b sirna (uaccuguauacguuagugaaauu) or with an on-targetplus human atg7 sirna-smart pool (catalog no. l-020112-00-0005). to study the onset of replication, huh7.5 cells were reverse transfected with sirna, as described above, in 6-well plates. forty-eight hours later, the cells were trypsinized, washed twice with cold pbs, resuspended in 100 μ l of cold ingenio electroporation solution (mirus) and then electroporated with 5 μg of in vitro-transcribed viral rna (jfh1/fluc or jfh1/fluc-gnd) in 2 mm gap electroporation cuvettes by using a btx harvard apparatus with the following settings: 820 v, 99 μ s, 4 pulses, 1.1 s interval. the cells were then resuspended in dmem-10% fbs and seeded in 96-well plates and further cultured for 24 h. the cells were then lysed in 20 μ l luciferase lysis buffer (rlb) and stored at − 80 °c until measurement of luciferase activity. for the determination of intra-and extracellular virus titers, jfh1-infected huh7 cells were reverse transfected with sirna in 6-well plates. two days later, the cells were washed three times with pbs and supplemented with fresh dmem. after 24 h, cells and supernatants were harvested. the cells were washed twice with pbs, trypsinized, resuspended in 1 ml culture medium and subjected to 3 rapid freeze-thaw cycles in a dry ice/ethanol bath and 37 °c water bath, respectively. cell debris was removed by centrifugation at 10,000 rpm for 3 minutes. samples were analyzed using a limiting dilution assay. production of hcvpp and cell entry assay. viral pseudotyped particles harboring the hcv glycoproteins (hcvpp) were produced by transfection in hek-293t cells of vectors encoding viral glycoproteins, packaging proteins and a luciferase marker. after 48 h, viral pseudoparticle supernatants were harvested and filtered through 45-μ m filters to remove the cell debris. for the entry assay, huh7.5 cells were reverse transfected with sirna in 96-well plates. after 48 h, the cells were infected with 50 μ l hcvpp containing supernatant. forty-eight hours post-infection, the cells were washed three times with pbs, lysed in 20 μ l luciferase lysis buffer (rlb) and stored at − 80 °c until measurement of luciferase activity. luciferase assay. cell lysates were prepared with reporter lysis buffer (rlb) (promega), and luciferase activity was measured with a luciferase assay system (promega), per the manufacturer's protocol. for the immunofluorescence experiment, huh7-lunet-t7 cells were reverse transfected with sirna as previously described 43 . forty-eight hours later, a second round of transfection with sirna was performed. after 48 h, cells were transfected with ptm-ns3-5b using lipofectamine 3000 (invitrogen). the coverslips were then fixed with 4% formaldehyde in pbs for 10 min, washed in pbs and incubated in blocking buffer (pbs, 3% bovine serum albumin, 10% fbs, 0.1% triton x-100) for 30 min at rt. after being washed three times with pbs, the coverslips were incubated with primary antibody in blocking buffer for 1 h at rt. then, the coverslips were washed with pbs and incubated with either alexa fluor ™ -(488 or 568) goat anti-mouse igg or alexa fluor ™ -(488 or 568) goat anti-rabbit igg (invitrogen) for 1 h at rt. after being washed, the coverslips were mounted on glass slides with prolong ™ . antifade (invitrogen) and examined with a laser scanning confocal zeiss lsm 780. for tem analysis, a similar setup was used, except that after transfection with ptm-ns3-5b, the cells were trypsinized and seeded into lab-tek chamber slides (thermo fisher). after 24 h, the monolayer of cells was washed with pbs, fixed with 2.5% glutaraldehyde (electron microscopy science) and incubated overnight at 4 °c. the cells were then washed in 0.1 m cacodylate (electron microscopy science) and incubated in 1% osmium tetroxide (mecalab) for 1 h at 4 °c. the cells were dehydrated in a graded series of ethanol/deionized water solutions (from 50% to 100%). the cells were then infiltrated with a 1:1 and 3:1 epon 812 for 1 h for embedding and polymerized overnight in an oven at 60 °c. the polymerized blocks were trimmed, and 100 nm ultrathin sections cut with an ultracut e ultramicrotome (reichert jung) and transferred onto 200-mesh copper grids (electron microscopy science) with formvar support film. the sections were stained with 4% uranyl acetate (electron microscopy science) for 8 min, then with lead citrate for 5 min (fisher scientific). the cells were imaged with an fei tecnai 12 transmission electron microscope (fei company) operating at an accelerating voltage of 120 kv and equipped with an amt xr80c ccd camera. vesicle size was measured using image j (nih). cell viability assay. cells were reverse transfected with different sirnas used in this study in a 96-well plate for 48 h. cell viability was then assayed using the celltiter 96 ® aqueous non-radioactive cell proliferation assay reagent (promega). the results shown represent the mean of at least three independent experiments. student's-t-test and one-way anova with dunnett's post-test (as indicated in the figure legends) were performed using graphpad prism 5. p-values below 0.05 were considered statistically significant. development of novel treatments for hepatitis c replication of hepatitis c virus identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus replication of hepatitis c virus rna on autophagosomal membranes autophagy in immunity and inflammation the atg16l complex specifies the site of lc3 lipidation for membrane biogenesis in autophagy subversion of cellular autophagosomal machinery by rna viruses autophagy is involved in the early step of japanese encephalitis virus infection flavivirus ns4a-induced autophagy protects cells against death and enhances virus replication dengue virus-induced autophagy regulates lipid metabolism autophagy sustains the replication of porcine reproductive and respiratory virus in host cells hcv induces the expression of rubicon and uvrag to temporally regulate the maturation of autophagosomes and viral replication hepatitis c virus inhibits akt-tuberous sclerosis complex (tsc), the mechanistic target of rapamycin (mtor) pathway, through endoplasmic reticulum stress to induce autophagy hepatitis c virus genotype 1a growth and induction of autophagy persistent expression of hepatitis c virus non-structural proteins leads to increased autophagy and mitochondrial injury in human hepatoma cells changes in autophagic response in patients with chronic hepatitis c virus infection hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro hcv infection selectively impairs type i but not type iii ifn signaling hepatitis c virus core protein activates autophagy through eif2ak3 and atf6 upr pathway-mediated map1lc3b and atg12 expression hepatitis c virus-induced autophagy is independent of the unfolded protein response irgm is a common target of rna viruses that subvert the autophagy network knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles dissection of autophagosome formation using apg5-deficient mouse embryonic stem cells autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection mouse apg16l, a novel wd-repeat protein, targets to the autophagic isolation membrane with the apg12-apg5 conjugate generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size molecular mechanism of autophagic membrane-scaffold assembly and disassembly formation of the approximately 350-kda apg12-apg5.apg16 multimeric complex, mediated by apg16 oligomerization, is essential for autophagy in yeast the autophagy protein atg12 associates with antiapoptotic bcl-2 family members to promote mitochondrial apoptosis methods in mammalian autophagy research characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins morphological and biochemical characterization of the membranous hepatitis c virus replication compartment recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment the lipid kinase phosphatidylinositol-4 kinase iii alpha regulates the phosphorylation status of hepatitis c virus ns5a dissecting the role of the atg12-atg5-atg16 complex during autophagosome formation mechanism and functions of membrane binding by the atg5-atg12/atg16 complex during autophagosome formation structures containing atg9a and the ulk1 complex independently target depolarized mitochondria at initial stages of parkin-mediated mitophagy novel hcv replication mouse model using human hepatocellular carcinoma xenografts ski-1/s1p inhibitor pf-429242 impairs the onset of hcv infection ski-1/s1p inhibition: a promising surrogate to statins to block hepatitis c virus replication we thank dr. takaji wakita for providing the hcv jfh-1 and the anti-ns5b antibody. the huh7-lunet, ns4b hareplication and canx ha cells were generously provided by dr. ralf bartenschlager. the huh7-lunet-t7 cells and the ptm-ns3-5b vector were provided by dr. volker lohmann. we are also grateful to dr. kouacou konan for providing the anti-ns4b antibody and dr. adi kimchi for providing the atg12 and atg12δ g140 plasmids. we also thank dr. hojatollah vali and his group for performing tem with af. in addition, we thank david paul, jessy tremblay and micheline letarte for technical assistance. this work was supported by a research grant from the nserc of canada. af received fellowships from the ncrtp-hepc. a.f. and p.l. designed the study. a.f. performed the experiments. a.f. and p.l. analyzed the data and wrote the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep key: cord-005138-u2lwgyyf authors: kou, yi-hen; chou, shang-min; wang, yi-ming; chang, ya-tzu; huang, shao-yong; jung, mei-ying; huang, yu-hsu; chen, mei-ru; chang, ming-fu; chang, shin c. title: hepatitis c virus ns4a inhibits cap-dependent and the viral ires-mediated translation through interacting with eukaryotic elongation factor 1a date: 2006-08-23 journal: j biomed sci doi: 10.1007/s11373-006-9104-8 sha: doc_id: 5138 cord_uid: u2lwgyyf the genomic rna of hepatitis c virus (hcv) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral ns3 and ns2-3 proteases. nonstructural protein 4a (ns4a) is a cofactor of the ns3 serine protease and has been demonstrated to inhibit protein synthesis. in this study, gst pull-down assay was performed to examine potential cellular factors that interact with the ns4a protein and are involved in the pathogenesis of hcv. a trypsin digestion followed by lc-ms/ms analysis revealed that one of the gst-ns4a-interacting proteins to be eukaryotic elongation factor 1a (eef1a). both the n-terminal domain of ns4a from amino acid residues 1–20, and the central domain from residues 21–34 interacted with eef1a, but the central domain was the key player involved in the ns4a-mediated translation inhibition. ns4a(21–34) diminished both cap-dependent and hcv ires-mediated translation in a dose-dependent manner. the translation inhibitory effect of ns4a(21–34) was relieved by the addition of purified recombinant eef1a in an in vitro translation system. taken together, ns4a inhibits host and viral translation through interacting with eef1a, implying a possible mechanism by which ns4a is involved in the pathogenesis and chronic infection of hcv. hepatitis c virus (hcv) is the major causative agent of human chronic hepatitis and is closely associated with hepatocellular carcinoma [1] . it is an enveloped virus and is classified as a separate genus in the family flaviviridae [2] . the genome of hcv is a singlestranded, positive sense rna of approximately 9.6 kb that encodes a polyprotein of approximately 3000 amino acid residues [3] [4] [5] . the polyprotein precursor is processed cotranslationally and post-translationally by cellular er peptidase and viral proteases to generate mature structural and nonstructural proteins [6] [7] [8] . cleavage at the junction of ns2-ns3 occurs autoproteolytically by the ns2-3 protease [9] , whereas cleavage of the downstream nonstructural proteins is performed by the viral ns3 serine protease [10] [11] [12] . nonstructural protein 4a (ns4a) is a multifunctional protein with 54 amino acid residues. it acts as a cofactor of ns3 serine protease and plays an essential role in the ns4a-dependent cleavage at the ns3-ns4a and ns4b-ns5a junctions [13, 14] . both ns4a and ns4b proteins were previously demonstrated to suppress translation in culture cells [15, 16] . however, neither ns4a nor ns4b affected the steady state level of canonical translational factors including eif4g, eif4e, pabp, and 4e-bp1 [16] . the mechanisms by which the hcv ns4a and ns4b involved in the translational inhibition remained unclear. in this study, we have demonstrated that ns4a specifically interacted with eukaryotic elongation factor 1a (eef1a) and inhibited both cap-and hcv ires-dependent protein synthesis. the inhibitory effect was mainly mediated by the central domain of ns4a and could be restored by the addition of recombinant eef1a in an in vitro translation system. (i) plasmids pcrii-topo-ns(3c-5an), pcrii-topo-ns4b, and pcdna-hcv-sg. plasmid pcrii-topo-ns(3c-5an) encompasses cdna sequences of the hcv genome (genotype 1b) from nucleotides 4458-6381 inserted into pcr ò ii-topo (invitrogen). the cdna fragment was generated from a serum sample of an hcv patient by reverse transcriptase-polymerase chain reaction (rt-pcr) with advantage ò one-step rt-pcr kit (bd biosciences clontech) and the primer sets 5¢-ctcgcag cgggcaggcaggactgg-3¢ and 5¢-cc catccacttccgtgaagaa-3¢ for the initial amplification and 5¢-gcggcgag-gcgcgactggtagg-3¢ and 5¢-cccat ccacttccgtgaagaa-3¢ for a further amplification. an ns4b cdna fragment was amplified from pcrii-topo-ns(3c-5an) with primer set 5¢-ggaattccatatggcctca cacctcccttacatcgaacaa-3¢ and 5¢-cgggatcctctagatcagcatggcgt ggagcagtcttcatt-3¢, and cloned into pcr ò ii-topo to generate plasmid pcrii-topo-ns4b. plasmid pcdna-hcv-sg represents a subgenomic replicon of hcv genomic type 1b and consists of the hcv-ires, the neo gene, and the emcv-ires fused to the hcv sequences from ns3 to ns5b and the 3¢ noncoding region. these plasmids were used to generate expression plasmids of hcv ns3, ns4a and ns4b proteins. (ii) plasmids pcmv-tag2c-ns4a and pgst-ns4a. for generation of pcmv-tag2c-ns4a, a full-length ns4a cdna was amplified from pcrii-topo-ns(3c-5an) with the primer set 5¢-cgggatccatatg agcacctgggtgcttgta-3¢ and 5¢-gga attcagcactcctccatttc-3¢, the pcr fragment was digested with bam-hi and ecori restriction endonucleases, and cloned into the bamhi-ecori site of pcmv-tag2c (stratagene). for generation of plasmid pgst-ns4a, plasmid pcmv-tag2c-ns4a was digested with bamhi and treated with the klenow fragment of dna polymerase i prior to a further digestion with xhoi restriction endonuclease. the resulting fragment was inserted into the pgex-6p-1 (ge healthcare bio-sciences) from which the polylinker sequences between bamhi and xhoi had been deleted and the bamhi site blunted. (iii) plasmids pcdna-ns4a-v5histopo, pc dna-ns3-v5histopo, and pcdna-ns4b-v5histopo. for generation of plasmid pcdna-ns4a-v5histopo, cdna sequence of the full-length ns4a was amplified from pcmvtag2c-ns4a with the primer set 5¢-caccatgagcacctgggtgcttgta3¢ and 5¢-gcactcctccatttcatc-3¢, and cloned into pcdna tm 3.1d/v5-his-topo ò (invitrogen). for generation of pc dna-ns3-v5histopo and pcdna-ns4b-v5his-topo, full length cdna fragments of the hcv ns3 and ns4b were amplified from pcdna-hcv-sg with the primer set 5¢-caccatggcgcccatcactgcctacg ctcaacaga-3¢ and 5¢-cgtgac-gacctccaggtcagctgccatgcat gt-3¢, and the primer set 5¢-cac catggcctgacacctcccttacatc gaacaa-3¢ and 5¢-gcatggcgt ggagcagtcttcatta-3¢, respectively, the resulting pcr fragments were cloned into pcdna tm (vi) plasmids pcdna-eef1a-v5histopo, pcd na-eef1a(1-240)-v5histopo, pcdna-eef1a(201-462)-v5histopo, and pcdna-eef1a-histopo. for generation of plasmids pcdna-eef1a-v5histopo and its deletion mutants, rna was isolated from huh7 cells by a single step extraction method as described previously [17] . the rna was used to perform rt-pcr with primers sets described below and the resulting cdna fragments were cloned into pcdna tm 3.1d/v5-his-topo ò . the primer sets used in the amplification were ef-k (5¢-cac catgggaaaggaaaaga ctc-3¢) and ef-r (5¢-tttagccttctgagctttct gg-3¢) for generating pcdna-eef1a-v5histopo that represents v5his-tagged full length eef1a, ef-k and ef-nr (5¢-acgagttggtggtaggat3¢) for generating pcdna-eef1a(1-240)-v5histopo that represents v5his-tagged n-terminal eef1a from amino acid residues 1-240, and ef-mk (5¢-caccatgctggagccaag tgctaa-3¢) and ef-r for generating pcdna-eef1a(201-462)-v5histopo that represents v5his-tagged c-terminal eef1a from amino acid residues 201-462. for generation of plasmid pcdna-eef1a-histopo, the v5-epitope was removed from pcdna-eef1a-v5histopo following a digestion with xhoi and agei restriction endonucleases and the ends were blunted with the klenow fragment of dna polymerase i prior to self-ligation. (vii) plasmids pgst-ns3, pgst-ns4b, and pgst-eef1a. for construction of pgst-ns3, plasmid pcdna-ns3-v5histopo was digested with ncoi and treated with the klenow fragment of dna polymerase i prior to a further digestion with xhoi restriction endonuclease. the resulting fragment was inserted into the pgex-6p-1 from which the polylinker sequences between ecori and xhoi had been deleted and the ecori site blunted. a similar approach was taken to generate pgst-eef1a from pcdna-eef1a-v5histopo. for generation of plasmid pgst-ns4b, pcrii-topo-ns4b described earlier was digested with ecori restriction endonuclease and the resulting ns4b-containing fragment was inserted into the ecori site of pgex-6p-1. (viii) deletion mutants of the plasmid pgst-ns4a. for construction of plasmids pgst-ns4a(1-34) and pgst-ns4a(21-54), cdna fragments were obtained by pcr-amplification from pgst-ns4a with primer set 5 ¢ -cgggatccatatgagcacctgggtgc ttgta-3 ¢ and 5 ¢ -ggaattcactt cccggacaagat-3 ¢ and primer set 5 ¢ -ggaattccatatgggcagcgtggtca ttgt-3 and 5 ¢ -ggaattcagcactcctc catttc-3 ¢ , respectively, and the resultant fragments were digested with ndei and ecori restriction endonucleases. for construction of plasmids pgst-ns4a(1-20), pgst-ns4a (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) , and pgst-ns4a(35-54), cdna fragments with ndei and ecori recognition sequences at the 5 ¢ and 3 ¢ ends, respectively, were generated by annealing the following synthetic oligonucleotide sets: 5 ¢ -tatgagcacctgggtgcttgtaggcg gggtccttgcagctctggccgcatac tg-3 ¢ and 5 ¢ -aattcacgttgtcagg cagtatgcggccagagctgcaaggac cccgcctacaagcacccaggtgctca-3 ¢ for pgst-ns4a(1-20), 5 ¢ -tatggg cagcgtggtcattgtgggcaggatc atcttgtcc gggaagtg-3 ¢ and 5 ¢ -aattcacttcccggacaagatgatc ctgcccacaatgaccacgctgccca-3 ¢ for pgst-ns 4a (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) , and 5 ¢ -ta tgccggctgtcattcctgataggga ggttctctaccgggagttcgatgaaa tggaggagtgctg -3 ¢ and 5 ¢ -aattmc agcactcctccatttcatcgaactcc cggtagagaacctccctatcaggaat gacagccggca-3 ¢ for pgst-ns4a (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) . these cdna fragments were used to replace the cognate fragment of pgst-ns4a to generate the deletion mutants of gst-ns4a. (ix) plasmids pcmv-luc and pjss12. plasmid pcmv-luc represents a cap-dependent monocistronic reporter of firefly luciferase that is driven by the promoter of cytomegalovirus. pjss12 represents a bicistronic luciferase reporter containing the structure components of cmv promoter-t7 promoter-renilla luciferase-stem loop-ires(hcv1-371)-firefly luciferase-poly a. transcription of the bicistronic reporter can be driven by the promoter of cytomegalovirus in culture cells or by the promoter of t7 rna polymerase in vitro. expression of the renilla luciferase and firefly luciferase genes are directed by the cap-dependent and hcv-ires-mediated mechanism, respectively. in addition, the stem loop structure is derived from the sequence 5¢-gtaccccggtacggcagtg ccgtacgacgaattcgtcgtacggca ctgcc gtaccggggtac-3¢ and was inserted into the bicistronic structure to prevent leaky scanning of ribosome. gst fusion plasmids were transformed into escherichia coli bl21, or bl21(de3) where indicated. the bacterial cells were grown in lb or 2x yt medium containing 50 lg ampicillin/ml to a density of 0.6-0.8 at 600 nm. following an induction of the expression of gst fusion proteins with isopropyl-b-d-thiogalactopyranoside (iptg), the bacterial cells were harvested and resuspended in lysis buffer consisting of 10 mm tris-hcl, ph 8.0, 150 mm nacl, 1 mm edta, 1.5% n-laurylsarcosine, and 2% triton x-100. after two freeze-thaw cycles and sonication, cell lysates were separated into soluble and insoluble fractions. for partial purification of gst fusion proteins, the soluble fractions were incubated with glutathione-sepharose 4b beads (ge healthcare bio-sciences) at 4°c for 2 h. following a centrifugation in a microcentrifuge for 5 min, gst fusion proteins immobilized on the beads were spun-down and washed for five times with pbs. as a control, plasmid pgex-6p-1 was transformed into the bacterial cells, and the expression and purification of gst protein were performed in parallel. in the in vitro translation inhibition assay, gst fusion proteins of the hcv nonstructural proteins were recovered from the beads with 10 mm glutathione in 50 mm tris-hcl (ph 8.0). in addition, eef1a was recovered from beads-immobilized gst-eef1a with prescission tm protease (ge healthcare bio-sciences). huh7 cells (a human hepatoma cell line) were maintained at 37°c in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum plus 100 units of penicillin, and 100 lg of streptomycin/ml. expression of recombinant proteins in huh7 cells was preformed by dna transfection with cationic liposomes (invitrogen) or by infecting cells with recombinant vaccinia virus (vtf7-3) harboring t7 rna polymerase gene followed by dna transfection as described previously [18] . two days posttransfection, the transfected cells were harvested for further analysis. preparation of huh7 cell lysates, in vitro translation products, and the ns4a(21-34) peptide to perform gst pull-down assay, cell lysates were prepared from huh7 cells grown to confluency with a lysis buffer containing 50 mm tris-hcl, ph 8.0, 150 mm nacl, 1% sodium deoxycholate, 1% np-40, and 0.1% sds. alternatively, in vitro translation products were used. in vitro translation was performed in the presence of [ 35 s]methionine (nen) by the t n t ò quick coupled transcription/translation system (promega). in addition, the ns4a(21-34) peptide used in the in vitro translation inhibition assay was synthesized and purified through hplc to > 85% purity. to perform gst pull-down assay, gst fusion proteins immobilized on glutathione-sepharose 4b beads were incubated independently with the huh7 cell lysates or in vitro translation products at 4°c for 2 h. the protein-bound glutathione beads were then washed for five times with pbs and boiled for 5 min prior to sds-page. proteins that copurified with gst fusion proteins were visualized by coomassie blue staining, silver staining, or autoradiography. identities of the copurified proteins were further examined by western blot analysis. to perform coimmunoprecipitation experiments, transfected cells were washed twice with pbs and lysed in a ripa buffer consisting of 50 mm tris-hcl, ph 7.6, 150 mm nacl, 1% sodium deoxycholate, 1% np-40, 0.1% sds, and complete tm protease inhibitors (roche). equivalent amounts of the cell lysates were incubated with specific antibodies for 16 h at 4°c and then with protein a sepharose cl-4b (ge healthcare bio-sciences) for additional 4 h. the immunoprecipitates were washed five times with ripa buffer and resuspended in a lysis buffer consisting of 1.7 mm urea, 3.3% sds, and 0.65 m b-mercaptoethanol. the resultant supernatants were resolved on polyacrylamide gel and subjected to western blot analysis. western blot analysis was performed as described previously [19] . the specific interactions between antigens and antibodies were detected by the enhanced chemiluminescence system (ge healthcare bio-sciences). mouse monoclonal antibody against v5 epitope (gkpipnpllgldst) was purchased from invitrogen. rabbit polyclonal antibody against his-tag was purchased from santa cruz. mouse monoclonal antibody against eef1a was purchased from upstate. goat polyclonal antibody against luciferase was purchased from promega. the level of luciferase mrna was examined by rt-pcr. in brief, total rna was isolated from culture cells by using trizol ò reagent (invitrogen). reverse transcription was performed with the rna template, amv reverse transcriptase (roche), and oligo-dt primer. the products were subjected to polymerase chain reaction with the primer set 5¢-cagaggacctatgattatgtc 3¢ and 5¢-cggtacttcgtccacaaacacaa c-3¢. in addition, primers 5¢-gaaggtgaagg tcggactc-3¢ and 5¢-tttagccttctga gctttctgg-3¢ were used in parallel to analyze the level of gapdh mrna as an internal control. to perform translation inhibition assay in culture cells, plasmids encoding v5-tagged hcv nonstructure proteins ns3, ns4a, ns4b, and ns4a mutants were independently cotransfected with the cap-dependent monocistronic luciferase reporter pcmv-luc or the bicistronic luciferase reporter pjss12 into huh7 cells. the cells were harvested 2 days posttransfection. luciferase activities were analyzed followed the procedures as described by the manufacturer (promega) and measured with a luminometer (orion ii, berthold). in addition, the levels of luciferase protein and luciferase mrna were examined by western blot analysis and rt-pcr, respectively. in-vitro-translation system was applied to study the inhibitory effects of ns4a on both cap-dependent and hcv ires-mediated translation. to perform cap-dependent in-vitro-translation inhibition assay, gst and gst-ns4a fusion proteins that were recovered from the sepharose 4b beads as described earlier were preincubated with 5 ll of rabbit reticulocyte lysate (rrl, promega) at 4°c for 1 h. translation reaction was then carried out at 30°c for 90 min after addition of 250 ng of in vitro-transcribed monocistronic luciferase mrna, amino acid mixtures, and rnase inhibitor, followed by luciferase activity assay. alternatively, the translation reaction was performed in the presence of [ 35 s]methionine (nen) and the inhibitory effects of ns4a on translation were analyzed by autoradiography following sds-page. on the other hand, in-vitro-translation inhibition assay was performed with synthetic ns4a(21-34) peptide and 500 ng of the bicistronic luciferase mrna in-vitro-transcribed from plasmid pjss12. luciferase activities of renilla and firefly that represent the capdependent and hcv ires-mediated translation, respectively, of the bicistronic reporter were analyzed using the dual-glo tm luciferase assay system (promega). in addition, to examine the ability of eef1a to restore the translation inhibition, eef1a was released from gst-eef1a by prescission tm protease, dialyzed to 50 mm tris-hcl (ph 8.0), and incubated for 30 min with the reaction mixture of gst-ns4a protein and rrl in the cap-dependent in-vitro-translation inhibition assay. luciferase activity was analyzed as described earlier. identification of cellular proteins specifically interact with the ns4a protein of hcv to learn the possible association of host factors with the hcv ns4a protein that may render ns4a pathogenic to the host, gst pull-down assay was performed to identify ns4a-interacting proteins. gst-ns4a protein was expressed in e. coli bl21 in the presence of iptg (figure 1a ). following purification with glutathione-sepherose 4b beads, the gst-ns4a protein was subjected to gst pull-down assay with huh7 cell extracts. silver staining identified several proteins that specifically pulled down by the gst-ns4a protein (figure 1b) . the most abundant ns4a-interacting candidate protein was subjected to trypsin digestion and lc-ms/ms analysis. thirty-four spectra that represent 19 independent tryptic fragments of 7-29 amino acid residues all identified the protein as human translation elongation factor 1 alpha-1 (eef1a) (figure 1c ). cellular factors that participate in protein synthesis are now well characterized. it is clear that eef1a interacts with gtp and is responsible for binding aminoacyl-trna to the ribosome during polypeptide elongation [20] . previous studies have demonstrated that both ns4a and ns4b inhibit protein synthesis [15, 16] , but the molecular basis involved in the inhibition was not clear. by performing cotransfection experiments with an hcv ns3-, ns4a-, or ns4b-encoding plasmid and a cap-dependent monocistronic luciferase reporter into huh7 cells, pecific inhibitory effects of ns4a and ns4b on the cap-dependent translation were detected in this study. the luciferase activity was significantly reduced when the reporter plasmid was coexpressed with ns4a or ns4b protein (figure 2a ). in addition, the effects correlated very well with the protein levels of luciferase (figure 2b ), whereas the luciferase mrna level was not affected by the hcv nonstructural proteins (figure 2c ). possible effects of the viral nonstructural proteins on hcv ires-mediated translation were further examined by cotransfecting into huh7 cells a bicistronic reporter that consists of the renilla luciferase, the hcv ires (genotype 1b), and the firefly luciferase genes. two days posttransfection, dual-glo tm luciferase assay (promega) was performed. renilla luciferase activity represents the cap-dependent translation and firefly luciferase activity represents the hcv ires-mediated translation. the results demonstrated that both ns4a and ns4b inhibited hcv ires-mediated translation to levels similar to those of the cap-dependent translation, whereas no effect was detected with the viral ns3 protein (figure 2d ). we proposed that through interacting with eef1a, hcv ns4a protein may deplete or interfere eef1a in forming functional complexes involved in both cap-dependent and ires-mediated translation. to test this hypothesis, we first examined the specificity of the interaction between ns4a and eef1a. gst fusion proteins of the viral ns3, ns4a, and ns4b were expressed in e. coli bl21(de3) in the presence of iptg, and immobilized on glutathione-sepharose 4b beads (figure 3a) . gst pull-down assay was performed with huh7 cell extracts followed by western blot analysis with anti-eef1a antibody. the results demonstrated that ns4a but neither ns3 nor ns4b interacted with eef1a in the gst pulldown system (figure 3b) . to examine the interaction in culture cells, cotransfection experiments were performed in huh7 cells with plasmids encoding v5-tagged ns4a (pcdna-ns4a-v5) or ns4b (pcdna-ns4b-v5) and a his-tagged eef1a (pcdna-eef1a-histopo). two days posttransfection, cell extracts were immunoprecipitated with anti-his antibody followed by western blot analysis with anti-v5 antibody. as shown in figure 3c , ns4a but not ns4b was coimmunoprecipitated with the eef1a. these results indicate that, although both ns4a and ns4b are capable of inhibiting protein synthesis, binding to eef1a is a unique characteristic of ns4a. it is likely that ns4a and ns4b inhibit protein translation through different mechanisms. to identify the subdomains of ns4a responsible for interacting with eef1a, deletion plasmids representing gst fusion proteins of various domains of the ns4a were generated and expressed in e. coli bl21. following a partial purification, the gst-ns4a mutant proteins were subjected to gst pull-down assay with huh7 cell extracts. as shown in figure 4a the functional domain of eef1a involved in the binding of ns4a was examined. full-length eef1a, its n terminus from amino acid residues 1-240 [eef1a(1-240)], and the c terminus from amino acid residues 201-462 [eef1a(201-462)] were synthesized in vitro by the t n t quick coupled transcription/translation system. the translation products were subjected to gst pull-down assay with partially purified gst and gst-ns4a fusion proteins. as shown in figure 4b , both the full-length eef1a and the eef1a(201-462) were pulled-down by the gst-ns4a fusion protein, but the eef1a(1-240) could not. these results indicate a specific interaction between ns4a and the c-terminal domain of eef1a. in figure 4a , we have demonstrated that ns4a interacted with eef1a through two independent domains within the n-terminal 34 amino acid residues. to link the interaction between ns4a and eef1a to the effect of ns4a on protein synthesis, translation inhibition assay was performed. huh7 cells were cotransfected with the monocistronic luciferase reporter and a plasmid representing ns4a(1-34) or ns4a (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) . two days posttransfection, cells were harvested and analyzed for the luciferase activity and the levels of luciferase protein and mrna. as shown in figure 5 , ns4a(1-34) inhibited both the luciferase activity and luciferase protein to levels compatible to the full-length ns4a protein did but had little effect on the level of luciferase mrna. these results indicated that the n-terminal 34 amino acid residues are responsible for the inhibitory effect of ns4a on translation. to determine which of the the inhibitory effects of ns4a and ns4b proteins on cap-dependent translation. plasmids pcdna-ns3-v5, pcdna-ns4a-v5, and pcdna-ns4b-v5 encoding hcv ns3, ns4a, and ns4b, respectively, were independently cotransfected into huh7 cells with the monocistronic luciferase reporter plasmid pcmv-luc. two days posttransfection, cell lysates were harvested and equal amounts of the proteins were used to perform luciferase assay (panel a) and western blot analysis (panel b). meanwhile, total rna was isolated to perform rt-pcr (panel c). luciferase activities derived from the transfected cells were normalized by the activity of the cells cotransfected with pcmv-luc and pcdna3.1(+) control vector. the results represent the average of four independent experiments. western blot analysis was performed with antibodies specific to luciferase and gapdh as indicated. intensities of the luciferase proteins were normalized in each set against the intensity of gapdh and compared to the control. relative intensities of the luciferase protein are shown. rt-pcr of luciferase mrna was performed with oligo-dt and primers as described in the materials and methods for 20 amplification cycles. intensities of the luciferase cdna fragment were normalized in each set against the intensity of gapdh and relative intensities as compared to the vector control are shown. (d) the inhibitory effects of ns4a and ns4b proteins on hcv ires-mediated translation. the bicistronic luciferase reporter plasmid pjss12 was cotransfected independently with the hcv ns3-, ns4a-, and ns4b-encoding plasmid. the activities of renilla luciferase and firefly luciferase that representing cap-dependent and hcv ires-mediated translation, respectively, were analyzed 2 days posttransfection with dual-glo tm luciferase assay system as described in the materials and methods. relative luciferase activities in the presence of the hcv nonstructural proteins were calculated by normalization of the activities in each set to that of the cells cotransfected with the reporter plasmid and pcdna3.1(+) control vector. two eef1a-interacting domains, ns4a(1-20) and ns4a (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) , is essential for the translation inhibition, gst-ns4a and its deletion mutant proteins were purified (figure 6a ) and used to perform a translation inhibition assay in a cell-free system. increasing amounts of the purified gst fusion proteins were pre-incubated with rrl followed by in vitro translation reaction with the monocistronic luciferase mrna as the reporter. the results demonstrated that both the gst-ns4a and gst-ns4a(21-34) significantly reduced the luciferase activity, whereas the effects of gst-ns4a(1-20) and gst-ns4a(35-54) proteins were negligible (figure 6b ). in addition, the gst-ns4a(21-34) protein had a stronger effect on the translation inhibition than that of the wild type. it required less than half amount of gst-ns4a(21-34) protein to reach a 50%-inhibition when compared with the full-length ns4a protein (4.29 â 10 13 moles for full-length and 1.81 â 10 13 moles for the subdomain 21-34). the effects of ns4a subdomains on the translation inhibition were also examined by analyzing the level of luciferase protein following an in-vitro-translation reaction in the presence of [ 35 s]methionine and 2 â 10 13 moles of the ns4a proteins. as shown in figure 6c , gst-ns4a(21-34) had a greater effect than the gst-ns4a(1-20) whereas gst-ns4a(35-54) had no effect on the translation inhibition. bl21(de3) . following a partial purification, the gst fusion proteins immobilized on the glutathione-sepharose 4b beads were analyzed by sds-page. coomassie blue staining is shown. (b) eef1a specifically copurified with gst-ns4a. huh7 cell extracts were incubated with partially purified gst-fusion proteins as indicated, followed by gst pull-down assay and western blot analysis with antibody specific to eef1a. cell extract control represents huh7 cell extract that was loaded directly onto the gel to serve as a control. (c) interaction between ns4a and eef1a in transfected culture cells. plasmids pcdna-ns4a-v5 and pcdna-ns4b-v5 that encode v5 epitope-tagged ns4a and ns4b proteins, respectively, were independently cotransfected into huh7 cells with plasmid pcdna-eef1a-histopo that encodes a his-tagged fusion protein of the fulllength eef1a (eef1a-his). cell lysates were prepared 2 days posttransfection to perform coimmunoprecipitation assay with antibodies against the his-tag of eef1a-his protein and western blot analysis with antibodies specific to the v5-epitope to detect v5-tagged ns4a and ns4b proteins. input lanes represent 5 % of the cell lysates used in the coimmunoprecipitation assay and were loaded directly onto the gel to serve as positive controls of western blot analysis. the effect of ns4a on the translation inhibition may be resulted from a competition between ns4a and translation factors that are involved in forming functional translation complexes with eef1a. we therefore examined gst and gst fusion proteins of the wild-type ns4a and its deletion mutants were eluted out from the glutathione-sepharose 4b beads. eef1a was purified from bead-bound gst-eef1a following a cleavage with prescission tm protease and dialyzed into 50 mm tris-hcl (ph 8.0) as described in the materials and methods. the purified proteins were analyzed by sds-page. coomassie blue staining is shown. (b)-(c) cap-dependent in-vitro-translation inhibition assay. various amounts of the purified gst fusion proteins as indicated in panel b or 2 â 10 13 moles of the gst fusion proteins as indicated in panel c were pre-incubated with rrl for 1 h at 4°c. translation reaction was then carried out at 30°c following addition of the monocistronic luciferase mrna, amino acid mixtures, and rnase inhibitor in the absence (panel b) or presence (panel c) of [ 35 s]methionine. the translation products were subjected to luciferase activity assay (panel b) or sds-page followed by autoradiography (panel c). relative luciferase activities were calculated by normalization of the luciferase activities in the presence of the gst and gst-ns4a proteins to that without any gst proteins. relative intensities of the luciferase protein were normalized against the intensity of luciferase protein in the presence of gst. (d) translation restore experiments. gst fusion proteins of 2 â 10 13 moles were preincubated with rrl for 1 h at 4°c followed by an additional incubation for 30 min in the presence of 0.5 lg purified recombinant eef1a. in-vitro-translation reaction was then performed. the ability of the purified eef1a to restore the translation inhibition was analyzed by luciferase activity assay. relative luciferase activities as compared to that of gst in the absence of exogenous eef1a are shown. the result represents the average of two independent experiments. (e) hcv ires-mediated in-vitro-translation inhibition assay. various amounts of the ns4a(21-34) peptide as indicated were preincubated with rrl for 30 min at 4°c prior to the in-vitro-translation reaction. in-vitro-translation reaction was performed as described in the legend to panel b except that the bicistronic luciferase reporter mrna and ns4a(21-34) peptide were used. luciferase activities of renilla and firefly that represent cap-dependent and hcv ires-mediated translation, respectively, were analyzed and normalized in each set against the luciferase activity of the reaction without addition of the ns4a(21-34) peptide. to eliminate the possibility that the translation inhibition effect of gst-ns4a(21-34) is resulted from a trace contamination of inhibitors present in the protein preparation, a synthetic ns4a (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) peptide purified by hplc to >85% purity was applied. meanwhile, the bicistronic luciferase reporter mrna was used in the in vitro translation system to examine the inhibitory effect of the ns4a(21-34) peptide on both cap-and iresdependent translation. the cap-dependent translation of renilla luciferase and hcv ires-mediated translation of firefly luciferase were analyzed by dual-glo tm luciferase assay. the results clearly demonstrated a dose-dependent inhibitory effect of the ns4a(21-34) peptide on both cap-dependent and hcv ires-mediated translation (figure 6e ). upon virus infection, the translation machinery of host cells is known to be down-regulated. viral proteins derive ability to modify or regulate the expression and function of translation factors that results in an inhibition of host protein synthesis [21] . poliovirus-encoded proteases 2a and 3c cleave eif4g (eif4gi and eif4gii) and pabp, respectively, of host cells [22, 23] . the nsp3 of rotavirus substitutes the function of cellular pabp. it binds to the 3¢ end of the nonpolyadenylated viral mrna and competes with pabp in binding to eif4g [24] . in both cases, the viral proteins selectively inhibit translation of capped host mrna but not the viral rna. hcv ns4a was previously demonstrated to inhibit protein synthesis through its n-terminal 40 amino acid residues [15] . but the steady state levels of eif4g, eif4e, pabp, and 4e-bp1 were not affected [16] . the molecular basis involved in the inhibitory effect of ns4a was not clear. in this study, we found that hcv ns4a protein specifically interacted with the translation elongation factor, eef1a (figures 1 and 3) . the n-terminal and the central domains of the ns4a were involved independently in the interaction (figure 4 ), but the central domain encompassing amino acid residues 21-34 played a major role in the inhibition of the cap-dependent and hcv ires-mediated translation (figures 6b, c, and e) . the translation inhibitory effect caused by the ns4a(21-34) could be relieved by the addition of purified recombinant eef1a into the translation system (figure 6d) . nevertheless, the translation inhibition was not fully restored, suggesting that binding of ns4a to the eef1a may simultaneously deplete other translation factors that are associated with eef1a. alternatively, other mechanisms may also involve in the inhibitory effect of ns4a. in addition, although both hcv ns4a and ns4b proteins were shown to inhibit translation ( figure 2 , and [15, 16] ), no interaction was detected between eef1a and hcv ns4b protein ( figure 3) . these indicate that ns4b inhibits translation through a mechanism different from that of the ns4a protein. recently, the interaction between eef1a and hcv ns4a protein was also identified by yeasttwo hybrid screening [25] . viral proteins including the ns5a protein of bovine viral diarrhea virus (bvdv) [26] , the rnadependent rna polymerase of vesicular stomatitis virus (vsv) [27] , and the gag protein of human immunodeficiency virus (hiv) have been demonstrated to interact with eef1a [28] . functional roles of the interactions are not completely understood, but the interactions were proposed to be involved in the regulation of viral replication, translation, and assembly. eef1a is a g protein that recruits aminoacyl-trna to the elongating ribosomes. however, accumulated information leads us to believe that components of the translational apparatus also play important roles beyond protein synthesis. eef1a couples the pathways of protein synthesis and degradation. it interacts with ubiquitinated proteins and proteasome following atp depletion and is involved in the proteasome-mediated cotranslational protein degradation [29] . it was also demonstrated that eef1a interacts with actin and is essential for the regulation of actin cytoskeleton and cell morphology [30] [31] [32] [33] . in addition, overexpression of eef1a is associated with oncogenic transformation and metastasis [34] [35] [36] . in this study, we found that hcv ns4a protein interacted with eef1a and inhibited protein synthesis. the interaction may result in a reduction of viral translation and replication leading to the escape of immune responses and establishment of chronic infection. on the other hand, the interaction may also link the un-conventional roles of eef1a to the pathogenesis of hcv ns4a protein. hepatitis c virus infection is associated with the development of hepatocellular carcinoma classification and nomenclature of viruses: fifth report of the international committee on taxonomy of viruses genetic organization and diversity of the hepatitis c virus molecular cloning of the human hepatitis c virus genome from japanese patients with non-a, non-b hepatitis structure and organization of the hepatitis c virus genome isolated from human carriers expression and identification of hepatitis c virus polyprotein cleavage products gene mapping of the putative structural region of the hepatitis c virus genome by in vitro processing analysis two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis c virus a second hepatitis c virus-encoded proteinase nonstructural protein 3 of the hepatitis c virus encodes a serine-type proteinase required for cleavage at the ns3/4 and ns4/5 junctions the hepatitis c virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor characterization of the hepatitis c virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites both ns3 and ns4a are required for proteolytic processing of hepatitis c virus nonstructural proteins hepatitis c virus ns3 serine proteinase: trans-cleavage requirements and processing kinetics inhibition of protein synthesis by the nonstructural proteins ns4a and ns4b of hepatitis c virus hepatitis c virus ns4a and ns4b proteins suppress translation in vivo single-step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction assembly of severe acute respiratory syndrome coronavirus rna packaging signal into viruslike particles is nucleocapsid dependent functional motifs of delta antigen essential for rna binding and replication of hepatitis delta virus regulation of peptide-chain elongation in mammalian cells hijacking the translation apparatus by rna viruses inhibition of hela cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex cleavage of poly(a)-binding protein by poliovirus 3c protease inhibits host cell translation: a novel mechanism for host translation shutoff rotavirus rna-binding protein nsp3 interacts with eif4gi and evicts the poly(a) binding protein from eif4f screening and cloning of hepatitis c virus non-structural protein 4a interacting protein gene in hepatocytes the ns5a protein of bovine viral diarrhea virus interacts with the alpha subunit of translation elongation factor-1 rna polymerase of vesicular stomatitis virus specifically associates with translation elongation factor-1 abc for its activity translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 gag polyprotein proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1a translation elongation factor 1a is essential for regulation of the actin cytoskeleton and cell morphology ef-1a and the cytoskeleton bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends identification of an actin-binding protein from dictyostelium as elongation factor 1a candidate metastasis-associated genes of the rat 13762nf mammary adenocarcinoma rat elongation factor 1 alpha: sequence of cdna from a highly metastatic fos-transferred cell line elongation factor-1a gene determines susceptibility to transformation we thank ching-yi chang and hsin-yi chang for technical assistance. this work was supported in part by research grants nsc 93-2320-b-002 key: cord-018220-8m11ig06 authors: duncan, coley b.; falsey, ann r. title: viral infections date: 2009-02-02 journal: infectious disease in the aging doi: 10.1007/978-1-60327-534-7_23 sha: doc_id: 18220 cord_uid: 8m11ig06 although influenza remains indisputably the most significant viral pathogen in adults, other viruses such as respiratory syncytial virus, parainfluenza viruses, and human metapneumovirus are now recognized as significant pathogens in older populations. oseltamivir and zanamivir are antiviral agents that are effective for the treatment and prophylaxis of influenza a and b. for treatment and for optimal effect, therapy should be initiated within 48 h of symptom onset. infection with hepatitis viruses may be more severe in older adults with more fulminate disease as observed with acute hepatitis a and a more rapid progression to cirrhosis with hepatitis c. outbreaks of viral gastroenteritis are common in long-term care facilities, and infection may lead to death due to dehydration and oliguria. the incidence of herpes zoster increases with advancing age and carries with it a significant risk of post herpetic neuralgia. the use of antiviral medications and corticosteroids may reduce the incidence and severity of chronic pain. parainfluenza viruses (piv), human metapneumoviruses (hmpv), coronaviruses (co-v), and rhinoviruses may cause severe disease in adults and result in hospitalization (2) . together pneumonia and influenza comprise the fifth leading causes of death in persons aged 65 years and older. influenza viruses are enveloped ribonucleic acid (rna) viruses that are classified as a, b, or c, based on stable internal proteins (3) . the virus contains two major surface proteins: hemagglutinin (h) and neuraminidase (n), which can undergo minor antigenic changes leading to yearly epidemics or major changes resulting in influenza pandemics. currently, there are two circulating influenza a viruses, h1n1 and h3n2, in addition to influenza b, present in the united states. h1n1 viruses do not appear to cause serious problems in older persons, possibly due to previous immunity. in 1997, influenza a (h5n1), which was previously seen only in birds, crossed the species barrier and human infection occurred in southeast asia (4) . this highly pathogenic avian influenza has spread in bird populations throughout asia and into europe. to date, human infection has been rare and transmission has occurred primarily by direct contact with infected birds. unlike seasonal influenza, illness due to avian influenza is more severe in young children as compared to older adults. in a community, peak influenza activity typically lasts 6-8 weeks, with attack rates highest in preschool and school-aged children and lowest in older persons (3) . despite lower attack rates, mortality from influenza rises dramatically with age and the presence of underlying medical conditions. the presence of one high-risk medical condition (cardiovascular, pulmonary, renal, metabolic, neurologic, or malignant disease) increases the risk of death from influenza 39-fold. despite increasing vaccine coverage, current centers for disease control and prevention (cdc) data indicate increasing influenza-related morbidity and mortality over the past decade (5) . in the united states, approximately 226,000 hospitalizations and 34,000 deaths occur each year in persons age 65 years and older (6, 7) . the devastating impact of influenza is most dramatically seen in long-term care facilities (ltcfs) where explosive epidemics may occur. during outbreaks, rates of pneumonia and hospitalization are as high as 52% and 29%, respectively, with case fatality rates of 30%. the classic presentation of influenza is an abrupt onset of fever, chills, headache, and myalgias (see table 1 ). dry cough, sore throat, and ocular pain are also common (3) . fever remains a common finding in the elderly, although the height of the fever may be lower compared with young persons. although many elderly adults have classic symptoms, a substantial number may have more nonspecific presentations such as fever and confusion or worsening of chronic medical conditions. in contrast to young healthy persons, the triad of fever, cough, and acute onset of symptoms has a positive predictive value of only 30% in elderly adults. given the protean manifestations of influenza in older persons, it is always important to consider influenza in the differential when evaluating an acutely ill elderly adult during the winter. influenza lower respiratory tract involvement increases steadily with advancing age with the rates of pneumonia 4-8% in persons aged 5-50 years and rising to 73% in persons over age 70 (8) . secondary bacterial pneumonia following acute influenza also occurs more frequently in older persons. although the rates of pneumonia rise with age, hospitalization most frequently results from exacerbation of chronic medical conditions. in addition to the immediate complications of influenza, residents of nursing homes who survive influenza experience a significant functional decline in activities of daily living. although many physicians use clinical features to make a diagnosis of "the flu," laboratory confirmation is best, especially if therapeutic decisions are needed because influenza may be difficult to distinguish from other respiratory viruses. winter-spring 1-2 non-specific none rsv, respiratory syncytial virus. *100 mg daily for severe hepatic dysfunction, renal failure (crcl 10 ml/min) and for elderly nursing home patients. **dosage should be reduced in persons with creatinine clearance of < 30 ml/min ***ribavirin is not fda approved for use in adults. bid twice a day; po orally older persons typically shed the virus for 3-5 days, although shedding up to 14 days has been documented in hospitalized patients (9) . rapid antigen testing may be done directly on nasopharyngeal specimens using an enzyme immunoassay (eia) (10) . although not as sensitive as viral culture, rapid tests offers quick turnaround times and may be useful for infection control and treatment decisions. sensitivity of rapid testing in adults is estimated to be 50-60% for influenza a strains and 10-20% for influenza b (11) . when available, rt-pcr offers rapidity while retaining excellent sensitivity. as of this writing, zanamivir or rimantadine can be used for the treatment or prophylaxis of influenza a h1n1 strains (*). for influenza a h3n2 or influenza b, oseltamivir or zanamivir can be used. if the strain of influenza is unknown, currently, zanamivir by itself or a combination of oseltamivir and rimantadine is appropriate coverage (3). resistance to zanamivir remains rare. zanamivir is not recommended for patients with reactive airway disease, because it may exacerbate bronchospasm. future influenza treatment and prophylaxis recommendations for influenza will need to be guided by close monitoring of cdc reports on influenza resistance patterns. treatment should begin < 48 h after onset of symptoms (table 1 ). in practice, physicians are often faced with the question of whether to treat patients who present outside the 48-h period. at present, only one observational study addresses this question in hospitalized adults (12) . in 327 adults hospitalized with influenza, mortality was significantly lower in those who received antiviral treatment as compared to those who did not receive antiviral treatment. of the 100 patients who were treated, only 29% had symptoms < 48 h. the cornerstone of reducing the morbidity and mortality of influenza in the elderly is vaccination (see also chapter "vaccinations"). although the degree of protective efficacy for current inactivated vaccines in the elderly has recently become a subject of controversy, older adults clearly benefit from vaccination (13) . a multi-layered approach for protecting the elderly from influenza is needed and includes vaccinating elderly persons, their close contacts and care givers, and providing oseltamivir if exposure to influenza has been documented (3, 14) . when staff was highly vaccinated, several studies have demonstrated a benefit to elderly residents of ltcfs (15) . the recommendations of the advisory committee on immunization practices (acip) 2007 relating to the elderly, include vaccination of all persons ³ 50 years, vaccination of residents of nursing homes and chronic-care facilities, vaccination of healthcare personnel, and vaccination of healthy household contacts (including children) and caregivers of adults ³ 50 years (3) . * http://emergency.cdc.gov/coca/ppt/antivirals_update_010809_fiore.pps at the present time, only trivalent inactivated virus (tiv) vaccine, which contains killed h1n1, h3n2 and b strain influenza, is recommended for use in persons 50 years and older (3) . mild acute local reactions occur in approximately one-third of vaccines and systemic reactions such as fever, and myalgias are uncommon in older persons. influenza vaccine may be safely given simultaneously with pneumococcal vaccine, and the only contraindications to vaccination are anaphylactic hypersensitivity to eggs or other components of the vaccine and a history of guillian-barré syndrome. by using adjuvants and higher doses of antigens, active research continues to improve the immunogenicity and efficacy of inactivate influenza vaccine. live attenuated influenza virus vaccine is not approved for persons over age 49; however, it may be given to healthcare workers and close household contacts of older adults. antiviral prophylaxis is recommended for all residents of nursing homes and chronic care facilities and to unvaccinated healthcare providers once influenza a has been documented in the institution (13, 14) . chemoprophylaxis is given regardless of vaccination status and is continued until 1 week after the onset of the last influenza case. rsv has long been recognized as the leading cause of lower respiratory tract disease in children; however, recently, it has been recognized as a serious adult pathogen (16) . it is estimated that rsv results in approximately 178,000 hospitalizations and 14,000 deaths annually in the united states yielding healthcare costs in excess of $1 billion. a number of epidemiologic studies and mathematical models indicate that rsv is second to influenza as a cause of serious viral respiratory disease in adults (7) . rsv was initially recognized as a pathogen in older persons when several outbreaks were described in long-term care facilities (17) . attack rates are variable and may be as high as 90% during outbreaks, but more commonly they range from 1 to 7% when residents are followed prospectively. in published reports, rates of pneumonia range from 0 to 53% and death from 0 to 55%. rsv appears to cause serious disease in community-dwelling older persons as well. in a 2-year prospective study of elderly persons in the united kingdom (uk) nicholson et al., identified rsv in 3% of illnesses using serology for diagnosis (18) . with the advent of sensitive molecular testing, a more accurate assessment of the true incidence of rsv has emerged. in a 3-year study from the united kingdom by zambon et al., rsv was identified by rt-pcr in 10-18% of adults age 65 years and older who were visiting a general practitioner during the winter for a respiratory illness (19) . in comparison, during the same period, influenza a was identified in 13-42% of subjects. in a prospective study from rochester, ny, using a combination of viral culture, rt-pcr and serology for diagnosis, rsv infection was documented in 3-7% of 608 healthy elderly and 4-10% of adults with chronic cardiopulmonary conditions over four winter seasons (16) . serious disease was more common in high-risk patients: 9% visited the emergency room, 16% required hospitalization, and 4% died. finally, a large study of community-acquired pneumonia in adults found rsv to be the third most commonly identified pathogen at 4.4% compared with 6.2% due to streptococcus pneumoniae and 5.4% due to influenza (20) . manifestations of rsv infection can be difficult to distinguish from other viral respiratory infections, particularly influenza. most individuals with rsv have nasal discharge, cough, sputum production, and constitutional symptoms. although overlap exists, there are some helpful clues to differentiate rsv from influenza. high fever, sore throat, myalgias, and gastrointestinal complaints are more characteristic of influenza, whereas rhinorrhea, dyspnea, sputum production, and wheezing are more frequently associated with rsv infection . unfortunately, because of the labile nature of the virus and low titers of virus in nasal secretions in adults, diagnosis of acute rsv by standard testing is difficult. under ideal circumstances, viral culture is only 50% sensitive when compared with serology using eia. commercial rapid antigen tests also have poor sensitivity in adults (21) . rt-pcr offers the best combination of sensitivity and specificity for the diagnosis of acute rsv in adults but is not widely available to most clinicians. the treatment of rsv infection in adults is largely supportive. supplemental oxygen and bronchodilators may be useful, and antibiotics should be considered if bacterial super-infection is suspected. ribavirin is a nucleoside analogue, which has broad antiviral activity, including rsv. anecdotal experience suggests it may be beneficial in selected cases, particularly in persons with immunosupression. however, due to lack of data in the elderly, general recommendations on its use cannot be made (22) . the major problems with ribavirin are its high cost and difficulty with administration. the recommended 12-18 h/day of aerosol at 20 mg/ml concentrations may be quite difficult for the elderly adult to tolerate. higher concentrations (60 mg/ml) given three times a day may also be effective and more tolerable (23) . in healthy elderly patients and in adults with chronic pulmonary disease, low serum neutralizing antibody titers are associated with increased risk of hospitalization with rsv infection suggesting a vaccine may be beneficial. although research is ongoing, an effective rsv vaccine has yet to be developed. rsv is spread primarily by large droplet inoculation and fomites, and handwashing is the single most important measure in the control of rsv. the parainfluenza viruses (piv) are most commonly thought of as the etiologic agents of croup, bronchiolitis, and pneumonia in young children (24) . four serotypes and two subgroups of piv are recognized (1, 2, 3, 4a, and 4b); piv-3 is endemic throughout the year, whereas piv-1 and piv-2 tend to occur during the fall. although piv infections are not commonly documented in older adults, several studies of community-acquired pneumonia and chronic obstructive pulmonary disease (copd) exacerbations implicate piv as a cause in 2-17% of cases (25, 26) . the piv-1 and 3 serotypes account for the majority of isolates in older persons, with piv-2 being relatively uncommon (27) . similar to rsv, outbreaks of piv infections in nursing homes have been described (27, 28) . variable morbidity and mortality has been reported. clinical characteristics of piv infection are not distinctive and include rhinorrhea, sore throat, hoarseness, and cough with high rates of pneumonia ranging from 20 to 30%. in an institutional outbreak of piv-3, the attack rates among residents and nursing staff were 31% and 11%, respectively. antecedent parainfluenza infection in long-term care residents has been linked to outbreaks of pneumococcal pneumonia. in clinical practice, a diagnosis of piv infection is usually made by viral culture, although rt-pcr is available in some settings (24) . diagnosis can also be made serologically; however, piv-1 and 3 infections result in cross-reactive antibody responses and cannot be distinguished. no antiviral agents have been approved for the treatment of piv infection. in 2001, human metapneumovirus (hmpv) was first identified in the netherlands from archived respiratory cultures collected from infants and young children in whom other pathogens could not be isolated (29) . it is an enveloped rna virus closely related to rsv and piv. since its discovery, infection has been widely reported each winter in young infants with an illness similar to rsv and characterized by wheezing and bronchiolitis (30) . however, as with many pediatric respiratory viral pathogens, hmpv infection induces incomplete immunity and reinfections occur later in life at all ages (31, 32) . in a 2-year study, hmpv infection was identified in 4.1% of elderly and high-risk adults, using rt-pcr and serology for diagnosis (32) . impact was greatest in subjects with cardiopulmonary diseases, who were ill twice as long as healthy elderly. in a study of adult pneumonia, 4% of subjects were diagnosed by rt-pcr with hmpv (33) . seventy-five percent of those infected were 65 years of age and older. such hmpv outbreaks can also occur in ltcfs. in a recent outbreak of severe hmpv illness in an ltcf in quebec, canada, 17% of those infected had pneumonia and 50% died (31) . autopsy material from an elderly woman with an extensive right middle and lower lobe pneumonia confirmed the presence of virus in the lower airways by immunohistochemical staining. the clinical characteristics of hmpv pneumonia in older adults do not appear to be distinctive from the other wintertime respiratory viruses. cough is universal and wheezing, dyspnea, and sputum production are common symptoms (32) . in part due to its fastidious growth characteristics, hmpv remained undetected for many years. although isolation by viral culture is possible, this method of diagnosis is not practical, and rt-pcr is the diagnostic method of choice (30) . rapid tests have been developed for direct antigen detection in respiratory secretions; however, there are little data regarding sensitivity in the elderly. currently, there is no known effective antimicrobial therapy against this virus. therapy is primarily symptomatic, supportive, and managing any complications. rhinoviruses are the most commonly identified cause of the "common cold," accounting for 25-50% of upper respiratory infections (34) . rhinoviruses circulate at all times of the year, but peak activity tends to be during the spring and fall. because the virus does not replicate well at 37°c, infection of the lower airways was previously considered rare. however, recent studies utilizing experimental challenge and specimens obtained at bronchoscopy clearly demonstrate rhinovirus infection of the lower respiratory epithelium (35) . a prospective study from the united kingdom indicates that rhinoviruses are an important cause of debility and lower respiratory disease in elderly people in the community (36) . rhinoviruses accounted for 24% of respiratory illnesses in a cohort of 533 persons over a 2-year period. although death and hospitalization rates were low, the mean length of illness was 16 days, and 26% of people were unable to perform their normal household activities. the presence of chronic medical conditions and smoking increased the likelihood of lower respiratory tract complications. because of the frequency of rhinovirus infection, the overall burden of disease in the elderly may approach influenza. rhinovirus has also been identified as the cause of 3.1%-19.6% of copd exacerbations (37) . lastly, outbreaks of severe respiratory illness due to rhinovirus have been described in nursing homes with attack rates as high as 56% and a mortality as high as 21% of those affected (38, 39) . in elderly persons, nasal congestion (79-89%), cough (71-94%), constitutional symptoms (43-91%), and sore throat (21-51%) characterize illnesses. rhinoviruses are now appreciated as a common trigger for asthma exacerbations and, therefore, in persons with preexisting lung disease, the dominant symptom may be wheezing (34) . the diagnosis of rhinovirus is usually made by a viral culture of nasopharyngeal secretions. if available, the use of rt-pcr greatly increases detection rates (34) . treatment is supportive and care should be exercised when prescribing "cold" medications to elderly persons because many of these "cold" medications contain combinations of sympathomimetics and antihistamines. coronaviruses are rna viruses of which two are major serotypes: human coronavirus 229e (hco-229e) and hco-oc43, which, for decades, have been known to cause respiratory disease in humans (34) . two recently discovered coronaviruses (hco-nl63 and hco-hku1) cause lower and upper respiratory tract infections with similar frequency (40) (41) (42) . in temperate climates, peak viral activity occurs in the winter. reinfections with coronaviruses are common throughout life, and, similar to rhinoviruses, illnesses are generally mild upper respiratory infections in healthy adults; symptoms include malaise, headache, sore throat, and nasal congestion. exacerbations of chronic obstructive pulmonary disease have been linked to coronavirus infections in several studies. coronavirus infections have been evaluated in the community-dwelling elderly and have, in one prospective study from the united kingdom, accounted for 9.5% of the respiratory illnesses (18) . coronavirus's were associated with lower respiratory tract symptoms in more than 40% of cases; infections have also been documented in ltcfs and in frail elderly people attending daycare centers (43) . coronaviruses have also been implicated in severe respiratory infections requiring hospitalization in older adults (44) . rt-pcr is now available for diagnosis of coronavirus infection but frequently a specific viral diagnosis is not made (34) . no antiviral agents are available, and treatment is supportive. hepatitis a virus (hav) is an rna virus in the picornavirus family (see table 2 ). the virus is easily transmitted by the fecal-oral route. in countries where the virus is endemic and sanitation is poor, most people become infected in early childhood when the disease is mild and life-long immunity results (45) . recently, a shift in the prevalence of cases from childhood to adulthood has occurred, presumably due to improved living conditions. the incidence of hepatitis a in the united states has fallen 88% from a peak in 1996 to an all-time low of presently 1.2 cases per 100,000 persons (46) . the prevalence of anti-hav antibodies increases with advancing age (47) . in a 1994 study from colorado, the prevalence of anti-hav antibodies at ages 60, 70, and 80 was 40%, 60%, and 80%, respectively (45) . an acute hepatitis a, advancing age correlates with more severe clinical manifestations, higher bilirubin levels and increased hospitalization rates (48) . liver failure and death are also more common with increased age (49) . in the united states, the overall case fatality rate for hav infection is 0.3%; however, it rises to 1.5% in persons 60 years or older (46) . an inactivated hepatitis a vaccine has been available since 1993, and clinical studies have shown the vaccine to be safe, very well tolerated, and highly immunogenic in all age groups. immunization of toddlers in israel resulted in a > 95% reduction of hepatitis a in the general population (50) . although the benefit was least for ages ³ 65 years, a 77.3% reduction in cases was observed in this age group. immunogenicity in frail elderly persons such as residents of long-term care has not been reported. although disease may be more severe in older adults, current vaccination policies do not specifically target the elderly. however, vaccination is recommended for older travelers who plan to visit countries endemic for hav. hepatitis b virus (hbv) is a complex deoxyribonucleic acid (dna) virus transmitted by percutaneous and mucous membrane exposure to infectious body fluids. serum, saliva, and semen have been shown to contain hepatitis b surface antigen (hbsag). since 1990, the incidence of acute hepatitis b has declined in all age groups with the largest decline (98%) being in children <15 years (46) . because the primary risk group in the united states and europe is intravenous drug abusers, acute infection is not common in the elderly. transfusion-related hbv infection is now an uncommon event with risk estimated to be 1 in 205,000 transfusions (51) . when several outbreaks occurred during the 1970s-1980s, ltcf's were thought to be a risk area for hbv (47) . however, recent surveys of geriatric hospitals indicate the prevalence of hbsag is similar to the general geriatric population (<1%) (47) . acute hbv in older adults is usually mild, and many cases are subclinical or presents with manifestations of cholestasis. in addition to the typical symptoms of jaundice, anorexia, and fatigue, diarrhea is a common complaint in elderly persons. complaints reflecting immune complex disease such as myalgias and arthalgias are rare in older adults. although acute hbv is generally not a severe disease in older adults, the mortality from fulminant hbv increases with age (47) . in a multivariate analysis of prognostic factors in 115 patients with hbv, age was an independent predictor of survival (46) . mortality for persons over age 60 years was 3.1% compared with 1.2% for persons ages 40-59. when individuals are infected at older ages, chronic carriage rates also increase. compared with a 10% carriage rate in young adults, approximately 60% of older persons become chronic carriers (47) . in addition to cirrhosis from chronic active hepatitis, one of the major complications of hbv infection is hepatocellular carcinoma (hcc). the length of time infected is an important factor in the development of cancer, and, thus, elderly persons who have been infected for many years are at the greatest risk. the rate of hcc rises from 197 per 100,000 in 30-to 39-year olds to 927 per 100,000 in 60-to 69-year-old chronic hbv carriers (52) . most cases of acute hepatitis b clear spontaneously and do not require treatment. in young patients with compensated disease, alpha-interferon is primarily used in the treatment of chronic hepatitis b. those who respond favorably may see suppression of viral replication and a decrease in the risk of progression to cirrhosis or to cancer. side effects of therapy are frequent and increase with advancing age (47) . therapy should be reserved for patients in overall good health, except for their liver disease, and who have evidence of active viral replication. five nucleotide/nucleoside analogs (lamivudine, adefovir, entecavir, tenofovir, and telbuvidine) are currently approved by the food and drug administration to treat chronic hepatitis b (53) . these drugs are usually better tolerated than interferon, although they have not been studied specifically in the elderly. patients with chronic hepatitis b should be tested for immunity to hepatitis a and, if seronegative, should be vaccinated against hepatitis a to prevent acute decompensation that could occur with hepatitis a superinfection. in addition, patients should be counseled to abstain from alcohol, to maintain a healthy body weight, and to use condoms to protect their sexual partners from infection. the currently licensed hepatitis b vaccine is a genetic recombinant vaccine; it is very well tolerated and highly immunogenic with excellent protective efficacy in children and young adults. however, response rates to hbv vaccine diminish significantly with increased age. ninety percent of persons under age 40 achieve an adequate seroresponse compared with only 50% in persons over age 60. hepatitis c virus (hcv) is an rna virus in the flavivirus family. exposures to contaminated blood, either through occupation or through intravenous drug abuse, may transmit hcv. although 40-50% of community-acquired hcv cases do not report a parenteral exposure, nonparenteral transmission of hcv is not well understood. sexual transmission, if it occurs, is not efficient. the major risk factor for hcv in older persons is transfusion, and most became infected prior to routine screening of the blood supply in 1990 (47) . for southern europe and japan, the peak era of transfusion-associated hcv was between 1940 and 1960 whereas in the united states and northern europe, the peak transmission was between 1960 and 1980. the current risk of acquiring hcv from transfusion in the united states is approximately 1 in 1,935,000 (51) . in southern europe, numerous population-based studies have shown that the prevalence of hcv is 16-42% in persons over 60 years and 33-50% in those over 80 years; infection is extremely rare in subjects <40 years (54) . the seroprevalence of 1.4-2.2% in ltcf residents is approximately that of the general elderly population (55) . the clinical manifestations of acute hcv are generally mild and nonspecific. in a series of 20 older people with acute non-a non-b hepatitis, approximately 30-40% had fever, abdominal pain, and jaundice (47) . fulminant hepatitis is rare with hcv, but development of chronic liver disease is very common (56) . virtually all persons become chronically infected, and a significant number develop chronic liver disease. on average, 20 years after initial infection, chronic active hepatitis or cirrhosis develops in 29-76% of persons. age affects the rate of progression to cirrhosis in two important ways: the younger a patient is when hcv is acquired, the slower the rate of progression to cirrhosis; however, the longer one is infected, the greater the risk of development of cirrhosis and hcc (54) . hepatocellular carcinoma is clearly associated with chronic hcv infection, and the relative risk of cancer from hcv may be even greater than that from hbv. in a study of 25 older persons with hcv in the united kingdom, 36% developed hcc (56) . chronic hcv infection is currently treated with pegylated interferon alpha and oral ribavirin. goals of therapy include clearance of viremia and prevention of decompensated cirrhosis and hcc. persons with high viral load and viral genotype 1 have a low response rate to a -interferon, and, many patients who initially respond, relapse when therapy is discontinued (47) . most studies of interferon treatment of hcv have not included older participants. in one study from japan, interferon was administered to 19 patients aged 65 and older with hcv, and the response rate was 26% compared with 33% in younger persons (57) . of note, the older subjects had higher hcv-rna titers and more severe fibrosis on their liver biopsy as compared to their younger subjects. response rates in elderly persons correlated with lower hcv-rna titers. because older persons have more side effects and a lower response rate to interferon, treatment should be carefully considered on an individual basis; it should be proposed only in patients up to 75 years who have a significant risk of progression of liver disease, no serious co-morbidities, and an otherwise good life expectancy (54) . patients with chronic hepatitis c should be tested for immunity to hepatitis a and b and, if seronegative to either, they should be vaccinated to prevent acute decompensations that could occur with hepatitis a or b superinfection. they should be counseled to abstain from alcohol, to maintain a healthy body weight, and to use condoms to protect their sexual partners from infection. although deaths related to diarrhea have traditionally been thought to be a problem of young children in developing countries, in the united states from 1979 to 1987, 51% of the 28,538 diarrhea-related deaths occurred in adults over the age of 74 as compared to 11% in children < 5 year old (58) . the odds ratio of dying during a hospitalization involving gastroenteritis was 52.6 for adults over the age of 70 as compared with children less than 5 years of age. residents of nursing facilities are at particular risk for infectious diarrhea illness because of the outbreaks that can occur in closed populations. the majority of nursing facility outbreaks of gastroenteritis are probably viral and include rotavirus, enteric adenoviruses, norovirus, snow mountain agent, and small round structured viruses (srsv) (58, 59) . rotaviruses are small rna viruses in the retrovirus family and are the most important cause of gastroenteritis in infants and young children worldwide. the mode of transmission is assumed to be fecal-oral, and the virus is relatively resistant to common disinfectants and facilitating nosocomial dissemination. several outbreaks of rotavirus infection in elderly residents of ltcfs have been reported with attack rates ranging from 36 to 66% and mortality rates of 1-10% (60, 61) . a typical illness lasts 2-3 days and includes voluminous vomiting and watery diarrhea with low-grade fever. death may result from dehydration progressing to oliguria and acidosis (60, 61) . in an analysis of specimens from gastroenteritis outbreaks in aged care facilities in australia, rotavirus was detected by electron microscopy in 18 of 29 individuals in 7 out of 53 outbreaks (62) . although rotavirus infections are most common in winter, outbreaks can occur at other times of the year (63) . rotavirus serum antibody titers offer some protection against severe disease and tend to diminish with increasing age (61) . two rotavirus vaccines were released in 2006 for use in children, a pentavalent human-bovine reassortant rotavirus vaccine called rotateq ® and a live-attenuated human rotavirus vaccine called rotarix ® . no data on safety or immunogenicity in the elderly exists; however, given the mortality rates in this age group, further study would be reasonable. norovirus (formerly called norwalk-like virus) is also a frequent cause of diarrhea in adults including the elderly (see also chapter "infectious diarrhea"). in a study of 20 gastroenteritis outbreaks in maryland, 52% of stool samples were positive for norwalk-like virus (nlv) (64) . the median duration of symptoms with nlv gastroenteritis is 2-3 days (65) . varicella-zoster virus (vzv) is a dna virus and a member of the herpes virus family. it causes two distinct clinical syndromes: primary disseminated infection (chickenpox) and reactivation of latent virus in the dorsal root ganglia, leading to herpes zoster or "shingles" (66) . herpes zoster is a painful, vesicular exanthem that erupts in one to two dermatomes after a prodrome of days to weeks and may take up to a month to heal. most patients report a deep aching or burning sensation, altered sensation to touch with paresthesias, dysesthesia, or hyperesthesia. herpes zoster is a common condition with a cumulative lifetime incidence of 20-30% with most of the risk concentrated in older age. the overall incidence is 1.2-3.4 per 1,000 personyears, but rates rise sharply with increasing age to 11.8 per 1,00,0 for persons age 65 years and older (67) . chapter "herpes zoster" is devoted to the topic of herpes zoster, and the reader is referred to this section for further details. the epstein-barr virus (ebv) is a herpes virus that establishes lifelong infection. primary infection may occur in childhood when infection is asymptomatic or during adolescence when the symptoms of classic mononucleosis are most often observed (66) . although primary infection is uncommon in old age, the manifestations may be different than those in youth, which makes diagnosis challenging. seroepidemiologic studies indicate that 3-10% of older adults are at risk for primary infection (66) . diagnosis is also often delayed because symptoms may be misleading. lymphadenopathy, pharyngitis, and splenomegaly are significantly less common and jaundice is more common in older persons as compared with the young persons (67) . fever is more common and often lasts more than 2 weeks (68, 69) . the neurologic manifestations of ebv infection are protean, and acute ebv encephalitis has been described (70) . adding to the difficulty of making a correct diagnosis, development of atypical lymphocytosis may be absent or delayed in the elderly. diagnosis of primary ebv is made by the presence of heterophile antibodies or ebv-specific igm. although acyclovir has in vitro activity against ebv, no benefit has been demonstrated in the treatment of acute ebv infection. trends in hospitalizations for pneumonia among persons aged 65 years or older in the united states improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction prevention and control of influenza. recommendations of the advisory committee on immunization practices (acip) current concepts: avian influenza a (h5n1) infection in humans impact of influenza vaccination on seasonal mortality in the us elderly population influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states lung involvement in influenza duration of influenza a virus shedding in hospitalized patients and implications for infection control rapid tests for influenza . current opinions in pediatrics performance of six influenza rapid tests in detecting human influenza in clinical specimens antiviral therapy and outcomes of influenza requiring hospitalization in ontario, canada effectiveness of influenza vaccine in the community-dwelling elderly antivirals and the control of influenza outbreaks effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus infection in adults acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults ribavirin therapy of adult respiratory syncytial virus pneumonitis high-dose, shortduration ribavirin aerosol therapy compared with standard ribavirin therapy in children with suspected respiratory syncytial virus infection parainfluenza viruses impact of respiratory virus infections on persons with chronic underlying conditions parainfluenza virus infection among adults hospitalized for lower respiratory tract infection parainfluenza infections in the elderly 1976-82 influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history, and health resource utilization a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus . seminar respiratory critical care medicine an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility human metapneumovirus infections in young and elderly adults human metapneumovirus pneumonia in adults: results of a prospective study rhinovirus and coronavirus infections quantitative and qualitative analysis of rhinovirus infection in bronchial tissues risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of copd rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus human coronaviruses: what do they cause? a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection the novel human coronaviruses nl63 and hku1 human coronavirus oc43 causes influenza-like illness in residents and staff of aged-care facilities in melbourne spectrum of clinical illness in hospitalized patients with "common cold" virus infections history and epidemology of hepatitis a virus surveillance for acute viral hepatitis -united states viral hepatitis in older adults hepatitis a epidemic in the elderly acute hepatitis a virus infection: a review of prognostic factors from 25 years experience in a tertiary referral center incidence of hepatitis a in israel following universal immunization of toddlers current prevalence and incidence of infectious disease markers and estimated window-period risk in the american red cross blood donor population liver disease in the elderly new drugs for chronic hepatitis b: a review hepatitis c virus infection in the elderly: epidemiology, natural history and management prevalence of hepatitis b surface antigen, hepatitis c antibody, and hiv-1 antibody among residents of a long-term care facility hepatitis c virus infection in the elderly interferon therapy for patients more than 60 years of age with chronic hepatitis c approach to acute diarrhea in the elderly outbreaks of gastroenteritis in elderly nursing homes and retirement facilities associated with human calciviruses rotavirus infection in a geriatric population an epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly rotavirus detection and characterisation in outbreaks of gastroenteritis in aged-care facilities report of a rotavirus outbreak in an adult nursing home population a predominant role for norwalk-like viruses as agents of epidemic gastroenteritis in maryland nursing homes for the elderly clinical manifestation of norovirus gastroenteritis in health care settings epstein-barr virus and the elderly host clinical and laboratory evaluation of elderly patients with heterophil-antibody positive infectious monoculeosis infectious mononucleosis in middle age infectious mononucleosis in patients aged 40 to 72 years: report of 27 cases, including 3 without heterophil-antibody responses epstein-barr virus causing encephalitis in an elderly woman hepatitis c virus infection in the elderly: epidemiology, natural history and management recommendations for the management of herpes zoster antiviral therapy and outcomes of influenza requiring hospitalization in ontario, canada key: cord-265592-r8nlef0h authors: teuber, g.; berg, t.; naumann, u.; raedle, j.; brinkmann, s.; hopf, u.; zeuzem, s. title: randomized, placebo‐controlled, double‐blind trial with interferon‐α with and without amantadine sulphate in primary interferon‐α nonresponders with chronic hepatitis c date: 2001-12-21 journal: j viral hepat doi: 10.1046/j.1365-2893.2001.00297.x sha: doc_id: 265592 cord_uid: r8nlef0h in primary interferon‐α (ifn‐α) nonresponders with chronic hepatitis c, retreatment with ifn‐α has only limited efficacy with sustained response rates below 10%. therefore, the aims of the present study were to compare the efficacy and safety of ifn‐α alone or in combination with amantadine sulphate in nonresponders to previous ifn‐α monotherapy. fifty‐five ifn‐α nonresponders with chronic hepatitis c (mean age: 46.6 years) received ifn‐α 6 miu thrice weekly for 24 weeks followed by 3 miu thrice weekly for additional 24 weeks. amantadine sulphate (n=26) or a matched placebo (n=29) was given orally twice daily for 48 weeks. because of a low initial response rate at week 12 (13/55 patients) and a high breakthrough rate (8/13 patients) after ifn‐α dose reduction in week 24, a virological end‐of‐treatment response with undetectable serum hcv‐rna (< 1000 copies/ml) was achieved in only five patients (ifn‐α/amantadine sulphate, one patient; ifn‐α/placebo, four patients). after 24 weeks follow‐up a sustained virological response was observed in only two patients receiving ifn‐α and placebo. health‐related quality‐of‐life analysis showed a substantial improvement of the profile of mood states (poms) scale concerning the subscales fatigue (p < 0.05) and vigor (p < 0.05) in patients receiving combined ifn‐α/amantadine sulphate treatment compared with those treated with ifn‐α alone. ifn‐α/amantadine sulphate combination therapy was well tolerated without any serious adverse events. in conclusion, retreatment with ifn‐α and amantadine sulphate does not increase the low sustained virological response rates of ifn‐α therapy in primary ifn‐α nonresponders with chronic hepatitis c, but may lead to a sustained improvement of health‐related quality‐of‐life. hepatitis c virus (hcv) infection frequently leads to chronic hepatitis and may progress to liver cirrhosis and possibly hepatocellular carcinoma [1±4] . hcv-related end-stage liver disease is currently one of the leading indications for liver transplantation. treatment with interferon-a (ifn-a) alone leads only to a sustained viral clearance in approximately 15±20% of patients [5, 6] . the addition of ribavirin to standard interferon-a treatment improves the sustained virological response rate to approximately 40% in previously untreated patients [7, 8] . several treatment strategies have been investigated in patients with chronic hepatitis c not responding to previous interferon-a monotherapy. however, retreatment of previous interferon-a nonresponders with standard regimens of interferon-a alone (3´3±6 miu/week s.c.) or in combination with ribavirin (1000±1200 mg/day orally) showed only limited therapeutic ef®cacy with sustained viral response rates below 10% [9, 10] . amantadine (1-aminoadamantanamine sulphate) is a tricyclic, symmetric amine with an antiviral activity against toga-, myxo-, arena-,¯avi-and coronaviruses [11±15] . the drug has been studied in detail in the in¯uenza a virus infection and the antiviral activity was found to be related to inhibition of viral uncoating and viral budding by interaction with the viral m2 protein [16±18] . the antiviral properties of amantadine have led to clinical trials evaluating the potential therapeutic role of amantadine alone or in combination with interferon-a in patients with chronic hepatitis c [19±23] . for retreatment of previous interferon-a summary. in primary interferon-a (ifn-a) nonresponders with chronic hepatitis c, retreatment with ifn-a has only limited ef®cacy with sustained response rates below 10%. therefore, the aims of the present study were to compare the ef®cacy and safety of ifn-a alone or in combination with amantadine sulphate in nonresponders to previous ifn-a monotherapy. fifty-®ve ifn-a nonresponders with chronic hepatitis c (mean age: 46.6 years) received ifn-a 6 miu thrice weekly for 24 weeks followed by 3 miu thrice weekly for additional 24 weeks. amantadine sulphate (n 26) or a matched placebo (n 29) was given orally twice daily for 48 weeks. because of a low initial response rate at week 12 (13/55 patients) and a high breakthrough rate (8/13 patients) after ifn-a dose reduction in week 24, a virological end-of-treatment response with undetectable serum hcv-rna (< 1000 copies/ml) was achieved in only ®ve patients (ifn-a/amantadine sulphate, one patient; ifn-a/placebo, four patients). after 24 weeks follow-up a sustained virological response was observed in only two patients receiving ifn-a and placebo. health-related quality-of-life analysis showed a substantial improvement of the pro®le of mood states (poms) scale concerning the subscales fatigue (p < 0.05) and vigor (p < 0.05) in patients receiving combined ifn-a/amantadine sulphate treatment compared with those treated with ifn-a alone. ifn-a/amantadine sulphate combination therapy was well tolerated without any serious adverse events. in conclusion, retreatment with ifn-a and amantadine sulphate does not increase the low sustained virological response rates of ifn-a therapy in primary ifn-a nonresponders with chronic hepatitis c, but may lead to a sustained improvement of health-related quality-of-life. nonresponders with chronic hepatitis c with the combination of interferon-a and amantadine, only data from a few uncontrolled studies with inconsistent response rates are available [24, 25] . therefore, the aims of the present study were to evaluate the therapeutic ef®cacy, tolerability and health-related quality of life of interferon-a2a (ifn-a2a) plus amantadine sulphate in comparison with ifn-a2a plus placebo in patients with chronic hepatitis c not responding to previous ifn-a treatment. patients with chronic hepatitis c not responding to previous ifn-a monotherapy with a minimal total ifn-a dose of 108 miu and a treatment duration of at least 12 weeks were eligible for enrolment when they met all of the following inclusion criteria: (1) nonresponse to previous ifn-a monotherapy with persistence of serum hcv-rna and a treatment-free interval of at least 24 weeks; (2) elevated alanine aminotransferase (alt) levels; (3) a positive anti-hcv test; (4) detectable serum hcv-rna; (5) compensated liver disease; (6) leukocyte count p 2500/ll, platelets count p 70000/ll; and (7) aged between 18 and 70 years. demographic, biochemical, serological and virological pretreatment characteristics of the 55 enrolled patients are summarized in table 1 . exclusion criteria were coinfection with hepatitis b virus or human immunode®ciency virus types 1 and 2, concomitant autoimmune disease, clinically signi®cant cardiovascular, metabolic, renal, haematological, rheumatological, neurological or psychiatric disease, organ grafts, systemic infections, bleeding disorders, anaphylactic reactions, a history of neoplastic disease within the last 5 years, systemic immunosuppressive treatment, average daily intake of alcohol > 50 g, drug abuse within the previous year, pregnancy and lactation period. liver biopsy was performed in all patients 1±6 months before the initiation of the primary ifn-a therapy. according to the study protocol, additional liver biopsies before retreatment were not required for enrolment in the present study. in the present prospective, randomized, double-blind, placebo-controlled trial, which was conducted at the university hospitals of berlin and frankfurt, germany, 55 eligible patients were randomly assigned to treatment with either the combination of ifn-a2a plus amantadine sulphate (n 26) or ifn-a2a plus placebo (n 29). randomization was performed with a random number generator in ®xed blocks of four with a ratio of 1 : 1. all patients were treated with 6 miu ifn-a2a (roferon a ò , hoffmann la-roche ag, grenzach-wyhlen, germany) thrice weekly subcutaneously for 24 weeks followed by 3 miu ifn-a2a thrice weekly subcutaneously for additional 24 weeks (fig. 1) . a daily dose of 200 mg amantadine sulphate (infex ò , merz + co. gmbh & co, frankfurt/m., germany) or matched placebo were given orally twice daily for 48 weeks. treatment was continued only in patients with undetectable serum hcv-rna (amplicor hcvä, roche diagnostic systems, branchburg, nj; lower detection limit: 1000 copies/ml) at treatment week 12 [27] . the follow-up period was 24 weeks in all patients. clinical examination, haematological and biochemical tests were performed within 2±12 weeks before treatment, at initiation of treatment, every 2 weeks for the ®rst 8 weeks of treatment, and every 4 weeks thereafter until the end of treatment. during the follow-up period patients were evaluated 4, 12 and 24 weeks after the end of treatment. serum hcv-rna was determined in all patients quantitatively before treatment (amplicor monitor hcv tm , version 2.0, roche diagnostic systems, branchburg, nj) and thereafter qualitatively at treatment weeks 4, 12, 24 and 48, and at the end of the follow-up period. genotyping was performed by a reverse hybridization assay (inno lipa hcv ii, innogenetics, gent, belgium) [28] . informed written consent was obtained from all patients before enrolment. the study was approved by the local ethics committees for medical research of the participating study centres and performed according to the declaration of helsinki, the german drug law, and the ich guidelines of`good clinical practice'. individual emotional and psychological states were determined at baseline, at treatment week 16, and at the end of the 24 weeks follow-up period by a german adapted and validated pro®le of mood states (poms) scale 1 assessing four factor scores for depression, fatigue, vigor and anger [29, 30] . at the same time points quality of life was evaluated by an`everyday life' 2 questionnaire [31, 32] . this german-validated questionnaire, related to the sf-36 health survey, assesses the following six subscales of health-related quality-of-life for subjective health: body (e.g. make demands on body, concentrate on a task), mind (e.g. cope with illness, accept oneself), everyday life (e.g. solve daily problems, personal hygiene), social activity (e.g. get along with family, count on partner's help), zest for life (e.g. enjoy life) and medical treatment (e.g. believe in success of treatment). sum scores were determined for every patient. missing items were replaced by the mean for the nonmissing items of the subscales. however, missing questionnaires were not replaced. the primary ef®cacy endpoint of the present study was de®ned as a sustained virological response with undetectable serum hcv-rna by qualitative serum hcv-rna at the end of the follow-up period. secondary ef®cacy parameters included the virological response at treatment weeks 12, 24 and 48 as well as the corresponding biochemical response de®ned as a normalization of alt. as an additional secondary ef®cacy parameter changes of hrqol during and after treatment were evaluated. based on an intent-to-treat analysis, 55 primary ifn-a nonresponders, who received at least one dose of the study medication, were included in the primary statistical analysis. virological response rates of the two treatment arms were compared with a one-tailed fisher's exact test on a signi®cance level of a 10%. corresponding biochemical response rates were analysed with a two-tailed mann±whitney u-test (a 0.05). statistical procedures were performed using sas procedures (version 6.12). for the evaluation of hrqol, the alt, alanine aminotransferase; ast, aspartate aminotransferase; c-gt, c-glutamyltransferase. *mean sd. normal reference ranges: 0±23 u/l for alt, 0±21 u/l for ast, 0±28 u/l for c-gt, and 0±25.7 lmol/l for bilirubin. àclassi®cation according to desmet et al. [26] . differences of the sum scores between the two treatment groups at treatment weeks 16 and 48, and at the end of follow-up were compared with a two-tailed wilcoxon rank sum test (a 0.05) using statxact4. an initial virological response with undetectable serum hcv-rna (< 1000 copies/ml) at treatment week 12 was achieved in ®ve of 26 (19%) and in eight of 29 (28%) patients treated with ifn-a2a and amantadine sulphate or placebo, respectively ( table 2 ). according to the protocol the antiviral therapy was discontinued at treatment week 16 in 42 patients because of detectable serum hcv-rna at treatment week 12. because of breakthrough events during treatment weeks 16±48, a virological end-of-treatment response was found only in one of 26 (4%) patients receiving combined ifn-a2a/amantadine sulphate treatment and in four of 29 (14%) patients on ifn-a2a and placebo. at the end of follow-up, a sustained virological response was observed in no patient treated with ifn-a2a and amantadine sulphate and in two patients treated with ifn-a2a plus placebo. initial biochemical responses with alt values within the normal range at treatment week 12 were comparable in both groups (38% for each treatment arm, table 2 ). a sustained according to the study protocol antiviral treatment was discontinued at week 16 in 42 of 55 patients with detectable serum hcv-rna at treatment week 12. therefore, healthrelated quality of life during treatment was evaluated in all patients at treatment week 16. assessment of health-related quality of live during treatment revealed a deterioration of the mean of all four factor scores of the poms scale in the patients treated with ifn-a plus placebo and in three of the four factor scores (depression, fatigue and anger) in patients treated with combined ifn-a/amantadine sulphate (fig. 2a) . the extent of the observed impairment of the poms scale was less pronounced for the subscales depression and fatigue in patients treated with ifn-a2a plus amantadine sulphate. at the end of follow-up, a sustained improvement of the mean factor scores for depression (p n.s.), fatigue (p 0.023), and vigor (p 0.025) in comparison with baseline levels was observed only in patients receiving combined treatment with ifn-a2a plus amantadine sulphate. irrespective of treatment, the evaluation of the`everyday life' questionnaire at treatment week 16 showed an impairment in the means of most subscales in our patients compared with the corresponding pretreatment scores (fig. 2b) . at the end of the follow-up period, a sustained improvement in the means of all subscales was observed only in patients receiving treatment with ifn-a plus amantadine sulphate. in the group of patients treated with ifn-a plus placebo the means of all subscales at the end of the follow-up period was worse than the corresponding pretreatment scores. adverse events (n 269) occurred in a similar frequency in both treatment arms and were mostly related to ifn. the spectrum and frequency of the observed adverse events in relation to treatment are listed in detail in table 3 . serious adverse events were not observed in this study. however, one patient treated with ifn-a2a plus placebo was discontinued prematurely from antiviral treatment at week 20 due to depression. a slight decrease of mean leukocyte and thrombocyte count was found in most patients irrespective of the treatment group from baseline to treatment week 4 (5.8 1.6/ nl to 4.3 1.4/nl, and 197 77/nl to 153 83/nl, respectively). at the end of the follow-up period leukocyte and thrombocyte counts returned to baseline levels with no signi®cant differences between the treatment groups. hyperthyroidism developed in one patient treated with ifn-a2a alone and in ®ve patients receiving ifn-a2a plus amantadine sulphate. in one patient, diabetes mellitus developed and in another patient diabetes mellitus was aggravated during treatment. both patients received ifn-a2a plus amantadine sulphate. during the last decade ifn-a therapy has been established as the standard treatment for chronic hepatitis c. sustained . with respect to the low sustained response rates to retreatment with ifn-a alone or in combination with ribavirin in ifn-a nonresponder, the need for the development of alternative, effective retreatment regimens in this subgroup of patients became apparent. in a 1997 published pilot study, a bene-®cial therapeutic effect of six months amantadine retreatment in ifn-a nonresponders with chronic hepatitis c was reported with a sustained biochemical and virological response in four of 22 patients (18%) after a 24 week followup period [19] . the postulated antiviral effect of amantadine was supported in vitro by a dose-dependent decrease of hcv-rna in the supernatant of isolated blood mononuclear cells from patients with chronic hepatitis c cultured with amantadine [33] . however, in vivo determination of hepatitis c viral load failed to show a direct synergistic antiviral effect of amantadine sulphate during combined ifn-a/amantadine treatment in previously untreated patients with chronic hepatitis c [23] . subsequently, mainly uncontrolled clinical trials of amantadine monotherapy in small cohorts of nonresponders with chronic hepatitis c showed con¯icting results with sustained virological response rates varying from 0% to 15%. as indicated by a recently published pilot study, the combination of ifn-a with amantadine may lead to an improvement of virological response rates in nonresponders with chronic hepatitis c [25] . the present study is the ®rst randomized, double-blind, placebo-controlled trial evaluating the ef®cacy of retreatment with ifn-a plus amantadine sulphate compared with ifn-a alone in previous ifn-a nonresponders with chronic hepatitis c. considering the demographic, biochemical, virological and histological patients' characteristics, signi®cant differences between both treatment groups were not present. the sustained virological response rates were similar in both treatment groups. thus, with respect to the results of the present controlled study, the addition of amantadine sulphate to ifn-a does not improve the sustained virological response rates in nonresponders with chronic hepatitis c. these results con®rm a larger controlled trial in 119 treatment naive patients with chronic hepatitis c which also showed no bene®cial virological effect of the addition of amantadine sulphate to ifn-a treatment in these patients [23] . in contrast, a recently published study in 60 nonresponders, treated with ifn-a, ribavirin and amantadine or ifn-a and amantadine, could demonstrate increased virological response rates in nonresponders with chronic hepatitis c who received amantadine [34] . however, these data have to be con®rmed by larger randomized, double-blind, placebo-controlled trials in nonresponders with chronic hepatitis c. despite a similar number of adverse events in both treatment groups, additional analysis of the individual emotional and psychological state as well as the evaluation of health-related quality of live revealed an improved outcome in patients treated with the combination of ifn-a plus amantadine sulphate compared with those patients receiving ifn-a alone. in the poms scale all four subscores deteriorated during treatment with ifn-a alone in comparison with the corresponding baseline levels and remained impaired during the follow-up period. the combined retreatment with ifn-a plus amantadine sulphate led to a smaller reduction of three of the four subscores, i.e. depression, vigor, and fatigue during treatment and interestingly, to a sustained improvement of the corresponding subscores in comparison with baseline levels at the end of the 24 week follow-up period. a similar improvement during amantadine sulphate treatment has also been shown for patients with central nervous disease, including multiple sclerosis and parkinson's disease [35] . in the present study, analysis of the sf-36 healthrelated`everyday life' questionnaire showed a sustained improvement of all six subscores in the group of patients treated with ifn-a plus amantadine sulphate at the end of the follow-up period in comparison with the corresponding baseline scores while ®ve of six subscores remained impaired in patients treated with ifn-a alone. a sustained improvement for the poms scale and the sf-36 health-related everyday life' questionnaire has also been reported in a previously published study in naive patients with chronic hepatitis c receiving ifn-a/amantadine sulphate combination therapy compared with those treated with ifn-a alone [23] . in summary, retreatment with ifn-a in combination with amantadine sulphate does not increase the low sustained virological response rates of ifn-a monotherapy in primary ifn-a nonresponders with chronic hepatitis c, but may lead to a sustained improvement of health-related quality of life. the recently suggested improved antiviral effect of triple retreatment with ifn-a, ribavirin and amantadine in previous ifn-a nonresponders has to be con®rmed in larger randomized, double-blind, placebo-controlled trials. hepatitis c virus infection is associated with the development of hepatocellular carcinoma the natural history of community-acquired hepatitis c in the united states clinical outcomes after transfusion associated hepatitis c therapy of hepatitis c: overview meta-analysis of interferon randomized trials in the treatment of viral hepatitis c: effects of dose and duration interferon-a2b alone or in combination with ribavirin as initial treatment for chronic hepatitis c an international randomized trial of interferon alfa-2b and ribavirin for 48 or for 24 weeks vs interferon alfa-2b, 48 weeks, for ®rst line treatment of chronic hepatitis c interferon-alpha retreatment in chronic hepatitis c retreatment with interferon-alpha and ribavirin in primary interferon-alpha nonreponders with chronic hepatitis c in vitro inhibition of rubella virus by 1-amantanamine hydrochloride inhibition of uncoating fowl plague virus by 1-adamantanamine hydrochloride arenaviruses: inhibition by amantadine hydrochloride inhibition of dengue virus replication by amantadine hydrochloride the effect of amantadine on mouse hepatitis virus replication a controlled trial of amantadine and rimantadine in the prophylaxis of in¯uenza a infection the molecular basis of the speci®c anti-in¯uenza action of amantadine ion channel activity of in¯uenza a virus m2 protein; characterization of the amantadine block treatment of chronic hepatitis c with amantadine the roles of amantadine, rimantadine, ursodeoxycholic acid, and nsaids, alone or in combination with alpha interferons, in the treatment of chronic hepatitis c amantadine hydrochloride decreases serum alt activity without effects on serum hcv-rna in chronic hepatitis c patients a pilot study of interferon plus amantadine versus interferon alone in the treatment of chronic hepatitis c randomized, doubleblind, placebo-controlled trial of interferon-alfa2a with and without amantadine as initial treatment for chronic hepatitis c amantadine plus interferon in non reponders or relapsing chronic hepatitis c patients: end of treatment response a controlled study of amantadine montherapy vs. amantadine combined with interferon-a in chronic hepatitis c patients nonresponders to interferon-a classi®cation of chronic hepatitis: diagnosis, grading and staging clinical evaluation of a new polymerase chain reaction assay (amplicor tm hcv) for detection of hepatitis c virus evaluation and comparison of different hepatitis c virus genotyping and serotyping assays 3 pro®le of mood states. educational and industrial testing service pro®le of mood states``(poms) und``psycohological general wellbeing index``(pgwi) sf-36 health survey manual and interpretation guide der fragebogen alltagsleben ± ein verfahren zur erfassung der gesundheitsbezogenen lebensqualita èt in vitro analysis of amantadine and interferon-a-2a on hepatitis c virus markers in cultured peripheral blood mononuclear cells from hepatitis c virus-infected patients triple antiviral therapy as a new option for patients with interferon nonresponsive chronic hepatitis c fatigue therapy in multiple sclerosis: results of a double-blind, randomized, parallel trial of amantadine, pemoline, and placebo key: cord-030361-0tepkjdl authors: mohammed abdul, mubeen khan; snyder, heather s.; chunduru, mythili; lee, susan m.k.; satapathy, sanjaya k. title: hepatitis c virus in the elderly in the direct-acting antiviral era: from diagnosis to cure date: 2020-08-11 journal: curr treat options infect dis doi: 10.1007/s40506-020-00231-8 sha: doc_id: 30361 cord_uid: 0tepkjdl purpose of review: hepatitis c (hcv) is the most common cause of viral hepatitis in elderly individuals. this patient population previously experienced suboptimal outcomes with interferon-based regimens. unfortunately, patients aged 65 years and older were underrepresented in phase 2 and 3 clinical trials with newer direct acting antiviral (daa) therapies. since the advent of second-generation daa in 2013, numerous robust real-world experiences highlighting the efficacy and safety of daa in the elderly have been published. this review article summarizes the cascade of care for hepatitis c from diagnosis to cure from an evidence-based perspective of the aging population. recent finding: in a large study from the veterans affairs healthcare system, the overall sustained virologic response (svr) of 15,884 patients treated with daa regimens was 91.2%. these newer therapies remained highly effective in the subset of patients aged 65 years and older with svr rates above 90%. a spanish national registry reported outcomes in patients ≥ 65 years old treated for hcv with oral daa regimens over a 2-year period. the overall svr was 94% in the study of 1252 subjects. summary: current real-world data imply daa treatment regimens remain highly effective and safe in elderly patients when compared to the general population. chronic hepatitis c affects 71 million people worldwide according to the world health organization (who) estimates [1] . who aims to decrease the incidence of hepatitis c by 80% and mortality by 65% globally by the year 2030. in the united states, chronic hepatitis c virus (hcv) has the highest mortality attributed to any infectious disease [2] . in 2012, the centers for disease control and prevention (cdc) added recommendations for one-time hcv screening in all persons born between 1945 and 1965, regardless of the presence or absence of hcv risk factors. [3] . this birth cohort accounted for 65% of all chronic hcv infections among adults in the united states [4] . infection with hcv should be identified early in this aging population as older adults tend to have rapid progression of hepatic fibrosis [5] [6] [7] [8] . historically, elderly patients experienced suboptimal outcomes with interferon-based regimens due to poor efficacy and tolerability [9] . newer direct-acting antivirals (daa) are highly effective with sustained virologic response (svr) rates above 90% [10] [11] [12] [13] [14] . these interferon-free treatment regimens also have improved safety profiles with the most common side effects including headache and fatigue. unfortunately, patients aged 65 years and older were underrepresented in phase 2 and 3 clinical trials for daa. thus, outcomes of newer hcv therapies in this patient cohort remained uncertain. in this review, we focus on the cascade of care for hcv treatment in elderly patients, including evidence surrounding the use of daa in persons greater than or equal to 65 years of age. screening & identification of elderly individuals infected with chronic hcv is a critical first step. studies have shown that 3.5% of the baby boomer birth cohort is hcv antibody positive, which is more than twice the prevalence seen in any other age cohort within the united states [15] . the initial hcv antibody test, if positive, requires another specimen for hepatitis c rna polymerase chain reaction (pcr) as a confirmatory test. infrequently, patients exposed to hcv can spontaneously clear the virus during the initial 6 months of the acute phase. a positive hcv antibody proves exposure whereas a positive hcv rna confirms diagnosis of active infection. most laboratories now offer reflex hcv rna testing, which allows a positive antibody test to trigger an automatic rna test from the same specimen. automatic reflex rna testing shortens the screening process allowing for quicker linkage to care. table 1 lists the cdc recommendations of who should be tested for hcv. updated recommendations by the cdc for routine screening of baby boomers, adults born between 1945 and 1965, have allowed for increased detection of disease burden in this difficult-to-treat population. hospitals and health systems throughout the country implemented automatic age cohort hcv testing alerts in the electronic medical records which have exponentially increased hcv screening capacity [8] . linkage to hcv care providers & despite accessible linkage to care in the past, innovative care models may further improve efforts for hcv eradication. • universal hepatitis c screening: hepatitis c screening at least once in a lifetime for all adults aged 18 years and older, except in settings where the prevalence of hcv infection (hcv rna-positivity) is less than 0.1% hepatitis c screening for all pregnant women during each pregnancy, except in settings where the prevalence of hcv infection (hcv rna-positivity) is less than 0.1% • one-time hepatitis c testing regardless of age or setting prevalence among people with recognized conditions or exposures: people with hiv people who ever injected drugs and shared needles, syringes, or other drug preparation equipment, including those who injected once or a few times many years ago people with selected medical conditions, including: ▪ people who ever received maintenance hemodialysis ▪ people with persistently abnormal alt levels prior recipients of transfusions or organ transplants, including: ▪ people who received clotting factor concentrates produced before 1987 ▪ people who received a transfusion of blood or blood components before july 1992 ▪ people who received an organ transplant before july 1992 ▪ people who were notified that they received blood from a donor who later tested positive for hcv infection healthcare, emergency medical, and public safety personnel after needle sticks, sharps, or mucosal exposures to hcv-positive blood children born to mothers with hcv infection • routine periodic testing for people with ongoing risk factors, while risk factors persist: people who currently inject drugs and share needles, syringes, or other drug preparation equipment people with selected medical conditions, including: ▪ people who ever received maintenance hemodialysis • any person who requests hepatitis c testing should receive it, regardless of disclosure of risk, because many persons may be reluctant to disclose stigmatizing risks management of hepatitis c can be challenging in the elderly population due to existing comorbidities and other aging-related factors. the availability of hepatologists may be scarce in certain communities. innovative care models have been established throughout the country from the emergence of necessity. project echo started in new mexico with the intention to expand access to liver specialists within rural areas in 2003. the interactive video platform is used to build knowledge and expertise in rural areas allowing greater access to specialists in real-time collaboration with local community providers. project echo effectively tele-mentors 50 programs in 39 countries spreading knowledge beyond borders [16] . alternatively, the veterans affairs (va) system, the largest hepatitis c care provider in the united states, developed an interdisciplinary model. a va medical clinic in indianapolis increased hepatitis c treating capacity by incorporating pharmacists. after the initial visit with an hcv clinic provider, subsequent visits were referred to the pharmacist-run hcv clinic. utilizing three pharmacists for five half days per week allowed up to 35 additional patient appointments. the implementation of this model doubled the number of hcv patients with comparable efficacy in regards to svr [17] . medication access is an unavoidable hurdle in treating hepatitis c due to high medication cost and lack of insurance. often times, the medication of choice is dependent on the patient's prescription insurance formulary unless medical necessity dictates alternative therapy. thus, for a successful hepatitis c treatment program, traditional healthcare models should be reevaluated to maximize their capacity to screen and treat hepatitis c. figure 1 summarizes the optimal cascade of hepatitis c care. & several patient-specific factors should be considered when evaluating for hcv treatment. prior to initiating hcv treatment, hcv genotype, treatment history, liver fibrosis, and potential concomitant drug-drug interactions must be assessed. baseline laboratory tests, including a complete blood count and comprehensive metabolic panel along with hepatitis c genotype and hcv pcr with quantitative levels, should be checked within 6 months before initiating hcv therapy [18] . patients who are cirrhotic need to be assessed for decompensated disease. international normalized ratio, albumin, total bilirubin, presence or absence of ascites, and hepatic encephalopathy are all components of the child-turcotte-pugh score required to evaluate the state of cirrhosis. screening for hepatitis b virus (hbv) is also a prerequisite to hcv treatment. hbv core total antibody, hbv surface antigen (hbsag), and hbv surface antibody should be assessed to evaluate the risk of hbv reactivation. there are two different approaches to hbsag positive patients with low or undetectable hbv dna. the conservative option is to initiate hbv treatment a week before starting hcv treatment. hbv treatment should be continued until 12 weeks after completing hcv therapy. the alternative option is to monitor monthly for hbv infection during and immediately after hcv treatment. if this pre-emptive strategy is utilized, hbv treatment should be initiated if there is a significant rise in hbv dna levels. given the complex medical comorbidities in the elderly, coupled with their cumulative progression to cirrhosis and hepatocellular carcinoma, early referral to providers with experience in the management of hcv is essential [19] [20] [21] . baseline hepatic fibrosis should be assessed with non-invasive modalities including transient electrography (fibroscan®) or more accessible indices such as a calculated fibrosis-4 (fib-4) score, aminotransferase to platelet ratio index (apri), or the fibrosure® blood test [18] . older adults with concomitant medications due to other underlying chronic comorbidities such as renal failure or cardiovascular disease should have a thorough drug-drug interaction analysis with potential hcv regimens. therefore, an interdisciplinary team approach is paramount to the safety and success of curing hcv. in clinical practice, there are currently five hcv daa therapies available. three of these regimens are pangenotypic, including glecaprevir/pibrentasvir, fig. 1 . cascade of linkage to care in elderly with hepatitis c virus sofosbuvir/velpatasvir, and sofosbuvir/velpatasvir/voxilaprevir. treatment duration usually spans 8-12 weeks, but may extend up to 16-24 weeks depending on hcv genotype, stage of fibrosis, and treatment history. there are three classes of direct acting antivirals used to disrupt the hcv life cycle. the ns3/4a protease inhibitors end with the suffix "previr", the ns5a inhibitors end with the suffix "-asvir", and the ns5b polymerase inhibitors (both nucleoside and nonnucleoside analogs) end with the suffix "-buvir". ledipasvir/sofosbuvir is indicated for hcv genotypes 1, 4, 5, and 6. weightbased ribavirin dosing should be added to this combination in patients with decompensated cirrhosis and in liver transplant recipients with compensated cirrhosis. this treatment regimen can be shortened to 8 weeks in patients infected with hcv genotype 1 who are treatment-naïve, noncirrhotic, and have a pretreatment hcv rna less than 6 million iu/ml. elbasvir/grazoprevir can be used to treat hcv genotypes 1 and 4 for a recommended treatment duration of 12 weeks. resistance testing is required prior to use in patients with genotype 1a. grazoprevir is a protease inhibitor; therefore, this combination therapy should not be used in patients with decompensated cirrhosis. glecaprevir/pibrentasvir is a pangenotypic regimen that may be used to treat hcv genotypes 1-6. this combination is administered as three tablets once daily, which differs from other single tablet daa regimens. on the other hand, glecaprevir/pibrentasvir has a shorter recommended duration of 8 weeks for patients who are treatment naïve, with or without compensated cirrhosis, regardless of genotype. this combination can also be used in patients who have previously failed treatment with daa therapies. glecaprevir is a protease inhibitor; therefore, this combination therapy should not be used in patients with decompensated cirrhosis. sofosbuvir/velpatasvir is a 12-week pangenotypic treatment regimen. this combination is an important option for the hard-to-treat genotype 3-infected patients with decompensated cirrhosis. the addition of weight-based ribavirin is recommended in any patient with decompensated cirrhosis. sofosbuvir/velpatasvir/voxilaprevir, a pangenotypic combination therapy, is reserved for use as a 12-week treatment in patients who previously failed daa regimens. this combination is also an option for patients infected with hcv genotype 3 who have compensated cirrhosis and have previously failed treatment with peginterferon/ribavirin. as the hcv population grows older, there will be an expected increase in liverrelated complications including cirrhosis and hepatocellular carcinoma [20] . achievement of hcv eradication will avoid significant healthcare costs caused by the burden of disease [22] . additionally, there is evidence demonstrating an association between chronic hcv infection with metabolic disorders, fatigue, depression, and poor quality of life [23] [24] [25] . achieving hcv cure has been shown to improve these extra-hepatic complications [22, 26] . current literature also suggests that anti-hcv treatment may reduce the risk of end-stage renal disease, coronary artery disease, and cerebrovascular accidents [24, 26] . evidence from phase 2 and phase 3 clinical trials saab and colleagues compared outcomes in patients g 65 years old to those ≥ 65 years old who received hcv treatment with ledipasvir/sofosbuvir with or without weight-based ribavirin for 8, 12, or 24 weeks [27] . the authors pooled data from four phase 3 clinical trials. the study included 264 patients aged 65 years or older, with 20% having compensated cirrhosis. the authors found similar rates of sustained virologic response at 12 weeks after completion of therapy (svr12) between patients g 65 years old and those ≥ 65 years old (97% vs. 98%, respectively). efficacy remained high among patients 75 years or older, with 100% achieving svr12. tolerability was similar between groups, with 78% of patients g 65 years old and 80% of patients ≥ 65 years old reporting adverse effects, the most common being headache and fatigue. this study reported a higher rate of study drug modification or interruption among patients treated with ribavirin in both age groups, with the elderly experiencing a higher incidence (6% g 65 years vs. 13% ≥ 65 years). in a study conducted by foster and colleagues, data was combined from nine phase 2 and phase 3 clinical trials to evaluate efficacy and safety outcomes in hcv patients ≥ 65 years old treated with the pan-genotypic regimen, glecaprevir/pibrentasvir, for 8, 12, or 16 weeks [28] . overall, the authors included 2369 subjects with 328 subjects ≥ 65 years old, including 47 patients aged 75 years or older. of the elderly individuals, 20% had cirrhosis at baseline. svr12 rates were similar between groups (97.3% in g 65 and 97.9% in ≥ 65 years old). rates of adverse effects and discontinuation due to daa-related adverse effects were also similar between groups. shiffman and colleagues reported outcomes of 123 patients aged 65 years or older enrolled in three phase 3 studies who received sofosbuvir/velpatasvir, a pan-genotypic daa, for 12 weeks for the treatment of chronic hcv [29] . fourteen of the patients were ≥ 75 years old. all of the elderly patients achieved svr12 compared to 97.8% of the younger subjects. similar to other daas, the most commonly reported side effects were headache, fatigue, nausea, and nasopharyngitis. none of the older patients required treatment discontinuation due to adverse effects. flamm et al. conducted an integrated retrospective analysis to evaluate the safety and efficacy of elbasvir/grazoprevir in the elderly population [30] . they pooled data from twelve phase 2 and 3 trials comprising 2478 subjects with hcv genotype 1 or 4. three hundred and thirty-nine patients were ≥ 65 years old, with 19% being cirrhotic. after 12 weeks of treatment, sustained virologic cure rates were similar between groups in the intent-to-treat population (95.4% g 65 years vs. 95.3% ≥ 65 years). serious adverse drug effects and discontinuations due to adverse effects were rare in both groups. a large veterans affairs healthcare system retrospective study utilized data from patients who completed hcv daa therapies over a 27-month period [31••] . daa treatments included sofosbuvir-based, and paritaprevir/ritonavir/ ombitasvir plus dasabuvir-based regimens. among the 15,884 patients with svr data, the overall svr was 91.2%. the percentage of patients over the age of 65 years was 27.8%. svr rates were comparable in the elderly age cohorts: 91.1% in patients between 65 and 69 years, 90% in patients 70-74 years, and 93.8% in patients ≥ 75 years of age. a multicenter observation study utilizing data from the spanish national registry reported outcomes in patients aged 65 years and older who were treated for hcv with oral daa regimens over a two-year time period [32••] . of the 1252 subjects, 76% were 65-74 years of age, 17% were 75-79 years of age, 7% were 80 years or older, and 74% of all patients had cirrhosis. overall, 33.3% took ledipasvir/sofosbuvir with or without ribavirin, 28% paritaprevir/ ritonavir/ombitasvir plus dasabuvir with or without ribavirin, and 26% received sofosbuvir plus simeprevir with or without ribavirin. the remaining subjects received other oral daa regimens. the overall svr12 rate in the intent-to-treat population was 94%. there was no significant difference in svr rates among age groups, but there was an increase in serious adverse effects among patients 75 years or older (8.8% in 65-74 years, 13% in 75-79 years, and 14% in ≥ 80 years). in a retrospective cohort study conducted by conti and colleagues, 556 patients (50.7% ≥ 65 years old) were treated for hcv infection with daa regimens at 11 centers in italy [33•] . more patients in the elderly group were cirrhotic (86.5% vs. 78.1% in the g 65 group, p = 0.010). svr12 rates were similar between age groups (90.5% in g 65 years vs. 94.7% in ≥ 65 years, p = 0.074). advanced fibrosis and cirrhosis did not hinder efficacy outcomes, but the severity of liver disease did affect svr12 rates (95.5% in child-turcotte-pugh a vs. 90.8% in child-turcotte-pugh b, p = 0.010). more patients in the elderly group reported adverse effects (54.6% in elderly vs. 38.7% in g 65 years group); however, the rates of serious adverse effects and discontinuation of therapy were similar between groups. two meta-analyses reviewed clinical trials and post-marketing studies to evaluate the efficacy and safety of daa regimens in the elderly population [34, 35] . overall, daa therapies are highly effective and well-tolerated in patients ≥ 65 years old. both studies also found that older patients treated with ribavirin are at an increased risk of developing anemia. there are currently no recommendations for an upper age limit for patients requiring hcv treatment [18] . current literature suggests that daa regimens are safe and effective for the treatment of hcv in the elderly population; however, the addition of ribavirin should be avoided if possible due to the risk of adverse effects. elderly patients have a higher prevalence of comorbidities, including cirrhosis, hypertension, diabetes, cardiovascular disease, chronic obstructive pulmonary disease, and chronic kidney disease [28, 30, 31 ••, 32••, 33•] . when compared to the younger population, patients ≥ 65 years old are also more likely to have lower albumin and hemoglobin levels at baseline. it does not appear that these comorbidities have an effect on treatment response; however, comorbid medical conditions should be considered when assessing the life expectancy of the patient. given their higher number of comorbidities, patients ≥ 65 years old generally require an increased number of medications [28, 30, 32••, 36] . prescribers and pharmacists should evaluate concomitant medications for potential drugdrug interactions when selecting daa regimens. one study compared possible drug-drug interactions between patients g 65 years and patients ≥ 65 years treated with various daa regimens [36] . there were 404 patients in the g 65 years group and 137 in the elderly group. there was a total of 152 different medications taken concomitantly with daa therapy, with the most common being proton pump inhibitors, beta-blockers, thyroid medications, loop diuretics, angiotensin-converting enzyme inhibitors, vitamin d supplements, and insulins. more patients in the ≥ 65 years group were taking concomitant medication therapies compared to the younger group (79% vs. 51%, respectively, p g 0.0001), with 35% of patients ≥ 65 years requiring at least four medications. there was a higher rate of clinically significant drug-drug interactions predicted in the elderly group (54% vs. 28% in the g 65 years, p g 0.0001). interactions were determined to be clinically significant if they required close monitoring, dose modifications, changes in administration times, or if co-administration was not recommended or contraindicated. comprehensive drug-drug interaction databases are encouraged if practitioners are uncertain of interactions between daa regimens and concomitant medication therapies. table 2 provides an overview of common drug-drug interactions with currently available daa regimens. practitioners should also address pharmacodynamic interactions, including the risk of hypoglycemia with anti-diabetic medications and more frequent monitoring of warfarin therapies due to changes in hepatic function throughout daa treatment. laboratory testing may be monitored at treatment week 4, the end of treatment, and 12 weeks after the treatment has been completed. the comprehensive metabolic panel and quantitative hcv rna pcr should be reviewed at each time point. in cirrhotic patients, particularly those with decompensation table 2 . drug-drug interactions with commonly prescribed medications in patients with chronic hcv infection. the color scheme represents the level of clinical significance according to the hep-druginteractions.org website: green = no interaction; yellow = weak interaction; orange = potential interaction; red = strong interaction/contraindicated ppi proton pump inhibitors, omeprazole equivalent, h2ra histamine 2 receptor antagonists, famotidine equivalent, ato atorvastatin, ros rosuvastatin, lov lovastatin, sim simvastatin, pra pravastatin, "↑" = increased exposure, "↓" = decreased exposure requiring treatment with ribavirin, a complete blood count should also be collected. it is recommended to monitor patients with decompensated cirrhosis more frequently than the aforementioned schedule. the quantitative hcv rna pcr should have a detection level of g 25 iu/ml to be deemed "undetectable." this number may differ depending on the laboratory-specific calibration. hepatitis c virologic cure can be concluded with an undetectable hcv rna 12 weeks after hcv treatment completion. hcv resistance most commonly occurs when the virus is exposed to subtherapeutic concentrations of the drug. transmission of an hcv strain with preexisting resistance may also occur despite the patient lacking previous exposure to hepatitis c therapy. the clinically impactful resistance-associatedsubstitutions (ras) mostly develop against ns5a inhibitors and ns3/4a protease inhibitor-containing regimens. ras against ns5a inhibitors can persist for years and are more fit than others with y93h being the most prevalent ras across genotypes [18] . the american association for the study of liver diseases recommends baseline testing for ns5a ras in genotype-specific cohorts. genotype 3 treatment-naïve patients with cirrhosis and treatment-experienced patients with or without cirrhosis, in whom sofosbuvir/velpatasvir is being evaluated, should receive ns5a ras testing at baseline. if y93h is present, weight-based ribavirin should be added or a different treatment regimen should be prescribed. with respect to ledipasvir/sofosbuvir, baseline resistance testing should be considered in those infected with genotype 1a who are treatment-experienced with or without cirrhosis. if y93h is present, a different treatment regimen should be prescribed. lastly, baseline resistance testing is recommended in all genotype 1a patients, including both treatment-naïve and treatment-experienced, for whom elbasvir/grazoprevir is being considered and a different regimen should be prescribed if y93h is present [18] . in summary, the specific genotype and presence of ns5a ras with certain treatment regimens may result in exponentially higher drug resistance, ultimately impacting svr. retrospective studies have shown regression in fibrosis after treatment of the original liver disease, including chronic hcv eradication [37] [38] [39] [40] . pre-treatment hepatic fibrosis staging by noninvasive methods can be compared against testing after the achievement of svr to assess for clinical response [41] . despite the cure of hcv, elderly patients remain at increased risk of liver-related complications, including advanced fibrosis and hepatocellular carcinoma, likely due to other concomitant comorbidities such as non-alcoholic steatohepatitis and other metabolic syndromes [42, 43] . older age is also associated with an increased risk of hepatocellular carcinoma without evidence of significant hepatic fibrosis after eradication of hcv [44, 45] . prescribers should maintain ongoing care for older adults with significant pre-treatment hepatic fibrosis. in these patients, routine surveillance imaging for hepatocellular carcinoma should be continued even after achieving hcv cure. patients with cirrhosis should be monitored every 6 months. elderly patients share the highest burden of chronic hcv worldwide with liverrelated complications, including advanced fibrosis and hepatocellular carcinoma, being more common among older adults. screening and treatment of chronic hcv in this patient population will facilitate the goal of hcv eradication by 2030. albeit previous experience with interferon-based therapies may have hindered hcv treatment in the elderly, current daa treatment regimens appear to be generally safe and highly effective in patients ≥ 65 years old. healthcare practitioners should continue to ambitiously work towards achieving hcv eradication by screening and safely treating the elderly population. conflict of interest dr. satapathy reports grants from gilead sciences, grants from conatus pharma, grants and other from intercept pharma, other from alexion, grants from genfit, grants and other from dova, grants and other from bayer, grants from exact sciences, grants and other from biotest, grants from shire nash, grants from enanta, outside the submitted work. the other authors declare no conflicts of interest relevant to this manuscript. this article does not contain any studies with human or animal subjects performed by any of the authors. have been highlighted as: • of importance •• of major importance 1 who guidelines. guidelines for the care and treatment of persons diagnosed with chronic hepatitis c virus infection. who rising mortality associated with hepatitis c virus in the united states recommendations for the identification of chronic hepatitis c virus infection among persons born during 1945-1965 centers for disease control and prevention initiatives to prevent hepatitis c virus infection: a selective update natural history of liver fibrosis progression in patients with chronic hepatitis c. the obsvirc, metavir, clinivir, and dosvirc groups progression to cirrhosis in hepatitis c patients: an agedependent process measurement and determinants of the natural history of liver fibrosis in hepatitis c virus infection: a cross sectional and longitudinal study implementation of a large system-wide hepatitis c virus screening and linkage to care program for baby boomers hepatitis c treatment in the elderly: new possibilities and controversies towards interferonfree regimens ledipasvir and sofosbuvir for untreated hcv genotype 1 infection ledipasvir and sofosbuvir for previously treated hcv genotype 1 infection grazoprevir-elbasvir combination therapy for treatment-naive cirrhotic and noncirrhotic patients with chronic hepatitis c virus genotype 1, 4, or 6 infection: a randomized trial glecaprevir and pibrentasvir yield high response rates in patients with hcv genotype 1-6 without cirrhosis sofosbuvir and velpatasvir for hcv genotype 1, 2, 4, 5, and 6 infection the changing epidemiology of hepatitis c virus infection in the united states: national health and nutrition examination survey, through 2010 outcomes of treatment for hepatitis c virus infection by providers improving veteran access to treatment for hepatitis c virus infection aasld/ idsa hcv guidance: recommendations for testing, managing, and treating hepatitis c viral hepatitis in older adults hepatitis c virus infection in the older patient fibrosis in patients with chronic hepatitis c: detection and significance treating medicaid patients with hepatitis c: clinical and economic impact health state utilities and quality of life in hepatitis c patients hepatitis c virus and metabolic disorder interactions towards liver damage and atherosclerosis assessment of fatigue in patients with chronic hepatitis c using the fatigue impact scale long-term outcome after interferon therapy in elderly patients with chronic hepatitis c safety and efficacy of ledipasvir/ sofosbuvir for the treatment of genotype 1 hepatitis c in subjects aged 65 years or older safety and efficacy of glecaprevir/pibrentasvir for the treatment of chronic hepatitis c in patients aged 65 years or older safety and efficacy of sofosbuvir/ velpatasvir for the treatment of chronic hepatitis c in patients aged 65 years or older: a retrospective analysis of phase 3 studies efficacy and safety of elbasvir/grazoprevir in hepatitis c virus gt1-and gt4-infected people aged 65 years or older directacting antivirals are effective for chronic hepatitis c treatment in elderly patients: a real-world study of 17,487 patients daas produce high rates of svr in all age groups interferon-free therapy in elderly patients with advanced liver diseas svr rates with interferon-free regimens in elderly patients are high and comparable to the general population safety and efficacy of direct-acting antivirals for the treatment of chronic hepatitis c in a real-world population aged 65 years and older efficacy and safety of direct-acting antivirals for hepatitis c in the elderly: a systematic review and meta-analysis direct-acting antivirals for hcv treatment in older patients: a systematic review and meta-analysis the efficacy and safety of direct acting antiviral treatment and clinical significance of drug-drug interactions in elderly patients with chronic hepatitis c virus infection histological outcome during long-term lamivudine therapy a morphometric and immunohistochemical study to assess the benefit of a sustained virological response in hepatitis c virus patients with cirrhosis reversal of fibrosis in patients with nonalcoholic steatohepatosis after gastric bypass surgery long-term liver stiffness assessment in hepatitis c virus patients undergoing antiviral therapy: results from a 5-year cohort study care of patients following cure of hepatitis c virus infection incidence of hepatocellular carcinoma in patients with hcv-associated cirrhosis treated with direct-acting antiviral agents hcv eradication induced by direct-acting antiviral agents reduces the risk of hepatocellular carcinoma development of hepatocellular carcinoma in patients with chronic hepatitis c who had a sustained virological response to interferon therapy: a multicenter, retrospective cohort study of 1124 patients treatment of elderly patients with chronic hepatitis c: a retrospective cohort study springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-000830-jiy4cp4n authors: cobo, fernando title: application of molecular diagnostic techniques for viral testing date: 2012-11-30 journal: open virol j doi: 10.2174/1874357901206010104 sha: doc_id: 830 cord_uid: jiy4cp4n nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. these techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. the increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. this technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. the introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. the main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. the use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. this review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory. molecular diagnostic techniques for viral testing have experimented a rapid development during the last years [1] , and have been introduced in the majority of laboratories as a new way for the diagnosis of human pathogens like viruses. this field of molecular microbiology presents many challenges to the practice of laboratory medicine, above all the implementation like automated methodology. the introduction of fully automated devices with faster turnaround times has allowed clinical laboratories the necessary tools to report sensitive and accurate results to physicians. the goals in performing microbiology nucleic acid tests (nat) are mainly to provide timely results useful for high-quality patient care at a reasonable cost. rapid results obtained by nat are associated with improvements in patient care. empiric data and modeling studies find that faster detection of enteroviral meningitis using nat is associated with reduced length of stay and duration of antibiotic administration, as well as substantial cost savings [2] . the use of amplification techniques such as polymerase chain reaction (pcr), real-time pcr or nucleic acid sequence-based amplification (nasba) [3] for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range [4, 5] . a great number of qualitative and quantitative molecular virus assays, mostly *address correspondence to this author at the microbiology unit (biotechnology area), hospital de poniente. ctra de almerimar s/n, el ejido 04700, almería, spain; tel: +34950022638; e-mail: fernando.cobo.sspa@juntadeandalucia.es based on pcr technology have been described [6, 7] . nasba assays could identify active infection by detecting viral messenger rna (mrna) but the most widely used tests in clinical virus diagnosis are quantitative real-time pcr techniques [8] . many molecular diagnostic methods have been replaced by automated devices that use less time, manipulate smaller volumes of liquids and provide quantified results with better precision. molecular techniques have revolutionated the diagnosis of infectious diseases, particularly the diagnosis of viral diseases. automation of these methods provides decrease in turnaround times, low contamination risk, ease of performance and speed, as well as the ability to have lower detection limits and to diminish cost per test. this review focuses on the application of molecular technology in the clinical virology laboratory. table 1 summarizes the main molecular techniques used in clinical virology. nucleic acid probes are segments of dna or rna labeled with radioisotopes, enzymes or chemiluminiscent molecules that can bind to complementary nucleic acid sequences of microorganisms. these probes can be used to identify some viruses. the commonly used formats for probe hybridization include liquid-phase, solid-phase and in situ hybridization [9] . in general, these techniques have a poor analytical sensitivity, so they could be used only to those situations in which the number of microorganisms is large. liquid-phase hybridization assay is a technique in which a single-stranded dna probe is labeled with an acridinium ester and is then incubated with the target nucleic acid. after hybridization step, the probe binding is measured in a luminometer. in solid-phase hybridization target nucleic acid bind to nitrocellulose or nylon and is hybridized with a probe solution. the identification is then carried out by means of fluorescence, luminescence, color development or radioactivity. the main limitation of this assay is the time consumption and the complexity of the technique, so application in clinical practice is very limited. finally, in situ hybridization is a solid-phase hybridization in which the nucleic acid is contained in cells or tissues fixed in microscope slides. the main disadvantage of in situ hybridization is the limitation of the accessibility of the target nucleic acid in the cells. the beginning of molecular diagnostics was initiated at the end of the eighties with the development of the pcr [10] . although this method is the most widely used nucleic acid amplification technique, other methodologies have been developed. the biochemical mechanisms of these techniques are based on target, signal or probe amplification. in signal amplification assays, the signal is directly proportional to the amount of the target sequence present in the clinical specimen, reducing false-positive results due to cross contamination; also, the development of quantitative assays is more reliable. the branched or bdna signal amplification system consists of a series of hybridization steps resulting in a "sandwich" complex of probes and target sequence with a branched structure [11] . in this technique, multiple targetspecific probes are used to capture the nucleic acid onto the surface of a microwell plate. the initial step in a bdna assay is to ensure that viral particles have been disrupted and that viral rna is present for analysis. target-specific oligonucleotides are then hybridized to the target nucleic acid, and other regions hybridize to multiple bdna amplifier molecules that create a branched structure. finally, the bdna signal is the chemiluminiscent product of the reaction [11] . the signal in the bdna assay is proportional to the number of labeled probes bdna assays for the quantitation of hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and human immunodeficiency virus type 1 (hiv-1) rna are commercially available (bayer healthcare, diagnostics division, tarrytown, n.y.). for the detection of hcv, the first-generation bdna assay (quantiplex hcv rna 1.0 assay, bayer) had a dynamic quantification range in human plasma (from 3.5 x 10 5 to 1.2 x 10 8 hcv rna copies/ml). genotypes 1-6 were detected by using this assay, although the sensitivity was lower for genotypes 2 and 3. to improve the detection rate for hcv genotypes 2 and 3, a second-generation assay was developed (quantiplex hcv rna 2.0 assay, bayer). however, a third-generation bdna assays both for hcv and hiv have been developed (versant hcv rna 3.0 and versant hiv-1 rna 3.0 assays, bayer) that uses isoc and isog-substituted oligonucleotides to reduce nonspecific hybridization. this system is a solution hybridization-antibody capture technique that uses a chemiluminescence detection system of the hybrid molecules. the dna from the sample is denatured and then hybridized with a specific rna probe. the dna-rna hybrids are captured by anti-hybrids antibodies. the antibody conjugate is detected with a chemiluminescent substrate and a luminometer serves as a device to measure the light emitted. hybrid capture assays for detection of human papillomavirus (hpv) and cytomegalovirus (cmv) in clinical specimens are currently commercially available (digene corp., gaithersburg, md) [12, 13] . these techniques use enzyme-mediated processes, in which the enzymes synthesize several copies of target nucleic acid. the amplification products are detected by two oligonucleotide primers that bind to complementary sequences. the final result is the production of millions of copies of the targeted sequence. there is possibility of contamination, so false positive results must be reduced through special laboratory design, practices and workflow. pcr allows the synthesis of million of copies of a targeted nucleic acid sequence. this chemical reaction occurs by means of the action of a dna polymerase that can copy a dna strand. pcr consists in a mixture of target dna, two oligonucleotide primers, a dna polymerase, a mixture of deoxyribonucleotide triphosphates (dntps), mgcl 2 , kcl and tris-hcl buffer. the reaction mixture is heated and cooled during several cycles in a programmable thermal cycler, and after n cycles the target sequence can be amplified 2 n -fold. after the pcr reaction, the detection of product of amplification should be done by means several techniques (e.g. gel analysis, colorimetric detection) [10] . rt-pcr was introduced to amplify rna targets. in this method, cdna is first produced from rna by reverse transcription and then the cdna is amplified by pcr. a thermostable dna polymerase derived from thermus thermophilus could function efficiently as both an rt and a dna polymerase [14] . commercially kits are available for detection of hcv rna and for quantitation of hiv-1 and hcv rna in clinical specimens (roche diagnostics, indianapolis, ind). this modality of pcr increases both the sensitivity and specificity [15] . this technique uses two pairs of amplification primers and two rounds of pcr. in the first round, it uses one primer pair for 15 to 30 cycles. the product of the first round of amplification is submitted to a second round of amplification with the second pair of primers. the major disadvantage of nested pcr is the high rates of contamination. in the same reaction mixture, two or more primer sets designed for amplification of different targets are used [16] . more than one target sequence in a clinical specimen can be co-amplified in a single tube. however, the primers used must be carefully selected in order that they have similar annealing temperatures and lack complementarity. this kind of pcr is less sensitive than pcr with single primer set. multiplex pcr assays for viral respiratory pathogens and for detection of viral infections of central nervous system have been developed and commercialized [17, 18] . in this method, the target amplification and detection steps occur simultaneously. these methods require special thermal cyclers that can monitor the fluorescence emission from the sample. the computer software supporting the thermal cycler monitors the data at every cycle and generates an amplification plot for each reaction [19] . the pcr product is detected by using fluorescent dyes that preferentially bind to double-stranded dna. the specificity of real-time pcr can also be increased by using fluorescent resonance energy transfer (fret) probes in the reaction mixture. another approach to real-time pcr is the use of dual hybridization probes that uses two specially designed sequence-specific oligonucleotide probes. finally, detection and quantitation of amplification products can be carried out with molecular beacons. real-time pcr decrease the time required to perform nucleic acid assays because there are no post-pcr processing steps. the main advantages of these methods are also the decrease of contamination and the possibility for quantitative applications. there are two methods included in this group: nucleic acid sequence-based amplification (nasba) and transcription-mediated amplification (tma). these are isothermal rna amplification methods modeled after retroviral replication [20, 21] . in both methods, the rna target is reverse transcribed into cdna and then rna copies are synthesized with a rna polymerase. transcriptionbased amplification systems have several characteristics such as no requirement for a thermal cycler, rapid kinetics and a single-stranded rna product that does not require denaturation prior to detection. gen-probe has developed tma-based assays for detection of hcv and hiv-1, while nasba-based kits (biomérieux) have developed commercially available kits for the detection and quantitation of hiv-1 rna and cmv rna and detection of enterovirus and respiratory syncytial virus rna. this method is an isothermal template amplification technique that can be used to detect trace amounts of dna or rna of a particular sequence (becton dickinson and company, sparks, md). in its current format, strand displacement amplification occurs in two phases, target generation and exponential target amplification [22] . recently, strand displacement amplification has been adapted to quantitate rna and rt-strand displacement amplification has been used for the determination of hiv viral load. these methods differ from those that use target amplification in which the amplification products contain only a sequence present in the initial probes. some examples of probe amplification methods are ligase chain reaction [23] , cycling probe technology [24] and cleavase-invader technology [25] . invader assays (third wave technologies, madison, ws) are based on a probe amplification method with specific recognition of particular dna structures by cleavase, a member of the fen-1 family of dna polymerases. in these assays, two primers are designed which hybridize to the target sequence. several methods can be used to detect the cleavage products, but fluorescence resonance energy transfer (fret) probes and a second invasive cleavage reaction to detect the target-specific products is the most common technique used. fret occurs due to the interaction between the electronic excited states of two dye molecules. fret probes are a pair of fluorescent probes placed in close proximity. the excitation is transferred from one dye molecule to the other without emission of a photon. the acceptor fluorophore emits light which is detected in specific channels. this technology is currently available for hcv genotyping. ligase chain reaction assay is based on the ligation of two adjacent synthetic oligonucleotide primers which hybridize to one strand of the target dna. a second pair of primers is used in a cycling reaction, using a thermostable dna ligase. both ligated products can then serve as templates for the next reaction cycle, leading to an exponential amplification process similar to pcr amplification. detection of ligase chain reaction product could be carried out by means of several methods such as autoradiography and fluorescence. one of the advantages of the fluorescent detection system is that it is relatively easy to quantitate the amount of the ligase chain reaction product. other methods for the detection of ligase chain reaction products in microtiter plates are been also used. lcr assays have been developed for the detection of viruses such as hpv, hsv and hiv. cycling probe technology is a method for detection and quantification of low amounts of target dna. the reaction is carried out at a temperature that allows the chimeric probe to anneal to the single-stranded target dna. rnase h, an enzyme that specifically degrades the rna portion of the dna-rna hybrids, cuts within the rna portion of the chimeric probe, and the shorter probe fragments dissociate from the target, regenerating the target for further cycling. the resulting accumulation of probe fragments can be detected. since the target dna is not amplified, this technique shows low background. moreover, cycling probe technology is fast, linear, isothermal and simple compared with other dna detection methods. a dna array (or dna chip) is a collection of spots attached to a solid support where each spot contains one or more single-stranded dna oligonucleotide fragment [26] . high density arrays, that permit attaching hundred or thousands of oligonucleotides, are referred to as microarrays. a labeled amplification product is hybridized to the probes, and hybridization signals are mapped to several positions within the array. the pattern of hybridization can identify the sequence of pcr, if the number of probes is sufficiently large. the results of hybridization between the bound probe and labeled sequences in the sample applied and tested are revealed by scanning or imaging the array surface. confocal microscopy is used to scan the chip, detecting fluorescent signals that reveal hybridization at precise locations on the chip. as many dna sequences can be present on a slide, it is possible for microarray analysis to test for multiple viruses simultaneously. the first application in diagnostic virology has been for rapid sequencing to detect hiv mutations associated with resistance to antiretroviral drugs [27] . since then, some research groups have developed microarrays that detect several viruses such as respiratory viruses [28] , hepatitis c virus [29] and virus causing cns infection [30] . microsphere-based suspension array technologies, such as the luminex ® xmap tm system, offer a platform for nucleic acid detection that have some advantages including rapid data acquisition, excellent sensitivity and specificity and multiplexed analysis capability [31] . as compared to planar microarrays, suspension arrays have the advantages of ease of use, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation [31] . this system incorporate microspheres dyed with two spectrally different fluorochromes. an array is created consisting of 100 distinct microsphere sets with specific spectrum. a third fluorochrome quantifies the biomolecular interaction that has occurred at the microsphere surface. microspheres pass through separate lasers in the luminex analyzer. high speed digital signal processing classifies the microsphere based on its spectral signal and quantifies the reaction on the surface. thousands of microspheres are analyzed per second resulting in an analysis system capable of analyzing and reporting a lot of different reactions. the assay can be used as direct dna hybridization, competitive dna hybridization and solution-based chemistries with microsphere capture. a multiplexed assay for detection and quantitation of viral nucleic acids using this system has been developed for hiv, hcv and hsv with high specific results [32] . since nucleic acid amplification methods have several disadvantages, such as the requirement for precision thermal cycling, isothermal techniques are being introduced as a diagnostic tool due to their simple operation, rapid reaction and easy detection. these new techniques do not require thermal cycler and can be performed by using a heating block and/or water bath. the main isothermal methods include loop-mediated isothermal amplification (lamp) and helicase-dependent amplification (hda) [33] . this method has improved classical pcr in its reaction simplicity, accuracy and higher amplification efficiency. the procedure is very rapid, and the amplification can be completed in less than 1 hour. the main advantage of lamp is that it does not require thermal cyclers, and the amplification can be carried out with a water bath or heating block. lamp is a one-step amplification reaction that proceeds at isothermal conditions. the chemistry of lamp amplification is based on the principle of strand displacement reaction, described previously [34] . the mechanism consists in three steps: an initial non-cycling step, a cyclic amplification step and an elongation step. the addition of reverse transcriptase makes it possible to amplify cdna from rna (rt-lamp). lamp method has been already used for emerging human viral pathogens such as dengue and sars viruses [35, 36] . also, rt-lamp assays have been developed for influenza a and b viruses' detection [37] , as well as for cmv, hsv, vzv, bk virus and hpv [38] [39] [40] [41] [42] . hda is an isothermal amplification method similar to dna replication in vivo by using a dna helicase to separate two complementary dna strands (dsdna) and further extension by a dna polymerase. the initial heat denaturation and subsequent thermocycling are not necessary, and the entire hda reaction can be performed at a single uniform temperature. this technique provides a useful tool to amplify dna in vitro under isothermal conditions. hda technique has been developed to detect several viruses in different clinical samples such as hiv-1 in human plasma [43] and hsv types 1 and 2 from genital lesions [44] . implementation of molecular techniques platforms in the clinical virology laboratory for diagnosis needs some requirements such as personnel and facility requirements and a correct work flow design. laboratory workers must be trained in both the preanalytical (specimen extraction and processing) and the analytical procedures. professionals that work in this kind of laboratory should have a correct training or experience in molecular methods and also should have theoretical knowledge of molecular virology. the majority of manufacturers provide overview presentations on molecular biology as well as technical information on their specific testing platform. the director of the virology laboratory should provide individualized training in molecular virology for all laboratory workers for success in performing correct molecular testing. it is very important to keep special attention to maintain strict adherence to standard operating procedures (sop) and avoid the samples contamination using aseptic techniques. the laboratory must have available detailed sop, training materials and checklists for each technique performed in the laboratory. respect to this fact, it would be very important to have a technical expert to provide a reference person in order to apply this methodology in the clinical laboratory. laboratory-developed tests require that the technical resources to resolve problems related to the assay are available within the laboratory. in order to minimize or decrease the risk of specimen contamination, it is necessary a physical separation of processes as well as to have reagents and equipment for use only in the molecular laboratory. each laboratory should define their work areas but, in general, four different work areas are recommended: a reagent preparation area to prepare pcr master mix, a sample processing area where different procedures are performed (like nucleic acid extraction), a target loading area where the specimen is added to the pcr master mix and an amplification area where thermocycling and probe detection is performed. the reagent preparation area should be kept free of all specimens and dna/rna extracts. the number of tubes that should be simultaneously opened must be minimizing in order to avoid cross-contamination between different samples. an important issue is that the different devices used in pcr such as pipettes, tubes, reagents should be dedicated exclusively to each working area. reagents should be prepared and aliquoted into single use or small volume. all working surfaces should be cleaned before and after each use with a reagent that eliminates nucleic acid. the manufacturer´s recommendations must be followed for cleaning of instruments, processing blocks and other instrument surfaces and parts. gloves should be changed frequently at least before beginning each procedure and must always be changed if moving from one to another work area. after selection and successful introduction of a molecular testing platform into the virology laboratory, work flow should be implemented at the same time that this technology is being introduced. the main factors to establish an effective work flow are determined by the arrival times of specimens, number of samples of each test, clinical urgency for the results and laboratory functionality. each laboratory should establish its own work flow depending of each technique and each viral determination, so this fact should be individualized. table 2 shows the main viruses that could be diagnosed by means of molecular techniques as well as the samples and the techniques more appropriate for them. cmv infection can have several clinical presentations such as non-specific viral syndrome, ocular and congenital disease. infection can also occur in the cns, and in the majority of them the clinical presentation is in the form of encephalitis, but also as myelitis, radiculomyelopathy and mononeuritis multiplex. studies suggest that the detection of cmv dna in the cerebrospinal fluid (csf) is highly sensitive and specific for cmv neurologic disease [45] . cmv dna could be detected by conventional pcr in the csf of hiv patients [46] but is rarely detected in hivinfected patients without clinical neurological disease. cmv viral load testing using pcr techniques (including real-time pcr) or hybrid capture assays can detect and quantify cmv dna or dna-rna hybrids in clinical specimens, including the csf [47] . cmv viral load assays can be performed quickly, but it is important to use the same assay while monitoring an individual patient. interpretation of the results of the viral load is sometimes problematic and unclear because cmv viral load have not been standardized, so it is not possible to define cutoff values. herpes simplex virus (hsv) is a cause of a wide spectrum of clinical manifestations such as cns, genital and dermal diseases. hsv is the most common cause of nonepidemic sporadic acute focal cns disease (mainly encephalitis). hsv can also cause aseptic meningitis, usually a self-limited disease that resolves without specific therapy. because of this, there is a need for a rapid and accurate diagnostic test for hsv cns diseases, so csf pcr testing has been evaluated as a diagnostic test for hsv encephalitis and meningitis, being a rapid and a very high sensitivity and specificity diagnostic tool [48] . however, because neither the sensitivity nor specificity is 100%, hsv pcr results should be interpreted with caution. false-negative hsv pcr results could be possible and false-positive hsv pcr results can be also possible due to eventual contamination. even so, currently it has been established that molecular amplification of hsv dna is the new gold standard for the laboratory diagnosis of these infections. pcr is positive early in the course of the illness (within the first 24 hours) and remains positive during the first week of therapy [49] . in some cases, viral genomes persist in the csf for two weeks or longer after the onset of antiviral therapy [49] . a real-time pcr assay is being used for the diagnosis of cmv, hsv-1 and hsv-2, ebv and vzv from csf specimens [50] . compared to conventional pcr, these realtime assays are rapid, simple and convenient for testing for herpesviruses dna in the routine laboratory. the similarity of clinical features of these viruses is a reason for include several targets for csf testing rather than for a single unique sequence of one virus. with respect to dermal and genital disease caused by hsv, real-time hsv pcr assays have emerged as a more sensitive method to confirm hsv infection in clinical specimen obtained from genital ulcers and muco-cutaneous lesions [51] . conventional pcr was not adapted for the detection of hsv in dermal or genital sources, because cell culture or direct staining techniques were relatively more sensitive for detecting hsv in these specimens. the main limiting factor in introducing real-time hsv pcr as the primary diagnostic tool in the laboratory is the cost of this assay. moreover, the lack of uniform validation of the pcr assay as a diagnostic method for detecting hsv in clinical specimens other than cerebrospinal fluid has limited its availability in some laboratories. vzv is a cause of cns disease such as encephalitis, myelitis and acute meningitis. in a retrospective study, vzv dna was detected from 5% of csf specimens [47] . realtime pcr assay provide rapid and sensitive confirmation of vzv from clinical specimens obtained from several samples such as exudate from skin lesions, broncho-alveolar lavage and csf [52, 53] . several studies comparing different assays against real-time pcr have demonstrated that vzv dna was detected in more samples that the rest of techniques [53, 54] . moreover, pcr-based testing was highly specific because no cross-reactivity was identified when tested against several other viruses. pcr testing is also useful for other indications such as the diagnosis of vzv infection in a patient with vaccine-modified infection [55] , as well as testing of serum or blood might also be helpful in the transplant patient who has visceral disease prior to the appearance of cutaneous lesions [56] . ebv has been implicated in the development of lymphomas, above all in immunosupressed patients. detection of ebv dna by molecular techniques in csf could be useful for the diagnosis of this infection. this technique has a sensitivity of 80-90% and a specificity that approaches 100% [57] . detection of ebv dna might also be useful in patients in whom a brain biopsy is not possible to do. on the other hand, detection of ebv dna in csf also provides a marker to monitor the response to treatment for cns lymphomas. ebv dna can be detected by conventional pcr [58] , by in situ hybridization [59] and also more recently has been introduced real-time pcr assays to detect ebv dna from cns lymphomas [60] . infection with parvovirus b19 might cause asymptomatic infection or a wide spectrum of disease (erythema infectiosum in children with arthropathy, severe anemia and systemic affectation) as well as hydrops fetalis, congenital anemia and abortion if the infection is produced in pregnant women. although parvovirus b19 infection is diagnosed by serologically detecting igm and igg class antibodies with elisa assays, the main application of dna detection by pcr is the control of transmission of the virus present in blood [61] . detecting b19 dna using molecular tests is now being used in many clinical laboratories, and these techniques are much more sensitive than antigen-based detection systems. most of these techniques detect genotype 1 dna but not genotypes 2 or 3. some real-time pcr assays such as lightcycler parvovirus b19 quantitative assay (roche diagnostics, indianapolis, in) and abi taqman (applied biosystems) have been developed for detecting b19 nucleic acids in association with infection during pregnancy or assessing the prevalence of the virus dna in blood products [62, 63] . the lightcycler parvovirus b19 quantitative assay is highly sensitive for genotype 1 but is not suitable for detecting genotypes 2 or 3. with respect to realart parvo b19 lc pcr (qiagen, hamburg), this assay can detect all three genotypes according some researchers, but no by others [64] . appropriate clinical specimens for nucleic acid analysis include plasma, serum, bone marrow, placental and fetal tissues and amniotic fluid. both viruses were recovered in cell cultures in 1971. bk virus is associated with nephropathy, above all in renal transplant patients as well as in patients with ureteral stenosis and hematuria [65] . on the other hand, it is well knowing the association of jc virus infection with progressive multifocal leukoencephalopathy in immunocompromised patients (such as those with aids) [66] . conventional pcr for detection of jc virus in the cerebrospinal fluid has replaced the brain biopsy for the diagnosis of presence of this virus in patients with leukoencephalopathy [67] . this technique had sensitivity that range 70-90% and a specificity that range 90-100% before the highly active antiretroviral therapy [68] . however, at the moment, the application of this therapy recovery the immune system, so the viral replication becomes to be decreased, being jc virus pcr negative in csf. currently, it has been developed a qualitative real-time pcr for jc virus detection in csf. bk virus can be detected in urine samples by conventional pcr; also, a quantitative real-time pcr technique has been developed to monitoring bk virus dna in renal transplant recipients [69] . active bk virus nephropathy is associated with high quantitative levels of bk virus dna, and resolution of nephropathy was correlated with decreased dna virus level in urine. however, although it is a sensitive test, the presence of bk virus dna in this kind of samples does not necessarily means a true infection because there is asymptomatic reactived infection in 10-45% of renal transplant patients. therefore, the result should be confirmed using blood samples [70] . in this sense, research has demonstrated that renal transplant recipients with higher urine dna levels are more likely to show detectable dna in blood. on the other hand, a negative result does mean no association of bk virus with nephritis. finally, pcr from kidney biopsy specimens is not an appropriate test for primary diagnosis of bk nephropathy, because persistent but low-level target dna in biopsy specimens can be detected in asymptomatic patients [71] . rapid laboratory diagnosis is critical for infection control, so several diagnostic tests have been developed and are available for the detection of influenza viruses. rapid influenza diagnostic tests are less sensitive and specific than fluorescent antibody assays and rt-pcr [72] . rt-pcr is the preferred diagnostic assay for influenza virus. these tests are the most sensitive and specific and can differentiate between influenza types (a or b) and subtypes [72] . the main problem of this technique is that it could not be available in all laboratories, so there is a need of other tests in these settings. real-time pcr is much more sensitive than other methods of detection and is available for detecting influenza virus [73] but is more expensive. with respect to parainfluenza viruses, types 1 to 4 have been associated with bronchiolitis, croup and pneumonia in children, but also in elderly and immunocompromised patients. pcr is an adequate technique for the detection of parainfluenza virus, above all in immunocompromised patients [74] . pcr has a sensitivity of 100% and specificity that range 95-98% if compared with culture method [75] . multiplex pcr assays can differentiate between a wide variety of respiratory pathogens [75] . also, a rapid and sensitive multiplex real-time pcr assay for detection of four serotypes of parainfluenza viruses has been developed [76] . for more information, it can see the article entitled "laboratory detection of respiratory viruses by automated techniques". pcr is a specific and sensitive assay for detecting adenovirus dna from a wide variety of clinical specimens, but results must be always interpreted in the context of the clinical findings of adenovirus disease. quantitative realtime pcr is being now used for the evaluation of adenovirus infections in immunocompromised patients [77] . moreover, quantification of adenovirus dna could be useful for assessing response to antiviral therapy [78] . real-time pcr assays mainly are used to detect adenovirus type 4 (subgroup e) that can cause respiratory, ocular and other infections. the sensitivity of real time pcr against conventional pcr is greater for the detection of adenovirus dna [79] . enteroviruses such as echoviruses, parechoviruses (echoviruses 22 and 23) and coxsackieviruses a and b could produce several diseases such as respiratory tract infections, aseptic meningitis, myocarditis and neonatal systemic enteroviral disease. cell culture has a low sensitivity, so molecular techniques like rt-pcr have been developed for the diagnosis of this kind of viruses [80] . moreover, rt-pcr has higher sensitivity than cell culture for detecting enteroviruses in the csf [81] . a rapid and sensitive detection of enterovirus in csf could be performed by the introduction of real-time pcr techniques in the laboratory. real-time pcr assays amplify conserved target nucleic acid sequences of the virus. sensitivity for detecting enterovirus is similar between conventional pcr and real-time pcr but the last one is less labor intensive and easier to implement in the clinical laboratory [82] . molecular techniques can be very useful for the diagnosis of viral hepatitis infections such as hepatitis a, b, c, d and e in cases in which serological assays are no conclusive. however, currently the main application of nucleic acid test in the management of these viruses, above all hepatitis b and c, is the detection of serum or plasma viral load of these viruses for monitoring therapeutic responses of infected patients. assays to quantify hepatitis b and c virus load in liver tissue have also been described [83, 84] . hbv dna detection and hbv dna viral load are also essential to explore a viral reactivation. real-time pcr quantification assays are widely used because of their sensitivity, specificity, accuracy, broad dynamic range and positive predictive values. the world health organization (who) has defined international standards [85] . however, because of these techniques do not use the same hbv primers, follow up of patients should be carried out with the same technical assay in order to compare viral dna load evolution. with respect to the hbv genotypes, there is currently a need to perform it due to several factors such as the prediction of clinical outcomes and the association with response to interferon treatment [86] . genotyping of chronic hbv infections can help practicing physicians identify those at risk of disease progression and determine optimal antiviral therapy. methods to determine the viral genotypes are based on hybridization and sequencing, and genotype affiliation rely on phylogenetic analyses. with respect to hcv, molecular diagnostic assays represent an essential approach in the management of hcv patients. qualitative and quantitative hcv molecular assays are used for the diagnosis of acute and chronic hcv infections, viral genotyping, viral-load determination, treatment, monitoring and prognosis. rt-pcr, transcriptionmediated amplification and branched dna amplification are commonly employed for detection of hcv rna. recently, new hcv molecular assays that employ nanostructures have emerged and have been proposed as suitable, without loss specificity and sensitivity [87] . for more information, it can see the article entitled "introduction of automated systems for the diagnosis and quantification of hepatitis b and hepatitis c viruses". hiv-1 and hiv-2 rna levels in the plasma of infected patients could be detected by quantitative rapid real-time pcr assays. there are some techniques for this detection that have similar sensitivity and specificity, but differ in the probe and in the amplification-detection systems used. quantitative assays for measurement of hiv-1 and hiv-2 proviral dna have also been developed, as well as qualitative real-time pcr assay for detection of proviral dna. for more information, it can see the article entitled "improving clinical laboratory efficiency: introduction of systems for the diagnosis and monitoring of hiv infection". molecular methods to detect hpv dna in clinical specimens have been introduced in clinical practice, and the majority of protocols for detection of this infection are currently based in the study of hpv dna. hpv dna detection could be carried out by several methods such as type-specific pcr, pcr with general primers and liquid hybridization (hybrid capture). several studies have shown the utility of hpv dna testing for management of women with abnormal pap smears [88] . identifying women at high risk by testing for hpv dna could avoid unnecessary colposcopy procedures. for more information, it can see the article entitled "human papillomavirus (hpv) genotyping: automation and application in routine laboratory testing". the main goal of a clinical virology laboratory is to assist clinicians in the diagnosis and treatment of viral diseases, and to support infection control specialists in their tasks. there is a need for a rapid identification of the etiological microorganisms for an effective patient management. since molecular techniques for detecting nucleic acids had been developed, the diagnosis of several viral diseases has been revolutionized in the clinical laboratories. in the majority of them, it has been introduced these methods for routine diagnosis, but some of those are being used only in reference settings. the main advantages of molecular techniques are its higher sensitivity and specificity compared with other diagnostic methods such as serological assays and culture methods, as well as its rapidity and possibility of automation. from an epidemiological and clinical point of view, these features are very important for the diagnosis of some diseases such as cns infections, in which the detection of microorganisms should be faster in order to treat rapidly the patients and isolate them to prevent viral transmission of disease. among this fact, the automation also permits the performance of much more assays and its rapidity helping to improve the patient diagnosis. virology laboratories for clinical diagnosis should introduce some of these techniques in order to determine the main viruses implicated in human diseases, after to do an analysis of cost. laboratory director and technical coordinator should establish the workflow for these techniques enhancing the efficiency of the testing methods. this workflow should be done in an individualized way, taking into account the assays introduced and the special characteristics of the laboratory. moreover, the personnel should be trained according the best practices for this methodology at this time. the author declares that they have no competing interests. diagnostic molecular microbiology review rapid enterovirus molecular testing in cerebrospinal fluid reduces length of hospitalization and duration of antibiotic therapy in children with aseptic meningitis nucleic acid amplification and related techniques in microbiological diagnosis and epidemiology molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time pcr assay typing of human adenoviruses in specimens from immunosupressed patients by pcr-fragment length analysis and real-time quantitative pcr monitoring of cytomegalovirus reactivation in bone marrow tranplant recipients by real-time pcr comparison of commercial and in-house real-time pcr assays for quantification of epstein-barr virus (ebv) dna in plasma cytomegalovirus quantification: where to next in optimising patient management? assay formats involving acridinium ester-labeled dna probes primer-directed enzymatic amplification of dna with a thermostable dna polymerase branched dna signal amplification for direct quantitation of nucleic acid sequences in clinical specimens comparison of the hybrid capture tube test and pcr for detection of human papillomavirus dna in cervical specimens multicenter comparison of the digene hybrid capture cmv dna assay (version 2.0), the pp65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia reverse transcription and dna amplification by a thermus thermophilus dna polymerase specific amplification with pcr of a refractory segment of genomic dna deletion screening of the duchenne muscular dystrophy locus via multiplex dna amplification dna microarrays for virus detection in cases of central nervous system infection rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3 and 4 kinetic pcr analysis: realtime monitoring of dna amplification reactions nucleic acid sequence-based amplification transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format strand displacement amplification and homogeneous real-time detection incorporated in a second-generation dna probe system, bdprobe tecet the ligation amplification reaction (lar) -amplification of specific dna sequences using sequential rounds of template-dependent ligation identification of oseltamivir resistance among pandemic and seasonal influenza a (h1n1) viruses by a his 275tyr genotyping assay using the cycling probe method polymorphism identification and quantitative detection from genomic dna by invasive cleavage of oligonucleotide probes diagnostic nucleic acid microarrays; proposed guideline. clsi document mmp12-p extensive polymorphisms observed in hiv-1 clade b protease gene using high-density oligonucleotide arrays oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay changes of ecm and cam gene expression profile in the cirrhotic liver after hcv infection: analysis by cdna expression array rapid virological diagnosis of central nervous system infectionsby use of a multiplex reverse transcription-pcr dna microarray applications of luminex ® xmap tm technology for rapid, high-throughput multiplexed nucleic acid detection a rapid, sensitive, multiplexed assay for detection of viral nucleic acids using the flowmetrix system alternative molecular tests for virological diagnosis loop-mediated isothermal amplification of dna rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplificaton assay development and evaluation of a novel loop mediated isothermal amplification (lamp) method for rapid detection of sars corona virus rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation rapid detection of varicellazoster virus infection by a loop-mediated isothermal amplification method rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method development of the loopmediated isothermal amplification method for rapid detection of cytomegalovirus dna development of a loopmediated isothermal amplification assay for rapid detection of bk virus loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16 and 18 nucleic acid assay system for tier ii labs and moderately complex clinics to detect hiv in low-resource settings a rapid and single isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2 diagnosis of cytomegalovirus (cmv) polyradiculopathy and documentation of in vivo anti-cmv activity in cerebrospinal fluid by using branched dna signal amplification and antigen assays detection of herpesvirus genomes by polymerase chain reaction in cerebrospinal fluid and clinical findings diagnosis of herpesvirus infections of the central nervous system diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease quantitation of herpes simplex virus type 1 dna in cells of cerebrospinal fluid of patients with herpes simplex virus encephalitis automated detection of five human herpes virus dnas by a set of light cycler pcrs complemented with a single multiple internal control development, implementation, and trend analysis of real-time pcr tests for the clinical microbiology laboratory routine use of a highly automated and internally controlled real-time pcr assay for the diagnosis of herpes simplex and varicella-zoster virus infections detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. comparison of real-time pcr, nested pcr and virus isolation a real-time pcr assay to identify and discriminate among wild-type and vaccine strains of varicella-zoster virus and herpes simplex virus in clinical specimens, and comparison with the clinical diagnoses evaluation of laboratory methods for diagnosis of varicella molecular diagnosis of visceral herpes zoster diagnosing central nervous system lymphoma in the setting of aids: a step forward detection of lymphotropic herpesvirus dna by polymerase chain reaction in cerebrospinal fluid of aids patients with neurological disease posttransplant primary cns lymphoma epstein-barr virus dna load in cerebrospinal fluid and plasma of patients with aids-related lymphoma high-titer screening pcr: a successful strategy for reducing the parvovirus b19 load in plasma pools for fractionation new lightcycler pcr for rapid and sensitive quantification of parvovirus b19 dna guides therapeutic decision-making in relapsing infections parvovirus b19 infection in pregnancy: quantitative viral dna analysis using a kinetic fluorescence detection system (taqman pcr) detection and diferentiation of human parvovirus variants by commercial quantitative real-time pcr tests bk virus and sv40 co-infection in polyomavirus nephropathy a prospective study demonstrates an association between jc virus specific cytotoxic t lymphocytes and the early control of progressive multifocal leukoencephalopathy detection of jc virus dna by polymerase chain reaction in patients with progressive multifocal leukoencephalopathy diagnosis of central nervous system complications in hiv-infected patients: cerebrospinal fluid analysis by the polymerase chain reaction correlates of quantitative measurement of bk polyomavirus (bkv) dna with clinical course of bkv infection in renal transplant patients monitoring for polyomavirus bk and jc in urine: comparison of quantitative polymerase chain reaction with urine cytology polyomavirus dna and rna detection in renal allograft biopsies: results from a european multicenter study evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak multiplex realtime pcr assay for detection of influenza and human respiratory syncytial viruses detection of respiratory viruses by molecular methods comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3 and 4 internally controlled real-time pcr monitoring of adenovirus dna load in serum or plasma of transplant recipients quantification of adenovirus dna in plasma for management of infection in stem cell graft recipients pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr laboratory diagnosis of enteroviral infections evaluation of a commercially available reverse transcription-pcr assay for diagnosis of enteroviral infection in archival and prospectively collected cerebrospinal fluid specimens rapid detection of enterovirus infection by automated rna extraction and real-time fluorescence pcr quantification of hepatitis c virus in human liver and serum samples by using lightcycler reverse transcriptase pcr real-time quantitation of hepatitis b virus (hbv) dna in tumorous and surrounding tissue from patients with hepatocellular carcinoma an international collaborative study to establish a world health organization international standard for hepatitis b virus dna nucleic acid amplification techniques the clinical implications of hepatitis b virus genotype: recent advances hepatitis c virus rna assays: current and emerging technologies and their clinical applications identifying women with cervical neoplasia: using human papillomavirus dna testing for equivocal papanicolaou results declared none. key: cord-001848-idmj2d7p authors: onabajo, olusegun o.; porter-gill, patricia; paquin, ashley; rao, nina; liu, luyang; tang, wei; brand, nathan; prokunina-olsson, ludmila title: expression of interferon lambda 4 is associated with reduced proliferation and increased cell death in human hepatic cells date: 2015-11-01 journal: j interferon cytokine res doi: 10.1089/jir.2014.0161 sha: doc_id: 1848 cord_uid: idmj2d7p interferon lambda 4 (ifn-λ4) is a novel type-iii interferon that can be generated only in individuals carrying a δg frame-shift allele of an exonic genetic variant (rs368234815-δg/tt). the rs368234815-δg allele is strongly associated with decreased clearance of hepatitis c virus (hcv) infection. here, we further explored the biological function of ifn-λ4 expressed in human hepatic cells—a hepatoma cell line hepg2 and fresh primary human hepatocytes (phhs). we performed live confocal imaging, cell death and proliferation assays, mrna expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. not only did we observe significant intracellular retention of ifn-λ4 but also detected secreted ifn-λ4 in the culture media of expressing cells. secreted ifn-λ4 induced strong activation of the interferon-stimulated genes (isgs) in ifn-λ4-expressing and surrounding cells in transwell assays. specifically, in phhs, secreted ifn-λ4 induced expression of the cxcl10 transcript and a corresponding pro-inflammatory chemokine, ip-10. in ifn-λ4-expressing hepg2 cells, we also observed decreased proliferation and increased cell death. all ifn-λ4-induced phenotypes—activation of isgs, decreased proliferation, and increased cell death—could be inhibited by an anti-ifn-λ4-specific antibody. our study offers new insights into biology of ifn-λ4 and its possible role in hcv clearance. w ith more than 170 million infected individuals, hepatitis c virus (hcv) infection represents a significant healthcare burden worldwide (mohd hanafiah and others 2013) . hcv infection is treated with interferon (ifn)-a-based regimens and recently approved ifn-a-free direct-acting antiviral agents (daa) (liang and ghany 2013) . genome-wide association studies identified a single nucleotide polymorphism rs12979860, located upstream of the ifnl3 (il28b) gene and thus initially referred to as the ''il28b marker,'' as one of several genetic variants strongly predictive of hcv clearance (ge and others 2009; thomas and others 2009) . further studies showed that rs12979860 is located in the intronic region of a novel gene, ifnl4, and is in high linkage disequilibrium (ld) with an ifnl4 exonic frameshift polymorphism rs368234815-tt/dg, initially designated as ss469415590 (prokunina-olsson and others 2013). the rs368234815-dg allele, which creates an open reading frame for a novel human interferon, interferon lambda 4 (ifn-l4), is associated with decreased hcv clearance (prokunina-olsson and others 2013) [reviewed in o'brien and others (2014) ]. the rs368234815-dg has allele frequency of *70% in individuals of african ancestry, *30% in europeans, while only 0%-5% in asians (prokunina-olsson and others 2013). in individuals of african ancestry, rs368234815 is more predictive of hcv clearance than rs12979860 (prokunina-olsson and others 2013; aka and others 2014); while in europeans and asians, these markers are in high ld and thus provide comparable predictive information (prokunina-olsson and others 2013). a genetic polymorphism rs117648444-c/t, which introduces an amino-acid substitution p70s in the ifn-l4 protein (prokunina-olsson and others 2013), is associated with reduced biological activity of ifn-l4 and increased hcv clearance (terczynska-dyla and others 2014), thereby supporting the critical role of ifn-l4 in this process. recent clinical trials showed that ifnl4 variants rs368234815 and rs12979860 are predictive of treatment efficacy even for daa therapies (fujino and others 2013; meissner and others 2014a; o'brien and pfeiffer 2015) and these markers, possibly together with p70s, could be used to optimize treatment regimens and duration in resource-limited settings. the functional importance of ifn-l4 is evidenced by the strong positive selection that favored elimination of ifn-l4 from human populations (key and others 2014) . although this selection cannot be explained by any known viral infection, it may reflect antiviral response to some extinct highly deadly infection. previously, we showed that transient transfection of an expression construct that generates ifn-l4 protein induced interferon signaling, with activation of interferon-stimulated genes (isgs) and generation of antiviral response in hepg2, a human hepatoma cell line (prokunina-olsson and others 2013). however, the function of ifn-l4 and its role in impaired hcv clearance remained unclear. here, we further explored this question by performing additional functional analyses of ifn-l4 transiently and stably overexpressed in human hepatic cells-fresh primary hepatocytes and hepg2 cells. the human hepatoma cell line hepg2 (atcc hb-8065) was purchased from the american tissue culture collection (atcc) and maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum (fbs). the custom isre-luc-hepg2 cell line stably expressing a luciferase reporter under control of the interferon-stimulated response element (isre) was previously described (prokunina-olsson and others 2013); the cells were maintained in dmem supplemented with 10% fbs and 2 mg/ml puromycin. fresh primary human hepatocytes (phhs) were purchased from bioreclamation ivt. the cells were received in suspension within 6 h after isolation and were maintained in in-vitrogro hi culture media with torpedo antibiotic mix. the liver donor 1 was a 55-year-old woman who died of cardiac arrest, and the liver donor 2 was a 40-year-old man who died of anoxia. both were hcv-free caucasians. on arrival, phhs had 88% and 90% viability for donor 1 and 2, respectively. dna extracted from the phhs was genotyped with a custom taqman assay (prokunina-olsson and others 2013) for the exonic ifnl4 genetic variant rs368234815 (dg/tt). both donors were homozygous for the rs368234815-tt allele, which is associated with higher hcv clearance (prokunina-olsson and others 2013). the rs368234815-tt allele introduces a frame shift within the first exon of ifnl4 gene and eliminates the ifn-l4 protein. thus, no ifn-l4 protein could be endogenously produced in these phh samples. the expression constructs (ifnl4-halo, p131-halo, and control-halo) based on the pfc14a vector (promega) were previously described (prokunina-olsson and others 2013). the control-halo construct generates only a halo-tag protein (33 kda), the ifnl4-halo construct generates the ifn-l4-halo protein (53 kda) consisting of the ifn-l4 protein (20 kda) c-terminally fused with the halo-tag protein (33 kda), and the p131-halo generates a protein of 47 kda, consisting of the halo-tag c-terminally fused with a splicing protein isoform of ifn-l4 (131 aa, 14 kda, designated as p131), which lacks exon 3 and is nonfunctional in induction of interferon response (prokunina-olsson and others 2013). all constructs have been validated by western blotting with an a-halo antibody (promega) and/or an a-ifn-l4 antibody (mabf227; emd millipore). the ifnl3-halo construct generating the secreted ifn-l3-halo protein was created and validated the same way. hepg2 cells and phhs were transfected for 24 or 48 h with corresponding constructs; hepg2 cells were transfected using lipofectamine/ltx and opti-mem with standard protocols (life technologies), while phhs were transfected using a nucleofection protocol optimized for primary cells (lonza). briefly, for each transfection, 2 · 10 6 fresh phhs were resuspended in 100 ml of optimized nucleofection buffer l3 and transfected with 5 mg of corresponding constructs using 4d-nucleofector (lonza). immediately after transfection, dead cells were separated by centrifugation (4 min, 570 rpm) of the transfection mix overlaid with 900 ml of ficoll (ge healthcare) and 750 ml of supernatant with dead cells was removed. the remaining cells were resuspended in invitro-gro cp media with torpedo antibiotic mix (bioreclamation ivt), plated onto collagen-coated plates (bd biosciences), and incubated overnight. unattached and dead cells were removed 12 h post-transfection by replacing the transfection media with invitrogro hi culture media with torpedo antibiotic mix (bioreclamation ivt). tetracycline-inducible hepg2 cell lines for ifn-l4-gfp, p131-gfp, and control-gfp were generated using the tet-on 3g expression system (clontech). first, a stable hepg2 cell line expressing the tet-on 3g transactivator was generated by lentiviral transduction and blasticidin selection (5 mg/ml). corresponding cdnas were cloned into a ptreg3g plasmid (clontech) to generate tetracyclineinducible ifn-l4-gfp, p131-gfp, and control-gfp constructs, which were transfected into the stable tet-on 3g-hepg2 using lipofectamine/ltx. positive clones were identified by limiting dilution under neomycin selection (1 mg/ml) over 3 weeks. expression of corresponding proteins was induced by treatment with 1 mg/ml doxycycline for indicated experiment-specific time periods. hepg2 cells were transfected with corresponding constructs in 4-well coverslip chambers (4 · 10 4 cells/well; labtek). after 24 h, transfection media was replaced with live cell imaging solution (life technologies), supplemented with 20 mm glucose. cell-permeant halo-tag ligands (tmr red or oregon green; promega) were added to live cells (1:2,000 for 15 min), and live imaging was performed using a lsm700 confocal laser scanning microscope (carl zeiss) fitted with a temperature and co 2 -controlled chamber. when applicable, nuclei were visualized using fluorescent hoechst 33342 dye (life technologies). for cell death analysis, live cells were imaged using inverted oil lens at 40· magnification, with 6 s scanning per minute per field of view for 18 h, generating 1,080 images per field; 6 fields were imaged in sequence using stage-positioning software. each field of view had at least 10 transfected cells. signs of cell death were scored visually by counting membrane rupture events in the videos representing the 18-h imaging periods. cell death rates were determined as the percentages of ruptured cells of the total counts of fluorescent cells in each field of view. hepg2 cells were transfected with corresponding constructs and imaged live as described earlier using oregon green halo-tag ligand. the mobility of the ifn-l4-halo and control-halo proteins was evaluated with fluorescence recovery after photobleaching (frap) method (snapp and others 2003) . briefly, after 10 prebleach scans, a selected region was bleached at 100% of imaging power with 3 iterations and 2 ms per iteration, with 1.94 s per scan. imaging was continued for a total of 150 s. fluorescence intensity was plotted with curve fitting to evaluate fluorescence recovery in the bleached areas over time. half-time fluorescence recovery (t½) and the ratio of immobile fraction were averaged for 7 individually scanned cells for each construct (ifnl4-halo and control-halo). recovery data were normalized to unbleached regions inside the cells and adjusted for background fluorescence; data acquisition and analyses were performed using lsm700 frap software. the stable hepg2-isre-luc cells were seeded in 96-well plates that were either untransfected or transfected with corresponding constructs. after 24 h, the cells were treated for 2 h with the following antibodies: 20 mg/ml of a blocking a-il10r2 antibody (r&d systems), 40 mg/ml of a goat isotype igg control (abcam), a range (20, 10, 5, 2.5, or 1.25 mg/ml) of a custom rabbit monoclonal a-ifn-l4 antibody (prokunina-olsson and others 2013), or 40 mg/ml of a rabbit isotype igg control (cell signaling). recombinant human interferons-10 ng/ml of custom ifn-l3 (prokunina-olsson and others 2013), ifn-a and ifn-b (pbl assay science), or ifn-g (r&d systems) were added to the untransfected cells pretreated with the antibodies. negative controls included nontreated cells, phosphate-buffered saline (pbs) in media, and mock transfection with control-halo constructs. the cells were assayed for luciferase expression of the isre-luc reporter 48 h post-transfection. all transfections and treatments were done in 8 biological replicates. for other experiments, inducible stable hepg2 cells were seeded into 96-well plates and protein expression was induced for 12 h. antibodies (20 mg/ml of rabbit monoclonal a-ifn-l4 or 40 mg/ml of rabbit igg control) were added to shared culture media for 48 h. hepg2 and phhs were transfected with corresponding constructs and seeded onto 6-well plates. separately, transwell inserts with 0.4 mm pores (corning) were seeded with untransfected cells and placed into 6-well plates with culture media, with 1 insert per well. for phhs, both the plates and transwell inserts were collagen coated (corning). after 24 h, media was replaced and transwell inserts with growing cells were added to the plates containing transfected cells. the pore size of transwell inserts allowed free circulation of the media but not of the cells. for phhs from donor 2, the transwell experiment was expanded to include antibody treatment. phhs transfected with ifnl4-halo, ifnl3-halo, and control-halo constructs were incubated with transwell inserts seeded with untransfected phhs. antibodies (20 mg/ml of rabbit monoclonal a-ifn-l4 or 40 mg/ml of rabbit igg control) were added to shared culture media for 24 h. after 24 h of coincubation, cells from both chambers that shared culture media (transfected vs. untransfected transwell cells) were collected separately for rna extraction and stored in rlt buffer; shared culture media was collected for protein analysis and stored at -80°c until use. for transient transfections, hepg2 cells were transfected with corresponding constructs for 48 h and media was changed after overnight incubation. expression of halo-tag in live cells was detected using cell-permeant halo-tag tmr red ligand. for stable hepg2 cells, expression of proteins was induced for 72 h. in both systems, dead cells were identified using near-ir fixable live/dead marker (life technologies). for analysis of apoptosis, cells were additionally stained for annexin v (bd biosciences). for proliferation assays, the cells that were induced for 72 h were treated with 10 mm 5-bromo-2¢-deoxyuridine (brdu) for 3 h. dead cells were excluded by removing nonadherent cells, and the remaining cells were stained using the apc brdu flow kit (bd biosciences). multiparametric flow cytometry analysis was performed on facs aria iii (bd biosciences) with flowjo.10 software (tree star). for viability assays, the protein expression was induced for 5 days in 96-well plates and viability was evaluated with the multi tox-glo assay (promega). in some experiments, viability assays were performed after protein induction for 72 h, with similar results. quantitative reverse-transcriptase-polymerase chain reaction expression analysis total rna was isolated using an rneasy kit with oncolumn dnase i treatment (qiagen). rna quantity and quality were evaluated by nanodrop 8000 (thermo scientific). cdna was prepared from 40 to 100 ng of total rna with the rt 2 first-strand cdna kit and random hexamers with an additional dna-removal step (qiagen). quantitative reverse-transcriptase-polymerase chain reaction (qrt-pcr) mrna expression analysis was performed using sybr green antiviral response qrt-pcr plates (qiagen). the plates included 88 expression assays for target genes, as well as positive, negative, and endogenous normalization controls (supplementary tables s1-s3; supplementary data are available online at www.liebertpub.com/jir). a custom expression assay for ifnl3 (prokunina-olsson and others 2013) and predesigned taqman expression assays for ifnl1 (hs00601677_g1) and ifng (hs00989291_m1; life technologies) were not included on the predesigned plates and were used separately (supplementary table s4 ). the qrt-pcr reactions (5 or 10 ml) included expression master mixes (qiagen or life technologies), cdna, and corresponding expression assays; the quantification was performed in 2-4 technical replicates on the quantsudio 7 instrument (life technologies). expression was measured in c t values (pcr cycle at detection threshold), which are distributed on log 2 scale. expression was normalized by the geometric means of 5 endogenous controls (actb, b2m, gapdh, hprt1, rplp0) included on the qrt-pcr plates or of 2 endogenous controls used separately (assay 4326317e for gapdh, and assay 4326315e for actb; life technologies). differences in expression were calculated according to the relative quantification method, as dc t = c t (control) -c t (target). fold differences between expression of any 2 samples or groups of samples were calculated as 2 (dct1 -dct2) . heat-map analysis of auto-scaled expression data for a panel of selected isgs was performed with the genex software (multid). protein levels of ip-10 (encoded by cxcl10) in culture media from hepg2 and phhs transfected with corresponding constructs were measured with an elisa kit (r&d systems), which has a protein detection range of 7.8-500 pg/ml. culture media was collected 24 h after transfection media change (48 h post-transfection). media samples were diluted 1:10 or 1:20, and each sample was measured in technical triplicates. a standard curve was generated using the protein provided with the kit, with a correlation coefficient >0.98. the plates were analyzed using glomax luminometer (promega). ifn-l4 was detected with a custom-developed electrochemical elisa on the meso quickplex sq120 instrument [mesoscale discovery (msd)]. briefly, standard capacity multi-array 96-well plates (msd) were coated overnight at 4°c with 30 ml of 2 mg/ml custom monoclonal rabbit a-ifn-l4 antibody. blocking was performed with bovine serum albumin (msd blocker a) for 1 h at room temperature with rotation at 400 rpm, followed by 3 washes with pbs and 0.05% tween. standards, controls, and experimental samples were prepared using diluent 2 (msd). culture media samples were diluted 1:10 and incubated at room temperature for 2 h with rotation at 400 rpm. after additional wash steps, 25 ml of the 4 mg/ml detection antibody [mouse monoclonal a-ifn-l4 antibody (buffer exchanged into pbs, mabf227; emd millipore)], conjugated with a sulfo-tag (msd) was added to the wells and incubated for 1 h at room temperature with rotation at 400 rpm. after additional washing, the plates were scanned in a 2· read buffer (msd) on the meso quickplex sq120 and the results were analyzed with the msd discovery workbench software. purified recombinant ifn-l4 protein (prokunina-olsson and others 2013) was used as a standard at 9 concentrations in the range of 46 pg/ml to 300 ng/ml, and the detection range for the standards was determined as 150 pg/ ml to 300 ng/ml. hepg2 cells were transfected for 48 h with 50 ng of corresponding constructs with/without cotransfection with 1.0 ml of the endoplasmic reticulum stress response element (erse)-luc cignal reporter (qiagen). transfections were done in a 96-well plate, with 8 replicates per transfection. media was changed 24 h post-transfection, and the plate was assayed for luciferase and renilla using the glomax luminometer. data were presented as relative luciferase units, which correspond to luciferase/renilla ratio. the stable hepg2-isre-luc cells were transfected with corresponding constructs in 96-well plates; untransfected cells were treated with human recombinant interferons-ifna (2 ng/ml; pbl assay science) or custom ifn-l3 (10 ng/ ml). all experiments were represented by 8 biological replicates. the media was replaced 24 h post-transfection by 100 ml of full culture media with 1 or 10 mm jak inhibitor [active against jak1, jak2, and jak3, #420097; emd millipore, in 0.1% dimethyl sulfoxide (dmso)]. for il10r2 blocking experiment, the transfection culture media was replaced by 100 ml of media with 20 mg/ml of an a-il10r2 blocking antibody (mab874; r&d systems) or 20 mg/ml of a goat isotype igg control (abcam). for treatment experiments, cells were pretreated for 1 h with the jak inhibitor or for 2 h with the a-il10r2 or igg control antibodies and then treated with ifn-l3 (10 ng/ml) or ifn-a (2 ng/ml). negative controls included untreated cells, cells treated with 0.1% dmso in media, and cells transfected with control-halo construct. the cells were assayed for isre-luc reporter 48 h post-transfection, which corresponds to 24 h posttreatment with jak inhibitor and antibodies. sirna silencing of ifnlr1 in hepg2-isre-luc cells the stable hepg2-isre-luc cells were transfected with corresponding constructs in 2 identical 96-well plates. the cells were co-transfected with 1 pmol/well of a scrambled negative control sirna (am4615; ambion) or a set of sirnas against ifnlr1 (m-007981-00; thermo scientific). after 24 h, the cells were treated with culture media, ifn-l3 (10 ng/ml), or ifn-a (2 ng/ml). after 48 h, the first plate was assayed for the isre-luc reporter on the glomax luminometer. the second plate was used for rna extraction (zymo research) and mrna expression analysis. cdna was synthesized using super script iii reverse transcriptase (life technologies). specific taqman assays for ifnlr1 (hs00417120_m1) and endogenous control actb (assay 4326315e) from life technologies were used for qrt-pcr analysis on the quantstudio 7 instrument (life technologies). expression of ifnlr1 was normalized to expression of actb and then expression in samples treated with ifnlr1 sirna was compared with untreated samples (no sirna) or treated with scrambled sirna. all samples were represented by 6 biological replicates. compared with sirna-untreated samples (100%), the expression of ifnlr1 was decreased to 76% in si-scr samples and to 42% in si-ifnlr1 samples ( supplementary fig. s1d ). unless specified, data plotting and statistical analyses were performed with prism 6 (graphpad), p values are for 2-sided unpaired t-tests. shown are means and standard errors of the mean. previously, by performing western blot analysis, we were unable to detect ifn-l4 in culture media of hepg2 cells transiently transfected with an ifn-l4-producing construct, even though this transfection resulted in strong activation of interferon signaling (prokunina-olsson and others 2013). however, ifn-l4 was detectable at low levels in culture media of transfected cells by western blot analysis after acetone precipitation (hamming and others 2013) . now, we developed a mesoscale assay (an electrochemical elisa) and were able to detect ifn-l4 in culture media of transfected hepg2 cells (fig. 1a) . treatment of these cells with a custom a-ifn-l4 antibody decreased the interferon signaling by 70% (fig. 1b) , suggesting that ifn-l4 generated in this experimental system is a secreted biologically active interferon. simultaneously, the a-ifn-l4 antibody did not affect signaling of the main interferons (ifn-a, ifn-b, ifng, and ifn-l3, fig. 1c ). signaling induced by ifn-l4 was strongly attenuated by the jak inhibitor and by blocking of the ifn-l family receptors (ifnlr1 and il10r2, supplementary fig. s1a -c), confirming these canonical components of the jak/stat pathway as essential elements for the ifn-l4 signaling. we evaluated the biological activity of the secreted ifn-l4 by its ability to induce expression of a set of isgs (supplementary tables s1-s3). we analyzed mrna ex-pression in cells transiently transfected with ifnl4-halo or control-halo constructs and in bystander nontransfected cells exposed to media from the corresponding transfected cells in transwell assays (ifnl4-trans and halo-trans, fig. 2a ). in both hepg2 and phhs, cells transfected with ifnl4-halo or exposed to media from those cells (containing secreted ifn-l4) showed strong induction of isgs, such as ddx58 (rig-i), dhx58, ifih1 (mda5), isg15, mx1, oas2, and stat1 and chemokines cxcl10 and cxcl11 ( fig. 2a) . expression of cxcl10 and cxcl11 was much higher in phhs compared with hepg2, highlighting cell-specific differences ( fig. 2a) . ifn-l4 was detectable in culture media of ifnl4-transfected phhs and hepg2 cells, but not in corresponding halo-transfected cells (fig. 2b) . we did not detect expression of other interferons (ifna1, ifna2, ifnb, ifng, ifnl1, ifnl3) in any of the experimental conditions (supplementary table s4 ). cxcl10 encodes ifn-g inducible protein 10 (ip-10), which is a chemotactic factor for neutrophils. high levels of ip-10 have been associated with inflammation and pathogenesis of chronic hcv infection (harvey and others 2003; lagging and others 2006) . we measured the levels of ip-10 in culture media of phhs and hepg2 transfected with different constructs. in phhs, ip-10 was detectable in the media from samples transfected with ifnl4-halo and ifnl3-halo (fig. 3b) , while it was undetectable in hepg2 cells transfected with ifnl4-halo (data not shown), in accordance with a much lower cxcl10 mrna expression observed in hepg2 compared with phhs (supplementary tables s1-s3) . importantly, the a-ifn-l4 antibody added to the shared media strongly decreased mrna expression of isgs, including cxcl10, in nonexpressing transwell phhs (fig. 3a ) and the amount of ip-10 secreted to the shared media (fig. 3a, b) . even though some of ifn-l4 gets secreted, as described earlier, ifn-l4 was also detected as a cytoplasmic protein by confocal imaging in fixed cells-in hepg2 cells transiently expressing ifn-l4-halo, and in phhs induced to express endogenous ifn-l4 (prokunina-olsson and others 2013). we next performed live imaging of intracellular movements of ifn-l4-halo and control-halo proteins (supplementary video s1). we also monitored frap (fig. 4a ). this method does not detect secreted proteins such as ifn-l3, but it helps characterize intracellular proteins based on their mobility (fig. 4b) , which is proportional to the speed of fluorescence recovery (t½, in seconds). the control-halo protein showed very high mobility, with a t½ of 4.46-1.19 s (fig. 4c and supplementary video s2), while ifn-l4-halo showed a much lower mobility, with a t½ of 34.69 -6.94 s (fig. 4c and supplementary video s3). intracellular mobility of proteins could be limited by their attachment to structural elements, involvement in proteinprotein interactions, or confinement within vesicles (lippincott-schwartz and others 2001; snapp and others 2003). therefore, we also estimated the fraction of immobile expression of mrna transcripts was quantified using an antiviral response qrt-pcr plate. the cells were transfected with corresponding constructs for 48 h, and transwell cells shared culture media with the corresponding transfected cells for 24 h. all samples were represented by 2 biological replicates. expression of each target assay on the plate was measured using the same amount of cdna; expression was normalized to a geometric mean of 5 endogenous controls included on the plate. the results are presented as dc t values for targets normalized by endogenous controls, on a log 2 scale; less negative dc t values correspond to higher levels of expression. full expression data for hepg2 cells and phh are available as supplementary tables s1 and s2. protein, which is represented by unrecoverable fluorescence-nearly 30% of ifn-l4-halo (28.43% -4.22%) compared with *10% of control-halo protein (9.58% -2.43%) was estimated to be immobile (fig. 4d) . we conclude that in our experimental system the control-halo was mostly present as a mobile unattached cytoplasmic protein, in agreement with other studies that used halo-protein (huybrechts and others 2009) . in contrast, we detected ifn-l4-halo both as an intracellularly retained protein with limited mobility and as a mobile protein potentially available for secretion or release. while conducting live confocal imaging in transiently transfected hepg2 cells, we observed dying cells that underwent morphologic changes of swelling, membrane blebbing, and rupture, followed by release of cytoplasmic content (fig. 5a, b and supplementary videos s4-s6), suggesting necrotic-type cell death (fiers and others 1999; festjens and others 2006; galluzzi and others 2012) . cell rupture events over the course of 18 h of imaging were significantly more frequent in ifn-l4 + compared with control-halo + hepg2 cells (fig. 5c) . flow cytometry analysis showed that necrotic cells (defined as annexin v + and live/dead + ) were significantly more common in cells transfected with ifnl4-halo compared with p131-halo (a nonfunctional protein isoform of ifn-l4), ifnl3-halo, and control-halo constructs (fig. 5d , e). transfection efficiency was comparable for all these constructs (fig. 5g , but could not be determined for the secreted ifnl3-halo), and thus could not explain the observed differences in cell death. we also evaluated cell death in phhs transiently transfected with ifnl4-halo or control-halo constructs. in general, in phhs, cell death rates were very high even in untransfected cells and similar for both constructs (data not shown), possibly due to physiologically high baseline cell death rates in primary cells unrelated to the effect of ifn-l4. cell death can also be triggered as a result of an endoplasmic reticulum response to unfolded recombinant proteins generated in vitro. previously, we showed that transient expression of ifnl4-halo and its splicing forms-p131, p107, and p170-induced the erse-luc reporter in hepg2 cells (prokunina-olsson and others 2013). we next repeated this experiment for the erse-luc reporter cotransfected with ifnl4-halo or p131-halo. transfection with p131-halo did not induce cell death (fig. 5d , e), despite considerable induction of the esre-luc reporter (fig. 5f ). we conclude that potential activation of the unfolded protein response to in vitro generated ifn-l4 cannot explain the observed ifn-l4-induced cell death. in an effort to define the mechanism of ifn-l4-induced cell death, we assayed for ripk1-dependent necrosis (necroptosis) and caspase-1-dependent cell death (pyroptosis), using specific inhibitors (necrostatin and yvad). however, we saw no evidence of the activation of these pathways after transient transfection with the ifnl4-halo construct (data not shown). treatment with zvad, a pan-caspase inhibitor, caused a significant decrease in ifn-l4-induced cell death, which is suggestive of apoptosis, but this effect was also ) . graphs represent 1 of 3 independent experiments, n = 3. (c) cell viability was analyzed with multi tox-glo assay and presented as the percentage of induced to uninduced cells, n = 6. **p < 0.01, ***p < 0.001, based on t-tests. onabajo et al. observed in controls (data not shown), implying that transfection itself might be contributing to apoptosis in this experimental system. to further explore our observations done in transient expression system, we developed inducible stable hepg2 cell lines expressing ifn-l4 and p131 fused with c-terminal green fluorescent protein (gfp) and a control-gfp cell line, all of which were under control of a tetracycline-inducible promoter. stable clones showed 70%-90% intracellular expression ( fig 6a) . secreted ifn-l4-gfp was detectable in media after 24 h of induction (fig. 6b ) and was biologically active (fig. 6c ). to assess cell death, protein expression was induced for 72 h and cells were stained for live/dead + and annexin v + markers. the ifn-l4-gfp-expressing cells showed a significant increase in cell death compared with cells expressing p131-gfp or control-gfp (fig. 7a, b) . in addition, cell viability analysis showed that 5 days of induced ifn-l4 expression resulted in a 30% reduction in the counts of viable cells compared with uninduced cells, with no change observed for the controls (fig. 7c) . although we observed increased annexin v + staining in the induced ifn-l4-expressing cells (fig. 7a) suggesting apoptotic cell death, we did not detect any caspase-3 activation (data not shown), which is expected in apoptosis. we reasoned that cell death might be associated with decreased proliferation, and we evaluated cell proliferation after 72 h of induced ifn-l4 expression by measuring brdu incorporation. compared with p131-gfp and gfp control, induction of ifn-l4 was associated with a significant reduction of proliferation by 40% (fig. 8a, b) . we induced ifn-l4 expression in the presence or absence of the rabbit monoclonal a-ifn-l4 antibody, which we previously found to be able to block the ability of ifn-l4 to induce interferon signaling (fig. 1) . the a-ifn-l4 antibody treatment attenuated ifn-l4-induced cell death (fig. 9a) , while it increased proliferation (fig. 9b ) and improved cell viability (fig. 9c ). since the antibody is not expected to enter the cells and can only block the secreted protein, these results indicate that the observed cell death and decreased viability and proliferation are caused by the secreted ifn-l4. interestingly, this effect was localized to ifn-l4-expressing cells and was undetectable in nonexpressing bystander cells in the transwell assays (supplementary fig. s2) . thus, the effect of ifn-l4 may be concentration dependent and primarily affecting the ifn-l4-expressing cells. fig. 9. a-ifn-l4 antibody inhibits ifn-l4-induced cell death and proliferation defect. protein expression of ifn-l4-gfp, p131-gfp, or control-gfp in stable hepg2 cells was induced with 1 mg/ml doxycycline for 72 h. at 12 h, cells were treated with 20 mg/ ml of rabbit a-ifn-l4 antibody or 40 mg/ml of rabbit igg control (rigg). cells in different treatment conditions were assessed for cell death (a), cell proliferation (b), and cell viability (c). graphs represent 1 of 2 independent experiments, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 based on t-tests. we suggest that ifn-l4 may have at least 3 functions in human hepatic cells-activation of interferon signaling, inhibition of cell proliferation, and induction of cell death. these roles may be overlapping, synergistic, or independent, and some or all of them may be relevant for hcv clearance. activation of isgs by ifn-l4 was expected based on our previous observations in transiently transfected hepg2 cells (prokunina-olsson and others 2013). early activation of a panel of isgs has also been reported in both in vitro hcvinfected and adjacent uninfected phhs (sheahan and others 2014) . however, we now determined the following about ifn-l4-induced isg activation: it is specifically caused by the ifn-l4 secreted from cells at low but detectable amounts; it has an effect on both primary hepatocytes and hepatoma cells; it similarly affects the expressing and nonexpressing bystander cells; and it can be specifically blocked by the a-ifn-l4 antibody. anti-proliferative and cell-death-inducing anti-tumor effects are, in general, characteristic of interferons (wang and others 2011) , including in hepatoma cell lines (murata and others 2006) . however, we now present the first evidence of these effects caused by ifn-l4, the newest addition to the interferon family. while activation of isgs was observed in both the ifn-l4-expressing and nonexpressing bystander cells, the decrease of proliferation and induction of cell death were detectable only in the expressing cells. this could mean that these effects require different concentration thresholds. the dose-dependent anti-proliferative effect in hepatic cells has already been shown for ifn-a (lim and others 2006) , and the same might be true for ifn-l4. anti-proliferative activity of ifn-l4 is isg dependent as it was blocked by the same a-ifn-l4 antibody that blocks ifn-l4induced activation of isgs. some of the isgs induced by ifn-l4, including 2¢5¢oas and ip-10, are known for their contribution to the anti-proliferative and pro-apoptotic effects of interferons (rysiecki and others 1989; hassel and others 1993; aksoy and others 2006; liu and others 2011) . at this point, we are unable to determine the mechanism of ifn-l4-induced cell death. although we did not detect activation of caspase-3 by ifn-l4, therapeutic anti-tumor mechanisms of interferons have been related to cell growth arrest and induction of caspase-3-independent apoptosis via the p53 pathway (vogelstein and others 2000; takaoka and others 2003; , and this pathway should be tested for ifn-l4 as well. the effect of ifn-l4 on activation of interferon signaling and cell death might be complex and intertwined, and further studies are warranted. the genetic association between the ability to generate ifn-l4 (in carriers of the rs368234815-dg and rs12979860-t alleles) and impaired spontaneous and treatment-induced clearance of hcv is well established, but the molecular understanding of this association is still limited. recently, it has also been reported that the same individuals who are more likely to clear the hcv infection, in fact, are significantly more likely to develop liver fibro-proliferative disease (fibrosis), which is associated with increased mortality (eslam and others 2015) . our new findings regarding ifn-l4 function may help reconcile these counterintuitive connections. we suggest that the induction of interferon signaling by ifn-l4 may have both positive and negative implications. the ifn-l4-induced isgs might provide some antiviral protection, explaining the lower pretreatment hcv loads in individuals with the rs368234815-dg allele, compared with those who do not carry this allele (uccellini and others 2012) . on the other hand, the increased pretreatment expression levels of these isgs have been associated with refractoriness to ifn-a therapy (dill and others 2011), especially in carriers of the ifnl4-rs368234815-dg or rs12979860-t alleles (urban and others 2010; prokunina-olsson and others 2013) . this could be because ifn-a and ifn-l4 compete for induction of the same set of isgs, and, once activated by ifn-l4, these isgs do not respond to ifn-a. the fact that despite having lower pretreatment hcv levels, individuals with rs368234815-dg allele are more likely to develop chronic hcv and fail ifn-a-based treatment, suggests that either negative effects of ifn-l4 outweigh its positive effect on lowering hcv viral load or antiviral effects induced by ifn-l4 are insufficient for complete viral clearance. the ifn-l4 effect on proliferation and cell death might also be positive and negative. increased cell death has been correlated with inflammation during hcv infection and was suggested as a factor contributing to liver damage (bantel and schulze-osthoff 2003) . high rates of cell death in ifn-l4-expressing cells could amount to substantial cell loss and inflammation, contributing to the development of liver disease. simultaneously, the anti-proliferative effect of ifn-l4 might limit the tissue remodeling ability that leads to liver fibro-proliferative disease (fibrosis), as has been reported by the largest study on this subject to date (eslam and others 2015) . individuals who are genetically unable to produce ifn-l4 or in whom ifn-l4 is not sufficiently induced by specific stimuli may be more likely to clear the infection, spontaneously or after treatment, especially with ifn-abased therapies. however, they may not have the benefit of an anti-proliferative effect also contributed by ifn-l4, and will be more likely to develop fibrosis. since in our system the a-ifn-l4 antibody efficiently blocked activation of isgs (including ip-10), and ifn-l4induced cell death, blocking of ifn-l4 with therapeutic agents in carriers of the rs368234815-dg allele might be a plausible therapeutic option before or in conjunction with other treatments. blocking of ifn-l4 might boost mechanisms of endogenous host immunity, which are important for efficient clearance of hcv and prevention of posttreatment relapse (meissner and others 2014b) , and also decrease liver damage due to inflammation and cell death. although the antiviral role of ifn-l4 has been shown for a number of viruses (hamming and others 2013; lu and others 2015) , it remains to be found what other biologically relevant factors can trigger endogenous ifn-l4 expression. identification of these triggers in hepatic and other human cell types could help explore the role of ifn-l4 in other relevant clinical conditions. so far, expression of endogenous ifnl4 has only been detected in liver biopsies from patients infected with hcv, with significant correlations between hcv titers and expression levels of ifnl4 and isgs (amanzada and others 2013; konishi and others 2013; meissner and others 2014b; murakawa and others 2015). however, ifnl4 was undetectable in liver biopsies from patients infected with hepatitis b virus or affected by 898 onabajo et al. unrelated liver diseases (amanzada and others 2013) . in vitro, ifnl4 expression could be induced in phhs by hcv infection or treatment with polyi:c, but polyi:c treatments failed to induce ifnl4 expression in hepg2 cells. the ifnl4 region is absent in the genomes of mouse and rat, precluding the development of relevant rodent models. we acknowledge the limitations of our experimental system, which is based on expression of ifn-l4 in human hepatic cells-a hepatoma cell line hepg2 and phhs. the observed effects might differ from the physiologic conditions in the hcv-infected hepatic cells in the complex whole-organ environment by the magnitude, duration, and efficiency of ifn-l4 induction. we also did not address the mechanism of intracellular retention and the function of the nonsecreted ifn-l4. however, we provide a comprehensive functional analysis of ifn-l4, a novel human interferon, and suggest its role in activation of interferon signaling, inhibition of cell proliferation, and induction of cell death in human hepatic cells. association of the ifnl4-deltag allele with impaired spontaneous clearance of hepatitis c virus cxcr3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation interferon-lambda4 (ifnl4) transcript expression in human liver tissue samples apoptosis in hepatitis c virus infection interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c interferon-lambda rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response more than one way to die: apoptosis, necrosis and reactive oxygen damage predictive value of the ifnl4 polymorphism on outcome of telaprevir, peginterferon, and ribavirin therapy for older patients with genotype 1b chronic hepatitis c molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death genetic variation in il28b predicts hepatitis c treatment-induced viral clearance interferon lambda 4 signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses expression of the chemokine ip-10 (cxcl10) by hepatocytes in chronic hepatitis c virus infection correlates with histological severity and lobular inflammation a dominant negative mutant of 2-5a-dependent rnase suppresses antiproliferative and antiviral effects of interferon peroxisome dynamics in cultured mammalian cells selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) interferon-lambda4 genetic polymorphism is associated with the therapy response for hepatitis c virus recurrence after a living donor liver transplant ip-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with hcv genotype 1 infection current and future therapies for hepatitis c virus infection antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo studying protein dynamics in living cells the emerging role of cxcl10 in cancer (review) interferon-lambda4 is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden ifnl4-deltag genotype is associated with slower viral clearance in hepatitis c, genotype-1 patients treated with sofosbuvir and ribavirin endogenous intrahepatic ifns and association with ifn-free hcv treatment outcome global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence impaired induction of interleukin 28b and expression of interferon lambda 4 associated with nonresponse to interferon-based therapy in chronic hepatitis c a comparison of the antitumor effects of interferon-alpha and beta on human hepatocellular carcinoma cell lines reply: subgroup differences in response to 8 weeks of ledipasvir/sofosbuvir for chronic hepatitis c ifn-lambda4: the paradoxical new member of the interferon lambda family a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus constitutive expression of a 2¢,5¢-oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness measuring protein mobility by photobleaching gfp chimeras in living cells integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence new aspects of ifn-alpha/beta signalling in immunity, oncogenesis and bone metabolism reduced ifnlambda4 activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes genetic variation in il28b and spontaneous clearance of hepatitis c virus hcv rna levels in a multiethnic cohort of injection drug users: human genetic, viral and demographic associations il28b genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis c surfing the p53 network interferon: current status and future prospects in cancer therapy the work has been supported by the intramural research program ( key: cord-256769-flfycl7i authors: stoermer, kristina a.; morrison, thomas e. title: complement and viral pathogenesis date: 2011-03-01 journal: virology doi: 10.1016/j.virol.2010.12.045 sha: doc_id: 256769 cord_uid: flfycl7i the complement system functions as an immune surveillance system that rapidly responds to infection. activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. however, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. this review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection. have been identified, such as interaction of c1q with the c-type lectin sign-r1 expressed on macrophages (kang et al., 2006) , c5 activation by thrombin (huber-lang et al., 2006) , and the properdin-activated pathway (kemper et al., 2010) . the classical pathway is primarily activated by igm and certain igg isotypes bound to antigen. these immune complexes interact with the complement component c1q. c1q binding leads to the activation of two serine proteases associated with c1q, c1r and c1s. c1s cleaves c4 into c4a and c4b resulting in the exposure of a reactive thioester that allows covalent attachment of c4b on surfaces. c2 binds c4b and is also cleaved by c1s to form the classical pathway c3 convertase (c4bc2a). c3 convertases cleave c3 to amplify complement activation and lead to the generation of ligands for a variety of complement receptors. the lectin pathway is initiated by pattern recognition receptors such as mannose-binding lectin (mbl) and the ficolins (fig. 1) . mbl and ficolins contain carbohydrate-recognition domains that recognize carbohydrate patterns on the surfaces of cells or invading microorganisms. mbl and the ficolins are in a complex with enzymes known as mbl-associated serine proteases (masps). similar to c1s, masp-2 activates the complement system by cleaving both c4 and c2 to form the c4bc2a c3 convertase. the alternative pathway is activated by spontaneous hydrolysis of c3 (c3-h 2 o) (fig. 1) . this pathway also functions as an amplification loop for the cleavage of c3 initially triggered by other mechanisms. c3-h 2 o or c3b bound to target surfaces are bound by the protease factor b (fb). factor d (fd) is a serine protease that cleaves c3-h 2 o or c3b-bound fb, resulting in the generation of bb and formation of the alternative pathway c3 convertase (c3bbb). both the classical and alternative convertases function to cleave c3 to c3a and c3b. similar to c4, cleavage of c3 exposes a reactive thioester bond in c3b that allows for the covalent attachment of c3b to target surfaces. in addition, c3b can bind to either the classical or alternative c3 convertases resulting in a change of the substrate specificity of the convertases from c3 to c5. these c5 convertases cleave c5 to c5a and c5b. release of c5b promotes assembly of the c5b-c9 membrane attack complex (mac) which can directly lyse pathogens or pathogen-infected cells. the anaphylatoxins c3a and c5a interact with specific receptors to promote chemotaxis and regulate effector functions of cells of both the innate and adaptive immune response. a variety of soluble and membrane-associated proteins regulate complement activation. factor h (fh), c4b-binding protein (c4bp), and c1 inhibitor (c1-inh) are soluble proteins that regulate activation of the complement system. fh promotes the dissociation of c3bbb convertases and acts as a cofactor for factor i (fi)-mediated cleavage of c3b. c4bp functions similar to fh in that it promotes dissociation of c4bc2a convertases and acts as a cofactor for fi-mediated cleavage of c4b. the c1-inh is a serine protease inhibitor that inactivates both the c1q-associated proteases of the classical pathway (c1r and c1s) as well as the masps of the lectin pathway. membrane-associated regulators of complement activation include decay accelerating factor (daf/cd55), membrane cofactor protein (mcp/cd46), complement receptor 1 (cr1/cd35), cd59, and crry (rodent-specific). these proteins function to destabilize both the classical and alternative complement convertases, act as cofactors for fi-mediated cleavage of c3b and c4b, and prevent assembly of the mac on cell surfaces. the complement system is increasingly recognized as a mediator of protection or pathology in a variety of viral infections. furthermore, the continued identification of novel mechanisms of viral antagonism of complement highlight the important role this system has in viral pathogenesis. here, we review recent studies of viral interactions with a variety of components of the complement system and emphasize fig. 1 . schematic of the complement system. complement is activated by three major pathways. the classical pathway is primarily activated when c1q interacts with igm and certain igg isotypes bound to antigen. c1q-associated c1s cleaves c4 and c2 to form the classical pathway (cp) c3 convertase (c4bc2a). the lectin pathway (lp) is initiated by carbohydrate pattern recognition receptors such as mannose-binding lectin (mbl) and the ficolins (f) which are in a complex with enzymes known as mbl-associated serine proteases (masps). masp-2 activates the complement system by cleaving both c4 and c2 to form the c4bc2a c3 convertase. the alternative pathway (ap) is activated by spontaneous hydrolysis of c3 (c3-h 2 o). this pathway also functions as an amplification loop for the cleavage of c3 initially triggered by other mechanisms. c3-h 2 o or c3b bound to target surfaces are bound by the protease factor b (fb). factor d (fd) is a serine protease that cleaves c3-h 2 o or c3b-bound fb, resulting in the generation of bb and formation of the alternative pathway c3 convertase (c3bbb). both the classical and alternative convertases function to cleave c3 to c3a and c3b. cleavage of c3 exposes a reactive thioester bond in c3b that allows for the covalent attachment of c3b to target surfaces. in addition, c3b can bind to either the classical or alternative c3 convertases resulting in a change of the substrate specificity of the convertases from c3 to c5. these c5 convertases cleave c5 to c5a and c5b. release of c5b promotes assembly of the c5b-c9 membrane attack complex (mac) which can directly lyse pathogens or pathogen-infected cells. c3b is further cleaved by factor i (fi), a function enhanced by factor h (fh), to generate degradation products such as ic3b and c3dg. c3b and its degradation products interact with cellular receptors to regulate effector functions such as phagocytosis and b cell activation. the anaphylatoxins c3a and c5a interact with specific receptors to promote chemotaxis and regulate effector functions of cells of both the innate and adaptive immune response. those findings that describe how these interactions impact the development of virus-induced disease. perhaps the best evidence that complement has an important role in the outcome of virus infection is the identification of specific mechanisms evolved by viruses to evade the complement system (fig. 2 ). one mechanism employed by viruses is to directly encode proteins that have structural and functional homology to host proteins that function as regulators of complement activation. gammaherpesviruses, including kaposi's sarcoma-associated herpesvirus, herpesvirus saimiri, and murine γ-herpesvirus 68 (γhv68) encode homologs of complement regulatory proteins (albrecht and fleckenstein, 1992; mullick et al., 2003a; spiller et al., 2003; virgin et al., 1997) . the γhv68 regulator of complement activation (rca) protein is expressed on the surfaces of infected cells and is detected in supernatants of γhv68infected cells, thus it has both membrane-associated and soluble forms (kapadia et al., 1999) . recombinant γhv68 rca protein and supernatants from γhv68-infected cells blocked c3 deposition on zymosan examples of viral evasion of the complement system. a.) virally encoded proteins allow viruses to evade complement-mediated destruction. human astrovirus (hastv) coat protein (cp) binds mbl and c1q, inhibiting the activation of both the lectin and classical pathways. influenza a matrix (m1) protein also binds c1q. flavivirus nonstructural protein 1 (ns1) binds c4 and c1s, leading to enhanced cleavage of c4 to c4b, as well as factor h (fh), leading to increased cofactor activity of fh for factor i (fi)-mediated cleavage of c3b into ic3b. additionally, membrane-bound flavivirus ns1 decreases deposition of c3b and the mac on cell surfaces. the murine gammaherpesvirus-68 (γhv68) regulator of complement protein (rca) blocks c3 deposition, whereas the hsv-1 glycoprotein (gc) binds c3b and blocks the binding of properdin and c5 to c3b. the variola virus inhibitor of complement enzymes (spice), the vaccinia virus complement control protein (vcp), the monkeypox virus inhibitor of complement enzymes (mopice), and the ectromelia virus inhibitor of complement enzymes (emice) function as cofactors for fi-mediated cleavage of c3b by binding to c3b and c4b. viruses, with their corresponding proteins that interfere with the complement cascade in parentheses, are indicated in red. b.) some viruses recruit host complement regulatory proteins into their virions. human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1), and human cytomegalovirus (hcmv) incorporate the host complement control proteins cd55/daf and cd59 into their virions, while simian virus 5 (sv5) and mumps virus (muv) recruit cd46/mcp into their virions. physiological complement regulatory proteins are shown in green. viruses that incorporate these regulatory proteins into their virions are indicated in red. beads by both the classical and alternative activation pathways (kapadia et al., 1999) . to directly test the role of the γhv68 rca protein in pathogenesis, kapadia et al. generated a γhv68 deleted for the rca protein and evaluated the outcome of infection in both wildtype and c3-deficient mice (kapadia et al., 2002) . deletion of the γhv68 rca protein resulted in decreased virulence following intracranial inoculation of weanling mice or intraperitoneal inoculation of adult ifn-γ −/− mice, models of acute γhv68-induced meningoencephalitis and γhv68 persistent infection, respectively. genetic deletion of c3 in the host was able to restore virulence of the rca protein-deleted virus in the model of acute meningoencephalitis and enhanced its persistent replication, confirming that rca protein interaction with the host complement system is a critical regulator of γhv68 pathogenesis. interestingly, although wild-type mice had a reduced number of latently infected cells compared to c3 −/− mice, indicating that complement regulates the establishment of γhv68 latent infection, this effect was not counteracted by the γhv68 rca protein (kapadia et al., 2002) . poxviruses, such as variola virus, vaccinia virus, monkeypox virus, and ectromelia virus, also encode complement regulatory proteins that have structural and functional homology to host encoded regulators of the complement pathway (mullick et al., 2003b) ( fig. 2a ). the variola virus inhibitor of complement enzymes (spice), the vaccinia virus complement control protein (vcp), the monkeypox virus inhibitor of complement enzymes (mopice), and the ectromelia virus inhibitor of complement enzymes (emice) all bind to c3b, as well as c4b, and function as cofactors for fi-mediated cleavage of c3b moulton et al., 2010; rosengard et al., 2002; sahu et al., 1998) . a vcp-deleted vaccinia virus produced smaller skin lesions in rabbits compared to wild-type vaccinia virus (isaacs et al., 1992) and pathogenesis studies in cynomolgus monkeys with different strains of monkeypox virus revealed that mopice is deleted from the less virulent strains (chen et al., 2005) . furthermore, spice is a much more potent inhibitor of human complement compared to vcp and mopice, correlating with the increased virulence of variola virus in humans compared to vaccinia virus and monkeypox virus rosengard et al., 2002) . taken together, these studies and the findings that multiple components of the complement pathway are required for mice to survive ectromelia virus infection (discussed below), indicate that complement activation and viral evasion of the complement system are critical determinants of poxvirus pathogenesis. in contrast to herpesviruses and poxviruses, flaviviruses encode a protein that antagonizes the complement system despite the lack of any sequence homology to known regulators of the complement system. the nonstructural protein 1 (ns1) encoded by flaviviruses is a glycosylated protein detected within infected cells, on cell surfaces, and secreted from infected cells (alcon-lepoder et al., 2006) . ns1 accumulates in the serum of dengue virus-infected individuals and high circulating levels are associated with severe disease (avirutnan et al., 2006; libraty et al., 2002) . in an attempt to purify wnv ns1 from cell supernatants, chung et al. observed that fh copurified with ns1 (chung et al., 2006) . soluble wnv ns1 was found to enhance the cofactor activity of fh for fi-mediated cleavage of c3b to ic3b, while cell surface-associated ns1 decreased deposition of c3b and the c5b-c9 membrane attack complex on cell surfaces ( fig. 2a) . flavivirus ns1 also binds to c4 and c1s (avirutnan et al., 2010) ( fig. 2a) . these activities enhanced the cleavage of c4 to c4b and resulted in reduced activity of the classical c3 convertase (c4b2a) and reduced c4b and c3b deposition on cell surfaces (avirutnan et al., 2010) , providing an additional mechanism by which flaviviruses can evade complement-dependent neutralization. soluble ns1 has also been reported to bind the complement inhibitory factor clusterin, which normally inhibits the formation of the c5b-c9 membrane attack complex (kurosu et al., 2007) , however, a functional consequence of this interaction has not been reported. many other viruses employ a similar complement evasion strategy by encoding proteins that bind and inhibit or sequester complement components. for example, the coat protein of human astrovirus type 1 (hastv-1) suppresses complement activation by binding c1q, functionally displacing the protease tetramer, and thus inhibiting classical pathway activation (bonaparte et al., 2008; hair et al., 2010) ( fig. 2a) . this was further demonstrated for serotypes 2 and 4 (bonaparte et al., 2008) . the astrovirus coat protein also bound mbl and inhibited mannan-mediated activation of the lectin pathway (hair et al., 2010) . as discussed below, c1q enhances the neutralizing and hemagglutination inhibition activity of anti-influenza antibodies. experimental evidence suggests that the matrix (m1) protein of influenza a virus has evolved to counteract this host response, as m1 prevents complement-mediated neutralization of influenza virus in vitro by binding c1q and blocking the interaction between c1q and igg (zhang et al., 2009 ). herpes simplex virus 1 (hsv-1) encodes several immune modulators, including glycoprotein c (gc), which inhibits activation of the complement cascade by binding c3 and c3 fragments (friedman et al., 1984; fries et al., 1986; kostavasili et al., 1997; tal-singer et al., 1991) and by blocking binding of properdin and c5 to c3b (fries et al., 1986; hung et al., 1994; kostavasili et al., 1997) . these effects are mediated by two distinct domains in gc: one domain blocks properdin and c5 binding to c3b, and the other directly binds c3 and c3 fragments (hung et al., 1994) . in vitro, gc protects hsv-infected cells from complement-mediated lysis (harris et al., 1990 ) and cell-free virus from complement-mediated neutralization (friedman et al., 1996; hidaka et al., 1991) . it was further demonstrated that natural igm antibody binds and neutralizes hsv-1 and hsv-2 gc-null viruses via a c1q-, c3-, and c5-dependent mechanism (hook et al., 2006) . studies in animal models have confirmed the importance of gc-mediated complement inhibition in hsv-1 pathogenesis. when inoculated intravaginally into wild-type guinea pigs, but not c3-deficient guinea pigs, a gc-null hsv-1 replicated less efficiently and caused significantly less severe vaginitis compared to wild-type hsv-1 (lubinski et al., 1998) . the c3-dependent attenuated phenotype of gc-null hsv-1 was confirmed by inoculating wild-type and c3-deficient mice via skin scratch and evaluating hsv-1-induced zosteriform disease (lubinski et al., 2002 (lubinski et al., , 1998 . further studies with this murine model demonstrated that both domains of gc that modulate complement activation are critical virulence factors; however, hsv-1 specifically lacking the c3 binding domain was more attenuated compared to hsv-1 specifically lacking the c5/ properdin inhibitory domain (lubinski et al., 1999) . importantly, the gc mutant viruses were as virulent as wild-type virus in c3-deficient mice, indicating that the gc interactions with the complement system regulate hsv-1 pathogenesis (lubinski et al., 1999) . most recently, the knowledge gained from these studies was utilized to improve vaccine efficacy against hsv-1 infection. awasthi and colleagues demonstrated that combined immunization with the hsv-1 glycoprotein d (gd), a potent immunogen, and gc prevented hsv-1 evasion from complement, due to the development of an anti-gc antibody response, and enhanced the protection provided by gd immunization (awasthi et al., 2009) . a number of viruses evade the complement system by recruiting host complement regulatory proteins into their virions (fig. 2b ). for instance, human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1), and human cytomegalovirus (hcmv) incorporate the complement control proteins cd55/daf and cd59 into their virions, thus protecting virions from complement-mediated destruction spear et al., 1995) . simian virus 5 (sv5) and mumps virus (muv), both paramyxoviruses, can be neutralized in a c3-dependent manner resulting in virion aggregation and virion lysis, respectively (johnson et al., 2008) . further studies revealed that both sv5 and muv virions contained cd46/mcp, a membrane-associated protein that has cofactor activity for fi-mediated cleavage of c3b and c4b. the incorporation of cd46 into sv5 and muv conferred virion-associated cofactor activity and increased resistance of sv5 and muv to complement-dependent neutralization ). thus, these viruses have evolved to usurp host complement regulatory proteins to avoid complement-mediated destruction. mannose binding lectin (mbl) is a c-type lectin that plays an important role in innate immunity by binding to carbohydrates on a wide range of pathogens (fujita, 2002) . recognition of carbohydrates is mediated via a c-terminal carbohydrate recognition domain. once bound, mbl can activate complement, due to its association with masps, or act directly as an opsonin. polymorphisms in the promoter and structural regions of the human mbl2 gene affect mbl oligomer formation and circulating levels of the protein (madsen et al., 1995) . due to these different mutations, humans exhibit a 1000-fold variation in circulating mbl levels that occur with varying frequencies in different populations. recently, a number of studies have demonstrated that direct interactions between mbl and virus particles can neutralize infection. mbl has been found to bind directly to virions from a number of different virus families, including human immunodeficiency virus (hiv), severe acute respiratory syndrome coronavirus (sars-cov), ebola virus, dengue virus (denv), and west nile virus (wnv) (fig. 3) . the finding that the hiv envelope glycoprotein, gp120, is modified with high mannose oligosaccharides led researchers to test the potential of hiv and gp120 to function as ligands for mbl. mbl was shown to bind directly to hiv-infected cells and recombinant gp120 (ezekowitz et al., 1989) . the importance of these interactions was demonstrated by experiments showing mbl could inhibit infection of specific target cells by cell culture-derived hiv. although primary isolates of hiv bound to mbl (saifuddin et al., 2000) , mbl binding inefficiently inhibited infection of peripheral blood mononuclear cells (ying et al., 2004) . however, mbl binding did lead to efficient opsonization and uptake of hiv by mononuclear phagocytes (ying et al., 2004) . despite these findings, the role of mbl in hiv pathogenesis is still unclear. different studies investigating the association between mbl levels and hiv infection have found no association, increased susceptibility among individuals with low mbl levels, and increased susceptibility among individuals with high mbl levels (ji et al., 2005a) . however, this area of research has led to an effort to identify lectins that interact with hiv and inhibit infection as a novel therapeutic approach to prevent hiv infection and disease (alexandre et al., 2010; hoorelbeke et al., 2010; huskens et al., 2010) . mbl has also been reported to bind to sars-cov or retroviral particles pseudotyped with the sars-cov spike glycoprotein (sars-s) (ip et al., 2005; zhou et al., 2010) (fig. 3) . similarly, mbl binds efficiently to retroviral particles pseudotyped with ebola virus or marburg virus glycoproteins (ji et al., 2005a) (fig. 3) . importantly, all of these studies showed direct mbl-mediated neutralization of virus infection (ip et al., 2005; ji et al., 2005b) . mbl binding to sars-s was dependent on a single n-linked glycosylation site in sars-s (n330) (zhou et al., 2010) . mbl binding blocked the interaction of sars-s with dc-sign, previously identified to interact with sars-s (yang et al., 2004) , but not with the major sars-cov receptor angiotensinconverting enzyme 2 (ace2) (li et al., 2003) . serum levels of mbl were reported to be significantly lower in patients with sars than in control patients; there was, however, no association between mbl genotypes and mortality related to sars (ip et al., 2005) . a second study also reported that mbl gene polymorphisms associated with reduced mbl levels were significantly associated with susceptibility to sars-cov infection (zhang et al., 2005) . however, a third study reported that mbl genotypes and allele frequencies do not influence the outcome of infection with sars-cov (yuan et al., 2005) . finally, studies showed that mbl binds n-linked glycans on the structural proteins of wnv and denv, resulting in neutralization through a c3-and c4-dependent mechanism that occurred, in part, by blocking viral fusion . these findings indicated that mbl opsonization was not sufficient for neutralization, but rather deposition of c3 or c4 onto virions was also required. for wnv, neutralization occurred only with virus produced in insect cells; for denv, neutralization occurred with insect and mammalian cellderived virus . experiments in mice demonstrated an accelerated intravascular clearance of denv or of a wnv mutant with two n-linked glycans on its e protein, but not wild-type wnv, that was mbl-dependent . the complement system enhances humoral immunity by a number of different mechanisms. complement regulates effector functions of both natural and immune antibodies, complement component c3 and its receptors participate in the capture and transport of antigen to the b cell compartments of secondary lymphoid tissue (gonzalez et al., 2010) , and complement receptor 2 (cr2, cd21) and complement receptor 1 (cr1, cd35) expression by follicular dendritic cells function to retain antigen in the lymphoid follicles, which is required for the generation of a normal humoral immune response (fang et al., 1998) . on b lymphocytes, cr2 forms a complex with other proteins, such as cd19, to activate signal transduction pathways that regulate b cell activation. coligation of the b cell receptor and cr2, which binds the cleavage products ic3b, c3dg, and c3d covalently attached to antigen, lowers the threshold of b cell activation (bradbury et al., 1992; hebell et al., 1991; heyman et al., 1990) . in fact, linking c3d to a model antigen generated a fusion protein that was 100-1000 fold more immunogenic (dempsey et al., 1996) . numerous studies have established a critical role for the complement system in regulating humoral immunity to virus infection. below, we highlight recent findings with a particular emphasis on studies that have investigated these pathways in the context of viral pathogenesis. natural antibody and complement neutralize virus. the humoral immunity of naïve individuals consists primarily of natural igm antibodies (ochsenbein and zinkernagel, 2000) . natural igm is polyreactive and it is thought that endogenous antigens drive the generation of natural igm. igm, which exists in circulation primarily as a pentamer, has a 1000-fold greater binding affinity for c1q compared with igg, making it a potent activator of the complement system (ehrenstein and notley, 2010) . initial studies revealed the presence of natural igm specific for various viral pathogens, including lymphocytic choriomeningitis virus (lcmv), vesicular stomatitis virus (vsv), and vaccinia virus (vv), in the sera of naïve mice. by reconstituting rag-1 −/− mice with sera from naïve mice, researchers demonstrated that natural antibodies protected mice from both vsv dissemination and disease (ochsenbein et al., 1999a) . earlier studies had shown that a natural igm antibody to a vsv antigen forms an immune complex that activates c1 and initiates complement activation via the classical pathway at the viral surface, thus neutralizing vsv (beebe and cooper, 1981) . this neutralization was associated with c3b deposition on the viral envelope that likely interferes with vsv attachment to susceptible cells. natural igm and components of the classical activation pathway have also been shown to neutralize influenza virus (jayasekera et al., 2007) . interestingly, rather than resulting in lysis of virions, these interactions resulted in coating and aggregation of virus particles. to test the significance of these interactions on influenza pathogenesis, rag-1 −/− mice were reconstituted with natural igm and then challenged with the mouse-adapted pr8 strain of influenza. reconstitution prolonged mouse survival following influenza challenge, suggesting that natural igm and complement also protect the host from influenza infection. furthermore, both complement and igm were required for the development of protective immunity against influenza virus after immunization (fernandez gonzalez et al., 2008) . similar to influenza virus, sera from naïve mice neutralizes ectromelia virus by a mechanism dependent on both complement and natural antibodies (moulton et al., 2008) . further analyses revealed that opsonization of ectromelia virus particles with c3b and c4b was the primary mediator of neutralization. consistent with these findings, ectromelia virus dissemination was more efficient and viral loads in tissues were greater in mice deficient in c3. in mortality studies, the genetic deficiency in the complement components c3, c4, or fb resulted in a higher mortality rate compared to wild-type mice. in addition, reconstitution of b cell-deficient μmt mice with sera containing natural antibodies significantly increased survival following inoculation of ectromelia virus. in sum, these studies have revealed the important role of natural antibodies and complement in protection from virus infection. complement-mediated enhancement of b lymphocyte responses protects from virus-induced disease. the study of humoral responses to herpesvirus infections has yielded important insights into complement-mediated regulation of b cell function. mice deficient in c3, c4, or cr2 (which in mice encodes both cr1 and cr2) had significantly diminished igg responses following herpes simplex virus-1 (hsv-1) infection, suggesting that activation of the classical pathway plays a key role in regulating the antibody response to hsv-1 infection (da costa et al., 1999) . interestingly, c3-deficient mice reconstituted with bone marrow from wild-type mice developed normal antibody responses following hsv-1 infection, while wild-type mice reconstituted with bone marrow from c3-deficient mice had significantly diminished igg responses (verschoor et al., 2003 (verschoor et al., , 2001 . these experiments indicated that c3 derived from bone marrow cells was essential to enhance b cell activation in response to hsv-1 infection and were some of the first studies to describe an important function for complement factors synthesized locally by a non-hepatic cellular source. in contrast to hsv-1 infection, igg responses to vsv infection, which is neurotropic in mice and can cause paralysis and death, were similar in c3 −/− mice compared to wild-type mice (ochsenbein et al., 1999b) . instead, researchers discovered that complement components c3 and c4, but not cr2, were critical for initial anti-vsv igm responses. further experiments suggested that complement was critical for targeting vsv antigens to marginal zone macrophages in lymphoid tissues to stimulate b cells and igm production. c3 −/− mice, but not cr2 −/− mice, were more susceptible to lethal vsv infection, suggesting that the depressed igm responses were critical to limit vsv spread and replication in the central nervous system. unlike vsv infection, the genetic absence of c3 or cr2 resulted in increased wnv-induced mortality, earlier wnv entry into the central nervous system, and greater viral loads of wnv in the brains of mice, suggesting complement-mediated regulation of b cell responses was critical to control wnv dissemination, replication, and disease (mehlhop et al., 2005) . in fact, c3 and cr1/cr2 were required for normal anti-wnv igm and igg responses in mice and passive transfer of immune serum protected c3 −/− mice from lethal wnv infection. in further studies, antibody responses to wnv were found to be normal in mice deficient in fb and fd, components of the alternative complement activation pathway (mehlhop and diamond, 2006) . these findings were consistent with previous findings in which igg responses to a t-dependent antigen were normal in fb −/− mice (matsumoto et al., 1997) and indicated that components of the alternative complement activation pathway are not required for the development of an anti-wnv antibody response. in contrast, components of the classical and lectin complement activation pathways, c4 and c1q, were required for normal humoral responses to wnv infection (mehlhop and diamond, 2006) . interestingly, c4, but not c1q, was required for normal anti-wnv igm responses, whereas both c4 and c1q were required for normal anti-wnv igg responses. these findings suggest that distinct complement pathways regulate igm vs. igg responses, at least in the context of a viral infection. importantly, similar to c3 and cr2 deficient mice, c4-and c1qdeficient mice were much more susceptible to lethal wnv-infection, providing further evidence that the host's complement system is critical to limit the severity of wnv disease (mehlhop and diamond, 2006) . c1q regulates anti-viral antibody effector mechanisms. a number of recent studies have identified a critical role for complement, and in particular the complement component c1q, in the regulation of antiviral antibody effector mechanisms. researchers investigating the serum-specific enhancement of influenza virus hemagglutinin (ha)specific monoclonal antibodies discovered that c1q could enhance both neutralization activity and hemagglutination inhibition (hi) activity (feng et al., 2002) . c1q more strongly influenced hi activity, and this enhancement did not require c3, suggesting that the effects were independent of c1q-mediated complement activation. c1qmediated enhancement of hi activity and neutralizing activity was dependent on the antibody isotype and epitope specificity (feng et al., 2002; mozdzanowska et al., 2006) . antibody-dependent enhancement (ade) of infection has been described for multiple viruses . in the case of dengue viruses, ade is associated with more severe disease (balsitis et al., 2010; kyle and harris, 2008; zellweger et al., 2010) . utilizing in vitro assays designed to measure ade of dengue virus infection, researchers discovered that the presence of fresh serum could completely abolish ade (mehlhop et al., 2007; yamanaka et al., 2008) . further analyses demonstrated that the inhibitory effect of fresh serum on ade was c1q-dependent and antibody isotypespecific (mehlhop et al., 2007; yamanaka et al., 2008) . however, in separate studies the suppression of ade by fresh serum was found to be c3-dependent or c3-independent, thus the extent to which other complement components participate in mediating these effects is unclear. in addition to suppressing ade, c1q was shown to enhance the neutralizing activity of anti-wnv antibodies (mehlhop et al., 2009) . mechanistic studies revealed that c1q reduced the number of antibodies required to bind a wnv virion and neutralize infectivity. interestingly, c1q reduced the number of antibodies required for neutralization to levels below the minimum number required for ade. importantly, the ability of an anti-wnv antibody to protect from lethal infection was decreased in c1q −/− mice compared to wild-type mice, indicating that c1q enhancement of neutralizing activity regulates wnv pathogenesis. in contrast to the effect of c1q on ade of flavivirus infection, ade of ebola zaire virus infection is c1qdependent . the glycoprotein of the reston strain of ebola virus, which is less pathogenic in humans, induces less enhancing activity compared to the zaire strain, suggesting that ade may also play a role in ebola virus pathogenesis. regulation of t cells by the complement system. increasing evidence from various experimental systems indicates that complement regulates cd4 + and cd8 + t cell activation and effector functions (dunkelberger and song, 2010; kemper and atkinson, 2007) . numerous complement receptors and membrane complement regulatory proteins can be expressed by antigen-presenting cells (apcs) and t cells. engagement of these receptors on apcs regulates apc effector functions, such as chemokine and cytokine gene expression, and modulation of apc function influences t cell activation during antigen presentation. direct action of complement on t cells is less well understood. ligation of cr1 on t cells has been shown to inhibit t cell proliferation (wagner et al., 2006) , while the c5a receptor (c5ar) has been shown to regulate t cell trafficking (tsuji et al., 2000) . in addition, several membrane complement regulatory proteins have been linked to t cell regulation. for example, cd46, which binds c3b and c4b and serves as a cofactor for their proteolytic inactivation, was found to regulate the proliferation and effector functions of cd4 + and cd8 + t cells (marie et al., 2002) . critically, complement regulates the effector function of activated t cells by regulating the development of th1, th2, and th17 helper cells. for example, c3ar-deficient mice were protected against th2-dependent airway hypereactivity and this has been linked to both decreased th2 cell responses and enhance-ment of il-17 producing helper t cells (drouin et al., 2001; lajoie et al., 2010) . in contrast c5 provides protection against airway hypereactivity by regulating th17 cytokine production (lajoie et al., 2010) . finally, evidence suggests that complement also regulates the termination of t cell responses by inducing the development of regulatory t cells (kemper et al., 2003) . utilizing mouse models, researchers discovered that c3 was required for normal t cell responses to influenza virus, lymphocytic choriomeningitis virus (lcmv) infection, and friend virus (fv) infection (banki et al., 2010; kopf et al., 2002; suresh et al., 2003) . in vitro, dendritic cells exposed to complement-opsonized fv or hiv were better inducers of cd8 + t cell activation (banki et al., 2010) . these effects were also observed with complement opsonized fv in mice. furthermore, c3-deficient mice had reduced numbers of fv-specific cd8 + t cells and higher numbers of infected spleen cells compared to wild-type controls, suggesting complement-mediated regulation of t cell responses functions to control fv replication (banki et al., 2010) . similarly, mice deficient in c3 had delayed clearance of influenza virus and increased titers in the lungs. these findings correlated with a dramatic decrease in the recruitment of virusspecific cd4 + and cd8 + t cells producing ifn-γ to the lung of influenza virus-infected c3 −/− mice. following either lcmv or influenza virus infection, c3 was required for normal priming of cd4 + and cd8 + t cells and mice deficient in cr1 and cr2 (cr2 −/− mice) did not show any of these defects, indicating that c3 regulates t cell responses in a cr1/cr2-independent manner (kopf et al., 2002; suresh et al., 2003) . in lcmv-infected mice, the effects of c3 on cd8 + t cell priming and expansion were epitopedependent and influenced by the mouse genetic background, suggesting that c3 may regulate epitope selection. subsequent experiments indicated that the c5ar may have an important role in the regulation of t cell responses to influenza virus infection as treatment of mice with a c5ar antagonist reduced the number of influenza virus-specific cd8 + t cells in both the lungs and lymphoid tissue (kim et al., 2004) . studies utilizing a mouse model of wnv infection and disease identified distinct complement pathways that regulate antiviral t cell responses (mehlhop and diamond, 2006) . mice deficient in fb were found to be highly susceptible to lethal wnv infection, despite normal anti-wnv antibody responses. instead, fb −/− mice, but not c1q −/− mice, had reduced cd8 + t cell responses in the spleen and impaired trafficking of cd4 + and cd8 + t cells to the cns. similar findings were observed in wnv-inoculated c4 −/− mice, suggesting that both the lectin and alternative complement activation pathways contribute to regulation of t cell functions. thus, similar to fv, influenza virus, and lcmv infections, complement activation was required for normal t cell priming and trafficking following wnv infection. the role of complement regulatory proteins in regulating t cell responses to viral infection has also been investigated. mice deficient in decay accelerating factor (daf; cd55), which regulates formation and inactivation of the c3 and c5 convertases, had an increased expansion of cd8 + t cells with greater killing capacity following lcmv infection and this correlated with lower viral titers. these enhanced cd8 + t cell responses were reversed in mice deficient for daf and c3 (daf-1 −/− ; c3 −/− ) or daf and the c5ar (daf-1 −/− ; c5ar −/− ). in contrast, following vaccinia virus infection, cd4 + , but not cd8 + , t cell responses were enhanced in mice deficient in cd59a, which blocks formation of the membrane attack complex (longhi et al., 2005) . taken together, these studies suggest that membrane complement regulators regulate both cd4 + and cd8 + t cell immunity. however, cd4 + t cell responses following vaccinia virus infection were enhanced in both cd59a −/− and cd59a −/− ; c3 −/− mice, indicating that the effects of cd59a on t cell responses were likely independent of complement activation, at least following vaccinia virus infection. hepatitis c virus (hcv) appears to exploit the complement system to establish persistence. hcv core protein is the first protein expressed during the early phase of hcv infection and free hcv core particles are found in the blood of hcv-infected patients (maillard et al., 2001) . by evaluating various vaccinia virus (vv)/ hcv recombinants in mice, large et al. discovered that a recombinant vv expressing the hcv core protein produced a lethal infection in mice and suppressed the vv-specific cd8 + t cell response (large et al., 1999) . using a yeast two-hybrid approach, kittlesen and colleagues identified that the hcv core protein bound the gc1q receptor (gc1qr) specific for the globular heads of the c1q protein (kittlesen et al., 2000) (fig. 3) . binding of gc1qr by the hcv core protein inhibited human peripheral blood t cell proliferation in standard one-way mixed lymphocyte reactions (mlr) or following mitogen stimulation (kittlesen et al., 2000) , a similar effect to the binding of c1q, the natural ligand for the gc1qr. this suppression was reversed by the addition of anti-gc1qr or anti-core antibodies in the t-cell proliferation assay (kittlesen et al., 2000) . more specifically, binding of hcv core protein to gc1qr on activated human peripheral blood t cells decreased il-2 and ifn-γ production and decreased il-2r and cd69 expression (yao et al., 2001) . a recent clinical study investigated the impact of hcv infection on t cell responses in acute as compared to resolved versus chronic infection (cummings et al., 2009) . during the acute phase of hcv infection, the frequency of gc1qr + cd4 + t cells increased compared to healthy controls. six months later, the frequency of gc1qr + cd4 + t cells remained elevated in chronic patients compared to that in resolved patients. chronic patients also had higher levels of circulating hcv core protein. tcr stimulation increased the frequency of gc1qr + cd4 + t cells, resulting in core-induced inhibition of t cell responses in both resolved and chronic patients. these results suggest that hcv infection expands gc1qr + cd4 + t cells, increasing the susceptibility to core-mediated immune dysregulation (cummings et al., 2009) . the gc1qr is also expressed by other immune cells, such as dendritic cells (dcs) and b cells. dcs isolated from patients chronically infected with hcv display a reduced capacity to induce t cell activation and to produce th1 cytokines (auffermann-gretzinger et al., 2001; bain et al., 2001; dolganiuc et al., 2003; kakumu et al., 2000; kanto et al., 1999 kanto et al., , 2004 . waggoner and colleagues demonstrated that binding of the hcv core protein to gc1qr on human monocytederived dcs (mddcs) inhibited tlr-induced il-12 production but not the production of other tlr-induced cytokines (waggoner et al., 2007) . in addition, hcv core protein engagement of gc1qr on mddcs promoted the production of th2 cytokines such as il-4 by cocultured cd4 + t cells. these results suggest that engagement of gc1qr on dcs by hcv core limits the induction of th1 responses (waggoner et al., 2007) . in contrast to the effects on t cells and dcs, hcv core protein binding to gc1qr on b cells leads to b cell activation and proliferation: increased costimulatory and chemokine receptor expression, cell proliferation, igm and igg production, and stat1 phosphorylation, and down-regulation of socs-1 expression (yao et al., 2008) . these data suggest that hcv exploits the complement system to dysregulate the host immune response via various mechanisms in order to establish persistence. to further investigate the role of the hcv core protein in hcv pathogenesis, transgenic mice were developed with tetracycline-regulated conditional hcv core protein expression (chang et al., 2009) . these mice develop liver inflammation, steatosis, and fibrosis. microarray analyses of inflamed liver showed induction of many components of both the complement and coagulation pathways, and administration of cd55 (daf) decreased hepatic inflammation, suggesting that the hcv core and the complement system contribute to hepatic inflammation associated with hcv infection. cryoglobulinemia is a systemic vasculitis that damages small and medium-sized arteries and veins of the skin, kidneys, and peripheral nerves (dammacco et al., 2001) , and evidence suggests that the deposition of immune complexes on the vessel wall activates complement and mediates this damage. mixed cryoglobulinemia (mc), which involves multiple immunoglobulin isotypes, is observed in 10-70% of hepatitis c virus (hcv)-infected patients and is associated with increased duration of hcv infection (charles and dustin, 2009 ). in hcv-positive patients with mc, circulating, nonenveloped hcv core protein was detected in cryoprecipitable immune complexes (sansonno et al., 2003) . as discussed above, the hcv core protein interacts with the gc1qr. the gc1qr can be proteolytically cleaved and released from the cell surface (kittlesen et al., 2000) . studies with hcv-positive mc patients revealed that mc following hcv infection correlated with significantly higher levels of circulating gc1qr compared to hcv-infected patients without mc (sansonno et al., 2009 ). in addition, higher serum gc1qr levels negatively correlated with circulating levels of the c4d fragment, which was instead sequestered in the vascular bed from skin biopsies of mc patients, indicative of complement activation in the vascular bed (sansonno et al., 2009) . these data suggest that following hcv infection, dysregulated shedding of gc1qr molecules contributes to vascular cryoglobulin-induced damage via a complement-mediated pathway (sansonno et al., 2009 ). hcv has also been linked to pulmonary diseases such as asthma and chronic obstructive pulmonary disease (copd) (moorman et al., 2005a) . in in vitro studies using normal human lung fibroblasts, moorman and colleagues found that the hcv core protein induced il-8 mrna and protein expression via gc1qr (moorman et al., 2005b) . taken together, these studies suggest that the hcv core protein interaction with the gc1q receptor of the complement system contributes to the pathogenesis of multiple diseases associated with hcv infection. further investigation into the proinflammatory role of the hcv core protein may provide a clearer understanding of the pathogenesis of hcv-associated diseases. chronic hcv infection is characterized by persistent complement activation, and can lead to liver fibrosis despite antiviral therapies. by interbreeding fibrosis-susceptible and fibrosis-resistant strains of mice, c5 was identified to be associated with hepatic fibrosis, and small molecule inhibitors of the c5a receptor had antifibrotic effects in vivo (hillebrandt et al., 2002) . polymorphisms of the human gene c5 were associated with advanced liver fibrosis, as compared with mild fibrosis, in chronic hcv infection (hillebrandt et al., 2005) (fig. 3) . moreover, the at-risk c5 haplotype in humans was associated with high serum c5 levels (hillebrandt et al., 2005) . these data suggest that c5 has a causal role in non-hcv and hcv-associated hepatic fibrosis across species and that c5-targeted therapeutics could be a beneficial treatment strategy. although denv ns1 can inhibit activation of the complement system (avirutnan et al., 2010) , evidence suggests that complement activation may contribute to increased disease severity following denv infection (fig. 3) . dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) are serious clinical conditions that typically occur after a second dengue infection by a different viral serotype or after a primary infection in infants born to dengue-immune mothers. lower levels of c3, c4, and fb, and higher levels of c3 cleavage products, were detected in severely ill dhf patients and correlated with signs of shock, suggesting that complement activation contributes to the more severe forms of dengue virus-induced disease (bokisch et al., 1973; churdboonchart et al., 1983; nascimento et al., 2009 ). in addition, dhf patients have been found to have higher levels of fd, which cleaves fb to yield the active (c3bbb) c3 convertase, and lower levels of fh, which inactivates the (c3bbb) c3 convertase, compared to patients with df (nascimento et al., 2009) . other studies have shown that plasma levels of sns1 and products of complement activation, including those with a known vascular effect such as c3a, c5a, and the terminal sc5b-9 complement complex, were present at higher levels in dhf patients before plasma leakage took place (avirutnan et al., 2006) , further supporting the hypothesis that complement activation is involved in the development of severe disease. comparison of global gene expression profiles in peripheral blood mononuclear cells (pbmcs) of patients with df or dhf discovered that the complement inhibitor cd59 was more strongly up-regulated in pbmcs from df patients than in pbmcs from dhf patients. furthermore, the wild-type mbl genotype, but not mbl genotypes associated with reduced mbl levels, was associated with the development of dengue-related thrombocytopenia and more severe disease (acioli-santos et al., 2008) . this is interesting given the recent findings that mbl binds denv resulting in complement activation and virus neutralization . these studies implicate the complement system in the pathogenesis of the more severe forms of dengue virus associated disease, dhf/dss; however, the mechanisms by which the complement system contributes to disease severity are not well understood. arthritogenic alphaviruses, including ross river virus (rrv) and chikungunya virus, are mosquito-borne viruses that cause debilitating musculoskeletal inflammation and pain in humans. evidence of complement activation was detected in synovial fluid from rrvinfected patients (morrison et al., 2007) . similarly, in a mouse model of rrv-induced disease, complement activation products were detected in inflamed joint and muscle tissues of wild-type mice (morrison et al., 2007) . rrv-infected c3-deficient mice exhibited less severe destruction of skeletal muscle tissue and less severe disease signs, indicating an important role for complement in rrv pathogenesis (fig. 3) . mice deficient in cr3 (cd11b −/− ) also developed less severe tissue damage and disease signs following rrv infection (morrison et al., 2008) (fig. 3) . interestingly, neither c3 nor cr3 deficiency prevented recruitment of inflammatory leukocytes to musculoskeletal tissues, suggesting that c3 and cr3 may act downstream of inflammatory cell invasion to promote tissue damage during rrv infection (morrison et al., 2007 (morrison et al., , 2008 . the genetic absence of c3 and cr3 significantly diminished rrv-induced expression of s100a9, s100a8, and il-6 within inflamed skeletal muscle tissue, indicating that the induction was regulated, at least in part, by cr3 interaction with its c3-derived ligand ic3b (morrison et al., 2008) . furthermore, mice deficient in mbl (mbl-dko) also developed less severe rrv-induced disease and these mice had reduced mbl and c3 deposition on tissues (unpublished results), providing evidence that rrv infection leads to complement activation via the lectin-dependent activation pathway. taken together, these studies suggest that interfering with the complement cascade and/or complement receptor signaling may represent a useful route for therapeutic intervention of rrv-induced disease. finally, recent evidence suggests that the complement system may contribute to seizures induced by virus infection. infection of wildtype mice with theiler's murine encephalomyelitis virus (tmev) results in acute seizures (libbey et al., 2008 (libbey et al., , 2010 . in contrast, c3 −/− mice developed far fewer seizures following tmev infection which correlated with reduced numbers of activated microglia and macrophages in the hippocampus and dentate gyrus as well as decreased neuronal cell loss (libbey et al., 2010) (fig. 3) . interestingly, depletion of systemic complement with cobra venom factor did not impact tmev-induced seizures, suggesting that locally produced complement within the central nervous system is sufficient to enhance tmev-induced seizures (libbey et al., 2010) . it is clear that the complement system is a critical determinant of the outcome of infection by a variety of different viruses. our understanding of the mechanisms by which complement protects from virus-induced disease has improved dramatically. research in this area will not only continue to contribute to our knowledge of viral pathogenesis, but will continue to provide insight into the regulation of immune responses, and lead to improved therapeutic and vaccine approaches for both viral and non-viral pathogens. perhaps less well understood are the mechanisms by which complement functions as a pathogenic effector in some virus-induced diseases. further progress towards identifying the signals and pathways that lead to complement activation, which are not understood for many viruses, particularly in vivo, and a deeper understanding of the impact of complement activation on host immune responses to viral infection may shed light. for example, recent studies have identified critical roles for the complement system in the regulation of th17 cells (lajoie et al., 2010) , regulation of myeloid-derived suppressor cells (markiewski et al., 2008) , and the induction of autophagy (joubert et al., 2009) , all of which have been linked to virus-induced disease. in addition, the complement system is intricately linked with other pathways that play important roles in viral pathogenesis, such as the toll-like receptor signaling network and the coagulation system markiewski et al., 2007) . thus, continued investigation of the role of complement in viral pathogenesis will provide important insights into virus-host interactions and strategies to prevent or treat virus-induced disease. mbl2 gene polymorphisms protect against development of thrombocytopenia associated with severe dengue phenotype new member of the multigene family of complement control proteins in herpesvirus saimiri secretion of flaviviral non-structural protein ns1: from diagnosis to pathogenesis mannose-rich glycosylation patterns on hiv-1 subtype c gp120 and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis c virus infection vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns1 and complement antagonism of the complement component c4 by flavivirus nonstructural protein ns1 immunization with hsv-1 glycoprotein c prevents immune evasion from complement and enhances the efficacy of an hsv-1 glycoprotein d subunit vaccine impaired allostimulatory function of dendritic cells in chronic hepatitis c infection lethal antibody enhancement of dengue disease in mice is prevented by fc modification complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirus-specific ctls neutralization of vesicular stomatitis virus (vsv) by human complement requires a natural igm antibody present in human serum the role of complement in hemorrhagic shock syndrome (dengue) human astrovirus coat protein inhibits serum complement activation via c1, the first component of the classical pathway the cd19/cd21 signal transducing complex of human b lymphocytes includes the target of antiproliferative antibody-1 and leu-13 molecules the complement system in regulation of adaptive immunity hepatic inflammation mediated by hepatitis c virus core protein is ameliorated by blocking complement activation hepatitis c virus-induced cryoglobulinemia virulence differences between monkeypox virus isolates from west africa and the congo basin west nile virus nonstructural protein ns1 inhibits complement activation by binding the regulatory protein factor h crossed immunoelectrophoresis for the detection of split products of the third complement in dengue hemorrhagic fever. i. observations in patients' plasma frequency of gc1qr + cd4 + t cells increases during acute hepatitis c virus infection and remains elevated in patients with chronic infection humoral response to herpes simplex virus is complementdependent the cryoglobulins: an overview c3d of complement as a molecular adjuvant: bridging innate and acquired immunity additive inhibition of dendritic cell allostimulatory capacity by alcohol and hepatitis c is not restored by dc maturation and involves abnormal il-10 and il-2 induction cutting edge: the absence of c3 demonstrates a role for complement in th2 effector functions in a murine model of pulmonary allergy role and mechanism of action of complement in regulating t cell immunity the importance of natural igm: scavenger, protector and regulator a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific igg response complement component c1q enhances the biological activity of influenza virus hemagglutinin-specific antibodies depending on their fine antigen specificity and heavy-chain isotype complement and natural antibody are required in the long-term memory response to influenza virus glycoprotein c of herpes simplex virus 1 acts as a receptor for the c3b complement component on infected cells immune evasion properties of herpes simplex virus type 1 glycoprotein gc glycoprotein c of herpes simplex virus 1 is an inhibitor of the complement cascade direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin evolution of the lectin-complement pathway and its role in innate immunity complement-dependent transport of antigen into b cell follicles complement driven by conformational changes human astrovirus coat protein binds c1q and mbl and inhibits the classical and lectin pathways of complement activation crosstalk pathways between toll-like receptors and the complement system glycoprotein c of herpes simplex virus type 1 prevents complement-mediated cell lysis and virus neutralization suppression of the immune response by a soluble complement receptor of b lymphocytes in vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody glycoprotein c of herpes simplex virus type 1 is essential for the virus to evade antibody-independent complement-mediated virus inactivation and lysis of virus-infected cells genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus hfib1 on chromosome 15 complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans the complement system as a therapeutic target in autoimmunity herpes simplex virus type 1 and 2 glycoprotein c prevents complement-mediated neutralization induced by natural immunoglobulin m antibody actinohivin, a broadly neutralizing prokaryotic lectin, inhibits hiv-1 infection by specifically targeting high-mannosetype glycans on the gp120 envelope generation of c5a in the absence of c3: a new complement activation pathway the interaction of glycoprotein c of herpes simplex virus types 1 and 2 with the alternative complement pathway microvirin, a novel alpha(1, 2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile mannose-binding lectin in severe acute respiratory syndrome coronavirus infection vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity mannose binding lectin (mbl) and hiv mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization differential mechanisms of complementmediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus the paramyxoviruses simian virus 5 and mumps virus recruit host cell cd46 to evade complement-mediated neutralization autophagy induction by the pathogen receptor cd46 decreased function of peripheral blood dendritic cells in patients with hepatocellular carcinoma with hepatitis b and c virus infection a dominant complement fixation pathway for pneumococcal polysaccharides initiated by sign-r1 interacting with c1q impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis c virus-infected individuals reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize t helper cells in chronic hepatitis c virus infection murine gammaherpesvirus 68 encodes a functional regulator of complement activation critical role of complement and viral evasion of complement in acute, persistent, and latent gamma-herpesvirus infection t-cell regulation: with complements from innate immunity activation of human cd4 + cells with cd3 and cd46 induces a t-regulatory cell 1 phenotype properdin: emerging roles of a patternrecognition molecule complement c5a receptor is essential for the optimal generation of antiviral cd8 + t cell responses interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation complement component c3 promotes t-cell priming and lung migration to control acute influenza virus infection mechanism of complement inactivation by glycoprotein c of herpes simplex virus secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1 global spread and persistence of dengue complement-mediated regulation of the il-17a axis is a central genetic determinant of the severity of experimental allergic asthma suppression of host immune response by the core protein of hepatitis c virus: possible implications for hepatitis c virus persistence angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus seizures following picornavirus infection role for complement in the development of seizures following acute viral infection high circulating levels of the dengue virus nonstructural protein ns1 early in dengue illness correlate with the development of dengue hemorrhagic fever structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation cutting edge: murine cd59a modulates antiviral cd4 + t cell activity in a complementindependent manner herpes simplex virus type 1 glycoprotein gc mediates immune evasion in vivo in vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gc herpes simplex virus type 1 evades the effects of antibody and complement in vivo interplay between promoter and structural gene variants control basal serum level of mannan-binding protein nonenveloped nucleocapsids of hepatitis c virus in the serum of infected patients linking innate and acquired immunity: divergent role of cd46 cytoplasmic domains in t cell induced inflammation complement and coagulation: strangers or partners in crime? modulation of the antitumor immune response by complement abrogation of the alternative complement pathway by targeted deletion of murine factor b protective immune responses against west nile virus are primed by distinct complement activation pathways complement activation is required for induction of a protective antibody response against west nile virus infection complement protein c1q inhibits antibody-dependent enhancement of flavivirus infection in an igg subclass-specific manner complement protein c1q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus hepatitis c virus and the lung: implications for therapy induction of p38-and gc1qr-dependent il-8 expression in pulmonary fibroblasts by soluble hepatitis c core protein complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease complement receptor 3 promotes severe ross river virus-induced disease surviving mousepox infection requires the complement system ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement enhancement of neutralizing activity of influenza virus-specific antibodies by serum components kaposi's sarcomaassociated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins herpes and pox viral complement control proteins: 'the mask of self alternative complement pathway deregulation is correlated with dengue severity natural antibodies and complement link innate and acquired immunity control of early viral and bacterial distribution and disease by natural antibodies protective t cell-independent antiviral antibody responses are dependent on complement complement: a key system for immune surveillance and homeostasis variola virus immune evasion design: expression of a highly efficient inhibitor of human complement interaction of vaccinia virus complement control protein with human complement proteins: factor i-mediated degradation of c3b to ic3b1 inactivates the alternative complement pathway role of virion-associated glycosylphosphatidylinositol-linked proteins cd55 and cd59 in complement resistance of cell line-derived and primary isolates of hiv-1 interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 non-enveloped hcv core protein as constitutive antigen of cold-precipitable immune complexes in type ii mixed cryoglobulinaemia role of the receptor for the globular domain of c1q protein in the pathogenesis of hepatitis c virus-related cryoglobulin vascular damage host cell-derived complement control proteins cd55 and cd59 are incorporated into the virions of two unrelated enveloped viruses. human t cell leukemia/lymphoma virus type i (htlv-i) and human cytomegalovirus (hcmv) complement regulation by kaposi's sarcoma-associated herpesvirus orf4 protein complement component 3 is required for optimal expansion of cd8 t cells during a systemic viral infection antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications antibody-dependent enhancement of ebola virus infection herpes simplex virus glycoprotein c is a receptor for complement component ic3b early local generation of c5a initiates the elicitation of contact sensitivity by leading to early t cell recruitment cutting edge: myeloid complement c3 enhances the humoral response to peripheral viral infection myeloid c3 determines induction of humoral responses to peripheral herpes simplex virus infection complete sequence and genomic analysis of murine gammaherpesvirus 68 hcv core protein interaction with gc1q receptor inhibits th1 differentiation of cd4 + t cells via suppression of dendritic cell il-12 production the complement receptor 1, cr1 (cd35), mediates inhibitory signals in human t-lymphocytes infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign hepatitis c virus core protein inhibits human t lymphocyte responses by a complement-dependent regulatory pathway differential regulation of socs-1 signalling in b and t lymphocytes by hepatitis c virus core protein interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection influenza a virus m1 blocks the classical complement pathway through interacting with c1qa a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms complement regulators and inhibitory proteins an enormous body of work has contributed to the knowledge of the complement system and viral interactions with the complement system. with that said, we acknowledge the research that was not specifically mentioned in this review. we thank michelle davis for her essential and outstanding contributions to the figures in this review. this work was supported by nih/niaid research grant k22 ai079163 awarded to t.e.m. k.a.s. was supported by the predoctoral nih institutional training grant t32 ai052066. key: cord-281941-97t45w73 authors: zhou, daijun; mei, qiang; li, jintao; he, haiyang title: cyclophilin a and viral infections date: 2012-08-10 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2012.07.024 sha: doc_id: 281941 cord_uid: 97t45w73 abstract cyclophilin a (cypa) is a peptidyl-prolyl cis/trans isomerase originally identified as the target of the immunosuppressive drug cyclosporine a. a number of reports have demonstrated that cypa plays a critical role in the successful replication of viruses such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), etc. however, recent studies demonstrated that cypa also possesses a repressive effect on the replication of some viruses like influenza a virus and rotavirus. moreover, cypa could also regulate host ifn-i response to viral infections. together, these evidences showed diverse roles of cypa in viral infection. cypa belongs to a family of highly conserved and ubiquitous proteins known as cyclophilins (cyp). this family possesses peptidyl-prolyl cis/trans isomerase activity [1] and acts as an acceleration factor in protein folding and assembly. till now more than 10 members of cyp subtypes have been found in mammals, including cypa, cypb, cypc, cypd, cype, cyp40, ranbp2, etc. among them, cypa is the most abundant subtype and is widely distributed in almost all tissues. in addition to its role in protein folding and assembly, the ppiase activity of cypa has recently been demonstrated to have other roles, including intracellular trafficking, signal transduction, transcription regulation, cell cycle regulation, and stress response [2] . cypa is commonly believed to be an important immune molecule. cypa was originally identified as the target of the immunosuppressive drug cyclosporine a (csa), which is used clinically for the prevention of graft rejection after organ transplantation [3] . in mammals, csa-cypa complex binds to and inhibits calcineurin (cn), prevents the dephosphorylation and nuclear translocation of nf-at, and at last leads to immunosuppression [4] . cypa plays a vital role in regulating immune response. cypaknockout mice have an ''allergic'' phenotype with increased serum igg1 and ige levels and tissue infiltration by mononuclear cells, eosinophils and mast cells. this phenotype is related to increased and dysregulated activity of th 2 cd4 + t cells [5] . interleukin-2 tyrosine kinase (itk) is a member of the tec family of sh 2 /sh 3 -containing tyrosine kinases and participates in the signal transduction cascade leading to t cell activation [6, 7] . cypa binds itk and negatively regulates its activity. in cypa-knockout cells, itk is constitutively activated. thus, cypa plays a suppressive role in the development of cd4 + t cell responses through its interaction with itk. in recent years many studies showed that cypa was involved in the pathogenesis of viral infection [8] , cardiovascular disease [9] and cancer [10] . for example, cypa expression can be upregulated in viral infections and correlates with final result of infection [11, 12] . cypa plays a critical role in the successful replication of viruses such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), etc. however, cypa possesses a repressive effect on the replication of some viruses like influenza a virus and rotavirus. in this review we focused our interest on these diverse roles of cypa in viral infections. moreover, the emerging function of cypa on regulating host ifn-i response to viral infections was also discussed. cypa gene polymorphisms have been demonstrated to influence susceptibility to hiv-1 and disease progression. a1604g and c1650g in the promoter region of the cypa gene might affect cypa expression levels and thus affect host susceptibility to hiv-1 and disease progression [13] . furthermore, the a1650g polymorphism in the regulatory region of cypa gene may also be associated with protection from hiv-1 infection in participants of the amsterdam cohort studies on hiv-1 infection and aids [14] . hiv-1 replication was decreased in human cd4 + t cells when cypa was knocked out [15] . these studies suggest cypa may play an important role in promoting hiv infection. the efficient incorporation of cypa into the hiv-1 virion is mediated through the direct binding of the prolyl peptide bond, located in a proline-rich loop of the fourth and fifth helices of the hiv-1 ca, and the active sites of cypa in the context of gag polyprotein [16] . importantly, the proline residue at position 90 (p 90 ) and the glycine residue at position 89 (g 89 ), which are both highly conserved among hiv-1 gag polyproteins, appear to be important for this interaction [17] . cypa is packaged into hiv-1 virions during viral replication at a molar ratio of 1:10 cypa/ca [18] . disruption of its incorporation by gag mutations or by treatment with csa attenuated the infectivity of progeny viruses [19] . therefore, packaging of host cypa into hiv particles is an important step in hiv morphogenesis and essential for hiv replication. the viral protein r (vpr) of hiv-1 is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the pre-integration complex into the nucleus and the induction of g2 host cell cycle arrest [20] . the investigation of conformational heterogeneity of the proline residues in the n-terminus of vpr suggested a functional interaction between vpr and a host cypa [21] . csa blocked expression of vpr without affecting vpr transcription, intracellular transport, or virus incorporation. similarly, vpr expression is also reduced in hiv-1 infected cypa-knockout t cells [22] . in the absence of cypa activity, the vpr-mediated cell cycle arrest is completely lost in hiv-1-infected t cells. however, other study showed an opposite opinion that the interaction with cypa is not essential for the induction of g2 cell cycle arrest by vpr [23] . nef is another hiv accessory protein. it is believed that optimal hiv-1 infectivity requires the presence of both nef and cypa during virion assembly and these factors facilitate a step in the viral life cycle between penetration and reverse transcription. a genetic dissection study of the relative contributions of nef and the cyclophilin a-gag interaction to hiv-1 infectivity demonstrated that nef was not required for incorporation of cypa into hiv-1 virions and vice versa. surprisingly, cypa-deficient virions remained sensitive to inhibition by csa, in a manner that was strongly dependent on the presence of a functional nef gene [24] . cypa-nef fusion protein enhanced the infectivity of nef-defective hiv-1 particles and was specifically incorporated into the virions via association with gag during particle assembly [25] . together, these results demonstrate that nef and cypa may act independently but complementary to render hiv-1 particles fully infectious. cd147 has been identified as the main signaling receptor for cypa on human leukocytes [26] . cd147-cypa interaction may regulate an early step in hiv-1 replication [27] . cypa-cd147 interaction might induce ma phosphorylation to regulate the detachment of the rt complex from the membrane or promote transition from the step of hemifusion to complete fusion, allowing liberation of the rt complex into the cytoplasm [28] . on the other hand it is proposed that cypa-cd147 interaction might indirectly affect ca conformation leading to destabilization of the ca shell [29] . cypa mediates hiv-1 attachment to target cells via heparans, and heparans facilitate cypa-cd147 interaction by first binding cypa and then presenting it to cd147 [30] . therefore, cypa-cd147 interaction may be downstream of cypa-heparan interaction. moreover, the facilitative effect of cypa-cd147 interaction on hiv-1 replica-tion is signaling-independent but probably through specific events mediated by the cytoplasmic domain of cd147 [31] . similar to hiv-1, there were several evidences that cypa is also required for hcv replication. knockdown of endogenous cypa significantly hampers hcv rna replication and viral protein expression [32] and mutation of the residues that reside in the hydrophobic pocket of cypa (histidine in position 126 and arginine in position 55) failed to restore hcv replication [33] . cypa increased the affinity of the polymerase to viral rna by interacting with the ns5b and enhanced hcv replication [34] . these data are in accordance with the finding that the isomerase pocket of cypa serves as a binding site for ns5b polymerase. these findings indicate that cypa enhances hcv replication by catalyzing the comformational change of ns5b. interestingly, recent studies suggest that cypa inhibition may also act on the hcv ns5a protein. ns5a is anchored to the cytoplasmic side of endoplasmic reticulum (er) membrane via an amphipathic n-terminal a-helix [35] and is composed of three cytoplasmic domains: d1 (residues 27-213), d2 (residues 250-342) and d3 (residues 356-447). although sequence conservation of ns5a-d2 and -d3 in hcv genotypes is significantly lower than d1, it was reported that ns5a-d2 from the hcv jfh1 strain was the substrate for the ppiase activities of cypa [36] . in addition, mutations in d2 and d3 of ns5a that interacts with cypa could significantly influence viral rna replication and thus confer resistance to cyclophilin inhibitor [37] . furthermore, verdegem et al. [38] revealed that cypa has in vitro ppiase activity toward some, but not all of the peptidyl-prolyl bonds in ns5a-d3. this finding provided novel insights into the structure of ns5a-d3 and suggested that the interaction with cypa might involve more than one specific ns5a domain. besides the role as a cis/trans prolyl isomerase, cypa has also been implicated in cholesterol transport. it has been shown that a complex consisting of cypa, heat-shock protein 56, cyclophilin 40 and caveolin transports newly synthesized cholesterol from the endoplasmic reticulum to caveolae [39] . hcv depends on cholesterol and any disturbance in cholesterol level or distribution decreases its replication [40] . furthermore, cypa has been involved in transcription regulation and over-expression of cypa was shown to interact with sin3a/rdp3 hdac complexes and regulate hdac-dependent gene silencing in yeast [41] . the orthologous complex msin3a/histone deacetylase 2 (hdac2) was further found to modulate the transcription of the myc target gene, carbamoyl-phosphate synthase-aspartate carbamoyltransferasedihydroorotase (cad) in humans [42] . cad is a crucial enzyme in the pyrimidine de novo biosynthesis that hcv replication is known to depend on [43] . so it is possible that cypa might play some important indirect role in hcv infection. the hbv genome encodes the envelope proteins, the core protein, the polymerase protein and a transactivating x protein [44] . hbv produces three envelope glycoproteins collectively known as hbv surface antigen (hbsag), including the large (lhbs), middle (mhbs), and small (shbs) surface proteins [45] . among them, the shbs protein is predominant and is an important viral component that reacts with the immune system of the host. cypa, as a target protein interacting with shbs proteins, was found to be markedly decreased not only in the liver of hbsag transgenic mice, but also in an hbsag expressing cell line. however, when supernatants from transfected cells expressing hbsag were assayed for cypa, the amount of cypa was found to be higher than that from the controls, suggesting that the decrease of cypa in hbsag positive cells was due to increased secretion of cypa [46] . since both shbs and cypa are secreted via the vesicular secretion pathway [47] , the interaction between shbs and cypa may be directly or indirectly bridged by some cellular components. it is likely that cypa binds to shbs and is secreted along with hbsag particles. under physiological conditions, cypa is an intracellular protein; however, during inflammation, cypa can be secreted [48] . therefore, the expression of hbsag could be speculated to affect cellular functions similar to that of inflammation, resulting in secretion of cypa from hbsag-positive hepatocytes. in addition, the decrease of cypa observed in hbsag expressing cell lines may also affect protein unfolding and contribute to the pathogenesis of hbv infections as in hiv [49] . matrix protein (m1) is the most abundant structural protein that plays a crucial role in the replication, assembly and budding of influenza a virus [50, 51] . cypa was found in the core of the influenza virion [52] and was up-regulated upon infection by avian h9n2 influenza virus in ags cells (a human gastric carcinoma cell line) [53] . liu et al. [12, 54] revealed that cypa interacted with the m1 protein both in vitro and in vivo and affected the early stage of viral replication. over-expression of cypa restricted influenza a virus replication, while the depletion of endogenous cypa resulted in enhanced production of influenza a virus. in addition, the infectivity of influenza virus increased in the absence of cypa. cypa had no effect on viral genome replication or transcription and it also did not impair the nuclear export of viral mrnas. however, it was found that cypa could significantly enhance the degradation of m1 protein through the ubiquitin/proteasome-dependent pathway, indicating that cypa restricts influenza virus replication through accelerating degradation of the m1 protein. notably, the cypa r55a mutation could still bind to the m1 protein and inhibit influenza virus replication, suggesting that the restrictive effect of cypa on influenza virus infection was independent of its ppiase activity. cypa is also associated with the life cycle of other viruses including vaccinia virus (vv), vesicular stomatitis virus (vsv), and severe acute respiratory syndrome coronavirus (sars-cov) [55] . vv infection led to an impressive increase in cypa stability whereas in vv infected cells, cypa relocalized to the peripheral region of the nucleus, colocalizing with sites of virus production. cypa was then incorporated into the virus particle during morphogenesis and localized specifically in the core [1] . in addition, cypa has been found associated with vsv, a negative stranded rna virus. cypa interacts with the nucleocapsid protein of vsv and, like vv, incorporated into vsv viral particles where it likely acts as a chaperone for the nucleocapsid protein that wraps the genomic rna to produce the functional template for transcription [56] . cypa was also reported to regulate sars-cov replication through binding to the nucleocapsid protein and being incorporated into particles [57, 58] . rotavirus (rv) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. our study showed that rv infection triggered a temporal increase of cypa protein. in rv infection, cypa was recruited to the viroplasm of rv in ma104 cells and herein interacted with rv structural protein vp2 to repress rv infectivity to ma104 cells and inhibit rv reproduction through its ppiase activity (unpublished data). it has been shown that cypa is excluded from wild-type simian immunodeficiency virus (sivagm) particles but is efficiently packaged into vif-deficient sivagm virions. the presence of cypa in vifdefective sivagm was correlated with reduced viral replication. infection of cypa-knockout jurkat cells or treatment with cyclosporine a eliminated the vif-sensitive inhibition and resulted in replication profiles similar for wild-type and vif-deficient sivagm [59] . therefore, cypa was predicted as a novel vif-sensitive antiviral factor that may limit zoonotic transmission of sivagm or hiv. dendritic cells have a central function in the host defence, linking innate immunity to microbes to activate pathogen-specific adaptive immunity. recently it has been reported that cypa in dendritic cells could recognize the newly synthesized hiv-1 ca domain and subsequently prompt a ifn-i response through activation of irf3, which is dependent on the ppiase activity of cypa. thus it is concluded that cypa functions as a cell-intrinsic sensor of viral infection able to recognize the newly synthesized viral capsid proteins [60] . in our study we also found that cypa was required for the host ifn-i response in rv infection of ma104 cells, however, this was not dependent on ppiase activity but on the jnk signaling pathway [61] . redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles acetylation regulates cyclophilin a catalysis, immunosuppression and hiv isomerization pro-and anti-cancer effects of immunosuppressive agents used in organ transplantation the cyclophilins cyclophilin a-deficient mice are resistant to immunosuppression by cyclosporine regulation of the tyrosine kinase itk by the peptidyl-prolyl isomerase cyclophilin a mechanistic insight into the role of transitionstate stabilization in cyclophilin a cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target cyclophilin a: promising new target in cardiovascular therapy cyclophilin a: potential functions and therapeutic target for human cancer cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication cyclophilin a restricts influenza a virus replication through degradation of the m1 protein polymorphisms in the regulatory region of the cyclophilin a gene influence the susceptibility for hiv-1 infection cyclophilin a regulates hiv-1 infectivity, as demonstrated by gene targeting in human t cells regulatory polymorphisms in the cyclophilin a gene, ppia, accelerate progression to aids absconding with the chaperone: essential cyclophilin-gag interaction in hiv-1 virions catalysis of cis/trans isomerization in native hiv-1 capsid by human cyclophilin a contribution of cyclophilin a to determination of simian immunodeficiency virus tropism: a progress update vpr and its interactions with cellular proteins structural characterization of the hiv-1 vpr n terminus: evidence of cis/trans-proline isomerism cyclophilin a interacts with hiv-1 vpr and is required for its functional expression induction of g2 arrest and binding to cyclophilin a are independent phenotypes of human immunodeficiency virus type 1 vpr mechanistic independence of nef and cyclophilin a enhancement of human immunodeficiency virus type 1 infectivity nef enhances hiv-1 infectivity via association with the virus assembly complex preferential chemotaxis of activated human cd4+ t cells by extracellular cyclophilin a dealing with the family: cd147 interactions with cyclophilins cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a, proc. nat. acad. sci human immunodeficiency virus type 1 gag protein binds to cyclophilins a and b host cyclophilin a mediates hiv-1 attachment to target cells via heparans cd147 stimulates hiv-1 infection in a signal-independent fashion the isomerase active site of cyclophilin a is critical for hepatitis c virus replication cyclophilin inhibitors for the treatment of hcv infection critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex structure and function of the membrane anchor domain of hepatitis c virus nonstructural protein 5a hepatitis c virus ns5a protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins a and b identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns5a protein domain 3 of ns5a protein from the hepatitis c virus has intrinsic {alpha}-helical propensity and is a substrate of cyclophilin a annexin 2-caveolin 1 complex is a target of ezetimibe and regulates intestinal cholesterol transport multiple cyclophilins involved in different cellular pathways mediate hcv replication cyclophilin a and ess1 interact with and regulate silencing by the sin3-crpd3 histone deacetylase msin3a/histone deacetylase 2-and prmt5-containing brg1 complex is involved in transcriptional repression of the myc target gene cad dynamics of subgenomic hepatitis c virus replicon rna levels in huh-7 cells after exposure to nucleoside antimetabolites the hepatitis b virus and common mutants entry of hepatitis b virus: mechanism and new therapeutic target hepatitis b virus (hbv) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of hbv infection hepatitis b virus subviral envelope particle morphogenesis and intracellular trafficking extracellular cyclophilins contribute to the regulation of inflammatory responses proteomic analysis of hepatitis b surface antigen positive transgenic mouse liver and decrease of cyclophilin a restriction of viral replication by mutation of the influenza virus matrix protein identification of hsc70 as an influenza virus matrix protein (m1) binding factor involved in the virus life cycle cellular proteins in influenza virus particles proteomics analysis of differential expression of cellular proteins in response to avian h9n2 virus infection in human cells cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication cd147/ emmprin acts as a functional entry receptor for measles virus on epithelial cells requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus vif counteracts a cyclophilin a-imposed inhibition of simian immunodeficiency viruses in human cells a cryptic sensor for hiv-1 activates antiviral innate immunity in dendritic cells cyclophilin a inhibits rotavirus replication by facilitating host ifn-i production this work was supported by grants from national natural science foundation of china (no. 81102233) and natural science foundation project of cq cstc (no. cstc2011jja10002). key: cord-268467-btfz6ye8 authors: schreiber, steven s.; kamahora, toshio; lai, michael m.c. title: sequence analysis of the nucleocapsid protein gene of human coronavirus 229e date: 1989-03-31 journal: virology doi: 10.1016/0042-6822(89)90050-0 sha: doc_id: 268467 cord_uid: btfz6ye8 abstract human coronaviruses are important human pathogens and have also been implicated in multiple sclerosis. to further understand the molecular biology of human coronavirus 229e (hcv-229e), molecular cloning and sequence analysis of the viral rna have been initiated. following established protocols, the 3′-terminal 1732 nucleotides of the genome were sequenced. a large open reading frame encodes a 389 amino acid protein of 43,366 da, which is presumably the nucleocapsid protein. the predicted protein is similar in size, chemical properties, and amino acid sequence to the nucleocapsid proteins of other coronaviruses. this is especially evident when the sequence is compared with that of the antigenically related porcine transmissible gastroenteritis virus (tgev), with which a region of 46% amino acid sequence homology was found. hydropathy profiles revealed the existence of several conserved domains which could have functional significance. an intergenic consensus sequence precedes the 5′-end of the proposed nucleocapsid protein gene. the consensus sequence is present in other coronaviruses and has been proposed as the site of binding of the leader sequence for mrna transcriptional start. this region was also examined by primer extension analysis of mrnas, which identified a 60-nucleotide leader sequence. the 3′-noncoding region of the genome contains an 11-nucleotide sequence, which is relatively conserved throughout the coronavirus family and lends support to the theory that this region is important for the replication of negative-strand rna. human coronavirus 229e (hcv-229e) belongs to one of two major antigenic groups of human coronaviruses (macnaughton, 1981) . it shares antigenic relationships with other coronaviruses, such as porcine transmissible gastroenteritis virus (tgev), feline infectious peritonitis virus (fipv), and canine coronavirus (ccv). the other well-characterized human coronavirus, hcv-oc43, is in a separate antigenic group which includes mouse hepatitis virus (mhv) and bovine coronavirus (bcv). both human coronaviruses are mainly respiratory pathogens and have been estimated to cause up to 25% of common colds (mcintosh et a/., 1974; wege et a/., 1982) . they have also been implicated in gastrointestinal diseases (resta et a/., 1985) . furthermore, the isolation of coronaviruses bearing an antigenic relationship to hcv-oc43 from the central nervous system of two patients with multiple sclerosis has suggested a possible etiologic relationship between human coronaviruses and multiple sclerosis (burks et a/., 1980) . this possibility is supported by the observation that neurotropic strains of mhv cause demyelination in the central nervous system of rodents (weiner and stohlman, 1978) . thus, human coronaviruses are important human pathogens. the structural and biochemical properties of several coronaviruses, particularly mhv and avian infectious sequence data from this article have been deposited with the embugenbank data libraries under accession no. jo441 9. ' to whom requests for reprints should be addressed. 2 present address: department of virology, tottori university, school of medicine, yonago 683, japan. peritonitis virus (ibv), have been well characterized (lai et a/., 1987; boursnell et a/., 1987) . the virion contains a single-stranded, positive-sense rna molecule (molecular weight 6-8 x 1 o6 da) (lai and stohlman, 1978) associated in a helical conformation with nucleocapsid proteins (n) . the viral nucleocapsid is enclosed by an envelope, in which are embedded at least two types of viral proteins, the peplomer (e2) and matrix (el) glycoproteins. coronavirus rna replication occurs in the cytoplasm of infected cells and is mediated by a virusencoded rna-dependent rna polymerase (brayton et a/., 1982) . the virus-specific mrna in infected cells comprises a genomic-sized rna plus six subgenomic mrna species. these mrnas are arranged in a nested-set structure, which is characterized by rnas having common 3'-termini but extending for varying lengths in the 5'direction (lai et al., 1981) . only the 5'proximal regions of each mrna are translated (rottier et a/., 1981) . a unique feature of the structure of coronavirus is the existence, at the 5'-end of each mrna, of an identical leader sequence. this sequence is derived from the 5'-end of the genomic rna and is of approximately 70 nucleotides in length (lai eta/., 1983 (lai eta/., , 1984 . recent evidence has supported a role for the leader sequence in mediating a novel type of discontinuous transcription of genomic rna (baric et a/., 1985; makino et al., 1986; . in contrast to other coronaviruses, the molecular biology of human coronaviruses is relatively poorly understood. the genomic rna of both hcv-229e and hcv-oc43 has a molecular weight of approximately 6 x 1 o6 da (hierholzer et al., 1981) . the six subgenomic rna species appear to have lower molecular weights than those of the corresponding mhv rnas (weiss and leibowitz, 198 1) . the structure of these mrnas is not yet known. analysis of purified hcv-229e virions has revealed three major polypeptides: a glycosylated protein with a molecular weight of 180 kda, a phosphorylated nucleocapsid protein of 50 kda, and a family of polypeptides with molecular weights of 25, 23, and 21 kda (kemp et al., 1984) . in addition, several minor nonstructural polypeptides of 107, 92, and 39 kda have been identified (kemp et al., 1984) . the functions of these proteins have not yet been characterized. to further understand the molecular biology of hcv-229e, we have initiated molecular cloning and sequence analysis of hcv-229e rna. in this paper we report the sequence analysis of the gene encoding the nucleocapsid protein of hcv-229e. in addition, the mrna leader sequence was also identified. the results are compared with sequences of other coronaviruses including mhv, bcv, ibv, and tgev. hcv-229e (obtained from dr. j. fleming, university of southern california) was propagated at low multiplicities of infection in human fetal lung cells l132 (kennedy and johnson-lussenberg, 197511976 ) using dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum. virus purification and preparation of virion rna following a virus adsorption period of 1 hr at 37", hcv-229e-infected l132 monolayers were incubated at 37" for 24 to 48 hr, at which time the cell culture fluid was harvested. viruses were precipitated from 2 liters of culture fluid with 50% ammonium sulfate and centrifuged at 8000 rpm for 30 min. the pellet was resuspended in nte buffer (0.1 m naci, 0.01 m tris-hydrochloride (ph 7.2), 1 mm edta) and then placed on a discontinuous sucrose gradient consisting of 60, 50, 30, and 20% (w/w) sucrose in nte buffer and centrifuged at 26,000 rpm for 13 hr at 4" in a beckman sw28.1 rotor. the virus band at the interface between 50 and 30% sucrose was collected and diluted threefold with nte buffer. the diluted virus suspension was centrifuged on a linear sucrose gradient at 26,000 rpm in an sw28.1 rotor for 4 hr at 4". the virus band was collected and treated with proteinase k (0.2 mg/ml) for 20 min at 37", followed by 1% sds for 30 min at 37". genomic rnawas extracted with phenol and then with phenol/chloroform, and precipitated with ethanol. monolayers of l132 cells grown in 100 x 20-mm culture dishes were infected with hcv-229e. cells were incubated in phosphate-free dmem containing 1% dialyzed fetal calf serum 4 hr prior to rna extraction. actinomycin d (1 pg/ml) (sigma) and [3zp] or-thophosphate (70 &i/ml) (icn radiochemicals) were added at 3 and 2 hr, respectively, prior to rna extraction at 15 hr postinfection (p.i.). cells were collected in cold phosphatebuffered saline and centrifuged at 5000 rpm for 3 min at 4". the pellet was mixed with cold 0.5% nonidet-p40 in nte buffer, incubated for 10 min at 4', and then centrifuged at 5000 rpm for 3 min. the supernatant was transferred to a fresh tube containing l/10 vol of 10% sds at room temperature and vortexed briefly. intracellular rna was extracted with phenol and phenol/ chloroform and precipitated with ethanol. poly(a)-containing rna was selected by oligo(dt)-cellulose chromatography as previously described (makino et al., 1984) . to examine the kinetics of viral mrna synthesis, intracellular rna was extracted from virus-infected l132 monolayers in 60 x 15-mm culture dishes at 7, 21, 29, 46, and 58 hr postinfection. cdna cloning cdna cloning was performed using a modified method of gubler and hoffman (1983) . the poly(a)containing rna extracted from 229e-infected l132 monolayers was precipitated, dried, and resuspended in 6.72 ~1 of autoclaved water. the rna was incubated with 10 mm methylmercuric hydroxide in an 8 ~1 total volume for 10 min at room temperature. first-strand cdna synthesis was carried out in a 50-~1 reaction mixture containing 60 units rnasin (promega biotec), 10 mm mgci,, 100 mm kci, 50 mm tris-hci (ph 8.3 at 42") 10 mm dtt, 1.25 mm dntps, 40 &i [a-32p]datp (3000 ci/mmol), 28 mm ,&mercaptoethanol, and 10 ng oligo(dt),2-,s primer. after 5 min at room temperature, 40 units of amv reverse transcriptase (life science) was added and the mixture was incubated for 1 hr at 42". the reaction was stopped by adding 4.4 ~1 of 250 mm edta. the products were extracted with phenol/ chloroform and precipitated with ethanol containing 0.3 m ammonium acetate. for second-strand synthesis, the loo-~1 reaction mixture contained 5 mm mgci,, 100 mm kci, 20 mm tris-hci (ph 7.5) 50 pgl ml bovine serum albumin (bsa), 10 mm ammonium sulfate, 0.15 mm p-nad, 100 pm dntps, 25 units of escherichia co/i dna polymerase i, 2 units of e. co/i dna ligase, and 0.8 units of rnase h. sequential incubations were for 1 hr at 12" and 1 hr at 22". the reaction was stopped by the addition of 8.7 ~1 of 250 mm edta and the products were extracted with phenol/ chloroform and precipitated with ethanol in the presence of 0.3 mammonium acetate. homopolymeric tailing of double-stranded cdna with poly(c) was carried out in a 1 ~-pi reaction mixture containing 10 units of terminal transferase, 200 mm potassium cacodylate, 0.5 mm co&, 25 mm tris-hci (ph 6.9), 2 rnn/l dlt, 250 pg/ml bsa, and 50 pm dctp at 37" for 4 min. the dc-tailed double-stranded dna was annealed to 200 pg of dg-tailed pstl-cut pbr322 plasmid in 20 ~1 of a buffer containing 10 mni tris-hci (ph 7.4), 100 mm naci, and 0.25 mh/l edta. the mixture was incubated for 5 min at 68" and then cooled slowly overnight. the annealed molecules were used to transform e. co/i mci061 as described (dagert and erhlich, 1979) . colonies grown on lb/tetracycline plates were incubated at 37" for 12 hr and transferred to colony/plaque screen disks (new england nuclear). bacterial lysis and dna fixation were carried out according to the methods previously described (grunstein and hogness, 1975) . the disks were prehybridized in a solution containing 0.2% polyvinylpyrrolidone (mw 40,000), 0.2% ficoll (mw 400,000), 0.2% bsa, 0.05 mtris-hci (ph 7.5) 1% sds, 1 l\/i naci, 10% dextran sulfate, and 100 pg/ml denatured salmon sperm dna at 65" for 6hr. fragments derived from either the 5'-or 3'-ends of gene 7 were labeled with 32p by nick-translation and added to the solution. hybridization was carried out for 20 hr at 65". the disks were then washed twice in 2~ ssc (0.3 lvi naci, 30 mlvi sodium citrate) at room temperature, twice in 2x ssc containing 1% sds for 30 min at 65", and twice in 0.1 x ssc at room temperature for 30 min. the disks were air-dried and exposed to xray film at -70". intracellular rna from virus-infected cells was denatured by glyoxal treatment and separated by electrophoresis on a 1% agarose gel containing 10 mll/l sodium phosphate (ph 7.0) as described previously (mc-master and carmichael, 1977) . rna transfer to biodyne nylon filters (icn radiochemicals) and subsequent hybridization were performed according to the method described by thomas (1980) . a synthetic oligodeoxyribonucleotide was 5'-end-labeled with [y-~'p]atp by polynucleotide kinase (pedersen and haseltine, 1980) . the total amount of poly(a)-containing rna extracted from 229e-infected cell monolayers in three 150 x 20-mm culture dishes was incubated in 8 pi of distilled water containing 10 mm methylmercuric hydroxide for 10 min at room tem-perature. a further incubation was carried out in a 50-~1 reaction volume containing 60 units of rnasin (promega), 10 mm mgc12, 100 mm kci, 50 ml\/l tris-hci (ph 8.3 at 42") 10 mm dlt, 1.25 mn/ldntps, 28 mm ,&mercaptoethanol, 5'-end-labeled synthetic oligodeoxyribonucleotides, and 20 units of amv reverse transcriptase (life science) for 1 hr at 42". reaction products were extracted with phenol/chloroform, precipitated with ethanol, and then analyzed by electrophoresis on a 6% polyacrylamide gel containing 8.3 m urea. the primer-extended product was identified by autoradiography and eluted from the gel according to the published procedure (maxam and gilbert, 1977) . sequencing was carried out by the dideoxyribonucleotide chain termination method (sanger et al., 1977) as well as the chemical modification procedure (maxam and gilbert, 1977) . in the first method, fragments of cdna inserts generated by various restriction endonucleases were cloned into the ml 3 vectors mp18 and mp19 (messing and vierira, 1982) . [(u-~~s]-datp was used as a label. sequence data were also obtained by chemical modification (maxam and gilbert, 1977) of various cdna fragments subcloned into the pt7-3 vector (tabor and richardson, 1985) . in the second method, cdna fragments were 3'-end-labeled with klenow fragment at internal restriction sites or, alternatively, at the polylinker cloning site of pt7-3. end-labeled cdna restriction fragments were separated by electrophoresis on preparative polyacrylamide gels (maxam and gilbert, 1980) and purified as described previously (hansen et a/., 1980; hansen, 1981) . sequencing of the primer-extended product of mrna7 was performed by the chemical modification procedure (maxam and gilbert, 1977) . sequence analysis was performed by the lntelligenetics and seqaid programs. hydropathy profiles were constructed using the pepplot program of the university of wisconsin computer genetics group, which employs both the kyle-doolittle (kd) and goldman, engelman, steitz (ges) algorithms. to determine the optimum time for extracting 229especific mrnas, we first studied the kinetics of virusspecific mrna synthesis. intracellular rna was extracted from infected l132 monolayers at specified times p.i. the rna was separated by agarose gel electrophoresis (fig. 1) . as can be seen, viral mrna synthesis could be detected as early as 7 hr p.i. and reached maximum at 29 hr p.i. thereafter, total rna synthesis gradually declined. by 46 hr p.i. onlythe most abundant mrna species were evident. the number and size of these mrna species are comparable to those of mhv mrnas and are in agreement with previously published results (weiss and leibowitz, 1981) . significantly, mrna 2a, which was previously found only in bcv-infected cells and proposed to encode hemagglutinins (king et a/., 1985; keck et a/., 1988) was not present. this is consistent with the finding that hcv-229e does not have hemagglutinating activity (hierholzer, 1976) . the relative amounts of the mrna species were the same throughout the replication cycle. therefore, in all of our subsequent experiments, the virus-specific intracellular rnas were extracted at 15 hr p.i. molecular cloning of hcv-229e genomic rna and intracellular virus-specific mrnas cdna cloning was initially performed using virion genomic rna as a template. the sizes of inserts in the resultant cdna clones ranged from 0.2 to 0.5 kb in length. one clone, a34, contained a 0.45-kb insert, which was subsequently characterized by restriction mapping and northern blot analysis. the 0.45kb fragment was labeled with 32p by nick-translation and hybridized with intracellular rna from 229e-infected cells. the result, shown in fig. 2 , revealed that the fragment hybridized to each of the mrna species. this result suggested that the hcv229e subgenomic mrnas possess a nested-set structure similar to other coronaviruses and that a34 represented a cdna clone of either the 3'-end of the genomic rna or the leader sequence. cloning was subsequently carried out using intracellular rna from 229e-infected cells as a template. the resulting cdna clones were screened by colony hybridization using the 0.45-kb fragment from clone a34 as a nick-translated probe (fig. 3) . several positive colonies were identified and characterized further. clone l8 contained a 3.6-kb insert but lacked a 3'-poly(a) tail. clone l37, which contained an insert of 1.7 kb, overlapped l8 but was 0.1 kb shorter at the 3'-end. this clone also lacked a poly(a) sequence (see below). therefore, additional cdna clones were isolated using a 0.24-kb bal i-ecori fragment of l8 (fig. 3a) as a probe. these latter clones were further characterized by southern blot analysis. clone slo contained an insert of 0.8 kb which overlapped the 3'-ends of the two previous clones and extended another 0.4 kb in that direction. figure 3b shows the orientation and sizes of clones l8, l37, sl 0, and a34 with reference to theviral genome. restriction enzyme sites used for sequencing are also shown. to determine the sequence of the 3'-end of hcv-229e genome, various restriction fragments of l8, l37, and slo were subcloned into ml 3 vectors. for l8, only the 1.2-kb fragment extending from an internal pstl site toward the 3'-end was sequenced. clone l37 was also sequenced in part. figure 3c shows the cdna fragments and strategy used in sequencing. each region a primer extension study was carried out using a synthetic oligodeoxyribonucleotide complementary to an 18.mer sequence underlined near the 5'.end of the gene. the 3'noncoding region contains a conserved sequence which is shown by the double line. the intergenic conserved sequence, tctaaact, is also shown (dotted line) was verified by dideoxy chain termination sequencing of both strands or by the chemical modification method. clone sl 0 was found to have a poly(a) stretch of 34 bases. figure 4 shows the complete dna sequence with a translation of the main open reading frame (orf) in one-letter amino acid code. this orf extends from base 147 to base 13 13 and predicts a 389 amino acid protein with a molecular weight of 43,366 da. this predicted molecular weight is slightly smaller than the measured molecular weight of the nucleocapsid protein of hcv-229e, which is 50 kda as determined by sds-polyacrylamide gel electrophoresis (macnaughton, 1980) . the difference is probably due to phosphorylation or other modification of the protein. the predicted protein shares features with the nucleocapsid proteins of tgev, mhv, bcv, hcv-oc43, and ibv (kapke and brian, 1986; skinner and siddell, 1984; armstrong er a/., 1983; lapps et a/., 1987; kamahora et al., 1988; boursnell et a/., 1985) . namely, the protein is highly basic and rich in serine residues. sixty percent of the amino acid residues are basic and 12% are acidic. there are 39 serine residues (10% of total), which are presumed to be sites of phosphorylation (stohlman and lai, 1979) . when compared to tgev, with which hcv-229e shares antigenic properties, both n proteins have identical amounts of basic and acidic amino acids and serine residues and similar molecular weights (kapke and brian, 1986) . figure 5 shows a schematic diagram of the possible orfs obtained by translating the nucleotide sequence. the orf in frame 3 is likely the one which encodes the nucleocapsid protein. in frame 2, the 5'-flanking region probably contains part of the sequence of the matrix protein encoded by gene 6. this possibility is sup--i1 i ii i iii -----__ 1 iii1 -i -llil i ii ii i i i iii i ii 2-j i i 111111 ii i1111111 lllll iii i ii, 1111 i ill1 iiii i 3 i i i i ill i i i11 i i i i i ported by the finding that reading frame 2 remains open at the extreme 5'-end. furthermore, the sequence tctaaact, which is found in the intergenic regions of several other coronaviruses (kapke and brian, 1986; skinner and siddell, 1984; armstrong et a/., 1983; lapps et al., 1987; kamahora et a/., 1988; budzilowicz eta/., 1985) is also present between the presumed initiation codon of the main orf and the 3'-end of gene 6. this sequence is the proposed site of fusion of the leader sequence with the mrna coding region makino et al., 1986; budzilowicz et al., 1985) . the 3'-noncoding region contains the sequence tggaagagcca, 75 nucleotides from the 3'-end (fig. 4) which is relatively conserved among coronaviruses and is found at approximately the same location in all of these viral genomes (kapke and brian, 1986; skinner and siddell, 1984; armstrong et a/., 1983; lapps et al., 1987; kamahora et a/., 1988; boursnell et al., 1985) ( table 1) . there is only one nucleotide difference in this conserved sequence when it is compared with that of tgev, bcv, and hcv-oc43. two and three nucleotide differences are found in ibv and mhv, respectively. this conservation of sequence and location suggests that it may be important for viral rna replication. in frame 1, there are several additional orfs of at least 30 amino acids. some of these, including one found in the 3'-noncoding region, lack appropriate translation start sites. another long internal orf is found from base 322 through 693. this contains an appropriate initiation sequence and encodes a hypothetical protein of 13,974 da, which is rich in leucine residues (17%). the significance of this orf remains to be defined. the mrnas of coronaviruses contain a stretch of leader sequence which is derived from the 5'-end of the viral genome and exhibits homologywith the intergenic consensus sequence budzilowicz et al., 1985) . since our cdna clones did not appear to contain leader sequences, we used primer extension studies to determine the sequence of the hcv-229e leader rna. a synthetic oligodeoxyribonucleotide which was complementary to an 18-mer sequence located near the 5'-end of the gene (fig. 4) was end-labeled and used in a primer extension study with poly(a)-selected intracellular mrna as a template. the reaction products, separated by agarose gel electrophoresis, revealed six bands (data not shown). since these bands were most likely to represent the primerextended products of the individual mrna species, the smallest and most abundant band, corresponding to the primer-extended product of mrna7, was eluted and sequenced by the chemical modification method (maxam and gilbert, 1977) . the sequence of the 3'-end of the primer-extended product was identical to the l8 sequence from nucleotides 129 to 17 1. at nucleotide 128, immediately 5' to the proposed leader mrna fusion site, the sequence diverged from the l8 sequence and revealed a putative 60-base leader sequence which is shown in fig. 6 . the figure also shows a degree of homology with the leader sequence of ibv. considerably less homology exists between the leader sequence of hcv-229e and those of hcv-oc43 and mhv-jhm (data not shown). this report presented the primary sequence of the nucleocapsid gene and leader sequence of hcv-229e. when compared to the known sequences of other coronaviruses (kapke and brian, 1986; skinner and siddell, 1984; armstrong et a/., 1983; lapps et al., 1987; kamahora et al., 1988; boursnell et al., 1985) , common features of coronavirus nucleocapsid proteins emerged; namely, they are highly basic and have a high proportion of serine residues, which have been shown 30 40 50 60 i i i i i i hcv-22 9e 5'-cttaag*taccttat*ctatcta*caaatagaaaag **ttgctttttagactttgtgtc*ta*cttc . . . . . . . . . . . . :: : : ::: :: : : :: : :: :::: :::. . ::: : :: : ibv 5'-acttaagatagatattaatatatatctattacactagccttgc**gctagatttttaa*cttaacaaa..... fig. 6. hcv-229e mrna leader sequence compared to the leader sequence of ibv. the ibv leader extends for at least 16 nucleotides in the 3' direction. to be sites of phosphorylation (stohlman and lai, region of 46% homology within the amino-terminal 1979). the relationship between the nucleocapsid one-third of the protein which extends from residues genes of wv-229e and tgev is particularly interest-29 to 134 in hcv-229e, and 41 to 146 in tgev. furing since the viruses are antigenically related (mac-thermore, approximately 10 amino acids downstream naughton, 1981). the predicted molecular weights of from the homologous region in both proteins lies an the n protein and the number of potential phosphoryla-area which is abundant in serine residues, suggesting tion sites of both viruses are almost identical. although that this may be an important functional domain of the these two viruses have little nucleotide sequence ho-molecule. to further examine such functional homolmology between their nucleocapsid genes, the amino ogy between the two proteins, hydropathy profiles acid sequences are homologous within a limited re-were constructed (fig. 7) . the contour of these plots gion. amino acid sequence analysis revealed several suggests that a certain degree of functional homology structural features common to both viruses, which may exists within the first and last one-third of each molehave functional significance. for instance, there is a cule, with an additional region around position 200. the peak around position 200 occurs just after the serine-rich region of the molecule. the relative conservation of these regions suggests a possible role in the interaction of the n protein with the viral genome. similar structural features exist among the n proteins of hcv-229e, ibv, mhv, hcv-oc43, and bcv (skinner and siddell, 1984; lapps et a/., 1987; kamahora et a/., 1988; boursnell et a/., 1985) . this is demonstrated by the hydropathy profiles of these proteins, which are also shown in fig. 7 . further studies are required to reveal the functional significance of the conserved domains. another interesting finding is the open reading frame internal to the main coding region of the hcv-229e n gene. thus far, two other coronaviruses, bcv and mhv-jhm, have been found to contain internal orfs in gene 7 (skinner and siddell, 1984; lapps eta/., 1987) which are preceded by optimum translation initiation signals according to kozak's consensus sequence (kozak, 1983) . the predicted amino acid sequences could encode hypothetical proteins of molecular weights 13,973; 14,842; and 23,057 for hcv-229e, mhv-jhm, and bcv, respectively. interestingly, all three sequences are abundant in leucine residues (17 to 19%). hcv-oc43 also has two smaller internal orfs encoding potential leucine-rich proteins of 8830 and 16,297 molecular weights (kamahora et a/., 1988) . further studies to determine whether this hypothetical protein can be detected in 229e-infected cells or by in vitro translation of a full-length cdna clone (i.e., l8) are in progress. finally, the 3'-noncoding conserved sequence of gene 7 lends additional support to a common ancestry for coronaviruses, regardless of antigenic subgroup. this sequence has been proposed as a recognition site for the virus-encoded rna-dependent rna polymerase prior to negative-strand synthesis (kapke and brian, 1986) . certainly future studies must focus on examining the role of this conserved region in the viral replication cycle. sequence of the nucleocapsid gene from murine coronavirus mhv-a59 characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna polymerase activities induced by mouse hepatitis virus. 1. viral three intergenic regions of coronavirus mouse hepatitis virus strain a59 genome rna contain a common nucleotide sequence that is homologous to the 3'end of the viral mrna leader sequence two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients prolonged incubation in calcium chloride improves the competence of escherichia coli cells colony hybridization: a method for the isolation of cloned dnas that contain a specific gene simple and very efficient method for generating cdna libraries use of solubilizable acrylamide disulfide gels for isolation of dna fragments suitable for sequence analysis chemical and electrophoretic properties of solubilizable disulfide gels purification and biophysical properties of human coronavirus 229e the rna and proteins of human coronaviruses sequence analysis of nucleocapsid gene and leader rna of human coronavirus oc43 sequence analysis of the porcine transmissable gastroenteritis coronavirus nucleocapsid protein gene temporal regulation of bovine coronavirus rna synthesis characterization of viral proteins synthesized in 229e-infected cells and effect(s) of inhibition of glycosylation and glycoprotein transport /76). isolation and morphology of the internal component of human coronavirus, strain 229e bovine coronavirus hemagglutinin protein comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles replication of coronavirus rna. /n "rnagenetits characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic virus mouse hepatitis virus a59: messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv-3 coronavirus: a jumping rna transcription presence of leader sequences in the mrna of mouse hepatitis virus the rnaof mouse hepatitis virus sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes the polypeptides of human and mouse coronaviruses structural and antigenic relationships between human, murine and avian coronaviruses leader sequences of murine coronavirus rna can be freely reassorted: evidence for the role of free leader rna in transcription analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus a new method for sequencing dna sequencing end-labeled dna with base-specific chemical cleavages coronavirus infection in acute lower respiratory tract disease of infants analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange a new pair of ml3 vectors for selecting either dna strand of double-digest restriction fragments a micromethod for detailed characterization of high molecular weight rna antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species isolation and propagation of a human enteric coronavirus translation of three mouse hepatitis virus strain a59 subgenomic rnas in xenopuslaevisoocytes dna sequencing with chain-terminating inhibitors the 5'-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderprimed transcription nucleotide sequencing of mouse hepatitis virus strain jhm messenger rna 7 phosphoproteins of murine hepatitis virus a bacteriophage t7 rna polymerase/promoter system for controlled exclusive expression of specific genes hybridization of denatured rna and small dna fragments transferred to nitrocellulose the biology and pathogenesis of coronaviruses viral models of demyelination comparison of the rnas of murine and human coronaviruses we thank carol flores for assistance in preparation of the manuscript. this work was supported by public health service research grants nsl8146 and all 9244 from the national institutes of health and grant 1449 from the national multiple sclerosis society. s.s.s. is supported by a postdoctoral training fellowship from the national institutes of health grant ns07149. key: cord-276364-zyw5aukk authors: wong, ho him; sanyal, sumana title: manipulation of autophagy by (+) rna viruses date: 2019-08-08 journal: semin cell dev biol doi: 10.1016/j.semcdb.2019.07.013 sha: doc_id: 276364 cord_uid: zyw5aukk autophagy is an evolutionarily conserved process central to host metabolism. among its major functions are conservation of energy during starvation, recycling organelles, and turnover of long-lived proteins. besides, autophagy plays a critical role in removing intracellular pathogens and very likely represents a primordial intrinsic cellular defence mechanism. more recent findings indicate that it has not only retained its ability to degrade intracellular pathogens, but also functions to augment and fine tune antiviral immune responses. interestingly, viruses have also co-evolved strategies to manipulate this pathway and use it to their advantage. particularly intriguing is infection-dependent activation of autophagy with positive stranded (+)rna virus infections, which benefit from the pathway without succumbing to lysosomal degradation. in this review we summarise recent data on viral manipulation of autophagy, with a particular emphasis on +rna viruses and highlight key unanswered questions in the field that we believe merit further attention. rna viruses have evolutionarily constrained genome sizes. at the same time they have co-evolved efficient means to manipulate host cellular processes to acquire nutrients while evading immune detection. multifunctional viral proteins, molecular mimicry of host components, and the intrinsically high mutagenicity of their rna genome converge to dysregulate host cellular pathways, and exploit metabolic processes to their advantage. one such target is the autophagy machinery. while this cellular degradative process has been historically described to restrict intracellular pathogens including bacteria, parasites and viruses, many have evolved mechanisms to circumvent and even actively benefit from it. autophagy is initiated by sequestration of cytoplasmic proteins and damaged organelles into crescent-shaped double-membrane vesicles known as isolation membranes, long-debated on their membrane source [1] . the best understood trigger for induction of autophagy is amino acid deprivation, whereby autophagy related proteins (atgs) are recruited to nucleate the isolation membrane, which forms a cup-shaped phagophore. current consensus on the source of autophagosomal membranes is the endoplasmic reticulum [2] . once contents are captured, the immature isolation membranes expand to forming autophagosomes, which subsequently fuse with lysosomes, thus forming autolysosomes. the contents undergo degradation within the autolysosomes to enable recycling during starvation. about 30 genes have been reported to participate in the process of autophagosomal degradation. the core autophagy proteins are broadly categorised into five complexes: (i) the unc-51 like kinase 1 (ulk1) complex, (ii) atg9, (iii) the class iii pi3k complex, (iv) wd repeat domain phosphoinositideinteracting proteins (wipi), and (v) ubiquitin-like atg12 and atg8 complexes. although a mechanistic understanding of the process is currently incomplete, formation of phagophore is believed to involve a cooperative activity of the ulk1 and pi3k complexes, along with local phosphatidylinositol synthesis. these activities are followed by recruitment of atg9-containing vesicles to phagophore assembly sites, which results in membrane expansion to form the autophagosomes (fig. 1 ). detailed analyses of the known regulatory mechanisms have been reviewed elsewhere [3, 4] . apart from turnover of organelles and primarily long-lived proteins, autophagy operates to defend host cells against intracellular pathogens -delivering trapped bacterial or viral products to lysosomes for degradation. besides, it is equipped to clear invasive pathogens through induction of cd8 + t-cell responses, and also initiates a primordial innate immune response by cooperating with pattern recognition receptor signalling to induce interferon production. this was recently described in the context of dna virus infections, where cyclic gmp-amp (cgamp) and sting-dependent activation of autophagy was necessary to remove viral dna from the cytosol [5] . however, in an ongoing evolutionary arms race, most pathogens have acquired the ability to hijack and subvert autophagy to evade degradation through this pathway. remarkably, +rna viruses have adapted to not only protect themselves from autophagic elimination but even harness the machinery to their own benefit, as will be covered in more detail in the subsequent sections. among the +rna viruses, data on favourable versus detrimental impact of autophagy is particularly confounding [4] . a link between autophagosomes and virus-induced vesicles was proposed by george palade by em imaging of poliovirus containing vesicles that resembled autophagosomal membranes [6] . over the past few decades, a growing body of research has defined the critical role of this pathway in facilitating infection by numerous +rna rna viruses, including poliovirus (pv) [7, 8] , coxsackievirus b3 (cvb3) [9, 10] , cvb4 [11] , enterovirus 71 (ev71) [12] , human rhinovirus (hrv) [13] , foot-and-mouth disease virus (fmdv) [14] , encephalomyocarditis virus (emcv) [15] , dengue virus (denv) [16, 17] , zika virus (zikv) [18, 19] , hepatitis c virus (hcv) [20] , mouse hepatitic virus (mhv), newcastle disease virus (ndv) [21] , severe and acute respiratory syndrome coronavirus (sars-cov) [22] , chikungunya virus (chikv) [23] , and japanese encephalitis virus (jev) [24] . in many of the above cases, pharmacological or genetic manipulation of autophagy in vitro confirmed an inhibition in replication and/or spread of these viruses, whereas induction of autophagy resulted in increased production of progeny virions [21, 25] . current evidence indicates that many, if not all +rna rna viruses depend on the initiation of the autophagic pathway for their optimal production. this is counterintuitive, since these viruses replicate in the cytosol, and autophagy serves to promote degradation of cytosolic contents. therefore, it is evident that +rna rna viruses in particular have evolved sophisticated mechanisms to circumvent or exploit this pathway. not surprisingly, most +rna rna viruses also trigger massive membrane remodelling within infected host cells to create membrane delineated structures, often referred to as replication organelles, vesicle packets, convoluted membranes or double membrane vesicles, depending on their morphology and ultrastructure [18, 26] . whether these replication organelles are pseudo-autophagosomes, has long been a point of contention. several genome-wide screens, e.g with crispr/cas9 libraries, haploid kbm7 cells, and shrna depletions, as well as proteomic studies have universally indicated the involvement of the autophagy pathway in +rna rna virus infections [25, 27, 28] . however, among the 30 odd autophagy-related genes, the functional contribution of the individual components in virus infection is far from clear. a recent targeted crispr/cas9 screen uncovered a fairly diverse range of involvement among autophagic components in three +rna rna virus infections -pv, denv and zikv [29] . the authors reported that all three viruses employed multiple proteins of the autophagy pathway while bypassing others, and each virus used a unique set of initiation components. a common feature among the tested viruses underscored the requirement of the lc3 protein but not its canonical cellular lipidation process, where lc3 was recruited to virally induced membranes by alternative means. this study highlights the importance of assessing the pathway in its entirety when seeking to understand how pathogens co-opt it for purposes of genome replication and spread, as well as to identify universal drug targets. many different mechanisms have been proposed on how autophagy is manipulated to facilitate infection while preventing degradation of +rna rna viruses (fig. 2 ). while in no way exhaustive, the following sections cover the salient features that are recurrent among several viral genera: the physical hallmark of the autophagy pathway is formation of lc3+ cytosolic double-membrane vesicles, also often observed in +rna rna virus infections. one of the long-running debates is whether these replication organelles are themselves immature autophagosomes or take advantage of the same machinery for their biogenesis. however, canonical autophagic vesicles are part of a degradative pathway, where they fuse with lysosomes for their contents to be hydrolysed by proteases and lipases. viruses from different families appear to possess a diverse set of strategies to prevent this from happening. flaviviruses, such as denv and zikv have been reported to trigger autophagy on the one hand, while utilising the er as a focal point for generating their replication organelles and assembly of progeny virions. consequently, both viruses have evolved means to suppress er-turnover via reticulophagy. the er-localised reticulophagy receptor fam134b was identified as a restriction factor for both denv fig. 1 . induction of the autophagy pathway. autophagy is initiated typically from cellular stress, such as starvation, whereby unc-51-like kinase 1 (ulk1) complex (comprising ulk1, autophagy-related protein 13 (atg13), fip200 and atg101 are activated. this complex triggers nucleation of the phagophore by phosphorylating components of the class iii pi3k (pi3kc3) complex i (consisting of class iii pi3k, vacuolar protein sorting 34 (vps34), beclin 1, atg14, activating molecule in beclin 1-regulated autophagy protein 1 (ambra1) and general vesicular transport factor (p115). this in turn activates local phosphatidylinositol-3-phosphate (pi3p) production at discrete er sites often referred to as omegasomes. wd repeat domain phosphoinositide-interacting proteins (wipis) and zincfinger fyve domain-containing protein 1 (dfcp1) are then recruited to these phagosome assembly sites followed by recruitment of atg12˜atg5-atg16l1 complex that enhances atg3-mediated conjugation of atg8 family proteins, including microtubule-associated protein light chain 3 (lc3) proteins to membrane-resident phosphatidylethanolamine (pe), thus forming the membrane-bound, lipidated form lc3-ii -the characteristic signature of autophagic membranes. atg8s are required for elongation and closure of the phagophore membrane, and in selective autophagy, are involved in sequestration of specific cargo into autophagosomes. several cellular membranes, most likely the er, contribute to elongation of the autophagosomal membrane by serving as membrane reservoir -delivered by atg9-containing vesicles. once sealed, autophagosomal membranes give rise to double-layered vesicles called autophagosomes, which mature and fuse with the lysosomes. autophagic cargo is hydrolysed and recycled back to the cytoplasm. and zikv. rnai-depletion of fam134b significantly enhanced denv and zikv replication at an early stage of the viral life cycle. the virusencoded ns3 protease from several flaviviruses directly cleaved fam134b at a single site within its reticulon homology domain to selectively suppress er degradation [30] , underscoring a sophisticated mechanism to differentially regulate specific arms of autophagy. coxsackievirus b3 (cvb3), an enterovirus belonging to the picornaviridae family, is known to rely on autophagosome formation for optimal replication [31, 32] ; however, both in vitro and in vivo evidence suggest that during infection, amphisome maturation and autophagic protein degradation are inhibited. an increase in autophagosomal abundance concomitant with a decrease in autophagic flux was reported with cvb3, prompting the hypothesis that infection selectively triggers autophagosome formation while preventing the terminal stages in degradation [7, 33] . the molecular determinants and mechanism by which cvb3 limits autophagic degradation is currently unknown. interestingly, treatment of cvb3-infected cells with inhibitors of autophagosome maturation resulted in increased virus production, indicating that canonical autophagy was not completely blocked in virus-infected cells, and at least a population of the virus remained sensitive. a similar finding was reported more recently with rotavirus where virus replication benefited from induction of autophagy while blocking degradation [34] . as with cvb3, the mechanism by which rotaviruses specifically inhibit autolysosomal degradation has not been elucidated, emphasising the importance of identifying the specific virus or host components that prevent degradation to provide fundamental insights on autophagic regulation in general. the case with hcv infection is more convoluted on account of contradictory data: whereas gfp-rfp-lc3 expressing cells infected with hcv displayed a complete maturation of autophagosomes followed by fusion with lysosomes [35, 36] , atleast one other study reported that hcv replication restricted autophagosomal fusion [37] . yet another study reported that the autophagy pathway in its entirety was necessary during hcv-infection for optimal replication; however, the advantage derived from it was primarily due to suppression of innate immune responses [36] . blocking fusion between autophagosomes and lysosomes has also been reported to increase denv2 yield [38] . however, this effect may be viral serotype-specific, since inhibiting lysosome fusion reduced denv3 production [39] . the mechanisms by which autophagy favors denv production was recently described where rather than replication, assembly and release of progeny virions was affected by blocking autophagy mediated lipid droplet hydrolysis [16, 40, 41] . with coronaviruses, initiation of autophagy appears to be through the er-derived, ptdlns3p-enriched omegasomes that normally operate during starvation. infectious bronchitis virus (ibv) -an avian coronavirus responsible for major losses to the poultry industry, is one such example where a significant portion of the genome encodes nonstructural proteins (nsp) dedicated to virus replication. expression of nsp6 proteins resulted in increased levels of ptdlns3p on er membranes, recruitment of ptdlns3p effector protein wipi2 and the generation of autophagosomes directly from the er [42, 43] . however, when compared to the properties of starvation-induced autophagosomes, those generated by coronavirus infection or nsp6 proteins presented significant differences. nsp6-induced autophagosomes displayed limited ability to undergo expansion, preventing formation of large autolysosomes, and hence circumvented degradation of viral particles through the lysosomal pathway [43] . results obtained with ibv, sars and mhv nsp6 was recapitulated with middle eastern respiratory syndrome coronavirus (mers-cov), where a similar phenomenon was observed for nsp6 [44] . a distinctive feature shared by +rna rna viruses is to assemble and replicate on intracellular membranes, which have been proposed to offer a two-fold advantage: (a) scaffold for anchoring and concentrating schematic illustration of the different pathways of selective autophagy that are triggered upon +rna rna virus infections. initiation of autophagosomes is through formation of an isolation membrane most likely derived from the er. depending on the molecular composition and function, they may form either omegasomes, edemosomes or amphisomes. flaviviruses such as zikv non-structural protein 4a (ns4a) and ns4b activate autophagy by inhibiting akt and mtorc1; autophagosomes generated are subverted to specialized functions to prevent viral degradation. turnover of organelles occur through convergence of specialised autophagosomes with lysosomes for their selective degradation: er via reticulophagy; mitochondria via mitophagy; lipid droplets via lipophagy and virions or viral proteins via virophagy. viruses that are known to upregulation specific autophagosomal pathways are depicted in black, those that suppress specific types or steps of autophagy are depicted in red. seminars in cell and developmental biology 101 (2020) [3] [4] [5] [6] [7] [8] [9] [10] [11] the replication complexes and (b) to insulate dsrna intermediates from innate sensing by cytosolic pattern recognition receptors [45] . the replication complexes are typically composed of the viral rna-dependent rna polymerase, accessory non-structural proteins, viral rna, and host factors. specialised autophagosomes, sometimes referred to as amphisomes (formed upon fusion with endosomes), omegasomes, and ede-mosomes (both er-derived) have been hypothesised to function as replication organelles [43, 46, 47] . given the resemblance of virustriggered double membrane vesicles with that of autophagosomes, it is plausible that generation of replication organelles are a result of mechanisms similar to autophagy [48] [49] [50] [51] . like autophagosomal membranes, many virus-induced vesicles are believed to be er-derived. recent data from several different studies have provided direct evidence of the association between viral replication complexes and autophagosome structures as summarized in table 1 . poliovirus (pv) vesicle clusters were found to contain lc3 and lysosomal markers, reminiscent of autolysosomes, and colocalised with the pv replication complex [7, 52] . furthermore, formation of infectious pv progeny virions was reported to depend on vesicular acidification, prompting the hypothesis that particle assembly, genome replication and virion maturation occurred in bona fide autophagosomal vesicles [46] . among the flaviviridae family, several non-structural proteins have been observed in lc3+ vesicles. denv non-structural protein ns1 and dsrna were reported to co-localise with lc3 and ribosomal proteins [38, 39] . another study described the induction of lc3+ vesicles, which colocalised with ns4a in infected cells, or when transfected with a combination of denv ns4a and ns4b [16] . this was independently corroborated by zikv infection in human fetal neural stem cells where expression of ns4a and ns4b were sufficient to block neurogenesis and promote autophagy, displaying partial colocalisation with lc3+ vesicles [53] . ultrastructural analysis of chikv virions also suggested their location in the lumen of autophagome-like vacuoles [23] . similar to other members of this family, hcv infection induces massive intracellular membrane rearrangements. competing hypotheses have been proposed as to whether autophagosomes themselves serve as sites for hcv replication. by sucrose gradient analysis, lc3-ii was found to co-sediment with hcv rna and non-structural proteins ns3 and ns5a [54] . however, in a separate study confocal microscopy showed little evidence of co-localisation of lc3 or atg5 with hcv proteins [55, 56] . along the same lines, depletion of either lamp2 or rab7, which allowed accumulation of autophagosomes by preventing fusion with lysosomes inhibited hcv viral replication, also suggesting that they are not the major sites for hcv genome replication [36] , while multiple reports indicate that lipid droplets are the more likely sites for replication and assembly, as reviewed elsewhere [40, 57] . conflicting evidence also exists for mhv-induced replication compartments. on the one hand, mhv replication complexes were found to be associated with lc3 and atg12 in embryonic stem cell lines [22] . on the other hand in primary macrophages and murine embryonic fibroblasts mhv replication did not require the autophagy gene atg5 [58] . differences in permisiveness to infection often exists between primary cells and transformed cell lines, as does viral tropism towards distinct cell types, either of which can account for these experimental discrepancies. among other +rna rna viruses, immunoelectron microscopy demonstrated co-localisation of ev71 capsid protein vp1 with autophagosomes in virus-infected mouse neurons [59] . similarly, during emcv infection, colocalisation of non-structural protein 3a and capsid protein vp1 was visualised by confocal and immunoelectron microscopy [60] . colocalisation of non-structural proteins 2b, 2c, and 3a with lc3, and structural protein vp1 with atg5 were also reported in fmdv-infected cells [14] . although direct evidence of the association of viral replication complexes with autophogasomes is lacking for cvb3, impaired maturation of autolysosomes brought about through pharmacological or genetic inhibition increased the accumulation of autophagosomes in virus-infected cells resulting in enhanced viral replication [31, 32] . these data implicated autophagosomes as virus anchoring and replication sites during cvb3 replication. delineating the process of viral assembly from replication is technically challenging, especially since both processes would very likely induces formation of autophagosome-like double-membrane liposomes [112] summary of interactions between proteins from positive strand rna viruses and host autophagy machinery. seminars in cell and developmental biology 101 (2020) [3] [4] [5] [6] [7] [8] [9] [10] [11] occur in concert at the same sites. although utilisation of autophagosomes as assembly sites has been recorded for some dna-viruses e.g., hepatitis b virus (hbv) [61] , experimental data with +rna rna viruses are scant. however, atleast for denv, recent evidence indicates that autophagy might actually assist in assembly of progeny virions, without them serving as replication organelles. one of the initial studies describing the induction of autophagy in flavivirus infections was performed by lee et al [62] . the authors demonstrated that denv2 infection in hepatocytes induced autophagy; targeting with either the inhibitor 3-methyladenine (3ma) or sirnas against autophagy genes compromised infection. denv-induced autophagosomes colocalised with lamp1, a marker of lysosomal fusion, which was independently validated by immunofluorescence assays and pharmacological inhibition. following this initial characterisation, a more mechanistic study emerged describing the role of selective autophagy facilitating hydrolysis of lipid droplets in an infection-specific manner [17] . apart from the relatively non-specific bulk macroautophagy, cellular organelles are turned over through several types of selective autophagy. this phenomenon occurs under normal physiological conditions and is hypothesised to initiate a physiological response to appropriately address a specific stress. in the context of denv infection, a type of selective autophagy of lipid storage organelles (lipid droplets) referred to as lipophagy was described that hydrolyses neutral fat deposits to free fatty acids and cholesterol, and supplements cellular energy reservoirs [40, 63, 64] . heaton et al performed a targeted sirna screen to identify cellular cofactors of denv2 replication in hepatocytes, which revealed, among others, genes involved in the induction of autophagy [65] , and were further characterised to reveal that denv induced autophagosomes not only acquired lamp1, but underwent complete maturation to become autolysosomes [17] . these did not colocalise with markers of the viral replication complex, suggesting that they may play an indirect, non-structural role in denv replication. a subsequent study described the involvement of aup1, a type-iii membrane protein, in the initiation of virus-induced autophagy. aup1 was regulated by monoubiquitin modification, where infection or a combined expression of viral ns4a and ns4b were necessary and sufficient to generate the unmodified form of aup1 -a step that was critical in induction of this pathway. interestingly, loss of aup1 did not affect viral replication; however, impaired autophagy was accompanied by degradation of viral proteins through the proteasomal pathway resulting in significantly reduced production of progeny virions, supporting a specific role of aup1-dependent lipophagy in assembly of virus particles [16] . these results were in agreement with an independent report demonstrating expression of dengue ns4a was sufficient to trigger autophagy and protect against cell death. a growing body of evidence indicates that mitochondrial function is altered during flavivirus infections. although the mechanistic underpinnings are currently not well understood, atleast for hcv, parkindependent mitophagy has a significant effect on virus propagation. this was verified by silencing parkin and pink1, which inhibited hcvtriggered mitophagy and in turn blocked virus replication. ultrastuctural analyses by electron microscopy and immunoelectron microscopy also confirmed the presence of damaged mitochondria in double-membrane vesicles in hcv-infected cells [66] . whether this pathway is activated during infection by other flaviviruses is currently not known. several reports on mechanisms of secretion and cell-to-cell transfer of intracellular pathogens indicate non-degradative autophagic vesicles as an efficient mode of transport. an recent study with mycobacterium demonstrated that autophagosomes chaperone an organelle referred to as the "ejectosome", facilitating cell-to-cell spread of cytosolic bacteria [67] . secretory autophagy is a newly discovered pathway in which autophagosomes fuse with the plasma membrane instead of lysosomes and release single membrane vesicles containing cytosolic content into the extracellular milieu [68] . non-degradative autophagy has been suggested to facilitate nonlytic egress of some +rna rna viruses. the initial characterisation was with enteroviruses, which appear to exploit this pathway to exit cells, and are released into the extracellular environment as particle populations contained within vesicles [69] . clusters of enteroviral particles were packaged with phosphatidyl serine into autophagic vesicles, which enabled efficient transfer to primary macrophages, significantly enhancing viral infectivity. this revealed a novel mode of transport where viral genomes were transferred en bloc to recipient cells, facilitating genetic cooperativity and enhancing infection. this mode of transfer had previously also been noted for cvb3, where a recombinant fluorescent virus was released into the extracellular medium in microvesicles containing autophagic markers [70] . poliovirus is often considered a lytic virus; however, non-lytic release of poliovirus has also been reported [8] . reduced levels of viral particles in extracellular medium in autophagy-deficient cells correlated with inhibition of non-lytic release of autophagic vesicles. more recently, an important role of the secretory autophagy pathway was implicated in zikv vertical transmission as well as cell-to-cell spread of denv. zikv-induced autophagic activity in human trophoblasts restricted by pharmacological inhibition, or by deficiency in an essential autophagy gene, atg16l1, limited zikv vertical transmission and improved placental and fetal outcomes, which supported a role for autophagic secretion in the process [71] . along the same lines, it was hypothesised that denv might evade neutralising antibodies and increase viral spread by exploiting autophagic vesicles for delivery to the extracellular medium [72] . double staining of denv e antigen and lc3 in a close-contact co-culture experimental set-up verified secretion of denv-containing autophagic vesicles from donor cells, which were subsequently taken up by recipient cells. in a parallel study, maturation of infectious denv virus particles was attributed to this process, when cleavage of pr peptide from prm by the furin protease was prevented upon blocking autophagy [73] . further investigation is needed to provide more direct evidence on the mechanism, regulation and molecular determinants of secretory autophagy in facilitating viral release. activation of autophagy represents a fairly ubiquitous response to eliminate intracellular pathogens. several studies have described mechanisms where pathogen recognition receptors trigger this response upon detection of microbe-specific pathogen associated molecular patterns. a diverse set of pathogens including bacteria, viruses and parasites have provided corroborating evidence supporting this phenomenon. whether this process occurs in parallel to non-degradative autophagy, or the cross-talk that might exist between the two flavours of autophagy during +rna rna viral infection merits further investigation. recent studies have shed light on how autophagy offers an advantage to hcv infection by suppressing innate immune responses [36, 74] . this contrasts with previous data on hcv-induced incomplete autophagy and defined a pathway where the entire process from initiation through lysosomal degradation is necessary for hcv replication largely to suppress anti-viral innate immune response. in hcv-infected cells, interferon-β (ifn-β) production could be modulated by uprmediated autophagy; activation of this pathway reduced ifn-β production and vice-versa [75] . inhibition of autophagy by suppressing beclin-1 or atg7 reduced hcv replication, which was accompanied by the activation of ifn signaling. a similar phenomenon was also proposed for denv where activation of autophagy not only facilitated viral h.h. wong and s. sanyal seminars in cell and developmental biology 101 (2020) [3] [4] [5] [6] [7] [8] [9] [10] [11] replication, but also suppressed ifn-i production, suggesting that both viruses may share the same mechanism to evade innate immune responses. interestingly, in west nile virus infections, perturbation of intracellular cholesterol levels dictated ifn-i responses [76, 77] . although the role of autophagy in wnv infection has been contested, replication was reported to occur independent of autophagy by mutiple groups -a clear difference from other flaviviruses; however, whether it plays a role in regulating free cholesterol and fatty acid levels is yet to be determined. together, these studies indicate a critical mechanism by which flaviviruses may avoid innate immune responses through activating the host autophagy pathway. the link between autophagy and apoptosis has been defined extensively in various contexts including cancer, neurodegenerative disorders, and infectious diseases [78] . a more comprehensive understanding on circumventing cell death has been recorded for bacterial rather than viral infections, amongst which most are dna viruses. premature cell death can function as an anti-viral host mechanism by providing an unfavorable environment and shorter timeframes for viral propagation. induction of autophagy has often been linked to inhibition of apoptosis [79, 80] . both denv2 and a murine flavivirus-induced autophagy was reported to prevent apoptosis mediated via the viral ns4a protein [81] . knockdown of autophagy-related gene expression abolished the protective role of autophagy against cell death and resulted in reduced viral replication. apart from denv, cross-talk between autophagy and apoptosis was also reported in cvb4 infection, where suppression of autophagy by 3ma triggered caspase activation and vice-versa [82] . an interesting question that arises from virus-triggered induction of autophagy in the context of flavivirus infections is the process of antigen presentation. particularly in major histocompatibility complex (mhc)-ii positive cells, autophagosomes are constitutively generated to deliver viral antigens on mhc-ii molecules for adaptive immune responses. virus infections therefore frequently impede maturation of antigen presenting cells and subsequent adaptive immunity as reviewed in detail elsewhere [4] . monocytes and monocyte-derived cells are a major target of flaviviruses, where antigen presentation is facilitated by autophagy. this implies that while autophagy favours production of viral progeny, it should simultaneously increase viral antigen presentation and t-cell responses, thus generating neutralising antibodies and promoting cytotoxic t-cell killing. information on these seemingly contradictory processes is currently limited. however, denv-infected human monocyte-derived dcs fail to upregulate mhc and co-stimulatory molecules and have an impaired ability to polarize cd4 + th type 1 (th1) effector properties [83] , contributing to inefficient adaptive immune responses observed in patients. in bulk cultures of dendritic cells, exposure to denv augments mhc-i and mhc-ii expression in non-infected bystander cells; however, infected monocyte-derived dendritic cells display an inhibition in this process within the same cultures [84] . in clinical studies gene expression analyses of denv patients revealed that severe cases expressed lower levels of genes linked to antigen processing, presentation and t-cell activation compared to mild cases. another related flavivirus, japanese encephalitis virus, inhibits expression of mhc-i and induces functional impairment of dcs, resulting in poor cd8 + t cell responses [85] . thus impaired antigen presentation and functionality of virus-infected dcs may reflect a viral immune escape strategy to dampen t-cell responses and impact disease severity. one study reported that denv activated autophagy only during the early infection stage, suggesting a biphasic response of autophagy to denv infection, where it shifted from a supporting to an antiviral role at later time points [86] . these results might enable us to reconcile how flaviviruses have evolved strategies to manipulate this pathway while subverting t-cell based immune responses. a quantitative and time resolved analyses of this process in virus-infected cells might shed light on its utilisation in the benefit versus detriment towards virus production. activation of autophagy in the presence of intracellular pathogens is a fairly universal cellular response. xenophagy as an intrinsic defence mechanism was first described through electron microscopy studies upon visualising hsv-1 and cytomegalovirus inside autophagosomes [87] . among viruses, autophagic protection has been recorded in a wideranging species and genera [88] [89] [90] . the mechanism of autophagymediated restriction of +rna rna viruses is less well-documented on account of its proviral influence in most cases. however, there are instances where autophagic degradation of virions (virophagy) or viral proteins has been observed, especially in neurons where it is a critical form of antiviral defence. during sindbis virus (sinv) infection, beclin 1 and p62-dependent degradation of the capsid protein protects against sinv-mediated encephalitis [91, 92] . moreover, atg5 deficiency results in delayed sinv clearance and accumulation of the autophagy receptor p62. more recently, fanconi anaemia group c protein (fancc) was found to interact with the sinv capsid protein and facilitate virophagy [28] . picornaviruses, such as pv and hrv permeabilise endosomes to release their genome into the cytosol. this step is detected by galectin 8, which restricts viral infection by initiating degradation of the viral rna genome [93] . as counterstrategy, the host protein hras-like suppressor 3 (pla2g16) is exploited by the virus to enable genome delivery. cvb3, also belonging to the same family, undergoes p62-dependent degradation and uses the viral protease 2a to cleave p62 and inhibit virophagy [94] . interestingly, although hcv has been demonstrated to induce autophagy to its own advantage by multiple groups, one study demonstrated that an er transmembrane protein, scotin, interacted with the viral protein ns5a, resulting in its autophagic degradation to suppress viral replication [95] . among flaviviruses, autophagosomal degradation of neurotropic viruses has been recorded. in the drosophila brain, zikv infection triggered nfκb-dependent inflammatory signaling, inducing expression of dsting which subsequently restricted infection by upregulating autophagy. defective or absence of autophagy resulted in increased infection in the fly brain and death [96] . sting-dependent induction of protective autophagy was independently reported for dna viruses and sea anemone, supporting an evolutionarily conserved role for sting in microbial autophagy [5] . despite major advances in elucidating molecular determinants of the autophagy pathway, the rules that govern its utilisation during infections are far from obvious. a complex interplay between viral manipulation and host innate immunity dictates disease outcomes. autophagy is expected to restrict viral infections at multiple levels by eliminating viruses, regulating inflammatory responses and promoting antigen presentation. however, +rna viruses have co-evolved to manipulate autophagy for immune evasion, replication, assembly and release from infected cells. the repertoire of universal and distinct mechanisms that these viruses draw on to interfere with autophagy are striking, often targeting the same pathway in unique ways with different functional implications. distinct viral strategies fine-tune the process to simultaneously escape destruction while capitalising on the structural and nutrient benefits that autophagy provides. several gaps remain in our understanding within the remit of viral manipulation of autophagy. first and foremost, more advanced strategies of isolating autophagic vesicles will be imperative for better characterisation of this process. emerging data indicate that many lc3-positive vesicles that h.h. wong and s. sanyal seminars in cell and developmental biology 101 (2020) [3] [4] [5] [6] [7] [8] [9] [10] [11] are induced upon virus infections are not autophagosomes. a combination of ultracentrifugation, density gradient separations and enrichment techniques will be necessary to characterise these vesicle populations. furthermore, in the context of virus infection, the equilibrium between degradative and secretory autophagy versus biogenesis of exosomal vesicles will need to be quantitated to arrive at firm conclusions regarding the functional outcome of autophagy. along the same lines, differences between infected host cells and neighbouring cells will become important to develop a more complete picture of its impact in viral pathogenesis versus immune responses. further understanding of the contribution of autophagy to the different stages in the viral lifecycle, subversion of its antigen presentation function and innate immune responses is therefore necessary to delineate the diverse functions of autophagy in virus pathogenesis. a current perspective of autophagosome biogenesis 24 endoplasmic reticulum and golgi complex: contributions to, and turnover by mechanism and medical implications of mammalian autophagy autophagy during viral infection -a doubleedged sword autophagy induction via sting trafficking is a primordial function of the cgas pathway electron microscopic study of the formation of poliovirus subversion of cellular autophagosomal machinery by rna viruses nonlytic viral spread enhanced by autophagy components coxsackievirus can exploit lc3 in both autophagy-dependent and -independent manners in vivo enteroviral infection inhibits autophagic flux via disruption of the snare complex to enhance viral replication coxsackievirus b4 uses autophagy for replication after calpain activation in rat primary neurons enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model human rhinovirus 2 induces the autophagic pathway and replicates more efficiently in autophagic cells foot-and-mouth disease virus utilizes an autophagic pathway during viral replication nonstructural proteins 2c and 3d are involved in autophagy as induced by the encephalomyocarditis virus flaviviruses exploit the lipid droplet protein aup1 to trigger lipophagy and drive virus production dengue virus-induced autophagy regulates lipid metabolism ultrastructural characterization of zika virus replication factories cytoarchitecture of zika virus infection in human neuroblastoma and aedes albopictus cell lines hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m, proc. natl. acad. sci autophagy benefits the replication of newcastle disease virus in chicken cells and tissues coronavirus replication complex formation utilizes components of cellular autophagy chikungunya triggers an autophagic process which promotes viral replication japanese encephalitis virus activates autophagy through xbp1 and atf6 er stress sensors in neuronal cells image-based genome-wide sirna screen identifies selective autophagy factors flaviviridae replication organelles: oh, what a tangled web we weave identification of zika virus and dengue virus dependency factors using functional genomics fanconi anemia proteins function in mitophagy and immunity differential and convergent utilization of autophagy components by positivestrand rna viruses dengue and zika viruses subvert reticulophagy by ns2b3-mediated cleavage of fam134b coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo autophagosome supports coxsackievirus b3 replication in host cells coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged lc3 activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response co-localization of constituents of the dengue virus translation and replication machinery with amphisomes a role for autophagolysosomes in dengue virus 3 production in hepg2 cells modulation of lipid droplet metabolism -a potential target for therapeutic intervention in flaviviridae infections dengue virus and autophagy severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles coronavirus nsp6 restricts autophagosome expansion targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus modification of intracellular membrane structures for virus replication intracellular vesicle acidification promotes maturation of infectious poliovirus particles generation of unique poliovirus rna replication organelles architecture and biogenesis of plus-strand rna virus replication factories rna replication of mouse hepatitis virus takes place at double-membrane vesicles non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex ultrastructural characterization of sars coronavirus remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles zika virus ns4a and ns4b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy ultrastructural and biochemical analyses of hepatitis c virus-associated host cell membranes the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles hepatitis c virus and lipid droplets:finding a niche coronavirus replication does not require the autophagy gene atg5 enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication autophagy promotes the replication of encephalomyocarditis virus in host cells hepatitis b virus subverts the autophagy elongation complex atg5-12/16l1 and does not require atg8/lc3 lipidation for viral maturation autophagic machinery activated by dengue virus enhances virus replication regulation of lipid stores and metabolism by lipophagy autophagy regulates lipid metabolism dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy the autophagic machinery ensures nonlytic transmission of mycobacteria phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses coxsackievirus b exits the host cell in shed microvesicles displaying autophagosomal markers inhibition of autophagy limits vertical transmission of zika virus in pregnant mice autophagyassociated dengue vesicles promote viral transmission avoiding antibody neutralization inhibition of cellular autophagy deranges dengue virion maturation knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro cholesterol manipulation by west nile virus perturbs the cellular immune response perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections autophagy in cell death: an innocent convict? lysosomes and autophagy in cell death control bcl-2 family members: dual regulators of apoptosis and autophagy flavivirus ns4a-induced autophagy protects cells against death and enhances virus replication impairment of cd4+ t cell polarization by dengue virus-infected dendritic cells differential effects of dengue virus on infected and bystander dendritic cells multifront assault on antigen presentation by japanese encephalitis virus subverts cd8+ t cell responses dengue virus inhibition of autophagic flux and dependency of viral replication on proteasomal degradation of the autophagy receptor p62 herpes simplex virus and human cytomegalovirus replication in wi-38 cells. iii. cytochemical localization of lysosomal enzymes in infected cells autophagy is an essential component of drosophila immunity against vesicular stomatitis virus hsv-1 icp34.5 confers neurovirulence by targeting the beclin 1 autophagy protein autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages protection against fatal sindbis virus encephalitis by beclin, a novel bcl-2-interacting protein autophagy protects against sindbis virus infection of the central nervous system pla2g16 represents a switch between entry and clearance of picornaviridae calcoco2/ndp52 and sqstm1/p62 differentially regulate coxsackievirus b3 propagation interferon-inducible protein scotin interferes with hcv replication through the autolysosomal degradation of ns5a inflammation-induced, sting-dependent autophagy restricts zika virus infection in the drosophila brain amino acid substitutions in the non-structural proteins 4a or 4b modulate the induction of autophagy in west nile virus infected cells independently of the activation of the unfolded protein response the role of secretory autophagy in zika virus transfer through the placental barrier japanese encephalitis virus replication is negatively regulated by autophagy and occurs on lc3-i-and edem1-containing membranes rab5 and class iii phosphoinositide 3-kinase vps34 are involved in hepatitis c virus ns4b-induced autophagy hepatitis c virus upregulates beclin1 for induction of autophagy and activates mtor signaling the autophagy elongation complex (atg5-12/16l1) positively regulates hcv replication and is required for wild-type membranous web formation coronavirus membraneassociated papain-like proteases induce autophagy through interacting with beclin1 to negatively regulate antiviral innate immunity coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired nfkb signaling protein 2b of coxsackievirus b3 induces autophagy relying on its transmembrane hydrophobic sequences enteroviruses remodel autophagic trafficking through regulation of host snare proteins to promote virus replication and cell exit foot-and-mouth disease virus capsid protein vp2 activates the cellular eif2s1-atf4 pathway and induces autophagy via hspb1 viroporin activity of the foot-and-mouth disease virus non-structural 2b protein modification of cellular autophagy protein lc3 by poliovirus double-membraned liposomes sculpted by poliovirus 3ab protein this work was supported by health and medical research funds (16150592 and 17161202 and 17161032), and partially by research grants council-general research funds (17112617). ss is supported by the croucher foundation. key: cord-284549-edliu3it authors: zhou, hui; qian, qi; shu, ting; xu, jiuyue; kong, jing; mu, jingfang; qiu, yang; zhou, xi title: hepatitis c virus ns2 protein suppresses rna interference in cells date: 2019-11-27 journal: virol sin doi: 10.1007/s12250-019-00182-5 sha: doc_id: 284549 cord_uid: edliu3it rnai interference (rnai) is an evolutionarily conserved post-transcriptional gene silencing mechanism and has been well recognized as an important antiviral immunity in eukaryotes. numerous viruses have been shown to encode viral suppressors of rnai (vsrs) to antagonize antiviral rnai. hepatitis c virus (hcv) is a medically important human pathogen that causes acute and chronic hepatitis. in this study, we screened all the nonstructural proteins of hcv and found that hcv ns2 could suppress rnai induced either by small hairpin rnas (shrnas) or small interfering rnas (sirnas) in mammalian cells. moreover, we demonstrated that ns2 could suppress rnai via its direct interaction with double-stranded rnas (dsrnas) and sirnas, and further identified that the cysteine 184 of ns2 is required for the rnai suppression activity through a serial of point mutation analyses. together, our findings uncovered that hcv ns2 can act as a vsr in vitro, thereby providing novel insights into the life cycle and virus-host interactions of hcv. electronic supplementary material: the online version of this article (10.1007/s12250-019-00182-5) contains supplementary material, which is available to authorized users. hcv is a positive-sense, single-stranded rna virus that belongs to the genus hepacivirus of the family flaviviridae. its genome is approximately 9.6 kb in length and encodes a polyprotein that is cleaved by host and viral proteases to produce ten mature viral proteins, including structural core protein, envelope proteins e1 and e2, the p7 ion channel protein, and nonstructural (ns) proteins ns2, ns3, ns4a, ns4b, ns5a and ns5b (yin et al. 2018) . hcv infection causes acute and chronic hepatitis in humans with a high propensity for chronicity. a large number of hcv-infected patients fail to clear this virus and remain asymptomatic for years to develop severe liver diseases such as cirrhosis and hepatocellular carcinoma (paboriboune et al. 2018) . the capability of establishing chronic infection by hcv is partly due to its ability to evade host innate immune responses (horner and gale 2013) . rnai is an evolutionarily conserved mechanism in all eukaryotes for post-transcriptional gene silencing. rnai serves as an important antiviral immune response in a wide range of organisms, including fungi, plants, and invertebrates (guo et al. 2019; maillard et al. 2019) . moreover, recent studies have demonstrated that rnai also exerts antiviral effects in mammals maillard et al. 2013; li et al. 2016; qiu et al. 2017; xu et al. 2019) . the antiviral rnai is triggered by the viral replicative intermediate double-stranded rnas (vri-dsrnas) produced in the course of viral replication. these vri-dsrnas can be recognized and cleaved by host endoribonuclease dicer into virus-derived small interfering rnas (vsirnas) of approximately 21-23 nucleotide (nt). argonaute (ago) protein within rna-induced silencing complexes (riscs) electronic supplementary material the online version of this article (https://doi.org/10.1007/s12250-019-00182-5) contains supplementary material, which is available to authorized users. incorporates one of the strands of vsirna duplex, which directs the risc-mediated degradation of cognate viral rnas (maillard et al. 2019) . to counteract the antiviral rnai, viruses have evolved to encode viral suppressors of rnai (vsrs) targeting various steps of the rnai pathway (wu et al. 2010) . one common strategy utilized by vsrs, including nodamura virus (nov) b2, ebola virus (ebov) vp35, influenza a virus (iav) ns1, and human enterovirus 71 (ev-a71) 3a, is to prevent viral dsrna from cleavage by dicer (li et al. 2004; sullivan and ganem 2005; haasnoot et al. 2007; qiu et al. 2017) . alternatively, multiple vsr proteins have been shown to directly target the key components of the rnai pathway, such as dicer or ago proteins. for example, wuhan nodavirus (whnv) b2 can directly bind to drosophila dicer-2 to inhibit vsirna production (qi et al. 2011 (qi et al. , 2012 , while cricket paralysis virus 1a has been found to directly inhibit drosophila ago2 (nayak et al. 2018) . in the case of hcv, the core protein was found to suppress rnai by interacting with dicer (chen et al. 2008) . moreover, hcv structural e2 protein was also identified to antagonize rnai by targeting ago2 (ji et al. 2008) . consistent with the findings in hcv, a number of rna viruses were shown to encode more than one viral protein to counteract rnai. for instance, vp35, vp30 and vp40 of ebov were found to suppress rnai through different mechanisms (haasnoot et al. 2007; fabozzi et al. 2011) . severe acute respiratory syndrome coronavirus (sars-cov) encoded 7a and nucleocapsid contain rnai suppression activities (karjee et al. 2010; cui et al. 2015) . besides, tat and nef proteins of human immunodeficiency virus-1 (hiv-1) were identified to suppress rnai through the dsrna-binding and ago interaction, respectively (bennasser et al. 2005; aqil et al. 2013) . the phenomena of viruses encoding multiple vsrs as well as vsrs targeting different steps of the rnai pathway highlight the importance of suppressing rnai during the viral life cycle. although the structural core and e2 proteins of hcv were shown to suppress rnai, it is unclear if any hcv nonstructural protein contains rnai suppression activity. in this study, we uncovered that hcv nonstructural ns2 protein possessed a potent in vitro vsr activity that suppressed the rnai induced by short hairpin rna (shrna) and sirna in mammalian cells. we also confirmed the conserved residue in hcv ns2 that could disrupt its activity to suppress rnai. overall, our findings demonstrated that hcv ns2 contains vsr activity, thereby providing insights into the life cycle of hcv. for expression of hcv nonstructural proteins including ns2, ns3, ns3/4a, ns4a, ns4b, ns5a and ns5b in hek293t cells, their orfs were cloned into the prk-flag, respectively. the templates expressing hcv proteins were kindly provided by prof. ying zhu (wuhan university, china). the point mutations were introduced into the ns2 coding region by pcr-mediated mutagenesis with the appropriate primers. for expression of recombinant ns2 in sf9 cells, the orfs of ns2 and its mutant were constructed into the vector pfastbac htb-mbp as previously described (yang et al. 2017) . the resulting plasmids were subjected to the bac-to-bac baculovirus expression system to express the fusion proteins with an mbp tag at the n-terminus. the egfp-sirna was chemically synthesized by guangzhou ribobio co., ltd., china. all the primers and oligonucleotides used in this study are shown in supplementary table s1 . hek293t, 293t-nodice (kindly provided by prof. bryan cullen, durham, nc, usa) and huh7.5 cells were maintained in dulbecco's modified eagle's medium (dmem, gibco) containing 7% fetal bovine serum (fbs) (gibco), 100 u/ml penicillin and 100 lg/ml streptomycin at 37°c in an incubator with 5% co 2 . cells were seeded in 6-well plates and grown overnight to reach 50% confluence. before transfection, the medium was changed to dmem containing without serum and antibiotic. cells then were transfected with the indicated plasmids by using fugene hd reagent (roche, basel, switzerland) according to the manufacturer's instructions. cells were harvested in lysis buffer [50 mmol/l tris-hcl (ph 7.4), 150 mmol/l nacl, 1% np40, 0.25% deoxycholate and a protease inhibitor cocktail (rhoche)]. then the lysates were subjected to 15% sds-page and western blotting analysis. the antibodies used in this study are as follow: anti-tubulin (proteintech group, 1:5000), anti-flag (proteintech group, 1:5000) and anti-myc (proteintech group, 1:3000). total cellular rnas were extracted using trizol reagent (thermo) according to the manufacturer's instructions. for virologica sinica the detection of egfp mrna, 5 lg of total rnas were subjected to denatured 1.5% agarose gels with 2.2 mol/l formaldehyde. the separated rnas were transferred onto the hybond-a nylon membrane (ge healthcare), and fixed at 120°c for 15 min. then the membranes were hybridized with dig-labeled probes in hybridization ovens at 65°c overnight. finally, the membranes were incubated with anti-dig antibody conjugated with alkaline phosphatase and exposed to the luminescent image analyzer las4000 (fuji film). the probes for detection of egfp and gapdh mrna were complementary to their orf region of 500-720 nt and 760-1060 nt, respectively. for detection of small rnas, 20 lg of total rnas were subjected to 7 mol/l urea-15% page and transferred to hybond-a nylon membrane (ge healthcare). the membrane was chemically cross-linked in 1-ethly-3-(3dimethylaminopropyl) carbodiimide (edc) at 60°c for 30 min. the dig-labeled rna probes targeting egfp sirna and u6 were synthesized by takara. the expression and purification of mbp alone and mbpfusion proteins were performed in sf9 cells using baculovirus expression system as previously described (yang et al. 2017) . briefly, cells were infected with mbp-tagged hcv ns2 expressing baculoviruses for 72 h. infected cells were resuspended, lysed via sonication and then centrifuged at 11,000 9g for 30 min to remove debris. the protein in the supernatant was purified using amylose affinity chromatography (new england biolabs, ipswich, ma) according to the manufacturer 0 s protocol and then concentrated using amicon ultra-15 filters (millipore, schwalbach, germany). all purified proteins were quantified with a bicinchoninic acid (bca) protein assay kit (cwbio, china) and stored at -80°c in aliquots. proteins were separated on 10% sds-page and visualized by coomassie blue. we generated 200-nt dig-labeled dsrna and 22-nt sirna via in vitro transcription using dig rna labeling mix (roche). mbp-fusion ns2 or mutant proteins were reacted with dig-labeled rnas (0.2 lmol/l 200-nt dsrna or 22-nt sirna) in a binding buffer [50 mmol/l hepes (ph 8.0), 15 mmol/l nacl, 0.5 mmol/l mgcl 2 , 10% glycerol and 1 u of rnase inhibitor (promega)] at 22°c for 45 min; the total volume was 10 ll. then the reaction mixtures were separated on 6% (for dsrna) or 12% (for sirna) tbe-page and transferred to hybond-a nylon membrane (ge healthcare). the membranes were incubated for 30 min with anti-dig antibody conjugated with alkaline phosphatase (roche). hek293t cells were lysed in a lysis buffer containing 20 mmol/l tris-hcl (ph 7.4), 200 mmol/l nacl, 2.5 mmol/l mgcl 2 , 0.5% triton x100, 0.5 u/ll rnase inhibitor (promega) and a protease inhibitor cocktail (roche). after centrifugation for 15 min at 12,000 9g, the supernatant was pre-cleared via incubation with protein-a/ g agarose beads (roche) at 4°c for 4 h. then the precleared lysates were incubated with antibodies (anti-flag, anti-myc or anti-igg as a negative control) together with protein-a/g agarose beads (roche) at 4°c for 12 h. the antibody-bound complexes were washed for five times with the same lysis buffer except that nacl concentration was raised to 600 mmol/l. finally, rnas were extracted using trizol reagent (thermo) according to the manufacturer 0 s protocol. to identify whether hcv has any nonstructural protein which works as a potential vsr, we screened them via the reversal-of-silencing assay in hek293t cells (fig. 1a) . in brief, cells were co-transfected with the plasmids encoding egfp and egfp-specific shrna (shegfp), together with the vectors for divers hcv nonstructural proteins. nov b2 (nb2), a well-characterized vsr was used as a positive control. the expression of the hcv-encoded proteins and nb2 were detected by western blotting with anti-flag and anti-myc antibodies (fig. 1b) . at 48 h post transfection (hpt), the mrna levels of egfp were detected by northern blotting with a digoxigenin (dig)-labeled rna probe targeting 500-720 nt of egfp orf. egfp-specific shrna can effectively eliminate the egfp transcripts (fig. 1a, lane 2) . as shown in fig. 1a , hcv ns2 protein can effectively restore the expression of rnai-silenced egfp (lane 4). expectedly, expression of nb2 or genetic ablation of dicer (nodice) suppressed shrna-induced rnai in hek293t cells (fig. 1a, lanes 3 and 11) . importantly, hcv ns2 did not affect the transcription efficiency of egfp in the absence of shrna (fig. 1c) , excluding out the possibility that hcv ns2 can directly promote egfp transcription. we sought to examine whether the rnai suppression activity of hcv ns2 is dependent on the protein expression levels. thus, hek293t cells were co-transfected with the plasmids for egfp and egfp-specific shrna, together with the increasing amount of ns2 plasmid as indicated. our findings showed that the reversal effect of egfp silencing increased progressively at the mrna levels, along with the growing amount of ns2 plasmid being transfected ( fig. 2a, 2b) . we further examined the vsr activity of hcv ns2 at different time points. our results showed that the reversal effect of egfp silencing could be observed at 48 hpt (fig. 2c) , indicating that the vsr activity was dependent on the expression level of hcv ns2 protein. taken together, our findings showed that hcv ns2 displays vsr activity in mammalian cells. having established that hcv ns2 contains rnai suppression activity, we sought to examine the detailed mechanism through which hcv ns2 inhibits rnai. in the process of rnai, dsrna/shrna requires the dicer-mediated cleavage into sirna (maillard et al. 2019) . to investigate whether hcv ns2 can inhibit this step, small rnas harvested from hek293t cells co-expressing egfp-specific shrna together with ns2 were subjected to northern blotting with a dig-labeled rna probe targeting egfp sirna produced from shrna by dicer. as shown in fig. 3a , the accumulation of dicer-cleaved sirna was reduced in the presence of ns2 compared to that in cells expressing empty vector, indicating that hcv ns2 can suppress dicer-mediated sirna production. these results are consistent with the previous findings that hcv ns2 effectively restored shrna-induced silencing of egfp transcript in hek293t cells (fig. 1a) . in the process of shrna-induced rnai, shrna is cleaved by dicer into sirna. after identifying that hcv ns2 suppressed the sirna production in cells expressing shrna, we performed rna immunoprecipitation (rna-ip) assay to test whether hcv ns2 sequestrated dsrna in hek293t cells. briefly, cells expressing flag-tagged ns2, myc-tagged nb2 or empty vector, together with egfpspecific dsrna (1-196 nt of egfp orf) were lysed and immunoprecipitated with the indicated antibodies, respectively. rnas extracted from the rna-ip precipitates were examined via northern blotting with the rna probe targeting the 196-nt dsrna of egfp. as shown in fig. 3b , our findings show that ns2 can associate with dsrna in cells (lane 9). we sought to examine whether hcv ns2 could directly bind to dsrna. to this end, we performed gel shift assays by incubating the recombinant mbp-fusion hcv ns2 (mbp-ns2; supplementary fig. s1a ) and the in vitrotranscribed 200-nt dig-labeled dsrna. as shown in fig. 3c , the mobility of dsrna was inhibited in a dosedependent manner by the hcv ns2 protein (lanes 4-7) , compared to the control reaction with mbp protein alone. of note, mbp-fusion whnv b2 (mbp-wb2), another well-characterized vsr that contains the dsrna-binding activity, was used as the positive control (fig. 3c, lane 3) . taken together, our findings indicate that hcv ns2 can suppress rnai by sequestrating dsrna. northern blotting with dig-labeled rna probe targeting the 500-720 nt of egfp orf. gapdh mrna was used as the loading control. b the expression of hcv proteins was detected by western blotting. arrow indicates the unrelated bands in the bot. c hek293t cells were co-transfected with the plasmid encoding egfp, together with hcv ns2 or nov b2 plasmid, respectively. at 48 hpt, egfp mrna levels were examined via northern blotting. we sought to examine whether hcv ns2 protein can inhibit rnai induced by sirna. to this end, hek293t cells were co-transfected the plasmid for egfp and the chemically synthesized egfp-specific sirna (siegfp, 50 nmol/l), together with the ns2 plasmid. egfp-specific sirna induces a significant reduction of egfp expression (fig. 4a, lane 2) . our results showed that hcv ns2 efficiently restored the egfp mrna levels, indicating that hcv ns2 can inhibit sirna-induced rnai in mammalian cells (fig. 4a, lane 4) . because hcv ns2 can directly bind to dsrna, we sought to determine whether ns2 suppressed sirna-induced rnai by directly binding to sirna. to this end, we conducted gel shift assay by incubating mbp-ns2 together with dig-labeled synthetic 22-nt sirna. as shown in fig. 4b , ns2 can directly bind to sirna in a dose-dependent manner. taken together, these results indicate that hcv ns2 can suppress rnai by sequestering sirna. after determining the vsr activity of hcv ns2, we sought to identify the critical residues required for its rnai suppression activity. thus, we performed multiple sequence alignments of ns2s encoded by multiple hcv isolates (supplementary fig. s1b ). because the dsrna/ sirna-binding and protein dimerization are critical for vsr's activity (maillard et al. 2019) , the positivelycharged and highly conserved residues, including arginine (r), lysine (k), histidine (h), glutamic acid (e) and cysteine (c) were subjected to single-point mutation to alanine (a), and the resulting mutant ns2 proteins were then examined via the reversal-of-silencing assay in hek293t cells (fig. 5a-5c ). our findings show that the c184a mutation (ns2 c184a ) significantly suppressed the activity of hcv ns2 to suppress rnai (fig. 5c, lane 7 and fig. 5d, lane 5) . moreover, we further confirmed that the c184 is critical for the vsr activity of hcv ns2 via the reversal-of-silencing assay in huh7.5 cell line (fig. 5e) , which is usually used for hepatitis c research. moreover, we found that c184a abolished the dsrna-and sirnabinding activities of hcv ns2 in vitro (fig. 6a, 6b ). hek293t cells were co-transfected with the plasmids encoding egfp (0.1 lg) and egfp-specific shrna (0.3 lg), together with the increasing amounts of the plasmid encoding hcv ns2 (0.5 lg, 1.0 lg and 1.5 lg, respectively). at 48 hpt, egfp mrna levels were determined by northern blotting (a). gapdh mrna was used as the loading control. the mrna levels of egfp normalized to that of gapdh were quantified with bio-rad quantity one software and were graphed in (b) with that in the presence of the hcv ns2 (0.5 lg) defined as 100%. error bars represent sd values from the results of three independent experiments. *p \ 0.05, as measured by two-way anova (graphpad prism). cell lysates were harvested and analyzed by western blotting with anti-flag, anti-myc and anti-tubulin antibodies. c hek293t cells were co-transfected with plasmids encoding egfp (0.1 lg) and egfp-specific shrna (0.3 lg), together with either empty vector or the plasmid encoding hcv ns2 (1 lg) or nb2 (1 lg) as indicated. at 24, 36, and 48 hpt, total rnas were extracted and the level of egfp mrna was examined by northern blotting. cell lysates were harvested and analyzed by western blotting with anti-flag, anti-myc and anti-tubulin antibodies. besides, we also found that ns2 c184a failed to suppress the processing of shrna into sirna in hek293t cells (fig. 6c ). rnai is an antiviral immune mechanism conserved in both invertebrates and mammals. as a counter-defense to rnai, viruses evolved to encode vsrs that were identified to target different steps of the rnai pathway. in the present study, we screened the viral nonstructural proteins of hcv for rnai suppression activity and found that hcv ns2 protein is a vsr. the gel shift and rna-ip analyses show that hcv ns2 possesses both dsrna-and sirna-binding activities that are required for rnai suppression. thus, we speculate that hcv ns2 may function as vsr by shielding virus-derived dsrna from dicer cleavage and binding to viral sirna to interfere with risc assembly. in addition to ns2, hcv structural proteins core and e2 were also shown to contain rnai suppression activity by directly targeting the protein components of the rnai pathway (chen et al. 2008; ji et al. 2008) . hcv core interacts with dicer and inhibits the sirna production, while hcv e2 can block the risc assembly by interacting with ago2. interestingly, we showed that hcv ns2 as a vsr can target the rna components of the rnai pathway by directly binding to dsrna and sirna. these results indicate that hcv can suppress antiviral rnai at different stages by encoding multiple vsrs. this phenomenon can also be found in other rna viruses, such as ebov, sars-cov, hiv-1 and denv (bennasser et al. 2005; karjee et al. 2010; fabozzi et al. 2011; aqil et al. 2013; kakumani et al. 2013; cui et al. 2015; kakumani et al. 2015) . thus, encoding multiple vsrs that target different steps of the rnai pathway may offer advantages in the mode of action required for efficient suppression of rnai, thereby highlighting the importance of the antiviral rnai in the hostvirus interactions. hcv ns2 protein is a * 23 kda transmembrane protein that has multiple functions during the hcv replication. it contains a protease domain that functions as a cysteine protease to catalyze a cleavage between ns2 and ns3 (pieroni et al. 1997) . besides, ns2 colocalizes with e1, e2 ns3 and ns5a near the core protein and lipid droplet during the virion assembly . moreover, ns2 can work as a potent interferon antagonist and apoptosis inhibitor (erdtmann et al. 2003; kaukinen et al. 2013 ). here we found that hcv ns2 contains an rnai fig. 5 c184 is critical for the vsr activity of hcv ns2. a-d hek293t cells were co-transfected with the plasmids encoding egfp (0.1 lg) and egfp-specific shrna (0.3 lg), together with either empty vector or the plasmids encoding different hcv ns2 mutants (1 lg each) as indicated, at 48 hpt, total rnas were extracted and the levels of egfp mrna were determined via northern blotting. 293t-nodice cells were used as a control. gapdh mrna was used as the loading control. cell lysates were also subjected to western blotting with anti-flag, anti-myc and anti-tubulin antibodies. e huh7.5 cells were co-transfected with the plasmids encoding egfp (0.25 lg) and egfp-specific shrna (0.75 lg), together with either empty vector or a plasmid encoding ns2 or ns2 c184a as indicated (1.5 lg each). at 48 hpt, total rnas were extracted and the level of egfp mrna was examined via northern blotting. suppression activity, adding a novel function to its multiple roles in the viral life cycle. hcv ns2 is identified to possess dsrna-and sirna-binding activities, similar to many other vsrs such as nov b2 and sars-cov nucleocapsid. however, although these vsrs use the same strategy of suppressing rnai by sequestrating dsrna and/ or sirna, they do not share sequence identity or structural conservation, suggesting that vsrs of distinct virus families are evolved independently during the viral evolution. hcv ns2 forms a dimer with an n-terminal alpha-helical subdomain and a c-terminal antiparallel beta-sheet (lorenz et al. 2006 ). in addition, the conserved residues h143, e163 and c184 have been found to important for hcv ns2's dimerization (lorenz et al. 2006) . our mutational analyses showed that mutation of c184 abolished the dsrna-and sirna-binding activities of hcv ns2 and eliminated its vsr activity, implying the dimerization of hcv ns2 is required for its rnai suppression activity. our findings are consistent with the previous observations that dimerization is important for vsr activities of many viruses. for example, ev-a71 3a forms a dimer and disrupting its dimerization blocks the vsr activity (qiu et al. 2017) . in conclusion, our findings demonstrate that hcv ns2 can act as a vsr by sequestrating dsrna from dicer cleavage and interfering with risc assembly by sirnabinding. moreover, these results add hcv ns2 as a new member of hcv-encoded vsrs, which suppressing rnai by a different mechanism. and it provides insight into the life cycles and virus-host interactions of hcv. fig. 6 the dsrna-and sirna-binding activities of hcv ns2 are required for its rnai suppression. mbp-ns2 wt and mbp-ns2 c184a were incubated with 0.2 lmol/l 200-nt dig-labeled dsrna (a) or 22-nt sirna (b) at 22°c for 45 min. complexes were separated on 6% (a) or 12% (b) tbe-page, respectively. c hek293t cells were co-transfected with egfp specific shrna (0.3 lg) and either the plasmid encoding ns2 wt or ns2 c184a (1 lg of each). total rnas were subjected to small rna northern blotting. u6 was used as the loading control. the hiv-1 nef protein binds argonaute-2 and functions as a viral suppressor of rna interference evidence that hiv-1 encodes an sirna and a suppressor of rna silencing hcv core protein interacts with dicer to antagonize rna silencing the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells the hepatitis c virus ns2 protein is an inhibitor of cide-b-induced apoptosis ebolavirus proteins suppress the effects of small interfering rna by direct interaction with the mammalian rna interference pathway small rna-based antimicrobial immunity the ebola virus vp35 protein is a suppressor of rna silencing regulation of hepatic innate immunity by hepatitis c virus suppression of short interfering rna-mediated gene silencing by the structural proteins of hepatitis c virus role of rna interference (rnai) in dengue virus replication and identification of ns4b as an rnai suppressor dengue ns3, an rnai suppressor, modulates the human mirna pathways through its interacting partner the 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an rna silencing suppressor hepatitis c virus ns2 protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk1 functions interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing rna interference functions as an antiviral immunity mechanism in mammals induction and suppression of antiviral rna interference by influenza a virus in mammalian cells structure of the catalytic domain of the hepatitis c virus ns2-3 protease hepatitis c virus ns2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins antiviral rna interference in mammalian cells slicing and dicing viruses: antiviral rna interference in mammals a viral protein restricts drosophila rnai immunity by regulating argonaute activity and stability hepatitis c in laos: a 7-year retrospective study on 1765 patients in vitro study of the ns2-3 protease of hepatitis c virus rna binding by a novel helical fold of b2 protein from wuhan nodavirus mediates the suppression of rna interference and promotes b2 dimerization targeting of dicer-2 and rna by a viral rna silencing suppressor in drosophila cells human virus-derived small rnas can confer antiviral immunity in mammals a virus-encoded inhibitor that blocks rna interference in mammalian cells viral suppressors of rna-based viral immunity: host targets zika virus infection induces rnai-mediated antiviral immunity in human neural progenitors and brain organoids cypovirus capsid protein vp5 has nucleoside triphosphatase activity nap1l1 regulates hepatitis c virus entry and interacts with ns3 acknowledgements we wish to thank prof. ying zhu (wuhan, china) and prof. bryan cullen (durham, usa) for kindly providing materials. this work was supported by the strategic priority research program of chinese academy of sciences (xdb29010300 to x.z.), the national natural science foundation of china (81873964 to y.q., 31670161 to x.z. and 31800140 to j.m.), the science and technology bureau of wuhan (2018060401011309 to x.z.), and the advanced customer cultivation project of wuhan national biosafety laboratory (2018accp-ms11 to y.q.). x.z. is supported by the newton advanced fellowship from the academy of medical sciences, uk (naf005\1002). author contributions hz performed the experiments, analyzed the data and drafted the manuscript; qq, ts, jx, jk and jm helped to perform the experiments; qq and ts helped to analyze the data; yq and xz designed the experiments, analyzed the data, and finalized the manuscript. all authors approved the final manuscript. conflict of interest the authors declare that there are no conflict of interest.animal and human rights statement this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-284978-vh1x6pg9 authors: jang, hongje; ryoo, soo‐ryoon; kim, young‐kwan; yoon, soojin; kim, henna; han, sang woo; choi, byong‐seok; kim, dong‐eun; min, dal‐hee title: discovery of hepatitis c virus ns3 helicase inhibitors by a multiplexed, high‐throughput helicase activity assay based on graphene oxide date: 2013-02-18 journal: angew chem weinheim bergstr ger doi: 10.1002/ange.201209222 sha: doc_id: 284978 cord_uid: vh1x6pg9 ein einfaches testsystem auf der basis von graphenoxid (go) ermöglicht das screening selektiver inhibitoren einer helicase des hepatitis‐c‐virus (hcv) zusammen mit inhibitoren eines sars‐coronavirus (sars‐cov) (siehe schema). in einem einzelnen screen wurden fünf hochselektive inhibitoren der hcv‐helicase gefunden, die ortholog zur sars‐cov‐helicase sind. einige dieser treffer wurden mit dem gleichen go‐assay validiert.[image: see text] worldwide, over 170 million people have hepatitis c virus (hcv) infections. [1] chronic infection with hcv leads to liver diseases such as cirrhosis and hepatocarcinoma and is the major reason of liver transplantation. [2] currently, the standard treatment for hepatitis c relies on a combination of interferon-a and ribavirin (an immune booster and a general inhibitor of virus replication, respectively) which is associated with serious side effects including hemolytic anaemia, depression, fatigue, flu-like symptoms, and birth defects. [3] the standard of care is frequently ineffective in clearing hcv infections and the virus often survives and thrives even under the treatment. to develop direct-acting antiviral agents for hepatitis c treatment, studies have been focused on the discovery of inhibitors of viral enzymes, specifically nonstructural protein 3 (ns3) serine protease and ns5b rna-dependent rna polymerase (rdrp). [4] last year, new drugs for treating hepatitis c, telaprevir (vertex) and boceprevir (merck) , which are ns3 serine protease inhibitors, were approved by the u.s. food and drug administration (fda), and over 30 drug candidates targeting the same protease or ns5b rdrp are currently in clinical trials. [5] although phase iii clinical trials of telaprevir and boceprevir showed notable increases in the cure rate, nearly every patient still suffered from at least one side effect of the new therapy. [5b,c, 6] in addition, hcv has strong drug resistance due to its high mutability. thus, further therapeutic options are urgently needed to treat hcv infections more effectively. the c-terminal two thirds of hcv ns3 forms a helicase, which has the ability to unwind double-stranded dna (dsdna) into single-stranded dna (ssdna) and is fueled by nucleoside triphosphates (ntps) hydrolyzed by its ntpase domain. [7] ns3 helicase is one of the essential enzymes of hcv along with ns3 serine protease and ns5b rdrp for processing hcv proteins and replication of hcv. thus, the inhibition of helicase activities is an important strategy for treating hcv infections. [8] however, discovery of helicase inhibitors has been much slower compared to that of other hcv drug targets. to date, only a few classes of ns3 helicase inhibitors have been reported, partly because high-throughput screens have yielded only a few successful hits. [8a,c] for example, the compounds discovered in the nih screen based on the molecular-beacon-based helicase assay (mbha) showed poor activity in cells and turned out to interfere with the assay, [9a] even though another mbha screening identified compounds that inhibited rna replication in cells. [9b] therefore, there is an urgent need for new assays to measure helicase activity that are suitable for high-throughput screens. herein, we developed a multiplexed helicase assay based on graphene oxide (go) for high-throughput screening of inhibitors of hcv ns3 helicase and severe acute respiratory syndrome coronavirus (sars cov) helicase. previously, we reported a go-based helicase assay (goha), which relied on the preferential binding of ssdna over dsdna to the go surface and subsequent quenching of the fluorescently labeled ssdna by energy transfer from the dyes to the go, and we validated the assay using sars cov helicase. [10] herein, we show that the goha can be used for measuring the activities of hcv ns3 helicase and sars cov helicase in a single mixed solution using two distinct dna substrates tethered to different fluorophores, and furthermore, for multiplexed high-throughput screening to discover highly selective small-molecule inhibitors of these helicases ( figure 1 ). one round of screening the chemical library using the multiplexed goha (mgoha) revealed three classes of inhibitors-specific inhibitors of hcv ns3 helicase, specific inhibitors of sars cov helicase, and general inhibitors of both helicases. to date, go has been used to develop various biosensors and enzyme assays, [10, 11] but concerns about the heterogeneity of the chemical structure and the physical dimensions of go and nonspecific binding of biomolecules to go hamper the application of go-based assays in high-throughput parallel assays for drug discovery that require high robustness and reproducibility. herein, we show that go can be used for the discovery of potent smallmolecule drug candidates and also determine the specificity and relative effectiveness towards each helicase of each of the identified hits. first, go sheets were prepared by using a modified hummers method. [10, 12] the thickness of the prepared go sheets was 0.97 nm and the width was 0.01-4 mm, which were determined by atomic force microscopy (afm; supporting information, figure s1 a). the ir spectrum of go showed peaks at 3395, 1716, 1225, and 1079 cm à1 corresponding to o à h stretching vibration, c=o stretching vibration, cào (epoxy) vibration, and cào (alkoxy) vibration (figure s1 b). the raman spectrum of go had strong d-and g-band absorptions at 1351 and 1589 cm à1 , respectively, which are characteristic for go (figure s1 c) . collectively, water-dispersible go sheets were successfully prepared. to measure the orthogonal activity of sars cov and hcv ns3 helicases, either or both helicases were added to a mixture of go and two dna substrates-a cy3-labeled dna duplex as the sars cov helicase substrate (cy3-dna-sh) and a cy5-labeled dna duplex as the hcv ns3 helicase substrate (cy5-dna-hh; figure s2 a). [13] fluorescence intensities corresponding to cy3 (ex/em = 550/570 nm) and cy5 (ex/em = 650/670 nm) showed little change without the addition of helicase over 60 min (figure 2 a) . the addition of sars cov helicase led to a dramatic decrease in fluorescence intensity for cy3 only, while cy5 fluorescence intensity only showed a very small change, suggesting that the cy3-labeled dna duplex was mainly unwound, and subsequently, the interaction of the generated cy3-labeled ssdna with go resulted in the quenching of the cy3 fluorescence (figure 2 b ). this data indicated that the unwinding activity of the sars cov helicase was specific to its corresponding dna substrate. conversely, the addition of hcv ns3 helicase caused a significant decrease in fluorescence intensity of cy5, but not cy3 ( to assess the quality of the mgoha for screening, the z'factor, a parameter widely used to evaluate the robustness and quality of screening assays, [14] was determined. generally, high-performance assays have z'-factors of 0.75 or higher. [15] to calculate the z'-factor, we ran 30 positive and 30 negative control reactions, calculated the mean (m c+ and m cà for the positive and negative controls, respectively) and standard deviations (s c+ and s cà ), and used an equation shown in figure s2b . the z'-factor of the mgoha platform for sars helicase was 0.95 and hcv helicase was 0.94, suggesting that the present goha is better and more robust than other established assays (figure 2 f) . a 96-well plate mgoha was used to screen a 10 000 compound library to discover inhibitors of sars cov helicase and hcv ns3 helicase (figure 3) . a mixture of library compounds and both helicases were prepared and added to mixed solutions of cy3-dna-sh, cy5-dna-hh, and go that were prepared separately. in the primary screen, the library compounds, helicases, and substrates were used at concentrations of 1 mm, 2 nm, and 10 nm, with a total volume of 60 ml for each mixture. after incubation for 30 minutes at 25 8c, the cy3 and cy5 fluorescence was measured using an ivis fluorescence imaging system (figure 3 ; figure s3 ). positive controls were performed with cy3-dna-sh and cy5-dna-hh and both helicases, in the absence of library, and negative controls were with cy3-dna-sh and cy5-dna-hh, excluding any helicases or compounds. out of the 10 000 small molecules, 26 specific inhibitors of sars cov helicase and 22 of hcv ns3 helicase were found, and 24 compounds were found to inhibit both helicases (! 50 % inhibition). we further characterized the discovered inhibitors to quantitatively evaluate their inhibitory effects towards the respective helicases. first, to increase the fidelity of the data and to eliminate false positive hits owing to poor solubility of the compounds, we performed a secondary screening in replicates of five with the selected compounds from the primary screen (72 total compounds) while carefully observing the reaction mixtures for precipitation. in the secondary screen, the selectivity towards each helicase and the degree of inhibition were comparable to the primary screen. out of 72, 25 compounds showed severe precipitation and were excluded from the final selection. finally, we selected the 15 most-potent compounds including five sars cov helicase selective inhibitors (antisars-hel-1-5), five hcv ns3 helicase selective inhibitors (antihcv-hel-1-5), and five inhibitors of both helicases (antisars-hcv-hel-1-5). the chemical formulas of the selected compounds are shown in figure s3 . dose-dependent inhibition of sars cov helicase and hcv ns3 helicase were measured with varying concentrations of inhibitor using goha, and the half-maximal inhibitory concentration (ic 50 ) was calculated (figure 4 a) . the antisars-hels and antihcv-hels had ic 50 values of 49.3-104.6 mm and 51.6-92.0 mm, respectively. we found that antisars-hcv-hel-1-5 had higher ic 50 values than the selective inhibitors, ranging from 103.8 to 537.5 mm for sars cov helicase and from 103.5 to 441.1 mm for hcv ns3 figure 3 . a small-molecule screen identified three sets of inhibitorsspecific for sars cov helicase, specific for hcv ns3 helicase, and nonspecific helicase inhibitors. top) representative fluorescent images from the primary screen carried out with 10 000 small molecules in 96well plates. cy3 and cy5 signals represent the inhibition of helicase activities for sars cov and hcv ns3, respectively. bottom) using the 72 compounds selected from the initial screens, a secondary screen was performed in quintuplicate revealing the top 15 inhibitors. blue bars = sars-hel; red bars = hcv-hel. . angewandte zuschriften helicase. previously known helicase inhibitors generally exhibit ic 50 values ranging from 1-1000 mm. [8b] we expect that a structure-activity relationship (sar) study could further optimize these hits. the goha system can also be used for an sar study. the dose-response data and fitted ic 50 curves are shown in figure s5 . we next investigated the mechanism of the inhibitory effects for the discovered compounds. first, we determined whether antisars-hcv-hels bind to dsdna because the low selectivity between the two types of helicases might come from nonspecific binding to dsdna and thus, slow down the unwinding process. however, isothermal titration calorimetry (itc) and nmr spectroscopy showed no interaction between the antisars-hcv-hel-1-5 and dsdna (figures s7-s9) . we next determined whether the 15 compounds had any inhibition of the atpase activities of the helicases (figure 4 b) . sars-hel and hcv-hel possess an atpase domain for the catalytic hydrolysis of atp. we used a commercial kit to analyze the atpase activities of both helicases in the presence of the 15 inhibitors. we found that all of the antisars-hels except for antisars-hel-2 showed a significantly higher inhibition of the atpase activity of sars cov helicase than hcv ns3 helicase; all the antihcv-hels showed a much higher inhibition of atpase activity of hcv ns3 helicase than sars cov helicase. antisars-hcv-hel-1 and -2 blocked the atpase activities of both helicases to a similar degree. the antisars-hcv-hel-3-5 inhibited the atpase activity of hcv ns3 helicase more effectively than that of the sars cov helicase; however, the overall inhibition of antisars-hcv-hels on the atpase activities were not as impressive as that of the antihcv-hels against the atpase activity of hcv ns3 helicase. inhibitors selective for each helicase could block the atpase activity of each helicase with high selectivity. further studies should be performed to investigate the mode of inhibition-whether the inhibitors bind to the atp binding sites or induce conformational change by allosteric regulation. we next measured the inhibition of hcv rna replication by the 15 selected inhibitors in the human hepatoma cell line huh-7 carrying the hcv replicon rna with luciferase as a reporter gene (huh-7 replicon cell). [16] the replicon huh-7 cells were treated with the 15 inhibitors at concentrations of 0-1000 mm and then lysed, and the luminescence intensity was measured after the addition of luciferin. separately, the cytotoxicity of each compound was measured in the same cell line using an mtt assay. relative luciferase signal intensities over relative mtt signal intensities (luc/ mtt) were plotted versus the concentrations of the inhibitors ( figure 5 ; see figure s6 for the complete data of the luciferase and mtt assays). [17] two compounds, antihcv-hel-2 and -3, showed a dose-dependent decrease in the luc/mtt values with the respective half-maximal effective concentrations (ec 50 ) of 188.1 ae 32.6 and 56.8 ae 7.4 mm, indicating that they dose-dependently blocked hcv rna replication in the cultured huh-7 cells (figure 5 b,c) . antihcv-hel-2 and -3 decreased the luciferase signal by more than 90 % at 1 mm. the other antihcv-hel compounds, antisars-hel-5 and all the antisars-hcv-hels, were highly cytotoxic showing less than 40 % cell viability at 1 mm. antisars-hel-1-4 showed little reduction in the luciferase signal even at 1 mm. we excluded antihcv-hel-5 because of its relatively high ec 50 % 400 mm. taken together, antihcv-hel-2 and -3, blocked hcv rna replication in the cells whereas all of the antisars-hels and antisars-hcv-hels showed no appreciable inhibitory effect on hcv rna replication in cultured cells. these results show that helicase inhibitors should be specific to the corresponding helicase to achieve an inhibitory effect in living cells. in addition, the nonselective inhibitors that moderately reduce the activity of both helicases had a relatively high cytotoxicity, indicating that those compounds should be excluded for further drug development. we further investigated whether the antihcv-hel-2 and -3 could reduce the expression of hcv ns3 serine protease by blocking hcv rna replication in huh-7 cells (figure 5 d) . the cells were first treated with 250 mm of antihcv-hel-2 and -3 at which the luc/mtt values were 0.39 and 0.24, respectively, and the cell viability based on the mtt assay was over 70 %. after cell lysis, the hcv ns3 serine protease activity was measured with a fçrster resonance energy transfer (fret) peptide substrate. treatment with antihcv-hel-2 and -3 reduced the protease activity in the huh-7 cells down to 30 % of that in the untreated cells. this indicates that ns3 serine protease activity was also decreased as a result of the inhibition of hcv rna replication in living cells by antihcv-hel-2 and -3. in conclusion, we demonstrated the capability of mgoha for assaying the activity of two helicases, hcv ns3 and sars cov, and applied this mgoha to a highthroughput inhibitor screen using a 10 000 small molecule library, which yielded drug candidate molecules. these results illustrate that mgoha is an important screening technique for drug discovery targeting helicases. goha allows for figure 5 . cell-based assay to test the inhibition of hcv rna replication in huh-7 liver cells by antihcv-hel-2 and -3. a) a luciferase reporter system based on the expression of the hcv subgenome in huh-7 cells with a neomycin antibiotic resistance marker. b, c) to characterize the inhibition of hcv rna replication in live cells, huh-7 liver cells were treated with inhibitors and luciferase signal intensity from the reporter shown in (a) divided by cell viability was plotted versus inhibitor concentrations (to correct for luciferase signal loss from cytotoxicity of the compounds). d) to confirm that the two inhibitors block hcv rna replication and thus, reduce the expression of hcv enzymes, the hcv ns3 serine protease activity of the huh-7 cell lysates was measured following incubation with the two inhibitors. more-accurate, real-time, quantitative monitoring of helicase activity by simply following the changes in fluorescence intensity, without requiring any additional separation steps or trapping agents. a multiplexed screen is highly advantageous not only because multiple rounds of screening are combined into one screen to reduce the overall cost and labor, but also because just one round of multiplexed screening provides information on the relative selectivity of the hit compounds towards each target helicase. given that various go-based activity assays have been developed for various enzymes, including nucleases, [18a] methyltransferases, [18] and caspases, [11c] the present work shows that a go-based assay can be used for high-throughput screening for new inhibitors of various essential enzymes. for the treatment of hcv infection, the addition of direct-acting antiviral agents, like inhibitors of ns3 protease, to the standard treatment was recently approved. the addition of multiple drugs acting against independent viral targets should be more effective in controlling infection with a rapidly mutating virus. we expect that the new inhibitors of hcv ns3 helicase discovered herin could be useful as antiviral drugs for the next generation of hcv treatment, with three or more active components. in addition, we expect that goha can be used to evaluate helicase activities in a highly parallel manner and of helicase-inhibitor-based drugs for various diseases, which has been difficult without a robust helicase assay. received: november 17, 2012 published online: january 25, 2013 . keywords: biosensors · graphene oxide · high-throughput screening · helicases · multiplex assays 360, 1839; b) key: cord-253825-d9borky8 authors: blaising, julie; polyak, stephen j.; pécheur, eve-isabelle title: arbidol as a broad-spectrum antiviral: an update date: 2014-04-24 journal: antiviral res doi: 10.1016/j.antiviral.2014.04.006 sha: doc_id: 253825 cord_uid: d9borky8 arbidol (arb) is a russian-made small indole-derivative molecule, licensed in russia and china for prophylaxis and treatment of influenza and other respiratory viral infections. it also demonstrates inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases such as hepatitis b and c, gastroenteritis, hemorrhagic fevers or encephalitis. in this review, we will explore the possibility and pertinence of arb as a broad-spectrum antiviral, after a careful examination of its physico-chemical properties, pharmacokinetics, toxicity, and molecular mechanisms of action. recent studies suggest that arb’s dual interactions with membranes and aromatic amino acids in proteins may be central to its broad-spectrum antiviral activity. this could impact on the virus itself, and/or on cellular functions or critical steps in virus-cell interactions, thereby positioning arb as both a direct-acting antiviral (daa) and a host-targeting agent (hta). in the context of recent studies in animals and humans, we will discuss the prospective clinical use of arb in various viral infections. arbidol (arb) has been administered for decades in russia and china against influenza, with no major adverse effects reported. its vast potential as a broad-spectrum antiviral agent, defined through in vitro and in vivo studies, lends hope for its clinical use against various infectious diseases that are at present not therapeutically controlled. however, evidence for beneficial effects in humans, especially in the perspective of long-term administration in chronic diseases, remains equivocal. this could be attributable to a relative lack of standardized animal studies and controlled clinical trials in healthy and infected subjects. in addition to influenza and pathogenic human respiratory viruses, arb shows mainly in vitro inhibitory activity against the hepatitis b virus (hbv), hepatitis c virus (hcv), chikungunya virus (chikv), reovirus, hantaan virus and coxsackie virus b5. in this paper, we update current knowledge about arb, linking its physico-chemical properties to its molecular mode of action, toxicity and possible pharmaceutical forms. we will outline recent studies on the molecular and cellular mechanisms by which arb may inhibit several steps of viral life cycles, and discuss how arb is emerging as both a direct-acting agent (daa) and a host-targeting agent (hta). china, toxicity arb, or ethyl-6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2 [(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate, is a small indole derivative (fig. 1a) . it is also called umifenovir. its invention is attributed to a joint consortium of russian scientists from the chemical-pharmaceutical scientific research institute of russia, the scientific research institute of medical radiology in obninsk and the leningrad-pasteur scientific research institute for epidemiology and microbiology, some 40 years ago, as described in: arbidol.net/robert-nikolaevich-glushkov.html arbidol.org/1973-4-arbidol-invented-way-to-the discovery.pdf arbidol.org/article1.html. one of the first descriptions of its chemical synthesis was published in 1993 (trofimov et al., 1993) , and modified later on miller and bergeron (1994) . the drug is manufactured by moscow-based masterlek™, a subsidiary of pharmstandard group (see below), and by shijiazhuang no.4 pharmaceutical™ in china (http://www.sjzsiyao.com/products_detail/&productid=46.html). arb has been marketed for 20 years in russia and has been used since 2006 in china for the prophylaxis and treatment of human pulmonary diseases caused by influenza a and b viruses and other human pathogenic respiratory viruses, as reviewed in boriskin et al. (2008) , brooks et al. (2004) . it is also used to prevent flu epidemics in poultry in china (berendsen et al., 2012) , and is available from chinese companies specialized in animal health products, such as: http://depond.b2bage.com/product-chemical-auxiliary-agent/ 1503414/arbidol-hydrochloride-pharmaceutical-rawmaterial.html the first reports on the clinical efficacy of arb were published in russian in the 1990s, in groups of students and industrial workers during epidemics of influenza a and outbreaks of acute respiratory diseases (gagarinova et al., 1993; obrosova-serova et al., 1991) . later studies performed in servicemen reported the efficacy and cost-effectiveness of prophylactic or curative treatments of arb against acute respiratory viral infections, where arb was shown to decrease the febrile period (shumilov et al., 2002; shuster et al., 2004) . when the information is available, the duration of arb treatment varies from 5 to 20 days. chinese clinical studies with similar design (patient inclusion criteria, arb doses and duration of administration) point to a comparable efficacy and tolerability of arb (wang et al., 2004) . arb efficacy compared well or even better with that of other commonly used antiviral molecules such as rimantadine (roflual ò ), oseltamivir (tamiflu ò ), ribavirin or interferon-alpha kolobukhina et al., 2008 kolobukhina et al., , 2009 leneva and shuster, 2006) . it potentiated the in vitro effect of rimantadine against influenza a and b viruses , enhanced the immunomodulatory properties of the anti-flu vaccine vaxigrip ò , administered in a cohort of 125 elderly patients (semenenko et al., 2005) , and had a beneficial effects on flu in patients with another infectious immunodeficiency (glushkov et al., 1999) . most of these studies point to a dual pharmacological action of arb: a specific effect on respiratory viruses and an immune-stimulating effect, with induction of serum interferon and activation of phagocytes. studies have also been conducted in children suffering from flu and other acute viral respiratory diseases (beliaev et al., 1996; drinevskii et al., 1998) . the latter study -and most documented one -was conducted on 158 children of 1-14 years old, infected with influenza a or b or both or with other respiratory viruses. over a 5-day treatment, arb was efficient at reducing the duration of infection and the occurrence of complications, and its immunomodulating action was again suggested. in 2002, masterlek™, the company currently marketing arb, sponsored a vast clinical trial conducted on 500 children from 6 to over 12 years old. arb was given (i) either in prophylaxis twice a week for 3 weeks or once daily for 12 days; (ii) or in treatment thrice daily for 3 days. in all cases, arb treatment led to a significant reduction of the duration of clinical signs, with no observed adverse effect or complications; see: http://arbidol.org/arbidol-childrens-study.pdf. interestingly, in studies comparing antivirals, most viral strains were sensitive to arb, whereas several resistant variants were found with rimantadine (cf also the recent iatsyshina et al., 2010; l'vov et al., 2013 ) (see also section 7.). since 2004, arb is also patented by masterlek™ for its medicinal use as an antiviral agent against atypical pneumonia induced by the severe acute respiratory syndrome coronavirus (sars-cov); see: http://www.arbidol.org/arbidol-patent-2004-sars-russian.pdf most recently, a double-blind, randomized, placebo-controlled phase iv clinical trial has been launched by pharmstandard/mas-terlek™, to assess whether arb is effective in the treatment and prophylaxis of flu and common cold: http://clinicaltrials.gov/ct2/show/ nct01651663?term=arbidol&rank=1. two dosages will be evaluated: 800 mg/day for 5 days, or 200 mg/day for 10 days. completion of this study is expected in 2015. apart from this recent trial and in spite of an abundance of studies in the 1990s, the overall language barrier renders difficult the precise evaluation of the number of subjects enrolled per study, the way clinical trials were designed, and subsequent statistical analyses performed. moreover, an official russian site exists for arbidol (arbidol.ru), where more information could be collected; but no english translation is available. as to studies specifically addressing arb toxicity issues, initial literature is mostly in russian, when available. acute toxicity data report oral ld50s of 340-400 mg/kg in mice, and >3000 mg/kg in rats and guinea pigs: http://img1.guidechem.com/msdspdf/131707-23-8.pdf; glushkov, 1992. these values are also reported elsewhere: http://arbidol.org/pre-1990-animal-human-test-results.pdf; http://arbidol.org/rat.html; (loginova et al., 2009; shi et al., 2007) . administered intravenously, arb exhibited ld 50 s of 109 mg/kg in mice and 140 mg/kg in rats (eropkin et al., 2009 ). on long-term per os administration of arb in rats, guinea pigs, rabbits or dogs from 2 to over 6 months (with doses ranging from 25 to 125 mg/ kg), no pathological changes were observed in animals. these doses would roughly correspond to 10-to 50-fold the therapeutic doses in humans. arb is also reported not to induce embryo toxicity in pregnant female rats, nor alter the reproductive function of animals, over a 20 day-administration period of 500 mg/kg doses (http://arbidol.org/rat.html). recent data from a chinese group showed a good tolerability of arb administered to rats per os, at daily doses ranging from 80 to 320 mg/kg over a 4-week period (wang et al., 2010) . but in fact this study assessed the toxicity of a 1:2.5 combination of arb with acetaminophen, which renders difficult to precisely evaluate the toxicity of each molecule individually. in healthy male volunteers receiving a single 200 mg-dose of arb, an excellent tolerability was reported . from these data, it appears that arb is a well-tolerated molecule with a high therapeutic index, when administered on periods ranging from a few days to a month. to date, however, no studies have addressed the long-term administration of arb, for example in the context of chronic infections. as an indole derivative, arb is expected to be poorly soluble in aqueous media. this is of major repercussion on its bioavailability, forms of administration and pharmacokinetics. efforts to improve arb water solubility were undertaken, through the chemical grafting of acrylamide polymers to the arb molecule (eropkin et al., 2009) . antiviral properties of these complexes were maintained compared to the parent molecule. they also displayed a better in vitro pharmacological index than arb, defined as ic 50 /vic 50 (vic, virus-inhibiting concentration). however these polymers were not further developed. arb is soluble in hot glycerol, where it remains soluble down to 23°c. it can then be diluted into aqueous media and administered in vitro or in vivo (brooks et al., 2012) . however no pharmacokinetic nor metabolite studies were performed from this mode of administration. a very selective, sensitive and accurate method of detection of arb from human plasma by high-pressure liquid chromatography was designed, and allowed to conclude that no interference existed between arb and its expected metabolites (metz et al., 1998) . studies based upon this hplc method then evaluated the pharmacokinetic parameters of arb after oral or intravenous administration in rats. comparable plasma elimination half-lives (t 1/2 ) and maximum concentrations (c max ) were obtained for oral doses six times higher than those injected; however this was only performed on a small number of animals (liu et al., 2007a) . the drug is manufactured in russia and china as tablets or capsules, each containing arb as its active ingredient. initial russian studies in humans revealed that the main site of arb metabolism is the liver. arb was rapidly distributed in tissues and organs with maximal accumulation in liver (3.1% w/w), pituitary gland, kidneys, lymphatic nodes and thyroid, adrenal gland, bone marrow, lungs, plasma, thymus and spleen (less than 1% each) (glushkov, 1992) ; see: http://www.chemeurope.com/en/encyclopedia/arbidol.html. plasma c max was reached within 1 h or 1.5 h after a 50 mgor 100 mg-dose, respectively, and t 1/2 was between 17 and 21 h. about 40% of the total intake dose of arb was excreted unchanged within 48 h via feces. more recent studies reported much shorter plasma c max and t 1/2 in chinese healthy volunteers, concluding that russian and chinese populations differed in arb elimination rates (liu et al., 2007b ). however doses administered differed, and technological improvements, especially in detection sensitivity, might also explain such discrepancies. single doses of 200 mg of arb administered to healthy volunteers from dispersible tablets or capsules were found bioequivalent and well tolerated . pharmacokinetics of oral single vs multiple doses of arb were compared in healthy subjects, from plasma samples analyzed by hplc (metz et al., 1998; sun et al., 2013) . c max increased linearly with the intake dose for single dose administrations, peaking at 2.16 lg/ml for a 800 mg-dose (sun et al., 2013) . arb exhibited little accumulation with repeated doses, and the pharmacokinetic profile differed from that observed after single dosage. based on arb's chemical structure, several metabolites can be predicted ( fig. 1a) : oxidation at the sulfur atom, loss of the 4-(dimethylamino)methyle group and n-demethylation, conjugation at the 5-hydroxyl moiety. in a pioneering study in rat urine, the formation of sulfone and sulfoxide forms was confirmed after hplc, from an oral administration of an arb/starch suspension (anisimova et al., 1995) . glucuronide or sulfate conjugations were also observed at position 5, and after demethylation of the (dimethylamino)methyle group (see also liu et al., 2012) . in human urine, after administration of a single 300 mg-dose of arb to healthy subjects, 17 metabolites could be identified, of which the major ones were arb glucuronide and sulfoxide-arb (or sulfinyl-arb; fig. 1b ) glucuronide (wang et al., 2008) . similar metabolites as identified in rats were observed. arb could also be glucuronidated in vitro, using purified human liver microsomes; this study also revealed that the microsomal (recombinant) udp-glucuronosyl-transferase (ugt) 1a9 was the major ugt isoform involved in arb glucuronidation (song et al., 2013) . conversely, arb was found to inhibit ugt1a9 (ibid.; liu et al., 2013) . since ugt1a9 is involved in the metabolism of several drugs (e.g. acetominophen, diclofenac), potential drug-drug interactions that could lead to adverse effects should be carefully examined if arb is administered with other molecules. a more complete picture could be obtained from a study in healthy volunteers receiving a single oral dose of 200 mg arb, where urine, feces and plasma metabolites were analyzed (deng et al., 2013) . most of the metabolites were sulfoxidated, dimethylamine n-demethylated, glucuronide-or sulfate-conjugated. about 32.4% of the total intake dose of arb was excreted unchanged via feces, as previously reported (boriskin et al., 2008) . sulfinyl-arb ( fig. 1b) was the major circulating component detected in plasma, followed by unmetabolized arb, n-demethyl-sulfinyl-arb and sulfonyl-arb ( fig. 1c ). urine samples contained mainly glucuronide and sulfate conjugates. in vitro experiments using human liver, intestine and kidney microsomes, and recombinant enzymes, showed that arb was metabolized by human microsomes from liver and intestines but not from kidney. in these organs, cyp3a4 was identified as a key metabolic enzyme of arb. still, questions remain about the pharmacokinetic properties of arb metabolites and their potential antiviral activity. the following parameters were reported (deng et al., 2013) : tmax for arb and dimethylamine n-demethylated arb were comparable (1.4 and 1.5 h, respectively), while it was much longer for sulfinyland sulfonyl-arb (13 and 19 h). plasma elimination half-lives (t 1/ 2 ) of all metabolites were longer than that of arb (26.3, 25, 25.7 and 15.7 h for n-demethylated, sulfinyl-, sulfonyl-arb and arb, respectively). exposure to metabolites is therefore greater than that to arb, and the main metabolite, sulfinyl-arb, is expected to accumulate on repeated arb doses. along these lines, sulfinyl-arb was reported to contribute for some of the pharmacological activities associated with arb, and sulfonyl-arb could inhibit protein kinase c ([ic50] = 7.78 lm) (demin et al., 2010) . therefore the potency and duration effect for the agent may be underestimated by measuring only arb concentrations. it is also predictable, based upon in vitro data with microsomes, that some of the in vivo metabolites could occur in cell cultures, especially in studies addressing the antiviral effect of arb on hepatotropic viruses using liver-derived cell lines. this could explain why arb demonstrated greater efficacy under pre-incubation conditions, where metabolites could already be produced and exert specific effects (see below section 4.). however, a recent study directly addressing the in vitro antiviral properties of sulfinyland sulfonyl-arb against the chikungunya alphavirus showed only moderate to weak activity as compared to that of the parent molecule (delogu et al., 2011) . this was reinforced by the observation that pre-incubation of cells with arb prior to infection did not improve antiviral effect, pointing to a minor role (if any) of arb metabolites against chikungunya infection, at least in vitro. in any case, further investigations will be necessary to: (i) understand the importance of metabolites in the efficacy and safety of arb, due to their high plasma exposure and long elimination half-lives; (ii) assess their stability in circulation, tissue binding and storage properties. arb has been shown to display antiviral in vitro and/or in vivo activity against a number of enveloped or non-enveloped rna or dna viruses, including influenza viruses a, b and c , respiratory syncytial virus, sars-cov, adenovirus, parainfluenza type 5, poliovirus 1, rhinovirus 14, coxsackievirus b5, hantaan virus, chikungunya virus, hbv and hcv [reviewed in boriskin et al. (2008) , brooks et al. (2004 brooks et al. ( , 2012 , liu et al. (2013a) ] (see also table 1 ). numerous reports in russian describe the antiviral potency of arb against human or avian influenza a viruses, and notably the highly pathogenic h5n1 leneva and shuster, 2006) and the pandemic 2009 h1n1 subtype (fediakina et al., 2011) . in vitro studies report ic50s in the 2.5-16 lm range, and state an effect of arb comparable to that of ribavirin, but superior to that of rimantadine, with rimantadine-resistant strains sensitive to arb romanovskaia et al., 2009; leneva et al., 2010; fediakina et al., 2011) . a few studies state a potentiating effect of arb and rimantadine (or amantadine) on influenza a and b viruses burtseva et al., 2007) . however adamantane antivirals are scarcely used against influenza viruses, due to low barrier to resistance. in these studies, accessible information does not allow to evaluate the stage of the viral life cycle targeted by arb nor its mode of action. shi and coworkers showed a greater inhibitory effect on influenza a h1n1 when arb was added before infection or when it was pre-incubated with the virus (shi et al., 2007) , suggesting that membrane impregnation and/or metabolites could underlie arb antiviral activity (see section 6.). arb demonstrated similar in vitro antiviral activity as the reference drug ribavirin (virazole ò ) against the respiratory syncytial virus (rsv), an enveloped virus of the paramyxoviridae family (leneva et al., 2002) . arb was most efficient when added before infection (shi et al., 2007) , with an ic50 of 16 lm. recently, tannock and coworkers reported a potent antiviral activity of arb on several virus families responsible of respiratory infections in animals and humans, in particular on influenza a h3n2 (ic50 12 lm), and the non-enveloped picornaviridae poliovirus 1 and rhinovirus 14 (brooks et al., 2012 ; see also brooks et al., 2004) . concerning rsv, only a reduction in plaque size and not in number could be observed, hampering the estimation of an ic50. in this study, arb was added to cells as an aqueous glycerol solution, instead of the classical dilution from dmso in other studies (leneva et al., 2002; shi et al., 2007) . this might explain the discrepancy of antiviral effect on rsv. arb also displayed an inhibitory effect on the coxsackievirus b5, another member of the picornaviridae family responsible for a variety of pathologies including respiratory infections, myocarditis or encephalitis . arb was most active on the virus itself (virucidal test) or when added after infection, through the inhibition of late stages of viral replication. indeed it was shown to prevent the viral rna synthesis in a dose-dependent manner, with maximal effect obtained at 5 lm. one study in russian describes in vitro antiviral activity of arb against the sars-cov, when added after viral infection and at high concentration (95 lm) (khamitov et al., 2008) . depending on the cell type, arb cc50 was reported to vary between 20 and 200 lm (e.g. boriskin et al., 2006; brancato et al., 2013; brooks et al., 2012; shi et al., 2007) . the dose exhibiting anti-sars-cov activity may likely be cytotoxic. studies conducted on mouse-adapted flu models showed that arb was effective when administered orally at doses from 15 to 30 mg/kg leneva et al., 2010) , or up to 100 mg/kg (shi et al., 2007) , especially when given in prophylaxis before infection. extrapolated to humans, these doses would correspond to 1-6 g per day, not evaluated clinically in terms of safety. recently, arb was found to be effective in vivo against two influenza a h1n1 strains, responsible for seasonal or pandemic flu (liu et al., 2013b) . reductions in lung viral titers and lesions were observed for oral doses of 90-180 mg/kg/day, and secretion of lung and macrophage cytokines was modulated, indicating an inhibitory effect of arb on virus-induced inflammation. however, no effect on interferon-alpha was observed, in line with (brooks et al., 2012) but contrary to initial reports (glushkov, 1992) . brooks et al. (2012) reported a minor effect of arb on flu a-infected mice at doses from 2 to 50 mg/kg/day. discrepancies between results from different groups might come from: (i) bioavailability issues, due to differences in solvents used to solubilize arb (dmso vs glycerol); (ii) animal models of flu, using viruses and viral strains adapted or not adapted to mice; (iii) doses administered to animals. however, an overall anti-flu effect of arb in vivo seems apparent. mice with rsv-induced pneumonia were responsive to arb at 10-50 mg/kg/day doses, with an observable but not significant reduction in lung infectious titers as compared to untreated animals (brooks et al., 2012) . while this study points to a potential promising effect of arb against rsv in vivo, the limited of global studies addressing the effect of arb against rsv should invite moderation and a call for additional studies. one study addressed the antiviral effect of arb in mice infected with the coxsackievirus b5 . mice developed interstitial pneumonia and myocarditis, and some received arb orally for 6 days. at a dose of 50 mg/kg, the drug prolonged survival and reduced viral propagation in lungs and heart. although this result completes the picture of the broad-spectrum antiviral activity of arb, it must again be taken with caution, since it is the sole study on this virus, conducted on a small number of animals and with a high dose of arb. recently, chinese studies demonstrated antiviral activity of arb against the hantaan virus, an enveloped virus from the bunyaviridae family wei et al., 2013) , causing an often lethal hemorrhagic fever with renal syndrome (hfrs). in vitro, arb was more efficient when added before infection, with an ic50 in the 2 lm range. a direct virucidal effect was noted only for arb concentrations over 15 lm. in vivo, it was able to increase the survival rate, reduce histopathological changes and viral loads in the lethal model of intracranially-infected suckling mice. also, serum levels of tnf-alpha were modulated. since these studies were performed by only one research group, with a limited number of animals, they should be reproduced by others before concluding to a beneficial effect of arb against hantavirus infection. however arb efficacy compared well in vivo with that of ribavirin, the reference treatment for such a disease (wei et al., 2013) . viruses from the rhabdoviridae family are known to induce neurological disorders, encephalitis or, more recently reported, hemorrhagic fever (grard et al., 2012) . the only study addressing the effect of arb against a virus of this family was conducted in our laboratory on the vesicular stomatitis virus (vsv) (blaising et al., 2013) . this enveloped rna virus mainly infects cattle and pigs, causing oral lesions, anorexia and lethargy. arb was shown to inhibit in vitro vsv infection in a very similar concentration range as that already shown to affect influenza a or rsv infection [ic50 of 14 (blaising et al., 2013) , 12 (brooks et al., 2012) or 16 lm (shi et al., 2007) , respectively]. again, arb displayed optimal antiviral activity when incubated with cells before infection. this family of double-stranded rna viruses comprises animal and human pathogens, such as the rotavirus, a major agent of (2007), teissier et al. (2011) gastroenteritis in children. we recently addressed the potential of arb against the mammalian reovirus t1l strain (blaising et al., 2013) . this virus is a prototypic member of the orthoreovirus genus, which infects a wide variety of host species without causing a significant pathology in humans. in spite of this, reovirus has proven to be a useful model for studying viral pathogenesis. in vitro, arb inhibited reovirus infection in the 10 lm range, but interestingly, did not exert any effect on infectious subvirion particles (isvps), intermediates of reovirus infection (chandran et al., 2002) that could also directly infect cells via a different entry mechanism from that of reovirus (martinez et al., 1996) . this points to the molecular mechanisms of action of arb (detailed in section 6.). this alphavirus is an enveloped single-stranded rna virus from the togaviridae family, loosely related to flaviviridae (see below hcv). it is responsible for recent outbreaks of a rheumatological disease. some neurological complications were described, together with meningo-encephalitis. arb demonstrated potent in vitro activity against chikv infection (delogu et al., 2011) . arb did not show virucidal activity, contrary to data on respiratory viruses (shi et al., 2007; zhong et al., 2009) , and displayed the highest efficiency when preincubated with cells 24 h before infection (ic50 7.5 lm). in this study, the main metabolites sulfinyl-and sulfonyl-arb were assayed and exhibited only weak antiviral activity, with ic50s > 55 lm. arb activity was not improved when a 12 h-preincubation with cells was performed, suggesting that metabolites or degradation products are not responsible for arb antiviral action. taken together, these results suggest an interference of the parent molecule arb with early steps of the viral life cycle, such as cell binding and entry. arb and derivatives demonstrated in vitro efficiency against the hepatitis b virus (hbv), an enveloped dna virus from the hepadnaviridae family zhao et al., 2006) . arb prevented hbv dna replication with an ic50 of 45 lm, and reduced the production of the virion surface antigen hbsag at 90 lm; however the 50% cytotoxic concentration was 140 lm, suggesting that inhibitory concentrations are most likely cytotoxic. this work will be further discussed below in the section structure/activity relationship (sar; section 5.). we showed that arb exerts in vitro antiviral activity against the hepatitis c virus (hcv) (reviewed in boriskin et al. (2008) ), a member of the flaviviridae family of enveloped viruses. more specifically, arb was most efficient when incubated with cells before infection and left during infection (pécheur et al., 2007) . as already shown with other viruses, arb also displayed virucidal activity (haid et al., 2009; pécheur et al., 2007) . in the 10 lm range, arb inhibited hcv entry, fusion in in vitro and in cellulo studies (blaising et al., 2013; haid et al., 2009; teissier et al., 2011) , and replication on longer times of cell treatment (boriskin et al., 2006; sellitto et al., 2010) . however, as previously reported in the case of influenza a infection in vivo (brooks et al., 2012) , arb was not found to induce interferon antiviral responses in vitro against hcv (boriskin et al., 2006) . from these studies, arb molecular mechanisms of action were proposed (see section 6 below). several studies aimed at gaining a better understanding of the structural features of arb important for its broad antiviral activity, improving arb therapeutic index, or identifying novel lead compounds active against emergent viruses. compounds derived from the chemical structure of arb were synthesized and assayed against various influenza a and b viruses (brancato et al., 2013) . the amine in position 4 and the hydroxyl moiety in position 5 were found important for arb antiviral action, whereas the presence or absence of br in position 6 had little effect (see fig. 1a ). insertion of a methyl group between the indole ring and 5-hydroxyl considerably increased antiviral potency of the resulting compound. this molecule was shown to directly bind ha2, with a greater affinity than arb. the presence or absence of the 6-bromo group had also no influence on hbv or hcv infections (sellitto et al., 2010; zhao et al., 2006) . more specifically, the introduction of particular azote-based heterocyclic groups at position 4 improved anti-hbv activity , while it had little effect against hcv (sellitto et al., 2010) . replacement of the s-phenyl group at position 2 by a phenyl-sulfonyl decreased the cytotoxicity and increased the anti-hbv activity of the compound zhao et al., 2006) , while removal of this group was without any influence against hcv (sellitto et al., 2010) . the 5-hydroxy group was found dispensable against hcv. thus, it appears that different substituents of the arb molecule play a role in the antiviral activity, depending on the virus considered, the cellular model used and the test conditions. the combination of in vitro, in cellulo and in silico analyses will help refine the sar of arb. in particular, in silico molecular docking studies allowed the precise identification of amino-acid(s) involved in arb (or derivative) interaction with ha2 (nasser et al., 2013) . also, three-dimensional quantitative sar (3d-qsar) helped design novel anti-hbv compounds based upon a 5-hydroxy-1h-indole-3-carboxylate skeleton, and predict their antiviral potency (chai et al., 2011) . this type of approach is also now conceivable to study the potential interactions of arb with hcv envelope glycoproteins and clarify structural requirements for antiviral activity, since the 3d-structure of hcv e2 has recently been released (kong et al., 2013) . arb's broad-spectrum antiviral activity suggests that the molecule acts on common critical step(s) of virus-cell interactions. evidence indicates that arb directly exerts a virucidal effect, and can then be considered as a direct-acting antiviral (daa). most studies also report an effect of arb on one or several stages of the viral life cycle, such as cell entry (attachment, internalization) and replication. arb could therefore also act as a host-targeting agent (hta). in the following section, we will examine the mechanisms by which arb could exert such dual antiviral activity (recapitulated in table 1 ). arb is an indole-based hydrophobic molecule susceptible to formation of supramolecular arrangements through aromatic stacking interactions with selective amino-acid residues of proteins (phenylalanine, tyrosine, tryptophan). by liquid-state nmr analysis, we showed that arb displays interfacial properties and intercalates in the shallow layer above the glycerol backbone of phospholipids (teissier et al., 2011) . it is even conceivable that arb could locally become more concentrated in viral or cellular membranes. it was also shown that arb interacts with aromatic residues within the viral glycoprotein involved in membrane interactions and destabilization necessary for fusion, aka the fusion protein (leneva et al., 2009 for influenza hemagglutinin; teissier et al., 2011 for hcv e2) . this could therefore underlie the virucidal (daa) effect of arb, interacting with the viral lipid envelope and/ or with key residues within structural proteins of virions (required for cellular receptor/captor recognition and/or membrane fusion). this effect has been described for enveloped [influenza a h1n1 virus (shi et al., 2007) ; hantaan virus ; hcv (haid et al., 2009; pécheur et al., 2007) ] and non-enveloped viruses [coxsackie virus b5 , consistent with arb's dual physico-chemical properties. arb could also locally impair viral attachment to cell plasma membrane by stabilizing the membrane, and/or by masking key residues in a viral protein involved in receptor recognition, in a sort of daa + hta effect. this would have consequences on viral entry. as shown by fluorescence spectroscopy and surface plasmon resonance analyses, arb affinity for lipid membranes is even more pronounced at acidic ph, the optimal ph for the fusion step of several enveloped viruses, influenza viruses and hcv in particular (fig. 2) (haid et al., 2009; pécheur et al., 2007; teissier et al., 2010 teissier et al., , 2011 . this interaction with phospholipids may perturb membrane fluidity, thereby rendering the lipid bilayer less prone to fusion. inhibition of viral entry and membrane fusion occurred in the 10 lm range, in agreement with arb affinity for membranes and the concentration range achieved in healthy volunteers (sun et al., 2013) . mechanistically, the dual binding capacity of arb to lipids and proteins might also underlie alterations of protein/protein and/or protein/lipid interactions at other stages of the viral life cycles, such as replication, assembly and budding. for a number of viruses, in particular in the flaviviridae family, replication occurs in a subcellular compartment called the membranous web (heaton and randall, 2011; moradpour et al., 2007) . the membranous web is an emanation of the endoplasmic reticulum induced by viral proteins such as hcv ns4b (gouttenoire et al., 2010; romero-brey et al., 2012) . since the web is created and maintained through interactions between viral and cellular proteins and lipids, it is plausible that arb could impair viral replication through its ability to bind proteins and lipids. concerning viral assembly and budding, intracellular membranes are obligate partners of nucleocapsids during packaging of enveloped viruses, and for the secretion of newly assembled viral particles. in the case of hcv, viral assembly is concomitant to the assembly of lipoproteins, giving rise to lipo-viro particles (bartenschlager et al., 2011) . arb could therefore interfere with these processes through its physico-chemical dual interactions with lipids and proteins. recently, we provided molecular details of how arb inhibits virus entry into target cells, in a study based on live-cell confocal imaging, using hcv as a model of an enveloped virus (blaising et al., 2013) (fig. 2) . first, arb was found to drastically impede virion attachment to cell plasma membrane. arb subsequently impaired the release of clathrin-coated pits (ccps) from the plasma membrane, resulting in a slowing of clathrin-coated vesicle (ccv) intracellular trafficking. the net result was an intracellular accumulation of ccvs containing trapped virions. arb was also shown to affect clathrin-mediated endocytosis (cme) by impeding dynamin-2-induced membrane scission, and thereby ccp formation. lastly, arb inhibited fusion between endocytic vesicles and endosomes and hindered viral intracellular trafficking. virions were not properly delivered to rab5-positive endosomal compartments where fusion occurs and/or rab5 was not recruited to virioncontaining vesicles. as a result, fusion was greatly impaired and virions trafficked to endo-lysosomal lamp-1-positive compartments for degradation. overall, the data suggest that arb's dual interactions with lipids and proteins may alter several aspects of intracellular trafficking with and maturation in endosomal compartments. arb may impede the recruitment and/or disassembly of machineries required for proper endosomal trafficking and viral entry. thus, arb inhibition of key actors of intracellular trafficking may be a likely explanation of its broad-spectrum antiviral activity (table 1 , fig. 2) , and suggest that arb acts as an hta. in most studies, arb exerted a maximal antiviral effect when used before infection, indicating an activity on early stages of viral infection and/or the requirement for arb to impregnate cells. in the current state of the literature, arb was shown to be active against viruses that enter cells by routes requiring at least one of these features: acidification, rab5, dynamin-2, actin. reovirus, vsv and hcv hijack cme (respectively: boulant et al., 2013; johannsdottir et al., 2009; meertens et al., 2006) , hbv also most likely enters via this pathway (yan et al., 2012) . chikv entry is mainly achieved via cme (leung et al., 2011) , but alternative clathrin-independent pathways have been described, dependent upon ph, dynamin-2, rab5 and actin cytoskeleton integrity (bernard et al., 2010) . viruses such as influenza or hantaan can enter through clathrindependent and -independent endocytotic pathways that have acidification in common (reviewed in mercer and helenius (2009), lozach et al. (2010) ). rsv entry is achieved by macropinocytosis and, as shown for hcv, the intracellular trafficking of virions is rab5-dependent (krzyzaniak et al., 2013) . in the picornaviridae family, group b coxsackieviruses entry in epithelial cells occurs via a complex process, combining caveolin-dependent endocytosis with features of macropinocytosis such as dependence upon rab5 (coyne et al., 2007) . also in this family, poliovirus 1 relies on an actin-and tyrosine kinase-dependent endocytic pathway to invade its target cells (brandenburg et al., 2007) , and rhinovirus 14 entry is ph-dependent and likely achieved by macropinocytosis (khan et al., 2010) . concerning sars-cov entry, the only consensus feature is its dependence on acidification in internal cell compartments (inoue et al., 2007; wang et al., 2008) . apart from lipid membranes, it is therefore conceivable that arb acts on several cellular targets common to the life cycle of various viruses. studies directly addressing arb as an hta and its interactions with proteins of intracellular trafficking are not available at present, but from our work with hcv, rab5, dynamin-2 and elements of the clathrin coat could be potential targets (blaising et al., 2013) . it is also conceivable that elements of the cytoskeleton could be targeted; indeed, molecules based on an aryl-thio-indole skeleton (fig. 1d) , closely related to arb chemically, are inhibitors of tubulin polymerization, and thereby potent anticancer agents (la regina et al., 2013) . arb was reported to inhibit influenza-and hcv-mediated membrane fusion (leneva et al., 2009; teissier et al., 2011) . in vitro studies showed that arb increases the stability of the influenza virus hemagglutinin (ha) and hinders low ph structural reorganizations necessary for ha to adopt its fusiogenic conformation, thus blocking infection at the viral fusion step (leneva et al., 2009 ; see also below section 7.). concerning hcv, fusion inhibition is dosedependent but does not depend on the hcv genotype or on the lipid composition of target membranes (liposomes); it predominantly prevails at low ph (boriskin et al., 2006; haid et al., 2009; pécheur et al., 2007; teissier et al., 2011) . arb was found to directly interact with peptides from the hcv e2 glycoprotein (teissier et al., 2011) and within a pocket of the influenza ha2 subunit of hemagglutinin (nasser et al., 2013) , thereby exerting its effect as a daa. interestingly, these peptides and pocket contain aromatic residues such as tyrosines and tryptophans, which could engage in aromatic stacking interactions with arb molecules, as described above. arb may therefore inhibit fusion by impairing conformational changes in viral fusion proteins during initiation of fusion (daa activity) and by increasing membrane rigidity, rendering membranes refractory to the destabilization that is required for fusion (hta activity). arb was shown to inhibit hcv replication in replicon systems, i.e. a cell culture context where virus replicates without any production of infectious viral particles (boriskin et al., 2006; sellitto et al., 2010) . a progressive decline in both viral protein and rna expression was observed in arb-treated cells, and cells could be cured of replicating viral rna after 10 weeks of arb treatment (boriskin et al., 2006) . since hcv modulates lipid metabolism (bassendine et al., 2013) and creates a lipid-rich internal membrane environment favorable for virus replication (i.e. the membranous web), arb could therefore impregnate these membranes to impede the formation and maintenance of the membranous web and in turn viral replication. chikv replication takes place in the host cell cytoplasm and is associated with cytoplasmic membrane alterations (solignat et al., 2009) . replication complexes are attached to the membrane of modified endosomes and lysosomes to form organelles characteristic of alphavirus replication called type 1 cytopathic vacuoles. these vacuoles consist in vesicles of 0.6-2.0 lm in diameter harboring numerous spherules (grimley et al., 1968) , which are positive for lysosomal markers (kujala et al., 2001) . the vacuoles produce viral rna until cell death. as already described for hcv, lipid bilayers are therefore essential for chikv replication. it is thus plausible that arb may also impede the formation and stability of these vacuoles, thereby perturbing chikv replication. in the absence of studies aimed at addressing the potential interactions between arb and cellular proteins involved in viral replication, one cannot exclude that such interactions might occur, as already suggested at the viral entry/maturation stage. to date, no report has been made concerning an effect of arb on viral assembly; however, further investigations are still needed to address this question directly. concerning viral budding, a recent study supports the notion that arb could inhibit influenza virus egress because viral rnas accumulate in cells at later stages of infection (brooks et al., 2012) . arb impregnation of cellular membranes and/or the targeting of proteins involved in intracellular trafficking that relate to viral morphogenesis/budding could again underlie this observation. in spite of its usage in russia and china for several years in flu, arb does not seem to generate a high degree of viral resistance. epidemic strains of influenza a/h1n1 and a/h3n2 isolated in russia in 2008-2009 revealed resistance to oseltamivir and/or rimantadine, but were all sensitive to arb ). the 2009 pandemic swine influenza a/h1n1 was found largely resistant to rimantadine, but had retained its sensitivity to oseltamivir (tamiflu ò ) and arb (iatsyshina et al., 2010) . in 2011-2012, influenza a/h3n2 and b viruses were found to be the cause of a vast epidemic in russia; all tested strains were sensitive to oseltamivir, zanamivir (relenza ò ) and arbidol, but resistant to rimantadine (l'vov et al., 2013) . however resistance to arb of various strains of influenza viruses has been reported, in particular in a population of influenza b . in a study aimed at understanding the anti-influenza mechanism of action of arb, leneva and colleagues isolated seven viral mutants from the influenza a/h7n7 ''weybridge'' strain, that were refractory to arb doses above 38 lm (leneva et al., 2009) . all mutants exhibited a single mutation in the ha2 subunit of the influenza hemagglutinin, the subunit involved in membrane fusion. this translated functionally into a 0.2-unit increase in the ph required to induce ha2 conformational changes. arb was found to directly interact with ha2, thereby increasing its stability to ph and impeding fusion in endosomes during virus infection. using an elegant proteomic approach, this interaction was further investigated by nasser and coworkers, and found confined to one peptide encompassing ha2 residues 104-120. this region contains the arb already identified mutation resistance k117r (leneva et al., 2009; nasser et al., 2013) . taken together, these data reveal that resistance of influenza viruses to arb mainly arises from mutations in the ha2 fusion protein, consistent with arb antiviral activity related to membrane fusion. addressing arb antiviral mechanism of action against chikv, delogu and coworkers isolated a mutant virus adapted to arb at 56 lm (delogu et al., 2011) . this virus was characterized by a single mutation in the e2 viral envelope glycoprotein, in a region most likely involved in cell-surface receptor recognition, and maybe indirectly to membrane fusion. clearly, additional studies on arb resistance in the context of other viral infections are warranted. in conclusion, the broad-spectrum activity of arb may arise through duality of function: a capacity to interact with both membranes and with viral and/or cellular proteins. arb therefore has features of both a daa and a hta. these interactions would impede cellular processes and pathways that are hijacked by several viruses to infect their host cells. regarding hcv, we have shown that arb inhibits most steps of hcv entry, from attachment to internalization, until the final step of membrane fusion. arb also inhibits hcv replication, which may arise via alteration of intracellular membrane-protein structures essential for intracellular trafficking (e.g. clathrin coat components, elements of the cytoskeleton) and virus replication (e.g. membranous web), and could hinder membrane rearrangements necessary for the viral budding step. the broad-spectrum activity and the cellular mechanisms affected by arb are summarized in fig. 2 . through these effects, arb could display an antiviral activity on viruses that hijack similar cellular pathways or have overlapping life cycles. in particular, endocytosis is used by several viruses and viral families including human immunodeficiency virus (von kleist et al., 2011) , adenoviridae, arenaviridae, coronaviridae, togaviridae to achieve productive infection (table 1) . moreover, all positive-strand rna viruses of eukaryotes are known to reorganize intracellular membranes to create specific virus replication organelles. for these reasons, efforts should be pursued in order to determine the potential inhibitory effect of arb on a large class of viruses. a better understanding of its molecular mechanisms of action would also contribute to refine the conditions at which it could be given in long-term regimens against chronic infections (e.g. hepatitis b or c). indeed current data on toxicity issues are insufficient to evaluate the safety of arb in chronic administration. nevertheless, most studies point to a good tolerability of this molecule. in the present state of our knowledge, arb could therefore constitute a cost-effective pharmacological approach, affordable for emerging countries in urgent need for effective antiviral therapies. study of metabolism of the antiviral drug arbidol by mass spectrometry, thin-layer and high-performance liquid chromatography assembly of infectious hepatitis c virus particles arbidole -a new drug for prevention of influenza and acute viral respiratory infections in children quantitative trace analysis of a broad range of antiviral drugs in poultry muscle using column-switch liquid chromatography coupled to tandem mass spectrometry endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments arbidol inhibits viral entry by interfering with clathrin-dependent trafficking arbidol: a broad-spectrum antiviral compound that blocks viral fusion arbidol: a broad-spectrum antiviral that inhibits acute and chronic hcv infection similar uptake but different trafficking and escape routes of reovirus virions and isvps imaged in polarized mdck cells design of inhibitors of influenza virus membrane fusion: synthesis, structure-activity relationship and in vitro antiviral activity of a novel indole series antiviral activity of arbidol, a broad-spectrum drug for use against respiratory viruses, varies according to test conditions antiviral chemotherapeutic agents against respiratory viruses: where are we now and what's in the pipeline? rimantadine and arbidol sensitivity of influenza viruses that caused epidemic morbidity rise in russia in the monitoring of the sensitivity of epidemic influenza virus strains isolated in russia to etiotropic chemical agents synthesis and in vitro anti-hepatitis b virus activities of some ethyl 6-bromo-5-hydroxy-1h-indole-3-carboxylates identification of novel 5-hydroxy-1h-indole-3-carboxylates with anti-hbv activities based on 3d qsar studies strategy for nonenveloped virus entry: a hydrophobic conformer of the reovirus membrane penetration protein micro 1 mediates membrane disruption coxsackievirus entry across epithelial tight junctions requires occludin and the small gtpases rab34 and rab5 in vitro antiviral activity of arbidol against chikungunya virus and characteristics of a selected resistant mutant protein kinase c inhibitors exhibiting an anti-inflammatory, anti-allergic and anti-asthma effect. russian patent efficacy of arbidol on lethal hantaan virus infections in suckling mice and in vitro pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans chemotherapeutics for treatment of influenza and other viral respiratory tract infections in children synthesis and biological activity of water-soluble polymer complexes of arbidol sensitivity of influenza a/h5 viruses isolated from wild birds on the territory of russia to arbidol in the cultured mdck cells susceptibility of pandemic influenza virus a 2009 h1n1 and highly pathogenic avian influenza virus a h5n1 to antiinfluenza agents in cell culture the new chemical preparation arbidol: its prophylactic efficacy during influenza epidemics viferon suppositories in the treatment of influenza in adults monograph: arbidol. antiviral, immunostimulant, interferon inducer mechanisms of arbidole's immunomodulating action hepatitis c virus nonstructural protein 4b: a journey into unexplored territory cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis low ph-dependent hepatitis c virus membrane fusion depends on e2 integrity, target lipid composition, and density of virus particles dengue virus and autophagy pandemic influenza a/h1n1 (sw2009) in russia: epidemiology, diagnosis, clinical picture, and treatment clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted host cell factors and functions involved in vesicular stomatitis virus entry antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures human rhinovirus 14 enters rhabdomyosarcoma cells expressing icam-1 by a clathrin-, caveolin-, and flotillin-independent pathway evaluation of the efficacy of wiferon and arbidol in adult influenza efficacy of ingavirin in adults with influenza hepatitis c virus e2 envelope glycoprotein core structure host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the f protein biogenesis of the semliki forest virus rna replication complex toward highly potent cancer agents by modulating the c-2 group of the arylthioindole class of tubulin polymerization inhibitors study of the effect of antiviral drugs on the reproduction of the respiratory syncytial virus by enzyme immunoassay sensitivity of various influenza virus strains to arbidol. influence of arbidol combination with different antiviral drugs on reproduction of influenza virus a antiviral etiotropic chemicals: efficacy against influenza a viruses a subtype h5n1 characteristics of arbidolresistant mutants of influenza virus: implications for the mechanism of antiinfluenza action of arbidol study of the antiviral activity of russian anti-influenza agents in cell culture and animal models replication of alphaviruses: a review on the entry process of alphaviruses into cells pharmacokinetic properties and bioequivalence of two formulations of arbidol: an open-label, single-dose, randomized-sequence, two-period crossover study in healthy chinese male volunteers characteristics of human infection with avian influenza viruses and development of new antiviral agents antiviral and anti-inflammatory activity of arbidol hydrochloride in influenza a (h1n1) virus infection determination of arbidol in rat plasma by hplc-uv using cloud-point extraction determination of arbidol in human plasma by lc-esi-ms identification of metabolites of arbidol by ultra-high performance liquid chromatography tandem mass spectrometry arbidol exhibits strong inhibition towards udp-glucuronosyltransferase (ugt) 1a9 and 2b7 therapeutic efficacy of ingavirin, a new domestic formulation against influenza a virus (h3n2) toxicity estimation of unspecific medicinal antiviral agents for prophylaxis and therapy of hazard and especially hazard viral infections entry of bunyaviruses into mammalian cells development of the influenza epidemic in season 2011-2012 in some areas of russia: results of activity of the influenza etiology and epidemiology center of the ivanovsky institute of virology the entry of reovirus into l cells is dependent on vacuolar proton-atpase activity virus entry by macropinocytosis hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles sensitive highperformance liquid chromatographic determination of arbidol, a new antiviral compound, in human plasma preparative liquid chromatographic isolation of unknown impurities in arbidol and si-5 replication of hepatitis c virus inhibition of influenza hemagglutinin with the antiviral inhibitor arbidol using a proteomics based approach and mass spectrometry the protective action of arbidol during a rise in respiratory diseases in 1990 biochemical mechanism of hepatitis c virus inhibition by the broad-spectrum antiviral arbidol investigation of susceptibility of influenza viruses a (h1n1), the cause of infection in humans in three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication synthesis and anti-hepatitis c virus activity of novel ethyl 1h-indole-3-carboxylates in vitro characteristics of the immune status in specific and nonspecific prophylaxis of influenza in elderly persons antiviral activity of arbidol against influenza a virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo efficacy of arbidol in prophylaxis and treatment of acute respiratory viral infections in servicemen arbidol used in the prophylaxis of acute respiratory viral infections and their complications in servicemen replication cycle of chikungunya: a re-emerging arbovirus glucuronidation of the broad-spectrum antiviral drug arbidol by ugt isoforms pharmacokinetics of single and multiple oral doses of arbidol in healthy chinese volunteers targeting cell entry of enveloped viruses as an antiviral strategy mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol synthesis of a new antiviral agent, arbidole role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition efficacy and safety of arbidol in treatment of naturally acquired influenza a 4-week oral toxicity study of an antiviral drug combination consisting of arbidol and acetaminophen in rats metabolite identification of arbidol in human urine by the study of cid fragmentation pathways using hplc coupled with ion trap mass spectrometry establishment of sybr green-based qpcr assay for rapid evaluation and quantification for anti-hantaan virus compounds in vitro and in suckling mice sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus synthesis and in vitro anti-hepatitis b virus activities of some ethyl 5-hydroxy-1h-indole-3-carboxylates antiviral activity of arbidol against coxsackie virus b5 in vitro and in vivo we thank steeve boulant for his invaluable contribution to live-cell imaging of hcv infection in blaising et al., 2013. j.b. is the recipient of a doctoral grant from the rhône-alpes region (arc 1 santé), and e-i. p. is supported by anrs (agence nationale pour la recherche sur le sida et les hépatites virales). key: cord-284551-j0nooxmd authors: shinohara, yoshiyasu; imajo, kento; yoneda, masato; tomeno, wataru; ogawa, yuji; kirikoshi, hiroyuki; funakoshi, kengo; ikeda, masanori; kato, nobuyuki; nakajima, atsushi; saito, satoru title: unfolded protein response pathways regulate hepatitis c virus replication via modulation of autophagy date: 2013-03-08 journal: biochem biophys res commun doi: 10.1016/j.bbrc.2013.01.103 sha: doc_id: 284551 cord_uid: j0nooxmd background: hepatitis c virus (hcv) induces endoplasmic reticulum (er) stress which, in turn, activates the unfolding protein response (upr). upr activates three distinct signalling pathways. additionally, upr induces autophagy (upr-autophagy pathways). on the other hand, it has become clear that some positive-single-strand rna viruses utilize autophagy. some groups have used the sirna silencing approach to show that autophagy is required for hcv rna replication. however, the mechanism of induction of the upr-autophagy pathways remain unclear in the cells with hcv. method and results: we used a genome-length hcv rna (strain o of genotype 1b) replication system (or6) in hepatoma cells (huh-7-derived or6 cells). as control, we used or6c cells from which the hcv genome had been removed by treatment with interferon-α. the upr-autophagy pathways were activated to a greater degree in the or6 cells as compared to the or6c cells. rapamycin, mtor-independent autophagy inducer, activated hcv replication in the or6 cells. on the other hand, hcv replication in the cells was inhibited by 3-methyladenine (3-ma), which is an inhibitor of autophagy. salubrinal (eukaryotic initiation factor 2(eif2)-alpha phosphatase inhibitor), 3-ethoxy-5, 6-dibromosalicylaldehyde (x-box binding protein-1 (xbp-1) splicing inhibitor) and sp600125 (c-jun n-terminal kinases (jnk) inhibitor) inhibited hcv replication and autophagy. additionally, hcv replication and autophagy were inhibited more strongly by combination of these inhibitors. conclusion: our results suggest that upr-autophagy pathways exert an influence on hcv replication. therefore, control these pathways may serve as a novel therapeutic strategy against replication of hcv. hepatitis c virus (hcv) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [1] . with over 170 million people chronically infected with hcv worldwide, this disease has emerged as a serious global health problem. hcv is an enveloped flavivirus with a 9.6-kb positive single-strand rna genome [2] . development of a hcv rna replicon capable of replication in the human hepatoma cell line huh7 has been a significant advance [3, 4] . hcv replication occurs in a ribonucleoprotein replication complex associated with an endoplasmic reticulum (er)-derived membranous web [5] . some groups have shown that hcv protein disrupts normal er functions and induces er stress [6, 7] . mammalian cells trigger a response called the unfolded protein response (upr) to cope with abnormal er functions and er stress [8] [9] [10] . upr activates three distinct signalling pathways, namely, the activating transcription factor 6 (atf-6) pathway, the inositol-requiring enzyme 1 (ire1) pathway and the double-stranded rna-activated protein kinase-like er kinase (perk) pathway [11] [12] [13] . some groups have reported the existence of some relationships among the three kinds of upr-autophagy pathways [14, 15] . autophagy is a major intracellular pathway involved in the degradation and recycling of long-lived proteins and cytoplasmic organelles, and plays an essential role in the maintenance of homeostasis in response to starvation and other cellular stresses [16] . on the other hand, autophagy also plays important roles in a variety of other cellular processes, including restriction of intracellular pathogen multiplication. recently, the significance of autophagy in the establishment of virus infection has become clear; in particular, some positive single-strand rna viruses utilize autophagy to generate the cytoplasmic membrane structures that they require for genome replication [17] . hcv has also been shown to induce er stress and trigger upr [18] [19] [20] [21] . furthermore, upr activation has been proposed to be responsible for subsequent induction of autophagy [22] . however, the induction of uprautophagy pathways by hcv has not been defined elucidated yet. in this study, we attempted to examine the influence on hcv replication of the upr-autophagy pathways, using a genomelength hcv rna (strain o of genotype 1b) replication system (or6) with luciferase as the reporter, which facilitated prompt and precise monitoring of hcv rna replication in hepatoma cells (huh-7-derived or6 cells). 3-ethoxy-5, 6-dibromosalicylaldehyde (3-e-5, 6-d), tunicamycin (tm), rapamycin, 3-methyladenine (3-ma), and interferon (ifn)-alpha were purchased from sigma (st. louis, mo, usa). sp600125 and salubrinal were purchased from calbiochem (los angeles, ca, usa). anti-core (cp11) was purchased from institute of immunology (institute of immunology, tokyo, japan), anti-nonstructure protein (ns) 4a from abcam (abcam, cambridge, uk); anti-microtubule-associated protein 1 light chain 3 (lc3) and anti-p62 were purchased from medical & biological laboratories (mbl, tokyo, japan), and anti-eif2-alpha, anti-phospholylated-eukaryotic initiation factor 2 (eif2)-alpha, anti-ire1, anti-c-jun n-terminal kinases (jnk), anti-phospholylated-jnk, anti-c-jun, anti-phospholylated-c-jun and anti-gapdh from cell signaling (cell signaling, inc.). or6 cells have been described in detail by ikeda et al. [23] . the or6 cell line is a clone of orn/c-5b/ke (strain o of genotype 1b) rna replicating huh-7 cells. or6 cells were cultured in dulbecco's modified eagle's medium supplemented with 10% fetal calf serum, penicillin, streptomycin and g418 (300 lg/ml; calbiochem, darmstadt, germany) and passaged twice a week at a 5:1 split ratio. control cells (or6c) were established by eliminating genomelength hcv rna from the or6 cells by ifn-alpha treatment (500 iu/ml for 2 weeks; sigma-aldrich, st. louis, mo, usa) without g418, as described previously. total rna was isolated using the rna prep mini kit (qiagen). template cdna synthesis was carried out with 1 lg of total rna using the high capacity rna-to-cdna kit (applyed biosystems, inc.). one-tenth of the synthesized cdna was subjected to pcr with the following primer pairs: xbp-1, 5 0 -ttacgagagaaaact-catggc-3 0 and 5 0 -gggtccaagttgtccagaatgc-3 0 ; hcv, 5 0 -aga-gccatagtggtctgcgg-3 0 and 5 0 -caagcaccctatcaggcagta-3 0 ; and gapdh, 5 0 -gactcatgaccacagtccatgc-3 0 and 5 0 -gag-gagaccacctggtgctcag-3 0 . for western blot analysis, 4à4.5 â 10 4 cells harboring or6 cells were plated on six-well plates and cultured for 24 h, and treated with each of the reagents for 72 h. samples were subjected to native gel and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis [24] . the proteins were transferred to a polyvinylidene difluoride membrane (millipore) and reacted with the appropriate antibodies. the immune complexes were visualized with super signal west femto substrate (pierce) and detected using an las-3000 image analyzer system (fujifilm). cells were cultured on glass slides and then fixed with 4% paraformaldehyde in phosphate-buffered saline (pbs) at room temperature for 30 min. after two washes with pbs, the cells were permeabilized at room temperature for 20 min with pbs containing 0.25% saponin, and then blocked with pbs containing 0.2% gelatin (gelatin-pbs) for 60 min at room temperature. the cells were incubated with gelatin-pbs containing appropriate antibodies at 37°c for 60 min and washed three times with pbs containing 1% tween 20 (pbst). the resulting cells were incubated with gelatin-pbs containing the corresponding fluorescent-conjugated secondary antibodies at 37°c for 60 min and then washed three times with pbst. the stained cells were covered with vecta-shield ò mounting medium containing dapi (4 0 , 6-diamidino-2-phenylindole) (vector laboratories inc.) and observed under a fluoview fv1000 laser scanning confocal microscope (olympus). to monitor the effects of the reagents, the rl assay was performed as described previously [25] . briefly, the cells were plated onto 24-well plates (2 â 10 4 cells per well) in triplicate and cultured with the medium in the absence of g418 for 24 h. the cells were then treated with each reagent at various concentrations for 72 h. after treatment, the cells were subjected to a luciferase assay using the rl assay system (promega, madison, wi). after these assays, the 50% effective concentration (ec50) of each reagent for hcv replication was determined. the cells were plated onto 96-well plates (1 â 10 3 cells per well) in triplicate and then treated with various concentrations of the reagents for 72 h. after the treatment, the cells were subjected to the wst-1 cell proliferation assay (takara bio, otsu, japan) according to the manufacturer's protocol. based on these assay results, the 50% cytotoxic concentration (cc50) of the reagents was estimated. the selective index (si) values of the reagents were also estimated by dividing the cc50 values by the ec50 values. the luciferase activities were statistically compared between the various treatment groups using student's t-test. p values of <0.05 were considered to signify statistically significant differences. the means ± standard deviations were determined from at least three independent experiments. to determine whether autophagy was induced in the or6 cells, we investigated the phosphoethanolamine (pe) conjugation of lc3 in the or6 cells showing autonomous replication of hcv rna. the amount of lc3-ii was significantly increased in the or6 cells as compared to the or6c cells (fig. 1a) . autophagy is involved in the response to er stress, which is represented by upr; in this case, double-membrane vesicles are formed but do not fuse with the lysosomes. accumulation of p62 has been reported to be caused by lysosomal fusion inhibition [14, 15] . also, in our experiment, significant accumulation of p62 was observed in the or6 cells (fig. 1a ). fig. 1b shows an increase in the amount of phosphorylated eif2-alpha (ser51) [9] in the or6 cells as compared to the or6c cells, suggesting that the perk pathway was activated by hcv. fig. 1c indicates the increase in the amount of spliced xbp1 in the or6 cells as compared to the or6c cells, suggesting that the atf-6 and ire1 pathways were activated too [10, 26] . fig. 1d shows an increase in the amount of ire1 in the or6 cells as compared to the or6c cells. moreover, increases in the amounts of phosphorylated jnk (thr183/tyr185) and phosphorylated c-jun (ser63) were also observed in the or6 cells as compared to the or6c cells (fig. 1d) , suggesting that the ire1 pathway was activated as well [8] . on the other hand, treatment of huh7 cells with tunicamycin, which triggers upr by inhibiting glycosylation, resulted in activation of the atf-6, perk and ire1 pathways. these results confirm that the delay in the appearance of upr was not due to an inherent defect in the huh7 cells (fig. s1b , s1c and s1d). tunicamycin treat-ment also induced lc3-ii and p62 accumulation (fig. s1a) . these data confirm that the hcv replicon cells showed activation of both autophagy and upr (fig. 1) . we investigated whether activation of autophagy had any influence on the hcv replication. rapamycin, a mtor-independent autophagy inducer [27] , enhanced hcv replication and formation of autophagosomes in the or6 cells, whereas 3-ma, an inhibitor of autophagy [27] , suppressed hcv replication and formation of autophagosomes in the or6 cells ( fig. 2a and b) . these data suggest that autophagy seems to somehow facilitate hcv replication. salubrinal is a selective inhibitor of the phosphatase complexes that dephosphorylate eif2-alpha in perk pathway [28] . 3-e-5, 6-d selectively inhibits xbp-1 splicing [29] . sp600125 down-regulates the expression of ire1 and dephosphorylates jnk [30] . to further investigate whether inhibition of the upr-autophagy pathway might affect the rate of hcv replication, we determined the ec50, cc50 and si values of salubrinal (eif2-alpha inhibitor), 3-e-5, 6-d (x-box binding protein-1 (xbp-1) inhibitor) and sp600125 (jnk inhibitor) using the or6 assay system. the results for the ec50, cc50 and si values were as follows: (salubrinal: ec50; 6.25 lm, cc50 > 100 lm, si > 16 (fig. 3a) , 3-e-5, 6-d: ec50; 2 lm, cc50; 20 lm, si; 10 ( fig. 3b ) and sp600125: ec50; 1 lm, cc50; 12.5 lm, si; 12.5 (fig. 3c) . meanwhile, the percentage of viable cells was determined by the wst-1 assay. cytotoxic effects were observed at >25 lm of the salubrinal (fig. 3a) , >6.25 lm of the 3-e-5, 6-d (fig. 3b) , and >3.13 lm of the sp600125 (fig. 3c) . on the other hand, no cytotoxic effects were observed in the presence of <12.5 lm of the salubrinal (fig. 3a) , <3.13 lm of the 3-e-5, 6-d (fig. 3b) , and <1.56 lm of the sp600125 (fig. 3c ). in addition, autophagosome formation in the or6 cells was decreased in the presence of ec50 concentrations of the salubrinal, 3-e-5, 6-d and sp600125 (fig. 3d) . we found these inhibitors resulted in efficient suppression of hcv replication and autophagy. we then attempted to clarify which of the three upr-autophagy pathways might be most closely involved in the activation of autophagy by investigating the effects of combined use of the salubrinal, 3-e-5, 6-d and sp600125 on the rate of hcv replication using the or6 assay system. salubrinal inhibited phasphorylated eif2-alpha, lc3-ii and hcv core protein, but not inhibited spliced xbp1 and phasphorylated jnk. 3-e-5, 6-d and sp600125 inhibited phasphorylated jnk, spliced xbp1, lc3-ii and hcv core protein, but not inhibited phasphorylated eif2-alpha. combined treatment with three inhibitors of salubrinal, 3-e-5, 6-d and sp600125 strongly enhanced the inhibition of both hcv replication and autophagy. these results indicated that combined use of the inhibitors was more effective than use of any of the inhibitors alone in suppressing hcv replication and autophagy (fig. 4) . in addition, the concentrations of each of the inhibitors used in combination did not affect the cell viability rate (fig. 4) . thus, in regard to the efficacy of inhibition for hcv replication and autophagy, combined use of 3-e-5, 6-d and sp600125 appears to be the most efficient as compared to use of other combinations. in this study, we showed, using a replicon system, activation by hcv of the three main signaling pathways from upr to autophagy ( fig. 1) in regard to the upr-atf6-xbp1 pathway, tardif kd et al. reported, from a study using subgenomic replicons, that hcv suppresses the ire1-xbp1 pathway to stimulate the synthesis of its viral proteins [31] . the differences from our results could be attributable to the fact that we used full-genome replicon or different hcv strain for our experiments. in addition, some other groups have shown that hcv induces er stress and upr activation [18] [19] [20] [21] . on the other hand, several groups have reported that autophagy and/or autophagy genes are likely to play both antiviral and proviral roles in the life cycles and pathogenesis of many different virus families. especially, replication and spread of some positive-strand rna viruses are regulated by their interactions with the host autophagy machinery [17] . components of the autophagic machinery seem to be subverted to promote replication of rna viruses, such as in the case of infections with the coronavirus [mouse hepatitis virus (mhv)], poliovirus, or rhinoviruses 2 and 14, by generation of a membrane scaffold for rna replication [32, 33] . we then attempted to determine if inhibitors of upr signaling might control hcv replication and autophagy by inhibiting the signaling pathways from urp to autophagy (figs. 2 and 3) . our results showed that salubrinal (eif2-alpha phosphatase inhibitor), 3-e-5, 6-d (xbp-1 inhibitor) and sp600125 (jnk inhibitor) could regulate hcv replication and autophagy in the or6 cells (fig. 3) . next, we attempted to determine combinated inhibitiors which upr-autophagy pathways might have the greatest influence in the regulation of hcv replication. when the concentrations of the inhibitors used were under the ec50, the cell viability of the hcv replicons was almost 100%. in particular, combined treatment with the 3-e-5, 6-d (xbp-1 splicing inhibitor) and sp600125 (jnk inhibitor) was the most efficient at reducing hcv replication (fig. 4) . some interesting findings have been reported previously. eif2-alpha phosphatase inhibits the assembly of the 80s ribosome and inhibits protein synthesis [34] . on the other hand, ire1 and atf6 promote transcription of the upr target genes. ire1 and atf6 process xbp1 mrna to generate mature xbp1 mrna. spliced xbp1 binds directly to the er stress response element and unfolded protein response elements and activates transcription of the molecular chaperones of the er. jnk activation, mediated by ire1, is required for autophagosome formation [35] . the perk pathway is independent of upr, however, the ire1 and atf6 pathways play complementary roles in upr signaling [36] [37] [38] . these findings indicate that inhibition of upr-autophagy pathways may have significant influence in the suppression of hcv replication, and 3-e-5, 6-d and sp600125 might inhibit strongly hcv replication and autophagy. in conclusion, hcv stimulates the three signaling pathways from upr to autophagy. salubrinal (eif2-alpha phosphatase inhibitor), 3-e-5, 6-d (xbp-1 inhibitor) and sp600125 (jnk inhibitor) acted as a potent anti-autophagy agent and limit both inhibition of autophagy and hcv replication. we lead to the notion that hcv may hijack the upr-autophagy pathway for hcv replication (fig. 5 ). our study demonstrates control of the upr-autophagy pathway machinery as a possible antiviral drug target. combined treatment with inhibitors of upr signaling enhanced the inhibition of hcv replication. the effects of combined treatment with salubrinal (eif2-alpha phosphatase inhibitor), 3-e-5, 6-d (xbp-1 splicing inhibitor) and sp600125 (jnk inhibitor) on hcv replication in the or6 assay system were examined. or6 cells were treated with the reagents for 72 h, following which the rl assay (bar graph) and wst-1 assay (line graph) were performed. the relative values (%) calculated at each point, with the level in the non-treated cells set at 100%, are presented here. western blot analysis of the treated cells for the hcv core protein and lc3 was also performed (lower panels). course and outcome of hepatitis c immunology of hepatitis b virus and hepatitis c virus infection replication of subgenomic hepatitis c virus rnas in a hepatoma cell line efficient initiation of hcv rna replication in cell culture expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex human hepatitis c virus ns5a protein alters intracellular calcium levels, induces oxidative stress, and activates stat-3 and nf-kb hepatitis c virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway the role of the unfolded protein response in tumour development: friend or foe? the mammalian unfolded protein response signal integration in the endoplasmic reticulum unfolded protein response xbp-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response atf6alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress transcriptional induction of mammalian er quality control proteins is mediated by single or combined action of atf6alpha and xbp1 linking of autophagy to ubiquitinproteasome system is important for the regulation of endoplasmic reticulum stress and cell viability signal integration in the endoplasmic reticulum unfolded protein response autophagy in health and disease: a double-edged sword viruses and the autophagy machinery hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro hepatitis c virus ns4b induces unfolded protein response and endoplasmic reticulum overload response-dependent nf-kappab activation induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response efficient replication of a full-length hepatitis c virus genome, strain o, in cell culture, and development of a luciferase reporter system establishment of a hepatitis c virus subgenomic replicon derived from human hepatocytes infected in vitro mechanism of action of ribavirin in a novel hepatitis c virus replication cell system focal distribution of hepatitis c virus rna in infected livers methods for monitoring autophagy a selective inhibitor of eif2alpha dephosphorylation protects cells from er stress potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease endoplasmic reticulum stress-mediated autophagy/apoptosis induced by capsaicin (8-methyl-n-vanillyl-6-nonenamide) and dihydrocapsaicin is regulated by the extent of c-jun nh 2 -terminal kinase/ extracellular signal-regulated kinase activation in wi38 lung epithelial fibroblast cells hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response subversion of cellular autophagosomal machinery by rna viruses coronavirus replication complex formation utilizes components of cellular autophagy er stress (perk/eif2a phosphorylation) mediates the polyglutamine-induced lc3 conversion, an essential step for autophagy formation autophagy is activated for cell survival after endoplasmic reticulum stress, autophagy is activated for cell survival after endoplasmic reticulum stress differential contributions of atf6 and xbp1 in activating endoplasmic reticulum stressresponsive cis-acting elements erse, upre and erse-ii a time-dependent phase shift in the mammalian unfolded protein response ire1 signaling affects cell fate during the unfolded protein response this study was supported by a grant-in-aid for research on the third term comprehensive control research for cancer from the ministry of health, labour and welfare, japan, to a.n., a grant from the national institute of biomedical innovation (nbio) to a.n., a grant from the ministry of education, culture, sports, science and technology, japan (kiban-c), to s.s., a grant from the yokohama foundation for advancement of medical to y.s. and s.s., and grants from the japanese ministry of health, labour and welfare science to s.s. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbrc.2013.01.103. key: cord-269294-vx7xr80t authors: kwong, ann d.; rao, b. govinda; jeang, kuan-teh title: viral and cellular rna helicases as antiviral targets date: 2005-09-23 journal: nat rev drug discov doi: 10.1038/nrd1853 sha: doc_id: 269294 cord_uid: vx7xr80t although there has been considerable progress in the development of antiviral agents in recent years, there is still a pressing need for new drugs both to improve on the properties of existing agents and to combat the problem of viral resistance. helicases, both viral and human, have recently emerged as novel targets for the treatment of viral infections. here, we discuss the role of these enzymes, factors affecting their potential as drug targets and progress in the development of agents that inhibit their activity using the hepatitis c virus-encoded helicase ns3 and the cellular helicase ddx3 adopted for use by hiv-1 as examples. in considering how to design antiviral drugs, one envisions two broad strategies. because viruses replicate by using self-encoded proteins or by seizing control of cellular factors, agents designed to interrupt viral replication could, in principle, target with equal effectiveness either a viral or cellular polypeptide 2 . interestingly, the two strategies hold different implications. in the case of a unique viral function, such as capsid formation, there is a lower risk of creating inhibitors with toxic effects on the host, similar to the advantage antibacterial drugs have in inhibiting bacterial targets that do not exist in their host. in the case of a target such as a viral helicase, there is a smaller window for specificity because the viral and cellular enzymes catalyse a similar enzymatic reaction. however, as the viral and cellular enzymes are not identical, traditional medicinal chemistry and structure-based drug design can exploit the difference between host and viral enzymes to create drugs with high specificity for the virus. the achilles heel of antiviral therapy is resistance 3 . unless a drug is incredibly potent (reducing the size of the replicating pool of virus rapidly), and therefore requires only a short duration of treatment (reducing the time for the resistant viruses to amplify), resistance to treatment will arise over time, as observed with hiv or hbv patients on therapy. targeting a cellular factor that is required for viral replication should help overcome the problem of viral resistance. theoretically, the drug could be pan-antiviral and inhibit all viruses that are dependent on the same host factor. operationally, we define pan-antiviral to mean that the inhibitor targets more than one family of viruses. this latter intervention strategy limits the development of resistant viruses, but it has a major disadvantage in generally causing greater toxicity to the host. empirically, how one focuses one's antiviral drug design can be influenced by whether the virus replicates largely autonomously of the host, using predominantly virally encoded genes (for example, herpes simplex virus (hsv)), or whether the virus is intimately associated with the host's metabolism (for example, integrated proviruses). in this review, we discuss in a non-exhaustive fashion the hcvencoded helicase ns3, and the cellular helicase ddx3 adopted for use by hiv-1, as two illustrative examples of potential antiviral targets. at the most basic level, helicases are motor enzymes that use energy derived from ntp hydrolysis to unwind double-stranded nucleic acids [4] [5] [6] [7] . further classification depends on whether the helicase can bind single-stranded nucleic acid, unwind rna or dna or both, and the direction of unwinding (3′ to 5′ or 5′ to 3′), and whether certain signature motifs are present in the primary sequence. helicases have been divided into three super-families (sf1, sf2 and sf3) and two small families based on sequence comparisons and conserved motifs 8 . examples of members of sf1, sf2 and sf3 are shown in table 1. figure 1a shows a schematic illustration of the seven highly conserved motifs among the two largest families (sf1 and sf2). although, as discussed later, the conserved motifs are associated with conserved helicase function, a comparison of the primary sequence of two sf2 helicases (fig. 1b) , hcv helicase and human ddx3 reveals a paucity of identical residues (highlighted in green) and a tremendous divergence in sequence outside of the conserved motifs (highlighted in red). the differences in primary sequence and tertiary structure between the helicase of a viral pathogen and that of cellular helicases can be exploited to confer specificity to an antiviral inhibitor. the structure of a number of helicases has been solved and a recent review on helicase structure and function is available 9 . the total number of domains can vary from four (that is, pcra/rep), to three (that is, hcv helicase) to two (that is, elongation initiation factor 4a (eif4a)). the different domains of a three-domain helicase (hcv helicase) are shown schematically in fig. 1c, in which domain 1 (magenta), domain 2 (yellow) and domain 3 (cyan) are clearly delineated. domains 1 and 2 form a core that is conserved in all helicases, and contain most of the conserved sequence motifs at the interface between the two domains, as seen in the example of hcv helicase (fig. 1d) . surprisingly, despite widely disparate protein sequences, similar structural elements are conserved in all the structures solved to date. this is illustrated by an overlay of the structures for hcv rna helicase 10 (red; three domains) and pcra dna helicase 11 (cyan; four domains) in fig. 2a. the position and length of the α-helices and the number and orientation of β-strands in the core of both enzymes (top two domains) are very similar. by contrast, a divergence is clearly seen in domain 3 (bottom left) and hcv helicase has no counterpart to domain 4 of pcra helicase (cyan, bottom right). all helicases bind ntp using two structural elements -a phosphate-binding p-loop, also known as motif i/walker a motif, and motif ii, a mg 2+ co-factorbinding loop also known as walker b 8, 11, 12 . sf2 rna helicases are also called dea(d/h)-box helicases after the signature sequence of walker b motif. as highlighted in orange in fig. 1b, the residues in the conserved motifs required for binding and hydrolysing ntp lie at the interface between of domains 1 and 2 in hcv helicase. there is considerable flexibility in the distance between the two domains, although ntp hydrolysis can only occur when the domains are in a 'closed' configuration. additional crystallographic and mutational analyses have identified motifs that contribute to oligonucleotide binding, such as ia and iv 8 . the txgx motif spans the bottom of the cleft between domains 1 and 2 and acts as a 'hinge' for the movement of domain 2. the exact changes in physical conformation and the path the rna or dna takes as one strand is destabilized and 'unwound' from the other are not well understood, but they do not seem to be conserved between different helicases. complicating matters further, some helicases seem to function as monomers (for example, hcv helicase), others as dimers (for example, hsv ul5) and yet others as hexamers (for example, simian virus 40 (sv40) t antigen). many putative helicases have so far only been identified by the presence of a dead/h box in the primary sequence and await biochemical verification of bona fide helicase function. however, not all of the crucial elements for helicase function can be detected from the primary sequence. structural analyses have identified crucial residues that are conserved only in space, but not in the primary sequence. an example is shown in fig. 2b, in which the crystal structures of the interface between domains 1 (left) and 2 (right) of hcv helicase 10 (in green) and pcra helicase 11 (in blue) are superimposed. two pairs of arginine residues on the inner face of domain 2 are highlighted (circles), which have been shown by mutational analyses to be essential for ntp hydrolysis. the lower pair, arg-610 of pcra (yellow sticks) and arg-464 of hcv helicase (white sticks) are both in motif vi and are structurally and functionally homologous to each other 13 . in the 1 m s h v a v e n a l g l d q -g f a g -l d l n s s d n q s g g s t a s k g r -hcvns3 1 ----i i t s l t g r d k n g v e g e v q v v s t a t q s f l a t c i n g v c ddx3 38 ---y i p p h l r n r e a t k g ----f y ---d k d s s g w s s s k d k d hcvns3 37 w t v f h g a g t k t l a g p k g p i t q n y t n v d g d l v g w q a p p --219 d l n a c a q t g s g k t a a f l l p i l s q i y s d g p g e a l r a m k e n g hcvns3 185 h l h --a p t g s g k a t k v p a a y a t g g y k --------------ddx3 259 r y g r r k q y p i s l v l s p t r e l a v q l y e e a r k f s y r s r v r p c 299 v v y g -g a d l g g g l r d l e r g c h l l v a t p g r l v --d m m e r g k hcvns3 229 -a y g t d p n l r t g v r t i t t g a p l t y s t y g k f l a d g g c s g g a 579 ------------h y k g s s r g r s k s s r f s g g f g a r d y r q s s hcvns3 517 e s v f t g l t h i d a h f l s q t k q a g d n f p y l v a y q a t v c a r a q ddx3 607 g a s s s s f s s s r a s s s r s g g g g h g s s r g f g g g g y g g f y n s d hcvns3 557 -a p p p s w d q m w k c l t n l k p t l h g p t --------p l l y r --ddx3 647 g y g g n y n s q q v d w w g n hcvns3 586 -l g a v q n a v ------upper pair, arg-467 of hcv helicase also shows close functional and structural homology with arg-287 of pcra, but they derive from two different sequence motifs. the hcv residue in the pair is encoded in motif vi, whereas the analogous pcra residue comes from motif iv, a relationship that cannot be derived by homology searches and sequence gazing 14 . splicing, mrna export, translation, rna stability and mitochondrial gene expression [16] [17] [18] [19] [20] . despite their large number, each rna helicase seems to be individually essential, because loss of one dead box protein in yeast cannot be complemented by another overexpressed family member 21 . unwinding of highly structured rnas might be reasoned to be important for eukaryotic transcription. however, direct evidence for such a role by of an rna helicase has been elusive. once transcribed, rnas are rapidly packaged into ribonucleoprotein complexes (rnps 22 ) for further processing. in such a setting, rna helicases can play roles in rna-rna and rna-protein 23 remodelling. there is evidence that helicases such as uap56, brr2, prp16, prp22, and prp43 have roles in rna splicing 24 , whereas others, such as dbp5 25, 26 and ddx3 27 , serve to chaperone rnas from the nucleus into the cytoplasm. translation of mrnas in the cytoplasm is facilitated by helicases such as eif4a and ded1, whereas rh1b, ski2, dob1 and dhh1 helicases modulate the stability of mrnas 24 . dead box helicases also act in ribosome biogenesis through regulation of small nucleolar (snornas) and ribosomal (rrnas) rna interactions 28, 29 . a correlation exists between genome size and the encoding of a helicase by (+ strand) rna viruses 30 . although all viruses are thought to functionally need helicase(s), in some cases the larger entities have the luxury of self-encoding such an enzyme, whereas size-constrained, smaller viruses can adapt to use a cellular helicase. in general, rna viruses that replicate in the cytoplasm mostly self-encode a helicase, whereas those that replicate in the nucleus often utilize a cellular helicase. consistent with the latter notion, hiv-1 was shown in two recent studies to co-opt the activity of a cellular helicase 27, 31 . human helicase members of superfamily 2, ddx1 31 and ddx3 are required for the viral rev protein to export unspliced/partially spliced hiv-1 mrnas from the nucleus into the cytoplasm. ddx1 or ddx3 can therefore be added to the list of potential treatment targets of hiv-1-encoded enzymes (that is, reverse transcriptase, protease and integrase). the potential of helicases as antiviral drug targets has recently been reviewed [32] [33] [34] [35] [36] . unlike retroviruses, two other human viruses, hsv and human papillomavirus (hpv), physically encode their own helicases. the hpv e1 protein 37 is a member of superfamily 3 table 1. a number of laboratories have developed high-throughput helicase or e1-dependent polymerase screens for inhibitors of hpv replication. to date, no successful clinical drug candidates derived from these efforts have been reported. the hsv ul5 and ul9 genes encode helicases in superfamily 1 and 2, respectively 38 . ul5 forms a heterotrimeric helicase-primase complex with ul8 and ul52 39 that is responsible for unwinding duplex viral dna at replication forks and laying down okazaki primers for elongation. preclinical proof of concept for helicase inhibitors as antiviral agents has been obtained for hsv 32, 33, 35 . using high-throughput screening (hts) 34 , thiazolylphenyl amino-thiazole 40,41 , (dichloroanilino)purines andpyrimidines 42 and thiazolylsulphonamide inhibitors 43 were identified by hts of the inhibition of hsv ul5/8/52 primase-helicase complex. optimization of the screening hits resulted in compounds that inhibited hsv growth in cell culture with little cytotoxicity and which were orally active in an animal model of hsv. mechanistically, the thiazolylphenylcontaining drugs seem to stabilize helicase-primase binding to polynucleotide substrates and halt further catalytic cycles. the mechanism of antiviral action was confirmed when several groups independently selected resistant viruses with single point mutations in the ul5 dna helicase gene for both classes of compounds, confirming the mechanism of action of the inhibitors. although the ul5 mutants showed little to no reduction in replicative fitness compared with wild-type virus in cultured cells 44 , the fitness of such mutants in the clinical setting is the most important measure of fitness. nonetheless, this is the most successful demonstration to date that it is possible to develop selective, potent inhibitors of a viral helicase as antiviral agents in a preclinical setting. the successful results of the hsv helicase inhibitors were not made public until several years after many companies had started screening for hcv helicase inhibitors. however, at the time there was widespread optimism that the hcv helicase would turn out to be a good target for antiviral drug discovery. bolstered by the successful examples of anti-hiv drug design, structure-based drug design efforts have focused on finding inhibitors of three essential viral enzymes: the [48] [49] [50] . in the ns3 bifunctional protein the first 181 amino acids form a serine proteinase, whereas the carboxy-terminal 181-631 amino acids form a helicase. from a biological standpoint, protease and helicase activities co-exist in vivo, and either could serve as a useful anti-hcv target. the helicase and polymerase form a complex with other host and viral proteins to create the viral replicase multi-protein complex [51] [52] [53] . in order for hcv to multiply, negative-stranded rna replicative intermediates must be synthesized by the replicase complex using incoming positive-stranded rna from the infecting virus as the template. the negative-stranded replicative intermediate is then used as an intermediate template to synthesize positive-stranded progeny rna, which is packaged into viral capsids. because the positive and negative rna strands are complementary, hcv helicase is thought to be required for strand separation. additional functions in hcv replication might include the melting of highly stable secondary structures, which are known to be present in hcv rna, in order to increase translational efficiency of the polyprotein or to increase replication rate and fidelity 54, 55 . currently, very little is known about the crucial protein-protein interactions in hcv, except that the ns4a cofactor interacts with ns3. microscopic studies indicate that replicase, as well as most of the hcv proteins, congregate on the er to form a 'membraneous web' . it is thought that cellular factors are probably present in this membrane-associated complex or complexes. future efforts at mapping protein-protein interactions, both between viral proteins and cellular factors, will be important for successful drug discovery. a consideration in assay design is the form of the protein to use in the enzymatic screen, particularly in the case of multi-domain or multi-functional proteins. in the native state, the helicase domain is part of the ns3•4a complex, which also contains the ns3 serine protease domain and its ns4a cofactor 56 . is coloured blue and the 4a cofactor is in green; the location of the active site is indicated by the position of vx-950, a peptidomimetic ns3•4a protease inhibitor created using structure-based drug design, which shows that the presence of the helicase domain is unlikely to block access of protease substrates or inhibitors. these structural studies support the notion that in the absence of other replicase proteins, the helicase domain can largely function independently of the protease domain and that use of the helicase domain alone could be a reasonable choice for an assay format. however, biochemical studies comparing the helicase activity of the full-length ns3 protein with the truncated helicase domain suggest that the full-length domain is more efficient at unwinding, and subtle differences exist in the mechanism of unwinding for the full-length and truncated domain [58] [59] [60] [61] [62] in the presence and absence of ns4 63 . it is therefore possible that different inhibitors might score positively in a screen using different forms of the enzyme. in considering drug designs against helicases, one can begin with several general conceptual strategies. helicases have multiple enzymatic activities and functional domains that present multiple potential mechanisms of action for the design of an inhibitor. this review will present a general description of helicase activity, suitable for the general reader. an in-depth description can be found in detailed physical and kinetic analyses of periodic cycles of rna unwinding and pausing by hcv ns3 helicase, which have been investigated by several laboratories 59,64-68 . as schematically shown in fig. 5a, the conformation of an active helicase can be broadly divided into an 'open' and a 'closed' complex of domains 1 and 2, with a transition between the two. as shown in fig. 5b, assays can be used to identify small-molecule helicase inhibitors that have the following effects: they inhibit ntpase activity by direct competition with ntp binding 69 ; competitively inhibit nucleic-acid binding; inhibit ntp hydrolysis or ndp release by blocking the movement of domain 2; inhibit the process that couples ntp hydrolysis to translocation and unwinding of nucleic acid; inhibit unwinding by sterically blocking helicase translocation 70 ; and inhibit unwinding (reviewed in refs 14, 71) . for all these mechanisms, unwinding and binding assays can be deployed. a separate category of potential inhibitors not depicted in fig. 5 are those that change the physical conformation of the helicase, inhibiting the natural range of motion required for unwinding or altering the interface between domains 1 and 2. other nonenzymatic mechanisms of action for new helicase inhibitors could include disruption of the turnover of ns3 protein or inhibiting crucial interactions with other proteins in the replicase complex. the solution of the crystal structure of hcv helicase complexed with oligonucleotide, as well as mutagenesis studies, have identified key residues that are essential for enzyme activity or translocation of the rna substrate 13, 72 . however, there is no consensus on the mechanism of unwinding, and three different models have been proposed by three different groups who have published crystal structures of hcv helicase. it is important to inhibit functions that are essential for helicase activity. as all helicases share some common enzyme reactions (for example, ntp hydrolysis and translocation) in their mechanisms of action, it might be difficult to avoid the possibility of hitting an unintended cellular helicase and generating unwanted side effects. electrostatic analysis of the hcv helicase shows both active-site and nonactive-site locations 73 that could be exploited for drug design. although it might be tempting to inhibit oligomerization, inhibition of protein-protein interactions with a small molecule could be difficult because such interactions usually have multiple points of contact over a broad surface area, and blocking one such contact with a small molecule is usually not sufficient to block binding of the two proteins. this kind of mechanism is more suitably targeted by antibodies and antibody mimics. likewise, targeting additional domains that are unique to a helicase in an attempt to gain specificity -such as the two novel conserved motifs in hcv helicase 74 -is not practical unless a specific assay is available for a function known to be associated with the domain or an approach is used to develop reagents that bind to the region of interest. recombinant human antibodies 75, 76 and rna aptamers 77, 78 have been developed that bind to hcv helicase and which inhibit hcv helicase activity. of all these potential assays, most laboratories have relied on a simple unwinding assay for primary hts, such as a scintillation proximity assay (spa) 79, 80 or a fluorometric assay 81 . theoretically, an unwinding assay should increase the odds of finding an inhibitor because inhibition of any of the potential mechanisms of action listed above, except those requiring a cellular environment (for example, turnover and replicase-complex formation), should result in 'hits' (for example, low-potency chemical starting points for medicinal chemistry optimization). the choice of a more stable dna substrate versus the more 'natural' rna substrate is available for hcv helicase because it can unwind both rna and dna homoand heteroduplexes. biochemical assays for detecting inhibitors of nucleic-acid binding or atpase activity can be run in hts mode, but are primarily used to elucidate the mechanism of action of an inhibitor. there is no reason why an unwinding assay could not also find inhibitors that cause conformational changes to the helicase. however, in most screenings the initial 'hits' or candidate inhibitors are usually not very potent (µm ic 50 ), and are useful in the sense that one hopes to evolve them into more potent compounds. empirically, in screens for hcv helicase, very few candidates were found; one explanation for this dearth of results could be that non-potent hits simply could not bind with sufficiently high affinity to helicase to inhibit the unwinding reaction. an alternative to an unwinding assay is to identify molecules that simply bind to the helicase and alter its conformation. accordingly, the strategy is to evolve initial binders that change conformation into tighter binders that can also inhibit unwinding. in scoring for binding and conformational perturbation, one could possibly find more hits than scoring for unwinding. of the three major hcv enzymatic targets for drug discovery, ns3 protease inhibitors have been the most successful to date. proof of concept for this class of inhibitors has been demonstrated by boehringer ingelheim and vertex pharmaceuticals using biln-2061 82, 83 and vx-950 84 , respectively. both compounds decreased hcv viral load in patients by ~2-3 logs in the first 3 days of dosing. in some patients treated with vx-950, the hcv viral load dropped from 10 6 infectious units (iu) per ml at the start of treatment to below the limit of detection (<10 iu per ml) during 14 days of dosing. several other hcv protease inhibitors from different companies are currently in clinical trials. the speed with which hcv protease inhibitors have approached the clinic is remarkable given that the active site of the enzyme is notorious for being 'flat and featureless'. in addition, most high-throughput protease screens of large compound libraries have not produced good hits. however, it has been possible to use the hcv protease substrate and cleavage products as starting points for structurebased design to create the peptidomimetic inhibitors that are in clinical development today. in contrast, hcv polymerase high-throughput assays for elongation have generally been fruitful and yielded a diverse group of active-site and non-active-site inhibitors with different scaffolds. the availability of multiple starting points for hcv polymerase inhibitors means that there is a wealth of companies with preclinical inhibitors, some of which have shown good potency and good bioavailability. in contrast to hcv protease inhibitors, a number of polymerase inhibitors have entered the clinic and failed to significantly decrease hcv viral load in patients. nm283 (idenix) has provided proof of concept for active-site polymerase inhibitors with ~1.2 log reduction in viral titre after treating patients for 15 days 85 . because there are multiple mechanisms of action that could inhibit hcv helicase, many have been surprised at the meagre array of hits from helicase screens or focused chemical libraries 54 , many of which were nucleic-acid binders or intercalators with low potency. the atp-binding site has been successfully targeted by kinase and gyrase inhibitors in which the binding site is a distinct and well-defined 'pocket' . by contrast, only weak inhibitors have been found that inhibit the hcv binding site 86 . one explanation might be that the hcv helicase ntp-binding site at the interface of domains 1 and 2 has little specificity -it binds all ntps primarily through the phosphate, not the base, of the nucleotide. in all helicases, the ntp-binding pocket forms transiently as domain 2 approaches domain 1, a movement that results in hydrolysis of atp and concomitant opening or separation of domain 2 from domain 1. most of the crucial residues for helicase activity and the conserved motifs are arrayed on the inside face between domains 1 and 2 in which the potential target-binding sites are only formed transiently when the two domains close. the constant movement of domains 1 and 2 between an 'open' and a 'closed' configuration and back again means that the enzyme is likely to often be in transition 87 . we have yet to understand the necessary principles required to inhibit such a dynamic protein system. it is not hard to imagine that the development of hcv helicase inhibitors for clinical use has been slow because the enzyme is a moving target and undergoes significant transient conformational changes that require the coupling of ntp hydrolysis to unwinding 67, 72, 74, 88 . the degree of movement is illustrated in fig. 6a , which is a superimposition of eight independent hcv helicase crystal structures, and fig. 6b, which is a superimposition of four of the above eight independent hcv helicase structures. domains 1 and 3 are relatively rigid with respect to each other (the traces lie on top of each other), whereas domain 2 has a large freedom of motion. this is indicated in fig. 6b by the silver ball identifying a single carbon atom in domain 2. finally, small-molecule drug design requires a reasonable starting point, whether it is a substrate mimic or a hit from screening. it is not obvious from the hcv helicase crystal structures that the 'gate keeper' residues, which are crucial for binding the (unwinding) nucleic acid, form a binding pocket suitable for structure-based inhibitor design. globally, hiv-1 infects more than 40 million individuals; hcv is estimated to have more than 170 million carriers. all cells and viruses require a helicase function, not necessarily their own, for nucleic-acid replication. the requirement for long-term treatment regimens for hiv allows for the generation of drug-resistant cellular enzymes, such as angiotensin-converting enzyme (ace) to treat hypertension, congestive heart failure, myocardial infarction, endothelial dysfunction and renal disease 89 . the potential for off-target-induced cytotoxicity is significant, irrespective of whether one is targeting a cellular or viral helicase, because of similarities in structure and function. however, the recently characterized requirement for a helicase in rev-dependent hiv-1 gene expression could be a new means to treat hiv-1 without generating drug-resistant viruses. in our laboratory (k.-t.j.), preliminary evidence has been found that ring-expanded nucleoside analogues, previously used successfully as ntpase/helicase inhibitors of west nile virus 90 , have substantial anti-hiv-1 activity in cultured cells at non-cytotoxic doses. the hsv helicase-primase inhibitors offer proof of principle that a viral helicase complex can be specifically differentiated from and selectively inhibited among a pool of related cellular helicases in cell-based experiments and in an infectious animal model without significant cytotoxicities. although the conserved helicase motifs within the helicases are very similar, individual helicases are widely divergent in their coding sequences, raising the possibility that each individual protein could be targeted with specificity in a knowledge-based manner. as highlighted in table 1, an emerging picture is that viruses either encode their own helicase or use a cellular helicase for replication. for example, poxviruses, papovaviruses and pathogenic rna viruses, such as west nile virus, severe acute respiratory syndrome (sars) coronavirus and dengue fever virus, all encode viral helicases that could potentially be future antiviral targets. regrettably, other than the rare drug candidates that we have mentioned in this review, there currently are no clinically useful candidate compounds for viral helicases. cumulated successes against virusencoded helicases are likely to precede effective drug targeting of cellular helicases used by viruses. antivirals and antiviral strategies references 1 and 2 are good overviews of antiviral drug discovery strategies the implications of drug resistance for strategies of combination antiviral chemotherapy mechanisms of helicasecatalyzed dna unwinding provides a detailed review of helicase mechanism a short, but very insightful, review of helicase mechanism of action modularity and specialization in superfamily 1 and 2 helicases in molecular motors helicases: amino acid sequence comparisons and structure-function relationships helicase structure and mechanism a review of helicase structure and function suitable for the general scientific reader hepatitis c virus ns3 rna helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding crystal structure of a dexx box dna helicase distantly related sequences in the α-and β-subunits of atp synthetase, myosin, kinases and other atp-requiring enzymes and a common nucleotide binding fold mutational analysis of the hepatitis c virus rna helicase structure and function of hepatitis c virus ns3 helicase birth of the d-e-a-d box discusses the first characterization of the dead protein motif rna chaperones exist and dead box proteins get a life dexd/h box rna helicases: from generic motors to specific dissociation functions the protein family of rna helicases mrna export: travelling with dead box proteins unwinding rna in saccharomyces cerevisiae: dead-box proteins and related families hrp1, a sequence-specific rnabinding protein that shuttles between the nucleus and the cytoplasm, is required for mrna 3′-end formation in yeast messenger-rnabinding proteins and the messages they carry protein displacement by dexh/d "rna helicases" without duplex unwinding dead-box proteins: the driving forces behind rna metabolism dbp5p, a cytosolic rna helicase, is required for poly(a)+ rna export dbp5, a dead-box protein required for mrna export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with can/nup159p requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function the first report to suggest a role for an rna helicase in the hiv-1 lifecycle dbp7p, a putative atpdependent rna helicase from saccharomyces cerevisiae, is required for 60s ribosomal subunit assembly the rrnaprocessing function of the yeast u14 small nucleolar rna can be rescued by a conserved rna helicase-like protein evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences a dead box protein facilitates hiv-1 replication as a cellular co-factor of rev new helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease references 32 and 33 discuss the development of hsv helicase inhibitors strategies for antiviral drug discovery helicases as antiviral drug targets minireview: virus-encoded rna helicases papillomavirus e1 proteins: form, function, and features a tale of two hsv-1 helicases: roles of phage and animal virus helicases in dna replication and recombination herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products t157602, a 2-amino-thiazole inhibits hsv replication by interacting with the ul5 component of the ul5/8/52 helicase primase complex inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the ul5-ul8-ul52 complex inhibition of herpes simplex virus type 1 helicase-primase by (dichloroanilino)purines andpyrimidines potent in vivo antiviral activity of the herpes simplex virus primase-helicase inhibitor bay 57-1293 aminothiazolyl-phenyl-based inhibitors of hsv helicase-primase: a novel class of orally active antiherpetic agents crystal structure of the hepatitis c virus ns3 protease domain complexed with a synthetic ns4a cofactor peptide reports the crystal structure of the hcv rna helicase domain crystal structure of rna helicase from genotype 1b hepatitis c virus crystal structure of the rnadependent rna polymerase from hepatitis c virus reveals a fully encircled active site structural analysis of the hepatitis c virus rna polymerase in complex with ribonucleotides crystal structure of the rna-dependent rna polymerase of hepatitis c virus viral rna replication in association with cellular membranes inhibition of native hepatitis c virus replicase by nucleotide and non-nucleoside inhibitors membrane association of the rnadependent rna polymerase is essential for hepatitis c virus rna replication biological evaluation of hepatitis c virus helicase inhibitors multiple enzymatic activities associated with recombinant ns3 protein of hepatitis c virus construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis c virus protease enzymatic characterization of hepatitis c virus ns3/4a complexes expressed in mammalian cells by using the herpes simplex virus amplicon system multiple full-length ns3 molecules are required for optimal unwinding of oligonucleotide dna in vitro the nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex rna efficiently hepatitis c virus ns3 rna helicase activity is modulated by the two domains of ns3 and ns4a polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis c virus ns3-ns4a complex isolated from transfected cos cells modulation of hepatitis c virus ns3 protease and helicase activities through the interaction with ns4a a kinetic analysis of the oligonucleotidemodulated atpase activity of the helicase domain of the ns3 protein from hepatitis c virus. the first cycle of interaction of atp with the enzyme is unique inhibition of the hepatitis c virus helicaseassociated atpase activity by the combination of adp, naf, mgcl 2 , and poly(ru) product release is the major contributor to kcat for the hepatitis c virus helicase-catalyzed strand separation of short duplex dna atp binding modulates the nucleic acid affinity of hepatitis c virus helicase periodic cycles of rna unwinding and pausing by hepatitis c virus ns3 helicase atp-binding domain of ntpase/helicase as a target for hepatitis c antiviral therapy nucleotide triphosphatase/helicase of hepatitis c virus as a target for antiviral therapy structure-based mutagenesis study of hepatitis c virus ns3 helicase electrostatic analysis of the hepatitis c virus ns3 helicase reveals both active and allosteric site locations two novel conserved motifs in the hepatitis c virus ns3 protein critical for helicase action inhibition of hepatitis c virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone abrogation of hepatitis c virus ns3 helicase enzymatic activity by recombinant human antibodies isolation of specific and high-affinity rna aptamers against ns3 helicase domain of hepatitis c virus in vitro selection of rna aptamers against the hcv ns3 helicase domain methods in molecular medicine expression and purification of a hepatitis c virus ns3/4a complex, and characterization of its helicase activity with the scintillation proximity assay system direct fluorometric measurement of hepatitis c virus helicase activity an ns3 protease inhibitor with antiviral effects in humans infected with hepatitis c virus short-term antiviral efficacy of biln 2061, a hepatitis c virus serine protease inhibitor, in hepatitis c genotype 1 patients initial results of a phase 1b multiple dose study of vx-950, a hepatitis c virus protease inhibitor phase i/ii dose escalation trial assessing tolerance, pharmacokinetics, and antiviral activity of nm283, a novel antiviral treatment for hepatitis c. digestive disease week synthesis and evaluation of atp-binding site directed potential inhibitors of nucleoside triphosphatases/helicases and polymerases of hepatitis c and other selected flaviviridae viruses hepatitis c virus ns3 atpase/helicase: an atp switch regulates the cooperativity among the different substrate binding sites double-stranded dna-induced localized unfolding of hcv ns3 helicase subdomain 2 angiotensin-converting enzyme inhibitors potent inhibition of ntpase/helicase of the west nile virus by ring-expanded ("fat") nucleoside analogues we would like to acknowledge m. sintchak and j. kim for their contribution to the hcv helicase structure figures and thank m. briggs, c. lin, m. murcko, s. lyons, j. thomson, a. elmo and v. yedavalli for helpful discussions and editorial assistance. the authors declare no competing financial interests. the following terms in this article are linked online to: entrez gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene ddx1 | ddx3 access to this interactive links box is free online. key: cord-263433-oldy0gta authors: barriocanal, marina; carnero, elena; segura, victor; fortes, puri title: long non-coding rna bst2/bispr is induced by ifn and regulates the expression of the antiviral factor tetherin date: 2015-01-09 journal: front immunol doi: 10.3389/fimmu.2014.00655 sha: doc_id: 263433 cord_uid: oldy0gta many long non-coding rnas (lncrnas) are expressed in cells but only a few have been well characterized. in these cases, lncrnas have been shown to be key regulators of several cellular processes. therefore, there is a great need to understand the function of more lncrnas and their regulation in response to stimuli. interferon (ifn) is a key molecule in the cellular antiviral response. ifn binding to its receptor activates transcription of several ifn-stimulated genes (isgs) that function as potent antivirals. in addition, several isgs are positive or negative regulators of the ifn pathway. this is essential to ensure a strong antiviral response and a later return of the cell to homeostasis. as the isgs described to date are coding genes, we sought to determine whether ifn also regulates the expression of long non-coding isgs. to this aim, we used rna sequencing to analyze the transcriptome of control and huh7 cells treated with ifnα2. the results show that ifn-treatment regulates the expression of several unknown non-coding transcripts. we have validated two lncrnas upregulated after treatment with different doses of type i ifnα2 in different cells or with type iii ifnλ. these lncrnas were also induced by influenza and vesicular stomatitis virus mutants unable to block the ifn response, but not by several wild-type lytic viruses tested. these lncrna genes were named lncisg15 and lncbst2 as they are located close to isgs isg15 and bst2, respectively. interestingly, inhibition experiments showed that lncbst2 is a positive regulator of bst2. therefore lncbst2 has been renamed bispr, from bst2 ifn-stimulated positive regulator. our results may have therapeutic implications as lncbst2/bispr, but also lncisg15 and their coding neighbors, are increased in cells infected with hepatitis c virus and in the liver of infected patients. these results allow us to hypothesize that several lncrnas could be activated by ifn to control the potency of the antiviral ifn response. the interferon (ifn)-mediated innate immune response provides a potent defense against pathogens (1) . upon invasion, pathogenassociated molecular patterns (pamps) are detected by specific receptors in the cells. these can be located on the surface of the cell, as in the case of toll-like receptors (tlrs), or intracellularly, as in the case of the retinoic acid-inducible gene i (rig-i). pamp recognition triggers a series of signaling cascades that lead to the production and secretion of type i ifn. type i ifn (ifnα, ifnβ, and others) binds to ifn receptors present on the surface of all cell types and activates janus-activated kinase/signal transducer and activator of transcription (jak/stat) signaling. this gives rise to the nuclear translocation of the stat1/stat2/irf9 (ifn regulatory factor 9) complex that binds ifn-stimulated response elements (isre) in the promoters of ifn-stimulated genes (isgs) and activates their transcription. a similar response is induced by type iii ifn (ifnλ) upon binding to its receptor (2, 3) . in contrast, type ii ifn or ifnγ, produced by cells of the immune system, binds to the widely expressed ifnγ receptor (4, 5) leading to nuclear translocation of stat1 homodimers, which bind to gamma-activated sequences (gas) in the promoter of immunoregulatory genes. ifn-stimulated genes are antiviral factors, positive regulators of the ifn pathway (stat1 and 2 and irf1) or negative regulators that help ifn-induced cells to return to cellular homeostasis (socs and ups18) (6) (7) (8) (9) (10) (11) . among antiviral genes, there are factors that function to increase cell sensitivity to pamps (oas and pkr) or true antiviral effectors that block viral entry (mx, ifitm, and trim), virus replication, translation and stability (ifit, oas, pkr, and isg15), or viral release (viperin and tetherin/bst2) (8) . while most ifn-induced factors known to date are proteins, ifn also activates the expression of several micrornas that contribute to the antiviral state or to the control of ifn response (12) . few studies have been performed to address whether ifn could also regulate expression of long non-coding rnas (lncr-nas) (13) (14) (15) . in recent years, viral infection has been reported to be able to induce the expression of cellular lncrnas. this has been shown for infection with enterovirus, influenza, hiv, hepatitis b and c viruses, and the sars coronavirus (13, (16) (17) (18) (19) (20) (21) (22) (23) (carnero et al., in preparation) . the lncrna signature found after infection www.frontiersin.org should be a mixture of transcripts induced by the virus and transcripts that respond to the cellular antiviral pathways activated by the infection. in fact, activation of tlrs by pamps induces the expression of several lncrnas. tlr2 signaling leads to the activation of lncrna-cox2, which regulates the expression of genes related to the immune system (24) . activation of tlr3 results in increased neat1, which increases the expression of genes such as il8 (19) . tlr4 controls il1b-erna and il1b-rbt46 lncr-nas whose downregulation diminishes il1b and accumulation of lps-induced rnas (25) . likewise, the lps-induced inflammatory response is controlled by lnc-il7r (26) . innate activation also induces linc1992/thril, which controls tnfα and other genes involved in the immune response (27) . in turn, tnfα induces lethe, a pseudogene that responds to nfκb and reduces inflammation by inhibiting nfκb dna binding activity (28) . lncrna responses are also critical for the functionality of dendritic cells, cd4+ and cd8+ t-cells (29) (30) (31) (32) . thus, nest lncrna controls ifnγ locus in cd8+ t-cells leading to decreased salmonella enterica pathogenesis (33, 34) . these studies illustrate the interest in identifying novel lncr-nas and elucidating their function and regulation. lncrnas are thought to be at least as numerous as protein-coding genes, but only a few are well characterized (35) (36) (37) (38) . lncrnas are transcripts similar to mrnas but with poor coding potential. they are more cell type-specific, less expressed, and less well conserved than mrnas (29, 39) . interestingly, lncrnas are cell regulators that can function in cis, co-transcriptionally, or in trans. some control the expression of coding genes located in the same genomic region. therefore, the genomic location of lncrnas can provide hints as to their functionality. they can be sense or antisense (when overlapping with one or more exons of another transcript in the same or in the opposite strand, respectively); intronic (when derived from an intron of another transcript); divergent or bidirectional (when they share a promoter with another transcript in the opposite strand and therefore are co-regulated); or intergenic (when they are independent, located in between two other genes). several mechanisms are involved in the regulation of neighboring or antisense genes by lncrnas. these include transcriptional activation or interference, recruitment of chromatin modifiers and remodelers, regulation of imprinting, editing, splicing or translation, and stability (40) (41) (42) (43) (44) . to address the issue of whether ifn could also regulate expression of lncrnas, which may play key roles in the antiviral response, we analyzed the transcriptome of cells treated or not with ifnα2 by rna sequencing (rnaseq). in this analysis, we identified two lncrnas upregulated in response to ifn in different cell lines. interestingly, these lncrnas are expressed from positions in the genome divergent from the well-characterized isgs isg15 and bst2. therefore, we have called them lncisg15 and lncbst2. these lncrnas and their coding counterparts are also induced in cells infected with mutants of influenza or vesicular stomatitis viruses (vsv) that fail to block the ifn response. surprisingly, they are also induced in culture cells infected with hepatitis c virus (hcv) and in the liver of patients with hcv infections. finally, according to hugo regulation, we have renamed lncbst2 bispr, from bst2 ifn-stimulated positive regulator, as we show that inhibition of lncbst2 expression by rnai leads to decreased levels of bst2 mrna, providing a new layer of regulation of the ifn response. the huh7 cell line, derived from a human hepatocarcinoma, was provided by dr. chisari's lab (scripps research institute, la jolla, ca, usa). a549 cells, from human non-small cell lung carcinoma, were kindly provided by estanislao nistal (cima, university of navarra, spain). human liver samples with or without hcv infection were obtained from the biobank of the university of navarra under approval from the ethics and scientific committees. liver tissue sections were snap frozen and stored at −80°c. the clinical data from hcv-infected subjects are shown in table s1 in supplementary material. cells were grown in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 1% penicillin-streptavidin and maintained at 37°c in a 5% co 2 atmosphere. twenty-four hours before treatment with ifn, huh7 and a549 cells were seeded in six-well plates. then, 0, 5, 50, 250, 1000, or 10000 units/ml of ifnα2 (sicor biotech, lithuania) were used in a final volume of 2 ml. huh7 cells were also treated with 250 ng/ml of il28b/ifn-λ3 (r&d systems) in a final volume of 2 ml. for treatment with ruxolitinib (selleckchem), cells were seeded out 24 h before and treated with 0.8 µm ruxolitinib in a final volume of 2 ml. one hour after treatment media were discarded and replaced by media containing 100 units/ml ifnα. cells were harvested for rna extraction at the indicated times post-treatment. sirnas targeting lncbst2/bispr were designed using iscore designer and rna scales (45, 46) and purchased from dharmacon. the lncbst2/bispr sirnas targeted the sequence gacuagugugagcaacaaa. for cell transfection with sir-nas, lipofectamine 2000 reagent (invitrogen) was used according to manufacturer's instructions. cells were seeded 24 h before transfection. for each well of a six-well plate, 80 pmoles sirna were used. the sirna was mixed with 50 µl optimem. furthermore, 6 µl lipofectamine were mixed with 250 µl optimem media and incubated for 5 min. then, lipofectamine and sirna solutions were mixed and incubated for 20-60 min at room temperature. after incubation, half of the volume of the cell media was discarded and 300, 150, or 75 µl of the lipofectamine mixture were added to each well of 6, 12, or 24-well plates, respectively. six hours post-transfection the media from the cells was discarded and substituted with dmem media enriched with 10% fbs and antibiotics. hepatitis c virus jfh-1 was obtained from an initial viral stock from the genotype 2a jfh-1 plasmid (pjfh-1) previously described by wakita et al. (47) . the virus was amplified as described (15) . influenza virus strain a/pr8/34 wt (pr8), a mutant lacking ns1 (∆ns1), vsv-gfp, and the mutant m51r frontiers in immunology | molecular innate immunity were kindly provided by estanislao nistal (cima, university of navarra, spain) (48) (49) (50) , semliki forest virus (sfv) was a gift from cristian smerdou (cima, university of navarra, spain), and adenovirus serotype 5 (ad5) was amplified as previously described (48) . vsv-egfp titration was performed in quadruplicates on a549 cells. the supernatant from infected cells was collected and 1:10 serial dilutions were performed. cells were seeded 24 h before infection in 96-well plates and infected with 50 µl of each dilution. twenty-four hours after infection, gfp expression was visualized by microscopy and used to determine the titer. cells were infected with hcv at a multiplicity of infection (moi) of 0.3, with vsv at a moi of 5 and with a moi of 10 of influenza a, ∆ns1, ad5, and sfv. in the case of the lytic viruses, we used a moi of 5 or 10 as this causes cytopathic effects at 24 h (for vsv, influenza and sfv) or 48 h (for ad) in huh7 or a549 cells. after infection, the virus was removed and fresh medium was added to the cells. cells were harvested for rna extraction at the indicated times post-infection. two million huh7 cells were incubated in 100 µl of cytoplasmic buffer (50 mm tris hcl ph7.4, 1 mm edta, and 1% np40) for 5 min at 4°c. then, cells were centrifuged for 5 min at 3000 g and the supernatant was used to isolate cytoplasmic rna. the pellet was washed with cytoplasmic buffer and centrifuged as before. the supernatant was discarded and the pellet was used to isolate the nuclear rna. rna from nuclear and cytoplasmic fractions was isolated with maxwell 16 research system (promega). human tissue was homogenized using the ultra-turrax dispersing machine (t25 basic ika-werke) (51) . total rna from the tissue was extracted in 1 ml trizol (sigma-aldrich) and recommendations of the supplier were followed (52) . dnase (fermentas) treatment was performed to eliminate dna from the samples before rt-pcr reactions. rna was extracted from cells with the maxwell 16 research system from promega following the manufacturer's recommendations. rna concentration was measured using nanodrop 1000 spectrophotometer. the quality of the rna was analyzed by bioanalyzer (agilent technologies). reverse transcription (rt) was performed as described (53) . the reaction was performed in the c1000 touch thermal cycler from bio-rad. the samples were incubated at 37°c for 60 min, then at 95°c for 60 s and then immediately cooled to 4°c. qpcr was performed in the cfx96 real-time system from bio-rad as described (54) . the results were analyzed with bio-rad cfxmanager software. gapdh levels were evaluated in all the cases as a reference. only the samples with similar gapdh amplification were analyzed further. the primers used are listed in table s2 in supplementary material and were designed with the primer3 program 1 . rna of excellent quality, as determined by bioanalyzer (agilent technologies) was treated with the ribo-zero rrna removal kit 1 http://frodo.wi.mit.edu (epicenter) to deplete from ribosomal rna. library preparation with truseq rna sample preparation kit (illumina) and sequencing was performed at the embl genomics core facility (genecore) in an illumina hiseq 2000. sequences were paired-end, 150 bases long, and strand specific. rnaseq data are available at the ncbi gene expression omnibus (geo) data repository 2 . rna sequencing data analysis was performed using the following workflow: (1) the quality of the samples was verified using fastqc software; (2) the preprocessing of reads was performed by elimination of contaminant adapter substrings with scythe and by quality-based trimming using sickle; (3) the alignment of reads to the human genome (hg19) was performed using the tophat2 mapper (55); (4) transcript assembly and quantification using fpkm of genes and transcripts was carried out with cufflinks 2 (56); (5) the annotation of the gene locus obtained was performed using cuffmerge with gencode v16 as reference; and (6) differential expression analysis was performed using cuffdiff 2 (56) . genes were selected as differentially expressed using a p-value threshold of 0.01. further analysis and graphical representations were performed using an r/bioconductor (57) . reads from all the differentially expressed sequences were visualized in the integrative genomics viewer (igv) 3 (58, 59) and the sequences were compared to the ensembl and encode databases and searched for in the genome browser from ucsc 4 for more information (60, 61) . candidates were divided into coding, non-coding (according to ucsc classification), or non-assigned, when the transcription of the sequence had not been annotated in the databases. functional enrichment analysis of gene ontology (go) categories was carried out using a standard hypergeometric test (62) . biological knowledge extraction was complemented through the use of ingenuity pathway analysis (ingenuity systems) 5 , with a database that includes manually curated and fully traceable data derived from literature sources. open reading frame finder (ncbi) was used to evaluate the length of all probable open reading frames (orfs) in lncisg15 and lncbst2/bispr. coding potential was assayed with the coding potential assessment tool (cpat) (63, 64) and by searching the lncipedia database (65) for the presence of our candidates in the pride archive (66) or in lists of transcripts associated with ribosomes (67, 68) . phylogenetic codon substitution frequencies (phylocsf) were also used to predict the coding potential of lncisg15 and lncbst2/bispr (69). statistical analysis of the rnaseq data has been already described. remaining analysis was performed using graph-path. statistical significance of infected versus non-infected samples was calculated using a two-tailed non-parametric mann-whitney t -test or with a two-tailed students t -test when the samples followed a normal distribution according to the shapiro-wilk test. welch's correction was applied for samples with heterogeneous variance. for correlation studies, a two-tailed non-parametric spearman analysis was used. p values lower than 0.05 were deemed as significant. to identify lncrnas that respond to ifn, we treated huh7 cells with 10000 units/ml of ifnα2 for 3 days. these conditions serve to induce the expression of well-known isgs such as gbp1, irf1, bst2, oas, or isg15 (15) . in addition, this treatment induces an antiviral effect, as hcv-infected huh7 cells treated with 10000 units/ml of ifnα2, show decreased levels of viral proteins and viral genomes compared to untreated infected cells (data not shown). finally, the rna isolated from huh7 cells treated with 10000 units/ml of ifnα2 for 3 days was used to hybridize an agilent array. analysis of the array showed that well-characterized isgs such as mx1, stat1, irf9, isg15, bst2, and several members of the gbp, oas, and ifi families were upregulated with a very high statistical significance (b > 7) (15) . ingenuity analysis of the data showed that ifn signaling was the pathway with the highest enrichment followed by other antiviral responses. the microarrays were used to identify lncrnas regulated by ifnα (15) . however, an array will only evaluate the expression levels of the transcripts that hybridize to probes spotted in the array. in the case of the lncrnas, the array used only addresses the expression of 7419 regions described as long intergenic noncoding rnas (lincrnas). however, it has been estimated that there could be as many lncrna genes as coding genes, and some authors consider that the number of lncrnas could be as high as 200000 (37, 38) . therefore, to achieve a more complete identification of lncrnas that respond to ifn, we analyzed the transcriptome by rnaseq. rna isolated from control cells or huh7 cells treated with ifn as described, was sequenced after ribodepletion. around 130 million reads were obtained per sample. analysis was performed using a bioinformatic workflow that includes tophat2 and cufflinks 2 as described in the methods section. the analysis showed that, among the genes upregulated in response to ifn, there were several isgs such as mx1, isg15, bst2, or members of the ifi and oas families ( figure 1a and table s3 in supplementary material). ingenuity analysis showed that ifn signaling is a top canonical pathway (p = 3.3 × 10 −3 ), the top upstream regulator is ifnα2 (p = 1.9 × 10 −8 ), and cell signaling and infectious and inflammatory diseases are among the main functions. the expression of~1000 coding genes was altered by ifn (table s3 in supplementary material). the rnaseq analysis also showed that the expression levels of many regions that do not correspond to coding genes were also significantly modified in response to ifn ( figure 1b) . out of the 890 putative non-coding genes whose expression was significantly altered, half were upregulated (table s3 in supplementary material). all candidates where visualized using igv ( figure s1 in supplementary material) (58, 59) . we also paid special attention to altered sequences located close to well-known isgs and to genomic regions that were highly expressed and deregulated in response to ifn. eight candidates that fulfill at least one of these two criteria were chosen for further validation ( table 1 and figure s1 in supplementary material). to validate the eight candidates chosen, we treated huh7 cells with different doses of ifnα2. rna was isolated from the cells at 6, 24, 48, or 72 h post-treatment and the expression levels of the candidates were evaluated by quantitative rt-pcr (qrt-pcr) (table 1; figure 2 ). all the candidates were induced after ifn-treatment from 2 to more than 1000-fold. however, many of the candidates were detected at very low cycles in the pcr amplification. a closer examination of their sequences indicated that they contained repetitive sequences or sequences similar to mitochondrial or ribosomal rnas that could have led to an erroneous alignment of the rnaseq reads to the human genome. we believe that, even when the oligonucleotides used for amplification were specific, a partial homology to other sequences could allow cross-amplification and thus increased possibilities of misleading results. these candidates were not studied further. we focused on two lncrnas with no repetitive sequences whose expression was highly upregulated in response to ifn (table 1; figure 2 ). interestingly, database analysis showed that they are expressed from positions in the genome located close to isg15 and bst2, both of which are well-characterized isgs. this may have functional relevance as some lncrnas have been described to regulate the expression of neighboring genes. therefore, we originally named these lncrnas after their neighbor, lncisg15 and lncbst2. later, lncbst2 was renamed bispr to follow hugo regulations. when we evaluated the expression of these lncrnas and their neighboring transcripts, we observed that both were strongly upregulated at early times in response to ifn (figure 2a) . furthermore, they responded to ifnα2 doses as low as 5 units/ml. these are similar levels to those found in the sera of some hcv patients (70) . the induction was also observed at late times post-ifn-treatment. to evaluate further the robustness of the effect www.frontiersin.org of ifn on these lncrnas, we tested whether they also respond to ifnλ, a type iii ifn. huh7 cells treated with ifnλ for 6, 12, 24, 48, or 72 h also showed increased levels of lncisg15, lncbst2/bispr, and their neighbors ( figure 2b) . in this case, all the transcripts showed a higher upregulation at later times post-ifnλ treatment. viruses activate the ifn response by several mechanisms. therefore, they have evolved to block ifn production and the activation of the ifn pathway. the molecular mechanisms involved in this ifn blockade have been characterized for many viruses. thus, for instance, ns1 protein from influenza virus and matrix protein from vsv are key factors in controlling ifn in infected cells (48) (49) (50) 71) . we sought to check whether lncisg15 and lncbst2/bispr were induced by the physiological ifn induced by an influenza virus that lacks ns1. therefore, we evaluated the expression of these lncrnas in cells infected with an influenza wild-type virus or a ns1 mutant. we also included cells infected with other rna viruses such as sfv and hcv or dna viruses such as adenovirus. all these viruses have developed mechanisms to block the cellular antiviral response and, with the exception of hcv, lead to a lytic infection. different times post-infection were evaluated. the last point was collected when the cytopathic effect was apparent. this occurred at 24 h post-infection in the case of influenza and sfv or 48 h post-infection, in the case of adenovirus. hcv-infected cells were collected at 48 and 72 h postinfection. the results showed that at later times post-infection with the influenza virus lacking ns1, there was increased expression of lncisg15, lncbst2/bispr, and their neighboring coding transcripts (figure 3a) . this increase was not observed in cells infected with wild-type influenza virus, or with other wildtype lytic viruses, suggesting that the induction may be mediated by ifn. most lncrnas are tissue-specific. to determine whether lncisg15 and lncbst2/bispr respond to infection only in huh7 cells or whether this effect is specific for influenza viruses, we infected alveolar epithelial a549 cells with vsv-gfp wild-type virus or with a m51r matrix mutant that fails to control ifn. we chose a549, because lung cells serve as the primary site for productive infection of vsv and many respiratory viruses (72) . infection with the wild-type virus did not increase the expression of lncbst2/bispr or bst2 (data not shown). however, a549 cells infected with the vsv mutant m51r for 4, 6, or 10 h did show increased levels of lncisg15, lncbst2/bispr, isg15, bst2, and other isgs such as gbp1 ( figure 3b) . surprisingly, infection with hcv also increased the expression of lncisg15, lncbst2/bispr, and other isgs, including isg15, bst2, and irf1 ( figure 3a and data not shown). to determine whether these genes were also upregulated in infected patients, we used liver samples from hcv-negative and hcv-positive donors. after quantification of the rna levels, we observed a significant increase in lncisg15, lncbst2/bispr, isg15, and bst2 in hcvinfected patients compared to controls (figure 4a ). with the number of patients evaluated, a significant correlation was not found between expression levels and infection with a particular genotype of hcv, presence of hcv-induced hepatocellular carcinoma (hcc), liver cirrhosis, or with a particular cirrhosis stage. therefore, there were no significantly different levels of these transcripts in hcv-infected livers without hcc compared with the peritumoral tissue of hcv-infected livers with hcc. although most of the samples belong to patients that are still alive, no significant correlation was observed between the levels of the evaluated transcripts and survival post-diagnosis. finally, we performed correlation studies to analyze whether in the patients, the expression level of lncisg15 or lncbst2/bispr correlates significantly with the expression level of their neighboring coding genes. the results show a highly significant positive correlation between lncisg15 and isg15 or lncbst2/bispr and bst2 (figure 4b ). the experiments performed so far suggest that a general correlation could exist between the expression of lncisg15 and isg15 or lncbst2/bispr and bst2. each lncrna and its neighboring coding gene have similar induction patterns in response to ifn or to viral infection (compare their levels in figures 2 and 3) . furthermore, the levels of each coding/non-coding pair correlate significantly in patient samples (figure 4b) . to analyze this in more detail, we performed correlation studies of the coding/noncoding pairs in all the samples evaluated in figures 2 and 3 . the results show that the correlation of each pair was highly significant ( figure s2 in supplementary material). this suggests that they could be co-regulated, and therefore, they could share similar functions. however, expression of lncisg15 and lncbst2/bispr also correlated significantly with the expression of other isgs such as oas, gbp1, or irf1 (data not shown). to obtain more information on the relationship between the coding/non-coding pairs, we searched several databases. lncisg15 and lncbst2/bispr genes are in head-to-head orientation with their coding neighbors ( figure s3 in supplementary material) and they could share the same promoter. this is based on the following facts: (i) the distance between the two genes is <1000 bp, a cut-off for bidirectional promoters (73, 74) ; (ii) there is a single dnase hypersensitivity region located between the genes, and (iii) polymerase ii (pol ii) chipseq analysis of k562 cells shows a single peak covering the h3k27ac region between both genes. interestingly, the peaks observed for pol ii chipseq are increased at 30 min or 6 h post-treatment with ifnα or ifnγ. finally, the promoter regions contain conserved isre sites and binding sequences for irf1, irf2, and irf7. to discriminate whether lncisg15 and lncbst2/bispr are induced directly by the jak/stat signaling pathway or by a secondary wave of the ifn response, we evaluated the expression of these lncrnas and their coding neighboring genes in huh7 or a549 cells incubated or not with the jak/stat inhibitor ruxolitinib. expression of gbp1, a bona fide isg, was also evaluated as a positive control (figure 5) . the results show that the levels of gbp1, bst2, and lncbst2/bispr are significantly reduced liver samples from hcv-negative and hcv-positive donors were used to quantify the levels of lncisg15, lncbst2/bispr, isg15, bst2, and gapdh mrnas. statistical significance was calculated using a two-tailed non-parametric mann-whitney t -test for lncbst2/bispr, isg15, and lncisg15 and with a two-tailed students t -test with welch's correction for bst2, which follows a normal distribution according to the shapiro-wilk test. (b) expression levels observed for lncisg15, lncbst2/bispr in patient samples were compared to the expression levels of their coding neighbors isg15 and bst2, respectively. a correlation analysis was performed and statistical significance was calculated using a two-tailed non-parametric spearman analysis. www.frontiersin.org in the presence of ruxolitinib, indicating that their expression is stat-dependent. levels of bst2 and lncbst2/bispr were also reduced in cells treated with sirnas targeting stat1 or by inhibition of irf1, a transcription factor that acts downstream of ifn (data not shown). this indicates that bst2 and lncbst2/bispr respond to stats but also to other transcription factors induced by ifn. these results agree with the possibility that bst2 and lncbst2/bispr share a bidirectional promoter. in contrast, the effect of the jak/stat pathway on isg15 and lncisg15 expression was less robust. treatment with ruxolitinib decreased the expression of isg15 and lncisg15, but in the latter, this effect was only observed in a549 cells. no effect on isg15 or lncisg15 expression was observed with a milder inhibition of stat1 or inhibition of irf1 using rna interference (data not shown). thus, although isg15 is induced very rapidly after ifntreatment, we do not observe a strong regulation of isg15 or lncisg15 by the stat pathway under the conditions used. in fact, it has been reported that a major regulator of isg15 is irf3, a transcription factor activated in response to pamps, but also a downstream effector of the ifn response (75) . we evaluated the coding capacity of lncisg15 and lncbst2/bispr bioinformatically. orf finder (ncbi) was used to determine all possible orfs in these lncrnas ( figure s4 in supplementary material). the analysis shows that all putative orfs are shorter than 50 amino acids. only two orfs could be translated according to their poor susceptibility to nonsense mediated decay. however, these orfs have non-consensus kozak sequences at the initiation codon and therefore a poor coding capacity. then, we evaluated the coding potential of lncisg15 and lncbst2/bispr with the cpat (63, 64) ( figure 6a) . cpat uses a model built with orf size and coverage together with codon (ficket score) and hexamer (hexamer score) usage bias. according to this program, lncisg15 and lncbst2/bispr are non-coding as they have a coding probability of 0.001 and 0.064, respectively, much lower than 0.364, used as a threshold with the highest sensitivity and specificity to differentiate between coding and non-coding transcripts in humans figure 6 | lncisg15, lncbst2/bispr have poor coding potential and accumulate preferentially in the nucleus. (a) bioinformatic analysis of the coding potential of lncisg15 and lncbst2/bispr. results obtained from cpat and lncipedia. two transcripts have been evaluated for lncbst2/bispr. lncisg15 and lncbst2/bispr have a coding probability and a coding label of "non-coding rnas" according to these analyses. "lists" indicated the number of times that these transcripts have been found in the pride archive or in lists containing ribosome-associated rnas published by lee or bazzini. see the text for other details. (b) subcellular localization of lncisg15 and lncbst2/bispr. huh7 cells were mock-treated or treated with 10000 units/ml of ifnα2 and divided into nuclear and cytoplasmic fractions. rna was isolated from each fraction and used to evaluate the expression levels of lncisg15 and lncbst2/bispr by qrt-pcr. malat1, gapdh, and isg15 mrna was also quantified and used as a reference to calculate the relative levels of each transcript and as a control to evaluate the subcellular fractionation. the ratio of cytoplasmic to nuclear levels is shown. the experiment was performed three times and each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. (64) . lncisg15 and lncbst2/bispr were also described as noncoding in lncipedia (65) . this lncrna database shows that these lncrnas are not found in the pride archive, a database for proteomic data, or in lists of transcripts associated with ribosomes in ribosome profiling experiments (66) (67) (68) . further, lncisg15 and lncbst2/bispr were also described as non-coding by the analysis of phylocsf, which uses multiple alignments to calculate the frontiers in immunology | molecular innate immunity phylogenetic conservation score and determines whether a multispecies nucleotide sequence alignment is likely to represent a protein-coding region (69) . finally, we evaluated the subcellular localization of lncisg15 and lncbst2/bispr in huh7 cells mock-treated or treated with 10000 units/ml of ifnα. rna was isolated from nuclear or cytoplasmic fractions and quantified by qrt-pcr. we found that the coding gapdh or isg15 mrnas accumulate preferentially in the cytoplasm while the nuclear rnas malat1 or u6 are preferentially nuclear (figure 6b data not shown) . similarly, lncisg15 and lncbst2/bispr, compared to mrnas, accumulate preferentially in the nucleus. this result, together with the bioinformatic analyses, strongly suggests that lncisg15 and lncbst2/bispr are non-coding rnas. to address the role of lncbst2/bispr, we used rna interference. huh7 cells treated or not with ifn, were transfected with sir-nas targeting lncbst2/bispr and rna expression was evaluated by qrt-pcr. the results show that expression of lncbst2/bispr was decreased compared to cells transfected with control sirnas ( figure 7a) . surprisingly, inhibition of lncbst2/bispr also led to decreased levels of bst2 mrna. expression of lncisg15, isg15, gbp1 or expression of genes located in the genome close to bst2 or lncbst2/bispr, such as gtpbp3 or mvb12a, was not affected (figure 7a and data not shown). to determine whether this was a general phenomenon, we transfected the sirnas targeting lncbst2/bispr into a549 cells infected or not with the vsv m51r mutant or treated with ifn. similarly to what has been observed in huh7 cells, the sirna that targets lncbst2/bispr leads to decreased levels of lncbst2/bispr and bst2 mrna while the levels of isg15 mrna are not significantly affected (figure 7b) . similar results were observed with a different sirna targeting lncbst2/bispr. rna sequencing analysis of human cells treated with ifnα2 and controls has allowed the identification of lncrnas induced in response to ifn (figure 1) . analysis of the rnaseq data shows that several of the upregulated genes are well-known coding isgs such as isg15 or oas ( figure 1a , table s3 in supplementary material). ingenuity analysis confirms the enrichment of genes involved in the ifn response among the regulated factors. we have used rnas from similar ifn-treated and control cells to hybridize expression arrays (15) . comparison of the datasets obtained in the analysis of the array and the rnaseq shows that only 13 coding genes were identified in both studies, including oas, isg15, mx1, and some members of the ifi family. generally, overlap between microarray and rnaseq analysis is not high (76) . furthermore, the overlapping decreases with sequencing depth and when low fold-changes or low abundance genes are analyzed. (77) . this is because sequencing of low transcript abundances is characterized by high variance, which impedes their identification in rnaseq analysis. we believe that this may explain the poor correlation found between the array and the rnaseq datasets. in fact, we have determined by qrt-pcr that some ifnrelated low abundance transcripts are detected only in the array analysis. these are early responders to ifn, which increased only marginally 3 days after ifn-treatment, when the analysis was performed. therefore, we believe that some lncrnas induced early post-ifn-treatment may have not been identified in our analysis. interestingly, in the process of writing this manuscript, a paper www.frontiersin.org was accepted describing the identification of ifn-induced lncr-nas by rnaseq in samples treated with ifn for short times (14) . we believe that this study will be complementary to our work. together, the datasets should contain lncrnas regulated at early and later times post-ifn-treatment. similarly, the lack of correlation between the microarray and rnaseq datasets also indicates that they can complement each other. we have identified two lncrnas whose expression is highly upregulated in response to different doses of ifnα (table 1; figure 2a ) or ifnλ (figure 2b) . our results show that induction of these lncrnas by ifnα seems faster than that observed for ifnλ. we cannot rule out the possibility that a fast response to ifnλ may also be observed when higher doses are used. encode analysis of polymerase ii binding to the promoters of these lncr-nas also shows that they may be induced by treatment with ifnα and ifnγ ( figure s3 in supplementary material). these lncrnas have been named lncisg15 and lncbst2/bispr after their neighboring genes, which play a key role in the antiviral ifn response. our molecular and bioinformatic analyses strongly suggest that lncisg15 and lncbst2/bispr are indeed lncrnas, as they accumulate preferentially in the nucleus of ifn-treated or untreated cells ( figure 6b ) and they have poor coding potential ( figure 6a and figure s4 in supplementary material). in general, the upregulation of lncisg15 and lncbst2/bispr mimics that of their coding counterparts (figures 2-4) . in fact, analysis performed with all the expression data obtained in our studies, shows a highly significant correlation between lncisg15 and isg15 and between lncbst2/bispr and bst2. significant correlations also exist between these lncrnas and other ifn-induced genes such as oas, gbp1, or irf1 ( figure s2 in supplementary material and data not shown). this may reflect the fact that all these genes are induced by ifn with a similar kinetics. in the case of the lncrnas and their coding counterparts, correlation of the expression may result from their transcriptional co-regulation. experimental and bioinformatic analyses indicate that bst2 and lncbst2/bispr are bona fide isgs strongly induced by the jak/stat pathway in response to ifn (figure 5 and figure s3 in supplementary material). furthermore, expression of bst2, isg15, and their neighboring non-coding genes is induced by downstream effectors of the ifn response. these studies allow us to suggest that lncisg15/isg15 and lncbst2/bispr/bst2 may share bidirectional promoters. other ifn-induced gene pairs may also be co-regulated by bidirectional promoters as these are enriched in stat1 binding (78) . bidirectional promoters often couple genes involved in the same process, allowing for coordinated temporal and environmental responses (73, (78) (79) (80) (81) (82) . non-coding rnas generated from bidirectional promoters may have functional roles that affect the bidirectional promoter, the neighboring protein-coding gene, or more distal genes (83) . these effects could lead to activation or repression of the expression and could be mediated by either the transcription process itself or by the produced ncrna transcript (84) . in this study, we show that post-transcriptional inhibition of lncbst2/bispr leads to reduced levels of bst2 mrna (figure 7) . therefore, lncbst2/bispr should increase transcription or stability of its coding neighboring gene. our results demonstrate that this regulation seems specific for bst2, as lncbst2/bispr downregulation does not affect the expression of genes located nearby, which has been described for compact genomes (85) . moreover, inhibition of lncbst2/bispr does not affect expression levels of other ifn-related genes such as isg15 or gbp1 (figure 7 and data not shown). we anticipate that inhibition of lncbst2/bispr, and therefore of bst2, could impact the antiviral effects of ifn. bst2 is also named tetherin, as it inhibits viral budding by using anchors that trap virions on the cell membrane (86) (87) (88) . several enveloped viruses have been shown to be susceptible to the action of bst2/tetherin and have evolved to develop evasion strategies (87) . interestingly, hiv, influenza, hcv, and vsv are among the susceptible viruses and could be used to test the antiviral role of lncbst2/bispr (49, (89) (90) (91) (92) (93) (94) (95) . in fact, we show that lncbst2/bispr and bst2 are induced after infection with hcv or influenza and vsv mutant viruses that activate the ifn response (figure 3) . upregulation of lncbst2/bispr, lncisg15, and their coding neighbors was also observed in patients infected with hcv compared to controls (figure 4) . similarly, a significant upregulation of lncbst2/bispr and isg15 was also detected in human tlymphocytes infected with hiv compared to controls (data not shown). a non-significant increase in bst2 and lncisg15 was also observed in these samples. this leads to the possibility that interference with these factors could have therapeutic relevance. it is unclear why cells infected by these viruses, which employ several viral proteins to block the ifn pathway, show activation of these ifn response genes (96) . in the case of hcv, it has been previously shown that patients with chronic hcv infections express isgs, including high levels of isg15 (97) (98) (99) . in fact, hcv has evolved to use some isgs for viral replication (100, 101) . this is the case for isg15. isg15 is an ubiquitin-like protein that attaches to its targets in a process called isgylation (102, 103) . protein isgylation may result in increased or decreased functionality depending on the target (104) . isg15 preferentially conjugates newly synthesized proteins affecting more strongly viral proteins or cellular proteins translated into ifn-induced cells (105) . viruses such as influenza, hiv, or vsv are susceptible to the action of isg15 (103, 106) . in the case of hcv, a pro-hcv role for isg15 has been reported (105, 107) . isg15 has been shown to negatively regulate rig-i and thus to inhibit the signaling process leading to ifn induction that affects hcv replication (108) . furthermore, isg15 expression in the liver of chronically infected patients is considered a negative predictive biomarker of the ability of the patients to respond to ifn therapy (97) (98) (99) (figure 4) . in our study, we cannot address whether lncisg15, bst2, or lncbst2/bispr are markers for the susceptibility of hcv patients to respond to ifn-treatment, as the hcv patients that we have studied are non-responders to ifn. we believe that, similar to lncbst2/bispr, lncisg15 could affect the expression of isg15 or other genes. this lncrnamediated control has also been described for a lncrna located close to the isg viperin, which has been shown to regulate the levels of many ifn-inducible genes (14, 109) . further experiments will be required to address the role of lncisg15 and to decipher the molecular mechanisms that allow the control exerted by lncbst2/bispr on bst2. we believe that these studies may be important to better understand the ifn response and its pro or antiviral functions on hcv and other viruses. frontiers in immunology | molecular innate immunity regulation of type i interferon responses dynamic expression profiling of type i and type iii interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression interferons alpha and lambda inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics interferon gamma: a master regulator of atherosclerosis the two faces of interferon-gamma in cancer complex modulation of cell typespecific signaling in response to type i interferons synergistic activation of inflammatory cytokine genes by interferon-gammainduced chromatin remodeling and toll-like receptor signaling interferon-stimulated genes: a complex web of host defenses socs proteins, cytokine signalling and immune regulation alpha interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp18/ubp43 fine tuning type i interferon responses interferons and micrornas annotation of long non-coding rnas expressed in collaborative cross founder mice in response to respiratory virus infection reveals a new class of interferon-stimulated transcripts negative regulation of the interferon response by an interferon-induced long non-coding rna type interferon regulates the expression of long noncoding rnas lncrna expression signatures in response to enterovirus 71 infection evidence for a crucial role of a host non-coding rna in influenza a virus replication unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling long noncoding rna neat1-dependent sfpq relocation from promoter region to paraspeckle mediates il8 expression upon immune stimuli hepatitis b virus x protein (hbx)-related long noncoding rna (lncrna) down-regulated expression by hbx (dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin long noncoding rna high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans elevation of highly up-regulated in liver cancer (hulc) by hepatitis b virus x protein promotes hepatoma cell proliferation via down-regulating p18 neat1 long noncoding rna and paraspeckle bodies modulate hiv-1 posttranscriptional expression a long noncoding rna mediates both activation and repression of immune response genes long noncoding rnas and enhancer rnas regulate the lipopolysaccharide-induced inflammatory response in human monocytes the human long noncoding rna lnc-il7r regulates the inflammatory response the long noncoding rna thril regulates tnfalpha expression through its interaction with hnrnpl a mammalian pseudogene lncrna at the interface of inflammation and antiinflammatory therapeutics chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals genome-wide identification of long noncoding rnas in cd8+ t cells role of micrornas and long-non-coding rnas in cd4(+) t-cell differentiation the stat3-binding long noncoding rna lnc-dc controls human dendritic cell differentiation the nest long ncrna controls microbial susceptibility and epigenetic activation of the interferon-gamma locus cutting edge: influence of tmevpg1, a long intergenic noncoding rna, on the expression of ifng by th1 cells an integrated encyclopedia of dna elements in the human genome the rise of regulatory rna human cancer long non-coding rna transcriptomes specific expression of long noncoding rnas in the mouse brain landscape of transcription in human cells molecular interplay of the noncoding rna anril and methylated histone h3 lysine 27 by polycomb cbx7 in transcriptional silencing of ink4a control of alternative splicing through sirna-mediated transcriptional gene silencing expression of a noncoding rna is elevated in alzheimer's disease and drives rapid feed-forward regulation of beta-secretase epigenetic silencing of tumour suppressor gene p15 by its antisense rna long noncoding rnas: fresh perspectives into the rna world thermodynamic instability of sirna duplex is a prerequisite for dependable prediction of sirna activities comparison of approaches for rational sirna design leading to a new efficient and transparent method production of infectious hepatitis c virus in tissue culture from a cloned viral genome adenovirus va rna-derived mirnas target cellular genes involved in cell growth, gene expression and dna repair induction and evasion of type i interferon responses by influenza viruses the vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter effect of adenovirus-mediated rna interference on endogenous micrornas in a mouse model of multidrug resistance protein 2 gene silencing combination of rna interference and u1 inhibition leads to increased inhibition of gene expression increased in vivo inhibition of gene expression by combining rna interference and u1 inhibition aav vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers tophat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation integrative genomics viewer (igv): high-performance genomics data visualization and exploration integrative genomics viewer the ucsc genome browser database: 2014 update gencode: the reference human genome annotation for the encode project data analysis tools for dna microarrays cpc: assess the proteincoding potential of transcripts using sequence features and support vector machine cpat: coding-potential assessment tool using an alignment-free logistic regression model lncipedia: a database for annotated human lncrna transcript sequences and structures the proteomics identifications (pride) database and associated tools: status in 2013 global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution identification of small orfs in vertebrates using ribosome footprinting and evolutionary conservation revisiting the protein-coding gene catalog of drosophila melanogaster using 12 fly genomes quantification of different human alpha interferon subtypes and pegylated interferon activities by measuring mxa promoter activation influenza a virus lacking the ns1 gene replicates in interferon-deficient systems inhibition of vesicular stomatitis virus infection in epithelial cells by alpha interferon-induced soluble secreted proteins an abundance of bidirectional promoters in the human genome complex loci in human and mouse genomes identification of a member of the interferon regulatory factor family that binds to the interferonstimulated response element and activates expression of interferon-induced genes comparison of microarrays and rna-seq for gene expression analyses of dose-response experiments large scale comparison of gene expression levels by microarrays and rnaseq using tcga data transcription factor binding and modified histones in human bidirectional promoters systematic analysis of headto-head gene organization: evolutionary conservation and potential biological relevance bidirectional gene organization: a common architectural feature of the human genome expression of the murine ranbp1 and htf9-c genes is regulated from a shared bidirectional promoter during cell cycle progression divergent transcription is associated with promoters of transcriptional regulators functional consequences of bidirectional promoters induced ncr-nas allosterically modify rna-binding proteins in cis to inhibit transcription antisense expression increases gene expression variability and locus interdependency tetherin inhibits hiv-1 release by directly tethering virions to cells bst-2/tetherin: structural biology, viral antagonism, and immunobiology of a potent host antiviral factor intrinsic cellular defenses against human immunodeficiency viruses bst-2 expression in human hepatocytes is inducible by all three types of interferons and restricts production of hepatitis c virus mechanism of hiv-1 virion entrapment by tetherin bst2/tetherin inhibits hepatitis c virus production in human hepatoma cells vpu binds directly to tetherin and displaces it from nascent virions influenza virus partially counteracts restriction imposed by tetherin/bst-2 tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu interferoninduced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms regulation of hepatic innate immunity by hepatitis c virus interferon type i gene expression in chronic hepatitis c interferon signaling and treatment outcome in chronic hepatitis c intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees hepatitis c virus controls interferon production through pkr activation hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation interferon-induced isg15 pathway: an ongoing virus-host battle the antiviral activities of isg15 positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg15 conjugation the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein 5a regulation of interferon-stimulated gene bst2 by a lncrna transcribed from a shared bidirectional promoter barriocanal contributed to the writing of the manuscript; victor segura was in charge of all the bioinformatic analyses; and puri fortes conceived the project and the required experiments, provided the budget, interpreted the data, and wrote the manuscript. we thank nerea razquin and celia prior for excellent technical assistance, paul miller for editorial work, estanis nistal, cristian smerdou, and rafael aldabe for influenza and vsv viruses, sfv, and hcv, respectively. we also thank pablo gastaminza for the huh7 cells sensitive to hcv infection used in all the experiments, esther larrea for ifnα2 and primers for isgs, elizabeth guruceaga for the initial studies on rnaseq data and ruben hernandez for helpful comments on the manuscript. we would like to thank patients for the generous donation of samples and virginia villar and the biobank of the university of navarra for their mediation. this work was supported by grants from ministerio de ciencia e innovacion bio2009/09295, and saf2012-40003, feder funding, funds from the "ute project cima" and by the project rnareg [csd2009-00080], funded by the ministry of science and innovation under the program consolider ingenio 2010. a manuscript from dr. valadkhan's laboratory has been accepted for publication that describes similar results (110) . the supplementary material for this article can be found online at http://www.frontiersin.org/journal/10.3389/fimmu.2014.00655/ abstract the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the review editor saba valadkhan declares that, despite having collaborated on a publication in the last 2 years with authors puri fortes, marina barriocanal, and elena carnero, the review process was handled objectively. key: cord-293790-7hyelm88 authors: guévin, carl; manna, david; bélanger, claudia; konan, kouacou v.; mak, paul; labonté, patrick title: autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection date: 2010-09-01 journal: virology doi: 10.1016/j.virol.2010.05.032 sha: doc_id: 293790 cord_uid: 7hyelm88 autophagy is an important cellular process by which atg5 initiates the formation of double membrane vesicles (dmvs). upon infection, dmvs have been shown to harbor the replicase complex of positive-strand rna viruses such as mhv, poliovirus, and equine arteritis virus. recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis c virus (hcv) infection. here, we identified atg5 as an interacting protein for the hcv ns5b. atg5/ns5b interaction was confirmed by co-ip and metabolic labeling studies. furthermore, atg5 protein colocalizes with ns4b, a constituent of the membranous web. importantly, immunofluorescence staining demonstrated a strong colocalization of atg5 and ns5b within perinuclear regions of infected cells at 2 days postinfection. however, colocalization was completely lacking at 5 dpi, suggesting that hcv utilizes atg5 as a proviral factor during the onset of viral infection. finally, inhibition of autophagy through atg5 silencing blocks hcv replication. there is compelling evidence that replication of all positivestranded rna viruses requires the formation of virus-induced membrane vesicles and that dmvs are the sites of genome replication for some positive-stranded rna viruses such as pv, mhv, eav, dengue virus, coronavirus, and coxsackievirus lee et al., 2008; pedersen et al., 1999; schlegel et al., 1996; suhy et al., 2000; wong et al., 2008) . in cells, dmvs derived from the endoplasmic reticulum membranes can be produced through the ubiquitous autophagy pathways (klionsky and emr, 2000) . autophagy, which has been well characterized in yeast, is an essential process by which bulk protein degradation and organelle turnover take place (kim and klionsky, 2000; mizushima et al., 1998) . in response to a limited supply of amino acids due to environmental depletion, the autophagy process will generate a new pool of amino acids required for cellular homeostasis. recently, the contribution of an autophagy protein, atg5, to viral replication has been demonstrated (prentice et al., 2004) . indeed, production of mhv particles is profoundly reduced (n99%) in atg5−/− knockout (ko) embryonic stem cells (prentice et al., 2004) . in this system, the expression of atg5 in atg5−/− cells restores virus production. the relationship between atg5 and mhv replication may come from the ability of atg5 to initiate the formation of dmvs, which have been observed in mhv-infected cells mizushima et al., 2001) . as seen for dengue virus-2 (dv2), when autophagy is blocked by atg5-ko mef cells, extracellular dv2 virus titers are reduced by 3fold compared to those from wild-type mef cells (lee et al., 2008) . altered vesicles, called the membranous web, have been observed in cells harboring hcv replicons gosert et al., 2003) . because hcv replicons replicate autonomously in huh-7, it has been proposed that the membranous web that contain the hcv replication complexes represents the genuine site of viral replication moradpour et al., 2003) . in hcv-infected cells, accumulation of lipid droplets, shown to be essential for hcv replication, has been observed in the proximity of the membranous web (miyanari et al., 2007) . given the involvement of autophagy at the site of replication of other positive-stranded rna viruses, are the autophagy proteins or structure involved in hcv replication? in that regard, tanida et al. (2009) recently showed that atg7 silencing decreased the levels of infectious hcvcc by about 40%, whereas intracellular hcv rna and protein levels remained unchanged. at the same time, another group demonstrated that autophagy proteins (atg4b and beclin-1) are required only for the initiation of incoming hcv rna translation/replication ). to identify novel cellular factors that may play an essential role in hcv rna replication, we have previously screened a human liver cdna library for proteins interacting with the hcv ns5b rnadependent rna polymerase (rdrp). here we report that atg5, a protein required for the formation of dmv in embryonic stem cell (mizushima et al., 2001) , specifically interacts with hcv ns5b. we propose that the ns5b/atg5 interaction may be required for the initial onset of hcv replication. using hcv ns5bδ21 protein as a bait in the yeast two-hybrid system, we identified positive clones from a human liver library. the clones that showed the strongest blue color (mel 1 gene activation) on sd (−leu, −trp, −his, −ade/+x α-gal) plates were chosen for further characterization. two of these positive clones matched the sequence of the human eukaryotic initiation factor 4aii (eif4aii), recently identified as an interacting protein for hcv ns5b (kyono et al., 2002) . two more clones corresponded to the embl/genbank/ ddbj accession number y711588a. this gene is highly homologous to the saccharomyces cerevisiae atg5 gene (hammond et al., 1998) . atg5/ns5b interaction was confirmed in the yeast two-hybrid system using the full-length human atg5 gene amplified from a human liver cdna library (clontech) (fig. 1) . as a control, a panel of proteins (rar-β, rar-α, hcv core, and nonstructural protein: ns3 prot , ns3 hel , ns4a, and ns4b) cloned in the gal4 dna binding domain was tested for interaction with atg5. from this panel, only hcv ns3 prot protein gave a weak signal, but the interaction was not characterized further (data not shown). to demonstrate a physical interaction between atg5 and ns5b proteins, we performed co-ip on radio-inert or metabolically labeled yeast cells coexpressing atg5 and ns5b. we used yeast cells in this study because cotransfection of huh-7 cells with c-myc-ns5b and hatagged atg5 resulted in a low level expression of atg5 protein. yeast extracts were immunoprecipitated with monoclonal antibody against either the c-myc or the ha tag, followed by separation on sds-page. as shown in fig. 2a , an immunoreactive band (lane 2) that corresponds to the size of the ns5b protein (68 kda; lane 4) was coimmunoprecipitated with atg5. this band was not present when the extracts were immunoprecipitated with monoclonal anti-5a antibody (lane 1) or without any antibody (protein a/g sepharose beads alone) (lane 3). the bands corresponding to the 50-and 25-kda molecular weight are the heavy and light chains, respectively, of the antibody. conversely, atg5 was coimmunoprecipitated with ns5b when the same extract was incubated with monoclonal anti-c-myc antibody. indeed, a distinct immunoreactive band (lane 5) that corresponds to the size of atg5 (lane 7) was detected. however, this band could not be immunoprecipitated by the monoclonal anti-5a antibody (lane 6). this proteinprotein interaction was further substantiated by the metabolic labeling experiments shown in fig. 2b in which atg5 was constitutively expressed and ns5b expression was under a copper-inducible promoter. as expected, when the labeled extracts were immunoprecipitated with monoclonal anti-ha, a prominent band corresponding to the size of atg5 was detected in yeast extracts under induced (lane 1) and noninduced (lane 2) conditions. however, the band corresponding to ns5b was only detected in lane 1 (induced condition). when the same extracts were immunoprecipitated with monoclonal anti-c-myc antibody, an intense band corresponding to the size of ns5b was apparent under the induced condition (lane 3). as expected, a band corresponding to the size of atg5 was also detected in the extracts under the induced condition (lane 3) but not under the noninduced condition (lane 4). taken together, these observations indicate that atg5 and ns5b interact. to map the interaction domains between ns5b and atg5, n-and c-terminally truncated mutants of both proteins were assessed by a yeast two-hybrid system. for that purpose, the truncated fragments were inserted into a pgbkt7 or pgadt7 plasmid. as illustrated in fig. 3 , all constructs containing the c-terminal end of the ns5b displayed interaction with atg5, while truncation of the ns5b cterminus completely abrogated such interaction. to confirm the data obtained by yeast two-hybrid screening, co-ip of the putative fig. 1 . the ns5bδ21 protein interacts with the full-length human atg5 in yeast twohybrid assay. ns5b and atg5 fused to the gal4 dna binding and activation domains, respectively, were double-transformed into ah109 cells. four independent colonies of recombinant ah109 cells were allowed to grow for a few days on −leu, −trp sd medium (left panel), after which they were replica-plated onto −leu, −trp, −his, −ade + x-αgal plates (right panel). a. soluble yeast extracts containing ns5bδ21 (n-terminal c-myc tag) and atg5 (n-terminal ha tag) were incubated with different monoclonal antibodies and the immunoprecipitates were pulled down using protein a/g beads. precipitated proteins were revealed by western blot using anti-c-myc (left panel) or anti-ha (right panel) monoclonal antibodies. note that ip of atg5 using anti-ha monoclonal antibody coprecipitated ns5bδ21 (lane 2). anti-hcv ns5a (lane 1) or no antibody (beads only, lane 3) did not precipitate ns5bδ21. soluble extract loaded on the gel was used as the size marker for ns5bδ21. ip of ns5bδ21 using antic-myc antibody precipitated atg5 (lane 5) but not anti-hcv ns5a antibody (lane 6). soluble extract loaded on the gel was used as the size marker for atg5 (lane 7). b. soluble [ 35 s]metlabeled protein extract was immunoprecipitated using anti-ha (lanes 1 and 2) or anti-c-myc (lanes 3 and 4). extracts were incubated in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of cupric sulfate, which induced ns5bδ21 expression. precipitated bands were analyzed by sds-page followed by autoradiography. interacting domains was performed. both techniques were able to identify the c-terminal end (aa 450-570) of ns5b as the interacting domain for atg5 (fig. 3) . the ns5b may have several amino acids involved in this interaction since the fragments 1-501, 500-535, and 536-570 independently interacted with atg5. thus, the binding domain of ns5b to atg5 corresponds to the back surface of the thumb domain which has been proposed to be a premium site for proteinprotein interaction due to the presence of a highly conserved patch of basic amino acids (bressanelli et al., 1999) . the interaction observed between atg5 and the hcv ns5b rdrp suggests that atg5 might be involved in hcv replication. in cells replicating hcv rna, ns5b has been localized to the membranous web gosert et al., 2003) . therefore, we analyzed the localization of atg5 and ns4b (a well-recognized marker of the membranous web) in genome-length c5b replicon cells (aligo et al., 2009; konan et al., 2003) . the results presented in fig. 4a indicate that fig. 3 . mapping of the ns5bδ21 and atg5 binding domains. results are indicated in the column on the right and were obtained by yeast two-hybrid assay (first two columns) or by co-ip (third column). deletion mutants of ns5b were assessed for interaction with full-length atg5. the binding area covered the n-terminal amino acids 450-570 as indicated by yeast two-hybrid assay and co-ip. note that none of the deletion mutants of ns5bδ21 self-activated in the yeast two-hybrid screen, as shown in the second column. nd indicates not done. ns4b indeed colocalized with the endogenous atg5 protein. it is known that atg5 and its conjugated form, atg5-apg12, are mostly cytoplasmic in mouse embryonic stem cells but that their association with the membrane increases upon starvation (mizushima et al., 2001) . thus, we looked at the subcellular membrane association of atg5 in replicon cells. as expected, both ns5b and atg5 proteins were excluded from the cytoplasmic fraction (fig. 4b) . indeed, both proteins appeared to reside in the microsomal/mitochondrial fractions, suggesting that atg5 is directed to membranes in replicon cells. note that the nuclear fractions contain both proteins most likely through contamination from the single-step purification protocol of the nuclear fraction. previously, we and others have unsuccessfully attempted to identify a hcv protein involved in the modulation of the autophagic response that occurs upon hcv infection (ait-goughoulte et al., 2008; ). it has recently been suggested that autophagy is required only early in infection . therefore, we evaluated the colocalization of atg5 and ns5b in infected huh7 cells at 2 days postinfection (2 dpi). the selection of 2 dpi, as our earliest time point, was based on the slow replication of jfh1 in huh7 cells for the first few days of infection zhong et al., 2005) . indeed, at 1 dpi, hcv core, ns5b, and ns5a were undetectable by immunofluorescence staining (data not shown). therefore, infected huh7 cells were transfected with pegfp-atg5 and analyzed at 2 or 5 dpi for the presence of ns5b and atg5 (fig. 5a) . to our surprise, a strong colocalization of gfp-atg5 and ns5b was evident in approximately 80% of infected cells at 2 dpi, but completely disappeared at 5 dpi. this result suggests that atg5-ns5b interaction occurs only during the initial onset of hcv replication and may explain why this interaction has not been detected previously. atg5 silencing is known to disrupt autophagy (matsushita et al., 2007; mizushima et al., 2001) . thus, we used atg5 sirna to evaluate the importance of atg5-ns5b interaction on hcv replication. as controls, a scramble sirna was used. the results indicate that silencing atg5 up to 2 dpi results in undetectable hcv core protein (fig. 5b) and in a marked reduction in intracellular viral replication as observed by qrt-pcr (fig. 5c ). this results suggest that atg5 is required for proper viral replication and this requirement is likely through atg5-ns5b interaction. recent reports suggest a role for autophagic proteins in hcv replication and/or secretion (ait-goughoulte et al., 2008; tanida et al., 2009 ). however, these reports are conflicting and no consensus has yet been reached. here we provide for the first time a link between a hcv protein, ns5b, and colocalization of atg5 and ns5b was observed at 2 dpi (magnified area in b) but not at 5 dpi (magnified area in c). b. silencing atg5 reduced viral replication in huh7 cells. huh7 cells were transfected with sirna targeting atg5 or with a scramble sirna as control. the cells were then infected for 2 days and analyzed for the presence of hcv core protein by western blot. as expected, scramble sirna had no effect on hcv replication, whereas atg5 greatly reduced hcv protein expression. c. quantification of intracellular hcv genome from samples in panel b. mock, mock-infected cells. the autophagy machinery. the specific interaction observed between atg5 and ns5b was through the thumb domain of the polymerase, a region with numerous basic amino acids that could favor proteinprotein interaction. using huh7 cells harboring hcv replicon, we showed that atg5 is associated with the membrane and colocalizes with the membranous web constituent, ns4b. we then used hcvcc to better define the subcellular distribution of atg5 and ns5b during the course of viral replication. interestingly, strong colocalization between the two proteins was only seen early in infection and was completely absent late in infection (fig. 5a) . this result may imply that the atg5-ns5b interaction is required for the onset of the viral replication. since the primary known function of atg5 is the formation of the crescent shape dmv, one could argue that hcv requires membrane import during the early stages of viral infection. indeed, atg5 is involved in other positive-strand rna virus replication, probably through the formation of dmv (khakpoor et al., 2009; lee et al., 2008; prentice et al., 2004) . however, we were unsuccessful in visualizing these crescent shaped vesicles or autophagosomes in hcv-infected cells. although unconjugated atg5 can be found on the crescent-shaped autophagosome precursor (mizushima et al., 2001) , maturation into the autophagosome requires the conjugate atg5-apg12 as well as a series of specific interactions with autophagy proteins (george et al., 2000; kim et al., 2001; mizushima et al., 2001) . because viruses such as mhv and perhaps hcv may utilize atg5 to initiate dmv formation but may not require further maturation of the dmv into autophagosomes, the function of apg12 in virus-induced dmvs remains to be determined. replicase proteins of positive-stranded rna viruses are localized in virus-induced membrane vesicles. in hcv replicon-harboring cells, a membranous structure that contains both viral proteins and rna, called the membranous web, has been identified . it has been shown that the formation of the membranous web can be induced by ns4b alone (konan et al., 2003) . another report has shown physical interaction between ns5b (or ns5a) and the snarelike protein, hvap-33 (tu et al., 1999) , leading to the localization of the hcv replicase complex on lipid rafts (aizaki et al., 2006; gao et al., 2004) . despite these findings, the role of the host factors in the formation and function of the hcv replication complex needs to be better defined. in that regard, we propose that autophagic proteins, and perhaps the resulting membranes, are indispensable during the onset of hcv replication. yeast strains for yeast two-hybrid screening were obtained from clontech (mountain view, ca, usa) as components of the pretransformed matchmaker cdna libraries and the matchmaker two-hybrid system 3. s. cerevisiae y187 (matα), which contained the pretransformed human cdna library (complexity n2-4 × 10 6 independent clones) cloned into the gal4 activation domain vector (pact2) was allowed to mate with s. cerevisiae ah109 (mata), which had been transformed with a gal4 dna-binding domain vector (pgbkt7) containing hcv ns5b as a bait. to construct the bait, the hcv ns5b gene lacking the region encoding the c-terminal 21 amino acid residues was amplified by pcr using the hcv-bk (genotype 1b) genomic cdna as a template and ns5b-h1 and ns5b-r1700 as primers (table s1 ). the resulting ns5bδ21 gene containing unique ecori and bamhi sites at the n-and c-termini, respectively, was cloned into the pgbkt7 expression plasmid to generate an in-frame fusion protein with a gal4 dna binding domain. the resulting plasmid, pgbkt7-5bδ21 was sequenced and subsequently used to transform the ah109 yeast strain. following mating, the diploid yeast strain (y187-ah109-ns5b) was selected on sd medium in the absence of leucine, tryptophan, and histidine (−lth). eight hundred potential positive yeast clones were obtained from two million screenable clones and were replica-plated onto x-α gal indicator plates in the absence of leucine, tryptophan, histidine, and adenine. three hundred blue colonies (positive for x-α gal as a result of mel1 gene activation) were selected by prototrophy for histidine and adenine. these clones were retested for positive interaction, and pact2/cdna plasmids were isolated from 20 strong positive clones as reflected by the intensity of the blue color formed on x-α gal indicator plates. these plasmids were retransformed into yeast strains carrying the bait construct, pgbkt7-5bδ21 or pgbkt7, to confirm true interactions. clones that gave a positive signal when cotransformed with pgbkt7-5bδ21 were chosen for dna sequencing. the dna sequences of the positive pact2/cdna clones were translated and compared with a nonredundant sequence database using the blast program through the national center for biotechnology information network service. clustal_x program was used to analyze statistically significant matches. to obtain the full-length coding region of the hatg5 gene (825 bp), the primers atg5-h1 and atg5-r800 (table s1) were prepared for pcr amplification using a human liver cdna library (clontech) as a template. pcr products with unique ndei and bamhi at the n-and c-termini, respectively, were cloned into the pgadt7 vector. the sequence of the resulting plasmid was confirmed by dna sequencing. the integrity of the hatg5 and ns5bδ21 genes inserted into the yeast plasmids (pgbkt7 and pgadt7) was confirmed in vitro by expressing the two proteins using the tnt® t7 coupled reticulocite lysate system (promega, madison, wi, usa) as described by the manufacturer. final confirmation of hapg5/ns5bδ21 interactions by yeast two-hybrid experiments in ah109 cells was carried out as described previously. for co-ip, n-terminal tag ha-hapg5 and c-myc-ns5bδ21 proteins were expressed in the bj2168 yeast strain. briefly, the hapg5 and ns5bδ21 genes were amplified using the tag-ha/apg5-r800-trp and tag-myc/ns5b-r1700-leu primer sets, respectively (table s1 ). the pcr products were then cloned into the yep c (ns5b) and yeptdh (hapg5) plasmids using unique restriction sites (table s1 ). the resulting expression plasmids, yep c -ns5bδ21 (copper-inducible) and yeptdh-hapg5, were used to transform a protease-deficient yeast strain (bj2168). the double transformant yeast strain, yep c -ns5bδ21/yeptdh-hapg5 was grown in sd medium depleted of tryptophan and leucine (−lt). for the induction of c-myc-ns5bδ21, cupric sulfate (1 μm) was added to the −lt medium and incubated overnight at 30°c. yeast extracts containing soluble hapg5 and ns5bδ21 proteins were prepared according to the protocol of mizushima et al. (2001) . briefly, yeast cells were washed and resuspended in ice-cold tes buffer (50 mm tris, 5 mm edta, and 150 mm nacl, ph 7.5). yeast cell walls were disrupted with acid-washed glass beads by vortexing vigorously for 10 minutes. after centrifugation at 3000 × g for 5 minutes, the supernatant (cytoplasmic and microsomal fractions) was mixed with 0.1 volume of 10% np40 and incubated for 15 minutes before centrifugation at 10,000 × g for 15 minutes. aliquots of the resulting supernatant (500-600 μg of protein) were incubated with or without 1 μl of monoclonal anti-myc antibody (9e10: santa cruz, ca, usa) or anti-ha antibody (f6: santa cruz) for 2 hours. a protein a/g sepharose bead mixture (pierce, rockford, il, usa) (10 μl) was added, and samples were incubated for an additional 2 hours. the sepharose beads were washed three times with tes buffer, and the bound proteins were eluted with 30 μl of laemmli buffer. samples (20 μl) were analyzed by sds-page and immunoblotting. proteins were detected by monoclonal anti-c-myc (9e10) or anti-ha (f2) and visualized by enhanced chemiluminescence (ecl; amersham pharmacia biotech; baie d'urfé, qc, canada). alternatively, co-ip was performed using metabolically labeled cell extract. in brief, bj2168 yeast cells carrying the yep c -ns5bδ21 and yeptdh-hapg5 plasmids were grown in sd (−lt) medium overnight at 30°c. when the cells reached mid-log phase, they were washed and incubated for 1 hour in sd (−lt) medium depleted in methionine. subsequently, 10 μm of cupric sulfate and 35 s-labeled methionine (50 μci) were added to the culture, and incubated for 3 hours. after washing the cells twice with sd medium, yeast extracts were prepared for co-ip study as described previously. immunoprecipitated proteins were separated on sds-page followed by autoradiography. the negative control included yeast extracts prepared from the yeast strain without cupric sulfate induction (i.e., expressing only ha-hapg5) and subjected to the same analysis. deletion mutants of ns5bδ21 were generated by pcr using the primers indicated in table s1 . pcr fragments were inserted in pgbkt7 or pgadt7 and the interactions were analyzed by yeast two-hybrid assay. to identify the interaction domains, we performed co-ip of the deletion mutants expressed in the bj2168 yeast strain using the yep c and yeptdh plasmids. transcribed full-length jfh1 rna (megascript, ambion, streetville, ontario, canada), and viral stocks were produced by infection of huh7 cells at a multiplicity of infection (moi) of 0.01, as described previously (guevin et al., 2009) . for subcellular fractionation, the hapg5 gene was cloned into the pegfp-c1 plasmid (clontech). the resulting pegfp-hapg5 plasmid was then transfected into clone-a and naïve huh7 cells using lipofectamine as suggested by the manufacturer (invitrogen; burlington, ontario, canada). the engineering and characterization of the clone-a cells, which constitutively expressed the hcv nonstructural proteins (ns3, 4a, 4b, 5a, and 5b), have been reported elsewhere (howe et al., 2006) . at 48 hours after transfection, cells were trypsinized and washed twice with pbs. after washing, 2 × 10 7 cells were homogenized in a hypotonic buffer containing 10 mm tris-hcl, ph 7.5, and 2 mm mgcl 2 , followed by centrifugation at 1000 × g for 5 min to yield the nuclear fraction. the supernatant was then centrifuged at 14,000 × g for 40 min to pellet the microsomal/ mitochondrial (mit/mic) fraction. the nuclear and the mit/mic pellets were resuspended in the same volume as the final supernatant using the hypotonic buffer, and 20 μl of each extract was resolved on sds-page and immunoblotting using either a rabbit polyclonal antiserum directed against the hcv ns5bδ21 or a mouse monoclonal anti-gfp (gfp-20; sigma). proteins were visualized by enhanced chemiluminescence (ecl; amersham pharmacia biotech). for indirect immunofluorescence, cells were transfected with the pegfp-hatg5 and infected with hcvcc jfh1 at a moi of 0.01. at 24 hours after transfection, the cells were trypsinized and grown on glass coverslips for another 24 hours. the coverslips were then fixed in pbs containing 4% formaldehyde for 10 min, washed three times in pbs, and incubated for 1 h at 4°c in blocking buffer (pbs, 3% bovine serum albumin, 0.1% triton x-100). after three washes in pbs, the coverslips were incubated with a rabbit polyclonal antibody directed against the hcv ns5b protein (generously provided by dr takaji wakita, national institute of infectious diseases, tokyo, japan) (dilution 1:200) in blocking buffer for 1 hour at room temperature (rt). the coverslips were then washed three times in pbs and incubated for 1 hour at rt with alexa fluor 488 or 568-conjugated secondary antibody goat anti-mouse or anti-rabbit igg (jackson immunoresearch laboratories inc., west grove, pa, usa) (dilution 1:500). coverslips were washed four times in pbs and mounted on glass slides with prolong™ antifade (molecular probes), and cells were examined with a laser scanning confocal biorad radiance 2000 microscope. sirna duplexes targeting human were purchased from ambion (sirna atg5 no. am16708a and scramble sirna no. 4611g). sirna duplexes (150 pmol) were transfected into 1 × 10 5 huh7 cells using the rnaimax transfection reagent (invitrogen) and infected 6 hours later with hcv jfh-1. protein knockdown was usually analyzed 48 hours after transfection. cells were washed three times in phosphate-buffered saline and lysed in ripa buffer (50 mm tris-hcl, ph 8.0, 1% (vol./vol.) nonidet p40, 0.5% sodium deoxycholate, 150 mm nacl and 0.1% (vol./vol.) sds) with a complete protease inhibitor mixture (roche applied science). after sds-page electrophoresis, protein samples were transferred to an immuno-blot pvdf membrane for protein blotting (bio-rad) for 45 min. nonspecific binding sites were blocked for 1 hour in pbs-5% skimmed milk, and the membrane was stained for 1 hour with the primary antibodies. the antibodies used were hcv polyclonal anti-core (obtained from dr denis leclerc, laval university, canada) (dilution 1:1000) and anti-atg5 (fl-275) polyclonal antibody (santa cruz biotechnology, ca, usa) (dilution 1:1000). after incubating with the primary antibody, the membranes were washed four times in pbs-0.1% tween-20. bound antibodies were detected by incubation for 45 min with a goat anti-rabbit hrp antibody (jackson immunoresearch) (dilution 1:10,000). the signals were developed with supersignal™ west pico chemiluminescent substrate (pierce). total cellular rna was prepared from sirna-transfected cells by using the rneasy mini kit (qiagen). the cdna were prepared from 250 ng of total cellular rna. briefly, rnas were incubated 3 min at 70°c then cooled on ice for 2 min before the addition of 4 μl of rt-buffer 5x (invitrogen), 2 μl of dtt (0.1 m), 1 μl of random primer p (dn6) (100 ng/μl), 1 μl of dntp (20 mm), 20 u of rnasin, and 100 u of mmlv reverse transcriptase. samples were incubated for 10 min at 25°c and 1 h at 37°c. to inactivate the mmlv, samples were incubated 15 min at 70°c, and cdnas were diluted to a final volume of 200 μl with rnase-free water. primers used for amplification were 5′utr-r: 5′-gagtgggttta tccaagaaag-3′ and 5′utr-f: 5′-tctgcggaaccggtgagt-3′. the mixture consists of 2.5 μl of cdna in a final volume of 25 μl of the reaction mixture containing 8.6 μl h 2 o, 0.5 μl of probe fam-utr (12.5 μm) ccggaattgccgggaagactg, and 0.25 μl (90 μm) of each hcv primers. for the internal control, the 18s ribosomal rna kit was used as suggested by the manufacturer (applied biosystem). the mixture was completed with 12.5 μl of the taqman universal master mix 2x (applied biosystem), and the amplification was performed as suggested by the manufacturer in a rotor-gene rg-3000 (corbet research). hepatitis c virus genotype 1a growth and induction of autophagy polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis formation and function of hepatitis c virus replication complexes require residues in the carboxy-terminal domain of ns4b protein crystal structure of the rna-dependent rna polymerase of hepatitis c virus autophagy proteins promote hepatitis c virus replication the autophagy machinery is required to initiate hepatitis c virus replication expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons novel hcv replication mouse model using human hepatocellular carcinoma xenografts homology between a human apoptosis specific protein and the product of apg5, a gene involved in autophagy in yeast molecular mechanism of a thumb domain hepatitis c virus nonnucleoside rna-dependent rna polymerase inhibitor a role for autophagolysosomes in dengue virus 3 production in hepg2 cells autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells membrane recruitment of aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires aut1p, aut2p, and the autophagy conjugation complex cell biology-autophagy as a regulated pathway of cellular degradation nonstructural protein precursor ns4a/b from hepatitis c virus alters function and ultrastructure of host secretory apparatus human eukaryotic initiation factor 4aii associates with hepatitis c virus ns5b protein in vitro autophagic machinery activated by dengue virus enhances virus replication structure of atg5.atg16, a complex essential for autophagy the lipid droplet is an important organelle for hepatitis c virus production a protein conjugation system essential for autophagy dissection of autophagosome formation using apg5-deficient mouse embryonic stem cells membrane association of hepatitis c virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex coronavirus replication complex formation utilizes components of cellular autophagy cellular origin and ultrastructure of membranes induced during poliovirus infection remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles hepatitis c virus rna polymerase and ns5a complex with a snare-like protein production of infectious hepatitis c virus in tissue culture from a cloned viral genome autophagosome supports coxsackievirus b3 replication in host cells robust hepatitis c virus infection in vitro we are grateful to takaji wakita and denis leclerc for reagents and nathalie fournier for technical assistance. this work was supported by nserc of canada (grant no. 312225-05). c. g. and c. b. were supported by a fellowship from the armand-frappier foundation (canada). supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2010.05.032. key: cord-285868-fz5utxss authors: jheng, jia-rong; ho, jin-yuan; horng, jim-tong title: er stress, autophagy, and rna viruses date: 2014-08-05 journal: front microbiol doi: 10.3389/fmicb.2014.00388 sha: doc_id: 285868 cord_uid: fz5utxss endoplasmic reticulum (er) stress is a general term for representing the pathway by which various stimuli affect er functions. er stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (upr), which compromises the stimulus and then determines whether the cell survives or dies. in recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell's response to various stressors. autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. however, the link between the upr and autophagy may be more complicated. these two systems may act dependently, or the induction of one system may interfere with the other. experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host's defense to their advantage; thus, this topic is a critical area in antiviral research. in this review, we summarize the current knowledge about how rna viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, japanese encephalitis virus, hepatitis c virus, and dengue virus, regulate these processes. we also discuss recent discoveries and how these will produce novel strategies for antiviral treatment. the endoplasmic reticulum (er) is a eukaryotic organelle in which an array of cell functions takes place. these include the transportation of cellular materials, provision of increased surface area for cellular reactions, and the production of proteins, steroids, and lipids. the er may be overloaded with molecular chaperones, folding enzymes, and massive protein products during normal processes, such as in the differentiation of b lymphocytes into antibody-secreting plasma cells (shaffer et al., 2004; ma et al., 2010) or in highly specialized cells for secretion (harding and ron, 2002) . in addition, dysfunction of the er, known as er stress, results from pathogenic stress signals, such as hypoxia (koumenis, 2006) , er-ca 2+ depletion, viral infections, or agents that affect ca 2+ balance (i.e., thapsigargin), protein glycosylation (i.e., tunicamycin), and er-golgi vesicular transport (i.e., brefeldin a), which lead to accumulation of misfolded and unfolded proteins (kaufman, 1999) . to reduce the adverse effects of accumulating misfolded or unfolded proteins, the cell operates an adaptive response known as the unfolded protein response (upr) to reduce the load of newly synthesized proteins within the er and eliminate inappropriately folded proteins through upregulation of er chaperone expression. in addition, proteins that fail to correctly fold are then deployed to the distal secretory pathway from the er by the er-associated protein degradation (erad) pathway of the upr (hampton, 2000; yoshida et al., 2003) . there are two erad models for protein degradation: ubiquitin-proteasome erad, designated as erad (i), and autophagy-lysosome erad, designated as erad (ii) (fujita et al., 2007; korolchuk et al., 2010) . both models depend on retrotranslocation of erad substrates from the er back to the cytoplasm with the help of the cdc48p-p97 complex. most soluble misfolded proteins are cleared through the ubiquitinproteasome system, which involves action of a cascade of three canonical ubiquitin enzymes: e1 ubiquitin-activating enzyme initiates the reaction by using atp to covalently activate and then conjugate the ubiquitin to an e2 ubiquitin-conjugating enzyme. ubiquitin is then transferred from the ubiquitin-charged e2 to the lysine residue of a specific target or a growing ubiquitin chain by e3 ubiquitin ligase, which results in a multiubiquitin chaintagged substrate. proteins that are ubiquitinated with k48-linked chains are specifically recognized by the 26s proteasome and subjected to degradation (hershko et al., 1983) . in contrast, erad (ii) degrades both soluble and insoluble misfolded protein aggregates in autolysosome. autophagy receptors and adaptors, called p62/sqstm1, nbr1, hdac6, and alfy, bind to proteins with k63-specific monoubiquitination or polyubiquitin chains and then guide them to the concave side of developing autophagosomes (behrends and fulda, 2012) . notably, p62 also recognizes k48 polyubiquitin-tagged proteins for autophagic clearance upon proteasome dysfunction. in addition to the protective role of upr, prolonged and/or excess er stress typically activates caspase-12, an er-resident caspase, leading to upr-mediated cell death (szegezdi et al., 2006) . basal autophagy plays a key role in maintaining cellular homeostasis through eliminating unwanted proteins and damaged organelles by cellular self-digestion in the lysosome to fulfill the demand for the building blocks required for cell survival (levine and klionsky, 2004; shintani and klionsky, 2004) . recently, the study of autophagy regulation has grown in different research areas, including regulation of cancer development and progression (mahoney et al., 2013a) , lipid metabolism (singh et al., 2009) , degenerative diseases (wang et al., 2006) , and the control of viral pathogenesis . the first step of autophagy relies on the formation of an isolation membrane at the so-called preautophagosomal site (pas) where a system of evolutionarily conserved proteins (atg proteins) comes together. recent reports have revealed that the er serves as a subcellular platform for autophagy initiation (axe et al., 2008) . the elongation of the initial autophagic membrane requires continued processing by two ubiquitin-like protein-conjugation systems, the atg12 and lc3 systems, which modify the autophagy proteins, atg5 and atg8/lc3, respectively (geng and klionsky, 2008) . the autophagosome then fuses with endosomal and/or lysosomal vesicles to create an autolysosome, where digestion of intracellular components occurs (eskelinen, 2005) . in addition, autophagy can be induced by various physiological and pathological conditions such as nutrient deprivation, oxidative stress, and pathogen infections. the live-or-dead signal is modulated by upr and autophagy and several lines of evidence suggest there is communication between these two pathways (bernales et al., 2006; ogata et al., 2006; yorimitsu et al., 2006; salazar et al., 2009) ; thus, it is believed that these two pathways could be a therapeutic target in certain circumstances (figure 1) . herein, we review recent findings, focusing on the regulation of the upr and autophagy involved in rna virus infection as a new antiviral strategy. viral virulence is determined by successful entrance, replication in the host cell, and release of mature virion. during the life cycle, er stress may arise from the exploitation of the er membrane, accumulation of misfolded proteins, imbalance of calcium concentration by viroporin, and the sabotage or depletion of the er membrane during virion release. details of viral effects are given as follows. many positive-strand rna viruses cause the rearrangement of host intracellular membrane compartments that house replication complexes. er, trans-golgi, or lysosomes are the likely origin of virally induced membranes (miller and krijnse-locker, 2008; korolchuk et al., 2010) . upon poliovirus (pv) and coxsackievirus b3 (cvb3) infection, clusters of vesicles have been considered to derive from er, although other cellular compartment marker proteins also colocalized with viral nonstructural proteins (schlegel et al., 1996; van kuppeveld et al., 1997) . consistent with these findings, our previous study indicates that enterovirus 71 (ev71) nonstructural 2c protein, which participates in viral replication, is associated with the er membrane through direct interaction with er membrane protein reticulon 3 (rtn3), which is required and sufficient for immediate early virus replication and translation (tang et al., 2007) . in the rtn3 sirna knockdown cells, synthesis of the 2c protein was ablated. however, in the rtn3 rescue cell line 2a3, figure 1 | diagram of the upr arms and their connection to autophagy. alteration of er functions results from stress signals by rna virus infection, by the exploitation of er membrane for viral replication, rapid accumulation of viral proteins, imbalance of calcium concentration by viroporin, and the sabotage or depletion of er membrane for viral release. this leads to the accumulation of misfolded and unfolded proteins, which triggers er stress. to alleviate this adverse effect, the cells operate an adaptive upr to reduce the load of the newly synthesized proteins in the er by activating the perk-eif2α branch and eliminating inappropriate protein accumulation by upregulating er chaperone proteins through ire1 and atf6 branches. in addition, the incurable misfolded proteins undergo retrotranslocation from the er into cytosol for degradation by an erad mechanism. er stress can contribute to autophagy via activation of jnk, xbp1, chop, and atf4. red dash arrows indicate the final outcome of the activated pathways, such as apoptosis and autophagy, caused by viral infection. red solid arrows indicate the upr pathways. the synthesis of viral protein and rna was restored. moreover, the interactions between rtn3 and two ev71 2c homologs of pv and cva16 have been confirmed (tang et al., 2007) . immunofluorescence studies reveal that replication of flaviviruses dengue virus (denv) and hepatitis c virus (hcv) may take place on perinuclear er membranes (el-hage and luo, 2003) . denv2 nonstructural protein 2 (ns2a) is a 22-kda hydrophobic protein containing five integral transmembrane segments that span the er membrane. functional analysis reveals that ns2a involves both denv rna synthesis and virion assembly/maturation (xie et al., 2013) . furthermore, denv infection induces rockdependent vimentin rearrangement and subsequent er redistribution (lei et al., 2013) . in addition, the hcv er integral membrane protein, ns4b, is responsible for rearranging the er membrane and inducing the formation of new er-derived membrane structures, and this is possibly negatively regulated by rtn3-ns4b interaction (lundin et al., 2003; wu et al., 2014) . the n-glycosylation pathway in the er modifies a mass of proteins at the asparagine residue of the consensus sequence asn-x-ser/thr, where x is any amino acid except pro (kornfeld and kornfeld, 1985; gavel and von heijne, 1990) . the modification influences protein folding and attributes various functional properties to the protein. thus, interference with host protein glycosylation by viral proteins competing for the modification process may cause er stress. viruses, including influenza a virus (iav), hepatitis virus, and japanese encephalitis virus (jev), use this host cell process to enhance viral pathogenesis through facilitating folding and trafficking, affecting receptor interaction, and modulating host immune responses (tatu et al., 1995; dubuisson and rice, 1996; zai et al., 2013) . hemagglutinin (ha) of iav is a type i transmembrane glycoprotein that determines viral antigenicity. throughout the glycosylation process, ha rapidly associates with calnexin in a monoglucosylated form. once folded, the ha monomers dissociate from calnexin and assemble into trimeric structures in the er or in the intermediate compartment (tatu et al., 1995) . hcv envelope glycoproteins e1 and e2 have been shown to cooperate for the formation of a functional noncovalent heterodimer (dubuisson et al., 1994; dubuisson and rice, 1996) . based on studies of hcv pseudoparticles, coexpression of both envelope glycoproteins has been shown to be necessary to produce infectious pseudoparticles (bartosch et al., 2003) . glycosylation also occurs in jev and wnv proteins, namely the precursor of membrane protein (prm), the envelope protein (e), and the nonstructural protein ns1, which affects the efficiency of virus release and infection (hanna et al., 2005; zai et al., 2013) . typically, viroporins are composed by integral membrane proteins to form a hydrophilic pore, which targets different cellular compartments and ions, thus affecting various viral functions (nieva et al., 2012) . for example, iav m2 reduces the acidity of vesicular compartments to trigger virus uncoating. it is also required for viral assembly and release. in the case of er-targeting viroporins, rotavirus-encoded nsp4 modifies the calcium homeostasis by enhancing the calcium permeability of the er membrane. this may be associated with virus-induced cell death and subsequent release of nsp4, which in turn causes activation of the phospholipase c-ip3 cascade in neighboring noninfected cells and is responsible for viral pathogenesis (tian et al., 1995 (tian et al., , 1996 dong et al., 1997) . on the other hand, 2b proteins of picornaviruses also participate in the remodeling of membrane structures and the formation of replication complexes (de jong et al., 2008) . among them, cbv3 2b, pv1, and rhinovirus 2b are present at the membranes of the er and golgi complex and are responsible for the release of ca 2+ and h + from these organelles. rotavirus studies propose that the double-layered particle (dlp)-vp4-nsp4 complex breaches the er membrane and penetrates into the er. the viral capsid protein, vp7, re-envelopes the immature particle (dlp) after removal of the er membrane and nsp4, and forms the infectious triple-layered particle (tian et al., 1996; trask et al., 2012) . the induction of individual branches or part of the upr by viruses was reported previously. viruses have also evolved different means to modulate the arms of the upr, which consequently expanded both the temporal and spatial superiority for virus replication or completion of the life cycle. it has been reported that viruses regulate the host translational machinery to promote viral protein synthesis by inhibiting the synthesis of proteins involved in host immune responses. in enteroviruses, 2a and 3c proteases target translation factors such as eif4gi and poly(a)-binding protein (pabp) to impede host translation (lloyd, 2006) . moreover, modulation of the integrated stress response (isr), which is determined by phosphorylation of eif2α to attenuate cellular translation, is another strategy for promoting virulence (figure 2 ) (sonenberg and hinnebusch, 2009) . four eif2α kinases have been identified: heme-regulated inhibitor (hri), which is a response to heme deficiency (chen, 2007) ; double-stranded rna-dependent protein kinase (pkr), which is induced by interferon (ifn) and activated by double-stranded rna (dsrna) during viral infection (meurs et al., 1990) ; general control nonderepressible-2 (gcn2), which is activated by serum and amino acid deprivation (harding et al., 2000) ; and finally, pkr-like er kinase (perk or pek), which is activated by unfolded proteins in the er (ron, 2002) . some researchers consider that eif2α phosphorylation plays a role in hampering viral protein synthesis. for example, upon vsv infection, the induction of activated perk only correlates with eif2α phosphorylation at the later stage of infection. in mef cells carrying a phosphorylation-insensitive eif2α s51a variant, viral protein synthesis increased compared with a wild-type control, indicating that eif2 phosphorylation is inhibitory to viral protein synthesis. as demonstrated by matrix (m) protein mutant virus (rm51rm), a viral protein (m protein) is involved in counteracting the antiviral response of the phosphorylation of eif2α (connor and lyles, 2005) . like vsv infection, chikungunya figure 2 | eif2 pathway under viral infection. the m protein of vsv, the e2 and ns5a proteins of hcv, and ns2a of jev counteract the phosphorylation of eif2α for viral replication. blue solid arrows indicate the direct target of the virus or viral proteins. iav also targets eif2α by inducing p58ipk, a cellular inhibitor of perk and pkr. ibv upregulates eif2α-atf4-chop-mediated apoptosis to benefit viral replication. august 2014 | volume 5 | article 388 | 3 virus (chikv) induces perk activation but delays eif2α phosphorylation. the expression of chikv nsp4, which is the rna-dependent-rna polymerase, contributed to suppression of eif2α phosphorylation, thus ensuring translation of viral proteins (rathore et al., 2013) . furthermore, viruses containing type i or type ii internal ribosomal entry sites (iress), such as pv, foot-and-mouth disease virus, mengovirus and emcv, require many canonical translation initiation factors for initial replication (beales et al., 2003; sarnow, 2003) . it is reported that pv switches translation mode from an eif2-dependent to an eif2independent one during the course of infection to ensure efficient proliferation. furthermore, studies have shown that the c terminal of the eif5b fragment, cleavage by 3c proteases, and proteolytic activity of 2a pro can stimulate virus ires translation of enteroviruses (de breyne et al., 2008; redondo et al., 2011) . interestingly, it is reported that phosphorylation of eif2α is required for activation of ires during cell differentiation (gerlitz et al., 2002) . thus, whether the phosphorylation level of eif2α positively correlates with ires-dependent viral mrna translational efficiency remains to be determined. some viruses regulate the eif2α pathway by interfering with the activation of eif2α kinases. hcv ns5a protein, containing an ifn sensitivitydetermining region (isdr), interferes with pkr activity by binding to a pkr dimerization domain (pkr residues 244-296) (gale et al., 1998) , while hcv e2 protein binds to perk and inhibits downstream eif2α phosphorylation by acting as a pseudosubstrate (pavio et al., 2003) . interestingly, it is reported that ns5a stimulates eif2α phosphorylation in the absence of pkr, implying that ns5a may activate other eif-2α kinases to regulate eif2α phosphorylation (tardif et al., 2002) . overexpression of hcv ns2 induces eif2α phosphorylation (von dem bussche et al., 2010) . taken together, these studies indicate that hcv proteins modulate eif2α pathway in a complex way, and the effect of regulation on virus replication cannot be established unequivocally. the n-terminal region of ns2a of jev contains a sequence that is highly similar to hcv ns5a isdr and also inhibits pkr-induced eif2α phosphorylation (tu et al., 2012) . denv2 infection triggers and then suppresses perk-mediated eif2α phosphorylation by elevating the expression of growth arrest and dna damage-inducible protein-34 (gadd34), which acts together with phosphatase 1 (pp1) to dephosphorylate eif2α-p (pena and harris, 2011) . influenza virus nonstructural protein ns1 interferes with dsrna binding to pkr, and the infection also induces and activates p58ipk, a cellular inhibitor of pkr and perk. both strategies deployed by ns1 and p58ipk prevent pkr dimerization and autophosphorylation, which limits eif2α phosphorylation (lee et al., 1992; lu et al., 1995; yan et al., 2002) . in some circumstances, such as when the host immune system specifically recognizes foreign viruses and kills them with cytotoxic t lymphocytes, or when cell death is directly induced in virus infected cells to prevent completion of the replication cycle, apoptotic cell death is considered to be a host strategy for fighting against viral infections. atf4 is a transcriptional activator of the isr, which is involved in the expression of isr target genes such as c/ebp homologous protein (chop) and gadd34 (ma and hendershot, 2003) . chop was originally identified as a transcriptional factor eliciting er stress-induced apoptosis. in cells subjected to west nile virus (wnv) infection, eif2α phosphorylation and chop-mediated apoptosis were induced. both viral protein expression level and virus titer are increased in chop-deficient cells (medigeshi et al., 2007) . on the other hand, a virus may induce apoptosis to facilitate replication or the spread of viral progeny. it is reported that coronavirus infectious bronchitis virus (ibv) upregulates eif2α-atf4-chop signaling in infected cells and that it relies on perk or pkr activation. knockdown of chop reduces ibv-induced apoptosis through activation of the extracellular signal-related kinase (erk). viral protein expression level is moderately suppressed in chop-knockdown cells, which suggests that upregulation of chop-mediated apoptosis during ibv infection probably promotes virus replication (liao et al., 2013) . in addition to regulation of cell death, it is reported that hcv induces the expression of chop at mrna and protein levels and is correlated with autophagy induction; knockdown of chop not only increases hcv pamp-mediated innate immune activation, but also elevates its inhibitory effect on virus replication (ke and chen, 2011) . however, upstream chop induction is a matter of debate. overexpression of hcv e1 and/or e2 induces the expression of chop in a perk-dependent manner (chan and egan, 2005) ; while upon hcv infection, chop protein is upregulated by perk, activating transcription factor (atf6), and inositol-requiring transmembrane kinase/endonuclease 1 (ire1) collectively. atf6 is a type 2 transmembrane protein of 670 amino acids and is constitutively expressed as a 90-kda protein (p90atf6). its c-terminal region is located in the er, whereas the n-terminal region is located on the cytosolic side (figure 3) . upon er stress, atf6 is cleaved to an n-terminal 50-kda protein (p50atf6) sequentially by the golgi site-1 and site-2 proteases (s1p and s2p) figure 3 | atf6 pathway under viral infection. many rna viruses activate the upr pathway by cleaving atf6 to release the p50 fragment. the n-terminal p50 with transcription activity enters the nucleus to activate the expression of er stress and erad genes, such as grp78/bip, chop, xbp1, or edem. however, the p50 fragment was not detected in the ev71 infection. august 2014 | volume 5 | article 388 | 4 (ye et al., 2000) . nuclear translocation of p50atf6, as a transcription factor, activates expression of er stress and erad genes including er chaperones, chop (aka gadd153), edem1, and x-box-binding protein 1 (xbp1) by targeting the cis-acting er stress response element (erse) (ccaat-n9-ccacg) and upr element (upre) (gatgacgtg(t/g) nnn(a/t)t), although atf6 has a much higher affinity for erse (yoshida et al., 1998) . in addition to directly regulating gene expression, atf6 also modulates the innate immune response. under subtilase cytotoxin (subab) treatment, cleavage and degradation of grp78/bip leads to activation of the akt-nf-κb pathway through atf6 activation (yamazaki et al., 2009) . based on its pivotal role of connecting the arms of the upr and converging the upr and immune response, many viruses preferentially regulate atf6 pathways to benefit replication. in wnv strain kunjin (wnv kun )-infected cells, expression of atf6-target genes increases, but viral production decreases in atf6 knockout mef cells. moreover, in atf6 knockout mef cells, phosphorylation of eif2α, downstream chop activity, and jak-stat1 phosphorylation induced by ifnα are upregulated upon infection, which implies that virusinduced atf6 activation is a prosurvival mechanism required for replication and inhibition of the antiviral signaling pathway (ambrose and mackenzie, 2013) . however, it is still unclear whether wnv kun ns4a and ns4b, potent inducers of the upr, inhibit ifnα-induced jak-stat signaling in an atf6-dependent manner (ambrose and mackenzie, 2011) . other flavivirus infections, including hcv, jev, and denv2, also induce cleavage of atf6, nuclear translocalization of atf6 and increases in chaperone proteins expression. in hcv replication, silencing of atf6 reduces hcv intracellular mrna levels (ke and chen, 2011) . however, in jev-infected cells, knockdown of the atf6-targeted gene, grp78, by sirna did not affect jev viral rna replication, although it did impair virus assembly or release. in sucrose gradient, mature jev viruses that do not cofractionate with gpr78 displayed a significant decrease in viral infectivity, indicating that jev acts with gpr78 to promote its infectivity (wu et al., 2011) . notably, denv2 triggers atf6 signaling in a celltype-specific manner. in a549 cells, nuclear-localized atf6 was observed (umareddy et al., 2007) ; however, no activating events can be detected in human fibrosarcoma 2ftgh cells, therefore, gpr78 upregulation may be mediated in an atf6-independent fashion (pena and harris, 2011) . this cell-type-specific regulation of atf6, also observed in iav infection, p50atf6, and its target gene erp57/grp58 expression (roberson et al., 2012) , has been shown to increase in murine primary tracheal epithelial cells infected with influenza a/pr/8/34, which is known to be involved in influenza virus ha protein folding (solda et al., 2006) . knockdown of erp57 abrogates viral progeny production. however, atf6 activity is not induced in infected human tracheobronchial epithelial (htbe) cells (hassan et al., 2012) . although atf6-mediated transcriptional activation is an ongoing research field, another role for atf6 in virus infection has emerged. we have previously demonstrated that ev71 infection results in the decline of p90atf6, while the grp78 promoter containing classical erse sites responsive to p50atf6 in ev71infected cells was not activated (jheng et al., 2010) . indeed, two potential 3c cleavage sites (glutamine-glycine; qg) located at adjacent amino acids 511-512 and 516-517 near the c terminus of p90atf6 were computationally predicted. it would be interesting to investigate the role of viral 3c in the regulation of atf6, for its possible contribution in manipulating virus infection. ire1 is an er-localized type i transmembrane protein containing an er luminal dimerization domain and cytosolic kinase and rnase domains (mori et al., 1993; sidrauski and walter, 1997) . during er stress, accumulation of unfolded proteins in the er stimulates ire1 oligomerization and autophosphorylation (figure 1) . its endoribonuclease activity initiates an unconventional splicing of the xbp1 mrna, excising a 26-nt sequence and shifts the reading frame to produce a functional isoform xbp1(s), which contains a c-terminal transactivation domain absent from the unspliced form, xbp1(u). xbp1(s) then translocates to the nucleus where it induces expression of target genes containing upre or erse. these target genes are involved in erad, chaperone protein production, and er membrane biosynthesis (shamu and walter, 1996; friedlander et al., 2000) . studies of the ire1 signaling pathway demonstrate its significant role in virus infection (figure 4) . hcv glycoprotein e2 is an example of a virus-derived erad substrate. hcv infection activates the ire1-xbp1-edem pathway, where edem1 and edem3, but not edem2, interact with hcv e2 to accelerate its degradation. either knockdown of edems or treating cells with kifunensine (kif), a potent inhibitor of er mannosidase, interferes with the binding of edems with sel1l, a component of erad complex, stabilizes e2 expression, and enhances virus replication and viral particle production. however, there is no interaction between edem proteins and the jev envelope protein and abolishing the erad pathway by kif does not affect jev production (saeed et al., 2011) . the results emphasize the pivotal role of the erad pathway in the life cycle of specific viruses. interestingly, upre reporter activity or erad of misfolded null hong kong α-antitrypsin is reduced in cells carrying hcv replicons, which lack structural proteins, even though upstream xbp1 splicing occurs (tardif et al., 2004) . this implies that hcv structural proteins play a key role in xbp1-mediated upre activation, and this is supported by a related study demonstrating that hcv e1 and/or e2 activates the xbp1-erad pathway (chan and egan, 2005) . furthermore, the ire1 signaling pathway also participates in viral protein retrotranslocation. hepatitis e virus (hev) orf2 is an n-linked glycoprotein which is cotranslationally translocated into the er while a significant fraction of it is also observed in the cytoplasm. based on the results of tunicamycin and kif treatment, it is believed that glycosylation and erad are essential for orf2 retrotranslocation from the er to the cytoplasm (surjit et al., 2007) . however, no ubiquitination of orf2 can be observed, and retrotranslocated orf2 protein was stable in the cytoplasm when the cells were treated with proteasome inhibitor mg132, which suggests that erad is required for orf2 access to the cytoplasm. microarray analysis reveals that orf2 overexpression causes upregulation of hsp70b, hsp72, and hsp40. hsp72 is an antiapoptotic heat shock protein that directly interacts with orf2 (john et al., 2011) . it is reported that expression of hsp72 enhances xbp1 mrna splicing and protects cells from xbp1 mrna splicing, studies demonstrated that ire1 activates ridd to promote the degradation of mrnas encoding er-targeted proteins to reduce the load of er client proteins during er stress. the mammalian ire1-traf2-jnk pathway, independent of xbp1 splicing, may lead to the activation of apoptosis after prolonged er stress. hcv and its structural proteins e1 and e2 play an important role in the activation of the ire1-xbp1-erad pathway. overexpression of orf2 of hev can upregulate antiapoptotic protein hsp72 to activate xbp1 splicing. however, further study is required to determine whether hev infection can activate xbp1 via hsp72. denv2 infection activates chop and gadd34 expression downstream of ire1-xbp1 signaling. however, apoptosis activation by jnk, but not chop, is essential for denv2 infection. er stress-induced apoptosis by association with ire1 (gupta et al., 2010) . thus, further investigation is needed to examine the correlations of orf2, ire1, and hsp72 in hev replication. under harsh er stress, the activation of ire1-xbp1 can also lead to the induction and expression of chop. denv2 infection induces chop, and gadd34 expression is a downstream event of ire1-xbp1 signaling. of note is that induction of chop does not lead to apoptosis markers such as decreased expression of bcl-2 or proteolytic cleavage of pro-caspase-9, pro-caspase-3, or parp, which indicates a role beyond guiding cell death in infected cells (pena and harris, 2011) . indeed, it has been reported that chop exhibits protective effects against radiation-induced apoptosis or has a role in autophagy induction (mayerhofer and kodym, 2003; ke and chen, 2011) . another er stress-induced cell death that relies on the ire1-traf2 pathway is implicated in jnk activation (see figure 4) . the role of this pathway is emphasized by denv2 infection; silencing of ire1 decreases the virus titer, but the viral progeny output is not affected by silencing of xbp1 (pena and harris, 2011) . however, jnk pathway inhibitors diminished virus yield significantly, which suggests that activation of jnk is essential for denv2 infection (ceballos-olvera et al., 2010) . our previous findings also demonstrated that ev71 phosphorylates ire1, but inhibits the expression of xbp1. the overexpression of xbp1 in cells appeared to inhibit viral entry, and therefore reduce viral rna and viral particle formation (jheng et al., 2012) . as previous studies have reported that picornavirus infections induce jnk activation (kim et al., 2004; peng et al., 2014) , further detailed studies of the ire1-jnk activation in ev71 infection would extend our understanding of the contributions of ire1-jnk in the virus life cycle. ire1 has also been linked to the mediation of the selective degradation of a subset of er-localized mrnas in a process known as regulated ire1-dependent degradation (ridd) (hollien and weissman, 2006) . mutation or removal of the signal sequences in targeted mrnas prevents their decay (kimmig et al., 2012) . however, it has been observed that drosophila mrna smt3, a homolog of a small ubiquitin-like modifier (aka sumo), lacks any er-targeting sequence, and is a noncanonical ridd target, which implies that unknown specific features other than er localization are involved in defining the ridd substrates (moore et al., 2013) . ridd has been suggested to play adaptive roles by reducing protein translocation load, such as decrease of proinsulin expression in pancreatic beta-cells faced with chronic high glucose, and protecting liver cells from acetaminopheninduced hepatotoxicity (lipson et al., 2008; hur et al., 2012) . alternatively, ridd has also been suggested to play destructive roles under unmitigated er stress because continued degradation of mrnas encoding secretory cargo proteins and proteins involved in er-resident protein folding occurs. in addition to ire1-xbp1 activation, jev also induces activation of the ridd cleavage pathway (bhattacharyya et al., 2014) . the addition of stf083010, a specific inhibitor of ire1 rnase activity, to infected cells decreases the tg-induced xbp1 splicing and potential ridd target transcripts. it also decreases viral protein expression as well as mature progeny formation, but does not affect viral rna synthesis, which indicates that jev viral rna is not a substrate of ridd, and ridd activation is beneficial for viral infectivity. it is not clear whether other viral infections trigger ridd. to extrapolate from the study of hcv, hcv replicons activate the phosphorylation of ire1 but impede xbp1 activation (tardif et al., 2004) . depletion of ire1 attenuates replicon translation, which implies that ridd may enhance viral protein synthesis. thus, the study of hcv replicon may have potential for deciphering the role of ridd in hcv infection because it could uncouple xbp1 signaling from ire1 activation. autophagy is a vesicular process that results in the degradation of the sequestered component, which can then be recycled by the cell. in mammalian cells, a complete autophagy includes the following four steps. (1) induction. induction is initiated by activation of the unc-51-like kinase 1 (ulk1) complex. the ulk1 complex contains ulk1, focal adhesion kinase (fak)family-interacting protein of 200 kd (fip200), atg13 and atg101 (mizushima, 2010) . ulk1 complex activity would be, at least, modulated by mtorc1, akt, and ampk (inoki et al., 2003; bach et al., 2011; egan et al., 2011; kim et al., 2011) . mtorc1 is a serine/threonine kinase complex, which phosphorylates ulk1 and atg13 and also inhibits autophagy. akt and amp-activated protein kinase (ampk) phosphorylate tsc2 at different residues, which results in the gtp hydrolysis of rheb and indirectly antagonizes the mtorc1 signaling pathway. recently, the combination of bioinformatic and proteomic approaches has identified ulk1 as a direct target of ampk and as involved in autophagy induction. (2) vesicle nucleation. the beclin1-pi3kc3 complex, generating pi3p, is essential for recruitment of pi3p effectors including dfcp1, wipis upstream of atg proteins and lipids recruitment to the pas, which is required for autophagosome construction (proikas-cezanne et al., 2004; axe et al., 2008) . importantly, the activity of the beclin1-pi3kc3 complex depends on its subunit composition. complexes containing atg14-like protein (atg14l or barkor) or ultraviolet irradiation resistanceassociated gene (uvrag) activate autophagy (itakura et al., 2008) ; nevertheless, the run domain and cysteine-rich domain containing beclin 1-interacting protein (rubicon) act as negative regulators of autophagy (matsunaga et al., 2009) . (3) vesicle expansion and completion. the cytosolic form of lc3 (lc3-i) is cleaved by the cysteine protease atg4, followed by conjugation with phosphatidylethanolamine (pe) assisted by the atg12-atg5-atg16l complex, which functions as an e3-like enzyme. lc3-pe leads to pas expansion, and cytosolic cargos are then enclosed into double membrane vesicles called autophagosomes (geng and klionsky, 2008) . (4) autophagosome maturation. an autophagosome matures into an autolysosome by sequential fusion with endosomes and with lysosomes, the contents of which are degraded by hydrolases therein. it is reported that autolysosome formation is related to uvrag and expression of lysosomal-associated membrane protein 2 (lamp-2) (liang et al., 2008; fortunato et al., 2009 ). previous studies suggest that autophagy may be an important antiviral defense mechanism (talloczy et al., 2006; orvedahl et al., 2010) ; however, the role of autophagy in virus infection is complicated and may have opposite consequences for the viral pathogenesis. many viruses manipulate autophagy for their own benefit by the following mechanisms. the exploitation of autophagy has been identified in many rna viruses including pv, cvb3, jev, and hcv wong et al., 2008; tanida et al., 2009; ke and chen, 2011; li et al., 2012) . increased amounts of autophagosomes, as well as colocalization of the autophagy marker protein lc3 and viral protein, were observed in virus-infected cells. in addition, cells treated with an autophagy inhibitor, or transfected with sirna specifically obstructed autophagic processes, which reduced virus replication or virus titer. for example, in pv infection, virus yield was correlated with the induction of autophagy. treating cells with sirna targeting lc3 or atg12 to block autophagy leads to reduced virus yield . in addition, based on the topology of a double membrane compartment, digestion of the inner membrane under the autolysosome formation would allow efficient fusion of the autophagosomal membrane with the cytoplasm membrane. thus, an emerging concept is that autophagy may also involve the nonlytic release of cytoplasm under autophagosome maturation, namely autophagic exit without lysis (awol), which may participate in the release of pv (kirkegaard and jackson, 2005; taylor et al., 2009) . virus-induced uncompleted autophagy was reported for cvb3-, rotavirus-, and iva-infected cells (gannage et al., 2009; kemball et al., 2010; alirezaei et al., 2012; crawford et al., 2012) . in cvb3infected pancreatic acinar cells, an increase in the number of double-membraned autophagy-like vesicles was observed upon infection. however, the accumulation of autophagy substrate p62 and the formation of large autophagy-related structures named megaphagosomes indicate that cvb3 blocks a later stage of the autophagic pathway (kemball et al., 2010) . further results highlight the impact of autophagy on cvb3 rna replication and translation (alirezaei et al., 2012) . it was reported that rotavirus nsp4 viroporin initiates autophagy to transport viral proteins to sites of virus replication for assembly of mature particles, which involves an increase of cytoplasmic calcium and subsequent activation of the camkk-β-ampk pathway. rotavirus also interferes with autophagy maturation; however, the mechanism is still unknown (crawford et al., 2012) . accumulated studies reveal that m2, ha, and ns1 proteins of iav are involved in the induction of autophagy, while only m2 has been identified as playing a critical role in impeding fusion of autophagosomes with lysosomes (gannage et al., 2009; sun et al., 2012; zhirnov and klenk, 2013) . autophagy-mediated immune responses that benefit virus replication have been reported in vsv, hcv, denv, and jev (jounai et al., 2007; ke and chen, 2011; jin et al., 2013) . in vsv infection, the atg5-atg12 conjugate targets rig-i/mda5-mavsdependent type i ifn production by directly interacting with the mavs and rig-i, and negatively regulates mavs-mediated nf-κb and type i ifn promoters, and permits vsv replication. furthermore, through an unknown mechanism, hcv-or denv-induced complete autophagy negatively regulates type i ifn production and promotes hcv replication (ke and chen, 2011) . recently, research about jev has shown that in autophagyimpaired cells, virus infection induces aggregates of mavs and activation of ifn regulatory factor 3 (irf3), markers for activation of innate immune responses, which suggests that autophagymediated immune responses are required for viral replication (jin et al., 2013) . as er proliferation, which paradoxically commits the cell to cell death or survival, is observed both in upr and autophagy, it is reasonable to propose a possible link between upr pathways and the autophagic response. indeed, many upr-related transcription factors manage atg expression ( table 1) . as demonstrated previously, yeasts with mutations in the gcn2-signaling pathway are defective in starvation-induced autophagy. gcn4, which undertakes gcn2-dependent transcriptional activation, is essential for autophagy induction (talloczy et al., 2002) . recently, august 2014 | volume 5 | article 388 | 7 gomez et al., 2007; margariti et al., 2013 results of multiple genetic models showed that the perk-eif2α-atf4 pathway affects cmyc-dependent tumorigenesis by evoking cytoprotective autophagy; while pharmacologic or genetic inhibition of autophagy resulted in enhanced myc-dependent apoptosis (hart et al., 2012) . thus, upr inhibition could provide new targets for the treatment of malignancies, characterized by cmyc overexpression. in addition, ire1 also mediates autophagy in huntington's disease under er stress. clearance of mutant huntingtin aggregates through autophagic flux was impaired via ire1-traf2 signaling, which results in neuronal cytotoxicity (lee et al., 2012) . although studies on upr autophagy mainly focus on the regulation of eif2α kinase and ire1, transcriptional regulation of autophagic genes by atf6 and srebp2, a membrane-bound transcription factor activated through proteolytic processing upon er stress, was noticed recently (ogata et al., 2006; seo et al., 2011; gade et al., 2012) . death-associated protein kinase 1 (dapk1), a positive mediator of ifn-regulated growth suppressor, is principally regulated by transcription factor c/ebp-β, one of the genes that increases expression during er stress (chen et al., 2004) . dapk1 promotes autophagy by phosphorylating beclin 1, and therefore dissociating it from autophagy negative regulator bcl2. an investigation found that activated atf6 could directly interact with c/ebp-β carrying an erk1/2 target site; this heterodimer then coacts to activate the dapk1 promoter, which in turn induces autophagy. additionally, xbp1, a downstream target of atf6, is essential for c/ebp-β expression (chen et al., 2004) . the role of srebp2 in autophagy was disclosed through gene ontology analysis (seo et al., 2011) . further study shows that srebp-2 activates autophagy gene expression, such as lc3b, atg4b, and atg4d, accompanied by increased lc3 puncta formation, while srebp-2 deficiency obtains an opposite result. in virus infection, hcv is a well-documented model illustrating upr autophagy regulation. induction of upr and incomplete autophagy was observed in cells transfected with hcv jfh1 rna. cells treated with sirna targeting perk, ire1, and atf6 showed a suppression of lc3 conversion and a decrease of hcv rna replication (sir et al., 2008) . in the hcv infection system, hcv induces complete autophagy and 23-(s)-2-amino-3phenylpropanoylsilybin impairs autophagy dai et al., 2013 chop plays a leading role in upr autophagy signaling (ke and chen, 2011) . further efforts to decipher how hcv activates autophagy revealed that perk-eif2α-atf4 and atf6 pathways activated chop expression in hcv core protein-transfected cells where the core protein had not been demonstrated to induce er stress previously. moreover, hcv core protein may promote atg12 and lc3 protein expression through transcriptional control by atf4 and chop, respectively (wang et al., 2014) . recent studies suggest that completed autophagy induced by chikv infection is mediated by the independent induction of the endoplasmic reticulum and oxidative stress pathways. knockdown of ire1 or treated cells with the ros inhibitor nacetyl-l-cysteine inhibits formation of autophagosomes as well as the conversion of lc3-i to lc3-ii. moreover, an additive inhibitory effect on autophagosome formation was observed in infected cells silenced for ire1mrna and treated with n-acetyl-l-cysteine (joubert et al., 2012) . because upr and autophagy play a role in viral pathogenesis, the regulation of upr and autophagy may be an important strategy for the future development of new therapeutic approaches to combat viruses. for example, we have demonstrated that overexpression of grp78 to relieve er stress decreases ev71 replication (jheng et al., 2010) . thus, agents such as grp78/bip inducer x (bix) (kudo et al., 2008) or chemical chaperone, tauroursodeoxycholic acid (tudca) (ozcan et al., 2006) , will be potentially useful in the treatment of ev71 ( table 2) . there are other established strategies to inhibit viruses by modulating upr target eif2α phosphorylation or ire1, e.g., salubrinal is a small molecule that prevents dephosphorylation of eif2α and 3,5-dibromosalicylaldehyde, an ire1 inhibitor, may cause restriction of iva (boyce et al., 2005; volkmann et al., 2011) . there is emerging evidence that pharmacological agents that directly activate or deactivate autophagy influence virus replication. evodiamine and 3-(s)-2-amino-3-phenylpropanoyl-silybin have been identified as anti-iva agents aimed at multiple processes of autophagy (dai et al., 2012 (dai et al., , 2013 . additionally, chloroquine-suppressed hcv replication has been proved (mizui et al., 2010) . because upr and autophagy are closely related, combination treatment may show a synergistic effect of their application, which was demonstrated in cancer research. the combination of nelfinavir (which induces upr autophagy) and chloroquine enhances cytotoxicity against cancer cells (mahoney et al., 2013b) ; therefore, the use of combination treatment with improved efficacy and decreased toxicity represents a promising strategy to fight viruses. although upr autophagy has been discussed in many research areas, its integrated response to virus infection is only now beginning to emerge. it needs to be experimentally proven whether virus-induced autophagy is associated with upr. furthermore, given what we know about the various means that viruses use to modulate upr or autophagy to advantage their own virulence, the development of specific inducers or inhibitors for these molecules is one of the major challenges in this field. pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion atf6 signaling is required for efficient west nile virus replication by promoting cell survival and inhibition of innate immune responses autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum the serine/threonine kinase ulk1 is a target of multiple phosphorylation events infectious hepatitis c virus pseudo-particles containing functional e1-e2 envelope protein complexes the eif2alpha/atf4 pathway is essential for stress-induced autophagy gene expression viral internal ribosome entry site structures segregate into two distinct morphologies receptor proteins in selective autophagy autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response regulated ire1-dependent decay pathway is activated during japanese encephalitis virus-induced unfolded protein response and benefits viral replication a selective inhibitor of eif2alpha dephosphorylation protects cells from er stress jnk phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response human ccaat/enhancer-binding protein beta gene expression is activated by endoplasmic reticulum stress through an unfolded protein response element downstream of the protein coding sequence regulation of protein synthesis by the heme-regulated eif2alpha kinase: relevance to anemias inhibition of host and viral translation during vesicular stomatitis virus infection. eif2 is responsible for the inhibition of viral but not host translation autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-beta signaling is required for rotavirus replication a drug screening method based on the autophagy pathway and studies of the mechanism of evodiamine against influenza a virus identification of 23-(s)-2-amino-3-phenylpropanoyl-silybin as an antiviral agent for influenza a virus infection in vitro and in vivo cleavage of eukaryotic initiation factor eif5b by enterovirus 3c proteases functional analysis of picornavirus 2b proteins: effects on calcium homeostasis and intracellular protein trafficking the rotavirus enterotoxin nsp4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase c-mediated inositol 1,4,5-trisphosphate production formation and intracellular localization of hepatitis c virus envelope glycoprotein complexes expressed by recombinant vaccinia and sindbis viruses hepatitis c virus glycoprotein folding: disulfide bond formation and association with calnexin the autophagy initiating kinase ulk1 is regulated via opposing phosphorylation by ampk and mtor replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna maturation of autophagic vacuoles in mammalian cells impaired autolysosome formation correlates with lamp-2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis a regulatory link between er-associated protein degradation and the unfoldedprotein response two endoplasmic reticulum-associated degradation (erad) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome erad(i) and autophagy/lysosome erad(ii) an ifn-gamma-stimulated atf6-c/ebp-beta-signaling pathway critical for the expression of death associated protein kinase 1 and induction of autophagy critical role for transcription factor c/ebp-beta in regulating the expression of death-associated protein kinase 1 control of pkr protein kinase by hepatitis c virus nonstructural 5a protein: molecular mechanisms of kinase regulation matrix protein 2 of influenza a virus blocks autophagosome fusion with lysosomes sequence differences between glycosylated and non-glycosylated asn-x-thr/ser acceptor sites: implications for protein engineering the atg8 and atg12 ubiquitin-like conjugation systems in macroautophagy phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation human x-box binding protein-1 confers both estrogen independence and antiestrogen resistance in breast cancer cell lines transactivation of atg4b by c/ebpbeta promotes autophagy to facilitate adipogenesis hsp72 protects cells from er stress-induced apoptosis via enhancement of ire1alpha-xbp1 signaling through a physical interaction er stress response: getting the upr hand on misfolded proteins n-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity regulated translation initiation controls stress-induced gene expression in mammalian cells endoplasmic reticulum stress and the development of diabetes: a review er stress-mediated autophagy promotes myc-dependent transformation and tumor growth influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ire1) stress pathway components of ubiquitin-protein ligase system. resolution, affinity purification, and role in protein breakdown decay of endoplasmic reticulum-localized mrnas during the unfolded protein response ire1alpha activation protects mice against acetaminopheninduced hepatotoxicity tsc2 mediates cellular energy response to control cell growth and survival beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian atg14 and uvrag subversion of cellular autophagosomal machinery by rna viruses inhibition of rnase l and rna-dependent protein kinase (pkr) by sunitinib impairs antiviral innate immunity endoplasmic reticulum stress is induced and modulated by enterovirus 71 inhibition of enterovirus 71 entry by transcription factor xbp1 japanese encephalitis virus activates autophagy as a viral immune evasion strategy hepatitis e virus orf2 protein activates the pro-apoptotic gene chop and anti-apoptotic heat shock proteins chikungunya virus-induced autophagy delays caspasedependent cell death the atg5 atg12 conjugate associates with innate antiviral immune responses stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo ampk and mtor regulate autophagy through direct phosphorylation of ulk1 coxsackievirus b3 infection induces cyr61 activation via jnk to mediate cell death the unfolded protein response in fission yeast modulates stability of select mrnas to maintain protein homeostasis topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm the unfolded protein response signals through high-order assembly of ire1 assembly of asparagine-linked oligosaccharides mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems er stress, hypoxia tolerance and tumor progression a molecular chaperone inducer protects neurons from er stress ire1 plays an essential role in er stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsrna-activated protein kinase rock is involved in vimentin phosphorylation and rearrangement induced by dengue virus development by self-digestion: molecular mechanisms and biological functions of autophagy autophagy is involved in the early step of japanese encephalitis virus infection beclin1-binding uvrag targets the class c vps complex to coordinate autophagosome maturation and endocytic trafficking upregulation of chop/gadd153 during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway the role of ire1alpha in the degradation of insulin mrna in pancreatic beta-cells translational control by viral proteinases binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor topology of the membrane-associated hepatitis c virus protein ns4b temporal orchestration of circadian autophagy rhythm by c/ebpbeta delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress plasma cell differentiation initiates a limited er stress response by specifically suppressing the perk-dependent branch of the unfolded protein response autophagy and er stress play an essential role in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol identification of endoplasmic reticulum stress-inducing agents by antagonizing autophagy: a new potential strategy for identification of anticancer therapeutics in b-cell malignancies xbp1 mrna splicing triggers an autophagic response in endothelial cells through beclin-1 transcriptional activation two beclin 1-binding proteins, atg14l and rubicon, reciprocally regulate autophagy at different stages gadd153 restores resistance to radiationinduced apoptosis after thiol depletion west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis molecular cloning and characterization of the human doublestranded rna-activated protein kinase induced by interferon the role of atf4 stabilization and autophagy in resistance of breast cancer cells treated with bortezomib modification of intracellular membrane structures for virus replication inhibition of hepatitis c virus replication by chloroquine targeting virus-associated autophagy the role of the atg1/ulk1 complex in autophagy regulation regulation of sumo mrna during endoplasmic reticulum stress a transmembrane protein with a cdc2+/cdc28-related kinase activity is required for signaling from the er to the nucleus viroporins: structure and biological functions autophagy is activated for cell survival after endoplasmic reticulum stress autophagy protects against sindbis virus infection of the central nervous system chemical chaperones reduce er stress and restore glucose homeostasis in a mouse model of type 2 diabetes identification of an ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e2 through the eukaryotic initiation factor 2alpha kinase perk dengue virus modulates the unfolded protein response in a time-dependent manner activation of jnk1/2 and p38 mapk signaling pathways promotes enterovirus 71 infection in immature dendritic cells transcriptional up-regulation of ulk1 by atf4 contributes to cancer cell survival wipi-1alpha (wipi49), a member of the novel 7-bladed wipi protein family, is aberrantly expressed in human cancer and is linked to starvationinduced autophagy differential unfolded protein response during chikungunya and sindbis virus infection: chikv nsp4 suppresses eif2alpha phosphorylation aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy translation without eif2 promoted by poliovirus 2a protease influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-jun n-terminal kinase-mediated transforming growth factor-beta release in lung epithelial cells translational control in the endoplasmic reticulum stress response the unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes map1lc3b and atg5 role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles cannabinoid action induces autophagy-mediated cell death through stimulation of er stress in human glioma cells viral internal ribosome entry site elements: novel ribosome-rna complexes and roles in viral pathogenesis cellular origin and ultrastructure of membranes induced during poliovirus infection genome-wide localization of srebp-2 in hepatic chromatin predicts a role in autophagy xbp1, downstream of blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation oligomerization and phosphorylation of the ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via er stress-related apoptosis autophagy in health and disease: a doubleedged sword the transmembrane kinase ire1p is a sitespecific endonuclease that initiates mrna splicing in the unfolded protein response autophagy regulates lipid metabolism induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response consequences of erp57 deletion on oxidative folding of obligate and facultative clients of the calnexin cycle regulation of translation initiation in eukaryotes: mechanisms and biological targets inhibition of autophagy ameliorates acute lung injury caused by avian influenza a h5n1 infection cytoplasmic localization of the orf2 protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum mediators of endoplasmic reticulum stress-induced apoptosis regulation of starvation-and virus-induced autophagy by the eif2alpha kinase signaling pathway pkr-dependent autophagic degradation of herpes simplex virus type 1 reticulon 3 binds the 2c protein of enterovirus 71 and is required for viral replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response hepatitis c virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment role of microtubules in extracellular release of poliovirus the rotavirus nonstructural glycoprotein nsp4 possesses membrane destabilization activity the rotavirus nonstructural glycoprotein nsp4 mobilizes ca2+ from the endoplasmic reticulum structural insights into the coupling of virion assembly and rotavirus replication blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a dengue virus serotype infection specifies the activation of the unfolded protein response inhibition of the vacuolar h(+)-atpase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in hep-2 cells coxsackievirus protein 2b modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease hepatitis c virus ns2 protein triggers endoplasmic reticulum stress and suppresses its own viral replication hepatitis c virus core protein activates autophagy through eif2ak3 and atf6 upr pathway-mediated map1lc3b and atg12 expression induction of autophagy in axonal dystrophy and degeneration autophagosome supports coxsackievirus b3 replication in host cells reticulon 3 interacts with ns4b of the hepatitis c virus and negatively regulates viral replication by disrupting ns4b self-interaction japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity membrane topology and function of dengue virus ns2a protein activation of the akt-nf-kappab pathway by subtilase cytotoxin through the atf6 branch of the unfolded protein response control of perk eif2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone p58ipk er stress induces cleavage of membrane-bound atf6 by the same proteases that process srebps induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured arpe-19 cells endoplasmic reticulum stress triggers autophagy identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. involvement of basic leucine zipper transcription factors a time-dependent phase shift in the mammalian unfolded protein response n-glycosylation of the premembrane protein of japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles influenza a virus proteins ns1 and hemagglutinin along with m2 are involved in stimulation of autophagy in infected cells conflict of interest statement: the authors declare that the research was con the authors are in debt to all previous and current laboratory members for their contributions and involvement in the study of ev71-induced er stress. this work was supported by the national science council [nsc-102-2325-b-182-016] and chang gung memorial hospital [cmrpd1b0422 and cmrpd1a0163]. key: cord-289321-ahl46ql9 authors: van buuren, nicholas; tellinghuisen, timothy l; richardson, christopher d; kirkegaard, karla title: transmission genetics of drug-resistant hepatitis c virus date: 2018-03-28 journal: elife doi: 10.7554/elife.32579 sha: doc_id: 289321 cord_uid: ahl46ql9 antiviral development is plagued by drug resistance and genetic barriers to resistance are needed. for hiv and hepatitis c virus (hcv), combination therapy has proved life-saving. the targets of direct-acting antivirals for hcv infection are ns3/4a protease, ns5a phosphoprotein and ns5b polymerase. differential visualization of drug-resistant and -susceptible rna genomes within cells revealed that resistant variants of ns3/4a protease and ns5a phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. confocal microscopy revealed that rna replication complexes are genome-specific, rationalizing the non-interaction of wild-type and variant products. no hcv antivirals yet display the dominance of drug susceptibility shown for capsid proteins of other viruses. however, effective inhibitors of hcv polymerase exact such high fitness costs for drug resistance that stable genome selection is not observed. barriers to drug resistance vary with target biochemistry and detailed analysis of these barriers should lead to the use of fewer drugs. in a recent triumph of modern science and medicine, patients chronically infected with hepatitis c virus (hcv) now receive multidrug regimens that are often curative and have low toxicity (lawitz et al., 2013; afdhal et al., 2014; dhaliwal and nampoothiri, 2014) . over the past two decades, researchers have developed and tested thousands of antiviral compounds with varying efficacies and toxicity profiles that have ultimately lead to the fda approval of powerful combination therapies (lawitz et al., 2013; scheel and rice, 2013) . several different direct-acting antivirals (daas) that target the ns3/4a protease, ns5a phosphoprotein, or ns5b rna-dependent rna polymerase of hcv have been approved for use in the clinic (afdhal et al., 2014; dhaliwal and nampoothiri, 2014; manns and von hahn, 2013; younossi et al., 2015) . ideally, the knowledge gained in developing hcv antivirals that are effective and not prone to the outgrowth of drug resistance will be applied to other viruses as well. the emergence of drug-resistant variants follows basic evolutionary principles, requiring spontaneous mutations as well as selective pressure, so that beneficial mutations increase the progeny size of genomes that bear them. the genetic diversity in rna viral genomes results from the high error frequencies incurred by rna-dependent rna polymerases, which occur at approximately 4 â 10 à5 errors for each nucleotide synthesized (sanjuán et al., 2010) . given the iterative copying of positive and negative strands, much higher cumulative error frequencies are observed, even during a single cycle of infection (sanjuán et al., 2010; acevedo et al., 2014) . when more than one mutation is required to confer drug resistance, the outgrowth of drug resistance can be delayed (bloom et al., 2010) . as a result, treatment with combinations of drugs can be extremely effective at suppressing siblings. if the drug target is, for example, a subunit of an oligomeric complex and subunits from different genomes have the opportunity to mix, chimeric oligomers often form. at the time of its creation, the drug-resistant genome will be a minority species, and such chimeras would be predominantly composed of the drug-bound, susceptible components thus incapacitating the entire oligomeric structure. such 'phenotypic masking' was originally invoked to explain the very low frequency of foot-and-mouth-disease escape variants following selection with neutralizing antibodies when passaged at high multiplicities of infection (mois) (holland et al., 1989) . our goal was to screen the hcv-encoded viral proteins that are current targets of antiviral compounds to determine the intracellular dominance relationships between drug-resistant and drug-susceptible genomes. the high cost to viral fitness of sofosbuvir-resistant variants is sufficient to explain its high barrier to resistance. there are currently no antivirals directed against hcv core protein; however, it is likely to be a dominant drug target. we used differential hybridization of rna probes to detect two different genomic rnas in a single cell by confocal microscopy and by flow cytometry. this analysis showed the cis-dominance of hcv viruses that are resistant to inhibitors of either ns3/ 4a protease or ns5a phosphoprotein, consistent with the rapid outgrowth of drug-resistance in patients of these two inhibitor classes. newly mutated drug-resistant genomes first arise within cells that are pre-populated by drug-susceptible genomes. to mimic such mixed infections, we have previously employed co-infection of cultured cells with drug-susceptible and drug-resistant viruses at high mois to ensure mixed infection (tanner et al., 2014; mateo et al., 2015) . for hcv, it is not practical to use high mois to achieve co-infection due to the difficulty of obtaining sufficiently high-titer viral stocks. thus, we needed to develop an approach to distinguish between uninfected, singly infected and co-infected cells in relatively sparsely infected cell populations ( figure 1a) . to detect individual genomes in infected cells, a single-molecule fluorescence in situ hybridization (fish) approach was used. a recently developed branched dna probe technology allows the generation of sufficiently sensitive rna probes to identify single molecules within cells, but requires approximately 1000 nucleotides of differential probe hybridization to achieve specificity (affymetrix inc, 2016) . to create a viral strain with this extreme dissimilarity from wild-type virus, we tested the viability of three different codon-altered versions of the jfh1 variant of hcv ( figure 1b) . each mutated version contained 200-300 nucleotide changes that did not alter the protein sequence (figure 1-figure supplements 1-3). of these codon-altered (ca) variants, ca-1 was inviable, ca-2 showed reduced viral protein accumulation, and ca-3 showed accumulation of both viral protein and rna to abundances equivalent to those of the wild-type virus ( figure 1c) . recently, detailed analysis of covarying nucleotides within the hcv coding region has identified the location of several previously unknown functional rna secondary structures (pirakitikulr et al., 2016) . interestingly, ca-1 contains two such regions and ca-2 contains one, which correlates with decreasing viability, while ca-3 contains no such regions (figure 1-figure supplements 1-3) (pirakitikulr et al., 2016) . thus, subsequent experiments were performed only with ca-3. this variant, now termed 'ca' virus, contains 247 synonymous mutations over a 918-nucleotide region that spans the coding sequences for most of ns2 and the n-terminus of ns3 (figure 1 to test the sensitivity of rna fish probes generated against the positive-and negative-strands of wild-type (wt) and codon-altered (ca) viruses, both confocal microscopy and flow cytometry analyses were employed. branched dna technology allowed the labeling of each target rna with as many as 8000 fluorophores ( figure 2a ) (affymetrix inc, 2016) . huh-7.5.1 cells were infected with either wt or ca viruses, subjected to fish and visualized by confocal microscopy. wt and ca probe sets specifically targeted either the positive-sense ( figure 2b ) or the negative-sense vrna ( figure 2c ) of their corresponding virus. additionally, we tested whether flow cytometry efficiently resolved cells transfected with different vrnas; transfection was used to maximize the yield of each population. we resolved cells transfected with wt vrna (figure 2di) , transfected with ca vrna (ii), a mixture of these two cell types (iii) and cells co-transfected with both wt and ca vrnas (iv). thus, figure 1 . construction of codon altered jfh1. (a) cell cultures coinfected with two strains of hcv result in four populations: uninfected, two types of singly infected, and coinfected cells. (b) three segments of the jfh1 genome, that were roughly 1000 nucleotides in length and had altered codon usage, were designed using geneart algorithms and synthesized. these genome fragments were then cloned into the jfh1 strain of hcv. specific rna probes could be used to monitor the fate of drug-susceptible and drug-resistant viruses in co-infected cells. to test the genetic properties of viruses that are resistant to ns3/4a inhibitors, we employed the original ns3/4a inhibitor, biln-2061 ( figure 3a ) (lamarre et al., 2003) . like other ns3/4a inhibitors, biln-2061 treatment rapidly allows the selection of drug resistant variants both in tissue culture and in patients (lamarre et al., 2003; lin et al., 2004) . given the ease of outgrowth of drug-resistant variants, we hypothesized that ns3/4a was not a dominant drug target and that drug resistance would be genetically dominant. ns3-d168a is the prototypic mutation associated with resistance to ns3/4a inhibitors. asp168 is in close proximity to the protease active site ( figure 3b ). the ability of the ns3-d168a mutation to confer resistance to biln-2061 was confirmed in both the wt and ca backgrounds ( figure 3 -figure supplement 1). as shown schematically in figure 3d , the ability to track cells that are uninfected (u), singly infected with drug-susceptible virus (s), infected with both susceptible and resistant virus (s + r) and singly infected with drug-resistant virus (r), can reveal dominance relationships during co-infection. in the absence of a drug, all viral populations should be present. however, in the presence of a drug, three outcomes are possible depending on the genetic outcome within the r + s population. if drug resistance were trans-dominant ( figure 3e ), the drug-resistant virus would rescue the drugsusceptible genomes and all viruses in r + s cells would survive in the presence of the drug. s cells would drop into the u population, and r cells would survive. if drug resistance were cis-dominant ( figure 3f ), only the r viruses in the r + s cells would survive, because the drug-resistant proteins would be unable to rescue the s viruses in the same cell. consequently, the r + s cells would drop into the r population. if drug susceptibility were dominant ( figure 3g ), all viruses in the r + s cells would be cleared, and the r + s cells, like the s cells, would drop into the u population, and only the r cells would continue to replicate. to determine the dominance relationship between biln-2061-susceptible and the biln-2061resistant virus, huh-7.5.1 cells were infected with ca and wt-d168a viruses ( figure 3c ). cells were infected for 72 hr at mois such that all four populations were represented, followed by 36 hr of continued incubation in the absence or presence of 2 mm biln-2061. cells were then harvested, fixed, co-stained with wild-type and codon-altered rna probe sets and analyzed by flow cytometry. all four cell types appeared in the absence of biln-2061 ( figure 3h ,i). in the biln-2061-treated samples ( figure 3j ,k), the susceptible s population shifted to the u cells as expected. the s + r cells, on the other hand, shifted to the r population upon drug treatment. thus, the drug-resistant viral genomes in the co-infected cells could replicate, but could not rescue the drug-susceptible ones. data from this and replicate experiments ( figure 3i ,k) confirmed the quantitative shift of s + r cells into the r population upon drug treatment. we conclude that, for the ns3/4a target, drug-resistant genomes are cis-dominant for the 1:1 ratio of s and r viruses tested here. we also tested whether over-expressed drug-resistant ns3/4a precursors could rescue biln-2061-susceptible virus ( proteins are drug targets, drug-resistant products will enhance the propagation of only those genomes that encode them, allowing powerful selection for drug resistance. for ns5b polymerase inhibitor sofosbuvir, the few resistant viral variants that arise in patients are highly attenuated. to investigate whether a related compound, r1479 (klumpp et al., 2006) , exacted a similar cost to viral fitness to drug-resistant variants, we attempted to recover r1479-resistant viruses for dominance testing. jfh1 was passaged for multiple rounds of infection in the . each mutation was introduced independently into the jfh1 genome and rna transfections were performed. the t481a genome was the only variant to show any viral rna production by 7 or 21 days post-transfection. we noticed that f427l and t481a were always isolated together. to test whether these mutations could together increase viral fitness, jfh1 viruses were generated that contained both mutations. viruses with the mutations separate or together were passaged extensively in the presence of r1479. occasional resistant outgrowths were observed, but none conferred sustained growth ( figure 3 -figure supplement 3c). thus, like sofosbuvir, the poor viability of mutant viruses resistant to r1479 precludes the ability to perform further genetic analysis but provides an excellent paradigm for antiviral development. transmission genetics and phenotypic dominance of drug-resistant ns5a variant y93n ns5a is highly oligomeric (sun et al., 2015; tellinghuisen et al., 2005) and we were curious as to whether drug resistance or drug susceptibility would be dominant during viral infections. this idea seemed promising because exogenously expressed ns5a has a dominant-negative effect on the growth of hcv replicons (graziani and paonessa, 2004) . additionally, the ns5a inhibitors, as a class, display ec 50 's in the low picomolar range (gao, 2013) , making them among the most potent antiviral compounds ever identified. assuming uniform inhibitor concentrations in cells and in medium, it has been estimated that only a small fraction of ns5a molecules should be bound to drugs under inhibitory conditions (sun et al., 2015; gao et al., 2016) . thus, it seemed mechanistically likely that drug-bound ns5a proteins from drug-susceptible viruses could be dominant inhibitors of ns5a encoded by newly arising drug-resistant ones. however, ns5a inhibitors have generally demonstrated low barriers to resistance in patients. our goal was gain mechanistic insight into this dichotomy. the structures of two such potent ns5a inhibitors, sr2486 (also known as bms-346) (lemm et al., 2011) and daclatasvir (gao et al., 2010) are shown in figure 4a . mutations of tyr93 to asp or his confer resistance to a broad array of ns5a inhibitors (gao et al., 2016) . tyr93 is located near an ns5a dimer interface shown in the crystal structure ( figure 4b ) . thus, this interface is postulated to be part of the binding site for the ns5a inhibitor class. the y93n and y93h mutations were introduced into both the wild-type and codon-altered viruses. as shown in figure 4c , the y93h mutation conferred resistance to both sr2486 and daclatasvir while the y93n mutation conferred resistance only to sr2486. to test whether susceptibility to ns5a inhibitors was dominant in the context of viral infections, we analyzed u, s, s + r and r cell populations by flow cytometry as previously performed for the ns3/4a inhibitor in figure 3 . huh-7.5.1 cells were coinfected with ca and wt-y93n viruses for 72 hr ( figure 4d ). cells were then treated with dmso or 500nm sr2486 for 24 hr, harvested, fixed, costained for wt and ca vrnas and analyzed by flow cytometry. in the absence of the ns5a inhibitor, all four populations, u, s, r + s and r were observed ( figure 4e ,f). in the presence of sr2486, the s population of cells dropped into the u population as expected. as was the case in figure 3 , the co-infected r + s population of cells dropped into the r population. thus, resistance to ns5a inhibitor sr2486 in the context of viral infection was genetically dominant and the lack of rescue of the s virus with which it was mixedly infected shows that drug resistance is also cis-dominant. hcv infected cells become resistant to superinfection upon expression of non-structural proteins (schaller et al., 2007; tscherne et al., 2007) . due to this superinfection exclusion, it is likely that all coinfected cells arise through nearly synchronous infection throughout the course of the experiment. to control for any effects on selection that may occur due to the differential timing of coinfections that occurs over the initial 72 hr incubation period, we performed the same experiment with higher titer virus and a single cycle of infection in the absence of drug. huh7.5.1 cells were infected at an moi of 1 focus-forming unit (ffu)/cell with ca and wt-y93n viruses and incubated for only 24 hr before drug treatment. cells were then incubated in the absence and presence of 500nm sr2486 for an additional 24 hr. in this case, we also observe cis-dominance of drug resistant wt-y93n genomes, indicating that asynchronous coinfection has no effect on selection ( figure 4g) . finally, the cis-dominance of daclatasvir-resistant wt-y93h was observed when coinfected with drug susceptible virus (s) in the absence and presence of daclatasvir ( figure 4h ). we conclude that ns5a, despite being an oligomeric species is not a dominant drug target. instead, genomes resistant to ns5a remain drug resistant in co-infected cells but do not rescue drug-susceptible viruses present in the same cell. this is consistent with the observed outgrowth of viruses that are resistant to ns5a both in cultured cells and in patients, and with an earlier report that at least some functions of ns5a act exclusively in cis (fridell et al., 2011) . one hypothesis that could mechanistically account for cis-dominant drug resistance is that ns5a molecules expressed from different alleles may not freely associate in mixed oligomers. as previously demonstrated, two different ns5as expressed from the same rna can associate, while ns5a molecules expressed from different constructs could not (berger et al., 2014) . we were curious whether the dominance phenotypes were altered if we forced ns5a alleles to mix. to test whether exogenously expressed drug-susceptible ns5a proteins could co-assemble with drug-resistant ns5a, we utilized the previously described hcv plasmid that expresses ha-tagged and gfp-tagged ns5a within the same polyprotein but does not support genome replication ( figure 5a) . constructs that contained all combinations of drug-susceptible ns5a (s) and the drug-resistant y93n variant (r) were created. upon transfection, all tagged proteins were expressed and can be observed in figure 5 (input, panels b,c). immunoprecipitation with anti-ha antibodies revealed that the gfptagged and ha-tagged ns5a proteins were present in the same complexes in the presence or absence of sr2486. therefore, as has been shown previously, mixed oligomers can form upon coexpression within the same polyprotein (berger et al., 2014) . furthermore, these interactions are not disrupted by drug treatment or by drug-resistant mutations ( figure 5b ,c). to determine whether there were any functional consequences of mixed oligomer formation, we visualized cells that expressed mixed oligomers using confocal microscopy. all s and r combinations of ns5a co-localized at discrete membrane-associated complexes characteristic of hcv infection in the absence of drug ( figure 5d , top panel). however, in the presence of sr2486, membrane-associated complex formation was inhibited in r:s and s:s expressing cells and observed only in r:r expressing cells ( figure 5d, bottom panel) . the dispersal of ns5a signal upon drug treatment in the presence of s protein makes ns5a protein appear less abundant ( figure 5d ). however, the immunoblots demonstrate that no such decrease in expression occurs as we observe equal levels of ns5a protein independent of allele or the presence of drug ( figure 5b ,c). one hallmark of hcv infection is the accumulation of cytoplasmic lipid droplets (miyanari et al., 2007; romero-brey et al., 2012) . electron microscopy performed by high-pressure freezing and freeze-substitution, to preserve membrane structure, revealed many lipid droplets in the cytoplasm of cells expressing s:s, r:r and s:r combinations of ns5as in the absence of inhibitor ( figure 5e,f) . however, in the presence of sr2486, only the r:r cells displayed the accumulation of lipid droplets ( figure 5e , g). therefore, using both assays, the presence of drug-susceptible ns5a prevented drug-resistant phenotypes from being displayed, and thus drug-susceptibility was genetically dominant. this confirmed our original hypothesis that ns5a had the potential to be a dominant drug target. one of the most likely mechanisms for cis-dominance, when the benefit of a particular gene product accrues only to the genome that encodes it, is physical isolation. we hypothesized that hcv genomes co-infecting the same cell might be physically isolated from each other. to test this possibility, confocal microscopy was used to identify and localize negative vrna strands in genome-specific rna replication complexes ( figure 6a ). the majority of negative strands of the two different viruses were found to be discrete. identification and quantification the vrna puncta in coinfected cells was determined computationally using volocity software. this program determined the number of negative strand puncta per cell per strain and quantified how many puncta overlapped ( figure 6b ). this value was low even for the positive stranded vrnas, which are present in the cytoplasm at much higher frequencies ( figure 6a,b) . as a positive control for colocalization, we performed a similar experiment but additionally stained for ns5a or core in addition to minus strand vrnas. we would expect minus strand vrna and ns5a to colocalize strongly, as ns5a is present inside replication complexes. alternatively, core is not localized directly within replication complexes, but is present within packaging complexes and on lipid droplets, which are nearby. volocity was used to count negative strand vrnas, and then asked, how many of those puncta were touching ns5a or core. representative images demonstrating each of the pairwise comparisons demonstrate that nearly 80% of all minus strand vrnas were touching ns5a while fewer than half of the minus strand vrnas were touching core ( figure 6c,d) . these data support the hypothesis that, upon co-infection, drug-resistant and drug-susceptible rna genomes create independent membranous web structures, limiting the mixing of ns3/4a and ns5a proteins and their precursors. this scenario is modeled schematically in figure 6e . failure of ns5a proteins to mix during infection is a likely explanation for the cis-dominance of drug resistance observed in cultured cells (figure 4) . these circumstances account for the ready outgrowth of drug resistance in patients (gao et al., 2016) , even though ns5a is highly oligomeric. (berger et al., 2014) were created to co-express ha-and gfp-tagged ns5a alleles. huh7-lunet-t7 cells were transfected with ptm-dual-ns5a constructs that contained two drug-resistant (r), two drug susceptible (s), or mixed alleles of ns5a. proteins from transfected cell extracts that were incubated in the absence (b) or presence (c) of sr2486 were subjected to sds-page without further fractionation (input) or after immunoprecipitation with anti-ha antibodies (ip aha). the gel was subjected to immunoblotting with gfp or ha antibodies as indicated. (d) cells were transfected with dual-ns5a constructs in the absence or presence of sr2486 for 24 hr. cells were then fixed, stained with anti-ha antibodies and visualized by confocal microscopy. representative images from over 25 cells are presented. (e) cells transfected with dual-ns5a constructs that expressed drug-susceptible (s) and drug-resistant (r) alleles as shown were prepared for electron microscopy by highpressure freezing and freeze-substitution and visualized by transmission electron microscopy. even when drug-resistant ns5a was overexpressed in a precursor form, no rescue of drug-susceptible virus was detected (figure 3-figure supplement 2c ). it has previously been shown that when ha-and gfp-tagged ns5a molecules were expressed on different constructs such as those depicted in figure 5a , no mixed complexes were formed (berger et al., 2014) . thus, even though high-order ns5a oligomers are formed in infected cells, it is unlikely that these are mixed-allele oligomers, preventing dominant inhibition of drug-resistant hcv. due to the highly mutagenic nature of rna viruses and the large number of genomes and antigenomes generated during infection, a high barrier to drug resistance is extremely difficult to achieve. this has led to abandoned usage and development of many otherwise promising antivirals. to decrease the frequency with which drug-resistant variants arise, combinations of antivirals that, individually, exhibit low barriers to resistance are often used. when drug-resistant variants are first formed intracellularly, through error-prone rna replication, they arise in a population that includes parental and sibling drug-susceptible viruses. several genetic relationships between drug-resistant and drug-susceptible genomes are possible. first, the drug resistance of the new variants has the potential to be genetically dominant, and rescue both resistant and susceptible viral genomes. alternatively, drug resistance can be cis-dominant, with the drug-resistant products rescuing only the genomes that encode them. finally, the drug-resistant genome can fail to benefit any genomes in the cell because the drug-susceptible products present in the same cell are dominant inhibitors. the daas targeting ns3/4a protease of hcv were the first to be discovered (lamarre et al., 2003) and the first to reach the clinic jacobson et al., 2011) . it was soon realized that, both during the growth of hcv replicons in cultured cells and in phase ii clinical trials, drug-resistant viruses were generated rapidly (lin et al., 2004) . nonetheless, in 2011, further advances led to fda approval of telaprevir and boceprevir poordad et al., 2011) . the anticipation, which proved to be correct, was that inhibitors of ns3/4a would prove useful in combination therapies (feld et al., 2014; lawitz et al., 2014) . we have used flow cytometry to identify cell populations that are co-infected with hcv that is susceptible or resistant at a 1:1 ratio, to protease inhibitors. within these cells, the drug-resistant genomes replicated, but the drugsusceptible genomes did not. we therefore conclude that ns3/4a inhibitor resistance is cis-dominant (figure 3) , which should allow the rapid and specific selection for outgrowth from its cell of origin. cis-dominance of drug resistance was also not originally anticipated while targeting ns3/4a. the original characterizations of the ns3/4a protease suggested that cleavage of the ns3/4a junction occurred in cis, but that cleavages at the 4a/4b, 4b/5a and 5a/5b junctions could all occur in trans (bartenschlager et al., 1994) . we felt that it was therefore, more likely, that drug-resistant ns3/4a could rescue drug-susceptible virus within the same cell. ns3/4a is not known to assemble into high-order oligomers in the same manner as ns5a, and we therefore did not anticipate drug-susceptible ns3/4a would be trans-dominant. furthermore, a trans cleavage assay demonstrated that a ns4b-5b polyprotein could be cleaved by ns3/4a supplied in trans (romero-brey et al., 2015) . however, the trans-cleavage system does not result in membranous web formation that would accompany genome sequestration. other groups have reported a different result, that defective ns3 mutants cannot be rescued in trans by replicons with functional ns3/4a (kazakov et al., 2015; appel et al., 2005) . our interpretation of these studies is that ns3/4a is likely physically able to figure 6 continued (c) huh7.5.1 cells were coinfected with ca and wt-y93n at a moi of 1 ffu/cell for 24 hr. cells were costained to visualize core or ns5a together with ca and wt vrnas. representative cells are displayed demonstrating all pairwise comparisons analyzed for colocalization. (d) volocity was used to quantify the number of vrnas per cell, the number of colocalized vrnas, as well as the number of vrna puncta touching ns5a or core. (e) depiction of the clonal nature of individual rna replication sites (adapted from figure 9 in zayas et al., 2016] ). membrane invaginations house either drugresistant (red) or drug-susceptible (blue) genomes. in the model, the rna replication sites are segregated and therefore only the rna from drugresistant virus is amplified in the presence of inhibitors of rna replication. it is visually suggested that ns5a molecules that bring core protein to lipid droplets for viral assembly mix on this surface and that this could lead to genetic dominance of drug susceptibility at a packaging step. cleave in trans in cells, but requires access to the alternate precursor proteins in order for this to occur. therefore, cis-dominance of drug-resistance is likely the result of a lack of free-mixing of ns3/ 4a encoded by different vrnas within the same cell. ns5a emerged as an hcv drug target through a chemical genetics screen for compounds that inhibited hcv growth but did not target the ns3/4a protease or the ns5b polymerase (gao et al., 2010) . the ease with which resistant viruses were selected suggested that drug resistance was either dominant or cis-dominant. this was somewhat surprising, given that ns5a is oligomeric and the ns5a inhibitors are extremely potent, and have been postulated to function at sub-stoichiometric ratios to ns5a protein (gao, 2013) . indeed, when drug-susceptible and drug-resistant ns5a protein were co-expressed in the present study, hetero-oligomers formed and the biological phenotypes of the drug-susceptible protein were dominant ( figure 5) . nonetheless, single-cell analysis of cells coinfected at a 1:1 ratio with ns5a inhibitor-susceptible and -resistant viruses showed, as with the ns3/4a inhibitor, that resistance to both sr2486 and daclatasvir was cis-dominant ( figure 4) . what does cis-dominant resistance mean mechanistically? one potential mechanism is physical sequestration of the rna replication complexes of the two co-infecting genomes. genome-specific rna probing of co-infected cells revealed that both the negative strands and positive strands from the two viruses were present at physically distinct locations ( figure 6 ). it is therefore highly probable that membrane-associated proteins such as hcv ns3/4a and ns5a do not mix within individual rna replication complexes. however, not all mutations in a particular viral product should lead to the same defect with the same genetic properties. for example, we show here that viruses that are defective in a function of ns5a in rna replication complexes are not rescued and have no effect on the outgrowth of drug-resistant variants. however, ns5a also plays an important role in packaging and assembly of mature hcv particles on lipid droplets (miyanari et al., 2007; boson et al., 2017) . lipid droplets are large and form adjacent to rna replication complexes. in figure 6e , we have depicted the possibility that ns5a molecules encoded by distinct rna replication complexes might mix on the surface of these lipid droplets. however, if replication of the drug-susceptible genomes is inhibited, contribution of their encoded proteins to any oligomers on the surface of lipid droplets should be minimal. in this vain, a hypothetical ns5a inhibitor that allowed rna replication but inhibited the function of ns5a in particle assembly might have different genetic properties than the ns5a inhibitors currently in use. viral capsids have especially interesting genetic properties, often intermixing within co-infected cells. defective capsid proteins of poliovirus, hbv and hiv have been shown to be dominant inhibitors of wild-type viruses (crowder and kirkegaard, 2005; tanner et al., 2016; tan et al., 2013; tan et al., 2015; trono et al., 1989; pettit et al., 2005; lee et al., 2009; müller et al., 2009; checkley et al., 2010) . thus, when antiviral targets are capsid proteins, drug susceptibility can be genetically dominant by suppressing the outgrowth of drug-resistant virus within the cell in which it is first generated (crowder and kirkegaard, 2005; tanner et al., 2014; kirkegaard et al., 2016) . for hcv, very few inhibitors of capsid function have been identified, and their inhibition of viral growth is not sufficiently robust to make genetic experiments possible (kota et al., 2010) . it is therefore not yet possible to test if, as we hypothesize, drug-susceptible virus will prove to be a dominant inhibitor of drug resistance. consistent with this hypothesis, however, epitope-tagged hcv core protein can form mixed disulfide-bonded core oligomers (kushima et al., 2010) . the success of combination therapy for hcv and the efficacy of the individual constituents illustrate some of the weapons in the arsenal of antiviral strategies. future directions are likely to include, as well, the rational design of antivirals with high barriers to resistance such as those that hyper-stabilize oligomers and the prediction of daa targets that impart a high fitness cost to drug resistance. huh7.5.1 cells were a gift from dr. michael gale jr (university of washington) and were cultured in dmem (sigma, st. louis, mo) supplemented with 10% fetal bovine serum (omega, tarzana, ca), penicillin/streptomycin (invitrogen, grand island ny), non-essential amino acids (invitrogen), and glutamax (invitrogen). huh7-lunet-t7 cells were a gift from dr. ralf bartenschlager (university of heidelberg) and were cultured in dmem supplemented with 10% fetal bovine serum, penicillin/ streptomycin, non-essential amino acids, glutamax and 5 mg/ml zeocin (invitrogen). cell line identification was performed using str profiling services available through the stanford functional genomics facility. alignments were generated using huh7 as a reference. cell lines were screened for mycoplasma contamination using the mycoalert mycoplasma detection kit (lonza). the plasmid pjfh1 was a gift from dr. michael gale jr (kato et al., 2006) . this plasmid contains a synthesized genome length copy of the jfh1 strain of hcv (genotype 2a). to produce cell-culturederived hcv particles (hcvcc), pjfh1 was digested with xbai (new england biolabs). the linearized plasmid was then used as a template for in vitro transcription with the megascript high yield transcription kit (ambion). vrna was purified using trizol (invitrogen) and electroporated into huh7.5.1 cells as previously described to generate hcvcc cultures (wakita et al., 2005) . following a period of amplification, hcvcc cultures were converted to human serum media as described previously (steenbergen et al., 2013) . human serum media comprised dmem supplemented with 2% heat inactivated human serum (omega), penicillin/streptomycin, non-essential amino acids and glutamax. antibodies recognizing hcv core (abcam), gapdh (santa cruz biotechnologies), gfp (life technologies) and ha (genscript) were purchased from the individual suppliers. antibodies recognizing ns5a were described previously (lindenbach et al., 2005) . to construct codon-altered strains of hcv, we subjected three approximately 1000-nucleotide fragments of the jfh1 genome through the geneart codon optimization algorithm offered by life technologies. the genome fragments were composed of nucleotides 2613-3530 (ca-3), 7441-8456 (ca-2), and 7867-8896 (ca-1). all three codon-altered genome fragments were synthesized by life technologies and cloned into the pjfh1 plasmid by restriction digestion and ligation with t4 dna ligase (invitrogen). the resulting plasmids: pjfh1-ca-1, pjfh1-ca-2 and pjfh1-ca-3, were used to produce hcvcc cultures as described above. to create drug-resistant hcvcc cultures, two subcloning plasmids were created by pcr by amplifying nucleotides 6395-8670 or 4584-6498 of the pjfh1 plasmid with taq polymerase (new england biolabs) and ligating the pcr products into pcr2.1 (invitrogen). the resulting plasmids, pcr2.1-6395-8670 and pcr2.1-4584-6498 were used as templates for site-directed mutagenesis using the quikchange site-directed mutagenesis kit (agilent technologies). pcr2.1-6395-8670-y93n was generated using the forward primer 5'-cctatcaattgcaatacggagggccag tgcgcgcc-3' and the reverse primer 5'-ggcgcgcactggccctccgtattgcaattgatagg-3'. pcr2.1-6395-8670-y93h was generated using the forward primer 5'-cctatcaattgccatacg-gagggccagtgcgcgcc-3' and the reverse primer 5'-ggcgcgcactggccctccgtatgg-caattgatagg-3'. pcr2.1-4584-6498-d168a was generated using the forward primer 5'-aaatccatcgccttcatcccc-3' and the reverse primer 5'-ggggatgaaggcgatggatttggc-3'. these mutated hcv genome fragments were cloned into pjfh1 or pjfh1-ca using restriction digestion and ligation with t4 dna ligase (invitrogen). hcvcc cultures were generated as described above. the plasmids ptm_ns3-5b_ns5a-ha_2a_ns5a-gfp_jfh1 (referred to as ptm-dual-ns5a) and ptm_ns3-5b_ns5a-gfp (referred to as ptm-ns3-5b) were the generous gifts of dr. ralf bartenschlager (university of heidelberg). the d168a and y93n mutations were cloned into the ptm-ns3-5b plasmid using the quikchange lightening mutagenesis kit using the primers described above. the ns5a alleles of the ptm-dual-5a plasmid were first separated by removing an rsrii fragment containing most of the ns5a-gfp allele to create ptm-dual-5a-drsrii and pcdna5-ns5a-gfp-rsrii. site-directed mutagenesis was performed using the quikchange lightening kit on ptm-dual-5a-drsrii or on pcdna5-ns5a-gfp-rsrii independently. the rsrii fragments containing wildtype ns5a or ns5a-y93n were then cloned back into the ptm-dual-5a-drsrii vectors to create all combinations of wild-type ns5a and ns5a-y93n ptm-dual-5a. vrna was harvested from cells using trizol (invitrogen) or collected from hcvcc culture supernatants using the qiaamp vrna mini kit (qiagen). a standard curve was generated using in vitro transcribed hcv vrna. qrt-pcr was performed using the quantitect sybr-green rt-pcr kit (qiagen) and the qrt-pcr forward 5'-ctggcgactggatgcgtttc-3' and reverse 5'-cgcattcctccatc tcatca-3' primers. alternatively, the following ca-specific primers were used: forward 5'-gtggtg tccatgaccggca-3' and reverse 5'-ggtcacggggcctctcagt-3', or the following wt-specific primers were used: forward 5'-gtggtgagtatgacggggc-3' and reverse 5'-cgtgaccg-gaccccgtaag-3'. samples were analyzed on a 7300 real-time pcr machine (applied biosystems). wt vrna target probes recognizing the ns2 region of either the positive or negative strand were designed and synthesized by affymetrix. these probes were specifically designed to avoid detection of codon-altered jfh1 viral rna. additionally, probes were designed to recognize the corresponding region of the negative or positive strand jfh1-ca vrna. these ca target probes were specifically designed not to recognize the wt vrna. huh7.5.1 cells were infected with wt or ca hcvcc particles for 72 hr. infected cells were fixed with 4% formaldehyde solution (sigma) and subjected to rna in situ hybridization (ish) using the viewrna cell assay kit (affymetrix) according to the manufacturer's protocol. cells were co-stained with both ca and wt vrna target probe sets in all experiments. cells were visualized on a leica sp8 confocal microscope. protein and vrna colocalization was performed on cells coinfected with jfh1-ca and jfh1-y93n for 24 hr. following infection, cells were fixed and stained using the view-rna cell plus assay reagents. core and ns5a were visualized using the antibodies described above at a 1 to 100 dilution followed by the anti-mouse-alexaflour-647 secondary antibody at 1 to 200 dilution. quantification of colocalization was performed using volocity software (perkin elmer). briefly, we defined vrna puncta as objects larger than 0.1 mm 2 . objects larger than 0.25 mm 2 were broken into subunits based on total volume. objects sharing 0.05 mm 2 of mutual space were quantified as mutual. due to the localization patterns of core and ns5a, spot counting algorithms were not appropriate. total vrna objects and as well as the total number of vrna objects touching ns5a or core were quantified. huh7-lunet-t7 cells were transfected with ptm-dual-ns5a constructs using branched polyethylenimine (sigma-aldrich) at a ratio of 1:3. at 4 hr post-transfection, cells were treated with 500nm sr2486 or a dmso control. at 24 hr post-transfection, cells were fixed with 4% paraformaldehyde, stained with anti-ha antibodies and dapi and visualized on a leica sp8 confocal microscope. a more detailed description of the rna fish microscopy methods are provided in bio-protocol (van buuren and kirkegaard, 2018) . huh7-lunet-t7 cells were transfected with ptm-dual-ns5a constructs using the polyethylenimine transfection reagent. at 4 hr post-transfection, cells were treated with dmso or 500nm sr2486. at 24 hr post-transfection, cells were harvested using an enzyme-free cell dissociation buffer (life technologies) and facs sorted for gfp-positivity on a facs aria cell sorter. gfp-positive cells were resuspended in 20% bsa in pbs then placed into a 200 mm deep hat and high-pressure frozen using a leica empact2. frozen samples were then freeze substituted in 1% osmium tetroxide and 0.1% uranyl acetate in acetone using a leica emafs at à90˚c for 72 hr, warmed to à25˚c in 16.3 hr at 4˚c/ hr and held for 12 hr then warmed to 0˚c in 5 hr at 5˚c/hr and held for 12 hr. the samples were then washed two times in acetone, then in propylene oxide for 15 min each. samples are infiltrated with embed-812 resin (ems cat#14120) mixed 1:2, 1:1, and 2:1 with propylene oxide for 2 hr each, leaving samples in 2:1 resin to propylene oxide overnight rotating at room temperature. the samples are then placed into embed-812 for three hours then placed into taab capsules with fresh resin and placed into a 65˚c oven overnight. sections were taken between 75 and 90 nm, picked up on formvar/carbon coated 100 mesh copper grids, then contrast stained for 30 s in 3.5% uranyl acetate in 50% acetone followed by staining in 0.2% lead citrate for 3 min. cells were visualized using the jeol jem-1400 120kv microscope and photos were taken using a gatan orius 4k â 4k digital camera. huh7.5.1 cells were either transfected with wt and/or ca vrna as previously described or infected with wt and/or ca hcvcc particles. coinfections were performed by infecting with each virus for 24 or 72 hr followed by treatment with either 2 mm biln-2061 for 36 hr or 500nm sr2486 for 24 hr. cells were harvested with trypsin and fixed with the flowrna fixation and permeablization kit. cells were then costained with ca and wt vrna target probe sets using the flowrna kit (affymetrix) and analyzed on the scanford facscan flow cytometer. data was analyzed and processed using flowjo software. mutational and fitness landscapes of an rna virus revealed through population sequencing ledipasvir and sofosbuvir for untreated hcv genotype 1 infection quantigene flowrna assay. a novel rna ish multicolor application for flow cytometry efficient rescue of hepatitis c virus rna replication by transcomplementation with nonstructural protein 5a boceprevir for previously treated chronic hcv genotype 1 infection kinetic and structural analyses of hepatitis c virus polyprotein processing daclatasvir-like inhibitors of ns5a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication permissive secondary mutations enable the evolution of influenza oseltamivir resistance daclatasvir prevents hepatitis c virus infectivity by blocking transfer of the viral genome to assembly sites the capsid-spacer peptide 1 gag processing intermediate is a dominant-negative inhibitor of hiv-1 maturation complete three-dimensional structures of picornaviral rna-dependent rna polymerases trans-dominant inhibition of rna viral replication can slow growth of drugresistant viruses coronaviruses resistant to a 3c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance daclatasvir plus sofosbuvir for hcv infection general catalytic deficiency of hepatitis c virus rna polymerase with an s282t mutation and mutually exclusive resistance towards 2'-modified nucleotide van oral direct-acting agent therapy for hepatitis c virus infection: a systematic review treatment of hcv with abt-450/r-ombitasvir and dasabuvir with ribavirin combining oncolytic virotherapy and cytotoxic therapies to fight cancer distinct functions of ns5a in hepatitis c virus rna replication uncovered by studies with the ns5a inhibitor bms-790052 chemical genetics strategy identifies an hcv ns5a inhibitor with a potent clinical effect antiviral activity and resistance of hcv ns5a replication complex inhibitors hcv ns5a replication complex inhibitors dominant negative effect of wild-type ns5a on ns5a-adapted subgenomic hepatitis c virus rna replicon virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions telaprevir for previously untreated chronic hepatitis c virus infection cell culture and infection system for hepatitis c virus hepatitis c virus rna replication depends on specific cis-and trans-acting activities of viral nonstructural proteins origins of combination therapy for tuberculosis: lessons for future antimicrobial development and application my cousin, my enemy: quasispecies suppression of drug resistance ná jera i. 2006. the novel nucleoside analog r1479 (4'-azidocytidine) is a potent inhibitor of ns5b-dependent rna synthesis and hepatitis c virus replication in cell culture ml322, a small molecule inhibitor of dimerization of the core protein of hepatitis c virus (hcv) a disulfide-bonded dimer of the core protein of hepatitis c virus is important for virus-like particle production an ns3 protease inhibitor with antiviral effects in humans infected with hepatitis c virus sofosbuvir for previously untreated chronic hepatitis c infection ribavirin, to treat chronic infection with hepatitis c virus genotype 1 in non-responders to pegylated interferon and ribavirin and treatment-naive patients: the cosmos randomised study a strongly transdominant mutation in the human immunodeficiency virus type 1 gag gene defines an achilles heel in the virus life cycle discovery of potent hepatitis c virus ns5a inhibitors with dimeric structures in vitro resistance studies of hepatitis c virus serine protease inhibitors, vx-950 and biln 2061: structural analysis indicates different resistance mechanisms complete replication of hepatitis c virus in cell culture novel therapies for hepatitis c -one pill fits all? suppression of drug resistance in dengue virus the r263k substitution in hiv-1 subtype c is more deleterious for integrase enzymatic function and viral replication than in subtype b the lipid droplet is an important organelle for hepatitis c virus production hiv-1 gag processing intermediates trans-dominantly interfere with hiv-1 infectivity processing sites in the human immunodeficiency virus type 1 (hiv-1) gag-pro-pol precursor are cleaved by the viral protease at different rates the coding region of the hcv genome contains a network of regulatory rna structures boceprevir for untreated chronic hcv genotype 1 infection three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication ns5a domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis c virus replication viral mutation rates analysis of hepatitis c virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes understanding the hepatitis c virus life cycle paves the way for highly effective therapies human serum leads to differentiation of human hepatoma cells, restoration of very-low-density lipoprotein secretion, and a 1000-fold increase in hcv japanese fulminant hepatitis type 1 titers resensitizing daclatasvir-resistant hepatitis c variants by allosteric modulation of ns5a l159f and v321a sofosbuvir-associated hepatitis c virus ns5b substitutions genetically altering the thermodynamics and kinetics of hepatitis b virus capsid assembly has profound effects on virus replication in cell culture the interface between hepatitis b virus capsid proteins affects self-assembly, pregenomic rna packaging, and reverse transcription dominant drug targets suppress the emergence of antiviral resistance exploiting genetic interference for antiviral therapy structure of the zinc-binding domain of an essential component of the hepatitis c virus replicase hiv-1 gag mutants can dominantly interfere with the replication of the wild-type virus detection and differentiation of multiple viral rnas using branched dna fish coupled to production of infectious hepatitis c virus in tissue culture from a cloned viral genome treatment with ledipasvir and sofosbuvir improves patient-reported outcomes: results from the ion-1, -2, and -3 clinical trials coordination of hepatitis c virus assembly by distinct regulatory regions in nonstructural protein 5a we thank drs. yury goltsev and garry nolan for advice on fluorescent cell sorting-based visualization of rna, drs. michael gale jr. and ralf bartenschlager for the generous donation of reagents and dr. peter sarnow for critical reading of the manuscript. we would like to acknowledge the work of drs. jeannie spagnolo and ernesto mendez for initiating hcv research in our laboratory. this work was supported by funding to kk from nih u19ai109662 (jeffrey glenn, pi), an nih director's pioneer award and the alison and steve krausz innovation fund. nvb was supported by the canadian institutes for health research ncrtp-hepc training program and the american liver foundation. electron microscopy was performed in a facility supported in part by arra award number 1s10rr026780-01 from the national center for research resources (ncrr). the cell sciences imaging facility used for confocal microscopy was supported by arra award number 1s10od010580 from the ncrr. the contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the ncrr or the national institutes of health. key: cord-285505-8norumv6 authors: vere hodge, r. anthony title: meeting report: 27th international conference on antiviral research, in raleigh, nc, usa date: 2014-09-16 journal: antiviral res doi: 10.1016/j.antiviral.2014.08.009 sha: doc_id: 285505 cord_uid: 8norumv6 the 27th international conference on antiviral research (icar) was held in raleigh, north carolina, usa from may 12 to 16, 2014. this article summarizes the principal invited lectures. john drach (elion award) described the early days of antiviral drugs and their novel modes of action. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept nucleoside analogs. replacing thymine by 5-chlorouracil led to the generation of a new form of escherichia coli. adrian ray (prusoff award) demonstrated how prodrugs can markedly improve both the efficacy and safety of potential drugs. the keynote addresses, by david margolis and myron cohen, tackled two emerging areas of hiv research, to find an hiv “cure” and to prevent hiv transmission, respectively. these topics were discussed further in other presentations – a cure seems to be a distant prospect but there are exciting developments for reducing hiv transmission. tdf-containing vaginal rings and gsk-744, as a long-lasting injection, offer great hope. there were three mini-symposia. although therapy with tdf/ftc gives excellent control of hbv replication, there are only a few patients who achieve a functional cure. myrcludex, an entry inhibitor, is active against both hbv and hdv. the recent progress with hbv replication in cell cultures has transformed the search for new antiviral compounds. the hbv capsid protein has been recognized as key player in hbv dna synthesis. unexpectedly, compounds which enhance capsid formation, markedly reduce hbv dna synthesis. the development of bcx4430, which is active against marburg and ebola viruses, is of great current interest. this article provides an overview of the invited lectures at the 27th international conference on antiviral research, sponsored by the international society for antiviral research (isar), which was held in raleigh, north carolina, usa from may 12 to 16, 2014 . it begins with reports of lectures by the recipients of isar's three major awards, held in memory of gertrude elion, antonín holý and william prusoff. these are followed by brief summaries of the keynote addresses and the three mini-symposia on ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. because this review article simply provides short accounts of oral presentations, it is not generally accompanied by references to the scientific literature. any descriptions of favorable treatment outcomes should not be taken as recommendations for clinical use. 2. gertrude elion memorial award lecture: collaborative antiviral studies for the discovery of drugs to treat cytomegalovirus infections john c. drach, ph.d., university of michigan, ann arbor, michigan, usa (fig. 1) . gertrude b. (trudy) elion was born in new york city and was pleased to work for the burroughs wellcome co. when based in new york but was concerned when it transferred to research triangle park, north carolina, not many miles from this year's meeting site. however, within just a few months she declared that she was ''at home'' in north carolina. she was awarded the nobel prize in physiology or medicine in 1988 for her pioneering work in purine biosynthesis which paved the way for the discovery of drugs to treat organ rejection, cancer and viral diseases. the focus of john's presentation was on the research conducted in his own and his collaborators' laboratories that ultimately led to the invention of three compounds which were discovered to have antiviral activity against human cytomegalovirus (hcmv) and which later entered clinical trials: bdcrb pyranoside (gw275175x) (phase i), maribavir (phases i, ii and iii) and cyclopropavir (phase i). his major collaborators included karen biron, charles shipman, leroy townsend, and jiri zemlicka. to date, there are only five fdaapproved drugs for treatment of hcmv infections: cidofovir, fomivirsen, foscarnet, ganciclovir and valganciclovir. being inspired by the presence of a naturally-occurring 5,6dimethylbenzimidazole nucleotide in vitamin b12, research on benzimidazole nucleosides was initiated by medicinal chemists in the 1950s and '60s. this led to the synthesis of a trichloro analog in townsend's laboratory at the university of utah and later the discovery of its activity against hcmv in john's laboratory. much work, in both their laboratories at the university of michigan, established that it and its 2-bromo analog (bdcrb) have excellent activity against hcmv with very low cytotoxicity. surprisingly, it was found to be inactive against other herpes viruses and it did not need conversion to a triphosphate to be active against hcmv. collaborative studies with karen biron at burroughs wellcome established that, unlike many other anti-virals that inhibit viral dna synthesis such as ganciclovir (gcv), these compounds acted by a novel mechanism, inhibition of viral dna processing. it was the viral resistance studies which revealed the viral targets, pul89 and pul56. these two proteins, with pul104, form a complex known as the terminase which cuts newly synthesised hcmv dna into unit lengths for packaging into virions. although bdcrb had many desirable properties in vitro, it had poor pharmacokinetics in mice and monkeys due to hydrolysis of its glycosidic bond; therefore it was not developed for human use. much additional work in drach's and townsend's laboratories at michigan and by biron's group at burroughs wellcome ultimately led to two potential drug candidates, bdcrb pyranoside and maribavir (fig. 2) . both compounds have excellent activity against hcmv, low toxicity, and excellent pharmacokinetics. clearly, their modes of action differed markedly from that of gcv. quite unexpectedly, they have different mechanisms of action. bdcrb pyranoside has a mechanism of action very similar to its parent compound bdcrb, inhibition of dna processing. in contrast, maribavir inhibits dna synthesis, albeit indirectly. it is a 2isopropylamine derivative of bdcrb except that it has the unnatural l-sugar configuration. its mechanism of action involves inhibition of the viral kinase (pul97), which phosphorylates another viral protein, pul44. phosphorylated pul44 is necessary for viral dna synthesis. thus inhibition of pul97 by maribavir inhibits viral dna synthesis. interestingly, pul97 is also the kinase that activates (phosphorylates) gcv. resistance studies confirmed that a single mutation in ul97, resulting in a mutation in the kinase (leu397arg), was necessary and sufficient for resistance to maribavir. in a further study of resistance, virus already resistant to bdcrb was passaged in increasing concentrations of maribavir and resistant virus was isolated. this strain grew at the same rate as the wild-type virus and was resistant to both bdcrb and maribavir. as expected, resistance to bdcrb was due to known mutations in ul56 and ul89. however, no mutations were found in ul97. further investigation showed that a single base change in ul27 (t1004c) was necessary and sufficient for resistance to maribavir. the role of the encoded protein was then unknown but the amino acid mutation (leu335pro) is in the middle of the protein. similarly, biron's group detected resistance due to mutations in the ul27 gene. further research studies on maribavir have been summarized in previous icar scientific reports. cyclopropavir (cpv, fig. 3 ) was synthesized in the laboratory of jiri zemlicka, karmanos cancer institute, detroit, michigan. it is a guanosine nucleoside analog which is very active against hcmv. unlike the benzimidazole nucleosides, it also inhibits epstein-barr virus (ebv) and human herpesvirus 8 (hhv-8). like gcv, it is phosphorylated by the kinase encoded by ul97. it is more potent in vitro and in vivo than ganciclovir but has a somewhat different pattern of resistance. in one resistant strain, the key mutation formed a stop codon resulting in a truncated pul97 kinase protein. the phosphorylation of cpv by pul97 is more efficient than that of gcv, with a considerably lower k m and higher v max . interestingly, the phosphorylation of cpv to its monophosphate (cpv-mp) by pul97 is stereoselective; only the (+) isomer of cpv-mp is formed. a single enzyme, gmp kinase, phosphorylates cpm-mp to both its di-and triphosphates. in contrast, acyclovir and gcv require additional cellular enzymes to convert their diphosphates to active triphosphates. cyclopropavir is currently in phase i clinical trials for the treatment of hcmv infections. 3. the antonín holý memorial award lecture: from modified nucleoside to a chemically modified genome piet herdewijn, rega institute for medical research, ku leuven, belgium (fig. 4) . the 2013 icar began with a symposium, on the legacy of the late antonín (tony) holý , at which the establishment of a new isar award in medicinal chemistry was announced. the awardee is to be a senior scientist of international stature in medicinal chemistry and who has made innovative contributions impacting antiviral drug discovery or development. piet is, therefore, the first to receive this award. in the late 1970s, the potent activities of bvdu and bvarau against herpes simplex virus type 1 (hsv-1) and varicella zoster virus (vzv) were discovered; this work motivated piet to start antiviral research with the synthesis of carbocyclic bvdu. through to the early 1990s, he synthesized several other nucleoside analogs with bicyclic bases having good activity against hsv-1 and vzv. during the 1990s, emphasis switched to investigating the effect of modifying the sugar ring, in particular the synthesis of six-membered rings containing an oxygen or a double bond. piet showed examples of compounds with activity against hsv-1, hsv-2, vzv and hcmv. back in 1984, erik de clercq showed piet a paper on aids, one of the authors being phil furman. this publication stimulated the search for anti-hiv compounds. many compounds were discovered with potent activities (and good selectivity indices) against hiv. piet worked out the first structure-activity relationships of anti-hiv dideoxy nucleosides. starting in the late 1980s, tony holý synthesised a series of phosphonates. at the 2013 icar, erik de clercq recalled how this work led, ultimately, to tenofovir, which was to become a major success for treating hiv-infected patients. from its first introduction in 2001, its market share has increased to well over 40%. in 2002, having a single-pill regimen was agreed as a way forward to simplify, and thereby enhance, hiv therapy. this led to atripla being approved in 2006, complera in 2011 and stribild in 2012. tenofovir, in its various prodrug forms, is now available in over 130 countries and is distributed widely to the known hivinfected population. in line with this research, piet synthesized phosphonate nucleosides, with a threose sugar moiety, which showed anti-hiv activity in the same range as 9-(2-phosphonylmethoxyethyl) adenine (pmea). piet's work had taken a different pathway. it is possible to link several nucleotides together to form aptamers. for example (fig. 5) , the above antiviral nucleosides, which have a 6-membered ring in place of the natural furanose, could be incorporated into hexitol nucleic acid (hna) aptamers. x-ray studies revealed the structures of hna-rna duplexes and hna-hna duplexes, the latter having a similar overall form to that of an rna-rna duplex with the same base sequence. hna-containing aptamers were shown to be potent and specific inhibitors of trans-activating region (tar)mediated transcription. normally, an hiv encoded protein, transactivator of transcription (tat), binds to cellular factors and to the viral tar rna regulatory element, resulting in a vastly increased rate of transcription of all hiv genes. hna-containing aptamers prevents this interaction and so inhibit hiv replication. it took four years to engineer a polymerase that would utilise hnas to assemble a strand complementary to a dna template. in line with this research, hexitol-modified sirna has shown good activity in an in vivo anti-hbv model. this success stimulated the concept that it may be possible to generate new forms of biologically active dna. in order to pursue this idea, a culture system with twin growth chambers was devised. alternative nutrient media could be fed into the chambers and the culture from one chamber could be used to seed the second chamber, the former culture being removed. in this example, the aim was to replace thymine with 5-chlorouracil ( fig. 6) using escherichia coli. initially, the nutrient contained 10% 5-chlorouracil and 90% thymine. with each cycle, seeding one chamber from the previous one, the proportion of 5-chlorouracil was increased. after 180 days, in which there had been about 4000 generations of e. coli, thymine had been replaced totally by 5-chlorouracil. an interesting outcome was that the alternative base led to a change not only in the genotype but also in the phenotype; the ''new'' e. coli cells were much longer than the original. this is the first example of a dna polymerase being adapted through evolutionary pressure to accept a nucleotide analog, resulting in the generation of a new living organism. prusoff young investigator award lecture: use of nucleotide prodrugs to enhance selectivity of anti-hiv and -hcv agents adrian s. ray, gilead sciences inc., foster city, ca, usa (fig. 7) . adrian started his lecture with photos of william (bill) prusoff and reminisced of his days with bill, raymond schinazi and yung-chi (tommy) cheng. adrian presented examples to illustrate two models of how a prodrug strategy can transform a potential drug into a much improved clinical candidate. in the first, the prodrug alters the distribution of the pharmacologically active nucleotide analog to tissues where viral infection is taking place (on-target) and away from tissues resulting in adverse events (off-target). in the second, the prodrug enables one to select a drug candidate based more directly on the intrinsic properties of the active nucleotide-triphosphate analog via by-passing an inefficient activation (phosphorylation) of the corresponding nucleoside analog. sofosbuvir (sovaldi ò ), a prodrug of 2 0 -f-2 0 -c-meump, was approved in the usa on 6th december, 2013 for treatment of patients with hepatitis c. this is a fine example of a prodrug enhancing the activity of the parent compound. the nucleoside analogue, 2 0 -f-2 0 -c-meu, is poorly active due to restricted phosphorylation to the monophosphate. sofosbuvir, a nucleotide analogue prodrug of 2 0 -f-2 0 -c-meu, delivers the monophosphate into the cell and this is then further phosphorylated efficiently to give high levels of the triphosphate which inhibits hcv rna polymerase. adrian recalled being much impressed by a result reported at the meeting in 2007 of the american association for the study of liver diseases (aasld). in a phase ii monotherapy trial in patients with hcv, at day 3, the viral loads were reduced by log 10 3.2 and log 10 1.1 for vx-950 (1250 mg bid, n=10) and rg-7128 (1500 mg bid, n=8), respectively. however, from day 4 to 13, the polymerase inhibitor (rg-7128) had continued to reduce the viral load, reaching a reduction of log 10 2.7. on the other hand, the protease inhibitor (vx-950) did not give a sustained reduction, with the viral load starting to increase from day 6. at day 13, the viral load was only log 10 2.2 less than baseline. nucleotide analogues have two advantages over other classes of inhibitors. there is a high genetic barrier to resistance selection, due to the hcv rna polymerase being highly specific for its natural substrates and template. this specificity can be altered but only under extreme evolutionary pressure (see section 3). also, nucleotide analogs often have pan-genotype activity because the active site of the hcv ns5b polymerase is so highly conserved. as an example of how prodrugs can impact a discovery program, allowing for more targeted delivery and for the optimization of the intrinsic properties of the triphosphate, adrian presented the history of the gs-6620 program. the c-adenine analogue (2 0 -c-me-4-aza-7,9-dideazaa, c-nuc1) was compared to the corresponding n-nucleoside, mk608. in a genotype1b replicon assay, the ec 50 values were 2.5 lm and 0.08 lm respectively. however, their triphosphates were equally effective against hcv ns5b polymerase (ic 50 values both 0.3 lm). in the replicon system, the triphosphate of the n-nuc (mk608) was formed more efficiently than that of the c-nuc1, thus explaining the lower activity of the c-nuc1. however, in primary human hepatocytes, c-nuc1 was phosphorylated to the triphosphate more efficiently than the n-nuc (mk608). this illustrates the importance of using primary human cells. c-nuc1 seemed to have a benign in vitro toxicity profile, including not inhibiting the mitochondrial dna polymerase-gamma, but it had very significant toxicity in animals. in a collaboration between gilead and craig cameron at pennsylvania state university, the researchers sought to identify the toxicity target(s) for ribonucleotide analogues, including c-nuc1 and others that had been stopped in phase ii trials. these studies showed a correlation between c-nuc1 and the phase ii candidates, r1626, nm283 and bms986094/idx184. all the latter were efficiently incorporated into rna by the mitochondrial rna polymerase (>70% of the corresponding natural nucleotide). the triphosphate of c-nuc1 was also an efficient substrate (22% the rate of atp). in contrast, the active nucleotide analogs, formed by drugs approved for the treatment of hcv, were poor substrates. ribavirin was poorly incorporated (about 5%) and sofosbuvir was below the limit of detection (= 0.02%). more extensive in vitro and cell culture evaluation of the compounds could have saved the expense of taking them into clinical trials. understanding that the mitochondrial rna polymerase is an important target for ribonucleotide toxicity, the gilead team sought analogs that were not incorporated by this polymerase. adding a cn group to the 1 0 position of c-nuc1 did not change its activity as an hcv ns5b polymerase inhibitor (ic 50 0.3 lm) but it did reduce incorporation in the mitochondrial rna assay (<0.02%). however, in the absence of a nucleotide prodrug to bypass the first phosphorylation step, the resulting di-substituted nucleoside analog would not be a drug candidate because it was not efficiently activated in cells. application of a nucleotide prodrug strategy allowed this nucleotide to be pursued further. oral absorption, delivery of the monophosphate into hepatocytes and high hepatic extraction were criteria used as part of the prodrug optimization process. a nucleotide prodrug, gs-464335 (a mixture of diastereoisomers at phosphorous) was well absorbed in dogs (>80%). comparing the pre-hepatic and post-hepatic plasma drug levels, about 80% of the absorbed drug was taken up by the liver. inside cells, gs-464335 was converted to the corresponding monophosphate which was efficiently converted to the triphosphate. at 24 h, the triphosphate levels remained about 2-fold above the ic 90 value. a pure stereoisomer was selected and later named gs-6620. in a phase ii trial (900 mg, bid 5 days), the mean reduction in hcv load was about log 10 1.5. two subjects achieved hcv rna <25 iu/ ml. however, the pharmacokinetics and antiviral responses were highly variable. whereas the activity results were disappointing, clinical proof of concept was observed in terms of safety. gs-6620 did have a markedly improved safety profile relative to c-nuc1, progressing through chronic toxicology studies in rats and dogs at relatively high doses. the story of gs-6620 illustrates both how nucleotide prodrugs enable further progression of candidates and also the complexity of predicting the behavior of nucleotide prodrugs across species. one wonders what cell culture test or animal model may have predicted such variability. when selecting famciclovir as the prodrug for penciclovir, one potential prodrug was rejected because the pharmacokinetics in rats varied widely between individual animals (vere hodge et al., 1989) . a recent publication by adrian and his team highlights the metabolism of gs-6620 by carboxylesterase 2, an enzyme highly expressed in the human small intestine but not uniformly expressed in different animal species, as a possible reason for the highly variable and suboptimal intestinal absorption of gs-6620 in humans (murakami et al., 2014) . the focus of adrian's talk then switched to hiv. over the last 15 or 20 years in north america, the hiv-infected population has been changing, becoming older (now 33% over 50 years old vs <10% in 1995) and more likely to be obese (in every usa state, >20% adults with bmip30). this has led to a shift in the focus of antiretroviral therapy (art), from solely control of hiv replication to now include tolerability in older, possibly obese, patients. the first example given for hiv was how application of a different prodrug strategy can markedly change the distribution even when delivering the same pharmacologically active nucleotide analog. the first approved prodrug of tenofovir (tfv) was tfv disoproxil fumarate (tdf). more recently, tfv alafenamide (taf) has been progressed into clinical development. a key difference in the properties of the two prodrugs is their stability in plasma, with half-lives of 0.4 and 90 min, respectively. even with a short halflife, tdf gave better delivery of tfv into cells, as indicated by the hiv ec 50 values in cell culture assays but there clearly was room for improvement; the ec 50 values for tfv, tdf and taf are 1.2, 0.015 and 0.003 lm respectively. whereas the gain in cell culture ec 50 value may be modest, this is not the only gain. the increased stability of taf allows it to load on-target cells and tissues (e.g., lymph nodes) for a longer period of time resulting in increased lymphoid cell and tissue levels at greatly reduced circulating tfv levels, leading to less exposure to off-target tissues (e.g., kidney). in monotherapy studies after oral dosing with tdf (300 mg) and taf (25 mg), the plasma tfv auc is reduced from 1920 to 268 ng.h/ml respectively whereas the reduction in hiv load from baseline is improved, from log 10 0.97 to log 10 1.46 copies/ml, respectively, reflecting the more efficient delivery of taf to target cells and tissues. clearly the lower dose of taf (25 mg) relative to tdf (300 mg) will give taf a marked advantage when considering combination pill therapy. understanding how marked a difference a prodrug can make from the taf example, adrian went on to describe how a prodrug approach transformed a new nucleotide project in which intrinsic properties of the pharmacologically-active nucleotide analog were optimized. their starting point was gs-2128 (d4api), which had good activity against both wild-type and resistant hiv strains but was an active inhibitor of mitochondrial polymerase-gamma. on comparing the known structures of hiv rt and mitochondrial polymerase-gamma, differences in the 2 0 -binding pocket were noted. this led to gs-9148 in which 2 0 -f was added to gs-2128 (fig. 8) . compared to tfv, gs-9148 was about 3-fold less active against wild-type hiv but maintained better activity against resistant strains (k65r and multiple thymidine analog resistance mutations). most importantly, it was inactive (ic 50 >300 lm) against mitochondrial polymerase gamma. more than 50 prodrugs were synthesized and evaluated in metabolism studies and in dogs (intravenous and oral administration). then the enantiomers were tested separately in dogs. this led to the selection of gs-9131. whereas tfv is efficiently utilised by renal uptake transporters, gs-9148 was poorly taken into the kidney. no adverse renal findings were observed with the prodrug (gs-9131) in 28-day studies in rats, dogs and monkeys at the highest doses tested (300 mg, 20 mg and 30 mg/kg daily, respectively). in summary, this work has given examples of the prodrug approach being used successfully both to increase selectivity (by loading on-target tissues vs off-target tissues) and to increase activity (via by-passing metabolic constraints). adrian presented cases in which a prodrug strategy was able to fulfil the full potential of a selective, active triphosphate analog and enable its further progression as a clinical candidate. the keynote speakers were david margolis and myron cohen (fig. 9) . david margolis, university of north carolina, nc, usa in hiv-infected patients, there is a long-lasting reservoir of hiv in the form of integrated viral dna in resting cd4+ memory cells of the host immune system. therefore, even if it were possible to eliminate 100% of viral replication, a reservoir of hiv would remain. there may be reservoirs in other long-lived cells. to date, there is only one known hiv patient who has been cured of his infection, the ''berlin patient''. he was treated for cancer by chemotherapy followed by a bone-marrow transplant. being ccr5 +/à, the chemotherapy had a greater chance to remove all the ccr5+ve cells. the bone marrow donor was ccr5àve. although this patient continues to have no sign of hiv infection, this is hardly a viable treatment option for most hiv-infected patients. even in subjects with hiv replication well controlled by therapy, 70% have detectable plasma viremia which does not appear to decay over time (at least two years). to improve the sensitivity of the assay for hiv, 4 billion lymphocytes are mixed with antibody attached to magnetic beads. this selects for the cd4+ t cells, about 0.2-1 billion cells. the limit of detection is 1 copy of hiv rna/million cells, limit of quantitation is 10 copies/million cells. to reduce the reservoir of hiv, it was suggested that activation of integrated hiv in resting cd4+ t cells would give renewed hiv rna synthesis and possibly result in cell death either due to viral cytopathic effects or resulting from hiv-specific immune responses. a small clinical trial was set up to test this hypothesis. vorinostat (vor), a clinically approved drug for treating certain cancers, has been shown to bind to the active site of histone deacetylases. after a single dose, there was an increase in hiv rna (1.5 to 5-fold, mean 2.6-fold). of these subjects, 5 elected to continue with multiple doses. from the 11th to 22nd vor dose, acetylation of histones and activation of hiv rna synthesis became refractory to therapy. also, it is not known what proportion of cells, with latent hiv, can be activated. whereas a single vor dose did increase the expression of hiv rna, this is not an effective therapy for removing the hiv reservoir. myron cohen, university of north carolina, nc, usa myron noted that there are 2.5 million new hiv infections each year. in this context, anal sex may be an important factor because just one or a few virions of hiv can be infective; within 3 weeks, there is rapid virus replication throughout the body and latent hiv reservoirs of ''founder virus'' are already formed. although anal sex has been associated with homosexual couples, myron pointed out that it is not uncommon amongst heterosexual couples. although behavioral education should be encouraged, it can never be the whole answer. various approaches to the prevention of hiv transmission are being evaluated. monoclonal antibodies, broad neutralising antibody (bnab) and vaccines may have potential for prevention of transmission, but most progress is being made with dapivirine rings containing tdf. these are designed to stay in the vagina for a month. phase iii trials are ongoing. a long-acting hiv integrase inhibitor, gsk 1265744 (generally known as gsk 744), is administered i.m. once every 3 months; a two-year safety trial will be required. phase i trial has been completed and phase ii trial is being planned. by analogy with tuberculosis therapy, in which the infectious state is disabled prior to a complete cure, one wonders if hiv transmission rates may decrease with effective art use. in 2005, the hiv prevention trials network (hptn) initiated a study (hptn 052) which enrolled 1,763 hiv sero-discordant couples (couples that have one member who is hiv-infected and the other who is hiv-uninfected), mostly (97%) heterosexual couples. the infected partner had to be well enough not to require immediate art. the couples were randomised to have either immediate or delayed art. both groups received the same care including counselling on safe sex practices, free condoms, treatment for sexually transmitted infections and regular hiv testing. in may 2011, it had been announced that there had been 27 hiv transmissions in the delayed art group (877 couples) compared to only 1 in the immediate art group (886 couples), a 96% reduction. in these 28 cases, the hiv strain was linked to the partner. this is the first randomised clinical trial to show that treating an hivinfected individual with art can reduce the risk of hiv transmission to an uninfected partner. even with ''safer-sex'' counselling, there were 60 pregnancies in the delayed art group, despite that group having more incentive for safer-sex. following the announcement of this result, all infected participants were offered art. myron reported the 10th annual review of this study. in the delayed art group, there had been a total of 28 cases of hiv transmission with the hiv strain linked to the partner and 11 cases of unlinked transmission. in the one case of hiv transmission in the immediate art group, infection had been detected at day 85 of the study and further investigation suggested that the infection event was on day 1. clearly, early art is highly beneficial. cdc guidelines now recommend that all hiv infected patients should have art. 6. mini-symposium: hepatitis b virus anna lok, university of michigan, mi, usa the number of people infected with hbv world-wide, as estimated by the who and cdc in 2007, was between 223 and 240 million, but was declining due to vaccination. in the usa, vaccine use has led to a steady decline in the rate of new infections, decreasing from about 10/100,000 residents in the 1980s to about 1/100,00 today. in contrast, the prevalence of chronic hepatitis b among immigrants remains high, with no decreasing trend. when infection is acquired early in life, chronic infection is the norm. high viral load is associated with progression to liver cancer. there are 7 fda-approved drugs to treat chronic hbv infection, including entecavir (etv), emtricitabine (ftc) and tdf. with several years of continuous therapy, hbeag loss is achieved in about 40% of patients but hbsag loss (the ultimate goal, seen as a ''cure'') is still a distant prospect for most patients. however, cirrhosis can be reduced by long-term antiviral treatment. in one tdf trial at 5 years, 344/348 patients had a liver biopsy which showed that 73% of patients had improved fibrosis scores (p2 units) and that most other patients had no worsening. tdf has now been used for 6 years without detecting hbv resistance, making it one of the first line drugs. tdf is generally well tolerated but its rare side effects include nephrotoxicity (see above for a possible switch to taf when it is approved), reduction in bone mineral density and very rarely lactic acidosis. despite the major progress made in hbv therapy, there remain various challenges. one is cost, about $60,000-$72,000 for 5-year tdf therapy. pharmacy claims show that adherence is a problem; doses used are less than doses prescribed. there is a lack of accurate prediction of how hbv disease will progress in individuals. hbv dna can be integrated into the human genome at an early stage of infection. fortunately, the integrated viral dna is usually not the complete viral genome and patients, who achieve hbsag loss, rarely relapse. stefan mehrle, university of heidelberg, germany (stephan urban, head of hepatitis b research group, university of heidelberg, was originally scheduled to give this presentation). some chronic hbv-infected subjects are co-infected with hepatitis delta virus (hdv). this is a defective virus that replicates only in the presence of hbv. current antiviral drugs do not inhibit hdv. recently, heparan sulphate proteoglycan (hspg) has been shown to be essential for binding both hbv and hdv to primary hepatocytes. in 2012, human sodium taurocholate co-transporting polypeptide (hntcp) was identified as a functional receptor for hbv and hdv. hntcp is also designated as a solute carrier protein 10a1 (slc10a1). hntcp was shown to be a binding factor for the pres1 domain of the hbv l envelope protein. this interaction was found to be essential for hbv and hdv infection. whereas hbv replication is poor in cell lines derived from hepatocytes (e.g. hepg2 and huh-7) in which hntcp is usually weakly expressed, hbv replication is possible in primary human hepatocytes. the critical discovery was that over-expression of hntcp in hepg2 or huh-7 cells conferred susceptibility to hbv and hdv infection. myrcludex-b is a lipopeptide derived from amino acid residues 2-48 of the pres1 region of the hbv l protein. because it quickly (within 5 min) targets the liver, it is being developed for liver imaging and for drug targeting. it also acts as an entry inhibitor for hbv and hdv by interrupting binding between the hbv l protein and hntcp. it specifically inhibits hntcp-mediated taurocholate transport but the effect on hbv replication is much greater. myrcludex-b activity has been investigated in vivo using scid mice reconstituted with human hepatocytes. with prophylactic treatment, not one infected hepatocyte was seen. following therapeutic treatment, at week 6 post-infection, there were a few isolated infected cells. after the end of therapy, the infection seems to spread but only to neighboring cells. myrcludex-b has been synthesised on a 100 g scale. toxicology evaluation in 3 chimpanzees has been completed and clinical trials have been initiated. in a phase i trial using a 20 mg dose, myrcludex-b was well tolerated. results of a further phase i trial are due to be reported later this year (2014). a dose-ranging phase ii trial has been started. kyong-mi chang, university of pennsylvania, pa, usa anti-hbs antibodies clearly play a critical role in controlling hbv disease. their presence has been accepted as an indication of an ''effective cure''. however, these antibodies appear late in the disease course and so they must have a limited role in the early stages of the disease. what is the role of t-cell responses? in contrast to other viruses, there is a delayed onset, about 4-8 weeks rather than days. cd4+ t cells regulate the adaptive response, cd8+ t cells attack hbv-infected cells. the national institute of diabetes and digestive and kidney diseases (niddk), part of the usa national institutes of health (nih), is supporting a prospective clinical trial to investigate hbv-specific t cell responses during the course of hbv disease. there are no clear t cell differences relative to hbv genotype. the t cell responses are highest during acute hbv infection. during the chronic phase of hbv disease, t cell responses remain suppressed. in conclusion, there are a lot of players in the immune control of hbv infection but their relative contributions and how they adapt to control hbv replication are still largely unknown. 6.4. diversifying the hepatitis b pipeline: current efforts to explore novel mechanisms andrea (andy) cuconati, institute for hepatitis & virus research, pennsylvania commonwealth institute, pa, usa current hbv therapy using nucleotide anti-virals has been highly effective in controlling the infection but a ''cure'', as defined by hbsag seroconversion, has remained elusive. at best after 5 years, the rate is about 25%. other approaches are needed. myrcludex-b (see section 6.2) is the lead entry inhibitor. nvr-1221, an encapsidation inhibitor, is entering clinical trials. in addition. studies with novel nucleotide analogues are ongoing. the hbv field has been transformed recently by the introduction of cell-based antiviral assays. stefan mehrle (see section 6.2) has been leading the way. the assay read-out will need to be optimized for high-throughput screening (hts) but, already, the assay has shown some ''hits''. a few compounds inhibited encapsidation of viral rna. (the hbv virion contains partly double stranded (ds) dna but the reverse-transcription from rna to dna occurs within the capsid.) within the cell, hbv dna is transported into the nucleus where the viral dna forms covalently closed circles (cccdna). two specific inhibitors of cccdna formation have been found. current nucleotide anti-hbv compounds do give large reductions of hbv dna in plasma but only a minimal reduction in levels of the hbs antigen (about log 10 0.1). in contrast, one ''hit'', hbf-0259 inhibited surface antigen production but not genomic replication. structure-activity-relationship (sar) studies have given the current lead compound, hbv-0215. in conclusion, the cell-based assay, with complete replication of hbv, has markedly improved the screening for anti-hbv compounds although further optimization is still needed to give hts capability. adam zlotnick, university of indiana, in, usa. over the last few years, there has been much progress towards understanding the critical role of the hbv core protein -it is much more than just a protective coat for the genome because it plays a major role in the hbv life cycle. the core protein, being 183 amino acids long, is known as cp 183 . the first 149 amino acids are involved in core assembly whereas the last 34 residues, rich in serines and arginines, bind to rna. phosphorylation of the serines, particularly s155, s162 and s172, is required for specific packaging of full length hbv rna complexed to the polymerase (reverse transcriptase -pregenomic rna; rt-pgrna). this rt-pgrna complex initiates encapsidation. the core consists mainly of cp 183 but also includes other proteins (about 0.5%). adam showed us a computer model of the core, using different colours to highlight the various critical components. inside the core, the area of highest density (highlighted in red) represented the polymerase which was attached to the inner surface of the core. the ''other proteins'' in the core were shown in blue. the current thinking is that the polymerase, initially acting as a reverse transcriptase, is attached to, and guided by, an ''inside railway track''. this enables the polymerase to jump to the other end of the rna to start the reverse transcription into dna and then jump again to the other end to start, but never complete, the replication of the complementary dna strand. the self-assembly of the core is an energetically ''downhill'' process. somewhat surprisingly, it is possible to get mutations in which the core is even more stable but the rt activity is reduced. the phenylpropenamide derivative, at-130, fills a pocket in the core and so stabilizes it, similar to the change in amino acids in the mutants. in the presence of at-130, core assembly occurs faster; hence it is known as a core assembly enhancer (as adam mentioned, not a term much loved by industry, their preference is for core assembly inhibitors). regardless, the whole capsid structure changes. the binding of only a few drug molecules is required to make the core non-functional. it seems that it is easier to find compounds to enhance core assembly than inhibitors. 6.6. targeting cccdna to cure chronic hepatitis b massimo levrero, sapeinza universita' di roma, italy. the current hbv therapies of choice are tdf alone or with etv. these drugs have an extensive safety record with use up to 7 years. however, as for other nucleoside/nucleotide analogs, there is only a limited (about 1 log 10 ) reduction in the levels of hbv cccdna. the half-life of hbv cccdna seems to be long, but is still unknown. hbv replication parallels host gene expression, in that they involve the acetylation of histones, for example h 3 and h 4 . both host transcription factors and viral proteins bind to the cccdna. massimo summarized various assays to study different stages of cccdna during the replication cycle. potentially, these assays would allow the study of various approaches: to reduce or clear cccdna, to silence cccdna or to prevent the formation of new cccdna so that it would eventually be removed by dilution and cell death. for proof-of-concept, known ''epigenetic'' compounds, which act as transcription inhibitors, have shown that cccdna can be silenced. by reducing histone acetylation, the cccdna becomes too compact to allow transcription. this approach mimics, partly, therapy with interferon. this research is still at an early stage. due to time constraints, the next two speakers were asked to present brief summaries. john morrey (utah state university, ut, usa) described four mouse models but all stages of the life cycle of hbv can be studied only in the chimeric mouse model, in which human hepatocytes are used. however, this model lacks the potential to study the immune system and it is very expensive. stephan menne (georgetown university, dc, usa) described the woodchuck model. woodchuck hepatitis virus (whv) resembles the human virus and the disease in animals has many similarities to that in humans. neonatal infection becomes chronic in about 60-75% of cases. these chronic cases have virtually a 100% life-time risk of developing cancer, the time scale being about 1 year of chronic infection, followed by cancer at years 3 to 4. the use of microbicides is an active area of research for the prevention of transmission of hiv. david katz (duke university, nc, usa) described how mathematical models may aid drug product design. for example, if it is assumed that the microbicide gel is 400 microns thick, the epithelium is 200 microns and the stroma (connective tissue) is 3000 microns and if the partition coefficient between gel and epithelium in known, then it is possible to model drug transfer and suggest how various other parameters, for example the size of the subject, may modify drug delivery. it is important that different disciplines work together, for example biophysicists with behavioral scientists. biophysics can help an understanding of complex physical phenomena but human behavior can be both complex and highly variable. ralph baric (university of north carolina, nc, usa) noted that a particular infective agent, for example norovirus (nov), may cause subclinical or serious disease in different individuals. in general, animal models are designed to give consistent outcomes rather than aiming to mimic the genetic diversity found in human subjects. in a collaborative effort, mice from 8 ''founder'' strains, including 3 wild-derived strains, were selected. the 5 founder laboratory strains were all derived ultimately from a single female mouse ca 1900. the susceptibility of the 8 founder strains to severe acute respiratory syndrome coronavirus (sars-cov) differed widely (ld 50 p10 6 -10 2 ). the founder strains were cross-bred. although ca 90% of the genes was equally distributed among the new mouse lines, there were gene combinations not seen previously. after infecting mice from the different founder strains with a constant sars-cov inoculum and measuring virus load at a set time after infection, there was a correlation between virus load and disease (as measured by vascular cuffing). it was possible to relate the effect to chromosomes 3 (27%) and 13 (20%). hopefully, identification of the important genes may be achieved. by keeping the virus inoculum constant, this system better represents the clinical spectrum of disease. when using this system to evaluate a potential vaccine, it was found that mice, under the age of one year, could be protected. however, there was a range of effectiveness, from good protection to inactive. these variations may give a representation of human diversity. angela kashuba, university of north carolina at chapel hill, nc, usa in four clinical studies, truvada [a combination pill containing tdf and emtricitabine (ftc)] was taken once daily to prevent hiv transmission, known as pre-exposure prophylaxis (prep). the adherence rates were unexpectedly poor in all four studies, particularly low in the study including at risk women. for example in one study, ''high adherence'' was defined as subjects taking at least 80% of drug doses and was achieved by only 54% of subjects. possible reasons may have been the apparent risk of side-effects (the long consent form included 7 pages of side-effects) and the perception that the subjects, as individuals, were not particularly at risk of infection by hiv. importantly, the trial did confirm the concept that prep could be effective. there was >90% protection in those subjects generally taking 7 doses/week and there was some protection, albeit much less, in subjects taking 2 doses/week. adherence rates, reported by subjects, were appreciably higher than the rates evidenced by drug blood level measurements taken just before the next dose (i.e. 24 h after previous dose). in an attempt to better understand and model these data, the drug concentrations (tdf/tfv and ftc) in various tissues were measured. the ratio between drug concentrations in blood and tissue samples differed greatly for tdf/tfv, with less variations for ftc. concentration ratios of tdf/tfv were about 50 in rectal tissue but only 0.2 in vaginal tissue. for ftc, the ratios were 2.6 and 1.3, respectively. when considering the possible consequences of missed doses, the time scale for hiv infection is an important factor. it is thought that hiv takes about 1-3 h to reach the epithelial cells. clearly, adherence is a critical factor for efficacy and so a real-time objective method for measuring adherence is urgently needed before further clinical studies are initiated. 7.4. the novel nucleoside analog bcx4430 exhibits broad-spectrum antiviral activity and confers post-exposure protection against ebola and marburg viruses travis k. warren, usamriid, fort detrick, md, usa ebola and marburg viruses are members of the filovirus family. even in recent outbreaks of these diseases, including the current ebola epidemic in west africa, care workers are becoming infected and dying. drugs, which are being investigated for treating these diseases, are progressed under the fda ''animal rule''. bcx4430 is a c-nucleoside adenine analog (fig. 10 ) which is being progressed by biocryst pharmaceuticals inc. (warren et al., 2014) . in cell culture assays, bcx4430 is active against ebola and marburg viruses, (ec 50 ca 1 lm). with bcx4430 at 30 lm, there was no detectable incorporation into host dna or rna. in rats, bcx4430 is efficiently activated (phosphorylated) to the triphosphate. in a primer-extension assay, there is some read-through beyond a single residue of bcx4430, but there is effective chain termination after the first bcx4430 residue where the template has two consecutive uridine residues. bcx4430 has been tested in rodent and nonhuman primate models of marburg hemorrhagic fever. in mice, there was a dose response (30, 20, 3.3 and 1.1 mg/dose, bid) with full protection at the two higher doses (survivors, 100%, 100%, 95% and 83% respectively). in an experiment with dosing starting at different times (4 h pre-infection, 24, 48, 72, 96 and 120 h post-infection vs placebo), the placebo-treated mice died on days 6, 7 and 8 with one survivor (10%). in the treated groups, the percent survival was 80, 100, 80, 100, 100 and 30, respectively. in guinea pigs, bcx4430 (bid) with treatment starting at different times (1 h pre-infection, 24, 48 and 72 h post-infection) there was full protection (100% survival) for the pre-infection and 24 h groups, with reduced efficacy at the later start times. in cynomolgus monkeys, bcx4300 treatment was started at 1, 24 and 48 h post-infection. in the placebo group, all 6 animals died within days 9 to 12. in all the treated groups, virus loads were reduced by more than log 10 3. there was one late death in the 1 h group but the other 17 monkeys survived. various markers of potential organ damage were reduced in all treated groups. encouraged by these results, 14-day toxicology trials have recently been completed without any serious concerns. biocryst is developing bcx4430 under the fda animal rule and indenabling work is ongoing. when asked about viral resistance, travis explained that it is not ethically permissible to create resistant strains of marburg virus, but samples collected from the monkeys are being sequenced to look for mutations indicative of drug resistance. as yet, mitochondrial toxicity has not been examined. had similar bone marrow transplants, initially seemed to have been ''cured'' but hiv was detected after 70 and 200 days, respectively. latent hiv can survive in various long-lived cells for decades, especially in memory t cells. when these cells proliferate, the integrated hiv genome is duplicated as the cell divides and the cells survive so long as hiv remains silent. compounds known to activate all t-cells are too toxic to become a clinical therapy. however, latency-reversing agents (lra) have greater specificity, ideally activating only the integrated hiv, leading to the death of hiv-containing t-cells. there remains another possibility (perhaps a less popular view) that there is continued low rate of hiv replication. two clinical studies have been initiated in subjects with undetectable plasma hiv levels. raltegravir, an hiv integrase inhibitor, was added to the background therapy. latent hiv is mostly integrated into host dna but hiv may also form episomal circular dna. the proportion of the circular form increases with raltegravir treatment. in the two clinical studies, 13/45 and 9/15 subjects, respectively, had detectable hiv circles which then decayed. this implies that some de novo infection of cells is ongoing. on the other hand, art works well, with no evidence of sequence evolution in the hiv circles at 48 weeks. is it possible that raltegravir is inducing a single round of hiv replication, to give an increase in hiv circles? derek sloan, gilead sciences, foster city, ca, usa like vorinostat, (vor), romidepsin (rmd) is a histone deacetylase inhibitor which is used clinically to treat cancer. memory cd4+ t cells were taken from hiv subjects on suppressive art; ex-vivo treatment with rmd (40 nm) induced a 6-fold increase in intracellular hiv rna which persisted for 48 h. in contrast, a much higher concentration of vor (1 lm) gave a 2 to 3-fold lower response which was only transient. rmd also increased levels of extracellular hiv rna and virions. encouragingly, this ex-vivo induction of latent virus was seen at rmd concentrations that are below the levels of drug achieved in humans by clinical doses of rmd. accordingly, in a phase i/ii trial in hiv-infected subjects on art, rmd gave a better and more sustained response than vor. about 1.5% of cells containing hiv provirus were activated. although this is far too low a percentage to eliminate the latent hiv reservoir, it is hoped that combination of such lra, which give improved results in ex-vivo cell assays, may give better clinical efficacy. gilead scientists have started screening for novel lras. ''gs-1'' has been identified as a hit by hts. research on this lead is at a very early stage. gilead workers are also investigating other approaches. for example, gs-9620 is a toll-like receptor 7 (tlr7) agonist and it acts as an immune stimulator. although it is being evaluated in phase ii studies for the treatment of chronic hbv infections, the potential effect on hiv reservoirs is being investigated. in siv-infected monkeys, oral dosing of tlr7 agonist induced the activation of immune effector cells such as cd8+ t cells and nk cells. based on these data, tlr7 agonists are being further investigated for their effect on latent siv reservoirs in monkeys which have good virological suppression. another approach is to use anti-envelope antibodies. broadly neutralising antibodies (bnabs) are very effective in preventing siv infection when the viral load is low but less effective against a high-load virus challenge. in addition, a prophylactic cmv-vector-based siv vaccine was effective in preventing siv infection in rhesus monkeys. this and similar vaccines are being tested in vivo for their effects on the latent siv reservoirs. in summary, lras are able to activate hiv provirus in memory cd4+ t cells and thereby may enhance the recruitment of immune effector cells to destroy provirus-containing cells. however, a ''cure'' for hiv infection is still a distant prospect. furthermore, latent hiv reservoirs are heterogeneous and so a combination of approaches will likely be required. gerardo garcia-lerma, centers for disease control and prevention, atlanta, ga, usa proof-of-concept studies for prep, are mostly conducted in nonhuman primates. these can be used either to model a single highdose infective challenge or repeated low inoculations, about 10-50 tissue culture infective doses (tcid 50 ). since 2005, rhesus macaque models have been used in a long series of investigations. in a study, in which the monkeys were treated daily with either oral tdf or tdf/ftc and given a weekly siv inoculum rectally, tdf/ftc gave a longer delay in infection than did tdf alone. when using the vaginal infection route, tdf/ ftc gave 100% protection. in contrast, there was far less protection in clinical trials -why? one possible reason may have been that women were having the contraceptive injection, depot medroxyprogesterone acetate (dmpa). a study, in macaque monkeys given dmpa, confirmed that dosing with tdv/ftc gave good drug levels in plasma and in vaginal secretions. therefore, this did not explain the poor protection in the clinical trial. the macaque model has been used successfully to investigate various situations that are presented in the clinic. when macaques were co-infected with siv and a bacteria and treated with tdf/ftc for 12 weeks, there was good, but not complete, protection (80%). with ftc-resistant virus, tdf/ftc remained protective. in this case, ftc-resistant virus has increased susceptibility to tdf. with the k65r mutant hiv, there was protection against a low inoculum but only partial protection (ca 50%) against a high inoculum. whereas daily dosing seems to be acceptable for patients living with hiv, another option for prep is desirable. gsk-1265744 (generally known as gsk-744) is an hiv integrase inhibitor. it can be formulated with nano-particles to provide an injectable drug depot. in the macaque model, gsk-744, injected once monthly, gave full protection against repeated rectal and vaginal exposures. because metabolism of gsk-744 is much slower in humans than macaques, it was expected to remain effective in humans for up to three months. a phase i study confirmed that drug levels remained above the predicted effective level with a 20-week dosing interval. a phase ii trial is planned. another approach is to use vaginal rings, which have been in clinical use as contraceptive devices for years. in the macaque model, tdf-containing rings, replaced every 4 weeks, gave full protection. a phase iii trial has just been initiated. another option, elvitegravir (evg) and taf are being evaluated in a biodegradable polymer. although daily dosing with tdf/ftc has not proved sufficiently successful as prep in clinical use, it has proved that prep is an achievable aim and this has encouraged the progression of other options. courtney fletcher, university of nebraska, omaha, ne, usa atripla was the first triple combination pill taken once daily for hiv therapy. it contained tdf, ftc and efavirenz (efv). the macaque model has been used to investigate the differing tissue distributions of these drugs and how viral replication may be continuing wherever the drug concentrations are lowest. there are two approaches: tissue homogenates and tissue cells. tissue homogenates give both the intracellular and extracellular drug amounts. from tissues, mononuclear cells (mncs) are collected and the intracellular drug concentration measured. this approach is preferred by courtney but this option may be constrained by sample size and the drug concentration may be underestimated. for exam-ple, with raltegravir, after the mncs have been washed 3 times, the drug concentration is very low. much higher raltegravir concentrations are found when the mncs are cleaned by a rapid spin through oil. comparing an oil spin and repeated washes, the oil process gives higher drug levels, typically about 50% higher. following initial studies in macaques, a clinical study, in 32 subjects, investigated distribution of the drugs from atripla in peripheral blood mononuclear cells (pbmc) and various tissues (see above). in 12/32 subjects, there are data on the time to reduce hiv load to <48 copies/ml. in plasma, the time was 3-4 months. in lymphoid tissues, there was a much slower rate of hiv decline. also, patient variability was noted, with the faster responders having the higher drug levels. a drug may be absorbed from the gastrointestinal tract either going via the portal vein to the liver and then into blood circulation or via the lymphoid system. blood flow is about 200 times faster than lymphoid flow. when the water/1-octanol partition-coefficient (logp) of a drug is <5, absorption tends to be via the blood route. the prodrug approach can be used to alter absorption or, as for tfv, stability of the prodrugs (tdf and taf) can influence the relative concentration in lymphoid tissues (see above). this year, the three major award lectures exemplified the strength of icar, covering very different areas of research. john drach (elion award) described his journey through the early days of antiviral research, which led to the identification of novel modes of antiviral action that had not been envisaged previously. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept novel nucleoside analogs. the replacement of thymine by 5-chlorouracil led to the generation of a new form of e. coli. i suggest that this work has important implications in conventional antiviral research. with hiv and hcv protease inhibitors, the genetic barrier is limited by the ability of the viral protease and its substrate (the viral polyprotein cleavage sites) to co-mutate so that the virus can become resistant to the antiviral drug. so far, polymerase inhibitors have not suffered the same fate but this work shows that a poor choice of nucleotide analog could result in a resistant virus with a new type of rna in which the drug replaces a natural nucleoside. adrian ray (prusoff award), describing work at gilead, demonstrated how the prodrug concept can markedly improve both the efficacy and safety of potential drugs. their progress with hiv and hcv therapies has been remarkable. the keynote addresses tackled two emerging areas of hiv research. david margolis summarized work aiming to eradicate hiv from infected subjects and myron cohen described current progress with approaches to prevent hiv transmission. i found both these presentations to be informative and stimulating. hiv ''cure'' still seems to be a distant prospect. in contrast, prior to exposure prophylaxis (prep) has been shown to be an achievable aim although the need for daily dosing is a barrier to success. gerardo garcia-lerma described recent progress which is likely to radically change the prospects for therapeutic convenience and success. tdf-containing vaginal rings, which need replacing only once a month, are being evaluated. another exciting prospect is gsk-744 which has been formulated as a long-lasting injection. a phase i trial confirmed that the drug may be administered at 3month intervals. in the absence of a proven hiv vaccine, prep with drugs has become the most promising strategy to reduce hiv infection rates among high-risk populations. this conference also included three interesting mini-symposia: ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. an innovation this year was a session devoted to the european training network, euvirna and introduced by frank van kuppeveld. all the 18 euvirna fellows, who attended icar, gave short presentations at this session. for further information, please see the isar news (24.1) in the september issue of antiviral research for an account by frank van kuppeveld. for many years, the clinical symposium was, for me, a major highlight of icar. in my report for the 2013 icar, i expressed a hope regarding hcv therapy: ''there is the prospect that the first nucleotide analogue will be licensed by the time of our next icar meeting. the combination of a nucleotide analogue and a ns5a inhibitor looks set to transform hcv therapy across all genotypes. as for hiv, single-pill, once-daily regimens are following on quickly''. on 6th december 2013, sofosbuvir (sovaldi ò ) was the first nucleotide analog to be approved in the usa by the food and drug administration (fda) for treatment of patients with hcv. approval by the european union followed soon afterwards, in january 2014. a ns5a inhibitor, ledipasvir, formulated as a single fixed-dose combination pill with sofosbuvir, is progressing quickly through clinical trials. with such remarkable progress being achieved since the 2013 icar, i was disappointed to discover that there was no presentation on this topic at this year's icar. a paper (sofia, 2014) , which was part of a symposium in antiviral research on ''hepatitis c: next steps toward global eradication'', emphasizes recent successes. after completing therapy, a sustained virological response for 12 weeks (svr12) is regarded as a cure for hcv-infected patients. the combination of sofosbuvir/ledipasvir has shown remarkable results in clinical trials, with svr12 in the range 95-100% across genotypes. this combination was well tolerated. a nda for the sofosbuvir/ledipasvir combination pill was submitted recently. i do not recall any previous antiviral trials in which the ''intention-to-treat'' analyses showed 100% success rates. perhaps similar to the hcv symposium in antiviral research, i hope that the 2015 icar, which will be held in rome, will have a mini-symposium which will include an account of this remarkable progress. it would be interesting to have an update on the clinical impact of this combination therapy for hcv and to have an assessment on the prospects for global eradication of hcv. beside this one disappointment, there were many excellent presentations and i would like to add my thanks to the isar officers and conference committee for organizing another interesting and successful icar. metabolism and pharmacokinetics of anti-hepatitis c virus nucleotide prodrug gs-6620 beyond sofosbuvir: what opportunity exists for a better nucleoside/nucleotide to treat hepatitis c? selection of an oral prodrug (brl 42810; famciclovir) for the antiherpesvirus agent brl 39123 protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 acknowledgements i wish to thank all those authors who have kindly provided me with copies of their presentations and for giving me valuable comments. also, i thank the president of isar for asking me to prepare this meeting report. key: cord-023033-tgt69ir6 authors: nan title: poster session (pp. 78a–178a) date: 2006-02-10 journal: hepatology doi: 10.1002/hep.1840380503 sha: doc_id: 23033 cord_uid: tgt69ir6 nan , ala-60 (n=4), tyr-77 (n=2), gly-42 ( n = l ) and lys-89 ( n = l ) . the mean time from onset of symptoms to diagnosis was 3.4 years (0-10) and patients were listed for olt an average of 10.8 months (0-60) after diagnosis. the mean time from listing to olt was 8.2 months (1-18). five patients died after olt, during a mean follow-up of 3.5 years (4 months -6 years). one-year patient survival was 100% and threeyear patient survival was 92%. one patient required retransplantation for hepatic artery thrombosis. neurologic symptoms were the initial clinical manifestation in the majority of patients (7/12), followed by cardiopulmonary (4112) and gastrointestinal symptoms (1112). most patients experienced multiple symptoms. subjective evolution of symptoms as assessed by chart review demonstrated that symptoms referable to amyloidosis worsened after olt in seven patients, and improved or stabilized in five. following olt, neuropathy symptoms improved in four patients, worsened in seven patients and were unchanged in one patient. pre and post-olt nerve conduction velocities were available for 5 patients and the post-olt studies showed progression of disease in 3, improvement in 1 and were unchanged in 1 patient. prior to olt, all twelve patients had an increased ivst on echocardiogram. post olt cardiac symptoms improved in six patients (five of whom also had cardiac transplantation), worsened in three patients, and were unchanged in three patients (one of whom also had cardiac transplantation backgr0und :accurate delineation of the scope and magnitude of peri-operative donor risk is necessary to better allow for informed consent, to maximize the potential for donor safety, for comparative outcome analysis and ultimately to serve as a key determinant of the utility of adult-to-adult living donor liver transplantation (aaldlt). however, at present, there is lack of uniformity regarding what constitutes a complication in this setting. moreover, a system to stratify adverse events with respect to their life altering (quantity or quality) impact is lacking. aims: 1) to define a graded, inclusive classification schema for both early (e) and late (l) adverse operation-related events in live liver donors a n d 2) to apply this system to a retrospective review of events in individuals undergoing partial hepatectomy for live liver donation at our center. two patients (6.5%) suffered cut-surface bile leaks and one (3.2%) a right colon injury (grade 3e complications). one patient (3.2%) developed a transient bilateral ulnar neuropathy (grade 2e). two patients (6.5%) were readmitted within 30 days of operation for nausea and dyspnea respectively (grade 1e). one patient underwent repair of an incisional hernia 6 months post donation (grade 2l). one patient suffered positioning-related brachial plexopathy (grade 3l). conclusions: the definition and adoption of a graded, scale-based system of operation-related adverse events in live liver donors will allow for an inclusive, consistent and universally applicable method to collect, analyze and report donor complications. all aaldlt programs must be encouraged to fully review and report their donor related morbidity, ideally through the creation of a national donor registry initiation of antiviral therapy in the early post-transplant period, prior to overt evidence of hcv recurrence (i.e. preemptive therapy) may enhance rates of hcv eradication. aims: to determine the efficacy and tolerability of preemptive ifn versus ifnlrbv in anti-hcv positive lt patients. methods: consecutive and eligible lt recipients from a single center were enrolled. the goal was to initiate treatment within 6 wks of lt. patients were randomized to ifn or ifn plus rbv (400mg daily x 2wks, then 800mg daily x 2 wks, then 1.0-1.2g daily based upon body weight 75 kg). induction therapy with daily ifn was used for the first 8 wks (1.5mu daily x 2wks, then 3mu daily x 6 wks) followed by 3 mu tiw (n=39) or peg-ifn 1.5 uglkglwk (n=10) for 40 wks. key inclusion criteria were stable clinical status, cr45,ooo and wbc>3.0. dose reductions for side effects, especially cytopenias were standardized. growth factors were given for neutropenia (anc<1000) and anemia (hgb<9.0gldl) beginning 7l2001. virological (vr) and biochemical responses (br) were evaluated at end-of-treatment (et) and 6 mos post-treatment (sustained virological (svr) and biochemical (sbr) responses (table below) were associated with response. histological disease was mild in the majority of patients at treatment end (78% stage 0 fibrosis and 72% grade 1 or less necroinflammatory activity). conclusions: preemptive antiviral therapy was applicable to -60% of transplanted patients. biochemical responses were frequent but svrs uncommon. treatment discontinuation and lowering of rbv doses due to side effects likely reduced svrs and may have limited our ability to detect differences between treatment groups. the only predictor of svr was a negative qhcvrna pre-treatment, which implies that patients with low vl early post-lt may be the best candidates for preemptive therapy. compared to historical reports, the severity of histological disease was very mild at 1-year post-lt in the majority of treated patients, suggesting preemptive antiviral therapy may provide important histological benefits. the cadaveric organ shortage has led to the development of alternative strategies for organ transplantation. living donor liver transplant (ldlt) is one strategy that has offered many individuals transplantation before they die or become too sick for transplant.chronic hepatitis c (hcv) is the leading indication for liver transplantation. preliminary results from small single center studies suggest that recurrent hcv is more common after ldlt compared to cadaveric transplant with concern that graft survival may be lower after ldlt. &: to compare patient and graft survival in hcv recipients who undergo ldlt to cadaveric liver transplant recipients. methods: we analyzed the united network for organ sharing (unos) liver transplant database from january, 1999 -december, 2002. inclusion criteria included liver transplant recipients 218 years old transplanted from 1999-2002 with the transplant diagnosis of chronic hepatitis c. exclusion criteria included subjects who were hepatitis b surface antigen positive, history of prior organ transplant, concurrent kidney or heart transplant. the logrank test was used to compare survival between the two groups. results: from 1999-2002 6.6% of adult liver transplants were ldlt. 279 ldlt recipients and 3,955 cadaveric recipients transplanted for end stage liver disease from hcv were identified. the results are shown in the table: ldlt ( a greater proportion of ldlt recipients were female and ldlt recipients were less ill at the time of transplant.1 and 3 year patient and graft survival were not significantly different between the 2 groups, (p=o.11). if only the pre-meld era is considered (01l99 thru 2/02) 1 year graft survival for ldlt (n=240) and clt (n=3322) are 78% and 83%, respectively, p=o.lo. conclusions: our analysis demonstrates that short-term patient and graft survival are equivalent in ldlt and cadaveric recipients with hcv suggesting recurrent hepatitis c does not adversely affect survival over this time period. ldlt recipients are less ill at transplantation which did not confer a short-term survival advantage. continued follow-up should determine the impact of less advanced liver disease at the time of transplant and recurrent hepatitis c after transplant on long-term graft and patient survival in ldlt recipients. disclosures: introduction: histologic injury due to recurrent hcv has been reported in up to 90% of hcv infected patients who undergo liver transplantation with a cadaveric graft. however, the natural history of hcv following living donor liver transplantation (ldlt) is not as well described. anecdotal evidence suggests that hcv recurrence may be more severe in ldlt recipients. we hypothesize that post-operative liver regeneration in the partial graft procured from living donors may facilitate hcv replication, or increase the vulnerability of hepatocytes to infection. methods: we performed a retrospective analysis comparing outcomes, and the incidence, timing, and severity of histologic recurrence of hcv following transplantation in patients who underwent living donor liver transplant compared to recipients of cadaveric organs. between 12/98 and 3/02,68 hcv infected adult patients (age > 18 years old) underwent liver transplantation for hcv associated cirrhosis. 45 patients received a cadaveric graft (cad) and 23 received a graft from a living donor (ldlt). mean time of patient follow up was 25 months, with a range of 6 to 39 months. elevated serum transaminases, positive hcv rna, and liver biopsy consistent with histologic evidence of hcv defined recurrence. cholestatic hepatitis c (chc) was confirmed if all of the following criteria were met: serum total bilirubin greater than lomg/dl with no evidence of extra-hepatic biliary obstruction on ultrasound and cholangiography, and histologic features on liver biopsy consisting of portal expansion, ductular proliferation, and bile stasis with or without hepatocyte ballooning. immunosuppression, definition and treatment of rejection were standardized for both cad and ldlt. results: when comparing cad to ldlt, both the incidence of hcv recurrence and time to recurrence were not different. the overall incidence of "severe" sequelae of hcv recurrence, either cholestatic hepatitis, grade 111-iv inflammation, and/or hcv induced graft failure requiring re-transplantation was also not different when comparing cad to ldlt. however, when comparing cad versus ldlt, no cad patient developed cholestatic hepatitis c, compared to 17% of ldlt who developed this complication (p = 0.001). conclusions: in this patient population, the timing and overall incidence of hcv recurrence were not different when comparing cad versus ldlt, but the incidence of cholestatic hepatitis was significantly greater in patients with hcv who underwent ldlt. further multicenter studies are warranted to determine the incidence and risk factors for cholestatic hepatitis c following liver transplantation. ucsf, sun francisco, ca; fabien zoulim, inserm, lyon, france; carol l brosgart, michael wulfsohn, michael d miller, shelly xiong, gilead sciences, inc., foster city, ca background lamivudine (lam) resistance occurs in approximately 40% of chronic hepatitis b (chb) patients after 2 years of lam monotherapy. in contrast, resistance to adefovir dipivoxil (adv) occurs infrequently with 1.6% of chb patients developing the adefovir resistance mutation rtn236t after 2 years of adv therapy. pre-existing lam resistance mutations or concurrent use of immunosuppressive therapy by lt patients may increase the risk of resistance to adv. objective: to determine the incidence of adv resistance in a clinical trial of liver transplantation (pre and post) patients with lam-resistant hbv treated with adv for 96 weeks (lam therapy was maintained in most patients). methods: the hbv reverse transcriptase domain was sequenced for lt patients with detectable hbv dna by pcr (>lo00 copieslml) after 96 weeks of adv therapy (n=114). in vih-o drug susceptibility was determined following transfection of hepg2 cells with patient-derived hbv clones from baseline and week 96 serum samples. results: the rtn236t mutation wa!j observed in 21114 patients (1.8%) at week 96. lam had been discontinued at weeks 16 and 28 after initiation of adv in these two patients respectively. the baseline lam-resistant ymdd mutation reverted to wildtype prior to week 96 in both patients. emergence of rtn236t was associated with rebound in serum hbv dna and alt elevation in both patients. in vitro phenotypic analysis showed approximately 4-fold reduced susceptibility to adefovir with rtn236t. however, these adefovir-resistant hbv clones were fully susceptible to lam and entecavir in vitro. lam therapy was re-initiated, in addition to the ongoing adv therapy, after emergence of rtn236t in these patients resulting in a > 2.9 loglo copies1ml reduction in serum hbv dna in both patients. one patient also had a significant reduction (>80%) in alt within 5 months of lam treatment. no other mutations potentially associated with adefovir resistance were detected. conclusions: emergence of the adefovir resistance mutation rtn236t was observed infrequently after 2 years in liver transplantation patients (1.8%, 2/114) infected with lam-resistant hbv, similar to observations in treatment nayve non-liver transplantation patients. the adv-resistant hbv was sensitive to lam and addition of lam resulted in clinical stabiliation in both patients. local liver immune responses are thought to play a major role in chronic autoimmune diseases directed at biliary epithelium.using the apical sodium dependent bile acid transporter (asbt) promoter to drive biliary epithelial cell -specific expression of a membrane form of ovalbumin (ova), we have previously developed ova-bil transgenic mice. because these mice are tolerant to ova, we use ova-specific t cells from ot-1 and ot-i1 transgenic mice, restricted by mhc class i and class 11, respectively, with well defined peptide epitopes specific for ova to induce biliary damage in a dose dependent manner. aim: 1) to determine the liver mononuclear cell populations (mnc) involved in necroinflammatory disease, and 2) to determine where adoptively transferred cells home and proliferate. methods: 10 million ot-i and 2 million ot-i1 nayve t cells were adoptively transferred to ova-bil mice by intraperitoneal injection. at days 0, 5, and 7, liver mnc were isolated by collagenase digestion, purified by discontinuous percoll gradient centrifugation, and analyzed by flow cytometry. tail bleeds were performed at days 0, 3, 5, and 7 to follow serum alt. in a subset of experiments, ot-1 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (cfse) and analyzed on day 3 and 5 by flow cytometry after adoptive transfer with unlabeled ot-i1 t cells into ova-bil mice. results: ova-bil mice develop normally without evidence of disease up to 2 years. after adoptive transfer of ova-specific t cells, there was a marked increase in serum alt. cd8+ ot-i t cells were required for liver damage and ova-specific cd4+ t cells markedly augmented this inflammation. adoptive transfer of ot-i1 cd4 t cells alone did not induce liver injury. there was extensive portal inflammation in every portal triad, centered around the bile ducts with infiltrating lymphocytes in the bile duct epithelia, apoptotic cells, loss of biliary epithelial cells as well as interface hepatitis. liver mnc were abundant in ova-bil mice and increased after adoptive transfer of ova-specific t cells. serum alt peaked at day 5 (mean 263 iulml), coincident with liver mnc peak. cfse labeling studies revealed robust homing of adoptively transferred ot-i cd8 cells to the liver, but not to the spleen, of ova-bil mice. the ova-specific cd8 cells, but not cd4, nk1.l, or cd19 cells, underwent cell division. conclu-sion: recognition of biliary epithelial antigen in ova-bil mice induces a necroinflammatory response in the liver as assessed by serum alt, liver mnc numbers and immunohistochemistry. the magnitude of this response correlates with influx of nahe ovaspecific cytotoxic t cells, which are activated and divide in the liver but not in the spleen. t cell recognition of antigen expressed on bile duct epithelium occurs rapidly, causes biliary specific inflammation with interface hepatitis, which may be a model of autoimmune bile duct injury or cholangiopathy. the etiology of autoimmune hepatitis (aih) is only poorly understood although the major autoantigens, such as cytochrome p450 2d6 (cyp2d6), could be identified and immunodominant epitopes have been mapped. one major reason for this lack of comprehension is the fact that there are currently only few valid animal models for aih and none of them involves the autoantigen targeted in humans. thus, we generated an animal model for human aih using a natural autoantigen (cyp2d6). self-tolerance in transgenic mice that express human cyp2d6 in the liver was challenged by infecting these cyp2d6-mice with an adenovirus-cyp2d6 vector (ad-2d6) in order to deliver large amounts of the critical antigen to the liver and to provide a inflammatory environment that would favour autoimmunity. infection with an empty adenovirus vector resulted in a transient form of focal necrosis 4 days after infection that subsequently disappeared after 2 weeks. in contrast, infection with ad-2d6 resulted in extended and persistent infiltration of cd4, cd8 lymphocytes as well as macrophages resulting in confluentlbridging necrosis at week 10 post-infection. in addition, the overall morphology of the liver was massively disturbed after ad-2d6 infection. at week 10 postinfection, the liver was approximately half the size of the control and its lobules were fused together and rounded up. first indications of fibrosis and hyperplasic nodules become apparent. the overall architecture of the liver was disrupted i disorganized and the liver parenchyma was partially collapsed. furthermore, theliver of ad-2d6 infected mice was surrounded by multiple layers of connective tissue as seen in some stages of liver cirrhosis. additional signs of cirrhosis started to become apparent in the form of nodules that are entrapped in fibrous tissue. in addition, accumulation of infiltrating cells are visible directly under the liver capsule. these observations indicate that the cyp2d6-mouse displays a persistent form of hepatitis after infection with ad-2d6 that may form the basis for a novel model system which would allow to study mechanisms involved in the initiation, propagation and precipitation of autoimmune processes involved in human autoimmune hepatitis. disclosures: urs christen -no relationships to disclose eric f johnson -no relationships to disclose michael p manns -no relationships to disclose antje rhode -no relationships to disclose matthias g von herrath -no relationships to disclose herkel, peter r galle, edgar schmitt, ansgar w lohse, johannes gutenberg university, mainz, germany background: in clinical hepatitis, hepatocytes express mhc class i1 molecules; we recently reported that mhc i1 expressing hepatocytes can function as antigen presenting cells that stimulate specific cd4 t cells (hepatology 2003; 37: 1079-85) . to understand the relevance of hepatocellular antigen presentation for hepatic immunity, we now examined whether hepatocytes may induce the differentiation of primary cd4 t cells. because inflammatory cd4 t cells in the liver are most likely primed by dendritic cells of the draining lymph nodes, we also examined whether hepatocytes may also re-differentiate dendritic cell-primed cd4 t cells. methods: primary cd4 t cells from ovalbumin-specific t cell receptor-transgenic mice were stimulated by antigen presenting hepatocytes from hepatocyte-specific ciita-transgenic mice or splenic dendritic cells.the response type was determined by measuring secreted interferon-gamma (ifn) and interleukin-4 (il-4). results: we found that antigen presenting hepatocytes by default induced differentiation of primary cd4 t cells to th2 cells (ifn: 200 ulml; 1l-4: 1800 ulml). in contrast, primary cd4 t cells stimulated by dendritic cells differentiated to thl effector cells (ifn: 2400 ulml; 1l-4 20 ulml). however, these dendritic cellprimed thl type cells, when re-stimulated by hepatocytes, had a decreased capacity to produce ifn (580 ulml vs. 2800 ulml after dentritic cell stimulation); and after repeated re-stimulation by hepatocytes, the dendritic-cell primed t cells even differentiated into th2 cells (ifn: 112 ulml; il-4: 1270 ulml). conclusions: these data show that antigen presenting hepatocytes induce th2 differentiation of undifferentiated cd4 t cells. most notably, even dendritic cell-primed cd4 t cells that are committed to thl differentiation could be reverted by hepatocytes to th2 type. thus, hepatocytes seem to have an extraordinary capacity to promote antiinflammatory hepatic immune responses. it is therefore conceivable that antigen presentation by hepatocytes associated with clinical hepatitis, in the absence of pro-inflammatory stimuli, seems to downregulate inflammatory infiltrating t cells. background nkt cells are a unique subset of regulatory lymphocytes with important immune modulatory effects. these cells recognize exogenous glycolipids anchored by a ceramide tail to the mhc-like cdld molecule, expressed by various antigen presenting cells. glycosylceramides, including the marine sponge-derived a-galactosylceramide can activate nkt cells, leading to exacerbation of hepatitis. concanavalin a induces immune mediated hepatitis in which nkt cells are key participants. glucocerebroside (gc) is a naturally occurring glycolipid. aims: to determine the immune modulatory effect of gc in a murine model of con a hepatitis. methods: five groups of balblc mice were studied group a and b mice were treated with gc (1.0 pg ip) two hours prior to and two hours following administration of 500 pg con a, respectively; group c mice were treated with con a alone; group d mice were treated with gc alone; group e mice did not receive any treatment. the degree of liver damage was evaluated by serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels, and by liver histology. the immunmodulatory effect of gc was determined by facs analysis of intrahepatic and intrasplenic lymphocytes for nkt markers, and by elisa measurements of serum ifny, il2, il4, il10, and il12. the effect of gc on nkt lymphocyte proliferation was assessed in vitro. results: treatment with gc markedly reduced serum ast and alt levels in group a compared to group c (143 vs. 600 iu in group a and group c, respectively, for ast; 57 vs. 801 iu in group a and group c, respectively, for alt, p<0.05). administration of gc alone did not change ast or alt levels compared to naive controls. histological sections of livers from group a mice revealed markedly attenuated damage compared to sections from group c livers, in which massive hepatocyte necrosis was present. the beneficial effect of gc on immune mediated hepatitis was associated with a 20% decrease in the intrahepatic nkt lymphocyte number, and with significant lowering of serum ifny levels (3725 vs. 5620 pglml in groups a and c, respectively, p<0.05); administration of gc alone led to increased serum il-12 levels (573 pglml vs. 92 pglml in group d vs. group e, respectively, p<0.05). in vitro, administration of gc decreased nkt cell pro-liferation by 42% in the presence of dendritic cells, but not in their absence. conclusions: administration of gc led to significant amelioration of con a hepatitis that was associated with a dendritic cell-dependent decrease of nkt cell proliferation in vitro, and with decreased intrahepatic nkt lymphocytes and serum ifny levels in vivo. these results suggest that the effect of gc may be mediated by inhibition of intrahepatic nkt cells, resulting from competitive displacement of activating elements from the cdld molecule on antigen presenting cells. glucocerebroside, a naturally occurring glycolipid, holds promise as a new immunomodulatory agent, with a possible role in treatment of autoimmune hepatitis and other immune-mediated liver disorders. disclosures: background cd4+ cells constitutively expressing cd25 have a regulatory function, their experimental removal leading to spontaneous autoimmune disease in normal rodents. cd4+cd25+ regulatory t cells suppress both th1 and th2 responses, competing with effector cd4+ cells in recognizing the same peptide antigens. their ability of expansion is key to the maintenance of tolerance. autoimmune hepatitis type 2 (aih-2) is characterized by t cell immune responses against 6 well-defined regions on cytochrome p4502d6, the autoantigen of aih-2. aims: to investigate the ability of expansion of cd4+cd25+ after exposure to non-antigen-specific and cypzd6-specific stimuli in aih-2. methods: cd4+cd25+ t cells were analysed by triple colour flow-cytometry of freshlcryopreserved peripheral blood mononuclear cells (pbmcs) befare and after culture in the presence of a t cell expander capable of maintaining the original t cell function (cd3kd28 dynabeads t cell expander, dynal biotech, norway) and of 28 synthetic cyp2d6 peptides (10 pmol), spanning the 6 antigenic regions known to induce proliferative cd4+ responses in aih-2. patients and controls: nine patients with aih-2 (7 female, median age 11.4 yrs, range 2.5 to 21 yrs) were investigated, 3 at diagnosis and 6 while on sustained remission. nine healthy laboratory workers served as normal controls. results: before stimulation, the level of cd4+cd25+ t cells was significantly lower in aih-2 patients (3.59 2 1.08) than in normal controls (6.39 f 0.87; p=o.o2), a difference present both at diagnosis (2.24 ? 0.56, p<0.005) and during remission (4.25 2 0.35, p=o.ol). following exposure to the t cell expander, the level of cd4+cd25+ t cells increased 4.5 times (28.6 2 30.8) in normal controls, but only 1.85 times in am-2 patients (6.62 ? 1.85, p=o.ol). upon exposure to cymd6 peptides, cd4+cd25+ t cells remained numerically unchanged, in contrast to the pbmcs from the same patients giving a strong proliferative response (up to 6.9 times). summary & conclusion: our data show a numerical and functional impairment of regulatory t cells in am-2. this defect is likely to be key to the initiation and perpetuation of the autoaggressive process in this condition. orth, mark a mcniven, nicholas f larusso, mayo medical school, clinic and foundation, rochester, mn cryptosporidium parvum (cp) opportunistically infects intestinal and biliary epithelia causing worldwide morbidity, especially in patients with aids. epithelial invasion by cp involves host cell membrane alterations resulting in a parasitophorous vacuole that envelops the parasite and a dense-band within the host cell underlying the attachment site; both processes require host cell actin remodeling. since recent studies in other systems have demonstrated that cdc42, an actin-associated gtp-binding protein, plays a central role in microbial-induced actin remodeling, we tested the hypothesis that cp activates host cell cdc42 and its downstream effectors to induce actin rearrangement and microbial invasion. methods: we exposed cholangiocytes derived from normal human liver and immortalized by sv40 transformation to freshly excysted cp sporozoites and applied molecular and morphofogical approaches to test our hypothesis. results by immunofluorescent and immunoelectron microscopy, we found accumulation of cdc42 in cholangiocytes at the parasite-host cell interface during cp invasion. we confirmed activation of cdc42 in infected cholangiocytes by immunoprecipitation using an antibody to pak4, a downstream effector molecule that binds exclusively to the activated form of cdc42. phosphatidylinositol 3-kinase (pi-3k), a membrane-associated kinase associated with cdc42 activation, was also recruited to the site of attachment, as were n-wasp, pak4 and p34-arc, downstream effectors of cdc42. inhibition of pi-3k by wortmannin or ly294002 blocked cp-induced cdc42 accumulation. while overexpression in cholangiocytes of a constitutively active mutant of cdc42 promoted cp invasion (up to 50%), overexpression of function-deficient mutants of pi-3k, cdc42 or of the wa fragment of n-wasp inhibited cp invasion by 60 -80 %. moreover, inhibition of host cell cdc42 activation by dominant negative mutation inhibited cp-induced actin accumulation, and parasitophorous vacuole and dense-band formation at the attachment site. conclusions: these combined data suggest that cp invasion of cholangiocytes (and perhaps other target epithelia) results from the organism's ability to activate a complex host cell cdc42 signaling pathway that induces host cell actin remodeling at the attachment site. background: hepatitis b (hbv) and hepatitis c (hcv) infections are a world-wide health concern. studies of these infections have been hampered by a lack of a convenient animal model. we previously have shown that immunocompetent rats tolerized and transplanted with human hepatocytes can support hbv infection for at least 15 weeks. aim: to demonstrate that the immunocompetent rat which has been tolerized and transplanted with human hepatocytes can be used to sustain and study hcv infection. methods: sprague-dawley rats were injected in utero at 16-17 days gestation with one hundred thousand huh 7 cells (human hepatocyte cell line) to tolerized them to human hepatocytes. one week after birth, the tolerized newborns were intrasplenically transplanted with 5 million huh 7 cells. one week after transplantation, rodents were inoculated with one hundred thousand hcv rna copies, genotype l b human serum. animals were sacrificed at 6 and 12 weeks. the presence of human cells was determined by immunofluorescent staining of cryosectioned liver tissue using antibodies to human albumin. pcr and rt-pcr was used to confirm the presence of human albumin in liver tissue. the presence of hcv was assayed using an antibody to ns5a viral protein, and visualized using a rhodamine labeled secondary antibody. hcv infection in the liver was confirmed by the presence of negative strand rna using nested pcr. results: functional huh7 cells in the chimeric livers were confirmed by the presence of human albumin mrna and dna using rt-pcr and pcr. the presence of human albumin protein in the liver was further demonstrated by immunofluorescence of human albumin in frozen liver sections. eighty-one percent (n=57) of the tolerized, huh 7 transplanted rodents were positive for human albumin in the livers. seventy-two percent (n=36) of the chimeric animals infected with hcv were positive for the presence of ns5a hcv protein from immunofluorescence studies. livers of control rats that were only tolerized and control rats that were tolerized and transplanted with huh7 cells, but not inoculated with hcv were negative for ns5a (n=12). hcv viral replication could be demonstrated in livers of tolerized, transplanted and hcv infected rats by the presence of hcv rna bands of the correct size from nested pcr using negative strand specific primers. conclusion immunocompetent rats tolerized and transplanted with human hepatocytes can be a useful laboratory model to study hcv infection. ( we have previously observed that allogeneic hepatocellular transplants are highly immunogenic and are resistant to immunosuppressive therapy with a variety of agents. donor specific transfusion in combination with anti-cd40l mab is highly effective in inducing prolonged survival of skin and myoblasts and inducing indefinite and donor-specific tolerance to heart and islet allografts. the proposed mechanism of action for dst and anti-cd40l mab suggests peripheral deletion of alloreactive cd8+ t cells and induction of a regulatory cd4+ t cell population. resistance to induction of indefinite acceptance has been attributed to peripheral reappearance of alloreactive cd8+ t cells. in preliminary studies, we observed that dst and anti-cd40l mab prolonged fvbln hepatocyte (hc) survival in c57e3l/6 mice to median survival time (mst) of 88 days and induced indefinite acceptance (>90 days) in 43% of mice. hc rejection occurs by cd4-dependent and (cd4-independent) cd8-dependent pathways. this model permits evaluation on these two pathways separately. the current studies address the hypothesis that dst and anti-cd40l mab induces prolonged hc allograft acceptance by inducing immunoregulation of both alloreactive cd4+ and cd8+ t cells. methods: fvbln (halat-fvbin, h-24) hcs were transplanted into cd4 ko, cd8 ko, and c57bl/6 (all h-2b) mice. hc survival was monitored by detection of reporter halat protein in serum by elisa. for comparison, donor-matched islets were transplanted into streptozotocin-induced diabetic cd8 ko (h-2b) mice, and survival was monitored by blood glucose. recipient mice received peritransplant administration of dst ( 1 0~1 0~ fvbln splenocytes, day -7) and anti-cd4ol mab (1.0 mg, ip, d-7, -4, 0, 4) . some cd8 ko hosts were reconstituted cd8+ t cells (2x107 iv, d-10). results: when the cd8-dependent rejection was studied in isolation (cd4 ko mice), dst and anti-cd4ol mab prolonged hc survival to mst of 35 days (n=4) compared to 10 days in untreated cd4 ko (n=7); however, this was not significantly different from anti-cd40l mab alone. when cd4-dependent rejection was studied in isolation (cd8 ko mice), hcs were unexpectedly rejected with mst of 7 days (n=8) despite dst and anti-cd40l mab treatment. in contrast, islets of the same strain were accepted indefinitely in cd8 ko mice (n=5, mst>70 days). since this siraiegy effectively controlled hc rejection in c57bl16 mice which have both cd4-and cd8-dependent pathways available, the collective results suggested that perhaps host cd8+ t cells were necessary for the beneficial effects of the dst and anti-cd40l mab. to determine whether cd8+ t cells were required for induction of longterm hc survival with dst and anti-cd40l mab, cd8 ko hc recipients were reconstituted with cd8+ t cells prior to dst and anti-cd4ol mab treatment. cd8reconstituted cd8 kos accepted hcs for mst of 56 days. conclusion: the effects of dst and anti-cd40l mab on combined cd4-and cd8-initated hc rejection are more pronounced than on either pathway alone. cd8+ t cells appear to be required for induction of longterm hc survival under cover dst and anti-cd40l mab, which offers an apparently distinct mechanism compared to the existing paradigm for the mechanism of action of dst and anti-cd40l mab. engraftment of transplanted cells in the liver is affected by cellcell interactions involving hepatic endothelial cells and kupffer cells. the proximity of transplanted cells to hsc suggested that hsc could promote cell adhesion and extracellular matrix remodeling during cell engraftment. to investigate cell-cell interactions in the liver, we analyzed hsc activation in dppn-rats transplanted intrasplenically with f344 hepatocytes. analysis of tissues by immunostaining from animals 6h, 24h, 3d and 7d after cell transplantation showed appearance of desmin +ve hsc in periportal areas, reaching a peak on day 3 (4-10 fold increase in desmin f v e hsc in cell recipients, p<o.ool). using combined immunostaining and dppiv histochemistry, desmin +ve hsc were often observed in the immediate vicinity of transplanted cells. however, a-smooth muscle actin (sma) was not expressed in hsc perturbed by cell transplantation, whereas ccl&reatment in control animals induced sma expression in hsc, as shown by tissue immunostaining, as well as western blots of tissue extracts. after cell transplantation, perturbed hsc expressed desmin extensively, especially in areas with cell transplantation-induced ischemia and consequential ggt expression. to examine whether desmin +ve hsc were perturbed following activation of kupffer cells by cell transplantation, we administered carbon particles to hepatocyte recipients intrasplenically. these studies established that the majority (up to 72%) of desmin +ve hsc were located directly adjacent to carbon containing activated kupffer cells. as kupffer cells are activated within 6 hrs after cell transplantation, whereas hsc were perturbed maximally at a later time, we reasoned that these cell-cell interactions were likely mediated by soluble factors. to demonstrate whether tnf-alpha plays a role in the stellate cell activation process, we transplanted hepatocytes into animals treated with the tnf-alpha-blocker etanercept. however, etanercept did not prevent perturbation of hsc, suggesting that factors other than tnf-alpha must be involved. to eliminate the component of ischemia-mediated cell activations, we treated animals with intrasplenic nitroglycerine infusion before and during cell transplantation, which resulted in marked attenuation of hepatic ggt expression. interestingly, desmin +ve hsc were now distributed throughout the entire liver lobule, with 3-5 fold greater prevalence, compared to rats treated with cells alone without nitroglycerine, p<o.ool, suggesting that circulating soluble factors reached hsc more effectively. in contrast, treatment of animals with nitroglycerine alone did not perturb hsc. to determine effects on cell integration, bile canalicular reconstitution was analyzed with dppiv and atpase co-stainings. nitroglycerine pretreatment resulted in superior parenchymal integration of transplanted cells, in proximity with desmin +ve hsc. conclusions: transplantation of cells in liver sinusoids activates desmin, but not sma, expression in hsc. activated kupffer cells are likely mediators of hsc perturbation. improved cell engraftment following perturbation of hsc indicates that extracellular matrix remodeling could facilitate this process. these findings offer ways to further optimize liver repopulation with transplanted cells. knudsen, timothy b hummer, kim a buffers, kiyoshi ogata, cody a koch, jefiey l platt, mayo clinic, rochester, m n background hepatocyte transplantation has been of much interest as a biological liver support. how to treat hepatocytes to achieve optimum function has been uncertain. cultured, cryopreserved and hypothermically preserved hepatocytes have been employed for clinical hepatocyte transplantation, but the efficacy was rarely compared between those cell sources. using a xenogeneic hepatocyte transplantation model, the function of transplanted hepatocytes was quantified in vivo. methods: porcine hepatocytes were isolated by a two-step collagenase technique. the hepatocytes were: (1) immediately used; (2) kept in uw solution at 4°c; (3) cultured in supplemented williams e medium; or (4) cryopreserved in ljw solution with 10% dmso before use. the viability of hepatocytes was determined by trypan blue exclusion, and 1 x lo6 viable hepatocytes were injected into the spleens of rag-2-defficient mice. to evaluate the function of transplanted hepatocytes, the porcine albumin level in the recipient plasma was determined by a sandwich elisa. the number of surviving porcine cells in the recipients' spleen was estimated by a quantification of porcine genomic dna sequences. results: the viability of hepatocytes was as follows: freshly isolated hepatocytes, 92.2 2 1.9%; 24 hours, 48 hours, and 72 hours at 4"c, 85.8 t 3.1, 74.3 i 1.9, 71.0 i 3.2%; cryopreserved hepatocytes after thaw, 65.0 2 2.6%; cultured hepatocytes after dissociation, 78.4 t 6.3% (means t se). after transplanting comparable numbers of viable hepatocytes, the porcine albumin concentration was found to be lower in recipients of hypothermically-stored (group 2) and cultured hepatocytes (group 3) than recipients of fresh hepatocytes (group 1). cryopreserved hepatocytes (group 4) failed to secrete any albumin. because the half-life of porcine albumin in rag-2deficient mice was 13.5 hours, the porcine albumin concentration in the recipient plasma reflected real-time albumin secretion from the transplanted hepatocytes. the porcine genomic dna in the recipient spleen was quantified 4 weeks after transplantation. fewer cells were found in the hypothermically preserved hepatocyte-recipients (group 2). therefore, the lower level of the porcine albumin secretion in the group 2 was mainly attributable to the failure of the transplanted hepatocytes to survive. the amount of porcine genomic dna was comparable between cultured hepatocyte-recipients and freshly isolated hepatocyte-recipients, although the plasma of cultured hepatocyte-recipients included less porcine albumin. it suggested that the hepatocytes had dedifferentiated during culture and secreted less albumin thereafter in vivo. the cultured hepatocytes did not seem to differentiate again in murine spleen. conclusions: both the survival and differentiation of hepatocytes were important to maintenance of good function in vivo. the hypothermically preserved and cryopreserved hepatocytes did not survive long after transplantation contrary to their good viable look. cultured hepatocytes showed relatively good survival, although their dedifferentiation was not reversible resulting in poor outcome. freshly isolated hepatocytes are recommended for cell transplantation. medicine, ube-yamaguchi, japan; hiroshi nishina, university of tokyo, tokyo, japan; kiwamu okita, yamaguchi university school of medicine, ube-yamaguchi, japan obiective; we had developed an in vivo model to monitor the trans-differentiation and proliferation of bmcs into functional hepatocytes via hepatoblast phenotype (gfpicc14 model). in this model, transplanted gfp-positive bmcs via tail vein efficiently migrated to the peri-portal area of liver lobules under cc14induced chronic liver damage condition. transplanted bmcs gradually proliferated and spread from the peri-portal area (zone 1) into the central area (zone 2). under persistent liver damage condition, bmcs trans-differentiated into functional hepatocytes. previous analysis had shown that inflammatory signals had been important for the trans-differentiatin of bmcs. in this study, we focused on growth factors and analyzed which affected the transdifferentiation and proliferation of bmcs into hepatocytes in gfpl cc14 model. materials and methods; immunohistochemical staining and immunofluorescent double staining with antibody of gfp, several growth factors and their receptors were performed in gfpiccl4 model. hepatocyte growth factor (hgf), epidermal growth factor (egf), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), and transforming growth factor (tgf) were analyzed. the percentage of stained area was quantified in the sections at thirty random fields (n=6 in each group), and statistical analysis was performed using plsd test (anova). results; the most significant result was shown in fibroblast growth factors (fgfi, fgf2) and their receptors (flg, bek). in immunohistochemical staining, the stained region migrated and increased from zone1 into zone2 of liver lobules with the time after bmt. in immunofluorescent double staining, co-expressions both gfp and fgfs, fgf-receptors were detected at the same distribution. the percentage of stained area of fgfs and fgfreceptors gradually increased with significant difference (the data was shown in table 1 ). although expressions of other growth factors and their receptors were detected, the significant differences were not shown. conclusions; fgfs are the most important growth factor to transdifferentiate and proliferate bmcs into hepatocytes. cholangiocytes are the target cells of chronic cholestatic liver diseases (i.e., cholangiopathies), characterized by the dysregulation of the balance between apoptosis and proliferation that leads to changes in ductal bile secretion. in rats with bile duct ligation (bdl), there is increased cholangiocyte proliferation and enhanced basal and secretin-stimulated ductal secretion. we have shown that both cholinergic (by vagotomy) and adrenergic b y administration of a single intraportal injection of 6-hydroxydopamine (60hda)i denervation impairs the proliferative and secretory responses of intrahepatic bile ducts to bdl through an increase in cholangiocyte apoptosis. we have shown that bile acids and nerves co-ordinately regulate the balance between cholangiocyte apoptosislproliieration in hyperplastic rat livers. in bdl rats, while ursodeoxycholate and tauroursodeoxycholate feeding prevents the loss of bile ducts caused by vagotomy in a caz+/pkc-dependent manner, taurocholate feeding prevents vagotomy-induced duct damage in a pi3k dependent manner. no information exists regarding the interaction between bile acids and adrenergic innervation in the regulation of cholangiocyte function. thus, we tested the hypothesis that taurocholate feeding protects from bile duct damage induced by adrenergic denervation by 6-ohda. methods: immediately after bdl, rats received a single intraportal injection of 6-ohda (which induces degeneration of adrenergic terminal fibers, 50 mglkglbody weight) or vehicle, and subsequently were fed 1% taurocholate or control diet in the presence of daily injections of wortmannin (a pi3k inhibitor, 0.7 mgl kg body weight) or a control solution. we used 1 week bdl rats as control animals. seven days later, we evaluated cholangiocyte apoptosis by : (i) tunel assay in liver sections; and (ii) measurement of caspase 3 activity and protein expression for cleaved pro-caspase 3, and the number of purified cholangiocytes positive for annexin-v. cholangiocyte proliferation was evaluated by measurement of the number of pcna-and ck-19-positive cholangiocytes in liver sections, and pcna immunoblots in pure isolated cholangiocytes. the changes in ductal functional activity were evaluated by measurement of (i) secretinstimulated camp levels and cl-ihcos exchanger activity in pure cholangiocytes; and (ii) secretin-stimulated bile flow and bicarbonate secretion in bile fistula rats. results: taurocholate feeding prevented the increase in the number of apoptotic cholangiocytes and the enhancement of caspase 3 activity and protein expression for cleaved pro-caspase 3 induced by 6-ohda. taurocholate feeding prevented 6-ohda-induced decrease in the number of pcna-and ck-19 positive cholangiocytes, and the loss of pcna protein expression. at the functional level, taurocholate feeding prevented the decrease in secretin-stimulated camp levels, c1-l hco3 exchanger activity and bile and bicarbonate secretion. consistent with the concept that pi3k regulates the interaction of bile acids with adrenergic nerves in cholangiocytes, we found that administration of wortmannin blocks 6-ohda-induced activation of cholangiocyte apoptosis, and inhibition of cholangiocyte proliferation and ductal secretion. summarylconclusion: taurocholate feeding prevents the increase in cholangiocyte apoptosis and the loss of cholangiocyte proliferation and ductal functional activity induced by adrenergic denervation by 6-ohda. taurocholate effects are mediated by the pi3k pathway, since the simultaneous administration of wortmannin reverses such effects. these novel findings suggest that bile acids play a major role in sustaining the growth and functional activity of the intrahepatic biliary tree following liver transplantation. hepatocyte apoptosis by death receptors is emerging as a critical determinant in the initiation of hepatic inflammation and fibrosis during cholestatic liver diseases. however, the cellular sources of death ligands to activate the death receptors is unknown. we postulated that kupffer cells, which contribute to hepatic inflammation, may also contribute to hepatocyte apoptosis and liver fibrosis by generating death ligands. thus, our aim was to ascertain whether kupffer cells express death ligands and contribute to hepatocyte apoptosis and liver fibrosis in the bile duct ligated mouse, an animal model of cholestasis. methods: mice were subjected to bile duct ligation (bdl) or a sham operation and treated with gadolinium chloride, a kupffer cell intoxicant, or saline for three days. hepatocyte apoptosis in liver sections was assessed using the tunel assay. serum alt values were quantitated using commercially available kits. kupffer cells were isolated using arabinogalactan gradient centrifugation. real time pcr was used to measure expression of death ligands and fibrosis indicators. results: hepatocyte tunel positive cells, and serum alt values were 3.3-fold, and 5.2-fold, respectively, greater in 3 day-(bdl) saline-treated vs. gadolinium chloride-treated mice. kupffer cell expression of the death ligands tnf-alpha and fas ligand was 14 and 20-fold greater when kupffer cells were obtained from bdl vs. sham operated mice. the enhanced expression of fas ligand was verified by immunoblot analysis and tnf-alpha by an elisa. these data confirm that kupffer cells generate death ligands and contribute to hepatocyte apoptosis in cholestatic liver injury. consistent with a role for kupffer cellassociated hepatocyte apoptosis in liver inflammation, neutrophil infiltration into the bdl liver was abrogated by gadolinium chloride. hepatic mrna transcripts for a -smooth muscle actin, and collagen a 1(i), markers for stellate activation were all significantly attenuated gadolinium chloride vs. vehicle-treated animals. finally, the mechanisms for kupffer cell activation were explored in cell culture. isolated kupffer cells phagocytosed apoptotic bodies, which induced death ligand expression. in contrast, bile acids had no effect on kupffer cell gene expression. infection is associated with vigorous, durable cd8+ t cell responses against nonstructural hcv proteins. hcv persistence has been attributed to poor priming of cellular immune responses. one of the factors that contributes to poor priming of cellular immune responses may be the noncytopathic nature of hcv and to the subsequent absence or scarcity of virus-infected apoptotic or dead cells as source of exogenous antigens for crosspresentation. because crosspriming contributes to the induction of cd8+ t cells in vivo (nature 1999; 39877-80), we hypothesized that cross-priming with dendritic cells (dcs) containing self-replicating rna might be useful to induce strong, hcv-specific cellular immune responses. methods: choosing hcv ns3 as a model antigen, we generated a recombinant cytopathic pestivirus self-replicating rna to amplify hcv ns3 in dcs. the principal characteristic of this vector is its ability to replicate in transfected cells which in turn enhances production, processing and presentation of the encoded hcv ns3 antigen. moreover, the availability of cytopathic and noncytopathic forms of the same replicon enabled us to study the immunologic implications of dc apoptosis and antigen reprocessing, leading to crosspriming in vivo. results: the murine dendritic cell lines dc2.4 was transfected with cytopathic and noncytopathic recombinant replicon rna, respectively. hcv ns3 expression was detectable in more than 95% of the transfected cells by immunofluorescence. the time kinetics of apoptosis induction was monitored by flow cytometry using annexin v and propidium iodide staining. in contrast to the noncytopathic replicon, the cytopathic replicon led to dc apoptosis 12 hours after transfection. dc2.4 transfected with the cytopathic recombinant replicon rna were then used to immunize hla-a2 transgenic mice subcutaneously. ifn-positive, proliferative cd4+ t cells and ifn--y positive, cytotoxic cd8+ t cell responses were detectable after a single subcutaneous immunization with replicon-transfected dendritic cells and significantly stronger than after conventional, intramuscular dna immunization, the previous gold standard. crosspriming was demonstrated when t cells, primed by injection of h-2b+ dcs into the h-2b+ hla-a2+ mice, were tested with hla-a2+ antigen-presenting cells. transfer of cellular material from the vaccine dcs to the endogenous apcs, a phenomenon that underlies crosspriming, was directly visualized in vivo when fragments of csfe-labeled, injected h2b+ dcs were demonstrated in hla-a2+ host cells in lymph nodes and spleens by flow cytometry and if microscopy. finally, the protective effect and the in vivo function of the induced hcv-specific t cells were evaluated in a surrorgate challenge model with recombinant, hcv ns3 encoding vaccinia virus. whereas lo4 to lo7 pfu were detected in nonvaccinated control mice, no vaccinia virus was detected in mice vaccinated with replicon-transfected dcs, indicating complete protection. summary and conclusion: these findings demonstrate and explain the immunologic potential of the proposed vaccination strategy. moreover, they support the hypothesis that experimental forcing of exogenous antigens into the mhc class i pathway and subsequent promotion of cross-priming is particularly important to generate t cell responses against noncytopathic and tissue tropic viruses such as hcv. disclosures: introduction: hepatitis c virus (hcv) infection leads to chronic liver disease in about 85% of infected individuals while only a minority achieves spontaneous viral elimination in the initial phase of acute hepatitis c. anti viral mechanisms clearing the infection in these patients, however, could contribute to the genera-tion of therapeutic or prophylactic vaccines. the strong association of a broad, hla restricted, vigorous and long lived anti viral cd4+/ th1 t-cell response with spontaneously resolving acute hepatitis c has been demonstarted in the past. only few epitopes recognized by anti hcv specific cd4+ t cells that are associated with spontaneous viral clearance have been characterized so far. the aim of the present study was to identify and characterize hcv specific cd4+ t cell epitopes and their hla restriction during acute self limited hcv infection andlor viral control. methods: proliferative cd4+ t cell responses (3h-thymidin incorporation) against viral proteins (hcv-ns3, -ns4) and against overlapping 20-mer pep-tides covering the entire ns3 and ns4 (aa 1027-1972) region were performed in 25 patients with acute self limited hcv infection and in two patients during temporary viral control (hcv rna negative). ten patients with chronic hcv infection served as control group. cd4+ t cell clones were generated by limit-ing dilution of hcv protein specific t cell lines. hcv specific cd4+ t cell clones were further characterized fine specificity was assessed by proliferation assay after stimulation with overlapping peptides of the corresponding protein region, then the minimal epitope was defined with n-and c-terminal truncated peptides for each epitope. hla restriction was assessed by inhibition experi-ments (anti-dp, -dq, -dr) and cd25+ expression in the presence of a panel of ebv blasts by flowcytometry. results: we studied 25 patients with acute self limited hcv infection. all pa-tients displayed significant proliferative t cell responses against hcv-ns3 or -ns4 protein, that was strongly associated with self-limited disease and absence of hcv rna. proliferative responses against 20-mer peptides showed multiple responses within the ns3 and ns4 region, possibly reflecting multiple epitopes in this region. in contrast no virus specific t cell responses were detectable in a control group of patients with chronic hcv. for further characterization of t cell epitopes hcv specific t cell clones were generated from 6 patients against eight different hcv epitopes located within the ns3 (aa1027-1657, two epi-topes) and ns4alb (aa1658-1972, six epitopes) region. the minimal epitopes consist of 8 to 12 amino acids. all t cell clones were hla class i1 restricted and could be stimulated by protein or peptide pulsed ebv blasts expressing the cor-responding hla-dr or -dq. conclusion: the hcv ns3 and ns4 proteins contain several immunodominant cd4+ t cell epitopes which are associated with spontaneous viral clearance. the detailed characterization of minimal epitopes and hla restriction are a pre-requisite for the synthesis of hla class i1 tetramers and for the development of prophylactic and therapeutic t cell vaccines. background a brief period of tissue ischemia followed by reperfusion renders tissue resistant against subsequent prolonged ischemia and reperfusion, a phenomenon called ischemic preconditioning. hepatic ischemic preconditioning protects sinusoidal endothelial cells against subsequent cold storagelwarm reperfusion injury and improves graft survival after liver transplantation in rats. aim ischemic preconditioning also reduces liver injury after warm ischemia and reperfusion, in which hepatocyte damage and kupffer cell activation are dominant features. however, it is incompletely elucidated which cell type ischemic preconditioning affects and how it works. therefore, our aim was to investigate the mechanisms of ischemic preconditioning against warm ischemia and reperfusion injury. method male sprague-dawley rats were injected intravenously with 20 mglkg gadolinium chloride (gdcls), 10000 ulkg superoxide dismutase (sod) or 300 mglkg n-acethyl-l-cyctein (nac) to suppress kupffer cells, superoxide formation or oxygen radical formation, respectively. livers were then preconditioned by clamping the hepatic artery and portal vein to the median and left lobes for 10 min followed by 10 min reperfusion. subsequently, livers were subjected to warm ischemialreperfusion injury in in vivo experiments and in liver perfusion experiments as follows. in vivo experiments: the blood flow to the median and left lobes was occluded with a vascular clamp for 40 min. one hour after removal of the clamp, blood was collected for determination of alt activity and hyaluronic acid (ha) concentration. liver perfusion experiments: livers were perfused through the portal vein for 10 min with physiological buffer and stored at 37°c in the same buffer. after 40 min, livers were reperfused with the buffer containing 500 ng/ml ha for 60 min in a recirculating system for determination of alt activity in the perfusate and hepatic ha uptake as a marker of sinusoidal endothelial cell injury. livers of other non-treated rats were preconditioned by perfusion with the buffer containing h202 instead of ischemic preconditioning, and then subjected to warm ischemialreperfusion similarly. in other experiments, livers were subjected to ischemic preconditioning and perfused with the buffer containing 0.5 mglml nitro blue tetrazolium (nbt) for 10 min and fixed with formalin. since nbt reacts with superoxide to form insoluble blue formazan, kupffer cell superoxide formation was evaluated in histological sections. results ischemic preconditioning decreased serum alt activity compared to control (168 ? 34 [mean 5 semi vs 333 ? 52, p < 0.05), but did not change serum ha concentration. pretreatment with gdc13 reversed the effect of ischemic preconditioning (262 2 41), though gdc13 itself did not change alt activity after ischemialreperfusion (318 t_ 33). similar results were obtained in liver perfusion experiments, suggesting that ischemic preconditioning protected hepatocytes, but did not change sinusoidal endothelial cell injury. it is also suggested that kupffer cells mediate hepatocytes protection by ischemic preconditioning. in rats treated with sod or nac, decrease in alt activity in the perfusate by ischbackground: living donor liver transplantation (ldlt) and split livers have emerged as a solution to ease the shortage of liver transplants and have achieved remarkable success in pediatric populations. in adults, ldlt is limited by the size of the graft that can be safely harvested and transplanted. similar limitations apply when extensive liver resections are needed for the treatment of hepatic tumors. we have previously demonstrated that the anti-apoptotic gene a20 is part of the physiologic response of hepatocytes to injury, protecting them from apoptosis and limiting inflammation by inhibiting nf-kb activation. our objective was to check whether a20 expression would help decrease the minimal liver mass required for mice survival. methods and results: two-thirds partial hepatectomy is well tolerated and is an accepted model of liver regeneration in rodents. we performed an extended (78%) liver resection (n=12/group) in non-infected (ni) mice (10-12 week balb/c) and mice infected with recombinant a20 adenovirus (rad.a20) or a control adenovirus (rad.p-gal). surviving mice suffer a delay in regeneration as assessed by the number of proliferation cell nuclear antigen (pcna) positive nuclei 48 h following resection (5-10 positive cellslhpf in mice with 78% liver resection vs. 60-70 positive cells/ hpf in mice with a 2/3 liver resection). interestingly, such a delay was not noted in mice expressing a20 (70-80 pcna positive cells/ hpf were detected 48 h following 78% resection). a20 also prevented the development of coagulopathy as assessed by prothrombin time. we extended our resection to a radical hepatectomy comprising 87% of the liver (n=12lgroup). we observed 100% lethality in ni and rad.p-gal infected mice. expression of a20 resulted in the survival of 60% of the animals and correlated with increased pcna positive hepatocytes as an indicator of increased cell proliferation. a20 mediated increase in hepatocyte proliferation was not merely dependent upon its anti-apoptotic function. expression of a20 in hepatocyte cultures, in a nonapoptotic milieu, increased their proliferative response to serum and hgf as compared to ni and rad.p-gal infected cells. conclusion: these results demonstrate a novel pro-proliferatif function for a20 in hepatocytes and argue for its critical role in accelerating liver regeneration and promoting hepatocyte survival and function, even when facing extreme metabolic demands. gene therapy with a20 may allow for successful transplantation with smaller ldlt, split liver allografts and even hepatocellular grafts. additionally, a20 based therapy to the liver may allow for extensive liver resections in the setting of cancer therapy. employee graeme currie -gilead sciences, inc.: employee samuel didier -gilead sciences, inc.: investigator stefanos hadziyannis -gilead sciences, inc.: investigator craig james -gilead sciences, inc.: employee ching-lung lai -gilead sciences, inc.: investigator nicole lama -gilead sciences, inc.: employee pietro lampertico -gilead sciences, inc.: investigator peter neuhaus -gilead sciences, inc.: investigator eugene schiff -gilead sciences, inc.: investigator norah terrault -gilead sciences, inc.: investigator hans tillmann -gilead sciences, inc.: investigator 24 prolonged survival of porcine hepatocytes in cynomolgus monkeys. h nagata aliberti -no relationships to disclose sven erik behrens -no relationships to disclose vito racanelli -no relationships to disclose barbara rehermann -no relationships to disclose 35 characterization of novel cd4+ t cell epitopes in acute selflimited hcv infection previous studies from our lab show that interleukin-6 (il-6) is a complete biliary epithelial cell (bec) mitogen that is normally produced by bec at low levels and upregulated by mechanical, infectious, or immunologic biliary tract injury. bile duct ligation (bdl) in il-6-deficient (il-6-/-) mice results in a reproducible phenotype that includes rapidly decompensating biliary cirrhosis, which is at least partially attributable to impaired biliary tree integrity. methods and results: oligonucleotide array analysis of primary mouse il-64-and wild type il-6+/+ bec cultures was used to search for possible molecular mechanisms that might account for impaired biliary tree integrity in il-64-mice. the results showed upregulation of trefoil factor family (tff) messenger rna (mrna) in the il-6+/+ bec compared to the il-64-bec. tff are mucin-associated protease-resistant peptides that greatly contribute to barrier and epithelial repair in the gastrointestinal tract. various molecular analyses in vitro using bec cultures confirmed that il-6-mediated gp130 signaling through the signal transducer and activator of transcription 3 (stat3) importantly contributes to tff3 mrna and protein expression. in vivo, tff3 mrna and protein was expressed predominantly in the medium and large bile ducts and peribfiary glands in normal mouse liver in il-6+/+ mice, but weakly present or absent in il-64-mice. within several weeks after bdl, tff3 mrna and protein levels decreased transiently then increased to near baseline levels by twelve (12) weeks after bdl in the il-6+/+. in contrast, the il-64-mice failed to show the secondary recovery of tff3 by 12 weeks when mrna and protein levels were significantly lower than il-6+/+ (figure) . the retarded recovery of tff3 mrna and protein in the il-6-imice was reversed by daily treatment of recombinant il-6. similar results were obtained in analysis of normal human liver and various biliary tract diseases. conclusions: tff3 is the predominant trefoil factor expressed in the mouse and human biliary tree; its expression appears to be upregulated by stat3 signaling and suppressed by the mitogen-activated protein kinases (mapk) signaling in the bec. tff expression in the biliary tree importantly contributes to biliary tree integrity and repair reactions. blackard, carolynne zander, massachusetts general hospital, harvard medical school, boston, ma; gregory beck, university of massachusetts, boston, ma; emmett v schmidt, raymond t chung, massachusetts general hospital, harvard medical school, boston, ma backgroundlaims: the innate immune response (type i ifn system) is postulated to play a critical role in control of viral infection.the role of the ifn system in controlling hcv replication has been inferred but not proven. in addition, hcv protein expression has been shown to antagonize the ifn response in vitro. until recently, it has been impossible to assess the interaction between hcv and the type i ifn system in vivo without a model that successfully supports viral replication. we have developed a cell-based binary hcv replication model that successfully recapitulates the early stages of the hcv life cycle (pnas 98 9847,2001) . we therefore used this system (1) to assess the role of type i ifn in controlling hcv replication; and (2) to assess the effect of hcv expression or replication on ifn-induced intracellular signaling. methods: full-length hcv cdna corresponding to the ph77 strain previously shown to be infectious in the presence of ?7 was transfected into huh-t7 cells (a t7 stably-transfected huh7 be), as well as mutant m g h human fibroblast cell lines null for the ifn signaling proteins tyk2 (ul), irf9 (u2), statl (u3), jakl (u4), and stat2 (us) respectively. to test the effects of known ifn antagoflists on hcv replication, the ifn signaling antagonist sendai virus y2 protein was cotransfected into huh-t7 cells. in addition, neutralizing antibodies (nab) to type i ifn were added to selected huh-ti cells. to study the effect of hcv on ifn signaling, huh-ti cells were cotransfected with the reporter plasmids pisre-luc and prl-tk. ifna (1000 iu/ml) was added to selected cells after transfection. reporter gene expression was monitored by the dual-luciferase reporter assay system and measured as relative luciferase activity. hcv core protein was measured using the trak-c hcv core ag elisa. results ifna significantly reduced hcv core ag production from 203.5 pg/ml (ph77+/ifn-) to 41.3 pglml (ph77+/ifn t) (p=o.ool). in contrast, y2 protein enhanced hcv core ag production to 407.6 pglml in ph771y2 cotransfected cells (p=o.oool). control experiments confirmed that y2 protein inhibited ifna-induced isre-mediated signaling in huh-t7 cells; relative luciferase activity was reduced from 653 (ph771 ifnp nab increased hcv core ag replication by 42% and 23% compared to no treatment (p=o.o1). the combination of anti-1fna nab and anti-ifnp nab significantly enhanced hcv core ag production by 73% (p=o.004). the u3 cell line enhanced hcv core ag replication by over two-fold compared to the parental m g h and other mutant cell lines. hcv (ph77) transfection suppressed ifn-induced relative luciferase activity by 50% compared to ifn-treated pgem transfected cells (p= 0.w). transfection with a replication defective h77 mutant, subgenomic hcv core, or hcv structural protein mutant constructs had effects on suppression of isre-luc similar to h77.conclusions: using a binary, full length hcv replication model, we found that (i) hcv replication is enhanced in the presence of the ifn antagonist y2 as well as by ifn neutralizing antibodies, confirming the criticality of the type i ifn system in innate control of hcv replication.(2) statl null cell lines enhanced hcv replication, indicating that this protein is the critical signaling component in innate antiviral immunity to hcv. (3) hcv expression inhibits ifn signaling. hcv protein expression and specifically hcv core expression appears to be su€ficient to suppress ifn signaling. these findings demonstrate that the innate immune response exerts direct antiviral effects on hcv, and that hcv protein expression subverts type i ifn signaling. our replication model has the potential to provide new insights into the mechanisms of host control of hcv replication as well as the mechanism of hcv persistence. disclosures:gregory beck -no relationships to disclose jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose yoshitaka kamegaya -no relationships to disclose wenyu lin -no relationships to disclose emmett v schmidt -no relationships to disclose carolynne zander -no relationships to disclose ifna) to 21.2 (ph77/ ifndy2) (p=o.o001). anti-ifna nab and anti-background cytotoxic t lymphocyte activator 4 downregulates t cell activation by ligating b7. single nucleotide polymorphisms (snps) at positions -318 in the promoter and +49 in exon 1 of the ctla4 gene have been associated with an increased risk for autoimmune diseases. the +49g allele has also been associated with a greater likelihood of response to interferon-alpha in patients treated for chronic hepatitis c. since some of the ctla4 snps either individually or linked together (haplotypes) may alter ctla4 expression or activity, we hypothesized that they may be associated with natural hepatitis c virus (hcv) clearance or persistence. methods: utilizing a nested case-control study design from an injection drug using cohort and a hemophiliac cohorts, we matched subjects with hcv clearance (anti-hcv positive and hcv rna negative on two occasions separated by a minimum of 6 months) to two persistently hcv-infected individuals (hcv rna positive on two occasions) based on h n status, race, and gender. five ctla4 snps, which define all common haplotypes, were genotyped using either sequence specific primers or a single base extension method. conditional logistic regression was used to determine odds ratios and p-values for these snps. haplotypes were constructed using the phase program and analyzed using the snpem program. results: included in this study were 190 and 370 subjects who either cleared or had a persistent infection with hcv, respectively. the study cohort was 47% black, 46% white, and 7% other. blacks who were homozygous for the mutant allele 49g were more likely to experience viral clearance (or 0.45, p=0.03). this association was not found in white subjects (or 0.92, p=0.81). since, hla-cw"o4 is associated with viral persistence in these cohorts (thio et a1 j virol2002), we stratified the 49g homozygous individuals by c w w . both blacks and whites who did not have cww4 were more likely to have viral clearance if they were homozygous for 49g (or 0.45, p=0.13 and or 0.49, p=o.11, respectively) . stratification by other hla alleles associated with hcv clearance or persistence did not reveal additional relationships. the 49g formed haplotypes with the wild type alleles at three of the four other snps examined, and those haplotypes did not have a stronger association than 49g alone. 49g was found in haplotypes with both the mutant and wild type alleles at the -1722 promoter position, but neither of these haplotypes had a stronger association with clearance. conclusions: homozygosity for the ctla4 49g snp is associated with clearance of hcv in blacks regardless of hla genotype, whereas in whites, homozygosity at this position trends toward an association with clearance in hla-cw*m-negative persons. since ctla4 49g has been reported to augment t ceil activation by decreasing ctla4 expression, these results point to the importance of t cell activation in hcv clearance. disclosures:jacquie astemborski -no relationships to disclose james g goedert -no relationships to disclose recurrence of hcv infection post liver transplant is universal. high levels of viraemia characterize the post transplant course. in this setting, acute rejection in association with hcv re-infection of the graft (ar (hcv)) and recurrent chronic hepatitis (chi) are recognized clinical entities. we aimed to 1) determine the intrahepatic gene profile in ar (hcv) and chi. 2) examine whether the gene profile in ar (hcv) is different to that of acute rejection in non-hcv infected allografts (ar non-hcv). 3) examine whether the gene profile of chi is different to chronic hepatitis c pre-transplant (ch). methods: genechip arrays were utilized to study pooled rna from 34 liver tissue; 6 patients with ar (hcv), 6 patients with ar (non-hcv), 8 patients with chi, 8 patients with ch and 6 normal livers. probe level data was analysed using the bioconductor affy library and genes with more than 2 fold changes were considered to be upregulated. results: ar (hcv) us ar (non-hcv): compared to normal tissue, both groups were similarly characterized by upregulation of several t cell dependent immune activation genes such as the lymphocyte antigen campath-1, mac-2 binding protein and ifn related genes. however, compared to ar (non-hcv), ar (hcv) was specifically characterized by upregulation of several ifn-alpha associated genes including 2-50as, and p27. chi vs c h compared to normal, both groups were characterized by upregulation of genes involved in antigen presentation (proteasome subunit 42, mdw), t cell activation (t cell receptor and ctl-17), ifn-gamma inducible genes (ifn-56kd, ip-10, humig and class i1 mhc) and ifn-alpha associated genes (p27 and 2-5 oas). however, the ifn-alpha and gamma genes were more upregulated in chi compared to ch. furthermore, compared to ch, the chi group displayed increased expression of genes involved in cellular proliferation (pcna, cyclin, beta integrin), apoptosis (cytochrome c, fas-l, caspase 4), inflammation (macrophage mannose receptor, complement factor h) and fibrosis (angiotensin i1 receptor, proteoglycan core protein). conclusions: at the transcriptome level, the pathological process in hcv infected allografts with acute rejection or recurrent chronic hepatitis is characterized by upregulation of several ifn-alpha and gamma related genes compared to other livers. moreover, chronic hcv post transplant is further characterized by an increased process of cellular proliferation, apoptosis and fibrosis compared to chronic hepatitis in the pretransplant setting. collectively the data suggest that the high levels of viraemia which characterize the post transplant setting drive the immune response to act at a higher level. in addition, the upregulation of several interferon related antiviral genes supports the notion that hcv is a strong inducer of intrahepatic interferons, but is relatively resistant to their antiviral activity. (hcv) infection is thought to be mediated by a multispecific immune response. hcv-specific t cells have been described in hcv-seronegative virus-exposed individuals such as i.v.-drug addicts, family members of chronic hcv-patients and lab-personnel. however, it is not known whether these individuals had recovered from previous asymptomatic acute hepatitis c. in this study, we prospectively followed 10 individuals who experienced an injury with an hcv-contaminated needle. methods: between january 2001 and march 2003 we collected pbmc from 10 individuals (medical health professionals at our institution; 3 females, 7 males; age 30-45 years) who experienced an injury with an hcv contaminated needle. blood samples were taken on the day or the day after the event and at different time points during follow-up for up to 12 months. low levels of hcv-rna were examined in serum by the highly sensitive transcription-mediated tma-assay (detection limit 5-50 iuiml) and in rna of pbmc. t cell-cytokine secretion (elispot-assays for ifn-gamma and il-10, flow-cytometry-based ifn-g capture assays), t cell-proliferation (3-thymidin incorporation, cfse-staining), and t cell-cytotoxicity (51-chromium release assays) were investigated directly ex vivo and in t cell lines. results: none of the individuals became positive for hcv-rna in serum (tma-assay) or pbmc and all of them remained anti-hcvnegative throughout follow-up. at the time of the needle stick injury, hcv-specific cd4+ t cell responses were already detectable in 2 of the individuals and became detectable thereafter in 2 additional persons. hcv-specific cd8+ t cell responses could be investigated in one hla-a2-positive individual in more detail at several time points. he was negative for hcv-specific interferon gamma-and il-10-producing cells on the day of the injury as determined by elispot assays. however, after 18 weeks, we detected for core-178-specific ifn-gamma producing cd8+ t cells. out of 7 mhc-class i-restricted hcv epitopes tested, one additional peptide (ns3-1406) became positive 8 weeks later. the presence of core-178 and ns3-1406-specific cd8+ t cells was confirmed by a second flow-cytometry-based assay. hcv-specific inf-gamma positive cells were cd8-dim and hla-dr-negative. the frequency of ns3-1406 and core-178-specific cd8+ t cells was about 114500 and 118500 pbmc, respectively, in the elispot assay and remained constant in this subject until month 11 of followup. the increase of cd8+ t cell responses was accompanied by the development of an hcv-specific cd4+ response targeting predominantly the hcv-core and hcv-helicase antigens. conclusions: we here demonstrate in a prospective study the development of hcv-specific t cells in hcv-exposed individuals after needle stick i n j q in the absence of viremia. surprisingly, these hcvspecific t cells became detectable rather late after the potential exposure. our data are in line with previous studies demonstrating hcvspecific t cell responses in chimpanzees inoculated with subinfectious doses of hcv. t cell immunity in medical health professionals against hcv may contribute to the low prevalence of hcv among doctors and nurses since hcv prevalence in medical health personnel has been shown to be equal or even lower as compared to the general population in most studies. aims: laparoscopic cholecystectomy is the treatment of choice for symptomatic gallstone disease at present. despite its wide spread application in the past decade, the incidence of iatrogenic bile duct injuries related to this procedure has remained unchanged around 0.3-0.6%. we review the investigation, management and outcomes of such injuries referred to a tertiary referral center in the united kingdom. methods: prospectively collected data on iatrogenic bile duct injuries following elective laparoscopic cholecystectomy referred to our center was analyzed. the strasberg classification was used for defining the types of injury. results: a series of 125 patients were referred for management of iatrogenic bile duct injuries between january 1991 and june 2003.median follow up period was 55 months. 87 patients had type e injuries (to%), followed by 20 with type a (16%) 14 with type d (11%) and 2 each of types b (1.6%) and c (1.6%). an uncomplicated primary operation was described in 74 patients (60%). despite the operation being converted to an open procedure in 27 patients only 18 injuries were recognized at the time of surgery.(l4.4%) although the median time to recognition of biliary tract injury was 2 days (1-21) the median time to referral increased to 10 days (0-99) for a type a injury, and 16 days (0-2200) for a type e injury. a total of 29 patients underwent surgical intervention prior to referral of which 20 had biliary reconstruction. nine of these patients required subsequent revision surgery. after referral 15(13%) patients were managed conservatively while 6(5%) had ercp and biliary stenting and 24(29%) had interventional radiology as the main procedure. surgical intervention was required in 79 patients (53%) at our center following referral. this included t-tube insertion (7), primary common bile duct repair (lo), biliary reconstruction (60) and liver resection (2) with three patients requiring subsequent revision surgery. major associated vascular injuries were present in eight patients. one patient suffers from recurrent cholangitis while five remain asymptomatic on follow up. two required liver resection due to severe ischaemic necrosis. bowel perforations were associated in three patients. both vascular and bowel injuries occurred in patients with type e bile duct injuries. nine patients (7%) had recurrent cholangitis following treatment. three patients developed end-stage liver disease due to secondary biliary cirrhosis with one undergoing orthotopic liver transplantation. there were five deaths related to bile duct injuries. two had associated vascular injuries and one had bowel perforation. a further two patients with type a injuries died following multiorgan failure due to sepsis. conclusions: iatrogenic bile duct injuries are associated with significant morbidity and mortality. delay in recognition and appropriate referral remain a continuing problem. a multi disciplinary approach in a specialized institution has a significantly better long term outcome. background radio-frequency ablation (rfa) is widely used as a treatment of inoperable tumor leasons in the liver. preliminary studies of a vx2 hepatoma model in rabbits showed tumor specific t lymphocytes after rfa treatment and a significantly prolonged survival of these treated animals compared to a control group. aim: the aim of the study was to assess whether the observed tumor specific t cell response after rfa treatment, found in the vx2 tumor model, is transferable to humans. methods: 12 patients were included into a pilot study. 6 patients had metastases of colonic cancer and 6 patients were suffering from hepatocellular carcinoma (hcc) with underlying cirrhosis. all patients were treated with rfa (elektrotom him 106, berchtold, germany).blood samples were taken before and 4 weeks after rfa. an interferon gamma cytokine secretion assay (miltenyi, germany) was carried out on whole blood and measured by a flowcytometer (facs calibur, bd science, germany). as test antigens served autologous liver tissue and tumor tissue received by biopsy. t cells were doublestained with cd 8 antibody to detect cytotoxic t cells. the basic ifn gamma secretion was also measured unstimulated. the activated/non activated cd8 positive t cell ratio (anr) was calculated and analysed statistically with spss. only rfa naive were included. results: before rfa patients had a mean stimulation of 0,022 anr (sd=0,001) against liver tissue and 0,027 anr (sd=0,002) against tumor tissue (see tab.1). 4 weeks after rfa a strong increase of anr was observed with 0,11 anr (hcc) and 0,15 anr (colorectal carcinoma) against tumor tissue. this increase was also detectable 8 weeks after rfa (0,14 anr (sd=0,003) (hcc) and 0,14 anr (0,003) (colorectal carcinoma)) (see fig. 1 and tab.1) . the anr levels were allways low against liver tissue with 0,03 anr (sd=0,002). discussion: rfa shows significant tumor specific t-cell stimulation in patients with primary and secondary tumors of the liver. rfa seems to overcome immune tolerance or presents alterated and/or cryptic tumor antigens, and causes a significant tumor specific cytotoxic tcell response. this effect could be exploided in multimodal antitumoral strategies. the strategy of primary hepatic resection followed by secondary liver transplantation (slt)was not evaluated in-term of operative risks and survival. between 1991-2001, among 88 cirrhotic patients transplanted for hcc with mazzafero's criteria on analysis of the specimen, 18 had liver resection prior to slt : for recurrence (n=11), deterioration of liver function (n=4) or high risk for recurrence (n=3) with a mean time between liver resection and listing for lt was 20 months .the comparison between slt group and patients who underwent primary liver transplantation (plt) showed similar median age (55 vs. 53 years), sex and etiology of liver disease (alcohol/viral biclother). overall time on lt waiting list of the two groups was similar (5 vs. 3 months). pathologic analysis of the specimen revealed similar number (1.7 vs. 1.6) and tumor size (2.3 vs. 2.2 cm). pen-and post-operative course were not different in terms of operative time (551 vs. 530 min), blood loss (1282 vs. 1191 ml), transfusion (3 vs. 2 units), icu (10 vs. 9 days) or hospital stay (32 vs. 31 days), morbidity (56% vs. 51%) or 30-day mortality (5.7% vs. 5.6%). during a median follow-up of 32 months (3 to 158 months), 3 patients recurred after plt and one after slt. after transplantation, 3 and 5-year overall survivals were not different between groups (82 vs. 82% and 59 vs. 61%). results of our study showed that liver resection for hcc prior to transplantation did not increased the morbidity or impair long-term survival following lt. therefore liver resection prior to transplantation can be integrated in the treatment strategy for hcc. aims: ct-1 is a member of the il-6 family of cytokines which protects myocardiocytes against thermal and ischemic insults. in this work we analyzed the expression of ct-1 by normal and diseased liver and whether this cytokine might protect the liver against ischemia-reperfusion injury. methods: rat ct-1 cdna was cloned, recombinat rat ct-1 was produced and used for generation of poly and monoclonal antibodies which were employed in immunochemistry, western blot and elisa. the recombinant cytokine was assayed for therapeutic use in rat models of ischemia-reperfusion injury. wistar rats were subjected to 60 min ischemia of 70% of the liver by clamping the corresponding branches of the hepatic and portal vein. after this period the clamp was released and serum and tissue samples were obtained at 6 hours. rats were divided into three groups: group 1 received 100 mcg ct-1 10 minutes prior to ischemia (n=8), group 2 received saline instead of ct-1 (n=8) and group 3 were sham operated (n=4). results: immunohistochemistry of normal livers demonstrated ct-1 expression selectively in hepatocytes of the centrolobular area. six hours after reperfusion of ischemic livers the values of serum alt were 10 times higher in group 2 than in group 1 (pco.01). severe focal hepatocellular necrosis was found in animals from group 2 while no apparent morpholgical changes were observed in rats from group 1. serum ct-1 levels were raised after reperfusion in untreated animals as compared with sham operated rats. western blot of liver samples showed an increase of ct-1 expression in the non-ischemic liver lobule as compared with the damaged part of the liver. conclusions: ct-1 is a hepatoprotective cytokine produced by hepatocytes in centrolobular areas. ct-1 synthesis increases after ischemic insults. administration of recombinant ct-1 protects very efficiently the liver against ischemia-reperfusion injury. ct-1 may be a valuable therapy to attenuate hepatic damage of the donor liver during orthotopic liver transplantation. disclosures: naiara beraza -no relationships to disclose matilde bustos -no relationships to disclose pun fortes -no relationships to disclose maria ifiiguez -no relationships to disclose eduardo martinez-ans6 -no relationships to disclose jesus prieto -no relationships to disclose cardiotrophin-1 (ct-1) is a potent background: in experimental and clinical acute liver failure, both a loss of cerebrovascular autoregulation and an increase in cerebral blood flow (cbf) have been implicated in the development of brain edema. it is unclear whether the pathogenesis of these two phenomena are linked. the accumulation of glutamine within astrocytes appears to be a critical first step in the development of cerebral hyperemia, as experimental inhibition of glutamine synthetase with methionine sulfoximine (mso) attenuates brain edema and corrects cerebral hyperemia. to study cerebrovascular autoregulation without the confounding variable of brain edema, we examined animals one day after portacaval anastomosis (pca), where a decreased cerebral blood flow has been noted in the setting of a hyperdynamic systemic circulation. material and methods: a pca or sham surgery was performed in male sld rats (300-400 gm). at 24 hrs, mean arterial pressure (map) was measured continuously in anesthetized rats. noradrenaline was infused at a dose of 1 pglkglmin for 60 minutes. mso or saline was given as an intraperitoneal injection (150 mglkg) three hours prior to na infusion. relative changes in cortical blood flow (cbf) were measured with a laser doppler probe placed over the parietal cortex. the animals were mechanically ventilated and arterial blood gases were monitored throughout the experiment. three groups of rats were studied (n= 18). results: the sham group had significantly higher map, in accordance with the systemic vasodilatation seen in pca rats. adding na to all groups significantly raised map and hr. the sham+na group did not exhibit an increase in cbf with a rise in map. the pca+na group had a significant increase in laser doppler flow (ldf) from baseline that coincided with an increase in map. pretreatment of the pca animals with mso prevented the rise in cbf despite a rise in map. there were no significant differences in pc02 among the three groups. as expected, arterial ammonia levels were higher in pca animals and increased further after inhibition of glutamine synthetase in the group that received mso. conclusions: a rise of map in rats after pca results in an increase of cbf, indicating a loss of cerebrovascular autoregulation. the restoration of autoregulation with mso, despite higher arterial ammonia levels, suggests that accumulation of glutamine within the brain is critical to the pathogenesis of altered cerebrovascular regulation in this model. as similar conclusions can be reached regarding the development of cerebral hyperemia in experimental acute liver failure, it appears that the mechanisms responsible for cerebral hyperemia and failed cerebrovascular autoregulation are linked. future studies should focus on the nature of the intracerebral signal that is responsible for the loss of cerebral autoregulation in this model. a decreased intra-hepatic production of nitric oxide (no) participates on the pathogenesis of portal hypertension in cirrhosis. although non-specific no donor substances, such as nitrates, are able to decrease the intra-hepatic vascular resistance to portal blood flow, the use of these vasodilators to treat portal hypertension in cirrhotic patients is limited by the occurrence of side effects (arterial hypotension, headaches etc). aim. to test the effect of a liver-specific no donor (ncx-1000), an ursodeoxycholic acid-derived compound, on both systemic and intra-hepatic hemodynamics in portal hypertensive cirrhotic rats. method. after the development of ascites, cirrhotic rats (cc4-induced) were treated with ncx-1000 (28 mglkg b.w.) or ursodeoxycholic acid (15 mglkg b.w., control group) by gavage once a day for 14 days and, then, used in 1 of 3 studies: 1) in vivo mean arterial pressure (map), portal pressure (pp), and superior mesenteric artery (sma) blood flow measurements before and during progressive blood volume expansion (blood infusion); 2) in situ liver perfusion to obtain doselresponse curves to methoxamine (alpha*-adrenergic agonist) or flowlpressure curves; and 3) cgmp concentration measurement in liver tissue. results. both map and heart rate were similar in both groups, indicating that ncx-1000 had no effect on systemic hemodynamics. pp was also similar in both groups. during blood infusion, ncx-1000 treated rats and control rats showed similar map (p=0.134) and sma blood flow (p=0.449) increases; however, the pp increase observed in control rats was blunted in ncx-1000 treated rats (p=0.015). flowlpressure curves obtained during liver perfusion were similar in both groups; however, livers from ncx-1000-treated rats showed a decreased response to methoxamine (p=0.016) than controls. the concentration of cgmp in ncx-1000-treated livers was significantly higher (p=0.015; ) than in ursodeoxycholic acid-treated livers (27.65 t 7.95 vs. 11.20 2 6.03 fmollmg of protein). conclusion. ncx-1000 improves the portal system adaptability to portal blood flow increase and decreases the intra-hepatic hyper-reactivity to methoxamine observed in cirrhotic rat livers without causing systemic side effects. these beneficial effects are probably due to increase in the intra-hepatic no bioavailability. the mechanisms behind the upregulation of endothelial nitric oxide synthase (enos) in portal hypertensive animals remain unknown. the aim of this study was to investigate the mechanism that initiates enos upregulation in the superior mesenteric artery (sma) in portal hypertension. it has been shown that the vasoconstriction of the sma with the consequent increase in the sma cyclic strain is the earliest event that occurs within hours after portal vein ligation (pvl). thus, we tested the hypothesis that this early vasoconstriction of the sma may initiate enos upregulation in pvl animals. methods: portal hypertension was induced by partial ligation of the portal vein (n=99). in a separate group of rats, mesenteric vasoconstriction was achieved also by renal artery ligation (ral, n=26). corresponding sham-operated rats were used as control groups (n=50). effects of vasoconstriction of the sma in pvl and ral rats were evaluated by measuring perfusion pressure changes in the isolated sma beds in response to methoxamine, nos activity and enos protein expression. data were compared to the corresponding sham group. hemodynamic features were also determined. mean arterial pressure (map), portal pressure ( i " ) and sma blood flow (qsma) were measured by catheterization and pulsed doppler flowmetry. sma vascular resistance was calculated from map, pp and qsma. results: there was a significant increase in the sma vascular resistance in pvl (2.33%0.13 vs 1.2220.03 rnmhgl %flow, k0.05) and ral rats (23220.18 vs 1.18%0.02 mmhgf%flow, p<0.05) compared to their corresponding sham-operated rats, demonstrating the presence of sma vasoconstriction in both pvl and ral groups. the mesenteric vasculature of pvl and ral animals showed hyporeactivity to methoxamine compared to their respective sham groups (p< 0.01). whiie pvl rats were portal hypertensive (p<o.ol), ral rats were not. the sma vascular hyporeactivity of pvl and ral were corrected by l-nmma and while there was not significant difference in enos protein expression nos enzyme activity was significantly higher in the pvl and ral rats compared to the corresponding sham-operated groups (see figure) . conclusions: mesenteric arterial vasoconstriction in itself leads to enos upregulation and therefore, plays a triggering role in the initial increase of enos catalyhc activity in sma of pvl rats. helen hendrickson, vijay shah, mayo clinic, rochester, m n background. diminished enos derived no production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in cirrhotic portal hypertension. unfortunately, prior studies aimed at delivering a constitutively active form of enos (s1179denos) using an adenoviral vector (ads1179denos) were unsuccessful in correcting hepatic vasoconstriction in the bile duct ligated (bdl) rat liver. aim. to better understand the mechanism of s1179denos dysfunction in bdl liver by examining the influence of the enos inhibitory protein caveolin on recombinant s1179denos function in transduced hepatic stellate cells, which do not express enos endogenously but are a prominent cell target of systemically delivered adenoviral vectors. methods. cellular localization of caveolin was determined from fixed sham and bdl liver sections by immunogold electron microscopy. in vitro gene transfer was performed in the lx2 hsc line with ads1179denos and an adenoviral vector encoding caveolin (ad-cav). protein interactions were determined by immunoprecipita-tion and western blot analysis. nos activity was assessed by arginine to citrulline conversion assay. results. caveolin immunogold analysis in bdl liver demonstrated prominent expression not only in liver endothelial cells, but also in stellate cells. western blot analysis from lx2 cell lysates confirmed caveolin protein expression in vitro. in lx2 cells transduced with ads1179denos, immunoprecipitation of caveolin co-precipitated recombinant s1179denos and conversely, immunoprecipitation of s1179denos coprecipitated caveolin. to examine the influence of excess caveolin protein levels on s1179denos, lx2 cells were co-transduced with ads1179deno.s and adcav. co-transduction of adcav and ads1179d promotes the enhanced binding between s1179denos and caveolin as well as a decrease in the activity of recombinant s1179denos by 25% (p<0.05; ads1179denos vs ads1179denos +adcav; n=5). conclusion. these studies demonstrate a functional effect of enhanced caveolin expression and binding with s1179denos in stellate cells and indicate that the lack of function of recombinant s1179denos protein delivered in vivo to cirrhotic liver may be due to inhibition of s1179denos by caveolin-1 in stellate cells. background and aims: the presence of actin-like microfilaments in the neighborhood of sinusoidal endothelial fenestrae (sef) indicates that the cytoskeleton of sinusoidal endothelial cells (sec)s plays an important role in the modulation of sef. we have reported that a potent vasoconstrictor endothelin (et) causes contractions of sef as well as hepatic stellate cells via the eta and etb receptors, leading to the elevations of sinusoidal microvascular resistance supposed to be one the essential factors in the pathogenesis of portal hypertension. rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. the present study aimed to examine how a rho stimulator; lysophophatidic acid (lpa), and a rho inhibitor; y-27632 rho-kinase inhibitor, affect the morphology of sef in vitro. methods: monolayers of cultured sec were obtained by collagenase infusion of a rat liver and then subjected to primary culture for 24 hours. the cells were separated into three groups; control, lpa-treated (lopm), and y-27632-treated (10pm) groups (treatment duration, 30 min). sef morphology was observed by scanning electron microscopy. formation of f-actin stress fibers was observed by confocal microscopy. actin cytoskeleton around sef was investigated by permealizing cells with streptolysin-0 (1.5 u) for 30 min and examined by transmission electron microscopy. rho a and phosphorylated myosin light chain kinase were analyzed by western blotting. rho activation was measured by rhotekin binding assay. results: following lpa treatment, f-actin stress fiber and active rho increased concomitant with a decrease in diameter of sef. however, following y-27632 treatment, f-actin stress fiber and active rho were reduced, concomitant with an increase in diameter of sef. actin cytoskeleton around sef increased in lpa-treated cell, but decreased in y-27632-treated cell. rho a levels were similar in control, lpa-treated, and y-27632treated secs. phosphorylated myosin light chain kinase levels were diminished gradually after 5 min in y-27632-treated secs and increased after 5 min in lpa-treated cells. lpa treatment increased the amount of active rho, compared to control and y-27632 treatment. conclusion: these results indicate that rho may play an essential role in the regulation of contraction and dilation of sef through the actomyosin system. disclosures: analysis of visual-evoked response patterns suggests that gabaergic tone is increased in brain in liver failure and that this is a major cause of hepatic encephalopathy (schafer et al., troenterolow 86:540-545,1984) . however, the precise mechanisms responsible have eluded researchers in this area for the last two decades. studies in brain tissue from both human and experimental animals with liver failure have consistently shown that gaba synthesis, gaba receptors and their associated benzodiazepine modulatory sites are intact and that expression of the subunit proteins of the gaba-a receptor complex are unchanged (zwingmann et al., hevatologv 37420-428,2003; ahboucha et al., neurochem. int., 2003) . on the other hand, there is emerging evidence to suggest that neurosteroids (ns), a novel class of compounds with potent gaba agonist properties are increased in brain in endstage chronic liver failure in humans (ahboucha et al., hepatola 36: 318a, 2002) . in order to further assess this possibility, ns concentrations were measured using a sensitive radioassay in samples of frontal cortex obtained at autopsy from 15 cirrhotic patients who died in hepatic coma compared to an equal number of controls matched for age, gender, and autopsy delay intervals. patient material contained up to 12-fold increases of ns and subsequent analysis by gas chromatography-mass spectrometry showed that the major component ns with positive allosteric properties at the gaba-a receptor was allopregnanolone. patient material did not contain significantly increased concentrations of either gaba or benzodiazepines. in a separate series of experiments, ns concentrations were found elevated up to 2-fold (p<0.02) in brains of rats following end-to-side portacaval anastomosis (pca) and 5.2-fold (p<o.ol) in brains of rats following hepatic devascularization (pca followed by hepatic artery ligation) compared to sham-operated controls. administration of inhibitors of the ns synthesis enzyme 3-hydroxysteroid dehydrogenase (trilostane or indomethacin) resulted in dose-dependent reductions in brain concentrations of allopregnanolone and a concomitant improvement in locomotor activity scores in pca rats. together, these findings offer unequivocal evidence that ns with positive allosteric modulatory properties are generated in brain in both human and experimental liver failure and that these substances contribute to the "increased gabaergic tone" characteristic of hepatic encephalopathy. pharmacological agents aimed at reduction of ns synthesis or modulation of the ns site on the gaba-a receptor complex could afford effective novel therapeutic strategies in the treatment of hepatic encephalopathy [funded by cihr canada] abstract primary hepatocellular carcinoma (hcc) is one of the most common malignancies, ranks fi&h in frequency among all cancers worldwide, and causes over 1 million deaths annually. although a number of oncogenes and alterations in signaling pathways have been identified in hcc, current treatment for this liver tumor is largely ineffective and has poor patient prognosis. one of the potentially curative approaches in hcc therapy is based on interfering with hcc progression by blocking cell division. in regenerating liver, expression of the proliferation-specific forkhead box m l b (foxmlb) transcription factor is reactivated prior to hepatocyte dna replication and its levels are sustained throughout the period of hepatocyte proliferation. deletion of the foxmlb fllfl (loxp targeted) allele using albumin promoterlenhancer driven-cre recombinase (alb-cre) transgene resulted in significant reduction in regenerating hepatocyte proliferation, which was associated with diminished expression of cell-cycle promoting cdc25a and cdc25b phosphatases and increased nuclear levels of cyclin dependent kinase (cdk) inhibitor p21cipl protein. in order to test the hypothesis that foxmlb is required for the development of hcc, we subjected alb-cre foxmlb -/-mice and foxmlb fllfl (control) mice to a diethylnitrosamine (den)lphenobarbital (w) liver tumor induction protocol. our data demonstrated that alb-cre foxmlb -imice are highly resistant to liver tumor formation as evidenced by their failure to develop either hepatic adenomas or hcc following 33 weeks of denlpb tumor induction protocol. in contrast, at 23 weeks following den/pb tumor induction protocol, foxmlb fllfl livers displayed a large number of adenomas that progressed to hcc by 33 weeks following den/pb treatment. the alb-cre foxmlb -iliver displayed enzyme-altered foci as evidenced by protein expression of the glutathione-s-transferase placental isoform. however, alb-cre foxmlb -/hepatocytes failed to develop hyper-proliferative foci, whereas abundant brdu labeling was found in hepatic adenomas and hcc of denlpb treated foxmlb fllfl mice. this reduced dna replication of foxmlb -/-hepatocytes was associated with increased nuclear levels of the cdk inhibitor p27kipl protein. furthermore, foxmlb fllfl tumors expressed high nuclear levels of m-phase promoting cdc25b protein, while undetectable nuclear levels of cdc25b were found in foxmlb -ihepatocytes after den/pb treatment. the cdc25b phosphatase is required for mitosis because it activates the m-phase promoting cyclin b-cdkl complex, and its increased expression is a prognostic marker for a variety of aggressive tumors. consistent with this finding, foxmlb -/-hepatocytes failed to enter mitosis following denlph tumor induction protocol and this was associated with accumulation of large polyploid hepatocytes. these studies demonstrate that foxmlb is required for the proliferative expansion of hepatic tumors, suggesting that foxmlb can be used as a potential target in treatment of patients with hcc. disclosures: robert h costa -no relationships to disclose vladimir v kalinichenko -no relationships to disclose joseph kuechle -no relationships to disclose brian shin -no relationships to disclose xinhe wang -no relationships to disclose yoder yoder -no relationships to disclose key: cord-274080-884x48on authors: rumlová, michaela; ruml, tomáš title: in vitro methods for testing antiviral drugs date: 2018-06-30 journal: biotechnology advances doi: 10.1016/j.biotechadv.2017.12.016 sha: doc_id: 274080 cord_uid: 884x48on abstract despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. a combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. we provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses. viral pandemics remain serious threats to humankind. every year, known viruses such as hiv-1 and hepatitis b virus newly infect millions of people across the globe. in addition, recent outbreaks of ebola virus, influenza virus, severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome-coronavirus (mers-cov) serve as a reminder of the silent danger. the world health organization reported in 2015 that over 1.34 million causalities per year are connected with hepatitis b and almost 500,000 with hepatitis c. occasional epidemic outbreaks of other viruses are also striking. these include outbreaks of noroviruses, flaviviruses (zika and dengue viruses), new strains of influenza viruses, the re-emergence of west nile virus in italy and the united states. despite relatively long pauses, viruses such as influenza virus can re-emerge and cause global pandemic health problems. unlike cellular genomes, which consist of double-stranded (ds) dna, viral genomes can be formed by a broad variety of nucleic acids (nas), including ds or single-stranded (ss) circular or linear dna or positive-, negative-or sometimes ambi-sense rna. as viral life cycles are dependent on cellular factors and cellular metabolic and signaling pathways, the number of possible antiviral drug targets is limited. however, almost all viruses encode unique proteins and enzymes that may serve as specific targets for antiviral therapy. the overarching goal of modern drug development efforts is to design compounds that specifically inhibit viral targets or cellular targets essential for virus replication. the purpose of this review is to provide insight into the broad variety of cell-based and biochemical assays used for identification and evaluation of antivirotics, including high-throughput screening (hts) methods. an overview of available inhibitors and vaccines, which has been reviewed elsewhere [e.g. in (de clercq and li, 2016) ], is beyond the scope of this paper. to package their genomes into particles of very limited size, viruses encode few genes. virions consist of an rna or dna genome that is protected by an outer shell called a capsid (also nucleocapsid) formed by a lattice of capsid proteins. in enveloped viruses, the capsid is additionally surrounded by a lipid bilayer spiked with viral proteins. the size of animal viruses ranges from approximately 25 nm to over 300 nm (cohen et al., 2011) . the capsids and virions of rna viruses adopt various shapes. viral capsids may be icosahedral (e.g. picornaviridae, astroviridae, reoviridae, togaviridae), bullet-shaped (rhabdoviridae), helical (coronaviridae), helical filamentous (filoviridae, orthomyxoviridae, paramyxoviridae), or filamentous (arenaviridae, bunyaviridae). the retroviral capsid core may be conical, spherical, or rod-shaped. the morphology of dna viruses is similarly diverse, ranging from icosahedral (enveloped -hepadnaviridae, herpesvridae; nonenveloped -adenoviridae, parvovirdae, polyomaviridae and papillomaviridae) to rodshaped (baculoviridae) or pleomorphic (poxviridae). the viral life cycle is the process of viral replication in a host cell. first, viruses enter the host cell and replicate their genomes. following translation of viral proteins by the host cell machinery, viruses package their genomic material into protective proteinaceous capsids and exit the cell to infect another host cell. nonenveloped viruses consist only of a protein capsid shell enclosing the viral genome and enzymes, while the capsid shell of enveloped viruses is enclosed in a lipid envelope derived from the host cell membrane. regardless of whether the virus is enveloped, its surface must display (glyco)proteins suitable for specific interactions with host cell receptors. in contrast to the surface proteins of nonenveloped viruses, those of enveloped viruses usually serve another function in addition to host cell recognition and attachment. for example, they may enable fusion of the viral and cellular membranes, usually through an interaction with a secondary receptor(s) or co-receptor(s). the fusion domains of viral surface glycoproteins thus can lower the kinetic barrier for the energetically demanding membrane fusion. the viral particle must be sufficiently stable to protect the genome until its delivery into the host cell. simultaneously, it must be metastable, to allow its disassembly and release of the genome for replication in the infected cell at the appropriate time. the energetic barrier that prevents viral disassembly outside the cell is lowered upon infection by structural changes in the viral components. these changes may be induced by binding of a cellular ligand or by changes in the environment, such as ph change upon entering a specific cellular compartment. numerous viruses preassemble immature particles that undergo irreversible (usually proteolytic) transitions into mature structures of fully infectious virions. mutual interactions of viral capsid proteins are typically different from those of cellular proteins, which predominantly create binary interactions. viral structural proteins interact with multiple neighboring partners to form multicomponent macromolecular structures [reviewed in (cheng and brooks, 2015; jayaraman et al., 2016) ]. the economy of the packaged viral genomes due to the limited capsid size implies formation of only a few types of structural proteins, which are usually symmetrically organized [reviewed in (prasad and schmid, 2012; raguram et al., 2017) ]. in the initial stage of infection, viruses must overcome several obstacles. the first is the cellular plasma membrane with an actin cortex. then, they must traffic through dense cytoplasm to reach their final destination for replication and assembly. these pathways are specific for different viruses and often are dictated by the size of the particle and its structure. the viral life cycle can be divided into several common stages, including entry, uncoating, genome replication, genome packaging and assembly, release, and maturation ( fig. 1) . in general, the first phase of viral infection is specific recognition of the target host cell and binding to a surface receptor displayed on the cell membrane. this process is common to both enveloped and nonenveloped viruses. in enveloped viruses, the binding is mediated by viral surface components, typically oligomers of integral glycoproteins. nonenveloped viruses bind receptors through sites or projections on the capsid surface. viruses can use either a single receptor (e.g. tim-1 for hepatitis a virus, gm1 for sv40, cd155 for poliovirus, low-density lipoprotein receptor for human rhinovirus) or multiple receptors with equivalent roles (e.g., nectin-1/2 or hvem for herpes simplex virus 1/ 2, ace or l-sign for sars coronavirus). for other viruses (e.g. hiv, hcv, adenoviruses, rotaviruses, picornaviruses, and some herpesviruses), the presence of at least two cytoplasmic membrane components is required. for example, hiv-1 binds to the primary receptor cd4 and one of two co-receptors (ccr5 or cxcr4) [reviewed in (cossart and helenius, 2014; grove and marsh, 2011) ]. to infect a cell, viruses must overcome the plasma membrane to deliver their genetic material into the cytosol. viruses enter cells by two main mechanisms. a majority of animal viruses, both enveloped and nonenveloped, enter cells by one or two types of endocytosis, such as clathrin-mediated endocytosis (e.g. vsv, influenza a virus, rhinovirus), caveolin-mediated uptake (echovirus, polyoma virus), clathrin/caveolin-independent endocytosis such as caveolar or lipid raft-mediated (e.g. sv40, polyomavirus mouse), or macropinocytosis (e.g. vaccinia virus, respiratory syncytial virus, ebola virus, hiv-1) (blaas, 2016; cossart and helenius, 2014; fields and knipe, 2013; kirkham and parton, 2005; marechal et al., 2001; mayor and pagano, 2007; mercer and helenius, 2009; parton and simons, 2007; rasmussen and vilhardt, 2015; saeed et al., 2010) . these endocytic mechanisms enable the virus to be transported by the cell's machinery through the plasma membrane and to pass through the dense actin cortex. cellular entry of some viruses is coupled with receptor-mediated signaling, resulting in activation of molecules that facilitate virus entry by cytoskeleton reorganization, induction of long-distance transport of the virus-containing vesicles, or actin cortex disassembly. the second type of entry is used by some enveloped viruses (paramyxo-, herpes-, and retroviruses), which upon cell surface receptor binding, infect the cell by direct fusion of viral and plasma membranes at neutral ph. upon internalization, most viruses become trapped and enclosed in vesicles that pinch off the inner side of the plasma membrane and transport their cargo into the cytoplasm. on their way through the cell, these transport vesicles undergo maturation by fusing with other vesicles. to fulfill their task of genome replication, viruses have to escape from these endosomes. the "great escape" is triggered by activation of a fusion or penetration mechanism, such as changes in conditions in the endosomal interior [e.g. ph, ionic environment (e.g. calcium ion concentration), oxido-reducing conditions], changes in membrane composition, and other physico-chemical cues (blaas, 2016; cossart and helenius, 2014; inoue and tsai, 2013) . depending on the requirements for a particular virus, these events can occur in early endosomes (ph 6.5-6.0; e.g. hepatitis c virus, vesicular stomatitis virus), late endosomes (ph 6.0-5.0; e.g. influenza a virus, dengue virus, sars coronavirus), recycling endosomes, macropinosomes, the endoplasmic reticulum, the golgi apparatus, or lysosomes (ph 5.0-4.5) (blaas, 2016; cossart and helenius, 2014; grove and marsh, 2011; inoue and tsai, 2013) . for the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. structural rearrangements in enveloped viruses usually mediate fusion of viral and endosomal membranes. in nonenveloped viruses, the structural changes uncover amphipathic or hydrophobic domains that may induce pore formation or disruption of endosomal membranes. to deliver genetic material to the replication site, these mechanisms ultimately release viral capsid structures from endosomal vesicles into the cytosol either by fusing with the endosomal membrane (enveloped viruses) or by penetrating the endosomal membrane (nonenveloped viruses). uncoating is the partial or complete disassembly of the protective capsid shell and/or lipid envelope to liberate the viral genome. for many viruses, this process is closely connected with conformational changes induced by the virus binding to the cell surface receptor; a low ph environment; or changes in oxido-reducing conditions, ion concentrations, or other factors. for enveloped viruses, uncoating involves a loss of the viral membrane by fusion either with the plasma membrane or with intracellular vesicles, followed by stepwise uncoating of the protective capsid shell. in nonenveloped viruses, the uncoating process typically involves conformational changes that result in the weakening of intermolecular interactions, loss of structural proteins, proteolytic cleavage, and so on (fields and knipe, 2013) . depending on the virus, uncoating can take place at the plasma membrane, in the cytoplasm, during endocytosis in early or late endosomes, in lysosomes, in the nucleus, or at the nuclear pore complex (npc). the ssrna genome of retroviruses, which is fully enclosed inside a protective capsid shell, must be reverse transcribed into dsdna and released from the capsid. although it is accepted that hiv-1 uncoating is linked to reverse transcription and nuclear import (ambrose and aiken, 2014) and is controlled by host factors (e.g. cyclophilin a, trim5α), the precise molecular mechanisms that trigger the uncoating remain unknown. several hypotheses have been proposed, including breakage of the capsid shell due to increased inner pressure caused by accumulation of the reverse transcription product (rankovic et al., 2017) , the requirement of intact microtubules and dynein and kinesin motors (lukic et al., 2014) , and phosphorylation of capsid shell protein by the host cell kinase melk (takeuchi et al., 2017) . in contrast to the majority of rna viruses, which replicate in the cytoplasm, most dna viruses (with the exception of large dna viruses including poxviridae, asfarviridae, and mimiviridae) and several negative stranded rna viruses enter the nucleus to replicate their genomes (kobiler et al., 2012; koonin and yutin, 2010) . passive diffusion into the nucleus is suitable for molecules smaller than 9 nm in diameter, but larger structures must enter through the nuclear pore complex (npc), which can accommodate molecules of up to 39 nm (pante and kann, 2002) . translocation through the npc is tightly regulated and requires the presence of a nuclear localization signal (nls) on the passing molecule and nuclear import receptors (importins or karyopherins) (cautain et al., 2015) . due to the diversity of viral particle sizes (from 25 nm to over 300 nm) and structures, viruses have evolved several strategies to export their dna or rna genome into the nucleoplasm. with regard to the npc, these pathways can be divided into npc-dependent and npcindependent mechanisms. due to size limitations, only a few, very small simplified scheme of common stages of viral life cycle targeted by antiviral drugs. these stages including: 1) attachment and entry, 2) uncoating, 3) genome replication, 4) genome packaging and assembly of viral particle and 5) virus release and maturation. m. rumlová, t. ruml biotechnology advances 36 (2018) 557-576 viral capsid particles, such as hepatitis b virus (hbv; 32-36 nm diameter), can pass through the npc (cohen et al., 2011; rabe et al., 2003) in an importin-dependent manner. hbv then disassembles at the nuclear side of the npc (the nuclear basket), releasing its genome into the nucleoplasm (fay and panté, 2015) . the capsid shells or ribonucleoprotein complexes (rnps) of some larger viruses, such as influenza a virus and hiv-1, disassemble within the cytoplasm. the viral genome, released from the shell and complexed with nls-containing components, is then translocated through the npc (ambrose and aiken, 2014; campbell and hope, 2015; cohen et al., 2011; fay and panté, 2015; hutchinson and fodor, 2013) . another mechanism, used by herpes simplex virus (hsv) and adenoviruses, involves cellular (importins, nup) or viral protein-mediated attachment (docking) of the capsid shell at the cytoplasmic side of the npc. this facilitates the passage of genomic dna into the npc either by ejection from an almost intact particle (through the capsid portal in hsv-1) or upon complete disassembly of the capsid shell (in adenoviruses) (kremer and nemerow, 2015; ojala et al., 2000; pasdeloup et al., 2009) . npc-independent mechanisms are used by some retroviruses (e.g. mlv) that enter the nucleus during the mitotic phase of the cell division cycle when the nuclear envelope is dissolved (matreyek and engelman, 2013; roe et al., 1993) . another mechanism, described for parvoviruses, involves partial disruption of the nuclear envelope (cohen and pante, 2005; fay and panté, 2015) . viral genomes can be encoded by various types of nas, as summarized in table 1 for dna viruses and table 2 for rna viruses. by convention, ssdna of equivalent polarity to mrna is designated as the positive (+) strand. the complementary ssnas are of minus polarity (−). the majority of dna viruses replicate in the nucleus, where cellular dna replication and transcription also occur. in contrast, rna viruses usually replicate in the cytoplasm. rna viruses are the only "organisms" that store their genetic information in the form of rna. replication of their genomes is accomplished either by rna-dependent rna synthesis ( fig. 2a , b) [reviewed in (ferron et al., 2017; lu and gong, 2017; menéndez-arias and andino, 2017; pietilä et al., 2017; tao and ye, 2010) ] or through rnadependent dna synthesis (reverse transcription) followed by dna integration, replication, and transcription ( fig. 2c ). as some of these enzymatic activities are not commonly found in uninfected host cells, rna viruses must encode enzymes to aid replication (table 2 ). rna viruses are divided according to the baltimore classification into dsrna viruses (birna-and reoviridae); positive-sense ssrna viruses (corona-, flavi-, and picornaviriade); negative-sense ssrna viruses (filo-, rhabdo-, paramyxo-, and orthomyxoviriade), and ambisense rna viruses (arena-and some members of bunyaviridae) with both positive-and negativesense rnas (nguyen and haenni, 2003 ) (see table 2 ). the nature of the genome not only dictates the mechanism of replication, but also has other important consequences. the genome of (+)rna viruses may serve directly as mrna for production of viral proteins. therefore, the mere introduction of genetic material (e.g. in exocytic vesicles) may result in productive infection. the rna polymerases that copy the genetic material of rna viruses are error-prone, which provides considerable genetic flexibility and the propensity to evolve drug-resistant mutants. these features are amplified in viruses with segmented genomes that undergo reassortment. in viruses with segmented genomes (orthomyxoviridae, arenaviridae and bunyaviridae), each segment is transcribed in an autonomous transcription-replication unit by viral rna polymerase that binds to the 5′ end cap structure. of note, the genomic rna (grna) is capped by a unique mechanism called cap-snatching (ferron et al., 2017) , in which the cap is cleaved from cellular mrna and transferred onto the viral grna by a subunit of viral rna polymerase (pflug et al., 2017) . some dsrna viruses (birnaviridae and reoviridae) also contain segmented genomes. upon replication, these viruses must ensure stoichiometric incorporation of single copies of each grna segment into new particles. this is guided by specific packaging signals on each segment of grna that interact with positively charged domains in the capsid proteins [reviewed in (isel et al., 2016; pohl et al., 2016) ]. although different models of packaging have been proposed for various segmented genome viruses, a common feature is co-assembly of the capsid proteins with the grna and rna polymerase. the genomes of dna viruses come in a considerable variety of sizes and shapes, from small ss to large ds molecules that may be linear or circular. the size range of these genomes (from 1.8 kb to 1200 kb) reflects the necessity for some viruses to encode specific proteins required for viral replication. small-genome dna viruses (polyoma-, papilloma-, and parvoviruses) use only host cell enzymes for replication and transcription. the only exceptions are some hepadnaviruses (e.g. hepatitis b virus) that despite having small genomes (approximately 3 kb), encode their own specific dna polymerase/reverse transcriptase that reverse transcribes pregenomic rna (pgrna) into genomic viral dna (fig. 2d , ii) (beck and nassal, 2007) . viruses with intermediate-size genomes (up to 35 kb) (e.g. adenoviruses) encode their own dna polymerase for genome replication, but they usually utilize cellular rna polymerases ii and iii for transcription (fields and knipe, 2013) . viruses with large genomes (larger than 100 kb) (e.g. herpesviruses) encode proteins for replication, including dna polymerase and dna helicase-primase, as well as some enzymes necessary for biosynthesis of deoxyribonucleotide triphosphates (dntps) and several transcription factors (boehmer and nimonkar, 2003) . poxviruses (e.g. vaccinia virus), are another type of virus with large dsdna genomes, and their replication, transcription, and translation take place entirely in the cytoplasm within discrete juxtanuclear sites called virus factories (moss, 2013) , (fig. 2d , iii). these viruses encode all enzymes and specific factors necessary for genomic replication and transcription. with their own replicative machinery, large-genome dna viruses can replicate in nondividing cells. in contrast, the replication of small-genome dna viruses, which depends on cellular dna polymerases, must occur simultaneously with the s-phase of the cell cycle (e.g. parvoviruses), or must express some viral protein/ oncogene to re-program the host cell-cycle regulatory proteins p53 or retinoblastoma protein (prb), triggering entry into the s-phase (e.g. polyomaviruses and papillomaviruses). by affecting the g1/s checkpoint (controlled by p53 and prb), the viruses ensure the production of host enzymes required for viral replication (fields and knipe, 2013) . production of viral proteins of dna viruses with intermediate and large size genomes is divided into early and late phases. in the early (prereplicative) phase, nonstructural proteins required for dna replication are translated. the late phase, during which the late structural proteins needed for assembly are translated, begins after viral dna replication ( fig. 2d , i). depending on the species, viruses assemble either in contact with the cellular membrane or independent of the membrane in either the nucleus or cytoplasm. the membrane-independent route is used by nonenveloped viruses and a few enveloped viruses (e.g. orthomyxoviruses, herpesviruses, and some retroviruses) that acquire the membrane envelope after intracellular assembly during budding from the cell. the limiting step for nuclear assembly is the size of npcs, which are large enough to transport rna and import proteins required for assembly into the nucleus; however, npc size limits the transport of larger assemblies. therefore, some viruses form assembly intermediates in the nucleus. these structures are then exported to the cytoplasm, where they come together to form viral particles. nuclear export is specific and depends on the presence of nuclear export signals (nes) in the transported proteins. one example of a nonenveloped icosahedral virus that assembles in the nucleus is adenovirus, the assembly of which has been studied intensely due to its potential use in gene therapies [reviewed in (ahi and mittal, 2016) ]. recent data suggest that upon accumulation of multiple copies of adenoviral dsdna genomes, coordinated assembly and packaging occur by two interlinked mechanisms that involve both capsid proteins and core components (condezo and san martín, 2017) . the assembly occurs in the so-called peripheral replicative zone with the assistance of scaffolding proteins that facilitate formation of adenoviral particles but are excluded from mature viruses. adenoviruses are finally released upon lysis of the infected host cell. orthomyxoviruses and herpesviruses are enveloped viruses that assemble their nucleoproteins in the nucleus. herpesviruses package their dsdna genome as head-to-tail concatemers and assemble icosahedral procapsids on scaffold proteins in the nucleus [reviewed in (heming et al., 2017) ]. however, subsequent steps of herpesvirus assembly proceed in the cytoplasm. the preassembled procapsid is too large to pass through the npc, but it exits the nucleus by viral proteindriven vesicular transport across the nuclear inner membrane leaflet. thus, the herpesvirus acquires an initial envelope from the inner nuclear membrane [for review see (fields and knipe, 2013; hellberg et al., 2016) ]. next, the herpesviral membrane fuses with the outer nuclear membrane, and the naked particle is released from the nucleus into the cytosol. here, the virus acquires tegument (a protein layer between the capsid and the envelope) and other proteins. final herpesvirus envelopment occurs at the golgi membrane containing the viral glycoproteins (fields and knipe, 2013) . preassembled orthomyxoviral ribonucleoproteins, upon their export from the nucleus, are driven to the plasma membrane to which they attach through electrostatic interactions of the matrix protein m1 with membrane phosphatidylserine. the virions then assemble simultaneously with budding during which they also acquire ha, na and m2 membrane proteins. poxviruses undergo an even more complicated pathway. they are enveloped with multiple membranes acquired from er/intermediate compartments and golgi or early endosomes (moss, 2015 (moss, , 2016 . these membranes also provide the poxviruses with their membrane proteins. most viruses assemble upon interaction of their structural proteins with cellular membranes. the target membrane for assembly differs according to the virus type. flaviviruses assemble at the surface of the er and then upon their budding into the er lumen, the immature particles are then transported into the tgn. some viruses, such as coronaviruses, assemble at the er-golgi intermediate compartment [reviewed in (ujike and taguchi, 2015) ]. the assembly of bunyaviruses occurs concurrently with replication of grna segments in virus factories located along the golgi (guu et al., 2012; strandin et al., 2013) . the presence of membrane glycoproteins at the golgi membrane determines the sites where assembling bunyavirus particles bud into the golgi lumen, similar to other enveloped viruses. numerous viruses, including paramyxoviruses (cox and plemper, 2017) , orthomyxoviruses (pohl et al., 2016) , alphaviruses (jose et al., 2009) , rhabdoviruses (okumura and harty, 2011) , and most retroviruses (freed, 2015) , assemble underneath the cytoplasmic membrane, which facilitates assembly by providing a scaffolding function. the virus then acquires a lipid envelope through budding across the plasma membrane. during this process, it also gains the envelope glycoproteins (env) that are anchored in the plasma membrane by hydrophobic transmembrane domains. env reaches the plasma membrane by a cellular secretory pathway upon synthesis in the er and subsequent glycosylation in the golgi. usually, a specific interaction between viral structural proteins and glycoproteins is required. this may be either direct or mediated through the interaction with matrix protein (e.g. in (-)rna viruses such as ortho-and paramyxoviruses or rhabdoviruses). most retroviruses, including hiv (so-called morphogenetic c-type), also assemble at the plasma membrane of the host cell. the interaction of the structural polyprotein precursor (gag) with the plasma membrane is usually facilitated by a bipartite signal in the n-terminal domain of gag (i.e. matrix protein) comprising both the basic patch and n-terminally linked myristoyl residue (added co-translationally to gag). in contrast to c-type assembly at the membrane, morphogenetic b/d-type retroviruses assemble at pericentriolar sites where gag polyproteins are transported along microtubules by dynein molecular motors (sfakianos et al., 2003; vlach et al., 2008) . for both morphogenetic pathways, the packaging of grna facilitates assembly. (+)rna viruses have adopted a mechanism of extensive rearrangement of intracellular membranes to provide a milieu for virus replication and assembly. this mechanism effectively protects dsrna intermediates from degradation by the host cell machinery (delgui and colombo, 2017; jackson, 2014) . this process has been well-documented for poliovirus, in which the newly formed membranous structures exclusively serve for virus production. various types of vesicles that are formed upon viral infection have different roles in viral replication (rossignol et al., 2015) . some nonenveloped mammalian viruses, such as reoviruses, assemble in the cytosol in so-called viroplasms or viral factories into icosahedral particles consisting of three concentric layers encircling segments of genomic dsdna (benavente and martinez-costas, 2006; jones, 2000; shah et al., 2017) . rotavirus seems to be the only exemption in the reovirus family, as it enters the endoplasmic reticulum to gain its outer protein shell (trask et al., 2012) . numerous viruses assemble from polyprotein precursors that must be specifically cleaved by a viral protease to generate infectious particles. this mechanism, which is an irreversible step in the virus life cycle, ensures equimolar packaging of structural proteins and proportional co-assembly of viral enzymes in the form of precursors. upon proteolytic release, the liberated proteins may undergo different trafficking pathways or fulfill various functions in the virus. maturation changes the energy of interaction forces among those interfaces required for intracellular assembly of immature particles to those suitable for viral stability in the environment and for disassembly and uncoating of genetic material for replication [reviewed in (veesler and johnson, 2012) ]. in poliovirus, the autocatalytic and subsequent viral proteasemediated cleavage of p1 precursor protein allows formation of pentamers that interact with grna. additional cleavage of vp0, yielding vp2 and vp4, is required to form infectious poliovirus particles. in retroviruses, proteolytic processing is initiated by the autocatalytic liberation of viral protease, which subsequently cleaves the polyproteins to trigger major structural rearrangements in the virus and release of other viral enzymes (reverse transcriptase and integrase) and structural proteins. in herpesviruses, maturation involves proteolytic processing of the scaffolding protein and recruitment of tegument proteins that stabilize the particle and mediate interactions with the membrane during envelopment (gibson, 2008; tandon and mocarski, 2012; tandon et al., 2015) . adenoviruses undergo a maturation process that involves processing of six structural components of the core and one non-structural precursor that initiates replication of gdna (gaba et al., 2017) . one interesting feature of adenoviral maturation is that dna is required as a co-factor for protease activity. this means that maturation occurs only in particles that have packaged gdna (mangel and san martín, 2014) . flaviviruses form icosahedral particles upon budding into the neutral milieu of the er. the particles then translocate to the more acidic environment of the trans-golgi network. this ph shift results in disassembly of the immature lattice and extensive rearrangement of the flaviviral particle. this is connected with the exposure of a viral structural glycoprotein (prm) that is specifically processed by the , retroviruses (c) and dna viruses (d) (the schemes a-c were modified from: (ahlquist, 2006) . a: following endocytosis, the genome of (+)rna viruses may directly serve as mrna for translation of virus encoded proteins. among them, there are proteins of rna-replication machinery that recruit (+)rna into a membrane-associated replication complex. the genomic rna is replicated by using (-)rna template, which is transcribed in a low copy number amplified (+)rna is then packaged into newly assembled virions that exit the host cell either through secretion or cell lysis. b: upon the virus attachment, a core containing both grna and virus encoded rna polymerase is delivered into the cytoplasm by endocytosis. cytoplasmic transcription of the (-)rna template provides (+)rna that serves as mrna for translation of viral proteins. in dsrna viruses, the (+)rna is packaged into new cores which undergo maturation by synthesizing (-) rna (dotted strand) and acquiring surface proteins. in the (-)rna viruses, the (+)rna strand is transcribed into genomic (-)rna in the cytoplasm and then packaged. the new virions egress the cell either through secretion or cell lysis. c: following fusion of viral and cell plasma membrane, the retroviral core is released into cytosol, rna genome is transcribed into dsdna by viral reverse transcriptase concomitantly with uncoating of the viral core, ds dna is transferred into nucleus where it is integrated into host cell genome by viral integrase, following translation of retroviral structural and enzymatic polyproteins, the unspliced grna is packaged into the immature particles that usually assemble at the plasma membrane and the viral particles bud from the cell. d: three mechanisms (i.-iii.) of dna viruses replication are shown: (i): following entry and uncoating, the dna genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. dna polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. herpesviruses). (ii) unique replication cycle of hepatitis b virus (hbv): following entry, the viral particle is internalized by endocytosis and the nucleocapsid is released into the cytoplasm; the genome (relaxed circular rcdna) is transported into the nucleus, where it is converted to a covalently closed circular form (cccdna); which serves as a template for transcription of pregenomic rna (pgrna) for translation of the core protein and the viral polymerase and three subgenomic mrnas used for translation of regulatory and envelope proteins; following viral transcription and translation, the hbv core proteins self-assemble in the cytoplasm into viral nucleocapsid with concurrent incorporation of pgrna and hbv polymerase, pgrna is reversely transcribed into a rcdna within the nucleocapsid; nucleocapsid containing rcdna can either re-enter the nucleus to amplify cccdna, or can be enveloped by hbv envelope proteins in the endoplasmic reticulum. the particles are then secreted from the infected hepatocyte by exocytosis. (iii) upon entering the cell, the replication, transcription and translation take place entirely in the cytoplasm, within discrete juxtanuclear sites called virus factories (e.g. poxviruses) adapted from: ahlquist, p., 2006. parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses. nat rev microbiol 4(5), 371-382. cellular protease furin in the trans-golgi network. the liberated globular heads of prm remain attached at the low ph, but are released when the virus enters neutral body fluids (rey et al., 2017) . one frequently used mechanism of release of nonenveloped and some enveloped viruses is lysis of the infected cell. this is the simplest release mechanism, although the transition to lysis from latent infection is delicately regulated (aneja and yuan, 2017; levings and roth, 2013; schmiedel et al., 2016) . however, viruses that are usually lysogenic may also use alternative release pathways (bird and kirkegaard, 2015a) . these include non-lytic spread of viruses mediated by vesicles, which has been observed for poliovirus (bird and kirkegaard, 2015b; jackson et al., 2005) , coxsackievirus (alirezaei et al., 2012) , and hepatitis a virus . another possibility is that vesicles released from a cell infected with (+)rna viruses contain naked viral grna. this (+)grna-containing vesicle functions itself as an infectious agent as it is transferred to another cell (bird and kirkegaard, 2015a) . the standard way for enveloped viruses to leave the host cell is budding, which includes membrane extrusion and separation of the viral and cellular membranes (so-called pinching off). the lipid envelope layer acquired during viral particle budding through the plasma membrane protects the virus particle. there are three basic mechanisms of budding: i) mediated by envelope glycoproteins, such as the e protein of coronaviruses; ii) independent of glycoproteins, in which the viral structural proteins trigger the extrusion of the plasma membrane, such as retroviral budding in which strong interactions between the gag n-terminal domain (matrix protein) and the plasma membrane induce bulging of the membrane; and iii) requiring interaction between the viral glycoproteins and the capsid for membrane extrusion, as in alphaviruses. the final step that results in the separation of virus from the cell surface is pinching off the particle. this is a controlled process that involves both viral protein domains and cellular factors. during this process, viruses apparently use cellular machinery similar to that used during the last step of cell division (the release of the daughter cell) called endosomal sorting complex required for transport (escrt) [reviewed in (campsteijn et al., 2016; hurley, 2015; olmos and carlton, 2016; scourfield and martin-serrano, 2017) ]. direct interactions of numerous viral domains with escrt complex subunits have been identified (bieniasz, 2006) . in retroviruses, short specific amino acid sequences (ptap or psap) interact with the escrt components, and the interaction of hiv with the escrt proteins nedd4 and alix is wellknown (freed, 2002; fujii et al., 2007; gomez and hope, 2005) . however, this mechanism has also been adopted by other viruses that interact with the same escrt components, such as filoviruses (han et al., 2015; jasenosky and kawaoka, 2004; liu and harty, 2010) . interactions with escrt proteins have also been reported for vesicular stomatitis virus luyet et al., 2008 ), rhabdovirus (taylor et al., 2007 , arenaviruses (ziegler et al., 2016) , picornaviruses , paramyxoviruses (park et al., 2016) , and hepatitis c virus, a representative of the flavivirus family (falcon et al., 2017) . the typical release pathways used by viruses may vary under some conditions. for example, during chronic infection, numerous viruses use alternative cell-to-cell transmission that may help the virus avoid host neutralization (hulo et al., 2017) . syncytium formation, an hivinduced cell fusion, was recognized in the early 1990s (callahan, 1994) . another type of cell-to-cell transmission through tight junctions was shown for hiv (hübner et al., 2009; wang et al., 2017) and murine leukemia virus (sherer et al., 2010) . receptors on tight junctions that specifically recognize hepatitis c virus (carloni et al., 2012; ploss et al., 2009 ) and reovirus (barton et al., 2001) have been identified. some viruses are able to modify cellular pathways to reprogram both the synthesis and metabolism of lipids and membrane compartmentalization for their transmission and to evade cellular defense mechanisms (mazzon and mercer, 2014) . despite a general understanding that poliovirus spreads through cellular lysis, it was recently found that it may also be transferred between cells by an autophagy-dependent mechanism, called autophagosome-mediated exit without lysis (bird and kirkegaard, 2015b; bird et al., 2014; lai et al., 2016; richards and jackson, 2012) . similar mechanisms have been described for varicellazoster virus and human cytomegalovirus (grose et al., 2016; meier and grose, 2017) . poxviruses encode several proteins that block the apoptotic cellular response to the presence of their dsdna in the cytoplasm. this allows cell-to-cell passage of poxviruses by a mechanism known as apoptotic mimicry (amara and mercer, 2015; nichols et al., 2017) . in this process, enveloped viruses expose surface phosphatidylserine to mimic apoptotic bodies. these cells are then macropinocytosed by either dendritic cells or macrophages (mercer and helenius, 2010) . among the plethora of compounds designed to inhibit infectious viruses, only a few (< 100) have been approved for clinical use (de clercq, 2004; de clercq and li, 2016) . nevertheless, some effective antiviral drugs have been on the market for several decades, such as the anti-influenza a virus drug amantadine, marketed under the trade name symmetrel (by dupont), which was approved in 1966. in 1982, burroughs-wellcome introduced acyclovir as an inhibitor of herpesviruses. its remarkable specificity is connected with its activation by viral thymidine kinase-catalyzed phosphorylation. however, due to development of drug resistance in a number of viruses, especially rna viruses, there is a continuous need to design and test new inhibitors, preferably targeting different steps of viral life cycles. here, we provide insights into the world of biochemical and cell-based assays that were developed to test antivirotics targeting various steps in the viral life cycle. different types of assays, including cell-cell fusion assays, cell-virus fusion assays with pseudotyped viral particles, and in vitro biochemical assays have been developed to screen inhibitors of viral entry. in enveloped viruses, entry is initiated by fusion of the viral envelope with the target cellular membrane. the entry mechanism has been well-described for hiv-1, in which it is mediated by env glycoprotein, consisting of transmembrane gp41 and surface gp120 subunits. binding of gp120 to its cellular receptor, cd4, and to one of two co-receptors, cxcr4 or ccr5, triggers a refolding of gp41 that promotes fusion of the viral and cellular membranes. the refolding involves oligomerization of the extracellular n-and c-terminal heptad repeat (hr) domains of gp41, which leads to the formation of a 6-helical bundle [reviewed in (melikyan, 2008) ]. jiang et al. established an in vitro system to quantify formation of the hiv-1 gp41 6-helical bundle (jiang et al., 1999 ). in their system, the bundle is formed by mixing equimolar concentrations of peptides derived from the n-and c-hr regions of gp41. elisa using the monoclonal antibody nc-1, which specifically recognizes and binds an epitope formed on the gp41 6-helix bundle but not the individual peptides, enables screening for compounds that prevent formation of fusion-active gp41. for hts of hiv-1 fusion inhibitors, this method was modified to use a fluorescence-linked immunosorbent assay (flisa), in which the c-hr peptide was replaced with fitc-conjugated c-hr peptide (boyer-chatenet et al., 2003) . cell-based assays are routinely used to screen viral entry inhibitors. high throughput formats have been developed for screening of hiv-1 fusion inhibitors. cell-cell fusion assays involve two types of cells: effector cells that stably (bradley et al., 2004) or inducibly (herschhorn et al., 2011; ji et al., 2006) express hiv-env glycoprotein and target cells that express cd4 and either cxcr4 or ccr5. co-cultivation of these cells leads to hiv-1 env-mediated cell membrane fusion, resulting in formation of multinucleated syncytia. membrane fusion induces expression of a reporter protein such as luciferase (herschhorn et al., 2011; ji et al., 2006) or β-galactosidase (bradley et al., 2004) . one such assay enables determination of both the efficiency and specificity of fusion inhibitors (herschhorn et al., 2011) . this approach uses effector cells that express both hiv-1 env and the renilla luciferase (r-luc) reporter protein using inducible tetracycline-controlled transactivator (tta) and target cells that express the hiv-1 receptor (cd4) and coreceptor (ccr5) and contain the firefly luciferase (f-luc) reporter gene under the control of a tta-responsive promoter. upon fusion of the effector and target cells, tta enters the target cells and activates the expression of the f-luc reporter. the inhibition of fusion of cellular membranes is determined as a decrease in f-luc luminescence, and the inhibitor specificity is measured as the r-luc activity (herschhorn et al., 2011) . based on an hiv-1 cell-cell fusion method that uses a computercontrolled digital image analysis system for automatic quantitation , a modified method was developed to screen inhibitors targeting mers-cov s protein (lu et al., 2014) . to test potential fusion inhibitors, effector cells stably expressing the mers-cov spike protein s2s and egfp were used to mediate fusion with target cells expressing the dpp4 receptor (lu et al., 2014) . virion-based fusion assays are another category of cell-based fusion assays. one such approach is based on production of chimeric hiv-1 virions carrying β-lactamase-vpr chimeric protein (blam-vpr). chimeric hiv released into the cell culture media is isolated by ultracentrifugation and used to infect target cells. entry of the virions into the cytoplasm is detected by cleavage of a fluorescent substrate by βlactamase. the fluorescence shift corresponds to the fusion efficacy and is measurable by fluorescence microscopy, flow cytometry, or fluorometric plate reader (cavrois et al., 2002; cavrois et al., 2004 cavrois et al., , 2014 . modification of this assay by constructing pseudotyped hiv-1 virions in which hiv-1 env was replaced with ebola virus glycoprotein (gp) has also been described (yonezawa et al., 2005) . tscherne et al. developed an approach to monitor viral entry using the blam reporter (tscherne et al., 2010) . their assay uses influenza virus-like particles (vlps) bearing the influenza membrane proteins hemagglutinin and neuraminidase. blam tagged with influenza matrix protein (m1) is incorporated into the vlps and delivered into target cells. upon release, blam can be detected by flow cytometry, microscopically, or fluorometrically. a rapid cell-based hts method was developed to assess sars-cov entry inhibitors (zhou et al., 2011) . this dual envelope pseudovirion (dep) assay employs two hiv pseudoviruses: one encodes an envelope protein from the target virus and firefly luciferase and the second encodes a control, unrelated viral envelope protein and renilla luciferase. reporter expression is determined with the dual-glo luciferase assay system (promega). inclusion of the unrelated envelope protein greatly reduced false positive hits (zhou et al., 2011) . the method was further used to screen compounds that inhibit entry of filoviruses, including ebola virus . a similar approach employing four pseudotyped hiv viruses, carrying marburg virus glycoprotein, hemagglutinin and neuraminidase isolated from a/goose/qinghai/59/05 (h5n1) influenza virus, ebolavirus zaire envelope glycoprotein, and lassa virus envelope glycoprotein, has been used for entry inhibitor screening . this screening identified multiple compounds with potent inhibitory activity against entry of both marburg and ebola viruses in human cancer cell lines, and confirmed their anti-ebola activity in human primary cells . other pseudotyped viral assays have been used to screen entry inhibitors of sars-cov, ebola, hendra, and nipah viruses. to establish infection, the glycoproteins of these viruses must be processed by the host intracellular lysosomal protease cathepsin l (catl). hts resulted in identification of several inhibitors that block the cleavage of viral glycoprotein but not catl itself (elshabrawy et al., 2014) . until recently, the development of anti-hbv therapeutics had been limited by the lack of an in vitro infection system. several aspects of the hbv life cycle have been elucidated using in vitro production of hbv particles after transfection of human hepatoma cell lines (hepg2) with recombinant hbv dna and by establishment of hepatoma cell lines with the entire hbv genome integrated, such as the hepg2.2.15 cell line (ladner et al., 1997; sells et al., 1987; sureau et al., 1986) . in addition to differentiated human (phhs) (gripon et al., 1988) and tupaia belangeri (glebe et al., 2003) hepatocytes, the heparg cell line, which exhibits hepatocyte-like characteristics, also supports hbv replication (gripon et al., 2002) . the identification of sodium taurocholate cotransporting polypeptide (ntcp) as an hbv receptor by yan et al. opened possibilities to use ntcp-complemented hepg2 cells not only for studies of hbv replication mechanisms but also for hts of inhibitors (yan et al., 2012) . in an infection competition assay, hbv particles were isolated and used to infect heparg and phhs cells that had been pre-incubated with hbv envelope protein-derived peptides to test their potential activity to block hbv infection. twelve days after infection, viral rnas were quantified by northern blot (gripon et al., 2002 (gripon et al., , 2005 . using this assay, researchers identified a peptide that specifically prevents hbv and hdv entry into heparg and phhs cell lines (gripon et al., 2005) , primary cultures of tupaia hepatocytes (glebe et al., 2005) , and cells in vivo (lütgehetmann et al., 2012; petersen et al., 2008; volz et al., 2013) . recently, a phase iia clinical trial of a first-in-class entry inhibitor (myrcludex-b) that functions as an ntcp inhibitor was promisingly completed (bogomolov et al., 2016) . development of cell culture systems producing hcv pseudoparticles (hcvpp) and infectious hcv particles (hcvcc) has shed light on hcv interactions and enabled discovery of antiviral drugs and vaccines (colpitts et al., 2016) . hcvpp consist of hcv e1 and e2 glycoproteins enveloping a retroviral particle that packages gfp mrna during assembly (bartosch et al., 2003) . the entry efficiency of hcvpp can be quantified by facs analysis as the number of gfp-positive cells. use of this screening system led to discovery of a triazine derivative that blocks the entry of hcv (baldick et al., 2010) . production of hcvcc is a robust system to generate infectious hcv in naïve cells (kato et al., 2006; zhong et al., 2005) . the anti-hcv activity of hundreds of compounds approved for a wide variety of indications was determined immunochemically with anti-hcv e2 antibody in 96-well plate format. over thirty compounds displayed anti-hcv activities, most of which were directed against hcv entry (gastaminza et al., 2010) . the majority of viruses enter cells by endocytosis. unfortunately, there are no suitable experimental techniques for endosome handling, making it difficult to study early steps in the viral life cycle such as uncoating and capsid disassembly. viruses that enter cells by direct membrane fusion, such as hiv-1, are an exception. there are several methods available to monitor and quantify hiv uncoating. as some of these were reviewed recently by campbell and hope (campbell and hope, 2015) , we will discuss them very briefly. two main techniques are used in these assays: ultracentrifugation or utilization of hiv-1 specific cellular restriction factors. the "in vitro core-stability assay" is based on ultracentrifugation of released hiv-1 virions through a detergent layer, where the viral membrane dissolves, into a sucrose gradient, where the viral cores are concentrated (shah and aiken, 2011) . the "fate of capsid" assay uses ultracentrifugation through a sucrose cushion to separate the hiv-1 core from a whole cell lysate prepared shortly after infection (stremlau et al., 2006) . the "csa-washout assay" involves specific cellular factors (trim-cyclophilin) that restrict hiv-1 infection by binding to the capsid core and cyclosporine a (csa), which can effectively turn off restriction (hulme et al., 2011) . recently, a novel entry/uncoating assay (eurt), an alternative to blam-vpr (described in section 3.1), was reported (da silva santos et al., 2016). it quantifies the protein product of a virion-packaged mrna reporter upon uncoating. a method to monitor the uncoating/disassembly of the capsid of influenza a virus, which enters the cell by endocytosis, also is based on ultracentrifugation (stauffer et al., 2016) . purified virions are separated using velocity gradient centrifugation through a two-layer glycerol gradient. similar to the "in vitro core stability" assay for hiv, the sedimenting viruses migrate through the detergent-containing layer of the gradient, which dissolves the membrane to release the viral core. moreover, modification of the detergent-containing glycerol layer-for example, by lowering ph, changing ionic strength, or adding putative viral uncoating factors-enables study of the factors or compounds that affect viral uncoating in vitro. depending on the virus type, either dna or rna polymerases replicate the viral genome. thus, these enzymes play a key role in viral life cycles. reverse transcriptase, an rna-dependent dna polymerase of retro-and hepadnaviruses, is unique among nucleic acid polymerases. despite the different mechanisms of viral replication, polymerases, which are essential for all viruses, are excellent targets for antiviral therapies. polymerase inhibitors represent the vast majority of clinically approved antiviral drugs, followed by protease inhibitors, immunostimulators, entry inhibitors, and integrase inhibitors [reviewed in (de clercq and li, 2016) ]. polymerases continue to be a preferred target for newly designed inhibitors (clercq, 2008; de clercq and li, 2016) . polymerase inhibitors may be nucleoside and nucleotide analogs, pyrophosphate analogs, or nonnucleosides, such as allosteric inhibitors (de clercq and li, 2016; öberg, 2006) . non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (tino et al., 1993; tisdale et al., 1993) . the activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (zhang et al., 2017) . more straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (coates et al., 1992; hirashima et al., 2006; joyce, 2010) ; these include hts methods using homopolymeric polycytidylic acid and [ 3 h]-gtp (amraiz et al., 2016) . alternatively, the pyrophosphate released during the polymerase reaction can be measured luminometrically by combining the primer extension with the commercial piper assay (malvezzi et al., 2015) . the pyrophosphate anion also can be detected with dna-attached magnetic nanoparticles (tong et al., 2015) or quantum dots (chai et al., 2015) . frequently used fluorescence methods avoid the use of radiochemicals. these methods exploit a fluorescent label, such as dsdna binding dyes like sybr green or pi-cogreen (driscoll et al., 2014; holden et al., 2009; zipper et al., 2004) , or fret between two nucleotides (schwartz and quake, 2009; weiss, 2000) . numerous kits for quantification of products of both dna and rna polymerases, including reverse transcriptase, are commercially available (bustin, 2000) . quantitative real-time pcr is a preferred method to monitor the activity of dna polymerases (cellular as well as viral) in the presence of inhibitors (beadle et al., 2016; zweitzig et al., 2012) , and quantitative real-time reverse-transcription pcr has become standard for screening inhibitors of viral rna polymerases okon et al., 2017; pelliccia et al., 2017; zhang et al., 2017) . white et al. described a hts method using a microfluidic apparatus combined with digital pcr for single-cell analysis (white et al., 2013) . in their method, a fluorescently labeled template also serves as a primer, due to stem formation. it emits a measurable polarization signal both upon binding of the polymerase and extension (mestas et al., 2007) . the assay has been used for hts of inhibitors of poliovirus rna polymerase (campagnola et al., 2011) . when available, viral genomes modified with reporter genes can be used for cell-based luminescent or fluorescent screening of viral inhibitors (beadle et al., 2016; edwards et al., 2015; fenaux et al., 2016; feng et al., 2014; lo et al., 2017; madhvi et al., 2017; wang et al., 2015c) . this approach can be extended to screen inhibitors of other enzymes. in the aptamer-based approach, a dna template encodes rna of interest joined to a fluorescence module and ribozyme sequence. when transcribed, the fluorescence module is released and detected (höfer et al., 2013) . hcv uses an rna-dependent rna polymerase (rdrp). a unique cell-based assay for monitoring hcv rna polymerase (ns5b) activity, based on the innate immune signaling molecule retionic acid-inducible gene i product (rig-i), has been developed (ranjith-kumar et al., 2011) . the rig-i-like receptors are cytosolic proteins that recognize viral rna and induce production of proinflammatory cytokines and interferon-activated genes (jensen and thomsen, 2012) . rig-i triggers cytokine production stimulated by various viral rnas from different families, including flaviviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, and arenaviridae, as well as ebola virus rna (jensen and thomsen, 2012) . the assay is based on recognition of hcv rna produced by active ns5b by rig-i, followed by rig-i-mediated activation of firefly luciferase expression controlled by the interferon b promoter. renilla luciferase expression is used for normalization of transfection efficacy. the viral grnas of some rna viruses are modified at the 5′ ends with cap structures, which may be either acquired from the host cell mrna (e.g. in influenza virus) or newly synthesized (e.g. flaviviruses). the methylation of viral grna mimics that of cellular mrna cap structures to enhance the chances of the virus to escape from cellular defense mechanisms and also to increase the efficiency of translation of viral proteins. virus-specific methyltransferases are thus a promising therapeutic target. virus-encoded methyltransferases have been identified and characterized in flaviviruses such as zika virus coutard et al., 2017; duan et al., 2017; munjal et al., 2017; zhao et al., 2017) , west nile virus, and dengue virus (dong et al., 2012) ; rhabdoviruses such as vesicular stomatitis virus (vsv) (rahmeh et al., 2009 ); coronaviruses such as sars (wang et al., 2015b) ; and roniviruses (zeng et al., 2016) . in flaviviruses, the n-terminal part of ns5 methyltransferase catalyzes cap formation via both guanine n-7 and ribose 2′-oh methylations at the 5′ end of grna, and the c-terminus of the enzyme is responsible for the rna polymerase activity (ray et al., 2006; zhao et al., 2015) . the c-terminal part of the sars nsp14 protein exhibits guanine n7-methyltransferase activity, forming the grna cap, while the 3′-5′ exoribonuclease activity of the n-terminus enhances the fidelity of viral replication (case et al., 2016; minskaia et al., 2006) . in vsv, the methyltransferase activity resides in the conserved region vi of the multifunctional large polymerase protein (li et al., 2005; ma et al., 2014) . some cellular methyltransferases regulate viral infections, as shown for herpes simplex virus, for which epigenetic control is involved in viral latency (cliffe and wilson, 2017) . inhibition of human histone methyltransferases such as histone-lysine n-methyltransferase (ezh2/ 1) (arbuckle et al., 2017) or lysine-specific demethylase-1 (lsd1) induces antiviral signaling pathways (liang et al., 2009) . the inhibitory effect of histone demethylase activity has been demonstrated for human cytomegalovirus (gan et al., 2017) and herpes simplex virus (hill et al., 2014; liang et al., 2013) . the activity of purified recombinant methyltransferases can be determined by measuring the methylation by-product s-adenosyl homocysteine (sah) using commercially available kits. in one such assay, conversion of s-adenosyl methionine (sam) to sah is monitored luminometrically via luciferase reaction, in which measurable atp is generated through a sequence of reactions with mtase-glo™ reagent (promega). the epigeneous™ methyltransferase kit (cisbio bioassays) is based on competition of produced sah with fluorescently labeled sah (sah-d2 tracer) for binding to a terbium cryptate-labeled anti-sah antibody. the decrease in fret between the tracer and antibody is then evaluated. elisa using anti-5-methylcytosine antibody can be used to quantify methylation of immobilized cytosine-rich dna substrate (e.g. epiquik™ dna methyltransferase activity/inhibition assay kit; epigentek). this method was modified with homogenous time resolved fret (degorce et al., 2009) to screen a library of inhibitors against sars-cov nsp14 . flaviviral and human cap n7-mtases have been screened with radioactive assays using 3 h-labeled sam and gpppac4 or 7m gpppac4 synthetic rnas. the 3 h methyl transferred onto deae filter-bound rna can be measured by scintillation after multiple washings to remove unincorporated 3 h-sam . alternatively, in vitro transcription can be carried out using 3 h-sam. the radioactively labeled products are then resolved by thin-layer chromatography and developed using a phosphorimager (li et al., 2007) . a yeast cell-based method was established based on the finding that coronavirus methyltransferase can functionally replace a methyltransferase essential for yeast viability sun et al., 2014) . in this method, sun et al. constructed recombinant yeasts producing the viral methyltransferase instead of the yeast one (sun et al., 2014) . this strain was used for a hts of methyltransferase inhibitor activity that negatively correlated with the cell density after 20 h incubation. virus-encoded atpase-driven helicases have been identified in numerous human pathogens. helicases from several (+)rna viruses have been characterized, including ns3 helicases from flaviviruses such as dengue virus, hcv, west nile virus, yellow fever virus, and japanese encephalitis virus (cao et al., 2016; gu and rice, 2016; jain et al., 2016; lin et al., 2017; nedjadi et al., 2015; wu et al., 2005) . sars and mers coronaviruses encode nsp13 with helicase activity (adedeji and lazarus, 2016; hao et al., 2017; seybert et al., 2000) . in semliki forest virus, a representative member of the togavirus family, helicase activity is encoded by the n-terminal domain of nsp2 protein. helicases are also common in some dna viruses, including poxviruses. these include the vaccinia virus helicase-primase d5 (bayliss and smith, 1996; hutin et al., 2016) , e1 protein of bovine papilloma virus 1 (yang et al., 1993) , and the large tumor antigen of sv40 (stahl et al., 1986) . helicases appear to be attractive targets for antiviral drugs (briguglio et al., 2011; frick, 2003; reynolds et al., 2015) , but development of such compounds is challenged by cytotoxicity and bioavailability issues (kwong et al., 2005) . traditional methods for monitoring the activity of rna helicases use radioactively labeled dsrna substrates and follow the unwinding reaction by electrophoretic separation (nondenaturing page) of the ss reaction products, which are detected autoradiographically (adedeji et al., 2012; utama et al., 2000) . to determine whether the inhibitor affects binding of helicase to nucleic acid, a standard gel mobility shift assay is usually used (adedeji et al., 2012) . helicase activity is fueled by atp hydrolysis; thus, inhibition of atpase activity became another possible antiviral strategy. atpase activity can be determined either by the decrease in atp or formation of adp (using commercially available fluorescent anti-adp antibodies) or inorganic pyrophosphate. phosphates released by atp hydrolysis can form a molybdophosphate complex that can be measured colorimetrically using malachite green, quinaldine red, or rhodamine b (baykov et al., 1988; debruyne, 1983; miyata et al., 2010) or by lightscattering (oshima et al., 1996) . the colorimetric methods can be miniaturized for hts (zuck et al., 2005) . the absorbance-based assay was also converted into a hts method based on fluorescence quenching by a colored quinaldine red complex ). an alternative method employs a europium-tetracycline complex for luminescent determination of inorganic phosphate (schäferling and wolfbeis, 2007) . a more complex coupled enzyme colorimetric assay (with maltose phosphorylase, glucose oxidase, and horseradish peroxidase) was used for hts of atpase inhibitors (avila et al., 2006) . assays for luminescent and fluorescent screening of atpase activity, including an immunochemical method using fluorescently labeled anti-adp antibodies, have been reviewed by shadrick and colleagues (shadrick et al., 2013) . in a radioactive method using [γ-32 p]atp, the amp product was separated from unreacted atp by thin-layer chromatography on polyethyleneimine-cellulose and visualized autoradiographically (adedeji et al., 2012) . several research groups have described fluorescence assays to identify inhibitors targeting sars-cov helicase (nsp13) (adedeji et al., 2012; cho et al., 2015; özeş et al., 2014) . the substrate is usually a dsdna oligonucleotide consisting of a fluorescently labeled strand annealed to the complementary strand carrying a quencher. this approach was also adapted for real-time determination of the rna helicase activity of hcv (tani et al., 2010) . in this assay, an ssdna capture strand complementary to the strand carrying the quencher was used to prevent reannealing of the unwound duplex. recently, a colorimetric assay for monitoring helicase activity using dna-conjugated gold nanoparticles was developed (deka et al., 2017) . this method is based on shifts in the optical properties of nanoparticles due to dna unwinding and allows simple screening of inhibitor activity. the dsdna melting curves can be determined spectrophotometrically (at 524 nm and 260 nm) or even by the naked eye. another fluorescence hts of dengue virus ns3 helicase inhibitors measures the unwinding of a double-labeled molecular beacon (basavannacharya and vasudevan, 2014) . other approaches involve graphene oxide-based fluorescence monitoring of viral helicase activity [reviewed in (jang et al., 2013) ]. a gquadruplex-based method for label-free determination of hcv helicase ns3 activity measures changes in the luminescence of transition metal complexes with dna upon helicase-mediated quadruplex melting (leung et al., 2015) . both protein tyrosine kinases and protein serine/threonine (s/t) kinases have been found in viruses. tyrosine kinase function is wellunderstood in connection with oncogenic retroviruses, [for review on retroviral oncogenes, see (vogt, 2012) ]. in contrast to tyrosine kinases from oncogenic viruses, such as rous sarcoma virus src tyrosine kinase, viral s/t kinases share little homology with cellular enzymes. they are exclusively encoded by large dna viruses (e.g. herpesviruses), in which they play important roles in viral virulence, helping the virus to escape defense mechanisms such as those regulated by cytokine signaling pathways (jacob et al., 2011; sato et al., 2017) . their autophosphorylation and transphosphorylation activities mimic those of cellular cyclin-dependent kinases such as cdc2. for example, viral kinases can phosphorylate translation elongation factor 1 delta (ef-1δ) (jacob et al., 2011; kawaguchi and kato, 2003) . all herpesviruses encode the s/t protein kinase ul13, and us3 s/t kinases have been described in the alphaherpesvirus subfamily (kato, 2016; kawaguchi and kato, 2003) . in addition to protein kinases, hsv encodes a thymidine kinase. unlike cellular thymidine kinase, hsv thymidine kinase has a wide substrate specificity that includes pyrimidine and purine phosphonate analogs (e.g. acyclovir, ganciclovir, penciclovir) (de clercq and li, 2016) . in the body, ganciclovir is phosphorylated by cellular kinases and penciclovir and acyclovir by virus-encoded thymidine kinases to the active nucleoside triphosphate forms of the drugs that inhibit viral dna synthesis (de clercq and li, 2016; kokoris and black, 2002) . some cellular protein kinases appear to support viral replication. for example, polo-like kinases induce early stages in the influenza virus life cycle (pohl et al., 2017) , and human protein kinase c regulates the assembly of the ribonucleoprotein complexes in influenza virus (mondal et al., 2017) . inhibitors of abelson tyrosine-protein kinase 2 are active against sars and mers coronaviruses (coleman et al., 2016) . several in vitro approaches can be used to determine kinase activity as well as the activity of kinase inhibitors. analyses of the cellular phosphoproteome, sometimes accompanied by phosphopeptide enrichment, have become standard to determine kinase activity (lea and simeonov, 2011; meyer et al., 2017; vyse et al., 2017) . these assays can be used to assess the impact of inhibitors on the overall phosphoproteome in mammalian cells. although these methods provide complex information about the overall array of kinases and phosphatases in the cell (olsen et al., 2006) , they are not applicable to screening inhibitors of a single viral kinase. for these purposes, in vitro assays with recombinant kinases have been developed [for review see ], including some hts fluorescence methods (zegzouti et al., 2009 ) that can replace radioactive techniques using [γ 32 p]atp (sanghera et al., 2009) . mass spectrometric analysis also can be used to identify in vitro kinase inhibitors. for these analyses, synthetic peptides, proteins, or phosphatase-and heat-treated tissue samples (to dephosphorylate the proteins and inactivate all enzymes, respectively) are subjected to kinase treatment in the presence of inhibitors (huang et al., 2007; meyer et al., 2017; xue et al., 2012) . recombinant kinase activity in the presence of inhibitors can be quantified as atp consumption or adp production in the phosphorylation reaction by numerous commercial kits. other methods monitor binding of inhibitors to phage-displayed kinases in ligand competition assays (fabian et al., 2005) . fluorescence methods including fret, fluorescence polarization or intensity endpoint measurement, and lifetime imaging of fluorescence including fluorescence biosensors (zhang and allen, 2007) have been reviewed elsewhere. sulfonamido-oxine labeled peptides can be used as chromophores that bind mg 2 + upon phosphorylation and emit chelation-enhanced fluorescence (devkota et al., 2013; luković et al., 2008) . kinase-catalyzed phosphorylation of fluorescent peptides promotes their binding to metal-coated nanoparticles, which decreases their mobility and enhances measurable fluorescence polarization (lea and simeonov, 2011; sportsman et al., 2004) . tbiii complexes, in which phosphotyrosine induces fluorescence emission (wang et al., 2015a) , may be used to evaluate protein tyrosine kinase activity (akiba et al., 2015; sumaoka et al., 2016) . fluorescence polarization methods also can be useful for drug screening [reviewed in (hall et al., 2016) ]. other alternatives are immunochemical methods that use antibodies specific to the phosphorylated amino acids, such as phosphotyrosine (li et al., 2001; youngren et al., 1997) or phosphoserine/ phosphothreonine. these antibodies can be used to detect protein/ peptide phosphorylation by western blotting, elisa, or immunoprecipitation of phosphorylated proteins for further mass spectrometry-based analysis (grønborg et al., 2002; zhang et al., 2002) . an elegant approach that limits the false-positive hits in screening of specific kinase inhibitors is based on an in situ proximity ligation assay using both an antibody against the target protein and an anti-phosphotyrosine antibody (leuchowius et al., 2010) . both antibodies are coupled with oligonucleotides, which when brought together due to antibody binding, can be enzymatically ligated and replicated through rolling circle amplification to form a long linear tandem repeat of sequences detectable by a complementary fluorescent oligonucleotide. in addition to protein kinases, some lipid kinases have been targeted by antivirals. one example is sphingosine kinase 1, which affects replication of dengue virus (aloia et al., 2017) . its activity can be determined by measuring the production of 32 p-labeled sphingosine-1phosphate from sphingosine and [γ 32 p]atp (clarke et al., 2016; pitman et al., 2012) . retroviral integrase inhibitors are a new type approved new type of inhibitors imposed by the emergence of drug-resistant mutants. hiv integrase activities, integrase inhibitors, and drug resistance have been discussed in detail elsewhere (andrake and skalka, 2015; anstett et al., 2017; hajimahdi and zarghi, 2016; liao et al., 2010; podany et al., 2017; thierry et al., 2016) . methods to assess the two major activities of integrase-end processing of the reverse transcription product and its joining to target chromosomal dna-have been reviewed in detail by several groups (engelman and cherepanov, 2014; marchand et al., 2001; merkel et al., 2009) . initial methods used radioactively labeled dna oligonucleotides comprising the terminal cis-acting sequences of linear viral dna required for integration. the joining of the processed strand to the other strand (self-integration) or to supplemented target dnas can be analyzed by page (katz et al., 1990; katzman et al., 1989) . a less time-consuming, non-radioactive method involves timeresolved fluorescence anisotropy measurement using a 21-meric oligonucleotide fluorescently labeled on the terminal gt dinucleotide. this assay monitors the binding of integrase to the substrate as well as the subsequent 3′-processing reaction, which both change the anisotropy (guiot et al., 2006) . alternatively, the yields of both the processing and joining reactions can be measured upon separation of the radioactively labeled product from the rest of the dna molecule using adsorption to pei-cellulose (muller et al., 1993) . a real-time hts method measures fluorescence emission resulting from removal of the 3′-terminal dinucleotide, labeled with a quencher, by integrase (he et al., 2007) . han et al. described a fluorescence method to screen molecules that inhibit binding of integrase to viral dna . methods evaluating the integrase strand transfer reaction have been modified to a high-throughput format using magnetic beads (he et al., 2008) or streptavidin-coated microplates (john et al., 2005) . a method to assess strand transfer by time-resolved fret with a europium-streptavidin-labeled substrate has been optimized for 384-and 1536-well plate formats (wang et al., 2005) . the hbv capsid protein is the building block of the viral core, surrounding the viral nucleic acid (pre-genomic rna, pgrna) and reverse transcriptase. the hbv core is icosahedral, formed by 240 copies of capsid protein dimers. in vitro hts of hbv core assembly inhibitors using a modified hbv capsid protein has been described (stray et al., 2006) . the capsid protein was modified by deleting the nucleic acid binding domain, which is dispensable for capsid assembly, and the nterminal assembly domain alone was used in the assay. to fluorescently label the hbv capsid protein, all cysteine residues dispensable for assembly were replaced with alanines. a unique cysteine residue (c150) was c-terminally joined to the assembly domain and labeled with fluorescent bodipy-fl dye (c150bo). during assembly, capsid proteins dimerize, bringing c150bo residues close together and resulting in c150bo fluorescence self-quenching. following incubation of the labeled hbv protein with inhibitors, fluorescence was measured in black 96-well microtiter plates. development of in vitro assembly systems has contributed greatly to current understanding of the structure of retroviral particles and mechanisms of virion formation. these systems also became the base for several high throughput assays for screening assembly inhibitors. during the last 20 years, a number of in vitro assembly assays have been established, mainly for hiv-1 (campbell et al., 2001; campbell and rein, 1999; ehrlich et al., 1992; gross et al., 2000; lanman et al., 2002) , mason-pfizer monkey virus (bohmova et al., 2010; klikova et al., 1995; rumlova-klikova et al., 2000; rumlova-klikova et al., 1999; ulbrich et al., 2006) , rous sarcoma virus vogt, 1995, 1997; purdy et al., 2008 purdy et al., , 2009 , and murine leukemia virus (dolezal et al., 2016; hadravova et al., 2012; cheslock et al., 2003) . hts assays for inhibitors of hiv-1 assembly include several methods using purified hiv-1 ca or ca-nc proteins. one of them, the turbidimetric assay, is based on the observation that direct dilution of the hiv-1 capsid protein (ca) into a high-salt solution (1.6-2.2 m nacl) leads to the formation of tubular structures. as the tube formation is accompanied by an increase in light scattering, assembly can be detected as an increase in turbidity, and the rate of turbidity change is proportional to the rate of the assembly (lanman et al., 2002) . other method published by lemke et al., exploits the affinity of nucleocapsid (nc) to a short tg-rich deoxyribooligonucleotide, d(tg25), which is used as a scaffold (lemke et al., 2012) . this arrangement enables ca to assemble at much lower protein and salt concentrations than in the turbidimetric assay (lanman et al., 2002) . biotin-labeled d(tg25) bound on the surface of neutravidin-coated microtiter well plates nucleates assembly of complexes of ca-nc and soluble fluorescein-labeled d(tg25). fluorescence is measured after washing to remove the unbound and unassembled material from the captured assembly products (lemke et al., 2012) . similarly, the faith assay uses a dually labeled oligonucleotide (tqon). however, in this case, the ssdna oligonucleotide tqon is labeled with the reporter dye fluorescein (fam) as well as black hole quencher (bhq); thus, it does not emit any fluorescence. the assembly reaction is triggered by mixing hiv-1 ca-nc or a gagtruncated assembly-competent version with tqon. following incubation, during which tqon is incorporated into the particles, exonuclease is added to degrade unbound tqon, while co-assembled tqon is protected from cleavage. degradation of free unbound tqon with exoi results in separation of fam from its quencher, and the emitted fluorescence is measured (hadravova et al., 2015) . phage display has been employed to screen peptide inhibitors of hiv-1 assembly (sticht et al., 2005) . a commercial library of m13-derived phages presenting random 12-amino-acid peptides was analyzed for specific binding to purified ca or ca-nc proteins. the specifically bound phages were sequenced, and corresponding peptides were chemically synthesized and re-tested in an in vitro assembly assay (gross et al., 2000) . late in the retroviral life cycle, grna is incorporated into the nascent particle during assembly at the plasma membrane. nc contains two zinc-finger domains that are responsible for specific binding of grna. to screen for compounds that would prevent nc-rna/dna interactions, a hts system consisting of two sequential screens was developed (breuer et al., 2012) . the primary screen uses fluorescence polarization (fp), while the secondary one uses differential scanning fluorimetry (dsf). the combination and order of these two techniques were selected to first identify compounds that disrupt interactions between dna and nc, and then identify the compounds binding to nc during the secondary screen. a rapid and simple turbidimetric method was developed to screen inhibitors of assembly of hcv core protein (fromentin et al., 2007) . for the in vitro assembly reaction, two components, an n-terminal part of the hcv core protein corresponding to the minimal assembly competent domain and rna corresponding to the full-length 5′utr of hcv, were used. assembly of hcv nucleocapsid-like particles was initiated by mixing the purified protein and nucleic acid, and the assembly process was monitored by measuring turbidity at 350 nm. numerous viruses, including picornaviruses, retroviruses, alphaviruses, and flaviviruses, encode proteases that are essential for their virulence. the majority of viral proteases specifically cleave viral polyprotein precursors to liberate the functional proteins of the virion. some viral proteases, such as the papain-like proteases of coronaviruses, also reprogram cellular signaling pathways, including ubiquitination mechanisms and interferon controlled responses, to prevent degradation of viral components (clementz et al., 2010; frieman et al., 2009; randow and lehner, 2009; xing et al., 2013) . numerous viruses, including papillomaviruses (bronnimann et al., 2016; buck et al., 2005) and retroviruses (hallenberger et al., 1992) , use also host cell proteases, mainly furin, to trim their envelope and surface proteins. this triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (laporte and naesens, 2017) ] such as influenza virus (böttcher-friebertshäuser et al., 2010; kühn et al., 2016) , newcastle disease virus (gotoh et al., 1992) , and respiratory syncytial virus (sugrue et al., 2001) . extensive research efforts have yielded detailed information about hiv-1 protease and its inhibitors [reviewed in (de clercq, 2004; konvalinka et al., 2015; midde et al., 2016) ]. large amounts of data also are available for hcv (foote et al., 2011; pawlotsky et al., 2007; razonable, 2011) and sars-cov 3cl protease inhibitors (pillaiyar et al., 2016) , although there is not yet an inhibitor of the latter target approved for clinical use. numerous approved drugs are synthetic peptides derived from the natural proteolytic substrates of target viruses modified to improve the in vivo effects related to bioavailability, stability, and so on. numerous in vitro assays to monitor the activity of proteases and their inhibitors, including commercial kits, have been developed. classical methods use synthetic peptides that mimic the target sites of the protease. although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. the cleavage yield is usually monitored either colorimetrically (ding and yang, 2015; zhou et al., 2014) or as a change in fluorescence triggered by the release of fluorescent labels such as 7-amido-4-methylcoumarin (amc) or rhodamine (grant et al., 2002) . fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (kuo et al., 2004; lee et al., 2014; park et al., 2017; song et al., 2014; tomar et al., 2015; wang et al., 2016; yang et al., 2005; zhao et al., 2008) . some recently described arrangements employ nanoparticles (feltrup and singh, 2012; khalilzadeh et al., 2016; udukala et al., 2016; wang et al., 2014; zeng et al., 2015) or quantum dot bioconjugates (lee and kim, 2015; li et al., 2014; medintz et al., 2006) with immobilized fluorescently or luminescently labeled peptide substrates. alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (hu et al., 2015; joshi et al., 2017; lathia et al., 2011; rumlová et al., 2003) , analytical hplc (teruya et al., 2016) , or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (gemene and meyerhoff, 2011; han et al., 1996) . to study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, x-ray or nmr structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of hiv-1 [reviewed in (ghosh et al., 2016) ], hcv (yilmaz et al., 2016) , and mers . cell-based assays can provide additional information, including the capability of the inhibitor to pass through the cell membrane and its stability in the cytoplasm. a general determination of infectivity, such as a plaque cytotoxicity assay in the presence of protease inhibitor, may be used for confirmation. one elegant example exploits the cytotoxicity of hiv protease. cells are transfected with a protease precursor fused to gfp. in the absence of inhibitor, hiv-1 protease is autocatalytically activated and cleaves a broad variety of cellular proteins, resulting in activation of apoptosis and cell death (cummins and badley, 2010; rumlova et al., 2014) . this toxic effect, when suppressed by active inhibitors, results in production of a gfp signal in surviving cells (lindsten et al., 2001) . another elegant approach for hiv and coxsackievirus b3 proteases, which both undergo autocatalytic cleavage, employs constructs in which the protease gene is inserted between sequences encoding the dna-binding domain and the domain that activates transcription of the gal1-lacz reporter gene (dasmahapatra et al., 1992; murray et al., 1993) . the protease-mediated cleavage separates the dna-binding domain from the trans-activating domain and results in failure of reporter gene transcription. this approach has also been modified with gfp as a reporter gene (hilton and wolkowicz, 2010) . co-expression of cleavage-activated luciferase substrate and mers-cov protease permits both live-cell imaging and quantification of the enzyme activity (kilianski et al., 2013) . the need to develop new antiviral compounds will likely persist over the long term, although there has been enormous progress in molecular biology methods, especially rna silencing, bioinformatics, imaging, and structural biology techniques. viruses present challenging targets for drug development due their flexibility and adaptability caused by the error-prone copying of their genomes, which can result in emergence of drug-resistant mutants. viral integration into the host genome and inhibitor toxicity are other obstacles. here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or cell-encoded proteins required for the infectivity. biochemical characterization of middle east respiratory syndrome coronavirus helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase components of adenovirus genome packaging click conjugation of a binuclear terbium(iii) complex for real-time detection of tyrosine phosphorylation pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo investigation of sphingosine kinase 1 in interferon responses during dengue virus infection viral apoptotic mimicry hiv-1 uncoating: connection to nuclear entry and regulation by host proteins development of robust in vitro rnadependent rna polymerase assay as a possible platform for antiviral drug testing against dengue retroviral integrase: then and now reactivation and lytic replication of kaposi's sarcoma-associated herpesvirus: an update hiv drug resistance against strand transfer integrase inhibitors toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay inhibitors of the histone methyltransferases ezh2/1 induce a potent antiviral state and suppress infection by diverse viral pathogens highthroughput screening for hsp90 atpase inhibitors a novel small molecule inhibitor of hepatitis c virus entry junction adhesion molecule is a receptor for reovirus infectious hepatitis c virus pseudoparticles containing functional e1-e2 envelope protein complexes suramin inhibits helicase activity of ns3 protein of dengue virus in a fluorescence-based high throughput assay format a malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay vaccinia virion protein i8r has both dna and rna helicase activities: implications for vaccinia virus transcription -phosphonomethoxy)ethyl]guanine (ode-bn-pmeg), a potent inhibitor of transient hpv dna amplification hepatitis b virus replication early steps in avian reovirus morphogenesis late budding domains and host proteins in enveloped virus release escape of non-enveloped virus from intact cells nonlytic spread of naked viruses nonlytic viral spread enhanced by autophagy components viral entry pathways: the example of common cold viruses herpes virus replication treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study effect of dimerizing domains and basic residues on in vitro and in vivo assembly of mason-pfizer monkey virus and human immunodeficiency virus cleavage of influenza virus hemagglutinin by airway proteases tmprss2 and hat differs in subcellular localization and susceptibility to protease inhibitors development and automation of a 384-well cell fusion assay to identify inhibitors of ccr5/cd4-mediated hiv virus entry identification of hiv-1 inhibitors targeting the nucleocapsid protein inhibition of rna helicases of ssrna(+) virus belonging to flaviviridae, coronaviridae and picornaviridae families furin cleavage of l2 during papillomavirus infection: minimal dependence on cyclophilins maturation of papillomavirus capsids absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays hiv-1 virion-cell interactions: an electrostatic model of pathogenicity and syncytium formation high-throughput screening identification of poliovirus rna-dependent rna polymerase inhibitors hiv-1 capsid: the multifaceted key player in hiv-1 infection in vitro assembly properties of human immunodeficiency virus type 1 gag protein lacking the p6 domain self-assembly in-vitro of purified ca-nc proteins from rous-sarcoma virus and human-immunodeficiency-virus type-1 in vitro assembly of virus-like particles with rous sarcoma virus gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles modulation of hiv-like particle assembly in vitro by inositol phosphates novel escrt functions in cell biology: spiraling out of control? molecular mechanism of divalentmetal-induced activation of ns3 helicase and insights into zika virus inhibitor design hcv infection by cell-to-cell transmission: choice or necessity? mutagenesis of s-adenosyl-l-methionine-binding residues in coronavirus nsp14 n7-methyltransferase m demonstrates differing requirements for genome translation and resistance to innate immunity components and regulation of nuclear transport processes a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes fluorescence resonance energy transfer-based hiv-1 virion fusion assay hiv-1 fusion assay a reversible fluorescence nanoswitch based on carbon quantum dots nanoassembly for detection of pyrophosphate ion functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase tsg101: a novel anti-hiv-1 drug target protein-protein interfaces in viral capsids are structurally unique identification of a coumarin-based antihistamine as an anti-filoviral entry inhibitor charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination identification of a novel small molecule inhibitor against sars coronavirus helicase reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases emerging antiviral drugs restarting lytic gene transcription at the onset of herpes simplex virus reactivation (-)-2'-deoxy-3'-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope how viruses access the nucleus abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion structures of ns5 methyltransferase from zika virus highthroughput approaches to unravel hepatitis c virus-host interactions localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center endocytosis of viruses and bacteria zika virus methyltransferase: structure and functions for drug design perspectives structure and organization of paramyxovirus particles mechanisms of hiv-associated lymphocyte apoptosis: 2010 a novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized hiv-1 infection a genetic system for studying the activity of a proteolytic enzyme antiviral drugs in current clinical use approved antiviral drugs over the past 50 years inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine b-phosphomolybdate complex htrf: a technology tailored for drug discovery -a review of theoretical aspects and recent applications dna-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of + srna viruses' replicases. front high-throughput screens for eef-2 kinase quantitative serine protease assays based on formation of copper(ii)-oligopeptide complexes functional and structural characterization of novel type of linker connecting capsid and nucleocapsid protein domains in murine leukemia virus 2′-o methylation of internal adenosine by flavivirus ns5 methyltransferase a quantitative fluorescence-based steadystate assay of dna polymerase the crystal structure of zika virus ns5 reveals conserved drug targets high-throughput minigenome system for identifying small-molecule inhibitors of ebola virus replication assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay retroviral integrase structure and dna recombination mechanism a small molecule-kinase interaction map for clinical kinase inhibitors ultrastructural and biochemical basis for hepatitis c virus morphogenesis nuclear entry of dna viruses development of a fluorescence internal quenching correction factor to correct bont/a endopeptidase kinetics using snaptide antiviral nucleotide incorporation by recombinant human mitochondrial rna polymerase is predictive of increased in vivo mitochondrial toxicity risk a pathogenic picornavirus acquires an envelope by hijacking cellular membranes inhibition of hepatitis c virus replication by gs-6620, a potent c-nucleoside monophosphate prodrug transcription and replication mechanisms of bunyaviridae and arenaviridae l proteins fields virology boceprevir: a protease inhibitor for the treatment of chronic hepatitis c viral late domains hiv-1 assembly, release and maturation helicases as antiviral drug targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling a method for in vitro assembly of hepatitis c virus core protein and for screening of inhibitors beyond tsg101: the role of alix in 'escrting' hiv-1 proteolytic cleavage of bovine m adenovirus 3-encoded pviii epigenetically repressing human cytomegalovirus lytic infection and reactivation from latency in thp-1 model by targeting h3k9 and h3k27 histone demethylases unbiased probing of the entire hepatitis c virus life cycle identifies clinical compounds that target multiple aspects of the infection detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode recent progress in the development of hiv-1 protease inhibitors for the treatment of hiv/aids structure and formation of the cytomegalovirus virion pre-s1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres1 lipopeptides and tupaia hepatocytes the ins and outs of hiv replication mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/pace to pc2 or pc1/ pc3 development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide infection of a human hepatoma cell line by hepatitis b virus efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein a mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, frigg, as a protein kinase a substrate varicella-zoster virus infectious cycle: er stress, autophagic flux, and amphisome-mediated trafficking a conformational switch controlling hiv-1 morphogenesis the cell biology of receptor-mediated virus entry the spring α-helix coordinates multiple modes of hcv (hepatitis c virus) ns3 helicase action relationship between the oligomeric status of hiv-1 integrase on dna and enzymatic activity bunyavirus: structure and replication in vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus xmrv faith -fast assembly inhibitor test for hiv progress in hiv-1 integrase inhibitors: a review of their chemical structure diversity fluorescence polarization assays in high-throughput screening and drug discovery: a review inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gpl60 selective monitoring of peptidase activities with synthetic polypeptide substrates and polyion-sensitive membrane electrode detection development of a fluorescence-based hiv-1 integrase dna binding assay for identification of novel hiv-1 integrase inhibitors alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp40: a role for alix in ebola virus egress crystal structure of middle east respiratory syndrome coronavirus helicase highthroughput real-time assay based on molecular beacons for hiv-1 integrase 3'-processing reaction a novel highthroughput format assay for hiv-1 integrase strand transfer reaction using magnetic beads nuclear egress of herpesviruses: the prototypic vesicular nucleocytoplasmic transport herpesvirus capsid assembly and dna packaging an inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of hiv-1 entry inhibitors inhibition of lsd1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes an assay to monitor hiv-1 protease activity for the identification of novel inhibitors in t-cells benzimidazole derivatives bearing substituted biphenyls as hepatitis c virus ns5b rna-dependent rna polymerase inhibitors: structure − -activity relationship studies and identification of a potent and highly selective inhibitor jtk-109 universal aptamer-based real-time monitoring of enzymatic rna synthesis factors affecting quantification of total dna by uv spectroscopy and picogreen fluorescence peptide code-on-a-microplate for protease activity analysis via maldi-tof mass spectrometric quantitation a systematic ms-based approach for identifying in vitro substrates of pka and pkg in rat uteri quantitative 3d video microscopy of hiv transfer across t cell virological synapses complementary assays reveal a relationship between hiv-1 uncoating and reverse transcription the ins and outs of eukaryotic viruses: knowledge base and ontology of a viral infection escrts are everywhere transport of the influenza virus genome from nucleus to nucleus domain organization of vaccinia virus helicase-primase d5 how viruses use the endoplasmic reticulum for entry, replication, and assembly experimental approaches to study genome packaging of influenza a viruses poliovirus-induced changes in cellular membranes throughout infection subversion of cellular autophagosomal machinery by rna viruses viral serine/threonine protein kinases structure of the ns3 helicase from zika virus a new helicase assay based on graphene oxide for anti-viral drug development filovirus budding oligomeric viral proteins: small in size, large in presence sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion development of a novel dual ccr5-dependent and cxcr4-dependent cell-cell fusion assay system with inducible gp160 expression a screening assay for antiviral compounds targeted to the hiv-1 gp41 core structure using a conformation-specific monoclonal antibody development and application of a highthroughput screening assay for hiv-1 integrase enzyme activities avian reovirus infections. revue scientifique et technique a structural and functional perspective of m. rumlová alphavirus replication and assembly the rational design of therapeutic peptides for aminopeptidase n using a substrate-based approach techniques used to study the dna polymerase reaction pathway molecular mechanism by which us3 protein kinase regulates the pathogenicity of herpes simplex virus type-1 cell culture and infection system for hepatitis c virus the avian retroviral in protein is both necessary and sufficient for integrative recombination in vitro the avian retroviral integration protein cleaves the terminal sequences of linear viral dna at the in vivo sites of integration protein kinases conserved in herpesviruses potentially share a function mimicking the cellular protein kinase cdc2 reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase-3 activity and apoptosis using peptide based biosensor assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers efficient in-vivo and in-vitro assembly of retroviral capsids from gag precursor proteins expressed in bacteria virus strategies for passing the nuclear envelope barrier characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity retroviral proteases and their roles in virion maturation origin and evolution of eukaryotic large nucleo-cytoplasmic dna viruses adenovirus tales: from the cell surface to the nuclear pore complex the proteolytic activation of (h3n2) influenza a virus hemagglutinin is facilitated by different type ii transmembrane serine proteases characterization of sars main protease and inhibitor assay using a fluorogenic substrate viral and cellular rna helicases as antiviral targets inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication the autophagic machinery in enterovirus infection kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro airway proteases: an emerging drug target for influenza and other respiratory virus infections multiplexed protease assays using element-tagged substrates fluorescence polarization assays in small molecule screening fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity identification of novel drug scaffolds for inhibition of sars-cov 3-chymotrypsin-like protease using virtual and high-throughput screenings characterization of the activity of 2′-c-methylcytidine against dengue virus replication distinct effects of two hiv-1 capsid assembly inhibitor families that bind the same site within the n-terminal domain of the viral ca protein high content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation label-free luminescence switch-on detection of hepatitis c virus ns3 helicase activity using a g-quadruplex-selective probe †electronic supplementary information (esi) available: compound characterisation and supplementary data immunity to bovine herpesvirus 1: i. viral lifecycle and innate immunity small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity vesicular stomatitis viruses resistant to the methylase inhibitor sinefungin upregulate rna synthesis and reveal mutations that affect mrna cap methylation fluorescence detection techniques for protein kinase assay quantum dots based molecular beacons for in vitro and in vivo detection of mmp-2 on tumor inhibition of the histone demethylase lsd1 blocks α-herpesvirus lytic replication and reactivation from latency a novel selective lsd1/kdm1a inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency authentic hiv-1 integrase inhibitors single-molecule imaging reveals the translocation and dna looping dynamics of hepatitis c virus ns3 helicase cellbased fluorescence assay for human immunodeficiency virus type 1 protease activity viral and host proteins that modulate filovirus budding rapid and automated fluorescencelinked immunosorbent assay for high-throughput screening of hiv-1 fusion inhibitors targeting gp41 gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. sci. rep. 7 a structural view of the rna-dependent rna polymerases from the flavivirus genus automatic quantitation of hiv-1 mediated cell-tocell fusion with a digital image analysis system (dias): application for rapid screening of hiv-1 fusion inhibitors structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor hiv-1 uncoating is facilitated by dynein and kinesin 1 recognition-domain focused (rdf) chemosensors: versatile and efficient reporters of protein kinase activity humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation the escrt-i subunit tsg101 controls endosome-to-cytosol release of viral rna the challenge of selecting protein kinase assays for lead discovery optimization mrna cap methylation influences pathogenesis of vesicular stomatitis virus in vivo a screen for novel hepatitis c virus rdrp inhibitor identifies a broad-spectrum antiviral compound quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors structure, function and dynamics in adenovirus maturation in vitro human immunodeficiency virus type 1 integrase assays human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes pathways of clathrin-independent endocytosis lipid interactions during virus entry and infection proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates variable effects of autophagy induction by trehalose on herpesviruses depending on conditions of infection common principles and intermediates of viral protein-mediated fusion: the hiv-1 paradigm viral polymerases virus entry by macropinocytosis apoptotic mimicry: phosphatidylserine-mediated macropinocytosis of vaccinia virus oligonucleotide-based assays for integrase activity a fluorescence polarization based screening assay for nucleic acid polymerase elongation activity multiplex substrate profiling by mass spectrometry for kinases as a method for revealing quantitative substrate motifs investigational protease inhibitors as antiretroviral therapies discovery of an rna virus 3′ → 5′ exoribonuclease that is critically involved in coronavirus rna synthesis high-throughput screen for escherichia coli heat shock protein 70 (hsp70/dnak): atpase assay in low volume by exploiting energy transfer influenza virus recruits host protein kinase c to control assembly and activity of its replication machinery poxvirus dna replication poxvirus membrane biogenesis. virology 479-480 membrane fusion during poxvirus entry rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant rous sarcoma virus integrases advances in developing therapies to combat zika virus: current knowledge and future perspectives inactivation of a yeast transactivator by the fused hiv-1 proteinase: a simple assay for inhibitors of the viral enzyme activity tackling dengue fever: current status and challenges expression strategies of ambisense viruses poxviruses utilize multiple strategies to inhibit apoptosis rational design of polymerase inhibitors as antiviral drugs herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro anchimerically activatable antiviral protides rabies virus assembly and budding the escrt machinery: new roles at new holes global, in vivo, and site-specific phosphorylation dynamics in signaling networks determination of phosphate as aggregates of ion associates by light-scattering detection and application to flow injection real-time fluorescence assays to monitor duplex unwinding and atpase activities of helicases nuclear pore complex is able to transport macromolecules with diameters of about 39 nm nipah virus c protein recruits tsg101 to promote the efficient release of virus in an escrt-dependent pathway evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors the multiple faces of caveolae herpesvirus capsid association with the nuclear pore complex and viral dna release involve the nucleoporin can/nup214 and the capsid protein pul25 the hepatitis c virus life cycle as a target for new antiviral therapies inhibition of dengue virus replication by novel inhibitors of rna-dependent rna polymerase and protease activities prevention of hepatitis b virus infection in vivo by entry inhibitors derived from the large envelope protein structural insights into rna synthesis by the influenza virus transcription-replication machine alphavirus polymerase and rna replication an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy isoform-selective assays for sphingosine kinase activity human occludin is a hepatitis c virus entry factor required for infection of mouse cells comparative clinical pharmacokinetics and pharmacodynamics of hiv-1 integrase strand transfer inhibitors late stages of the influenza a virus replication cyclea tight interplay between virus and host identification of pololike kinases as potential novel drug targets for influenza a virus principles of virus structural organization critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly retroviral capsid assembly: a role for the ca dimer in initiation nuclear import of hepatitis b virus capsids and release of the viral genome a chiral pentagonal polyhedral framework for characterizing virus capsid structures ribose 2'-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n-7 methylation by the large polymerase protein viral avoidance and exploitation of the ubiquitin system a cell-based assay for rna synthesis by the hcv polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by ns5a reverse transcription mechanically initiates hiv-1 capsid disassembly macropinocytosis is the entry mechanism of amphotropic murine leukemia virus west nile virus 5′-cap structure is formed by sequential guanine n-7 and ribose 2′-o methylations by nonstructural protein 5 antiviral drugs for viruses other than human immunodeficiency virus flavivirus structural heterogeneity: implications for cell entry melting of duplex dna in the absence of atp by ns3 helicase domain through specific interaction with a single-strand/ double-strand junction intracellular vesicle acidification promotes maturation of infectious poliovirus particles integration of murine leukemia virus dna depends on mitosis the role of electron microscopy in studying the continuum of changes in membranous structures during poliovirus infection specific in vitro cleavage of mason-pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection hiv-1 protease-induced apoptosis conditions resulting in formation of properly assembled retroviral capsids within inclusion bodies of escherichia coli analysis of mason-pfizer monkey virus gag domains required for capsid assembly in bacteria: role of the n-terminal proline residue of ca in directing particle shape cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes comparison of the luminescent adp-glo assay to a standard radiometric assay for measurement of protein kinase activity involvement of herpes simplex virus type 1 ul13 protein kinase in induction of socs genes, the negative regulators of cytokine signaling europium tetracycline as a luminescent probe for nucleoside phosphates and its application to the determination of kinase activity human herpesvirus 6b downregulates expression of activating ligands during lytic infection to escape elimination by natural killer cells single molecule measurement of the "speed limit" of dna polymerase growing functions of the escrt machinery in cell biology and viral replication production of hepatitis b virus particles in hep g2 cells transfected with cloned hepatitis b virus dna the human coronavirus 229e superfamily 1 helicase has rna and dna duplex-unwinding activities with 5′-to-3′ polarity the m-pmv cytoplasmic targeting-retention signal directs nascent gag polypeptides to a pericentriolar region of the cell discovering new medicines targeting helicases: challenges and recent progress in vitro uncoating of hiv-1 cores genome packaging of reovirus is mediated by the scaffolding property of the microtubule network directional spread of surface-associated retroviruses regulated by differential virus-cell interactions papain-like protease (plpro) inhibitory effects of cinnamic amides from < i > tribulus terrestris < /i > fruits immobilized metal ion affinity-based fluorescence polarization (imap): advances in kinase screening targeting zoonotic viruses: structure-based inhibition of the 3c-like protease from bat coronavirus hku4 -the likely reservoir host to the human coronavirus that causes middle east respiratory syndrome (mers) dna helicase activity of sv40 large tumor antigen in vitro disassembly of influenza a virus capsids by gradient centrifugation a peptide inhibitor of hiv-1 assembly in vitro cytoplasmic tails of bunyavirus gn glycoproteins-could they act as matrix protein surrogates an in vitro fluorescence screen to identify antivirals that disrupt hepatitis b virus capsid assembly specific recognition and accelerated uncoating of retroviral capsids by the trim5alpha restriction factor furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells selective sensing of tyrosine phosphorylation in peptides using terbium(iii) complexes yeast-based assays for the high-throughput screening of inhibitors of coronavirus rna cap guanine-n7-methyltransferase production of hepatitis b virus by a differentiated human hepatoma cell line after transfection with cloned circular hbv dna phosphorylation of the hiv-1 capsid by melk triggers uncoating to promote viral cdna synthesis viral and host control of cytomegalovirus maturation the a, b, cs of herpesvirus capsids real-time monitoring of rna helicase activity using fluorescence resonance energy transfer in vitro ubiquitin depletion and dominant-negative vps4 inhibit rhabdovirus budding without affecting alphavirus budding structural basis for the development of sars 3cl protease inhibitors from a peptide mimic to an aza-decaline scaffold different pathways leading to integrase inhibitors resistance synthesis and antiviral activity of novel isonucleoside analogs rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the ymdd region of reverse transcriptase ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl(pro)): implications for nsp5 regulation and the development of antivirals fluorescent sensing of pyrophosphate anion in synovial fluid based on dna-attached magnetic nanoparticles structural insights into the coupling of virion assembly and rotavirus replication an enzymatic virus-like particle assay for sensitive detection of virus entry early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection incorporation of spike and membrane glycoproteins into coronavirus virions distinct roles for nucleic acid in in vitro assembly of purified mason-pfizer monkey virus canc proteins identification and characterization of the rna helicase activity of japanese encephalitis virus ns3 protein virus maturation d-retrovirus morphogenetic switch driven by the targeting signal accessibility to tctex-1 of dynein retroviral oncogenes: a historical primer the entry inhibitor myrcludex-b efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis b virus advances in mass spectrometry based strategies to study receptor tyrosine kinases homogeneous highthroughput screening assays for hiv-1 integrase 3β-processing and strand transfer activities nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases a terbium (iii)-complex-based on-off fluorescent chemosensor for phosphate anions in aqueous solution and its application in molecular logic gates coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis establishment of a high-throughput assay to monitor influenza a virus rna transcription and replication structure of main protease from human coronavirus nl63: insights for wide spectrum anti-coronavirus drug design mathematical analysis of an hiv latent infection model including both virus-to-cell infection and cell-to-cell transmission measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy highthroughput microfluidic single-cell digital polymerase chain reaction structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus the e1 protein of bovine papilloma virus 1 is an atp-dependent dna helicase design of wide-spectrum inhibitors targeting coronavirus main proteases improving viral protease inhibitors to counter drug resistance studies of ebola virus glycoproteinmediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha decreased muscle insulin receptor kinase correlates with insulin resistance in normoglycemic pima indians adp-glo: a bioluminescent and homogeneous adp monitoring assay for kinases compact, programmable, and stable biofunctionalized upconversion nanoparticles prepared through peptide-mediated phase transfer for high-sensitive protease sensing and in vivo apoptosis imaging identification and characterization of a ribose 2′-o-methyltransferase encoded by the ronivirus branch of nidovirales fret-based biosensors for protein kinases: illuminating the kinome phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs cell-based high-throughput screening assay identifies 2′,2′-difluoro-2′-deoxycytidine gemcitabine as a potential antipoliovirus agent structure of the main protease from a global infectious human coronavirus, hcov-hku1 molecular basis for specific viral rna recognition and 2′-oribose methylation by the dengue virus nonstructural protein 5 (ns5) structure and function of the zika virus full-length ns5 protein robust hepatitis c virus infection in vitro inhibitors of sars-cov entryidentification using an internally-controlled dual envelope pseudovirion assay a new colorimetric strategy for monitoring caspase 3 activity by hrp-mimicking dnazyme-peptide conjugates protease inhibitors targeting coronavirus and filovirus entry the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles investigations on dna intercalation and surface binding by sybr green i, its structure determination and methodological implications miniaturization of absorbance assays using the fluorescent properties of white microplates characterization of a novel dna polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes this work was supported by ga čr (cz) ga17-25602s to mr, ga17-24281s to tr and the ministry of education, youth and sport of the czech republic(oppc cz.2.16/3.1.00/24503), through its "national program of sustainability i" npu lo 1601. key: cord-267867-q52nvn0n authors: chevalier, christophe; saulnier, aure; benureau, yann; fléchet, dorian; delgrange, david; colbère-garapin, florence; wychowski, czeslaw; martin, annette title: inhibition of hepatitis c virus infection in cell culture by small interfering rnas date: 2016-12-14 journal: mol ther doi: 10.1038/sj.mt.6300186 sha: doc_id: 267867 cord_uid: q52nvn0n hepatitis c virus (hcv) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. in this study, we investigated rna interference (rnai), a specific gene silencing process mediated by small interfering rna (sirna) duplexes, as an antiviral strategy against hcv. synthetic sirnas were designed to target conserved sequences of the hcv 5′ nontranslated region (ntr) located in a functional, stem–loop structured domain of the hcv internal ribosome entry site (ires), which is crucial for initiation of polyprotein translation. several sirnas dramatically reduced or even abrogated the replication of selectable subgenomic hcv replicons upon cotransfection of human hepatoma cells with viral target and sirnas, or upon transfection of cells supporting autonomous replication of hcv replicon with sirnas. importantly, three sirnas also proved capable of strongly inhibiting virus production in cell culture. one sirna, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. these results indicate that the hcv life cycle can be efficiently blocked by using properly-designed sirnas that target functionally important, highly conserved sequences of the hcv ires. this finding offers a novel approach towards developing ires-based antiviral treatment for chronic hcv infections. hepatitis c virus (hcv) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. in this study, we investigated rna interference (rnai), a specific gene silencing process mediated by small interfering rna (sirna) duplexes, as an antiviral strategy against hcv. synthetic sirnas were designed to target conserved sequences of the hcv 5 nontranslated region (ntr) located in a functional, stem-loop structured domain of the hcv internal ribosome entry site (ires), which is crucial for initiation of polyprotein translation. several sirnas dramatically reduced or even abrogated the replication of selectable subgenomic hcv replicons upon cotransfection of human hepatoma cells with viral target and sirnas, or upon transfection of cells supporting autonomous replication of hcv replicon with sirnas. importantly, three sirnas also proved capable of strongly inhibiting virus production in cell culture. one sirna, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. these results indicate that the hcv life cycle can be efficiently blocked by using properly-designed sirnas that target functionally important, highly conserved sequences of the hcv ires. this finding offers a novel approach towards developing iresbased antiviral treatment for chronic hcv infections. hepatitis c virus (hcv) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis, and, potentially, liver cirrhosis and hepatocellular carcinoma at later stages. chronic hcv infection affects 2.2% of the world's population and is known to be the leading factor necessitating liver transplantation in patients in developed countries. no vaccine is available for hcv and the current treatment, which consists of administering pegylated interferon α and ribavirin, has limited efficacy against certain hcv genotypes, and also produces significant adverse effects (reviewed in ref. 1) . the development of alternative, specific therapies for chronic hcv infection is therefore a major public health objective. hcv, a member of the flaviviridae family, contains a singlestranded positive-sense rna genome that encodes a unique precursor polyprotein; this polyprotein is further processed into structural proteins and nonstructural proteins, thereby ensuring genome replication. the long open reading frame is flanked by 5 and 3 nontranslated regions (ntrs) that are highly conserved among the majority of hcv genotypes and contain elements that are essential for genome replication (reviewed in ref. 1 ). in addition, hcv 5 ntr contains a highly structured element, namely, an internal ribosome entry site (ires), which is essential for the initiation of hcv polyprotein translation. 2 anti-hcv drug development has been hampered both by the lack of efficient cell culture systems that support virus replication and by the unavailability of accessible animal models. hcv subgenomic rna replicon systems 3 have permitted the assaying of the inhibitory effect of antiviral candidates on hcv genome replication in vitro in human hepatoma cells. the recent development of stable cell culture systems permitting robust production of infectious hcv particles in vitro, based on the jfh-1 strain of hcv genotype 2a, 4 will facilitate the investigation and testing of new antiviral strategies. in addition, alternative animal models that have recently been developed seem to show promise in evaluating candidate antiviral therapeutics. these animal models include transgenic mice engrafted with human hepatocytes, 5 as well as nonendangered primate species (tamarins, marmosets) infected by gb virus b, a hepatotropic virus that is closely related to hcv, 6, 7 or by gb virus b derivatives containing functional hcv sequences. 8 among possible therapeutic strategies, rna interference (rnai) is an attractive path to explore. first described as a natural defense mechanism against plant viruses, rnai was subsequently shown to be a universal phenomenon of post-transcriptional gene silencing, which is initiated by double-stranded rna and leads to specific degradation of homologous rnas. this process involves the generation of 21-nucleotide small interfering rnas (sirnas) which, in association with a multiprotein complex named "rna induced silencing complex", are used as guides to target specific rna substrates by watson-crick base-pairing. such sirnas, when introduced directly into mammalian cells or expressed from viral vectors, can lead to the degradation of targeted sequences (reviewed in ref. 9 ). during the past few years, rnai has been extensively investigated as an alternative specific therapy to treat cancers, genetic diseases, and infections by various human pathogens of medical importance, using both in vitro and in vivo model systems (reviewed in refs. 10, 11) . rna viruses, in particular viruses with liver tropism such as hcv, are ideal candidates for nucleic acid-based therapies that have been shown to efficiently target the liver (reviewed in ref. 12) . recently, various studies have reported variable inhibitory effects of hcv-specific synthetic sir-nas or small hairpin rnas (shrnas) expressed from viral vectors, that target sequences encoding various hcv nonstructural proteins as well as sequences within the 5 ntr. [13] [14] [15] [16] [17] [18] [19] these studies all relied on the use of hcv subgenomic or genomic replicon models in cell culture. in this study we designed sirnas targeting highly conserved sequences within the hcv 5 ntr. the efficiency of these hcvspecific sirnas was first evaluated using previously described self-replicating hcv rnas. 20, 21 this allowed us to select four sir-nas that abrogated or substantially reduced genome replication. by using these sirnas in recently described cell culture systems that support the production of genotype 2a jfh-1 4 in order to investigate the potency of virus-specific sirnas in inhibiting hcv infection, we selected seven sirnas that target conserved sequences of the pivotal domain (domain iii) of the hcv ires within the 5 ntr 2 (figure 1a) . this domain is particularly important for initiation of polyprotein translation, as it directly contacts 40s ribosomal subunits 22 and binds translation initiation factor 3, eif3. 23 domain iii has also been reported to be involved to some extent in genome replication. 24 sirnas were designed essentially according to previously described criteria; in particular, whenever possible, the asymmetric thermostability of sirna duplexes was respected. 25, 26 as a messenger, hcv positive strand rna genome is an ideal target for sir-nas, but it harbors strong secondary and tertiary structures, in particular within the ires. this makes it difficult to design optimal sirnas according to the criteria referred to earlier. on the other hand, the high nucleotide conservation of this region the footprints of the seven hcv-specific sirnas selected to target the internal ribosome entry site (ires) are represented on the schematic structure of domains iii-iv of the 5 nontranslated region (ntr) from the h77 strain of hcv genotype 1a. 2 sirna si240 targets a viral sequence that contains a nucleotide change in hcv genotype 1a and 1b sequences, as indicated. (b) the two replicons used in this study, ntat2aneo 21 and nneo 20 with 5 ntrs derived from hcv genotype 1a and 1b strains, respectively, are schematically represented, with cell culture adaptive mutations (s2005i in ns5a and r2889g in ns5b, respectively) indicated by arrowheads. both replicons encode neomycine phosphotransferase (neo) c-terminally fused to either human immunodeficiency virus tat protein followed by foot-and-mouth disease virus autocatalytically cleaved 2a protein (ntat2aneo), or to a few amino acid residues from core protein ( c in nneo). permitted the identification of sirnas that were homologous to several hcv genotypes ( table 1) . selected sirnas were named according to the nucleotide position within the genome of the h77 strain of hcv genotype 1a 27 that corresponds to the first 5 nucleotide targeted by the sirna antisense strand: si205, si214, si240-1a, si240-1b, si244, si284, si313 ( table 1) . sirna si313 targets the 3 end of domain iii and 5 end of domain iv involved in a pseudo-knot located upstream from the initiator codon (figure 1a) . the sequences of all sirnas, with the exception of si240, matched both hcv genotype sequences 1a and 1b that were present in the subgenomic replicons used in the first part of the study (figure 1b) . two sirnas si240 (si240-1a and si240-1b) were synthesized with sequences homologous to genotypes 1a and 1b, respectively. when targeted to genotype 1b 5 ntr, si240-1a formed a g:u wobble base-pairing at position 18 of the sirna antisense strand, whereas si240-1b formed an a:c mismatch with the genotype 1a replicon (figure 1c ). the g:u non-canonical base-pairing is known to be non-disruptive in double-stranded rna structures and both g:u wobble basepairing and a:c mismatch, if present at certain positions of the sirna, were recently reported to be well tolerated for sirnamediated gene silencing. [28] [29] [30] [31] therefore, we proceeded to analyze the effects of si240-1a and si240-1b on both homologous and heterologous replicon targets. finally, as a non-specific, negative control in these experiments, we used an irrelevant sirna, referred to as siirr ( table 1) , that had previously been used for targeting a sequence within the 5 ntr of a poliovirus strain, 30 and that did not share homology with the hcv sequence. first we evaluated whether selected sirnas were able to inhibit the replication of an hcv subgenomic replicon (ntat2aneo) in cell culture. we did this by using a previously described system that allowed simple monitoring of rna replication by measurement of an enzymatic activity, namely, secreted alkaline phosphatase (seap) activity 21 (see materials and methods and figure 1b) . all hcv sequences of ntat2aneo replicon are derived from the n strain of genotype 1b, with the exception of the 5 ntr, which is derived from the h77 strain of genotype 1a. following coelectroporation of en5-3 cells with 5 μg of in vitro-transcribed ntat2aneo rna and 2 μg of each synthetic sirna, the culture supernatant was replaced daily with fresh medium during the first 4 days after transfection and then on day 7, and seap activity was measured in all culture supernatants collected. during the first 2 days, seap signals essentially reflected translation of the input rnas, since seap levels were roughly similar in cells transfected with ntat2aneo replicon and those transfected with a replication-defective rna encoding an inactive ns5b rna polymerase deleted in its active site (δgdd, see materials and methods). rna replication was then clearly detected between days 3 and 7 after transfection (figure 2a , compare seap levels in cells transfected with ntat2aneo and δgdd). cotransfection of the irrelevant sirna (siirr) did not significantly affect ntat2aneo replication (figure 2a) . hcvspecific sirnas si214 and si284 showed only transient and nil effect on hcv replication, respectively, and seap expression levels on day 7 after transfection were similar to those induced by transfection of ntat2aneo alone (figure 2a) . these two sirnas were therefore not further utilized in the course of our study. in sharp contrast, sirnas si205, si240-1a, si240-1b, si244, and si313 proved capable of abolishing hcv replication in this system, yielding seap levels as low as those obtained with replication-defective rna δgdd. in the presence of these sirnas, seap levels were even slightly lower at days 1, 2, and 3 after transfection than those obtained with δgdd, as a result of sirna-mediated degradation of input rnas, rendering them unavailable for translation (figure 2a) . the inhibitory effect of these five sirnas on hcv replication was shown to be prolonged to 14 days after transfection (data not shown). we next monitored dose-response effects of each of the five selected sirnas using decreasing sirna doses in the range of 2,000-4 ng (67-0.13 nmol/l per μg replicon) in coelectroporation experiments with 5 μg of ntat2aneo replicon (figure 2b ). from these experiments, inhibition doses 50 were calculated for each sirna (figure 2b) . the sirnas si240-1a and si244 proved to be the most efficient inhibitors of hcv rna replication with inhibition doses 50 of 19 ng (0.64 nmol/l) and 33 ng (1.11 nmol/l), respectively, and all sirnas exhibited inhibition doses 50 within the 19-141 ng range. the inhibition dose 50 of si240-1b (127 ng) was shown to be substantially higher than that of si240-1a (19 ng), in agreement with the fact that si240-1b is not fully homologous to ntat2aneo 5 ntr (genotype 1a). this demonstrates the sequence-specificity of these sirnas. we also found that the co-administration of two sirnas targeting nonoverlapping hcv sequences, in suboptimal doses, resulted in additive inhibitory effects (data not shown). this might prove useful in future studies in which a strategy based on combined sirnas is sought to be developed in order to prevent the occurrence of escape mutations. the five sirnas (si205, si240-1a, si240-1b, si244, and si313) that specifically target domain iii of the hcv ires and strongly inhibit subgenomic rna replicon replication when used in the nanomolar range were retained in the remaining part of the study. next, in order to determine whether sirnas are capable of curing replicon-containing cells, we investigated whether hcv replicon rna was eliminated in sirna-treated cells in which seap activities were at basal level. total rna was extracted from cells cotransfected with replicon and sirna on day 10 after transfection and analyzed for hcv rna. this was done by semi-quantitative reverse transcription-polymerase chain reaction (rt-pcr) using an hcv primer pair that allows amplification of a 1630 base-pair fragment spanning the ns3 coding region, as well as a primer pair that allows the detection of a housekeeping messenger rna (glyceraldehyde-3-phosphate dehydrogenase) for the purpose of rna quantity normalization (figure 2c ). hcv rna was readily detected in similar abundance in cells transfected with ntat2aneo or cotransfected with ntat2aneo and siirr (figure 2c , lanes 4, 5). very low levels of residual transfected δgdd rna molecules could be occasionally detected under these experimental conditions (figure 2c, lane 3) . in duplicate experiments, using cells cotransfected with ntat2aneo and si240-1a, si244, or si313, viral rna was either not detected or detected at very low levels, comparable to the results obtained with δgdd rna (figure 2c , lanes 8-13). hcv rna was consistently detected in cells cotransfected with ntat2aneo and si205 (figure 2c, lanes 6, 7) , but at levels lower than in cells cotransfected with ntat2aneo and siirr. these results correlated well with inhibition levels of reporter seap activity, and confirmed that hcv rna replication is strongly inhibited, if not abolished, in cells treated with 2 μg of the three most efficient sirnas (si240-1a, si244, and si313). in order to work with a cell culture system mimicking an established persistent infection, we analyzed the inhibitory capacity of sirnas in en5-3 cells that stably contain and continuously replicate ntat2aneo replicon. the ongoing replication of ntat2aneo replicon in these cells was reflected by seap levels that were more than tenfold higher than in transient experiments after hcv replicon electroporation. two micrograms of each sirna were electroporated into these cells, and their effect on hcv rna replication was determined at different timepoints. data obtained at day 7 after transfection, corresponding to cumulative seap levels secreted between days 4 and 7, are represented in figure 3 . the seap activity generated by the hcv replicon in the presence of either of the homologous sirnas was reduced by 50% or more relative to that of ntat2aneo in mock-transfected cells. when a higher dose (5 μg) of sirnas was used, seap activity was reduced by 70-93% (data not shown). the sirna-mediated inhibitory effect was not as dramatic as in cotransfection experiments, probably due, in part, to limited transfection efficiency (~70%, data not shown), thereby permitting ongoing rna replication in cells that did not receive sirna in the transfected culture. in addition, viral rnas undergoing replication within replication complexes may be less accessible than transfected viral rnas to sirnas. nonetheless, we demonstrated strong, dose-dependent inhibitory potency of hcv domain iii-specific sirnas in cells supporting continuous hcv rna replication. we next addressed the question of the efficacy of sirnas in inhibiting hcv genome replication in cells placed under selective pressure, and examined whether there was any hcv escape from specific sirna treatment. in these experiments, we used the hcv replicon-free subclone 2-3c of huh7 cells, 32 and either ntat2aneo or nneo replicon. in contrast to ntat2aneo, nneo replicon (see figure 1b and ref. 20) contains a 5 ntr derived from the n strain of genotype 1b and encodes neomycine phosphotransferase gene in the first cistron. 2-3c cells were coelectroporated with either ntat2aneo or nneo replicon and each sirna, then placed under g418 selective pressure. g418resistant cell clones were counted on day 21 after transfection. data from two or three experiments carried out with each replicon were pooled for sirnas homologous to both replicons (si205, si244, si313), and the mean of these data is shown in figure 4 . a mean of 34,000 ± 23,800 resistant cell clones was obtained after transfection of hcv replicon alone. the data are represented with respect to 10,000 g418-resistant clones in each experiment. treatment with each of the hcv-specific sirnas resulted in a substantial reduction in the number of g418-resistant cell clones (figure 4) . sirna si205 reduced the formation of g418-resistant cell clones by ~2 logs, whereas si244 and si313 reduced cell clone formation by 3 logs or more (figure 4a) . this is in good correlation with results obtained in the seap reporter system. both si240-1a and si240-1b were more efficient on their homologous target than on heterologous targets (figure 4b) . the nature and the position of the mismatch between si240 and its target (g:u and a:c for si240-1a on hcv genotype 1b and si240-1b on genotype 1a, respectively, at nucleotide 18 of the sirna antisense strand) are reasonably well tolerated by the rnai machinery, resulting in ~2 log reduction in g418-resistant cell clone formation, an inhibition that was, however, 1-1.5 log less efficient than on homologous targets. these experiments confirmed, as already described, 28, 29, 31 that perfect base-pairing between sirnas and targeted messenger rna is required for most effective silencing. to look for potential emergence of sirna escape mutants, several g418-resistant cell clones, obtained after a single treatment with each sirna, were isolated and independently amplified. the 5 ntrs of the hcv replicon rnas recovered from 3 to 13 cell clones for each sirna were reverse-transcribed and pcr-amplified. the resulting pcr products were subjected to sequencing. no nucleotide substitution was found within or in the vicinity of the sirna-targeted region of the hcv replicon rnas. these data suggest that either a single sirna treatment may not be sufficient to select for hcv replicon escape mutants in this system, or that replication competence does not tolerate nucleotide variation in this genomic region. we sought to determine whether the sirnas si240-1a, si244, and si313, shown to be the most efficient in inhibiting the replication of hcv subgenomic replicons both transiently and under selective pressure, are also capable of efficiently blocking virus infection in cell culture. we utilized the jfh-1 strain of hcv genotype 2a that was shown to infect huh7 and huh7-derived cell lines and produce infectious particles. 4 this in vitro infectious system therefore recapitulates the entire hcv life cycle. sequences of si240-1a and si313 were perfectly homologous to the 5 ntr sequence of this genotype 2a strain. in contrast, si244 was shown to hybridize to the jfh-1 5 ntr with a c:a mismatch at the third position of the sirna antisense strand (see figure 1c) . such a mismatch at the 5 end of the antisense strand is expected to hamper rnai. 29, 33 for this reason, we also used an engineered chimeric virus that contains 5 ntr and core-coding sequences derived from genotype 1a within the backbone of the jfh-1 genome (jfh-1/c(+) 6 (figure 5a) . in contrast, hcv-specific sirna si313 considerably reduced the number of cells infected with either jfh-1 or the chimeric virus (figure 5a) . similarly, si244 efficiently inhibited infection with the 1a/2a chimeric virus (figure 5a, left) , but had only a weak effect, if any, on jfh-1 infection (figure 5a , right), consistent with the existing mismatch between si244 and genotype 2a 5 ntr (see figure 1c) . interestingly, si240-1a sirna also appeared to have a higher inhibitory effect on the infection with the chimeric virus (figure 5a , left) than with jfh-1 (figure 5a, right) , in spite of perfect homology with the 5 ntr sequences of both viruses. we hypothesized that these differential effects of si240-1a might be linked to delayed replication kinetics of the chimeric 1a/2a virus, as compared to jfh-1 (d. delgrange, a. pillez, l. cocquerel, g. paranhos-baccala, y. rouillé, j. dubuisson et al., unpublished results), a phenotype that may impact on target/sirna ratios at early time-points following infection. the effects of the three sirnas on both viruses were also monitored by western blot analysis of e2 expression in infected cells (figure 5b) . the data obtained confirmed that si313 is the most efficient sirna, causing complete inhibition of e2 expression in cells infected with either jfh-1 or jfh-1/ c(+)6-1a. in order to verify whether virus production from sirnatreated cells is reduced accordingly, viral particles were titrated in supernatants from cells transfected with each sirna and infected with either jfh-1 or the chimeric virus, both by realtime quantitative rt-pcr (genome equivalents, figure 5c ) and by determination of infectious focus-forming units ( table 2) . for each of the sirnas, quantification of virus production in sirnatreated cultures (figure 5c, table 2 ) was in good agreement with core and e2 intracellular expression levels (figure 5a and b) . in particular, supernatants from si313-treated, hcv-infected cells exhibited a >92% reduction in genome equivalents/ml titer and a >96% reduction in focus-forming units/ml infectious titer, as compared to mock-treated hcv-infected cells. taken together, these data demonstrate that hcv 5 ntr-specific sirnas are capable of strongly inhibiting virus production in cell culture. in exploring rnai as a potential new therapeutic approach against hcv infection, one of the reasons for our choice to target domain iii of the hcv 5 ntr was that domain iii contains well-conserved nucleotide sequences across all hcv genotypes ( table 1) . 34 the high degree of conservation seen in these sequences may be required for preserving virus function, since this 5 ntr domain controls the initiation of polyprotein translation and modulates rna replication. 23, 24 recent nuclear magnetic resonance and electron cryomicroscopy studies of domain iii 22, 35, 36 have helped identify the structure-function relationships of the various subdomains of domain iii. the results of these studies suggest that subdomains iiia/c and iiid, as well as subdomain iiif that forms a pseudoknot structure (figure 1a) , directly contact the 40s subunit body and act synergistically for the proper positioning of the aug codon. in the present study, si240 and si244 (that hybridize to subdomain iiid) and si313 (that essentially hybridizes to the iiif pseudoknot), therefore target regions that are essential for ires structural integrity and functioning. these three sirnas proved able, at low doses in the nanomolar range, to substantially reduce or even abolish hcv subgenomic rna replication, as measured by reporter seap activities and g418-resistant cell clone formation (figures 2 and 4) , and resulted in the elimination of hcv replicons from treated cells (figure 2c) . importantly, we also demonstrated that these sirnas, particularly si313, considerably limit hcv infection in cell culture. we did this using two hcv strains that can be propagated in huh7-derived cells: (i) the jfh-1 strain of hcv genotype 2a, 4 and (ii) a chimeric derivative of jfh-1 carrying 5 ntr and core sequences of hcv genotype 1a (d. delgrange, a. pillez, l. cocquerel, g. paranhos-baccala, y. rouillé, j. dubuisson et al., unpublished results). our data contrast with those obtained by others who concluded that domains ii 17 and iii 17, 19 of hcv 5 ntr are relatively resistant to sirna, because there is reduced accessibility to these domains, given their association with different proteins and factors involved in translation. our data indicate that regions involved in complex secondary and tertiary structures should not be disregarded as targets for rnai. the data also underscore the importance of sirna design. 25, 26 in contrast, two of the sirnas tested in the present study (si214 and si284, targeting domains iiic and iiie, respectively) exhibited poor or only transient inhibitory effect on hcv rna replication (figure 2a) . interestingly, two other studies reported that sirnas having sequences identical to that of si284 showed relative discrepancies in their effects in cells supporting autonomous replication of hcv subgenomic replicon. 13, 19 these conflicting results may be explained by the different strategies used for sirna synthesis and/or delivery. in addition, the sensitivity of the replicon systems used may have an impact on the sirna efficiencies reported. we observed some differences in sirna efficiencies between: (i) transient replication systems in which viral target rna and sirna were co-introduced into cells (figures 2 and 4) , and (ii) stable replication systems in which sirna was introduced in cells supporting ongoing replication of the viral target, whether dealing with a subgenomic replicon (figure 3 ) or genomelength infectious rna (figure 5, table 2 ). two micrograms of sirna did not inhibit viral rna replication in the stable subgenomic replicon system as efficiently as in cotransfection experiments (compare figures 2 and 4) . in addition, si240-1a and si244 caused elimination of viral rna upon cotransfection with subgenomic replicon (figure 2a) , whereas they substantially reduced, but did not abolish the production of virus particles ( figure 5, table 2 ). this was in spite of the fact that the molar ratio of sirna to targeted hcv rna was higher in cells stably containing hcv replicons or infected with virus than in cells cotransfected with replicon and sirna. in another study, high doses (4,000 pmol) of sirnas proved necessary in order to strongly inhibit hcv rna replication in subgenomic replicon-containing cells. 18 we speculate that viral rna templates engaged in the replicase complex and nascent rnas are probably not as accessible to the rnai machinery as transfected viral rnas are. our data can also be explained in part by the fact that a proportion of cells in the culture were infected or supported ongoing viral rna replication but did not receive sirna, given that the efficiency of electroporation is ~70% in en5-3 and 2-3c cells (data not shown). the specificity of hcv sirnas was demontrated by studying their dose-effect responses (figure 2b) , as well as by using si240 to target the replicon 5 ntr from two hcv genotypes (1a and 1b; figures 2-4 ) and si244 to target the 5 ntr of genotype 1a or 2a virus strains ( figure 5, table 2 ). when used with heterologous targets, the sirnas si240-1a and si240-1b exhibited a noncanonical wobble base-pairing and a mismatch at the 3 end (position 18) of the sirna antisense strand, respectively (figure 1c) . for both 1a and 1b replicons, perfect base-pairing was preferred for maximum rnai efficacy, but a g:u wobble and an a:c mismatch were both tolerated (figure 4) . this is consistent with the fact that wobble base-pairing is known to be well tolerated in double-stranded rna helices, providing stability similar to a watson-crick base-pairing. in agreement with our data, wobble base-pairs, especially when located at the 3 end of the sirna antisense strand, were recently suggested to have no disruptive effect on rnai. 28, 29, 33 more surprisingly, but also consistent with our data, it was reported that an a:c mismatch was not deleterious in double-stranded rna structures. 29 contrasting with these tolerated mismatches between sirna and viral target rna, we found that a c:a mismatch at the 5 end of the sirna antisense strand strongly reduces the inhibitory effect of si244 on jfh-1 infection in cell culture ( figure 5, table 2) , and this is consistent with the rnai mechanism. 29, 37 taken together, these data provide strong evidence against the involvement of a spurious interferonmediated mechanism of inhibition in the suppression of viral replication we observed. the sequence of rna viruses, such as hcv, is known to evolve continuously, resulting in the production of many quasispecies, because of the high error rate of rna-dependent rna polymerases with no proof-reading activity. this property allows rna viruses to escape rapidly to a selective pressure such as antiviral treatments, when sequence changes are compatible with virus functions. this holds true for anti-protease and antipolymerase compounds that are currently under development for hcv treatment. 38, 39 however, we did not observe, after a single sirna treatment, the emergence of escape mutations within sirna-targeted genomic regions in rna extracted from several g418-resistant cell clones. in other studies, sirna-resistant viral mutants harboring single nucleotide substitutions or deletions within or at the vicinity of the sirna-targeted sequence were reported to have emerged in cells expressing constitutively an shrna (in the case of human immunodeficiency virus infections), 40, 41 and after repeated treatments with an sirna targeting the polymerase coding sequence (in the case of hcv subgenomic replicons). 16 the lack of escape mutations observed in our study could either be due to the fact that a single sirna was used in the treatment, or to the fact that sirnas target highly conserved, functionally important genomic sequences, in which substitutions would not be compatible with translation/replication competence. additional studies will be needed in order to determine which of these hypotheses holds true. from the point of view of evaluating the potency of sirnabased antiviral strategies to eradicate persistent infections, it was recently shown that sirnas may be used to cure in vitro persistent infections caused by rna viruses such as poliovirus or lymphocytic choriomeningitis virus. 30, 42 for assaying such a therapeutic strategy in animal models, synthetic sirnas need to be further chemically stabilized and formulated to be efficiently delivered. successful utilization of stabilized, lipid-encapsulated sirnas was reported in a mouse model of hepatitis b virus replication. 43 in addition, organ or cell-type specific delivery of sirnas has been achieved through sirna binding to cholesterol 44 or to antibodies directed against cell surface antigens. 45 synthetic shrnas also showed a more prolonged inhibitory effect in murine liver than sirnas did. this was demonstrated upon co-delivery of shrna or sirna targeting domain iv of the hcv ires and plasmid dna encoding a reporter protein placed under the translational control of the hcv ires. 46 alternatively, optimization of sirna delivery may rely on the use of viral vectors that allow continuous synthesis of shrnas in cells, thereby leading to sustained rnai. 31, 47 it is crucial, however, to control intracellular shrna production levels, since it has recently been reported that constant, high expression of shrnas in the liver could be lethal in mouse models. 48 nevertherless, controlled doses of sirna or expression of shrna have already been shown to be efficient and safe in several animal models of viral infection, including in a mouse model of hepatitis b virus infection, 47 a rhesus macaque model of severe acute respiratory syndrome-associated coronavirus infection, 49 and mouse models of west nile virus and japanese encephalitis virus infections. 31 in the present study, we have identified promising anti-hcv sirnas (si313 and, to a lesser extent, si244 and si240) that target highly conserved sequences in domain iii of the 5 ntr and efficiently silence hcv infection in cell culture. it has now become possible to monitor the effect of these sirnas in vivo using a tamarin/marmoset primate model of infection with a chimeric gb virus b-containing hcv domain iii of the hcv ires, that we have previously described. 8 during revision of this manuscript, kanda et al. 50 reported that delivery of an hcv 5 ntr-specific shrna to hepatoma cell lines infected by hcv resulted in the reduction of viral production. hepatoma cell lines, 2-3c, 32 en5-3, 21 and huap are derived from the hepatocarcinoma cell line huh7, and were cultured in dulbecco's modified eagle medium (invitrogen, cergy-pontoise, france) supplemented with 10% fetal calf serum, 100 u/ml penicillin and 100 μg/ml streptomycin. en5-3 cells stably express seap under the control of the human immunodeficiency virus long terminal repeat promoter, 21 and were cultured in the presence of 2 μg/ml blasticidin (invivogen, toulouse, france). en5-3 cells supporting autonomous replication of ntat2aneo hcv replicon were cultured in the presence of 2 μg/ml blasticidin and 0.5 mg/ml geneticin (g418, invitrogen, cergy-pontoise, france). genotype 2a jfh-1 and chimeric jfh-1/c(+)6-1a virus stocks were generated by transfection of huap cells with corresponding in vitro transcribed genomic rnas. plasmid pjfh-1 containing the genome-length complementary dna (cdna) of the jfh-1 isolate of hcv genotype 2a (genbank accession no. ab047639, ref. 4) was generously provided by t. wakita. plasmid pjfh-1/c(+)6-1a was derived from pjfh-1 by substituting nucleotides 154-341 of the 5 ntr, as well as the capsid coding sequence by corresponding sequences of the h77 strain of genotype 1a (d. delgrange, a. pillez, l. cocquerel, g. paranhos-baccala, y. rouillé, j. dubuisson et al., will be described elsewhere). plasmids pjfh-1 and pjfh-1/c(+)6-1a were linearized with xbai and treated with mung bean nuclease prior to in vitro transcription using megascript t7 kit (ambion, courtaboeuf, france). huap cells were electroporated with in vitro transcribed rna, as previously described, 51 and supernatants collected 8-10 days after transfection were used to re-infect naïve huap cells. supernatants collected at 10 days after infection were used as virus stocks and stored at 80 °c. design and synthesis of sirnas. sirnas targeting hcv ires were designed using previously described criteria. 25, 26 the sequence of the sense-strand of each sirna selected is shown in table 1 . sirnas are referred to by the nucleotide position within the genome of the h77 strain of hcv genotype 1a 27 that corresponds to the first nucleotide targeted by the sirna antisense strand. si240 was synthesized with a sequence homologous to hcv subtype 1a or 1b, i.e., containing a u (si240-1a) or a c (si240-1b) residue at position 18 of the sirna antisense strand. an irrelevant sirna, named siirr, targets the 5 ntr of the sabin strain of poliovirus type 3 30 and was used as negative control in this study. the sequences of all sirnas were compared with known genes by using basic local alignment search tool within the genbank database, and no significant homology to other genes was found. the two strands of each sirna were chemically synthesized at the institut pasteur ("plate-forme 7") and annealed at a final concentration of 500 pmol/μl, as previously described. 25 the quality and quantity of hybridized sirnas were analyzed by electrophoresis on nondenaturing 3% agarose gels. hcv subgenomic replicons and in vitro transcriptions. two dicistronic, subgenomic hcv replicons that encode a selectable reporter gene, neomycine phosphotransferase (neo) were used in this study (see figure 1 ). these replicon cdnas, kindly provided by s.m. lemon, are inserted downstream of the t7 rna polymerase promoter and have been previously described as pnneo/3-5b 20 and pntat2aneo. 21 we will refer to these replicons as nneo and ntat2aneo, respectively, in this study. nneo and ntat2aneo each carries a cell culture adaptive mutation, r2889g and s2005i, respectively. both replicon cdnas are derived from the cdna of the n strain of genotype 1b hcv with the exception of the 5 ntr sequence of the ntat2aneo, which is derived from the h77 strain of hcv genotype 1a. ntat2aneo rna encodes human immunodeficiency virus tat protein fused to the 2a protease of foot-and-mouth disease virus, followed by neo. 21 upon transfection of en5-3 cells with ntat2aneo replicon, the expression of tat-2a drives the synthesis of secreted seap. 21 cdnas bearing a 30-nucleotide deletion, including the three codons (gdd) of the active site of the rna polymerase ns5b within the backbone of nneo and ntat2aneo, 20, 21 and referred to as δgdd, were used as replicationdeficient rnas. plasmid dnas were linearized with xbai prior to in vitro transcription using megascript t7 kit. the dna template was removed by treatment with turbo dnase (ambion, courtaboeuf, france) and rnas were purified by phenol-chloroform extractions and precipitated with ethanol. the quality and quantity of replicon rnas were analyzed by electrophoresis on a non-denaturing 0.8% agarose gel and by optical density measurements. cell transfections. en5-3 or 2-3c cells (2 × 10 6 ) were electroporated with 5 μg replicon rna, or coelectroporated with 5 μg replicon rna and various doses of each sirna, in a 4 mm-gap width cuvette by applying one pulse at 240 v, 900 μf (easyject plus, equibio, kent, uk). cells were resuspended in complete medium immediately after the pulse and seeded at various concentrations. en5-3 cells supporting autonomous replication of ntat2aneo replicon were similarly transfected by electroporation with each sirna. en5-3 transfected cells (4 × 10 5 ) were seeded in 6-well plates and supernatants were collected at 1, 2, 3, 4, and 7 days after electroporation and replaced with fresh medium. seap activity was measured in supernatant aliquots with the phospha-light system (applied biosystems/tropix, courtaboeuf, france) using a luminescent substrate according to the manufacturer's recommendations. luminescent signals were read for 1 second using a lumat lb 9507 luminometer (berthold technologies, thoiry, france). to select for g418-resistant cell clones, variable fractions of 2-3c transfected cells were seeded in 100 mmdiameter dishes and supplemented with nneo-δgdd or ntat2aneo-δgdd transfected cells to adjust the final number of cells to 5 × 10 5 cells per dish. twenty-four to forty-eight hours following transfection, cells were placed under the selective pressure of 0.5 mg/ml geneticin (g418, invitrogen, cergy-pontoise, france) for 3 weeks, with the medium being replaced twice a week. g418-resistant cell clones supporting viral rna replication were fixed and stained with a 0.1% crystal violet solution, or selected and expanded under selective pressure for viral rna sequencing. viral rna sequencing. total rna was extracted from expanded sirnaresistant clones with trizol reagent (invitrogen, cergy-pontoise, france). viral rna was reverse transcribed and amplified with hcv specific primers designed to generate pcr products spanning nucleotides 100-451, using the superscript one-step rt-pcr kit with platinum taq (invitrogen, cergy-pontoise, france). sequencing reactions were carried out on uncloned rt-pcr products using big dye terminator version 1.1 kit (applied biosystems, courtaboeuf, france) and analyzed on a abi 3700 capillary dna sequencer (applied biosystems, courtaboeuf, france). en 5-3 transfected cells (2 × 10 5 ) were seeded in 6-well plates (duplicate wells per transfection) and cultured for 4 days, then trypsinized and passaged at a 1:5 dilution in new 6-well plates. on day 10 after transfection, total rna was extracted novel insights into hepatitis c virus replication and persistence internal ribosome entry site-mediated translation in hepatitis c virus replication replication of subgenomic hepatitis c virus rnas in a hepatoma cell line production of infectious hepatitis c virus in tissue culture from a cloned viral genome hepatitis c virus replication in mice with chimeric human livers comparison of tamarins and marmosets as hosts for gbv-b infections and the effect of immunosuppression on duration of viremia chronic hepatitis associated with gb virus b persistence in a tamarin after intrahepatic inoculation of synthetic viral rna a chimeric gb virus b with 5 nontranslated rna sequence from hepatitis c virus causes hepatitis in tamarins the silent revolution: rna interference as basic biology, research tool, and therapeutic silencing viruses by rna interference antiviral rnai therapy: emerging approaches for hitting a moving target therapeutic short hairpin rna expression in the liver: viral targets and vectors small interfering rna-mediated inhibition of hepatitis c virus replication in the human hepatoma cell line huh-7 interference of hepatitis c virus rna replication by short interfering rnas inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas hepatitis c virus replicons escape rna interference induced by a short interfering rna directed against the ns5b coding region alternative approaches for efficient inhibition of hepatitis c virus rna replication by small interfering rnas clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas suppression of hepatitis c virus replicon by rna interference directed against the ns3 and ns5b regions of the viral genome selectable subgenomic and genome-length dicistronic rnas derived from an infectious molecular clone of the hcv-n strain of hepatitis c virus replicate efficiently in cultured huh7 cells subgenomic hepatitis c virus replicons inducing expression of a secreted enzymatic reporter protein hepatitis c virus ires rna-induced changes in the conformation of the 40s ribosomal subunit mechanism of ribosome recruitment by hepatitis c ires rna sequences in the 5 nontranslated region of hepatitis c virus required for rna replication analysis of gene function in somatic mammalian cells using small interfering rnas rational sirna design for rna interference transcripts from a single full-length cdna clone of hepatitis c virus are infectious when directly transfected into the liver of a chimpanzee sirnas can function as mirnas a systematic analysis of the silencing effects of an active sirna at all single-nucleotide mismatched target sites complete cure of persistent virus infections by antiviral sirnas a single sirna suppresses fatal encephalitis induced by two different flaviviruses virus-host cell interactions during hepatitis c virus rna replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral rna synthesis risc is a 5 phosphomonoester-producing rna endonuclease genetic diversity and evolution of hepatitis c virus-15 years on structures of two rna domains essential for hepatitis c virus internal ribosome entry site function structure of the hepatitis c virus ires bound to the human 80s ribosome: remodeling of the hcv ires specificity of microrna target selection in translational repression anti-hcv therapies in chimeric scid-alb/upa mice parallel outcomes in human clinical application mutations conferring resistance to a hepatitis c virus (hcv) rna-dependent rna polymerase inhibitor alone or in combination with an hcv serine protease inhibitor in vitro human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome rna interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus potent and persistent in vivo anti-hbv activity of chemically modified sirnas therapeutic silencing of an endogenous gene by systemic administration of modified sirnas antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors small hairpin rnas efficiently inhibit hepatitis c ires-mediated gene expression in human tissue culture cells and a mouse model clearance of hepatitis b virus from the liver of transgenic mice by short hairpin rnas fatality in mice due to oversaturation of cellular microrna/short hairpin rna pathways using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque small interfering rna targeted to hepatitis c virus 5 nontranslated region exerts potent antiviral effect subcellular localization of hepatitis c virus structural proteins in a cell culture system that efficiently replicates the virus functional analysis of cell surface-expressed hepatitis c virus e2 glycoprotein courtaboeuf, france). five micrograms of total cellular rna were heatdenatured at 65 °c for 5 minutes and used as template for reverse transcription with 50 u of superscript ii reverse transcriptase (invitrogen, cergy-pontoise, france) and 250 ng of random hexanucleotide primers (roche, meylan, france) for 50 minutes at 42 °c. the resulting cdnas were treated with 2 u of rnaseh (invitrogen, cergy-pontoise, france) for 30 minutes at 37 °c, and purified using qiaquick pcr purification kit (qiagen, courtaboeuf, france). one fifth of the cdna was used for programming pcrs with either hcv-specific primers that allowed amplification of a fragment spanning nucleotides 3457-5085 of the n strain of hcv genotype 1b, or primers specific to cellular housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, so as to normalize for total rna content. pcrs were performed using platinum taq dna polymerase (invitrogen, cergy-pontoise, france) under the following cycling conditions: 1 cycle at 94 °c for 3 minutes, followed by 35 cycles of: (i) 30 seconds at 94 °c; (ii) 30 seconds at 55 °c; and (iii) 1.5 minutes at 72 °c. pcr products were analyzed by electrophoresis on 1% agarose gels. huap cells (4 × 10 6 ) were electroporated with 2 μg of sirna, and 5 × 10 4 electroporated cells were seeded on coverslips in 24-well plates and subsequently infected at 16 hours after transfection with jfh-1 or jfh-1/c(+)6-1a virus stocks at ~1 focus forming unit per cell. after 2 hours of incubation at 37 °c, virus inoculum was washed off and cells were fed with culture medium. at 42 hours after infection, cells were fixed and processed for core detection by indirect immunofluorescence, as previously described, 51 using anti-core monoclonal antibody acap27 (kindly provided by j.f. delagneau, bio-rad, marnes-la-coquette, france). cells were counterstained with hoechst dye to enable nuclei to be detected. for titration of infectious viral particles, 5 × 10 4 huap cells were infected with 1:10 dilutions of supernatants from cells transfected with sirnas and infected with hcv. the foci of infected cells were detected by immunofluorescence with anti-core monoclonal antibody at 72 hours after infection and counted to determine titers in focus forming units/ml. huap cells (4 × 10 6 ) seeded in 24-well plates were lyzed in 50 mmol/l tris-hcl (ph 7.5), 150 mmol/l nacl, 5 mmol/l edta, and 0.5% (vol/vol) igepal buffer containing a mixture of protease inhibitors (complete, roche, meylan, france) and processed for e2 or β-actin detection by immunoblot analysis as previously described, 51 using anti-e2 monoclonal antibody 3/11 52 or anti-β-actin monoclonal antibody (sigma-aldrich, saint quentin fallavier, france), respectively. rnas were isolated from cell culture supernatants using qiaamp viral rna kit (qiagen, courtaboeuf, france) and quantified by real-time quantitative rt-pcr using primer pair and probe targeting a sequence spanning nucleotides 130-290 within the hcv 5 ntr : fp 5 -cgggagagc catagtgg-3 ; rp 5 -agtaccacaaggcctttcg-3 ; probe 5 -fam-ctgcggaaccggtgagtacac-tamra-3 . assays were performed using taqman one-step rt-pcr master mix reagents kit and an abi prism 7700 sequence detector instrument (applied biosystems, courtaboeuf, france), according to the manufacturer's instructions. key: cord-284693-mgpxnnk0 authors: jothimani, dinesh; venugopal, radhika; vij, mukul; rela, mohamed title: post liver transplant recurrent and de novo viral infections date: 2020-09-26 journal: best pract res clin gastroenterol doi: 10.1016/j.bpg.2020.101689 sha: doc_id: 284693 cord_uid: mgpxnnk0 survival following liver transplantation has changed dramatically owing to improvement in surgical techniques, peri-operative care and optimal immunosuppressive therapy. post-liver transplant (lt) de novo or recurrent viral infection continues to cause major allograft dysfunction, leading to poor graft and patient survival in untreated patients. availability of highly effective antiviral drugs has significantly improved post-lt survival. patients transplanted for chronic hepatitis b infection should receive life-long nucleos(t)ide analogues, with or without hbig for effective viral control. patients with chronic hepatitis c should be commenced on directly acting antiviral (daa) drugs prior to transplantation. daa therapy for post-lt recurrent hepatitis c infection is associated with close to 100% sustained virological response (svr), irrespective of genotype. de novo chronic hepatitis e infection is an increasingly recognised cause of allograft dysfunction in lt recipients. untreated chronic hev infection of the graft may lead to liver fibrosis and allograft failure. similarly, cmv and ebv can reactivate leading to systemic illness following liver transplantation. with covid-19 pandemic, post-transplant patients are at risk of sars-co-v2 infection. majority of the lt recipients require hospitalisation, and the mortality in this population is around 20%. early recognition of allograft dysfunction and identification of viral aetiology is essential in the management of post-lt de novo or recurrent infections. optimising immunosuppression is an important step in reducing the severity of allograft damage in the treatment of post-transplant viral infections. viral clearance or control can be achieved by early initiation of high potency antiviral therapy. liver transplantation (lt) is the only curative therapy for patients with decompensated cirrhosis, with an one year and 5-year survival of around 90% and 70%, respectively 1 survival following olt has improved over the years from a 1 and 5 year survival of 70% and 50% to 90% and 70%, respectively. significant improvement in surgical techniques, peri-operative care and immunosuppression therapy has translated in to improved survival. however, in this review, we focus on the prevalence, natural history and clinical outcomes of hepatitis c (rhcv), hepatitis b (rhbv), hev and other viral infections such as cmv, hsv and ebv in lt recipients. in addition, given the current covid-19 pandemic, we discuss impact of sars-cov-2 in the liver allograft and their outcomes. recurrent hepatitis c (rhcv) infection of the liver allograft is universal in untreated patients transplanted for this indication. allograft colonisation by viral particles occurs at the time of portal reperfusion inducing liver damage as early as 72 hours post-liver transplantation. a study on hcv viral kinetics demonstrated an initial sharp decline in the rna levels within 24 hours of reperfusion followed by a rapid increase to nearly 20 fold higher than the pretransplant values during the first week 6 . untreated rhcv may progress with necroinflammation, leading to allograft injury and fibrosis. in a study to evaluate the natural course of 183 hcv liver transplanted patients using protocol liver biopsies, fibrosis score progressed from 1.2 to 2.2 over 10 year period. in addition, patients with severe fibrosis at 1 j o u r n a l p r e -p r o o f year due to rhcv was associated with poor survival. furthermore, donor age >33 years and hcv genotype 1 or 4 developed rapid fibrosis 7 . post-transplant immunosuppression accelerates rhcv related liver damage with fibrosis progression at the rate of 0.3%-0.8% per year leading to cirrhosis and graft loss in 20-40% of patients within 5 years 8, 9 . up to 40% patients with rhcv develop hepatic decompensation within a year of cirrhosis 10 . these patients follow an accelerated course with a three year survival of only 10% following hepatic decompensation unlike 60% in those with native cirrhotic liver. over all patients transplanted for hcv have a lower 3 and 5 year survival of 73% and 67%, respectively 11 . rarely (<10%), rhcv can present in a severe form, fibrosing cholestatic hepatitis (fch), characterized by bilirubin >6 mg/dl, alp and ggt >5x upper limit normal, high hcv rna typically occurring in the first 6 months post liver transplantation. liver histology characterised by severe cholestasis, hepatocyte ballooning spotty necrosis and kupffer cell hypertrophy. progressive fch is associated with severe allograft dysfunction leading to graft failure leading to death within 12 months. in summary, hcv lt recipients had worst long term survival until 5 years ago. hcv viral clearance described as sustained virological response (svr) defined as undetectable hcv rna at 24 weeks following antiviral therapy, but recently reduced to 12 weeks (svr12). svr has a major implication in disease progression in rhcv. similar to chronic hepatitis c in pre-transplant patients, achieving svr in post-transplant patients results in stability of the disease or even regression of fibrosis in 75% of patients 12 . whereas, failure to achieve svr results in worsening of fibrosis. unlike, in patients with chronic liver disease, fibrosis in rhcv progresses at faster pace. in patients untreated rhcv, fibrosis score ≥2 progress rapidly to cirrhosis and graft failure than patients with absent or mild fibrosis 13 . in a 10 year follow up italian study of 358 lt patients for hcv, patients who did not achieve svr had the worst 10-year survival 39.8% compared to 84.7% in those with svr 14 . a number of studies evaluated recipient, donor and virus related risk factors associated with rhcv. advanced recipient age, diabetes mellitus, severe liver disease (child pugh >10), il-28b polymorphism, high hcv rna >10 7 iu/ml, ischemic/reperfusion injury, cmv, donor age >65 years, cold ischaemic time over 8 hours and warm ischemia over 90 minutes, marginal graft, dcd donor, higher immunosuppression in particular high dose corticosteroids for acute cellular rejection, use of anti-thymocyte globulin were significantly associated with rhcv in the liver allograft 15, 16 . calcineurin inhibitors (cni) such as tacrolimus and cyclosporine are the most commonly used immunosuppressive drugs in lt recipients. ideal immunosuppressive regimen in hcv lt recipients to avoid rejection and at the same time to reduce the risk of rhcv is not established. corticosteroid was considered to be the main drug associated with increased risk of rhcv. in particular, use of steroid boluses and rapid tapering has been shown to reactivate hepatitis c in the graft 17 . various immunosuppressive modifications were adopted in the past to reduce rhcv of the allograft. low dose slow steroid tapering has shown to reduce rhcv in the graft. a study by berenguer et al., showed 29% rhcv in patients with cni and tapering steroids over 9-12 months compared to 48% in the historical controls 18 . studies attempted to eliminate corticosteroids using il-2receptor antagonist induction regimens to reduce rhcv. in a prospective randomised multi-centre trial evaluation dacluzimab induction followed by tacrolimus and mmf (n=153) vs standard treatment rhcv occurred in 33% vs 40% (p not significant) 19 with increasing demand for cadaveric organs, dcd organ harvest has been increasingly utilized. studies showed increased rhcv in patients undergoing lt from a dcd compared to donation after brain death (dbd) liver, probably related to ischaemic reperfusion injury 22 . interestingly, a large case control study on hcv patients who received dcd liver showed no significant differences in the hcv rna titres (p=0.7), severe rhcv with fibrosis 8% vs 15%, p=0.38) and graft loss (5% vs 9%, p=0.6) between the two groups at 12 months posttransplant 23 . previously, interpretation of abnormal lfts in patients transplanted for hcv infection was difficult to distinguish between a rejection episode and rhcv infection, in the absence of liver biopsy. therefore, management of graft dysfunction in hcv transplant recipients was a challenge. this led to the development of non-invasive markers of liver fibrosis. fibroscan using transient elastography technique plays a major role in the assessment of disease severity in these patients 24 . liver histology is definitive in the diagnosis and management of rhcv. acute hepatitis c in the liver allograft shows mild lobular activity with mononuclear inflammatory infiltrate, necroinflammatory foci and apoptotic bodies. nodular lymphoid aggregates may also be present. fch is characterised histologically by irregular portal expansion, portal fibrosis with immature pericellular/sinusoidal fibrous bands, extensive hepatocyte ballooning and degeneration, bile ductular reaction, marked canalicular and intracellular bilirubinostasis, j o u r n a l p r e -p r o o f and mild to moderate mononuclear inflammation. confluent or bridging necrosis may be present. pre-lt hcv rna level strongly predicts post-transplant rhcv severity. therefore, treatment should be aimed at making the patient virus free prior to liver transplantation.. svr has consistently shown to improve several aspects of hcv related complications. achieving svr in patients awaiting liver transplantation has shown to decrease the rate of disease progression, symptomatic improvement, improvement in meld score and importantly improves post-transplant survival 25, 26 treatment of hcv in the previous era: a decade ago, the treatment of hcv in patients with decompensated cirrhosis was nearly impossible. in the absence of advanced liver disease, the use of peg ifn and ribavirin achieved 50-70% svr depending on the genotype. unfortunately, both these drugs are contraindicated in patients with decompensated cirrhosis due to higher adverse events, like anaemia and sepsis. subsequent introduction of first generation directly acting antiviral (daa) therapy, protease inhibitors telaprevir and boceprevir achieved svr around 70% given with peg ifn and ribavirin. however, protease inhibitors were effective only against hcv genotype 1 and similarly these were contraindicated in advanced liver disease. both these drugs were discontinued in 2015. treatment of hcv in patients with decompensated cirrhosis changed significantly following the introduction of sofosbuvir, the second generation daa with high efficacy, shorter treatment course, better safety profile and importantly interferon free regime. sofosbuvir with ribavirin for up to 48 weeks in 61 hcv hcc patients awaiting liver transplantation, showed an undetectable hcv rna at the time of lt was associated with j o u r n a l p r e -p r o o f 70% svr12 in the post-transplant period. moreover, patients with undetectable hcv rna at least 4 weeks prior to lt never developed rhcv. adverse events leading to drug discontinuation was noted in 2 patients (sepsis and kidney injury) which was unrelated to sofosbuvir 27 . the success of this therapy changed the perspective of hcv management in patients undergoing liver transplantation. subsequent studies were conducted in hcv patients with decompensated cirrhosis with second generation daa. solar-1 was a phase 2 open label multi-centre study on 12 or 24 weeks of ledipasvir (ns5a polymerase inhibitor), sofosbuvir and ribavirin in patients with advanced cirrhosis (child pugh b or c, n=108). svr12 for child pugh b was 87% and 89%, and for child pugh c was 86% and 87%, with 12 and 24 weeks treatment irrespective of previous therapy. adverse events occurred in 23% of patients but only 4% discontinued treatment 28 . similarly, solar-2 study was conducted across european and new zealand sites on hcv child pugh b (n=56) and child pugh c (n=51) with svr12 of 87% and 96%, and 85% and 78% for 12 and 24 weeks, respectively. adverse events occurred in 11-50% of patients, predominantly in child pugh c, including 3 (12%) patients death. these two studies clearly demonstrated high svr rates in patients with 12 weeks of this combination therapy with effective hcv clearance in patients with decompensated cirrhosis. with the higher viral eradication rates, meld score improved in these patients over a period of time; however, in many of the study centres patients have undergone liver transplantation in view of shorter waiting times in different parts of the study sites 29 .data from our liver unit showed a weeks daclatasvir 60 mg daily in combination with sofosbuvir 400 mg twice a day and weight based ribavirin in patients with decompensated cirrhosis (n=60) with an svr12 of 83% 30 . patients with child pugh a and b had a higher svr12 than c, 94%, 94% and 56%, respectively. likewise, genotype 1b had a better svr12 than genotype 1a (100% vs 76%). in patients with decompensated cirrhosis, meld score improved by -0. higher rates of svr with daa has shown to reduce hcv related death by 74% 31 . achieving svr has shown to reduce the chance of liver disease progression with improvement in portal pressure 32 . in several cases there were significant improvement in meld score leading to delisting of patients waitlisted for lt. in a study involving 409 patients with child b and c cirrhosis, the mean meld declined by 0.85 within 6 months compared to untreated patients (p<0.0001) and they encountered reduced episodes of hepatic decompensation (3.7% vs 10%, p=0.009) in patients with svr 33 . importantly, a cohort study from srtr database showed 32% reduction in the hcv lt waitlist after they cleared hcv with daa therapy 34 . in the previous decade, management of post-transplant rhcv has been posed several difficulties. introduction of peg-ifn and ribavirin in the treatment of rhcv slightly improved clinical outcome. unfortunately, these drugs were associated with significant side effects including graft rejection. therefore, treatment was recommended only in patients with moderate and severe rhcv. fatigue, diarrhoea and anaemia occurred in 30%, 28% and 20% of patients 37 . initial studies with sofosbuvir and ribavirin combination therapy for post-transplant rhcv showed poor drug tolerance, however, the main adverse event was anaemia related to ribavirin in 62% of patients, and subsequent hepatic decompensation related to the low haemoglobin 38 . solar 1 and solar 2 also evaluated the efficacy of the combination of sofosbuvir and ledipasvir also included post liver transplantation rhcv patients. solar-1 study involving 12 or 24 weeks of ledipasvir, sofosbuvir and ribavirin in post-lt rhcv (n=229), the svr12 was 96%, 96%, 86%, 60%, 100% in patients with no cirrhosis, child a, child b, child c and fch patients with 12 weeks and, 98%, 96%, 88%, 75% and 100% for 24 weeks therapy. adverse events such as mild hyperbilirubinemia and anaemia however, mostly managed by reducing median dose of ribavirin to 600 mg daily 37 . solar-2 trial evaluated 226 post-transplant rhcv patients with ledipasvir sofosbuvir combination however, with child pugh score above 13 were excluded. svr12 was 93%, 100% and 95% in patients with no cirrhosis, child a and child b cirrhosis with 12 weeks j o u r n a l p r e -p r o o f therapy and 100%, 96%, 100% with 24 weeks therapy. these drugs were well tolerated with 5% adverse events, 2% discontinuation rates, and 2% viral relapse rates 39 .the success of solar trial with all oral daa in this challenging population opened the avenue for patients post lt rhcv. these two studies clearly demonstrated high svr rates in patients with 12 weeks of this combination therapy with effective hcv clearance post-transplant rhcv, than the previous generation drugs. ally-1 study also recruited patients with post-transplant rhcv infection (n=53) to assess the efficacy of 12 weeks daclatasvir 60 mg daily in combination with sofosbuvir 400 mg twice a day and weight based ribavirin. svr12 was 94% in lt recipients. viral relapsed in 3 posttransplant patients and were subsequently treated with 24 weeks of daclatasvir, sofosbuvir and ribavirin. there was no significant drug interactions with cni requiring dose adjustments 30 . interestingly, a real world analysis of daclatasvir and sofosbuvir showed 87-100% svr12 in patients with child b and c cirrhosis, and 58% of decompensated cirrhotic patients showed improvement in meld score 40 . a large multicentre study on daclatasvir in combination with sofosbuvir or simeprevir with or without ribavirin for 24 weeks was carried out on 97 lt recipients with severe rhcv including 37% patients with fch and 31% with cirrhosis. svr12 with dac + sof was 91% and dac + smv was 72%. there was a significant improvement in child pugh score from 6.8 to5.7 (p<0.001) and meld score from 12.1 to 9.7 (p<0.001) following svr. however, these scores worsened in 13% patients despite antiviral therapy 41. introduction of pangenotypic velpatasvir-sofosbuvir combination further simplified hcv therapy with maximal efficacy. a phase 3 study on 624 patients with hcv increased svr to 99% including in cirrhotic patients 42 . higher alt was shown to accelerate disease progression and cirrhosis. alt>100 u/l was shown to predict cirrhosis at 5 years due to rhcv (35% vs 6%). similarly, serum bilirubin >3.5 mg/dl was strongly associated with development of cirrhosis following rhcv (prieto 1999 hepatology, rosen 1999 transplantation) 43, 44 . practice of protocol liver biopsies at regular intervals post transplantation were carried out in the past. presence of moderate to severe lobular inflammation was associated with disease progression (30% vs 0.10%) compared to no or minimal inflammation over 5 years post transplantation. hcv rna titres has a direct correlation with the severity of post-transplant rhcv infection. in a study by shakel et al., 45 .peak hcv rna in the first year of untreated patients was associated with poor patient survival. a level of less than 10 7 , 10 7 -10 8 and >10 8 49 .in a recent innovative approach non-liver recipients of hcv positive donors were prescribed a short 7 day course of newer daa glecaprevir/pibrentasvir along with ezetimibe (hcv entry inhibitor) showed 100% viral clearance at week 12 50 . in summary, long term survival of hcv lt recipients has improved dramatically following second generation daa. with current svr close 100%, excellent safety profile daa therapy are increasingly used in decompensated cirrhosis and for post-transplant rhcv treatment. most centres treat hcv decompensated cirrhosis because it is associated with significant improvement in meld and may help reducing transplant waitlist. there was a debate j o u r n a l p r e -p r o o f whether to treat these patients while waiting for transplant or to treat in the posttransplant period, because some studies observed an increased risk of hcc following svr. in the management of rhcv, the timing of antiviral therapy is not well established. most experts recommend starting daa after first 3 months with stable immunosuppression onboard. a study by pellicelli et al., showed significant adverse events including hepatic decompensation and 25% mortality in those with advanced disease following treatment with daclatasvir and sofosbuvir for post-transplant rhcv 51 . similarly, forns et al, provided compassionate access sofosbuvir and ribavirin to 104 patients with severe rhcv with life expectancy less than 12 months. svr was 59% and much higher (73%) in patients with severe rhcv. however, severe adverse event occurred in 47% of patients including 13% mortality 52 . therefore, it is advisible to commence antiviral therapy prior to hepatic decompensation. post-transplant rhcv management previously included allograft biopsy to assess the severity of rhcv before commencing anti-viral therapy. however, the necessity of biopsy may be arguable with the currently available daa. abnormal liver enzymes in the presence of high hcv rna level may suffice to commence daa. retransplantation for graft failure secondary to rhcv had much worse outcomes. retransplantation for rhcv related graft failure is associated with prolonged hospitalization, increased cost and reduced survival. however, outcome and patients selection differed amongst various studies. in a multi-centred study from the us showed 1 and 3 year survival of hcv retransplantation was 69% and 49% with no difference in survival compared to other indications 53 .however, the currently available daa, the need for retransplantation likely to have reduced. hepatitis b is the major cause of chronic liver disease across the world affecting 350 million population, with higher prevalence in asian and african countries despite universal vaccination. hepatitis b was a considered an absolute contraindication in the initial years of liver transplantation due to early graft reinfection and poor survival benefit (less than 50%, j o u r n a l p r e -p r o o f therefore, most liver transplant centres adopted lifelong entecavir or tenofovir to reduce viral relapse following liver transplantation, particularly in low risk patients such as those with decompensated cirrhosis and low viral load (<100 copies/ml), alf presentation. low dose and short course hbig with antivirals may be useful in patients with high hbv dna, hbeag positive patients. tenofovir alafenamide (taf) is the most recently introduced formulation of tenofovir, to overcome renal and bone related adverse events related to tenofovir disoproxil fumarate. the efficacy is similar to its prodrug tenofovir disoproxil fumarate with a good safety profile. it is recommended in patients with chronic kidney disease and osteoporosis. data in posttransplant patients with taf is scarce. small case series found switching from tdf to taf hepatic involvement occurs in 14-53% of patients with covid-19, particularly in severe cases 73 . ace2 receptor is highly expressed in cholangiocytes (59.7%), vascular endothelial cells. interestingly, only 2% hepatocytes express ace2 and no expression observed in sinusoidal endothelial cells 72 . in a recent multicentre study on covid-19 patients with underlying cirrhosis (n=50), 97% required hospitalization and 71% required respiratory support. the number of patients with meld>15 increased from 13% to 26% (p=0.037) and aclf occurred in 28% of patients. in that study the 30-day mortality was 34% 74 . sars-cov-2 in decompensation in patients is associated with higher mortality 75 . analysis of apasl covid-19 liver injury spectrum study (apcolis) on 228 patients (43 cirrhotics), hepatic decompensation occurred in 9% and aclf in 11.6% of patients. child pugh score >9 predicted higher mortality (roc 0.94, hr 19.2). in patients with decompensated cirrhosis, mortality was 33% compared to 16.3% in compensated cirrhosis 76 to the non-transplant setting, however, chronic presentation defined as detectable hev rna or hev igm in the serum for 6 months, is exclusively observed in the post-transplant patients 86 . chronic hev following in transplant recipients was first recognised by kamar et al, including in lt patients. these patients continued to have allograft dysfunction following an acute infection and persistent positive hev rna in serum or stool for 10 to 24 months 87 . exact prevalence of hev seroprevalence in lt recipients is unknown. tests based on hev rna or hev igg level, a retrospective analysis of frozen sera showed 1% to 16% seroprevalence of hev in lt recipients 88 . in a french study, hev seroprevalence (hev igg) was observed in 12.9% of patients during pre-lt evaluation, interestingly one third of these patients became hev negative in the post-transplant period 89 late onset cmv disease occurs after completion of prophylaxis. especially in (r-/d+) recipients and its incidence is 25% which when compared to 8.3% in preemptive treatment 3-6 months reduces the advantage of universal prophylaxis. some studies report mitigation of this by extending the prophylaxis to 200 days, but data are insufficient 118 varicella zoster, that causes chicken pox remains latent in the dorsal root ganglion. this can reactivate following immunosuppression leading to herpes zoster, characterized by painful vesicular rash along the nerve distribution, usually restricted to one side. the 1-year incidence is 3% whereas the 5-and 10-year incidences are 14% and 18% respectively 146 human herpes virus 6 (hhv-6) belongs to beta herpesviridae subfamily under roseolovirus genus. this is a common name for 2 similar viruses hhv-6a and hhv-6b, the latter accounting in most cases 149 . asymptomatic exposure to hhv-6 occur in childhood such that 90-95% adult population are seropositive 150 . the circular dna of the virus integrates with the host genome and may remain latent for several years in the mononuclear cells 151, 152 . hhv-6 infection occurs as a result of reactivation in the post-transplant state. occasionally, donor derived infections or rarely through blood products have been reported 153 . incidence of hhv6 varies between 14% to 82% 154 , commonly occurring in the first 2-8 weeks after liver transplantation. sporadic cases were reported as early as 10 days and rarely after 5 years following liver transplantation 155 . in a prospective study of 51 lt patients, 11 (21.5%) developed hhv-6b reactivation, of which 4 had fever and abdominal pain 156 . reactivation rates are less when patients are covered up for cmv prophylaxis with ganciclovir. most hhv-6 reactivation are asymptomatic with low viral load 156 and they usually do not require treatment. clinically, hhv-6 reactivation presents with fever, and skin rash and raised liver enzymes 158, 159 . histologically, hhv-6 hepatitis may mimic acute cellular rejection with elevated liver enzymes and portal lymphocytic infiltration and confluent periportal necrosis on liver 153, 158 . in 170 lt patients with graft hepatitis, high levels of intra-hepatic hhv-6 dna and hhvantigenemia was significantly associated with decreased graft survival 160 . hhv-6 j o u r n a l p r e -p r o o f encephalitis though rare in lt recipients occurs usually within 4-6 weeks of transplantation. hhv-6a is neurotrophic and is detected in plasma as well as csf. brain imaging shows characteristic signal intensity in medial temporal lobes involving amygdala and hippocampi. hhv-6 infection may also present as post-transplant colitis in some 161 . it can also occur as co infection with cmv where hhv-6 antigenemia precedes cmv antigenemia 162 to summarise, hhv-6 infection after liver transplantation is rare, but its reactivation is associate with significant increase in graft failure, mortality, hepatitis c progression and cmv disease 169 . hhv infections in lt patients can be treated successfully with cmv antivirals ganciclovir, cidofovir and foscarnet. post lt viral infections can cause significant allograft dysfunction. early recognition, diagnosis and systematic approach can ameliorate the infective process and preserve allograft function. newly evolving less familiar viral infections such as covid-19 should also be considered in the differential diagnosis of post-transplant viral infections. management j o u r n a l p r e -p r o o f of post-transplant viral infection has improved over the last decade due to significant changes in immunosuppression protocols with optimal usage, and introduction of effective anti-viral drugs with high genetic barrier for resistance. these high potency anti-viral drugs have translated in to better long-term allograft and patient survival. • patients with decompensated cirrhosis due to chronic hepatitis b or hepatitis c infection should be commenced on high efficacy antiviral therapy • pathogenesis of covid-19 in post-liver transplant patients requires larger studies. • ideal immunosuppressive regimen to reduce the chance of recurrent or de novo viral infection needs to be studied. j o u r n a l p r e -p r o o f liver transplantation around the world. curr opin organ transplant hepatitis b virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures global epidemiology of hepatitis c virus infection: an up-date of the distribution and circulation of hepatitis c virus genotypes estimation of stage-specific fibrosis progression rates in chronic hepatitis c virus infection: a meta-analysis and meta-regression natural history of hepatitis c hepatitis c virus kinetics during and immediately after liver transplantation fibrosis progression after liver transplantation in patients with recurrent hepatitis c hcv-related fibrosis progression following liver transplantation: increase in recent years recent advances in liver transplantation natural history of clinically compensated hepatitis c virus-related graft cirrhosis after liver transplantation natural history of clinically compensated hepatitis c virus-related graft cirrhosis after liver transplantation comparison of two non-contemporaneous hcv-liver transplant cohorts: strategies to improve the efficacy of antiviral therapy hepatic venous pressure gradient identifies patients at risk of severe hepatitis c recurrence after liver transplantation hepatitis c virus recurrence after liver transplantation: a 10-year evaluation risk factors for hepatitis c recurrence after liver transplantation natural history and management of hepatitis c infection after liver transplantation model for end-stage liver disease limbo, model for end-stage liver disease purgatory, and the dilemma of treating hepatitis c in patients awaiting liver transplantation treat chronic hepatitis c virus infection in decompensated cirrhosis -pre-or post-liver transplantation? the ironic conundrum in the era of effective and well-tolerated therapy sofosbuvir and ribavirin prevent recurrence of hcv infection after liver transplantation: an open-label study sofosbuvir and ribavirin for treatment of compensated recurrent hepatitis c virus infection after liver transplantation pre-and post-transplant treatment of viral hepatitis c daclatasvir with sofosbuvir and ribavirin for hepatitis c virus infection with advanced cirrhosis or post-liver transplantation recurrence impact of sustained virologic response with direct-acting antiviral treatment on mortality in patients with advanced liver disease effects of all-oral anti-viral therapy on hvpg and systemic hemodynamics in patients with hepatitis c virus-associated cirrhosis sofosbuvir and velpatasvir for hcv genotype 1, 2, 4, 5, and 6 infection high incidence of allograft cirrhosis in hepatitis c virus genotype 1b infection following transplantation: relationship with rejection episodes timing and severity of initial hepatitis c recurrence as predictors of long-term liver allograft injury early high peak hepatitis c viral load levels independently predict hepatitis c-related liver failure post-liver transplantation the use of hepatitis c-infected grafts in liver transplantation impact of donor age on survival and fibrosis progression in patients with hepatitis c undergoing liver transplantation using hcv+ allografts long-term follow-up and outcome of liver transplantation from anti-hepatitis c virus-positive donors: a european multicentric case-control study liver transplantation from anti-hepatitis c virus-positive donors: our experience short-course, direct-acting antivirals and ezetimibe to prevent hcv infection in recipients of organs from hcv-infected donors: a phase 3, single-centre, open-label study sofosbuvir plus daclatasvir for post-transplant recurrent hepatitis c: potent antiviral activity but no clinical benefit if treatment is given late. dig liver dis sofosbuvir compassionate use program for patients with severe recurrent hepatitis c after liver transplantation retransplantation for hepatitis c: results of a u.s. multicenter retransplant study hepatitis b virus reinfection after orthotopic liver transplantation. serological and clinical implications orthotopic liver transplantation for patients with hepatitis b virus-related liver disease liver transplantation in european patients with the hepatitis b surface antigen lamivudine plus low-dose hepatitis b immunoglobulin to prevent recurrent hepatitis b following liver transplantation transmission of hepatitis b infection from hepatitis b core antibody--positive liver allografts is prevented by lamivudine therapy failure of reactivation of hepatitis b after liver transplantation in hepatitis b surface antigen-negative, core antibody-positive recipients rapid early hdv rna decline in the peripheral blood but prolonged intrahepatic hepatitis delta antigen persistence after liver transplantation a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization. situation report -120 evaluating new evidence in the early dynamics of the novel coronavirus covid-19 outbreak inwuhan, china with real time domestic traffic and potential asymptomatic transmissions. medrxiv specific ace2 expression in cholangiocytes may causeliver damage after 2019-ncov infection covid-19 and the liver high rates of 30-day mortality in patients with cirrhosis and covid-19 covid-19 in decompensated cirrhosis pre-existing liver disease is associated with poor outcome in patients with sars cov2 infection impact of covid-19 on liver transplantation in europe: alert from an early survey of european liver and intestine transplantation association and european liver transplant registry covid-19 in solid organ transplant recipients: initial report from the us epicenter covid-19 in an international european liver transplant recipient cohort covid-19 in long-term liver transplant patients: preliminary experience from an italian transplant centre in lombardy determining risk factors for mortality in liver transplant patients with covid-19 chronic infection with camelid hepatitis e virus in a liver transplant recipient who regularly consumes camel meat and milk prevalence of antibodies to hepatitis e virus in veterinarians working with swine and in normal blood donors in the united states and other countries prevalence and clinical consequences of hepatitis e in patients who underwent liver transplantation for chronic hepatitis c in the united states hepatitis e virus and chronic hepatitis in organ-transplant recipients prevalence of hepatitis e virus infection in pediatric solid organ transplant recipients--a single-center experience prevalence of hepatitis e virus infection in liver transplant recipients hepatitis e virus infection without reactivation in solid-organ transplant recipients, france. emerg infect dis hepatitis viruses and liver transplantation: evolving trends in antiviral management liver transplant from a donor with occult hev infection induced chronic hepatitis and cirrhosis in the recipient factors associated with chronic hepatitis in patients with hepatitis e virus infection who have received solid organ transplants ribavirin for chronic hepatitis e virus infection in transplant recipients sofosbuvir inhibits hepatitis e virus replication in vitro and results in an additive effect when combined with ribavirin sofosbuvir monotherapy fails to achieve hev rna elimination in patients with chronic hepatitis e -the hepnet sofe pilot study management of cmv infection and disease in transplant patients european association for the study of the liver. electronic address: easloffice@easloffice.eu. easl clinical practice guidelines: liver transplantation randomised trial of efficacy and safety of oral ganciclovir in the prevention of cytomegalovirus disease in liver-transplant recipients. the oral ganciclovir international transplantation study group dynamics of cytomegalovirus replication during preemptive therapy with oral ganciclovir who among cytomegalovirus-seropositive liver transplant recipients is at risk for cytomegalovirus infection? liver transpl cytomegalovirus hepatitis in liver transplantation: prospective analysis of 93 consecutive orthotopic liver transplantations infections and allograft rejection -intertwined complications of organ transplantation cytomegalovirus infection and donor/recipient hla antigens: interdependent co-factors in pathogenesis of vanishing bile-duct syndrome after liver transplantation severe ductopenic rejection with features of vanishing bile duct syndrome: clinical, biochemical, and histologic evidence for spontaneous resolution hepatic artery thrombosis after orthotopic liver transplantation: a review of nonsurgical causes clinical utility of cytomegalovirus (cmv) serology testing in high-risk cmv d+/r-transplant recipients cytomegalovirus in solid organ transplantation clinical utility of cytomegalovirus viral load testing for predicting cmv disease in d+/r-solid organ transplant recipients preemptive therapy for human cytomegalovirus detection by real-time pcr and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries. pathog glob health the transplantation society international cmv consensus group. the third international consensus guidelines on the management of cytomegalovirus in solidorgan transplantation effect of preemptive therapy vs antiviral prophylaxis on cytomegalovirus disease in seronegative liver transplant recipients with seropositive donors: a randomized clinical trial evaluation of clinical outcomes of prophylactic versus preemptive cytomegalovirus strategy in liver transplant recipients cytomegalovirus infection in liver transplant recipients: current approach to diagnosis and the use of antibiotics: a clinical review of three volume set new developments in the management of cytomegalovirus infection after solid organ transplantation. drugs the human cytomegalovirus terminase complex as an antiviral target: a close-up view the efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients impact of a preemptive strategy after 3 months of valganciclovir cytomegalovirus prophylaxis in kidney transplant recipients. transplantation mycophenolate mofetil increases cytomegalovirus invasive organ disease in renal transplant patients selection and use of immunosuppressive therapies after liver transplantation: current german practice use of everolimus-based immunosuppression to decrease cytomegalovirus infection after kidney transplant antiviral drug resistance of human cytomegalovirus epstein-barr virus infections: biology, pathogenesis, and management lymphoproliferative disorders and de novo malignancies in intestinal and multivisceral recipients posttransplant lymphoproliferative disease following liver transplantation occurrence of post-transplant lymphoproliferative disorders among over thousand adult recipients: any role for hepatitis c infection? associations between ebv serostatus and organ transplant type in ptld risk: an analysis of the srtr national registry data in the united states diagnosis of post-transplant lymphoproliferative disorder in solid organ transplant recipients -bcsh and bts guidelines pcr in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial world health organization. who classification of tumours of haematopoietic and lymphoid tissues lymphomas occurring late after solid-organ transplantation: influence of treatment on the clinical outcome posttransplantation lymphoproliferative disorder--the great mimic in liver transplantation: appraisal of the clinicopathologic spectrum and the role of epstein-barr virus virus and posttransplant lymphoproliferative disorder in solid organ transplantation sequential treatment with rituximab followed by chop chemotherapy in adult b-cell post-transplant lymphoproliferative disorder (ptld): the prospective international multicentre phase 2 ptld-1 trial therapeutic options in post-transplant lymphoproliferative disorders age-specific prevalence of infection with herpes simplex virus types 2 and 1: a global review herpes simplex virus principles and practice of infectious diseases infections with cytomegalovirus and other herpesviruses in 121 liver transplant recipients: transmission by donated organ and the effect of okt3 antibodies herpes simplex virus and varicella-zoster virus infections in hematopoietic stem cell or solid organ transplantation transplant infections infections after liver transplantation infections in solid-organ transplant recipients oral acyclovir therapy for mucocutaneous herpes simplex virus infections in immunocompromised marrow transplant recipients absolute bioavailability and metabolic disposition of valaciclovir, the l-valyl ester of acyclovir, following oral administration to humans herpes simplex virus in solid organ transplantation incidence and risk factors for herpes zoster in patients undergoing liver transplantation herpes zoster after liver transplantation: incidence, risk factors, and complications varicella zoster virus in solid organ transplantation classification of hhv-6a and hhv-6b as distinct viruses update on human herpesvirus 6 biology, clinical features, and therapy latent human herpesvirus 6 infection of human monocytes/macrophages the prevalence of chromosomally integrated human herpesvirus 6 genomes in the blood of uk blood donors et pediatric liver transplant patients hhv-6a, 6b, and 7: persistence in the population, epidemiology and transmission human herpesvirus-6 infection after liver transplantation clinical impact of human herpesvirus 6 infection after liver transplantation in syncytial giant-cell hepatitis detection of hhv-6 in over a thousand samples: new types of infection revealed by an analysis of antigenemia related to cmv infection after liver transplantation co-infection and clinical impact of human herpesvirus 5 and 6 in liver transplantation human herpesvirus-6 in liver transplant recipients: role in pathogenesis of fungal infections, neurologic complications, and outcome and cytomegalovirus dna in liver donor biopsies and their correlation with hla matches and acute cellular rejection acute hepatitis with periportal confluent necrosis associated with human herpesvirus 6 infection in liver transplant patients encephalitis caused by human herpesvirus-6 in a liver transplant recipient treating hhv-6 infections diagnosis and clinical management hhv-6 in liver transplantation: a literature review key: cord-299747-qovrstak authors: deval, jerome; symons, julian a; beigelman, leo title: inhibition of viral rna polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand rna viruses beyond hepatitis c virus date: 2014-09-17 journal: curr opin virol doi: 10.1016/j.coviro.2014.08.004 sha: doc_id: 299747 cord_uid: qovrstak a number of important human infections are caused by positive-strand rna viruses, yet almost none can be treated with small molecule antiviral therapeutics. one exception is the chronic infection caused by hepatitis c virus (hcv), against which new generations of potent inhibitors are being developed. one of the main molecular targets for anti-hcv drugs is the viral rna-dependent rna polymerase, ns5b. this review summarizes the search for nucleoside and nucleotide analogs that inhibit hcv ns5b, which led to the fda approval of sofosbuvir in 2013. advances in anti-hcv therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand rna viruses. although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by rna viruses represents an important paradigm shift and a new frontier for future antiviral therapies. inhibition of viral rna polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand rna viruses beyond hepatitis c virus jerome deval, julian a symons and leo beigelman a number of important human infections are caused by positive-strand rna viruses, yet almost none can be treated with small molecule antiviral therapeutics. one exception is the chronic infection caused by hepatitis c virus (hcv), against which new generations of potent inhibitors are being developed. one of the main molecular targets for anti-hcv drugs is the viral rna-dependent rna polymerase, ns5b. this review summarizes the search for nucleoside and nucleotide analogs that inhibit hcv ns5b, which led to the fda approval of sofosbuvir in 2013. advances in anti-hcv therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand rna viruses. although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by rna viruses represents an important paradigm shift and a new frontier for future antiviral therapies. introduction: the rna polymerase of hcv as the target for nucleoside analogs hepatitis c virus (hcv) is a member of the flaviviridae family. viruses from this family all contain a single-strand, positive-sense rna genome of about 9.5 kb. the viral genome encodes only one open-reading frame translated into a polyprotein of approximately 3000 amino acids. hcv is estimated to have infected approximately 175 million individuals worldwide, with 2-4 million new infections occurring each year [1] . until recently, treatment options for chronic hcv infections were largely suboptimal due to limited efficacy and substantial toxicity. the standard of care (soc) was a 24-week or 48-week course of pegylated interferon alpha (peg-ifn-a) in combination with ribavirin. effective clearance or sustained virologic response (svr) rate of the virus was achieved in less than 50% cases of genotype-1 infection, the most prevalent strain of hcv in the united states and europe. since 2011, two inhibitors of the viral serine protease, ns3/4a, boceprevir and telaprevir, were approved for use in combination with peg-ifn-a and ribavirin. these molecules are called direct-acting antivirals (daa) because they specifically bind to, and inhibit, a viral protein required for virus replication. although the toxicity burden of these newer treatment options remains high, the svr rate in the presence of protease inhibitors has improved to 70-80% in difficult-to-treat genotype-1 infections [2, 3] . other daas that specifically block hcv enzymatic functions have been intensely studied over the last decade, and the polymerase function of ns5b has emerged as one of the most attractive targets for the next generation of anti-hcv therapy. the hcv ns5b protein is an rna-dependent rna polymerase (rdrp). ns5b is required both for replication of the viral genome by synthesis of the minus-strand intermediate and at the transcription level for synthesis of viral mrna. the rdrp enzymatic activity of ns5b is unique to viruses and not found in human cells, which makes ns5b an attractive target for antiviral drug development (see [4] for a more detailed review on the structure and functions of ns5b). the ns5b protein is composed of 591 amino acids. similar to other known rdrps, the hcv ns5b contains six conserved motifs designated a-f. the amino acids involved in the catalytic activity of ns5b are located within motif a (aspartate at position 220) and the catalytic triad gdd at position 318-320 in motif c [5 ] . the orientation of these residues in the active site of ns5b and their contribution to the catalytic activity are supported by the crystal structure of the protein [5 ,6,7 ]. using the polymerase right-hand analogy model, the hcv ns5b protein features the fingers, palm, and thumb subdomains (figure 1a ). unlike the traditional open-hand conformation shared by many dna polymerases, the hcv ns5b features an encircled active site due to extensive interactions between the fingers and thumb subdomains. these contacts restrict the flexibility of the subdomains and favor the first steps -or initiation -of rna synthesis leading to the formation of the primer strand. therefore, important structural changes involving an opening of the thumb and the fingers are required for primer extension during the elongation steps [8,9 ,10] . another unique feature of ns5b is its b-hairpin loop that protrudes into the active site located at the base of the palm subdomain (figure 1a ). this 12 amino acid loop located within the thumb (residues 443-453) was suggested to interfere with binding to double-stranded rna due to steric hindrance. its deletion allows the enzyme to favor primer-dependent rna synthesis [11,12 ,13] , and the resulting truncated protein was cocrystallized in the elongation mode with double-stranded rna [14] . primer extension also requires the c-terminal part of ns5b to move away from the catalytic site, a structural feature shared with other rna polymerases [15] . once these important conformational changes take place, the enzyme becomes processive and the efficiency of rna synthesis increases considerably [16 ,17] . it is precisely during the elongation phase of rna synthesis that hcv ns5b is inhibited by nucleotide analogs acting as chain terminators (figure 1b) . the initial major class of nucleoside analogs of therapeutic potential to demonstrate potent inhibition of hcv rna polymerase activity were 2 0 c-methyl-ribonucleosides, the first 2 0 c-methyl ribonucleosides were originally synthesized in the 1960s [18] . later, 2 0 c-methyluridine triphosphate was found to act as a chain terminator of escherichia coli rna polymerase [19, 20] . in antiviral assays, 2 0 c-methyl-cytidine was originally described as an inhibitor of bovine diarrhea virus (bvdv), a virus closely related to hcv and used as a surrogate [21] [22] [23] [24] . although the compound was highly potent and selective in tissue culture, its low bioavailability made it unsuitable for oral dosing. this limitation was overcome by adding an l-valine ester group at the 3 0 -oh position on the sugar ( figure 2a ). the resulting drug, valopicitabine (nm283), was efficacious when dosed orally in hcv-infected chimpanzees [25] . although this nucleoside significantly reduced hcv viral load in patients, its development was discontinued in phase ii clinical trials due to doselimiting gastrointestinal (gi) toxicity [26] . other 2 0 cmethyl nucleosides such as 2 0 c-methyl-adenosine or 2 0 c-methyl-7-deaza-adenosine have been reported to inhibit hcv replication [27 ,28] , but none were evaluated in clinical trials presumably due to tissue retention issues in preclinical species [29] . in vitro, prolonged culture of hcv replicon-containing hepatocytes with 2 0 c-methyl-nucleosides results in the selection of a single s282t mutation within ns5b, and the resulting polymerase is resistant to this class of nucleosides [30] [31] [32] . the second class of anti-hcv nucleosides is the 4 0azido-nucleoside scaffold. molecules in this class resemble 3 0 -azido-thymidine (azt) and were originally synthesized for testing against human immunodeficiency virus [33 ] . during compound library screening in the sub-genomic replicon assay, 4 0 azido-cytidine was later identified as a potent inhibitor of hcv [34] . in its 5 0 -triphosphate form, the inhibitor was recognized as a substrate for hcv ns5b, and its incorporation to the growing rna strand resulted in immediate chain termination. one advantage of 4 0azido-cytidine over the 2 0 c-methyl-nucleosides was the lack of cross-resistance associated with the presence of the s282t mutation [34, 35] . the uridine analog counterpart was inactive in the replicon assay due to lack of intracellular phosphorylation. however, the 5 0triphosphate forms of both cytidine and uridine analogs were equally potent as chain terminators against hcv ns5b. the pharmacokinetic properties of 4 0 -azido-cytidine were further improved with the triester prodrug balapiravir (figure 2a ), which achieved a 3.7 log 10 reduction in viral rna at the highest dose in a 14day phase 1b monotherapy clinical trial [36] . four weeks of treatment with balapiravir in combination 2 virus replication in animals and plants with soc resulted in a further decrease in viral load, but also in hematologic adverse events such as lymphopenia, which led to the discontinuation of development of balapiravir for hcv infection [37] . analogs of balapiravir with similar 4 0 -modification scaffolds have also been reported, but none have progressed into further development [38] [39] [40] [41] . recently it was shown that 4 0 -azido-ctp is a good substrate for human mitochondrial rna polymerase, one of the proteins considered to be responsible for the mitochondrial toxicity of several other ribonucleosides [42 ] . the third major class of nucleoside analogs is the 2 0 -fluoro-2 0 c-methyl modification, which includes sofosbuvir. the double substitution at the 2 0 -position on the ribose evolved from the earlier 2 0 c-methyl scaffold, combined with further change resulting from the observation that 2 0deoxy-2 0 -fluoro-cytidine was weakly active in the hcv replicon [43] . compared with its 2 0 -fluoro mono-substituted counterpart, 2 0 -fluoro-2 0 c-methyl-cytidine (psi-6130) was significantly more potent in the hcv replicon assay and less toxic to the huh-7 hepatocarcinoma cells in vitro [44] . the parent 2 0 -fluoro-2 0 c-methyl-cytidine nucleoside was also developed as the orally bioavailable diisobutyrate ester prodrug, mericitabine, which is currently under phase ii clinical development. in an important series of experiments, it was found that 2 0 -fluoro-2 0 c-methylcytidine is metabolized also to its uridine 5 0 -triphosphate form as a result of intracellular deamination [45 ,46] . as the parent uridine analog was not readily converted to its monophosphate form by intracellular kinases, a series of monophosphate forms of 2 0 -fluoro-2 0 c-methyl-uridine were designed to bypass the first and most limiting kinase step [47] . phosphoramidate prodrugs were made to mask the charges of the alpha-phosphate with an amino acid ester and an aryl group, both protecting groups being removed in the cytoplasm of hepatocytes after cell penetration [47] . optimization of the leaving groups of the prodrug and separation of stereoisomers led to the selection of sofosbuvir (psi-7977), as one of the most potent and selective inhibitors in this series (figure 2a ) [48] . in addition to forming high levels of the nucleoside 5 0 -triphosphate (ntp), the exquisite potency of sofosbuvir can be explained in vitro by the fact that its active form 2 0 -fluoro-2 0 c-methyl-uridine 5 0triphosphate is a very efficient substrate and chain terminator for hcv ns5b (figure 2b ) [49] . this study also showed that the 2 0 -fluoro substitution contributes less to chain termination than the 2 0 -c-methyl moiety. the ntp derivative of sofosbuvir is a very poor substrate for human mitochondrial rna polymerase, one of the proteins considered to be responsible for the mitochondrial toxicity of several other ribonucleosides [42 ] . similar to the two former classes of inhibitors, many other 2 0 -fluoro-2 0 cmethyl-nucleosides have been evaluated, including the very potent monophosphate guanosine analog, psi-353661, that progressed to phase ii clinical trials, before being discontinued due to elevated alanine aminotransferase levels (see complete reviews of recent hcv nucleoside and nucleotide development in [4, 50, 51] ). the quest for structurally novel nucleoside inhibitors of hcv ns5b continues to be an active area of pharmaceutical research, and other chemical scaffolds have been recently discovered (e.g. [52, 53] ). to this date, none of the other classes of nucleoside analogs have advanced beyond phase ii clinical trials. several important and sometimes severe human diseases are caused by rna viruses in the flaviviridae, picornaviridae, caliciviridae, and coronaviridae families. all these viruses contain a positive-strand rna genome, and their rna-dependent rna polymerases share significant amino acid similarities based on sequence alignment and phylogenetic analysis [54, 55] . since hcv belongs to the flaviviridae family, some of the nucleoside analogs originally developed against hcv would also be expected to inhibit related pathogens within the same family or even viruses in other positive-strand rna families. this prediction was confirmed by counter-screening anti-hcv molecules against representative panels of viruses from other families and sub-families (table 1 ). in particular, 2 0 c-modified nucleosides are known to inhibit multiple positive-strand rna virus families. in one of the first reported examples, 2 0 c-methyl-cytidine was found to be potent in cell-based in vitro assays against flaviviruses such as west nile, yellow fever, and dengue virus [56] . this result is not entirely surprising since the same molecule was already known to inhibit bvdv, which also belongs to the flaviviridae family, and was used as a surrogate for hcv antiviral screening. in an in vivo efficacy model, 2 0 c-methyl-cytidine protected hamsters challenged with a lethal dose of yellow fever virus even when administered up to 3 days post-infection [57] . the cytidine analog also inhibits the in vitro replication of tickborne, hemorrhagic fever-associated flaviviruses [58] . in addition, 2 0 c-methyl-cytidine inhibits the replication of the norwalk virus [59, 60] and foot-and-mouth disease virus [61] from the caliciviridae and picornaviridae family, respectively. although it has not been as extensively profiled, the purine analog 7-deaza-2 0 c-methyl-adenosine similarly displays broad antiviral activity against positive-strand rna viruses, while being inactive against single-strand negative-sense rna viruses [27 ,28] . this broad spectrum activity was further profiled with the chemically related 7-deaza-2 0 c-ethynyl-adenosine. although it was found to also inhibit hcv, the molecule was potent enough to be developed specifically against dengue virus infection [62, 63 ] . to date, it is the only 4 virus replication in animals and plants table 1 inhibition of positive-strand rna viruses by nucleoside analogs nucleoside analog known to inhibit dengue virus replication in a mouse efficacy model [63 ] . however, its safety profile was insufficient to enable further drug development toward human clinical trials. on the basis of the finding that 4 0 -azido cytidine was potent against dengue virus replication in vitro, the ester prodrug, balipiravir, was also repurposed toward treatment of dengue virus infection [64] . for reasons that remain to be clarified, the drug did not reduce either viral load or symptoms when administered to hospitalized dengue-infected patients. challenges for discovering novel nucleoside analogs targeting positive-strand rna viruses other than hcv although many anti-hcv nucleoside analogs may potentially inhibit other positive-strand rna viruses in vitro, there are currently no obvious drug candidates for direct repurposing from hcv infection to other disease indications. in particular, sofosbuvir, the only fda-approved anti-hcv nucleotide analog, is a phosphoramidate prodrug that has been optimized to specifically deliver high levels of the nucleoside 5 0 -triphosphate as the active species through release of the prodrug moiety by firstpass effect, the process by which drugs get metabolized into the liver before reaching systemic circulation [65] . therefore, the active metabolite of sofosbuvir is likely not significantly distributed to organs and tissues other than the liver and targeted by most positive-strand rna viruses. in comparison, 2 0 c-methyl-cytidine and 7-deaza-2 0 c-ethynyl-adenosine are among the only known broadspectrum nucleoside analogs with potential for systemic organ exposure of the 5 0 -monophosphate and 5 0 -triphosphate forms in levels sufficient to achieve in vivo efficacy. however, their relatively poor safety profiles and narrow dose margins make them poor candidates for further clinical development. what are the main challenges to designing and optimizing new inhibitors of non-hcv positive-strand rna viruses? the search for such molecules has been hampered by several factors, the first one being the need to achieve in vivo pharmacokinetic properties compatible with delivery of the ntp to the site of infection, which differs by virus and includes, for example, the gi tract (norwalk virus), the lungs (rhinovirus, middle east respiratory syndrome virus), the brain (west nile virus), and lymphoid organs (dengue virus). as mentioned before, the only organspecific prodrugs that have been successfully developed to date for nucleosides target the liver, and will likely not be useful for non-liver infections. the second important limitation to finding new nucleoside analogs is also related to 5 0 -triphosphate formation and the choice of the immortalized cell lines used for in vitro infection experiments. the metabolic kinase activation pathways of many common laboratory strains and species of cell lines differ from natural human tissues or human primary cells. although this is not generally a problem for small molecule drug testing, the metabolic activation of nucleoside analogs to ntps entirely relies on the presence of specific nucleoside and nucleotide kinases that are sometimes deficient in common lab-adapted cell lines. finally, it will be important to thoroughly assess the selectivity and toxicity of new nucleoside analogs to ensure that they do not interfere with cellular mechanisms at efficacious doses. in the case of acute infections, the safety requirements for short-term treatments may differ from those of anti-hcv nucleotides. despite these challenges, the prospect of using nucleoside analogs to treat acute infections caused by rna viruses represents an important paradigm shift and a new frontier for future antiviral therapies. lesburg ca, cable mb, ferrari e, hong z, mannarino af et al.: crystal structure of the rna-dependent rna polymerase from hepatitis c virus reveals a fully encircled active site. nat struct biol 1999, 6:937-943. together with bressanelli et al. [7 ], this study reports the first crystal structure of hcv ns5b showing that the polymerase is in a closed fingers conformation that serves to encircle the enzyme active site. replication of hepatitis c virus a new era of hepatitis c therapy begins telaprevir for retreatment of hcv infection hepatitis c virus and its inhibitors: the polymerase as a target for nucleoside and nucleotide analogs. cancer-causing viruses and their inhibitors a novel mechanism to ensure terminal initiation by hepatitis c virus ns5b polymerase hepatitis c virus rna-dependent rna polymerase (ns5b) as a mediator of the antiviral activity of ribavirin first transient kinetic study with hcv ns5b showing how deletion of the b-hairpin loop enables the polymerase to readily extend a primer multiple interactions within the hepatitis c virus rna polymerase repress primer-dependent rna synthesis structure of hepatitis c virus polymerase in complex with primer-template rna structural insights into mechanisms of catalysis and inhibition in norwalk virus polymerase klumpp k: assembly, purification, and pre-steady-state kinetic analysis of active rna-dependent rna polymerase elongation complex describes a method to isolate a stable ns5b-rna elongation complex contribution of a mutational bias in hepatitis c virus replication to the genetic barrier in the development of drug resistance 3 0 -deoxynucleosides. iv. pyrimidine 3 0 -deoxynucleosides a new synthesis of 2 0 -c-methylnucleosides starting from d-ribose substrate properties of c 0 -methylnucleoside triphosphates in a reaction of rna synthesis catalyzed by escherichia coli rna-polymerase bovine viral diarrhea virus as a surrogate model of hepatitis c virus for the evaluation of antiviral agents nucleoside derivatives as inhibitors of rna-dependent rna viral polymerase methods and compositions for treating hepatitis c virus. patent number wo methods and compositions for treating flaviviruses and pestiviruses. patent number wo nm 283 has potent antiviral activity against genotype 1 chronic hepatitis c virus (hcv-1) infection in the chimpanzee efficacy and safety of valopicitabine in combination with pegylated interferon-alpha (pegifn) and ribavirin (rbv) in patients with chronic hepatitis c this report describes the identification of 2 0 -substituted nucleosides as inhibitors of hcv replication. the study sets the basis for anti-hcv nucleoside discovery by establishing a correlation between inhibition potency of the ntp analogs against hcv polymerase, amount of intracellular ntp formation a 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis c virus replication with excellent pharmacokinetic properties robust antiviral efficacy upon administration of a nucleoside analog to hepatitis c virus-infected chimpanzees general catalytic deficiency of hepatitis c virus rna polymerase with an s282t mutation and mutually exclusive resistance towards 2 0 -modified nucleotide analogues replication fitness and ns5b drug sensitivity of diverse hepatitis c virus isolates characterized by using a transient replication assay characterization of resistance to non-obligate chainterminating ribonucleoside analogs that inhibit hepatitis c virus replication in vitro first synthesis of 4 0 -azido-cytidine aimed to inhibit hiv as an analog of azt 0 -azidocytidine) is a potent inhibitor of ns5b-dependent rna synthesis and hepatitis c virus replication in cell culture in vitro selected con1 subgenomic replicons resistant to 2 0 -cmethyl-cytidine or to r1479 show lack of cross resistance robust antiviral activity of r1626, a novel nucleoside analog: a randomized, placebo-controlled study in patients with chronic hepatitis c balapiravir plus peginterferon alfa-2a (40 kd)/ribavirin in a randomized trial of hepatitis c genotype 1 patients 0 -deoxy-4 0 -azido nucleoside analogs are highly potent inhibitors of hepatitis c virus replication despite the lack of 2 0 -alpha-hydroxyl groups the application of phosphoramidate protide technology to the potent anti-hcv compound the design, synthesis, and antiviral activity of monofluoro and difluoro analogues of 4 0 -azidocytidine against hepatitis c virus replication: the discovery of 4 0 -azido-2 0 -deoxy-2 0 -fluorocytidine and 4 0 -azido-2 0 -dideoxy-2 0 ,2 0 -difluorocytidine design, synthesis, and antiviral properties of 4 0 -substituted ribonucleosides as inhibitors of hepatitis c virus replication: the discovery of r1479 sensitivity of mitochondrial transcription and resistance of rna polymerase ii dependent nuclear transcription to antiviral ribonucleosides comprehensive evaluation of 2 0 -c-methyl, 4 0 -methyl and 4 0 -azido anti-hcv ribonucleotide analogues as substrates for human mitochondrial rna polymerase. this study establishes a link between human mitochondrial rna polymerase as an off-target for ribonucleotide analogs and adverse effects resulting from mitochondrial toxicity observed in recent anti-hcv clinical trials inhibition of the subgenomic hepatitis c virus replicon in huh-7 cells by 2 0 -deoxy-2 0 -fluorocytidine inhibition of hepatitis c replicon rna synthesis by beta-d-2 0 -deoxy-2 0 -fluoro-2 0 -c-methylcytidine: a specific inhibitor of hepatitis c virus replication characterization of the metabolic activation of hepatitis c virus nucleoside inhibitor beta-d-2 0 -deoxy-2 0 -fluoro-2 0 -cmethylcytidine (psi-6130) and identification of a novel active 5 0 -triphosphate species this study shows that 2 0 -fluoro-2 0 c-methyl-uridine is the deaminated metabolic product of 2 0 -fluoro-2 0 c-methyl-cytidine. the corresponding phosphorylated uridine species was identified in primary hepatocytes, and shown to be a potent inhibitor of hcv ns5b the mechanism of action of beta-d-2 0 -deoxy-2 0 -fluoro-2 0 -c-methylcytidine involves a second metabolic pathway leading to beta-d-2 0 -deoxy-2 0 -fluoro-2 0 -c-methyluridine 5 0 -triphosphate, a potent inhibitor of the hepatitis c virus rnadependent rna polymerase nucleoside analog inhibitors of hepatitis c viral replication: recent advances, challenges and trends discovery of a beta-d-2 0 -deoxy-2 0 -alpha-fluoro-2 0 -beta-c-methyluridine nucleotide prodrug (psi-7977) for the treatment of hepatitis c virus efficiency of incorporation and chain termination determines the inhibition potency of 2 0 -modified nucleotide analogs against hcv polymerase current race in the development of daas (directacting antivirals) against hcv nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis c virus ns5b rna-dependent rna-polymerase synthesis of 2 0 -o,4 0 -c-alkylene-bridged ribonucleosides and their evaluation as inhibitors of hcv ns5b polymerase 0 -substituted nucleoside derivatives and methods of use thereof for the treatment of viral diseases the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses te velthuis aj: common and unique features of viral rnadependent polymerases 0 -c-methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of rna virus replication efficacy of 2 0 -c-methylcytidine against yellow fever virus in cell culture and in a hamster model inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses inhibition of norovirus replication by the nucleoside analogue 2 0 -c-methylcytidine the viral polymerase inhibitor 2 0 -cmethylcytidine inhibits norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model proof of concept for the inhibition of foot-and-mouth disease virus replication by the anti-viral drug 2 0 -cmethylcytidine in severe combined immunodeficient mice biochemical characterization of the inhibition of the dengue virus rna polymerase by beta-d-2 0 -ethynyl-7-deazaadenosine triphosphate an adenosine nucleoside inhibitor of dengue virus proof-of-concept study demonstrating in vivo efficacy of a ribonucleoside analog against dengue virus. to date, it is the only nucleoside analog known to inhibit dengue virus replication in a mouse efficacy model a randomized, double-blind placebo controlled trial of balapiravir, a polymerase inhibitor, in adult dengue patients phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery: application to anti-hcv agents we wish to thank peggy korn for her editorial review of the manuscript. papers of particular interest, published within the period of review, have been highlighted as:of special interest of outstanding interest key: cord-266105-8avkjc84 authors: li, qiang; feng, wei; quan, ying-hui title: trend and forecasting of the covid-19 outbreak in china date: 2020-02-27 journal: j infect doi: 10.1016/j.jinf.2020.02.014 sha: doc_id: 266105 cord_uid: 8avkjc84 by using the public data from jan. 20 to feb. 11, 2020, we perform data-driven analysis and forecasting on the covid-19 epidemic in mainland china, especially hubei province. our results show that the turning points of the daily infections are predicted to be feb. 6 and feb. 1, 2020, for hubei and china other than hubei, respectively. the epidemic in china is predicted to end up after mar. 10, 2020, and the number of the total infections are predicted to be 51600. the data trends reveal that quick and active strategies taken by china to reduce human exposure have already had a good impact on the control of the epidemic. a novel coronavirus from wuhan in central china, named 2019-ncov, has recently caused an epidemic of pneumonia in humans and posed a huge threat to global public health. 1 , 2 to the date 06/02/2020, 2019-ncov has led to more than 31,0 0 0 confirmed cases and 637 deaths in china according to national health commission of the people's republic of china ( http://en.nhc.gov. cn/index.html ). cases have also been documented in a growing number of other international locations, including the united states ( https://www.cdc.gov/coronavirus/2019-ncov/index.html ). as a consequence, it is urgent to develop effective measures to control this novel coronavirus on the basis of its pathogenesis. host receptor recognition is a determinant for virus infection. during the time of this letter preparation, three works have just been published to explore the receptor usage of 2019-ncov. a work by zheng-li shi et al. has shown that angiotensin-converting enzyme 2 (ace2), the receptor for severe acute respiratory syndrome coronavirus (sars-cov), 3 from human, rhinolophus sinicus bat, civet, swine but not mouse mediate 2019-ncov infection in vitro , while the detailed mechanisms are not yet determined. 4 the other two works have reported or predicted human ace2 usage of 2019-ncov in a similar way to sars-cov mainly based on the coronavirus spike (s) glycoproteins. 5 , 6 considering the fact that the s proteins mutate and gain capability to recognize host receptors among species, 7 , 8 there is still a lack of analyses on receptor usage of 2019-ncov from the receptor perspective, which does not evolve as quickly as viruses. here, we firstly performed amino acid sequence alignment of ace2 from different species, including human, five non-human primates (gibbon, green monkey, macaque, orangutan and chimpanzee), two companion animals (cat and dog), six domestic animals (bovine, sheep, goat, swine, horse and chicken), three wild animals (ferret, civet and chinese horseshoe bat) and two rodents (mouse and rat). the alignment by clustal w 2.1 shows that they share a high sequence similarity except chicken (data not shown). the result suggests that 2019-ncov of probable bat origin may not interact with chicken ace2 and subsequently infect them, which were not considered in the following analyses. in ace2, the regions at position 30-41, 82-84 and 353-357 are demonstrated to be involved in the interaction with sars-cov s protein, where the residues at positions 31, 35, 38, 82 and 353 are critical. 9 therefore, we took a close comparison in these regions and residues. as shown in fig. 1 , human and non-human primates share the identity sequences in the regions and residues, implying that ace2 from non-human primates may recognize 2019-ncov and medi-ate its infection. as a result, non-human primates may be susceptible to 2019-ncov and serve as animal models for antiviral research or intermediate hosts for cross-species transmission. in fig. 1 , the residues of most companion, domestic and wild animals are conserved, especially for the critical ones stated above, while certain ones are variable. for example, lys31, glu35, asp/glu38 and lys353 are conserved, which probably form salt bridges. interestingly, the changes at positions 31, 38 and 82 are observed. these changes suggest steric hindrance and electrostatic interference for host-virus interaction. taking civet ace2 as an example, the change of lys31 to thr31 is likely to form a hydrogen bond instead of a salt bridge. in addition, the polar side chain of thr82 may influence the hydrophobic interaction of the original met82. all these changes may result in a lower binding affinity. however, an additional region covering residues 90-93 has been shown to be involved in civet ace2 binding to sars-cov and enhance their interaction. 10 consequently, we can't preclude the existence of other regions to compensate for the residue changes. with most residues in human ace2, the ones from these compaion, domestic and wild animals may be favorable for 2019-ncov recognition, which is in consistent with the recent work by zheng-li shi et al. in case cross-species transmission, close contact with sick or asymptomatic companion, domestic and wild animals should be cautious, such as for workers in livestock farming and travellers in the wild. in contrast, certain significant changes occur in the mouse and/or rat ace2 compared to the human one ( fig. 1 ) . the asn31 and ser82 in mouse ace2 may not form favorable interactions with 2019-ncov due to their electrostatic or hydrophilic characteristics. importantly, the change into his353 in both mouse and rat ace2 does not form a strong salt bridge as lys353 does. since the structural information for mouse and rat ace2 is unavailable, we carried out homology modeling using human ace2 (pdb code 2ajf) as template on online ( https://swissmodel.expasy.org ) for further analyses. in fig. 2 a, the change into ser82 in mouse ace2 may interfere with the hydrophobic interaction of the original met82. additionally, the changes into asn31 and his353 definitely affect the salt bridge formation and electrostatic potential. the change into his353 in rat ace2 is similar in the effect on receptor-virus interaction ( fig. 2 b) . these analyses partially explain why mouse ace2 does not mediate 2019-ncov infection reported by zheng-li shi et al. and assume that rodents are not likely to be the susceptible host. in conclusion, we conducted sequence and structural analyses of angiotensin-converting enzyme 2 (ace2) from different species, which sheds some light on cross-species receptor usage of 2019-ncov. all these analyses raise an alert on a potential interspecies transmission of 2019-ncov and propose further surveillance in other animal populations. structural studies on human and other species ace2 in complex with 2019-ncov spike protein will con the authors declare no conflict of interest. very recently, a letter in journal of infection reported the outbreak of the novel cornonavirus from dec. 2019 in china, especially in hubei province. 1 this novel cornonavirus may originate from the bat, 2 is just named as the covid-19 by the world health organization (who). the covid-19 outbroke from wuhan, the capital of hubei province, has spread to other provinces of china and even other countries. 3 strong human-to-human transmission is established. 4 until feb. 11, 2020, there have been 44653 cases of covid-19 infections confirmed in mainland china, including 1113 deaths. to prevent and control the spread of the epidemic, many strategies are needed. 5 predicting the trend of the epidemic are quite important to the allocation of medical resources, the arrangement of production activities, and even the domestic economic development all over china. therefore, it is very urgent to use the latest data to establish an efficient and highly suitable epidemic analysis and prediction model according to the actual situation, and then to give reliable predictions, which could provide an important refer-ence for the government to formulate emergency macroeconomic decisions and medical resources allocation. recently, the susceptible-exposed-infectious-recovered (seir) or other similar models 6,7 are used to forecast the potential domestic and international spread of this covid-19 epidemic with parameters estimated from other sources.the real situation could be much more complicated and changing all the time. especially, with the implementation of the chinese government's multiple epidemic control policies, the control of nationwide epidemic has become obvious. however, the medical supplies in hubei will still affect the implementation of national policies. in this letter, we present the current situation of the epidemic, predict the ongoing trend with data driven analysis, and estimate the outbreak size of the covid-19 in both hubei and other areas in mainland china. the data of the epidemic are listed in table 1 and also graphically shown in fig. 1 , in which "china" is used to denote the mainland china, and "other" mainland china other than hubei province. the data includes the daily confirmed(suspected) infections, totally confirmed(suspected) infections, daily deaths, and total deaths from jan. 20, to feb. 11, 2020, reported by the national health commission of the republic of china (nhc), 8 , and health commission of hubei province (hch). 9 jan. 20, 2020, containing all the cases reported from 0 to 24, is the zeroth day in this letter, and then others are implied. the total number of suspected cases reaches the peak value on the 19th day (feb. 8), and then drops rapidly. notice that, until feb. 11, 2020, almost all the cases of deaths (1068/1113, 96%,) locates in hubei province, which reveals the epidemic in hubei is much more serious than that in the other areas of china. on the hand, it states the strict quarantine and limitation on population mobility have effectively prevented outbreaks in other provinces of china. scribe the data of daily infections and deaths in hubei, where x = (t + 0 . 5 − t t ) with t denoting the day, and t t representing the turning point; a and k are the parameters and determined by the data together with t t . the cumulative data of infections or deaths are obtained by the integration over h ( t ). for the epidemic in the other areas of china, the data of infections shows an asymmetric character, and then will be described as where x = t − t t ; the parameters b, k 1 , and k 2 together with t t , are then determined by fitting to the data. fig. 2 shows the fit and trend predictions to the total infections and deaths in hubei and china other than hubei. the extracted turning point of the infections in hubei is the 17th day, namely, feb. 6, 2020. the epidemic in hubei is predicted to end after mar. 10, 2020. we estimated that the epidemic is to end up with a total of 39, 0 0 0 infections in hubei, not including the clinically diagnosed cases since feb. 12, which may enlarge the prediction by 1.4 times. with considered data, namely, data from jan. 20 to feb. 11, the average errors are bout 166 and 190 for the fits to describe the daily and cumulative infections in hubei, respectively, corresponding to 8.6% and 1.6% for the average relative errors, respectively. fig. 2 (b) and (e) shows the estimations of the total and daily deaths in hubei. the predicted turning point is feb. 12, 2020. the total deaths is estimated to be 2250. notice the distribution of the daily deaths is delayed about 5 ∼6 days compared with the that of the daily infections. the average errors are bout 4 and 22 for the model to describe the daily and cumulative death numbers, respectively, corresponding to the relative errors 8.6% and 6.2%, respectively. the numbers of the daily and total infections in china other than hubei are showed in fig. 2 (c) and 2 (f), respectively. the extracted turning point is feb. 1, 2020 and the epidemic is expected to end on the 45th day, namely, on mar. 5, 2020. the estimated number of cumulative infections is about 12,600 in china other table 1 the data of epidemic caused by the covid-19 pneumonia in the mainland china and hubei, including (a) daily infections, (b) daily deaths, (c) total infections, (d) total deaths, (e) daily and (f) total suspected cases. china hubei a b c d e f a b c d 2020/1/20 77 2 291 6 27 54 72 2 270 6 2020/1/21 149 3 440 9 26 37 105 3 375 9 2020/1/22 131 8 571 17 257 393 69 8 4 4 4 17 2020/1/23 259 8 830 25 680 1072 105 7 549 24 2020/1/24 4 4 4 16 1287 41 1118 1965 180 15 729 39 2020/1/25 688 15 1975 56 1309 2684 323 13 1052 52 2020/1/26 769 24 2744 80 3806 5794 371 24 1423 76 2020/1/27 1771 26 4515 106 2077 6973 1291 24 2714 100 2020/1/28 1459 26 5974 132 3248 9239 840 25 3554 125 2020/1/29 1737 38 7711 170 4148 12,167 1032 37 4586 162 fig. 1 (f)), we did not parameterize this data, and hence did not give a trend prediction. the covid-19 epidemic in china is predicted to end after mar. 20, 2020, and cause 52,0 0 0-68,0 0 0 infections and about 240 0 deaths. however, the data trends show that the quick and active strategies to reduce human exposure taken in china, such as limi-tation on population mobility and interpersonal contact rates, strict quarantine on migrants, have already had good impacts on control of the epidemic. now the outbreak and deaths of the covid-19 epidemic are mainly in hubei province. after this letter has been written, the hubei reported 14,840 confirmed infections (including 13,332 clinically diagnosed cases) on feb. 12, 2020, which is almost 9 times greater than the data of the previous day. the huge fluctuation is due to the changing of diagnostic criteria in hubei. and this clinical criteria taken in hubei is expected to play an active and important role in controlling the outbreak and death rate. the authors declare no conflict of interest. dear editor , recently, several studies in this journal have highlighted the threat of avian influenza virus (aiv) to humans, poultry, and other animals. [1] [2] [3] [4] [5] [6] in equines, there was only one reported influenza outbreak caused by aiv, which occurred in 1989-1990 in china. however, aiv still poses a potential threat to equines. in contrast to aiv, equine influenza virus (eiv) is a commonly known causative pathogen of acute respiratory disease in equines. to date, two subtypes of eivs have been determined in the equine population worldwide: h7n7 and h3n8. 7 h7n7 eiv was initially identified in prague in 1956, and the last outbreak caused by h7n7 eiv occurred in 1979. 7 h3n8 eiv was first isolated in miami in 1963 and is currently responsible for ei outbreaks worldwide. during continuous transmission and evolution in equines, h3n8 eiv strains diverged genetically into three distinct lineages: predivergent, european, and american. 7 historically, there have been four main ei outbreaks in mainland china, occurring in 1974 china, occurring in , 1989 china, occurring in -1990 china, occurring in , 1994 , and 20 07-20 08. in the first, third, and fourth outbreaks, the isolated eiv strains were of the h7n7 subtype, h3n8 subtype european lineage, and h3n8 subtype american lineage, respectively. in the second ei outbreak, the causative pathogen (a/equine/jilin/1/1989) was determined to be h3n8 subtype influenza a virus (iav) by antigenicity characterization. however, after genetic sequencing, all eight segments of a/equine/jilin/1/1989 were genetically closer to those of avian influenza virus than those of eiv, indicating an interspecies transmission event. the 1989-1990 ei outbreaks in china contain two major outbreaks. the first ei outbreak occurred in jilin and heilongjiang provinces between march and june 1989, with a morbidity of 81% and a mortality of up to 20% in some herds. the second ei outbreak occurred in heilongjiang province in april 1990, with a morbidity of 41% and no mortality. the high mortality and unique antigenicity of a/equine/jilin/1/1989 attracted the attention of eiv researchers. however, after the 1989-1990 ei outbreaks in china, the virus disappeared from the equine population for unknown reasons. although no evidence in the epidemiological investigation worldwide supports the continuous circulation of a/equine/jilin/1/1989-like eiv in equines, we should not underestimate the potential of interspecies aiv transmission to equines and the possibility of future ei outbreaks caused by aiv. the haemagglutinin (ha) of iavs recognizes and binds the cell surface sialic acid (sa) receptor of the host respiratory tract, and then the virus enters into cells and replicates. the distribution of the sa receptor influences the host range of iavs. human influenza viruses preferentially bind the saa2,6 gal receptor, while aivs preferentially bind the saa2,3 gal receptor. it has been reported that the saa2,3 gal receptor is abundant in the epithelial cells of the horse trachea, and animal experiments indicate that iavs with an ha recognizing the saa2,3 gal receptor could replicate in horses. this finding provides a prerequisite for cross-species transmission of aiv to equines. in fact, there were several pieces of direct molecular evidence supporting the interspecies transmission of aiv to equines, in addition to the 1989-1990 ei outbreaks in china. in 2010, abdel-moneim et al. reported one h5n1 eiv strain (a/equine/egypt/ av1/2009) isolated from donkeys with cough, fever and serous nasal discharge in egypt. 8 sequencing results indicated that the virus had a close genetic relatedness to h5n1 aiv. in addition, h5 seroconversion was observed in 25.71% (27/105) of the examined donkeys. in 2011, he collected 284 equine lung tissue samples in china and isolated one aiv-derived h9n2 eiv strain (a/equine/guangxi/3/2011) ( https://kns.cnki.net/ kcms/detail/detail.aspx?dbcode=cmfd&dbname=cmfd201301& filename=1012496506.nh&v=mtmxmzc3ck5wrji2sexleedove1x wkviuelsogvymux1efltn0romvqzcvryv00xrnjdvvjmt2vazvpt rknua1u= ). among the tested equine serum samples in china, 0.54% (7/1110) of samples were positive for anti-h9n2 antibody. in the 2015-2016 ei outbreaks in pakistan, khana et al. reported that the isolated eiv strain (a/equine/pakistan/16) was reassorted from aiv. 9 the common characteristic for the reported cross-species transmission events of aiv to equines in china, pakistan, and egypt is that they occur in farming equines. 8 , 9 in the mixed farming system, equines and domestic poultry often live in close proximity. compared with racehorses, farming equines have more opportunities to contact domestic poultry and experience long-term environmental exposure to poultry. aiv infections in poultry increase exposure risks to equines. recently, frequent reports of cross-species transmission events of aiv to farming dogs in china and korea also indicate the potential threat of aiv to farming animals with close contact with poultry. 10 another problem is that the farming equines in china, pakistan, and egypt had no vaccination history. even in some racehorse populations, vaccinations for eiv are not routinely performed as recommended by the world organization for animal health (oie) expert surveillance panel on ei vaccine composition. although h3n8 eiv is antigenically distinct from a/equine/jilin/1/1989, animal experiments indicated that high doses of ei vaccines still provided complete protection against challenge with a/equine/jilin/1/1989. accordingly, routine vaccination with h3n8 ei vaccines in equines might prevent aiv infection to some degree, at least for h3n8 subtype aiv. several strategies may help reduce the threat of aiv to equines, including reducing exposure of equines to poultry, birds, and other hosts of iav, especially animals with clinical signs of influenza virus infection; monitoring aiv prevalence in domestic poultry around equines and routinely vaccinating domestic poultry with aiv vaccines; vaccinating susceptible equines with ei vaccines, especially farming equines in close contact with domestic poultry; and monitoring the prevalence of multiple aiv subtypes in equines, not merely that of those restricted to h3n8 subtype. none. we read with interest recent articles in this journal regarding the utility of next-generation sequencing for the diagnosis bacterial meningitis. 1 , 2 bacterial meningitis causes substantial morbidity and mortality worldwide. 3 rapid identification of the microorganisms is essential for early initiation of appropriate antimicrobial therapy, thereby improving clinical outcome. yet routine diagnostic methods fail to identify the bacteria in the majority of patients. over the last decade, advanced sequencing technologies have greatly improved our capacity to detect the causative agents of infectious diseases in clinical samples. 4 , 5 of these, the single molecule real-time sequencing developed by oxford nanopore technologies (ont) is a promising tool for diagnostic setting because of its short turnaround time. in late april 2019, a 59-year old seller of fish-noodles was referred to our hospital with a 1-day history of headache, fever and vomiting. he had a history of heavy alcohol use and hepatitis c infection, and had cirrhosis and diabetes mellitus. on admission, he was unconsciousness (a glasgow coma scale of 8), with a body temperature of 40 °c, a blood pressure of 140/80 mmhg and neck stiffness. initial gram-stain and microscopy of csf showed grampositive cocci, 8449 white cells/ul with 91% neutrophils, elevated protein and low glucose level, and high lactate concentration ( fig. 1 a) . routine bacterial culture, plus streptococcus pneumoniae and s. suis pcrs were all negative. he was diagnosed with bacterial meningitis, and given a combination of ceftriaxone (2 g/12 h) and dexamethasone (0.4 mg/kg/12 h). his clinical condition steadily improved. his second and third csf samples became negative by gram stain. the other csf parameters also improved, except the glucose, which remained low ( fig. 1 a) . on day 20 of hospitalization, the patient suddenly became unconsciousness with fever. brain magnetic resonance imaging showed bifrontal abscesses ( fig. 1 b) . after consulting a local neurosurgeon, aspiration of the brain abscesses was not advised and the patient was treated empirically with meropenem (2 g/8 h) and vancomycin (1 g/8 h). due to continued diagnostic uncertainty, we performed 16s rrna sequencing of the admission csf, stored as part of an going clinical study (supplementary materials), using an established sangersequencing based 16s rrna method. 6 subsequently, analysis of the obtained sequences revealed evidence of s. agalactiae (supplementary figure 1 ). given this new diagnostic result of the admission csf and because the patient had recovered clinically, the patient was given 24 million units of penicillin g for every 4 h. after day 43 of hospitalization, all csf parameters had normalised ( fig. 1 a) . likewise, on ct scan the brain abscess was now significantly improved ( fig. 1 c) . the patient was discharged with full clinical recovery. additionally, minion sequencing of complete 16s rrna gene was retrospectively carried out on the extracted nucleic acid of the admission csf yielded a total of 14,848 reads after 100 min of sequencing run. of these, 11,556 reads (79%) were successfully aligned to s. agalactiae ( fig. 1 d) . the remaining reads were assigned to other streptococcus species (mostly s. dysgalacticiae ( n = 2.145, 14%)), likely attributed to a combination of the high level of sequence similarities of the 16s rrna region between them and the sequencing errors introduced by the minion systems. analysis of sequencing data generated during the 25, 50 and 75 min of sequencing run time also yielded the same results (supplementary figure 2 ). details about the minion procedure are presented in supplementary materials. to further assess of the utility of csf minion sequencing of 16s rrna gene for the detection of bacterial meningitis pathogens, six csf samples from patients with confirmed bacterial meningitis enrolled in the abovementioned clinical study were tested ( table 1 ) . analysis of the minion reads obtained after two hours of the sequencing run showed that the majority of reads were correctly assigned to the corresponding bacterial species ( s. pneumoniae and s. suis) or genus ( neisseria ) found in the csf samples by diagnostic work up of the clinical study ( fig. 1 e and table 1 ). additional analysis of the obtained reads generated at two earlier time points (20 min and 60 min) of the sequencing run generated the same results ( table 1 ) . collectively, we report the first application of minion sequencing of 16s rrna gene to detect bacterial meningitis causing pathogens in csf samples from a low and middle-income country. the assay was able to detect the bacterial causes in all of the seven tested csf samples. meanwhile, gram stain and culture, the two most commonly used methods in clinical microbiology laboratories worldwide, were negative in 3/7 samples. ( fig. 1 and table 1 ). in addition to csf samples described in the present study and a recent pilot study from korea, 7 successful detections of haemophilus influenzae in sputum and campylobacter fetus in culture materials by minion sequencing of 16s rrna have recently been reported. 8 together, the data suggest that minion sequencing of 16s rrna is a sensitive method for rapid and accurate detection of pan-bacterial pathogens, including unexpected microorganisms, in clinical samples. additionally, the bacterial species information generated by the analysis of 16s rrna sequences can be useful for disease surveillance and vaccine evaluation. thus, the application of the method would be relevant for both patient management and epidemiological research. indeed, to the best of our knowledge the present study represents the first report of s. agalactiae associated meningitis in vietnam. because the incidence of invasive diseases (including meningitis) caused by s. agalactiae has been reported with increased frequency in recent years, 9 s. agalactiae should be considered as an important differential owing to the unavailability of the reagents at the time of patient admission, we were not able to perform real-time diagnosis using minion sequencing on the collected csf samples. however, same day diagnosis is theoretically achievable, because the current workflow takes 5 -6 h to operate. prospective study is urgently needed to assess its translational potential in the diagnosis of bacterial meningitis. since september 2017, a prospective observational study aiming at exploring the utility potential of next-generation sequencing in patients presenting with central nervous system (cns) infections has been conducted in the brain infection ward of the hospital for tropical diseases (htd) in ho chi minh city, vietnam. htd is a tertiary referral hospital for patients with infectious diseases from southern provinces of vietnam, serving a population of > 40 million. any patient ( ≥16 years) with an indication for lumbar puncture was eligible for enrolment. patient was excluded if no written informed consent was obtained. as per the study protocol, csf, plasma and urine samples were collected at presentation alongside demographic, meta-clinical data and results of routine diagnosis. after collection, all clinical specimens were stored at −80 °c until analysis. the clinical study received approvals from the institutional review board of the htd and the oxford tropical research ethics committee of the university of oxford. written informed consent was obtained from each study participant or relative (if the patient was unconsciousness). sequencing of complete 16s rrna gene was retrospectively performed using minion nanopore sequencer (ont), following the manufacturer's instructions. in brief, amplification of the complete 16s rrna gene and library preparation were carried out on extracted nucleic acid using 16s barcoding kit (sqk-rab204, ont) and primers (27f 5 -agagtttgatcctggctcag-3 and 1492r 5 -ggttaccttgttacgactt-3 ), followed by the sequencing of the amplified product using r9.4 flow cells (ont). minion reads were first basecalled using albacore v2.1.7 (ont), followed by demultiplexing using porechop ( https://github.com/rrwick/porechop ). determination of bacterial genus/species composition in the obtained reads was then carried out using epi2me interface (metrichor, oxford, uk), a platform for cloud-based analysis of minion data. overall, the whole procedure of minion sequencing of 16s rrna gene takes 5-6 h to complete (supplementary figure 3) . we, the author of the submitted manuscript declare that we do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding). dear editor, several aspects of influenza have been highlighted recently, including its global, comparative seasonality, 1 and issues around rapid point-of-care testing. 2 in addition, the uk has a national surveillance programme, the uk severe influenza surveillance system (usiss) to monitor and investigate severe cases of influenza across the country, 3 including severe cases of influenza admitted to intensive care (icu) and high dependency units (hdu). 4 specifically, the aim of this latter arm was to "monitor and estimate the impact of seasonal influenza on the population" and to "describe the epidemiology of severe disease." this surveillance began in the 2011-2012 influenza season and has continued to the present, with mandatory participation by all nhs trusts. leicester is one of 5 nhs commissioned centres in the uk providing extra-corporeal membrane oxygenation (ecmo) support for severe acute respiratory failure in adults. this process involves draining deoxygenated venous blood from the superior and inferior vena cavae, pumping this blood through a membrane lung, where oxygenation and carbon dioxide elimination take place. oxygenated blood is delivered back into the right atrium, therefore replacing the function of the native lung. in this way, ecmo can be used to support patients with respiratory failure of any cause. acceptance for admission for ecmo support follows referral to the ecmo service via structured questionnaire and discussion with the ecmo consultant on-call. patients are commenced on ecmo where benefits are deemed to outweigh the risks in patients with potentially reversible respiratory failure, who are already on maximal conventional therapy at their referring centre, and who are not achieving lung protective ventilation. 5 here, as part of our national usiss role, we describe severe influenza cases that required ecmo support in whom the predominant indications were severe hypoxia with a pao 2 :fio 2 ratio of < 100 despite maximal conventional therapy, and/or hypercapnoeic respiratory failure with a ph < 7.2 despite ventilation pressures > 30 cm h 2 o. during the 2018-2019 influenza season 33 cases of severe influenza were admitted to glenfield hospital for ecmo from our referring centres. most were male (23/33, 69.7%, 28-62 years, bmi: 17-47; female 10/33, 27-51 years, bmi: 21-38), and of white british ethnicity (30/33, 91.0%; with 1 each of chinese, asian, african ethnicity). comorbidities included, obesity, hypertension, asthma, copd, diabetes, anxiety, depression, epilepsy, a history of smoking and alcohol use (or abuse). all cases except for one influenza a(h3n2) infection (possibly two as subtyping was not performed for another sample) were due to influenza a(h1n1)pdm09. only one case had a history of influenza vaccination. various 'on referral' ecmo-related parameters were extracted, as well as contemporaneous laboratory results. these were statistically compared between patients who died ( n = 8) and those who survived using t -test or mann-whitney test for continuous variables and fisherexact test for categorical variables. correlation between duration on ecmo and laboratory parameters was assessed using spearman's rank correlation coefficient. ( n = 24) ( tables 1 and 2 ) . surprisingly, it was found that on direct comparison, most of the referral parameters for patients starting ecmo were not statistically different between those influenza-infected patients that eventually survived ( n = 24) versus those who died ( n = 8) -a case fatality rate of 25%. one patient was dropped from this analysis due to some missing data. only the respiratory rate (rr, p = 0.041) and the lactate ( p = 0.008) showed statistically significant differences between the two groups, with higher values being found in the patients who ecmo -extra-corporeal membrane oxygenation; sofa -sequential organ failure assessment; peep -positive end expiratory pressure; h 2 o -water; bpm -breaths per minute; pao 2 /paco 2 -partial pressure of arterial oxygen/carbon dioxide; altalanine aminotransferase. * rank correlation coefficient measures the strength and direction of a relationship between two variables. the coefficient ranges from −1 to 1 with 1 indicating a strong positive relationship between two variables and −1 indicating a strong negative relationship. a correlation coefficient close to 0 means the relationship between the two variables is very weak. died ( table 1 ) . however, clinically, these differences are of doubtful significance, as the rr is at the discretion of the parent clinical team prior to referral to ecmo, and the lactate levels are normal in the survivors and only marginally elevated in those who died which will again be dependent on use of cvvh. the higher pao 2 :fio 2 (p:f) ratio was statistically significantly correlated ( p = 0.020) with a shorter duration of ecmo, which suggests those with less severe disease recover quicker ( table 2 ) . many studies have reported on intensive care patient outcomes of the 2009 influenza a(h1n1)pdm09 pandemic, with or without the use of ecmo. in one study from australia, where ecmo was used, of 68 patients with confirmed influenza a (53 a(h1n1)pdm09, 8 un-subtyped) infection, 14 (21%) had died mainly due to intracranial and other forms of haemorrhage ( n = 10), or intractable respiratory failure ( n = 4). 6 another study from the usa, where influenza a(h1n1)pdm09-infected patients had non-ecmo icu admission, of 154 cases, 48 (37%) developed acute respiratory distress syndrome (ards), of whom 37 (24%) died. 7 a more recent study from spain that reviewed influenza a(h1n1)pdm09 cases admitted to icu (non-ecmo) from 2009-2015, found that of a total of 2421 cases, the mortality ranged from 18.8% (for community-acquired influenza) to 32.9% (for hospitalacquired influenza). 8 these ecmo-icu case fatality rates of severe influenza a(h1n1)pdm09 infection are similar to ours of 25% reported here, though compared to the specific ecmo patient cohort, 6 the causes of death in our patients were more variable, including intracranial and other haemorrhage ( n = 3), sepsis and multi-organ failure ( n = 2), respiratory failure ( n = 1), post-cardiopulmonary resuscitation hypoxic brain injury ( n = 1), and ischaemic bowel associated with atrial fibrillation ( n = 1). thus, even after 10 years of experience with this 'new' pandemic influenza a(h1n1)pdm09 virus, across almost the entire adult age-range ( ∼25-70 years in these previous studies), 6 -8 it appears that for severe cases, globally, the survival of such patients appears not to have improved. this may be somewhat surprising as the world's populations have become more immunologically experienced with this virus, which is now considered as a seasonal influenza virus that should be conferring some degree of persisting, cross-reactive individual and herd immunity over consecutive seasons. 9 this may be due to some predisposing genetic or environmental factors in individual patients, which should reinforce the general message that seasonal influenza immunisation is still recommended to minimise the number of people needing icu or ecmo support for severe influenza infection. more detailed monitoring on how these physiological parameters change over time (perhaps including more complex cytokine studies), in these severely ill, influenza a(h1n1)pdm09-infected patients admitted to icu-ecmo units, may eventually yield data to improve their management and clinical outcomes. none. we read with interest the report by stalenhoef and colleagues in this journal who show that biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection (ref). 1 here we report on a prospective observational analysis of the biomarker: cd5 antigen like protein (cd5l), in serum samples collected from 64 patients diagnosed with pneumonia 2 and 44 healthy adults between 2018 and 2019. the demographic and clinical characteristics of the 64 adults (males 70.3%; mean age 66 ± 20 years) with pneumonia were summarized in table 1 . 57 (89.1%) bacterial pneumonia patients (including 34 patients with confirmed bacterial pneumonia 2 and 23 patients with suspected bacterial pneumonia 3 ) and 7(10.9%) viral pneumonia patients were studied. there were significant differences in age, sex, wbc, neu%, crp, pct, cd5l, apache ii scores and length of icu stay between bacterial pneumonia and viral pneumonia. the pathogens responsible for pneumonia were described in supplementary table 1 . globally, gram-negative bacteria infection is more common than gram-positive bacteria infection in pneumonia. among them, acinetobacter baumannii (16, 59.3%) was considered the major pathogen in gram-negative bacteria and staphylococcus aureus (3, 42.9%) was considered the major pathogen in gram-positive bacteria. in viral pneumonia, influenza a (h1n1) was detected as the unique pathogen. in the study of the diagnostic performance of serum cd5l to identify etiology of pneumonia, as we found in supplementary fig. 1 , no matter in total bacterial pneumonia, suspected bacterial pneumonia or confirmed bacterial pneumonia, the serum of cd5l levels on day of pneumonia diagnosis were significantly higher than those in viral pneumonia and healthy control subjects. for evaluating the diagnostic performance of cd5l to differentiate bacterial from viral infection in pneumonia, roc analysis was conducted for the above bacterial pneumonia (supplementary fig. 2 ) and compared with routine laboratory markers ( table 2 ). by horizontal comparison, we found that the best auc was for cd5l (auc = 0.89), better than neu% (auc = 0.79), wbc (auc = 0.79), crp (auc = 0.78) and even pct (auc = 0.85). interestingly, by longitudinal comparison, the auc was observed highest in confirmed bacterial pneumonia (auc = 0.92), intermediate in all bacterial pneumonia (auc = 0.89), and lowest in suspected bacterial pneumonia (auc = 0.84), which may better elucidate the diagnostic performance of cd5l for etiology diagnosis in pneumonia patients. although there are 23 patients with suspected bacterial pneumonia for which no definitive pathogen was found, our result here may still provide a new treatment for bacterial infection identification in pneumonia patients for the current limitations in direct pathogen testing make it difficult to identify the pathogen at the time of diagnosis 4 . besides, as a potential biomarker to distinguish pathogens 5-8 , cd5l levels were also significantly correlated with wbc, neu%, crp and pct ( supplementary fig. 3 ) in patients with pneumonia. in the study of the diagnostic performance of serum cd5l to predict mortality in adults with pneumonia, mortality was defined as death occurring within 30 days after the onset of pneumonia. as we observed in supplementary fig. 4a , serum cd5l levels on day of pneumonia diagnosis were significantly higher in non-survivors ( n = 18) than survivors ( n = 46) ( p < 0.001). to ensure that serum cd5l levels were not influenced by the class of the infection in the specimens, spearman's rank correlation analysis was performed between serum cd5l levels and poor prognosis related scores 9 , 10 (sofa and apache ii scores) in patients with total bacterial pneumonia, suspected bacterial pneumonia, confirmed bacterial pneumonia and viral pneumonia, respectively. our correlation analysis between serum cd5l levels and sofa or apache ii scores shows that no matter for bacterial pneumonia or viral pneumonia, the cd5l levels showed positive correlation with sofa and apache ii scores ( supplementary figs. 5 and 6) . furthermore, the auc of cd5l for identifying 30-day mortality in adult pneumonia patients was 0.79 ( supplementary fig. 4b) , a value means good diagnostic performance, which is consistent with gao's study 9 before. therefore, we found that determining serum cd5l concentrations on day of pneumonia diagnosis was of great value in identifying bacterial infection from viral infection and predicting 30day mortality in adult patients with pneumonia, which suggests that cd5l may work for the etiologic diagnosis and could represent a novel biomarker for identification of a group of patients with pneumonia presenting with higher risk of death. these findings encourage further effort s aimed at exploring the clinical value of circulating cd5l to help early clinical decision-making in human pneumonia. none. high morbidity and mortality (up to 100%). asf is a devastating threat to pig agriculture and is responsible for serious production and economic losses. the asfv genome is 170-193 kilo base pairs in length 2 and has been divided into 24 different genotypes based on their b646l gene sequence, a gene which encodes the capsid protein p72. 3 , 4 our previous study showed that the p72 gene located in a very low genetic diversity region of the genome. 5 ye and colleagues, however, identified two novel genotypes xxv and xxvi, with genotype xxvi being especially divergent from all other genotypes. 1 to examine this unexpected result, in this study, we reanalyzed their data to evaluate the reliability of these two genotypes. we collected 716 available p72 gene sequences from ncbi ( http://www.ncbi.nlm.nih.gov/ ), which we then aligned with mafft software, a fast multiple sequence alignment program. 6 the alignments show that genotype xxv (accession number fr668409) has a single amino acid change at position 506 compared to genotype i, while for genotype xxvi (accession numbers fr668404 and fr668418), the region coding for amino acid residues 509 to 528 differs greatly compared to all other genotypes ( fig. 1 a) . to examine this in greater detail, we calculated genetic distance across the p72 coding sequence in 60 bp sliding windows, with a step size of 10 bp, between these two sequences and the 24 other genotypes ( fig. 1 b) . this sliding window analysis shows these two sequences for genotype xxvi have a region that is very divergent, with genetic distance up to 0.5067 from the other genotypes, while the genetic distances of the remaining regions are less than 0.1 ( fig. 1 b) . genetic distances for asfvs were calculated for 182 coding gene sequences, from 42 available complete genomes of asfvs, to assess the overall divergence. pairwise genetic distances of each gene for all asfv strains were calculated. the mean pairwise genetic distance for asfv genes was 0.041 ( fig. 2 ) , which is much lower than this divergent region of these two sequences for genotype xxvi (0.5067). therefore, it seems highly unlikely that a gene has such a highly divergent region. since recombination frequently occurs in asfvs, 5 we conducted a recombination analysis with the rdp4 program, which used the 3seq, bootscan, chimaera, genecov, lard, maxchi, rdp and siscan detection methods, 7 to determine whether recombination might have occurred in the xxvi genotype. we found reliable evidence for recombination events in both p72 genotype xxvi sequences ( fig. 1 c) . this suggests that the very highly divergent region of this p72 genotype is not homologous with other p72 genotypes. we used blastn, from ncbi ( https://blast.ncbi.nlm.nih.gov/blast.cgi ), to identify a source for this highly divergent region, however, no similarity sequence was found. we then mapped the multiple amino acid changes found in genotype xxvi to the 3d structure of p72 ( fig. 3 ) . the changed sites (marked in red in fig. 3 ) are located in the dec loop, a region which plays an important role in the formation of the trimer spike. 8 the amino acid mutations result in changes of electrostatic potential, hydrophobicity, and steric hindrance. it seems unlikely to have so many changes in such a functionally important region. in our previous studies we noticed that sequences directly submitted by individual laboratories to genbank often contain errors such as misidentification of species, sampling error, contamination, or are pseudogenes, which can lead to sequence analysis problems and erroneous conclusions. 9 , 10 sequences that deviate from the overall intraspecific pairwise divergence are potentially erroneous. 9 , 10 since genotype xxvi deviates from other genotypes not only in genetic distance, but also in protein structure, and mutations occur in the flanking regions of the sequences, we deduced that these reported mutations might be due to low quality sequencing. we identified the original manuscript reporting the three sequences of genotypes xxv and xxvi, 11 where the authors stated that the "alignment and translation of sequences obtained from sardinian isolates revealed that the c-terminal end of p72 gene was completely conserved between the sequences compared". this statement indicates that these sequences did not considerably diverge from previously reported asfv p72 sequences, and thus, suggests that an error in these sequences had been introduced upon submitting them to genbank. further examination of the original samples and sequences is needed. we contacted the corresponding author of this manuscript to check these three sequences, and were told that these three p72 sequences were all genotype i, and had some errors when submitted to genbank. in conclusion, the two novel genotypes of asfvs (xxv and xxvi) identified by ye and colleagues 1 are misled by problematic sequences. as reminded by our previous studies, many problematic sequences are present in genbank, which can lead to problems in downstream analyses. 9 , 10 thus, when published data is used for new analyses, the first set in the process of data analyses should be to filter these sequences for potential errors to reduce the possibility of reaching incorrect conclusions. if conclusions are based on obviously strange sequences, then we should trace back the data to their original source, and manuscript, and contact the corresponding authors before making conclusions based on these sequences. the authors declare no conflict of interest. fig. 1 . the analyses of phylogenetic tree, recombinant, and nucleotide divergence between 6xi and the other known 6 subtypes (6a-6xh) based on full-length hbv genome sequences. (a) the known hcv subtype 6 reference sequences (6a-6xh) from the previous report were used. phylogenetic analysis was performed by the maximum-likelihood method, based on the gtr + g + i substitution model, with 10 0 0 bootstrap replicates using the software mega v6. the sequences of hcv 6xi (ynkh261 and ynkh284) are marked in red dot. (b) bootscan plots were constructed using simplot 3.5.1 software based on 100 replicates with a 300-bp sliding window moving in steps of 50 bases. (c) pairwise comparisons of nucleotides similarities between 3 hcv 6xi strains and 30 reference genotype 6 sequences. 399,0 0 0 deaths per year. 2 currently, daa treatment regimens that target ns3/ns4a protease, ns5a phosphor-protein and the ns5b polymerase have shown high safe and high rates of sustained virologic response in hcv chronically infected patients ( > 90%). 3 however, under selective pressure from these drugs, drug resistance-associated substitutions (ras) can emerge during this therapy and result in treatment failure in 2 −10% of patients. 4 therefore, hcv infection is still a major global health concern. to date, eight confirmed genotypes have been characterized based on > 30% sequence divergence in the complete hcv genome, and genotypes are further classified into > 80 subtypes with a sequence divergence of > 15% to other subtypes of the same geno-type. 5 in the current study, we characterized a new hcv subtypes among chronic hepatitis c patients in yunnan, china, initially designated as 6xi, further analyzed its evolutionary history and investigated its baseline ras by next generation sequencing (ngs) method. plasma samples were collected between january 2018 and october 2018 from 160 chronic hepatitis c patients from kunming city in yunnan, china (fig. s1a) . the samples met the following inclusion criteria: (1) hepatitis c antibody-positive for 6 months with normal serum alanine aminotransferase (alt) levels; (2) subject was residing in yunnan province and was over 18 years old; (3) complete demographic information and clinical data were available; (4) consented to the use of patient information in studies on hcv epidemics; and (5) were treatment-naïve during sampling. there was no epidemiologic link among these individuals. the study was approved by the first people's hospital of yunnan province ethics committee. written informed consent was obtained from all participants prior to the study. out of a total of 160 chronic hepatitis c patients, 152 partial ns5b gene fragments were successfully amplified and sequenced with a success rate of 95.0% (152/160). multiple subtypes were identified in 152 subjects, including subtype 3b (36.2%, 55/152), 3a (23.7%, 36/152), 1b (19.1%, 29/152), 2a (8.5%, 13/152), 6n (7.9%, 12/152), 6a (2.0%, 3/152), and 1a (1.3%, 2/152) (fig. s1b) . interestingly, the remaining two strains (1.3%, 2/152) involving ynkh261 and ynkh284 together with the isolate km35 reported formed a novel separate cluster in the genotype 6 with an 82% bootstrap value, indicating a potential new hcv subtype 6. to confirm that the two strains belong to a novel hcv subtype 6, their complete genome sequences were successfully amplified and sequenced with 12 overlapping fragments. further, phylogenetic analysis was performed along with hcv reference sequences of representative subtypes 6a-6xh. the result showed that the two strains and isolate km35 formed a distinct monophyletic cluster supported by a high bootstrap value of 100%. the three strains were isolated from three hiv-1 infected patients without obvious epidemiological linkage in yunnan and showed no evidence of recombination using bootscan analysis ( fig. 1 (b) ). moreover, the intergroup nucleotide divergence (mean ± sd) % over the fulllength genome sequences of the isolates (ynkh261, ynkh284, and km35) were compared to that of representative subtypes (6a-6xh) ( fig. 1 (c) ). the results revealed that the three strains were different from known hcv subtypes of 6a-6xh by 16.5-28.5%. therefore, the three strains are initially designated 6xi. to better understand the time of emergence of hcv 6xi, we performed bayesian molecular clock analyses using full-length genome sequences to estimate the time to the most recent common ancestor (tmrca). as shown in fig. 2 (a) , the estimated tmr-cas for the genotype 6xi was 1971.1 [95% highest probability density (hpd): 1951. 1, 1991.4 ]. in addition, to further investigate baseline ras of subtype 6xi, naturally occurring resistance-associated substitutions (ras) were analyzed for the ns3, ns5a and ns5b sequences using next generation sequencing (ngs) method. strikingly, hcv 6xi strains contain the substitution 28 v with a 100% frequency of mutations in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor, 6 suggesting that the subtype 6xi maybe basically resistant to ns5a inhibitors ( fig. 2 (b) ). among the hcv eight genotypes, genotype 6 exhibits a high degree of genetic complexity and diversity, and 31 subtypes have been confirmed by the international committee on taxonomy of viruses. 7 in china, hcv genotype 6 is common, and subtype 6a is the most prevalent subtype, primarily distributed in guangdong, 6n is the second most prevalent subtype, mainly found in yunnan, followed by subtypes 6xa, 6 g, 6v, 6 w, 6e, 6b, 6j, 6q,and 6r among genotype 6 isolates. 8 -10 to our knowledge, 6xi is the eighth detection of novel hcv subtypes 6 in china combined with previously identified 6a, 6e, 6n, 6v, 6xa, 6xe and 6xh, resulting in genotype 6 to expand to 32 subtypes in the world. our findings again demonstrated that hcv genotype 6 was more complex and diverse. in summary, we characterized a new hcv subtype 6xi based on the characteristics of a monophyletic cluster, > 15% genetic distances, no significant evidence of recombination, and no epidemiologic link among individuals. in addition, bayesian analyses showed that 6xi may originate around the year 1971, and the strains of hcv 6xi naturally contain the substitution 28 v in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor. the present finding again highlights the genetic characteristics and hcv strains in yunnan, and the urgent need for continuous molecular screening and epidemic surveillance in yunnan to implement effective measures to reduce hcv transmission. the authors declare no competing financial interests. we recently read the article by corma-gómez a. et al. on which the authors described a higher probability of relapses with sofosbuvir/ledipasvir 8 weeks compared with 12 weeks of hcv (hepatitis c virus) among hiv (human immunodeficiency virus)/hcv coinfected patients. 1 in this regard, coinfections of hiv/ hcv also with hepatitis b virus (hbv) is associated with high mortality and comorbidity too. the persistence of hbv dna within the core cell in the absence of hbsag and even after clearance of the infection has been described previously in immunosuppressed patients, where hbv screening and prophylaxis is recommended. 2 interestingly, hbv reactivation has recently emerged during or after hcv treatment with direct-acting antivirals (daa). [3] [4] [5] the us food and drug administration issued a warning about this risk in 2016, 6 until that moment 24 cases were reported, two of which died. although specific mechanisms of this event are not well known, it has been suggested that hcv core proteins could inhibit hbv replication and hbsag production as well as production of envelope proteins, being hbcab the only marker of the presence of hbv. 2 in this context, treatment with daa would produce a drastic and rapidly blocking of hcv replication providing the opportunity to hbv to emerge and produce an immune reconstitution syndrome. the interference hbv-hcv has been outlined in 24% of patients with chronic hbv infection (hbsag-positive serology) and 1.4% of patients with resolved hbv infections (hbsag-negative and hbcabpositive) in a recently published systematic review and metaanalysis of patients with hcv-hbv. 7 in most cases, these reports have focused on coinfection hcv-hbv, but there is still a concern about patients triple-infected with hcv, hbv and hiv. although most cases reported occur in hbsag-positive, the main concern affects to patients with a basal serology showing hbcab-positive, hbsag-negative and hbsab-negative. hbsagpositive was associated with higher rates of hbv reactivations compared with hbsag-negative and hbcab-positive patients. 8 there is no clear information about this issue in hbsag-negative, hbcab-positive, and, when occurs, it is considered an uncommon event. 9 reactivation of hbv is characterized by reappearance or increase in hbv dna levels, and could also be accompanied by symptoms of hepatitis. the cases reported previously did show difference in the time of presentation of clinical symptoms, they used to start either during or after daa treatment. it seems that hbv reactivation usually occurs early after daa initiation treatment (4-8 weeks) while otherwise can occur after treatment completion. in that way, it seems necessary that hbv infection should be monitored early after daa initiation. the most recent european association for the study of the liver (easl) guideline about hbv infection, recommends that patients hbsag-negative and hbcab-positive undergoing daa treatment should be monitored and tested for hbv reactivation only in case of alt elevation. 10 they also recommend performing hbv dna levels only in case of alt increase. at the same time, the american association for the study of liver diseases (aasld) and the infectious disease society of america (idsa) guideline has been recently updated recommendations to monitor hbv dna levels only in patients hbsag-positive, but they emphasize that there is not enough data to monitoring dna among patients hbcabpositive or hbcab-positive and hbsab-positive. they remark that a reactivation should be considered if an unexplained increase in liver enzyme is present. in hiv patients, they provide the same recommendations. 11 in a recent review of the literature, the authors also recommend to perform an hbv dna only in patients with altered alt. 9 a meta-analysis recommended not to perform an hbv dna test in this case due to low rate of incidence and the associated cost which needs to be considered especially in an endemic hbv areas. 7 few data exist in hiv patients. the fact that many triple-infected patients are receiving antiretroviral therapy including (art) nucleoside/nucleotide analogous, as tenofovir disoproxil fumarate (tdf) or tenofovir alafenamide (taf), both active against hbv, suggest that hbv reactivation rate could be underestimated. in a recently published review, chang et al., recommend to perform an hbv dna test at baseline in triple-infected patients. if positive, an hbv-active antiretroviral therapy (art) should be started and hbv dna needs to be monitored every 4 weeks during treatment and until week 12 after completion of treatment. however, if baseline hbv dna is negative they match with aasld/idsa and easl recommendations. 12 one of our patients has a basal serology with hbcab-iggpositive, both hbsag-and hbsab-negatives, and undetectable hbv dna levels, prior to treatment with daas. regarding hiv infection, he has an analogues-free regimen. one month after having finished treatment with daas, the patient consulted because of abdominal pain, nausea and jaundice. he had a total bilirubin level of 11 mg/dl, ast 1025 iu/l, alt 642 iu/l and inr 1.30. a hbv viral load of 6,193,455 iu/ml was detected; hbsag, hbcab-igm and hbeag were positive. there were no reasons to believe that he was re-infected. treatment with entecavir was initiated; however, the clinical evolution was unfavorable and died as a result of an acute liver failure. the hbv viral load was requested from the stored samples drawn during the treatment of hcv showing that hbv viral load was undetectable at the beginning of treatment and in week 2, but progressively increased to 98 iu/ml in week 8 and up to 82,700 iu/ml in week 4 after treatment completion. during the follow-up of daa treatment alt and ast remained normal. this case changed our daily practice, since then all hiv patients and more specifically those not treated with either tdf or taf and presenting a previous hbcab-positive, are followed using dna levels and not only monitoring hepatic enzymes, as recommended by guidelines. we consider that this case may reflect the necessity of change the current guidelines. we recommend to perform a periodic monitoring of hbv reactivation using hbv dna during and after daa therapy in hbsag-negative and hbcab-positive patients, independently of hbsab presence, hepatic enzymes and clinical symptoms, particularly in hiv patients who are not receiving active treatment of hbv. the study was not funded. authors declare that there is no conflict of interest. recently, a notable pattern of synchrony of influenza a and b virus, and respiratory syncytial virus incidence peaks globally was reported in this journal. 1 a previous study characterized seasonal pattern of influenza a and b in china and identified three epidemiological regions featured by distinct seasonality. 2 on the basis of laboratory surveillance data from 30 chinese provinces spanning about 12 years from october 2004 through january 2017, our study further characterized seasonal patterns of circulating influenza a subtypes and influenza b lineages in the three defined epidemiological regions. our study revealed that pre-2009 a(h1n1) and a(h3n2) displayed wintertime and summertime epidemics in midlatitude and southernmost chinese provinces with subtropical climate, wavelet analysis demonstrated the two subtypes displayed twice-annual cycle in some years in mid-latitude chinese provinces ( fig. 1 (a)-(h) ). however, the two subtypes peaks in the winter with annual or longer cycle in northern chinese provinces. influenza a(h1n1)pdm09 b/victoria and b/yamagata virus all displayed epidemics in the winter or winter-spring with annual or longer cycle in all three epidemiological regions. we developed univariate and multivariate regression models to evaluate the association between climatic factors and the presence or absence of epidemics of each influenza subtype and lineage (positive proportion ≥5%) in the southernmost provinces where heating system is not generally used in the winter so temperature and relative humidity in external conditions in winter are close to those in indoor environment where people spend most of the time. we fitted the mixed-effects logistic regression model to control for the repeated measurements in each province of the region cluster. we kept one of temperature and absolute humidity (representative of vapor pressure) with smaller akaike information criterion in the model to reduce of multi-collinearity due to a high degree of correlation between the two factors. our analysis indicated temperature, humidity and rainfall were environmental predictors of influenza subtype/lineage-specific epidemics in the southernmost provinces when a 1-week lag of influenza epidemics behind climate was considered, which was similar to the findings from some ecological studies ( table 1 ). 3 our study indicated the u-shaped relationship between absolute humidity (ah) and pre-2009 a(h1n1) or a(h3n2) epidemics in the southernmost provinces, and suggest that high levels of ah in the summer, and low levels of ah in the winter increased the possibility of epidemics of the two subtypes. 3 in our analysis, lower temperature was an environmental driver of a (h1n1)pdm09 and b/yamagata epidemics in the southernmost provinces while there were bimodal associations between temperature, rainfall and b/victoria epidemics with highest probability of b/victoria epidemics at 11.7 °c of daily average temperature and 11.3 mm of daily average rainfall. although seasonal changes in human behaviors, such as school attendance or crowding indoors, and seasonal variations in immunity, such as melatonin and vitamin d levels have been proposed to account for the seasonal nature of influenza, 4 our findings suggest that the heterogeneity in influenza subtype/lineage-specific seasonality patterns could be driven by seasonal variations in virus survival, transmission and adaptive immunity by influenza subtype and lineage because of the same behavior modes and background of non-adaptive immunity in the same regions and seasons. we propose a hypothesis: under humid and hot condition the dominant transmission mode(s) for a(h1n1)pdm09, b/victoria and b/yamagata might have reduced efficiency, however, there could be effective transmission mode(s) for a(h3n2) and pre-2009 a(h1n1) virus. some experimental studies were performed to establishing a causal link between humidity, temperature and influenza virus survival/transmission. 5 transmission of influenza a (h3n2), a(h1n1)pdm09, b/victoria and b/yamagata virus by respiratory droplets or aerosols in the guinea pig model proceeds most readily under cold, dry conditions. 5 low humidity and temperature increased the stability of influenza virus in aerosols and on surfaces. furthermore, aerosol transmission of a (h3n2) virus in the guinea pig model was almost completely blocked at 30 °c, but contact transmission of a (h3n2) virus seemed to be efficient at different level of humidity and 30 °c. 5 it remains unclear if high temperature and humidity levels have effect on aerosol and contact transmission of pre-2009 a(h1n1), a(h1n1)pdm09, b/victoria and b/yamagata virus among hosts and their stability on surfaces. of note, an experimental study found the survival durations of a(h3n2) strains on swiss banknotes were significantly longer than pre-2009 a(h1n1) and b/victoria virus. 6 further studies are needed to understand how efficient are these transmission modes for different influenza subtypes or lineages, which is/are dominant mode(s) of transmission among hosts, and which of potential mechanisms is at play under humid and hot condition. one of our findings was the mutual inverse association between a(h3n2) and a(h1n1)pdm09 epidemics, which provided the evidence on interference between the two influenza a subtypes perhaps possibly due to multiple immune mechanisms. 7 however, our study showed b/yamagata epidemics were positively correlated with a(h3n2) epidemics, suggesting b/yamagata epidemics across over 12 study years that were weak could get well along with simultaneous weak h3n2 epidemics. understanding of influenza seasonality is important to define optimal timing of influenza vaccination campaigns. our study indicated that a(h3n2) virus brought about twice-annual epidemics in some years in mid-latitude chinese provinces and more frequent summertime epidemics in southernmost chinese provinces, which questions if a single annual influenza vaccination campaign starting in october can offer optimal protection against summertime epidemics of a (h3n2) virus in mid-latitude and southernmost chinese provinces. in recent years, it has been reported that mismatches of a(h3n2) virus between the influenza vaccine strains and circulating strains were identified frequently, 8 and vaccine effectiveness of a(h3n2) virus declined within 4-6 months postvaccination. 9 in conclusion, we identified the heterogeneity of seasonality pattern of pre-2009 a(h1n1) or a(h3n2) virus in three epidemiological regions of china, and different environment predictors for influenza subtypes and lineages in the southernmost provinces. further work should focus on understanding difference in virus survival, transmission by influenza subtype and lineage under humid and hot conditions. bjc has received research funding from medimmune inc. and sanofi pasteur, and consults for crucell nv. the authors report no other potential competing interests. as this study included data from the national influenza surveillance system, ethics approval was not required. not applicable. the datasets at national level analyzed during the current study are available in the world health organization flunet. the datasets with more specific information analyzed during the current study are available in chinese national influenza surveillance informatio system, but they are not open-access datasets. these influenza surveillance data can be available from chinese national influenza center on reasonable request. this study was supported by the national mega-projects for infectious diseases (grant number 2018zx102010 02-0 08-001 ), national natural science foundation of china (grant number 81302476 ) and emergency prevention and control project of ministry of science and technology (grant number 10600100000015001206 ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. reported in zhejiang in 2017. 8 in this study, we identified ten cases of imported chikv infection in travelers returning to yunnan from southeastern asia in 2019. out of the ten patients with imported chikv infection examined in this study, nine patients had traveled back from myanmar and one patient had travelled back from thailand ( fig. 1 (a) ). all the patients displayed different degrees of symptoms, such as fever, cough, muscle pain, and rash. the details of all the symptoms are shown in table 1 . chikv infection was diagnosed using specific real-time reverse transcription-pcr. serum specimens were collected from the ten patients that tested positive for chikv by real-time pcr analysis, in yunnan between may 7, 2019 and august 1, 2019. the current study was approved by the medical ethics committee of kunming university of science and technology. written informed consent was obtained from all the participants. a new coronavirus associated with human respiratory disease in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin emergence of sars-like coronavirus poses new challenge in china bat origin of a new human coronavirus: there and back again transmission of 2019-ncov infection from an asymptomatic contact in germany early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia what to do next to control the 2019-ncov epidemic? nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) national health commission of people's republic of china prevention and control of novel coronavirus pneumonia a hospital cluster combined with a family cluster of avian influenza h7n9 infection in anhui province first human infection by a novel avian influenza a(h7n4) virus contact reductions from live poultry market closures limit the epidemic of human infections with h7n9 influenza comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds death of a very young child infected with influenza a (h5n6) equine influenza-a global perspective isolation and characterization of highly pathogenic avian influenza virus subtype h5n1 from donkeys molecular epidemiology of a novel re-assorted epidemic strain of equine influenza virus in pakistan in 2015-16 avian-origin h3n2 canine influenza virus circulating in farmed dogs in guangdong detection of pediatric bacterial meningitis pathogens from cerebrospinal fluid by next-generation sequencing technology next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center community-acquired bacterial meningitis rapid bacterial identification by direct pcr amplification of 16s rrna genes using the minion tm nanopore sequencer campylobacter fetus meningitis confirmed by a 16s rrna gene analysis using the minion nanopore sequencer bacterial diversity assessment in antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation rapid diagnosis of bacterial meningitis by nanopore 16s amplicon sequencing: a pilot study diagnosis of haemophilus influenzae pneumonia by nanopore 16s amplicon sequencing of sputum epidemiology of invasive group b streptococcal infections among nonpregnant adults in the united states comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses impact of a poorly performing point-ofcare test during the 2017-2018 influenza season uk severe flu surveillance system icu-hdu influenza surveillance nhse) service specification 170110s. extra corporeal membrane oxygenation (ecmo) for respiratory failure in adults australia and new zealand extracorporeal membrane oxygenation (anz ecmo) influenza investigators extracorporeal membrane oxygenation for 2009 influenza a(h1n1) acute respiratory distress syndrome intensive care unit patients with 2009 pandemic influenza a (h1n1pdm09) virus infection -united states characteristics of patients with hospital-acquired influenza a (h1n1)pdm09 virus admitted to the intensive care unit cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection guidelines for the management of adults with community-acquired pneumonia. diagnosis, assessment of severity, antimicrobial therapy, and prevention cytokine markers as predictors of type of respiratory infection in patients during the influenza season procalcitonin as a marker of etiology in adults hospitalized with community-acquired pneumonia cd5l contributes to the pathogenesis of methicillin-resistant staphylococcus aureus-induced pneumonia orchestrating role of apoptosis inhibitor of macrophage in the resolution of acute lung injury aim inhibits apoptosis of t cells and nkt cells in corynebacterium-induced granuloma formation in mice the macrophage soluble receptor aim/api6/cd5l displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression assessment of apoptosis inhibitor of macrophage/cd5l as a biomarker to predict mortality in the critically ill with sepsis plasma long pentraxin 3 (ptx3) concentration is a novel marker of disease activity in patients with community-acquired pneumonia the updated analysis of african swine fever virus genomes: two novel genotypes are identified african swine fever virus replication and genomics phylogeographic patterns of the african swine fever virus genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype the recombination hot spots and genetic diversity of the genomes of african swine fever viruses parallelization of the mafft multiple sequence alignment program rdp4: detection and analysis of recombination patterns in virus genomes structure of the african swine fever virus major capsid protein p72 detection of potential problematic cytb gene sequences of fishes in genbank assessing dna barcoding as a tool for species identification and data quality control genetic characterisation of african swine fever viruses from recent and historical outbreaks in sardinia consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group hepatitis c virus infection in children and adolescents challenges and perspectives of direct antivirals for the treatment of hepatitis c virus infection whole-genome characterization and resistance-associated substitutions in a new hcv genotype 1 subtype identification of 19 novel hepatitis c virus subtypes-further expanding hcv classification hepatitis c virus drug resistance associated substitutions and their clinical relevance: update identification of a new hcv subtype 6xg among injection drug users in kachin hepatitis c virus genotypes and subtypes circulating in mainland china higher relapse rate among hiv/hcv-confected patients receiving sofosbuvir/ledipasvir for 8 vs 12 weeks significance of anti-hbc alone serological status in clinical practice evaluation of hepatitis b reactivation among 62,920 veterans treated with oral hepatitis c antivirals acute fulminant hepatitis b during hepatitis c virus therapy with direct-acting antivirals in a patient co-infected with hiv spontaneous remission of hepatitis b virus reactivation during direct-acting antiviral agent-based therapy for chronic hepatitis c adverse events reporting system (faers): the public's stake in adverse event reporting hepatitis b virus reactivation during direct-acting antiviral therapy for hepatitis c: a systematic review and meta-analysis reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals controversies in hepatitis c therapy: reactivation of hepatitis b virus clinical practice guidelines on the management of hepatitis b virus infection monitoring patients who are starting hcv treatment, are on treatment, or have completed therapy. hcv guidance: recommendations for testing, managing, and treating hepatitis c. electronic access significance and management of isolated hepatitis b core antibody (anti-hbc) in hiv and hcv: strategies in the daa era comparative global epidemiology of inßuenza, respiratory syncytial and parainßuenza viruses characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data environmental predictors of seasonal influenza epidemics across temperate and tropical climates global influenza seasonality: reconciling patterns across temperate and tropical regions roles of humidity and temperature in shaping influenza seasonality survival of influenza virus on banknotes a systematic approach to virus-virus interactions comparing influenza vaccine efficacy against mismatched and matched strains a systematic review and meta-analysis i-move multicentre case-control study 2010/11 to 2014/15: is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination? metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers chikungunya virus infections in military deployments in tropical settings-a narrative minireview chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen east/central/south african genotype in a chikungunya outbreak caribbean and la réunion chikungunya virus isolates differ in their capacity to induce proinflammatory th1 and nk cell responses and acute joint pathology new insights into chikungunya virus emergence and spread from southeast asia mosquito-associated viruses in china chikungunya fever outbreak enhanced molecular surveillance of chikungunya virus chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes this work was supported by henan emergency project for prevention and control of 2019 novel coronavirus, the earmarked fund for modern agro-industry technology research system of china (cars-35) and the special fund for henan agriculture research system (s2012-06). the funders had no role in study de-sign, data collection and interpretation, or the decision to submit the work for publication. we thank jing li and hao-nan wang for the helpful discusses and suggestions. this work is supported by the open research fund of key laboratory of digital earth science ( 2019lde005 ), and by the fundamental research funds for the central universities under grant no. 310201911qd054 . this work was supported by the national natural science foundation of china ( 31702271 ) and the guangdong provincial natural science foundation ( 2017a030310367 ). we thank le kim thanh, le nguyen truc nhu, and lam anh nguyet for their logistic support. we are indebted to patients for their participations in this study.this study was funded by the wellcome trust of great britain ( 10 6 680/b/14/z and 204904/z/16/z ). supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.011 . we would like to thank the patients and healthy volunteers in the first affiliated hospital of chongqing medical university for their cooperation and support. this work has been financially supported by national natural science foundation of china (no. 81902134 and no. 81722001 ). we thank chinese national influenza surveillance network for contribution in influenza epidemiological and laboratory surveillance. we thank the members of the yunnan international travel healthcare center and kunming changshui airport customs for the data and sample collection. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.003 . supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.11.019 . recent correspondence in this journal has highlighted the current threat posed by recently-emerging imported chikungunya virus (chikv) in febrile returning travellers. 1 chikungunya fever is infection caused by the chikv and is characterized by fever, arthralgia, myalgia, headache, and rash. chikv belongs to the genus alphavirus within the togaviridae family and is transmitted to humans by the bite of infected mosquitoes-ae. aegypti and ae. albopictus . 2 since the first report of chikv infection in humans in tanzania, intermittent outbreaks have been documented in africa, south america, southern and southeastern asia, and the indian ocean islands; thus, its outbreak has become a global public health problem. 3 to date, three evolutionary distinct chikv genotypes, namely west african (wa), east/central/south african (ecsa), and asian have been identified, based on phylogenetic analyses. 4 a novel lineage of chikv-the indian ocean lineage (iol)-has also been reported, which descended from the ecsa genotype during an outbreak on the island of la reunion between 2005 and 2006. 5 recently, it has been reported that iol of chikv has spread to malaysia, singapore, thailand, and indonesia. 6 in china, the first case of imported chikv infection was found in yunnan in 1987. subsequently, several sporadic cases of nonindigenous chikv infection have been described. in 2010, the first outbreak of chikv fever with 129 cases was documented in guangdong. 7 recently, another outbreak of chikungunya fever was table 1 epidemiological information on ten febrile returning travelers infected with chikv in this study. in this study, nine complete genome sequences isolated from serum samples were successfully amplified and sequenced with 12 overlapping fragments, and then the sequence obtained was deposited in genbank under accession no. mn402883-mn402892. compared with the nucleotide sequences of the available from the ncbi database, the nine strains shared the highest 99.61-99.74% nucleotide identity with the east/central/south african lineage strain thail 2019 reported previously in thailand, 2019. further, bayesian maximum-clade-credibility tree for full-length nucleotide sequences were constructed using the beast package v.1.7.3. phylogenetic analyses revealed that the ten chikv strains clustered into the homogeneous indian ocean clade of the ecsa genotype ( fig. 1 (b) ).notably, the ten chikv strains isolated from serum samples possessed the mutation k211e in the e1 gene and v264a in the e2 gene; these mutations are associated with significant increase in viral infectivity in ae. aegypti . 9 the strains also possessed g60d and i211t substitutions in the e2 gene; these mutations contribute to increased chikv fitness in ae. albopictus. 9 however, the a226v mutation in the e1 gene that is related to significant increase in viral infectivity in ae. albopictus was not observed in any of the strains. 10 in summary, we characterized ten cases of human infection caused by imported ecsa genotype chikv in yunnan, china and successfully isolated nine infectious chikvs from the chikvpositive serum samples. the mutations associated with significant increase in viral infectivity for ae. aegypti or ae. albopictus were also observed in these strains. geographically, the yunnan province is in southeastern china and shares its border with southeast asian countries (laos, vietnam, and myanmar) that are most affected by chikv. with the increase of tourism and trade with southeast asian countries, cases of imported chikv infection are constantly increasing and may have the potential for re-emergence and autochthonous transmission to yunnan. the present study highlights the urgent need for continuous molecular screening and epidemic surveillance for chikv and its vectors to prevent future outbreaks of chikv infection among the human population of yunnan. this work was supported by the national natural science foundation of china (nsfc) ( u1702282 ), the reserve talents project for young and middle-aged academic and technical leaders of yunnan province (2019hb012), and youth talent program of yunnan "ten-thousand talents program" (ynwr-qnbj-2018-054). the authors declare no competing financial interests. key: cord-022380-49oti4zg authors: panlilio, adelisa l; gerberding, julie louise title: occupational infectious diseases date: 2009-05-15 journal: textbook of clinical occupational and environmental medicine doi: 10.1016/b978-0-7216-8974-6.50026-9 sha: doc_id: 22380 cord_uid: 49oti4zg nan infections acquired in the work setting represent an eclectic group that is seldom, if ever, considered together as a single category. occupational infections involve several organ systems, respiratory, enteric, and skin infections being particularly common. transmission involves not only casual person-to-person contact, but also a variety of other routes in special work environments. it is thus important to consider what unique features characterize the infectious diseases that can be considered occupational. perhaps one useful way of thinking about these diseases is that, as a group, they tend to be transmitted during work schedules or practices that are systematized. therefore, they can be anticipated, and to the extent that unsafe infectionprone practices can be identified and modified, they can be systematically prevented. another common feature is that many of the occupational infectious diseases can be regarded as behavioral. to the extent that unsafe practices have been defined, and practice policies modified to reduce infection risk, continued transmission often represents failure to follow accepted standards. although certain occupational infections can be prevented by vaccines (e.g., hepatitis b), prevention often depends on simple behavioral changes, such as hand hygiene, use of gloves, and not working while ill. finally, the anthrax cases during the fall of 2001 in the united states demonstrate the possibility of intentional (and criminal) exposure to infectious agents in the workplace, in this instance among those handling mail. 1 these intentional exposures, fortunately, are rare events, but should be considered in assessing sources of exposure for unexpected illnesses. prevention depends primarily on defining risky occupational practices or environments, clearly articulating policies for preventing communicable disease acquisition; removing structural barriers to compliance with policies (e.g., providing soap, hand cleansers, and gloves; allowing time away from work during periods of illness); and promoting healthy practices through behavioral change. because infectious diseases may represent the most common cause of time lost from work, it is important for the clinician concerned with occupational medicine to understand the relationship of specific infections to specific work environments and practices, and to give at least as much attention to prevention as to diagnosis and treatment. occupationally acquired infections have historically been associated with animal exposures and unsanitary work environments. modernization of agrarian techniques and improvement in sanitation have markedly decreased the incidence of these infections in the developed world, though they remain problematic in developing countries. while nearly all infectious diseases could conceivably be transmitted in the workplace, the emphasis here is on those that can be transmitted by casual contact or by specific workrelated exposures, with emphasis on diseases that are most common, most serious, or most readily prevented. healthcare settings pose a unique challenge because of the proximity of infectious patients, susceptible patients, and healthcare personnel. infections transmitted from personnel may have devastating effects on certain groups of patients, particularly the immunosuppressed. likewise, certain infections transmitted to personnel, such as multidrug-resistant tuberculosis, may have serious or even fatal, consequences. table 22 .1 summarizes the microbial etiology, sources, routes of infection, categories of workers at risk, and clinical manifestations of selected infectious diseases that have occupational predilections. a detailed discussion of waterborne microbial diseases is also provided in chapter 54. because the treatment of occupationally acquired infections does not differ from that of infections acquired non-occupationally, 2 the emphasis of this chapter is on the recognition and prevention of these infections. etiologic category, as well as the potential for recurrences, account for the high incidence of the common cold, even among healthy adults. colds are more common in the fall, winter, and early spring, perhaps because of increased crowding among children during the colder seasons. workers are most apt to acquire colds from exposure to young children in the home. secondary cases among coworkers may then develop. adults experience two to four colds each year, although the incidence among adult women exceeds that of men by a small margin, and smokers have a substantially increased risk. the modes of transmission of cold viruses are not entirely elucidated. for rhinoviruses, transmission among experimental subjects occurs most readily by direct handto-hand contact, with a case followed by autoinoculation of the mucous membranes of the eye or nose. such finger-tomucous membrane contact is ubiquitous and unavoidable. other viruses are transmissible by aerosolized droplets. the importance of fomites (such as drinking glasses, telephone receivers, and shared office equipment) as vectors of transmission has not been determined. clinical manifestations typical cold symptoms include nasal congestion, coryza, non-productive cough, sneezing, pharyngitis, and laryngeal irritation. fever is often low grade, or may be absent. viral upper respiratory infections usually resolve within 7-10 days, but longer durations are not uncommon. treatment, prevention, and control treatment for uncomplicated infections is symptomatic. decongestants are more useful in relieving symptoms than are antihistamines. expectorants, saline gargles, and other nonprescription remedies are useful in some cases. antibiotics should not be prescribed for the treatment of colds. colds are difficult to prevent. a policy of work restriction until symptoms improve may prevent the spread of colds but is likely to be impractical (table 22. 2). the cost-benefit analysis of such an approach could be useful, especially in childcare and healthcare settings. hand washing after contact with nasal secretions may be helpful. care should be taken to use tissues when coughing or sneezing and to dispose of soiled tissues after use. epidemiology influenza is a self-limited respiratory illness caused by types a and b influenza virus. epidemics of influenza occur annually in the winter months. adults remain susceptible to the illness despite prior episodes of infection because the antigenic structure of influenza viruses changes frequently, leading to new epidemics. influenza is spread from person to person, primarily by the coughing and sneezing of infected persons or sometimes by direct contact, either with infected persons or a contaminated surface. the disease is easily transmitted, and a single index case may transmit to a large number of susceptible persons in a short period of time. adults and children typically are infectious from 1-2 days before through 5-6 days after the onset of symptoms. clinical syndromes an attack of influenza starts abruptly with fever, malaise, myalgia, and headache. respiratory symptoms mimicking those of the common cold and lower respiratory symptoms including dry cough also are frequent. fever resolves in uncomplicated cases in 48-72 hours, but other symptoms may persist for days to weeks. influenza pneumonia, associated with hypoxemia, cough, and interstitial infiltrates, is not common in healthy adults. elderly patients and those with underlying immunodeficiencies and chronic pulmonary diseases are at high risk for secondary bacterial pneumonias, often caused by streptococcus pneumoniae and less often by haemophilus influenzae and staphylococcus aureus. the diagnosis of influenza frequently is made on the basis of clinical symptoms and signs. however, influenza is very difficult to differentiate from respiratory illnesses caused by other pathogens on the basis of clinical symptoms alone. other pathogens that can cause similar symptoms include, but are not limited to, mycoplasma pneumoniae, adenovirus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza viruses, and legionella species. many pathogens, including influenza, rsv, and parainfluenza, cause outbreaks in a seasonal pattern. laboratory confirmatory tests can be performed to differentiate influenza from other illnesses. appropriate patient samples to collect for laboratory testing can include a nasopharyngeal or throat swab from adults, or nasal wash or nasal aspirates, depending on which rapid test is used. samples should be collected within the first 4 days of illness. rapid influenza tests provide results within 24 hours; viral culture provides results in 3-10 days. most of the rapid tests are more than 70% sensitive for detecting influenza and more than 90% specific. because as many as 30% of samples that would be positive for influenza by viral culture may give a negative rapid test result, negative rapid tests should be followed by viral culture in a sample of the swabs collected. viral culture can also identify other causes of influenza-like illness when influenza is not the cause. serum samples can be tested for influenza antibody to diagnose acute infections. two samples should be collected per person: one sample within the first week of illness and a second sample 2-4 weeks later. if antibody levels increase from the first to the second sample, influenza infection likely occurred. because of the length of time needed for a diagnosis of influenza by serologic testing, other diagnostic testing should be used for rapid detection of possible outbreaks. during community outbreaks, specific virologic or serologic diagnosis is not necessary once the type(s) of influenza virus causing the outbreak have been identified. treatment, prevention, and control persons at high risk for serious morbidity (persons aged 65 and older, persons with chronic underlying diseases) should receive influenza vaccine annually. immunization also is recommended for healthcare personnel and others at risk for transmitting influenza to high-risk patients ( had been associated with reduced work absenteeism and fewer deaths among nursing home patients. 3, 4 most employers do not provide influenza prevention programs for workers outside the healthcare field. 3 amantadine and rimantadine can reduce the duration of uncomplicated influenza a illness when administered within 2 days of onset of illness in otherwise healthy adults. zanamavir and oseltamivir can reduce the duration of uncomplicated influenza a and b illness by approximately 1 day compared with placebo. none of these antiviral agents has been shown to be effective in preventing serious influenza-related complications. to reduce the emergence of antiviral drug-resistant viruses, the duration of therapy should typically be no longer than 5 days. both amantadine and rimantadine are indicated as prophylaxis for influenza a, but not for influenza b infection. oseltamivir has been approved as prophylaxis for influenza a and b. zanamivir has not been approved for prophylaxis, but has been shown to be as effective as oseltamivir in preventing febrile, laboratory-confirmed influenza illness. they are approximately 70-90% effective in preventing illness from influenza a. chemoprophylaxis can be a component of influenza outbreak control programs. 3 epidemiology the incidence of measles (rubeola) has steadily declined in the united states during the last decade and is no longer considered endemic. measles is a major cause of morbidity and mortality worldwide. the majority of cases in the united states in recent years have been imported or secondary cases epidemiologically linked to imported cases. 5 infected persons are highly contagious by the air-borne route during the viral prodrome, and when cough and coryza are prominent, until about 2 days after the rash appears. infection confers lifelong immunity. although most adults born prior to 1957 experienced childhood infection and are no longer susceptible, up to 10% may lack natural immunity. in recent epidemics, cases occurred among unimmunized children, as well as children and young adults who had received a single vaccination with live virus, and among older adults. 5 clinical syndromes measles progresses in several phases. initial virus replication occurs in the respiratory tract and leads to a primary viremic phase, which usually is asymptomatic. release of virus from infected reticuloendothelial cells produces secondary viremia and infection of the entire respiratory system, accompanied by symptoms of coryza, cough, and in some, bronchiolitis or pneumonia. koplik's spots, a bluish-gray enanthem most prominent on the buccal mucosa, precede development of the rash. in a typical case of measles, the rash begins on the face, then progresses to the trunk and distal extremities, and disappears in the same sequence after 5-6 days. treatment, prevention, and control live-attenuated vaccine for prevention of measles became available in the early 1960s. all healthy children should receive the vaccine at age 15 months. because 10% do not respond to a single dose of vaccine, a second dose is now recommended to improve vaccine efficacy. 7 all healthy adults born after 1957 who have not received two doses of live virus vaccine or have not experienced measles also are advised to receive vaccine (table 22. 3). persons who received killed vaccine have a risk of developing atypical measles, and require re-immunization with live virus vaccine. live virus vaccine is contraindicated in infants, pregnant women, and immunosuppressed persons. passive immunization with γ globulin is available for unimmunized persons exposed to infected individuals, but is not routinely recommended for adults. measles rarely can exacerbate tuberculosis and cause a temporary inhibition of delayed hypersensitivity. vaccine administration should be delayed for 1 month after tuberculin testing, and until treatment is under way in persons with active tuberculosis. epidemiology mumps is a viral illness transmitted by the oral or respiratory route during contact with contaminated fomites or aerosolized droplet secretions. mumps is less contagious than measles or rubella but produces significant morbidity, especially among adults. the incubation period ranges from 2 to 3 weeks. virus is detectable for 6 days prior to and 9 days after the appearance of symptoms. most adults are immune to mumps, but 10-20% of unimmunized adults have no serologic evidence of prior infection and are considered susceptible. mumps incidence is now very low in all areas of the united states. the substantial reduction in mumps incidence during the past few years likely reflects the change in the recommendations for use of measles mumps rubella (mmr*) vaccine. 6 clinical syndromes parotitis typically is bilateral, but unilateral disease and involvement of other salivary glands occurs in some persons. localized parotid tenderness and swelling, fever, and painful swallowing suggest the diagnosis. aseptic meningitis is common but benign. encephalitis is a rare but serious manifestation. about 25% of affected postpubescent men develop orchitis, epididymitis, or both, which is bilateral in 15% of cases and may be the sole manifestation of mumps infection. about 50% of cases of mumps orchitis result in testicular atrophy, but neither sterility nor impotence are common sequelae. oophoritis occurs in about 5% of women with mumps. vaccine separated by at least 1 month (i.e., a minimum of 28 days), and administered on or after the first birthday, are recommended for all children and for certain highrisk groups of adolescents and adults. 6 adult men and healthcare personnel with no history of mumps or mumps immunization should be screened for immunity and vaccinated if they are susceptible. immunization is contraindicated in persons with immunosuppression and in pregnant women (table 22. 3). passive immunization with mumps immune globulin decreases the incidence of orchitis and is recommended for mumps in adult men with a single testis. individuals with active mumps should be excluded from work until 9 days after the onset of parotitis to avoid transmission to others in the workplace (table 22. 2). epidemiology fifth disease, also called erythema infectiosum or 'slapped cheek disease', is an infection caused by parvovirus b19. it is a common rash illness that is usually acquired in childhood, but can be an occupational risk for school and childcare personnel. it has been transmitted to personnel in healthcare settings. clinical syndromes symptoms begin with mild fever and symptoms of fatigue. after a few days, the cheeks take on a flushed 'slapped' appearance. there may also be a lacy rash on the trunk, arms, and legs. not all infected persons develop a rash. the child is usually not very ill, and the rash resolves in 7-10 days. most persons who get fifth disease are not very ill and recover without any serious consequences. an adult who is not immune can be infected with parvovirus b19 and either have no symptoms or develop the typical rash of fifth disease, joint pain or swelling, or both. usually, joints on both sides of the body are affected. the joints most frequently affected are the hands, wrists, and knees. the joint pain and swelling usually resolve in a week or two, but they may last several months. about 50% of adults, however, have been previously infected with parvovirus b19, have developed immunity to the virus, and cannot get fifth disease. fifth disease is believed to be spread through direct contact, fomites, or large droplets. the period of infectivity is before the onset of the rash. once the rash appears, a person is no longer contagious. the incubation period is 4-14 days but may be as long as 20 days. treatment, prevention, and control symptomatic treatment for fever, pain, or itching is usually all that is needed for fifth disease. adults with joint pain and swelling may need to rest, restrict their activities, and take anti-inflammatory medications to relieve symptoms. transmission can be prevented by careful attention to hygiene, especially hand washing. no special precautions are necessary. excluding persons with fifth disease from work, childcare centers, or schools is not likely to prevent the spread of the virus, since people are contagious before they develop the rash. epidemiology pertussis, or whooping cough, is an acute infectious disease caused by the bacterium bordetella pertussis. pertussis continues to be an important cause of mortality in the united states. a dramatic decline in the incidence followed the widespread use of whole-cell pertussis vaccines in the mid-1940s. however, since the early 1980s, the reported pertussis incidence has increased cyclically with peaks occurring every 3-4 years. 7, 8 contributing to this increase in incidence is the waning of immunity over time following vaccination, particularly in older age groups. transmission most commonly occurs by contact with respiratory secretions or large aerosol droplets from the respiratory tracts of infected persons and less frequently by contact with freshly contaminated articles of an infected person. analysis of national surveillance data for pertussis during 1997-2000 indicates that pertussis incidence continues to increase in infants too young to receive three doses of pertussis-containing vaccine and in adolescents and adults. 7 clinical syndromes the incubation period of pertussis is commonly 7-10 days. pertussis begins insidiously with non-specific upper respiratory symptoms including coryza, sneezing, low-grade fever, and a mild, occasional cough, similar to the common cold. the cough gradually becomes more severe, and after 1-2 weeks, the second, or paroxysmal stage, begins. characteristically, the patient has paroxysms of numerous, rapid coughs generally with a characteristic high-pitched whoop, commonly followed by vomiting and exhaustion. the patient usually appears normal between attacks. older persons (i.e., adolescents and adults), and those partially protected by the vaccine may become infected with b. pertussis, but usually have milder atypical disease. pertussis in these persons may present as a more persistent cough of greater than 7 days duration, and may be indistinguishable from other upper respiratory infections. inspiratory whoop is uncommon. b. pertussis is estimated to account for up to 7% of cough illnesses per year in older persons. even though the disease may be milder in older persons, these infected persons may transmit the disease to other susceptible persons, including unimmunized or underimmunized infants. the medical management of pertussis cases is primarily supportive, although antibiotics are of some value, with erythromycin being the drug of choice. this therapy eradicates the organism from secretions, thereby decreasing communicability and, if initiated early, may modify the course of the illness. there is no pertussis-containing vaccine (including dtap) currently licensed for persons 7 years of age or older, and vaccination with dtap currently is not recommended after the 7th birthday. vaccine reactions are thought to be more frequent in older age groups, and pertussis-associated morbidity and mortality decrease with increasing age. studies are currently under way to determine if a booster dose of acellular pertussis vaccine administered to older children or adults may reduce the risk of infection with b. pertussis. this may in turn reduce the risk of transmission of b. pertussis to infants and young children who may be incompletely vaccinated. studies among older children, adolescents, and adults examining pertussis disease burden and transmission of disease to infants might guide future policy decisions on the use of acellular pertussis vaccines among persons more than seven years of age. currently, vaccination of children more than 7 years of age, adolescents, and adults is not recommended either routinely or as an outbreak control measure. in the future, licensure of pertussis vaccines for adolescents or adults may lead to new recommendations for the use of vaccines in outbreaks. 9 epidemiology most epidemics of bacterial pneumonia in the workforce are due to community-acquired infections. however, legionellosis is one type of pneumonia which can be transmitted in the workplace. legionella pneumophila is an important cause of both epidemic and endemic adult pneumonia, and it can be associated with outbreaks in the workplace. this organism colonizes aquatic ecosystems and potable water, and it is transmitted to humans by the air-borne route. contaminated air conditioners, humidifiers, and shower heads have been implicated in outbreaks among workers and hospital patients. outbreaks of legionellosis have occurred after persons have breathed mists that come from a water source (e.g., air conditioning cooling towers, whirlpool spas, showers) contaminated with legionella bacteria. persons may be exposed to these mists in homes, workplaces, hospitals, or public places. legionellosis is not passed from person to person. a careful occupational history should be obtained from all adults who present with pneumonia, because occupational exposures cause many otherwise rare pneumonias. public health authorities should be notified if an occupational source is suspected so that an epidemiologic investigation to identify transmission routes and other susceptible individuals may commence. clinical pneumonia syndromes community-acquired bacterial pneumonia usually is exhibited acutely, with fever, chills, productive cough, and often, pleurisy. chest examination demonstrates signs of consolidation that may be confirmed radiologically. sputum examination may aid implementation of empiric therapy by suggesting the etiologic pathogen. blood cultures should be obtained when invasive disease is suspected, and lumbar puncture to evaluate meningeal fluid is indicated when symptoms or signs of meningitis are present. patients with legionnaire's disease usually have fever, chills, and a cough, which may be dry or productive. some patients also have muscle aches, headache, tiredness, loss of appetite, and, occasionally, diarrhea. chest x-rays often show pneumonia but are not pathognomonic. it is difficult to distinguish legionnaire's disease from other types of pneumonia by symptoms alone; other tests are required for diagnosis. the definitive test is culture isolation of the organism in sputum, bronchoalveolar fluid, or pleural fluid. other useful diagnostic tests detect the bacteria in sputum by specialized stains, identify legionella antigens in urine samples, or compare antibody levels to legionella in two blood samples obtained 3-6 weeks apart. the time between the patient's exposure to the bacterium and the onset of illness for legionnaire's disease is 2-10 days. treatment, prevention, and control empiric ambulatory therapy of acute community-acquired bacterial pneumonia, not requiring hospitalization, should include coverage for pneumococcus and h. influenzae, if the patient has a history of chronic obstructive lung disease. amoxicillin, trimethoprim-sulfamethoxazole and cefixime are reasonable choices, unless atypical pneumonia caused by m. pneumoniae or c. pneumoniae is suspected, in which case erythromycin is preferred. erythromycin is the antibiotic currently recommended for treating persons with legionnaire's disease. in severe cases, a second drug, rifampin, may be added. preventing bacterial pneumonia is a difficult challenge. workers at risk for pneumococcal and haemophilus infections should be immunized, although the efficacy of this approach among patients at highest risk is debated (table 22. 3). influenza immunization could eliminate a major risk factor for both primary and secondary bacterial pneumonias. occupational exposures to potential pathogens should be minimized with proper ventilation. prevention of legionellosis is achieved by maintaining an environment that is not conducive to survival or multiplication of legionella. the necessary preventive measures may involve water treatment or modification of air conditioning and ventilation systems. 10 these preventive steps, which may be costly, should be directed at healthcare facilities, and occupational settings where cases have been identified. 11 epidemiology rubella (german measles) virus is transmitted person to person by mucosal exposure to infected droplets of respiratory secretions. since 1969, children in the united states have been routinely immunized against rubella at age 15 months, so that the majority of recognized cases today occur in adults and unimmunized children. since 1992, reported indigenous rubella has continued to occur at a low but relatively constant endemic level with an annual average of less than 200 rubella cases. 7 recent data indicate that the rate of rubella susceptibility and risk for rubella infection are highest among young adults. no large epidemics have occurred since the vaccine was licensed for use in 1969. however, outbreaks continue to occur among groups of susceptible persons who congregate in locations that increase their exposure, and among persons with religious and philosophic beliefs against vaccination. several recent outbreaks have occurred in workplaces where most employees are foreign born, particularly from latin america. 6 reinfection can occur following natural or acquired immunity, but it is usually asymptomatic and only rarely accompanied by viremia. rubella virus is shed from the respiratory tract of infected persons beginning 10 days before the development of rash and for several days after the rash appears. the onset of the rash coincides with the period of maximal contagiousness, and infected persons are not considered infectious for more than 7 days after the rash appears. infected infants shed virus for several months despite the presence of antibody. clinical syndromes adult rubella is often asymptomatic. symptoms occur 12-23 days after exposure. following a prodrome of fever and malaise, adults exhibit a maculopapular rash that begins on the face and extends downward, persists for 3-5 days, and often is accompanied by regional lymphadenopathy of the head and neck, which persists for days to weeks. one-third of adult women may develop arthritis in the fingers, knees, and wrists during the exanthematous phase of illness. children develop hemorrhagic complications more often than adults. in contrast, encephalitis, albeit rare, is more common in adults and is fatal in 20-50% of cases. maternal rubella infection acquired in the first 20 weeks of gestation frequently results in congenital rubella. the earlier in pregnancy rubella occurs, the more severe the fetal consequences. infection in the first trimester results in deafness, congenital heart disease, cataracts or glaucoma, endocrine abnormalities, and mental retardation in up to 60% of newborns. spontaneous abortion also occurs commonly. treatment, prevention, and control immunization of children and susceptible adults with live attenuated rubella virus effectively prevents rubella and accounts for the dramatic decline in the incidence of this disease in the united states. however, many adult women of childbearing age remain susceptible to rubella and require immunization prior to conception to prevent congenital rubella. the hemagglutination-inhibition serologic assay detects natural or acquired immunity. the advisory committee on immunization practices (acip) recommends screening of healthcare personnel who have not been vaccinated, and immunization of susceptible individuals. 7 complications of rubella vaccine occur among adults and include lowgrade fever, symmetric polyarthralgias, distal paresthesias, lymphadenopathy, and rash. vaccine is contraindicated in immunosuppressed persons and pregnant women. pregnancy should be avoided for 3 months after vaccination (table 22. 3). susceptible household contacts of infected adults and children pose a transmission risk in the workplace during the period of virus shedding, beginning about 10 days before the development of rash (about 1 week after exposure) until 7 days after rash appears. therefore, susceptible individuals should not report to work during this time interval (table 22. 2). epidemiology tuberculosis (tb) is caused by mycobacterium tuberculosis and, rarely today, by m. bovis. the incidence of tuberculosis (tb) in the united states declined steadily until the mid-1980s, but then sharply increased, especially in urban areas. the resurgence of tb in the united states in the late 1980s and early 1990s was associated with the emergence of multidrug-resistant tb (mdr-tb) and the hiv/aids epidemic. with this resurgence of tb in the united states came several high-profile nosocomial outbreaks associated with lapses in infection control practices and delays in diagnosis and treatment of persons with infectious tb, as well as the appearance and transmission of mdr-tb strains. since 1992, the declines in the overall number of reported tb cases, including the level of mdr-tb, appear to reflect successful efforts to strengthen tb control following the resurgence of tb and the emergence of mdr-tb. 12 activities emphasizing the first priority of tb control (i.e., promptly identifying persons with tb, initiating appropriate therapy, and ensuring completion of therapy) have been the most important factors in achieving this improvement. such activities reduced community transmission of m. tuberculosis, particularly in areas with a high incidence of aids. improvements in implementation of infection control measures in healthcare settings, concurrent with mobilization of the nation's tb control programs, succeeded in reversing the upsurge in reported cases of tb, and case rates have declined to their lowest levels to date. the threat of mdr-tb is decreasing, and the transmission of tb in healthcare facilities continues to abate due to implementation of infection controls and reductions in community rates of tb. nevertheless, some healthcare personnel are at risk for acquiring tb. pulmonary tb is most commonly transmitted in healthcare settings by inhalation of aerosolized droplet nuclei derived from the respiratory secretions of patients with active respiratory tb. close contact usually is required. most other categories of workers generally are not at risk without close and sustained workplace contact with a person who has active untreated disease. ingestion of unpasteurized milk from cows infected with m. bovis is no longer an important source of tb in most industrialized countries. clinical syndromes primary infection usually is asymptomatic in adults. teenagers and young adults are at higher risk for rapid progression to active disease, usually characterized by apical cavitary disease, than are older adults. primary infection in the elderly usually is exhibited as lower lobe consolidation with hilar adenopathy. primary tuberculosis in persons with advanced hiv infection is commonly symptomatic and progressive. once infection occurs, the organism may disseminate from the lungs to other sites, including the gastrointestinal and genitourinary tracts, and bone. normally, the infection is contained by the host's immune response at this stage. the risk for reactivation is highest in the first year after exposure and declines thereafter. however, aging and stressors such as immunosuppression, intercurrent illness, and chronic malnutrition may increase the risk for reactivation or dissemination of the disease later in life. clinically, reactivation tuberculosis usually is exhibited as upper lobe pulmonary cavitary disease, but virtually any organ system may be involved. treatment, prevention, and control tuberculin skin testing allows determination of prior exposure to tb in immunologically healthy adults, by assessing delayed hypersensitivity to tuberculin antigens using purified protein derivative. the tuberculin skin test (tst) is the only proven method for identifying infection with m. tuberculosis in persons who do not have tb disease. although the available tst antigens are neither 100% sensitive nor specific for detection of infection with m. tuberculosis, no better diagnostic methods have yet been devised. the preferred skin test for diagnosing m. tuberculosis infection is the mantoux test. it is administered by injecting 0.1 ml of 5 tuberculin units (tu) ppd intradermally into the dorsal or volar surface of the forearm. tests should be read 48-72 h after test administration, and the transverse diameter of induration should be recorded in millimeters. there are three cut-off levels recommended for interpretation of the tst results. 14 in hiv-infected persons, any reaction resulting in an induration larger than 5 mm is read as positive. among others, the presence of 15 mm or more of induration always indicates a positive test, 5-10 mm indicates a positive result in persons at risk for tb, and less than 5 mm is negative. a positive tst means an individual has been exposed to tb in the past and is at risk for reactivation. a baseline chest radiogram should be performed on all persons with newly diagnosed tst positivity. if the x-ray study suggests active disease, sputum samples should be obtained, stained for acid-fast bacilli, and cultured for mycobacteria. treatment should be implemented immediately if the index of suspicion is high. public health officials should be notified to institute case management and evaluation of contacts in the home and work environment. if the x-ray study is negative, treatment with isoniazid to suppress or eradicate latent organisms may be recommended, especially in persons younger than age 35 and for those who have recently converted to positive tsts. 13 although bcg vaccine is the most widely administered of all vaccines in the world, and has the highest coverage of any vaccine in the who expanded programme on immunization, it appears to have had little epidemiologic impact on tb. despite its shortcomings, and because of its beneficial effect in children and against leprosy, bcg vaccine likely will remain a component of childhood vaccination strategies in developing countries. however, because of questions about the vaccine's efficacy, and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, bcg has never been recommended for programmatic use in the united states. 14 healthcare providers should follow appropriate infection control procedures, including use of isolation rooms and respiratory protection, when caring for patients with active tuberculosis. 15, 16 varicella/zoster epidemiology varicella virus, the causative agent of chickenpox and zoster, is a highly contagious herpes virus spread by the respiratory route from person to person. the incubation period is about 14 days, and the period of infectivity begins a few days prior to the onset of the rash to about 6 days after the first crop of vesicles appears. immunosuppression usually prolongs the period of infectivity, especially if varicella zoster immune globulin (vzig) has been administered. zoster represents reactivation of varicella virus that is latent in sensory nerve ganglia, and it is not a manifestation of primary infection except in newborns infected in utero. the incidence of zoster increases with age and immunosuppression. susceptible persons in direct contact with zoster lesions risk developing primary varicella. in the prevaccine era, varicella was endemic in the united states, and virtually all persons acquired varicella by adulthood. as a result, the number of cases occurring annually was estimated to approximate the birth cohort, or approximately 4 million per year. this incidence has likely decreased since licensure of the vaccine in 1995. varicella is not a nationally notifiable disease, and surveillance data are limited. 17 clinical syndromes varicella in otherwise healthy children usually is a benign, self-limited disease characterized by low-grade fever and vesicular rash, often preceded by a viral prodrome. varicella vesicles of primary infection appear first on the scalp and trunk and disseminate in crops showing various stages of development over the next 3-4 days. healing results in crusting accompanied by intense pruritus. manifestations of varicella are more severe in adults than in children. about 15% of adults with varicella show radiographic evidence of pulmonary involvement, but this is rarely clinically significant. however, cough, tachypnea, and impaired gas exchange can occur and persist for months after infection. varicella during pregnancy can produce congenital varicella. in its most severe form, this infection can result in mental retardation, blindness, growth retardation, deafness, chorioretinitis, and a peculiar dermatomal lesion of the upper or lower extremity associated with limb atrophy. zoster, the most common manifestation of varicella infection among adults, characteristically produces unilateral vesicular eruptions preceded by pain in one to three dermatomes. disseminated zoster, which is more likely in immunosuppressed patients, probably poses the same risk of infection transmission as primary varicella infection. the major complication of zoster is postherpetic neuralgia, which is especially common in the elderly and may be extremely debilitating. zoster frequently produces cerebrospinal fluid pleocytosis and occasionally encephalitis. immunologically healthy persons may experience recurrences of zoster, usually in the same dermatome as the initial outbreak. zoster is a marker of deteriorating cellmediated immunity among hiv-infected patients, and it may disseminate. treatment, prevention, and control passive immunization with vzig is recommended for immunosuppressed susceptible persons, children with leukemia and other malignancies, and neonates exposed in utero within 5 days before delivery. 18 several antiviral drugs are active against varicella zoster virus, including acyclovir, valacyclovir, famciclovir, and foscarnet. 18 famciclovir and valacyclovir are approved for use only in adults. clinical studies indicate that these drugs may be beneficial if given within 24 hours of onset of rash, resulting in a reduction in the number of days new lesions appeared, in the duration of fever, and in the severity of cutaneous and systemic signs and symptoms. antiviral drugs have not been shown to decrease transmission of varicella, reduce the duration of absence from school, or reduce complications. oral acyclovir can be considered in otherwise healthy adolescents and adults or secondary cases in the household, because of the increased risk of severe illness in these groups. antiviral therapy may also be considered for persons with chronic cutaneous or pulmonary disorders, persons receiving long-term salicylate therapy, and for children receiving short, intermittent or aerosolized courses of corticosteroids. antiviral drugs are not recommended for routine postexposure prophylaxis. systemic steroids in older adults (more than 60 years old) may reduce the incidence and severity of postherpetic neuropathy if started early (within 5 days of skin manifestations). varicella has been difficult to prevent because of the high degree of contagion in households, schools, and healthcare settings. live attenuated virus vaccines have demonstrated efficacy in preventing primary infection, and one was licensed for use in the united states in 1995. routine immunization is now recommended for children less than 18 months of age. 17 varicella vaccination should be given to susceptible adolescents and adults who are at high risk of exposure to varicella. this group includes persons who live or work in environments in which there is a high likelihood of transmission of varicella, such as teachers of young children, residents and staff in institutional settings, and military personnel. varicella vaccination is also recommended for susceptible adolescents and adults who will have close contact with persons at high risk for serious complications of acquired varicella, including healthcare personnel and susceptible family contacts of immunocompromised individuals. the acip recommends that all healthcare personnel be immune to varicella, either from a reliable history of prior varicella infection or vaccination, to reduce the risk of infection and its complications, and to decrease the possibility of transmission of varicella zoster virus to patients (table 22. 3). susceptible adults exposed to children with varicella or with disseminated zoster pose a risk of transmitting varicella to non-immune coworkers, and they should not work until the incubation period is over or, if they become ill, until all lesions are crusted (table 22. 2). dermatomal zoster is not spread efficiently by the air-borne route and otherwise healthy adults afflicted with this illness may be allowed to work if they can avoid touching the lesions and contaminating the work environment. the role of vaccine in the postexposure management of susceptible employees needs to be elucidated. data from the united states and japan in a variety of settings indicate that varicella vaccine is effective in preventing illness or modifying the severity of illness if used within 3 days, and possibly up to 5 days, of exposure. acip recommends vaccine be used in susceptible persons following exposure to varicella. 17 personnel should be excluded from work who have onset of varicella until all lesions have dried and crusted (table 22. 2). following exposure to varicella, personnel who are not known to be immune to varicella (by history or serology) should be excluded from duty beginning on the 10th day after the first exposure until the 21st day after the last exposure (28th day if vzig was given). epidemiology acute gastrointestinal infection follows upper respiratory illness as the next leading category of infectious diseases causing absenteeism among adult workers. a wide array of pathogens, including viruses, bacteria, and protozoa, can result in acute infections of the stomach, small bowel, or colon. a comprehensive discussion of enteric pathogens is provided in chapter 54 (waterborne microbial diseases). most of the etiologic agents are acquired by the fecal-oral route; produce mild, self-limited diseases; and resolve without specific therapy. agents of dysentery (e.g., shigella spp.) often are highly transmissible through low-inoculum exposures. occupational transmission of food-borne or water-borne illnesses occurs; person-to-person transmission has propagated outbreaks of many of these illnesses in healthcare settings, daycare and nursery schools, and institutions where sanitation is poor. instances of such transmission have generally involved food handlers, who are often poorly trained and short-term employees, serving as sources of transmission to others. occupations requiring travel to countries with poor sanitation present a major risk for gastrointestinal infections. poultry workers are frequently exposed to salmonella infections. avoidance of oral contact with sources of fecal contamination is the most important strategy for preventing transmission of pathogens associated with intestinal infections. maintaining good personal hygiene, including careful hand hygiene after using restrooms and before food preparation; proper cooking and storage of foods; and avoidance of contaminated foods and water when traveling are essential prevention strategies. food handlers with diarrheal illnesses should not work until symptoms have resolved, and cure of bacterial infections should be documented by obtaining negative stool cultures more than 48 hours after antimicrobial therapy is completed (table 22. 2). the only vaccines for any of the etiologic agents for acute enteric infections are for typhoid and hepatitis a, which are recommended for personnel in laboratories who frequently work with salmonella typhi or hepatitis a virus (table 22. 3). epidemiology cytomegalovirus (cmv) is a ubiquitous herpes virus transmitted by direct inoculation with infected body fluids (including blood, blood products, respiratory secretions, saliva, and urine) and through sexual contact with infected partners. at least 40% of healthy adults have serologic evidence of prior cmv infection. infection can be acquired perinatally, in utero during maternal primary infection, during birth by passage through infected vaginal secretions, or through ingestion of infected breast milk. cmv is known to be highly transmissible in daycare centers and nursery schools. sexually active adults and recipients of blood products are also at high risk for infection. infants and young children excrete cmv in their urine, saliva, and respiratory secretions for several months after infection. virus is much less readily detected in healthy adults, but intermittent shedding has been documented. like all herpes viruses, cmv remains latent in the host after initial infection. previously infected persons may be reinfected with new strains of cmv. occupational transmission of cmv has been documented in childcare settings, where person-to-person spread through exposure to infected secretions and urine is believed to provide an efficient mode of transmission. up to 50% of seronegative workers in preschool daycare centers have acquired cmv infection in some studies, indicating a potentially serious risk to women of child-bearing age, because of the adverse effects of primary maternal cmv infection on the fetus. at one time, employment in healthcare settings also was believed to pose a high risk for cmv acquisition. however, epidemiologic investigations suggest that most infections in healthcare personnel are acquired sexually, or from exposure to young children in the home, and not from work-related contact. is asymptomatic in healthy persons. a self-limited mononucleosis-like illness occurs in a minority, which may be complicated by hepatitis, pneumonitis, hematologic abnormalities, and myocarditis. immunosuppressed children and adults with primary cmv infection, reactivation, or reinfection may develop severe sequelae. organ transplant recipients, hiv-infected patients, and persons with malignancies have a risk of developing cmv viremia, pneumonia, hepatitis, pancreatitis, enteritis, and retinitis. primary cmv infection at any stage of pregnancy carries a greater risk to the fetus than does recurrent cmv infection during pregnancy. symptoms of congenital cmv infection may be present at birth, and are due to the consequences of active virus replication and resultant end-organ damage. congenital cytomegalic inclusion disease, the most severe form of this entity, includes central nervous system disease, respiratory distress, hepatitis, hepatosplenomegaly, rash, and multi-system failure. infection acquired from exposure to cervical cmv during birth usually is asymptomatic and detected by the onset of virus shedding 4-8 weeks postpartum. with ganciclovir or foscarnet for cmv infection is reserved for immunosuppressed persons at high risk for severe complications. the safety and effectiveness of these agents in preventing congenital cmv have not been established. avoidance of mucosal contact with infected body fluids is the best strategy for preventing cmv transmission. hand washing after contact with secretions and fomites is essential, especially in nurseries and daycare settings. the presence of persons at risk for cmv shedding in the workplace does not pose a hazard to other employees unless direct contact with infected secretions is anticipated. isolation of infected neonates or children is not essential if hand washing is performed after contact with secretions, blood, and urine. pregnant healthcare providers compliant with hand washing protocols can generally safely care for patients with cmv infection. [19] [20] [21] no work restriction is necessary for individuals with cmv infection (table 22 .2). direct exposure to blood and other infected body fluids. children born to infected mothers are at high risk for hbv infection. persons parenterally exposed to blood, including multi-transfused patients, hemophiliacs, dialysis patients, and injection drug users, also are at significant risk. sexual contact with infected partners is another efficient mode of hbv spread. in most industrialized countries, adult infections usually are acquired sexually or by injection drug use. hbv is a relatively hardy virus capable of surviving on environmental surfaces and fomites. transmission in households is well documented and may, in part, be attributable to mucosal contact with fomites contaminated with secretions or blood from infected persons. healthcare personnel and others at risk for occupational blood exposure through percutaneous, mucosal, or dermal routes can acquire hbv infection. the risk associated with accidental needle-stick inoculation of infected blood to susceptible healthcare personnel varies between 5% to 35%, depending on the hepatitis b e antigen (hbeag) status, and hence the viral titer of the source. in up to 50% of occupational infections, a discrete exposure cannot be identified. hepatitis b has an incubation period of 40-180 days. the period of infectivity precedes the development of jaundice by 2-7 weeks and correlates with the presence of hepatitis b surface antigen (hbsag) in the serum; 5-10% of persons with acute (but often clinically silent) infection develop chronic antigenemia. in the united states, up to 1% of adults are carriers of hbv, and provide a reservoir for maintenance of the disease in the population. apparent hepatitis in about one-third of acutely infected adults. clinical hepatitis may be preceded by a prodrome of fever, malaise, urticarial or maculopapular rash, and arthralgias for several days. fever usually resolves before the onset of jaundice. jaundice, dark urine, and scleral icterus usually are present by the time patients seek medical attention. right upper quadrant tenderness, mild hepatic enlargement, and occasionally, splenomegaly are signs that should suggest the diagnosis. the most striking laboratory abnormality is the finding of extreme elevations in the aminotransferase enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) may be elevated to more than 10 times the normal levels, whereas the bilirubin and alkaline phosphatase levels are increased to a much lesser extent. fulminant liver involvement occurs in about 1% of adults and may be complicated by more serious abnormalities, including hypoglycemia, coagulopathy, and hypoalbuminemia. hepatic encephalopathy, hepatorenal syndrome, and bleeding diatheses are life-threatening complications seen in these patients. about 10% of adults with clinically apparent acute hbv infection proceed to chronic hbs-antigenemia, and are at risk for chronic hepatitis, postnecrotic cirrhosis, and primary hepatocellular carcinoma. patients with asymptomatic primary hbv infection are at higher risk for chronic infection than those with symptomatic infection. while chronic persistent hepatitis, a benign illness of little clinical consequence except for the potential for hbv transmission to susceptible individuals, may occur, the major health concern is chronic active hepatitis, which eventually may produce cirrhosis, liver failure, and hepatoma. hepatitis b is differentiated from other causes of hepatitis by serologic assays. 23 a positive hepatitis b surface antigen (hbsag) test identifies patients with current infection and correlates with infectivity during acute and chronic infection. titers of hbsag in the chronic phase of illness may wax and wane and occasionally fall below the limits of laboratory detection, so sequential testing should be performed if chronic hbv is suspected. the presence of hbeag correlates with active virus replication and is a marker of high infectivity and high titer of hbv in the liver and blood. antibody to hepatitis b surface antigen (hbsab) appears when hbsag is cleared and is positive in individuals with immunity after recent or prior infection or immunization. persons with hbsab are not susceptible to acute infection or chronic hepatitis b, except in the very rare case in which reinfection occurs with a strain of hepatitis b against which the normal antibody response does not provide cross-protection. hepatitis b core antibody (hbcab) appears before hbsab and just after hbsag is cleared from the serum, and this is a useful test for diagnosing acute hepatitis b in the window period before hbsab appears. high titers of hbcab persist in chronically infected persons and obviously do not predict immunity from further liver disease. there currently is no treatment for acute hepatitis b. alpha interferon and lamivudine have been licensed for the treatment of persons with chronic hepatitis b. these drugs are effective in up to 40% of cases. hbv infection is largely preventable. inoculation with recombinant vaccines containing hbsag components is safe and highly immunogenic, and appears to confer protection from infection for at least 12 years (table 22. 2). postvaccination testing should be done 1-2 months after completion of the three-dose series to document an appropriate response (i.e., > 10 miu/ml). 23 more than 90% of persons immunized with three properly timed doses (e.g., 0, 1, and 6 months) of vaccine administered intramuscularly in the deltoid region develop protective hbsab levels. factors associated with a lack of response include improper vaccination (improperly stored vaccine, gluteal inoculation, subcutaneous injection), obesity, older age, and smoking. persons who do not respond to the primary vaccine series should receive a second three-dose series or be evaluated for hbsag positivity. since 1982, substantial progress has been made toward eliminating hbv transmission in children and reducing the risk for hbv infection in adults. 24 recommendations of acip have evolved from universal childhood vaccination, to prevention of perinatal hbv transmission, vaccination of adolescents and adults in high-risk groups, and catch-up vaccinations for susceptible children in high-risk populations. 25, 26 the acip vaccination strategies for children and adolescents have been implemented successfully in the united states, and routine immunization of all children is now recommended. the occupational safety and health administration's (osha's) blood-borne pathogen standard mandates provision of vaccine at no cost to all healthcare employees and others at occupational risk for blood exposure. 27 substantial declines in the incidence of acute hepatitis b have occurred among highly vaccinated populations, such as young children and healthcare personnel. vaccine should also be provided to susceptible individuals before sexual maturity, particularly to teenagers in those settings (e.g., inner cities, concentration of poverty) where hbv is highly prevalent, and to all adults at risk for sexual or occupational exposure. preimmunization screening for evidence of prior or persistent infection usually is not cost effective. however, postimmunization testing for antibody response is recommended 1-2 months after the 3 rd dose to detect nonresponders among persons at high risk for exposure. titers of hbsab fall over time and may be undetectable after 5-10 years. the duration of vaccine protection is under investigation. most data suggest that protection persists even when hbsab titers fall below the level of detection, and routine screening and boosting are not recommended. the need for prophylaxis for persons sustaining accidental percutaneous or mucosal exposures to blood should be based on several factors, including the hbsag status of the source, and the hepatitis b vaccination and vaccineresponse status of the exposed person. such exposures usually involve persons for whom hepatitis b vaccination is recommended. any blood or body fluid exposure to an unvaccinated person should lead to initiation of the hepatitis b vaccine series. a summary of prophylaxis recommendations for percutaneous or mucosal exposure to blood according to the hbsag status of the exposure source and the vaccination and vaccine-response status of the exposed person is shown in table 22 .4. 23 when hepatitis b immune globulin (hbig) is indicated, it should be administered as soon as possible after exposure (preferably within 24 hours). the effectiveness of hbig when administered more than 7 days after exposure is unknown. when hepatitis b vaccine is indicated, it § hepatitis b immune globulin; dose is 0.06 ml/kg intramuscularly. ¶ a responder is a person with adequate levels of serum antibody to hbsag (i.e., anti-hbs ≥10 miu/ml). ** a non-responder is a person with inadequate response to vaccination (i.e., serum anti-hbs <10 miu/ml). † † the option of giving one dose of hbig and reinitiating the vaccine series is preferred for non-responders who have not completed a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. § § antibody to hbsag. should also be administered as soon as possible (preferably within 24 hours) and can be administered simultaneously with hbig at a separate site (vaccine should always be administered in the deltoid muscle). for exposed persons who are in the process of being vaccinated but have not completed the vaccination series, vaccination should be completed as scheduled, and hbig should be added as indicated (table 22 .4). persons exposed to hbsag-positive blood or body fluids who are known not to have responded to a primary vaccine series should receive a single dose of hbig and reinitiate the hepatitis b vaccine series with the first dose of the hepatitis b vaccine as soon as possible after exposure. alternatively, they can receive two doses of hbig, one dose as soon as possible after exposure, and the second dose 1 month later. the option of administering one dose of hbig and reinitiating the vaccine series is preferred for non-responders who did not complete a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. 23 states. it is estimated that 1.8% of americans have been infected with hcv. hcv-associated end-stage liver disease is the most frequent indication for liver transplantation among us adults. 28 the incubation period for acute hcv infection ranges from 2 to 24 weeks (averaging 6-7 weeks). hcv transmission occurs primarily through exposure to infected blood, such as through injection drug use, blood transfusion, solid organ transplantation from infected donors, unsafe medical practices, occupational exposure to infected blood, and birth to an infected mother (i.e., vertical transmission). hcv may also be acquired through sexual contact, but the importance of this mode of transmission in the united states is not well characterized. hcv is not transmitted efficiently through occupational exposures to blood. healthcare personnel who are parenterally exposed to infected blood through needlestick injuries may acquire hcv infection, but the magnitude of risk (approximately 2 in 100 hcv needlesticks) is less than that associated with hbv exposure. one epidemiologic study indicated that transmission occurred only from hollow-bore needles compared with other sharps. transmission rarely occurs from mucous membrane exposures to blood, and no transmission in hcv has been documented from skin exposures to blood. data are limited on survival of hcv in the environment. in contrast to hbv, the epidemiologic data for hcv suggest that environmental contamination with blood containing hcv is not a significant risk for transmission in the healthcare setting, with the possible exception of the hemodialysis setting where hcv transmission related to environmental contamination and poor infection control practices have been implicated. the risk for transmission from exposure to fluids or tissues other than hcv-infected blood also has not been quantified but is expected to be low. hcv is not known to be transmissible through the airborne route, through casual contact in the workplace, or by fomites. clinical syndromes hepatitis c virus infection produces a spectrum of clinical illness similar to hbv and is indistinguishable from other forms of viral hepatitis based on clinical symptoms alone. serologic tests are necessary to establish a specific diagnosis of hepatitis c. most adults acutely infected with hcv are asymptomatic. after acute infection, 15-25% of persons appear to resolve their infection without sequelae as defined by sustained absence of hcv rna in serum and normalization of alt levels. 28 chronic hcv infection develops in most persons (75-85%), with persistent or fluctuating alt elevations indicating active liver disease developing in 60-70% of chronically infected persons. no clinical or epidemiologic features among patients with acute infection have been found to be predictive of either persistent infection or chronic liver disease. moreover, various alt patterns have been observed in these patients during follow-up, and patients might have prolonged periods (greater than or equal to 12 months) of normal alt activity even though they have histologically confirmed chronic hepatitis. thus, a single alt determination cannot be used to exclude ongoing hepatic injury, and long-term follow-up of patients with hcv infection is required to determine their clinical status and prognosis. the course of chronic liver disease is usually insidious, and progresses slowly without symptoms or physical signs in the majority of patients during the first two or more decades after infection. chronic hepatitis c frequently is not recognized until asymptomatic persons are identified as hcv positive during blood donor screening, or elevated alt levels are detected during routine physical examinations. most studies have reported that cirrhosis develops in 10-20% of persons with chronic hepatitis c over a period of 20-30 years, and hcc in 1-5%, with striking geographic variations in rates of this disease. however, when cirrhosis is established, the rate of development of hcc might be as high as 1-4% per year. longer follow-up studies are needed to assess lifetime consequences of chronic hepatitis c, particularly among those who acquired infection at young ages. although factors predicting severity of liver disease have not been well defined, recent data indicate that increased alcohol intake, being aged greater than 40 years at infection, and being male are associated with more severe liver disease. in particular, among persons with alcoholic liver disease and hcv infection, liver disease progresses more rapidly; among those with cirrhosis, a higher risk for development of hcc exists. in addition, persons who have chronic liver disease are at increased risk for fulminant hepatitis a. screening enzyme immunoassay (eia) and supplemental confirmatory immunoblot tests are licensed and commercially available to detect antibodies to hcv (anti-hcv). anti-hcv may be detected within 5-6 weeks after the onset of infection but a single anti-hcv test cannot distinguish between acute, chronic, or past infection. hcv rna can be detected within 1-2 weeks of exposure to the virus and several weeks before elevations of alt and detection of anti-hcv. testing for anti-hcv by eia is recommended 4-6 months after an exposure to detect infection; testing for hcv rna may be performed 4-6 weeks after exposure if earlier detection of infection is desired. 23 treatment, prevention, and control hcv-positive patients should be evaluated for the presence and severity of chronic liver disease. initial evaluation for presence of disease should include multiple measurements of alt at regular intervals, because alt activity fluctuates in persons with chronic hepatitis c. patients with chronic hepatitis c should be evaluated for severity of their liver disease and for possible treatment. alpha interferon (with or without ribavirin) treatment of hcv appears to prevent hcv replication, decrease hepatic inflammation, and improve symptoms among chronically infected persons. persons with chronic hcv have undergone successful liver transplantation, although recurrences have been documented in this setting. antiviral therapy is recommended for patients with chronic hepatitis c who are at greatest risk for progression to cirrhosis. these persons include anti-hcv-positive patients with persistently elevated alt levels, detectable hcv rna, and a liver biopsy that indicates either portal or bridging fibrosis or at least moderate degrees of inflammation and necrosis. therapy for hepatitis c is a rapidly changing area of clinical practice and consultation with a knowledge specialist (e.g., hepatologist) is recommended. 29 no clinical trials have been conducted to assess postexposure use of antiviral agents (e.g., interferon with or without ribavirin) to prevent hcv infection, and antivirals are not fda approved for this indication. available data suggest that an established infection might need to be present before interferon can be an effective treatment. 23, 29 because there is currently no postexposure prophylaxis (pep) for hcv, the intent of recommendations for postexposure management is to achieve early identification of infection and, if present, referral for evaluation of treatment options. in addition, no guidelines exist for administration of therapy during the acute phase of hcv infection. however, limited data indicate that antiviral therapy might be beneficial when started early in the course of hcv infection. when hcv infection is identified early, the person should be referred for medical management to a specialist knowledgeable in this area. 23 at present, avoidance of parenteral exposure to blood is the only available strategy for preventing hcv infection. epidemiology it is estimated that more than 60 million persons worldwide had been infected by hiv and that 40 million were living with hiv/aids by the end of the year 2000. 30 in the united states, almost 1 million persons are living with hiv. 30 most individuals with hiv infection are active adults employed in the workforce. the primary means of acquiring infection among adults is either through behaviors such as unprotected homosexual or heterosexual intercourse with an infected partner, involving the exchange of body fluids, or injecting drug use involving shared needles and syringes. the virus also is perinatally transmitted to approximately 20-40% of children born to infected mothers, (e.g., vertical transmission). breastfeeding is a bidirectional mode of transmission, to nursing infants of infected mothers and, rarely, to mothers of nursing infants when nipple maceration and biting occur. since 1985, all donated blood in the united states has been screened for hiv infection. the risk of hiv infection due to transfusion of blood products screened by current methods is estimated to be 1 in 1,900,000 units transfused. 32 screening does not completely eliminate the potential for a seronegative but infected unit from a recently infected donor to escape detection. hiv is not transmitted by the air-borne route, by household or workplace contact with infected persons, by exposure to contaminated environmental surfaces, or by insect vectors. the virus is easily inactivated by most common disinfectants, including household bleach (diluted 1:100). commercial sex workers are at the greatest risk of acquiring hiv infection occupationally. the other group of workers at risk for acquiring hiv infection occupationally is healthcare personnel. healthcare providers and other workers in contact with blood or other body fluids who sustain accidental percutaneous or mucosal inoculations with virus-infected material are at risk for infection. the magnitude of risk depends on the severity of exposure, but on the average, about 1 in 300 hiv needlesticks results in infection. the risk for infection following mucosal exposures is estimated to be lower at approximately 0.09%. in the absence of direct exposure, healthcare providers are not at occupational risk for hiv infection. in the united states, through december 2002, there have been 57 cases of occupationally acquired hiv infection reported with an additional 139 possible cases. 32 clinical syndromes the clinical course of hiv infection is variable and changing with the advent of antiretroviral therapy, as well as treatment and prophylaxis for infectious complications. early after infection, within a few weeks to months, an acute febrile illness characterized by malaise, pharyngitis, lymphadenopathy, maculopapular rash, and headache may occur. the frequency of this mononucleosis-like illness has varied widely in reports of seroconverting individuals. at initial presentation of such patients, hiv antibody screening tests (enzyme immunoassay (eia)) may be negative, but viral antigen (p24 antigen) and serologic reactivity to one or more viral components (western blot test) allows the diagnosis to be established at this stage. hiv infection should be suspected in any person with a mononucleosis syndrome lacking a positive heterophil antibody response (monospot test). following initial infection, most persons have generalized asymptomatic lymphadenopathy and appear well. however, laboratory tests document a gradual decline in the number of circulating t-helper lymphocytes (cd4 cells), beginning soon after infection and continuing over the next several years. t-helper cells are essential components of the immune system and mediate aspects of both cellular and humoral immunity. symptoms, signs, and illness suggestive of mild to moderate immunodeficiency appear after about 5 years, when cd4 cells decrease by about 50%, to less than 500 cells/dl. intermittent fever, oral thrush, bacterial pneumonia, enteric infections, and reactivated tb are typically diagnosed at this time. signs suggestive of more rapid deterioration include oral hairy leukoplakia (a wart-like white growth in the oral cavity), shrinking lymphadenopathy, fever, weight loss, and elevated erythrocyte sedimentation rate. when cd4 cell counts fall below 200, serious opportunistic infections can be anticipated. pneumocystis pneumonia (pcp) was the most common index diagnosis in the first 5 years of the epidemic, but the advent of effective pcp prophylaxis has altered the picture. other opportunistic infections and malignancies, including kaposi's sarcoma, lymphoma, disseminated tb, toxoplasmosis, and cryptococcal meningitis, now account for the majority of index hiv diagnoses. with the exception of tb, the infectious complications of hiv infection generally are not transmissible to healthy individuals and pose no risk in the workplace. indeed, the causative organisms are ubiquitous and most adults have already been exposed. opportunistic infections in hivinfected patients usually represent reactivation of dormant organisms when the immune system can no longer keep them inactive. be offered to all patients with symptoms ascribed to hiv infection. 34 recommendations for offering antiretroviral therapy in asymptomatic patients require analysis of many real and potential risks and benefits. information regarding treatment of acute hiv infection from clinical trials is very limited. ongoing clinical trials are addressing the question of the long-term clinical benefit of potent treatment regimens for primary infection. in general, treatment should be offered to individuals with fewer than 350 cd4 t cells/mm or plasma hiv rna levels exceeding 55,000 copies/rnl (by rt-pcr or bdna assay). once the decision has been made to initiate antiretroviral therapy, the goals should be maximal and durable suppression of viral load, restoration and/or preservation of immunologic function, improvement of quality of life, and reduction of hivrelated morbidity and mortality. 33 hiv-infected individuals found to have latent tb infection should be treated with antituberculous therapy to prevent activation of disease. 13 persons at risk for direct contact with blood and other potentially infected materials should receive specific instruction in universal/standard precautions for infection control, as recommended by the centers for disease control and prevention 34, 35 and mandated by the occupational safety and health administration. 27 for most environments outside healthcare settings, common sense and attention to personal hygiene are adequate to protect workers. gloves should be worn to clean up visible sites of blood contamination. environmental surfaces can then be decontaminated with disinfectant solutions or household bleach (diluted 1:100). 34, 36 individuals sustaining accidental parenteral exposures to hiv should be counseled to undergo baseline and followup testing for 6 months after exposure (e.g., 6 weeks, 3 months, and 6 months) to diagnose occupational infection. postexposure chemoprophylaxis with antiretroviral agents has been recommended by the us public health service since 1996 after certain exposures to hiv-infected sources which pose a risk of infection transmission, such as needlesticks, mucous membrane, and non-intact skin exposures (tables 22.5 and 22.6). data from animal models of prophylaxis with these agents suggest that antiviral activity is diminished when treatment is delayed for more than 24 hours. for this reason, immediate reporting and access to chemoprophylaxis is recommended. occupational exposure is a frightening experience. consultation with clinicians knowledgeable about hiv transmission risks who can provide supportive counseling to the worker is essential during the follow-up interval. cdc recommends that occupationally exposed workers refrain from unsafe sexual practices, pregnancy, and blood and organ donation for 6 months after exposure to minimize the risk of transmission. zoonoses are infections that are maintained in nature by transmission between vertebrate animals, and they can be transmitted from other vertebrates to humans or from humans to other vertebrates. zoonotic pathogens can be divided into two major groups: (1) those transmitted primarily among wild animals (e.g., yersinia pestis, rabies), and (2) those transmitted primarily among domestic animals (e.g., sporothrix schenkii, non-typhoid salmonella spp.). other infections not properly classified as zoonoses can result from working directly with animals (e.g., infected wounds resulting from animal bites) or with animal products (e.g., anthrax in carpet weavers). many zoonotic infections present occupational risks, not only to those who work with live or dead vertebrate animals or animal products but also to workers exposed to certain environments contaminated by animals or animal products. thus, workers in veterinary medicine, animal husbandry, and animal research are at risk for acquiring a host of zoonotic infections specific to the type of live animal exposure, just as those involved in healthcare work with humans are at risk for infections acquired from humans. examples of such zoonotic infections include q fever in veterinarians, psittacosis (caused by chlamydia psittaci) in duck farmers, orf (contagious ecthyma) in shepherds, lymphocytic choriomeningitis (e.g., leptospirosis) in laboratory workers who handle rodents, fatal herpes virus simiae infection in primate handlers, and more recently, monkeypox in veterinarians and pet store owners. 37, 38 influenza a (h5nl) (avian flu) infection was shown to have been transmitted from ducks and chickens to poultry workers in hong kong and has become an important source of epidemic infection in various international settings; 39 lyssavirus (related to rabies virus) infections have been transmitted from bats to humans in australia, 41 and a large outbreak of febrile encephalitic and respiratory illnesses among workers who had exposure to pigs was shown to be due to infection with a previously unrecognized paramyxovirus (formerly known as hendra-like virus, now called nipah virus). 41 brucellosis is an example of a zoonotic infection in abattoir workers exposed to live or dead animals or animal products. examples of zoonotic infections acquired by workers exposed to environments harboring or contaminated by contagious animals include leptospirosis in rice field workers, and argentine hemorrhagic fever typically acquired by adult males harvesting corn in cornfields inhabited by rodents, which serve as the reservoir for junin or machupo virus. it is beyond the scope of this chapter to review the large number of zoonoses (about 200 have been described) that could pose a risk to workers in unique jobs that involve contact with various animals or environments. for each type of occupation that involves regular animal contact, it is important to recognize the types of infectious disease risks involved, consider baseline studies and storage of serum for future serologic tests if risks are high, plan preventive measures when possible, and prepare for early diagnosis and treatment of such infections when illness occurs. 42 some of the zoonoses, the occupational groups they affect, and their clinical presentations are included in table 22 .1. infectious diseases continue to emerge, posing threats to the health of workers in numerous settings. a prime example of such a threat is severe acute respiratory syndrome or sars, first identified in early 2003 and responsible for illness and death primarily among exposed healthcare personnel. 43 emerging infectious issues which may prove to be challenges for occupational health include those posed by bioterrorism, biotechnology, 44 and emerging and reemerging infections. these emerging infections emphasize the need for continued vigilance and for careful history taking about occupational exposures when evaluating individuals for illnesses that could possibly be occupationally acquired. timeliness in identification and reporting of cases assists in the accurate estimation of the magnitude of the infectious disease problem and in the development of additional preventive and therapeutic measures. screening of employees for infection with or susceptibility to infectious diseases is an important part of healthcare maintenance, especially when the occupational setting poses a significant risk of transmitting or acquiring infections. screening also is warranted if specific interventions are available to prevent disease transmission among workers. assessment of behaviors, such as smoking, that increase the risk of acquiring infections also is valuable so that employees can be provided educational and other interventions to modify risks. preventing infectious diseases in workers can decrease absenteeism and financial costs associated with disability, sick leave, and health insurance, even if the primary source of infection is non-occupational. attending to these issues at the time of employment obviates the need for ongoing surveillance of many infections and simplifies outbreak investigations by documenting the pre-exposure immune status of contacts. tst screening for active disease identifies those persons who would benefit from prophylaxis (tables 22.5 and 22.6). 14 tb vaccination with bacillus of calmette-guerin (bcg) vaccine, a live attenuated strain of m. bovis, is provided for children and some workers in most european countries, but it is not recommended in the united states because of its unproven efficacy when used in adults and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, impeding the usefulness of tst as a screening tool. persons age 65 years and older, persons with chronic diseases or pulmonary disorders, and healthcare personnel should be offered pneumococcal vaccine and annual influenza vaccine (table 22. 3). 45 rubella immune status should be ascertained and men and women immunized in settings where women of childbearing age are employed (table 22. 3). even though rubella is not often transmitted in the workplace, outbreaks can occur among susceptible individuals and vaccination is an important public health intervention. medical personnel should demonstrate proof of rubella immunity or vaccination prior to patient contact. measles vaccine should be provided to all workers born after 1957 with no documented history of measles who have not received two injections of live virus vaccine (table 22. 3). screening for immunity to varicella and mumps is not routinely recommended, except for healthcare providers and adults with no history of infection with these agents who are exposed to young children. all adults require tetanus immunization. tetanus diphtheria toxoid (td) boosters should be administered every 10 years to adults who have completed primary immunization (table 22. 3). employees with no prior history of tetanus diphtheria immunization or with uncertain histories should receive a series of three primary vaccine injections. 45 similarly, adults with no history of polio immunization should undergo primary immunization with inactivated polio vaccine, especially if they are employed in healthcare settings or when travel to endemic areas is anticipated. persons employed in occupations that pose a risk for parenteral contact with blood and other body fluids should be offered hepatitis b immunization (table 22. 3). healthcare personnel, laboratory workers, animal handlers, first responders, and personal service workers such as barbers, tattooists, and cosmetologists are included in this category. adults with multiple sexual partners also should be encouraged to undergo immunization. serum banking to allow documentation of baseline serostatus is useful for laboratory and healthcare personnel at risk for other bloodborne infections such as hiv or more exotic infections. laboratory workers and animal handlers may be at risk for unusual infectious diseases. q fever, a rickettsial disease transmitted by the air-borne route, is a special risk encountered by handlers of sheep and similar animals. serologic testing for q fever titers prior to occupational exposure is important to document baseline status and to detect seroconversion at follow-up testing. although smallpox vaccine is no longer recommended routinely, genetically engineered vaccines prepared from vaccinia may pose a risk to researchers and clinicians treating patients enrolled in vaccine trials. laboratory workers who handle vaccinia or recombinant vaccinia preparations in culture or in animals should receive vaccinia vaccine. healthcare personnel caring for patients immunized with vaccinia or other orthopoxviruses or tissues and specimens from patients with these infections also should be immunized. a program for smallpox vaccination for selected individuals who may be in the frontline for responding to a bioterrorist attack has recently been initiated in the united states. 46 consultation should be obtained to determine the need for screening, immunization, and testing for other exotic infections. some animal handlers are at risk of acquiring rabies through bites or exposure to infected secretions and tissues. immunization with human diploid cell vaccine (three 1-ml intradermal doses on day 0, 7, and 21 or 28) should be provided to workers at risk for rabies and for persons traveling for more than 1 month to areas where rabies is endemic (table 22. 3). booster injections should be provided every 2 years for those with continuing exposure. surveillance of infectious diseases is conducted to detect increased occurrence of disease so that preventive interventions can be initiated. surveillance can be passive (based on employee health consultations or reports from contractual providers or supervisors) or active (actual monitoring of disease occurrence). active surveillance for infectious diseases is not required in most occupational settings. in work environments where exposure to m. tuberculosis may occur -such as healthcare settings, residential care facilities, shelters, and correctional facilities -active tst surveillance among susceptible individuals is indicated. periodic tsts are especially important in the wake of several recent outbreaks associated with drug-resistant strains of m. tuberculosis in hospitals, adult care settings, and home healthcare settings. 16 skin testing should be performed at least annually in these settings, and perhaps as often as every 6 months, for personnel at high risk for exposure to active tb. surveillance of teachers, travelers to endemic areas, and employees in other institutional settings where close contact with infected individuals is possible also may be warranted, depending on the local prevalence of tb. 14 surveillance for infections among laboratory workers and animal handlers exposed to specific pathogens should be individualized in accordance with standard guidelines for biosafety in microbiologic and biomedical laboratories. 47 maintaining standardized records of reportable infectious diseases is an important component of passive surveillance in the workplace. centralized collection and assessment of these records at regular intervals may allow early detection of outbreaks of occupational infections amenable to specific control interventions. geographic or temporal clusters of cases or clustering among persons with similar attributes or occupational tasks suggest a common source of exposure and infection and warrant investigation. local public health officials and regulatory agencies should be consulted promptly when an outbreak is initially suspected. reporting of occupationally acquired infections permits public health agencies to identify clusters of old and emerging illnesses and ultimately prevent them. these events should be reported as mandated by state and local regulations. 48, 49 return-to-work criteria employees diagnosed with communicable infectious diseases should not return to work until the period of infectivity is past. specific guidelines should be consistent with local public health regulations. some workers, for example food handlers with certain diarrheal illnesses, cannot resume their duties until culture evidence of cure is obtained. employees should be advised of the return-towork policies at the time of employment and when illness is diagnosed. a table for length of work restriction for healthcare personnel can be used to guide return-to-work policies for the workplace (table 22. 2). 19 common sense dictates attention to personal hygiene among all workers. hand washing after using the bathroom and before handling food is essential. the mouth should be covered while sneezing or coughing, and soiled tissues and dressings should be disposed of in trash containers. employers have a responsibility to minimize crowding in the work setting. facilities for hand washing should be available in bathrooms and food preparation areas. proper ventilation also is important. trash should be emptied at regular intervals, and work areas should be clean and free of pests. smoking should be prohibited in common work areas. spills of blood, body fluids, and other potentially infectious substances should be removed with disposable paper towels or other suitable procedures. contaminated areas should then be disinfected with commercial products or with a solution of household bleach (diluted 1:100). 34, 36 infection control in healthcare settings infection control programs in healthcare settings are necessary to prevent transmission of healthcare-related infections to patients and healthcare personnel. the cdc has established a two-tiered system of infection control precautions. 16 the first tier consists of 'standard precautions' which are precautions recommended for delivery of care to all patients regardless of diagnosis or presumed infection status. they are designed to limit exposure to blood or other body substances and include elements such as hand hygiene and use of appropriate protective barriers, e.g., masks, eye protection, and gloves, as needed to prevent direct contact. the second tier of precautions recommended by cdc are 'transmission-based precautions', designed for the management of patients known or suspected to be infected with pathogens whose transmission can be limited by the adoption of additional measures beyond those which are part of standard precautions. they apply to pathogens transmitted by the air-borne or aerosol routes, droplets, and by direct and indirect contact. respiratory precautions are employed for patients with infections communicable by the air-borne route. such patients are housed in private rooms with special ventilation and should wear surgical masks when leaving their rooms. respiratory protection (i.e., n-95 respirators) also are advised for providers in close contact with patients on respiratory precautions. however, the re-emergence of epidemic and mdr-tb has led to a re-emphasis of other fundamentals for prevention of transmission of tuberculosis in healthcare and other settings. early identification of tb allows early indication for therapy, and requires alertness in considering tb in high-risk patients with pulmonary symptoms, especially those with hiv infection. special ventilation measures and respiratory protection are especially important for cough-inducing procedures, such as sputum induction and aerosolized pentamidine administration. healthcare personnel who have the potential for being exposed to m. tuberculosis should be screened on employment and at least annually thereafter by ppd skin testing, comparing previous test results to current results to identify those who have converted to skin test positivity. 15 procedures for disposal of infectious wastes have been developed by the cdc. 36 needles and other sharp objects should be sterilized prior to disposal. liquid and laboratory wastes may be dumped into sewage systems. materials heavily contaminated with bacteria or blood should be placed in special bags that are specifically labeled for infectious waste and should be disposed of according to community standards for such materials. 36 employers have a responsibility to educate employees about infection control. barriers to prevent exposure, including masks, gowns, eye protection, and gloves, should be readily available to workers at risk. hand washing facilities and hand hygiene supplies are essential. where access to sinks or running water is not feasible, alcohol hand rubs or packaged towels containing disinfectants should be provided. impervious containers for the disposal of needles and other sharp objects are essential. 50 such containers should be made available on ambulances and provided to home health aides and other visiting healthcare personnel. personal service workers who use needles, razors, and other sharp objects also should have access to safe disposal units. persons receiving care at home who require injections or other procedures that demand the use of needles also should be provided with impervious disposal containers and instructed in proper disposal methods to protect sanitation workers and others in contact with waste. despite improvements in engineering controls, work practices, and personal protective equipment, laboratory personnel are nevertheless at risk for occupationally acquired infections. laboratory personnel may acquire infection by aerosolization of specimens, mouth pipetting, or percutaneous injury or mucocutaneous contact. methods of infection control applicable to laboratory settings are described in the cdc document entitled 'biosafety in microbiological and biomedical laboratories'. 47 by 1995, it was estimated that 14.5 million children were attending out-of-home daycare in a variety of settings including licensed child daycare centers, regulated daycare homes, and unregulated family daycare homes. 51 many serious infections occur as endemic or microepidemic problems in the daycare setting. these include h. influenzae type b, hepatitis a, cytomegalovirus, parvovirus b 19, and enteric infections (shigella, giardia, rotavirus, clostridium difficile, campylobacter, cryptosporidium, calcivirus, salmonella, enteric adenovirus, astrovirus, and several types of e. coli infections). in addition, the high rate of acute respiratory infections leads to early onset of otitis media, frequent antibiotic use, and emergence of multidrug-resistant enteric pathogens. thus, workers in close contact with children risk exposure to a wide variety of communicable pathogens contained in secretions, urine, and stool. all such personnel, as well as all children in schools and daycare centers, should be screened for immunity to common childhood infections and vaccinated if immunity is not present (table 22. 2). regulation of childcare facilities is essential to reduce risks to children and workers. national standards for infection control in childcare facilities were promulgated in 1990. 52 hand washing facilities and policies are the most important component of disease prevention in school and daycare settings. hands should be washed after contact with mucous membranes and potentially infected body fluids. older children should be instructed in personal hygiene. children with fever or diagnosed infections should be excluded from attending daycare or school until transmission risk is no longer present, and policies for such exclusion should be in place. prompt reporting of disease outbreaks and prompt involvement of public health authorities are essential. employees should be instructed in common-sense first aid procedures for handling wounds, bites, and other situations in which exposure to infected blood or tissues is possible. barrier protection is rarely required in schools, but gloves should generally be available for emergencies requiring first aid. infection with a variety of agents during pregnancy has the potential to cause fetal damage, especially when primary infection occurs. while a number of these infections can be community acquired, the likelihood of exposure to certain of these pathogens can be greater in healthcare settings. infections with as rubella, cmv, and parvovirus are among the infectious agents which may be of special concern to pregnant healthcare personnel. in general, adherence to standard precautions as well as preexposure immunizations when available and appropriate are the best way of preventing the devastating effects of such infections (table 22. 2). 19, 21 immunodeficient workers are at increased risk of devastating infections, particularly with opportunistic agents. the greatest risk for such workers is likely in the healthcare setting where there can be ample opportunity for exposure to these agents. many immunocompromising illnesses would be viewed by the us legal system as disabilities and therefore, individuals with those conditions would be covered under the provisions of the americans with disabilities act of 1990 (see chapter 57.1). 53 such persons should be informed about their risks and furthermore, their employers should make reasonable accommodations to allow their employees to continue to perform their jobs, taking into consideration the provisions of applicable federal, state, and local regulations. occupational infectious diseases encompass a large variety of infections which can involve many organ systems. they include some common infections, such as influenza, that pose a special problem in the workplace because of close interpersonal contact and crowding, and that taken together account for a large proportion of time lost from work. many of these infections are preventable by policies that promote hygiene and provide exclusion from work during periods of contagion. in addition, a variety of less common, but sometimes serious, infections are particularly associated with specific occupations. recognition of the types of infection risk associated with specific occupations can, in most cases, lead to effective, often simple steps for primary prevention, as well as opportunities for early diagnosis and treatment. bioterrorismrelated inhalational anthrax: the first 10 cases reported in the united states principles and practice of infectious diseases centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza vaccination in long-term-care hospitals reduces the mortality of elderly patients measles, mumps, and rubella -vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) changing epidemiology of pertussis in the united states: increasing reported incidence among adolescents and adults guidelines for the control of pertussis outbreaks ashrae guideline 12-2000: minimizing the risk of legionellosis associated with building water systems guideline for prevention of nosocomial pneumonia. the hospital infection control practices advisory committee progressing toward tuberculosis elimination in low-incidence areas of the united states: recommendations of the advisory council for the elimination of tuberculosis centers for disease control and prevention. targeted tuberculin testing and treatment of latent tuberculosis infection the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices guidelines for preventing the transmission of tuberculosis in health-care facilities guideline for isolation precautions in hospitals. the hospital infection control practices advisory committee prevention of varicella: update recommendations of the advisory committee on immunization practices (acip) epidemiology and prevention of vaccine-preventable diseases guideline for infection control in healthcare personnel guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force infection control precautions for the pregnant healthcare worker. bailliere's risk and management of bloodborne infections in healthcare workers updated us public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis achievements in public health: hepatitis b vaccination -united states hepatitis b virus: a comprehensive strategy for eliminating transmission in the united states through universal childhood vaccination: recommendation of the immunization practices advisory committee (acip) protection against viral hepatitis: recommendations of the immunization practices advisory committee (acip) occupational safety and health administration, department of labor. 29 cfr part 1910.1030, occupational exposure to bloodborne pathogens; final rule recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease national institutes of health consensus development conference panel statement. management of hepatitis c an unequal epidemic in an unequal world the risk of transfusion-transmitted viral infections surveillance of healthcare personnel with hiv/aids, as of panel on clinical practices for the treatment of hiv infection. guidelines for the use of antiretroviral agents in hiv-infected adults and adolescents. us department of health and human services recommendations for prevention of hiv transmission in healthcare settings update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis b virus, and other bloodborne pathogens in health-care settings guideline for environmental control in healthcare facilities centers for disease control and prevention. multistate outbreak of monkeypox -illinois risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 emerging viral diseases: an australian perspective outbreak of hendra-like virus -malaysia and singapore, 1998-1999 risks and prevention of nosocomial transmission of rare zoonotic diseases sars -looking back over the first 100 days occupational standards for the protection of employees in biotechnology centers for disease control and prevention. general recommendations on immunization: recommendations of the advisory committee on immunization practices (acip) and the american academy of family physicians (aafp) recommendations for using smallpox vaccine in a pre-event vaccination program: supplemental recommendations of the advisory committee on immunization practices (acip) and the healthcare infection control practices advisory committee (hicpac) us department of health and human services centers for disease control. case definitions for public health surveillance mandatory reporting of infectious diseases by clinicians. mandatory reporting of occupational diseases by clinicians selecting, evaluating, and using sharps disposal containers. dhhs (niosh) publication no. 97-111 child care arrangements for preschoolers by family characteristics: fall national standards for infection control in out-ofhome child care americans with disabilities act of 1990, 104 stat. 327, 42 u.s.c. sec. 12101 et seq immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infections control practices advisory committee (hicpac) known responder ¶ known non-responder** antibody response unknown hbig § × 1 and initiate hepatitis b vaccine series no treatment hbig x 1 and initiate revaccination or hbig × 2 † † test exposed person for anti-hbs: § § if drug resistance is a concern, obtain expert consultation. initiation of postexposure prophylaxis (pep) should not be delayed pending expert consultation, and, because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. § source of unknown hiv status (e.g., deceased source person with no samples available for hiv testing). ¶ unknown source (e.g., splash from inappropriately disposed blood). ** small volume (i.e., a few drops). † † the designation 'consider pep' indicates that pep is optional and should be based on an individualized decision between the exposed person and the treating clinician. § § if pep is offered and taken, and the source is later determined to be hiv negative, pep should be discontinued. ¶ ¶ large volume (i.e., major blood splash). key: cord-027860-s97hdhh6 authors: zeimet, anthony; mcbride, david r.; basilan, richard; roland, william e.; mccrary, david; hoonmo, koo title: infectious diseases date: 2020-06-22 journal: textbook of family medicine doi: 10.1016/b978-1-4377-1160-8.10016-8 sha: doc_id: 27860 cord_uid: s97hdhh6 nan infections of the upper respiratory tract accounted for more than 36 million ambulatory medical visits in 2005, according to the national ambulatory medical care survey (cherry et al., 2007) . although a large percentage of these infections are viral in origin, antibiotics are still prescribed for more than 50% of patients with acute respiratory tract infection (arti). acute bronchitis, in the arti category, is defined as a respiratory infection in which cough is the predominant symptom and there is no evidence of pneumonia. antibiotics are often prescribed despite limited evidence that they shorten the duration of acute bronchitis. with increasing incidence of antibiotic resistance, bronchitis allows physicians to practice "prescriptive restraint" and to provide supportive therapy. consider using the phrase "chest cold" to help patients understand the viral and benign nature of this infection. chronic bronchitis is one of the manifestations of chronic obstructive pulmonary disease (copd) and is defined clinically as cough and sputum production on most days for 3 months annually for 2 years. chronic bronchitis is thought to be primarily inflammatory in origin, although infection may be associated with acute exacerbations; with increased sputum production and worsening dyspnea, antibiotics have proved effective in acute episodes. however, systemic corticosteroids are the mainstay of copd exacerbation management. the patient with acute bronchitis presents with cough, often productive. patients may report clear or colored mucus in association with the presumed diagnosis of acute bronchitis. despite what many patients believe, the color of sputum, even purulent sputum, is not predictive of bacterial infection. the cough of bronchitis can last up to 4 weeks, sometimes even longer. typically, acute bronchitis is associated with other manifestations of infection, such as malaise and fever. respiratory viruses are thought to cause the majority of cases of acute bronchitis. influenza a and b, parainfluenza, respiratory syncytial virus (rsv), coronavirus, adenovirus, and rhinovirus are common pathogens in the viral category. clues to a specific virus may be found in the patient history; for example, rsv might be considered when there is household exposure to infected children. influenza typically presents with sudden onset of symptoms, including fever, myalgias, cough, and sore throat. neuraminidase inhibitors are modestly effective in shortening the duration of influenza in ambulatory and healthy patients (by about 1 day), if initiated in the first 48 hours of illness. the resistance patterns of influenza a and b have shifted in the last several years and may vary based on yearly viral strains. influenza b has remained, as of 2010, sensitive to zanamivir (relenza) and oseltamivir (tamiflu). currently circulating strains of influenza a, both h1n1 and h3n2, and influenza b have generally remained sensitive to both oseltamivir and zanamivir (fiore et al., 2011) . family physicians are advised to consider restraint in the prescribing of these agents, since resistance is of great concern. yearly influenza immunization and cough etiquette and hygiene are likely the most useful techniques for influenza management. studies have identified other pathogens, such as mycoplasma pneumoniae and chlamydophila pneumoniae, in a small minority of cases of clinical acute upper respiratory illness with cough as the predominant symptom. no significant benefit has been found in treating these infections with antibiotics. an exception in the treatment of acute bronchitis-like illness with antibiotics is when confirmed or probable bordetella pertussis is present. early treatment with a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) . although common upper respiratory bacterial pathogens, such as moraxella (branhamella) catarrhalis, streptococcus pneumoniae, and haemophilus influenzae, may be isolated from patients with acute bronchitis, their relevance is questionable because these bacteria can be present in the respiratory tract of healthy individuals. obtaining sputum for culture when bronchitis is the diagnosis generally is not useful. antibiotics may offer a modest benefit in the treatment of acute bronchitis, with many studies showing no statistical significance in the outcome of treated versus not-treated groups. measures of function, such as duration of illness, loss of work, and limitation of activity, have not shown clinically significant improvement in those with acute bronchitis taking antibiotics. coupled with cost and the potential for side effects, the use of antibiotics for acute bronchitis is not recommended. if a provider decides to use an antibiotic in a specific patient situation, narrow-spectrum respiratory agents are preferred, such as a first-generation macrolide or doxycycline. treating the symptom of cough in acute bronchitis is an important concern for patients. in adults with acute bronchitis with signs of airway obstruction, evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough. these agents are not helpful for children with acute cough or adults with cough and no evidence of airway obstruction. side effects of tremor and an anxious feeling must be weighed against this benefit. patients often are primarily interested in alleviating symptoms caused by respiratory illness. unfortunately, there is mixed evidence for the use of over-the-counter (otc) and prescription cough medications. dextromethorphan and codeine may be somewhat effective, although they have not been evaluated in randomized, double-blinded, placebo-controlled trials for acute bronchitis. combination first-generation antihistamine-decongestant products may be effective for the cough associated with colds. naproxen showed efficacy against cough in one upper respiratory model study (sperber et al., 1992) . guaifenesin acts as an expectorant and may have some effect on cough by its mucus-thinning properties. community-acquired pneumonia (cap) is defined as an acute infection of the pulmonary parenchyma and, along with influenza, is the seventh leading cause of death in the united states. fever, cough, sputum production, pleuritic chest pain, and dyspnea are common symptoms of cap. nausea, vomiting, and diarrhea also may occur, and in elderly patients, cap may present with mental status changes. although its absence usually makes pneumonia less likely, fever can be absent in the elderly patient. other physical examination findings include an elevated respiratory rate, conversational dyspnea, tachycardia, and rales. egophony and dullness to percussion may be noted with focal consolidation. typical laboratory findings include leukocytosis. the diagnosis of pneumonia is based on the presence of symptoms and the presence of an infiltrate on chest radiograph. if infiltrate is not present, consider obtaining a chest tomography scan (which has higher sensitivity) to rule in or rule out cap. if negative, other diagnoses should be considered. the most common microbiologic agent of pneumonia is often not isolated (table 16 -1). furthermore, studies have shown that bacteriologic causes of pneumonia cannot be determined by radiographic appearance (i.e., "typical" vs. "atypical"). in the proper clinical setting, certain clinical microbes should be considered because they can affect treatment considerations and epidemiologic studies. these include legionella spp., influenza a and b, and communityacquired methicillin-resistant staphylococcus aureus (mrsa). certain diagnostic tests are performed based on clinical setting. blood cultures are not routinely done in the outpatient setting but should always be done if the patient is being admitted to the hospital, ideally before antibiotics are given. the use of gram stain and sputum culture remains controversial but can provide more evidence of a bacterial cause (e.g., many pmns). if sputum cultures are being obtained, it is recommended that the physician have the patient expectorate directly into a specimen cup and have it sent immediately for processing. this can increase the yield of isolating streptococcus pneumoniae among antibiotics for the treatment of bronchitis is not recommended because of the cost, potential for side effects, and lack of clinical benefit (braman, 2006; smith et al., 2009 ) (sor: a). in the treatment of bordetella pertussis, early administration of a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) (sor: a). in adults with acute bronchitis with signs of airway obstruction, as evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough (braman, 2006) (sor: b). for acute exacerbation of copd associated with purulent sputum and increased shortness of breath, treatment with antibiotics decreases mortality by 77% and treatment failure by 53% (ram et al., 2009 ) (sor: a). 210 other respiratory pathogens. other tests include urine antigen tests for s. pneumoniae, legionella pneumophila serogroup 1, and nasal swab for influenza a and b. in young children, rsv, adenovirus, and parainfluenza in addition to influenza are common causes. nasal swab for rsv and influenza can be rapidly done, but the other causes can be determined with viral cultures, serology, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr), although results usually are received after resolution of the acute symptoms. perhaps the most important decision for clinicians is to determine the location of treatment. the american thoracic society (ats) and the infectious diseases society of america (idsa) recommend use of the pneumonia severity index (psi), which uses 20 variables to risk-stratify the patient into five mortality classes, or the curb-65, which measures five clinical variables in this decision making. the curb-65 may be the easiest and most convenient to use at the site of decision making. a score of 0 or 1 indicates treatment as an outpatient; a score of 2 requires hospital admission to the general medical ward; and a score of 3 or more indicates admission to an intensive care unit (icu) (box 16-1). treatment of cap should be targeted toward the most likely etiology (table 16 -2). outpatient therapy for patients who have no comorbidities and have not received antibiotics within the last 3 months includes doxycycline or a macrolide antibiotic. use of a fluoroquinolone antibiotic (levofloxacin or moxifloxacin) should be reserved for patients with more complicated pneumonia and those requiring hospitalization. patients who have comorbid conditions or recent antibiotic exposure, or who will be hospitalized, should receive a respiratory fluoroquinolone or combination therapy with a betalactam drug plus a macrolide, for 48 to 72 hours after fever abates (usually 5-7 days' total therapy). if an organism is isolated, therapy may be narrowed to cover the causative agent. the clinician should consider longer therapy and appropriate antibiotics to cover for infection by less common organisms such as staphylococcus aureus or pseudomonas aeruginosa. if the patient has no more than one abnormal value (temperature <37.8° c, heart rate <100, respiratory rate <24, sbp >90, o 2 saturation >90%, po 2 >60 on room air) and the patient is able to maintain oral intake and has a normal mental status, the clinician can safely switch to oral therapy and discharge the patient from the hospital. unless the etiology of the pneumonia is known, the physician should switch to oral antibiotics in the same class as the intravenous antibiotics used. the u.s. preventive services task force (uspstf) along with idsa and ats recommend annual influenza vaccinations to those over 50 years of age, those who are (or who reside with those who are) at high risk for influenza complications, and all health care workers. furthermore, the pneumococcal vaccine should be given to all those over age 65. smoking cessation is also important and should be discussed at each clinic visit. • concerns about development of resistant seasonal and h1n1 swine-derived influenza virus should be considered in the decision to administer antiviral medications to healthy patients with these infections. • the abrupt onset of fever with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season" is sufficient to diagnose influenza. • prevention of influenza is generally with vaccination. influenza deserves special mention because it is an important cause of pneumonitis and can precede a bacterial pneumonia. influenza viruses are medium-sized enveloped ribonucleic acid (rna) viruses that consist of a lipid bilayer with matrix proteins with spiked surface projections of glycoproteins (hemagglutinins, neuraminidase) on the outer surface ( figure 16-1) . both influenza a and influenza b have eight segmented pieces of single-stranded rna. the only difference between influenza a and b is that b does not have an m2 ion channel. hemagglutinins, three types of which typically infect humans (h1, h2, h3), bind to respiratory epithelial cells and allow fusion with the host cell. neuraminidase, consisting of two types (n1, n2), allows release of virus from the infected cells. a unique aspect of influenza is that antigenic variation occurs annually. antigenic shift is caused by a genetic reassortment between animal and human influenza strains, producing a novel virus that generally causes the worldwide pandemics. influenza viruses circulate mostly among humans, birds, and swine. sometimes; a human strain and an animal strain can intermingle and create a new, unique virus. this is what happened during spring 2009, heralding the most recent pandemic and creating "novel h1n1 influenza" (swine influenza). genotype analysis • score 0 or 1: outpatient treatment • score 2 : inpatient treatment on a general medical floor • score >3: inpatient treatment in an intensive care unit bun, blood urea nitrogen. locally adapted guidelines should be implemented to improve the processing of care variables and relevant clinical outcomes in pneumonia (mandell et al., 2007) (sor: b) . objective criteria or scores should always be supplemented with physician determination of subjective factors, including the patient's ability to take oral medication safely and reliably and the availability of outpatient support resources (sor: b). for patients with curb-65 score of 2 or higher, more intensive treatment (i.e., hospitalization or, where appropriate and available, intensive in-home health care services) is usually warranted (sor: c). of this strain determined that components came from an influenza virus circulating among swine herds in north america that combined with a virus circulating among ill swine in eurasia, creating a new influenza strain capable of causing disease in humans. because this virus had not previously infected humans, it had the potential to cause widespread morbidity and mortality worldwide. during pandemics, the u.s. centers for disease control and prevention (cdc) estimates an additional 10,000 to 40,000 deaths caused by influenza. although higher than in nonpandemic years, mortality was significantly less than initially predicted in 2009. no recent antibiotic therapy a respiratory fluoroquinolone alone or an advanced macrolide plus a β-lactam † † an advanced macrolide plus a β-lactam, or a respiratory fluoroquinolone alone (regimen selected will depend on nature of recent antibiotic therapy) intensive care unit (icu) a β-lactam † † plus either an advanced macrolide or a respiratory fluoroquinolone pseudomonas infection is not an issue but patient has a β-lactam allergy a respiratory fluoroquinolone, with or without clindamycin pseudomonas infection is an issue ‡ ‡ (cystic fibrosis, impaired host defenses) either (1) copd, chronic obstructive pulmonary disease; mrsa, methicillin-resistant staphylococcus aureus. * azithromycin or clarithromycin. † that is, the patient was given a course of antibiotic(s) for treatment of any infection within the past 3 months, excluding the current episode of infection. such treatment is a risk factor for drug-resistant streptococcus pneumoniae and possibly for infection with gram-negative bacilli. depending on the class of antibiotics recently given, one or another of the suggested options may be selected. recent use of a fluoroquinolone should dictate selection of a nonfluoroquinolone regimen, and vice versa. ‡moxifloxacin, levofloxacin, or gemifloxacin. § dosage: 1 g orally (po) three times daily (tid). ¶ dosage: 2 g po twice daily (bid). ** high-dose amoxicillin (1 g tid), high-dose amoxicillin-clavulanate (2 g bid), cefpodoxime, cefprozil, or cefuroxime. † † cefotaxime, ceftriaxone, ampicillin-sulbactam, or ertapenem. ‡ ‡the antipseudomonal agents chosen reflect this concern. risk factors for pseudomonas infection include severe structural lung disease (e.g., bronchiectasis) and recent antibiotic therapy, health care-associated exposures or stay in hospital (especially in the icu). for patients with cap in the icu, coverage for s. pneumoniae and legionella species must always be considered. piperacillin-tazobactam, imipenem, meropenem, and cefepime are excellent β-lactams and are adequate for most s. pneumoniae and h. influenzae infections. they may be preferred when there is concern for relatively unusual cap pathogens, such as p. aeruginosa, klebsiella spp., and other gram-negative bacteria. § § piperacillin, piperacillin-tazobactam, imipenem, meropenem, or cefepime. ## data suggest that older adults receiving aminoglycosides have worse outcomes. ¶ ¶ dosage for hospitalized patients, 750 mg/day. the abrupt onset of fever, along with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season," is sufficient to diagnose influenza. as the fever resolves, a dry cough and nasal discharge predominate. a rapid nasal swab or viral cultures can be used to confirm the diagnosis of influenza but is rarely needed. in fact, the sensitivity of these rapid tests can range from 50% to 70%, so a negative test does not rule out influenza. the primary care physician needs to determine if the patient has influenza or the common cold, because symptoms of both illnesses generally overlap (table 16-3) . treatment of influenza is generally not necessary because it is usually a self-limiting condition. treatment should be reserved for those with comorbidities who present within 48 hours of symptom onset. neuraminidase inhibitors (zanamivir and oseltamivir) prevent the release of virus from the respiratory epithelium and are approved for both influenza a and influenza b. the m2 inhibitors (amantadine and rimantadine) are approved by the u.s. food and drug administration (fda) for the treatment of influenza a because these drugs block the m2 ion protein channel, preventing fusion of the virus to host cell membrane (influenza b has no m2 ion channel). the use of m2 inhibitors is limited because of increasing resistance among influenza a viruses, as well as causing central nervous system (cns) problems that are usually exacerbated in elderly persons, who are more likely to seek treatment for influenza (table 16 -4). the major complication of influenza is a secondary bacterial pneumonia or exacerbation of underlying copd. initial improvement in clinical symptoms followed by deterioration usually suggests a secondary bacterial pneumonia, which can usually be confirmed with a chest radiograph showing an infiltrate. other, less common complications of influenza include myositis, myocarditis, pericarditis, transverse myelitis, encephalitis, and guillain-barré syndrome. prevention of influenza is generally with vaccination. box 16-2 outlines patients at risk for influenza complications who should be vaccinated yearly. although anyone wanting an influenza vaccine should be vaccinated, during periods of vaccine shortage, high-risk groups have priority. a well-matched vaccine can prevent influenza among 70% to 90% of adults and decrease work absenteeism. conversely, a poorly matched vaccine only prevents influenza in 50% of healthy adults. proper hand hygiene and covering one's cough are two additional important components in preventing the spread of influenza virus. • population-based vaccination programs have been highly effective in decreasing the incidence of many viral infections. • acyclovir can be used in adults and children with varicella to decrease symptoms if given in the first 48 hours after rash onset, but its benefit must be weighed against its cost and the possibility of development of viral resistance. • antiviral medications should be considered to decrease the incidence of postherpetic neuralgia, particularly in older patients. early treatment (within 48 hours of onset of symptoms) with oseltamivir or zanamivir is recommended for influenza a (jefferson et al., 2006) (sor: a). use of oseltamivir and zanamivir is not recommended for patients with uncomplicated influenza with symptoms for more than 48 hours (kaiser and hayden, 1999) (sor: a). oseltamivir and zanamivir may be used to reduce viral shedding in hospitalized patients or to treat influenza pneumonia (sor: c). (from treanor jj: influenza viruses, including avian influenza and swine influenza. in mandell gl, bennett je, dolin rd (eds) . mandell, douglas, and bennett's principles and practices of infectious diseases, 7th ed. philadelphia, churchill livingstone, 2010, p 2266.) • measles has had a resurgence in recent years and should be suspected when a patient presents with cough, coryza, conjunctivitis, and head-to-toe rash. • epstein-barr virus and cytomegalovirus infections are generally not clinically distinguishable, and their treatment is primarily supportive. vaccinations have dramatically decreased the incidence of a number of historically common viral infections; smallpox has been eradicated through widespread vaccination. however, recent outbreaks of measles and mumps on college campuses underscore the need to remain vigilant in administering vaccines at the population level, even though no vaccine is available for many common viruses. varicella is one of the classic viral exanthems of childhood. before routine vaccination, having chickenpox was one of childhood's "rites of passage." the virus, a herpesvirus (human herpesvirus 3), is effectively transmitted, causing outbreaks in schools and households. patients with primary varicella present with fever, headache, and sore throat. generally within 1 to 2 days of onset of symptoms, a papulovesicular rash erupts diffusely. the classic description of the chickenpox lesion is "a dewdrop on a rose petal," suggesting a central vesicle on an erythematous base. lesions continue to appear for 5 to 7 days. all lesions going from papule to vesicle to crusted lesion takes about 2 weeks. patients are considered to be infectious, primarily through respiratory secretions, during the 2 days before symptoms appear and until all lesions are crusted. treatment of varicella is generally supportive. control of spread may be a concern in group-living environments such as schools or residence halls. isolation of the infected patient away from those susceptible to varicella infection is standard practice. acyclovir can be started within the first 24 hours after rash eruption to achieve an attenuation of the infectious course. in children, this means a decrease in the duration of fever by about 1 day and a decrease in the number of lesions (swingler, 2010) . in adults, acyclovir decreases rash duration and the number of lesions, although the results are less significant than for children. adult dosing of acyclovir for varicella is 800 mg five times daily. the marginal benefit must be weighed against the possible development of resistance at a population level and the cost of the medication. complications of varicella can include secondary infection of skin lesions, pneumonitis, encephalitis, and dehydration from vomiting and diarrhea. varicella is prevented primarily through administration of vaccine. the vaccine is highly effective in children, with recommended dosing at 12 to 15 months with a second dose at 4 to 6 years. varicella is now included in a measles-mumpsrubella (mmr) vaccine, which can be given between 12 months and 12 years of age. the varicella vaccine is a live, attenuated virus and should not be given to certain immunocompromised patients. the vaccine can also be administered to exposed immunocompetent contacts, although the benefit is clearer for children than adults. severely immunocompromised patients exposed to varicella (particularly those with advanced hiv disease) may be given high-dose acyclovir to prevent development of disease. herpes zoster is a reactivation of the neurotropic varicella virus, typically in a dermatomal distribution. this is more common in elderly or immunocompromised patients but can occur in healthy people as well. patients with zoster may note generalized malaise, hyperesthesia, numbness, tingling, and pain in the skin before development of a rash. the appearance of the rash is the same as for chickenpox, although most often isolated to a unilateral dermatome. the diagnosis of herpes zoster is clinical based on the history and the classic appearance of the rash. in immunocompromised patients, however, the rash may not be dermatomally isolated. when the diagnosis is unclear, viral culture can be obtained from the base of a lesion. antiviral medications are likely to decrease the incidence of postherpetic neuralgia and are recommended, particularly in elderly patients (wareham, 2010) . valacyclovir (1 g three times daily) or famciclovir (500 mg every 8 hours) for 7 days is likely more effective than acyclovir in achieving this result. either drug should be started as soon after the diagnosis as possible, preferably within 48 to 72 hours of rash onset. when patients have established postherpetic neuralgia, gabapentin and tricyclic antidepressants are helpful in alleviating the pain. the rash of zoster is infectious to the touch. patients should be advised to keep the rash covered until all the lesions have crusted. zoster of the trigeminal nerve can extend to the eye and warrants immediate ophthalmologic intervention. a vaccine to prevent herpes zoster in adults was released in 2006. the zoster vaccine differs from the varicella vaccine in that the amount of attenuated virus is 14 times higher in the zoster vaccine. the vaccine decreases the incidence of zoster by 50%. it is recommended for administration by the american academy of family physicians (aafp) to adults over age 60, regardless of prior varicella or zoster history. although generally well tolerated, the vaccine is somewhat costly. in 2008, more measles cases were reported than in any other year since 1997 (cdc, 2010) . measles is the "first disease" of childhood from the history of medicine. in adults, measles infection may be acquired in the face of waning immunity from remote immunization. a booster dose of mmr vaccine is recommended before college entry. clinically, measles presents with cough, coryza (nasal irritation and congestion), and conjunctivitis. fever is common several days before the onset of the rash. the rash of measles typically spreads from head to toe and has an erythematous, papular appearance with a "sandpaper" feeling. koplik's spots are erythematous papules with a bluish center on the oral mucosa and appear early in measles. measles is highly contagious through droplets. lymphopenia and neutropenia are common laboratory findings with measles infection. complications of measles include primary infections such as pneumonia, gastroenteritis, encephalitis, and the rare subacute sclerosing panencephalitis. secondary infections such as otitis media, pneumonia, and adenitis may also occur. treatment is supportive, and the implications of measles infection are primarily in the public health realm. patients with measles should be isolated for at least 4 days after the appearance of the rash. it is important to recognize that patients are contagious for 2 days before the development of symptoms. careful verification of immunization status for close contacts is essential. clinical infectious mononucleosis is a common infection in adolescents and early adults. the clinical syndrome is most often caused by epstein-barr virus (ebv), although cytomegalovirus (cmv) may also be the source in this clinical syndrome, which includes fever, exudative tonsillitis, adenopathy (often including posterior cervical or occipital nodes), and fatigue. ebv is transmitted in oral secretions and may be transmitted sexually as well. b cells are infected with ebv either directly or after contact with epithelial cells, resulting in diffuse lymphoid enlargement. the diagnosis of infectious mononucleosis is made by recognizing the clinical symptoms of fever, pharyngitis, and adenopathy along with the laboratory findings of greater than 50% lymphocytes with 10% or more atypical lymphocytes (hoagland, 1952) . also, a positive serologic test for heterophile antibody assists the family physician in the diagnosis. to differentiate ebv from cmv mononucleosis, serology (igg and igm) may be obtained. results of these tests are generally not available in time to have a significant benefit clinically. splenic enlargement as part of this lymphoid hypertrophy can lead to splenic rupture (0.1% risk) (dommerby et al., 1986) . athletes with infectious mononucleosis must be managed carefully to avoid their participation in sports that could result in abdominal trauma. other risks associated with infectious mononucleosis include upper airway obstruction, asymptomatic transaminase elevation, thrombocytopenia, and rash after the administration of ampicillin or amoxicillin. routinely obtaining transaminase levels in patients without clinical hepatitis is of little value and can increase the overall cost of management. treatment of infectious mononucleosis is largely supportive. patients should be instructed to treat fever with antipyretics, rest, and expect symptom duration of 2 to 4 weeks, although symptoms can last for several months. the use of steroids, such as prednisone, has shown limited benefit. data suggest an initial benefit 12 hours after steroid administration, although this is lost within several days (candy and hotopf, 2006) . combination of steroid and an antiviral (valacyclovir) may have some positive effect on fatigue. • the most common presentation of tuberculosis is pulmonary disease. • tuberculosis is diagnosed by acid-fast bacilli smears and cultures. • standard first-line agents to treat tb are isoniazid, rifampin, pyrazinamide, and ethambutol. • high-risk patients with a positive purified protein derivative skin test or quantiferon-tb gold test should be treated for latent tb infection. • the current recommendation for first-line treatment for latent tb is 9 months of oral isoniazid. tuberculosis skin testing should be interpreted without regard to bacille calmette-guérin (bcg) history, because bcg is administered in areas where tb is endemic and bcg does not provide complete protection from tb infection. tuberculosis (tb) is a disease that has plagued humans since antiquity, with evidence of spinal tb in neolithic and early egyptian remains. at present, tb affects approximately one third of the world's population. tb is the world's second most common cause of death from infectious disease after human immunodeficiency virus or acquired immunodeficiency syndrome (hiv/aids). tuberculosis is caused by mycobacterium tuberculosis, an acid-fast bacillus. tb is acquired by inhalation of respiratory droplets. these respiratory droplets are spread by coughing. brief contact carries little risk for acquiring tb, and infection generally does not occur in open air; open-air sanatoriums were the cornerstone of tb treatment before antimicrobial therapy. in the united states, tb incidence rates have been on the decline since 1992, coinciding with the control of hivinduced aids by antiretroviral therapy. however, tb remains prevalent in certain high-risk groups (i.e. immigrants, iv drug use, homeless persons). most cases of tb are in people age 15 to 49 years. tb in elderly persons is generally caused by a reactivation of latent infection acquired in the remote past, whereas tb in young children indicates ongoing active transmission in the community. infection in children is more likely to progress to active tb and disseminated disease. persons with hiv infection have a disproportionately higher risk for acquiring tb than the general population. tuberculosis is most frequently manifested clinically as pulmonary disease, but it can involve any organ. extrapulmonary tb accounts for about 20% of disease in hiv-seronegative persons but is more common in hiv-seropositive persons. pulmonary tb typically manifest with fever, night sweats, chronic cough, sputum production, hemoptysis, anorexia, and weight loss. chest radiographs in patients with pulmonary tb typically reveal upper-lobe cavitary lesions and can reveal infiltrates or nodular lesions, as well as lymphadenopathy ( figure 16 -2). tb in the setting of advanced hiv co-infection does not generally manifest in the typical manner (table 16-5) . acyclovir started within the first 24 hours after varicella rash eruption can attenuate the infectious course, decreasing duration of fever by 1 day and reducing the number of lesions (sor: a). administration of varicella vaccine to a susceptible child within 3 days of exposure will likely modify or prevent disease (macartney and mcintyre, 2008) the diagnosis of pulmonary tb is made by the demonstration of acid-fast bacilli (afb) in sputum and the growth of m. tuberculosis in culture. these patients typically have an abnormal chest radiograph, as previously described. m. tuberculosis is a slow-growing bacterium, and cultures can take up to 6 weeks to grow. a pcr assay developed for m. tuberculosis can be run on afb smear-positive sputum to hasten the diagnosis of pulmonary tb. a positive pcr on afb-positive sputum is diagnostic of pulmonary tb, but a negative test does not rule out the diagnosis. patients with afb positive smears from sputum samples should be started on anti-tb therapy while awaiting results of pcr and cultures. the treatment of tb always uses multiple agents with anti-tb activity. single agents should never be used. the standard first-line agents are isoniazid (inh), rifampin (rif), pyrazinamide (pza), and ethambutol (emb) (figure 16 -3 and table 16-6). if administered, inh should be given with pyridoxine (vitamin b 6 ; 25-50 mg orally daily) to prevent neuropathy. treatment of active pulmonary tb is generally for 6 months regardless of hiv status, but treatment may need to be extended in certain situations. directly observed therapy (dot) is the preferred mechanism of administration to ensure compliance. many local county and state health departments have systems for dot. treatment of hiv-seropositive patients with tb who are receiving an antiretroviral (arv) regimen that contains a protease inhibitor is complicated by the latter's interaction with rifamycins (particularly rifampin). management of such patients should be coordinated with an infectious diseases specialist, who also should manage drug-resistant tb treatment. in the united states, latent tuberculosis infection (ltbi) is the most prevalent form of tuberculosis. ltbi is the term given to patients with a positive purified protein derivative (ppd) skin test without evidence of active tb. ppd has been used for more than 100 years and relies on delayed-type hypersensitivity (dth) to m. tuberculosis cellular proteins. early late figure 16 -3 treatment algorithm for tuberculosis. patients in whom tb is proved or strongly suspected should have treatment initiated with isoniazid, rifampin, pyrazinamide, and ethambutol for the initial 2 months. a repeat smear and culture should be performed when 2 months of treatment has been completed. if cavities were seen on the initial chest radiograph or the acid-fast smear is positive at completion of 2 months of treatment, the continuation phase of treatment should consist of isoniazid and rifampin daily or twice weekly for 4 months to complete a total of 6 months of treatment. if cavitation was present on the initial chest radiograph and the culture at completion of 2 months' therapy is positive, the continuation phase should be lengthened to 7 months (total of 9 months of treatment). if the patient has hiv infection and the cd4+ cell count is less than 100/mm 3 , the continuation phase should consist of daily or three-times-weekly isoniazid and rifampin. in hiv-uninfected patients having no cavitation on chest radiograph and negative acid-fast smears at completion of 2 months of treatment, the continuation phase may consist of either once-weekly isoniazid and rifapentine, or daily or twice-weekly isoniazid and rifampin, to complete a total of 6 months (bottom). patients receiving isoniazid and rifapentine, and whose 2-month cultures are positive, should have treatment extended by an additional 3 months (total of 9 months). *emb may be discontinued when results of drug susceptibility testing indicate no drug resistance. †pza may be discontinued after it has been taken for 2 months (56 doses). ‡rpt should not be used in hiv-infected patients with tb or in patients with extrapulmonary tb. therapy should be extended to 9 months if 2-month culture is positive. afb, acid-fast bacilli; cxr, chest radiograph (x-ray); emb, ethambutol; inh, isoniazid; pza, pyrazinamide; rif, rifampin; rpt, rifapentine. because ppd relies on dth, any factor that reduces the dth affects the host response to ppd. the most common clinical example is use of corticosteroids, which blunt the dth response and can complicate ppd interpretation. therefore, ppd testing should not be performed while a patient is taking corticosteroids. also, tb testing should be targeted to those with higher risk of infection and should not routinely be done in those with low risk (ats/cdc, 2000) . the ppd can also give false-positive results in patients with previous bacille calmette-guérin (bcg) vaccination or with infection by other mycobacterial infections. in the united states, this may cause difficulties in testing immigrants from countries who routinely use bcg vaccination programs. however, previous bcg vaccination should not change the interpretation of the ppd or willingness to treat such individuals accordingly. ‡when dot is used, drugs may be given 5 days per week and the necessary number of doses adjusted accordingly. although there are no studies that compare five with seven daily doses, extensive experience indicates this would be an effective practice. § patients with cavitation on initial chest radiograph and positive cultures on completion of 2 months of therapy should receive a 7-month (31 weeks, either 217 doses [daily] or 62 doses [twice weekly]) continuation phase. ¶ five-days-a-week administration is always given by dot. rating for 5 day per week regimens is aiii. ¶ ¶ not recommended for hiv-infected patients with cd4+ cell counts <100 cells/μl. ** options 1c and 2b should be used only in hiv-negative patients who have negative sputum smears at completion of 2 months of therapy and who do not have cavitation on initial chest radiograph. for patients started on this regimen and found to have a positive culture from the 2-month specimen, treatment should be extended an extra 3 months. the dth response can wane over time. to overcome this problem, nonreacting patients may undergo repeat ppd 1 week after their initial ppd. the diagnosis of ltbi is made by interpretation of a ppd and by ascertaining the patient's risk factors for progression to active tb if left untreated . interpretation of the ppd should be based on the area of induration and not the area of surrounding erythema. persons whose ppds have converted from negative to positive within 2 years are presumed to have been infected recently. the decision to use ppd means treating the patient for ltbi if the ppd test is positive. patients at increased risk for progression to active tb include those who have been recently infected (recent ppd converters); patients who are hiv seropositive; patients who have silicosis, diabetes, or chronic renal failure (including those receiving hemodialysis); solid-organ transplant recipients; patients with gastrectomy or jejunoileal bypass or head and neck cancer; injection drug users; patients with chest radiograph evidence of prior tb; and patients who weigh at least 5% less than ideal body weight. patients taking chronic corticosteroid therapy and those who are to receive tumor necrosis factor alpha (tnf-α) blockers (e.g., infliximab) are also at risk. patients taking corticosteroids also have higher risk of progression to active tb with larger doses and longer courses of corticosteroids. standard therapy for ltbi is inh, 300 mg orally daily for 9 months, regardless of hiv status. again, inh should always be administered with pyridoxine to prevent neuropathy. to overcome the false-positive results and confusion of ppd testing in certain populations, newer interferon-gamma (ifn-γ) release assays such as the quantiferon-tb gold (qft-g) test have been developed to detect latent m. tuberculosis. qft-g quantifies the release of ifn-γ from lymphocytes of the host's blood in response to three m. tuberculosis target antigens that are absent from bcg and most other nontuberculous mycobacterium spp. the advantages of using qft-g include one-time blood testing without the need for followup visit, no triggering of amnestic responses, and possibly more specific response to m. tuberculosis. however, qtf-g use in immunocompromised or anergic patients is limited, with indeterminate results. some studies also show discordant results in individuals tested with both ppd and qtf-g. in general, qtf-g may be used in all circumstances in which the ppd is used. however, whether the qtf-g is truly more specific or sensitive than the ppd in latent or active tb is yet to be determined. • the u.s. preventive services task force recommends "highintensity" behavioral counseling to at-risk adults and adolescents to prevent sexually transmitted infections. • be specific in addressing patients' sexual practices so as to provide appropriate prevention advice. hiv-positive persons recent contacts of tuberculosis patients fibrotic changes on chest radiography consistent with prior tuberculosis patients with organ transplants and other immunosuppressed patients (receiving equivalent of ≥15 mg/day of prednisone for at least 1 month) development in the primary prevention of stis is immunization against human papillomavirus (hpv). the vaccine can prevent infection with certain strains of hpv that cause cervical cancer and genital warts. trials are ongoing to determine the effectiveness of daily arv therapy in preventing transmission of hiv. vaccination investigation is ongoing for herpes simplex, chlamydia trachomatis, and hiv. this breadth of research effort holds promise for the future in the prevention of stis. the uspstf recommends "high-intensity" behavioral counseling to at-risk adults and adolescents to prevent stis. highintensity counseling involves multiple sessions and often is delivered to groups of patients. unfortunately, this type of intervention has limitations in its practicality for population-based delivery. no risk of harm was discovered in the delivery of counseling for sti prevention. vaccination is the most important form of primary prevention of common infectious diseases. two vaccines are currently on the market for hpv prevention-one that protects against four viral subtypes (6, 11, 16, 18) and is licensed for use in males and females 9 to 26 years of age, and the other against two subtypes (16,18), licensed for females 10 to 25 years of age. hepatitis b is a sexually transmitted infection, and immunization is recommended for adolescents who have not been previously inoculated. this is a requirement in many states for school entry. hepatitis a can be transmitted by oro-anal sexual contact, and vaccination should be offered to patients who are contemplating engaging in this sexual practice. recommendations surrounding the use of barrier methods for sti prevention should be tailored to the sex practices of the client. for example, a percentage of women use anal sex as a method of birth control but may not consider the need for condom use with this practice. the question, "do you regularly use condoms?" has little relevance to infection control for many sexual practices. evidence supports the advice to use barrier methods of latex or other approved material in a manner that prevents the exchange of blood and body fluids in decreasing stis. condoms confer a 30% risk reduction for herpes simplex and up to an 80% risk reduction for hiv, when used correctly (weller and davis-beaty, 2002; martin et al., 2009) . the secondary prevention of stis is achieved through direct and nonjudgmental patient assessment and screening and avoiding assumptions about patient sexual practices. screening is a tool to prevent the inadvertent spread of infection as well as the sequelae of undetected disease. infectious genital ulcers are associated with herpes simplex virus (hsv), syphilis, chancroid, lymphogranuloma venereum, and granuloma inguinale. hsv is by far the most common, affecting 50 million people in the united states. hsv-1 and hsv-2 are chronic, neurotropic viral infections that enter through epithelium and come to rest in the dorsal root ganglia. therefore, infection leads to lifetime presence of the virus, but the clinical manifestation of this condition is variable. a small percentage of those with serologic evidence of hsv-2 (10%-25%) have had symptoms of clinical herpes infection. in addition, patients with hsv infection can shed the virus in the absence of symptoms, creating a prime opportunity for spread. herpes simplex outbreak may be followed by a prodrome of malaise, fever, and regional lymphadenopathy before the appearance of grouped vesicles on an erythematous base. the vesicles are typically quickly broken and become ulcerated in appearance, with each vesicle usually less than several millimeters in size. true first-time infections tend to present more severely than secondary presentations of previously infected individuals, with a prodrome present in 80% of cases. the lesions can be in any location around the genitals or rectum, on the proximal thighs and buttocks, inside the vagina, and in and around the mouth. the lesions are most who to screen? often painful, particularly when on mucosal surfaces, or itchy. in women, herpes simplex can present with cervicitislike symptoms with bleeding and discharge and cervical ulcerations on examination, or simply mucopurulent cervicitis. herpetic lesions around the urethra tend to be extremely painful and can make urination difficult. rectal hsv can be confused with irritation, perianal fissure, and even candidiasis because of its often beefy-red appearance and itching. vesicles typically appear 6 days after infection and can last up to 2 weeks in an initial infection. subsequent outbreaks tend to have a shorter duration and to be less uncomfortable for patients. confirmation of infection is helpful, but the diagnosis can be made primarily on the clinical appearance of the exanthema. vigorous sample collection from an ulcer (which the patient may not appreciate) to be sent for pcr identification and typing is the most readily available method of laboratory diagnosis. serum antibody testing is not useful in the initial hsv diagnosis because antibody levels will not be appreciable early in infection. the appearance of convalescent immunoglobulin g (igg) and igm levels several weeks after a suspected outbreak might help to support the diagnosis of hsv infection. the value of screening for hsv immunity is debatable and should generally not be recommended for asymptomatic individuals. in addition, the uspstf recommends against screening asymptomatic pregnant women for hsv to prevent transmission to the newborn. given that many patients with hsv infection never manifest symptoms, the value of knowing that one is hsv seropositive is questionable. in addition, hsv-1 and hsv-2, although classically oral and genital, respectively, can "mix and match" based on sexual practices. it is often confusing for asymptomatic individuals to know that they have hsv antibody (do i have cold sores? do i have genital herpes? how should this change the way i live my life?). in monogamous couples with one partner known to be hsv positive and the other with unknown status, testing of the latter may indicate suppressive therapy in the seropositive partner if the other is found to be negative. regular barrier method use decreases transmission of herpes in both men and women, with patients using condoms 100% of the time having a 30% reduction in hsv acquisition from those who never use condoms (martin et al., 2009) . serodiscordant couples may also decrease transmission through antiviral suppressive therapy to the hsv-positive partner (table 16-8) . syphilis is a spirochetal infection that has resurged since 2001, the nadir year since 1996. syphilis infection rates are highest in men who have sex with men. syphilis is much less common than the other stis, with an infection rate of 5.6 per 100,000 population in the united states (vs. 496 per 100,000 for chlamydia). syphilis presents in several stages. the primary phase of syphilis is a painless ulcer called a chancre (figure 16-4) . the chancre may be visible on the genitals, although it can also be inside the vagina, mouth, or rectum, making it difficult to find. this lesion will appear within 3 weeks of transmission and will last for several weeks untreated. the secondary phase of infection is disseminated and involves a diffuse macular rash, typically with palm and sole lesions, generalized lymphadenopathy, fever, and condyloma latum (smooth, moist lesions on genitals without cauliflower appearance of condyloma acuminatum). tertiary syphilis is often asymptomatic but affects the heart, eyes, and auditory system and can be associated with gumma formation. gummas are soft, granulomatous growths in organs that can cause mechanical obstruction and weakening of blood vessel walls. latent infection often involves the cns. diagnosis of primary syphilis is challenging. the test of choice is darkfield microscopy, which is not readily available. direct fluorescent (monoclonal) antibody (dfa) testing may be available. antibody tests for syphilis, such as the rapid plasma reagin (rpr) and the less frequently used venereal disease research laboratories (vdrl), are often not positive early in infection and thus cannot be used to rule out primary syphilis based on a single reading. treponemal antigen testing (eia) may be available in some laboratories. the fluorescent treponemal antibody absorption (fta-abs) test may also be negative in the early infection. direct pcr for primary syphilis lesions has been tested but is not yet fda approved. a physician may choose to treat presumptively if a painless chancre and risk factors are present and may then do a convalescent rpr test in 1 to 2 weeks to confirm the infection by the appearance of a positive reaction. one would expect a fourfold change in titer of either test to indicate the presence of disease. primary and secondary syphilis are treated with a single injection of penicillin g, 2.5 million units. other regimens do not have proven effectiveness but can be used in the penicillin-allergic patient, including doxycycline, 100 mg twice daily for 14 days; ceftriaxone, 500 mg to 1 g intramuscularly (im) daily for 8 to 10 days; or azithromycin, 2 g as a single oral dose, although resistance to azithromycin has been observed. patients treated for primary syphilis should have periodic clinical follow-up and serologic testing to determine a fourfold decrease in rpr reactivity within 6 months. latent syphilis can be either early, meaning infection within the last year, or late, meaning infection beyond a year. early latent syphilis is treated with a single injection of penicillin g, 2.4 million units. syphilis of late latency or unknown duration is treated with three injections of penicillin g, 2.4 million units, in 3 consecutive weeks. for penicillin-allergic patients, doxycycline, 100 mg twice daily for 28 days, is required. those with latent syphilis should have ophthalmic examination as well as evaluation for vascular gumma formation. suspected neurologic involvement of latent syphilis must be evaluated with cerebrospinal fluid (csf) examination and treatment with aqueous penicillin g, 3-4 million units intravenously (iv) every 4 hours for 10 to 14 days. partners of patients with newly diagnosed syphilis are at risk for infection. partners within 90 days of a diagnosis of primary syphilis should be tested, but treated presumptively even if serologic testing is negative. for partners prior to 90 days before diagnosis, serology is generally reliable in detecting presence of infection and may guide treatment. patients with secondary syphilis should inform partners within 6 months before diagnosis, or 12 months for those diagnosed with tertiary syphilis (table 16 -9). chancroid may occur in regional outbreaks and presents with a painful genital ulcer and suppurative regional adenopathy. herpes and syphilis should both be ruled out in the patient suspected of having chancroid infection. chancroid is caused by haemophilus ducreyi and there is currently no fda approved test to directly detect this organism. treatment with azithromycin (1 g as single dose), ceftriaxone (250 mg im as a single dose), ciprofloxacin (500 mg twice daily for 3 days), or erythromycin (500 mg three times daily for 7 days) are all alternatives (table 16 -10). it may be necessary to perform incision and drainage on fluctuant inguinal nodes. patients should be reexamined in 1 to 2 weeks to ensure healing of the primary ulcer(s) and resolution of the adenopathy. partners who had contact with the infected patient starting 10 days before development of the patient's symptoms should be treated, regardless of the presence of symptoms. less common ulcerating stis include lymphogranuloma venereum (lgv) and granuloma inguinale ( figure 16 -5). lgv causes regional adenopathy and often an ulcer at the point of entry. rectal lgv may cause a proctocolitis with anal pain, discharge, bleeding, and diarrhea. lgv is caused by chlamydia trachomatis serotypes and can be detected by testing swabbed material from open lesions or aspirates from lymph nodes with culture, dfa, or nucleic acid detection. treatment is noted above (table 16-10) . granuloma inguinale, caused by klebsiella granulomatis, is rare in the united states and causes progressive ulcerative disease of the genitals. a second sti category includes those causing the clinical presentation of vaginal discharge, pelvic pain, dyspareunia, and dysuria in women and penile discharge and dysuria in men, as well as possible rectal pain and discharge in men and women. of this group, chlamydia trachomatis is the most common, causing 1.2 million infections in the united states in 2008 (cdc, 2009 ). in fact, chlamydia is the most frequently reported reportable infection. the majority of women with chlamydia infection are without symptoms. many men are asymptomatic as well. regular screening for chlamydia, as recommended by the uspstf, can significantly reduce the incidence of pelvic inflammatory disease (pid), one of the most serious sequelae of untreated infection. in women with untreated chlamydia infection, in addition to pid, tubo-ovarian abscess, tubal scarring and ectopic pregnancy, and infertility can all result. as previously mentioned, regular screening is currently recommended for all sexually active women under age 24, all pregnant women under 24, and at-risk pregnant and nonpregnant women over 24. chlamydia testing can be performed on several liquid-based papanicolaou (pap) tests. endocervical swabs for nucleic acid amplification are acceptable when a conventional pap smear is being used. given the recent liberalization of recommendations about pap testing for women under 21 years of age, urine nucleic acid amplification is a readily available alternative for chlamydia testing. this can easily be done at a contraceptive counseling clinic. urine testing is also an acceptable method of testing for men, in addition to a urethral swab. rectal chlamydia infection can occur in individuals who practice receptive anal intercourse. an fda-approved method of testing should be used for screening and diagnosis of this infection. asymptomatic chlamydia infection is treated with either a single dose of azithromycin, 1 g orally, the drug of choice, or doxycycline, 100 mg twice daily, for 7 days (table 16-11) . patient-delivered partner therapy (pdpt), the practice of dispensing treatment to diagnosed patients to treat their partner(s), has proved effective in reducing reinfection rates and further spread of infection. ept is legally allowable in 21 states and potentially allowable in another 21. chlamydia infection may present symptomatically in men or women with symptoms of dysuria and with discharge and with pelvic pain and dyspareunia in women. the discharge of c. trachomatis, versus that of neisseria gonorrhoeae, is said to be more mucoid than purulent, although this characteristic is not specific enough to provide diagnostic accuracy. symptomatic chlamydia, without evidence of pid, is treated the same as asymptomatic infection. many practitioners will treat presumptively for chlamydia and gonorrhea in patients who present with the symptoms previously mentioned while they wait for confirmatory testing. neisseria gonorrhoeae infection may be asymptomatic in both men and women. the current uspstf recommendation is for screening women at risk. men with penile gonorrhea typically present with purulent penile discharge and dysuria with n. gonorrhoeae infection. mucopurulent discharge, dysuria, pelvic pain, and dyspareunia are typical symptoms in women. in patients who engage in anal intercourse, anal discharge, rectal pain, and bleeding can be presenting symptoms. gonococcal pharyngitis is within the differential of exudative pharyngitis in sexually active patients. when symptomatic, throat pain, tonsillar exudates, and anterior cervical adenopathy may be present. testing for gonorrhea can be done using liquid-based pap technologies, cervical or urethral swabs, or urine for nucleic acid amplification. in men with visible discharge, a gram stain with white blood cells (wbcs) and gram-positive intracellular diplococci has a high degree of sensitivity. culture testing may be preferred for suspected pharyngeal and rectal specimens pending fda approval of other methods. again, physicians may opt to treat patients with mucopurulent cervicitis or urethritis presumptively for gonorrhea and chlamydia while waiting for confirmatory testing. fluoroquinolone therapy is no longer recommended because of widespread resistance (table 16-11) . because reinfection with gonorrhea is common for several months after treatment, it may be advisable to retest patients with confirmed gonorrhea in the 3 months after treatment. similarly, stis may be an indicator of risk behavior, and a complete risk history and testing for other stis is advisable if not completed at the initial visit. in male patients with symptomatic urethritis, a causative agent may not be identified, a situation often referred to as nongonococcal urethritis (ngu). technically, chlamydia is included in this category. organisms such as ureaplasma urealyticum and mycoplasma genitalium may be the cause and may be difficult to detect. treatment for these infections is the same as for symptomatic chlamydia, with azithromycin or doxycycline (table 16 -11). it is recommended that partners of patients with ngu should be evaluated and treated. in some cases, testing of partners may detect a specific organism as the cause of infection (e.g., chlamydia). trichomonas vaginalis causes vaginitis in women, who may have a stereotypic frothy, green, and foul-smelling discharge. many women are asymptomatic with trichomoniasis. in addition to causing asymptomatic infection in men, t. vaginalis may cause urethritis. this organism may be suspected in men when patients have repeated treatment failures and no other explanation for symptoms. microscopic examination of vaginal discharge is 60% to 70% sensitive in women. a first voided urine specimen or urethral swab for microscopic exam may be helpful in identifying the protozoa. culture for trichomonas, which requires a special medium, may be necessary to identify this infection accurately in men. trichomonas is effectively treated with a single 2-g dose of metronidazole (table 16-11) . for non-sti causes of vaginal discharge, see the online discussion at www.expertconsult.com. pelvic inflammatory disease can be caused by a number of organisms, including chlamydia, and presents with pelvic pain and discharge. findings that contribute to the diagnosis of pid include fever greater than 101° f, cervical or vaginal mucopurulent discharge, abundant wbcs on saline preparation of vaginal discharge, elevated erythrocyte sedimentation rate (esr), elevated c-reactive protein (crp), and evidence of n. gonorrhoeae or c. trachomatis infection. hospitalization with parenteral antibiotics may be necessary in pregnant patients, patients in whom surgical emergency cannot be ruled out, those who do not respond to oral treatment, those who cannot tolerate oral treatment, and patients who have severe illness or tubo-ovarian abscess. when treating pid parenterally, improvement of symptoms for 24 hours may prompt a change to oral therapy (table 16-12) . conversely, if oral therapy is not producing significant improvement within 2 to 3 days, admission for parenteral therapy may be necessary. patient awareness of human papillomavirus infection has greatly increased in recent years, in large part related to the patient-directed advertising of the hpv vaccine. hpv is likely the most common sti. thirty types of hpv can infect the genital area, some causing genital warts, some causing malignancies of the genital organs, and most being asymptomatic. the gross categories most often used are "high risk" (most often types 16 and 18) and "low risk" (types 6 and 11) hpv infection, the former more often associated with genital cancer. prevention of hpv infection and cervical cancer was revolutionized with the release of the hpv vaccine, which is effective in reducing the incidence of hpv-associated disease. currently, two vaccines are licensed in the united states. gardasil (merck), released in 2006, includes protection against viral types 6, 11, 16, and 18. it is approved for the prevention of vulvar and vaginal cancer and for the prevention of cervical cancer, cervical dysplasia, and genital warts in females age 9 to 26. the vaccine was recently approved for males of the same age range for the prevention of genital warts. more recently, cervarix (glaxosmithkline) was approved for the prevention of cervical cancer and cervical dysplasia from hpv types 16 and 18 in women age 10 to 25. ideally, the vaccine should be administered before initiation of sexual activity to prevent initial acquisition of these hpv types. patients who are already sexually active may also receive the vaccine. the transmission of hpv to men decreases with consistent condom use, from 53.9% in men who never use condoms to 37.9% in men who "always" use them. unfortunately, hpv can infect skin that is not covered by the use of traditional barrier methods (nielson et al., 2010) . male circumcision may decrease the transmission of hpv. patients have many questions about hpv, in particular about screening for asymptomatic infection. hpv infection occurs with high frequency in the sexually active population; up to 50% or more of sexually active individuals have hpv at some point in their life. in addition, hpv is effectively transmitted, even if contact does not involve genital-togenital touching (i.e., manual stimulation can transmit the virus). again, most hpv infections are without symptoms and resolve spontaneously through eradication by the intact immune system. for all these reasons, screening for the mere presence of hpv infection has minimal utility. there is no treatment for asymptomatic hpv infection. the most common presentation of hpv infection is in the context of an abnormal pap smear. hpv is directly linked to cervical dysplasia. for women over age 21 and under 35, hpv testing with high-risk viral detection is common. the presence of high-risk hpv informs further management of the pap result. it is currently recommended that women over 35 be automatically tested for high-risk hpv infection at the pap smear. patients may present with visible warts, or these may be detected at routine or sti screening. genital warts are often cosmetically unacceptable to patients, even though they are infrequently functionally problematic. in some circumstances, wart burden can be high enough to cause physical discomfort or relative obstruction of the vagina or rectum. vulvovaginal candidiasis and bacterial vaginosis are generally not thought to be sexually transmitted, although they are often in the differential diagnosis of sexually transmitted infection (sti). both these infections likely are related to changes in the vaginal ph and the normal flora distribution. it is not always clear which of these factors is primary and which is secondary, because at diagnosis, both ph and normal vaginal flora will often be abnormal. vulvovaginal candidiasis is a common infection causing typically white, curdlike discharge, itching, and sometimes dysuria. the causative organism is usually candida albicans but can be other candida spp. antibiotics can alter normal vaginal flora, so the recent use of antibiotics may predispose women to candidiasis. physical examination may reveal erythematous external genitalia as well as external and internal white, clumping discharge. usually, no distinctive odor is associated with vaginal yeast. wet preparation of vaginal specimen or treatment with potassium hydroxide (koh) may reveal branching pseudohyphae and yeast. when ph is performed, it should be directly on the vaginal discharge and not on the saline-diluted specimen because the saline will alter the ph of the specimen. typically, the ph of yeast discharge is less than 4.5 (normal vaginal ph,3.8-4.5). bacterial vaginosis (bv) is the most common cause of infectious vaginal discharge (spence and melville, 2007) . many different organisms are associated with the diagnosis of bv, including gardnerella vaginalis and mycoplasma hominis. women with bv may report discharge, vaginal irritation, vaginal odor, and at times, dysuria. findings of bv are often detected during a normal screening pap smear or pelvic examination. physical findings may reveal signs of vaginal irritation. the discharge is usually thin and gray. an amine (fishy) odor may be produced with the application of koh. the finding of clue cells, or epithelial cells with adherent bacteria, under saline preparation microscopy and a decrease in normal lactobacilli are common findings. the amsel criteria are useful in bv diagnosis; other scoring systems (e.g., nugent criteria) have been used but require gram staining. the specific amsel criteria are (1) milky, homogeneous, adherent discharge; (2) discharge ph greater than 4.5; (3) positive whiff test (fishy smell with addition of koh); and (4) at least 20% clue cells on microscopic examination. if three of the four criteria are present, the likelihood of bv is 90%. in routine vaginal examination and bimanual examination for patients with vaginal discharge, signs and symptoms of vaginitis are poor predictors of the microbiologic cause of infection (schaaf et al., 1990) . the clinical examination and office testing, in fact, are fair predictors of the true cause of infection (lowe et al., 2009 ). many patients with vaginal discharge will use over-the-counter preparations before consulting a physician, which can delay correct diagnosis of the etiology of symptoms. patient-collected, low vaginal swabs may be as useful as provider-collected specimen in making a diagnosis for the patient with vaginal discharge. the purpose of bimanual examination is to evaluate for signs of pelvic inflammatory disease and is not necessary in the low-risk patient with vaginal discharge. treatment of asymptomatic bv or vaginal yeast is not necessary in the nonpregnant patient or usually is not needed to test or treat partners of patients with isolated yeast or bv. when infection is recurrent, particularly when a woman's male partner is uncircumcised, treatment of the male partner for carriage of either infection may be warranted. options for treatment of recurrent infections are presented in etable 16-1. the treatment of warts is destructive and may serve to stimulate an immune response to the hpv-infected cells, which are typically "above" the surveillance mechanisms of the immune system in the epidermis. office methods of treatment include cryotherapy and trichloroacetic acid or podophyllin resin application. patients may apply podofilox 0.5% solution or gel or imiquimod 5% cream (table 16-13) . for more extensive cases of warts or intra-anal or intravaginal infections that are difficult to treat using the previous methods, surgical techniques may be necessary to achieve resolution. untreated, warts may resolve spontaneously, remain the same, or worsen. patients with pediculosis pubis, or pubic lice, most often present with pruritus or with visible nits. pubic lice are visible on inspection of the pubic area, as are nits, which are adherent to the hair shaft. partners of patients with pubic lice should also be treated to prevent reinfection. linens and clothing should be laundered or dry-cleaned or kept in a closed plastic container or bag for 72 hours. scabies diagnosis can be challenging. again, patients present with itching that can be anywhere on the body, although often in the genital area or on the buttocks when infection is sexual in origin. the pruritus associated with sarcoptes scabiei is a result of sensitization to the mite droppings underneath the skin as the mite burrows. the classic "burrow" or linear papular eruption is not always present. scraping of lesions with microscopic examination may be performed to identify the mite. as with pediculosis, close contacts should be treated. linens and clothing should be laundered or dry-cleaned or isolated in plastic containers for 72 hours. the pruritus-associated with scabies can take several weeks to resolve after treatment. patients living in group settings (dormitories or apartments) may reinfect one another as a result of inadequate primary treatment of all contacts ( cryotherapy trichloroacetic acid (tca): small amount applied until wart whitens podophyllin resin, 10% to 25% all these may be repeated every 1 to 2 weeks until warts are resolved. podofilox 0.5% solution or gel applied twice daily for 3 days, followed by 4 days of no therapy. imiquimod 5% cream applied once daily at bedtime three times a week for up to 16 weeks; washed off 6 to 10 hours after application. urinary tract infection (uti) is defined as significant bacteriuria in the presence of symptoms. uti accounts for a significant number of emergency department visits; an estimated 20% of women experience a uti in their lifetime. the urinary tract is normally sterile. uncomplicated uti involves the urinary bladder in a host without underlying renal or neurologic disease. the bladder mucosa is invaded, most often by enteric coliform bacteria (e.g., e. coli) that ascend into the bladder via the urethra. sexual intercourse can promote this migration, and cystitis is common in otherwise healthy young women. frequent and complete voiding has been associated with a reduction in the incidence of uti. complicated uti occurs in the setting of underlying structural, medical, or neurologic disease. signs and symptoms of a uti include dysuria, frequency, urgency, nocturia, enuresis, incontinence, urethral pain, suprapubic pain, low back pain, and hematuria. fever is unusual. up to 30% of patients with symptoms of cystitis have a smoldering pyelonephritis, especially when symptoms have been present for more than 1 week. a patient with pyelonephritis usually appears ill, with fever, sweating, and prostration, along with costovertebral angle (flank) tenderness in most cases. the differential diagnosis of uncomplicated uti includes use of diuretics or caffeine, interstitial cystitis, vaginitis, pregnancy, pelvic mass, pid, and benign prostatic hypertrophy (bph). if a uti is suspected, the initial test of choice is urinalysis, although with classic signs and symptoms of infection in women, this test is not always necessary. pyuria, as indicated by a positive result on the leukocyte esterase dip test, is found in the majority of patients with uti. the presence of urinary nitrites is fairly specific for uti. the combination of positive leukocyte esterase and nitrites improves sensitivity. on urine microscopy, levels of pyuria as low as two to five leukocytes per high-power field (2-5 wbcs/hpf) in a centrifuged specimen are significant in the female patient with appropriate symptoms, as is the presence of bacteriuria. urine culture and sensitivity are not needed in simple utis. cultures should be done in patients with recurrent utis, patients with pyelonephritis, and pregnant patients. antibiotic therapy can be given in a 3-day regimen for young, sexually active women. a 7-to 10-day course of antibiotics should be used in pregnant patients and patients with complicated utis. all the drugs listed in table 16 -15 can be used in a 3-day or 7-to 10-day course. clinical practice guidelines that include telephone assessment and treatment have shown a decrease in unnecessary laboratory utilization while maintaining quality of care (saint et al., 1999) . trimethoprim-sulfamethoxazole (tmp-smx) has been a mainstay of uti therapy, but in some localities, resistance of e. coli to tmp-smx is 20% (mehnert-kay, 2005) . if a urine culture is done and the organism is resistant to the drug prescribed, a change in antibiotics is indicated only if the patient is still symptomatic. for symptomatic treatment, a bladder anesthetic can be used, such as phenazopyridine (pyridium), 200 mg three times daily for 2 days. patients should be warned that this produces an orange tinge in tears and urine. patients should also be instructed to increase fluid intake. pyelonephritis is suggested by a failure of a short course of antibiotics. signs and symptoms of pyelonephritis include shaking chills and fever higher than 38.5° c (101.3° f), flank pain, malaise, urinary frequency and burning, and costover-tebral angle tenderness. the infection can produce septic shock. a patient who is unable to tolerate oral intake should be hospitalized and given empiric iv antibiotics aimed at broad-spectrum gram-negative coverage, such as third-generation cephalosporins, fluoroquinolones, or aminoglycosides, while awaiting results of blood and urine cultures. a 14-day course of antibiotic therapy (iv or po) is recommended. although the most common bacterial infection during pregnancy, the incidence of uti in pregnancy is similar to that reported in sexually active nonpregnant women of childbearing age. up to 40% of pregnant women with tmp-smx, 160/800 mg q12h trimethoprim, 100 mg q12h fluoroquinolones ‡ ciprofloxacin, 100-250 mg q12h ciprofloxacin xr, 500 mg qd gatifloxacin, 200 mg qd levofloxacin, 250 mg qd nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid amoxicillin, 250 mg q8h or 500 mg q12h cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. amoxicillin, 250 mg q8h or 500 mg q12h nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid cephalexin, 250 mg q6h, or other cephalosporin tmp-smx, 160/800 mg q12h male gender, diabetes, symptoms for 7 days, recent antimicrobial use, age > 65 tmp-smx, § 160/800 mg q12h fluoroquinolones, as per 3-day regimens cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. from hooton tm, stamm we. diagnosis and treatment of uncomplicated urinary tract infection. infect dis north am 1997;11:551. tmp-smx, trimethoprim-sulfamethoxazole; qd, every day; q12h, every 12 hours; q6h, every 6 hours; q8h, every 8 hours; qid; four times daily. * treatments listed to be prescribed before etiologic agent is known (gram stain may help); therapy can be modified when cause is identified. † characteristic pathogens are escherichia coli (85%-90%) and staphylococcus saprophyticus (5%-15%); other organisms account for less than 5% of cases and include proteus mirabilis, klebsiella pneumoniae, and enterococcus spp. ‡fluoroquinolones should not be used in pregnancy. § although classified as pregnancy category c, tmp-smx is widely used; however, avoid its use in the first and second trimesters. untreated bacteriuria in the first trimester develop acute pyelonephritis later in pregnancy. premature births and perinatal mortality are increased in pregnancies complicated by uti. therefore, in pregnant women, asymptomatic bacteriuria should be actively sought and aggressively treated with at least one urinalysis, preferably toward the end of the first trimester. nitrofurantoin, ampicillin, and the cephalosporins have been used most extensively in pregnancy and are the regimens of choice for treating asymptomatic or minimally symptomatic uti. tmp-smx should be avoided in the first trimester because of possible teratogenic effects and should be avoided near term because of a possible role in the development of kernicterus. fluoroquinolones are avoided because of possible adverse effects on fetal cartilage development. for pregnant women with overt pyelonephritis, admission to the hospital for parenteral therapy should be the standard of care; beta-lactam agents with or without aminoglycosides are the cornerstone of therapy. prevention of uti, including pyelonephritis, can be accomplished during pregnancy with nitrofurantoin or cephalexin taken prophylactically after coitus or at bedtime without relation to coitus. such prophylaxis should be considered for patients who have had acute pyelonephritis during pregnancy, patients with bacteriuria during pregnancy who have had a recurrence after a course of treatment, and patients who had recurrent uti before pregnancy that required prophylaxis. catheter-associated utis are associated with increased mortality and costs. risk factors for catheter-associated utis include the duration of catheterization, lack of systemic antibiotic therapy, female gender, age older than 50 years, and azotemia. to help prevent infection, urinary catheters should be avoided when possible and used only as long as needed. the catheter should be inserted with strict aseptic technique by trained persons, and a closed system should be used at all times. treatment of catheter-associated uti depends on the clinical circumstances. symptomatic patients (e.g., those with fever, chills, dyspnea, and hypotension) require immediate antibiotic therapy along with removal and replacement of the urinary catheter if it has been in place for a week or longer. in an asymptomatic patient, therapy should be postponed until the catheter can be removed. patients with long-term indwelling catheters seldom become symptomatic unless the catheter is obstructed or is eroding through the bladder mucosa. in patients who do become symptomatic, appropriate antibiotics should be administered and the catheter changed. therapy for asymptomatic catheterized patients leads to the selection of increasingly antibiotic-resistant bacteria. recurrence of uncomplicated cystitis in reproductive-age women is common, and some form of preventive strategy is indicated if three or more symptomatic episodes occur in 1 year. however, risk factors specific to women with recurrent cystitis have received little study (sen, 2008) . several antimicrobial strategies are available, but before initiating therapy, the patient should try such simple interventions as voiding immediately after sexual intercourse and using a contraceptive method other than a diaphragm and spermicide. ingestion of cranberry juice has been shown to be effective in decreasing bacteriuria with pyuria, but not bacteriuria alone or symptomatic uti, in an elderly population. cranberry juice may be effective for preventing uti in young, otherwise healthy women. if simple nondrug measures are ineffective, continuous or postcoital-if the infections are temporally related to intercourse-low-dose antimicrobial prophylaxis with tmp-smx, a fluoroquinolone, or nitrofurantoin should be considered. typically, a prophylactic regimen is initially prescribed for 6 months and then discontinued. if the infections recur, the prophylactic program can be instituted for a longer period. an alternative approach to antimicrobial prophylaxis for women with less frequent recurrences (<4 a year) is to supply tmp-smx or a fluoroquinolone and allow the patient to self-medicate with short-course therapy at the first symptoms of infection. a minority of patients have relapsing uti, as evidenced by finding the same bacterial strain within 2 weeks after completion of antimicrobial therapy. two factors can contribute to the pathogenesis of relapsing infection in women: (1) deep tissue infection of the kidney that is suppressed but not eradicated by a 14-day course of antibiotics and (2) structural abnormality of the urinary tract, particularly calculi. patients with true relapsing utis should undergo renal ultrasound, intravenous pyelogram (ivp), or voiding cystourethrogram, and longer-term therapy should be considered. urinary tract infection is one of the most common infections of childhood. factors predisposing to uti include taking broad-spectrum antibiotics (e.g., amoxicillin, cephalexin), which are likely to alter gastrointestinal and periurethral flora; incomplete bladder emptying or infrequent voiding; voiding dysfunction; and constipation. uti in young children serves as a marker for abnormalities of the urinary tract. imaging of the urinary tract is recommended in every febrile infant or young child with a first uti to identify children with abnormalities that predispose to renal damage. imaging should consist of urinary tract ultrasonography to detect dilation of the renal parenchyma. voiding cystourethrography is often ordered but does not appear to improve clinical outcomes in uncomplicated utis (alper and curry, 2005) . a common complication of uti in men is prostatitis. bacterial prostatitis is usually caused by the same gram-negative bacilli that cause uti in female patients; 80% or more of such infections are caused by escherichia coli. the pathogenesis of this condition is poorly understood. antibacterial substances in prostatic secretions probably protect against such infections. a national institutes of health (nih) expert consensus panel has recommended classifying prostatitis into three syndromes: acute bacterial prostatitis, chronic bacterial prostatitis, and chronic pelvic pain syndrome (cpps). acute bacterial prostatitis is a febrile illness characterized by chills, dysuria, urinary frequency and urgency, and pain in the perineum, back, or pelvis. the bladder outlet can be obstructed. on physical examination, the prostate is found to be enlarged, tender, and indurated. pyuria is present, and urine cultures generally grow e. coli or another typical uropathogen. chronic bacterial prostatitis is a clinically more occult disease and may be manifested only as recurrent bacteriuria or variable low-grade fever with back or pelvic discomfort. urinary symptoms usually relate to the reintroduction of infection into the bladder, with both pyuria and bacteriuria. a chronic prostatic focus is the most common cause of recurrent uti in men. cpps is the diagnosis for the large group of men who present with minimal signs on physical examination but have a variety of irritative or obstructive voiding symptoms; perineal, pelvic, or back pain; and sexual dysfunction. these men can be divided into those with and those without inflammation (defined as >10 wbcs/hpf in expressed prostatic secretions). the etiology and appropriate management in these patients, regardless of inflammatory status, is unknown. • laboratory findings in acute tick-borne infection often include a normal or low wbc count, thrombocytopenia, hyponatremia, and elevated liver enzymes. • doxycycline is the drug of choice for patients with rmsf. • appropriate antibiotic treatment should be initiated immediately with strong suspicion of ehrlichiosis. • if left untreated, lyme disease can progress to cognitive disorders, sleep disturbance, fatigue, and personality changes. in the united states, more vector-borne diseases are transmitted by ticks than by any other agent. tick-borne diseases can result from infection with pathogens that include bacteria, rickettsiae, viruses, and protozoa. most tick-borne diseases are transmitted during the spring and summer months when ticks are active. a knowledge of which species of tick is endemic in an area can help narrow the diagnosis (table 16-16) . rocky mountain spotted fever (rmsf) is the most severe and most often reported rickettsial illness in the united states. it is caused by rickettsia rickettsii, a species of bacteria that is spread to humans by ixodid (hard) ticks (figure 16-6) . initial signs and symptoms include sudden onset of fever, headache, and muscle pain, followed by development of rash. the disease can be difficult to diagnose in the early stage. rmsf is most common among males and children. risk factors are frequent exposure to dogs and living near wooded areas or areas with high grass. the presentation of rsmf is nonspecific, following an incubation of about 5 to 10 days after a tick bite. initial symptoms can include fever, nausea, vomiting, severe headache, muscle pain, and lack of appetite. later signs and symptoms include rash, abdominal pain, joint pain, and diarrhea. the rash first appears 2 to 5 days after the onset of fever. most often it begins as small, flat, pink, nonitchy spots on the wrists, forearms, and ankles. the characteristic red spotted rash of rmsf is usually not seen until the sixth day or later after onset of symptoms. as many as 10% to 15% of patients never develop a rash (figure 16-7) . no widely available laboratory assay provides rapid confirmation of early rmsf, although commercial pcr testing is available. therefore, treatment decisions should be based on epidemiologic and clinical clues. treatment should never be delayed while waiting for confirmation by laboratory results. routine clinical laboratory findings suggestive of rmsf include normal wbc count, thrombocytopenia, hyponatremia, and elevated liver enzyme levels. serologic assays are the most often used methods for confirming cases of rmsf. doxycycline is the drug of choice for patients with rmsf. therapy is continued for at least 3 days after fever subsides and until there is unequivocal evidence of clinical improvement, generally for a minimum total course of 5 to 10 days. tetracyclines are usually not the preferred drug for use in pregnant women. whereas chloramphenicol is typically the preferred treatment for rmsf during pregnancy, care must be used when administering chloramphenicol late during the third trimester of pregnancy because of risks associated with gray baby syndrome. three species of ehrlichia in the united states are known to cause disease in humans. ehrlichia chaffeensis, the cause of human monocytic ehrlichiosis, occurs primarily in southeastern and south-central regions and is primarily transmitted by the lone star tick, amblyomma americanum ( figure 16-8) . human granulocytic ehrlichiosis is caused by anaplasma phagocytophila or anaplasma equi and is transmitted by ixodes ticks. ehrlichia ewingii is the most recently recognized human pathogen, with cases reported in immunocompromised patients in missouri, oklahoma, and tennessee. after an incubation period of about 5 to 10 days following the tick bite, initial symptoms generally include fever, pregnant women should be screened for asymptomatic bacteriuria in the first trimester of pregnancy (wadland and plante, 1989) (sor: a). pregnant women who have asymptomatic bacteriuria should be treated with antimicrobial therapy for 3 to 7 days (nicolle et al., 2005) (sor: b) . pyuria accompanying asymptomatic bacteriuria should not be treated with antimicrobial therapy (nicolle, 2003) (sor: c1). a 3-day course of tmp-smx (bactrim, septra) is recommended as empiric therapy of uncomplicated utis in women, in regions where the rate of resistant e. coli is less than 20% (warren et al., 1999) (sor: c). fluoroquinolones are not recommended as first-line treatment of uncomplicated utis, to preserve their effectiveness for complicated utis (warren et al., 1999) (sor: c). a randomized, placebo-controlled trial of 150 women over 12 months found that cranberry juice and cranberry extract tablets significantly decreased the number of patients having at least one symptomatic uti per year (stothers, 2002) appropriate antibiotic treatment should be initiated immediately when there is a strong suspicion of ehrlichiosis on the basis of clinical and epidemiologic findings. the treatment recommendations are the same as for rocky mountain spotted fever. rifampin has been used successfully in a limited number of pregnant women with documented ehrlichiosis. babesiosis is caused by hemoprotozoan parasites of the genus babesia. the white-footed deer mouse is the main reservoir in the united states, and the vector is ixodes ticks. most infections are probably asymptomatic. manifestations of disease include fever, chills, sweating, myalgias, fatigue, hepatosplenomegaly, and hemolytic anemia. symptoms typically occur after an incubation period of 1 to 4 weeks and can last several weeks. the disease is more severe in immunosuppressed, splenectomized, or elderly patients. diagnosis can be made by microscopic examination of thick and thin blood smears stained with giemsa, looking for the parasite in red blood cells (rbcs). options for treatment include clindamycin plus quinine or atovaquone plus azithromycin. lyme disease is caused by the spirochetal bacterium borrelia burgdorferi. ixodes ticks are responsible for transmitting lyme disease bacteria to humans. in the united states, lyme disease is mostly localized to states in the northeastern, mid-atlantic, and upper north-central regions, as well as northwestern california. lyme disease most often manifests with a characteristic bull's-eye rash (erythema migrans) accompanied by nonspecific symptoms such as fever, malaise, fatigue, headache, muscle aches, and joint aches (figure 16-9) . lyme disease spirochetes disseminate from the site of the tick bite, causing multiple (secondary) erythema migrans lesions. other manifestations of dissemination include lymphocytic meningitis, cranial neuropathy (especially facial nerve palsy), radiculoneuritis, migratory joint and muscle pains, myocarditis, and transient atrioventricular blocks of varying degree. if left untreated, the disease can progress to intermittent swelling and pain of one or a few joints (usually large weight-bearing joints such as the knee), cognitive disorders, sleep disturbance, fatigue, and personality changes. the diagnosis is based primarily on clinical findings, and it is often appropriate to treat patients with early disease solely on the basis of objective signs and a known exposure. serologic testing may provide valuable supportive diagnostic information in patients with endemic exposure and objective clinical findings that suggest later-stage disseminated lyme disease. treatment for 3 to 4 weeks with doxycycline or amoxicillin is generally effective in early disease. cefuroxime axetil or erythromycin can be used for persons allergic to penicillin or who cannot take tetracyclines. later disease, particularly with objective neurologic manifestations, can require treatment with intravenous ceftriaxone or penicillin for 4 weeks or more, depending on disease severity. tularemia is caused by francisella tularensis, one of the most infectious pathogenic bacteria known. most cases in the united states occur in south-central and western states. humans can become infected through diverse environmental exposures, including bites by infected arthropods; handling infectious animal tissues or fluids; direct contact with or ingestion of contaminated food, water, or soil; and inhalation of infective aerosols. inhaled f. tularensis causes pleuropneumonitis. some exposures contaminate the eye, resulting in ocular tularemia; penetrate broken skin, resulting in ulceroglandular or glandular disease; or cause oropharyngeal disease with cervical lymphadenitis. untreated, bacilli inoculated into skin or mucous membranes multiply, spread to regional lymph nodes, multiply further, and then can disseminate to organs throughout the body. the onset of tularemia is usually abrupt, with fever, headache, chills and rigors, generalized body aches, coryza, and sore throat. a dry or slightly productive cough and substernal pain or tightness often occur with or without objective signs of pneumonia. nausea, vomiting, and diarrhea can occur. sweats, fever, chills, progressive weakness, malaise, anorexia, and weight loss characterize continuing illness. rapid diagnostic testing for tularemia is not widely available. respiratory secretions and blood for culture should be collected in suspected patients and the laboratory alerted to the need for special diagnostic and safety procedures. streptomycin (1 g im bid for 10 days) is the drug of choice, and gentamicin is an acceptable alternative. tetracyclines and chloramphenicol can also be used. colorado tick fever is an acute viral infection transmitted by the bite of the dermacentor andersoni tick (figure 16-10) . the disease is limited to the western united states and is most prevalent from march to september. symptoms start about 3 to 6 days after the tick bite. fever continues for 3 days, stops, and then recurs 1 to 3 days later for another few days. other symptoms include excessive sweating, muscle aches, joint stiffness, headache, photophobia, nausea, vomiting, weakness, and an occasional faint rash. routine blood tests might show a low wbc count, mildly elevated liver function, and mildly elevated creatine phosphokinase (cpk). diagnosis is confirmed by testing blood for complement fixation immunofluorescent antibody staining to colorado tick virus. treatment is removal of the tick and treatment of symptoms. physicians should advise patients who walk or hike in tickinfested areas to tuck long pants into socks to protect the legs and wear shoes and long-sleeved shirts. ticks show up on white or light colors better than dark colors, making them easier to remove from clothing. if attached, ticks should be removed immediately by using a tweezers, pulling carefully and steadily. insect repellents such as deet, alone or in combination with permethrin, may be helpful. • most cases of cellulitis are caused by staphylococci or streptococci, but other causes should be considered by clinical situation. • physicians must rule out more ominous causes of skin inflammation, such as necrotizing fasciitis and pyomyositis, when considering cellulitis. • edema-associated cellulitis is best treated by mobilizing edema fluid. cellulitis is an acute, spreading inflammation of the derma and subcutaneous issue. patients complain of tenderness, warmth, swelling, and spreading erythema. in contrast to erysipelas, cellulitis usually lacks sharp demarcation at the border. factors that predispose to cellulitis include trauma, an underlying skin lesion (furuncle, ulcer), or a complication arising from a wound, ulcer, or dermatosis. occasionally, cellulitis results from a blood-borne infection that metastasizes to the skin. pain and erythema usually develop within several days and are often associated with malaise, fever, and chills. the area involved is often extensive, red, hot, and swollen. patchy involvement with skip lesions can be seen. regional lymphadenopathy is common, and bacteremia can occur. several clinical entities resemble cellulitis, including pyoderma gangrenosum, gout, and insect bites. necrotizing fasciitis and gas gangrene are surgical emergencies. given that the predominant organism involved in most cases of cellulitis is a grampositive coccus, clinical history and morphology on physical examination usually suffice in the diagnosis and treatment of cellulitis. a history of freshwater exposure may implicate aeromonas hydrophila as the causative organism; saltwater appropriate antibiotic therapy should be initiated immediately when there is suspicion of rocky mountain spotted fever, ehrlichiosis, or relapsing fever rather than waiting for laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). treatment with doxycycline (vibramycin) or tetracycline is recommended for rmsf, lyme disease, ehrlichiosis, and relapsing fever (bratton and corey, 2005; spach et al., 1993) (sor: c). recommended actions to prevent tick-borne disease include avoidance of tick-infested areas; wearing long pants and tucking the pant legs into socks; applying diethyltoluamide (deet) insect repellents; using bed nets when camping; and carefully inspecting oneself frequently while in an at-risk area (bratton and corey, 2005; spach et al., 1993) (sor: c). antibiotic prophylaxis is not routinely recommended for a tick bite to prevent lyme disease, unless the risk of infection is high (wormser et al., 2006) (sor: b). recommended treatment for suspected tularemia is streptomycin or gentamicin given empirically before evidence of laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). exposure suggests vibrio spp. cellulitis in a patient with liver disease and shellfish ingestion moves vibrio vulnificans to the top of the differential. patients with soft tissue infection should have blood drawn for laboratory testing if signs and symptoms of systemic toxicity are present (e.g., fever or hypothermia, tachycardia, hypotension). laboratory testing should include blood culture and drug susceptibility tests; wbc count with differential; and measurement of creatinine, bicarbonate, cpk, and crp levels. hospitalization should be considered for patients with hypotension or an elevated creatinine level, low serum bicarbonate level, elevated cpk level (i.e., 2-3 times upper limit of normal), marked left shift, or crp level greater than 13 mg/l (123.8 nmol/l). gram stain with culture and culture of needle aspiration or punch biopsy specimens should be performed to determine a definitive etiology, and a surgical consult should be considered for inspection, exploration, and drainage. findings that may signal potentially severe, deep, soft tissue infection and that may require emergent surgical evaluation include cutaneous hemorrhage, gas in the tissue, pain disproportionate to physical findings, rapid progression, skin anesthesia, skin sloughing, and violaceous bullae. radiologic studies may be helpful if abscess or osteomyelitis is a possibility. ultrasonography is helpful in detecting a subcutaneous collection of fluid. magnetic resonance imaging (mri) is also useful in differentiating cellulitis from necrotizing fasciitis. the diagnosis of necrotizing cellulitis is by direct surgical examination or by frozen pathology sections. empiric antibiotics for immunocompetent patients with cellulitis should be targeted toward gram-positive cocci (table 16-17) . broader coverage should be initiated for diabetic patients to include gram-positive aerobes, gram-negative aerobes, and anaerobes. patients who present with severe infection or whose infection is progressing despite empiric antibiotic therapy should be treated more aggressively; the treatment strategy should be based on results of appropriate gram stain, culture, and drug susceptibility analysis. in the case of staphylococcus aureus, the physician should assume that the organism is resistant, and agents effective against mrsa, such as vancomycin, linezolid (zyvox), or daptomycin (cubicin), should be used. the antibiotic may be switched from an intravenous drug to an oral drug when fever has subsided and the skin lesion begins to resolve, usually in 3 to 5 days. the total duration of therapy should be 7 to 14 days. longer duration may be required if the response is slow or is associated with abscess, tissue necrosis, or underlying skin processes (infected ulcers or wounds). treatment of cellulitis should include elevation and immobilization to decrease swelling. patients with interdigital dermatophytic infections should be treated with a concomitant topical antifungal applied once or twice daily. topical antifungals can also help reduce the risk of recurrence of the cellulitis. support stockings, good skin hygiene, and prompt treatment of tinea pedis helps with prevention of cellulitis in patients with peripheral edema, who are predisposed to recurrence. in patients who continue to have frequent episodes of cellulitis or erysipelas, prophylactic treatment with penicillin v, 250 mg or 500 mg orally twice daily, or erythromycin, 250 mg once or twice daily (for penicillin-allergic patients), may be indicated. • the majority of furuncles and carbuncles are caused by staphylococcus spp., increasingly, community-acquired methicillin-resistant s. aureus. • drainage of pus is of primary importance in treating skin and soft tissue infections. • culture of sstis is important in guiding antibiotic treatment when initial measures of drainage are not effective. • for recurrent boils, consider referral to infectious disease specialist, possibly to eradicate carriage state. furuncles, or boils, are infections of the skin and soft tissue usually associated with a hair follicle. carbuncles are an extension of this skin and soft tissue infection continuum and involve more of the surrounding and subcutaneous tissue. the broad category skin and soft tissue infections (sstis) is used to describe this continuum that includes furuncles and carbuncles. sstis are common in both healthy and immunocompromised patients and likely initiate with some breach of the skin integrity, such as irritation of hair follicles from friction or microscopic trauma to the skin. up to 74% of furuncles and carbuncles are caused by community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) (cdc, 2010). other potential causative organisms include nonresistant staphylococcus spp. and streptococcus spp. it has become increasingly important to obtain culture of a lesion to direct antibiotic coverage given the increase in ca-mrsa. there is no reliable historical or examination element that will distinguish a ca-mrsa from a methicillin-sensitive staphylococcal skin lesion. stereotypically, patients report ca-mrsa lesions starting like a spider bite. furuncles and carbuncles can occur anywhere on the body, although the axillae, groin, and buttocks are particularly common sites. in addition, practices that cause skin trauma (e.g., shaving, waxing) are often noted in patients with these sstis. fever and malaise are uncommon with milder lesions but become more frequent with the increasing scope of localized infection. of primary importance in the management of carbuncles and furuncles is facilitation of drainage of any purulent material. with smaller lesions, this may be accomplished by heat application by the patient at home. as lesions increase in size and fluctuance, surgical drainage is essential to facilitate resolution of an ssti. it is important to consider culture penicillin, given parenterally or orally depending on clinical severity, is the treatment of choice for erysipelas (sor: a). for cellulitis, a penicillinase-resistant semisynthetic penicillin (amoxicillin/clavulanate) or a first-generation cephalosporin should be selected, unless streptococci or staphylococci resistant to these agents are common in the community (sor: a). for suspected mrsa skin infections, oral treatment options include trimethoprim-sulfamethoxazole, clindamycin, and doxycycline of purulent material when performing incision and drainage in the event that the patient fails to improve and antibiotic coverage becomes necessary. cure rates of lesions with drainage alone exceed 90%. careful follow-up after drainage is essential to ensure clinical improvement; daily dressing changes in the office after surgical drainage is effective. the addition of postdrainage antibiotics has not shown much added benefit. to prevent the spread of infection to others who come into contact with the patient recovering from an ssti, an occlusive dressing to prevent leakage of lesion fluid and careful hygiene are indicated. there is no evidence that extensive cleaning of common spaces (e.g., locker rooms) prevents the spread of ssti-causing bacteria more than routine cleaning measures. towels and soiled clothing should be laundered in hot water, and any common equipment should be cleaned per manufacturer recommendations. when lesions do not respond to heat, or when lesions are larger yet not amenable to drainage, antibiotics may be used. reasonable first-line antibiotic coverage for nonfluctuant lesions may include dicloxacillin, first-or secondgeneration cephalosporins, macrolides, or clindamycin. in patients with suspected ca-mrsa, better choices include tmp-smx, tetracycline, or clindamycin. it is important to note that up to 50% of ca-mrsa species will be resistant to clindamycin, particularly if the patient has been treated with other antibiotics in the previous weeks to months . oral administration of these antibiotics is acceptable in the nontoxic patient. patient signs and symptoms that would warrant hospital admission include fever or hypothermia, tachycardia, or hypotension as signs of sepsis and lesions greater than 5 cm in size (table 16-18) . for patients with recurrent sstis, evaluation for the presence of nasal carriage with a nasal culture is indicated. the value of eradication of bacterial carriage is unclear. referral for infectious disease specialist evaluation may be indicated to guide decision making in the patient with recurrent furuncles and carbuncles. • the existence, severity, and extent of infection, as well as vascular status, neuropathy, and glycemic control, should be assessed in patients with a diabetic foot infection. • visible bone and palpable bone on probing suggest underlying osteomyelitis in patients with a diabetic foot infection. • before an infected wound of a diabetic foot infection is cultured, any overlying necrotic debris should be removed to eliminate surface contamination and to provide more accurate results. patients with diabetes are prone to skin ulcers caused by neuropathy, vascular insufficiency, and diminished neutrophil function. minor wounds can be secondarily infected, leading to ulcer formation. these ulcers often have extensive undermining with necrotic tissues and are often close to the anus, thus promoting an environment suitable for multiple species of microorganisms, including anaerobes. diabetic foot infections range in severity from superficial paronychia to deep infection involving bone. non-limb-threatening infections involve superficial ulcers with minimal cellulitis (<2 cm from portal of entry), no signs of systemic toxicity, and no significant ischemia in the limb. cure rates of fluctuant skin lesions with drainage alone is over 90%. postdrainage antibiotics do not significantly improve outcomes rajendran et al., 2007) (sor: a). trimethoprim-sulfamethoxazole (tmp-smx), clindamycin, and tetracycline are first-choice antibiotics when ca-mrsa is suspected. up to 50% of ca-mrsa species will be resistant to clindamycin, particularly in the patient previously treated with other antibiotics (sor: c). subcutaneous tissues, and prominent ischemia. infection in patients who have recently received antibiotics or who have deep, limb-threatening infection or chronic wounds are usually caused by a mixture of aerobic gram-positive, aerobic gram-negative (e.g., escherichia coli, proteus spp., klebsiella spp.), and anaerobic organisms (e.g., bacteroides, clostridium, peptococcus, and peptostreptococcus spp.) . surgery is necessary to unroof encrusted areas, and the wounds need to be examined and probed to determine the extent of the infection and check for bone involvement (dinh et al., 2008) . debridement or drainage should be promptly performed. deep wound cultures should be obtained if possible. if deep culture is not feasible, gram stain and culture from the curettage of the base of the ulcer or from purulent exudates may be needed to guide antibiotic therapy (figure 16-11) . plain radiography of the foot is indicated for detection of osteomyelitis, foreign bodies, and soft tissue gas. when plain radiography is negative but osteomyelitis is clinically suspected, radionuclide scan or mri should be performed. mri provides more accurate information regarding the extent of the infectious process. the presence of peripheral artery disease and neuropathy should be assessed. the antibiotic regimen should be based on meaningful bacteriologic data. however, the initial regimen for a previously untreated patient with non-limb-threatening infection should focus on s. aureus and streptococci. mild infections may be treated with dicloxacillin or cephalexin for 2 weeks. amoxicillin/clavulanate may be used if polymicrobial infection is suspected. if msra is suspected, oral treatment options include tmp-smx or doxycycline. for limb-threatening infections, broad-spectrum antibiotics are recommended for coverage of group b streptococci, other streptococci, enterobacteriaceae, anaerobic gram-positive cocci, and bacteroides spp. treatment regimens include ampicillin-sulbactam or ertapenem (invanz), clindamycin plus a third-generation cephalosporin, and clindamycin plus ciprofloxacin. intravenous vancomycin should be added if mrsa infection is suspected. ciprofloxacin as a single agent is not recommended. in addition to antibiotic treatment, good glycemic control should be obtained and open wounds gently packed with sterile gauze moistened with ¼-strength povidone-iodine (betadine) solution. edema should be reduced by bed rest, elevation, and diuretic therapy as indicated. for prevention of diabetic foot ulcers, all patients with diabetes should have an annual foot examination that includes assessment for anatomic deformities, skin breaks, nail disorders, loss of protection sensation, diminished arterial supply, and inappropriate footwear. • the use of prophylactic antibiotics may be necessary in the initial management of bite wounds, particularly if the bite is on the hand or face or from a cat. • first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy for bite wounds because of resistance issues. • avoid primary wound closure in the management of bite wounds. it is estimated that bites account for 800,000 medical visits annually in the united states, making up 1% of emergency department visits. bite wounds consist of lacerations, evulsions, punctures, and scratches. the microbiology of bite wounds is generally polymicrobial, with an array of potential bacteria from the environment, the victim's skin flora, and the biter's oral flora. dog bites account for approximately 80% of all animal bites requiring medical attention, in which 85% are provoked attacks. most dog bites occur on the distal extremities, but children tend to sustain facial bites. patients who present for medical attention are often concerned about the care of disfiguring wounds or the need for appropriate vaccination (i.e., tetanus, rabies). however, up to 30% of medically treated wounds may become infected. these wounds are often contaminated with multiple strains of aerobic and anaerobic bacteria. local signs of infection with erythema, edema, pain, and purulent drainage are common with animal bite wounds. although the most frequently isolated pathogen related to dog and cat bite wounds is pasteurella multocida, the array of potential organisms is much greater. anaerobes such as bacteroides tectum, prevotella spp., fusobacteria, and peptostreptococci can be isolated from animal bite wounds 75% of the time, mostly from wounds with abscess formation. capnocytophaga canimorsus has also been associated with fatal infection from fulminant sepsis in asplenic patients. wounds inflicted by cats are often scratches or tiny punctures located on the extremity and are likely to become infected and lead to abscess formation. in the united states, venomous snakes bite approximately 8000 people yearly. envenomation in such snakebites account for the majority of morbidity and mortality associated with such bites. however, infection of soft tissue structures may also occur as a result of oral flora from the snake, which tends to be fecal in nature because live prey usually defecate in the snake's mouth with their ingestion. human bites are not uncommon, especially in children. human bites have a higher complication and infection rate than do animal bites. human bite wounds most often affect the hand and fingers and in some cases may present as "love routine wound swabs and cultures of material from sinus tracts are unreliable and strongly discouraged in the management of diabetic foot infection (pellizzer et al., 2001; senneville et al., 2006) nips" to the breast and genital areas. self-inflicted bites often include wounds of the lip and tissues surrounding the nail, such as paronychia. also included in this are clenched-fist injuries or "fight bites," which result in small lacerations to the knuckles when striking a person in the mouth. normal human oral flora, rather than skin flora, is the source of most bacteria isolated from human bite wound cultures (viridans streptococci, eikenella corrodens). management of bite wounds is the same as for any other wound: good wound care in the form of adequate irrigation and debridement of nonviable tissue as needed (table 1619) . bite wounds in general do not require primary closure, but after adequate irrigation and debridement, wounds may be approximated and closed by delayed primary or secondary intention. an exception to this rule may include bite wounds to the face. general wound management measures such as tetanus toxoid administration should also be employed. bite wounds involving the hands should be evaluated by a hand surgeon, given the risk of adjacent tendon sheath, bone, or joint involvement and the dire consequences if such structures are involved. the transmission of rabies through the bites of domestic pets in the united states and developed countries is rare. in fact, the dog strain of rabies is considered eliminated in the u.s. dog population, and cat bites are often managed through observation of the animal, without the immediate need for rabies postexposure treatment (pet). however, wild mammal exposure, especially bat, skunk, or raccoon, often warrants pet, which involves thorough cleaning of the bite wound, ideally with povidone-iodine solution, along with rabies immune globulin given at the wound site and rabies vaccine given on days 0, 3, 7, and 14. bite wounds should be considered contaminated wounds from presentation, given the oral microbial flora of humans and animals, and most patients should probably receive antibiotics early. empiric antibiotics are used to eradicate oral flora inoculated from the mouth of the biter, whether human or animal, into the wound. all moderate to severe animal bite wounds, or wounds that have an associated crush injury or that are close to a bone or joint, should be considered contaminated with potential pathogens, and these patients should receive 3 to 5 days of "prophylactic" antimicrobial therapy. gram stains with culture of bite wounds are specific but not sensitive indicators of bacterial growth. nonetheless, gram stain can be used to help guide initial empiric antibiotic therapy. amoxicillin-clavulanic acid (amoxicillin-clavulanate; augmentin) or penicillin plus a penicillinase-resistant penicillin are normally first-line agents for empiric therapy directed at bite wounds. first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy because of resistance of some anaerobic bacteria and e. corrodens. a 5-to 10-day course of antibiotics is usually adequate for infections limited to the soft tissue, and a minimum of 3 weeks of therapy is required for infections involving joints or bones. close follow-up is required in all bites to ensure adequate healing. of special consideration in human bite wounds is the potential for spread of viral pathogens, most notably hepatitis b virus (hbv) and hiv, if the source person is positive. hbv exposure in this setting should be handled in the same manner as other exposures, with administration of hbig and hbv vaccination. with regard to hiv, cdc guidelines for managing nonoccupational hiv exposure recommend handling each case individually in consultation with an infectious diseases specialist. • the diagnosis of osteomyelitis is based on radiographic findings (plain radiograph or mri) showing bony destruction along with histologic analysis and culture results. • chronic osteomyelitis is not an emergency, and antibiotics can be safely withheld until an etiologic diagnosis is established. • diabetic foot infections require a careful evaluation to assess perfusion and vascular supply, and corrective measures should be undertaken to reestablish adequate perfusion if necessary. • in diabetic foot ulcers, if one can probe to bone, the patient most likely has osteomyelitis. • orthopedic hardware infections are best managed in conjunction with an infectious diseases specialist and orthopedic surgeon. osteomyelitis is defined as progressive, inflammation leading to destruction of the bone, usually secondary to an infectious agent. bacteria can enter bone through hematogenous seeding or a contiguous focus after trauma, implantation of a foreign device, or a local soft tissue infection. acute osteomyelitis is defined as infection that evolves over a few weeks. chronic osteomyelitis implies persistent infection of several weeks to months. hematogenous osteomyelitis occurs primarily in children within the metaphyses of long bones (tibia and femur) and vertebrae in adults. in addition to local signs of inflammation and infection, patients generally have various systemic signs, including fever, irritability, and lethargy. physical findings include tenderness over involved area and decreased range of motion in adjacent joints. chronic osteomyelitis usually occurs in adults, caused by an open injury to bone and surrounding soft tissue. erythema, drainage around area, and bone pain are usually present on physical examination. systemic symptoms occur less frequently. the diagnosis of osteomyelitis is based on the clinical picture and supporting laboratory and radiologic findings. leukocytosis and elevations in crp and esr may use of antibiotic prophylactic after bites of the hand reduces the incidence of infection (medeiros and saconato, 2005) (sor: b) . antibiotic prophylaxis after bites by humans reduces incidence of infection (sor: c). animal bite: ascertain the type of animal, whether the bite was provoked or unprovoked, and the situation/environment in which the bite occurred. if the species can be rabid, locate the animal for 10 days' observation or sacrifice. patient: obtain information on antimicrobial allergies, current medications, splenectomy, mastectomy, liver disease, and immunosuppression. record a diagram of the wound with the location, type, and depth of injury; range of motion; possibility of joint penetration; presence of edema or crush injury; nerve and tendon function; signs of infection; and odor of exudate. infected wounds should be cultured and a gram stain performed. anaerobic cultures should be obtained in the presence of abscesses, sepsis, serious cellulitis, devitalized tissue, or foul odor of the exudate. small tears and infected punctures should be cultured with a minitipped (nasopharyngeal) swab. copious amounts of normal saline should be used for irrigation. puncture wounds should be irrigated with a "high-pressure jet" from a 20-ml syringe and an 18-gauge needle or catheter tip. devitalized or necrotic tissue should be cautiously debrided. debris and foreign bodies should be removed. radiographs should be obtained if fracture or bone penetration is possible to provide a baseline for future osteomyelitis. wound closure may be necessary for selected, fresh, uninfected wounds, especially facial wounds, but primary wound closure is not usually indicated. wound edges should be approximated with adhesive strips in selected cases. prophylaxis: consider prophylaxis (1) for moderate to severe injury less than 8 hours old, especially if edema or crush injury is present; (2) if bone or joint penetration is possible; (3) for hand wounds; (4) for immunocompromised patients (including those with mastectomy, liver disease, or steroid therapy); (5) if the wound is adjacent to prosthetic joint; and (6) if the wound is in the genital area. coverage should include pasteurella multocida, staphylococcus aureus, and anaerobes. treatment: cover p. multocida, s. aureus, and anaerobes. use oral medication if the patient is seen early after a bite and only mild to moderate signs of infection are present. the following can be considered for cat or dog bites in adults: • first choice: amoxicillin/clavulanic acid, 875/125 mg bid or 500/125 mg tid with food. • penicillin allergy: no alternative treatment for animal bites has been established for penicillin-allergic patients. the following regimens can be considered for adults: 1. clindamycin (300 mg po qid) plus either levofloxacin (500 mg po daily) or trimethoprim-sulfamethoxazole (2 double-strength tablets po bid). 2. doxycycline, 100 mg po bid. 3. moxifloxacin, 400 mg po daily. 4. in the highly penicillin-allergic pregnant patient, macrolides have been used, but the wounds must be watched carefully. on emergency department discharge, a single starting dose of parenteral antibiotic, such as ertapenem (1 g), may be useful in selected cases. if hospitalization or closely monitored outpatient follow-up is required, intravenous agents should be used. current choices include ampicillin/sulbactam and cefoxitin. the rising incidence of community-acquired s. aureus isolates that are methicillin resistant and therefore resistant to the drugs recommended here emphasizes the importance of susceptibility-testing any s. aureus isolates. indications include fever, sepsis, spread of cellulitis, significant edema or crush injury, loss of function, a compromised host, and patient noncompliance. give tetanus booster (td; tetanus and diphtheria toxoids for adults) if original three-dose series has been given but none in the past 5 years. adults who have not received acellular pertussis vaccine (tdap), should be given this instead of td. give a primary series and tetanus immune globulin if the patient was never immunized. rabies vaccine (on days 0, 3, 7, 14, and 28) with hyperimmune globulin may be required, depending on the type of animal, ability to observe the animal, and locality. elevation may be required if any edema is present. lack of elevation is a common cause of therapeutic failure. be seen but can also be normal. blood cultures may be positive in up to half of children with acute osteomyelitis. if plain radiographs show bone destruction and inflammation; the diagnosis of osteomyelitis is confirmed. typical findings on plain-radiographs will include osteolysis, periosteal reaction, and sequestra (segments of necrotic bone separated from living bone by granulation tissue). findings seen on plain radiographs usually denote a process that has been ongoing for at least 2 weeks. bone scintigraphy (bone scan) is often performed on patients with suspected osteomyelitis; however, sensitivity is quite low, and a negative result can offer false reassurance to the physician, so its routine use is not recommended. if the plain-radiographs are negative but the suspicion for osteomyelitis is still high, an mri scan should be considered. once the diagnosis of osteomyelitis has been made, the next step is to obtain an etiologic diagnosis. histopathologic and microbiologic examination of bone is the "gold standard." cultures of sinus tracts are not reliable for identifying the causative organism. common causative bacteriologic organisms in neonates include staphylococcus aureus, group b streptococci, and escherichia coli. later in life, s. aureus is most common, and in elderly persons, gram-negative organisms such as pseudomonas aeruginosa and serratia spp. have increased incidence. empiric antibiotics are rarely required for chronic disease but are often necessary for acute osteomyelitis. ideally, surgical debridement of all necrotic tissue and inflammatory debris (pus) should be undertaken and multiple surgical cultures with bone histology samples obtained. antimicrobial therapy will be dictated by test results. generally, treatment is for 4 to 6 weeks. with the exception of the fluoroquinolone class of antibiotics, which achieve high serum levels with oral administration, bone antibiotic levels cannot exceed the minimum inhibitory concentration (mic) for the infecting organism; therefore, antibiotics must be given intravenously. this underscores the importance in obtaining a bacterial diagnosis so that the appropriate antibiotic can be used for the duration of treatment. acute osteomyelitis is usually readily curable; however, chronic osteomyelitis is generally more refractory to therapy and requires repeat debridement and antibiotic courses. patients with uncontrolled diabetes are at increased risk for development of osteomyelitis, especially in the presence of neuropathy or venous or arterial insufficiency. s. aureus and beta-hemolytic streptococci are the predominant organisms, although other gram-positive or gram-negative aerobic or anaerobic bacteria may also be seen. plain radiographs should be the initial test to evaluate for the presence of osteomyelitis, followed by mri if negative. if there is a draining sinus, the "probe to bone" test should be performed with a sterile probe; if bone is palpated, the diagnosis of osteomyelitis is highly likely. further evaluation of the diabetic patient should be to assess for vascular insufficiency with the use of ankle-brachial indices and transcutaneous oximetry. if significant compromise is found, arteriography followed by revascularization should be undertaken. surgical debridement is again the cornerstone of treatment, along with antibiotics directed toward the causative microorganism. infections secondary to orthopedic hardware devices have become common problems with the increasing incidence of hip, knee, and shoulder replacement surgeries. also, patients with traumatic injury resulting in a fracture often have hardware implanted to stabilize the bone. these patients present in one of the three following ways: 1. early: symptoms develop less than 3 months after surgery and have an acute presentation with pain, erythema, and warmth, usually caused by s. aureus and gram-negative bacilli. 2. delayed: symptoms develop 3 to 24 months after surgery, generally with subtle signs of infection, including implant loosening and persistent pain, and usually caused by less virulent organisms such as coagulasenegative staphylococci and propionibacterium acnes. 3. late: symptoms develop 24 months after surgery and are usually caused by hematogenous seeding from skin, dental, respiratory, and urinary infections. treatment requires debridement of the surrounding tissue and hardware removal, although this cannot always be done in patients with bone instability. it is recommended that treatment follow-up should occur at 24 hours and perhaps 48 hours for outpatients. reporting the incident to a local health department may be required. from goldstein ejc. bites. in mandell gl, bennett je, dolin rd (eds). mandell, douglas, and bennett's principles and practice of infectious diseases, 7th ed. philadelphia, churchill livingstone--elsevier, 2010. po, orally; bid, twice daily; tid, three times daily; qid, four times daily. of these infections be done in conjunction with an infectious diseases specialist working with the orthopedic surgeon. septic arthritis is defined as infection within the joint space of two bones. the major causative organisms include s. aureus and in the sexually promiscuous individual, neisseria gonorrhoeae. intravenous drug users are likely to develop septic arthritis within unusual joints (e.g., sternoclavicular, sacroiliac). rheumatoid arthritis, presence of joint prostheses, and steroid use are predisposing factors for development of septic arthritis. diagnosis is usually based on clinical presentation of a warm, swollen joint with limitation in range of motion. a joint aspiration should be completed and the synovial fluid sent for gram stain with culture, wbc count with differential, and crystal analysis to rule out gout and pseudogout. blood cultures should also be drawn before initiation of antibiotics. gonococcal arthritis usually presents as an acute arthritis involving one or more joints in a sexually active individual. two thirds of patients have dermatitis with one or multiple, usually asymptomatic, lesions that progress from macular to papular and finally vesicular or pustular. joint fluid, urethral, and rectal cultures should also be obtained. treatment is generally with a third-generation cephalosporin intravenously until improvement, followed by oral therapy to complete a 1-week course of therapy. treatment of nongonococcal arthritis requires proper draining of the infected joint. this is often done surgically, although repeat needle drainage may also be successful if the joint is easily accessible. treatment generally depends on the gram stain and includes a third-generation cephalosporin, with the addition of vancomycin if gram-positive cocci in clusters are seen. duration of therapy is 3 to 4 weeks. • a comprehensive history and physical examination with laboratory and radiologic evaluation are important in the workup for fever of unknown origin (fuo). • if routine information is unrevealing, more specific testing for fuo is undertaken based on the patient's age, travel history, and disease process to develop a differential diagnosis. • the serum ferritin level (often elevated with malignancy) and naproxen test (reduces fever with malignancy) may be helpful in determining an underlying malignant process. • initiation of empiric antibiotics should be done only in specific fuo situations to prevent skewing culture results, thus maximizing isolation of the causative organism. patients who have a persistent fever despite workup are generally classified as having a "fever of unknown origin" (fuo). in 1961, petersdorf and beeson described 100 patients with persistent fever, otherwise known as fever of unknown origin. they introduced the standard, classic definition of fuo: fever higher than 38.3° c (101° f) on several occasions, persisting without diagnosis for at least 3 weeks, with 1 week of investigational study in the hospital setting. with advancing technology, this definition has been revised to allow for more than two outpatient visits, or 3 days if investigation is in the hospital setting. most patients with fuo have chronic or subacute symptoms and can be safely evaluated in the outpatient setting, with a median time to diagnosis of 40 days. the differential diagnosis of fuo is quite broad and extensive. determining an etiologic diagnosis of an fuo depends on generating a differential diagnosis compatible with the patient's history and physical examination. the principal disease categories for fuo include infection (30% overall), neoplasms (18%), collagen vascular diseases (12%), and miscellaneous (14%) (box 16-5). because of this broad differential, a newer classification system divides fuo into four groups: classic, nosocomial, neutropenic, and hiv associated, which helps narrow the differential diagnosis. furthermore, classic fuo can be broken down into three subgroups: infants and children, elderly, and travelers. despite an extensive workup, the etiologic diagnosis usually remains elusive in 7% to 30% of patients (box 16-4) . the diagnostic workup of fuo should begin with a thorough history and physical examination, including documentation of the fever. the patient may provide a diary noting the date and time of fever. routine noninvasive investigations are recommended in all patients before diagnosing fuo (box 16-6). acute febrile illness is never called an fuo. the patient's medication profile is reviewed because numerous drugs can be the cause. if unrevealing, a workup is initiated based on the differential diagnosis for the patient's age, travel history, geographic location, and disease process. dukes criteria for infective endocarditis have 99% specificity in patients with fuo. when the initial investigations are not helpful in identifying a cause, imaging should be considered, such as computed tomography (ct) scans of the chest, abdomen, and pelvis; ct may reveal an abscess or suggest an underlying malignancy. an elevated serum ferritin level can suggest a neoplasm or myeloproliferative disorder and, if normal, greatly decreases the chance that the patient has an underlying malignancy. lower-extremity doppler ultrasound should be considered in the sedentary or obese patient to rule out deep venous thrombosis. a temporal artery biopsy should be considered in the elderly patient to rule out temporal arteritis. liver biopsy has a high diagnostic yield with minimal toxicity, whereas bone marrow cultures usually have a low yield and should be considered only in special situations. empiric therapy with antibiotics is rarely appropriate for the patient with fuo. a diagnosis is essential to guide treatment of osteomyelitis requires surgical debridement followed by a 4-to 6-week course of intravenous antibiotic therapy (sor: c). septic arthritis is usually caused by a gonococcus in a sexually active adult, and use of a third-generation cephalosporin is the mainstay of therapy (sor: a). nongonococcal arthritis should be treated with surgical debridement or repeated needle aspirations, with a third-generation cephalosporin and vancomycin if gram-positive cocci are seen (goldenberg, 1998) (sor: b). treatment, and use of antibiotics may delay determining a causative infectious agent. the naproxen test (naprosyn; 375 mg po every 12 hours for 3 days) is helpful in determining if the fever is secondary to infection or malignancy. a dramatic decrease in the patient's temperature during the test generally indicates a malignant focus, whereas minimal or no response indicates an infectious etiology. the prognosis of fuo depends on the etiologic category. undiagnosed fuo has a very favorable outcome. patients in whom diagnostic investigations fail to identify an etiology should be followed clinically with serial history reviews and physical examinations until the fever resolves or new diagnostic clues are found. connective tissue diseases are more prominent. infections: malaria, hepatitis, pneumonia/bronchitis, uti/pyelonephritis, dysentery, dengue fever, enteric fever, tb, rickettsial infection, acute human immunodeficiency virus (hiv) infection, amebic liver abscess. postoperative urinary and respiratory tract instrumentation; use of intravascular devices; drug therapy; immobilization. septic thrombophlebitis, pulmonary embolus, clostridium difficile colitis, drug fever. fungal: 40% susceptible to empiric antifungals, 5% will be resistant to empiric therapy. bacterial: 10% not responding to empiric antimicrobial therapy and usually with cryptic focus. unusual pathogens: 5% will be toxoplasmosis (toxoplasma gondii) reactivation, atypical mycobacterial, tb, fastidious pathogens (legionella, mycoplasma, chlamydophila). viral: 5% of causes (hsv, cmv, ebv, hhv-6, vzv, rsv, influenza, parainfluenza). other: 10% will be transplant related (e.g., gvhd) following stem cell transplant, 25% will be undefined. infections: mycobacterium avium complex (mac), pneumocystis carinii pneumonia (pcp), cytomegalovirus (cmv), histoplasmosis, viral (hcv, hbv, adenovirus, hsv esophagitis, vzv encephalitis), tb, other fungi, cerebral toxoplasmosis, disseminated cryptosporidiosis. neoplasms: lymphoma, kaposi's sarcoma. other: drug fever, castleman's disease. hsv, herpes simplex virus; ebv, epstein-barr virus; hhv, human herpesvirus; vzv, varicella-zoster virus; rsv, respiratory syncytial virus; gvhd, graft-versus-host disease; hcv, hepatitis c virus; hbv, hepatitis b virus. abscesses: hepatic, subhepatic, gallbladder, subphrenic, splenic, periappendiceal, perinephric, pelvic, and other sites. granulomatous: extrapulmonary and miliary tuberculosis, atypical mycobacterial infection, fungal infection. intravascular: catheter-related endocarditis, meningococcemia, gonococcemia, listeria, brucella, rat-bite fever, relapsing fever. viral, rickettsial, and chlamydial: infectious mononucleosis, cytomegalovirus, human immunodeficiency virus, hepatitis, q fever, psittacosis. parasitic: extraintestinal amebiasis, malaria, toxoplasmosis. collagen vascular diseases: rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis (particularly still's disease), vasculitis (all types). granulomatous: sarcoidosis, granulomatous hepatitis, crohn's disease. tissue injury: pulmonary emboli, sickle cell disease, hemolytic anemia. familial mediterranean fever fabry's disease cyclic neutropenia intra-abdominal infections may either be uncomplicated (limited to the gut lumen, such as gastroenteritis or colitis) or complicated (extending through to the peritoneum) . the clinical presentation of complicated intra-abdominal infections can range from mild symptoms such as nausea, mild abdominal pain, and cramping to lifethreatening septic shock. clinical findings result from local or diffuse inflammation with or without abscess formation. fever and abdominal pain are typically present, with additional symptoms depending on the organ involved. elderly and immunocompromised patients present with atypical, usually milder symptoms. imaging studies form an important adjunct to diagnosis. management involves empiric antibiotic coverage for bowel flora-mainly streptococci, enterococci, enteric gram-negative rods, and anaerobes-as well as controlling the source of infection, usually through surgery. • spontaneous bacterial peritonitis usually occurs in the setting of ascites and chronic liver disease. • spontaneous bacterial peritonitis is a diagnosis of exclusion. • ascitic fluid culture yield improves with inoculation into blood culture bottles at bedside. spontaneous bacterial peritonitis (sbp) is a form of infectious peritonitis without a surgically correctable cause and is therefore a diagnosis of exclusion. the route of infection in sbp is usually not apparent and is often presumed to be hematogenous, lymphogenous, by transmural migration through an intact gut wall from the intestinal lumen, or in women, from the vagina via the fallopian tubes (levison and bush, 2010) . sbp occurs in the setting of ascites in most cases, and it is particularly common in patients with cirrhosis. in pediatric populations, those with postnecrotic cirrhosis or nephrotic syndrome are more often affected. in adults, almost 70% of patients who develop sbp have child-pugh class c liver disease, and 10% to 30% of hospitalized patients with cirrhosis and ascites have sbp (mowat and stanley, 2001) . sbp is almost always caused by a single organism, typically enteric gram-negative rods, most often e. coli, followed by klebsiella pneumoniae. gram-positive cocci account for about 25% of episodes of sbp, and streptococci are isolated most often. sbp caused by anaerobes is rare. growth of more than one organism should raise the suspicion of secondary peritonitis. signs and symptoms of sbp are subtle and require a high index of suspicion. fever greater than 100° f (38° c) is the most common presenting sign, occurring in 50% to 80% of cases. abdominal pain, nausea, vomiting, and diarrhea are usually present. peritoneal signs (abdominal tenderness or rebound tenderness) are common but may be absent in patients with ascites. in adults, mental status changes may also occur. sbp is often confused with acute appendicitis in children. in adults, sbp should be suspected in any patient with previously stable chronic liver disease who undergoes acute decompensation in clinical status. spontaneous bacterial peritonitis is diagnosed by analysis of ascitic fluid obtained by abdominal paracentesis. infection has been typically defined as an ascitic fluid wbc count higher than 250 cells/mm 3 , which is considered diagnostic even when the culture of the ascitic fluid is negative. in cases where bloody fluid is obtained ("traumatic paracentesis"), the wbc count should be corrected by 1 wbc per 250 rbcs/mm 3 . the use of bedside dipstick for leukocyte esterase has a high false-negative rate and is not recommended (nguyen-khac et al., 2008) . ascitic fluid culture yield can be increased by inoculating blood culture bottles with 10 ml of ascitic fluid at the bedside. blood cultures should also be obtained as part of the workup. after the diagnosis of peritonitis is established, secondary peritonitis should be ruled out. ct of the abdomen with oral and intravenous contrast can help direct the surgeon to a particular source of infection, as opposed to doing a full exploratory laparotomy. a high ascitic fluid total protein (>1 g/dl) or amylase level is suggestive of secondary peritonitis. the treatment of choice is generally a third-generation cephalosporin such as cefotaxime (2 g iv every 8-12 hours) or ceftriaxone (2 g iv once daily). patients who have an ascitic fluid wbc count higher than 250 cells/mm 3 should be given empiric intravenous antibiotics without delay. oral amoxicillin-clavulanic acid can be used for mild, uncomplicated cases (navasa et al., 1996) . duration of treatment varies diagnosis of fuo may be assisted by the dukes criteria for endocarditis, ct scan of the abdomen, nuclear scanning with a technetiumbased isotope, and liver biopsy (mourad et al., 2003) (sor: b) . routine bone marrow cultures are not recommended in the fuo workup (mourad et al., 2003) (sor: b) . empiric antibiotics should be initiated only in specific situations, to avoid skewing culture results and thus maximizing potential isolation of the causative organism (mourad et al., 2003) (sor: b). from 5 to 14 days depending on clinical response. patients usually respond to appropriate antibiotic therapy within 48 to 72 hours; otherwise, a repeat paracentesis should be performed. if the ascitic fluid wbc count does not decrease by more than 25%, alternative diagnoses should be considered. prophylaxis with a fluoroquinolone or trimethoprim-sulfamethoxazole should be considered, particularly in high-risk patients (garcia-tsao and lim, 2009 • bacterial meningitis is life threatening and requires urgent medical attention and treatment. • viral encephalitis should be treated with acyclovir until herpes simplex virus is ruled out. • most brain abscesses are caused by streptococci and staphylococcus aureus. • the cns infections most likely to be encountered in clinical practice include meningitis, encephalitis, and abscess. • all cns infections can be difficult to diagnose, and a high index of suspicion by the health care provider is sometimes indicated to ensure patient survival. • mri is the most sensitive neuroimaging test for encephalitis. • acyclovir should be started immediately and continued until hsv pcr testing is obtained. meningitis can be acute, subacute, or chronic. in otherwise healthy children, the three most common organisms causing acute bacterial meningitis are streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae type b (hib). isolation of an organism other than these three organisms from the csf of a child older than 2 months always requires an explanation or evaluation for unusual host susceptibility. children with cochlear implants, asplenia, hiv infection, or csf leak from basilar skull or cribriform fracture are at greater risk for pneumococcal meningitis. deficiencies in terminal components of complement lead to greater risk for meningococcal infection (saez-llorens and mccracken, 2008) . in adults, the common etiologic agents of acute meningitis include s. pneumoniae, n. meningitidis, and listeria monocytogenes. patients with acute meningitis most often present with fever, headache, meningismus, and altered mental status. infants can present with nonspecific symptoms such as inconsolable crying, irritability, nausea, vomiting, and diarrhea. lethargy, anorexia, and grunting respirations indicate a critically ill infant. older children may complain of headache, vomiting, back pain, myalgia, and photophobia; may be confused or disoriented; and may verbalize specifically that the neck is stiff or sore. seizures are noted in up to 20% to 30% of children before hospital admission or early in the course of the illness. in contrast, patients with subacute or chronic meningitis may have the same symptoms with a much more gradual onset, lower fever, and associated lethargy and disability. mycobacterium tuberculosis, treponema pallidum (syphilis), borrelia burgdorferi (lyme disease), and fungi (e.g., cryptococcus neoformans, coccidioides spp.) are the most common agents (tunkel et al., 2010) . physical examination should look for papilledema, middle ear and sinus infections, petechiae (common with n. meningitidis), nuchal rigidity, and in infants, a bulging fontanel. blood cultures should be taken. a lumbar puncture (lp) for csf analysis should be done as soon as possible. a brain ct scan before lp is not necessary if the patient has no evidence of immunocompromise, cns disease, new seizure, papilledema, altered consciousness, or focal neurologic deficit, and if a subarachnoid hemorrhage is not suspected. if neuroimaging is necessary, blood cultures should be taken and antibiotics given before the study; a delay in administration of antibiotics leads to a worse outcome. csf should be sent for cell count, wbc differential, glucose, protein, and gram stain with culture. acid-fast bacilli stain and cryptococcal antigen may be obtained when indicated. empiric antibiotics for the initial treatment of bacterial meningitis are listed in table 16 -20, but these should be tailored to the isolated organisms whenever possible. adjunctive dexamethasone is recommended for children and infants with hib meningitis, but not if they have already received antibiotics. in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) . close contacts of patients with n. meningitidis should receive rifampin, 20 mg/kg (not to exceed 600 mg) twice daily for 2 days, or ciprofloxacin, 500 mg as a single dose, or ceftriaxone, 250 mg im as a single dose. unimmunized persons exposed to h. influenzae meningitis should receive rifampin (turkel et al., 2010) . pregnant women should not receive rifampin or doxycycline. a repeat lp should be done if no clinical response is seen after 48 hours of appropriate antibiotic therapy, particularly for patients with resistant pneumococcal disease and those who received dexamethasone. neonates with gram-negative bacilli and patients with ventriculoperitoneal (vp) shunts require documentation of csf sterility. the duration of antimicrobial therapy is 7 days for patients with n. meningitidis or hib, 10 to 14 days for pneumococcal meningitis, and 14 to 21 days for streptococcus agalactiae. spontaneous bacterial peritonitis is treated with third-generation cephalosporins (cefotaxime or ceftriaxone), with ampicillin-sulbactam, fluoroquinolones, or carbapenems as alternative agents (solomkin et al., 2010) (sor: b) . patients with diffuse peritonitis should undergo an emergency surgical procedure as soon as possible, even if ongoing measures to restore physiologic stability need to be continued during the procedure (sor: b). viral meningitis viral meningitis manifests similar to bacterial meningitis, although its course is rarely aggressive. the diagnostic process and examination are similar to those for bacterial meningitis. viral meningitis is usually caused by enteroviruses, hsv, mumps virus, and hiv. along with the signs of meningitis, signs that suggest a viral etiology include genital lesions (hsv-2), diarrhea, or a maculopapular rash (enteroviruses). diagnosis is made by the history, examination, and csf results. early in the course, the csf might show predominantly neutrophils that can resemble bacterial meningitis. treatment is symptomatic. suppressive therapy should be offered to patients with recurrent hsv meningitis. although encephalitis can also be caused by bacteria and fungi, the great majority of cases are caused by viruses. herpes simplex accounts for 10% of cases. patients present with fever, acute decreased level of consciousness, and occasionally, seizures and language, memory, or behavior disturbances. mri is the most sensitive neuroimaging test for encephalitis and might show temporal lobe inflammation in early hsv encephalitis. csf studies and electroencephalography (eeg) are also recommended for all patients with encephalitis. herpes simplex pcr should be done, and acyclovir should be given immediately until hsv encephalitis is ruled out. during late summer and early fall, doxycycline should be considered to cover for tick-borne illnesses, and testing should include the mosquito-borne encephalitides such as west nile, st. louis, eastern equine, and western equine. treatment depends on the suspected etiologic agent but is generally supportive (tunkel et al., 2008) . a brain abscess is a focal, intracerebral infection that develops into a collection of pus surrounded by a well-vascularized capsule. although fungi and protozoa (particularly toxoplasma) can also cause brain abscesses, bacterial causes are much more common. streptococci are found in 70% of bacterial abscesses and are usually from oropharyngeal infection or infective endocarditis, whereas staphylococcus aureus accounts for 10% to 20% of isolates and is more often found after trauma. community-associated mrsa strains have been increasing. enteric gram-negative bacilli (e.g., e. coli; proteus, klebsiella, and pseudomonas spp.) are isolated in 23% to 33% of patients, often in patients with ear infection, septicemia, or immunocompromise and those who have had neurosurgical procedures. most clinical symptoms are caused by the size and location of the abscess rather than the systemic signs of an infection. headache is the most common complaint and may be accompanied by fever, mental status changes, evidence of increased intracranial pressure (nausea, vomiting, papilledema), or focal neurologic deficits. diagnosis is usually made by ct scan with iv contrast showing the characteristic hypodense center with a peripheral uniform ring enhancement, with or without a surrounding area of brain edema. mri is becoming the preferred imaging modality because of increased sensitivity, particularly for detecting satellite lesions. additional testing depends on risk factors and the likely underlying source of infection and may include blood cultures, chest imaging, testing for hiv and antibodies to toxoplasma, and transesophageal echogram. empiric therapy typically involves vancomycin, ceftriaxone, and metronidazole. optimal management also includes surgical drainage for most abscesses, both to find an etiologic microorganism and to improve chances of cure (turkel, 2010) . • most acute diarrheal illness is viral and can be managed symptomatically and with appropriate attention to hydration. • travelers' diarrhea is usually caused by diarrheogenic escherichia coli. • the infection in travelers' diarrhea is usually self-limited. • antibiotics may shorten the duration of diarrhea by 1 to 3 days. • the most common cause of antibiotic-associated diarrhea is clostridium difficile. • treatment of antibiotic-associated diarrhea involves discontinuing the offending agent, if possible. adjunctive dexamethasone is recommended for children and infants with h. influenzae type b meningitis, but not if they have already received antibiotics (tunkel et al., 2004) (sor: a). in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) (sor: b). diarrhea is a common presenting complaint in the primary care physician's office. not all causes of diarrhea are infectious, and not all infectious causes of diarrhea require specific antibiotic therapy. diarrhea remains a major cause of morbidity and mortality, particularly for children in the developing world. diarrhea is an alteration of normal bowel function, characterized by an increase in the water content, volume, or frequency of stools. acute diarrhea is typically defined as present less than 14 days, and diarrhea is considered chronic when symptoms persist longer than 30 days (figure 16-12 ). infectious diarrhea seen in the primary care physician's office is most frequently caused by viruses. a number of viral agents can cause diarrheal illness (box 16-7). rotaviruses are the principal enteric pathogens in children less than 5 years of age and the most important cause of hospitalization and infant mortality related to diarrheal illnesses. noroviruses evaluate severity and duration obtain history and physical examination [1] [2] [3] [4] [5] treat dehydration report suspected outbreaks 6 check all that apply: 7 are the most common cause of food-borne disease worldwide. viral gastroenteritis is usually an acute self-limited illness, referred to as the "stomach flu." enteric viruses are easily spread by fecal-oral transmission, through contamination of food and water, fomites, and person-to-person spread. secondary attack rates can be high. nausea and vomiting are the most prominent symptoms of viral gastroenteritis. diarrhea, fever, headache, and constitutional symptoms may also be experienced. these viral infections can occur at any time during the year, but tend to occur more often in the winter. there is no specific therapy. treatment is supportive, with particular emphasis on adequate replacement of fluids and electrolytes. if rehydration can be accomplished enterally, it is preferred. both the pentavalent bovine-human reassortment (rv5) and the oral, live-attenuated monovalent (rv1) rotavirus vaccines are effective for prevention of severe gastroenteritis. the rv5 vaccine series is recommended for children at ages 2, 4, and 6 months, whereas the rv1 vaccine should be administered to children 2 and 4 months of age. approximately 40% of travelers to developing regions of the world will develop diarrhea. bacteria are responsible for approximately 80% of diarrhea acquired by travelers. other important causes include viruses and parasites. the onset of the majority of cases of travelers' diarrhea is usually within 5 to 15 days after arrival. the presentation is typically a noninflammatory, nonbloody diarrhea associated with abdominal discomfort, fever, nausea, or vomiting. the duration is usually 1 to 5 days. enterotoxigenic e. coli is responsible for approximately 30% of travelers' diarrhea. enteroaggregative e. coli is the second most common bacterial agent and causes 20% of cases. salmonella, shigella, and campylobacter spp. are less often detected but are important causes of dysentery, particularly in asia and africa. dysentery is severe inflammatory diarrhea manifested by fever and bloody stools. most cases of travelers' diarrhea are self-limited, but chronic postinfectious irritable bowel syndrome may occur in up to 10% of those who experience diarrhea. prevention of travelers' diarrhea is an important component of pretravel counseling for high-risk countries. food should be boiled, cooked, or peeled and water boiled to avoid consumption of fecal contamination. if a person develops travelers' diarrhea, a short course of antibiotics with rifaximin, ciprofloxacin, or azithromycin can shorten the duration of illness by 1 to 3 days. antibiotic therapy is recommended for persons with bloody diarrhea or fever. rifaximin, a nonabsorbed antibiotic, is not effective against invasive pathogens and should not be administered for dysentery. ciprofloxacin or azithromycin should be used for dysenteric symptoms based on local antimicrobial susceptibilities. antibiotics are frequently prescribed in the primary care physician's office for a variety of infections. unfortunately, antibiotics can alter the normal host microflora that can be protective against other infections. antibiotic effects on the normal gastrointestinal tract microbiome can lead to antibiotic-associated diarrhea, which causes significant morbidity and mortality. administration of antibiotics usually precedes symptoms of antibiotic-associated diarrhea by about 1 week but can be as distant as 2 or 3 months. strong associations with clindamycin (cleocin), cephalosporins, penicillins, and fluoroquinolones have been demonstrated, but any antibiotic can lead to antibiotic-associated diarrhea. the most important cause of antibiotic-associated diarrhea is clostridium difficile, an anaerobic, gram-positive, spore-forming rod. c. difficile is implicated as the cause in up to 25% of antibiotic-associated diarrhea cases, in 50% to 75% of antibiotic-associated colitis cases, and in more than 90% of antibiotic-associated pseudomembranous colitis cases. risk factors for c. difficile diarrhea include antibiotics, health care exposure (recent stay in hospitals or long-term care facilities), older age (>60), and comorbid conditions. the clinical presentation of c. difficile colitis is usually diarrhea, abdominal pain or cramping, and fever in a patient who recently received antibiotics. leukocytosis is common and may be profound; levels can be consistent with leukemoid reaction. a rare but potentially fatal complication is toxic megacolon. toxic megacolon manifests as acute colonic dilation to a diameter greater than 6 cm, associated with systemic toxicity and the absence of mechanical obstruction. with its high associated mortality, any patient who develops toxic megacolon requires immediate surgical evaluation for possible colectomy. diagnosis of c. difficile diarrhea is achieved by demonstration of c. difficile toxin a or b in the stool by enzyme immunoassay (eia) or cell culture cytotoxicity assay in a symptomatic patient with a previous history of antibiotic use. asymptomatic patients should not be tested. with the improved sensitivities of these diagnostic assays, one stool sample is usually sufficient to test for c. difficile, unless symptoms recur. test of cure after therapy with repeat stool for c. difficile toxin is not recommended because stools may remain positive for c. difficile toxin despite clinical resolution. endoscopy can demonstrate pseudomembranes in the colon. pseudomembranes are diagnostic of c. difficile infection, but are often not present. endoscopy may only reveal the presence of nonspecific colitis. clostridium difficile colitis is treated by discontinuing the offending agent(s) if possible and initiating antibiotic therapy (box 16-8). antimotility agents should be avoided. oral metronidazole (flagyl), 500 mg three times daily for 10 to 14 days, is recommended for mild-moderate c. difficile diarrhea. severe diarrhea should be treated with oral vancomycin. oral vancomycin is currently not recommended for all patients with c. difficile diarrhea because of concerns for the promotion of vancomycin-resistant enterococci (vre) and its expense. about 10% to 20% of patients experience relapse in travelers' diarrhea, in which enterotoxigenic e. coli or other bacterial pathogens are likely causes, prompt treatment with a fluoroquinolone, azithromycin, or rifaximin or, in children, azithromycin 10 mg/kg/day once daily can reduce the duration of an illness from 3 to 5 days to 1 to 2 days (dupont, 2010) (sor: a). after therapy. for relapse, a repeat course of the original c. difficile treatment should be administered. patients who have mild to moderate cases without volume depletion or systemic toxicity can be treated as outpatients. discussions of the following infections can be found online at www.expertconsult.com: • infectious viral hepatitis • endocarditis treat mild-moderate c. difficile diarrhea with metronidazole (zar et al., 2007) evidence-based reviews of the diagnosis and treatment of many common clinical problems. www.mdcalc.com/curb-65-severity-score-community-acquired-pneumonia curb-65 score calculator to determine the severity of communityacquired pneumonia and need for hospitalization. the complete reference list is available online at www.expertconsult.com. anthony zeimet hepatitis is defined as inflammation of the liver that is commonly induced by viruses that include the hepatitis viruses a through e, which will be the focus of this discussion. other viruses that can induce hepatitis include epstein-barr virus (ebv), cytomegalovirus (cmv), herpes simplex virus (hsv), varicella zoster virus (vzv), adenovirus, and coxsackievirus. various medications and alcohol abuse are two important nonviral causes. most infectious causes of hepatitis are self-limiting; however, hepatitis b and c viruses can cause a chronic infection that may lead to cirrhosis and eventual liver failure, as well as hepatocellular carcinoma. hepatitis a virus (hav) and hepatitis e virus (hev) are spread by the fecal-oral route and only cause an acute infection. hepatitis b, c, and d viruses (hbv, hcv, hdv) are spread through the blood and have an acute form of disease that sometimes can become chronic. the clinical presentation of hepatitis is clinically indistinguishable. asymptomatic infections are more common than symptomatic infection. symptoms generally include right upper quadrant (ruq) abdominal pain, anorexia, nausea, vomiting, diarrhea, dark-colored urine, pale stools, and generalized malaise; patients may notice a yellow hue to their skin or eyes. pruritus is common, caused by deposition of bilirubin in the skin. the physical examination generally reveals jaundice and sclera icterus in addition to ruq pain. hepatomegaly is seen in 85% and splenomegaly in 15% of patients with hepatitis. liver function tests reveal elevated levels of aspartate transaminase (ast), alanine transaminase (alt), and bilirubin, and to a lesser extent, alkaline phosphatase (alp). hepatitis a virus is the most common cause of viral hepatitis worldwide. poor hygiene practices in both the industrial and the developing world account for its prevalence. in the united states, hav is common among lower socioeconomic groups, daycare attendants and workers, men who have sex with men (msm), and illicit drug users. hepatitis a is often acquired by travelers to endemic areas. the incubation period is 15 to 45 days (mean, 30 days). hav is highly contagious, and peak fecal shedding generally occurs at the onset of illness in most infected patients. viremia averages 30 to 90 days. hav infection manifests as an acute, self-limited illness, with the prodromal symptoms lasting about a week before the onset of jaundice. jaundice generally resolves after 2 weeks, and most patients recover. fulminant hepatic failure is possible but extremely rare. diagnosis of acute hav infection is made by demonstration of anti-hav immunoglobulin m (igm) in the patient's serum. this may be negative if the patient presents early, and repeat testing may be necessary if hav is strongly suspected. anti-hav igg in the serum indicates remote infection or immunization (efig 16-1). treatment is primarily supportive, except in patients with fulminant liver failure, who may require a liver transplant. vaccination should be administered to all patients who are seronegative and to persons at increased risk for acquiring hav, including those about to travel to endemic areas, patients with chronic liver disease or receiving clotting factor concentrates, msm, hiv-positive patients, and illicit drug users. certain areas of the united states now require mandatory vaccination of children as well as those who work in the restaurant industry. the vaccine is safe and highly efficacious and is given as a two-dose series at 0 and at 6 to 12 months. passive immunization with immune globulin is recommended for those exposed to the virus by a known contact, including household and sexual contacts, and those who are traveling to an endemic area for less than 4 weeks but never vaccinated. any person who receives immune globulin should also start the vaccination series. hepatitis b virus infection can be acute or chronic. about 40,000 people die from acute hbv infection annually, and 500,000 die of cirrhosis and hepatocellular carcinoma caused by chronic infection. about 400 million people worldwide are living with chronic hbv infection. in the united states, an estimated 1.25 million residents have chronic hbv infection, with 4000 to 5500 deaths each year. significant burdens of disease are seen in asia, pacific islands, sub-saharan africa, amazon basin, and eastern europe. most adults with acute hbv will clear the virus, with less than 5% progressing to chronic infection. chronic infection will develop in almost all children infected perinatally and in 50% of those who become infected at 1 to 5 years of age. hbv is transmitted through exchange of body fluids, sexually and perinatally. in the united states, most hbv cases are acquired during adolescence and early adulthood with onset of sexual activity, experimentation with drug use, and sometimes occupational exposure. fever, polyarthralgia, rash, and a serum sickness-like illness are features of hbv infection in addition to jaundice and may be seen in association with polyarteritis nodosa. clinicians have the most difficulty in interpreting the various serologic tests for diagnosis of hepatitis b (etable 16-3) . the mean incubation period is 60 to 70 days, with a range of 30 of 180 days after infection. diagnosis of acute infection can be detected by obtaining hbv surface antigen (hbsag), which can appear as early as 1 week after exposure but generally by day 50. in a patient strongly suspected to have hbv infection, the clinician can consider checking the hbv dna viral load; which can be detected as early as 1 week after exposure. eventually the patient will develop an anti-hbv surface antibody, which indicates recovery from the illness. the other viral serologies for hbv are rarely obtained in acute illness. in chronic hbv infection, there are three major phases of infection: 1. immune tolerant. active viral replication in the liver with high levels of hbv dna levels but essentially normal or minimal elevation of ast and alt. most patients eventually progress to the next stage. 2. immune active. more robust liver inflammation with alt elevation, and liver biopsy shows inflammation with or without fibrosis. hbv early antigen (hbeag) is detected along with hbsag. 3. inactive carrier state. as patients enter this phase, they clear the hbeag and develop anti-hbe antibody and have undetectable or low levels of hbv dna, with normalization of alt and liver inflammation. if patients become hbsag negative, they then develop anti-hbs and have resolved their infection; otherwise, they are considered a chronic carrier. treatment of acute hbv is primarily supportive. in the last decade, however, there have been significant advances in the treatment of chronic hbv infection. the use of interferon has long been the mainstay of treatment and has a defined, limited course but is generally poorly tolerated. with the advent of the hiv/aids epidemic and research into treatment of hiv disease, antiviral medications are now starting to replace interferon as the preferred treatment option for hbv patients. nucleoside/nucleotide analogs such as lamivudine, adefovir, entecavir, tenofovir, and telbivudine are generally given for long-term, indefinite therapy to prevent progression of liver disease and development of hepatocellular carcinoma. any patient with chronic hbv infection should be referred to an infectious diseases specialist or a hepatologist to determine the appropriate treatment course. universal vaccination of newborns and infants is routine in the united states since 1991, and the incidence of hbv infection has declined. during primary care visits, the vaccination status of any adult or adolescent born before 1991 should be reviewed and the vaccine offered. the vaccine requires three doses given at 0, 1, and 6 months. an unvaccinated person or neonate who is exposed to the body fluids of a hbv-infected individual should start the vaccination series in addition to receiving the hepatitis b immune globulin (hbig). hepatitis c virus infection is the most common cause of chronic viral hepatitis in the united states. hcv does have an acute form of infection but is usually subclinical and rarely diagnosed. the cdc estimates that there are more than 2.7 million people with hcv infection. hcv is generally transmitted parenterally, as in injection drug users who share needles. before 1992, those who received a blood transfusion may have contracted hcv. sexual transmission acute hbv has been reported in monogamous couples, with one partner who has hcv infection and the other without infection who eventually acquires the virus. this occurs in 3% to 5% of couples and represents a rare mode of transmission. because the most common mode of acquisition is sharing needles, any patient who is hcv positive should be screened for hiv because these two infections often occur together (ebox 16-1). the diagnosis of acute hcv infection can be made by obtaining a hcv rna viral load; although this is rarely done because the initial infection is subclinical. chronic disease is generally discovered by a positive anti-hcv antibody along with an elevated hcv rna viral load. hcv genotype should also be obtained in any positive individual, because this has important prognostic factors with regard to therapy, with genotype 1a and 1b the predominant type in the united states and unfortunately having a poor response to therapy. as with hbv, chronic hcv infection can lead to cirrhosis and the development of hepatocellular carcinoma. treatment consists of 24 to 48 weeks of interferon and ribavirin therapy. any patient being considered for therapy should be referred to an infectious diseases specialist or hepatologist. a liver biopsy is often needed to determine appropriate treatment candidates. also known as the hepatitis delta antigen virus, hdv is a defective virus that requires the presence of hbv to be infectious. hdv should be suspected in any patient with chronic hbv who develops acute hepatitis. hepatitis d is endemic in the mediterranean, balkans, africa, middle east, and amazon basin. diagnosis is made through an anti-hdv antibody in the presence of someone with positive hbsag or anti-hb core antibody igm or igg. treatment is supportive. any person vaccinated against hbv cannot become infected with hdv. similar to hav infection, hev is spread by the fecal-oral route. hev only has an acute form and does not progress to chronic infection. most reported epidemics have been related to consumption of contaminated drinking water. hev is endemic to southeast and central asia, north africa, middle east, mexico, brazil, venezuela, and cuba. hepatitis e can be considered a cause of infectious hepatitis in the united states in the traveler returning from an endemic area. the incubation period is 40 days. infection is of major concern during pregnancy, which can cause death in late pregnancy. diagnosis is made by demonstration of anti-hev antibody in serum. treatment is supportive. • endocarditis prophylaxis is now recommended solely for patients at high risk of a complicated course with a more narrow range of cardiac conditions. • routine prophylaxis for gi and gu procedures is no longer recommended. • the duke criteria represent a reliable scoring system for diagnosing endocarditis. • echocardiography is indicated to confirm suspected endocarditis. bacterial endocarditis is one of the most feared infections; although uncommon, it carries high morbidity and mortality. increase in antibiotic resistance among bacteria causing this infection has created challenges for effective treatment. the fundamental view of the american heart association (aha) in preventing infective endocarditis has shifted in recent years. views on pathophysiology have not changed substantially, but it is now recognized ebox 16-1 persons for whom hepatitis c virus (hcv) screening is recommended persons who have injected illicit drugs in the recent and remote past, including those who injected only once and do not consider themselves to be drug users. persons with conditions associated with a high prevalence of hcv infection, including: persons with human immunodeficiency virus (hiv) infection persons with hemophilia who received clotting factor concentrates before 1987 persons who have ever received hemodialysis persons with unexplained abnormal transaminase (aminotransferase) levels prior recipients of transfusions or organ transplants before july 1992, including: persons who were notified that they had received blood from a donor who later tested positive for hcv infection persons who received a transfusion of blood or blood products persons who received an organ transplant children born to hcv-infected mothers health care, emergency medical, and public safety workers after a needle stick injury or mucosal exposure to hcv-positive blood current sexual partners of hcv-infected persons * modified from centers for disease control and prevention. recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease. mmwr 1998;47(rr):1-39. *although the prevalence of infection is low, a negative test in the partner provides reassurance, making testing of sexual partners of benefit in clinical practice. • universal vaccination of infants with hepatitis b vaccine reduces the risk of acute hepatitis, chronic carrier state, and complications of chronic infection and may be more effective than selective vaccination of high-risk individuals (lee et al., 2006) (sor: a). • as part of a comprehensive health evaluation, all persons should be screened for behaviors that place them at high risk for hepatitis c infection (ghany et al., 2009 ) (sor: b). • liver biopsy may be considered in patients with chronic hcv infection to determine fibrosis stage for prognostic purposes or to make a treatment decision (ghany et al., 2009 ) (sor: b). that cumulative daily episodes of bacteremia likely carry more risk than the transient bacteremia caused by dental procedures. infective endocarditis likely begins with turbulent flow and damaged endothelium around heart valves, which allow platelet aggregation and thrombus formation, causing a "nonbacterial thrombotic endocarditis" (wilson et al., 2007) . the presence of bacteremia then allows this vegetation to become seeded with infection. bacterial "adhesins" are present to a greater degree in some species and allow for more effective attachment to the injured area of endothelium. with high concentrations of bacteria in the mouth, vagina, gi tract, and perhaps gu system, antibiotic prophylaxis was initiated when these anatomic locations were manipulated. recommendations for infective endocarditis prevention changed in 2007-2008, with aha recognizing more likely benefit from providing adequate population-based dental care and good oral hygiene, and thus less significant ongoing bacteremia at home in brushing, flossing, and "toothpicking," than in providing antibiotic prophylaxis to patients undergoing a dental procedure. no prospective rct has shown that dental prophylaxis prevents infective endocarditis. with recognition of the risk associated with administration of antibiotics (gi upset, diarrhea, rash, anaphylaxis) and the risk of contributing to increasing antibiotic resistance, versus the likely negligible benefit, aha has substantially changed its advice on this long-held practice. a preexisting cardiac condition produces a predisposition to the development of infective endocarditis (ebox 16-2). for example, those who have valve replacement for infection of an infected native valve carry a lifetime risk of 630 per 100,000 patient-years. the risk in the general population without known heart disease is 5 per 100,000 patient-years. more concerning, however, is the risk to a given patient of poor outcome if the patient develops endocarditis, which drives current aha recommendations. those with an infected mechanical valve have a mortality rate of about 20%, versus 5% or less for patients with an infected native valve (wilson et al., 2007) . a summary of current recommendations for endocarditis prophylaxis is provided in etable 16-4. of note, gi and gu procedures have been removed from those for which antibiotics are recommended, unless those systems are actively infected at the time of the procedure. the same is true for skin and soft tissue procedures, in that only infected tissue would warrant antibiotics to prevent infective endocarditis. it is still recommended to provide prophylaxis for respiratory tract procedures, if the respiratory wall will be invaded through biopsy or the procedure. in addition, respiratory procedures to treat infections (e.g., empyema) should be combined with antibiotic administration (nishimura et al., 2008) . antibiotic regimens for prophylaxis for dental procedures are still based primarily on synthetic penicillins as their cornerstone. this is with recognition that streptococcus viridans is both a mouth floral inhabitant and a common agent causing infective endocarditis. with other procedures, antibiotics should be targeted to bacterial pathogens causing any active infection in the system being manipulated. ebox 16-2 cardiac conditions associated with the highest risk of adverse outcome from endocarditis for which prophylaxis with dental procedures is reasonable prosthetic cardiac valve or prosthetic material used for cardiac valve repair previous ie congenital heart disease (chd) * unrepaired cyanotic chd, including palliative shunts and conduits completely repaired congenital heart defect with prosthetic material or device, whether placed by surgery or by catheter intervention, during the first 6 months of the procedure † repaired congenital heart defect with residual defects at the site or adjacent to the site of a prosthetic patch or prosthetic device (which inhibit endothelialization) cardiac transplantation recipients who develop cardiac valvulopathy *except for the conditions listed above, antibiotic prophylaxis is no longer recommended for any other form of chd. †prophylaxis is reasonable because endothelialization of prosthetic material occurs within 6 months after the procedure oral antibiotic william osler discussed "malignant endocarditis" in 1885 and its great diagnostic challenge. in 2009 the modified duke criteria remains a reliable tool for assessing patients with endocarditis. endocarditis is suspected in febrile patients without an obvious source, in those with recent bacteremia (including iv drug use), in those with underlying cardiac predisposition, and perhaps in patients with the clinical finding of a new cardiac murmur. in establishing a diagnosis of infective endocarditis, a patient is considered to have definite disease if two major or one major and three minor or five minor criteria are present. possible disease is defined as one major and one minor or three minor criteria (ebox 16-3). pathologic specimens showing changes consistent with endocarditis would make a definitive diagnosis. echocardiography is indicated in making the diagnosis of infective endocarditis. transthoracic echocardiography (tte) is helpful if vegetations are seen, although size of the patient and other disease (e.g., copd) may limit the ability of tte to view the cardiac valves adequately. if tte is negative and suspicion remains, transesophageal echocardiography (tee) is indicated. tte may be more widely available, depending on regional and institutional variation, and should be used rather than delaying this diagnostic test. bacteria present within valvular vegetations are often less metabolically active, which partly explains the requirement for longer courses of antibiotics for this type of infection. clearly, therapy for endocarditis should be targeted at the organism identified on blood culture, if any. the counting of antibiotic days should begin when the blood culture becomes negative and not at the start of the particular agent. recommendations for antibiotic use in infectious endocarditis are highly variable and based on the presence or absence of synthetic valvular material and the infectious agent (etable 16-5). generally speaking, a minimum of 4 weeks of iv antibiotics is indicated. in cases of resistant organisms, up to 8 weeks may be required. in either case, synergistic use of agents such as gentamicin may be indicated for the first several weeks of treatment, which then can be discontinued. the ability of a given patient to complete this course at home versus in a health care facility is dependent on the dosing frequency of the antibiotic, availability of inhome nursing services, and the type of intravenous access through which the antibiotic will be delivered. at the completion of endocarditis therapy, echocardiography should be repeated to re-assess the function of the valve(s) in question. valvular dysfunction at the completion of therapy is a good indication that the patient will need valve replacement in the future. there are circumstances, like the development of congestive heart failure in the face of endocarditis, in which primary surgery is indicated. typical microorganisms consistent with ie from 2 separate blood cultures: viridans streptococci, streptococcus bovis, hacek group, staphylococcus aureus; or community-acquired enterococci in the absence of a primary focus; or microorganisms consistent with ie from persistently positive blood cultures, defined as follows: at least 2 positive cultures of blood samples drawn >12 hours apart; or all of 3 or a majority of ≥4 separate cultures of blood (with first and last sample drawn at least 1 hour apart). single positive blood culture for coxiella burnetii or anti-phase 1 igg antibody titer >1:800 echocardiogram positive for ie (tee recommended for patients with prosthetic valves, rated at least "possible ie" by clinical criteria, or complicated ie [paravalvular abscess]; tte as first test in other patients) defined as follows: oscillating intracardiac mass on valve or supporting structures, in the path of regurgitant jets, or on implanted material in the absence of an alternative anatomic explanation; or abscess; or new partial dehiscence of prosthetic valve; new valvular regurgitation (worsening or changing or preexisting murmur not sufficient) predisposition, predisposing heart condition, or idu fever, temperature >38° c vascular phenomena, major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial hemorrhage, conjunctival hemorrhages, and janeway's lesions immunologic phenomena: glomerulonephritis, osler's nodes, roth's spots, and rheumatoid factor microbiologic evidence: positive blood culture but does not meet a major criterion as noted above * or serological evidence of active infection with organism consistent with ie echocardiographic minor criteria eliminated echocardiography should be performed in all patients with suspected infective endocarditis (baddour et al., 2005) there is no evidence that antibiotic prophylaxis is effective or ineffective for preventing infectious endocarditis after dental procedures in patients at risk (chung, 2009 ) (sor: c). regimen dosage * and route duration (wk) chronic cough due to acute bronchitis: accp evidencebased clinical practice guidelines interim recommendations for the use of influenza antiviral medications in the setting of oseltamivir resistance among circulating influenza a (h1n1) viruses, 2008-09 influenza season national ambulatory medical care survey: 2005 summary short-course antibiotic treatment in acute exacerbations of chronic bronchitis and copd: meta-analysis of doubleblind studies antibiotics for exacerbations of chronic obstructive pulmonary disease antibiotics for acute bronchitis effects of naproxen on experimental rhinovirus colds: a randomized, double-blind, controlled trial infectious diseases society-american thoracic society consensus guidelines on management of community-acquired pneumonia in adults emergence of a novel swineorigin influenza a (h1n1) virus in humans acute pneumonia influenza. in: schlossberg d, ed. clinical infectious disease seasonal influenza in adults and children: diagnosis, treatment, chemoprophylaxis, and institutional outbreak management. clinical practice guidelines of the infectious diseases society of america antivirals for influenza in healthy adults: systematic review hospitalizing influenza in adults infectious diseases society-american thoracic society consensus guidelines on management of community-acquired pneumonia in adults influenza viruses, including avian influenza and swine influenza steroids for symptom control in infectious mononucleosis hepatosplenomegaly in infectious mononucleosis, assessed by ultrasonic scanning infectious mononucleosis vaccines for post-exposure prophylaxis against varicella (chickenpox) in children and adults centers for disease control and prevention (ats/cdc). targeted tuberculin testing and treatment of latent tuberculosis infection centers for disease control and prevention. extensively drug-resistant tuber culosis-united states short-course therapy with rifampin plus isoniazid, compared with standard therapy with isoniazid, for latent tuberculosis infection: a meta-analysis genital warts expedited partner therapy in the management of sexually transmitted diseases. atlanta: us department of health and human services center for disease control and prevention. sexually transmitted disease surveillance. division of std prevention accuracy of the clinical diagnosis of vaginitis compared with a dna probe laboratory standard a pooled analysis of the effect of condoms in preventing hsv-2 acquisition consistent condom use is associated with lower prevalence of human papillomavirus infection in men the limited value of signs and symptoms in the diagnosis of vaginal infections cervical cancer condom effectiveness in reducing heterosexual hiv transmission sexually transmitted diseases treatment guidelines genitourinary infections urinary tract infection in children diagnosis and management of uncomplicated urinary tract infections american geriatric society. guidelines for the diagnosis and treatment of asymptomatic bacteriuria in adults asymptomatic bacteriuria: when to screen and when to treat the effectiveness of a clinical practice guideline for the management of presumed uncomplicated urinary tract infection in women recurrent cystitis in non-pregnant women a randomized trial to evaluate effectiveness and cost effectiveness of naturopathic cranberry products as prophylaxis against urinary tract infection in women screening for asymptomatic bacteriuria in pregnancy: a decision and cost analysis guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women tick-borne infections tick-borne diseases tick-borne diseases in the united states the clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis practice guidelines for the diagnosis and management of skin and soft tissue infections furuncles and carbuncles centers for disease control and prevention. health-care-associated methicillin-resistant staphylococcus aureus (mrsa) double-blind, placebo-controlled trial of cephalexin for treatment of uncomplicated skin abscesses in a population at risk for community-acquired methicillin-resistant staphylococcus aureus infection practice guidelines for the diagnosis and management of skin and soft-tissue infections bacteriological study of diabetic foot infections diabetic foot infection diagnostic accuracy of the physical examination and imaging tests for osteomyelitis underlying diabetic foot ulcers: meta-analysis diagnosis and treatment of diabetic foot infections deep tissue biopsy vs. superficial swab culture monitoring in the microbiological assessment of limb-threatening diabetic foot infection culture of percutaneous bone biopsy specimens for diagnosis of diabetic foot osteomyelitis: concordance with ulcer swab cultures bite infections antibiotic prophylaxis for mammalian bites (cochrane review) sexually transmitted diseases treatment guidelines fever of unknown origin a comprehensive evidence-based approach to fever of unknown origin fever of unexplained origin: report on 100 cases complicated intra-abdominal infections; spontaneous bacterial peritonitis; and secondary bacterial peritonitis and intra-abdominal abscesses members of veterans affairs hepatitis c resource center program. management and treatment of patients with cirrhosis and portal hypertension: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program mandell, douglas, and b ennett's principles and practices of infectious diseases spontaneous bacterial peritonitis: diagnosis, treatment and prevention review article: the utility of reagent strips in the diagnosis of infected ascites in cirrhotic patients randomized, comparative study of oral ofloxacin versus intravenous cefotaxime in spontaneous bacterial peritonitis diagnosis and management of complicated intra-abdominal infection in adults and children. guidelines by the surgical infection society and the infectious diseases society of america acute bacterial meningitis beyond the neonatal period brain abscess practice guidelines for the management of bacterial meningitis the management of encephalitis: clinical practice guidelines of the infectious disease society of america acute meningitis guidelines on acute infectious diarrhea in adults. the practice parameters committee of the american college of gastroenterology a comparison of vancomycin and metronidazole for the treatment of clostridium difficileassociated diarrhea, stratified by disease severity acute cholecystitis nett's principles and practices of infectious diseases diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the surgical infection society and the infectious diseases society of america infectious viral hepatitis national institutes of health consensus development conference statement: management of hepatitis b acute viral hepatitis hepatitis b virus infection chronic viral hepatitis chronic hepatitis diagnosis, management and treatment of hepatitis c: an update acute viral hepatitis hepatitis c virus infection effect of hepatitis b immunisation in newborn infants of mothers positive for hepatitis b surface antigen: systematic review and meta-analysis chronic hepatitis b: update infective endocarditis: diagnosis, antimicrobial therapy, and complications prescription of antibiotics for prophylaxis to prevent bacterial endocarditis experience with a oncedaily aminoglycoside program administered to 2184 adult patients acc/aha 2008 guideline update on valvular heart disease: focused update on infective endocarditis prevention of infective endocarditis: guidelines from the american health association. circulation key: cord-256118-gxhhwqdd authors: abadie, r.; dombrowski, k. title: “caballo”: risk environments, drug sharing and the emergence of a hepatitis c virus epidemic among people who inject drugs in puerto rico date: 2020-10-23 journal: harm reduct j doi: 10.1186/s12954-020-00421-z sha: doc_id: 256118 cord_uid: gxhhwqdd background: sharing drug injection equipment has been associated with the transmission of hcv among pwid through blood contained in the cooker and cotton used to prepare and divide up the drug solution. while epidemiologists often subsume this practice under the sharing of “ancillary equipment,” more attention should be paid to the fact that indirect sharing takes place within the process of joint drug acquisition and preparation. methods: we employed an ethnographic approach observing active pwid (n = 33) in four rural towns in puerto rico in order to document drug sharing arrangements involved in “caballo”, as this practice is locally known. we explored partners’ motivation to engage in drug sharing, as well as its social organization, social roles and existing norms. findings: findings suggest that drug sharing, is one of the main drivers of the hcv epidemic in this population. lack of financial resources, drug packaging, drug of choice and the desire to avoid the painful effects of heroin withdrawal motivates participants’ decision to partner with somebody else, sharing injection equipment—and risk—in the process. roles are not fixed, changing not only according to caballo partners, but also, power dynamics. conclusion: in order to curb the hcv epidemic, harm reduction policies should recognize the particular sociocultural contexts in which people inject drugs and make decisions about risk. avoiding sharing of injection equipment within an arrangement between pwid to acquire and use drugs is more complex than assumed by harm reduction interventions. moving beyond individual risk behaviors, a risk environment approach suggest that poverty, and a strict drug policy that encourage users to carry small amounts of illicit substances, and a lack of hcv treatment among other factors, contribute to hcv transmission. while hcv-related deaths in the united states seem to have declined recently [1] , research suggests the existence of an emerging hepatitis c virus (hcv) epidemic among people who inject drugs (pwid) in non-urban areas [2] , which is likely associated with the transition from oral prescription opioid use to injection and often with the transition from prescription opioids to heroin [3] . rates of hiv and hcv among pwid are on a divergent trajectory. hiv prevalence has been declining for almost a decade, while hcv has increased over the same period [4] . nowhere in the united states are these trends more prevalent than in puerto rico, a us territory, where recent epidemiological data suggest epidemic levels of hcv among pwid, with 89% prevalence in metropolitan san juan and 78.4% in rural areas [5, 6] . in the us, hcv prevalence among pwid ranks highest in puerto rico, at a similar level as the worst cases in the global south [7, 8] . epidemiological studies of hcv transmission routes among pwid show that the virus can be transmitted not only though blood contained in shared syringes but also by sharing the cooker and cotton used to prepare the drug solution [9] [10] [11] . yet, epidemiologists often misunderstand the dynamics of hcv transmission among pwid, subsuming the common use of a cooker, or a filtering cotton ball, as the shared use of "ancillary equipment" [12, 13] . as social scientists have noted, equipment sharing is often a proxy for preparing and dividing drugs [14, 15] . based on a study of pwid in colorado, koester (2005) suggests that indirect sharing, which happens when powder drugs are diluted in water before been divided up in a cooker with the help of a syringe, is an efficient way of distributing drugs among injection partners. using the calibration on the syringe barrel allows participants to compare the syringe contents' , effectively ensuring an equitable distribution. this type of indirect sharing which happens more often than direct syringe sharing, when one syringe is shared from one user to another [16] (friedman et al. 1997 ) has been found to be a common feature among pwid in a variety of social contexts [17] [18] [19] [20] [21] [22] [23] [24] . in a social network study of pwid in brooklyn, new york curtis and colleagues (1995) found that indirect sharing is extensively practiced within a network particular social networks, contributing to define a user position in it. those that engage in drug sharing often, are the "regulars" or insiders who are placed at the center of the network, while those that do engage occasionally sometimes coming from other neighborhoods or sporadically over the weekends, tend to occupy a peripheric position [25] . a similar study by zule with pwid in san antonio shows that participants drug sharing arrangements are asymetric relationships, with the person that provides the drug in a position to direct drug preparation and the person receiving the drug solution unable to avoid the indirect sharing of injection equipment [26] . a qualitative study of pwid in tajikistan shows that while few users report direct syringe sharing, joint drug acquisition and preparation is common, particularly in outdoor settings or if participants experience withdrawal symptoms [27] . another study in viet nam shows that drug sharing practices are driven by heroin price and accessibility as well as a punitive approach to drug use that penalizes drug possession [28] . in a multi-year study of puerto rican pwid in new york city and bayamon, in san juan found that, recent migrants from the island exhibit a much higher frequency of indirect drug sharing than native users [29] . an ethnographic study conducted among pwid in san juan extends these observations by exploring the motivations and social roles involved in drug sharing arrangements [30] . finlinson (2011) shows that the price and packaging of drugs and access to drug preparation materials along with power differences among partners shape the process by which drugs are prepared and injected. other ethnographic studies demonstrated that drug sharing is facilitated by the type of heroin available. for example, bourgois documents how homeless pwid in san francisco use "black tar, " a sticky variety of heroin, originating in mexico, that is extremely hard to divide up without preparing it, which, in turn, encourages the sharing of injection equipment [31, 32] . departing from epidemiological views that focus on individual behaviors, these authors make a valuable contribution to the understanding of hcv risk among pwid by showing how particular social contexts, from the ways in which drugs are acquired, to social roles, cultural norms, drug policies and power dynamics among those involved in "caballo" shape hcv risk. these findings complicate traditional epidemiological views shaping harm reduction initiatives, suggesting that forms of indirect sharing within the process of jointly acquiring and using drugs, are not easily modified by knowledge about hcv transmission, or the access to new injection equipment alone. as critics have suggested, the focus on individual behaviors at the expense of local social contexts in which pwid live and make decisions about risk, might obscure how social, structural and environmental contexts shape drug use and related harms [33] [34] [35] [36] [37] . nested within a study of social networks and hiv/ hcv risk among pwid in rural puerto rico, we propose an ethnographically informed approach to "caballo", the joint acquisition and sharing of drugs, as a window into the social production of an hcv epidemic among pwid. while drug sharing arrangements among pwid have been amply documented, an ethnographic study of caballo in puerto rico will illuminate the social context behind the joint acquisition and use of drugs and its related epidemiological risk. this paper utilizes ethnographic data from pwid recruited into a multi-phase study of social networks and hiv/hcv risk in four rural towns in the mountainous area of central puerto rico. in the first phase of this study we collected demographic and sociometric data on pwid in rural puerto rico, as well as information on injection behaviors, particularly on the sharing of syringes and injection equipment. in addition, rapid blood testing for hiv and hcv were conducted using insti rapid hiv antibody tests (biolytical laboratories) and oraquick hcv rapid antibody tests (orasure technologies). it was during the administration of a national health behavioral survey (nhbs) questionnaire [38] that we learned for the first time about "caballo". consistently, we heard from participants that while they carried their own syringe and avoided sharing it with others, they could not avoid using the same cooker within the process of drug sharing. participants described the ways in which they divided up the drug solution in the cooker, using a backloading method to distribute it among partners. all participants insisted that they "always used their own cooker" during the preparation. of course, since there are two or more caballo partners and only one cooker available, this was not possible. after this phase of data collection, we were left with many questions about the social organization and the meaning of drug sharing among this population. to explore these issues, we conducted an ethnographic study with (n = 33) participants randomly recruited from the first phase. during the participant observation, each participant was followed -with their consent-for up to two weeks, documenting practices related to joint drug acquisition and use. yet, our ethnographic observations were broadly constructed, focusing not only on drug sharing practices, but in describing their everyday lives and the strategies they used in order to afford their drugs of choice. for example, we had ample opportunity to observe participants' hustling, in car washes, or guarding a parking lot, or begging at the entrance of banks or dollar stores or other venues with heavy foot traffic. after partnering with el punto en la montana, the only syringe exchange provider in the area, we earned participants' trust and were allowed to join them at the "chutin" a spanglish deformation of shooting gallery. in so doing, we were able to collect data on their drug sharing practices. we paid particular attention to the ways in which caballo partners talked about acquiring and sharing drugs in an attempt to convey the norms or hidden scripts regulating caballo arrangements. in addition, we conducted in depth interviews to understand how pwid in our study described the social practice of "caballo", the joint acquisition and later use of drugs. some of the questions we posed were what is caballo, when do participants do it and with whom. we used our initial observations to iteratively refine our research questions. since we had observed during the course of the ethnographic data collection that sometimes arguments emerge during caballo, we asked participants about potential problems or conflicts associated with this practice and we also inquired about participants' views on drug sharing related hcv risk and their perceived ability to enact changes to prevent its transmission. one limitation of our data collection is that for security reasons, ethnographic observations were conducted during the day and until dusk and only during working days. abandoned and dilapidated houses without electricity, shooting galleries are seldom used at night but are more used early in the day -as soon as drug selling spots are open-and on weekends when "regulars' are joined by more occasional users. despite the limits imposed by fieldwork dynamics we believe that our observations of caballo and the social context in which it occurs were not affected by these constraints. fieldnotes were transcribed while in-depth interviews were transcribed and translated. all personal identifiers were removed. maxqda software was employed to manage coding. codes were developed to convey the wide arrange of themes in the data set: caballo, drug acquisition, motivations, social roles, norms and expectations and injection setting, among others. these codes were then iteratively revised and re-organized until they represented higher-level axial codes describing participants' caballo experiences [39, 40] . following strauss' grounded theory approach [41] (strauss and corbin 1998) the interpretation of the data emerged intuitively without imposing a pre-existing theoretical framework. the study received irb approval through the (omitted due to blind review) and the (omitted due to blind review). participants provided written consent at the study office prior to enrollment in the study and were compensated for their time and travel expenses. participants' sociodemographic background (table 1) . participants had a mean age of 44.15 years and the sample had a standard deviation of 9.2 years. the sample is overwhelmingly male (87.8%) and heterosexual (96.97%). three-quarters were unemployed at the time, and almost one-half lived in poverty and had completed high school or a higher educational level. one in five were married or living together and the same number had been homeless during the past year. almost all participants (90.91%) were currently covered by health insurance, with a large majority having "la reforma, " the local version of medicare/medicaid. around one in six (15.15%) had participated in a drug treatment program but only one participant had enrolled during the past year. participants had been injecting for a mean of 22.91 years with a standard deviation of 10.54. age at first injection is 22.27 years with a standard deviation of 7.97. almost four out of ten reported injecting two or three times a day while one in three injected four times or more a day. while hiv prevalence in this population is extremely low (3.03%), hcv prevalence reaches epidemic levels, with almost nine out of ten study participants testing positive reactive for the virus (84.8%). less than one in ten (7.14%) declared having used a syringe after somebody else had used it the last time they injected with somebody, and a large majority (84.6%) admitted using a sterile syringe. almost three-quarters (71.43%) used a cooker, cotton, or water that somebody had previously used, while one in three (32.14%) divided drugs with a syringe that had been previously used by somebody else. in rural puerto rico, two or more pwid often pool funds necessary to acquire and later share drugs. most participants in our study have as a drug of choice heroin "droga" and cocaine "perico", "speedball". speedballs have more heroin than cocaine, a usual way in which participants talk about their drug mix is by identifying it by the ratio of heroin to cocaine. for example, they would say "1-2" meaning one bag of cocaine and two of heroin. other users might prefer three bags of heroin and one of cocaine "1-3". in turn, this preference is also reflected in drug sharing arrangements. the drugs are mixed together in a cooker dissolved in water, and the resulting drug solution is shared usually through backloading, removing the plunger in a syringe and squirting the content using the tip of the needle of a loaded syringe, before placing it back. this practice is locally known as "caballo" (literally, horse). participants do not recall the origin of the name, "caballo" but suggest that the same expression is used on the island in situations where people pool resources to acquire and later consume goods together, usually food but also transportation. an ethnographic fieldnote taken at the shooting gallery a few blocks away from where our office was located, describes two study participants, pablito and cesar cayey engaging in a "caballo": pablito and cesar had bought their drugs of choice at the one of the local "puntos", the drug selling spot, only a few blocks away from the dilapidated house that served as shooting gallery. they briefly discussed how the drugs would be divided up agreeing that since they had contributed the same amount of money, each would receive equal parts of the drug solution. however, since pablito, was helping cesar inject because his partner was unable to find his own veins, pablito would be in charge of the preparation. the cooker, a small sized tin cup provided by the local syringe exchange provider, el punto en la montana and a plastic water bottle were laid out on the cement floor by pablito, who was in charge of the preparation. the plastic clear blue bag containing cocaine was opened and placed carefully on the cooker along with the contents of three colored metallic paper envelopes with heroin. water from a plastic bottle was loaded into a syringe and then discharged into the cooker. the content was then mixed with the back of a syringe making sure it was completely diluted. although the cocaine is a white powder and the heroin a light brown, grey or creamy color, when mixed with water, the solution turned into a brown color. without heating-it is believed to "eat up" the drug because when heated some of the content evaporates leaving a more powerful concentration, but less quantity-pablito skillfully "recogio" or loaded the preparation with the help of a syringe with its needle inserted into a small cotton ball no larger than the head of a match to filter any impurities. with the syringe "full", half of its content was then "echado" distributed into cesar's syringe by removing the plunger on the back. after verifying that both syringes had exactly the same amount by holding them side by side, using the lines on the side of the syringes as a guide, the plunger in cesar's syringe was placed back. both syringes were now, half full, having divided a loaded syringe used to divide drugs up, in two equal parts. far from a ritual practice to strengthen a bond between injection partners, caballo seems to be a consciously made strategic decision to maximize drug access among pwid. drugs are jointly acquired and then divided up among pooling partners. the most common arrangement is to distribute the drug solution according to the monetary contributions of each member proportionally. partners can share drugs at "brazo partido" a local expression translated as half and half, or 50%-50%, if they contributed equally, although it is possible that the partner that contributes the most agrees to divide drugs equally, particularly if they have a long-standing relationship with the caballo partner. josephine, a 34-year-old woman who started injecting in her teens, provides a description of the advantages of this practice: "look, let's suppose that i want to use two and one [two bags of heroin and one bag of cocaine] and that you have and i put [in for] the heroin. ' we put everything together in the cooker, and then we divide it in the syringe, half and half, and we get cured. that's it. " while most pwid in the study would prefer to avoid caballo if they could, particularly, for high frequency users, the economic demands make it extremely hard to go during the day without partnering with another user to acquire and use drugs. with a large proportion in unemployment, receiving meager social security checks, working at the lower levels of the local drug trade, or engaging in side hustles, it becomes extremely difficult for pwid in our area to secure the whole amount every time they need to inject. opioid users dread the painful effects of heroin withdrawal, or what they call "being sick, " characterized by bodily pain and discomfort, nausea, coldness, shivers, and diarrhea that leave them "unable to function. " only "la cura", the cure, another dose of heroin, would stop or prevent these symptoms form occurring. faced with limited resources to "get cured" the user has to make a choice between partnering with somebody in a caballo or going it alone and hustling until they can afford the whole dose they need. entering into a caballo arrangement, enables them to feel normal again, while they can keep hustling to get their next dose. while the rewards of going alone might be higher because participants get their full dose, so are the associated costs because users have to battle their withdrawal symptoms while they come up with the money. and the longer it takes for participants to secure the resources to afford their dose, the worst their withdrawal symptoms become, offering a powerful incentive to enter into a drug sharing agreement with another user. to avoid heroin withdrawal, if possible, caballo partners prefer to acquire their drug of choice in a punto close to where they can use it, without delay. but in relatively small rural locations, drug choices are restricted, particularly in relation to package size. locally, heroin can be bought in bags of five or ten dollars and cocaine comes in "cinquillos" fives, or $5, but if caballo partners can get a hold on a car, they usually pool up $2 or $3 each for gas and head to nearby caguas o san juan where bags cost the same but are two or up to three times larger. this drug packaging makes the trip worthwhile, even considering the gasoline costs and that time spent on the hour round trip could have been used "revuleando", hustling, at home. the frequency of caballo changes from participant to participant; some engage in caballo almost every time they use, while others do it less frequently. since caballo partners tend to inject smaller drug amounts, they also need to engage in this practice more often. speedball also provides more opportunities to share drugs because one partner might have cocaine but not heroin, while other might be in the opposite situation, in this situation, one solution is pooling resources and then dividing up the drug solution. pwid in our study declared that they prefer not to do caballo for their first dose of the day, as it would significantly decrease the amount of heroin received and thus stave off withdrawal symptoms for a shorter amount of time. walter, a 48-year-old user who injects two or three times a day, explains this choice with the use of a metaphor: "it's like pizza: if you eat two pieces you are going to feel full but if you eat only one, or just a bite, you will feel hungrier sooner. " however, here economic considerations play a role. if access to a sufficient dose to prevent the onset of withdrawal symptoms is not available, users might resort to caballo early in the day. those who have been successful in securing their own first dose might decide to engage in caballo later in the day in order to maximize resources, and can afford to engage in the practice more selectively. caballo is also affected by a particularly punitive version of the war on drugs adopted by the island since the 1980′s. most of our study participants have been jailed at least once in their lifetime, often for non-violent drug offences [42] . having no more than a few bags can lead to heavy prison sentences under the "possession with intent" to distribute charges. to avoid problems, pwid tend to carry small drug amounts with them, which, in turn, encourages drug sharing. caballo can be structured along defined social roles, with important epidemiological repercussions. a primary partner directs the preparation and distribution of the drug solution, usually keeping the cooker and cotton used to share drugs. the soaked filter and the drug residue left in the cooker can be later re-used adding a little bit of water for another shot. usually, this role is occupied by the user that contributed the most to the caballo. these roles are not static, it is possible for one user to be a primary partner in one caballo but engage in another drug sharing arrangement later on, either with the same partner or a different one, without being in charge of the process. in addition, partners might follow different strategies while seeking to jointly acquire and use drugs. "fixers" do caballo with a limited number of trusted injection partners in their network, usually kin, or others with whom they have close relationships, from school age friends, to neighbors or those with whom they have shared drugs extensively in the past. by minimizing the number of partners and routinizing sharing expectations, this strategy ensures access to resources while limiting the potential problems associated with doing caballo with strangers. other pwid take on the role of "maximizers, " entering into caballo with as many partners as possible, increasing their opportunities to access drugs by multiplying potential partners. sometimes maximizers only know their caballo partners because they have seen them around, in puntos, or shooting galleries, or because they have done a caballo in the past. the downside is that this choice also increases the potential problems associated with the transaction-robbery, cheating, hoarding. yet, neither of these are fixed strategies. a pwid might have been a maximizer but, over time, begun doing caballo with a limited number of partners, and the opposite also happens. jail, drug treatment, quitting drug use, and migration can all affect a person's social networks and their ability to engage in caballo. of course, this is only an approximate typology, and some users are neither "fixers" nor "maximizers" but operate in between these extremes. whether users might be primary partner or not, or rely on a "fixer" or a "maximizer" strategy, they all seek in their interactions some kind of fair play, adhering to the norms that regulate drug sharing in the community. bebe, in his late 30 s, washes cars at a local gas station in addition to taking turns as a drug dealer in the only drug selling spot in town. he explains the need for reciprocity: "i tend to avoid caballo, if i have all the money i need for my dose i get the drug and i get your money and even if you don't have enough, i share with you because i know what it is to be 'sick. ' how much do you have, i would ask? four dollars [an insufficient amount for a 50/50 caballo]. fine! let's go! and we share. but then i remember that in other occasions it was me that had only four bucks and i were really sick and he had his full dose and he decided not to help me out, leaving me sick to fend for myself. i still decided to help him but i tell him right away: 'see, before you told me you couldn't help me because you had enough for yourself and now you are desperate, see how the world is round? yesterday it was me [that needed] and today it's you. come here, i'll fix it!'". caballo partners who consistently demand more than their fair share are labeled "problematic" and tend to be excluded. "tricksters" are also avoided. according to participants, tricks are very common among pwid, such as pocketing the money that participants have pooled to do caballo. this is viewed very negatively, not only because it causes a lack of trust among partners but also because it deprives users of the "cure" they need, forcing them to hustle for money again before they can have their dose. tampering with "bags, " for example by taking a cut, is also negatively viewed but is judged less severely than the first situation. participants also worry about others pulling a "water shoot, " a trick in which an injector uses deception to substitute drugs with water. a similar and more frequent trick involves placing not one but two cotton balls in the cooker, hiding the one used to filter the drug under the finger holding the cooker, and leaving the other with only a trace of drug for the other injector to use. personality issues and past disagreements also play a role in selecting potential partners. those users with more social connections will be able to find more suitable partners for a caballo while being able to reject those partners deemed less desirable. the reverse is also true: those users with fewer social connections might not have as many options. caballo partnerships are often shaped by asymmetric dynamics involving gender and other forms of power disparities among prospective members. these in turn affect risk taking and drug related harms. caballo partners try to manage hiv/hcv by serosorting, but most assume that there is no point in asking about their partners' hiv status, because "they will lie to you. " perhaps because they are in a more vulnerable position, women tend to ask their injection partners about their hcv status more often than men do. women are also more likely to avoid entering in a caballo with somebody if they know the prospective partner has a positive status. this study outlines how the social practice of caballo has contributed to the production of an hcv epidemic among pwid in rural puerto rico. results show that 84.84% tested positive reactive to hcv, a result that has been confirmed by other epidemiological surveys in the area [43] . pwid tend to avoid direct sharing of syringes; only 7.14% reported having used a needle after somebody else had employed it, and 84.62% used a sterile needle the last time they used drugs with somebody. on the other hand, participants often engaged in indirect sharing: 71.43% divided drugs with a cooker or cotton that had been used by somebody else, and 32.14% divided drugs with a syringe that had been used by somebody else. these forms of indirect sharing are linked to the practice of caballo and the need to divide up jointly acquired drugs, which in turn increases the risk of hcv among this population. while pwid in our study tend to avoid direct sharing, indirect sharing is driven by the mode of drug acquisition, drug packaging and pricing, a reliance on speedball, the need to avoid heroin withdrawal, and a strict drug policy that encourage users to carry small amounts of illicit substances, among other factors. the finding that participants attempt to manage hiv/ hcv transmission risk during caballo by serosorting has been replicated in other studies of pwid [44] [45] [46] . however, the epidemic levels of hcv in this population suggest that this strategy has serious limitations and that individual behaviors alone are insufficient to curb hcv transmission. findings illustrate the need to understand social epidemics such as hcv among pwid, leaving behind individual-centered models prevalent in public health. decades of ethnographic studies with this population have shown that what appears to be the product of individual behaviors is better comprehended as a result of the particular social contexts in which pwid live and make decisions about risk [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] . moving beyond public health's emphasis on individual behaviors, the notions of "risk environment" [57] and "syndemics" [58] have recently been used to analyze the interplay of micro-level dynamics and larger structural forces in accounting for risk outcomes. while both approaches share some features, rhodes (2009) suggests that a risk-environment approach-combining insights from political economy, social epidemiology, and the sociology of health-can be productive for understanding how the relationship between individuals and environment can produce drug harms, thus contributing to a social science of "harm reduction. " [59] . our findings show that poverty and economic dispossession have been found to be an important driver of indirect sharing among pwid. high frequency users might spend $100 or even more a day, not an insignificant sum considering that puerto rico has a per capita income more than half of the poorest us states like missouri or mississippi [60] . the same relationship between poverty and drug sharing has been replicated in numerous studies [27, 31, 61] . in a study of homeless pwid in san francisco bourgeois (1997) finds that if users cannot afford their dose in full, indirect sharing allows users to regulate their dose intake in order to minimize the risk of experience heroin withdrawal, with some participants using half or even a third of a bag each time. in turn, using small doses encourages higher frequency of drug injection, making drug sharing more likely. our findings have shown that roles within drug sharing arrangements are not fixed. this aspect has been documented also by finlinson [30] suggesting that roles change within the process of acquiring and dividing up drugs. according to this author, a primary partner in charge of dividing up the drug solution using their own equipment, might become a secondary partner receiving a drug solution that has been divided using up somebody else's equipment in another caballo arrangement. these practices facilitate the transmission of hcv within a population, complicating harm reduction efforts. one of the most distinctive features of indirect sharing in our study is that the drug preparation is usually loaded from the cooker using a single syringe, and then distributed to the injecting partners through backloading. while indirect sharing offers a fair method for equitable distribution [62] , participants in our study do not seem to favor drawing the drug solution directly from the cooker, because of the perception that it makes it harder to ensure an equal share of the drug. it is possible that this preference might be driven by a lack of trust among injection partners and that in different social contexts, other ways of dividing up the drug solution might be preferable. a study of puerto rican pwid in new york and puerto rico show that pwid in new york city take turns to draw the drug solution from the cooker while those on the island distributed it through backloading as well as taking turns [63] . the fact that our findings correspond not to metropolitan san juan, but rural puerto rico might account for extensive use of backloading. as other authors have shown, the joint acquisition and use of drugs appear to be shaped by particular cultural norms, which in turn are an adaptive response to a particular risk environment [64] . examining the social context in which pwid use drugs and make decisions about risk is critical for the successful adoption of harm reduction strategies to reduce hcv [65] [66] [67] [68] . while harm reduction policies that emphasize the distribution of injection equipment are a valuable component in syringe exchange programs, findings suggest that preventing the sharing drug preparation equipment while sharing drugs might be more complex that behavioral health models suggests [69] . indirect sharing occurs not because pwid lack information about hcv risk or do not possess knowledge about safe injection practices. this perspective has been criticized for blaming the victim [70] while obscuring the role of social and risk environments in shaping drug sharing arrangements [71, 72] . in addition, local implementation of effective harm reduction recommendations regarding hcv transmission among pwid is hampered by larger political and socio economic processes such as the colonial status of puerto rico that deprives the island of the resources it needs to tackle an hcv epidemic, while enacting an aggressive version of the war on drugs and subsequent incarceration that furthers the impact of hcv within its population [73] . furthermore, the subjection to us patent laws and drug pricing ensures that despite the fact that a new hcv treatment is available (in the form of a three-month-long direct-acting antiviral regimen), the cost of that treatment is approximately $50,000. in the continental us, the cost of this treatment is covered by most private insurers or by medicare/medicaid; in puerto rico, few pwid have access to private insurance, and hcv treatment is not covered by la reforma, a local version of medicare/medicaid for hiv-negative individuals who constitute the majority of the pwid population [74] . the extremely low hcv coverage among pwid [75] continues to fuel the epidemic as hcv-negative users are more likely to enter caballo arrangements with somebody infected by the virus. expanded hcv treatment coverage would not only reduce the incidence of hcv among this population but also would contribute to provide certain measure of "herd immunity" protecting those that are hcv negative [76] . similarly, access to harm-reduction-based interventions such as medically assisted treatment and syringe exchange provision have been shown to be protective against hcv transmission [77] [78] [79] [80] [81] [82] [83] . unfortunately, these resources are severely lacking in puerto rico, particularly in rural areas [84] . a recent study showed that rural pwid have less access to syringe exchange in rural areas than those in urban settings, although both have had to resort to acquiring syringes from drug stores, peers, and drug dealers [85] . understanding the social factors behind the hcv epidemic in puerto rico has a renewed urgency. as the island was starting to recover from the aftermath of hurricane maria in september 2017 it was hit by the arrival of covid-19, a pandemic with an epicenter in wuhan china that has currently stricken most of the world. while the lasting effects of covid19 are not yet well known, it is likely the pandemic will reinforce the devastating effects of hurricane maria which furthered a protracted economic crisis on the island, while exposing deep political corruption and mismanagement practices, [86, 87] severely affecting the provision of health services pwid rely on, like medically assisted treatment and syringe exchange providers, leading the way to the worsening of an already existing hcv epidemic. increasingly, harm reduction programs will need to consider the impact of large-scale natural disasters, or pandemic events, and its effects on the risk environment of pwid, already afflicted by severe forms of poverty, social suffering, and structural violence [88] . one limitation of this study is that findings are based on a sample that is overwhelmingly male. while this distribution mirrors not only the composition of the parent study but also of other studies of pwid in puerto rico that seem to be gendered, the relative lack of female participants raises questions about how gender dynamics and power imbalances might shape drug sharing arrangements among pwid in rural puerto rico. more research is needed to explore this issue. we believe that despite this limitation, the study of the social organization of drug sharing arrangements makes an important contribution to the understanding of hcv risk among this population. this study shows that drug sharing plays an important role in the hcv transmission among pwid in rural puerto rico. while participants avoided direct sharing of syringes, they were forced to share the cooker and cotton used to divide and inject the drug solution. drug sharing occurs not only within the joint acquisition and use of injection drugs, but in a particular risk environment that contributes to hcv risk. poverty, drug packaging and pricing, a reliance on speedball, the need to avoid heroin withdrawal, and a strict drug policy that encourage users to carry small amounts of illicit substances, among other factors fuels the hcv transmission. this finding complicates harm reduction interventions based on the distribution of injection equipment or information about safe injection practices alone, suggesting that preventing indirect sharing practices should consider the social context in which pwid acquire and use drugs. deaths associated with hepatitis c virus infection among residents in 50 states and the district of columbia estimating the number of persons who inject drugs in the united states by meta-analysis to calculate national rates of hiv and hepatitis c virus infections risk factors for hcv infection among young adults in rural new york who inject prescription opioid analgesics hiv infection among persons who inject drugs: ending old epidemics and addressing new outbreaks understanding differences in hiv/hcv prevalence according to differentiated risk behaviors in a sample of pwid in rural puerto rico prevalence and correlates of hepatitis c virus infection among street-recruited injection drug users in global estimates of prevalence of hcv infection among injecting drug users county-level vulnerability assessment for rapid dissemination of hiv or hcv infections among persons who inject drugs, united states sharing of drug preparation equipment as a risk factor for hepatitis c des jarlais dc. meta-analysis of hepatitis c seroconversion in relation to shared syringes and drug preparation equipment prevalence and correlates of indirect sharing practices among young adult injection drug users in five u risk of hepatitis c virus transmission through drug preparation equipment: a systematic and methodological review risk of transmission associated with sharing drug injecting paraphernalia: analysis of recent hepatitis c virus (hcv) infection using cross-sectional survey data sharing cookers and cottons are surrogates for drug sharing drug sharing among heroin networks: implications for hiv and hepatitis b and c prevention social capital or networks, negotiations, and norms? a neighborhood case study ethnography, epidemiology, and public policy: needle-use practices and hiv-1 risk reduction among injecting drug users in the midwest migration and hiv risk behaviors: puerto rican drug injectors in new york city and puerto rico syringemediated drug sharing among injecting drug users: patterns, social context and implications for transmission of blood-borne pathogens injecting equipment sharing in russian drug injecting dyads it's not what you know but who you know": role of social capital in predicting risk injection drug behavior in a sample of people who inject drugs in baltimore city hiv risk behaviors associated with the injection process: multiperson use of drug injection equipment and paraphernalia in idu networks prescription opioid injection among young people who inject drugs in new york city: a mixedmethods description and associations with hepatitis c virus infection and overdose street-level drug markets: network structure and hiv risk. soc netw risk and reciprocity: hiv and the injection drug user drug preparation, injection, and sharing practices in tajikistan: a qualitative study in kulob and khorog drug injecting and hiv risk among injecting drug users in hai phong, vietnam: a qualitative analysis trends in hiv seroprevalence and needle sharing among puerto rican drug injectors in puerto rico and new york: 1992-1999 injecting shared drugs: an observational study of the process of drug acquisition, preparation, and injection by puerto rican drug users social misery and the sanctions of substance abuse: confronting hiv risk among homeless heroin addicts in san francisco the textures of heroin: user perspectives on "black tar" and powder heroin in two us cities addressing the "risk environment" of injection drug users: the mysterious case of the missing cop syndemics and public health: reconceptualizing disease in a bio-social context risk environment": a framework for understanding and reducing drug related harm the social production of hepatitis c risk among injecting drug users: a qualitative synthesis back then" and "nowadays": social transition narratives in accounts of injecting drug use in an east european setting needle acquisition patterns, network risk and social capital among rural pwid in puerto rico etnographer's toolkit: designing and conducting ethnographic research treatment scaleup to achieve global hcv incidence and mortality elimination targets: a cost-effectiveness model grounded theory research: procedures, canons, and evaluative criteria it ruined my life": the effects of the war on drugs on people who inject drugs (pwid) in rural puerto rico hepatitis c serosorting among people who inject drugs in rural puerto rico injection-related hepatitis c serosorting behaviors among people who inject drugs: an urban/rural comparison hepatitis c virus serosorting in people who inject drugs: sorting out the details stories of pain and the problem of aids prevention: injection drug withdrawal and its effects on risk behavior beyond prevention? injecting drug user narratives about hepatitis c do practice approaches go far enough in shifting focus from the individual? addiction the pastoral clinic: addiction and dispassion along the rio grande addicted, pregnant, poor needle exchange and the geography of survival in the south bronx enabling environments and the reduction of drug related harm: re-framing australian policy and practice intravenous drug use and hiv infection in miami social and structural violence and power relations in mitigating hiv risk of drugusing women in survival sex work perception of risk and safety within injection settings: injection users' reasons for attending a supervised injecting facility in vancouver structural violence and structural vulnerability within the risk environment: theoretical and methodological perspectives for social epidemiology of hiv risk among injection drug users and sex workers critical medical anthropology risk environments and drug harms: a social science for harm reduction approach small area income and poverty estimates drug sharing among intravenous drug users attitudes toward needle "sharing" among injection drug users: combining qualitative and quantitative research methods joint drug purchases and drug preparation risk behaviors among puerto rican injection drug users injection norms and practices among migrant puerto rican people who inject drugs in new york city: the limits of acculturation theory social structural factors that shape assisted injecting practices among injection drug users in vancouver, canada: a qualitative study navigating environmental constraints to injection preparation: the use of saliva and other alternatives to sterile water among unstably housed pwid in london safer environment interventions": a qualitative synthesis of the experiences and perceptions of people who inject drugs a systematic review of rural-specific barriers to medication treatment for opioid use disorder in the united states the theory of planned behaviour: reactions and reflections the political economy of aids among drug users in the united states: beyond blaming the victim or powerful others the process of drug injection: applying ethnography to the study of hiv risk among idus commentary on harris & rhodes: discouraging syringe re-use by addressing drug injectors' everyday suffering puerto rican syndemics: opiates, overdoses, hiv, and the hepatitis c virus in a context of ongoing crisis to enroll or not to enroll?: a researcher struggles with the decision to involve study participants in a clinical trial that could save their lives treatment access is only the first step to hepatitis c elimination: experiences of universal anti-viral treatment access in australia convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? effectiveness of structural-level needle/syringe programs to reduce hcv and hiv infection among people who inject drugs: a systematic review state hcv incidence and policies related to hcv preventive and treatment services for persons who inject drugs-united states des jarlais dc. a systematic review and meta-analysis of interventions to prevent hepatitis c virus infection in people who inject drugs the promise of treatment as prevention for hepatitis c: meeting the needs of people who inject drugs? recommendations for the management of hepatitis c virus infection among people who inject drugs combination interventions to prevent hcv transmission among people who inject drugs: modeling the impact of antiviral treatment, needle and syringe programs, and opiate substitution therapy retention in buprenorphine treatment is associated with improved hcv care outcomes tackling the health challenge posed by hepatitis c in puerto rico: a call for immediate public health actions differential access to syringe exchange and other prevention activities among people who inject drugs in rural and urban areas of puerto rico the battle for paradise: puerto rico takes on disaster capitalists aftershocks of disaster: puerto rico before and after the storm. chicago: haymarket books the everyday violence of hepatitis c among young woman who inject drugs in san francisco aids and accusation: haiti and the geography of blame the social value of drug addicts: uses of the useless springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-279202-iyteg4h9 authors: shesheer kumar, munpally; venkateswara rao, khareedu; mohammed habeebullah, chittor; dashavantha reddy, vudem title: expression of alternate reading frame protein (f1) of hepatitis c virus in escherichia coli and detection of antibodies for f1 in indian patients date: 2008-01-05 journal: infect genet evol doi: 10.1016/j.meegid.2007.12.008 sha: doc_id: 279202 cord_uid: iyteg4h9 apart from the core (21 kd), a novel hepatitis c virus (hcv) frame shift protein (f1) is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frame shift into the −2/+1 reading frame. to date, no information is available on f1 protein of indian isolates, and hence detection of antibodies for f1 protein in indian patients assumes great relevance. specific primers have been designed to amplify sequence coding for 120aa of truncated f1 (tf1). the amplified tf1 has been cloned in bacterial expression vector, pet21b for expression in escherichia coli. partially purified expressed protein has been subjected to western blot analysis using patients’ sera. three hcv positive sera employed in western analysis showed positive signals to tf1, while sera from uninfected individuals failed to give any signals. further, results of western blots, carried out with patients sera titrated with purified core protein, confirmed the presence of antibodies specific to f1. the positive signal observed for f1 in western analysis with hcv infected sera suggests that f1 protein is synthesized in the natural course of hcv infection in indian patients as well. presence of antibodies against f1 protein of subtype 1c has been demonstrated, for the first time, in indian patients. hepatitis c virus (hcv) is the major causative agent of posttransfusion and parenterally transmitted, non-a, non-b hepatitis throughout the world (alter and seeff, 2000) . hcv is an enveloped rna virus that is classified in the family flaviviridae (robertson et al., 1998) . hcv has high genomic variability and at least six different genotypes and an increasing number of subtypes have been reported (simmonds, 1999) . the genome of hcv comprises a single stranded positive-sense rna of $9.6 kb in length and contains a single open reading frame (orf) that encodes for a non-functional polyprotein of about 3000 aminoacids (grakoui et al., 1993) . this polyprotein is cleaved co-and post-translationally by cellular and viral proteases to yield 10 different functional proteins. structural proteins are the major components of the mature virion, which are coded by the 5 0 quarter of the orf and arranged as c-e1-e2 and p7, while the non-structural proteins are coded by the 3 0 three-quarters of the orf in the order ns2, ns3, ns4a, ns4b, ns5a and ns5b (barbara and contreras, 1991) ; these proteins are involved in polyprotein processing and replicative functions of the virus (suzuki et al., 1999; suzuki et al., 2007) . translation of the hcv polyprotein sequence was reported to be regulated by a cap-independent mechanism that requires most of the 5 0 -non-coding region and the first nine codons of the polyprotein-coding sequence to serve as the internal ribosomal entry sequence (ires) (rijnbrand and lemon, 2000) . initial expression studies indicated that, besides the core protein (21 kd), another protein (17 kd) also expressed from the same core protein coding sequence both in vitro and in mammalian cells and it was thought to be a truncated core protein (lo et al., 1994 (lo et al., , 1995 basu et al., 2004) of family flaviviridae, revealed that a novel translation mechanism of a ribosomal frame shift exists within the capsid-encoding region, which results in a frame shift protein (varaklioti et al., 2002; walewski et al., 2001; xu et al., 2001) . the frame shift protein was named as f1 or arfp (alternative ribosomal frame shift protein) based on translation initiated at a non-aug codons in a à2/+1 reading frame relative to the polyprotein of hcv (baril and brakier-gingras, 2005) . similar synthesis of capsid protein via frame shift was also observed in various other viruses such as sars-cov (baranov et al., 2005) . it was first demonstrated that the 17 kd protein was synthesized by ribosomal frame shift and was mostly derived from the coding sequence that overlaps the hcv core protein reading frame (xu et al., 2001; choi et al., 2003) . the expressed f1 protein was localized in the cytoplasm of hepg2 cells, with a notable perinuclear localization (roussel et al., 2003) , and was found to be associated with the endoplasmic reticulum . this subcellular localization of hcv f1 protein is similar to that of the hcv core and ns5a proteins, raising the hypothesis that the f1 protein may participate in hcv morphogenesis or replication . in addition, sera from patients who were positive for hcv genotype 1a or 1b were shown to react differently to synthetic peptides of f1 (boulant et al., 2003) . the present study mainly deals with the cloning and expression of f1 coding sequence of the hcv indian isolate belonging to genotype 1c. further, antibodies against f1 protein have been detected in indian patients. the recombinant plasmid containing hcv core coding sequence, belonging to genotype 1c (genbank acc. no. ay051292), was used as template for amplification of truncated f1 (tf1) coding sequence employing f1f 5 0 att cat atg gca cga atc cta aac c 3 0 and f1r 5 0 att aag ctt acc caa att gcg tga cct gc 3 0 as forward and reverse primers, respectively. the pcr amplification was performed using conditions of 94 8c/30 s, 52 8c/45 s, 72 8c/1 min for 35 cycles and a final extension of 72 8c/5 min. pcr product was gel purified and subjected to restriction digestion using ndei and hindiii and subsequently cloned at same sites of pet21b. the pet21b-tf1 was subjected to automated dna sequencing. e. coli bl21(de3) competent cells were transformed with pet21b-tf1 to carryout the expression studies. western blot analysis was carried out using three positive (anti-hcv and hcv rna positive) and three negative sera (anti-hcv and hcv rna negative). westerns were also performed employing patients' sera titrated with purified core protein. the deduced aminoacid sequences in +1 reading frame of the standard reference set representing various genotypes were utilized in multiple alignment of f1 sequences. the phylogenetic tree was generated based on alignment using clustalw program (http:// swift.embl-heildelberg.de). analyses of f1 coding à2/+1 reading frame indicated a protein product of 142aa in the indian isolate ay051292 belonging to genotype 1c. different genotypes of hcv were reported to code for varied lengths of f1-the genotype 1a encoded 162aa, 1b coded for 144aa and 2a coded for 126aa fig. 1 . nucleotide sequence of clone pet21b-tf1 with deduced aminoacids. the deduced aminoacid sequence of f1 is represented in red and the sequence in black indicate the aminoacids derived from pet21b plasmid. the sequence in bold represents the sequence of cloning sites. fig. 2 . phylogenetic analysis based on deduced aminoacid sequences of f1 of various genotypes of hcv. the phylogenetic analysis was done using clustalw and the tree was constructed using treeview program. scale bar shows number of nucleotide substitutions per site. (kolykhalov et al., 1997; lohmann et al., 1999; yanagi et al., 1999; xu et al., 2001) . pcr amplification product of $370 bp region coding for truncated f1 (tf1) was cloned at ndei and hindiii sites of pet21b. the clone pet21b-tf1 released a fragment of $370 bp upon double digestion with ndei and hindiii. the clone having the tf1 insert when subjected to sequencing revealed the presence of f1 coding sequence (fig. 1) . the deduced aa of tf1 sequence subjected to blast search exhibited domain based identity of 74-77% with poco45, a core frame shift product of hcv isolate h belonging to type 1a. sequence alignment of deduced aa of f1 belonging to different genotypes displayed substantial diversity in f1 sequences. despite these variations, presence of various conserved aa clusters in f1 indicates the conserved nature of its secondary structure among isolates. phylogenetic analysis of f1 showed close clustering of sequences belonging to various subtypes of specific genotype (fig. 2) implicating that f1 sequences are genotype specific. motif search analysis of f1 revealed the presence of caesin kinase 2-phosphorylation site; protein kinase c-phosphorylation site and ldl class b (ldlrb) receptor binding site. the function of f1 protein in the life cycle of hcv remains unknown (baril and brakier-gingras, 2005) . presence of a binding site for ldlrb indicates the possibility of interaction of f1 with lipids in the natural course of infection. the protein expressed upon iptg induction yielded $17 kd band which was absent in un-induced samples (fig. 3a) . the expressed protein tf1 was in the insoluble fraction as inclusion bodies. inclusion bodies were purified by washing the pellet after lysis using 0.1% triton-x100 and subsequently the pellet was dissolved in phosphate buffer (ph 8.0) containing 0.5% sodium laural sarcosine (sls). the partially purified tf1 was employed in western blot analyses. three hcv positive sera employed in western analysis showed the presence of antibodies to tf1, while sera from uninfected individuals failed to give any signals (fig. 3b) . similar results were observed with patients' sera titrated with purified core protein (fig. 3c ). purified core protein was electro-transferred on to nitrocellulose membrane from sds-page. several strips of the membrane containing purified core protein were used to titrate out anti-core antibodies in three different positive sera. finally a western blot without a signal for purified core protein, when titrated sera were used, confirmed the absence of anticore antibodies. the positive signal observed for f1 in western blot analysis with hcv infected sera and its absence with uninfected sera suggests that f1 protein is plausibly synthesized in the natural course of hcv infection in indian patients as well. an overview of the results amply indicates that f1 protein is also synthesized in the natural course of hcv in indian patients. phylogenetic analysis of f1 of various hcv isolates revealed that aminoacid sequences of f1 are genotype specific. 1n, 2n, 3n : three different negative sera were used as negative controls. (c) western blot analysis of tf1 using patients' sera titrated with purified c120. 1p, 2p, 3p: three different positive sera used as primary antibody after titration with purified core protein. n represents negative sera and p for positive sera. establishment of the presence of antibodies to f1, in this investigation, emphasizes the need for further studies dealing with the role of f1 in hcv pathogenesis. recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome programmed ribosomal frameshifting in decoding the sars-cov genome non-a, non-b hepatitis and the anti-hcv assay translation of the f protein of hepatitis c virus is initiated at a non-aug codon in a +1 reading frame relative to the polyprotein functional properties of a 16 kda protein translated from an alternative open reading frame of the core encoding genomic region of hepatitis c virus unusual multiple recoding events leading to alternative forms of hepatitis c virus core protein from genotype 1b triple decoding of hepatitis c virus rna by programmed translational frameshifting expression and identification of hepatitis c virus polyprotein cleavage products transmission of hepatitis c by intrahepatic inoculation with transcribed rna comparative studies of the core gene products of two different hepatitis c virus isolates: two alternative forms determined by a single amino acid substitution differential subcellular localization of hepatitis c virus core gene products replication of subgenomic hepatitis c virus rnas in a hepatoma cell line internal ribosome entry site-mediated translation in hepatitis c virus replication classification, nomenclature, and database development for hepatitis c virus (hcv) and related viruses: proposals for standardization characterization of the expression of the hepatitis c virus f protein viral heterogeneity of hepatitis c virus processing and functions of hepatitis c virus proteins molecular biology of hepatitis c virus alternate translation occurs within the core coding region of the hepatitis c viral genome evidence for a new hepatitis c virus antigen encoded in an overlapping reading frame synthesis of a novel hepatitis c virus protein by ribosomal frame shift hepatitis c virus f protein is a short lived protein associated with the endoplasmic reticulum hepatitis c virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras msk is highly grateful to the csir, govt. of india, new delhi, for the award of fellowship. authors extend their thanks to prof. t. papi reddy, former head, department of genetics, o.u, for his helpful suggestions in improving the manuscript. key: cord-305263-fgwf6wy3 authors: wang, ben x.; fish, eleanor n. title: the yin and yang of viruses and interferons date: 2012-02-07 journal: trends immunol doi: 10.1016/j.it.2012.01.004 sha: doc_id: 305263 cord_uid: fgwf6wy3 interferons (ifns)-α/β are critical effectors of the innate immune response to virus infections. through activation of the ifn-α/β receptor (ifnar), they induce expression of ifn-stimulated genes (isgs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. many highly pathogenic viruses have evolved mechanisms to evade an ifn response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. here, we discuss ifns as broad-spectrum antivirals for treatment of acute virus infections. in particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (daas) have limited utility due to daa-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak. interferons (ifns)-a/b are critical effectors of the innate immune response to virus infections. through activation of the ifn-a/b receptor (ifnar), they induce expression of ifn-stimulated genes (isgs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. many highly pathogenic viruses have evolved mechanisms to evade an ifn response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. here, we discuss ifns as broad-spectrum antivirals for treatment of acute virus infections. in particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (daas) have limited utility due to daa-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak. ifns-a/b: host-derived broad spectrum antivirals virus infections range from mild and benign to highly virulent epidemics and pandemics, and significantly affect global health. daas, which target specific steps of virus replication, and vaccines, are currently the most effective therapeutic intervention strategies used against virus infections. newly emerging or re-emerging viruses that have undergone mutations may, however, be resistant to the effects of daas, whereas vaccines require that the virus strain be identified before vaccine production, precluding their use at the onset of any new virus infection outbreak. broad-spectrum antivirals, capable of modulating the innate immune response regardless of the infecting virus, present as ideal candidates as a first-line treatment for acute virus infections such as respiratory tract or sexually transmitted infections. ifns-a/b are produced by plasmacytoid dendritic cells (pdcs), macrophages, fibroblasts and endothelial cells, and are critical effectors in an innate immune response to virus infections [1] . ifns-a/b are induced following pattern recognition receptor (prr) activation by viruses (box 1, as reviewed in [2] ) and target many different stages of viral replication: for example, viral entry, envelope uncoating, genome replication, protein assembly, and release of viral progeny [3, 4] . ifns-a/b also activate different cell types in the immune system to promote viral clearance and induce apoptosis of cells to prevent viral replication [1, 3] . as host-derived innate immune response factors, ifns are, therefore, broad-spectrum antivirals, crucial for the primary host response to viral infection. ifns-a/b bind to and activate the ifnar complex, resulting in the rapid induction of transcription and translation of isgs ( figure 1 , as reviewed in [3] ). notably, ifnars are ubiquitously expressed on all cell lineages; probably an evolutionary consequence of different viruses being able to target and infect different cell types. ifnar expression on any and all cell types ensures that an ifn response to a virus may be induced upon infection. as an ifn response is central to a robust innate immune response, viruses have evolved a variety of mechanisms to interfere with ifn production and signaling, to disrupt innate host antiviral factors. successful viral clearance is determined by the balance between virus-encoded molecules that antagonize the host innate immune response and the robustness of the host innate immune response. here, we discuss how ifn therapy presents as a viable treatment option for a range of acute virus infections that target a variety of tissues, including respiratory tract infections by severe acute respiratory syndrome coronavirus (sars-cov) and influenza a viruses, infections of the liver by hepatitis c virus (hcv) and hepatitis b virus (hbv), and mucosal infections by herpes simplex virus (hsv). this may be particularly important given the paucity of broad-spectrum antivirals for treating newly emerging and re-emerging virus infections, which present a major threat to human health. yin: ifn activates the immune system in addition to the induction of isg expression in all cell types, ifns shape the landscape of the immune system in response to virus infection by promoting neutrophil survival [3, 5] and the activation of macrophages [6] , natural killer cells [7] , dcs [1, 8] , b cells [9] and cd8 + t cells [1] , and t helper (th)1 polarization of effector cd4 + t cells [8] ( figure 2 ). ifn therapy therefore has the advantage over daa treatments in that, in addition to stimulating genes that block viral replication in infected cells, ifns activate other innate and adaptive immune responses to combat the virus. polymorphisms in genes encoding factors involved in different stages of the ifn response can lead to marked differences in susceptibility to virus infection and severity of disease, and can also serve as predictive markers for the outcome of ifn treatment. for example, polymorphisms in host genes encoding proteins associated with regulation of an ifn response such as interferon receptor a-chain (ifnar1) [10] , the ifn-inducible myxovirus resistance gtpase protein, mx [11] , the ifn-inducible 2 0 ,5 0 -oligoadenylate synthetase (oas) [12] and the suppressor of cytokine signaling (socs) 3 associated with regulation of an ifn response [13] , are predictive markers linked with the rate of sustained virological response (svr) to hcv infection following ifn-a treatment. this highlights the importance of an intact ifn response during viral infection and indicates that genetic variations among patients can present as a challenge for optimizing ifn therapy. yang: virulence factors antagonize the ifn response it is not surprising that many pathogenic viruses, including sars-cov, influenza a viruses, hcv and hsv, have developed mechanisms to disrupt and limit the ifn-a/b response (table 1) . many viruses are able to evade the innate immune system by directly targeting pathways required for the induction of ifn-a/b production [14] [15] [16] [17] [18] . these viruses are also able to inhibit an ifn response, by interfering with effectors in ifn-inducible signaling cascades [19] [20] [21] . understanding the basis of these antagonistic mechanisms is essential for optimizing the timing and dosage of ifn treatments as a viable therapy for acute virus infections. box 1. ifn-a/b induction by viral pathogen-associated molecular patterns (pamps) prrs, including membrane-associated toll-like receptors (tlrs), and cytosolic rna and dna sensors such as rig-i, are able to detect both extracellular, endosomal or cytosolic viral pamps: viral genomic material. the primary outcome of this nonspecific surveillance system that detects any and all viruses, regardless of the target cell or tissue tropism of the virus, and independent of where the virus is located, is the transcriptional activation of ifn-a/b genes and the rapid production and secretion of these ifns. upon viral pamp recognition, prrs are able to trigger a phosphorylationdependent signaling cascade to activate irf3 and/or irf7. for instance, endosomal rna/dna-sensing tlr7 and tlr9 activate irf7 via myeloid differentiation primary response gene 88 (myd88), whereas endosomal dsrna-binding tlr3 and rig-i, are able to activate irf3 and irf7 through tbk1 and inhibitor of ikke. irf3 and irf7 activation results in their nuclear translocation where they act as transcription factors and up-regulate ifn-a/b gene expression. ifns-a/b bind with high affinity to the ifnar complex, composed of an a-chain, ifnar1, which is structurally modified by cell membrane glycosphingolipids, galabiosylceramide (gb2) and globotriaosylceramide (gb3) to promote efficient ifn binding [71, 72] , and a b-chain, ifnar2. ifn binding to ifnar induces phosphorylation of the receptor-bound tyrosine kinases, tyrosine kinase 2 (tyk2) and jak1, leading to the subsequent regulation of: (a) protein synthesis via the activation of pi3k and mammalian target of rapamycin (mtor); (b) histone modification, via the activation of p38 mapk; and (c) gene expression via the phosphorylation of stat proteins and mapk activation. tyk2-and jak1-mediated activation of insulin receptor substrate (irs) 1 and irs2 is required for recruitment and activation of pi3k. pi3k phosphorylates akt, which inactivates inhibitors of mtor, tuberous sclerosis protein (tsc) 1 and tsc2 [73] . mtor activates the serine/ threonine kinase p70s6k, and inactivates eukaryotic translation initiation factor 4e-binding protein 1 (4e-bp1), to upregulate cap-dependent mrna translation and protein synthesis. p38 is activated downstream of mapk kinases 3 and 6 (mkk3/6) and regulates histone modification and gene expression through mitogen-and stress-activated protein kinase (msk) 1 and msk2. in addition, ifn signaling invokes promyelocytic leukemia zinc finger (plzf) protein-mediated histone modification to regulate isg expression [74] . phosphorylation of stat proteins results in their dimerization and ifn-stimulated gene factor (isgf) formation. stat complexes translocate to the nucleus and bind to specific gene elements in the promoters of isgs, ifn-g activated sequence (gas) and ifn-sensitive response element (isre), to induce expression of antiviral genes. ifn therapy as a first-line treatment against newly emerging or re-emerging virus outbreaks: sars-cov the sars-cov outbreak originated in hong kong in late 2002-2003 and resulted in >8000 cases of disease worldwide, with a 9.6% mortality rate between november 2002 and july 2003 (http://www.who.int/csr/sars/country/ table2004_04_21/en/index.html). sars-cov is a singlestranded rna virus that encodes in its genome virulence factors that antagonize the ifn-a/b response. in infected host cells, sars-cov expresses the nonstructural protein (nsp) 1 and nsp3. nsp1 suppresses host gene expression by disrupting mrna translation and by upregulating mrna degradation [14, 22] . immunoprecipitation and luciferase reporter studies have shown that nsp1 directly associates with the 40s ribosomal subunit to inhibit its translational activity [22] . in addition, the nsp1-40s complex is able to modify mature 5 0 -capped rnas to limit translation and promote degradation [22] . in the context of an ifn response, these antagonistic mechanisms of nsp1 on host gene expression and protein synthesis inhibit ifn-a/b expression and production [14] . in vitro, nsp1 also inhibits signal transducer and activator of transcription (stat) 1 protein phosphorylation induced by ifn-a treatment [23] . both nsp1 and nsp3 inhibit interferon regulatory factor (irf) 3 and irf7 activation to downregulate ifn production in response to viral infection [23, 24] . specifically, nsp3 inhibits irf3 in human bronchial epithelial cells via its papain-like protease (plp) domain, which [4, 76] , tripartite motif-containing protein 5a (trim5a) and trim22, which are antiviral factors that limit hiv-1 infection [77] , and transcription factor jun-d (jund) and claudin 4 (cldn4) [78] . ifn treatment primes cells for apoptosis by modulating the expression of proteasome subunits, major histocompatibility complex (mhc) class i, and fas receptor (cd95) [79] [80] [81] [82] . ifns-a/b also contribute to the activation and differentiation of cells involved in the (b) innate and (c) adaptive immune responses to virus infection. ifn-a/b induces production of interleukin (il)-6, il-12, and il-15 by dcs, and il-10 by macrophages to modulate b and t cell differentiation (th1 polarization) and activation [79] . ifn-b signaling in pdcs leads to altered cd69 and sphingosine-1-phosphate 4 (s1p4) receptor expression, thereby affecting pdc retention in lymph nodes [83] . ifns-a/b increase mhc class ii, cd40 and cd86 expression on antigen presenting cells. ifn-a/b treatment induces macrophage and neutrophil phagocytosis [79, 84] . moreover, ifns-a/b promote neutrophil survival by activating cellular inhibitor of apoptosis 2 (ciap2) [5] . natural killer (nk) cells respond to ifns-a/b with increased fas ligand (fasl) and perforin expression, and ifn-g production [85, 86] . in response to ifns-a/b, b cells upregulate l-selectin and igg production [9, 79] . trends in immunology april 2012, vol. 33, no. 4 interacts with irf3 to inhibit irf3 phosphorylation and nuclear translocation [24] . in addition to nsp1 and nsp3, the open reading frame 6 (orf6) and matrix (m) proteins of the sars-cov also inhibit an ifn response [19, 25] . orf6 localizes to the host endoplasmic reticulum (er) and blocks the transcription factor function of phosphorylated stat1, by binding to nuclear import factors to prevent its translocation to the nucleus [19] . the m protein interacts with rna sensor retinoic acid-inducible gene 1 (rig-i), an rna helicase and key intracellular prr associated with induction of irf-dependent ifn production following detection of viral rnas. the m protein also interacts with the signaling effectors serine/threonine-protein kinase 1 (tbk1), inhibitor of nuclear factor-kb kinase subunit e (ikke), and tumor necrosis factor (tnf)-associated factor 3 (traf3), again associated with ifn gene induction [25] . thus, there are multiple mechanisms by which sars-cov might inhibit the host ifn response. the implications are that an ifn response to sars-cov infection must be dramatically limited for virus replication to proceed, suggesting that the dominant immune response is the ifn response. different recombinant ifn-as and ifn-b are now approved for various clinical indications, including the treatment of chronic hcv infections [26, 27] . through a comprehensive analysis of how structural features in the ifn-a/b moleculescrucial clusters of amino acidsaffect the sensitivity of target cells to ifn-induced biological responses, specific epitopes on the exposed surface of the ifn molecule have been identified that are associated with receptor recognition [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] . accumulating evidence suggests that the affinity of a particular ifn-a/b subtype for ifnar determines the biopotency of the ifn, specifically in the context of antiviral and antiproliferative responses [28, 39] . a direct consequence of this was the design and development of a synthetic ifn-a, ifn alfacon-1, that exhibits optimized affinity for ifnar [40] [41] [42] [43] . initially, treatment for sars-cov infection focused on the use of a daa, ribavirin, in combination with corticosteroid therapy [44, 45] . however, in a pilot clinical study, the therapeutic potential of ifn alfacon-1 was evaluated in individuals infected with sars-cov and hospitalized in toronto, canada [46] . ifn alfacon-1 treatment together with corticosteroids is associated with reduced disease-associated impaired oxygen saturation, more rapid resolution of radiographic lung abnormalities and lower levels of disease-associated creatine kinase. in vitro studies to examine the mechanism of action of ifn against the sars-cov have revealed that ifn-inducible janus kinase 1 (jak1), protein kinase c (pkc)-d and p38 mitogen-activated protein kinase (mapk) activation mediate ifn antiviral protection. target genes downstream of activation of these kinases are differentially expressed in the peripheral blood cells of sars patients treated with ifn alfacon-1 compared with patients not treated with ifn, and functionally these genes are associated with antimicrobial activity [47] . treatment of a human bronchial epithelial cell line, calu-3, with ifn alfacon-1 before infection with the sars-cov results in inhibition of virus infection and a reduction in overall virus yield, further supporting the idea that ifn alfacon-1 demonstrates antiviral activity against the sars-cov [40] . these data demonstrate that despite the inherent ability of the sars-cov to inhibit ifn production and limit an ifn response, treatment with exogenous ifn-a overrides these inhibitory effects. these results support the further evaluation of ifn alfacon-1 as a first-line treatment for acute sars-cov infection and approved inhibits the function of pkr and 2 0 ,5 0 -oas via its dsrna-binding domain. [64] review trends in immunology april 2012, vol. 33, no. 4 randomized clinical trial protocols are in place in the usa and canada should there be outbreaks of sars-cov. seasonal influenza a virus infections are a considerable health burden and vaccine programs are currently implemented in most developed countries. vaccines, however, are not relevant during an outbreak involving an emergent variant. the 2009 h1n1 swine-origin influenza a virus is a prime example of how quickly a pandemic can develop given the potential for genetic shift and mutation of influenza a viruses among natural hosts. during the 2009 h1n1 pandemic, daas such as the neuraminidase inhibitors oseltamivir and zanamivir were widely used before a vaccine became available [48] . not surprisingly, however, daa-resistant variants of pandemic h1n1 emerged [48, 49] . avian h5n1 influenza virus outbreaks, now affecting populations throughout asia and europe, are associated with mortality rates around 60% [50] . notably, a number of h5n1 strains are resistant to oseltamivir [51] . to date, there have been no reported cases of human-to-human transmission of this lethal h5n1 influenza virus infection, but if a newly emerging strain capable of human-to-human transmission appears, daa resistance will develop and until a vaccine becomes availableprobably 4-6 monthspopulations will be at risk in the absence of access to broad-spectrum antivirals. ns1 is the primary virulence factor encoded by influenza a viruses and it is expressed in host cells during the earliest stages of infection [15] . in comparison with sars-cov nsp1, influenza virus ns1 has both overlapping functions as well as unique mechanisms to inhibit the ifn response. ns1 acts both in the nucleus and cytoplasm of an infected cell, and is the primary antagonist of the host innate immune response. remarkably, ns1 has evolved to inhibit virtually all stages of the ifn response to virus infection, including inhibition of ifn production, interference with ifn signaling events, and inhibiting the function of antiviral factors induced by ifn signaling. ns1 inhibits the activity of rig-i (box 1) where the ns1 dsrna-binding domain interacts directly with rig-i [15] . within the nucleus of an infected cell, ns1 inhibits the processing and synthesis of host mrnas, including ifn-a/b mrnas, by binding to and inhibiting both cleavage and polyadenylation specific factor 30 kda (cpsf30) and poly(a)-binding protein ii (pabpii), via its proteinbinding domain [15] . the expression of avian h5n1 ns1 disrupts ifn signaling events by downregulating the surface expression of one of the ifnar subunits, ifnar1, and by upregulating socs1 protein expression, leading to a reduction in ifn-inducible stat phosphorylation and stat homo/heterodimer nuclear translocation [20] . ns1 is able to block directly the antiviral activities of ifninducible antiviral proteins such as protein kinase rnaactivated (pkr) and 2 0 ,5 0 -oas/rnasel, via its proteinbinding domain and dsrna-binding domain, respectively [15] . the src homology 2 (sh2)-binding domain within the protein-binding region of ns1 permits interaction with the internal sh2 domain of p85b, the inhibitory subunit of phosphoinositide 3-kinase (pi3k). this leads to activation of the pi3k-akt pathway [15] . activation of pi3k, a downstream target of ifn-a/b signaling, by a specific ns1 promotes cell survival during the early stages of infection, illustrating the complex interplay between virus encoded factors and the ifn-a/b response [15] . remarkably, distinct highly pathogenic respiratory viruses, namely influenza viruses and the sars-cov, encode nonstructural proteins in their genomes that function as virulence factors that specifically target the host innate ifn response, further emphasizing the importance of ifns as broad-spectrum antivirals. a recently completed randomized controlled trial has examined the safety and efficacy of recombinant ifn-a (rifn-a) treatment, administered in the form of a nasal spray, in military recruits, in the context of protection from respiratory virus infections [52] . serum igm levels were measured as evidence of virus infection. subjects receiving rifn-a had lower concentrations of serum igm specific for h3n2 influenza a virus, influenza b virus, adenovirus (species b), and parainfluenza virus types 1, 2 and 3 [52] . specifically with regard to influenza a virus, only 30 recruits treated with rifn-a had detectable levels of influenza a virus igm compared with 104 recruits in the untreated control group [52] . no adverse events were reported in the treatment group, the data demonstrating that ifn was well tolerated and was effective in preventing a variety of common viral respiratory infections. thus as for the sars-cov, the implications are that treatment with ifn-a can override the inhibitory effects of ns1 on an ifn response during influenza a virus infection. ifn therapy for highly pathogenic and oncogenic viral infections: hbv and hcv worldwide, >170 million people are infected with hcv, resulting in approximately 350 000 deaths each year (http://www.who.int/mediacentre/factsheets/fs164/en/). more than an estimated 350 million people are chronically infected with hbv, resulting in approximately 600 000 deaths each year (http://www.who.int/mediacentre/factsheets/fs204/en/). hcv and hbv target the liver and cause both acute and chronic infections, resulting in liver cirrhosis and eventually, hepatocellular carcinoma [16, 53] . the current approved standard-of-care treatment for hcv infection comprises daily ribavirin in combination with weekly pegylated ifn-a (peg-ifn-a) . the covalent linkage of polyethylene glycol to ifn-a increases the halflife of ifn-a in the circulation. common side effects associated with ifn therapy include a range of flu-like symptoms (fatigue, fever, myalgia), that often diminish spontaneously during the first few weeks of therapy. more severe neuropsychiatric disturbances including sleep disturbances and depressive mood changes have their onset within the first months of ifn therapy. hematological disturbances such as neutropenia or anemia may occur and are responsive to ifn dose reduction or treatment [granulocyte colony-stimulating factor (g-csf), erythropoietin (epo), respectively]. this combination ifn/ribavirin therapy has been very successful in patients infected with hcv genotypes 2 or 3, and 70-90% of patients go on to achieve an svr, characterized by undetectable hcv rna following 24 weeks of treatment [53] . the rate of svr falls to 40-60% in patients infected with hcv genotypes 1 or 4, trends in immunology april 2012, vol. 33, no. 4 following 48 weeks of treatment with peg-ifn-a and ribavirin [53] . the incomplete response to ifn treatment is partially attributable to virally encoded virulence factors that interfere with an ifn response: ns3/4a and ns5a. ns3/4a is an hcv serine protease that targets mitochondrial antiviral signaling (mavs) proteins required for rig-i-mediated irf3 activation and subsequent ifn production [16] . recently, the secondary structure of the hcv genotype 1b ns3 n-terminal region was identified as a predictive marker for the virological response in patients who had received ifn and ribavirin combination therapy for 48 weeks. specifically, polymorphisms in the secondary structure of the ns3 amino-terminal region segregate hcv genotype 1b infected individuals into two groups and are predictive of the virological response to peg-ifn plus ribavirin therapy [54] . ns5a associates with intracellular membranes and its expression is vital for hcv genome replication. ns5a is able to interact with ifn-inducible pkr to evade an ifn-induced antiviral response. polymorphisms in amino acid residues 70 and 91 in the hcv core and in ns5a are also predictive markers of the virological response in patients receiving ifn and ribavirin therapy [55] . notably, ns5a is a target of ifn, because the ifn-activated gene 15 (ifi15)/interferon stimulated gene (isg15) encoding a 17-kda protein, promotes isgylation of ns5a to enhance its degradation, thereby inhibiting hcv replication [56] . in addition to hcv ns5a, both the hcv envelope protein e2 and the internal ribosome entry site (ires) are able to inhibit ifn-inducible pkr activity [57] . e2 contains a eukaryotic translation initiation factor 2 (eif-2a) phosphorylation homology domain through which it is able to interact with pkr, whereas the hcv ires binds to pkr, precluding dsrna binding, thereby preventing pkr activation [57] . despite these potent inhibitory effects of hcv-encoded factors on an ifn response, clinical data provide direct evidence that peg-ifn-a treatment in combination with ribavirin is effective at limiting hcv infection and, dependent on the hcv genotype, may invoke a svr. the mechanisms by which hbv evades an innate immune response are less well understood. the hbv polymerase (pol) blocks irf signaling and subsequent ifn production by inhibiting tbk1/ikke activity, associated with prr signaling [17] . this inhibition is mediated by direct protein-protein interactions between pol and the host dead box (d-e-a-d amino acid sequence motif) rna helicase, ddx3, that enhances tbk1/ikke activity [17] . peg-ifn-a is an effective treatment for hbv infection, again suggesting that ifn treatment can overcome virus-imposed inhibition of the innate immune response, specifically ifn production. peg-ifn-a is an effective treatment option for hepatitis b e core antigen (hbeag)positive disease, where detection of hbeag in the blood is indicative of viral replication. up to 40% of hbeag-positive patients treated with peg-ifn-a are able to develop hbeag-specific antibodies (seroconversion) by 6 months after the end of treatment. this percentage rises to 60% at 5 years after the end of treatment [58] . in comparison to monotherapy with the daa lamivudine, which can lead to the emergence of mutant lamivudine-resistant hbv strains, peg-ifn-a alone or in combination with lamivudine is up to 50% more effective for inducing hbeag seroconversion, although more side effects are reported in patients receiving peg-ifn-a [59] . in contrast to hbeag-positive disease, peg-ifn-a, alone or in combination with ribavirin, has limited effect in patients with late stage hbeag-negative disease, where the hbv mutation has resulted in loss of hbeag expression [60] . in a randomized clinical trial, the percentage of hbeag-negative patients with hbv dna levels <10 000 copies/ml, receiving peg-ifn-a monotherapy, dropped from 36% to 20%, from the end of treatment to 24 weeks later [60] . the stage of viral disease can therefore affect the efficacy of ifn therapy and the timing of treatment contributes to the capacity to resolve an infection. moreover, as for influenza viruses and sars-cov, despite hbv and hcv encoding viral factors that antagonize an ifn response, exogenous ifn therapy has proven to be an effective treatment for establishing an svr. ifn therapy for highly transmissible viral infections: hsv-1 hsv-1 is a highly contagious virus, prevalent among sexually transmitted infections. hsv-1 is able to establish a latent infection in immunocompetent individuals by evading the immune system and is only reactivated when the host immune system is weakened [61, 62] . the hsv-1 genome encodes a number of virulence factors, namely infected cell protein (icp) 34.5, icp0 and icp27, which are associated with immunoevasion and suppression of the innate immune response to virus infection [18, 21, 63, 64] . specifically, icp34.5 dephosphorylates eif-2a to reverse pkr-mediated inactivation of eif-2a [63] , icp0 localizes to the cytoplasm and inhibits irf3 activity [18] , and icp27 blunts the ifn-inducible jak-stat signaling pathway by inhibiting ifn-inducible stat1 phosphorylation and nuclear translocation [21] . furthermore, hsv structural protein us11, which has a dsrna-binding domain, disrupts the activation of the ifninducible antiviral proteins 2 0 ,5 0 -oas and pkr [64] . for immunocompromised individuals infected with hsv-1, viral pathogenesis can lead to serious life-threatening disease; more so in the context of emergent drug-resistant hsv-1 strains [65] [66] [67] . different daas have been used to control hsv-1 infection, including acyclovir, penciclovir and foscarnet, resulting in the emergence of daa-resistant hsv-1 strains [65] [66] [67] . ifn-g is able to exert antiviral activity by stimulating a t cell response. however, ifn-g alone may have limited efficacy in immunocompromised hsv-1-infected individuals lacking a robust adaptive immune response. recent studies have shown that when immunocompromised nude mice are infected with a daa (acyclovir)-resistant hsv-1 variant and treated with ifn-b in combination with ifn-g, viral infection is reduced [65] . these preliminary data are in further support of the broadspectrum antiviral activities of ifns-a/b. shifting the balance to favor the host innate immune response: the future of ifn antiviral therapy mechanisms for viral evasion of the host immune response include both the expression of many virulence factors by a review trends in immunology april 2012, vol. 33, no. 4 single virus to target different stages of the ifn response, or the expression of a single, highly specialized molecule that alone targets multiple facets of an ifn response. a priori, the widespread existence of these virally encoded virulence factors that target an ifn response highlights the critical role of a robust ifn response to limiting virus infection. the ability of ifns-a/b to target multiple types of viruses at different stages of viral replication, and the ubiquitous expression of ifn receptors on cells that are susceptible to different virus infections with different tissue tropisms, as well as the ability of ifns to activate innate immune cells and influence the adaptive immune response, emphasizes the relevance of ifns-a/b as broadspectrum antivirals. understanding the viral strategies for evasion of an ifn will permit the design of strategic ifn treatment regimens to both protect from and clear virus infections. the opportunity to limit virus infections even in the absence of characterizing the specific infecting virus, a reality during an outbreak of unknown etiology, or during a pandemic of a newly emerging or re-emerging virus strain, has profound implications for global health. indeed, early data indicate that ifn therapy may be effective in treating west nile virus [68] , hemorrhagic yellow fever virus [69] and ebola virus infections [70] . moreover, short-term ifn therapy for an acute virus infection may not invoke the debilitating side effects associated with long-term ifn therapy for chronic infections such as hbv and hcv. preliminary data from pilot clinical trials of ifn treatment for sars-cov and for influenza a viruses showed this to be the case [46, 52] . cognizance of the yin and yang of viruses and ifns opens the door to the widespread clinical application of these broad-spectrum antiviral ifns. type i interferons in host defense pathogen recognition by the innate immune system mechanisms of type-i-and type-ii-interferonmediated signaling ifitm proteins mediate the innate immune response to influenza a h1n1 virus, west nile virus and dengue virus ) type i and type ii interferons delay human neutrophil apoptosis via activation of stat3 and up-regulation of cellular inhibitor of apoptosis 2 reversible inhibition of murine cytomegalovirus replication by gamma interferon (ifn-g) in primary macrophages involves a primed type i ifn-signaling subnetwork for full establishment of an immediate-early antiviral state direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells require a systemic type i interferon response to mature and induce cd4 + th1 immunity with poly ic as adjuvant type i ifn enhances follicular b cell contribution to the t cell-independent antibody response the dinucleotide microsatellite polymorphism of ifnar1 gene promoter correlates with responsiveness of hepatitis c patients to interferon identification of a single nucleotide polymorphism in the mxa gene promoter (g/t at nt -88) correlated with the response of hepatitis c patients to interferon polymorphisms of interferon-inducible genes oas associated with interferon-a treatment response in chronic hbv infection elevated expression and polymorphisms of socs3 influence patient response to antiviral therapy in chronic hepatitis c severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells the multifunctional ns1 protein of influenza a viruses mavs dimer is a crucial signaling component of innate immunity and the target of hepatitis c virus ns3/4a protease hepatitis b virus polymerase blocks pattern recognition receptor signaling via interaction with ddx3: implications for immune evasion cellular localization of the herpes simplex virus icp0 protein dictates its ability to block irf3-mediated innate immune responses severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane influenza virus non-structural protein 1 (ns1) disrupts interferon signaling role for herpes simplex virus 1 icp27 in the inhibition of type i interferon signaling a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex clinical application of interferons interferon: current status and future prospects in cancer therapy definition of receptor binding domains in interferonalpha a three-dimensional model of consensus sequence interferon-alpha domains of interaction between alpha interferon and its receptor components mutational and structural analysis of the binding interface between type i interferons and their receptor ifnar2 mapping of ifn-beta epitopes important for receptor binding and biologic activation: comparison of results achieved using antibody-based methods and alanine substitution mutagenesis interferon-a/b-receptor interactions: a complex story unfolding mutational analysis of the ifnar1 binding site on ifna2 reveals the architecture of a weak ligand-receptor binding site the human type i interferon receptor: nmr structure reveals the molecular basis of ligand binding mutation of the ifnar-1 receptor binding site of human ifn-alpha2 generates type i ifn competitive antagonists human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells the role of three domains in the biologically active configuration of human interferon alpha structural linkage between ligand discrimination and receptor activation by type i interferons interferon alfacon 1 inhibits sars-cov infection in human bronchial epithelial calu-3 cells interferon alfacon-1 and ribavirin versus interferon alpha-2b and ribavirin in the treatment of chronic hepatitis c interferon alfacon-1: a review of its pharmacology and therapeutic efficacy in the treatment of chronic hepatitis c the biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon sars: systemic review of treatment effects identification of severe acute respiratory syndrome in canada interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome characterization of the antiviral effects of interferon-alpha against a sars-like coronavirus infection in vitro the potential for multidrug-resistant influenza oseltamivir-resistant pandemic (h1n1) 2009 virus infection in england and scotland avian influenza a(h5n1) in humans: new insights from a line list of world health organization confirmed cases oseltamivir resistance during treatment of influenza a (h5n1) infection a randomized controlled trial of low-dose recombinant human interferon alpha-2b nasal spray to prevent acute viral respiratory infections in military recruits peginterferon and ribavirin treatment for hepatitis c virus infection secondary structure of the amino-terminal region of hcv ns3 and virological response to pegylated interferon plus ribavirin therapy for chronic hepatitis c amino acid substitutions in core and ns5a regions of the hcv genome can predict virological decrease with pegylated interferon plus ribavirin therapy inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns5a inhibition of the protein kinase pkr by the internal ribosome entry site of hepatitis c virus genomic rna durability of peginterferon alfa-2b treatment at 5 years in patients with hepatitis b e antigen-positive chronic hepatitis b peginterferon alfa-2a, lamivudine, and the combination for hbeag-positive chronic hepatitis b a randomized trial of peginterferon alpha-2a with or without ribavirin for hbeag-negative chronic hepatitis b increasing proportion of herpes simplex virus type i as a cause of genital herpes infection in college students herpes simplex virus encephalitis: new infection or reactivation? replication of herpes simplex virus 1 depends on the g 1 34.5 functions that facilitate virus response to interferon and egress in the different stages of productive infection inhibition of cellular 2 0 -5 0 oligoadenylate synthetase by the herpes simplex virus type 1 us11 protein beta interferon plus gamma interferon efficiently reduces acyclovir-resistant herpes simplex virus infection in mice in a t cell-independent manner development of drug-resistant herpes simplex virus infection after haploidentical hematopoietic progenitor cell transplantation herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy use of interferon-a in patients with west nile encephalitis: report of 2 cases treatment of yellow fever virus with an adenovirus-vectored interferon, def201, in a hamster model evaulation of immune globulin and recombinant interferon-a2b for treatment of experimental ebola virus infections evidence for glycosphingolipid modification of the type 1 ifn receptor a structural basis for interferon-a-receptor interactions tsc2 is phosphorylated and inhibited by akt and suppresses mtor signalling promyelocytic leukemia zinc finger protein regulates interferon-mediated innate immunity the isg56/ifit1 gene family distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus association of trim22 with the type 1 interferon response and viral control during primary hiv-1 infection interferon-inducible stat2 activation of jund and cldn4: mediators of ifn responses direct effects of type i interferons on cells of the immune system virus-induced type i ifn stimulates generation of immunoproteasomes at the site of infection ifn-a secretion by type 2 predendritic cells upregulates mhc class i in the hiv-1-infected thymus involvement of fas-mediated apoptosis in the inhibitory effects of interferon-alpha in chronic myelogenous leukemia dynamic accumulation of plasmacytoid dendritic cells in lymph nodes is regulated by interferon-b effect of interferon-alpha(2a) on neutrophil adhesion and phagocytosis in chronic myeloid leukemia and behçet's disease ifnalpha regulates nk cell cytotoxicity through stat1 pathway induction of interferon-gamma from natural killer cells by immunostimulatory cpg dna is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha inhibition of the interferon-inducible protein kinase pkr by hcv e2 protein key: cord-302295-nblmshni authors: savva, athina; roger, thierry title: targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases date: 2013-11-18 journal: front immunol doi: 10.3389/fimmu.2013.00387 sha: doc_id: 302295 cord_uid: nblmshni toll-like receptors (tlrs) are pattern recognition receptors playing a fundamental role in sensing microbial invasion and initiating innate and adaptive immune responses. tlrs are also triggered by danger signals released by injured or stressed cells during sepsis. here we focus on studies developing tlr agonists and antagonists for the treatment of infectious diseases and sepsis. positioned at the cell surface, tlr4 is essential for sensing lipopolysaccharide of gram-negative bacteria, tlr2 is involved in the recognition of a large panel of microbial ligands, while tlr5 recognizes flagellin. endosomal tlr3, tlr7, tlr8, tlr9 are specialized in the sensing of nucleic acids produced notably during viral infections. tlr4 and tlr2 are favorite targets for developing anti-sepsis drugs, and antagonistic compounds have shown efficient protection from septic shock in pre-clinical models. results from clinical trials evaluating anti-tlr4 and anti-tlr2 approaches are presented, discussing the challenges of study design in sepsis and future exploitation of these agents in infectious diseases. we also report results from studies suggesting that the tlr5 agonist flagellin may protect from infections of the gastrointestinal tract and that agonists of endosomal tlrs are very promising for treating chronic viral infections. altogether, tlr-targeted therapies have a strong potential for prevention and intervention in infectious diseases, notably sepsis. sepsis is one of the leading causes of death worldwide. incidence of severe sepsis is increasing and mortality rates remain significantly high despite early care management (1) . moreover, more than 30% of survivors develop long-term functional disabilities and cognitive impairments (2) . the surviving sepsis campaign is a global initiative incepted in early 2000s with the aim to improve sepsis diagnosis and treatment in order to enhance the awareness of sequelae and to decrease high mortality rates associated with sepsis 1 . in collaboration with many countries in europe and the united states, the surviving sepsis campaign suggests evidencebased guidelines and bundles. the most recent guidelines recommend acute resuscitation of septic patients, administration of antibiotics and support of organ failure. yet, no treatment targeting the underlying mechanism of sepsis is actually available (3) . recombinant human activated protein c (rhapc, xigris®, eli lilly), the only drug specifically registered for sepsis, has recently been withdrawn from the market following the negative results from the prowess-shock study that did not show reduction in mortality at 28 or 90 days in patients with septic shock (4) . it is generally admitted that sepsis results from a dysregulated host response to an initial insult, characterized by inflammation mediating tissue damage and organ failure and an immune suppression state responsible for the development of secondary infections (5) (6) (7) . the immune response to an infection is initiated by the sensing of microbial structures through families of receptors collectively called pattern recognition receptors (prrs). the most well-described families comprise toll-like receptors (tlrs), nucleotide binding oligomerization domains (nods)-like receptors (nlrs), c-type lectin receptors (clrs, such as dectin-1, dectin-2, dc-sign), rig-i-like receptors (rlrs, rig-i, and mda5), and intra-cytosolic dna sensors. prrs are expressed by innate immune cells like dendritic cells and macrophages. the binding of microbial ligands to prrs promotes the release of mediators, among which cytokines, that initiate and regulate the inflammatory response necessary to eliminate invasive pathogens and coordinate the development of the adaptive immune response (8, 9) . innate immune cells are also triggered by damage (or danger)associated molecular patterns (damps), known as alarmins. damps are endogenous components commonly released by injured or stressed cells, such as nucleic acids, histones, uric acid crystals, atp, cytochrome c, s100 molecules, and hmgb1. damps are primarily sensed through the nlrp3 inflammasome, which controls the secretion of il-1β and il-18 (10) . the concept of prrs sensing microbial-associated molecular patterns (mamps) and discriminating self from non-self molecular structures was proposed by janeway (11) . two major cornerstone discoveries largely confirmed janeway's concept. the first one was the demonstration of the essential antifungal role of www.frontiersin.org the toll protein in drosophila (12) . the second one arose from the positional cloning linking lps (commonly called endotoxin) unresponsive phenotype of c3h/hej and c57bl/10sccr strains of mice to missense and null mutations of the toll-like receptor 4 (tlr4) gene (13) . the importance of these discoveries and of the role of dendritic cells as central regulators of innate and adaptive immunity has been acknowledged by the 2011 nobel prize in physiology or medicine attributed to bruce a. beutler and jules a. hoffmann "for their discoveries concerning the activation of innate immunity" and to ralph m. steinman "for his discovery of the dendritic cell and its role in adaptive immunity" (14) . toll-like receptors belong to the most studied family of prrs, due to their central role in host defenses and involvement in a number of pathological processes that include sepsis. tlrs are type i trans-membrane proteins composed of an extracellular leucine-rich repeat (lrr) domain involved in ligand recognition, a trans-membrane domain, and a toll-interleukin 1 receptor (tir) domain involved in signaling (9, 15) . about 10 functional human tlrs (tlr1-10) and 12 functional mouse tlrs (tlr1-9, tlr11-13) have been described, each one being involved in the sensing of distinct microbial products ( table 1) . expressed at the cell surface, tlr4 detects lps from gram-negative bacteria. tlr4 shuttles to late endosome to induce alternative signaling following lps sensing. tlr2 as heterodimers in association with either tlr1 or tlr6 (and possibly tlr10) senses a variety of microbial products, such as lipopeptides, lipoproteins, peptidoglycan, porins, β-glucan, glycosylphosphatidylinositol (gpi) anchors, and glycoproteins from gram-positive bacteria, gram-negative bacteria, mycoplasma, mycobacteria, fungi, parasites, and viruses. tlr5 senses flagellin of bacterial flagella. tlr3, tlr7, tlr8, and tlr9 are strategically expressed in endosomal compartments to recognize microbial nucleic acids: double-stranded rna (dsrna) by tlr3, single-stranded rna (ssrna) by tlr7 and tlr8, and unmethylated cpg motif containing dna by tlr9 ( table 1) . it is therefore not surprising that endosomal tlrs have been primarily involved in host defenses against viruses, whereas tlr1, tlr2, and tlr4-6 have been mainly involved in host response to bacterial and fungal infection. tlrs cooperate with other molecules to recognize microbial ligands. for example, tlr2 requires cd14, cd36, and dectin-1 for the recognition of peptidoglycan, lipopeptides, and β-glucan, respectively. of note, tlrs are also triggered by damps released by injured or stressed cells during infection (16) ( table 1) . tlr activation enables pathogen elimination by promoting bactericidal activity of leukocytes, and maturation and function of antigen presenting cells, thus orchestrating the development of adaptive immune responses (17) . the signaling pathways resulting from tlr triggering engage adaptors that are recruited by tir/tir domain interactions ( table 1) : myeloid differentiation primary response gene (18) (myd88), tir domain-containing adaptor protein (tirap, also known as mal), tir domain-containing adaptor inducing interferon (ifn)β (trif), and trif related adaptor molecule (tram) (19) . myd88 is essential for signaling through all tlrs except tlr3 and is involved in early nuclear factor-κb (nf-κb) and mitogen-activated protein kinases (mapks) activation and pro-inflammatory gene expression. tirap serves as a bridge to recruit myd88 to tlr2 and tlr4. trif initiates myd88independent ifn regulatory factor 3 (irf3) and late nf-κb activation involved in the production of type i ifns and ifn-inducible genes (19, 20) . trif is recruited to the cytoplasmic domain of tlr3 and, in late endosome, through tram that bridges trif to tlr4. a fifth tir domain-containing adaptor, sterile α-, and armadillo-motif containing protein (sarm) acts as a negative regulator of tlr3 and tlr4 signaling. sarm interacts with trif and inhibits the induction trif-dependent genes. the signaling pathways activated downstream tlrs have some redundancy. yet, the engagement of multiple tlrs, especially myd88 and trif-dependent tlrs, have synergistic effects on host responses (21, 22) . intracellular cross talk between signaling pathways may also occur when different families of prrs are involved. for example, dectin-1 synergizes with tlr2 and tlr4 and increases cytokine production through canonical and noncanonical nf-κb pathways (23, 24) . moreover, a single mamp can be detected by different prrs. this differential sensing is primarily depending on the localization of the mamp. indeed, flagellin is sensed by tlr5 expressed at the cell surface and by the naip5/nlrc4 (also known as ipaf) inflammasome when localized in the cytoplasm (25) (26) (27) . similarly, peptidoglycans stimulate membrane tlr2 and intra-cytosolic nod1/nod2dependent cell activation (28) . interestingly, some cpg and non-cpg oligodeoxynucleotides directly stimulate and polarize t-cells through tlr9 and myd88-independent mechanisms (29), possibly through intracellular dna sensors. recently, hagan et al. and kayagaki et al. demonstrated non-canonical tlr4-independent recognition of intracellular lps through an uncharacterized receptor (30, 31) . this unconventional mode of lps sensing activates caspase-11-dependent il-1β secretion and sensitizes mice to endotoxic shock. all these observations indicate that the host has evolved different strategies to sense invading microorganisms. ideally, all possible interactions should be characterized and/or anticipated, so that the effect of treatment application can be predicted and/or translated. this mandates carefully planned experiments that represent real-life conditions and a detailed knowledge of compound's mode of action. obviously, the redundancy of microbial sensing pathways should be taken into consideration when developing or applying targeted-treatment strategies to a single prr. experimental animal models and human clinical studies support a crucial role for tlrs in infectious diseases. the first evidence came from the observation that tlr4 defective c3h/hej and c57bl/10sccr mice are hyporesponsive to lps and susceptible to otherwise non-lethal infection with escherichia coli and salmonella typhimurium. subsequent studies with mice knockout in tlrs or tlr adaptor molecules have demonstrated the importance of the tlr pathway in host defenses. for example, tlr2 knockout mice are highly susceptible to infections by staphylococcus aureus and streptococcus pneumoniae (32) . more recently, human association studies have linked polymorphisms affecting tlr expression or tlr structure with an augmented propensity to develop infections (32) (33) (34) (35) . the discovery of tlrs and their involvement in innate immune responses has attracted much interest into the development of drugs for controlling infections and improving sepsis management. this field of research has been very dynamic, and (36) . these two particular aspects of the tlr-targeting field will not be addressed in this review. herein, we will review the most popular agonist (tlr3, tlr5, tlr7, tlr8, tlr9) and antagonist (tlr2, tlr3, tlr4, tlr9) agents used in pre-clinical and clinical models of acute and chronic infections, including sepsis. relevant registered clinical trials 2 are listed in table 2 . frontiers in immunology | microbial immunology lps is the main pro-inflammatory molecule anchored in the outermembrane of gram-negative bacteria (51) . neutralization of bacterial lps, inhibition of its recognition by host cells or inhibition of signaling downstream lps binding to its receptor has long been considered a promising approach for the treatment of severe sepsis and septic shock. interestingly, endotoxemia is prevalent in septic patients, not only in those with gram-negative infection. indeed, translocation of viable bacteria and lps from the gastrointestinal tract has been proposed to participate in the pathophysiology of sepsis. tlr4 was identified 15 years ago as the signal-transducing molecule of the lps receptor complex (13) , which also comprises md-2 and cd14. thus, tlr4 is regarded as a primary target for treating sepsis (52) . tlr4 expression is increased in human monocytes of healthy volunteers challenged with lps (53), as well as in patients with sepsis (54) . moreover, polymorphisms in the tlr4 gene have been associated with gramnegative sepsis (33, 35) . in the following sections, we present the most advanced tlr4 antagonists developed for the treatment of sepsis. strategies to inhibit lps-mediated toxic effects have been initiated years before the discovery of tlr4 (13) and the unraveling of the crystal structure of the tlr4-md-2-lps complex (55) . lipid a, the toxic moiety of lps, is highly conserved among endotoxins and constitutes an ideal therapeutic target (56) . e5531, developed by eisai research institute of boston (andover, ma, usa), was the first-generation lipid a antagonist derived from rhodobacter capsulatus endotoxin. e5531 conferred protection in experimental models of endotoxemia and lethal infection with e. coli (57) . the protective effect likely occurred through the binding of e5531 to the tlr4-md-2 complex and the inhibition of the interaction between lps and tlr4-md-2 (58) . e5531 also blocked endotoxin response in human healthy volunteers challenged intravenously with lps (59). e5531 development went through phase 2 clinical trial, but was stopped due to issues of bioavailability. a second-generation lps antagonist drug candidate developed by eisai is eritoran tetrasodium (known as eritoran or e5564), a synthetic lipid a analog of rhodobacter sphaeroides (60) . eritoran blocked lps-induced cytokines in vitro and in experimental animal models (61) (62) (63) . in a phase 1 clinical trial enrolling healthy volunteers challenged with lps, eritoran inhibited pro-inflammatory cytokine production and diminished clinical symptoms of sepsis, including fever, chills, tachycardia, and headache. additionally, creactive protein levels and white blood counts were significantly decreased (64) (65) (66) (67) . the only adverse event observed was a dosedependent phlebitis, due to the fact that high doses of eritoran were used to achieve stable activity of the drug over time. a phase 2 randomized control trial recruiting critically ill septic patients as assessed by the acute physiology and chronic health evaluation ii (apache ii) score disclosed a trend toward decreased mortality in the eritoran treated group (37) . phase 3 access (a controlled comparison of eritoran and placebo in patients with severe sepsis) clinical trial for severe sepsis started in 2006, and results were published in 2013. about 1304 patients were treated with eritoran and 657 patients with placebo within 12 h after the onset of the first organ dysfunction. unfortunately, analyses did not reveal reduced all-cause mortality in primary and secondary end-points (i.e., 28 days and 1 year mortality) (38) . eisai (tokyo, japan) waived to submit eritoran to marketing authorization for the treatment of severe sepsis in january 2011, based on preliminary results of the access trial. several reasons may account for the lack of efficacy of eritoran (68) (69) (70) . for instance, patients were not enrolled or monitored based on the circulating levels of lps, questioning about the appropriateness of inclusion criteria. it is also possible that eritoran would be more efficient if administrated rapidly, before septic shock is underway, pointing the early and aggressive sepsis management as a possible interfering factor. other factors to take into account include the heterogeneity of patients for genetic background, underlying diseases, inflammatory and immune status, sepsis severity, infectious agent, and site of infection. as mentioned earlier, intracellular lps sensed in a tlr4-independent manner sensitizes mice to endotoxic shock (30, 31) . this non-canonical lps detection may have limited the efficacy of the anti-tlr4 strategy. moreover, upon infection, innate immune cells will likely sense several mamps via several tlrs and non-tlr prrs. for example, gram-negative bacteria express mamps that may trigger redundant inflammatory pathways through tlr2 (lipopeptides), tlr4 (lps), tlr5 (flagellin), tlr7 (ssrna), and tlr9 (bacterial dna). all these observations suggest that blocking one single pathway may be insufficient to interfere with the deleterious cascade of events observed in sepsis. the positive side of the access trial failure was a rethink of the design of sepsis clinical trials (68) (69) (70) . clearly, a drug like eritoran should be tested in selected patients and treatment efficacy examined and adjusted according to predefined appropriate biomarkers (such as lps blood levels and genetic polymorphisms affecting the tlr4 pathway). a rigorous approach combining the power of "omics" technologies would allow the selection of homogeneous cohorts and the follow-up of the response to treatment, both of which are mandatory for the successful development of anti-sepsis drugs. another anti-sepsis agent that exhibited promising therapeutic properties is tak-242 [ethyl-(6r)-[n -(2-chloro-4-fluorophenyl) sulfamoyl] or resatorvid] from takeda pharmaceutical company (osaka, japan). tak-242 was originally characterized as a suppressor of nitric oxide (no) and cytokine production by lpsstimulated macrophages and during endotoxic shock in mice (71) . tak-242 binds to cysteine 747 in the intracellular domain of tlr4, thereby inhibiting both myd88-dependent and myd88independent pathways activated by lps (72) . when administered in conscious guinea pigs following lps challenge, tak-242 significantly improved septic shock symptoms, decreasing hmgb1 systemic levels, and increasing survival in a dose-dependent manner (73) . tak-242 also increased survival rates from 17 to 50% and improved organ dysfunction when co-administered with antibiotics in a mouse model of cecal ligation and puncture (clp). no effect on circulating bacterial counts was observed (74) . a double-blind, randomized, placebo-controlled trial was initiated with tak-242 (39) . inclusion criteria comprised symptoms of severe sepsis accompanied with either shock and/or respiratory frontiers in immunology | microbial immunology failure. the study was stopped prematurely due to failure to achieve significant decrease of systemic cytokine levels at stage 1 of the analysis (39) . a phase 3 clinical study was designed but never launched based on business decision and not due to safety or efficacy concerns. antibodies directed against tlr4 or the tlr4-md-2 complex have been generated and showed promising results in several pre-clinical studies. we engineered a soluble chimeric protein composed of the n-terminal and central domains of mouse tlr4 (amino acid 1-334) fused to the fc domain of human igg1 (75) . the chimeric molecule was used to generate high titer anti-mouse tlr4 rabbit polyclonal antibodies. the anti-tlr4 antibodies powerfully inhibited nf-κb and mapk activation and cytokine production by lps-stimulated cells in vitro. the antibodies also hampered cytokine production and protected mice from lethal endotoxemia when administered both prophylactically and therapeutically 4 h after lps. prophylactic administration of anti-tlr4 antibodies blunted tnf production and strikingly increased survival in e. coli sepsis, from 0% in the control antibody group to 80% in the anti-tlr4 group. even more impressive, anti-tlr4 therapy initiated as much as 13 h after the onset of infection in a model of e. coli peritoneal infection improved survival from 30 to 75% (75) . our studies demonstrate that anti-tlr4 antibodies are efficient as adjunctive therapy for e. coli sepsis, with a window of clinical application comprising prophylactic and therapeutic intervention opportunities. several anti-tlr4 monoclonal antibodies have been produced. the group of miyake (university of tokyo, japan) reported in 2000 the generation of mts510, the first rat monoclonal antibody specific of the mouse tlr4-md-2 complex (76) . mts510 was shown to inhibit lps-induced nf-κb activation and tnf production by macrophages. 5e3 is a rat monoclonal antibody produced by novimmune sa (geneva, switzerland) that reacts with the tlr4-md-2 complex (77). 5e3 inhibited lps-induced cell activation, and protected mice from lethal endotoxemia when injected up to 7 h after lps challenge. moreover, administration of 5e3 at the time of surgery improved the outcome of mice with colon ascendens stent peritonitis, a model of polymicrobial abdominal sepsis. finally, the rat monoclonal antibody 1a6, that recognizes both mouse and human tlr4-md-2 complexes, conferred protection in a model of e. coli sepsis, but not salmonella enterica, sepsis (78) . although some tlr4 inhibitors have entered clinical and pre-clinical trials, others remain in the developmental stage. lps-trap-fc antibodies (comprising the extracellular domain of mouse tlr4 fused with md-2 and linked to human igg fc) dose-dependently decreased il-6 release by macrophages, opsonized gram-negative bacteria, and enhanced phagocytosis and complement-mediated bacterial killing (79) . cell-penetrating peptides comprising the translocating segment of drosophila antennapedia homeodomain fused with bb loop sequences of tlr4 (i.e., tlr4-bb peptides) inhibited lps-induced nf-κb and mapk activation and cytokine production (80, 81) . further studies will be required before advancing these products toward the clinical level. altogether, the experimental data reported above provided strong support for the concept of tlr4-targeted therapy for gram-negative sepsis. in the gloomy context following the withdrawn of rhapc and eritoran from the sepsis field, it is hopeful that ni-0101 has entered clinical development. ni-0101 is an anti-tlr4 monoclonal antibody produced by novimmune able to block tlr4 dimerization and tlr4-mediated signaling triggered by lps and endogenous and chemical ligands of tlr4. data from pre-clinical studies in models of arthritis, respiratory inflammation, and organ injury have highlighted the potential favorable action of this agent 3 . a phase 1 clinical study is currently recruiting participants to evaluate drug safety and tolerance in healthy volunteers before and after ex vivo and in vivo lps challenge. pharmacokinetics and pharmacodynamics will also be assessed. results from these studies are eagerly awaited. albeit less well characterized, tlr4 has been implicated in the sensing of non-bacterial microorganisms such as viruses and fungi. tlr4 recognizes o-linked mannan from candida albicans, and human studies have linked asp229gly tlr4 polymorphism with susceptibility to bloodstream candidiasis and pulmonary aspergillosis (82) (83) (84) . in a model of disseminated infection with c. albicans, c3h/hej tlr4-deficient mice exhibited a 10-fold increased fungal load in the kidneys, which was associated with reduced production of the chemokines kc and mip-2 and an impaired recruitment of neutrophils (85) . treatment with hta 125, an anti-human tlr4 mouse monoclonal antibody (86), interfered with neutrophil-mediated protection against c. albicans invasion and cell injury in an in vitro epithelial model of oral candidiasis (87) and inhibited tnf production by human pbmcs stimulated with aspergillus hyphae (88) . it is still unclear whether targeting tlr4 may be beneficial in the context of fungal infections. a more clear yet unexpected picture has arisen from viral infection studies. reactive oxygen species (ros) produced by the nadph oxidase generates oxidized host phospholipids that stimulate tlr4 and the production of cytokines involved in acute lung injury (89) . using a mouse model of lethal infection with influenza, the group of stephanie vogel (university of maryland, baltimore, ma, usa) reported that eritoran significantly increases survival in a dose-dependent manner even when administered 6 days after viral challenge. lung pathology and clinical symptoms were improved while viral titers and influenza-induced cytokine gene expression in lung homogenates were decreased compared to the placebo-treated group. these data suggest that the therapeutic effect of eritoran in a more practical timing of severe sepsis treatment remains substantial (90) . they also suggest that, despite the failure of eritoran in the access trial, new therapeutic potentials might still emerge for this agent. toll-like receptor 2 has been implicated in the recognition of an amazingly broad spectrum of microbial ligands originating from bacteria, fungi, viruses, and parasites (91) . this property is at least partly due to the fact that tlr2 forms heterodimers with tlr1, tlr6, and possibly tlr10 (9, 15, 92) . the biological relevance of tlr2 homodimers is controversial. indeed, some ligands have been reported to trigger cells through tlr2 independently of tlr1 and tlr6. yet, only tlr2/tlr1 and tlr2/tlr6 heterodimers have been successfully crystallized (93, 94) . tlr2 represents an interesting target for numerous conditions, but clinical development of tlr2-targeting drugs has been less extensive than that of tlr4. t2.5 is a tlr2 neutralizing mouse monoclonal antibody. t2.5 blocked pam 3 csk 4 lipopeptide (a tlr1/tlr2-ligand)stimulated nf-κb nuclear translocation and mapk phosphorylation in vitro. in models of pam 3 csk 4 -induced toxic shock and microbial challenge with a high inoculum of heat-inactivated bacillus subtilis, t2.5 prevented lethal shock-like syndrome and increased survival when administered 1 h before or up to 3 h after infection (95) . furthermore, t2.5 used in combination with the 1a6 anti-tlr4/md-2 antibody and antimicrobial therapy protected mice from sepsis caused by s. enterica and e. coli (78) . intracellular antibodies, i.e., intrabodies, have been designed to block the intracellular translocation of tlrs from the endoplasmatic reticulum to the cell surface. αt2ib is a functional anti-tlr2 scfv intrabody comprising the variable domains of the heavy and light chains of t2.5 linked together by a synthetic (gly 4 ser) 3 amino acid sequence. αt2ib bound intracellularly to tlr2 and led to retention and accumulation of tlr2 inside the endoplasmatic reticulum. adenovirus-mediated expression of αt2ib in raw 264.7 macrophages and mouse bone marrow derived macrophages inhibited tlr2 surface expression and tlr2-ligand-driven tnf production (96) . these data suggest for a therapeutic potential of t2.5 or αt2ib in microbial infections. many studies have attempted to elucidate the pathogenesis of acute kidney injury associated with sepsis, which involves mechanisms similar to those occurring during ischemia/reperfusion (97) . damps released during infection are detected through tlr2 by immune cells recruited to the ischemic tissue and/or by cells of the ischemic tissue itself, amplifying the inflammatory response and inducing injury upon reperfusion (98, 99) . blocking tlr2 under these conditions may be cytoprotective (18) . opn-305 is a humanized anti-tlr2 igg4 monoclonal antibody [derived from opn-301 (98) developed by opsona therapeutics (dublin, ireland)]. opn-305 reduced tlr2-driven pro-inflammatory cytokine production through blocking of tlr2/1 and tlr2/6 mediated signaling. in a porcine model of myocardial ischemia/reperfusion injury, pretreatment with opn-305 or administration of opn-305 1 h after ischemia was associated with a 50% decrease in infarct size (100) . results from a first in human phase 1 trial evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending doses of opn-305 given intravenously in healthy adult subjects have just been released (101) . tlr2 occupancy and inhibition of il-6 secretion induced by heat-killed listeria monocytogenes were assessed in whole blood collected up to 90 days after treatment with either the antibody or placebo. opn-305 was well tolerated, with no significant toxicity even at the highest dose tested. impressively, opn-305 at doses of 0.5 and 10 mg/kg occupied 100% of tlr2 molecules expressed on monocytes collected 14 and 90 days after challenge, respectively. il-6 release was inhibited in a parallel manner. these results suggest that treatment with opn-305 could provide short-term protection against ischemia/reperfusion and be adjusted to confer long-lasting blockage in the case of tlr2-mediated chronic diseases (101) . a phase 2 trial assessing safety, tolerability, and efficacy of opn-305 in kidney transplant patients has been initiated (nct01794663). new techniques are continuously implemented to facilitate the identification of therapeutic targets for adjunctive treatment in sepsis. immunoprecipitation with systematic evolution of ligands by exponential enrichment (selex) was developed to screen and identify high-affinity dna and rna molecules that bind to tlr2 and could be used to detect other molecules influencing tlrdriven activity. a most promising candidate, ap-177, was shown to interact with tlr2, thereby obstructing ligand binding to the receptor and inhibiting tlr2-ligand-induced nf-κb activity and il-6 and il-8 production in thp-1 and hek 293 cells (102) . cellpenetrating tlr2-bb peptides have been generated and shown to interfere with tlr2-ligand-induced activation of nf-κb and mapk and cytokine production (80) . whether these compounds will undergo clinical evaluation is unknown. toll-like receptor 3 is an endosomal prr that senses dsrna typically produced during viral infection (103) . experimental models comparing tlr3 wild-type and tlr3 knockout mice revealed either a protective role (west nile virus, encephalomyocarditis virus, poliovirus, coxsackievirus, murine cytomegalovirus, herpes simplex virus), a deleterious role (west nile virus, influenza a virus, phlebovirus), or no influence (lymphocytic choriomeningitis virus, vesicular stomatitis virus, murine cytomegalovirus, reovirus) of tlr3 on anti-viral responses (104) . therefore, tlr3 agonists and antagonists might be efficient adjunctive therapies for viral infections depending on the context. in the following sections, we describe the development of synthetic dsrna tlr3 agonists (see tlr3 agonists) and of synthetic ssdna tlr3 antagonists and anti-tlr3 neutralizing antibodies (see tlr3 antagonists). the dsrna synthetic analog polyinosinic:polycytidylic acid [poly(i:c)] is a potent immunostimulant. for clinical development, poly(i:c) was stabilized with polylysine and carboxymethylcellulose (poly-iclc) (hiltonol, oncovir, washington, dc, usa) and used to generate poly(i:c 12 u) (rintatolimod, tradename ampligen, hemispherx biopharma, philadelphia, pa, usa) by substituting an uridylic acid at a molar ratio of 12:1 in the synthesis of the polycytidylic acid strand (105) . poly(i:c) and its derivatives have been tested in several clinical trials as adjuvants for vaccines (for both infectious diseases and cancer) and complement to haart (highly active anti-retroviral therapy) in human immunodeficiency virus (hiv) infected patients, topics that we do not discuss here. poly(i:c 12 u) is highly specific for tlr3 and, unlike its parental molecule poly(i:c), does not require melanoma differentiationassociated protein 5 (mda5, a cytosolic prr for viruses), for the induction of the signaling cascade leading to type i ifn production (106) . poly(i:c 12 u) has some anti-viral activity against hiv, hepatitis b virus (hbv), coxsackie b3 virus, and several flaviviruses. results from animal models of lethal respiratory viral infection by severe acute respiratory syndrome coronavirus (sars-cov) and punta toro virus highlighted the favorable impact of intranasal treatment with poly(i:c) or poly(i:c 12 u) on survival and viral loads in infected mice (107, 108) . the mode of action of poly(i:c) in the respiratory tract was linked to the induction of caspase-mediated apoptosis and ros, which are involved in the cleavage and shedding of soluble tnf receptor blocking tnf bioactivity (109) . interestingly, poly(i:c) protected against bacterial infections in the respiratory tract as well as in the central nervous system (cns). in a mouse model of pseudomonas aeruginosa pneumonia secondary to clp, intranasal administration of poly(i:c) improved immune activation and lowered bacterial load in the lungs compared to the untreated animals (110) . corroborating results showing enhanced phagocytosis and killing of e. coli by microglial cells suggest that tlr3 activation is crucial for the immune response of cns against invading pathogens (111, 112) . these data support the development of tlr3 agonists as adjuvant therapies to prevent or reduce the severity of respiratory tract infections caused by viruses and possibly bacteria. in connection with that particular field, a phase 1 safety, tolerability, and pharmacokinetic trial of nasally applied poly-iclc in human volunteers is ongoing and will explore immune activation markers. a phase 1/2 clinical trial is assessing the immunogenicity and safety of flumist®(live attenuated influenza vaccine, med-immune, gaithersburg, md, usa) intranasal influenza vaccine administered with and without a poly(i:c 12 u). more recently, tlr3 antagonists have been developed taking into consideration that tlr3 over-activation by viral dsrna may have detrimental consequences in some situations. indeed, it has been reported that administration of poly(i:c) in mice prior to intratracheal challenge with s. pneumoniae impaired bacterial clearance and increased mortality. excessive production of type i ifn was involved in this phenomenon (113) . single-stranded dna oligonucleotides (ssdna odns) efficiently competed with dsrna for binding to tlr3, thus inhibiting cytokine production and costimulatory molecule expression by epithelial cells, pbmcs, and dendritic cells (114, 115) . the efficacy of ssdna odns was demonstrated in cynomolgus macaques, where intranasal injection of ssdnas odns inhibited poly(i:c)-induced cytokine production in nasal secretions (115) . in addition to microbial ligands, tlr3 senses damps released from injured tissue during inflammation, for example rna from necrotic cells, promoting an excessive inflammatory response. interestingly, administration of a tlr3 neutralizing antibody to mice reduced cecal damage induced by gut ischemia and improved survival of animals with polymicrobial sepsis when the antibody was given 6 and 24 h after clp surgery (116) . collectively, these data demonstrate that tlr3 works as an endogenous sensor of necrosis and a regulator of the immune response, pointing to receptor modulation as a possible adjuvant therapy for sepsis. work remains to be done to clearly delineate the precise role of tlr3 in viral and bacterial infections and to appraise the benefit afforded by tlr3 agonistic or antagonistic strategies for infectious diseases, especially septic shock. a single tlr5 agonist has had clinical development, namely cblb502. flagellin is the only ligand of tlr5 described to date. detailed structural basis of flagellin recognition by tlr5 has been obtained through crystallographic analyses, unraveling a unique mode of interaction between the two molecules as depicted from stoichiometry, ligand arrangement, and binding interfaces (117) . the role of structural constraints for induction of the nf-κb signaling cascade downstream tlr5 was supported by structure-guided mutagenesis and deletion analyses on cblb502 (entolimob), a therapeutic agent derivative of s. enterica flagellin implemented by cleveland biolabs (buffalo, ny, usa). cblb502 is currently tested in a phase 1 trial in late stage cancer patients (nct01527136). several clinical trials have investigated the safety and adjuvant efficacy of recombinant flagellin in a number of vaccine settings (against influenza virus, helicobacter pylori, campylobacter, yersinia pestis, west nile virus, etc) (118) . cblb502 plays a protective role against radiation-induced tissue injury, probably by suppressing apoptosis, attenuating ros generation, and promoting tissue regeneration (119) . these properties could explain the beneficial effect of this agonist in a murine model of acute ischemic renal failure when administered 30 min after reperfusion (120) . highlighting the favorable role of flagellin against tissue damage, results from two mouse studies suggested that protection and repair of the intestinal mucosa that serves as a first line defense barrier is the key mode of action of flagellin. in the first study, flagellin was shown to induce the expression of regiiiγ, a c-type lectin with bactericidal activity, and to restrict small intestine colonization with vancomycin-resistant enterococcus (vre) in animals inoculated with vre via oral gavage (121) . in the second study, treatment with flagellin reduced intestinal epithelium destruction induced by dextran sodium sulfate (a chemical used to induce severe acute colitis) and increased survival of mice inoculated with s. typhimurium by oral gavage (122) . these data suggest that flagellin or tlr5 agonists may represent attractive tools for treating pathologies that injure the intestinal tract, including severe sepsis. no tlr5 antagonists have been reported. toll-like receptor 9 agonists tested in clinical trials are synthetic cpg oligodeoxynucleotides (cpg odns) among which cpg10101, imo-2125, sd-101, and cpg 7909 that mimic unmethylated cpg dinucleotide-rich sequences enriched in microbial dna. cpg odns are powerful immunostimulants, exploited for their adjuvant properties in vaccines against infectious diseases (flu, malaria, hiv infection, pneumococcal and meningococcal diseases) and cancer (melanoma, leukemia, glioblastoma, and colorectal, prostate and breast cancer) (123) . the adjuvant properties of cpg odns have been used to enhance www.frontiersin.org the phagocytosis and the killing of bacteria (s. typhimurium and s. pneumoniae) by phagocytic cells (124, 125) . interestingly, a recent study showed that cpg odn given 1 h prior to clp surgery prevented clp-induced cardiac dysfunction in mice. the authors proposed that targeting tlr9 could be a useful approach for the management of cardiovascular dysfunction in severe sepsis patients (126) . therapeutic strategies focusing on tlr9-mediated immunomodulation are currently being implemented for chronic viral infections, such as chronic hepatitis c (hcv). plasmacytoid dendritic cells are the main cells producing type i ifn and are therefore considered to play an important role in viral infections. tlr9 agonists stimulate plasmacytoid dendritic cells to produce large amounts of type i ifn, especially ifnα, which is the backbone of therapy for hcv. indeed, ifnα powerfully inhibits viral replication and promotes innate and adaptive host immune responses. moreover, ifn production appears to be impaired in plasmacytoid dendritic cells of hcv patients (127, 128) . cpg10101 was originally developed under the trade name actilon by coley pharmaceuticals (wellesley, ma, usa), a company recently incorporated by pfizer (new york city, ny, usa). cpg10101 has undergone two phase 1 studies with promising results. a phase 1a study for drug safety and pharmacokinetics conducted in 48 healthy volunteers revealed well tolerated immunostimulatory effects without serious adverse events even when using high doses of cpg10101 (129) . cpg10101 was also tested in 60 hcv patients, 50 infected with genotype 1 hcv. cpg10101 was administered subcutaneously to four randomized groups at different doses twice per week for 4 weeks alone or in combination with pegylated ifnα and ribavirin. the tlr9 agonistic effect of cpg10101 was associated with the induction of ifnγ and ifnα and the decrease of viral loads. the only serious adverse events were urticaria and pruritis, without manifestation of respiratory complications (130) . a phase 2 study enrolling 113 non-responders genotype 1 hcv patients has been completed, but results have not been released yet. imo-2125, manufactured by idera pharmaceuticals (cambridge, ma, usa), has undergone two phase 1 trials: one for dose estimation and one for safety, pharmacokinetics, and pharmacodynamics, enrolling 60 and 40 treatment-naïve genotype 1 hcv patients, respectively. imo-2125 administration dosedependently decreased viral loads, increased the production of anti-viral cytokines and chemokines especially ifnα, and activated nk and t-cell responses (41, 42) . a phase 2 trial was planned, but in april 2011 the company postponed its initiation. the decision was made based on histological data from a 26-week non-clinical toxicology study of imo-2125 in rodents and non-human primates 4 . preliminary analyses suggested evidence of atypical lymphocytic proliferation, although no adverse events were reported in humans. thorough analysis results are pending. a phase 1b study sponsored by dynavax (berkeley, ca, usa) investigated the safety and efficacy of sd-101 in chronic hcv. sd-101 was administered as monotherapy or in combination with ribavirin to 34 chronically infected, treatment-naïve, genotype 1 hcv patients. results released in 2010 indicated that sd-101 was well tolerated and safe without any serious adverse events. the drug had significant anti-viral activity based on dose-dependent anti-viral response, with 100% of patients at the highest dose showing more than one log reduction in viral load, and increased expression of type i ifn-dependent anti-viral genes (ip-10, mcp-1 mx-b, isg-54k). these data comfort results from in vitro studies showing that sd-101 stimulated human pbmcs to produce 20-fold higher levels of both ifnα and ifnλ in comparison with first-generation tlr9 agonists (40) . bacterial dna released during infection is a mamp, and exuberant activation of tlr9 may participate to the sepsis pathophysiology. hence, drug inhibitors of tlr9 may have therapeutic potential in human sepsis. as a proof of concept, administration up to 12 h after surgery of a single dose of an inhibitory cpg odn blocking tlr9 signaling protected mice from polymicrobial sepsis following clp (131) . hmgb proteins are essential for triggering nucleic acid receptor-mediated innate immune responses. hmgb-binding non-immunogenic-odns have been designed to inhibit hmgb-mediated pathologies. a non-immunogenic odn termed ism odn was tested in a mouse model of endotoxemia. impressively, 70% of mice treated with ism odn survived up to 72 h after lps challenge, while all mice from the control group died within 48 h (132). these data argue for a possible use of non-immunogenic-odns in therapeutic interventions. antimalarial drugs such as chloroquine are well known for their anti-inflammatory properties in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (133) . chloroquine blunted cytokine production and protected mice from toxic shock induced by cpg odn and lps. chloroquine down-regulated the expression of both tlr9 and tlr4, suggesting that it acts at multiple levels to inhibit inflammation (134, 135) . additionally, when administered 6 h after clp in elderly mice treated with fluids and antimicrobials, chloroquine significantly improved survival, strengthened renal function and protected from multiple organ dysfunction (136) . these results support clinical evaluation of chloroquine in patients with severe sepsis, especially those presenting with acute renal failure. based on its anti-inflammatory activity and because it inhibits endosomal acidification which are important for cell infection by viruses, chloroquine is also tested in multiple trials for prevention and/or treatment of viral infections (hiv, influenza, dengue, chikungunya). yet, the mode of action of chloroquine is multi-factorial and not only through tlr9. toll-like receptor 7 and 8 are closely related tlrs well known for their capacity to recognize ssrna from viruses such as hcv, hbv, hiv, influenza virus, herpes simplex virus, epstein-barr virus, vesicular stomatitis virus, papilloma virus, respiratory syncytial virus, and sendai virus. in agreement, tlr7 is primarily expressed in plasmacytoid dendritic cells. more recently, tlr7 and tlr8 were shown to sense bacterial rna released within phagosomal vacuoles (137) . tlr7 and tlr8 triggering induces potent antiviral immune responses characterized by the production of type frontiers in immunology | microbial immunology i ifns and nf-κb-dependent cytokines. tlr7/8 agonists are primarily developed for treating viral diseases, but also as adjuvants for cancer and infectious disease vaccines. imiquimod (aldara, originally developed by 3m pharmaceuticals, maplewood, mn, usa) is the only tlr7 agonist marketed for anti-viral treatment, i.e., external ano-genital warts caused by human papilloma virus. numerous tlr7/8 agonists are in clinical development, like cl097, isatoribine, ana975, ana773, pf-04878691 (852a), r-848 (resiquimod), and gs-9620. although therapeutic strategies for hcv have evolved in the recent years, quest of new immunomodulatory targets remains mandatory. treatment with new agents such as protease inhibitors appears to be efficient but presents with issues of resistance in the long run (138) . moreover, current protocol therapy with ifnα provides replenishment with a specific subtype of this cytokine. however, use of tlr7 agonists is able to induce a variety of ifn subtypes, possibly providing a more radical and integrated anti-viral activity (139) . finally, administration of tlr7/8 agonists may overcome the adverse events caused by ifnα, like the suppression of granulocyte colony stimulating factor (g-csf) leading to neutropenia. indeed, cl097 reversed ifnα-mediated inhibition of g-csf production by pbmcs obtained from hcv patients and healthy volunteers (140) . cl097 also restored defective cytokine production by myeloid dendritic cells from hiv patients (141) . isatoribine (anadys pharmaceuticals, san diego, ca, usa) was one of the first guanosin analog selective tlr7 agonists to be implemented and tested on humans (142) . intravenous administration of isatoribine to 12 hcv patients during a 7day treatment plan resulted in reduced plasma concentration of hcv rna, regardless virus genotype. adverse events comprised dose-dependent joint pain, decreased white blood cells, and platelets counts, insomnia, and headache (143) . the development of an oral prodrug that could lack the detrimental effects of isatoribine, especially in the gastrointestinal tract, pointed to a new candidate, ana975. preliminary results from a study conducted with ana975 were promising. oral administration of ana975 presented with elevated plasma levels of isatoribine at a concentration able to reduce hcv rna in the plasma of infected patients (144) . unfortunately, anadys pharmaceuticals and novartis (basel, switzerland) announced in 2007 discontinuation of drug development due to unacceptable toxicity in pre-clinical animal studies (145) . subsequent elaboration of an oral prodrug of isatoribine by anadys pharmaceuticals led to the generation of ana773. ana773 exhibits efficient induction of endogenous type i ifns. a double-blind, placebo-controlled study was conducted in 34 patients with chronic hcv, either treatment-naïve or relapsed from ifn-based therapy. interestingly, ana773 was safe and well tolerated and presented only with grade 1 and 2 adverse events. moreover, ana773 dose-dependently decreased hcv rna levels (48) . in another study, repeated treatment with ana773 was associated with transient decrease of myeloid and plasmacytoid dendritic cells and increased levels of ifnα and ip-10 in the blood of patients achieving a reduction in the viral load, suggesting an impairment in ifnα production in the case of non-responders (49) . pf-04878691, formerly known as 852a, is a tlr7 agonist generated by pfizer and implemented so that repeated low doses of the drug would be accompanied with the benefits of the agonistic activity without adverse events. a proof of concept study was conducted to evaluate safety and tolerability of the drug and to determine pharmacokinetics and pharmacodynamics. twentyfour healthy volunteers received orally increasing doses of pf-04878691. pf-04878691 induced immune response in a dosedependent and frequency-related manner. however, two of the subjects exhibited severe lymphopenia along with flu-like symptoms and hypotension (50) . in an attempt to decide on the future perspectives of this compound, a model was used to predict the safety and efficacy of pf-04878691 in hcv patients. this model exploited clinical results from the former study in healthy volunteers along with those reported from the use of cpg10101. further optimization will be required before entering the drug in a phase 2 study (146) . other tlr7/8 agonists have reached phase 3 trials, but demonstrated lack of efficacy and serious adverse effects. when monocytes from hiv patients were stimulated with resiquimod, il-12 secretion was augmented while tnf production was decreased compared to the control group. additionally, hiv replication in cultured monocytes was inhibited (147) . these promising in vitro results were reproduced in a phase 2 trial that enrolled patients with herpes simplex virus type 2. topical application of resiquimod protected from viral lesion spreading (148) . however, a phase 3 trial disclosed lack of efficacy of the drug and, although a phase 2 study for the treatment of hcv demonstrated decreased viral loads, adverse side effects similar to those resulting from ifnα treatment were of serious concern (149). two phase 1 clinical trials for the safety and pharmacokinetics of a novel compound, gs-9620 (gilead sciences, foster city, ca, usa), are currently enrolling treatment-naïve and viral suppressed hbv patients respectively, while another one enrolling hcv patients has been recently approved. gs-9620 is a tlr7 agonist tested originally in hbv infected chimpanzees. drug administration was associated with reduced viral loads both in plasma and liver, probably through enhanced apoptosis of hepatocytes (150) . testing of ascending dosages of the drug in healthy volunteers presented with flu-like adverse events at a dose of 8-12 mg, but cytokine induction was achieved even at administration of 2 mg pointing to a promising adjunctive treatment for chronic viral infections (151). all the above data illustrate the efforts devoted to the development of tlr7/8 agonists for treating viral pathologies. taking into account that bacterial rna triggers tlr7 and tlr8 pathways, one may speculate that tlr7/8 agonists would impact on bacterial sepsis. unfortunately, there are almost no data available on that subject. in one report, intravenous injection of r-848 prior to sepsis induction using the colon ascendens stent peritonitis model increased cytokine release and bacterial clearance, but the effect on survival was not reported (152) . finally, considering that excessive tlr activation induced by viruses or bacteria may have www.frontiersin.org detrimental effects for the host, it might be of interest to generate tlr7/8 antagonists to counteract overwhelming immune activation in conditions of severe infections. targeting tlrs is a promising field for sepsis management and infection control. tlr agonists are widely used to optimize vaccine efficacy, taking advantage of their powerful adjuvancity. whether tlr agonists and antagonists will have such success for treatment therapies, especially for sepsis, has to be established. agonists of tlr3 and tlr7-9 yielded very promising results for treating viral infections. only few studies tested antagonistic drugs. mainly for historical reasons and because of their ligand specificity, tlr4 and to a lesser extent tlr2 are the favorite targets for developing antisepsis drugs. this domain of research has monopolized important resources, but unfortunately many drugs tested in animal models have not entered human studies. those that proceeded, like eritoran and tak-242, did not achieve primary endpoint goal and/or were accompanied with manifestation of adverse events. animal models provide invaluable information about the role of tlrs and the mechanism underlying infection pathogenesis, but lack complexity in terms of comorbidities and host response compared to human disease (153) . so, how to explore more efficiently new treatment strategies? improving the design of clinical studies is mandatory. we should discriminate those patients that could benefit from therapy based on their genotype (for example affecting tlr pathways) and the expression of the targeted molecule, and select homogeneous, well-described population bearing the same underlying conditions and disease severity (68) (69) (70) 154) . ideally, sepsis studies should use specific biomarkers to help patient enrollment and weigh treatment efficacy in realistic conditions (155) . the failure of the recent trials in patients with severe sepsis has taught us valuable lessons regarding patient selection, time of intervention, and follow-up (68) (69) (70) . blocking one single mediator may also not be sufficient to interfere with sepsis. developing sepsis-specific tlr-targeted therapies for patients is a path strewn with obstacles, but an exciting and promising area of research. these studies offer new possibilities for prevention and intervention in infectious diseases, but also for other conditions, such as cancer, allergy, asthma and autoimmune diseases either directly or through improvement of vaccines. benchmarking the incidence and mortality of severe sepsis in the united states long-term cognitive impairment and functional disability among survivors of severe sepsis surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock drotrecogin alfa (activated) in adults with septic shock epigenetics in sepsis: targeting histone deacetylases sepsis: something old, something new, and a systems view harmful molecular mechanisms in sepsis regulation of adaptive immunity by the innate immune system toll-like receptors and their crosstalk with other innate receptors in infection and immunity inflammasomes and their roles in health and disease approaching the asymptote? evolution and revolution in immunology the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene bridging innate and adaptive immunity newly described pattern recognition receptors team up against intracellular pathogens dampening inflammation by modulating tlr signalling toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system renal-associated tlr2 mediates ischemia/reperfusion injury in the kidney the family of five: tir-domain-containing adaptors in toll-like receptor signalling pattern recognition receptors and inflammation synergy and cross-tolerance between toll-like receptor (tlr)2-and tlr4-mediated signaling pathways selected toll-like receptor agonist combinations synergistically trigger a t helper type 1-polarizing program in dendritic cells syk kinase is required for collaborative cytokine production induced through dectin-1 and toll-like receptors dectin-1 directs t helper cell differentiation by controlling noncanonical nf-kappab activation through raf-1 and syk cytosolic flagellin requires ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages innate immune recognition of bacterial ligands by naips determines inflammasome specificity the nlrc4 inflammasome receptors for bacterial flagellin and type iii secretion apparatus signalling pathways and molecular interactions of nod1 and nod2 cpg and non-cpg oligodeoxynucleotides directly costimulate mouse and human cd4+ t cells through a tlr9-and myd88-independent mechanism cytoplasmic lps activates caspase-11: implications in tlr4-independent endotoxic shock noncanonical inflammasome activation by intracellular lps independent of tlr4 how important are toll-like receptors for antimicrobial responses? innate immunogenetics: a tool for exploring new frontiers of host defence human genetic susceptibility to infectious disease genetic variation in toll-like receptors and disease susceptibility targeting toll-like receptors: emerging therapeutics? phase 2 trial of eritoran tetrasodium (e5564), a toll-like receptor 4 antagonist, in patients with severe sepsis effect of eritoran, an antagonist of md2-tlr4, on mortality in patients with severe sepsis: the access randomized trial a randomized, double-blind, placebo-controlled trial of tak-242 for the treatment of severe sepsis phase 1b dose-secalation study of sd-101, a novel therapeutic tlr-9 agonist, in treatment-naive chronic hepatitis c patients a phase 1, multi-center, randomized, placebo-controlled, dose-escalation study of imo-2125, a tlr9 agonist, in hepatitis c-nonresponders imo-2125 plus ribavirin gives substantial first-dose viral load reductions, cumulative anti-viral effect, is well tolerated in naive genotype 1 hcv patients: a phase 1 trial chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine use improves dengue-related symptoms imiquimod 3.75% cream applied daily to treat anogenital warts: combined results from women in two randomized, placebo-controlled studies imiquimod 5% cream for external genital or perianal warts in human immunodeficiency virus-positive patients treated with highly active antiretroviral therapy: an open-label, noncomparative study first-line therapy for human cutaneous leishmaniasis in peru using the tlr7 agonist imiquimod in combination with pentavalent antimony randomised clinical trial: anti-viral activity of ana773, an oral inducer of endogenous interferons acting via tlr7, in chronic hcv potent immune activation in chronic hepatitis c patients upon administration of an oral inducer of endogenous interferons that acts via toll-like receptor 7 the innate immune response, clinical outcomes, and ex vivo hcv antiviral efficacy of a tlr7 agonist (pf-4878691) innate immune sensing and its roots: the story of endotoxin toll-like receptor 4 modulation as a strategy to treat sepsis expression of tumour necrosis factor receptor and toll-like receptor 2 and 4 on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating increased expression of tolllike receptor-2 and -4 on leukocytes from patients with sepsis the structural basis of lipopolysaccharide recognition by the tlr4-md-2 complex bacterial endotoxin: molecular relationships of structure to activity and function lipopolysaccharide interaction with cell surface toll-like receptor 4-md-2: higher affinity than that with md-2 or cd14 a lipid a analog, e5531, blocks the endotoxin response in human volunteers with experimental endotoxemia eritoran tetrasodium (e5564) treatment for sepsis: review of preclinical and clinical studies inhibition of endotoxin response by e5564, a novel toll-like receptor 4-directed endotoxin antagonist toll-like receptor 4 antagonist (e5564) prevents the chronic airway response to inhaled lipopolysaccharide physiologic, biochemical, and imaging characterization of acute lung injury in mice pharmacokinetics of e5564, a lipopolysaccharide antagonist, in patients with impaired hepatic function extended in vivo pharmacodynamic activity of e5564 in normal volunteers with experimental endotoxemia safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (e5564) during continuous intravenous infusion into healthy volunteers continuous pharmacodynamic activity of eritoran tetrasodium, a tlr4 antagonist, during intermittent intravenous infusion into normal volunteers sepsis studies need new direction recombinant human activated protein c as a therapy for severe sepsis: lessons learned? trial watch: sepsis study failure highlights need for trial design rethink discovery of novel and potent small-molecule inhibitors of no and cytokine production as antisepsis agents: synthesis and biological activity of alkyl 6-(n-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate analysis of binding site for the novel small-molecule tlr4 signal transduction inhibitor tak-242 and its therapeutic effect on mouse sepsis model the novel selective toll-like receptor 4 signal transduction inhibitor tak-242 prevents endotoxaemia in conscious guinea-pigs combination of imipenem and tak-242, a tolllike receptor 4 signal transduction inhibitor, improves survival in a murine model of polymicrobial sepsis protection from lethal gram-negative bacterial sepsis by targeting toll-like receptor 4 cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-md-2 complex on mouse peritoneal macrophages tlr4/md-2 monoclonal antibody therapy affords protection in experimental models of septic shock tlr4-induced ifn-gamma production increases tlr2 sensitivity and drives gramnegative sepsis in mice lipopolysaccharide-trap-fc, a multifunctional agent to battle gram-negative bacteria cutting edge: differential inhibition of tlr signaling pathways by cell-permeable peptides representing bb loops of tlrs targeting tlr4 signaling by tlr4 toll/il-1 receptor domain-derived decoy peptides: identification of the tlr4 toll/il-1 receptor domain dimerization interface immunity to fungal infections interplay between candida albicans and the mammalian innate host defense genetic susceptibility to candida infections the role of toll-like receptor (tlr) 2 and tlr4 in the host defense against disseminated candidiasis md-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor human epithelial cells establish direct antifungal defense through tlr4-mediated signaling involvement of cd14 and toll-like receptors in activation of human monocytes by aspergillus fumigatus hyphae identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury the tlr4 antagonist eritoran protects mice from lethal influenza infection the role of tlr2 in infection and immunity human tlr10 is a functional receptor, expressed by b cells and plasmacytoid dendritic cells, which activates gene transcription through myd88 crystal structure of the tlr1-tlr2 heterodimer induced by binding of a tri-acylated lipopeptide recognition of lipopeptide patterns by toll-like receptor 2-toll-like receptor 6 heterodimer antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes generation of anti-tlr2 intrabody mediating inhibition of macrophage surface tlr2 expression and tlr2-driven cell activation searching for mechanisms that matter in early septic acute kidney injury: an experimental study myocardial ischemia/reperfusion injury is mediated by leukocytic tolllike receptor-2 and reduced by systemic administration of a novel anti-toll-like receptor-2 antibody ischaemia-induced up-regulation of toll-like receptor 2 in circulating monocytes in cardiogenic shock treatment with opn-305, a humanized anti-toll-like receptor-2 antibody, reduces myocardial ischemia/reperfusion injury in pigs randomized, double-blind, placebo-controlled, dose-escalating phase i, healthy subjects study of intravenous opn-305, a humanized anti-tlr-2 antibody identification and characterization of oligonucleotides that inhibit toll-like receptor 2-associated immune responses toll-like receptor, rig-i-like receptors and the nlrp3 inflammasome: key modulators of innate immune responses to double-stranded rna viruses antiviral responses induced by the tlr3 pathway structural requirements of the ri n-rc n complex for induction of human interferon essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus tlr3 is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c12u), but not poly(i:c): differential recognition of synthetic dsrna molecules intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections double-stranded rna induces shedding of the 34-kda soluble tnfr1 from human airway epithelial cells via tlr3-trif-rip1-dependent signaling: roles for dual oxidase 2-and caspasedependent pathways tlr3 agonist improves survival to secondary pneumonia in a double injury model the viral tlr3 agonist poly(i:c) stimulates phagocytosis and intracellular killing of escherichia coli by microglial cells microglia recognize doublestranded rna via tlr3 poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3 singlestranded dna oligonucleotides inhibit tlr3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques tlr3 is an endogenous sensor of tissue necrosis during acute inflammatory events structural basis of tlr5-flagellin recognition and signaling flagellin as an adjuvant: cellular mechanisms and potential an agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models tlr5 agonist inhibits acute renal ischemic failure bacterial flagellin stimulates toll-like receptor 5-dependent defense against vancomycin-resistant enterococcus infection flagellin treatment protects against chemicals, bacteria, viruses, and radiation cpg still rocks! update on an accidental drug tlr 9 activation in dendritic cells enhances salmonella killing and antigen presentation via involvement of the reactive oxygen species toll-like receptor stimulation enhances phagocytosis and intracellular killing of nonencapsulated and encapsulated streptococcus pneumoniae by murine microglia the toll-like receptor 9 ligand, cpg oligodeoxynucleotide, attenuates cardiac dysfunction in polymicrobial sepsis, involving activation of both phosphoinositide 3 kinase/akt and extracellular-signal-related kinase signaling rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells a class c cpg toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis c safety, pharmacokinetics and immune effects in normal volunteers of cpg 10101 (actilon), an investigational synthetic toll-like receptor 9 agonist phase 1b, randomized, double-blind, dose-escalation trial of cpg 10101 in patients with chronic hepatitis c virus toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (hmgbs) mechanism of endosomal tlr inhibition by antimalarial drugs and imidazoquinolines chloroquine inhibits proinflammatory cytokine release into human whole blood chloroquine protects mice from challenge with cpg odn and lps by decreasing proinflammatory cytokine release chloroquine and inhibition of toll-like receptor 9 protect from sepsisinduced acute kidney injury detection of prokaryotic mrna signifies microbial viability and promotes immunity telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis c virus replication in patients stimulation of interferon and cytokine gene expression by imiquimod and stimulation by sendai virus utilize similar signal transduction pathways interferon-alpha suppressed granulocyte colony stimulating factor production is reversed by cl097, a tlr7/8 agonist tlr7/tlr8 activation restores defective cytokine secretion by myeloid dendritic cells but not by plasmacytoid dendritic cells in hiv-infected pregnant women and newborns molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor 7 isatoribine, an agonist of tlr7, reduces plasma virus concentration in chronic hepatitis c infection discovery of ana975: an oral prodrug of the tlr-7 agonist isatoribine masked oral prodrugs of toll-like receptor 7 agonists: a new approach for the treatment of infectious disease use of modelling and simulation techniques to support decision making on the progression of pf-04878691, a tlr7 agonist being developed for hepatitis c r-848 triggers the expression of tlr7/8 and suppresses hiv replication in monocytes topical resiquimod 0.01% gel decreases herpes simplex virus type 2 genital shedding: a randomized, controlled trial oral resiquimod in chronic hcv infection: safety and efficacy in 2 placebocontrolled, double-blind phase iia studies gs-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees safety, pharmacokinetics and pharmacodynamics of gs-9620, an oral toll-like receptor 7 agonist stimulation of tlr7 prior to polymicrobial sepsis improves the immune control of the inflammatory response in adult mice the search for effective therapy for sepsis: back to the drawing board? sepsis: time to reconsider the concept biomarkers in sepsis we would like to thank all our collaborators that participated in our studies. thierry roger is supported through grants from the swiss national science foundation (310000_114073, 310030_132744, 310030_138488, and 310030_146838), an interdisciplinary grant from the faculty of biology and medicine of the university of lausanne (switzerland) and the european com the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. frontiers in immunology | microbial immunology key: cord-266738-8xx1xm2d authors: feng, zhan-hui; cheng, yong-ran; chen, juan; ye, lan; zhou, meng-yun; wang, ming-wei title: chinese medical personnel against the 2019-ncov date: 2020-05-31 journal: journal of infection doi: 10.1016/j.jinf.2020.02.011 sha: doc_id: 266738 cord_uid: 8xx1xm2d nan in this journal, zhu et al. recently reported the results of their genomic analysis of multidrug-resistant klebsiella pneumoniae isolates from individual patients before and after colistin treatment highlighting the rapid emergence and multifaceted molecular mechanisms of colistin resistance in k. pneumoniae. 1 this work highlights the therapeutic and public-health challenges of colistinresistance (cr), which is increasingly used as a large resort antibiotic, despite its unattractive toxicity profile and narrow therapeutic window. 2 oral non-absorbed colistin has been proposed as a decontamination strategy in intensive care units and for patients carrying multidrug resistant enterobacterales (mdr-e). 3 , 4 the impact of decolonization strategies in terms of emergence of cr has rarely been monitored because no reliable selective medium existed and cr was not considered a public-health problem. recently, reliable universal culture media have been developed to screen for cr. 5 here, we studied the impact of non-absorbed oral colistin on the emergence of cr in the gut microbiota of patients from the rgnosis-wp3 randomized controlled trial. 6 thirty-nine subjects colonized with mdr-e were randomized to receive oral colistin sulfate 2 miu 4 times a day + neomycin sulfate 500 mg bid for 5 days followed by a fecal microbiota transplant (fmt) from healthy donors, or no intervention. stool samples were collected on visit v0 (screening sample), v2 (after 5 days of oral decontamination and before fmt for the intervention group), v3, v4 and v5, respectively 5-10 days, 30-40 days and 150-210 days later. 6 stool samples from donors and 15 subjects from the intervention group and 15 from the control group were available for this work and plated on drigalski plates (control) and superpolymyxin r plates. 5 colony forming units (cfu) counts of all gram-negative rods were determined. isolates growing on superpolymyxin r plates were identified by maldi-tof; cr was confirmed by the culture-based rapid polymyxin np test and mic determined by the microdilution method. the limit of detection was 10 2 cfu/g of stool. cr-e. coli were sequenced using the illumina hiseq technology. to determine whether cr isolates were present before the intervention, a specific mcr-1 pcr was performed on patients stool prior to intervention (v0) and on the donor's stool. 7 electroporation of plasmids was performed to localize the gene conferring resistance to colistin and neomycin and molecular typing of the electroporants was performed using pcr based replicon typing (pbrt). ✩ université de paris, iame, inserm, umr-1137 no patient or donor included in the trial carried cr isolates on v0. among the 15 patients in the intervention group two (13.3%, [ic90 −1; 3], p = 0.24) carried cr isolates at least at one visit after the intervention ( fig. 1 ) . no cr-enterobacterales was detected in the stools of subjects from the control group. among both subjects with cr-enterobacterales, one carried 10 log cfu/g of hafnia paralvei , a species which is intrinsically resistant to colistin (mic = 8 mg/l), also resistant to neomycin (mic = 32 mg/l) on visit 4 and the other carried 1 log cfu/g and 3 log cfu/g cr-e. coli at visits 4 and 5, respectively, with a colistin mic at 8 mg/l. relative abundance of cr-e. coli increased between v4 and v5 from 0.01% to 1% of the total enterobacterales population. the cr-e. coli recovered at v4 and v5 both belonged to phylogroup c st23 group and carried the serotype o176:h9. a plasmid-borne mcr-1.1 gene encoding for cr as well as a aph(3 )-ia gene conferring resistance to neomycin were identified, both being co-located on the same inchi2 plasmid. in addition, resistance genes conferring resistance to hygromycin b ( aph(4)-ia ), sulfonamides ( sul2 ), tetracyclines ( tet(a) ) and phenicols ( flor and cata1 ), all antibiotics used in veterinary medicine, were evidenced. for both subjects, cr strains could not be retrieved in the initial stool of the subject or in the donor's stool. pcr experiments performed with specific primers to detect mcr-1 gene directly on the pre-therapeutic stool were also negative. to our knowledge, this is the first report of the in-vivo selection of cr-enterobacterales in the gut microbiota of patients after oral decontamination by colistin. the selection of cr strains (a naturally-resistant h. paralvei and a mcr-1 producing e. coli ), both resistant to colistin and neomycin, may be the result either of the enrichment process by sod of preexisting cr strains that had not been initially detected because of very low abundances, or of an exogenous acquisition, either from other individuals or through fmt. 8 indeed the transmission from fmt of mdr strains from positive donors is a potential risk. 8 despite our efforts to decrease the limit of detection of mcr producers by using a pcr technique directly on the pre-therapeutic stool sample and the donors' stools, we failed to detect the parental strain, either because cr strains were in intestinal niches, the limit of detection remained too high, or the strain was acquired exogenously. however, the mcr-1 -positive e. coli is likely of animal origin according to its genetic features and its co-resistance profile. 9 indeed, phylogroup st23 is frequently encountered among avian pathogenic e. coli (apec) and co-resistances to many antibiotics used specifically in veterinary medicine is striking. 10 furthermore, the aph(3 )-ia gene confers resistance to neomycin and paromomycin, the latter commonly used in cattle and pigs. the selection of the mcr-1 producer is an illustration of the "one health" problem of resistance: a strain likely to have been selected by veterinary antibiotics among animals ended up in a patient's gut, later enriched by the use of colistin and neomycin as decontaminant. although the small number of subjects is a clear limitation, this observation is a "proof-of-concept" of the risk of selection of cr-enterobacterales after oral colistin treatment and fmt, at a time when colistin is one of the last resort antibiotics to treat mdr-enterobacterales infections. the selection of commensal cr-e. coli is especially worrying, given the pathogenic potential of e. coli and its ability to widely colonize animals and humans. given the controversial results of oral decontamination by colistin, we believe it should only be used with precautions and with thorough monitoring of cr. we read with interest a recent paper in this journal by luzatti and colleagues, 1 who explored the significance of the presence of herpes simplex virus (hsv) dna in lower respiratory tract (lrt) specimens for the diagnosis of hsv pneumonia in immunocompromised patients. the authors underlined the difficulty in gauging the clinical relevance of such a laboratory finding in the absence of histopathological data, as hsv shedding in the lrt may occur in the absence of disease. the interpretation of real-time pcr results for diagnosis of pneumocystis jirovecii (pj) pneumonia (pjp) faces an analogous challenge, since the presence of pj dna in lrt may reflect colonization (carriage) rather than infection. 2 there is limited information on the clinical value of pj real-time pcr in diagnosing pjp in patients with hematological diseases; 3-6 this is exceedingly challenging as the sensitivity of direct examination procedures is suboptimal due to low fungal burdens. 3 here, we report on our experience on this matter. a total of 219 episodes of pneumonia occurring in 192 consecutive patients with hematological disorders in which pjp was considered in the differential etiological diagnosis were included. of these, 127 episodes developed in patients undergoing either allogeneic hematopoietic stem cell transplantation-allo-hsct-( n = 86) or autologous-hsct ( n = 19), and 92 in non-transplant patients (acute leukemia, n = 61; lymphoma, n = 16; chronic leukemia, n = 8; others, n = 2). the patients were attended at the hospital clínico universitario-hcu-( n = 100) or at the hospital universitario politécnico "la fe" -hlf-( n = 92) between june 2014 and august 2019. no patients in the cohort tested positive for hiv. this study was approved by the respective hospital ethics committee and informed consent was obtained from all patients. a single specimen per episode was available for diagnosis (bal fluids, n = 179; sputa, n = 22; ta, n = 17 and bronchial biopsy, n = 1). the realcycler pjir kit r (progenie molecular, spain) was used at hcu, and the pneumocystis jirovecii real time pcr detection (certest biotech; zaragoza, spain) was employed at hlf (see footnote in table 1 ). both assays target the large sub-unit of ribosomal (mtlsu) rna gene. preliminary experiments using 5 bal specimens indicated that both assays yield comparable pcr cycle thresholds (c t s) (median, 28.2, range, 26.4-32.3 vs. median 27.5; range, 26.3-33.1, respectively; p = 0.89). all specimens tested negative by direct examination for pj, whereas 27 were positive by real-time pcr (bal, n = 18; sputa, n = 7, and ta, n = 2); following stringent clinical, microbiological and imaging criteria ( table 1 ) , pjp was deemed to be the most probable diagnosis in 12 episodes occurring in unique patients. no histopathological confirmation of pjp was available for any patient. pcr c t values inversely correlate with fungal burden in the sample. 6 which is higher in patients with pjp than in colonized individuals. 2 here, overall, pj pcr c t s in specimens from patients with pjp tended to be lower than in pj carriers ( p = 0.39); when only bal fluid specimens were considered, the difference reached statistical significance (median, 29.0; range, 26.4-34.7 vs. median 34.6; range, 30.0-41.0; p = 0.04). this finding is likely related to use of more standardized procedures for bal fluid sampling. receiver operating characteristic (roc) curve analysis showed that a threshold c t value of 30.0 in bal specimens displayed a sensitivity of 85.7% (95% ci, 45.0-100%) and a specificity of 80% (95% ci, 40.8-100%) for pjp diagnosis. a number of studies have established different c t s cut-offs for that purpose, [6] [7] [8] [9] . in our view, however, the variability in the performance of different pcr assays and sampling conditions, heterogeneity of patient populations, and in particular the lack of a pj international standard material for pcr result normalization precludes defining a consensus universal threshold nowadays. the absence of anti-pj prophylaxis, treatment with corticosteroids and serum ldh levels ≥400 u/l have been shown to be associated with pjp. 3 here, patients not undergoing anti-pj prophylaxis were more likely to display a clinically significant pj pcr result ( table 1 ). in turn, roc curve analysis indicated that a cut-off ldh value ≥400 u/l had a sensitivity of 81.8% (ci 95%, 59.0-100%) and specificity of 67% (95% ci, 34.0-99.3%) for pjp diagnosis. in univariate regression logistic models, serum ldh values ≥400 u/l were associated with a clinically significant positive pcr pj result (or, 9.0; 95% ci, 1.2-63.8; p = 0.02). in contrast, corticosteroid use within the month before sampling was not different between the probability of pneumocystis jirovecii (pj) pneumonia (pjp) for each patient was retrospectively evaluated by an expert committee including infectious diseases and microbiology specialists at both centers, on the basis of (i) documented pj presence in respiratory specimens by microscopy; (ii) compatibility of clinical signs and symptoms (at least 2 of the following: subtle onset of progressive dyspnea, pyrexia, nonproductive cough, hypoxaemia and chest pain), (iii) compatible (suggestive) radiological findings (chest radiograph and/or high-resolution computed tomographic scan detection of interstitial opacities and/or diffuse infiltration infiltrates); (iv) complete resolution of symptoms after a full course of anti-pjp treatment; (v) absence of alternative diagnosis. the efficacy of therapy was assessed on a daily basis. pjp was ruled out if real-time pcr for pj tested negative, or if clinical recovery occurred in the absence of pj-targeted antimicrobial therapy. pj colonization (carriage) was the most likely possibility when patients did not meet the above criteria and an alternate diagnosis was made. b frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. two-sided exact p values were reported and p values ≤ 0.05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. c respiratory tract specimens were obtained following conventional procedures. specimens were examined for presence of ascus or trophic forms of pj by microscopy following blue toluidine, calcofluor white or grocott's methenamine silver staining. cytospin preparations were prepared from bal specimens for direct examination. sputa and ta samples were mixed v/v with sputasol (oxoid, uk) and vortexed for 5 min. all samples were centrifuged at 30 0 0 g for 10 min, and the pellets were resuspended 1/10 in 0.9% nacl for further processing. for real-time pcr, dna was extracted from 200 μl of specimens using the qiaamp dna blood mini kit (qiagen, hilden, germany) on either qia symphony or ez-1 platforms (qiagen), following the manufacturer's instructions. at hcu, a commercially-available real-time pcr assay previously evaluated by others, the realcycler pjir kit r (progenie molecular, spain), which targets the mitochondrial large sub-unit of ribosomal (mtlsu) rna gene, was used according to the manufacturer's instructions ( http://www.progenie-molecular.com/pjir-u-in.pdf ). at hlf, the commercially-available pneumocystis jirovecii real time pcr detection. (certest biotech; zaragoza, spain), which also targets the large sub-unit of ribosomal (mtlsu) rna gene, was employed following the manufacturer instructions ( https://www.certest. es/wpontent/uploads/2019/02/viasure _ real _ time _ pcr _ pneumocystis _ jirovecii _ sp1.pdf ). at both centers pcr were performed in the applied biosystems 7500 fast real-time pcr platform (applied biosystems, ca, usa). pcr results were reported as positive or negative. for positive samples, threshold cycle (c t ) values were also recorded. no standard curve was generated with a positive control for quantitative estimations. d antimicrobial prophylaxis for pjp was performed with trimethoprim-sulfamethoxazole (tmp/smx), one double-strength tablet (160 mg tmp/800 mg smx) given 2 (in allogeneic hsct patients) or 3 times a week with oral folic acid (15, 16) . patients with suspicion of pjp according to the attending physician were treated with tmp/smx 15-20 mg/kg (tmp) 75-100 mg/kg (smx) per day for 2-3 weeks. e in all these cases, death was attributable to pjp. patients with clinically significant pj detection and pj carriers ( table 1 ) . detection or recovery of other microbial agents (one or more) was documented in 17 of the 27 specimens testing positive by pj pcr ( table 2 ). in line with a previous report, 9 this microbiological finding was significantly less frequent ( p = 0.001) in specimens from patients with pjp than in colonized patients; in fact, microbial co-detection was inversely associated with pjp in univariate logistic regression models (or, 0.024; 95% ci, 0.002-0.26; p = 0.002). strengths of the current study are the following: (i) clinical categorization of pjp was based upon stringent criteria defined by a multidisciplinary team; (ii) only hematological patients were included; (iii) a comprehensive routine investigation of microbial causes of pneumonia other than pj was conducted; (iv) the experience of two centers was collected. in addition to its retrospective nature, our study also has some limitations: (i) we cannot completely rule out that some patients categorized as being pj carriers did in fact have pjp, as most of these patients received full courses of tmp/smx in combination with antimicrobials targeting other microbial agents. the lack of standardized criteria for pjp diagnosis makes clinical misclassification of patients a potential drawback in studies such as ours, particularly when no positive microscopy or histopathology findings are available; (ii) although we evaluated bal, bronchoalveolar lavage; pjp, pneumocysis jirovecii pneumonia; ta, tracheal aspirate. a as per our routine protocol, all specimens were examined by gram and acid-fast bacilli stain. samples were also examined for presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, or the clart® pneumovir assay (genomica, coslada, spain) at both centers, as previously reported. 10 semiquantitative (sputa) and quantitative (bal and ta) cultures for bacteria were performed on conventional media: bacterial loads > 10 4 cfu/ml or > 10 5 cfu/ml were deemed to be clinically relevant on bal fluids and ta samples, respectively. specimens were cultured on bcye-alpha agar, bd (becton dickinson) mgit® ( mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and other fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. all bal fluid specimens underwent cytomegalovirus (cmv) pcr testing using the realtime cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf, as previously reported. 10 over 200 patients, only 12 presumptively had pjp; (iii) two different commercially-available pcr assays were used across centers. nevertheless, we found them to yield rather comparable c t s. in summary, we found that a positive pj pcr result in respiratory specimens from transplant and non-transplant hematological patients with pneumonia frequently reflects colonization rather than infection; pcr c t values in bal fluids, serum ldh levels and lack of co-detection of other microorganisms potentially involved may be helpful in clinical categorization in the absence of positive by pj microcopy results. we have no conflict of interest to declare. dear editor , poller et al., in this journal, provided a useful consensus for use of personal protective equipment for managing high consequence infectious disease 1 . although this was driven largely by recent ebola virus disease emergencies, we should remind your readers of the continuing problem of lassa fever (lf) in west africa. lf is a febrile infectious disease caused by lassa virus. the clinical presentation of the disease is nonspecific and includes fever, fatigue, hemorrhage, gastrointestinal symptoms, respiratory symptoms, and neurological symptoms 2 . the observed case fatality rate among patients hospitalized with severe lf is 15-20% 3 , 4 . the disease is mainly spread to humans through contamination with the urine or faeces of infected rats 2 . human-to-human transmission can occur through contact with the body fluids of infected per-sons. therefore, health care workers are at high risk for infection when the standard precautions for infection prevention and control including appropriate personal protective equipment are inadequate 5 . it is estimated that there are approximately 30 0,0 0 0 lf cases annually, resulting in approximately 50 0 0 deaths in west african countries 4 . in 2018, nigeria had a large lf outbreak, and we previously reported epidemiological characteristics of the outbreak, analyzing data collected between january and may 2018. 6 however, information on laboratory-negative suspected cases was not enough to conduct a case-control study to fully determine the risk factors and clinical characteristics of the disease. nigeria had a lf outbreak in 2019 as well. 7 here we report the epidemiological and clinical characteristics of the outbreak including case-control analysis against laboratory-negative suspected cases using data collected between 1st january 2018 and 6th october 2019. from january to december 2018, there were 3498 suspected cases, including 633 laboratory-confirmed lf cases. in 2019, there were 4019 suspected cases reported by 6th october, including 721 laboratory-confirmed lf cases. details on the case definition, laboratory test, surveillance, and data collection have been described previously. 6 of the confirmed lf cases, there were 171 fatalities (case fatality rate, 27.0%) in 2018 and 154 fatalities (case fatality rate, 21.4%) in 2019. the number of laboratory-confirmed lf cases and positivity rate peaked in the dry season (january-march) in both 2018 and 2019 ( fig. 1 (a) ). the largest number of laboratory-confirmed lf cases were reported from the neighboring edo and ondo states in both 2018 and 2019 ( fig. 1 (b) ). there were laboratory-confirmed lf cases in states such as kebbi and zamfara that had no reported cases previously, in 2019. during the study period, the detailed demographic and clinical information was collected for 1305 laboratory-confirmed lf cases (of 1354 cases, 96.4%) and 3350 laboratory-negative suspected cases (of 6163 cases, 54.4%). chi-square tests were conducted to compare the distribution of age, sex, and each symptom between the laboratory-confirmed lf cases and laboratory-negative suspected cases ( table 1 ). the proportion of children was significantly lower in laboratory-confirmed lf cases compared with that in laboratory-negative suspected cases. the proportion of males was significantly higher in laboratory-confirmed lf cases than that in laboratory-negative suspected cases. fever was the most prevalent symptom in both laboratoryconfirmed lf cases and laboratory-negative suspected cases, followed by headache ( table 1 ) . gastrointestinal symptoms, such as abdominal pain, vomiting, and diarrhea, were observed in more than 30% of laboratory-confirmed lf cases, whereas hemorrhaging was observed in 21.1% of laboratory-confirmed lf cases. while the prevalence of face/neck edema was low even in laboratoryconfirmed lf cases (6.4%), nonetheless, the odds ratio of having face/neck edema was 6.0 times high for laboratory-confirmed lf cases. we here reported the lf outbreak in 2018-2019 largest recorded in history. while previous studies have focused on laboratory-confirmed lf cases and mainly compared fatal cases and survived cases, 6 , 8 , 9 our observation revealed the difference between laboratory-confirmed lf cases and laboratory-negative suspected cases. the age and sex distribution differed significantly between laboratory-confirmed lf cases and laboratory-negative suspected cases. fever, headache, and gastrointestinal symptoms were the most common symptoms in laboratory-confirmed lf cases, which are similar to those reported previously. 6 , 8 however, these symptoms were also prevalent in laboratory-negative suspected cases. clinical guidelines for lf state that edema in the face and neck is a specific sign of the disease. 10 the present study found that the symptom had a significantly high odds ratio for confirmed lf although the prevalence of this symptom was low. unfortunately, we did not determine the differential diagnosis for the laboratory-negative suspected cases. laboratory tests for the differential diagnoses are now underway for the lf-negative samples collected during the outbreak. the results would provide us further insight for better clinical management of patients with febrile illnesses in lf-endemic areas. in addition to the standard precautions for infection prevention and control including appropriate personal protective equipment pointed out by poller et al., 1 it is important to know epidemiological and clinical characteristics of high consequence infectious diseases such as lf. that would help healthcare workers and public health officers increase an index of suspicion of the diseases, further leading to better clinical management and surveillance. the authors have declared that no conflicts of interest exist. this work was partially supported by the leading initiative for excellent young researchers from the ministry of education , culture, sport, science & technology of japan and the japan society for the promotion of science (grant number, 16809810 ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 these authors contributed equally to this article. accepted 31 december 2019 available online 15 january 2020 https://doi.org/10.1016/j.jinf.2019.12.020 © 2020 the british infection association. published by elsevier ltd. all rights reserved. recently, several studies in this journal have demonstrated the threat of animal-derived viruses to humans. [1] [2] [3] since 2018, an increase in human pseudorabies virus (prv) infection cases has been reported in china, indicating a new animal-derived virus threat to human health. porcine pseudorabies (pr), also known as aujeszky's disease, is one of the most economically important viral diseases in pigs globally. its causative agent is prv, which is classified into the genus varicellovirus of subfamily alphaherpesvirinae , family herpesviridae . prv is almost always fatal in newborn piglets, is frequently accompanied by neurological symptoms, and may cause abortions and/or stillbirths in pregnant sows. prv primarily infects members of the suidae family and can also infect other domestic and wild mammals, including horses, cattle, sheep, goats, dogs, cats, etc. currently, vaccination is the most effective strategy for pr prevention and control in pigs worldwide. in china, prv infections in pigs were first recorded in 1947. in the 1970s, an inactivated vaccine consisting of the bartha-k61 vaccine strain was imported into china. since then, this vaccine has been widely used in pig vaccination for pr prevention and control. before 2011, no large pr outbreaks were reported in pigs in china. however, after late 2011, novel prv wild-type variants emerged in nearly all regions of china and affected a number of swine herds vaccinated regularly with the bartha-k61 vaccine, resulting in significant economic losses. 4 subsequent animal experiments indicated that the bartha-k61 vaccine could not provide complete protection for pigs against a challenge with novel prv wild-type variants in china. for control and eradication of pr, the disease was listed in the "mid-and long-term animal disease prevention and control program in china (2012-2020)" by the chinese government, with the aim of eliminating pr in china by 2020 ( http://www.gov.cn/zwgk/2012-05/25/content _ 2145581.htm ). however, vaccination for prv is still voluntary and not required in china. a nationwide epidemiological investigation in 2014 demonstrated a high prevalence of 23.26% of prv among swine herds in china. 5 humans were previously regarded as refractory for prv infection, although serological prv antibody positivity was found in three cases. in 2018, the first human prv infection case with direct molecular evidence was reported in china (case 1, table 1 ). 6 in this case, the eyes of a 46-year-old woman were directly exposed to sewage on a hog farm. in the following two weeks, symptoms of fever, headache, coma, and endophthalmitis were observed in the patient. next-generation sequencing (ngs) indicated that prv dna was detected in her vitreous humor samples but not in her cerebrospinal fluid (csf). after surgery, the patient was discharged, but her vision remained impaired. in a subsequent study, zhao et al. table 1 clinical characteristics and other information on the twelve human prv infection cases in china. analyzed csf samples from four patients with encephalitis of unknown etiology using ngs (cases 2-5, table 1 ) and found molecular evidence of prv infection. 7 in addition, retinitis and blindness was observed in two cases (cases 2, 5, table 1 ), and the patient in case 3 died. the occupation of the four patients was all associated with pork production/sale/cooking. in 2019, six other human prv infection cases involving encephalitis were reported in china, and all patients were pork/pig handlers or veterinarians. [8] [9] [10] it was noted that all patients still suffered from various sequelae after discharge, except for in one case where the patient died. increasing reports on human prv infection cases in china have recently indicated that prv poses a significant threat to public health in china, especially in people in close contact with sick pigs and/or related pork products/contaminants. to reduce the risk of prv infection in susceptible workers, it is necessary to promulgate relevant policies by the chinese government to promote pr vaccine development to protect pigs from infection with novel prv wild-type variants currently circulating in china. in addition, relevant policies should be updated by the chinese government to monitor vaccination status and virus variation in pigs nationwide. moreover, it seems that prv can infect humans via injury to the skin or eyes. until now, no effective drugs to prevent the progression of the disease caused by prv infection have been reported. therefore, it is necessary to improve biosafety and self-protection awareness in susceptible populations that have contact with sick pigs and work in jobs related to handling pork products/contaminants. promoting drug development for curing prv-related disease in infected patients may also help reduce the currently increasing threat of prv to human health in china. all authors declare that they have no competing interests. dear editor, the emergence and spread of gram-negative bacteria, for example, klebsiella pneumoniae , co-producing carbapenemases and mobilized colistin resistance ( mcr ) genes limit our choice for treating multidrug-resistant infections, posing significant threats to public health. herein, we reported the discovery of mcr-9 gene in 28 k. pneumoniae strains isolated from patients in eight european countries, including belgium ( n = 2), denmark ( n = 1), montenegro ( n = 13), poland ( n = 1), romania ( n = 1), serbia ( n = 1), slovenia ( n = 1), and spain ( n = 7). notably, the co-existence of mcr-9 and the carbapenemase-encoding genes, ndm-1, vim-1, and oxa-48 were confirmed in k. pneumoniae isolates of human origin. phylogenetic analysis suggested that mcr-9 -carrying k. pneumoniae isolates, including 23 carbapenem-resistant and five susceptible k. pneumoniae strains, show a highly geographically clustered pattern. genetic environment analysis revealed the presence of insertion element is903, is1 or a cupin fold metalloprotein, wbuc, in the mcr-9 flanking. taken together, these findings indicated that mcr-9 has existed for a long time and already spread among crkp isolates of human origin in europe since 2013, further increasing the significant threat of public health through either the nosocomial spread or environmental routes. the mobilized colistin resistance ( mcr ) gene mcr-9 was detected in 28 human gut microbiomes, which has been disseminating across three continents, including asia, europe and america, recently published in the journal of infection. 1 the rapid increase in carbapenem resistance among gram-negative bacteria worldwide has greatly compromised the efficacy of carbapenem antibiotics, which has gotten renewed attention to the importance of polymyxin antibiotics for multidrug-resistant (mdr) infections. recently, sophia david and colleagues 2 reported the epidemic of carbapenem-resistant klebsiella pneumoniae (crkp) in europe, which raised concern that mobile carbapenemase resistance determinants were widely spread in european hospital settings, and inter-hospital spread is far more frequent within, rather than between, countries. however, limited information regarding the co-occurrence of carbapenemases and mcr genes in the klebsiella pneumoniae ( k. pneumoniae ) isolates have been provided. plasmid-mediated resistance genes mcr as well as tet (x3/x4) have been widespread in bacterial species of animal, human, and environment origin as well as human and animal gut microbiomes worldwide, where is a huge arg reservoir with a high horizontal gene transfer possibility. 1 , 3 -5 several studies 6 -8 illustrated that the newly described mcr-9 has spread beyond the united states into europe and asia, and into other enterobacteriaceae species. of clinical concern is the inevitable spread of a plasmid harboring the mcr-9 gene into a crkp isolate, which has been listed by the world health organization as a critical priority antibiotic-resistant bacterial pathogen for which new antibiotics are urgently needed. 9 in response to this potential clinical problem, we download > 1717 k. pneumoniae genomes isolated from 244 hospitals in 32 european countries 2 and the 14,764 complete bacterial genome sequence (accessed 26 july 2019), and explored the distribution of plasmid-mediated resistance genes mcr and tet (x3/x4). we are surprised to find that the complete mcr-9 gene (nucleotide = 100%) was present in 28 k. pneumoniae isolates of 1717 k. pneumoniae strains from belgium ( n = 2), denmark ( n = 1), montenegro ( n = 13), poland ( n = 1), romania ( n = 2), serbia ( n = 1), slovenia ( n = 1), and spain ( n = 7) (table s1 ). additionally, 23 of the 28 k. pneumoniae isolates of human origin were crkp strains and only five were carbapenem-susceptive isolates (table s1 ). as reported, these mcr-9 -harbouring strains were isolated from patients in europe between 2013 and 2014. these results suggest that mcr-9 gene might have been presented in europe for a long time and already spread to the crkp isolates, which is a major cause of both hospital-and community-acquired infections. 2 to further analyze these mcr-9 -positive k. pneumoniae isolates, the resistome of the draft genome was analyzed using the comprehensive antibiotic resistance database. 10 interestingly, the mcr-9 gene was co-existed with various carbapenemase-encoding genes: in eleven isolates with ndm-1, eight with vim-1, and two with oxa-48 ( fig. 1 (a) ). it should be noted that the mcr-9 -and ndm-1-carrying isolates were distributed in denmark ( n = 1), montenegro ( n = 8), romania ( n = 1), and serbia ( n = 1), as well as several other beta-lactam resistance determinants (for example, tem-1, cmy-16, oxa-10 and shv-11) ( fig. 1 (a) and table s1 ). moreover, the mcr-9 -and vim-1-harbouring isolates were dominant in spain ( n = 7) and slovenia ( n = 1), as well as two beta-lactamase-encoding genes (non-carbapenemases) (ctx-m-9 and shv-11) ( fig. 1 (a) and table s1 ). it is worrying that a crkp isolate from spain in 2014 was carrying mcr-9 , vim-1, and oxa-48 genes simultaneously. the presence of mcr-9 in crkp isolates from patients is of critical importance as mcr-9 could be present in hospital-borne outbreaks cre strains in the future. from whole-genome shotgun (wgs) data of the 28 mcr-9 -positive k. pneumoniae isolates, sequences types (sts) were extracted and assigned to nine different types, i.e. , 274, 461, 15, 16, 416, 1890, 37, 1942, and 147 ( fig. 1 (a) ). phylogenetic analysis suggested that mcr-9 -carrying k. pneumoniae isolates show a highly geographical clustering pattern 2 ( fig. 1 (a) ). isolates from patients in the same hospital were clustered into one clade, for example, in spain and montenegro. overall, the k. pneumoniae isolates from different countries were genetically diverse, suggesting that the mcr-9 -positive k. pneumoniae isolates were also genetically diverse and that mcr-9 could disseminate among different k. pneumoniae isolates, mainly by nosocomial transmission. 2 nowadays, all known mcr genes have been detected in various gram-negative bacterial species, whereas a small number of studies have shown the presence of mcr-1, mcr-3, mcr-7 , and mcr-8 in k. pneumoniae isolates from animal and human origin at relatively low detection rate. 11 -14 the presence of mcr-9 in the crkp isolates indicated that this novel mcr gene may already be widely spread among k. pneumoniae isolates of human origin in europe. we subsequently searched mcr-9 gene in 14,764 complete bacterial genome sequence and ncbi-nr database (14 october 2019) in the ncbi, to fully understand the prevalence of mcr-9 gene in klebsiella species isolates. interestingly, the mcr-9 gene (identity > 99% and 100% query coverage) was present in various bacterial genomes, including three klebsiella species isolates consisting of k. pneumoniae ( n = 4), k. quasipneumoniae ( n = 1), and k. oxytoca ( n = 3) (table s3) . therefore, further studies focusing on the epidemiology and transmission mechanism of mcr genes, in particular mcr-9 in klebsiella species of human origin are warranted to better understand the public health threat of emergence of antibiotic resistance among clinical k. pneumoniae . contigs carrying mcr-9 in 28 k. pneumoniae isolates could be classified into two groups (for example, gca_900513855.1 and gca_900501685.1) (table s2) . genetic environments analysis indicated that the presence of insertion element is903 and wbuc (a cupin fold metalloprotein), in the mcr-9 (gca_900513855.1, ∼28 kb) upstream and downstream flanking, respectively, similar to (identity > 99%) the plasmid sequences of pme-1a, pctxm9_020038, and pmrvim0813, and contigs from of e. coli isolate 68a and nz_naan010 0 0 063 from salmonella 15 ( fig. 1 (b) ). additionally, mcr-9 in another contig ∼8.7 kb was in the upstream of two insertion element is1 and is30 0 0, as well as a beta-lactamase-encoding resistance gene ctx-m-9, which similar to the plasmid sequence of pmrvim0813. we did not detect the downstream regulatory genes (qsec and qseb) found in the isolates that harbor mcr-9 . 8 , 16 moreover, we were unable to determine whether a complete is903 element is upstream due to a short mcr-9 -bearing contig that is available for comparison ( fig. 1 (b) ). therefore, a long-read sequencing coupled with a hybrid assembly method is needed to fully evaluate and monitor the transfer and development of args, especially mcr-9 among crkp isolates. although two unique plasmid-mediated tigecycline resistance genes firstly discovered in bacteria of animal origin in china and subsequently identified in many bacterial isolates of human, animal and environment origin, including klebsiella species, as well as human and animal gut microbiomes, 1 , 4 , 5 none of them was detected in the 1717 k. pneumoniae strains in europe. in summary, we reported the discovery of mcr-9 gene in 28 clinical k. pneumoniae strains of human origin in eight european countries. importantly, the mcr-9 gene was co-existed with different carbapenemase-encoding genes in the same strains. the spread of mcr-9 , ndm-1, vim-1, and oxa-48 and other beta-lactam resistance determinants (non-carbapenemase) carrying by crkp appears likely to be by plasmid dissemination, as the genes identified in isolates belonging to a diverse set of sts distributed in different hospitals in europe. it is noteworthy that all these mcr-9 -positive crkp strains were isolated between 2013 and 2014, highlighting an earlier presence of mcr-9 among crkp around the world than previously known. these findings raise the likelihood of ongoing undetected mcr-9 gene spread among cre strains. therefore, further study is urgently needed to understand the prevalence and dissemination of mcr-9 , especially in cre and crkp strains, and effective measures should be taken to control its spread. g.f.g. designed the study. y.n.w. and f.l. collected and downloaded the datasets. y.n.w., f.l., y.f.h., b.l.z., g.p.z., and g.f.g. analyzed and interpreted the data. y.n.w. and g.f.g. wrote the draft of the manuscript. all authors discussed, reviewed and approved the final report. supplementary information is available for this paper. correspondence and requests for materials should be addressed to g.f.g. the authors declare no competing interests. the interesting systematic review by amin-chowdhury and colleagues provides information about outbreaks of severe pneumococcal disease (spd) in closed settings that occurred in the conjugate vaccines era 1 . it shows that vaccine-type spd outbreaks are still occurring and it highlights the lack of consensus on how to manage such outbreaks. in the following, we will describe how we managed a recent outbreak of spd in norway. in march 2019, møre and romsdal hospital trust notified the norwegian institute of public health (niph) about a cluster of spd amongst men working in shipyards in møre and romsdal county. serotype data from niph were available for nine of the cases -all were serotype 4. the majority of cases had been working at one specific shipyard. municipal medical officers (mmo), the norwegian labour inspection authority (nlia), and niph formed a multidisciplinary outbreak team to investigate and control the outbreak. we formed specific case definitions: each case had to have resided in møre and romsdal county in the period from 1. january 2019 onwards and: confirmed : had invasive pneumococcal disease (ipd) with serotype 4 isolated from a normally sterile site. probable : worked at the specific shipyard and had a clinical presentation compatible with lower respiratory tract infection or ipd, but without microbiological confirmation or serotype 4 isolated from a non-sterile medium (e.g. nasopharyngeal swab or sputum culture). we identified 20 cases, ten confirmed and ten probable in the period between 28. january and 3. april ( fig. 1 ). all available isolates were serotype 4 (10 confirmed, 7 probable) and were susceptible to penicillin. fifteen isolates were sequence type (st) 801, while two were a single locus variant of 801, st 15,063. all cases were men between 20 and 60 years, with a mean age of 47 years. fifteen were hospitalized. four were norwegian citizens, the remaining came from other european countries. seven cases smoked. one case had an underlying medical risk condition. immunization history against pneumococci were unknown for all. the cases had several professions; mostly related to interior outfitting and metal welding. approximately 1800 individuals worked at the shipyard in the time period. many of them lived in temporary accommodation. at an on-site inspection of the shipyard, nlia observed a polluted atmospheric work environment and little use of personal protective equipment. several measures were put in place to control the outbreak, including information and advice to raise symptom awareness and to reinforce hand and respiratory hygiene, vaccination and occupational corrections. local medical clinics and hospital were alerted about the outbreak and advised to have a low threshold to admit and treat suspected cases. mmo held information meetings with shift leaders, and written information about spd in several languages was distributed to workers to increase spd awareness. intensified hygiene measures were implemented at the ship yard and housing quarters. nlia ordered immediate occupational corrections related to controlling the atmospheric work environment. niph recommended vaccination with the 13-valent conjugate vaccine (pcv13) to interrupt transmission and prevent disease. both the pcv13 and the 23-valent polysaccharide vaccine provide protection against serotype 4, but pcv13 was preferred as this may also affect colonization. as several work tasks were conducted in parallel process in confined spaces with suboptimal ventilation, we were unable to identify a single target group for vaccination. hence, the shipyard offered vaccination to all workers. occupational health service promptly vaccinated all workers during a four-day period. contrary to the majority of studies included in the systematic review, niph did not recommend chemoprophylaxis. as the workers were otherwise healthy (i.e. no high risk group like old age, immunocompromising conditions etc.), and since it was impossible to target a specific group of workers, niph deemed it undesirable to distribute antibiotics to 1800 asymptomatic workers, with the possibility of inducing antimicrobial resistance and possible side effects. due to high turnover of personnel it was not possible to calculate an attack rate. we did not find any new cases after control measures were implemented. no deaths have been reported in relation to the outbreak. this outbreak closely resembles one of the outbreaks described in the systematic review; between april and june 2015, an outbreak with serotype 4, st 801 occurred at a shipyard in belfast 2 . we are also aware of an outbreak this fall, 2019, at a shipyard in finland with serotype 4 (st 801), 8 and 12f 3 . although welders are a known risk group for spd, 4 in all these three outbreaks, people who worked closely alongside welders were also infected. in addition to exposure to welding fumes, the crammed and poorly ventilated working conditions, and possibly housing conditions, may have increased the risk of developing spd and facilitated the transmission of pneumococci in this closed setting. overall, this norwegian outbreak extends the knowledge about how to manage and control outbreaks of spd in closed settings. none. in this journal brunet and colleagues 1 discussed reactivation of latent infections in the context of chronic disease, solid organ transplantation or long-term immunosuppressive treatment. we recently observed the reactivation of leishmania infection in a 46-year-old patient receiving methotrexate for psoriasis, who was diagnosed with visceral leishmaniasis (vl) showing a mucocutaneous involvement. we analyzed the epidemiologic and clinical characteristics of all cases of leishmaniasis in patients with psoriasis found through a review of the literature. our patient was admitted into the infectious disease unit of paolo giaccone hospital, in palermo, with a painless and ulcerated lesion onto the oral mucosa ( fig. 1a ) , two nodular ulcerated lesions on the right knee and another one on instep of the right foot appeared one month before ( fig. 1b ) . the patient did not travel outside italy during the last year. he had been suffering from lowgrade fever in the last month. considering the above findings leishmaniasis was suspected and a needle aspiration of oral and cutaneous lesions was arranged in order to perform microscopy and leishmania-pcr, which were positive for leishmania. laboratory tests exhibited: wbc 4970/mmc, hb 13.9 g/dl, c reactive protein, 26.9 mg/l; positive serology for leishmania (igg 1/3200) and positive leishmania-pcr test on peripheral blood. abdominal us examination revealed splenomegaly (14 cm); methotrexate was suspended and liposomal amphotericin b, 4 mg/kg per day for 10 days, followed by two further administrations two weeks later was started. cutaneous and mucosal lesions improved at the end of the first 10 days of therapy and completely vanished after two further administrations, 40 days from the beginning of treatment. leishmania-pcr on peripheral blood after 10 days of therapy was negative. table 1 shows the literature data about characteristics, therapy and outcome of patients with psoriasis and leishmaniasis. leishmaniasis is a vector-born chronic infectious disease caused by protozoa of the genus leishmania and transmitted to humans by the bite of phlebotomine sandflies. in europe, the mediterranean countries are the most affected areas. leishmania parasite establishes chronic intracellular parasitism, survives for an infected person's lifetime and, in the event of major immune deficiency, may be reactivated from sites of latency. leishmaniasis can present with a spectrum of clinical manifestations and three patterns of infection are described: cutaneous (cl), mucosal or mucocutaneous (ml or mcl) and visceral leishmaniasis (vl). the infecting species of leishmania is very important in determining the clinical manifestations and the host immune response is crucial in determining the clinical outcome of infection 2 . today, non-hiv related immunosuppressive conditions are becoming increasingly prevalent, mainly because of better medical care of patients with chronic illnesses and the therapeutic use of immunosuppressive drugs. in the field of rheumatology, leishmaniasis has been reported in association with the use of various immunosuppressive drugs. 3 the introduction of tumor necrosis factor-alpha (tnf-α) antagonist drugs has received much attention recently and several cases of vl have been reported in rheumatic patients who do anti-tnf α drugs. 4 psoriasis is a chronic inflammatory autoimmune disease affecting 2-3% of the world's population and characterized by an aberrant hyper-proliferation of keratinocytes. the pathogenesis of psoriasis is complex. genetic susceptibility, environmental triggering factors and an over-reaction of local innate immune response initiate inflammation. subsequent involvement of adaptive immune response with production of th 1 cytokines, chemokines and growth factors lead to epidermal hyperplasia. 5 recently, a functional role of interleukin-17-producing t helper cells (th17) in psoriasis has been suggested by their reduction during successful anti-tnf treatment. 6 it is also known that th17 lymphocytes play an essential role in protecting against intracellular protozoa and in the successful clearance of leishmania by strengthening the th1 response. 7 in view of this, it could be argued that psoriasis may represent a protective factor for leishmania infection. indeed, in our review we did not found any case of leishmaniasis in psoriatic subjects who were not under immunosuppressive therapies. biological agents, which are powerful immunosuppressive drugs, have been more and more used in rheumatic patients and leishmania infections have been reported among anti-tnf-agents users. 8 recently maritati et al. found higher prevalence of subclinical leishmaniasis in patients with inflammatory rheumatic diseases receiving biological drugs than those treated with other immunosuppressive drugs. 9 however, leishmaniasis has also been reported in psoriatic patients not receiving biological drugs, as occurred to our patient ( table 1 ) . diagnosis of cl in psoriatic patients is challenging, as it mimics many other infections or a flare-up of psoriasis itself that can lead to ineffective and harmful changes of therapy. immunosuppressive therapies cause atypical manifestations of leishmaniasis with large lesions spread over large cutaneous areas and associated to a possible mucosal involvement. ml by l. infantum is very rare and only sporadically described in patients receiving powerful immunosuppressive therapies or in hiv-coinfected patients. mcl is mostly observed in latin america where l. braziliensis accounts for most cases, but l. panamensis, l. guyanensis, and l. amazonensis have also been implicated. only rarely cutaneous lesions extend to areas of skin distant from the mucosa involved, as in our case in which two lesions on the foot and knee were associated with the oral lesion. in the context of impaired immunity, it is also advisable to rule out vl by pcr-leismania on peripheral blood so as to establish the most appropriate therapy: intralesional or intravenous. finally, there is no agreement on appropriate screening for leishmaniasis before immunosuppressive treatments and on the strategy to be followed after the diagnosis of leishmaniasis in rheumatic patients taking immunosuppressive drugs. 10 molecular methods are highly sensitive and specific tools for the diagnosis of visceral leishmaniasis and a screening with leishmania-pcr in immunosuppressed patients living in endemic areas could be useful to identify patients at highest risk of reactivation. 9 specific leishmaniasis treatment followed by suspension of the immunosuppressive therapy was adopted by most of the authors. overall even if the treatment response is not as good as seen in the immunocompetent population, our review reports a good outcome in all cases and patients remained relapse-free without maintenance therapy and despite the ongoing use of immunosuppressive medication. in conclusion physicians must be alert to the possibility of development of leishmaniasis in immunosuppressed rheumatic patients. adequate screening for vl should be incorporated into the list of baseline studies to carry out before initiating biologic therapies, at least in endemic areas. the authors declare that there is no conflict of interest. as demonstrated in several studies in journal of infection , herpesviruses pose an increasing threat to human health. [1] [2] [3] according to international committee on taxonomy of viruses (ictv), equine herpesviruses (ehvs) belong to the family herpesviridae . until now, a total of 9 ehv species types have been determined in equines, viz. ehv1-ehv9. 4 among them, ehv1 and ehv4 are the most relevant herpesviruses affecting equines. both ehv1 and ehv4 infection are associated with upper respiratory tract disease, but only ehv1 infection could cause abortion and myeloencephalitis. ehv1 and ehv4 are prevalent in equines on all continents and have considerable economic impact on the horse industry. 5 in china, the number of equines is very large, reaching to be ∼ 6.8 million in 2018 ( http://www.stats.gov.cn/tjsj/zxfb/ ). ehv infection in equines was first reported in china in 1980, and the epidemiological investigation since then indicated ehv was prevalent in the equine population in all the studied 16 provinces in mainland china, with a seroprevalence ranging 8% −92%. [6] [7] [8] vaccination is commonly used to prevent and control ehv. however, china has not developed a commercially available ehv vaccine so far. ehv vaccine has a limited market application potential in china currently. due to the lack of relevant knowledge on ehv, most of the chinese horse owners always erroneously identified it as other common pathogen of equine respiratory diseases, and didn't realize its potential threat to equine health and reproduction. although the number of equines in china is large, most of them are labor/farming horses. to the best of our knowledge, even for racehorses, vaccination with ehv vaccine has not been performed in mainland china. considering the wide distribution and high prevalence of ehv in china, it is urgently to popularize knowledge on ehv in horse owners and promote market application prospects of ehv vaccine. in china, few veterinary researchers are currently investigating equine virus, including ehv. this is mainly caused by the change of equines' historical role. in the last century, a great number of equines were used for military in china. however, there is only one military equine farm in mainland china at present. considering a more important economic role of other domestic animals (e.g., pigs, chickens, and cattle) compared with equines, investigating equine virus (including ehv) is not a priority in the related guide policies issued by the chinese government. though epidemiological studies on ehv in china are limited, it still could be concluded that epidemic status of ehv is very complicated in china, which increases the difficulty in ehv vaccine development. in most provinces, ehv1 and ehv4 were co-circulating in equines with a high seroprevalence. 8 until now, a total of 7 ehv1 strains have been isolated from tissue samples of aborted equine fetuses (5 from farming horses in northeast china in 1980, 1 from asian wild horses in western china in 1999, 1 from farming horses in western china in 2016). 6 , 8 in addition, a novel ehv8 strain was isolated from one horse with serious respiratory disease in northern china in 2010. 9 recently, our laboratory firstly determined the molecule evinces for ehv4 and ehv5 in racehorses in sothern china (data not shown). however, a more large-scale and surveillance of ehv in equines is necessary to fully understand epidemic status of ehv in china, which could establish a foundation for updating the composition of ehv vaccine developed in china in future. in other countries, much effort has been made to develop ehv vaccine, and modified-live and inactivated virus vaccines have been registered for sale. 10 before an ehv vaccine is developed successfully in china by itself, it is necessary to vaccinate the susceptible equine population with an ehv vaccine commercially available from other countries to prevent and control ehv in china. however, a well-designed case-control animal challenge study still needs to estimate the protective efficacy of different vaccines against the field prevalent ehv strains in china. all authors declare that they have no competing interests. a recent review article on the treatment of hepatitis c with directly-acting antiviral (daa) drugs, makes numerous recommendations for baseline drug resistance testing. 1 in our local practice, we have been performing baseline drug resistance testing for some years now, prior to the publication of these guidelines. we present a recent retrospective hcv kinetics analysis of these patients' changing viral loads in response to daa therapy below. such studies have been used previously to compare viral suppressive responses in different hcv genotypes and treatment regimens. 2 , 3 the patients were a mixture of treatment-naive and treatment-experienced (including with interferon-based, ns3 protease inhibitor-based and daa-based regimens) cases. the current standard of care for hepatitis (hcv) patients is a combination of direct acting antivirals (daas), for which there are three different hcv viral protein targets (ns3, ns5a and ns5b). table 1 ns3, ns5a, ns5b resistance associated substitutions (ras), by hcv genotype, found in this patient cohort at baseline drug resistance testing (viral sequencing performed at imperial college, london, uk). the patients included a mixture of treatment-naïve and treatment experienced (i.e. interferon-based, ns3 protease inhibitor-based and more recent daa-based regimens) cases. resistance associated substitution (ras) by hcv genotype treatment with daas cure the vast majority of hcv-infected patients, with oral regimens having > 95% efficacy in most patient groups. 1 , 4 , 5 treatment failure currently affects approximately 5% of treated patients and is often associated with the selection of resistance associated substitutions (ras). 1 we performed hcv drug resistance testing both retrospectively (following treatment failures) and prospectively (prior to treatment) in our cohort of hcv genotype (g) 1-3-infected patients, during march 2015-june 2018. viral extraction, pcr and sequencing were performed at imperial college, using qiagen viral rna mini kits (qiagen pn:52906, qiagen ltd., manchester, uk), and inhouse pcr and sanger sequencing methods on an abi prism 3100-avant genetic analyser (thermo fischer scientific, loughborough, uk). the prediction of hcv genotype and drug sensitivities is derived from the geno2pheno algorithm [ www.geno2pheno.org ]. 6 treatment regimens used during this period complied with contemporaneous nhs rate cards: for non-cirrhotic or compensated cirrhotic patients: g1-treatment-naive: omb/par/rit + das + r; g1-treatment-experienced: elb/grz + /-r; g2-treatmentnaive/experienced: gle/pib; g3-treatment-naive/experienced: gle/ pib; g1-decompensated cirrhotic patients sof/led + r; g2/g3decompensated cirrhotic patients: sof/vel + r. we assessed the impact of any ras across g1-g3 on hcv rna kinetics by analysing viral load (realtime hcv viral load, abbott m20 0 0, abbott molecular uk, maidenhead, england) decline rates. we applied linear mixed regression to model the viral loads and assumed a linear decline (log 10 scale) over time, using sas statistical software (sas institute inc., nc, usa). in this cohort of 54 patients (n: g1 = 21, g2 = 6, g3 = 27), hcv ras were found as shown in table 1 . hcv rna viral load decline rates were found to be similar and not statistically different ( p = 0.760) at: −2 log 10 and −1.8 log 10 per month, respectively, for g1 and g2/g3 ( fig. 1 ). this suggests that these viral load decline rates were similar across g1-g3 infections despite baseline differences in viral load, ras profile, or a history of any previous treatment (i.e. interferon-based, older ns3 protease inhibitor-based, or more recent daa regimens). these results demonstrate similar hcv rna clearance efficacies of the various daa treatment regimens for g1-g3, in this patient cohort. although other studies on hcv kinetics have been published, they do not usually compare multiple hcv genotypes. 7 similar studies on patients infected with g4-6 viruses, and/or undergoing other daa treatment or retreatment combinations, 8 , 9 will be with great interest we have read the report of zhang et al. concerning the increased susceptibility to pertussis in chinese adults at childbearing age, as determined in a comparative seroprevalence study using samples collected from 2010 to 2016. 1 the authors describe that about 5% of the individuals had pt-igg antibodies, which is indicative of a recent infection. in the adults 20-39 years of age, 29.1% subjects had undetectable pt-igg antibodies in 2010 but 57.4% in 2015/2016. it is well known that adolescents and adults have become the reservoir of pertussis and an important source of transmission to vulnerable infants. bordetella pertussis is commonly associated with atypical pneumonia as determined in hospitalized children. 2 several seroprevalence studies conducted in different regions of china indicate that the incidence of pertussis is most likely underestimated. 3 , 4 this may be due to the use of insensitive diagnostics. at present, the diagnosis of pertussis in china is mainly based on culture. however, both the cdc and the world health organization (who) use pcr as the gold standard for diagnosis, in addition to culture. 3 oropharyngeal or nasopharyngeal swabs were obtained from 17,104 inpatients aged between 10 days and 14 years of age with clinical suspicion of pertussis, enrolled from march 2014 to february 2018 in shenzhen children's hospital. more than 50% of all patients were younger than 6 months of age. the hospitalized patients included 10,894 males and 6210 females (sex ratio, 1.75). all patients recovered after the treatment. a real time pcr assay targeting ptxa-pr was used to detect b. pertussis . of the 17,104 samples, 772 (4.51%) tested positive for b. pertussis by rt-pcr. our results indicate that despite vaccination pertussis remains a major health problem in china, since the prevalence of infection by b. pertussis in hospitalized children was high. the majority of patients were admitted because of pneumonia. the detection rate in hospitalized patients was lower than the rates reported earlier in shanghai and ji'nan. 3 , 4 this may be due to lower number of samples collected in these studies and due to the use of serology or culture methods. the overall prevalence rates were 3.97% and 5.48%, respectively. however, b. pertussis infection in female patients was significantly higher than in male patients (x 2 = 20.913, p < 0.001). this has earlier been reported by the ecdc 5 and haberling et al. 6 and may point to a genetic association with susceptibility to b. pertussis . the detection rates were dependent on age in patients (x 2 = 139.8, p < 0.001). the prevalence decreased with age: 8.71% newborns, 5.33% in infants, 2.39% in toddlers, 1.28% in (pre-) schoolers ( fig. 1 ) . the high vulnerability of newborns and young infants for b. pertussis infection may be related to a combination of insufficient herd immunity and suboptimal protection against b. pertussis infection in children too young to be fully vaccinated. since vaccination rates in infants are already at 99%, it will be difficult to improve this further. therefore, other measures must be considered, including booster vaccination at pre-school age and vaccination during pregnancy. because young infants are mainly cared for by mothers and other adults, the most important cause of infection with b. pertussis is their close contact with parents and siblings. 7 in general, b. pertussis was detected more often during seasonal changes, especially from late summer to early autumn. in hospitalized children the number of b. pertussis infections increased in march and september as compared to other months ( fig. 2 ) . the seasonal infection rates were 4.66% in spring, 4.75% in summer, 4.62% in autumn and 4.00% in winter, respectively. the prevalence in the winter season was lower but not statistically different than in other seasons (x 2 = 3.388, p = 0.336). in this study, we used real-time pcr, the most accurate method to detect b. pertussis . the detection rate may be significantly lower than the actual level, because oropharyngeal samples in most patients were collected instead of nasopharyngeal samples, and the pcr target gene was ptxa-pr instead of is4 81. is4 81 is present in high-copy numbers in b. pertussis whereas ptxa-pr is a single-copy target. however, the ptxa-pr pcr is more specific and will not detect b. parapertussis , which contributes to more than 5% of pertussis cases. 8 many studies have shown that adolescents and adults with b. pertussis infections, causing chronic cough, are an important reservoir for transmission, putting newborns at high risk. 9 maternal pertussis immunization prevents infant pertussis, as recently shown by amirthalingam et al. vaccine effectiveness against infant deaths was estimated at 95%, and disease incidence in infants < 3 months of age has remained low. 10 according to our results, vaccination of pregnant women and adults, especially those in close contact with infants and young children, may help to prevent pertussis in infants and young children in china. the authors have declared that no competing interests exist. this study was supported by the sanming project of medicine in shenzhen ( szsm201512030 ) and by the shenzhen science and technology project ( jcyj20170303155012371 ). tang and colleagues reported in this journal their experience with covid-19 disease 1 , the outbreak of which began in december 2019 in wuhan, hubei province, china 2 , 3 with spread to 33 additional countries 4-8 as of the 21st february 2020. here we report the clinical features and outcome of the first two cases of disease caused by sars-cov-2 infection in the united kingdom (uk) -the first imported and the second associated with probable person-toperson transmission within the uk. public health management will be reported separately. the index case (a) entered the uk on 23/01/20 from hubei province in china. initially asymptomatic, this individual, a 50 year-old female with no past medical history and on no regular medications, developed symptoms of fever and malaise on 26/01/20, accompanied by sore throat and dry cough. she had travelled with her partner and reported no infectious contacts prior to travel. on 28/01/20, a close household contact of the index case, a resident of the uk, developed symptoms of fever (38.5 °c), followed the next day by diffuse myalgia and a dry cough. this patient (case b) is a previously fit and well 23 year-old male. he had returned to the uk from hubei province on 06/01/20. case b promptly sought advice via the national health service (nhs) self-referral service nhs 111, and he and case a were assessed as being possibly at risk of covid-19, and were admitted to the regional infectious diseases unit at castle hill hospital, hull university teaching hospitals for isolation, assessment and diagnostic sampling. they were managed in separate negative pressure cubicles with anterooms. nursing and medical staff donned personal protective equipment (ppe) as recommended by public health england (phe). the clinical observations of each of the patients, together with their initial blood tests, are shown in table 1 . ( fig. 1 ) . clinical examination findings were unremarkable. initial investigations revealed only mild lymphopenia and elevation of crp, with mild neutrophilia in case b. periodic fever of 38-38.5 °c was observed in case b until d3 of admission. repeat blood tests in this individual on d3 demonstrated mild acute kidney injury (aki, serum creatinine 144 μmol/l). the aki was thought most likely due to dehydration, and resolved within 24 h with administration of intravenous infusion of crystalloid at 125 ml/h. cxr was normal. empirical oral antibiotic therapy (co-amoxyclav 500/125 mg p.o. t.d.s.) was administered on d3, to cover the possibility of secondary bacterial infection, but was subsequently discontinued. symptoms resolved in case a by d3 and in case b by d4 of admission. pcr testing of sars-cov-2 from nose and throat swabs taken daily was negative from d2 onwards in case a and from d5 in case b (throat swabs from this individual were negative throughout). there was no clinical indication for the use of experimental antiviral therapies. patients were deisolated according to current phe guidance, based on complete resolution of symptoms and two sequential negative respiratory pcr tests at least 24 h apart. rooms were decontaminated with 0.1% hypochlorite followed by uv light treatment. the contact of these individuals remained asymptomatic throughout the 14 days incubation period but was isolated as a precaution and to be close to family these first cases of sars-cov-2 are informative for clinicians caring for suspected and confirmed cases in the uk and elsewhere. reassuringly, illness in both individuals was relatively mild and short-lived, with no evidence of parenchymal lung disease (reflected by normal oxygenation and the absence of radiological infiltrates) or of the late-stage deterioration that has been reported in case series 9 , possibly due to the absence of comorbidities. experimental antiviral therapeutic options for severe disease were not considered necessary given the mild clinical nature of the illness. clinical illness correlated with the presence of viral rna in upper airway samples ( fig. 1 ) , with no evidence of prolonged asymptomatic shedding, although discordance between nose and throat samples in case b highlights the need to sample both areas. it was reasonably assumed that the source of infection in case b was close contact with symptomatic case a, given that the time from travel to china to onset of symptoms in case b was 22 days, although this cannot be proven. based on this assumption, the period from exposure to disease onset appeared short, at approximately 48 h, consistent with recent reports of the incubation period of sars-cov-2 6 . co-occurrence of respiratory viral infection, as we observed in case b with rhinovirus, has been described in the context of sars-cov-2 ( https://www.medrxiv.org/ content/10.1101/2020.02.12.20022327v1 ) as it has with many other respiratory viruses spread by similar routes, and may have contributed to the increased symptomatology in case b. interestingly the partner of case a, who was a close household contact, remained asymptomatic throughout and had negative tests for sars-cov-2 shedding. it will be of interest to investigate the serological responses in this individual to ascertain evidence of subclinical infection. isolation, minimisation of contacts and use of appropriate ppe is a cornerstone of management of high consequence respiratory viral infection. in the cases reported here, phe recommendations for ppe were followed 10 and there were no breeches in ppe or nosocomial transmission. this should provide reassurance to healthcare workers managing patients with suspected covid-19 in the uk that current ppe is both feasible and effective. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. dear editor , as reported in this journal 1 and elsewhere, 2 an outbreak of atypical pneumonia caused by the zoonotic 2019 novel coronavirus (sars-cov-2) is on-going in china and has spread to the world. as of feb 16, 2020 (24:00, gmt + 8), there have been 70,548 confirmed patients and more than 1700 deaths from sars-cov-2 infection in china, and 58,182 confirmed patients and 1696 deaths in the most affected province, hubei province. much research progress has been made in dissecting the evolution and origin of sars-cov-2 and characterizing its clinical features. [3] [4] [5] [6] [7] while the outbreak is on-going, people raise grave concerns about the future trajectory of the outbreak, especially given that the working and schooling time has been already dramatically postponed after the chinese lunar new year holiday was over (scheduled on jan 31). in particular, a precise estimation of the potential total number of infected cases and/or confirmed cases is highly demanding. earlier studies based on susceptible-exposed-infectious-recovered metapopulation and susceptible-infected-recovered-dead models revealed the number of potentially infected cases and the basic reproductive number of sars-cov-2. 3 , 8 , 9 these traditional epidemiological models apparently require much detailed data for analysis. 3 , 8 here we explored a simple data-driven, boltzmann functionbased approach for estimation only based on the daily cumulative number of confirmed cases of sars-cov-2 (note: the rational for boltzmann function-based regression analysis is presented in supporting information (si) file). we decided to collect data (initially from jan 21 to feb 10, 2020) in several typical regions of china, including the center of the outbreak (i.e. wuhan city and hubei province), other four most affected provinces (i.e., guangdong, zhejiang, henan, hunan) and top-4 major cities in china (i.e., beijing, shanghai, guangzhou, shenzhen). during data analysis on feb 13, 2020, the number of new confirmed cases on feb 12 in hubei province and wuhan city suddenly increased by 14,840 and 13,436, respectively, of which 13,332 and 12,364 are those confirmed by clinical features (note: all the number of confirmed cases released by feb 12 were counted according to the result of viral nucleic acid detection rather than by referring to clinical features). we thus arbitrarily distributed these suddenly added cases to the reported cumulative number of confirmed cases from jan 21 to feb 14 for hubei province by a fixed factor (refer to table s1 ), assuming that they were linearly accumulative in those days. it is the same forth with the data for wuhan city. regression analyses indicate that all sets of data were well fitted with the boltzmann function (all r 2 values being close to 0.999; figs. 1 a, b, s1 , and table 1 ). the potential total number of confirmed cases for mainland china, hubei province, wuhan city, and other provinces were estimated as 72,80 0 ±60 0, 59,30 0 ±60 0, 42,10 0 ±70 0 and 12,80 0 ±10 0; respectively; those for provinces guangdong, zhejiang, henan and hunan were 1300 ±10, 1170 ±10, 1260 ±10, 1050 ±10, 1020 ±10 and 940 ±10, respectively ( table 1 ) ; those for beijing, shanghai, guangzhou and shenzhen were 394 ±4, 328 ±3, 337 ±3 and 397 ±4, respectively. in addition, we estimated the key date, on which the number of daily new confirmed cases is lower than 0.1% of the potential total number as defined by us subjectively (refer to table 1 ). the above analyses were performed assuming that the released data on the confirmed cases are precise. however, there is a health commission, the state administration of traditional chinese medicine, the academy of chinese medical sciences, 29 provinces and cities, as well as the army ( fig. 1 ) . huoshenshan hospital is a specialized hospital established in the wuhan staff sanatorium. patients with confirmed coronavirus pneumonia have been admitted to our hospital. it has a total of 10 0 0 beds, and includes an intensive care unit, an ordinary care unit, a laboratory, and radiology and other auxiliary departments. according to the national health commission of the people's republic of china, the related design scheme of the institute was completed on january 24, 2020. construction of the hospital began on january 29th, and the hospital was completed and put into use on february 2nd. the chinese people's liberation army has transferred 1400 medical personnel to undertake the task of helping people infected with the virus. we firmly believe that chinese medical personnel and people throughout the country can work together to win this defensive battle with one heart and one mind. herpes simplex virus (hsv) pneumonia in the non-ventilated immunocompromised host: burden and predictors colonization by pneumocystis jirovecii and its role in disease ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients pcr diagnosis of pneumocystis pneumonia: a bivariate meta-analysis evaluation of pcr in bronchoalveolar lavage fluid for diagnosis of pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review molecular diagnosis of pneumocystis pneumonia in immunocompromised patients real-time pcr assay-based strategy for differentiation between active pneumocystis jirovecii pneumonia and colonization in immunocompromised patients detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients diagnosis of pneumocystis jirovecii pneumonia in immunocompromised patients by real-time pcr: a 4-year prospective study pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia a unified personal protective equipment ensemble for clinical response to possible high consequence infectious diseases: a consensus document on behalf of the hcid programme lassa fever: epidemiology, clinical features, and social consequences world health organization. lassa fever fact sheet review of cases of nosocomial lassa fever in nigeria: the high price of poor medical practice epidemiologic and clinical features of lassa fever outbreak in nigeria measures to control protracted large lassa fever outbreak in nigeria clinical and laboratory predictors of lassa fever outcome in a dedicated treatment facility in nigeria: a retrospective, observational cohort study molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation national guidelines for lassa fever case management pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds first human infection by a novel avian influenza a(h7n4) virus comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings investigation on pseudorabies prevalence in chinese swine breeding farms in 2013-2016 human endophthalmitis caused by pseudorabies virus infection clinical experience and next-generation sequencing analysis of encephalitis caused by pseudorabies characteristics of human encephalitis caused by pseudorabies virus: a case series study human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: a case report a case of human viral encephalitis caused by pseudorabies virus infection in china metagenomic data screening reveals the distribution of mobilized resistance genes tet (x), mcr and carbapenemase in animals and humans epidemic of carbapenem-resistant klebsiella pneumoniae in europe is driven by nosocomial spread antibiotic resistance gene reservoir in live poultry markets emergence of plasmidmediated high-level tigecycline resistance genes in animals and humans plasmid-encoded tet(x) genes that confer high-level tigecycline resistance in escherichia coli first report of blavim-4-and mcr-9-coharboring enterobacter species isolated from a pediatric patient a link between the newly described colistin resistance gene mcr-9 and clinical enterobacteriaceae isolates carrying blashv-12 from horses in sweden mcr-9, an inducible gene encoding an acquired phosphoethanolamine transferase in escherichia coli , and its origin discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis expansion and model-centric curation of the comprehensive antibiotic resistance database emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study novel plasmid-mediated colistin resistance gene mcr-7.1 in klebsiella pneumoniae emergence of a novel mobile colistin resistance gene, mcr-8 , in ndm-producing klebsiella pneumoniae novel plasmid-mediated colistin resistance gene mcr-3 in escherichia coli identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible salmonella enterica serotype typhimurium isolate coproduction of mcr-9 and ndm-1 by colistin-resistant enterobacter hormaechei isolated from bloodstream infection outbreaks of severe pneumococcal disease in closed settings in the conjugate vaccines era serious pneumococcal disease outbreak in men exposed to metal fume -detection, response and future prevention through pneumococcal vaccination outbreak of invasive pneumococcal disease among shipyard workers welders are at increased risk for invasive pneumococcal disease reactivation of dormant/latent fungal infection visceral leishmaniasis: host-parasite interactions and clinical presentation in the immunocompetent and in the immunocompromised host visceral leishmaniasis among immunosuppressed patients with rheumatic diseases tumor necrosis factor alpha antagonist drugs and leishmaniasis in europe psoriasis as a systemic disease the inflammatory response in psoriasis: a comprehensive review the equivocal role of th17 cells and neutrophils on immunopathogenesis of leishmaniasis leishmaniasis and biologic therapies for rheumatologic diseases subclinical leishmania infection in patients with rheumatic diseases under biological drugs appropriate screening for leishmaniasis before immunosuppressive treatments human herpesvirus 6 infection after autologous stem cell transplantation: a multicenter prospective study in adult patients next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center a different response to cytomegalovirus (cmv) and epstein-barr virus (ebv) infection in uk people with multiple sclerosis (pwms) compared to controls complete genome sequence of equine herpesvirus type 9 equine herpesviruses type 1 (ehv-1) and 4 (ehv-4)-masters of co-evolution and a constant threat to equids and beyond isolation and identification of equine herpesvirus type 1 identification of equine herpesvirus type 1 infection in a horse farm in shanghai epidemiological status of equine rhinopneumonia in china complete genomic sequence of an equine herpesvirus type 8 wh strain isolated from china equine herpesviruses 1 (ehv-1) and 4 (ehv-4)-epidemiology, disease and immunoprophylaxis: a brief review consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group differences in viral dynamics between genotypes 1 and 2 of hepatitis c virus the paradox of highly effective sofosbuvir-based combination therapy despite slow viral decline: can we still rely on viral kinetics? therapy of chronic hepatitis c virus infection in the era of direct-acting and host-targeting antiviral agents oral direct-acting agent therapy for hepatitis c virus infection: a systematic review hcv] -a web-based interpretation system to support hepatitis c treatment decisions in the era of direct-acting antiviral agents antiviral therapies for chronic hepatitis c virus infection with cirrhosis real life efficacy of ledipasvir/sofosbuvir in hepatitis c genotype 4-infected patients with advanced liver fibrosis and decompensated cirrhosis systematic review with meta-analysis: efficacy and safety of directacting antivirals for chronic hepatitis c genotypes 5 and 6 increased susceptibility to pertussis in adults at childbearing age as determined by comparative seroprevalence study ritm-tohoku collaborative research group. bordetella pertussis infection in children with severe pneumonia rapid detection of respiratory organisms with the filmarray respiratory panel in a large children's hospital in china seroprevalence of diphtheria and pertussis immunoglobulin g among children with pneumonia in ji'nan. china european centre for diseases prevention and control. annual epidemiological report infant and maternal risk factors for pertussis-related infant mortality in the united states clinical features, genotype variations of antigenic genes and macrolides resistance pertussis outbreak in a primary school in china: infection and transmission of the macrolide-resistant bordetella pertussis seroprevalence of pertussis among adults in china where whole cell vaccines have been used for 50 years sustained effectiveness of the maternal pertussis immunization program in england 3 years following introduction emergence of a novel coronavirus causing respiratory illness from wuhan the ncov outbreak joint field epidemiology investigation team an outbreak of ncip (sars-cov-2) infection in china -wuhan , hubei province a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia importation and human-to-human transmission of a novel coronavirus in vietnam transmission of sars-cov-2 infection from an asymptomatic contact in germany first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan public health england. www.gov.uk/government/publications/wuhan-novelcoronavirus-infection-prevention-and-control/wuhan-novel-coronavirus-wncov-infection-prevention-and-control-guidance accessed 3rd emergence of a novel coronavirus causing respiratory illness from wuhan a novel coronavirus outbreak of global health concern epidemiological and clinical features of the 2019 novel coronavirus outbreak in china a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical characteristics of 2019 novel coronavirus infection in china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study data-based analysis, modelling and forecasting of the novel coronavirus (2019-ncov) outbreak a data driven time-dependent transmission rate for tracking an epidemic: a case study of 2019-ncov emergence of a novel coronavirus causing respiratory illness from wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan the novel coronavirus originating in wuhan. china: challenges for global health governance emergence of sars-like coronavirus poses new challenge in china we are indebted to all colleagues who contributed to this substudy in paris and geneva. in particular, we would like to thank no public or private funds were used for the current study. eliseo albert holds a río hortega research contract from the carlos iii health institute (ref. cm18/00221 ). estela giménez holds a juan rodés research contract from the carlos iii health institute (ref. jr18/0 0 053 ). we thank all the staff of the domestic and international organizations who fought against this outbreak, including those at the various health care facilities, lassa fever diagnostic laborato-ries, nigeria centre for disease control, world health organization, african field epidemiology network, public health england, ehealth africa, pro health international, university of maryland baltimore, us centers for disease control and prevention, alliance for international medical action, médecins sans frontières, and numerous other partners. we also express our sincerest condolences to the families and friends of those who died during the outbreak. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.016 . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.01.019 . this work was supported by the national natural science foundation of china ( 31702271 ), and the guangdong provincial natural science foundation ( 2017a030310367 ). we are grateful to the patients for providing their written informed consent to publish this report. our thanks go to nursing, laboratory and medical colleagues in hull university teaching hospitals nhs trust and the newcastle upon tyne hospitals nhs foundation trust who contributed directly and indirectly to patient care, and to many colleagues in public health england and across the hcid network who contributed their time and expertise to the management of these cases. cjad is supported by a clinical research career development fellowship from the wellcome trust ( 211153/z/18/z ). we thank graduate students (boyan lv, zhongyan li, zhongyu chen, yu cheng, mengmeng bian, shuang zhang, zuqin zhang, and wei yao; all from prof. xinmiao fu's research group at fujian normal university) for data collection. this work is support by the national natural science foundation of china (no. 31972918 and 31770830 to xf). the reported cumulative number of confirmed cases may have uncertainty. assuming the relative uncertainty follows a single-sided normal distribution with a mean of 1.0 and a standard deviation of 10%, the potential total number and key dates were estimated at 95% ci. for detail, refer to the methods section and figs. 1 c, d, s2 and s3.b key date is determined when the number of daily new confirmed cases is less than 0.1% of the potential total number. tendency to miss-report some positive cases such that the reported numbers represent a lower limit. one typical example indicating this uncertainty is the sudden increase of more than 14 0 0 0 new confirmed cases in hubei province on feb 12 after clinical features were officially accepted as a standard for infection confirmation.another uncertainty might result from insufficient kits for viral nucleic acid detection at the early stage of the outbreak. we thus examined the effects of such uncertainty using a monte carlo method (for detail, refer to the methods section in si file). for simplicity, we assumed that the relative uncertainty of the reported data follows a single-sided normal distribution with a mean of 1.0 and a standard deviation of 10%. under the above conditions, the potential total numbers of confirmed cases of sars-cov-2 for different regions were estimated ( figs. 1 c, d, s2 and s3 ) and summarized in table 1 16 ,092), respectively, indicating that overall the outbreak may not be so bad as previously estimated. 9 such uncertainty analysis also allowed us to estimate the key dates at 95% ci. as summarized in table 1 , the key dates for mainland china, hubei province, wuhan city, and other provinces would fall in (2/28, 3/10), (2/27, 3/10), (2/28, 3/10) and (2/27, 3/13), respectively.finally, the ongoing sars-cov-2 outbreak has undoubtedly caused us the memories of the sars-cov outbreak in 2003. we thus collected the data from the who officiate website for analysis, and found that the cumulative numbers of confirmed cases of 2003 sars-cov both in china and worldwide were fitted well with the boltzmann function, with r 2 being 0.999 and 0.998, respectively ( figs. 1 e and f) .in summary, we found that all data sets, including both the on-going outbreak of sars-cov-2 in china and the 2003 sars-cov epidemic in china and worldwide, were well fitted to the boltzmann function ( fig. 1 and s1 ). these results strongly suggest that the boltzmann function is suitable for analyzing the epidemics of coronaviruses like sars-cov and sars-cov-2. one advantage of this model is that it only needs the cumulative number of confirmed cases, somehow as simple as the recently proposed model. 10 in addition, the estimated potential total numbers of confirmed cases and key dates may provide valuable guidance for chinese central and local governments to deal with this emerging threat at current critical stage. none. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.02.019 . we appreciate the work tang et al. have report emergence of a novel coronavirus in china. 1 the 2019-ncov broke out in wuhan, china at the end of 2019, and has attracted worldwide attention. [2] [3] [4] although the chinese government has taken active measures to control this epidemic, the virus is very infectious. according to the real-time data of the national health commission of the people's republic of china up until february 5, 2020, within a short period of half a month, the number of confirmed cases and the number of deaths were 24,443 and 493, respectively. the epidemic is progressing rapidly. 2019-ncov poses new public health challenges in china. 5 in wuhan, china, the number of local medical staff is insufficient for the demand resulting from the explosive increase in the number of infected patients. therefore, many medical personnel are needed to devote themselves to the front line of combating the virus.medical personnel throughout the country are led under the unified leadership of the chinese government. although the epidemic in wuhan is serious, a large number of medical staff rushed to wuhan to supplement the shortage of manpower in wuhan hospitals. this is a battle without smoke, the heroes of which are our medical staff. according to the national health commission of the people's republic of china, as of january 30, 2020, hubei province opened 11,0 0 0 isolated patient beds, and about 170,0 0 0 healthcare professionals from all kinds of medical institutions are working on the front lines and providing care for patients with fevers, and for suspected or confirmed patients. in this time of emergency, under the unified deployment of the chinese government, there are 52 medical teams including 6097 medical personnel from the national key: cord-305303-82n96ukr authors: shapira, assaf; shapira, shiran; gal-tanamy, meital; zemel, romy; tur-kaspa, ran; benhar, itai title: removal of hepatitis c virus-infected cells by a zymogenized bacterial toxin date: 2012-02-16 journal: plos one doi: 10.1371/journal.pone.0032320 sha: doc_id: 305303 cord_uid: 82n96ukr hepatitis c virus (hcv) infection is a major cause of chronic liver disease and has become a global health threat. no hcv vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. moreover, there is no preventive therapy for recurrent hepatitis c post liver transplantation. the ns3 serine protease is necessary for hcv replication and represents a prime target for developing anti hcv therapies. recently we described a therapeutic approach for eradication of hcv infected cells that is based on protein delivery of two ns3 protease-activatable recombinant toxins we named “zymoxins”. these toxins were inactivated by fusion to rationally designed inhibitory peptides via ns3-cleavable linkers. once delivered to cells where ns3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. the zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting mazf ribonuclease, the toxic component of the e. coli chromosomal mazef toxin-antitoxin system, into an ns3-activated zymoxin that is introduced to cells by means of gene delivery. we constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, maze, linked via an ns3-cleavable linker. while covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. in contrast, activating proteolysis that is induced by even low levels of ns3, results in an eradication of ns3 expressing model cells and hcv infected cells. zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases. hepatitis c virus (hcv) is a small, enveloped rna virus belonging to the hepacivirus genus of the flaviviridae family. hcv has been recognized as a major cause of chronic liver disease and affects approximately 200 million people worldwide at the present time. persistent infection is associated with the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. a protective vaccine for hcv is not yet available and even the most recent combination of antiviral therapy is often poorly tolerated [1] . the hcv genome encodes one large open reading frame that is translated as a polyprotein which is proteolytically processed to yield the viral structural and nonstructural (ns) proteins. the non-structural proteins include the p7 ion channel, the ns2-3 protease, the ns3 serine protease/rna helicase and its co-factor ns4a, the ns4b and ns5a proteins and the ns5b rna-dependent rna polymerase (rdrp) [2, 3] . two virally encoded proteases participate in polyprotein processing, the ns2-3 autoprotease (which cleaves in cis at the ns2-3 junction) and the ns3-4a serine protease (which cleaves at four downstream ns protein junctions). ns3 is an extensively studied hcv protein that possesses multiple enzymatic activities that are essential for hcv replication, making ns3-4a the most attractive target for anti hcv drug development [4] . the n-terminus of ns3, in complex with its endoplasmic reticulum (er) membrane-anchored cofactor ns4a, primarily functions as a serine protease, which cleaves the viral polyprotein precursor downstream to ns3. the remaining 2/3 of the protein has a helicase and ntpase activities, both of which are essential for hcv replication [5] . zymogens are inactive enzyme precursors that are converted to their active form following a biochemical modification, such as proteolytic processing. among the known and important groups of enzymes that are proteolytically activated are secreted digestive enzymes like pepsin and trypsin [6, 7] , the cysteine aspartic acid proteases (caspases) which play an essential role at various stages of the apoptotic process [8] ; and blood coagulating factors [9] . recently we described a proof of concept for potential new antiviral agents that can specifically eradicate virally infected cells (thus limiting virus production and spread). these agents, which we named ''zymoxins'' (for ''zymogenized toxins''), were composed of a fusion between the binding and translocation domains of pseudomonas exotoxin a and ns3-activable modified catalytic domains of the bacterial or the plant toxins diphtheria toxin (dta) or ricin toxin (rta), respectively. upon binding and translocation into cells cytoplasm by virtue of the corresponding pseudomonas exotoxin a domains, activation of these toxins is mediated by hcv-ns3 protease cleavage that separates between the toxic domains and a fused, rationally designed inhibitory peptide. these zymoxins showed a higher level of cytotoxicity when applied to ns3 expressing cells or to hcv infected cells, demonstrating a potential therapeutic window. still, the zymoxins we described had two limitations: first, they required high levels of ns3 protease for efficient activation, and second, they were not totally inactive in the zymogenized state, and their basal cytotoxic activity without proteolytic activation resulted in quite narrow therapeutic windows [10] . apparently, the zymoxins were only partially inhibited by the rationally designed inhibitory peptides. toxic proteins that are completely inhibited by a natural inhibitor can be found in bacterial ''toxin-antitoxin'' systems. natural bacterial plasmids ensure their survival within the bacterial host by replicating inside their host cell and using mechanisms that function to segregate them prior to cell division [11] . in addition, several low copy number plasmids use a unique system called ''addiction module'' or ''toxin-antitoxin (ta) system'' which functions to prevent the proliferation of plasmidfree cells by executing the so called ''post-segregational killing effect''. the system consists of a pair of genes encoding for a stable toxin and an unstable antitoxin organized in a bicistronic operon that is transcriptionally autoregulated either by the toxin-antitoxin complex itself or by the antitoxin alone. when coexpressed in plasmid harboring cells, the antitoxin component interferes with the lethal action of the toxin. if a cell loses the plasmid, the cellular concentration of the labile antitoxin (that is degraded faster than the more stable toxin) is rapidly diminished, enabling the toxin to exert its action which eventually results in cell death (reviewed by [12, 13, 14, 15, 16] ). toxin-antitoxin systems have also been found integrated to the chromosome of various bacteria species where their function has been the subject of considerable speculation [14, 17, 18, 19, 20, 21] . one of the most studied ''genomic'' ta modules was found on the chromosome of e. coli as a negatively autoregulated bicistronic operon. the system, which is activated by several stress conditions, was denoted mazef after its two active protein components: the long lived mazf toxin and the labile maze antitoxin. mazf induced toxicity is executed by blocking denovo protein synthesis through its endoribonuclease activity that catalyze the cleavage of single-stranded mrnas at aca sequences [22] . when coexpressed with mazf, the maze antitoxin complexed with the toxin and a catalytically inactive heterohexamer is formed in which a maze dimer is sandwiched between two mazf dimers (mazf 2 -maze 2 -mazf 2 ) (reviewed by [21, 23] ). the crystal structure of the maze-mazf complex indicates that the interactions between the toxin and the antitoxin are primarily mediated by the acidic c terminus of maze which wraps around the mazf homodimer crossing the edge of the dimer interface [24] . later on, li et al had discovered that a short acidic peptide corresponding to maze c-terminal 23 amino-acids, which they denoted ''mazep'', binds strongly to the homodimer of mazf which possesses two identical active sites. interestingly, it was found that one inhibitory peptide, occupying a single active site on the mazf homodimer, affects the conformation of both sites that consequently become catalytically inactive. this unique mechanism also explains the inhibitory activity of maze toward mazf at a 1:2 molar ratio [25] . the discovery of the mazf mechanism of action was soon followed by demonstrations that it is also toxic to eukaryotic cells, causing bak-dependent programmed cell death in mammalian cells, suggesting it may be used as a tool for gene therapy against diseases such as cancer and aids [26, 27] . here we describe the design of a different zymoxin than those described in [10] , based on ns3-activable mazf ribonuclease that is delivered as a transgene by an adenoviral vector. the delivered transgene encodes for a fusion between mazf, and a potent inhibitory peptide derived from its natural antidote maze, through an ns3 cleavable linker. we show that the self-inhibited, zymogenized toxin is well tolerated when expressed in naïve cells. in contrast, in ns3 expressing or in hcv infected cells, ns3mediated cleavage separates between the toxin and its inhibitor which results in inhibition of protein synthesis followed by death of the cells. finally, we demonstrate that treatment with the mazf based zymoxin has a ''curing effect'' when applied to mixed culture of healthy and hcv infected cells, leading to specific eradication of the infected cell population. for the construction of ns3-activated mazf based zymoxin, the mazf coding sequence was fused through its c terminus to the hcv p10-p10' ns3 cleavage sequence derived from the 2a genotype (strain jfh1) ns5a/b junction. a short inhibitory peptide corresponding to maze c-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (mazep) that has been described by li et al. [25] ) was fused, preceded by a short flexible linker, to the c terminus of the mazf-ns3 cleavage site sequence. a flexible linker, followed by the c-terminal er membrane anchor of the tyrosine phosphatase ptp1b [28] , was than fused to the c terminus of the inhibitory peptide and the whole construct was fused through its n terminus to the monomeric red fluorescence protein mcherry [29] (see figure 1 ). the rationale behind the design of this construct, which was denoted ''mcherry-ns3 activated mazf'', was that the coupling between the ribonuclease and its antidote may enable high level of expression of the non-toxic fusion on the er membrane of uninfected mammalian cells without causing any deleterious effect. in contrast, in hcv infected cells, the fusion protein is expected to colocalize with the er membrane-bound viral ns3 protease (in infected cells, ns3 is localized to the cytosolic side of the er membrane and membranes of er-like modified compartments [30, 31, 32, 33, 34] ). as a result, the ns3cleavable linker between the toxin and its inhibitory peptide is expected to be cleaved. the toxic ribonuclease, no longer covalently tethered to its er membrane-anchored inhibitor, is now free to diffuse into the cytoplasm (which lacks the antidote) and exert its cytotoxic activity. finally, fusion to the fluorescent protein mcherry makes the whole construct trackable and facilitates the determination of its expression level and intracellular localization by fluorescence microscopy. as a control, an uncleavable construct (denoted ''mcherry-uncleavable mazf'') was constructed in which the ns3 cleavage sequence was replaced by a mutated 14 amino acids cleavage sequence (p10-p4') from hcv genotype 1a ns5a/b junction in which the p3 valine was substituted by alanine, the p2 cysteine by glycine, the p1 cysteine by glycine and the p4' tyrosine by alanine. a schematic representation of the ns3-activated mazf-based zymoxin (''mcherry-ns3 activated mazf'') and the hypothetical mechanism of its cleavage by ns3 protease on the cytoplasmic side of the er membrane are shown in figure 1 . the aminoacid sequence of the mazf based zymoxins can be found in text s1. to verify that expression of ''mcherry-ns3 activated mazf'' is tolerated by cells that do not express the ns3 protease, a colony formation assay was carried out. in this assay, the toxicity of an expressed transgene can be comparatively and qualitatively assessed by testing its effect on the competency of transfected cells to evolve into colonies under selection. hek293 t-rex cells where transfected with plasmids encoding either mcherry-ns3 activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide). 48 hours (h) later, expression of the encoded transgenes was confirmed by fluorescence microscopy (data not shown) and transfected cells were seeded in 3 fold dilutions and were treated with g418 (to which all three plasmids confer resistance). after 10 days of selection, surviving colonies were stained. as shown in figure 2 , similar numbers of surviving colonies were observed when the cells were transfected with the plasmids encoding mcherry-ns3 activated mazf or the red fluorescent protein alone, suggesting that expression of ns3-activable ribonuclease in naïve hek293 t-rex cells (that do not express ns3) cause minimal toxicity, if any. as expected, growth was severely inhibited when cells were transfected with the egfp-fused active (uninhibited) toxin. the er membrane-targeted zymoxin colocalizes with ns3 protease in vivo previously we described a hek293 cell line which inducibly expresses (by addition of tetracycline) a fusion between egfp and the coding sequence of the full length ns3 (including the helicase domain) followed by ns4a from hcv 1a genotype [10] . these cells, denoted ''tet-inducible full ns3-4a expressing cells'', were stably transfected with a plasmid encoding the ns3-activated zymoxin ''mcherry-ns3 activated mazf'' or with the uncleavable control (''mcherry-uncleavable mazf'') encoding plasmid. following selection, stable clones that constitutively express high level of the cleavable construct (denoted ''tet-ns3/activated mazf cells'') or the uncleavable control (denoted ''tet-ns3/uncleavable mazf cells'') were isolated. in order to characterize the intracellular distribution of the mcherry fused zymoxins, tet-ns3/activated or uncleavable mazf cells were subjected to immunofluorescence microscopy analysis. as shown in figure 3 (a and b), both zymoxins have a similar cellular distribution, colocalizing with the er marker calnexin at the juxtanuclear region of the er. importantly, upon induced expression of ns3-a4 in tet-ns3/uncleavable mazf cells, a colocalization of the two fluorescent fusion proteins could be observed ( figure 3 , c and d).these observations confirm that indeed both the protease and the modified ribonuclease are tethered to a common cellular compartment, presumably the cytoplasmic side of the er membrane (see scheme in figure 1 ). since expression of active mazf was found to inhibit de-novo protein synthesis in mammalian cells [27] , we hypothesized that such an effect may be observed also in cells in which mazf based zymoxin is proteolytically activated. in order to validate this assumption, tet-ns3/activated mazf and tet-ns3/uncleavable mazf cells were supplemented with tetracycline for 24 or 48 h, or left untreated. levels of de-novo protein synthesis were than determined by [ 3 h]-leucine incorporation assay. as shown in figure 4 (upper panel), a complete shutoff in protein synthesis was observed as soon as 24 h post ns3 induction in cells that express the cleavable construct, indicating proteolytic activation of the zymoxin. as expected, protein synthesis was not impaired following ns3 induction in cells that express the uncleavable toxin. figure 1 . schematic representation of the construct ''mcherry-ns3 activated mazf'' and hypothetical mechanism of activation by ns3 protease. the ns3-activated mazf zymoxin was constructed by fusing 5 elements in the following order (from the n terminus): monomeric red fluorescence protein mcherry, e. coli mazf ribonuclease, hcv p10-p10' ns3 cleavage sequence derived from 2a genotype (strain jfh1) ns5a/b junction, a short inhibitory peptide corresponding to maze c-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (mazep) that has been described by li et al. [25] ) and the c-terminal er membrane anchor of the tyrosine phosphatase ptp1b [28] . after being anchored to the er membrane, the ns3 cleavage site that is located between the ribonuclease and the inhibitory peptide in the ''mcherry-ns3 activated mazf'' construct (which is active as a dimer but for convenience is illustrated here in its monomeric form) is cleaved by the hcv-ns3 protease which is also localized to the cytoplasmic side of the er membrane. the toxic ribonuclease, no longer covalently tethered to its er-anchored inhibitor, is now free to diffuse to the cytoplasm (which lacks the antidote) and exert its destructive activity. doi:10.1371/journal.pone.0032320.g001 in support of these findings, an immunoblot assay revealed a near complete cleavage of the zymoxin following tetracycline-induced expression of ns3 protease in ns3-activated mazf expressing cells ( figure 4 , lower panel). presumably, this proteolytic activation of the ribonuclease toxin resulted in a cessation of cellular protein synthesis soon after induction, what explains the detection of relatively low levels of ns3 protease in these cells. as expected, no zymoxin cleavage could be detected in cells expressing the uncleavable form of the toxin. a faint band, corresponding to a cleaved zymoxin, can also be detected in uninduced cells that express the ns3 cleavable construct. this cleavage can be attributed to the ''leakiness'' of the tet inducible system, allowing a very low figure 2 . colony formation assay for the assessment of ''mcherry-ns3 activated mazf'' cytotoxicity toward naïve cells. a day before transfection, 7.5610 5 hek293 t-rex cells where seeded per well in 6 wells plate and subsequently transfected with 2 mg of plasmids encoding either mcherry-ns3 activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide). 48 hours later, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. transfected cells were than trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of g418 (to which all three plasmids confer resistance). surviving colonies were fixed and stained with giemsa (upper panel). number of surviving colonies from wells that were seeded with 5556 cells was determined by manual counting. each bar represents the mean 6 standard deviation (sd) of a set of data from two wells (lower panel). doi:10.1371/journal.pone.0032320.g002 zymoxin-mediated removal of hcv infected cells plos one | www.plosone.org basal transcription of the protease in the absence of externally added tetracycline. such cleavage could not be detected in ''naïve'' cells following transfection with ns3-cleavage substrate encoding transgene ( [10] , data not shown). apparently, this very low, basal proteolytic activity is well tolerated by ns3 activated zymoxin expressing cells that shows no indication of de-novo protein synthesis inhibition in comparison to cells that express the uncleavable toxin (see cpm values in the upper panel of figure 4 ). to evaluate the potential of the ns3-cleavable mazf based zymoxin in eradication of ns3 expressing cells, three 96 well plates were seeded with tet-inducible full ns3-4a, tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells and were supplemented with 3 fold serial dilutions of tetracycline starting with 1000 ng/ ml. after 72 h, the relative fraction of viable cells was determined using an enzymatic mtt assay. as shown in figure 5 (lower panel), the expression level of ns3 can be roughly tuned by modulation of the final tetracycline concentration in the growth figure 4 . inhibition of de-novo protein synthesis by ns3-activated mazf based zymoxin in ns3-expressing cells. 1610 5 tet-ns3/ activated mazf or tet-ns3/uncleavable mazf cells were seeded per well in 24-wells plate. 24 or 48 h later, cells were supplemented with tetracycline to a final concentration of 1000 ng/ml, or left untreated (48 h tet, 24 h tet and no tet, respectively). 72 h after seeding, levels of de-novo protein synthesis were determined by [ 3 h]-leucine incorporation assay, as described in ''materials and methods''. results are expressed as percent of the value obtained for cells which were not induced to express the ns3 protease (no tet). each bar represents the mean 6 sd of a set of data determined in triplicates. numbers above each bar represent mean counts per minute (cpm) values for 7 micrograms total protein samples (upper panel). 30 micrograms of total protein from lysates of the described cells were analyzed by immunoblotting with mouse anti-mcherry (for detection of the zymoxin), mouse anti-gfp (for the detection of egfp-ns3) and mouse anti actin antibodies (loading control) followed by hrp-conjugated secondary antibodies and ecl development. solid arrow: full length zymoxin. hollow arrow: n' terminal portion of ns3-cleaved zymoxin (lower panel). doi:10.1371/journal.pone.0032320.g004 media, with around 10 ng/ml as an intermediate concentration for induction of low ns3 expression level. indeed, strong cytotoxicity was clearly evident when tet-ns3/activated mazf cells where treated with tetracycline concentrations of down to 4 ng/ml. no cytotoxic effect was detected when the controls tet-ns3/uncleavable mazf or tet-inducible full ns3-4a (protease only) expressing cells were similarly treated ( figure 5 , upper panel). these findings demonstrate the deleterious effect of the mazf based zymoxin specifically toward ns3 protease expressing cells, as well as its competence to be activated by very low cellular levels of protease. in order to obtain a visual insight at the cell level, tet-ns3/activated mazf and tet-ns3/uncleavable mazf cells were supplemented with tetracycline to a final concentration of 10 ng/ml or 1000 ng/ml (for low and high induction levels of ns3 expression, respectively), or left untreated. 36 h later, nuclei were stained and cells were examined under a fluorescence microscope. the results show that both lower and higher induction levels of ns3 protease caused growth inhibition and rounding of cells that constitutively expresses the cleavable mazf. furthermore, both green and red fluorescence were faint in these cells, probably as a result of the destructive ribonuclease activity of the cleaved toxin toward the ns3 protease and its own mrna. as expected, none of the above observations was evident when these cells were not supplemented with tetracycline or when ns3 expression was induced to high level in cells that constitutively express the uncleavable toxin ( figure 6 ). adenovirus-mediated delivery of mcherry-ns3 activated mazf encoding cassette specifically eradicates ns3 expressing hepatocytes to achieve efficient dna delivery into mammalian cells, the expression cassette encoding mcherry-ns3 activated mazf was cloned into a ''first generation'' de1/de3 human type 5 adenoviral vector plasmid dna by homologous recombination in bacteria [35] . virus particles were propagated in hek293 packaging cells as described in the ''materials and methods'' section. in addition, a control adenoviral vector was constructed for the delivery of a similar cassette that encodes for the . after 24 h, cells were supplemented with 3 fold dilutions of tetracycline starting with concentration of 1000 ng/ml, or left untreated. 72 hours later, the fraction of viable cells (relatively to the untreated controls) was determined using an enzymatic mtt assay. each bar represents the mean 6sd of a set of data from six wells. lower panel: 30 ng of total protein from lysates of tet-ns3/uncleavable mazf cells that were supplemented with 3 fold dilutions of tetracycline for 48 h were analyzed by immunoblotting with mouse anti-gfp (for the detection of egfp-ns3) and mouse anti-actin antibodies (loading control) followed by hrp-conjugated secondary antibodies and ecl development. doi:10.1371/journal.pone.0032320.g005 uncleavable version of the construct (mcherry-uncleavable mazf). red-fluorescent comet-like adenovirus-producing foci were apparent upon infection of packaging cells with both recombinant viruses (encoding cleavable or uncleavable constructs) (see figure s1 ). the production yields for both viruses were ,3610 8 plaque forming units (pfu)/ml, after two ''cycles'' of virus amplification (see text s2). in order to evaluate the ability of adenovirus-mediated delivery of ns3 activated mazf encoding cassette to eradicate ns3 expressing hepatocytes, wild-type huh7.5 hepatoma cells and our previously described egfp-full ns3-4a expressing huh7.5 cells [10] were infected with the ns3-activated or uncleavable mazf encoding viruses. since our previous unpublished observations and studies of others have indicated that high level of transgene expression and adenoviral infection, per se, may adversely affect cell viability and growth [36, 37, 38, 39] ; the cells were infected with a series of multiplicity of infection (moi) ratios in order to find an optimal moi that leads to eradication of ns3-expressing cells while maintaining minimal toxicity to naïve cells. as shown in figure 7a (left panel), infection with the ns3activated zymoxin resulted in a considerable cytotoxic effect against ns3-expressing hepatocytes, leading to their almost complete eradication at moi's$12. however, infection at these ratios also adversely affected the growth of wild-type hepatocytes. in contrast, infection at moi of ,3 decreased the viability of the ns3-expressing huh7.5 cells to about 35% of the untreated control without affecting the viability of the wildtype hepatocytes (see also microscopic examination in figure 7b ). therefore, infection at this moi was applied during the next experiments. as expected, no substantial enhancement in cytotoxicity against ns3 expressing huh7.5 cells (relatively to wild-type huh7.5 cells) was observed, at any moi, upon infection with the uncleavable mazf encoding viruses ( figure 7a , right panel). adenovirus-mediated delivery of ns3-activated mazf encoding cassette specifically eradicates hcv infected hepatocytes to test the competence of the mazf based zymoxin to specifically eradicate hcv infected hepatocytes, we utilized the infectious chimeric virus hj3-5 [40] . this is one of the currently used models to study hepatitis c virus in which recombinant infectious hcv particles are produced in cell culture (hcvcc) (for review, see [41, 42] ). as the main purpose of zymoxins based treatment is the specific eradication of hcv infected cells from a background of healthy cells, ''mixed culture'' experiments were carried out. in these experiments, huh7.5 hepatoma cells were infected with the hcv 1a/2a chimeric virus hj3-5 (encoding the ns3 protease of genotype 2a strain jfh1 [40] ). when infection reached ,50% (about 50% of the cultured cells showed expression of the hcv-core protein, as detected by immuno-staining and fluorescence microscopy), the mixed culture and a culture of uninfected cells were treated with ns3 activated mazf or uncleavable-mazf encoding adenoviruses at moi of ,3. control cells remained untreated. 72 h post adenoviral infection; viability assay and microscopic examination that included immunostaining for hcv-core protein were performed. as shown, treating the mixed culture with ns3-activated mazf zymoxin-encoding adenovirus reduced the viable cells population to about 65% relatively to untreated control; while viability of the uninfected cells (''hcv negative'') was barely affected by this treatment (figure 8, upper panel) . microscopic examination of the treated mixed culture revealed two cell populations that differ in their appearance. while one population is characterized by a ''typical'' huh7.5 cell morphology (hollow arrows, figure 8, lower panel) , the other is composed of partially detached cells with round, condensed or distorted shape (filled arrows) that are hypothesized to be zymoxin-intoxicated hcv infected cells. in order to validate this assumption, the fraction of hcv infected cells from the general population was evaluated by immunofluorescence analysis using anti-hcv core protein specific antibodies. indeed, treatment with the ns3activated zymoxin showed a ''curing effect'' upon the partially infected culture, considerably reducing the fraction of the hcv infected cells from the general population ( figure 9 ). as expected, no significant effect upon the hcv infected cell population was observed following treatment with the uncleavable zymoxin. fight against viral infections is considered as one of the most challenging areas in modern medicine. while vaccination is considered to be the most efficient method for fighting viral infections; for some viral pathogens which cause world-wide health problem like human immunodeficiency virus (hiv) and hepatitis c virus (hcv), no efficient vaccine has yet been developed. over the last years, efforts have been focused on the discovery and development of anti-viral agents that target crucial steps in the viral replication cycle which includes viral entry, rna translation and post-translational processing, reverse transcription, genome integration, viral assembly and release [43, 44, 45] . an essential step in the replication cycle of many viruses is the processing of a polyprotein precursor by a viral-encoded protease. a partial list of human diseases associated viruses encoding protease(s) in their genome include flaviviruses such as hepatitis c virus (hcv), west nile virus (wnv), dengue fever virus (dfv) and yellow fever virus (yfv); retroviruses such as hiv-1; picornaviruses such as coxsackievirus, poliovirus and hepatitis a virus; nidoviruses such as coronaviruses (cov), including the severe acute respiratory syndrome (sars) causative sars-cov and herpesviruses such as varicella-zoster virus (vzv) and epstein-bar virus (ebv) [46, 47] . therefore, a large part of antiviral drug discovery is focused on inhibiting viral proteases [46] . our group too has published several studies where hcv replication was inhibited by intracellular expression of antibodies and peptide aptamers [48, 49, 50] . the opposite ''side of the coin'' -taking advantage of the activity of a viral protease per-se as an antiviral approach has been rather neglected. recently we described a study that was based on the concept of ''sitoxins'' which are anti-viral agents that are designed to eradicate viral-infected cells by taking advantage of a specific viral activity instead of inhibiting it [51, 52] . specifically, a sitoxin is comprised of an effector domain (e.g. a toxin); a domain bearing an intracellular signaling moiety (e.g. a degradation or an intracellular localization signal); and a domain located between the effector domain and the domain bearing the intracellular signaling moiety which specifies a cleavage site for a predetermined protease (e.g. a viral encoded protease). following the introduction of the sitoxin into the target cell (that expresses the specific protease), cleavage by the predetermined protease separates the toxic effector domain of the sitoxin from the intracellular signaling moiety, resulting either in a longer-lived (and therefore more toxic) effector domain or in an effector domain that moves from a cellular compartment where the domain is nontoxic to a cellular compartment where the domain is able to exert its effect [51] . we developed and successfully evaluated two rationally designed viral-protease-activated chimeric toxins which we named ''zymoxins'' for ''zymogenized toxins'' [10] . in contrast to sitoxins, in which the inhibitory activity is mediated by intracellular components which recognize a cleavable signaling polypeptide fused to a constitutively active toxin; the concept of zymoxins is based on the idea of reengineering a toxic enzyme into an inactive zymogen which is specifically activated by a predetermined protease. this general approach was demonstrated by other elegant studies in which several enzymes, including bovine rnase a, vip2 and the maize ribosome inactivating protein (maize-rip) were converted into protease activated forms [53, 54, 55, 56, 57] . our previous work focused on the design of protein-delivered toxins that were converted into protease-activated forms by means of fusion to specific, rationally designed inhibitory peptides through hcv-ns3 protease cleavable linker. when tested in-vitro and on ns3-overexpressing or hcv infected cells, a clear ns3 protease cleavage-dependent enhancement in activity/cytotoxicity was observed. however, these constructs had two major drawbacks, as mentioned in the introduction: the first is the incomplete inhibition of the toxic enzymatic activity that is conferred by the rationally designed fused peptide. the second relates to the necessity of relatively high level of expressed viral protease for achieving adequate zymoxin activation inside the cells [10] . in the current study, we turned to evaluate a similar strategy for eradicating hcv-infected cells, namely, by converting a constitutively active enzyme into an ns3 activated zymogen. as a toxic moiety, we chose mazf, an endoribonuclease that together with its polypeptidic antidote, maze, constitute one of the most studied ''genomic'' toxin-antitoxin systems in e. coli. in order to convert mazf into a zymoxin, a fusion polypeptide was constructed in which an inhibitory peptide derived from the maze antitoxin was fused to the c terminus of the mazf toxin via an ns3-cleavable linker. regarding the issue of incomplete inhibition of zymoxin's activity in uncleaved form, it should be mentioned that in contrast to our previously described constructs, in which a ''rationally designed'' peptide is appended to provide the inhibition of the toxin's activity; in the mazf based-zymoxin, a ''natural'' inhibitory polypeptide was chosen for that purpose. antidote polypeptides in toxin-antitoxin systems were ''evolutionary shaped'' to strongly inhibit the destructive activity of their toxic counterparts. thus, a very efficient inhibition may be achieved when using them as inhibitory peptides in the construction of zymoxins. indeed, our results show that the fused maze-derived inhibitory peptide diminishes the toxin's activity to such an extent that enables its non-lethal overexpression in naïve cells. in order to improve the responsiveness of the zymoxin to the presence of low levels of cellular-expressed viral protease, an er membrane ''anchoring peptide'' was fused to the c terminus of the construct, subsequently to the maze derived inhibitory peptide. by using that design, a colocalization between the er-bound ns3 protease and its zymoxin substrate might be achieved, resulting in an improved cleavage efficiency. indeed, such a colocalization could be observed, as shown above (figure 3) . moreover, activation of the zymoxin was evident also upon expression of very low cellular levels of the viral protease ( figures 5 and 6) . it should also be mentioned that while being extremely sensitive to minute amounts of expressed ns3 protease, zymoxin expressing cells can still tolerate some degree of activating proteolysis (as shown above for ns3-activated mazf expressing cells that show a very low, basal ns3 proteolytic activity without tetracycline induction (figure 4) ). this property may, in fact, contribute to the specificity and general safety of zymoxin-based therapeutics, even though ''spontaneous'', unspecific proteolytic activity toward ns3 substrates could not be detected in naïve cells during our experiments (data not shown). as discussed, tethering the zymoxin to the er membrane through its inhibitory peptide may also enhance its cleavage-dependent toxicity. this might be achieved by enabling spatial separation between the activated toxin and its antidote following ns3-cleavage and diffusion of the active mazf to the inhibitory peptide-free cytoplasm. supporting this assumption, we found that although cleaved, a cytoplasmic version of the ns3-activated mazf based zymoxin is barely toxic to ns3 protease-expressing cells (data not shown). in contrast to our previous study, in which the toxins were delivered into mammalian cells as purified recombinant proteins; in the current research an adenoviral vector gene-delivery system was used. widely applied in gene therapy, recombinant viral vaccines and basic science studies, this system enables efficient delivery and high level of transgene expression in a wide variety of human cells. using this system, we have demonstrated specific eradication of full ns3-4a expressing huh7.5 cells, sparing ''naïve'' cells which do not express the protease. furthermore, specific eradication of hcv infected cells has also been achieved, demonstrating a prominent ''curing effect'' when tested on mixed cultures of healthy and hcv infected hepatocytes. the use of viral gene delivery for treating hcv infection by eradication of infected cells has been proved successful in previous study on mice with chimeric human livers. in that study, hsu et al designed a modified proapoptotic bid molecule in which its endogenous cleavage sites were replaced by ns3 recognition site. as bid requires proteolytic processing in order to activate its apoptotic function, infection with adenoviral vector that delivers a transgene encoding the engineered bid molecule was demonstrated to induce activation of apoptosis in cells expressing the hcv ns3 protease. moreover, a significant reduction in hcv titer in the serum of the hcv infected mice was detected following injection of the engineered adenoviral vector into the jugular vein [58] . although such an approach had proved successful; it should be taken into account that a delivered transgene which encodes for a modified form of a protein that is naturally expressed in the target cell has the potential to negatively influence normal cellular processes in which its endogenous counterpart plays a role [59, 60, 61, 62, 63] . for example, when overexpressed in noninfected cells, an ns3-cleavable apoptotic molecule, such as that described above, may act as a ''dominant negative'' by competing with the natural, endogenous protein for interactions with activators, regulators or substrates. delivery of a transgene encoding for a foreign protein which has no endogenous counterpart in target cells, such as mazf, may reduce that risk. furthermore, it should be noted that mazf possess a ribonuclease activity that is also capable of processing multiple potential cleavage sites in the hcv genome. although not evaluated in this study, a direct attack on the parasite's genetic material may represent an additional mode of anti-viral activity that works in parallel with host-cell protein synthesis shutoff. of note, the applicability of the described zymoxin may be extended by replacing the protease cleavage site that separates between the toxic moiety and the inhibitory peptide. this could facilitate eradication of cells that are infected with other protease expressing viruses other than hcv (a partial list of protease expressing viruses is given at the beginning of the discussion) or different strains of hcv that differ in their ns3 protease cleavage specificity. in addition, one may replace the c terminal er anchoring peptide with sequence that tethers the construct to a different intracellular location in which the viral protease resides. in that way, a colocalization between the zymoxin and the predetermined viral protease may be achieved. in conclusion, the presented anti-viral agent was designed following our previously described ''zymoxins'' concept in which a constitutively active toxin is converted into a ''zymogenized'', viral-protease activated from. the mazf-based zymoxin, that is introduced to target cells by means of adenovirus mediated gene delivery, shows very low toxicity to naïve cells and enhanced responsiveness to low viral-protease expression level, when compared to our previously presented constructs. as evident from our results, the mazf based zymoxin eradicates ns3-expressing model cells and hcv infected cells with remarkable efficiency and specificity, providing further proof to the concept of zymoxins and a potential new means of fighting viral diseases. obviously, further optimizations and pharmacological assessments in animal models may be required in order to determine the safety and efficiency of zymoxins treatment in the context of the whole organism. the following escherichia coli (e. coli) strains were used: xl-1 blue and dh5a (stratagene, usa) for plasmid propagation and bj5183 (stratagene, usa) for the generation of recombinant adenovirus plasmid dna. recombinant dna techniques were carried out according to standard protocols or as recommended by the suppliers. nucleotide sequences were determined using the prism 3100 genetic analyzer (applied biosystems, usa) according to the supplier's recommendations. the eukaryotic cmv promoterbased gfp-fusion expression vector pegfp c2, which was used for expression of mcherry, mazf and mazf-based zymoxins, was from clontech (usa). the adeasy plasmid system (pshuttle and padeasy-1) [35] , that was used for generation of recombinant human type 5 adenoviral vectors for gene delivery of the zymoxins expression cassettes, was a generous gift from prof. nadir arber, integrated cancer prevention center, tel aviv sourasky medical center, israel. all plasmid and dna fragment purifications were carried out with high-speed plasmid mini kit and gel/pcr dna fragments extraction kit (geneaid biotech ltd., taiwan) unless mentioned otherwise. t4 dna ligase and restriction enzymes were purchased from new england biolabs (usa). dna ligations were carried out at 16uc overnight. oligonucleotides. all the oligonucleotides that were used in this study were purchased from hylabs, israel. oligonucleotides that were used in this study are listed in table s1 . construction of the vector encoding for ''mcherry-ns3 activated mazf''. a polymerase chain reaction (pcr) was carried out using a single colony of escherichia coli strain xl-1 as a template, the forward primer: 40-clvmazf and the reverse primers: 41-clvmazf, 42-clvmazf, 43-clvmazf, 44-clvmazf, 45-clvmazf, 46clvmazf and 47-clvmazf. the pcr product, encoding for a fusion polypeptide composed of (from the n terminus) mazf, hcv p10-p10' ns3 cleavage sequence derived from 2a genotype (strain jfh1) ns5a/b junction, a short flexible linker, a short inhibitory peptide corresponding to maze c-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (mazep) that has been described by li et al. [25] ), a flexible linker and the cterminal er membrane anchor of the tyrosine phosphatase ptp1b [28] was digested with xhoi and ecori and was cloned between the corresponding sites in the plasmid pegfp-c2, generating plasmid ''pegfp-ns3 activated mazf''. next, the sequence of the red fluorescent protein mcherry [29] was amplified by pcr from an expression cassette (kindly provided by prof. adi avni, department of molecular biology and ecology of plants, tel-aviv university, israel) using the forward primer: 48clvmazf and the reverse primer: 49-clvmazf. the pcr product was digested with nhei and xhoi and was cloned between the corresponding sites of the plasmid ''pegfp-ns3 activated mazf'' (replacing the egfp coding sequence), generating plasmid: ''pmcherry-ns3 activated mazf''. the amino-acid sequence of the encoded cleavable zymoxin can be found in text s1. construction of the vector encoding for ''mcherryuncleavable mazf''. a pcr was carried out using dna of plasmid ''pmcherry-ns3 activated mazf'' as a template, the forward primer: 40-clvmazf and the reverse primers: 50-unclmazf and 51-unclmazf. the pcr product was digested with ecorv and nrui, and the digestion product of 233 bp was cloned between the corresponding sites of the same plasmid that has been used as a template, generating the plasmid: ''pmcherry-uncleavable mazf''. this plasmid encodes for an uncleavable construct in which the ns3 cleavage sequence was replaced by a mutated 14 amino acids cleavage sequence (p10-p4') from hcv genotype 1a ns5a/b junction in which p3 valine was substituted by alanine, p2 cysteine by glycine, p1 cysteine by glycine and p4' tyrosine by alanine. the amino-acid sequence of the encoded uncleavable zymoxin can be found in text s1. construction of the vector encoding for ''egfp-mazf''. this is a mutated variant of an ''intermediate'' vector used in the construction process of the ''mcherry-ns3 activated mazf'' encoding vector. in this variant, a nonsense mutation was inserted instead of the tyrosine in the smsy sequence of the ns3 recognition site, generating the plasmid ''pegfp-mazf'' that encodes for a truncated egfp-mazf fusion protein that lacks the maze derived inhibitory peptide and the er anchor. construction of the vector encoding for mcherry. the sequence of the red fluorescent protein mcherry [29] was amplified by pcr from an expression cassette (see construction of the vector encoding for ''mcherry-ns3 activated mazf'') using the forward primer: 48-clvmazf and the reverse primer: 49clvmazf. the pcr product was digested with nhei and bglii and was cloned between the corresponding sites of the plasmid pegfp c2 (replacing the egfp coding sequence), generating the plasmid ''pmcherry''. construction and propagation of recombinant adenoviral vectors. construction and propagation of recombinant human type 5 adenoviral vectors for gene delivery of the mcherry-ns3 activated mazf and mcherry-uncleavable mazf expression cassettes was carried out using the adeasy system essentially as described in [35, 64] . a more detailed description of the procedure is provided in text s2. human embryonic kidney cells hek293, stably expressing the tetracycline repressor protein (t-rex hek293 cell line, invitrogen, usa), and human hepatoma cells huh7.5 [65] were used throughout this study. cell lines were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal calf serum (fcs), 2 mm l-glutamine, 100 u/ml penicillin and 100 mg/ml streptomycin (biological industries, israel) in a humidified 5% co 2 incubator at 37uc. the calcium-phosphate transfection method was applied for introducing 2 mg of the plasmid ''pmcherry-ns3 activated mazf'' or the plasmid ''pmcherry-uncleavable mazf'' into t-rex hek293 cells inducibly expressing egfp-full ns3-4a [10] seeded 1.5610 6 cells per 60 mm plate 24 h before transfection. stable transfectants, inducibly expressing egfp-full ns3-4a and constitutively expressing mcherry-ns3 activated mazf (denoted ''tet-ns3/activated mazf cells'') or mcherry-uncleavable mazf (denoted ''tet-ns3/uncleavable mazf cells'') were selected in a medium containing 1 mg/ml of g418 (a.g. scientific, usa). cell clones that express high level of the cleavable construct or the uncleavable control were identified by fluorescence microscopy and isolated. for protein extraction, 48 h post-transfection the cells were washed with pbs, scraped and lysed in a buffer containing 150 mm nacl, 5 mm edta, 0.5% np-40, 10 mm tris(hcl) ph 7.5, and protease inhibitors cocktail (sigma, israel). following 30 minutes of incubation on ice, lysates were cleared by centrifugation at 20,000 g for 10 minutes, at 4uc. for immunoblotting, protein samples were electrophoresed on 12% sds/polyacrylamide gel, transferred to nitrocellulose and detected using mouse monoclonal anti-mcherry antibody (clontech, usa), mouse monoclonal anti-gfp antibody (santa-cruz, usa) or mouse monoclonal anti-actin antibody (abcam, usa), followed by horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (jackson immunoresearch laboratories, usa) and enhanced chemiluminescence (ecl) detection using supersignal west pico chemiluminescent substrate (thermo scientific/pierce, usa). virus assays were carried out with an inter-genotypic chimeric hepatitis c virus (hcv) produced by replacing the core-ns2 segment of the jfh-1 virus genome with the comparable segment of the genotype 1a h77 virus. this chimeric virus, hj3-5 (kindly provided by prof. stanley lemon, university of texas at galveston), contains two compensatory mutations that promote its growth in cell culture as described previously [40] . hcv rnas were transcribed in vitro and electroporated into cells essentially as described previously [66, 67] . in brief, 10 mg of in vitro-synthesized hcv rna was mixed with 5610 6 huh7.5 cells in a 2-mm cuvette and pulsed twice at 1.4 kv and 25 mf. cells were seeded into 12well plates or 25-cm 2 flasks, and passaged at 3-to 4-day intervals posttransfection by trypsinization and reseeding with a 1:3 to 1:4 split into fresh culture vessels. when infectivity reached ,50%, as was monitored by immunofluorescent staining with anti hcv core protein mouse monoclonal antibody (affinity bioreagents, usa) and cy2-conjugated goat anti-mouse igg (jackson immunoresearch laboratories, usa) (see ''visualizing hcv infected cells (infectivity assay)'' below), the mixed culture (of uninfected and hcv infected cells in 1:1 ratio) was taken for cytotoxicity assays. confocal fluorescence microscopy analysis of t-rex hek293 cells inducibly expressing egfp-full ns3-4a and constitutively expressing mcherry-ns3 activated or uncleavable mazf. 1610 5 tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded on poly-l-lysine coated cover-slips in a 24 well-plate. 12 h later, the cells were supplemented with 1 mg/ml of tetracycline, or left untreated. 24 h later, the cells were fixed with 4% formaldehyde in pbs. uninduced cells were permeabilized with triton x-100 (0.1% in pbs) for 5 minutes, blocked with 90% fetal calf serum/10% pbs at room temperature for 25 minutes, incubated with 1:200 diluted rabbit-polyclonal anti-calnexin antibody (sigma, usa) as primary antibody for 1 h and followed by 1:200 diluted cy2conjugated anti-rabbit igg (jackson immunoresearch laboratories, usa) secondary antibody for 30 minutes. nuclei of induced and uninduced cells were then stained by hoechst 33258 for 1 h at room temperature. slides were washed with pbs, mounted in mowiol 4-88 solution (calbiochem, usa) (immunostained uninduced cells) or immuglo mounting medium (immco diagnostics, usa) (induced tet-ns3/uncleavable mazf cells) and examined using a zeiss lsm 510 meta laser scanning confocal microscope. visualizing t-rex hek293 cells inducibly expressing egfp-full ns3-4a (supplemented with different tetracycline concentrations) and constitutively expressing mcherry-ns3 activated mazf or mcherry-uncleavable mazf. 1610 5 tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded on poly-l-lysine coated cover-slips in a 24 well-plate. 12 h later, cells were supplemented with 10 ng/ml or 1000 ng/ml of tetracycline, or left untreated. 36 h later, cells were fixed with 4% formaldehyde in pbs. following nuclear staining by hoechst 33258 for 1 h at room temperature, slides were washed with pbs, mounted in immuglo mounting medium and examined using a fluorescence microscope. visualizing hek293 cells infected with recombinant adenovirus encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf. 3610 5 hek293 cells were seeded per well in 6 wells plate. when the culture reached 90% confluence, the growth medium was replaced by fresh medium containing 10 fold dilutions of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf, starting from 2.5610 6 pfu per well. after 36 h, cells were fixed with 4% formaldehyde in pbs and examined using a fluorescence microscope. visualizing hcv infected cells (infectivity assay). huh7.5 cells infected with hcv hj3-5 chimeric virus were seeded into 8well chamber slides (nalge nunc, usa). after 24 h, cells were fixed and permeabilized with 1:1 acetone/methanol mixture and stained with 1:300 diluted mouse monoclonal antibody c7-50 (affinity bioreagents, usa) specific for the hcv core protein followed by staining with 1:100 diluted cy2-conjugated goat anti-mouse igg (jackson immunoresearch laboratories, usa). nuclei were then stained with dapi (sigma, israel) and slides were washed with pbs, mounted (southernbiotech, usa) and examined using a fluorescence microscope. visualizing zymoxin-treated mixed culture of uninfected and hcv infected cells. see ''cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf on a mixed culture of uninfected and hcv-infected huh7.5 cells'' below. colony formation assay 7.5610 5 hek293 t-rex cells were seeded per well in 6 wells plate. 24 h later, cells were transfected with 2 mg of the plasmids ''pmcherry-ns3 activated mazf'', ''pmcherry'' or ''pegfp-mazf'' encoding for mcherry-ns3 activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide), respectively. transfection was carried out using fugene 6 reagent (roche, germany) according to the manufacturer instructions. after 48 h, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. transfected cells were then trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of g418 (to which all the three plasmids confer resistance). surviving colonies were fixed with 4% formaldehyde in pbs and stained with giemsa (sigma, usa). number of surviving colonies from wells that were seeded with 5556 cells was determined by manual counting. the cell-killing activities of ns3-activated mazf and uncleavable mazf zymoxins were measured by a thiazolyl blue tetrazoliam bromide (mtt) assay as described below: cytotoxicity assay of intracellularly expressed mcherry-ns3 activated mazf or mcherry-uncleavable mazf in t-rex hek293 cells inducibly expressing egfp-full ns3-4a. tet-inducible full ns3-4a, tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded in 96 well plates (2610 4 cells per well). after 24 h, cells were supplemented with 3 fold dilutions of tetracycline starting with concentration of 1000 ng/ml, or left untreated. 72 h later, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml mtt (thiazolyl blue tetrazoliam bromide (sigma, israel) dissolved in pbs) reagent and the cells were incubated for further 30 minutes. mtt-formazan crystals were dissolved by the addition of extraction solution (20% sds, 50% n, n-dimethyl formamide (dmf), ph 4.7) (100 ml per well) and incubation for 16 h at 37uc. absorbance at 570 nm was recorded on an automated microtiter plate reader. the results were expressed as percentage of living cells relatively to the untreated controls. cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf on full ns3-4a expressing huh7.5 cells. 1610 4 wildtype or egfp-full ns3-4a expressing huh7.5 cells [10] were seeded per well in 96 well plates. after 24 h, growth media were replaced by fresh media containing recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf at indicated multiplicity of infection (moi) ratios. control cells remained untreated. four days post infection, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml mtt (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further 60 minutes. the next steps were identical to theses described above. cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf on a mixed culture of uninfected and hcv-infected huh7.5 cells. uninfected huh7.5 cells and mixed culture of hcv infected and uninfected cells at 1:1 ratio (50% infected culture) were seeded in 96-well plates (1610 4 cells/well). after 24 h, cells were treated with recombinant adenoviruses (at moi of ,3) encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf zymoxins. control cells remained untreated. 72 h later, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml mtt (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further 60 minutes. the next steps were identical to these described above. for hcv-infection immunofluorescence analysis, 3610 4 cells from the hcv infected and uninfected mixed culture were seeded per well into 8-well chamber slides (nalge nunc, usa). 24 h later, cells were treated with recombinant adenoviruses (moi of ,3) encoding for the mcherry-ns3 activated mazf or mcherryuncleavable mazf zymoxins. control cells were remained untreated. 72 h post treatment, cells were fixed and permeabilized with 1:1 acetone/methanol mixture and stained with 1:300 diluted mouse monoclonal antibody c7-50 (affinity bioreagents, usa) specific for the hcv core protein followed by staining with 1:100 diluted fitc-conjugated goat anti-mouse igg (jackson immu-noresearch laboratories, usa). nuclei were then stained with dapi and slides were mounted (southernbiotech, usa) and examined using a fluorescence microscope. for each treatment, evaluation of the fraction of the hcv-infected cells from the general cell population was performed by dividing the number of the green, hcv-core positive cells by the general number of cells (dapi stained) from five representative microscopic fields. [ 3 h]-leucine incorporation assay 1610 5 tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded per well in 24-wells plate. 24 or 48 h later, cells were supplemented with tetracycline to a final concentration of 1000 ng/ml, or left untreated. 72 h after seeding, cells were supplemented with [ 3 h]-leucine (perkin elmer, usa) to a final concentration of 1 mci/ml and returned to incubation. after 6 h, cells were scraped, washed with pbs and lysed by four freeze/thaw cycles. 7 mg total protein form the lysate of each treatment were then add to a solution containing pbs and a final concentration of 150 mg bovine serum albumin (bsa) in a total volume of 75 ml. the solution was then mixed with an identical volume of ice-cold 10% trichloro acetic acid (tca). mixtures were then incubated on ice for 30 minutes and centrifuged for 10 minutes at 20,000 g, 4uc, after which the pellet was washed with ice-cold 5% tca, followed by washing with ice-cold 80% ethanol. the pellet was then dissolved in 300 ml of 0.1 m naoh, transferred to a scintillation tube and neutralized with 200 ml 1 m hcl. 4 ml of scintillation liquid was added and radioactivity was counted by a beta-counter device. figure s1 fluorescence microscopy analysis of adenovirus producing foci. 3610 5 hek293 cells were seeded per well in 6 wells plate. when reached 90% confluence, cells were infected with 10 fold dilutions of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf, starting from 2.5610 6 pfu per well. after 36 h, cells were fixed and examined under a fluorescence microscope. red fluorescent adenovirus-producing foci from wells infected with 2.5610 3 pfu are shown. the bar represents 200 mm. table s1 oligonucleotides that have been used in this study. text s1 amino-acid sequences of mazf based zymoxins. text s2 construction and propagation of recombinant adenoviral vectors. stanley lemon (university of texas at galveston, usa) for the hj3-5 hcvcc clone. we thank prof. nadir arber (integrated cancer prevention center, tel aviv sourasky medical center, israel) for the adeasy plasmid system used for generation of recombinant human type 5 adenoviral vectors. treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus replication of hepatitis c virus hepatitis c viral life cycle advances in the development of new therapeutic agents targeting the ns3-4a serine protease or the ns5b rnadependent rna polymerase of the hepatitis c virus the ns3/4a proteinase of the hepatitis c virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy role of proteolytic enzymes in biological regulation (a review) mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin structure and zymogen activation of caspases a brief historical review of the waterfall/cascade of blood coagulation engineered toxins ''zymoxins'' are activated by the hcv ns3 protease by removal of an inhibitory protein domain bacterial mitotic machineries bacterial death by dna gyrase poisoning addiction modules and programmed cell death and antideath in bacterial cultures prokaryotic toxin-antitoxin stress response loci toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest programmed cell death in bacterial populations hypothetical functions of toxin-antitoxin systems occurrence of mazef-like antitoxin/toxin systems in bacteria toxin-antitoxin loci are highly abundant in freeliving but lost from host-associated prokaryotes bacterial toxin-antitoxin systems: more than selfish entities? mazef: a chromosomal toxinantitoxin module that triggers programmed cell death in bacteria mrna interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems bacterial programmed cell death and multicellular behavior in bacteria crystal structure of the maze/mazf complex: molecular bases of antidote-toxin recognition characterization of dual substrate binding sites in the homodimeric structure of escherichia coli mrna interferase mazf the discovery of mrna interferases: implication in bacterial physiology and application to biotechnology nbk/ bik antagonizes mcl-1 and bcl-xl and activates bak-mediated apoptosis in response to protein synthesis inhibition characterization of the c-terminal er membrane anchor of ptp1b improved monomeric red, orange and yellow fluorescent proteins derived from discosoma sp. red fluorescent protein subcellular localization, stability, and trans-cleavage competence of the hepatitis c virus ns3-ns4a complex expressed in tetracycline-regulated cell lines membrane association of hepatitis c virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex structural determinants for membrane association and dynamic organization of the hepatitis c virus ns3-4a complex expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex a dynamic view of hepatitis c virus replication complexes a simplified system for generating recombinant adenoviruses adenoviral vector cytotoxicity depends in part on the transgene encoded recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells recombinant e1-deleted adenovirus vector induces apoptosis in two lung cancer cell lines compensatory mutations in e1, p7, ns2, and ns3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis c virus hepatitis c virus molecular clones and their replication capacity in vivo and in cell culture studying hepatitis c virus: making the best of a bad virus review article: investigational agents for chronic hepatitis c novel targets for hiv therapy perspectives on antiviral drug development antiviral drug discovery targeting to viral proteases protease inhibitors as antiviral agents inhibition of protease-inhibitor-resistant hepatitis c virus replicons and infectious virus by intracellular intrabodies inhibition of hepatitis c virus rna replicons by peptide aptamers hcv ns3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen the n-end rule toxins that are activated by hiv type-1 protease through removal of a signal for degradation by the nend-rule pathway a ribonuclease zymogen activated by the ns3 protease of the hepatitis c virus creation of a zymogen design and characterization of an hiv-specific ribonuclease zymogen from enzyme to zymogen: engineering vip2, an adp-ribosyltransferase from bacillus cereus, for conditional toxicity a switch-on mechanism to activate maize ribosome-inactivating protein for targeting hivinfected cells modified apoptotic molecule (bid) reduces hepatitis c virus infection in mice with chimeric human livers mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes caspase-8 prevents sustained activation of nf-kappab in monocytes undergoing macrophagic differentiation cytochrome c and datp-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade a c-jun dominant negative mutant protects sympathetic neurons against programmed cell death structure and activation mechanism of the drosophila initiator caspase dronc a protocol for rapid generation of recombinant adenoviruses using the adeasy system highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication adaptive mutations producing efficient replication of genotype 1a hepatitis c virus rna in normal huh7 cells production of infectious genotype 1a hepatitis c virus (hutchinson strain) in cultured human hepatoma cells we thank prof. matti sä llberg (karolinska institute, sweden) for the plasmid carrying the ns3-4a gene of the 1a genotype. we thank prof. key: cord-293653-u2qrxq6t authors: watashi, koichi; shimotohno, kunitada title: cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target date: 2007-02-05 journal: drug target insights doi: nan sha: doc_id: 293653 cord_uid: u2qrxq6t cyclophilin (cyp) is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. cyp plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. in addition to these cellular events, a number of reports demonstrated that cyp plays a critical role in the life cycle of viruses, especially human immunodeficiency virus (hiv) and hepatitis c virus (hcv). these two viruses are significant causes of morbidity and mortality worldwide, but current therapies are often insufficient. cyp may provide a novel therapeutic target for the management and/or cure of these diseases, in particular hcv. cyclophilin (cyp) and fk506 binding protein (fkbp) are peptidyl-prolyl cis-trans isomerases (ppiases), enzymes that catalyze the cis-trans interconversion of peptide bonds amino terminal to proline residues (fischer et al. 1989; harding et al. 1989; takahashi, 1999; takahashi et al. 1989) . cyp and fkbp are originally identifi ed as cellular factors that bind csa and fk506, respectively, both of which are immunosuppressants used clinically for the prevention of graft rejection following organ transplantation (handschumacher et al. 1984; harding et al. 1989) . therefore, these ppiases are also called immunophilin. the action of ppiases leads to changes in protein conformation (takahashi, 1999) , but the binding of csa and fk506 to cyp and fkbp, respectively, inhibits the activity of these enzymes (fischer et al. 1989; rosen et al. 1990; takahashi et al. 1989 ). however, the inhibition of ppiase activity by csa and fk506 is an insuffi cient requirement for their immunosuppressive function (bierer et al. 1990; schreiber, 1991) . the csa/cyp or fk506/fkbp complex, subsequently interacts with and inhibits calcineurin (cn), a phosphatase involved in the activation of the transcription factor nf-at. proper nf-at function is essential for the generation of a productive t cell response (clipstone and crabtree, 1992; fruman et al. 1992; liu et al. 1991) . in the absence of immunosuppressants, cn dephosphorylates cytoplasmic nf-at, leading to nf-at nuclear translocation and transactivation of downstream genes participating in the immune response (liu et al. 1992; mccaffrey et al. 1993) . csa and fk506 prevent the dephosphorylation and subsequent nuclear translocation of nf-at leading to immunosuppression. more than 10 cyp subtypes are found in mammals (table 1 ). the subcellular localization of cyps varies. cypa is primarily found in the cytoplasm, while cypb, cypd, cype, and ranbp2 are distributed in the endoplasmic reticulum (er), mitochondria, nucleus, and nuclear pore, respectively. members of the cyp family play roles in a variety of cellular processes including the immune response, transcription, mitochondrial function, cell death, and chemotaxis, as described below. while a number of cyp family members have been ideitifi ed, intensive functional analysis has been performed on only a few including cypa, cypb, cypd, and cyp40. cypa is the most abundant cyp subtype found in the cells (waldmeier et al. 2003) , and it is the primary factor mediating the immunosuppressive effects of csa (colgan et al. 2005) . however, even in the absence of csa, cypa plays an important role in regulating the immune responses as seen in cypa-defi cient mice. cypa-knockout mice have an "allergic" phenotype with increased serum igg1 and ige levels and tissue infi ltration by mononuclear cells, eosinophils, and mast cells (colgan et al. 2004 ), related to increased and dysregulated activity of th2 cd4+ t cells. in cypa-knockout cells, interleukin-2 tyrosine kinase (itk), a signaling molecule crucial for the development of a th2 response, is constitutively activated. itk is a member of the tec family of sh2/sh3-containing tyrosine kinases, and it participates in the signal transduction cascade leading to t cell activation. cypa can bind itk, and this negatively regulates itk activity (brazin et al. 2002) . thus, cypa plays a suppressive role in the development of cd4+ t cell responses through its interaction with itk. other studies have reported several non-immune system roles for cypa. cypa interacts with apoptosis-inducing factor (aif) and promotes aif-mediated chromatinolysis during apoptosis (cande et al. 2004) . additionally, cypa interacts with membrane-bound guanylate cyclase-a (gc-a), a receptor for atrial natriuretic factor (anf) ). gc-a and anf are involved in cardiovascular homeostasis, and cypa appears to function as an endogenous inhibitor of gc-a activation by competing for anf binding. further interactions of cypa with prolactin receptor (syed et al. 2003 ) and transcription factor yy1 (yang et al. 1995) have been observed, but the exact role of cypa in these processes remains unclear. cypa was also observed to bind dna in a zinc-dependent manner in a mouse macrophage cell line (krummrei et al. 1995) . however, the best-characterized role identified for cypa is not in normal cellular physiology, but rather as co-factor during the human immunodefi ciency virus-1 (hiv-1) viral life cycle (see below). cypb was originally identifi ed as a cyp family member bearing a signal sequence leading to the er lumen or the secretory pathway (price et al. 1991) , but the specifi c function of cypb is poorly understood. a yeast two-hybrid screening using cypb as a bait identified an interaction with calcium-signal modulating cyclophilin ligand (caml) (bram and crabtree, 1994) . caml is located on the cytoplasmic face of the er membrane (holloway and bram, 1998) . caml participates in calcium signal transduction pathway and it is essential for peripheral t cell development (tran et al. 2005) . however, the importance of cypb binding to caml function remains unknown. cypb also enhances prolactin-driven cell proliferation (rycyzyn et al. 2000) and promotes the nuclear retrotranslocation of prolactin through a direct interaction. additionally, cypb potentiates prolactin-induced stat5 transactivation by promoting the dissociation of pias3, a stat5 repressor (rycyzyn and clevenger, 2002) . cypb can also associate with interferon regulatory factor (irf)-3 (obata et al. 2005) . extracellular cypb can bind platelets (allain et al. 1999 ) and yokoyama et al. 1995 this initiates a transmembranous infl ux of calcium ion, kinase activation, and platelet adhesion to collagen. accumulating evidence suggests that cyps, in particular cypa and cypb, can mediate intercellular communication similar to cytokines. cyps are secreted from cells in response to infl ammatory stimuli or oxidative stress (jin et al. 2000; seko et al. 2004; sherry et al. 1992; xu et al. 1992) and they can act as potent chemoattractants for neutrophils (sherry et al. 1992) , eosinophils (xu et al. 1992) , and t cells (allain et al. 2002) . cypa and cypb are recognized by the cell surface receptor cd147, and cyp binding leads to erk activation and chemotaxis (pushkarsky et al. 2001; yurchenko et al. 2001; yurchenko et al. 2002) . cypd plays a critical role in mitochondrial function and cell death (tanveer et al. 1996) . during ischemia-induced necrosis, e.g. following a heart attack or stroke, the accumulation of calcium and increase of reactive oxygen species (ros) trigger the opening of a pore in the inner mitochondrial membrane, known as the membrane permeability transition pore (mptp) (halestrap, 1999) . calcium overload and ros induce a conformational change in adenine nucleotide translocase (ant), a key component regulating the opening of mptp at the inner mitochondrial membrane. the opening of mptp leads to mitochondrial swelling, rupture of the outer membrane, and the release of small molecules (waldmeier et al. 2003) . cypd is located within the matrix of the mitochondria and it binds ant facilitating its conformational change (crompton et al. 1998; woodfi eld et al. 1998 ). in cypd-knockout cells, necrosis induced by calcium and ros was decreased, but apoptotic cell death induced by cytokines or anticancer agents was unaffected (baines et al. 2005; nakagawa et al. 2005) . cypd-knockout mice also experienced reduced cardiac injury following reperfusion after ischemia. thus, cypd is a key molecule involved in the cell death process. cyp40 regulates the activity of steroid receptors (srs) (duina et al. 1996; owens-grillo et al. 1995; ratajczak et al. 1993) . srs including the glucocorticoid receptor, estrogen receptor, androgen receptor and progesterone receptor are nuclear hormone receptors that exert transcriptional activity following steroid ligand binding and nuclear translocation. in the absence of steroid ligands, srs form complexes with heat shock protein 90 (hsp90) together with the immunophilins cyp40, fkbp51, or fkbp52 in the cytoplasm. these immunophilins control sr activity by increasing receptor avidity for hormone ligands through ppiase-dependent conformational changes. upon hormone binding, this sr/hsp90/ immunophilin complex dissociates, leaving homodimeric sr, which then translocates into the nucleus to transactivate downstream genes. although there are some reports on other cyp subtypes (table 1 ), the precise functions and signifi cances of them are largely unknown. as described above, cyps play essential roles in diverse cellular processes. interestingly, several viruses have evolved to use cyps during their life cycles. in particular, cyps are demonstrated to be involved in the proliferation of hiv-1 and hepatitis c virus (hcv). other viruses using cyps during their life cycle include vaccinia virus (vv), vesicular stomatitis virus (vsv), and sars-coronavirus. a signifi cant role of cyp in vv replication was fi rst identifi ed through the analysis of several csa analogs. the ability of cyclosporins to suppress vv replication correlated with the inhibition of cyp function (damaso and moussatche, 1998) . vv infection stabilizes cypa, leading to the accumulation of cypa (castro et al. 2003) . in vv infected cells, cypa relocalizes to the peripheral region of the nucleus, colocalizing with sites of virus production. cypa is incorporated into viral particles and is located in the viral core. cypa interacted with the nucleocapsid protein of vsv (bose et al. 2003) , and, like vv, cypa is incorporated into vsv viral particles. although the binding and incorporation of cypa occurred beyond the virus serotypes, the functional role of cypa in the viral life cycle appears to be straindependent. inhibition of cyp activity by csa reduced primary transcription of vsv-new jersey (vsv-nj) but not vsv-indiana (vsv-ind) serotype, and cypa activity was required for the replication of vsv-nj to a greater extent than vsv-ind. the authors suggest that differential requirements of cypa are likely the results of evolutionary pressure during lineage development. the nucleocapsid protein (np) of severe acute respiratory syndrome coronavirus (sars-cov) binds cypa (luo et al. 2004) , and another group reported that cypa is incorporated into sars-cov particles (chen et al. 2005) . extracellular cypa binds cd147 on the cell surface, and treatment with a peptide that blocks cd147 binding inhibits viral infection. thus, cypa may be involved in sars-cov invasion into host cells through interaction with np and cd147, respectively. cypa plays an important role in the viral life cycle of hiv-1. in 1993, cypa was found to interact with hiv-1 gag (luban et al. 1993) , and in 1994, cypa was reportedly incorporated into viral particles (franke et al. 1994; thali et al. 1994) . a gene targeting study demonstrated that only cypa among cyp subtypes was essential for hiv-1 proliferation (braaten and luban, 2001) . within the hiv-1 life cycle, cypa plays multiple roles through different interaction partners, including an early step prior to reverse transcription (braaten et al. 1996; mlynar et al. 1997; steinkasserer et al.1995) . although cypa is incorporated into virions through binding to the ca domain of the gag polyprotein (franke et al. 1994; ott et al. 1995; thali et al. 1994 ), this incorporation is not required for viral infection. instead, target cell expressed cypa is important for productive infection and viral replication (hatziioannou et al. 2005; sokolskaja et al. 2004 ). it has been known for several decades that host cells express different restriction factors to prevent infection by certain retroviruses (cullen, 2003) , and several recent studies have suggested that cypa modulates sensitivity to such a restriction factor early in the hiv-1 life cycle prior to reverse transcription. trim5α is a host restriction factor originally identifi ed using expression cloning that recognizes ca limiting retrovirus proliferation (stremlau et al. 2004 ). towers et al. showed that cypa regulates the activity of a host restriction factor (towers et al. 2003) . disruption of cypa-ca binding by introducing of point mutation into ca or treating human cells with csa decreases hiv-1 infectivity. conversely, the loss of cypa-ca binding greatly enhanced hiv-1 infectivity in simian cells (berthoux et al. 2005; kootstra et al. 2003; sayah et al. 2004) . from the results, the hypothesis was proposed by luban et al. that ca binding by cypa prevented normal antiviral effects mediated by trim5α during hiv-1 infection of human cells, but this same interaction mediated hiv-1 restriction in nonhuman primate cells (sokolskaja et al. 2006; luban, in press ). both the mechanism of trim5α restriction of hiv-1 and the modulation of ca recognition by cypa remain unclear, and further studies are clearly needed to resolve these important issues in the hiv-1 life cycle and the host response to hiv-1 infection. cypa may be important for other aspects of hiv-1 infection. cypa interacts with cd147 (pushkarsky et al. 2001) , heparans (saphire et al. 1999) , vpr (zander et al. 2003) , and envelope glycoprotein gp120 (endrich and gehring, 1998) , although their relevances of the interactions should be further verifi ed. hcv is a major causative agent of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (liang et al. 1993) . hcv infection is a serious health problem affecting approximately 170 million individuals worldwide (poynard et al. 2003) . the current standard therapy for hcv is restricted to interferon (ifn) or pegylated-ifn either alone or in combination with ribavirin. because treatment with these agents, however, fails to produce sustained virus elimination in about one half of the patients (di bisceglie et al. 2002) , alternative and effective strategies to combat against hcv are greatly needed. hcv encodes a single polypeptide that is cleaved by host and hcv-encoded protease including ns3 to generate a set of functional proteins. its genome is replicated by the hcv-encoded rna-dependent rna polymerase (rdrp) ns5b. both of these proteins, ns3 and ns5b, are essential for hcv genome replication and are possible targets for the development of anti-hcv therapeutics (di bisceglie et al. 2002) . small molecule compounds targeting ns3 and ns5b have been developed, and their effi cacy has been examined in clinical trials (di bisceglie et al. 2002) . in addition to these viral enzymes, host cell factors are required for viral replication, and these may provide other options for the development of novel anti-viral agents. disrupting the function of host cell derived factors is particularly appealing as the mutation rate of host proteins is much less than that of viral encoded proteins and should less give rise to drug-resistant viruses. however, while some host factors required for viral genome replication have been identifi ed, other host proteins need to be identifi ed to develop optimal anti-viral therapies with few side effects. at this time, only a limited number of host proteins have been found to be involved in hcv genome replication with biological relevance. hvap-33 is one of snare family proteins that regulate vesicle biogenesis, protein sorting, and membrane fusion. hvap-33 binds hcv ns5a and ns5b, and this interaction regulates the presence of hcv proteins in the subcellular compartment performing viral genome replication (evans et al. 2004; gao et al. 2004; tu et al. 1999) . fbl2 is a member of the f-box protein family, involved in the ubiquitination pathway. fbl2 is geranylgeranylated in cells , and associates with ns5a in a geranylgeranylation-dependent manner to regulate hcv genome replication. however, the mechanism of action of fbl2 in hcv genome replication is not known. additionally, we recently found that cypb is a cofactor for hcv replication in host cells, and this may represent a new target for anti-hcv therapeutics (watashi et al. 2005) . we will fi rst discuss the role of cypb in hcv genome replication followed by the therapeutic implications of this discovery in hcv treatment. we identifi ed csa as an anti-hcv agent using a hcv replicon system, a cell culture system supporting hcv genome replication (lohmann et al. 1999) , and csa inhibits hcv genome replication as potently as ifnα (watashi et al. 2003) . since that time, several groups have made similar observations (firpi et al. 2006; nakagawa et al. 2004; paeshuyse et al. 2006) . as shown in fig.1a , cellular treatment with 1 µg/ml csa decreases hcv rna levels by approximately 1/500 (watashi et al. 2003) . csa also reduces the expression of hcv-encoded proteins to undetectable levels (fig. 1b) . in contrast, fk506 has no effect on the production of hcv rna or proteins ( fig. 1a and b ). the differences between csa and fk506 suggest that csa prevents viral genome replication independently of cn, an effector (copy/pg total rna) amount of hcv rna figure. 1 csa suppresses hcv genome replication. (a) hcv rna was quantifi ed in total rna isolated from hcv replicon-bearing cells treated with various concentrations of csa, fk506, or ifnα for 7 days. the amount of hcv rna per 1 pg total rna was plotted against the concentration of csa (µg/ml), fk506 (µg/ml), or ifnα (× 100 iu/ml). (b) the expression of hcv ns5a and protein disulfi de isomerase (pdi) as a cellular protein was examined in the hcv replicon-bearing cells treated without (control) or with 100 iu/ml ifnα, 1 µg/ml csa, or 1 µg/ml fk506 for 7 days. common to both csa and fk506 mediated immunosuppression. and the anti-hcv effects of csa are mediated by pathway(s) distinct from those of ifnα (watashi et al. 2003 ). the ability of csa to inhibit hcv genome replication correlates with the inhibition of cyp activity (watashi et al. 2005) . moreover, an alternative cyp inhibitor, sanglifehrin, also decreases the levels of hcv rna. thus, the inhibition of cyp activity is essential for the anti-hcv effect of csa, and this strongly suggests that cyp plays a direct, important role in hcv genome replication. moreover, the specifi c knockdown of cypb by rnai reduced hcv rna titer, but knockdown of cypa, cypc, cype, or cyph had no effect on hcv replication activity. these data indicate that cypb plays a critical role in hcv genome replication. the effects of cypb on hcv genome replication are mediated through a direct interaction with ns5b as demonstrated in both in vitro and in cells (watashi et al. 2005) (fig. 2a) . cypb do not bind any other hcv proteins involved in viral replication. ns5b binds hcv genome rna in order to function as a rdrp. cypb but not cypa promotes the rna binding activity of ns5b and stimulates hcv genome replication in cells ( fig. 2a) . this functional support by cypb to ns5b is essential for the effi cient replication of the hcv genome, and csa blocks the interaction of cypb with ns5b, leading to reduced rna binding (fig. 2b ). thus, cypb serves as a cellular cofactor for hcv genome replication. the anti-hcv activity of csa analogs correlates with their ability to inhibit cyp function (watashi et al. 2005) . the dissociation of cypb and ns5b greatly reduces the extent of hcv genome replication. these observations suggest that the inhibition of cypb may represent a novel therapeutic strategy against hcv. this possibility has been examined by two reports using stronger cyp inhibitors than csa. paeshuyse et al. used the csa analog debio-025 to inhibit cyp activity (paeshuyse et al. 2006) , and this compound inhibited hcv replication 10-fold more potently than csa. the authors speculated that debio-025 might be an attractive drug candidate for the treatment of individuals with hcv/hiv coinfection because csa derivatives should also inhibit hiv-1 replication. we used the non-immunosuppressive csa derivative nim811 to target cyp ishii et al. 2006) . nim811 inhibits cyp enzymatic activity two-fold more than csa , and this increased inhibition correlates with greater suppression of hcv genome replication than csa, especially at lower doses. cotreatment of cells with nim811 and ifnα led to a synergistic anti-hcv effect at higher doses of nim811. treatment of nim811 for three weeks eliminated hcv rna from host cells to under detectable level. because the immunosuppression in patients during a viral infection is undesirable, these non-immunosuppressive variants of csa that inhibit cyp activity are likely to offer great promise for the treatment of patients with chronic hcv infection. cyps are cellular ppiases that catalyze conformational changes in proteins, but the role(s) and substrates for this protein family in cells are not well-characterized. however, discoveries of the signifi cance of cyp in life cycles for several viruses make this protein virologically notable and present novel therapeutic anti-viral targets. further analyses of the role of cyps in viral life cycles should reveal novel functions for these proteins as well as provide mechanistic insight into possible therapeutic targets. cyclophilin b binding to platelets supports calcium-dependent adhesion to collagen interaction with glycosaminoglycans is required for cyclophilin b to trigger integrin-mediated adhesion of peripheral blood t lymphocytes to extracellular matrix loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death the cyclophilin multigene family of peptidyl-prolyl isomerases. characterization of three separate human isoforms cyclophilin a is required for trim5{alpha}-mediated resistance to hiv-1 in old world monkey cells probing immunosuppressant action with a nonnatural immunophilin ligand requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype cyclophilin a is required for an early step in the life cycle of human immunodefi ciency virus type 1 before the initiation of reverse transcription cyclophilin a regulates hiv-1 infectivity, as demonstrated by gene targeting in human t cells calcium signalling in t cells stimulated by a cyclophilin b-binding protein regulation of the tyrosine kinase itk by the peptidyl-prolyl isomerase cyclophilin a aif and cyclophilin a cooperate in apoptosis-associated chromatinolysis redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus cyclophilin a functions as an endogenous inhibitor for membrane-bound guanylate cyclase-a. hypertension identifi cation of calcineurin as a key signalling enzyme in t-lymphocyte activation cyclophilin a regulates tcr signal strength in cd4+ t cells via a proline-directed conformational switch in itk cyclophilin a-defi cient mice are resistant to immunosuppression by cyclosporine cyclophilin-d binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore hiv-1 infection: fooling the gatekeeper inhibition of vaccinia virus replication by cyclosporin a analogues correlates with their affi nity for cellular cyclophilins new therapeutic strategies for hepatitis c a cyclophilin function in hsp90-dependent signal transduction the v3 loop of human immunodefi ciency virus type-1 envelope protein is a high-affi nity ligand for immunophilins present in human blood phosphorylation of hepatitis c virus nonstructural protein 5a modulates its protein interactions and viral rna replication cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins specifi c incorporation of cyclophilin a into hiv-1 virions two cytoplasmic candidates for immunophilin action are revealed by affi nity for a new cyclophilin: one in the presence and one in the absence of csa calcineurin phosphatase activity in t lymphocytes is inhibited by fk 506 and cyclosporin a interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft evaluation of the anti-hepatitis c virus effects of cyclophilin inhibitors, cyclosporin a and nim811 the mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury cyclophilin: a specifi c cytosolic binding protein for cyclosporin a a receptor for the immunosuppressant fk506 is a cis-trans peptidyl-prolyl isomerase cyclophilin interactions with incoming human immunodefi ciency virus type 1 capsids with opposing effects on infectivity in human cells co-localization of calcium-modulating cyclophilin ligand with intracellular calcium pools a new cyclophilin and the human homologues of yeast prp3 and prp4 form a complex associated with u4/u6 snrnps diverse effects of cyclosporine on hepatitis c virus strain replication a novel leucine-rich repeat protein (lrr-1): potential involvement in 4-1bb-mediated signal transduction cyclophilin a is a secreted growth factor induced by oxidative stress isolation and characterization of a 40-kda cyclophilin-related protein abrogation of postentry restriction of hiv-1-based lentiviral vector transduction in simian cells cyclophilin-a is a zinc-dependent dna binding protein in macrophages viral pathogenesis of hepatocellular carcinoma in the united states cloning, expression, and purifi cation of human cyclophilin in escherichia coli and assessment of the catalytic role of cysteines by sitedirected mutagenesis inhibition of t cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk506 complexes replication of subgenomic hepatitis c virus rnas in a hepatoma cell line human immunodefi ciency virus type 1 gag protein binds to cyclophilins a and b cyclophilin a, trim5 and resistance to hiv-1 infection nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a nf-atp, a t lymphocyte dna-binding protein that is a target for calcineurin and immunosuppressive drugs a nuclear rna-binding cyclophilin in human t cells the non-immunosuppressive cyclosporin a analogue sdz nim 811 inhibits cyclophilin a incorporation into virions and virus replication in human immunodefi ciency virus type 1-infected primary and growth-arrested t cells specifi c inhibition of hepatitis c virus replication by cyclosporin a drug target insights cyclophilin d-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death rs cyclophilins: identifi cation of an nk-tr1-related cyclophilin role of cyclophilin b in activation of interferon regulatory factor-3 analysis and localization of cyclophilin a found in the virions of human immunodefi ciency virus type 1 mn strain the cyclosporin a-binding immunophilin cyp-40 and the fk506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor cloning, expression and chromosomal mapping of a novel cyclophilin-related gene (ppil1) from human fetal brain the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro viral hepatitis c human cyclophilin b: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence cd147 facilitates hiv-1 infection by interacting with virus-associated cyclophilin a the cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (fkbp59) inhibition of fkbp rotamase activity by immunosuppressant fk506: twisted amide surrogate inhibition of human immunodefi ciency virus type 1 replication by sdz nim 811, a nonimmunosuppressive cyclosporine analog the intranuclear prolactin/cyclophilin b complex as a transcriptional inducer role of cyclophilin b in prolactin signal transduction and nuclear retrotranslocation host cyclophilin a mediates hiv-1 attachment to target cells via heparans cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 chemistry and biology of the immunophilins and their immunosuppressive ligands. science hypoxia followed by reoxygenation induces secretion of cyclophilin a from cultured rat cardiac myocytes identifi cation of cyclophilin as a proinfl ammatory secretory product of lipopolysaccharide-activated macrophages target cell cyclophilin a modulates human immunodefi ciency virus type 1 infectivity cyclophilin a, trim5, and innate immunity to hiv-1 mode of action of sdz nim 811, a nonimmunosuppressive cyclosporin a analog with activity against human immunodefi ciency virus type 1 (hiv-1): interference with early and late events in hiv-1 replication the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys a novel and functional interaction between cyclophilin a and prolactin receptor pharmacodynamics of ciclosporin a (cyclosporine) peptidyl-prolyl cis-trans isomerase is the cyclosporin a-binding protein cyclophilin involvement of cyclophilin d in the activation of a mitochondrial pore by ca2+ and oxidant stress functional association of cyclophilin a with hiv-1 virions cyclophilin a modulates the sensitivity of hiv-1 to host restriction factors caml is a p56lck-interacting protein that is required for thymocyte development hepatitis c virus rna polymerase and ns5a complex with a snare-like protein cyclophilin d as a drug target identifi cation of a nuclear-specifi c cyclophilin which interacts with the proteinase inhibitor eglin c identifi cation of fbl2 as a geranylgeranylated cellular protein required for hepatitis c virus rna replication cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes cyclophilin b is a functional regulator of hepatitis c virus rna polymerase drug target insights direct demonstration of a specifi c interaction between cyclophilin-d and the adenine nucleotide translocase confi rms their role in the mitochondrial permeability transition leukocyte chemotactic activity of cyclophilin cyclophilin a and fkbp12 interact with yy1 and alter its transcriptional activity a giant nucleopore protein that binds ran/tc4 cd147 is a signaling receptor for cyclophilin b active site residues of cyclophilin a are crucial for its signaling activity via cd147 cyclophilin a interacts with hiv-1 vpr and is required for its functional expression molecular cloning, structure and expression of a novel nuclear rna-binding cyclophilin-like gene (ppil4) from human fetal brain molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (ppil3) from human fetal brain key: cord-271635-tydlyc1q authors: abdel-hamid, nabil m.; abass, shimaa a.; mohamed, ahmed a.; muneam hamid, daniah title: herbal management of hepatocellular carcinoma through cutting the pathways of the common risk factors date: 2018-11-30 journal: biomedicine & pharmacotherapy doi: 10.1016/j.biopha.2018.08.104 sha: doc_id: 271635 cord_uid: tydlyc1q abstract hepatocellular carcinoma (hcc) is considered the most frequent tumor that associated with high mortality rate. several risk factors contribute to the pathogenesis of hcc, such as chronic persistent infection with hepatitis c virus or hepatitis b virus, chronic untreated inflammation of liver with different etiology, oxidative stress and fatty liver disease. several treatment protocols are used in the treatment of hcc but they also associated with diverse side effects. many natural products are helpful in the co-treatment and prevention of hcc. several mechanisms are involved in the action of these herbal products and their bioactive compounds in the prevention and co-treatment of hcc. they can inhibit the liver cancer development and progression in several ways as protecting against liver carcinogens, enhancing effects of chemotherapeutic drugs, inhibiting tumor cell growth and metastasis, and suppression of oxidative stress and chronic inflammation. in this review, we will discuss the utility of diverse natural products in the prevention and co-treatment of hcc, through its capturing of the common risk factors known to lead to hcc and shed the light on their possible mechanisms of action. our theory assumes that shutting down the risk factor to cancer development pathways is a critical strategy in cancer prevention and management. we recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid hcc progression and decrease its global incidence. the mortality rate due to hepatocellular carcinoma (hcc) increased rapidly during the past decade. unluckily, the clinically satisfactory and successful treatment for hcc patient is still absent [1] . several risk factors are involved in hepatocarcinogenesis like non-alcoholic fatty liver disease (nafld), hepatitis b virus (hbv) and hepatitis c virus (hcv) infection, alcoholism, obesity, aflatoxin b1, iron accumulation and diabetes [2] . there are several protocols used in the treatment of hcc, including, surgical resection, ablation, chemotherapy and embolization. the use of these methods is limited due to their side effects and the development of resistance to the available chemotherapy and their complexities. due to the limited treatment options to hcc, other than surgery and the poor prognosis of the disease, there is a critical need for additional therapies to enhance the survival or the quality of life. complementary and alternative medicine (cam) is considered as one way that may improve the anticancer drug efficacy and reduce their toxic effects [3] . the use of herbal medicines can be traced back to more than 4000 years ago in ancient china and egypt. over recent decades, an increasing number of herbal products, including medicinal herbs and phytochemicals, have been used for treating chronic liver diseases worldwide due to cost effectiveness, higher safety margins, long-lasting curative effects and few adverse effects. according to the previous studies, medicinal herbs and phytochemicals could protect the liver by several mechanisms such as eliminating the virus, blocking fibrogenesis, inhibiting oxidative injury and suppressing tumorigenesis [4] . in this review, we discuss several factors that lead to hcc development, focusing on the role of different herbal medicines that used in the treatment of hcc by alleviating these risk factors. is similar to the fatty liver damage that caused by alcoholism, but, it happens in non-alcohol abuse people. nafld is characterized by the accumulation of triglycerides within hepatocytes, which is usually associated with metabolic syndrome and obesity [5] . the prevalence of nafld was established by the histological features found in ∼70% of obese individuals suffering from steatosis, while steatosis was present in ∼ 35% of lean individuals. nafld was also present in about18.5% of obese individuals and in about 2.7% of leans [6] . fatty liver is also found in about 13-22% of lean non-alcoholic individuals through several studies based on ultrasound imaging [7, 8] . nafld is present in 20-35% of adult individuals and 5-17% of children in the western world [9] . nafld is considered as one of the important reasons leading to chronic liver diseases in hong kong (∼27.3%) [10] and china (∼15%) [11] . this is because of the high-fat content in the modern diet and individual's lifestyle. nafld is considered as one of the most important reasons that cause chronic liver disease in developing and developed countries. nafld increases the risk of hepatocarcinogenesis similar to other pot cirrhotic liver diseases. hcc is now the end stage as a leading cause of obesity-related cancer deaths in middle-aged men in the usa [12] . an increasing number of case reports showed that hcc arises in non-cirrhotic individuals suffering from nafld [13] . other hcc risk factors may be synergistically involved in hcc development besides nafld, such as alcoholic liver injury and chronic hepatitis c. several mechanisms are involved in nafld-related hcc development ( fig. 1 ) [14] [15] [16] . obesity participates in increasing the risk of cancer development through a low-grade, chronic inflammatory impact [17, 18] . the expansion of adipose tissue stimulates the generation of pro-inflammatory cytokines such as tumor necrosis factor (tnf) and interleukin-6 (il-6) [19] . tnf and il-6 derived from adipose tissue play an important role in hcc development. this role has been supported in an experimental model, assuming that obesity enhances the growth of diethylnitrosamine-induced malignant liver tumor in mice [20] . the prevalence of hcc due to nafld is increasing around the world [21] , where, 4-22% of hcc patients in western countries are attributable to nafld [22] . in asia, viral hepatitis remains endemic, so that, 1-2% of hcc patients are attributed to nafld [23, 24] . wolfberry, a famous traditional chinese supplement or drug, is the fruit part of lycium barbarum plant, family solanaceae. it has valuable benfits in both eyes and liver [26] . the most important part of wolfberry is the lycium polysaccharide portion(lpp), which has a numerous biological actions, like immunoregulation, antioxidant effect, neuroprotection, control of glucose metabolism and anti-tumor activities. the lymphocyte number, interleukin-2 and immunoglobulin g level, were found to be increased upon the intake of polysaccharide juice in human beings in one of the clinical trials. it was reported also that it increases the levels of serum antioxidants and decreases the lipid peroxide level [27, 28] . in the liver, lpp was found to inhibit hepatocyte proliferation and induce apoptosis of hepatoma cells, which indicate its possible application as anti-tumor [29, 30] . another study demonstrated that lpp causes a restoration of the activities of antioxidant enzymes and reduction of oxidative stress products caused by high-fat diet induced liver injury [31] . the co-treatment of lpp with ethanol administration markedly enhanced the liver injury in an alcohol-induced liver injury rat model by decreasing the oxidative stress and the accumulation of lipid in the liver [32] . in acute liver injury, lpp was found to keep the normal hepatic histology, decrease the hepatic inflammation/chemoattraction, stimulate the partial regeneration of the liver through the nuclear factor kappa b (nf-κb)-dependent pathway, and reduce the oxidative stress when used prior ccl 4 intoxication in mice [33] . lpp is helpful in nafld due to its useful properties in decreasing the inflammation and the oxidative stress. the co-treatment with lpp, orally, in nafld in rats, showed a significant improvement in the hepatic histology, reduction in the fibrosis, oxidative stress, inflammation, accumulation of fats and apoptosis, through modulating the transcriptional factors nf-κb and activator protein-1 (ap-1). furthermore, the uptake of lpp for long-term did not have any unwanted side effects on the liver of healthy rats. so lpp can be useful in nafld treatment with minimal side effects [25] . green tea (camellia sinensis) leaves, has been reported to be used in the prevention of liver diseases. the origin of green tea is china, which then distributed to asian countries, including korea, vietnam and japan. in the last years, green tea also spread to western countries that traditionally consume black tea. the beneficial or therapeutic properties of green tea extracts have been reported by several studies. the major polyphenol of green tea, epigallocatechin-3-gallate (egcg), was used in ccl 4 -treated mice and showed a significant therapeutic potential in hepatic damage, inflammation and oxidative stress induced by ccl 4 in a dose-dependent manner at both biochemical and histological levels [34] . it was also reported that co-treatment of the whole green tea extract with alcohol administration showed an effective reduction of the hepatic oxidative stress and reduced form of nicotinamide adenine dinucleotide phosphate (nadph) oxidase systems in experimental alcohol-induced liver injury [35] . green tea extract also showed an enhancement of the nash features, such as oxidative stress, inflammation and lipid accumulation in obese mice [36] . a recent study also demonstrated that green tea extract showed significant improvement in inflammatory, chemical, metabolic, and radiological parameters of nafld patients who were dyslipidemic and non-diabetic [37] . it also showed an improvement in the liver enzymes of nafld patients in another study [38] . numerous in vitro studies demonstrated the chemoprevention and anti-tumor properties of green tea in hcc [39] . the growth and cell proliferation of hcc-derived cells are inhibited by egcg by induction of apoptosis [40] . the human hcc cell line, hepg2, growth was also inhibited by egcg, through suppressing the phosphorylation of insulinlike growth factor-1 receptor (igf-1r) and reducing the activation of its signaling molecules like extracellular signal-regulated kinase (erk), stat-3, akt and gsk-3β [41] . drinking of egcg caused a significant inhibition of liver cell adenoma development in contrast to the egcguntreated control group. this is related to decreased phosphorylation of erk, akt and igf-1r proteins, activation of the hepatic amp-activated kinase protein and the treatment of liver steatosis. the administration of egcg in drinking water also caused a significant reduction of serum levels of insulin, igf-1, igf-2, tumor necrosis factor (tnf)-α, and free fatty acid. it also caused a decrease in the hepatic expression of tnf-α, interleukins(il-1β,il-6), and il-18 mrnas [42] . the development of glutathione s-transferase placental form (gst-p + ) foci was also inhibited after the administration of egcg in drinking water through the reduction of hepatic fibrosis, triglyceride content, inflammation, oxidative stress and inhibition of excessive hepatocyte proliferation [43] . resveratrol (rsv; 3, 5 4′-trihydroxystilbene), a phytoalexin that extracted from red grapes, is considered as one of the most accepted and recognized herbal derivatives worldwide as it has a powerful antioxidant and anti-inflammatory properties [44] . recently, rsv has been found to be helpful in the treatment of nafld. when rsv is given after nafld induction by using high fat-diet, it causes a reduction of lipogenic genes as srebp-1c and fas [45] . the treatment with rsv also reduced the inflammation and oxidative stress in rats [46] . rsv causes a dysregulation in the metabolism of lipids in nafld through sirtuin 1 (sirt1) pathway [47] and the up-regulation of hepatic low-density lipoprotein receptor [48] . rsv increases apoptosis in hcc cells, which is associated with the reduction of hexokinase 2 expression. additionally, rsv improved the inhibition of cell growth induced by sorafenib in aerobic glycolytic hcc cells. the inhibition of hexokinase 2 by rsv can be considered to be a new trend to treat hcc and prevent its progression [49] . rsv was also found to decrease the expression of myosin light chain kinase (mlck), which inhibited liver tumorigenesis and promoted cell apoptosis in hcc rats induced by dena. the expression of mlck was found to be higher in hcc rats than normal rats, which is responsible for the proliferation and anti-apoptotic properties [50] . the latin word "inflammation" means set a light or ignite, which describes exactly its effect on cancer. inflammation increases the resistance to chemotherapy and promotes oncogenes and genes that convert healthy cells to tumors. it also stimulates cancer cell spreading and improves the cancer cells' ability for angiogenesis. because cancer is defined as an inflammation, the anti-inflammatory drugs can be useful in treating cancers as the relation between cancer development and inflammation has been appreciated [51, 52] . the hepatic damage may be due to either chronic or acute inflammation. in response to inflammation, several kinds of hepatic cells are activated, such as hepatic stellate cells (hscs), liver sinusoidal endothelial cells (lsecs), kupffer cells (kcs) and dendritic cells (dcs) to produce several types of cytokines, immune mediators and chemokines. one of the important pro-inflammatory cytokines is interleukin-6 (il-6) that can inhibit apoptosis and tissue inflammation [53] . the inflammatory response mediates a defense mechanism against the microbial infection and stimulates tissue regeneration and repair. there is a relation between the inflammation and cancer development as chronic inflammation stimulates the development of dysplasia. about 15% of cancer occurrence is associated with microbial infection. in immunocompetent patients, chronic inflammation, like hepatitis b and c viral infection or human papilloma virus may result in the development of hepatocellular and cervical carcinoma, respectively [54] . cancer also may result from an opportunistic infection like kaposi's sarcoma, which results from human herpes virus (hhv)-8 infection. inappropriate immune responses to microbes may also lead to cancer development as gastric cancer, which may result from chronic inflammation due to helicobacter pylori colonization. the long-standing inflammatory bowel disease may lead to colon cancer. long-term exposure to asbestos, silica and cigarette smoke, may lead to chronic irritation and subsequent inflammation that result in cancer development [51, 52] . the promotion of tumor cells requires both the survival of the initiated cells and their expansion. numerous inflammatory mediators like chemokines, eicanosoids, and cytokines have the ability to stimulate the proliferation of both untransformed and tumor cell [51] . inflammation plays an important role in tumor growth through angiogenesis mediation. it also plays a critical role in other aspects of tumor progression like metastasis and tissue invasion. matrix metalloproteinases and their inhibitors are important for remodeling of extracellular matrix and angiogenesis, which enhances vascular invasion of migrating cells [55] .the mechanisms contributed to the role of inflammation in hepatocarcinogenesis was showed in fig. 2 [56] . xanthorrhizol (xnt), a sesquiterpenoid complex, obtained from curcuma xanthorrhizza rhizome, family zingiberaceae. the anti-inflammatory effect of xnt was reported for the first time in lipopolysaccharide-mediated mouse leukemic monocyte macrophage cell raw 264.7. it caused a significant reduction in the activities of inducible nitric oxide synthase (inos) and reduced cyclooxygenase-2 (cox-2), through the inhibition of nitric oxide (no) and prostaglandin e2 (pge 2) production [57] . xnt was also found to inhibit pro-inflammatory cytokine interleukin-6 (il-6), tnf-α, inos and cox-2 in activated microglial cells [58] . the anti-inflammatory property of xnt was also postulated in another study as it blocks the inflammatory and neurogenic pain response in pain test that induced by formalin in rats [59] . xnt has also anticancer activities, which may be due to its antiinflammatory effect by inhibiting the activity of nf-kb, cox-2 and inos release [60] . it also inhibited the tumor development and formation in different in vivo studies. it decreased the expression of cox-2, ornithine decarboxylase and suppressing nf-kb signaling activity. an in vivo study also demonstrated that xanthorrhizol has anti-metastatic activity through inhibiting matrix metallopeptidase 9 and cox-2 in a mice lung metastasis model [61] . it has a potent anti-proliferative effect on hepg2 cells through apoptosis induction via bcl-2 family members [62] . berberine is a small alkaloid molecule isolated from coptidis rhizome. it possesses anti-inflammatory and anticancer activities [63] . it down-regulates numerous hepatic pro-inflammatory genes such as il-6, serum amyloid a3 (saa3), nf-kb and tnf-α. these genes play a vital role in steatohepatitis development [64] . it was reported that berberine has an anti-inflammatory activity for hepatic cells in different animal models. it has also been showed to decrease tnf-α and cox-2 expression in cyclophosphamide-induced hepatotoxicity in a rat model [65] . it also has the ability to inhibit tnf-α and il-6 production in hepg2 cells. the anti-inflammatory effect of berberine may be due to the inhibition of erk1/2 activation [66] . its anti-inflammatory effect was postulated through the inhibition of lps-induced inflammatory response in macrophages [64] . berberine has anti-cancer activity on the human hcc cell lines through the induction of apoptosis and inhibition of tumor cell proliferation [67] . berberine induces both cell death and apoptosis in hepg2 cells. this is related to the down-regulation of cd147, which is highly expressed in hcc cells [68] . it was shown to selectively inhibit the human hepatocellular cancer cell growth through the induction of ampk-mediated caspase-dependent mitochondrial pathway cell apoptosis in addition to suppressing p53 [69] . the expression of p53 was found to be up-regulated by berberine through suppression of mdm2, the inner p53 inhibitor, at the post-transcriptional level [70] . the combination of berberine and vincristine showed a helpful effect against hepatoma cell lines through the potentiation of the pro-apoptotic effect of the single drug [71] . alpinia officinarum, known as lesser galangal, belongs to family zingiberaceae. it has a variety of pharmacological actions as antioxidant, anti-inflammatory, antimicrobial, antiemetic and cytotoxic properties [72] . different mechanisms are involved in the anti-inflammatory effect of alpinia officinarum, including the regulation of nf-κb, mapks pathway and the inhibition of prostaglandin e2 synthase and cox-2, the important enzymes involved in the inflammation. this effect is usually due to its content of diarylheptanoids and flavonoids [73] . two compounds of alpinia officinarum rhizome extract, galangin and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone, exhibited antioxidant and anti-inflammatory activities due to their phenolic content. these compounds showed a high affinity toward cox-2 active site through a molecular docking study [74] . our recent study also demonstrated that five compounds isolated from the alpinia officinarum rhizome extract showed a powerful anti-inflammatory effect on hepg2 cells stimulated by lipopolysaccharide. these compounds are galangin, isorhamnetin, two diarylheptanoids and kaempferide. these compounds down-regulated the gene expression of il-6, il-1β, pro-inflammatory cytokines and tnf-α in a dose-dependent manner. this indicates that these isolated compounds may be a promising treatment for other inflammatory diseases [75] . alpinia officinarum rhizome extract and its components showed anticancer activities against numerous cancer cell lines, such as breast, neuroblastoma, lung, and liver [76] [77] [78] . our previous study suggested that alpinia officinarum rhizome extract can be used as a promising natural chemopreventive agent against hcc in rats. it is also a helpful chemosensitizing agent when used in combination with cisplatin in the treatment of hcc. additionally, alpinia officinarum rhizome extract improved hepatic functions and decreased alpha-fetoprotein concentration in experimental hcc model. it also protected the hepatic tissue of the treated rats [79] . oxidative stress (os) occurs when the body is exposed to either a harmful exogenous or endogenous per-oxidative stimuli. one of these stimuli is free radicals, including reactive nitrogen species (rns) and reactive oxygen species (ros). free radicals are produced during numerous oxidation-reduction (redox) reactions that happened in the cells. os is usually associated with the development of various diseases as cardiovascular and nervous system diseases, diabetes and cancer through the induction of oxidative damage of dna and abnormal expression of proteins [80] . os may be a risk factor for hcc as it elevates hepatocyte dna oxidative damage [81] . chronic viral infections may increase the production of ros and rns by causing inflammation and necrosis of hepatocytes that accompany immune cell infiltration [82] . the main cause of dna damage is mutation, caused by increasing ros and the failure in dna repair resulting in an increase in the mutation of cancer-related genes, meaning that chronic inflammation is considered the main risk factor for hcc [83, 84] . increasing os and the degree of dna damage has elevated the incidence of hcc related to viral infection [85] . the elevated level of transforming growth factor beta (tgfβ) and also tnf-α in patients with chronic hepatitis c is mainly related to oxidative stress. tgf-β is used as an indicator of how tissues are damaged and the extent to which they are injured [86] . ros is elevated due to increased oxidative stress, which stops the electron transport chain in damaged mitochondria. the excessive release of tnf-α not only causes severe damage to the mitochondrial respiratory chain but also causes damage to cytochrome oxidase. the high level of ros increases lipid peroxidation and inhibits the respiratory electron transport chain [87] . ros not only alter the mitochondrial metabolic activity but also affects the apoptotic pathways, changes the membrane permeability and causes damage to mitochondrial dna [88] . telomere shortening is accelerated by os that leads to an increase in the cytoplasmic migration of reverse transcriptase telomerase subunits [89] . damaging of the cells, mainly by os, causes the release of a unique type of dna that is called free circulating dna (fcdna), produced as oxides released by dna from dead cells. it can be used as an indication for a wide group of diseases like cancer. fc dna also can be present by low value in normal cells [90] . during the exposure to os, different types of genes and immune system mediator expression is altered. among these mediators, is micro rna (mirna), which is a small non-coding rna molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in rna silencing and post-transcriptional regulation of gene expression. mirna dysfunction is related to different types of cancer including hcc [91] . the mechanisms of hcc development caused by os can be summarized in fig. 3 [89]. 4.1.1. vitamin c and diallyl sulfide vitamin c, (l-ascorbate, l-ascorbic acid), is known by its antioxidant activity and the ability to capture free radical with singlet oxygen such as oh − or hoo − and superoxide ion o 2 − and produce dehydroascorbate. vitamin c activity is related to its free electron that interacts with electron deficient ions. several in vitro studies reported the antioxidant activity of vitamin c [92] . in vivo studies also showed the antioxidant properties of vitamin c in a dose-dependent manner. vitamin c administration protects guinea pigs, that do not form vitamin c, and osteodystrophy syndrome rats from oxidative stress when given oxidizing agents as endotoxin [93] and carbon tetrachloride [94] . it also protects rats exposed to cigarette smoke [95] . parquet is highly toxic nitrogen herbicide and has strong oxidizing stress. the administration of vitamin c reduces oxidative stress before (not after) parquet administration [96] . vitamin c has a promising role in cancer treatment through its selective cytotoxic activity for diverse cancer cell lines [97, 98] . in 1970s, a clinical study was performed and proved that ascorbate has an important role in increasing the survival of cancer patients at late stages [99] . combination of methotrexate with vitamin c is promising in cancer treatment through the reduction of h 2 o 2 produced from methotrexate in hep3b cells [100] . it was shown that vitamin c helped in the treatment of hepatotoxicity induced by dena in smp30 ko mice [101] . garlic contains diallyl sulfide (das) as a major active constituent. das is characterized by its anti-inflammatory activity through the modulation of cytokines as it inhibits the activity nf-kb [102] . das' anti-inflammatory activity is related to the nuclear factor erythroid 2-related factor 2 (nrf2) transcription activation and it also has antioxidant activity. nrf2 is an emerging regulator of cellular resistance to oxidants [103] . a combination of vitamin c and das was shown to offer several benefits, as inhibition of circulatory tnf-α and il-6 in dena-induced hcc in rats [104] and increases the sensitivity to chemotherapy as cisplatin in the treatment of hcc [105] . ginger (zingiber officinale) is one of the predominant herbaceous plants. it is a perennial plant and the main active part is the rhizome. ginger not only used as a condiment but also it has antiemetic and anticancer activity [106] . ginger has strong antioxidant and cell protection activity. this action of ginger is due to its potent active constituents as sesquiterpenoids, tannin, gingerols, shogaols and anthocyanin. several in vitro and in vivo studies documented the antioxidant activity of ginger. the protective effect of ginger was showed against several toxic agents, like bromobenzene and cisplatin [107] . another study displayed the chemopreventive effect of ginger against cancer [108] . ginger has a great activity in the treatment of experimental cancer in a rat model. it decreased the level of growth factors and αfetoprotein (liver tumor marker) after giving rats a daily dose about 50 mg/kg of ginger extract [109] . the anticancer effect of ginger is due to it's proapoptotic and anti-inflammatory properties. the anti-inflammatory activity of ginger was confirmed by inhibiting nf-κb and tnf-α after administration of 100 mg/kg of ginger in hcc rat model [110] . ginger contains other different constituents as clavatol, pinostrobin, and geraniol. these active components were detected by gas chromatography and mass spectrometry. ginger was found to inhibit the proliferation of cells in hepg-2 cell line (ic50, 900 μg/ml) [111] . one of the most popular active components in ginger is 6-shogaol which showed an anticancer effect against hepatoma cell line through the activation of ros-mediated caspase-dependent apoptosis in a multidrug resistance [112] . one of the most popular cultivated plants is broccoli which is distinguished by its high content of the antioxidant content. the most active antioxidant components in this plant are vitamins, flavonoids and carotenoids. isothiocyanates, the hydrolytic product of glucosinolate, is considered one of the antioxidant components, which motivates dna protection from damage through its antioxidant activity [113, 114] . the antioxidant property of broccoli may be direct by contributing in biochemical, cellular and physiological steps that inhibit free radical production, or indirect by inducing phase ii enzymes that have a protective effect against os [115] . the antioxidant activity of broccoli was observed in the human colon mucosa that exposed to oxidative stress [115] . broccoli showed a potential anticancer activity due to its high content of glucosinolates [116] . broccoli also contains a distinct component, sulforaphane, which is characterized by its activity as antioxidant and its ability to protect dna from breaking down by highly reactive electrophiles through increasing the antioxidant system activity and inhibition of inflammation [117] . antioxidant activity of sulforaphane is related to certain pathways, including the reduction of inflammation through inhibiting nf-κb and overexpression of transcription of nrf2, which has a very important role in keeping healthy cells and protect them from toxic chemicals and lifestyle-related factors [118] . previous in vivo studies registered that sulforaphane has abroad activity against different types of liver diseases related to toxic chemicals [119, 120] , consumption of alcohol [121] and using high calorie food [122] . broccoli has a major role in the suppression of different types of cancers, including liver hep-g2 and colon cancer. sulforaphane has different pathways related to its anticancer effect as it has anti-inflammatory, proapoptotic and cell cycle arresting action [123] . hbv and hcv infections are considered the chief reasons for hcc. usually, there are no symptoms for people with chronic infection but lately; cirrhosis and hcc are developed [124] . treating and overcoming hcv and hbv infection can help in the prevention of hcc development as they are oncogenic viruses [125] . the association between hcv infection and hcc varies worldwide. in western countries and africa, hcv is considered as the main cause of hcc, it also contributes to 80% to 90% of hcc cases in japan [126, 127] . as well as,80% of patients infected with hcv can progress to chronic hepatitis, with about 20% developing cirrhosis [128] . the hcv -related liver cirrhosis can increase the risk of liver cancer, with17-fold higher risk of developing hcc than in chronic hepatitis c infection alone, although this risk differs and depends on the degree of liver fibrosis caused by hcv [129] . the elevation of the risk of hcc development in patients infected with hcv arises from chronic inflammation, which results from the progression of liver fibrosis and cirrhosis. these inflammations cause alteration of the architecture of hepatocyte and defects of both cellular functions and the microcirculation of liver. hcv rna does not integrate into the host genome. alternatively, hcv viral proteins like hcv core protein and their induced host response have been involved in reactive oxygen species (ros) production, apoptosis, activation of transcription and modulation of immunity through up-regulation of tnf-α, il-6, and il-1, which participate in the transformation into malignancy [130] . hepatitis b virus (hbv) is a circular genome. chronic hbv infection can be confirmed by the presence of serum hbsag for a period not less than 6 months [131] . about 10%-25% of hepatitis b patients have a high risk of hcc development during their life. chronic hepatitis differs than other causes of hcc, as hcc occurs in the absence of cirrhosis [132] . after tobacco smoking, hbv is considered the second environmental carcinogen that affects individuals, resulting in about 55% of all hcc cases around the world [133] . the association of chronic hbv infection and hcc that now widely recognized was first explained by beasley and colleagues in 1981 [134] in taiwanese patients with positive serum hb surface antigen (hbsag). serum hbsag can be detected in 24%-27% of patients with hcc in japan, 41% of patients with hcc in the united states and 70% of patients with hcc in china in the absence of other risk factors [135] . once hbv arrives at the liver cell, transcription of messenger viral rnas occurs, followed by a translation into viral proteins and then, synthesis of dna of the virus. dna of the virus is then capable of integrating into the host genome in infected hepatocytes. cancer can be facilitated via this process through numerous ways, like rapid cell cycling of hepatocytes and viral dna integration into the host genome which causes instability and it may insert into, or adjacent to, genes that code proteins required for cancer development. it also leads to a chronic inflammation with fibrosis and proliferation of hepatocyte, which ultimately result in cirrhosis and cancer development [136] [137] [138] . andrographis paniculata nees (a. paniculata) is a medicinal plant, which belongs to family acanthaceae. it is used in japan, india, korea, china and other asian countries for a long time in the treatment of several diseases like inflammations, viral and bacterial infections and high blood pressure [139] . the most abundant di-terpene lactone found in the leaves and stems of a. paniculata is andrographolide. the andrographolide treatment was found to reduce the replication of hcv markedly and have a synergistic effect with the clinical trial drug psi-7977 or current antiviral drugs like telaprevir and ifn-α when used in combination to treat hcv. the mechanism of action of andrographolide fig. 3 . the mechanism of hcc development due to oxidative stress [89] . may be due to its ability to induce the p38 mapk/nrf2/ho-1 pathway, where, mapk stands for mitogen activated protein kinase and ho-1 is heme oxygenase-1. it was shown that andrographolide can be used as natural product or potential drug that is helpful in the treatment of hcv [140] . the treatment with a. paniculata aqueous extract was found to enhance the activity of hepatic enzymes and normalizes histopathological changes of malignant hepatic tissue induced by hexachlorocyclohexane [141] . it showed indirect and direct effects on tumor cells by inducing apoptosis and cancer cell necrosis, improving body's own immune system against tumor cells and inhibiting cell-cycle arrests and cancer cells proliferation [142] . the ethanolic extract of a. paniculata showed a cytotoxic effect against diverse human cancer cell lines like pc-3 (prostate), hepg2 (hepatoma), colon 205 (colonic) cancer cells and jurkat (lymphocytic) [143] . the inhibitory effect of andrographolide and its analogs on tumor cells may be due to their ability to depress cyclin-dependent kinase and induce the expression of inhibitory proteins of the cell cycle that result in blocking the cell cycle progression at g0/g1 [144] . andrographolide causes induction of apoptosis by several mechanisms including, the activation of caspase cascade, the release of cytochrome c from mitochondria and the activation of proapoptotic bcl-2 family members bax conformational change [145] . it also causes activation of ros-dependent c-jun nh 2 -terminal kinase (jnk) resulting in the activation of tumor suppressor p53 and thereby increasing p53 phosphorylation and protein stabilization [146] . silymarin is extracted from milk thistle seeds, silybum marianum l. gaertn., which belongs to asteraceae family [147] . the extract of silymarin contains silibinin, which consists of a mixture of two flavonolignans called silybin a (sa) and silybin b (sb). it has diverse pharmacological activities including, antioxidant, antiproliferative, immunomodulatory, antiviral activities and antifibrotic in different tissues and organs [148] [149] [150] . silymarin and its component silibinin possess antiviral activity against hcv infection in cell culture. their antiviral activity is due to their ability to block the entry of the virus, the synthesis of viral rna and protein, viral fusion, virus transmission and the activity of hcv n5sb rna dependent rna polymerase [148, 149, 151, 152] . one study showed that the treatment with a soluble form of silibinin in the form of daily intravenous injection causes a significant inhibition of hcv viral loads by 3-4 logs in 1-2 weeks in previous ifn non-responder patients [153] . several studies reported that silymarin has a potential anticancer effect against hcc. in a dose-dependent manner, silymarin inhibits the population growth of the human hepatocellular cancer cells (hepg2) as it elevates the percentage of apoptotic cells [154] . the antiproliferative activity of silymarin was also reported by another study without affecting the nontumor hepatic cells. in the g0/g1 phase, silymarin caused an increase in the percentage of cells, while in the s-phase it decreased the cell percentage associated with down-regulation of cyclin e, cyclin d1, phospho-rb and cdk4 and up-regulation of p53, retinoblastoma protein (rb), p27kip1, and p21cip1 [155] . silymarin also showed in vivo preventive and therapeutic efficiency against liver cancer. ramakrishnan et al. [156] reported that silymarin has a protective effect against dena-induced hcc in rats. silymarin also showed a potent preventive effect against spontaneous hcc in hbv x protein transgenic mouse model. oral silymarin in a dose-responsive manner causes a restoration of the early stage hepatic damage and fatty changes that lead to the recovery of hepatic tissue [157] . the oral administration of silibinin showed a significant reduction of hcc xenograft growth by inducing histone acetylation, apoptosis and expression of sod1 and inhibiting cell cycle progression, cell proliferation (ki-67 expression), erk and pten/p-akt signaling [158] . glycyrrhiza glabra, a perennial herb, originates from south-western and central asia and the mediterranean region. it showed numerous pharmacological activities like antioxidant, anti-inflammatory and immunomodulatory activities. the main constituent of glycyrrhiza glabra root is glycyrrhizin 1-9%, w/w [159] . glycyrrhizin has anti-viral, antiinflammatory, hepatoprotective and anti-tumor activities [160] . the antiviral activity was reported for glycyrrhizin and other components that isolated from glycyrrhiza species against different viruses, such as herpes simplex, hiv, severe acute respiratory syndrome, influenza virus, coronavirus, enteroviruses and hepatic viruses [161] [162] [163] . glycyrrhizin has been reported to be used in the treatment of hepatic diseases like chronic hepatitis c and b [164] . a preparation that contains glycyrrhizin was reported to reduce hepatic steatosis in transgenic mice expressing the full-length hcv poly-protein [165] . it was shown to have an inhibitory effect on hcv core gene expression and hcv fulllength viral particle both at protein and rna level and have a synergistic effect with interferon [166] . glycyrrhizin and other components of glycyrrhiza glabra showed antitumor activity in different kinds of cancers such as skin, liver and breast cancer, through inhibition of cellular proliferation, development and growth of cancer cells [167] . glycyrrhizic acid, a major bioactive component of the extract of glycyrrhiza glabra, has the ability to inhibit hcc occurrence in dena-treated mice [168] . another study showed that glycyrrhiza glabra extract has a potent effect in the treatment of hcc induced by dena/ccl4 in rats and this effect is more potent than the effect of cisplatin alone or cisplatin combined with glycyrrhiza glabra, so that cisplatin has several side effects and glycyrrhiza glabra is not associated with that side effects [169] . apoptosis, or programmed cell death, has a great interest in the field of oncology [170] . the recognition of each pathway of apoptosis is very important not only in understanding cancer development but also in the prevention and treatment of the disease. the normal tissue homeostasis maintained by keeping the balance between the proliferation of cells and their death. the imbalance between these two processes may lead to dysregulated clonal expansion, the cause of all neoplastic diseases [171, 172] . the mechanism of action of numerous cytotoxic agents includes apoptosis. several experimental approaches aimed to stimulate apoptosis that leads to the improvement of therapeutic response. numerous natural products play a vital role in the regulation of cellular proliferation and differentiation. the chemopreventive and chemotherapeutic activities of natural products may be due to their role in mediating different pathways involved in cancer development and progression [173] . 6.1. herbals with cytotoxic activity 6.1.1. nigella sativa nigella sativa (n. sativa), an annual flowering plant, originates from south and southwest asia and northern africa is grown almost all over the world [174] . n. sativa and its main constituent thymoquinone (tq) possess numerous therapeutic and pharmacological activities like antiinflammatory [175] , anticancer [176] , antioxidant [177] and immunomodulatory activities [178] . it has cytotoxic activity, as the ethyl acetate column chromatographic fraction of the ethanolic extract of n. sativa showed a cytotoxic effect against diverse cell lines such as molt4, hep g2 and ll/2 [179] . another in vitro studies showed about 50% cytotoxicity of a crude methanolic extract of n. sativa against dalton's lymphoma ascites (dla), ehrlich ascites carcinoma (eac) and sarcoma-180 cells (s-180 cells) [180] . its anti-cancer activity is due to its ability to exhibit powerful pro-apoptotic, anti-proliferative, anti-mutagenic, anti-metastatic and anti-oxidant effects. it also can inhibit tumor initiation and progression and has an anti-inflammatory and immunomodulatory effect. n. sativa can regulate signaling pathways like p53, inos and caspases [181] . the anti-tumor activity of n. sativa was reported in several in vivo and in vitro studies. a decoction that consists of seeds of n. sativa, smilax glabra rhizome, and hemidesmus indicus roots showed a significant improvement in the hepatocarcinogenesis induced by dena(4-6 g/kg/day) in rats [182] . the ethanolic extract of n. sativa showed a marked enhancement of dena-induced histopathological variations of the hepatic tissue [183] . another in vivo study showed that the administration of a methanolic extract of n. sativa in hcc albino rat model showed modulation of glucoregulatory enzymes [184] . aqueous extract of n. sativa showed in vitro antiproliferative activity and morphological changes like membrane damage and cell shrinkage in hepg2 cells, which lead to dna damage, cell death and a decrease in cell proliferation [185] . the mechanisms that explain the pharmacological effects of nigella sativa can be summarized in fig. 4 [186]. illicium verum (i. verum) hook belongs to family illiciaceae. it is commonly known as chinese star anise or star anise. it is an aromatic evergreen tree that originates from pakistan, china and other asian countries. due to its low toxic effects to humans, it was classified as "food and medicine" in 2002 by the ministry of health, people's republic of china [187] . the main active constituents that present in i. verum are sesquiterpenoids, monoterpenoids, lignans, phenylpropanoids, volatile compounds, and flavonoids. it also contains tannins, bitter principles and essential oils. these essential oils include transanethole, limone, α-pinene, β-phellandrene, farnesol, safrol and α-terpineol [188] . it also possesses antimicrobial, antioxidant, antifungal, analgesic, anti-inflammatory, sedative, insecticidal and anticonvulsive activities [189] . its cytotoxic activity was also reported in several studies [190] [191] [192] . it showed numerous mechanisms that involved in cell death such as apoptosis induction, scavenging of free radicals and tumor metastasis inhibition [191] . it was reported that alcoholic extract of i. verum showed a significant in vitro antiproliferative activities [190] . similar in vitro study also showed a marked inhibition of cell proliferation by its alcoholic extract by promoting apoptosis, growth inhibition and modulating the pro-apoptotic gene expression like bax and p53 [193] . separately, the cytotoxic effect of i. verum extract was studied in liver cancer model,it exhibited a significant anticancer outcome in hepatic tissue of rats with a significant improvement of tumor burden (decrease of nodule incidence, multiplicity, size, volume and liver weight). it also up-regulated phase ii detoxifying enzymes (glutathione-s-transferase) and decreased oxidative stress by restoration of superoxide dismutase activity [194] . sex has a vital role in hcc development as males are more diagnosed for hcc than females, with a ratio of 2:1-4:1. this may be due to the higher susceptibility of males to be infected with viral hepatitis, smoking, consuming higher amounts of alcohol and have a higher body mass index than females. the higher level of testosterone is associated with advanced hepatic fibrosis in males infected with hcv and hcc in hepatitis b carriers [195, 196] . another potent hepatocarcinogen is aflatoxin, which produced by aspergillus species found on corn, grains, soybeans or peanuts that stored in warm humid conditions [197] . several genetic and metabolic diseases are associated with hcc development such as wilson's disease, hemochromatosis, α-1 antitrypsin disease, glycogen-storage disease types i and ii, porphyrias and tyrosinemia [198] . other factors are also reported to be associated with the marked elevation of hcc development such as cigarette smoking and prolonged use of contraceptive pills [199] . diabetes mellitus is now considered as an independent risk factor for hcc [200, 201] . it causes liver cell damage through hyperinsulinemia and insulin resistance [202] . hyperinsulinemia induces hcc through inflammation, cellular proliferation and apoptosis inhibition. as well, the increase in insulin levels can lead to a decline in the synthesis of insulin growth factor binding protein 1 by the liver, which is supposed to cause an increase in the bioavailability of insulin-like growth factor 1, in addition to the fig. 4 . the cellular and molecular mechanisms of n. sativa that explain its pro-apoptotic, antiproliferative, cytotoxic, anti-oxidant, anti-metastatic, anti-mutagenic and nk-mediated effects. (lsa: lipid-bound sialic acid, tsa: total sialic acid, tnfα: tumor necrosis factor α, mda: malondialdehyde, afp: α-fetoprotein, il-6: interleukin-6, ros: reactive oxygen species, no: nitric oxide, gsh: glutathione, t-pa: tissue-type plasminogen activator, ifn-γ: interferon-γ, u-pa: urokinase-type plasminogen activator, pai-1: plasminogen activator inhibitor type 1) [186] . increase in apoptosis inhibition and cellular proliferation [203] . insulin has also been related to increased oxidative stress and the ros production, participating in dna mutation [204] . efficient storage of cereals liable to aspergillus attack, continuous inspection of both male and female sex hormones in suspected individuals, managing storage diseases as wilson's and hemochromatosis and controlling diabetes mellitus and other leading diseases will certainly contribute to a decline in hcc liability among risky individuals. the plants and their activities mentioned in this review could be summarized in table 1 : hcc is a prevalent disease in many countries around the world. it is highly related to the increase in deaths rate. the development of hcc passes through several intermediate steps such as molecular and transcriptional events that end finally to malignant transformation of hepatocytes. several factors are involved in these steps including; nafld, hcv, hbv, oxidative stress, chronic inflammation, some inborn metabolic errors, environmental toxins and some drugs, etc. accumulating evidence suggested that many dietary and natural products could be potential sources for prevention and treatment of liver cancer. these natural products (summarized in table 1 ) and their active ingredients can inhibit the liver cancer development and progression, through cutting the roads in front of the known leading risk factors for hcc. we here call urge the ministry of health in each country to establish records of liver patients, especially liver tumors. we recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid hcc progression and decrease its global incidence. we also draw attention of specialists in the food industry to add some of these natural products in different recipes to reduce the probabilities of liver diseases infections. none. none. the anti-inflammatory, anti-oxidant, cytotoxic and immunomodulatory [175, 176, 177, 178, 179] illicium verum cytotoxic activity, antioxidant, anti-inflammatory, induction of apoptosis, and inhibition of tumor metastasis [189, 190, 191, 192] therapeutic applications of herbal medicines for cancer patients molecular pathogenesis of hepatic fibrosis and current therapeutic approaches traditional herbal medicine: a review of potential of inhibitory hepatocellular carcinoma in basic research and clinical trial current status of herbal medicines in chronic liver disease therapy: the biological effects, molecular targets and future prospects prevalence of nonalcoholic fatty liver disease in the united states: the third national health and nutrition examination survey fatty liver hepatitis (steatohepatitis) and obesity: an autopsy study with analysis of risk factors prevalence of and risk factors for hepatic steatosis in northern italy frequency of abnormalities detected by abdominal ultrasound among japanese adults prevalence of fatty liver in children and adolescents prevalence of non-alcoholic fatty liver disease and advanced fibrosis in hong kong chinese: a population study using proton-magnetic resonance spectroscopy and transient elastography epidemiology of non-alcoholic fatty liver disease in china risk of cancer in patients hospitalized with fatty liver: a danish cohort study non-alcoholic fatty liver disease as a risk factor for hepatocellular carcinoma: mechanisms and implications nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace obesity, inflammation, and insulin resistance inflammation and metabolic disorders lipid homeostasis, lipotoxicity and the metabolic syndrome dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing il-6 and tnf expression liver transplantation in patients with nonalcoholic steatohepatitis-related hepatocellular carcinoma hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis hepatocellular carcinoma in japanese patients with nonalcoholic fatty liver disease, alcoholic liver disease, and chronic liver disease of unknown etiology: report of the nationwide survey recent advances in the herbal treatment of non-alcoholic fatty liver disease use of anti-aging herbal medicine, lycium barbarum, against aging-associated diseases. what do we know so far? lycium barbarum (goji) juice improves in vivo antioxidant biomarkers in serum of healthy adults immunomodulatory effects of a standardized lycium barbarum fruit juice in chinese older healthy human subjects hot water-extracted lycium barbarum and rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells effect of lycium barbarum polysaccharide on human hepatoma qgy7703 cells: inhibition of proliferation and induction of apoptosis chemical characterization of lycium barbarum polysaccharides and its inhibition against liver oxidative injury of high-fat mice the effect of lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats lycium barbarum polysaccharides protect mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation green tea polyphenols prevent toxin-induced hepatotoxicity in mice by down-regulating inducible nitric oxide-derived prooxidants depression by a green tea extract of alcohol-induced oxidative stress and lipogenesis in rat liver green tea extract protects against nonalcoholic steatohepatitis in ob/ob mice by decreasing oxidative and nitrative stress responses induced by proinflammatory enzymes therapeutic benefits of green tea extract on various parameters in non-alcoholic fatty liver disease patients, pak the effect of green tea extract supplementation on liver enzymes in patients with nonalcoholic fatty liver disease chemopreventive and therapeutic potential of tea polyphenols in hepatocellular cancer green tea constituent (-)-epigallocatechin-3-gallate inhibits hep g2 cell proliferation and induces apoptosis through p53-dependent and fasmediated pathways egcg inhibits activation of the insulin-like growth factor (igf)/igf-1 receptor axis in human hepatocellular carcinoma cells chemopreventive potential of green tea catechins in hepatocellular carcinoma epigallocatechin-3-gallate suppresses hepatic preneoplastic lesions developed in a novel rat model of non-alcoholic steatohepatitis therapeutic potential of resveratrol: the in vivo evidence resveratrol improves non-alcoholic fatty liver disease by activating amp-activated protein kinase resveratrol inhibits nonalcoholic fatty liver disease in rats resveratrol inhibits the expression of srebp1 in cell model of steatosis via sirt1-foxo1 signaling pathway alleviative effects of resveratrol on nonalcoholic fatty liver disease are associated with up regulation of hepatic low density lipoprotein receptor and scavenger receptor class b type i gene expressions in rats by reducing hexokinase 2, resveratrol induces apoptosis in hcc cells addicted to aerobic glycolysis and inhibits tumor growth in mice resveratrol down-regulates myosin light chain kinase, induces apoptosis and inhibits diethylnitrosamine-induced liver tumorigenesis in rats inflammation and cancer: back to virchow? inflammation and cancer stats in cancer inflammation and immunity: a leading role for stat3 infections as a major preventable cause of human cancer new functions for the matrix metalloproteinases in cancer progression role of nonresolving inflammation in hepatocellular carcinoma development and progression, npj precis suppressive effect of natural sesquiterpenoids on inducible cyclooxygenase (cox-2) and nitric oxide synthase (inos) activity in mouse macrophage cells xanthorrhizol inhibits 12-o-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor-kappa b evaluation of the antinociceptive activity and acute oral toxicity of standardized ethanolic extract of the rhizome of curcuma xanthorrhiza roxb xanthorrhizol: a review of its pharmacological activities and anticancer properties antiproliferative property and apoptotic effect of xanthorrhizol on mda-mb-231 breast cancer cells xanthorrhizol induced dna fragmentation in hepg2 cells involving bcl-2 family proteins advances in the study of berberine and its derivatives: a focus on anti-inflammatory and anti-tumor effects in the digestive system berberine suppresses proinflammatory responses through ampk activation in macrophages berberine mitigates cyclophosphamide-induced hepatotoxicity by modulating antioxidant status and inflammatory cytokines berberine inhibits inflammatory response and ameliorates insulin resistance in hepatocytes berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: the cellular mechanism berberine induces cell death in human hepatoma cells in vitro by downregulating cd147 berberine induces selective apoptosis through the ampkmediated mitochondrial/caspase pathway in hepatocellular carcinoma berberine inhibits p53-dependent cell growth through induction of apoptosis of prostate cancer cells the combinational effect of vincristine and berberine on growth inhibition and apoptosis induction in hepatoma cells alpinia: the gold mine of future therapeutics a review on the pharmacological activities and phytochemicals of alpinia officinarum (galangal) extracts derived from bioassayguided fractionation and isolation isolates of alpinia officinarum hance as cox-2 inhibitors: evidence from anti-inflammatory, antioxidant and molecular docking studies isolates from alpinia officinarum hance attenuate lps-induced inflammation in hepg2: evidence from in silico and in vitro studies diarylheptanoids from the rhizomes of alpinia officinarum and their anticancer activity diarylheptanoids derived from alpinia officinarum induce apoptosis, s-phase arrest and differentiation in human neuroblastoma cells galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-bid mitochondrial pathway chemosensitizing effect of alpinia officinarum rhizome extract in cisplatin-treated rats with hepatocellular carcinoma autophagy, mitochondria and oxidative stress: crosstalk and redox signalling survival rate in patients with hepatocellular carcinoma: a retrospective analysis of 389 patients hepatitis, cirrhosis, and hepatoma radical causes of cancer bagging survival trees viralinduced human carcinogenesis: an oxidative stress perspective role of oxidative stress and dna damage in human carcinogenesis hesperidin alleviates acetaminophen induced toxicity in wistar rats by abrogation of oxidative stress, apoptosis and inflammation is mitochondrial dna content a potential biomarker of mitochondrial dysfunction? mitochondrion oxidative stress and liver cancer: etiology and therapeutic targets circulating tumor cells and circulating tumor dna oxidative dna damage correlates with cell immortalization and mir-92 expression in hepatocellular carcinoma vitamin c as an antioxidant: evaluation of its role in disease prevention endotoxin increases oxidative injury to proteins in guinea pig liver: protection by dietary vitamin c the effect of vitamin c on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production vitamin c supplementation on hepatic oxidative stress induced by cigarette smoke in vivo dual effects of vitamin c on paraquat-induced lung damage: dependence on released metals from the damaged tissue pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer vitamin c enhances anticancer activity in methotrexatetreated hep3b hepatocellular carcinoma cells preventive effects of vitamin c on diethylnitrosamine-induced hepatotoxicity in smp30 knockout mice garlic (allium sativum l.) modulates cytokine expression in lipopolysaccharide-activated human blood thereby inhibiting nf-kappab activity diallyl sulfide enhances antioxidants and inhibits inflammation through the activation of nrf2 against gentamicin-induced nephrotoxicity in wistar rats abdel-bakey, polyol profile as an early diagnostic and prognostic marker in natural product chemoprevention of hepatocellular carcinoma in diabetic rats vitamin c and diallyl sulfide as chemosensitizers to cisplatin in treating hepatocellular carcinoma cancer chemoprevention effects of ginger and its active constituents: potential for new drug discovery hepatoprotective, antioxidant, and ameliorative effects of ginger (zingiber officinale roscoe) and vitamin e in acetaminophen treated rats aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells ginger ingredients inhibit the development of diethylnitrosoamine induced premalignant phenotype in rat chemical hepatocarcinogenesis model ginger extract (zingiber officinale) has anti-cancer and anti-inflammatory effects on ethionine-induced hepatoma rats induction of apoptosis by ginger in hep-2 cell line is mediated by reactive oxygen species 6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism antioxidant functions of sulforaphane: a potent inducer of phase ii detoxication enzymes phenolic compounds in brassica vegetables antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion increasing antioxidant content of broccoli sprouts using essential oils during cold storage protective effect of sulforaphane against oxidative stress: recent advances sulforaphane-rich broccoli sprout extract improves hepatic abnormalities in male subjects protective effect of sulforaphane pretreatment against cisplatin-induced liver and mitochondrial oxidant damage in rats sulforaphane increases the survival rate in rats with fulminant hepatic failure induced by dgalactosamine and lipopolysaccharide sulforaphane induces nrf2 and protects against cyp2e1-dependent binge alcohol-induced liver steatosis sulforaphane attenuates obesity by inhibiting adipogenesis and activating the ampk pathway in obese mice antiproliferative effects and metabolism of sulforaphane and glucoraphanin from broccoli sprouts in human colon and liver cancer cells hepatitis b virus infection predictive value of antiviral effects in the development of hepatocellular carcinoma in the general korean population with chronic hepatitis b the global health burden of infection-associated cancers in the year 2002 high prevalence of multinodular hepatocellular carcinoma in patients with cirrhosis attributable to multiple risk factors management of hepatocellular carcinoma alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women epidemiology of hepatocellular carcinoma reactivation of hepatitis b: pathogenesis and clinical implications current management of hepatocellular carcinoma hepatocellular carcinoma and hepatitis b virus. a prospective study of 22 707 men in taiwan hepatocellular carcinoma: global epidemiology award lecture, viruses, immunity, and cancer: lessons from hepatitis b the role of hepatitis b virus integrations in the pathogenesis of human hepatocellular carcinoma integrations of the hepatitis b virus (hbv) and human papillomavirus (hpv) into the human telomerase reverse transcriptase (htert) gene in liver and cervical cancers antifungal activity of andrographis paniculata extracts and active principles against skin pathogenic fungal strains in vitro andrographolide exerts anti-hepatitis c virus activity by up-regulating haeme oxygenase-1 via the p38 mapk/nrf2 pathway in human hepatoma cells effect of aqueous extract of andrographis paniculata on liver tumor regulatory t cells, a potent immunoregulatory target for cam researchers: modulating tumor immunity, autoimmunity and alloreactive immunity (iii) cytotoxic constituents from andrographis paniculata induce cell cycle arrest in jurkat cells inhibition of cell-cycle progression in human colorectal carcinoma lovo cells by andrographolide 123 poster critical role of pro-apoptotic bcl-2 family members in andrographolide-induced apoptosis in human cancer cells andrographolide sensitizes cancer cells to trail-induced apoptosis via p53-mediated death receptor 4 upregulation hepatoprotective and antiviral functions of silymarin components in hcv infection inhibition of t-cell inflammatory cytokines, hepatocyte nf-kappab signaling, and hcv infection by standardized silymarin identification of hepatoprotective flavonolignans from silymarin anticancer potential of silymarin: from bench to bed side silymarin inhibits in vitro t-cell proliferation and cytokine production in hepatitis c virus infection differential in vitro effects of intravenous versus oral formulations of silibinin on the hcv life cycle and inflammation silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells synergistic anti-cancer effect of baicalein and silymarin on human hepatoma hepg2 cells suppression of n-nitrosodiethylamine induced hepatocarcinogenesis by silymarin in rats chemopreventive effect of silymarin on liver pathology in hbv x protein transgenic mice runx3 directly interacts with intracellular domain of notch1 and suppresses notch signaling in hepatocellular carcinoma cells studies on mechanism of action of glycyrrhizin against hepatitis a virus replication in vitro therapeutic basis of glycyrrhizin on chronic hepatitis b review of pharmacological effects of glycyrrhiza sp. and its bioactive compounds glycyrrhizic acid inhibits virus growth and inactivates virus particles glycyrrhizin, an active component of liquorice roots, and replication of sarsassociated coronavirus herbal medicine in the treatment of liver diseases a glycyrrhizin-containing preparation reduces hepatic steatosis induced by hepatitis c virus protein and iron in mice glycyrrhizin as antiviral agent against hepatitis c virus the long term efficacy of glycyrrhizin in chronic hepatitis c patients inhibition of hepatocellular carcinoma by glycyrrhizin in diethylnitrosamine-treated mice therapeutic efficacy of licorice and/or cisplatin against diethylnitrosamine and carbon tetrachloride-induced hepatocellular carcinoma in rats rehabilitation of cancer through oncogene inactivation discovering early molecular determinants of leukemogenesis expansion of a mutated clone: from stem cell to tumour natural products as mechanism-based anticancer agents: sp transcription factors as targets structure-activity studies on therapeutic potential of thymoquinone analogs in pancreatic cancer immunomodulatory and therapeutic properties of the nigella sativa l. seed anticancer activity of nigella sativa (black seed) -a review nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats, free radic potential immunomodulation effect of the extract of nigella sativa on ovalbumin sensitized guinea pigs cytotoxic and immunopotentiating effects of ethanolic extract of nigella sativa l. seeds antitumour principles from nigella sativa seeds recent advances on the anti-cancer properties of nigella sativa, a widely used food additive protection against diethylnitrosoamine-induced hepatocarcinogenesis by an indigenous medicine comprised of nigella sativa, hemidesmus indicus and smilax glabra: a preliminary study in vivo modulation of inos pathway in hepatocellular carcinoma by nigella sativa can methanolic extract of nigella sativa seed affect glyco-regulatory enzymes in experimental hepatocellular carcinoma? evaluation of the effect of nigella sativa extract on human hepatocellular adenocarcinoma cell line (hepg2) in vitro chemical composition and biological activity of star anise illicium verum extracts against maize weevil, sitophilus zeamais adults biological activities and chemical constituents of illicium verum hook fruits (chinese star anise) investigation of the antioxidant activity of illicium verum extracts in vitro screening for cytotoxic activity of herbal extracts anticancer attributes of illicium verum essential oils against colon cancer, south afr a new flavane acid from the fruits of illicium verum in vitro screening for cytotoxic activity of herbal extracts chemo-preventive effect of star anise in n-nitrosodiethylamine initiated and phenobarbital promoted hepato-carcinogenesis a cohort study of serum testosterone and hepatocellular carcinoma in shanghai, china el-serag, higher serum testosterone is associated with increased risk of advanced hepatitis c-related liver disease in males global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment hepatocellular carcinoma: a review el-serag, oral contraception and the risk of hepatocellular carcinoma the association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence diabetes and cancer: a consensus report liver disease in patients with diabetes mellitus an evaluation of the role of insulin-like growth factors (igf) and of type-i igf receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against druginduced apoptosis the major lipid peroxidation product, trans-4-hydroxy-2-nonenal, preferentially forms dna adducts at codon 249 of human p53 gene, a unique mutational hotspot in hepatocellular carcinoma key: cord-271091-ffn59sgf authors: galao, rui p; scheller, nicoletta; alves-rodrigues, isabel; breinig, tanja; meyerhans, andreas; díez, juana title: saccharomyces cerevisiae: a versatile eukaryotic system in virology date: 2007-10-10 journal: microb cell fact doi: 10.1186/1475-2859-6-32 sha: doc_id: 271091 cord_uid: ffn59sgf the yeast saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. less known is its value for virus research, an area in which saccharomyces cerevisiae has proven to be very fruitful as well. the present review will discuss the main achievements of yeast-based studies in basic and applied virus research. these include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production. the yeast saccahromyces cerevisiae has been successfully used for many years as a model organism to unravel biological processes in higher eukaryotes. because it is easy to grow and to manipulate genetically it has been always at the forefront of technical advances. for example, s. cerevisiae was the first eukayotic organism whose genome was sequenced. the complete yeast genome is known since 1996 and comprises 6000 genes, from which more than 60% have an assigned function. remarkably, comparative genomic analysis have shown that approximately 40% of yeast genes share conserved amino acid sequences with at least one known or predicted human protein [1] . moreover, 30% of human genes with a recognized involvement in human diseases have orthologs in yeast [2] . due to this notable gene homology and the high conservation of fundamental biochemical pathways, studies in yeast have been essential for understanding fundamental cellular processes such as mrna translation and degradation [3, 4] , dna repair mechanisms [5] and the cell cycle [6] . many of these studies were carried out using classical genetic tools in which a few number of genes can be analyzed at a time. however new technological platforms and tools have been recently created that allow large-scale functional analysis in yeast. these platforms and tools are commercially available and comprise (i) gene-deletion mutant collections that include ~6000 heterozygous diploid strains, each of which contains a deletion of a single copy of one specific gene, and ~5000 homozygous diploid and haploid strains in which each of the non-essential yeast genes is deleted [7, 8] , (ii) essential gene mutant collections that include an extensive set of promoter-shutoff strains in which the essential genes are placed under the control of a tetracycline(tet)-repressible promoter [9] , and a set of heat inducible shutoff strains to generate ts alleles of essential genes [10] . both systems allow the study of the ~1000 essential genes in a homozygous background; (iii) geneexpression libraries that include plasmids in which each yeast gene is cloned under the control of the galactoseinducible gal1 promoter. an additional set of plasmids have been created in which the yeast proteins are fused with different tags such as the flag epitope, the glutathione s. transferase (gst) or the histidine 6 repeat (his6) [11] [12] [13] [14] ; (iv) dna microarrays have been created that allow to assess the level of expression of thousands of yeast genes in parallel [15, 16] , and (v) protein chips containing approximately 5800 yeast proteins fused to gst-his6 [14] . all these tools have opened new avenues in medical research in which systematic genome-wide screenings can be used to characterize disease-related molecular events and to discover novel medical compounds [17] [18] [19] . viruses remain a major thread for human and animal health as well as in agriculture. nowadays, there is still no vaccine or curative therapy available for main virus pathogens such as the human immunodeficiency virus (hiv) and the hepatitis c virus (hcv). in this respect, a detailed understanding of the biology of pathogenic viruses together with new and systematic screening tools for novel antiviral compounds will be most helpful. as for the cellular biology studies mentioned above, virologist have turned to the use of yeast as a model system to approach fundamental issues in basic and applied research of higher eukaryotic viruses. these studies have exploited the classical yeast genetics and also the recent yeast technological platforms that allow to apply a system biology approach to fundamental questions in virus biology. the present review will discuss first the expression of individual proteins from important pathogenic viruses in yeast to elucidate their function. the vast literature on the yeast two-hybrid system as a method to explore protein-protein interactions however is not a subject of this review. second, the establishment of yeast/virus systems that allow the replication of higher eukayotic viruses in yeast. these systems have made groundbreaking contributions in the dissection of the life cycle of viruses and the host factors involved. finally, the third and fourth sections will focus on recent advances in applied virus research, the use of yeast as a tool for antiviral drug development and as a vaccine vehicle, respectively. although the expression of viral proteins in yeast not always necessarily reflects their role in higher eukaryotes, here we selected some examples in which the analysis in yeast of their effect on highly conserved cellular processes such as cell cycle control, apoptosis or mrna degradation have contributed to the current understanding of the pathogenesis of important viral pathogens such as hiv-1 and hcv. with around 40 million people infected worldwide and no effective vaccine or curative therapy, hiv remains a major human thread. a better understanding of the hiv replication cycle and the viral and host factors involved are essential to develop new therapy options. yeast-based studies have brought to light key aspects of the function of three hiv proteins, the viral protein r (vpr), the protease (pr) and the regulatory viral protein (rev). the vpr protein plays a pivotal role in the pathogenesis of hiv-1. it is involved in the suppression of immune functions and the depletion of infected cd4 + t cells, which finally leads to the progression to aids [20] . however, the mechanism underlying its effects on pathogenesis was unknown. observations initially made in yeast and then corroborated in mammalian cells demonstrated that vpr acts as an inducer of g2 arrest and cd4 + t cell killing by induction of mitochondrial-dependent apoptosis [21] . the hiv protease is needed to cleave the precursor pr55 gag to the mature proteins matrix (p17), capsid (p24), nucleocapsid (p7) and p6 proteins [22] . studies in s. cerevisiae demonstrated that the pr specifically arrested cell growth and induced cell lysis mediated by the loss of membrane integrity [23] . the contribution of hiv-1 pr to hivinduced necrosis was later confirmed with a cd4 + human t cell line, where a pr-specific inhibitor led to prevention of virus-induced cell lysis [24] . these results highlight the importance of pr-inhibitors to prevent cd4 + t cell loss during infection. finally, the rev protein plays an essential role in the hiv replication cycle. rev mediates the export of unspliced and incompletely spliced hiv mrnas to the cytoplasm and thus, enables the production of hiv-1 particles. the important finding showing that the rev protein mediates the export of hiv pre-mrna by interacting with it via cis-acting rev responsive elements (rre) in the context of the spliceosome was first shown in yeast [25] . these observations led to the identification in s. cerevisiae of the small-nucleoporin-like protein rip1p as the responsible cellular component for rev-mediated export [26] and of the crm1p protein which mediates the interaction between rip1p and rev [27] . hcv has chronically infected more than 170 million people worldwide and is a major cause of liver cirrhosis and hepatocarcinoma. in spite of the great advances accomplished since its identification in 1989, there are still limited therapy options and no vaccine. the expression in s. cerevisiae of two hcv proteins, the non-structural protein 5a (ns5a) and the core protein, together with the construction of hcv chimeras to study hcv rna translation have made yeast a valuable tool in hcv research. in yeast and human cells, the ns5a protein predominantly associates with the er membrane through the highly conserved amphiphatic alpha-helix in its n-terminus [28, 29] . this is consistent with its proposed role as a key regulator for membrane-associated viral replication. moreover, studies in yeast and human cells showed that n-terminally truncated ns5a mutants were transported to the nucleus due to a functional nls and exhibited trans-activating properties [29] [30] [31] [32] . interestingly, the caspase-mediated cleavage of ns5a, occurring during apoptosis or after interaction of ns5a with the core protein during infection, resulted in similar truncations which translocated to the nucleus [33] [34] [35] . however, in which way the ns5a-dependent transactivation contributes to hcv pathogenesis still remains to be elucidated. the hcv core protein interacts with ns5a and is linked to many processes in hcv pathology. from mammalian studies it is known that the hcv core protein influences the transcription of cellular and viral promotors [36] , however the mechanisms involved were mainly unknown. studies in yeast demonstrated the effect of the core protein on the transcription factor ap1-like. the core protein is processed in yeast as in mammalian cells and localizes in the nucleus and the cytoplasm [37] [38] [39] [40] . the transport of the mature core protein to the nucleus was shown to be mediated by direct interaction with the ortholog of human importin 4 (kap123). this interaction in turn abrogates the transport of the ap1-like transcription factor (yap-1) into the nucleus [37, 41] and consequently contributes to the regulation of transcription by the core protein. a bicistronic vector has been recently designed to study hcv rna translation in yeast [42] . the hcv rna is not capped and thus translation depends on an internal ribosomal entry site (ires) located at the 5'non-translated region (ntr). hcv ires-mediated translation is modified by the 3'-ntr and several viral and probably also host proteins bind to the 5'-ntr of hcv [43] . since hcv ires is functional in yeast [44, 45] the bicistronic construct generated will allow to fully explore the effect of viral and cellular factors on hcv translation. an understanding of the fundamental steps of virus life cycles including virus-host interactions is essential for the design of novel effective antiviral strategies. such understanding has been hampered by the complexity of higher eukaryotic host organisms. to overcome these experimental difficulties, systems have been developed that allow the replication of higher eukaryotic viruses in s. cerevisiae. the first higher eukaryotic virus shown to replicate and encapsidate its genome in s. cerevisiae was the bromo mosaic virus (bmv), a positive strand rna ((+)rna) virus from plants [46, 47] . this pioneer work opened new avenues in virus research and allowed to apply the versatile yeast genetic tools in the elucidation of fundamental steps in virus replication. the list of higher eukaryotic viruses shown to replicate in yeast has been expanded ever since and include (i) other viruses with rna genomes that infect plants (carnation italian ringspot virus (cirv), tomato bushy stunt virus (tbsv) and cymbidium ringspot virus (cymrsv)) or animals (flock house virus (fhv) and nodamura virus (nov)), and (ii) viruses with dna genomes that infect humans (human papillomavirus), animals (bovine papillomavirus) or plants (mungbean yellow mosaic india virus (mymiv)) [48] [49] [50] [51] [52] [53] . importantly, these yeast systems reproduce the known features of virus replication in their corresponding natural hosts. since a detailed description of each of these systems and contributions has been recently reviewed [54, 55] , we will only highlight some main achievements in virus research. in particular, we will focus on studies carried out with yeast/ rna virus systems since they are the most advanced. all rna viruses that have been shown to replicate in yeast belong to the group of (+)rna viruses. this large virus group includes many important plant, animal and human pathogens such as hcv and the severe acute respiratory syndrome coronavirus (sars). all (+)rna viruses share common features in their replication cycles. first, the genomic rna serves as messenger rna (mrna) for translation of the viral proteins and as template for replication. since these two functions are mutually exclusive, the transfer of the genomic rna from the cellular translation machinery to the replication complex must be highly regulated. second, viral replication complexes are associated with intracellular host membranes which proliferate and rearrange in response to the expression of viral protein. and third, host factors are required at multiple steps of the viral replication cycle [56] [57] [58] . all the yeast/(+)rna virus systems developed show common characteristics. (i) the viral rna-dependent rna polymerase (rdrp) and additional proteins required for viral replication are expressed in trans from yeast plasmids, (ii) the genomic rna with authentic 5' and 3' ends is also transcribed from a yeast plasmid or transfected directly into the cell, and (iii) replication is monitored by detecting viral rna via northern blot analysis or by measuring the expression of a reporter gene inserted into the viral genome, such that its expression depends on viral rna replication. the fact that in these systems the individual viral proteins are given in trans from separate plasmids is of great advantage since it allows simplifying and controlling functional studies of each player on viral replication. with this, important aspects of the viral life cycles were studied like translation of viral proteins, the process of template selection, formation of the replication com-plex, viral rna replication, encapsidation and recombination (reviewed in detailed in [54, 55, 59] . these studies have resulted in an impressive progress in the understanding of these fundamental steps of viral rna replication and the viral proteins involved. furthermore, due to the common replication strategy of (+)rna viruses, the results obtained go much beyond the specific virus studied. the identification of the host factors involved in viral rna replication is a priority area of research in virology because it can provide new targets for antiviral drug development. the use of traditional yeast genetics and genomewide screenings have resulted in the groundbreaking identification of multiple host factors required for viral rna replication and recombination. the gene-deletion mutant collection in which all the non-essential yeast genes are deleted was used to carry out genome-wide screenings of host factors affecting the replication of bmv and tbsv [60, 61] . in the bmv studies each of the single deletion strains was transformed with plasmids expressing the bmv replicases (the polymerase 2a and the helicase 1a) and a bmv rna replication template harbouring a luciferase reporter gene which expression was screened as a marker for bmv rna replication. in the tbsv studies, a similar approach was applied; however, effects on replication were followed by direct visualization in ethidiumbromide-stained gels of a shorter form of the tbsv rna, which accumulates at a very high level in the wild-type strain. these analyses revealed that from the approximately 5000 non-essential yeast genes analyzed, around 100 were found to affect the replication of each virus. these results uncovered the involvement in viral replication of previously unconsidered cellular pathways, such as mrna turnover, stress response and the ubiquitin pathway of protein degradation. surprisingly and against all previous predictions, only 4 genes were found to be common in both studies. this was discussed to be possibly related to the fact that bmv and tbsv replicate in association with cellular membranes from different compartments. in spite of the impressive achievement in identifying essential host facors for replication, the above mentioned approach has the limitation that only the non-essential genes can be screened. to overcome this, an additional genome-wide screening was performed in the yeast tet promoter hughes collection (ythc) with tbsv [62] . in the ythc, which covers 800 of the ~1000 essential genes, each essential gene is under the control of a tet-titratable promoter in the genome [9] . in this way, the expression of the essential gene can be turned off by the addition of doxycycline to the yeast growth medium. with this approach 30 additional cellular genes were identified that are involved in the replication of tbsv [62] . tbsv not only replicates in yeast but also undergoes recombination [49] . to elucidate the eventual role of host factors in the viral rna recombination process, genomewide screenings were performed in both yeast collections mentioned above [63, 64] . since the recombinant and non-recombinant tbsv forms vary in length, a screening was performed by direct visualization of tbsv rna in agarose gels in combination with northern blot analysis and rt-pcr sequencing. the genetic screens identified 11 nonessential and 16 essential genes that affected tbsv recombination. the exciting observation that host factors can influence viral rna recombination and thereby evolution has brought a totally new perspective to the field. major viral pathogens still remain without effective vaccine or therapy options, thus safer and more effective antiviral drugs are urgently needed. for this, improved screening processes that enable to assess the mode-ofaction of the tested compounds are essential. in this section we will discuss the exciting contributions of yeast genetics and high-throughput screenings to drug development. we will present first the principal characteristics of these global approaches and then their application to the antiviral research field. the process that leads to the discovery and development of new drugs is long and costly. two approaches have been traditionally used. if the target is already known, the drug discovery search classically starts with in vitro assays to identify small molecule inhibitors. these assays give only a partial view of the effect of the compounds since, for instance, they cannot fully explore drug effects on other additional targets, which can be the source of undesirable side effects. putative toxic effects will be tested subsequently in cell culture and in animal models. however, if toxicity is detected, the underlying mechanism will still be unknown and no rational approach is possible to modify the drug to improve safety. an alternative drug discovery approach directly performs cell-based screens for molecules that produce a specific desired effect. this has the advantage that the molecule encounters natural physiological conditions and allows selecting those molecules that are stable within the metabolic environment and discarding those that show toxic effects. however, also with this approach the exact mechanism of action of the selected drug remains unknown. this is a great disadvantage, since its elucidation could lead to the design of new compounds with improved safety and efficacy profiles. both above approaches will benefit from the large-scale chemical and genetic tools developed in s. cerevisiae that allow to systematically screen putative targets and effects of the selected drugs [17, 19] . since the effect of a drug on every yeast gene is analyzed, a broad view of all the proteins and related metabolic pathways affected can be accomplished. thus, including yeast-based screenings at an early stage in drug development programmes, crucial information will be rapidly obtained that can be used to discard or to improve the tested compounds. the large-scale chemical and genetic yeast screens use the mutant strain collections and gene expression plasmids described above. there are five main screening approaches [19] . first, the drug-induced haploinsufficiency approach uses the heterozygous mutant collection and is based on the finding that reducing the copy number of a drug target gene from two to one copy can significantly sensitize a diploid cell to that drug [65] [66] [67] . the second approach uses the haploid or the homozygous diploid deletion collection and is based on the principle that the deletion of a gene that renders cell-hypersensitivity to a specific drug identifies pathways that buffer the cell against the toxic effect of the drug and thus provides clues about its mode of action [68, 69] . the third approach uses gene expression libraries and is based on the principle that increasing the concentration of a protein that is the target of an inhibitory drug should increase resistance to the drug. these three approaches can be performed in two different ways, (i) by growth measurement of arrayed strains, and (ii) by competitively growing pooled strains. the fourth approach is based on gene expression profiling and consists of comparing changes in mrna expression elicited by a drug of interest with those induced by other drugs or by gene deletions. finally, the plasmid collection of all yeast proteins expressed as gst-fusions was used to create protein chips that allow the screening of direct drug-protein interactions. the combination of all the five powerful techniques together with the rapidly growing knowledge of yeast genetic networks [70] has made yeast a recognized system in drug development that until today has not been fully exploited in antiviral research. after more than two decades of searching for antiviral drugs, a rather limited number of compounds is on the market. from these, almost all focus on hiv and herpesviruses. furthermore, most antivirals are not curative and produce major side effects. thus, there is an urgent need of novel and high-quality targets and drugs together with improved screening systems to identify them. two kinds of targets can be used in antiviral research, viral factors or host factors. classically, viral proteins have been used as targets for therapeutic interventions because the developed inhibitor molecules were expected not to produce significant side effects. however, this does not seem to be the case and, for instance, all the antiretrovirals currently available produce important side effects. since drug-resistant mutants are rather easily selected, researchers have shown again interest in host factors. they have the advan-tage of being genetically stable and may even be efficient as targets against groups of viruses. the above mentioned screening options could be helpful to select those host factors that when targeted by drugs will be lethal for the virus but have minor effects on the cell. two yeast cell-based assays have been established to screen for viral inhibitors. the protein m2 of influenza virus has a fundamental role in the disassembly of the influenza virion. in fact, some of the commercially available drugs target this protein. since expression of the m2 protein in yeast produced a slower growth rate, a screening procedure was set up based on the rescue of wt cell growth in the presence of test compounds [71] . more recently, a parallel approach has been applied to the serine protease encoded by the human cytomegalovirus (hcmv) [72] . this protease is essential for the virus and recognizes a 40 amino acid sequence, which was inserted into the coding sequence of a yeast enzyme involved in the biosynthesis of tryptophan. coexpression of the hcvm protease with the engineered enzyme resulted in an arrest of cell growth in media without tryptophan that could be rescued in the presence of protease inhibitors. this system provides the basis for the high-throughput screening of inhibitory molecules. related to the analysis of host factors as antiviral targets, the genome-wide screenings in the yeast/virus systems described in the previous section have provided many new candidates for which the therapeutic potential remains to be explored. few studies in antiviral research make fully use of the yeast platforms and tools available. because of their innovation in the field, we would like to point out two of them that explore the mode of action of antivirals plus other therapeutic compounds. in the first study the heterozygote yeast deletion collection was used to asses the cellular effects of 78 different compounds [67] . it is important to mention that in the construction of this collection, each yeast gene was replaced with a cassette containing the selectable marker gene kanmx plus two unique tag sequences located up-and down-stream of the marker gene. these tag sequences enabled rapid analysis of strains in a pool by using hybridization to high-density nucleotide arrays. importantly, the tag sequences were flanked with universal primer sets for polymerase chain reaction amplification [8] . thus, a pool of ~3500 heterozygous deletion strains were competitively grown for 20 generation in media containing the selected compounds. then, dna was extracted from the cell culture at generation 0 and 20 and amplified by pcr using universal primers and cy3 (green) or cy5 (red) labels, respectively. the amplified dna was hybridized to microarrays containing each specific tag sequence. since signal intensities from micro-array hybridizations are proportional to the relative tag abundance in the pool population, for each yeast strains red, green or yellow colours in the array will correlate with an enrichment, depletion or unchanged representation of the strain in the population. in this way the effect of a drug on each of the yeast deletion strains could be rapidly analyzed [67] . in a second study a complementary approach was followed. the ~5000 yeast haploid deletion mutant collection was tested for hypersensitivity to 82 different compounds including some molecules with antiviral activity [69] . since it is considered that compounds with similar biological effects lead to similar chemical profiles, clustering of the obtained results provided a very useful data set that allowed the organization of the compounds into functionally relevant groups. the results obtained with both studies helped to identify new modes of action of the tested compounds and revealed numerous insights into the cellular pathways involved, yeast has been utilized in vaccine development. the classical example is the recombinantly expressed hepatitis b surface antigen that has become a safe and efficient prophylactic vaccine worldwide [73] . however, in this section we will focus on its use not as a mere production tool for recombinant antigens but as a vaccine vehicle itself. immunization against bacterial pathogens and viruses is an attractive strategy in combating infectious diseases by stimulation of the immune system. traditional vaccines like soluble antigens formulated with adjuvants efficiently activate the humoral immune response. the production of neutralizing antibodies that bind to invading microorganisms inhibits their entry into target cells, thereby preventing an infection. however, cell-mediated immunity, not humoral immunity alone, is required to control infections with intracellular pathogens, especially viruses like hiv and hcv. in the classical view of cellular immunity, antigen-presenting cells (apc) present peptides from endogenously synthesized pathogen proteins in the context with mhc class i molecules on their cell surface. antigen-specific cd8-positive t lymphocytes become activated by recognizing these peptides and subsequently differentiate into a mature effector cell population. these cytotoxic t cells (ctl) are able to recognize and kill the respective infected cells. additionally, dendritic cells (dc) which are the most potent apc use the mechanism of cross-priming for antigen presentation, wherein peptides from exogenous antigens are also presented via mhc class i molecules [74] . "danger" signals as given by the interaction of pathogen components (e.g. cell wall components of yeast) with pattern recognition receptors on the surface of apc, such as toll-like receptors (tlr), mannose or glu-can receptors, leading to the maturation of the apc and an efficient antigen presentation [75, 76] . whole recombinant yeast cells, known as tarmogens™ (targeted molecular immunogens) represent a novel vaccination tool for the induction of potent cell-mediated immune responses against target antigens [77] . in most cases, the non-pathogenic baker's yeast saccharomyces cerevisiae has been used as antigen carrier because this genus combines several advantages. (1) products with s. cerevisiae, e.g. bread and beer, are consumed in large amounts worldwide and healthy individuals show only moderate t cell responses against s. cerevisiae, probably due to mechanisms of oral tolerance [78] . (2) important for primeboost immunization strategies is the observation that there is no neutralization of yeast vectors by the host immune system following multiple immunizations in mice and rabbits [79, 80] . remarkable low titres of antiyeast antibodies were found after repeated injections of either live or heat-killed antigen-expressing s. cerevisiae [77] . (3) yeast cells themselves have potent adjuvant effect via "danger" signals that lead to the maturation of dc without being dangerous, so no exogenous adjuvant is needed [80, 81] . (4) facilitated by the particulate structure of yeast, antigens are efficiently presented via the mhc class i pathway resulting in the activation of antigen-specific cd8 t lymphocytes [81] [82] [83] . stubbs et al. showed for the first time that recombinant yeasts expressing hiv-1 antigens and tumour antigens potently elicit antigen-specific ctl responses, including those mediating tumour protections in mice [80] . further studies with the animal tumour challenge model could demonstrate that yeast-based immunotherapy using mutated ras oncoprotein as target antigen caused a mutant-selective tumour remission in animals [77, 79] . this concept is now being tested in a phase i clinical trial in patients with colorectal, pancreatic and non-small cell lung cancers [77] . most recently, the same group has evaluated a recombinant yeast cell vaccine that expresses the hcv ns3-core fusion protein as an immunotherapeutic vaccine in chronic hcv-infected individuals [84] . one advantage of using whole recombinant yeasts as antigen carriers is the oral application route. feeding mice with s. cerevisiae expressing actinobacillus pleuropneumoniae antigen led to the induction of protective systemic and mucosal immune responses [85] . the fission yeast schizosaccharomyces pombe represents, like s. cerevisiae, a physiologically and genetically well characterized eukaryote that has been used in africa for hundreds of years in brewing of millet beer, from which it was first isolated. recombinant sz. pombe cells expressing the human cytomegalovirus pp65 protein were able to stimulate cd4-and cd8-positive memory t cells in human blood [82] . instead of using recombinant yeast expressing an antigen of choice as a vaccine, attempts have been made to express mhc proteins plus the desired antigenic peptide on the yeast surface and to use the modified cell as a kind of apc. brophy and colleagues succeeded in expressing mouse mhc i protein heavy chain, beta2-microglobulin plus an antigenic peptide from a single gene cassette. the resulting complex assembled in a functional conformation on the cell surface and even induced the activation of naïve t cells [86] . however whether this approach will be of practical use in vaccination has yet to be demonstrated. the strategy to use yeast cells for vaccination could also be used to elicit protective immune responses against human pathogenic yeasts. for example, the genus candida is able to cause a variety of infections from mucosal candidiasis to life-threatening disseminated and bloodstream infections, especially in immunocompromised individuals. unfortunately, the interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated candidiasis in mice receiving a heatinactivated whole-cell c. albicans vaccine [87] . this observation may explain why subjects with elevated anti-candida antibody titers remain nonetheless susceptible to invasive candidiasis [87] . however, ibrahim and colleagues could show that vaccination with the recombinant n-terminal domain of the adhesin als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity in both, immunocompetent and immunocompromised mice [88, 89] . in conclusion, the yeast system has been extremely valuable in virus research. however, the full potential from the remarkable progress in systemic yeast biology over the last years has yet to be exploited. with the increasing options for analysing protein interaction networks at an unprecedented level of complexity and the rising number of viral replication systems in yeast that correctly mimic the fundamental properties of the virus life cycle in higher eukaryots, the era of system virology is expected to become a new focus in virus research. subsequently, the better understanding of the virus protein/host cell protein interaction networks will allow the search for and development of more efficient and safer antiviral drugs. moreover, the acquired information about drug functioning down to the molecular level will help in the search for new drug applications and might significantly reduce the time frame for drug development when a new virus appears to threaten the human population. yeast genomics and proteomics in drug discovery and target validation human genetic diseases: a cross-talk between man and yeast eukaryotic mrna decapping interaction of mrna translation adn mrna degradation in saccharomyces cerevisiae chromatin remodelling at a dna double-strand break site in saccharomyces cerevisiae functional characterization of the s. cerevisiae genome by gene deletion and parallel analysis exploration of essential gene functions via titratable promoter alleles heat-inducible degron and the making of conditional mutants microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in tor pathway biochemical and genetic analysis of the yeast proteome with a movable orf collection systematic identification of protein complexes in saccharomyces cerevisiae by mass spectrometry global analysis of protein activities using proteome chips expression monitoring by hybridization to high-density oligonucleotide arrays quantitative monitoring of gene expression patterns with a complementary dna microarray from drug to protein: using yeast genetics for high-throughput target discovery yeast as a model for medical and medicinal research yeast as a tool to uncover the cellular targets of drugs the vpr protein from hiv-1: distinct roles along the viral life cycle viral infections and cell cycle g2/m regulation hiv-1 replication cell killing by hiv-1 protease involvement of hiv-1 protease in virus-induced cell killing a functional interaction between rev and yeast pre-mrna is related to splicing complex formation identification of a novel nuclear pore-associated protein as a functional target of the hiv-1 rev protein in yeast the yeast nucleoporin rip1p contributes to multiple export pathways with no essential role for its fg-repeat region an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein 5a the amino terminal deletion mutants of hepatitis c virus nonstructural protein ns5a function as transcriptional activators in yeast hepatitis c virus nonstructural protein 5a contains potential transcriptional activator domains characterization of the nuclear localization signal and subcellular distribution of hepatitis c virus nonstructural protein ns5a hepatitis c virus nonstructural region 5a protein is a potent transcriptional activator the hepatitis c virus core protein interacts with ns5a and activates its caspase-mediated proteolytic cleavage use of an in vitro model and yeast two-hybrid system to investigate the pathogenesis of hepatitis c cleavage of hepatitis c virus nonstructural protein 5a by a caspase-like protease(s) in mammalian cells hepatitis c virus core protein: an update on its molecular biology, cellular functions and clinical implications the core protein of hepatitis c virus is imported into the nucleus by transport receptor kap123p but inhibits kap121p-dependent nuclear import of yeast ap1-like transcription factor in yeast cells differential subcellular localization of hepatitis c virus core gene products. virology nuclear localization of the truncated hepatitis c virus core protein with its hydrophobic c terminus deleted the native form and maturation process of hepatitis c virus core protein importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains pospisek m: hepatitis c virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast the hepatitis c virus life cycle as a target for new antiviral therapies. gastroenterology hepatitis c virus internal ribosome entry site-dependent translation in saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rc)-binding protein 2, and la protein internal initiation in saccharomyces cerevisiae mediated by an initiator trna/eif2-independent internal ribosome entry site element rna-dependent replication, transcription, and persistence of brome mosaic virus rna replicons in s. cerevisiae rnacontrolled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein stable replication of papillomavirus genomes in saccharomyces cerevisiae yeast as a model host to study replication and recombination of defective interfering rna of tomato bushy stunt virus replication of carnation italian ringspot virus defective interfering rna in saccharomyces cerevisiae complete replication of an animal virus and maintenance of expression vectors derived from it in saccharomyces cerevisiae the dna-a component of a plant geminivirus (indian mung bean yellow mosaic virus) replicates in budding yeast cells replication of bovine papillomavirus type 1 (bpv-1) dna in saccharomyces cerevisiae following infection with bpv-1 virions saccharomyces cerevisiae: a useful model host to study fundamental biology of viral replication saccharomyces cerevisiar as a model host for studying gene expression and rna replication of positivestrand rna viruses host factors in positive-strand rna virus genome replication intracellular determinants of picornavirus replication viral rna replication in association with cellular membranes yeast as a model host to dissect functions of viral and host factors in tombusvirus replication ahlquist p: systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of rna viruses identification of essential host factors affecting tombusvirus rna replication based on the yeast tet promoters hughes collection screening of the yeast ythc collection identifies essential host factors affecting tombusvirus rna recombination genome-wide screen identifies host genes affecting viral rna recombination chemogenomic profiling: identifying the functional interactions of small molecules in yeast genomic profiling of drug sensitivities via induced haploinsufficiency shoemaker dd: discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast exploring genetic interactions and networks with yeast growth impairment resulting from expression of influenza virus m2 protein in saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus novel yeast cell-based assay to screen for inhibitors of human cytomegalovirus protease in a high-throughput format synthesis and assembly of hepatitis b virus surface antigen particles in yeast selective transport of internalized antigens to the cytosol for mhc class i presentation in dendritic cells the danger model: a renewed sense of self. science decoding the patterns of self and nonself by the innate immune system yeasts encoding tumour antigens in cancer immunotherapy extensive mhc class i-restricted cd8 t lymphocyte responses against various yeast genera in humans mutation-selective tumor remission with ras-targeted, whole yeast-based immunotherapy whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity human dendritic cell interactions with whole recombinant yeast: implications for hiv-1 vaccine development specific activation of cmv-primed human t lymphocytes by cytomegalovirus pp65 expressed in fission yeast cross-presentation of hla class i epitopes from influenza matrix protein produced in saccharomyces cerevisiae whole recombinant yeast-based immunotherapy induces potent t cell responses targeting hcv ns3 and core proteins induction of antigen-specific immune responses by oral vaccination with saccharomyces cerevisiae expressing actinobacillus pleuropneumoniae apxiia a yeast display system for engineering functional peptide-mhc complexes interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated candidiasis in recipients of a candida albicans vaccine vaccination with recombinant n-terminal domain of als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity the anti-candida albicans vaccine composed of the recombinant n terminus of als1p reduces fungal burden and improves survival in both immunocompetent and immunocompromised mice this work was supported by the spanish ministerio de educación y ciencia (grants bfu2004-00654 and ha2006-0110), and the daad. i. a-r was supported by fundação para a ciência e tecnología (sfrh/bd/9630/2002), portugal. the author(s) declare that they have no competing interests. all authors have contributed to the content and structure of the present review. key: cord-303189-ktl4jw8v authors: coccia, eliana m.; battistini, angela title: early ifn type i response: learning from microbial evasion strategies date: 2015-03-31 journal: seminars in immunology doi: 10.1016/j.smim.2015.03.005 sha: doc_id: 303189 cord_uid: ktl4jw8v abstract type i interferon (ifn) comprises a class of cytokines first discovered more than 50 years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. as such, their induction downstream of germ-line encoded pattern recognition receptors (prrs) upon recognition of pathogen-associated molecular patterns (pamps) is a hallmark of the host antiviral response. the acknowledgment that several pamps, not just of viral origin, may induce ifn, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. acting in both autocrine and paracrine manner, ifn interferes with viral replication by inducing hundreds of different ifn-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. on the other hand an inverse interference to escape the ifn system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the ifn pathway, that result in progression of infection or establishment of chronic disease. in this review we discuss the interplay between the ifn system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies. the ability of the host to respond to invading pathogens relies on the activation of the innate immune system that orchestrates adaptive immune responses for pathogen clearance. in recent years, our understanding of the mechanisms involved in the activation of the innate response has evolved significantly with the identification and characterization of the mammalian system of pathogen recognition. the innate immune system detects the presence of a pathogen through a set of germline-encoded membrane-associated or cytoplasmic receptors, termed pattern recognition receptors (prrs) that are engaged by microbial-derived products named pathogenassociated molecular patterns (pamps). major classes of prr include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlr), c-type lectin receptors (clrs), retinoic-acid inducible gene (rig)-i-like receptors (rlrs) and a growing list of cytosolic dna-sensing receptors [1] [2] [3] [4] [5] [6] [7] . upon engagement, these receptors recruit a number of adaptor proteins to signal downstream and activate three major pathways: the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b), the mitogen-activated protein kinases (mapks) and the ifn regulatory factor (irf) pathway [8, 9] . downstream tlrs and rlrs, type i ifn and pro-inflammatory cytokines are mainly produced, while nlrs predominantly activate inflammasomes, resulting in the release of il-1 and multiple inflammatory cytokines (fig. 1) . based on the structure of their receptors, interferons are broadly classified into three groups, type i, ii and iii ifns. type i ifn comprises the largest ifn class that includes ifn-␣, constituted by several partially homologous genes and ifn-␤, ifn-, ifn-, ifnrepresented by a single gene. the ifn-␣ and ifn-␤ are the best characterized as antiviral and the most broadly expressed [10] [11] [12] and will be referred as ifn-i from now on. the ifn-␥, the only type ii ifn, is released by activated t and nk cells; type iii ifns, which include ifn-1-4, similarly to ifn-i are believed to regulate the antiviral response [13] . according to the most common view, ifn-i exerts primarily an anti-viral action while ifn-ii acts predominantly on macrophages to induce a microbicidal state against ingested intracellular, non-viral pathogens. anti-microbial ifn-i activity is not intrinsic, but mediated, in both autocrine and paracrine manner, by a unique set of induced genes named ifn-stimulated genes (isgs) [10, 11, 14] . secreted ifn-i, indeed, binds to a common heterodimeric ubiquitously expressed receptor composed of ifnar1 and ifnar2 chains, and initiates a signaling cascade that has been characterized in detail and reviewed elsewhere [10] [11] [12] . briefly, canonical ifn-i pathway results in activation of the janus kinase (jak) family (cytoplasmic tyrosine kinases) that, in turn, activate by phosphorylation the signal transducers and activators of transcription (stats) [15] . activated stats complex with irf9 to form the heterocomplex ifn-stimulated gene factor 3 (isgf3), that translocates in the nucleus, binds to upstream sequence elements named ifn-stimulated response elements (isre), and activates the transcription of isgs (fig. 2) . these genes act to promote viral clearance and establish an antiviral state in uninfected bystander cells, or to induce apoptosis and several anti-microbial mechanisms in infected cells. they also stimulate cells at the interface of innate and adaptive immunity, such as macrophages and dendritic cells (dcs) that trigger the adaptive response [16, 17] . the ability to break in innate immunity before the onset of the adaptive response is, thus, crucial for the survival of virtually all mammalian pathogens. on the other side of the coin, an essential part of the early response to pathogens is aimed at limiting their ability to hijack host cellular machinery and evade the ifnmediated antimicrobial mechanisms. nowadays, while it is largely accepted that also bacteria can induce the production of ifn-i, the role of the ifn system in the pathogenesis of bacterial infections can be either detrimental (mainly in intracellular bacterial infections) or protective (mainly in extracellular bacterial infections). the route and tropism of bacterial infection, viral co-infections and last, but not least, the balance between ifn-i and ifn-ii effects are all determinants of the different outcome [18] . the ratio between ifn-i and ii species produced in response to infection, might differ as consequence of the capacity of the pathogen to stimulate specific cell types in the infected tissues. moreover, taking into consideration that several antibacterial ifn-ii-induced genes are stimulated to a much lesser degree by ifn-i, it is quite difficult in vivo to separate the activities strictly dependent on one of the two ifn families. a more reliable view consists of a crosstalk between the two pathways: if the bacterium is able to stimulate ifn-i production, generally it occurs early after the infection as an immediate innate immune response, while ifn-ii intervenes later on when immune cells (such as t cell subsets and nk cells) are activated. also due to this complex picture, while a number of viral evasion strategies have been mechanistically defined so far, studies aimed at characterizing the bacterial components that inhibit the ifn system are only recently starting to be elucidated in molecular details [19] [20] [21] . main strategies that pathogens have evolved to disarm the ifn-i response include: (i) blocking ifn-i production by modifying, curtailing or limiting production of their pamps to make them inaccessible to prrs and/or hitting the components of prrs signaling pathways; (ii) interfering with the ifn-i signaling by inhibiting signal transducers; (iii) blocking or disturbing the action of isgs; (iv) hijacking host proteins or components of the ifn system. each of these strategies involves a number of different molecular mechanisms and the combination of more strategies may be necessary to overcome the ifn-i response by a single pathogen. depending on the nature of the pathogen, these countermeasures that tip the balance toward the pathogen, may result in increased replication or in the establishment of a persistent infection. leaving out strategies that envision ifn-i repression due to a general inhibition of cellular gene expression, reviewed elsewhere [22, 23] , here we summarize recent findings on evasion tricks utilized by pathogens to specifically subvert the ifn system. a special focus is put on few highly pathogenic bacteria and emerging or re-emerging viruses, which represent a major threat to human health due to the lack of effective vaccines and/or therapeutics. for an in-depth coverage of other pathogens and strategies to escape the host immune response, the reader is referred to more comprehensive and specific reviews [19] [20] [21] [23] [24] [25] [26] . recent years have seen major advances in our understanding of the innate response to infectious pathogens and specifically of how pathogen recognition promotes ifn-i release [8, 27, 28] . cytoplasmic and membrane-associated prrs recognize a variety of pathogen components. viral pamps mainly consist of nucleic acids originating from the uncoating of infecting virions, the transcription of viral genomes and the replication of genomic intermediates, in the form of single-stranded (ss) and double-stranded (ds) dna, and ss and ds rna, as well as viral glycoproteins. bacterial pamps include various molecules, ranging from lipoproteins, lipopolysaccharide (lps), flagellin and peptidoglycan to unique bacterial nucleic acid structures, such as cyclic dinucleotides (cdns). multiple endosome-associated tlrs (tlr3, 7, 8 and 9) are specialized in the detection of viral and bacterial nucleic acids. tlr3 recognizes dsrna, tlr7/8 are bound by ssrna and tlr9 by cpgcontaining dna. tlr2, 4 and 13, previously considered sensors only for bacterial components, are now been involved also in recognition of viral ligands and in the induction of ifn-i [29] . all tlrs contain an intracellular domain, the toll-interleukin-1 receptor (tir), which recruits one or more tir-containing adaptor proteins to transmit signals downstream. tlr3 signals through the adaptor tir domaincontaining adaptor inducing ifn-␤ (trif) to activate the two related kinases, inhibitor of kb kinase (ikk)-and tank-binding kinase 1 (tbk1) that mediates activation by phosphorylation of irf3 and irf7. tlr7/8 and tlr9 use, as adaptor, myeloid differentiation primary-response protein 88 (myd88) that then initiates signaling cascades involving il-1r-associated kinases (irak1-4) and tnfrassociated factor (traf) 3/6 proteins, which finally converge at the activation of the ib kinase (ikk) family members ikk-␣, ikk-␤, ikk-and tbk1 responsible for activation of nf-b and irf3/7 [4] . in the cytoplasm, two closely related helicases, retinoic acidinducible gene 1 (rig-i) and melanoma differentiation-associated gene-5 (mda5), recognize dsrna of many replicating viruses in a tlr-independent manner. rig-i preferentially senses short dsrna and ssrna with a 5 -triphosphate (5 -ppp rna), while mda5 recognizes long dsrna and poly i:c [30] [31] [32] . like viral rnas, bacterial rnas can possess 5 -ppp termini and secondary structures that make them rig-i agonists. consistent with this notion, rig-i can act as a sensor of bacterial rna and may help maintain homeostasis to gut microbiota [33, 34] . upon recognition of non-self rna, rig-i and mda5 are recruited to the mitochondrial antiviral signaling protein (mavs; also known as cardif, visa or ips1), which triggers a signaling cascade that leads to the activation of ikk-and tbk1 and, in turn, irf3/7 phosphorylation [35] . stimulator of ifn genes (sting), initially identified as a cytosolic dna sensor, also participates in the rig-i signalling [36] . sting interacts with the adaptor mavs at the mitochondrial associated membranes (mam) facilitating the recruitment of tbk1 and the activation of irf3 [37] . a more general role of sting in innate immune responses, not only limited to its function as adaptor of rna and dna sensors, has been now established [38] . in addition to these rna sensors, viral rnas may also be recognized by effector molecules that are themselves ifn-induced proteins with antiviral functions. these molecules include dsrnaactivated protein kinase (pkr) that binds and is activated by dsrna from viruses and bacteria [39] , ifn-induced tetratricopeptide repeat proteins 1/2/3 (ifit1, ifit2, and ifit3) that, as rig-i, bind 5 -ppp rna and may recognize viral mrnas that lack 2 -omethylation [1, 40] . a growing list of cytosolic dna sensors then recognizes dna from different sources including viruses, bacteria and apoptotic cells [3, 6, 7] . more than ten dna cytosolic receptors have been proposed so far that include dai (dna-dependent activator of irf), rna polymerase-iii, ifn-inducible interferon gamma-interferoninducible protein 16 (ifi16), sting, extrachromosomal histone h2b, leucine rich repeat (in flii) interacting protein (lrrfip1), ku70, deah box protein (dhx) 9 and dhx36, cyclic gmp-amp synthase (c-gas). as other sensors, dna sensor activation results in the production of ifn-i and proinflammatory cytokines and chemokines via the sting-tbk1-irf3 axis. in addition to the activation of irf3-and nf-b-dependent signaling cascades, cytosolic dna can also promote an apoptosis-associated speck-like protein containing a card (asc)-dependent inflammasome-mediated response resulting in the secretion of proinflammatory cytokines [3, 7, 25, 27, 28] . the intracellular nlrs scaffold large signaling complexes to mediate innate immunity and inflammatory responses. they may trigger the assembly of inflammasomes and modulate the nf-b, mapk and irf signaling pathways. in particular, nod1 and nod2 are important for immune detection of intracellular bacterial pathogens and are also involved in a variety of immune homeostatic functions [41] . upon the recognition of bacterial peptidoglycans by nod1 and nod2, receptor-interacting serine-threonine kinase 2 (rip2) is activated via cellular inhibitors of apoptosis 1 and 2 (ciap1 and 2), subsequently leading to ubiquitination of nf-b essential modulator (nemo) and the activation of the proinflammatory nf-b pathway. in parallel, the recognition of muramyl dipeptide present in all peptidoglycans, can also lead to the activation of mapk pathway via rip2, which contributes to cytokine production. for the induction of ifn-i, nod2 activated by viral rna, signals through the mitochondrial mavs independently of rip2. then, some sensors can aggregate with adaptors as apoptosisassociated speck-like protein containing a caspase recruitment domain (asc) and caspase 1 to form multimeric structures named inflammasome [42] (fig. 1) . as first line strategy to face these sensing pathways, pathogens hide detection by modifying their pamps. they may also degrade/inactivate target key signal transduction hubs by counteracting host-induced post-translational modifications required for signaling molecule activity and use concomitant different strategies as better described below and also discussed by thomas kufer and igor brodsky in this issue. we focus here on some members of coronaviruses, flaviviruses, hepaciviruses, filoviruses and retroviruses as well as bacteria whose pamps could in principle activate the ifn system, but that efficiently betray host sensing and effector pathways (fig. 3) . coronaviruses (cov) are large enveloped rna viruses, in the family coronaviridae, of both veterinary and clinical importance. two newly emerging viruses in the family, the severe acute respiratory syndrome cov (sars-cov) and the middle east respiratory syndrome cov (mers-cov), have been recently responsible for severe disease in humans (reviewed in [43] [44] [45] [46] [47] ). a common trait in their pathogenesis is the lack of induction of a robust ifn-i response in infected cells [48, 49] . to do this, cov have developed multiple strategies (recently reviewed in [50, 51] ). rig-i, mda5 and the host isgs ifit1 and 2 are critically involved in sensing of cov infection. however, as first line of hiding from recognition, cov encode several highly conserved nonstructural proteins (nsps) implicated in viral rna capping activity in order to examples of viral and bacterial antagonists that block subvert or exploit the ifn system. pathogens affect at every step the ifn system by multiple mechanisms. sites of intervention by several antagonists are indicated. ifn antagonists may prevent prr recognition by hiding or modifying pamps, may inhibit prr signaling by directly targeting adaptors and signaling effectors, may interfere with the ifn signaling by impairing signaling transducers and may block or disturb the action of isgs. some antagonists have more than one cellular target while others target common signaling molecules, effectively blocking ifn induction from a variety of pamps. see the text for more details and references. mimick n7-and 2 -o-methylated 5 cap structure of cellular mrnas [52] . these viral proteins include a rna-triphosphatase, a guanine-n7-methyltransferase, and a 2 -o-methyltransferase encoded by nsp13, 14 and 16, respectively [50, 53] . consistently, human and mouse cov mutants lacking 2 -o-methyltransferase activity induce higher expression of ifn-i and are highly sensitive to ifn-i effects [54, 55] . sars-cov nsp14 also encode a 3 ,5 exoribonuclease that is involved in rna-proofreading, but probably it also functions in degrading viral pamps, further hiding immune detection [50, 56] . similarly, nsp11 encodes a ribonuclease that may degrade viral pamps [57] . another strategy used to avoid detection is the replication in protected sites as the double membrane vesicles (dmvs) that the virus induces in the host cytoplasm. in the case of sars-cov these membranes contain the replicase complex and the viral genomic rna suggesting that replication occurs in these sites and the generated nucleic acids are soon after shielded [58, 59] . in addition, the highly basic nucleocapsid (n) protein of sars-cov has been reported to directly inhibit ifn-i production induced by both poly(i:c) or sendai virus, by a mechanism that involves steps upstream rig-i. thus, it is conceivable that by binding dsrna, n protein prevents rig-i/mda5 activation [60] . others sars-covencoded proteins, as orf-3b and orf6, may also interfere with the rlr recruitment of the adaptor mavs based on their preferential localization at the mitochondrial membrane even if their mechanism of action is not yet elucidated [61, 62] . the sars-cov membrane (m) protein blocks transcription of ifn-i when stimulated by dsrna or members of the rig-i signaling pathway including rig-i, mavs, ikk-, and tbk1, but it does not influence the transcriptional activity of the ifn promoter when irf3 or irf7 are overexpressed. the physical association of sars-cov m with rig-i, tbk1, ikk-, and traf3 suggests that m protein may prevent the formation of the functional complex with tbk1 thereby inhibiting activation of irf3/irf7 and ifn-i transcription [63] . similarly, the papain-like protease (plp) domain contained in the sars-cov nsp3 protein, an essential component of the viral replicase complex, interacts with the adaptor sting blocking the binding to mavs and the recruitment of the tbk1/irf3 complex [64] [65] [66] . finally, through its deubiquitinating activity, the plp protein removes ubiquitin from several components of the rlr pathway blocking their activation (reviewed in [50, 51, 67, 68] ). recently, two mers-cov accessory proteins, the orf-4a and orf-4b products, have also been identified as immunosuppressive factors. mers-cov 4a is a rna-binding protein that interacts in a mrna-dependent manner with pkr-associated activator (pact), a cellular dsrna-binding protein, which potently stimulates rig-iinduced ifn production by binding to the c-terminal repression domain of rig-i [69] . so, orf-4a inhibits rig-i/mda5 pathway without a direct binding to the sensor, but by perturbing the function of a stimulator of rig-i signaling as pact [70] . the orf-4bencoded accessory protein is also able to inhibit ifn-i induction in vitro, however, the mechanism involved is not yet elucidated [71] . the genus flavivirus, of the flaviridae family, includes west nile virus (wnv), dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), tick-borne encephalitis virus (tbev) and several other viruses all causing serious medical problems in humans [72] [73] [74] [75] . currently, vaccines for humans are available only for yfv, jev, and tbev and no clinically approved antiviral therapy is available for the treatment of flavivirus infections [75] . in particular, denv and wnv are re-emerging as global life-threatening human pathogens [75, 76] . both viruses are sensitive to ifn antiviral effects and the severity of the disease is mostly dependent on the ability to avoid and/or attenuate induction of ifn-i and its effector responses through several viral encoded ifn-antagonists [73, 74] . interestingly, some of these strategies are shared with cov. the most relevant prrs for the detection of wnv and denv products, described so far, are the tlr3, 7, 8 and the rlrs, rig-i/mda-5. suppression of both tlr-and rlr-mediated ifn induction has been shown to be important for viral replication [77, 78] . as cov, flaviviruses contain a cap structure that is generated by a methyltransferase mapped to the n-terminal region of the ns5 protein [74, 79] . through 2 -o-methylation of the viral mrna cap wnv, denv and jev evade ifit1-dependent and -independent mechanisms of host restriction in vitro and in vivo [80, 81] . moreover, to hide their nucleic acids during the replicative cycle, both denv and wnv induce the formation of convoluted membranes in the endoplasmic reticulum (er) and golgi apparatus that envelop the virus replication complex [82, 83] protecting viral nucleic acids from both tlr and rlr recognition, as it occurs along the infection with cov. so far, an antagonism at the level of sensing signaling has been clearly defined only for denv that fails to induce an ifn response in myeloid cells where it replicates, despite other pro-inflammatory cytokines and chemokines are produced [84, 85] . the ability to inhibit ifn-i production is due to the viral ns2b/3 protease that binds and cleaves the adaptor/sensor sting [85] [86] [87] . interestingly, first evidences indicate that the ns2b/3 proteases of other flaviviruses, as jev or yfv, are not able to cleave sting, while the ns4b of yfv can do it [68] . to the same flaviviridae family belongs the hepacivirus genus, distantly related to flaviviruses, that includes hepatitis c virus (hcv) a major cause of chronic liver disease [88, 89] . hcv uses several strategies to efficiently evade innate immunity and this escape is considered the main determinant of viral persistence that leads to a chronic infection in 70-80% of infected people [88, 90] . hcv rna can be sensed by different prrs, namely rlrs, tlrs, nlrs, and, as recently reported, by protein kinase r (pkr). rig-i is the best described sensor for the poly u/uc region located within the 3untranslated region (utr) of the viral rna, along with a 5 -ppp. this region is essential for viral replication and, thus, highly conserved among hcv genotypes. the key viral protein involved in the evasion strategies is the ns3/4a protease, which consists of ns3 and ns4a. the complex is essential for several steps in the viral cycle including viral rna replication, polyprotein processing and viral assembly [91] . taking advantage of its serine protease activity, ns3/4a cleaves the adaptor mavs, preventing its dimerization and downstream signaling [92, 93] . after cleavage, mavs dissociates from the mitochondrial associated endoplasmic reticulum membranes (mam) where upon hcv-induced rig-i activation it is recruited and colocalizes with ikk[94] , thus impairing ifn-i expression [95] . importantly, the cleavage of mavs by the viral protease has been confirmed in patients [96] . to antagonize ifn-i production, ns4b protein instead targets sting. hcv ns4b, indeed, contains a sting homology domain and interacts with sting in the er blocking sting interaction with mavs and tbk1 [97, 98] . even if the exact molecular mechanism involved in ns4b inhibition of sting signaling has not yet been defined in the context of viral infection, ns4b likely cooperates with ns3/4a in targeting the rig-i signaling pathway. interestingly, ns2b/3 and ns4b of other members of the flaviviridae family including denv and yfv as mentioned above also block sting signaling and possess the same sting homology domain, indicating a conserved mechanism of sting antagonisms between flaviviruses and hepaciviruses. intracellularly, both tlr3 and tlr7 have been shown to sense hcv rna, depending on the infected cell type considered [88] . sensing by tlr3 may occur in liver cells, as hepatocytes, and liver resident macrophages kupffer cells, while tlr7 sensing occurs predominantly in plasmacytoid dcs (pdcs) and macrophages. as reported above for the mavs adaptor, the serine protease ns3/4a also cleaves the key adaptor of tlr3, trif [99] , thus preventing an ifn response in productively-infected hepatocytes. in these cells, the hcv-induced mir-21 has been recently reported to be involved in evasion of ifn-i production and stimulation of hcv replication, upon suppression of myd88 and irak1 expression, that is required for the tlr7-mediated sensing of the virus [100] . in macrophages, myd88 signaling is instead targeted by the ns5a protein that, upon the direct binding to the adaptor, impairs the recruitment of irak-1 and cytokine production in response to tlr ligands [101] . interestingly, tlr3 and tlr7 levels are decreased in patients chronically infected with hcv [102, 103] and this has been recently correlated with increased levels of mir-758 [104] . hcv-infected cells, however, can trigger ifn-i production in non-productively-infected pdcs in a cell-cell contact-and tlr7dependent manner depending on the intracellular hcv rna level of cocultured infected cells [105] . this production of ifn-i by pdcs may thus account for the strong ifn-i response observed in the liver of infected people [106] . interestingly, a robust expression of isgs correlated with a decreased response to ifn therapy [107] supporting a pathogenetic role of a high ifn signature in chronically infected individuals where the virus has established a persistent infection. in contrast, in monocytes and macrophages upon clathrinmediated endocytosis and recognition of the virus by tlr7, hcv activates the inflammasome and not ifn-i production in an infection-independent process [108] . an association between tlr-polymorphisms and cytokine production in response to tlr7 agonist in vitro has also been reported, supporting a pathogenic role of tlr7-mediated sensing in immune cells [109] . interestingly, this cell-type dependent stimulation of ifn-i and inflammasome in response to hcv infection is also observed in human immunodeficiency virus type 1 (hiv-1) infection that as hcv can establish a persistent infection (see discussion below). pkr has been shown to sense hcv rna very early in infection even prior to rig-i sensing [110] . pkr is a dsrna binding protein that upon activation phosphorylates the ␣ subunit of the eukaryotic translation initiation factor 2 (eif2 ␣) to inhibit translation of host capped mrna but not of non-capped mrna, as that of hcv. pkr, upon binding hcv rna and independently of its kinase-activity, interacts with mavs to induce the transcription of a number of early isgs including ifn stimulated gene 15 (isg15), but not ifn-i. isg15, in turn, deubiquitinates rig-i inhibiting its functions [110] . by doing so, hcv blocks sensing by pkr and reinforces hcv evasion from rig-1 signaling. amongst rna viruses that, as hcv, can establish a persistent infection, hiv-1, a lentivirus from the retroviridae family, represents a paradigm for its ability to prevent or circumvent the innate immune response mediated by ifn-i. in spite of evidence that a sustained ifn-i response occurs in hiv-infected patients, it fails to clear the infection in the first place and to prevent the early establishment of long-lived hiv-1 reservoirs [111] [112] [113] . hiv-1 has, indeed, developed a number of strategies to block the ifn signaling and the activity of ifn-induced host restriction factors. here, we only briefly summarize these strategies some of which have been only recently discovered, leading to the identification of immune pathways, thus far, unrecognized (as recently reviewed elsewhere [114] [115] [116] [117] [118] [119] [120] ). in the past few years, the knowledge on innate immunity against hiv-1 has evolved enormously with the recent identification and the characterization of the molecular basis of retroviral recognition by prrs [116, 118, 121] . retroviral replication generates several structural and intermediate molecules as ssrna, hybrids rna/dna, ss and ds dna produced upon reverse transcriptase, that are potentially available for recognition by cellular prrs as "non-self". with the exception of pdcs, which produce high levels of ifn-i upon detection of hiv-1 ssrna by tlr7, in all other immune cells ifn-i production is prevented or barely detectable unless viral countermeasures are disabled. in conventional dcs (cdcs), monocytes and resting cd4 + t cells, indeed, hiv-1 sensing is prevented by the restriction factor sterile alpha motif (sam) and the hystidine/aspartic acid (hd) domain-containing protein1 (samhd1) that blocks reverse transcription or directly degrades genomic rna, thus preventing prr recognition [122, 123] . this samhd1mediated restriction is overcome by the viral protein vpx [124, 125] that is a hiv-2 and siv accessory protein absent in hiv-1. this apparent disadvantage is, however, effectively exploited by the virus that maintains a reward by not replicating in myeloid cells and by reducing the impact of ifn-i production in these cells. the block of productive infection in non-cycling cells where samdh1 is active, particularly in cdcs, results in lack of maturation and thus in impairment in priming of naive, hiv-1-specific t cells for optimal anti-hiv-1 immunity [126] . furthermore, in the small fraction of cdcs that become infected, the recognition of hiv-1 genomic rna by tlr8 paradoxically licenses hiv-1 transcription [127] . in this case, productive dc infection allows an increased transmission to t cells while inhibiting ifn-i production. in monocytes instead, recognition of viral rna by tlr8, does not trigger ifn-i production but, as in the case of hcv, leads to the formation of the nlrp3 inflammasome with activation of caspase-1 and il-1␤ production, favoring the establishment of an inflammatory milieu that fuels hiv-1 replication [108, 126] . in contrast, in cells that are target of a productive infection, such as macrophages and t lymphocytes, ifn-i production is prevented by an escape mechanism mediated by the hiv-1 protease that drives rig-i to the lysosomes [128] . the ssdna derived from proviral dna upon rt can, instead, be sensed by the newly identified dna sensor interferon gammainterferon-inducible protein 16 (ifi16) [129] , however, hiv exploits the host cytosolic nuclease 3 repair exonuclease1 (trex1) to digest hiv-1 dna generated during infection that, thus, does not accumulate at levels sufficient to be detected by ifi16, unless trex1 activity is blocked [130] . finally, resting cd4 + t cells are not permissive to virus replication due to the expression of an active samhd1 that, as mentioned before, degrades genomic rna and prevents efficient rt and recognition of ssdna. however, this prevention of sensing may not be complete and partial recognition of rt intermediates by the ifi16 sensor not only leads to the initiation of ifn-i production, but also to the activation of the inflammasome, triggering cell death mechanisms including pyroptosis and apoptosis [131] . finally, the viral capsid exploits two cellular proteins, cyclophilin a and cpsf6, and binds just a right amount of both to allow opening of the capsid and rt process, while preventing sensing of the viral cdna before integration with the following production of ifn-i [132, 133] . overall, viruses as hcv and hiv-1 have evolved nifty strategies to dampen the host innate response in cells where a productive infection may take place, while they induce infection-independent mechanisms in non-permissive cells to facilitate the viral life cycle and promote a chronic inflammation. the genus filoviruses from the filoviridae family are among the most virulent known human pathogens and comprise one species of marburg virus and five species of ebola viruses, including zaire ebolavirus (ebov) that is the most lethal and responsible of the recent severe outbreak of hemorrhagic fevers causing up to 90% mortality in untreated humans [134] . several immunoevasion strategies that result in total impairment of the innate immune system are responsible for most of ebov virulence [135] . a major target of these strategies that are exerted by few viral proteins, is the ifn-i response that controls in vivo filoviruses infection [136] . at the level of viral sensing, the ebov vp35 inhibits rig-i signaling [137, 138] . vp35 binds with high affinity to dsrna and 5 -ppp dsrna in a sequence-independent manner. four crystallographic structures of ebov vp35 rbd/iid from zaire and reston viruses and of marv vp35 rbd/iid have elucidated how vp35 rbd/iid dimers bind to rna strands and how the dimers mimic the rlr shape, hiding the rna recognition site [139, 140] . interestingly, the residues involved in both protein-protein interactions at the rbd/iid dimer interface and involved in dsrna binding are highly conserved among all known ebov and marv species. moreover, all residues that are important for dsrna binding are also crucial for ifn-i inhibition. as the knowledge on the importance of ifn-i in controlling the immunity against bacteria increases, studies aimed at characterizing the evasion mechanisms that these pathogens employ to evade, inhibit, or otherwise manipulate the innate immune response are arising. the mechanisms induced by bacteria for ifn-i expression in different cell types are various and reflect the heterogeneity of host-pathogen interactions established along bacterial infections. while the extracellular bacteria activate ifn signaling mainly through the interaction with molecules present on the cell surface, the intracellular bacteria are recognized as they enter a cell by cell-surface or endosome/phagosome bound receptors or by cytoplasmic pathogen sensors once they escape from these compartments [141] . here, we report only few examples of bacterial evasion mostly related to signaling pathways converging in ifn-i stimulation. a more exhaustive discussion of bacterial evasion strategies from prr-signaling pathways and inflammasome is provided by the contribution of thomas kufer and igor brodsky in this issue. to elude prr recognition many bacterial pathogens, like viruses, have modified the molecular structure of their pamps. lps, an ubiquitous component of gram-negative bacterial cell wall, is a pamp that is recognized by the tlr4/md-2 complex present on the cell surface. some bacterial species have evolved an alternative form of lps resulting in a weak antagonist of tlr4/md-2 signaling. this strategy is utilized by yersinia pestis, the causative agent of pestis infection. the acylation status of lipid a from hexa-to tetra-acylated is reduced when the temperature increases from 21 • c (flea temperature) to 37 • c (human temperature). this alteration renders lipid a less recognizable lps by tlr4 and, following transmission from fleas to humans, contributes predominantly to the virulence of the bacterium [142] . another example is shigella that, after internalization and proliferation within epithelial cells, hypoacylates lipid a to become less visible to the immune system once leave the infected epithelial cells [143] . in addition to lps, other bacterial components that may induce ifn-i, include bacterial nucleic acids and peptidoglycans. these pamps can be recognized outside or inside the host cells, leading to the activation of distinct signaling pathways [141] . intracellularly bacterial rna and dna nucleic acids are recognized by the several intracellular receptors that also sense viral pamps, while bacterial peptidoglycans are detected by nod1 and nod2 system. most of these pathways then converge in the activation of the sting/tbk1/irf3 axis [6] . as viruses, bacteria also encode proteins with enzymatic activity that interfere with the activation of adaptor molecules involved in prr signaling. this is the case of the yersinia pestis virulence factor yopj that, besides being a potent inhibitor of the nf-b and mapk signaling pathways, also inhibits tlr-mediated ifn response. as a deubiquitinating protease, yopj prevents or removes the k63polymerized ubiquitin conjugates, which are required for traf3 and traf6 activation in the signal transduction pathway leading to irf3 activation [144] . similarly, the type iii effector ospi of shigella flexneri inactivates by deamination the e2 ubiquitin ligase ubc13, a factor important for traf6 auto-polyubiquitinylation and activation [145] . another interesting example is represented by the translocated intimin receptor (tir), which is one of the first type iii effector proteins discovered in a/e pathogens including the enteropathogenic e. coli (epec), enterohemorrhagic e. coli o157:h7 (ehec), and citrobacter rodentium. in addition to the role played in the attachment to the host membrane, this factor shares sequence similarities with conserved regions present in the cytoplasmic tails of inhibitory receptors of the host immune system, such as the immunoreceptor tyrosine-based inhibition motifs (itims). tir utilizes these itimlike motifs to mimic an endogenous innate immunoregulatory mechanism. in particular, tir recruits the host src-homologyregion-2-domain-containing phosphatase 1 to the adaptor traf6 and thus prevents polyubiquitinylation and activation of traf6 in the ifn-stimulated pathway [146] . adaptor molecules downstream sensors transmit signals to classical ib kinase complex, including nemo/ikk-␥, tank and to the atypical ikk-related kinases ikk-and tbk1 that trigger activation of nf-b and irfs, respectively [147, 148] . ifn-i production downstream of these signaling pathways depends essentially on the presence and activation of irfs and their contribution changes depending on the cell type considered [9] . the irf family is presently composed of nine mammalian members namely irf1 to 9, coded by distinct but related genes that exert a number of functions in the regulation of innate and adaptive immune responses. the irfs with intrinsic antiviral function include irf3 and irf7 that are essential for the prr-mediated ifn gene transcription, but also induce some ifn effectors in an ifn-independent manner. irf9, as mentioned before, is part of the heterocomplex isgf3 that drives the expression of most isgs including irf1 (fig. 2) . although irf1 is itself an isgs, which affects different aspects of the immune response even independently from ifn-i production [149, 150] , it also represents a positive regulator of ifn-i gene expression in response to specific stimuli in a cell type specific manner [151] . moreover, irf1 plays a crucial role in regulating mavs-dependent signaling from peroxisomes [152] . in this respect, irf1 regulates the transcriptional profile of antiviral genes unique to that induced by ifn-i and cooperatively promotes an effective antiviral program against a broad spectrum of viruses [152, 153] . irf5 is instead specifically involved in inflammatory cytokines induction [9] . given the unique functions exerted by irfs, viruses have evolved strategies aimed at the specific destruction of these transcription factors. with regard to the viruses covered here, most of them inhibit irf activation either indirectly by acting on sensors and elements of the signaling pathway that activate them, as described above, or directly by impairing/hijacking irf activity (fig. 3) . as described above, the sars-cov plp affects the activation of both irf3 and nf-b not directly, as initially suggested [66, 154] , but by targeting rig-i, mavs, traf3 [63] and, as more recently reported, sting [64, 65] . interestingly, this activity is independent from plp protease activity. recently, it has been reported that the plp of mers-cov also suppresses ifn-i transcription by interfering with irf3 phosphorylation and nuclear translocation [155] . as sars-cov, mers-cov plp is a viral deubiquitinating enzyme that acts on both k48-and k63-linked ubiquitination and isg15-linked isgylation, two posttranslational modifications that play important roles in regulating the rig-i and sting/irf3 and nf-b activation [156] . whether the deubiquitination and deisgylation activity of mers-cov plp are directly responsible for inactivation of irf3/nf-b or upstream signaling pathway, it remains unclear. the wnv ns1 protein inhibits the tlr3-induced activation of ifn-i and il-6 transcription through inhibition of nuclear translocation of irf3 and nf-b [157] . recent studies indicate that this effect seems to be dependent on ns1 domains that control viral replication [158] . interestingly, in the draining lymph nodes the protein released predominantly from macrophages and dcs can inhibit the innate immune signaling pathways in uninfected cells and impairs cytokine production in response to infection [159] , thus suggesting that ns1 could also influence the development of the adaptive immune response directed to wnv. the ns2b/3 serine protease of denv, instead, blocks the serine 386 phosphorylation and nuclear translocation of irf3 by directly interacting with ikk-and masking the kinase domain [160] . two tick-borne flaviviruses, lgtv and tbev, have been recently reported to inhibit irf1 independently of their ability to antagonize ifn signaling. in particular, a weak expression of irf1 protein and nuclear localization, without reduction in irf1 mrna expression, was observed in dcs, an early cellular target of infection [161] . several hcv proteins interfere with irf activity. the hcv ns3 protease impairs irf3 activation by blocking the interaction with tbk1 and irf3 [162] . in addition, the ns2 protein inhibits, in a dose-dependent manner, ikk--and especially tbk1-induced irf3 phosphorylation [163] . the basic amino acid region 1 (br1) in the n-terminal region of the core protein is also crucial in inhibiting irf3 dimerization as well as phosphorylation induced by ndv infection and poly (i:c) [164] . interestingly, this domain has been identified as the binding region for a dead box protein the ddx3, which has been recently found to enhance the tbk1/ikk--induced ifn-␤ promoter activity upon binding to the adaptor mavs [165] . the hcv core also decreased the expression levels of ddx3 suggesting that the irf3 inhibition may be mediated by the core effect on ddx3. moreover, through binding to ddx3, the hcv core protein also promotes hcv replication. thus, the core protein appears to switch ddx3 from an ifn-inducing mode to an hcv-replication mode [166] . irf1 expression is, instead, suppressed by the hcv core at the transcriptional level. this event blocks the expression of several antiviral and immunomodulatory genes of both innate and adaptive immunity and, in doing so, facilitates the establishment of hcv persistent infection [167] . in line with this hypothesis, accumulating evidence suggests that hcv also targets dcs to control the host antiviral response and trigger persistence. as wnv ns1, hcv core is, indeed, a secreted protein found in the peripheral blood of patients with chronic infection that may thus affect directly dc functions. in this context, it has been recently reported that the core protein suppresses ifn-i production in response to tlr agonists and to rig-i stimulated by hcv pamp in a cell culture model of pdcs, through the reduced levels of irf7 and of phosphorylated stat1 protein [168] . the effect on irf7 is, however, not direct but probably mediated by the reduced levels of ifn-i production by core-stimulated pdcs. whether or not this also occurs along the natural infection and contributes to hcv persistency remains to be determined. to increase virus replication and establish viral persistence and latency, hiv-1, besides to dismantle or exploit almost all cell intrinsic innate recognition pathways, as discussed above, also directly hits irfs [113, 119, 169] . in t cells, the virion-associated accessory proteins, vif, vpr and vpu, directly target irf3 for ubiquitin-associated proteasome degradation [170] [171] [172] . recently, this vpu effect on irf3 degradation has been, however, challenged and it has been reported that vpu, instead, mediates a partial cleavage of irf3 in a caspase-dependent manner. interestingly, this cleavage produces a c-terminal fragment that can act as a negative regulator of irf3-dependent gene activation [173] . thus, hiv-1, as already reported for several viruses, can also exploit the apoptotic machinery to interfere with irf3 function [174] . in myeloid cells, instead, hiv-1 does not inhibit but, rather, stimulates both irf1 and irf7 expression. irf1 activity is, however, exploited by the virus to induce a distinct subset of isgs that despite displays intrinsic and unique antiviral actions, does not restrict viral infection [175] . irf1 is also induced in hiv-1-productively infected t cells where it may regulate viral promoter activity even in the absence of the viral transactivator tat driving initial transcription of the viral genome [176, 177] . later on, however, when viral replication is mostly accomplished by the viral transactivator, irf1 is sequestered by tat to accelerate proteasomal-mediated irf1 degradation (remoli al and battistini a, unpublished) and to quench irf1 transcriptional activity on target genes [178] . by so doing, tat disarms the unique antiviral response against viral infections that irf1 could exert [153] . a block of ifn transcription in primary cd4 + t cells may also depend on cd3/cd28-mediated activation of ikk-. we, indeed, recently reported that ikk-activation results in a peculiar pattern of irf phosphorylation in t cells, including a splicing isoform of irf3, which may function as an inhibitor of ifn-␤ expression, and phosphorylation of irf1 that blocks its activity on ifn-i promoter [179] . the ebov vp35, in addition to prevent rlr recognition, inhibits ifn-i promoter activation mediated by tbk-1/ikk-overexpression but not by a constitutively active irf3, strongly suggesting a specific inhibition of the kinase activity of tbk-1/ikk-. indeed, vp35 interacts with the tbk-1/ikk-kinase domain and functions as a substitute substrate, thus inhibiting both the kinase activity and the binding of the physiological irf3/7 substrate [180, 181] . notably, mutations of vp35 residues, involved in this ifn antagonism, do not alter the function of vp35 in viral replication and transcription [182] . in dcs, vp35 targets irf7. by interacting with the small ubiquitinlike modifier sumo e2 enzyme ubc9 and the e3 ligase pias1, vp35 promotes irf7 sumoylation, a post-translational modification that prevents irf translocation into the nucleus and, in turn, ifn-i gene transcription. a similar effect of vp35 was also reported for irf3 [183] . this vp35 activity is independent of its ability to recognize dsrna and maps to the n-terminus, which is essential for interactions with both irf7 and pias1. interestingly, sumo modification of irf3/7 is a part of the negative feedback loop of normal ifn-i signaling [184] that is exploited by ebov to weaken host innate immunity. downstream prrs, bacteria mainly target the mapk pathway and the nemo-ikk-nf-b signaling axis, which primarily induces inflammatory cytokines (reviewed in [19] and thomas kuper and igor brodsky in this issue). as an example of bacteria that target the irf pathway. listeria monocytogenes suppresses ifn-i gene induction downstream of tlr-triggered myd88 signaling pathway, acting on irf3. indeed, a mapk phosphatase renders irf3 hypophosphorylated by enhancing the formation of a mapk phosphatase-irf3-tbk-1 ternary complex in response to infection [185] . in fig. 2 is illustrated the ifn-i signaling pathway downstream the ifn-i receptor (ifnar) (a heterodimer of ifnar1 and ifnar2) that is activated upon binding of virus-infected cell-secreted ifn-i, and some isgs, including positive and negative feed-back regulators. most of the viruses here covered and some bacteria also target these pathways (fig. 3) . upstream the jak/stat pathway, the sars-cov 3a promotes serine phosphorylation within the ifnar1 degradation motif and increases ifnar1 ubiquitination [186] . the plp from sars-cov has a complex mechanism of interference with the jak/stat pathway. through its de-ubiquitinase activity upregulates the expression of the ubiquitinating enzyme e2-25k, leading to degradation of the erk kinase that, in turn, interferes with stat1 phosphorylation [187] . the orf6-encoded protein, instead, antagonizes stat1 function by interacting and sequestering in the er components of the nuclear import complex, as karyopherin alpha 2 and karyopherin beta 1. by doing so, orf-6 competes for the binding of the nuclear import complex to stat1, thus inhibiting stat1 nuclear import [188] . however, the majority of these evidences have been obtained by overexpression or stably expression of individual viral components in cell culture, which represents an experimental setting that may not accurately reflect the innate immune signaling occurring during sars-cov infection in vivo. wnv interferes with ifnar complex by promoting phosphorylation-dependent ubiquitination and degradation of ifnar1. this effect is mediated by the hydrophobic ns4a and ns4b proteins that potently induce the unfolded protein response (upr). this pathway is physiologically induced by different stimuli, including the accumulation of misfolded proteins in the er [189, 190] that, as mentioned before, represents the site of flaviviruses replication. activation of the upr pathway inhibits ifn activation and induces a general er stress response, thus facilitating viral replication. the methyltransferase domain of ns5 from langat virus and jev, instead, binds directly to ifnar through its methyltransferase domain and inhibits the activation of kinases associated to the receptor [191, 192] . several wnv non structural proteins, as ns2a, ns2b, ns3, ns4a, ns4b and ns5, have been reported to prevent the phoshorylation of jak1, tyk2 and, as a consequence, the activation of stat1/2 (recently reviewed in [73] ). likewise, expression of denv nonstructural protein ns2a, ns4a, or ns4b proteins impairs the jak/stat signaling pathway by reducing the phosphorylation and nuclear translocation of stat1 [193] . phosphorylation of stat2 is also blocked by denv ns5 through inhibition of jak1 and tyk2 activity [194] . ns5 also binds to the coiled-coil region in the first half of the human stat2 protein and acts as a bridge between ubr-4, a member of the n-recognin family, and stat2 leading to stat2 ubiquitination and proteasomal-mediated degradation [195, 196] . interestingly, only proteolytically-processed ns5 can efficiently mediate stat2 degradation, though both unprocessed and processed ns5 proteins are able to bind stat2. the jev ns5 protein greatly reduces tyk2 and, partially, stat1 phosphorylation, probably through its phosphatase activity [197] . the tbev ns5, instead, blocks stat1 phosphorylation by promoting the association with the pdz membrane protein scribble [198] . by activating the ras/raf/mek pathway, hcv replication has been shown to increase the phosphorylation of a motif contained in the cytoplasmic tail of ifnar1, which is involved in controlling the receptor ubiquitin-dependent endocytosis and attenuation of stat1/2 phosphorylation [199] . several hcv proteins have also been implicated in the regulation of the ifn response pathway interfering directly with the jak/stat signaling. however, contrasting results have been reported that probably stem from the use of different cell lines or different hcv expression/replication systems [200] [201] [202] [203] [204] . the core protein has been reported to upregulate the expression of socs3, thereby inhibiting tyrosine phosphorylation of stat1 [201] , although the decreased stat1 phoshorylation has not been detected in other studies [200, 205] . the hcv core protein, expressed alone, has been reported to directly bind to stat1 and to prevent its phosphorylation and subsequent expression of downstream isgs [206] . the ns5a, similarly, binds and prevents stat1 phosphorylation specifically in hepatocyte-derived cell lines [207] . two strategies are used by ebov vp24 to limit the jak1/tyk2mediated activation of stat1/2 and their subsequent nuclear localization. vp24 binds within the tyrosine-phosphorylated-stat1 binding region located in the c terminus of members of the npi-1 subfamily of karyopherin alpha nuclear localization signal receptors preventing their binding and shuttling to the nucleus of tyrosine-phosphorylated-stat1 [208, 209] . the crystal structure of human kpnalpha5 c terminus in complex with vp24 has been recently resolved and a unique nonclassical nuclear localization signal binding site on kpna5 has been identified. this motif is necessary for binding and efficient nuclear import of tyrosinephosphorylated-stat1 [210] . ebov vp24 can also directly bind stat1; whether the binding occurs with the unphosphorylated or phosphorylated stat1 or both isoforms it is not yet clear [211] . however, also unphosphorylated stat1 enters the nucleus to activate and sustain the expression of a number of ifn-induced immune regulatory genes, which are distinct from those activated by the phosphorylated stat1. thus, ebov vp24 binding and inhibition of either forms of stat1 may be important in the suppression of an antiviral state. notably, in spite of the high similarity of ebov and marv genome organization and high sequence homology between many of the ebov and marv encoded proteins, marv vp24 unlike ebov vp24, does not inhibit jak/stat signaling [212] . this is consistent with the observation that regions important for karyopherin alpha binding are different between these two vp24s [210] . in contrast, tyrosine phosphorylation of jak1 and stat is inhibited by the marv matrix protein vp40 that also inhibits this ifn-ii signaling. interestingly, also jak1-dependent and il-6-induced tyrosine phosphorylation of stat1 and stat3 are targeted by marv vp40 suggesting that marv may globally inhibit jak1dependent cytokine signaling with mechanisms different from that employed by ebov [212] . among the escape mechanisms used by bacteria to evade immune responses, some have been recently reported to target ifn-i signaling molecules. having found that influenza viruses replicate to a higher efficiency in cells co-infected with staphylococcus aureus, warnking et al. [213] demonstrated that an impaired stat1/stat2 dimerization is responsible for a poor induction of isg transcription in spite of an abundant secretion of ifn-i driven by the flu virus infection. similarly, the inhibition of the response to ifn-i by mycobacterium tuberculosis was observed in human macrophages and correlated with mycobacterial pathogenicity [214] . in primary cells and thp-1 cells, indeed, mycobacterium tuberculosis specifically inhibits ifn-i signal transduction pathway by impairing the activation of stat1, while the avirulent mycobacterium bovis bcg fails to do so. alteration in isgf-3 complex formation was instead observed in human macrophages infected with nonpathogenic lactobacillus rhamnosus gg where only stat1 homodimers were found. in contrast, the pathogenic streptococcus pyogenes led to formation of not only stat1 homodimers but also of isgf-3 [215] . based on these finding the authors speculated that the efficient induction of ifn-i production and related transcription factor activation by streptococci would lead to fast and effective immune responses that, however, could play a role in the pathogenesis. although the first antiviral isgs were discovered decades ago, until recently the mechanisms of action was defined for only a limited number of isg-encoded proteins. the renewed interest in the innate immune response to retroviruses with the identification of how several host restriction factors may limit retroviral infection [118] [119] [120] , as well as large-scale functional screening of isgs, have identified genes that coordinately control the infection of a range of rna and dna viruses and have begun to dissect their mechanism of action [153, 216] . interestingly, some of the most potent antiviral effectors reinforce the system by further inducing ifn or isgs. thus, by directly disarming and/or making the use of individual ifn-induced effector proteins, the antiviral effect of host cells may still be attenuated even though ifn-i is induced, and viruses can ensure protection from both autocrine and paracrine effects of secreted ifn-i. here, we will primarily focus on isgs for which viral countermeasures have been identified in the context of viruses covered in the present review. recent reviews report more extensively on isg viral antagonists and specifically on ifn-induced hiv restriction factors that are not covered here [117] [118] [119] [120] [217] [218] [219] . pkr is one of the major effectors of the ifn-i-induced antiviral state. upon activation from binding dsrna molecules via a dsrna binding domain, pkr phosphorylates the translation initiation factor, eif2␣, thus blocking cellular and viral protein synthesis in infected cells. due to its ability to bind dsrna molecules pkr is also a nucleic acid sensor, as mentioned above [220] . viruses have evolved specific mechanisms to inhibit pkr activity or escape its action downstream. these include: the production of small and highly structured rna molecules that prevent the dsrnainduced dimerization and activation of pkr; expression of proteins that bind directly to and inhibit the activity of pkr or pkr activators; proteins that behave as pseudosubstrate and competitive inhibitors of pkr [221] [222] [223] . amongst viruses covered in this review, both antiviral and proviral roles of pkr have been reported for hcv. the hcv ns5a and e2 proteins directly interfere with the antiviral action of pkr. ns5a binds directly to pkr, while the glyco-protein e2 acts as competitive substrate with eif2 ␣ for pkr binding, resulting in inhibition of pkr kinase activity and in increased hcv replication. the full length ires of hcv rna, which is recognized by pkr, may mediate either activation or inhibition of pkr [224] . moreover, ns5a, by binding to various domains of the ires, can alter the activation of pkr [225] . another indirect inhibitory strategy mediated by the hcv ires has been recently reported. upon hcv rna sensing, pkr activates the mavs/traf3/irf3 pathway that, however, does not induce ifn but a set of isgs including isg15. as mentioned, isg15 deubiquitinates rig-i to negatively control the rig-i/mavs pathway and prevent uncontrolled detrimental ifn-i expression in physiological conditions. thus, hcv hijacks a protective cellular pathway to curtail host innate response [110] . through a specific ires-mediated inhibition of eif2␣-dependent translation, the hcv ires also regulates the translational activity of pkr [224] . eif2 ␣ is, indeed, essential to the translation of capped mrna, as those of isgs, while non-capped mrnas, as those of hcv, are translated independently from this factor. a general attenuation of isgs expression by hcv ires can be achieved also through a direct activation of pkr [226] . interestingly, this attenuation of isg expression is observed in acute but not persistent infection, where, instead, a sustained isg expression occurs [206, 227] . other proteins from flaviviruses have been shown to inhibit pkr including the ns2a protein of jev that physically interacts with pkr and blocks its activation in response to several stimuli [228] . ebov vp35 also interferes with the pathway regulated by pkr by blocking and also reversing pkr activation, thereby preventing translational arrest of viral mrnas. this pkr antagonism seems to be functionally different from dsrna binding and irf3 inhibition [229] [230] [231] . ebov vp 35 also associates with pact (pkr-associated activator). this complex abolishes pact interaction with the cterminal domain of rig-i, which is required for full activation of rig-i [232] . as for retroviruses, hiv-1 infection does not activate pkr due to both viral and cellular controls (reviewed in [233] ). in vitro, pkr is activated by low amounts of tar rna, whereas high concentrations inhibit the kinase function. the viral tat protein also counteracts pkr activation by several other mechanisms: it sequesters the activating dsrna; it can act as a substrate homologue of eif2␣ preventing the pkr mediated inhibition of protein synthesis; it prevents pkr auto-phosphorylation and exploits pkr activity to get phosphorylated and increase its binding to tar rna. moreover, hiv-1 replicates only in cells that have high levels of the tar rna binding protein (trbp), a strong inhibitor of pkr activation. interestingly, during hiv-1 infection of lymphocytes, when hiv-1 replicates at high levels, increased amounts of adar1, an ifn-induced rna editing enzyme that binds to pkr to inhibit its activation, have been observed. moreover, pact contributes to pkr dephosphorylation during hiv-1 replication probably due to its binding to adar1 [234] . thus, hiv-1 has evolved to replicate in cells with high levels of trbp, to induce the expression of adar1 and to change the function of pact for pkr inhibition [235] . isg15 is an ubiquitin-like modifier that is induced rapidly by ifn-i and possesses antiviral activity against a number of viruses. isg15 antiviral functions include inhibition of virus release, isgylation of both viral and host proteins and immunomodulatory cytokine-like properties in its unconjugated and secreted form, as recently reviewed [236, 237] . more than 160 host proteins that are isgylated have been identified including irf3, pkr and rig-i. isgylation preferentially targets newly translated proteins and, as a consequence of isgylation, degradation of the target protein is reduced by competition with ubiquitin conjugation [238, 239] . to date, only few viral proteins have been shown to be isgylated and functional characterizations of isg15 conjugation has not been always verified under conditions of endogenous protein expression [237] . moreover, often, isg15-mediated protection might not be a result of direct antagonism of virus replication. as an example, isg15 has been shown to inhibit the release of hiv-1 and ebov. this effect is mediated by an ubiquitin antagonism. isg15 disrupts the ubiquitin-mediated regulation of ebov vp40, necessary to produce budding and release of vp40 vlps [240] , as well as the ubiquitination of the hiv-1 gag protein, which is required for the interaction with the cellular protein tsg101 to mediate hiv-1 budding and release [241] . several viruses have, thus, developed countermeasures against isg15 and/or its conjugation. strategies, identified so far, include viral proteins that bind isg15 or that remove isg15 from target proteins (reviewed in [237] ). sars-cov plp has both deubiquitinating and deisgylating activities [242, 243] . recently, the structural basis of recognition and processing of deubiquitin and isg15 by plp has been reported [244] . interestingly, despite mers-cov encodes a single plp similar to sars-cov, there is little to no sequence conservation among residues important for the deubiquitinating and deisgylating activity, suggesting that mers-cov plp is likely to recognize and process ubiquitin and isg15 substrates differently than sars-cov plp [155, 244] . however, by affecting this post-translational modification, both viruses may modify cellular protein localization, protein activity and stability as well as signal transduction in order to increase viral replication and severity of infection. nevertheless, although these results stem from in vitro overexpression or mutant studies, the direct evidence for isg15 antagonism by these proteins remains to be demonstrated during viral infection. despite the well-characterized role in restricting replication of several viruses, isg15 may actually also promote the replication of specific viruses, as hcv. in hcv infections both anti-viral or proviral effects exerted by isg15 could be related to the net effect of isgylation on the various viral and host proteins targeted by isg15. an antiviral effect has been reported when isg15 could conjugate to hcv ns5a, thereby enhancing the inhibitory effect of ifn-i on hcv replication [245] . in contrast, several groups have reported that isg15 and isgylation promote hcv production in a cell culture model independently of upstream ifn signaling [246, 247] . although counter-intuitive, this finding may be explained by the observed inhibition of ifn-i induction by hcv upon isg15 overexpression that negatively controls the rig-i/mavs pathway at the level of rig-i ubiquitination [110] . a negative regulation of ifn-i expression may also be mediated by usp18, an isg displaying a potent inhibitory effect on the ifn-i pathway, to prevent autoinflammatory consequences of uncontrolled ifn-i production. similarly, usp18 may dampen the detrimental role that the hyperactivation of ifn-i signaling plays in the pathogenesis of some viral and bacterial infections, including hiv-1, hcv and mycobacterium tuberculosis. usp18 is, indeed, stabilized by isg15 in an unconjugated free form [248] . in this respect, the suggested potential role of the isg15/usp18 pathway in hcv persistence is consistent with the observation that increased expression of hepatic isgs before ifn treatment is associated with an absent or poor response in patients chronically infected with hcv [249] . thus, isg15/usp18 pathway might explain the paradox that the preactivation of the endogenous ifn system, while fails to clear the infection, instead, may stimulate hcv production and blunt the effect of exogenous ifn-i. usp18 is similarly induced during some bacterial infections, including salmonella and mycobacterium tuberculosis. the decreased survival of mice that carry a point mutation in usp18 results from higher salmonella load in the spleen and liver, an increased inflammatory response and increased ifn-i signaling. similarly, these usp18 mutant mice are more susceptible to mycobacterium tuberculosis infection and have increased bacterial load in the lung and spleen, elevated inflammatory cytokine production and more severe lung pathology [250] . in line with these findings, the results of dorhoi et al. [251] have shown that ifnar1 deficient mice were protected from death upon aerogenic infection with mycobacterium tuberculosis. moreover, a rather detrimental effect of ifn-i was also found in whole blood of patients with tuberculosis, where a neutrophil-driven, ifn-inducible transcriptional signature was associated with clinical severity [252] [253] [254] . these results thus reveal that some viruses and bacteria, to push replication and persistence, utilize an opposite, but as much as effectual strategy, consisting in enhancing/perpetuating an ifn-i response by targeting negative regulators of ifn-i expression. anti-microbial molecules, such as nitric oxide (no) radicals and reactive oxygen species (ros) mediate the antibacterial properties of ifn-i. the key producers of no is inducible nitric oxide synthase (inos), an enzyme that can be induced by both ifn-i and -ii, although the latter is the conventional inducer that plays a crucial role in fighting the infection of intracellular bacteria [255] . similarly, ifn-i and -ii induce the subunits of the phagocyte nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which generates ros for killing organisms [256] . thus, pathogens have evolved several ways of avoiding ros-and no-mediated killing. in spite of the fact that no data are available, so far, on the strategies exploited by bacteria to contrast the ifn-mediated transcriptional regulation of inos and nadph oxidase, a common theme for successful intracellular pathogens is the ability to avoid the colocalization with these harmful host enzymes. intracellular salmonella, which resides within a specialized membrane compartment called the salmonella-containing vacuole (scv) in macrophages, uses a t3ss called salmonella pathogenicity island 2 (spi2) to mediate protection from no and ros intermediates [257] . intracellular organisms have also developed mechanisms to detoxify and repair no-mediated damage [255] , as well as to avoid the induction of inos activity [258] . given the crucial role played by these antimicrobial enzymes in contrasting bacterial infection, it is likely that strategies pinpointed by pathogens to inhibit the ifn-driven expression of these molecules will be discovered in the nearest future. to effectively resist the continual microbial threat from the environment, vertebrates possess several defense mechanisms including innate and adaptive immunity. as an essential component of the innate immunity, the ifn system constitutes the first line of defense against a number of pathogens to clear an incoming infection and instructing an ensuing adaptive response. successful pathogens have, thus, evolved sophisticated strategies to subvert and/or exploit the host immune system where blocking the ifn response, in the first place, is required to replicate and survive. the elucidation of some of these strategies has led to the identification of several, thus far, poorly recognized features of the innate immune response. in parallel, with the enormous recent advances in the comprehension of the molecular mechanisms of innate immune responses to pathogens, specific processes by which pathogenic microorganisms subvert these innate immune pathways, including the ifn system, is becoming progressively appreciated and it is reasonable to assume that many more will be discovered in the near future. by learning from the anti-immune strategies of pathogens we can, thus, not only identify key pathogen regulators as useful target to exploit to the host advantage, but we can also unveil weaknesses of host defenses and intervene to more precisely tune the immune response. the number and diversity of pathogen strategies for counteracting at each step the ifn system is stupefying. although beyond the scope of this review to discuss all antagonisms in detail, the ones that we have here reported, represent common and recurrent strategies used by a number of pathogens. this is illustrated by the existence of both viral and bacterial examples of pamp modifications as well as of viral and bacterial proteins that share cellular-like domain or cellular-like enzymatic properties that can compete with the host counterparts to dampen their physiological activities. in this respect, common hubs in the signaling pathways downstream pathogen sensors that trigger ifn-i, as few common adaptors or cofactors and transcription factors, are attractive targets of pathogen antagonism. the recognition of the mechanisms involved in microbial countermeasures may have several translation implications. the definition of microbial ability to elude the detection, as the methylation of their rna caps, can suggest strategies to utilize methyltransferase mutants as successful vaccine candidates against a number of different viruses as already suggested for denv [259] . many of the pathogen proteins responsible for ifn-i antagonism are also determinants of virulence and pathogenesis and, as such, they are highly conserved and may, thus, constitute attractive targets for the development of promising therapeutics against various clinically relevant pathogens reducing the bias of resistance mutations. in turn, some of the cellular identified targets of pathogen proteins as well as protein interacting partners might turn out to be new drug targets for treating a range of different diseases that disarme common components of the ifn pathway. in this respect, the resolution of the crystallographic structures of viral antagonists in complex with their different viral and cellular ligands, is then crucial for the rational design of new drugs. the system biology approach and the ability to simultaneously investigate diverse pathways has led to appreciate the interconnection between these pathways also in terms of shared components and stimulation by different pathogens. thus, a single therapeutic strategy could modulate multiple pathways to the host benefit. nevertheless, each approach needs to be complemented with effective treatments that also overcome other concurrent strategies that often the same pathogen put in place. on the other side of the coin, recovery of a full innate response must be finely tuned. ifn-i is, indeed, not always protective but can instead play a pathogenic role as reported for some bacterial and viral infections, where an uncontrolled ifn production is a determinant of disease progression. even in these cases, however, insights in the mechanisms involved in turning an ifn protective response into a pathogenetic one, may be as well relevant for nonpathogen-induced diseases, as autoimmunity and inflammatory diseases. in this context, cell death and inflammasome activation have been described as crucial ifn-i-regulated events exploited by both pathogen and host to get their own advantage. despite inflammasome facilitates pathogen clearance and is beneficial to the host, in some instances, ifn-induced non-canonical nlrp3 inflammasome activation and pyroptosis appear to be detrimental due to excessive cell death, inflammation, and collateral tissue damage in vital organs [260] . so pathogen proteins themselves or modified versions of them could be used as therapeutics working in suppressing inappropriate immune activation. this would be a bright way of hijacking molecules evolved during pathogen adaptation and associated fitness, to shift the balance to the host advantage. similarly, some identified targets of viral proteins in prr signaling pathways might be turned out to be new targets for treating a range of diseases. to find the way to generally induce an ifn response that is protective against a number of different infectious diseases, may be particularly relevant during an outbreak of unknown etiology or during the arising of newly emerging and re-emerging strains. likewise, finding the key to unlock the detrimental outcome of excessive ifn-i production during an infectious disease can open the way to cure autoimmune and inflammatory diseases. the authors declare no competing financial interests. innate immune sensing and signaling of cytosolic nucleic acids signaling by myeloid c-type lectin receptors in immunity and homeostasis immune sensing of dna intracellular toll-like receptors advances in nod-like receptors (nlr) biology the interferon response to intracellular dna: why so many receptors? intracellular sensing of viral dna by the innate immune system pathogen recognition and innate immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors regulation of type i interferon responses interferon-stimulated genes: a complex web of host defenses mechanisms of type-i-and type-ii-interferon-mediated signalling charleston, type i and iii interferon production in response to rna viruses interferome: the database of interferon regulated genes the jak-stat pathway at twenty type i interferons in host defense ifn regulation and functions in myeloid dendritic cells confounding roles for type i interferons during bacterial and viral pathogenesis bacteria fighting back: how pathogens target and subvert the host innate immune system anti-immunology: evasion of the host immune system by bacterial and viral pathogens bacterial subversion of host innate immune pathways inverse interference: how viruses fight the interferon system viral tricks to grid-lock the type i interferon system viral evasion and subversion of patternrecognition receptor signalling cellular sensing of viral dna and viral evasion mechanisms microbial strategies for antagonizing toll-likereceptor signal transduction toll-like receptors and their crosstalk with other innate receptors in infection and immunity a virological view of innate immune recognition microbial sensing by toll-like receptors and intracellular nucleic acid sensors length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 rig-i-mediated antiviral responses to single-stranded rna bearing 5 -phosphates 5 -triphosphate rna is the ligand for rig-i rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids mitochondrial antiviral signaling protein (mavs) monitors commensal bacteria and induces an immune response that prevents experimental colitis identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation mita/sting: a central and multifaceted mediator in innate immune response dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection ifit1: a dual sensor and effector molecule that detects non-2 -o methylated viral rna and inhibits its translation nod proteins: regulators of inflammation in health and disease mechanisms of inflammasome activation: recent advances and novel insights advancements in the battle against severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group coronaviruses: important emerging human pathogens from sars to mers: 10 years of research on highly pathogenic human coronaviruses severe acute respiratory syndrome vs. the middle east respiratory syndrome human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential to sense or not to sense viral rna -essentials of coronavirus innate immune evasion sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon ribose 2 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 coronavirus non-structural protein 16: evasion, attenuation, and possible treatments 2 -o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2 -o-methyltransferase activity rna 3 -end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit ifn-beta induction sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars-cov nucleocapsid protein antagonizes ifnbeta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response middle east respiratory syndrome coronavirus 4a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling flaviviral rnas: weapons and targets in the war between virus and host immune evasion strategies of flaviviruses flavivirus rna cap methyltransferase: structure, function, and inhibition flaviviruses and flavivirus vaccines emerging viral diseases: confronting threats with new technologies innate immunity evasion by dengue virus west nile virus infection and immunity flavivirus methyltransferase: a novel antiviral target 2 -o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo ifit1 inhibits japanese encephalitis virus replication through binding to 5 capped 2 -o unmethylated rna the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites dengue virus inhibits the production of type i interferon in primary human dendritic cells inhibition of the type i interferon response in human dendritic cells by dengue virus infection requires a catalytically active ns2b3 complex denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity activation and evasion of antiviral innate immunity by hepatitis c virus the global burden of hepatitis c interferon-stimulated genes and their role in controlling hepatitis c virus nonstructural protein 3-4a: the swiss army knife of hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus dissociation of a mavs/ips-1/visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns3-4a proteolytic cleavage cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the tolllike receptor 3 adaptor protein trif hcv-induced mir-21 contributes to evasion of host immune system by targeting myd88 and irak1 hepatitis c virus nonstructural protein 5a modulates the toll-like receptor-myd88-dependent signaling pathway in macrophage cell lines expression of toll-like receptors in chronic hepatitis c virus infection distinct toll-like receptor 3 and 7 expression in peripheral blood mononuclear cells from patients with chronic hepatitis c infection hepatitis c virus infection decreases the expression of toll-like receptors 3 and 7 via upregulation of mir-758 plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c hiv and hcv activate the inflammasome in monocytes and macrophages via endosomal toll-like receptors without induction of type 1 interferon sex-specific association between x-linked toll-like receptor 7 with the outcomes of hepatitis c virus infection hepatitis c virus reveals a novel early control in acute immune response redefining the viral reservoirs that prevent hiv-1 eradication therapeutics for hiv-1 reactivation from latency hiv-1 latency: an update of molecular mechanisms and therapeutic strategies hiv-1, interferon and the interferon regulatory factor system: an interplay between induction, antiviral responses and viral evasion innate antiviral immune signaling, viral evasion and modulation by hiv-1 innate immune recognition of hiv-1 the interface between the innate interferon response and expression of host retroviral restriction factors unmasking immune sensing of retroviruses: interplay between innate sensors and host effectors type i ifn -a blunt spear in fighting hiv-1 infection interactions between hiv-1 and the cellautonomous innate immune system innate immune sensing of hiv-1 by dendritic cells the ribonuclease activity of samhd1 is required for hiv-1 restriction samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein dendritic cells in progression and pathology of hiv infection hiv-1 exploits innate signaling by tlr8 and dc-sign for productive infection of dendritic cells rig-i-mediated antiviral signaling is inhibited in hiv-1 infection by a proteasemediated sequestration of rig-i ifi16 senses dna forms of the lentiviral replication cycle and controls hiv-1 replication lieberman, the cytosolic exonuclease trex1 inhibits the innate immune response to human immunodeficiency virus type 1 cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv-1 evades innate immune recognition through specific cofactor recruitment transmission dynamics and control of ebola virus disease (evd): a review characterization of host immune responses in ebola virus infections the role of the type i interferon response in the resistance of mice to filovirus infection evasion of interferon responses by ebola and marburg viruses filoviral immune evasion mechanisms marburg virus vp35 can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp35-mediated immune evasion mechanisms the yin and yang of type i interferon activity in bacterial infection structural modifications of bacterial lipopolysaccharide that facilitate gram-negative bacteria evasion of host innate immunity intracellular shigella remodels its lps to dampen the innate immune recognition and evade inflammasome activation yopj targets traf proteins to inhibit tlr-mediated nf-kappab, mapk and irf3 signal transduction the shigella flexneri effector ospi deamidates ubc13 to dampen the inflammatory response inhibition of tlr signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs triggering the interferon antiviral response through an ikk-related pathway ikkepsilon and tbk1 are essential components of the irf3 signaling pathway regulation of immunity and oncogenesis by the irf transcription factor family interferon regulatory factors in immune cell development and host response to infection evidence for licensing of ifn-gamma-induced ifn regulatory factor 1 transcription factor by myd88 in toll-like receptor-dependent gene induction program peroxisomes are signaling platforms for antiviral innate immunity a diverse range of gene products are effectors of the type i interferon antiviral response regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease immunity by ubiquitylation: a reversible process of modification west nile virus nonstructural protein 1 inhibits tlr3 signal transduction abrogation of tlr3 inhibition by discrete amino acid changes in the c-terminal half of the west nile virus ns1 protein modulation of innate immune signaling by the secreted form of the west nile virus ns1 glycoprotein dengue virus subverts the interferon induction pathway via ns2b/3 protease-ikappab kinase epsilon interaction tick-borne flaviviruses antagonize both irf-1 and type i ifn signaling to inhibit dendritic cell function interaction between the hcv ns3 protein and the host tbk1 protein leads to inhibition of cellular antiviral responses hepatitis c virus ns2 protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk1 functions impairment of interferon regulatory factor-3 activation by hepatitis c virus core protein basic amino acid region 1 dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-beta-inducing potential hepatitis c virus core protein abrogates the ddx3 function that enhances ips-1-mediated ifn-beta induction repression of interferon regulatory factor 1 by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes hepatitis c virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (stat) 1 protein expression battistini, hiv-1 targeting of ifn regulatory factors human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells hiv-1 accessory proteins vpr and vif modulate antiviral response by targeting irf-3 for degradation vpu-deficient hiv strains stimulate innate immune signaling responses in target cells hiv-1 vpu induces caspase-mediated cleavage of irf3 apoptosis as an hiv strategy to escape immune attack hiv infection of dendritic cells subverts the ifn induction pathway via irf-1 and inhibits type 1 ifn production irf-1 is required for full nf-kappab transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer modulation of human immunodeficiency virus 1 replication by interferon regulatory factors intracellular hiv-1 tat protein represses constitutive lmp2 transcription increasing proteasome activity by interfering with the binding of irf-1 to stat1 ikappab kinase epsilon targets interferon regulatory factor 1 in activated t lymphocytes the ebola virus vp35 protein inhibits activation of interferon regulatory factor 3 ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk-1 basic residues within the ebolavirus vp35 protein are required for its viral polymerase cofactor function ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery virus infection triggers sumoylation of irf3 and irf7, leading to the negative regulation of type i interferon gene expression beneficial innate signaling interference for antibacterial responses by a toll-like receptor-mediated enhancement of the mkp-irf3 axis the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferoninduced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type i interferon receptor a conserved peptide in west nile virus ns4a protein contributes to proteolytic processing and is essential for replication inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist identification of residues critical for the interferon antagonist function of langat virus ns5 reveals a role for the rna-dependent rna polymerase domain inhibition of alpha/beta interferon signaling by the ns4b protein of flaviviruses dengue virus ns5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation ns5 of dengue virus mediates stat2 binding and degradation dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatase-mediated mechanism tick-borne encephalitis virus ns5 associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling activation of the ras/raf/mek pathway facilitates hepatitis c virus replication via attenuation of the interferon-jak-stat pathway expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling-3 hcv structural proteins interfere with interferon-alpha jak/stat signalling pathway expression of hcv structural proteins impairs ifn-mediated antiviral response identification of the nonstructural protein 4b of hepatitis c virus as a factor that inhibits the antiviral activity of interferonalpha hepatitis c virus inhibits interferon signaling through up-regulation of protein phosphatase 2a intracellular innate immune cascades and interferon defenses that control hepatitis c virus hcv ns5a inhibits interferon-alpha signaling through suppression of stat1 phosphorylation in hepatocyte-derived cell lines ebola virus vp24 proteins inhibit the interaction of npi-1 subfamily karyopherin alpha proteins with activated stat1 ebolavirus vp24 binding to karyopherins is required for inhibition of interferon signaling ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 the ebola virus interferon antagonist vp24 directly binds stat1 and has a novel, pyramidal fold marburg virus evades interferon responses by a mechanism distinct from ebola virus super-infection with staphylococcus aureus inhibits influenza virusinduced type i ifn signaling through impaired stat1-stat2 dimerization inhibition of response to alpha interferon by mycobacterium tuberculosis lactobacilli and streptococci activate nf-kappa b and stat signaling pathways in human macrophages interferon-stimulated genes: roles in viral pathogenesis host restriction factors in retroviral infection: promises in virus-host interaction intrinsic antiviral immunity evolutionary conflicts between viruses and restriction factors shape immunity protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response the dsrna protein kinase pkr: virus and cell control inhibition of pkr by rna and dna viruses activation of the antiviral kinase pkr and viral countermeasures hepatitis c virus controls interferon production through pkr activation regulation of pkr by hcv ires rna: importance of domain ii and ns5a hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation failure of innate and adaptive immune responses in controlling hepatitis c virus infection blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a ebola virus vp35 antagonizes pkr activity through its c-terminal interferon inhibitory domain the vp35 protein of ebola virus inhibits the antiviral effect mediated by double-stranded rna-dependent protein kinase pkr ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome multiple levels of pkr inhibition during hiv-1 replication the pkr activator, pact, becomes a pkr inhibitor during hiv-1 replication hiv-1 translation and its regulation by cellular factors pkr and pact the antiviral activities of isg15 interferon-induced isg15 pathway: an ongoing virus-host battle positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification the isg15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg15 isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns5a the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment human intracellular isg15 prevents interferon-alpha/beta overamplification and auto-inflammation activation of endogenous type i ifn signaling contributes to persistent hcv infection contribution of increased isg15, isgylation and deregulated type i ifn signaling in usp18 mutant mice during the course of bacterial infections type i ifn signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis an interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis common patterns and disease-related signatures in tuberculosis and sarcoidosis inducible nitric oxide synthase and control of intracellular bacterial pathogens nadph oxidases: an overview from structure to innate immunity-associated pathologies salmonella pathogenicity island 2-dependent evasion of the phagocyte nadph oxidase modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice rational design of a live attenuated dengue vaccine: 2 -o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques role of type i interferons in inflammasome activation, cell death, and disease during microbial infection we apologize to the many colleagues whose data and influence have been overlooked due to space or our knowledge limitations. a special thank to members of the e.m. coccia's and a. battistini's laboratory for helpful discussion and critical reading of the manuscript and eugenio morassi for preparing drawings. our work is supported in part by grant rf-2010235199 from italian ministry of health (to emc) and from istituto superiore di sanità (to ab). key: cord-269194-b1wlr3t7 authors: engstrom-melnyk, julia; rodriguez, pedro l.; peraud, olivier; hein, raymond c. title: chapter 5 clinical applications of quantitative real-time pcr in virology date: 2015-12-31 journal: methods in microbiology doi: 10.1016/bs.mim.2015.04.005 sha: doc_id: 269194 cord_uid: b1wlr3t7 abstract since the invention of the polymerase chain reaction (pcr) and discovery of taq polymerase, pcr has become a staple in both research and clinical molecular laboratories. as clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has pcr performance. through optimisation, the present-day uses of real-time pcr and quantitative real-time pcr are enumerable. the technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time pcr provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. all of these serve vital roles in the continuum of care to enhance patient management. beyond this, quantitative real-time pcr facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. processes, small sample volumes and can be utilised in a wide variety of applications, making it the method of choice in today's molecular laboratories. through the aid of fluorescent signalling probes to measure amplification of dna at each pcr cycle, at the point of exponential dna accumulation, real-time pcr is able to provide broader linear dynamic ranges and increased assay performance as determined by sensitivity, specificity, precision, and reproducibility. due to the consistency in signal intensity changes during the exponential growth phase of pcr, it is also easily adaptable for quantitative reporting. however, there are three properties that are uniquely associated with quantitative real-time pcr: quantification, standardisation, and lower limit resulting. the accumulation of fluorescence signal is measured at each pcr cycle of the reaction and the cycle at which this signal exceeds a predetermined background fluorescence threshold during the logarithmic phase of amplification is referred to as the cycle threshold (c t ). the c t value is inversely proportional to the viral copy number in the specimen, and through comparisons of this value to an external calibration curve or an internal quantitation standard, the initial nucleic acid target concentration can be calculated (heid, stevens, livak, & williams, 1996; livak & schmittgen, 2001) . however, accurate quantitation within each sample is hindered when relying solely on an external standard as amplification efficiencies for each individual sample may be variable and inconsistent. by utilising a standard internal reference template, with the rationale that any variable influencing amplification efficiency should example of amplification and detection of target nucleic acid by real-time pcr. affect both template and target similarly, inhibition and amplification effects are compensated for which allows for more accurate quantitation ( figure 2 ). this control can be further enhanced when incorporating an internal reference that utilises the same primer sequence as the target since any potential additional effects on pcr efficiency for each of the two targets is eliminated. thus, the competitive real-time pcr strategy is the most reliable approach for nucleic acid quantitation (diviacco et al., 1992; gilliland, perrin, & bunn, 1990; stieger, demolliere, ahlborn-laake, & mous, 1991; wang, doyle, & mark, 1989; zentilin & giacca, 2007) and is the basis for the majority of present-day virology assays. it is equally important to utilise appropriate quantitation standards, when available, to ensure accurate quantitative results, inter-laboratory correlation, and overall standardisation. standardisation of reported viral loads ensures not only interlaboratory consistency but also high clinical utility of viral load monitoring, sets the foundation for establishing clinical correlations and critical thresholds leading to better management of infections and treatments, and are critical for the development of clinical guidelines (miller et al., 2011) . with the wide availability of assay methods, viral targets, specimen type, and lack of standard reference material (hayden et al., 2012) , viral load variability across laboratories can range significantly, as high as 4.3 log copies/ml . specifically, results from proficiency testing/external quality assessment programmes as well as interlaboratory specimen exchange studies have demonstrated that there is significant variability in quantitative results for assays that lack appropriate standards quantitation of viral target using competitive quantitation standard (qs). the qs compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen. the competitive qs contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons. the competitive qs is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous pcr amplification, and detection. viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the qs signal (dashed line) for each specimen and control (a, b) . in the presence of inhibitors, both qs and viral target are equally suppressed and yield accurate viral load calculations (c). 1 introduction (hayden et al., 2008; pang et al., 2009; preiksaitis et al., 2009; wolff, heaney, neuwald, stelrecht, & press, 2009 ). findings such as these reinforce the fact that with this high degree of variability and discrepancy, clinicians are unable to compare test results between two different laboratories and, further, clinically relevant cut-offs set by one test would not apply to results of another . without standardisation, the quality of patient care is dramatically impacted, preventing meaningful inter-laboratory comparison of patient results and influencing disease prevention and management programmes (kraft, armstrong, & caliendo, 2012) . this is especially critical for transplant patients, who may be initially monitored at one institution and then transferred to another for longer-term follow-up receiving results that no longer correlate. therefore, whenever possible, viral load monitoring tests must report results in iu/ml and be fully traceable to the higherorder first who international standard. they must generate highly accurate and reliable results based on a robust calibration methodology and have excellent reproducibility across the dynamic range of the test with demonstrated co-linearity to the who standard. lastly, there exist two distinct end-points with quantitative real-time pcr, which should be of consideration for result interpretation and reporting: the lower limit of detection (llod/lod) and the lower limit of quantitation (lloq/loq). these two limits are assessed differently and are not equivalent in either definition or, in some cases, their assigned values. the lod (also referred to as analytical sensitivity) represents the lowest viral load level at which !95% of tested samples are detected (clsi ep17-a, 2004); theoretically, viral levels at or below the lod are not detected !5% of the time. it differentiates between 'detectable' and 'undetectable' results. the lloq, on the other hand, is the lowest viral level that is within the linear and analytically acceptable range of the assay (clsi ep17-a, 2004) . in other words, the lloq is the lowest point at which an accurate viral load can be assigned and determines which 'detectable' sample will have a reported viral load. a common misconception is that the lod of the assay is the minimum viral level for a 'detected' result but 'undetectable' and 'detectable' viral levels are never differentiated by a single theoretical viral threshold as viral levels less than the lod may still have a high probability of being detected. this probability spans a broad range in which the lower the viral titre, the more likely the 'undetectable' result. ultimately, the statistical probability will favour the 'undetectable' result ( figure 3 ). and because the lloq can be equal or greater than the lod on some viral load assays, it is not unusual for 'detectable but below the loq' (detectable/bloq) result reporting (cobb et al., 2011) . further, the 'detectable/bloq' results should not be inferred that the actual viral concentration of the sample is between the lod and loq. the clinical demand has driven and shaped the evolution of pcr and continues to do so as we gain a greater understanding of the infections we monitor and treat. through the study of the natural history and disease progression attributed to specific viral infections, the need for sensitive, accurate, precise, reproducible, and reliable quantitative measurements of viral levels has become a necessity. with the deeper understanding of the natural history of human immunodeficiency virus (hiv) infections, it is now well understood that progressive immunosuppression and the onset and development of clinical disease are strictly associated with increasing viral burden (furtado, kingsley, & wolinsky, 1995; ho, moudgil, & alam, 1989; mathez et al., 1990; nicholson et al., 1989; schnittman et al., 1990) . thus, quantitative real-time pcr is critical for monitoring patients infected with hiv (hufert et al., 1991; mellors et al., 1995) and those undergoing antiretroviral therapy (art) to ensure viral replication is sufficiently and effectively suppressed and to monitor potential for viral resistance to the medication (dhhs hiv, 2014) . this monitoring and maintained viral suppression is absolutely necessary not only to maintain progression-free survival of hiv-infected patients but also to reduce subsequent hiv transmission (cohen et al., 2011; diffenbach, 2012) . due to the significance of viral load monitoring and maintaining viral suppression, the demand for likelihoods of different test results given different viral concentration. when the viral concentration tends to 0, the proportion of 'target not detected' increases to 1 (dotted line), increasing the likelihood of 'not detected' results. as the concentration tends to lloq (dashed line), the likelihood of 'detected but <lloq' results peaks. when the concentration tends to infinity, the proportion of quantitative results tends to 1 (solid line), resulting in a continual increase in the likelihood of 'detected: quantitative' results. at any concentration, the sum of the three types of reported results is always 100% and throughout the concentration continuum, variations in result reporting exist. as the concentration of viral levels approaches the lloq, near equal likelihood of 'detected: quantitative' and 'detected but <lloq' results are possible, as are 'not detected' results. decreasing concentrations will further shift the likelihoods and increase the chance of 'detected but <lloq' or 'not detected' results. diagram assumes lloq ¼ llod. increasingly more sensitive assays for hiv has driven the innovation of diagnostic tests and continues to push the limits of the lod and loq ever lower (glaubitz et al., 2011; sizmann et al., 2010) . additionally, the amount of viral dna in samples from either hepatitis b virus (hbv) or cytomegalovirus (cmv) chronic carriers is indicative of active viral replication in liver cells and is correlated with liver disease progression (chen, lin, et al., 2006; chen, yang, et al., 2006; emery et al., 2000; humar et al., 1999; humar, kumar, boivin, & caliendo, 2002; iloeje et al., 2006; wursthorn, manns, & wedemeyer, 2008) , highlighting the need for quantitative viral levels in chronic disease monitoring. similarly, hepatitis c virus (hcv) levels have been linked to prognosis and treatment outcomes (trepo, 2000) , treatment response (pearlman & ehleben, 2014; zeuzem et al., 2009) , as well as assessing sustained virologic response (svr), or 'cure', following treatment (pearlman & traub, 2011) . more recently, as new antiviral therapies for hcv treatment rely on targeting specific biological steps of the viral replication cycle, reliable quantitative monitoring of the viral burden at specific time-points to ensure treatment compliance and resistance emergence during treatment is recommended (au & pockros, 2014 ; aasld/idsa/ias-usa, 2014). alongside the changing clinical needs, several instrument and manufacturing innovations have been introduced to meet these newer requirements. the first pcrbased diagnostic test and the first automated system were both introduced in the early 1990s, allowing for improved standardised technique, increased efficiency, and reduction in error and contamination. in 1996, to meet the clinical needs of monitoring patients infected with hiv, the first 'personalised healthcare' diagnostic test was fda approved, that monitored whether art was working and whether a patient was on optimal treatment. entire systems were introduced in the early 2000s that automated the up-front sample preparation step, leading to further reduction of hands-on time, increased reliability and reproducibility. with the introduction of real-time pcr techniques to further enhance assay performance, the first pcr tests that allowed for simultaneous amplification and detection were introduced in 2003. throughout the decade, improvements in assays and instrumentation began pushing the boundary for hiv-1 detection, achieving greater and greater sensitivity, a feat that has proven to be incredibly relevant in understanding risks of viral load rebound and virologic failure (doyle et al., 2012; estevez et al., 2013; pascual-pareja et al., 2010) . more recently, shortly after the release of two new higher-order standards for cmv (first who international standard and nist cmv standard), fully automated real-time quantitative pcr assays were fda approved to meet laboratory and clinical demands for standardisation. not only does the instrumentation reduce assay design variability and inconsistency across laboratories, but also the assays are standardised to the who and report in international units, providing accuracy across the entire dynamic range that is only achieved by calibration and traceability to these higher-order standards. but it is not until these fda-approved tests gain widespread use will inter-laboratory agreement for cmv viral load results truly improve (kraft et al., 2012) . the objective of test innovation is to evolve and adapt to clinical and laboratory needs. as patient outcome gaps are identified, and as we gain greater understanding and insight into the clinical progression of disease and disease management, technological achievements facilitate advancements in quality of diagnostics and patient outcomes. life-threatening illnesses convert to chronic/manageable disease with the aid of viral load monitoring. and pressure exists to develop more precise, accurate, sensitive assays, which, in turn, drives the development of more efficacious drugs. this synergy demonstrates the value of diagnostics: it is an integral part of the patient care continuum. applications of quantitative real-time pcr for virology are extensive. it is especially necessary when antibody seroconversion is delayed after an acute infection and early diagnosis are essential (dibiasi & tyler, 2004; thomson et al., 2009) , in immunocompromised patients that may not have an optimal antibody response (kadmon et al., 2013) , and for the diagnosis of congenital or perinatally acquired viral infections (park, streicher, & rothberg, 1987; young, nelson, & good, 1990) . additionally, it is critical in maintaining a high level of patient care at each stage of the disease and infection (figure 4 ). screening tests are designed with one key parameter in mind: exceptional sensitivity (herman, gill, eng, & fajardo, 2002) . they must ensure that disease is not missed in a population that is generally free from risk to allow for appropriate early intervention, thereby effectively reducing mortality and morbidity. although traditional applications of real-time pcr in the continuum of care and patient management. population-based screening programmes and recommendations do not often utilise nucleic acid testing (nat) for virology targets, relying more on immunoassays and antigen testing, nats and real-time pcr assays are still integral components. donor-eligibility determination ensures that a donor is eligible to donate cells or tissues to be used as human cells, tissues, and cellular and tissue-based products (hct/ps), which can include haematopoietic stem/progenitor cell, organ, semen, and other types of donations. in part, living and cadaveric hct/p donor eligibility is granted if screening shows that the donor is free from risk factors for, and clinical evidence of, infection due to relevant communicable disease agents and diseases such as hiv type 1 and 2, hbv, hcv, and west nile virus (wnv) (fda testing, 2014) . the fda testing recommendations stipulate that hct/ps tests for hiv and hcv may include fda-licensed nat blood donor screening and that, specifically, an fda-licensed nat should be used to assess infection with wnv. despite the fact that these nat tests provide qualitative as opposed to quantitative results, the molecular technology utilised is often real-time pcr due to its enhanced accurate and reliable resulting (fda assays, 2014) . additionally, pre-emptive virology screening post-transplant may reduce subsequent complications and provide a more cost-effective management strategy (evers, 2013) . pre-emptive therapy utilises routine viral screening to initiate therapy at the first indications of viraemia, prior to clinical manifestation of disease. this strategy reduces the overall morbidity and mortality post-transplant compared to strategies in which treatment is initiated at the onset of clinical disease (sch€ onberger et al., 2010). the pre-emptive treatment model utilising routine screening by quantitative realtime pcr of asymptomatic post-transplant patients has been shown to be especially effective for paediatric patients undergoing haematopoietic stem cell transplant, who are uniquely at a high risk of cmv and epstein-barr virus (ebv) infections (evers, 2013) . this strategy and prospective screening utilising a quantitative real-time pcr assay for bk polyoma virus (bkv) is recommended as part of routine posttransplant follow-up of all kidney transplants since early identification and management of bkv infection may prevent future incidence of polyoma virus-associated nephropathy (hirsch et al., 2005; kdigo, 2009) . nats and real-time pcr are also utilised as vital parts of certain screening algorithms. the center for disease control (cdc) currently recommends that hcv rna testing be utilised for anyone who may have been exposed to hcv within the preceding 6 months. in addition, nats would identify active hcv infection among persons who have tested anti-hcv positive or those with an indeterminate antibody test indicating need for referral for further medical evaluation and care (cdc hcv, 2013). viral diagnostic tests are used to determine presence or absence of current or previous infection. because many viral infections present with similar symptoms, accurate diagnosis is critical as each requires unique and vastly different interventions and/or management strategies. historically, diagnosis of viral infections has relied on viral growth in cell culture, immunoassays, antigen assays (elisa), haemagglutination testing, and electron microscopy (krishna & cunnion, 2012) . the introduction of molecular methods including nats and real-time pcr assays has vastly improved viral diagnosis with their superior sensitivity, specificity, and rapid result reporting (emmadi et al., 2011) . nat-based infectious disease testing, providing rapid results, aids in outbreak detection (ebola), genotype identification (hcv), and identification of possible drug resistance (hiv-1), which can lead to rapid clinical therapeutic decisions and early infection control to prevent spread of disease (espy et al., 2006) . viral culture was traditionally considered to be the 'gold standard' for viral diagnosis because of increased sensitivity compared to rapid antigen-testing methods. however, a significant limitation of viral culture was a long time to result, reaching 14 days for cmv (gleaves, smith, shuster, & pearson, 1985) . improvements in culture included the introduction of shell viral culture, which reduced the result turnaround-time (tat) for cmv to 24 h. although considered to be a vast improvement, certain viruses require immediate treatment intervention to prevent life-threatening infection and therefore, a 24-h tat is simply much too long. further, reliance on viral culture for diagnostics testing introduces other pronounced drawbacks, the most noteworthy being that not all routine viruses grow in culture and that virus viability, and thus its culture ability, could be impacted by sample collection, transport conditions, or prior patient treatment. with these limitations in mind, nat testing has tremendous advantages and has replaced culture for most viruses due to its greatly reduced tat (typically less than 8 h) while still retaining high sensitivity. the applications of nat testing, and more specifically, real-time quantitative pcr technology, in viral diagnostics and confirmatory testing continue to expand. the cdc-updated recommendations for hiv testing state that specimens that are reactive on the initial antigen/antibody combination immunoassay and non-reactive or indeterminate on the hiv-1/hiv-2 antibody differentiation immunoassay should be tested with an fda-approved hiv-1 nat test (branson, 2010; branson et al., 2014) . additionally, nats have demonstrated utility in high-risk populations, in which antibody testing alone might miss a considerable percentage of hiv infections that are otherwise detectable by nat virologic tests (priddy et al. 2007; stekler et al., 2009) . particularly, immunoassays for hiv diagnosis are limited by the marked delay between infection and seroconversion, a time when hiv viral levels are at their peak ( figure 5 ). for this reason, nats are the recommended method for diagnosis of hiv during the acute phase of infection (10-50 days post-infection). hiv viral rna is the first marker to manifest itself approximately 10 days post-infection at initiation of the acute phase of infection (lindback et al., 2000) . not until 4-10 days after initial detection of hiv rna do the hivp24 antigen levels rise to detectable levels using fourth-generation immunoassays. this marked delay and the need for rapid diagnosis by the identification of hiv rna is especially critical when an early diagnosis is medically warranted. during the acute phase of the hiv infection, patients typically present with flu-like symptoms, which can often lead to a misdiagnosis. misdiagnosing those at the highest risk of infection such as prison inmates, iv drug users, and men who have sex with men (msm), also increases the risk of further disease spreading (chu & selwyn, 2010) . therefore, the use of a real-time pcr testing method that is able to identify the presence of the hiv virus at this initial stage is critical. additionally, nats are critical for early diagnosis of congenital or perinatally acquired viral infections, especially infants born to hiv-infected mothers (tang & ou, 2012) . the maternal antibodies against hiv can persist in exposed infants for up to 18 months of age, which, in turn, prevents the use of antibody-based assays for early diagnosis of infection. there is a high level of morbidity and mortality during the first 2 years of life for infected infants; therefore, it is of high importance to determine infection status quickly in the exposed infant to implement the appropriate art therapy early (van rossum, fraaij, & de groot, 2002) . in addition to hiv, additional viruses have been demonstrated to exhibit perinatal transmission including respiratory virus, cmv, herpes simplex virus (hsv), varicella zoster virus (vzv), hbv, enterovirus, rotavirus, and human papilloma virus (prober et al., 1987) . for all these, nat testing may serve as a valuable tool for early intervention, especially in this critical neonate population that lacks fully developed immune systems. in addition to their applications for hiv diagnosis, nat testing is also utilised as a diagnostic for hcv. although diagnostic hcv rna real-time pcr tests are predominately used as confirmation of a positive hcv antibody result (cdc hcv, 2012), nat testing is recommended for the confirmation of a non-reactive hcv antibody test for patients suspected of having recent hcv exposure. hcv rna can be detected as early as 2 weeks post-exposure, whereas hcv antibodies are not detectable until 8-12 weeks post-infection (ghany, strader, thomas, & seeff, 2009 ). therefore, similar to strategies outlined for hiv, nat testing is the most suitable for detection and diagnosis during the acute phase of hcv infection. further, immunocompromised patients, those with weakened immune systems can include those that are hiv positive, on immunosuppressive drugs, or undergoing chemotherapy, may lack the ability to generate an appropriate immune response required for an anti-hcv test. in these special circumstances, the cdc recommends considering hcv rna testing for diagnosing viral infections. rapid diagnostic testing is also very important for viruses with high potential to create an epidemic or outbreak. throughout history, influenza has caused many such outbreaks, the most notable being the 1918 spanish flu thought to be responsible for an estimated 50 million deaths (taubenberger & morens, 2006) . in response to the possibility of future outbreaks, the world health organization (who) has established that pcr-based influenza testing is now the first-choice diagnostic test for both humans and animals (who influenza, 2011) . the conversion to molecular tests was based on the fact that these assays are more sensitive and specific for detecting influenza viruses compared to other non-molecular methods. nat methods also have a lower likelihood of false positive or false negative results, and therefore, result interpretation is less impacted by community-based influenza prevalence (cdc influenza, 2014) . the introduction of rapid molecular testing, which can provide results in as little as 15 min, is extremely beneficial especially in hospitals, nursing homes, and chronic care facilities where early influenza identification can prevent outbreaks. the number of molecular-based diagnostic tests has expanded even further with the introduction of tests for wnv, respiratory syncytial virus, hsv, rotavirus, and even more recently, ebola. as development and improvements continue to be made to real-time pcr technology that will reduce both cost and time to result even further, the number of nat diagnostic tests will continue to increase and the applications for real-time pcr in diagnostics will also expand. once a patient is appropriately diagnosed and linked to care, partly through the aid of quantitative real-time pcr, clinicians will often consider several baseline factors, which collectively help to guide therapeutic decision. these decisions are based on a series of questions including: are treatment options available? what treatment regimen should be prescribed? for how long will the patient need to be on therapy? factors influencing these therapeutic decisions include disease complications, patient predisposition, prior treatment experience, viral genotype, viral resistance profile, and host genetic profile, among others. and depending on the viral infection, a quantitative baseline viral load may also serve an important role in helping to guide treatment decision (table 1) . over the past 25 years, pharmaceutical development and clinical trials investigating cutting-edge antiviral treatments have relied heavily on the data generated from pcr-based-and eventually quantitative real-time pcr-based-technology to determine safe and effective drug use (cobb et al., 2011; mackay, arden, & nitsche, 2002) . practice guidelines continue to reference registrational and nonregistrational studies utilising quantitative real-time pcr to guide both clinicians and laboratories in the proper implementation of treatment and testing in order to deliver the most effective personalised care to patients (aasld/idsa/ias-usa, kotton et al., 2013) . because of this extensive co-utilisation of real-time pcr, it is widely accepted as the gold-standard technology for measuring a patient's quantitative viral load before, during, and after the course of treatment. once the decision to initiate treatment for chronic hcv infection is made, several baseline factors are routinely considered (aasld/idsa/ias-usa, 2014). among these are complications like liver fibrosis stage, co-infection with hiv, hepatocellular carcinoma, end-stage liver disease, genetic variations like hcv genotype, subtype and resistance markers, and prior treatment experience and outcome. the association of baseline viral load, measured by quantitative real-time pcr, to chronic hcv treatment outcome has been well documented with earlier therapies (pegylated-interferon plus ribavirin) but, given the lack of alternative therapeutic options, has not been recommended as a therapeutic decision factor (jensen et al., 2006; pawlotsky, 2012) . historically, practice guidelines have recommended the measurement of baseline viral load to serve only as an initial time-point required for effective monitoring of treatment without any prognostic indication (yee, currie, darling, & wright, 2006) . currently, tremendous advancements in the treatment of chronic hcv infection that employ direct-acting antivirals (daas) reported cure rates (as determined by svr) of >90% for even the once most difficult to treat hcv genotype-1 patients, the most predominant in the united states. because of this high potency of these drugs across patient populations and the greater importance of numerous other factors, including hcv genotype and prior treatment experience, in determining the appropriate course of treatment, the most recent aasld/idsa practice guidelines still do not recommend a baseline quantitative viral load as a therapeutic decision factor. however, in the rapidly evolving field of hcv treatment, the recent fda approval of a fixed-dose combination drug consisting of two daas (sofosbuvir and ledipasvir) for the treatment of hcv genotype-1, the manufacturer's drug label now includes a new indication for quantitative real-time pcr. it is indicated that treatment naïve and non-cirrhotic patients with a specific baseline viral load are eligible for shortened therapy, an indication with tremendous implications. according to the prescribing information, patients with a baseline viral load below 6 million iu/ml are eligible to have shorter therapy duration of 8 weeks, much shorter than the 12-or 24-week duration for other patient populations (harvoni, 2014) . this therapeutic decision practice is the first of its kind in treatment of chronic hcv infection and is likely to be a recurring theme as daa manufacturers strive to develop high efficacy regimens requiring shorter treatment durations. additionally, shorter treatment durations are more favourable to patients and payers when considering the cost of achieving svr with daas and may improve patient drug adherence and completion of therapy (hep c online, 2014) . as much as quantitative real-time pcr helped to develop this claim for this particular regimen, this technology will also be employed by numerous laboratories to aid in this part of therapeutic decision. in contrast to chronic infection, treatment of patients presenting in the acute phase of hcv infection, within the first 6 months after exposure, is not recommended by aasld/idsa for patients in whom hcv infection spontaneously clears (aasld/ idsa/ias-usa, 2014). therefore, careful monitoring of hcv rna by a sensitive nucleic acid test is required in order to confirm spontaneous clearance, defined as hcv rna negative at two specific measurements. quantitative and qualitative real-time pcr assays are both widely used for this purpose, given their comparable sensitivity. factors influencing art decision for hiv-infected patients include determination of pregnancy, aids-defining conditions, acute opportunistic infections, low cd4 counts, hiv-associated nephropathy, potential drug interactions, co-infection with hcv or hbv, hiv resistance testing, and prior treatment experience (dhhs hiv, 2014). plasma hiv rna viral load, performed widely by quantitative real-time pcr, is also recommended as a pre-art decision factor specifically for treatment naïve patients. the department of health and human services (dhhs hiv) recommends that only art-naïve patients with a plasma hiv viral load below 100,000 cp/ml can be prescribed various regimen options, which they otherwise should be restricted from taking with higher viral load. this is primarily due to inferior virologic responses in patients with higher viral loads observed in clinical studies (sax et al., 2009) . these clinical trial studies employed quantitative real-time pcr in order to help determine this cut-off and many labs have utilised the same technology to help guide hiv-treating clinicians in this decision. in the case of chronic hbv infection, several studies have shown that hepatitis b 'e' antigen (hbeag) and high levels of hbv dna are independent risk factors for the subsequent development of cirrhosis and hepatocellular carcinoma (chen, lin, et al., 2006; chen, yang, et al., 2006; iloeje et al., 2006) . however, due to the fluctuating nature of chronic hbv infection, the prognostic utility of one high hbv dna level at a single time-point is limited. thus, hbv baseline dna viral load, along with hbeag, alanine aminotransferase (alt) levels, and fibrosis, collectively aids in the decision to treat with antiviral agents as well as which hbv antiviral regimen to choose and duration of treatment (lok & mcmahon, 2009 ). typically, patients with an hbv dna viral load >20,000 iu/ml, signs of liver disease (i.e. high alt levels and/or significant fibrosis), and loss of hbeag are considered for immediate treatment with antivirals, whereas patients <2000 iu/ml are closely monitored for viral load changes prior to treatment. patients who fall in between this range are monitored for persistent viraemia and signs of liver disease before deciding to treat. quantitative real-time pcr, therefore, plays a crucial role in the care of chronic hbv patients who, if not treated at the appropriate time with the appropriate regimen and duration, are at greater risk of liver complications. unlike treatment guidelines for hcv, hiv, and hbv, management of cmv after solid organ transplant is not associated with specific quantitative cmv viral load cutoffs in order to make therapeutic decisions (kotton et al., 2013) . this is partly due to the historical lack of an international standard and varying assay designs, which has led to poor inter-institutional correlation of quantitative nats. in addition, the widespread practice of universal prophylaxis, where cmv antiviral medication is administered to patients early in the post-transplant period and continued for a finite period of time, has diminished the clinical utility of baseline viral loads for making therapeutic decisions. however, with the recent availability of the who cmv international reference standard, the establishment of viral load cut-offs that can be applied to pre-emptive monitoring of patients prior to treatment initiation may soon become more widely accepted . until then, institutions are required to determine their own test performance characteristics and clinical cut-offs. several studies have shown that a low cmv virologic threshold (e.g. detectable viraemia) using quantitative real-time pcr should be used for starting pre-emptive therapy especially in high-risk cases where the organ donor screens positive and the receptor screens negative for cmv serology (atabani et al., 2012; couzi et al., 2012; sun, cacciarelli, wagener, & singh, 2010) . among a variety of baseline risk factors that may indicate longer cmv treatment duration, significant predictive value has been demonstrated with higher baseline viral loads where longer treatment duration may prevent cmv disease relapse (kotton et al., 2013; sia et al., 2000) . clinical trial studies supporting the recent fda approval of a quantitative real-time pcr cmv test calibrated to the who international standard also demonstrated clinical value for baseline testing of patients with cmv disease who are undergoing treatment with the anti-cmv drugs ganciclovir or valganciclovir (razonable et al., 2013) . data from this study suggested that patients with a baseline cmv viral load <18,200 iu/ml are likely to resolve cmv disease more rapidly than those who have a higher baseline viral load. further studies are needed to determine universal thresholds for pre-emptive therapy initiation and predictive value for cmv baseline viral load in defining optimal treatment duration. there exists a clear application for quantitative real-time pcr technology in baseline determination of patients with significant viral infections, and in fact, quantitative viral load determination plays a critical role in therapeutic decision for many other viral infections. high baseline viral load has been shown to correlate with advanced disease during infection with numerous viruses such as bkv, hsv-1, ebv, and adenovirus and may potentiate the need for longer duration therapies in certain scenarios (cincinnati children's hospital medical center, 2012; domingues, lakeman, mayo, & whitley, 1998; gustafson et al., 2008; randhawa et al., 2004) . after the patient's baseline assessment or pre-emptive monitoring suggests if treatment is available, which treatment regimen to choose and perhaps the duration of therapy, the patient can move on to therapeutic administration. quantitative realtime pcr has helped and continues to set the stage for decisions that potentially saves lives, reduces complications, decreases morbidity, and lessens the economic burden to both the patient and the healthcare system. serial measures of viral load serve as an individualised map of a viral infection through the estimation of the amount of virus found within an infected person. tracking viral load in the continuum of care is a vital tool used predominantly to monitor treatment response and its effectiveness, early signs of resistance emergence during therapy of chronic viral infections, and viral activation or reactivation in immunocompromised patients following bone marrow or solid organ transplantation. while the goal of treatment for chronic hcv infection is svr, patients may fail therapy due to non-response, on-treatment breakthrough, or post-treatment relapse ( figure 6 ). the early change in quantitative viral load over time may be predictive of treatment efficacy and a shorten therapy for patients who respond rapidly to treatment (yee et al., 2006) . this 'response-guided therapy' (rgt) is best exemplified during treatment of chronic hcv patients. specifically, the sooner a patient becomes hcv rna undetectable during treatment, the lower the relapse rate when treatment is shortened. conversely, the longer it takes for a patient to become hcv rna undetectable, the longer they need to remain on treatment to limit relapse. however, given the poorer efficacy of earlier regimens, not all patients who received therapy achieved svr. for this reason, 'futility rules' or 'stopping rules' were also developed, which required that failure of a patient to respond (target not detected or viral load cutoff ) by a given time-point indicated the need to immediately discontinue therapy. monitoring hcv viral loads during treatment. despite advances in treatment for hcv patients, failure to achieve svr is still a reality. patients who do not achieve svr fall into four categories: (1) null responders (black line) achieve less than 2-log decrease in hepatitis c viral load upon treatment; (2) partial responders (red line; light grey in the print version) experiences at least a 2-log decrease in hepatitis c viral load during hcv treatment but fail to proceed to an undetectable viral load level; (3) breakthrough patients (orange line; light grey in the print version) have an undetectable hcv viral load, but the virus rebounded during treatment; (4) relapsers (blue line; dark grey in the print version) have had an undetectable hcv viral load, but the virus rebounded after they completed hcv treatment. although these rgt notions were originally developed from observations made during treatment with the older therapies, peg-ifn and ribavirin, rgt was also required during treatment with the much more potent first-generation daas, telaprevir and boceprevir, and stopping rules were put in place during treatment with the second-generation daa, simeprevir (aasld/idsa/ias-usa, 2014; ghany et al., 2009; yee et al., 2006) . newer ifn-free daa regimens targeting hcv, which are better tolerated by patients and by virtue of the targets they inhibit, have a higher barrier to resistance, yield more rapidly declining viral kinetics, and, thus, do not contain treatment indications for rgt in their prescribing information (harvoni, 2014; olysio, 2014; sovaldi, 2013; viekira, 2014) . while rgt was a major driver for regular viral load monitoring during antiviral therapy, it is not the only reason to monitor hcv viral load. in the interval between baseline measurement and assessment of svr, the 2014 aasld/idsa guidelines also include recommendations for monitoring initial response (week 4 on treatment with a repeat at week 6 if detectable) and end of treatment in order to provide an assessment of drug compliance/early efficacy and predict treatment outcomes, respectively (aasld/idsa/ias-usa, 2014). in the most recent revision to these web-based guidelines, it is recommended that an hcv viral load increase of greater than 10-fold on repeat testing at week 6 (or thereafter) should prompt a discontinuation of hcv treatment. many clinicians also closely monitor and report the declining viral loads to their patients in order to demonstrate treatment efficacy, motivating patients to continue treatment and remain adherent to the drug regimen until the next follow-up appointment (fusfeld et al., 2013) . regardless of monitoring during hcv treatment for rgt, adherence/compliance, patient motivation, early treatment efficacy, etc., quantitative real-time pcr is widely used by laboratories due to its sensitivity, accuracy, and reproducibility of each consecutive viral load test. for patients infected with chronic viral infections, such as hiv, the lifelong regimen of highly active art aims to suppress hiv viral levels to near undetectable levels, ensuring progression-free survival (delay or all together prevention of the progression to aids) and reducing potential transmission. alongside monitoring immune function and immunologic efficacy through cd4 t-cell count, hiv viral levels are critical in the clinical evaluation and assessment of hiv-infected patients undergoing art. determining a patient's hiv viral load is indicated prior to entry into care, at the initiation of art, at 2-8 weeks after art initiation, and then typically every 3-4 months while on treatment: (1) to establish a baseline level of hiv viral load; (2) to establish viral response to the therapy to assess the virologic efficacy of art; and (3) to monitor for abnormalities that may be associated with antiretroviral drugs (dhhs hiv, 2014) . the baseline hiv viral load is not only linked to treatment options (sax et al., 2009) but also helps to establish the magnitude of viral load decline after initiation of art and provides prognostic information about the probability of progression to aids or death (marschner et al., 1998; murray, elashoff, iacono-connors, cvetkovich, & struble, 1999; thiebaut et al., 2000) . once treatment is initiated, the goal is to reach and maintain suppressed hiv replication as determined by undetected viral levels utilising highly sensitive nat tests, which is generally achieved within 8-24 weeks after art initiation. the need for sensitive assays to effectively assess viral suppression hinges on the need to suppress hiv replication to the extent that viral evolution and drug resistance mutations do not emerge, which typically do not occur in patients whose hiv rna levels are maintained below the llod of current real-time quantitative pcr assays (kieffer et al., 2004) . due to the introduction of more sensitive real-time pcr assays, which can detect as few as 20 viral copies/ml, natural variability in hiv viral levels over time, even in patients with effective suppression, is much more evident (lima, harrigan, & montaner, 2009; gatanaga et al., 2009; willig et al., 2010) . although controversy exists between the clinical significance of viral loads between llod and <200 copies/ ml, there are reports suggesting that this low-level viraemia is predictive of virologic rebound (doyle et al., 2012; eron et al., 2013; laprise, de pokomandy, baril, dufresne, & trottier, 2013) , virologic failure (estevez et al., 2013) , and indication of drug resistance (taiwo et al., 2010) , signifying the need for highly sensitive assays. viraemic blips, a single detectable hiv viral load (<500 copies/ml) in an otherwise seemingly suppressed patient (figure 7) , however, do not indicate subsequent virologic failure or development of resistance mutations (castro et al., 2013; lee, kieffer, siliciano, & nettles, 2006; nettles et al., 2005) . blips are not unusual (havlir et al., 2001) and appear to be more common in winter, suggesting that host-related and seasonal factors are associated with the occurrence of viraemia (van sighem et al., 2008) . on the other hand, persistent hiv rna levels !200 copies/ml are often evidence of viral evolution and accumulation of drug resistance on-treatment hiv patient monitoring. (a) hiv viral loads will fluctuate as patients are on treatment, and, in most instances, will remain 'undetectable' (at or below dotted line); viral 'blips' are not uncommon and will result in transient 'detectable' and even quantifiable results (above the dashed line). (b) virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time. mutations (aleman, soderbarg, visco-comandini, sitbon, & sonnerborg, 2002; karlsson et al., 2004) . once treatment failure is confirmed, immediate intervention is recommended to avoid progressive accumulation of resistance mutations and effective response of new regimen (dhhs hiv, 2014), which is benefited by low hiv rna levels and/or higher cd4 cell counts (eron et al., 2013) , and even a brief interruption in therapy may lead to a rapid increase in hiv rna and a decrease in cd4 cell count and increases the risk of clinical progression (deeks et al., 2001; lawrence et al., 2003) . with the development and administration of newer drugs that target specific biological processes of hiv, routine and clinical monitoring of viral loads using a real-time quantitative pcr assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success. viral load monitoring is also essential when the recipient of a solid organ transplant is cmv seropositive and the decision is made to initiate treatment only once the cmv levels predictive of disease are reached. this strategy, known as pre-emptive therapy, utilises intensive monitoring for cmv activity by sensitive real-time quantitative pcr methods and short-term antiviral treatment is given only to those with significant viral counts before symptoms occur. cmv is one of the most common opportunistic pathogens that infect solid organ transplant recipients (fishman, 2007) and is associated with increased morbidity and mortality (sagedal et al., 2004; schnitzler et al., 2003) . following primary infection, the virus establishes a lifelong latent infection in several sites of the body and may reactivate in the presence of immunosuppression, such as in transplant recipients. once reactivated, cmv is able to modulate the immune system and is known to be a potent upregulator of alloantigens (razonable, 2008) , increasing the risk of chronic allograft dysfunction (reischig, 2010; sagedal et al., 2002; smith et al., 2010) and acute rejection (sagedal et al., 2004) . pre-emptive therapy reduces the incidence of cmv disease (khoury et al., 2006; reischig et al., 2008) , which has been documented as a serious problem in randomised trials upon completion of universal antiviral prophylaxis therapy (kalil, levitsky, lyden, stoner, & freifeld, 2005; lowance et al., 1999; paya et al., 2004) . long-term studies have demonstrated that patients receiving preemptive therapy, when compared to prophylaxis therapy, were less likely to develop moderate-to-severe kidney scaring and atrophy and significantly better survival of the transplanted organ (reischig et al., 2012) . however, challenges still exist around defining appropriate thresholds to initiate pre-emptive therapy (humar & snydman, 2009) . but with new standardised real-time pcr assays, widespread adoption, and utilisation of these tests, pre-emptive therapy relying in intensive viral load monitoring may become the standard for certain at-risk patients. test of cure, or end of treatment response, is assessed following a given therapeutic regimen for signs of treatment efficacy. in few cases, a quantitative viral load measurement serves as a way to establish a cure rate, but, in others, may only be used as a confirmation of virologic suppression as clinical cure may not yet be possible with current therapies or technical limitations by real-time pcr that limits the overall sensitivity of viral detection. regardless of the clinical utility for measuring a virologic suppression, quantitative real-time pcrs with their current limits of detection and limits of quantitation are valuable tools in measuring low-level viraemia and establishing undetectable viral loads. utilisation of quantitative real-time pcr to assess virologic cure is perhaps best exemplified by treatment of patients with chronic hcv. according to the aasld/ idsa guidelines, patients who have 'undetectable' hcv rna in the serum, when assessed by a sensitive pcr assay, 12 or more weeks after completing treatment, are deemed to have achieved a sustained virologic response . achieving an svr is considered a virologic cure of hcv infection since, in these patients, hepatitis c-related liver injury stops and recurrence of infection is marginal, detected in <1% of patients after 5 years post-treatment (aasld/idsa/ias-usa, 2014; manns et al., 2013) . in agreement with these guidelines, the fda recommendation to pharmaceutical daa manufacturers also stipulates that viral rna clearance at svr-12 be measured in clinical trials using an fda-approved sensitive and specific quantitative hcv rna assay (fda hcv, 2013) . according to prescribing information accompanying the current daas, the threshold of svr-12 is defined as a quantitative threshold of hcv rna <25 iu/ml at 12 weeks after the end of treatment (feld et al., 2014; kowdley et al., 2014; lawitz et al., 2013) . this is somewhat dissimilar to the aasld/idsa guidelines as 'undetected' viral levels are not equivalent to 'detected but below the limit of quantitation' (figure 3 ). but, with the benefit of high sensitivity and reproducibility, quantitative real-time pcr has a clear established role in assessment of hcv virologic cure in both clinical trials and clinical practice and is able to meet the needs for assessing svr. quantitative real-time pcr may also play a critical role in the assessment of cmv disease resolution. the consensus guidelines recommend that two consecutive negative samples be obtained with a minimum treatment course of 2 weeks before treatment is discontinued, which is thought to minimise the risk for development of resistance and disease recurrence (asberg et al., 2009; chou, 2001; sia et al., 2000) . still, some transplant centres may extend treatment (secondary prophylaxis) in patients with compartmentalised disease for as long as necessary to reduce the likelihood of recurrent cmv infection (kotton et al., 2013) . resolving cmv disease has the long-term benefits of reducing mortality, potential allograft rejection, and the risk of bacterial, fungal, or viral opportunistic infections, among many other transplantand non-transplant-specific effects (arthurs et al., 2008; fishman, 2007) . although there is currently no cure for hiv infection, highly sensitive quantitative and qualitative real-time pcr tests targeting total hiv dna and rna have been used in clinical studies for both sterilisation (elimination of hiv-infected cells) and functional (controlled hiv in the absence of art) cures (kibirige, 2013; lewin & rouzioux, 2011) . improvements in real-time pcr technology may lead to profound increases in assay sensitivity and the ability to achieve single-copy detection (1 cp/ml) may lead us to a better understanding of hiv virology and what may be needed therapeutically to achieve a cure (alidjinou, bocket, & hober, 2014) . if therapeutic strategies are one day able to achieve an hiv cure, these highly sensitive tests will no doubt play a key role in the continuum of care for patients and, most importantly, in the confirmation of cure. clinical laboratories have undergone changes to become more efficient and flexible while delivering the same high-quality results. when choosing to implement new testing, even beyond viral targets, laboratories have to consider first and foremost the performance and medical value of the test and then factors such as tat, ease of use, and cost. real-time pcr with its wide dynamic range, high specificity, and high sensitivity is considered the gold standard for the quantification and identification of a variety of targets including bacteria, fungi, viruses, or oncological mutations (klein, 2002) . furthermore, the multiplexing capability of real-time pcr increases the number of targets and information gathered from the same test, further improving laboratory workflow, tat, and costs (deshpande & white, 2012) . while novel technologies have entered clinical laboratories including mass spectrometry and next-generation sequencing, real-time pcr remains a staple and an attractive option for clinical laboratories aiming to create molecular laboratory-developed tests (ldts). in addition, pcr can quickly be adapted to provide a robust test for the identification of emerging disease and molecular testing is now able to reach beyond the clinical laboratory and further enhance healthcare (farrar & wittwer, 2015; foudeh, didar, veres, & tabrizian, 2012) . most molecular tests used in clinical laboratories are fda-approved and commercially available. there are instances, however, when a test may not be available for a specific virus or the sample type and/or clinical indication used by the laboratory differs from those of the fda-approved assay, typically leading a laboratory to design its own pcr-based test or modify existing assays. fda defines an ldt as 'a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory' and recognises that 'ldts are important to the continued development of personalised medicine' (fda ldt, 2014) . laboratory developed tests can be grouped into three categories, fda-cleared or approved test that have been modified, tests that are not subject to fda clearance or approval, and tests for which no performance specifications have been provided by the manufacturer (e.g. analytespecific reagents or asrs) (burd, 2010; code of federal regulations, 2009 ). with alternative sample types or applications, fda-approved tests are often modified to fit the testing needs of laboratories, including alternative collection media and sample types or expanded clinical applications. as an example, a recent gap was created in the hcv-screening algorithm for the confirmation of a positive enzyme immunoassay result following the discontinuation of the only fdaapproved confirmatory test (alter, kuhnert, & finelli, 2003) . in response, the cdc published recommendation for the use of fda-approved tests detecting hcv viraemia (cdc hcv, 2013), despite the fact that most of these assays did not have specific claims for confirmatory testing; as a result, several laboratories chose to validate these assays as ldts to meet the screening needs for hcv. additionally, ldts are the only option for the identification of the aetiologic agents of viral infections that can occur in transplant patients, such as ebv, adenovirus, vzv, and bk virus, that often present with non-specific clinical manifestations (razonable, 2011) and for which fda-approved assay options are lacking. ldts are an integral part of molecular laboratory testing. whether created from the ground-up or modified from fda-approved assays, ldts are answering the clinicians' needs for information as an aid for diagnosis or treatment of patients. as with any clinical tests, ldts have to meet the minimum standards set forth by clia prior to report patient results (code of federal regulations, 2009). in july 2014, fda informed congress of the agency's ldt regulatory oversight framework (fda ldt, 2014) . fda aims to address concerns over high-risk ldts with inadequately supported claims, lack of appropriate controls, and falsification of data that may lead to inadequate treatment, possible harm to patients, and unnecessary healthcare cost. presently, there is still a high degree of uncertainty as to what the final regulation scope will be and the possible impact on molecular laboratories will have to be seen. palaeopathology confirmed the truism that humanity, since its inception, has been exposed to genetic and infectious diseases with early documentation of trachoma (8000 b.c.e.), tuberculosis (7000 b.c.e.), and pneumonia (ca. 1150 b.c.e.) (aufderheide & rodreguez-martin, 1998; hershkovitz et al., 2008; roberts & manchester, 1995; webb, 1990) . even today, emerging infectious diseases (eids) continue to appear unpredictably driven by changes in human demographic, land use, and population behaviour (lederberg, hamburg, & smolinski, 2003; sehgal, 2010; taylor, latham, & woolhouse, 2001) . these infections can be classified as either newly emerging/a previously unknown disease or re-emerging infectious diseases/a previously known disease, that reappears after a significant reduction in incidence or elimination (morens & fauci, 2013) . eids are a threat not only to human health but also to global stability and economy. efforts to monitor these eids are in place both at the global level spearheaded by the who and at the national level. in the united states, governmental agencies (department of health and human services, united states agency for international development, department of defense) are supporting activities to detect, assess, and respond to potential outbreaks. specifically, pcr and real-time pcr are easily adaptable to detect nucleic acid targets that are unique to each given pathogen, and as such, they play essential roles in the identification and detection of infectious pathogens and have been routinely used by health organisation agencies during epidemic outbreaks such as severe acute respiratory syndrome (sars), h5n1, h1n1, and ebola (shuaib et al., 2014; who influenza, 2011) . the sars epidemic appeared in november 2002, in the chinese province of guangdong before reaching the adjacent hong kong in 2003 (who sars, 2003 . this sars eventually spread to 26 countries and resulted in more than 8000 cases. in response, the cdc triggered its emergency operations centre and issued a draft genome in april 2003, 33 days after the initial who global alert (cdc sars, 2013) . soon after, real-time quantitative pcr assays were described and put in use for the diagnosis of sars (drosten et al., 2003; peiris et al., 2003) . a host of measures were taken in order to contain this epidemic, and the molecular identification and diagnosis of the infectious agent by pcr played a key role in providing critical information to address the situation and contributed to the care of the patients infected. additionally, the re-emerging 2014 ebola epidemic (cdc ebola, 2014; who ebola, 2014) started in guinea in march of 2014 before spreading to nearby west african countries and eventually reaching the united states and europe (who ebola, 2014) . at the height of the epidemic, fda issued an emergency use authorisation (eua) for the use of the first real-time rt-pcr assay (fda eua, 2014) and less than 4 months later, five additional realtime pcr tests were authorised under an eua (fda eua, 2014) to provide an early diagnosis of the ebola viral disease (cdc ebola, 2014). eids remain a constant and unpredictable threat to human health. the flexibility of real-time pcr technology continues to show how promptly it can be used for the detection of infectious agents. by providing a rapid diagnostic, real-time pcr can help in starting the appropriate treatment right away and maximise the chances of a positive outcome. the goal of point-of-care testing (poct) is to quickly obtain a test result that will be used to implement the appropriate treatment for an improved clinical outcome. by definition, poct is laboratory testing that takes place at or near the site of patients (cap poc, 2013) . the advantages of poct are an improved tat and result availability regardless of normal core laboratory hours, access to care in remote areas, and greater patient involvement. the fight against aids largely contributed to the development of poct devices with viral load capabilities (hong, studer, hang, anderson, & quake, 2004; lee et al., 2010; marcus, anderson, & quake, 2006; tanriverdi, chen, & chen, 2010; unitaid, 2014; vulto et al., 2009) . originally developed to meet the difficult conditions associated with remote places, far from any core laboratory facility often found in the developing world, the design and convenience of a portable poct device with fast turnaround and accurate results extends the reach of healthcare. with this in mind, these poct systems could easily be used in developed nations at hospitals, within clinics a physicians' offices, pharmacies, correction facilities, or mobile health units, to target pathogens that benefit from immediate actionable results, for which not only accurate but also quick results are critical (kiechle & holland, 2009 ). ultimately, test menu available on these platforms will drive its implementation as a complement for the clinical laboratory core testing. the ideal molecular poct system that includes medical value, simplicity, fast tat, and ruggedness remains an ongoing engineering challenge. however, the latest advances in microfluidics are a great example of the potential of these devices and brings the real-time pcr lab-on-a-chip closer to mainstream diagnostic use. this is an exciting time for molecular poct and the upcoming years should bring new systems and perhaps a paradigm change in the world of healthcare. as the needs of the clinicians, laboratory, and patients continue to evolve, so do the applications of molecular diagnostics and pcr. over the past decade, quantitative real-time pcr technology has been increasingly phased into clinical practice and all of the potential present-day applications of real-time pcr-based methods are enumerable. they serve to advance experimental approaches within biological fields, pushing the boundaries of what we know and what we can learn, as well as to diminish empiric medical identification and management of viral diseases. the high sensitivity of the technology has reduced risks of the most commonly transmitted transfusion illnesses and has become an integral part of managing a variety of viral infections by providing pretreatment prognostic information, therapeutic effectiveness through monitoring, and end of treatment response assessment. quantitative real-time pcr complements serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses and are able to predict therapeutic failures sooner than traditional methods, allowing for a more timely management response. real-time pcr assays can be rapidly developed in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. the next few years are likely to see an even further increase in the expansion of the clinical applications of nucleic acid quantification, particularly following bone marrow and solid organ transplantation for which the newest standardised assays may provide an avenue for the development of consensus management guidelines for initiating pre-emptive anti-cmv treatment. further, with the drive towards hiv eradication and complete elimination of the virus from within cells of infected patients, innovations in quantitative real-time pcr assay design will continue to push the boundaries of detection and introduce assays with progressively lower limits of detection. thus, quantitative real-time pcr has and will facilitate advancements in the quality of diagnostics and of what we can achieve in research, medicine, and patient outcomes. recommendations for testing, managing, and treating hepatitis c drug resistance at low viraemia in hiv-1-infected patients with antiretroviral combination therapy quantification of viral dna during hiv-1 infection: a review of relevant clinical uses and laboratory methods guidelines for laboratory testing and result reporting of antibody to hepatitis c virus delayed-onset primary cytomegalovirus disease and the risk of allograft failure and mortality after kidney transplantation long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy novel therapeutic approaches for hepatitis c the cambridge encyclopedia of human paleopathology the future of hiv testing centers for disease control and prevention and association of public health laboratories. laboratory testing for the diagnosis of hiv infection: updated recommendations validation of laboratory-developed molecular assays for infectious diseases a commutable cytomegalovirus calibrator is required to improve the agreement of viral load values between laboratories point of care testing influence of episodes of intermittent viremia ("blips") on immune responses and viral load rebound in successfully treated hiv-infected patients centers for disease control and prevention centers for disease control and prevention. recommendations for the identification of chronic hepatitis c virus infection among persons born during 1945-1965 testing for hcv infection: an update of guidance for clinicians and laboratorians guidance for clinicians on the use of rt-pcr and other molecular assays for diagnosis of influenza virus infection centers for disease control and prevention past hbv viral load as predictor of mortality and morbidity from hcc and chronic liver disease in a prospective study risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus dna level cytomegalovirus drug resistance and clinical implications diagnosis and initial management of acute hiv infection evidence based clinical practice guideline for management of ebv-associated post-transplant lymphoproliferative disease (ptld) in solid organ transplant protocols for determination of limits of detection and limits of quantitation: approved guideline. pennsylvania: clinical and laboratory standards evolution in the sensitivity of quantitative hiv-1 viral load tests health care financing administration, department of health and human services, part 493. laboratory requirements, section 493.1253. standard: establishment and verification of performance specifications prevention of hiv-1 infection with early antiretroviral therapy high incidence of anticytomegalovirus drug resistance among d+r-kidney transplant recipients receiving preemptive therapy virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in hiv-infected patients with detectable viremia multiplexed nucleic acid-based assay for molecular diagnostics of human disease panel on antiretroviral guidelines for adults and adolescents. guidelines for the use of antiretroviral agents in hiv-1-infected adults and adolescents. department of health and human services molecular methods for diagnosis of viral encephalitis preventing hiv transmission through antiretroviral treatmentmediated virologic suppression: aspects of an emerging scientific agenda a novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates application of competitive pcr to cerebrospinal fluid samples from patients with herpes simplex encephalitis plasma hiv-1 rna detection below 50 copies/ml and risk of virologic rebound in patients receiving highly active antiretroviral therapy identification of a novel coronavirus in patients with severe acute respiratory syndrome application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation molecular methods and platforms for infectious disease testing: a review of fda-approved and cleared assays efficacy and safety of raltegravir for treatment of hiv for 5 years in the benchmrk studies: final results of two randomised, placebo-controlled trials. the lancet infectious diseases real-time pcr in clinical microbiology: applications for routine laboratory testing quantification of viral loads lower than 50 copies per milliliter by use of the cobas ampliprep/cobas taqman hiv-1 test, version 2.0, can predict the likelihood of subsequent virological rebound to >50 copies per milliliter pre-emptive virology screening in the pediatric hematopoietic stem cell transplant population: a cost effective analysis extreme pcr: efficient and specific dna amplification in 15-60 seconds complete list of donor screening assays for infectious agents and hiv diagnostic assays emergency use authorizations guidance for industry chronic hepatitis c virus infection: developing direct-acting antiviral drugs for treatment, draft guidance fda laboratory developed tests fda testing hct/p donors for relevant communicable disease agents and diseases treatment of hcv with abt-450/r-ombitasvir and dasabuvir with ribavirin. the new england infection in solid-organ transplant recipients microfluidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics changes in the viral mrna expression pattern correlate with a rapid rate of cd4 + t-cell number decline in human immunodeficiency virus type 1-infected individuals assessment of motivating factors associated with the initiation and completion of treatment for chronic hepatitis c virus (hcv) infection detection of hiv type 1 load by the roche cobas taqman assay in patients with viral loads previously undetectable by the roche cobas amplicor monitor aasld practice guidelines, diagnosis, management, and treatment of hepatitis c: an update competitive pcr for quantitation of mrna accuracy to 2nd international hiv-1 rna who standard: assessment of three generations of quantitative hiv-1 rna nucleic acid amplification tests comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in culture specimens quantification of adenovirus dna in unrelated donor hematopoietic stem cell transplant recipients gilead sciences, foster city prevalence and predictive value of intermittent viremia with combination hiv therapy multicenter comparison of different real-time pcr assays for quantitative detection of epstein-barr virus factors contributing to variability of quantitative viral pcr results in proficiency testing samples: a multivariate analysis real time quantitative pcr medications to treat hcv screening for preclinical disease: test and disease characteristics detection and molecular characterization of 9,000-year-old mycobacterium tuberculosis from a neolithic settlement in the eastern mediterranean polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations quantitation of human immunodeficiency virus type 1 in the blood of infected persons detection of specific polymerase chain reaction product by utilizing the 5 0 -3 0 exonuclease activity of thermus aquaticus a nanoliter-scale nucleic acid processor with parallel architecture progression of hiv-1 infection. monitoring of hiv-1 dna in peripheral blood mononuclear cells by pcr clinical utility of quantitative cytomegalovirus viral load determination for predicting cytomegalovirus disease in liver transplant recipients cytomegalovirus (cmv) virus load kinetics to predict recurrent disease in solid-organ transplant patients with cmv disease ast infectious diseases community of practice. cytomegalovirus in solid organ transplant recipients predicting cirrhosis risk based on the level of circulating hepatitis b viral load early identification of hcv genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy polymerase-chain-reaction-based diagnosis of viral pulmonary infections in immunocompromised children meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients immunologic and virologic evolution during periods of intermittent and persistent low-level viremia kidney disease: improving global outcomes transplant work group. kdigo clinical practice guideline for the care of kidney transplant prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients the use of ultra-sensitive molecular assays in hiv cure-related research point-of-care testing and molecular diagnostics: miniaturization required genotypic analysis of hiv-1 drug resistance at the limit of detection: virus production without evolution in treated adults with undetectable hiv loads quantification using real-time pcr technology: applications and limitations updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation ledipasvir and sofosbuvir for 8 or 12 weeks for chronic hcv without cirrhosis interpreting quantitative cytomegalovirus dna testing: understanding the laboratory perspective role of molecular diagnostics in the management of infectious disease emergencies virologic failure following persistent low-level viremia in a cohort of hiv-positive patients: results from 12 years of observation sofosbuvir for previously untreated chronic hepatitis c infection structured treatment interruption in patients with multidrug-resistant human immunodeficiency virus microbial threats to health: emergence, detection, and response simple amplification-based assay: a nucleic acid-based point-of-care platform for hiv-1 testing hiv-1 viral load blips are of limited clinical significance hiv cure and eradication: how will we get from the laboratory to effective clinical trials? increased reporting of detectable plasma hiv-1 rna levels at the critical threshold of 50 copies per milliliter with the taqman assay in comparison to the amplicor assay viral dynamics in primary hiv-1 infection. karolinska institutet primary hiv infection study group analysis of relative gene expression data using real-time quantitative pcr and the 2 àddct method chronic hepatitis b: update international valacyclovir cytomegalovirus prophylaxis transplantation study group: valacyclovir for the prevention of cytomegalovirus disease after renal transplantation survey and summary: real-time pcr in virology long-term clearance of hepatitis c virus following interferon alpha-2b or peginterferon alpha-2b, alone or in combination with ribavirin parallel picoliter rt-pcr assays using microfluidics use of changes in plasma levels of human immunodeficiency virus type 1 rna to assess the clinical benefit of antiretroviral therapy productive human immunodeficiency virus infection levels correlate with aids-related manifestations in the patient quantitation of hiv-1 rna in plasma predicts outcome after seroconversion roadmap for harmonization of clinical laboratory measurement procedures emerging infectious diseases: threats to human health and global stability specific synthesis of dna in vitro via a polymerasecatalyzed chain reaction the use of plasma hiv rna as a study endpoint in efficacy trials of antiretroviral drugs intermittent hiv-1 viremia (blips) and drug resistance in patients receiving haart serial determinations of hiv-1 titers in hiv-infected homosexual men: association of rising titers with cd4 t cell depletion and progression to aids interlaboratory comparison of cytomegalovirus viral load assays transmission of human immunodeficiency virus from parents to only one dizygotic twin detection of hiv-1 at between 20 and 49 copies per milliliter by the cobas taqman hiv-1 v2.0 assay is associated with higher pretherapy viral load and less time on antiretroviral therapy chronic hepatitis c virus: advances in treatment, promise for the future valganciclovir solid organ transplant study group: efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients hepatitis c genotype 1 virus with low viral load and rapid virologic response to peginterferon/ribavirin obviates a protease inhibitor sustained virologic response to antiviral therapy for chronic hepatitis c virus infection: a cure and so much more coronavirus as a possible cause of severe acute respiratory syndrome interlaboratory comparison of epstein-barr virus viral load assays detection of acute hiv infections in an urban hiv counseling and testing population in the united states low risk of herpes simplex virus infections in neonates exposed to the virus at the time of vaginal delivery to mothers with recurrent genital herpes simplex virus infections correlates of quantitative measurement of bk polyomavirus (bkv) dna with clinical course of bkv infection in renal transplant patients cytomeglavirus infection after liver transplantation: current concepts and challenges management of viral infections in solid organ transplant recipients virologic suppression measured by a cytomegalovirus (cmv) dna test calibrated to the world health organization international standard is predictive of cmv disease resolution in transplant recipients cytomegalovirus-associated renal allograft rejection: new challenges for antiviral preventive strategies long-term outcomes of pre-emptive valganciclovir compared with valacyclovir prophylaxis for prevention of cytomegalovirus in renal transplantation valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation the archaeology of disease impact of early cytomegalovirus infection and disease on long-term recipient and kidney graft survival the impact of cytomegalovirus infection and disease on rejection episodes in renal allograft recipients primer-directed enzymatic amplification of dna with a thermostable dna polymerase enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia abacavir-lamivudine versus tenofoviremtricitabine for initial hiv-1 therapy increasing viral burden in cd4 + t cells from patients with human immunodeficiency virus infection reflects rapidly progressive immunosuppression and clinical disease the association of cytomegalovirus sero-pairing with outcomes and costs following cadaveric renal transplantation prior to the introduction of oral ganciclovir cmv prophylaxis prospective, comprehensive, and effective viral monitoring in children undergoing allogeneic hematopoietic stem cell transplantation deforestation and avian infectious diseases ebola virus disease outbreak-nigeria cytomegalovirus (cmv) dna load predicts relapsing cmv infection after solid organ transplantation improved hiv-1 rna quantitation by using a novel dual-target approach subclinical viremia increases risk for chronic allograft injury in pediatric renal transplantation hiv testing in a high-incidence population: is antibody testing alone good enough competitive polymerase chain reaction assay for quantitation of hiv-1 dna and rna preemptive therapy for cytomegalovirus based on real-time measurement of viral load in liver transplant recipients hiv drug resistance evolution during persistent near-target viral suppression past, present and future molecular diagnosis and characterization of human immunodeficiency virus infections. emerging microbes & infections, 1, e19 a rapid and automated sample-to-result hiv load test for near-patient application 1918 influenza: the mother of all pandemics risk factors for human disease emergence clinical progression of hiv-1 infection according to the viral response during the first year of antiretroviral treatment delayed anti-hcv antibody response in hiv-positive men acutely infected with hcv genotype and viral load as prognostic indicators in the treatment of hepatitis c hiv/aids diagnostics technology landscape efficacy of highly active antiretroviral therapy in hiv-1 infected children immunologic, virologic, and clinical consequences of episodes of transient viremia during suppressive combination antiretroviral therapy viekira pak™ (ombitasvir, paritaprevir, and ritonavir tablets a microchip for automated extraction of rna from gram-positive bacteria. transducers quantitation of mrna by the polymerase chain reaction prehistoric eye disease (trachoma?) in australian aborigines world health organization. global alert and response: ebola outbreak world health organization. the use of pcr in the surveillance and diagnosis of influenza world health organization. severe acute respiratory syndrome cost ramifications of increased reporting of detectable plasma hiv-1 rna levels by the roche cobas ampliprep/cobas taqman hiv-1 version 1.0 viral load test multi-site pcr-based cmv viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology natural history: the importance of viral load, liver damage and hcc management and treatment of hepatitis c viral infection: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program office discordant human immunodeficiency virus infection in dizygotic twins detected by polymerase chain reaction. the pediatric infectious competitive pcr for precise nucleic acid quantification expert opinion on the treatment of patients with chronic hepatitis c key: cord-286719-1xjmlwqr authors: draz, mohamed shehata; shafiee, hadi title: applications of gold nanoparticles in virus detection date: 2018-02-15 journal: theranostics doi: 10.7150/thno.23856 sha: doc_id: 286719 cord_uid: 1xjmlwqr viruses are the smallest known microbes, yet they cause the most significant losses in human health. most of the time, the best-known cure for viruses is the innate immunological defense system of the host; otherwise, the initial prevention of viral infection is the only alternative. therefore, diagnosis is the primary strategy toward the overarching goal of virus control and elimination. the introduction of a new class of nanoscale materials with multiple unique properties and functions has sparked a series of breakthrough applications. gold nanoparticles (aunps) are widely reported to guide an impressive resurgence in biomedical and diagnostic applications. here, we review the applications of aunps in virus testing and detection. the developed aunp-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-aunp hybrid structures, virus detection targets, and assay modalities and formats. we pay particular attention to highlighting the functional role and activity of each core au nanostructure and the resultant detection improvements in terms of sensitivity, detection range, and time. in addition, we provide a general summary of the contributions of aunps to the mainstream methods of virus detection, technical measures, and recommendations required in guidance toward commercial in-field applications. viruses are remarkable pathogens that are causing prominently increasing morbidity and mortality worldwide. their highly contagious nature and the absence of immediate and efficient control systems are the main reasons behind their potential health impacts. currently, viral infections and associated diseases are major causes of death in mankind, and under the present context of industrialization and immigration, they continue to emerge at a rapid pace, causing significant human, social, and financial costs [1] . additionally, gaps in currently applied detection systems potentially contribute to increasing the likelihood of international incidences and outbreak of viral infections [2] [3] [4] [5] . the implementation of highly sensitive and specific diagnostic tools has the potential to rapidly identify viral infections, initiate and guide judicious controls, and subsequently curtail their dissemination. toward this endeavor and beyond the pitfalls of current immunological and molecular techniques commonly applied to virus detection, several new approaches based on nanoparticles (nps) have recently been developed. aunps are widely described to be suitable for numerous biosensing functions and applications. their unique photonic, electric, and catalytic properties, coupled with the molecular interaction specificity of various biomolecules (e.g., antibodies, single-stranded (ss) dna, and rna aptamers, among others), represent the design principles of a wide range of virus detection systems [6] [7] [8] . the advantages of being simple, rapid, and sensitive and facilitating quantitative detection with excellent multiplexing capabilities have greatly promoted these systems to be envisioned as state-of-the-art technologies for virus ivyspring international publisher detection [9, 10] . however, there are no available reviews of the applications of bio-aunp hybrid structures for sensing and detecting viruses. in this article, we provide a current review of aunp-based virus detection at the level of virus type, including the types and structures of the applied aunps. we highlight their role in enhancing sensory and detection performance in comparison with current techniques in terms of analytical sensitivity, detection range, and time. furthermore, this review specifically summarizes detection designs, formats, functions, and contributions, which are of special importance to both scientific and applied research. viruses are particulate in nature and usually exist in different morphological forms that generally range in size from 20 to 900 nm [11] [12] [13] . their intact, mature infectious particles are typically composed of definite units of proteins and nucleic acid that self-assemble to form nanoparticulate structures called virions. the viral proteins are usually arranged in a surface layer called the capsid and sometimes in an outer envelope surrounding an inner core of nucleic acids. this core of viral nucleic acids can be single-or double-stranded (ds), dna or rna, one or several molecules, linear or circular in shape, and a few thousands of nucleotides to one million base pairs in size. the nanoscale size and the relatively simple structure of viruses tend to impose technical difficulties in establishing wide-use and long-term systems for virus detection. the small size of viruses increases the difficulty of their isolation and visualization compared with other microbes, such as bacteria and fungi that can be readily examined using ordinary light microscopes. only electron microscopy (em) with a high-magnification power of ~100,000× can allow the direct visualization of viruses and the study of their structures [14, 15] . therefore, em remains crucial for many purposes in virus research. however, em is certainly inappropriate for routine clinical diagnosis because of the required time and cost, as well as many safety concerns. furthermore, although the simple structure of virus particles allows the features of diagnostic relevance to be easily defined and tested, it presents limitations on the use of their characteristics for practical applications, especially with the increasing number of discovered viruses and recorded viral infections [14, 16, 17] . in addition, this structural simplicity confers viruses with rapid rates of spontaneous adaptation and evolution that may occur through direct genetic mutation, genetic substitution, or recombination. in this way, viruses not only outpace our attempts to develop sustainable control strategies but also raise more questions about the appropriateness and validity of current diagnostic techniques for long-term use [18, 19] . along with these limitations in virus detection and control, the possibility of viral infection emergence or reemergence has become increasingly prevalent, and severe pandemics and epidemics have occurred around the world. in the past, many outbreaks of viruses, such as human immunodeficiency virus (hiv), severe acute respiratory syndrome (sars), influenza virus (h5n1 and h1n1) and zika virus, started as local events and then expanded to have global consequences. hiv was discovered in central africa as a new virus causing acquired immune deficiency syndrome (aids) three decades ago, and it now represents one of the most significant public health threats worldwide [20, 21] . the highly contagious respiratory virus sars has fueled global fears of pandemics since its first appearance in china [22, 23] . in late 2012, the world health organization (who) raised a global alert for a new sars-like respiratory coronavirus, which is now called middle east respiratory syndrome (mers) coronavirus. mers is now classified as one of the most prominent viral threats to the middle east and has caused hundreds of deaths and thousands of infections in a short time. moreover, in the past few years, the world witnessed the worst ebola outbreak ever that started to hit west africa in early 2014 and the pandemic reemergence of zika virus in 2016 [11, 12, 24] . with these evident possibilities of massive outbreaks and implications, the situation is rapidly growing more serious, and the demand for the development of rapid diagnostics and effective control strategies is becoming more urgent. the first studies on virus isolation and detection were started early in the 1950s when the first cell culture system and electron microscope were developed [25, 26] . for decades, these techniques represented the main tools for studying and investigating the biochemical and morphological properties of viruses that remain the main foundation of all known classification and detection systems. however, their practical use in virus detection has remained greatly debated due to several considerations, including laboratory equipment expenses and time and safety concerns [27, 28] . in the early 1980s, the field of diagnostic virology was boosted with two other major developments: 1) the birth of various immunoassays; and 2) the invention of polymerase chain reaction (pcr). this was followed by the development of a very wide range of serological and molecular detection techniques, which rapidly evolved to constitute the mainstream approaches of both laboratory research and the clinical diagnosis of viruses ( fig. 1) [24, 29] . serology remains the standard method for virus detection. it primarily relies on testing for the presence of specific viral antigens or the corresponding antibody responses of the immune system. the most common types of serological tests include the neutralization assay, complement-fixation test, immunoprecipitation assay (ipa), hemagglutination-inhibition (hai) assay, enzyme immunoassay (eia), radioimmunoassay (ria), chemiluminescent immunoassay (cia), particle agglutination, immunostaining, immunofluorescence assay, single radial hemolysis, immunoblotting assay (iba), and immunochromatographic test (ict). the main working principle of these techniques is the use of specific antibodies in conjugation with different signal reporting systems, such as red blood cells, enzymes, and radioactive or fluorescent materials [30, 31] . most of these techniques are relatively easy to perform and flexible in their timing of specimen collection, and most of the needed reagents are usually commercially available. furthermore, they are generally unrestricted by the practical limitations of virus isolation and propagation usually involved with other direct detection methods, such as cell culture and em [9, 32] . therefore, serological methods are widely accepted and recognized techniques for simple, safe, and cheap virus detection [25, 30] . in addition, they are usually described as the first choice for large-scale testing in epidemiological studies and for evaluating antiviral therapies and vaccinations. however, the accuracy and reliability of serological methods are usually challenged by the cross-reactivity of the used antibodies, and the risk of false-positive results is usually very high. in addition, most immune responses can only be detected for a period of time after the initial virus infection; thus, serological detection does not normally benefit patients. molecular techniques are attracting more interest and have found an increasing number of applications in virus detection. the discovery of the genetic enzyme systems involved in the cellular machinery of nucleic acid replication and the stunning invention of an in vitro nucleic acid amplification system, commonly called pcr, by mullis in the early 1980s, opened new frontiers in nucleic acid-based detection [33] . in addition, the known high specificity of nucleic acid hybridization and the absolute availability of its synthesis and modification guided the development of many figure 1 . the onset of nanotechnology in virus detection applications compared with the development of the most common virus detection techniques. cell culture and electron microscopy techniques that are now commonly applied in the direct testing for and detection of viruses were discovered in the mid-20th century [25] . then, different serological and molecular techniques were developed. the serological detection of viruses with immunoassays was first reported in 1970: a radioimmunoassay was applied for the detection of the australia antigen, later called hepatitis b virus surface antigen [218] . pcr was discovered in the 1980s and first reported in virus detection in 1988 for acquired immune deficiency syndrome detection [219] . later, many molecular techniques, including amplification-and nonamplification-based techniques, were reported in virus detection. the concept of nanotechnology was envisioned as early as 1959 by the renowned physicist richard feynman [36] . however, nanotechnology was only applied to virus detection in 1997, when gold nanoparticles were employed for the detection of single-copy human papillomavirus [39] . nanotechnology has recently come to represent one of the most outstanding trends in virus detection and diagnosis via the wide variety of assays described in this review. detection and genotyping techniques known for the rapid and specific detection of viruses. these techniques can be classified as amplification-or nonamplification-based molecular detection techniques. amplification-based molecular techniques usually employ one or more forms of nucleic acid amplification to allow the indirect detection of the target virus. these techniques represent the majority of molecular techniques applied in virus detection and include various types of target amplification techniques (e.g., pcr, loop-mediated isothermal amplification (lamp), transcription-mediated amplification, and nucleic acid sequence-based amplification), signal amplification techniques (e.g., branched dna and hybrid capture), and probe amplification techniques (e.g., ligase chain reaction and strand-displacement amplification). nonamplification-based techniques are mainly applied to the direct testing for the presence of a specific virus in clinical samples (e.g., in situ hybridization, southern blot hybridization, and dot blot hybridization). generally, molecular methods are relatively rapid and more sensitive than immunoassays and can be applied either in a simple form for the manual detection of viruses or as an embedded component of more advanced systems. numerous fully automated and high-throughput detection systems are now available and widely used in clinical settings. in fact, the development of advanced molecular detection systems has revolutionized the way in which diagnostic tests are delivered and greatly enhanced the control of many viral infections, whether in hospitals or communities. in addition, these systems have eliminated the biosafety and time concerns usually associated with the clinical and academic study of viruses. however, despite these wide and promising applications, most molecular techniques still have several potential limitations in repeatability, accuracy, sensitivity, and specificity that are mainly caused by the high genetic variability of some viruses. furthermore, these assays are expensive and time consuming and typically call for specialized laboratory instruments and skilled personnel [34, 35] . the concept of nanotechnology was introduced in early 1959 [36] . subsequently, nanotechnology was realized via various types of new materials that rapidly emerged as promising tools for biological and chemical analyses. nanomaterials are known to possess multiple unique optical, electronic, magnetic, and mechanical properties enabling very attractive applications, especially in the fields of biomedical imaging and clinical diagnosis [37, 38] . the first reported application of nanomaterials in the detection of viruses was attempted in the late 1990s: aunps were coupled with silver staining and applied for the detection of human papillomavirus in cervical carcinoma cells (fig. l) [39] . currently, there is a very wide range of nanomaterials, including metal nps, carbon nanotubes, silica nps, quantum dots (qds), upconversion nps, and polymeric nps, that are being heavily investigated for virus detection [37, 38, 40] . one of the most common approaches for exploiting these nanostructures in virus detection is the development of nanobio hybrid systems that contain one or more biomolecules derived from viruses (e.g., dna, rna, antibody, pentabody, antigen, or peptide) conjugated to the surface of different np forms. these systems leverage the significant labeling properties and signal transduction functions of nps and the specific activity of the conjugated biomolecules to act as multivalent-np probes [37, 38, 41] . such virus-specific np probes have surprisingly been used to build up various optical, fluorometric, electrochemical, and electrical assays that have been extensively reported for single and multiple detection modes ( table 1 ). the results of most of these studies clearly demonstrate the inherent potential of these probes, along with numerous advantages over traditional approaches, in terms of size, performance, specificity, signal sensitivity, and stability. additionally, these studies have extensively described their application to allow simple, rapid, highly sensitive and label-free detection. aunps are a leading class of metal nanostructures that is widely known for its chemical stability, water solubility, and broad size and shape controllability. aunps can range from 1 to 800 nm in size and have different morphological shapes, including spheres, rods, prisms, tetrapods, dog bones, cubes, shells and several hollow structures [42, 43] . the synthesis of aunps can be performed using different methods such as chemical reduction of salts, ultraviolet irradiation, lithography, aerosol technologies, laser ablation, ultrasonic fields, photochemical reduction of au, and biological synthesis [9, 32] . aunps possess a high surface density of free electrons that results in inherent optical, electrical, and catalytic properties. the excitation of aunps with light can cause these free surface electrons, i.e., "plasmons," to oscillate to one side away from the atomic core, which remains as a positive charge on the other side, thereby creating a dipole or plasmon polariton [44, 45] . this dipole plasmon can change its direction in accordance with the frequency of incident light, and the resonance condition is reached when their frequency is approximately the same. this condition has been referred to as surface plasmon resonance (spr). the most widely used aunps exhibit intense spr bands that usually exist between 510-1100 nm and are known to be weakly dependent on the size of the aunps and the refractive index of the surrounding media but very sensitive to both the shape of the nps and the interparticle distance [46] . the spr of aunps has been observed to cause intense enhancing or quenching effects upon interactions with nearby photon emitters. these distance-dependent coupling effects are dipole-dipole interactions that usually include surface plasmon-mediated energy nanotransfer processes similar to fluorescence resonance energy transfer (fret) and may be either destructive (resulting in quenched emission) or constructive (resulting in enhanced emission). compared to other types of nanomaterials, metal nanoparticles, particularly aunps, constitute ideal tools in virus detection for numerous reasons, including the ease of synthesis, characterization, and surface modification, outstanding stability, biocompatibility, and exceptionally high absorption coefficients [47, 48] . furthermore, as labeling agents, aunps are easily visualized due to their intense colors and are known to form stable and highly active bioconjugates with common targeting biomolecules, such as dna and proteins, thereby enabling highly sensitive and specific sensing and detection applications [48] . aunps have been preferentially employed to perform numerous optical signal transduction functions in virus detection, such as resonance light scattering [49] [50] [51] [52] [53] , color amplification [54] [55] [56] [57] [58] [59] , and fluorescence quenching or enhancing [60] [61] [62] [63] [64] [65] (fig. 2) . resonance light-scattering-based detections usually involve measuring the amount of light scattered by different light spectroscopy techniques, including localized surface plasmon resonance (lspr) [52] , raman spectroscopy [49, 50] and dynamic light scattering (dls) [51, 53] . the colorimetric detection of viruses using aunps usually relies on two main techniques: 1) a color amplification technique in which aunps are applied to act as direct coloring labels with their characteristic, intense red color [55, 58, 59, 66] ; and 2) color change technique in which a color change from red to purple occurs in response to particle aggregation. aunp aggregation can be noncrosslinked aggregation caused by the target-triggered removal of stabilizing ligands from the surfaces of aunps [54, 56] or interparticle-crosslinked aggregation caused by the binding of ligands on modified aunps with target analytes [57, 67] . analogous to fret, aunp-based fluorescence quenching is a distance-dependent process in which aunps act to reduce the radiative rate of fluorophores in their proximity. thus, as efficient acceptors, aunps have been paired with dyeand qd-based fluorophores in different analyte-induced donor-acceptor crosslinking and coupling protocols [60-62, 64, 65] . aunps are widely described as electroactive and catalytic tags in various electrochemical assays applied to virus detection (fig. 2 ). based on their redox and electrical properties, aunps can directly act as electrochemical tags detected either by their acid dissolution followed by electrochemical stripping measurements of gold ions [68] or by their direct deposition on the surface of electro transducers, allowing an enhanced electrical conduction and resistance change [69] [70] [71] [72] [73] . aunps can indirectly perform electrochemical transduction functions based on their catalytic activity toward some chemical reduction reactions of metal ions (e.g., silver and copper) [72, 74, 75] or other species, such as h2o2 [76] [77] [78] . this potential to catalyze metal deposition is preferentially called np-metal enhancement amplification, and it represents the basic function of multiple scanometric [50, 70, [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] , photoelectric [89] , light-scattering [90] , and colorimetric schemes [91] for virus detection. additionally, aunp-based catalysis for the oxidation-reduction reaction of hydroquinone has been reported in another simple, colorimetricbased virus detection method [92] . other studies have exploited aunps to enhance the detection sensitivity of some bioanalytical techniques, such as quartz crystal microbalance (qcm) [93] , inductively coupled plasma mass spectrometry (icp-ms) [94] , and atomic force microscopy (afm) [95] in virus detection. through these schemes, aunps have been applied as effective nanoparticulate amplification tags to enhance mass, elemental, and topographic signal transduction, respectively (fig. 2) . interestingly, the enhanced surface area-to-volume ratio of aunps has allowed them to further act as exquisite scaffolds for biorecognition and detection reactions (fig. 2 ); in addition, it has granted these structures unprecedented capabilities for bioimmobilization, which is a basic principle of most aunp-based diagnostic designs. by coupling these bioimmobilization and signal transduction functions, aunps can achieve virus detection with highly improved analytical sensitivity and specificity. furthermore, the direct increase in loading efficiency allows new possibilities of controlled multifunctionalization with different biomolecules. based on this principle, developed aunp tags are characterized by enhanced reactivity and stability. increased loading efficiency also allows the design of new probes modified with multiple structures for biotargeting (e.g., antibodies and dna), together with other signal amplification structures (e.g., enzymes and dna oligonucleotides) [69, 76, 77, [96] [97] [98] , both types of which are stridently included in the development of new diagnostic schemes and designs. for example, the aunpfigure 2 . a generalized scheme for the basic characteristics and functions of aunps applied in virus detection. the size and metal nature of aunps are the key characteristics that enable them to directly transduce multiple types of signals, including optical, catalytic and electrical signals that can be detected by dynamic light scattering (dls), localized surface plasmon resonance (lspr), surface-enhanced raman scattering (sers), ultraviolet-visible (uv-vis) spectroscopy, fluorometry and other electrochemical analysis techniques. in addition, aunps act as nanoparticulate tags to enhance the signal detection of several analytical techniques, such as quartz crystal microbalance (qcm), atomic force microscopy (afm), and inductively coupled plasma mass spectrometry (icp-ms). their large surface area-to-volume ratio is known to permit them to function as an excellent scaffold for bioimmobilization and target-probe interactions with highly enhanced specificity and sensitivity. barcoding assay and its descendent technique of immuno-pcr universally rely on aunp tags modified with antibodies and dna oligonucleotides [81, 96, 97] , and aunp-based enzymatic electrochemical assays are based on using aunps dually modified with dna and antibodies together with an enzyme to induce a chemical reduction reaction allowing electrochemical signal transduction [76, 77] . several recent studies have attempted to harness the unique properties of aunps to develop various advanced schemes for virus detection. the developed assays are greatly variable in design and underlying principle. however, the utilization of aunps conjugated with specific virus-targeting biomolecules is a key component in most of these assays. various aunp bioconjugates have been widely employed in many colorimetric, scanometric, electrochemical, and fluorometric systems for the detection of many groups of well-known human viruses (table 1 and fig. 3 ). each of these groups will be discussed in more detail in the following sections. the bunyaviridae family comprises more than 300 members that are primarily organized into four main genera: hantavirus, orthobunyavirus, phlebovirus and nairovirus [99] . bunyaviruses are spherical, enveloped rna viruses 80-100 nm in size. their genome is composed of three segments of negative-sense, ssrna: a large segment (l, 6.3-12 kb) that encodes rna polymerase, a medium segment (m, 3.5-6 kb) that encodes viral glycoprotein, and a small segment (s, 1-2.2 kb) that encodes nucleocapsid protein [100] . these viruses are diverse in their host range and are frequently involved in a wide range of diseases in plants, animals, and humans. human pathogenic bunyaviruses, such as crimean-congo hemorrhagic fever virus, hantaan virus, la crosse virus, oropouche virus, rift valley fever virus, and toscana virus, continue to present increasingly important health concerns worldwide [101, 102] . the genus hantavirus was recently expanded to include more than 24 antigenically and genetically distinct htnvs [99] . among them, rodent-borne htnvs can cause serious diseases in humans, including hemorrhagic fever with renal syndrome, and hantavirus cardiopulmonary syndrome, and hospitalizes more than 150,000 persons each year with a mortality rate reaching 10%. recently, the health impacts of htnvs are expected to dramatically increase in the near future due to the increasing number of reports on newly discovered htnvs [102] [103] [104] . aunps were utilized to develop a novel immuno-pcr assay for the immunological detection of htnv nucleocapsid protein [96] . this assay mainly relies on the enhanced surface area of aunps to prepare dually functionalized antibody-oligonucleotide conjugates that exceptionally carry specific monoclonal antibodies to label the target htnv antigen; the assay also involves barcoding dna for signal amplification (fig. 4a ). this assay could optimally detect concentrations as low as 200 am of purified or spiked antigen samples, which is ~7 orders of magnitude more sensitive than conventional elisa. the high detection sensitivity together with the targeting of htnv nucleocapsid protein [105] , which is the most abundant viral component synthesized post virus infection, makes this assay a promising candidate method for the early diagnosis and control of htnv. rvfv is an arthropod-borne pathogen primarily known to affect animals and later discovered to infect humans. rvfv infection in humans can progress to serious hemorrhagic fevers that often lead to death, with prominent mortality rates of up to 10-12%. infected persons can also develop other clinical manifestations, including retinitis, encephalitis, and paralysis [106, 107] . rvfv is historically endemic to africa and has had a long history of outbreaks ranging from its reemergence in egypt in 1977 to the most recent outbreak in south africa in 2011 [108, 109] . thus far, rvfv outbreaks are unpredictable and usually associated with very high socio-economic and public health consequences [110, 111] . therefore, diagnostic methods that can rapidly identify the virus have become crucial not only for avoiding and preventing such outbreaks but also for monitoring cross-border dissemination. a novel immunoassay based on aunps coupled with a surface-enhanced raman scattering (sers) technique has been developed for the detection of rvfv capsid antigen [49] . in this assay, aunps are applied as both immobilization scaffolds for the target-capturing antibodies and metal promoters to enhance the raman reporter dyes. the target antigen is first captured by antibody-magnetic particle conjugates and then labeled with dye/antibody-aunp conjugates, forming a three-component immunocomplex that is magnetically concentrated and finally detected by sers (fig. 4b) . this approach has shown an outstanding sensitivity level down to 5 fg/ml of the target antigen. this high sensitivity with the possibility for the direct detection of rvfv in complex media or samples allowed by magnetic particles support the application of this assay for the rapid and sensitive detection of rvfv and the control of any future outbreaks. immuno-pcr assay for htnv detection using aunp probes dually functionalized with antibody (ab) and double-stranded (ds)dna. au-nanoprobes are directly applied to label virus antigens precaptured on a microplate. the aunps are carrying dsdna that includes barcode single-stranded (ss) dna for signal amplification. the barcode dna is separated, amplified and detected by gel electrophoresis [96] . additionally, this assay can be modified in a more complex detection scheme that has been reported for human immunodeficiency virus detection [81] . (b) surface-enhanced raman spectroscopy (sers)-based assay for detection of rvfv using raman reporter dye-coated aunps and magnetic nps (mnps). aunps and mnps are conjugated with a polyclonal ab specific for the target virus antigen forming aunp/virus antigen/mnp complexes; then, a 785 nm laser excites the magnetically concentrated aunp/virus antigen/mnp complexes. the presence of the target antigen yields a reduction in the intensification of raman dye signature spectrum peaks, thereby providing an estimation of its concentration [49] . this assay has been applied for the detection of wnv infection [49] . the coronaviridae family comprises two subfamilies, coronavirinae and torovirinae, that were recently expanded to include many viruses. coronavirinae are classified into four genetically distinct genera (alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus) [112] . coronaviruses are enveloped rna viruses that are pleomorphic in shape (spherical and 120-160 nm or bacilliform and 170-200 nm by 75-88 nm). they have a relatively large, positive-sense, ssrna genome of 27-32 kb that encodes four to five structural proteins (s, the spike glycoprotein; m, the membrane glycoprotein; n, the nucleocapsid interrupt phosphoprotein; e, the envelope protein; and he, the hemagglutinin-esterase glycoprotein) and 2 nonstructural polyprotein precursors (pp1a, polyprotein 1a; and pp1ab, polyprotein 1ab), which are later processed into several other nonstructural proteins and viral polymerase [113, 114] . coronaviruses are clinically significant to humans and usually associated with different respiratory, intestinal, hepatic, or neurological diseases [115] . hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 are among the most disseminating coronaviruses that usually infect the upper respiratory tract in humans, causing mild common cold-like diseases [116] [117] [118] [119] . other coronaviruses, such as sars-cov identified in china in 2002 and mers-cov discovered in the middle east in 2012, are notorious pathogens able to cause widespread outbreaks of pneumonia and pneumonialike conditions with very high morbidity and mortality rates of up to 35% [120, 121] . due to their high clinical significance and contagious nature, there has recently been great interest in testing and developing advanced sars detection methods. sars detection using aunps is primarily focused on developing rapid and specific molecular detection through two main assays: 1) a colorimetric assay for pp1ab gene detection; and 2) an electrochemical assay for nucleocapsid protein gene detection ( table 1 ). the colorimetric assay involves the ability of aunps to preferentially adsorb ssdna over dsdna and specifically sense the presence of the target dna [54] . specific, short ssdna probes adsorb to the surface of aunps, resulting in an increased particle colloidal stability and an increased ability to withstand slightly elevated salt concentrations without significant aggregation or color changes. upon addition, the target dna specifically forms dsdna with the adsorbed ssdna probes, which then easily detach, leaving the aunps to aggregate due to the salt. the particle aggregation causes the red color of the solution to change to blue, indicating the presence of target sars nucleic acids; this change can be directly assessed by the naked eye or precisely quantified by ultraviolet-visible (uv-vis) spectroscopy in correlation to the target dna concentration (fig. 5a ). in the electrochemical assay, aunps are applied to enhance the electrode conductivity and increase the surface area available for detection probe immobilization [72] . sars-specific dna-capturing probes are first immobilized on the surface of an electrode structured with aunps; then, they are allowed to hybridize with the biotinylated targets. then, streptavidin-labeled alkaline phosphatase is applied to catalyze the indirect reduction and deposition of silver ions, which are eventually measured by anodic stripping voltammetry in correlation with the target dna concentration (fig. 5b) . the aunp-based assays developed for the molecular detection of sars are relatively rapid and simple; especially the colorimetric assay, which completely eliminates the need for instrumentation or trained personnel and yields results exclusively in the liquid phase that can be rapidly identified as positive/negative by the naked eye within 5 min. this technique can allow the detection of target sars nucleic acids with a sensitivity limit down to 100 fm; thus, it can be very beneficial for the early diagnosis of the sars virus, which is critical for such a contagious pathogen. in addition, the simplicity and very high sensitivity of aunp-based colorimetric assays for nucleic acids have promoted the application of aunps for the detection of other viruses either using slightly modified procedures or in combination with other sensing characteristics of aunps [56] . the family filoviridae can be classified into two main genera, ebolavirus [119, 122] . filoviruses are filamentous, enveloped rna viruses 80 nm in diameter and approximately 800-1000 nm in length. their genome is nonsegmented, negative-sense, ssrna 19 kb in size that encodes for at least 7 proteins (nucleoprotein, viral proteins vp24, vp30, vp35, and vp40, glycoprotein, and polymerase protein) [123] . aunps stabilized by single-stranded (ss)dna probes. in the presence of the target dna sequence, double-stranded (ds)dna is formed and desorbs from the surface of citrate-reduced aunps, leaving them to aggregate. this aggregation results in a color change from red to blue, indicating the presence of target nucleic acids [54, 56] . (b) enzymatic electrochemical detection of sars by anodic stripping voltammetry using a aunp-screen-printed carbon electrode. the aunp-modified electrode is modified with capture dna probes that are allowed to hybridize with biotinylated targets. streptavidin-alkaline phosphatase (s-ap) is then applied for the indirect reduction of silver ions in the solution into a metallic deposit. the formed silver metals are then electrochemically measured to determine the target virus dna concentration [72] . most ebovs primarily originate from africa, where they are frequently associated with large outbreaks of ebola hemorrhagic fever (ehf), which is now known to be one of the most deadly and virulent diseases affecting humans, with a case fatality approaching 90% [124] . the early outbreaks of ehf were confined to sudan and its neighbor, democratic republic of the congo, from 1976 to 1978, followed by several independent outbreaks in different countries: [124, 125] . most of these outbreaks were caused by zebov and sebov, while some were caused by new species later named ciebov and bebov [125] . these heavy series of ehf outbreaks are continuing, and most recently, multiple countries in west africa have struggled against a massive outbreak of ebov beginning in march 2014. ebola is expected to remain a major global public health threat, especially with the current absence of effective treatments; therefore, it is necessary to have flexible rapid-detection schemes in place to diagnose and control ebov outbreaks [126] . a very interesting aunp-based molecular assay has been developed for the bimodal scanometric and sers-based detection of ebov by using aunps to promote silver staining on their large surface area [50] . the target ebov dna sequence is first captured on chip surfaces by specific, short dna probes and labeled with aunp-dna-cy3 probes. then, the formed 3-component hybridization complexes are subjected to a silver enhancement step. the deposited silver metal eventually enables scanometric detection (based on a silver-caused dark color) and sers detection (based on using cy3 as a raman-active tag) (fig. 6) . this assay has additional benefits beyond its high specificity and sensitivity that reach down to 20 fm of the target rna. because a very large number of probes can be designed based on using different raman tags, this assay can be readily adapted for multiplex detection, allowing the simultaneous detection of different diagnostic targets of the same virus or even completely different viruses. furthermore, it does not require dna amplification and eliminates the need for specific thermal cyclic equipment. in addition, the capability for bimodal detection simplifies evaluation of the results, as the output can be positive/negative either by traditional scanner systems or by sers when the probes are labeled with raman dyes. such simplicity based on bimodal detection without the need for complicated thermal amplification equipment, multiplexing, and high sensitivity supports the application of this technique for ebov, which is very contagious and usually necessitates the detection of several targets. it is a chip-based detection scheme in which the target dna is captured by immobilized, specific dna oligonucleotides and then directly labeled by aunp-dna probes, followed by silver enhancement. the aunps enhance the surface area available for silver deposition and dna immobilization to achieve highly sensitive scanometric detection signals. modification of the aunp probes with cy3 allows the additional sers-based detection of target dna. this scheme has been reported for the detection of a wide range of viral nucleic acids, including those of hcv, hbv, and hav, which belong to different virus groups [50] . furthermore, the scanometric detection reported in this scheme has been considered a universal step in other detection schemes described for other viruses, such as hev [82] and hiv [81, 84] . this assay has been reported by the same authors for multiplex detection of hepatitis a virus (hav), hepatitis b virus (hbv), hiv, and variola virus (smallpox, vv), which confirms the broad capability of this assay for virus detection [50] . flaviviridae is a large virus family comprising three main genera: flavivirus (53 species, type species is yellow fever virus), hepacivirus (one species, hepatitis c virus; hcv), and pestivirus (four species, type species is bovine virus diarrhea) [127] . flaviviruses are spherical, enveloped rna viruses 40-65 nm in size. their genome is nonsegmented, positive-sense ssrna approximately 10-11 kb in size and includes one open reading frame (orf) encoding three structural proteins (c capsid; prm, premembrane; and e, envelope), and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). several well-known human pathogens are included in this family, such as dengue virus (denv), hcv, yellow fever virus, and west nile virus (wnv), japanese encephalitis virus, and tick-borne encephalitis virus. these viruses have become increasingly serious and have been frequently involved in causing many endemics and epidemics around the world [128, 129] . denv is the most rapidly spreading vector-borne virus in the world. denv is now known to be epidemic in more than 100 subtropical and tropical countries, threatening up to 2.5 billion people. denv causes more than 350 million new annual infections, including 90 million with symptoms varying from flu-like, mild, undifferentiated fever to classic dengue fever (df) or df with hemorrhagic manifestations [130, 131] . although many of these infections are self-limiting and resolve without hospitalization, some progress to severe disease that needs to be quickly identified and treated [132, 133] . currently, there are no specific treatments or protective vaccines available against denv, and as a result of its rapid expansion, accurate and sensitive diagnostic techniques have become crucial for effective control and treatment applications [134] . aunps have been combined with the well-known analytical techniques qcm and icp-ms to develop two novel molecular assays for denv detection. for the first time, these assays implemented aunps to increase the mass of target dna to allow its detection by qcm in a highly quantitative and sensitive manner (fig. 7a) or to amplify the detection signal by releasing many gold ions during icp-ms analysis of the target dna (fig. 7b ). in the aunp-based qcm assay, the presence of target dna initiates a layer-by-layer hybridization of the target dna and several specific multivalent aunp-dna probes, resulting in significant mass changes detected by qcm [93] (fig. 8a) . whereas, in the icp-ms-based assay, two types of probes prepared from specific aunp-dna and mnp-dna conjugates are applied to specifically recognize and sandwich the target virus dna sequences. the mnp probes then help to magnetically separate the formed sandwich complexes, and the target dna concentration is indirectly estimated by detection of the au concentration existing in the sample [94] (fig. 8b) . it is worth noting that both techniques are among the most novel applications of aunps in virus detection. the implementation of aunps extends the ability of qcm and icp-ms to detect dna. moreover, aunp-based icp-ms is among the most sensitive molecular dna assays applied for virus detection, with a detection limit down to 80 zmol, which can greatly help to control denv and other viruses [94] . the bio-barcode ssdna is released and employed to establish multilayered aunps over the electrodes to enhance the produced electric current [72] . (b) dry-reagent strip-based dna assay using aunp-oligo (dt) reporters. aunps with poly (dt) are applied to label biotinylated target dna modified with a poly (da)-tail pre-captured by immobilized streptavidin in the test zone of the strip and generate characteristic red bands [55] . (c) single-particle fluorometric detection of hcv using cy3-tagged ssrna that is electrostatically adsorbed or covalently coupled through thiol (sh) chemistry to the surface of aunps. the coupling of aunps and ssrna probes brings cy3 in the vicinity of the aunp surface and eventually results in emission quenching; upon hybridization with the target rna, the quenched emission is released [55] . hcv is one of the leading causes of chronic liver diseases in humans. approximately 3% of the global population has experienced hcv infection. currently, there are 170 million chronic carriers, and more than three million new infections occur globally each year [135, 136] . chronic hcv infection is frequently associated with marked hepatic injuries, including cirrhosis, liver failure or hepatocellular carcinoma. these types of injuries can be very severe and often end in death or liver transplantation [135, 137] . hcv infection is generally resilient and usually requires treatment that combines numerous medications. the most popular treatment regimen for hcv is a combination of pegylated interferon alfa (peg-ifnα) and ribavirin (rbv) [138] . other three-drug regimens based on the addition of the serine protease inhibitor telaprevir (tvr) or boceprevir (bec) to the standard peg-ifnα/rbv treatment have also been described [139] . in addition, several recently developed oral drugs, such as simeprevir and sofosbuvir, have been approved by the fda for more effective care and shorter treatment duration [140] [141] [142] [143] . despite these numerous drugs and due to the lack of efficient vaccines against hcv, reliable and sensitive detection methods are essential for viral infection prevention and therapeutic responses. numerous aunp-based scanometric, fluorometric, electrochemical, and colorimetric assays have been reported for the molecular detection of hcv (table 1 ). in these assays, aunps are utilized for different sensing functions: 1) aunps have been modified with poly(dt)-tailed dna and directly applied to label target dna sequences terminally modified with a poly(da)-tail in a lfa assay to allow the colorimetric detection of hcv (fig. 7b) [55] . 2) aunps electrostatically or covalently modified with cy3-tagged ssrna sequences have been applied for the fluorometric detection of target hcv sequences, in which the fluorophores are quenched in a process analogous to fret. the presence of cy3 in the vicinity of the surface of aunps eventually results in cy3-emission quenching. upon hybridization with target viral rna, the electrostatically adsorbed probes labeled with cy3 are detached from the surface of the particles, releasing the quenched fluorescence. the covalently bound probes form rigid dsrna with a linear configuration, causing cy3 to move away from the particle surface and release the quenched emission [60] (fig. 7c) . 3) aunps have also been applied to enhance electrical conduction and catalyze a reduction reaction on the surface of detection electrodes. a specifically designed hairpin dna probe terminally modified with aunps and bound to the surface of a glassy carbon electrode is utilized to detect the amplified viral rna. the presence of target dna results in the formation of rigid dsdna, and the hairpin conformation consequently changes, moving the aunps away from the electrode surface, which eventually appears as a detectable decline in the current value in proportion to the target dna concentration [78] . 4) aunps decorated with a specific nucleic acid probe were used for colorimetric detection of hcv. the assay is based on using cationic aunps to induce the aggregation of aunps probes. in the absence of hcv, the cationic aunps electrostatically bind to negatively charged aunps-dna probes causing their aggregation and change of the solution color to blue. the presence of hcv nucleic acid protects the probes and prevents their aggregation by cationic aunps and the solution color remains red. this assay was specific and showed a sensitivity of 93.3% and a detection limit of 4.57 iu/μl [13] . in the immunological detection of hcv, aunps dually modified with capturing antibodies and barcoding dna, similar to those described for the immuno-pcr technique, in combination with magnetic np (mnp)-antibody conjugates, have been applied to capture and separate the target antigen. subsequently, the barcode dna is further allowed to act as a bridge, guiding the formation of a multilayer of aunp-dna probes over the surface of a nanogap electrode (fig. 7a) . the formation of such barcode dna-guided aunp multilayers significantly increases the detected electrical current and is utilized to quantitatively measure the target hcv antigen concentration in applied solutions. the results of this immunoassay indicate a detection limit of 1 pg/µl [69] . in a similar protocol, the barcoding dna was further applied to hcv core antigen detection based on an enzyme to release the barcode dnas to be quantified with rt-pcr. this assay showed a detection limit of 1 fg/ml, which is one magnitude greater than the standard elisa (2 ng/ml) [14] . it is worth mentioning that the primary focus of the developed aunp-based assays was the detection of hcv rna rather than hcv antigens or antibodies. this is due to the broader potential of molecular assays for the detection and management of active hcv infections. hepadnaviridae comprises two main genera, orthohepadnavirus and avihepadnavirus. the genus orthohepadnavirus includes 4 species (hbv, woodchuck hepatitis virus, ground squirrel hepatitis virus, and woolly monkey hbv) that infect mammals, while the genus avihepadnavirusincludes2 species (duck hbv and heron hbv) infect birds [144] . hepadnaviruses are spherical, enveloped dna viruses 40-48 nm in size. they possess a unique gapped genome of 3.2-kb circular dsdna, which contains 4 partly overlapping orfs encoding the different viral proteins: the core protein (c), e antigen, polymerase protein (pol), envelope proteins, and transcriptional transactivator protein [144, 145] . the prototype member of the family hepadnaviridae, hbv, is a health threat to ~88% of the global population [145, 146] . hbv is the etiological agent of hepatitis type b in humans. it is estimated that nearly 2 billion people around the world have been exposed to hbv infection, and there are more than 360 million chronic carriers suffering from the serious risk of developing liver cirrhosis and cancer [146, 147] . the availability of an effective protective vaccine against hbv has greatly reduced the number of new infections. in addition, there have been significant advances in the available treatments for chronic hbv infection. viral dna polymerase inhibitors and peg-ifnα are now well known to significantly control hbv infection and prevent its progression to chronic hepatitis b-associated liver failure and hepatocellular carcinoma [148, 149] . however, chronic hbv infections remain highly contagious, and the virus can easily be transmitted to other persons through any close contact allowing the transfer of infected bodily fluids. the highly contagious nature of hbv and its ability to spread among people are the main reasons why this pathogen continues to threaten the public. therefore, diagnostic methods that are highly sensitive to low concentrations of virus are needed to curtail this virus and prevent its dissemination. hbv is by far the most common virus reported in the published literature regarding the applications of aunps in virus detection (fig. 3) . interestingly, the developed assays cover most of the known diagnostic markers of hbv through many different electrochemical, scanometric, fluorometric, lightscattering, colorimetric, and sers detection schemes (table 1 and fig. 6, 14-18) . several aunp-based electrochemical assays have been developed for the detection of hbv antigens and dna. two enzyme-amplified electrochemical assays have been reported for the detection hbv surface antigen (hbsag) using conjugates of aunps and horseradish peroxidase (hrp)-labeled antibodies against hbv surface antigen (hbsabs) [76, 77] . the target hbsag is captured and labeled with aunp-hbsab/hrp as secondary antibody conjugates. then the reduction of h2o2 catalyzed by the bound hrp is measured either by using an aunp/thionin/dna-modified au electrode coupled with a cyclic voltammeter [76] or through using a nanoporous au electrode coupled with differential pulse voltammeter [77] (fig. 9a) . alizadeh et al. reported aunps modified with horseradish peroxidase mimicking dnazyme to label hbsag magnetically captured and concentrated on the surface of a au sheet-like electrode. due to the efficient catalytic activity of hrp-mimicking dnazyme, the proposed immunoassay allowed quantitative detection of hbsag with a linear concentration range of 0.3-1000 pg/ml and detection limit of 0.19 pg/ml [15] . other studies described the electrochemical detection of hbv based on aunp-metal enhancement amplification, including copper and silver metal enhancement [74, 75] . using copper metal enhancement, hbsag is sandwiched by mnp-hbsab conjugates and aunp-hbsab probes to form a 3-component immunocomplex, which is magnetically separated and stained through a copper enhancement step (fig. 9b ). after copper acidic dissolution, the resulting ions are measured by anodic stripping voltammetry (asv) [75] . using silver enhancement, hbv dna is detected using probes prepared of streptavidin-modified aunps. biotinylated hbsag gene sequences are magnetically pre-separated and concentrated using magnetic bead-dna conjugates. the deposited silver ions are then analyzed by an electrochemical stripping technique to detect the target hbv dna quantitatively [74] . hbv detection through aunp-based scanometric assays mainly relies on the well-described aunp-promoted silver-staining protocol, in which aunp probes are applied to facilitate the reduction of silver ions into metallic silver that is deposited as visible black spots, indicating the presence of hbv. following this protocol, aunps modified with staphylococcal protein a (spa) [88] or specific dna probes [50, 80] have been utilized in multiple chip-based assays to permit the detection of different hbv antibodies and dna (fig. 6 ). in addition, wang et al. combined this aunp-promoted silver-staining protocol integrated with a bio-barcode-amplification (bca) technique to allow an enhanced scanometric detection of hbv dna [85] . this bca-based scanometric assay employs two different sets of dna conjugates to capture and detect a specific hbv sequence. the first set is a group of aunps modified with the short dsdna of a signal amplification dna strand (barcode dna) partially complemented with a detection probe strand, while the second set is a group of magnetic microparticles (mmps) modified with ssdna specific to the target hbv dna. the presence of the complementary target sequences of dna guides the assembly of aunp/dna/mmp sandwich hybrids. subsequently, the sandwiched hybrids are magnetically isolated and washed, and the barcode dna is eventually released from the aunps. the barcode dna is then added to the surface of a chip modified with specific dna-capturing probes and directly labeled with aunp-dna conjugates, followed by silver staining to further amplify the detection signal (fig. 10) . the aunps-fluorometric assays for hbv are performed through different schemes that basically depict the quenching efficiency of aunps in a manner remarkably similar to the traditional fret protocol. zheng et al. developed a multiplex detection system based on applying gold nanorods (aunrs) as acceptors and qds of different colors as donors to simultaneously detect hbsag and hbv e antigen [62] . this assay follows the direct sandwich immunoassay format, and the presence of target antigens is manifested by the fret-induced quenching of qd fluorescence in the formed sandwich nanostructure of aunr-ab1/ag/qd-ab2 (fig. 9c ). draz et al. further coupled multiple aunp-peptide conjugateacceptors with single-core qd-antibody fab conjugate-donors, forming a preassembled hybrid nanocluster plasmonic resonator complex for hbsag and hbv particle detection (fig. 9c) . this scheme follows a competitive assay format, and the addition of hbv target surface antigen or particles to the preassembled nanocluster releases the quenched fluorescence signal of the qds in proportion to the in this assay, the target hbsag is captured and labeled with aunp-ab/hrp, and the detection signal is measured by using an ab-modified au electrode to assess the reduction of h2o2 catalyzed by the bound hrp using differential pulse voltammetry (dpv) technique [76] . (b) copper (cu)-enhanced electrochemical immunoassay. aunps serve as a scaffold for a horseradish peroxidase (hrp) enzyme that catalyzes a redox reaction in the first scheme or enhances metal deposition in the second scheme [75] . (c) fluorometric detection by the fret-induced quenching of fluorophores on the surface of aunps. aunps and gold nanorods (aunrs) are applied to quench the fluorescence of fluorophores, such as (i and ii) quantum dots (qds) [62, 65] and (iii) fluorescein amidite (fam) [61] , through a fret-based interaction in the presence of the target virus. a similar aunp fret-based scheme has been reported for the detection of h1n1, which belongs to the family orthomyxoviridae [63] . (d) light-scattering assays using aunp-antibody probes. (i) light-scattering immunoassay of virus antigen based on using aunps with different sizes (10 nm and 50 nm) conjugated with antibodies (abs) to sandwich target antigens and enhance the dynamic light scattering [51] . additionally, this scheme has been reported with the use of aunps of the same size for h1n1 detection [53] . (ii) plasmonic immunoassay based on allowing aunr-ab conjugates to interact with the target antigen, thus changing the aunr localized surface plasmon resonance (lspr) behavior [52] . target concentration [65] . furthermore, lu et al. developed a fluorometric assay for hbv dna detection in which positively charged aunrs are employed to chelate fluorescein amidite (fam)tagged ssdna electrostatically onto their surface, forming fam-ssdna-aunr ternary complexes [61] . the formation of these complex results in the fret-based quenching of fam fluorescence, and when complementary target dna is present in the system, a fam-ssdna/cdna duplex is formed. this dna duplex is comparatively more negative than the fam-ssdna and can mediate stronger electrostatic interactions with aunrs, leading to increased fret efficiency, which manifests as a further decline in the fluorescence intensity of fam. this change in fluorescence intensity is utilized to sense and estimate the exact concentration of the added target dna (fig. 9c) . aunp-based light-scattering assays have been described for the detection of hbsag through two main schemes: the first scheme applies spherical aunps of different diameters (10 nm and 50 nm) modified with hbsab to sandwich hbsag, and the presence of hbsag results in specific immuno-based aggregations that can be estimated by measuring the increases in hydrodynamic diameter using dls (fig. 9d) [47, 51] ; the second scheme relies on the lspr characteristic behavior of aunrs to directly sense the binding of hbsag to the antibodies conjugated to their surface by measuring lspr peak shifts via uv-vis spectroscopy (fig. 9d) [52] . a aunps-based colorimetric lateral flow assay (lfa) assay was developed for the detection of hbsag. this assay relies on the characteristic red color of different sized aunps to label hbsag on an immunochromatographic test strip. a strip composed of a sample pad, conjugation pad, control line, test line and absorbent pad was used to capture hbsag labeled with aunps and the test line becomes red in color. the results showed that the signal visibility of lfa could be improved by increasing the diameter of aunps, and ~43 nm aunp enabled lfa assay with a detection limit of 100 ng/ml [16] . a aunps-based sers assay for hbv dna detection was developed following a sandwich assay format performed on a heat-responsive hybrid silicon substrate modified with aunps. the target hbv dna is captured on the surface of the silicon substrate and forms a sandwich complex with specifically designed aunps modified with dnaindocyanine green proximally to the surface, leading to high sers signals. this heat-responsive hybrid silicon substrate-based sers platform can detect remarkably low hbv dna concentrations of ~0.44 fm at 25 °c and ~0.14 fm at 37 °c [17] . aunp-based fluorometric detection is the most sensitive among the reported methods for hbv detection, with a detection limit of 1 particle/μl. due to the highly infectious nature of hbv, this limit of detection can be very useful for exploiting these assays for efficient control and management of hbv infection. in addition, the simplicity of scanometric and colorimetric detections is very interesting, allowing flexibility in the detection design and target, and was reported for single and multiple target detection for more efficient and accurate screening of hbv. on the other hand, the developed dls assay is very novel to hbv research, simple and sensitive; however, the need for bulky equipment for dls measurement remains the major challenge for its wide use and real application in hbv detection. the family hepeviridae is monogeneric with one exclusive member called hev, which is a spherical, small, nonenveloped rna virus with a ~7.2 kb ssrna genome [150] . the viral rna is a positive-sense molecule organized into 3 partially overlapping orfs flanked by short untranslated regions at both ends. the first orf encodes 4 mature nonstructural proteins (methyltransferase, protease, helicase and replicase), the second orf encodes 2 structural proteins (c and vp1), and the third orf encodes a small protein of unknown function [150] . hev is known for causing human liver inflammation figure 10 . aunp-based nucleic acid assay for hepatitis b virus (hbv) detection. hbv dna was detected using a scanometric detection that integrates magnetic separation and aunps-based bio-barcode-amplification method. aunps provide a large surface area for silver deposition and dna immobilization, thereby achieving highly sensitive scanometric detection signals through a chip-based silver deposition scheme [85] . called hepatitis e, which is widely disseminated in developing countries with poor sanitation conditions and has recently become apparently endemic in some industrialized countries, including the usa [151] . one aunp-based colorimetric assay has been reported for the molecular detection of hev [152] . this assay targets a conservative fragment of hev in orf1 that is pre-amplified by one-step rt-pcr/pcr amplification, and the generated cdna is labeled by aunp-dna conjugates, forming three-component sandwich hybrids in a manner very similar to that previously depicted in fig. 6 . this hybridization is followed by a silver-staining step to allow the scanometric detection of target dna either by the naked eye or using simple scanners, with a detection limit of 100 fm of hev amplicon. one of the major advantages of this hev detection assay is its sensitivity, which can help control hev. however, this assay suffers from the same disadvantages of conventional pcr because of the requirements of the amplification step. herpesviridae is a taxonomically large family of viruses, including 3 major subfamilies (alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae), which together comprise nearly 9 genera with over 100 viruses [153] . repeats. the rearrangements of these repeated sequences result in different genome isomers. in epstein-barr virus (ebv) and kaposi sarcoma-associated herpes virus (kshv), from the subfamily gammaherpesvirinae, there are repeated sequences at the termini of their genomes that differ from those of other human pathogenic herpesviruses, including herpes simplex virus type 1 (hsv-1) and type 2 (hsv-2), human cytomegalovirus (hcmv), and varicella zoster virus (vzv); these viruses are characterized by the definite presence of us and ul regions in their genomes [153] [154] [155] . herpesviruses can infect humans and many animals. they account for significant and recurrent human cutaneous diseases, including oral herpes, genital herpes, and many cancers. eight herpesviridae family members are commonly involved in human infection: hsv-1 (human herpesvirus1; hhv-1), hsv-2 (hhv-2), vzv (hhv-3), ebv (hhv-4), hcmv (hhv-5), hhv-6, hhv-7, and kshv (hhv-8). hsvs are among the most prevalent viruses in humans. there are two serotypically and genetically different hsvs, types 1 (hsv-1) and 2 (hsv-2) [156] . hsv-1 is estimated to infect up to one-third of the world population, while hsv-2 infects nearly 500 million people around the globe, with more than 20 million new cases occurring every year [157] [158] [159] . both hsv types are renowned for infecting epithelial cells of the skin or mucosal surfaces and then infecting the central nervous system, causing lifelong, incurable infections in humans. these infections are primarily asymptomatic but can progressively result in serious health complications. for example, hsv-1 infection is the main cause of infectious blindness in the world and has emerged as an important cause of genital disease in the developed world, and hsv-2 infection is the leading cause of genital ulcers worldwide [159, 160] . nevertheless, these viruses are involved in many other clinical conditions, such as encephalitis, conjunctivitis, zosteriform skin lesions, pneumonia, and systemic infections that compromise vital organs in the body [161] . therefore, desperate efforts have been directed toward developing an effective vaccine against hsvs. however, there are no protective vaccines against hsvs, and only a few anti-herpes drugs, such as acyclovir, valacyclovir, and famciclovir, are commercially available [162] [163] [164] . these drugs are only helpful to control symptoms and signs of infection and do not eradicate the virus or even decrease the frequency or severity of infection recurrence and due to the drug resistance rapidly developed by hsvs during treatment, the efficacy of anti-herpes drugs is increasingly hampered over time. thus, the definitive diagnosis of hsv virus infection is essential for effectively controlling its severity and applying treatment regimens. with their characteristic red color, aunps have been exploited as colorimetric labels in a lateral-flow immunochromatographic assay (lfia) to allow the immunological detection of hsv antigen. in this assay, the lfia strip is preloaded with the labeling aunp-anti-human igg conjugates onto the conjugate pad, and the capturing igg-1 and igg-2 antigens, along with the anti-igg antibody, are immobilized onto separate test and control lines directly next to the sampling area (fig. 11a) . the sample is loaded and allowed to migrate across the strip with the chase buffer to react with the capturing antigens (igg-1 and igg-2) loaded on the test lines, as well as the anti-igg antibody loaded on the control line. as a result, hsv antibodies are captured on the test lines and labeled with the loaded aunp-dna conjugates, causing characteristic red lines to appear. according to the color of the test lines, the samples can be qualitatively determined to be positive or negative within 20 min [59] . lateral-flow immunochromatographic assay of hsv-2 antibodies using aunp anti-igg probes. sample addition followed by chase buffer addition causes gold nanoparticle (aunp)-anti-igg conjugates and the sample to migrate, contacting the test lines sequentially and resulting in the capture of type-specific antibodies to hsv-2. the concentration of hsv-2 antibody-aunp complexes on the test line causes a corresponding pink color to form [59] . this scheme is similar to that reported for the detection of h5n1 [58] and hiv-1 [91] . (b) electrochemical detection of hcmv by the acidic dissolution of aunps. hcmv dna is directly labeled with specific aunp-dna probes. after the acidic dissolution of aunps, au iii ions are measured by anodic stripping voltammetry (asa) [68] . (c) colorimetric one-pot np-based assay of kshv. this assay has been reported for the differential diagnosis of kshv dna and dna from a frequently confounding bartonella species. upon the addition of sample dna to a mixture of silver np (agnp)-dna and aunp-dna conjugates specific for kshv and bartonella dna, respectively, complementary dna hybridization triggers the aggregation of bare np conjugates and the subsequent decrease or disappearance of its corresponding color [60] . hcmv/hhv-5 is the largest known endemic virus in humans. it persistently infects over 70% of the population worldwide [165] . hcmv can infect a broad range of cells, including endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, neuronal cells, hepatocytes, and many types of immune cells [165, 166] . nearly all hcmv infections are asymptomatic, except in immunocompromised individuals and neonates, and can lead to severe symptoms. hcmv infection is the leading cause of congenital malformations in live births worldwide, while in immunocompromised individuals hcmv is usually opportunistic, causing very serious clinical manifestations, such as pneumonia, hepatitis, encephalitis, colitis, and retinitis [167, 168] . moreover, hcmv has been described to be associated with several human malignancies, especially in the brain, breast, prostate, skin and colon [169] . numerous hcmv vaccine candidates have been developed and tested, but a vaccine has yet to be licensed. antiviral therapy with several anti-hcmv drugs, such as ganciclovir, valganciclovir, foscarnet, and cidofovir, which target the viral dna polymerase, is currently the main strategy for controlling hcmv infection [166, 170, 171] . unfortunately, these drugs result in substantial hematotoxicity and nephrotoxicity in patients, and hcmv remains a considerable public health threat without an efficient treatment or control strategy. in such conditions, the need for new diagnostic methods that can help to control the dissemination of this pathogen is very important. a novel aunp-based electrochemical assay for the molecular detection of hcmv dna has been reported [68] . in this assay, aunps are applied to amplify the detected electrochemical signal by releasing many au ions upon exposure to acid dissolution. typically, the immobilized target hcmv ssdna is initially labeled with oligonucleotidemodified aunp probes followed by a metal dissolution step, releasing many gold ions. the concentration of solubilized gold ions is then indirectly assessed by asv (fig. 11b ). this assay allows the highly sensitive detection of target dna concentrations down to 5 pm [68] . kshv/hhv-8 is a leading cancer-causing virus. it is commonly responsible for inducing a considerable range of neoplastic diseases in immunocompromised patients, such as kaposi sarcoma, primary effusion lymphoma, and some kinds of multicentric castleman disease [172, 173] . owing to the dramatically increasing rates of immunocompromised patients over the past two decades, the clinical significance of kshv is clearly increasing; additionally, kshv has a prevalence of 3-40% in this class of patients worldwide [173, 174] . while kshv is a relatively newly discovered virus, there are no standard diagnostic approaches, and the detection of this virus through traditional serological and molecular methods remains challenging. this difficulty is mainly due to the inconsistency observed among serological assays targeting different antigens, and the low levels of viral dna that are usually present in the peripheral blood of infected individuals [175, 176] . hence, there is a continued interest in developing new reliable and sensitive methods for kshv detection. through using a mixture of aunps and silver nps (agnps), a colorimetric assay has been established for the multiplex, one-pot molecular detection of kshv and its frequently confounding disease bacillary angiomatosis [57] . in this assay, two types of probes are prepared from aunps and agnps separately functionalized with kshv and bartonellaspecific dna-capturing dna probes, respectively, and mixed with the target dna. then, the color of the reaction mixture changes in a manner dependent on the type of target dna added to the reaction solution: the addition of kshv dna causes aunp conjugates to aggregate and the solution to appear murky yellow-orange, i.e., the specific color of the nonaggregated agnps, while the addition of bartonella dna causes agnp conjugates to aggregate and the solution to appear pink, i.e., the color of nonaggregated aunps (fig. 11c ). in addition to being simple, this multicolor-change system successfully allows the multiplex detection of targets present in nanomolar concentrations. the family orthomyxoviridae includes five genera: influenzavirusa, influenzavirus b, influenzavirus c, isavirus, and thogotovirus [177] . the first three genera are widely known to infect birds, humans, and other mammals, causing influenza. the influenza-virusa genus contains many strains and can be further divided into several antigenic subtypes according to the antigenic properties of their envelope glycoproteins, i.e., hemagglutinin (ha) and neuraminidase (na). in contrast, the influenzavirusb genus has no distinct antigenic subtypes of hemagglutinin or neuraminidase. influenzavirusc is similar to types a and b but less common and lacks the na protein. the genus isavirus is known to infect salmon, while thogotoviruses usually cause infection in some vertebrates and invertebrates [177, 178] . orthomyxoviruses can be spherical (80-120 nm) or filamentous (20 nm in diameter and 200-300 nm long) in shape, and their genome contains 7 or 8 segments of linear negative-sense ssrna with a total length of 12-15 kb encoding different structural and nonstructural proteins, including nucleoprotein, three large proteins (pb1, pb2, and pa), matrix proteins (m1 and m2), the glycoproteins ha and na, and two nonstructural proteins (ns1 and ns2) [177, 179, 180] . several strains of the genus influenzavirusa cause seasonal and occasional acute respiratory diseases called influenza, which is profoundly and rapidly spread in humans. influenza is reported to affect up to 5% of adults and 20% of children of the global population each year [181, 182] . over the past decade, the world witnessed two major influenza outbreaks caused by newly emerging influenzavirusa strains: h5n1 in 2003 (avian influenza), and h1n1 in 2009 (swine influenza). both were severe enough to incite the who to issue a pandemic warning alert, which reached the highest level, i.e., "phase 6," for h1n1 in 2009 [21, 183] . the severity of influenza infection depends on both the virus strain and the age of the infected person. in elderly persons, influenza can result in hospitalization or death, while in younger adults, influenza-associated morbidity usually results in restricted activity, impaired work performance, and increased health care use [184, 185] . most people with the flu recover within one to two weeks without treatment. however, serious complications usually require medications to reduce the severity and duration of infection. some antiviral drugs, such as oseltamivir and zanamivir, can effectively treat or prevent influenza [186] . on the other hand, immunization with vaccine preparations of the most-circulating virus strains (trivalent or quadrivalent vaccines) remains the key strategy for preventing both influenza and its serious complications [187, 188] . in fact, both the treatment and management of influenza infection, either by antivirals or vaccination, depend on the influenza virus strains and the timing and severity of the infection. therefore, the rapid and accurate testing and typing of influenza is critical for treating infection and plays an important role in public health measures. the application of aunps in iav detection has primarily been focused on developing assays that enable the direct detection of whole iav particles [53, 58, 92] or viral rna [64, 66, 67, 70, 71, 83, 86] through numerous aunp-based fluorometric, sers, scanometric, light-scattering, colorimetric, and electrochemical detection schemes ( table 1) . the aunp-based fluorometric assays developed for h5n1 detection have simply exploited the intense plasmonic-based quenching properties of aunps through different detection formats. one of these assays targets the detection of the whole virus particle through a lateral-flow immunoassay format that is similar to that previously described for the detection of hsv-2, as shown in fig. 11a . in this assay, the virus is first captured on a lateral-flow test strip by pre-immobilized antibodies; then, aunp-antibody conjugates are applied to label the captured virus particles. after washing, the aunps are collected and dissolved to release gold ions that are eventually utilized to quench the fluorescence of qds in an additional, separate step [58] . in addition, ganbold et al. have developed a bimodal assay for the fluorometric and sers detection of viral rna using aunps modified with ssdna-dye probes [64] . the surface ssdna-dye probes are weakly adsorbed to the surface of aunps by electrostatic interactions, and with the addition of the complementary target dna, perfectly matched dsdna forms and desorbs from the surface of aunps, resulting in measurable changes in the conjugated dye fluorescence and raman fingerprint that are more substantial than those resulting from either mismatched dsdna or ssdna (fig. 12a) . aunp-based colorimetric assays have been developed for iav detection through four main detection schemes: 1) the first scheme involves the application of aunps conjugated with anti-digoxigenin as colorimetric labels to detect the target viral dna premodified with digoxigenin and electrophoresed through a membrane-based lateral-flow technique [66] . 2) the second scheme is simply based on the electrostatic interactions between the negatively charged phosphate groups of the target dna amplified with lamp and the positively charged ctab bilayer on aunrs, resulting in the aggregation of aunrs and a color change from red to purple [67] . 3) the third scheme relies on a magnetic-based hydroquinone oxidation reaction involving two metal np bioconjugates: iav-specific pentabody-mnps as capture probes and anti-aiv monoclonal antibody-aunps as detection probes. in the presence of virus particles, an immunocomplex forms and is separated by a magnetic field; after washing, aunps in the separated complex act to catalyze the oxidation of hydroquinone, which is detected optically in correlation with the aiv sample concentration [92] . 4) the fourth scheme relies on the peroxidase-like catalytic activity of au np films and colloidal aunps toward tmb-h2o2 mixtures. the virus is captured on a 96-well plate modified with aunp film biofunctionalized with ha antibody. then it is labeled with aunps modified with na antibody for signal generation. tmb-h 2 o 2 is added, and rapid color changes are observed because of the oxidation of peroxidase substrate tmb. using h1n1, the response of the developed assay was linear in the range from 10 pg/ml to 10 μg/ml and the limit of detection was 50.5 pg/ml, while with h3n2, the optical density of the developed color was dependent on the virus concentration in the range of 10-50,000 pfu/ml and the limit of detection was 24.3 pfu/ml [22] . aunp-based electrochemical assays rely on using aunps to directly modify the target dna or electron transducer surfaces, allowing enhanced electrical conduction and increased surface area for probe immobilization and target interaction. following this protocol, liu et al. [71] successfully detected aiv ha gene sequences using a dna aptamer immobilized onto an electrode; its surface was modified with a composite of carbon nanotubes, polypyrrole nanowires and aunps (fig. 12b) . furthermore, bonanni et al. [70] developed an impedimetric electrochemical assay for the detection of aiv m gene sequences based on measuring changes in the impedimetric behavior of the electrode when the target dna hybridizes with the capture dna probes immobilized onto its surface and is subsequently labeled by aunps via streptavidin/ biotin interaction (fig. 12c) . aunp-based scanometric assays for detecting aiv are mainly performed using the well-described aunp-promoted metal staining protocol shown in fig. 12d . in this protocol, the target nucleic acid is recaptured on the surface of glass chips modified with specific capture dna probes [86] . then, the captured probes are labeled with aunps either modified with specific dna probes or with the renowned dsdna intercalator daunorubicin [83] to mediate the metal staining. following this labeling step with aunp probes, the presence of the target nucleic acids is visually determined with an additional silver-or gold-staining step. specific dna aptamers immobilized on a gold electrode (ge) modified with a layer of multiwall carbon nanotubes (mwcnts), polypyrrole nanowires (ppnws) and aunps are collectively applied as an electrochemical platform for the detection of dna hybridization using dpv [71] . (c) impedimetric electrochemical assay using streptavidin-aunps probes. the direct/sandwich hybridization of biotinylated target dna with capture probes immobilized on the surface of a screen-printed electrode (spe) and additional conjugation with streptavidin-aunps results in impedimetric signal enhancement [70] . (d) label-free scanometric detection using positively charged aunps. the electrostatic interaction between positively charged aunps and negatively charged dna is utilized to visualize the double-stranded (ds)dna formed by the hybridization of single-stranded probe dna and complementary target dna on the array of chips through aunp-mediated metal staining [83, 86, 87] . the light-scattering detection of h1n1 virus using aunps relies on dls to measure the aggregation of aunp-antibody conjugates induced by the addition of target virus particles (similar to fig. 9d ). the magnitude of aunp aggregation and the corresponding increase in their hydrodynamic size is correlated with the concentration of virus particles [53] . finally, the immunological detection of h1n1 hemagglutinin (ha) antigen has only been reported through a novel lspr-based immunoassay. in this assay, the presence of target antigen induces a sandwich immunoreaction between the fluorophorelabeled detection antibodies and the aunp-capturing antibody conjugates, directly resulting in enhanced fluorescence caused by the coupling of fluorophore emission with the localized surface plasmons of aunps in a manner similar to that shown in fig. 9c . the sensitivity of this method has been described to be 10 3 -fold, improved over that of the conventional elisa technique, with a reported limit of detection of 13.9 pg/ml [63] . papillomaviridae is a widely diverse virus family that includes hundreds of recognized viruses classified into 16 taxonomically distinct genera designated with specific greek alphabet letters, from alpha to pi. the first five genera (alphapapillomavirus, betapapillomavirus, gammapapillomavirus, mupapillomavirus and nupapillomavirus) include the human papillomaviruses, while the remaining genera specifically include other mammalian and avian papillomaviruses [189, 190] . papillomaviruses are spherical, nonenveloped dna viruses 40-55 nm in size. their genome consists of a single molecule of circular, supercoiled, dsdna 5.3-8 kbp in size and encodes 6 early expressed (e) and 2 late (l) expressed proteins, in addition to a noncoding long control region [189, 191] . hpv infections are the most epidemic sexually transmitted viral infections worldwide [192, 193] . hpvs are highly epitheliotropic pathogens that usually infect the deepest layer of the skin or the genital surfaces. most hpv infections cause no symptoms and usually self-clear without causing clinical problems, but persistent infections lead to serious clinical complications [194] . this persistence typically depends on the type of hpv causing the infection. among more than 100 types of hpvs, approximately 40 types infect the mucous membranes and can be either high-or low-risk hpvs. high-risk types, such as hpvs 16, 18, 31, 51 and 52, are usually associated with many cancers of the cervix, vagina, vulva, penis, anus, neck, and head, as well as oropharyngeal cancer, while low-risk types, such as hpvs 6 and 11, are mainly related to anogenital warts and recurrent respiratory papillomatosis [195, 196] . currently, hpv is considered the most important cancer-causing virus, as it is associated with ~4.8% of all human cancers [197] . despite this high clinical significance of hpv, there are no available treatments, and vaccination using the bivalent vaccine against hpvs 16 and 18 or the quadrivalent vaccine against hpvs 6, 11, 16, and 18 remains the first and only choice for controlling the global hpv disease burden [198] [199] [200] . in fact, these vaccines are relatively expensive, and their efficacy is very restricted to specific hpv types. therefore, the development of accurate diagnostic techniques for hpv detection and typing is essential for acquiring surveillance data and initiating appropriate vaccination programs. aunp-based assays have been developed for the molecular detection of hpv through different photoelectric, fluorometric, electric and colorimetric schemes [89, 98] . the photoelectric assay essentially leverages aunps to enhance the deposition of silver metal on their surfaces, as previously described for several other assays. however, the enhanced precipitation of silver metal in this assay is used to block the light generated from a photodiode array (pda), and the target dna concentration is measured in proportion to the decrease in light intensity as electrically detected using a bipolar integrated circuit pda chip [89] . specifically, pcr-amplified biotinylated target dna is captured on the surface of the pda chip, and then labeled with aunp-biotin antibody conjugates; subsequently, a silver enhancement step decreases the light intensity, which is quantitatively translated into an electrical signal depending on the amount of target dna (fig. 13a) . on the other hand, a fluorometric assay integrating aunps and a microfluidic bead-based array has been developed as a rapid and controlled hpv dna detection system [98] . in this assay, aunps dually functionalized with hrp enzyme and capture dna strands are applied to label the target dna captured on the surface of microbeads (fig. 4a) , causing hrp to be in proximity to biotin-tyramine loaded on the surface of the microbeads and catalyze their oxidation by hydrogen peroxide. this process eventually increases the deposition of biotin moieties on the surface of the beads, subsequently increasing the labeling efficiency with qd-streptavidin conjugates and yielding an enhanced fluorescence signal that varies significantly based on the target dna concentration (fig. 13b) . in the electrical detection of hpv, aunps were used as an immobilization scaffold for dna immobilization on a silicon substrate carrying nanogapped interdigitated electrodes. this aunps deposition technique was shown to reduce the size of the nanogaps, allowing enhanced electric detection of the target dna captured on the silicon substrate by specific hpv dna probes. the results demonstrated that this assay was able to specifically detect hpv dna within 30 min and at concentrations down to 1 pm [201] . based on the color change of aunps upon aggregation, aunps carrying hpv dna probe were coupled with the lamp technique for colorimetric detection of hpv. lamp reaction was performed at a temperature of 65°c for 20 min and the generated products were hybridized with aunp-dna probes for 5 min. then the presence of the target dna was detected by the addition of magnesium salt. the addition of the salt causes aunps aggregation and the color changes from red to blue. the presence of lamp amplicons can protect aunps from aggregation and prevents aunps aggregation. the sensitivity of the developed lamp-aunp assay was greater than the lamp turbidity assay by up to 10-fold with detection limits of 10 2 and 10 0 copies for hpv16 and hpv18, respectively [27] . the family picornaviridae is taxonomically very diverse and has recently been expanded to involve 12 genera: eight are originally identified genera (aphthovirus, cardiovirus, enterovirus, erbovirus, hepatovirus, kobuvirus, parechovirus, and teschovirus), and four newly proposed genera (avihepatovirus, sapelovirus, senecavirus, and tremovirus) [202] . picornaviruses are spherical, relatively small (22-30 nm) , nonenveloped rna viruses. their genome is positive-sense ssrna that is 7.0-9.5 kb in size and terminally bonded to a noncapsid viral protein (vpg) at its 5' end and to a polyadenylated tail at its 3' end. the expression of this genome produces a single large polyprotein that undergoes a cascade of cleavage and processing reactions to form 10-12 final structural (vp1-4) and nonstructural (2a-c and 3a-d) proteins [203] . picornaviruses can infect a broad spectrum of hosts, including birds, humans, and other mammals [202, 204] . the genera enterovirus, hepatovirus, parechovirus, kobuvirus, and cardiovirus include several important species that affect humans. particularly, the genus hepatovirus encompasses hav, which is the most likely causal agent of acute viral hepatitis in humans. hav infection is usually asymptomatic and self-limiting, and the related symptoms can be mild or sporadically progress to fulminant hepatic failure [205, 206] . the availability of an effective vaccine against hav has substantially reduced the number of people who become infected with hav [207, 208] . however, the ability of this virus to survive harsh environmental conditions and remain in seawater, fresh water, wastewater, and soil for extended periods increases the possibility of outbreaks, especially in developing countries where sanitation is not typically available [205] . the transmission of the virus usually occurs through the consumption of contaminated materials. as such, the virus does not replicate in the environment, water, or foods, and traces of viral contamination are difficult to identify, which remains a challenge for controlling hav. the need for sensitive assays for hav remains an urgent public health demand. hav detection using aunps was accomplished through scanometric and sers-based assays. both assays rely on the extensively described virtue of aunps to enhance silver staining, thereby allowing the molecular detection of hav dna in a microarray chip format (table 1 and fig. 6 ). in the scanometric aunps modified with anti-biotin igg are applied to label biotinylated target dna captured on the pda chip. after a silver enhancement step, the precipitated silver metal at the surfaces of the aunps blocks the light irradiated from above by light-emitting diodes (leds) and decreases the resulting photocurrent [89] . (b) microfluidic bead-based enzyme-aunp amplification method for the fluorometric detection of hpv. streptavidin-coated beads are dually functionalized with biotin-electron-rich protein and biotin-dna capture probes. the captured target dna first reacts with horseradish peroxidase (hrp)-functionalized aunp-dna labels, bringing hrp close to the surface of the microbeads. the oxidation of tyramine-biotin by hydrogen peroxide results in the deposition of multiple biotin moieties onto the surface of the beads. this deposition is markedly increased in the presence of immobilized electron-rich proteins. streptavidin-labeled quantum dots (qds) are then allowed to bind to the deposited biotin moieties and display amplified fluorescence signals in relation to the target dna concentration [98] . detection scheme, aunp-dna conjugates are applied as specific detection probes to label target hav rna captured by specific vp1 probes on the surface of a microarray chip. a silver metal enhancement step is subsequently used for signal amplification, followed by the visual detection of nucleic acids [79] . in the sers detection, the captured hav dna is labeled by aunp-dna probes loaded with the raman reporter dye cy3; this approach has been applied as previously described for ebov and depicted in fig. 6 of this review [50] . these assays are very simple and can detect target concentrations ranging from 3.37×10 -7 -3.37×10 -8 m, with 100 fm as a limit of detection, which can be superior to the relatively expensive pcr-based approaches currently used for hav detection and management. the family retroviridae is broadly classified into 7 genera (alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus, lentivirus, and spumavirus), which include nearly 51 viruses [209] . retroviruses are spherical, small (80-100 nm), enveloped rna viruses. their viral genome is typically bipartite and composed of two positive-sense ssrna molecules (10 kb in total length). they comprise 4 major genes that encode the different structural and nonstructural proteins of the virus: gag encodes the large protein form matrix, capsid, and nucleocapsid; pro encodes the protease enzyme; pol encodes the reverse transcriptase and integrase enzymes; and env encodes the envelope glycoproteins and transmembrane proteins [209] [210] [211] . retroviruses infect a wide variety of vertebrates, including humans. their infection in humans is usually very serious and can potentially cause oncogenesis and variable hematopoietic and neurological conditions. among retroviruses, hiv is a well-known, formidable human pathogen responsible for the most devastating viral pandemic that has ever occurred in human history [212] . hiv infection is usually correlated with the progression of what is clinically called aids, in which the immune system is harshly destroyed, and the entire body becomes vulnerable to terrible, life-threatening opportunistic infections and cancers. people with aids also suffer from other systemic symptoms, such as fevers, sweats, swollen glands, chills, weakness, and weight loss [213] . currently, there is no effective vaccine against hiv, and most of the treatment strategies rely on using antiretroviral drugs, which in most cases maintain the viral load at low levels to prevent the formation of acute hiv infection rather than cure the virus [214] . therefore, the accurate detection of hiv is a key determinant for monitoring hiv treatment and reducing its transmission and dissemination. currently, aunps have been extensively tested for the immunological and molecular detection of hiv through various scanometric, colorimetric, electrochemical, ipcr, and afm assays ( table 1) . the scanometric assays target the detection of the hiv p24 antigen and hiv nucleic acid using the silver metal enhancement protocol. for hiv p24, the target antigen is precaptured by magnetic particles [81] or isolated in a microplate [84] , followed by a silver enhancement step for the quantitative visual immunological detection of hiv (fig. 4a, b and fig. 6 ). for hiv nucleic acid detection, aunp-dna probes specific to the hiv pol gene are applied to detect hiv nucleic acids precaptured and isolated by magnetic particles, followed by a silver metal enhancement step (fig. 14a) . the deposition of silver on the aunp surface increases the np size and scattering efficiency and additionally allows the target detection via dls [90] . in aunp-based colorimetric detection of hiv, the large surface area of aunps was exploited to amplify the detection signal by enhancing the gold metal staining on their surfaces, allowing naked-eye detection of hiv nucleic acids (hiv gag gene) in a lateral-flow format, similar to the scheme presented in fig. 11a [91] . on the other hand, the ability of aunps to enhance electron transfer has been applied to develop an electrochemical assay for hiv detection (fig. 14b) . in this electrochemical assay, the target hiv genome is detected in correlation with the change in electrical impedance of graphene sheets decorated with spherical aunp-dna probes specific to the hiv pol gene [73] . the developed aunp-based rna detection of hiv using the aunp-lfa technique has a sensitivity limit of ~11 log10 copies/ml, while the best dna detection can be achieved through the light-scattering scheme with a sensitivity limit of 10 fm. furthermore, afm coupled with aunps as nanoparticulate labels has been applied for the detection of hiv p24 as follows: the target antigen is precaptured on the surface of a nanoarray specifically coated with anti-p24 and labeled with aunp-antibody conjugates that significantly changes the surface topography of the array, allowing the detection of p24 (fig. 14c) [95] . so far the developed aunps-based assays for hiv are among the most innovative in virus detection and offer a significantly improved detection limit down to attomolar range, which is hundreds of times lower than that of conventional techniques used in hiv detection, including pcr and elisa. therefore, aunps assays can be very beneficial in detecting hiv early, preventing its dissemination, and treatment monitoring. furthermore, simple detection methods such as lateral flow and scanometric assays can be easily incorporated into the point-of-care detection of hiv, which is currently of great interest and more suitable for developing countries. aunps have made a tremendous impact in the detection of viruses, as demonstrated by the numerous assays for most of the clinically relevant groups of human viruses (fig. 3) . coupled with different biomolecules, aunps have enabled the development of a completely new class of probes that are specifically composed of a aunp core (spherical or rod-like in shape) for signal readout and a surface layer of one or multiple types of biomolecules for target recognition and interaction. these probes have mostly been incorporated as a new set of functional and highly interactive components in different traditional techniques (e.g., eias and pcr) and have derived substantial improvements in design, detection performance, sensitivity, specificity and stability. this created a new trend in applying aunps to overcome the limitations of traditional techniques. aunp-based immunological and serological detection methods are the most common and have been accomplished through multiple scanometric, colorimetric, dls, immuno-pcr, electrochemical, electrical, sers, lspr, fluorometric, and afm-based approaches (table 1) . aunp-based dls and lspr assays are especially novel, simple and among the most sensitive reported assays for the immunodetection of viruses with a detection limit that is hundreds of times lower than that of the standard elisa technique [51, 52] . in contrast, the developed aunp-based molecular assays are not as sensitive as the conventional pcr technique or other pcr-based molecular techniques. however, the addition of aunps greatly simplifies the detection procedures of viral nucleic acids, and largely eliminates the need for complex procedures and expensive thermal cycling machines, which greatly reduces the detection time and total cost. in particular, colorimetric assays based on the intense red color of aunps represent an interesting qualitative shift in nucleic acid detection due to the complete elimination magnetic np (mnp)-dna probes scavenge target hiv sequences, which are then magnetically collected and labeled with aunp-dna probes. after repetitive washing, aunps are released into solution by a dehybridization step and subjected to silver metal enhancement, resulting in particle growth and increased scattering [90] . (b) electrochemical impedance assay of hiv rna. graphene oxide sheets decorated with aunps are applied for the immobilization of dna detection probes. hybridization with specific dna sequence results in increased impedance [73] . (c) atomic force microscopy (afm)-based immunoassay of hiv. aunp-antibody probes form a three-component sandwich immunocomplex with the target virus antigen captured on the surface of a specific antibody nanoarray; eventually, the increase in the surface height of the array is detected by afm [95] . of the pcr amplification step and the possibility of direct and rapid detection of the target virus within a few minutes. other assays rely on well-known analytical techniques, such as icp-ms and qcm, which have been introduced to virus nucleic acid detection for the first time through the application of aunps and have achieved detection sensitivity down to femtomolar concentrations [93, 94] . aside from these immunological and molecular assays, aunps have also been described for viral load testing by detecting intact virus particles, which are of special interest as a substitution to traditional cell culture technique [58, 65, 92] . in this context, the small size of aunps probes that is comparable to virus particles, allows rapid, and highly dynamic interaction with viruses for gaining information about infectivity. the application of aunps, especially in biology and medicine, require specific structural and physicochemical characteristics, and any subtle alterations to the intended characteristics can compromise the function and performance of the whole system. for bioanalytic and diagnostic applications, aunps exhibiting homogeneous morphological and fine structural characteristics are important specifically for effective and accurate response to target detection. therefore, many pre-analytical factors related to np synthesis and surface modification are critical to the development of an efficient virus detection assay. the synthesis of aunps commonly relies on methods that basically employ wet chemistry reduction techniques, as presented in other reviews [9, 32] . although these techniques are generally simple and fast and can support the large-scale production of aunps, the synthesis of uniform aunps with a discrete size and shape remains challenging [215] . recently, some approaches have been reported for the controlled synthesis and modification of aunps. however, these methods remain unsuitable either due to the protocol complexity or difficulty of particle separation, which might require additional steps or specific surface modifications. therefore, the development of more stringent and advanced methods for the facile and controlled synthesis of uniform aunps with the option of performing multiple surface modifications is strongly recommended. because of the direct relationship between aunp size and shape and their function as labels, such characteristics are also of inherent importance to both the performance of au nanoprobes and their efficiency for diagnostic applications. for instance, small aunps are preferred in colorimetric and light scattering-based assays due to their spr band sensitivity. in contrast, large aunps are preferentially employed in assays such as bca, immuno-pcr, and enzymatic-based electrochemical detection techniques, which rely on the surface functionalization of aunps with targeting or signaling biomolecules. additionally, a large np size is highly desirable in assays that depend on aunps as metal labels, such as electrochemical stripping, qcm, and icp-ms detection methods. in addition, spherical aunps are more commonly applied than rod-shaped nps. however, the latter exclusively enable a very interesting class of lspr-based detection methods that have shown an unprecedented detection sensitivity range. since aunps by themselves are "blind" with respect to sensing the target, the addition of specific biomolecules to their surfaces is a key step for detecting viruses [37, 38, 40] . unfortunately, most biomolecules have been reported to be very sensitive to harsh chemical modifications that can adversely affect their reactivity and specificity. thus, the type of targeting biomolecule and the surface conjugation reaction method applied are crucial and require optimization. short oligonucleotides and other dna sequences are physically rigid and more stable than protein biotargeting structures, including antibodies, which usually have several secondary and tertiary conformations that are important for their full, specific interaction with a target molecule. furthermore, the full reactivity of surface biomolecules via a suitable conjugation reaction is critical and should be retained and confirmed with additional assessment steps before use in virus detection. while the application of traditional conjugation techniques, such as electrostatic adsorption, is still widely reported, it is strongly recommended to be avoided due to the incident loss of conjugated biomolecule activity and large inequality in loading efficiency, which directly result in decreased assay sensitivity and reproducibility [216] . in this regard, aunps have an enhanced surface area-to-volume ratio that greatly improves the efficacy of current conjugation techniques, and several directional and controlled conjugation methods based on covalent and noncovalent interactions have been thoroughly described in the literature [9, 32, 35] . although aunps are widely employed in various sensing functions and formats, their use for monitoring of virus infection is challenged by long-term stability and reusability of the developed aunp-based sensors. the reusability of sensing platforms has a crucial role in developing applications that are reliable, economic and sustainable. nonetheless, the reusability of aunps in virus detection is limited to electrical and electrochemical assays, in which the main function of aunps is limited to enhancing the performance of the modified electrodes [68, [70] [71] [72] [73] [75] [76] [77] [78] . the long-term stability of aunps is reported and well investigated in numerous studies. however, their conjugates to different biomolecules (i.e., dna, rna, antibody, enzymes) are sensitive to storage time and conditions and the reusability of these conjugates is usually hampered by the loss of activity of surface molecules and irreversible aggregations. it was demonstrated that exposure to elevated temperatures, high salt concentrations, and reducing agents such as dithiothreitol (dtt), mercaptoethanol, and glutathione (commonly existing in biological fluids) results in the loss of stability and activity of aunp-conjugates [9, 41, 43] . thus, the development of new chemistries and novel conjugation strategies for the synthesis of stable aunp-conjugates becomes very crucial for practical use of aunps in virus detection. considering these limitations and challenges, important analytical parameters, such as the use of positive and negative controls, follow-up patient specimens, and internal reference tests, are recommended to confirm the overall accuracy and practicality of developed schemes. a restricted set of measures related to the assay detection sensitivity, specificity, and performance is required for the quality assurance and assessment of these applications [217] . due to the rapid development rate of aunp-based assays, international data analysis measures and standards are also critical to reflect the true assay sensitivity and specificity and to allow the evaluation of their ability and potential to substitute current detection techniques. furthermore, more efforts are needed to test the practicality and generalizability of these assays through clinical sample testing and in-field evaluation of the newly developed aunp-based methods along with the conventional methods using standard evaluation systems and protocols. aunps are a powerful addition to the range of techniques available for virus testing and control. when used as probes, aunps enable simple, rapid, and sensitive detection with an unprecedented multiplexing capability. importantly, aunp-based probes have matured from tools being added to improve traditional techniques, such as pcr, eliza, and many others, to the basis of completely novel techniques developed and introduced for the first time for virus detection. with aunps, virologists can create an unlimited number of detection assays that match the diagnostic needs of each virus in terms of simplicity, speed and cost with sensitivity levels that reach zeptomolar concentrations. aunps can be further developed to become components of more routine techniques, and they will likely engage the interest of scientists from all the disciplines, whether fundamental, environmental, clinical or industrial, in the future. the challenge of emerging and re-emerging infectious diseases globalization and infectious diseases in women globalization, international law, and emerging infectious diseases globalization and infectious diseases globalization of infectious diseases: the impact of migration gold nanoparticles in biomedical applications: recent advances and perspectives gold nanoparticles: opportunities and challenges in nanomedicine biomedical applications of plasmon resonant metal nanoparticles a review on functionalized gold nanoparticles for biosensing applications optical resonator biosensors: molecular diagnostic and nanoparticle detection on an integrated platform ebola virus disease (the killer virus): another threat to humans and bioterrorism: brief review and recent updates insights from clinical research completed during the west africa ebola virus disease epidemic gold aggregating gold: a novel nanoparticle biosensor approach for the direct quantification of hepatitis c virus rna in clinical samples an improved gold nanoparticle probe-based assay for hcv core antigen ultrasensitive detection a highly sensitive electrochemical immunosensor for hepatitis b virus surface antigen detection based on hemin/g-quadruplex horseradish peroxidase-mimicking dnazyme-signal amplification development of lateral flow assay based on size-controlled gold nanoparticles for detection of hepatitis b surface antigen sers detection of hepatitis b virus dna in a temperature-responsive sandwich-hybridization assay cross-species virus transmission and the emergence of new epidemic diseases genetic variability: the key problem in the prevention and therapy of rna-based virus infections factors in the emergence of infectious-diseases updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic in situ self-assembly of gold nanoparticles on hydrophilic and hydrophobic substrates for influenza virus-sensing platform avian influenza virus (h5n1): a threat to human health re-emergence of zika virus: a review on pathogenesis, clinical manifestations, diagnosis, treatment, and prevention diagnostic virology protocols clinical and diagnostic virology high sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18 the brazilian zika virus strain causes birth defects in experimental models molecular diagnosis of influenza diagnostic virology viral serology and detection functionalized gold nanoparticles: synthesis, properties and applications-a review molecular techniques for clinical diagnostic virology molecular diagnosis of medical viruses nanoparticle-mediated systemic delivery of sirna for treatment of cancers and viral infections there's plenty of room at the bottom engineering nanomaterial surfaces for biomedical applications nanostructures in biodiagnostics sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-nanogold, and silver acetate autometallography: detection of single-copy human papillomavirus the use of nanocrystals in biological detection enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity various methods of gold nanoparticles (gnps) conjugation to antibodies chemical synthesis of novel plasmonic nanoparticles a molecular ruler based on plasmon coupling of single gold and silver nanoparticles gold and silver nanoparticles in sensing and imaging: sensitivity of plasmon response to size, shape, and metal composition review: synthesis of fluorescent metallic nanoclusters toward biomedical application: recent progress and present challenges gold nanoparticles for the development of clinical diagnosis methods surface-enhanced raman scattering (sers) detection of multiple viral antigens using magnetic capture of sers-active nanoparticles nanoparticles with raman spectroscopic fingerprints for dna and rna detection detection of hepatitis b surface antigen by target-induced aggregation monitored by dynamic light scattering gold nanorod-based localized surface plasmon resonance biosensor for sensitive detection of hepatitis b virus in buffer, blood serum and plasma one-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles colorimetric detection of dna sequences based on electrostatic interactions with unmodified gold nanoparticles oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for dna analysis by hybridization direct detection of unamplified hepatitis c virus rna using unmodified gold nanoparticles multiplexed colorimetric detection of kaposi's sarcoma associated herpesvirus and bartonella dna using gold and silver nanoparticles a fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin g antibodies in serum and whole blood sizeand distance-dependent nanoparticle surface-energy transfer (nset) method for selective sensing of hepatitis c virus rna a gold nanorods-based fluorescent biosensor for the detection of hepatitis b virus dna based on fluorescence resonance energy transfer multiple homogeneous immunoassays based on a quantum dots-gold nanorods fret nanoplatform detection of swine-origin influenza a (h1n1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor aggregation effects of gold nanoparticles for single-base mismatch detection in influenza a (h1n1) dna sequences using fluorescence and raman measurements hybrid nanocluster plasmonic resonator for immunological detection of hepatitis b virus electrophoresis-enhanced detection of deoxyribonucleic acids on a membrane-based lateral flow strip using avian influenza h5 genetic sequence as the model nanomolecular detection of human influenza virus type a using reverse transcription loop-mediated isothermal amplification assisted with rod-shaped gold nanoparticles gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus dna using disposable microband electrodes ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based dna amplification impedimetric detection of influenza a (h1n1) dna sequence using carbon nanotubes platform and gold nanoparticles amplification electrochemical detection of avian influenza virus h5n1 gene sequence using a dna aptamer immobilized onto a hybrid nanomaterial-modified electrode genosensor for sars virus detection based on gold nanostructured screen-printed carbon electrodes green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance dna hybridization biosensing nanoparticle-based electrochemical detection of hepatitis b virus using stripping chronopotentiometry highly sensitive electrochemical stripping detection of hepatitis b surface antigen based on copper-enhanced gold nanoparticle tags and magnetic nanoparticles gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis b surface antigen in human serum electrochemical immunoassay of hepatitis b surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode catalytic signal amplification of gold nanoparticles combining with conformation-switched hairpin dna probe for hepatitis c virus quantification development of array-based technology for detection of hav using gold-dna probes visual gene diagnosis of hbv and hcv based on nanoparticle probe amplification and silver staining enhancement detection of hiv-1 p24 gag in plasma by a nanoparticle-based bio-barcode-amplification method array-based nano-amplification technique was applied in detection of hepatitis e virus scanometric analysis of dna microarrays using dna intercalator-conjugated gold nanoparticles nanoparticle-based immunoassays for sensitive and early detection of hiv-1 capsid (p24) antigen detection of hepatitis b virus deoxyribonucleic acid based on gold nanoparticle probe chip multiplexed, rapid detection of h5n1 using a pcr-free nanoparticle-based genomic microarray assay label-free and naked eye detection of pna/dna hybridization using enhancement of gold nanoparticles rapid and simultaneous detection of human hepatitis b virus and hepatitis c virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes a lateral flow assay for quantitative detection of amplified hiv-1 rna influenza virus detection with pentabody-activated nanoparticles a method of layer-by-layer gold nanoparticle hybridization in a quartz crystal microbalance dna sensing system used to detect dengue virus gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific rna sequence the use of nanoarrays for highly sensitive and selective detection of human immunodeficiency virus type 1 in plasma gold nanoparticle enhanced immuno-pcr for ultrasensitive detection of hantaan virus nucleocapsid protein two types of nanoparticle-based bio-barcode amplification assays to detect hiv-1 p24 antigen multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using au nanoparticle probes and quantum dots as labels virus taxonomy: classification and nomenclature of viruses fields virology bunyaviruses and the type i interferon system hantaviruses: a global disease problem hantavirus infections: epidemiology and pathogenesis hantavirus infection: a review and global update isolation of the causative agent of hantavirus pulmonary syndrome rift valley fever virus rift valley fever virus(bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention epidemiologic investigations into outbreaks of rift valley fever in humans outbreak of rift valley fever affecting veterinarians and farmers in south africa an assessment of the regional and national socio-economic impacts of the 2007 rift valley fever outbreak in kenya rift valley fever and a new paradigm of research and development for zoonotic disease control supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron ciyomicroscopy coronaviruses post-sars: update on replication and pathogenesis coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus a previously undescribed coronavirus associated with respiratory disease in humans identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia no novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide codehop-based primers isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease filoviruses in bats: current knowledge and future directions marburg and ebola viruses evolutionary history of ebola virus ebola virus outbreaks in africa: past and present efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial. the lancet virus taxonomy: ninth report of the international committee on taxonomy of viruses opinion -architects of assembly: roles of flaviviridae non-structural proteins in virion morphogenesis phylogeny of the genus flavivirus perspectives for the treatment of infections with flaviviridae a brief review on dengue molecular virology, diagnosis, treatment and prevalence in pakistan world health organization. dengue guidelines for diagnosis, treatment, prevention and control: new edition the global distribution and burden of dengue the burden of hepatitis c in the united states estimating the future health burden of chronic hepatitis c and human immunodeficiency virus infections in the united states histological and clinical outcome after liver transplantation for hepatitis c peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection telaprevir for previously untreated chronic hepatitis c virus infection sofosbuvir for previously untreated chronic hepatitis c infection sofosbuvir and ribavirin in hcv genotypes 2 and 3 simeprevir with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients with chronic hepatitis c virus genotype 1 infection (quest-1): a phase 3, randomised, double-blind, placebo-controlled trial quest for a cure for hepatitis c virus: the end is in sight virus taxonomy: classification and nomenclature of viruses (ninth report of the international committee on taxonomy of viruses) hepatitis b virus infection--natural history and clinical consequences natural history and clinical consequences of hepatitis b virus infection world health organization best practice in the treatment of chronic hepatitis b: a summary of the european viral hepatitis educational initiative (evhei) diagnosis and management of chronic hepatitis b in children, young people, and adults: summary of nice guidance virus taxonomy: ninth report of the international committee on taxonomy of viruses hepatitis e virus --an update array-based nano-amplification technique was applied in detection of hepatitis e virus virus taxonomy: ninth report of the international committee on taxonomy of viruses herpesvirus entry: an update herpesviruses remodel host membranes for virus egress genital herpes. the lancet the nine ages of herpes simplex virus an estimate of the global prevalence and incidence of herpes simplex virus type 2 infection epidemiology, clinical presentation, and antibody response to primary infection with herpes simplex virus type 1 and type 2 in young women herpesviruses: latency and reactivation-viral strategies and host response herpes simplex virus 2 infection: molecular association with hiv and novel microbicides to prevent disease hsv-2 vaccine: current status and insight into factors for developing an efficient vaccine immune evasion by herpes simplex virus evasion of early antiviral responses by herpes simplex viruses review of cytomegalovirus seroprevalence and demographic characteristics associated with infection cytomegalovirus: pathogen, paradigm, and puzzle international consensus guidelines on the management of cytomegalovirus in solid organ transplantation neonatal cytomegalovirus blood load and risk of sequelae in symptomatic and asymptomatic congenitally infected newborns is hcmv a tumor promoter? vaccine prevention of maternal cytomegalovirus infection identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma kshv infection and the pathogenesis of kaposi's sarcoma kaposi's sarcoma-associated herpesvirus kaposi's sarcoma-associated herpesvirus infection prior to onset of kaposi's sarcoma detection of kaposi sarcoma associated herpesvirus in peripheral blood of hiv-infected individuals and progression to kaposi's sarcoma. the lancet virus taxonomy: ninth report of the international committee on taxonomy of viruses avian influenza viruses and their implication for human health influenza: lessons from past pandemics, warnings from current incidents the viruses and their replication seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america influenza and rsv: know the diagnostic options world health organization. pandemic h1n1 2009 update 112 cost-benefit analysis of a strategy to vaccinate healthy working adults against influenza burden of influenza-like illness and effectiveness of influenza vaccination among working adults aged 50-64 years antiviral drugs for seasonal influenza efficacy of high-dose versus standard-dose influenza vaccine in older adults acip releases updated recommendations on influenza vaccination to include the 2014-2015 season virus taxonomy: ninth report of the international committee on taxonomy of viruses cross-roads in the classification of papillomaviruses the biology and life-cycle of human papillomaviruses chapter 1: hpv in the etiology of human cancer screening to prevent cervical cancer: guidelines for the management of asymptomatic women with screen detected abnormalities: commonwealth of australia epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update multi-site study of hpv type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in england global burden of human papillomavirus and related diseases efficacy and safety of prophylactic vaccines against cervical hpv infection and diseases among women: a systematic review & meta-analysis safety and persistent immunogenicity of a quadrivalent human papillomavirus types 6, 11, 16, 18 l1 virus-like particle vaccine in preadolescents and adolescents: a randomized controlled trial prophylactic hpv vaccines: prospects for eliminating ano-genital cancer gold nanoparticle mediated method for spatially resolved deposition of dna on nano-gapped interdigitated electrodes, and its application to the detection of the human papillomavirus natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily clinical significance, diagnosis, and treatment of picornavirus infections hepatitis a virus. mandell, douglas, and bennett's principles and practice of infectious diseases hepatitis a acute liver failure: follow-up of paediatric patients in southern brazil centers for disease c, prevention. surveillance for acute viral hepatitis -united states hepatitis a in the era of vaccination virus taxonomy, ninth report of the international committee on taxonomy of viruses historical introduction to the general properties of retroviruses translational control of retroviruses global report: unaids report on the global aids epidemic abc of aids: natural history and management of early hiv infection new paradigms for hiv/aids vaccine development synthetic approaches to metallic nanomaterials aspects of recent development of immunosensors towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative radioimmunossay of australia antigen detecting the aids virus directly with pcr nanoparticle-based electrochemical detection of hepatitis b virus using stripping chronopotentiometry development of array-based technology for detection of hav using gold-dna probes we would like to thank frank fanqing chen the authors have declared that no competing interest exists. key: cord-324054-d71rj29o authors: zhang, xuming; kousoulas, konstantin g.; storz, johannes title: the hemagglutinin/esterase gene of human coronavirus strain oc43: phylogenetic relationships to bovine and murine coronaviruses and influenza c virus date: 1992-01-31 journal: virology doi: 10.1016/0042-6822(92)90089-8 sha: doc_id: 324054 cord_uid: d71rj29o abstract the complete nucleotide sequences of the hemagglutinin/esterase (he) genes of human coronavirus (hcv) strain oc43 and bovine respiratory coronavirus (brcv) strain g95 were determined from single-stranded cdna fragments generated by reverse transcription of virus-specific mrnas and amplified by polymerase chain reaction. an open reading frame of 1272 nucleotides was identified as the putative he gene by homology to the bovine coronavirus he gene. this open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kda. ten potential n-linked glycosylation sites were predicted in the he protein of hcv-oc43 while nine of them were present in brcv-g95. fourteen cysteine residues were conserved in the he proteins of both viruses. two hydrophobic sequences at the n-terminus and the c-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. the predicted he protein of hcv-oc43 was 95% identical to the hes of brcv-g95 and other bovine coronaviruses, and 60% identical to the hes of mouse hepatitis viruses. phylogenetic analysis suggests that the he genes of coronaviruses and influenza c virus have a common ancestral origin, and that bovine coronaviruses and hcv-oc43 are closely related. coronaviruses possess a single-stranded, nonsegmented rna genome of positive polarity (1, 2) and are associated with a variety of diseases in man and animals (3-5). coronaviruses are divided into two major antigenic groups. the first group includes human coronavirus strain oc43 (hcv-oc43), bovine coronavirus (bcv), mouse hepatitis virus (mhv), and hemagglutinating encephalomyelitis virus of swine (hev) (2, 5). hcv-oc43 causes respiratory infection of man similar to those of influenza viruses (6). bcv causes enteritis of newborn calves and is also considered to be an etiological factor of respiratory diseases of calves (7, 8) . mhv can infect different organs, causing enteric, respiratory, and neurological diseases (5, 9) . a unique property of coronaviruses within this antigenic cluster is the presence of the hemagglutinin/esterase (he) gene. the genome of mhv-a59 contains an open reading frame (orf), which may code for an he protein. however, the he is not expressed in infected cells (10, 11, 23 binding) and acetylesterase (receptor-destroying) activities similar to the he (or hef) glycoprotein of influenza c virus (icv) (12) (13) (14) (15) (16) (17) (18) (19) (20) . it was shown that the he glycoprotein of bcv strain quebec induces neutralizing antibodies both in vitro and in viva and thus, is important in viral infectivity (21, 22) . it is evidently not required for viral infectivity in mhv-a59 and mhv-jhm (11). the role of the he gene and its protein in coronavirus evolution, replication, and pathogenesis remains unclear. the exact genomic organization of hcv-oc43 is not known. antigenic and nucleic acid hybridization studies indicate that the hcv-oc43 is closely related to bcv (23-25). by analogy to bcv, the order of the genes coding for the structural proteins probably is 5'-he-s-m-n-3'. recently, the n gene of hcv-oc43 was sequenced, and it was found to be similar to bcv n gene (97.59/o amino acid sequence homology) (26). the origin and evolutionary relationships among the he genes of hemagglutinating coronaviruses isolated from different species are poorly understood. to elucidate the molecular evolution of the coronavirus he genes, we sequenced the he genes of hcv-oc43, a bovine respiratory coronavirus (brcv), a virulent, and an avirulent bcv strains. we report here the complete nucleotide sequence of the he genes of hcv-oc43 and brcv-g95, and their phylogenetic relatedness to bcvs, mhv, and icv. hcv-oc43 was obtained from the american type culture collection (atcc, 759-vr) and propagated in human rectal tumor (hrt-18) cells as described previ-ously (27). a bovine respiratory coronavirus strain giessen 89-4595 (g95) was kindly provided by dr. w. herbst, institute of hygiene and infectious diseases of animals, justus liebig university giessen, germany. this virus was originally isolated from nasal swabs of a calf suffering from respiratory disorder, and propagated in hrt-18 cells. isolation and purification of viral rna, cdna synthesis, double-stranded (ds) cdna amplification and single-stranded (ss) cdna production by polymerase chain reaction (pcr), as well as dna sequencing were performed as described previously (28, 44) . primers were designed to generate cdna fragments from virus-specific mrnas by reverse transcription and pcr amplification, based on the high degree of genomic similarity between hcv-oc43 and bcvs (25, 26). these primers were previously used for amplification and sequencing of bcv s and he genes (28,44). pcrgenerated cdna fragments were directly sequenced in both directions. analysis of the sequences revealed that a large orf of 1272 nucleotides was identical in size to the he genes of bcvs (29, 30, 44) . this orf terminated 14 nucleotides upstream from the s gene (zhang eta/., unpublished data) , and encoded a protein of 424 amino acid residues with an estimated molecular weight of 47.7 kda (figs. 1 and 2). two identical sequences (ctaaac), similar to the consensus intergenie sequence upstream of the hcv-oc43 n gene (ctaaat) (26) and identical to the consensus sequence upstream of bcv he and s genes, were found 16 nt upstream of the predicted initiation codon (at nucleotides 16 to 18) for the he protein and 8 nt downstream from the termination codon, respectively. hydropathic analysis of the predicted amino acid sequence indicated that the putative he protein possessed the characteristics of a membrane protein. specifically, a hydrophobic stretch of 15 amino acids at the n-terminus may serve as signal peptide with a cleavage site between amino acids 15 and 16 (30, 31, 44) . another hydrophobic amino acid sequence near the c-terminus (amino acids 389 to 414) may serve as the transmembrane domain anchoring the protein in the viral envelope. a hydrophilic sequence of 10 amino acids at the c-terminus may serve as an intravirion-domain. ten potential a/-linked glycosylation sites were predicted in the he protein of hcv-oc43 while nine of them were present in that of brcv-g95. two internal orfs were predicted within the large orf extending from nt 107 to 517 and from 976 to 1228. by analogy to bcv he gene, these results suggest that the predicted large orf 2b represents mrna 2-l of hcv-oc43 and brcv-g95, encoding the he glycoprotein. the predicted amino acid sequences of the he genes from hcv-oc43 and brcv-g95 (fig. l) , bcv(29), bcv-l9, bcv-ly138 (44), mhv-a59 (io), mhv-jhm (11) were aligned using the programs of the university of wisconsin genetic computer group software package, version 6.1. the alignment revealed that the he gene of hcv-oc43 was more closely related to brcv-95 and bcvs than to mhvs. nucleotide and amino acid sequences among hcv-oc43, brcv-g95, and bcvs were 95.8 to 96.3% and 94.1 to 94.8% identical, respectively, while the amino acid sequence identity between hcv-oc43 and mhvs was approximately 60%. fourteen cysteine residues were strictly conserved in the he proteins of hcv-oc43, brcv-g95, bcvs, and mhvs. the mhv-jhm had 15 amino acids and two cysteine residues more than hcv-oc43 and brcv-g95. the alignment indicated that the eight he genes among coronaviruses can be divided into two groups. the first group includes hcv-oc43, brcv-g95, and all bcvs, and the second group includes mhv-jhm and mhv-a59. to identify a possible evolutionary pathway for the he gene of coronaviruses, we compared the coronavirus he genes with the icv he gene. an alignment of the predicted amino acid sequences is shown in fig. 2 . in this alignment, the icv he1 subunit shows a sequence identity of approximate 28.2% with the he protein of hcv-oc43, brcv-g95, and bcvs, and 26.3% with the he protein of mhvs. the alignment shows that several regions are completely identical. most importantly, the putative acetylesterase active site (f-g-d-s) (at amino acids 72 to 75 in fig. 2) is conserved in all he proteins of human, bovine, and murine coronaviruses and icv. ten of the 14 cysteine residue positions of hcv-oc43 are conserved among all he proteins compared. these data suggest that these proteins may be evolutionarily related to each other. dna sequences for each gene were optimally aligned based on the alignment of their respective amino acid sequences (fig. 2) . a maximum parsimony analysis was per-formed on the aligned dna sequences to predict possible phylogenetic relationships among coronaviruses (detailed methodology for the phylogenetic, computer-assisted analysis is described in the legend of fig. 3 ). cladistic analysis of the dna sequence data resulted in a single phylogenetic hypothesis (phylogram) with a total length of 1503 steps and a resealed consistency index of 0.962. this analysis suggested that all coronaviruses were divided in two clades. one clade included hcv-oc43, brcv-g95, and bcvs. the other clade consisted of mhv-jhm and mhv-a59. neither coronaviral clade is derived from the other. within the clades, all bcvs were closely related taxa to hcv-oc43, and the mhv-jhm and mhv-a59 were sister taxa. the phylogram sug-g tc ga ctaaactcagtgaaaa tgtttitgctl'cc!xgatltattctagttagctgcataattggtagcitaggttlttacaaccckctaccaatgttg'lltcgc ****** metpheleuleuproarqpheileleuvalsercysileileglyser~uglyphe~r~nprop~o~rasnvalvalserh """""""""""""""""""""""""""""""""""""""""""""""""""""" -ualaphephetrpalaleuarqleu---__""____"_"""""""_""""""""""""""""""""""""""""""-"-""""""""" the sequences for bcv-l9, bcvly138 were obtained from recent work (44). gested a common ancestor of this antigenic group of and assuming icv as outgroup. the highly variable recoronaviruses. the highly cell-adapted strains bcv-gions (positions 19-l 85, 235-365,451-l 046, 1255 -l9, bcv-quebec, and bcv-mebus are closely related 1295 , 1351 -1430 , and 1459 -1503 were excluded to the wild-types bcv-ly138 and brcv-g95. we ex-because they were not aligned with confidence. we cluded these strains in the final phylogenetic analysis, identified 125 phylogenetically informative sites (varibecause their close relationships resulted in collapsed able sites with at least two taxa potentially sharing a branches in the phylogenetic tree. we further at-derived base) from 439 aligned base positions. the tempted to analyze the relationship among the he phylogram shows an almost identical topology for the genes of selected coronaviruses and icv based on coronaviral ingroup obtained by the previous analysis these results using limited dna sequence information, (fig. 3) . (io), and bcv-ly138 (44) and a partial he gene sequence of icv (32) were used for this phylogram. the dna sequences were aligned based on their deduced amino acid sequence alignment as shown in fig. 2 since the hcv-oc43 and icv infect similar tissues in human subjects, the significant sequence homology between the he genes of the two viruses suggests that coinfection of an ancestral coronavirus with icv followed by recombination may have given rise to hcv-oc43. this was also proposed by luytjes et al. (10) . phylogenetic analysis also suggests that the he genes of coronaviruses and icv may originate from a common ancestor. it is worth noting that the he protein of icv contains receptor-binding, acetylesterase and fusion activities while that of coronaviruses contains only the receptor-binding and acetylesterase activities. the fusion function of icv is associated with the /v-terminal hydrophobic region of the he2 subunit of the he protein (17) (18) (19) 32) . a similar hydrophobic domain was not found in the coronavirus he protein. the high similarity between the he proteins of hcv-oc43 and bcvs (95% identity on the average) suggests that both viruses are very closely related. this hypothesis is also supported by the tree branch distance in the phylogenetic analysis shown in fig. 3 . interestingly, the he of hcv-oc43 is more closely related to those of brcvg95 and the wild-type, virulent strain bcv-ly138 than to that of the cell-culture adapted avirulent strain bcv-l9. the wild-type strain bcv-ly138 does not replicate in numerous bovine cells in vitro, but it grows readily in human cells (hrt-18) without requiring prior adaptation (27, 33, 34) . since these polarized human cells retain many features of primary epithelial cells, infection by bcv suggests that bcv may also infect humans, and therefore, it is a zoonotic virus (23, 35). we previously reported a case of human diarrhea caused by bcv-ly 138, in which the virus was identified from the infected patient (35). recently, we found that brcv-g95 exhibited almost identical cytopathology in vitro to the wild-type virulent strains bcv-ly 138 (data not shown). the hcv-oc43, bcvs, and brcv-g95 differed only in few amino acids in the he, and their putative acetylesterase active sites were conserved (see fig. 2 ). 0-acetylneuraminic acid was shown to be the major determinant for icv (36-38). hcv-oc43, bcvs, and brcv-g95 probably recognize this receptor on the surface of many different epithelial cells. they may be able to replicate in epithelial cells of both respiratory and intestinal tracts, and to cross species-barriers causing diseases in heterologous hosts. however, hcv-oc43 primarily causes respiratory diseases and bcvs cause enteritis. the ability of these viruses to replicate in different organs and to cause different clinical symptoms is probably due to multiple amino acid differences occurring within several viral proteins. the s protein of mhv was shown to be important in tissue tropism (39). recently, it was reported that turkey enteric coronavirus is antigenically and genomically closely related to bcvs (40) and similar functions were found in the he protein of hev (20). whether swines or turkeys may also serve as reservoir (mixing-vessel) for coronavirus recombination in nature, as it was proposed for influenza a viruses (41) remains to be elucidated. it is worth noting that icv was also isolated from pigs (42). it will be worthwhile to compare the he genes among these coronaviruses. comparison of the remaining genes with hcv-oc43 and bcvs will provide further insight into their evolution and host cell tropism. proc. nat/. acad. sci. usa proc. nat/. acad. sci. usa key: cord-292940-kg1rl6rb authors: butt, adeel a.; yan, peng title: rates and characteristics of sars‐cov‐2 infection in persons with hepatitis c virus infection date: 2020-10-02 journal: liver int doi: 10.1111/liv.14681 sha: doc_id: 292940 cord_uid: kg1rl6rb background: rate of sars‐cov‐2 infection and impact of liver fibrosis stage upon infection rates in persons with hepatitis c virus (hcv) infection are unknown. methods: we retrospectively analyzed the electronically retrieved cohort of hcv infected veterans (erchives), a well‐established database of hcv infected veterans in care. we excluded those with missing fib‐4 score and those with hiv or hepatitis b virus coinfection. we determined the number of persons tested, proportion who tested positive for sars‐cov‐2, and the infection rate by age and liver fibrosis stage. results: among 172,235 persons with hcv, 14,305 (8.3%) were tested for sars‐cov‐2 infection and 892 (6.2%) tested positive. those with sars‐cov‐2 infection were older, more likely to be black (55.2% vs. 37.8%), obese (body mass index >30kg/m(2) 36.2% vs. 29.7%) and have diabetes or stroke (p<0.0001 for all comparisons). mean fib‐4 scores and proportion of persons with cirrhosis (based on a fib‐4 > 3.25) were similar in both groups. incidence rate/1,000 tested persons was much higher among blacks (88.4; 95% ci 81.1,96.2) vs. whites (37.5; 95% ci 33.1,42.4) but similar among those with cirrhosis (fib‐4>3.25). the rates were also similar among those who were untreated for hcv vs. those treated with or without attaining a sustained virologic response. conclusions: testing rates among persons with hcv are very low. persons with infection are more likely to be black, have a higher body mass index and diabetes or stroke. the degree of liver fibrosis does not appear to have an impact on infection rate. rate of sars-cov-2 infection and impact of liver fibrosis stage upon infection rates in persons with hepatitis c virus (hcv) infection are unknown. we retrospectively analyzed the electronically retrieved cohort of hcv infected veterans (erchives), a well-established database of hcv infected veterans in care. we excluded those with missing fib-4 score and those with hiv or hepatitis b virus coinfection. we determined the number of persons tested, proportion who tested positive for sars-cov-2, and the infection rate by age and liver fibrosis stage. among 172,235 persons with hcv, 14,305 (8.3%) were tested for sars-cov-2 infection and 892 (6.2%) tested positive. those with sars-cov-2 infection were older, more likely to be black (55.2% vs. 37.8%), obese (body mass index >30kg/m 2 36.2% vs. 29.7%) and have diabetes or stroke (p<0.0001 for all comparisons). mean fib-4 scores and proportion of persons with cirrhosis (based on a fib-4 > 3.25) were similar in both groups. incidence rate/1,000 tested persons was much higher among blacks (88.4; 95% ci 81.1,96.2) vs. whites (37.5; 95% ci 33.1,42.4) but similar among those with cirrhosis (fib-4>3.25). the rates were also similar among those who were untreated for hcv vs. those treated with or without attaining a sustained virologic response. testing rates among persons with hcv are very low. persons with infection are more likely to be black, have a higher body mass index and diabetes or stroke. the degree of liver fibrosis does not appear to have an impact on infection rate. this article is protected by copyright. all rights reserved as of august 9, 2020, >20 million persons have been infected and over 730,000 have died of sars-cov-2 infection worldwide. while infection rates appear to have diminished significantly in some countries, the pandemic rages on in others, particularly in the americas. while primarily a respiratory illness, sars-cov-2 may affect multiple organ systems. effects on cardiovascular, 1 renal, 2 gastrointestinal, 3 hepatic, 3,4 endocrine 5 and neurologic systems have been reported. gastrointestinal symptoms may be present in up to 15% of patients and abnormal liver enzymes in up to 36% of the hospitalized patients. 6, 7 the incidence of sars-cov-2 infection among persons with hepatitis c virus (hcv) infection, and its association with degree of liver fibrosis is unknown. whether persons with hcv experience more severe disease or higher mortality is also unknown. we undertook this study to determine infection rate and clinical characteristics of sars-cov-2 infection among persons with hcv infection. we used the electronically retrieved cohort of hcv infected veterans (erchives) for the current study. creation of erchives has been described in numerous previous publications. [8] [9] [10] [11] [12] [13] briefly, all for the current study, we identified all veterans in the erchives database with a positive hcv antibody as well as a positive hcv rna. we excluded those with missing lab values to calculate the fib-4 score as the marker for liver fibrosis stage. we also excluded those with hiv or hepatitis b virus coinfection. comorbidities were defined using established and published definitions. [8] [9] [10] [11] [12] [13] the diagnosis of sars-cov-2 infection was confirmed from the va cdw, where a standard nasopharyngeal swab is tested using rt-pcr to confirm the diagnosis. liver fibrosis stage was calculated using the fib-4 score using an average of two values closest to but before baseline. this article is protected by copyright. all rights reserved we compared the baseline characteristics of all veterans with hcv in the erchives cohort who were diagnosed with sars-cov-2 with those who tested negative for sars-cov-2. we calculated incidence rates per 1,000 persons with 95% confidence intervals by age group, sex, race, liver fibrosis stage and hcv treatment status. we used sas® (version 9.4, sas institute inc., cary, nc, usa) for analyses. the study was approved by the institutional review board at va pittsburgh healthcare system, pittsburgh, pa. were similar among those who were untreated for hcv compared with those treated and attained sustained virologic response and those treated and did not attain a sustained virologic response. we compared the incidence rate of sars-cov-2 infection in our hcv positive group with persons without hcv infection. the latter group consisted of all persons in erchives with a negative hcv antibody test and a negative hcv rna (if done). similar to the group with hcv, we excluded those with hiv and hbv coinfection and retained those with lab data to calculated fib-4 score at baseline. among 347,117 persons without hcv infection, the overall sars-cov-2 incidence rate per 1,000 persons was 3.7 (95% ci 3.5,3.9; p-value = 0.8 compared with hcv+ group). all-cause mortality rate during the follow-up period was 0.3/1,000 persons for both groups (p-value=0.6). to our knowledge, this is the first study describing the testing rate and the incidence of sars-cov-2 infection in a large national population of persons with hcv infection. we also describe factors associated with infection. we found that the persons with sars-cov-2 infection were more likely to be black, have a higher body mass index and have diabetes or stroke. higher rates of infection and poorer clinical outcomes have been described in racial/ethnic minorities and those with comorbidities. more recently, obesity has emerged as an independent risk factor for more severe disease. these associations have not been previously reported in persons with hcv infection. while our current study did not determine the impact of these factors upon mortality or severity of illness, the higher rate of infection coupled with existing literature of their impact upon outcomes should prompt an aggressive testing and monitoring strategy in these persons. several small studies (50-363 patients) have assessed the impact of liver disease and/or cirrhosis upon severity of sars-cov-2 infection and overall mortality. [14] [15] [16] [17] [18] presence of advanced fibrosis as this article is protected by copyright. all rights reserved measured by the fib-4 score, clinical liver disease or cirrhosis are all associated with a higher risk of more severe disease or higher mortality. the cause of liver disease, and more specifically presence of hcv infection were not uniformly reported. furthermore, there are no estimates of testing for sars-cov-2 and infection rates among persons with hcv and the impact of liver fibrosis or cirrhosis upon the testing or infection rates. we found that the degree of liver fibrosis as measured by the noninvasive fib-4 score was similar and a similar proportion had advanced fibrosis or cirrhosis. incidence rate was also similar regardless of the liver fibrosis stage at baseline. additionally, treatment of hcv and attainment of sustained virologic response did not seem to affect the rate of infection. strengths of our study include a large national sample with relatively easy and free access to testing. however, lack of widespread testing limits the generalizability of our findings. some veterans may have been tested outside the va healthcare system which may have skewed the results. effect of various factors upon severity of disease and mortality require further study. this study provides compelling data regarding sars-cov-2 infection in persons with hcv infection with several notable points for clinicians and policy makers. the overall testing rate in persons with hcv is extremely low. when tested, infection appears to disproportionately affect minorities, obese persons and those with certain comorbidities. however, degree of liver fibrosis does not appear to increase the risk of infection. similarly, infection rates are comparable among those who never received hcv treatment and those who received treated with or without attainment of viral eradication. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved outcomes of cardiovascular magnetic resonance imaging in patients recently recovered from coronavirus disease 2019 (covid-19) renal complications in covid-19: a systematic review and metaanalysis gastrointestinal involvement in covid-19: a systematic review and meta-analysis gastrointestinal and hepatic abnormalities in patients with confirmed covid-19: a systematic review and meta-analysis new-onset diabetes in covid-19 correlation between gastrointestinal symptoms and disease severity in patients with covid-19: a systematic review and meta-analysis incidence, risk factors, and prognosis of abnormal liver biochemical tests in covid-19 patients: a systematic review and meta-analysis atorvastatin and fluvastatin are associated with dose-dependent reductions in cirrhosis and hepatocellular carcinoma, among patients with hepatitis c virus: results from erchives incidence and progression of chronic kidney disease after hepatitis c seroconversion: results from erchives the short-term incidence of hepatocellular carcinoma is not increased after hepatitis c treatment with direct-acting antivirals: an erchives study direct-acting antiviral therapy for hcv infection is associated with a reduced risk of cardiovascular disease events hepatitis c virus (hcv) treatment with directly acting agents reduces the risk of incident diabetes: results from electronically retrieved cohort of hcv infected veterans (erchives) treatment of hcv reduces viral hepatitis-associated liver-related mortality in patients: an erchives study high rates of 30-day mortality in patients with cirrhosis and covid-19 elevation of liver fibrosis index fib-4 is associated with poor clinical outcomes in patients with covid-19 comparison of mortality risk in patients with cirrhosis and covid-19 compared with patients with cirrhosis alone and covid-19 alone: multicentre matched cohort pre-existing liver disease is associated with poor outcome in patients with sars cov2 infection; the apcolis study (apasl covid-19 liver injury spectrum study) impact of chronic liver disease on outcomes of key: cord-298033-kzdp9edn authors: domingo, esteban title: quasispecies dynamics in disease prevention and control date: 2019-11-08 journal: virus as populations doi: 10.1016/b978-0-12-816331-3.00008-8 sha: doc_id: 298033 cord_uid: kzdp9edn medical interventions to prevent and treat viral disease constitute evolutionary forces that may modify the genetic composition of viral populations that replicate in an infected host and influence the genomic composition of those viruses that are transmitted and progress at the epidemiological level. given the adaptive potential of viruses in general and the rna viruses in particular, the selection of viral mutants that display some degree of resistance to inhibitors or vaccines is a tangible challenge. mutant selection may jeopardize control of the viral disease. strategies intended to minimize vaccination and treatment failures are proposed and justified based on fundamental features of viral dynamics explained in the preceding chapters. the recommended use of complex, multiepitopic vaccines, and combination therapies as early as possible after initiation of infection falls under the general concept that complexity cannot be combated with simplicity. it also follows that sociopolitical action to interrupt virus replication and spread as soon as possible is as important as scientifically sound treatment designs to control viral disease on a global scale. medical interventions have dramatically increased over the last century, and in the case of infectious diseases, the discovery and development of antibiotics and antiviral agents have represented a very powerful external selective constraint imposed upon replicating microbes. hundreds of antiviral agents have been developed since the second half of the 20th century, and viruses can generally find evolutionary pathways to continue replication in their presence. the same is true of antibiotics and replication of bacteria. the belief that bacterial diseases were on their way toward extinction was quite widespread in the middle of the 20th century. sir m. burnet wrote in his 1966 textbook: "and since bacterial infections are, with unimportant exceptions, amenable to treatment with one or other of the new drugs, our real problems are likely to be concerned with virus diseases" (burnet, 1966) . in 1932, a prominent spanish medical doctor, g. marañ on, declared: "in the year 2000 cancer will be a historical disease. infections will be almost entirely absent as a cause of mortality." (on a personal note, when i joined the university of california, irvine in 1969, to work as postdoctoral student with r.c. warner, i attended some biology courses in which the teachers expressed to students that infectious diseases would disappear in a few decades as a consequence of the use of antibiotics and antiviral agents). predictions in science tend to fail. the optimistic view was not unanimous. a. fleming, the discoverer of penicillin, recognized the adaptive capacity of bacteria and suggested virus as populations https://doi.org/10.1016/b978-0-12-816331-3.00008-8 that bacteria would inevitably find ways of resisting the damage to them caused by antimicrobial drugs [quoted from the document "new antimicrobial drugs" from the european academies science advisory council, november 2014 (www.easac.eu); see also chapter 10]. furthermore, there was early evidence of selection of mycobacterium tuberculosis mutants resistant to streptomycin (mitchison, 1950) . antibiotic resistance in bacteria has similarities and differences with antiviral resistance in viruses, and they are compared in chapter 10. we are now very aware that one of the major problems in antiviral therapy is the nearly systematic selection of drug-resistant virus mutants, which is often associated with treatment failure. other external influences, such as vaccination or immunotherapy, particularly using monoclonal antibodies, can also evoke the selection of viral subpopulations capable of replicating in the presence of those components inherent to an immune response. thus, selective constraints intended to limit rna virus replication meet with the broad and dynamic repertoire of variants ingrained in quasispecies dynamics. two space-time levels of the effects of drugs or vaccines are distinguished in coming sections: (i) short-term consequences for the individual in the form of treatment or vaccination failure and (ii) long-term consequences at the population level in the field, or vaccine-driven evolution of the antigenic properties of viruses. there are other medical interventions that may alter virus survival. individuals who are immunocompromised as a consequence of treatment after organ transplantation or those subjected to anticancer chemotherapy become particularly vulnerable to viral infections. enhanced viral replication can favor pathological manifestations in the affected individual as well as the spread of a large number of viruses into the environment, with consequences for the emergence and reemergence of viral disease (section 7.7 in chapter 7). viral diseases are an important burden for human health and agriculture (bloom and lambert, 2003) . virus evolution, through the basic mechanisms exposed in previous chapters, can influence the two major strategies to combat viral infections: prevention by vaccination and treatment by antiviral inhibitors. for the design of new antiviral vaccines, a critical issue is the diversity displayed in the field by the virus to be controlled. the natural evolution of the virus may result in the circulation of one major antigenic type or the cocirculation of multiple antigenic forms. the vaccine composition (independently of the type of vaccine; see section 8.3.1) must match the antigenic composition of the virus to be controlled. hepatitis a virus (hav) circulates as a single serotype, while foot-and-mouth disease virus (fmdv) circulates as seven serotypes and diverse subtypes, and the antigenic types are unevenly distributed in different geographical locations. a monovalent vaccine made of the prevailing antigenic type of hav should be sufficient to confer protection, while a multivalent vaccine composed of several types or subtypes is required to confer protection against fmdv, and the antigenic composition of the vaccine should match the circulating viruses in each geographical region. this is why antifmd vaccines of different compositions are used in different world areas at a given time, and vaccine composition must be periodically updated to maintain its efficacy. thus, one effect of virus evolution relevant to vaccine design derives from the necessity to prepare a vaccine that mirrors the antigenic composition of the virus to be controlled. in the case of live-attenuated antiviral vaccines, the evolution of the vaccine virus while it replicates in the vaccinee is a risk factor to produce virulent derivatives. the invasion of a susceptible host by a virus and the ensuing viral replication can be regarded as a step-wise process during which the virus must adapt to a series of selective pressures presented by the host, notably the immune response. the outcome can be either viral clearance (elimination of the infection) or virus survival and progression toward an acute or a persistent infection. administration of antiviral agents is an additional selective constraint that limits viral replication. evolutionary mechanisms may either succeed in the selection of mutants resistant to the antiviral agent that will permit the infection to continue or fail in sustaining the infection, resulting in the clearing of the virus from the organism. treatment planning, one of the aims of the new antiviral pharmacological interventions, based on information of viral genomic sequences present in each infected patient, has parallels with vaccine composition design. for vaccines, the information comes from the analyses of antigenic composition of circulating viruses, and for antiviral agents, the information comes from the mutant spectrum composition of the virus to be controlled in the infected patient. world-wide vaccination campaigns made possible the eradication of human smallpox [with the official declaration by the world health organization (who) in 1980] and animal rinderpest [with the official declaration by the world organization for animal health, office international des epizooties (oie) in 2011]. the number of new cases has decreased as a result of vaccination programs against several viral diseases, including measles or hepatitis b (bloom and lambert, 2003) , and substantial progress has been made toward the eradication of poliomyelitis (chumakov and kew, 2010; himman, 2017) . only the deliberate decision not to vaccinate (for religious reasons or misinformation campaigns) or lack of vaccine accessibility (for socioeconomic circumstances) jeopardizes vaccine efficacy. these facts demonstrate that at least some viral diseases can be controlled on a global basis by vaccination, an unprecedented achievement of human and animal health. despite the huge economic investment, however, there are important viral diseases such as acquired immunodeficiency syndrome (aids), hepatitis c, or viral hemorrhagic fevers for which no effective vaccines are available. for some diseases such as human influenza or animal fmd, vaccines are accessible, but they require periodic updating to approximate the antigenic composition of the vaccine to that of the circulating virus (section 8.2). in the case of influenza virus (iv), a major change in antigenic composition can occur through antigenic shift, in which the virus acquires new hemagglutinin and neuraminidase genes by genome segment reassortment (section 7.4 in chapter 7), with the first evidence obtained by g. laver as early as 1971 (for the early history of influenza, its causative virus, and vaccine designs, see beveridge, 1977; kilbourne, 1987) . antigenic variation of viruses, whatever the mechanism might be, can affect vaccine efficacy and in some cases, the extreme rapid intra and interhost evolution of a virus may render an effective vaccine unfeasible at least with the current tools of vaccinology. cd8 þ t cell responses may act soon after infection and promote the selection of escape mutants (bull et al., 2015) . the difficulties for the control of virus disease derived from the adaptive potential of viruses (domingo, 1989; domingo and holland, 1992; bailey et al., 2004; hamelaar et al., 2019) require the judicious application of existing tools and innovative approaches that are still in their infancy. a first basic requisite for the preparation of a vaccine against a viral agent is the understanding 8.3 antiviral vaccines and the adaptive potential of viruses of the immune response evoked by the virus when it infects the organism to be protected (activation of b and t lymphocytes for antibody production, cellular responses, and generation of memory cells) and correlates of protection (bloom and lambert, 2003; van regenmortel, 2012; hagan et al., 2015; cunningham et al., 2016; rolland, 2019) . despite social pressure to rapidly obtain a vaccine, for each virus-host system, well-designed experiments are necessary to try to establish the determinants of protection, which is not a simple issue. the discussions in coming paragraphs are focused on the relevance of virus evolution in vaccine efficacy, irrespective of the type of protection afforded by the vaccine. what we term "protection" may mean the total absence of replication of the infecting virus (termed "sterilizing" immunity) or absence of disease manifestations despite infection and virus replication. as a general initial statement, which is widely accepted by vaccinologists, a vaccine is likely to be effective when it evokes an immune response that is similar to the response elicited by the authentic viral pathogen when it produces disease successfully overcome by the infected organism (evans and kaslow, 1997; bloom and lambert, 2003) . we refer to this as the basic principle of vaccinology. when infection by an antigenically constant virus produces lifelong immunity (i.e., measles virus infection), a vaccine is likely to evoke longlasting protection. in contrast, if a patient cured of a virus can be reinfected by the same (or a closely related) virus (i.e., hepatitis c virus infection) a vaccinedat least one prepared by standard methodologydis unlikely to evoke protection. some points to be considered in the design of antiviral vaccines are listed in box 8.1. they are intended to minimize the selection of vaccineescape mutants and favor the success of vaccination campaigns. some of the recommendations deserve further comment. first, a basic knowledge of virus evolutionary dynamics and how it affects virus antigenic stability (or lack of) is essential. the fact that a methodology is available (i.e., vectors that can express large amounts of based on domingo and holland (1992) . 8 . quasispecies dynamics in disease prevention and control antigens displaying good immunogenicity) does not guarantee vaccine efficacy, and even less if correlates of protection are not understood. the order of efficacy of different vaccine designs proposed in box 8.1 is justified both by the basic principle of vaccinology and by the mechanisms of selection of antibody (ab)-and cytotoxic tcell (ctl)-escape mutants by viruses. single amino acid substitutions at b-and t-cell epitopes in viral proteins are often sufficient to elude neutralization by the corresponding cognatespecific antibody or to escape recognition by a clonal ctl population. for many viruses, the frequency of monoclonal antibody-escape mutants has been measured in 10 à4 to 10 à6 , even in clonal populations obtained under controlled laboratory conditions and that have undergone a limited number of replication rounds (section 7.4.2 in chapter 7). generation of immune-escape variants can result in lack of vaccine efficacy, contribute to viral persistence (pircher et al., 1990; weidt et al., 1995; ciurea et al., 2000 ciurea et al., , 2001 richman et al., 2003; pawlotsky, 2006) , and provoke vaccination-induced virus evolution (section 8.3.2). in human immunodeficiency virus type 1 (hiv-1), antibody-escape variants are incessantly being produced in vivo to the point that virus replication continues despite the antibody response (richman et al., 2003; bailey et al., 2004) . the frequency of selection of mutants that can escape a number (n) of components in which we could hypothetically separate a global immune response is far lower than the frequency of escape to a single (a, b, c, etc.) of the i components of the response. making a simple mathematical abstraction that is applicable also to antiviral-escape mutants (section 8.4), the frequency of mutants that escape n components of an immune response is the product of frequencies of escape to each individual component [10 àa â 10 àb â 10 àc â . . this is an oversimplification because it is not realistic to dissect the selective impact of a complex immune response into discrete components. a virus generally includes multiple antigenic sites and each of them is often composed of several overlapping or nonoverlapping epitopes; in addition, a virus has several t-cell epitopes in different structural and nonstructural proteins, and each epitope displays a different degree of relative dominance. however, the above abstraction reflects the advantage of stimulating the host immune system with a sufficiently broad array of b-and t-cell epitopes to prevent selection of vaccineescape mutants due to a high genetic and phenotypic barrier (compare with the barrier to drug resistance described in section 8.4.2). therefore, selection of vaccine-escape viral mutants is more likely with synthetic peptidic vaccines, than with whole virus-attenuated or inactivated vaccines because the latter present a broad epitope repertoire to the immune system. selection of escape-mutants by peptidic vaccines that evoked partial protection of cattle was documented with fmdv (taboga et al., 1997; tami et al., 2003) . the arguments in favor of multiepitopic presentation are also endorsed by a notorious scarcity of licensed peptidic vaccines for viral diseases despite horrendous economic investments (orders of magnitude greater than investments in quasispecies research!). use of a complex, multiepitopic vaccine, however, need not prevent long-term selection of antigenic virus variants as a result of vaccine usage, an important still largely underexplored topic discussed in section 8.3.2. experimental evolution with fmdv has opened the way to a new generation of antiviral vaccines that share features of attenuated and inactivated vaccines. this new design is based on the conversion of the monopartite fmdv genome into a segmented genome version that dominated the population after extensive high moi passages (section 2.12 in chapter 2 and section 6.6 in chapter 6). for the segmented virus version to be able to produce progeny, the two genome classes (which are encapsidated into separate particles) must reach the same cell. therefore, administration of the segmented virus preparation should lead to an 8.3 antiviral vaccines and the adaptive potential of viruses immune response akin to that evoked by inactivated viral particles, followed by a self-limiting infection. the vaccine was tested successfully in mice and swine, the authentic host of the parental virus (rodriguez-calvo et al., 2010) . potential concerns with this type of vaccine are that the standard (monopartite) genome can be reconstructed by recombination in the early stages of replication in the animal. a safety level is included in that particular fmd vaccine because of multiple mutations that accumulated during the transition toward genome segmentation, which deviated the genome sequence from the one present in the original swine isolate . exploration of evolutionary mechanisms that result in altered forms of viruses may open new possibilities for vaccine design. new prospects for attenuated vaccines have been opened with the engineering of viruses with suboptimal replication fidelity or deoptimized codon or codon pair usage (coleman et al., 2008; vignuzzi et al., 2008; cheng et al., 2015) . altered polymerase copying fidelity often leads to virus attenuation (gnadig et al., 2012; graham et al., 2012; korbouk et al., 2014; rozen-gagnon et al., 2014; van slyke et al., 2015) . a critical issue with live-attenuated vaccines based on deviation from the standard mutation rate is the stability of the attenuation trait. not only true revertants or other site revertants of the polymerase may arise and displace the vaccine virus, but other viral proteins may affect nucleotide incorporation (smith et al., 2013 stapleford et al., 2015; agudo et al., 2016 ). when a virus circulates in a population where vaccinated and unvaccinated host individuals coexist, and the vaccine does not induce sterilizing immunity, viruses with an altered antigenic profile might be selected. the larger the overall effective population size of the circulating virus, and the longer the virus is allowed to replicate in such a scenario, the higher the probability of incorporation of compensatory mutations that yield high-fitness antigenic variants. these events in the case of vaccines used in veterinary medicine are particularly significant because they may alter the cell tropism and host range of viruses, thus increasing the possibilities of their zoonotic transmission into humans (schat and baranowski, 2007) . evidence of vaccination-induced dna and rna virus evolution is increasing, and it has been documented with bovine respiratory syncytial virus, bovine herpesvirus-1, marek's disease virus, porcine circovirus 2, and classical swine fever virus, among others [ (valarcher et al., 2000; muylkens et al., 2006; ji et al., 2014; kekarainen et al., 2014; constans et al., 2015; yoo et al., 2018) ; reviews in gandon et al., 2003; schat and baranowski, 2007) ]. the timing of dominance of ctl-escape mutants of the simian immunodeficiency virus (siv) was influenced by vaccination, and the process could be analyzed by penetration into the mutant spectra of the relevant viral populations (loh et al., 2008) . for human viruses, evidence of vaccineescape mutants has been obtained for hepatitis a and b viruses. vaccination-associated escape mutants of hav with substitutions around the immunodominant site of the virus were identified in a cohort of hiv-1, hav doubly infected individuals (perez-sautu et al., 2011) . the study suggested that an incomplete vaccination schedule, combined with the hiv-1-produced immunosuppression might have contributed to high-hav loads, thus facilitating the generation and dominance of antigenic variants. in taiwan, the prevalence of mutants at a major antigenic determinant of the surface antigen of hepatitis b virus (hbv) tripled in 1 decade, and it has been suggested that this increase of prevalence might be due to the ample vaccination coverage in the region (hsu et al., 1999) . other studies also suggest the circulation of hbv mutants 8. quasispecies dynamics in disease prevention and control associated with vaccine escape and diagnosis failures (di lello et al., 2019) . vaccines can rarely afford protection to all vaccinated individuals due to many factors that include variations in vaccine receptivity factors due to polymorphisms in genes involved in the adaptive immune response, immunosuppression of the vaccine recipient, the insufficient time between vaccination and exposure to the viral pathogen, and antigenic differences between the vaccine strain and circulating viruses. in addition, for massive vaccinations in veterinary medicine, damage to the vaccine (during transport, storage, etc.) and improper administration are additional problems. vaccination may occasionally promote the selection not only of antigenic variants but also host cell tropism, host range, or virulent variants (swayne and kapczynski, 2008; kirkwood, 2010; read et al., 2015; rolland, 2019) . it is not known to what extent the widespread use of vaccination can contribute to antigenic variation relative to other factors (persistence of antibodies from previous infections, genetic drift due to genetic bottlenecks, etc.). however, our current understanding of virus dynamics should encourage investigations on the genetic and antigenic modifications of breakthrough viruses that arise from vaccinated individuals as compared with changes in viruses from unvaccinated host populations. reversion of live-attenuated vaccine viruses into virulent forms is a cause of disease derived from the evolutionary potential of viruses. in the case of attenuated sabin poliovirus vaccine, the rate of vaccine-associated poliomyelitis among those vaccinated for the first time was one per 500,000 to 750,000 vaccinees, and the rate of those receiving the second vaccine dose was about one in 12 million (reviewed in rowlands and minor, 2010) . attenuated antifmd vaccines were used in some countries during the second half of the 20th century, but a reversion to virulence forced the halting of the vaccination programs. vaccine-escape mutants may arise due to ineffective vaccines, and concomitant factors, such as immunosuppression. the escape mutants may remain confined to the unsuccessfully vaccinated host or may spread to other susceptible individuals, and attain different degrees of epidemiological relevance. escape mutants may be direct mutants of the infecting virus or may originate by recombination between the infecting virus and other coinfecting related viruses, as observed with poliovirus and bovine herpesvirus-1 [kew et al., 2002; thiry et al., 2006 ; among other studies]. reiteration of vaccine selection and fitness increase processes over many generations of vaccinees (be it humans or animals) may result in accelerated virus evolution. since systematic use of vaccines for humans and animals in intensive production units is relatively recent in terms of evolutionary time (less than 100 years, and in some cases even only a few decades), it is still premature to evaluate whether vaccination is a significant factor in promoting long-term virus evolution. the first description of virus resistant to an antiviral inhibitor was by j. barrera-oro, h.j. eggers, i. tamm, and colleagues working with enteroviruses and guanidine hydrochloride and 2-(alpha-hydroxybenzyl)-benzimidazole as inhibitors (eggers and tamm, 1961; melnick et al., 1961) . these early results that suggested that antiviral-resistant mutants could be readily selected have been amply confirmed with many viruses and inhibitors in cell culture and in vivo. indeed, the selection of viral mutants resistant to antiviral agents is an extremely frequent occurrence that has been known for decades, although it became widely recognized in the course of development and clinical use of antiretroviral agents to treat hiv-1 infections and aids. the description of drug-escape mutants has been based on three main groups of observations: • detection of antiviral-resistant mutants in patients during treatment. when a reverse genetics system is available, the suspected mutation should be introduced in an infectious clone and resistance ascertained and quantified in cell culture or in vitro enzyme assays. • selection of resistant mutants in cell culture, by subjecting the viruses to passages in the presence of inhibitors. the viral population size is an important variable in this type of experiment (section 8.4.1). • calculation of the frequency of resistant mutants by plating a virus in the absence and presence of the antiviral agent similar to the assays to calculate the frequency of monoclonal antibody (mab)-resistant mutants (described in chapter 7, section 7.4.2). in the three groups of observations, the frequency at which a specific escape mutant is found depends on a number of barriers to resistance (section 8.4.2). traditionally, the fact that a drug can select virus-resistant mutants is regarded as a proof of the selectivity of the drug, as opposed to unspecific or toxic effects on the host cell that indirectly impair virus replication (herrmann and herrmann, 1977; golan and tashjian, 2011) . selection of viral mutants resistant to antiviral inhibitors is a major problem for the control of viral disease for two main reasons: (i) because it often results in virus breakthrough (increase of viral load) resulting in treatment failure and (ii) because resistant virus variants may become epidemiologically relevant, with the consequent decrease of inhibitor efficacy at the population level (domingo and holland, 1992; huang et al., 2019) . increasing numbers of antiviral agents have been developed based on the three-dimensional structure of viral proteins and their complexes with natural and synthetic ligands, in efforts that have engaged academic institutions and pharmaceutical companies. others have been incorporated as a result of drug repositioning, that is, the discovery of antiviral activity of compounds licensed for other medical purposes, often with the help of computational tools (ab ghani et al., 2019) . antiviral agents may target viral or cellular proteins involved in any step of the virus life cycle. they may interact with virions and inhibit an early step of infection, such as the attachment to the host cell, penetration into the cell, or uncoating to liberate the genetic material of the virus inside the cell. other agents interfere with the synthesis of viral nucleic acids or viral protein processing, particle assembly, or virus release from cells. selection of resistant mutants has been described for virtually any chemical type of antiviral agent directed to any step of the infectious cycle of dna or rna viruses, including important pathogens, such as herpesviruses, picornaviruses, iv, hbv, and hepatitis c virus (hcv). several reviews and articles have covered the theoretical basis of drug resistance, and consequences for treatment management [as examples see (domingo et al., 2001b) and previous versions in progress in drug research (richman, 1994 (richman, , 1996 ribeiro and bonhoeffer, 2000; domingo et al., 2001a domingo et al., , 2012 menendez-arias, 2013; perales, 2018; mokaya et al., 2018; nitta et al., 2019; pawlotsky, 2019) , and the articles in the current opinion of virology volume edited by l. menendez-arias and d. richman (menendez-arias and richman, 2014) ]. therefore, the general mechanisms that confer adaptability to viruses are very effective in finding drug-escape pathways through molecular mechanisms that are summarized in section 8.5. considering the implications of quasispecies dynamics explained in previous chapters, the 8 . quasispecies dynamics in disease prevention and control following statement will be obvious to the reader: "if a single mutation is able to confer resistance to an antiviral agent, and the mutation does not cause a significant selective disadvantage to the virus (fitness decrease) in the considered environment, a drug-resistant virus mutant will be present in most, if not all, virus populations" (domingo, 1989) . if a virus replicates in such a way that a population size of 10 4 can never be achieved in a single population, it is extremely unlikely that any drug-resistance mutation (or any mutation associated with a phenotypic change) that is generated at a frequency of 10 à4 or lower will be propagated from that viral population (perales et al., 2011) . selection of escape mutants depends on the replicative load, and the concentration of inhibitor attained at the sites of virus replication. consider different cell or tissue compartments in which an antiviral inhibitor reaches different concentrations (exerts different intensity of selection) ( fig. 8.1 ). in each compartment, there are multiple replication complexes. a mutation conferring resistance to the inhibitor will occur at the same rate in each of them, assuming that the mutation rate is independent of the presence of the inhibitor. however, after its occurrence, the proportion of viral rnas harboring the mutation will decrease depending on the inhibitor concentration. the time at which the effect of the inhibitor will be manifested depends on the inhibitor target. in the example of fig. 8 .1 we assume that the concentration of inhibitorresistant mutants will decrease in the replication complexes, reaching resistant mutant frequencies of 10 à3 , 10 à4 , and 10 à5 in compartments 1, 2, and 3, respectively. the frequency of inhibitor-resistant mutants in the entire cell, tissue, or organism at that time will be given by the weighted average of mutation frequencies at the individual compartments. in the case of a virus-producing viremia, assuming no bottleneck effects or differential selection for other figure 8 .1 frequency of a drug-resistant mutant in different compartments (subcellular site, cell, tissue, or organ) of the same organism. three compartments labeled 1, 2, and three are drawn. replication complexes are depicted as ellipses and replicating genomes as lines. a replicative unit is defined here as a set of replication complexes. the inhibitor-resistant mutant is represented by a red circle in a genome. compartments 1, 2, and 3 reach increasing concentration of the inhibitor, rendering inhibitor-resistant mutant frequencies of 10 à3 , 10 à4 , and 10 à5 , respectively. see text for the difference between occurrence and presence of the resistant mutant, and implications of compartmentalization. 8.4 resistance to antiviral inhibitors traits, the frequency of resistant mutants calculated for the virus in blood should reflect the average frequency in all compartments that supply virus to blood. low inhibitor concentration in a compartment will favor the selection of the resistant mutant that can either be archived as an adaptive reservoir or penetrate other compartments, depending on the sequence of events of virus spread. if two or more independent mutations can confer resistance to an inhibitor, the probability of occurrence of an inhibitor-resistant mutant is equal to the sum of probabilities of occurrence of each mutation. for multiple mutations, the probability will be the sum of probabilities of the different mutations, a frequent case in viruses since they often display several evolutionary pathways to drug resistance. the probability of finding a viral genome resistant to two or more inhibitors directed to different targets is given by the product of probabilities of resistance to each of the individual inhibitors. the basic probability considerations regarding the frequency of occurrence of inhibitor-resistant viral mutants are summarized in box 8.2. when two or more mutations occur in the same genome, they may be subjected to epistatic effects, meaning either increase (positive epistasis) or decrease (negative epistasis) of viral fitness (see section 2.3 of chapter 2 for the concept of epistasis). the diversity of chemical structures of the antiviral compounds that can select for escape mutants is illustrated in figs. 8.2 and 8.3 with the formulae of some antiviral agents in current or historical use. they include relatively simple organic molecules, nucleoside analogs, and complex heterocyclic compounds with a variety of residues (ch 3 -, c═ o, nh, nh 2 , f, and cl) that may contribute to interactions with viral proteins or alter the electronic structure of neighbor bonds thus modifying the interaction behavior of some atoms. for all of them, resistant viral mutants have been identified, despite barriers imposed upon the virus to reach a drug-resistance phenotype. • if there are two or more different mutations that produce the same inhibitor-resistance phenotype, and once one of the mutations is present additional mutations are no longer necessary to produce the phenotype, the probability of achieving the phenotypic change is equal to the sum of probabilities of finding each mutation individually. • if two or more independent mutations must happen to produce resistance to an inhibitor, the probability of occurrence of the necessary mutations is equal to the product of probabilities of occurrence of each mutation individually. • if a virus is inhibited by an inhibitor combination, and the mutations that confer resistance to each inhibitor are independent (no cross-resistance is involved), the probability of a combination-resistant mutant to arise is equal to the product of probabilities of resistance to the individual mutations. these probability calculations are applicable to other mutation-dependent virus variations. the impediments for a virus to attain resistance to an inhibitor are divided into genetic, phenotypic, and mutant swarm (population) barriers to resistance (box 8.3). the genetic barrier to resistance to a specific inhibitor is not a universal value for a virus group, since it may be affected by genetic differences among natural viral isolates. the diversification of hcv into genotypes 1a and 1b influenced the genetic barrier to resistance to the ns3/4a protease inhibitor telaprevir [formula (20) in fig. 8 .3]. one of the amino acid substitutions that confer resistance to telaprevir is r155k in ns3. in genotype 1a, the triplet encoding r155 is aga; therefore, a single nucleotide transition g / a can yield the triplet aaa, which encodes k. in genotype 2b, the triplet encoding r-155 is cga; therefore, two nucleotide changes (transversion c / a and transition g / a) are required to reach aaa, the triplet encoding k. reaching the alternative aag codon for k would require the same or a larger number of mutations (see section 4.3.1 in chapter 4 for another example of how the synonymous codon usage can influence an 7) rimantadine, (a-methyl-1-adamantane methylamine). (8) oseltamivir (trade name tamiflu), ethyl (3r, 4r, 5s)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate. (9) zanamivir (trade name relenza), (2r, 3r, 4s)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1r, 2r)-1,2,3-trihydroxypropyl)-3,4-dihydro-2h-pyran-6-carboxylic acid. 8.4 resistance to antiviral inhibitors figure 8.3 some inhibitors of human immunodeficiency virus type 1 (antiretroviral agents) and hepatitis c virus. the inhibitors are (10) zidovudine (azt), 1-[(2r, 4s, 5s)-4-azido-5(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione. (11) zalcitabine (ddc), 4-amino-1-((2r, 5s)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1h)-one. (12) 2r,3r ,4r,5r)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4methyl-tetrahydrofuran-2-yl]methoxy-phenoxy-phosphoryl]amino]propanoate. additional drugs currently used in antiviral therapy can be found in the references quoted in the text and in chapter 9. 8 . quasispecies dynamics in disease prevention and control evolutionary outcome). the requirement of transitions versus transversions to reach the resistant phenotype will affect the genetic barrier to resistance. most viral polymerases tend to produce transition mutations more readily than transversions presumably because in the course of rna elongation it is easier to misincorporate a purine by another purine than by a pyrimidine, and the same for pyrimidine misincorporations (chapter 2). mutation preference is one of several factors that determine the frequency of drug-escape mutants. thus, evolution may diversify viruses to display different genetic barriers to the same drugs. since in many cases, several independent mutations may confer resistance to the same drug to complicate matters even more, it also has to be considered that the genetic barrier to one inhibitor may be affected by the presence of other inhibitors (beerenwinkel et al., 2005) . the phenotypic barrier to drug resistance is equivalent to the fitness cost inflicted upon the virus by the mutations and corresponding amino acid substitution(s) required for resistance [fitness cost is treated in chapter 4 (section 4.6) and in chapter 7 (section 7.4.2) in connection with the frequency of monoclonal antibody-or cytotoxic t-cell-escape mutants in viral populations]. when a drug-resistance mutation inflicts a high fitness cost, a likely result is a reversion of the mutation when the virus replicates in the absence of the drug. an alternative outcome is that compensatory mutations are introduced in the genome so that viral fitness increases while maintaining the inhibitor-resistance mutation. the two outcomes are not mutually exclusive and may contribute to the multiple, transient selection pathways observed by the application of deep sequencing to monitor the response of a viral population to specific selective force (tsibris et al., 2009; fischer et al., 2010; cale et al., 2011; kortenhoeven et al., 2015) (see section 6.3 in chapter 6). a high fitness cost may prevent or delay the selection of escape mutants. sofosbuvir [formula (21) in fig. 8 .3] is a very effective ns5b (viral polymerase) inhibitor of hcv. amino acid substitution s282t in ns5b has been associated with sofosbuvir resistance, and the substitution has been detected in patients and in some natural isolates of hcv. in one of several clinical studies on sofosbuvir efficacy, the mutant spectrum composition of hcv genotype 2b in an infected patient treated with the drug was followed by deep sequencing of the virus at baseline (prior to initiation of treatment), in the course of treatment, and posttreatment. the frequency of s282t was 0.05% at baseline, indicating preexistence of resistance mutations despite no exposure of the virus to the drug (section 8.6). two days after initiation of sofosbuvir treatment, the level of s282t decreased to 0.03%, and viral breakthrough was detected 4 weeks later when 99.8% of the viral population included s282t. during the posttreatment period, genomes with the wild-type s282 amino acid regained dominance that was attributed to the true reversion of mutant genomes rather than the outgrowth of baseline wild-type genomes (hedskog et al., 2015) . this result suggests a high phenotypic barrier for sofosbuvir, and that hcv has mechanisms to overcome the barrier. the complexities of virus-host interactions render the elucidation of the pathways exploited by a virus to overcome the phenotypic barrier to a drug a highly empirical endeavor. the hope is that a combination of inhibitors that display a high barrier to resistance may impede escape and drive the virus to extinction (chapter 9). the mutant swarm barrier to resistance is a consequence of the interfering interactions that operate within quasispecies, and that are described in chapter 3 (section 3.8). it is a particular case of interference that can delay or impede the increase of frequency of a resistance mutation (crowder and kirkegaard, 2005; kirkegaard et al., 2016) . the possible contribution of mutant swarms to facilitate or prevent the dominance of drug-resistant mutants in infected patients is still largely unexplored. it is difficult to anticipate how the three types of barrier listed in box 8.3 may result in a level of drug resistance for a particular virus, in a particular host individual, in a specific target organ, at a given time. additional influences are drug pharmacokinetics, drug penetration into different cells, tissues, and organs where the virus replicates (see fig. 8 .1), and prior history of virus replication in the infected host. it is not surprising that the study of drug resistance in viruses remains fundamentally descriptive. when independent amino acid substitutions can lead to resistance to the same drug, alternative evolutionary pathways may be followed depending on trna abundances, mutational preferences, and relative nucleotide substrate concentrations at the virus replication sites. if resistance requires two or more amino acid substitutions, the genetic barrier will be correspondingly increased (section 8.4.1 and box 8.2). quantification of barriers to resistance in experiments in cell culture requires a prior characterization of the drug to be tested when acting on the cell culture-adapted virus as it infects a specific cell line. the two basic parameters to be determined are the toxicity of the drug for the host cell, and its capacity to inhibit the production of infectious virus. toxicity is quantified by the concentration of drug that kills a given percentage (generally 50%, but sometimes another value) of cells under the conditions used in the infection. it is expressed as the cytotoxic concentration 50 (cc 50 ), as depicted in fig. 8 .4. toxicity may depend on the cell concentration, the extent of confluence in a cell monolayer, and the metabolic state of the cell (resting vs. actively dividing). the capacity of inhibition is quantified by the concentration of inhibitor that reduces the infectious progeny production by a given percentage (generally 50%, but sometimes another value) under the defined conditions of the infection, including a multiplicity of infection (moi). it is expressed as the inhibitory concentration 50 (ic 50 ), as depicted in fig. 8 .4. the therapeutic index (ti) is given by the quotient cc 50 /ic 50 , and although generally used for in vivo experiments of drug efficacy testing, it can also be applied to cell culture measurements. the three parameters, cc 50 , ic 50, and ti, are not universal for a virus and a drug since they may be influenced by the composition of the viral population and environmental factors, as repeatedly expressed for other features of viruses in the present book. as a guide, ti values of 100 or more suggest the excellent performance of an antiviral agent; values higher than ten are acceptable, but values lower than ten predict limited efficacy. the quantitative effects of a drug may vary when analyzing a single round of infection versus multiple rounds in serial passages, or when comparing in vivo versus cell culture experiments. cc 50 and ic 50 values serve as a guide to decide the range of the drug concentration to be used in serial passage experiments to evaluate the possible selection of inhibitor-resistant mutants and to estimate the genetic barrier. the possibility to overcome a genetic barrier depends on the virus population size. for viruses that replicate in cell culture, it is possible to estimate the minimal viral population size needed to select a drug-resistant mutant which is generally positively correlated with the genetic barrier ( fig. 8.5 ). in the hypothetical example of the figure, a viral population is composed of inhibitor-sensitive viruses (blue spheres), and a low level of inhibitor-resistant viruses (red spheres). the proportion of inhibitorresistant viruses is given by the mutational pressure (e.g., at a frequency of 10 à4 , which is increased in the picture for clarity). passage of a small amount of virus (e.g., 10 2 infectious virus in the small circle at the upper part of the figure) will exclude the mutant virus (red spheres) that will be maintained at the basal level dictated by mutational pressure in the course of passages (limited to two in the figure for simplicity). selection of escape mutants is precluded by the limited population size at each transfer. in contrast, if the population size used for the successive infections is sufficiently large (>10 4 , larger circles at the bottom that surround both sensitive and resistant viruses), the resistant mutant can become figure 8.4 schematic representation of two experiments to determine the concentration of an inhibitor needed to kill 50% of cells in culture (cc 50 value, left) and the concentration of inhibitor that reduces the viral production to 50% (ic 50 value, right). the therapeutic index is the quotient between cc 50 and ic 50 (box at the bottom). similar tests can be performed with tissue explants or animals, under controlled environmental conditions. see text for pharmacological implications. 8.4 resistance to antiviral inhibitors gradually dominant and can be isolated for further studies. to give another example, a single amino acid replacement that requires two mutations (the change from cag to aug to attain substitution q151m in hiv-1 reverse transcriptase, associated with resistance to multiple antiretroviral nucleosides) will occur at a lower frequency than replacements that require a single mutation. if each of the two mutations reaches a frequency of 2 â 10 à4 , the expected frequency of the drug-resistant genomes (ignoring fitness effects) will be (2 â 10 à4 ) â (2 â 10 à4 ) ¼ 4 â 10 à8 . thus, at least 4 â 10 8 viral genomes must undergo one round of copying (or a lower number of genomes a proportionally higher number of rounds of copying) to approach a good probability to obtain a drug-resistant genome in that viral population. population size limitation of a drug selection event is a specific example of how random events may intervene in the process of positive selection (compare with section 3.2 and fig. 3 .2 in chapter 3; in that figure, the random event that excludes the positively selected population is conceptually equivalent to the insufficient population size depicted in the upper infection series of fig. 8 .5). when two or more mutations are needed to confer the resistance phenotype, drug resistance will be less likely not only due to the lower probability of generating the two required mutations but also because of the increased chances of two mutations in the same genome entailing a fitness cost. a virus that requires three or more mutations to overcome a selective constraint may occur at a frequency in the range of 10 à 12 or lower which will often be insufficient for the mutant to be present in the mutant spectrum of the infected host ( fig. 8.6 ). failure to select for a drug-resistant mutant in cell culture does not necessarily mean that the resistant mutant is not present in the population. it may mean that due to a high genetic barrier, the selection experiment was designed to infect with an insufficient amount of virus. similar and even more accentuated problems are encountered in selection experiments in vivo, since not only the phenotypic barrier or fitness cost inflicted by a drug-resistance mutation (box 8.3) is often estimated empirically from the frequency of the relevant substitution in patients treated with the drug or in cell culture assays. an adequate procedure to quantify the phenotypic barrier to resistance is to determine the fitness of the virus expressing the protein with the wild-type amino acid (the one that confers drug sensitivity) relative to the virus expressing the protein with the substituted amino acid that confers resistance; fitness is measured in the absence and presence of the drug (double assay). this is an extension of the determination of fitness vectors described in section 5.1.1 of chapter 5, as depicted in fig. 8.7 ; the assays are best performed in cell culture, although the use of explants or in vivo assays is also feasible. two parameters can be calculated: the fitness cost inflicted by the amino acid substitution associated with resistance, in the absence of the drug (reflected in a fitness value f à drug 1 relative to the wild type), and the selective advantage conferred by the substitution in the presence of the drug (reflected in a fitness value f þ drug > 1 relative to the wild type). the lower the value of f à drug , the higher the fitness cost. we define the selective strength of the resistance mutation as f þ drug /f à drug . for example, if we put arbitrary numbers (unrelated to values shown in ordinate) to the fitness values in the first graph of fig. 8 .7, figure 8.6 decreased frequency and fitness of mutant genomes resistant to one, two, or three inhibitors. the genome frequency level decreases by several orders of magnitude when resistance to one inhibitor (red asterisk in the upper mutant spectrum), or two inhibitors (red and green asterisks in the middle-mutant spectrum), or three inhibitors (red, green, and blue asterisk in the bottom mutant spectrum) must occur in the same genome (left part of the figure and numerical values at the center). increased number of mutations generally implies fitness decrease (right part of the figure) . see text for implications. 8.4 resistance to antiviral inhibitors f þ drug ¼ 1.4 and f à drug ¼ 0.8, we obtain a selective strength of 1.7. for the vectors in the second graph, if f þ drug ¼ 3.6 and f à drug ¼ 0.1, the selective strength is 36. high selective strength means an important selective advantage conferred by the amino acid substitution for virus replication in the presence of the drug despite a high fitness cost inflicted by the substitution (compare with section 4.2 in chapter 4 for the trade-off and "no free lunch" concepts). if the substitution does not entail any fitness cost (f à drug ¼ 1), the fitness value in the presence of the drug equals the selective strength. selective strength can be calculated for a mutation or group of mutations that confer resistance to a drug used at a given concentration in a defined environment [example in (de la higuera et al., 2017) ]. the limitations of fitness measurements (environment dependence, etc.) described in section 5.1.2 of chapter 5 apply here. since viral genomic sequences may vary in the course of fitness assays, a limited number of passages and triplicate parallel assays are recommended. if a substitution entails a high fitness cost, direct reversion of the substitution or incorporation of compensatory mutations may occur. nucleotide sequence monitoring in the course of the assay should reinforce the conclusions. most drug-resistance mutations inflict a fitness cost upon the virus and yet very rarely drug resistance represents an unsurmountable barrier to maintain viral infectivity. several possibilities can account for the pertinacious occurrence and selection of drug-resistant, viable viral mutants. one possibility, supported by some experimental and clinical observations, is that a drug-resistant phenotype may be achieved through a number of alternative genetic modifications. even if a specific amino acid substitutiondthat would serve as the most direct and effective determinant of drug resistancedwere highly detrimental or lethal for the virus, alternative mutations can often be found that lead to a similar resistance phenotype, or at least a sufficient resistance to permit virus replication and exploration of sequence space to find compensatory mutations. the connectivity among points of sequence space and the fact that several space positions map into the same (or similar) drug-resistance phenotype contribute to the extended occurrence of drug resistance. this phenotypic redundancy applies to both standard nonmutagenic inhibitors and to mutagenic inhibitors. the cascade of mutations that confer resistance of picornaviruses to the mutagenic purine analog ribavirin illustrates how alternative amino acid substitutions in the viral polymerase (some being genuine resistance mutations and others acting as compensatory substitutions to maintain polymerase function) can lead to the ribavirin-resistance phenotype (discussed in chapter 9). a speculative interpretation of the systematic occurrence of drug-resistant viral mutants is that the majority of the chemicals used in antiviral therapy (figs. 8.2 and 8.3) have a structure which may be related to natural compounds that viruses and their ancestral replicative machineries encountered in their continuous struggle to survive. in this view, drug resistance would have been gradually built as a consequence of coevolution (section 4.5 of chapter 4) between virus replicative and gene expression machineries and the "space" of chemical compounds that interacted with them. mechanisms of drug resistance might have had their roots in molecular events repeatedly experienced as viruses evolved in an interactive manner with protocellular and cellular metabolites in our biosphere. discrimination in favor of small molecule substrates compatible with a flow of genome replication and gene expression and avoidance of perturbing intruders that could alter catalytic activities should have been positively selected. unfortunately, this is a possibility we will never be able to test. whatever the reasons behind, the unfortunate reality is that drug resistance is an extremely frequent event that complicates enormously the control of the viral disease. multiple drug evasion mechanisms have been identified or proposed for rna and dna viruses, and they can operate depending on basic replicative parameters in connection with drug levels and their variation with time. delayed lysis of bacteriophage vx174 that reduced the replication rounds in the presence of 5-fluorouracil (fu) produced resistance to this analog (pereira-g omez and sanju an, 2014). in this line, synchronization of a virus life cycle so that replication occurs when drug levels are minimal has been proposed as a potential resistance mechanism (neagu et al., 2018) . virus plasticity favors modification of life cycle parameters for virus survival in the presence of drugs, provided time for the relevant selection events is allowed once the treatment has been implemented. the great majority of inhibitor-resistance mechanisms involve amino acid substitutions in viral proteins that directly or indirectly diminish the binding of the drug to the viral target. the following major mechanisms have been documented: • substitutions in the protein targeted by the drug that decreases the affinity of the protein for the drug. mutations that modify nucleotide selectivity in viral polymerases belong to this group. substitutions may affect the neighborhood of the polymerase catalytic site, other polymerase domains, or even nonstructural proteins that interact with the polymerase. • if the inhibitor acts on a viral protein that itself has some other viral protein or genomic structure as a target, amino acid substitutions, or mutations that affect that target may also contribute to resistance. correlated mutations 8.5 molecular mechanisms of antiviral resistance in the first and second target may also yield the resistance phenotype. this is the case of some protease inhibitor-resistance mutations in hiv-1. • in the case of viral polymerases, some mutations permit the excision of a chainterminating nucleotide at the 3 0 -end of the primer. it is achieved through phosphorolysis mediated by a pyrophosphate donor, probably atp. resistance to nucleoside/nucleotide reverse transcriptase (rt) inhibitors (nrtis) is achieved by one of at least two mechanisms: (i) discrimination against the incorporation of the triphosphate form of the nrti and (ii) excision of the chain-terminating nucleotide once incorporated at the 3 0 -end of the growing dna chain. this occurs with thymidine analog resistance mutation (or tams) that are typically selected under treatment with azt or d4t [formulae (10) and (13) in fig. 8.3 ]. groups of amino acid substitutions may yield a multidrugresistance phenotype. a well-studied example in hiv-1 is the q151m complex in the reverse transcriptase, which includes substitutions a62v, v75i, f77l, f116y, and q151m. the phenotype consists of the limitation of incorporation of several nucleotide analogs. these and other mutants are characterized by a decrease in the catalytic rate constant (k pol ) of incorporation of the analog or analogs relative to standard nucleotides (see section 2.6 in chapter 2 for the basic kinetic parameters for polymerase activity). the combination of enzymological and structural studies has provided a molecular interpretation of the mechanism of inhibition of hiv-1 by nrtis (reviews in men endez-arias, 2013; men endez-arias and alvarez, 2014; clutter et al., 2016; g€ unthard et al., 2019) ; for a predictive model that includes nucleotide levels, see von kleist et al. (2012) . nonnucleoside rt inhibitors (nnrtis) bind to an rt pocket 10å away (a considerable distance) from the catalytic site composed of residues of the p66 and p51 subunits of the enzyme. the hydrophobic nature of nnrtis is illustrated in fig. 8.3 with the structures of nevirapine, efavirenz, and delavirdine [formulae (15), (16), and (17), respectively]. several mechanisms have been proposed to explain their inhibitory activity, including alteration of the catalytic amino acids ymdd at the rt active site, distortion of the nucleotidebinding site, or modification of the position of the primer that receives the incoming nucleotides (men endez-arias, 2013). amino acid substitutions that confer resistance to nnrtis block the access of the inhibitors to their binding sites or alter the conformation and volume of the binding pocket. the essential viral proteases are a target for the development of specific antiviral inhibitors. examples are saquinavir and ritonavir for the hiv-1 [formulae (18) and (19), respectively, in fig. 8.3 ]. multiple resistance mutations have been described for protease inhibitors. they can affect the substrate-binding site or neighbor positions, often accompanied of compensatory substitutions at distant positions including sites of the target viral protein [e.g., the gag cleavage sites in hiv-1 (fun et al., 2012; flynn et al., 2015) ]. some hiv-1 protease inhibitor combinations display a high genetic barrier to resistance but, significantly for the capacity of viruses to explore sequence space, resistant mutants with 20e30 amino acid substitutions in the proteasecoding region have been isolated (rhee et al., 2010) . the reviews by men endez-arias (2013) and men endez-arias and alvarez (2014) provide excellent and detailed accounts of mechanisms of resistance of hiv-1 and hiv-2 to inhibitors that include virus entry and integrase inhibitors, in addition to the rt and protease inhibitor briefly described here. 8 . quasispecies dynamics in disease prevention and control because of the distance effects that can be exerted among amino acids on the structure of proteins, substitutions that confer resistance to inhibitors of viral enzymes may lie far from the catalytic site. the effect of drug-resistance mutations on the general catalytic efficiency of a viral enzyme is one of the determinants of the functional barrier to resistance, since it may diminish replicative fitness. when an enzyme activity assay in vitro is available, the effects of specific drug-resistance amino acid substitutions on enzyme activity can be tested, although the observed alteration may not be the only influence on the fitness modification of the corresponding mutant virus. the reason is that most viral proteins (including viral enzymes) are multifunctional, and an enzyme activity assay may not capture the range of influences exerted by the enzyme. regarding direct-acting antiviral (daa) agents for hcv, the inhibitors that target the hcv polymerase (ns5b) generally display a higher functional barrier to resistance than the protease inhibitors and manifest a broader genotype coverage. fitness decreases entail reductions in viral load and, consequently, lower probability of viral breakthrough (treatment failure). nucleotide analogs that bind to conserved residues at or near the active site of the viral polymerase tend to show subtypeindependent antiviral activity. because amino acid substitutions at or near the active site of viral enzymes are likely to inflict a fitness cost, such substitutions may not preexist in treatment-naive patients (margeridon-thermet and shafer, 2010; sarrazin and zeuzem, 2010) . interestingly, in the case of hiv-1, mutations conferring resistance to rt inhibitors inflict a lower fitness cost than mutations that confer resistance to protease inhibitors (reviewed in martinez-picado and martinez, 2008) . thus, enzymes that perform similar functions for different viruses may have evolved to display different tolerance to amino acid substitutions. it is not possible to generalize which types of resistance mutations will display high or low functional barriers. there is a broad range of frequencies of antibody-and drug-escape mutants in viral populations, although values of 10 à4 to 10 à6 mutants per infectious unit are frequent for many rna and dna viruses (see table 7 .2 in chapter 7 for antibody-escape mutants). a few estimates for drug-escape mutants are listed in table 8 .1; several observations and characterization of escape mutants have not been accompanied by frequency measurements. a point worth emphasizing is that quantification of viruses harboring biologically relevant mutations has been possible because an adequate biological assay is available. an antiviral agent or a neutralizing antibody measures the proportion of infectious viral particles that differ from the majority of the population in the relevant resistance trait. there is no reason to suspect that the viral amino acid residues (that are the target of an inhibitor or an antibody) that are substituted to confer the resistance phenotype are more prone to accept variations than many other amino acids in viral proteins. if we had additional selective agents to probe other viral sites, we expect a similar range of variant amino acids than using inhibitors or antibodies. this quite straightforward prediction is another way to state that there is general agreement in the mutation rates and frequencies for viruses being in the range of 10 à3 to 10 à5 substitutions per nucleotide (s/nt), calculated using a variety of biochemical and genetic methods (chapter 2). a quite general observation is that in antibody neutralization experiments, a fraction of the virus population remains infectious despite the 8.5 molecular mechanisms of antiviral resistance addition of high antibody concentrations. although the resistance mechanism is unclear, a possibility is that the heterogeneous viral population includes a small proportion of antigenic variants with decreased affinity for antibodies. incomplete neutralization with the nonsigmoidal slope in the neutralization curves has been characterized for broadly neutralizing antibodies directed to hiv-1 (mccoy et al., 2015) . this general observation is an added complication to preventive designs that consider administration of neutralizing antibodies either as vaccine additives or in combination with antiviral inhibitors (section 9.2 in chapter 9). the calculated frequencies of antibody-or drug-resistant mutants in viral populations may be lower than the rate at which they originate by mutation due to the fitness cost of the mutation (section 8.4.4) . the argument is parallel to that used to justify why mutation rates and frequencies differ due to the fitness effects of mutations (chapter 2). another prediction derived from the above considerations is that mutations conferring resistance to antiviral agents are expected to be detected in viral populations never exposed to the relevant drugs. all forms of genetic variation of viruses can contribute to dominance and spread of drugresistant mutants, including the combined effect of mutation, recombination, and genome segment reassortment (richman, 1996; neher and leitner, 2010; rogers et al., 2015) . the basal level of mutational pressure may be sufficient to provide a detectable proportion of escape mutants without the need for selection by the selective agent. this is an important aspect of antiviral therapy that is addressed next. the first demonstration that the baseline mutation level in viral quasispecies can include a detectable level of mutations that confer resistance to inhibitors in the absence of selection by the inhibitors, was obtained by d.d. ho, i. n ajera, c. l opez-galíndez, and their colleagues working with hiv-1 (mohri et al., 1993; n ajera et al., 1994 , 1995 . one of the studies examined the pol gene of 60 hiv-1 genomes obtained directly from lymphocytes of infected patients. mutation frequencies for independent viral isolates were in eggers and tamm (1965) amantadine and rimantadine iv 1 â 10 à3 to 4 â 10 à4 measurements in cell culture appleyard (1977) , lubeck et al. (1978) rimantadine a iv 27% percentage of children treated with rimantadine that shed resistant iv belshe et al. (1988) disoxaril a hrv14 1 â 10 à3 to 4 â 10 à4 low-level resistance in cell culture heinz et al. (1989) 4 â 10 à5 high-level resistance in cell culture guanidine d pv 1.8 â 10 à5 to 4 â 10 à8 measurements in cell culture pincus and wimmer (1986) a the formula of these drugs is included in figure 8 .2 with the following number in parenthesis: amantadine (6); rimantadine (7); disoxaril (2) the range of 1.6.10 à2 to 3.4.10 à2 s/nt, while for mutant spectrum components of individual isolates the values were 3.6.10 à3 to 1.1.10 à2 s/nt. in the virus from these patients, mutation frequencies at the codons for amino acids involved in antiretroviral resistance were very similar to the average mutation frequency for the entire pol gene. consistently with the mutation frequency values, several mutations that led to amino acid substitutions that conferred resistance to reverse transcriptase inhibitors were identified in patients not subjected to therapy. at the time of the study, the number of antiretroviral agents was still limited, and a considerable number of patients were not treated. the authors gave convincing epidemiological arguments that the background of mutations related to antiretroviral resistance was a consequence of high mutation rates and quasispecies dynamics, and not due to the transmission of resistant virus from individuals that had been subjected to therapy (primary resistance) (n ajera et al., 1995) . the presence of inhibitor-resistance mutations in viral populations never exposed to the corresponding inhibitor has been confirmed for hiv-1 and for several other viruses, including hcv, and it is supported by the calculated mutant frequencies in viral quasispecies (havlir et al., 1996; lech et al., 1996; ribeiro et al., 1998; ribeiro and bonhoeffer, 2000; cubero et al., 2008; johnson et al., 2008; toni et al., 2009; tsibris et al., 2009; peres-da-silva et al., 2017) . ample support has also come from deep sequencing analyses of mutant spectra, opening a point of debate on the basal frequency of inhibitor-resistance mutations that constitutes an indication to avoid the use of the corresponding inhibitors in therapy. at least in the case of hcv (and probably applicable to other viruses), there is no basis to suggest that the presence of a drug resistance mutation below a certain level in a patient will not have relevance for treatment failure when the inhibitor is administered. the understanding of quasispecies dynamics cautions against such reductionist arguments as evidenced by clinical cases (perales et al., 2018) . the data underline the relevance of mutant spectra as phenotypic reservoirs to confront selective constraints before constraints are in operation. for treatments, including a drug that has already been administered to a patient in the past, the influence of quasispecies memory should also be considered (section 5.5.1 in chapter 5). mutant spectra can be viewed as an anticipatory reservoir of phenotypes. in addition to the presence of drug resistance mutations in viral populations due to mutant frequency levels, the transmission of drug-resistant mutants from treated to na € ive patients may contribute to epidemiological relevance of resistance mutations. such primary resistance has been amply documented with hiv-1, and it appears to increase in the case of hcv (franco et al., 2014; echeverría et al., 2016; huang et al., 2019) . higher levels of resistance mutations as a function of time in untreated patients is an indication that the mutations are not due to basal mutant frequencies but to the epidemiological expansion of virus mutants that originated in treated patients. from the clinical data available, the rate of expansion of virus harboring resistance mutations may vary depending on transmission and epidemiological features of each pathogen, but it seems unavoidable in the face of extended treatments for genetically variable pathogenic viruses. we confront a situation with parallels with antibiotic resistance in bacteria (chapter 10). the major mechanism of drug resistance in viruses is based on amino acid substitutions that render the drug ineffective through the several molecular mechanisms summarized in section 8.5. application of the cell culture system of hcv replication in human hepatoma cells (lindenbach et al., 2005; wakita et al., 2005; zhong et al., 2005) to examine the effects of 8.7 fitness or a fitness-associated trait as a multidrug-resistance mechanism long-term evolution has indicated that viral fitness can be an additional mechanism of drug resistance. the evidence was obtained when addressing the important issue of hcv resistance to interferon-alpha (ifn-a). ifn-a and ribavirin were the two components of the standard of care treatment against hcv infections until the advent of new therapies based on daa agents in 2014. natural hcv isolates differ in ifn-a sensitivity, and the molecular basis of the difference is largely unknown. the study in cell culture consisted in subjecting a clonal population of hcv (termed hcvp0, prepared by electroporation of hepatoma cells with rna encoding the viral genome, transcribed from a plasmid) to 100 serial passages (of the type described in section 6.1 of chapter 6) in the absence or presence of increasing concentrations of ifn-a added to the culture medium. several mutations scattered throughout the hcv genome were associated with ifn-a resistance (perales et al., 2013) . the selection of multiple alternative mutations is most likely influenced by the fact that ifn-a evokes a multicomponent antiviral response, which is not focused toward a single viral protein (perales et al., 2014) . unexpectedly, even the control hcv populations (those passaged 100 times in the absence of ifn-a) displayed a partial (but statistically significant) resistance to ifn-a that could not be attributed to endogenous ifn production by the hepatoma cells (perales et al., 2013) . in view of this intriguing result, the initial hcvp0 population and the hcv population passaged 45 and 100 times in the absence of ifn-a (termed hcvp45 and hcvp100, respectively) were tested for their resistance to other inhibitors of hcv replication: the protease inhibitor telaprevir [formula (20) in fig. 8 .3], the ns5a inhibitor daclatasvir, the cellular protein cyclophilin a inhibitor cyclosporin a, the mutagenic purine nucleoside ribavirin, and the high barrier inhibitor sofosbuvir [formula (21) in fig. 8.3 ]. hcvp45 and hcvp100 displayed significantly increased resistance to all inhibitors tested, as compared with the parental population hcvp0 gallego et al., 2016) (fig. 8.8) . passage of hcv entailed a 2-to 3-fold increase of viral fitness and a broadening of the mutant spectrum that might have increased the frequency of mutations associated with drug resistance, thus explaining the behavior of the multiply passaged hcv populations. the search for the resistant mutations was easier for telaprevir, daclatasvir, and cyclosporin a than for the other drugs because amino acid substitutions in the target protein had been previously identified as responsible for drug resistance. (in the case of cyclosporin a resistance, substitutions map in ns5a and ns5b, because the drug binds to cyclophilin a, which in turn interacts with ns5a). analysis of the mutant spectra of hcvp45 and hcvp100 by molecular cloning and sanger sequencing and by deep sequencing failed to identify specific drugresistance mutations. since it could not be excluded that the broadening of the mutant spectrum might have increased the frequency of resistance mutations still to be characterized, two additional tests were performed. one was to determine the kinetics of viral production over a 1000-fold range of moi in the absence and presence of telaprevir. both the unpassaged and multiply passaged hcv displayed parallel kinetics at the different mois, which excludes that drug resistance was due to the presence of resistance mutations in minority components of the mutant spectrum ( fig. 8.8 ). to further substantiate the findings, biological clones obtained by end-point dilution of the corresponding hcvp0 and hcvp100 populations were tested regarding drug resistance. a biological clone should have eliminated the minority genomes that harbored drug-resistance mutations since biological cloning is the most severe form of bottleneck event (sections 6.2 and 6.5 in chapter 6). the biological clones did not display any decrease in drug resistance as compared 8. quasispecies dynamics in disease prevention and control moreno, e., gallego, i., pineiro, d., et al., 2014 . increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus. j. virol. 88, 12098e12111, with permission from the american society for microbiology, washington dc, usa. 8.7 fitness or a fitness-associated trait as a multidrug-resistance mechanism with their corresponding parental, uncloned populations . the above observations have established viral fitness as a multidrug-resistance determinant in hcv that may also apply to other viruses. one possible molecular mechanism may consist of competition between replicative complexes and inhibitory molecules inside the infected cells. this model implies that fitness increase is reflected either in more replicating molecules per each replicative unit or in an increase in the number of replicative units per cell, without any influence on the number of inhibitor molecules that reach the replication sites. exploration of this competition model and alternative models and the extension to other viral-host systems are important challenges in the field of antiviral research. to sum up, mutant spectra and quasispecies dynamics can mediate antiviral resistance by at least two mechanisms: (i) by increases in the proportion of resistance mutations in the mutant spectra and (ii) by a fitness increase promoted by continued viral replication in the same environment. both mechanisms may act conjointly during viral infections in vivo. some studies with hcv have documented drugresistance phenotypes in infected patients, in the absence of specific drug-resistance mutations (sullivan et al., 2013; svarovskaia et al., 2014; sato et al., 2015; stross et al., 2016; di maio et al., 2017; dietz et al., 2018) . in fact, prolonged chronic hcv infections represent an adequate scenario for fitness increase due to extended rounds of infections in the same host liver. as a consequence, chronic infections may be prone to display fitness-associated multidrug-resistance phenotypes in the absence of drug-resistance mutations. the multiple mechanisms of drug resistance related to quasispecies dynamics justify even further the need for new antiviral strategies, as presented in chapter 9. high viral loads are predictors of disease progression. for hiv-1 and other lentiviruses, efficient early control of virus replication by the host immune response is generally associated with limited disease severity. the viral load that follows after the initial immune response to hiv-1 is referred to as the "viral set point." in the absence of early therapy, low set points in hiv-1 are generally attributed to a strong cellular immune response, likely influenced by additional host and viral factors. [this and other aspects of hiv-1 replication and pathogenesis have been reviewed in excellent monographs by levy (2007 levy ( , 2015 ]. a low set point predicts an asymptomatic outcome, and this is generally the case for viruses that establish persistent rather than acute infections. high viral fitness during the early stages of viral replication can promote disease manifestations. this was suggested by the progression toward the disease of a cohort of individuals that were infected during blood transfusion with an hiv-1 containing a large deletion in nef, an adaptor protein that mediates replication and pathogenesis (reviewed in arien and verhasselt, 2008) . after more than 15 years, some of the infected individuals showed clinical signs, probably as a result of the accumulation of mutations in the hiv-1 genome that compensated for the lack of nef. more generally, fitness-decreasing (but not lethal) genetic lesions in a viral genome may be compensated by additional genomic mutations that become increasingly dominant in the course of further viral replication. the kinetics of fitness gain will depend on the nature of the lesion and the functional implications of the altered protein or genomic regulatory region (chapter 5). fitness, replicative capacity, and viral load are directly interconnected parameters, and they 8. quasispecies dynamics in disease prevention and control affect disease progression (domingo et al., 2012) ( fig. 8.9 ). fitness gain will be more effective with a high load of actively replicating virus in the infected organism. elevated replicative capacity and fitness sustain high viral loads. the reason for this basic feature of viral population dynamics is that given a basal mutation rate, a large number of replicating genomes entails a correspondingly higher probability that a required mutation for fitness gain can be produced. the events involved are a specific case of search for adaptive mutations in terms of exploration of sequence space, as discussed in section 3.7 of chapter 3. while active viral replication, high load, and high fitness favor progression of the infection and disease manifestations, the fourth parameter included in the large arrow of fig. 8 .9, mutant spectrum diversity, has an optimal range. too low or too high intrapopulation diversity is detrimental to virus adaptability. insufficient diversity limits adaptability to complex environments (pfeiffer and kirkegaard, 2005; vignuzzi et al., 2006) , while excess diversity may lead the virus to cross an extinction threshold, and this is the basis of lethal mutagenesis as an antiviral therapy (chapter 9). an additional implication of the parameters shown in fig. 8 .9 for antiviral interventions is that fitness decrease is recognized as an alternative to inhibition of viral replication to control viral infections[ (clementi, 2008; clementi and lazzarin, 2010) ; reviewed in (domingo et al. 2012) ]. thus, key features of quasispecies dynamics have a direct implication on the management of viral infections. external interventions that have been applied or have been envisaged to limit or suppress virus infection include not only vaccination and administration of antiviral agents as described in previous sections, but also passive immunotherapy, antisense rnas, or oligonucleotides with various chemical modifications, interfering rnas, ribozymes, or their combinations. biotechnological developments have favored the design of chemically defined vaccines (consisting of expressed immunogenic proteins, synthetic peptides, or peptide arrays), without the need to handle or administer live virus. one of the most na € ive manifestations of trust in biotechnology in the middle of the 20th century was the belief that a catalog of plasmids encoding the antigenic proteins of the circulating types of pathogenic viruses would suffice to prepare the required vaccine as needed. concerning influenza vaccines, w.i. beveridge wrote the following: "the first objective would be to capture the full range of influenza a subtypes. their antigens would be studied by specialists at central laboratories and made available for the preparation of particular vaccines if and when required. it might be feasible to stockpile some vaccine against all the principal hemagglutinin antigens to be used in a figure 8.9 a schematic representation of interconnected parameters of viral replication that often relate to disease progression. an understanding of quasispecies dynamics has made it evident that the aim of antiviral therapy need be not only to directly diminish the viral load but also to affect other parameters that can then reduce the viral load. see text for justification and references and chapter 9 for new antiviral strategies that follow the concept expressed in this figure. 8.9 limitations of simplified reagents and small molecules as antiviral agents fire brigade type of action as soon as an incipient pandemic is spotted" (beveridge, 1977) . naivety is also perceived in current designs of universal vaccines based on conserved antigens, considering the different escape mechanisms that operate in viruses to elude neutralization by antibodies (chai et al., 2016) . it is remarkable how our present understanding of viral populations renders obsolete the views expressed in the w.i. beveridge book, and in other writings at the boom of implementation of dna recombinant techniques. yet, attempts to produce vaccines against highly variable viruses, based on antigenic structures or some viral isolates, are ongoing (christiansen et al., 2018) . a critical issue is if the host immune response against a vaccine engineered with "universal" (conserved) antigenic motifs will be sufficient to prevent disease upon infection by other forms of the same pathogen (freeman and cox, 2016) . from our current knowledge of viral population dynamics, it seems unlikely but time will tell, since efforts toward the manufacturing of universal antiviral vaccines are under way. with a conceptual similarity to vaccines, in medical practice, monotherapy with an antiviral agent was traditionally preferred over drug mixtures. (the change of paradigm was largely a consequence of the aids epidemic, and it was publicly expressed by the pioneer hepatologist s. sherlock in a summary address of an international symposium on viral hepatitis held in madrid in 1998). the change of perspective is clear. some antiviral strategies, such as antisense nucleic acids or virus-directed ribozymes, were intensely investigated decades ago. it is unlikely that when used in isolation, they can be converted into useful antiviral therapies because resistant mutants are likely to be selected. yet, they could be part of combinations with other antiviral inhibitors to provide a larger antiviral barrier (chapter 9). a similar fate is likely for interfering rnas (boden et al., 2003; gitlin et al., 2005; herrera-carrillo and berkhout, 2015; mcdonagh et al., 2015) . to a large extent, the failures of defined chemical entities (oligonucleotides, ribozymes, small molecule inhibitors, etc.) to control virus replication and spread are a consequence of their targeting a very defined viral genomic sequence combined with the adaptability of viral populations. combination of such multiple elements have been envisaged and tested, but off-target effects and the adaptive potential of viruses are likely to limit their efficacy. unfortunately, biotechnological developments that have been so positive for many research areas and practical applications tend to simplify the types of agents to prevent disease or inhibit virus replication, ignoring the inherent complexity of the object to be controlled. success is unlikely when "complexity" is combated with "simplicity." an increased understanding of viral population dynamics over the last decades has changed the picture dramatically by providing an interpretation of "virus escape" as a general and largely unavoidable phenomenon. such awareness has pushed the development of new antiviral designs, which are fundamentally centered in two strategies: a combination of multiple, independently acting elements or fitness decrease through excess mutations (chapter 9). a pronouncement by p. ehrlich at the international congress of medicine held in london in 1913, reflected old traditions on how to treat microbial infections ["here, therefore, the old therapeutic motto is applicable: frapper fort et frapper vitte" and "therefore, it is in my opinion necessary to allow the therapeutic treatment to come into action as early as possible, ..."; sentences taken from (ehrlich, 1913) , with emphases as in the original text]. this pronouncement fits our current understanding of viral populations. following ehrlich, the full implications of quasispecies-mediated adaptation of viruses for 8. quasispecies dynamics in disease prevention and control antiviral therapy were expressed by d.d. ho in an influential article entitled "time to hit hiv, early and hard" (ho, 1995) . the article title captures what is needed to prevent adaptation of a virus in the infected host. any opportunity to replicate is exploited by the virus to increase its fitness and to become less vulnerable to internal (intrahost) or external interventions such as antiviral therapy. treatment interruptions during chronic infections, such as "drug holidays" that in the case of hiv-1-infected patients were justified to alleviate side effects associated with administration of antiretroviral agents, provided an opportunity for the virus to gain fitness. in principle, given our current understanding of viral quasispecies dynamics, the proposal of p. ehrlich and d.d. ho is applicable to other viral pathogens. one argument that tones down the strength of the "hit early and hit hard" proposal is that some infected patients may not progress to disease, but maintain an asymptomatic lifelong persistent infection. this is the case with elite controllers in the case of hiv-1 infection, and individuals infected with hcv who will not progress toward liver disease. in cases in which such nonprogression can be anticipated by viral and host parameters, it may be justified to exclude some patients from aggressive interventions (suthar and harries, 2015; casado et al., 2018) . as a general rule, however, the potential benefits of early treatment are obvious not only to avoid disease on an individual basis, but also to diminish the chances of virus transmission (reviewed in suthar and harries, 2015; see also sections 7.1 and 7.7 in chapter 7 regarding the relevance of viral population numbers in transmission). restricting the number of treated patients for economic reasons will result in more expensive public health interventions when infected individuals develop the disease. box 8.4 includes recommendations for the use of antiviral agents and recapitulates concepts explained in this and preceding sections. despite the emphasis on evolutionary aspects, prevention and treatment of viral disease have many other angles some of which were box 8.4 • avoid monotherapy. ideally, use two or more antiviral agents which do not share a mechanism of action ( fig. 8.6 ). • treat as soon as possible after virus diagnosis, to avoid virus adaptability associated with high virus population size and to minimize transmission of inhibitor-resistant mutants. • individual patients should be treated only during the time at which the drug proves effective. when viral load rebounds, treatment should be discontinued. • use deep sequencing methodology to determine mutant spectrum composition for an adequate choice of inhibitors. the aim is to design personalized treatments that consider the probability of drug combination efficacy with minimal side effects. • consider temporary shelving of effective drugs when resistant mutants acquire epidemiological relevance. considered in chapter 7 in connection with factors of disease emergence (smolinski et al., 2003) . three of them should be mentioned here because they are as important as the adequate treatment designs described in this chapter: (i) adequacy of public health measures, (ii) public information about virus sources and means of contagion and (iii) need of global political action. information to the public should aim at limiting the spread of disease that is, undertaking personal and collective actions to reduce the r 0 value for a given virus (chapter 7, section 7.2). as an illustration of this key point, there was a quite extensive information campaign on hiv-1 and aids in developed countries during the early decades of hiv-1 spread, while the information about other potentially threatening viruses such as ebola or the severe acute or the middle east respiratory syndrome (sars and mers, respectively) coronaviruses was more limited. the need of a global response to limit the extension of disease episodes at the sites where they are initiated has been recognized for a long time, but it became obvious with the 2014e15 west african ebola epidemic (see siedner et al., 2015) . there is a need for international organizations and governments of developed countries to provide the health-care workforce to assist low-and middle-income countries to control viral episodes at an early stage. "help early, help effectively" is the recognized need at a global scale, which is the parallel to "hit early, hit hard" for the treatment of infected patients. global early action and adequate information can be as important as an adequate treatment design to control viral disease. it can restrict viral replication rounds and consequent adaptability. information is thought to have been critical for the control of ebola epidemic in nigeria (siedner et al., 2015) . however, information must also be planned to reach the target population in a convincing manner, as learned from the poliovirus vaccination and eradication campaign (renne, 2010) . the uncertainties regarding whether an initial, limited episode of viral disease will expand or die out do not help in decision making. however, the best choice in the case of emerging and reemerging infections is to act assuming the worst scenario. medical interventions represent a totally new set of selective constraints that viruses are facing only since decades ago, an infinitesimal time of their existence as biological entities. however, the evolutionary mechanisms available to viruses have successfully coped with many selective pressures, notably the effect of vaccination when a broad immune response is not evoked, or treatment with antiviral agents. a common way to proceed is to test a new vaccine with an animal host, be it the authentic host or an animal model, and obtain full protection when the animal is challenged with a virus that matches the antigenic composition of the vaccine. following the initial excitement, very often the vaccine displays only partial protection when tested in the natural environment. somewhat parallel arguments can be made about clinical trials for antiviral agents, usually performed initially with selected groups of patients. clinicians have coined the term "reallife" treatment studies when patients are not selected for optimal results (often pushed by commercial and political interests). this chapter has emphasized how the complexity of viral populations is a serious (often underestimated) difficulty to prevent and treat viral disease. examination of the molecular mechanisms exploited by viruses to survive despite antiviral interventions suggests two major lines of action: first, more judicious use of existing tools that should consider the complexity of viral populations and their dynamics; complexity 8. quasispecies dynamics in disease prevention and control cannot be combated with simplicity. second, the need to design new antiviral strategies, a topic addressed in chapter 9. several interconnected parameters determine the probability of success of an antiviral intervention. most of them follow from the general concepts of darwinian evolution explained in preceding chapters. it is important, however, to quantify as much as possible the evolutionary events that determine therapy success or failure. for this reason, the importance of viral population size, basic probability calculations of developing resistance, and the selective strength of mutations, have been explained with numerical examples. hopefully, these simple quantifications will permit a higher awareness of when and why treatment may succeed or fail. we live in a very unequal society. the chapter closes with the recognition that there are many social economic issues that are as important as scientific planning to combat the pathogenic viruses around us (see summary box). • medical interventions represent a new class of selective constraints acting on viral populations. • viral evolution affects antiviral preventive and treatment strategies in two different ways: (i) through the molecular mechanisms of short-term response in treated individuals that select escape mutants, and (ii) through the epidemiological impact of viruses that have acquired escape mutations. • ineffective vaccines can contribute to the selection of antigenic viral variants. • selection of viral mutants resistant to antiviral agents is a general phenomenon. selection is favored by suboptimal treatments and is delayed by the combined administration of multiple inhibitors. resistance may also occur in the absence of specific resistance mutations, and it is associated with viral fitness. • the aim of therapy should be to increase the functional barrier to resistance and to give no opportunity to the virus to pursue replication that increases its replicative fitness. • replication rate, viral load, fitness, and mutant spectrum complexity are interconnected parameters that may tip the balance toward either control of the infection or disease progression. each of these parameters can be targeted in an antiviral design. • health care resources, adequate public information, and firm political action are as important as antiviral designs to control virus infections at a global level. drug reposer: a web server for predicting similar amino acid arrangements to know drug binding interfaces for potential drug repositioning involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism amantadine-resistance as a genetic marker for influenza viruses hiv nef: role in pathogenesis and viral fitness mechanisms of hiv-1 escape from immune responses and antiretroviral drugs estimating hiv evolutionary pathways and the genetic barrier to drug resistance genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection influenza: the last great plague; and unfinished story of discovery the vaccine book transmitted/founder viruses rapidly escape from cd8 þ t cell responses in acute hepatitis c virus infection natural history of infections disease epitope-specific cd8 þ t lymphocytes cross-recognize mutant simian immunodeficiency virus (siv) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during siv infection two escape mechanisms of influenza a virus to a broadly neutralizing stalk-binding antibody development of live-attenuated arenavirus vaccines based on codon deoptimization immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent hcv vlp vaccine the poliovirus eradication initiative viral persistence in vivo through selection of neutralizing antibody-escape variants cd4þ t-cell-epitope escape mutant virus selected in vivo perspectives and opportunities for novel antiviral treatments targeting virus fitness human immunodeficiency virus type 1 fitness and tropism: concept, quantification, and clinical relevance hiv-1 drug resistance and resistance testing antigenic determinants of possible vaccine escape by porcine circovirus subtype 2b viruses trans-dominant inhibition of rna viral replication can slow growth of drugresistant viruses naturally occurring ns3-protease-inhibitor resistant mutant a156t in the liver of an untreated chronic hepatitis c patient vaccine development: from concept to early clinical testing molecular and functional bases of selection against a mutation bias in an rna virus molecular epidemiology of hepatitis b virus mutants associated with vaccine escape, drug resistance and diagnosis failure patterns of resistanceassociated substitutions in patients with chronic hcv infection following treatment with direct-acting antivirals multiclass hcv resistance to direct-acting antiviral failure in real-life patients advocates for tailored secondline therapies rna virus evolution and the control of viral disease complications of rna heterogeneity for the engineering of virus vaccines and antiviral agents quasispecies dynamics in disease prevention and control quasispecies and rna virus evolution: principles and consequences virus population dynamics, fitness variations and the control of viral disease: an update viral quasispecies evolution. microbiol naturally occurring ns3 resistance-associated variants in hepatitis c virus genotype 1: their relevance for developing countries. virus res spectrum and characteristics of the virus inhibitory action of 2-(alpha-hydroxybenzyl)-benzimidazole coxsackie a9 virus: mutation from drug dependence to drug independence address in pathology on chemotherapy viral infections of humans. epidemiology and control. plenum medical book company transmission of single hiv-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing deep sequencing of protease inhibitor resistant hiv patient isolates reveals patterns of correlated mutations in gag and protease detection of a sexually transmitted hepatitis c virus protease inhibitor-resistance variant in a human immunodeficiency virus-infected homosexual man lessons from nature: understanding immunity to hcv to guide vaccine design human immunodeficiency virus gag and protease: partners in resistance barrier-independent, fitness-associated differences in sofosbuvir efficacy against hepatitis c virus imperfect vaccination: some epidemiological and evolutionary consequences poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches coxsackievirus b3 mutator strains are attenuated in vivo principles of pharmacology: the pathophysiologic basis of drug therapy a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease systems vaccinology: enabling rational vaccine design with systems biological approaches global and regional molecular epidemiology of hiv-1, 1990-2015: a systematic review, global survey, and trend analysis nevirapine-resistant human immunodeficiency virus: kinetics of replication and estimated prevalence in untreated patients evolution of the hcv viral population from a patient with s282t detected at relapse after sofosbuvir monotherapy genetic and molecular analyses of spontaneous mutants of human rhinovirus 14 that are resistant to an antiviral compound the impact of hiv-1 genetic diversity on the efficacy of a combinatorial rnaibased gene therapy a working hypothesisevirus resistance development as an indicator of specific antiviral activity time to hit hiv, early and hard changes of hepatitis b surface antigen variants in carrier children before and after universal vaccination in taiwan comparison of naturally occurring resistanceassociated substitutions between 2008 and 2016 in chinese patients with chronic hepatitis c virus infection vaccination influences the evolution of classical swine fever virus minority hiv-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy genetic variability of porcine circovirus 2 in vaccinating and non-vaccinating commercial farms outbreak of poliomyelitis in hispaniola associated with circulating type 1 vaccinederived poliovirus influenza. plenum medical book company my cousin, my enemy: quasispecies suppression of drug resistance genetic and antigenic diversity of human rotaviruses: potential impact on vaccination programs rna virus population diversity, an optimum for maximal fitness and virulence in vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects hiv and the pathogenesis of aids dispelling myths and focusing on notable concepts in hiv pathogenesis vaccination and timing influence siv immune escape viral dynamics in vivo susceptibility of influenza a viruses to amantadine is influenced by the gene coding for m protein comparison of the mechanisms of drug resistance among hiv, hepatitis b, and hepatitis c hiv-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo incomplete neutralization and deviation from sigmoidal neutralization curves for hiv broadly neutralizing monoclonal antibodies combination sirna therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro rapid development of drug-resistant mutants of poliovirus molecular basis of human immunodeficiency virus type 1 drug resistance: overview and recent developments antiretroviral therapy and drug resistance in human immunodeficiency virus type 2 infection development of streptomycin resistant strains of tubercle bacilli in pulmonary tuberculosis; results of simultaneous sensitivity tests in liquid and on solid media quantitation of zidovudine-resistant human immunodeficiency virus type 1 in the blood of treated and untreated patients a systematic review of hepatitis b virus (hbv) drug and vaccine escape mutations in africa: a call for urgent action quasispecies dynamics in disease prevention and control exploration of sequence space as the basis of viral rna genome segmentation pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy impact of novel ns5a resistance-associated substitutions of hepatitis c virus detected in treatment-experienced patients hepatitis c virus population dynamics during infection retreatment of hepatitis c virus infected patients with direct-acting antiviral failures influence of mutagenesis and viral load on the sustained low-level replication of an rna virus response of hepatitis c virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype molecular basis of interferon resistance in hepatitis c virus baseline hepatitis c virus resistance-associated substitutions present at frequencies lower than 15% may be clinically significant delayed lysis confers resistance to the nucleoside analogue 5-fluorouracil and alleviates mutation accumulation in the single-stranded dna bacteriophage vx174 hepatitis a virus vaccine escape variants and potential new serotype emergence increased fidelity reduces poliovirus fitness under selective pressure in mice production of guanidineresistant and -dependent poliovirus mutants from cloned cdna: mutations in polypeptide 2c are directly responsible for altered guanidine sensitivity imperfect vaccination can enhance the transmission of highly virulent pathogens the politics of polio in northern nigeria hiv-1 protease mutations and protease inhibitor cross-resistance production of resistant hiv mutants during antiretroviral therapy resistance, drug failure, and disease progression antiviral drug resistance rapid evolution of the neutralizing antibody response to hiv type 1 infection new vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment and natural selection hiv-1 phylogenetics and vaccines vaccine strategies alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models resistance to direct antiviral agents in patients with hepatitis c virus infection deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype-1b hepatitis c virus animal vaccination and the evolution of viral pathogens increased replicative fitness can lead to decreased drug sensitivity of hepatitis c virus coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity microbial threats to health. emergence, detection and response natural hcv variants with increased replicative fitness due to ns3 helicase mutations in the c-terminal helix a18 evolution of treatmentemergent resistant variants in telaprevir phase 3 clinical trials a public health approach to hepatitis c control in low-and middle-income countries infrequent development of resistance to genotype 1-6 hepatitis c virus-infected subjects treated with sofosbuvir in phase 2 and 3 clinical trials strategies and challenges for eliciting immunity against avian influenza virus in birds a large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants evidence of the coevolution of antigenicity and host cell tropism of foot-and-mouth disease virus in vivo recombination in the alphaherpesvirus bovine herpesvirus 1 detection of human immunodeficiency virus (hiv) type 1 m184v and k103n minority variants in patients with primary hiv infection quantitative deep sequencing reveals dynamic hiv-1 escape and large population shifts during ccr5 antagonist therapy in vivo basic research in hiv vaccinology is hampered by reductionist thinking sequence-specific fidelity alterations associated with west nile virus attenuation in mosquitoes quasispecies diversity determines pathogenesis through cooperative interactions in a viral population hiv-1 polymerase inhibition by nucleoside analogs: cellular-and kinetic parameters of efficacy, susceptibility and resistance selection production of infectious hepatitis c virus in tissue culture from a cloned viral genome emergence of virus escape mutants after immunization with epitope vaccine genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine robust hepatitis c virus infection in vitro key: cord-307817-2vy28i4m authors: lou, zhiyong; sun, yuna; rao, zihe title: current progress in antiviral strategies date: 2014-01-14 journal: trends pharmacol sci doi: 10.1016/j.tips.2013.11.006 sha: doc_id: 307817 cord_uid: 2vy28i4m the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. virus-targeting antivirals can function directly or indirectly to inhibit the biological functions of viral proteins, mostly enzymatic activities, or to block viral replication machinery. host-targeting antivirals target the host proteins that are involved in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. here we review key targets and considerations for the development of both antiviral strategies. the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. virus-targeting antivirals can function directly or indirectly to inhibit the biological functions of viral proteins, mostly enzymatic activities, or to block viral replication machinery. host-targeting antivirals target the host proteins that are involved in the viral life cycle, regulating the function of the immune system or other cellular processes in host cells. here we review key targets and considerations for the development of both antiviral strategies. viruses comprise a large group of pathogens that are responsible for causing severe infectious diseases. over the past 30 years, antiviral agents that target viral proteins or host factors have been successfully developed. based on their inhibitory mechanisms, antiviral reagents can be divided into two groups: (i) inhibitors that target the viruses themselves or (ii) inhibitors that target host cell factors. virus-targeting antivirals (vtas) can function directly (dvtas) or indirectly (indvtas) to inhibit biological functions of viral proteins, mostly enzymatic activities, or they block the correct formation of the viral replication machinery (table 1) . host-targeting antivirals (htas) include reagents that target the host proteins that are involved in the viral life cycle (figure 1 ), regulating the function of the immune system or other cellular processes in host cells. with increased knowledge of viral protein and host factors, the scientific community has achieved great progress in mechanism-based antiviral discovery against chronic viral infectious diseases, and in understanding of the emergence of new viral diseases and of the resistance to traditional antivirals. this review will highlight recent achievements in antiviral development and discuss various strategies for preventing virus attachment and entry into the host cell, as well as strategies for preventing virus replication and transcription within the host cell. the first step in viral invasion is the attachment to host cells via an interaction with functional receptor(s). for enveloped viruses, the viral proteins located on the outer envelope of the virion are responsible for the recognition of receptors and the attachment to host cells. hiv (a member of the retroviridae family) is a typical enveloped virus, and its invasion is mediated by the envelope proteins gp120 and gp41, which are arranged on the viral membrane as a trimer of three trans-membrane gp41 and three noncovalently attached gp120 surface subunits [1] (figure 2 ). gp120 recognizes the cd4 receptor and launches the conformational changes that expose the binding sites for the binding of a co-receptor, (i.e., ccr5 and cxcr4 [2] ). antagonists that block the interactions between hiv and its receptor and co-receptors have therefore been developed as anti-hiv therapeutics. attempts to find specific inhibitors that block the interaction between hiv-1 and cd4 were initiated using a soluble extracellular domain of cd4 protein that retained the ability to bind gp120. although the preliminary results revealed that either soluble cd4 protein or a cd4-immunoglobulin fusion protein showed good in vitro anti-hiv activity, all failed in clinical trials due to poor pharmacokinetic features (e.g., the half-life of cd4-immunoglobulin fusion protein in mice is only 2.4 h) [3] [4] [5] . small molecule inhibitors that occupy a specific region within the cd4-binding pocket of gp120 were subsequently developed to block the gp120-cd4 interaction (figure 2a ). for example, bms488043 [6] and bms663068 [7] were found to significantly reduce hiv-1 proliferation and have good pharmaceutical characteristics. another success is influenza neuraminidase (na) inhibitor (nai). influenza na is a surface glycoprotein and functions at two steps of the viral life cycle: (i) cleaves the cell receptor sialic acid residues, which bind to influenza hemagglutinin (ha), and allows the release of the progeny virus; and (ii) cleaves the sialic acid moieties on the mucin that bathes the airway epithelial cells or cobinds the receptor with ha [8] . in line with the structure of na [9, 10] , several nais have been successfully developed to competitively occupy the sialic acid-binding pocket of na. among these nais, oseltamivir and zanamivir were first used clinically as an anti-flu therapy [11] . oseltamivir is a prodrug that is readily absorbed by the gastrointestinal tract and is converted by hepatic esterases to the active compound (oseltamivir carboxylate). zanamivir has poor oral bioavailability and is currently available as a dry powder mixed with lactose. moreover, laninamivir and peramivir were also approved in north asia recently. laninamivir has excellent in vitro activity against wild type, as well as oseltamivir-resistant, influenza viruses currently circulating [12] . additionally, peramivir is another nai that differs structurally from other inhibitors through novel substitutions that result in multiple binding interactions with the active site and allows the antiviral to be active against nai-resistant viruses [13] . non-enveloped viruses, such as the picornavirus (picornaviridae family) and human papillomavirus (hpv) (papillomaviridae family), interact with their functional receptors through viral capsid proteins. picornaviruses are typical non-enveloped viruses, and some members, including enterovirus 71 (ev71) and human rhinoviruses (hrvs), are responsible for causing severe human infection diseases. the non-enveloped capsids of picornaviruses are icosahedral structures comprising 60 copies of viral structural proteins vp1-4 [14, 15] . vp1-3 each adopt a b-barrel configuration and are arranged with icosahedral symmetry such that vp1 surrounds the 5-fold axes and vp2 and vp3 alternate around the 2-and 3-fold axes [16] . although the receptor-binding sites on the surface of picornavirus capsids are not conserved [17] , these sites have been used to discover inhibitors that block virus-receptor interactions. for example, the canyon structure on the surface of the hrv capsid serves to bind to the hrv receptor, and the soluble portion of the intercellular adhesion molecule-1 (icam-1) [18] and numerous compounds that compete with the putative hrv receptor binding site have been shown to bind in a nearby hydrophobic pocket to inhibit virus attachment to the receptor [19] . however, none of these compounds have been clinical successes to date. after attaching to host cells, a virus will release its genome into the cytoplasm through endocytosis or direct membrane fusion. because this viral entry is one of the key early steps in the viral life cycle (figure 1 ), entry inhibitors have been successfully developed for antiviral therapies. as an enveloped virus, hiv-1 uses gp41 to facilitate its entry process after gp41 is activated by the binding of gp120 to the receptor. the extracellular portion of gp41 contains two heptad repeat domains (hr1 and hr2) separated by a loop region and a hydrophobic fusion peptide (fp) at the n terminus. during the fusion process, the fp of gp41 is inserted into the host cell membrane, and hr1 adopts a triple-stranded coiled-coil structure, forming a meta-stable prefusion intermediate. hr2 subsequently folds into the hydrophobic grooves of the hr1 coiled-coil to form a stable six-helix bundle that juxtaposes the viral and cellular membranes for fusion ( figure 2c ). fusion inhibitors are designed to block the conformational changes that are required for membrane fusion. the t20 peptide (enfuvirtide), which is a peptidic mimic of hr2 and acts by competitively binding to hr1, is the first and the only clinically approved fusion inhibitor [20, 21] . t20 can inhibit a broad range of hiv strains at the nanomolar level, but the poor bioavailability of the drug (it has a plasma half-life of 4 h) make the clinical application of this drug difficult [22, 23] . to overcome this pharmacokinetic disadvantage, a series of modified peptides including cp32m [24] , sifuvirtide [25] , and t2635 [26] have been generated to stabilize the helical structure of the hr2-like peptide by incorporating intrahelical salt bridges between the helix turns. in particular, the plasma half-life of sifuvirtide is 5-fold greater than that of t20 [27] . moreover, chemical modifications including the pegylation [28] , glycosylation [29] , and more have also been introduced to improve the pharmacokinetics of hr2-based fusion inhibitors. alternatively, a subset of bioavailable small molecule fusion inhibitors have been developed targeting the hr2binding pocket on the hr1 trimer [30] [31] [32] or other undefined regions [33] . a similar strategy has been successfully used against many viruses that use a class i fusion mechanism, including influenza virus, ebola virus (filoviridae family), and severe acute respiratory syndrome coronavirus (sars-cov) (coronaviridae family) [34] , but this approach is rare in antiviral development targeting viruses with a class ii/iii fusion mechanism. however, the difficulty in production and delivery means that none of these small molecule fusion inhibitors can be approved for clinical use. non-enveloped viruses use a different strategy for virus entry. after attaching to their receptors, a non-enveloped virus releases its genome into the host cell through a conformational shift of its capsid protein(s). for example, the capsid of ev71 harbors 60 copies of a hydrophobic 'pocket factor', a natural lipid (sphingosine), at the base of the canyon in the capsid protein vp1 [35] (figure 3 ). the expulsion of sphingosine following the binding of the virus to its receptor triggers the opening of the capsid to release the viral genome. pleconaril [36] and bta798 are two examples of hydrophobic compounds that can replace the natural pocket factor and inhibit picornaviral uncoating by generating resistance to the expulsion of pocket factor [37] [38] [39] . using the skeletons of pleconaril and related molecules, a novel class of imidazolidinones has been further synthesized with significant anti-ev71 activity (ic 50 in the range of 0.001-25 mm) [40] . most viruses encode one or several proteases that play crucial roles in the viral life cycle. the viral proteases carry out the proteolysis of a polyprotein precursor and release functional viral proteins, allowing them to function correctly and individually in replication/transcription and maturation [41, 42] . viral proteases also effectively protect viral proteins by modulating host cell pathways, including ubiquitination and isgylation [43] [44] [45] [46] [47] [48] . in contrast to the diversity of viral protease functions and structures, the catalytic active site of viral proteases generates stringent substrate specificity in protein cleavage. synthetic substrate peptides, which can be designed according to the natural substrates of individual viral proteases, usually generate high-affinity binding and thus provide potent candidates for further drug discovery. one of the great successes is the hiv-1 protease inhibitors (pis). there are ten pis currently approved to treat hiv-1 infection: amprenavir (apv), atazanavir (atz), darunavir (tmc114), fosamprenavir (lexiva), indinavir (idv), lopinavir (lpv), nelfinavir (nfv), ritonavir (rtv), saquinavir (sqv), and tipranavir (tpv) [49] . all hiv-1 pis share relatively similar chemical structures derived from its natural peptidic substrate and, therefore, the cross-resistance to pis occurs at the active site of hiv-1 protease [50] . as a result, pis are commonly used as the combination with other anti-hiv drugs to avoid drug resistance. because most of the pharmaceutical disadvantages have been overcome, pis have become the most potent types of antiviral drugs. current progress in generating antiviral pis has been systematically reviewed elsewhere [51, 52] . almost all viruses encode polymerases in the central steps of replication and transcription. thus, polymerases are becoming the most attractive and suitable targets for antiviral development. based on the function and structure of viral polymerases, there are two major types of polymerase inhibitors: (i) nucleoside and nucleotide substrate analogs and (ii) allosteric inhibitors. nucleoside/ nucleotide analogs play a dominant role in antiviral drugs targeting viral polymerases. nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids. the disadvantage of nucleoside analogs is that the initial phosphorylation step, that is, production of the monophosphorylated form, required for activation to a triphosphate may not correctly occur in the host cell [53] . therefore, monophosphate nucleotide analogs were developed as polymerase inhibitors to avoid this problem. to date, most of the approved antiviral drugs for anti-hiv therapy utilize this mechanism, including zidovudine (azt, , and others. the same strategy was also successfully used in the development of antivirals against a wide range of viruses, including cytomegalovirus (cmv) [54] and herpes simplex virus (hsv) [55] (herpesviridae family), hepatitis b virus (hbv) (hepadnaviridae family) [56] , and hcv [57] . during the course of a polymerase cycle, the relative orientation of the polymerase domains undergoes a slight shift and this shift causes the conformational change of a specific site, the allosteric site, in the viral polymerase. therefore, compounds that bind to the allosteric site could conceivably block the structural movement of polymerase domains and thus inhibit the function of viral polymerases. antiviral inhibitors that work by this mechanism are known as 'allosteric inhibitors'. the allosteric inhibitors of hiv reverse transcriptase (rt) are also known as nonnucleoside reverse transcriptase inhibitors (nnrtis). nnrtis and the hydrophobic binding site on hiv rt were first identified by screening compound libraries against hiv-1 rt combined with structural biological analysis [58] [59] [60] [61] . the binding of nnrti to hiv-1 rt prevents the flexibility and movement of rt required for the elongation of the nucleic acid. to date, there are five nnrtis, that is, nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine, that have been approved by the fda for clinical use to treat hiv-1 infection. furthermore, the development of nnrtis against other viral polymerases, such as that of hcv, is ongoing, and other polymerases are likely to have suitable sites for allosteric inhibitors [62] . integrase is an enzyme that helps the retrovirus to facilitate the incorporation of proviral dna into the host cell genome and catalyzes a vital function in viral replication. inhibitors of integrase represent the newest class of figure 4) . raltegravir, an integrase strand transfer inhibitor (insti), was the first generation drug of this class to be approved and is a potent and welltolerated antiviral agent [63] . dolutegravir is the most advanced second generation insti, and it possess good tolerability, once-daily dosing with no need for a pharmacological enhancer, and relatively little cross-resistance with raltegravir [64] . another insti, elvitegravir, was recently approved for the treatment of hiv infection as part of a fixed dose combination known as stribild [65] . furthermore, inhibitors of the lens epithelium-derived growth factor (ledgf)/p75 binding site of integrase (led-gins) have also been developed, but these are still in a very early stage. each of these drugs contributes a new benefit to the class and will extend the treatment options for patients with hiv-1 infection. the 5 0 terminus of the genomic rna in a subset of rna viruses requires a cap structure, which can be directly taken from the mrna of host cells, as in influenza virus, or synthesized by viral enzymes, as in flavivirus and coronavirus. in the latter cases, a cap structure is generated by a series of enzymes and attached to the first nucleotide of the genome rna via a 5 0 -5 0 triphosphate linker. for example, the genome of flavivirus is capped by a type 1 cap structure (m 7 gpppam), which is methylated at the n-7 position of the guanine (m 7 g) and on the 2 0 oh of the ribose of the first nucleotide (am) of the genome rna [66] . three enzymatic steps are required to form the cap structure, including an rna triphosphatase (ns3), a guanylyl transferase (gtase), and a methyltransferase (mtase), provided by the n terminus of ns5 protein. based on the structure of mtase and its complex with different substrates, three key ligand-binding sites were identified ( figure 5 ). the binding site for s-adenosyl methionine (sam), which acts as the methyl donor, is located at the core domain of mtase, whereas the binding site for the receiver gtp molecule and the guanine moiety of the rna cap is formed by the core domain and the n-terminal extension. moreover, there is another site that binds rna between the sam and gtp pockets; this site is known to be a second, lower affinity binding site that is formed by the core domain and by a channel between two helices of the nterminal extension. because these sites are crucial for mtase activity and virus replication, all are valid sites for the design of dvtas (figure 4) . ribavirin was first shown to inhibit the binding of the rna cap to dengue virus (denv) mtase by competitively binding to the gtp/rna cap pocket [67] . through structure-based drug design, aurintricarboxylic acid was found to inhibit the 2 0 -o activity of denv mtase with an ic 50 of 2 mm by binding to the lower affinity site [68] . another inhibitor, sinefungin, which is an analog of sam with the methylated sulfur of sam replaced by a carbon and amine, can inhibit flavivirus activity with an ic 50 as low as 0.7 mm [69] . recently, the compound bg323 has been discovered using high-throughput screening (hts) methods as an in vitro inhibitor of the guanylyltransferase activity of denv mtase (and thus denv replication) [70] . however, these inhibitors must be used at a high concentration to outcompete the high-affinity endogenous ligand, and it is necessary to solve this problem before this mechanism can be clinically exploited. some viruses utilize a viral helicase to separate one strand of dna or rna from the complementary strand in a process driven by adenosine triphosphate (atp) hydrolysis. the most widely studied viral helicase is the flaviviral helicase, which is the helicase domain of nonstructural protein 3 (ns3) encoded by hcv. sars-cov (nsp13 protein), simian virus 40 (sv40) (polyomaviridae family) (tag protein), and hpv (e1 protein) are also known to encode helicases for their replication [71] . the inhibitors of helicase-catalyzed atp hydrolysis are the most straightforward class, and several nucleotide and nucleobase analogs have been discovered targeting this mechanism. biphenyls and triphenylmethanes have been studied as inhibitors of sv40 tag [72] , hpv e1 [73] , and hcv ns3 helicase [74] . biphenyls and biphenysulfonacetic acid were found to inhibit hpv growth, an inhibitor of hpv e1-catalyzed atp hydrolysis with an ic 50 value >2 mm [75] . compounds similar to triclocarban (cid7547) and bisphenol a (bpa; cid6623) were found to inhibit the helicase activity of sv40 tag. based on the complex structure of the hcv ns3 helicase domain and soluble blue ht (pdb code 2zjo), triphenylmethanes, a more potent triphenylmethane [76] , and aurintricarboxylic acid (ata) [77] were developed to inhibit the helicase activity of hcv ns3. the nucleic acid binding site of helicase is also a promising site for the discovery of antiviral compounds that directly inhibit helicase-catalyzed unwinding [78] . for example, many dna-binding pharmacophores, such as anthracyclines, acridones, tropolones, and amidinoanthracyclines, have been optimized as hcv helicase inhibitors [78] . although there are numerous efforts that have been launched to find antivirals targeting viral helicase, little progress had been made to push them into clinical usage, and these projects have met great challenges on cytotoxicity, bioavailability, and pharmacokinetic properties [79] . replication and transcription complex blockers following viral entry, viral proteins, together with a number of host cell factors, assemble a viral replication and transcription complex (rtc) that is responsible for the production of the viral genome or other nucleic acids [80] . therefore, reagents that can efficiently block the formation of the viral rtc could conceivably inhibit viral proliferation. for example, once hcv enters the host cell, the genome of hcv will work as mrnas to produce viral proteins and form an rtc in which the host factors account for viral replication and transcription. ns5a is a membrane-associated nonstructural phosphoprotein, and it is believed that ns5a has no enzymatic activity but plays a critical role in regulating the formation of the hcv rtc. gao et al. identified a potent hcv inhibitor, bms790052, that targets ns5a and produced few side effects in a phase i clinical study [81] . although the exact mechanism by which bms790052 exerts its effects is yet to be defined, the resistance profile reveals that inhibitor sensitivity maps to the n terminus of domain 1 of ns5a. this inhibitor is further shown to block the hyperphosphorylation of ns5a, which is believed to play an essential role in the viral life cycle. this work, for the first time, proved the concept that small molecules targeting a non-traditional viral protein without any known enzymatic activity can also have profound antiviral effects with considerable promise for the treatment of hcv infection. during the replication of ev71 and other picornaviruses, polymerase (also named 3d pol ), 3b (also named vpg), and protease (also named 3c pro ) participate in the formation of viral rtc, together with host polya-binding protein 1 (pabp-1) and polyc-binding protein 2 (pcbp-2) [15] . unlike many other viruses, the replication of the picornavirus genome is initiated by the 5 0 end of genome covalently linked to vpg through a so-called vpg uridylylation process [82] . although vpg-binding sites vary in picornaviruses [14, 15, 83, 84] , the binding of vpg to 3d pol is known to be critical for vpg uridylylation and virus replication. in our recent study, we demonstrated that a ten amino acid peptide of vpg can effectively inhibit the vpg uridylylation process with an ec 50 as low as 50 nm (z. lou et al., unpublished). these results shed light on discovering future indvtas. throughout the life cycle of a negative sense single-stranded rna (ssrna) virus, the genome length rna is encapsidated by a virally encoded nucleoprotein (np), instead of a naked rna, and associated with rdrp (polymerase complex) to form a stable ribonucleoprotein (rnp) complex, which is responsible for virus replication, transcription, and assembly [85] . during this process, np can protect the rna against exogenous nucleases or the innate immune system in the host cell. based on the crystallographic achievements regarding the ssrna virus rnp complex [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] over the past few years, great progress has been made in this new putative antiviral strategy. kao et al. first identified a compound, nucleozin, that triggers the aggregation of and inhibits the nuclear accumulation of np; this compound can inhibit the replication of influenza virus with a nanomolar median effective concentration (ec 50 ) [97] . in a parallel effort, gerritz et al. discovered a series of influenza replication inhibitors and showed that they interfere with np-dependent processes via the formation of higher order np trends in pharmacological sciences february 2014, vol. 35, no. 2 oligomers with an ec 50 of 60 nm [98] . notably, the structure of the np in complex with a representative compound from this class of inhibitors revealed that two molecules of an inhibitor in an antiparallel orientation lock two adjacent np protomers. this unexpected quaternary complex explained the viral inhibition via ligand-induced formation of stable np oligomers [98] . in addition to inhibiting np, the disruption of the polymerase complex of influenza virus has been proposed for antiviral strategies. based on the complex structure of the pa c-terminal domain (pa c ) and the first 25 amino acids of pb1 [99] , a subset of modifications on n-terminal peptide of pb1 was shown to diminish the binding affinity of pa and pb1, inhibit polymerase activity, and attenuate the replication of influenza virus [100] [101] [102] . moreover, the structure of sftsv np in complex with suramin, an antiviral inhibitor, revealed that the blockers bind to the rna-binding cavity and can attenuate sftsv replication; this indicated that targeting rnp formation may be a new therapeutic antiviral approach [103, 104] . because both the polymerase complex and np show significant conservation between different influenza viruses, these results demonstrated that targeting the formation of viral rnp is a valid approach to the development of small molecule therapies against serious antiviral resistance to currently available drugs, such as adamantanes or neuraminidase inhibitors. other key interactions between viral proteins and/or host factors play essential roles in virus entry, replication, and maturation. a very interesting example is the interaction between hpv e1 and e2 protein, helping e1 helicase to tether to the hpv origin of replication. this interaction can therefore be used to guide the development of antivirals treating hpv infection. the disruption of the hpv e1-e2 interaction was first facilitated by a very potent inhibitor, chembl1207308. this inhibitor can effectively diminish the interaction between hpv e1 and e2 and thus inhibit hpv proliferation with an ec 50 of 6 nm [105] . although a few indvtas have been discovered for antiviral therapy, their mechanisms of working are still required to be further investigated. we cannot conclude the possibility that indvtas may have additional functions (or may be the major function) and the category of antivirals will be refined according to further knowledge. viruses utilize many host factors for their efficient proliferation. the correct functions of these host factors are crucial for virus replication and, therefore, compounds that regulate the function of host factors can be introduced as antiviral agents. next we discuss two host factors that are involved in broad spectrum virus infection. apart from the acquired immune response, host cells mount a number of defenses, such as the innate immune response, to virus infection. interferon (ifn) is one of the most crucial molecules in the innate immune response and acts as the primary switch for initiating antiviral immunity in vertebrates. upon being infected by a virus, host cells produce and secrete type i (mainly ifn-a and ifn-b) and type iii (ifn-l) ifns. these secreted ifns interact with the membrane-anchored ifn receptors (ifnars), mainly ifnar1 and infar2, and subsequently stimulate and upregulate the expression of hundreds of ifn-stimulated genes (isgs) to inhibit the replication of viruses [106] . among these isgs, ifn-inducible transmembrane (ifitm) proteins restrict the entry of influenza virus, west nile virus (wnv), and denv. additionally, mx (myxovirus resistance) gtpases can inhibit the correct function of viral nucleocapsids and polymerases, such as influenza virus [107] . due to the significance of ifn in the host cells to restrict viral infections, ifn is used in certain instances as a primary antiviral therapy, particularly in the absence of an effective antiviral or vaccination strategy. the use of ifn for antiviral therapy was first developed for treating hbv infection [108] and subsequently applied to treat hcv in combination with ribavirin [109] . ifn-b [110] and ifn-g [111] have also been evaluated for use in anti-hcv treatment. to further improve the therapeutic efficacy of ifn-a therapy, several options are being investigated. some clinical benefits were observed in pilot studies with ofloxacin [112, 113] and an immunomodulatory peptide, a1-thymosin [114] . in particular, peg-modified ifn-a [115] combined with ribavirin is now standard treatment for hcv infection. cyclophilins (cyps) are key cellular factors that function in numerous cellular processes, including transcriptional regulation, the immune response, protein secretion, and mitochondrial function [116, 117] . cyclophilin a (cypa) is a key member of the cyp family and was first identified as a mediator of the immunosuppressive function of cyclosporin a (csa) through the formation of a csa-cypa complex. this complex binds to and inhibits the function of the protein phosphatase calcineurin [118] , which normally functions to dephosphorylate nf-at, a transcription factor important for t cell activation. cypa is also known to play critical roles in the proliferation of viruses, including hiv-1, influenza virus, hcv, vesicular stomatitis virus (vsv), vaccinia virus, sars-cov, rotavirus (rv), and hpv [117, 119] , by interacting with viral proteins or facilitating ifn-b production [14, [120] [121] [122] [123] . cypa was first revealed to be incorporated into hiv-1 virions by interacting with the capsid protein (ca), and an interaction between newly synthesized hiv-1 ca and cypa is required for hiv-1 to induce dendritic cell maturation [114, 124] . cypa also interacts with other hiv-1 proteins, such as vpr and p6, to regulate hiv infection [88, 91, [125] [126] [127] [128] . several lines of evidence indicate that cypa and cyclophilin b (cypb) [129] function in the replication of hcv by either increasing the affinity of hcv polymerase ns5b for viral rna to enhance hcv replication [130] or binding to the hcv ns5a protein to aid viral replication [131] . the discovery of cypa inhibitors as antiviral agents started with the immunosuppressive drug csa that inhibits hcv [129] and hiv [132] . on the basis of these results, cypa antagonists, which are often derived from review trends in pharmacological sciences february 2014, vol. 35, no. 2 csa, have been developed, including alisporivir (debio-025), nim811, and scy635; these compounds lack immunosuppressive effects but retain high-affinity cypa binding and show very good antiviral effects against hiv or hcv infection [133] [134] [135] . hiv-1 co-receptors antagonists hiv-1 co-receptor antagonists that block the interactions between hiv-1 and ccr5 and/or cxcr4 have also been introduced to the anti-hiv efforts, and a few of these have been successful. for instance, ccr5 antagonists that potently inhibit hiv-1 replication and have good pharmaceutical properties, including aplaviroc [136] , maraviroc (the only clinically used anti-hiv drug as a co-receptor antagonist) [137] , vicriviroc [138] , and cenicriviroc [139, 140] , have been successfully advanced to phase ii/iii clinical trials or approved for clinical usage in the past 5 years. however, the precise mechanisms of these co-receptor antagonists are still not clear. recently, the crystal structures of ccr5 [141] and cxcr4 [142] have been reported. these two structures provided the atomic information of the ligand-binding pocket and the sites for co-receptor oligomerization. further structural study on the complex of ccr5 and cxcr4 with their antagonists will promote the investigation into the inhibitory mechanisms of these inhibitors and help us to increase their anti-hiv efficacy. at present, diverse antiviral drugs are clinically approved or in the later stages of clinical trials. most are based on the conserved mechanisms described in this review, in particular through targeting of viral polymerases and proteases. however, the majority of drug-resistant infection cases have been reported with the usage of antivirals based on this strategy. the requirement for new antiviral drugs to treat chronic infectious diseases and the emergence of more efficient new viruses serve as catalysts for research to find additional targets and mechanisms for antiviral development. a few novel strategies have been introduced for antiviral research, including inhibitors of viral mtase and helicase, blockages of viral rtc formation (e.g., nucleozin to influenza), and host factor antagonists or agonists. however, protease and polymerase inhibitors still occupy a dominant place among antivirals. this is because we still do not have very precise knowledge on the mechanisms underlying alternative strategies, and this requires further investigations on their mechanism before these strategies can be widely used for antiviral development, in particular those targeting drug-resistant viral infections. in our opinion, protease and polymerase inhibitors will still be the first, and probably the major, choice in the development of therapies against emerging novel viruses. in addition, we must consider another important aspect for antiviral development. for some viruses, currently available drugs can effectively eliminate the virus in the host, but genetic components are left behind. thus, the host still suffers from the infection because the virus can integrate its genetics into the host cell. for example, treatment with the combination of inf and adefovir (or other antivirals) can effectively, if not completely, reduce hbv titer in human liver. however, the covalently closed circular dna (cccdna) cannot be removed and still exists in the nuclei of infected liver cells, where it continuously coordinates the expression of hbv antigens. therefore, new mechanisms and strategies to completely remove viral components integrated in host cells, as well as to kill the virus itself, are required in future antiviral development. molecular architecture of the uncleaved hiv-1 envelope glycoprotein trimer elicitation of hiv-1-neutralizing antibodies against the cd4-binding site biological and pharmacokinetic properties of a novel immunoglobulin-cd4 fusion protein recombinant soluble cd4 therapy in patients with the acquired immunodeficiency syndrome (aids) and aids-related complex. a phase i-ii escalating dosage trial treatment of advanced human immunodeficiency virus type 1 disease with the viral entry inhibitor pro 542 antiviral activity, pharmacokinetics, and safety of bms-488043, a novel oral small-molecule hiv-1 attachment inhibitor, in hiv-1-infected subjects in vitro antiviral characteristics of hiv-1 attachment inhibitor bms-626529, the active component of the prodrug bms-663068 a mutant influenza virus that uses an n1 neuraminidase as the receptor-binding protein structure of the catalytic and antigenic sites in influenza virus neuraminidase structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 a resolution antivirals and resistance: influenza virus laninamivir and its prodrug, cs-8958: longacting neuraminidase inhibitors for the treatment of influenza clinical experience in adults and children treated with intravenous peramivir for 2009 influenza a (h1n1) under an emergency ind program in the united states crystal structure of enterovirus 71 rnadependent rna polymerase complexed with its protein primer vpg: implication for a trans mechanism of vpg uridylylation enterovirus 71 vpg uridylation uses a twomolecular mechanism of 3d polymerase rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen crystal structure of equine rhinitis a virus in complex with its sialic acid receptor a soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection inhibitory effect of dibenzofuran and dibenzosuberol derivatives on rhinovirus replication in vitro; effective prevention of viral entry by dibenzosuberenone peptides corresponding to a predictive a-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry the safety, plasma pharmacokinetics, and antiviral activity of subcutaneous enfuvirtide (t-20), a peptide inhibitor of gp41-mediated virus fusion, in hiv-infected adults a phase ii clinical study of the long-term safety and antiviral activity of enfuvirtide-based antiretroviral therapy potent hiv fusion inhibitors against enfuvirtideresistant hiv-1 strains synthesized peptide inhibitors of hiv-1 gp41-dependent membrane fusion design of helical, oligomeric hiv-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus design and evaluation of sifuvirtide, a novel hiv-1 fusion inhibitor pegylation, successful approach to drug delivery glycoengineering: the effect of glycosylation on the properties of therapeutic proteins ads-j1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation design, synthesis, and biological activity of novel 5-((arylfuran/1h-pyrrol-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thi azolidin-4-ones as hiv-1 fusion inhibitors targeting gp41 design, synthesis, and structure-activity relationship of a novel series of 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans as hiv-1 entry inhibitors a low-molecular-weight entry inhibitor of both ccr5-and cxcr4-tropic strains of human immunodeficiency virus type 1 targets a novel site on gp41 viral membrane fusion picornavirus uncoating intermediate captured in atomic detail in vitro and in vivo evaluation of ribavirin and pleconaril antiviral activity against enterovirus 71 infection treatment of picornavirus infections a novel basis of capsid stabilization by antiviral compounds stabilization of poliovirus by capsidbinding antiviral drugs is due to entropic effects design, synthesis, and structure-activity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus 71 inhibitors jr (1968) evidence for large precursor proteins in poliovirus synthesis translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme mechanisms and enzymes involved in sars coronavirus genome expression identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes deubiquitinating function of adenovirus proteinase the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism function from tmc114 to darunavir: five years of data on efficacy implications of antiretroviral resistance on viral fitness protease inhibitors for the treatment of chronic hepatitis c genotype-1 infection: the new standard of care hiv protease inhibitors in pregnancy: pharmacology and clinical use rational design of polymerase inhibitors as antiviral drugs drug targets in cytomegalovirus infection entecavir (bristol-myers squibb) new drugs for chronic hepatitis b: a review antiviral drug advances in the treatment of human immunodeficiency virus (hiv) and chronic hepatitis c virus (hcv) a novel lead for specific anti-hiv-1 agents: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine a new class of hiv-1-specific 6-substituted acyclouridine derivatives: synthesis and anti-hiv-1 activity of 5-or 6-substituted analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (hept) pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity structure-activity relationship studies on clinically relevant hiv-1 nnrtis non-nucleoside inhibitors of the hcv ns5b polymerase: progress in the discovery and development of novel agents for the treatment of hcv infections next-generation integrase inhibitors: where to after raltegravir? clinical use of hiv integrase inhibitors: a systematic review and meta-analysis co-formulated elvitegravir, cobicistat, emtricitabine, and tenofovir versus co-formulated efavirenz, emtricitabine, and tenofovir for initial treatment of hiv-1 infection: a randomised, double-blind, phase 3 trial, analysis of results after 48 weeks structural biology of dengue virus enzymes: towards rational design of therapeutics a structural basis for the inhibition of the ns5 dengue virus mrna 2 0 -o-methyltransferase domain by ribavirin 5 0 -triphosphate flaviviral methyltransferase/rna interaction: structural basis for enzyme inhibition structural and functional analyses of a conserved hydrophobic pocket of flavivirus methyltransferase identification of a novel antiviral inhibitor of the flavivirus guanylyltransferase enzyme development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target a screen for modulators of large t antigen's atpase activity uncovers novel inhibitors of simian virus 40 and bk virus replication biphenylsulfonacetic acid inhibitors of the human papillomavirus type 6 e1 helicase inhibit atp hydrolysis by an allosteric mechanism involving tyrosine 486 discovering new medicines targeting helicases: challenges and recent progress discovery of small-molecule inhibitors of the atpase activity of human papillomavirus e1 helicase structure-based discovery of triphenylmethane derivatives as inhibitors of hepatitis c virus helicase characterization of aurintricarboxylic acid as a potent hepatitis c virus replicase inhibitor ns3 helicase inhibitors viral and cellular rna helicases as antiviral targets the dependence of viral rna replication on co-opted host factors chemical genetics strategy identifies an hcv ns5a inhibitor with a potent clinical effect protein is linked to the 5 0 end of poliovirus rna by a phosphodiester linkage to tyrosine the structure of a protein primerpolymerase complex in the initiation of genome replication the crystal structure of coxsackievirus b3 rnadependent rna polymerase in complex with its protein primer vpg confirms the existence of a second vpg binding site on picornaviridae polymerases architecture and regulation of negative-strand viral enzymatic machinery nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and rna polymerization the structure of native influenza virion ribonucleoproteins crimean-congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses structure of the lassa virus nucleoprotein reveals a dsrna-specific 3 0 to 5 0 exonuclease activity essential for immune suppression crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation organization of the influenza virus replication machinery cap binding and immune evasion revealed by lassa nucleoprotein structure phleboviruses encapsidate their genomes by sequestering rna bases structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation structural basis for encapsidation of genomic rna by la crosse orthobunyavirus nucleoprotein identification of influenza a nucleoprotein as an antiviral target inhibition of influenza virus replication via small molecules that induce the formation of higherorder nucleoprotein oligomers crystal structure of the polymerase pa(c)-pb1(n) complex from an avian influenza h5n1 virus identification of high-affinity pb1-derived peptides with enhanced affinity to the pa protein of influenza a virus polymerase identification of a pa-binding peptide with inhibitory activity against influenza a and b virus replication disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza a virus the nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate rna encapsidation structure of crimean-congo haemorraghic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage optimization and determination of the absolute configuration of a series of potent inhibitors of human papillomavirus type-11 e1-e2 protein-protein interaction: a combined medicinal chemistry, nmr and computational chemistry approach structural linkage between ligand discrimination and receptor activation by type i interferons intrinsic antiviral immunity natural killer cell activity and function in chronic hcv-infected patients during peg interferon and ribavirin: early effects of active substance use interferon-a-2b plus ribavirin: a review of its use in the management of chronic hepatitis c dynamics of hepatitis c virus monitored by real-time quantitative polymerase chain reaction during first 2 weeks of ifn-b treatment are predictive of long-term therapeutic response interferon-g brings additive anti-viral environment when combined with interferon-a in patients with chronic hepatitis c treatment of chronic hepatitis c with ainterferon plus ofloxacin in patients not responding to a-interferon alone effects of combination therapy with interferon and ofloxacin on chronic type c hepatitis: a pilot study interferon and thymosin combination therapy in naive patients with chronic hepatitis c: preliminary results development of pegylated interferons for the treatment of chronic hepatitis c cyclophilin: a specific cytosolic binding protein for cyclosporin a insights into the roles of cyclophilin a during influenza virus infection calcineurin is a common target of cyclophilincyclosporin a and fkbp-fk506 complexes target cell cyclophilins facilitate human papillomavirus type 16 infection hepatitis b virus (hbv) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of hbv infection requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a cyclophilin a inhibits rotavirus replication by facilitating host ifn-i production binding of the human immunodeficiency virus type 1 gag polyprotein to cyclophilin a is mediated by the central region of capsid and requires gag dimerization cyclophilin a interacts with hiv-1 vpr and is required for its functional expression structural characterization of the hiv-1 vpr n terminus: evidence of cis/trans-proline isomerism hiv-1 p6-another viral interaction partner to the host cellular protein cyclophilin a the host-pathogen interaction of human cyclophilin a and hiv-1 vpr requires specific n-terminal and novel c-terminal domains cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns5a protein the effect of cyclosporine a on infection of susceptible cells by human immunodeficiency virus type 1 debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis c virus (hcv) repliconcontaining cells when used alone or in combination with specifically targeted antiviral therapy for hcv (stat-c) inhibitor scy-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis c virus rna replication in vitro inhibition of human immunodeficiency virus type 1 replication in human cells by debio-025, a novel cyclophilin binding agent antiviral activity and safety of 873140, a novel ccr5 antagonist, during short-term monotherapy in hiv-infected adults maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naive subjects with ccr5-tropic hiv-1 infection vicriviroc plus optimized background therapy for treatment-experienced subjects with ccr5 hiv-1 infection: final results of two randomized phase iii trials pharmacokinetics and pharmacodynamics of tbr-652, a novel ccr5 antagonist, in hiv-1-infected, antiretroviral treatment-experienced, ccr5 antagonist-naive patients safety, efficacy, and pharmacokinetics of tbr-652, a ccr5/ccr2 antagonist, in hiv-1-infected, treatmentexperienced, ccr5 antagonist-naive subjects structure of the ccr5 chemokine receptor-hiv entry inhibitor maraviroc complex structures of the cxcr4 chemokine gpcr with small-molecule and cyclic peptide antagonists parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses structure of a v3-containing hiv-1 gp120 core a sensor-adaptor mechanism for enterovirus uncoating from structures of ev71 this work was supported by the ministry of science and technology of china '973' project (grant numbers 2013cb911103 and 2014cb542800) and the national natural science foundation of china (grant numbers 81322023, 31000332, 31100208, 31170678, and 31370733). key: cord-286334-d9v5xtx7 authors: li, rui; qiao, songlin; zhang, gaiping title: analysis of angiotensin-converting enzyme 2 (ace2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov date: 2020-04-30 journal: journal of infection doi: 10.1016/j.jinf.2020.02.013 sha: doc_id: 286334 cord_uid: d9v5xtx7 nan a novel coronavirus from wuhan in central china, named 2019-ncov, has recently caused an epidemic of pneumonia in humans and posed a huge threat to global public health. 1 , 2 to the date 06/02/2020, 2019-ncov has led to more than 31,0 0 0 confirmed cases and 637 deaths in china according to national health commission of the people's republic of china ( http://en.nhc.gov. cn/index.html ). cases have also been documented in a growing number of other international locations, including the united states ( https://www.cdc.gov/coronavirus/2019-ncov/index.html ). as a consequence, it is urgent to develop effective measures to control this novel coronavirus on the basis of its pathogenesis. host receptor recognition is a determinant for virus infection. during the time of this letter preparation, three works have just been published to explore the receptor usage of 2019-ncov. a work by zheng-li shi et al. has shown that angiotensin-converting enzyme 2 (ace2), the receptor for severe acute respiratory syndrome coronavirus (sars-cov), 3 from human, rhinolophus sinicus bat, civet, swine but not mouse mediate 2019-ncov infection in vitro , while the detailed mechanisms are not yet determined. 4 the other two works have reported or predicted human ace2 usage of 2019-ncov in a similar way to sars-cov mainly based on the coronavirus spike (s) glycoproteins. 5 , 6 considering the fact that the s proteins mutate and gain capability to recognize host receptors among species, 7 , 8 there is still a lack of analyses on receptor usage of 2019-ncov from the receptor perspective, which does not evolve as quickly as viruses. here, we firstly performed amino acid sequence alignment of ace2 from different species, including human, five non-human primates (gibbon, green monkey, macaque, orangutan and chimpanzee), two companion animals (cat and dog), six domestic animals (bovine, sheep, goat, swine, horse and chicken), three wild animals (ferret, civet and chinese horseshoe bat) and two rodents (mouse and rat). the alignment by clustal w 2.1 shows that they share a high sequence similarity except chicken (data not shown). the result suggests that 2019-ncov of probable bat origin may not interact with chicken ace2 and subsequently infect them, which were not considered in the following analyses. in ace2, the regions at position 30-41, 82-84 and 353-357 are demonstrated to be involved in the interaction with sars-cov s protein, where the residues at positions 31, 35, 38, 82 and 353 are critical. 9 therefore, we took a close comparison in these regions and residues. as shown in fig. 1 , human and non-human primates share the identity sequences in the regions and residues, implying that ace2 from non-human primates may recognize 2019-ncov and medi-ate its infection. as a result, non-human primates may be susceptible to 2019-ncov and serve as animal models for antiviral research or intermediate hosts for cross-species transmission. in fig. 1 , the residues of most companion, domestic and wild animals are conserved, especially for the critical ones stated above, while certain ones are variable. for example, lys31, glu35, asp/glu38 and lys353 are conserved, which probably form salt bridges. interestingly, the changes at positions 31, 38 and 82 are observed. these changes suggest steric hindrance and electrostatic interference for host-virus interaction. taking civet ace2 as an example, the change of lys31 to thr31 is likely to form a hydrogen bond instead of a salt bridge. in addition, the polar side chain of thr82 may influence the hydrophobic interaction of the original met82. all these changes may result in a lower binding affinity. however, an additional region covering residues 90-93 has been shown to be involved in civet ace2 binding to sars-cov and enhance their interaction. 10 consequently, we can't preclude the existence of other regions to compensate for the residue changes. with most residues in human ace2, the ones from these compaion, domestic and wild animals may be favorable for 2019-ncov recognition, which is in consistent with the recent work by zheng-li shi et al. in case cross-species transmission, close contact with sick or asymptomatic companion, domestic and wild animals should be cautious, such as for workers in livestock farming and travellers in the wild. in contrast, certain significant changes occur in the mouse and/or rat ace2 compared to the human one ( fig. 1 ) . the asn31 and ser82 in mouse ace2 may not form favorable interactions with 2019-ncov due to their electrostatic or hydrophilic characteristics. importantly, the change into his353 in both mouse and rat ace2 does not form a strong salt bridge as lys353 does. since the structural information for mouse and rat ace2 is unavailable, we carried out homology modeling using human ace2 (pdb code 2ajf) as template on online ( https://swissmodel.expasy.org ) for further analyses. in fig. 2 a, the change into ser82 in mouse ace2 may interfere with the hydrophobic interaction of the original met82. additionally, the changes into asn31 and his353 definitely affect the salt bridge formation and electrostatic potential. the change into his353 in rat ace2 is similar in the effect on receptor-virus interaction ( fig. 2 b) . these analyses partially explain why mouse ace2 does not mediate 2019-ncov infection reported by zheng-li shi et al. and assume that rodents are not likely to be the susceptible host. in conclusion, we conducted sequence and structural analyses of angiotensin-converting enzyme 2 (ace2) from different species, which sheds some light on cross-species receptor usage of 2019-ncov. all these analyses raise an alert on a potential interspecies transmission of 2019-ncov and propose further surveillance in other animal populations. structural studies on human and other species ace2 in complex with 2019-ncov spike protein will con the authors declare no conflict of interest. very recently, a letter in journal of infection reported the outbreak of the novel cornonavirus from dec. 2019 in china, especially in hubei province. 1 this novel cornonavirus may originate from the bat, 2 is just named as the covid-19 by the world health organization (who). the covid-19 outbroke from wuhan, the capital of hubei province, has spread to other provinces of china and even other countries. 3 strong human-to-human transmission is established. 4 until feb. 11, 2020, there have been 44653 cases of covid-19 infections confirmed in mainland china, including 1113 deaths. to prevent and control the spread of the epidemic, many strategies are needed. 5 predicting the trend of the epidemic are quite important to the allocation of medical resources, the arrangement of production activities, and even the domestic economic development all over china. therefore, it is very urgent to use the latest data to establish an efficient and highly suitable epidemic analysis and prediction model according to the actual situation, and then to give reliable predictions, which could provide an important refer-ence for the government to formulate emergency macroeconomic decisions and medical resources allocation. recently, the susceptible-exposed-infectious-recovered (seir) or other similar models 6,7 are used to forecast the potential domestic and international spread of this covid-19 epidemic with parameters estimated from other sources.the real situation could be much more complicated and changing all the time. especially, with the implementation of the chinese government's multiple epidemic control policies, the control of nationwide epidemic has become obvious. however, the medical supplies in hubei will still affect the implementation of national policies. in this letter, we present the current situation of the epidemic, predict the ongoing trend with data driven analysis, and estimate the outbreak size of the covid-19 in both hubei and other areas in mainland china. the data of the epidemic are listed in table 1 and also graphically shown in fig. 1 , in which "china" is used to denote the mainland china, and "other" mainland china other than hubei province. the data includes the daily confirmed(suspected) infections, totally confirmed(suspected) infections, daily deaths, and total deaths from jan. 20, to feb. 11, 2020, reported by the national health commission of the republic of china (nhc), 8 , and health commission of hubei province (hch). 9 jan. 20, 2020, containing all the cases reported from 0 to 24, is the zeroth day in this letter, and then others are implied. the total number of suspected cases reaches the peak value on the 19th day (feb. 8), and then drops rapidly. notice that, until feb. 11, 2020, almost all the cases of deaths (1068/1113, 96%,) locates in hubei province, which reveals the epidemic in hubei is much more serious than that in the other areas of china. on the hand, it states the strict quarantine and limitation on population mobility have effectively prevented outbreaks in other provinces of china. scribe the data of daily infections and deaths in hubei, where x = (t + 0 . 5 − t t ) with t denoting the day, and t t representing the turning point; a and k are the parameters and determined by the data together with t t . the cumulative data of infections or deaths are obtained by the integration over h ( t ). for the epidemic in the other areas of china, the data of infections shows an asymmetric character, and then will be described as where x = t − t t ; the parameters b, k 1 , and k 2 together with t t , are then determined by fitting to the data. fig. 2 shows the fit and trend predictions to the total infections and deaths in hubei and china other than hubei. the extracted turning point of the infections in hubei is the 17th day, namely, feb. 6, 2020. the epidemic in hubei is predicted to end after mar. 10, 2020. we estimated that the epidemic is to end up with a total of 39, 0 0 0 infections in hubei, not including the clinically diagnosed cases since feb. 12, which may enlarge the prediction by 1.4 times. with considered data, namely, data from jan. 20 to feb. 11, the average errors are bout 166 and 190 for the fits to describe the daily and cumulative infections in hubei, respectively, corresponding to 8.6% and 1.6% for the average relative errors, respectively. fig. 2 (b) and (e) shows the estimations of the total and daily deaths in hubei. the predicted turning point is feb. 12, 2020. the total deaths is estimated to be 2250. notice the distribution of the daily deaths is delayed about 5 ∼6 days compared with the that of the daily infections. the average errors are bout 4 and 22 for the model to describe the daily and cumulative death numbers, respectively, corresponding to the relative errors 8.6% and 6.2%, respectively. the numbers of the daily and total infections in china other than hubei are showed in fig. 2 (c) and 2 (f), respectively. the extracted turning point is feb. 1, 2020 and the epidemic is expected to end on the 45th day, namely, on mar. 5, 2020. the estimated number of cumulative infections is about 12,600 in china other table 1 the data of epidemic caused by the covid-19 pneumonia in the mainland china and hubei, including (a) daily infections, (b) daily deaths, (c) total infections, (d) total deaths, (e) daily and (f) total suspected cases. china hubei a b c d e f a b c d 2020/1/20 77 2 291 6 27 54 72 2 270 6 2020/1/21 149 3 440 9 26 37 105 3 375 9 2020/1/22 131 8 571 17 257 393 69 8 4 4 4 17 2020/1/23 259 8 830 25 680 1072 105 7 549 24 2020/1/24 4 4 4 16 1287 41 1118 1965 180 15 729 39 2020/1/25 688 15 1975 56 1309 2684 323 13 1052 52 2020/1/26 769 24 2744 80 3806 5794 371 24 1423 76 2020/1/27 1771 26 4515 106 2077 6973 1291 24 2714 100 2020/1/28 1459 26 5974 132 3248 9239 840 25 3554 125 2020/1/29 1737 38 7711 170 4148 12,167 1032 37 4586 162 fig. 1 (f)), we did not parameterize this data, and hence did not give a trend prediction. the covid-19 epidemic in china is predicted to end after mar. 20, 2020, and cause 52,0 0 0-68,0 0 0 infections and about 240 0 deaths. however, the data trends show that the quick and active strategies to reduce human exposure taken in china, such as limi-tation on population mobility and interpersonal contact rates, strict quarantine on migrants, have already had good impacts on control of the epidemic. now the outbreak and deaths of the covid-19 epidemic are mainly in hubei province. after this letter has been written, the hubei reported 14,840 confirmed infections (including 13,332 clinically diagnosed cases) on feb. 12, 2020, which is almost 9 times greater than the data of the previous day. the huge fluctuation is due to the changing of diagnostic criteria in hubei. and this clinical criteria taken in hubei is expected to play an active and important role in controlling the outbreak and death rate. the authors declare no conflict of interest. dear editor , recently, several studies in this journal have highlighted the threat of avian influenza virus (aiv) to humans, poultry, and other animals. [1] [2] [3] [4] [5] [6] in equines, there was only one reported influenza outbreak caused by aiv, which occurred in 1989-1990 in china. however, aiv still poses a potential threat to equines. in contrast to aiv, equine influenza virus (eiv) is a commonly known causative pathogen of acute respiratory disease in equines. to date, two subtypes of eivs have been determined in the equine population worldwide: h7n7 and h3n8. 7 h7n7 eiv was initially identified in prague in 1956, and the last outbreak caused by h7n7 eiv occurred in 1979. 7 h3n8 eiv was first isolated in miami in 1963 and is currently responsible for ei outbreaks worldwide. during continuous transmission and evolution in equines, h3n8 eiv strains diverged genetically into three distinct lineages: predivergent, european, and american. 7 historically, there have been four main ei outbreaks in mainland china, occurring in 1974 china, occurring in , 1989 china, occurring in -1990 china, occurring in , 1994 , and 20 07-20 08. in the first, third, and fourth outbreaks, the isolated eiv strains were of the h7n7 subtype, h3n8 subtype european lineage, and h3n8 subtype american lineage, respectively. in the second ei outbreak, the causative pathogen (a/equine/jilin/1/1989) was determined to be h3n8 subtype influenza a virus (iav) by antigenicity characterization. however, after genetic sequencing, all eight segments of a/equine/jilin/1/1989 were genetically closer to those of avian influenza virus than those of eiv, indicating an interspecies transmission event. the 1989-1990 ei outbreaks in china contain two major outbreaks. the first ei outbreak occurred in jilin and heilongjiang provinces between march and june 1989, with a morbidity of 81% and a mortality of up to 20% in some herds. the second ei outbreak occurred in heilongjiang province in april 1990, with a morbidity of 41% and no mortality. the high mortality and unique antigenicity of a/equine/jilin/1/1989 attracted the attention of eiv researchers. however, after the 1989-1990 ei outbreaks in china, the virus disappeared from the equine population for unknown reasons. although no evidence in the epidemiological investigation worldwide supports the continuous circulation of a/equine/jilin/1/1989-like eiv in equines, we should not underestimate the potential of interspecies aiv transmission to equines and the possibility of future ei outbreaks caused by aiv. the haemagglutinin (ha) of iavs recognizes and binds the cell surface sialic acid (sa) receptor of the host respiratory tract, and then the virus enters into cells and replicates. the distribution of the sa receptor influences the host range of iavs. human influenza viruses preferentially bind the saa2,6 gal receptor, while aivs preferentially bind the saa2,3 gal receptor. it has been reported that the saa2,3 gal receptor is abundant in the epithelial cells of the horse trachea, and animal experiments indicate that iavs with an ha recognizing the saa2,3 gal receptor could replicate in horses. this finding provides a prerequisite for cross-species transmission of aiv to equines. in fact, there were several pieces of direct molecular evidence supporting the interspecies transmission of aiv to equines, in addition to the 1989-1990 ei outbreaks in china. in 2010, abdel-moneim et al. reported one h5n1 eiv strain (a/equine/egypt/ av1/2009) isolated from donkeys with cough, fever and serous nasal discharge in egypt. 8 sequencing results indicated that the virus had a close genetic relatedness to h5n1 aiv. in addition, h5 seroconversion was observed in 25.71% (27/105) of the examined donkeys. in 2011, he collected 284 equine lung tissue samples in china and isolated one aiv-derived h9n2 eiv strain (a/equine/guangxi/3/2011) ( https://kns.cnki.net/ kcms/detail/detail.aspx?dbcode=cmfd&dbname=cmfd201301& filename=1012496506.nh&v=mtmxmzc3ck5wrji2sexleedove1x wkviuelsogvymux1efltn0romvqzcvryv00xrnjdvvjmt2vazvpt rknua1u= ). among the tested equine serum samples in china, 0.54% (7/1110) of samples were positive for anti-h9n2 antibody. in the 2015-2016 ei outbreaks in pakistan, khana et al. reported that the isolated eiv strain (a/equine/pakistan/16) was reassorted from aiv. 9 the common characteristic for the reported cross-species transmission events of aiv to equines in china, pakistan, and egypt is that they occur in farming equines. 8 , 9 in the mixed farming system, equines and domestic poultry often live in close proximity. compared with racehorses, farming equines have more opportunities to contact domestic poultry and experience long-term environmental exposure to poultry. aiv infections in poultry increase exposure risks to equines. recently, frequent reports of cross-species transmission events of aiv to farming dogs in china and korea also indicate the potential threat of aiv to farming animals with close contact with poultry. 10 another problem is that the farming equines in china, pakistan, and egypt had no vaccination history. even in some racehorse populations, vaccinations for eiv are not routinely performed as recommended by the world organization for animal health (oie) expert surveillance panel on ei vaccine composition. although h3n8 eiv is antigenically distinct from a/equine/jilin/1/1989, animal experiments indicated that high doses of ei vaccines still provided complete protection against challenge with a/equine/jilin/1/1989. accordingly, routine vaccination with h3n8 ei vaccines in equines might prevent aiv infection to some degree, at least for h3n8 subtype aiv. several strategies may help reduce the threat of aiv to equines, including reducing exposure of equines to poultry, birds, and other hosts of iav, especially animals with clinical signs of influenza virus infection; monitoring aiv prevalence in domestic poultry around equines and routinely vaccinating domestic poultry with aiv vaccines; vaccinating susceptible equines with ei vaccines, especially farming equines in close contact with domestic poultry; and monitoring the prevalence of multiple aiv subtypes in equines, not merely that of those restricted to h3n8 subtype. none. we read with interest recent articles in this journal regarding the utility of next-generation sequencing for the diagnosis bacterial meningitis. 1 , 2 bacterial meningitis causes substantial morbidity and mortality worldwide. 3 rapid identification of the microorganisms is essential for early initiation of appropriate antimicrobial therapy, thereby improving clinical outcome. yet routine diagnostic methods fail to identify the bacteria in the majority of patients. over the last decade, advanced sequencing technologies have greatly improved our capacity to detect the causative agents of infectious diseases in clinical samples. 4 , 5 of these, the single molecule real-time sequencing developed by oxford nanopore technologies (ont) is a promising tool for diagnostic setting because of its short turnaround time. in late april 2019, a 59-year old seller of fish-noodles was referred to our hospital with a 1-day history of headache, fever and vomiting. he had a history of heavy alcohol use and hepatitis c infection, and had cirrhosis and diabetes mellitus. on admission, he was unconsciousness (a glasgow coma scale of 8), with a body temperature of 40 °c, a blood pressure of 140/80 mmhg and neck stiffness. initial gram-stain and microscopy of csf showed grampositive cocci, 8449 white cells/ul with 91% neutrophils, elevated protein and low glucose level, and high lactate concentration ( fig. 1 a) . routine bacterial culture, plus streptococcus pneumoniae and s. suis pcrs were all negative. he was diagnosed with bacterial meningitis, and given a combination of ceftriaxone (2 g/12 h) and dexamethasone (0.4 mg/kg/12 h). his clinical condition steadily improved. his second and third csf samples became negative by gram stain. the other csf parameters also improved, except the glucose, which remained low ( fig. 1 a) . on day 20 of hospitalization, the patient suddenly became unconsciousness with fever. brain magnetic resonance imaging showed bifrontal abscesses ( fig. 1 b) . after consulting a local neurosurgeon, aspiration of the brain abscesses was not advised and the patient was treated empirically with meropenem (2 g/8 h) and vancomycin (1 g/8 h). due to continued diagnostic uncertainty, we performed 16s rrna sequencing of the admission csf, stored as part of an going clinical study (supplementary materials), using an established sangersequencing based 16s rrna method. 6 subsequently, analysis of the obtained sequences revealed evidence of s. agalactiae (supplementary figure 1 ). given this new diagnostic result of the admission csf and because the patient had recovered clinically, the patient was given 24 million units of penicillin g for every 4 h. after day 43 of hospitalization, all csf parameters had normalised ( fig. 1 a) . likewise, on ct scan the brain abscess was now significantly improved ( fig. 1 c) . the patient was discharged with full clinical recovery. additionally, minion sequencing of complete 16s rrna gene was retrospectively carried out on the extracted nucleic acid of the admission csf yielded a total of 14,848 reads after 100 min of sequencing run. of these, 11,556 reads (79%) were successfully aligned to s. agalactiae ( fig. 1 d) . the remaining reads were assigned to other streptococcus species (mostly s. dysgalacticiae ( n = 2.145, 14%)), likely attributed to a combination of the high level of sequence similarities of the 16s rrna region between them and the sequencing errors introduced by the minion systems. analysis of sequencing data generated during the 25, 50 and 75 min of sequencing run time also yielded the same results (supplementary figure 2 ). details about the minion procedure are presented in supplementary materials. to further assess of the utility of csf minion sequencing of 16s rrna gene for the detection of bacterial meningitis pathogens, six csf samples from patients with confirmed bacterial meningitis enrolled in the abovementioned clinical study were tested ( table 1 ) . analysis of the minion reads obtained after two hours of the sequencing run showed that the majority of reads were correctly assigned to the corresponding bacterial species ( s. pneumoniae and s. suis) or genus ( neisseria ) found in the csf samples by diagnostic work up of the clinical study ( fig. 1 e and table 1 ). additional analysis of the obtained reads generated at two earlier time points (20 min and 60 min) of the sequencing run generated the same results ( table 1 ) . collectively, we report the first application of minion sequencing of 16s rrna gene to detect bacterial meningitis causing pathogens in csf samples from a low and middle-income country. the assay was able to detect the bacterial causes in all of the seven tested csf samples. meanwhile, gram stain and culture, the two most commonly used methods in clinical microbiology laboratories worldwide, were negative in 3/7 samples. ( fig. 1 and table 1 ). in addition to csf samples described in the present study and a recent pilot study from korea, 7 successful detections of haemophilus influenzae in sputum and campylobacter fetus in culture materials by minion sequencing of 16s rrna have recently been reported. 8 together, the data suggest that minion sequencing of 16s rrna is a sensitive method for rapid and accurate detection of pan-bacterial pathogens, including unexpected microorganisms, in clinical samples. additionally, the bacterial species information generated by the analysis of 16s rrna sequences can be useful for disease surveillance and vaccine evaluation. thus, the application of the method would be relevant for both patient management and epidemiological research. indeed, to the best of our knowledge the present study represents the first report of s. agalactiae associated meningitis in vietnam. because the incidence of invasive diseases (including meningitis) caused by s. agalactiae has been reported with increased frequency in recent years, 9 s. agalactiae should be considered as an important differential owing to the unavailability of the reagents at the time of patient admission, we were not able to perform real-time diagnosis using minion sequencing on the collected csf samples. however, same day diagnosis is theoretically achievable, because the current workflow takes 5 -6 h to operate. prospective study is urgently needed to assess its translational potential in the diagnosis of bacterial meningitis. since september 2017, a prospective observational study aiming at exploring the utility potential of next-generation sequencing in patients presenting with central nervous system (cns) infections has been conducted in the brain infection ward of the hospital for tropical diseases (htd) in ho chi minh city, vietnam. htd is a tertiary referral hospital for patients with infectious diseases from southern provinces of vietnam, serving a population of > 40 million. any patient ( ≥16 years) with an indication for lumbar puncture was eligible for enrolment. patient was excluded if no written informed consent was obtained. as per the study protocol, csf, plasma and urine samples were collected at presentation alongside demographic, meta-clinical data and results of routine diagnosis. after collection, all clinical specimens were stored at −80 °c until analysis. the clinical study received approvals from the institutional review board of the htd and the oxford tropical research ethics committee of the university of oxford. written informed consent was obtained from each study participant or relative (if the patient was unconsciousness). sequencing of complete 16s rrna gene was retrospectively performed using minion nanopore sequencer (ont), following the manufacturer's instructions. in brief, amplification of the complete 16s rrna gene and library preparation were carried out on extracted nucleic acid using 16s barcoding kit (sqk-rab204, ont) and primers (27f 5 -agagtttgatcctggctcag-3 and 1492r 5 -ggttaccttgttacgactt-3 ), followed by the sequencing of the amplified product using r9.4 flow cells (ont). minion reads were first basecalled using albacore v2.1.7 (ont), followed by demultiplexing using porechop ( https://github.com/rrwick/porechop ). determination of bacterial genus/species composition in the obtained reads was then carried out using epi2me interface (metrichor, oxford, uk), a platform for cloud-based analysis of minion data. overall, the whole procedure of minion sequencing of 16s rrna gene takes 5-6 h to complete (supplementary figure 3) . we, the author of the submitted manuscript declare that we do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding). dear editor, several aspects of influenza have been highlighted recently, including its global, comparative seasonality, 1 and issues around rapid point-of-care testing. 2 in addition, the uk has a national surveillance programme, the uk severe influenza surveillance system (usiss) to monitor and investigate severe cases of influenza across the country, 3 including severe cases of influenza admitted to intensive care (icu) and high dependency units (hdu). 4 specifically, the aim of this latter arm was to "monitor and estimate the impact of seasonal influenza on the population" and to "describe the epidemiology of severe disease." this surveillance began in the 2011-2012 influenza season and has continued to the present, with mandatory participation by all nhs trusts. leicester is one of 5 nhs commissioned centres in the uk providing extra-corporeal membrane oxygenation (ecmo) support for severe acute respiratory failure in adults. this process involves draining deoxygenated venous blood from the superior and inferior vena cavae, pumping this blood through a membrane lung, where oxygenation and carbon dioxide elimination take place. oxygenated blood is delivered back into the right atrium, therefore replacing the function of the native lung. in this way, ecmo can be used to support patients with respiratory failure of any cause. acceptance for admission for ecmo support follows referral to the ecmo service via structured questionnaire and discussion with the ecmo consultant on-call. patients are commenced on ecmo where benefits are deemed to outweigh the risks in patients with potentially reversible respiratory failure, who are already on maximal conventional therapy at their referring centre, and who are not achieving lung protective ventilation. 5 here, as part of our national usiss role, we describe severe influenza cases that required ecmo support in whom the predominant indications were severe hypoxia with a pao 2 :fio 2 ratio of < 100 despite maximal conventional therapy, and/or hypercapnoeic respiratory failure with a ph < 7.2 despite ventilation pressures > 30 cm h 2 o. during the 2018-2019 influenza season 33 cases of severe influenza were admitted to glenfield hospital for ecmo from our referring centres. most were male (23/33, 69.7%, 28-62 years, bmi: 17-47; female 10/33, 27-51 years, bmi: 21-38), and of white british ethnicity (30/33, 91.0%; with 1 each of chinese, asian, african ethnicity). comorbidities included, obesity, hypertension, asthma, copd, diabetes, anxiety, depression, epilepsy, a history of smoking and alcohol use (or abuse). all cases except for one influenza a(h3n2) infection (possibly two as subtyping was not performed for another sample) were due to influenza a(h1n1)pdm09. only one case had a history of influenza vaccination. various 'on referral' ecmo-related parameters were extracted, as well as contemporaneous laboratory results. these were statistically compared between patients who died ( n = 8) and those who survived using t -test or mann-whitney test for continuous variables and fisherexact test for categorical variables. correlation between duration on ecmo and laboratory parameters was assessed using spearman's rank correlation coefficient. ( n = 24) ( tables 1 and 2 ) . surprisingly, it was found that on direct comparison, most of the referral parameters for patients starting ecmo were not statistically different between those influenza-infected patients that eventually survived ( n = 24) versus those who died ( n = 8) -a case fatality rate of 25%. one patient was dropped from this analysis due to some missing data. only the respiratory rate (rr, p = 0.041) and the lactate ( p = 0.008) showed statistically significant differences between the two groups, with higher values being found in the patients who ecmo -extra-corporeal membrane oxygenation; sofa -sequential organ failure assessment; peep -positive end expiratory pressure; h 2 o -water; bpm -breaths per minute; pao 2 /paco 2 -partial pressure of arterial oxygen/carbon dioxide; altalanine aminotransferase. * rank correlation coefficient measures the strength and direction of a relationship between two variables. the coefficient ranges from −1 to 1 with 1 indicating a strong positive relationship between two variables and −1 indicating a strong negative relationship. a correlation coefficient close to 0 means the relationship between the two variables is very weak. died ( table 1 ) . however, clinically, these differences are of doubtful significance, as the rr is at the discretion of the parent clinical team prior to referral to ecmo, and the lactate levels are normal in the survivors and only marginally elevated in those who died which will again be dependent on use of cvvh. the higher pao 2 :fio 2 (p:f) ratio was statistically significantly correlated ( p = 0.020) with a shorter duration of ecmo, which suggests those with less severe disease recover quicker ( table 2 ) . many studies have reported on intensive care patient outcomes of the 2009 influenza a(h1n1)pdm09 pandemic, with or without the use of ecmo. in one study from australia, where ecmo was used, of 68 patients with confirmed influenza a (53 a(h1n1)pdm09, 8 un-subtyped) infection, 14 (21%) had died mainly due to intracranial and other forms of haemorrhage ( n = 10), or intractable respiratory failure ( n = 4). 6 another study from the usa, where influenza a(h1n1)pdm09-infected patients had non-ecmo icu admission, of 154 cases, 48 (37%) developed acute respiratory distress syndrome (ards), of whom 37 (24%) died. 7 a more recent study from spain that reviewed influenza a(h1n1)pdm09 cases admitted to icu (non-ecmo) from 2009-2015, found that of a total of 2421 cases, the mortality ranged from 18.8% (for community-acquired influenza) to 32.9% (for hospitalacquired influenza). 8 these ecmo-icu case fatality rates of severe influenza a(h1n1)pdm09 infection are similar to ours of 25% reported here, though compared to the specific ecmo patient cohort, 6 the causes of death in our patients were more variable, including intracranial and other haemorrhage ( n = 3), sepsis and multi-organ failure ( n = 2), respiratory failure ( n = 1), post-cardiopulmonary resuscitation hypoxic brain injury ( n = 1), and ischaemic bowel associated with atrial fibrillation ( n = 1). thus, even after 10 years of experience with this 'new' pandemic influenza a(h1n1)pdm09 virus, across almost the entire adult age-range ( ∼25-70 years in these previous studies), 6 -8 it appears that for severe cases, globally, the survival of such patients appears not to have improved. this may be somewhat surprising as the world's populations have become more immunologically experienced with this virus, which is now considered as a seasonal influenza virus that should be conferring some degree of persisting, cross-reactive individual and herd immunity over consecutive seasons. 9 this may be due to some predisposing genetic or environmental factors in individual patients, which should reinforce the general message that seasonal influenza immunisation is still recommended to minimise the number of people needing icu or ecmo support for severe influenza infection. more detailed monitoring on how these physiological parameters change over time (perhaps including more complex cytokine studies), in these severely ill, influenza a(h1n1)pdm09-infected patients admitted to icu-ecmo units, may eventually yield data to improve their management and clinical outcomes. none. we read with interest the report by stalenhoef and colleagues in this journal who show that biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection (ref). 1 here we report on a prospective observational analysis of the biomarker: cd5 antigen like protein (cd5l), in serum samples collected from 64 patients diagnosed with pneumonia 2 and 44 healthy adults between 2018 and 2019. the demographic and clinical characteristics of the 64 adults (males 70.3%; mean age 66 ± 20 years) with pneumonia were summarized in table 1 . 57 (89.1%) bacterial pneumonia patients (including 34 patients with confirmed bacterial pneumonia 2 and 23 patients with suspected bacterial pneumonia 3 ) and 7(10.9%) viral pneumonia patients were studied. there were significant differences in age, sex, wbc, neu%, crp, pct, cd5l, apache ii scores and length of icu stay between bacterial pneumonia and viral pneumonia. the pathogens responsible for pneumonia were described in supplementary table 1 . globally, gram-negative bacteria infection is more common than gram-positive bacteria infection in pneumonia. among them, acinetobacter baumannii (16, 59.3%) was considered the major pathogen in gram-negative bacteria and staphylococcus aureus (3, 42.9%) was considered the major pathogen in gram-positive bacteria. in viral pneumonia, influenza a (h1n1) was detected as the unique pathogen. in the study of the diagnostic performance of serum cd5l to identify etiology of pneumonia, as we found in supplementary fig. 1 , no matter in total bacterial pneumonia, suspected bacterial pneumonia or confirmed bacterial pneumonia, the serum of cd5l levels on day of pneumonia diagnosis were significantly higher than those in viral pneumonia and healthy control subjects. for evaluating the diagnostic performance of cd5l to differentiate bacterial from viral infection in pneumonia, roc analysis was conducted for the above bacterial pneumonia (supplementary fig. 2 ) and compared with routine laboratory markers ( table 2 ). by horizontal comparison, we found that the best auc was for cd5l (auc = 0.89), better than neu% (auc = 0.79), wbc (auc = 0.79), crp (auc = 0.78) and even pct (auc = 0.85). interestingly, by longitudinal comparison, the auc was observed highest in confirmed bacterial pneumonia (auc = 0.92), intermediate in all bacterial pneumonia (auc = 0.89), and lowest in suspected bacterial pneumonia (auc = 0.84), which may better elucidate the diagnostic performance of cd5l for etiology diagnosis in pneumonia patients. although there are 23 patients with suspected bacterial pneumonia for which no definitive pathogen was found, our result here may still provide a new treatment for bacterial infection identification in pneumonia patients for the current limitations in direct pathogen testing make it difficult to identify the pathogen at the time of diagnosis 4 . besides, as a potential biomarker to distinguish pathogens 5-8 , cd5l levels were also significantly correlated with wbc, neu%, crp and pct ( supplementary fig. 3 ) in patients with pneumonia. in the study of the diagnostic performance of serum cd5l to predict mortality in adults with pneumonia, mortality was defined as death occurring within 30 days after the onset of pneumonia. as we observed in supplementary fig. 4a , serum cd5l levels on day of pneumonia diagnosis were significantly higher in non-survivors ( n = 18) than survivors ( n = 46) ( p < 0.001). to ensure that serum cd5l levels were not influenced by the class of the infection in the specimens, spearman's rank correlation analysis was performed between serum cd5l levels and poor prognosis related scores 9 , 10 (sofa and apache ii scores) in patients with total bacterial pneumonia, suspected bacterial pneumonia, confirmed bacterial pneumonia and viral pneumonia, respectively. our correlation analysis between serum cd5l levels and sofa or apache ii scores shows that no matter for bacterial pneumonia or viral pneumonia, the cd5l levels showed positive correlation with sofa and apache ii scores ( supplementary figs. 5 and 6) . furthermore, the auc of cd5l for identifying 30-day mortality in adult pneumonia patients was 0.79 ( supplementary fig. 4b) , a value means good diagnostic performance, which is consistent with gao's study 9 before. therefore, we found that determining serum cd5l concentrations on day of pneumonia diagnosis was of great value in identifying bacterial infection from viral infection and predicting 30day mortality in adult patients with pneumonia, which suggests that cd5l may work for the etiologic diagnosis and could represent a novel biomarker for identification of a group of patients with pneumonia presenting with higher risk of death. these findings encourage further effort s aimed at exploring the clinical value of circulating cd5l to help early clinical decision-making in human pneumonia. none. high morbidity and mortality (up to 100%). asf is a devastating threat to pig agriculture and is responsible for serious production and economic losses. the asfv genome is 170-193 kilo base pairs in length 2 and has been divided into 24 different genotypes based on their b646l gene sequence, a gene which encodes the capsid protein p72. 3 , 4 our previous study showed that the p72 gene located in a very low genetic diversity region of the genome. 5 ye and colleagues, however, identified two novel genotypes xxv and xxvi, with genotype xxvi being especially divergent from all other genotypes. 1 to examine this unexpected result, in this study, we reanalyzed their data to evaluate the reliability of these two genotypes. we collected 716 available p72 gene sequences from ncbi ( http://www.ncbi.nlm.nih.gov/ ), which we then aligned with mafft software, a fast multiple sequence alignment program. 6 the alignments show that genotype xxv (accession number fr668409) has a single amino acid change at position 506 compared to genotype i, while for genotype xxvi (accession numbers fr668404 and fr668418), the region coding for amino acid residues 509 to 528 differs greatly compared to all other genotypes ( fig. 1 a) . to examine this in greater detail, we calculated genetic distance across the p72 coding sequence in 60 bp sliding windows, with a step size of 10 bp, between these two sequences and the 24 other genotypes ( fig. 1 b) . this sliding window analysis shows these two sequences for genotype xxvi have a region that is very divergent, with genetic distance up to 0.5067 from the other genotypes, while the genetic distances of the remaining regions are less than 0.1 ( fig. 1 b) . genetic distances for asfvs were calculated for 182 coding gene sequences, from 42 available complete genomes of asfvs, to assess the overall divergence. pairwise genetic distances of each gene for all asfv strains were calculated. the mean pairwise genetic distance for asfv genes was 0.041 ( fig. 2 ) , which is much lower than this divergent region of these two sequences for genotype xxvi (0.5067). therefore, it seems highly unlikely that a gene has such a highly divergent region. since recombination frequently occurs in asfvs, 5 we conducted a recombination analysis with the rdp4 program, which used the 3seq, bootscan, chimaera, genecov, lard, maxchi, rdp and siscan detection methods, 7 to determine whether recombination might have occurred in the xxvi genotype. we found reliable evidence for recombination events in both p72 genotype xxvi sequences ( fig. 1 c) . this suggests that the very highly divergent region of this p72 genotype is not homologous with other p72 genotypes. we used blastn, from ncbi ( https://blast.ncbi.nlm.nih.gov/blast.cgi ), to identify a source for this highly divergent region, however, no similarity sequence was found. we then mapped the multiple amino acid changes found in genotype xxvi to the 3d structure of p72 ( fig. 3 ) . the changed sites (marked in red in fig. 3 ) are located in the dec loop, a region which plays an important role in the formation of the trimer spike. 8 the amino acid mutations result in changes of electrostatic potential, hydrophobicity, and steric hindrance. it seems unlikely to have so many changes in such a functionally important region. in our previous studies we noticed that sequences directly submitted by individual laboratories to genbank often contain errors such as misidentification of species, sampling error, contamination, or are pseudogenes, which can lead to sequence analysis problems and erroneous conclusions. 9 , 10 sequences that deviate from the overall intraspecific pairwise divergence are potentially erroneous. 9 , 10 since genotype xxvi deviates from other genotypes not only in genetic distance, but also in protein structure, and mutations occur in the flanking regions of the sequences, we deduced that these reported mutations might be due to low quality sequencing. we identified the original manuscript reporting the three sequences of genotypes xxv and xxvi, 11 where the authors stated that the "alignment and translation of sequences obtained from sardinian isolates revealed that the c-terminal end of p72 gene was completely conserved between the sequences compared". this statement indicates that these sequences did not considerably diverge from previously reported asfv p72 sequences, and thus, suggests that an error in these sequences had been introduced upon submitting them to genbank. further examination of the original samples and sequences is needed. we contacted the corresponding author of this manuscript to check these three sequences, and were told that these three p72 sequences were all genotype i, and had some errors when submitted to genbank. in conclusion, the two novel genotypes of asfvs (xxv and xxvi) identified by ye and colleagues 1 are misled by problematic sequences. as reminded by our previous studies, many problematic sequences are present in genbank, which can lead to problems in downstream analyses. 9 , 10 thus, when published data is used for new analyses, the first set in the process of data analyses should be to filter these sequences for potential errors to reduce the possibility of reaching incorrect conclusions. if conclusions are based on obviously strange sequences, then we should trace back the data to their original source, and manuscript, and contact the corresponding authors before making conclusions based on these sequences. the authors declare no conflict of interest. fig. 1 . the analyses of phylogenetic tree, recombinant, and nucleotide divergence between 6xi and the other known 6 subtypes (6a-6xh) based on full-length hbv genome sequences. (a) the known hcv subtype 6 reference sequences (6a-6xh) from the previous report were used. phylogenetic analysis was performed by the maximum-likelihood method, based on the gtr + g + i substitution model, with 10 0 0 bootstrap replicates using the software mega v6. the sequences of hcv 6xi (ynkh261 and ynkh284) are marked in red dot. (b) bootscan plots were constructed using simplot 3.5.1 software based on 100 replicates with a 300-bp sliding window moving in steps of 50 bases. (c) pairwise comparisons of nucleotides similarities between 3 hcv 6xi strains and 30 reference genotype 6 sequences. 399,0 0 0 deaths per year. 2 currently, daa treatment regimens that target ns3/ns4a protease, ns5a phosphor-protein and the ns5b polymerase have shown high safe and high rates of sustained virologic response in hcv chronically infected patients ( > 90%). 3 however, under selective pressure from these drugs, drug resistance-associated substitutions (ras) can emerge during this therapy and result in treatment failure in 2 −10% of patients. 4 therefore, hcv infection is still a major global health concern. to date, eight confirmed genotypes have been characterized based on > 30% sequence divergence in the complete hcv genome, and genotypes are further classified into > 80 subtypes with a sequence divergence of > 15% to other subtypes of the same geno-type. 5 in the current study, we characterized a new hcv subtypes among chronic hepatitis c patients in yunnan, china, initially designated as 6xi, further analyzed its evolutionary history and investigated its baseline ras by next generation sequencing (ngs) method. plasma samples were collected between january 2018 and october 2018 from 160 chronic hepatitis c patients from kunming city in yunnan, china (fig. s1a) . the samples met the following inclusion criteria: (1) hepatitis c antibody-positive for 6 months with normal serum alanine aminotransferase (alt) levels; (2) subject was residing in yunnan province and was over 18 years old; (3) complete demographic information and clinical data were available; (4) consented to the use of patient information in studies on hcv epidemics; and (5) were treatment-naïve during sampling. there was no epidemiologic link among these individuals. the study was approved by the first people's hospital of yunnan province ethics committee. written informed consent was obtained from all participants prior to the study. out of a total of 160 chronic hepatitis c patients, 152 partial ns5b gene fragments were successfully amplified and sequenced with a success rate of 95.0% (152/160). multiple subtypes were identified in 152 subjects, including subtype 3b (36.2%, 55/152), 3a (23.7%, 36/152), 1b (19.1%, 29/152), 2a (8.5%, 13/152), 6n (7.9%, 12/152), 6a (2.0%, 3/152), and 1a (1.3%, 2/152) (fig. s1b) . interestingly, the remaining two strains (1.3%, 2/152) involving ynkh261 and ynkh284 together with the isolate km35 reported formed a novel separate cluster in the genotype 6 with an 82% bootstrap value, indicating a potential new hcv subtype 6. to confirm that the two strains belong to a novel hcv subtype 6, their complete genome sequences were successfully amplified and sequenced with 12 overlapping fragments. further, phylogenetic analysis was performed along with hcv reference sequences of representative subtypes 6a-6xh. the result showed that the two strains and isolate km35 formed a distinct monophyletic cluster supported by a high bootstrap value of 100%. the three strains were isolated from three hiv-1 infected patients without obvious epidemiological linkage in yunnan and showed no evidence of recombination using bootscan analysis ( fig. 1 (b) ). moreover, the intergroup nucleotide divergence (mean ± sd) % over the fulllength genome sequences of the isolates (ynkh261, ynkh284, and km35) were compared to that of representative subtypes (6a-6xh) ( fig. 1 (c) ). the results revealed that the three strains were different from known hcv subtypes of 6a-6xh by 16.5-28.5%. therefore, the three strains are initially designated 6xi. to better understand the time of emergence of hcv 6xi, we performed bayesian molecular clock analyses using full-length genome sequences to estimate the time to the most recent common ancestor (tmrca). as shown in fig. 2 (a) , the estimated tmr-cas for the genotype 6xi was 1971.1 [95% highest probability density (hpd): 1951. 1, 1991.4 ]. in addition, to further investigate baseline ras of subtype 6xi, naturally occurring resistance-associated substitutions (ras) were analyzed for the ns3, ns5a and ns5b sequences using next generation sequencing (ngs) method. strikingly, hcv 6xi strains contain the substitution 28 v with a 100% frequency of mutations in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor, 6 suggesting that the subtype 6xi maybe basically resistant to ns5a inhibitors ( fig. 2 (b) ). among the hcv eight genotypes, genotype 6 exhibits a high degree of genetic complexity and diversity, and 31 subtypes have been confirmed by the international committee on taxonomy of viruses. 7 in china, hcv genotype 6 is common, and subtype 6a is the most prevalent subtype, primarily distributed in guangdong, 6n is the second most prevalent subtype, mainly found in yunnan, followed by subtypes 6xa, 6 g, 6v, 6 w, 6e, 6b, 6j, 6q,and 6r among genotype 6 isolates. 8 -10 to our knowledge, 6xi is the eighth detection of novel hcv subtypes 6 in china combined with previously identified 6a, 6e, 6n, 6v, 6xa, 6xe and 6xh, resulting in genotype 6 to expand to 32 subtypes in the world. our findings again demonstrated that hcv genotype 6 was more complex and diverse. in summary, we characterized a new hcv subtype 6xi based on the characteristics of a monophyletic cluster, > 15% genetic distances, no significant evidence of recombination, and no epidemiologic link among individuals. in addition, bayesian analyses showed that 6xi may originate around the year 1971, and the strains of hcv 6xi naturally contain the substitution 28 v in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor. the present finding again highlights the genetic characteristics and hcv strains in yunnan, and the urgent need for continuous molecular screening and epidemic surveillance in yunnan to implement effective measures to reduce hcv transmission. the authors declare no competing financial interests. we recently read the article by corma-gómez a. et al. on which the authors described a higher probability of relapses with sofosbuvir/ledipasvir 8 weeks compared with 12 weeks of hcv (hepatitis c virus) among hiv (human immunodeficiency virus)/hcv coinfected patients. 1 in this regard, coinfections of hiv/ hcv also with hepatitis b virus (hbv) is associated with high mortality and comorbidity too. the persistence of hbv dna within the core cell in the absence of hbsag and even after clearance of the infection has been described previously in immunosuppressed patients, where hbv screening and prophylaxis is recommended. 2 interestingly, hbv reactivation has recently emerged during or after hcv treatment with direct-acting antivirals (daa). [3] [4] [5] the us food and drug administration issued a warning about this risk in 2016, 6 until that moment 24 cases were reported, two of which died. although specific mechanisms of this event are not well known, it has been suggested that hcv core proteins could inhibit hbv replication and hbsag production as well as production of envelope proteins, being hbcab the only marker of the presence of hbv. 2 in this context, treatment with daa would produce a drastic and rapidly blocking of hcv replication providing the opportunity to hbv to emerge and produce an immune reconstitution syndrome. the interference hbv-hcv has been outlined in 24% of patients with chronic hbv infection (hbsag-positive serology) and 1.4% of patients with resolved hbv infections (hbsag-negative and hbcabpositive) in a recently published systematic review and metaanalysis of patients with hcv-hbv. 7 in most cases, these reports have focused on coinfection hcv-hbv, but there is still a concern about patients triple-infected with hcv, hbv and hiv. although most cases reported occur in hbsag-positive, the main concern affects to patients with a basal serology showing hbcab-positive, hbsag-negative and hbsab-negative. hbsagpositive was associated with higher rates of hbv reactivations compared with hbsag-negative and hbcab-positive patients. 8 there is no clear information about this issue in hbsag-negative, hbcab-positive, and, when occurs, it is considered an uncommon event. 9 reactivation of hbv is characterized by reappearance or increase in hbv dna levels, and could also be accompanied by symptoms of hepatitis. the cases reported previously did show difference in the time of presentation of clinical symptoms, they used to start either during or after daa treatment. it seems that hbv reactivation usually occurs early after daa initiation treatment (4-8 weeks) while otherwise can occur after treatment completion. in that way, it seems necessary that hbv infection should be monitored early after daa initiation. the most recent european association for the study of the liver (easl) guideline about hbv infection, recommends that patients hbsag-negative and hbcab-positive undergoing daa treatment should be monitored and tested for hbv reactivation only in case of alt elevation. 10 they also recommend performing hbv dna levels only in case of alt increase. at the same time, the american association for the study of liver diseases (aasld) and the infectious disease society of america (idsa) guideline has been recently updated recommendations to monitor hbv dna levels only in patients hbsag-positive, but they emphasize that there is not enough data to monitoring dna among patients hbcabpositive or hbcab-positive and hbsab-positive. they remark that a reactivation should be considered if an unexplained increase in liver enzyme is present. in hiv patients, they provide the same recommendations. 11 in a recent review of the literature, the authors also recommend to perform an hbv dna only in patients with altered alt. 9 a meta-analysis recommended not to perform an hbv dna test in this case due to low rate of incidence and the associated cost which needs to be considered especially in an endemic hbv areas. 7 few data exist in hiv patients. the fact that many triple-infected patients are receiving antiretroviral therapy including (art) nucleoside/nucleotide analogous, as tenofovir disoproxil fumarate (tdf) or tenofovir alafenamide (taf), both active against hbv, suggest that hbv reactivation rate could be underestimated. in a recently published review, chang et al., recommend to perform an hbv dna test at baseline in triple-infected patients. if positive, an hbv-active antiretroviral therapy (art) should be started and hbv dna needs to be monitored every 4 weeks during treatment and until week 12 after completion of treatment. however, if baseline hbv dna is negative they match with aasld/idsa and easl recommendations. 12 one of our patients has a basal serology with hbcab-iggpositive, both hbsag-and hbsab-negatives, and undetectable hbv dna levels, prior to treatment with daas. regarding hiv infection, he has an analogues-free regimen. one month after having finished treatment with daas, the patient consulted because of abdominal pain, nausea and jaundice. he had a total bilirubin level of 11 mg/dl, ast 1025 iu/l, alt 642 iu/l and inr 1.30. a hbv viral load of 6,193,455 iu/ml was detected; hbsag, hbcab-igm and hbeag were positive. there were no reasons to believe that he was re-infected. treatment with entecavir was initiated; however, the clinical evolution was unfavorable and died as a result of an acute liver failure. the hbv viral load was requested from the stored samples drawn during the treatment of hcv showing that hbv viral load was undetectable at the beginning of treatment and in week 2, but progressively increased to 98 iu/ml in week 8 and up to 82,700 iu/ml in week 4 after treatment completion. during the follow-up of daa treatment alt and ast remained normal. this case changed our daily practice, since then all hiv patients and more specifically those not treated with either tdf or taf and presenting a previous hbcab-positive, are followed using dna levels and not only monitoring hepatic enzymes, as recommended by guidelines. we consider that this case may reflect the necessity of change the current guidelines. we recommend to perform a periodic monitoring of hbv reactivation using hbv dna during and after daa therapy in hbsag-negative and hbcab-positive patients, independently of hbsab presence, hepatic enzymes and clinical symptoms, particularly in hiv patients who are not receiving active treatment of hbv. the study was not funded. authors declare that there is no conflict of interest. recently, a notable pattern of synchrony of influenza a and b virus, and respiratory syncytial virus incidence peaks globally was reported in this journal. 1 a previous study characterized seasonal pattern of influenza a and b in china and identified three epidemiological regions featured by distinct seasonality. 2 on the basis of laboratory surveillance data from 30 chinese provinces spanning about 12 years from october 2004 through january 2017, our study further characterized seasonal patterns of circulating influenza a subtypes and influenza b lineages in the three defined epidemiological regions. our study revealed that pre-2009 a(h1n1) and a(h3n2) displayed wintertime and summertime epidemics in midlatitude and southernmost chinese provinces with subtropical climate, wavelet analysis demonstrated the two subtypes displayed twice-annual cycle in some years in mid-latitude chinese provinces ( fig. 1 (a)-(h) ). however, the two subtypes peaks in the winter with annual or longer cycle in northern chinese provinces. influenza a(h1n1)pdm09 b/victoria and b/yamagata virus all displayed epidemics in the winter or winter-spring with annual or longer cycle in all three epidemiological regions. we developed univariate and multivariate regression models to evaluate the association between climatic factors and the presence or absence of epidemics of each influenza subtype and lineage (positive proportion ≥5%) in the southernmost provinces where heating system is not generally used in the winter so temperature and relative humidity in external conditions in winter are close to those in indoor environment where people spend most of the time. we fitted the mixed-effects logistic regression model to control for the repeated measurements in each province of the region cluster. we kept one of temperature and absolute humidity (representative of vapor pressure) with smaller akaike information criterion in the model to reduce of multi-collinearity due to a high degree of correlation between the two factors. our analysis indicated temperature, humidity and rainfall were environmental predictors of influenza subtype/lineage-specific epidemics in the southernmost provinces when a 1-week lag of influenza epidemics behind climate was considered, which was similar to the findings from some ecological studies ( table 1 ). 3 our study indicated the u-shaped relationship between absolute humidity (ah) and pre-2009 a(h1n1) or a(h3n2) epidemics in the southernmost provinces, and suggest that high levels of ah in the summer, and low levels of ah in the winter increased the possibility of epidemics of the two subtypes. 3 in our analysis, lower temperature was an environmental driver of a (h1n1)pdm09 and b/yamagata epidemics in the southernmost provinces while there were bimodal associations between temperature, rainfall and b/victoria epidemics with highest probability of b/victoria epidemics at 11.7 °c of daily average temperature and 11.3 mm of daily average rainfall. although seasonal changes in human behaviors, such as school attendance or crowding indoors, and seasonal variations in immunity, such as melatonin and vitamin d levels have been proposed to account for the seasonal nature of influenza, 4 our findings suggest that the heterogeneity in influenza subtype/lineage-specific seasonality patterns could be driven by seasonal variations in virus survival, transmission and adaptive immunity by influenza subtype and lineage because of the same behavior modes and background of non-adaptive immunity in the same regions and seasons. we propose a hypothesis: under humid and hot condition the dominant transmission mode(s) for a(h1n1)pdm09, b/victoria and b/yamagata might have reduced efficiency, however, there could be effective transmission mode(s) for a(h3n2) and pre-2009 a(h1n1) virus. some experimental studies were performed to establishing a causal link between humidity, temperature and influenza virus survival/transmission. 5 transmission of influenza a (h3n2), a(h1n1)pdm09, b/victoria and b/yamagata virus by respiratory droplets or aerosols in the guinea pig model proceeds most readily under cold, dry conditions. 5 low humidity and temperature increased the stability of influenza virus in aerosols and on surfaces. furthermore, aerosol transmission of a (h3n2) virus in the guinea pig model was almost completely blocked at 30 °c, but contact transmission of a (h3n2) virus seemed to be efficient at different level of humidity and 30 °c. 5 it remains unclear if high temperature and humidity levels have effect on aerosol and contact transmission of pre-2009 a(h1n1), a(h1n1)pdm09, b/victoria and b/yamagata virus among hosts and their stability on surfaces. of note, an experimental study found the survival durations of a(h3n2) strains on swiss banknotes were significantly longer than pre-2009 a(h1n1) and b/victoria virus. 6 further studies are needed to understand how efficient are these transmission modes for different influenza subtypes or lineages, which is/are dominant mode(s) of transmission among hosts, and which of potential mechanisms is at play under humid and hot condition. one of our findings was the mutual inverse association between a(h3n2) and a(h1n1)pdm09 epidemics, which provided the evidence on interference between the two influenza a subtypes perhaps possibly due to multiple immune mechanisms. 7 however, our study showed b/yamagata epidemics were positively correlated with a(h3n2) epidemics, suggesting b/yamagata epidemics across over 12 study years that were weak could get well along with simultaneous weak h3n2 epidemics. understanding of influenza seasonality is important to define optimal timing of influenza vaccination campaigns. our study indicated that a(h3n2) virus brought about twice-annual epidemics in some years in mid-latitude chinese provinces and more frequent summertime epidemics in southernmost chinese provinces, which questions if a single annual influenza vaccination campaign starting in october can offer optimal protection against summertime epidemics of a (h3n2) virus in mid-latitude and southernmost chinese provinces. in recent years, it has been reported that mismatches of a(h3n2) virus between the influenza vaccine strains and circulating strains were identified frequently, 8 and vaccine effectiveness of a(h3n2) virus declined within 4-6 months postvaccination. 9 in conclusion, we identified the heterogeneity of seasonality pattern of pre-2009 a(h1n1) or a(h3n2) virus in three epidemiological regions of china, and different environment predictors for influenza subtypes and lineages in the southernmost provinces. further work should focus on understanding difference in virus survival, transmission by influenza subtype and lineage under humid and hot conditions. bjc has received research funding from medimmune inc. and sanofi pasteur, and consults for crucell nv. the authors report no other potential competing interests. as this study included data from the national influenza surveillance system, ethics approval was not required. not applicable. the datasets at national level analyzed during the current study are available in the world health organization flunet. the datasets with more specific information analyzed during the current study are available in chinese national influenza surveillance informatio system, but they are not open-access datasets. these influenza surveillance data can be available from chinese national influenza center on reasonable request. this study was supported by the national mega-projects for infectious diseases (grant number 2018zx102010 02-0 08-001 ), national natural science foundation of china (grant number 81302476 ) and emergency prevention and control project of ministry of science and technology (grant number 10600100000015001206 ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. reported in zhejiang in 2017. 8 in this study, we identified ten cases of imported chikv infection in travelers returning to yunnan from southeastern asia in 2019. out of the ten patients with imported chikv infection examined in this study, nine patients had traveled back from myanmar and one patient had travelled back from thailand ( fig. 1 (a) ). all the patients displayed different degrees of symptoms, such as fever, cough, muscle pain, and rash. the details of all the symptoms are shown in table 1 . chikv infection was diagnosed using specific real-time reverse transcription-pcr. serum specimens were collected from the ten patients that tested positive for chikv by real-time pcr analysis, in yunnan between may 7, 2019 and august 1, 2019. the current study was approved by the medical ethics committee of kunming university of science and technology. written informed consent was obtained from all the participants. a new coronavirus associated with human respiratory disease in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin emergence of sars-like coronavirus poses new challenge in china bat origin of a new human coronavirus: there and back again transmission of 2019-ncov infection from an asymptomatic contact in germany early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia what to do next to control the 2019-ncov epidemic? nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) national health commission of people's republic of china prevention and control of novel coronavirus pneumonia a hospital cluster combined with a family cluster of avian influenza h7n9 infection in anhui province first human infection by a novel avian influenza a(h7n4) virus contact reductions from live poultry market closures limit the epidemic of human infections with h7n9 influenza comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds death of a very young child infected with influenza a (h5n6) equine influenza-a global perspective isolation and characterization of highly pathogenic avian influenza virus subtype h5n1 from donkeys molecular epidemiology of a novel re-assorted epidemic strain of equine influenza virus in pakistan in 2015-16 avian-origin h3n2 canine influenza virus circulating in farmed dogs in guangdong detection of pediatric bacterial meningitis pathogens from cerebrospinal fluid by next-generation sequencing technology next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center community-acquired bacterial meningitis rapid bacterial identification by direct pcr amplification of 16s rrna genes using the minion tm nanopore sequencer campylobacter fetus meningitis confirmed by a 16s rrna gene analysis using the minion nanopore sequencer bacterial diversity assessment in antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation rapid diagnosis of bacterial meningitis by nanopore 16s amplicon sequencing: a pilot study diagnosis of haemophilus influenzae pneumonia by nanopore 16s amplicon sequencing of sputum epidemiology of invasive group b streptococcal infections among nonpregnant adults in the united states comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses impact of a poorly performing point-ofcare test during the 2017-2018 influenza season uk severe flu surveillance system icu-hdu influenza surveillance nhse) service specification 170110s. extra corporeal membrane oxygenation (ecmo) for respiratory failure in adults australia and new zealand extracorporeal membrane oxygenation (anz ecmo) influenza investigators extracorporeal membrane oxygenation for 2009 influenza a(h1n1) acute respiratory distress syndrome intensive care unit patients with 2009 pandemic influenza a (h1n1pdm09) virus infection -united states characteristics of patients with hospital-acquired influenza a (h1n1)pdm09 virus admitted to the intensive care unit cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection guidelines for the management of adults with community-acquired pneumonia. diagnosis, assessment of severity, antimicrobial therapy, and prevention cytokine markers as predictors of type of respiratory infection in patients during the influenza season procalcitonin as a marker of etiology in adults hospitalized with community-acquired pneumonia cd5l contributes to the pathogenesis of methicillin-resistant staphylococcus aureus-induced pneumonia orchestrating role of apoptosis inhibitor of macrophage in the resolution of acute lung injury aim inhibits apoptosis of t cells and nkt cells in corynebacterium-induced granuloma formation in mice the macrophage soluble receptor aim/api6/cd5l displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression assessment of apoptosis inhibitor of macrophage/cd5l as a biomarker to predict mortality in the critically ill with sepsis plasma long pentraxin 3 (ptx3) concentration is a novel marker of disease activity in patients with community-acquired pneumonia the updated analysis of african swine fever virus genomes: two novel genotypes are identified african swine fever virus replication and genomics phylogeographic patterns of the african swine fever virus genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype the recombination hot spots and genetic diversity of the genomes of african swine fever viruses parallelization of the mafft multiple sequence alignment program rdp4: detection and analysis of recombination patterns in virus genomes structure of the african swine fever virus major capsid protein p72 detection of potential problematic cytb gene sequences of fishes in genbank assessing dna barcoding as a tool for species identification and data quality control genetic characterisation of african swine fever viruses from recent and historical outbreaks in sardinia consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group hepatitis c virus infection in children and adolescents challenges and perspectives of direct antivirals for the treatment of hepatitis c virus infection whole-genome characterization and resistance-associated substitutions in a new hcv genotype 1 subtype identification of 19 novel hepatitis c virus subtypes-further expanding hcv classification hepatitis c virus drug resistance associated substitutions and their clinical relevance: update identification of a new hcv subtype 6xg among injection drug users in kachin hepatitis c virus genotypes and subtypes circulating in mainland china higher relapse rate among hiv/hcv-confected patients receiving sofosbuvir/ledipasvir for 8 vs 12 weeks significance of anti-hbc alone serological status in clinical practice evaluation of hepatitis b reactivation among 62,920 veterans treated with oral hepatitis c antivirals acute fulminant hepatitis b during hepatitis c virus therapy with direct-acting antivirals in a patient co-infected with hiv spontaneous remission of hepatitis b virus reactivation during direct-acting antiviral agent-based therapy for chronic hepatitis c adverse events reporting system (faers): the public's stake in adverse event reporting hepatitis b virus reactivation during direct-acting antiviral therapy for hepatitis c: a systematic review and meta-analysis reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals controversies in hepatitis c therapy: reactivation of hepatitis b virus clinical practice guidelines on the management of hepatitis b virus infection monitoring patients who are starting hcv treatment, are on treatment, or have completed therapy. hcv guidance: recommendations for testing, managing, and treating hepatitis c. electronic access significance and management of isolated hepatitis b core antibody (anti-hbc) in hiv and hcv: strategies in the daa era comparative global epidemiology of inßuenza, respiratory syncytial and parainßuenza viruses characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data environmental predictors of seasonal influenza epidemics across temperate and tropical climates global influenza seasonality: reconciling patterns across temperate and tropical regions roles of humidity and temperature in shaping influenza seasonality survival of influenza virus on banknotes a systematic approach to virus-virus interactions comparing influenza vaccine efficacy against mismatched and matched strains a systematic review and meta-analysis i-move multicentre case-control study 2010/11 to 2014/15: is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination? metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers chikungunya virus infections in military deployments in tropical settings-a narrative minireview chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen east/central/south african genotype in a chikungunya outbreak caribbean and la réunion chikungunya virus isolates differ in their capacity to induce proinflammatory th1 and nk cell responses and acute joint pathology new insights into chikungunya virus emergence and spread from southeast asia mosquito-associated viruses in china chikungunya fever outbreak enhanced molecular surveillance of chikungunya virus chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes this work was supported by henan emergency project for prevention and control of 2019 novel coronavirus, the earmarked fund for modern agro-industry technology research system of china (cars-35) and the special fund for henan agriculture research system (s2012-06). the funders had no role in study de-sign, data collection and interpretation, or the decision to submit the work for publication. we thank jing li and hao-nan wang for the helpful discusses and suggestions. this work is supported by the open research fund of key laboratory of digital earth science ( 2019lde005 ), and by the fundamental research funds for the central universities under grant no. 310201911qd054 . this work was supported by the national natural science foundation of china ( 31702271 ) and the guangdong provincial natural science foundation ( 2017a030310367 ). we thank le kim thanh, le nguyen truc nhu, and lam anh nguyet for their logistic support. we are indebted to patients for their participations in this study.this study was funded by the wellcome trust of great britain ( 10 6 680/b/14/z and 204904/z/16/z ). supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.011 . we would like to thank the patients and healthy volunteers in the first affiliated hospital of chongqing medical university for their cooperation and support. this work has been financially supported by national natural science foundation of china (no. 81902134 and no. 81722001 ). we thank chinese national influenza surveillance network for contribution in influenza epidemiological and laboratory surveillance. we thank the members of the yunnan international travel healthcare center and kunming changshui airport customs for the data and sample collection. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.003 . supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.11.019 . recent correspondence in this journal has highlighted the current threat posed by recently-emerging imported chikungunya virus (chikv) in febrile returning travellers. 1 chikungunya fever is infection caused by the chikv and is characterized by fever, arthralgia, myalgia, headache, and rash. chikv belongs to the genus alphavirus within the togaviridae family and is transmitted to humans by the bite of infected mosquitoes-ae. aegypti and ae. albopictus . 2 since the first report of chikv infection in humans in tanzania, intermittent outbreaks have been documented in africa, south america, southern and southeastern asia, and the indian ocean islands; thus, its outbreak has become a global public health problem. 3 to date, three evolutionary distinct chikv genotypes, namely west african (wa), east/central/south african (ecsa), and asian have been identified, based on phylogenetic analyses. 4 a novel lineage of chikv-the indian ocean lineage (iol)-has also been reported, which descended from the ecsa genotype during an outbreak on the island of la reunion between 2005 and 2006. 5 recently, it has been reported that iol of chikv has spread to malaysia, singapore, thailand, and indonesia. 6 in china, the first case of imported chikv infection was found in yunnan in 1987. subsequently, several sporadic cases of nonindigenous chikv infection have been described. in 2010, the first outbreak of chikv fever with 129 cases was documented in guangdong. 7 recently, another outbreak of chikungunya fever was table 1 epidemiological information on ten febrile returning travelers infected with chikv in this study. in this study, nine complete genome sequences isolated from serum samples were successfully amplified and sequenced with 12 overlapping fragments, and then the sequence obtained was deposited in genbank under accession no. mn402883-mn402892. compared with the nucleotide sequences of the available from the ncbi database, the nine strains shared the highest 99.61-99.74% nucleotide identity with the east/central/south african lineage strain thail 2019 reported previously in thailand, 2019. further, bayesian maximum-clade-credibility tree for full-length nucleotide sequences were constructed using the beast package v.1.7.3. phylogenetic analyses revealed that the ten chikv strains clustered into the homogeneous indian ocean clade of the ecsa genotype ( fig. 1 (b) ).notably, the ten chikv strains isolated from serum samples possessed the mutation k211e in the e1 gene and v264a in the e2 gene; these mutations are associated with significant increase in viral infectivity in ae. aegypti . 9 the strains also possessed g60d and i211t substitutions in the e2 gene; these mutations contribute to increased chikv fitness in ae. albopictus. 9 however, the a226v mutation in the e1 gene that is related to significant increase in viral infectivity in ae. albopictus was not observed in any of the strains. 10 in summary, we characterized ten cases of human infection caused by imported ecsa genotype chikv in yunnan, china and successfully isolated nine infectious chikvs from the chikvpositive serum samples. the mutations associated with significant increase in viral infectivity for ae. aegypti or ae. albopictus were also observed in these strains. geographically, the yunnan province is in southeastern china and shares its border with southeast asian countries (laos, vietnam, and myanmar) that are most affected by chikv. with the increase of tourism and trade with southeast asian countries, cases of imported chikv infection are constantly increasing and may have the potential for re-emergence and autochthonous transmission to yunnan. the present study highlights the urgent need for continuous molecular screening and epidemic surveillance for chikv and its vectors to prevent future outbreaks of chikv infection among the human population of yunnan. this work was supported by the national natural science foundation of china (nsfc) ( u1702282 ), the reserve talents project for young and middle-aged academic and technical leaders of yunnan province (2019hb012), and youth talent program of yunnan "ten-thousand talents program" (ynwr-qnbj-2018-054). the authors declare no competing financial interests. key: cord-325989-nf6ouaq3 authors: es-saad, salwa; tremblay, nicolas; baril, martin; lamarre, daniel title: regulators of innate immunity as novel targets for panviral therapeutics date: 2012-09-24 journal: curr opin virol doi: 10.1016/j.coviro.2012.08.009 sha: doc_id: 325989 cord_uid: nf6ouaq3 interferons (ifns) have long been used as an immunomodulatory therapy for a large array of acute and chronic viral infections. however, ifn therapies have been plagued by severe side effects. the discovery of pathogen recognition receptors (prr) rejuvenated the interest for immunomodulatory therapies. the successes obtained with toll-like receptor (tlr) agonists in activating immune cells and as adjuvant for prophylactic vaccines against different viruses paved the way to targeted immunomodulatory therapy. better characterization of pathogen-induced immune disorders and newly discovered regulators of innate immunity have now the potential to specifically withdraw prevailing subversion mechanisms and to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than ifn therapies. salwa es-saad 1,3 , nicolas tremblay 1,3 , martin baril 1 and daniel lamarre 1, 2 interferons (ifns) have long been used as an immunomodulatory therapy for a large array of acute and chronic viral infections. however, ifn therapies have been plagued by severe side effects. the discovery of pathogen recognition receptors (prr) rejuvenated the interest for immunomodulatory therapies. the successes obtained with toll-like receptor (tlr) agonists in activating immune cells and as adjuvant for prophylactic vaccines against different viruses paved the way to targeted immunomodulatory therapy. better characterization of pathogen-induced immune disorders and newly discovered regulators of innate immunity have now the potential to specifically withdraw prevailing subversion mechanisms and to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than ifn therapies. the innate immune system is the first line of defense for organisms that possess an adaptive immune system. it relies on the presence of specific receptors able to recognize recurring pattern in molecules associated with pathogens but not with host cells, allowing discrimination between self and non-self. these receptors are named pattern recognition receptors (prr) and recognized pathogen-associated molecular patterns (pamp) to induce the expression of cytokines and chemokines that restrict dissemination, eliminate pathogens and instruct pathogenspecific adaptive immune responses. in the recent years, tremendous advances in the characterization of prr families, nucleic acid sensing, downstream signaling pathways and effector responses have revealed essential role of novel proteins and dynamic protein interactions network in the triggering of immune responses to intracellular pathogen such as viruses. in the near future, targeting specific regulators of prr-mediated innate response to withdraw viral subversion mechanisms, and access to novel surrogate measurable effector markers, hold the promise of new panviral therapeutics that will minimize adverse effects associated with type i ifn therapy. this review briefly summarizes strategies and challenges of present and future targeted immunomodulatory therapies according to our increasing knowledge in regulation of innate immunity and of virus-induced immune host dysfunction. signaling pprs include the major families of toll-like receptors (tlrs), retinoic acid-inducible gene i (rig-i)like receptors (rlrs) and nucleotide-binding oligomerization domain (nod)-like receptors (nlrs). pathogen sensing takes place in all nucleated cells to generate cellintrinsic innate immunity and in professional antigen presenting cells (apcs) to promote specific adaptive immune responses. while tlrs sense pamps in the extracellular space and endosomes, rlrs and nlrs function as pathogen sensors in intracellular compartments [1] . interestingly, only a few of the known 13 tlrs have the ability to recognize viral molecules: tlr3 for viral dsrna, tlr7/8 for viral ssrna and tlr9 for viral unmethylated cpg dna. three cytosolic sensors of viral rna have been characterized thus far: rig-i for the sensing of 5 0 triphosphate structure and blunt-end base paring, mda5 for the sensing of long dsrna and lgp2 a cardless regulator of its counterparts [2] . following their activation, the card domain of rig-i and mda5 interacts with the card domain of the signaling adaptor mavs (mitochondrial antiviral signaling protein) [3 ] . both tlr and rlr viral sensing pathways converge to activate ifn regulatory factor irf3-mediated and irf7-mediated type i ifn (a/b) antiviral response and nf-kb-mediated inflammatory pathway [4] (figure 1 ). recent studies aims at better defining innate immune responses have identified several novel signaling and regulatory molecules [5] . global proteomic analysis has further revealed signaling modules with high interconnectivity and adaptor proteins regulating signalosome assembly upon antiviral response and type i ifn production [6 ] . cytokines and type i ifns increase expression of mhc class ii antigens, cd40 and cd86 on apcs [7] . cytokines produced at sites of infection play a key role in the activation and differentiation of dendritic cells (dc), macrophages, neutrophils and nk cells, all major players of the innate immune response [8] (figure 1 ). when mature dcs detect virus derived antigens, they migrate to the lymph nodes to present antigens to cd4+ and cd8+ t cells and b cells, inducing their activation [9] . thus, modulation of prr-mediated antiviral responses can have important ripple effects on both qualitative and quantitative aspects of the specific adaptive immune responses to maximize the therapeutic potential of immunomodulatory drugs [10] . negative regulation of innate immune response and pathological consequences antiviral innate response must be tightly regulated in order to prevent uncontrolled production of cytokines that might have deleterious effects on the host. type i ifn signature induced by prr activation has been observed in diverse autoimmune disorders including diabetes, and is believed to play a role in the induction of chronic inflammatory disorders such as asthma and rheumatoid arthritis. in the recent years, a better picture has emerged in the biology of regulators illustrating the existence of numerous negative regulators that often play a nonredundant role and target the same positive regulator [5] . many negative regulators have been characterized that are either involved in direct interaction with prrs, dissociation of adaptors complexes, degradation of signal proteins or transcriptional regulation [12] . posttranslational modifications (phosphorylation and ubiquitination) have emerged as key mechanisms to regulate innate immune responses. degradation of signal proteins mediated by the ubiquitin-proteasome and autophagy systems plays crucial roles in negative regulation of tlr signaling, and unlike disruption of adaptors regulators of innate immunity as novel targets for panviral therapeutics es-saad et al. 623 contributes to termination of signaling as these degradations are irreversible [11] . examples include proteins socs and pin1 that promote polyubiquitination and proteasomal degradation of mal adaptor and irf3/7 respectively, to suppress type i ifn and antiviral responses. recently, mirnas have also emerged as fine tuners of innate immune responses, which target mrnas encoding tlrs, intracellular signaling proteins and cytokines. examples include mir-146 that targets irak1 and traf6, and mir-155 that targets myd88, tab2 and ikke [12] . thus, targeting specific negative regulator of the innate immune response may offer a new immunotherapeutic strategy to treat a range of infectious and inflammatory diseases [13] . cellular defence has evolutionarily challenged viruses that in turn have developed strategies to counteract innate immune response. indeed tlr and rlr sensing pathways are fundamental targets for virus-encoded immune suppression. these viral subversion mechanisms include recruitment of ubiquitin proteasome system, mimicry of the host cell components and sequestration and cleavage of key components of the immune system. one notable example is mavs adaptor that is targeted by numerous viruses through proteolytic cleavage by hepatitis c virus (hcv), hepatitis a virus (hav), coxsackievirus b3 (cvb3), human rhinovirus 1a (hrv1a) and gb virus b (gbv-b), through decrease of the mitochondrial membrane potential by influenza a virus (flu) or through inhibition of its interaction with rig-i by hepatitis b virus (hbv). processes of viral evasion are varied and are beyond the scope of this review, but are recapitulated in figure 1 (reviewed in [14] ). importantly, host proteins targeted by multiples viruses highlight key players of innate immunity, which represent potential therapeutic targets to restore antiviral response and eventually cure cells from viruses. however, these specific viral evasion strategies must also be taken into account when developing immunomodulatory therapeutics to provide the greatest clinical benefits. type i ifns were rapidly used as a therapeutic agent against hbv and hcv, and demonstrated antiviral activity against infection with sars-cov [15] , flu [16] , west nile virus (wnv) [17] , yellow fever virus (yfv) [18] and ebola virus [19] . refinement of therapies was explored with the development of improved ifn molecules like consensus interferon (cifn: a completely synthetic interferon) [20] , albinterferon (a fusion protein between ifna2a and human albumin) [21] and y shape interferon [22] . recently, virus-induced type iii ifns (ifn-l1-3: il-29, il28a, il28b) have gained a lot of interest to treat viral infections since naturally occurring variants of the il28b gene have been a major prediction factor in spontaneous and treatment-induced clearance of hcv [23, 24] . early clinical trials of recombinant pegylated-ifn-l1 in hcv-infected patients showed reduced adverse effects compared to ifn-a, likely linked to minimal expression of ifn-l receptors in hematopoietic cells [25, 26] . tlr targeted therapies (table 1) the discovery of tlrs heralded the rebirth of interest in innate immunity. their specificity in recognizing most classes of pathogens, as well as their role in the pathogenesis of multiple diseases represent the strongest evidences that tlrs are valuable therapeutic targets. tlr targeted drugs have been approved and small-molecule compounds are being investigated in the treatment of viral infections as stand-alone treatment or adjunct to direct acting antivirals (daas). the most advanced examples of tlr agonists are imiquimod (aldara, 3m) and resiquimod (r-848, 3m), which are members of the imidazoquinolinamines [27 ] . imiquimod is the only approved tlr7 agonist and is used for topical treatment of external genital and perianal warts resulting from human papillomavirus (hpv) infection [28] . resiquimod is a mixed tlr7/8 agonist that reached phase iii trial for the treatment of genital herpes before being suspended due to a lack of efficacy [29] . isatoribine ana-773 (anadys pharmaceuticals) is a second generation of orally bioavailable prodrug of isatoribine that signals through tlr7, which is expressed in b cells and dcs [30] . in hcv infected patients, ana-773 was generally well tolerated and resulted in a significant à1.26 log 10 decrease in hcv rna levels following 10 days of treatments [31] . ana-773 is now assessed in phase iia, and its efficacy will be evaluated in combination with ribavirin and daas as an ifn replacement. synthetic cytosine-phosphate-guanine containing oligodeoxynucleotides (cpg-odns) are potent tlr9 agonists, which interact directly with dcs to stimulate cytokine release and induce adaptive immune responses [32] . in phase i clinical trials, subcutaneously administration of imo-2125 (idera pharmaceuticals) as monotherapy resulted in a more than à1 log 10 decrease in hcv rna levels in prior nonresponders to peg-ifn/ribavirin after 4 weeks [33] , and in combination with ribavirin to a à2.4 log 10 decrease in hcv rna in treatment-naïve patients at day 29 [34 ,35] . on the basis of its efficacy, imo-2125 could provide an alternative to ifns for hcv therapy. however, idera pharmaceuticals delayed a phase ii study after the observation of atypical lymphocytic proliferation in preclinical toxicology study. vaccine adjuvants using tlr agonists tlr agonists have been an extensively explored area in the development of vaccine adjuvants for prophylactic and therapeutic applications by linking innate and adaptive immune systems. the proof-of-concept of this approach was made with the as04 adjuvant system that combines monophosphoryl lipid a (mpla), an agonist of the tlr4 receptor and aluminium salt [36] [37] [38] . as04 has been approved in prophylactic vaccine against hbv (fendrix, glaxosmithkline) [39] and hpv 16 and 18 (cervarix, glaxosmithkline) [40] . the mechanism of action of as04 is mediated by a transient and local activation of nf-kb activity and cytokine production, thus providing an innate immune signal for optimal activation of apcs [41] . other notable examples of adjuvants in clinical development are heplisav and vaxinnate. heplisav is a hbv vaccine comprised of an immunostimulatory sequence (iss-1018, dynavax technologies) that targets tlr9 receptor and hbv surface antigen. in phase iii clinical trials, heplisav demonstrated earlier and higher protection with fewer doses than currently licensed vaccines [42 ] . vax-innate corporation is developing vaccines using highly conserved influenza immunogens fused to tlr5 agonist salmonella typhimurium flagellin type 2 as an adjuvant to potentially protect against all strains of seasonal and pandemic flu strains (vax102, vax125, vax128 and vax168) [43] [44] [45] . future immunomodulatory targeted therapy and panviral approaches (table 2) in the past decade, many newly emerging or re-emerging virus infections and fear of future pandemics have accentuated the need for novel antiviral therapy. panviral therapeutics with a targeted therapy approach would be an ideal treatment for acute and chronic viral infections, either as a standalone treatment or in combination with daas. the major challenge in developing future immunomodulatory therapy will be to minimize adverse effects. the aggravation of psoriatic plaques in hpv-infected patients treated with imiquimod illustrates that triggering innate immune responses can lead to uncontrolled activation of the inflammatory response. furthermore, immunomodulatory molecules, such as peptidoglycans, that bind to multiple prrs (tlr2, nod proteins and peptidoglycan recognition proteins) increase the risk of undesired side effects. development of therapeutics will require more extensive structural information of receptor-ligand interaction to maximize the specificity and avoid undesired interactions. the selection of specific targets will require a comprehensive knowledge of innate immunity signaling pathways and regulators that are induced by and common to numerous viral infections. the mapping of an innate regulators of innate immunity as novel targets for panviral therapeutics es-saad et al. 625 table 1 development status of tlr-targeting molecules for treatment of viral infections. immune protein interaction network regulating ifnb1 has revealed signaling modules with high interconnectivity including mavs, tbk1 and irak [6 ] . each module interacts with many signaling proteins of the pathway offering multiple drug targets with specific immune effector function. using a genome-wide rnai screen assessing virus-induced ifnb1 transcription in human cells, we identified novel proteins and pathways capable of negatively and positively regulating innate immune responses (unpublished data). comprehensive epistasis analysis of the various regulators acting at different steps of the antiviral responses from virus sensing, signal propagation/amplification up to feedback regulation, offers valuable information for selection of drug targets. in principle, strategies of targeted therapy could include small molecule-mediated activation of positive regulators or inhibition of negative regulators. an example of targeting a negative regulator could be the immuno-mirna mir-155, which is induced by virus infection and downregulate myd88, irak3, tab2 and ikke gene expression to suppress tlr signaling [12] . silencing mir-155 function using antagomirs or locked nucleic acid (lna) in infected cells could potentially restore tlr signaling. a better knowledge of surrogate end point measurable makers of immune effector function (correlating with pan antiviral efficacy) in relevant infected biological material will undoubtedly enhance selection process and therapeutic value of drug targets. indeed, microarray analysis of infected primary cells can be used to identify early and late response innate immune genes, as well as virus-mediated inhibition of these genes [46] [47] [48] . finally, the knowledge of virus-induced immune host dysfunction and of immune proteins targeted by multiples viruses will validate key viral host interfaces, leading to hypothesis-driven selection of therapeutic targets intended to restore innate immune responses. tlrs agonists reflect substantial promise as therapeutic targets and demonstrate the huge potential of targeting innate immunity in fighting viral infections. in the future, integration of structural, proteomics and functional genomics data will pave the way to the identification of key regulators of innate immunity. targeting immune regulators that promote prr signaling to maintain transient activation of innate immune responses upon viral infection should pioneer the discovery of panviral therapeutics. such targeted immunomodulatory therapy approach could change the way we treat infectious diseases by allowing a single treatment to be effective against numerous viruses, with minimal viral breakthrough. in the near future, the increasing availability and potency of new targeted immunomodulatory panviral therapeutics could allow the re-thinking of temporal aspects of treatments that, in combination with available daas, could achieve viral eradication. the ultimate goal is to shape tlr-dependent and rlr-dependent innate immune responses to restore antiviral effects and to generate an optimal global immune response, while controlling inflammation. table 2 current and future development of immunomodulatory targeted therapy [49 ,50-56] . activation leads to pro-inflammatory cytokines and type i ifns production set the pace for an adaptive immune response via t and b cells. activation leads to pro-inflammatory cytokines and type i ifns production modulation of the adaptive immunity through dc and nk cells extensive structural and functional studies available [49] tlr agonists marketed or in phase i-iii clinical trials structure recently identified [51, 52] ssrna rig-i ligand reduces infection with flu [53] rig-i agonist as adjuvant in flu vaccine [54] restore immunity by counteracting viral evasion processes papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest pattern recognition receptors and inflammation immune signaling by rig-i-like receptors mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response describes how viral infection induces the formation of prion-like aggregates of mavs on mitochondria that potently activate irf3 toll-like receptors and their crosstalk with other innate receptors in infection and immunity regulation of rlr-mediated innate immune signaling -it is all about keeping the balance mapping a dynamic innate immunity protein interaction network regulating type i interferon production proteomic mapping of ligand-dependent interactions reveals dynamic remodeling of the human innate immunity interactome direct effects of type i interferons on cells of the immune system the yin and yang of viruses and interferons dynamic accumulation of plasmacytoid dendritic cells in lymph nodes is regulated by interferon-beta nucleic acid sensing at the interface between innate and adaptive immunity in vaccination rig-i-like receptors: cytoplasmic sensors for non-self rna micrornas: the fine-tuners of toll-like receptor signalling dissecting negative regulation of toll-like receptor signaling rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity interferon alfacon 1 inhibits sars-cov infection in human bronchial epithelial calu-3 cells a randomized controlled trial of low-dose recombinant human interferons alpha-2b nasal spray to prevent acute viral respiratory infections in military recruits use of interferon-alpha in patients with west nile encephalitis: report of 2 cases treatment of yellow fever virus with an adenovirus-vectored interferon def201, in a hamster model evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental ebola virus infections comparative efficacy and overall safety of different doses of consensus interferon for treatment of chronic hcv infection: a systematic review and meta-analysis albinterferon-alpha 2b: a new treatment option for hepatitis c clinical trial of the efficacy, dosing, safety and tolerability of y-shaped pegylated interferon (ypeg-ifna-2a) plus ribavirin in egyptian patients with untreated chronic hepatitis c genetic variation in il28b predicts hepatitis c treatment-induced viral clearance genetic variation in il28b and spontaneous clearance of hepatitis c virus phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis c virus infection interferon-lambda: a new addition to an old family targeting of toll-like receptors: a decade of progress in combating infectious diseases comprehensive review on the therapeutic targeting of tlrs in infectious diseases either as antimicrobial treatment or vaccine adjuvants topical imiquimod: a review of its use in the management of anogenital warts, actinic keratoses, basal cell carcinoma and other skin lesions oral resiquimod in chronic hcv infection: safety and efficacy in 2 placebo-controlled, doubleblind phase iia studies current and future treatment options for hcv randomised clinical trial: anti-viral activity of ana773, an oral inducer of endogenous interferons acting via tlr7, in chronic hcv safety, pharmacokinetics and immune effects in normal volunteers of cpg 10101 (actilon), an investigational synthetic toll-like receptor 9 agonist a phase 1, multicenter, randomized, placebo-controlled, dose-escalation study of imo-2125, a tlr9 agonist antiviral applications of tolllike receptor agonists update on the development of antiviral tlr agonists including description of the chemistry applied in the develpment of the therapeutic agents imo-2125 plus ribavirin gives substantial first-dose viral load reductions, cumulative antiviral effect, is well tolerated in naïve genotype 1 hcv patients: a phase 1 trial the vaccine adjuvant monophosphoryl lipid a as a trif-biased agonist of tlr4 adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling triggering tlr signaling in vaccination new hepatitis b vaccine formulated with an improved adjuvant system clinical update of the as04-adjuvanted human papillomavirus-16/18 cervical cancer vaccine, cervarix as04, an aluminum salt-and tlr4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity vaccine adjuvants: scientific challenges and strategic initiatives immunopotentiation of trivalent influenza vaccine when given with vax102, a recombinant influenza m2e vaccine fused to the tlr5 ligand flagellin development of vax128, a recombinant hemagglutinin (ha) influenza-flagellin fusion vaccine with improved safety and immune response safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (vax125) in healthy young adults targeted impairment of innate antiviral responses in the liver of chronic hepatitis c patients distinct antiviral signaling pathways in primary human hepatocytes and their differential disruption by hcv ns3 protease poly(i:c) and lipopolysaccharide innate sensing functions of circulating human myeloid dendritic cells are affected in vivo in hepatitis c virus-infected patients the structural biology of toll-like receptors highlights the variation in the nature of the interactions of tlr paralog extracellular domains following ligand-induced dimerization rig-i like receptors in antiviral immunity and therapeutic applications structural basis of rna recognition and activation by innate immune receptor rig-i structural insights into rna recognition by rig-i gold nanorod delivery of an ssrna immune activator inhibits pandemic h1n1 influenza viral replication coexpressed rig-i agonist enhances humoral immune response to influenza virus dna vaccine a signaling polypeptide derived from an innate immune adaptor molecule can be harnessed as a new class of vaccine adjuvant viral and cellular micrornas as determinants of viral pathogenesis and immunity this work was supported by grants from the canadian institutes for health research (cihr-ci6-103135) and by the novartis/canadian liver foundation hepatology research chair to dl. nt is a recipient of a ph.d. scholarship from the fonds de recherche en santé du qué bec (frsq). key: cord-307556-k2lavvca authors: jang, hongje; ryoo, soo‐ryoon; kim, young‐kwan; yoon, soojin; kim, henna; han, sang woo; choi, byong‐seok; kim, dong‐eun; min, dal‐hee title: discovery of hepatitis c virus ns3 helicase inhibitors by a multiplexed, high‐throughput helicase activity assay based on graphene oxide date: 2013-02-18 journal: angew chem int ed engl doi: 10.1002/anie.201209222 sha: doc_id: 307556 cord_uid: k2lavvca a go‐to solution: a simple graphene oxide (go)‐based assay to screen for selective inhibitors of a hepatitis c virus (hcv) helicase along with inhibitors of a severe acute respiratory syndrome coronavirus (sars cov) helicase was tested (see scheme). a single screen found five inhibitors highly selective for the hcv helicase orthologous to the sars cov helicase. some of these hits were validated using the same go‐based assay.[image: see text] worldwide, over 170 million people have hepatitis c virus (hcv) infections. [1] chronic infection with hcv leads to liver diseases such as cirrhosis and hepatocarcinoma and is the major reason of liver transplantation. [2] currently, the standard treatment for hepatitis c relies on a combination of interferon-a and ribavirin (an immune booster and a general inhibitor of virus replication, respectively) which is associated with serious side effects including hemolytic anaemia, depression, fatigue, flu-like symptoms, and birth defects. [3] the standard of care is frequently ineffective in clearing hcv infections and the virus often survives and thrives even under the treatment. to develop direct-acting antiviral agents for hepatitis c treatment, studies have been focused on the discovery of inhibitors of viral enzymes, specifically nonstructural protein 3 (ns3) serine protease and ns5b rna-dependent rna polymerase (rdrp). [4] last year, new drugs for treating hepatitis c, telaprevir (vertex) and boceprevir (merck) , which are ns3 serine protease inhibitors, were approved by the u.s. food and drug administration (fda), and over 30 drug candidates targeting the same protease or ns5b rdrp are currently in clinical trials. [5] although phase iii clinical trials of telaprevir and boceprevir showed notable increases in the cure rate, nearly every patient still suffered from at least one side effect of the new therapy. [5b,c, 6] in addition, hcv has strong drug resistance due to its high mutability. thus, further therapeutic options are urgently needed to treat hcv infections more effectively. the c-terminal two thirds of hcv ns3 forms a helicase, which has the ability to unwind double-stranded dna (dsdna) into single-stranded dna (ssdna) and is fueled by nucleoside triphosphates (ntps) hydrolyzed by its ntpase domain. [7] ns3 helicase is one of the essential enzymes of hcv along with ns3 serine protease and ns5b rdrp for processing hcv proteins and replication of hcv. thus, the inhibition of helicase activities is an important strategy for treating hcv infections. [8] however, discovery of helicase inhibitors has been much slower compared to that of other hcv drug targets. to date, only a few classes of ns3 helicase inhibitors have been reported, partly because high-throughput screens have yielded only a few successful hits. [8a,c] for example, the compounds discovered in the nih screen based on the molecular-beacon-based helicase assay (mbha) showed poor activity in cells and turned out to interfere with the assay, [9a] even though another mbha screening identified compounds that inhibited rna replication in cells. [9b] therefore, there is an urgent need for new assays to measure helicase activity that are suitable for high-throughput screens. herein, we developed a multiplexed helicase assay based on graphene oxide (go) for high-throughput screening of inhibitors of hcv ns3 helicase and severe acute respiratory syndrome coronavirus (sars cov) helicase. previously, we reported a go-based helicase assay (goha), which relied on the preferential binding of ssdna over dsdna to the go surface and subsequent quenching of the fluorescently labeled ssdna by energy transfer from the dyes to the go, and we validated the assay using sars cov helicase. [10] herein, we show that the goha can be used for measuring the activities of hcv ns3 helicase and sars cov helicase in a single mixed solution using two distinct dna substrates tethered to different fluorophores, and furthermore, for multiplexed high-throughput screening to discover highly selective small-molecule inhibitors of these helicases ( figure 1 ). one round of screening the chemical library using the multiplexed goha (mgoha) revealed three classes of inhibitors-specific inhibitors of hcv ns3 helicase, specific inhibitors of sars cov helicase, and general inhibitors of both helicases. to date, go has been used to develop various biosensors and enzyme assays, [10, 11] but concerns about the heterogeneity of the chemical structure and the physical dimensions of go and nonspecific binding of biomolecules to go hamper the application of go-based assays in high-throughput parallel assays for drug discovery that require high robustness and reproducibility. herein, we show that go can be used for the discovery of potent smallmolecule drug candidates and also determine the specificity and relative effectiveness towards each helicase of each of the identified hits. first, go sheets were prepared by using a modified hummers method. [10, 12] the thickness of the prepared go sheets was 0.97 nm and the width was 0.01-4 mm, which were determined by atomic force microscopy (afm; supporting information, figure s1 a). the ir spectrum of go showed peaks at 3395, 1716, 1225, and 1079 cm à1 corresponding to o à h stretching vibration, c=o stretching vibration, cào (epoxy) vibration, and cào (alkoxy) vibration (figure s1 b). the raman spectrum of go had strong d-and g-band absorptions at 1351 and 1589 cm à1 , respectively, which are characteristic for go (figure s1 c) . collectively, water-dispersible go sheets were successfully prepared. to measure the orthogonal activity of sars cov and hcv ns3 helicases, either or both helicases were added to a mixture of go and two dna substrates-a cy3-labeled dna duplex as the sars cov helicase substrate (cy3-dna-sh) and a cy5-labeled dna duplex as the hcv ns3 helicase substrate (cy5-dna-hh; figure s2 a). [13] fluorescence intensities corresponding to cy3 (ex/em = 550/570 nm) and cy5 (ex/em = 650/670 nm) showed little change without the addition of helicase over 60 min (figure 2 a) . the addition of sars cov helicase led to a dramatic decrease in fluorescence intensity for cy3 only, while cy5 fluorescence intensity only showed a very small change, suggesting that the cy3-labeled dna duplex was mainly unwound, and subsequently, the interaction of the generated cy3-labeled ssdna with go resulted in the quenching of the cy3 fluorescence (figure 2 b ). this data indicated that the unwinding activity of the sars cov helicase was specific to its corresponding dna substrate. conversely, the addition of hcv ns3 helicase caused a significant decrease in fluorescence intensity of cy5, but not cy3 ( to assess the quality of the mgoha for screening, the z'factor, a parameter widely used to evaluate the robustness and quality of screening assays, [14] was determined. generally, high-performance assays have z'-factors of 0.75 or higher. [15] to calculate the z'-factor, we ran 30 positive and 30 negative control reactions, calculated the mean (m c+ and m cà for the positive and negative controls, respectively) and standard deviations (s c+ and s cà ), and used an equation shown in figure s2b . the z'-factor of the mgoha platform for sars chemie helicase was 0.95 and hcv helicase was 0.94, suggesting that the present goha is better and more robust than other established assays (figure 2 f) . a 96-well plate mgoha was used to screen a 10 000 compound library to discover inhibitors of sars cov helicase and hcv ns3 helicase (figure 3) . a mixture of library compounds and both helicases were prepared and added to mixed solutions of cy3-dna-sh, cy5-dna-hh, and go that were prepared separately. in the primary screen, the library compounds, helicases, and substrates were used at concentrations of 1 mm, 2 nm, and 10 nm, with a total volume of 60 ml for each mixture. after incubation for 30 minutes at 25 8c, the cy3 and cy5 fluorescence was measured using an ivis fluorescence imaging system (figure 3 ; figure s3 ). positive controls were performed with cy3-dna-sh and cy5-dna-hh and both helicases, in the absence of library, and negative controls were with cy3-dna-sh and cy5-dna-hh, excluding any helicases or compounds. out of the 10 000 small molecules, 26 specific inhibitors of sars cov helicase and 22 of hcv ns3 helicase were found, and 24 compounds were found to inhibit both helicases (! 50 % inhibition). we further characterized the discovered inhibitors to quantitatively evaluate their inhibitory effects towards the respective helicases. first, to increase the fidelity of the data and to eliminate false positive hits owing to poor solubility of the compounds, we performed a secondary screening in replicates of five with the selected compounds from the primary screen (72 total compounds) while carefully observing the reaction mixtures for precipitation. in the secondary screen, the selectivity towards each helicase and the degree of inhibition were comparable to the primary screen. out of 72, 25 compounds showed severe precipitation and were excluded from the final selection. finally, we selected the 15 most-potent compounds including five sars cov helicase selective inhibitors (antisars-hel-1-5), five hcv ns3 helicase selective inhibitors (antihcv-hel-1-5), and five inhibitors of both helicases (antisars-hcv-hel-1-5). the chemical formulas of the selected compounds are shown in figure s3 . dose-dependent inhibition of sars cov helicase and hcv ns3 helicase were measured with varying concentrations of inhibitor using goha, and the half-maximal inhibitory concentration (ic 50 ) was calculated (figure 4 a) . the antisars-hels and antihcv-hels had ic 50 values of 49.3-104.6 mm and 51.6-92.0 mm, respectively. we found that antisars-hcv-hel-1-5 had higher ic 50 values than the selective inhibitors, ranging from 103.8 to 537.5 mm for sars cov helicase and from 103.5 to 441.1 mm for hcv ns3 figure 3 . a small-molecule screen identified three sets of inhibitorsspecific for sars cov helicase, specific for hcv ns3 helicase, and nonspecific helicase inhibitors. top) representative fluorescent images from the primary screen carried out with 10 000 small molecules in 96well plates. cy3 and cy5 signals represent the inhibition of helicase activities for sars cov and hcv ns3, respectively. bottom) using the 72 compounds selected from the initial screens, a secondary screen was performed in quintuplicate revealing the top 15 inhibitors. blue bars = sars-hel; red bars = hcv-hel. helicase. previously known helicase inhibitors generally exhibit ic 50 values ranging from 1-1000 mm. [8b] we expect that a structure-activity relationship (sar) study could further optimize these hits. the goha system can also be used for an sar study. the dose-response data and fitted ic 50 curves are shown in figure s5 . we next investigated the mechanism of the inhibitory effects for the discovered compounds. first, we determined whether antisars-hcv-hels bind to dsdna because the low selectivity between the two types of helicases might come from nonspecific binding to dsdna and thus, slow down the unwinding process. however, isothermal titration calorimetry (itc) and nmr spectroscopy showed no interaction between the antisars-hcv-hel-1-5 and dsdna (figures s7-s9) . we next determined whether the 15 compounds had any inhibition of the atpase activities of the helicases (figure 4 b) . sars-hel and hcv-hel possess an atpase domain for the catalytic hydrolysis of atp. we used a commercial kit to analyze the atpase activities of both helicases in the presence of the 15 inhibitors. we found that all of the antisars-hels except for antisars-hel-2 showed a significantly higher inhibition of the atpase activity of sars cov helicase than hcv ns3 helicase; all the antihcv-hels showed a much higher inhibition of atpase activity of hcv ns3 helicase than sars cov helicase. antisars-hcv-hel-1 and -2 blocked the atpase activities of both helicases to a similar degree. the antisars-hcv-hel-3-5 inhibited the atpase activity of hcv ns3 helicase more effectively than that of the sars cov helicase; however, the overall inhibition of antisars-hcv-hels on the atpase activities were not as impressive as that of the antihcv-hels against the atpase activity of hcv ns3 helicase. inhibitors selective for each helicase could block the atpase activity of each helicase with high selectivity. further studies should be performed to investigate the mode of inhibition-whether the inhibitors bind to the atp binding sites or induce conformational change by allosteric regulation. we next measured the inhibition of hcv rna replication by the 15 selected inhibitors in the human hepatoma cell line huh-7 carrying the hcv replicon rna with luciferase as a reporter gene (huh-7 replicon cell). [16] the replicon huh-7 cells were treated with the 15 inhibitors at concentrations of 0-1000 mm and then lysed, and the luminescence intensity was measured after the addition of luciferin. separately, the cytotoxicity of each compound was measured in the same cell line using an mtt assay. relative luciferase signal intensities over relative mtt signal intensities (luc/ mtt) were plotted versus the concentrations of the inhibitors ( figure 5 ; see figure s6 for the complete data of the luciferase and mtt assays). [17] two compounds, antihcv-hel-2 and -3, showed a dose-dependent decrease in the luc/mtt values with the respective half-maximal effective concentrations (ec 50 ) of 188.1 ae 32.6 and 56.8 ae 7.4 mm, indicating that they dose-dependently blocked hcv rna replication in the cultured huh-7 cells (figure 5 b,c) . antihcv-hel-2 and -3 decreased the luciferase signal by more than 90 % at 1 mm. the other antihcv-hel compounds, antisars-hel-5 and all the antisars-hcv-hels, were highly cytotoxic showing less than 40 % cell viability at 1 mm. antisars-hel-1-4 showed little reduction in the luciferase signal even at 1 mm. we excluded antihcv-hel-5 because of its relatively high ec 50 % 400 mm. taken together, antihcv-hel-2 and -3, blocked hcv rna replication in the cells whereas all of the antisars-hels and antisars-hcv-hels showed no appreciable inhibitory effect on hcv rna replication in cultured cells. these results show that helicase inhibitors should be specific to the corresponding helicase to achieve an inhibitory effect in living cells. in addition, the nonselective inhibitors that moderately reduce the activity of both helicases had a relatively high cytotoxicity, indicating that those compounds should be excluded for further drug development. we further investigated whether the antihcv-hel-2 and -3 could reduce the expression of hcv ns3 serine protease by blocking hcv rna replication in huh-7 cells (figure 5 d) . the cells were first treated with 250 mm of antihcv-hel-2 and -3 at which the luc/mtt values were 0.39 and 0.24, respectively, and the cell viability based on the mtt assay was over 70 %. after cell lysis, the hcv ns3 serine protease activity was measured with a fçrster resonance energy transfer (fret) peptide substrate. treatment with antihcv-hel-2 and -3 reduced the protease activity in the huh-7 cells down to 30 % of that in the untreated cells. this indicates that ns3 serine protease activity was also decreased as a result of the inhibition of hcv rna replication in living cells by antihcv-hel-2 and -3. in conclusion, we demonstrated the capability of mgoha for assaying the activity of two helicases, hcv ns3 and sars cov, and applied this mgoha to a highthroughput inhibitor screen using a 10 000 small molecule library, which yielded drug candidate molecules. these results illustrate that mgoha is an important screening technique for drug discovery targeting helicases. goha allows for figure 5 . cell-based assay to test the inhibition of hcv rna replication in huh-7 liver cells by antihcv-hel-2 and -3. a) a luciferase reporter system based on the expression of the hcv subgenome in huh-7 cells with a neomycin antibiotic resistance marker. b, c) to characterize the inhibition of hcv rna replication in live cells, huh-7 liver cells were treated with inhibitors and luciferase signal intensity from the reporter shown in (a) divided by cell viability was plotted versus inhibitor concentrations (to correct for luciferase signal loss from cytotoxicity of the compounds). d) to confirm that the two inhibitors block hcv rna replication and thus, reduce the expression of hcv enzymes, the hcv ns3 serine protease activity of the huh-7 cell lysates was measured following incubation with the two inhibitors. chemie more-accurate, real-time, quantitative monitoring of helicase activity by simply following the changes in fluorescence intensity, without requiring any additional separation steps or trapping agents. a multiplexed screen is highly advantageous not only because multiple rounds of screening are combined into one screen to reduce the overall cost and labor, but also because just one round of multiplexed screening provides information on the relative selectivity of the hit compounds towards each target helicase. given that various go-based activity assays have been developed for various enzymes, including nucleases, [18a] methyltransferases, [18] and caspases, [11c] the present work shows that a go-based assay can be used for high-throughput screening for new inhibitors of various essential enzymes. for the treatment of hcv infection, the addition of direct-acting antiviral agents, like inhibitors of ns3 protease, to the standard treatment was recently approved. the addition of multiple drugs acting against independent viral targets should be more effective in controlling infection with a rapidly mutating virus. we expect that the new inhibitors of hcv ns3 helicase discovered herin could be useful as antiviral drugs for the next generation of hcv treatment, with three or more active components. in addition, we expect that goha can be used to evaluate helicase activities in a highly parallel manner and of helicase-inhibitor-based drugs for various diseases, which has been difficult without a robust helicase assay. received: november 17, 2012 published online: january 25, 2013 . keywords: biosensors · graphene oxide · high-throughput screening · helicases · multiplex assays 360, 1839; b) key: cord-299719-bvdsz626 authors: fournier, c.; duverlie, g.; castelain, s. title: are trans‐complementation systems suitable for hepatitis c virus life cycle studies? date: 2013-02-14 journal: j viral hepat doi: 10.1111/jvh.12069 sha: doc_id: 299719 cord_uid: bvdsz626 complementation is a naturally occurring genetic mechanism that has been studied for a number of plus‐strand rna viruses. although trans‐complementation is well documented for flaviviridae family viruses, the first such system for hepatitis c virus (hcv) was only described in 2005. since then, the development of a number of hcv trans‐complementation models has improved our knowledge of hcv protein functions and interactions, genome replication and viral particle assembly. these models have also been used to produce defective viruses and so improvements are necessary for vaccine assays. this review provides an update on hcv trans‐complementation systems, the viral mechanisms studied therewith and the production and characterization of trans‐encapsidated particles. complementation is a naturally occurring genetic mechanism that enables the functional rescue of defective or mutant genomes. this phenomenon can be used for in vitro assays. in conventional complementation experiments, a defective genome or protein is rescued by a wild-type (wt) copy (fig. 1 ). in virology, complementation is a genomic tool used to investigate protein functions and viral particle assembly or establish whether genome replication occurs preferentially through a cis-acting mechanism. moreover, this model can also be used to produce defective viruses for various purposes. flaviviridae are positive-strand rna viruses and replicate their genomes in virus-induced vesicular compartments. these membranous complexes are rather enclosed structures, which tends to limit to exchange of viral rna and proteins by trans-complementation [1] . moreover, for a number of plus-strand rna viruses from a range of virus families (such as the picornaviridae (poliovirus), alphaviridae (sindbis virus, semliki forest virus, and venezuelan equine encephalitis virus), coronaviridae (human coronavirus e229) and flaviviridae (tick-borne encephalitis virus, kunjin virus, west nile virus, and yellow fever virus)), assembly of progeny viruses can be achieved when structural proteins are expressed in trans independently of the rna molecule that encodes the replicase proteins [2] [3] [4] [5] [6] [7] . the particles are infectious but are only capable of a single round of infection. these viral transencapsidation systems may also be useful vaccine delivery systems [8] [9] [10] . concerning hcv, some researchers have reported the presence of natural hcv subgenomic rnas in the serum and liver tissue of infected patients. most of these subgenomic rnas contain large, in-frame deletions (from e1 up to ns2) and are always found together with the full-length wt rnas [11] [12] [13] [14] . the mutant viral genomes persist for at least 2 years and sequence analysis suggests that subgenomic and full-length hcv rnas evolve independently [14] . the relative abundance and persistence of these subgenomic rnas in vivo suggests that they are capable of autonomous replication and can be packaged and secreted as infectious viral particles. the first hcv trans-complementation studies concerned the replication complex. next, the development of cell-culture-grown hepatitis c virus (hcvcc) systems that can assemble and release of infectious viral particles has made it possible to the study structural regions using trans-complementation systems. furthermore, various trans-packaging systems for hcv subgenomic replicon rnas have been developed. the first trans-complementation studies were performed in 2005 by appel et al. by using subgenomic replicons, such as con-1 (hcv type 1b, embl accession number aj238799) [15] . the researchers looked at whether lethal mutations in non-structural (ns) hcv genes could be rescued by trans-complementation [1] . a series of replicon rnas carrying mutations (in the ns3, ns4b, ns5a and ns5b regions) that abolished replication were transfected into huh-7 hepatoma cells harbouring autonomous replicating hcv helper rnas. in this context, only ns5a mutations in the low complexity sequence i (lcs i) domain have been efficiently rescued ( table 1 ). the required proteins for complementation were ns3-ns5a, whereas ns5a expressed alone did not restore rna replication. these findings strongly suggested that other ns hcv proteins specifically act in cis on hcv rna and, indeed, the hypothesis was confirmed in 2006 by tong and malcom [16] . nevertheless, trans-acting function of ns5a likely would require protein hyperphosphorylation [17] . in 2008, additional studies showed that deletion of ns5a domain iii from the jc1 genome (hcv chimeric type 2a/2a) [18] does not abolish replication but that pseudoinfectious particles are not produced [19] . therefore, cotransfection of deleted jc1 with a jfh1 ns3-ns5b replicon rescues hcv particle production (table 1) . taken as a whole, these results have elucidated the key role of ns5a domain iii in the hcv assembly process. in 2009, jones et al. [20] performed the firstly ns4b transcomplementation using various replicon rnas carrying various replication-abolishing mutations in the ns4b c-terminal domain. the rnas rescued replication of two g196a and e226a mutants (table 1) . nevertheless, the complementation efficiency was lowabout 1%, vs 24% for the ns5a s232a mutant. both g196 and e226 are highly conserved amino acids that are predicted to lie within unstructured regions flanking helix 1. the researchers concluded that ns4b complementation was limited. trans-complementation assays have also been used to study the role of ns2 in viral particle assembly [21] . a number of different deletions within trans-membrane segments (tms1-3) or the protease domain of the jc1 (j6/ jfh1) chimera genome have been examined (table 1) . again, replication was seen in the absence of pseudo-infectious particle production. three bicistronic helper rnas (spe2-p7-ns2, spp7-ns2 and spns2) were built by combining the jfh1 replicase module (utrs and the ns3-ns5b coding region) and a 5′-terminal expression module under control of the poliovirus internal ribosomal entry site (ires). various cotransfections between jc1 ns2 mutants and bicistronic helper rnas were performed in huh-7.5 cells. all three ns2 mutants could be rescued by transcomplementation. infectivity titres obtained in rescue experiments were about 10-fold lower than in the jc1 wt. at the same time, yi et al. [22] studied the role of the ns2 serine amino acid (aa) at position 168 (ser-168) in the viral assembly and maturation process. they found that substitution of ser-168 by an alanine or glycine aa abolished infectious virus production but not polyprotein processing or genome replication (table 1 ). however, transfection of ser-168 mutant replicon rna into a cell line expressing wt ns2 rescued the production of infectious virus. in contrast, transfection into a wt h77 (type 1a) ns2-expressing cell line was unable to rescue virus production. although h77 and jfh1 ns2-expressing cells could rescue viral production in the h77-jfh1 chimera ser-168 mutant, this rescue was only partial with wt jfh1 ns2expressing cells. the same experiment was performed in a cell line expressing ns2 carrying a 71-aa deletion in the cterminal domain. infectious particle production was not obtained, indicating that ns2's c-terminal domain is a major determinant of hcv production. all these results indicate that wt ns2 expression is able to trans-complement ns2-defective constructs and is genotype-and straindependent. fig. 1 schematic representation of the transcomplementation principle in virology. mutant or defective genomes can be rescued by a wild-type copy that allows the production of infectious particles capable of a single round of infection. appel et al. [19] jc1-ns5ad2328-2435 jones et al. [20] luc-jfh1-e226a yi et al. [22] jfh1-ns2-s168g hj3-5-ns2-s168g in conclusion, trans-complementation based on ns regions has shown that (i) ns5a region carrying mutations in the lcs1 domain or deletions in domain iii can be complemented by the minimal sequence ns3-ns5a, (ii) ns2 can be efficiency trans-complemented by wt ns2 and (iii) ns4b trans-complementation exists for mutations in unstructured regions but is weak. the use of the hcvcc system to produce infectious viral particles has enabled investigation of the core protein's role in the assembly process. it has been shown that core and 60-90% of the ns proteins were localized within lipid droplets (lds) [23] . conversely, the lack of core expression in replicating jfh1 cells (jfh1dc) was associated with a diffuse distribution of ns proteins on the endoplasmic reticulum (er) and the absence of concentration around the lds. moreover, ld levels were lower in jfh1dc replicating cells than in wt jfh1 replicating cells. interestingly, ns proteins again accumulated around lds when jfh1dc replicating cells were transfected with a plasmid expressing wt core. these results showed that ld-associated core recruits ns proteins from the er to the lds. moreover, core activity could be restored by trans-complementation. in parallel, kopp et al. [24] used trans-complementation assays to investigate the role of the core c-terminal region and to identify the essential residues involved in infectious particle production. mutations were introduced between residues 170-191 (table 1 ). it was found that mutations at positions 170 and 174-177 abrogated pseudo-infectious particle production, despite the presence of wild-type replication levels. in contrast, cotransfection of the j6/jfh1 replicon (containing a large deletion in the sequence coding for core protein, that is residues 57-160) with a fulllength hcv core protein plasmid led to significant levels of pseudo-infectious particle release. no pseudo-infectious particles were obtained when using core 170 and 174-177 mutants plasmids. in this context, trans-complementation systems have highlighted the importance of these residues in pseudo-infectious particle production. brohm et al. [25] developed a complementation system for investigating key determinants that are essential for the optimal function of p7 in hcv infectious particle production. three jc1 replicons were constructed: one with a deletion of the entire p7 coding region (jc1dp7 full ), a second lacking residues 1-32 of p7 (jc1dp7 half ) and a third carrying a dual mutation of the highly conserved basic residues kr33/35 to qq (jc1-kr33.35qq) ( table 1 ). the replicons jc1dp7 full and jc1dp7 half were unable to generate infectious particles. with the jc1-kr33.35qq replicon, 100-fold fewer infectious particles were released than with the parent jc1 genome. these defective hcv p7 replicons were then cotransfected with bicistronic jfh1-derived helper replicons expressing either p7 alone, p7-ns2, e2-p7, e2 alone or e2-p7-ns2 in the first cistron and jfh1 ns proteins in the second. jc1dp7 half infectivity was rescued by all helper rnas expressing p7, whereas the e2 signal sequence had no influence. however, cotransfection of e2-p7 and e2-p7-ns2 helper rnas yielded higher virus titres. in the case of the jc1dp7 full replicon, complementation was achieved only by the co-expression of e2-p7-ns2. jc1-kr33.35qq infectivity was rescued by all helper rnas, although pseudo-infectious particle production was only increased in the presence of e2-p7 and e2-p7-ns2 helper rnas. lastly, no production of infectious particles was seen with the con1-p7 (hcv type 1b) helper replicon, which indicated genotype specificity. these results indicate that a p7 protein-deficient replicon could only be rescued by e2-p7-ns2 expression and not by p7 protein alone. in conclusion, trans-complementation of the structural region has shown that core can be trans-complemented by wt core and p7 can be trans-complemented by the e2-p7-ns2 sequence and not by p7 expressed alone. hcv trans-encapsidation systems the first trans-encapsidation systems were described for packaging replicons for flavivirus, including kunjin virus [2, 3] , yellow fever virus [4] , tick-borne encephalitis virus [5] and west nile virus [6, 7] . these virus-like-particle-generating systems enabled the development of vaccine models and the analysis of some aspects of viral assembly and entry [6] . based on these studies, various systems for packaging hcv replicons have been created. two types of constructs have been used in trans-encapsidation systems: a minimal replicase complex and the fulllength genome carrying deletions in envelope-encoding genes ( table 2 ). the first studies used jfh1-ns3-ns5b replicons associated either with a resistance gene (such as neomycin in sgr-jfh1) [26] [27] [28] [29] or a reporter gene (such as renilla luciferase in luc-ns3-ns5b) [30] . the sgr-jfh1 replicon enabled the establishment of stable cell lines [26] , the titration of colony-forming units (cfu/ml) [26, 27] and the selection of efficiently replicating cell clones [28] . rapid infectious particle assays [30] could been performed with the luc-ns3-ns5b replicon. the sgr-jfh1 ns2-ns5b construct was used by adair et al. [26] to study ns2's role in the particle assembly process and has yielded the highest infectious titres. masaki et al. and suzuki et al. chose to use a jfh-1 cdna clone plasmid containing the pol i promoter, terminator sequences and luciferase gene reporter sequences for directly transfecting cells without the need for a transcription step [31, 32] (table 1) . another approach was adopted by pacini and sugiyama, using replicons based on the defective genomes found in both serum and liver tissue from chronically hcv-infected patients. sugiyama et al. generated a 1b/jfh1-2a chimera based on defective 1b patient isolate and that carried deletions in the genes coding for the envelope, p7 and ns2 proteins [33] . likewise, pacini et al. used a j6/jfh1 chimera to build constructs with various deletions in the e1-e2 glycoprotein region (448 aa) and the e1 to ns2 (674 aa), e2 to p7, core to p7 and e2 to ns2 regions, respectively (table 2) . lastly, several research groups have studied replicons carrying deletions (351 aa) in the envelope gene (jfh1de1e2 or j6/jfh1-de1e2) [29, 30, 34, 35] (table 2) . encapsidation of subgenomic replicons with partially or totally deleted structural regions has been performed in various systems expressing structural proteins in trans (table 3) in the presence or absence of p7 and ns2 proteins. some researchers have focused on replicons, helper viruses [29, 30] and direct expression vectors [29, 31] , whereas others have established stable cell lines [27, 30, 33] and baculovirus or lentivirus delivery systems [26, 28] . luc-cored212-ns2 luc-nocore-ns2 luc-cored212-ns3 pacini et al. [29] pacini et al. [29] pacini et al. [ the first trans-encapsidation assays were performed in huh7.5 cells via transient transfection with replicons or helper viruses, allowed reproducing in vitro natural transencapsidation mechanism in vivo [29] . similar experiments with heterologous helper viruses (such as con1/c3 and jc1 [30] ) have been attempted. however, given the competition between defective replicons and helper viruses, it has been difficult to obtain equal replication and high trans-encapsidation efficiencies. moreover, two different markers must been introduced to identify the two encapsidated replicons. other researchers have chosen structural proteins expressing plasmids (in the presence or absence of p7 and ns2) to perform trans-encapsidation and determine the minimal sequence required for efficient encapsidation. various plasmid constructs have been used, such as the pef6 plasmid (encoding c-ns2, c-p7, c-e2, e1-ns2, e1-p7 or e1-e2 from j6/jfh1 chimeras [29] ), the pcagc plasmid (expressing c-p7 and c-ns2 regions [31, 32] ), pcdna-e1-p7 [34] and pcdna3-jfh1-e1/e2 [35] (table 2) . in parallel, the use of stable cell lines has improved the yield of pseudo-infectious particles. with a pef4 plasmid, ishii et al. [27] established stable cell lines expressing jfh1 core-p7 and core-ns2 proteins, respectively. steinmann et al. [30] cotransfected lentiviral vectors expressing jc1 core-e1 and e2-ns2 proteins into huh7.5 and huh7-lunet cells, respectively, and obtained high levels of structural protein expression. conversely, stable cell lines based on defective replicons with resistant genes (such as sgr-jfh1) can be used to select more efficient cell clones and achieve high levels of replication. the latter models to have been developed for the expression of complementing structural proteins are baculovirus systems expressing jfh1 core to p7 or core to ns2 proteins [26] and lentivirus systems expressing core to ns2 [28] . although trans-encapsidation assays have preferentially been performed in human liver-derived cell lines (i.e. huh7 and subclones), murine hepatic cell line have also been studied [28] . in a two-step development process, deleted replicons and helper replicons are first validated in transient experiments and then adapted to stable cell lines for standardizing and improving the system. various researchers have thus shown that trans-packaging systems are functional (table 3) . it has been proven that the ns3-ns5b replicon can be packaged by core-ns2 or core-ns3 proteins expressed in trans but not by core-p7 [26] [27] [28] 30, 31] . therefore, ns2 protein is essential for infectious particle production. the most efficient trans-packaging has been obtained with core-ns2 [28] . in parallel, it was found that the ns2-ns5b replicon could be packaged by the core-p7 region. however, the trans-packaging efficiency was higher with the ns3-ns5b replicon and the core-ns2 sequence [26] . in the case of full-length defective genomes, trans-packaging has been observed with core-ns2, core-ns3 and e1-ns2 constructs [29, 33] . the efficiency was greatest with core-ns2 [33] . in turn, the pseudo-infectious particles' physical and antigenic properties have been compared with those observed for wt virus [26] [27] [28] 30, 35] . the particle density was similar to that seen in hcvcc particles. neutralization assays with anti-cd81 [27] [28] [29] [30] [31] 35] , anti-e2 [26] , anti-scarb-1 and anti-claudin-1 [28] antibodies have shown that pseudo-infectious particles use both e2 glycoprotein and hcv entry receptors for cell entry. ishii et al. [27] have observed particles under the electron microscope and have confirmed similarities with hcvcc particles. these pseudo-infectious particles were capable of a only single round of infection and the defective genome had been clearly encapsidated [27, 30, 35] . several research groups have developed assays for transpackaged particles [26] [27] [28] 30, 35] , whereas others have only observed the particles' features and estimated the efficiency of trans-encapsidation [29, 31, 33] . various titres of pseudo-infectious particles have been obtained: 3.4 ae 0.6 10 2 focus-forming units (ffu)/ml according to ishii et al., 10 4 tissue culture infectivity dose (tcid) 50 /ml according table 3 summary of infectious particles generated by cotransfection of deleted subgenomic replicons with structural proteins expressed in trans. packaging feasibility is expressed as positive (+) or negative (à). nt: not tested. [26, 27, 30, 34, 35] ( table 4 ). the comparison of the respective systems' yields is difficult because of heterogeneity of the infectious units used and differences in detection experiments. several groups have tried to improve their trans-packaging systems by introducing the above-mentioned adaptive mutations into subgenomic replicons [30, 32, 35] or complemented structural protein regions [26] . for example, the v2440l mutation has been introduced into c-terminal region of ns5a and was found to enhance production [36] . using jfh1de1e2 replicons, li et al.'s group inserted an m1051l mutation into ns3 region and a c2219r mutation into ns5a. they obtained a peak titre of 5 9 10 3 iu/ml [35] . similar observations have been obtained with baculovirus systems and the introduction of f172c and p173s mutations in the c-terminal part of core [26, 37] . lastly, mutations in e2 (n417s), p7 (n765d) and ns2 (q1012r) were introduced into the core-ns2 expression plasmid pcagc and resulted in a more than fourfold increase in pseudoparticle production [32] . in parallel, heterologous trans-packaging assays have been developed. the first group to do so was steinmann et al., who tested the jfh1-luc-ns3-ns5b replicon with con1/c3 (1b/jfh1) and jc1 (j6-2a/jfh1) chimeras. infectious, trans-packaging particles were obtained with each of the two chimeras but jc1 gave the best yield [30] . li et al. [35] chose heterologous trans-encapsidation of jfh1de1e2 replicons with hcv j6 [2] , h77 (1a) and con1 (1b) envelope glycoproteins. only j6 (2a) envelope proteins were able to rescue pseudo-infectious particle production. intergenotype compatibility between envelope proteins and the other proteins may be essential in the assembly process [35] . the new jfh1-based culture system has enabled the development of trans-complementation systems that have facili-tated studies of (i) genomic and protein functions and (ii) interactions between hcv proteins. trans-complementation based on ns regions has shown that (i) ns5a region carrying mutations in the lcs1 domain or deletions in domain iii can be complemented by the minimal sequence ns3-ns5a, (ii) ns2 can be efficiency trans-complemented by wt ns2 and (iii) ns4b trans-complementation exists for mutations in unstructured regions but is weak. as expected, genomic trans-complementation in hcv replication systems is limited and ns hcv proteins act preferentially in cis. these trans-complementation studies have increased our knowledge of non-structural proteins' functions and interactions in hcv. they have demonstrated that ns5a domain iii is a major determinant of hcv assembly. ns4b is involved in rna replication but also contributes to virus assembly and release. ns2 is involved in the viral assembly process; in a late-post assembly maturation step (perhaps in concert with ns5a) and, it confers infectivity on the hcv particle. moreover, ns2 generates a strict cis requirement for the core region, to allow efficient trans-packaging of the subgenomic rnarevealing the complex interplay between ns2 and core genes [29] . overall, these results have confirmed that ns proteins are involved in viral assembly and release. trans-complementation of the structural region has shown that core can be trans-complemented by wt core. core recruits ns proteins and replication complexes to ldassociated membranes. this recruitment is critical for producing infectious viruses and residues 170 and 174-177 are important for the production of infectious viruses. likewise, p7 can be trans-complemented by the e2-p7-ns2 sequence. p7 is absolutely essential for the production of pseudo-infectious hcv particles; it can operate independently of an upstream signal sequence and a tyrosine residue close to its conserved, dibasic motif is important for optimal virus production in genotype 2a viruses. these results have clarified the role of core, p7 and lds in pseudo-infectious particle production and have indicated that some steps of virus assembly take place around lds. various trans-packaging systems have been developed with subgenomic replicons. the structural proteins were expressed in trans using helper replicons, helper plasmids, stable cell lines or viral vectors. although the respective [34] 1100 ffu/ml pcdna-e1-p7 transient expression system efficiencies of these systems cannot be compared directly, the titres of trans-packaged infectious particles (around 10 2 ffu/ml) were generally lower than wt particle titres (about 10 3 -10 4 ffu/ml for the jfh1 strain). nonetheless, these titres have been improved by the introduction of adaptive mutations that favour particle production with defective replicons or structural regions expressed in trans. however, some limits exist with heterologous glycoproteins and intergenotype compatibility seems to be required for some proteins. flaviviridae trans-complementation studies have enabled the production of vaccine-like preparations based on transpackaging particles. in particular, it has been reported that the envelope glycoprotein erns in classical swine fever virus (a pestivirus) can be complemented in trans by an sk6 cell line that constitutively expresses this protein [38] . pigs vaccinated with these defective viral particles were protected against a lethal challenge with the virulent brescia strain. this observation opened up new perspectives in the development of modified, live-attenuated vaccines. however, there are no reports of in vivo vaccine activity or neutralizing antibody production with hcv trans-complementation particles. one possible explanation is that immunization studies require high infectious titres (in the order to 10 7 or 10 8 ffu/ml) but trans-packaging particle production is low (about 10 2 ffu/ml). additional studies are needed to increase pseudo-infectious particle yields for vaccine assays. lastly, hcv trans-complementation is a natural phenomenon that can be observed with native defective subgenomic rnas identified in infected patients and encapsidated by helper viruses. in conclusion, hcv trans-complementation systems have been a valuable tool for improving our knowledge of the hcv life cycle. nevertheless, these systems need to be perfected for use in vaccine assays. efficient rescue of hepatitis c virus rna replication by trans-complementation with nonstructural protein 5a encapsidation of the flavivirus kunjin replicon rna by using a complementation system providing kunjin virus structural proteins in trans kunjin virus replicon vectors for human immunodeficiency virus vaccine development construction and applications of yellow fever virus replicons incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line n-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity high-throughput assays using a luciferase-expressing replicon, viruslike particles, and full-length virus for west nile virus drug discovery kunjin virus replicon vaccine vectors induce protective cd8 + tcell immunity replicon-helper systems from attenuated venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo selfreplicating semliki forest virus rna as recombinant vaccine high levels of subgenomic hcv plasma rna in immunosilent infections identification of novel defective hcv clones in liver transplant recipients with recurrent hcv infection characterization of hepatitis c virus deletion mutants circulating in chronically infected patients identification of novel hcv subgenome replicating persistently in chronic active hepatitis c patients replication of subgenomic hepatitis c virus rnas in a hepatoma cell line trans-complementation of hcv replication by non-structural protein 5a distinct functions of ns5a in hepatitis c virus rna replication uncovered by studies with the ns5a inhibitor bms complete replication of hepatitis c virus in cell culture essential role of domain iii of nonstructural protein 5a for hepatitis c virus infectious particle assembly the hepatitis c virus ns4b protein can trans-complement viral rna replication and modulates production of infectious virus structural and functional characterization of nonstructural protein 2 for its role in hepatitis c virus assembly trans-complementation of an ns2 defect in a late step in hepatitis c virus (hcv) particle assembly and maturation the lipid droplet is an important organelle for hepatitis c virus production genetic analysis of the carboxy-terminal region of the hepatitis c virus core protein characterization of determinants important for hepatitis c virus p7 function in morphogenesis by using trans-complementation expression of hepatitis c virus (hcv) structural proteins in trans facilitates encapsidation and transmission of hcv subgenomic rna trans-encapsidation of hepatitis c virus subgenomic replicon rna with viral structure proteins mouse hepatic cells support assembly of infectious hepatitis c virus particles naturally occurring hepatitis c virus subgenomic deletion mutants replicate efficiently in huh-7 cells and are trans-packaged in vitro to generate infectious defective particles efficient trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles production of infectious hepatitis c virus by using rna polymerase imediated transcription trans-complemented hepatitis c virus particles as a versatile tool for study of virus assembly and infection genetic analysis of hepatitis c virus with defective genome and its infectivity in vitro vps4 and the escrt-iii complex are required for the release of infectious hepatitis c virus particles production of hepatitis c virus lacking the envelope-encoding genes for singlecycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans cell culture adaptation of hepatitis c virus and in vivo viability of an adapted variant robust production of infectious viral particles in huh-7 cells by introducing mutations in hepatitis c virus structural proteins classical swine fever virus e(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, liveattenuated marker vaccines this study was funded by the conseil r egional de picardie (hepanovir, grant no. aap08-12). key: cord-259916-gr6v098c authors: wang, hongliang; tai, andrew w. title: mechanisms of cellular membrane reorganization to support hepatitis c virus replication date: 2016-05-20 journal: viruses doi: 10.3390/v8050142 sha: doc_id: 259916 cord_uid: gr6v098c like all positive-sense rna viruses, hepatitis c virus (hcv) induces host membrane alterations for its replication termed the membranous web (mw). assembling replication factors at a membranous structure might facilitate the processes necessary for genome replication and packaging and shield viral components from host innate immune defenses. the biogenesis of the hcv mw is a complex process involving a concerted effort of hcv nonstructural proteins with a growing list of host factors. although a comprehensive understanding of mw formation is still missing, a number of important viral and host determinants have been identified. this review will summarize the recent studies that have led to our current knowledge of the role of viral and host factors in the biogenesis of the mws and discuss how hcv uses this specialized membrane structure for its replication. hepatitis c virus (hcv) is a globally prevalent human pathogen. more than 170 million people are chronically infected worldwide, among whom many will develop cirrhosis and hepatocellular carcinoma. hcv is an enveloped, single-stranded positive-sense rna virus classified in the hepacivirus genus within the flaviviridae family. the 9.6 kb genome contains one open reading frame (orf) that is flanked by non-translated regions, which are necessary for viral rna translation and replication [1] [2] [3] . a single polyprotein is translated from the orf, which is co-and post-translationally processed by cellular and viral proteases to generate ten mature proteins: core, e1, e2, p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b. the structural proteins (core, e1, and e2) are incorporated into virus particles, whereas the nonstructural proteins p7 to ns5b coordinate the intracellular processes of the virus life cycle [1] [2] [3] . while p7 and ns2 are dispensable for genome replication, they are required for particle assembly [4, 5] . ns3 through ns5b are necessary and sufficient for hcv genome replication. as we will discuss extensively in this review, hcv replication is associated with the induction of host membrane alterations that are thought to support sites of viral rna replication. the induction of altered host membranes for viral replication is characteristic of all positive sense rna viruses [6] . a negative sense replicative intermediate synthesized from the positive sense rna genome serves as template for the generation of progeny positive sense rna genomes. the newly-synthesized positive sense rna can either enter a new translation/replication cycle or be packaged into virions [2] . this review will summarize our current knowledge on hcv-induced membrane alterations, as well as the role of viral nonstructural proteins and host factors in this process. hcv-induced membrane alterations, as well as the role of viral nonstructural proteins and host factors in this process. even before the identification and molecular cloning of hepatitis c virus, electron microscopy (em) studies of liver tissue from chimpanzees infected with "non-a, non-b hepatitis" demonstrated membrane alterations in hepatocytes [7, 8] . the successful isolation of the first hcv cdna clone [9] enabled studies to determine the effects of expressing viral proteins in hepatocytes in cell culture. egger et al. reported that expression of the entire hcv polyprotein in u2-os human osteosarcoma cells was associated with the formation of membrane alterations described as vesicles within a membranous matrix, which collectively was termed "membranous webs" (mws) [10] . in this study, expression of ns4b alone also induced membrane alterations similar to those seen with the whole viral polyprotein. the subsequent establishment of the replicon model of hcv replication made it possible to visualize hcv induced membrane alterations in the context of viral genome replication. by using cell lines harboring persistent subgenomic replicons, gosert et al. [11] found altered membrane structures similar in ultrastructural morphology to those observed by egger et al. [10] ; furthermore, by using immunogold em, the authors reported that the membranous web could be labeled by antibodies against each of the hcv nonstructural proteins. at the light microscope level, immunofluorescence labeling for ns3 or ns5a was visible as dot-like cytoplasmic structures [11] [12] [13] [14] . a later study of cells containing a subgenomic hcv replicon reported that the membrane alterations induced by hcv replication consisted in part of double-membrane vesicles (dmvs; figure 1 ) with a diameter around 200 nm that were also positive on immunoelectron microscopy with antibodies against ns5a and double-stranded rna (dsrna) [15] . this was a notable observation, as a number of other rna viruses also induce dmvs in infected cells [6, 16] . the morphology of mws described above was confirmed and extended by using the cell culture infectious, full-length jc1 clone of hcv in conjunction with high pressure freezing with freeze substitution and electron tomography [17] . in this study, romero-brey et al. found that hcv infection was initially associated with accumulation of dmvs with an average diameter of 150 nm. the kinetics of dmv accumulation correlated with viral rna replication; at later time points of infection, multi-membrane vesicles (mmvs) with larger diameter (~330 nm) became more predominant [17] . 3d reconstructions of em tomographic series showed that most of the dmvs were tightly opposed to er membranes, and some of them were identified as protrusions from the er membrane into the cytosol, suggesting that mws originate from er membranes [17] . in addition, most of the dmvs were closed structures, with only a minority possessing either a visible opening towards the cytosol or a short neck-like structure connecting the dmv to the er membrane bilayer [17] . the identification of sites within hcv proteins that tolerate the insertion of heterologous sequences, such as epitope tags and even fluorescent proteins (for example, domain iii of ns5a tolerates the insertion of green fluorescent protein (gfp)), made it possible to study the dynamics of mw in live cells. ns5a-gfp was found in the cytoplasm as brightly fluorescing dots and in a reticular staining pattern [18] , similar to the distribution of ns5a observed in fixed and immunostained replicon cells. live cell imaging revealed two populations of ns5a-gfp foci in cells: larger, relatively static structures and smaller structures with saltatory microtubule-dependent movements over long distances, both of which contain other hcv replicase components [19] . however, the relationship of the two structures to one another is not well understood; for example, it is not known whether the smaller structures are precursors of or arise from larger static structures, or whether either structure participates preferentially in genome replication versus particle assembly. another approach to visualizing hcv replication organelles dynamics employed snap (a mutant of the human dna repair protein o6-alkylguanine-dna alkyltransferase) tagging of ns5a [20] , which permitted labeling of temporally-distinct populations of ns5a. this approach revealed that ns5a synthesized 48-72 h before imaging was located on structures distinct from those associated with ns5a synthesized 0-48 h before imaging, which provides an upper bound for the duration of viral polyprotein translation at a given replication organelle. a fundamental shortcoming of light microscopy is its limited resolution. correlative light electron microscopy (clem), which integrates the molecular specificity of fluorescent light microscopy with the resolution of em, has been employed to study the ultrastructural morphology of mws [17] and has also been used to analyze the effect of ns5a small molecule inhibitors [21] or ns5a mutants [22] on the morphology of mws. the imaging techniques to study mw morphology and dynamics summarized above have been complemented by biochemical studies. unfortunately, in vitro reconstitution of a functional hcv replicase is still beyond our technical reach. early studies showed that crude membrane fractions isolated from hcv subgenomic replicon cells or selectively permeabilized replicon cells could use endogenous replicon rna as a template to synthesize new viral rna [12, 14, [23] [24] [25] [26] . viral rna synthesis was found to be resistant to protease and nuclease treatment, suggesting that replication occurs in membrane structures [12] . additionally, membrane fractions containing hcv rna and nonstructural proteins were found to have properties similar to detergent-resistant membranes (drms) in that they were resistant to solubilization by cold triton x-100 and that these drms co-fractionated with cellular markers of drms on density gradient centrifugation [14, 26] . the specialized lipid and protein composition of the hcv replication organelle might, therefore, facilitate the generation of membrane curvature necessary for dmv formation [27] . in addition, as drms are important platforms for cellular membrane trafficking and signal transduction [27, 28] , it is conceivable that this property of hcv replication membranes may also regulate signal transduction and membrane trafficking at viral replication sites. while the viral rna is largely resistant to nuclease treatment, only a small fraction (2%-3%) of viral ns protein is resistant to protease treatment [26, 29] . a likely explanation for this observation is the large excess of hcv protein compared to positive-and negative-strand hcv rna; indeed, a quantitative analysis of hcv rna and protein content of replicon cells estimated that there is a 1000-fold excess of hcv protein over hcv rna [29] . in addition to subcellular fractionation, attempts have been made to isolate mws or replicase components by affinity capture of tagged ns proteins [30] [31] [32] or viral rna with associated proteins [33, 34] . one of these studies using epitope-tagged ns4b identified dmvs in the affinity-purified fraction and the presence of replicase activity associated with dmvs [30] , providing direct evidence that dmvs are a site of hcv rna synthesis. although hcv has long been known to induce membrane rearrangements, it is only recently that some of the mechanisms that are responsible for the formation of these structures have begun to be unraveled. despite the substantial progress that has been made during the past few years, we are still far from understanding this complex process in detail. in the following sections, we summarize what is known about the role of both viral and cellular proteins in hcv-induced membrane reorganization. by using hcv polyprotein overexpression, egger et al. [10] first showed that expression of viral nonstructural proteins resulted in membrane alterations that morphologically resemble those observed in replicon cells, demonstrating that viral rna replication is not required for membranous web formation. by expressing individual hcv proteins, these authors found that expression of ns4b was sufficient to induce membrane alterations resembling mws. later studies have shown that the n-terminal alpha helix ah2 [35] as well as c-terminal sequences [36] are important for hcv replication, ns4b oligomerization, and dmv morphogenesis. mutations of either of the two positively-charged lysine residues flanking the n-terminal alpha helix ah1 abrogate hcv replication and are associated with the formation of significantly larger dmvs when expressed in the context of ns3-5b using a non-replicative system [37] . subsequent studies, however, have provided evidence that ns5a is the only hcv ns protein capable of forming dmvs when expressed in isolation [17, 22] , albeit much less efficiently than when expressed in the context of ns3-5b. in contrast, expression of ns4b alone leads to the exclusive formation of single-membrane vesicles rather than dmvs [17] . the amino-terminal "domain 1" of ns5a is necessary and sufficient for the formation of dmvs when expressed in the context of the ns3-5b polyprotein [22] . this work also identified roles of other hcv ns proteins in efficient dmv formation, notably the ns3 helicase domain, and the expression of the ns3-4a protease in cis with ns4b-5b. finally, mutations that accelerate the normally slow polyprotein cleavage kinetics at the ns4b-5a junction or constructs that do not express any ns4b-5a precursor impair or abrogate dmv formation [22] , suggesting that a ns4b-5a precursor is somehow essential for dmv biogenesis. further evidence for a functional interaction between ns4b and ns5a comes from the identification of mutations in ns5a that rescue mutations flanking ns4b ah1 [37] or help rescue a ns4b c-terminal mutant [36] . overall, these studies suggest that most, if not all, nonstructural proteins are required to work in concert for the efficient formation of dmvs. in addition to viral proteins, an increasing list of host factors has also been shown to contribute to membranous web formation. here, we well briefly discuss the mechanisms of several selected host factors in mw formation. several rna interference screens have identified the cellular lipid kinase pi4ka (also known as phosphatidylinositol 4-kinase iii alpha, pi4kiiiα, and pik4ca) as essential for hcv replication [38] [39] [40] [41] [42] . inhibition of pi4ka, either by rna interference [40, [42] [43] [44] or by pharmacologic inhibitors [45] , leads to accumulation of large 'clusters' of ns5a-positive membranes at the light microscopic level. at the ultrastructural level, these 'clusters' correspond to clusters of dmvs with reduced diameter [42] , suggesting that pi4ka is essential for the proper formation and/or integrity of mws. ns5a and ns5b can interact with pi4ka and ns5a activates its lipid kinase activity, giving rise to elevated intracellular phosphatidylinositol 4-phosphate (pi4p) levels [42, 43] . pi4p has a highly negatively-charged headgroup and has been reported to cause membrane curvature at physiologically relevant concentrations [46] , so local production of pi4p at nascent replication organelles might facilitate membrane curvature and dmv formation. another function of pi4p is to recruit specific viral and/or host proteins with pi4p-binding domains [47] . in particular, two pi4p effectors, oxysterol-binding protein (osbp) and four-phosphate adaptor protein 2 (fapp2) are essential for hcv replication [32, 48] , and inhibition of either osbp or fapp2 results in altered mw morphology [48, 49] . interestingly, osbp and fapp2 are both lipid transfer proteins (ltps), which are responsible for non-vesicular sterol and glycosphingolipid trafficking, respectively [50] ; both of these lipids are important components of drms generally and, likely, also of hcv replication organelles specifically. inhibition of osbp leads to reduced trafficking of cholesterol to hcv replication organelles [49] . similarly, the ltp ceramide transfer protein cert has also been reported to be involved in the hcv life cycle [51] . these findings suggest a model in which pi4p recruits ltps such as osbp and fapp2 to hcv replication organelles, which in turn result in the trafficking of cholesterol and glycosphingolipids to hcv replication membranes. dmvs are highly-curved structures; it is likely that proteins and/or lipids with membrane-deforming properties are involved in mw biogenesis. one such protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein 2 (pstpip2), has been identified as a host factor essential for hcv viral replication and mw formation [52] . pstpip2 is a member of the pombe cdc15 homology (pch) family proteins with membrane-deforming properties, likely mediated by their f-bar domain. pstpip2 co-fractionates with detergent-resistant membranes regardless of the presence of hcv, interacts with both ns4b and ns5a, and co-localizes with ns5a on mws by immunoelectron microscopy. mutations in pstpip2 predicted to ablate its membrane-deforming function rendered it less effective in rescuing hcv replication in cells silenced for endogenous pstpip2 relative to expression of wild-type protein. another member of the pch family, bridging integrator 1 (bin1), has been reported to be possibly involved in the hcv life cycle through an interaction with ns5a [53] , though it is not known whether bin1 is essential for viral replication or participates in mw biogenesis. the precise contribution of pch family proteins and other membrane-deforming proteins to mw formation remains to be determined. as a positive-sense rna virus, hcv is not known to require the nucleus for any step in its infection cycle. however, putative nuclear localization signals and nuclear export signals have been reported in hcv proteins (reviewed in [54] ), and some reports have described localization of core protein and ns5a to the cell nucleus during viral infection [55] [56] [57] [58] . furthermore, hcv and other positive-sense rna viruses appear to interact with nucleocytoplasmic transport factors [59] . more specifically, hcv infection has been reported to directly interact with and relocate nuclear transport components, including karyopherins and nucleoporins, to sites enriched for hcv replication and assembly [60] . furthermore, knockdown of a few of these karyopherins and nucleoporins impairs viral replication and/or virion assembly [60] . these findings raises the intriguing hypothesis that relocation of nuclear transport components to the hcv replication organelles might influence membrane curvature and/or transport factors across membranes of dmvs and other replication organelle structures. however, this remains to be functionally demonstrated. autophagy is a cellular response to a variety of stimuli, including nutrient depletion, hormone treatment, and viral or bacterial infection in eukaryotic cells [61] . one of the most distinguishing features of autophagy is the formation of double-membrane vesicles called autophagosomes, which engulf cytoplasmic macromolecules and damaged organelles and deliver them to lysosomes for degradation and recycling. while the cellular origin of the autophagosome membrane is not completely established, the endoplasmic reticulum may be one of its membrane sources [62] . given these similarities between cellular autophagosomes and the dmvs seen in hcv infection, multiple studies have examined the possibility that dmv formation in hcv infection exploits the cellular autophagocytic machinery (reviewed in [63] ). several studies have reported that the expression of hcv replicons e.g., [15, 64] or hcv infection [65] induces the accumulation of autophagosomes in cultured cells. other studies have demonstrated that ectopic expression of hcv ns4b or ns5a is also sufficient to induce autophagic vesicles and upregulate markers of autophagy induction, such as lipidated lc3 [66, 67] , though whether these findings reflect effects of protein overexpression is unclear. in addition, an important question that arises from these experiments is whether autophagy and autophagosome induction are byproducts of hcv infection or whether autophagy itself is necessary for hcv infection and replication. while multiple studies have indicated that autophagy is somehow important for productive hcv infection, there is controversy regarding the precise steps of hcv infection that are facilitated by autophagy. several groups have reported that autophagy plays an important role in hcv rna replication [15, [68] [69] [70] [71] . however, dreux et al., found that autophagy specifically modulates the onset of translation of incoming hcv rna and, therefore, the initial establishment of hcv replication [72] , and this observation was supported by another study [69] . in addition to a possible role for autophagy in establishing hcv replication, tanida et al. observed that the release of hcv core and infectious particles from infected cells is reduced when autophagy is inhibited, and they proposed that in addition to facilitating the initiation of viral replication, autophagy proteins also contribute to hcv particle assembly and/or egress [73] . we still do not understand the molecular mechanisms by which the autophagy machinery supports either of these processes; in particular, why autophagy should be required only for the establishment of hcv replication but not for the maintenance of ongoing replication remains to be elucidated. the biogenesis of membranous web is a complex process involving a concerted effort of hcv nonstructural proteins and a growing list of host protein and lipid factors. twenty-seven years after the molecular cloning of hcv, we all still far from understanding the molecular processes that lead to mw formation in the hcv-infected cell. based on our current state of knowledge, we will discuss candidate general mechanisms that direct mw formation. it is generally believed that hcv induced mws are derived primarily from the host cell er membrane [10, 15, 17, 18] . as mmvs appear later in hcv infection than dmvs, after the peak of hcv rna replication [17] , and as several other rna viruses also induce the formation of dmvs, most investigators have focused on the mechanisms of dmv morphogenesis rather than on mmvs. studies of other viruses that also induce the formation of er-derived dmvs have led to the proposal of several models for the formation of virus-induced double-membrane vesicles, including but not limited to a protrusion and detachment model, a double-budding model, and a model of exvagination, followed by invagination [6, 17, 74] (figure 2 ). these models are not necessarily mutually exclusive and, in theory, could operate simultaneously in infected cells. the first 'protrusion and detachment model' invokes local bending/deformation of part of an er cisterna with tight apposition of the two lipid bilayers, followed by pinching off and sealing to form a double-membrane vesicle. in the 'double budding model', a single-membrane vesicle buds by invagination into the er lumen, from which it is subsequently released by a second budding event into the cytosol to give rise to a dmv. in the last model, exvagination or tubulation of the er membrane is followed by partial invagination to form a cup-like structure that is then sealed to form a dmv. in the case of hcv, 3d reconstruction of electron tomographic images reveals that virus-induced dmvs are exvaginations connected via a short neck-like structure to the er membrane bilayer, and most dmvs are linked to the er only via their outer membrane [17] . reconstruction of electron tomographic images reveals that virus-induced dmvs are exvaginations connected via a short neck-like structure to the er membrane bilayer, and most dmvs are linked to the er only via their outer membrane [17] . at this point in time none of these models has been convincingly demonstrated to be a mechanism of dmv formation. however, kinetic analysis of the ultrastructural membrane alterations following hcv [17, 75] infection have identified single-membrane vesicles early in hcv infection, while similar studies of enterovirus-infected cells have identified single-membrane tubules [76] , which could be precursors of dmvs and thus might argue for models a and/or c presented above. the membranous web appears to be the site of hcv rna genome replication. by immunofluorescence microscopy, newly synthesized viral rna metabolically labeled with 5-bromouridine 5'-triphosphate (brutp) co-localizes with ns5a protein as a marker of the membranous web [11, 14, 18] . furthermore, negative strand hcv rna, which is a necessary intermediate of hcv replication, has been detected at ns5a-positive foci by confocal microscopy [77] . the precise localization of the hcv replicase complex at the different membranous structures that make up the membranous web (e.g., smvs, dmvs, and mmvs) has not yet been unequivocally determined, as localization of nascent hcv rna or the hcv negative strand has yet to be clearly demonstrated at the ultrastructural level e.g., [17, 28] . this may be due to poor accessibility of the interior of membrane structures to rna labeling reagents and/or to incompatibility between em sample preparation techniques that preserve ultrastructural detail and currently available rna labeling methods. we will discuss three lines of evidence that hcv rna replication occurs in association with dmvs. at this point in time none of these models has been convincingly demonstrated to be a mechanism of dmv formation. however, kinetic analysis of the ultrastructural membrane alterations following hcv [17, 75] infection have identified single-membrane vesicles early in hcv infection, while similar studies of enterovirus-infected cells have identified single-membrane tubules [76] , which could be precursors of dmvs and thus might argue for models a and/or c presented above. the membranous web appears to be the site of hcv rna genome replication. by immunofluorescence microscopy, newly synthesized viral rna metabolically labeled with 5-bromouridine 5'-triphosphate (brutp) co-localizes with ns5a protein as a marker of the membranous web [11, 14, 18] . furthermore, negative strand hcv rna, which is a necessary intermediate of hcv replication, has been detected at ns5a-positive foci by confocal microscopy [77] . the precise localization of the hcv replicase complex at the different membranous structures that make up the membranous web (e.g., smvs, dmvs, and mmvs) has not yet been unequivocally determined, as localization of nascent hcv rna or the hcv negative strand has yet to be clearly demonstrated at the ultrastructural level e.g., [17, 28] . this may be due to poor accessibility of the interior of membrane structures to rna labeling reagents and/or to incompatibility between em sample preparation techniques that preserve ultrastructural detail and currently available rna labeling methods. we will discuss three lines of evidence that hcv rna replication occurs in association with dmvs. first, immunoelectron microscopy using an anti-dsrna monoclonal antibody suggests that dsrna labeling is associated with dmvs in hcv-replicating cells [17, 75] and with dmvs immunoisolated from hcv-infected cells [30] . while it is assumed that dsrna-containing replicative intermediates are formed as a result of viral negative-strand rna synthesis and thus indicative of sites of viral rna replication, it is likely that some fraction of dsrna-containing foci is not actively engaged in rna synthesis and instead represents inactive replication complexes or products of replication. second, analysis of the kinetics of hcv rna and dmv accumulation in acutely infected cells has shown a correlation between the two, while the appearance of mmvs lags significantly behind both [17] , suggesting that dmvs might be the principal site of hcv rna replication and that mmvs are not a major site of hcv rna replication. there are two caveats to this interpretation: a role for single-membrane vesicles (smvs) in hcv replication was not specifically evaluated in this study, and correlation between dmv and viral rna accumulation does not exclude the possibility that dmvs serve as storage sites for replication-inactive hcv rna molecules that have been synthesized elsewhere. a similar study of the kinetics of poliovirus-induced membrane alterations found that the appearance of smvs correlated best with the exponential phase of viral rna synthesis, while dmvs appeared only later in infection [78] . third, as already mentioned above, perhaps the most direct evidence that dmvs are sites of hcv rna synthesis is a study of replicon cells expressing epitope-tagged ns4b [28] . dmvs are presented in affinity-purified membranes from these cells, and about half of these dmvs can be labeled by brutp in in vitro replicase assays. however, the immunogold labeling of brutp was observed both on the exterior and in the interior of dmvs, leaving unresolved the question of whether the hcv replicase is located on the interior or on the exterior of the dmv. in favor of the former model is the observation that hcv rna is sensitive to nuclease digestion only in the presence of detergents e.g., [12, 14, 79] , and this is also true of in vitro replicase activity of membranes isolated from hcv replicon cells [29] . on the other hand, replicase localization within a membrane-enclosed compartment raises the question of how ribonucleotides and other molecules necessary for rna synthesis gain access to the replicase complex and how progeny rna genomes exit the membrane structure. in an ultrastructural study using cryoelectron tomography of hcv-infected cells, only about 8% of all dmvs had an identifiable opening connecting the interior to the cytosol [17] . the outer membrane bilayer of 45% of dmvs was contiguous with the er membrane, but the inner bilayer appeared to be closed. a spherical dmv of 125 nm in diameter [17, 49] , assuming a ribonucleoside tri-phosphate (rntp) concentration of 5 mm [80] and no exchange with the cytosol, will contain only about 3000 molecules of each rntp, which is enough to synthesize only about one complete hcv genome. therefore, any model of hcv replicase localization within a dmv or membrane structure of comparable volume must also allow for a mechanism for rntp replenishment. it may be that only the minority of dmvs with an opening to the cytosol are engaged in active genome replication. another possibility is that protein channel(s) in both dmv membrane bilayers mediate the entry of rntps and other necessary factors, though this has not been experimentally demonstrated. an alternative model is that the hcv replicase complex is located on the cytosolic surface of dmvs or another membrane compartment. this would be analogous to the poliovirus replicase complex, which is associated with the cytosolic face of virus-induced vesicles [81] . coronaviruses also generate dmvs during infection [82, 83] ; the nonstructural protein 2 (nsp2) of murine hepatitis coronavirus has been shown to localize to the cytosolic face of dmvs [82] . although this would appear to be inconsistent with the known nuclease resistance of hcv rna and hcv replicase activity, it would resolve the problem of accessibility of rntps and other factors to the replicase complex. membrane association of the hcv replicase complex has also been shown to shield viral rna from innate immune recognition. the hcv rna genome and its replicative intermediates are thought to encode potent pathogen-associated molecular patterns (pamps) recognized by host cell pattern recognition receptors (prrs) such as retinoic acid-inducible gene 1 (rig-i) and melanoma differentiation-associated protein 5 (mda5) (reviewed in [84] ). recently, neufeldt et al. showed that both rig-i and mda5 are excluded from the hcv replication organelles, and that addition of a nuclear localization signal to rig-i or mda5 resulted in their replicase complex localization and stimulation of immune response [85] . these results would appear to support a model of hcv replicase and viral rna localization inside dmvs, and suggest that enclosure of the hcv replicase and hcv rna within membranous structures restricts access of prrs to hcv-encoded pamps and by doing so, protect viral rna from innate immune recognition. despite important recent advances in our understanding of the molecular requirements of the hcv replication process, many important questions remain unresolved. it is still not clear how viral proteins and host factors work in concert to alter the host er membranes to initiate the formation of mws. it is also not known whether the replication of hcv occurs on the exterior or in the interior of dmvs. advances in microscopy techniques, including cryo-electron tomography, focused ion beam scanning electron microscopy, and superresolution microscopy, will probably contribute to future breakthroughs in this field. finally, while this review focuses on the replication of hcv, how genome replication is coordinated with polyprotein translation or virion assembly is not well known. as all of these are general questions shared by all positive-sense rna viruses, answers to these questions will benefit not only the study of hcv but also of positive-sense rna viruses. unravelling hepatitis c virus replication from genome to function hepatitis c virus rna replication hepatitis c virus replication cycle hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus hepatitis c virus p7 protein is crucial for assembly and release of infectious virions modification of intracellular membrane structures for virus replication acute non-a, non-b hepatitis: specific ultrastructural alterations in endoplasmic reticulum of infected hepatocytes non-a, non-b hepatitis: ultrastructural evidence for two agents in experimentally infected chimpanzees isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna hepatitis c virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons hepatitis c virus rna replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2 ultrastructural and biochemical analyses of hepatitis c virus-associated host cell membranes architecture and biogenesis of plus-strand rna virus replication factories three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication insertion of green fluorescent protein into nonstructural protein 5a allows direct visualization of functional hepatitis c virus replication complexes a dynamic view of hepatitis c virus replication complexes dynamic imaging of the hepatitis c virus ns5a protein during a productive infection daclatasvir-like inhibitors of ns5a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication ns5a domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis c virus replication cell-free replication of the hepatitis c virus subgenomic replicon hepatitis c virus rna synthesis in a cell-free system isolated from replicon-containing hepatoma cells in vitro rna replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis c virus hepatitis c virus non-structural proteins in the probable membranous compartment function in viral genome replication lipid rafts and membrane traffic lipid rafts and signal transduction quantitative analysis of the hepatitis c virus replication complex morphological and biochemical characterization of the membranous hepatitis c virus replication compartment rab18 binds to hepatitis c virus ns5a and promotes interaction between sites of viral replication and lipid droplets role of oxysterol binding protein in hepatitis c virus infection affinity capture and identification of host cell factors associated with hepatitis c virus (+) strand subgenomic rna two-step affinity purification of the hepatitis c virus ribonucleoprotein complex amphipathic a-helix ah2 is a major determinant for the oligomerization of hepatitis c virus nonstructural protein 4b ns4b self-interaction through conserved c-terminal elements is required for the establishment of functional hepatitis c virus replication complexes aminoterminal amphipathic a-helix ah1 of hepatitis c virus nonstructural protein 4b possesses a dual role in rna replication and virus production roles for endocytic trafficking and phosphatidylinositol 4-kinase iii alpha in hepatitis c virus replication class iii phosphatidylinositol 4-kinase alpha and beta are novel host factor regulators of hepatitis c virus replication a functional genomic screen identifies cellular cofactors of hepatitis c virus replication identification of a lipid kinase as a host factor involved in hepatitis c virus rna replication recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment hepatitis c virus stimulates the phosphatidylinositol 4-kinase iii alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication the role of the phosphatidylinositol 4-kinase pi4ka in hepatitis c virus-induced host membrane rearrangement metabolism of phosphatidylinositol 4-kinase iii alpha-dependent pi4p is subverted by hcv and is targeted by a 4-anilino quinazoline with antiviral activity lipid membrane curvature induced by distearoyl phosphatidylinositol 4-phosphate membrane-trafficking sorting hubs: cooperation between pi4p and small gtpases at the trans-golgi network modulation of hepatitis c virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2 oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for hcv replication membrane integrity and cholesterol trafficking non-vesicular lipid transport by lipid-transfer proteins and beyond protein kinase d negatively regulates hepatitis c virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein proline-serine-threonine phosphatase interacting protein 2, a host membrane-deforming protein, is critical for membranous web formation in hepatitis c virus replication the sh3 binding motif of hcv ns5a protein interacts with bin1 and is important for apoptosis and infectivity hepatitis c virus and host cell nuclear transport machinery: a clandestine affair molecular determinants for subcellular localization of hepatitis c virus core protein functional characterization of nuclear localization and export signals in hepatitis c virus proteins and their role in the membranous web regulation of hepatitis c virus replication by nuclear translocation of nonstructural 5a protein and transcriptional activation of host genes identification of a functional, crm-1-dependent nuclear export signal in hepatitis c virus core protein nuclear proteins hijacked by mammalian cytoplasmic plus strand rna viruses hepatitis c virus-induced cytoplasmic organelles use the nuclear transport machinery to establish an environment conducive to virus replication autophagy: process and function the puzzling origin of the autophagosomal membrane hepatitis c virus and autophagy induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response hepatitis c virus genotype 1a growth and induction of autophagy rab5 and class iii phosphoinositide 3-kinase vps34 are involved in hepatitis c virus ns4b-induced autophagy hepatitis c virus upregulates beclin1 for induction of autophagy and activates mtor signaling induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection replication of hepatitis c virus rna on autophagosomal membranes activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection the transformation of enterovirus replication structures: a three-dimensional study of single-and double-membrane compartments spatiotemporal analysis of hepatitis c virus infection complex dynamic development of poliovirus membranous replication complexes characterization of the hepatitis c virus rna replication complex associated with lipid rafts visualization and measurement of atp levels in living cells replicating hepatitis c virus genome rna reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral rna synthesis dynamics of coronavirus replication-transcription complexes sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum hepatitis c virus. strategies to evade antiviral responses the hepatitis c virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites the authors declare no conflict of interest. the following abbreviations are used in this manuscript: key: cord-314148-5xisw9au authors: hasony, hassan j.; macnaughton, malcolm r. title: prevalence of human coronavirus antibody in the population of southern iraq date: 2005-12-06 journal: j med virol doi: 10.1002/jmv.1890090308 sha: doc_id: 314148 cord_uid: 5xisw9au sera from adults in southern iraq were collected during winter and screened by an enzyme–linked immunosorbent assay for the presence of antibodies to the two antigenic groups of human coronaviruses, the 229e and the oc43 groups: 91% of the sera had antibodies to at least one of the groups, whereas 4 and 5% of the sera had antibodies to only the 229e or oc43 groups, respectively. there was significant correlation between the levels of antibody to the 229e and oc43 group coronaviruses in these sera. human coronaviruses (hcvs) are an important cause of common colds in man and up to 25% of colds may be caused by these viruses [bradburne and tyrrell, 1971; larson et al, 1980; mcintosh, 1974 ; monto, 19741 . recent reports have suggested that these viruses may be associated with more severe diseases, and in particular gastroenteritis, but as yet there is no clear evidence to support this [macnaughton and davies, 19811 . several reports have analyzed the extent of infections in various populations in america and europe by estimates of hcv antibodies in sera from patients and volunteers [bradburne and somerset, 1972; candeias et al, 1970; cavallaro and monto, 1970 ; hamre and beem, 1972; hendley et al, 1972; hovi et al, 1979 ; kaye et al, 1971; mcintosh et al, 1970; zakstelskaya et al, 19721 . a number of assays have been used, including neutralization, complement fixation, hemagglutination inhibition, and radioimmunoassay. the numbers of subjects with significant hcv antibody in their sera varied considerably depending on the population studied, time of assay, assay system used, and method used to determine significant levels of antibody. in one report most of the adult population was found to have serum antibodies to hcv oc43 as detected by radioimmunoassay [hovi et al, 19791 indicating that infections with theseviruses are very common. two serological groups of human coronaviruses, the hcv 229e and the hcv oc43 groups, have been recognized and show no antigenic cross-reaction with each other [mcintosh et al, 1969; mcintosh, 1974; pedersen et al, 19781 . all hcvs, so far identified, fall into one or other of these groups, suggesting that only two antigenically distinct hcv antigens should prove adequate in detecting all hcv infections [macnaughton et al, 19811. in this study representative viruses from the 229e and oc43 antigenic groups were used in elisa to measure the antibodies to these viruses in sera collected during the winter from patients and volunteers from the basrah area of southern iraq. the distribution of antibodies to these two hcv groups in the sera of the iraqi population and the correlation between the amount of antibody to the two hcv groups in individual sera are described. the importance of these results is discussed in the light of previous work. two prototype hcv strains, 229e [hamre and procknow, 19661 and oc43 [mcintosh et al, 1%7b] , and seven hcvs, ad, gi, ho, pa, pr, to, and ro, isolated from the nasal washings of subjects with natural colds [larson et al, 19801 were used. in addition, a coronavirus of possible human origin, called cv paris, was also used. this virus was isolated from the feces of a neonate with necrotizing enterocolitis [sureau et al, 19801 , although it shows close morphological and structural similarities to a bovine coronavirus [macnaughton and davies, 19811 also studied in that laboratory. the hcv strains, 229e, pr, and to, were readily adapted to growth in monolayer cultures of mrc continuous cells, which were originally obtained from dr. a.f. bradburne. cv paris readily grew in hrt 18 cells, a cell line derived from a human rectal adenocarcinoma. hcv oc43 grew to only low titers in tissue culture [macnaughton et al, 19811 , although readily in suckling mouse brain [mcintosh et al, 1967al . the other five hcv isolates, strains ad, gi, ho, pa, and ro, could only be passaged in human fetal tracheal and nasal organ cultures [larson et al. 19801 . hcv 229e was grown in mrc continuous cells as described previously [macnaughton and madge, 19781 . cv paris was obtained from dr. j. laporte and grown in hrt 18 cells in rpmi-1640 with 10% fetal calf serum containing antibiotics at 37°c and harvested after 30 hr. for both viruses, the cells were subjected to one freeze-thaw cycle, and the resulting suspension was clarified at 2000g for 30 min. control antigens were prepared from uninfected cells by the same method. hcv oc43, grown in suckling mouse brain, was obtained from dr. s.e. reed of the common cold unit, salisbury and used as a 10% suspension of suckling mouse brain. uninfected suckling mouse brain was used as a control antigen. preparations of hcv 229e and cv paris containing between lo7 and 108 particles/ml and hcv oc43 containing between los and lo9 particles/ml, as determined by electron microscopy [macnaughton et al, 19801 , were used. sera were obtained during 1976 from adult volunteers taking part in experiments at the common cold unit, salisbury. the paired sera used in this study were collected from volunteers who developed colds after an inoculation of hcv or from controls given a saline inoculation. postinoculation sera were collected about 3 weeks after virus inoculation. the sera were mixed with equal volumes of bovine calf serum and held at 4°c for 16 hr and stored at -20°c. the experiments were approved by the ethical committee of northwick park hospital, harrow. 258 serum samples were collected during february and march, 1981, from students at the agricultural college, basrah university, iraq, or from patients' samples sent to the central public health laboratory at basrah, the al-jumhury hospital or to the basrah medical college teaching hospital, iraq. sera were absorbed for 16 hr at 4°c with equal volumes of bovine calf serum or tissue culture fluid from uninfected cell monolayers and stored at -20°c before testing by elisa. the elisa method used for detection of antibodies to hcvs in human sera was based on a previously described method [kraaijeveld et al, 1980bl . flat-bottomed polystyrene microtiter plates (dynatech) were coated with duplicate 0.2-ml amounts of antigen diluted in 0.1 m carbonate-bicarbonate buffer (ph 9.6) and incubated overnight at room temperature. after incubation the plates were washed four times with phosphatebuffered saline containing 0.05% tween 20 and 0.02% sodium azide (pbst) and shaken dry. portions of 0.2 ml of sera diluted in pbst were added to the wells and incubated for 4 hr at room temperature. after incubation, the plates were washed four times in pbst and shaken dry. anti-human immunoglobulin g , directed against heavy and light chains, and labeled with alkaline phosphatase conjugate (miles laboratories) at a dilution of 1 :800, was added in 0.2-ml quantities and left overnight at room temperature. after four additional washes with pbst, 0.2 ml of phosphatase substrate, consisting of a 0.1 yo solution of p-nitrophenylphosphate in 10% (wthol) diethanolm i n e buffer (ph 9.8) with 0.02% sodium azide and 0.01% mgc126h,0, was added to each well. absorbance values were read after 30 min at 405 nm in a flow titertek multiscan photometer. hcv strains 229e and oc43 and cv paris, which were representative members of the two hcv antigenic groups, were used. cv paris was tested by elisa against serum pairs from 46 volunteers, given oc43 group virus strains oc38/43, gi, ho, and ro; 229e group virus strains 229e, ad, pa, and to; or saline ( table i) . the highest ratio of postinoculation to preinoculation absorbance values, obtained at serum dilutions of 1:50,1 :loo, and 1 :200, was called the elisa ratio, and ratios of 2 or more were considered to represent significant antibody rises [kraaijeveld et al, 198obl . significant elisa ratios (antibody rises) were detected with cv paris antigen in the serum pairs from volunteers given oc43 group viruses, but not in those given 229e group viruses or saline. this indicates that cv paris is antigenically related by elisa to the oc43 group. "elisa ratios of 2 or more were considered to be positive. cv paris was the only oc43 group virus that grew to high titers in tissue culture: hcv oc43 grew only to low titers in tissue cultures, although to high titers in suckling mouse brain [macnaughton et al, 19811 . tissue culture-grown viruses were more suitable for elisa than suckling mouse brain-grown material, as less nonspecific reactions were observed and more material was available. thus in this study cv paris and hcv 229e, respectively, were used in elisa as representative members of the oc43 and 229e antigenic groups. the 258 iraqi serum samples were tested against hcv 229e and cv paris (fig. 1) . for comparative purposes all sera were tested at the same dilution of 150. controls were absorbance values obtained with pbsa or 1:50 dilutions of tissue culture fluid from uninfected cell monolayers. we considered an individual serum sample to contain significant antibody to hcvs when it had an absorbance value of at least twice the average of the controls, represented by the dotted lines in figure 1 . however, this criterion for deciding that a particular serum sample was positive was very rigorous, and some positive sera may therefore have been missed. there was no marked variation in the number of positive sera or their relative absorbance values on using different serum dilutions or on reading the absorbance values after different times after developing the plates. these absorbance values were related to elisa titers defined as the reciprocal of the highest dilution to produce an absorbance value of twice the control absorbance value at the same dilution. thus, absorbance values in the range of 1-40 to 1.99 corresponded to antibody titers of 2000 to 4000 for both viruses, and lower absorbance values corresponded to proportionately lower antibody titers. figure 1 shows that 86 and 87% of the sera contained antibody to hcv 229e and cv paris, respectively, with a wide range in the amount of antibody to both viruses in positive sera. 50 of the sera, taken at random, were tested in elisa against the prototype hcv oc43 at the same dilution of 150 as for cv pans, and the same number of sera were positive with each virus. figure 2 showed a strong correlation between the distribution of antibody to hcv 229e and cv paris in the sera (spearman's rank correlation coefficient of 0.73, p c 0.001). all points below and to the left of the lines in the figure represent sera with no hcv antibody, the serum samples showing significant antibody levels to either hcv group formed 91% of the total. some sera in figure 2 had significant antibody levels to hcv 229e (4%) and none to cv paris, whereas other samples showing significant antibody levels to cv (paris 5%) were negative for hcv 229e. 67 random sera were collected from healthy adult volunteers at the common cold unit, salisbury, before inoculation with any viruses, and tested by elisa for antibodies to hcv 229e and cv paris. similar antibody levels as those from the iraqi sera were observed, and 100 and 94% of these volunteers had serum antibodies to hcv oc43 and 229e group viruses, respectively. this is the first report to describe the prevalence of hcv antibodies in a middle eastern population. our results show that clinical or subclinical hcv infections in an adult population from the basrah region of southern iraq are widespread with a high proportion, 91% of this population, having serum antibodies to hcv 229e and/or oc43 group viruses. however, it is not clear how frequently individuals are infected with hcvs, as we do not know how long hcv serum antibodies remain after infection and how much antibody is produced by natural hcv infections. serum samples were taken during the winter when hcv infections have been reported to occur most frequently [mcintosh et al, 1970; cavallaro and monto, 1970; mcintosh, 1974; monto, 19741 . a similar prevalence of hcv antibodies was detected in sera of volunteers at the common cold unit, salisbury, england. however, in this study a smaller number of serum samples was examined and they were taken randomly throughout the year. these results imply that hcv infections occur frequently in both populations. we cannot say at present that hcv infections are less common in iraq than in england, as considerable variation has been reported in hcv antibody prevalence in sera depending upon when samples are taken [bradburne and tyrrell, 1971; monto, 19741. most previous studies have revealed no antigenic relationship between the hcv 229e and hcv oc43 group viruses [mcintosh et al, 1969; monto, 1974; macnaughton et al, 19811 , although generally only reactions involving external antigens were examined in these studies. however, one group has described an antigenic relationship between these virus groups [bradburne and tyrrell, 1971; bradburne and somerset, 19721 , which may reflect the type of sera used and the presence of contaminating serum and other components [kraaijeveld et al, 1980al . in our study we observed a strong correlation between the amount of antibody to the two hcv groups in individual iraqi sera. this may be due to the detection of antibody directed against antigenically related internal antigens: such antigens are present in significant amounts in human sera (macnaughton, unpublished results). however, further studies are required into both the antigenic relatedness of hcv antigens and into the nature, duration, and role of hcv serum antibodies produced during hcv infection, in order to resolve this problem. previous studies have analyzed the prevalence of hcv antibodies in the sera of various populations in north america [cavallaro and monto, 1970; hamre and beem, 1972; hendley et al, 1972; kaye et al, 1971; mcintosh et al, 19701, south america [candeias et al, 19721, and europe [bradburne and somerset, 1972; hovi et al, 1979; zakstelskaya et al, 19721 and shown that hcv antibodies are widespead in these populations. several of these reports describe variations in the incidence of antibodies to hcvs depending on a number of factors including the population studied and the season the samples were taken. most previous studies have reported considerably lower prevalence of hcv antibodies, than our study. this probably does not reflect significant differences in antibody prevalence in these populations, but rather the sensitivity of the assays used; our elisa is more sensitive for detecting hcv antibody than many other assays [kraaijeveld et al, 1980bl and as such should prove to be a useful technique for hcv antibody prevalence studies. future studies are required to determine the variation in the levels of hcv antibodies in sera from iraq from season to season, to identify epidemics, and to relate the severity of infections with those from other parts of the world. coronavirus antibody titres in sera of healthy adults and experimen coronaviruses of man seroepidemiologic study of coronavirus infection in brazilian children and civilian adults community-wide outbreak of infection with a 229e-like coronavirus in virologic studies of acute respiratory disease in young adults a new virus isolated from the human respiratory tract coronavirus infections in working adults. american re oc43 strain-related coronavirus antibodies in different seroepidemiologic survey of coronavirus (strain oc43) related enzyme-linked immunosorbent assay for coro enzyme-linked immunosorbent assay for detection tally infected volunteers 229e infections during six years of surveillance of antibody in volunteers experimentally infected with human coronavirus 229e group viruses isolation of rhinoviruses and coronaviruses from 38 colds in adults human enteric coronaviruses the genome of human coronavirus strain 229e infectivity of human coronavirus strain coronaviruses: a comparative review growth in suckling mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease recovery in tracheal organ cultures of novel viruses from patients with respiratory disease antigenic relationships among the coronaviruses of man and between human and animal coronaviruses seroepidemiologic studies of coronavirus infection in adults and children antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species une epidtmie d'enttrocolites ulcirontcrosantes en maternitt occurrence of antibody to coro-286-293. navimses in sera of people living in the ussr we thank staff at the agriculture college, basrah university, the central public health laboratory, basrah, and the department of microbiology, basrah medical college teaching hospital for assistance during this study. in particular we are grateful to dr. a. jabbar for help in the collection of samples. we also thank dr. s.e. reed for supplying us with sera from adult volunteers taking part in experiments at the common cold unit, salisbury. key: cord-305393-96mrxt8a authors: lai, yvonne; yi, guanghui; chen, alice; bhardwaj, kanchan; tragesser, brady j.; rodrigo a. valverde,; zlotnick, adam; mukhopadhyay, suchetana; ranjith-kumar, c. t.; kao, c. cheng title: viral double-strand rna-binding proteins can enhance innate immune signaling by toll-like receptor 3 date: 2011-10-10 journal: plos one doi: 10.1371/journal.pone.0025837 sha: doc_id: 305393 cord_uid: 96mrxt8a toll-like receptor 3 (tlr3) detects double-stranded (ds) rnas to activate innate immune responses. while poly(i:c) is an excellent agonist for tlr3 in several cell lines and in human peripheral blood mononuclear cells, viral dsrnas tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow tlr3 to respond to viral dsrnas. tlr3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. recombinant 1b hepatitis c virus polymerase was found to enhance tlr3 signaling in the lung epithelial beas-2b cells when added to the media along with either poly(i:c) or viral dsrnas. the polymerase from the genotype 2a jfh-1 hcv was a poor enhancer of tlr3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed rna. the 1b polymerase also co-localizes with tlr3 in endosomes. rna-binding capsid proteins (cps) from two positive-strand rna viruses and the hepadenavirus hepatitis b virus (hbv) were also potent enhancers of tlr3 signaling by poly(i:c) or viral dsrnas. a truncated version of the hbv cp that lacked an arginine-rich rna-binding domain was unable to enhance tlr3 signaling. these results demonstrate that several viral rna-binding proteins can enhance the dsrna-dependent innate immune response initiated by tlr3. viral nucleic acids are agonists for innate receptors that can activate anti-viral responses [1, 2, 3, 4] . the receptors include the rig-i-like receptors that are localized to the cell cytoplasm and several membrane-associated toll-like receptors [5, 6] . mutations in these receptors have been correlated with pathologies in viral infections in humans [7, 8, 9, 10] . understanding how the innate immune receptors can interact with agonists will be important for the detection and response to viral infections. toll-like receptor 3 (tlr3), the focus of this study, is composed of a large ligand-binding ectodomain, a transmembrane helix, and a signaling toll/il-1 receptor homology (tir) domain [11, 12] . the ectodomain contains 23 leucine-rich repeats flanked with cysteine-rich caps and a bipartite motif, both of which are critical for ligand binding [13, 14] . tlr3 localizes to either the plasma membrane or acidic endosomes, the latter was presumed to be the site of high affinity binding to dsrna [15, 16] . ligand binding induces the tlr3 dimer to orient the two tir domains to recruit adaptor proteins and activate signal transduction [6, 14] . karioko et al. [17] have observed the activation of tlr3 signaling in cultured cells with complex ligand mixtures, such as nucleic acids fractions from necrotic cells. however, we have anecdotally observed that highly purified rnas, including necrotic rnas, are poor activators of tlr3 in cultured cells when compared to the synthetic dsrna analog, poly(i:c). these observations suggest that intrinsic features within dsrnas can influence signaling by tlr3. also, component(s) in addition to the dsrnas are needed to activate tlr3. the antimicrobial peptide ll37 and other peptides that can bind dsrna were recently found to enhance the tlr3 response to poly(i:c) and viral dsrnas [18] . since viral rnas exist as complexes with proteins, we wanted to determine whether viral proteins could act to modulate tlr3 signaling. in this work, we determined that viral dsrna-binding proteins could substitute for ll37 in activating signaling by tlr3. furthermore, the proteins promoted more efficient recognition of viral dsrnas. the recombinant polymerases from the hepatitis c virus (hcv) and three viral capsid proteins were characterized in this study. hcv, a member of the hepacivirus genus in the flaviviridae family, infects approximately 2% of the world's population, with up to 80% of untreated individuals progressing to chronic infection and severe liver damage [19, 20] . the hcv-encoded polymerase, ns5b, is a validated drug target and has been extensively characterized [21] . as with polymerases in general, ns5b resembles a closed right hand that contains thumb, palm and finger subdomains [19] . in vitro, recombinant ns5b proteins can bind and initiate rna synthesis by a de novo initiated mechanism or by extension from a template annealed to a primer [22, 23, 24, 25, 26, 27, 28] . de novo initiated rna synthesis uses a single-stranded (ss) rna template and a purine nucleotide as the first nucleotide, while primer extension uses a partially duplexed rna [28] . the de novo initiated versus primer-extended modes of rna synthesis is regulated by a flexible loop named d1 that extends from the fingers subdomain to contact hydrophobic residues in the thumb subdomain [27] . the release of the d1 loop is needed to sterically accommodate the ternary complex during elongative synthesis. the polymerase from the 2a genotype of hcv exists in a structurally more closed conformation than that of the 1b polymerase in part due to a tighter contact between the d1 loop and the thumb subdomain [27, 29, 30] . this feature has been proposed to allow the 2a polymerase to be more efficient at rna synthesis by the de novo initiated mechanism since it cannot initially accommodate a partially duplexed rna. two of the viral capsid proteins we examined are from members of the alphavirus-like superfamily with positive-stranded rna genomes: ross river virus (rrv) and brome mosaic virus (bmv) [31, 32] . the third is from the orthohepadnavirus, hepatitis b virus (hbv), which has a partially dsdna genome and replicates by reverse transcription [33, 34] . the capsids of all three viruses contain a positively-charged arginine-rich region that is required for rna binding [35, 36, 37, 38, 39] . we have anecdotally observed that cultured cell lines that express tlr3 respond poorly to viral dsrnas when compared to the response to poly(i:c). to document this observation, beas-2b cells that endogenously express tlr3, tlr4, and rig-i were examined for their response to several dsrnas. beas-2b cells have been used extensively to study the effects of viral infection and activation of innate immunity on cytokine production, including the production of interleukin 6 (il6) [40, 41, 42] . the dsrnas tested include the reovirus genomic rna, the s4 dsrna made by annealing in vitro transcribed positive-and negative-sense strands of the reovirus s4 rna, and the bell pepper endornavirus dsrna, bpev, purified from bell pepper plants [43] , and other viral and synthetic dsrnas. transcripts of the jfh-1 genome were used as a ssrna control. all the rnas were added to the media of beas-2b cells and the amount of cytokine il6 secreted into the medium quantified by elisa (fig. 1a) . at 20 h, il6 levels induced by poly(i:c) (0.13 mg/ml) was at least six-fold higher than that of dsrnas or ssrna (0.3 mg/ ml; fig. 1a and fig. s1a ). the production of il6 in response to poly(i:c) was rapid and could be observed within 2 h of poly(i:c) addition (fig. s1b ). in contrast, cells treated with s4 dsrna did not yield level of il6 significantly above the background even after 20 h (fig. s1b) . hek 293t cells do not detectably express tlr3 but can be transfected to express either wild-type or mutant tlr3 [44] . transfection of two plasmids, one containing an interferon stimulated response element (isre) promoter-driven firefly luciferase and a second encoding a constitutively expressed renilla luciferase allow the analysis of tlr3 activation by different rnas. hek 293t cells expressing wt tlr3 responded to poly(i:c) (1-2 mg/ml), better than viral dsrnas (1-2 mg/ml) purified from reovirus and bpev (fig. 1b) . annealed s4 reovirus dsrna, ssrnas from jfh-1 (fig. 1b) , endornavirus dsrna from rice plants and other viral ssrnas (fig. s1c ) also had no effect on tlr3 signaling. none of rna tested induced reporter activity in hek 293t cells expressing a tlr3 mutant h539e that had a substitution at the c-terminal ligand-binding domain [16, 45] ( fig. 1b) or in cells transfected with the empty vector (data not shown), demonstrating that the observed responses are from tlr3. the results from 293t cells are consistent with those from the beas-2b cells and support our observation that viral dsrnas are poor agonists in comparison to poly(i:c). to determine whether tlr3 was responsible for the increased il6 levels, we treated cells with sirnas specific to tlr3, rig-i, or a nonspecific control for 48 h prior to the addition of bpev dsrna to the cells (fig. 1c) . rig-i is a suitable control in this experiment since it also recognizes dsrna [46] . only cells treated with sirnas to tlr3 had reduced il6 levels in the presence of bpev dsrna (fig. 1c) . with poly(i:c)-induced cells, sirnas to tlr3 also reduced il6 production by 5163% (n = 4) (data not shown; [18] ). rt-pcr confirmed that the sirnas did knock down tlr3 mrna levels in both untreated and poly(i:c) treated cells (fig. 1d) . odn2006, which we previously shown to inhibit tlr3 signaling [47] reduced the il6 levels in the presence of poly(i:c) to less than 20% of the control reaction (data not shown) while egcg, an inhibitor of rig-i [48] had no effect on il-6 production in the presence of poly(i:c) (data not shown). thus, while the viral dsrnas are poor tlr3 agonists, the low level of cytokine production by beas-2b cells was primarily due to the activation of tlr3. these results form the basis of the hypothesis to be tested in this work: the activation of tlr3 by viral dsrna requires additional factors. several viral dsrna-binding proteins can enhance poly(i:c)-dependent signaling addition of the peptide ll37 to the cell culture media along with poly(i:c) or viral dsrnas was found to enhance signaling by tlr3 [18] . ll37 binding to the dsrna was correlated with enhancement of tlr3 signaling [18] . since rnas in cells exist in complex with proteins, we postulate that viral rna-binding proteins could also enhance the tlr3 response to the viral dsrnas. several highly purified rna-binding proteins were selected to test this idea. these include the recombinant nsp15 protein from the sars coronavirus that binds ssrnas [49] , two versions of the hepatitis b virus capsid protein (h-cp149 and h-cp183) [38] , the capsid protein from the ross river virus (r-cp) [39] , the capsid from the plant-infecting brome mosaic virus (b-cp) [50] and polymerases from the 1b and 2a genotypes of the hepatitis c virus (1bd21 and 2ad21) [27] . except for the b-cp, which was extracted from cesium-chloride purified bmv virions, the other proteins were produced in e. coli. while the cytokine induction by the various rna-binding proteins in the absence of poly(i:c) varied depending on individual experiments, fold inductions of il6 above basal across multiple experiments showed no statistically significance in paired t-tests ( fig. 2 and table s1 ). these results, including those from b-cp that was produced in plants also suggest that common contaminants such as lipopolysaccharides which would induce tlr4 signaling, is not a significant factor in the assays. incubation of poly(i:c) with the corresponding buffers in which the proteins were purified also did not result in a significant change of cytokine production (data not shown). the presence of poly(i:c) (0.13 mg/ ml) alone induced il6 and il8 levels at least five-fold above background (fig. 2) . several of the proteins added to a final concentration of 0.15 mm along with poly(i:c) resulted in additional increases in il6 and il8 levels of two-to five-fold ( fig. 2a and 2b ; table s1 ). the level of enhancement by the recombinant proteins were comparable to that observed with ll37 (3 mm). the viral proteins that did not significantly increase cytokine production include the polymerase from hcv genotype 2a figure 1 . a comparison of viral dsrnas and poly(i:c) for activation of tlr3. a) effects of various potential tlr3 ligands on il6 production by beas-2b cells. all of the rnas were added to the culture media. poly(i:c) (pic) was added to 0.13 mg/ml and the other rnas were added at 0.13 to 0.3 mg/ml. il6 was quantified in an aliquot of clarified cell medium, and the total amount of il6 was normalized to the total medium volume. the data shown is the mean 6 sem, with the number of assays in parentheses: pic (n = 23), reovirus dsrna (reov, n = 9), reovirus s4 dsrna (s4; n = 28), bpev (n = 6), jfh1 (n = 3). b) a comparison of the effects of viral dsrnas and poly(i:c) in hek 293t cells transfected to express the wt tlr3 or a ligandbinding mutant, h539e. activation of tlr3 was measured by the ratio of the firefly luciferase driven by an isre promoter element to a constitutivelyexpressed renilla luciferase [47] . each bar represents the mean value of three replicates, with the range for one standard error shown above the bars. c) tlr3 is required for the increase in il6 production in response to bpev dsrna. beas2b cells were transfected with a control nonspecific sirna (nsrna), a set of three sirnas to tlr3 (sitlr3), or a sirna to rig-i (sirig-i), grown for 48 h before addition of bpev dsrna (0.3 mg/ml) to the culture media. d) rt-pcr results demonstrating that the sirna targeting tlr3 decreased the abundance of the tlr3 message. beas2b cells were transfected with either control nonspecific sirna or sirnas to tlr3. after 48 h, cells were exposed to media alone (f) or to poly(i:c) at 0.13 mg/ml. rt-pcr was performed to determine fold induction of tlr3 mrna above media 18 h later. data is presented as a percent of the result from the nonspecific sirna. doi:10.1371/journal.pone.0025837.g001 (2ad21), the truncated hbv cp (h-cp149), and the sars-cov nsp15 ( fig. 2a and 2b ). the nsp15 used in this experiment is a catalytic-inactive version of an endoribonuclease that binds and preferentially cleaves ssrnas [49] . identical results were obtained with the wt nsp15, hence the enzymatic activity of nsp15 is not a factor in the response (data not shown). these results show that a subset of the rna-binding proteins is more competent in enhancing tlr3 signaling. the activities of the hcv polymerases and the viral capsids will be analyzed in turn below. the hcv 1b polymerase can enhance dsrna-induced signaling by tlr3 we seek to determine whether the observed il6 production in the presence of the 1b hcv polymerase was due to signaling by tlr3. sirnas to tlr3, to rig-i, or to a nonspecific target were transfected into beas-2b cells prior to the addition of either poly(i:c) alone or poly(i:c) and 1bd21 (fig. 3a) . only the cells transfected with the sirnas to tlr3 had reduced il6 production compared to cells transfected with nsrna ( fig. 3a ; p,0.05). we reproducibly observed using rt-pcr that tlr3 mrna treated with the sirnas were reduced to between 8 and 26% of the level in cells treated with the nsrna control (table s2 ). the presence of the polymerase did not change lps-induced tlr4 response or change the response from cells treated with only polyinosinic acid, suggesting specific induction of tlr3 (fig. 3b) . moreover, odn2006, an inhibitor of tlr3 signaling [47] reduced il6 levels to 4067% of control while egcg, an inhibitor of rig-i [48] had no effect, further suggesting that the enhancement of dsrna induced signaling is dependent on tlr3 (data not shown). the hcv polymerase also enhanced the response to poly(i:c) lengths that ranged from 45 to 125 bp (fig. s2a ). these results indicate that tlr3 is required for 1bd21 and poly(i:c) to enhance cytokine production. 1bd21 is less active for rna synthesis in vitro than is the 2ad21 [27, 29, 30] . therefore, it is unlikely that rna synthesis by 1bd21 is responsible for the induction of tlr3 signaling. however, to establish this directly, we tested a catalytic mutant named gda wherein the divalent metal-binding motif required for polymerization was mutated. we also tested three additional mutants that are defective for de novo initiation, including a five-residue deletion of the d1 loop name m26-30 [26] . the locations of the key mutations in the 1b polymerase structure are shown in fig. 3c . since m26-30 has a major change in conformational to 1bd21, we tested it for the ability to bind a dsrna of ca. 900 bp derived from reovirus genome and found that retained the ability to bind dsrna (fig. 3d ). all of these mutant polymerases enhanced il6 production over the level of poly(i:c) alone by at least two-fold ( fig. 3e and fig. s2b ). figure 3 . the 1b hcv polymerase can enhance il6 production by beas-2b cells. a) enhanced il6 production in the presence of 1bd21 can be knocked down by sirna to tlr3. sirnas specific to tlr3 (sitlr3), rig-i (sirig-i), or a nonspecific control (nsrna) were transfected into at a concentration of 30 nm into cells 48 h prior to the addition of 1bd21 (0.15 mm) and poly(i:c) (0.13 mg/ml) to the culture medium. il6 in the medium was collected 24 h later and quantified by elisa. the data from 3 to 4 sets of samples are presented as a percentage of the il6 in the sample treated with nsrna (100%). b) il6 production by beas-2b cells in response to stimulation by the single-stranded polyinosinic acid (0.13 mg/ml), poly(i:c) (0.13 mg/ml) or lipolysaccharide (lps) (1 mg/ml), the agonist for tlr4 in the absence (ã�) or presence of 1bd21 (0.15 mm). c) a model of the 1b hcv polymerase that illustrates the location of the d1 loop (in light blue), the gdd active site (in yellow), and the short helix deleted in mutant m26-30 (in red). d) the 1bd21 polymerase and 1b.m26-30 can form a complex with the double-stranded s4 rna. the s4 dsrna was labeled with a-32 p-ctp and used in an electrophoretic mobility shift assay. the gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. e) effects of mutations in 1bd21 on il6 production induced by poly(i:c). f denotes a reaction with no added proteins. proteins 1bd21, 1b.m26-30, and the active site mutant gda were added to the culture media (final concentrations 0.01, 0.1 or 0.5 mm) in the absence or presence of 0.13 mg/ml poly(i:c). ll37 was added to a final concentration of 3 mm. f) the 1b hcv polymerase (1bd21, 0.1 mm), but not the 2a polymerase (2ad21, 0.1 mm), allowed tlr3 to produce cytokines in response to viral dsrnas. in addition to poly(i:c), the rna ligands used were those extracted from reovirus virions (reov), from the endornavirus bpev, and the annealed transcripts of the sense and antisense strands made from the s4 cdna of reovirus (s4), and the transcript of the hcv 2a jfh-1. doi:10.1371/journal.pone.0025837.g003 next, we examined whether 1bd21 could enhance il6 production in concert with viral rnas (fig. 3f ). in the absence of 1bd21, the single-stranded jfh-1 rna and three different viral dsrnas were poor inducers of il6 production in beas2b cells when compared to poly(i:c). this result is consistent with our initial observation that tlr3 does not respond efficiently to viral rnas ( fig. 1a & 3f, fig. s1a ). 1bd21 (0.1 mm) increased il6 levels induced by the s4 dsrna and the bpev dsrna by 4 to 6fold (fig. 3f) . 1bd21 had only a modest increase when jfh-1 ssrna was used as a ligand. in contrast, the activation of signaling was approximately 20-fold higher with dsrna than with jfh-1 (fig. 3f) . consistent with our previous observation, 2ad21 (0.1 mm) was unable to enhance il6 production when added to cells along with either the ssrna or dsrna (fig. 3f ). the conformation of 2ad21 can affect dsrna-dependent tlr3 signaling 2ad21 differs from 1bd21 in the relative amounts of product generated from de novo initiated rna synthesis or extension from a primed template (fig. 4a ). 2ad21 produced high levels of de novo initiated rna when compared to the primer extension products while 1bd21 produced similar amounts of the two products (fig. 4a) . in this assay, de novo initiated rna synthesis was determined with template le19p, whose 39 terminal puromycin prevents primer extension, while primer extension used template pe46, which contains a partially double-stranded hairpin structure that can be extended by the polymerase. the products of the two modes of rna synthesis demonstrate whether a single or a partially duplexed rnas are bound in the template channel of the polymerase [24] [25] [26] . furthermore, products from the two templates are associated with two distinct polymerase conformations: a structurally closed conformation can initiate better by a de novo mechanism or a more open conformation that favors primer extension [26] . notably, the crystal structure of 2ad21 has a more closed conformation than that of 1bd21 due to additional interactions between the d1 loop and the thumb subdomain of the polymerase [29] . if the closed conformation of 2ad21 decreases its ability to bind dsrna and to induce tlr3, a five-residue deletion at the tip of the d1 loop which results in a more open conformation should the templates used, le19p and pe46, can, respectively, produce de novo initiated products (dn) as well as those extended from a partial duplex rna (labeled as ''pe'') as described in chinnaswamy et al. [27] . b) the denaturation profile of the proteins (5 mm) analyzed by differential scanning fluorimetry. the reactions were performed in a stratagene mx3005p realtime pcr machine in the presence of sypro orange, which fluoresces when complexed to denatured proteins. the ramp for temperature increase was at 0.5uc per min. c) the mutant hcv 2a polymerase, 2am26-30, can enhance poly(i:c) (0.13 mg/ml)-induced il6 production by beas-2b cells. d) the sequence and likely structure of a fluorescently-labeled rna, sl26, used to analyze polymerase-dsrna interaction. e) binding of sl26 by the three hcv polymerases. the proteins were mixed together with a 1:1 molar ratio of sl26 then crosslinked with a stratalinker prior to analysis by sds-page. the gel image on the left shows the fluorescently-labeled rna shifted above the free sl26 at the bottom of the gel. the complexes that contain a monomer or a dimer of the hcv polymerase are labeled to the left of the gel image. the same gel was then stained with coomassie blue to allow visualization of the proteins, the image of which is shown to the right. doi:10.1371/journal.pone.0025837.g004 increase dsrna binding and thus the rna synthesis from a primed template. a protein with such a deletion named 2a.m26-30 was found to be debilitated in de novo initiated rna synthesis while retaining the ability to extend from a primed template (fig. 4a ). 2a.m26-31 also showed a distinct thermal denaturation profile compared to that of 2ad21, when analyzed by differential scanning fluorimetry, consistent with the two proteins having different conformation(s) (fig. 4b) . protein 2a.m26-30 added to beas-2b cells had no effect on il6 production (fig. 4c) . when added along with poly(i:c) to beas-2b cells, 2a.m26-30 dramatically enhanced il6 production to levels comparable to reactions containing 1bd21 (fig. 4c, n = 4) . these results clearly demonstrate that the conformations of the hcv polymerase that impact interaction with single or double-stranded rnas affect the ability of the polymerase to enhance signaling by tlr3. the deletion of the d1 loop in the 1b polymerase makes the template channel more accessible to dsrnas [27] . to determine whether 2a.m26-30 was altered in rna binding in comparison to 2ad21, we performed a uv-crosslinking assay with a fluorescently-labeled rna sl26, which contains a gnra tetraloop with an 11-bp stem (fig. 4d ). the 26-nt sl26 was used to minimize non-specific interaction by the polymerase so as to better analyze rna binding potential of polymerase mutant. the rna-protein complex was visualized with a phosphorimager and the position of the protein identified by staining with coomassie blue (fig. 4e) . the purified hcv polymerase usually exists as a mixture of monomers and higher order oligomers [27] . both the monomeric and dimeric forms of 2a.m26-30 bound sl26 better than those from 2ad21 (fig. 4e) . taken together, these results show that the changes in 2a polymerase to increase its binding to a largely dsrna is correlates with increased enhancement of tlr3 signaling. we sought to determine whether the recombinant hcv polymerase could enter cells and co-localize with tlr3 in endosomes. 1bd21 added to the medium of beas-2b cells in the absence of poly(i:c) was localized to punctate spots within 30 min. of addition (fig. 5a) . some of these spots co-localized with lysotracker that stains acidic compartments in the cell where tlr3 is located ( fig. 5b ; [15] ). the observation that not all of the dsrna co-localized with tlr3 is consistent with previous reports that dsrna could traffic into cells independent of tlr3 [47, 51] . to determine whether 1bd21 co-localized with tlr3, cells exposed to 1bd21 for 6 h were stained with the antibodies to tlr3 and the hcv polymerase. signals to both proteins overlapped significantly, although not at every location where tlr3 was detected (fig. 5c) . the co-localization of the hcv polymerase to endosomes containing tlr3 may factor into the mechanism to enhance tlr3 signaling. the localization of 1bd21 in beas-2b cells suggests that it is taken up by endocytosis. arginine-rich cell penetrating peptides and vaccinia virus had been reported to enter cells by several pathways, including macropinocytosis [52, 53, 54] . to examine whether macropinocytosis is likely involved in the uptake of 1bd21, we tested the inhibitor 5(n-ethyl-n-isopropyl)-amiloride (eipa) on tlr3 signaling in the presence of poly(i:c) and either ll37 or recombinant 1bd21 (fig. s3) . eipa dose-dependently inhibited tlr3 signaling by either poly(i:c) alone, or poly(i:c) added to the cell culture medium along with ll37 or 1bd21. these results are consistent with macropinocytosis playing a role in the uptake of either the dsrna and/or the recombinant protein into endosomes. to extend the analysis of viral protein-rna interaction further, we characterized the effects of three capsid proteins (cps) that enhanced the dsrna-specific innate immune signaling (fig. 2) . knockdowns of tlr3, rig-i, or a nonspecific target prior to stimulation with poly(i:c) and the cps (50 nm) showed that tlr3 was the receptor primarily responsible for il6 production for h-cp184 and r-cp (fig. 6a) . sirnas to tlr3 also decreased b-cp induced il6 production (data not shown). in addition, all three wt viral cps increased il6 production in a concentrationdependent manner (fig. s4) . h-cp149 that lacks the c-terminal sequence of h-cp183 (fig. s4a ) was unable to modulate il6 production (fig. s4b) . we examined whether the cp can enhance signaling by viral ssrna or dsrna. il6 level were enhanced when the reovirus genomic rnas and the s4 dsrna (all at 0.15 mg/ml) were added to the medium in the presence of h-cp183. the single-stranded jfh-1 rna was not significantly enhanced by h-cp183. furthermore, h-cp149 did not result in statistically significant enhancement of il6 in the presence of viral dsrnas or the jfh1 ssrna (fig. 6b, fig. s4b ). the amount of enhancement with the 150 nm of h-cp183 was comparable to that of 3 mm ll37. the rna-binding is required to enhance dsrna-dependent signaling by tlr3 the differential effects of h-cp183 and h-cp149 should reveal a requirement important for the enhancement of dsrna-dependent signaling by tlr3. the hbv cp contains sixteen arginines in its c-terminal thirty-four residues (fig. s4a ). h-cp149 is competent for the formation of virus-like particles, but is defective for binding to rna [38] . it is therefore likely that the altered interaction with rna is responsible for the inability of h-cp149 to enhance tlr3 signaling when compared to the other capsid proteins. again, to reduce the effects of nonspecific rna binding inherent to many rna-binding proteins, sl26 containing an 11-bp dsrna was used to examine whether h-cp183 and h-cp149 are distinguishable in their ability to bind rna. sl26 bound h-cp183 as well as b-cp, and the r-cp (indicated by shifts to higher molecular mass smears). however, no shift occurred with h-cp149 (fig. 6c) . these results provide another set of example wherein a viral protein's ability to bind dsrna is linked to the enhancement of tlr3-dependent signaling. the hcv polymerase 1bd21 co-localized with tlr3 in endosomes (fig. 5) . the bmv cp has also been shown to enter barley cells through macropinocytosis and localize to punctate spots within the cytoplasm that are suggestive of endosomes [53] . the hbv capsid can also enter cells, although permeabilization of the cells with digitonin was required [55] . we seek to determine whether the capsid proteins can localize to endosomes that contain tlr3, using the r-cp as an example. 2 h after the addition of rcp to the media of beas-2b cells, r-cp was found close to the plasma membrane, with a small proportion of the signal localizing in endosomes (fig. s5 ). at 6 h after r-cp addition, the majority of the signal was within endosomes (fig. 7) . immunostaining to detect tlr3 revealed an extensive co-localization of r-cp (fig. 7) . the effects of the hcv polymerases and the viral capsid proteins have thus far been examined in beas-2b cells. we seek to determine whether they could enhance signaling in hek 293t cells transiently transfected to express tlr3. tlr3specific activation of luciferase production from an isre promoter element was used to assess signal transduction. reporter production in the presence of poly(i:c), and reovirus genomic dsrna were all reproducibly enhanced in the presence of 1bd21 or 2a.m26-30, but not with 2ad21 (fig. 8a) . as with the beas-2b cells, jfh1 rna was unable to induce tlr3 in hek 293t cells. the activation of reporter production was dependent on the concentration of 1bd21 added to the 293t cell culture media (fig. 8b) . the effects of the viral cps on tlr3 signaling in hek cells were also examined using the reovirus dsrna as the tlr3 ligand. b-cp, h-cp183, and the r-cp all increased reporter levels in a concentration-dependent manner when compared to cells transfected with only the empty plasmid vector (fig. 8c ). this result shows that all five recombinant proteins that enhanced tlr3 signaling with viral dsrnas in beas-2b cells enhanced tlr3-dependent signaling in hek 293t cells. while tlr3 binds to dsrna to activate innate immune responses, viral dsrnas tend to be poor tlr3 agonists, especially when compared to the synthetic dsrna analog, poly(i:c). in a companion manuscript [18] , we demonstrated that ll37 is a potent enhancer of dsrna-dependent tlr3 signaling. herein, we demonstrate that a subset of viral proteins can significantly enhance both poly(i:c) and viral dsrna-induced tlr3 signaling. these viral proteins include polymerases from two genotypes of hepatitis c virus and three viral capsid proteins. requirements for the polymerases and capsid proteins (1bd21, 2a.m26-30, b-cp, rcp, and h-cp183) to activate tlr3 signaling were examined and a common property is the ability to bind dsrnas. two proteins (2ad21 and h-cp149) that bind dsrna less well are also less effective in enhancing tlr3 signaling. these results suggest that features that affect dsrna binding modulate the ability of proteins to activate tlr3 signaling. a second requirement elucidated with the 1b hcv polymerase and the rrv cp is the ability of the proteins to enter cells and co-localize with tlr3 in endosomes. the focus of the discussion below will be on the requirements for activating tlr3 signaling, with a final section on the conformation of the hcv polymerase. the chemical features in dsrna that are recognized by tlr3 are poorly understood. the difference between what makes poly(i:c) a good tlr3 agonist in the absence of rna-binding proteins and what makes the viral dsrnas inferior ones will require in-depth analysis. we believe that this work identifies conditions that will facilitate these efforts. based on our current results, reovirus s4 dsrna made by in vitro transcription is as potent a tlr3 agonist as the genomic dsrna extracted from reovirus virions, suggesting that no obvious posttranscriptional modifications are required to activate tlr3 signaling. another feature of poly(i:c) that may obviate the need for viral proteins or ll37 may be that the inosine-cytosine base pair has only two hbonds. we observed that poly(a:u) is also an excellent agonist for tlr3 in the absence of ll37 and viral protein (fig. s1a) . however, weaker base pairing alone is unlikely to provide the full explanation for poly(i:c) being such a potent agonist; poly(g:u), which also has two h-bonds per base pair, is a poor agonist for tlr3 signaling in the absence of viral dsrna proteins (fig. s1a) . we can offer the following observations on proteins and peptides that can enhance tlr3 signaling. the proteins can be produced in e. coli or extracted from virions (as in the case of bmv), suggesting that post-translational modifications are not required for the enhancement of the tlr3 response. similarly, peptides such as ll37 that are potent enhancers of tlr3 signaling can be synthesized chemically without specific modifications [18] . in addition, both enzymes and structural proteins can be potent enhancers of tlr3 signaling as long as they interact with either dsrna or partially duplexed rnas. the sars-cov nsp15 preferentially binds ssrna and not dsrna [49] , and could not efficiently enhance tlr3 signaling. a deletion that increased interaction between the 2a hcv polymerase and a partially duplexed rna template made the polymerase a better enhancer for tlr3 signaling (fig. 4c) . the second feature of the tlr3 enhancers is that they have the ability to form oligomers. this is intuitive with the viral capsid proteins, but viral rdrps have surfaces that mediate oligomerization that may facilitate rna binding [56, 57, 58] . indeed, oligomerization of the hcv polymerase can affect the mode of rna synthesis [27] . ll37 also exists in higher order complexes in the absence of rna and is required at micromolar concentrations in contrast to the viral proteins that are active in our assays at nanomolar concentrations. this is likely due to multiple ll37 molecules being needed to bind and/or coat the dsrnas and mediate tlr3 activation [18] . the fact that bmv and rrv encapsidate ssrnas may initially seem to incompatible with their ability to enhance signaling by dsrnas. however, the encapsidated viral rnas are highly compacted and contain a high percentage of dsrnas with non-watson-crick base pairing, close to 70% [59, 60] . this likely explains how the cp can bind the dsrna needed to activate tlr3. the cp's of bmv and rrv are capable of binding to the double-stranded s4 rna in an electrophoretic mobility shift assay (fig. s4c ). in the context of a viral infection, however, we note it is likely that fully assembled virions may not result in activation of tlr3 signaling if the dsrnas are not exposed. we have added highly purified bmv virions and reovirus virions to beas-2b cells and 293t cells expressing tlr3 and have not observed induction of cytokines or tlr3-specific reporters (kao and ranjith-kumar unpublished results). we also demonstrated that two dsrna-binding proteins that can enhance tlr3 signaling can colocalize with tlr3 in endosomes. whether the dsrna-binding proteins facilitate binding of the dsrna by the tlr3 or the uptake of dsrna to endosomes remain to be determined. however, it is known that tlr3 binds to linear dsrna [61] . tlr3, unlike the rig-i-iike receptors [62] , lacks helicase activity to unravel the tertiary interactions that could stabilize the dsrna. the dsrna-binding proteins could disrupt the tertiary structure in the dsrnas to facilitate recognition of the dsrna by tlr3. electron microscopy revealed that ll37 decreases the particle size of the primarily globular poly(i:c) and results in more filamentous structures [18] . with the second scenario, the dsrna-binding proteins could facilitate entry of the viral dsrnas into endosomes, perhaps by increasing the interaction with trafficking chaperones, such as scavenger receptors [51] , but these rna-binding proteins likely need to complex and bring dsrna to endosome in order for tlr3 activation to occur. the net effect is an increase in the concentration of ligand available to tlr3. further studies will be required to unravel the complex requirements whereby the dsrna-binding protein activate tlr3 signaling. our demonstration that ll37 and viral dsrna-binding proteins can enhance tlr3 signaling raises the possibility that inappropriately expressed cellular dsrna-binding proteins could activate tlr3 signaling from cellular rnas. autoimmunity has been linked to antibodies that recognize rnp complexes [63, 64] , and tlr signaling can affect the production of autoantibodies associated with lupus erythematosis [65] . the results in this work are also informative for the function of the hcv polymerase. we note that the enhancement of tlr3 signaling by hcv polymerase is independent of rna synthesis since the active site mutant is capable of enhancing tlr3 signaling (fig. 3d ). all polymerases undergo structural transitions between open and closed conformations in response to the stages of nucleic acid synthesis. indeed, the monomer of the hcv polymerase contains a template channel that can sterically accommodate ssrna, but not dsrna [26, 66] . during the formation of a nascent rna from the template, however, the polymerase needs to release the interaction between the d1 loop and the thumb subdomain in order to accommodate the elongating nascent rna-template rna duplex. the 1bd21 can both extend from a primed template as well as initiate from the 39 terminus of a single-stranded rna. we believe that this is a manifestation of the 1b polymerase existing in an equilibrium between the open and closed forms in vitro, likely accounting for better dsrna-binding by the 1b polymerase and its enhanced ability for dsrna-induced signaling by tlr3. in contrast, the 2a polymerase exists predominantly in a closed conformation and thus is less able to bind to dsrna and enhance signaling by tlr3 [29] . increasingly, the open and closed conformations of the hcv polymerases are being linked to polymerase function. for the 1b hcv polymerase, the open conformation also exposes a binding pocket for the cell cycle regulator retinoblastoma and mediates the ubiquitinylation and degradation of retinoblastoma to increase cell cycle progression during hcv infection [67, 68] . during viral infection, viral polymerases and rnas are expressed inside the cell and depending on the cellular locations of the polymerase protein and viral or cellular dsrnas they recognize, could activate cytoplasmic innate immunity receptors such as rig-i or endosomal tlrs. previous study by naka et al [69] found that immortalized liver cells stably transfected with hcv ns5b and hek 293 cells stably transfected with both ns5b and tlr3 genes show increased activation of tlr3-depedent ifnb signaling, although the activation in hek 293 cells was only 2-fold. in a recent study from our laboratory, transient overexpression of ns5b and rig-i resulted in 8 to 14-fold induction of ifnb promoter-driven luciferase activity in huh7 and in hek 293t cells while overexpression of ns5b and tlr3 had no effect, suggesting that expression of ns5b in these two cell lines predominantly activates cytoplasmic innate immunity receptors such as rig-i and mda5 [70] . in both these studies, the ns5b polymerase was expressed from transfected plasmids and catalytic activity of the polymerase is required. in the present study, exogenously added hcv polymerase protein activates tlr3 without requiring rna synthesis since polymerase mutated at the catalytic site still activates tlr3. in addition three viral capsid proteins as well as ll37 that bind rna but do not have catalytic activity also activate tlr3 signaling. addition of both dsrna and the rna-binding protein are required for this activation. rnabinding protein alone has no effect since rnas synthesized by the hcv polymerase in cells may not enter efficiently into acidic endosomes, the site of tlr3 signaling [15] . thus the effect and mechanism of tlr3 activation by rna-binding protein are likely different from that observed by naka et al. [69] or ranjith-kumar et al. [70] . during viral infection and subsequent cell lysis, viral proteins and rnas may be released to the cell media and these viral proteins and rnas may be taken up by cells to activate innate immunity. exogenously added hcv core and ns3 proteins enters cells to activate tlr2 signaling in the absence of any added ligand, although the mechanism(s) of activation is not known [71] . in the present study, addition of ns5b protein to beas2b or hek293t cells has minimal effect on its own but significantly activates tlr3 signaling in combination with dsrna ligands, including viral dsrnas (table s1 ). indeed, ns5b and the rrv capsid protein have the ability to enters beas-2b cells within 30 min and colocalize with endosomal tlr3 by 6 h. the cellular entry of released viral proteins and rnas during viral infection may contribute to the cellular innate immune responses and could provide a point for therapeutic intervention. recombinant genotype 1b (con1 strain) and 2a (jfh-1) hcv polymerases lacking the c-terminal transmembrane helix were purified from e. coli as described by chinnaswamy et al. [28] . the preparations were devoid of detectable contaminating rna, dna, or lps. the hbv and rrv capsid proteins were expressed in e. coli and purified according to porterfield et al. [38] and mukhopadhyay et al., [39] . poly(i:c) was purchased from amersham biosciences (piscataway, nj) and reconstituted in phosphate-buffered saline. rna from purified reovirus virions was extracted with trizol, followed by ethanol precipitation and a wash with 70% ethanol. the purified rna was stored in rnasefree water. s4 dsrna from reovirus was synthesized by in vitro transcription from cdna (kind gift of p. danthi, indiana university, indiana) that has been engineered to have t7 promoters at termini of the two complementary strands. the annealed rnas were then analyzed for quantity and concentration before use. bpev dsrna was purified according to the protocol of valverde and gutierrez [43] and stored in rnase-free water. beas2b and hek293t cells were from atcc and cultured in begm media with supplements (lonza, basel, switzerland). culture media from beas-2b cells were collected 24 h after treatments, centrifuged at 2000 g for 2 min, and the supernatants were assayed for il6 secretion using a human il6 elisa kit along with quantification controls (bd systems) as per the manufacturer's protocol. hek293t cells were plated in costar white 96-well plates in dmem amended with 10% fbs at 4.4610 4 cells per well and transfected at ,85 to 90% confluency with a mixture of the lipofectamine 2000 and plasmids pisre-luc, puno-hutlr3 and phrl-tk as described in sun et al. [44] . the transfected cells were induced 18 to 22 h later by the addition of poly(i:c) or viral dsrnas to the culture medium to a final concentration of 1 mg/ ml in the presence or absence of 100 nm of rna binding proteins. the cells were harvested for analysis of luciferase 14 to 18 h after poly(i:c) addition. rdrp assays were performed as 20 ml reactions containing 20 mm sodium glutamate (ph 8.2), 12.5 mm dtt, 4 mm mgcl 2 , 1 mm mncl 2 , 0.5% triton x-100, 0.2 mm gtp, 0.1 mm atp and utp, 250 nm [a-32 p] ctp (mp biomedicals), 2 pmol pe46 and 1 pmol le19p as templates [26] . the reaction mixture was incubated at 30uc for 1 h and terminated by phenolchloroform extraction, followed by precipitation of the rna in the presence of two volumes of ethanol, 5 mg glycogen and 0.3 m naoac (ph 5.2). rna products were separated by 20%-7.5 m urea polyacrylamide gels. the signals were detected and quantified by using a phosphorimager. each reaction contained 400 ng of purified protein mixed with 1 pmol cy5-labeled sl26 or s4 dsrna radiolabled by a polynucleotide kinase reaction in a buffer containing 50 mm tris-hcl (ph 7.5), 4 mm mgcl 2 , 50 mm nacl, and 1 mm dithiothreitol. the reaction was irradiated with uv at 1200 mj for 3 min. and then subjected to sds-page. the signals were detected and quantified with a phosphorimager. the gel was then stained with coomassie blue to visualize the locations of proteins. dsf was performed in a stratagene mx3005p real-time pcr machine according to the protocol of niesen et al. [72] . each sample (5 mm final concentration) was prepared in a total volume of 50 ml containing sypro orange (molecular probes) in 100 mm tris, ph 7.0, 50 mm kcl, and 5 mm mgcl 2 ). the ramp condition was from 25 to 75uc, at increments of 0.5uc/min and the change in fluorescence intensity used to determine the t m values with the use of kaleidagraph software (synergy, reading, pa). beas-2b cells were seeded at 1.0610 5 cells/well in begm amended with supplements (lonza, switzerland) in a 48-well tissue culture plate or 1.0610 4 cells/well in a 96-well plate. 6 h later, the cells were transfected with a pool of three sirnas specific to tlr3, nonspecific control sirna (santa cruz biotechnology inc.), or sirna to rig-i (qiagen, valencia ca). 30 nm sirnas were transfected using lipofectamine rnaimax (invitrogen, carlsbad ca) according to the manufacturer's protocol. the cells were incubated for 48 h prior to treatment with ligands and proteins. culture media was collected 24 h later and assayed for il6 production using elisa. rt-pcr analysis of the tlr3 mrna to confirm the effects of the sirna knockdown was performed. after treatments total rna samples pooled from either six wells of a 96-well plate or three wells of a 48-well plate were isolated from beas2b cells using rneasy kit (qiagen, valencia, ca). 0.5 mg of total rna was then reverse transcribed to cdna with mmlv reverse transcriptase (ambion, austin, tx) using random decamers (new england biolabs, ipswich, ma). cdna generated from 16 ng of total rna was amplified using sybr green supermix (bio-rad, hercules, ca) with an initial 3 min denaturing temperature of 95uc, followed by a total of 40 cycles of 30 s of denaturation at 95uc and 30 s of annealing at 55uc and elongation at 72uc. tlr3-specific primers were previously used by homma et al. [73] : (forward: 59-gatct-gtctcataatggcttg-39; reverse: 59-gacagattcc-gaatgcttgtg-39; and gapdh primers (59-gagtcaacg-gatttggtcgt-39; reverse: 59-tgggatttccattgat-gaca-39) were used. for each sample, tlr3 ct values were subtracted from corresponding gapdh ct values. tlr3 mrna levels for each treatment were calculated as %tlr3 mrna for samples treated with control nonspecific sirna. beas-2b cells were seeded on coverslips in 6 well plates. highly purified recombinant ns5b (0.2 mm) that was free of pyrogen was added to the cells at the same time as the dsrnas and incubated for 30 min at 37uc. after washing with pbs, cells were fixed with 4% paraformaldehyde at room temperature for 30 min. after two additional washes with pbs, they were permeabilized with 0.5% triton x-100 at room temperature for 10 min. the cells were then washed twice more with pbs amended with 0.02% tween-20 (tbs-t) and incubated with blocking buffer (1% bsa in pbs) for 1 h at room temperature. after washing three times with pbs-t, slides were incubated with anti-ns5b antibody (mab; 5b-3b1 from alexis biochemicals) and goat anti-tlr3 (r&d systems, minneapolis, mn) overnight at 4uc. slides were then probed with a texas red-labeled bovine anti-goat mab (santa cruz biotechnology) for 1 h at rt, washed, then incubated with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (santa cruz biotechnology) for 1 h at room temperature. coverslips were washed with pbs-t three times, air dried for 1 h and mounted in vectashield mounting medium with dapi (vector laboratories). the images were obtained on a leica tcs sp5 scanning confocal microscope with an hcx pl apo lambda blue 63 x 1.4 oil objective lens (leica microsystems). excitation was at 20% of the maximum laser power. images were captured with a scanning speed of 200 hz and image resolution of 5126512 pixels and then analyzed using leica application suite 2.02. ross river virus capsid protein (0.2 mm) was added the culture media of beas-2b cells seeded on coverslips for up to 6 hours at 37uc. the cells were washed and fixed as described above. the cells were incubated with anti r-cp rabbit polyclonal antibody (custom prepared by cocalico biologicals, reamstown, pa) and anti-tlr3 (mab, a kind gift of l. san mateo of centocor inc) diluted in pbs+0.1% bsa+0.01% tx100 overnight at 4uc. slides were then probed with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (santa cruz biotechnology) for tlr3 and bovine anti-rabbit texas red (santa cruz biotechnology) for r-cp for 1.5 h at room temperature. the slides were imaged and the results analyzed as described above. figure s1 effects of various rnas on il6 production. a) results from beas-2b cells. the endornaviral dsrnas labeled a-c were extracted from several rice isolates collected in lousiana and have distinct migrations in agarose gels. however, the genomes have not been determined. pic, pau, and pgu are three homopolymeric rnas. the single-stranded viral rnas were all extracted from purified virions. all rnas used were at a final concentration of 0.5 mg/ml. b) a time course of il6 production by beas-2b cells in response to poly(i:c) and s4 dsrna. the addition of ll37 to 3 mm final concentration resulted in tlr3 enhancing signaling in response to the s4 dsrna or poly(i:c). c) results from hek 293t cells expressing recombinant tlr3. all rnas were used at 1 mg/ml. the ratios denote the ratio of the isre-driven firefly luciferase to the renilla luciferase produces in the same cells. (tif) figure s2 additional characterization of the 1b hcv polymerase that could affect il6 production in beas-2b cells. a) effects of poly(i:c) of different lengths on il-6 production. the poly(i:c) fragments were separated on a denaturing gel and eluted from the gel fragments. the eluted fragments were annealed and their lengths determined by comparison to an dna ladder. b) effects of several recombinant hcv polymerase proteins on tlr3 induction of il6. the final concentrations of the proteins added are shown on the horizontal axis. the amount of poly(i:c) in each reaction was at 0.13 mg/ml. c) a demonstration that the mutant hcv polymerases retain the ability to bind the sl26 rna. dsrna. the gel is from a nondenaturing stacked polyacrylamide gel of 5 and 20%. the s4 dsrna was radiolabeled during in vitro transcription of the plus-and minus-strands and then annealed at a 1:1 ratio. (tif) figure s5 localization of the r-cp in beas-2b cells 2 h after addition into the cell culture medium. r-cp is stained red. the boxed area in the upper right is enlarged to show that small punctate spots of r-cp can be found in the cell's cytoplasm. (tif) plant and animal sensors of conserved microbial signatures innate immune recognition of viral infection intracellular pattern recognition receptors in the host response preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing sensing and signaling in antiviral innate immunity structure and function of toll receptors and their ligands a missense mutation of the toll-like receptor 3 gene in a patient with influenzaassociated encephalopathy tlr3 deficiency in patients with herpes simplex encephalitis effects of single nucleotide polymorphisms on toll-like receptor 3 activity and expression in cultured cells the l412f variant of toll-like receptor 3 (tlr3) is associated with cutaneous candidiasis, increased susceptibility to cytomegalovirus, and autoimmunity dissecting tlr3 signalling in dendritic cells the toll-like receptor 3:dsrna signaling complex leucinerich repeats and pathogen recognition in toll-like receptors predicting toll-like receptor structures and characterizing ligand binding the tlr3 signaling complex forms by cooperative receptor dimerization biochemical and functional analyses of the human toll-like receptor 3 ectodomain mrna is an endogenous ligand for toll-like receptor 3 ll37 and cationic peptides enhance tlr3 signaling by viral doublestranded rnas replication of hepatitis c virus hcv ns5b polymerase inhibitors template requirements for rna synthesis by a recombinant hepatitis c virus rnadependent rna polymerase de novo initiation of rna synthesis by the rna-dependent rna polymerase (ns5b) of hepatitis c virus mechanism of de novo initiation by the hepatitis c virus rna-dependent rna polymerase: role of divalent metals requirements for de novo initiation of rna synthesis by recombinant flaviviral rna-dependent rna polymerases de novo initiation pocket mutations have multiple effects on hepatitis c virus rnadependent rna polymerase activities a locking mechanism regulates rna synthesis and host protein interaction by the hepatitis c virus polymerase regulation of de novo-initiated rna synthesis in hepatitis c virus rna-dependent rna polymerase by intermolecular interactions de novo initiation of viral rna-dependent rna synthesis structural and functional analysis of hepatitis c virus strain jfh1 polymerase a comprehensive structure-function comparison of hepatitis c virus strain jfh1 and j6 polymerases reveals a key residue stimulating replication in cell culture across genotypes ross river virus transmission, infection, and disease: a cross-disciplinary review the coat protein leads the way: an update on basic and applied studies with the brome mosaic virus coat protein hepatitis b virus infection-natural history and clinical consequences virus assembly, allostery and antivirals effects of deletions in the n-terminal basic arm of brome mosaic virus coat protein on rna packaging and systemic infection biological significance of the seven aminoterminal basic residues of brome mosaic virus coat protein fulllength hepatitis b virus core protein packages viral and heterologous rna with similarly high levels of cooperativity in vitro-assembled alphavirus core-like particles maintain a structure similar to that of nucleocapsid cores in mature virus cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-idependent antiviral responses in human lung epithelial cells cytokine induction by respiratory syncytial virus and adenovirus in bronchial epithelial cells human rhinovirus-induced epithelial production of cxcl10 is dependent upon ifn regulatory factor-1 transmission of a dsrna in bell pepper and evidence that it consists of the genome of an endornavirus structural and functional analyses of the human toll-like receptor 3. role of glycosylation the dsrna binding site of human toll-like receptor 3 crystal structure of rig-i c-terminal domain bound to blunt-ended double-strand rna without 59 triphosphate single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3 green tea catechin, epigallocatechin gallate, suppresses signaling by the dsrna innate immune receptor rig-i structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp15 gold nanoparticles as spectroscopic enhancers for in vitro studies on single viruses scavenger receptor class-a is a novel cell surface receptor for doublestranded rna cellsurface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis cell-penetrating peptides derived from viral capsid proteins vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells nuclear entry of hepatitis b virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids oligomeric structures of poliovirus polymerase are important for function oligomerization and cooperative rna synthesis activity of hepatitis c virus rna-dependent rna polymerase oligomeric interaction of hepatitis c virus ns5b is critical for catalytic activity of rnadependent rna polymerase ordered duplex rna controls capsid architecture in an icosahedral animal virus comparison of the native ccmv virion with in vitro assembled ccmv virions by cryoelectron microscopy and image reconstruction structural basis of toll-like receptor 3 signaling with double-stranded rna cytosolic viral sensor rig-i is a 59-triphosphate-dependent translocase on doublestranded rna two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus anti-5s rna/protein (rnp) antibody levels correlate with disease activity in a patient with systemic lupus erythematosus (sle) nephritis endosomal tlr signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus structural analysis of the hepatitis c virus rna polymerase in complex with ribonucleotides hepatitis c virus induces e6ap-dependent degradation of the retinoblastoma protein down-regulation of the retinoblastoma tumor suppressor by the hepatitis c virus ns5b rnadependent rna polymerase hepatitis c virus ns5b delays cell cycle progression by inducing interferon-beta via toll-like receptor 3 signaling pathway without replicating viral genomes a cell-based assay for rna synthesis by the hcv polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by ns5a hepatitis c core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation the use of differential scanning fluorimetry to detect ligand interactions that promote protein stability corticosteroid and cytokines synergistically enhance toll-like receptor 2 expression in respiratory epithelial cells we thank members of the kao lab and jarrat jordan and lani san mateo of centocor inc. for helpful discussions and jing wang for some assays for il-8. we thank laura kao for editing the manuscript and the indiana university metacyte microscopy center and jim powell for the use of the facility and help with confocal microscopy. key: cord-306934-29ljbl7g authors: tonelli, michele; vazzana, iana; tasso, bruno; boido, vito; sparatore, fabio; fermeglia, maurizio; paneni, maria silvia; posocco, paola; pricl, sabrina; colla, paolo la; ibba, cristina; secci, barbara; collu, gabriella; loddo, roberta title: antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives date: 2009-07-01 journal: bioorganic & medicinal chemistry doi: 10.1016/j.bmc.2009.05.020 sha: doc_id: 306934 cord_uid: 29ljbl7g abstract twelve aminoarylazocompounds (a–c) and 46 aryltriazene 7 derivatives (d–g) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 rna and dna viruses. eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high level, against one or more viruses. a marked activity against bvdv and yfv was prevailing among the former compounds, while the latter type of compounds affected mainly cvb-2 and rsv. none of the active compounds inhibited the multiplication of hiv-1, vsv and vv. arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are the following; bvdv: 1 > 7 > 8 > 4; yfv: 7 > 5; cvb-2: 25 > 56 > 18; rsv: 14 > 20 > 55 > 38 > 18 > 19; hsv-1: 2. for these compounds the ec50 ranged from 1.6μm (1) to 12μm (18), and the s. i. from 19.4 (1) to 4.2 (2). thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for developing antiviral agents against ss rna viruses, particularly against rsv and bvdv, which are important human and veterinary pathogens. finally, molecular modeling investigations indicated that compounds of structure a–c, active against bvdv, could work targeting the viral rna-dependent rna-polymerase (rdrp), having been observed a good agreement between the trends of the estimated ic50 and the experimental ec50 values. we have recently shown that some arylazoenamines (fig. 1) are endowed with antiviral activity in vitro against rna viruses, particularly cvb-2, rsv, bvdv, yfv and sb-1, in many cases with ec 50 in the range from 0.8 to 10 lm. 1 arylazoenamines can be considered as substructures of ortho-(and para-) aminoazocompounds, many of which are endowed with antimicrobial and antiparasitic activities, but additional pharmacological activities have been also shown in particular cases (fig. 2) . 2,6-diamino-3-phenylazopyridine (pyridium) inhibits various cocci and coli bacilli, 5-(4-amino-1-naphthylazo)uracil is highly effective against schistosoma mansonii, 2 while 1-[3-(4-phenylazo-5,6,7,8-tetrahydronaphth-1-yl)amino]propylpiperidine 3 and 1-[3-(4-phenylazo-5,6,7,8-tetrahydro naphth-1-yl)amino]lupinane 4 are very active against mycobacterium tuberculosis. antifungal and anti-proliferative activities have been found in a peculiar class of cyclic aminoazocompounds (3,3-disubstituted-3,4-dihydro-1,2,4-benzo triazines), which, moreover, can display several other pharmacological activities depending on the substituents that are present on the bicyclic system. 5, 6 very potent relaxant activity on intestinal and uterine smooth muscles was displayed at nanomolar concentration by n-dialkylaminoalkyl derivatives of another cyclic aminoazo system, namely 11h-dibenzo-[1,2,5]-triazepine, which exhibited also local anesthetic and antithrombotic activities. 7 arylazoenamines could be also seen as vinylogues of aryltriazenes (fig. 2) , a class of compounds which hold an important position as carcinogens and/or anticancer agents. 3,3-dimethyl-1phenyltriazene and, even better, dacarbazine are able to methylate dna; thus the former resulted a powerful carcinogen, while the latter is in clinical use for the treatment of malignant melanoma and hodgkin's lymphoma. 8 temozolamide, 9 closely related to dacarbazine, is currently used to treat malignant glyomas. a recent development of this cyclic aryltriazene is represented by benzotriazepinones, 10 that are very promising agents against breast cancer. it is worth noting that these compounds are weak alkylating agents and may damage dna by a novel mechanism. the triazene pharmacophore has been linked to other bioactive moieties in order to obtain chymeric compounds which should be able to exert a double cytotoxic mode of action. thus, the triazene group was linked to 4-anilino-quinazoline backbone, 11 which is known to inhibit the epidermal growth factor receptor tyrosine kinase (egfr-tk), and also to a benzophenone residue, 12 that is present in an inhibitor of pharnesyl-transferase and of tubulinpolymerization. the introduction of a triazene group on the molecule of pyrimethamine, a dihydrofolate reductase inhibitor, generated a compound that combines antitumoral potential with inhibition of pneumocystis jirovecii 13 (p. carinii). the insertion of triazene group between two benzamidine units produced diminazene (berenil), a powerful agent against the african trypanosomiasis. to the best of our knowledge, no antiviral activity has been seen with either amino arylazocompounds or aryltriazenes, with the only exception for a patent claiming the activity of 1-phenyl-3,3dimethyltriazene against the tobacco mosaic virus. 14 figure 2 . some biologically active aminoarylazo compounds and aryltriazene derivatives. antiviral activity has been shown by several benzotriazole derivatives, which could be formally considered as cyclic triazenes, though the resonance stabilization of the heterocycle could imply even substantial differences in the chemical and hence the biological behavior of the two kinds of compounds. interestingly, some halogenated benzotriazoles have been shown to inhibit the ntpase/helicase activities of hepatitis c and related viruses. 15 aromatic and heteroaromatic esters of 1-hydroxybenzotriazole were found to strongly inhibit (k i = 7.5 nm) the 3cl-protease, which is essential for the replication of the coronavirus responsible of sars (severe acute respiratory syndrome). 16 more recently, a set of benzotriazole derivatives have been found endowed with potent activity against rsv and moderate activity against yfv, bvdv and cvb-2. 17 on this base we deemed interesting to investigate the possible antiviral activity of some assorted aminoarylazo compounds and aryltriazene derivatives, allotted in seven groups a-g (fig. 3) . on the whole 12 amino arylazo compounds (a-c) and 46 triazene derivatives (d-g) were evaluated in cell based assays for cytotoxicity and antiviral activity against a large panel of rna and dna viruses. thirty out of the 58 compounds, that were evaluated for antiviral activity, were already known and were prepared according to the literature. compounds 7-12 were already described by some of us. 4 (48) is reported as known, r but we failed to find out any data concerning its characterization. references, a-r concerning the previously described compounds, are available as supplementary data. the 28 novel compounds were prepared according to the following schemes 1 and 2. all new compounds were characterized by elemental analyses and 1 h nmr spectra. the n-(3'-dimethylaminopropyl)-3-trifluoromethylaniline and the n-(lupinyl)-3-trifluoromethylaniline, required by scheme 1, were already described by some of us. 18 it is observed that the presence of a 3-trifluoromethyl group prevented completely the coupling of diazonium salt on the para position of the n-substituted anilines giving place only to the triazene derivatives e. the prepared compounds (12 amino arylazocompounds a-c and 46 triazene derivatives d-g) were evaluated in vitro in parallel cell-based assays for cytotoxicity and antiviral activity (tables 3-5) against viruses representative of two of the three genera of the flaviviridae family, that is, flaviviruses (yfv) and pestiviruses (bvdv), as hepaciviruses can hardly be used in routine cell-based assays. title compounds were also tested against representatives of other virus families. among ssrna+ were a retrovirus (human immunodeficiency virus type 1, hiv-1), two picornaviruses (coxsackie azt (3 0 -azido-thymidine), nm 108 (2 0 -c-methyl-guanosine), nm 176 (2 0 -c-ethynyl-cytidine), ribavirin, nm 299 (6-azauridine), m 5255 (mycophenolic acid) and acg (acyclovir) were used as reference inhibitors of ssrna+, ssrnaà and dna viruses, respectively. interestingly, 35 (8 aminoazocompounds and 27 triazene derivatives) over 58 tested compounds exhibited antiviral activity against one or more viruses; in particular 16 compounds exhibited a selective activity against a single virus, while 13, 4 and 2 were, respectively, active against two, three and four viruses. on the other hand, 23 compounds (4 aminoarylazo and 19 triazene derivatives) were not able to inhibit the multiplication of any virus at concentrations up to 100 lm. none of the active compounds inhibited the multiplication of hiv-1, vsv and vv, but an increasing number of them exhibited antiviral activity against, in the order, reo-1 (4), sb-1 (7), hsv-1 (8), bvdv (9), yfv (9), rsv (12) and cvb-2 (13) (tables 1 and 2 therefore, both the [(dialkylaminoalkyl)amino]azobenzene and the aryltriazene molecular patterns represent interesting pharmacophore for developing novel antiviral agents, particularly against ssrna viruses. cytotoxicity and antiviral activities of tested and reference compounds are reported in tables 3-5. in these tables, viruses for which no active compounds have been found are not indicated. test compounds showed different degrees of cytotoxicity against the confluent cell monolayers (in stationary growth) used to support the multiplication of the different viruses. the most susceptible to toxicity were the exponentially growing lymphoblastoid human cells (mt-4) used to grow hiv-1, while the non-human host cell lines exhibited a progressively reduced sensitivity in the order vero-76 > bhk > mdbk. on the other hand, it is observed that the toxicity is not evenly distributed among the different types of test compounds, being mainly shown by the triazene derivatives of structure e and by the amino azo compound a-c, which are characterized by the presence of the basic dimethylaminopropyl and lupinyl moieties. indeed the most toxic compounds were 29 and 12, with a mean of the cc 50 values for the host cells of 17.5 and 21.5 lm, respectively. the other groups of triazene derivatives of structure d, f and g are relatively non toxic, exhibiting in most cases cc 50 p 100 lm. as already seen, eight [(dialkylaminoalkyl)amino]azobenzenes (a-c) and 27 triazene derivatives (d-g) have shown antiviral activity, sometimes of high level against one or more viruses. from tables 1 and 3 appears that compounds of structure a-c have a marked activity against bvdv and yfv, with only occasional, though still of high level, activity against hsv-1, reo-1, cvb-2 and rsv. on the whole the triazene derivatives d-g are characterized by the most frequent activity against cvb-2 and rsv, to which are associated, with decreasing frequency, the activity on sb-1, hsv-1, yfv, reo-1 and bvdv (tables 2, 4 and 5). however, considering separately each subgroup of triazene derivatives, it is observed that the activity against sb-1 is mainly associated to the pyrrolidine-related triazenes f, while activity against hsv-1 is associated with the diaryltriazenes that bear a dimethylaminopropyl chain. the importance of a basic chain for the activity against hsv-1 is underlined by the high activity shown by compounds 2 and 4, bearing the same dimethylaminopropyl chain, even linked to the aminoazobenzene pharmacophore (a), but also by the moderate activity of triazene 53, which contains a basic n-methylpiperazine residue. conversely it is worth noting that the activity against bvdv, largely diffused among the amino azo compounds a-c, is very rarely present (2 over 46 compounds) among the triazene derivatives d-g, even among those (e) sharing the basic chain of the former groups. active and inactive compounds are found side by side in all groups a-g, thus their molecular patterns seem to address the activity against the different type of viruses, while the substituents on the aromatic nucleus are the determinants for activity or inactivity. however, attempts to define general structure-activity relationships resulted rather frustrating, since observations that hold for the activity against most viruses may not hold for some others. indeed the meta substitution with halogens, trifluoromethyl and nitro groups favors the display of activity in the groups a-e and g, but in compounds of group f is the para substitution that promotes the activity; however occasional exceptions are observed in both cases. an unsubstituted aromatic ring can be found in active (1, 7, 9, 21, 25, 52, 54-56) and inactive (13, 34, 45) compounds, while the presence of electron-releasing groups as ch 3 and och 3 (32, 42) or of a benzyl residue (15, 22) is always detrimental for the expression of activity. despite the high antiviral activity of many compounds, the corresponding selectivity index (s. i.) are generally low, due to the rather high cytotoxicity exerted on the host cells. only few compounds exhibited a s. i. higher than 10 for one or more viruses, while 13 compounds had a s. i. in the range from 9 to 5 and the remaining had s. i. below this value. taking into account the ec 50 and s. i. values, the best compounds were the following, which are arranged in the order of decreasing potency and selectivity; bvdv: 1 > 7 > 8 > 4; yfv: 7 > 5; cvb-2: 25 > 56 > 18; rsv: 14 > 20 > 55 > 38 >18 > 19 > 23; sb-1: 41; hsv-1: 2. for these compounds the ec 50 ranged from 1.6 lm (1) to 15 lm (23, 41) , while the corresponding s. i. values were in the range from 19.4 (1) to 4.2 (2) . though the activity shown by the above compounds may appear as not particularly impressive, it must be noted that, at the present, very few experimental agents are known to act against those viruses and that, moreover, they often display high cytotoxicity. a particular interest is deserved by compounds displaying good activity against rsv and bvdv. rsv, is responsible of serious respiratory tract infections in infants, elderly and immunocompromised patients with high mortality rate. very potent anti-rsv agents, interfering with the virus-host fusion process, have been developed in the last years, as the benzimidazole derivative bms-433771 which exhibited an ec 50 as low as 21 nm; however, attempts to demonstrate therapeutic efficacy were not successful, 19 probably due to the rapid emergence of resistance. quite recently a chiral 1,4-benzodiazepine derivative, acting through a different mechanism, though much less potent than bms-433771, showed very promising characteristics and is being examined in phase ii clinical studies. 20 nevertheless the chirality-associated developability issue cannot be underestimate, since the antiviral activity was found to reside mainly in the s-enantiomer (rsv-604), that exhibited an ec 50 = 0.9 lm, while racemate had ic 50 = 3.5 lm, a value comparable with that of compound 14. on the other hand, bvdv is the prototype of pestiviruses, which commonly affect cloven-hoofed animals (cattle, swine, sheep). bvdv infection is responsible of reduced dairy production and increased (even up to 30%) cattle mortality throughout the world. 21 thus, the availability of effective and inexpensive anti-pestivirus drugs is of importance to relieve such an heavy economic burden. the development of anti-bvdv drugs and the understanding of their mechanism of action is of further interest because it may provide valuable informations for the design of agents active against hepatitis c virus (hcv). 22 hcv, which like bvdv belongs to the flaviviridae family, infects about 3% of world population. since chronic hcv infection can progress to fatal cirrhosis and hepatocellular carcinoma, the development of effective and safe anti-hcv agents is urgently needed. the current combination therapy with pegylated interferon and ribavirin is effective in less than 60% of the treated patients and is associated with heavy side effects. for the understanding of some aspects of virus replication, bvdv has been considered even more advantageous than the currently used hcv subgenomic replicon system. 23 in the last years, several potent anti-bvdv agents have been developed and shown to target the rna-dependent rna-polymerase (rdrp), even if resulting almost inactive on the purified enzyme. compounds vp32947 (3-[(dipropylaminoethyl)thio]-5h-1,2,4-triazino [5,6-b] indole) 24 and bpip (523 exhibited ec 50 as low as 30-40 nm. other compounds (as the thiazolylurea dpc-a69280-29, 25 table 3 cytotoxicity against mt-4, mdbk, bhk and vero-76 cell lines and bvdv, yfv, reo-1, cvb-2, rsv and hsv-1 inhibitory activity of amino azocompounds of structure a-c the benzimidazolone 1453 26 and 7-amino-1,3-dihydroxy-10methyl-6-[4-(2-pyridinyl)-1-piperazinyl]-9(10h)acridone 27 ) have ec 50 in the range 0.6-1.5 lm, that are comparable with those of the presently described aminoaryl azocompounds 1 and 7 (1.6 and 2.5 lm, respectively). the structural simplicity of these compounds, which allows a wide range of easy and inexpensive molecular modifications, make them an attractive model to develop more active and less toxic anti-pestivirus agents. in view of these considerations, we deemed interesting to perform some molecular modeling investigations to study wether the bvdv rdrp could be the target of also our compounds 1, 2, 4 and 7-10. bvdv, the best-studied pestivirus, has a genome that consists of an approximately 12.6-kb positive sense ssrna. the bvdv genome is translated into a single polyprotein which is processed into at least four structural and six nonstructural (ns) proteins required for viral assembly and replication. among the nonstructural proteins, the ns5b is an rna-dependent rna-polymerase (rdrp) enzyme responsible for genome replication as a part of a larger, membrane associated replicase complex. the crystal structure of rdrp from several families of single-or double-strand rna viruses, including hcv [28] [29] [30] and bvdv, 31, 32 have been recently made available in the protein data bank (pdb) repository. despite the low sequence identity between different polymerases, the crystal structures of these proteins (from bvdv, hcv and other families of ss and dsrna viruses) all present the shape of a right hand with fingers, palm, and thumb domains. in particular, the bvdv rna-dependent rna-polymerase core domain (residues 139-679) has a dimension of approximately 74 â 60 â 58 å around a central cavity, 31 which serves as for rna template bind-ing, nucleotides recruitment, and polymerization reaction (see fig. 4a ). in addition, there is an n-terminal region (residues 71-138) of which residues 71-91 are disordered in the relevant crystal structure. a thorough search of a putative binding site for our molecules onto bvdv rdrp was conducted following our successful recipe developed for studying allosteric inhibitors of polio-virus helicase. 33 the portion of the enzyme making up the binding site interacting with the inhibitors is located in the fingers domain (residues 139-313 and 351-410), consisting of 12 a-helices and 11 b-strands (see fig. 4a ). in bvdv rdrp, as in other viral rdrps, the n terminus of the fingers domain, together with a long insert in the fingers domain (residues 260-288), form the fingertip region that associates with the thumb domain. this region is characterized by a three-strand conformation, and since the fingers and the thumb domains are associated through this fingertip region, the conformational change induced by the rna template binding into the central channel is somewhat limited. the remainder of the fingers domain is comprised of a b-strand rich region (b-fingers) and an a-helix rich region (a-fingers) close to the palm domain. according to the procedure adopted, all compounds were characterized by a similar docking mode in the putative binding site of the bvdv rdrp, as exemplified by compound 1 in figure 5 . in particular, the residues lining the pocket (see fig. 5d ) include the side chains of residues from n217 to f224 belonging to a loop between the two strands b3 and b4, and those of residues from k263 to e265 of a loop between strands b5 and b6. amino acids from i287 to e291 contribute to binding from the b-sheet portion b8, whilst another loop, connecting two a-helical motifs (a19 and a20, respectively), concurs with residues from r529 and l530. going into details, the aminoalkyl part of the molecule is engaged in stabilizing nonbonded interactions with the apolar side chains of residues i261, i287, a221, and a222, while the diarylazo moiety is nicely encased in a subsite lined by the side chains of n217, n264, e265, k266, r529, and l530. more importantly, however, this molecular scaffold is anchored in place by three persistent hydrogen bonds (hbs). a first hb bridge involves the nitrogen atom of the n(ch 3 ) 2 substituent group on 1 and the -oh group of y289 side chain, with an average dynamic length (adl) of 3.08 ± 0.3 å. the second hb interaction engages the terminal amino group of k263 and the n atom of the inhibitor secondary amino group (adl = 3.18 ± 0.2 å). finally, one of the nitrogen atoms belonging to the azo group characterizing compound 1 is involved in the third hb interaction with the backbone -nh group of e265 (adl = 2.92 ± 0.3 å). importantly, quantitative information about the affinities of our ligands towards bvdv rdrp can be inferred by applying the mm/ pbsa methodology to estimate the free energy of binding, dg bind , and its components. the calculated dg bind values for the dimethylaminopropyl derivatives 1, 2, 4, and the quinolizidinylmethyl derivatives 7-10 are listed in table 6 . generally speaking, and in harmony with our previous findings, 33,34 both the nonbonded mechanical energy components of dg bind , de vdw and de el , afford a substantial, favorable contribution to binding for both series of compounds. on the other hand, due the polar character of the azo group, the desolvation penalty paid by these molecules upon binding (dg pb ) is also quite substantial, so that the net, resulting electrostatic contribution to the affinity of these inhibitors to their enzyme receptor are notably unfavorable. specifically, for this series of compounds, the mean value of the electrostatic energy (de el + dg pb ) is 26 kcal/mol, whilst the corresponding mean values of the van der waals and hydrophobic overall interaction energies (de vdw + dg np ) are à46 kcal/mol. accordingly, it follows that the association between the ligands and the rdrp is mainly driven by more favorable nonpolar interactions in the complex than in the solution, in harmony with a proposed general scheme for noncovalent association. 33, 34 however, as indicated for instance by the energy components of compound 2, this driving force can be weakened when the polar groups on the molecule do not find an adequate bonding pattern in the protein compared to water. the free energy penalty for this (de el + dg pb ) term is least for compound 1 and this, together with its substantial van der waals contribution and moderate entropic unfavorable term, consequently leads to one of the highest binding affinity in this set of inhibitors. one of the most important benchmark in this study, however, is the correspondence between the estimated free energies of binding and the experimental measured ec 50 values. indeed, we can observe a good agreement between the trend exhibited by the ic 50 values reported in table 6 and the corresponding biological activity determined for these compounds in bvdv infected cell line (see table 3 ). although we obviously cannot directly compare the computed binding free energy (and hence the corresponding ic 50 ) with the ec 50 values deriving from experiment, we can observe that, in most cases, the rank of the inhibitors with respect to their activity towards their putative targets, the rdrps of bvdv, is maintained, although with some discrepancies. in fact, all estimated ic 50 values are systematically higher than those determined by in vitro experiments with infected cell lines. the overall results obtained from molecular modeling deserve some more comments. first of all, the binding site identified by our procedure is very close to the putative binding site proposed for two allosteric inhibitors of bvdv rdrp, vp32947 and bpip, 23 table 5 cytotoxicity against mt-4, mdbk, bhk and vero-76 cell lines and yfv, reo-1, cvb-2, rsv, hsv-1 and sb-1 inhibitory activity of triazene derivatives of structure f and g tables 3 and 4. and involves residue f224 (see fig. 5d ), a residue found mutated into s224 in bvdv bpip-resistant clones. the binding pocket is located in a turn of the fingers domain between two b-sheets which is believed to be involved in finger flexibility for rna template/product translocation, 35 dimerization of the rdrp in the replication complex, or protein/protein interactions, 36, 37 enabling the assembly of an active replication complex. according to our docking results, then, the hypothetical binding of our compounds to the polymerase could affect the finger flexibility or impair the capacity of the protein to translocate its template/product during rna polymerization. alternatively, the diarylazo moiety could possibly act by hampering the entrance of the template rna in the template binding cavity. although our in silico data clearly show the same trend of the corresponding experimental ec 50 values, at the same time they yield no evidence that binding of these inhibitors onto the bvdv rdrp result in a real allosteric inhibition of the enzyme or in a reduced template-binding ability. one of the explanations why we find ic 50 values higher than the corresponding ec 50 could be explained by the following rationale. recently an attempt of co-crystallize the bvdv rdrp with the inhibitor vp32947 failed do to a dimer interface near the putative binding site of vp32947 in the bvdv rdrp crystal. the authors of the work proposed that the for-mation of this dimer could constitute an important point in the replication complex. moreover, it was also hypothesized that the top of the fingers domain could be a protein-docking site pivotal to the interactions with other enzymes of the replicase complex. as the vp32947 binding site is part of the putative binding pocket for our molecular series, it could be that, upon binding, our compounds hinder the interactions among different proteins making up the replicase complex. last but not least, it is important to recall that rdrp functions in virus-infected cells in the context of membrane-bound replication complexes, that usually consist of several virus-encoded proteins, host proteins, and various forms of viral rna. the rna-dependent rna polymerase may then have a role in interacting with these other components and/or in the stability of the replication complexes. if one or more of these putative actions of the rdrp in infected cells is sensitive to our inhibitors, viral rna synthesis may be disrupted in a manner which is distinct from the one estimated by our in silico assays. now, given the similarity among the polymerases of pestiviruses, hepaciviruses, and flaviviruses, any knowledge gained from any one of these viral systems is likely to foster progress in the others. given the high importance of the hepatitis c pandemic and the stringent need for efficient therapeutics for this pathology, it is expected that new molecular entities such as those described in this work can provide meaningful insights towards the identification of possible anti-hcv agents. interestingly, the overall sequence identity between bvdv and hcv rdrp proteins is quite low, approximately 30%. if we consider the alignment of bvdv and hcv rdrp reported in figure 4c , and in particular focus on the highlighted fingers domain part, we can immediately speculate that: (i) the f224 residue in the bvdv protein has no counterpart in the corresponding hcv enzyme; accordingly, if the drug would bind in the same binding pocket with the same binding mode, there should in principle be no problem of resistant mutant at this position. (ii) the important region of k263-k266 in the bvdv rdrp is highly conserved in the corresponding hcv polymerase (knek in bvdv and knev in hcv, respectively, see fig. 4c ); importantly, both k263 and e265 are engaged in fundamental stabilizing hydrogen bonds with the inhibitors in the bvdv case but, being conserved, they are able to establish the same interactions with the compounds also in the case of hcv. (iii) the residue y289 of bvdv rdrp is not conserved in the corresponding hcv polymerase, being replaced by a phenylalanine. therefore, the anchoring hydrogen bond between the y289 -oh group and, for instance, the tertiary nitrogen of 1 can no longer take place. taken all together, these evidences can justify a minor potency of this molecular series towards the hcv rdrp as a target with respect to the bvdv counterpart. at the same time, in the case of both viruses, the binding pockets on the respective rdrps do share a sufficient degree of structural and sequence homology; thus, since these regions involve the fingers domains, which overhang the palm domains of the enzymes, they might be involved in rna substrate recognition and, hence, the the side chains of all residues that form the primary binding pocket interacting with compound 1 are shown as stick models, and the atom color-coding is as follows: n217, firebrick; a221, orange; a222, dark kaki; f224, red; e258, dim gray; t259, rosy brown; i261, green; k263, hot pink; n264, tan; e265, sienna; k266, dark magenta; i287, gold; q288, navy blue; y289, purple; p290, dark slate blue; e291, pink; r295, olive drab; r529, coral; l530, kaki. hydrogen bonds are highlighted as light gray broken lines. hydrogen atoms, counterions, and water molecules are omitted for clarity. free energy components and total binding free energies for compounds of the series a-c on bvdv rdrp inhibitor molecules may affect the rdrps interactions with the incoming rna molecule. twelve aminoarylazocompounds of structures a-c, and 46 aryltriazene derivatives d-g have been synthesized and assayed for antiviral activity against a panel of 10 rna and dna viruses. thirty-five (8 aminoazocompounds and 27 triazene derivatives) over 58 tested compounds exhibited antiviral activity against one or more viruses and many of them had shown an ec 50 6 10 lm against at least one virus. none of the active compounds inhibited the multiplication of hiv-1, vsv and vv. aminoazocompounds a-c have a marked activity against bvdv and yfv, while the triazene derivatives d-g are characterized by the most frequent activity against cvb-2 and rsv. despite the high antiviral activity of many compounds, the corresponding selectivity index (s. i.) are generally low, due the rather high cytotoxicity exerted on the host cell lines. taking into account the ec 50 a particular interest is deserved by compounds displaying good activities against rsv (as 14 and 20) and bvdv (as 1 and 7) . the former virus is responsible of serious respiratory tract infections in humans, with high mortality rate, while the latter is responsible of severe epidemic outbreaks in livestock, but is also a surrogate model for the evaluation of novel, urgently needed agents against hcv, an emerging threat to human health worldwide. the above compounds represent valuable starting models for developing better (more potent and less toxic) and inexpensive antiviral agents through the variation of the dialkylaminoalkyl chain and/or the substituents on the aromatic rings. in view of these considerations, molecular modeling investigations were performed to study wether the active compounds of structures a-c could target the bvdv rna-dependent rna-polymerase (rdrp), which shares some structural similarity with hcv rdrp. indeed a good agreement was observed between the trend exhibited by the ic 50 (calculated from the estimated free energies of binding) and the corresponding biological activities determined for these compounds in bvdv infected mdbk cell line. the overall sequence identity between bvdv and hcv rdrp proteins is approximately 30%, but the important region k263-k266 in the bvdv rdrp is highly conserved in the corresponding hcv polymerase, thus targeting of hcv rdrp by the a-c molecular series could be expected, even if with a minor potency in respect to the bvdv counterpart. a solution of arylamine (8 mmol) in 1.56 ml of conc. hydrochloridric acid and 1 ml of water was cooled to 0°c and slowly diazotized with a solution of sodium nitrite (8 mmol) in 2 ml of water. the diazonium salt solution was added dropwise, during about 10-20 min, to a cold water solution of the n-(3-dimethylaminopropyl/lupinyl)aniline monoacetate (8 mmol) , and maintaining carefully ph 7 with the simultaneous addition of a saturated solution of sodium acetate. after stirring the reaction mixture for 45 min at 0-5°c, the oil product was extracted with et 2 o, dried, then vacuum-distilled (0.1-0.2 torr) at 80-90°c to remove the unreacted n-substituted aniline. the residue was fractionated by cc, eluting firstly with et 2 o the triazene derivative (e) and then with et 2 o + 5%-meoh the isomeric azocompound (a) in quite lower yield. the isolated compounds were further purified either by crystallization or, when oily, by a second cc or distillation. the aromatic amine (25 mmol) was dissolved in 7.3 ml of conc. hydrochloridric acid and 10 ml of water, cooling in a ice bath. the solution was kept at 0-5°c, while adding drop by drop a solution of sodium nitrite (25 mmol) in 10 ml of water. the diazonium salt was then coupled with the relevant amine (25 mmol) dissolved in 45 ml of 1 n naoh (ph 7-8). after 20 min of stirring, the precipitate was collected by filtration. the solid was crystallized from the suitable solvent. in the case of compounds 14, 18 and 19 the proper oily aniline was added to the diazonium salt solution, followed by a solution of sodium acetate to reduce acidity. when the coupling-product separated as an oil (compounds 37, 43, 53) it was extracted with et 2 o and after removing the solvent the oil was purified by cc. the cytisine derivative 58 was extracted with chcl 3 , and the solid obtained was crystallized from acetone. cell lines were purchased from american type culture collection (atcc). the absence of mycoplasma contamination was checked periodically by the hoechst staining method. cell lines supporting the multiplication of rna and dna viruses were the following: cd4 + human t-cells containing an integrated htlv-1 genome (mt-4); madin darby bovine kidney (mdbk); baby hamster kidney (bhk-21) and monkey kidney (vero 76) cells. for cytotoxicity tests, run in parallel with antiviral assays, mdbk and bhk cells were resuspended in 96 multiwell plates at an initial density of 6 â 10 5 and 1 â 10 6 cells/ml, respectively, in maintenance medium, without or with serial dilutions of test compounds. cell viability was determined after 48-96 h at 37°c in a humidified co 2 (5%) atmosphere by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (mtt) method. 38 vero 76 cells were resuspended in 24 multiwell plates at an initial density of 4 â 10 5 cells/ml. the cell number of vero 76 monolayers was determined by staining with the crystal violet dye. for cytotoxicity evaluations, exponentially growing cells derived from human hematological tumors [cd4 + human t-cells containing an integrated htlv-1 genome (mt-4)] were seeded at an initial density of 1 â 10 5 cells/ml in 96 well plates in rpmi-1640 medium supplemented with 10% fetal calf serum (fcs), 100 units/ml penicillin g and 100 lg/ml streptomycin. cell cultures were then incubated at 37°c in a humidified, 5% co 2 atmosphere in the absence or presence of serial dilutions of test compounds. cell viability was determined after 96 h at 37°c by the mtt method. activity of compounds against human immunodeficiency activity of compounds against yellow fever virus (yfv) and reo virus type-1 (reo-1) was based on inhibition of virus-induced cytopathogenicity in acutely infected bhk-21 cells. activities against bovine viral diarrhoea virus (bvdv), in infected mdbk cells, were also based on inhibition of virus-induced cytopathogenicity. bhk and mdbk cells were seeded in 96-well plates at a density of 5 â 10 4 and 3 â 10 4 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37°c in a humidified co 2 (5%) atmosphere. cell monolayers were then infected with 50 ll of a proper virus dilution (in serum-free medium) to give a m.o.i = 0.01. one hour later, 50 ll of mem earle's medium, supplemented with inactivated fetal calf serum (fcs), 1% final concentration, without or with serial dilutions of test compounds, were added. after 3-4 days of incubation at 37°c, cell viability was determined by the mtt method. activity of compounds against coxsackie virus type b2 (cvb-2), polio virus type-1 sabin strain (sb-1), vesicular stomatitis virus (vsv), vaccinia virus (vv), herpes virus 1 (hsv-1) and respiratory syncytial virus (rsv), a-2 strain, in infected vero 76 cells, was determined by plaque reduction assays in vero 76 cell monolayers. to this end, vero 76 cells were seeded in 24-well plates at a density of 2 â 10 5 cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium at 37°c in a humidified co 2 (5%) atmosphere. then, monolayers were infected with 250 ll of proper virus dilutions to give 50-100 pfu/well. following removal of unadsorbed virus, 500 ll of dulbecco's modified eagle's medium supplemented with 1% inactivated fcs and 0.75% methyl-cellulose, without or with serial dilutions of test compounds, were added. cultures were incubated at 37°c for 2 (sb-1 and vsv), 3 (cvb-2, vv and hsv-1) or 5 days (rsv) and then fixed with pbs containing 50% ethanol and 0.8% crystal violet, washed and air-dried. plaques were then counted. ec 50 (50% effective concentration) was calculated by linear regression technique. et al. coupling algorithm 47 with separate coupling of the solute and solvent to the heat, an integration time step of 2 fs, and the applications of the shake algorithm to constrain all bonds to their equilibrium values, thus removing high frequency vibrations. longrange nonbonded van der waals interactions were truncated by using a dual cutoff of 9 and 13 å, respectively, where energies and forces due to interactions between 9 and 13 å were updated every 20 time steps. the particle mesh ewald method was used to treat the long-range electrostatics. for the calculation of the binding free energy between the rdrp and each inhibitor in water, a total of 100 snapshots were saved during the md data collection period described above, one snapshot per each 0.1 ns of md simulation. the binding free energy dg bind of each rdrp/drug complex in water was calculated according to the procedure termed molecular mechanic/poisson-boltzmann surface area (mm/pbsa), and originally proposed by srinivasan et al. 48 in the mm/pbsa framework of theory, the binding free energy of a given ligand to its receptor protein can be evaluated as the individual terms of the mm/pbsa approach that contribute to the free energy of a molecule are where e mm denotes the sum of intra-and intermolecular mechanical (mm) energies of a molecule in the gas phase, g solv is its solvation free energy, and -ts solute represents an estimate of the solute entropy. e mm can be further divided into terms arising from electrostatic (e el ), van der waals (e vdw ), and internal (e int ) (i.e., bond, angle, and torsional energies): the solvation free energy: consists of a polar solvation energy component, g pb , which is calculated in a continuum solvent, usually a finite-difference poisson-boltzmann (pb) model, and a nonpolar term, g np , which is proportional to the solvent-accessible surface area (sa). the ensemble of structures for the uncomplexed reactants are generated either running separate md simulations for them, or by using the trajectory of the complex, simply removing the atoms of the protein or ligand. in this work we applied the latter variant. accordingly, the term e int in eq. 2 cancels out in the calculation of the free energy of binding. the calculations of the polar solvation term g pb were done with the delphi package, 49 with interior and exterior dielectric constants equal to 1 and 80, respectively. a grid spacing of 2/å, extending 20% beyond the dimensions of the solute, was employed. the non-polar component g np was obtained using the following relationship: g np = csa + b, in which c = 0.00542 kcal/(mol å 2 ), b = 0.92 kcal/mol, and the surface area was estimated by means of the msms software. 50 the last parameter in eq. 1, that is, the change in solute entropy upon association -ts solute , was calculated through normal-mode analysis. in the first step of this calculation, an 8-å sphere around the ligand was cut out from an md snapshot for each ligand-protein complex. this value was shown to be large enough to yield converged mean changes in solute entropy. on the basis of the size-reduced snapshots of the complex, we generated structures of the uncomplexed reactants by removing the atoms of the protein and ligand, respectively. each of those structures was minimized, using a distance-dependent dielectric constant e = 4r, to account for solvent screening, and its entropy was calculated using classical statistical formulas and normal mode-analysis. to minimize the effects due to different conformations adopted by individual snapshots we averaged the estimation of entropy over 50 snapshots. -tolylazo)phenylpropane-1,3-diamine (6) yield: 10%. mp 78-79°c (et 2 o) 96 (s, ch 3 ) bp (p = 0.1-0.2 torr) 100-105°c 3-trifluoromethylphenyl)-3-phenyltriazen-3-yl]-3-propanamine (26) yield: 13%. oil (cc: al 2 o 3 , et 2 o) et 2 o + 2%et 2 nh). 1 h nmr (cdcl 3 ): 1.83 (q, 2h, c(2)); 2.20 (s, n(ch 3 ) 2 ); 2.31 (t tlc: r f = 0.7 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr anal. calcd for c 17 h 21 n 5 o 2 : c, 62 tlc: r f = 0.8 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr found: c, 62 tlc: r f = 0.7 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr found: c, 64.30 n-dimethyl-3-propanamine (30) yield: 57%. oil (cc: al 2 o 3 , et 2 o) tlc: r f = 0.7 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr found: c, 63.98 1-phenyl-3-(3 0 -trifluoromethylphenyl)triazene (14) yield: 92%. mp 95-96°c (hexane). 1 h nmr 5-difluorophenyl)-3-phenyltriazene (18) yield: 30%. mp 125-126°c (pentane). 1 h nmr 0 -trifluoromethylphenyl) triazene (19) yield: 61%. mp 103-104°c (hexane). 1 h nmr 5°c (hexane). 1 h nmr (cdcl 3 ): 1.96 (br s, 4h, c(3, 4)); 3.73 (br s, 4h, c(2, 5)) oil (cc: al 2 o 3 , et 2 o) 1 h nmr calcd for c 11 h 12 f 3 n 3 : c, 54.32 5-difluorophenyl)azopyrrolidine (43) yield: 89%. oil (cc: al 2 o 3 , et 2 o). 1 h nmr calcd for c 10 h 11 f 2 n 3 : c, 56 4-dichlorophenyl)azopyrrolidine (44) yield: 65%. mp 63-65°c (hexane). 1 h nmr anal. calcd for c 10 h 11 found: c, 49 -chlorophenyl)azopiperidine (46) yield: 30%. mp 32-33°c (hexane). 1 h nmr calcd for c 11 h 14 cln 3 : c, 59 -bromophenyl)azopiperidine (47) yield: 28%. mp 30°c (hexane). 1 h nmr mp 33-35°c (hexane). 1 h nmr 5-difluorophenylazo)piperazine (53) yield: 71%. oil (cc: al 2 o 3 , et 2 o). 1 h nmr mp 74-75°c (hexane). 1 h nmr found: c, 60 financial support from italian miur (firb rbne01j3sk01) and biomedicine project is gratefully acknowledged. the authors thanks o. gagliardo for performing the elemental analyses. all molecular dynamics simulations were carried out using the sander and pmemd module within the amber 9 suite of programs, 39 and the parm99 all-atom force field, 40 working in parallel on 32 processors of the tartaglia cluster at the university of trieste (trieste, italy). the crystallographic coordinates of the rna-dependent rna-polymerase (rdrp) of bvdv (pdb entry 1s48.pdb) 31 and hcv (pdb entry 1csj.pdb) 28 were employed as starting geometries for protein simulations in complex with the most active compounds (1, 2, 4, 7-10). missing hydrogen atoms were added to the protein backbone and side chains with leap module of amber 9. all ionizable residues were considered in the standard ionization state at neutral ph. the geometry of added hydrogens and ions was refined for 200 steps (steepest descent) in vacuum using the parm94 force field. 40 further protein geometry refinement was carried out using the sander module of amber 9 via a combined steepest descent-conjugate gradient algorithm, using as a convergence criterion for the energy gradient the root-mean-square of the cartesian elements of the gradient equal to 0.01 kcal/(mol å). the generalized born/surface area (gb/sa) continuum solvation model 41 was used to mimic a water environment. as expected, no relevant structural changes were observed between rdrp relaxed models and the original 3-d structure.the putative binding site for our compounds on the bvdv rdrp was determined using the activesite_search option of the binding site module of insightii (v. 2001, accelrys, san diego, usa). 33 active-site_search identifies protein active sites or binding sites by locating cavities in the protein structure. according to the site_search algorithm employed, the protein is first mapped onto a grid which covers the complete protein space. the grid points are then defined as free points and protein points. the protein points are grid points, within 2 å from a hydrogen atom or 2.5 å from a heavy atom. then, a cubic eraser moves from the outside of the protein toward the center to remove the free points until the opening is too small for it to move forward. those free points not reached by the eraser will be defined as site points. after a site is located, it can be modified by expanding or contracting the site. one layer of grid points at the cavity opening site will be added or removed by each expand or contract operation, respectively.the model structures of all ligand molecules were built and subjected to an initial energy minimization. the convergence criterion was set to 10 à4 kcal/(mol å). a conformational search was carried out using a well-validated, ad-hoc developed combined molecular mechanics/molecular dynamics simulated annealing (mdsa) protocol. 33, 34 accordingly, the relaxed structures were subjected to five repeated temperature cycles (from 310 k to 1000 k and back) using constant volume/constant temperature (nvt) md conditions. at the end of each annealing cycle, the structures were again energy minimized to converge below 10 à4 kcal/ (mol å), and only the structures corresponding to the minimum energy were used for further modeling. the atomic partial charges for the geometrically optimized compounds were obtained using the resp procedure, 42 and the electrostatic potentials were produced by single-point quantum mechanical calculations at the hartree-fock level with a 6-31g * basis set, using the merz-singh-kollman van der waals parameters. 43 eventual missing force field parameters for the compounds considered were generated as follows: am1 geometry optimization of the structure was followed by rhf/6-31g * single point calculation to obtain the electrostatic potentials. next, the resp method was used for charge fitting. the missing bond, angle torsion or van der waals parameters not included in the parm99 were generated using the antechamber module of amber 9.the optimized structures of the inhibitors were docked into the putative enzyme allosteric binding site by applying a consolidated procedure; 33, 34 accordingly, it will be briefly reported below. the software autodock 3.0 44 was employed to estimate the possible binding orientations of all compounds in the receptor. in order to encase a reasonable region of the protein surface and interior volume, centered on the binding site, the grids were 60 å on each side. grid spacing (0.375 å), and 120 grid points were applied in each cartesian direction so as to calculate mass-centered grid maps. am-ber 12-6 and 12-10 lennard-jones parameters were used in modeling van der waals interactions and hydrogen bonding (n-h, o-h and s-h), respectively. in the generation of the electrostatic grid maps, the distance dependent relative permittivity of mehler and solmajer was applied. 45 for the docking of each compound to the proteins, three hundred monte carlo/simulated annealing (mc/ sa) runs were performed, with 100 constant temperature cycles for simulated annealing. for these calculations, the gb/sa implicit water model 41 was used to mimic the solvated environment. the rotation of the angles u and /, and the angles of side chains were set free during the calculations. all other parameters of the mc/ sa algorithm were kept as default. following the docking procedure, the structure of all compounds were subjected to cluster analysis with a tolerance of 1 å for an all-atom root-mean-square deviation from a lower-energy structure representing each cluster family. in the absence of any relevant crystallographic information for our compounds, the structure of each resulting complex characterized by the lowest interaction energy in the prevailing cluster was selected for further evaluation.each best substrate/rdrp complex resulting from the automated docking procedure was further refined in the amber 9 suite using the quenched molecular dynamics (qmd) method. 33, 34 in this case, 1 ns md simulation at 310 k were employed to sample the conformational space of the substrate-enzyme complex in the gb/sa continuum solvation environment. the integration step was equal to 1 fs and the parm99 force field parameters were applied in the simulations. after each ps, the system was cooled to 0 k, the structure was extensively minimized, and stored. to prevent global conformational changes of the enzyme, the backbone of the protein binding site was constrained by a harmonic force constant of 100 kcal/å, whereas the amino acid side chains and the ligands were allowed moving without any constraint.the best energy configuration of each complex resulting from the previous step was allowed to relax in a 55-å radius sphere of tip3p water molecules. 46 the resulting system was minimized with a gradual decrease in the position restraints of the protein atoms. at the end of the relaxation process, all water molecules beyond the first hydration shell (i.e., at a distance > 3.5 å from any protein atom) were removed. finally, to achieve electroneutrality, a suitable number of counterions were added to neutralize the system using the leap module of amber 9, removing eventually overlapping water molecules. to reduce computational time to reasonable limits, all proteins residues with any atom closer than 20 å from the center of mass of each bounded ligand were chosen to be flexible in the dynamic simulations. subsequently, a spherical tip3p water cap of radius equal to 20 å was centered on each inhibitor in the corresponding rdrp complex, including the hydrating water molecules within the sphere resulting from the previous step. after energy minimization of the new water cap for 10 ps, keeping the protein, the ligand, and the pre-existing waters rigid, followed by a md equilibration of the entire water sphere with fixed solute for 1 ns, further unfavorable interactions within the structures were relieved by progressively smaller positional restraints on the solute (from 25 to 0 kcal/(mol å 2 ) for a total of 2 ns. each system was gradually heated to 310 k in three intervals, allowing a 1 ns interval per each 100 k, and then equilibrated for 5 ns at 310 k, followed by 10 ns of data collection runs, necessary for the estimation of the free energy of binding (vide infra). the md simulations were performed at constant t = 310 k using the berendsen supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmc.2009.05.020. key: cord-281883-l9yshyc7 authors: alekseeva, ekaterina; sominskaya, irina; skrastina, dace; egorova, irina; starodubova, elizaveta; kushners, eriks; mihailova, marija; petrakova, natalia; bruvere, ruta; kozlovskaya, tatyana; isaguliants, maria; pumpens, paul title: enhancement of the expression of hcv core gene does not enhance core-specific immune response in dna immunization: advantages of the heterologous dna prime, protein boost immunization regimen date: 2009-06-08 journal: genet vaccines ther doi: 10.1186/1479-0556-7-7 sha: doc_id: 281883 cord_uid: l9yshyc7 background: hepatitis c core protein is an attractive target for hcv vaccine aimed to exterminate hcv infected cells. however, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. we aimed to design an hcv vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. methods: plasmids encoding core with no translation initiation signal (pcmvcore); with kozak sequence (pcmvcorekozak); and with hcv ires (pcmvcoreires) were designed and expressed in a variety of eukaryotic cells. polyproteins corresponding to hcv 1b amino acids (aa) 1–98 and 1–173 were expressed in e. coli. c57bl/6 mice were immunized with four 25-μg doses of pcmvcorekozak, or pcmv (i). balb/c mice were immunized with 100 μg of either pcmvcore, or pcmvcorekozak, or pcmvcoreires, or empty pcmv (ii). lastly, balb/c mice were immunized with 20 μg of core aa 1–98 in prime and boost, or with 100 μg of pcmvcorekozak in prime and 20 μg of core aa 1–98 in boost (iii). antibody response, [(3)h]-t-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. results: plasmids differed in core-expression capacity: mouse fibroblasts transfected with pcmvcore, pcmvcoreires and pcmvcorekozak expressed 0.22 ± 0.18, 0.83 ± 0.5, and 13 ± 5 ng core per cell, respectively. single immunization with highly expressing pcmvcorekozak induced specific ifn-γ and il-2, and weak antibody response. single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong t-cell proliferation (pcmvcoreires), and antibodies in titer 10(3)(pcmvcore). boosting with pcmvcorekozak induced low antibody response, core-specific t-cell proliferation and ifn-γ secretion that subsided after the 3rd plasmid injection. the latter also led to a decrease in specific il-2 secretion. the best was the heterologous pcmvcorekozak prime/protein boost regimen that generated mixed th1/th2-cellular response with core-specific antibodies in titer ≥ 3 × 10(3). conclusion: thus, administration of highly expressed hcv core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. instead, the latter is induced by a heterologous dna prime/protein boost regimen that circumvents the negative effects of intracellular core expression. (pcmvcoreires), and antibodies in titer 10 3 (pcmvcore). boosting with pcmvcorekozak induced low antibody response, core-specific t-cell proliferation and ifn- secretion that subsided after the 3rd plasmid injection. the latter also led to a decrease in specific il-2 secretion. the best was the heterologous pcmvcorekozak prime/protein boost regimen that generated mixed th1/th2cellular response with core-specific antibodies in titer  3 × 10 3 . thus, administration of highly expressed hcv core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. instead, the latter is induced by a heterologous dna prime/protein boost regimen that circumvents the negative effects of intracellular core expression. globally, an estimated 170 million people are chronically infected with hepatitis c virus (hcv), and 3 to 4 million persons are newly infected each year [1, 2] . the human immune system has difficulties in clearing the virus in either the acute, or chronic phase of the infection with up to 40% of patients progressing to cirrhosis and liver failure [3] [4] [5] [6] . extensive studies have unraveled important reliable correlates of viral clearance [7] [8] [9] [10] [11] . this, together with the growing need to diminish the magnitude of hcv associated liver disease served as a basis for intensive hcv vaccine research. a series of hcv vaccine candidates have moved into clinical trials [11] . one such is the peptide vaccine ic41 consisting of a panel of mhc class i and class ii restricted epitopes adjuvanted by poly-l-arginine administered to healthy volunteers [12] and to chronic hcv patients including non-responders to the standard therapy [13, 14] . another therapeutic vaccine employed peptides chosen individually for their ability to induce the strongest in vitro cellular response [15] . in a further vaccine trial, chronic hepatitis c patients received the recombinant hcv envelope protein e1 [16] . the first clinical trial of an hcv dna vaccine consisting of a codon-optimized ns3/4a gene administered to chronic hepatitis c patients is currently ongoing (chronvac-c ® ; http:// www.clinicaltrials.gov/ct2/results?term=nct00563173; http://www.bion.no/moter/vaccine/ matti_s%e4llberg.pdf). so far, none of the peptide or protein vaccines were able to induce a significant improvement in the health conditions of chronic hcv patients, or a significant decrease of hcv rna load, specifically if compared to the conventional ifn-based therapy [13, 15, 16] . the vaccine trials have, however, demonstrated that when achieved, hcv rna decline in the vaccine recipients correlates with induction of strong ifn-gamma t-cell response [13] . such a response can best be recruited by dna vaccines, either alone or with the aid of heterologous boosts [11, 17] . indeed, vaccination of chimpanzees showed the ability to elicit effective immunity against heterologous hcv strains using t-cell oriented hcv genetic vaccines that stimulated only the cellular arm of the immune system [17, 18 ]. an attractive target for hcv vaccine is the nucleocapsid (core) protein [19] [20] [21] . it is highly conserved among various hcv genotypes with amino acid homology exceeding 95% [21, 22] . core binds and packages the viral genomic rna, regulates its translation [23] [24] [25] [26] and drives the production of infectious viruses [27] [28] [29] . core contributes to hcv persistence also indirectly by interfering with host cell transcription, apoptosis, lipid metabolism, and the development of immune response [30] [31] [32] [33] . extermination of core expressing cells and inhibition of the activity of extracellular core (non-enveloped particles containing hcv rna [34]) could be highly beneficial. ideally, hcv core could be eliminated by a specific vaccine-induced immune response. it is a strong immunogen with anti-core immune response evolving very early in infection [35, 36] . early and broad peripheral and intrahepatic cd8+ t-cell and antibody response to core/core epitopes is registered in chimpanzees controlling hcv infection hcv, but not in chimpanzees that become chronically infected [37] [38] [39] . in mice, potent experimentally induced anti-core immune response conferred partial protection against challenge with core expressing recombinant vaccinia virus [40] . however, despite high immunogenicity in the natural infection, core does not perform well as an immunogen, specifically if introduced as naked dna [2, [41] [42] [43] . attempts to enhance core immunogenicity by targeting hcv core protein to specific cellular compartments [44] , co-immunization with cytokine expressing plasmids [2, 41] , adjuvants as cpg [45] , or truncated core gene versions [46] had limited or no success. prime-boost strategies have been used to increase immune responses to a number of dna vaccines. immunization regimens comprised of a dna prime and a viral vector boost for instance for vaccinia virus [47, 48] , adenovirus [49] , fowlpox [50, 51] , and retrovirus [52] . priming with dna and boosting with protein is another promising approach. this regimen has been studied for hiv [53, 54] , hepatitis c virus [55, 56] , anthrax [57] , mycobacteria [58, 59] , streptococcus pneumoniae [60] and bvdv [61] . dna vaccines and recombinant protein vaccines utilize different mechanisms to elicit antigen-specific responses. due to the production of antigen in transfected cells of the host, a dna vaccine induces robust t-cell responses, which are critical for the development of t-cell-dependent antibody responses [62] . dna immunization is also highly effective in priming antigen-specific memory b cells. in contrast, a recombinant protein vaccine is generally more effective at eliciting antibody responses than cell-mediated immune responses and may directly stimulate antigen-specific memory b cells to differentiate into antibody-secreting cells, resulting in production of high titer antigen-specific antibodies [63] . therefore, a dna prime plus protein boost is a complementary approach that overcomes each of their respective shortcomings. certain improvement of the immune response was reached after co-delivery of hcv core dna and recombinant core [2, 40, 64] . in this study, we have shown that in dna immunization, poor core-specific immune response can be a consequence of high levels of intracellular core expression, and that such a response can be improved by using low-expressing core genes, or single core gene primes in combination with recombinant core protein boosts. region encoding aa 1-191 of hcv core was reverse-transcribed and amplified from hcv 1b isolate 274933ru (genebank accession #af176573) [65] using oligonucleotide primers: sense gatccaagcttatgagcac-gaatcc and antisense gatccctcgagtcaagcggaagctgg containing recognition sites of hindiii and xhoi restriction endonucleases. the amplified dna was cleaved with hindiii/xhoi and inserted into pcdna3 (invitrogen, usa) cleaved with hindiii/xhoi resulting in pcmvcore. region encoding aa 1-191 of hcv core was also reverse-transcribed and amplified from hcv isolate 274933ru using another set of primers that carried kozak consensus sequence sense agctgctagcgccgccaccatgagcacgaatcct and antisense gatcgttaactaagcggaagctggatgg primers containing recognition sites of restriction endonucleases nhei and xhoi, respectively. the amplified dna was cleaved with nhei/kspai and inserted into the plasmid pcmve2/p7-2 [66] cleaved with nhei/xhoi, resulting in pcmvcorekozak. the region corresponding to hcv 5'utr, and coding sequences for aa 1-809 was reverse-transcribed and amplified from hcv 1b isolate ad78p1 (genebank accession #aj132997) [67] , kindly provided by prof. m. roggendorf (essen, germany) using sense-gacccaagcttcgtagaccgtgcaccat and antisense catgctcgagttaggcgtatgctcg primers. the amplified dna was cleaved with hindiii/xhoi and inserted into pcdna3 cleaved with hindiii/xhoi resulting in pcmvcoreires. hcv 274933ru core differed from hcv ad78p1 core in positions 70 (h versus r), 75 (t versus a), and 147 (v versus t), respectively. growth of pcdna3, pcmvcore, pcmvcorekozak, and pcmvcoreires was accomplished in the e. coli strain dh5alpha. plasmid dna was extracted and purified by endo free plasmid maxi kit (qiagen gmbh, germany). the purified plasmids were dissolved in the phosphate buffered saline (pbs) and used for in vitro expression assays and for dna immunization. bhk-21, cos-7, and nih3t3 cells were seeded into plates (3 × 10 5 cells/well) and transfected by plasmid dna (2 g) using lipofectamine (gibco-brl, scotland) or exgen 500 (fermentas, lithuania) as described by the manufacturers. hcv core expression was analyzed 24, 48 and 72 h post transfection. cells were lysed for 10 min at 0°c in the buffer containing 50 mm tris-hcl, ph 7.5, 1 mm edta, 1 mm pmsf and 1% np-40. lysates were cleared by 10 min centrifugation at 6000 g, resolved by 12% sds-paag, and transferred to pvdf membranes (amersham pharmacia biotech, ireland). hcv core expression was detected by immunostaining with polyclonal rabbit anti-core antibodies [68] , and secondary horseradish peroxidase (hrp)-conjugated anti-rabbit immunoglobulins (amersham pharmacia biotech, ireland) followed by ecl™ detection (ecl plus, amersham pharmacia biotech, ireland). nih3t3 cells were transfected with either pcdna3, pcm-vcore, pcmvcorekozak, pcmvcoreires, or pegfp-n1 (clontech, ca, usa). the percent of transfection was evaluated by counting the number of gfp expressing cells per 500 transfected nih3t3 cells using a fluorescence leica dm 6000 microscope (leica camera ag, germany). cells were harvested 48 h post-transfection, counted, and 10 4 cells were lysed in 2× sds sample buffer. lysates and samples containing 1 to 50 ng of recombinant core aa 1-173 (corresponding to p21) were run simultaneously on 12% sds-paag and transferred onto pvdf membrane for calibration. blots were blocked overnight in pbs-t with 5% non-fat dry milk, stained with polyclonal anti-core antibodies #35-6 (1:5000) followed by the secondary antirabbit hrp-conjugated antibodies (dakopatts ab, denmark). signals were detected using the ecl™ system (amersham pharmacia biotech, ireland). x-ray films were scanned, and processed using image j software http:// rsb.info.nih.gov/ij. the data are presented as the mean grey values (mgv). the core content was quantified by plotting the mgv of each sample onto a calibration curve prepared using recombinant core aa 1-173. after core detection, blots were striped according to the ecl protocol and re-stained with monoclonal anti-tubulin antibod-ies (sigma, usa) and secondary anti-mouse hrpconjugated antibodies (dakopatts ab, denmark). core content per transfected cell was evaluated after accounting for the percent of transfection and normalization to the tubulin content per well. immunofluorescence staining bhk-21 cells were seeded on the chamber slides (nunc international, denmark) and transfected as above. 24 h post transfection, the slides were dried, fixed with acetic acid and ethanol (1:3) for 15 min and rinsed thoroughly in distilled water. fixed cells were re-hydrated in pbs, and incubated for 24 h at 4°c with anti-hcv core rabbit polyclonal antibodies (1:50) in the blocking buffer (pbs with 2.5 mm edta and 1% bsa). secondary antibodies were goat anti-rabbit immunoglobulins labeled with tritc (1:200; dako, denmark). slides were then mounted with permafluor aqueous mounting medium (immunon, pa., usa) and read using a fluorescence microscope. peptides covering core amino acids 1-18, 1-20, 23-43, 34-42, 133-142 and a control peptide ttavpwnas from gp41 of hiv-1 were purchased from thermo electron gmbh (germany). core proteins representing aa 1-152 of hcv 274933ru and aa 1-98, and 1-173 of ad78p1 were expressed in e. coli and purified by chromatography as was described earlier [69, 70] . purified proteins were dissolved in pbs. the following immunizations were performed: scheme i groups of 12 female 8-week old c57bl/6 mice (stolbovaya, moscow region, russia) were immunized with a total of 100 g of pcmvcorekozak, or empty vector, split into four i.m. injections done with 3-4 week intervals. control mice were mock-immunized with pbs. scheme ii female 6-8 week old balb/c mice (animal breeding centre of the institute of microbiology and virology, riga) had injected into their tibialis anterior (ta), 50 l of 0.01 mm cardiotoxin (latoxan, france) in sterile 0.9% nacl five days prior to immunization. groups of 6 to 7 mice were immunized with a single 100 g dose of either pcm-vcoreires, or pcmvcore, or pcmvcorekozak, or empty vector, all dissolved in 100 l pbs, applied intramuscularly (i.m.) into the cardiotoxin-treated ta. control mice were left untreated. groups of 5 to 6 female 6-8 week old balb/c mice pretreated with cardiotoxin, were injected i.m. with 100 g of pcmvcorekozak and boosted three weeks later with 20 g of core aa 1-98 in pbs, or primed and boosted subcutaneously with 20 g of core aa 1-98 in pbs. control animals were left untreated. mice were bled from retro-orbital sinus prior to, and 2 to 3 weeks after each immunization, or 5 weeks post a single gene immunization (in scheme ii). peptides corresponding to core aa 1-20, 23-43 or 133-142 were coated onto 96-well maxisorp plates (nunc, denmark), and recombinant core aa 1-98, 1-152, or 1-173, on the 96-well polysorp plates (nunc, denmark). coating was done overnight at 4°c in 50 mm carbonate buffer, ph 9.6 at antigen concentration of 10 g/ml. after blocking with pbs containing 1% bsa for 1 h at 37°c, serial dilutions of mouse sera were applied on the plates and incubated for an additional hour at 37°c. incubation was followed by three washings with pbs containing 0.05% tween-20. afterwards, plates were incubated with the horseradish peroxidase-conjugated anti-mouse antibody (sigma, usa) for 1 h at 37°c, washed, and substrate opd (sigma, usa) added for color development. plates were read on an automatic reader (multiscan, sweden) at 492 nm. elisa performed on plates coated with core aa 1-98, 1-152, or 1-173 showed similar results (data not shown). immune serum was considered positive for anti-core antibodies whenever a specific od value exceeded, by at least two-fold, the signals generated by: pre-immune serum reacting with core-derived antigen, and by immune serum reacting with bsa-coated plate, the assays performed simultaneously. for t-cell proliferation tests, mice were sacrificed and spleens were obtained two weeks after each immunization in scheme i; and three and five weeks after the last immunization in schemes ii and iii. murine splenocytes were harvested using red blood cell lysing buffer (sigma, usa), single cell-suspensions were prepared in rpmi 1640 supplemented with 2 mm l-glutamine and 10% fetal calf serum (gibco brl, scotland) at 6 × 10 6 cells/ml. cell were cultured in u-bottomed microculture plates at 37°c in a humidified 5% co 2 chamber (gibco, germany). cell stimulation was performed with peptides representing core aa 1-20, 23-43, 34-42 and recombinant core aa 1-98, 1-152, and aa 1-173 at dilutions to 3.1, 6.25, 12.5, 25.0, 50.0, and 100 g/ml, all in duplicate. concanavalin a (cona) was used as a positive control at 2 g/ml. cells were grown for 72 h, after which [ 3 h]-thymidine (1 ci per well; amersham pharmacia biotech, ireland) was added. after an additional 18 h, cells were harvested onto cellulose filters and the radioactivity was measured on a beta counter (beckman, usa). the results were presented as stimulation indexes (si), which were calculated as a ratio of mean cpm obtained in the presence and absence of a stimulator (protein or peptide). emptyvector immunized and control mice showed si values of 0.8 ± 0.4. si values  1.9 were considered as indicators of specific t-cell stimulation. for detection of cytokines, cell culture fluids from t-cell proliferation tests were collected, for il-2 -24 h, and for il-4 and ifn- -48 h post the on-start of t-cell stimulation. detection of cytokines in the cell supernatants was performed using commercial elisa kits (pharmingen, bd biosciences, ca, usa) according to the manufacturers' instructions. plasmids were constructed encoding core of hcv 1b isolate 274933ru without translation initiation signals (pcmvcore); and with kozak translation initiation signal (pcmvcorekozak). core with viral translation initiation signal ires taken in the natural context was derived from hcv 1b isolate ad78p1 [67] . viral cores had a minimal sequence difference in positions 70, 75, and 147, all three cases representing homologous substitutions. expression from these plasmids was tested both in vitro and in cell cultures. plasmids pcmvcore and pcmvcore-kozak were used as the templates for the t7-driven mrna transcription; mrna was translated in vitro in the rabbit reticulocyte lysate system. both mrnas generated a translation product of approximately 23 kda corresponding to the molecular mass of unprocessed hcv core (p23; data not shown). next, core-expressing vectors were used to transfect a series of mammalian cell lines. western blotting of bhk-21 and cos-7cells transfected with pcmvcore, pcmvcorekozak and pcmvcoreires using corespecific antibodies demonstrated an accumulation of proteins with the expected molecular mass of 21 kda that corresponds to core aa 1-171 cleaved from the full-length core by cellular proteases [71, 72] (fig. 1) . minimal amounts of p23 were also detected, specifically after transfections of bhk-21 with pcmvcore and pcmvcoreires (fig. 1) . the overall level of hcv core synthesis in bhk-21 cells was somewhat higher than in cos-7 cells (fig. 1) . in both cell lines, the highest level of core expression was achieved with pcmvcorekozak ( fig. 1, 2) . all cells expressing core and immunostained with core-specific antibodies demonstrated cytoplasmic granular staining characteristic of the processed p21 form of hcv core [72] [73] [74] (fig. 2) . the expression capacity of the vectors was quantified in murine fibroblasts to reproduce dna immunization that was to be done in mice. core expression was assessed on western blots of sds-paag resolving lysates of nih3t3 transfected with core expressing and control plasmids ( fig. 3a and 3b) . images of western blots were processed using the imagej software and individual bands were represented in arbitrary units (mean grey values, mgv). their correspondence to core quantity was established using calibration curves built with the use of recombinant core aa 1-173 (see additional file 1) after normalization to the percent of transfection and protein content of the samples. plasmid pcmvcore with no translation initiation signals provided the lowest level of core expression (fig. 3b) . ires promoted a two-fold increase, and the kozak sequence, a 35-fold increase of core expression with > 15 ng of protein produced per expressing cell (fig. 3b) . immunization of mice with hcv core dna all plasmids were purified by standard protocols in accordance with a glp practice for preparation of dna vaccines, and used in a series of mouse immunization experiments. plasmid directing the highest level of core expression was selected and a pilot experiment defining the strategy of dna immunization was performed. c57bl/6 mice were immunized four times with 25 g of pcmvcorekozak, and core-specific antibody and cellular responses were evaluated. no specific response was registered after the 1 st injection (data not shown). the immune response generated after the following three boosts is illustrated by fig. 4 . three injections of 25 g led to no increase of core-specific igg response over the initial levels achieved after the first two plasmid injections (fig. 4a) . three plasmid injections generated a better t-cell proliferative response to core and core-derived peptides than two. however, the response could not be boosted further (fig. 4b) . ifn- and il-2 response to core was also boostable. however again, no boosting was seen after the initial two pcmvcorekozak injections (fig. 4c) . furthermore, the repeated injections led to a significant decrease of il-2 secretion in response to splenocyte stimulation by recombinant core and peptides representing core n-terminus (p < 0.05; fig. 4b , and data not shown). core-specific il-4 secretion was not detected. thus, the development of core-specific immune responses occurred within six weeks after the on-start of immunization; repeated boosts with hcv core gene did not lead to a significant enhancement of core-specific immunity. in the next series of experiments, we selected balb/c mice as a strain that is expected to support a better th2-type response with stronger antibody production [75] . plasmid pcmvcore kozak was given as a single 100 g injection with the effect of repeated intramuscular dna boosts substituted by pre-treatment of the injection sites by cardiotoxin [76] . t-cell proliferative response, antibody production and cytokine secretion were monitored two and five weeks after immunization. significant responses in the form of core-specific ifn- and il-2 secretion exceeding the background levels in empty-vector-immunized mice were detected five weeks after a single administration of hcv core gene (fig.5) . immunization generated no core-specific t-cell response and a low titer of core-specific igg. antibody response against hcv core has already been shown to develop slowly [46] , mirroring the development of anti-core antibody response in hcv infected individuals [77] . here as well, a slow increase in the level of anti-core antibodies expression of hcv core proteins plasmids used for nih 3t3 transfection core expression (units) was observed 35 days after a single gene injection as compared to levels detected at day 21 (data not shown). there was no difference between balb/c and c57bl/6 mice with respect to core-specific ifn- secretion (fig. 4c versus fig. 5c ), or core-specific igg production (p > 0.05 mann whitney u-test; fig. 4a versus fig. 5a and additional file 1). the magnitude of anti-core response suggested that the increase of hcv core gene dose either by one-time large dose injection, or by repeated injections of smaller doses, did not significantly enhance core-specific immunity. to delineate if that could be influenced by core expression level, balb/c mice were immunized with a single dose of low-expressing core genes with no translation initiation signals (pcmvcore), or with ires (pcmvcoreires). the results were compared to immunization with core gene regulated by the kozak sequence (pcmvcoreires) (fig. 5) . the t-cell proliferative response to core-and corederived peptides was stronger in mice immunized with pcmvcoreires (fig. 5) . the highest anti-core igg response was raised in mice immunized with pcmvcore that directed the lowest level of hcv core expression ( fig. 3; fig. 5a ). it was significantly higher than the antibody response induced by pcmvcorekozak (p < 0.05); the immune response in pcmvcoreires-immunized mice was intermediate (fig. 5a) . the t-cell proliferative response to core-and core-derived peptides was stronger in mice immunized with pcmvcoreires ( fig. 5b ; p < 0.05). while il-2 secretion was somewhat higher in mice immunized with highly expressing pcmvcorekozak, both dna immunogens provided a similar level of core-specific ifn- secretion (fig. 5c) . we aimed to see if core-specific immune response could be enhanced without increasing core gene doses, but instead by using the heterologous prime-boost immunization regimens. hcv core protein aa 1-98 and pcmv-corekozak were used to immunize balb/c mice either separately, or in the dna prime-protein boost regimen. a high titer of core-specific antibodies was achieved only after the heterologous boost (fig. 6a) . the heterologous regimen effectively induced a proliferative response, both in si values (p levels 0.034, mann whitney u-test) and in the number of positive t-cell proliferation tests (p level 0.014; fig. 6b ); and potent core-specific ifn- and il-2 secretion (fig. 6c) . core-specific il-4 secretion was, in all cases, very low. heterologous regimen induced significant anti-core antibody production (fig. 6a) . sera of mice primed with pcmvcorekozak and boosted with core aa 1-98 were analysed for the presence of anti-core antibodies of igg, igg1, igg2a, igg2b and igm subclasses, and the results were compared to seroreactvivity in mice immunized with single injection of core or core expressing plasmids (fig. 7) . mice primed with pcmvcorekozak and boosted with core protein had significantly higher levels of anti-core igg than mice immunized with pcmvcorekozak (p = 0.0006, mann-whitney u-test) or pcmvcore (p = 0.002) (immunization with pcmvcore gave higher level of igg than immunization with pcmvcorekozak, p < 0.05). group with heterologous prime/boost regimen had also an increased levels of anti-core igg1, although the difference with the control group did not reach the level of significance (p < 0.1). antibodies of igg2a or igg2b subclasses were not found. low specific anti-core igm were observed only in mice immunized with recombinant core aa 1-98 (p < 0.1; fig. 7) . it was higher than in mice primed with core dna and boosted with core protein (p level 0.05). at the same time, core-immunized mice had no anti-core igg1 or igg2 (fig. 7) . thus, the heterologous core dna prime/core protein boost regimen preferentially induced anti-core igg, while protein immunization triggered mostly low-level anti-core igm. the immune response in dna immunization depends on the amount of antigen produced from the immunogen in vivo as predetermined by the gene dose and by the gene capacity to direct an efficient antigen expression [78, 79] . normally, the response increases with the increase of the dose and efficacy of gene expression (for examples, see [80] [81] [82] [83] ). however, the dna immunogen used here encodes not just a structural component of the virus, but also a pathogenic factor. hcv core protein interacts with spectrum of core-specific immune response figure 7 spectrum of core-specific immune response. 39, 71, 84, 85] . of importance for hcv vaccine design was to find to what extent the immune response to hcv core in dna immunization is influenced (positively, or negatively) by the level of core expression as determined by gene dose (i), and gene expression efficacy (ii). the first issue was addressed in a series of immunizations in which the same dose of hcv core was given as a single or split into multiple injections. we and others have earlier observed that repeated hcv core gene boosts do not lead to an enhancement of core-specific immune response [42, 46, 68] . on the contrary, both core-specific ifn- and il-2 production [68] and anti-core antibody response [2, 46, 64] appear to be down-regulated. here as well, the overall comparison between immunizations carried out by single and multiple core gene injections in different mouse strains demonstrated that the outcomes of immunization with one 100 g versus two to four 25 g core gene doses were quite similar ( fig. 4 ; see also the summary in additional file 2). furthermore, antibody response was not boosted; t-cell proliferative response and core-specific ifn- secretion could not be boosted beyond the levels reached after the initial two injections, and core-specific il-2 secretion even appeared to be suppressed. thus, core-specific immune response can be achieved after single dna immunization, while repeated core gene administration may actually suppress core-specific immunity. the issue of translation efficacy was assessed in singledose immunizations with plasmids directing different levels of hcv core expression. there are different ways to increase the level of gene expression efficacy such as the use of strong promoters, optimal species-specific codons, and manipulations with rna folding [78, 78, 79] . an important factor is the efficacy of translation initiation. in the cap-dependent translation of mammalian genes it is determined by sequences flanking the aug initiator codon. high levels of translation are achieved with the kozak sequence, a guanine at position +4 and an adenine at -3 from aug [86, 87] . the alternative mechanisms of initiation site selection on eukaryotic cellular and viral mrnas, also of hcv, include the translation initiation from iress (internal ribosome entry sites/segments) [88] . located in the 5'-utr region of viral genome, hcv ires is optimized to hijack the ribosomes and translation factors from the host for the translation of hcv polyprotein [89] . core is tightly involved in the ires-mediated regulation of hcv translation with several regulatory signals localized in both core protein and core coding sequence [24, 90, 91] . thus, the 5'-end of hcv genome incorporating 5'-utr and core coding sequence were harmonized during evolution to provide for the levels of core expression essential for the virus. both cap and ires translation initiation options were employed in the design of core dna immunogens. eukaryotic expression vectors were constructed encoding core of hcv 1b without translation initiation signal (pcmvcore), core preceded by the 5'-utr of hcv 1b isolate ad (pcmvcoreires), and core preceded by the consensus kozak sequence (pcmvcorekozak). the latter directed the expression of 35-fold more core than the gene devoid of the translation initiation signals, and 16-fold more core than the gene regulated by ires. however, despite a considerable difference in the core expression capacity, kozak-and ires-regulated dna immunogens induced similar levels of core-specific ifn- secretion (fig. 5c ). more so, while il-2 secretion was somewhat higher in mice immunized with highly expressing pcmvcorekozak, a t-cell proliferative response to core-and corederived peptides was stronger in mice immunized with pcmvcoreires ( fig. 5b) . thus, high core expression levels did not promote a better core specific cellular response. dna-based immunization can induce potent antibody response including virus neutralizing antibodies [92] [93] [94] [95] [96] . however, no significant antibody response has ever been induced in core gene immunization unless it was followed by the protein boost [2, 64] . anti-core antibody titers obtained here after immunization with both cap-and ires-regulated core genes were also low. interestingly, however, significantly higher titers of anti-core antibodies were obtained in mice that received the least expressed core gene devoid of any translation regulation signals (pcmv core; figs. 5a, 7). thus, the use of highly expressing hcv core dna did not promote an effective core-specific antibody response. altogether, this points to possible adverse effects of the high-level as well as of the prolonged hcv core gene expression. we have additional data in support of this concept from immunization of c57bl/6 mice with a synthetic truncated hcv core gene devoid of hcv core nucleotide-sequence dependent regulatory signals. the latter expressed hcv 1b core at five to six-fold lower levels than the viral full-length core gene [97] , but nevertheless, was capable of inducing potent core-specific cellular and antibody response http://www.meetingsmanagement.com/ dna_2004/index.htm [98] . dna immunization with antigens co-expressed in natural virus infection can result in inhibition of both protein expression and specific immune response [99] . more so, pathological effects were reported of the repeated immunization with certain microbial genes, for example the hsp60 gene of mycobacterium that causes necrotizing bronchointerstitial pneumonia and bronchiolitis in healthy mouse recipients, and multifocal regions of cellular necrosis in lungs when applied therapeutically [100, 101] . hcv core is the factor of hcv pathogenicity. it activates cellular and viral promoters [102] , induces er-and mitochondrial stress [103, 104] , regulates apoptosis [105, 106] , tumorigenesis [107, 108] , and induces abnormal lipid metabolism [109] . in experimental systems, core expression leads to the development of diverse pathological effects including cd4+ t-cell depletion, liver steatosis, insulin resistance, and hepatocellular carcinoma [33, 110] . one of the notable although controversial features is the capacity of hcv core to suppresses host immunity [32, 39, 84, 85] . these features of hcv core may explain why here a better immune response was achieved after single immunization with vectors providing for comparatively low hcv core expression. altogether, this points to the necessity to devise alternative immunization regimens that would help to circumvent possible adverse effects of hcv core. many approaches can be pursued, with dna vaccination combined with heterologous protein or recombinant viral boosts considered as the most promising [11] . the principle of this strategy is to prime t-cells to be antigen-specific and then, upon repeated exposure to a specific antigen, induce a rapid t-cell expansion. in heterologous boosts, the encoded antigen is delivered in a different form/different vehicle [111] . dna plasmids are perfectly fit for priming since they are internalized by antigen presenting cells and can induce antigen presentation via mhc class i or class ii. such heterologous regimens can be effective when infection occurs with both viral particles and virusinfected cells, and neither cellular, nor antibody response is sufficient for sterilizing protection or viral clearance, if acting alone. this approach may help to circumvent the negative effects of intracellular core expression. indeed, here, the heterologous dna-prime/protein boost strategy was shown to be advantageous to both immunizations with core dna and with the recombinant core protein (figs. 6, 7) . protein alone performed even worse than single dna injections (figs. 6, 7) . only the heterologous dna-prime-protein boost regimen induced a significant core-specific antibody production and potent t-cell response of mainly th1-profile. this may be beneficial since most correlates of spontaneous hcv clearance are th1-oriented [32, 39, 84, 85] . this data suggests that the administration of highly expressed hcv core gene, as well as repeated core gene injections may hamper core-specific immune response. the boosting effect of repeated core gene injections is transient as it disappears with subsequent injections. one possible way to enhance core-specific response is to deliver limited intracellular amounts of core, either by giving lower plasmid doses, or by giving vectors with low expression efficacy. an additional option is the use of heterologous dna prime/protein boost regimen that leads to potent immune response of a mixed th1/th2-type. we are currently testing if transient hcv core gene expression and acquisition of anti-hcv core immunity affect the immune status and functionality of the immune system in gene recipients. hepatitis c virus infection expression and immune response to hepatitis c virus core dna-based vaccine constructs syringe exchanges: a public health response to problem drug use intrahepatic cytotoxic t lymphocytes specific for hepatitis c virus in persons with chronic hepatitis natural history and disease manifestations of hepatitis c infection liver-derived ctl in hepatitis c virus infection: breadth and specificity of responses in a cohort of persons with chronic infection mutational escape from cd8+ t cell immunity: hcv evolution, from chimpanzees to man the role of hepatitis c virus specific cd4+ t lymphocytes in acute and chronic hepatitis c neutralizing antibody response during acute and chronic hepatitis c virus infection quantitative analysis of anti-hepatitis c virus antibodysecreting b cells in patients with chronic hepatitis c. hepatology vaccines to prevent chronic hepatitis c virus infection: current experimental and preclinical developments immunogenicity and safety of a novel therapeutic hepatitis c virus (hcv) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects therapeutic vaccination of chronic hepatitis c nonresponder patients with the peptide vaccine ic41 functional and phenotypic characterization of peptide-vaccine-induced hcv-specific cd8+ t cells in healthy individuals and chronic hepatitis c patients phase i clinical study of a personalized peptide vaccination for patients infected with hepatitis c virus (hcv) 1b who failed to respond to interferon-based therapy a pilot study of ther characterization of the immune response against hepatitis c infection in recovered, and chronically infected chimpanzees viral and immunological determinants of hepatitis c virus clearance, persistence, and disease hepatitis c virus (hcv) core protein enhances the immunogenicity of a co-delivered dna vaccine encoding hcv structural antigens in mice enhancement of cellular and humoral immune responses to hepatitis c virus core protein using dna-based vaccines augmented with cytokine-expressing plasmids immune responses to plasmid dna encoding the hepatitis c virus core protein an epitope in hepatitis c virus core region recognized by cytotoxic t cells in mice and humans targeting of hepatitis c virus core protein for mhc i or mhc ii presentation does not enhance induction of immune responses to dna vaccination cpg immuno-stimulatory motifs enhance humoral immune responses against hepatitis c virus core protein after dna-based immunization a truncated variant of the hepatitis c virus core induces a slow but potent immune response in mice following dna immunization a dna prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malarianaive adults against plasmodium falciparum sporozoite challenge enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans enhancing th2 immune responses against amyloid protein by a dna prime-adenovirus boost regimen for alzheimer's disease heterologous prime/boost immunization of rhesus monkeys by using diverse poxvirus vectors prime-boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in pekin ducks challenged with asian h5n1 hpai recombinant retrovirus-like particle forming dna vaccines in prime-boost immunization and their use for hepatitis c virus vaccine development dna-vlp prime-boost intra-nasal immunization induces cellular and humoral anti-hiv-1 systemic and mucosal immunity with cross-clade neutralizing activity the safety and tolerability of an hiv-1 dna prime-protein boost vaccine (dp6-001) in healthy adult volunteers elicitation of strong immune responses by a dna vaccine expressing a secreted form of hepatitis c virus envelope protein e2 in murine and porcine animal models advances in tuberculosis vaccine strategies improved protective efficacy of a species-specific dna vaccine encoding mycolyl-transferase ag85a from mycobacterium ulcerans by homologous protein boosting enhanced protective immunity against pneumococcal infection with pspa dna and protein littel-van den hurk s: dna prime protein boost strategies protect cattle from bovine viral diarrhea virus type 2 challenge littel-van den hurk s: immunization with plasmid dna encoding a truncated, secreted form of the bovine viral diarrhea virus e2 protein elicits strong humoral and cellular immune responses immunogenicity of dna vaccines in humans: it takes two to tango characterization of the humoral and cellular immune responses against hepatitis c virus core induced by dna-based immunization signal sequences modulate the immunogenic performance of human hepatitis c virus e2 gene cloning and characterization of a complete open reading frame of the hepatitis c virus genome in only two cdna fragments immunization with hepatitis c virus core gene triggers potent t-cell response preparation of hepatitis c virus structural and non-structural protein fragments and studies of their immunogenicity properties of the hepatitis c virus core protein: a structural protein that modulates cellular processes the native form and maturation process of hepatitis c virus core protein intracellular localization of full-length and truncated hepatitis c virus core protein expressed in mammalian cells nuclear localization of the truncated hepatitis c virus core protein with its hydrophobic c terminus deleted th1 and th2 cells: different patterns of lymphokine secretion lead to different functional properties dna-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants the hepatitis c virus: identification, epidemiology, and clinical controversies dna vaccines: improving expression of antigens a nonviral cytoplasmic t7 autogene system and its applications in dna vaccination wagner r: multiple effects of codon usage optimization on expression and immunogenicity of dna candidate vaccines encoding the human immunodeficiency virus type 1 gag protein influence of codon usage on the immunogenicity of a dna vaccine against tetanus dose dependence of ctl precursor frequency induced by a dna vaccine and correlation with protective immunity against influenza virus challenge comparison of various expression plasmids for the induction of immune response by dna immunization viral persistence, antibody to e1 and e2, and hypervariable region 1 sequence stability in hepatitis c virus-inoculated chimpanzees immunology of hepatitis b virus and hepatitis c virus infection role of atp in binding and migration of 40s ribosomal subunits point mutations close to the aug initiator codon affect the efficiency of translation of rat preproinsulin in vivo alternative mechanisms of initiating translation of mammalian mrnas structural and mechanistic insights into hepatitis c viral translation initiation amino acids 1-20 of the hepatitis c virus (hcv) core protein specifically inhibit hcv ires-dependent translation in hepg2 cells, and inhibit both hcv core protein-coding sequence, but not core protein, modulates the efficiency of cap-independent translation directed by the internal ribosome entry site of hepatitis c virus dna inoculation induces protective in vivo immune responses against cellular challenge with hiv-1 antigenexpressing cells heterologous protection against influenza by injection of dna encoding a viral protein multi-subtype gp160 dna immunization induces broadly neutralizing anti-hiv antibodies a single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies field trials of a very potent rabies dna vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions gene immunization may induce secondary antibodies reacting with dna bacterialized" gene for the c-terminus truncated version of human hepatitis c virus core induces potent th2-type immune response in mice interactions of single and combined human immunodeficiency virus type 1 (hiv-1) dna vaccines pulmonary necrosis resulting from dna vaccination against tuberculosis lack of protection in mice and necrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of mycobacterium tuberculosis transcriptional regulation of cellular and viral promoters by the hepatitis c virus core protein cellular response to conditional expression of hepatitis c virus core protein in huh7 cultured human hepatoma cells oxidative stress in the absence of inflammation in a mouse model for hepatitis c virus-associated hepatocarcinogenesis the hcv core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid t cell line suppression of apoptotic cell death by hepatitis c virus core protein the core protein of hepatitis c virus induces hepatocellular carcinoma in transgenic mice hepatitis c virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype hepatitis c virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver hepatitis c virus core protein induces hepatic steatosis in transgenic mice the prime-boost strategy: exciting prospects for improved vaccination we thank prof. eva stankevica and her group for the oligonucleotide synthesis and automatic sequencing and ms. natalija gabrusheva and ms. irena timofeeva for technical assistance. this work was supported by grants from the latvian council of science 05.1626, eraf vpd1/eraf/cfla/05/ apk/2.5.1./000021/010, eu #05-1000004-7748, the european social fund (esf), the new visby program of the swedish institute, compuvac grant #lshb-ct-2004-005246 and the russian foundation for basic research #08-04-01107-a. the authors declare that they have no competing interests. is and ek constructed plasmids and screened their immunogenicity; ea did experiments on expression and wrote a draft of the manuscript; es and ei carried out quantifications of core expression; ds and np did immunological experiments; rb conducted the immunocytochemistry; mi was involved with the immunological experiments, statistical evaluations and worked with the manuscript; tk and pp give useful scientific advice and revised the manuscript. all authors read and approved the final manuscript. establishment of calibration curves for quantification of core expression in vitro. recombinant core aa 1-173 in serial dilutions in the range of 10 to 25 ng (a), 10 to 100 ng (b), or 12.5 to 100 ng (c) was loaded on 15% sds-paag and resolved by gel electrophoresis together with the study samples. proteins were transferred to pvdf membrane and subjected to western blotting with core-specific rabbit antibodies, and secondary anti-rabbit hrp-conjugated antibodies (dakopatts). signals were registered using x-ray films and ecl detection system, images were saved, scanned, and signal of individual band corresponding to core was quantified by image j http://rsb.info.nih.gov, and calibration curves were built (d key: cord-294842-aesiff1f authors: romero-brey, inés; bartenschlager, ralf title: membranous replication factories induced by plus-strand rna viruses date: 2014-07-22 journal: viruses doi: 10.3390/v6072826 sha: doc_id: 294842 cord_uid: aesiff1f in this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) rna viruses. we discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. a general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (er) and double membrane vesicles, representing extrusions also originating from the er, respectively. we hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. members of the family flaviviridae are enveloped viruses with a single stranded rna genome of positive polarity. this family contains four different genera: hepacivirus (from the greek hepar, hepatos, -liver‖), flavivirus (from the latin flavus, -yellow‖), pestivirus (from the latin pestis, -plague‖) and the recently included genus pegivirus [1, 2] (figure 1 ). hepatitis c virus (hcv) is the prototype species of the genus hepacivirus. it was first discovered by choo et al. [3] in the serum and tissues of a chimpanzee experimentally inoculated with serum from an individual with chronic, non-a, non-b hepatitis. this virus was associated with a mild form of chronic hepatitis frequently observed in recipients of blood transfusions [4] and was called hcv. a second species within the genus hepacivirus is gbv-b which was first identified in tamarins that developed hepatitis following inoculation with the serum from a surgeon (initials g.b.) with acute hepatitis. additional gb-like viruses were discovered later on and have been assigned to the new genus pegivirus (an acronym derived from pe, persistent; g, gb or g) within the family flaviviridae [1] . and cms may represent the site of denv replication and rna translation/polyprotein processing, respectively [14] . the most complete characterization of denv-induced intracellular membrane rearrangements elucidated their 3d architecture as well as their spatial connection with viral assembly sites [15] . tem of resin-embedded infected cells revealed a complex collection of convoluted and vesicular structures, including cms that were usually surrounded by multiple vesicles, often appearing as longitudinal vesicle arrays. by using electron tomography (et), the latter were found to correspond to er tubules containing 80-90 nm single-membrane vesicles (ve) that result from the invagination of the er membrane into the er lumen. by conventional em, these vesicles appeared as double membrane vesicles, likely corresponding to the vps described earlier [13] . immuno-em confirmed that the vesicles visible in resin-embedded cells were induced by denv infection and contained all ns proteins. however, only ns3 was detectable within the cms, which could be due to lower affinity of the antibodies or poor accessibility of the other ns proteins in the cms. double-stranded rna (dsrna) detected by immunostaining appeared as discrete electron-dense structures inside or on the cytosolic surface of a subset of vesicles, suggesting that dsrna might be present only in some of the vesicles at a given time point. furthermore, the vesicles contain rather uniform pores of ~10 nm diameter towards the cytosol ( figure 2a ). thus, both the topology of the vesicles and the immunolabeling results support the idea that the vesicles might be the site of rna replication. moreover, these results showed that replication factories are a continuous membrane network that provides a platform for the transport of viral proteins and genomes between sites of rna replication, ribosome-containing compartments (rna translation) and virus assembly sites. in fact, virus budding sites were found in close proximity to the pores of the replication vesicles. this topological link may ensure efficient production and delivery of viral rna for the assembly of infectious virus progeny. consistent with these findings, a very recent publication using et showed that these virally modified structures were also observed in denv-infected mosquito cells, with one exception: cms were absent from denv-infected c6/36 mosquito cells [16] . in addition, after multiple rounds of virus replication, tubular structures were also observed in the vicinity of vps. these structures might represent a hallmark of chronically infected insect cells, since these structures are also induced by tbev in tick cells (see below). the first reports on wnv-infected cells described the visualization of virions [17] . an extensive characterization of kunjin virus-the australian variant of wnv (wnv kun )-infected cells has been carried out more recently [18] [19] [20] . three well-defined structures were found, corresponding to large cms, paracrystalline arrays (pcs) and vps that appeared as membrane sacs containing small vesicles (ve) [18, 21] . based on immunolocalization studies, a distinct redistribution of the trans-golgi network (tgn) and colocalization of tgn markers with dsrna has been observed, suggesting that the replication factories of wnv kun were derived from the tgn [22] . three-dimensional reconstructions of the wnv kun replication sites revealed an intimate association of the rough er (rer) with the bounding membrane of the vps [20] (figure 2b ), resembling the vesicles observed in denv-infected cells. these results argue for an additional role of the rer in the formation of the wnv kun replication factories. similar to denv, individual necks were observed in the vesicles as well as the majority of the viral rna, as detected by immunolabeling with a dsrna-specific antibody, resided within these vesicles [18, 20, [22] [23] [24] . in most cases, viral rna spanned the breadth of the vesicles and was juxtaposed to the necks open to the cytoplasm [20] . . slices through tomograms of infected cells (on the left) and 3d top and lateral (90° rotation) views of the same tomograms (on the right) are depicted, showing the characteristic virus-induced structures. the replication vesicles (ve) of denv, wnv and tbev (genus flavivirus) correspond to invaginations of er membranes that remain connected to the cytosol via 10 nm-pores (highlighted with white arrows in the 3d lateral views), forming vesicle packets (vps). the replication factory of hcv (genus hepacivirus) is primarily composed of double membrane vesicles (dmvs) that seem to be formed aser protusions connected to er membranes via neck-like structures (highlighted with white arrows in the 3d lateral view). the er is shown in yellow (denv, tbev and hcv) or in red (wnv) and the replication organelles in brown (denv, tbev and hcv) or in white (wnv). the outer and inner membranes of dmvs are depicted in different shades of brown (outer membrane in dark brown and inner membrane in light brown). figure 2b is reproduced with permission from [20] . in cells infected with tbev, one of the most important tick-transmitted viruses in europe and asia, virus particles and membrane-connected vesicles were also observed inside the er [25] , similar to what was described for denv and wnv kun . the viral dsrna was only detected inside the vesicular structures within rer, suggesting that tbev rearranges internal cell membranes to generate a compartment that protects viral rna from detection by cytoplasmic pathogen recognition receptors (prrs) [26] [27] [28] . this localization of dsrna might suffice to delay the onset of the ifn response [25] . for tbev [25] and wnv kun [19] it was shown that treatment with brefeldin a (bfa), a drug which disrupts the golgi apparatus, did not interfere with viral replication. however, this treatment rendered wnv kun sensitive to the antiviral action of the ifn-induced protein mxa, indicating that bfa might have disrupted the membranous wnv kun replication compartments, thus leading to exposure of dsrna and its detection by prrs. in contrast, treatment of tbev-infected cells with bfa neither affected viral replication, nor the level of ifn production. these findings indicate that tbev dsrna might be stored inside bfa-resistant membrane vesicles that robustly protect the viral rna from recognition by cellular sensors. vector-borne flaviviruses like denv and tbev must replicate in both mammalian and arthropod cells. a few comparative studies have been published describing virus-induced structures such as cytoplasmic membrane proliferations and vesicle formation, also in insect cells [11,16,21,29,30,] . a detailed comparative ultrastructural analysis of tbev-induced modifications revealed that the extent of membrane expansion and the abundance of vesicles were lower in insect cells [31] . single-membrane vesicles, ranging in diameter from 60-100 nm were frequently found within proliferated er areas, often occurring in large groups contained within er cisternae. pore-like openings connected these vesicles to the cytoplasm and to other vesicles. apart from these vesicles, in tick-infected cells elongated vesicles or tubules were found that were much more prevalent in persistently than in acutely infected cells. these tubules were only occasionally noted in infected mammalian cells, similar to what was found with denv-infected cells [15] . the tubular structures had a cross-sectional diameter of 60-100 nm, similar to the one of vesicles, reached up to 800 nm in length, were closed at the ends and often arranged in fascicle-like bundles, shrouded with the er membrane. however, no pores between the tubules or towards the cytoplasm were observed [31] . the function of these tubules is unclear and it is not known whether they represent bona fide features of replication factories, aberrant structures as a result of incorrect membrane remodeling, or the result of a cellular process to restrict infection [31] . in any case, the tubules might be a feature of persistent infection, eventually linked to the high number of defective virus particles, because the lack of pores could prevent proper replication or packaging of the viral genome [32] [33] [34] . further studies are required to shed light on the biogenesis and biological significance of these membranous tubular structures. a recent study identified 80 nm-diameter vesicles within the er lumen of tbev-infected bhk-21 cells and in cells transfected with a tbev replicon [35] . et revealed that these vesicles are invaginations of the er within a highly organized network of interconnected membranes with half of vesicles containing pore-like connections to the cytoplasm ( figure 2c ). however, no pore-like openings were observed between adjacent/neighboring vesicles, in contrast to what has been described for cells infected with langat virus (lgtv), a naturally attenuated tick-borne flavivirus [31] or in wnv kun -infected cells [20] . interestingly, in tbev replicon cells, the number of pore-containing vesicles was slightly larger (~75%) and they were found in much more fragmented er tubules as compared to tbev-infected cells. however, despite more extensive er rearrangements in replicon cells, they contained fewer vesicles, consistent with the lower level of viral replication [36] [37] [38] . conventional em analysis of neurons infected with murray valley encephalitis virus (mvev) revealed several ultrastructural features, including proliferation of er and golgi complex membranes as well as the appearance of membrane-bound spherical vesicles (75-145 nm diameter) [39] , similar to those observed for the related flaviviruses japanese encephalitis virus (jev) [40, 41] and st. louis encephalitis virus (slev) [42] . in the latter case, cylindrical membranous structures (or tubules) were also observed [43] . the presence of vesicles was also detected in monkey liver cells infected with yellow fever virus (yfv) [44] . these findings indicate that all members of the genus flavivirus utilize the er as a source of membranes for the formation of their replication factories, whereas assembly of new virions seems to occur at er sacs in close proximity to the replication sites [15, 35] , thus creating an optimized membranous environment to support efficient viral replication and assembly. maturation of the newly synthesized virions takes place in the golgi apparatus, where flavirirus virions are often observed [15] . in stark contrast to flaviviruses, hcv, the prototype of the genus hepacivirus, provokes an alternative rearrangement of intracellular membranes, originally designated -membranous web‖ (mw). this term referred to compact vesicle accumulations embedded into a membranous matrix [45] as detected in cells inducibly expressing the hcv polyprotein. by using different em methods, we and others have recently found that the mw is primarily composed of double membrane vesicles (dmvs) [46] [47] [48] . the fact that the kinetics of their appearance correlates with hcv replication suggests that these structures play an important role for viral rna amplification [48] . indeed, immunolabeling of purified dmvs revealed an enrichment for viral proteins as well as dsrna [46, 49] . importantly, dmvs contain enzymatically active viral replicase [49] and they originate from er membranes, similar to what has been found for other members of the family flaviviridae. et analysis showed that most of the dmvs remain connected to the er via their outer membrane [48] ( figure 2d ). although dmvs are primarily closed structures, ~10% of them have an opening towards the cytosol. late in infection, multi-membrane vesicles (mmvs) with an average diameter of 390 nm are generated, likely originating from dmvs by secondary enwrapping events [48] . by using huh7.5 cells infected with the highly replicative hcv strain jfh-1, ferraris and coworkers observed three different types of membrane alterations: vesicles in clusters (vics), contiguous vesicles (cvs) and dmvs [47] . the vics were small single-membrane vesicles of variable size (100-200 nm) , grouped together in well-delimited areas. most of them had an internal invagination. the cvs were small single-membrane vesicles, present in large numbers and widely distributed throughout the cytoplasm, with a more homogeneous size (around 100 nm). they were tightly associated to each other and tended to form a collar around lipid droplets (lds). dmvs were heterogeneous in size (150-1000 nm) and had a thick, electron-dense membrane consisting of two closely apposed membranes. the increase of cvs' number correlated with an increase of intracellular hcv rna levels, arguing for a possible role of cvs in the early stages of viral replication. the presence of ns5a in cvs, as demonstrated by immunogold staining, is consistent with this hypothesis. alternatively, cvs might constitute the membranous platform for viral assembly. in fact, the core protein is present in these structures (16%) as well as on the ld surface (81%). however, so far visualization of virus particles in infected cells has not been possible, making this hypothesis difficult to prove. while most of the dsrna signal was located within dmvs or at dmv membranes, vics were free of viral components and rna and these structures as well as cvs were very rarely observed in cells with a subgenomic jfh-1 replicon [46] or absent in cells infected with a jfh-1 variant designated jc1 [48] . the first 3d reconstruction of a complete hcv-infected cell revealed that all these three membrane structures were tightly connected and closely associated with ld clusters [47] . taken together, these findings indicate a fundamental role of dmvs in hcv replication. an in-depth comparison of the study by ferraris and coworkers [47] and our publication [48] suggests that cvs might be also dmvs for several reasons: first, cvs have electron dense tightly apposed membranes; second, by using correlative light and electron microscopy, we also detected dmv accumulations around lds, reminiscent of the cvs described by ferraris and coworkers [47] ; third, taking into consideration the density of content and morphology, some of the structures described as dmvs by ferraris and colleagues might correspond to mmvs according to our nomenclature. this might account for the differences in size between the dmvs reported in both studies (up to 1000 nm versus 150 nm, respectively). alternatively, the difference might be due to the use of distinct virus strains (jfh-1 and jc1) that differ in their capacity to produce infectious virus particles by ~3 orders of magnitude [50] , which might also explain the presence of vics only in jfh-1 infected cells. much less about membranous replication factories is known for pestiviruses. tem-based studies from the times in which the genus pestivirus was still belonging to the family togaviridae reported that pestivirus-infected cells exhibited ultrastructural modifications of rer and contained small numbers of virus-like particles (vlps) [51, 52] . gray and nettleton (1987) reported that border disease virus (bdv)-infected cells contained several profiles of er and many dense lamellar bodies, which when transversely sectioned appeared as multiple rows of tubules, 33 nm in diameter [52] . these lamellae were often found in association with rer and in one occasion vlps appeared to be budding within them. bovine viral diarrhea virus (bvdv)-infected cells contained rer modified into tubules, in which electron-dense vlps were present. more recent studies on bvdv-infected cells revealed cytoplasmic vacuolization and vlps in dilated er cisternae [53, 54] . in addition, membrane structures consisting of vesicles of various sizes enclosed in much larger vesicles have been reported [55] . these structures that morphologically resemble multivesicular bodies (mvbs) are distinct from the hcv-induced membranous web and more reminiscent of the flavivirus-induced vps. studies on the morphogenesis of pestiviral particles were hampered by a low rate of virion production. in a recent study, schmeiser and colleagues have overcome this problem by using high multiplicity of infection in mdbk cells with a distinct virus strain, the giraffe-1 strain [56] . obtained results define the er as the site of pestivirus particle assembly, where budding of virions was observed. virus particles were also found inside the lumen of the golgi and in vesicles associated with the golgi compartment, suggesting that virus egress occurs via the conventional secretory pathway. interestingly, replication kinetics of pestiviral rna did not correlate with distinct membrane rearrangements and only slight dilatation of the er lumen was noticed. the absence of significant membrane rearrangements argues for a major difference between pestiviruses and other members of the flaviviridae family. interestingly, the authors detected the capsid protein and dsrna, the marker for viral replication intermediates, mainly in mvbs, indicating that pestiviruses are either using this compartment for replication or that viral rna and proteins are transferred to this compartment for degradation. similar assumptions have been made for hiv [57] and marburg virus, a member of the filoviridae family [58, 59] . alternatively, pestiviral rna and protein in mvbs might be intermediates of the entry process, prior to fusion of the envelope with the endosomal membrane. indeed, particles inside mvbs matching the morphological criteria of pestivirus virions were detected [56] . however, mvbs of non-infected cells also contain vesicles for lysosomal degradation termed intraluminal vesicles (ilvs) that display a very similar morphology to pestiviral virions. thus, unambiguous discrimination between ilvs and pestivirus particles will require detailed immunolabeling approaches. the first visualization of the 3d architecture of a (+) strand rna virus replication factory was reported for flock house virus (fhv), a member of the family nodaviridae [60] . this insect nodavirus induces the formation of invaginations at the outer mitochondrial membrane (omm) with an average diameter of ~50 nm [60, 61] ( figure 3a ). the interior of these vesicles (called spherules) is connected to the cytoplasm by a necked channel of ~10 nm diameter, which is wide enough to allow import of ribonucleotides and export of synthesized rna (diameter < 2 nm) [60] . furthermore, metabolically labeled fhv rna localized between inner and outer mitochondrial membranes inside these spherules, thus validating the spherules as bona fide fhv-induced compartments for viral rna synthesis [60] . conventional tem analyses of coronavirus-infected cells identified large numbers of isolated dmvs [62] . at least in case of the severe acute respiratory syndrome (sars)-coronavirus, these dmvs are part of an elaborate reticulovesicular network (rvn) of modified er that consists of convoluted membranes, numerous dmvs (diameter 200-300 nm) ( figure 3b ) [63] , and -vesicle packets‖ apparently arising from merging of dmvs. the cms were most intensively immunolabeled for viral replicase subunits whereas dmvs labeled abundantly for dsrna. while this result argues that dmvs might be the site of viral rna synthesis, et analyses failed to detect dmv connections to the cytoplasm to allow transport of nascent rna. instead, dmvs are connected to each other, to cms and to the er via their outer membranes. also, in case of another coronavirus, the mouse hepatitis virus (mhv), er membranes are thought to be the lipid donor of the membranous replication compartment [64, 65] . qualitative and quantitative analyses by (immuno)-electron microscopy of mhv-induced membrane rearrangements revealed the appearance, in strict order, of dmvs (diameter 200-350 nm), cms, large virion-containing vacuoles, tubular bodies and cubic membrane structures [65] . the recently identified coronavirus middle east respiratory syndrome coronavirus (mers-cov) induces extensive membrane rearrangements in the perinuclear region, including the formation of dmvs and cms [66] . the diameter of mers-cov induced dmvs ranged from 150-320 nm, comparable to the sars-cov induced dmvs. in addition, cms were always surrounded by dmv clusters and were only observed in cells that appeared to be more advanced in infection. this observation strengthens the notion that dmv formation precedes the development of cms, as postulated previously for sars-cov [63] . in addition to the betacoronaviruses (sars-cov, mhv and mers-cov), er-derived dmvs with a diameter of ~200 nm have been observed in primary avian and mammalian cells infected with infectious bronchitis virus (ibv), an important poultry pathogen belonging to the genus gammacoronavirus [67] . however, the most striking structures induced by ibv are zippered er membranes ( figure 3c ). the zippered er was associated to 60-80 nm diameter spherules, structures that are not present in cells infected with betacoronaviruses. et showed that these ibv-induced spherules are tethered to the zippered er and contain a 4.4 nm long channel connecting their interior to the cytoplasm of the cell, making them the ideal candidates for the site of ibv rna synthesis. er membranes are also targeted by nidovirales that belong to the family arteriviridae. cells infected with the prototypic arterivirus, equine arterivirus (eav), also contain dmvs associated to er tubules. these dmvs are ~2-3 times smaller (~95 nm) as compared to coronaviruses [68] . a recent in-depth ultrastructural analysis revealed that the outer membranes of eav-induced dmvs are interconnected with each other and with the er (figure 3d ), thus forming a reticulovesicular network (rvn) resembling the one previously described for the distantly related sars-cov [69] . despite significant morphological differences, a striking parallel between the two virus groups, and possibly all members of the order nidovirales, is the accumulation of dsrna, the presumed intermediate of viral rna synthesis, in the dmv interior. along these lines, dmvs visualized by means of electron spectroscopy imaging contained phosphorus in amounts corresponding on average to a few dozen copies of the eav rna genome. like in sars-coronavirus infected cells, connections between dmv interior and cytosol could not be unambiguously identified, suggesting that dsrna is compartmentalized by the dmv membranes. in addition, et revealed a network of nucleocapsid protein-containing protein tubules, intertwined with the rvn. this potential intermediate in nucleocapsid formation, which was not observed in coronavirus-infected cells, suggests that arterivirus rna synthesis and assembly are spatially coordinated. membrane remodeling in picornavirus-infected cells has been studied for more than 50 years. massive virus-induced membrane modifications have been reported already in 1958 [70] , but the origin of these membranes is still a matter of controversy. several lines of evidence, including biochemical and structural data, suggest that the er must play a major role in the formation of those structures [71] [72] [73] . early in infection membranous replication factories contain markers of the golgi [74, 75] , whereas markers of the er, golgi and lysosomes were all found to be associated with poliovirus replication sites late in infection [76] . initial reports identified membrane rearrangements as u-bodies because of their horseshoe-like shape [70] . later, bienz et al. described rearranged membranes as clusters of single-membrane vesicles [71, 77] , while other reports [76, 78] noticed the double-membrane morphology of picornavirus-induced vesicles. the single-versus double-membrane morphology of the vesicles was first interpreted as two different models of their formation. however, several recent publications suggest that picornavirus-induced membrane rearrangements might occur in a consecutive manner. thus, early in poliovirus infection small clusters of single-membrane vesicles predominate that are transformed into bigger irregularly shaped single-membrane structures ( figure 3g ) and, late in infection, replaced by either round or irregularly shaped dmvs [79] . interestingly, the small clusters of single-membrane vesicles of ~100-200 nm diameter contain gm130, a cis-golgi marker, but did not stain positive for calnexin, an er marker. however, this does not exclude a role of the er for biogenesis of these vesicles, because er-resident proteins might be sorted out as these membranes are transformed. although dsrna and metabolically labeled viral rna were detected in single-membrane vesicles and dmvs, the exponential phase of viral rna synthesis correlates with the appearance of single-membrane and intermediate structures [79] arguing that these structures are most relevant for high level poliovirus rna synthesis. similar results have been obtained with another member of the family picornaviridae, coxsackie b3 virus (cvb3) that also induces the formation of single-and double-membrane compartments, whose relative abundance correlates with the stage of the replication cycle [80] (figure 3f ). based on the observation that the golgi apparatus disappears in cvb3-infected cells, the membrane rearrangements might originate from this organelle (montserrat bá rcena, personal communication). similar to poliovirus, single-membrane tubular clusters occur predominantly early in infection, whereas the number of dmvs increases as infection progresses. a budding event could account for the formation of the tubules, depicting an average length of 654 ± 291 nm and an average diameter of 81 ± 7 nm. a subsequent enwrapping of these single-membrane tubules via an -autophagy-like‖ mechanism could then lead to the formation of dmvs that have an average diameter of 159 nm ± 47 nm. this transformation may require several steps: (i) membrane pairing; (ii) induction of curvature; and (iii) membrane fusion [80] . this scenario would be consistent with the membrane surface of dmvs as an average-sized dmv with a diameter of 160 nm would be equivalent to a tubule with a length of 632 nm and a diameter of 81 nm. er membranes were found near dmv clusters. however, in contrast to previous observations in nidovirus-infected cells, these dmvs were not connected to neighboring structures. in addition to these compartments, a third type of modification was detected in cvb3-infected cells: multilamellar structures, which are typical for the late phase of infection and that correspond to enwrapped dmvs: despite their various shapes and degrees of complexity, in all instances they contained one dmv, surrounded by one or several layers of curved cisternae. in conclusion, these results, and similar observations made for foot and mouth disease virus (fmdv) (genus aphthovirus) [81] suggest that members of the picornaviridae family induce singleand double-membrane vesicles. they appear in a time-dependent manner and seem to evolve from each other, possibly in coordination with the progression of the viral replication cycle [80] . these membrane rearrangements occur independently from the used virus strain and cell line. rubivirus (family togaviridae) [82] . rubv anchors its rna synthesis machinery to membranes of a cell organelle known as -cytopathic vacuoles‖ (cpvs) that is derived from modified endosomes or lysosomes and has an average diameter of 600-2000 nm [83] [84] [85] . freeze-fracture and et analysis of rubv-infected cells revealed a high complexity of cpvs that are composed of stacked membranes, rigid sheets, small vesicles and large vacuoles ( figure 3i ) [86] . the cpvs are interconnected and linked to the endocytic pathway, as deduced from labeling experiments with endocytosed bsa-gold. furthermore, rer cisternae, mitochondria and golgi stacks are recruited around cpvs to build up rubv factories. cpvs have several contacts with cellular organelles: they are coupled to the rer through protein bridges of ~10-15 nm and closely apposed membranes and they are attached to golgi vesicles, whereas contacts with mitochondria were not detected [86] . it has been proposed that rna synthesis occurs on vesicular membranes within the cpvs, which are linked to the cytosol and that the viral replicase molecules are associated with vesicles that transform with time into large vacuoles and straight elements [86] . this is supported by immunogold labeling revealing replicase components and dsrna within the cpvs [83, 84, 87] . the modification of late endosomes and lysosomes is a feature that rubv shares with alphaviruses, the other genus of the family togaviridae [88, 89] . cells infected with alphaviruses like semliki forest virus (sfv), sindbis virus and western equine encephalitis virus (weev) contain large cpvs with a diameter ranging between 600 and 2000 nm. the inner surface of these cpvs is covered with small invaginations or spherules that originate at the plasma membrane [90] [91] [92] ( figure 3j ). these spherules are comprised of a single membrane forming a vesicle with a diameter of ~50 nm. in addition, the spherules were shown to be the site of viral rna synthesis as deduced from metabolic labeling and detection by em [89, 91, 92] . importantly, the inside of the spherule is connected to the cytoplasm by a pore with a diameter of 5-10 nm. the spherules are formed at the plasma membrane by the concerted action of the viral nonstructural proteins (nsp1-nsp4) and genomic viral rna [89] . furthermore, froshauer et al. [88] showed that the cpvs that contain the spherules possess endosomal and lysosomal markers. time course studies revealed that the spherules of sfv undergo an unprecedented large-scale movement between cellular compartments [93] . the spherules first form as blebs (exvaginations) at the plasma membrane. then, they are internalized by an endocytic process requiring a functional actin-myosin network. the spherules therefore represent an unusual type of endocytic cargo. after endocytosis, spherule-containing vesicles, namely cpvs-i fuse with acidic endosomes and move along microtubules. this leads to the formation of a very stable compartment, where the spherules accumulate as invaginations on the outer surface of unusually large, acidic vacuoles localized in the pericentriolar region [93] . members of the genus norovirus (novs, family caliciviridae) are major agents of acute gastroenteritis [94] . ultrastructural examination of murine norovirus 1 (mnv-1)-infected cells revealed a striking change in their overall morphology and intracellular organization [95] . structures resembling virus particles were observed within or next to single-or double-membrane vesicles in the cytoplasm. the vesiculated areas increase in size with time and by 24 hpi, large numbers of these vesicles and viral particles occupy most of the cytoplasm and displace the nucleus ( figure 3h ). in addition, a complete rearrangement of the er and loss of intact golgi apparatus was observed. both dsrna and mnv-1 nonstructural protein 7, the rna dependent rna polymerase, localize to the limiting membrane of individual vesicle clusters by immuno-em [96] . immunofluorescence-based double-labeling showed that mnv-1 appears to recruit membranes derived from multiple cellular organelles and/or compartments: the er, trans-golgi apparatus and endosomes. however, despite extensive efforts, human norovirus cannot be grown in cultured cells [97] . thus, detailed studies have not been possible, but it is assumed that replication structures are similar to those of mnv-1. feline calicivirus (fcv), a member of the genus vesivirus within this family, is a major agent of respiratory disease in cats, which replication originates also membranous rearrangements and vesicles [98] . brome mosaic virus (bmv, family bromoviridae) generates its replication factory by hijacking er membranes, similar to what has been described for other plant viruses like tobacco mosaic virus (tmv, family virgaviridae) [99] , tobacco echt virus (tev, family potyviridae) [100] and red clover necrosis mosaic virus (family tombusviridae) [101] . however, other plant viruses such as alfalfa mosaic virus (amv) [102] and cucumber mosaic virus (cmv), both belonging to the family bromoviridae, and turnip yellow mosaic virus (tymv, family tymoviridae) [103] anchor their replication sites on chloroplasts. cucumber necrosis virus (cnv), family tombusviridae, utilizes peroxisomal membranes as replication platforms [104] , while other plant viruses replicate on the surface of mitochondria [105] . although the 3d architecture of these membranous replication sites remains largely unknown, their characteristics are strikingly similar to those for fhv (family nodaviridae). best studied is bmv that induces spherules, of similar size as the insect nodavirus fhv, in the er close to the nucleus, where viral rna synthesis and viral replication proteins are localized [106] [107] [108] . in the case of beet yellows closterovirus (byv, family closteroviridae), tem of infected plant cells revealed the formation of ~100 nm-diameter dmvs and multivesicular complexes (single-membrane vesicles surrounded by a common membrane) ( figure 3e ) [109] . these multivesicular complexes often reside next to stacks of aligned filamentous byv particles [110, 111] and resemble the dmvs and vps produced by nidoviruses and flaviviruses. several byv replication-associated proteins (l-pcp, mtr and hel) colocalize with dmv and vp membranes, supporting the role of these structures as replications platforms [112, 113] . the membranes in closterovirus dmvs and vps are likely to be derived from er for members of the genus crinivirus [114] or mitochondria in case of ampelovirus [115, 116] . whether these structures are -closed‖ or -necked‖ remain unknown. based on amino acid sequence homologies of their rna-dependent rna polymerases, (+) rna viruses have been classified into three large supergroups [113, 117, 118] : supergroup i (picornavirata or picorna-like group), including picorna-, corona-, arteri-and nodaviruses; supergroup ii (flavivirata or the flavi-like group), including tombus-, diantho-, pesti-, hepaci-and flaviviruses as well as single-strand rna bacteriophages; supergroup iii (rubivirata or the alpha-like group), including tobamo-, hordei-, alpha-and rubiviruses as well as hepatitis e virus (hev). these higher-order taxonomic units encompass diverse viruses infecting different hosts from almost all kingdoms of life. as discussed earlier [119] , amongst these viruses two main architectures of remodeled membranes (morphotypes) can be found that may reflect two alternative strategies to induce the membranous microenvironments required to allow virus replication (summarized in table 1 ). the first morphotype involves the formation of negatively curved membranes, initiated by invaginations of the pre-existing membrane bilayer and giving rise to spherules, vesicles or vacuoles towards the lumen of the targeted cell organelle. these structures have been identified in a broad range of mammalian, plant and insect cells infected with viruses belonging to supergroups ii and iii. the second strategy involves the formation of membranes with positive curvature, giving rise to double-membrane structures that are the predominant characteristic of the replication factories of the picorna-like virus supergroup. the conservation of these two sorts of morphotypes in distantly related viruses supports the assumption of an evolutionary conserved mechanism. a striking finding in this regard was the observation that hcv, despite belonging to the flavi-like supergroup, induces dmvs whereas flaviviruses induce the formation of negatively curved membranes. to our current knowledge, hcv is the only member of the family flaviviridae inducing the formation of membrane structures with positive curvature, suggesting that hcv might share common host cell pathways to induce membranous replication compartments with distantly related viruses such as corona-, arteri-, picorna-, calivi-or closteroviruses (table 1) . however, it still remains to be elucidated whether other members of the family flaviviridae, belonging to the genera pestivirus and pegivirus, also utilize a picorna-like membrane remodeling strategy. a common feature associated with the spherule/vesicle/vacuole/-type of rearranged membranes is their size (50-150 nm diameter) and the presence of a pore connecting the interior of the vesicle with the cytoplasm [15, 20, 60, 88] . since rna replication occurs in the vesicle interior, the pore allows exchange of nucleotides and rna products with the cytoplasm. the size of the pore is variable, ranging from 4.4 nm in case of ibv-induced spherules to ~10 nm in case of membrane invaginations induced by flaviviruses. in contrast, in the majority of dmvs no such channel or pore has been detected. nevertheless, as exemplified with nidoviruses, the inner compartments enclosed by interconnected dmvs contain the bulk of dsrna [63] , and in some cases they depict an electron-dense core assumed to correspond to viral rna (ibv and eav) [67, 69] . although this represents a functional enigma in terms of rna synthesis and transport, the presence of dsrna in the dmv interior does not necessarily indicate active rna replication. assuming a temporal regulation, it is possible that dmvs might be sites of rna synthesis as long as they are linked to the cytoplasm, but replication would stop upon closure of the vesicles. yet, another strategy appears to be used by enteroviruses, where active rna replication has been detected on the cytosolic side of the membranous structures [71, 120] , consistent with the membrane topology of the nonstructural proteins catalyzing rna replication [121] [122] [123] . as described above, studies conducted with picornaviruses revealed that the exponential phase of viral rna synthesis coincides with the accumulation of single-membrane tubules [79, 80] . importantly, pulse-radiolabeling experiments localized sites of active rna replication to the outer surface of single-membrane tubules [71] and isolation of the membranous replication factories and their subsequent visualization by em revealed that they form rosette-like structures composed of virus-induced cytoplasmic vesicles [124] . rna replication is thought to occur at sites where the vesicles cluster, whereas rna translation probably takes place on the exposed periphery of the vesicles. this raises the question, what the role of dmvs in the replication cycle of picornaviruses might be. it is possible that dmvs either support rna synthesis or serve as rna storage sites (especially in case of closed dmvs). in this manner, dmvs might be involved in regulating viral rna replication: by complete sealing of the viral replicase inside the vesicle, it would be inactive, thus regulating overall rna copy number in the infected cell. alternatively, dmvs might be an epiphenomenon, resulting from the over-expression of membrane-active proteins that accumulate especially during the late stages of infection. the mechanism responsible for dmv formation is not clear. in case of picornaviruses, it is thought that single-membrane structures are the precursors of dmvs [79, 80] . nevertheless, dmv formation might also involve the autophagy machinery, or at least several components thereof, by a process analogous to the formation of autophagic vacuoles [76] . this hypothesis is supported by the morphological resemblance of dmvs and autophagosomes. it has been shown that the inhibition or stimulation of autophagy results in a modest inhibition or stimulation of poliovirus and coxsackie b3 virus yield, respectively, and there are also data supporting the involvement of autophagy in the replication of rhinovirus 2 and 14 [125, 126] . however, brabec-zaruba et al. [127] reported that replication of rhinovirus 2 was insensitive to pharmacological manipulation of autophagy and did not induce detectable modification of lc3. this discrepancy might be due to the use of different cell types and experimental conditions. the mechanism of dmv formation in case of hcv and coronaviruses is also unclear. biochemical analysis of isolated host cell membranes associated with hcv rna and proteins identified markers of the autophagy machinery, including lc3-ii, the lipidated form of lc3 (lc3-ii) that is generated upon activation of the autophagy machinery [46] . however, the role of autophagy in the hcv replication cycle is also a matter of controversy. for instance, immunolabeling did not identify lc3-ii at those sites where nonstructural proteins accumulate [48] . moreover, different roles of autophagy for the hcv replication cycle have been proposed. these include a role of autophagy in hcv rna translation [128] , initiation of rna replication [129, 130] , production of infectious virus particles [131] or suppression of the innate antiviral defense [132, 133] . to clarify these discrepancies, future studies should combine biochemical and cell biological approaches with ultrastructural analyses. the autophagy machinery might also be involved in the formation of virus-induced membrane invaginations/spherules. for instance, lee and coworkers provided evidence that denv infection enhances autophagolysosome formation and that inhibition of the autophagy machinery by 3-methyladenine (3-ma) reduces denv particle production [134] . however, the effects were moderate, arguing that this pathway may contribute to denv replication to only a minor extent. moreover, autophagy includes membrane wrapping, leading to double-membrane compartments involved in lysosomal degradation whereas denv-induced vesicles are invaginations. finally, immunolabeling experiments failed to detect lamp-1 at these vesicles [15] , arguing against the involvement of lysosomes in the formation of the denv replication vesicles. it remains to be determined whether autophagy is actively induced by these viruses to provide a compartment favoring replication or induced as a bystander defense against infection leading to degradation of the replicase proteins [135] . another membrane compartment frequently induced by (+) rna viruses are convoluted membranes (cms) that were observed e.g., in sars-cov-, mhv-, wnv-or denv-infected cells [13, 15, 18, 63, 65, 66, 136] . morphologically, cms resemble smooth er membranes, lack ribosomes and in case of denv are induced by the sole expression of ns4a [137, 138] . cms are often associated with late stages of infection, suggesting that dmv formation might precede the development of cms. in sars-cov-infected cells, dmvs appear to be connected with cms [63] , while in mhv-infected cells no such connections have been observed [65] . the role of cms for the viral replication cycle is not well understood. in case of wnv kun , cms are supposed to be the site of polyprotein processing [18, 136] . this conclusion is based primarily on the strong immunolabeling for ns2b and ns3 and the absence of ns1 and ns4b. since polyprotein cleavage occurs co-translationally and thus, should happen at the rer, this model would require the formation of rather stable processing intermediates that are transferred from the rer to the cms where further cleavage would occur. alternatively, at least in case of denv, cms might represent a storage site for proteins and lipids involved in viral replication that can be recruited to vesicles upon demand. the fact that cms are physically linked with er-containing invaginations and contain ns3 would be consistent with this assumption [15] . along these lines, the fact that insects are cholesterol auxotrophs and lack several enzymes in the cholesterol biosynthesis pathway [139] , suggests that cholesterol might be a key component of cm structures, which would explain their absence in infected insect cells [16] . viral replication complexes are targeted to the respective membranous organelle primarily by nonstructural (ns) proteins rather than viral rna [140] . these ns proteins seem to have some specificity in recognizing organelle subpopulations and often contain multiple hydrophobic domains implicated in membrane targeting and rearrangement. the molecular mechanisms orchestrating the formation of these complex structures are still poorly understood, but it is clear that ns proteins, often working in a concerted action, are key players in replication factory biogenesis. one well-studied example among the single-membrane vesicle inducers is bmv where it was shown that the sole expression of the ns protein 1a is sufficient to induce the formation of single-membrane spherules resembling the ones observed in infected cells. these spherules had a diameter of 50-70 nm, resided in the er lumen and were shown to be the site of viral rna synthesis [108] . in case of the insect nodavirus fhv, protein a and replication competent rna were required for induction of the spherules. expression of protein a alone induced only -zippering‖ of the surfaces of adjacent mitochondria, but did not induce spherules. thus, protein a is necessary, but not sufficient for spherule formation. moreover, spherules were not formed when replication-competent fhv rna templates were expressed with a protein a mutant lacking polymerase activity or when wild-type protein a was expressed with a replication-incompetent fhv rna template. thus, the membranous fhv replication compartment requires both a viral protein and active rna synthesis [141] . feline calicivirus (fcv) infection results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. expression of individual fcv nonstructural proteins revealed that p30 induces significant reorganization of the er into large, fenestrated membrane networks, resembling the structures found in infected cells [142] . moreover, expression of p39 and p32, two additional fcv ns proteins, induced extensive reorganization of the er and the nuclear envelope suggesting that the er is the primary source of the membranous replication factory [142] . flavivirus membrane rearrangements are mainly induced by ns4a, as suggested by recent studies with wnv kun and denv [137, 138] , but it is unknown whether the same applies to ns4a of tbev. in case of denv, ns4a is thought to contain a central peripheral membrane domain that intercalates into the luminal leaflet of the er membrane [138] . it is tempting to speculate that ns4a oligomers [143] might dilate the luminal leaflet, resulting in membrane invaginations towards the er lumen. however, the sole expression of ns4a is not sufficient to induce er membrane invaginations that have been detected in infected cells. instead, expression of ns4a lacking the c-terminal 2k fragment (corresponding to fully processed ns4a) induced er membrane rearrangements reminiscent of cms, whereas unprocessed ns4a/2k did not induce membrane alterations [138] . these results provide strong evidence that processing at the ns4a-2k site is required for the induction of membrane alterations. the critical role of polyprotein cleavage for induction of membrane rearrangements is supported by studies conducted with wnv kun . there it was shown that a regulated cleavage of a ns4a/2k/4b precursor by the viral ns2b/3 protease is needed for induction of membrane rearrangements [137] . however, the same study reported that expression of full-length (uncleaved) wnv kun ns4a/2k led to membrane alterations similar to those induced in infected cells whereas the 2k fragment impaired the ability of ns4a to induce membrane rearrangements. whether this reflects a biological difference between wnv kun and denv ns4a or is due to the use of alternative experimental approaches remains to be determined. one of the most fascinating mechanisms employed by (+) rna viruses to induce their replication factories is used by sfv. it was shown that the spherules of sfv arise by blebbing at the surface of the plasma membrane [93] . these blebs are internalized and after fusion with lysosomes give rise to large cytoplasmic vacuoles. formation of these membrane alterations requires the viral protein nsp1, which has several functions. it has guanine-7-methyltransferase and guanylyltransferase activities and thus is critically involved in capping of the viral rnas [144] [145] [146] , but at the same time has affinity to lipids [89] . in fact, of the four ns proteins of sfv, only nsp1 has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the plasma membrane [147] . nsp1 is a monotopic membrane protein and its affinity for membranes is dictated by an amphipathic α-helix, located in the central region of the protein [148, 149] . nsp1 has a specific affinity for negatively charged phospholipids, which might account for its prevalent localization to the plasma membrane, where such lipids are enriched. membrane binding of nsp1 via its amphipathic α-helix is essential for alphavirus replication [93] . however, nsp1 is not sufficient for cytoplasmic vacuole formation. for instance, it was found that nsp3 contributes to the transport of the replicase polyprotein from the plasma membrane to the surface of endosomes [150] . these results indicate that nsp1 has to cooperate with other viral and cellular factors to allow formation of the cytoplasmic vacuoles. furthermore, in a recent study kallio et al. [151] have shown that the size of the spherule is dependent on the length of the rna template, in contrast to what has been observed for fhv, another spherule-inducer [141] . these results indicate that in addition to the ns proteins, the viral rna template itself critically determines the morphology of the membranous vesicles. in order to induce the variety of membrane alterations observed in cvb3 -infected cells ( figure 3f ), several membrane-remodeling mechanisms are required: induction of membrane curvature, membrane fusion and membrane-membrane interactions [80] . these rearrangements require the enteroviral proteins 2bc and 3a; their coexpression generates er membrane-derived structures mimicking those observed during viral infection [78] . importantly, 2b and 2c both contain an amphipathic α-helix [152] [153] [154] , a well-known curvature-inducing motif [155] . along the same lines, fmdv 2b and 2bc locate to the er when expressed on their own and cause a swelling of er cisternae [156] . in case of hcv, we recently found that a concerted action of ns3/4a, ns4b, ns5a and ns5b is required to generate the membranous web. furthermore, all these replicase components were capable of inducing membrane vesiculation with ns5a having the highest potential to trigger membrane curvature. importantly, some of these ns5a-induced structures corresponded to dmvs [48] . in addition, ns4b also plays an important role in triggering rearrangements of intracellular membranes [45] . ns4b is an integral membrane protein containing two n-terminal amphipathic α-helices, a highly hydrophobic central core domain composed of four putative transmembrane segments, and a highly conserved c-terminal domain that is thought to harbor two α-helices (reviewed in [157] ). a recent study has demonstrated that ns4b oligomerizes through multiple conserved determinants and that oligomerization appears to be required for membranous web induction [158] . indeed, mutations affecting the highly conserved c-terminal domain impairing ns4b self-interaction resulted in the formation of aberrant dmvs arguing for a central role of ns4b in formation of functional replication compartments [159] . studies on arteriviruses revealed that the sole expression of eav nsp2 and nsp3 is sufficient to induce membrane structures similar to those generated during eav infection [160] . mutations within nsp3, which is a tetra-spanning integral membrane protein, alter membrane rearrangements, highlighting the importance of this protein for the biogenesis of eav-induced dmvs [161] . in case of sars-cov, nsp3, nsp4 and nsp6 were found to be sufficient to induce the formation of dmvs that are similar to those observed in sars-cov-infected cells [162] . these dmvs were, however, smaller in diameter, suggesting a role for other viral proteins or the presence of viral rna in determining the dmv morphology. importantly, em analysis of nsp4 mutants that are impaired in rna replication and virus growth, revealed an aberrant morphology of dmvs as well as an increased prevalence of cms [163] . another important viral protein involved in inducing the sars-cov membranous replication factory is nsp6, which is predicted to contain seven transmembrane segments and a hydrophilic cytoplasmic domain [164] . nsp6 was shown to induce vesicles containing atg5 and lc3-ii as well as phosphatidylinositol-3-phosphate, thus sharing many features with omegasomes, which are omega-shaped membrane compartments that are formed during activation of autophagy [164] . this result suggests that autophagy might contribute to the formation of the membranous replication site of sars-cov. in the last couple of years, our knowledge of the architecture of the replication factories induced by (+) rna viruses has increased substantially. this is primarily due to the more widespread use of et and other high-resolution imaging methods. nevertheless, our knowledge is still rather restricted to descriptions of the morphologies of these complex structures, whereas our understanding of their biogenesis in most cases is very rudimentary. more efforts are required to elucidate the role of the viral proteins in the formation of the replication vesicles, to identify the involved cellular components and the mechanisms used by these proteins to subvert and exploit cellular pathways to establish membranous replication factories. this includes determination of the 3d structure of involved (viral) proteins as well as evaluation of host cell factors and lipids contributing to biogenesis and activity of the replication compartment. in addition, further studies are needed to understand how viruses utilize these compartments to coordinate the different steps of their life cycle (replication, assembly and release) in space and time to achieve efficient replication. work in the authors' laboratory was supported by the deutsche forschungsgemeinschaft (sonder-forschungsbereich 638 the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae the origin of hepatitis c virus isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome transfusion-associated hepatitis not due to viral hepatitis type a or b the viruses and their replication emerging flaviviruses: the spread and resurgence of japanese encephalitis, west nile and dengue viruses virus taxonomy: classification and nomenclature of viruses. ninth report of the international committee on taxonomy of viruses hepatitis c virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups studies on the nature of dengue viruses. v. structure and development of dengue virus in vero cells dengue virus-induced modifications of host cell membranes an electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: maturation events ultrastructural studies on the reproductive system of male aedes aegypti (diptera: culicidae) infected with dengue 2 virus intracellular localisation of dengue-2 rna in mosquito cell culture using electron microscopic in situ hybridisation improved membrane preservation of flavivirus-infected cells with cryosectioning composition and three-dimensional architecture of the dengue virus replication and assembly sites ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells west nile virions aligned along myelin lamellae in organotypic spinal cord cultures ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex immunolocalization of the dengue virus nonstructural glycoprotein ns1 suggests a role in viral rna replication markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells west nile virus strain kunjin ns5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral rna synthesis subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and ns4a tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles reis e sousa, c. innate recognition of viruses reis e sousa, c. activation of mda5 requires higher-order rna structures generated during virus infection interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures maturation site of dengue type 2 virus in cultured mosquito c6/36 cells and vero cells differences in maturation of tick-borne encephalitis virus in mammalian and tick cell line a three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines persistent infection of cultured mammalian cells by japanese encephalitis virus a replication-efficient mutant of west nile virus is insensitive to di particle interference persistent infection of vero cells by the flavivirus murray valley encephalitis virus three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated rna analysis of the effects of alterations in the tick-borne encephalitis virus 3'-noncoding region on translation and rna replication using reporter replicons subgenomic replicons of the flavivirus kunjin: construction and applications construction and applications of yellow fever virus replicons morphological features of murray valley encephalitis virus infection in the central nervous system of swiss mice entry and replication of japanese encephalitis virus in cultured neurogenic cells ultrastructural changes of mouse brain neurons infected with japanese encephalitis virus louis encephalitis virus infection in mice. electron microscopic studies of central nervous system louis encephalitis virus: an ultrastructural study of infection in a mosquito vector electron microscopic and immunofluorescent observations on monkey liver and tissue culture cells infected with the asibi strain of yellow fever virus liver and tissue culture cells infected with the asibi strain of yellow fever virus expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex ultrastructural and biochemical analyses of hepatitis c virus-associated host cell membranes sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication morphological and biochemical characterization of the membranous hepatitis c virus replication compartment construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras virus-like particles in bovine turbinate cells infected with bovine virus diarrhoea/mucosal disease virus the ultrastructure of cell cultures infected with border disease and bovine virus diarrhoea viruses alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (bvdv) cytoplasmic vacuolization responses to cytopathic bovine viral diarrhoea virus bovine viral diarrhea virus ns4b protein is an integral membrane protein associated with golgi markers and rearranged host membranes morphogenesis of pestiviruses: new insights from ultrastructural studies of strain giraffe-1 hiv-1 buds predominantly at the plasma membrane of primary human macrophages multivesicular bodies as a platform for formation of the marburg virus envelope role of the transmembrane domain of marburg virus surface protein gp in assembly of the viral envelope three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle visualizing flock house virus infection in drosophila cells with correlated fluorescence and electron microscopy ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum rna replication of mouse hepatitis virus takes place at double-membrane vesicles qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral rna synthesis fine structure of changes produced in cultured cells sampled at specified intervals during a single growth cycle of polio virus association of polioviral proteins of the p2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography intracellular location and translocation of silent and active poliovirus replication complexes cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures viral reorganization of the secretory pathway generates distinct organelles for rna replication cellular origin and ultrastructure of membranes induced during poliovirus infection kinetics and location of poliovirus macromolecular synthesis in correlation to virus-induced cytopathology remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles complex dynamic development of poliovirus membranous replication complexes the transformation of enterovirus replication structures: a three-dimensional study of single-and double-membrane compartments the ultrastructure of the developing replication site in foot-and-mouth disease virus-infected bhk-38 cells molecular biology of rubella virus novel replication complex architecture in rubella replicon-transfected cells characterization of rubella virus replication complexes using antibodies to double-stranded rna rubella virus replication complexes are virus-modified lysosomes three-dimensional structure of rubella virus factories structural maturation of rubella virus in the golgi complex alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes biogenesis of the semliki forest virus rna replication complex membrane-associated replication complex in arbovirus infection cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis specific membranous structures associated with the replication of group a arboviruses phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes human caliciviruses replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway laboratory efforts to cultivate noroviruses isolation of enzymatically active replication complexes from feline calicivirus-infected cells replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral rna formation of plant rna virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein red clover necrotic mosaic virus replication proteins accumulate at the endoplasmic reticulum localization and biochemical characterization of alfalfa mosaic virus replication complexes complete replication of a eukaryotic virus rna in vitro by a purified rna-dependent rna polymerase the role of the p33:p33/p92 interaction domain in rna replication and intracellular localization of p33 and p92 proteins of cucumber necrosis tombusvirus mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells brome mosaic virus helicase-and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral rna synthesis identification of sequences in brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes a positive-strand rna virus replication complex parallels form and function of retrovirus capsids beet yellows virus replicase and replicative compartments: parallels with other rna viruses ultrastructural features of beta leaves infected with beet yellows virus relation of beet yellows virus to the phloem and to movement in the sieve tube ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of beet yellows virus processing and subcellular localization of the leader papain-like proteinase of beet yellows closterovirus lettuce infectious yellows virus (liyv) rna 1-encoded p34 is an rna-binding protein and exhibits perinuclear localization ultrastructure and mitochondrial vesiculation associated with closterovirus-like particles in leafroll-diseased grapevines cyotchemistry and immuno-cytochemistry of the inclusion bodies induced by grapevine leafroll-associated closteroviruses glrav-1 and glrav-3 the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences architecture and biogenesis of plus-strand rna virus replication factories initiation of poliovirus plus-strand rna synthesis in a membrane complex of infected hela cells complete protein linkage map of poliovirus p3 proteins: interaction of polymerase 3dpol with vpg and with genetic variants of 3ab structure-function analysis of the coxsackievirus protein 3a: identification of residues important for dimerization, viral rna replication, and transport inhibition membrane topography of the hydrophobic anchor sequence of poliovirus 3a and 3ab proteins and the functional effect of 3a/3ab membrane association upon rna replication structural and functional characterization of the poliovirus replication complex subversion of cellular autophagosomal machinery by rna viruses autophagosome supports coxsackievirus b3 replication in host cells induction of autophagy does not affect human rhinovirus type 2 production the autophagy machinery is required to initiate hepatitis c virus replication induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles autophagy: a novel guardian of hcv against innate immune response knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes autophagic machinery activated by dengue virus enhances virus replication aggresomes and autophagy generate sites for virus replication proteins c and ns4b of the flavivirus kunjin translocate independently into the nucleus regulated cleavages at the west nile virus ns4a-2k-ns4b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns4a protein the non-structural protein 4a of dengue virus is an integral membrane protein inducing membrane alterations in a 2k-regulated manner the srebp pathway--insights from insigs and insects viral rna replication in association with cellular membranes nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, rna templates, and polymerase activity feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation an n-terminal amphipathic helix in dengue virus nonstructural protein 4a mediates oligomerization and is essential for replication reaction in alphavirus mrna capping: formation of a covalent complex of nonstructural protein nsp1 with 7-methyl-gmp critical residues of semliki forest virus rna capping enzyme involved in methyltransferase and guanylyltransferase-like activities the effects of palmitoylation on membrane association of semliki forest virus rna capping enzyme the alphavirus replicase protein nsp1 is membrane-associated and has affinity to endocytic organelles semliki forest virus mrna capping enzyme requires association with anionic membrane phospholipids for activity membrane binding mechanism of an rna virus-capping enzyme properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment template rna length determines the size of replication complex spherules for semliki forest virus studies of a putative amphipathic helix in the n-terminus of poliovirus protein 2c amino terminal regions of poliovirus 2c protein mediate membrane binding structure-function analysis of coxsackie b3 virus protein 2b modification of intracellular membrane structures for virus replication inhibition of the secretory pathway by foot-and-mouth disease virus 2bc protein is reproduced by coexpression of 2b with 2c, and the site of inhibition is determined by the subcellular location of 2c hepatitis c virus nonstructural protein 4b: a journey into unexplored territory amphipathic alpha-helix ah2 is a major determinant for the oligomerization of hepatitis c virus nonstructural protein 4b bartenschlager, r. ns4b self-interaction through conserved c-terminal elements is required for the establishment of functional hepatitis c virus replication complexes non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate the authors are very grateful to erik j. snijder (leiden university medical center, leiden, the netherlands) and kè vin knoops (european molecular biology laboratory, grenoble, france) for providing the unpublished pictures depicted in figure 3b and 3d and to montserrat bá rcena (leiden university medical center, leiden, the netherlands) and tero ahola (university of helsinki, helsinki, finland) for providing the unpublished pictures depicted in figure 3f and 3j, respectively. figures 3a, 3c , 3e, 3g, 3h and 3i are reproduced with permission from [60] , [67] , [109] , [79] , [95] and [86] , respectively.we also would like to thank jason m. mackenzie the authors declare no conflict of interest. key: cord-311823-85wj08gr authors: katze, michael g.; fornek, jamie l.; palermo, robert e.; walters, kathie-anne; korth, marcus j. title: innate immune modulation by rna viruses: emerging insights from functional genomics date: 2008 journal: nat rev immunol doi: 10.1038/nri2377 sha: doc_id: 311823 cord_uid: 85wj08gr although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus–host interactions and provide new insights into how rna viruses cause disease. viruses can have a devastating effect despite their small genomes. all rna viruses encode proteins that are essential for structural components and replication, and most encode proteins that function to circumvent host antiviral responses [1] [2] [3] . this limited number of proteins is sufficient to ensure the entry, replication and subsequent spread of the virus. however, viruses do not self-propagate and depend on various host-cell functions to complete their life cycle. the processes of viral entry, the triggering and regulation of the host antiviral response and subsequent viral replication together result in an intricate series of interactions between virus and host. much can be learnt about the nature and complexities of these interactions by global profiling of the transcriptional changes in host cells that occur during viral infection (box 1) . in this review, we discuss how functional genomic and systems-biology approaches are contributing to our understanding of interactions between rna viruses and the host, of viral pathogenesis and of host immunity to infection. rather than providing a comprehensive literature review, we present examples of how these approaches are providing insight into the interaction of viruses with innate immune defence mechanisms, the evaluation of therapeutics that target these pathways and the crucial balance between protective immune responses and immunopathology. in addition, we describe how genomic approaches are being applied to vaccine evaluation and design, and how these approaches can be combined with other high-throughput technologies to provide an improved and integrated systems-biology view of virus infection. although genomic approaches are being used to study a wide variety of viruses, we highlight the current literature through discussion of a select few. among these is influenza virus, for which the looming threat of a new pandemic and concerns regarding therapeutic and vaccine preparedness have stimulated exciting new research efforts. we also review findings relating to hepatitis c virus (hcv) infection, for which genomic analyses are being used to shed light on the response of patients to treatment with type i interferons (ifns) and the relationship between hcv replication and liver disease. in addition, we highlight studies of west nile virus, severe acute respiratory syndrome-associated coronavirus (sars-cov) and ebola virus, all of which have revealed previously undescribed strategies used by these viruses to regulate innate immunity. finally, we discuss how genomic approaches are being applied to vaccine evaluation and how genomics is being combined with other high-throughput approaches to provide a systems-biology view of virus-host interactions. viruses and innate immunity a variety of cellular signalling networks have evolved in host cells to detect and respond to viral infection. one area in which genomics-based analyses are being put to abstract | although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus-host interactions and provide new insights into how rna viruses cause disease. these genes contain interferon (ifn)-responsive promoters and are responsible for the antiviral, antiproliferative and immunomodulatory properties of ifn. over 400 such genes have been identified by microarray analysis. some, such as protein kinase r, ribonuclease l, mx1 (myxovirus resistance 1) and isg15 (ifn-stimulated protein of 15 kda), have well documented antiviral activities, but the precise biological function of the majority of these genes is unknown. particularly good use is in shedding new light on the components of innate antiviral defence mechanisms and the viral strategies used to overcome them. in this section, we review recent studies in which genomic approaches have been used to provide new information on how viruses trigger and regulate innate immune pathways, and to evaluate the use of type i ifn-based therapy as a means to enhance the innate immune response to hcv. mammalian cells have specialized proteins that are responsible for the recognition of virus infection, and other proteins that elicit responses to combat the invading virus. the antiviral response is triggered when host pathogen-recognition receptors (prrs) are engaged by pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids (reviewed in refs 4, 5) . prrs that function in virus recognition include the cytosolic double-stranded rna helicases retinoic-acid-inducible gene i (rig-i) and mda5 (melanoma differentiation-associated gene 5) and certain toll-like receptors (tlrs) that are present on the cell surface or in endosomal membranes. after binding to viral pamps, prrs initiate intracellular signalling cascades that result in the activation of transcription factors, including ifn-regulatory factors (irfs) and nuclear factor-κb (nf-κb). these transcription factors in turn regulate the expression of hundreds of genes, such as ifns and ifn-stimulated genes (isgs) 6, 7 , and pro-inflammatory cytokines and chemokines that are involved in the orchestration of the adaptive immune response (fig. 1) . one way in which gene-expression profiling has been used to examine this aspect of the antiviral response is through the use of mouse embryonic fibroblasts deficient in rig-i or mda5. a recent study demonstrated that west nile virus infection of wild-type cells led to the induction of irf3 target genes and isgs, including several subtypes of ifnα (ref. 8) . this was followed by a second phase of ifn-dependent antiviral gene expression that occurred at a later stage of infection. by contrast, cells lacking rig-i had delayed or inhibited initial and secondary gene-expression responses to the virus, indicating that rig-i has an essential but not exclusive role in initiating innate immune responses to west nile virus (fig. 2) . the additional deletion of mda5 in these cells was found to further block their ability to respond to infection, indicating that the host immune response to west nile virus also involves mda5. this is a noteworthy finding, as previous studies suggested that rig-i and mda5 recognized a specific subset of viruses, rather than acting cooperatively as found in the response to west nile virus 9 . the role of rig-i in the response to influenza virus infection has also been assessed 10 . similar to west nile virus, genomic analysis of influenza virus-infected wild-type and rig-i-deficient mouse embryonic fibroblasts revealed that rig-i is necessary for the type i ifn response to this virus (fig. 2) . in rig-i-deficient cells, influenza virus fails to elicit the expression of ifnβ and of many isgs, including key antiviral mediators such as irf3, stat1 (signal transducer and activator of transcription 1), ifit1 (ifn-induced protein with tetratricopeptide repeats 1; also known as isg56) and isg54 (also known as ifit2). this study also showed that, unlike during infection with west nile virus, mda5 does not function as a secondary mediator of the response to infection with influenza virus 10 . important next steps in these studies will be to compare the profiles of genes induced by each of these viruses -and to determine whether some genes are specific for rig-i or mda5 signalling -and to begin to define the involvement of these genes in innate immunity. although this biological validation process will be necessary to follow-up genomic analyses, few studies so far have included such experiments. functional genomic analyses have also been helpful in elucidating the complex transcriptional events triggered following tlr signalling. tlrs are expressed by various immune cells, including macrophages, dendritic cells and lymphocytes, and a subset of these receptors are involved in viral recognition. so far, genomic studies have largely focused on the analysis of macrophages treated with tlr ligands, such as lipopolysaccharide (lps; a component of the cell wall of gram-negative bacteria) or polyinosinic-polycytidylic acid (a synthetic mimic of viral double-stranded rna, dsrna) [11] [12] [13] . to obtain a comprehensive view of the transcriptional programmes that are induced by tlr activation, elkon et al. used a computational approach to analyse geneexpression data sets derived from four studies in which human or mouse macrophages were stimulated with pathogen-mimetic agents that engage various tlrs 14 . this analysis identified one transcriptional profile that is universally activated by all tlrs and a second profile that is specific to both tlr3 (which specializes in the recognition of viral dsrna) and tlr4 (which recognizes genomics is broadly defined as the study of genomes. the term was first adopted nearly 20 years ago to describe the emerging discipline of using nucleotide sequencing, gene mapping and computational biology to define the structure and organization of a genome 110 . as ever increasing amounts of nucleotide sequence information have become available, the focus of genomics has expanded to include gene function 93 . the human genome project was a driving force in advancing both structural and functional genomics, and the nucleotide sequence information generated by this project has fuelled tremendous advances in our understanding of human health and disease. one way in which this has occurred is through the convergence of comprehensive genome sequence information with advances in high-throughput technology. today, the standard technology in functional genomics is the oligonucleotide microarray [111] [112] [113] . several alternative platforms are available, with the most common being microarrays for which thousands of oligonucleotide 'probes', each corresponding to an mrna transcript, are synthesized in situ directly on a glass slide. such microarrays enable researchers to simultaneously measure the expression of virtually all genes in a genome. for 'target' preparation, mrna is extracted from experimental samples and labelled with fluorescent dyes by reverse transcription. the labelled target is then hybridized with the microarray, and the fluorescence of the features is determined using an array scanner. following image analysis, the data are subjected to a variety of bioinformatic processes to identify statistically significant changes in gene expression between samples. because each comparison yields tens of thousands of data points, mining the data for biological meaning is a formidable challenge. a variety of sophisticated commercial and open-source analysis tools are therefore used to find relationships between differentially expressed genes, to identify networks or signalling pathways that are activated or repressed and to compare gene-expression profiles between experimental samples. envelope components of viruses and cell-surface components of bacteria (such as lps)). a computational analysis of promoter sequences identified nf-κb as the key regulator of the universal response, which occurs early after tlr stimulation, and the ifn-stimulated response element (isre) as the key component of the tlr3 and tlr4 response, which is induced after the nf-κb response. this computational approach provided additional knowledge regarding the kinetics of the tlr3 and tlr4 response, the regulatory circuitry involved and the identity of the genes figure 1 | stimulation of interferon-stimulated gene expression and initiation of antiviral activity. pathogenassociated molecular patterns (pamps) in viral proteins and nucleic acids are recognized by cellular pathogen-recognition receptors (prrs) that include rig-i (retinoic-acid-inducible gene i), mda5 (melanoma differentiation-associated gene 5) and certain toll-like receptors (tlrs). prr-pamp interactions trigger signalling cascades that result in the activation of transcription factors, including interferon (ifn)-regulatory factor 3 (irf3) and nuclear factor-κb (nf-κb), which induce the production of type i ifns, ifn-stimulated genes (isgs) and pro-inflammatory cytokines and chemokines. the specific process differs between antigen-presenting cells, in which both the tlr pathway and the rig-i or mda5 pathway are operative, and other cell types, in which only the rig-i or mda5 pathway is present. activation of prr signalling induces an antiviral state in all cell types, and in antigen-presenting cells it can also induce the production of pro-inflammatory cytokines and chemokines. this normally results in an innate antiviral response that controls infection until it is resolved by the adaptive immune response. however, some viruses, such as the 1918 pandemic influenza virus, elicit an aberrant or disproportional response that results in immunopathology. alternatively, viruses that suppress the type i ifn response can subvert the mechanisms of innate surveillance and diminish the potential adaptive immune response, resulting in a chronic infection. for vaccine strategies, the best induction of a broad adaptive immune response might require some degree of type i ifn response in the initial stages of infection. dcs, dendritic cells; dsrna, double-stranded rna; ifnar, ifnα receptor; il, interleukin; ips1, ifnb-promoter stimulator 1; oas, 2′,5′-oligoadenylate synthetase; pkr, protein kinase r; ssrna, single-stranded rna; stat, signal transducer and activator of transcription; tap1, transporter associated with antigen processing 1; tnf, tumour-necrosis factor. chimeric scid-alb/upa mouse model a chimeric mouse model of severe combined immunodeficient (scid) mice that contain a urokinase plasminogen activator transgene driven by an albumin promoter (alb/upa). these mice can be transplanted with human hepatocytes to generate chimeric mousehuman livers, providing the only small-animal infection model for hepatitis c virus infection. activated in both the universal and tlr3-and tlr4mediated responses. although these studies have provided considerable information regarding the genes activated downstream of tlr activation, it will be advantageous to extend genomic analyses in the context of viral infection using cells lacking the expression of specific tlrs. the ability of a virus to establish an infection depends, at least to some extent, on its ability to block the host innate immune response or to modulate the activity of antiviral effector proteins. hcv is one example of a virus that has devised a means to block the initial triggering of the host innate immune response. several studies have shown that the hcv ns3-ns4a serine protease blocks the tlr3-dependent activation of irf3 (refs 17, 19) . this is achieved by ns3-ns4a-mediated cleavage of trif (toll/interleukin-1 (il-1) receptor-domain-containing adaptor protein inducing ifnβ), an adaptor protein that links tlr3 to kinases that are responsible for activating irf3 and nf-κb 17, 19 . hcv also inhibits the ability of rig-i to activate irf3 (refs 15,16,18,20), which is achieved through ns3-ns4a-mediated cleavage of ips1 (ifnb-promoter stimulator 1; also known as visa, cardif, mavs), a recently identified rig-i adaptor protein [21] [22] [23] [24] [25] . in light of these findings, it is both perplexing and paradoxical that virtually all gene-expression profiling carried out using hcv-infected tissue shows the induction of isg expression, including irf3 target genes [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . the induction of isg expression is observed in liver tissue from hcv-infected patients 30, 32, 37 and during the initial host response in acutely infected chimpanzees 26, 28 , and is a major part of the transcriptional response to hcv infection in the chimeric scid-alb/upa mouse model 33 . this poses an interesting question about the source of both type i ifns and isg expression. it is possible that isgs are mainly expressed in uninfected hepatocytes and are induced in response to exogenous type i ifn released from adjacent hcv-infected cells. alternatively, it has been suggested that t cells and plasmacytoid dendritic cells that infiltrate the liver are a possible source of hepatic type i ifns 37 . although this is possible, it is relevant to note that hcv infection in the scid-alb/upa mouse model is also associated with the induction of hepatic isg expression in the absence of these immune cell types 33 . other genomic studies have revealed examples of highly virulent viruses that are relatively successful at inhibiting isg expression. perhaps the best example is a characterization of the host transcriptional response of human liver cells infected with filoviruses 38 . this study demonstrated the marked suppression of genes in key innate antiviral pathways, including those mediated by irf3. interestingly, this study also suggested a correlation between the antagonism of the type i ifn response and filovirus virulence. highly virulent viruses, such as zaire ebola virus and marburgvirus, inhibit the expression of most isgs that are induced in uninfected ifn-treated cells. by contrast, the relatively non-pathogenic reston ebola virus is less inhibitory and induces the expression of more than 20% of these genes. the suppression of the type i ifn response by the pathogenic viruses is associated with more rapid viral spread and higher rate of viral replication than that observed during reston ebola virus infection. a comparable trend was seen in a study evaluating the host transcriptional response and inflammation in the brains of mice infected with rabies virus 39 . this study revealed that infection with an attenuated virus results in both inflammation and the induction of expression of key isgs. however, these events are either absent or diminished during infection with a highly pathogenic rabies virus. on the basis of results with filoviruses, it would follow that attenuation of the type i ifn response would be associated with higher viral replication and spread in the case of pathogenic infection with rabies virus; however, this was not measured in the study. similarly, infection with highly virulent pseudorabies virus suppressed the induction of a subset of isgs, even in type i ifn-treated cells 40 . together, these data suggest that the virulence of acute, highly pathogenic viruses is at least partially related to their ability to suppress the host antiviral response, which seems to allow higher levels of viral replication. genomic analyses using cells that lack rig-i (retinoic-acid-inducible gene i) show the requirement for this pathogen-recognition receptor in the induction of interferonregulatory factor 3 (irf3) target genes and interferon-stimulated genes (isgs) by west nile virus and influenza virus. a | the infection of rig-i-deficient cells by west nile virus results in the delay and partial inhibition of isg expression. deletion of mda5 (melanoma differentiation-associated gene 5) further blocks the response to infection (not shown), indicating that the response to west nile virus also involves mda5. b | by contrast, the infection of rig-i-deficient cells by influenza virus results in a near complete inhibition of isg expression that is not further blocked by the absence of mda5, suggesting that mda5 does not mediate influenza virus-induced gene-expression changes. pamps, pathogen-associated molecular patterns. images generated from data in refs 8, 10. suppression of innate immunity and persistent infection. evidence discussed in this review suggests that suppression of elements of the innate immune response enables extensive viral replication and increased pathogenesis. does the converse hold true for a virus such as hcv, which typically establishes a persistent infection characterized by mild (or slowly progressing) disease? some evidence suggests that this might be the case; for example, studies using the chimeric scid-alb/upa mouse model indicate that an attenuated type i ifn response is associated with higher levels of intrahepatic hcv replication together with a greater induction of lipid metabolism and oxidative-stress genes, which have the potential to cause cytopathic effects 33 . similarly, gene-expression profiling of serial liver biopsies obtained from patients that had received an hcv-infected liver transplant shows that rapid progression of fibrosis following transplantation is associated with the suppression of genes involved in the type i ifn response, antigen presentation and the cytotoxic t-cell response 30 . although in these studies the apparent defect in the host antiviral response is probably related to host genetics rather than viral factors, the concept that a defective innate immune response correlates with enhanced pathogenesis is still evident. it is possible that the selective pressures on persistent viruses never resulted in a need for a complete subversion of host innate antiviral responses, so such viruses use these responses to limit their replication to a level that does not significantly affect the normal functions of the host cell. conversely, acute viruses, such as filoviruses, highly pathogenic influenza virus and rabies virus, seem to have evolved to antagonize these responses following cell entry to allow immediate, high levels of replication, which subsequently facilitate virus spread and transmission. given the importance of the innate immune response in regulating virus infection, there is considerable interest in enhancing or modulating this response for therapeutic benefit. one role for genomics in this area is assisting in the evaluation of type i ifn treatment of hcv infection. combination therapy with ifnα and the antiviral drug ribavirin results in virus clearance in only ~50% of individuals infected with hcv genotype 1 and ~80% of individuals infected with hcv genotypes 2 or 3 (refs 41-44). as ifnα is the only approved treatment for chronic hcv, there is strong interest in improving this therapy, in understanding the molecular mechanisms that underlie treatment failure and in identifying markers to accurately predict a patient's response to treatment (that is, responders or non-responders). several groups that have used transcriptional profiling of patient hepatic tissue to address these issues have found that higher levels of expression of isgs before treatment are associated with treatment failure. for example, chen et al. carried out microarray experiments on pretreatment liver tissue obtained from a cohort of 31 patients with chronic hcv infection who subsequently underwent ifnα and ribavirin therapy 45 . this analysis identified a set of 18 genes, many of which are known isgs; in general these genes were more highly induced in the livers of patients that did not respond to therapy. although the authors suggest that this set of genes could therefore be used to predict the response to therapy, it remains to be determined whether they can be used to accurately predict the response in other patient cohorts. similarly, feld et al. showed that non-responders have significantly higher intrahepatic pretreatment expression levels of isgs than patients who respond to type i ifn therapy 46 . although these studies are intriguing, it is still unclear whether there is a causal relationship between higher pretreatment levels of isgs and therapy failure. other factors, such as viral quasispecies diversity, may also be important. owing to the technical and ethical issues of obtaining sufficient liver material for gene-expression studies, investigators have also used peripheral-blood mononuclear cells (pbmcs) to evaluate the response to treatment 47,48 . an example is virahepc, a multicentre study designed to define the differences in response rates among caucasian and african americans and to identify host and viral parameters associated with a lack of response to treatment 48 . overall, this study showed that, during the first 28 days of treatment, a lower level of induction of known isgs is associated with non-responsiveness to type i ifn treatment. however, in many cases, these differences are not strikingly dissimilar between responders and non-responders. the implication of such minor differences with respect to antiviral function is uncertain and the feasibility of using them for predicting a patient's response is questionable. in addition, analyses using pbmcs should be interpreted with caution, as a recent study showed that the transcriptional response to type i ifn treatment is significantly different in the blood and the liver of hcv-infected chimpanzees, presumably owing to the absence of hcv replication in pbmcs 49 . although it has not yet been evaluated, this will almost certainly hold true for humans as well. an alternative mechanism of a failed response to type i ifn treatment could involve the induction of genes associated with ifn inhibitory pathways 46 . walsh et al. found significantly increased intrahepatic expression of the gene encoding suppressor of cytokine signalling 3 (socs3) in patients who did not respond to type i ifn treatment 50 . enhanced intrahepatic socs3 expression is also thought to contribute to the non-responsiveness of hcv-infected chimpanzees to type i ifn therapy 51 . however, a separate evaluation of 21 patients for intrahepatic socs3 mrna expression before antiviral therapy actually found higher levels of expression in those patients who went on to respond successfully to type i ifn treatment 51 . therefore, the relationship between treatment failure and induction of type i ifn inhibitory pathways is currently less clear than that between higher pretreatment levels of expression of isgs and treatment failure. there are still surprisingly few answers to the fundamental question of how virus infection results in disease pathology. although the mechanisms are certain to be different for each virus, a common theme is that there is abarrently high and sustained nature reviews | immunology infection resolves immunopathology a crucial balance between protective immune responses and immunopathology 52,53 . although the innate immune response is designed to target and eliminate invading pathogens, genomic analyses have indicated that some viruses, such as the highly virulent influenza virus that was responsible for the 1918 pandemic, elicit aberrant or disproportional innate immune responses that may also harm the host. the 1918 influenza virus pandemic (known as the spanish flu) killed as many as 50 million people worldwide 54 , and several studies have begun to provide clues to what made this virus so deadly (reviewed in . although genomic analyses have previously been carried out using engineered viruses containing one or more genes from the 1918 pandemic virus 58,59 , a major advance in the ability to study this virus came from its reconstruction based on nucleotide sequence information 60 . genomic analyses of lung or bronchial tissue derived from mice or macaques that were infected with the reconstructed 1918 virus indicate how the beneficial role of the innate immune response can be tipped towards immunopathology. mice infected with the reconstituted 1918 influenza virus show severe pulmonary pathology and an increased and accelerated transcriptional activation of immuneresponse genes 61 . this includes a marked activation of genes associated with pro-inflammatory and cell-death pathways by 24 hours after infection (fig. 3) , which remain unabated until the death of the animals. this response is in contrast to the less dramatic and delayed host immune responses (and less severe disease pathology) in mice that were infected with influenza viruses containing only subsets of genes from the 1918 virus, including the haemagglutinin (ha) and non-structural protein (ns) genes, or the ha, neuraminidase (na), matrix (m) and nucleoprotein (np) genes. these findings suggest that enhanced pro-inflammatory and cell-death responses can contribute to severe immunopathology. an additional study that evaluated the host response to the 1918 influenza virus using a cynomologus macaque (macaca fascicularis) infection model produced similar results 62 . in macaques, the 1918 virus replicates to high levels and spreads rapidly throughout the respiratory tract of infected animals, causing severe lung damage and the massive infiltration of immune cells throughout the course of infection. functional genomic analyses of bronchial tissue revealed that the 1918 virus triggers the aberrantly high and sustained expression of numerous genes involved in the innate immune response, including pro-inflammatory cytokines and chemokines. although the timing of the response is somewhat different, the increased and sustained host response in macaques that were infected with the 1918 virus is similar to that observed in mice. these studies reveal similarities and differences in the host response to contemporary and 1918 pandemic influenza virus infection. first, contemporary and 1918 viruses each trigger an innate immune response that includes the expression of nf-κb and irf3 target genes, which is expected to occur if the virus triggers the rig-i pathway in infected respiratory cells. second, both viruses trigger a robust cytokine response that probably attracts immune-cell infiltration to infected tissues. unlike contemporary virus strains, in which the early response to infection is resolved, the innate immune response triggered by the 1918 virus is characterized by a strong and sustained induction that is associated with massive tissue damage and death of the infected animal. however, in preliminary genomic analyses carried out with lung tissue from macaques that were infected with avian h5n1 viruses, we have found that there are significant differences in the regulation of antiviral responses by the 1918 pandemic and h5n1 viruses (j. c. kash and m.g.k., unpublished observations). therefore, there may be differences in the ways in which highly pathogenic influenza viruses regulate the innate immune response and cause disease. the enhanced pathogenicity of the 1918 and h5n1 influenza viruses might be attributed to distinct components of their genomes. although much emphasis has been placed on the ns1 protein of the 1918 virus acting as an inhibitor of the type i ifn response, recent evidence suggests that the viral proteins pb1 (a polymerase), ha and na contribute to its pathogenicity 63 . likewise, the polymerases of h5n1 viruses have been linked to increased viral pathogenesis 64 , suggesting that the increased pathogenesis of these viruses may be related to their replicative fitness. another respiratory virus, sars-cov, has emerged recently and has caused great concern among the public health and research communities. it has been suggested that disease pathology associated with sars-cov is caused by a disproportional immune response, illustrated by increased levels of pro-inflammatory cytokines and chemokines [65] [66] [67] . studies carried out in our laboratory have combined the use of functional genomics with a cynomologus macaque infection model to study the host response to this virus 68 . we observed that sars-cov-infected macaques show a strong increase in the expression of innate immune response genes early after infection and that this response wanes after 4 days. conversely, genes that are induced later in infection tend to be involved in the cell cycle and in cell repair. none of the animals used in this study succumbed to infection, and sars-cov-induced pathology in these macaques resembled the pathological changes seen in the majority of human patients with sars who recover from the disease 68 . unlike the findings of the 1918 pandemic influenza virus study, these data suggest that early immune responses to sars-cov infection are productive and enable the host to properly fight the virus, allowing a return to cellular homeostasis. however, in the 10% of human infections in which sars-cov infection is fatal (mostly in the elderly), it is possible that the timing or magnitude of the response results in immunopathology. studies using aged macaques might help to address this possibility. viruses such as sars-cov, h5n1 influenza virus and 1918 influenza virus are all zoonotic infections, in which a virus that was adapted to another host was transferred to humans. because the type i ifn response is somewhat different in different hosts, it is possible that these viruses, which have adapted to their normal animal hosts, elicit an aberrant response when infecting a human host in which adaptation has not occurred, resulting in immunopathology. this possibility also raises the question of how appropriate the various animal infection models (such as mice and macaques) are for the understanding of human pathogenesis. as reviewed elsewhere 56 , there are both advantages and disadvantages associated with different animal models, and it is important to keep in mind that responses observed using an animal model may not always accurately reflect the response in humans. genomics in vaccine evaluation and design genomic information and high-throughput technologies are beginning to have an impact on the field of vaccine development, but the main focus has been directed towards identifying important conserved features of pathogens that could serve as immunogens and characterizing host genotypes associated with strong protective responses [69] [70] [71] . in recent years, it has become evident that the type i ifn response has a significant role in the development of the adaptive immune response. this commences with the influence of type i ifns on the activation, maturation and migration of dendritic cells 72, 73 . the development of the antibody response is also enhanced by type i ifns through the direct effect of ifn on b cells and on the priming or function of cd4 + t helper cells 74 . there is now also evidence that type i ifns act directly on cd8 + t cells to promote clonal expansion and indirectly by stimulating cross-priming by antigen-presenting cells that have engulfed infected cells to acquire antigen [75] [76] [77] . so, viruses that suppress the type i ifn response not only subvert the mechanisms of innate surveillance, but also diminish the potential adaptive immune response that could mediate viral clearance or establish a quiescent, non-pathogenic state. for vaccine strategies, the implication is then that the best induction of a broad adaptive immune response will require some degree of type i ifn response in the initial stages. just as dna microarray technology spurred the development of functional genomics, the development of immunomic microarray technology is driving the emerging field of functional immunomics (reviewed in ref. 114) . the goal of immunomics is to provide a detailed understanding of host immunological responses to foreign antigens through the use of high-throughput technologies and computational methods. the technologies that are central to this effort include antibody microarrays (consisting of antibodies as probes and antigens as targets), peptide microarrays (consisting of antigen peptides as probes and serum antibodies as targets) and more recently peptide-mhc microarrays (consisting of recombinant peptide-mhc complexes and co-stimulatory molecules as probes and populations of t cells as targets). antibody microarrays are used to measure the concentration of specific antigens (such as cancer antigens), whereas peptide-mhc microarrays can map mhc-restricted t-cell epitopes which are involved in helper and regulatory functions of the immune system. peptide microarrays are used in various applications, including b-cell epitope mapping and detection and diagnostic assays. peptide microarrays are also being used in vaccine studies for mapping epitopes associated with effective immune responses and for testing the ability of experimental vaccines to generate specific antibody responses against those epitopes after immunization and challenge 115 . studies of immune responses that are associated with different clinical outcomes, such as those of patients who are hiv positive and who rapidly progress to aids and those of infected long-term survivors, can also provide direction for the development of vaccines 116 . it is probable that immunomics will become an increasingly integral part of a systems-biology approach to vaccine development and of obtaining a better understanding of host immunity to virus infection. animal models. we have used functional genomics to evaluate a live influenza virus vaccine in a macaque model, in which attenuation of the virus was accomplished by truncation of the gene encoding ns178. this modification eliminates or reduces the ability of the ns1 protein to antagonize type i ifn production 79 and, in mouse and swine models, such attenuated live viruses are immunogenic and protective 80, 81 . gene-expression profiling of tracheal and bronchial epithelial cells from macaques immunized with the ns1-truncated virus show clear evidence of a robust type i ifn response. compared with immunization with a traditional killedvirus vaccine, the attenuated live-virus-vaccine group had higher antibody titres before and after challenge and a broader range of influenza virus-specific t-cell responses. following challenge with infective virus, the protection afforded by the attenuated live-virus vaccine was evident by the limited viral replication and minor pathology observed in treated animals. in addition, gene-expression profiles of lung tissue from animals that received the attenuated live-virus vaccine show less upregulation of innate and pro-inflammatory response genes compared with animals immunized with the killed-virus vaccine or untreated animals. at the same time, the transcriptional profiles for the attenuated live-virus-vaccine animals showed a stronger induction of genes that are associated with b-cell and t-cell responses. the general picture overall is that the truncated-ns1containing influenza virus vaccine undergoes minimal replication but induces sufficient type i ifns to galvanize the adaptive immune response, leaving the host in a state of adaptive preparedness after just one immunization. the early induction of type i ifns in response to the truncated-ns1-containing vaccine might be especially important in the local b-cell response that is crucial for viral clearance. a relevant observation in this regard is that early stimulation of the respiratory-tract b cells (within 48 hours of influenza virus infection) was shown to be strongly driven by virus-induced type i ifns 82, 83 . human studies. at present, there are only limited examples in which gene-expression profiling applied to vaccine design supports a picture consistent with that described above for the influenza virus model. the standards for prevention of measles and yellow fever are immunizations with attenuated live-virus vaccines. to assess the impact of infection on primary target cells, gene-expression profiling was carried out in tissue-culture systems comparing wild-type and vaccine strains. for both measles and yellow fever, it was clear that the attenuated vaccine strains led to a greater induction of the type i ifn response than the pathogenic wild-type virus 84, 85 . although in the case of measles virus this disparity in the ifn response has previously been shown by serological techniques 86 , expression analysis indicated that the antagonism of the response by the wild-type virus originated at the level of transcription. this early induction of the type i ifn response was also evident in microarray studies examining chimeras of the yellow fever vaccine strain that were devised as attenuated live-virus vaccines against other flaviviruses such as dengue virus 87 . this contrasted with the low-level induction of type i ifns by dengue virus infection as seen by expression profiling using infection of primary cells or macaque disease models 87, 88 . it is interesting to note that the measles and yellow fever vaccine strains are attenuated by passage in cells from other species. therefore, with suitable molecular understanding, the ability of some viruses to induce type i ifns might be optimized by directed molecular techniques, as was done for the truncated-ns1 influenza virus strain. as an alternative, one might consider using recombinant type i ifns as vaccine adjuvants instead of inducing them with the vaccine constituents 89 , but at our present level of understanding, these approaches have yet to prove clinically tenable 90 . functional genomics for the evaluation of immunological memory. functional genomic studies have been more equivocal in assessing the significance of type i ifn production during the immunological memory response. in the aforementioned macaque influenza virus study, animals receiving the attenuated live-virus vaccine showed upregulation of type i ifn pathways in tracheobronchial cells 2 days after challenge, and this coincided with the development of a strong memory response 78 . this type i ifn induction seems to be weaker than that observed at the corresponding time after the primary exposure to the vaccine, but is far lower than the type i ifn induction observed after challenge of animals receiving the killed-virus vaccine or of naive animals. this would suggest some role of this innate pathway in stimulating immunological recall. in contrast to this, examination of transcriptional profiles observed shortly after rechallenge of human pbmcs from individuals previously immunized against influenza virus are more in accord with early production of ifnγ, possibly arising from antigenic stimulation of memory cells 91 . dhiman et al. also did not see evidence of a type i ifn response in a microarray study of whole blood taken from individuals immunized with measles virus after rechallenge with an attenuated live-virus vaccine strain, although genes associated with lymphocyte activation and survival were upregulated 92 . it could be considered that technical issues might hamper the relevance of these studies in assessing the role of type i ifns in the memory response. in the case of the first study 91 , pbmcs are not a primary target of influenza virus, so virus internalization might have been inefficient and a type i ifn response might have been poor. in the measles study 92 , the earliest time point examined was 7 days after rechallenge rather than early, when the type i ifn response would be expected to be strongest. therefore, further functional genomic experiments, with appropriately designed models, are required to address whether an early innate immune response is a key stage in triggering immunological memory. functional genomics has proven to be a highly efficient method for providing broad views of the host response in studies of virus-host interactions. as we have discussed, these techniques have revealed the activation or nature reviews | immunology repression of innate immune signalling pathways, crosstalk between pathways, the timing and magnitude of the immune response and, depending on the experimental system, the degree to which the immune response varies among individuals. conversely, functional genomics has been less effective in pinpointing the role of specific host genes in the antiviral response or, somewhat surprisingly, in identifying previously undiscovered genes and pathways that are important in the infection process, despite this being one of its early goals 93 . indeed, the early assumption that functional genomics would provide quick answers to the complexities of virus-host interactions has proved naive. how then can greater benefits be gained from using functional genomics to study virus-host interactions? rather than being used as a singular approach, the future of functional genomics in virology will be in the integration of genomic data with data derived from other high-throughput technologies (fig. 4) . the obvious complementary approach to functional genomics is proteomics, which will provide much needed information regarding the correlation of gene expression with protein abundance [94] [95] [96] . our group has begun to integrate genomic and proteomic data to better understand the host response to influenza virus infection 97 . other possibilities for data integration are also beginning to unfold. for example, micrornas, which regulate both transcription and translation, might have an important role in mediating virus-host interactions 98 . the discovery of micrornas in certain large dna viruses, such as herpesvirus, suggests that some viruses may encode micrornas to regulate cellular functions 99 . in addition, immunomic strategies (box 2) will provide additional opportunities to interrogate the host immune response; screens using small interfering rnas are currently being combined with genomic data to identify specific cellular proteins that are used by viruses during infection [100] [101] [102] . together with virology, clinical and pathology data, this integrated set of information might provide the systems-biology view that will be needed to clearly understand the role of specific host genes and pathways involved in the development of immunity or disease after virus infection. another use for genomics that will no doubt expand is expression quantitative trait loci (eqtl) mapping 103 . the combination of global gene-expression data with eqtl mapping provides greater power in elucidating complex genetic traits in addition to providing insights into specific genes or mutations that might be responsible for the trait in question. this approach is currently being used to better understand the genetic basis for various disease conditions in mice [104] [105] [106] , and it is likely that it will also be useful in increasing our understanding of virus-host interactions. for example, using recombinant inbred strains of mice derived from parental strains that react differently to infection with a given virus, it should be possible to use eqtl mapping to determine chromosomal locations for potential traitcontributing factors and highlight genes of interest for the trait. with this increased level of complexity, however, it will be important to work closely with the bioinformatics and computational-modelling communities, and to make best use of the sophisticated bioinformatics tools, data-mining schemes and mathematical-modelling strategies that are continually being developed 107, 108 . it might also be necessary to take a step back to simpler experimental systems (such as cell-culture models) to dissect cellular events before moving on to more complex in vivo models. the use of combined computational approaches that can account for gene-regulatory networks and cell-to-cell interactions will also facilitate the move to whole animal physiological modelling. functional genomics is clearly providing advances in our understanding of virus-host interactions, and the evolution to an integrated systems-biology approach holds even greater promise for the field. in addition to providing new insights into viral pathogenesis and host immunity, this approach provides a host-oriented antiviral discovery paradigm with the potential for discovering the benefits of functional genomics will be further enhanced by integrating genomic data with data derived from other high-throughput technologies. the potential information and biological insights provided by these technologies are shown. together, these approaches will help to provide a systems-biology view of virus-host interactions that spans the flow of biological information from dna (genetics) to mrna (genomics) to protein (proteomics) to protein function (immunomics). new targets for broad-spectrum antiviral therapies 109 and for improving vaccine evaluation and design. we are optimistic about continuing advancements in the technologies and computational methods used to study virus-host interactions and in improved capabilities to identify, characterize and circumvent the strategies used by viruses to outsmart their long-suffering hosts. recent studies have shown that rig-i preferentially recognizes single-stranded rna (ssrna) with polyu motifs, whereas mda5 recognizes long dsrna molecules. these differences might help to explain the differential recognition and innate immune signalling induced by different rna viruses [117] [118] [119] . pathogenic viruses: smart manipulators of the interferon system pathogen subversion of cell-intrinsic innate immunity viruses and interferon: a fight for supremacy principles of intracellular viral recognition toll-like receptors, rig-i-like rna helicases and the antiviral innate immune response functional classification of interferon-stimulated genes identified using microarrays identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda5 signaling through ips-1 in this study, genomic analyses show that rig-i and mda5 operate cooperatively to establish an antiviral state and mediate an ifn amplification loop that supports immune effector gene expression during west nile virus infection differential roles of mda5 and rig-i helicases in the recognition of rna viruses distinct rig-i and mda5 signaling by rna viruses in innate immunity systems biology approaches identify atf3 as a negative regulator of toll-like receptor 4 transcriptional profiling of the lps induced nf-κb response in macrophages human macrophage activation programs induced by bacterial pathogens functional genomic delineation of tlrinduced transcriptional networks regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling immune evasion by hepatitis c virus ns3/ 4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i molecular determinants of trif proteolysis mediated by the hepatitis c virus ns3/4a protease inhibition of rig-i-dependent signaling to the interferon pathway during hepatitis c virus expression and restoration of signaling by ikkε viral and therapeutic control of ifn-β promoter stimulator 1 during hepatitis c virus infection ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf3 visa is an adapter protein required for virus-triggered ifn-β signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus dna microarray analysis of chimpanzee liver during acute resolving hepatitis c virus infection intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees genomic analysis of the host response to hepatitis c virus infection hepatitis c virus and liver disease: global transcriptional profiling and identification of potential markers gene expression patterns that correlate with hepatitis c and early progression to fibrosis in liver transplant recipients protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic use of functional genomics to understand influenza-host interactions a question of self-preservation: immunopathology in influenza virus infection cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza global host immune response: pathogenesis and transcriptional profiling of type a influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus characterization of the reconstructed 1918 spanish influenza pandemic virus genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus genomic analyses indicate that cynomologus macaques infected with the 1918 influenza virus mount an immune response that is characterized by dysregulation of the antiviral response and that is insufficient for protection. this indicates that atypical host innate immune responses might contribute to lethality single gene reassortants identify a critical role for pb1, ha, and na in the high virulence of the 1918 pandemic influenza virus molecular basis for high virulence of hong kong h5n1 influenza a viruses interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome expression profile of immune response genes in patients with severe acute respiratory syndrome an interferon-γ-related cytokine storm in sars patients functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques immunomics: discovering new targets for vaccines and therapeutics heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics bridging the knowledge gaps in vaccine design these authors show the importance of type i ifns in plasmacytoid dendritic-cell activation and migration using an elegant series of in vivo mouse models that exploited ifnα-receptor-deficient mice and synthetic tlr ligands type i ifns enhance the terminal differentiation of dendritic cells cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd8 t cells for clonal expansion and memory formation a role for the transcription factor relb in ifn-α production and in ifn-α-stimulated crosspriming functional genomic and serological analysis of the protective immune response resulting from vaccination of macaques with an ns1-truncated influenza virus influenza virus evades innate and adaptive immunity via the ns1 protein immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns1 genes efficacy of intranasal administration of a truncated ns1 modified live influenza virus vaccine in swine type i ifn receptor signals directly stimulate local b cells early following influenza virus infection influenza virus infection causes global respiratory tract b cell response modulation via innate immune signals measles virus-induced modulation of host-cell gene expression despite a limited technical capacity to measure only 1,176 genes host-cell interaction of attenuated and wild-type strains of yellow fever virus can be differentiated at early stages of hepatocyte infection evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of α/β interferon production innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4. am transcriptional activation of interferon-stimulated genes but not of cytokine genes after primary infection of rhesus macaques with dengue virus type 1 type i ifn as a vaccine adjuvant for both systemic and mucosal vaccination against influenza virus the relevance of cytokines for development of protective immunity and rational design of vaccines transcriptional analysis of human peripheral blood mononuclear cells after influenza immunization immune activation at effector and gene expression levels after measles vaccination in healthy individuals: a pilot study virology in the 21st century: finding function with functional genomics hepatoproteomics: applying proteomic technologies to the study of liver function and disease viral proteomics: global evaluation of viruses and their interaction with the host integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics microarray analysis shows that some micrornas downregulate large numbers of target mrnas identification of micrornas of the herpesvirus family identification of host proteins required for hiv infection through a functional genomic screen copi activity coupled with fatty acid biosynthesis is required for viral replication identification of host genes involved in hepatitis c virus replication by small interfering rna technology genetical genomics: combining genetics with gene expression analysis an integrative genomics strategy for systematic characterization of genetic loci modulating phenotypes integrating genetic and gene expression data: application to cardiovascular and metabolic traits in mice uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics' computational methodologies for modelling, analysis and simulation of signalling networks the model organism as a system: integrating 'omics' data sets systems biology and the host response to viral infection a new discipline, a new name microarray analysis: basic strategies for successful experiments dna microarray technology for the microbiologist: an overview chips with everything: dna microarrays in infectious diseases from functional genomics to functional immunomics: new challenges, old problems, big rewards microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen microarray profiling of antiviral antibodies for the development of diagnostics, vaccines, and therapeutics nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses innate immunity induced by compositiondependent rig-i recognition of hepatitis c virus rna the length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiationassociated gene 5 we thank b. paeper and s. proll for discussions and assistance with preparation of the original figures. research in the authors' laboratory is supported by public health service grants (r01ai022646, r01hl080621, r21ai017892, r 2 4 r r 01 6 3 5 4 , p 01 a i 0 5 210 6 , p 01 a i 0 5 811 3 , p30da015625 and p51rr000166) from the national institutes of health, usa. this study uses gene-expression profiling of serial liver-biopsy samples from patients that had received a liver transplant to demonstrate that rapidly progressive fibrosis is associated with an impaired immune response, as indicated by a lack of induction of genes associated with the ifnmediated antiviral response, antigen presentation and cytotoxic t-cell response. 31 key: cord-300154-3p7tjp5j authors: pezacki, john paul; blake, jessie a; danielson, dana c; kennedy, david c; lyn, rodney k; singaravelu, ragunath title: chemical contrast for imaging living systems: molecular vibrations drive cars microscopy date: 2011-02-14 journal: nat chem biol doi: 10.1038/nchembio.525 sha: doc_id: 300154 cord_uid: 3p7tjp5j cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-stokes raman scattering (cars) microscopy without the need for labels. here we review the application of cars microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. high-resolution cars microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. the cars imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules. a ll known living organisms are composed of complex and molecularly diverse sets of biopolymers, small molecules and ions, as well as, of course, water 1 . these biomolecules serve both structural and functional purposes and together give rise to different types of cells and tissues with varying physical attributes and primary functions 2, 3 . a large proportion of living matter is composed of polymers that rely on a rather small set of monomers to create a large degree of molecular diversity. for example, polynucleotides that are made up of four simple building blocks (nucleotides) are simultaneously the source of genetic material, key players in the transcription and translation of their respective genomes [4] [5] [6] [7] [8] , and capable of catalyzing chemical transformations 6, 9, 10 . other highly abundant biomolecules include proteins, lipids [11] [12] [13] [14] and carbohydrates 15, 16 , which play bifunctional roles as energy stores and recyclable structural components. given that polymeric and self-assembled materials are key to both the structure and function of living organisms, the ability to observe them in their native environment is highly valuable. most methods currently available for tracking molecules in living systems involve the addition of labels containing unnatural functionalities (for example, fluorophores) that provide contrast in imaging experiments. there are many strategies for the incorporation of these groups into biomolecules 1, [17] [18] [19] ; however, all require perturbation of the cellular environment. for this reason, it is very important to develop new label-free imaging methods based on the natural biochemical make-up of the living system under study. label-free methods for cellular imaging rely on inherent electronic or vibrational resonances, which provide natural chemical contrast of cellular biomolecules. excitation of these resonances leads to phenomena such as autofluorescence, infrared absorption and raman scattering 20 . other label-free imaging modalities commonly used in clinical settings, including magnetic resonance imaging (mri) and ultrasound, achieve tissue penetration but suffer from poor resolution. as such, these techniques only provide whole tissue rather than molecular information and are not useful for cell culture-based experiments. vibrational absorption spectroscopies (for example, infrared spectroscopy) john paul pezacki 1-3 *, jessie a blake 1,2 , dana c danielson 1,3 , david c kennedy 1 , rodney k lyn 1,2 & ragunath singaravelu 1, 3 cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-stokes raman scattering (cars) microscopy without the need for labels. here we review the application of cars microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. high-resolution cars microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. the cars imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules. yield detailed information about the chemical bonds within a given sample. however, the low energy of the transitions involved requires excitation by longer wavelength radiation relative to fluorescence experiments. as a result, vibrational absorption microscopies generally have inherently low spatial resolution. vibrational scattering spectroscopies including raman scattering techniques offer slightly better resolution with similar information about the unique chemical bonds within a given sample but suffer from low sensitivity; thus, imaging requires high laser power and long acquisition times, which are incompatible with live-cell imaging 21 . poor sensitivity in raman scattering detection can be overcome using enhancement techniques such as surfaceenhanced raman scattering [22] [23] [24] [25] , which gives near-single molecule sensitivity for biological imaging but involves labeling, typically with functionalized metal nanoparticles [26] [27] [28] [29] . on the other hand, cars, which is a nonlinear optical version of raman scattering (box 1, fig. 1 ), offers the same natural chemical contrast as linear raman but with much better sensitivity 21, [30] [31] [32] . as a result of the pioneering work in this field by x.s. xie and coworkers, it is now possible to obtain raman images of live cells nondestructively 32 . cars microscopy, being a multiphoton method, also has very high spatial resolution typical of other multiphoton microscopies. although there are distinct raman scattering signals for peptides and nucleic acids that are clearly visible when using raman imaging techniques on cells (box 1), the strongest signal arises from lipidic c-h bond stretches. thus the vast majority of applications of cars microscopy to date have involved lipid imaging. although this may seem limiting at first, the roles of lipids are so numerous and diverse in biology that the ability to image them has provided insight into a vast diversity of processes. lipids make up all cellular membranes, control phenomena that are critical for cell signaling and function and mediate processes such as entry and secretion within a cell [11] [12] [13] [33] [34] [35] [36] . lipids are also an important source of raw materials and energy. the dysregulation of lipid biogenesis, storage and metabolism can give rise to a number of disease states including obesity, diabetes, cardiovascular disease, neurodegeneration and cancer 37 . the details of these processes are not fully understood, their study having been stagnated by the inherent difficulty in studying lipids. past methods such as nile red staining or lipid extraction have allowed visualization and component analysis separately, but researchers have never easily been able to track lipids dynamically in living systems. thus, cars microscopy fills a very important and long-vacant niche in studying lipid biology. as cars microscopy can image lipids and other molecules in cells with excellent chemical contrast, this technique has been used in a wide variety of applications. cars microscopy has proven to be an invaluable tool for examining metabolic changes in cells including changes in lipid storage, organelle transport and aspects of lipid droplet biology [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] . it is well known that metabolism is affected during the development of various types of cancer, and cars microscopy has recently emerged as a way to image these nonstatic changes 51 . cars microscopy has also proven to be an important tool for imaging neurons and brain slices as well as investigating diseased states associated with demyelination [52] [53] [54] [55] [56] [57] [58] . this tool has also been used to investigate stem cell differentiation 59 , host-pathogen interactions 39, 43, 48, [60] [61] [62] [63] [64] [65] and the effects of drugs on target cells and tissues 38, 40, 43, 53, [66] [67] [68] [69] . over the last decade we have seen cars microscopy begin to have an impact on the field of chemical biology. as the details of the technique have been reviewed elsewhere 21, 30, 70 , this review highlights the applications of cars microscopy to studies in chemical biology and suggests potential opportunities for the future. as cars microscopy is a relatively new and promising method for imaging with chemical contrast, it is tempting to propose that it may be a solution to many long-standing challenges in imaging and microscopy. however, it is important to realize the limitations of this tool. in this section, we outline the scope of cars raman scattering spectroscopy detects the vibrations inherent in molecules without the need for dyes or fluorescent labeling. the signals generated result from inelastic scattering of photons with an energy shift that is characteristic of specific molecular vibrations (fig. 1a , upper panel, and fig. 1b , left panel). despite the obvious appeal for biological imaging, single-photon raman imaging has been limited by low signals because the majority of photons are scattered elastically (no energy shift). the advent of coherent raman spectroscopy brings many advantages, prominent among them a thousand-fold improvement in signal intensity. the phenomenon of coherent anti-stokes raman scattering occurs when a target molecule is irradiated using two short-pulse laser beams, the pump beam and the stokes beam. importantly, the frequencies of these two beams must be tuned such that the frequency difference corresponds to a vibration of the target. when this condition is met, coherently vibrating molecules in the probe volume (that is, the focal point of the pump and stokes beams) will scatter the probe beam, resulting in a coherent signal with a higher frequency than that of the probe beam and a much higher intensity than the signal from spontaneous raman scattering (fig. 1a , lower panel, and fig. 1b , right panel). the greatest advantage of cars, its basis in inherent molecular vibrations, also represents one of its greatest limitations. to achieve natural contrast with cars, it is necessary to have a high concentration of the same molecular moiety. for this reason, lipids, with their long aliphatic chains full of -ch 2 groups, are perfectly suited to cars imaging. this is quite apparent when examining the individual raman spectra of cellular components (fig. 1c) . the largest signal present in the raman spectrum of the whole cell is from the c-h stretch of each component but is predominantly from the lipid (fat) component. conveniently, lipids are also one of the biomolecules that can benefit from label-free imaging by cars microscopy. their size is on par with or smaller than the size of most common fluorescent labels, so lipid labeling is likely to perturb both lipid function and transport. also apparent from the spectra in figure 1c is the potential to use smaller raman labels with raman signals in the "silent region, " for example -cd or -cn. microscopy so that an appreciation of its true capabilities and potential applications can be highlighted. with the high spatial resolution typical of multiphoton microscopies and fast image acquisition rates, cars microscopy has proven to be an excellent imaging modality for monitoring c-h vibrational stretches in lipids and o-h stretches in water. however, because this is a nonlinear technique that depends on three photons to coherently stimulate a resonance frequency, the cars signal depends on the square of the concentration of vibrational oscillators in the focal volume of the material being imaged. for this reason, the enhanced sensitivity associated with cars drops off quickly with decreasing oscillator concentration, and the imaging of less abundant biomolecules is difficult. although it is difficult to define a detection limit because of the different methods for performing cars microscopy, typically 10 5 to 10 6 oscillators per focal volume produce sufficient contrast, making this a much less sensitive technique than fluorescence imaging. cars images often show some spectral distortion and limited sensitivity arising from unwanted background signals (referred to as nonresonant background) 21, 30, 70 . because of these issues, quantifying cars signals can present a challenge. there are, however, tools available to achieve relative quantification. changes in signal intensity relative to control samples, even if semiquantitative in nature, can give important information about biological phenomena over time in living systems. in some of the examples we will discuss, image analysis algorithms have been applied to correct for the nonlinear effects of cars microscopy 38, 62, 71 . another approach that has been used for the analysis of cars images is called voxel analysis 39, 40, 43, 72 . this tool calculates signal intensity within a field of view as a function of pixel volume, and only those pixels that meet a minimum threshold signal are counted 39, 40, 43, 72 . using methods such as these for the systematic analysis of cars images allows for the quantification of changes that are independent of unwanted background signals. for most applications of cars microscopy, the intensity of the cars signal is not the only important observable criterion in studying biological processes. changes in features such as size, spatial distribution and morphology do not necessarily involve significant changes in the concentration of vibrational oscillators in the focal volume. these features are often as important, if not more important, than the intensity of the signal in terms of understanding a chemical or biochemical event. many of the examples highlighted within this review fall into this category. recently another coherent raman microscopy called stimulated raman scattering (srs) has emerged 73, 74 . although many of the issues that are of concern with cars microscopy, such as the nonlinear dependence of the signal with respect to analyte concentration and high background signals, are overcome with srs, it remains a very difficult approach to employ, with significant technical barriers, and no commercial srs microscope currently exists. on the other hand, because a commercial cars microscope based on a set-up described by pegoraro et al. is currently available 75 , cars microscopy is broadly accessible to researchers in a number of fields including chemical biology; this opens up new avenues for label-free imaging with chemical contrast. the potential of cars microscopy to improve our understanding of lipid metabolism and fat storage has been demonstrated in multiple ways with lipid droplets 38 , cells 42, 47 , tissue samples 71,75-77 and even live organisms 39, 78 (fig. 2) . as described in box 1, the imaging of lipids can be accomplished by simply tuning the resonance frequency to the spectral region in which c-h bond vibrations are observed, thus allowing visualization of lipids and lipid-rich structures as well as direct measurement of the effects small molecules and metabolites have on lipids. for instance, a recent study compared the growth of lipid droplets in the liver by cars microscopy in mice fed a diet lacking methionine and choline compared with those fed a regular diet 76 . methionine and choline deprivation is (i) lipid droplet detection imaging the �ch 2 stretch at 2,850 cm �1 identifies fatty acyl chains of neutral lipids compacted into lipid droplets. (iii) chemical probes cars spectral features provide contrast to visualize localization of biomolecules. (iv) capturing lipid metabolism the effect of small molecules (for example, metals and inhibitors) and cellular processes (for example, adipocyte differentation and steatosis) can be imaged with cars microscopy. known to impair very low-density lipoprotein (vldl) production, which leads to impaired triglyceride secretion and an accumulation of hepatic fat 76 . cars microscopy made it possible to directly measure the growth of lipid droplets caused by lowered levels of methionine and choline for intact tissue sections. the effects of drugs that target pathways involved in lipid metabolism have also been investigated using cars microscopy, including a study of lysophosphatidylcholine and other pharmacological agents on demyelination and neurodegeneration 55, 79 . similarly, specific gene knockdown through rna interference (rnai) can be used in conjunction with cars microscopy to elucidate the role of specific gene products involved in lipolysis. rnai and cars microscopy revealed that cgi-58, a protein that facilitates lipolysis of lipid droplets, is not involved in hormone-stimulated lipid droplet remodeling 80 . by dynamically tracking the cellular response to gene knockdown and lipolytic stimulation with cars microscopy, the authors obtained previously unattainable time-course data with high resolution that revealed new insights into de novo lipid-droplet formation and clarified the role of cgi-58 (ref. 80) . another study used cars microscopy to follow adipogenesis and gene regulation induced by the small molecules isobutylmethylxanthine and dexamethasone during differentiation 81 . all of these studies demonstrate the potential of cars microscopy to elucidate the function and cellular consequences of small molecules in live tissue. in our lab, cars microscopy has been used to probe the link between copper and lipid metabolism 40 . this link has been previously observed for wilson's disease using gene knockout studies with mice; however, our study is the first direct observation of the effect copper has on lipid density 82 . cars images revealed that cells exposed to uncomplexed copper (cucl 2 ) exhibited only a slight increase in lipid density, whereas exposure to either copper complexed with edta (cu(edta)) or histidine (cu(his) 2 ), a bioavailable form of copper, resulted in a marked increase in lipid density. this demonstrated the significant effect that overexposure to complexed copper has on lipid metabolism as well as the relevance of the ligand to cellular uptake of the different copper complexes 40 . in another study, we used cars microscopy to probe the cellular consequences of exposure to copper catalysts commonly used in [3+2] cycloadditions between alkynes and azides ('click' reactions) 1,83 for bioconjugation. toxicity, the effects on lipid metabolism, the level of copper uptake and the ability to catalyze reactions on live cells were characterized for cuso 4 , cu-sbp (sulfonated bathophenanthroline), cu-tbta (tris((1-benzyl-1h-1,2,3-triazol-4-yl)methyl)amine) and cu(his) 2 (unpublished data). by exploring the biological effects and potential utility of copper catalysis in living systems, these results open the possibility of developing tailored catalysts specifically for applications in living systems. in some cases, it is desirable to follow specific exogenous molecules rather than endogenous lipid features. introduction of a label into these molecules makes it possible to track them with cars imaging. ideally the label should possess a vibration in the raman silent region of the cell (box 1) without altering any other properties. perhaps the best example of such a label is the replacement of hydrogen by deuterium. this strategy was employed to help elucidate the health benefits imparted by fish oil that arise from polyunsaturated fatty acids such as oleic acid and eicosapentaenoic acid (epa) 84 . the -cd 2 vibration in deuterated oleic acid is 2,105 cm -1 , significantly shifted from the -ch 2 in nondeuterated oleic acid, which appears at 2,850 cm -1 (ref. 84 ). in the presence of epa, deuterated oleic acid and epa were found to colocalize within lysosomes in the form of triglycerides. in the absence of epa, oleic acid did not accumulate in lysosomes, thus shedding light on how such polyunsaturated acids participate in lipid metabolism and what processes lead to the observed health benefits. deuterated small molecules have also been used to show the signal-to-noise benefits of frequency modulation cars 85 for studying membrane partitioning in a nonperturbative manner 86 and for monitoring aquaporin function in the diffusion of water 87 . in general, isotopic labeling provides a highly convenient way of tracking exogenous molecules. one of the significant advantages cars microscopy offers is the wealth of information it can provide for one system under study. for example, as will be discussed in a subsequent section, whole-tissue imaging can be complemented by simultaneous subcellular imaging. in addition, multiplex cars spectroscopic imaging provides detailed information on the chemical composition of the sample, measuring the raman spectrum for each submicron pixel of the cars image. by using multiplex cars tuning to two different vibrational signatures (c=c and c-h stretches), one study successfully quantified the degree of fatty acid unsaturation and the amount of acyl chain order in hela cells and adipocytes 71 . recently the enzymatic functions of proteins involved in lipolysis have also been monitored by multiplex cars 38 . triglycerides within lipid droplets were shown to be distinguishable from their lipolysis products on the basis of the characteristic raman signals for each molecule 38 . multiplex cars was used to carry out component analysis of the digestion of glyceryl trioleate by porcine pancreatic lipase into its lipolytic products in vitro. changes in the cars spectrum between 2,800-3,100 cm -1 gave quantitative information about the rate of digestion within a lipid droplet 38 . hyperspectral analysis of the raman spectrum of each pixel allowed quantification of the different chemical components, whereby the spectra are deconvoluted (treated as a linear combination of the component spectra) and the relative concentration of each component is visualized as a false color image (see fig. 3 (ref. 38) ). this technique was also used to distinguish between the bioactive molecules ergosterol, progesterone and vitamin d contained within lipid droplets to aid in understanding the physiological transport and absorption of these molecules as well as their effects on lipolysis 38 . it should be noted that raman spectra obtained via cars imaging are more complex than those from linear raman spectroscopy and therefore require higher level analysis. however, the data treatment used in the above examples should be able to distinguish any set of molecules with distinct raman signals. as such, multiplex cars is becoming an increasingly attractive tool in chemical biology, providing all the benefits of cars microscopy with detailed raman spectroscopic data. although the majority of applications in chemical biology using cars microscopy involve lipid imaging, the technique is certainly not limited to lipids. any molecular oscillator can be monitored by cars, provided it is found in sufficiently high local concentrations to give chemical contrast. for example, cars microscopy has been applied to monitoring drug release from polymer and bead samples used as drug delivery agents. in this case, both the delivery agent and drug must have unique vibrational modes that can be used to individually identify and monitor them. paclitaxel release has been studied in this manner 66 . raman vibrations that arise from the aromatic groups within the drug and that are absent in the surrounding poly(ethylene glycol)/ poly(lactic-co-glycolic acid) (peg/plga) polymer coatings were used to follow the drug changes in the release over time from a coated polymer by cars microscopy. this allowed researchers to study how changes in the polymer composition affected the rate of drug release into solution. recently, this approach was applied to monitoring the release of theophylline (dimethylxanthine), a drug commonly used to treat respiratory conditions, from solid lipid-based tablets upon dissolution in an aqueous medium 69 . as cars microscopy is well suited to live-cell imaging, we have only begun to realize its potential to visualize dynamic processes during drug delivery and to aid in characterization in physiologically relevant settings. there remain a wealth of possibilities for its use in many other imaging applications in which chemical information and contrast are useful. the above examples highlight the application of cars microscopy to monitoring dynamic biochemical processes in response to chemical perturbation. a powerful asset of cars microscopy is its ability to be used in conjunction with other two-photon imaging modalities. in particular, when a femtosecond laser is employed, the excitation source is amenable to inducing both the cars and the twophoton fluorescence signals. thus, both systems can be combined into one microscope, thereby allowing the collection of real-time information from the same sample window using both modalities, as well as streamlining data collection. there is particular benefit from this approach in monitoring host-pathogen interactions. by summarizing the most recent discoveries in the field, we aim to highlight the possibilities offered by multimodal cars to chemical biology. the interactions of pathogenic organisms such as viruses and bacteria necessarily involve interactions at cellular membranes for, at the minimum, the entry of the pathogen into its host cell to begin infection 88 . many pathogens also alter their environment in a way that changes the internal make-up, metabolic processes and energy requirements of an infected cell 88 . indeed, nearly all viruses of concern to human health have some specific interactions with lipids, including human immunodeficiency virus (hiv), hepatitis c virus (hcv) and the influenza virus 88 . viruses that modify the host cell to further their propagation are also common 88 . for instance, infection with most positive-sense rna viruses, including dengue virus, severe acute respiratory syndrome (sars) virus and hcv, results in modification of the membranous er 89 . as hcv also modulates many other facets of lipid metabolism to facilitate its viral life cycle 61 , including viral particle assembly around lipid droplets 90 and viral particle secretion using components of the vldl pathway 91 , there is great potential for applying cars microscopy to study host-virus interactions in hcv and other viral infections. cars microscopy in combination with two-photon fluorescence (tpf) microscopy has been applied to the visualization of spatiotemporal relationships between hcv rna and alterations in host-cell lipid metabolism 62 . in this study, replicationcompetent hcv rna was chemically labeled at the 5′ end with a fluorophore and subsequently monitored in living cells using tpf microscopy while changes to the host cell lipid content were simultaneously monitored using cars microscopy; changes in lipid metabolism as a function of hcv rna were then quantified 62 . increasing lipid density was observed in hepatoma cells expressing the hcv rna 62 . this initial study demonstrated the multimodal approach's value for simultaneous dynamic tracking of the subcellular localization of viral rna while establishing the perturbed lipid phenotype. it became apparent that cars microscopy would have similar utility in studying the effects of host and viral genes as well as the effects of small-molecule inhibitors on the viral life cycle in real time. cars microscopy in conjuction with tpf microscopy was subsequently used to examine the effects of lipid metabolismassociated host gene expression on hcv propagation. peroxisome proliferator-activated receptor alpha (pparα) antagonism was shown to create an antiviral state, judging from the observation that both sirna and chemical knockdown created a hyperlipidemic condition that correlated with an antiviral state 64 . similarly, cars microscopy was instrumental in understanding the role of host factor carboxylesterase 1 (ces1) as a proviral gene that is upregulated upon hcv infection (fig. 4) 92 . the tool allowed for correlation between ces1 levels in hepatoma cells and the size and density of lipid droplets, which are necessary for the functional vldl pathway and are a likely vehicle for hcv particle assembly and secretion. the multimodal strategy allowed for a better understanding of ces1's role, as the host factor was found to concentrate around lipid droplets near the endoplasmic reticulum, suggesting the protein loading of neutral lipids to er-associated lipid droplets is crucial for hcv replication and maturation of hcv virion. several studies have shown the liver-specific microrna mir-122 is a key regulator of cholesterol biosynthesis as well as of hcv replication [93] [94] [95] [96] . thus, we are currently using cars microscopy to investigate the role of mir-122 and other mirnas in modulating hepatic lipid content and lipid droplet morphology. cars and tpf microscopies were also used to examine the hcv antiviral mechanism of small-molecule modulators, including modulators of the mevalonate pathway 97 and ppar signaling pathways 43 . hcv viral replication and particle assembly is thought to be localized to the interface between ers and lipid droplets. using live cell imaging techniques, it was shown that hcv replication complexes are dispersed by modulating the mevalonate pathway using lovastatin and the ppar signaling pathway using a pparα antagonist, most likely by preventing the lipidation of a host protein, fbl2, that has a known association with the hcv replication complex 43 . this multimodal strategy lent itself to a kinetics-based comparison of the drugs' effects, as the study revealed the pparα antagonist mediated its dispersion of hcv rna at a much faster rate than lovastatin did 43 . more recently, this bimodal strategy was adapted to include differential interference contrast microscopy, which enabled examination of the dynamics of the hcv core protein's interaction with lipid droplets 72 . it was shown that the hcv core protein can rapidly induce lipid droplet biogenesis in living cells and that the hcv core protein also has a dramatic influence on the speed and directionality of lipid droplet movement, presumably as part of a mechanism by which hcv viral particle assembly is initiated 72 . although the application of cars microscopy to host-pathogen interactions is in its infancy, researchers are beginning to apply this technique to viruses other than hcv. for example, a recent study used cars microscopy to examine the infection of fibroblast cells by cytomegalovirus (hcmv), also known as human herpesvirus 5 (hhv-5) 48 . hcmv is a double-stranded dna virus that is potentially life threatening in immunocompromised patients. cars microscopy was used to examine mouse hcmv infection of nih/3t3 fibroblast cells, tracking morphological changes in cells such as expanded nuclei and altered lipid droplet distributions 48 . infection with hcmv-gfp fusion constructs and imaging with tpf microscopy allowed for the simultaneous tracking of viral proteins during infection so that morphological changes could be correlated with the degree of hcmv infection 48 . these results clearly show the potential of cars microscopy to elucidate new mechanistic details, correlate metabolic changes in a cell with the levels of infection and measure dynamics in the detailed examination of host-pathogen interactions. it is expected that many more applications in this field will be discovered in the near future. cars presents unique opportunities for tissue imaging that allow for more detailed studies of biological systems and the effects of small-molecule treatment and may also have clinical applications in the future. as a nondestructive, label-free modality that achieves subcellular resolution with chemical specificity, cars is quite suitable for tissue imaging. furthermore, deep penetration of thick and turbid specimens is possible because of near infrared excitation, and the nonlinear dependence of the signal on excitation intensity gives inherent three-dimensional optical sectioning. already the usefulness of cars microscopy has been demonstrated in tissue imaging, predominantly using the high contrast of the c-h stretch for lipid imaging. live animal imaging using cars microscopy was first applied to the imaging of skin tissue in mice using video rate imaging methods 78 . this was accomplished with backscattering of the cars signal in a given tissue that yields an 'epi-cars' signal. cars imaging and spectroscopy of lipid-rich tissue structures in the skin of living mice allowed for the direct observation of sebaceous glands, corneocytes and adipocytes with high contrast and subcellular resolution 78 . notably, this technique allowed for the observation of dynamic processes. diffusion of oils into the skin of mice could be observed without the addition of a label because of the contrast achievable using the c-h vibrational band 78 . the changes in the distribution of the oil were monitored, and it was found that penetration only occurred through the stratum corneum, not into dermis, suggesting that the oil could not diffuse farther than the epidermal layer of the mouse skin. this represents the first direct, label-free imaging of an exogenously added small molecule in the tissues of a live animal by cars microscopy. live animal imaging has been conducted with the organism caenorhabditis elegans 39 . c. elegans serves as an excellent model organism because of its small size and the ease of wholebody imaging and also because its genetics have been extensively studied. as a result, there are a number of functionally relevant knockout strains available that perturb lipid metabolism through mechanisms similar to those that occur in higher order eukaryotes. c. elegans was used to examine the details of metabolic diseases that involve changes in lipid stores on the single-cell level in vivo 39 . interestingly, cars microscopy was able to visualize hypodermal lipid droplets that were not visible or quantifiable by other methods (fig. 5) 39 . specifically, the technique was used to quantify changes in lipid stores in genetic mutants in which metabolic pathways affecting lipid storage had been previously established. a feeding-defective mutant called pha-3 showed a decrease in lipid content of approximately one-third compared to that of control organisms, as measured by lipid volume fraction 39 . in contrast, other mutants such as daf-2 and daf-4 with impaired insulin growth factor and transforming growth factor signaling pathways, respectively, showed significantly increased lipid volumes, a change in lipid phase and an increased number of lipid droplets in hypodermal cells 39 . this example highlights one of the advantages label-free imaging has over fluorescent labeling and staining. the researchers were unable to observe hypodermal lipid droplets via nile red fluorescence. the authors attributed this to one of two factors: either the nile red reaches these lipid droplets less efficiently (metabolic effect) or the fluorescence is less efficient in the environment of the hypodermal lipid droplets (photophysical effect). further mutants have subsequently been studied 65 , and the results emphasize cars microscopy's utility in measuring lipid phenotype and also in predicting genotypes in mutant organisms that are models for genetic diseases. cars microscopy is also well suited to imaging nerve tissue; the myelin sheath that coats nerve axons is lipid rich and thus gives excellent contrast 57 (fig. 6a,b) . real-time imaging of intact myelin by cars has helped elucidate the cellular mechanisms of demyelination disorders, which would not be possible with methods that require fixed tissue, such as electron microscopy and immunofluorescence 55 . additionally, cars has potential for in situ brain imaging. thus far, high-resolution images of brain structures from an unfixed mouse brain slice have allowed identification of a large brain tumor with the same spatial accuracy of tumor margins as in histological staining 54 . other clinically relevant applications of cars microscopy include the quantification of lipid-content for intact tissues. as mentioned previously, cars microscopy has been successfully applied to visualizing mouse liver tissue 76 . assessing tissue lipid content with cars microscopy is also relevant in cancer pathology 51 . cars is currently being used to understand the link between obesity and breast cancer. using this technique, it has been shown that the mammary glands of obese rats contain elevated levels of adipocytes with increased sizes of lipid droplets 98 . by combining cars, second harmonic generation and tpf, the authors were able to image several components of the mammary stroma, thus lending insight into the dynamics of the tumor microenvironment. furthermore, cars has been used to probe the association of lipid-rich tumors with increased tumor metastasis, tracking intracellular lipid accumulation in primary, circulating and metastasized cancer cells 51 . lipid metabolism also plays a key role in atherosclerosis 41, 77, 99 . as such, cars microscopy may prove to be very valuable in the study of the disease, improvement in diagnosis and discovery of new treatments. to aid in understanding the pathogenesis of plaque vulnerability, multiplex cars spectroscopy has been used to identify and characterize the chemical profiles of four types of atherosclerotic lipids ( fig. 6c-f) 41 . prior to this application of cars, such information was only available through indirect analyses after sample removal and processing or via dissection imaging. the effect of statin drugs on the chemical profiles of the plaques was also measured with specific reference to plaque stability. although the morphologies of lipid-rich features remained unchanged, the chemical composition of each feature was quite different for cells treated with statins 41 . after eight weeks of treatment with simvastatin, lipid crystals were less solidified, as evidenced by a decrease in the signal intensities assigned to the asymmetrical -ch 2 and -ch 3 vibrations 41 . cars is currently capable of tissue penetration from 30-120 µm, but constant improvements in optics are achieving greater penetration with lower levels of nonresonant background. also, cars endoscopy has been on the horizon since the recent development of a fiber-delivered probe able to collect the back-scattered, forward-generated cars signal in biological tissues 100 . a cars endoscope will provide access to deeper tissues in vivo, inviting a wealth of applications in clinical diagnostics such as in situ imaging of tumor margins and atherosclerotic plaques, among others. it is clear that cars has a future in clinical imaging. cars is able to fill the role of histological staining and biochemical analysis in several applications, achieving the same result while avoiding tissue fixation and use of reagents. furthermore, cars complements mri applications, as it is a label-free and nondestructive modality. although cars cannot match the tissue penetration that mri provides, it achieves far greater resolution. the ability of cars to be used simultaneously with other imaging modalities makes it particularly attractive. the most powerful aspect of cars microscopy is that it can report with high resolution on the chemical and biological nature of any sample using inherent raman vibrational resonances, provided the resonances are sufficiently distinct and in high enough local concentration. many of the applications of cars microscopy to date have focused on imaging lipids and monitoring their phenotypic changes in response to a chemical or biological perturbation. the themes highlighted in this review speak to the relevance of results obtained with cars microscopy to problems in health and disease and highlight the opportunities for further investigation both in terms of imaging and discovery. cars microscopy increases the repertoire of today's chemical biologist and, notably, provides the previously unavailable ability to dynamically monitor biological systems with chemical specificity. additionally, there remains great potential for this technique to image exogenous molecules, to perform high-throughput screening and to visualize chemical reactions that are of relevance to biology. constant improvements in methods related to cars microscopy (such as srs microscopy) and in optics are increasing its capacity to image less abundant target molecules and expanding the possible applications of this technique. these advances are also driving the progress toward exciting clinical imaging applications. given the recent commercialization of a cars microscope, based on the design by pegoraro et al. 75 , and thus the increased accessibility of the technique, we envision its routine usage in a wide variety of fields in the near future. chemistry in living systems therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration regulatory phases of early liver development: paradigms of organogenesis the complete atomic structure of the large ribosomal subunit at 2.4 angstrom resolution ribosome dynamics and trna movement by time-resolved electron cryomicroscopy initiation of translation in prokaryotes and eukaryotes what recent ribosome structures have revealed about the mechanism of translation principles of stop-codon reading on the ribosome focus on function: single molecule rna enzymology controlling ribozyme activity by metal ions an update on sphingosine-1-phosphate and other sphingolipid mediators translation of the phosphoinositide code by pi effectors lipid rafts: bringing order to chaos sphingosine 1-phosphate signalling in mammalian cells brick by brick: metabolism and tumor cell growth glycosylation in cellular mechanisms of health and disease in situ click chemistry: probing the binding landscapes of biological molecules chemical approaches to perturb, profile, and perceive glycans innovation: a chemical toolkit for proteins-an expanded genetic code introduction to biophotonics coherent anti-stokes raman scattering microscopy: chemical imaging for biology and medicine surface-enhanced raman scattering sers-a single-molecule and nanoscale tool for bioanalytics single-molecule and single-nanoparticle sers: from fundamental mechanisms to biomedical applications localized surface plasmon resonance spectroscopy and sensing sers detection and boron delivery to cancer cells using carborane labelled nanoparticles nanoscale aggregation of cellular beta(2)-adrenergic receptors measured by plasmonic interactions of functionalized nanoparticles development of nanoparticle probes for multiplex sers imaging of cell surface proteins mammalian cell surface imaging with nitrile-functionalized nanoprobes: biophysical characterization of aggregation and polarization anisotropy in sers imaging coherent anti-stokes raman scattering microscopy: instrumentation, theory, and applications scanning coherent anti-stokes raman microscope three-dimensional vibrational imaging by coherent anti-stokes raman scattering this seminal study is a landmark demonstration of cars microscopy using near-infrared co-linear laser pulses phosphatidylinositol transfer proteins and cellular nanoreactors for lipid signaling endocytosis and molecular sorting structure of lipid bilayers trafficking and signaling by fatty-acylated and prenylated proteins lipid metabolism and health label-free imaging of lipophilic bioactive molecules during lipid digestion by multiplex coherent anti-stokes raman scattering microspectroscopy this paper represents the first example of the imaging of enzyme catalysis involving the lipolysis of triglycerides within lipid droplets monitoring of lipid storage in caenorhabditis elegans using coherent anti-stokes raman scattering (cars) microscopy this paper contains first examples of live whole organism imaging using cars microscopy by examining processes involved in lipid storage in caenorhabditis elegans cellular lipid metabolism is influenced by the coordination environment of copper multiplex coherent anti-stokes raman spectroscopy images intact atheromatous lesions and concomitantly identifies distinct chemical profiles of atherosclerotic lipids differential association of adipophilin and tip47 proteins with cytoplasmic lipid droplets in mouse enterocytes during dietary fat absorption in this study, cars microscopy is used to show drug-induced modulations of the host lipid environment and dispersal of hcv replication complexes biophotonic probing of macromolecular transformations during apoptosis vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes raman scattering microscopy quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy intracellular imaging of host-pathogen interactions using combined cars and two-photon fluorescence microscopies high-speed vibrational imaging and spectral analysis of lipid bodies by compound raman microscopy a dynamic, cytoplasmic triacylglycerol pool in enterocytes revealed by ex vivo and in vivo coherent anti-stokes raman scattering imaging coherent anti-stokes raman scattering imaging of lipids in cancer metastasis in vivo optical monitoring of tissue pathologies and diseases with vibrational contrast polyethylene glycol protects injured neuronal mitochondria chemically-selective imaging of brain structures with cars microscopy coherent anti-stokes raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-ptdcho-induced demyelination this study showed that demyelination of neurons can be detected using cars microscopy in vivo coherent anti-stokes raman scattering imaging of sciatic nerve tissue coherent anti-stokes raman scattering imaging of axonal myelin in live spinal tissues molecular composition and orientation in myelin figures characterized by coherent anti-stokes raman scattering microscopy in situ analysis of living embryonic stem cells by coherent anti-stokes raman microscopy synthesis and characterization of cn-modified protein analogues as potential vibrational contrast agents host-virus interactions during hepatitis c virus infection: a complex and dynamic molecular biosystem intracellular imaging of hcv rna and cellular lipids by using simultaneous two-photon fluorescence and coherent anti-stokes raman scattering microscopies evaluation of chemical labeling strategies for monitoring hcv rna using vibrational microscopy peroxisome proliferator-activated receptor alpha antagonism inhibits hepatitis c virus replication label-free quantitative analysis of lipid metabolism in living caenorhabditis elegans in situ visualization of paclitaxel distribution and release by coherent anti-stokes raman scattering microscopy this report describes how the rate of drug release from a delivery polymer for paclitaxel can be monitored using cars microscopy three-dimensional molecular mapping of a multiple emulsion by means of cars microscopy imaging receptor-mediated endocytosis with a polymeric nanoparticle-based coherent anti-stokes raman scattering probe chemical imaging of oral solid dosage forms and changes upon dissolution using coherent anti-stokes raman scattering microscopy shedding new light on lipid biology with coherent anti-stokes raman scattering microscopy imaging of chemical and physical state of individual cellular lipid droplets using multiplex cars microscopy dynamics of lipid droplets induced by the hepatitis c virus core protein label-free biomedical imaging with high sensitivity by stimulated raman scattering microscopy video-rate molecular imaging in vivo with stimulated raman scattering this study is the first demonstration of cars microscopy with a single femtosecond laser light source, a photonic crystal fiber for generating the stokes light and optical chirping to improve spectral resolution quantitative assessment of hepatic fat of intact liver tissues with coherent anti-stokes raman scattering microscopy nonlinear optical measurements of the artery wall: parameters related to the progression of atherosclerosis chemical imaging of tissue in vivo with video-rate coherent anti-stokes raman scattering microscopy in this report, live tissue is imaged at video rate to visualize rapid processes such as the penetration of oils into skin glutamate excitotoxicity inflicts paranodal myelin splitting and retraction cgi-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation single-cell profiling reveals the origin of phenotypic variability in adipogenesis high copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of wilson disease click chemistry: diverse chemical function from a few good reactions perspective-living cells as test tubes high-sensitivity vibrational imaging with frequency modulation coherent anti-stokes raman scattering (fm cars) microscopy quantitative coherent anti-stokes raman scattering imaging of lipid distribution in coexisting domains real-time visualization of intracellular hydrodynamics in single living cells this paper is one of the first to demonstrate imaging of water and the effects that aquaporins have on water movement into cells by cars implications for lipids during replication of enveloped viruses modification of intracellular membrane structures for virus replication lipid droplets and hepatitis c virus infection cellular determinants of hepatitis c virus assembly, maturation, degradation, and secretion activity-based protein profiling identifies a host enzyme, carboxylesterase 1, which is differentially active during hepatitis c virus replication lna-mediated microrna silencing in non-human primates modulation of hepatitis c virus rna abundance by a liver-specific microrna microrna-122 modulates the rhythmic expression profile of the circadian deadenylase nocturnin in mouse liver silencing of micrornas in vivo with 'antagomirs' the influence of cholesterol and lipid metabolism on host cell structure and hepatitis c virus replication nonlinear optical imaging to evaluate the impact of obesity on mammary gland and tumor stroma multimodal cars microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in apoe-deficient mice fiber delivered probe for efficient cars imaging of tissues slepkov (nrc) for advice and help with cars microscopy. r.s. thanks nserc for a vanier graduate scholarship and the cihr national canadian research training program in hepatitis c for additional support and training. d.c.d. thanks nserc for a canadian graduate scholarship. r.k.l. thanks the nserc collaborative research and training experience program in medicinal chemistry and biopharmaceutical development for support and training the authors declare no competing financial interests. reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. correspondence should be addressed to j.p.p. key: cord-343902-hzh3cjzh authors: peel, michael; scribner, andrew title: cyclophilin inhibitors as antiviral agents date: 2013-08-15 journal: bioorg med chem lett doi: 10.1016/j.bmcl.2013.05.101 sha: doc_id: 343902 cord_uid: hzh3cjzh cyclophilins (cyps) are ubiquitous proteins that effect the cis–trans isomerization of pro amide bonds, and are thus crucial to protein folding. cypa is the most prevalent of the ∼19 human cyps, and plays a crucial role in viral infectivity, most notably for hiv-1 and hcv. cyclophilins have been shown to play key roles in effective replication of a number of viruses from different families. a drug template for cypa inhibition is cyclosporine a (csa), a cyclic undecapeptide that simultaneously binds to both cypa and the ca(2+)-dependent phosphatase calcineurin (cn), and can attenuate immune responses. synthetic modifications of the csa scaffold allows for selective binding to cypa and cn separately, thus providing access to novel, non-immunosuppressive antiviral agents. since the initial development of cyclosporin a (csa) as an immunosuppressive agent for preventing allograph rejection, 1 cyclophilin (cyp) inhibitors have been explored extensively over the last four decades for treating a wide variety of medicinal conditions, including autoimmune diseases, 2 myocardial infarction ischemia reperfusion, 3 and viral diseases, 4 the latter of which will be the focus of this review. an overview of cyp structure and function will first be presented, followed by evidence implicating the role of cyps in viral diseases, notably hiv-1 and hcv. this will be followed by a discussion of csa-based cyp inhibitors, including in vivo and in vitro structure-activity relationships, leading up to a discussion of cyp inhibitors currently in development for the treatment of hepatitis c virus. cyclophilins: the cyclophilin (cyp) class of proteins shares a common enzymatic activity, that of a peptidyl-prolyl isomerase (ppiase), which effects a cis-trans isomerization of amide bonds n-terminal to proline (pro) residues, and thus play a regulatory role in protein folding. 5 at least 20 members of the cyp class have been identified in humans, 6 with sizes ranging from small, single ppiase-domain cyps (cypa, cypb, cypc, cypd, cypj, ppil1, ppial4, and usa-cyp) to large multi-domain proteins in which a catalytic ppiase activity is linked to various additional activities (fig. 1) . 7 cypa is the most abundantly expressed member of the class, representing the majority of the ppiase activity in a cell, with cypb being the only other cyp found above trace level. in addition to the cyps, two other families of proteins, fkbps and parvulins ( fig. 1) , have been described with similar ppiase activity. 8 inhibi-tion of the ppiase activity of fkbps by either fk506 or rapamycin was shown to be required for the immunosuppressive activity of these compounds. 9 the parvulins are responsible for the cis-trans isomerization of po 3 h 2 ser (po 3 h 2 thr)-pro bonds; however, they are much less well studied. 10 one key member of the parvulin class, pin1, interacts with phosphoproteins involved in the cell cycle of some cancer cells; 10 however, the lack of potent and selective inhibitors for parvulins has limited research in this area. 10, 11 cyps show widely differing expression profiles, with cypa being found predominantly in the cytoplasm, while more restricted localization is observed for cypb (endoplasmic reticulum), cypd (mitochondrial inner membrane), cype (nucleus), and ranbp2 (nuclear pore). 6 studies directed at the functional characterization of cypa, cypb, cypd and cyp40 have been reported over the last 20 years; 5b however, native roles for the majority of the class, and potential roles in disease states, have not yet been elucidated. several mechanisms by which cyps catalyze the cis-trans isomerization of an xaa-pro bond have been proposed; however, the currently accepted mode of catalysis is based on distortion of the planar amide bond (fig. 2) . 12 binding of a pro-containing substrate in a manner that twists the amide bond leads to a lower barrier to rotation due to loss of amide resonance. 13 an important hydrogen bond between an active site residue (side chain of arg55 in cypa) and the prolyl amide nitrogen facilitates the formation of a pyramidal nitrogen, which is a key feature of the isomerization. 14 notably, mutation of arg55 results in a 'catalytically inactive' form of cypa, which has been used extensively as a mechanistic probe. 15 crystal structure analysis of several pro-containing substrate peptides bound to human cypa revealed that all ligands evaluated bound with the xaa-pro bond in the cis conformation and with no distortion from planarity. 16 since most xaa-pro amide bonds reside preferably (5-to 6-fold preference) in the trans conformation, ke proposed that the principle role of cyps is to facilitate a trans to cis isomerization. 14, 16 while the ppiase activity of cyps has been well studied, several additional roles for cyps have also been described. binding of either cypa or cypb to cd-147/emmprin, an extracellular receptor for cypa, leads to activation of map kinase pathways and induction of matrix metalloproteinase (mmp) production. 17 a role for cypa as an inflammatory chemotactic cytokine, acting through the cd-147/emmprin receptor, was revealed using a murine model of asthma in which blocking the cypa-cd-147 interaction with anti-cd-147, or csa, resulted in a significant decrease in neutrophil and eosinophil migration, and improved lung pathology. 18 further roles for extracellular cyps in renal fibrosis 19 and ischemia-reperfusion injury 20 have been described with cd-147/emmprin being a key activating receptor. 21 the discovery that cypa is a requisite binding protein for the immunosuppressive activity of csa served to place cypa firmly within the sphere of immunology research. the demonstration that this binary cypa:csa complex inhibits the calcium-dependent phosphatase activity of calcineurin (cn), leading to inhibition of key activators of t-cells, provides a compelling explanation of the immunosuppressive activity of csa. 22 sar studies performed on csa allowed two discrete 'faces' to be ascribed to csa, reflecting how affinity for each protein is affected by modification of various regions of csa (1, fig. 3 ). however, an expanding range of activity demonstrated by csa, including mitochondrial function, cell death, chemotaxis and motility, suggested mechanisms that were not easily ascribed to cn inhibition. 2 the discovery of new csa analogs that retained potent binding to cypa but prevented ternary complex formation with cn allowed the biology of cyps to be probed independent of immunosuppressive activities. 23 furthermore, the use of knockdown and gene-silencing techniques have revealed a rich and expanding biology associated with cyps, with virology emerging as an important focus of research. 24 human immunodeficiency virus-1: hiv-1, the parasitic virus causing aids, depends heavily on host cellular machinery for its survival, replication, and infectivity. cypa is the first cellular protein ever found to be incorporated into hiv-1 virions. 4a subsequently, a significant number of publications have suggested several roles of cypa in the hiv life cycle, 4a all of which are independent of cn inhibition. initial reports of csa inhibiting hiv-1 surfaced in 1988, 25 although the first report specifically implicating cypa involvement in the hiv-1 life cycle was published five years later when it was reported that endogenous cypa colocalizes with the hiv-1 gag protein in the cytoplasm, binding specifically to a pro-rich sequence in the hiv-1 capsid domain of gag. 26 x-ray crystallography later revealed that residues 85-93 of hiv-1 capsid bind to the active site of cypa, 27 and subsequent studies have shown that virions produced in the presence of gag mutated in the cypa binding region, or under pressure from competitive cypa inhibitors, were less infectious than wild type virions. 27 cypa is then incorporated into nascent hiv-1 virions during the assembly and budding process. 4a facilitates uncoating of the capsid and supports sufficient reverse transcription of the viral genome. 28 more recent studies, however, have suggested that cypa in the infected target cell, and not cypa from hiv-1 virions, is more important to hiv-1 infectivity. 29 it has been further shown that cypa in the target cell increases hiv-1 infectivity by inhibiting host cell restriction factors mounted as part of an innate immune response that ordinarily combats invading retroviruses. 30 reducing the cypa-capsid interaction either by mutations altering binding of cypa to capsid, or by the introduction of a cypa inhibitor such as csa, has been shown to increase hiv-1 susceptibility to trim5a restriction. 30 other recent studies have suggested that cypa binds to hiv-1 viral protein r (vpr), which undergoes cis-trans isomerization of pro residues in its nterminal region. this activation by cypa ultimately governs the functional expression of vpr, which is needed for translocation of virus to the nucleus, and induction of cell cycle arrest and apoptosis in infected t cells. 31 in summary, there are several stages in the hiv-1 life cycle that involve cypa, some understood more clearly than others. clearly, a cypa inhibitor that bound to cypa alone and not cn would potentially be an effective anti-hiv therapeutic. such compounds have been developed, and will be discussed in section c. hepatitis c virus: the first report of hcv inhibition by csa emerged in 1988, even predating hcv being named as such. 32 clinical studies in hcv infected patients undergoing liver transplantation showed a superior virologic response to csa in combination with interferon a2b versus interferon a2b alone, 33 implicating cyp inhibition as an approach to hcv therapy. it was not until 2003 that csa was found to inhibit hcv in a cell-culture-based replicon system; 34 however, the immunosuppressive activity of csa still obscured the role of cyps in hcv replication. initial studies identified cypb and/or cypc as being crucial for hcv replication; 33b,35 however, subsequent reports have indicated that cypa alone is crucial for replication of hcv genotypes 1a, 1b, and 2a. 24 to complicate matters, a recent study suggests hcv replication is influenced at different stages by several cyps, including cypa (regulation of transcription and translation), cypb and cypc (protein conformation and transport), cype and cyph (regulation of mrna splicing, generation of host proteins necessary for hcv), cyp40 (regulation of translation, non-vesicular transport of cholesterol, co-chaperone of hsp90). 36 further support for cypa being the primary cyp involved in hcv rna replication include a demonstration that the catalytic ppiase activity of cypa is required for efficient hcv rna replication, and the finding that a specific binding between cypa and ns5a is dissociable by addition of csabased cyp inhibitors. 24c,37 while a recent consensus has emerged that cypa is likely the principal and perhaps exclusive cyp involved in the hcv replication complex, 38 roles for other cyps in different aspects of hcv propagation should be considered. evidence to support the binding of cypa to several hcv proteins, including ns5a, ns5b, and ns2, has been published, 4b which is consistent with the finding that in vitro resistance to cyp inhibitors, while slow to emerge, is associated with mutations in both ns5a and ns5b. the demonstration that knockdown of cypa inhibited replication of a full length hcv genome significantly more effectively than a sub-genomic replicon system (ns3-ns5b) supports the idea that cypa is involved in interactions with hcv proteins other than ns5a and ns5b. 24b details of the specific role of cypa in the hcv replication complex have recently appeared; however, a consensus mechanism has not emerged. while it is clear that cypa is an essential component of the hcv replication complex, different models have been proposed to explain this requirement, including a role in actively incorporating ns5b into the replication complex 37a versus a model whereby cypa, pre-residing in crude replication complex membrane fractions, serves to associate ns5a-ns5b into an assembling replication complex. 39 clues toward an understanding of the role that cypa ppiase activity might play in hcv rna replication were presented by bartenschlager 24b in a study of the rate of hcv polyprotein processing. a resistance mutation to the cyp inhibitor alisporivir, occurring near the cleavage site of the ns5a-ns5b polyprotein, resulted in delayed processing of the protein and impaired replication activity. 24b this suggests that cypa may be required to allow the ns5a-ns5b protein to achieve the required conformation to allow effective cleavage. the virus may escape the need for cypa at a specific location by mutation of residues close to the cleavage site that introduce more conformational flexibility, however this comes at a cost to the virus of impaired replication capacity. the high barrier to resistance to a cyp inhibitor may result from the need for multiple virus mutations to appear to escape the need for all cypa mediated reorganizations, with each mutation incurring a replication 'fitness' cost. most recently, ciesek and co-workers explored genetic variants of cypa via nonsynonymous single nucleotide polymorphisms (snps), and how this impacted hcv replication. 40 these studies showed that several snps present in human populations that cause depletion of intracellular cypa also reduce hcv infectivity. 40 in summary, there is ample evidence that the hcv life cycle is dependent on host cell cyp for perhaps multiple processes, as was the case with hiv-1. a cyp inhibitor, and in particular a cypa inhibitor that is non-immunosuppressive, would be an effective anti-hcv therapy. such compounds have been developed, and will be discussed later in this review. dengue, west nile and other flaviviruses: isoform-specific knockdown of cypa, cypb, or cypc in huh-7. double knockdown of cypa and cypb, and the ppiase activity of cypa was shown to be essential for effective replication of dengue and wnv. tang further showed that cypa directly interacts with the ns5 protein of wnv; however, this interaction did not affect the enzymatic activity of ns5 in vitro. 41 consistent with these findings, csa was shown to block the interaction of cypa with ns5 of wnv, suppress viral rna synthesis, and inhibit a broad spectrum of rna viruses at non-toxic concentrations. a role for cypb in the replication of japanese encephalitis virus (jev) was demonstrated by the rescue of jev replication by expression of wild type cypb, but not ppiase-deficient cypb, in various mammalian cells in which endogenous cypb was suppressed. 42 this finding was again supported by the finding that csa potently inhibits the replication of jev in vitro. 42 non-flaviviruses: while cyp inhibition has been demonstrated in restricting flavivirus replication, the role of cyps in other viruses is less clear. a convincing argument for cyp involvement in the replication of vaccinia virus (vv) was provided by the demonstration that csa analogs with strong binding affinity for cypa were potent inhibitors of vv replication, while analogs with weak affinity for cypa lost antiviral activity. 43 subsequent studies revealed an incorporation of cypa into vv virions as an essential event for successful maturation. 44 a similar incorporation of cypa into viral particles has been reported for vesicular stomatitis virus 45 and sars coronavirus; 46 however, in each case additional roles for cypa have been proposed. a novel function of a cyp in viral entry was described for human papillomavirus type 16 involving a cell-surface fraction of cypb which, following virus attachment, serves to effect a conformational change in capsid proteins l1 and l2, leading to efficient infection. 47 incorporation of cypb into virions of measles virus (mv) has been shown to be an important requirement for expanding the trophism of the virus to include epithelial and neuronal cells. 48 neutralizing antibodies directed at cd-147/emmprin (receptor for cypa and cypb) served to limit infection of epithelial cells by mv. 48 inhibition of mv infection was also demonstrated by treatment with csa, and has led to the proposal that mv incorporates cypb, but not cypa, into mature virions, and that this cypb serves to allow effective infection of cd-147/emmprin expressing epithelial cells. 48 a role for cyps in the replication of herpes simplex virus has been suggested by the finding that csa inhibits virus production in vitro; 49 however, the immunosuppressive properties of csa need to be taken into consideration when assessing such results. while a supportive function of cyps in the replication of numerous viruses has been identified, some evidence of cyps being restrictive toward viral replication has been reported. cypa was shown to bind to the m1 protein of influenza a and to restrict viral replication by inducing ubiquitin/proteosome-dependent degradation of the m1 protein. 50 notably, a ppiase deficient cypa protein could still bind m1 and inhibit viral replication, while depletion of cypa was found to enhance the replication of influenza a virus in vitro. 50 a similar finding was reported by wu 51 as a result of studies on the effect of cypa on rotavirus replication. silencing cypa resulted in a decrease in interferon-b production and an increase in viral replication, with both effects being reported to be independent of the ppiase activity of cypa. 50 a role for cyps in signaling pathways that are activated following viral infection was first suggested by the demonstration that cypb is involved in regulation of irf-3. 52 knockdown of cypb was found to suppress newcastle disease virus-induced phosphorylation of irf-3, which blocked irf-3 dimerization and inhibited the production of interferon-b. a recent report from hopkins 53 describes a novel induction of type i and iii interferons in hcv-infected patients following treatment with the non-immunosuppressive cyp inhibitor scy-635. these results point to a possible role for cyps in virus-induced blocks on innate immune responses that would otherwise serve to clear the infection and place cyp inhibition as a potentially important immunotherapy approach to virology. factors affecting cyclophilin and calcineurin binding: the cyp binding domain of csa comprises of residues 9, 10, 11, 1 and 2; hence, altering these residues tends to have a significant effect on cyp binding. modifications to residues in the cn binding domain (4, 5, 6, and 7), on the other hand, influence cyp binding indirectly through effects on conformation, but often exert a more profound affect on cn binding. the (4r)-4-[(e)-2-butenyl]-4methyl-l-threonine (mebmt) group at position 1 is unique in that it resides in the cyp binding domain, yet its sidechain drapes across the csa scaffold and ultimately into the cn binding domain (fig. 4) . in general, modifications to mebmt tend to decrease cyp binding. removing or altering the stereochemistry of the 2-methyl group reduces activity, as does introducing a second methyl group at this position. 54 likewise, removing, replacing, or capping the 3 0hydroxy as an ester also reduces potency. 55 one case in which cyp binding is actually improved is when the c@c double bond is replaced with a bioisosteric sulfur atom ('methiabmt'), which was found to bind to cyp with 178% of the affinity of csa itself. 54b the 3 csa bind to cyp with affinity comparable to that of csa, 55a while [d-meser] 3 csa binds to cyp with over threefold greater affinity than csa. 57 this is an important attribute that has been exploited in the design of csa-based therapeutics. an essential feature of cyp inhibitors designed to be used as antiviral agents is that the immunosuppressive activity due to cn inhibition is reduced or removed. this has been most effectively achieved by modification of the side chain of the [meleu] 4 residue, which is involved in a tight 'aromatic sandwich' with cn residues trp 352 and phe 356 (fig. 4) . surprisingly, moving one methyl group at the [meleu] 4 residue to give [meile] 4 csa (nim-811), was found to remove immunosuppressive activity, while retaining the in table) . 62 in view of the well-described loss of cypa binding potency as a result of esterification of the mebmt-hydroxyl, this points to the importance of an appropriate substituent at [sar] 3 in antiviral activity. no hcv data is yet available for these derivatives. (9) . compounds of high interest that have evolved from these include those possessing both optimal [sar] 3 substitution for cyp binding and/or drugability with [meleu] 4 functionality to attenuate immunosuppressive potential, such as 4-7 and 12-14. both the 5-and 8-positions have also been modified to both improve drug-like properties and diminish immunosuppressive potential, resulting in potent antivirals such as 16 and 17. other scaffold positions that have been successfully modified to yield antivirals include position 2 (19), 9 (11, 19) , and 10 (18). from these, alisporivir (7) and scy-635 (4) have proceeded the furthest into clinical development. building on data showing that replacement of the 4-position leucine residue with a valine resulted in a loss of immunosuppressive activity, along with metabolism data from csa indicating that demethylation of the amide of [meleu] 4 is a major clearance mechanism, debiopharm identified [meala] 3 [etval] 4 csa (alisporivir, 7) as a very potent cypa binder with good anti-hiv activity. 74 this drug has demonstrated good hcv activity in combination with peg-ifna2a and ribavirin, and was active as monotherapy against hcv genotypes 2 and 3. combination of the [sar] 3 modifications that result in potent anti-hiv activity with a [4 0 -homeleu] 4 residue results in scy-635 (4) that has potent anti-hiv and anti-hcv activity, and is non-immunosuppressive. 75 scy-635 was shown to be active against multiple hcv genotypes, to have a high barrier to resistance, and to be less inhibitory than csa or alisporivir toward key transporters (mrp-2) involved in bilirubin control. 76 the finding that scy-635 has lower in vitro cyp450 and transporter interactions than csa or alisporivir is likely to be important for diseases such as hcv, for which a polypharmacy approach is likely. while alisporivir and scy-635 represent the most clinically advanced non-immunosuppressive cyp inhibitors, a number of comparable structures beyond those shown in tables 1 and 2 have appeared in the patent literature, many of which involve modification of the [meleu] 4 residue. 77 figure 6 shows synthetic pathways towards alisporivir (7) 68 and scy-635 (4). 63a efforts over the last 10 years to identify non-csa-based inhibitors of cyps have met with some success. sanglifehrin a (fig. 7) is a natural product that was first described in 1999 and shown to be a potent inhibitor of cypa. 78 derivatives of sanglifehrin a have been described with good anti-hcv activity; however, these compounds have not yet progressed to clinical studies. 79 in conclusion, cyp inhibition has emerged, over the last 10 years, from a supporting role required for immunosuppression via cn inhibition, into a viable approach towards treating a wide variety of disease states of which virology is the most mature. host cyps are clearly hijacked by several viruses to enable effective replication, and cyp inhibition offers a promising new approach to antiviral therapy. the additional finding that cyp inhibition plays a role in uncloaking a virus (hcv) to immune surveillance is potentially exciting; however, discovery efforts in this field are likely to be challenging, as many in vitro virus infection screening systems are successful due to an immunological defect that renders the host cell permissive to viral infection. the discovery of non-immunosuppressive cyp inhibitors, such as alisporivir, allows cyp inhibition to be considered in disease states wherein immunosuppression would be prohibitive. furthermore, the demonstration of scy-635 to have good oral bioavailability and ddi potential clearly indicates that relatively large, macrocyclic peptides could, in some cases, be considered as 'drug-like'. as the biology of cyps continues to unfold, the challenges to medicinal chemistry will be to discover compounds that can selectively inhibit members of the cyp family, and to use these tools to validate the role of cyps in disease states and as leads for drug discovery efforts. while csa has served admirably as the starting point for cyp research, little selectivity within the cyp family has been described for derivatives of this compound. new inhibitory templates are beginning to appear that may deliver improved selectivity; however, the application of fragment-based screening, with the aid of available structural data to support the location of binding, should allow inhibitors to be designed that exploit regions of the cyp protein that differ among family members. future virol. 2007, 2, 65; (b) tang, h. viruses us 6255100 b1 ep 1086124 b1 61st gordon research conference on natural products 57th annual meeting of the american association for the study of liver diseases 45th annual meeting of the european association for the study of the liver key: cord-293857-o8rlqsq5 authors: ghosh, arun k.; brindisi, margherita title: organic carbamates in drug design and medicinal chemistry date: 2015-01-07 journal: j med chem doi: 10.1021/jm501371s sha: doc_id: 293857 cord_uid: o8rlqsq5 [image: see text] the carbamate group is a key structural motif in many approved drugs and prodrugs. there is an increasing use of carbamates in medicinal chemistry and many derivatives are specifically designed to make drug–target interactions through their carbamate moiety. in this perspective, we present properties and stabilities of carbamates, reagents and chemical methodologies for the synthesis of carbamates, and recent applications of carbamates in drug design and medicinal chemistry. carbamate-bearing molecules play an important role in modern drug discovery and medicinal chemistry. organic carbamates (or urethanes) are structural elements of many approved therapeutic agents. structurally, the carbamate functionality is related to amide-ester hybrid features and, in general, displays very good chemical and proteolytic stabilities. carbamates are widely utilized as a peptide bond surrogate in medicinal chemistry. this is mainly due to their chemical stability and capability to permeate cell membranes. another unique feature of carbamates is their ability to modulate inter-and intramolecular interactions with the target enzymes or receptors. the carbamate functionality imposes a degree of conformational restriction due to the delocalization of nonbonded electrons on nitrogen into the carboxyl moiety. in addition, the carbamate functionality participates in hydrogen bonding through the carboxyl group and the backbone nh. therefore, substitution on the o-and n-termini of a carbamate offers opportunities for modulation of biological properties and improvement in stability and pharmacokinetic properties. carbamates have been manipulated for use in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamate derivatives are widely represented in agricultural chemicals, such as pesticides, fungicides, and herbicides. they play a major role in the chemical and paint industry as starting materials, intermediates, and solvents. furthermore, organic carbamates serve a very important role as optimum protecting groups for amines and amino acids in organic synthesis and peptide chemistry. in recent years, carbamate derivatives have received much attention due to their application in drug design and discovery. however, there are hardly any reviews on this subject in the literature. in the present perspective, we plan to provide an overview of the leading role of organic carbamates in medicinal chemistry, with particular focus on therapeutic carbamates and carbamate-based prodrugs. in this context, we will highlight the chemical methodologies adopted for the synthesis of these carbamate derivatives. also, we will outline successful designs of organic carbamates, including a variety of cyclic ether-derived carbamates, as suitable amide bond surrogates leading to a wide range of novel organic carbamates as potent hiv-1 protease, βsecretase, serine protease, and cysteine protease inhibitors. this information may be useful in further design of carbamate-based molecules as drugs or prodrugs. peptide-based molecules are an important starting point for drug discovery, especially in the design of enzyme inhibitors. because of their high affinity and specificity toward biological functions, peptide-based molecules also serve as valuable research tools. however, the poor in vivo stability, inadequate pharmacokinetic properties, and low bioavailability have generally limited their broader utility. hence, a variety of peptide mimics are being developed to improve drug-like character along with increased potency, target specificity, and longer duration of action. 1−3 to this end, several classes of peptidomimetics are tailored by replacing the native amide bond with unnatural linkages 4−6 such as retro-amide, 7 urea, 8−12 carbamate, 13 and heterocycles 14,15 as peptide bond surrogates. these functionalities confer metabolic stability toward aminopeptidases, the enzymes involved in the metabolism of peptidelike drugs. the carbamate's emerging role in medicinal chemistry is also due to its chemical stability and to its capability to increase permeability across cellular membranes. these attributes of organic carbamates have been exploited in drug design. as a result, the carbamate motif is becoming the choice for peptide bond surrogates. other uses of carbamates are well-known. particularly, the employment of carbamates in various industries as agrochemicals, in the polymer industry, and also in peptide syntheses. 16−18 in addition, among the various amineprotecting groups, carbamates are commonly used to enhance their chemical stability toward acids, bases, and hydrogenation. 19 one important feature of organic carbamates is represented by the amide resonance. the amide resonance in carbamates has been studied in detail employing both experimental and theoretical methods by estimating the c−n bond rotational barriers. 20−25 the amide resonance in carbamates has been shown to be about 3−4 kcal mol −1 lower than those of amides, owing to the steric and electronic perturbations due to the additional oxygen. 26 three possible resonance structures (a, b, and c, figure 1 ) contribute to the stabilization of the carbamate moiety. carbamate motifs are characterized by a pseudo double bond. this implies the potential deconjugation of the heteroatom-(σ-bond)-carbon-(π-bond)-heteroatom system that restricts the free rotation about the formal single σ-bond. therefore, two isomers, syn and anti, may coexist in carbamates ( figure 2) . 23, 27 although carbamates display close similarity to amides, they show preference for the anti-isomer conformation. 28 the anti rotamer is usually favored by 1.0−1.5 kcal mol −1 for steric and electrostatic reasons with respect to the syn counterpart. 22 in many cases, the energy difference may be close to zero. as a result, those carbamates are found as an approximately 50:50 mixture of syn and anti isomers, as in the case of a number of boc-protected amino acid derivatives. this issue is of key importance since this balanced rotamer equilibria and the low activation energies render carbamates as optimal conformational switches in molecular devices. 23 the influence of the r and r 1 substituents on the free-energy difference between the two conformations has been investigated. beyond steric effects, electronegativity of r 1 must be considered since it may affect the conformation in many ways, including changes in the dipole moment and bond angles. 28 only the anti conformation would be expected in five-, six-, and seven-membered cyclic carbamates. calculations of the dipole moment for the carbamate group support this expectation. 29 solvent, concentration, salts, and ph strongly influence the free energy difference of the syn and anti isomers of carbamates as well. intra-and intermolecular hydrogen bonding may also perturb the syn−anti isomer equilibrium of carbamates. 22, 25, 30 a representative example of hydrogen bonding and concentration dependence was provided by gottlieb, nudelman, and collaborators. 28 the authors took into consideration n-boc-amino acids and their corresponding methyl esters. an unusual abundance of syn-rotamer for n-boc-amino acids was detected. n-boc-amino acid esters give the expected spectra, consistent with previous reports of only a single species being observed at room temperature. concentration-dependent 1 h nmr spectra indicate that the proportion of the syn-rotamers increases with concentration, supporting the existence of an aggregation process. 28 since decreasing temperature is another method for stabilizing oligomerization, nmr experiments were also performed at different temperatures. as expected, when the temperature increases, the favored rotamer switches from syn to anti. overall, the collected data strongly supports the concept that the syn rotamers of n-carbamoylated amino acids form intermolecularly h-bonded species and the oh of the carboxylic acid must be involved in this process, as the corresponding esters do not behave similarly. to explain this phenomenon, the formation of a dimer was suggested ( figure 3 ). support of this hypothesis was provided by adding increasing amounts of acetic acid to a solution of a carbamoylated amino acid ester. as expected, the syn rotamer appeared, and its concentration increased as a function of the amount of acid added. in contrast, addition of acetic acid to a solution of the corresponding carbamoylated amino acid did not affect the anti/syn ratio. in this context, moraczewski and co-workers designed a more effective hydrogen-bonding system that selectively perturbs the syn/anti rotamer equilibrium of a target carbamate group. 22 the authors examined the abilities of acetic acid and 2,6-bis(octylamido)pyridine (3) to perturb the syn/ anti ratio of carbamates 1 and 2 ( figure 4 ). 22 in a cdcl 3 solution, acetic acid moderately stabilizes double hydrogen bonding of the syn rotamer of phenyl carbamate 1 ( figure 4a) , with no relevant effect on the syn/anti ratio for 2pyridyl carbamate 2 ( figure 4b ). in the second case, the carboxylic acid favors donation of a hydrogen bond to the more basic pyridyl nitrogen and forms the complex shown in figure 4b . on the contrary, in the case of the donor−acceptor−donor triad 3, it strongly stabilizes the syn rotamer of 2 ( figure 4d ) over the anti rotamer ( figure 4c ). there is no effect on the syn/anti ratio for 1, presumably because of a steric deterrent to the formation of a hydrogen-bonded complex ( figure 4e ). the carbamate moiety plays a noteworthy role in medicinal chemistry, not only because it is found in drugs but also for its presence in a number of prodrugs. 31 the rate and level of their hydrolysis is a key issue for the duration and intensity of their pharmacological activity. fast hydrolysis of carbamate-bearing drugs may result in weak or shortened activity. on the contrary, carbamate-based prodrugs must undergo extensive hydrolysis at a suitable rate for releasing an active drug and obtaining the expected activity profile. vacondio et al. recently proposed an interesting study in which they compiled a large number of reliable literature data on the metabolic hydrolysis of therapeutic carbamates. 32 the authors were able to exploit the collected data to gain a qualitative relationship between molecular structure and lability to metabolic hydrolysis. a trend was extrapolated, according to which the metabolic lability of carbamates decreased in the following series: aryl-oco-nhalkyl ≫ alkyl-oco-nhalkyl ∼ alkyl-oco-n(alkyl) 2 ≥ alkyl-oco-n(endocyclic) ≥ aryl-oco-n(alkyl) 2 ∼ aryl-oco-n(endocyclic) ≥ alkyl-oco-nharyl ∼ alkyl-oco-nhacyl ≫ alkyl-oco-nh 2 > cyclic carbamates. 32 therefore, carbamates derived from ammonia or aliphatic amines are sufficiently long-lived. an example is represented by cefoxitin (4), a second-generation cephalosporin antibiotic ( figure 5 ). cyclic fiveor six-membered carbamates are quite stable and do not usually undergo metabolic ring opening. the antibacterial agent linezolid (5) is a representative example of this class ( figure 5 ). for these drugs, carbamate hydrolysis is not necessarily the half-lifedetermining metabolic reaction. on the contrary, fatty acid amide hydrolase (faah) inhibitor 6 (urb524) 33 showed significant hydrolysis in buffer at physiological ph after 24 h ( figure 5 ). other representative therapeutic carbamate drugs and prodrugs will be discussed in sections 4 and 5, respectively. organic carbamates play an important role in organic synthesis, especially as subunits of biologically active compounds. accordingly, simple and efficient methods for the synthesis of carbamates are of great interest. a number of methods have been developed for the synthesis of carbamates. 3.1. carbamate synthesis via traditional methods. over the years, a variety of carabamates have been prepared by utilizing the hofmann rearrangement of amides, 34−36 the curtius rearrangement of acyl azides, 37,38 the reductive carbonylation of nitroaromatics, 39 the carbonylation of amines, 40 the reaction of alcohols with isocyanates, 41 and carbon dioxide alkylation. 42−45 the hofmann rearrangement (method i, scheme 1) is wellrecognized as a useful method to convert primary carboxamides to amines or carbamates, characterized by the reduction of one carbon in the structure. 46 much effort has been devoted to the development of modified reagents to optimize the hofmann rearrangement since the classical method for this transformation, involving the use of an alkaline solution of bromine, is unsatisfactory and unreliable. 35 a variety of oxidants and bases have been proposed as modified agents, e.g., iodine(iii) reagents such as phi(oac) 2 , 47 meobr, 48 nbs-ch 3 ona, 49 nbs-koh, 46 lead tetraacetate, 50 and benzyltrimethylammonium tribromide. 51 these modified methods, however, require more than 1 equiv or an excess amount of the oxidizing reagent, which is not very convenient. the curtius rearrangement (method ii, scheme 1) is the thermal decomposition of acyl azides into the isocyanate intermediate. this method is widely employed in the transformation of carboxylic acids into carbamates and ureas. acyl azides are usually prepared from carboxylic acid derivatives such as acyl chlorides, 52,53 mixed anhydrides, 54,55 and hydrazides. 56, 57 subsequent isocyanate intermediates can be trapped by a variety of nucleophiles to provide the carbamate derivatives. the acid chloride method is not suitable for acidsensitive functionalities. one-pot transformations of carboxylic acids into carbamates avoids the isolation of unstable acyl azides. however, protocols involving the use of diphenylphosphoryl azide (dppa) for the one-pot curtius reaction are also characterized by issues related to toxicity and the high boiling point of dppa, which creates difficulties during workup and purification. 58−60 other general methods for carbamate preparation involve the use of the highly toxic phosgene, 61 phosgene derivatives, 62, 63 or isocyanates. 64 significant efforts have been made to find an alternative to the phosgene process. a very attractive substitute for phosgene is carbon dioxide because it is a classic renewable resource (method iii, scheme 1). in addition, its use is also very attractive due to its environmentally benign nature (nontoxic, noncorrosive, and nonflammable). 65 carbon dioxide is wellknown to react rapidly with amines to form carbamic acid ammonium salts. the majority of the approaches in this context rely on the creation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles. nevertheless, since the nucleophilicity of the carbamate anion is lower than that of the amine formed in the equilibrium of the salt formation, the subsequent reaction of the carbamate salts with alkyl halides does not selectively provide urethanes. 44, 66 the formation of carbamates from isocyanates (method iv, scheme 1) is fundamentally important to polyurethane industries. synthetic limitations and toxicity issues, however, are associated with the use of phosgene, the most common route to obtain isocyanates. 64 the readily available alkyl chloroformates are the most frequently used reagents for the preparation of carbamates (method v, scheme 1). however, these reagents display major drawbacks, as a large excess of base and a long reaction time are required in order to gain acceptable reaction efficiency. moreover, excess reagents are not suitable for the synthesis of molecules bearing multiple functionalities in which the chemoselectivity is critical. 67 3.2. carbamate synthesis via activated mixed carbonates. a number of organic carbonates have been developed as low-cost and benign alternatives to the phosgenebased routes for the synthesis of organic carbamates. in this context, several new alkoxycarbonylating agents (7−11) based on mixed carbonates have been developed ( figure 6 ). these methods are often used for the synthesis of carbamates in drug design. 68−72 mixed carbonates with a p-nitrophenyl moiety are frequently used for the preparation of a large range of carbamates. 73−76 for this, p-nitrophenyl chloroformate (7, pnpcocl), when treated with the suitable alcohol in the presence of base, furnishes the corresponding activated carbonates, which have been shown to be useful and effective alkoxycarbonylating reagents for suitable amines (scheme 2). examples of carbamate derivatives are shown in table 1 . several alkoxycarbonylating reagents for amino groups having heterocyclic groups, such as n-hydroxyimide, have scheme 1. traditional synthetic methodologies adopted for the synthesis of carbamates been reported. moreover, the utility and versatility of carbonates and oxalates containing an electron-withdrawing group, such as n-hydroxyimide and benzotriazole derivatives as reagents for various tranformations, have been described. 72, 81, 82 takeda et al. reported that 1-alkoxy[6-(trifluoromethyl)benzotriazolyl]carbonates easily derived from 1,1-bis[6-(trifluoromethyl)benzotriazolyl]carbonate (8, btbc) showed high acylating reactivity toward alcohols as well as amino groups. 83 btbc was prepared from 6-trifluoromethyl-1hydroxybenzotriazole and trichloromethyl chloroformate and purified by washing with dry ether. moreover, it can be stored for several months in a freezer. btbc was allowed to react with primary alcohols in acetonitrile at room temperature to give stable activated carbonates. the carbonates were treated with amines in the presence of 4-dimethylaminopyridine (dmap), providing the corresponding carbamates (scheme 2 and table 2 ). 83 in connection with our research work aimed at synthesizing biologically active polyfunctional molecules for probing enzyme active sites, we required a more general and synthetically reliable method for the synthesis of various carbamate derivatives. in 1991, we described the utility of di(2-pyridyl) carbonate (9, dpc) 84 as an efficient, high-yielding, and convenient alkoxycarbonylation reagent for amines overcoming many of the limitations of existing methodologies. 85 dpc was readily prepared from commercially available 2-hydroxypyridine and triphosgene in the presence of triethylamine and subsequently reacted with the suitable primary or secondary alcohol (e.g., (+)-menthol) to provide a mixed carbonate. alkoxycarbonylation of primary and secondary amines with the mixed carbonates was carried out in the presence of triethylamine and furnished the corresponding carbamates in good yields (scheme 2, method a, and table 3 ). potassium hydride was used in the place of triethylamine in the preparation of the mixed carbonates containing tertiary alcohols (scheme 2 and table 3 ). 85 subsequently, we investigated the scope of n,n′-disuccinimidyl carbonate (10, dsc) 81 promoted alkoxycarbonylation of amines with a host of alcohols under mild conditions. 86 rich and co-workers highlighted the convenience of succinimidylbased mixed carbonates for the high-yielding introduction of a 2-(trimethylsilyl)ethoxycarbonyl (teoc) protecting group to amino acids, without oligopeptide byproduct formation. 87 dsc was found to be a highly effective alkoxycarbonylating reagent for a variety of primary and sterically hindered secondary alcohols. dsc is commercially available, or it can be conveniently prepared from n-hydroxysuccinimide following a procedure tracing out the synthesis of dpc. 85 the ready availability of dsc, the stability of the mixed carbonates, and the mildness of the reaction procedure render this method a reliable route to organic carbamates (scheme 2 and table 4 ). 86 since azides were extensively employed as incipient amines in the context of amino sugar and amino acid syntheses, their conversion into the corresponding carbamate derivatives could provide a novel, effective route for medicinal chemistry applications. in this context, a facile synthetic protocol to transform various azides into the corresponding functionalized journal of medicinal chemistry urethanes in high yields has been developed. 90 in general, mixed carbonates of variously protected alcohols were prepared by reaction of excess dsc or dpc, as described previously. exposure of mixed carbonates to catalytic hydrogenation conditions with azides in the presence of 10% palladium on charcoal in tetrahydrofuran furnished the corresponding carbamates. interestingly, the use of triethylamine as a promoter has a notable effect on the yield and the rate of the alkoxycarbonylation process (scheme 2 and table 5 ). 90 more recently, yoon and co-workers exploited 2-substitutedpyridazin-3(2h)-ones as electrophilic transfer reagents. 91, 92 in particular, the authors investigated the carbonylation potency of phenyl 4,5-dichloro-6-oxopyridazine-1(6h)-carboxylate (11) to amines for the preparation of phenylcarbamates (scheme 2 and table 6 ). compound 11 is stable in air and in organic solvents at high temperature and is prepared easily from cheap and commercially available 4,5-dichloropyridazin-3(2h)-one (12) in the presence of phenylchloroformate and triethylamine (scheme 2). 3.3. recent methodologies for carbamate synthesis. the application of carbon dioxide in organic synthesis has recently attracted much interest. most of the approaches rely on the generation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles, usually alkyl halides. 93−95 in this context, a mild and efficient preparation of alkyl carbamates on solid supports was described by jung et al. 96 amines and anilines were coupled with merrifield's resin through a co 2 linker in the presence of cesium carbonate and tetrabutylammonium iodide (tbai). carbon dioxide was supplied by bubbling it into the reaction suspension, where n,n-dimethylformamide (dmf) was the solvent of choice (scheme 3). 96 the reaction conditions are convenient for purification, and the reactions undergo complete conversions. the method is convenient for the generation of large combinatorial libraries for rapid screening of bioactive molecules. chiral substrates susceptible to racemization have survived the conditions (table 7) . later, these authors reported a one-pot synthesis of n-alkyl carbamates starting from primary amines (scheme 4). 96 carbamates were generated via a three-component coupling of primary amines, co 2 , and an alkyl halide in the presence of cesium carbonate and tbai in anhydrous dmf (scheme 4 and table 8 ). direct n-alkylation of the intermediate carbamate a in the presence of additional cesium carbonate by using a different alkyl halide gave rise to the desired n-alkyl carbamate b (scheme 4). isolation of the intermediate a proved to be unnecessary, offering shortened synthetic sequences. 40 it is interesting to note that tbai helps to minimize the overalkylation of the produced carbamate, presumably by enhancing the rate of co 2 incorporation and/or stabilizing the incipient carbamate anion through conjugation with the tetrabutylammonium cation. 97 sakakura and co-workers reported urethane synthesis by the reaction of dense carbon dioxide with amines and alcohols by a procedure that is not only phosgene-free but also completely halogen-free (scheme 5). 98 dialkyl carbonate synthesis from an alcohol and co 2 is catalyzed by metal complexes such as dialkyl(oxo)tin and dialkyl(dichloro)tin. however, the alcohol conversion is very poor. similarly, the direct reaction of an amine, an alcohol, and carbon dioxide in the presence of dialkyltin compounds produced urethane only in a poor yield. the low conversion observed was attributed by the authors to thermodynamic limitations and catalyst deactivation by coproduced water. in order to overcome this issue, a new reaction system utilizing acetals as a chemical dehydrating agent, with subsequent alcohol regeneration (scheme 5), was developed. in order to obtain urethane in good yields, dense-phase co 2 under high pressure was necessary to lower the major side reactions, namely imine formation from acetone and alkylation of amines by alcohols. however, developing less toxic and more active catalysts based on metals other than tin was required. later, these authors reported novel nickel-based catalytic systems for dehydrative urethane formation from carbon dioxide, amines, and alcohols (scheme 6). 99 interestingly, adding nitrogenbased bidentate ligands efficiently improved the catalytic activity of ni(oac) 2 -based catalysts (scheme 6 and table 9 ). bipyridines and phenanthrolines with strong coordinating abilities (low steric hindrance and high electron densities) were the better choice for obtaining urethanes in high yields. it is important to note that the ni-phenanthroline system is more active and less toxic than dialkyl(oxo)tin under the same reaction conditions. it is also noteworthy that the catalytic activity of the ni(oac) 2 -(4,4′-dimethylbipyridine) system is highly dependent on the ligand/metal ratio (table 9 ). peterson and co-workers proposed a method for rapid sar development of compounds bearing urea or carbamate functionalities (scheme 7). 100 for carbamate formation, an journal of medicinal chemistry amine, in principle, could proceed through the carbamic acid− isocyanate reaction, and subsequent reaction with an alcohol may provide a carbamate product. while this is precedented by an intramolecular reaction variant to produce cyclic carbamates, 101 the desired intermolecular coupling was not fruitful under the proposed reaction conditions. carbamic acids produced from secondary amines, however, did react with alcohols under mitsunobu conditions (dibenzyl azodicarboxylate, dbad, and tributylphosphine) in a dbu-catalyzed reaction with gaseous carbon dioxide, providing the corresponding carbamates (scheme 7 and table 10 ). this reaction did not proceed through the isocyanate intermediate but rather through an s n 2 displacement of the activated alcohol. this hypothesis is supported by the observed inversion of stereochemistry upon conversion of a chiral secondary alcohol to the corresponding carbamate (table 10) . 100 very recently, jiao and co-workers reported a practical, pdcl 2 -catalyzed efficient assembly of organic azides, carbon monoxide, and alcohols for the direct synthesis of carbamates via isocyanate formation and application in situ (scheme 8). 102 mild and neutral reaction conditions and generation of harmless n 2 as the byproduct render this protocol very useful, particularly for the synthesis of bioactive compounds. moreover, the employment of co at atmospheric pressure and the use of a small amount of pdcl 2 catalyst (2 mol %) in the absence of any ligand represent a real alternative to customary carbamate synthetic methods (table 11) . 102 the synthesis of carbamates through the generation of carbamoyl chlorides is not convenient because of the requirement of the toxic phosgene. also, such carbamoyl chlorides are highly reactive, prone to hydrolysis, unstable, and not suitable for long-term storage. for these problems, batey and co-workers identified the use of carbamoylimidazolium salts as convenient n,n′-disubstituted carbamoyl transfer reagents, showing increased reactivity over carbamoylimidazoles as a result of the imidazolium effect (scheme 9). 103−105 these salts are readily prepared by the sequential treatment of secondary amines with n,n′-carbonyldiimidazole (cdi) and iodomethane (scheme 9). authors envisaged that the carbamoylimidazolium salts, while relatively unreactive with alcohols, would react with nucleophlic alkoxides to produce the corresponding carbamates (table 12 ). in the case of phenols, tertiary amines are appropriate bases for the in situ generation of the reactive phenoxides. the lower acidity of aliphatic alcohols presumably prevents the formation of the alkoxide anion, which would serve as the reactive nucleophile. less acidic alcohols react with carbamoylimidazolium after their conversion into more nucleophilic sodium alkoxides (scheme 9). 106 the use of solid-supported reagents has become ubiquitous due to enhanced reactivity and selectivity, milder reaction conditions, convenient work-ups, and decreased solvent waste. the modified hofmann rearrangement, proposed by gogoi et al., is operationally simple, inexpensive and applicable to a variety of aliphatic and aromatic amides for the synthesis of methyl carbamates (scheme 10). 36 kf/al 2 o 3 represents a useful and interesting solid-supported strong base, which replaces organic bases in a variety of reactions. 107 sodium hypochlorite is an inexpensive, convenient, and safe alternative to the currently employed oxidants. 108 this prompted the authors to investigate kf/al 2 o 3 along with naocl as an efficient reagent system for hofmann rearrangement. kf/al 2 o 3 basicity stems from the formation of koh in the initial preparation of the solid-supported material by the reaction of kf with alumina supports. under these highly basic reaction conditions, hypochlorite ion is the predominant form of chlorine, reacting with the amide to form an n-chloroamide, which later undergoes rearrangement to the isocyanate. in the presence of methanol, the isocyanate is rapidly converted into the corresponding methyl carbamate (table 13) . 36 modifications of the curtius rearrangement have also been explored. lebel and co-workers have reported a useful protocol for the preparation of tert-butyl carbamates from the corresponding carboxylic acids. 109 their reaction with di-tert(scheme 11a and table 14) . these authors extended the same methodology to the direct synthesis of carbamates of aromatic amines using aromatic carboxylic acids (scheme 11b and table 14) . 110 in particular, the reaction of a chloroformate or di-tert-butyl dicarbonate and sodium azide with an aromatic carboxylic acid produced the corresponding acyl azide, presumably through the formation of an azidoformate. in contrast to what was observed with aliphatic carboxylic acids, using similar reaction conditions, aromatic carboxylic acids led mainly to the formation of the corresponding tert-butyl ester, likely via the displacement of an azide leaving group with tert-butoxide. this may be ascribed to the higher stability of aromatic acyl azides with respect to their aliphatic counterparts. therefore, for these substrates, the curtius rearrangement can be promoted only at higher temperatures (40 vs 75°c). 110, 111 as mentioned, alkyl chloroformates are the most frequently used reagents for the preparation of carbamates, although the need of an excess amount limits their usefulness. a promising method for preparing carbamates involves the use of a catalytic promoter. 112−115 lately, indium-mediated reactions have gained significant consideration due to the high reactivity and unique properties of indium reagents, among them nontoxicity and inertness toward air and water. 116−118 moreover, pretreatment is not required for activating indium metal. in this context, jang and co-workers developed a simple, efficient, and selective method for synthesizing carbamates from amines, employing a catalytic amount of indium and only an equimolar amount of alkyl chloroformate (scheme 12). 67 the method shows the generality for a wide variety of sterically diverse amines and alcohols and can also be applicable for the selective protection of amino groups under mild conditions (table 15) . arndtsen et al. proposed another application of indium-based reagents for the generation of n-protected amines in a single step (scheme 13 and table 16 ). 119 since organoindium reagents readily transfer their organic groups to an imine carbon, only one-third of an equivalent is required, and the only byproduct is represented by indium trichloride. tetraorganoindium reagents can also be employed in a similar fashion for transferring all four organic groups. therefore, one-fourth of an equivalent of indium is necessary for their reaction with imines. copper(i) chloride (10%) was found to be the most efficient catalyst. sodeoka and colleagues reported the use of 1-alkoxycarbonyl-3-nitro-1,2,4-triazole reagents as useful intermediates for the preparation of carbamates (scheme 14). 121 to achieve a rapid and clean reaction, the features of the leaving group have a key role. an ideal leaving group should have a highly electronwithdrawing element in order to increase the electrophilicity of the carbonyl carbon, and the nucleophilicity should be low to avoid side reactions. it should also be easily separated from the reaction product. 3-nitro-1,2,4-triazole (nt), 120 although showing nucleophilicity, could be easily removed from the table 9 . nickel-catalyzed urethane synthesis from co 2 99 a conversion of amine. b urethane/consumed amide × 100. c ni/l = 1:1. d ni/l = 1:5. reaction due to its insolubility in dichloromethane or chloroform. nt-based reagents have a series of benefits such as high stability, since they can be stored for long periods without decomposition. reactions of these nt reagents with primary and secondary amines proceeded quickly to give the corresponding carbamates in >95% yield (scheme 14a and table 17 ). in contrast to aliphatic amines, aromatic amines were less reactive. however, the addition of triethylamine was found to be effective in promoting the reactions (scheme 14b and table 17 ). 121 the reductive carbonylation of aromatic nitro compounds to the corresponding carbamates has remained a subject of great interest both from mechanistic and application standpoints (scheme 15). in this section, we will briefly mention the methodologies involving the use of an alcohol, although other procedures employing chloroformates have also been recently reported. 122, 123 cheng and collaborators report the use of ru(co) 4 − and ru 3 (co) 12 complexes for the catalysis of this reaction and highlighted the key effect of alcohol on the selectivity of carbamates (table 18) . 124 the results clearly indicate that low selectivity of carbamate is closely related to the ability of the alcohol to reduce nitroarenes to amino derivatives. therefore, the employment of an alcohol that cannot reduce nitroarene greatly increases the selectivity of carbamate. later, the binuclear rhodium complex [(ph 3 p) 4 rh 2 (μ-oh) 2 ]·2c 6 h 6 was employed as an effective catalyst for the reductive carbonylation of nitrobenzenes to carbamate esters (table 18) . 125 palladiumbased catalysts have also been explored (table 18) . 126−128 carbamate synthesis via transfunctionalization of substituted ureas and carbonates in the presence of di-n-butyltin oxide (dbto) as the catalyst was reported by chaudhari and colleagues (scheme 16a and table 19 ). 129 the carbonate reactivity pattern seems to be driven by the leaving group ability of the alkoxides and phenoxide to form the carbamate observed in aminolysis of carbonates. it has been shown that basicity of reacting urea plays a vital role in the catalytic activity of this reaction. indeed, aliphatic ureas show higher reactivity compared to aromatic ureas due to their higher basicity. the basic dbto is supposed to work as a nucleophile by attacking the carbonyl carbon of the carbonate, thus generating the catalytically active species dibutyl alkoxy carbonato tin [a]. 130 use of dialkyl carbonates as environmentally friendly and nontoxic phosgene substitutes in alkoxycarbonylation reactions has also been exploited by porco et al. (scheme 17). 132 particularly, the authors examined the scope of zr(iv)catalyzed carbonate−carbamate exchange processes to prepare carbamates from dialkyl carbonates employing 2-hydroxypyridine (hyp) as a catalytic additive (table 20) . recently, padiya and co-workers reported a useful method for preparing carbamates in an aqueous media (scheme 18). 133 interestingly, they found that 1,1′-carbonyldiimidazole (cdi), although unstable in water, rapidly reacts in aqueous media with amine to give good yields of the corresponding nsubstituted carbonylimidazolide. carbonylimidazolide derived from the primary amine reacts in situ with a nucleophile such as phenol, providing the corresponding carbamate. the product precipitates out from the reaction mixture and can be obtained in high purity by filtration, making the method simple and scalable (table 21) . 133 cdi was also found to mediate the lossen rearrangement, which occurs in the transformation of an activated hydroxamic acid into the corresponding isocyanate (scheme 19). 134 the proposed methodology is experimentally efficient and mild, being characterized by imidazole and co 2 as the only stoichiometric byproducts. this method is a green and unconventional alternative to the curtius and hofmann rearrangements (table 22) . 135 another method based on the lossen rearrangement was recently proposed. 136 the methodology envisaged the reaction of a hydroxamic acid with an alcohol, promoted by 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride; tct) in the presence of an excess of n-methyl morpholine (nmm) (scheme 20 and table 22 ). carbamates are inherent to many fda approved drugs. this structural motif is also a key functionality in numerous medicinal agents with clinical potential. in this section, a series of therapeutic carbamates with a variety of applications is outlined. 4.1. miscellaneous carbamates with clinical relevance. 4.1.1. rivastigmine. rivastigmine (194, figure 7 ) tartrate (exelon, novartis pharma) is a carbamate derivative that reversibly inhibits the metabolism of acetylcholinesterase (ache) and butyrylcholinesterase (buche) preferentially in the central nervous system (cns). it is used for the treatment of mild-to-moderate alzheimer's disease (ad) dementia and dementia due to parkinson's disease. 137, 138 the drug can be administered orally or via a transdermal patch. the transdermal patch reduces side effects such as nausea and vomiting. rivastigmine undergoes extensive metabolism by che-mediated hydrolysis to the decarbamylated metabolite, without involvement of the major cytochrome p450 (cyp450) isozymes. the metabolite may undergo n-demethylation as well as conjugation. the pharmacokinetic half-life of rivastigmine in ad patients is around 1.5 h. when given orally, rivastigmine is well-absorbed, with a bioavailability of about 40% administered as a 3 mg dose. 137, 139 4.1.2. muraglitazar. muraglitazar (195) contains a carbamate functionality. it is a potent, novel nonthiazolidindione peroxisome proliferator-activated receptor dual agonist (pparα/γ) that demonstrated highly efficacious glucose and lipid lowering activities in vivo, along with an excellent adme profile. 140 in a double-blind randomized clinical trial, muraglitazar resulted in a statistically significant improvement in plasma triglyceride, hdl cholesterol, apob, and non-hdl cholesterol concentrations at week 12. muraglitazar reduced triglyceride concentrations to a larger extent than did pioglitazone, regardless of baseline triglyceride levels. muraglitazar and pioglitazone treatment was associated with slight (3−4%) increases in ldl cholesterol. however, muraglitazar development was discontinued due to major adverse cardiovascular side effects. 141 4.1.3. roxifiban. roxifiban (196) is a carbamate derivative with a methyl ester prodrug. it is a potent, nonpeptide antagonist of the glycoprotein iib/iiia receptor. 142, 143 the free acid resulting from roxifiban hydrolysis blocks the binding of fibrinogen to the receptor, thereby inhibiting platelet aggregation and providing a mechanism for antithrombotic mebendazole (199) is a methyl carbamate derivative showing broad-spectrum anthelmintic properties. it demonstrated efficacy in the oral treatment of ascariasis, uncinariasis, oxyuriasis, and trichuriasis. like other benzimidazole anthelmintics, mebendazole's primary mechanism of action is consistent with tubulin binding. 148 mebendazole was discontinued in 2011. 149 4.1.6. flupirtine and retigabine. flupirtine (200) and retigabine (201) are ethyl carbamate derivatives. flupirtine is a centrally acting nonopioid analgesic 150 that was identified within an antiepileptic drug discovery program by the u.s. national institutes of health. the doses used in a small clinical trial exceeded those established for analgesic activity. 151 on the basis of this data, subsequent structural optimization resulted in retigabine. 152 retigabine has anticonvulsant properties that appear to be mediated by opening or activating neuronal voltage-gated potassium channels. flupirtine showed n-methyl-d-aspartate (nmda) receptor antagonist properties. 4.1.7. felbamate. felbamate (202, felbatol, meda pharmaceuticals) is an alkyl carbamate derivative. it is an antiepileptic drug. the mechanism of action of felbamate involves a dual mechanism involving inhibition of n-methyl-d-aspartate (nmda) receptor response and positive modulation of γamino butyric acid subtype a (gaba a ) receptor, thus decreasing neuronal excitation. 153 felbamate is rapidly absorbed (t max = 2−6 h) with an oral bioavailability > 90%. 154 felbamate undergoes moderate metabolism via cyp3a4 and cyp2e1 isoenzymes, which are amenable to inhibition and induction effects. 155, 156 the clinical use of scheme 11. synthesis of carbamates by modified curtius rearrangement it is a nonnucleoside reverse transcriptase inhibitor (nnrti). the drug is used as part of highly active antiretroviral therapy (haart). 157, 158 however, its use is associated with variable treatment response and adverse effects, in most part because of the large differences in pharmacokinetics. 159 cyp2b6 is the main enzyme catalyzing the major clearance mechanism of efavirenz (8-hydroxylation to 8-hydroxyefavirenz) in vivo. 160, 161 4.1.9. zafirlukast. zafirlukast (204, accolate, astrazeneca) is a cyclopentyl n-aryl carbamate derivative. it is a selective and competitive receptor antagonist of the cysteinyl leukotrienes d-4 and e-4, which is indicated for the prophylaxis and treatment of mild-to-moderate persistent and chronic asthma. 162 both o → ch 2 and o → nh bioisosteric analogues of zafirlukast were found to be potent. the carbamate moiety present in zafirlukast provided an excellent in vitro and in vivo profile and high oral bioavailability. 163 zafirlukast undergoes hepatic metabolism, where hydroxylation by cytochrome cyp2c9 is the major biotransformation pathway. the metabolites of zafirlukast do not significantly contribute to its overall activity. 164 4.1.10. mitomycin c. mitomycin c (205, mmc, mutamycin) is a complex carbamate derivative. it is an antitumor antibiotic that was identified in the 1950s in fermentation cultures of the gram-negative bacteria streptomyces caespitosus. 165 mmc is a site-specific, nondistorting dna cross-linking agent. 166, 167 however, recent reports suggest that dna may not be the primary target of the drug. in particular, interaction of mmc with rrna and subsequent inhibition of protein translation has been proposed. 168 mmc is customarily used as a chemotherapeutic agent in the treatment of several types of cancer, such as bladder, colon, and breast cancers. 169 4.2. therapeutic carbamates as hiv protease inhibitors. hiv protease is an aspartic acid protease responsible for the cleavage of the gag−pol polyprotein into functional proteins essential for the production of infections progeny virus. inactivation of hiv-1 protease either by site-directed mutagenesis or by chemical inhibition results in the formation of immature, noninfections virus particles. as a consequence, hiv-1 protease is an attractive target in antiviral therapy. hiv protease is a c 2 -symmetric, 198-amino acid homodimeric aspartyl protease in which each protein subunit contributes one asp-thr-gly motif to the single active site. 170 the x-ray crystallographic analysis of the native protein and subsequent protein−ligand complexes and extensive research programs on other aspartyl proteases, including human renin, 171 provided a path toward accelerated drug discovery programs targeting hiv protease. 172−174 a number of fda-approved hiv protease inhibitor drugs contain an important carbamate functionality. in this section, currently approved protease inhibitor drugs are discussed (figure 8) . 4.2.1. ritonavir. ritonavir (206, norvir, abt-538, a-84538, abbvie, inc.) structure possesses a thiazolyl methyl carbamate functionality. it is a peptidomimetic inhibitor of both the hiv-1 and hiv-2 proteases and was approved by the fda in march 1996. 175 this first-generation protease inhibitor was developed at abbott laboratories. the discovery of ritonavir was based on 50 of 0.025 μm, bioavailability of 78%, and a plasma half-life of 1.2 h. ritonavir has a high molecular weight; however, it showed excellent pharmacokinetic properties. this is possibly due to the increased stability of the thiazole groups to oxidative metabolism and also due to its effect on cytochrome p450 oxidative enzymes. ritonavir is a type ii heme ligand that fits into the cyp3a4 active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen. 176 amprenavir was identified as a potent, orally bioavailable hiv-1 protease inhibitor with a low molecular weight and a mean ic 50 of 12 nm. 177 it is marketed with a twice-a-day dosing format. amprenavir structure bears a stereochemically defined tetrahydrofuranylcarbamate engaging in a weak backbone interaction with the protease. 178, 179 in vitro and in vivo studies have shown that amprenavir is primarily metabolized by cyp3a4, and the two major metabolites result from oxidation of the tetrahydrofuran and aniline moieties. properties. drv also maintains high potency against multidrugresistant hiv-1 strains. the design of drv originated from the backbone binding concept envisaging that an effective protease inhibitor maximizes rich networks of hydrogen-bonding interactions with the backbone atoms throughout the active site of the protease. 185 the bis-thf moiety present in drv was designed based on the x-ray structure of inhibitor-hiv-1 protease complexes. the bis-thf carbamate moiety of drv was found to be essential for enzyme affinity (see figure 14 for details). drv demonstrated exceptional potency against both wild-type hiv isolates and a wide range of resistant variants. 186 hiv-1 variants. in 2008, drv received full approval for the treatment of therapy-naive adults and children. 188 drv is metabolized by the isoenzyme cyp3a4. 189, 190 however, in the presence of a low dose of ritonavir, drv exhibits very good pharmacokinetic properties in patients. 191 metabolism prodrugs are chemically modified forms of the actual pharmacologically active drug that undergo in vivo transformation to release the active drug molecule. this is a wellestablished strategy to improve drug disposition properties (physicochemical, biopharmaceutical, or pharmacokinetic properties) of pharmacologically relevant compounds and thereby increase their drug-like profile. 192, 193 a prodrug strategy helps to overcome a variety of hurdles in drug formulation and delivery such as (i) poor oral absorption and aqueous solubility, (ii) poor lipid solubility, (iii) chemical instability, (iv) rapid presystemic metabolism, (v) toxicity and local irritation, and (vi) lack of site-selective delivery. 193 a functional group on the parent drug may be used to form a chemical bond with the promoiety. generally, the linker should be self-removing or cleavable so that the parent drug can be released spontaneously or under a certain triggering condition, such as the presence of an enzyme or a change in ph. the promoiety coupled to the parent drug provides the ability to improve the drug-like properties or overcome the barriers in delivering the drug to its target cells. 194 carbamates are the esters of carbamic acid, preferentially used in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamates are typically enzymatically more stable than the corresponding esters. they are, in general, more susceptible to hydrolysis than amides. 195 thus, bioconversion of carbamate prodrugs requires esterases for the release of the parent drug. upon hydrolysis, carbamate esters release the parent phenol or alcohol drug and carbamic acid, which, due to its chemical instability, breaks down to the corresponding amine and carbon dioxide. carbamates of primary amines can also fragment into isocyanates and alcohols on treatment with bases, a further potential pathway for metabolic degradation. 192, 195 the oh-catalyzed hydrolysis of these carbamate esters (r′-nhco-or) is strongly dependent on both the pk a of the proton on the leaving group (roh) and the degree of substitution on the nitrogen of the carbamate ester. 196 since phenols have a lower pk a with respect to alcohols, carbamate esters of phenols are generally more chemically labile than those of alcohols. in the case of alcohols, both the n-monosubstituted and n,n-disubstituted carbamates are chemically stable toward hydrolysis. in phenols, n,ndisubstituted carbamates are chemically stable, whereas nmonosubstituted carbamates are the most labile toward chemical hydrolysis. short-lived carbamates have also been used as prodrugs of heteroaromatic amines (e.g., capecitabine, 217) and amidines (lefradafiban (221), dabigatran). 195 5.1. alcohol and phenol carbamate prodrugs. most of the therapeutically relevant carbamate prodrugs have been designed as substrates of specific enzymes. antibody-directed enzyme prodrug therapy (adept) 197, 198 and gene-directed enzyme prodrug therapy (gdept) 199 are new strategies for targeting tumors. carboxypeptidase g2 (cpg2), an enzyme of bacterial origin, has been shown to catalyze the cleavage of an amide, carbamate, or urea linkage between glutamic acid and an on the basis of this specificity, a large number of prodrugs have been designed and synthesized for cpg2. as shown in figure 9 , the prodrug 208 (zd2767p) 200 is activated by hydrolysis at the carbamate bond by cpg2 to the corresponding potent di-iodophenol mustard (209) . 201 208 was found to possess the best profile in terms of enzymatic kinetics, cytotoxicity, and in vivo efficacy. it was selected for clinical development. the half-life (t 1/2 ) of the drug is approximately 2 min, which is enough for diffusion into the tumor cell from the local release site and to minimize peripheral toxicity. 192, 202 irinotecan was designed to deliver camptothecin as a predominant topoisomerase i inhibitor for anticancer therapy. irinotecan hydrochloride salt 210 (cpt-11, camptosar; pfizer) is a parenteral aqueous soluble carbamate prodrug of antineoplastic topoisomerase i inhibitor 211 (sn-38, 7-ethyl-10-hydroxy-camptothecin). the potent antitumor activity of irinotecan is due to rapid formation of active metabolite 211 in vivo (figure 9 ). in this molecule, a dipiperidino ionizable promoiety is linked to the phenol functionality by a carbamate bond, thus improving the overall aqueous solubility. 203−205 the bioconversion back to 211 occurs primarily by human liver microsomal carboxylesterases, ces 1a1 and ces2, which release the ionizable piperidinopiperidine promoiety and 211, the active form of the drug. 205 beyond minimizing the rate of enzymatic hydrolysis of its prodrug, sustained drug action can also be provided by decreasing the rate of drug metabolism. this is the case of bambuterol (212, bambec, astrazeneca), a bis-dimethyl carbamate prodrug of the β2-agonist terbutaline (213), which is used as a bronchodilator in the treatment of asthma. the phenolic moiety of terbutaline is subjected to rapid presystemic metabolism. in bambuterol, protection of this functionality also avoids first-pass intestinal and hepatic metabolism. this prodrug is inactive, however, after oral administration; it is slowly converted to terbutaline, mainly outside the lungs, by a series of hydrolysis and oxidation reactions (maily catalyzed by plasma cholinesterase, pche, and by cyp450, figure 9 ). 206, 207 this allows a once-daily bambuterol treatment with respect to the three daily terbutaline administrations. 208 an n,n′-dimethyl ethylenediamine spacer, used for the evaluation of cyclization-elimination-based prodrugs of phenols 209 and alcohols, 210 has been used for the development of prodrugs as a part of the adept activation strategy. when activated by a specific enzyme, the terminal amino group on the spacer activates and initiates an intramolecular cyclization reaction to eliminate a phenol 211 or alcohol 212 parent drug with parallel release of the cyclized spacer. in one such application, scherren et al. 213 explored paclitaxel-2′-carbamates. this is particularly interesting because a free 2′-hydroxyl group is important for biological activity. in general, carbamate linkages are more stable in vivo than esters and carbonates. since the proteolytic active form of plasmin is located in the tumor, linking a cytotoxic drug to a plasmin substrate may result in tumor-selective delivery. on the basis of this rationale, following plasmin hydrolysis, the spacer is expected to undergo spontaneous cyclization to yield a cyclic urea derivative (imidazolidinone), thereby releasing paclitaxel (214) , as illustrated in scheme 21. 192, 214 5.2. amine and amidine carbamate prodrugs. the amine group is one of the most common functional groups in many approved drugs. amines in drugs can cause physicochemical hurdles that have the potential to limit their safety and effective delivery to desired sites of action. therefore, a variety of prodrugs of amines have been designed to overcome formulation and delivery barriers. the carbamate functionality has been utilized in many prodrug strategies designed for amines. short-lived carbamates are also used as prodrugs of heteroaromatic amines and amidines. 192 gabapentin (216, neurontin; pfizer, figure 10 ) is a structural analogue of γ-aminobutyric acid (gaba). it is marketed as an anticonvulsant and an analgesic agent. gabapentin shows a number of limitations, including saturable absorption, high interpatient variability, lack of dose proportionality, and a short half-life. gabapentin enacarbil (215, horizant, previously known as xp13512) is a carbamate prodrug of gabapentin. the prodrug is benefited by a monocarboxylate transporter type 1 (mct1). mct1 is expressed in all segments of the colon and upper gastrointestinal tract. the prodrug also helps the sodium-dependent multivitamin transporter (smv t), responsible for absorption of multiple essential nutrients. 215, 216 following absorption via these pathways, the prodrug is rapidly converted to gabapentin by nonspecific esterases, mainly in enterocytes and to a lesser extent in the liver. during conversion to gabapentin, each molecule of 215 also generates carbon dioxide, acetaldehyde, and isobutyrate (figure 10 ). 217 the oral bioavailability of 215 was improved from 25 to 84% in monkeys. it showed doseproportional gabapentin exposure in humans. 193 in 2011, xenoport received fda approval (horizant) for the treatment of moderate-to-severe restless legs syndrome. in 2012, horizant was also approved for the management of postherpetic neuralgia (phn) in adults. 218 capecitabine (217, xeloda, roche) was designed to achieve greater selectivity than its active form, 5-fluorouracil (220, 5-fu). 219 it is an orally administered carbamate prodrug of 5-fu, belonging to the fluoropyrimidine carbamate class. it requires a cascade of three enzymes for the bioconversion to the active drug. 220 as shown in figure 10 , the enzymatic bioconversion starts in the liver, where human carboxylesterases 1 and 2 (ces1 and ces2) cleave the carbamate ester bond. 219 intact capecitabine is absorbed in the intestine, and its bioconversion in the liver releases the parent drug. to some extent, its bioconversion proceeds in tumors, thus avoiding any systemic toxicity. in particular, the remaining transformations to 5-fu are catalyzed by cytidine deaminase and thymidine phosphorylase. the latter enzyme is highly enriched in tumors, thus providing selective release of 5-fu in cancer cells. 220, 221 the absorption of capecitabine is evident since 95% of an orally administered dose is recovered in urine and the t max of 5-fu is reached in approximately 1.5−2 h. 193 capecitabine is currently approved as a first line of therapy for colorectal and breast cancers and is also approved for use in combination with other anticancer drugs. 192, 222 alkoxycarbonyl derivatives can serve as useful prodrugs for benzamidines. for example, the methoxycarbonyl methyl ester lefradafiban (221, bibu104, boehringer ingelheim, germany) is effectively converted to the active platelet aggregation inhibitor fradafiban (222, bibu 52) after oral administration. this was revealed by monitoring the plasma concentrations of 222 and by ex vivo platelet aggregation studies. lefradafiban is the orally active prodrug of fradafiban, a glycoprotein iib/iiia receptor antagonist. 192 esterases, but not cyp450-dependent enzymes, are involved in the conversion of lefradafiban to fradafiban in vivo (figure 10 ). 223 over the years, we have developed a series of novel hiv-1 protease inhibitors incorporating cyclic ether-derived carbamates designed based on the x-ray structures of inhibitor-hiv-1 protease complexes. 224, 225 in this endeavor, we have specifically developed stereochemically defined cyclic ether templates, where the cyclic ether oxygen could effectively replace a peptide carbonyl oxygen. the advantage of such replacement is to reduce peptidic features and improve metabolic stability of compounds. these cyclic ligands have been incorporated as carbamate derivatives. the evolution of the carbamate structural template is shown in figure 11 . on the basis of the x-ray crystal structure of saquinavir (223)-bound hiv-1 protease, we first investigated 3-(r)-tetrahydrofuranylglycine so that the 3-(r)-thf ring oxygen would interact with the asp30 nh, similar to the asparagine side chain carbonyl oxygen of saquinavir (compound 224). 226−228 in an effort to reduce molecular weight, the p3 quinoline was removed, and the amide bond was replaced with a carbamate to provide inhibitor 225 with significant reduction of molecular weight (515 da from 670 da). the x-ray crystal structure of 225bound hiv-1 protease revealed that the ring oxygen of the 3-(s)-tetrahydrofuran (3-(s)-thf) is within proximity to form a hydrogen bond with the asp29 nh bond in the s2 subsite. the importance of the carbamate moiety is evident. the carbamate nh forms a hydrogen bond with the backbone carbonyl of gly27, and the carbamate carbonyl functionality makes a tightly bound water-mediated hydrogen bond with the backbone nh's of the flap ile50 and ile50′ in the active site. our further investigation of the 3-(s)-thf in inhibitors containing a hydroxyethylene isostere led to a series of exceptionally potent inhibitors. 178 as shown in table 23 , 3-(s)-thf-containing carbamate drivatives (compounds 226− 231) provided very potent inhibitors in antiviral assays. the potency enhancing effect of 3-(s)-thf carbamate was subsequently demonstrated in inhibitors containing the (r)-(hydroxyethyl)sulfonamide isostere. 229 clinical development of inhibitor 46 (vx476) led to fda approval of amprenavir for the treatment of hiv/aids patients. 179, 230 further development of carbamate-derived novel hiv-1 protease inhibitors is shown in figure 12 . we have designed a variety of inhibitors incorporating cyclic sulfones and bicyclic ligands (figure 12, compounds 232−237) . 230, 231 these ligands were conceived in order to maximize hydrogen-bonding interactions with the protease backbone as well as to fill in the hydrophobic pocket in the s2 subsite. on the basis of the x-ray structure of saquinavir-bound hiv-1 protease, we then designed a fused bicyclic tetrahydrofuran (bis-thf) ligand to form hydrogen bonds with backbone aspartates in the s2 subsite as well as to fill in the hydrophobic site adjacent to the p3-quinoline ring of saquinavir ( figure 12) . 185, 232 an x-ray structural analysis of 236-bound hiv-1 protease revealed that the bis-thf carbamate mimics the majority of p2−p3-amide bonds of saquinavir. a detailed structure−activity study also established that the stereochemistry of the bis-thf ring, and the position of the ring oxygens is critical to potency. with the development of a bis-thf carbamate that could form a network of hydrogen bonds in the s2 subsite of hiv-1 protease, we investigated transition state isosteres that can be functionalized to form hydrogen bonds in the s2′ subsite. our basic hypothesis was to design inhibitors that form a network of hydrogen bonds with the protease backbone atoms throughout the active site of hiv-1 protease, from s2 to s2′ subsites. this backbone binding strategy to combat drug resistance led to the development of a series of very potent carbamate-derived protease inhibitors. 185, 225, 226 as shown in figure 13 , we incorporated the bis-thf ligand in the (r)-hydroxyethylsulfonamide isostere bearing p-methoxysulfonamide as the p2′ ligand so that the methoxy oxygen can interact with aspartate backbone atoms in the s2′ subsite. the resulting inhibitors exhibited notable potency. 233, 234 inhibitor 239 with a (3r,3as,6ar)-bis-thf as the p2 ligand is significantly more potent in an antiviral assay than corresponding inhibitor 238 with an enantiomeric bis-thf ligand. an x-ray structure of 239-bound hiv-1 protease revealed that the carbamate nh formed a hydrogen bond with the backbone gly27 carbonyl group and that carbamate carbonyl of 239 is involved in an interesting tetra-coordinated hydrogen-bonding interaction with the structural water molecule, inhibitor sulfonamide oxygen, and the flap ile 50 nh residues. also, the structure revealed interactions with the backbone atoms in both the s2 and s2′ subsites. 235, 236 further replacement of the p-methoxy group at the s2′ to a p-amino group led to inhibitor 48 ( figure 14) . this inhibitor showed marked enzyme inhibitory activity as well as antiviral activity. an in-depth antiviral study revealed that 48 maintained excellent antiviral activity against multidrug-resistant hiv-1 variants. 237−239 the x-ray structural studies of darunavir-bound hiv-1 protease showed extensive active site interactions ( figure 14) . particularly, it formed a network of hydrogen bonds with the protein backbone throughout the active site. darunavir also exhibited favorable pharmacokinetic properties. subsequently, clinical development led to its fda approval as darunavir for the treatment of hiv/aids patients. 240, 241 the carbamate functionality of darunavir (48) was assembled as shown in scheme 22. (3r , 3 a s , 6 a r ) -3 -hydroxyhexahydrofuro[2,3-b]furan (bis-thf) 47 was treated with disuccinimidyl carbonate to provide activated mixed carbonate 240. reaction of this activated carbonate with hydroxyethylsulfonamide isostere 45 provided darunavir. 242, 243 the backbone binding inhibitor design strategies to combat drug resistance have been further utilized by us and others to advance a number of other preclinical and clinical inhibitors with carbamates. 185, 225 figure 15 shows selected bis-thfderived carbamates (241−244) with marked enzyme and antiviral activities. 244−247 like darunavir, inhibitor-bound x-ray structures of these inhibitors showed a network of hydrogen bonds in both s2 and s2′ subsites of hiv-1 protease. the inhibitor side chains as well as the bis-thf bicyclic framework also effectively filled the hydrophobic pockets in the active site. we have outlined a selected number of cyclic ether-derived carbamates that have been developed based on the backbone table 22 . carbamates from cdi-and tct-mediated lossen rearrangement 135, 136 scheme 20. tct-mediated lossen rearrangement for carbamate synthesis binding concept in figure 16 . 185, 225 particularly, incorporation of these stereochemically defined oxacyclic ligands such as cp-thf, tp-thf, tris-thf, and fluoro-bis-thf provided exceptionally potent inhibitors (51 and 245−249) with clinical potential. 247−252 the importance of the carbamate functionality in these inhibitors is particularly worthy of note. x-ray crystal structures of these inhibitors in complex with hiv-1 protease provided the ligand-binding site interactions responsible for their respective antiviral potency against wild-type and multidrug-resistant viruses. in general, inhibitors are involved in hydrogen-bonding interactions with asp29, asp30, gly27, asp25, asp25′, and asp30′ in the hiv-1 protease active site. furthermore, the ring cycles adequately fill the hydrophobic pockets in the active site. 251 inhibitors the search for an effective treatment for alzheimer's disease (ad) remains a major challenge in medicine. one of the pathological hallmarks of ad is the formation of β-amyloid (aβ) peptides in the cortex of ad patients. aβ-peptides are generated from β-amyloid precursor protein (app) by sequential cleavage by β-secretase (also known as bace1 or memapsin 2) and γ-secretase. due to this central role of aβproduction, both β-secretase and γ-secretase have been implicated as important therapeutic targets for ad intervention. 253,254 as a result, design and synthesis of selective βsecretase and γ-secretase inhibitors have become an intense area of research over the years. 7.1. development of β-secretase inhibitors. following the discovery of β-secretase, the first-generation β-secretase inhibitors were designed and synthesized by ghosh, tang, and co-workers. 255 as shown in figure 17 , utilizing a carbamate derivative of the leu−ala isostere 250, potent pseudopeptide inhibitors 251 and 252 were identified. the x-ray crystal structure of 252-bound β-secretase was determined to provide molecular insight into the ligand binding site interactions. 256 the in-depth structural analysis thus provided critical drug design templates and led to the beginning of structure-based design approaches to peptidomimetic/nonpeptide β-secretase inhibitors. 254, 255 the x-ray structure of 252-bound β-secretase revealed that the p2 asparagine side chain carboxamide nitrogen formed an intermolecular hydrogen bond with the p4 glutamic acid carbonyl group. on the basis of this molecular insight, a number of 14−16membered cycloamide-carbamate-based macrocyclic inhibitors were designed and synthesized. 257 as shown in figure 18 , acyclic carbamate derivatives (253 and 254) were less potent than their corresponding cyclic inhibitors. inhibitor 255, with a 16-membered macrocycle containing a trans-olefin, amide and carbamate functionalities within the macrocycle, showed good β-secretase inhibitory activity. saturated inhibitor 256 is less potent against bace1, but it showed enhanced potency for bace2. x-ray structural studies of inhibitor 256-bound secretase revealed that the carbamate carbonyl forms a hydrogen bond with the gln73 side chain carboxamide residue. interestingly, unsaturated inhibitor 255 showed slight selectivity against memapsin 1 (k i = 31 nm). the design of a selective inhibitor is important for reducing toxicity through off-target effects. particularly, selectivity over other aspartic proteases, such as bace2, pepsin, renin, cathepsin d (cat-d), and cathepsin e, may be important for the reduction of side effects and drug efficiency. 258 on the basis of our detailed structure−activity studies and xray structural analysis, we have designed a variety of highly selective and potent bace1 inhibitors. in this perspective, we will highlight only the development of bace1 inhibitors bearing carbamate functionalities. as shown in figure 19 , inhibitor 257 is a potent bace1 inhibitor. however, it did not show selectivity against bace2 or cat-d. subsequent structure-based design led to the development of selective inhibitors 258 and 259, which contain a pyrazolylmethyl and oxazolymethyl carbamate at the p3 position, respectively. 259 inhibitor 258 showed excellent bace1 potency and selectivity over bace2 and cathepsin d. the x-ray crystal structure of 258-bound β-secretase revealed that the carbamate carbonyl formed a hydrogen bond with the thr-232 backbone nh. also, figure 9 . examples of phenol carbamate prodrugs and their metabolic activation. ethylenediamine spacer and release of paclitaxel the pyrazole nitrogen formed a strong hydrogen bond with the thr-232 side chain hydroxyl group. the p2-sulfonyl functionality formed a number of hydrogen bonds in the s2 subsite as well. on the basis of this molecular insight, oxazole-derived 259 was designed to provide a more stable and selective inhibitor. the synthesis of inhibitors 258 and 259 is outlined in scheme 23. urethanes 263 and 264 were prepared by treatment of 2,5-dimethylpyrazolylmethanol (260) or 2,5dimethyl-4-oxazolemethanol (261) with triphosgene in the presence of triethylamine, followed by l-methionine methyl ester hydrochloride (262) . saponification of the resulting methyl esters provided the corresponding acids. coupling of amine 265 with acids 263 and 264, as described previously, and subsequent oxidation of the sulfides with m-chloroperbenzoic acid furnished inhibitors 258 and 259. 259 freskos and co-workers have reported a series of β-secretase inhibitors that incorporated polar carbamate derivatives as the p2 ligand. 260, 261 this strategy led to improve the cat-d selectivity. it was hypothesized that the s2 subsite of cat-d is more lipophilic and less tolerant of polar groups. as can be seen in figure 20 , benzyl carbamate derivative 266 displayed 6-fold selectivity over cat-d. however, polar 3-pyridylmethyl derivative 267 improved selectivity nearly 90-fold. the corresponding 4-pyridyl methyl compound 268 provided a reduction in selectivity (∼50-fold). 3-(s)-tetrahydrofuranyl carbamate 269 showed a nearly 30-fold selectivity over cat-d. these inhibitors have also shown good to excellent ic 50 values in hek cells. 7.2. development of γ-secretase inhibitors. over the years, many structural classes of potent and selective γ-secretase inhibitors have been reported. a number of inhibitors displayed drug-like properties and also inhibited aβ production in animal models. in this section, we will review inhibitors with carbamate functionality. on the basis of γ-secretase inhibitor 270 (ly-411575), peters and co-workers designed a series of carbamate derivatives of dibenzazepinone as potent and metabolically stable γ-secretase inhibitors. 265 as shown in figure 21 nm. absolute stereochemistry of the active enantiomer was not determined. piperidine carbamate 274 also showed good potency. carbamate derivative 275 displayed a good membrane aβ ic 50 value; however, it showed poor cyp properties. further modification led to compound 276 with good inhibitory activity and improved cyp properties. bergstrom and co-workers reported a series of carbamateappended n-alkyl sulfonamides as γ-secretase inhibitors. 269, 270 figure 23 depicts selected examples that show potent aβ inhibitory activity. sulfonamide derivative 277 was identified as a potent γ-secretase inhibitor. exploration of carbamateappended n-alkylsulfonamides resulted in potent inhibitors such as 278−280. tertiary carbamate 280 showed significant reduction of brain aβ in transgenic mice compared to that of its benzyl derivative. this compound also showed improved brainto-plasma ratio and good absolute brain concentration. hepatitis c virus (hcv) is a bloodborne virus that is found worldwide. there are multiple strains or genotypes of the hcv virus. hcv infections lead to progressive liver damage, cirrhosis, and liver cancer. in recent years, there have been a number of new and effective antiviral drugs developed for the treatment of hepatitis c. these include the development of hcv ns3/4a protease inhibitors and inhibitors hcv ns5a. in this section, carbamate-derived therapeutics will be discussed. 8.1. carbamate-derived serine protease inhibitors. serine proteases are a large family of proteolytic enzymes that play a variety of critical roles in many physiological processes. 271, 272 deregulation of serine proteases has been related to the pathogenesis of diseases such as stroke, inflammation, alzheimer's disease, cancer, and arthritis. therefore, significant research efforts have been focused in the discovery of serine protease inhibitors. the active site of all serine proteases consists of a catalytic triad of ser, his, and asp. the nucleophilic attack by the hydroxyl group of serine at the carbonyl carbon of the scissile bond of the substrate, via general base catalysis by histidine, leads to the tetrahedral transition state. the tetrahedral intermediate ultimately collapses, leading to cleavage products. 273−275 these key active residues are conserved in all serine proteases. x-ray structural studies revealed that these residues are superimposable in the majority of serine proteases. 273, 276 therefore, selectivity over other serine proteases represents a key issue to be taken into consideration during inhibitor design. most early inhibitors acted via a covalent mechanism in which an electrophilic group formed a covalent bond with the serine hydroxyl of the catalytic triad. the electrophilic groups are commonly referred to as serine traps or warheads. however, covalent inhibitors lack selectivity and specificity against other proteases in the same class or clan. the rational design of covalent serine protease inhibitors usually involves the selection of a good substrate to be linked to a serine trap/warhead. chloromethyl ketones, diphenyl phosphonate esters, trifluoromethyl ketones, peptidyl boronic acids, α-ketoheterocycles, and β-lactam derivatives are usually employed as warheads. on the basis of these warheads, a variety of irreversible and reversible covalent serine protease inhibitors were designed. 275 in this section, representative serine protease inhibitors containing a carbamate functionality will be outlined. carbamate derivative 281 (figure 24 ), a diphenyl phosphonate ester containing a cbz group and bearing a single amino acid side chain, showed very good inhibitory activity against human plasma kallikrein, useful for the treatment of hereditary angioedema. 277, 278 thrombin is an attractive therapeutic target for drug development against pulmonary embolism, thrombosis, and related diseases. 279, 280 compound 282 showed good potency and selectivity against human thrombin. it is stable and displayed no activity against acetylcholinesterase and no selectivity over cysteine proteases. peptidyl boronic acid-based thrombin inhibitors were developed by dupont-merck. in particular, n-boc derived inhibitor 283 is a potent inhibitor (k i = 0.004 nm). 281,282 imperiali and co-workers introduced trifluoromethyl ketones as specific serine protease inhibitors, particularly for chymotrypsin and elastase. 283 researchers at astrazeneca designed numerous peptidyl trifluoromethyl ketone derivatives as potent human elastase inhibitors. 284−286 further optimization of features resulted in the development of a number of orally active inhibitors. in particular, methyl carbamate derivative inhibitor 284 was shown to be a very potent inhibitor (k i = 13 nm) with excellent oral bioavailability in laboratory animals. 286, 287 optically pure compound 284 with an (s)-configuration at the p1 isopropyl side chain became a candidate for clinical development for potential treatment of elastase-implicated respiratory diseases. peptidomimetic boronic acid-based hepatitis c virus (hcv) ns3/4a protease inhibitors were designed and synthesized for the treatment of chronic hcv infections. hcv infections can lead to progressive liver damage, cirrhosis, and liver cancer. 288 the ns3/4a serine protease plays a critical role in virus replication and has become an antiviral drug development target. 289, 290 the first specific and potent hcv protease inhibitor with good oral bioavailability was ciluprevir, which contains a carbamate functionality (285, biln 2061, figure 25 ). this noncovalent macrocyclic peptidic inhibitor was the result of a substrate-based approach for the design of active site inhibitors. this inhibitor is very active in enzymatic (ic 50 = 3 nm) and cell-based replicon assays (ic 50 = 1.2 nm) of hcv genotype 1. 291 ciluprevir was later discontinued due to cardiac toxicity in animal models, but its development paved the way to boceprevir (victrelis, schering-plough, approved by fda in may 2011) and telaprevir (vx-950, vertex pharmaceuticals and johnson & johnson). 292, 293 in particular, for the development of boceprevir, the introduction of a ketoamide moiety, together with p2 and p3 optimization, led to inhibitor 286 showing a k i of 66 nm. 294 its x-ray crystal structure in complex with the enzyme also provided insight for further optimization. indeed, a cyclopropylalanine residue was found to be optimal at p1, and the resulting carbamate derivative 287 showed a k i of 15 nm. although inhibitors 286 and 287 displayed good enzyme inhibitory potency, they did not display cellular activity in a subgenomic hcv replicon assay, possibly because of their strong peptidic character. 295 the discovery that an nmethylated leucine at p2 was critical for both enzymatic potency and cellular activity led to the potential of cyclopropylfused proline being envisaged as an optimum, conformationally constrained surrogate for this part of the inhibitor. combination of the p2-optimized ligand with previously optimized p1 and p3 residues provided carbamate derivative 288 with a k i = 3.8 nm and ic 90 = 100 nm. 296 finally, truncation and p1 optimization, by the employment of a cyclobutyl moiety, led to compound 289 (k i = 76 nm), the direct boceprevir ancestor. 297 subsequently, compounds 290 and 291 ( figure 26 ) with a carbamate containing p2 proline core showed very potent inhibitory activity (ic 50 = 2 nm for 290 and 23 nm for 291). 298 similarly, macrocyclic inhibitor 292 with an α-amino cyclic boronate showed good potency (ic 50 = 43 nm). 299 the electron-withdrawing effect of the ester and amide functionalities was also utilized in the design of α-ketoesteror α-ketoamide-derived transition state inhibitors. 300 a range of hcv ns3/4a protease inhibitors were designed and synthesized, incorporating α-ketoamide templates at the scissile site. structure-based design led to a variety of potent acyclic and cyclic inhibitors with ketoamide templates, as exemplified in compounds 293 (ic 50 tripeptidyl α-ketobenzoxazole 295 inhibited human neutrophil elastase (hne) with an ic 50 of 3 nm. the ketooxazolinederived inhibitor 296 displayed very potent activity against hne (ic 50 = 0.6 nm). 304 8.2. hcv ns5a inhibitors. carbamate derivatives also play a key role as inhibitors of hcv ns5a, which represents a new and promising target for hcv therapy. hcv ns5a is a zincbinding phosphoprotein, and its role in the hcv virus life cycle is still not clear. 305 however, it plays a critical role in hcv rna replication. also, it is involved in virion morphogenesis. 306 due to the lack of enzymatic function, inhibitors of this viral-encoded protein have been pursued. 307 researchers at bristol-myers squibb screened a library of compounds for their ability to inhibit hcv rna replication. this led to the identification of a lead compound specifically interfering with rna replication and later proving to inhibit the activity of ns5a protein. subsequent optimization was focused on broadening the genotype specificity and improving pharmacokinetic properties of compounds. symmetry of the molecule played an important role in inhibitory potency. this finally led to the discovery of daclatasvir (297, bms-790052, figure 28 ) a first-in-class inhibitor of the hcv ns5a replication complex. 308 daclatasvir was approved in europe in august, 2014. ledipasvir (298, gs-5885, gilead sciences, figure 28 ) is another carbamate-containing hcv ns5a inhibitor with potent antiviral activity against hcv genotypes 1a and 1b. 309 harvoni, a combination of ledipasvir and sofosbuvir (a nucleotide polymerase inhibitor), was approved by the fda in october 2014 for the treatment of chronic hcv genotype 1 infection. this also represents the first approved regimen that does not require administration with interferon or ribavirin. 310, 311 9. carbamates as cysteine protease inhibitors cysteine proteases, also known as thiol proteases, are proteolytic enzymes responsible for the degradation of proteins. 312 these enzymes are divided into three classes based on their sequence homology: the papain, caspase, and picornaviridae families. the papain family of proteases is the most known and studied. 313 cysteine proteases have been identified in a variety of diverse organisms, such as bacteria, eukaryotic micro-organisms, plants, and animals and are divided into the clans ca, cd, ce, cf, and ch in the merops peptidase database. the largest subfamily among the class of cysteine proteases is the papain-like cysteine proteases, originating from papain as the archetype of the cysteine proteases. clan ca proteases utilize catalytic cys, his, and asn residues that are invariably in this order in the primary sequence of the protease. clan ca, family c1 (papain-family) cysteine proteases are wellcharacterized for many eukaryotic organisms. also, the best characterized plasmodium cysteine proteases, namely, the falcipains, belong to papain-family (clan ca) enzymes. clan cd presents two catalytic residues, his and cys, in sequence; clan ce has a triad formed by his, glu, or asp and cys at the c-terminus; in clan cf, the asparagine residue of the catalytic triad is replaced by a glutamate residue and the catalytic triad is ordered as glu, cys, and his; clan cg has a dyad of two cysteine residues, and clan ch presents a cys, thr, and his triad with the catalytic cysteine at the n-terminus. the proteolytic mechanism involves the formation of a thiolate−imidazolium ion pair, which provides a highly nucleophilic cysteine thiol. over the years, many cysteine protease inhibitors have been designed by appropriately linking electrophilic warheads to the specific recognition sequence of peptide substrates. reversible inhibitor warheads include aldehydes, α-ketoamides, α-ketoesters, and α-ketoacids. these inhibitors interact with the protease active site, forming the tetrahedral intermediate, but are eventually hydrolyzed, regenerating both the enzyme and the inhibitor in an equilibrium reaction. irreversible inhibitors of cysteine proteases include epoxides, aziridines, haloketones, vinyl sulfones, and acyloxymethylketones. these inhibitors inactivate the target through alkylation of the active site cysteine thiol, permanently disabling enzyme function. 273,313,316−318 the occurrence of severe acute respiratory syndrome (sars) in 2003 and the subsequent identification of a novel coronavirus as the etiological agent recognized cysteine proteases sars-cov 3clpro and sars-cov plpro (papainlike protease) as possible targets for drug design. 319, 320 subsequent structure-based design based on a previous inhibitor's x-ray co-crystal structure with the enzyme 321 provided carbamate derivative 299 ( figure 29 ) as a potent sars-cov 3clpro inhibitor (ic 50 = 80 μm). 322 a wide variety of human rhinovirus 3c (hrv 3c) protease inhibitors were developed by the incorporation of α,βunsaturated carbonyl moieties as warheads. hanzlik et al. reported the first hrv 3c protease inhibitors containing a peptide portion and incorporating α,β-unsaturated esters. 323 the peptide parts were selected based on the substrate cleavage site. the representative carbamate-containing inhibitor 300 ( figure 29 ) showed an ic 50 value of 130 nm. human cathepsin k plays a critical role in bone resorption. in an effort to block bone resorption, noncovalent cathepsin k inhibitors were developed. kim et al. provided carbamate derivative 301 ( figure 29 ) as a noncovalent and reversible cathepsin k (ic 50 = 0.01 μm) and l inhibitor (ic 50 = 0.002 μm). 324 glaxowellcome scientists developed carbamate-containing ketoamide-based cathepsin k inhibitors such as 302 ( figure 29 ) (ic 50 = 0.072 nm). 325 starting from a potent ketone-based inhibitor with unsatisfactory drug-like properties, 326,327 incorporation of p 2 −p 3 elements from the ketoamide-based inhibitor 302 led to a hybrid series of ketone-based cathepsin k inhibitors with improved bioavailability, as exemplified in inhibitor 303 ( figure 29 ) (ic 50 = 4 nm). 328 cathepsin s has been suggested for the development of agents against a range of immune disorders. a new class of nonpeptidic and noncovalent cathepsin s inhibitors was reported in 2007. 329 subsequent structural optimization resulted in a very potent and competitive noncovalent carbamate-containing inhibitor 304 ( figure 29 ) (ic 50 = 20 nm). metabolizing enzyme inhibitors carbamates have been employed in the design of serine hydrolase inhibitiors. in this section, we will focus on inhibitors of endocannabinoid metabolizing enzymes, in which the carbamate functionality plays an important role. the endocannabinoid system is known to be a ubiquitous neuromodulatory system with a wide range of action that can be found in every primitive organism. it is composed of cannabinoid receptors (cbrs), endogenous cannabinoids (endocannabinoids, ecs), and the enzymes responsible for their production, transport, and degradation. 330, 331 ecs are a class of signaling lipids, such as n-arachidonoyl ethanolamine (anandamide, aea), oleamide, and 2-arachidonoyl glycerol (2-ag), that exert their biological actions through the interaction with two g-protein coupled receptors, cb1 and cb2. they modulate a range of responses and processes including pain, inflammation, appetite, motility, sleep, thermoregulation, and cognitive and emotional states. 332 the actions of these signaling lipids are rapidly terminated by cellular reuptake and subsequent hydrolysis operated by a number of enzymes. an attractive approach involved the modulation of the ec system and aimed at eliciting the desirable effects of cbrs activation through the pharmacological inactivation of the main endocannabinoid metabolizing enzymes, namely, monoacylglycerol lipase (magl) and α/β-hydrolase domain containing 6 and 12 (abhd6 and abhd12). these three serine hydrolases account for approximately 99% of 2-ag hydrolysis in the cns, 333 whereas fatty acid amide hydrolase (faah) is responsible for aea inactivation. 330 inactivation of these enzymes would elevate the endogenous concentrations of all of its substrate and consequently prolong and potentiate their beneficial effects on pain and anxiety without evoking the classical cb1r agonists side effects (hypomotility, hypothermia, and catalepsy). monoacylglycerol lipase (magl) is the primary enzyme responsible for the hydrolysis of 2-ag in the cns. 333 about 85% of the total 2-ag hydrolysis in the brain is ascribed to magl. magl is a 33 kda membrane enzyme belonging to the superfamily of the serine hydrolases with a catalytic triad represented by ser122, his269, and asp239. 334 it is ubiquitously present in the brain (cortex, hippocampus, cerebellum, thalamu, and striatum), where it localizes to presynaptic terminals, even if lower levels are found in the brainstem and hypothalamus. a concomitant distribution in membranes as well as in the cytosol has been reported. magl shares a common folding motif called the α/β-hydrolase fold. studies in recent years have shown that magl inhibitors elicit antinociceptive, anxiolytic, and antiemetic responses. magl inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. 335 recently, the potential of magl inhibitors for the therapy of fragile x syndrome has been reported. 336 the early discovered magl inhibitors were molecules able to target the cysteine residues present in the active site of the enzyme. later, the research has been focused on the synthesis of compounds covalently binding to ser241 of the catalytic triad. among them, carbamate 305 (urb602, figure 30 ) was the first selective inhibitor of 2-ag degradation, although its potency remained limited (ic 50 = 28 μm on rat brain). 337 selective magl inhibitors bearing a carbamate scaffold were developed by cravatt and coworkers. 338 inhibitor 306 (jzl184, figure 30 ) exhibited selectivity toward faah in vitro (ic 50 = 3.9 nm and 4 μm for human recombinant magl and faah, respectively). 338 more recently, cravatt and co-workers reported a distinct class of o-hexafluoroisopropyl (hfip) carbamates bearing a reactive group that is bioisosteric with endocannabinoid substrates. 339 the representative compound, 307 (kml29, figure 30 , ic 50 = 5.9 nm, human magl), displays excellent potency and in vivo. in comparison to previously described o-aryl carbamates, inhibitor showed enhanced selectivity over faah and other serine hydrolases. 339 abhd6 gene encodes a ∼35 kda protein containing an nterminal transmembrane region followed by a catalytic domain that includes the canonical gxsxg active-site motif of serine hydrolases. abhd6 is a unique and highly conserved enzyme in mammals and is mainly expressed in the brain, liver, kidney, and brown adipose tissue. as a member of the serine hydrolase class, abhd6 is predicted to hydrolyze esters, amides, or thioester bonds in substrates that could include small molecules, lipids, or peptides. although, the full range of substrates regulated by this enzyme in vivo is currently unknown. 340 recent studies have also shown that abhd6 carbamate inhibitors produce anti-inflammatory and neuroprotective effects in a mouse model of traumatic brain injury. 341 among them, optimized inhibitor 308 ( figure 30 ) displayed an ic 50 value of 70 nm and notable selectivity. 342 faah is a membrane-bound enzyme belonging to the amidase family. the analysis of its crystal structure revealed a core composed of a characteristic ser-ser-lys catalytic triad. 343 the catalytic residues of faah are buried deep within the enzyme and are accessible by two narrow channels. the importance of faah was demonstrated by the generation of faah knockout mice. faah −/− mice showed an elevated resting brain concentration of aea and manifested (i) an analgesic phenotype in both the carrageenan model of inflammatory pain and in the formalin model of spontaneous pain, 344 (ii) a reduction in inflammatory responses, 345 and (iii) improvements in slow wave sleep and memory acquisition. 346, 347 the urb class of compounds was the first class of inhibitors identified for faah, and it is well-represented by 309 (urb597, figure 30 , ic 50 = 4.6 nm). 348 the n-(6phenyl)hexylcarbamate analogue 310 (jp83, figure 30 , ic 50 = 14 nm) is another very potent compound representative of the biphenyl series of inhibitors. 349 gattinoni and co-workers developed a series of oxime carbamate inhibitors. compound 311 ( figure 30 , ic 50 = 8 nm) displayed good affinity and selectivity toward faah. 350 more recently, butini et al. developed a new class of potent and selective faah reversible carbamate inhibitors. 351 among them, compound 312 (nf1245, figure 30 , k i = 0.16 nm on mouse brain faah) showed excellent activity. the compound showed impressive selectivity toward all the enzymes and receptors of the endocannabinoid system. 351 in this perspective, the role of carbamates in drug design and medicinal chemistry has been highlighted. in particular, the perspective covers physical properties of carbamates and the development of novel chemical methodologies overcoming the historical safety and toxicity issues related to their preparation. furthermore, the importance of carbamate-derived compounds in medicinal chemistry and their widespread employment as drugs and prodrugs have been discussed. also showcased is the exploitation of organic carbamates in the development of numerous aspartic acid, serine, and cysteine protease inhibitors. we hope that this perspective will stimulate further use of organic carbamate as a structural motif in drug design and medicinal chemistry. the authors declare no competing financial interest. financial support of this research by the national institutes of health (gm53386) and purdue university is gratefully acknowledged. we would like to thank our colleagues anthony tomaine, heather osswald, and anindya sarkar (purdue university) for helpful discussions. ad, alzheimer's disease; cat-d, cathespsin d; faah, fatty acid amide hydrolase; ppar, peroxisome proliferator-activated receptor; gaba, γ-amino butyric acid; haart, highly active antiretroviral therapy; bis-thf, bis-tetrahydrofuran; bace1, beta-site amyloid precursor protein cleaving enzyme 1; mdr, multidrug resistant; nnrti, non-nucleoside reverse transcriptase inhibitors; adept, antibody-directed enzyme prodrug therapy; gdept, gene-directed enzyme prodrug therapy; mct1, monocarboxylate transporter type 1; smvt, sodiumdependent multivitamin transporter; ces1, carboxylesterases 1; magl, monoacylglycerol lipase ■ references unusually low barrier to carbamate c−n rotation amide resonance in thioand seleno-carbamates: a theoretical study role of conjugation in the stabilities and rotational barriers of formamide and thioformamide. an ab initio valence-bond study on the stabilization of the syn-rotamer of amino acid carbamate derivatives by hydrogen bonding the dipole moment and structure of the carbamate group chemical aspects of the restricted rotation of esters, amides, and related compounds hydrolysis in drug and prodrug metabolismchemistry qualitative structuremetabolism relationships in the hydrolysis of carbamates design, synthesis, and structure−activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors a mild amide to carbamate transformation electrochemically induced hofmann rearrangement an efficient modification of the hofmann rearrangement: synthesis of methyl carbamates hydrazide und azide organischer saüren i azides: their preparation and synthetic uses ruthenium carbonyl catalyzed reductive carbonylation of aromatic nitro-compoundsa selective route to carbamates an efficient one-pot synthesis of n-alkyl carbamates from primary amines using cs 2 co 3 isocyanates from primary amines and carbon-dioxidedehydration of carbamate anions mechanistic aspects of the copolymerization of co 2 and epoxides by soluble zinc bis(phenoxide) catalysts as revealed by their cadmium analogues selective conversion of carbon dioxide to dimethyl carbonate by molecular catalysis catalytic production of urethanes from amines and alkyl halides in supercritical carbon dioxide efficient cs 2 co 3 -promoted solution and solid phase synthesis of carbonates and carbamates in the presence of tbai nature of n-bromosuccinimide in basic-mediathe true oxidizing species in the hofmann rearrangement preparation of methyl carbamates from primary alkylcarboxamides and arylcarboxamides using hypervalent iodine a versatile modification of the hofmann rearrangement preparation of methyl carbamates via a modified hofmann rearrangement oxidative rearrangement of amides with lead tetraacetate oxidation using quaternary ammonium polyhalides 0.1. an efficient method for the hofmann degradation of amides by use of benzyltrimethylammonium tribromide curtius conversion of acids to amines under neutral conditions via an anthrylmethyl carbamate a concise synthesis of tamiflu: third generation route via the diels− alder reaction and the curtius rearrangement -1,3-dienes: preparation from dienoic acids curtius rearrangement and wolff homologation of functionalized peroxides a convenient route to 1-benzyl 3-aminopyrrolidine and 3-aminopiperidine regioselective synthesis of the 3,4-dihydrofuro[3,2-d]pyrimidin-2(1h)-one skeleton: a new class of compound general route to 2,4,5-trisubstituted piperidines from enantiopure β-amino esters. total synthesis of pseudodistomin b triacetate and pseudodistomin f a solid-phase synthesis of n,n′-disubstituted ureas and perhydroimidazo[1,5-a]pyrazines via the curtius rearrangement reliable protocol for the large scale synthesis of diphenylphosphoryl azide (dppa) an improved method for the synthesis of enantiomerically pure aminoacid ester isocyanates a safe and efficient method for preparation of n,n′-unsymmetrically disubstituted ureas utilizing triphosgene an efficient new protocol for the formation of unsymmetrical tri-and tetrasubstituted ureas recent advances in isocyanate chemistry halogen-free process for the conversion of carbon dioxide to urethanes by homogeneous catalysis a direct synthesis of carbamate ester from carbon dioxide, amine and alkyl halide indium-catalyzed reaction for the synthesis of carbamates and carbonates: selective protection of amino groups evolution of a series of peptidoleukotriene antagonists: synthesis and structure/ activity relationships of 1,3,5-substituted indoles and indazoles rational design of peptide-based hiv proteinase inhibitors triphosgene, a crystalline phosgene substitute 2′-carbonyl-bis(3,5-dioxo-4-methyl-1,2,4-oxadiazolidine): a new reagent for the preparation of carbamates and amides, application to the synthesis of dipeptides 1,1′-bis[6-(trifluoromethyl)benzotriazolyl] oxalate (btbo): a new reactive coupling reagent for the synthesis of dipeptides, esters, and thio esters a new method for protecting amines alkoxycarbonylation of .alpha.,.omega.-diamino acids with 2-(trimethylsilyl)ethyl 4-nitrophenyl carbonate solid phase synthesis of hydantoins using a carbamate linker and a novel cyclization/cleavage step prodrugs for amidines: synthesis and anti-pneumocystis carinii activity of carbamates of 2,5-bis(4-amidinophenyl)furan syntheses of fda approved hiv protease inhibitors substituent effects on p2-cyclopentyltetrahydrofuranyl urethanes: design, synthesis, and x-ray studies of potent hiv-1 protease inhibitors synthesis and biological evaluation of novel allophenylnorstatine-based hiv-1 protease inhibitors incorporating high affinity p2-ligands design and synthesis of stereochemically defined novel spirocyclic p2-ligands for hiv-1 protease inhibitors a novel active ester synthesis reagent (n,n′-disuccinimidyl carbonate) β-elimination of βhydroxyamino acids with disuccinimido carbonate a synthesis of a new type of alkoxycarbonylating reagents from 1,1-bis[6-(trifluoromethyl)benzotriazolyl] carbonate (btbc) and their reactions di-2-pyridyl carbonate: a new efficient coupling agent for the direct esterification of carboxylic acids carbonate promoted alkoxycarbonylation of amines: a convenient synthesis of functionalized carbamates dissuccinimidyl carbonate: a useful reagent for alkoxycarbonylation of amines synthesis and evaluation of novel activated mixed carbonate reagents for the introduction of the 2-(trimethylsilyl)ethoxycarbonyl(teoc)-protecting group re-and si-face-selective nitroaldol reactions catalyzed by a rigid chiral quaternary ammonium salt: a highly stereoselective synthesis of the hiv protease inhibitor amprenavir (vertex 478) enantioselective synthesis of cyclopentyltetrahydrofuran (cp-thf), an important high-affinity p2-ligand for hiv-1 protease inhibitors an efficient synthesis of functionalized urethanes from azides n-nitration of secondary amines with 4-chloro-5-methoxy-2-nitropyridazin-3-one (6h)-carboxylate as carbonyl source: facile and selective synthesis of carbamates and ureas under mild conditions preparation and characterization of copper(i) amides a convenient method for the synthesis of carbamate esters from amines and tetraethylammonium hydrogen carbonate palladium-catalyzed generation of o-allylic urethanes and carbonates from amines/alcohols, carbon dioxide, and allylic chlorides cs 2 co 3 -promoted efficient carbonate and carbamate synthesis on solid phase efficient cs 2 co 3 -promoted solution and solid phase synthesis of carbonates and carbamates in the presence of tbai halogen-free process for the conversion of carbon dioxide to urethanes by homogeneous catalysis nickel-catalyzed dehydrative transformation of co 2 to urethanes parallel synthesis of ureas and carbamates from amines and co 2 under mild conditions the mechanism of the mitsunobu reaction and its application to co 2 fixation pdcl 2 catalyzed efficient assembly of organic azides, co, and alcohols under mild conditions: a direct approach to synthesize carbamates an efficient new protocol for the formation of unsymmetrical tri-and tetrasubstituted ureas a new protocol for the formation of carbamates and thiocarbamates using carbamoyl imidazolium salts carbamoylimidazolium salts as diversification reagents: an application to the synthesis of tertiary amides from carboxylic acids carbamoylimidazolium and thiocarbamoylimidazolium salts: novel reagents for the synthesis of ureas, thioureas, carbamates, thiocarbamates and amides kf/al 2 o 3 mediated organic synthesis competing reactions of secondary alcohols with sodium hypochlorite promoted by phase-transfer catalysis boc-protected amines via a mild and efficient one-pot curtius rearrangement curtius rearrangement of aromatic carboxylic acids to access protected anilines and aromatic ureas one-pot curtius rearrangement processes from carboxylic acids an environmentally benign access to carbamates and ureas synthesis of carbamates using yttria−zirconia based lewis acid catalyst group 3 metal (sc, la) triflates as catalysts for the carbomethoxylation of aliphatic amines with dimethylcarbonate under mild conditions synthesis of carbamates from aliphatic amines and dimethyl carbonate catalyzed by acid functional ionic liquids indium in organic synthesis indium-and galliummediated carbon−carbon bond-forming reactions in organic synthesis recent advances in indium-promoted organic reactions general approach to the coupling of organoindium reagents with imines via copper catalysis 1,2,4-triazoles containing nitro groups convenient method for the preparation of carbamates, carbonates, and thiocarbonates ultrasound promoted 'one pot' conversion of nitrocompounds to carbamates facile procedure for the synthesis of n-aryl-n-hydroxy carbamates the role of alcohol in the catalytic reductive carbonylation of nitrobenzenes to carbamates in the presence of rh reductive carbonylation of nitrobenzenes catalyzed by a new binuclear rhodium complex reductive carbonylation of aromatic dinitro compounds with a palladium(phenanthroline)2(triflate)2 catalyst and an aromatic carboxylic acid as cocatalyst the synthesis of carbamate from the reductive carbonylation of nitrobenzene over pd-based catalysts catalytic activity of pdcl 2 complexes with pyridines in nitrobenzene carbonylation carbamate synthesis via transfunctionalization of substituted ureas and carbonates dialkyl and diaryl carbonates by carbonate interchange reaction with dimethyl carbonate investigation of dialkyltin compounds as catalysts for the synthesis of dialkyl carbonates from alkyl carbamates synthesis of carbamates and ureas using zr(iv)-catalyzed exchange processes unprecedented "in water" imidazole carbonylation: paradigm shift for preparation of urea and carbamate introducing catalytic lossen rearrangements: sustainable access to carbamates and amines carbonyldiimidazole-mediated lossen rearrangement cyanuric chloride: an efficient reagent for the lossen rearrangement a review of rivastigmine: a reversible cholinesterase inhibitor rivastigmine for dementia associated with parkinson's disease the tolerability and safety of cholinesterase inhibitors in the treatment of dementia a novel peroxisome proliferator-activated receptor alpha/gamma dual agonist with efficacious glucose and lipid-lowering activities squibb announces discontinuation of development of investigational oral treatment for type 2 diabetes oral antiplatelet, antithrombotic efficacy of dmp 728, a novel platelet gpiib/iiia antagonist discovery of an orally active series of isoxazoline glycoprotein iib/iiia antagonists a synthetic inhibitor of histone deacetylase, ms-27-275, with marked in vivo antitumor activity against human tumors the histone deacetylase inhibitor ms-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21cip1/waf1 1 enantioselective renal excretion of albendazole metabolites in patients with neurocysticercosis treatment of hydatid disease with high oral doses of mebendazole. long-term follow-up of plasma mebendazole levels and drug interactions teva pharmaceuticals product line changes: mebendazole tablets new anticonvulsant drugs synthesis and quantitative structure-activity relationships of anticonvulsant 2,3,6-triaminopyridines a neuropharmacological evaluation of felbamate as a novel anticonvulsant drug interactions with the newer antiepileptic drugs (aeds)part 1: pharmacokinetic and pharmacodynamic interactions between aeds discontinuation of phenytoin and carbamazepine in patients receiving felbamate effects of felbamate on the pharmacokinetics of phenobarbital efavirenzstill first-line king? prospective, randomized, open label trial of efavirenz vs lopinavir/ ritonavir in hiv+ treatment-naive subjects with cd4+<200 cell/mm 3 in mexico efavirenz primary and secondary metabolism in vitro and in vivo: identification of novel metabolic pathways and cytochrome p450 2a6 as the principal catalyst of efavirenz 7-hydroxylation the cytochrome p450 2b6 (cyp2b6) is the main catalyst of efavirenz primary and secondary metabolism: implication for hiv/aids therapy and utility of efavirenz as a substrate marker of cyp2b6 catalytic activity impact of cyp2b6 polymorphism on hepatic efavirenz metabolism in vitro a randomized, doubleblind, placebo-controlled trial of the effect of zafirlukast on upper and lower respiratory responses to cat challenge evolution of a series of peptidoleukotriene antagonists: synthesis and structure/ activity relationships of 1,3,5-substituted indoles and indazoles pharmacokinetic profile of zafirlukast crosslinking of dna by enzymatically or chemically activated mitomycins and porfiromycins, bifunctionally "alkylating" antibiotics sequence preferences of dna interstrand cross-linking agentsimportance of minimal dna structural reorganization in the cross-linking reactions of mechlorethamine, cisplatin, and mitomycin-c dna interstrand cross-linking by 2,5-bis(1-aziridinyl)-1,6-benzoquinonenucleotide-sequence preferences and covalent structure of the dg-to-dg crosslinks at 5′-d(gn(n)c) in synthetic oligonucleotide duplexes mitomycin c inhibits ribosomal rna: a novel cytotoxic mechanism for bioreductive drugs mitomycin c: mechanism of action, usefulness and limitations active human immunodeficiency virus protease is required for viral infectivity structure-based design: from renin to hiv protease three-dimensional structure of aspartyl protease from human immunodeficiency virus hiv-1 conserved folding in retroviral proteases: crystal structure of a synthetic hiv-1 protease x-ray analysis of hiv-1 proteinase at 2.7 a resolution confirms structural homology among retroviral enzymes new drugsreports of new drugs recently approved by the fda. ritonavir structure and mechanism of the complex between cytochrome p4503a4 and ritonavir in vitro antiviral activity of 141w94 (vx-478) in combination with other antiretroviral agents 3-tetrahydrofuran and pyran urethanes as high-affinity p2-ligands for hiv-1 protease inhibitors crystal structure of hiv-1 protease in complex with vx-478, a potent and orally bioavailable inhibitor of the enzyme safety and pharmacokinetics of amprenavir (141w94), a human immunodeficiency virus (hiv) type 1 protease inhibitor, following oral administration of single doses to hiv-infected adults new aza-dipeptide analogues as potent and orally absorbed hiv-1 protease inhibitors: candidates for clinical development bms-232632, a highly potent human immunodeficiency virus protease inhibitor that can be used in combination with other available antiretroviral agents clinical pharmacokinetics and summary of efficacy and tolerability of atazanavir design of hiv protease inhibitors targeting protein backbone: an effective strategy for combating drug resistance novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (pi) uic-94017 (tmc114) with potent activity against multi-pi-resistant human immunodeficiency virus in vitro tmc114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitor-resistant viruses, including a broad range of clinical isolates fda approves new hiv treatment for patients who do not respond to existing drugs clinical pharmacokinetics of darunavir p-glycoprotein mediates efflux transport of darunavir in human intestinal caco-2 and abcb1 gene-transfected renal llc-pk1 cell lines absorption, metabolism, and excretion of darunavir, a new protease inhibitor, administered alone and with lowdose ritonavir in healthy subjects challenges and rewards prodrugs: design and clinical applications 659−670. (195) florencio zaragoza, d. lead optimization for medicinal chemists: pharmacokinetic properties of functional groups and organic compounds niculescu-duvaz, i. optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (zd2767) for antibody-directed enzyme prodrug therapy (adept) niculescu-duvaz, i. i. antibody-directed enzyme prodrug therapy (adept): a review introduction to the background, principles, and state of the art in suicide gene therapy a phase i trial of antibody directed enzyme prodrug therapy (adept) in patients with advanced colorectal carcinoma or other cea producing tumours zd2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts a higher yielding synthesis of the clinical prodrug zd2767p using di-protected 4-[n,n-bis(2-hydroxyethyl)amino]-phenyl chloroformate structural insights into cpt-11 activation by mammalian carboxylesterases piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases ces1a1, ces2, and a newly expressed carboxylesterase isoenzyme bioactivation of the anticancer agent cpt-11 to sn-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions hydrolysis of 3h-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro oral bambuterol versus terbutaline in patients with asthma cyclization-activated prodrugs. basic carbamates of 4-hydroxyanisole cyclization-activated prodrugs. basic esters of 5-bromo-2′-deoxyuridine synthesis of self-immolative glucuronide-based prodrugs of a phenol mustard. anti-cancer drug des bioreduction activated prodrugs of camptothecin: molecular design, synthesis, activation mechanism and hypoxia selective cytotoxicity synthesis and biological evaluation of 2′-carbamate-linked and 2′-carbonate-linked prodrugs of paclitaxel: selective activation by the tumor-associated protease plasmin elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release alpha-isobutanoyloxyethoxy)-carbonyl] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: ii. improved oral bioavailability, dose proportionality, and colonic absorption compared with gabapentin in rats and monkeys ] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: i. design, synthesis, enzymatic conversion to gabapentin, and transport by intestinal solute transporters clinical pharmacokinetics of xp13512, a novel transported prodrug of gabapentin hydrolysis of capecitabine to 5′-deoxy-5-fluorocytidine by human carboxylesterases and inhibition by loperamide design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue rational development of capecitabine capecitabine: a review profound and sustained inhibition of platelet aggregation by fradafiban, a nonpeptide platelet glycoprotein iib/iiia antagonist, and its orally active prodrug, lefradafiban, in men bis-tetrahydrofuran: a privileged ligand for darunavir and a new generation of hiv protease inhibitors that combat drug resistance enhancing protein backbone bindinga fruitful concept for combating drug-resistant hiv crystal structure of an in vivo hiv-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance potent hiv protease inhibitors: the development of tetrahydrofuranylglycines as novel p2-ligands and pyrazine amides as p3-ligands anti-hiv protease inhibitor darunavir inhibitors of hiv-1 protease containing the novel and potent (r)-(hydroxyethyl)sulfonamide isostere design and synthesis of amprenavir, a novel hiv protease inhibitor the development of cyclic sulfolanes as novel and high-affinity p2 ligands for hiv-1 protease inhibitors nonpeptidal p2 ligands for hiv protease inhibitors: structure-based design, synthesis, and biological evaluation structure-based design of non-peptide hiv protease inhibitors potent hiv protease inhibitors incorporating high-affinity p2-ligands and (r)-(hydroxyethylamino)sulfonamide isostere high resolution crystal structures of hiv-1 protease with a potent non-peptide inhibitor (uic-94017) active against multi-drug-resistant clinical strains ultra-high resolution crystal structure of hiv-1 protease mutant reveals two binding sites for clinical inhibitor tmc114 a potent human immunodeficiency virus type 1 protease inhibitor, uic-94003 (tmc-126), and selection of a novel (a28s) mutation in the protease active site potent inhibition of hiv-1 replication by novel nonpeptidyl small molecule inhibitors of protease dimerization design of hiv-1 protease inhibitors active on multidrug-resistant virus darunavir, a conceptually new hiv-1 protease inhibitor for the treatment of drugresistant hiv darunavir, a new pi with dual mechanism: from a novel drug design concept to new hope against drug-resistant hiv stereoselective photochemical 1,3-dioxolane addition to 5-alkoxymethyl-2(5h)-furanone: synthesis of bis-tetrahydrofuranyl ligand for hiv protease inhibitor uic-94017 (tmc-114) synthesis and optical resolution of high affinity p2-ligands for hiv-1 protease inhibitors a novel bistetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (pi), grl-98065, is potent against multiple-pi-resistant human immunodeficiency virus in vitro ultra-potent p1 modified arylsulfonamide hiv protease inhibitors: the discovery of gw0385 suppression of hiv-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring tmc310911, a novel human immunodeficiency virus type 1 protease inhibitor, shows in vitro an improved resistance profile and higher genetic barrier to resistance compared with current protease inhibitors flexible cyclic ethers/polyethers as novel p2-ligands for hiv-1 protease inhibitors: design, synthesis, biological evaluation, and protein-ligand x-ray studies design and synthesis of potent hiv-1 protease inhibitors incorporating hexahydrofuropyranol-derived high affinity p(2) ligands: structure-activity studies and biological evaluation probing multidrug-resistance and protein-ligand interactions with oxatricyclic designed ligands in hiv-1 protease inhibitors grl-04810 and grl-05010, difluoride-containing nonpeptidic hiv-1 protease inhibitors (pis) that inhibit the replication of multi-pi-resistant hiv-1 in vitro and possess favorable lipophilicity that may allow blood-brain barrier penetration the discovery of β-secretase and development toward a clinical inhibitor for ad: an exciting academic collaboration design of potent inhibitors for human brain memapsin 2 (βsecretase) structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin 2 (beta-secretase) cathepsin d is membrane-associated in macrophage endosomes design, synthesis and x-ray structure of protein-ligand complexes: important insight into selectivity of memapsin 2 (beta-secretase) inhibitors design of potent inhibitors of human beta-secretase design of potent inhibitors of human beta-secretase. part 2 chronic treatment with the gammasecretase inhibitor ly-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation studies of abeta pharmacodynamics in the brain, cerebrospinal fluid, and plasma in young (plaque-free) tg2576 mice using the gammasecretase inhibitor n2-[(2s)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-n1-[(7s)-5-methyl-6-oxo-6,7-dihydro-5h-dibenzo quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor ly-411575 novel orally active, dibenzazepinone-based gamma-secretase inhibitors design, synthesis, and evaluation of tetrahydroquinoline and pyrrolidine sulfonamide carbamates as gamma-secretase inhibitors discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors 2,6-disubstituted n-arylsulfonyl piperidines as gamma-secretase inhibitors nitrogen-appended n-alkylsulfonamides as inhibitors of gamma-secretase carbamate-appended nalkylsulfonamides as inhibitors of gamma-secretase serine proteases of the human immune system in health and disease proteases: multifunctional enzymes in life and disease irreversible inhibitors of serine, cysteine, and threonine proteases strategies for the inhibition of serine proteases protease inhibitors: current status and future prospects structural basis of substrate specificity in the serine proteases novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases synthesis and kinetic studies of diphenyl 1-(npeptidylamino)alkanephosphonate esters and their biotinylated derivatives as inhibitors of serine proteases and probes for lymphocyte granzymes thrombin inhibitors: a new generation of antithrombotics the current status of coagulation the solution conformation of (d)phe-pro-containing peptides: implications on the activity of ac-(d)phe-pro-boroarg-oh, a potent thrombin inhibitor synthesis of conformationally-restricted boropeptide thrombin inhibitors inhibition of serine proteases by peptidyl fluoromethyl ketones nonpeptidic inhibitors of human leukocyte elastase. 5. design, synthesis, and x-ray crystallography of a series of orally active 5-aminopyrimidin-6-one-containing trifluoromethyl ketones nonpeptidic inhibitors of human leukocyte elastase. 6. design of a potent, intratracheally active, pyridone-based trifluoromethyl ketone discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase orally active trifluoromethyl ketone inhibitors of human leukocyte elastase clinical consequences of hepatitis c virus infection challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection hepatitis c virus ns3-4a protease inhibitors: countering viral subversion in vitro and showing promise in the clinic an ns3 protease inhibitor with antiviral effects in humans infected with hepatitis c virus discovery of (1r,5s)-n-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl sch 503034, a mechanism-based inhibitor of hepatitis c virus ns3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells hepatitis c virus ns3-4a serine protease inhibitors: sar of p′2 moiety with improved potency depeptidization efforts on p3-p2′ alpha-ketoamide inhibitors of hcv ns3-4a serine protease: effect on hcv replicon activity discovery of sch446211 (sch6): a new ketoamide inhibitor of the hcv ns3 serine protease and hcv subgenomic rna replication discovery of boceprevir, a direct-acting ns3/ 4a protease inhibitor for treatment of chronic hepatitis c infections synthesis and antiviral activity of hcv ns3/4a peptidomimetic boronic acid inhibitors novel macrocyclic hcv ns3 protease inhibitors derived from alpha-amino cyclic boronates inhibition of cathepsin b and papain by peptidyl alpha-keto esters, alpha-keto amides, alpha-diketones, and alpha-keto acids design, synthesis, and biological activity of m-tyrosine-based 16-and 17-membered macrocyclic inhibitors of hepatitis c virus ns3 serine protease in vitro antiviral activity of sch446211 (sch6), a novel inhibitor of the hepatitis c virus ns3 serine protease discovery and structure−activity relationship of p1−p3 ketoamide derived macrocyclic inhibitors of hepatitis c virus ns3 protease synthetic inhibitors of elastase hcv-targeted antivirals: current status and future challenges small molecule inhibitors of the hepatitis c virus-encoded ns5a protein chemical genetics strategy identifies an hcv ns5a inhibitor with a potent clinical effect modeling shows that the ns5a inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis c virus half-life a phase 1, randomized, placebocontrolled, 3-day, dose-ranging study of gs-5885, an ns5a inhibitor, in patients with genotype 1 hepatitis c ledipasvir and sofosbuvir for untreated hcv genotype 1 infection fda approves first combination pill to treat hepatitis c evolutionary lines of cysteine peptidases thiol proteases: inhibitors and potential therapeutic targets review: novel cysteine proteases of the papain family cysteinyl proteinases and their selective inactivation michael acceptors as cysteine protease inhibitors use of cysteine-reactive small molecules in drug discovery for trypanosomal disease identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov 3clpro inhibitors synthesis and evaluation of peptidyl michael acceptors that inactivate human rhinovirus 3c protease and inhibit virus replication 4-piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin k inhibitors p2−p3 conformationally constrained ketoamide-based inhibitors of cathepsin k azepanone-based inhibitors of human and rat cathepsin k an azepanone-based inhibitor of human cathepsin k with improved oral bioavailability in the rat and the monkey acyclic, orally bioavailable ketone-based cathepsin k inhibitors nonpeptidic, noncovalent inhibitors of the cysteine protease cathepsin s discovery and development of fatty acid amide hydrolase (faah) inhibitors the endocannabinoid system: drug targets, lead compounds, and potential therapeutic applications cannabinoid receptors: where they are and what they do a comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol crystal structure of the human monoacylglycerol lipase, a key actor in endocannabinoid signaling therapeutic potential of monoacylglycerol lipase inhibitors uncoupling of the endocannabinoid signalling complex in a mouse model of fragile x syndrome urb602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects highly selective inhibitors of monoacylglycerol lipase bearing a reactive group that is bioisosteric with endocannabinoid substrates discovery and optimization of piperidyl-1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of alpha/beta-hydrolase domain containing 6 (abhd6) selective inhibition of alpha/betahydrolase domain 6 attenuates neurodegeneration, alleviates blood brain barrier breakdown, and improves functional recovery in a mouse model of traumatic brain injury a functional proteomic strategy to discover inhibitors for uncharacterized hydrolases structural adaptations in a membrane enzyme that terminates endocannabinoid signaling mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptormediated phenotypic hypoalgesia the endogenous cannabinoid system protects against colonic inflammation characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase inhibition of fatty-acid amide hydrolase accelerates acquisition and extinction rates in a spatial memory task. neuropsychopharmacology design, synthesis, and structure-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors mechanism of carbamate inactivation of faah: implications for the design of covalent inhibitors and in vivo functional probes for enzymes a new group of oxime carbamates as reversible inhibitors of fatty acid amide hydrolase discovery of potent inhibitors of human and mouse fatty acid amide hydrolases key: cord-006856-b1w25ob5 authors: nan title: 19th meeting of the austrian society of transplantation, transfusion, and genetics, october 26–28, 2005 date: 2005 journal: eur surg doi: 10.1007/s10353-005-0216-6 sha: doc_id: 6856 cord_uid: b1w25ob5 nan background. nowadays lung transplantation is an established standard procedure for the treatment of most end-stage respiratory disorders. in the last years, like for all solid-organ transplantations, the demand for donor lungs exceeded significantly the existing organ pool. thus, most specialised centers face a high mortality on the waiting list. this retrospective study compares the outcome of marginal versus ideal donor lungs. methods. we performed a retrospective analysis of 98 consecutive primary lung transplantations from 94 donors from 1/2001 to 12/2002 . recipients were divided in two groups (standard versus "extended") according to the donor lung acceptance criteria (age, >55 years; pao 2 at fio 2 /peep 5, <300 mmhg; positive tobacco anamnesis [>20 packages per year]; inhalation of noxious agents; presence of infiltration on chest x-ray or purulent secretions at bronchoscopy). results. twenty-three donors (24.5%) were extended. twenty-six recipients (26.55%) received organs from extended donors. according to our data, differences in intubation times, icu stay, and hospital stay were not statistically significant. furthermore, postoperative bleeding rates were comparable as well as bronchial anastomotic complications. we encountered no significant statistical difference in the 3-month (standard 88.89% vs. extended 92.31%) and 1-year (standard 81.94% vs. extended 84.62%) survival between the two groups. conclusions. our study suggests that the use of selected marginal donor lungs has no influence on the outcome after transplantation. background. female donor gender has been described to be an independent risk factor for primary graft failure. we performed this study to evaluate the impact of donor gender on outcome and complications after lung transplantation. methods. we retrospectively reviewed the impact of donor gender on outcome of 163 primary lung transplant recipients (93 recipients were male [57%], 70 were female [43%]) from january 2001 to december 2003. recipients were stratified whether they received a female-or male-donor organ. both groups were compared with regard to duration of intubation time, icu stay, postoperative complications and survival. both groups were comparable with regard to mean age, indications, and mean waiting list time. results. mean time until extubation was 8 days in the group receiving organs from male donors and 16 days in the group receiving organs from female donors (p = 0.041). mean icu stay of 12 days for the male-donor recipient group was significantly shorter than that for the female-donor recipient group, 20 days (p = 0.044). 3-month survival rates were comparable in both groups: 89.53% (male-donor recipi-lunge 19th meeting of the austrian society of transplantation, transfusion and genetics background. nowadays lung transplantation is an established therapeutic option for most end-stage respiratory diseases and one of the fastest-growing solid-organ transplantation procedures in the world, reflected by the enormous demand for lungs. this retrospective review of the vienna lung transplantation group demonstrates data about waiting time and mortality rate for various end-stage respiratory diseases for the years 2003 and 2004. methods. . different variables specific for pretransplant period such as probability of transplantation, death on the waiting list, and time till transplantation were analysed for most frequent end-stage respiratory diseases. results. we found no significant changes in the average mortality rate (constantly 8%) on the waiting list during the studied period. however, patients suffering from pph have the highest probability of dying while being listed for lung transplantation (2003, n = 2 [20%] ; 2004, n = 2 [14.3%]), followed by patients with idiopathic fibrosis (2003, n = 3 [15%]; 2004, n = 4 [11.4%] ). subsequently the next subgroup is represented by cystic fibrosis patients, who are characterized by a moderate mortality rate (2003, n = 1 [3.8%] ; 2004, n = 1 [5%]). in contrast, patients who were suffering from copd showed the lowest probability of dying (2003, n = 0 [0%]; 2004, n = 1 [2.9%]). the average waiting time for the observed 2 years amounts to 97.5 days (2003, 99 ± 98 [min, 0; max, 464] days; 2004, 96 ± 96 [min, 0; max, 400] days). regarding the disease-specific waiting time, pph patients had to wait a longer period (133 ± 128 days) for their transplantation than patients with other diagnosis. conclusions. waiting time and mortality on the waiting list are showing remarkable differences within the disease-specific subgroups. v05 increased recipient vegf serum level is a risk factor for severe reperfusion edema after lung transplantation k. krenn, s. taghavi, w. klepetko, s. aharinejad laboratorium für kardiovaskuläre forschung, zentrum für anatomie und zellbiologie, medizinische universität wien, wien, österreich background. primary graft dysfunction (pgd) due to ischemia-reperfusion injury is a severe complication in lung transplantation (ltx) . therapeutic strategies are limited and there exist no preoperative markers to predict the risk for reperfusion-induced edema. vascular endothelial growth factor (vegf) is a key regulator of vascular permeability. methods. preoperative vegf serum levels were measured by elisa for 76 patients undergoing ltx. underlying diseases in ltx patients were copd (n = 22), cystic fibrosis (n = 15), idiopathic pulmonary fibrosis (n = 11), emphysema (n = 6), primary pulmonary hypertension (n = 6), sarcoidosis (n = 4), and others (n = 12). the ischemia time of the grafts and the blood gas parameters in donors were comparable. reperfusion edema was diagnosed and scored by characteristic changes in chest radiographs and deteriorating blood gases according to the guidelines of the international society for heart and lung transplantation, grading pgd from 0 to 3 (0, none; 1, only radiographic evidence; 2, moderate, pao 2 /fio 2 ratio of 200-300; 3, severe, pao 2 /fio 2 ratio of <200 or ecmo support necessary). results. grade 3 pgd occurred in 15%, grade 2 in 23%, grade 1 in 43%, and grade 0 in 19% of the patients. the preoperative vegf serum levels were significantly higher in patients with pgd grade 2 and 3 versus those without clinically relevant pgd (grade 0 and 1) following ltx (p = 0.0007). preoperative recipient vegf serum levels significantly predicted pgd in receiver operating characteristic analysis (p = 0.0002, auc = 0.755, ci = 1.001-1.003). conclusions. preoperative serum vegf levels in patients awaiting ltx could identify those at risk for reperfusion-induced edema following transplantation. background. airway stenoses are well known after lung transplantation although most occur due to surgical problems of the bronchial sutures. we wanted to analyse the use of endobronchial stenting in stenoses not related to the bronchial anastomoses. methods. we performed a retrospective analysis in 12 patients after bilateral lung transplantation with consecutive stent placement aside the bronchial anastomoses. the indication for stent implantation was central bronchial stenoses due to bronchomalacia, granulation tissue with bronchial wall destruction, and endoluminal stenosis with airflow limitation or occurrence of segmental or lobar atelectasis. we used boston ultraflex (23 of 26), rüsch polyflex (2 of 26), rüsch dynamic (1 of 26) stents. results. in 12 lung allograft recipients, we implanted in total 26 stents between 2 and 72 month after bilateral lung transplantation. the predominant locations were the right (20%) and left lower lobe bronchus (25%) and the bronchus intermedius (33%). the patient had granulation tissue proliferation due to ischemia and/or chronic bacterial or fungal bronchitis. only one patient had concomitant anastomoses dehiscence. the stents remained in situ from 1 day to 850 days after placement. in 18 of 26 stent placements no severe complications occurred. stent migration was observed in 6 of 26; severe granulation tissue that revealed further interventional treatment, in 4 patients. one patient died because of necrotizing vasculitis and letal hemoptysis. all other stents are still in place (10 of 26), were in place at the time of death due to other reasons (4 of 26), or were explanted regularly (5 of 26). conclusions. endobronchial stent placement is an effective treatment for bronchial stenoses that are not related to the bronchial anastomoses. complications occurred in about 40% of all stents. v07 preoperative oral corticosteroids predict the risk of late postoperative bleeding and perioperative mortality in lung transplantation conclusions. patients listed for lung transplantation with high-dose preoperative oral cs intake have a significantly increased risk of late postoperative bleeding. the perioperative mortality and the probability of 1-year survival of recipients with late bleeding are severely affected compared with patients with no or early bleeding. background. the efficacy of induction therapy after lung transplantation remains controversial and data on its use are limited. we hypothesized that induction therapy would have an impact on incidence of early rejection after lung transplantation and may cause a higher rate of infectious complications during the first 6 months post transplantation. methods. all patients who underwent lutx between jannuary 2003 and march 2004 and received induction therapy with rabbit antithymocyte globulin (2.5 mg/kg/d for 3-6 days) were analysed retrospectively. basic immunosuppression consisted of tacrolimus, mycophenolate mofetil, and prednisone. primary end point was patient survival after 6 months. secondary end point was histologically proven grade i or higher grade of rejection within the first 6 months, incidence of infectious episodes and bronchiolitis obliterans syndrome (bos). a total of 29 adult patients who underwent single (n = 2), heart-lung (n = 2), or bilateral lung (n = 26) transplantation entered the study. female, 10 (35%); male, 19 (65%). mean age was 37 ± 12.9 years (range, 19.3-59.9 years). results. the 6-month survival using kaplan-meier analysis was 92%; 27 patients are alive, two died 151 and 160 days after transplantation. one patient was retransplanted 3 days after primary transplantation because of graft dysfunction. follow-up ranged from a minimum of 151 days to 492 days (mean, 279 ± 85 days). 14 patients had one histologically proven rejection episode. rejections were graded ai for 10 patients, grade ai-ii for 2, and grade aii for 2 patients. 7 rejection episodes were treated with iv methylprednisone, no recurrent or ongoing rejections were observed. incidence of bacterial infections requiring treatment was 21/100 patients days. 2 cmv infections, 1 non-cmv viral infection, and 4 fungal infections (2 candida, 2 aspergillus) were diagnosed and treated. no patient developed signs of bos of grade ≥1. no lymphoma occurred. conclusions. this retrospective analysis suggests that induction therapy with atg in combination with tacrolimusbased triple immunosuppressive regimen results in excellent survival rates and a low rate of acute early rejections. however, this high immunosuppression efficacy was paralleled by a considerably high rate of bacterial infections during the first 6 postoperative months. rin inhibitor based quadruple drug is. an extensive infectious monitoring was used in this cohort. results. one-year patient survival was 71.4%, perioperative rejection rate 35%. infection incidence during first hospitalization was 79.6% (1.3 episodes per transplant): pneumonia, 74%; sepsis, 13%; wound infection, 17%; hsv/vzv, 28%; uti, 2%. during follow-up, cmv-associated complications were observed in 50% of patients including cmv infection (n = 9), cmv disease (n = 16). there were nine patients with cmv syndrome, ten patients with cmv graft pneumonitis and two patients with cmv gastrointestinal disease. excluding the 3 retransplants and the 2 perioperative deaths, the incidence of aspergillosis was 27%, six patients with aspergillus tracheobronchitis and seven patients with invasive disease. a total of 33 patients died during follow-up, 28 from infectious complications. as part of our centers' microbiological monitoring, 2773 specimens (51/transplant) were collected. these specimens were taken on 1276 observation days. 1426 investigated specimens were sterile and in 993 specimens microorganisms could be isolated (354 normal flora, 639 pathogens). a total of 1155 pathogens were identified: 673 gram-positive cocci, 177 gram-negative rods, 155 pseudomonas/acinetobacter, 124 candida. >60% of staphylococcus aureus and 55% of coagulase-negative staphylococci were methicillin resistant. other multiresistant organisms were e. faecium (no vre), n = 75; corynebacterium jk, n = 2; stenotrophomonas maltophila/burkholderia cepacia, n = 31. conclusions. infection remains the most common complication and the most common cause of death following lung transplantation. further refinement of infectious prophylaxis is required to improve results. background. valgancyclovir is routinely used for prophylaxis against cytomegalovirus (cmv) reactivation in lung transplant recipients. problems may arise due to its myelotoxic properties. it is unclear whether cytotect ® , a human igg cmv antibody preparation, offers similar protection against cmv reactivation without causing neutropenia. methods. we report on a female patient (63 years) who received a double lung transplant in 2002. cmv status was d+r+. four months postoperatively, she developped cmv pneumonitis and gastroduodenitis and was treated with gancyclovir and cytotect. thereafter, valgancyclovir was instituted but had to be stopped repeatedly because of neutropenia; each withdrawal of valgancyclovir prompted further reactivation episodes as detected by pp65 monitoring. in july 2004, she was started on cytotect, 150 mg q.d. over five days, followed by weekly doses of 50 mg for twelve weeks. results. cytotect was tolerated without side effects. of note, leucocyte counts remained within normal limits. since she has been started on cytotect, she has not experienced any further episodes of cmv reactivation until to date. conclusions. the observations made in this patient suggest that long-term therapy with cytotect ® has the potential to prevent cmv reactivation in selected patients who do not tolerate valgancyclovir due to its myelotoxic side effects. v12 die kombinationsprophylaxe verbessert die cmv bedingte morbidität und mortalität und reduziert das risiko der bronchiolitis obliterans (bos) nach lungentransplantation e. ruttmann, c. geltner, b. bucher, h. ulmer, d. höfer, h. b. hangler, s. semsroth, h. bonatti, r. margreiter, g. laufer, l. müller klinische abteilung für herzchirurgie, universitätsklinik für chirurgie, medizinische universität innsbruck, innsbruck, österreich grundlagen. die opportunistische cmv-infektion stellt ein schwerwiegendes problem nach lungentransplantation dar. ziel dieser untersuchung war, den einfluss der cmv-kombinationsprophylaxe mittels ganciclovir und cmv-hyperimmunglobulinen (cmv-ig) bezüglich patientenüberleben, cmv-reaktivierung, klinischer cmv-erkrankung und entwicklung der bronchiolitis obliterans (bos) zu evaluieren. methodik. eine konsekutive serie von 68 cmv-hochrisikopatienten (d+/r-, d+/r+) mit einem minimalen followup von mindestens 1 jahr post-transplant wurden analysiert. dreißig patienten (44,1 %) erhielten eine alleinige ganciclovir-prophylaxe für 3 monate (kontrollgruppe), 38 transplantationsempfänger (55,9 %) erhielten eine zusätzliche prophylaxe mit cmv-ig (cytotect ® , biotest pharma) in 5 dosen während des 1. postoperativen monats (studiengruppe). das mediane follow-up betrug 16,5 monate in der kontrollgruppe und 23,8 monate in der studiengruppe (p = 0,54). ergebnisse. insgesamt 5 cmv-assoziierte todesfälle (16,7 %) ereigneten sich in der kontrollgruppe, jedoch keiner in der studiengruppe (p = 0,014). in der kontrollgruppe wurden 13 fälle mit klinischer cmv-erkrankung beobachtet (43,3 %), in der studiengruppe 5 patienten (13,2 %) (p = 0,007). zusätzlich zeigte sich ein signifikant verbessertes patientenüberleben in der studiengruppe (log-rank, p = 0,01). die 1-jahresfreiheit von cmv-reaktivierungen betrug 52,1 % in der kontrollgruppe und 71,5 % in der studiengruppe (logrank, p = 0,027). die 3-jahresfreiheit von bos war signifikant höher in der studiengruppe (54,3 % vs. 82 %, log-rank, p = 0,024). schlussfolgerungen. eine zusätzliche cmv-hyperimmunglobulinprophylaxe senkt die cmv-assoziierte morbidität und mortalität. weiters kann das auftreten der bos mittels augmentierter cmv-prophylaxe reduziert werden. durch die dadurch reduzierte morbidität ist die kosteneffizienz gegeben. v13 extracorporeal photoimmune therapy (ecp) with uvadex in conjunction with standard therapy compared to standard therapy alone for the prevention of rejection in lung transplantation patients p. jaksch, r. knobler, b. schlechta, s. guth, w. klepetko klinische abteilung für herz-thoraxchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich background. extracorporeal photopheresis has been shown to be beneficial in acute and chronic rejection in heart transplant patients and has also been used in lung transplant recipients with acute rejection or bronchiolitis obliterans. methods. we performed a prospective study to document the efficacy of photoimmune therapy in the prevention of acute rejections in the first 12 months after lung transplantation. 12 lung transplant recipients with copd were randomized in 2 groups. group a (6 pat) received in total 16 ecp treatments (starting 2 weeks after tx) and group b without ecp or other kind of induction therapy, both groups receiving standard triple immunosuppression with tacrolimus, mycophenolate, and steroids. surveillance bronchoscopies with biopsies were performed after 2, 4, 8, 12, 26, and 52 weeks. primary objectives were acute biopsy-proven rejections of ishlt grade >1, secondary objectives were number of infections (cmv, bacterial, fungal, viral non-cmv, tuberculosis, parasitic), patients and graft survival. results. demographics in both groups were similar (gender, age, underlying disease, cmv mismatch, and type of tx). fev 1% and mef 50% values after 1 year were equal (88.3 ± 7.9% vs. 90 ± 28. 1 and 113.5 ± 41.7% vs. 114.5 ± 42.2%, respectively) . the number of rejections in group a (with ecp) was lower than in group b (0.17 ± 0.41 vs. 0.83 ± 0.75, p = 0.094), as well as the median rejection grade (0.16 ± 0.4 for group a vs. 0.66 ± 0.51 for group b, p = 0.092). after one year post tx, all patients are alive, none developed bo(s) within the first year post tx. conclusions. our preliminary data show a clear trend towards reducing the number and severity of acute rejections in lung transplant recipients. the number of infections was similar in both groups. adding ecp to a standard triple-drug immunosuppressive regimen seems to be a safe and efficient tool in reducing rejection rates without increasing the rate of bacterial, fungal, or viral infection. grundlagen. erhöhte natriumspiegel bei organspendern können mit der bildung von reperfusionsödemen und transplantatdysfunktion assoziiert sein. unklarheit besteht allerdings über die klinischen auswirkungen von erhöhtem spender-natrium nach herztransplantation (htx). in dieser studie wurde der einfluss von hohem spender-natrium auf die frühund 1-jahresmortalität nach htx in einem großen patientenkollektiv analysiert. methodik. es wurden 3157 herztransplantationen der eurotransplant-region aus dem zeitraum jänner 1997 bis dezember 2001 analysiert. entsprechend der spender-natrium-spiegel (sns) wurde das kollektiv in drei gruppen unterteilt: gruppe a, <160 mmol na + je liter n = 2903; gruppe b, 160-170 mmol na + je liter n = 218; gruppe c, ≥170 mmol na + je liter n = 54. eine kaplan-meier-überlebensanalyse und eine multivariate analyse bezüglich des einflusses des sns wurden durchgeführt. endpunkte waren die mortalität ein monat und ein jahr nach htx. ergebnisse. der sns hatte in der univariaten analyse keinen einfluss auf die frühmortalität und einen grenzwertig signifikanten einfluss auf die 1-jahres-mortalität (p = 0,06). in der multivariaten analyse war dieser effekt nicht signifikant (p = 0,2). die kombination aus hohem spenderalter mit hohem sns hatte jedoch in der multivariaten analyse einen hochsignifikanten einfluss auf die früh-und 1-jahresmortalität (p = 0,004). schlussfolgerungen. diese daten zeigen, dass hohe sns mit einer erhöhten früh-und 1-jahresmortalität nach htx einhergehen. diese ergebnisse stehen im gegensatz zu früheren arbeiten mit geringeren patientenzahlen. vor allem die kombination aus höherem spenderalter und hohem sns zeigt einen deutlichen risikoanstieg. herzen von spendern mit einem natriumspiegel von >170 mmol/l sollten nicht elektiv transplantiert werden, bei gleichzeitig hohem spenderalter sollte das herz nicht verwendet werden. die organempfänger wurden entsprechend dem spenderalter in 2 gruppen geteilt: gruppe 1, <35 a, n = 12; gruppe 2, >35a, n = 10. die gruppen waren hinsichtlich organischämiezeit, geschlechts-und cmv-mismatch sowie lipidstatus, nierenfunktion und medikamentöse therapie (immunsuppresion, ace-hemmer, statine, ca-antagonisten) 1 jahr post-htx vergleichbar. pv1 war in gruppe 1 tendenziell niedriger als in gruppe 2 (25,2 ± 17,3 mm 3 vs. 34 ± 22 mm 3 ; p > 0,05). umgekehrt wies gruppe 1 im verlauf des 1. jahres nach htx einen zuwachs an plaquevolumen auf, während in gruppe 2 eine abnahme (∆pv, 2,6 ± 17,6 mm 3 vs. 3,0 ± 10,2 mm 3 ; p > 0,05) festgestellt wurde. von den oben angeführten risikoparametern zeigte lediglich der triglyzeridspiegel 1 jahr post htx eine signifikante korrelation mit ∆pv (r = -0,613, p = 0,002). ∆pv und spenderalter waren nicht miteinander korreliert. schlussfolgerungen ergebnisse. die mortalität beträgt 0 %. wegen gastrointestinaler beschwerden (übelkeit, erbrechen) mussten 3 patienten (12%) auf ein anderes immunsuppressives schema umgestellt werden. bei 2 patienten (everolimus-talspiegel, >8 ng/ml) zeigte sich eine schwere pneumonie (pseudomonas), welche stationär behandelt wurde. es gab keine stationäre behandlung wegen cmv-infekten. die nierenfunktion war in allen patienten stabil (mittleres crea, 1,84 ± 0,85), außer in 2 patienten, welche bereits vor der umstellung erhöhte kreatininwerte zeigten und in denen sich eine weitere erhöhung der kreatininwerte (+20,25%) feststellen ließ. eine bei den meisten patienten auftretende hyperlipidämie konnte unter erhöhung der statintherapie eingestellt werden. in den routinemäßig durchgeführten endomyocard-biopsien fanden sich einen monat nach umstellung und danach keine akuten zellulären abstoßungen mit grad von >1b nach dem ishlt-grading. schlussfolgerungen. certican erwies sich als sicher und verträglich, die umstellung auf das neue immunsuppressivum war in allen patienten komplikationslos. everolimus-talspiegel von 8 ng/ml scheinen ausreichend, höhere spiegel könnten das infektionsrisiko erhöhen. bezüglich der nierenfunktion bleibt abzuwarten, wie sich ein cyclosporin-a-talspiegel von 60-80 ng/ml auswirkt. eine aussage bezüglich der cav steht zu diesem zeitpunkt noch aus. v17 20 jahre herztransplantation in wien (eine retrospektive über 1000 transplantationen) a. zuckermann empfänger-und spenderalter sind im laufe der jahre signifikant gestiegen (recipient age: 42,5 ± 11,8 vs. 52,0 ± 13,8, p < 0,05; donor age, 28,8 ± 10,3 vs. 35,2 ± 12,9, p < 0,01) . mehr patienten wurden präoperativ stationär aufgenommen (28 % vs. 44 %), jedoch hat sich die zahl der intensivpflichtigen patienten signifikant reduziert (21 % vs. 7 %). die zahl der patienten, die zur transplantation "gebridged" werden, ist ebenfalls massiv angestiegen (28 vs. 63 %, p < 0,01). innerhalb dieser gruppe hat die gruppe der patienten mit mechanischer herzunterstützung am stärksten zugenommen (7 % vs. 19 %, p < 0,01). pharmakologisches bridging (55 % vs. 42 %) und aicd (38 % vs.40 %) blieben stabil. pharmakologisches bridging wird heutzutage vermehrt mit prostaglandinen und levusimendan als mit dopamin oder dobutamin durchgeführt. mehr patienten sind am herzen voroperiert (28 % vs. 52 %, p < 0,01), patienten warten länger auf die transplantation (75,9 ± 91,6 vs. 289,8 ± 368,8, p < 0,01) . trotzdem hat sich die mortalität auf der warteliste stark verbessert (27,6 % vs. 12,0 %, p < 0,01), was ein klares zeichen der verbesserten überbrückungsmaßnahmen ist. ischämiezeiten sind ebenso angestiegen (129,3 ± 49,8 vs. 190 ,0 ± 49,2, p < 0,01) wie die liegezeiten auf der intensivstation (4,7 ± 5,7 vs. 7,6 ± 7,7, p < 0,01). dies ist ein klares indiz dafür, dass heute ältere, kränkere und komplexere patienten transplantiert werden. diese veränderungen sind international bei allen zentren zu be-obachten. dies hat dazu geführt, dass mit der zunehmenden erfahrung und verbessertem überleben die nachbeobachtungszeit der patienten stark gestiegen ist und damit die behandlung dieser patienten kostenintensiver geworden ist, was aber mit der guten lebensqualität der herztransplantierten patienten zu rechtfertigen ist. v18 neoplastic diseases after heart transplantation: a retrospective study d. kammerstätter, a. aliabadi, j. ankersmit, d. dunkler, g. wieselthaler, e. wolner, m. grimm, a. zuckermann klinische abteilung für herz-thoraxchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich background. prolonged immunosuppression after solidorgan transplantation is associated with an increased risk for development of neoplasms. the purpose of this analysis was to investigate neoplasm incidence and outcome in patients with induction therapy. methods. 921 cardiac recipients were included in this retrospective analysis. all patients received antibody induction therapy with either polyclonal-atg or monoclonal antibodies. neoplasms were devided into 3 groups: (1) skin cancer, (2) ptld, (3) other neoplasms. overall incidence of neoplasms, patient survival after diagnosis of neoplasms were analysed by the kaplan-meier method. results. a total of 143 tumors were diagnosed at a mean follow-up of 59.6 ± 51.2 months after cardiac transplantation. freedom from neoplasms was 96.7%, 86.6%, and 71.5% after 1-, 5-, and 10-year respectively. 5-year survival after diagnosis of tumor was 50.3%. 65 patients developed skin cancers after 62 ± 40.6 months. 1-and 5-year survival after diagnosis was 96% and 71% respectively. there were 7 tumor-related deaths in this group. 19 patients developed ptld 40.7 ± 33.7 months after transplantation. 1-and 5-year survival was 63% and 40% with 12 deaths associated with the neoplasm. in the third group, a total of 59 patients were included. this group consisted of lung cancer (n = 17), abdominal cancer (n = 15), urogenital cancer (n = 7), and other tumors (n = 20). neoplasms were diagnosed at an average follow-up of 53.5 ± 42.7 months. 1-year and 5year survival was 57% and 25%. 31 deaths were associated with tumor. conclusions. although all patients received antibody induction therapy, overall incidence of neoplasms was comparable to centers using no induction therapy. especially incidence of ptld was low. as long-term survival after cardiac transplantation increases steadily and the risk of cancer increases continuously, patients in long-term follow-up should be checked for malignant diseases on a routine basis. background. while the predictive value of n-terminal pro-b-type natriuretic peptide (nt pro-bnp) in nontransplant cardiac patients is well recognized, its value in heart transplantation (htx) is incompletely understood. certain graft factors (e.g., isolated diastolic dysfunction) may affect both, nt pro-bnp levels and peak exercise tolerance. we therefore hypothesized a relationship between these variables in long-term htx recipients. methods. we measured nt pro-bnp levels of 27 htx patients before a symptom-limited upright bicycle exercise test was performed. graft function was stable in all patients and there were no signs of rejection. patients were divided according to a cut-off value of 70% exercise tolerance predicted normal into "low" and "normal" htx fitness groups. results. in 12 patients (11 m, 1 f; 57 ± 10 years; 6.5 ± 4 years posthtx; donor age, 36 ± 10 years; bmi, 28.6 ± 2.8 kg/m 2 ; creatinine clearance, 42 ± 10 ml/min) peak exercise tolerance was "low" (93 ± 25 w), while in 15 patients (11 m, 4 f; 57 ± 11 years; 7.4 ± 3.6 years posthtx; donor age, 32 ± 10 years; bmi, 26.6 ± 2.7 kg/m 2 ; creatinine clearance, 42 ± 8 ml/min) it was "normal" (136 ± 37 w). in the "low" htx fitness group, nt pro-bnp levels were 702 ± 778 pg/ml, in the "normal" htx fitness group, 324 ± 250 pg/ml (p = 0.08 between groups). regression analysis of peak exercise tolerance, achieved percentage of exercise tolerance predicted "normal", and renal function with nt pro-bnp levels failed to demonstrate a significant relationship. conclusions. the findings confirm previous studies that nt pro-bnp levels are increased in asymptomatic long-term htx recipients. a larger sample size is warranted, however, to support the hypothesis that nt pro-bnp might be a useful indicator to predict physical fitness in these patients. calcineurin inhibitor therapy is an important cause of renal dysfunction in heart transplant patients. sirolimus (srl) is a novel immunosuppressive (is) drug without nephrotoxic side effects. however, cases of proteinuria associated with srl have been reported following renal transplantation. in cardiac transplantation the potential incidence of proteinuria is not known. 29 long-term cardiac transplant patients (age, 60.9 ± 7.3 years) were switched from cyclosporine-based immunosuppression to a srl-based is 8.8 ± 4.5 years after transplantation. concomitant is consisted of mycophenolate-mofetil with or without steroids. two patients died 14 and 37 months post switch due to infection. both patients were dialysis dependent at time of death. one other patient was switched back to cni-based is due to interstitial nephritis. 24 h collections of urine were performed in all patients before switch and at several time points post switch to measure proteinuria. proteinuria increased significantly from 0.45 ± 1.0 mg/dl (median, 0.17) pre switch to 1.03 ± 2.04 mg/dl (median, 0.21) post switch (p = 0.017). proteinuria of 0.21-1.0 mg/dl was seen in 21% of patients before switch and in 28% after switch. proteinuria of >1.0 mg/dl was seen in 10% of patients before switch and in 24% after switch (n.s.). three patients developed severe proteinuria (>3.5 mg/dl) after switch. one died on dialysis, one was switched back to cni and one still remains on srl. in conclusion, proteinuria may develop in cardiac transplant patients after switch to srl-based is. srl seems to have a potential adverse renal effect in these patients. srl should be used cautiously with close monitoring for proteinuria or increased renal dysfunction. v21 early growth-response factor-1 is involved in cellular injury of transplanted hearts background. we have shown a persistent mitochondrial pathology in patients with idiopathic dilative (dcm) but not ischemic (icm) cardiomyopathy following cardiac transplantation. early growth response factor (egr)-1 that is stimulated by cytokines and hypoxia is suggested to induce inflammation and tissue injury. whether egr-1 mediates the persistent cellular pathology in hearts transplanted to dcm patients is unknown. methods. egr-1 and hypoxia-inducible factor-1 (hif-1) gene expression was examined in left ventricular biopsies of explanted failing hearts in 28 icm and 42 dcm patients, as well as in 12 donor grafts before reperfusion (control), at 10, 30, 60 minutes after reperfusion, and at 1, 2, 3, 4, 6, 12 posttransplant weeks, using real-time rt-pcr. hif-1 binding activity was examined using emsa. results. egr-1 myocardial gene expression was upregulated in dcm and icm (p < 0.01). hif-1 mrna levels were unchanged in both groups, whereas hif-1 binding activity was increased in icm only (p < 0.01) vs. controls. egr-1 and hif-1 myocardial expression increased during reperfusion in donor grafts (p < 0.01) vs. control hearts. in icm group, graft egr-1 and hif-1 expression returned to and remained at the baseline level of control hearts one week after transplantation. in contrast, in dcm group, egr-1 levels remained significantly upregulated during the follow-up period in transplanted hearts (p < 0.01), although hif-1 expression returned to the control baseline level one week after transplantation. conclusions. chronic hypoxia specifically triggers hif-1 binding activity in icm, and reperfusion upregulates egr-1 and hif-1 mrna expression in heart grafts. the persisting egr-1 overexpression in grafts transplanted to dcm patients could mediate mitochondrial impairment. targeting egr-1 overexpression that bypasses hif-1 might be beneficial to counteract acute reperfusion-induced injury, and the chronic cellular pathology in cardiac grafts transplanted to dcm patients. introduction. giant-cell myocarditits (gcm) is a rare and frequently fatal disorder of unknown origin that is defined histopathologically as diffuse myocardial necrosis with multinucleated giant cells in the absence of sarcoid-like granuloma. patients usually have ventricular arrhythmias or congestive heart failure. although a variety of systemic disorders have been seen in association with giant cell mycocarditis, most cases occur in previously healthy adults. conclusive diagnosis requires histologic analysis of myocardial tissue obtained by endomyocardial biopsy (emb). congestive heart failure (chf) is the most common cardiac presentation (75%), with sustained, refractory ventricular tachycardia. the case presented here is that of a 65-old-man suffering of gcm in whom an extracorporeal membrane oxygenation (ecmo) had to be implanted to overcome cardiogenic shock. antithymocyte gobuline (ratg, thymoglobuline, sangstat), respectively ciclosporine (inn: cyclosporine), mycophenolate and steroids were utilized to bridge the time to complete myocardial recovery. we are reporting the first successful implantation and bridging to myocardial regeneration in a patient suffering of gcm by means of ecmo and initiation of immunomodulating drugs including polyclonal ratg. clinical summary. a 65-year-old man was admitted to a public hospital because of thoracic pain, positive heart enzymes, and a highly pathologic electrocardiogramm. echocardiography demonstrated a pericardial effusion and reduced left ventricular function (lvf, ef 15%). performed angiography evidenced and a high-grade stenosis of the left anterior descending (lad) which led to stent implantation. despite stent placement, the clinical condition worsened leading to cardiogenic shock including incipient shock liver. in this clinical condition the patient was transferred to our institution. in addition, the patient developed malign ventricular tachycardia (lowen iv) and had to undergo repetitive defibrillation. in this clinical scenario it was decided to implant a femoral veno-arterial ecmo. the patient's metabolic data improved noticeably; however, due to repeated ventricular tachycardia, the patient had to be defibrillated multiple times (max. 30×/d). to define exact cardiac pathology, we performed a heart biopsy with the pathology of gcm. immunosuppressive therapy with cyclosporine (50 mg/d), mycophenolate and prednisolon (250 mg/d) was initiated. in addition, rabbit atg (ratg, thymoglobuline, sangstat) at the dosage of 75 mg/d was added to the therapy. this drug therapy was continued for 5 days. of note is the fact that with initiation of ratg, cardiac fibrillation episodes abated immediately. routinely performed echo-cardiography (tee) revealed an improvement in ventricular function, and one week after ecmo support, we were able to wean the patient from extracorporeal support. moreover, a routine biopsy after 14 days revealed complete remission of gcm in the heart tissue. an intracardial defibrillator (icd) was additionally implanted. three months after emergency admission to our department the patient was discharged. continuous medication of prednisolon 5 mg/d, mycophenolat mofetil 500 mg/d, and plavix 75 mg/d was prescribed. 12 month after initial event the patient describes nyha class i heart function and echocardiography reveals an moderate impairment of heart function (ef 55%). immunosuppressive with low-dose steroid and mycophenolate drug regimen is continued as the patient describes no side effects. discussion. this report adds to the available knowledge of giant-cell myocarditis by providing that (a) ecmo implantation is feasible if the patient is demonstrating acute haemodynamic deterioration because of biventricular dilation and medically intractable ventricular fibrillation and (b) after verified histological diagnosis of gcm immunemodulation with cyclosporine/mycophenolate with additional application of ratg is feasible with favourable outcome. in various studies, patients with gcm were treated with immunosuppression (cyclosporine, steroids, murine monoclonal antibodies [okt-3]) and even assist device as bridge to transplantation. heart transplantation has shown to be successful as method of treatment. autoimmune diseases and its mechanisms were suggested to be involved in the pathogenesis of gcm. most recently, a novel mechanism of action of immunoglobulin was proposed to be due to anti-inflammatory activities through the inhibitory fc receptos (fcrs). it has been suggested that t-cell-mediated autoimmune diseases is the result of inappropriate antigen presentation of either a self-antigen or an antigen with the capacity to mimic a self-antigen in the peripheral lymphoid tissues. relevant to this novel application of ratg, polyclonal suspensions like igm/g, ivig containing fc receptors and have been demonstrated to be beneficiary in autoimmune myocarditis. in an elegant study by shioji et al. ivig was highly effective in ameliorating experimental myocarditis. however it has to be mentioned that immunoglobulin treatment failed to ameliorate myocarditis. in respect to our patient suffering of rcm fc containing ratg provided remarkable clinical benefit. background. the criteria for liver donation have been widely extended due to the increasing waiting time and waiting list mortality. marginal donors provide an upcoming option to diminish the number of waiting patients. methods. the criteria for marginal were icu stay of >7 days, age of >65 years, bmi of >27, alcohol or oral drug abuse, infection, hypernatremia (na, >150 mg/dl), high liver enzymes (ast, alt 2 times the normal), liver parenchymal damage and metabolic diseases. from 01/00 until 06/05 our center reported 55 donors, who fulfilled at least 3 of the above criteria. results. all livers were transplanted either in standard or in piggyback technique with a cold ischemic period between 130 minutes and 12 hours. the mean recipient age was 45 (13-75) years. 6 livers were used for hu patients, 5 for a retransplantation, 2 were implanted in combination with kidneys, and 37 organs were transplanted electively. primary diseases were cryptogenic cirrhosis, hepatocellular carcinoma, post-hepatitis cirrhosis, polycystic liver disease, scleroting bile ducts, hepatic artery thrombosis, and acute liver failure. 2 month after transplantation, 54 recipients were alive, 1 died 1 month after tx not transplant related. 16 livers were implanted at our center in piggyback technique with retrograde reperfusion. 15 patients were elective patients in good or moderate condition, 1 patient was a hu patient suffering a hepatic artery thrombosis. the initial graft function was good (got, <1000 mg/dl pod 1) in 8, moderate (got, 1000-2000 mg/dl pod 1) in 5, and delayed (got, >2000 mg/dl pod 1) in 3 cases, all patients survived. conclusions. marginal livers are eligible for transplantation. delayed graft function has to be taken into account. hospital between january 1993 and december 2003. we employed the local registry of the department of transplant surgery, where variables of all patients are routinely and prospectively recorded. primary outcome was initial graft function, secondary outcome was patient survival. results. cumulative number of marginal donor criteria was significantly and linearly associated with an increased rate of primary dysfunction (p = 0.005). in patients with more than three cumulative marginal donor criteria the rate of primary dysfunction was 36 percent. patient survival was not influenced by the cumulative number of donor criteria (log-rank test, p = 0.81). independent marginal donor criteria to predict primary dysfunction were cold ischemia time of >10 hours (or, 0.56; 95% ci, 0.32 to 0.98) and donor peak serum sodiumof >155 meq/l (or, 0.44; 95% ci, 0.26 to 0.77), as assessed in a multivariate regression model. conclusions. the use of marginal liver donors with more than three marginal donor criteria shows deleterious effects on initial graft function. noteworthy, patient survival was not associated with marginal donor criteria, which may be explained by early and successful retransplantation of liver recipients with primary nonfunction. ergebnisse. alle transplantate zeigten eine gute initiale organfunktion, der transaminasenverlauf und das gesamtbilirubin, gemessen am 1. postoperativen tag, am 7. postoperativen tag und bei entlassung (mittelwerte mit standardabweichung) waren wie folgt: got 1334 u/l (±848), 43 u/l (±13), 25 u/l (±9); gpt 877 u/l (±245), 420 u/l (±649), 54 u/l (±40); ggt 118 u/l (±86), 355 u/l (±232), 232 u/l (±205); gesamtbilirubin 5,1 mg/dl (±2,2), 3,5 mg/dl (±2,5), 1,9 mg/dl (±0,9) . background. meld score is a useful tool in predicting mortality in patients awaiting liver transplantation. its capacity to predict patient survival seems to be relatively poor and is still discussed controversially. the purpose of the study was to analyse the impact of alterations of the meld score during waiting time on the posttransplant survival rate. additionally, the impact of donor quality on posttransplant survival was investigated. methods. 242 adult patients were transplanted between 1997 and 2003 because of end stage liver disease without malignancy. the meld scores at time of listing (meld on) and of transplantation (meld tx) were gathered. additionally the delta-meld was calculated from listing to transplantation. results. a high meld tx showed only a trend to poorer survival. patients who died within the 1 st year after transplantation showed a significant increase in the meld score during waiting time (p < 0.01). patients with a delta-meld higher than 4 during waiting time had a 50% 1-year mortality after transplantation, patients with a meld increase not higher than 4 had only a 23.1% 1-year mortality (p < 0.01). patients with a meld score higher than 24 who received a marginal graft showed a trend to poorer posttransplant survival. conclusions. patients with a substantial increase of the meld score during waiting time had a significantly poorer 1year posttransplant survival. in contrast, the meld score at time of listing or transplantation had no impact on the posttransplant survival rate. the use of marginal grafts in patients with a higher meld score has to be evaluated carefully. there is no significant difference of serum sodium levels in ltx candidates with or without ascites crease in serum creatinine is a late event in patients with ascites and is not directly reflected within the meld formula. for this purpose we compared patients who died on the waiting list with patients who finally were transplanted. the impact of serum sodium and ascites on death on the waiting list was evaluated. methods. from 1997 to 2005, 621 adult patients with end-stage liver disease were listed for orthotopic liver transplantation (olt). only patients without hepatoma who died on the waiting list (123 patients) or were finally transplanted (300 patients) were evaluated. in addition to the meld score, the serum sodium and the ascites were investigated at time of listing and of coming off the list (transplantation or death). results. transplanted patients had a significantly lower meld on (p < 0.01) than the patients who died while on the waiting list. patients who died while on the waiting list had a significant increase in the meld score during waiting time (p < 0.01). patients who underwent transplantation showed a stable meld score during their waiting time. refractory ascites and spontaneous bacterial infection were evaluated as independent risk factors for death on the waiting list as well as the meld on. 47.2% of the patients (58 of 123 patients) who died on the waiting list were suffering from ascites, in contrast to only 28.7% of the transplanted patients (86 of 300 patients). there was no significant difference in the mean meld on between the patients who were suffering from ascites and those who were not (p = 0.72). nor was any significant difference found in the meld off (p = 0.77). the serum sodium of patients suffering from ascites showed no significant difference to those who showed no signs of ascites. conclusions. ascites was evaluated as independent risk factor for death on the waiting list. no significant difference in the serum sodium levels were found between patients suffering from ascites or not. therefore complications of portal hypertension should be treated adequately and rigorously, especially in patients with lower meld scores. renal failure is an established risk factor for impaired patient outcome after orthotopic liver transplantation (olt). as the endothelin pathway is known to be involved in the development of acute renal failure (arf), we designed a study to clarify its role in arf following olt. 20 consecutive patients with intact kidney function scheduled for their first olt were prospectively studied. plasma big endothelin-1 (et-1) levels were measured before surgery, after graft reperfusion, and on the first and second postoperative days. according to postoperative gfr, patients were assigned to the acute renal dysfunction group (ardf) and the non-ardf group. each patient's gfr was estimated according to the four variable formula used in the modification of diet in renal disease before surgery, daily within the first postoperative week, and at 1, 3, 12 and 24 months after surgery. postoperative mean big et-1 lev-els correlated significantly with the maximum percent decrease of gfr within 3 days after olt (p < 0.01). the proportion of patients who developed ardf was significantly correlated to mean postoperative big et-1 quartiles (p < 0.01). in the ardf group, the percent decrease of gfr within 24 months was significantly higher (p < 0.05) as compared to the non-ardf group. in conclusion, patients who develop acute renal dysfunction immediately after olt do not fully recover to baseline regarding long-term kidney function. short-term gfr was significantly correlated with postoperative big et-1 plasma levels, suggesting renal dysfunction is mediated by the activated endothelin system. background. with improved survival of liver transplant recipients, chronic renal failure has become an important cause of morbidity and is associated with a high mortality. serum creatinine is widely used as marker for renal function, but it depends on various nonrenal factors and major changes will occur late in the course of progressive renal impairment. we evaluated cystatin c and urine microscopy for detection of renal dysfunction in patients after liver transplantation. methods. from november 2003, 70 liver transplant recipients at various intervals from liver transplantation were included to our follow-up. every three months we investigated serum creatinine, urea, renal creatinine clearance and cystatin c as marker for renal function. furthermore urinary sediment was examined by urinary test, automated urinary sediment analyser, and urine microscopy. in patients with reference to renal deterioration we tried to decrease immunosuppressive therapy, to optimize the blood pressure, and to discontinue nephrotoxic medication. infections were detected early by urine microscopy and treated, even when there was no clinical appearance and the urinary test was negative. results. the results of our study showed that concerning the renal function, cystatin c is more sensitive than creatinine and creatinine clearance. the microscopy of the urine sediment showed the highest sensitivity compared with the other methods. concerning damages of the kidney, urine microscopy offered the best possibility to identify the etiology. during the follow-up and after adequate and early therapy, fifteen patients (21.4%) showed an amelioration of renal function after a few months. in 3 patients (4.2%) there was a marked deterioration. two of them had to receive a higher dose of immunosuppressive therapy and one had an infection which was treated with nephrotoxic medication. conclusions. the early identification of renal failure and its etiology are necessary in patients after liver transplantation. the results of our study confirmed cystatin c as early prognostic marker for patients with renal dysfunction. in combination with urine microscopy, renal dysfunction could be detected in time and renal function could be protected with an adequate therapy. background. hcv-infected patients and their grafts have short-term survival rates similiar to other indications. recent data on long-term outcome are contradictory, showing a trend towards poorer outcome in patients transplanted for hcv cirrhosis. in this study we present a retrospective analysis of our experience with patients who underwent liver transplantation (lt) due to hcv-associated end stage liver disease. methods. patients' charts were reviewed for survival, histologically defined hcv recurrence, genotype, presence of cirrhosis, donor and recipient age as well as type of immunosuppression (cyclosporine and tacrolimus; azathioprine and mycophenolate mofetil). survival was analysed by the kaplan-meier procedure, the influence of baseline variables was analysed by binary logistic regression. results. between 1986 and october 2004, 162 patients were transplanted for hcv-related cirrhosis. ten pts. received one and 3 pts. two relts. in 3 (23%) pts. recurrent hepatits c was the cause for relt, in 10 vascular and/or biliary complications. hepatitis b coinfection was present in 6 patients. median follow-up was 44 months (range, 0.6-221). the overall, 1-, 2-, 3-, 5-, and 10-year survival rates were 86%, 81%, 78%, 71%, and 59%, respectively, which were comparable to other indications. the probability of recurrent hcv was 34%, 51%, 62%, 70%, and 83% after 1, 2, 3, 5, and 10 years, respectively, post lt. nine (6.6%) pts. developed cirrhosis after a median of 50 months (28-116). hcv recurrence did not negatively influence patient and graft survival. concerning genotype, cmv status, presence of hcc before lt, rejection episodes, immunosuppression, donor and recipient age only immunosuppression had a significant effect on survival. in cyclosporine-treated patients (lt after 1995) 1-, 2-, and 5-year survival rates were 79%, 72%, 64% compared to 93%, 91%, 77%, respectively, for tacrolimus-based regimens (p = 0.04, log rank test). conclusions. on the basis of our data, the overall survival of hcv transplanted patients were similiar to other indications. recurrent hcv infection did not influence patient and graft survival. immunosuppression may have an impact on survival in hcv-positive recipients, but optimal regimens need to be better defined by prospective studies. the advent of highly sensitive molecular techniques has revealed the possible persistence of hepatitis b virus (hbv) genomes in hbsag-negative patients with or without serologic markers of previous infection, a state called occult hbv infection. the highest prevalence of such infection has been shown in patients infected with hepatitis c virus (hcv). some studies suggested that occult hbv infection might favor or accelerate the progression of hcv infection towards cirrhosis. hcv infection recurs almost in all patients after liver transplantation (lt). about 5-10% of lt patients develop a fibrosing cholestatic hcv recurrence, which is associated with a very poor outcome. no specific risk factor for this pattern of recurrence has been described so far. the aim of this study was to determine the prevalence of occult hbv infection in patients presenting with fibrosing cholestatic hcv recurrence after lt. between 1986 and 2004, 151 hcv patients (104 m; 47 f) underwent lt at our institution. the mean follow-up was 51 months (range, 1-228 months). the diagnosis of recurrence was based on biochemical and histologic parameters. genotype 1 was predominant (75%), followed by 2 (15%), 3 (8%), and 4 (3%). eleven patients (7.3%; 10 m, 1 f) developed a fibrosing cholestatic pattern of recurrence characterized by highly elevated cholestatic parameters and typical histologic findings. also in this group, genotype 1 was predominant (n = 7), three had type 3, and one type 4. serum hbv dna was tested with the taqman test (roche, austria). five patients were hbsag negative, whereas six had serologic markers (antihbc positive). interestingly, hbv dna could not be detected in the sera of any of these patients with fibrosing cholestatic hcv recurrence. the actuarial 1-, 2-, 5-, and 10-year survival rates of all hcv patients were 83%, 79%, 68%, and 60%. hcv recurrence did not show a negative impact on patient and graft survival; however, the outcome of the patients with the fibrosing cholestatic pattern was less favorable. seven out of 11 patients died due to hcv recurrence, one patient had to be retransplanted secondary to recurrent disease. only three patients are alive with a functioning first allograft. this study indicates that occult hbv infection is not associated with fibrosing cholestatic hepatitis c recurrence after lt. background. the use of grafts from hepatitis b core antibody (anti-hbcab)-positive donors for liver transplantation (lt) is associated with the risk of de novo hepatitis b. patients who test positive for anti-hbcab pretransplant are also theoretically at risk to develop graft hepatitis b. methods. the outcome of 467 consecutive lts performed in 402 individuals between 1998 and 2001 was retrospectively analyzed with regard to the presence of anti-hbcab in donors and recipients. patients with hepatitis b and recipients of known anti-hbcab-positive grafts received hbig/lamivudine prophylaxis. results. a total of 111 recipients (28%) tested positive for anti-hbcab including 24 patients (7%) with hbv-associated cirrhosis and three patients with fulminant hepatitis b. a total of 20 allografts from anti-hbcab-positive donors were utilized, 14 of those (70%) were allocated to anti-hbcab-positive recipients. anti-hbcab-positive recipients were significantly more likely to have concomitant hcv (62% vs. 34%, p < 0.0001) and hepatocellular carcinoma (30% vs. 14%, p < 0.002). anti-hbcab-positive donors were more frequently non-caucasian (60 vs. 24%, p < 0.001) and cmv seropositive (85% vs. 65%, p < 0.035). survival of anti-hbcab-positive individuals and recipients of allografts from anti-hbcab-positive donors did not differ from their anti-hbcab-negative counterparts. there were no reported cases of recurrent hepatitis b in anti-hbcab recipients or patients receiving lt for hbv-associated liver disease. three cases of de novo acute posttransplant hepatitis b were identified, one being acquired during unprotected intercourse and two being transmitted through the graft. the two with graft-transmitted disease were anti-hbcab negative, treated initially with lamivudine and were switched to adefovir due to the emergence of ymdd mutants. conclusions. the frequency of anti-hbcab-positive recipients in our series was higher than expected. these individuals seem at minimal risk for posttransplant hepatitis b. recurrence of hbv after lt in the setting of hbig/lamivudine prophylaxis is extremely rare with 5-year median follow-up. the risk of transmission of hbv through anti-hbcab-positive livers despite prophylaxis cannot be neglected and the emergence of an ymdd mutant is of particular concern. anti-hbcab-positive grafts should be preferably given to anti-hbcab-positive recipients. v36 the response to preoperative transarterial chemoembolisation predicts outcome of patients with hepatocellular carcinoma after liver transplantation division of gastroenterology and hepatology, department of internal medicine, medical unversity of innsbruck, innsbruck, austria liver transplantation (lt) is the only curative therapy for patients with early-stage hepatocellular carcinoma (hcc). the impact of prelt chemoembolisation (ce) on patient survival and incidence of hcc recurrence has been controversially discussed and remains undetermined. the aim of this study was to evaluate the efficacy of ce prior to lt in hcc patients with regard to tumor recurrence and patient outcome. between 1984 and 2004, 167 hcc patients (142 m; 25 f) underwent lt at our institution. the underlying liver disease was viral hepatitis in 87 (hcv 69, hbv 18), alcoholic liver cirrhosis in 49, and other diseases in 18 patients. according to child-pugh classification, 75 patients presented with child a, 79 with stage b and 9 with stage c. hccs were diagnosed according to the easl guidelines. on the basis of prelt radiology, 23 patients were diagnosed with stage i, 79 stage ii, 34 stage iii, and 31 stage iv according to the modified uicc criteria. ce was performed in 120 patients prior to lt. in 47 patients no treatment was performed prior to lt. patients underwent multiple cycles of ce (mean, 1.6 ce/patient). ce response prior to ce was assessed by ct scan. on explant histology, complete response with no evidence of vital tumor was found in 38 of 120 (32%) patients, 55 (46%) patients showed a partial remission (tumor necrosis, >50%), and 27 (22%) patients showed a poor response or even progression. the intention-totreat analysis showed that patients with early-stage hcc showed an excellent survival with a 1-, 5-, and 10-year rate of 98%, 69% and 69%, respectively. ce prior to lt had no positive effect on overall patient outcome and rate of recurrence. however, patients with complete response to ce, on the basis of both pre lt and post lt histology, had a significantly bet-ter long-term survival (1-, 5-, and 10-year rates of 98%, 91%, and 91%) and rate of recurrence compared to those with partial or no response, but only in patients within milan and not san francisco criteria. the 1-, 5-, and 10-years survival rates of patients with advanced hcc were 95%, 67%, and 35%. hcc recurrence was found in 24 patients, 11 of 24 presented with advanced stage (iii and iv). only 13 had undergone ce prior to lt, and 9 of those were nonresponders to ce. this study indicates that response to prelt ce may predict the outcome of hcc patients after lt. patients with early-stage hcc, who responded to ce pre lt, showed an excellent outcome with 5-and 10-year survival rates around 90%. patients with early-stage hcc and only partial or no response to ce had a higher risk of recurrence of hcc after lt, but outcome was still favorable compared with advanced-stage tumors regardless of ce response. the roles of the regenerative factors hepatocyte growth factor (hgf), transforming growth factor a (tgf-a), and of vascular endothelial growth factor (vegf) were described in the context of hypertrophy and regeneration after liver resection but not well known in the transplantation situation. in the recipients of 63 consecutive liver transplantations with a graft survival of >2 weeks, the factors hgf, tgf-a and vegf were determined postoperatively (day 1, 3, 5, 7, 10, 14) by an el-isa in the serum and correlated to graft survival (kaplan-meier). the median concentrations of hgf were constant in total during the observation period (day 1, 2591 pg/ml; day 7, 2434 pg/ml; day 14, 2490 pg/ml). an individual increase to levels above 4000 pg/ml in the middle of the observation period correlated to a significantly decreased one-year graft survival (54% vs. 85%). similar was the course of tgfa. an increase from the median concentration of 39 pg/ml to levels above 80 pg/ml was observed in the context of a decreased primary function. regarding vegf, an almost linear increase of the concentration from 60 pg/ml via 177 pg/ml to 424 pg/ml (day 1, 7, and 14) was observed. here it became obvious, that an extensive increase of the vegf concentration correlated to a good transplant function. under the premise that the systemically determined concentrations were in relevant correlation to the secretion and thus to the local concentration in the liver, it can be concluded that vascular regeneration induced by vegf substantially contributes to graft survival, whereas a temporal increase of hgf and tgfa rather has to be interpreted as an indicator of an injured graft with a decreased functional prognosis. in total there are about 1650 patients on dialysis in bh and percentage of transplanted is 9.24. the aim of the paper is to analyse survival of grafts, patients, and grafts and patients over a 5-year period of living-related kidney transplantation history at university medical center tuzla. the results obtained were compared with the results of the centers with great number of transplants. methods. recipients and donors as family members were admitted after informative discussions and after the test results had been obtained from primary health care laboratories. the protocol was the standard one, in accordance with the recommendations of esot and eurotransplant. donors were tested first and then recipients. lumbotomy was done for nephrectomy. colins solution was used for kidney rinsing and perfusion, reconstruction of blood vessels was done as well as kidney biopsy. then, kidneys were stored at +4°c and grafts implantations were done on the contralateral iliac fossa. terminolateral anastomosis between external iliac artery and vein was done. implantation of ureters was done by modified lich-gregore technic, with or without dj stent. all patients received basic immunosuppressive protocol. the peculiarity was introduction of basiliximab (simulect) in therapy on the first and fourth postoperative day. descriptive statistics was done using spss software for windows 8.0. survival is presented by kaplan-meier curve. results are shown as means with standard deviations (sd). results. the first living-related kidney transplantation was done at university medical center tuzla on 15. 09.1999 09. . until 15.09.2004 . as many as 52 transplants have been done. donors were related to recipients as follows (in parentheses, mean age [years] with sd): mother, n = 24 (55.0 ± 11.3); father, n = 15 (61.4 ± 6.9); sister, 5 (44.5 ± 6.4); brother, n = 6 (46.0 ± 2.9); others, n = 2 (38.5 ± 5.5). table 1 shows mean dtpa clearance rate in donors. average separate dtpa clearance rates were within normal limits. as a rule, left-side donor nephrectomy was done; and in five cases, right-side nephrectomy. three donors showed borderline dtpa clearance rates of about 40 ml/min. bladder neck sclerosis was found in one patient and the air expansion of the bladder had to be done in the other one, at least to achieve its minimal capacity. two renal arteries were found in 5 patients (3 mothers, 1 father, 1 sister), two arteries were found with left kidney transplantation. termino-lateral anastomosis was done in 2 cases. the average age of the donors was 51.08 ± 6.58 years. as many as 16 donors were over 60 years old in our transplants, which reflected in our results. the average donor age of 36 males and 16 females was 32.4 ± 8.1 years. the data on hypertension before transplants were not reliable. the average hemodialysis time was 21.48 ± 10.36 months. the most common recipients' diseases were chronic glomerulonephritis, pyelonephritis, interstitial nephritis, nondefined renal disease, diabetes mellitus, systemic lupus, vesico-ureteral reflux. there were only 8 biopsies done before transplantation so that their histological background was known. the average serum creatinine after 5 years is 153.56 ± 24.65 µmol/l. cumulative graft survival after 5 years is shown by kaplan-meier curve: graft survival, 75.1%; patients survival, 83.9%; graft and patients survival, 67.1%. conclusions. the results on 5year living-related kidney transplantation at university medical center tuzla, bosnia and herzegovina, are similar or identical to the results of the developed centers in the world. the existing program has got to be improved, especially with respect to the donor selection from the standpoint of biological and chronological age. the experience gained so far is the basis for the development of cadaveric kidney transplantation in bosnia and herzegovina. background. shortage of available organs has increased the demand for living kidney donation. whereas donation for relatives is well accepted, there remains some controversy in the setting of emotionally related donation. there might be a survival benefit for grafts donated by relatives due to better hla matching. a retrospective analysis of 162 living donor kidney transplants which were performed at the university hospital of innsbruck was made. we divided the transplants in two groups according to the date of transplant: group 1 (1975-1993), 41 transplants; group 2 (1994-2005) , 89 transplants. the time period from 1994 until 2004 was analyzed in detail and data additionally compared to a cohort of 940 cadaveric renal transplants. during these eleven years, five liv-ing donations were carried out in patients who were not eligible to receive a cadaveric graft within eurotransplant. results. overall there were 128 lrt (53 siblings, 70 parents, 5 off-springs) and 34 ert (22 spouses, 12 others). mean donor age was 45 years (range, 21-70); mean recipient age was 40 (range, 2-72) years. in group 1 only 2 transplants were emotionally related (5%), whereas in group 2 the proportion increased to 25%. graft survival of living donated kidneys was better when compared with cadaveric kidneys (95% versus 92% at one and 89% and 83% at five years), but the difference did not reach statistical significance (p = 0.06). lrts and erts produced equal outcome. overall graft loss rate after a median follow-up of six (range, 0.5-11.5) years was 11% (lrts) vs. 16% (erts). the rejection rate was slightly higher in the lrt group with 31% vs. 22% (p > 0.05, n.s.). ten living donated grafts were lost and seven recipients of a living donated graft died. causes of death were cardiac ischemia (n = 1), pulmonary embolism (n = 1), fungal infections (n = 1), suicide (n = 1), and 3 causes were not specified. conclusions. the frequency of living-related and unrelated kidney donation has increased during the past two decades at our institution. equal results for cadaveric grafts were achieved when compared to lrt and erl. background. the 20s proteasome plays an important role in the nonlysosomal pathway of intracellular protein degradation and apoptosis, thus being a possible marker for ischemic and reperfusion injuries. the aim of this study was to monitor the proteasome levels in patients receiving kidney transplants to detect a relationship with the return of renal function. methods. we examined 12 patients with end stage renal disease, receiving kidney transplants: blood samples were collected intraoperatively and postoperatively on 5 consecutive days and 20s proteasome levels were measured for each sample with a sandwich elisa as described by dutaud et al. anesthesia and immunosuppressive medications were standardized, creatinine clearance and urine output were assessed daily on a routine basis. results. patients who had no adequate urine output (430 ± 300 ml) after the 4th postoperative day had significantly higher proteasome levels intraoperatively than patients with high urine output (4032 ± 1076 ml; 1.7 ± 1.5 µg/ml low vs. 0.5 ± 0.4 µg/ml high urine output, means with standard deviations, p = 0.02). this difference in proteasome levels seemed to even out during the follow-up period. conclusions. patients with impaired renal function after kidney transplant had significantly higher proteasome levels intraoperatively. a higher plasma level of proteasomes intraoperatively may therefore be a negative prognostic marker for postoperative return of renal function of the transplanted kidney. background. dendritic cells (dcs) are the most potent antigen-presenting cells and are pivotal for initiating allograft immunity. recently, however, particular dc subsets have also been implicated in allogeneic tolerance induction. campath-1h (alemtuzumab) is a novel t-cell-depleting antibody that is currently under investigation for the use in allogeneic organ transplantation and may confer tolerogenic properties. here we study the effect of alemtuzumab on peripheral dc subsets in kidney transplant patients under fk506 monotherapy in comparison to patients under conventional triple therapy. methods. patients receiving their first renal allograft were recruited within the tacam trial and randomly assigned to receive either alemtuzumab as induction agent followed by fk506 monotherapy (n = 7) or to receive conventional immunosupression consisting of fk506, mycophenolate mofetil and steroids (n = 7). absolute numbers of peripheral blood dcs and their different subpopulations were assessed by four-colour, single-platform fluorocytometry at the day before and 1, 4, 12, and 24 weeks after the transplant procedure, respectively. peripheral dcs were identified as hla-dr + and lineage-negative cell population. the respective dc subpopulations were cd11c + dcs (myeloid dcs or dc1), cd123 + dcs (plasmacytoid dcs or dc2), cd11c + and further, bdca1 + and bdca3 + dcs. results. induction with campath-1h led to a strong and sustained reduction of the total number of peripheral dcs compared to controls. while the absolute number of peripheral dcs in control patients recovered within 6 months after transplantation, campath-1h-treated patients exhibited a profound reduction of their circulating dcs. interestingly, a prominent shift of the ratio of myeloid to plasmacytoid dc subsets (dc1/dc2 ratio) was observed as early as one month after transplantation in campath-1h-treated patients. conclusions. employment of campath-1h as induction therapy in renal transplant patients is associated with a peculiar alteration of the peripheral dc repertoire. since plasmacytoid dcs have been linked to tolerance induction, the presented data suggest that the use of campath-1h in solidorgan transplantation creates an opportunity to safely introduce novel strategies to achieve alloantigen-specific hyporesponsiveness. background. detection of c4 complement split product c4d in peritubular capillaries represents a valuable diagnostic marker for antibody-mediated rejection (amr). numerous studies have demonstrated inferior allograft function and survival in c4d-positive as compared with c4d-negative kidney allograft recipients. however, anecdotal data implicate that in selected cases stable long-term function can be maintained despite detectable c4d deposits. as recently dicussed in the literature, c4d deposits could also reflect a state of graft acceptance (accommodation), rather than rejection. aim. the objective of this study was to investigate individual clinical outcomes in a large cohort of c4d-positive kidney transplant recipients. our emphasis was thereby to identify and characterize a subpopulation of c4d-positive recipients with only mild graft dysfunction and stable long-term graft function. methods. this retrospective analysis focused on 74 out of 878 adult kidney transplant recipients (transplantation between between 1999 and 2003) included on the basis of a positive c4d result early after transplantation. results. at the time of c4d-positive biopsy (median, 12 days post-tx) median serum creatinine was 6 mg/dl (range, 1.1 to 7 mg/dl) with 49% of the patients dialysis-dependent. according to our local standard, patients with severe graft dysfunction (n = 15) were subject to immunoadsorption treatment (ia). in another 20 highly sensitized recipients, c4d-positive graft dysfunction was diagnosed during or after pre-emptive peritransplant ia. furthermore, intense treatment included antilymphocyte antibody therapy (32%) and/or high-dose steroids in a high proportion of c4d-positive recipients (46%). analyzing all 74 c4d-positive recipients, 1-month post-biopsy se-rum creatinine was 2.1 mg/dl (30% of the patients dialysis-dependent). 1-year graft and patient survival was 73% (serum creatinine, 1.84 mg/dl). in a subsequent subanalysis we focused on twelve patients (two were biopsied under ia/atg induction) with only mild to moderate graft dysfunction (serum creatinine, 1.4 to 2.45 mg/dl) at the time of c4d-positive biopsy (banff borderline lesion in five, and banff i rejection in two biopsies). within this patient subgroup, we were able to identify five recipients in whom stable long-term graft function could be achieved following steroid bolus therapy only, without further therapeutic measures. in this particular subgroup excellent 1-year allograft function (serum creatinine levels between 1.0 to 1.8 mg/dl) was observed. conclusions. our results demonstrate that in the majority of patients peritubular capillary c4d deposits are associated with severe graft dysfunction necessitating aggressive treatment. nevertheless, in a small subgroup of recipients stable graft function for a long period of time can be achieved without intense therapy despite capillary c4d deposition in biopsy. background. combined kidney pancreas transplantation (ptx) evolved as excellent treatment for diabetic nephropathy with infections remaining common and serious complications. methods. 217 consecutive enteric drained ptxs performed from 1997 to 2004 were retrospectively analyzed with regard to bloodstream infection. immunosuppression consisted of antithymocyteglobuline induction, tacrolimus, mycophenolic acid, and steroids for the majority of cases. standard perioperative antimicrobial prophylaxis consisted of pipercillin/tazobactam in combination with ciprofloxacin and fluconazole. results. one-year patient, pancreas and kidney graft survival were 96.4%, 88.5%, and 94.8%, surgical complication rate was 35%, rejection rate 30%, and rate of infection 59%. in total, 46 sepsis episodes were diagnosed in 35 patients (16%) with a median onset on day 12 (range, 1-45) post transplant. sepsis source was intra-abdominal infection (iai) (n = 21), a contaminated central venous line (n = 10), wound infection (n = 5), urinary tract infection (n = 2), and graft transmitted (n = 2). nine patients (4%) experienced multiple sepsis episodes. overall, 65 pathogens (iai sepsis, 39; line sepsis, 15; others, 11) were isolated from blood. gram-positive cocci accounted for 50 isolates (77%): coagulase negative staphylococci (n = 28 [43%]) (nine multiresistant), staphylococcus aureus (n = 11 [17%]) (four multiresistant), enterococci (n = 9 [14%]) (one e. faecium). gram-negative rods were cultured in twelve cases (18%). patients with blood borne infection had a two-year pancreas graft survival of 76.5% versus 89.4% for those without sepsis (p = 0.036), patient survival was not affected. conclusions. sepsis remains a serious complication after ptx with significantly reduced graft but not patient survival. the most common source is iai. background. mobilized allogeneic peripheral blood stem cells (pbsc) are increasingly used as graft source instead of bone marrow. although the short-term safety profile of recombinant human granulocyte colony-stimulated factor (rhg-csf) seems acceptable, minimal data exist regarding long-term safety. methods. we reviewed data of 196 allogeneic pbsc donors (siblings, n = 159; unrelated donors, n = 37) with respect to side effects of rhg-csf administration, adverse events of leukapheresis and late effects. written informed consent was signed before pbsc mobilization and collection. donors (m/f, 119/77) had a median age of 40 years (range, 11-72). they received rhg-csf at a dose of 2 times 5 µg/kg of body weight a day for 4 consecutive days starting on day 1. routinely, pbsc collection was started on day 5. on condition that donors' white cell count exceeded 70.000/µl or cd34-positive cells exceeded 50/µl, pbsc harvest was started on day 4. rhg-csf was administered until end of the apheresis period unless white blood cell count did not exceed 70.000/µl. pbsc were collected with a continuous-flow blood cell separator processing 3-3.5 times total blood volume. citrate was used as anticoagulation and in the majority of donors a continuous calcium infusion (2.25 mmol [89.4 mg] of calcium per h) was given. reinfusion of autologous platelet-rich plasma from pbsc collection was performed in donors with post-donation thrombocytopenia of <100 g/liter if further collection were necessary or in donors with platelet counts of <80 g/liter, respectively. pbsc harvest procedures were repeated until the predicted cd34 + cell yield of 4 × 10 6 /kg of body weight of recipient was collected. however, not more than 3 consecutive collections were performed. flowcytometric analyses were performed using a becton-dickinson facs-scan or facs-calibur, respectively. for follow-up we assessed peripheral blood counts, electrolytes, lactic dehydrogenase (ldh), alkaline phosphatase (ap), total protein, albumin, on days 1, 7, 30, 100, 365 and then yearly for 5 years. donors received a questionnaire for evaluation of medical history and quality of life, results and evaluation are pending. results. the main adverse events related to rhg-csf administration were bone pain (63 of 196, 32%), myalgia (61 of 196, 31%), headache (44 of 196, 22%), fatigue (36 of 196, 18%), sleep disturbance (30 of 196, 15%) and were rated as moderate to severe by 35% of the donors. due to continuous calcium infusion, incidence of citrate toxcicity was low (34 of 196, 17%) and consisted only of mild paraesthesia. in 10 of 196 (5%) donors post donation platelet count decreased below threshold and required reinfusion of autologous rich plasma. eighty-eight of 196 donors (44%) were lost for follow-up. eighty-two were sibling (82 of 159, 52%) and 6 (6 of 37, 16%) were matched unrelated donors, respectively. from the remaining 108 donors, unrelated donors (31 of 37, 84%) had a median of 4 check-ups (range, 1-8) during the first 100 days (median; range, 1 day to 3 years) after donation, whereas siblings (77 of 159, 48%) had a median of 2 check-ups (range, 1-6) during the first 30 days (median; range, 1 day to 5 years), respectively. two donors (1%), both siblings, were hospitalized within 2 weeks after donation due to severe enteritis and subarachnoidal haemorrhage. the latter donor never had platelet counts below 100 g/liter after pbsc donation and recovered without neurological deficit. follow-up of peripheral blood counts showed a loss of platelets during donation and early post-donation period and a decrease of white blood cells 7 days after donation, both returning to normal within 30 days after, respectively. ldh and ap showed a significant increase during pbsc mobilization, they returned to normal within 1 week after donation, other blood parameters remained unaffected. conclusions. due to the fact that we observed hospitalization of 2 donors within 14 days after pbsc collection, a close monitoring of donors in the early post-donation period seems advisable. although reported anomalies in medical history of donors beyond 30 days after pbsc harvest could not be associated with rhg-csf administration, a regular followup for at least 5 years should be considered. with the particular focus on donor safety, a standardized approach to data collection of follow-ups to monitor short-and long-term effects in all centers should be established. background. donor-recipient sex mismatch is an established risk factor for poor outcome after allogeneic myeloablative hematopoietic stem cell transplantation (hsct). the risk of transplant-related mortality (trm) due to graft-versushost disease (gvhd) is higher in male recipients of female stem cells compared with female patients receiving a graft from a female donor. with longer follow-up, however, the graft-versus-leukemia (gvl) effect due to hy minor histocompatibility antigen mismatch may predominate. the contribution of donor-patient sex on outcome after nonmyeloablative hsct, however, has not been examined in detail yet. methods. we therefore analyzed a single-center cohort of 72 high-risk patients transplanted with a related or unrelated stem cell graft after nonmyeloablative conditioning for outcome (acute and chronic gvhd, trm, relapse, and survival). results. of the 72 patients, 19 male patients received a graft from a female donor, 21 males a graft from a male donor. sixteen female donors were transplanted with a male donor and 16 with a female donor. around 30% of the patients with a sex-mismatched donor received stem cells with an hla mismatch compared to 10% of the patients without sex-mis-stammzellen matched donor, the other clinical differences were similar between all groups. the highest cumulative incidence for acute and chronic gvhd was detected in male patients receiving a stem cell graft from a male donor (52.4% and 53.3%, respectively). the highest relapse incidence (55.6%) was detected in male patients transplanted with a female donor. this was borderline significant (p = 0.0845) to female patients receiving a female graft (20% relapse incidence) and argues against an effective anti-hy response in this patient cohort. the mean cumulative incidence for trm was 57.3%; however, female recipients receiving a graft from a female donor displayed an unexpectedly high incidence for trm (87.5%) which could not be explained by clinical characteristics. the overall survival of 41% 2.5 years after transplant in this group, however, was not different from male patients receiving a female graft (43.7% at 1.4 years after transplant). the overall survival from male patients with a male donor was slightly lower (33% at 2.5 years after transplant) compared with female patients transplanted with male stem cells (47.4% at 1.4 years after transplant). conclusions. these data, analyzed in a small cohort of patients, show that a sex mismatch between patient and donor may have a negative impact also on outcome after nonmyeloablative hsct. however, studies with larger and homogeneous cohorts have to be performed to prove these findings. between 1995 and 2004, 20 patients with chronic lymphoid leukemia (cll), binet stage b or c (n = 19) or a with risk factors (n = 1) with a median age of 53 (range, 27-62) years underwent autologous (n = 11) or allogeneic (n = 9) hematopoietic stem cell transplantation (hsct) at the medical university of vienna. the median time from diagnosis to hsct was 29 (range, 6-77) months. eleven patients underwent autologous stem cell transplantation (asct) in partial remission (n = 9) or complete remission (cr) (n = 2) and received bcell-depleted peripheral blood stem cell (pbsc) grafts. nine patients with refractory disease (n = 6) or chemosensitive relapse (n = 3) underwent allogeneic hsct with unmanipulated bone marrow (n = 3) or pbsc (n = 6) from sibling (n = 8) or unrelated (n = 1) donors. in the majority, conditioning therapy consisted of total-body irradiation (tbi) of 12-13.2 gy and cyclophosphamide. three patients underwent reduced-intensity conditioning (ric) with fludarabine and tbi of 2 gy. graft-versus-host disease (gvhd) prophylaxis consisted mainly of cyclosporine (csa) and methotrexate for myeloablative and csa and mycophenolate mofetil for ric-hsct. complete clinical remission was attained in 10 patients (91%) after asct and in 6 (67%) after allogeneic hsct. molecular remission assessed by immunoglobulin heavy chain gene (igh) rearrangement pcr was attained in 8 patients after asct and 5 after allogeneic hsct. in seven patients we noticed persistence of the igh rearrangement, six of these patients died of disease progression or relapse 1-29 months after asct (n = 2) or allogeneic hsct (n = 4). after a median follow-up of 58 (range, 21-122) months, nine autologous (82%) and four allogeneic (44%) graft recipients are alive and 10 patients (asct, n = 6; allogeneic hsct, n = 4) are in clinical and molecular remission. the median time to clinical relapse was 20 (range, 1-102) months. treatment-related death occurred only in one patient 27 months after myeloablative hsct. probability of overall survival is 82% after asct and 42% after allogeneic hsct. in summary, all cll patients with long-term cr after asct and allogeneic hsct also attained sustained molecular remission of the igh rearrangement. . the incidences of the observed genotypes in this small group of patients and donors tested were in the range as published: il10 with 54% c/c, 41% c/a, 5% a/a; nod2 with 12% recipient or donor; 5% recipient and donor, and mpo with 56% g/g, 43% g/a, 1% a/a. since the patient population was very heterogeneous concerning diagnosis (34 aml, 12 all, 9 nhl, 6 cml, 5 mds, 4 saa, 4 solid tumors, 3 mm, 2 omf, 1 cll, and 1 et), course of disease, and condition regimen, patients were divided into four groups for evaluation: 23 with aml and identical high-dose induction therapy (bu/cy), 23 with reduced condition regimen, 6 with unrelated donors, and 29 others. concerning all 81 patients, 19 of them (23%) died through trm (3 aml, 6 reduced, 10 others), but none of them was at high risk determined by nod2 polymorphism (mutated donor and recipient) and only one determined by mpo a/a genotype (aml). four patients (3 reduced, 1 others) with nod2 mutations in donor and recipient dna did not die from trm. twenty-three patients developed severe (grade 3 or 4) acute gvhd (7 aml, 5 reduced, 11 others), 10 of them had il10 c/c genotype (5 aml, 2 reduced, 3 others) and in three patients nod2 was mutated in donor and/or recipient dna (1 aml, 2 others). on the other hand, 34 patients with il10 c/c genotype and 13 patients with nod2 mutations in donor and/or recipient dna developed no or mild gvhd only. in conclusion, so far we were not able to find a correlation between gvhd/trm risks and polymorphisms of il10, nod2, and mpo. this might be due to the small number and the heterogeneity of the patients; however, a panel of additional snps may increase the accuracy of risk assessment prior to allogeneic sct. background. allogeneic stem cell transplantation is a curative therapy for patients with lymphoproliferative disorders as a result of the intensity of the conditioning regimen and the application of a graft-versus-lymphoma effect. however, conventional conditioning regimens have been associated with a 24-61% risk of transplant-related mortality (trm) in advanced hodgkin's lymphoma (hl). in an attempt to reduce the high trm reported with allografting in lymphoma, reduced-intensity regimens have been investigated. methods. four patients between the age of 34 and 44 years underwent allogeneic peripheral blood stem cell (pbsc) transplantation (sct) from hla-identical sibling or unrelated donors at our institution. age, sex, manifestation of disease, and donor were as follows: patient 1-37 years, female, pulmonary bulk, sibling; patient 2-34 years, male, pulmonary bulk, unrelated donor; patient 3-42 years, male, pulmonary bulk, sibling; patient 4-44 years, male, abdominal bulk, sibling. all patients had received multiple courses of polychemotherapy (table 1 ) and local radiotherapy prior to sct. patients 1 and 4 had undergone autologous stem cell transplantation. we administered a reduced-intensity conditioning consisting of beam (bcnu, etoposide, cytarabine and melphalan) over 6 days. to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with an anti-cd52 antibody (campath) at a total dose of 50 mg over 5 days and graft-versus-host disease (gvhd) prophylaxis of cyclosporine a. pbsc of donors were collected after g-csf stimulation at 10 µg/kg of body weight given for 4 days. results. patient 2 rejected his unrelated-donor graft and received an autologous stem cell infusion 44 days thereafter resulting in sustained hematologic recovery (anc, >0.5 g/l on day 10). all other patients engrafted uneventfully (anc, >0.5 g/l on days 14-16; platelets, >20 g/l on days 10-13). none of the patients showed evidence of acute or chronic gvhd. two patients achieved complete donor chimerism in myeloid and lymphoid cell populations, another one had prolonged mixed chimerism and was given a donor leukocyte infusion of 1 × 10 6 cd3-positive cells per kg. one patient experienced a cmv reactivation on day 51 after sct and a sarcoidosis on day +150. three months after sct, all patients showed marked regression of disease. after a follow-up of 5 to 11 months (median, 8 months), 2 patients experienced progression undergoing salvage chemotherapy. time to progres-sion was 5 and 6 months, respectively. two patients remained progression free for 7 and 11 months, respectively. overall survival is 5 to 11 months. conclusions. our data demonstrate that this regimen was well tolerated with a low risk of gvhd and transplant-associated morbidity. an increased dosage of campath could be considered to prevent rejection in unrelated-donor transplantation. longer follow-up and larger patient numbers are warranted for assessment of the efficacy of campath-beam with regards to durable remissions. background. as long-term survivors of hematopoietic cell transplantation (hct) become more numerous, studies addressing the issue of long-term follow-up are necessary. in this study, we report on the quality of life (qol) of hct patients who were alive at least at 5 years after transplantation in comparison with an age-and sex-matched sample of healthy controls assessed in the same time period and the same geographical region. methods. the eortc-quality of life questionnaire (eortc-qlq c30) was sent by post to 39 hct survivors. thirty-four patients answered the questionnaire. patients were compared with 68 healthy controls from the same geographical region. patients and controls completed the eortc in the same time period. results. mann-whitney u tests identified significantly lower qol on the dimensions of physical and social functioning and on the financial impact symptom scale. conclusions. patients who had survived their hct for more than 5 years did generally well in terms of global qol. this is consistent with kiss et al. (2002) who found that cml patients who were alive at least 10 years after hct report lower physical functioning in comparison with healthy controls. problems in the areas of social functioning and financial difficulty can possibly be addressed by intensive rehabilitation processes integrating patients, family members, and significant others. interdisciplinary (medical, psychological, and social) treatment of patients should not come to an end after the acute phase of the illness but should continue during checkups following transplantation. background. bone marrow transplantation (bmt) together with costimulation blockade can reliably induce mixed chimerism and tolerance in certain experimental models. natural killer t cells play an important role in the induction of tolerance in several transplantation and autoimmunity models. it has been reported, for instance, that activation of nk t cells by α-galactosylceramide (α-gal) can prevent the onset and recurrence of autoimmune type i diabetes. in recent experiments we wanted to investigate the role of nk t cells in a model of tolerance induction through bmt with costimulation blockade. to delineate the role of donor and recipient nk t cells, we used nk-t-cell-deficient mice (jα281-/-c57bl/6 and jα281-/-balb/c) as recipient and/or donor strain. we also evaluated whether in vivo stimulation of nk t cells with α-gal has a beneficial effect. methods. c57bl/6 mice and c57bl/6 jα281-/-received a total-body irradiation (tbi) of 3 gy or 1 gy (day -1), approximately 20 × 10 6 fully mismatched balb/c or balb/c jα281-/-bone marrow cells (day 0) and costimulation blockade consisting of anti-cd154 mab (mr1, day 0) and ctla4ig (day +2). groups were additionally treated with α-gal or a vehicle (5 µg on day -1, +2, +7, +14, and +21). multilinage chimerism and skin graft survival were followed for more than 120 days. results. with our standard protocol, 12 of 18 mice developed long-term multilinage chimerism and permanent donor skin graft survival. when using recipients deficient in nkt cells, 5 of 5 became chimeric and showed long-term skin graft survival; using nkt knockouts as donors 6 of 6 and using nkt knockouts both as recipients and donors 5 of 6 became chimeric (p = n.s. for all comparisons). unexpectedly, stimulation of nk t cells by α-gal (using wild-type recipients and donors) prevented chimerism induction after 3 gy tbi (0 of 8 vs. 4 of 6 and 4 of 4 in the vehicle group, p < 0.01) and did not promote engraftment after 1 gy tbi (0 of 8 vs. 0 of 8). conclusions. nk t cells do not play a critical role in tolerance induction after bmt with costimulation blockade. their stimulation with α-gal even prevents induction of mixed chimerism and tolerance. background. phospholamban (plb) is a critical regulator of sarcoplasmic reticulum ca 2+ -atpase activity and myocardial contractility. in this study we investigated the role of plb phosphorylation in ischemia and reperfusion following cardiac transplantation. methods. gene expression of plb was investigated in a syngeneic heterotopic cardiac transplant model in mice. grafts underwent 10 h of cold ischemia or were tranplanted immediately after harvest. gene expression was analysed at various time points employing dna microarray and rt-pcr. for in vitro experiments, hl-1 cardiomyocytes were submitted to a protocol of global normothermic hypoxia for 6 h and reoxygenation in the absence or presence of the ca 2+ /calmodulin kinase ii inhibitor aip (1 µm) or the beta-adrenergic blocker dl-propranolol (1 µm) vs. beta-adrenergic stimulator isoproterenol (1 µm). results. at 24 h, gene expression of plb was diminished by 14.1 and 3.6-fold in groups with and without ischemia, respectively. basal phosphorylation of plb at ser16 (protein kinase a site) and at thr17 (ca 2+ /calmodulin kinase ii site) was present in cultured cardiomyocytes and heart lysates. in the mouse system, increase in plb phosphorylation is observed during early (up to 10 min) reperfusion. thereafter, plb phosphorylation drops below that of control levels. addition of aip diminishes reperfusion-induced thr17 phosphorylation; propranolol significantly decreases ischemia-induced ser16 phosphorylation. in contrast, isoproterenol enhances plb-ser16 and thr17 phosphorylation. conclusions. ischemia regulates phospholamban by two different mechanisms, decrease in expression levels and alterations in the phosphorylation of critical regulatory sites. modulation by aip and dl-propranolol will help for investigation of the role of pbl phosphorylation in ischemia and reperfusion in cardiac transplantation in the future. background. the p38 mitogen-activated protein kinase (p38-mapk) pathway plays a crucial role in pathological events like oxidative stress, inflammation, and abnormal cellular proliferation, resulting in activation of several signalling cascades, involving overexpression of tumor necrosis factor alpha (tnf-α). tnf-α is known to play an important role in chronic rejection. currently, pharmacological inhibitors of p38-mapk are being tested clinically for the treatment of experimentelle transplantation chronic inflammatory diseases such as rheumatoid arthritis, morbus crohn and psoriasis as well as inhibition of vascular smooth muscle cell (vsmc) proliferation after balloon angioplasty. to date, there are no findings that address the role of p38-mapk in the context of chronic allograft vasculopathy, which is characterized by vascular lesions in the graft that consist of concentric myointimal proliferation, resulting in deterioration of allograft function and organ loss. the purpose of this study was to understand the role of p38-mapk in abnormal vsmc proliferation associated with chronic rejection and to investigate the potential therapeutic role of a specific inhibitor of p38-mapk activation in chronic allograft vasculopathy. methods. in vivo, a mouse model of heterotopic aorta transplantation in an allogenic setting has been used. aortas were allografted into recipient mice by a carotid artery cuff technique, using c57bl/6 (h-2 b ) mice as donors and balb/c (h-2 b ) mice as recipients. four weeks after transplantation, the aortic segments were harvested and immunofluorescence was performed using anti-vsmc-α-actin and anti-phospho-p38 antibodies. tnf-α serum levels were measured by elisa. in vitro, vsmcs were isolated from c57bl/6 aortas. expression levels of total and phosphorylated forms of p38-mapk as well as key cell cycle regulators were detected by western blot. immunocytochemistry was performed with primary antibodies directed against phospho-p38-mapk. proliferation of vsmcs was measured by [ 3 h]thymidine incorporation in the presence or absence of sb 203580, a specific inhibitor of p38-mapk activation. cell cycle progression was monitored by dna content analysis; apoptosis by the annexin v binding assay. cell lysates were probed with antibodies directed against cyclindependent kinase 2 (cdk2) and yin yang 1 (yy1). results. in vivo, 4 weeks after the transplant aortic segments were significantly narrowed due to neointimal hyperplasia. the neointimal lesions mainly consisted of vsmcs and showed profound activation of p38-mapk as demonstrated by immunofluorescence. further, serum tnf-α levels were significantly increased even 4 weeks after allogeneic aortic transplantation, suggesting an important role of p38 activation in this model. in vitro, stimulation of vsmcs with 10% fcs resulted in a rapid increase of phosphorylated forms of p38-mapk (8.0 ± 0.7 fold increase) when compared with the nonstimulated quiescent state. immunocytochemistry showed higher levels of phospho-p38-mapk in the nuclei as well as in the cytosol after stimulation. sb 203580 in a dose-dependent manner significantly inhibited vsmc proliferation, which was due to inhibition of cell cycle progression at the g 0 /g 1 phase. we did not observe apoptosis in the sb 203580-treated vsmcs at 20 µm, the highest dose being tested. blockade of p38-mapk activation decreased protein levels of cdk2 and the transcription factor yy1, which plays an important role in vsmc dna replication and protein synthesis. conclusions. p38 mapk activation appears to play an important role in an in vivo allogeneic model of aorta transplantation as well as in vsmc proliferation in vitro, blocking of which prevents the cells from entering the s phase of the cell cycle, thus abrogating cell proliferation. targeting p38-mapk might become a potent strategy in the treatment of vascular proliferative diseases like chronic allograft vasculopathy. background. ctla4ig, a costimulation blocker which is currently under advanced clinical development, has been used for years as part of mixed chimerism protocols. recent data suggest that ctla4ig also functions via modulation of tryptophan metabolism by upregulating indoleamine 2,3-dioxygenase (ido) through b7 signals. we thus investigated the role of ido in our ctla4ig-based mixed chimerism protocol. methods. c57bl/6 mice received a total body irradiation (tbi, d -1) of 3 gy, approx. 20 × 10 6 fully allogeneic balb/c bone marrow cells (d 0) and costimulation blockade consisting of anti-cd154 mab (1 mg, d 0) and ctla4ig (0.5 mg, d 2). different groups of mice were additionally implanted with 1-methyltryptophan pellets on d -1 (1-mt, which is a competitive inhibitor of ido, 7-day release at 0.9 mg/h) or on d -1 and d 6 (14-day release) or placebo pellets. macrochimerism and deletion of donor-reactive cells were followed by flow cytometry. levels of tryptophan, kynurenine, and 1-mt were measured at several timepoints in serum by hplc. results. 8 of 10 mice which received bmt, tbi, mr1 plus ctla4ig but only 2 of 10 mice without ctla4ig developed lasting multilineage chimerism (p < 0.05, measured at week 32 post bmt). 8 of 10 mice with ctla4ig treatment accepted donor skin grafts for more than 150 days, whereas only 1 of 10 mice, which got no ctla4ig injection accepted donor skin long-term (p < 0.05; representative data from two separate experiments), demonstrating that ctla4ig is critical for our protocol. 3rd-party grafts were promptly rejected in all groups. long-term multilineage chimerism developed in 6 of 8 mice with 1-mt treatment, which is not significantly different from treatment with placebo pellets or standard protocol alone (7 of 11, pooled data from both groups). 2-week treatment with 1-mt also did not lead to a significant difference in the rate of multilineage chimerism (4 of 5 with 1-mt vs. 4 of 7 without 1-mt). deletion of donor-reactive t cells was neither blocked nor enhanced by treatment with 1-mt. the kynurenine-to-tryptophan ratio in serum was similar in groups with (11.8 ± 2.7) and without (12.1 ± 3.3) ctla4ig (p = n.s., measured on d 3). substantial serum levels of 1-mt were detected in mice with 1-mt treatment but not in untreated mice, indicating that ido was indeed inhibited in the 1-mt groups. conclusions. ctla4ig plays an essential role for tolerance induction in this model but its mechanisms of action does not critically depend on ido. background. induction of antigen-specific tolerance remains the ultimate goal in clinical organ and cell transplantation, as it would eliminate the need for continuous immunosuppression. one strategy leading to tolerance induction against a transplanted organ consists of infusing blood from the organ donor, an approach known as donor-specific transfusion (dst). although the mechanisms underlying tolerance induction by dst are not fully understood, clonal deletion of alloreactive t cells and generation of immunoregulatory cd25 + cd4 + t cells are important in the process. it is well established that expression of heme oxygenase-1 (ho-1) can promote the survival of transplanted organs. however, the mechanisms underlying this effect remain to be elucidated. we hypostesized that ho-1 is required for tolerance induction involving dsts and that ho-1 can magnify the tolerogenic effect of dsts. methods. allograft survival has been tested by using a well established model of costimulatory blockade (anti-cd40l-ab) plus dst (day -7) in c57bl/6 (h-2 b ) heart allografted balb/c (h-2 d ) wt and ho-1 ko mice. further, ho-1 activity has been induced (by copp) or suppressed (by znpp) in b6af1 (h-2 k/d,b ) mice receiving dba/2 (h-2 d ) allografts plus dst on day 0 or day -7. donor-specific tolerance was tested by challenging the mice with a second dba/2 or third-party fvb (h-2 q ) allograft. leukocytes (depleted or undepleted of cd25 + cd4 + t cells) from mice carrying allografts for >100 days were adoptively transferred to sublethally irradiated b6af1 mice receiving dba2 heart allografts. rna levels of foxp3, tgf-β, il-10 and ctla-4 have been assesed by using rt-pcr. cd25 + cd4 + t cells have been enumerated by flow cytomerty. results. anti-cd40l-ab plus dst treatment resulted in balb/c recipients tolerizing fully mismatched c57bl/6 allografts (n = 6). however, by using ho-1-deficient recipients, this effect was abrogated, in that c57bl/6 allografts have been rejected in a similar manner as in untreated wt recipients (mst = 17.5 d, n = 4). further, dst plus copp (in contrast to dst plus znpp) treatment resulted in donor-specific tolerance of dba/2 allografts in b6af1 recipients (n = 7). tolerant animals showed significantly increased percentage of cd25 + cd4 + t cells and increased levels of foxp3, tgf-β, il-10, and ctla-4 mrna. adoptively transferred b6af1 leukocytes retrieved from the lymph nodes and spleens transferred to sublethally irradiated b6af1 recipients of dba2 allografts led to subsequent allograft loss (mst = 16.7 d, n = 6); in contrast, transfer of leukocytes of tolerant (copp plus dst treated) mice led to indefinite allograft survival in this model. however, when those leukocytes were depleted of cd25 + cd4 + t cells, allografts were immediately rejected (mst = 17.7 d, n = 5). conclusions. ho-1 in a graft recipient can be critical for long-term graft survival and for induction of tolerance. this mainly is mediated by generation of cd25 + cd4 + t cells (t regs). modulation of ho-1 expression and activity may be used therapeutically to promote graft tolerance. background. human immunoglobuline (ivig) has been advocated in the treatment of acute rejection in allograft transplantation and treatment of sepsis. mechanisms describing the immune modulatory activity are however scarce. we sought to investigate immune suppressive properties of pooled human immuneglobuline (ig), unspecific fc and fab fragments and their respective ligands by allogeneic blastogenesis assays. methods. human mixed lymphocyte reactions (mlr) were performed. in detail peripheral blood mononuclear cells (pbmcs) were purified from 5 donors by ficoll density gradient centrifugation (amersham biosciences, buckinghamshire, england). 100,000 cells per well were stimulated with 100,000 radiated pbmc (60 gy) and incubated for 5 days at 37°c together with unspecific igg, igm (both sigma-alderich, st. louis, mo), anti-cd32 (lab vision, fremont, ca) and anti-cd64 (chemicon, temecula, ca) in a dose-dependent fashion. before harvesting cells were pulsed for 18 h with [ 3 h]thymidine (3.7 × 10 4 bq/well) and the [ 3 h]thymidine uptake was measured in a liquid scintillation counter. results. the addition of pooled human igg and igm to allogeneic stimulated cells both resulted in a significant and dose dependent decrease of proliferation, although the suppressive properties of igm was greater as compared to igg (both, p < 0.0001). to investigate whether this effect was partly related to fc receptor involvement we blocked cd32 and cd64 on antigen presenting cells (apcs), known receptors in the activation of mlr. surprisingly, this assay demonstrated that sole fc blocking (high-and low affinity fc receptors) resulted in a significant downregulation of allogeneic response in vitro (both, p < 0.001). conclusions. our data evidence that the addition of unspecific immune globulines results in a reduction of proliferation in in vitro allogeneic blastogenesis assays and is partly fc receptor dependent. these results corroborate the clinical success of ivig and pooled igm in the treatment of solid organ allograft rejection and cautions the utilization of immune globulines in immune suppressed septic patients. die interaktion professioneller antigen-präsentierender zellen mit allogenen t-zellen resultiert in der ausbildung der sog. immunologischen synapse (is), welche für die effiziente aktivierung von t-zellen und damit letztlich für die transplantatabstoßung essentiell ist. im rahmen der is werden der t-zellrezeptor/cd3-komplex, kostimulatorische wie adhäsionsmoleküle und komponenten des zytoskeletts in die kontaktzone des mhc-tcr/cd3-komplexes der is rekrutiert. der einfluss von gängigen immunsuppressiva, die zur prävention der allogenen organabstoßung klinisch verwendet werden, auf die is-evolution ist bislang unbekannt. in dieser studie zeigen wir erstmals, dass kalzineurinhemmer wie csa oder fk506, aber nicht mtor-inhibitoren, selektiv die rekrutierung des tcr/cd3-komplexes in die is blockieren. ein ähnlicher effekt wurde für kortikosteroide, aber nicht mycophenolat beobachtet, was auf eine essentielle rolle des calcineurin-nf-κb-signalweges für die tcr/cd3-regulation im rahmen der t-zellaktivierung hinweist. interessanterweise blockierte das neue immunsuppressivum fk778 nicht nur die tcr/cd3-, sondern auch die lfa-1-und f-aktin-rekrutierung in die is. die bedeutung dieser globalen interferenz mit der is-ausbildung für die spezifische immunantwort muss in weiterführenden studien geklärt werden. diese daten zeigen, dass klassiche immunsuppressiva nicht nur simple blocker der zytokinsynthese sind, sondern schon sehr früh die t-zell-apz-interaktion stören und damit einen weiteren immunologischen mechanismus besitzen, der ihre klinische potenz erklärt. background. ischemia (i) and reperfusion (r) trigger a series of events, which culminate in severe injuries to the affected organs in organ transplantation. cell death, metabolic alterations, and inflammation result in impairment of shortand long-term function. the group of mitogen-activated protein kinases (mapks) are central regulators of these events, they have been implicated through aberrant activation in many pathophysiological settings ranging from autoimmune diseases, cancer, to i/r-associated organ damage. methods. intracellular signaling pathways were analysed in vivo and in vitro employing a cardiomyocyte cell line and a murine heart transplant model. hl-1 cardiomyocytes are a cardiac muscle cell line derived from at-1 mouse atrial cardiomyocytes. syngeneic cardiac transplants were carried out us-ing male inbred balb/c mice, hearts were transplanted heterotopically into the neck of recipients. results. in summary, (i) reoxygenation was characterized by a dramatic increase in the activity of all mapks at the end of the observation period; (ii) growth factor abrogation together with hypoxia (h) and reoxygenation (r) had a substantial effect on the course of signaling events; (iii) signaling processes in response to ischemia and reperfusion in vivo are in line with observations made in cardiomyocytes. conclusions. data obtained so far in our study demonstrate the suitability of the chosen experimental approaches for investigation of i/r-associated alterations in intracellular signaling and cellular responses. preliminary data suggest that h/r treatment of hl-1 results in significant apoptotic cell death, the intracellular signaling pathways involved are therefore currently analyzed. background. ischemia and reperfusion (i/r) in cardiac transplantation results in inflammation and cell death. to gain further insight into the regulation of these processes, we investigated the role of lipocalin-2 (24p3, ngal, uterocalin), a potential regulator of the acute inflammatory response and cellular apoptosis in vitro and in vivo. methods. c57bl/6 hearts were heterotopically transplanted to syngeneic recipients immediately and after 10 hours of prolonged cold ischemia with the grafts harvested at various timepoints (2 min, 2 h, 12 h, 24 h, 48 h, 10 d) after transplantation. gene expression analysis on mrna extracts was performed employing cdna microarray and rt-pcr. protein synthesis was investigated by western blotting and apoptosis by using tunel assay. for the identification of the cellular source of lipocalin-2 and its function in i/r-associated cell death, the effect of recombinant lipocalin-2 was investigated in the hl-1 cardiomyocyte cell line. hl-1 cardiomyocytes undergoing ischemia/reoxygenation as well as purified mononuclear cells and granulocytes were analyzed for lipocalin-2 expression by rt-pcr. results. in the cardiac transplants, high levels of lipocalin-2 gene and protein expression were detected at 12 h of reperfusion, whereby transcription was higher in groups without cold ischemia (22-versus 8.8-fold). he staining demonstrated dense mononuclear infiltrates, cellular edema, and small focal necrosis in groups with and without prolonged cold ischemia. upregulation of gene transcription was confirmed by pcr. apoptotic cells were first detectable at day 2 and peaked 10 days after transplantation. expression of lipocalin-2 was also detected in hl-1 cells by rt-pcr and western blotting following i/r, demonstrating for the first time the presence of lipocalin-2 in this cell lineage. lipocalin-2-transfected hl-1 cardiomyocytes showed a higher cell viability especially under ischemic condition. megalin, known as the lipocalin-2 receptor, was detected in hl-1 cells by rt-pcr. conclusions. this study demonstrates the time-dependent expression of lipocalin-2 in a cardiomyocyte culture as well as in transplanted hearts in response to ischemia and reperfusion/reoxygenation. lipocalin-2 is suggested to target cardiomyocytes with ameliorating effects on cell viability during ischemia. obvious alterations in lipocalin-2 expression at the protein level suggest a possible involvement of lipocalin-2 during i/r-induced cell death probably as a self-limiting process for inflammation. background. protease activation as well as inflammatory responses contribute to organ damage in response to ischemia and reperfusion. in this study we investigated the role of the protease inhibitor slpi in ischemia and cardiac transplantation. methods. hearts from slpi knockout mice (slpi-/-) were heterotopically transplanted to slpi-/-recipients. grafts underwent 10 hours of cold ischemia (ci) prior to transplantation or were transplanted immediately. c57bl/6 wild-type isografts (wt) undergoing the same procedure served as controls. in selected groups, 200 µg of recombinant slpi (rslpi) were added to the preservation solution or given i.v. after reperfusion. after evaluation of graft function, hearts were removed at 15 min, 12 h, 24 h, and 10 days. morphology was investigated by histology, slpi gene expression was analysed using quantitative rt-pcr. slpi protein expression was studied by immunohistochemistry (ihc). slpi, tnf-α, tgf-β, and nf-κb, cathepsin g, and elastase activity were analysed employing elisa and western blot. results. at 15 min, recovery of graft function was normal in wt and slpi-/-mice transplanted without ci (4.0 ± 0.0). in contrast, slpi-/-hearts transplanted after 10 h of ci showed no or marginal recovery of organ function (0.6 ± 0.65). at 24 h, cardiac function in slpi-/-(2.8 ± 0.89) was less when compared with wt (3.36 ± 0.55). single administration of rslpi i.v. had no effect; however, when slpi was added to the preservation solution, organ function comparable to wt mice was observed (3.36 ± 0.55). a mild mononuclear cell infiltrate and small focal necrosis where found in all groups at 24 h. at 10 days, postischemic inflammation as well as myocyte necrosis were significantly higher in the slpi-/group (2.5 ± 0.5 vs. 1.8 ± 0.4 and 1.6 ± 0.5 vs. 0.2 ± 0.4). myocyte vacuolisation as a sign of sublethal ischemic injury was present at high level in slpi-/-mice undergoing ci only. slpi gene expression was detected in wt mice at 12 and 24 h after reperfusion. gene transcription at 12 h was significantly higher after prolonged ci (7.99 vs. 1.57 orders of magnitude) and was associated with significantly decreased nf-κb, tgfβ, and tnf-α activity. slpi protein was first observed at 24 h, high levels of slpi protein were found at 10 days. slpi-positive cells were mainly identified as macrophages (ihc). high intragraft levels of slpi activity were found early as well as 10 days after application of recombinant protein. high slpi levels correlate with decreased cathepsin g early and decreased nf-κb, tgf-β, and elastase activity late after reperfusion. conclusion. herein we demonstrate that slpi has a substantial effect in prevention of inflammation and myocyte damage in response to ischemia and reperfusion of the heart via inhibition of nf-κb, tgf-β, and elastase. in addition, slpi seems to be crucial for recovery of organ function early after heart transplantation -inhibition of protease activity seems to be the underlying mechanism. perfusion with rslpi ex vivo represents a promising therapeutic option for modulating the destructive processes of postischemic inflammation while preserving its restorative nature. methodik. die studie wurde an 5 hausschweinen durchgeführt. die tiere wurden in allgemeinnarkose versetzt und intubiert. alle hämodynamischen daten wurden invasiv gemessen. die parameter (abp, pap, zvd und lap) wurden wie in der klinischen praxis mit einem hp-patientenmonitor registriert und gespeichert. das herzzeitvolumen (co) wurde mit hilfe eines flowmeters der firma transsonic systems inc. gemessen. die hämodynamischen ausgangswerte von 4 patienten vor ecmo wurden retrospektiv mit den experimentellen daten verglichen. ergebnisse. in der frühphase des tierversuchs nach erfolgter abklemmung blieb ein eindeutiger anstieg des pap bei 4 von 5 versuchstieren aus. auch ein rascher abfall des co war nicht zu beobachten. auch der pvr zeigte keine signifikante veränderung. die pap/ap-druckratio reagierte sehr rasch und in allen fällen mit einem anstieg auf das ereignis. von 4 patienten, die mittels ecmo erfolgreich therapiert worden waren, hatten 3 eine pap/ap-ratio über 0,3, ein patient hatte eine pap/ap-ratio von 0,26. die patienten mit pap/ap über 0,3 wurden mittels va-ecmo therapiert, der patient mit pap/ap von 0,26 mittels vv-ecmo. schlussfolgerungen. isolierte veränderungen der pulmonalen hämodynamik repräsentieren offenbar nur bedingt den schweregrad der vorhandenen beeinträchtigung. als wichtiger parameter für eine rasche einschätzung des grades der kompromittierung kann die druckratio zwischen pulmonalem und systemischem kreislauf angesehen werden. als leicht zu erhebender parameter könnte die pap/ap-ratio eine entscheidungsgrundlage für die zu wählende ecmo-konfiguration darstellen. nach den bisherigen erfahrungen wäre die indikationsgrenze bei einer pap/ap-ratio von etwa 0,3 zu ziehen. background. pancreas transplantation in the mouse is an extremely demanding procedure and severe technical problems have limited its widespread use. since the mouse, however, would be a good model for the study of various transplantation-related problems, such as ischemia-reperfusion injury or graft pancreatitis, we designed a new surgical strategy for cervical heterotopic vascularized pancreas transplantation using a cuff technique. methods. male syngeneic c57bl6 (h-2 b ) mice (n = 27) 10-to 12-week-old were used as size-matched donor and recipient pairs. recipients were intraperitoneally injected with 312.5 mg of streptozotocin per kg in order to become hyperglycemic (blood glucose, >300 mg/dl) and transplantation was performed 4 days later. recipient operation: the right external jugular vein (ejv) and common carotid artery were dissected free. by using a polyethylen cuff (od, 0.63 mm) it became possible to evert the artery over the cuff body and finally fix the vessel with 8-0 silk ligatures. similarly, the ejv could be everted over a 0.94 mm cuff. donor operation: after a complete midline incision the pancreas was isolated using a no-touch technique on a segment of the aorta, including the celiac axis and the superior mesenteric artery. the venous outflow was provided by the portal vein. all grafts were flushed with 4 °c saline solution. implantation: the graft was placed in the right cervical region and vascular anastomoses completed by pulling the pv over the ejv cuff and the donor aortic segment over the carotid cuff and held in place with a 8-0 silk ligature. after releasing venous and arterial clamps, all grafts immediately returned to their normal pink color with the arterial stump pulsating. results. out of 27 recipients, surviving over 50 days, 2 animals died from haemorrhage (survival rate, 93%). donor operation lasted 40 ± 5 min and dissection of recipient vessels took 20 ± 4 min. implantation time was 4 to 6 min, resulting in a total pancreas ischemia time of 33 ± 6 min. no thromboembolic complications at the cuff side were observed. preoperative glucose levels were 518 ± 59 mg/dl and could all be normalized by po day 1 (88 ± 13 mg/dl). histopathological examination on po day 10 and 30 showed almost normal islet cell and acinar architecture of all grafts. conclusions. for the first time a method of cervical heterotopic pancreas transplantation using a non-suture cuff technique in the mouse is described. major advantages are a short ischemia time, lack of arterial thrombosis or venous stenosis, and short operation time, and thus a very high survival rate. this model is especially applicable for investigating preservation, reperfusion injury, and graft pancreatitis. background. as human islet transplantation is limited by the lack of sufficient numbers of human donor organs, xenotransplantation with the use of porcine islet cells seems to be a promising therapeutic option to cure diabetes. in order to achieve sufficient numbers of viable islet cells, better protocols for organ preservation, isolation, and purification are needed. recent studies showed that the two-layer method (tlm) of pancreas preservation prior to isolation significantly improved islet yield. the tlm oxygenates pancreata and activates metabolism to generate atp and leads to resuscitation of ischemically damaged organs. another possibility to achieve a higher partial pressure of oxygen levels in fluids is the use of hyperbaric oxygenation (hbo). the aim of this study was to assess the influence of preoxygenated preservation solutions on the porcine pancreas. methods. university of wisconsin solution (uw), celsior, perfadex, custodiol, and a preservation solution especially designed for this study on the basis of ketoglutarate are oxygenated with 100% oxygen for 50 minutes at 1.5 bar using a hyperbaric chamber. porcine pancreata are harvested at a local slaughter house and stored in preoxygenated and not oxygenated preservation solutions at 4 °c for 4 hours. tissue cuts are performed to assess the occurrence of apoptosis and to determine the oxidative stress. atp-to-adp ratio is measured and immunohistochemistry is performed. results. it is feasible to preoxygenate preservation solutions. the oxygen levels can be raised up to 100 times in the preservation solutions using hbo and can be maintained for at least 12 hours after hbo treatment. mda and carbonylated protein levels are not significantly elevated in organs stored in preoxygenated solutions. atp-to-adp ratio as a sign of viability is significantly higher in organs stored in preoxygenated solutions. conclusions. preoxygenation of preservation solutions using 100% oxygen and a hyperbaric chamber is feasible. hbo has a positive impact on porcine organ preservation. as ischemically damaged islet cells are likely to undergo cell death or lose functionality due to hypoxia, the use of preoxygenated preservation solutions is a promising method to achieve better yields after islet isolation and transplantation. alternative zur humanen pankreastransplantation und inselzelltransplantation stellt die xenotransplantation von porcinen inselzellen dar. um xenogene zellen erfolgreich transplantieren zu können, müssen sie vom immunsystem des empfängers abgeschirmt werden. die verwendung von mikrokapseln scheint eine vielversprechende methode dazu zu sein, wobei natrium zellulose sulfat (nacs) in graz verwendet wird. darüber hinaus muss eine ausreichende anzahl an vitalen zellen mit hoher reinheit aus dem porcinen pankreas isoliert werden. methodik. porcine pankreata werden von einem lokalen schlachthof erhalten. die organe werden kanüliert, und ein enzymgemisch aus neutraler protease und kollagenase nb wird infundiert. die digestion erfolgt in einer modifizierten ricordi-kammer mechanisch und enzymatisch, und die anschließende purifikation wird mit einem ficoll-dichtegradienten durchgeführt. vitalität der zellen wird mit fluorescein-diacetat/propidium jodid, mtt und der bestimmung der atp/adp-ratio überprüft. die reinheit wird mit einer dithizon-färbung festgestellt. insulinkonzentrationen werden mittels elisa detektiert, der dna-gehalt der inselzellen mit dem dneasy-kit festgestellt. inselzellen werden in kooperation mit der firma austrianova mit nacs mikroverkapselt. ergebnisse. die organe wurden durchschnittlich 35 min digestiert, es wurde eine reinheit von 96% und eine zellzahl von durchschnittlich 2 × 10 5 zellen isoliert. beste resultate wurden mit dem lymphoprep tm -dichtegradienten erzielt. isolierte porcine inselzellen überleben bei 37 °c im durchschnitt 12 tage in vivo und produzieren glucose-abhängig insulin. die funktionalität der zellen bleibt über den gesamten zeitraum erhalten. mikroverkapselung mit nacs ist machbar. schlussfolgerungen. die isolation porciner inselzellen ist eine viel versprechende methode den mangel an humanen spenderorganen in der pankreastransplantation und humanen inselzelltransplantation zu umgehen. nacs als verkapselungsmaterial ist weniger immunogen und weitaus biokompatibler als alle bisher verwendeten materialien und die mikroverkapselung xenogener inselzellen mit nacs scheint eine innovative methode zur therapie des diabetes mellitus zu sein. background. to achieve its full potential, transplantation of pancreatic islets has to overcome a number of obstacles. one of the obstacles are the still lacking read-out parameters to assess the quality of human islets after the isolation procedure and prior to transplantation. being able to predict the functional potential of the pancreatic islets after isolation or even short-term culture would greatly enhance the success of islet transplantation. therefore one of the primary challenges in islet transplantation is to identify and understand the changes taking place in islets after isolation or culture. life confocal microscopy is a powerful tool to identify such changes in living islets not achievable by use of fixed-cell techniques. methods. islets were isolated according to the method of ricordi et al., using a continuous ficoll gradient. 3 fluorescent dyes, dichlorodihydrofluorescein diacetate (dcf), tetramethylrhodamine methyl ester (tmrm), and fluorescent wheat germ agglutinin were used to assess either overall oxidative stress, time-dependent mitochondrial membrane potentials, or localisation of oligosaccharides. confocal microscopy was performed with an microlens-enhanced nipkow disk-based confocal system ultraview rs (perkin elmer, wellesey, ma, usa) mounted on an olympus ix-70 inverse microscope (olympus, nagano, japan). results. with the above described confocal system we were able to identify differences in the localisation and amount of oligosaccharides in endocrine vs. exocrine cells of freshly isolated pancreatic islets. the staining pattern changed during the course of culture, suggesting a remodeling of cell surface oligosaccharides. the study of the mitochondrial membrane potential proved to be very useful in order to early identify damaged or stressed cells and thereby gain insights into the vitality of the isolated islets. conclusions. this makes us believe that, especially in the light of the many other fluorescent dyes which can be used as subcellular markers, a combination of these with a powerful live confocal imaging system will be of great value for a better islet assessment after isolation and culture. background. therapeutic drug monitoring (tdm) of immunosuppressants is a well established concept supporting the work of clinicians from the historical onset of the use of these drugs. within the last years the combination of hplc (highperformance liquid chromatography) with tandem mass spectrometry (ms/ms) provides an alternative to antibody-based immunoassays. this new analytical method to quantify immunosuppressants affords a robust and rapid separation technique with high selectivity. up to now, the quantification of wholeblood levels of cyclosporin a, fk 506 (tacrolimus), and 42-o-(2-hydroxyethyl)-rapamycin (everolimus) at our institute have relied upon three different, indirect assays from different companies. however, these systems have drawbacks caused by cross reactions with active and inactive drug metabolites, fluctuations in assay performance, and comparatively high prices. in contrast, hplc-ms/ms platforms are now becoming frequently used to measure circulating drug levels and their metabolites with lower associated costs and higher specificity, accuracy, and precision. methods. the setup chosen at our laboratories consists of two independent mass spectrometers (one machine at the zimcl, a backup machine at biocrates) and allows a sample throughput of about 25 samples per hour. all immunosup-varia pressants currently monitored in whole blood can be quantified within one analysis from a sample volume of less than 100 µl. the sample workup (cell lysis and protein precipitation) is performed in a bar-code-supported parallel setup, minimizing the risk of sample mix-up. an online solid-phase extraction (spe) strategy has been chosen to reduce matrix interferences and ion suppression. daily calibrations and quality control samples performed with certified reference materials assure a high level of accuracy and precision. results. the hplc-ms/ms assay established for cyclosporin a, tacrolimus, everolimus, and sirolimus covers the analyte concentration range needed for tdm (e.g., up to 2000 mg/ml for cyclosporin a). intraday and interday repeats of the assay and data from quality control measurements were sufficiently accurate and precise (all cvs, <15%; bias, <15%). comparison of the quantitative results did show a linear relationship between antibody-based immunoassays and the hplc-ms/ms-derived data with bias values (ca. 10% for cyclosporin a and ca. 30% for tacrolimus on the basis of bland-altmann plots) in agreement with the literature. conclusions. the established hplc-ms/ms platform will allow replacement of the current antibody-based assays. the major advantage of this technique is the ability to simultaneously acquire absolute quantitative concentrations of each of the therapeutic drugs administered from one sample. therefore, the requirement for different sample preparation schemes or parallel measurements on different analytical instruments are no longer needed. the high sample throughput also assures timely tdm data report to the ward. a significant reduction of costs is now expected due to the lower consumable expenses in hplc-ms/ms assays. background. a clinical trial towards improved safety of the application of everolimus (certican) in heart transplantations (crad001a2403, data collection started in august 2004 and is ongoing) allowed us to compare therapeutic drug monitoring (tdm) data (e0 and e2 levels) measured on two different platforms -an lc-ms/ms method in an external laboratory and a immunochemical method recently established at our institute. methods. lc-ms/ms measurements of everolimus were performed in a reference laboratory. the immunochemical measurements were performed with a fluorescence polarization immunoassay (innofluor certican immunoassay by seradyn) measured on a tdx instrument from abbott. method comparison included analysis of the time series of the patients (n = 7, 65 observation pairs) and the quality control samples of the immunochemical assay gathered over a year as well as inter-and intra-assay data comparisons based on bland-altmann plots and regression data. results. comparison of the quantitative results obtained from the lc-ms/ms and the immunochemical assay showed good agreement between these methods. the bias between the methods (bland-altmann plot) was found to be rather small, with the immunochemical method measuring in average approximately 5% (±25%) lower values as the lc-ms/ms method, which is in good agreement with current reports. the coefficient of variation (cv) (measured over several months) of the innofluor quality control measurements was <7% for the medium (ca. 12 ng/ml) and high (ca. 25 ng/ml) quality control samples, whereas the lower quality control level (ca. 4.5 ng/ml) showed an increased cv (<13%). however, especially during the phase of method establishment, higher cv values and bias deviations have been found conclusions. the method comparison did show that the immunochemical everolimus assay provided by seradyn is a good alternative to hplc-ms/ms measurements. the assay bias was found to be rather low and the assay uncertainty was within acceptable ranges. however, a stringent quality control network must be provided to assure stable assay performance over time. grundlagen. die inzidenz der endokarditis beträgt ca. 6/100 000 einwohner pro jahr in der gesamtbevölkerung. obwohl die immunsuppression das auftreten systemischer infektionen begünstigt, sind endokarditisstudien an transplantationsempfängern nicht verfügbar. ziel dieser epidemiologischen studie war, das auftreten der endokarditis und ihrer risikofaktoren nach organtransplantation zu evaluieren. methodik. insgesamt 2556 patienten, welche sich zwischen 1989 und 2004 einer soliden organtransplantation an unserem zentrum unterzogen, wurden untersucht. der mbds unserer klinik wurde zum patientenscreening herangezogen. ergebnisse. insgesamt wurden im beobachtungszeitraum 27 endokarditisfälle beobachtet. neun endokarditisfälle (33,3 %) konnten erst post mortem mittels autopsie diagnostiziert werden, 8 patienten (29,6 %) konnten durch alleinige antibiotische therapie geheilt werden. insgesamt 10 transplantationsempfänger (37,1 %) mussten sich einem kardiochirurgischen eingriff unterziehen. die gesamtmortalität betrug 44,4 % (12 patienten). staphylococcus aureus konnte in 16 fällen (59,3 %) und pilze konnten in 4 fällen als ursächliche keime isoliert werden. die inzidenz der endokarditis in transplantationsempfängern beträgt 1 % (95 % ci, 0.67-1.49) und zeigt ein 171-fach erhöhtes risiko verglichen mit der gesamtbevölkerung. schlussfolgerungen. die endokarditis stellt ein signifikantes problem nach organtransplantation dar und ist mit einer exzessiv hohen mortalität assoziiert. eine erhöhte aufmerksamkeit ist daher indiziert, da wir durch den einsatz der transösophagealen echokardiographie vermehrt derartige fälle in der zukunft diagnostizieren werden. ein 47-jähriger patient erhält 1995 eine herztransplantation aufgrund einer dilatativen kardiomyopathie bei altersentsprechender nierenfunktion. die immunsuppression besteht aus cyclosporin a und mycophenolat mofetil. innerhalb der ersten drei monate nach der operation steigt das kreatinin auf 2,0 mg/dl an, bleibt dann aber konstant. ab 2003 nimmt die nierenfunktion weiter ab, weshalb im august 2004 wegen des verdachtes auf eine chronische calcineurininhibitor-nephropathie auf sirolimus und mmf umgestellt wird. das serum-kreatinin beträgt zu diesem zeitpunkt 3,0 mg/dl. zwei monate später ist das serum-kreatinin auf 7,0 angestiegen, der inzwischen 57-jährige patient leidet unter einer renalen anämie, hyperphosphatämie und hypokalziämie. im harn finden sich leukozyten und erythrozyten, die allerdings nicht deformiert sind (keine dysmorphen erythrozyten und akanthozyten), sowie eine geringgradige proteinurie (300 mg/24 h). in der harn-polyacrylamidgel-elektrophorese zeigt sich vor allem alpha-1-mikroglobulin als indikator einer tubulointerstitiellen schädigung. nach erneuter umstellung der immunsuppression von sirolimus/mmf auf niedrigdosis-cyclosporin/mmf kommt es zum raschen rückgang des serum-kreatinins, der harnbefund sowie die elektrolyte normalisieren sich, einzig die erythropoetin-substitution muss beibehalten werden, um den hämoglobin-zielwert von 11 g/dl zu erreichen. acht wochen nach der zweiten therapieumstellung führen wir eine nierenbiopsie durch. es findet sich eine akute interstitielle nephritis ohne glomeruläre schädigung. unseres wissens ist dies der erste dokumentierte fall einer akuten interstitiellen nephritis auf sirolimus. background. group milleri streptococci (gms) are a heterogeneous group of streptococci including the species streptococcus intermedius, s. constellatus, and s. anginosus. due to their ability of producing toxins, they tend to cause chronic intra-abdominal and intrathoracic abscesses, which are difficult to treat, as gms are able to escape conventional antibiotic therapy. aim. evaluation of epidemiology, clinical significance, and impact on the outcome in all solid-organ recipients with gsm infections during a 4-year period. patients and methods. retrospective analysis comprising 45 solid-organ recipients with gms. results. between 2001 and 2004, 45 solid organ recipients (88 isolates) including 34 liver, four kidney and pancreas, one kidney, two small bowel, three combined liver and kidney, and one combined kidney and small bowel transplant re-cipients developed infection with gms. in 42 cases, gms caused intra-abdominal infection; in two cases, pleural empyema; and in one case, soft tissue infection. in only one case, gms were cultured from blood. in 54 of the 88 specimens (61%) which grew gms, also other pathogens could be isolated. gms frequently caused recurrent cholangitis (n = 17) associated with anastomotic and anastomotic biliary strictures. these cases were managed by repeated stenting or surgical intervention and prolonged antibiotic therapy. no patient died directly related to gms infection. all responded to combined surgical and antibiotic treatment. one pancreas graft was lost due to erosion haemorrhage associated with an abscess. all isolated strains of gms were susceptible to penicillin g, carbapenems, and clindamycin, whereas cephalosporins and quinolones showed intermediate activity or resistance in some cases and gms in general were found resistant to aminoglycosides. conclusions. gms are frequent pathogens in transplant surgery and are capable of causing difficult to treat infections. their prevalence in transplant surgical site infection thus far might have been underestimated. therefore, we recommend empiric antibiotic treatment for sufficiently long time in combination with surgical intervention when necessary. background. by using intensified immunosuppressive protocols the incidence of immunological complications after solid-organ transplantation has constantly declined. however, the incidence of some infections, in particular complicated fungal infections, seems to increase. candida krusei (ck) is resistant to fluconazole and recently this pathogen has been more commonly isolated in severe infections, particularly in the immunocompromised host. methods. between 1. 1. 2004 and 30. 6. 2005 , a total of 400 solid-organ transplants were performed at the innsbruck medical university. this included 150 renal, 70 liver, 50 pancreas, 5 intestinal, 30 cardiac, 20 lung, and 10 islet transplants. prophylactic immunosuppression consisted of calcineurin inhibitor-based triple drug therapy for the majority of cases. antifungal prophylaxis was given to all pancreatic and intestinal recipients (fluconazole 400 mg per day) and to all patients considered at high risk for filamentous-fungal infections (ambisome 3 mg/kg). results. a total of five patients with ck infection were identified within this series (2%). five patients developed infection with candida tropicalis and three with candida glabrata. within this time period, another two patients with ck infection were identified, one had a pleural empyema following esophageal perforation and the other was treated with a ventricular assist device and developed pulmonary infection with ck. both patients were treated on the transplant intensive care unit. the transplant patients were three pancreas, one liver, and one lung recipient. all three pancreas recipients were diagnosed intra-abdominal infection, the liver recipient had an ischemic cholangiopathy with ck cholangitis and required retransplantation, and the lung recipient developed postoperative hemorrhage and subsequently ck pleural empyema. all patients presented also with other infectious complications. treatment of ck infection consisted in four cases of caspofungin or voriconazole or combination of the two, and in one case, ck was isolated from infected hematoma and did not require antifungal therapy after surgical removal. all infected collections were evacuated either surgically (n = 3) or through pig-tail drainage (n = 2). ck infection was successfully managed in all cases and patients are currently alive, only one pancreas graft was lost. conclusions. candida krusei infections now represent frequent severe complications in solid-organ recipients. however, rapid diagnosis and treatment with new antifungal agents such as voriconazole, caspofungin, or ambisome allow successful therapy of these infections. solid-organ recipients seem to be at increasing risk to acquire non-albicans candida infections. background. after solid-organ transplantation immunosuppression (is) and concomitant infection with cmv, ebv, hhv-6, -8, or papillomavirus put patients at risk for developing malignant diseases (14% cumulative risk at 10 years; adami et al. 2003) . posttransplant lymphoproliferative disorders (ptld) and skin cancers are known to have the highest incidence in immunocompromised patients. reports on colorectal cancer after different types of organ transplantation are rare and the incidence was 0.01% to 3.9% and did not differ from that of the normal population. we analysed our database with regard to the incidence and course of colorectal malignancies following treatment and introduction of tor inhibitor-based is. methods. medical records of solid-organ transplants performed between 1986 and 2005 at our center were analysed retrospectively. in a total of 3568 patients 247 heart, 118 lung, 2074 kidney, 757 liver, 367 pancreas, and 9 combined heart-lung transplantations were performed (27 small bowel and 4 hand transplantations not included). immunosuppressive therapy consisted of triple therapy comprising steroids, azathioprine/mmf, and cya/tac. some of them received induction therapy with antithymocyte globulin. results. a total of 206 patients (5.72%) developed malignancies. of them, 9 patients (1 female, 8 male; median age at diagnosis, 66.1 years; 2 kidney, 3 heart, 4 liver recipients) had colorectal malignancies (0.25%) during a mean follow-up period of 7.3 years. on average, diagnosis was made 5.8 years after transplantation. four carcinomas were located in the rectum or at the rectosigmoid junction and five were colon cancers (five pt3, one pt2, and three pt1 stages). r0 resection was performed in all 9 patients plus radio-and/or chemotherapy in all t3 stages. five patients (55%) died 7.2 years post transplant due to cardiovascular disease (n = 4) and recurrent tumor disease (n = 1). the 1-year survival rate was 67% for t3 and 100% for t1 rectal cancers, 50% for t3 and t1 each and 100% for the only t2 colon cancer. three anal neoplasms (one ain iii°, two anal cancers, pt1 and pt2; median age at diagnosis, 50 years) developed on average 7.2 years after transplantation (0.08% vs. 0.001% in the general population) with a 100% 1-year survival rate. all patients were switched to rapamycin or everolimus after completion of primary therapy. conclusions. the incidence of anal but not of colorectal cancers in our transplant patient population differed from that of immunocompetent patients of corresponding age (0.08% vs. 0.001% and 0.25% vs. 0.2% tyrol tumor registry and 0.3% seer). the 1-year survival rate was significantly decreased in the transplant group with t3 tumors (67% [rectum] and 50% [colon] vs. 80%). potential antineoplastic effects of rapamycin and overall less immunosuppression long-term may improve prognosis of colorectal malignancies following transplantation. background. solid-organ transplantation is the treatment of choice for terminal organ failure. due to the scarcity of organs, the mandate to utilize extended-criteria donors has dramatically increased over time and achievable results are good. this harbors the risk of transmission of infections from the donor to the recipient. aim. an overview on the magnitude of possible transmittable diseases that could accompany donor organs is given. possible preventive strategies are discussed. overview. the faith of an organism that is transmitted by a donor allograft depends on the virulence and the quantity of the transmitted pathogen and the site of infection and furthermore is impacted by prophylactic steps undertaken during donor procurement, organ perfusion, implantation, and post transplant course and lastly depends on the ability of the recipient to control the infection by the immune system. in the best case, the microorganism is cleared before it can do any harm; however, in the worst case scenario, transmitted pathogens can lead to fatality. there is a multitude of pathogens that can potentially be transmitted by an allograft including viruses, bacteria, fungi, and protozoa. there are microorganisms that can be transmitted by any type of graft and there are pathogens which due to a certain tropism can be transmitted by certain organs only. there are common pathogens such as cmv and ebv as opposed to extremely rare pathogens such as lyssa, west nile fever, or lymphochoriomenigitis virus. intracellular pathogens are preferably targeted by mhc restricted cytotoxic t-cell reaction. as in organ transplantation hla matching is in general not performed, donor-derived antigen-presenting cells cannot be recognised by recipient-specific cd8 + lymphocytes. therefore, either donor-specific cytotoxic t cells must function as counterparts or alternative tar-gets must be defined by the immune system. bacterial and/or fungal contamination must be considered in lung transplantation (bronchial stump), pancreas transplantation (duodenal segment), and small bowel allografts. cardiac, renal, and liver grafts can be considered sterile but can become contaminated during procurement. rare transmitted organisms are toxoplasma gondii, trypanosoma cruzii, and schistosoma mansoni. conclusions. when looking at allocation of latently virus-infected organs, they should be preferably given to recipients who have antibodies against the particular agent. in terms of bacterial or fungal contamination of graft and/or preservation solution, routine monitoring seems mandatory in order to assure quality. keeping blood and serum samples from donors should be carried out from an epidemiological and legal standpoint. bacterial and fungal prophylaxis should be used to prevent not only recipient-but also donor-derived pathogens. antiviral prophylaxis is standard for cmv and hbv, for all other viruses no final recommendations are available. methods. the aim of the current review was to investigate the impact of several risk factors on ptld. therefore, patients developing ptld after heart transplantation at our institution were screened. results. 8 patients have been identified with ptld, all cases occurring during the last decade. mean age at ptld onset was 46.4 ± 20.0 years and time between transplant and development of ptld was 26.4 ± 30.4 months. there were 5 ebv-associated ptlds, 2 non-ebv-associated, cd20-negative b-cell lymphomas, and 1 t-cell lymphoma. immunosuppression at ptld onset was calcineurin inhibitor based (cyclosporine a, 5 patients; tacrolimus, 3 patients). initial immunosuppression included atg induction. six patients received perioperative antiviral prophylaxis with either valgancyclovir/gancyclovir (n = 4) or acyclovir (n = 2) in combination with anti-cmv hyperimmunoglobulin (n = 1). two patients experienced a total of five episodes of acute rejection (ishlt ii°), all were treated with bolused steroids. four patients are still alive (50%), three of them in current remission of ptld, one patient is under therapy recently. median survival was 27 months in survivors and 3.4 months in nonsurvivors. conclusions. these data show that ptld is associated with a high mortality rate. the majority of ptlds are ebvassociated. therefore, screening for ebv infection and prophylactic treatment may help to prevent a potentially fatal consequence of heart transplantation. background. late acute cellular rejection is associated with a decrease of survival and the development of cav. new immunosuppressice drugs have been introduced into clinical practice. everolimus, as one of these, has shown to be safe in cardiac transplantation. we report of our experience with everolimus in heart transplant recipients who developed late cardiac rejection. methods. patients with a history of previous rejection episodes who experienced cardiac rejection after at least 2 months postoperative were switched to an everolimus, cyclosporine a, and corticosteroid-based immunosuppressive regime. all patients already received statins and antihypertensive medication before. everolimus, cyclosporine a trough levels and laboratory values were controlled monthly. drug administration was adapted to an everolimus trough level between 3 and 8 ng/ml, mean maintenance dosage was at 0.25 to 1.5 mg/day. death, safety, side effects, biopsy-proven acute rejection, laboratory values and blood levels were evaluated retrospectively. results. 4 cardiac allograft recipients (2 male, 2 female), at a median of 1473.25 days post orthotopic heart transplantation (ohtx) (65-3045), received 1 mg to 1.5 mg everolimus per day. in a follow-up period of at least 2 month (2-10) mortality was 0%. the drug was well tolerated and no acute cellular rejection greater than grade 1a (ishlt grading) was observed after two month. in one patient raised cholesterol values and in two others elevated triglyceride levels were seen but were controlled with higher statine therapy. no obvious raised creatinine values were seen with certican ® . conclusions. in conclusion, conversion to an everolimus-based immunosuppressive regimen after late cardiac rejection is safe and effective. no major side effects were observed. p02 collagen iii in transplanted heartdonor or recipient derived m. pichler, b. tessaro, d. kniepeiss, r. kleinert, g. hoefler institute of pathology, medical university of graz, graz, austria background. transplantation of donor hearts is often associated with progressive development of interstitial myocardial fibrosis and alterations in composition and organisation of the extracellular matrix. changes in cardiac interstitial collagen network are thought to contribute to abnormal stiffness and loss of function of the myocardium. fibroblasts are the main producers of type i and type iii collagens, the major in-poster terstitial collagens found in the heart. in transplanted hearts, intragraft fibroblasts may consist of two cell populations. donor-derived fibroblasts preexist in donor organs, whereas hostderived fibroblasts may progressively immigrate as mesenchymal progenitors from the circulation to the allograft. purpose of the study. to determine the contribution of these two distinct fibroblast populations to progression of myocardial fibrosis, we studied endomyocardial biopsies over a time period of some years from a male patient who had received a heart from a female donor. methods. in these sex-mismatched patients two frequent genetic polymorphisms at the collagen iii locus were determined by polymerase chain reaction-based restriction enzyme digestion, both in donor allograft and in the corresponding explanted recipient heart. on the basis of differences of the collagen genotype in donor and recipient tissue, we selected 10 endomyocardial biopsies by hematoxylin eosin staining covering an overall follow-up period of nine years since the transplantation event. immunohistochemistry, chromogene in situ hybridisation, and population-specific collagen expression using single nucleotide polymorphisms were used to determine the course of fibrosis. results. we developed an analytical system generally applicable to measure population-specific differences of collagen iii synthesis in transplanted organs. the amounts of interstitial collagen type i and type iii increased in a time-dependent manner within cardiac allograft. years after transplantation, a number of y-chromosome-positive recipient-derived cells consisted of noninflammatory spindle-shaped fibroblast-like cell types. conclusions. our data confirm the existence of a substantial number of fibrosis-mediating immigrated recipient-derived fibroblasts in cardiac allografts. furthermore, this suggests a potential, future therapeutic approach for reduction the cardiac fibrosis process. methods. between 1993 and 1998 a total of 32 lung transplants (31 patients) and six combined heart-lung transplants were performed at our center. immunosuppression consisted of atg induction, cyclosporine a, azathioprine, and steroids. all patients with rti underwent a meticulous micro-biological screening and underwent bronchoscopy with bronchiolo-alveolar lavage (bal) and transbronchial biopsies (tbb). rsv was detected from bal specimens by an immunoassay. results. a total of five lung recipients (31% of 19 survivors) and one of the two alive heart-lung recipients developed rsv infection of the lung allograft. all cases were observed during an rsv epidemic in children in the area between october 1998 and may 1999. the patients were 47-55 years old, all were females. one patient experienced two episodes. onset of rsv was median 3 (range, 4-29) months post transplant. clinical symptoms included cough (6 of 7), rhinitis (6 of 7), and fever (4 of 7). deterioration of lung function occurred in six of the seven episodes with one deteriorating to respiratory failure requiring ventilator support. two individuals developed pulmonary infiltrates. in all patients, immunosuppression was significantly tapered, 86% required hospitalization and antibiotic therapy. the patient with rsv recurrence received inhalative ribavirin therapy during the second episode. all patients recovered and survival of this cohort was 86% after 1 year and 72% at 4 years after rsv infection. none of the patients developed bronchiolitis obliterans syndrome (bos) or rejection during follow-up. conclusions. rsv is a severe but benign complication following lung transplantation with a wide range of clinical presentations. routine use of antiviral treatment is not necessary; however, reduction in the level of immunosuppression is required. no long-term effects were observed in this cohort. a. stamatelopoulos 1,2 , s. guth 2 , a. abrahim 2 , g. m. marta 2 , l. tsourelis 3 , p. jaksch 2 , c. konaris 4 , s.taghavi 2 , w. klepetko 2 due to cni nephrotoxicity in a substantial number of ntx patients. we therefore studied the effects of a switch from a cni regimen (cni + mpa + p) to a dual regimen of sirolimus (srl) plus prednisolon (p) in 26 pts with moderately impaired kidney function (s-crea, 1.5+0.4 mg%) due to either cni toxicity or clinical evidence for chronic allograft disease. 13 pts received regimen 1 consisting of srl 12 mg dosage on day 1 and from days 2 to 5 srl 4 mg plus csa at half of maintenance dosage. from day 6 on, pts received srl 8 mg and csa was withdrawn. mpa or aza dose was continued halfed for 4 to 6 weeks. p was kept constant. target level for srl was 12-20 ng/ml. another 13 pats received regimen 2 consisting of srl 12 mg and csa withdrawal on day 1 and from days 2 to 6 srl 6 mg was administered for a target level of 7 to 10 ng/ml. mpa or aza were continued halfed for 4 to 6 weeks, p was kept constant. with regimen 1 there were 6 dropouts due to adverse events, whereas with regimen 2 only 2 dropouts occurred. 45% of the patients showed a decrease of s-crea after 2-year observation period, 20% were unchanged and only 25% showed an increase. overall, there was a slight but significant increase of cholesterol and triglycerides, whereas other parameters were unchanged. conclusions. switch from cni containing immunosuppression to a dual regimen of sirolimus plus prednisolon results in an improved kidney function after 2 years in the majority of pts. a regimen of sirolimus with target levels of 12 to 20 ng/ml and an overlap of immunosuppression shows a high rate of adverse events and is associated with a 3-fold dropout rate as compared to a regimen with a target level of 7-10 ng/ml. background. certain anatomical variations mainly concerning the portal system preclude living donor liver transplantation (ldlt). to the best of our knowledge, two left lateral segments with two arteries have never been transplanted so far. case report. a 6-month-old girl was diagnosed with endstage liver cirrhosis secondary to biliary atresia and therefore scheduled for ldlt. preoperative evaluation of the donor including ct with 3-d reconstruction revealed normal vascular supply of the liver with a left and a right hepatic artery. during donor operation, two tiny arteries with a diameter of 2 mm for segments ii and iii were identified. they were found to arise from the left hepatic artery right behind the bifurcation of the proper hepatic artery, which made it impossible to preserve a common trunk. venous and portal venous reconstruction was performed in an end-to-end fashion. the left graft artery was directly anastomosed to the proper hepatic artery with 8/0 pds interrupted sutures using a microscope. the 2nd graft artery was revascularised with the help of saphenous vein from the recipient as interposition graft to the gastroduodenal artery but failed. therefore, the trunk of the inferior mesenteric vein was used for reconstruction with 8/0 pds with excellent outcome. under fk-based immunosuppression, postoperative course was uneventful with both arteries patent. conclusions. multiple arteries for the left lateral segments are not a contraindication for paediatric ldlt. the inferior mesenteric vein can be used as an interposition graft. combined renal-pancreas transplantation is an established treatment for patients with diabetic nephropathy, producing excellent results. we report on a patient who suffered from long-standing multiple sclerosis and underwent successful combined renal-pancreas transplantation. post transplant course was complicated by yeast intra-abdominal infection, which was treated with antifungal agents. using tacrolimus and sirolimus, both grafts are well functioning at two years and the patient is without any symptoms of disseminated encephalitis and does not require specific medication for multiple sclerosis. for diabetic patients suffering from multiple sclerosis, pancreas transplantation offers an excellent treatment option. whether normalisation of carbohydrate metabolism or chronic immunosuppression or both lead to complete response of multiple sclerosis is not clear. background. until recently, the peripheral blood stem cell (pbsc) donation procedure was used only infrequently among unrelated allogeneic donors. nowadays, both related and unrelated donors are expected to consider this alternative donation. to promote pbsc donation both safety and well-being of healthy unrelated volunteer donors must be protected and data are to be collected to establish the long-term safety of g-csf stimulation. methods. from 2000 to 2004, 16 pbsc aphereses on unrelated allogeneic donors have been carried out in our center. all pbsc donors were treated with 5 µg of g-csf per kg twice a day from day -4 to day -1. aphereses were performed using peripheral venous access on day 0 and -if indicatedon day 1. since 2003, all donors have annually received a questionnaire about their actual state of health and medication, as well as their physical and mental conditions. detailed questions concerned donors' anamnesis for epistaxis, bruises, thrombosis or embolia, as well as infections and fever, night sweat, and weight loss of unclear origin. results. 11 male and 2 female donors (81%) with an average age of 44.59 (29-56) years responded to the questionnaire. the observation periods were between 3 and 53 months (mean, 28.75 months) after g-csf stimulation. 5 pbsc donors (38%, all male) reported that they had been severely ill during the observation period: one donor developed an exostosis of the 5th rib, one was operated on an umbilical hernia, one suffers from recurrent articular and muscle pain accompanied by night sweat and weight loss, one has a chronic compensated renal failure, one had diarrhoe and a common cold and suffered from fatigue, nausea, sleeping problems, and circulatory disorders. one female donor recognized dizziness and an increased tendency for bruises as well as paraesthesia in both arms. the disorders these donors reported occurred between 3 and 22 months after g-scf stimulation. all the other pbsc donors (62%) have never been severely ill or under medical observation. no donor had fever of unclear origin, phlebitis, thrombosis, or embolia, no donor recognized an increased tendency for epistaxis. three donors need medication they did not have before g-csf stimulation, which are to lower blood lipids and anti-inflammatory ones. one donor estimates that he is getting ill more easily than others, all the other donors feel themselves in best physical and mental condition. background. hematopoietic stem cell transplantation (hsct) has been successfully performed in patients with otherwise incurable malignant diseases. however, relapse after hsct is one of the main reasons for treatment failure and further therapeutic strategies with acceptable toxicity are warranted. since myeloablative (ma) conditioning after prior hsct has been associated with high treatment-related mortality (trm), reduced-intensity conditioning (ric) regimens have been developed as salvage therapies for these patients. so far, encouraging results have been achieved with ric; however, a direct comparison with standard conditioning has never been performed. therefore, we retrospectively analysed these two conditioning strategies in patients experiencing relapse after prior stem cell grafting. methods. we analyzed 45 patients with relapsed disease (acute myeloid leukemia, n = 16; indolent lymphoma, n = 9; multiple myeloma, n = 7; chronic myeloid leukemia, n = 4; myelodysplastic syndrome, n = 2; chronic lymphocytic leukemia, n = 2; acute lymphocytic leukaemia, n = 2; aggressive lymphoma, n = 1; hodgkin's disease, n = 1; ovarian cancer, n = 1) after prior hsct who received either reduced-intensity or myeloablative conditioning for allogeneic hsct between 1986 and 2005. ric consisted of fludarabine 90 mg/m 2 and total-body irradiation (tbi) of 2 gy according to the seattle protocol (n = 18) or alemtuzumab in combination with the beam regimen (n = 1). myeloablative therapy consisted of cyclophosphamide (cy) and tbi of 12 to 13 gy (n = 13), cy plus busulfan (bu) (n = 4), c plus antithymocyte globulin plus bu (n = 2), or bu alone (n = 7). donors were syngeneic in 4, related in 21, and unrelated in 20 patients. stem cell source was bone marrow in 13 (29%) and peripheral blood in 32 (71%) patients. for graft-versus-host disease (gvhd) prophylaxis, 22 patients received cyclosporine a (csa) plus mycophenolate mofetil, 16 csa plus methotrexate, 2 mtx alone, and 1 csa alone. results. all patients achieved complete hematopoietic engraftment by day 28 after stem cell transplantation with complete donor chimerism. all patients conditioned with ric presented complete donor chimerism of t cells, myeloid progenitor cells, and nk cells 28 to 81 days after hsct. the in-cidence of acute gvhd was 37% and comparable in both groups consisting of grade i in 6 patients, grade ii in 3, grade iii in 6, and grade iv in 2. fourteen patients died after ma conditioning of acute gvhd (n = 2), infections (n = 5), or severe toxicity (n = 7), while only one patient died due to infection after ric. probability of transplant-related mortality (trm) at 1 year after hsct was with 54% significantly (p = 0.001) higher in patients given myeloablative conditioning compared to 5% after ric. incidence of therapy requiring chronic gvhd was with 64% versus 33% significantly (p = 0.001) higher in patients who received ric. response rates were comparable between patients who received ric or ma conditioning (90% versus 100%). relapses occurred in 37% of patients after ric and 32% after ma conditioning. after a median follow-up of 44.5 (range, 8-204) months 3 (11%) of patients of the ma group and 11 (58%) of the ric group are currently alive. probability of survival at 1 and 4 years after hsct is with 73% versus 24% and 48% versus 12% significantly (p = 0.008) higher after ric than after myeloablative conditioning. conclusions. allogeneic stem cell transplantation is a highly effective treatment option in patients relapsing after prior hsct. durable hematologic engraftment and sustained complete remissions can be achieved in patients with otherwise poor prognosis. since transplant-related mortality of dose-reduced conditioning is in comparison to myeloablative hsct considerably lower, overall survival can be significantly improved with this new treatment modality. background. patients with comorbidities such as organ dysfunctions or preexisting infections experience a high treatment-related mortality which makes them ineligible for conventional conditioning therapy. for these patients, reduced-intensity conditioning (ric) is an option which offers the benefits of an allogeneic transplant with lower extramedullary toxicity. we report on 8 pediatric and young adult patients who were considered for ric because of severe cumulative pretreatment or substantial comorbidities treated at our institution between 2001 and 2005. methods. eight patients (median age, 11 years; range, 1-28 years; m, 4; f, 4) diagnosed with aml (cr 1, n = 4; cr 2, n = 1), all (cr 3, n = 1), alps (n = 1), and relapsed ewing's sarcoma (n = 1). 7 of 8 patients were planned to receive ric, in one patient the regime was changed during conditioning due to an acute viral infection. all patients received fludarabine (n = 7, 150 mg/m 2 ; n = 1, 60 mg/m 2 ) combined with melphalan (n = 6, 100-140 mg/m 2 ), busulfan (n = 1, 13 mg/kg), or total-body irradiation (n = 1, 2 gy). 3 patients received atg (45-60 mg/kg), two patients campath (40 mg/m 2 ). post-transplant immunosuppression consisted of cyclosporine a in all patients combined with mycophenolate mofetil (n = 1) or methotrexate (n = 2). 7 of 8 patients received hla identical grafts (sibling, n = 3; hla identical mother, n = 2; mud, n = 2), one patient received a c-locus mismatched graft. stem cell sources were bone marrow in 6 patients and peripheral blood stem cells in 2 containing a median of 5.13 × 10 6 cd34 + cells per kg of body weight of the recipient. results. all patients had primary engraftment; the median time to neutrophil recovery (n > 1.0 × 10 9 /l) was 15 days. complete donor chimerism as evidenced by vntr was achieved in median on day +22 (+11 to +28). acute mild graftversus-host disease (gvhd) of the skin occurred in 5 of 8 patients and responded to prednisolone; one patient required additional immunosuppression with mmf. one patient progressed to extensive chronic gvhd; one patient developed chronic gvhd of the skin, another patient shows clinical evidence of chronic gvhd of liver and gut. the remaining 3 patients showed no gvhd at all. 5 of 8 patients were positive for one or multiple viruses on routine viral pcr monitoring, requiring virostatic treatment. no treatment-related mortality occurred. the patient with ewing's sarcoma died 4 months posttransplant from tumor progression; the patient with extensive chronic gvhd died of sepsis on day +400. all 5 patients with aml are in remission. the patient with alps is still positive for various autoantibodies. 3 patients developed a posttransplant macrophage-activation syndrome (mas). all of them required a stem cell boost because of pancytopenia. conclusions. ric followed by allogeneic hsct was successful in terms of achieving primary engraftment and complete donor chimerism as well as avoiding transplant-related mortality in all patients; in our patients with myeloid malignancies it was also successful in terms of maintaining stable remission. as for posttransplant problems, we encountered acute gvhd of the skin in 5 of 8 patients, three of whom developed chronic gvhd; and viral infections in 5 of 8 patients, three of whom developed mas and eventually required a stem cell boost. background. due to an aggressive course mantle cell lymphoma is characterized by poor prognosis with a median survival of 3 years and only 10-15% long-time survivors. recent improvements in therapy have been made by combined immunochemotherapy and intensification with high-dose chemotherapy. methods und results. ten patients (4 female, 6 male) with a median age of 56 (49-65) years were treated with rituximab plus chop for four or six cycles. furtheron, patients received 2 or 3 cycles of claeg-d (3 days cladribine 0.2 mg/kg, ara-c 1.5 g/m 2 , etoposid 60 mg/m 2 , and on day 1 daunoxome 80 mg/m 2 ) including stem cell collection. as a result, 5 out of 7 newly diagnosed patients reached cr1 and 2 pr. out of 3 patients treated after first relapse, 2 reached again cr and 1 pr. autologous transplantation was performed at a median of 9 (5-11) months after diagnosis. high-dose conditioning consisted of beam chemotherapy plus the addition of 4 doses of rituximab (days -9 and -1 of the conditioning regimen and days +48 and +55 after transplantation). four patients could not receive the rituximab therapy at days +48 and +55 due to complications. a median number of 5.71 × 10 6 cd34+ cells (1.65-21.21 ) per kg were reinfused. all patients had a short haematologic regeneration time (granulocytes, <0.5 g/l day +10 [9-11]; platelets, day +13 [10-17]) and received a median number of 3 erythrocyte (2-6) and 2 platelet (2-5) concentrates. all patients suffered from mucositis grade i-ii, 3 of 10 had emesis grade i-ii. infectious complication of short duration appeared in 7 patients (4 fuo, 2 pneumonia, and 1 sepsis) early after transplantation. one patient developed late complications (hypothyreosis on day +88 and sarcoidosis on day +105). following transplantation, all patients reached clinical and molecular cr lasting for a median time of 35 (1-53) months. two patients relapsed 17 and 26 months after transplantation. despite continuous salvage immunochemotherapy, one of these patients died 34 months after transplantation, the other one is still in pr (+48 months). median observation periode after diagnosis is 46 (11-105) months. conclusions. treatment intensification with immunochemotherapy and high-dose consolidation is accompanied by acceptable toxicity and seems to be an effective treatment procedure for mantle cell lymphoma. background. bone marrow transplantation (bmt) together with costimulation blockade can reliably induce mixed chimerism and tolerance. in recent studies, we showed that regulation by cd4 + cd25 + t cells plays an important role in this model. stimulation of cd4 + cd25 + t regs by an anti-cd3 mab can inhibit and reverse the outbreak of certain autoimmune diseases, and the maintenance and function of t regs was demonstrated to critically depend on il-2. we thus investigated if stimulation of regulatory cells by anti-cd3 or interleukin-2 facilitates bm engraftment and reduction of tbi in our model. methods. c57bl/6 mice received a total-body irradiation (day -1) of 3 gy or 1.5 gy, approximately 20 × 10 6 fully mismatched balb/c bone marrow cells (day 0) and costimulation blockade consisting of anti-cd154 mab (mr1, day 0) and ctla4ig (day +2). additional groups received further treatment with (n = 4-8 per group): (1) anti-cd3 mab (5 µg, day 0 to +4), (2) il-2 (4000 ie/day, day 0-27), (3) rapamycin (0.3 mg/kg/day, day 0-27), and (4) rapamycin and il-2 (day 0-27). multilinage chimerism and skin graft survival were followed for more than 120 days. results. with our standard protocol, 10 of 10 (3 gy) and 8 of 14 (1.5 gy) mice developed long-term multilinage chimerism and accepted donor skin grafts permanently. while the majority of mice treated with anti-cd3 and 3 gy (7 of 8), rapamycin plus 1.5 gy (4 of 6), and il-2 and rapamycin plus 1.5 gy (4 of 5) developed multilinage chimerism and longterm skin graft survival, groups treated with anti-cd3 plus 1.5 gy tbi (0 of 8, p < 0.01) and il-2 plus 1.5 gy (2 of 6, p = 0.1) showed markedly reduced rates of chimerism and tolerance. we investigated if these negative effects might be correlated with a cytokine shift caused by anti-cd3 treatment, but we did not observe such a shift towards th1 or th2 (as measured on day 13 post-bmt). conclusions. unexpectedly neither anti-cd3 nor il-2 had a positive effect in this model, in some groups anti-cd3 treatment even showed a negative effect. thus, other strategies for augmenting the effect of regulatory cells need to be developed. the role of minor antigen disparities for the induction of mixed chimerism and tolerance through bmt plus costimulation blockade s. bigenzahn 1 , i. pree 1 , p. nierlich 1 , e. selzer 2 , f. mühlbacher 1 , t. wekerle 1 long-term donor skin grafts looked macroscopically intact in the b10.d2 group but showed shrinking and scabs in the balb/c group. conclusions. minor antigen disparities pose a substantial barrier for the induction of mixed chimerism and tolerance. for increased clinical relevance, tolerance models should preferably use strain combinations including major plus minor mismatches. p20 immunohistochemical and confocal analysis of pancreatic tetranectin d. pirkebner 1 , m. hermann 1 , a. draxl 1 , w. mark 2 , r. margreiter 2 , p. hengster 2 background. islet transplantation is not yet widely used in part because of the shortage of human islet cells. gaining detailed knowledge of the physiological basis governing the processes of differentiation of pancreatic stem or progenitor cells that have the capacity to self-renewal and to generate differentiated beta cells is instrumental for the ambitious goal of engineering new pancreatic islets in order to cure type i diabetes. the aim of our study was to cultivate and characterize a pancreatic cell population expressing tetranectin (tn). the ability of tn to bind plasminogen indicates that it may have a role in regulating pericellular proteolysis and proteolytic activation of latent forms of metalloproteinases and growth factors. methods. islets were isolated according to the method of ricordi et al. (1992) using a continuous ficoll gradient. immunohistochemistry and immunofluorescence were performed with a monoclonal antibody against human tetranectin (amino acids 17-181 of human tetranectin monomer; antibodyshop, grusbakken, denmark). to determine in vitro cell proliferation, cells were labeled with brdu (roche, basel, switzerland). results. we describe the localization of tn-positive cells in the human pancreas and their growth in vitro. interestingly, individual positive cells are present within the exocrine acini. we were able to isolate human and murine islets and cultivate these tetranectin-positive cells under adherent and nonadherent conditions as shown by immunohistochemistry and confocal immunofluorescence. the possibility to culture these cells is a first step towards their better characterisation. conclusions. together with its above mentioned ability to bind plasminogen-like hepatocyte growth factor and tissuetype activator, tn may thereby play an important role in the survival of islets after islet transplantation. as we could show, tn positive cells can be isolated and maintained in culture after human islet isolation, thereby providing the possibility to further clarify their role and function in vivo as well as in the course of islet transplantation. p21 fty720 interferes with effector functions and downregulates protein expression of s1p1 and s1p4 in human dendritic cells tures with fty720/fty720-p-treated dc illustrated a cytokine production profile with a lower ifn-? (22% vs. 25% relative reduction) and a higher il-4 (64% vs. 92% relative increase), indicating a shift from th1 toward th2 differentiation as previously evinced for s1p. dc yields, phenotypic differentiation into idc and mdc (besides a minor reduction in cd18 surface expression), as well as the investigated mechanisms of idc antigen uptake (bacterial phagocytosis, mannose receptor-mediated, and fluid-phase endocytosis), were not affected at therapeutically relevant concentrations. conclusions. we conclude that treatment of human dc with fty720 and fty720-p interferes with dc effector functions that are essential for dc to serve their pivotal duty as professional antigen-presenting cells and that dc can therefore be added to the potential list of target cells of fty720. impairment of dc migration and th1-priming capacity due to downmodulation of dc-expressed s1p 1 and s1p 4 might represent a new aspect in the overall mechanism of action and hence contribute, in part, to fty720-mediated immunosuppression. the ibal-fresenius: bioartificial liver innsbruck (ibal) utilizing fresenius standalone, rotating bioreactor m. wurm, v. lubei, p. hengster in order to establish a suitable environment for cultivation of hepatocytes serving as bioartificial liver (bal), we were testing and further developing fresenius standalone, rotating bioreactor. to create optimal conditions for cultivation of hepatocytes, a special environment of nearly gravity-free, low shear force and high mass transfer is needed. furthermore, basic parameters like stability of heating, gas exchange, and sufficiency of nutrition have to be evaluated allowing utilization of various breeding chambers. in summary, we have established a standalone bioreactor capable of quick mass transfer between small and big chambers and external media supply, in nearly gravity-free environment minimizing shear force thus allowing for cultivation of various cell types. background. laparoscopic donor nephrectomy is a less invasive alternative to open nephrectomy for living kidney donation. this study presents the results of the first 58 laparoscopic donor nephrectomies in our center. methods. from june 2000 to may 2005, 58 patients underwent laparoscopic donor nephrectomy for living-related renal transplantation. patient demographic, intraoperative, and postoperative parameters and complications, as well as renal allograft outcome, were evaluated. results. 58 patients (40 female and 18 male) donated their left kidney. the mean donor age was 45 years (24-69 years). the mean surgical time was 233 ± 54 minutes. mean warm ischemia time was 179 ± 63 seconds. patients could be discharged from hospital after a mean time of 5.8 ± 1.5 days. in four patients (6.9%) conversion to open surgery had to be performed. reasons for conversions were lack of operative progression in two cases, in one case venous bleeding, and in one case lesion of the renal artery. there were no reoperations required in the donors. in the recipients, 3 (5.2%) delayed graft functions and 1 (1.7%) primary nonfunction were observed. mean serum creatinine level in the recipients was 1.3 mg/dl 3 months after transplantation. conclusions. laparoscopic live donor nephrectomy is safe for the donor and the transplant kidney. we believe that offering this technique for living renal donation can safely and effectively increase the pool of donor organs available to patients with end-stage renal disease. background. liver transplantation is the treatment of choice for end-stage acute and chronic liver failure. some liver diseases are associated with diseases of the intestinal tract such as primary sclerosing cholangitis and inflammatory bowel disease. moreover, post transplant immunosuppressive agents might cause colonic diseases and there is an abundance of opportunistic pathogens that can manifest in the large intestine. aim. we retrospectively analyzed the incidence and spectrum of colonic disorders in a cohort of 402 liver recipients and determined the impact of these complications on survival. methods. a total of 467 consecutive lts in 402 individuals were performed between 1998 and 2001 at the mayo clinic, jacksonville, florida. standard immunosuppression consisted of tacrolimus, mycophenolic acid, and rapidly tapered steroids. results. there were 29 patients transplanted for psc and 19 were also diagnosed with inflammatory bowel disease (ulcerative colitis, n = 16; crohn's disease, n = 3), with eight having undergone colonic resection prior to lt. in four patients, colitis persisted post transplant. six patients had a history of colonic resection for malignant (n = 3) or infectious diseases. five patients had pre-transplant endoscopic polypectomy. combined colon resection and transplantation were done in 2 patients; one with peritonitis and multiple colonic perforations during retransplantation and the other for ischemic colitis leading to fulminant liver failure. in another case a preexisting transverse colostomy had to be reinforced. there were 32 cases of clostridium difficile-associated enterocolitis. nine patients developed cmv gastrointestinal complications with three cases of colitis, one leading to perforation, intra-abdominal sepsis and death. two patients developed sigmoid diverticulitis and one appendicitis. colonic polyps were endoscopically removed in seven patients and three patients were diagnosed with colorectal cancer (one cecal, two rectal cancers), which all were surgically treated. chronic unexplained diarrhea was observed in fifteen patients, which led to withdrawal of mycophenolic acid. one patient developed a hemorrhage of the terminal ileum/cecal region in the course of intra-abdominal sepsis and was treated by endovascular embolization of the ileocolic artery. four patients had ongoing ulcerative colitis. one herpetic rectal ulcer and two perianal hsv-associated lesions were diagnosed. two patients developed hemorrhoids requiring surgical interventions, and two patients had perianal fistulas. conclusions. the frequency of colonic disorders in our series was higher than expected, with infections accounting for majority of cases. the high incidence of clostridial colitis warrants improvement in screening and preventive measurements. screening for polyps pre-transplant and annually post-transplant might be recommended. background. bartonella has been identified as causative agent of cat scratch disease but is also inflicted in other diseases in the immunocompromised host. case reports. we describe two cases of bartonella henselae-associated diseases in liver transplant recipients who both had contact with cats. the first recipient developed localized skin manifestation of bacillary angiomatosis in association with granulomatous hepatitis. he tested positive for igg antibodies against bartonella henselae. the second patient developed axillary lymphadenopathy, with biopsy showing necrotizing granulomatous inflammation and pcr studies were positive for bartonella henselae dna. her serology for bartonellosis showed a fourfold rise in antibody titers during her hospitalization. both patients responded to treatment with azithromycin in combination with doxycyclin. these were the only cases within a series of 467 liver transplants in 402 patients performed during a four-year period. conclusions. although bartonellosis is a rare infection in lt recipients, one should consider this disease in patients presenting with fever, cns symptoms, skin lesions, lymphadenopathy or hepatitis in particular if contact with cats is reported. background. new immunosuppressive protocols and advanced surgical technique resulted in a major improvement in the outcome of pancreatic transplantation. by reducing the incidence of immunological complications using intensified immunosuppressive protocols, the incidence of some infections, in particular complicated fungal infections, might increase. methods. 217 enteric drained whole-pancreas transplants (ptx) performed at the innsbruck university hospital between march 1997 and october 2004 were retrospectively analysed. prophylactic immunosuppression consisted of atg induction, tacrolimus, mmf, and steroids for the majority of cases. perioperative antimicrobial prophylaxis consisted of amoxicillin/clavulanic (30 ptx), pipercillin/tazobactam (157 ptx), and others (30 ptx). 168 patients additionally received fluconazole. results. actuarial patient, pancreas and kidney graft survival at one year were 96.4%, 88.5% and 94.8%, rejection rate was 30%. within this series, a total of 13 patients developed invasive fungal infections. of those, four had aspergillosis, one zygomycosis, and the remaining ten were caused by yeast. two patients had aspergillosis and later pulmonary infection with candida albicans and candida glabrata. the zygomycosis was the only fungal infection that was diagnosed post mortem and this patient had received pretreatment with caspofungin for non-albicans candida wound infection. one patient died due to aspergillosis following his second pancreas retransplant. three cases of aspergillosis were successfully treated using liposomal amphotericinb in one and a combination of caspofungin and voriconazol in two cases. this combination was also used in a patient who developed intra-abdominal infection with candida krusei. the remaining infections were due to candida albicans including six cases of intra-abdominal infection, one urinary tract infection, and one mucocutaneous candidiasis. type ii diabetics were found at increased risk for fungal infection. conclusions. fungal infections represent frequent and life-threatening complications after ptx. they are amongst the most common causes of graft loss and death. non-albicans candida strains are increasingly isolated and the incidence of filamentous fungal infections has increased during the study period parallel to a decreasing rejection rate. c57bl/6 (h-2 b ) mice received a total-body irradiation (tbi, d-1) of 3 gy and costimulation blockade consisting of anti-cd154 mab (1 mg, d0) vß11 + cd4-cells, p < 0.05). donor skin acceptance fty720 is the first agent in a new class of immunomodulators termed sphingosine-1-phosphate (s1p) human dc, which are known to express mrna for s1p 1 , s1p 2 , s1p 3 and s1p 4 , have not been described so far. methods. to elucidate for the first time the influence of s1p receptor agonists on human monocyte-derived dc (mo-dc), we used therapeutically relevant concentrations (2-200 ng/ml) of fty720 and its phosphorylated metabolite fty720-p and investigated their effects on dc surface marker expression (lineage markers, costimulatory and adhesion molecules, chemokine receptors), protein levels of s1p receptors and dc effector functions: antigen uptake ccl19/elc; or fty720-p-treated mdc (53% vs. 49%; untreated dc, 72%) 24-25 datenkonvertierung und umbruch: manz crossmedia druckerei ferdinand berger & söhne gesellschaft m. b. h., 3580 horn, österreich. -verlagsort: wien. -herstellungsort: horn. printed in austria p. b. b. / erscheinungsort: wien / verlagspostamt 1201 wien -fachkurzinformation rapamune 1mg bzw. 2mg überzogene tablette; rapamune 1 mg/ml bzw. 1mg/ 1ml bzw. 2 mg/2ml lösung zum einnehmen wirkstoff: sirolimus zusammensetzung: 1 tablette enthält 1 mg bzw. 2 mg sirolimus. 1 ml lösung enthält 1 mg sirolimus. 1beutel zu 1 ml bzw. 2 ml enthält: 1 mg bzw. 2 mg sirolimus. sonstige bestandteile: tablettenkern: laktose-monohydrat, macrogol, magnesiumstearat, talkum, tablettenüberzug: macrogol engmaschige überwachung der sirolimus-talspiegel im vollblut bei: leichter bis mittelgradiger leberfunktionsstörung; gleichzeitiger verabreichung starker cyp3a4-induktoren oder -inhibitoren sowie nach deren absetzen; bei absetzen oder deutlicher dosisreduktion von ciclosporin. begrenzte exposition gegenüber sonnen-und uv-strahlung bei patienten mit einem erhöhten risiko für hautkrebs. antimikrobielle prophylaxe gegen pneumocystis carinii pneumonie während der ersten 12 monate nach der transplantation sowie eine zytomegalievirus (cmv)-prophylaxe über 3 monate nach der transplantation ( insbesondere für patienten mit einem erhöhten risiko für eine cmv-erkrankung ) empfohlen. in kombination mit einem hmg-coa-reduktaseinhibitor oder fibrat überwachung auf entwicklung einer rhabdomyolyse und anderen nebenwirkungen dieser präparate. bei kombinierter gabe mit ciclosporin nierenfunktion überwachen, ggf. bei erhöhten serumkreatininspiegeln eine angemessene dosisanpassung erwägen. vorsicht bei gleichzeitiger anwendung von anderen substanzen, die bekanntermaßen eine schädigende wirkung auf die nierenfunktion haben erhöhte laktat-dehydrogenase (ldh), arthralgie, akne, infektion des harntraktes gelegentlich: pankreatitis, lymphom/lymphoproliferative erkrankung nach transplantation, panzytopenie. die immunsuppression erhöht die anfälligkeit, lymphome oder andere bösartige neubildungen, vor allem der haut, zu entwickeln fachkurzinformation zu inserat von umschlagseite 2 p08 liver transplantation for patients with hepatitis b-related liver disease: a single-center experience l. hinterhuber, i. w. graziadei background. liver transplantation (lt) is the only effective therapy for end-stage liver disease due to hepatitis b (hbv). before introduction of passive immunoprophylaxis with hepatitis b immunoglobulin (hbig) and new antiviral nucleoside analogues, hepatitis b recurrence was seen in the majority of the patients resulting in an inferior graft survival. in this study we analyzed the different clinical courses of hbv recurrence, the impact of hbv recurrence on patient survival, and potentially contributing factors for long-term outcome of hbv patients after lt.methods. between 1983 and 2003, 57 out of 692 patients (50 m, 7 f; mean age, 51 years) were transplanted secondary hbv cirrhosis at our center. the mean follow-up was 44 months (range, 1-246 months). immunosuppression (is) consisted of cya/fk 506, prednisolone and/or azathioprine/mmf. fourteen patients received no hbig (prior to 1993), 14 patients received hbig alone, and 29 patients hbig in combination with lamivudine (lam).results. the actuarial overall 1-, 5-, 10-year survival rates were 83%, 69%, and 65%, comparable to those of other indications. patients with combined prophylaxis showed the best survival rates (88%, 77%, 77%), compared to patients treated with hbig (82%, 51%, 51%) and patients without treatment (76%, 70%, 63%). five patients required reltx, one patient two reltx. in total, 17 patients (33.5%) developed recurrent hbv infection after lt: 50% (6 of 12) in the non-hbig group, 42% (6 of 14) in the hbig mono, and 18% (5 of 28) in the combined prophylaxis group. four of the five patients in the hbig/lam group were hbv dna positive prior to lt, two presented with lam mutants. hbv recurrence, however, did not negatively impact patient outcome. all patients with recurrent disease were treated with antivirals (famcyclovir, lam, adefovir). forty-seven percent of patients responded to the treatment and remained hbv dna negative. only one patient was retransplantated due to hbv recurrence. no possible risk factors for overall survival were found to be significant.conclusions. our study showed that patients with combined hbig/lam prophylaxis had excellent long-term survival. recurrent hbv in the allograft could be effectively treated in the majority of patients and did not influence longterm survival. background. liver failure is associated with reduced synthesis of clotting factors, consumptive coagulopathy, and platelet dysfunction. the aim of the study was to evaluate the effects of liver support using the molecular adsorbent recirculating system (mars) on the coagulation system in patients at high risk of bleeding.methods. 61 mars treatments in 33 patients with acute liver failure (n = 15), acute on chronic liver failure (n = 8), sepsis (n = 5), liver graft dysfunction (n = 3), and cholestasis (n = 2) have been studied. standard coagulation tests, standard thromboelastography (teg), heparinase-modified and abciximab-fab-modified teg were performed immediately before and 30 minutes after commencement of mars and after the end of mars treatment. to all patients, prostaglandin i2 was administered extracorporeally. 17 patients additionally received unfractioned heparin.results. three moderate bleeding complications in three patients, requiring 3-4 units of packed red blood cells, were observed. all were sufficiently managed without interrupting mars treatment. although there was a significant decrease in platelet counts (median, 9 g/l; range, -40 to 145 g/l) and fibrinogen concentration (median, 15 mg/dl; range, -119 to 185 mg/dl) with a consecutive increase in thrombin time, the platelet function, as assessed by abciximab-fab-modified teg, remained stable. mars did not enhance fibrinolysis.conclusions. mars treatment appears to be well tolerated in patients with marked coagulopathy due to liver failure. although mars leads to a further decrease in platelet count and fibrinogen concentration, platelet function, measured as contribution of the platelets to the clot firmness in teg, remains stable. according to teg-based results, mars does not enhance fibrinolysis. methodik. ein 69 jahre alter patient war vor 13 jahren aufgrund einer post-hepatitischen zirrhose (phcc) lebertransplantiert worden. nun war es im verlauf der letzten zeit zu einer deutlichen gewichtszunahme gekommen, sodass der patikey: cord-321053-lgae22f8 authors: gerold, gisa; pietschmann, thomas title: opportunities and risks of host-targeting antiviral strategies for hepatitis c date: 2013-10-04 journal: curr hepat rep doi: 10.1007/s11901-013-0187-1 sha: doc_id: 321053 cord_uid: lgae22f8 hepatitis c virus (hcv) infects more than 2 % of the world population with highest prevalence in parts of africa and asia. past standard of care using interferon α and ribavirin had adverse effects and showed modest efficacy for some hcv genotypes spurring the development of direct acting antivirals (daas). such daas target viral proteins and are thus better tolerated but they suffer from emergence of vial resistance. furthermore, daas are often hcv genotype specific. novel drug candidates targeting host factors required for hcv propagation, so called host-targeting antivirals (htas), promise to overcome both caveats. the genetic barrier to resistance is usually considered to be high for htas and all hcv genotypes presumably use the same host factors. recent data, however, challenge these assumptions, at least for some htas. here, we highlight the most important host-targeting strategies against hepatitis c and critically discuss their opportunities and risks. hepatitis c virus (hcv) chronically infects more than 2 % of the world's population with highest incidents in africa and asia [1] . out of these patients 20 % will develop severe liver disease 10 to 25 years after contraction [2] . as a result, chronic hepatitis c is the number one indication for liver transplantation in many countries. as a member of the family of flaviviridae hcv particles bear a plus strand rna genome and a glycoprotein decorated envelope. hcv can be classified into six epidemiologically relevant genotypes and many subtypes with intergenotypic sequence variability at the nucleotide level of greater than 30 % [3] . this high diversity of hcv is caused by a fast replication rate combined with an error prone replication machinery. owing to its pronounced genomic variation, hcv can evade immune recognition and become resistant to antiviral drugs. after the discovery of hcv as the etiological agent causing non-a non-b hepatitis in 1989 [4] , chronic hepatitis c patients were initially treated with interferon-α (ifnα). this original treatment was effective in only a fraction of treated patients, was poorly tolerated and required medication for 48 weeks. during the 1990s, ifn based therapy regiments were refined by addition of the nucleoside analogue ribavirin, and usage of pegylated ifnα (peg-ifnα) derivatives increasing viral response rates [5] . after construction of the first infectious clone in 1997 [6] and the creation of the hcv replicon system [7] development of novel improved therapeutic strategies significantly gained momentum. a search for better drugs concentrated on direct acting antivirals (daa), i.e., agents that specifically interfere with viral proteins required for propagation. new cell culture models for a genotype 2a isolate that permit analysis of the complete viral replication cycle in cell culture aided these efforts [8] [9] [10] . since 2011, the first two daas, which are inhibitors of the viral protease ns3/4a, boceprevir and telaprevir, are on the market and achieve response rates of8 0 % in genotype 1 patients, when combined with ribavirin and peg-ifnα. however, although this triple therapy has clearly improved response rates and shortened treatment duration, side effects, costs and exclusive licensing for genotype 1-infected patients limit its application. fortunately, other promising virus-targeting drugs are in clinical trials, e.g., inhibitors of the ns5a phosphoprotein and the ns5b viral rna-dependent rna polymerase. two major shortcomings of virus-targeting daas are the emergence of resistance mutations and -at least for several classes of daas (e.g., protease inhibitors)-a pronounced genotype-dependent efficacy. numerous in vitro studies in combination with a growing number of hcv sequencing data from patients undergoing daa treatment underline that the virus can develop drug-resistance and fitness restoring compensatory mutations [11] . thus, daas will typically be used in combination therapy as in the current standard of care, which includes one of the two available protease inhibitors combined with peg-ifnα and ribavirin. again, as ribavirin and ifn are contraindicated in many patients and moreover cause harsh side effects that limit compliance, current drug development aims for future ifn-sparing and possibly also ribavirin-free combination-therapy regimens. in fact, numerous combination therapies involving daas with different targets on the virus are in clinical development (compare www. clinicaltrials.gov). it is expected that these trials will identify optimal drug combinations. given the variability of hcv combined with the heterogeneity of patients with regards to co-morbidities, degree of liver disease and genetic background, it is likely that several combination therapy regimen will evolve, which are tailored toward specific patient and virus groups. an emerging third group of antivirals, so called hosttargeting antivirals (hta), may be part of such future combination therapies, in particular as htas hold the promise of overcoming some of the caveats of daas. htas are antibodies, rnas or small molecules, which interfere with host factors needed for hcv propagation. intense research in the past decade revealed many molecular details of the hcv life cycle, including the usage of human proteins and a microrna during entry, genome replication, particle assembly and/or release [12, 13•, 14] . this knowledge allowed targeted design of numerous htas, some of which with promising clinical trial results (table 1) . host-targeting antiviral strategies are assumed to have two major advantages: first, the resistance barrier to host-targeting therapies is supposed to be high since host factors are genetically stable. exceptions to this will be critically discussed in the specific chapters below. second, usage of host factors is thought to be independent of the hcv genotype and thus htas should have pan-genotypic activity. importantly, this assumption has not been fully supported by experimental evidence yet, as culture systems for genotypes other than genotype 1 and 2 were not available until interference with hcv entry factors is a strategy to block de novo infection of naïve cells (fig. 1) . therefore, they are promising drugs for preventive therapy in chronic hepatitis c patients, who undergo liver transplantation. indeed, re-infection of liver graft tissue is universal and unfortunately disease progression after transplantation is commonly accelerated. for hepatitis c treatment, virus-targeting entry blockers, i.e., neutralizing antibodies against the hcv glycoprotein e2, are unfavorable, as e2 is the most variable among the hcv proteins. due to the error prone viral replication, an infected individual carries a swarm of related viral variants (quasispecies), which may comprise viruses that escape neutralizing antibodies. consequently, there is a strong need for the development of htas interfering with virus entry. the most advanced entry-blocking compound is a small molecule inhibitor of scarb1 termed itx 5061 [44] . this orally bioavailable drug showed a good safety profile in clinical trials with 280 subjects [44] . in a clinical phase 1b study in treatment naive chronic genotype 1 patients only 1 of 7 patients showed a virological response [45] . however, in a post-transplant setting a better efficacy profile is possible. in preclinical tests using hcv pseudotypes, itx 5061 had broad specificity for genotypes 1 through 6 confirming that scarb1dependence is conserved among viral genotypes and raising the hope that scarb1-targeting molecules like itx 5061 could be pan-genotypic hcv entry inhibitors [44] . binding studies involving soluble truncated e2 protein indicate that itx 5061 interferes with the interaction of e2 with scarb1, which is likely the reason for its antiviral activity [44] . in vitro resistance selection revealed that a single nucleotide change leads to an amino acid substitution in hcv e2 (n415d) which renders hcv insensitive to itx 5061 [46] . on the one hand, this demonstrates that hcv can in theory evade hta therapy by mutating the viral binding partner of the targeted host factor and in fact suggests a low genetic barrier to resistance. on the other hand, the n415 residue is highly conserved among sequenced hcv isolates and only one in 1300 reported sequences in the los alamos national laboratories hcv database shows aspartic acid at this position [46] . notably, the same mutation was reported upon long term passage of the jfh1 virus in tissue culture [47] and further characterization revealed that it rendered the virus highly susceptible to neutralization by serum igg from chronically hcv-infected patients. therefore, immunemediated constraints may prevent the virus from developing this type of itx 5061 resistance in vivo. moreover, the n415d itx 5061 resistant virus was still sensitive to protease inhibitors, arguing that itx 5061 could be used in combination with virus-targeting daas. finally, as patients will receive itx 5061 only for a comparably short duration in a post-transplant setting, the risk for resistance emergence may be low. nevertheless, careful monitoring of viral variants emerging during in vivo application of this compound will be critical. apart from drug resistance, an expected caveat of targeting host molecules and possibly their endogenous function are side effects. those appear minimal for itx 5061. the only reported but not adverse side effect of itx 5061 is the inhibition of scarb1 mediated high density lipoprotein (hdl) uptake leading to increased serum hdl levels [48] . in fact, itx 5061 was originally tested as an anti-atherogenic compound. lastly, it remains to be determined whether host genetic variance of scarb1 could affect itx 5061 inhibition. a recent study suggests that several lab-generated variants of scarb1 all efficiently support hcv cell entry [49] . given that itx 5061 competes with hcv for scarb1 binding, it is likely that the compound inhibits all scarb1 variants, but future work, in particular on naturally occurring scarb1 variants, will clarify this assumption. in summary, itx 5061 is a promising small molecule, pangenotypic hta for preventive combination therapy of posttransplant hepatitis c patients. apart from small molecules, entry factor blocking antibodies efficiently inhibit hcv entry and are in preclinical development [41] [42] [43] 50 ]. the most advanced antibodies target scarb1 and cd81. in a recent report, two neutralizing antibodies against scarb1 (mab8 and mab151) blocked hcv infection and direct cell-to-cell spread in vitro and in vivo [43] . notably, in a fig. 1 hcv life cycle and critical host factors for intervention. hcv requires host proteins and rnas during its life cycle offering several points of intervention using htas. initially, hcv-lipoviroparticles attach to ldl-r and heparan sulfate proteoglycans (hspg) on the surface of hepatocytes, followed by a coordinated uptake requiring the essential entry factors scarb1, cd81, cldns and ocln. the latter four entry factors can be targeted by specific antibodies or small molecules, like itx 5061, which blocks hcv-scarb1 interactions and is in clinical phase 1b development. accessory factors including receptor tyrosine kinases, transferrin receptor 1 (tfr1) and the cholesterol receptor npc1l1 provide alternative targets for entry blockage. after internalization and particle uncoating, the hcv rna genome replicates in specialized cytosolic compartments, termed membraneous webs. formation of these replication complexes is aided by host and viral proteins. inhibition of the host chaperone cypa by cyclosporine and its derivatives (alisporivir, scy-635, nim811) affects function of the viral ns5a protein and thereby inhibits hcv replication. alisporivir, scy-635, nim811 are currently tested in clinical phases 3, 2 and 1, respectively. pi4kiiiα is a host lipid kinase required for membraneous web formation and its inhibition by small molecules, like al-9, abolishes hcv replication. a third class of replication htas is comprised by antagomirs of the host mir-122. this microrna binds to and stabilizes the hcv rna genome and facilitates its translation. moreover, an involvement of mir-122 in hcv rna replication is being discussed. sequestration of mir-122 by miravirsen, a clinical phase 2 hta, strongly suppresses viral titers human liver xenotransplant mouse model (upa-scid) both anti-scarb1 antibodies prevented hcv infection in a prophylactic setting, when five antibody dosages were given over a period of two weeks starting one day prior to infection. however, viral rebound occurred at least one week after termination of antibody treatment, suggesting that a two-week treatment is insufficient to eliminate virus from peripheral reservoirs. in a three-day postinfection setting, the antibodies prevented infection in three out of five mice and reduced viral dissemination in the remaining two mice. similar to itx 5061, scarb1 antibodies are pangenotypic and show no adverse side effects, thus presenting an alternative option for preventive therapy. future studies will have to show whether anti-scarb1 therapy is efficient in a broad range of orthotropic liver transplant patients. in contrast to scarb1 antibodies, cd81 antibodies only block hcv infection in a prophylactic setting in the xenotransplant upa-scid mouse model [41] , suggesting that targeting scarb1 is superior to blockage of cd81. although the reason for this is not clear, this discrepancy may point to a differential role of these entry factors during virus transmission. in vitro data indicate that hcv is transmitted by cell-free, secreted virus particles and also by direct cell-to-cell transmission between neighboring cells [51] [52] [53] . this latter mode of infection may be particularly relevant in vivo and could facilitate viral escape from neutralizing antibodies. notably, while cd81 is absolutely essential for infection by cell-free virus, its importance for direct cell-to-cell transmission has been disputed [52, 53] . on the one hand, fofana et al. identified a cd81 antibody, whichat least in vitro-also interferes with cell-to-cell spread [54] . the epitope for this antibody currently remains elusive, but this finding raises the hope that development of cd81 antibodies for post-infection treatment could be possible. on the other hand, recent evidence confirmed that cd81 facilitates, but is not absolutely essential for, cell-to-cell transmission in vitro [55] . if that holds true in vivo, the absence of an essential role for cd81 during cell-to-cell spread may limit efficacy of cd81 antibodies compared to antibodies targeting entry factors critical for both cell-free and cell-to-cell transmission. as mentioned above, scarb1 neutralizing antibodies potently repress cell-to-cell transmission in vitro [53] and in humanized mice [42, 43] . taken together, these results suggest that scarb1 is more important for direct cell-to-cell spread than cd81, which in turn explains why targeting scarb1 blocks hcv infection in vivo more effectively than cd81. antibodies binding cldn1 efficiently block hcv entry in vitro [50] and may thus be another promising avenue for clinical development. notably, cldn6 and cldn9 can substitute for lack of cldn1 in human non-liver cells [56, 57] . although it was originally assumed that this broad tropism toward different members of the cldn protein family is common to all hcv isolates, this notion was recently challenged: using cell lines expressing either only cldn1, cldn6 or both molecules, haid et al. showed that all tested viral strains efficiently used cldn1 while only some strains also used cldn6 [58] . importantly, viruses capable of using both cldn1 and cldn6 were not fully neutralized by cldn1-specific antibodies, if the same cell co-expressed a modest level of cldn6. these findings point toward a possible viral escape from cldn1-targeting agents for viral strains with broad cldn-tropism. however, fofana et al. were unable to observe an additive inhibitory effect when combining cldn1 and cldn6-specific antibodies on huh-7.5.1 cells challenged with hcv [54] . this discrepancy might result from the use of host cell lines with different cldn1 and six expression levels. notably, primary human hepatocytes of some donors express low but detectable cldn6 at the cell surface and cldn6 transcript expression is highly variable in hcv patient derived liver biopsies [54, 58] . thus, the potential risk of viral escape from cldn1-targeting strategies via use of cldn6 may not only depend on the viral strain but also on host determinants, i.e., differential abundance of cldn6. clearly, the relevance of viral cldn tropism and its potential implication for development of cldn1-targeting strategies requires further investigation, e.g., in xenotransplanted mice with human hepatocytes from donors with distinct cldn6 expression. aside from these aspects related to efficacy and potential viral escape, possible side effects of anti-cldn treatments should be carefully considered. currently, in vitro data suggests that cldn1 endogenous functions remain unaltered upon antibody treatment [54] . consequently, side effects of anti-cldn1 treatment are expected to be low. confirmation of this assumption in preclinical models will be critical before finally evaluating cldn1-targeting therapy. for the fourth essential entry factor, ocln, no neutralizing antibodies have been reported to date. however, as ocln deficient mice have a severe phenotype including growth retardation, infertility and bone thinning, preclinical tests need to carefully investigate possible side effects of ocln-targeting in humans [59] . in conclusion, small molecule inhibitors and antibodies targeting scarb1 are the most advanced hcv entry-targeting agents. given the essential and pan-genotypic role of scarb1 for both cell-free and cell-to-cell transmission, scarb1 blockage holds the promise for a well-tolerated therapy to prevent allograft infection of chronic hepatitis c transplant patients. apart from the four essential entry factors, additional molecules have been shown to be involved in hcv cell entry. these include attachment factors like glucosaminoglycans [34, 60] and the low density lipoprotein receptor (ldl-r) [61, 62] , receptor tyrosine kinases [63•] , and nieman-pick c1-like 1 and transferrin receptor 1 [64, 65] . this multitude of host factors offers numerous levels for interference (fig. 1) . among the most prominent entry co-factors are the two receptor tyrosine kinases epidermal growth factor receptor (egfr) and ephrin a2 (epha2) [63•] . inhibition of egfr and epha2 using the small molecules erlotinib and dansatinib blocks hcv entry in vitro with an ic50 of 500 nm for both compounds. in a human liver chimeric mouse model daily preventive erlotinib treatment reduces serum titers of hcv approximately ten-fold compared to placebo treated animals. viral titers rebound to serum titers of control animals shortly after treatment termination. together, these findings provide evidence that erlotinib at least partially represses hcv infection in vivo. moreover, in vitro erlotinib inhibits both cell-free and cell-to-cell transmission of hcv and silencing of egfr suggest that the usage of egfr is hcv genotype independent [63• ]. if, however, erlotinib is able to fully ablate hcv cell entry in vivo and whether the virus can escape this type of treatment by resistance mutations is currently not clear. adverse effects of erlotinib, which is an approved anti-cancer drug, include rashes, diarrhea, lung, liver and kidney problems [66] . occurrence of side effects is in line with the fact that egfr is ubiquitously expressed and has important physiological functions including cell proliferation, migration and adhesion. while the intensity of side effects associated with erlotinib and many other anti-cancer agents may be acceptable in malignant disease, it may preclude their use for a not quite as immediately life threatening condition such as chronic hcv infection. lastly, polymorphisms in egfr seem to correlate with the outcome of erlotinib treatment in non-small cell lung cancer, suggesting that host genetic variance could also influence antiviral therapy outcome [67] . in summary, targeting of conserved host factors with less critical endogenous functions seems favorable over interference with receptor tyrosine kinases. novel mechanistic studies on the role of egfr revealed that it signals through the gtpase hras and the kinase braf for hcv receptor complex assembly [68] . small molecule inhibitors of both signaling molecules exist and are licensed for anti-cancer therapy. we currently lack preclinical in vivo data for their usefulness in anti-hcv therapy. in principle, targeting hras and braf offers an additional mode of intervention with hcv cell entry, possibly with superior efficacy and/or reduced side effects when compared with erlotinib. a second and less frequently discussed hcv entry modulator is the cholesterol receptor niemann-pick c1-like 1 (npc1l1) [64] . this protein is a 13 transmembrane molecule, which is expressed on the apical side of hepatocytes and mediates cholesterol absorption. in vitro the small molecule npc1l1 inhibitor ezetimibe decreased hcv infection at least five-fold at a 30 μm dose. in a human liver chimeric mouse model ezitimibe prevented infection of two out of seven mice when a three-week dosing was started two weeks prior to infection. treatment onset two days prior to infection surprisingly did not prevent hcv infection of mice. although npc1l1 seems to reduce entry of all hcv genotypes and although ezitimibe is a licensed cholesterol-lowering drug with little side effects [69] , the overall modest efficacy in mice does not favor application of npc1l1 inhibitors in monotherapy. however, in conjunction with other htas or daas ezitimibe may be useful to improve treatment efficacy. taken together, targeting hcventry modulators seems less efficient than targeting of bona fide entry factors, likely due to their accessory rather than essential role during hcv cell entry. still, compounds with high tolerability could be a valuable addition in combination therapy, in particular after orthotropic liver transplantation. targeting hcv rna replication: cyclophilin a while blockage of hcv entry into hepatocytes is a promising strategy to prevent de novo infection of naïve cells, targeting hcv replication, i.e., amplification of the viral genome, holds the promise of efficiently eradicating hcv from already infected tissue. hcv replication takes place in the cytoplasm of the host cell, where virus encoded rna-dependent rna polymerase (ns5b) amplifies the plus strand rna genome. to this end, hcv induces with the aid of host factors specialized membranous compartments [70] [71] [72] [73] , at which multiple viral proteins including non-structural proteins ns3, ns4a, ns4b, ns5a and ns5b and host factors assemble the hcv replication complex [74] . hence, induction, assembly and function of hcv replication complexes involve numerous host factors offering multiple targets for intervention. cyclophilin b (cypb) was one of the first host factors reported to be crucial for hcv replication [75] . cyclophilins are highly conserved peptidyl-prolyl isomerases, which catalyze the isomerization of peptide bonds at proline residues from trans to cis. such transformations either aid folding of newly synthesized proteins or change the structure of already folded proteins. interestingly, cyclophilins are essential replication factors for a number of viruses, including hiv, herpes simplex virus, vaccinia virus, vesicular stomatitis virus and coronavirus. in the context of hcv replication, more recent evidence indicates that cyclphilin a (cypa) rather than cypb is used by hcv as co-factor and that the isomerase activity of cypa is essential for this function [76] [77] [78] [79] . moreover, biochemical and genetic analyses revealed an interaction between subdomains of ns5a and cypa [80, 81] , which could promote viral protein folding, regulate polyprotein processing and thereby facilitate rna replication. targeting of cypa by the cyclic polypeptide immunosuppressant agent cyclosporine a (csa) prevents the interaction of cypa and ns5a across all viral genotypes and has strong anti-hcv activity in vitro (fig. 1) [82, 83] . csa is not only antiviral, but also immunosuppressive, since the csa-cypa complex inhibits calcineurin and thereby suppresses t helper cells [84, 85] . this dual role stimulated the development of derivatives, which retain antiviral activity without being immunosuppressive. currently, numerous cypa inhibitors with exclusive antiviral effect are in preclinical and clinical trials [86] . among these, alisporivir (debio 025), nim811 and scy-635 are the most extensively studied drug candidates. alisporivir showed efficient reduction of viral load (2-log to 4-log depending on the study) for genotype 1, 2, 3 and 4 during monotherapy [87] [88] [89] . moreover, combination with infα/ribavirin resulted in additive effects. in a phase 2 clinical trial with such combined triple therapy the incidence of adverse effects was low. thereafter, a phase 3 trial in treatment-naïve genotype 1 patients was initiated, but soon halted by the fda due to occurrence of pancreatitis with one fatal outcome upon treatment with ifn and alisporivir. therefore ifn/alisporivir combinations are precluded from future clinical development. however, trials with ifn-free alisporivir treatment regimens with improved safety profile will resume. apart from its broad hcv genotype specificity alisporivir seems to act independently of the host genetic background. a recent study investigated host variability of cypa and found that rare nonsynonymous snps in cypa not only rendered cells largely resistant to hcv infection, but also residual replication was still sensitive to cypa inhibition [90] . lastly, cypa inhibitors appear to have a high genetic barrier to development of viral resistance in vivo. however, in vitro resistance toward anti-cyclophilin compounds can be selected either by long term viral passage in the presence of the drugs or by selecting viruses in host cells with reduced cyclophilin abundance [78, 91, 92] . currently described resistance mutations map to domain 2 and 3 of ns5a supporting the concept that cypa facilitates hcv replication via modification of ns5a function. interestingly, some of these resistance mutations are located at the c-terminus of ns5a close to the cleavage site between ns5a and ns5b. moreover, these alterations were shown to modulate polyprotein cleavage at the ns5a-ns5b site. this supports the notion that cyclophilin a fine tunes protein folding processes that are required for optimal polyprotein cleavage and in turn replication complex assembly [78] . although some of the mutations described in vitro confer a high degree of viral resistance (in part shifting the ec50 value by more than 40-fold [91] ), so far no resistance mutations have been described during treatment of hcv patients. in fact, during alisporivir monotherapy no viral breakthrough was observed suggesting that in vivo the barrier to viral resistance is high [93] . notably, in the same study one alisporivir null responder was identified. if, however, absence of viral response in this case was due to viral or host resistance is not clear and merits further investigation. a possible explanation for this rare treatment failure could be a recently reported second antiviral mechanism of cyclophilin derivatives, which is independent from cypa-ns5a complex disruption. the alternative non-immunosupressive cypa inhibitor, scy-635, which is currently in clinical phase 2a trials, seems to reconstitute ifn signaling and in turn increase innate antiviral defenses. first evidence for this stems from a clinical trial with scy-635 monotherapy in chronic hcv genotype 1 infected patients. in this context, scy-635 not only dose dependently repressed viral load but also caused increased plasma levels of ifnα, ifn λ 1 and 3 as well as 2´5´oligoadenylate synthase 1 (2´5´oas-1), a key ifn stimulated gene (isg) [94••] . meanwhile, two possible molecular links between scy-635, ifn and isg induction were disclosed: bobardt and colleagues reported that cypa binds ifn regulatory factor 9 (irf9) and that ns5a competes with irf9 for cypa binding [95] . as irf9 is the dna binding part of the transcription factor ifn-stimulated gene factor 3 (isgf3), it is critical to transmit ifn-induced jak/ stat signaling to the nucleus for expression of isgs [96] . importantly, inhibition of cypa by cyclosporine prevents irf9-cypa complex formation and thereby enhances ifninduced expression of isgs [95] . on the other hand, watashi et al. noted that in ifn-treated hcv infected cells, scy-635 decreases phosphorylation of protein kinase r (pkr) [97] . since phosphorylated pkr downregulates expression of isg at the level of translation, it is possible that scy-635-dependent repression of pkr phosphorylation enhances translation of isgs [98] . therefore, blockade of cypa by scy-635 may increase expression of isgs and antiviral activity of ifn by both transcriptional and post-transcriptional mechanisms. it will be interesting to dissect to what extent these mechanisms contribute to the antiviral activity of cypa-targeting strategies and if both antiviral mechanisms are shared by the different compounds targeting cypa. provided concerns regarding the safety of cypa-targeting htas can be eliminated, these agents could be attractive pan-genotypic therapeutics. targeting hcv rna replication: phosphatidylinositol 4-kinase iii alpha (pi4kiiiα) genome wide rna interference screens and in depth cell culture replication assays with hcv replicons and full length infectious virus have revealed numerous additional host dependency factors, that could in principle serve as antiviral targets [99] [100] [101] [102] [103] [104] [105] [106] [107] . one of the most prominent and most consistently identified host factors for hcv replication is pi4kiiiα [101] [102] [103] [104] [105] [106] . this protein belongs to a family of enzymes that catalyze phosphorylation of lipids at position four of their inositol moiety. the resulting phosphoinositides (pis) reside at the cytosolic leaflet of vesicle and organelle membranes, where they play a critical role in the recruitment and activity of signaling proteins [108, 109] . to date, four mammalian phosphatidylinositol 4-kinases are known (pi4kii α and β and pi4kiii α and β), which differ in their subcellular localization and create distinct pi pools, thus contributing to vesicle trafficking and lipid transport [108] . pi4kiiiα is located at the endoplasmic reticulum and the plasma membrane and was found to be the primary mammalian pi4k that influences hcv replication [102] . notably, silencing of pi4kiiiα dramatically reduces hcv rna replication and concomitantly results in a clustered distribution of viral non-structural proteins and aberrant ultrastructure of the membranous web [105] , which is an accumulation of membrane vesicles and the site of hcv rna replication [70, 71] . interestingly, hcv ns5a directly interacts with pi4kiiiα and this interaction stimulates kinase activity of the enzyme [102, 105] . more recently, the binding site of pi4kiiiα was mapped to a highly conserved region within domain 1 of ns5a. moreover, pi4kiiiα, although being a lipid kinase, was reported in the same study to modulate the phosphorylation status of ns5a [110] . although it is currently unclear if this effect is direct or indirect, likely both the pi4kiiiα-dependent regulation of ns5a phosphorylation and local accumulation of phosphatidylinositol 4-phosphate pools are important for hcv replication [110] . since reduced levels of pi4kiiiα and viral ns5a mutations ablating the interaction with pi4kiiiα both cause aberrant ultrastructure of hcv replication complexes, it is reasonable to assume that the pi4kiiiα-ns5a interplay is essential for proper assembly and function of membrane bound hcv replication complexes. to date, two studies have addressed if targeting the interaction between ns5a and pi4kiiiα or the enzymatic activity of pi4kiiiα itself would be a useful strategy to control hcv rna replication. bianco et al. reported that al-9, a 4-anilino quinazoline, targets the kinase activity of pi4kiiiα in vitro and within liver cells [111] . interestingly, 4-anilino quinazoline compounds had previously been reported as hcv inhibitors [111] [112] [113] [114] , and based on putative resistance mutations within ns5a, presumed to target ns5a. until now, none of these mutations was however shown to confer resistance to quinazolines [111, 112] . nevertheless, given the interaction between pi4kiiiα and ns5a and the observation that aniline quinazoline moieyties are frequently present in kinase inhibitors, it seems likely that al-9 targets pi4kiiiα and thereby inhibits hcv replication. additional compelling evidence that pi4kiiiα-inhibitors interfere with hcv replication was reported by vaillancourt [115•] . using a pi4kiiiα kinase assay to screen more than 500,000 compounds various specific inhibitors of this enzyme were identified. molecules belonging to three different chemotypesall of which are unrelated to the 4-anilino quinazoline compounds described abovepotently repressed kinase activity in vitro and hcv replication in replicon assays. importantly, inhibition of other kinases was excluded by in vitro assays involving various kinases and lipid kinases. these studies also revealed 15 to 20-fold selectivity toward pi4kiiiα compared to pi4kiiiβ. besides this, the antiviral activity of the compounds correlated with the degree of pi4kiiiα inhibition further supporting the notion that the compounds are antiviral due to blockade of pi4kiiiα. finally, viral resistance was selected and revealed that specific mutations within the c-terminus of ns4b and domain 1 of ns5a reduce sensitivity of hcv to these molecules by up to 20-fold. interestingly, the resistance mutations permitted efficient hcv replication in cells with silenced pi4kiiiα highlighting that they confer reduced dependence of hcv on functional pi4kiiiα and indeed that the compounds target pi4kiiiα. combining these observations with the ultrastructural analyses of replication complexes from cells with pi4kiiiα silencing or mutant viruses with reduced pi4kiiiαbinding, it is reasonable to assume that recruitment of pi4kiiiα by ns5a and the enzymatic activity of pi4kiiiα are crucial for proper assembly/morphology and function of hcv replication complexes (fig. 1) . inhibitors of pi4kiiiα disturb ns5a binding and viral resistance to these molecules is attained by reduced dependence on pi4kiiiα due to altered function of ns5a and/or ns4b. of note, kinase inhibitor resistance mutations decreased hcv rna replication in huh-7.5 with endogenous levels of pi4kiiiα. thus, resistance is likely linked to a decrease in viral fitness, which may contain emergence of these mutations in vivo. to explore the physiological role of pi4kiiiα in vivo, villaincourt et al. created knockout mice with conditional lesion of the pi4kiiiα gene locus [115•] . unfortunately, induction of the gene defect in homozygous animals caused lethal gastrointestinal disorders. therefore, the critical physiological role of pi4kiiiα and possible side effects of therapies targeting this enzyme will probably limit further development of this class of inhibitors for future hcv therapy. targeting hcv rna replication: microrna-122 apart from proteinaceous host factors, hcv requires a microrna for efficient replication. micrornas (mirnas) are 20 to 22 nucleotides long non-coding rna molecules, which typically bind to mrna and arrest translation or induce mrna cleavage and degradation, thus having emerged as powerful regulators of gene expression [116] . not surprisingly, viruses have evolved to exploit this cellular machinery. in fact, several dna viruses such as herpesviruses encode viral mirnas and use these for tuning expression of both host and viral rnas [117] . although hcv, like most rna viruses, does not encode mirnas itself, it depends on these rna molecules in a unique way, thus offering a potential target for antiviral intervention. specifically, mirna-122 (mir-122), a liver-specific mirna, that regulates numerous genes involved in fatty acid and cholesterol metabolism, binds to the 5´non translated region of the hcv rna genome. two tandem binding sites for mir-122 have been characterized [118] and the site specific binding of mir-122 has been reported to facilitate translation of the viral rna [119] and to stabilize the hcv rna leading to an accumulation of vial genomes [120] [121] [122] [123] . although presence of mir-122 is not absolutely essential for hcv rna replication, its high abundance is crucial for efficient replication [124] . thus, the liver-specific expression of mir-122 likely contributes to the overt hepatotropism of hcv [125, 126] . inactivation of mir-122 using a complementary locked nucleic acid-modified oligonucleotide (miravirsen or spc3649) reduces hcv titers in vitro and in hcv infected chimpanzees by 2-3 logs (fig. 1) [127••] . moreover, broad hcv genotype specificity in vitro suggests a wide usage for a miravirsenbased therapy in patients [128] . genetically engineered variants of hcv, which lack the mir-122 binding site, are resistant to miravirsen and still viable although showing fitness loss [128] . notably, in monotherapy clinical trials and in cell culture so far no resistance mutations to miravirsen emerged [129] . therefore, the barrier to viral resistance to this drug seems high. with regard to side effects, preclinical studies in chimpanzees suggest that miravirsen treatment reduces serum cholesterol levels, but neither induces toxicity nor histopathological changes. more importantly, in a recent phase 2a clinical trial five weekly injections of miravirsen reduced viral titers up to 3 logs without adverse side effects or resistance emergence [130••, 131••] . in fact, some treated patients cleared hcv rna during miravirsen monotherapy. furthermore, miravirsen treatment both in chimpanzees and humans elicited a continuous and prolonged antiviral effect that lasted for several weeks after cessation of therapy [127••] . while miravirsen administration is currently only possible through the less attractive parenteral route, an advantage of miravirsen therapy could be the longlasting effect. pharmacokinetic patient studies reveal a 37 day plasma half-life and suggest that miravirsen may be administered only once per months [132] . collectively, in vitro and in vivo data provide firm evidence that targeting mir-122 is an efficacious and -at least in these transient treatment regimens-well tolerated future therapeutic option. in spite of the encouraging initial results, recent findings regarding the physiological role of mir-122 warrant caution: mice lacking mir-122 are viable but develop steatohepatitis, fibrosis and hepatocellular carcinoma. importantly, reconstitution of mir-122 reduces tumor incidence demonstrating that mir-122 acts as tumor suppressor in mice [133] . to date, the molecular details of mir-122´s anti-tumor activity are unclear. certainly, more research is needed to exclude that transient sequestration of mir-122 could promote tumorigenesis. if severe adverse side effects occur, the long half-life of miravirsen will make its serum levels difficult to control, in particular since there is no readily available means of terminating the drug's action. apart from this note of caution regarding mir-122 as host target, it will be interesting to explore if the association of chronic hcv infection with hepatocellular carcinoma is connected with the virus usurping and sequestering mir-122. in conclusion, miravirsen is a pan-genotypic, effective hta with low risk of resistance emergence. critical evaluation of adverse effects will clarify if miravirsen will be part of future inf-free regimen against hcv. cell based assays to dissect the pathways and steps of hcv particle assembly and virus release have been available only for a relatively short time [8] [9] [10] . therefore, host-targeting antiviral strategies focusing on these late stages of the viral replication cycle are least advanced. the first reported assembly blockers were iminosugars, which target α-glucosidase i, an er enzyme required for hcv glycoprotein folding and maturation [134] [135] [136] . however, comparably modest efficacy of the iminosugar celgosivir in genotype 1 patients led to termination of clinical trials [137] . nevertheless, more recent reports have highlighted several cellular co-factors that assist virus production and that may be future targets for antiviral therapies. it has been known for a long time that hcv travels through the blood stream in tight association with lipoproteins [8] . moreover, careful proteomic analysis of serum-derived hcv revealed the presence of apolipoprotein e (apoe), apoc1, apob, and apoa1 in the lipoviroparticle [8, 138, 139] . for cell culture derived hcv (hcvcc) apoe, and apoc1, have been reported to associate with particles and the lipid composition of hcvcc was found to resemble the one of very low density lipoprotein (vldl) [140] . given this interplay of hcv with lipoproteins, it was not surprising that host factors involved in assembly and release of vldl seem to aid production of infectious viral progeny: specifically, microsomal triglyceride transfer protein (mttp), which is involved in loading of lipids onto nascent apob, as well as apob and apoe, both components of vldl, were reported to contribute to virus production. as a consequence, modulators of vldl production and secretion emerged as potential antivirals. for some of these compounds including mttp and apob inhibitors, preclinical results are available and show modest antiviral activities. notably, recent evidence suggests that among the different factors of the vldl pathway implicated in hcv virus production, only apoe is absolutely essential, since production of infectious hcv can be reconstituted in an engineered human kidney derived cell line (293 t), which does not produce vldl and lacks endogenous expression of mttp and apob [141] . although these hcv particles with minimal lipoprotein coat may not be as infectious as natural hcv it is therefore possible that hcv could escape mttp and apob targeting strategies. on the other hand, these findings favor development of apoetargeting strategies. moreover, apoe associated with hcv particles plays a critical role during viral cell entry by facilitating attachment of virions to cellular heparin sulfate proteoglycans [142] [143] [144] , so that apoe-targeting compounds may arrest both assembly and entry of hcv particles. besides host factors of the vldl pathway additional cellular proteins have recently been shown to contribute to virus production. for instance, cellular lipid modifying enzymes like diacylglycerol acyl transferase 1 (dgat1) and the cytosolic phospholipase a2 (pla2ga4) contribute to production of infectious hcv progeny [145, 146] . specifically, dgat1 catalyzes triglyceride biosynthesis and thereby promotes lipid droplet formation. hcv is thought to assemble on the surface of lipid droplets and in vitro dgat1 inhibition or silencing was shown to reduce hcv genotype 2a assembly and release by limiting the trafficking of hcv core to these orgenelles [145] . intriguingly, upon inhibition of dgat1, the related enzyme dgat2 seemed to compensate the endogenous function of dgat1. this in vitro finding indicates that adverse effects of dgat1 inhibition may be low. moreover, dgat1 inhibitors are in development for treatment of obesity and display little adverse effects [147] . the second lipid modifying enzyme reported to be involved in hcv assembly, pla2ga4, cleaves glycerophospholipids with arachidonic acid at the sn2-position. thereby pla2ga4 alters membrane fluidity and curvature and releases arachidonic acid, which is a precursor for inflammatory mediators. hence, pla2ga4 inhibitors like pyrrolidine-2 (py-2) are in preclinical development for treatment of inflammatory disorders. in a recent study by menzel et al. py-2 inhibited assembly and release of hcv genotypes 1, 2, 3 and 5 in vitro and treatment with arachidonic acid restored hcv infectivity [146] . thus pla2ga4 inhibitors present a possible avenue for assembly blockage and future preclinical tests could connect to the existing pipelines of pyrrolidines as anti-inflammatory agents. in summary, the identification of host lipoproteins and enzymes required for hcv assembly and release provides novel hta development options. as we currently lack knowledge of possible side effects and efficacy of such htas in vivo, future preclinical work needs to elucidate if compounds targeting the last step of the hcv life cycle merit further development as possible anti-hcv therapeutics. after 15 years of ifnα/ribavirin based therapies against hepatitis c, novel treatment regimens developed rapidly since the approval of the first daas in 2011. an increased understanding of the molecular virology of hcv and of the usage of host factors for propagation led to the discovery of a multitude of antiviral targets. initial efforts focused on the inhibition of viral enzymes or viral structural proteins. this is reflected by the overall distribution of fda-approved antiviral drugs. in 2012, the fda listed 47 virus-targeting drugs including those against hiv and influenza virus and only 10 htas [148] . the latter will, however, gain increasing attention as they often have a high genetic barrier for resistance emergence and display a broad specificity for various genotypes and subtypes of a given virus. the most prominent htas against hcv include entry inhibitors and replication inhibitors. preclinical studies are, however, starting to reveal that some of the proposed advantages of htas do not apply. in rare cases genotype specificity is observed as different genotypes can engage different host factors as exemplified by the usage of cldn1 and cldn6 for hcv entry. in other cases the virus can acquire resistance mutations, which lead to usage of an altered binding site on the same host factor. for instance, ns5a mutations can confer resistance to cypa-targeting drugs. these findings suggest that even htas should be used in combination therapy with other drugs. an obvious caveat of htas is the possibility of side effects. consequently, preclinical and early clinical studies need to carefully evaluate dosing and treatment duration of host-targeting therapies. it should be noted that the reported side effects for some htas in clinical phases like itx 5061 and scy-635 are low. however, caution is warranted as the termination of alisporivir/ifn clinical trials shows. in summary, at least some hta therapies appear to have a superior side effect profile compared to past ifnα-based standard of care, which showed strong, sometimes intolerable adverse effects over a 24 to 48 week treatment period. lastly, we are just beginning to understand the role of host genetics in hcv infection. the best example is a polymorphism upstream of the il28b gene, which correlates with development of chronicity and ifnα-based treatment response [18] [19] [20] [21] . future research needs to address whether or not hcv host factors are genetically diverse in the human population and what impact polymorphisms have on hcv infection and response to hta treatment. nonetheless, the era of virus-and host-targeting antivirals against hcv holds a big promise to chronic hcv patients. many previous nonresponders can now be treated with the new protease inhibitors in combination with ifnα/ribavirin and patients undergoing treatment benefit from reduced side effects and shortened therapy duration. future development of htas and novel infα-free possibly all-oral combination therapy is expected to further increase quality of life of chronic hcv patients. certain patient groups, e.g., hiv co-infected individuals and patients with late stage liver disease, might require individualized therapy. thus, intense research on daas and htas is needed to find the most effective drug combinations with least adverse effects. finally, a better understanding of host and virus genetic diversity and their influence on drug efficacy might allow personalized treatment in the future and thereby guarantee cost effective use of novel drugs and optimal therapy outcome for individual patients. conflict of interest gisa gerold has no conflicts of interest to declare. she received grants from the german academy of science leopoldina, the human frontier science program and the german liver foundation. thomas pietschmann is a paid board member for janssen and biotest, and his institution receives grants from erc, dfg, and helmholtz association. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by the authors. papers of particular interest, published recently, have been highlighted as: natural history of chronic hepatitis c the origin of hepatitis c virus isolation of a cdna clone derived from a bloodborne non-a, non-b viral hepatitis genome treating viral hepatitis c: efficacy, side effects, and complications transmission of hepatitis c by intrahepatic inoculation with transcribed rna replication of subgenomic hepatitis c virus rnas in a hepatoma cell line robust hepatitis c virus infection in vitro complete replication of hepatitis c virus in cell culture production of infectious hepatitis c virus in tissue culture from a cloned viral genome the role of resistance in hcv treatment hepatitis c virus entry this is the first study describing a small molecule inhibitor for hcv entry virion assembly and release development and characterization of hepatitis c virus genotype 1-7 cell culture systems: role of cd81 and scavenger receptor class b type i and effect of antiviral drugs genetics of il28b and hcv-response to infection and treatment a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus screening for il28b gene variants identifies predictors of hepatitis c therapy success impact of donor and recipient il28b rs12979860 genotypes on hepatitis c virus liver graft reinfection genetic variation in il28b and spontaneous clearance of hepatitis c virus genetic variation in il28b predicts hepatitis c treatment-induced viral clearance productive hepatitis c virus infection of stem cellderived hepatocytes reveals a critical transition to viral permissiveness during differentiation modeling hepatitis c virus infection using human induced pluripotent stem cells human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis c virus new targets for antiviral therapy of chronic hepatitis c. liver int off j int assoc stud liver understanding the hepatitis c virus life cycle paves the way for highly effective therapies novel therapies for hepatitis c -one pill fits all? nat rev drug discov the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus binding of hepatitis c virus to cd81 hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies cd81 is dispensable for hepatitis c virus cell-tocell transmission in hepatoma cells neutralizing antibody-resistant hepatitis c virus cell-to-cell transmission a novel monoclonal anti-cd81 antibody produced by genetic immunization efficiently inhibits hepatitis c virus cell-cell transmission different requirements for scavenger receptor class b type i in hepatitis c virus cell-free versus cell-to-cell transmission claudin-6 and claudin-9 function as additional coreceptors for hepatitis c virus the tight junction proteins claudin-1, -6, and −9 are entry cofactors for hepatitis c virus isolate-dependent use of claudins for cell entry by hepatitis c virus complex phenotype of mice lacking occludin, a component of tight junction strands viral and cellular determinants of the hepatitis c virus envelope-heparan sulfate interaction hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor apolipoprotein e on hepatitis c virion facilitates infection through interaction with low-density lipoprotein receptor egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor identification of transferrin receptor 1 as a hepatitis c virus entry factor safety profiles of erlotinib therapy in patients with advanced non-small-cell lung cancer egf receptor-targeted therapy in non-small-cell lung cancer: role of germline polymorphisms in outcome and toxicity hras signal transduction promotes hepatitis c virus cell entry by triggering assembly of the host tetraspanin receptor complex ezetimibe: cholesterol lowering and beyond identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex morphological and biochemical characterization of the membranous hepatitis c virus replication compartment three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication quantitative analysis of the hepatitis c virus replication complex cyclophilin b is a functional regulator of hepatitis c virus rna polymerase the isomerase active site of cyclophilin a is critical for hepatitis c virus replication critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro cyclophilin a interacts with domain ii of hepatitis c virus ns5a and stimulates rna binding in an isomerase-dependent manner domain 3 of ns5a protein from the hepatitis c virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin a hcv resistance to cyclosporin a does not correlate with a resistance of the ns5a-cyclophilin a interaction to cyclophilin inhibitors cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes cyclophilin a-deficient mice are resistant to immunosuppression by cyclosporine biological effects of cyclosporin a: a new antilymphocytic agent curing a viral infection by targeting the host: the example of cyclophilin inhibitors the cyclophilin inhibitor debio 025 combined with peg ifnalpha2a significantly reduces viral load in treatment-naive hepatitis c patients suppression of viral rna binding and the assembly of infectious hepatitis c virus particles in vitro by cyclophilin inhibitors alisporivir plus ribavirin is highly effective as interferon-free or interferon-add-on regimen in previously untreated hcv-g2 or g3 patients: svr12 results from vital-1 phase 2b study hepatocytes that express variants of cyclophilin a are resistant to hcv infection and replication a major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis c virus identified by a genetic approach deb025 (alisporivir) inhibits hepatitis c virus replication by preventing a cyclophilin a induced cis-trans isomerisation in domain ii of ns5a this study describes a successful phase ii study for the cyclophilin inhibitor alisporivir (debio 025) in combination with peg ifnalpha2a. in a randomized, placebo-controlled, multicenter trial alisporivir co-administration induced an additive rna reduction in patients with genotypes 1 and 4 during a 4 week treatment window the cyclophilin inhibitor scy-635 suppresses viral replication and induces endogenous interferons in patients with chronic hcv genotype 1 infection importantly, it correlates the efficacy of scy-635 treatment with the host il28b genotype and serum ifn-lambda levels, suggesting that scy-635 acts through restoring host innate immunity hcv ns5a and irf9 compete for cypa binding isgf3, the transcriptional activator induced by interferon alpha, consists of multiple interacting polypeptide chains cyclophilin inhibitors potentiate interferon signaling through diminished pkr phosphorylation in hcv-infected cells hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation identification of human kinases involved in hepatitis c virus replication by small interference rna library screening a functional genomic screen identifies cellular cofactors of hepatitis c virus replication identification of a lipid kinase as a host factor involved in hepatitis c virus rna replication roles for endocytic trafficking and phosphatidylinositol 4-kinase iii alpha in hepatitis c virus replication class iii phosphatidylinositol 4-kinase alpha and beta are novel host factor regulators of hepatitis c virus replication kinases required in hepatitis c virus entry and replication highlighted by small interference rna screening recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment a genome-wide genetic screen for host factors required for hepatitis c virus propagation identification of host genes involved in hepatitis c virus replication by small interfering rna technology phosphatidylinositol 4-kinases: old enzymes with emerging functions the multiple roles of ptdins(4)p -not just the precursor of ptdins(4, 5)p2 the lipid kinase phosphatidylinositol-4 kinase iii alpha regulates the phosphorylation status of hepatitis c virus ns5a metabolism of phosphatidylinositol 4-kinase iiialpha-dependent pi4p is subverted by hcv and is targeted by a 4-anilino quinazoline with antiviral activity hepatitis c: antiviral drug development ns5a-from obscurity to new target for hcv therapy. recent patents anti-infect drug discov a-831, a novel hcv inhibitor targeting ns5a this extensive study identifies several pi4kiiiα small molecule inhibitors using an in vitro kinase assay and shows inhibition of hcv rna replication by these compounds. importantly, the authors generate conditional pi4kiiiα knockout mice and demonstrate that knockout induction is lethal molecular mechanisms of rna interference virus-encoded micrornas position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome microrna-122 stimulates translation of hepatitis c virus rna competing and noncompeting activities of mir-122 and the 5' exonuclease xrn1 in regulation of hepatitis c virus replication masking the 5' terminal nucleotides of the hepatitis c virus genome by an unconventional micrornatarget rna complex stabilization of hepatitis c virus rna by an ago2-mir-122 complex modulation of hepatitis c virus rna abundance by a liverspecific microrna requirements for human dicer and trbp in microrna-122 regulation of hcv translation and rna abundance reconstitution of the entire hepatitis c virus life cycle in nonhepatic cells recapitulation of the hepatitis c virus life-cycle in engineered murine cell lines this landmark paper reports the first in vivo usage of a microrna antagonist to treat viral infection. lanford et al. demonstrate in chronically hcv infected chimpanzees that the mir-122 antagonist spc3649 strongly reduces viral titers without side effects microrna-122 antagonism against hepatitis c virus genotypes 1-6 and reduced efficacy by host rna insertion or mutations in the hcv 5' utr sequence analysis of hcv variants from a phase iia trial of miravirsen (mir), an oligonucleotide targeting mir-122, in treatment naive patients with chronic hcv infection final results -randomized, double-blind, placebo-controlled safety, anti-viral proof-of-concept study of miravirsen, an oligonucleotide targeting mir-122, in treatmentnaive patients with genotype 1 chronic hcv infection clinical study demonstrating the efficacy of miravirsen in chronic genotype 1 patients without viral resistance emergence pharmacokinetics of miravirsen, a mir-122 inhibitor, predict the prolonged viral load reduction in treatment naive genotype 1 hcv infected patients essential metabolic, anti-inflammatory, and antitumorigenic functions of mir-122 in liver reduction of the infectivity of hepatitis c virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors antiviral effect of alpha-glucosidase inhibitors on viral morphogenesis and binding properties of hepatitis c virus-like particles antiviral effects of amantadine and iminosugar derivatives against hepatitis c virus an alpha-glucosidase i inhibitor for the potential treatment of hcv infection highdensity lipoprotein binding rate differs greatly between genotypes 1b and 2a/2b of hepatitis c virus association between hepatitis c virus and verylow-density lipoprotein (vldl)/ldl analyzed in iodixanol density gradients biochemical and morphological properties of hepatitis c virus particles and determination of their lipidome ferritin heavy chain is the host factor responsible for hcv-induced inhibition of apob-100 production and is required for efficient viral infection hepatitis c virus attachment mediated by apolipoprotein e binding to cell surface heparan sulfate syndecan-1 serves as the major receptor for attachment of hepatitis c virus to the surfaces of hepatocytes apolipoprotein e mediates attachment of clinical hepatitis c virus to hepatocytes by binding to cell surface heparan sulfate proteoglycan receptors efficient hepatitis c virus particle formation requires diacylglycerol acyltransferase-1 map-kinase regulated cytosolic phospholipase a2 activity is essential for production of infectious hepatitis c virus particles validation of diacyl glycerolacyltransferase i as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor new horizons for antiviral drug discovery from virushost protein interaction networks key: cord-291321-ao80xk11 authors: khan, mohsin; syed, gulam hussain; kim, seong-jun; siddiqui, aleem title: mitochondrial dynamics and viral infections: a close nexus date: 2015-10-31 journal: biochimica et biophysica acta (bba) molecular cell research doi: 10.1016/j.bbamcr.2014.12.040 sha: doc_id: 291321 cord_uid: ao80xk11 abstract viruses manipulate cellular machinery and functions to subvert intracellular environment conducive for viral proliferation. they strategically alter functions of the multitasking mitochondria to influence energy production, metabolism, survival, and immune signaling. mitochondria either occur as heterogeneous population of individual organelles or large interconnected tubular network. the mitochondrial network is highly susceptible to physiological and environmental insults, including viral infections, and is dynamically maintained by mitochondrial fission and fusion. mitochondrial dynamics in tandem with mitochondria-selective autophagy ‘mitophagy’ coordinates mitochondrial quality control and homeostasis. mitochondrial dynamics impacts cellular homeostasis, metabolism, and innate-immune signaling, and thus can be major determinant of the outcome of viral infections. herein, we review how mitochondrial dynamics is affected during viral infections and how this complex interplay benefits the viral infectious process and associated diseases. this article is part of a special issue entitled: mitophagy. viruses are obligate parasites completely reliant on host cell energy and molecular machinery to multiply. in order to attain a cellular environment conducive for proliferation, the viruses strategically modulate host cells' metabolism and physiology, resulting in a dramatic alteration of cellular and sub-cellular architecture and functions [1] . mitochondria have emerged as one of the key organelles in the maintenance of cellular homeostasis, metabolism, aging, innate immunity, apoptosis and other signaling pathways [2, 3] . in the last decade, work on the mitochondria has expanded our knowledge of its roles in cellular homeostasis in many parallel ways. at subcellular level, the size, shape and motility of the mitochondria are governed by mitochondrial dynamics that has emerged as a key event which influences many cellular processes [4] . once believed to be a solitary and rigid organelle, the mitochondria constitute a population of organelles that continuously elongate (by fusion), divide (by fission) and undergo a controlled turnover (by mitophagy) [4] . the processes of fusion, fission and mitophagy set a fundamental framework of mitochondrial dynamics. the mitochondrial dynamics (fusion and fission) in concert with mitophagy sustains mitochondrial homeostasis and constitutes an important arm of mitochondrial quality control. this dynamic process is sensitive to the changes in the cellular physiological or metabolic conditions and guards against detrimental stresses that thwart cellular homeostasis [4] . the mitochondrial dynamics and mitophagy play crucial roles in metabolism, cellular differentiation, cancer, neurodegeneration and numerous pathologies [4] . in the past, the aspects of mitochondrial dynamics were broadly studied in the context of neurodegenerative disorders like parkinson's, alzheimer's and huntington's diseases [5, 6] . the role of mitochondrial dynamics in viral infections is scant and described only for few viruses. currently, our understanding of mitochondrial dynamics and mitophagy during viral infections is still in its infancy, but certainly represents an emerging avenue of molecular investigations in virology. the integral roles of the mitochondria as a hub of innate immune signaling and metabolism implicate its role in viral pathogenesis. the mitochondria are either directly targeted by viral proteins or influenced by the physiological alterations to cellular environment during viral pathogenesis like deregulated calcium homeostasis, endoplasmic reticulum stress, oxidative stress and hypoxia. recent work by kim et al., revealed intriguing aspects of how hepatitis b virus (hbv) and hepatitis c virus (hcv) utilize the alterations in mitochondrial dynamics for the maintenance of persistent infection [7] [8] [9] . further investigation of the effect of mitochondrial dynamics in viral infections will enhance our understanding of virus-host interactions and their role in pathogenesis. such an understanding will be useful in the therapeutic design of new antiviral strategies. in this review article, we discuss the events of virus-regulated mitochondrial dynamics and membrane components. therefore, this process serves to alleviate the damage by fusing a partially impaired mitochondrion to a fully healthy, tubular mitochondrial network by simple exchange of matrix proteins. at first, based on the above explanation, it is easy to make out the inverse correlation between the mitochondrial fusion and mitophagy, however in cellular context, both processes are complementary and together they aid in the maintenance of homeostasis [12] . the mitochondria that are highly depolarized and irreversibly damaged are destined to form a pre-mitophagic pool and are eliminated selectively by mitophagy. the mitochondria that undergo fusion are selectively rescued from this pre-mitophagic pool and incorporated back into the functional mitochondrial population. the mitochondrial membrane potential (δψ m ) plays an important role in the process of sorting out and segregation of defective mitochondria [18] . depending on the extent of damage, the impaired and segregated daughter mitochondria are either restored, undergo repair and incorporated back or are completely eliminated by mitophagy, if the δψ m is not repairable [18, 19] . the events of fusion and fission are highly dynamic. in a cellular cycle, the mitochondria follow a kiss-and-run pattern, which entails a brief fusion event (for seconds) followed by fission [20] . in eukaryotic cells, mitochondrial life cycle can be seen in two distinct periods i.e. pre-fusion period and post-fusion period [21] . in pre-fusion stage, the the key events associated with mitochondrial dynamics and mitophagy affecting the mitochondrial quality control are depicted here. under normal physiological conditions, the mitochondria are usually tubular (a). physiological stress induces mitochondrial injury and causes mitochondrial impairment (b). the impaired part of the mitochondria is segregated by the fission process (c). the impaired mitochondria are selectively flagged by the pink1 (d) that facilitates parkin recruitment and mitophagy (e). all the repaired mitochondria are recruited to the functional mitochondrial network by fusion with other mitochondria (f). the factors that orchestrate the fission, fusion and mitophagy activities are shown in the inset. mitochondria are solitary, while post-fusion stage structures them in a complex network [21] . sometimes, in the solitary stage of the mitochondria, even asymmetric fission can induce remarkable alteration in the δψ m because of the unequal distribution of the membrane surface and nucleoid structure between the two daughter mitochondria. thus, it is presumed that the depolarized mitochondria can also result from the spontaneous depolarization during solitary stage [12] . whether it is spontaneous or stress-induced event, such depolarized mitochondria are destined for either repair or clearance. studies that correlate the size of the mitochondria with the rate of mitophagy show that the elongated mitochondria exhibit reduced rate of mitophagy and the mitochondria inside the autophagosomes undergoing mitophagy are smaller in size (b1 μm) [22] [23] [24] . mitophagy of the larger elongated mitochondria appears less likely. fission facilitates the sorting of impaired mitochondria from the healthy mitochondrial pool. if the damage in a sorted mitochondrion is repairable, it may follow the fusion phase but those in which the damage is irreversible are eliminated by mitophagy [19] . in this manner, the mitochondrial dynamics and mitophagic machinery work in concert to prevent circulation of severely damaged mitochondria into the active and healthy pool ( fig. 1c -e). mitochondrial dynamics i.e. fission, and fusion, is coordinated by dynamin-related proteins. dynamin has the capacity to oligomerize into spirals and mechanically remodel membranes by utilizing energy from gtp hydrolysis. they commonly promote membrane scission however their role in mitochondrial fusion is quite unique. mitochondrial function and quality are determined by the subtle balance between continuous fission and fusion. below, we discuss the central players of these processes. the fuzzy onion (fzo) was the first fusion regulating protein identified in drosophila. fzo regulates mitochondrial fusion and its deletion results in fragmented mitochondria [25] . in the fzo mutant flies, the mitochondria aggregate and fail to fuse leading to the accumulation of mitochondria in spermatid [26] . the molecules commandeering fusion pathway in mammals are mitofusins (mfn1 and mfn2) and optic atrophy 1 protein (opa1). knockdown of any of these proteins leads to embryonic lethality and mitochondrial dysfunction [27] . mfn1 and mfn2 are homologs of fzo and have related but discrete roles in controlling mitochondrial morphology. mitofusins are the members of the membrane anchored dynamin family expressed in a wide variety of mammalian cells and uniformly localize to the outer mitochondrial membrane (omm) [28] . mitochondrial fusion involves fusion of the outer and inner mitochondrial membranes resulting in the merger of two individual mitochondria and concomitant mixing of the mitochondrial contents [28] . the omm fusion is coordinated by mfn1 and mfn2. mutations in either of the mfns lead to abnormalities in mitochondrial morphology associated with dramatic reduction in mitochondrial fusion. depletion of both mfn1 and 2 in cells results in poor cell growth, decreased cellular respiration associated with the heterogeneity of mitochondrial membrane potential [29] . inner mitochondrial membrane (imm) fusion is orchestrated by opa1. opa1 is a dynamin-related gtpase expressed in many mammalian cell types and localizes on imm [30] . opa1 is considered a multifunctional protein with pivotal roles in mitochondrial cristae architecture, mitochondrial bioenergetics and apoptosis [13, 31] . overall the mitochondrial fusion is a multistep process with mfns mediating the outer membrane fusions followed subsequently by inner membrane fusions by opa1 [13] . the presence of mfns on both the fusing mitochondria is a prerequisite for outer membrane fusion. the mfns on adjacent mitochondria oligomerize bringing the opposing membranes within close proximity and eventually promoting fusion by membrane merger [27] . both mfn1 and mfn2 physically interact homotypically and heterotypically resulting in mfn1 homotypic oligomers, mfn2 homotypic oligomers and mfn1-mfn2 heterotypic oligomers (fig. 1f) . interestingly, inner membrane fusion by opa1 does not require its presence on adjacent fusing membranes [32] . both mfns and opa1 undergo post-translational modifications and proteolysis. due to differential rna splicing and precursor protein processing, opa1 exists as distinct isoforms. the long isoform has membrane anchor but the short isoform lacks membrane anchor but does interact with membranes [33] . usually both long and short isoforms drive membrane fusion but under conditions of stress, the long isoform solely promotes mitochondrial fusion. the activity of opa1 is regulated by sirt3-mediated deacetylation [34] . moreover, it has recently been shown that decrease in mitochondrial membrane potential triggers the proteolysis of opa1 by the isoenzymes of the adenosine triphosphate (atp)-dependent matrix aaa (atpase associated with diverse cellular activities [m-aaa]) protease and oma1 [35, 36] . oma1 is constitutively active under normal cellular conditions. however, various stress stimuli can enhance its catalytic activity several fold leading to enhanced turnover. in a recent study, baker et al., have identified a stress sensor n-terminal domain of oma1 that is responsible for stress-induced activation of oma1 [37] . this mechanism probably drives selective fusion of normal mitochondria resulting in isolation of defective/dysfunctional mitochondria. jnk-mediated phosphorylation of mfn2 during genotoxic stress leads to interaction with huwe1, an e3 ubiquitin ligase, resulting in proteasomal degradation of mfn2, mitochondrial fragmentation, and apoptotic cell death [38] . similarly recruitment of parkin to damaged mitochondria also results in the ubiquitination and degradation of mfns, which prevents the fusion of damaged mitochondria with the healthy mitochondrial pool [39] . the central player in mitochondrial fission is a cytosolic dynaminrelated gtpase, dynamin-related protein 1 (drp1). drp1 is localized primarily in the cytosol and in mammalian cells the accessory proteins mid49, mid51, and mitochondrial fission factor (mff) mediate its recruitment to the mitochondria by acting as ligands for drp1 on the mitochondria. once recruited to the mitochondria, drp1 oligomerizes as rings on mitochondrial tubules and causes scission of both outer and inner mitochondrial membranes [40, 41] . our current understanding of the mitochondrial fission mechanism is incomplete, however recent studies suggest that er tubules wrap around and constrict the mitochondria, marking them for subsequent scission by drp1 [42] . these sites of er-mitochondrial contact are enriched with drp1 suggesting that er contact with mitochondria directs mitochondrial division. interestingly, mitochondrial constriction at er contact sites was also apparent in cells depleted of drp1 and mff suggesting that the er-mediated flagging of mitochondrial division sites occurs prior to the mitochondrial recruitment of drp1 [42] . the mechanical force required for the constriction of mitochondrial tubules by the er seems to be generated by actin assembly at the er-mitochondria contact sites via interaction with er-associated formin inf2 [43] . genetic or chemical inhibition of drp1 leads to elongated mitochondria. the first molecule to be identified as drp1 receptor was the fission1 (fis1) in yeast. fis1, is a small c-tail protein anchored on omm and displays uniform distribution [44] . however, the role of mammalian fis1 in mitochondrial fission is enigmatic and mitochondrial morphology or mitochondrial recruitment of drp1 was unaffected in fis1 knockout cells, raising the concern about the role of mammalian fis1 in mitochondrial fission [45] . mitochondrial fission factor mff is also a c-tail anchored protein on omm and a well-recognized receptor of drp1. it transiently interacts with drp1 through its n-terminal cytoplasmic region. in contrast to uniform localization of fis1 on omm, mff mostly colocalizes with drp1 foci on the omm. overexpression of mff stimulates mitochondrial recruitment of drp1 and mitochondrial fragmentation and silencing of mff results in mitochondrial elongation [46, 47] . the mids can mediate mitochondrial fission in the absence of fis1 and mff. knockdown of either gene results in the increment of mitochondrial length and interconnectivity suggesting that these proteins positively regulate mitochondrial fission. interestingly, overexpression of mids also results in the elongation of the mitochondria. the mitochondrial elongation caused by mid overexpression is associated with enhanced phosphorylation of drp1 at s637 site, which negatively regulates the drp1 function [40, 41] . although the cells mutated for drp1 resist mitochondrial fragmentation induced by depolarization, they exhibit substantial mitochondrial fragmentation when treated with apoptotic stimuli such as actinomycin d and etoposide, suggesting that drp1-independent mechanisms of mitochondrial fission also occur. the mitochondrial fragmentation induced by the pore-forming toxin listeriolysin o, secreted by the bacterium listeria monocytogenes, is independent of traditional fission protein drp1 but dependent on actin cytoskeleton [48] . several posttranslational modifications of drp1 regulate mitochondrial fission dictated by various physiological cues. in normal cells, drp1 is mostly cytoplasmic with about~3% of total drp1 associated with the mitochondria. overexpression of drp1 does not usually lead to enhanced fission but stimulation of drp1 mitochondrial recruitment, gtpase activity, and spiral assembly directly influences mitochondrial fission ( fig. 1a ) [49] . during mitosis cdk1/cyclin b phosphorylates drp1 at s616 residue (amino acid numbering corresponds to human drp1), resulting in the upregulation of mitochondrial recruitment of drp1 and fission activity. this regulation is essential in cells undergoing mitosis to ensure equal distribution of the mitochondria to daughter cells [50] . during mitosis, aurora a kinase also promotes mitochondrial fission by phosphorylating small ras-like gtpase, rala leading to its relocation to mitochondria, followed by the recruitment of its effector ralbp1 to the mitochondria. ralbp1 then facilitates cdk1-mediated drp1 s616 phosphorylation and recruitment to the mitochondria [51] . interestingly, a study indicates that high-glucose stimulation of liverderived cells induces ca 2+ -mediated map kinase signaling leading to erk1/2-dependent drp1 s616 phosphorylation and enhanced mitochondrial fission [52] . phosphorylation of drp1 at s637 residue leads to inhibition of fission activity. protein kinase a (pka) or campdependent protein kinase phosphorylates drp1 on s637 located in the gtpase effector domain (ged) and impairs intramolecular interaction of ged and gtpase domains [53] . during hypoxia, pka-mediated phosphorylation of drp1 is affected due to the proteasomal degradation of mitochondrial a-kinase anchoring protein 121 (akap1) that anchors pka to mitochondria [54, 55] . calcium-dependent phosphatase calcineurin dephosphorylates drp1 at s637 and facilitates mitochondrial recruitment of drp1 followed by fission [56] . the same residue is also dephosphorylated by the phosphatase, pp2a/bβ 2 [55] . in contrast, phosphorylation of same residue in response to rise in intracellular calcium by ca 2+ /calmodulin-dependent protein kinase α (camkiα) in neuronal cells or by rock1, results in enhanced fission [57, 58] . surprisingly, recent reports reveal the importance of upstream kinases, rather than the site of phosphorylation as an important factor that determines the activity of drp1. for example, the s616-drp1 phosphorylation by cdk1 or pkcδ, is reported to enhance fission by activating drp1 but if the same site is phosphorylated by cdk5, it was explained to enhance the dissociation of drp1 from its oligomeric to monomeric form and severely reduce its fission-prompting activity [59] . calcineurin mediated dephosphorylation of s637 as well as phosphorylation of s637 by camkiα also enigmatically leads enhanced fission suggesting that different cellular contexts may have different effects of drp1 phosphorylation on same serine residue. however, further studies are needed to clarify how phosphorylation at the same site, by different kinases can modulate drp1 activity with different consequences. in addition, ubiquitination, sumoylation, and s-nitrosylation have also been shown to modulate drp1 activity [60] [61] [62] . parkin and mitochondria ubiquitin ligase (mitol) facilitate drp1 degradation via proteasomal pathways [63, 64] . all the post-translational modifications of the key molecules involved in the processes of fission and fusion are outlined in table 1 . autophagy is a catabolic process, in which a destined cargo is actively engulfed by phagophore and subsequently followed by fusion of autophagosome with lysosomes to deliver the cargo to the lysosome for degradation [65] . autophagy is a nonselective process, but recent advancements in this field have established the presence of selective autophagy in which specific cargo or damaged organelles are selectively eliminated. here, we discuss the selective autophagy of the mitochondria, termed mitophagy. mammalian mitophagy can be broadly classified in two distinct groups; parkin-dependent, and independent forms of mitophagy. parkin-dependent mitophagy involves selective tagging of damaged mitochondria by pten-induced putative kinase 1 (pink1) followed by parkin recruitment to the impaired mitochondria and subsequent mitophagy [66] . the serine/threonine kinase, pink1 contains a mitochondrial targeting sequence (mts) and is sequentially imported into the healthy mitochondria via the translocase of outer mitochondrial membrane (tom) and translocase of inner mitochondrial membrane (tim). at the imm, pink1 is processed by the mitochondrial processing protease (mpp) to excise the mts, and further cleaved by the presenilinassociated rhomboid-like protease (parl) to generate a 52 kda protein, which is then rapidly degraded. however in the impaired mitochondria, the loss of membrane potential (δψm) rapidly perturbs the import of pink1 to the imm and prevents its degradation by mpp, parl and other proteases (fig. 1d ). this leads to the display of pink1 on the surface of the mitochondria, which thereby marks the damaged mitochondria for parkin recruitment [66, 67] to initiate the process of mitophagy. several studies have explained how pink1 recruits parkin to the mitochondria. pink1 auto-phosphorylation on depolarized mitochondria is required for parkin recruitment and direct phosphorylation of parkin by pink1 at s65 residue has been shown to stimulate parkin's e3 ligase activity [68] . pink1 also phosphorylates s65 residue of ubiquitin that subsequently unlocks parkin's autoinhibition at the active cysteine site [69] . ubiquitin mutant s65a inhibited parkin translocation to impaired mitochondria [70] . based on these premise small molecules that mimic the s65 phospho-ubiquitin can be used as potential therapeutic tool against the parkinson's disease [71] . together these studies propose a feed-forward mechanism for parkin recruitment to the mitochondria involving pink1 mediated phosphorylation of both parkin and poly-ub chains synthesized by parkin to the inner mitochondrial membrane [70] [71] [72] . some studies suggest a direct physical interaction between parkin and pink1 [66] . it is likely that other substrates of pink1 kinase activity are involved in parkin recruitment to the mitochondria. although pink1 facilitates parkin recruitment to the mitochondria, in flies it is not the only means for parkin recruitment. mitochondrial morphology in pink-1 deficient flies can be rescued by parkin overexpression and parkin deficient flies had more severe phenotype than the later, suggestive of pink1 independent functions of parkin in flies [73] . can parkin be recruited to the mitochondria independent of pink1 in mammalian cells remains to be determined. however, in one study, pink1 mediated phosphorylation of mfn2 was shown to promote parkin recruitment on depolarized mitochondria [74] . parkin ubiquitinates and facilitates degradation omm proteins such as mfn1, mfn2 and tom complex which seems to be essential for subsequent autophagic removal of the damaged mitochondria [39] . in agreement with this notion, inhibition of proteasomal activity impairs parkindependent mitophagy. the accumulation of proteasomal subunits on omm following mitochondrial recruitment of parkin, suggests that the proteasomal machinery is directly recruited to the mitochondria to facilitate degradation. the aaa + atpase p97, which structurally remodels or unfolds ubiquitinated client proteins destined for degradation is shown to be recruited to the mitochondria along with parkin and regulate protein degradation at omm [39] . currently, it is unclear how proteins are targeted by parkin and which of these targets are critical for initiation of mitophagy. parkin not only promotes k-48 linked polyubiquitination which leads to proteasomal degradation but can also induce the formation of various other lysine-linked polyubiquitin chains conjugated to its target proteins [72] . considering parkinmediated k-63 linked ubiquitination, it is likely that p62 adaptor protein that binds to k-63 linked polyubiquitin conjugated proteins, plays a role in recruiting mitochondria to autophagosomes [75] . however, mitochondrial elimination has been reported even in the absence of p62 clustering at the mitochondria [76] . the histone deacetylase 6 (hdac6), also involved in binding ubiquitinated proteins, is recruited to the mitochondria in parkin-dependent manner. hdac6 has been shown to play a role in autophagosome maturation [77] . pink1 and parkin have also been shown to directly interact with beclin-1 and pi3k (phosphatidylinositol-4,5-bisphosphate 3-kinase) complex to promote autophagy. parkin also facilitates the recruitment of activating molecule in beclin-1 regulated autophagy (ambra-1) to the mitochondria, which could activate beclin1 and promote the nucleation of isolation membranes (phagophore) around the mitochondria [78] . mutations in both parkin and pink1 are the most prevalent cause of autosomal recessive genetic disorder parkinsonism, which is usually associated with accumulation of impaired mitochondria leading to neurodegeneration in parkinson's disease (pd). this lends support to the view that pink1/parkin-mediated mitophagy is important for mitochondrial quality control [5] . pink1/parkin pathway, not only initiates the mitophagic removal of damaged mitochondria, but also facilitates the segregation of the damaged mitochondria by preventing their fusion with healthy functional mitochondria. this segregation is achieved in two different ways; making the impaired mitochondria fusion defective by parkin-mediated degradation of mfns and by restricting cytosolic motility of the damaged mitochondria by targeting of miro (rho-gtpase), a mitochondrial adapter molecule that anchors kinesin motor complex to the outer surface of the mitochondria. pink1 phosphorylates miro, which is recognized and degraded by the parkin. miro degradation releases the kinesin motor from the mitochondria, which hinders their motility [39, 79] . these events clearly illustrate that mitochondrial dynamics and mitophagy are interlinked and regulated in a coordinated manner. several deviations to the pink1-parkin pathway support the existence of pink1-parkin-independent pathways of mitophagy. pink1-parkin pathway may not represent the only pathway for steady state turnover of the mitochondria, because parkin deficiency does not lead to dramatic loss of mitochondrial mass and mice deficient in parkin are normal unless exposed to stress [80] . allen et al., observed that iron chelation induces mitophagy independent of pink1 stabilization and parkin activation in primary human fibroblasts as well as those isolated from pd patients with parkin mutations [81] . ambra-1 is reported to induce parkin-independent mitophagy by directly interacting with lc3 via its lc3-interacting region (lir). ambra-1 can induce mitophagy in parkin-deficient cells as well as potentiate the parkin-mediated mitophagy [82] . in response to energy depletion, ampk stimulates autophagy by phosphorylating and activating ulk1 [83] . loss of ampk or ulk1 in mammalian cells leads to accumulation of the autophagy adaptor p62 and deficient mitophagy suggesting that ulk1 is required for mitochondrial and cellular homeostasis during starvation [83] . a recent study suggests that ulk1 translocates to the mitochondria and phosphorylates the fun14 domain-containing protein (fundc1), which directly binds to lc3 via its lir motif to regulate mitophagy [84] . similarly, bcl2/adenovirus e1b 19 kda protein-interacting protein 3-like (bnip3l) or nix facilitates the elimination of the mitochondria from reticulocytes during erythrocyte maturation by directly interacting with lc3 via its lir motif [85] . another study implicates fundc1, an omm protein as mediator of hypoxia-induced mitophagy [86] . in neurons, cardiolipin externalization to the outer mitochondrial membrane serves as a signal for elimination of the damaged mitochondria. the phospholipid cardiolipin, present in the imm of the mitochondria when externalized to the omm, interacts with lc3 to facilitate mitochondrial degradation [87, 88] . it is still unknown whether the parkindependent and -independent pathways function in parallel during depolarization-induced mitophagy. over the past decade, it has become evident that mitochondrial dynamics and mitophagy constitute the two main pathways required to maintain mitochondrial quality control and cellular homeostasis. perturbations in these pathways have physiological consequences and are among the most common reason underlying the pathogenesis of many neurodegenerative disorders, cancer, inflammation, metabolic syndrome and cardiac dysfunctions. we discuss, below, the roles of mitochondrial dynamics and mitophagy in cellular physiology. perturbation in mitochondrial dynamics and mitophagy usually generates an unhealthy pool of the mitochondria that create an energy-deprived condition in the cytosol that can eventually lead to apoptosis. mitochondrial fragmentation has been implicated in apoptosis. enhanced fragmentation increases the sensitivity of the cells for apoptotic stimuli while enhanced fusion has been associated with delay in bax activation, cytochrome c release and cell death [89] . drp1-mediated fission has been shown to facilitate cytochrome c release from the mitochondria [90] . the drp-1-dependent remodeling of the omm facilitates the oligomerization and insertion of bax into the mitochondria [91] . conversely, both bax/bak facilitate drp-1 sumoylation and stabilize drp1 on the mitochondria thereby promoting fission [92] . in the neuronal cell model of nitric oxide-induced oxidative stress, mitochondrial fission preceded apoptosis, and inhibition of fission conferred protection [93] . enhanced mitochondrial fusion has been shown to protect cells from mitochondria-triggered apoptosis but not the mitochondriaindependent apoptotic stimuli [94] . contradicting the common notion, cytochrome c release was also observed in drp1 defective cells upon induction of apoptosis, suggesting that mitochondrial fission and mitochondrial outer membrane permeabilization are independent of each other. currently, the precise role of mitochondrial fission in apoptosis is not clearly defined, however the anti-apoptotic bcl2 family members in their pursuit to promote cellular adaptability to various stressful conditions may modulate mitochondrial dynamics. defective mitochondria are a potential source of reactive oxygen species (ros), which can also lead to damage of the healthy mitochondria. hence, perturbation in the rapid clearance of damaged mitochondria will initiate a vicious cycle of ros generation and mitochondrial damage, which eventually deteriorates other cellular components and promotes cell death [95] [96] [97] . defective mitochondrial quality has been associated with induction of inflammatory responses via the activation of inflammasome [98] . pre-conditioning the cells to upregulate mitochondrial quality control pathways has been shown to enhance resistance to stressful stimuli. interorganelle contacts are essential for mitochondrial functions and are implicated in mitochondrial dynamics. one such example is the involvement of the er-mitochondria encountered structure (ermes) in maintenance of mitochondrial morphology in budding yeast [99] . in mammals, mitochondrial-associated membranes (mams) represent the er-mitochondria contact sites [89] . mfn2 is enriched in mams and it is proposed that mfn2 on the er is required to stabilize interaction between er and mitochondria by engaging in homotypic and heterotypic complexes with mfn1/2 on the surface of the mitochondria. alterations in mfn2 expression affect er-mitochondria contact followed by hindrance to lipid exchange and calcium signaling between er and mitochondria [100] . in mammals many other interactions between er and mitochondrial proteins have been reported, however it remains to be determined if these interactions are an integral part of a physical complex [101] . the role of mitochondrial dynamics in innate immune signaling is an upcoming field. pattern recognition receptors (prrs), the components of the innate immune system, recognize pathogen associated molecular patterns (pamps) and activate a cascade of complex pathways to eliminate infection, sometimes involving the apoptosis of infected cells [1] . many prrs have been characterized and each of them has a specific ligand. many viral pamps are recognized by the retinoic acid-inducible gene i (rig-i) and rig-i like receptors (rlrs), which then activate the mitochondria associated antiviral signaling protein (mavs) via binding facilitated by the card domains of each protein [102] . recent studies implicate the role of mitochondrial dynamics and mitophagy in modulation of this intrinsic defense strategy [103] . mitochondrial fusion and fission play a central role in amplifying or dampening the rlr signaling. mitochondrial fusion serves to increase mavs interactions with downstream signaling molecules resulting in enhanced interferon (ifn) synthesis upon polyi:c stimulation or sendai virus infection [103] . in contrast, the mitochondrial fission serves to block mavs downstream signaling resulting in reduced ifn synthesis [103] . in agreement with this notion, fibroblasts lacking both mfn1 and 2 were impaired in interferon and pro-inflammatory cytokine synthesis in response to viral infection [104] . absence of fusion leads to the loss of mitochondrial membrane potential in these double-knockout cells, which correlated with reduced mavs signaling suggesting that mitochondrial membrane potential may be required for mavs signaling [104] . it is also envisaged that mitochondrial fusion and the resultant tubular mitochondrial network formation around viral replication sites assist in concentrating the mavs signalosome around sites of viral replication [103, 105] . a recent report claims that mfn1 promotes dramatic redistribution of mavs on the mitochondria after rlr activation and that mfn1 is required for recruitment of mavs-enriched mitochondria to the rig-i enriched sites of viral replication [106] . the effect of mitochondrial morphology is also implicated in regulating the signaling of er-associated innate immunity molecule sting (stimulator of interferon genes). tubular elongated mitochondria tend to interact more with the er and therefore assist in sting's downstream interaction with mavs [103, 105] . we observed that inhibition of mitochondrial fission by silencing drp1 during hcv infection enhanced ifn production indicating that hcv-mediated altered mitochondrial dynamics serves to perturb the antiviral defense [8] . this may operate in addition to the hcv ns3/ 4a-orchestrated proteolytic cleavage of mavs to cripple host innate immunity [107] . viruses interfere with the mitochondrial pathways and distort mitochondrial functions to facilitate their proliferation. viral strategy to impede mitochondria-associated antiviral signaling mechanism is one such paradigm of the virus-mitochondria interactions [102] . the functional implication of mitochondrial dynamics in various mitochondrial and cellular functions suggests that alteration of mitochondria dynamics can serve as an efficient viral strategy to promote interference of cellular signaling pathways [103, 105] . in addition, the sensitivity of mitochondrial dynamics to subtle physiological perturbations in cellular environment makes the effect of viral infections on mitochondrial dynamics a likely inevitable consequence. the viral infection causes many physiological alterations in host cell and many of those alterations can directly affect the mitochondrial dynamics and mitophagy. the overall effects of viral infection on mitochondrial dynamics and its consequences on viral pathogenesis are illustrated in fig. 2 . we discuss, below, few of the viruses, which have been characterized for their effect on mitochondrial dynamics and highlighting the significance of these alterations in the viral infectious process. a brief description of few viruses and their effects on mitochondrial dynamics and mitophagy are summarized in table 2 . chronic infection with hepatitis c virus (hcv) and hepatitis b virus (hbv) has long been associated with prominent mitochondrial injury of the liver [108] . although hbv and hcv differ in their genomic organization and life cycle, they share similar pathologies, and promote chronic hepatitis, which subsequently progresses into liver fibrosis, cirrhosis and hepatocellular carcinoma [108, 109] . histopathological features commonly observed in hbv/hcv infection include mitochondrial swelling and loss of mitochondrial cristae and number [108] . hbv and hcv infection-associated er-stress triggers leakage of er-calcium stores and their subsequent uptake by the adjacent mitochondria promotes mitochondrial depolarization and dysfunction, associated with mitochondrial ros generation [108] . we have recently investigated the effect of hbv and hcv infections on host cell mitochondrial dynamics and established a direct relationship between viral infections, mitochondrial dynamics, mitochondrial homeostasis, and viral persistence. hcv: hcv is a positive strand rna virus belonging to the genus hepacivirus in the flaviviridae family. the 9.5 kb rna genome is translated via an ires element located in the 5′ untranslated region (utr) producing a~3000 amino acid precursor polyprotein, which is processed by cellular and virus-encoded proteases to produce 3 structural and 7 non-structural viral proteins with various functions [110] . most of the viral proteins remain associated with the er membrane and promote er stress. hcv induces er stress and promotes autophagy which plays a crucial role in hcv rna translation and hcv replication [111, 112] . hcv infection promotes mitochondrial abnormalities, and it is assumed that the er-stress mediated depletion of er ca 2+ stores and uptake by mitochondria paves the way for mitochondrial dysfunctions [113, 114] . hcv proteins have also been shown to directly associate with the mitochondria and localize to the omm and mam [107] . hcv core and ns5a proteins have been shown to perturb complex 1 activity and promote mitochondrial ca 2+ uptake, ros production, and mitochondrial permeability transition [115, 116] . hcv ns3/4a protease localizes to mam and cleaves mam-associated mavs to facilitate evasion from innate immune response [117] . hcv infection leads to elevation of ros in the mitochondria that is induced by calcium signaling [113] . alteration of mitochondrial dynamics by viruses and its physiological significance. list of viruses known to disrupt mitochondrial dynamics and the consequences on cell physiology and viral pathogenesis. effect on mitochondrial dynamics affected protein(s) consequences on cell physiology hepatitis c virus [8, 9] enhanced fission and mitophagy core, e1-e2 activation of drp-1, and mitochondrial translocation of parkin inhibition of apoptosis and innate immune response, facilitates persistent infection pseudorabies virus [136] fragmented mitochondria glycoprotein b (gb) altered functioning of miro protein affects intracellular calcium signaling and mitochondrial motility human cytomegalovirus [130] enhanced fission vmia affects mechanism of bax inhibition of apoptosis epstein-barr virus [128] enhanced fission lmp2a up regulation of drp-1 cell migration and apoptosis hepatitis b virus [7] enhanced fission and mitophagy hbx parkin and pink1 up-regulation and drp-1 phosphorylation inhibition of apoptosis and innate immune response, facilitates persistent infection influenza a virus [98] induction of mitophagy unknown the nod2 and ripk2 promote ulk1 phosphorylation to induce mitophagy inhibits inflammasome activation and reduces disease severity influenza a virus [137, 138] induction of mitochondrial fragmentation inhibition of mitochondrial β-oxidation of fatty acids [109] . this is in conjunction with enhanced lipogenesis and lipid uptake during hcv infection ultimately leads to intracellular accumulation of lipid droplets. hepatosteatosis is characterized by the accumulation of lds which accelerate progression into end stage liver diseases in chronic hepatitis c [118] . other effects on mitochondrial functions during hcv infection include reduced oxidative phosphorylation and atp generation [119] . hcv infection induces perinuclear clustering of the mitochondria, a notable phenotype in cells exposed to oxidative stress [9] . our studies have shown that hcv promotes mitochondrial fragmentation by inducing drp-1 s616 phosphorylation. drp1 phosphorylation at s616 promotes its mitochondrial translocation and subsequent fission/fragmentation of the mitochondria. hcv-infected cells also displayed enhanced expression of parkin and pink1 and mitochondrial recruitment of parkin. mitochondrial recruitment of parkin in hcv-infected cells was associated with concomitant increase in autophagic removal of the damaged mitochondria by parkin-dependent mitophagy which was elegantly demonstrated using the mitophagy reporter assay (box-1) [8, 9] . depletion of drp-1 and parkin in hcv-infected cells hindered hcv-induced mitochondrial fission and mitophagy respectively, but led to a marked increase in mitochondrial apoptotic signaling as evidenced by robust cytochrome c release and increase in caspase-3 activity. our study suggested that hcv-induced mitochondrial fission followed by complete mitophagy is required to sustain mitochondrial homeostasis during the infection and promote viability of infected cells. the upregulation of mitochondrial quality control pathway likely attenuates the impending cell death due to the mitochondrial injury incurred during infection. in addition to promoting viability of infected cells, hcv-induced alteration of mitochondrial dynamics also subverted the mitochondria-associated antiviral signaling which was evident by a marked increase in interferon (ifn) production by silencing drp-1 expression in hcv-infected cell [8] . hcv cripples the innate immune response by ns3/4a proteasemediated cleavage of mavs protein suggesting that the effect of altered mitochondrial dynamics on mavs signaling, further complements this effective viral strategy [120] . these studies clearly show that hcvmediated alteration of mitochondrial dynamics and mitophagy is a major determinant of viral persistence and sheds light on the novel aspect of viruses exploiting the mitochondrial dynamics and quality control mechanism to favor viral propagation. inhibition of mitochondrial fission affects hcv secretion that presumably occurs due to altered rate of glycolysis and atp synthesis. alternatively, reactive oxygen species emanating from accumulated dysfunctional mitochondria can damage lipids and proteins that could play a crucial role in hcv secretion [8] . hbv: unlike hcv, the genetic makeup of hepatitis b virus (hbv) consists of dna. hbv replicates via an rna intermediate and amplifies its genome by reverse transcription, a strategy similar to retroviruses [121, 122] . a successful recombinant vaccine against hbv is available with significant level of protection (about 98%) against infection. hbv encodes a regulatory protein, termed hbx or hbv x protein. hbx is localized to the mitochondria via its association with vdac, alters membrane potential, and elevates the levels of calcium and ros, ultimately causing damage to the mitochondria [108, 123, 124] . hbx-regulated calcium signaling and ros are involved in activation of latent transcription factors such as stat-3, nf-kb and nfat [125] . hbv also induces early autophagic pathways that subsequently help the viral dna replication [126] . hbv, like hcv also induced drp1-dependent mitochondrial fission, and parkin-dependent mitophagy [7] . disruption of mitochondrial dynamics was observed in cell expressing full-length hbv genome or only hbx but hbx-defective (hbv-δx) genome had no effect on mitochondrial dynamics reinforcing the issue that hbx is sufficient to promote mitochondrial damage and alter mitochondrial dynamics [7] . hbv-altered mitochondrial dynamics and mitophagy effectively contribute to mitochondrial quality control resulting in mitochondrial homeostasis. this promotes the maintenance of persistent infection by inhibition of apoptosis of hbv infected cells. in parkin-silenced cells the reporter plasmid encodes mrfp-gfp tandem flourochrome fused in frame with mitochondrial localization signal of human cytochrome c oxidase subunit viii the reporter exploits the differential stability of the rfp and gfp flourochrome in the acidic environment of the lysosomes. when the rfp-gfp tagged mitochondria are delivered to the lysosomes, gfp gets rapidly degraded but rfp persists for a while, hence rfp-gfp tagged mitochondria in lysosomes display only red fluorescence. rfp plus gfp uninfected cell the mrfp-egfp tagged mitochondria when delivered to the lysosomes display only red fluorescence, due to higher stability of rfp compared to gfp in the acidic environment of the lysosomes. lysosomes the mrfp-egfp tagged mitochondria in the cytosol display both green and red fluorescence and merger yields only yellow mitochondria box 1. mitophagy reporter assay. hbv infection promoted apoptosis as evidenced by the burst of cytochrome c release, caspase-3 activation and poly (adp-ribose) polymerase (parp) cleavage [7] . these observations collectively implicate mitochondrial dynamics in the maintenance of persistent phenotype in hbv infected hepatocytes [7] . depletion of parkin also modestly affected hbv dna synthesis, presumably due to the impaired mitochondrial functions or enhanced innate immunity [7] . ebv is an oncogenic virus belonging to the herpes virus family and is implicated in various lymphoid and epithelial cancers [127] . a recent study has shown that the viral latent membrane protein 2a (lmp2a) causes elevated mitochondrial fission in gastric and breast cancer cells. their observations revealed that lmp2a-triggered notch pathway leads to induction in drp1 expression and manifests in enhanced mitochondrial fission [128] . however, it has to be noted that drp1 overexpression per se did not lead to enhance fission, but the mitochondrial recruitment of drp1 and trigger of its gtpase activity is essential to promote fission. drp1 has been implicated in the invasive ability and enhanced epithelial-mesenchymal transition of breast cancer cells. concurrently, this study establishes that altered mitochondrial dynamics plays a critical role in the metastatic behavior of ebv-associated gastric and breast carcinomas. cytomegaloviruses are ubiquitous viruses and pose an important health problem because of the high frequency of congenital infections [129] . severe infections are associated with immunosuppression [129] . in an interesting report by mccormick et al., it has been observed that fibroblast infected with human cytomegalovirus exhibits a peculiar characteristic of punctate and dispersed mitochondria reminiscent of mitochondrial fission at 24 h post infection. the factor responsible for this alteration is the product of ul37x1 gene, which is an early antiapoptotic gene and also referred to as viral mitochondrion localized inhibitor of apoptosis (vmia) [130] . vmia contains two domains. the first domain is located between amino acids 5 and 34 (30 amino acids), overlaps with the mitochondrial signal sequence that targets vmia to the mitochondria [131] . the second domain located between amino acids 118 and 147 displays anti-apoptotic properties [131] . a recombinant fusion protein derived from vmia consisting of these two domains is fully functional. vmia is conserved in all human cmv strains as well as in other primate cmvs that have been examined [132] . the vmia-mediated inhibition of apoptosis is dependent on its interaction with the pro-apoptotic bax and bak proteins [133, 134] . but the primary sequence of vmia has very low similarity with bcl-xl (anti-apoptotic). the ectopic bax is able to rescue the vmia-mediated fragmented phenotype, whereas the bh3 mutant bax did not show any rescuing effect [135] . this study indicates involvement of the functions of the bax and bak bh3 domains in both promoting apoptosis as well as regulating mitochondrial morphology in healthy cells. pseudorabies virus (prv) and herpes simplex virus type 1 (hsv-1) infection disrupts mitochondrial motility and morphology in the superior cervical ganglion (scg) neurons of rodents [136] . during prv infection the fusion events mediated by glycoprotein b (gb) resulted in electrical coupling of neurons and increased action potential firing rates that were causes of intracellular ca 2+ increase and altered mitochondrial dynamics [136] . this mechanism is governed by the ca 2+ sensitive cellular protein, miro, which inhibits the recruitment of kinesin-1 hc to the mitochondria. prv-mediated disruption in mitochondrial dynamics is required for efficient growth and spread of prv, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection. this study has opened the possibility that kinesin-1 hc is hijacked and recruited for the movement of viral particles or protein cargo during prv assembly and/or egress [136] . infections by influenza viruses account for millions of deaths worldwide. an interesting study demonstrates that nod2 and receptor interacting protein kinase 2 (ripk2) respond to influenza a virus (iav) infection by promoting ulk1 phosphorylation and inducing mitophagy [98] . ripk2 −/− cells exhibited defective mitophagy and triggered greater inflammasome activation because of the accumulation of the damaged mitochondria. this suggests that upregulation of mitochondrial quality control during iav infection dampens inflammasome activation and il-18 production [98] . this induction in mitophagy was independent of parkin but was associated with ulk1 phosphorylation at serine 555. however, it is not clear if ripk2 directly phosphorylates ulk1 or whether other intermediates are involved. overall this study suggests that nod2-ripk2 signaling protects against virally triggered immunopathology by upregulating ulk1-dependent mitophagy [98] . the influenza a viral protein pb1-f2 binds to mavs and affects ifn synthesis [137] . pb1-f2 also induces mitochondrial fragmentation, modulates innate immune response and nlrp3 inflammasome activation [138] . pb1-f2 translocates to mitochondrial inner membrane space via tom40 channel and leads to the reduction of mitochondrial membrane potential. the pb1-f2 variant lacking c-terminal domain does not affect mitochondrial function [138] . measles is an acute, highly infectious disease. the edmonston strain of measles virus (mv-edm) triggers p62-mediated mitophagy in nonsmall cell lung cancer (nsclc) cells [139] . defect in autophagy decreases viral titers and cell death induced by mv-edm in nsclc cells. mv-edm triggered mitophagy results in the reduction of mavs protein levels and subsequent dampening of the innate immune response. silencing p62 expression inhibited mitophagy and resulted in the preservation of mitochondrial mass in mv-edm-infected cells. overall this study suggests that mv usurps mitophagy to mitigate the innate immune response mediated by rig-i/mavs signaling [139] . interestingly, a similar study by the same group suggests that mv-edm triggered p62-mediated mitophagy that subverts apoptosis of infected nsclc cells by preventing cytochrome c release and leads to enhanced viral replication [140] . autophagy-sustained persistent viral replication and eventually promotes necrotic cell death due to atp exhaustion and inhibition of autophagy resulted in marked decline in mv-edm associated oncolytic activity. though both the studies agree that mv-edm induces p62-dependent mitophagy, and that this induction is required to promote viral replication, both differ on the physiological outcome of mitophagy. the same group also showed that the newcastle disease virus (ndv) uses a similar strategy and exploits p62-mediated mitophagy to promote viral propagation. induction of mitophagy attenuated cytochrome c release and intrinsic apoptotic signaling in ndv-infected nsclc cells and inhibition of mitophagy enhanced the oncolytic effect of ndv [140] . it is also likely that inhibition of mitophagy promotes innate immune signaling leading to cell death or both mitochondrialapoptosis andinnate immune signaling contribute to cell death/oncolysis. severe acute respiratory syndrome-coronavirus (sars-cov) is a novel coronavirus, which has emerged as a lethal pathogen and threat to human health. how sars-cov escapes the innate immune signaling is poorly understood. a recent study revealed that sars-cov protein, open reading frame-9b (orf-9b) localizes to the mitochondria and promotes proteasomal degradation of drp1 leading to mitochondrial fusion [141] . expression of 9b in hek 293 cells led to~70% reduction in drp1 levels and this reduction was sensitive to proteasome inhibition but unaffected by inhibition of autophagy [141] . interestingly, in this study, lowering drp1 expression was associated with impaired mavs signaling, although this reduction was not as effective in comparison to exogenous expression of orf-9b protein suggesting that other mechanisms may be operative. the notion that reduced drp1 expression or enhanced mitochondrial fusion hampers mavs signaling is in sharp contrast to the common belief that mavs signaling and ifn production are enhanced and dampened by mitochondrial fusion and fission respectively [141] . it is further shown that orf-9b also promotes degradation of mavs and its interacting partners by targeting poly(rc) binding protein 2 (pcbp2) and the hect domain e3 ligase aip4 to trigger the degradation of mavs, traf3, and traf6 leading to disruption of mavs signaling and ifn production. the localization of orf-9b to the mitochondria led to the translocation of pcbp2 from the nucleus to the mitochondria, which subsequently interacts with aip4 and targets it to the mitochondria. it remains to be clarified if traf3 and -6, are direct targets of aip4. silencing pcbp2 or aip4 expression restored mavs signaling and ifn production [141] . these results indicate that sars-cov orf-9b manipulates mitochondrial dynamics and targets mitochondrial innate immune signaling to evade host innate immunity. however, more studies will be required to elucidate the physiological significance of enhanced mitochondrial fusion in the viral infectious processes or disease pathogenesis. despite the efforts made in the last few decades in research focusing on the role of the mitochondria and viral infections, many issues remain unresolved. recently, the mitochondrial dynamics and mitophagy have gained substantial attention as these events modulate mitochondrial functions during viral infections. current findings indeed have placed the mitochondrial dynamics and viral infections at the crossroad. undoubtedly, there is a close relationship between the mitochondrial dynamics and viral infections but in-depth characterizations and their relevance to pathogenesis may help in understanding the pathogenic processes. it is important to determine how the players of mitochondrial dynamics and mitophagy are differentially regulated during viral infections and elucidate the different ways viruses exploit these alterations to their own benefits. elucidation of the functional relevance of mitochondrial dynamics and viral pathogenesis will open new avenues for therapeutic design of strategies to combat viral infections and associated diseases. greater understanding of the critical mitochondrial functions like bioenergetics, apoptosis, innate anti-viral signaling and inter-organelle cross talk needs to be pursued in future investigations to elucidate the impact of viral infections on mitochondria and mitochondrial dynamics. pathogen recognition and innate immunity mitochondria: more than just a powerhouse mitochondria: a historical review mitochondria: dynamic organelles in disease, aging, and development functions and dysfunctions of mitochondrial dynamics abnormal mitochondrial dynamics and neurodegenerative diseases hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis hepatitis c virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy viruses as modulators of mitochondrial functions a renewed focus on the interplay between viruses and mitochondrial metabolism the interplay between mitochondrial dynamics and mitophagy mitochondrial dynamics-fusion, fission, movement, and mitophagy-in neurodegenerative diseases autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis autophagy in embryonic erythroid cells: its role in maturation parkin overexpression selects against a deleterious mtdna mutation in heteroplasmic cybrid cells fusion of mitochondria in mammalian cells is dependent on the mitochondrial inner membrane potential and independent of microtubules or actin mitophagy selectively degrades individual damaged mitochondria after photoirradiation fission and selective fusion govern mitochondrial segregation and elimination by autophagy mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view the mitochondrial permeability transition initiates autophagy in rat hepatocytes high levels of fis1, a pro-fission mitochondrial protein, trigger autophagy giant mitochondria do not fuse and exchange their contents with normal mitochondria mitochondrial fusion in yeast requires the transmembrane gtpase fzo1p developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted gtpase mitofusins mfn1 and mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development control of mitochondrial morphology by a human mitofusin disruption of fusion results in mitochondrial heterogeneity and dysfunction primary structure of a dynamin-related mouse mitochondrial gtpase and its distribution in brain, subcellular localization, and effect on mitochondrial morphology fusion and fission: interlinked processes critical for mitochondrial health mitofusins and opa1 mediate sequential steps in mitochondrial membrane fusion opa1 processing controls mitochondrial fusion and is regulated by mrna splicing, membrane potential, and yme1l sirt3 deacetylates and activates opa1 to regulate mitochondrial dynamics during stress inducible proteolytic inactivation of opa1 mediated by the oma1 protease in mammalian cells regulation of opa1 processing and mitochondrial fusion by m-aaa protease isoenzymes and oma1 stress-induced oma1 activation and autocatalytic turnover regulate opa1-dependent mitochondrial dynamics stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis proteasome and p97 mediate mitophagy and degradation of mitofusins induced by parkin fis1, mff, mid49, and mid51 mediate drp1 recruitment in mitochondrial fission mid49 and mid51, new components of the mitochondrial fission machinery er tubules mark sites of mitochondrial division an actin-dependent step in mitochondrial fission mediated by the er-associated formin inf2 the mitochondrial protein hfis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein dlp1 roles of the mammalian mitochondrial fission and fusion mediators fis1, drp1, and opa1 in apoptosis the novel tail-anchored membrane protein mff controls mitochondrial and peroxisomal fission in mammalian cells mff is an essential factor for mitochondrial recruitment of drp1 during mitochondrial fission in mammalian cells atypical mitochondrial fission upon bacterial infection dynamin-related protein drp1 is required for mitochondrial division in mammalian cells mitotic phosphorylation of dynamin-related gtpase drp1 participates in mitochondrial fission rala and ralbp1 regulate mitochondrial fission at mitosis high-glucose stimulation increases reactive oxygen species production through the calcium and mitogen-activated protein kinase-mediated activation of mitochondrial fission cyclic amp-dependent protein kinase phosphorylation of drp1 regulates its gtpase activity and mitochondrial morphology mechanism of neuroprotective mitochondrial remodeling by pka/akap1 pka/akap1 and pp2a/bbeta2 regulate neuronal morphogenesis via drp1 phosphorylation and mitochondrial bioenergetics dephosphorylation by calcineurin regulates translocation of drp1 to mitochondria cam kinase i alpha-induced phosphorylation of drp1 regulates mitochondrial morphology mitochondrial fission triggered by hyperglycemia is mediated by rock1 activation in podocytes and endothelial cells cdk5-dependent inhibitory phosphorylation of drp1 during neuronal maturation s-nitrosylation of drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury mapl is a new mitochondrial sumo e3 ligase that regulates mitochondrial fission the sumo protease senp5 is required to maintain mitochondrial morphology and function parkin ubiquitinates drp1 for proteasome-dependent degradation: implication of dysregulated mitochondrial dynamics in parkinson disease a novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics autophagy: process and function pink1 is selectively stabilized on impaired mitochondria to activate parkin the mitochondrial intramembrane protease parl cleaves human pink1 to regulate pink1 trafficking pink1 is activated by mitochondrial membrane potential depolarization and stimulates parkin e3 ligase activity by phosphorylating serine 65 ubiquitin is phosphorylated by pink1 to activate parkin pink1 phosphorylates ubiquitin to activate parkin e3 ubiquitin ligase activity parkin is activated by pink1-dependent phosphorylation of ubiquitin at ser65 quantitative proteomics reveal a feedforward mechanism for mitochondrial parkin translocation and ubiquitin chain synthesis mitochondrial dysfunction in drosophila pink1 mutants is complemented by parkin pink1-phosphorylated mitofusin 2 is a parkin receptor for culling damaged mitochondria broad activation of the ubiquitin-proteasome system by parkin is critical for mitophagy p62/sqstm1 is required for parkin-induced mitochondrial clustering but not mitophagy; vdac1 is dispensable for both disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and hdac6-dependent mitophagy the pathways of mitophagy for quality control and clearance of mitochondria pink1 and parkin target miro for phosphorylation and degradation to arrest mitochondrial motility parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction loss of iron triggers pink1/parkinindependent mitophagy ambra1 is able to induce mitophagy via lc3 binding, regardless of parkin and p62/sqstm1 ampk and mtor regulate autophagy through direct phosphorylation of ulk1 ulk1 translocates to mitochondria and phosphorylates fundc1 to regulate mitophagy role of bnip3 and nix in cell death, autophagy, and mitophagy mitochondrial outer-membrane protein fundc1 mediates hypoxia-induced mitophagy in mammalian cells lc3 binds externalized cardiolipin on injured mitochondria to signal mitophagy in neurons: implications for parkinson disease cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells the role of dynamin-related protein 1, a mediator of mitochondrial fission inhibiting drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis membrane remodeling induced by the dynamin-related protein drp1 stimulates bax oligomerization bax/bak promote sumoylation of drp1 and its stable association with mitochondria during apoptotic cell death nitric oxide-induced mitochondrial fission is regulated by dynamin-related gtpases in neurons opa1 controls apoptotic cristae remodeling independently from mitochondrial fusion mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner mitochondrial dysfunction indirectly elevates ros production by the endoplasmic reticulum physiological roles of mitochondrial reactive oxygen species receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection erassociated mitochondrial division links the distribution of mitochondria and mitochondrial dna in yeast mitofusin 2 tethers endoplasmic reticulum to mitochondria mitochondrial fission: regulation and er connection identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 mitochondrial dynamics regulate the rig-i-like receptor antiviral pathway mitochondrial membrane potential is required for mavs-mediated antiviral signaling mitochondria in innate immune responses virus-infection or 5′ppp-rna activates antiviral signal through redistribution of ips-1 mediated by mfn1 mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus hepatitis b and c virus hepatocarcinogenesis: lessons learned and future challenges hepatitis c virus hijacks host lipid metabolism unravelling hepatitis c virus replication from genome to function the autophagy machinery is required to initiate hepatitis c virus replication induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response hepatitis c virus, er stress, and oxidative stress endoplasmic reticulum (er) stress: hepatitis c virus induces an er-nucleus signal transduction pathway and activates nf-kappab and stat-3 hepatitis c virus proteins: direct link to hepatic oxidative stress, steatosis, carcinogenesis and more human hepatitis c virus ns5a protein alters intracellular calcium levels, induces oxidative stress, and activates stat-3 and nf-kappa b hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity abnormalities of lipid metabolism in hepatitis c virus infection hepatitis c virus protein expression causes calciummediated mitochondrial bioenergetic dysfunction and nitro-oxidative stress viral and therapeutic control of ifnbeta promoter stimulator 1 during hepatitis c virus infection hepatitis b virus and hepatitis delta virus hepatitis b virus x protein colocalizes to mitochondria with a human voltage-dependent anion channel, hvdac3, and alters its transmembrane potential calcium signaling by hbx protein in hepatitis b virus dna replication mitochondrially associated hepatitis b virus x protein constitutively activates transcription factors stat-3 and nf-kappa b via oxidative stress the early autophagic pathway is activated by hepatitis b virus and required for viral dna replication epstein-barr virus: 40 years on epstein-barr virus latent membrane protein-2a alters mitochondrial dynamics promoting cellular migration mediated by notch signaling pathway human cytomegalovirus cell tropism and pathogenesis disruption of mitochondrial networks by the human cytomegalovirus ul37 gene product viral mitochondrionlocalized inhibitor of apoptosis the sequence and antiapoptotic functional domains of the human cytomegalovirus ul37 exon 1 immediate early protein are conserved in multiple primary strains vmia, a viral inhibitor of apoptosis targeting mitochondria cytomegalovirus cell death suppressor vmia blocks bax-but not bak-mediated apoptosis by binding and sequestering bax at mitochondria cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vmia role of bax and bak in mitochondrial morphogenesis alphaherpesvirus infection disrupts mitochondrial transport in neurons influenza virus protein pb1-f2 inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential influenza a virus protein pb1-f2 translocates into mitochondria via tom40 channels and impairs innate immunity mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-i-like receptor signaling mitophagy switches cell death from apoptosis to necrosis in nsclc cells treated with oncolytic measles virus sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome aberrant mitochondrial fission in neurons induced by protein kinase c{delta} under oxidative stress conditions in vivo march5-mediated quality control on acetylated mfn1 facilitates mitochondrial homeostasis and cell survival mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells research in the laboratory (a.s.) is supported by grants from nih (dk077704, dk08379 and ai085087) and michael j fox foundation. key: cord-316703-8kxx3034 authors: parera, mariona; martrus, gloria; franco, sandra; clotet, bonaventura; martinez, miguel angel title: canine hepacivirus ns3 serine protease can cleave the human adaptor proteins mavs and trif date: 2012-08-01 journal: plos one doi: 10.1371/journal.pone.0042481 sha: doc_id: 316703 cord_uid: 8kxx3034 canine hepacivirus (chv) was recently identified in domestic dogs and horses. the finding that chv is genetically the virus most closely related to hepatitis c virus (hcv) has raised the question of whether hcv might have evolved as the result of close contact between dogs and/or horses and humans. the aim of this study was to investigate whether the ns3/4a serine protease of chv specifically cleaves human mitochondrial antiviral signaling protein (mavs) and toll-il-1 receptor domain-containing adaptor inducing interferon-beta (trif). the proteolytic activity of chv ns3/4a was evaluated using a bacteriophage lambda genetic screen. human mavsand trif-specific cleavage sites were engineered into the lambda ci repressor. upon infection of escherichia coli cells coexpressing these repressors and a chv ns3/4a construct, lambda phage replicated up to 2000-fold more efficiently than in cells expressing a chv protease variant carrying the inactivating substitution s139a. comparable results were obtained when several hcv ns3/4a constructs of genotype 1b were assayed. this indicates that chv can disrupt the human innate antiviral defense signaling pathway and suggests a possible evolutionary relationship between chv and hcv. the origin of hepatitis c virus (hcv) infections in humans has remained unknown, because related animal virus homologs had not been identified [1, 2] . recently, a flaviviridae rna genome that was isolated from domestic dogs with respiratory illness was found to be the virus most closely related to hcv [3] . this flaviviridae agent is known as canine hepacivirus (chv). the discovery of chv may shed light on the origin of hcv, and may also serve as a new model system with which to study this deadly human virus. chv-like viruses (also known as nonprimate hepaciviruses [nphv] ) were also recently identified in horses [4] . hcv belongs to the genus hepacivirus, one of the four genera in the family flaviviridae [2] . hcv infects more than 170 million people worldwide (http://www.who.int/mediacentre/factsheets/fs164/ en/) and is one of the leading causes of liver cirrhosis and failure [5] . although the discovery of the close homology between chv and hcv is intriguing, there are barriers preventing viral transmission across species. cross-species transmission of chv to humans would most likely require evasion of the human cellular innate immune response, which leads to type i interferon production through rna composition-dependent activation of retinoic acid inducible gene-i (rig-i) and toll-like receptor (tlr) [6] . a number of host-encoded restriction factors can protect human cells from viral infections. a well characterized model is retroviruses. the human genome has evolved a constellation of antiviral genes with the potential to target retroviruses. these genes are part of the innate immune system, and in the retrovirus literature, they are often called restriction factors. well-character-ized restriction factors are apobec3g [7] , trim5-a proteins [8] , tetherin (bst2) [9] , and samhd1 [10, 11] , which inhibit human immunodeficiency virus (hiv) propagation at various steps in its replication cycle. the evolution of the genes encoding these restriction factors affected the adaptation of hiv to humans in the current pandemic, and the adaptation of other primate lentiviruses to their modern hosts. in the case of hcv, signalling pathways leading to type i interferon production are the first line of defence employed by the host to combat viruses and represent a barrier that invading viruses must overcome in order to establish infection [12, 13] . a major strategy employed by hcv to subvert the host innate immune response is disruption of the rig-i and tlrsignalling pathway through hcv ns3/4a protease cleavage of the adaptor proteins mavs and trif [14, 15] . mavs from multiple primates are resistant to inhibition by the hcv ns3/4a protease [16] . this resistance maps to single changes within the protease cleavage site in mavs, which protect mavs from getting cleaved by the hcv ns3/4a protease. these changes were likely driven by ancient hepaciviruses, providing insights into hepaciviral infections over the course of primate evolution [16] . because the propagation of chv and nphv in humans would most likely have had to overcome the human innate host defence mediated by type i interferon, we hypothesized that chv ns3/ 4a protease could specifically process the human adaptor proteins mavs and trif. to test this hypothesis, we coexpressed the chv ns3/4a protease with specific mavs and trif cleavage sites that are known to be processed in vivo by the hcv ns3/4a protease. using this approach, we demonstrated that the chv ns3/4a protease can cleave human mavs and trif. the catalytic activity of chv ns3/4a protease was assayed using a bacteriophage lambda-based genetic screen. we previously demonstrated that the bacteriophage lambda-based genetic screen employed here can be used to characterize site-specific proteases, including hcv ns3/4a, hiv, and respiratory syndrome (sars) coronavirus proteases [17, 18, 19, 20, 21, 22, 23, 24] . this genetic screen is based on the bacteriophage lambda ci-cro regulatory circuit, in which the viral repressor ci is specifically cleaved to initiate the switch from lysogeny to lytic infection [25] . this genetic screen is a simple alternative approach to the purification and characterization of expressed proteases by in vitro classical methodologies. the specific cleavage sites engineered in the lambda ci repressor and assayed in this study are depicted in fig. 1 . these sites included specific mavs and trif cleavage sites known to be processed in vivo by the hcv ns3/4a protease, as well as chv and hcv ns5a/ns5b viral polyprotein cleavage sites. a mutated version of the trif cleavage site was also included as a control. the target specificity for the mavs and trif cleavage sites was tested by coexpressing them with chv or hcv ns3/4a protease constructs. the ns3/ 4a protease constructs contained ns4 residues 21-34 fused in frame via a short linker to the amino terminus of the ns3 protease domain (residues 1-181) (fig. 2) . the chv ns3/4a protease construct was chemically synthesized following the nucleotide sequence reported by kapoor et al. [3] . upon infection of e. coli cells coexpressing the lambda ci repressor with either mavs or trif cleavage site and a chv ns3/4a construct, lambda phage replicated up to 2,000-fold more efficiently than in cells expressing a chv protease variant that included a substitution in catalytic residue s139 ( fig. 3a and 3b). equivalent results were obtained when four hcv ns3/4a of genotype 1b were assayed. three assayed hcv ns3/4a proteases were isolated from three treatment-naïve hcv-infected patients [18, 24] . the fourth hcv ns3/4a protease was amplified from the hcv subgenomic replicon i389/ns3-3 [26] . to investigate the influence of the ns4 protein on the catalytic efficiency of chv ns3 protease, the chv ns4 residues i25 and v29 were replaced with a residues (fig. 1 ). hcv ns4 residues 25 and 29 are critical for ns4 activation of hcv ns3 protease [18, 27] . similar to hcv ns3 protease, a mutated version of chv ns4 reduced the catalytic efficiency of the chv ns3 protease, indicating that the chv ns4 peptide is essential for efficient processing of mavs and trif cleavage sites ( fig. 3a and 3b) . nevertheless, the effect of the mutated ns4 residues 25 and 29 on chv ns3/4a protease activity was less drastic than that observed on hcv ns3/4a protease ( fig. 3b ) (see below). we further tested the target specificity of the mavs and trif cleavage sites by analyzing a control mutant trif target site. a mutant trif cleavage site was constructed in which the c residue at the p1 position was substituted with a y residue (fig. 1 ). as shown in fig. 3c , the mutant trif cleavage site prevented phage replication, indicating that trif processing was specifically mediated by chv ns3/4a protease. western blot analysis also demonstrated ( fig. 4 ) that the expression of the chv ns3/4a protease resulted in nearly complete cleavage of the lambda ci repressor with either mavs (fig. 4a ) or trif cleavage site (fig. 4b ). expression of ns3/4a proteases that included a substitution in catalytic residue s139 (fig. 4) completely abolished the cleavage of lambda ci repressor with either mavs or trif cleavage site. expression of ns3/4a proteases with a mutated version of ns4 partially abolished wildtype mavs or trif repressor cleavage (fig. 4) ; specifically, mutations in ns4 had less impact in the activity of chv ns3/4a protease than in the activity of hcv ns3/4a protease (fig. 4b) . as expected, a control mutant trif target site ( fig. 1) was not cleaved by wild-type ns3/4a proteases. overall, these results demonstrate that chv ns3/4a protease is able to specifically cleave the human adaptor proteins mavs and trif. we next investigated whether chv and hcv ns3/4a proteases could specifically process heterologous viral polyprotein cleavage sites. to do so, the chv and hcv ns5a/ns5b cleavage sites were engineered ( fig. 1 ) and tested against both proteases. interestingly, the chv and hcv ns3/4a proteases processed the heterologous viral cleavage sites with comparable efficiency (fig. 5 ), suggesting that chv and hcv ns3/4a proteases may share the same evolutionary linage. finally, two macrocyclic hcv ns3/4a protease inhibitors, 25a [28] and danoprevir [29] , were assayed to test their ability to inhibit chv ns3/4a protease. macrocyclic inhibitors were chosen because they have a higher potency against different hcv genotypes compared to linear inhibitors [30] . the inhibitory capability of these two macrocyclic inhibitors was assessed with the mavs and trif cleavage sites. as expected, 25a and danoprevir inhibited hcv ns3/4a protease (fig. 6 ) when tested with either cleavage site. in contrast, no significant inhibition was observed when either of the two inhibitors was assayed with the chv ns3/4a protease. of note, the positions associated with resistance to macrocyclic hcv ns3/4a protease inhibitors, hcv residues q80, r155, a156, and d168 [31] , are mutated in the chv ns3/4a protease sequence (fig. 2) . the specificity of this inhibition was tested by including an hcv ns3/4a protease mutant carrying the protease inhibitor resistance substitution d168v; this substitution reduced the inhibitory capacity of the two tested protease inhibitors (fig. 6 ). recent studies have identified viruses, in domestic dogs and horses, which are genetically very closely related to hcv [3, 4] . these findings suggest that hcv could have been introduced into the human population through contact with dogs and/or horses. nevertheless, adaptation of chv and nphv to human cells would likely require these viruses to evade the human cellular innate immune response, which responds to viral pathogens by rna composition-dependent activation of rig-i and subsequent signalling via interferon regulatory factor (irf)-3 [6] . the response to hcv infection is regulated by hepatic immune defences triggered by cellular rig-i [14, 32] . in human liver cells, hcv interferes with rig-i-dependent signalling to irf-3 by ns3/4a-dependent cleavage of the crucial adaptor protein mavs [14] . likewise, tlr-3-dependent signalling to irf-3 is ablated by cleavage of trif by the hcv ns3/4a protease [15] . in this study, we tested the ability of chv ns3/4a protease to specifically cleave the human adaptor proteins mavs and trif. we found that chv ns3/4a protease could process, with comparable efficiency, the specific mavs and trif cleavage sites known to be processed in vivo by hcv ns3/4a protease [14, 15] . these findings extend previous reports [3, 4] , confirming a possible evolutionary relationship between chv and hcv. the results of our study indicate that chv can disrupt the human innate antiviral defense signaling pathway. in addition, our study also showed that, similar to hcv ns3/4a protease, chv ns3 protease efficiency is regulated by the viral ns4 protein [33] , suggesting that the mechanism of action and substrate specificity is similar between the two proteases. chv ns4 polypeptide improved cleavage of mavs-and trif-specific cleavage sites. however, chv ns3/4a protease was not susceptible to macrocyclic inhibitors of hcv ns3/4a protease. important hcv genotype-specific differences in the efficacy of ns3/4a protease inhibitors were reported recently [29] . most of the hcv ns3/4a protease inhibitors now in clinical development were identified using hcv replicon systems of genotype 1 and are being licensed for treatment of genotype 1 infection. in vitro studies performed with hcv chimeras have shown that genotypes 2a, 5a, and 6a had similar sensitivity, whereas genotype 3a was comparatively resistant to macrocyclic hcv ns3/4a protease inhibitors [30] . chv ns3/4a protease has amino acid changes at positions associated with resistance to macrocyclic hcv ns3/4a protease inhibitors [31] , which would explain why this protease is resistant to macrocyclic hcv ns3/4a protease inhibitors. 1-181) . it was previously demonstrated that the kinetic parameters of a single-chain protein containing the ns4a cofactor and the hcv ns3 protease are identical to those of the bona fide ns3/4a (ns3 residues 1 to 631 and ns4a residues 1 to 54 protein complex generated in eukaryotic cells (5, 38) . asterisks represent the stop codons. mutated residues generated by site-directed mutagenesis are marked in red. doi:10.1371/journal.pone.0042481.g002 gb virus b (gbv-b) is another hepacivirus that before the discovery of chv/nhv was the virus phylogenetically most closely related to hcv. gbv-b does not infect humans or chimpanzees, but it causes acute hepatitis in experimentally infected tamarins (a type of new world monkey) [2] . although there are important differences in the outcomes of hcv and gbv-b infections (gbv-b rarely causes persistent infections), gbv-b has evolved a strategy similar to that employed by hcv, resulting in the disruption of rig-i signalling. indeed, gbv-b ns3/4a proteolytically cleaves human mavs. more distantly related hepacivirus such as gbv-c, which infects humans and chimpanzees, and gbv-a are also able to antagonizing mavs [16] . in contrast, proteases from the more distantly related pestivirus bovine viral diarrhea virus and flavivirus yellow fever virus do not antagonize mavs [16, 34] . overall these data suggest that the ability of ns3/4a proteases to antagonize mavs is a phylogenetically discrete characteristic exclusive to hcv and the three gbv viruses. based on these observations, chv ns3/4a should be able to cleave the canine's mavs and trif proteins. canine orthologs of human mavs and trif differ in sequence at the cleavage site processed by hcv ns3/4a protease; therefore, they were not tested in this study. inspection of the canine mavs and trif proteins did not reveal the presence of apparent substrate specificities for hcv ns3/4a protease. although chv was initially found in dogs, subsequent efforts to find similar viruses in canids has remained largely unsuccessful [4] . recently, evidence of a chv-like virus infection was confirmed by the detection of diverse viral genomes, termed nphv, in horses [4] . horses can support replication of several other flaviviruses, including the vector-borne west nile, japanese encephalitis, dengue, and st. louis encephalitis viruses. these flaviviruses can be transmitted among several mammalian species, including humans. most of the nphv strains detected in horses are genetically distinct from chv; however, one variant is almost expression of the protease was induced with iptg for 3 h. the western blot proved that the lambda ci repressor with either mavs or trif cleavage site was not cleaved by ns3/4a proteases that included a substitution in catalytic residue s139. similarly, a control mutant trif target site (fig. 1) was not cleaved by wild-type ns3/4a proteases. the hcv 1 ns3/4a protease was also tested without iptg. doi:10.1371/journal.pone.0042481.g004 identical to chv, suggesting that chv/nphv viruses may be able to jump species [4] . this is the first study to our knowledge to demonstrate that at least parts of the identified and sequenced chv/nphv rna genomes are biologically active. initial attempts to culture chv were not successful [3, 35] . only a single strain of hcv, the genotype 2a strain jfh1 from a japanese patient, has been found to grow robustly in culture, in human hepatoma cell lines [36] . however, viable jfh1-based recombinants with gene elements specific to other hcv genotypes have been developed [30, 37, 38] . given the difficulties in culturing hcv, our results suggest that it may be possible to generate hcv/chv chimeras that can be cultivated. our study has some limitations that are worth noting. first, this study only tested the functionality of a small chv genomic region; other chv/nphv genomic regions that have been previously described [3, 4] should be analyzed to guarantee the viability of future hcv/chv chimeras. second, the in vitro approach used here to measure the ability of the protease to cleave human adaptors mavs and trif only partially mimics what occurs in vivo. third, it has not been determined here whether chv ns3/ 4a protease can efficiently cleavage the mavs and trif proteins equivalent of horses and canines, their primary hosts. nevertheless, the similarity observed between hcv and chv ns3/4a proteases in their capability to process mavs and trif, as well as hcv/chv ns5a/ns5b cleavage sites, strongly supports the hypothesis that chv might be able to disrupt the human innate antiviral defense signaling pathway. further work should include an evaluation of ability of the chv ns3/4a protease to process both the human and canine adaptor proteins mavs and trif in human and canine hepatoma cell lines, as well as the construction of hcv/chv chimeras to explore this hypothesis or to perform other functional studies related to hepacivirus pathogenesis and treatment. insertion of the mavs and hcv ns5a/ns5b-specific cleavage sites (fig. 1) into the lambda ci repressor has been described elsewhere [20, 24] . in this study, trif and chv ns5a/ns5b-specific cleavage sites (fig. 1) were inserted into the lambda ci repressor. briefly, the nucleotide sequence for trif and chv ns5a/ns5b-specific cleavage sites (fig. 1 ) was chemically synthesized (integrated dna technologies). the constructs included two restriction sites, nsii and hindiii, at the ends, which are present in the lambda ci repressor [39] . after digestion with nsii and hindiii, the construct was ligated into pci.hcvns5a/ns5bcro [20] , previously digested with nsii and hindiii, to generate the pci.trifcro and pci.chvns5a/ ns5bcro plasmids sequence analysis of this plasmid confirmed the accuracy of the synthetic construct. e. coli jm109 cells containing plasmid pci.mavscro, pci.trifcro, pci.hcvns5a/ ns5bcro or pci.chvns5a/ns5bcro were then transformed with the plasmid expressing the corresponding protease. transformed cells were grown overnight at 30uc in the presence of 0.2% maltose, 12.5 mg/ml tetracycline, and 20 mg/ml ampicillin; harvested by centrifugation; and resuspended to an optical density at 600 nm of 2.0/ml in 10 mm mgso4. to induce expression of the protease, 20 ml of cells were incubated in 100 ml of luria-bertani (lb) medium containing 12.5 mg/ml tetracycline, 20 mg/ ml ampicillin, 0.2% maltose, 10 mm mgso4, and 0.1 mm isopropyl-b-d-thiogalactopyranoside (iptg) for 1 h. the cell cultures were then infected with 10 5 plaque-forming units (pfu) of phage. after 3 h at 37uc, the titer of the resulting phage was determined by coplating the cultures with 200 ml of e. coli xl-1 blue cells (adjusted to an optical density at 600 nm of 2.0/ml in 10 mm mgso4) on lb plates using 3 ml of top agar containing 12.5 mg of tetracycline/ml, 0.2% maltose, and 0.1 mm iptg. after incubation at 37uc for 6 h, the resulting phage plaques were counted in order to score growth. western blots were performed as previously described [19] . briefly, e. coli jm109 cells containing plasmid pci.mavscro or pci.trifcro were transformed with the plasmid expressing the corresponding protease. transformed cells were then grown overnight at 30uc in the presence of 0.2% maltose, 12.5 mg/ml tetracycline, and 20 mg/ml ampicillin; harvested by centrifugation; and resuspended to an optical density at 600 nm of 2.0/ml in 10 mm mgso4. to induce expression of the protease, 200 ml of cells were incubated in 1 ml of lb medium containing 12.5 mg/ml tetracycline, 20 mg/ml ampicillin, 0.2% maltose, 10 mm mgso4, and 0.1 mm iptg for 3 h. the ods of the cultures after 3 h (in the presence of iptg) were measured to assure that equivalent amounts of total cell protein were blotted. no significant differences were observed when the ods of the different cultures were compared, suggesting that the expression of the ns3/4a proteases did not affect the growth of the bacteria. cultures were lysed in sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis sample buffer, resolved in 12% gradient sds polyacrylamide gels (invitrogen), transferred to nitrocellulose membranes, and blocked in phosphate-buffered saline-0.1% tween 20-10% nonfat dry milk. for immunochemical detection of the lambda repressor, membranes were subsequently incubated with rabbit serum containing polyclonal anti-ci antibodies (anti-ci sera; invitrogen). bound antibodies were visualized with peroxidaselinked anti-rabbit antybody hrp-linked igg (cell signaling technology) and the supersignal west pico chemiluminescent substrate (thermo scientific). the nucleotide sequence for chv ns3/4a protease (fig. 2 ) was chemically synthesized (integrated dna technologies) following the nucleotide sequence reported by kapoor et al. [3] . the construct included two restriction sites, ecori and xhoi, at the ends. after digestion with ecori and xhoi, the construct was cloned into pbluescript sk-(agilent technologies) to generate a b-galactosidase-chv ns3/4a protease fusion protein. sequence analysis of this plasmid confirmed the accuracy of the synthetic construct. control mutant constructs (red residues in figs. 1 and 2) were generated by site-directed mutagenesis using the quik-change kit (agilent technologies) and following the manufacturer's instructions. three hcv ns3/4a proteases used in this study, 1, 50, and 51, were obtained from hcv genomic rna extracted from the plasma of three different hcv-infected patients. the nucleotide sequences and amplification procedures of these proteases have been published previously [18, 24] . the fourth hcv ns3/4a protease employed in this study was amplified from the hcv subgenomic replicon i389/ns3-3 [26] . macrocyclic protease inhibitor 25a [28] was kindly provided by merck, sharp and dohme. donoprevir was purchased from selleck chemicals (houston, tx, usa). genetic diversity and evolution of hepatitis c virus-15 years on the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae characterization of a canine homolog of hepatitis c virus serology enabled discovery of genetically diverse hepaciviruses in a new host hepatitis c virus: virology, diagnosis and management of antiviral therapy evasion of intracellular host defence by hepatitis c virus isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein evasion and disruption of innate immune signalling by hepatitis c and west nile viruses induction and evasion of innate antiviral responses by hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif convergent evolution of escape from hepaciviral antagonism in primates a bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease genetic screen for monitoring hepatitis c virus ns3 serine protease activity genetic screen for monitoring severe acute respiratory syndrome coronavirus 3c-like protease genetic and catalytic efficiency structure of an hcv protease quasispecies fitness landscape of human immunodeficiency virus type 1 protease quasispecies hiv-1 protease catalytic efficiency effects caused by random single amino acid substitutions epistasis among deleterious mutations in the hiv-1 protease complexity and catalytic efficiency of hepatitis c virus (hcv) ns3 and ns4a protease quasispecies influence responsiveness to treatment with pegylated interferon plus ribavirin in hcv/hiv-coinfected patients a genetic switch replication of subgenomic hepatitis c virus rnas in a hepatoma cell line identification of the sequence on ns4a required for enhanced cleavage of the ns5a/5b site by hepatitis c virus ns3 protease molecular modeling based approach to potent p2-p4 macrocyclic inhibitors of hepatitis c ns3/4a protease preclinical characteristics of the hepatitis c virus ns3/4a protease inhibitor itmn-191 (r7227) differential efficacy of protease inhibitors against hcv genotypes 2a, 3a, 5a, and 6a ns3/ 4a protease recombinant viruses treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna multiple determinants influence complex formation of the hepatitis c virus ns3 protease domain with its ns4a cofactor peptide gb virus b disrupts rig-i signaling by ns3/4a-mediated cleavage of the adaptor protein mavs hepatitis c homolog in dogs with respiratory illness production of infectious hepatitis c virus in tissue culture from a cloned viral genome development and characterization of hepatitis c virus genotype 1-7 cell culture systems: role of cd81 and scavenger receptor class b type i and effect of antiviral drugs efficient culture adaptation of hepatitis c virus recombinants with genotype-specific core-ns2 by using previously identified mutations dna sequence of the bacteriophage gama ci gene we thank merck, sharp and dohme for providing the 25a protease inhibitor. key: cord-300642-c7adeis1 authors: lai, andrew sh; lai, kar neng title: viral nephropathy date: 2006 journal: nat clin pract nephrol doi: 10.1038/ncpneph0166 sha: doc_id: 300642 cord_uid: c7adeis1 viral infections can cause many glomerular diseases. the diagnostic criteria for virus-related nephropathy include detailed clinical and laboratory data, and tissue molecular analysis. several mechanisms are involved in the pathogenesis of virus-related nephropathy, including tropism of the virus in the kidney, induction of abnormal immune complexes, direct cytopathogenic effects, and multiorgan failure. hepatitis b virus is associated with membranous nephropathy and mesangiocapillary glomerulonephritis in endemic areas. hepatitis c virus causes various forms of glomerulonephritis, including cryoglobulinemia-mediated glomerulonephritis. infection with hiv is associated with a collapsing focal segmental glomerulosclerosis, a distinctive disease that affects mainly africans and african americans. in the course of hiv infection, other types of immune complex glomerulonephritis can occur, most frequently in whites. recent reports indicate a role for parvovirus b19 in 'idiopathic' collapsing focal segmental glomerulosclerosis. both hantaviruses, and coronaviruses associated with severe acute respiratory syndrome, can lead to acute renal failure. renal biopsy followed by appropriate serological and molecular testing is essential for defining virus-related glomerular lesions and guiding prognostic and therapeutic evaluation. the pathogenetic links between viral infection and renal disease are often difficult to establish. the criteria for proving causality are complex and include (besides recognition of the clinical syndrome) serological diagnosis, identification of specific viral antigenemia, and detection in glomerular structures of viral antigens and host antibodies. according to koch's postulates, the etiological link should be confirmed by complete cure following eradication of the virus, but this is not always possible. the concentration of viral antigens is higher in tissue than in the circulation, where antigens are complexed with specific autoantibodies. virological and molecular analy sis of pathologic tissues by in situ hybridiza tion, polymerase chain reaction and ultra structural analysis led to successful detection and identifica tion of the virus. it should be noted that tubular uptake of viral particles is common and does not necessarily establish an etiological link with renal disease. improvement of the renal disease concomitant with clearance of the suspected antigen, or recurrence of glomerulonephritis following reinfection, are additional clinical criteria. different mechanisms are operative in different viral nephropathies (box 1). in acute glomerulonephritis, direct viral infection of the glomerulus induces proliferative changes following release of cytokines. 1 the nephropathy is reversible in most cases if the virus is rapidly cleared. in chronic forms of glomerulonephritis, persistent viral infection provides continuous antigenic stimulation, resulting in antibody production and formation of immune complexes. studies indicate a role in the disease pathogenesis for these immune complexes, which can be derived from the circulation or formed in situ. 2,3 viral proteins cause inflammatory renal diseases via synthesis of various mediators that can cause sclerosis and worsen glomerulopathy. 4, 5 viral infections can cause many glomerular diseases. the diagnostic criteria for virus-related nephropathy include detailed clinical and laboratory data, and tissue molecular analysis. several mechanisms are involved in the pathogenesis of virus-related nephropathy, including tropism of the virus in the kidney, induction of abnormal immune complexes, direct cytopathogenic effects, and multiorgan failure. hepatitis b virus is associated with membranous nephropathy and mesangiocapillary glomerulonephritis in endemic areas. hepatitis c virus causes various forms of glomerulonephritis, including cryoglobulinemia-mediated glomerulonephritis. infection with hiv is associated with a collapsing focal segmental glomerulosclerosis, a distinctive disease that affects mainly africans and african americans. in the course of hiv infection, other types of immune complex glomerulonephritis can occur, most frequently in whites. recent reports indicate a role for parvovirus b19 in 'idiopathic' collapsing focal segmental glomerulosclerosis. both hantaviruses, and coronaviruses associated with severe acute respiratory syndrome, can lead to acute renal failure. renal biopsy followed by appropriate serological and molecular testing is essential for defining virus-related glomerular lesions and guiding prognostic and therapeutic evaluation. a direct cytopathic effect of viral proteins has also been postulated. 6 in hepatitis c virus (hcv)-induced mesangiocapillary glomerulonephritis (mcgn), production of circulating cryo globulins is induced as an abnormal host response to infection. cryoglobulins are either type ii or type iii. at least two classes of immuno globulins are involved, one of which is polyclonal. 7 in acute renal failure associated with infection by hantavirus or severe acute respiratory syndrome coronavirus, the pathogenetic mechanisms of interstitial nephritis, disseminated intravascular coagulopathy, and multiorgan failure-rather than formation of immune complexes-are predominant. box 2 lists the viruses that are known to induce renal diseases. globally, the most frequent and well recognized virus-related glomerulonephropathies are those associated with hepatitis b virus (hbv), in which formation of immune complexes is important. hcv is the etiological agent of cryoglobulinemiarelated mcgn in most cases. infection with hiv can induce a broad spectrum of glomerular lesions via multiple pathogenic mechanisms. parvovirus b19 (pvb19) is associated with non-hiv collapsing glomerulopathy, 8 idiopathic focal segmental glomerulosclerosis (fsgs), 9 and immune complex glomerulonephritis. 10 polyoma bk virus and hantavirus most frequently cause tubulointerstitial damage; occasionally, virus is simultaneously localized to the glomerulus. a rare or speculative role in glomerulonephritides is currently attributed to other viruses, such as those causing yellow fever, 11 mumps, 12 measles, 13 varicella, 14 and herpes. 15 hbv is a hepatotropic, double-stranded dna virus of the hepadnaviridae family. hbv itself is not cytopathic; hepatitis develops as a result of the host's immune reaction to infected hepatocytes. hbv uses reverse transcriptase to transcribe rna into dna. unlike retroviruses, however, hbv dna is not integrated into host cell dna during replication. after an hbv particle binds to and enters a hepatocyte, hbv dna enters the cell's nucleus and is converted into covalently closed circular dna. this highly stable genetic material acts as the intermediate template for transcription of rna copies. this pregenomic messenger rna is transported to the cytoplasm. it has dual functions: acting as a template for synthesis of new hbv dna, and carrying genetic information to direct synthesis of viral proteins. today, an estimated 350-400 million people worldwide are infected with hbv. in endemic areas, transmission is usually vertical-that is, from infected mother to child. horizontal transmission occurs via direct contact with blood (e.g. during blood transfusions) or mucous membranes (e.g. during sexual contact), or via the percutaneous route upon contact with blood or body fluids (e.g. during intravenous drug use and needle sharing). familial clustering of the virus occurs in some regions. the reported prevalence of hbv-associated nephropathy closely parallels the geographic patterns of prevalence of hbv. 16 frequently reported in asian populations 17 and in children, 18 particularly male children. 16 by contrast, mesangial proliferative forms with iga deposits seem to be most common in adults. 19 three types of glomerulonephritis with pathologic characteristics similar to the human subtypes have been described in woodchucks chronically infected with hepatitis virus. 20 as in humans, the membranous pattern of injury most frequently affects young woodchucks, whereas the mesangial proliferative pattern of injury tends to affect older animals. the male : female ratio of affected woodchucks was significantly greater than that of the chronic carrier population. 20 in most reports, diagnosis of hbv-asso ciated glomerulonephritis has been based on persistence of circulating hbv or hbv dna, absence of other causative agents, and presence of hbvspecific antigen(s) or viral genome in the glomerulus. one major difference between the human and woodchuck studies is that the hepatitis b e antigen (hbeag) system has not been characterized in the latter. in clinical practice, regression of pathology following viral eradication is not easy to demonstrate because of ethical concerns relating to repeat renal biopsies in humans subsequent to clinical remission. as such, the diagnosis of hbv-associated renal disease usually relies heavily upon detection of hbv-specific antigen(s) in glomeruli. laboratory testing for diagnosis and assessment of response to treatment should include standard liver biochemistries (serum alanine aminotransferase, γ-glutamyltransferase, and bilirubin levels), and hbv serologies (hepatitis b surface antigen, hbeag, anti-hepatitis b e, and anti-hepatitis b core antigen antibodies). hbeag is present in 80% of patients, who might also have high titers of anti-hepatitis b core antigen. 21 subjects with biochemical hepatitis should be tested for circulating hbv dna 22 and undergo liver biopsy. an α-fetoprotein assay could be an important adjunct. 23 serum c3 and c4 levels can be low in 20-50% of patients. hbv-related membranous nephropathy tends to manifest slightly differently in pediatric and adult patients. in children, there is a strong male preponderance, and the most frequent presentation is nephrotic syndrome, microscopic hematuria, and normal or mildly impaired renal function. 24 pediatric chronic hbv carriers often do not have overt liver disease, and transaminase levels are usually normal. in adults, proteinuria or the nephrotic syndrome are the most common manifestations. adult male predominance is less obvious than in pediatric populations. adults are more likely than children to have hypertension, renal dysfunction, and clinical evidence of liver disease. the prognosis of hbv-associated membranous nephropathy in children is favorable. stable renal function and high rates of spontaneous remission have been reported in several geographical areas in which disease prevalence is high. by contrast, adults with hbvassociated membranous nephro pathy typically develop progressive disease. in hong kong, up to 29% of patients had progressive renal failure, and another 10% developed terminal uremia over 5 years. 21 the prognosis is even worse for patients with nephrotic-range proteinuria and abnormal liver function tests at presentation. over 50% of these patients require renal replacement therapy within 3 years. 25 vertical transmission is associated with poorer outcomes than horizontal transmission, as is endemic versus sporadic infection. 21,26 box 2 viral infections that cause nephropathy. as mentioned above, hbv infection can also cause mcgn (with or without cryoglobulinemia), mesangial proliferative glomerulonephritis, and igan. 19,27 polyarteritis nodosa has been reported in some patients with hbv and might respond to treatment with corticosteroids and interferon-α. 28 occasional concomitance of the pathologic subtypes can lead to 'double' glomerulopathies. for instance, membranous nephropathy and igan have been reported to coexist in an hbv carrier. 29 unlike affected children, who have a high rate of spontaneous remission, 30 adults with hbvassociated membranous nephropathy typically develop progressive disease. 21 various management strategies have been tried, but an ideal agent is yet to be found. treatment for hbvassociated renal disease should ideally achieve the following objectives: (i) amelioration of nephrotic syndrome and its complications; (ii) preservation of renal function; (iii) normalization of liver function and prevention of hepatic complications of hbv; and (iv) permanent eradication of hbv. because of the involvement of immune complexes in the disease, immunosuppressive therapy-similar to that used in the idiopathic form of the disease-was once fashionable. corticosteroids were reported to provide symptomatic relief in isolated cases. the contemporary view, however, is that steroid and cytotoxic agents can cause deleterious hepatic flares or even fatal decompensation by enhancing viral replication when the drugs are withdrawn. 31 another approach is treatment with an antiviral agent. interferon-α is a naturally occurring cytokine produced by b lymphocytes, null lymphocytes, and macrophages that exerts antiviral, antiproliferative and immuno modulatory effects. while reportedly useful in children, 32 interferon-α has produced mixed results in adults with hbv-associated membranous nephropathy. 21, 26 introduction of the nucleoside analog lamivudine has revolutionized the treatment of chronic hbv infection. 33 lamivudine is the (-)-enantiomer of 3'-thiacytidine. this analog inhibits dna synthesis by terminating the nascent proviral dna chain through inter ference with the reverse transcriptase activity of hbv. in children and adults with hbv-asso ciated membranous nephropathy, lamivudine has been anecdotally reported to induce remission of nephrotic syndrome and to suppress viral replication. 24, 34 in a recent analysis comparing 10 adult nephrotic patients with hbv-related membranous nephropathy who received lamivudine with 12 matched historical control subjects who presented in the pre-lamivudine era, lamivudine significantly improved proteinuria, aminotransferase levels, and renal outcome over a 3-year period. 25 randomized studies in a larger cohort of patients are needed to prove this effect. a potential limitation of prolonged treatment with lamivudine is emergence of drugresistant virus strains resulting from induction and selection of hbv variants with mutations at the tyrosine-methionine-aspartate-aspartate (ymdd) motif of dna polymerase. one agent that might be useful in lamivudine-resistant cases is adefovir dipivoxil, an acyclic nucleotide analog that is effective against both lamivudineresistant hbv mutants and wild-type hbv. 35 this agent does have nephrotoxic potential, and there are no clinical data on its efficacy in hbvrelated membranous nephro pathy that does not respond to lamivudine treatment. data do indicate, however, that the recommended dose of 10 mg adefovir dipivoxil is associated with a relatively low risk of nephro toxicity. 36 while awaiting an ideal agent for treatment of hbv-associated glomerulopathy, active immuniza tion remains the most effective means of immunoprophylaxis. 37 in taiwan, active immunization of all newborns since 1984 has led to a dramatic (10-fold) decline in the incidence of neonatal hbv infection and its sequelae. 38 in the us, universal vaccination of infants began in 1991, and a 67% reduction in hbv infection was recorded 10 years later. in 2003, the who recommended that all countries establish universal hbv immunization programs for infants and adolescents. 39 hcv is a small rna virus in the flaviviridae family. evolution of hcv has been characterized by the emergence of six major genotypes (based on sequence homology) and more than 50 subtypes. to date, around 170-200 million individuals worldwide are estimated by the who to be chronically infected with hcv. although viral replication is primarily confined to the liver, a variety of extrahepatic disease manifestations are associated with hcv infection. the principal renal manifestation of hcv infection is mcgn type i, usually in the context of type ii (mixed) cryoglobulinemia. 40 the prevalence of mcgn in hcv type ii cryoglobulinemia is approximately 30%. mcgn is occasionally observed in patients with hepatitis c in the absence of cryoglobulinemia. 40 type ii mcgn (e.g. dense deposit disease) has not been described in association with hcv infection. two immunologic features of hcv might underlie predisposition to extrahepatic disease manifestations. first, hcv is known to evade immune elimination, leading to chronic infection and accumulation of circulating immune complexes. mcgn associated with hcv infection might result from this phenomenon. second, hcv stimulates production of monoclonal rheumatoid factors. this feature causes type ii cryoglobulinemia, which accounts for most symptomatic cryoglobulinemic vasculitis. although this manifestation occurs relatively infrequently, as do all the extrahepatic disease manifestations, it accounts for much of the increased morbidity and mortality that accompanies the disease. between 35% and 90% of hcv-infected patients have been reported to have mixed cryoglobulinemia. 41, 42 prevalence increases with duration of hepatitis. it should be noted, however, that the prevalence of mixed cryo globulinemia has not been determined in popula tions of unselected hcv-infected patients. frank symptomatic cryoglobulinemia affects 1% or less of patients and is usually associated with high levels of rheumatoid factor and cryo globulins. testing of unselected patients with cryo globulinemia has shown that up to 90% have anti-hcv antibody. type i mcgn has long been regarded as idiopathic, but a consider able proportion of patients has concomitant chronic hcv infection. the exact proportion of patients with type i mcgn who are anti-hcv-antibody-positive is unknown. the true prevalence of mcgn without detectable cryoglobulinemia is difficult to assess. such cases might represent a subclinical form of cryoglobulinemia in which circulating cryoglobulins have not been detected by standard laboratory techniques. further, production of igm antibodies with anti-igg activity might induce formation of immune complexes that lack cryoprecipitable properties. 43 finally, these patients might develop detectable circulating cryoglobulinemia only later in the course of the disease. 44 laboratory testing coupled with renal biopsy establishes the diagnosis of hcv-related mcgn. most patients will have anti-hcv antibody, as well as hcv rna, in serum. serum transaminase levels are elevated in 70% of patients. cryoglobulins are detected in 50-70% of patients. serum electrophoresis and immunofixation detects type ii (mixed) cryo globulins. monoclonal rheumatoid factor, almost in variably an igmκ, is a distinguishing feature of cryo globulinemic glomerulonephritis. the amount of cryoglobulins, usually measured as a cryocrit, varies between patients and with time in a given patient (range 2-70%). κ light chains are also commonly present in the urine. the serum complement pattern, which does not change greatly with clinical disease activity, is also discriminative. characteristically, early complement components (c4 and c1q) and ch50 are present at very low or undetectable levels in these patients, whereas the c3 level tends to remain normal or is only slightly depressed. renal disease associated with hcv is rare in children. the typical age of disease onset is the fifth or sixth decade of life after longstanding infection, often in association with mild subclinical liver disease. patients might have other symptoms of cryoglobulinemia, such as palpable purpura and arthralgias. renal manifestations include nephrotic (20%) or non-nephrotic proteinuria and microscopic hematuria. 45 acute nephritic syndrome is the presenting feature in about a quarter of cases. renal insufficiency, frequently of mild severity, occurs in about half of patients. over 80% of patients have refractory hypertension at presentation, which might be responsible for a considerable number of cardiovascular deaths. the clinical course of hcv-associated renal disease can vary dramatically. this disease does not frequently progress to uremia, despite the persistence of urinary abnormalities in the majority of patients. when such progression does occur, it tends to be in males and those of older age. according to an italian series, around 15% of patients eventually require dialysis. 46 other forms of glomerular injury, including membranous nephropathy, fsgs, mesangial proliferative glomerulonephritis, and crescentic glomerulonephritis, have been reported in hcv carriers as individual case reports and small series. notably, membranous nephropathy in hcv carriers is characterized by the absence of cryoglobulin and male predominance. 47 in general, therapy can be directed at two levels: removal of cryoglobulins by plasmapheresis; and inhibition of cryoglobulin synthesis by attenuating immune responses (using steroids or cytotoxic agents) or suppressing viral replication (using interferon and ribavirin). before the association between hcv and cryo globulinemic mcgn was established, steroids and cyclophosphamide were the mainstays of treatment. our awareness of this link has facilitated a more rational approach to management of this condition. controlled trials have shown that antiviral therapy with interferon-α is asso ciated with improvements in systemic symptoms of immune complex disease. unfortunately, posttherapy relapse occurs in a large pro portion of patients, particularly when interferon monotherapy is administered in short courses. introduction of combination therapy with interferon-α2b plus ribavirin was an important milestone in the treatment of chronic hepatitis c. 48 this cocktail has also produced favorable results in mixed cryo globulinemia, although non-responses and relapses after initial improvements still occur. 49 the introduction of pegylated forms of interferon (peginterferon) in 2000 was another breakthrough in treatment of chronic hepatitis c. recent data on peginterferon and ribavirin combination therapy are encouraging. 50 an increased rate of treatment failure in carriers of hcv genotype 1 has been recognized. 51 observational studies support the effectiveness of peginterferon and ribavirin combination therapy in hcv-associated cryoglobulinemic mcgn. one therapeutic drawback is the hemolytic effect that complicates ribavirin therapy, particularly in patients with functional renal impairment. this difficulty has been overcome by adjusting the dose according to glomerular filtration rate instead of body weight alone, and utilizing recombinant erythropoietin to combat anemia. post-treatment renal biopsy showed histological improvement in two of three patients who received combination therapy for 12 months. 52 the wide spectrum of glomerulopathies occurring in the course of hiv infection can be classified into four groups. the first group is the classical hiv-associated nephropathy (hivan), a distinct entity with histological features of fsgs with tuft collapse or, more rarely, mesangial hyperplasia. hivan seems to be related to a direct effect of hiv or viral proteins on renal epithelium. 53 the second group is a diffuse proliferative-mesangiocapillary or lupuslike glomerulonephritis, with predominantly mesangial immune deposits, also known as hiv immune-complex-mediated disease. 54 this group also includes other immune-complex-mediated glomerulonephritides with more-heterogeneous histological features. the third group (which includes immunotactoid glomerulonephritis) is heterogeneous with regard to glomerular lesions and pathogenic mechanisms, some of which are still undefined. the true role of hiv infection in glomerulopathies of this type is also uncertain. the final group includes hiv-associated thrombotic microangiopathy/hemolytic uremic syndrome, in which hiv is the main, but not sole, etiological factor. ethnic/geographic background is an important determinant of the type of glomerulopathy associated with hiv; for example, collapsing fsgs is prevalent in patients of african descent. the fsgs variant of hivan is the most commonly reported chronic renal disease associated with hiv infection. hivan affects up to 10% of hiv-infected patients of african descent-mainly males at risk of drug abuse, and often african americans. glomerular changes associated with this variant are capillary wall collapse of varying severity, with widening of bowman's space. visceral epi thelial cells undergo hyperplasia and hypertrophy, and develop protein inclusions in their swollen cytoplasm surrounding the collapsed lobules. sclerosis affects segments of capillary tuft or the whole glomerular surface. tubular cells might undergo degenerative changes, necrosis or flattening. large dense casts can develop in dilated tubules. detection of hiv rna in renal tissue from patients with hivan, and of hiv dna in patients with and without nephropathy, 55 raises the question of whether renal cells can be infected in vivo (tubular epithelial cells can be infected in vitro). 4 renal uptake of viral gene products might induce transactivation of host lai and lai may 2006 vol 2 no 5 www.nature.com/clinicalpractice/neph genes. in renal cells, hiv proteins can cause apoptosis, 4 phenotypic modifications, 56 and subsequent tubulointerstitial fibrosis. clinical manifestations of hivan with fsgs are nephrotic-range proteinuria and renal insufficiency. hypertension and edema are uncommon. in overt cases, ultrasonography typically reveals enlarged, highly echogenic kidneys, which probably develop in response to microcystic tubular dilatation. before effective antiretroviral treatment was available, clinical progression was rapid. intensive antiretroviral treatment delays progression. 57 pathology of the hiv-associated disease mediated by immune complexes resembles lupus nephropathy. the clinical presentation is nephrotic syndrome with microscopic hematuria. progression to renal failure occurs, but more slowly than in hivan. patients often have hcv coinfection, but hiv seems to have the prevailing role. viral antigen has been detected in glomeruli, and antibodies eluted from the kidney react with hiv antigens in circulating immune complexes (iga-p24 antigen, igg-p24 and igg-gp120). in cases of hiv-related igan, circulating immune complexes containing iga idiotype antibodies have been detected. 58 most patients with hiv-associated thrombotic microangiopathy/hemolytic uremic syndrome present with acute renal failure, microscopic hematuria, and non-nephrotic proteinuria. multiorgan involvement is frequent and prognosis is poor, with a high rate of mortality. multifactorial etiologies encompass drugs, neoplasia, lymphoma and infection. pvb19 has been associated with acute glomerulonephritis. typical life stage of onset is the second or third decade. patients present with mild proteinuria and microhematuria, with low levels of serum complement 3. the pathology of this disease is characterized by endo capillary glomerulonephritis, mcgn, or both, with sub endothelial deposits. pvb19 capsid protein is found in the glomeruli. 10 spontaneous recovery is the norm. pvb19 is also associated with collapsing glomerulopathy. prevalence of pvb19 dna in renal biopsies (78%) and peripheral blood (87%) is significantly higher in patients with collapsing glomerulopathy than in those with other nephropathies. 10 glomerular and tubular infection with pvb19 might trigger collapsing glomerulopathy, but only in patients with immune defects and a racial pre disposition (african descent). 8 most intriguingly, tanawattanacharoen and coworkers 9 detected pvb19 dna in 80% of patients with 'idiopathic' fsgs, and frequently also in controls. these results possibly reflect the presence of latent dna from past infection. failure to localize pvb19 nucleic acid within kidney is evidence against ongoing, high-level viral replication. hantaviruses are responsible for 'hemorrhagic fever with renal syndrome' , an acute inter stitial nephritis resulting from direct vascular injury of renal tissue. 59 the severe form leads to acute renal failure in 50% of cases. less severe forms occur in nonendemic areas, and present primarily as fever, hepatitis, and mild renal impairment. in most cases, glomeruli are not affected and the pathology is tubulointerstitial. isolated cases with immune complex glomerular disease have been described, in association with diffuse proliferative glomerulonephritis and complete recovery after remission of the systemic clinical syndrome. 60 six percent of patients suffering from severe acute respiratory distress syndrome had acute renal impairment. 61 despite detection of viral dna in the urine, there was no evidence of viral tropism of the kidney. the pathology is exclusively tubulointerstitial nephritis. the mechanism of disease is probably related to multiorgan failure, rhabdomyolysis, and hemodynamic disturbance. renal infection with bk virus affects kidney allograft recipients, leading to renal dysfunction and sometimes graft loss. 62 more rarely, acute interstitial nephritis is observed in immunocompromised patients. 63 hepatitis a virus infection can present as acute post-infectious glomerulopnephritis with pathology resembling that of igan. 64 more commonly, hepatitis a virus induces acute renal failure secondary to acute fulminant hepatitis. 65 acute renal failure complicating other viral infections, such as epstein-barr virus 66 and dengue fever, 67 is related to multiorgan failure, rhabdomyolysis, and hepatorenal syndrome. diverse mechanisms of glomerular and tubulointerstitial injury and heterogeneous clinicopathologic patterns underlie the relationship between viral infection and glomerular disease. the etiological role of some viruses is still un defined. molecular biology techniques are vital in elucidating the precise location and role of viruses in the pathogenesis of virus-related nephropathy. treatment of hepatitis b virus-associated membranous nephropathy with recombinant alphainterferon a one-year trial of lamivudine for chronic hepatitis b: asia hepatitis lamivudine study group hbv associated nephrotic syndrome: resolution with oral lamivudine adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis b mutants adefovir dipivoxil in chronic hepatitis b infection clinical practice: prevention of hepatitis b with the hepatitis b vaccine universal hepatitis b vaccination in taiwan and the incidence of hepatocellular carcinoma in children: taiwan childhood hepatoma study group safety of immunisation and adverse events following vaccination against hepatitis b membranoproliferative glomerulonephritis associated with hepatitis c virus infection immunological disorders in c virus chronic active hepatitis: a prospective casecontrol study hepatitis c, cryoglobulinemia, and cirrhosis: a meta-analysis secondary membranoproliferative glomerulonephritis hepatitis c virus infection and acute or chronic glomerulonephritis: an epidemiological and clinical appraisal renal involvement in hepatitis c infection: cryoglobulinemic glomerulonephritis long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis membranous glomerulonephritis associated with hepatitis c virus infection: case report and literature review randomised trial of interferon α2b plus ribavirin for 48 weeks or for 24 weeks versus interferon α2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis c virus: international hepatitis interventional therapy group (ihit) treatment of refractory, symptomatic, hepatitis c virus related mixed cryoglobulinemia with ribavirin and interferon-alpha peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection peginterferon-α2a and ribavirin combination therapy in chronic hepatitis c: a randomized study of treatment duration and ribavirin dose hepatitis c virus-related cryoglobulinemic glomerulonephritis: long-term remission after antiviral therapy renal complications of human immunodeficiency virus type 1 hiv-associated immunemediated renal disease renal epithelium is a previously unrecognized site of hiv-1 infection the dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and hiv-associated nephropathy cohort study of the treatment of severe hiv-associated nephropathy with corticosteroids brief report: idiotypic iga nephropathy in patients with human immunodeficiency virus infection mechanism of disease in hemorragic fever with renal syndrome different pathohistological presentations of acute renal involvement in hantan virus infection: report of two cases acute renal impairment in coronavirus-associated severe acute respiratory syndrome bk-virus nephropathy in renal transplants-tubular necrosis, mhc-class ii expression and rejection in a puzzling game bk virus renal infection in a patient with the acquired immunodeficiency syndrome iga-dominant glomerulonephritis associated with hepatitis a renal failure in otherwise uncomplicated acute viral hepatitis epstein-barr virus-associated acute renal failure: diagnosis, treatment, and followup clinical characteristics and risk factors for concurrent bacteremia in adults with dengue hemorrhagic fever some of the authors' work cited in this review was supported by the l & t charitable fund and indocafe. the authors declared they have no competing interests. key: cord-265005-e6rpryrh authors: tomasello, elena; pollet, emeline; vu manh, thien-phong; uzé, gilles; dalod, marc title: harnessing mechanistic knowledge on beneficial versus deleterious ifn-i effects to design innovative immunotherapies targeting cytokine activity to specific cell types date: 2014-10-30 journal: front immunol doi: 10.3389/fimmu.2014.00526 sha: doc_id: 265005 cord_uid: e6rpryrh type i interferons (ifn-i) were identified over 50 years ago as cytokines critical for host defense against viral infections. ifn-i promote anti-viral defense through two main mechanisms. first, ifn-i directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. second, ifn-i orchestrate innate and adaptive anti-viral immunity. however, ifn-i responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. we will review the proposed nature of protective versus deleterious ifn-i responses in selected diseases. emphasis will be put on the potentially deleterious functions of ifn-i in human immunodeficiency virus type 1 (hiv-1) infection, and on the respective roles of ifn-i and ifn-iii in promoting resolution of hepatitis c virus (hcv) infection. we will then discuss how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the subtypes and dose of ifn-i produced, (ii) the cell types affected by ifn-i, and (iii) the source and timing of ifn-i production. finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. specifically, we will discuss how induction or blockade of specific ifn-i responses in targeted cell types could promote the beneficial functions of ifn-i and/or dampen their deleterious effects, in a manner adapted to each disease. type i interferons (ifn-i) were the first cytokines discovered, over 50 years ago, based on their potent anti-viral effects (1, 2) . ifn-i play a crucial, non-redundant role in vertebrate anti-viral defenses (3) (4) (5) . ifn-i also mediate protective effects in other physiopathological contexts, including cancer (6) and multiple sclerosis (ms) (7) . on the contrary, ifn-i responses can be deleterious in a number of circumstances, including bacterial or fungal infections (8-10), many autoimmune diseases (11) , and, paradoxically, certain chronic viral infections (12) (13) (14) . it is only recently that an integrated picture has emerged of the cellular and molecular mechanisms regulating the production of ifn-i and underlying their functions. much knowledge was gained recently on another class of potent innate anti-viral interferons, the lambda, or type iii ifns (ifn-iii). we will review knowledge on ifn-i/iii (ifns) and discuss how it could be harnessed to develop innovative therapeutic strategies aimed at surgically tuning ifn activity toward protective responses in a manner adapted to each disease. we will focus on ifn-α/β/λ because they are the best characterized ifns and already used therapeutically. recent reviews are covering information on other ifn-i subsets including ifn-ε, which is produced at mucosal sites and promotes local anti-viral defenses (15, 16) . dendritic cells (dcs) are rare heterogeneous mononuclear phagocytes functionally characterized by their unique efficacy for antigen-specific activation of naïve t lymphocytes. dcs are sentinel cells of the immune system, able to sense and integrate a variety of danger input signals for delivery of output signals instructing the activation and functional polarization of effector immune cells. in mammals, five major dc subsets exist, which differ in their expression of innate immune recognition receptors (i 2 r 2 s) and in their functional specialization: monocyte-derived dcs (modcs), langerhans cells, cd11b + dcs, xcr1 + dcs, and plasmacytoid dcs (pdcs) (17) . a recurrent theme of this review will be the intricate relationships between ifns and dcs, since these cells can be a major source and/or target of these cytokines under various conditions. www.frontiersin.org the first section will synthesize current knowledge on ifn production and protective anti-viral functions. the i 2 r 2 s and downstream signaling pathways responsible for ifn-i production during viral infection will be listed. the roles of different cell types for this function will be discussed. the two main mechanisms through which ifn-i promote anti-viral defense will be reviewed, succinctly for direct anti-viral effects and in greater details for immunoregulatory functions. the second section will focus on the detrimental functions of ifn-i. selected diseases will be discussed to illustrate how different, and sometimes opposite, processes underlie deleterious ifn-i responses depending on the physiopathological contexts. ifn-i induction of unbridled inflammatory responses causing lethal tissue damage will be discussed as a major pathological mechanism during bacterial encounters secondary to influenza infection or in some autoimmune diseases. inappropriate functional polarization of immune responses by ifn-i will be discussed as one potential cause for enhanced susceptibility to bacterial or fungal infections. the complex and disputed role of ifn-i in chronic viral infections will be reviewed, with emphasis on the physiopathology of the infections by human immunodeficiency virus type 1 (hiv-1) and human hepatitis c virus (hcv), with an outlook for the development of novel immunotherapeutic strategies to combine with anti-viral drugs. the third section will recapitulate how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the source and timing of ifn-i production, (ii) the cell types affected by ifn-i, and (iii) the signaling pathways activated by ifn-i. in the last section, we will speculate how integration of all the knowledge discussed before combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments, based on induction or blockade of specific ifn-i responses in targeted cell types. this"activity-by-targeting"concept is based on the design of novel "immuno-ifns" consisting in covalent association between a mutated ifn-i with decreased affinity for its receptor and an antibody with high avidity for a molecule specifically expressed on target cell types (18) . this design ensures lack of activity of the immuno-ifns on all cell types but those targeted, contrary to previous strategies using ifns with close to maximal potency that were still able to mediate strong off-target effects despite their coupling to cell-type specific antibodies and/or their local delivery. type i interferons expression is not detectable under steady state conditions in vivo using classical methods such as gene expression analysis by rt-pcr or protein titration by elisa or bioassays. however, mice deficient for the expression of the alpha chain of the ifn-i receptor (ifnar1) harbor alteration in the ontogeny or functions of various cell types (19) (20) (21) (22) (23) (24) (25) (26) . hence, extremely small or localized but functionally relevant quantities of ifn-i must be produced under steady state conditions (27) . indeed, the existence of steady state responses to ifn-i in various organs in vivo was demonstrated by using reporter mice expressing the firefly luciferase under the control of the promoter of ifnb1 (28) or of mx2 (29) , a canonical ifn-i-stimulated gene (isg). steady state ifn-i responses are promoted by gut commensals (30) . early and transiently after many viral infections, large amounts of ifns can be detected, in blood and spleen in the case of systemic infections or locally in the case of confined infections. ifn induction during viral infections results from the detection of specific danger signals by specialized i 2 r 2 s. this includes the detection of pathogen-associated molecular patterns as well as the sensing of stress signals or damage-associated molecular patterns (31, 32) . based on the nature and intracellular location of the danger signals that induce the production of the cytokines, the cellular sources of ifns during viral infection can be classified in two main groups. infected cells often contribute to ifn production as a response to their sensing of endogenous viral replication, or consecutive to the metabolic stress induced during massive translation of viral structural proteins, or as a result of plasma membrane perturbations upon viral entry. specific subsets of uninfected cells can also significantly contribute to ifn production upon engulfment of material containing viral-derived nucleotide sequences and sensing of these molecules in endosomes by specific i 2 r 2 s. all sensing pathways leading to ifn induction converge on the activation of interferon response factors 3 or 7 (irf3/7), which are the master transcription factors inducing ifn genes. most cell types constitutively express irf3 but not irf7 or only at low levels. irf7 expression requires ifn-i stimulation. ifn-β can directly be induced by irf3. all but one of the ifn-α subtypes require irf7 for their induction. hence, ifn-β secretion promotes its own production and that of ifn-α in an autocrine manner (33, 34) . this positive feedback loop strongly amplifies ifn production during viral infections, promoting fast and widespread induction of cell-intrinsic anti-viral defenses in uninfected cells to prevent virus dissemination. other feedback loops tightly regulate ifn-i production positively or negatively. this section reviews different mechanisms controlling ifn production and how they could play different roles in host/virus interactions. different innate immune recognition receptors are involved in sensing various types of viral nucleic acids in distinct categories of cells during viral infections, which may promote different types of anti-viral defenses. for each selected sensor shown, the types of viral nucleic acids recognized and the downstream signaling cascade induced are represented in a simplified, schematic manner. the potential specific role of each cell type in anti-viral defenses is also indicated at the bottom of each panel. (a) potentially all types of infected cells can detect endogenous viral replication through cytosolic sensors triggering their local production of ifn-β/λ to control viral replication in an autocrine and paracrine fashion in infected tissues. (b) uninfected xcr1 + dcs selectively produce high levels of ifn-λ and ifn-β upon engulfment of materials containing dsrna and the consecutive triggering of tlr3 in endosomes. the receptor of ifn-λ is mostly expressed by epithelial cells. hence, xcr1 + dcs might be involved in inducing local ifn responses in virally infected epithelial tissues. since xcr1 + dcs are especially efficient at producing ifn-iii upon hcv stimulation, they might contribute to local or systemic ifn production during infection with this virus, to promote ifn-λ-mediated protection of hepatocytes. uninfected xcr1 + dcs and other uninfected cells may produce some ifn-β upon engulfment of materials containing ssrna and the consecutive triggering of tlr8 in endosomes. the contribution of this pathway to anti-viral defense is not well understood yet, in part because mouse tlr8 is deficient for this function. (c) uninfected pdcs selectively produce high levels of all subsets of ifns upon engulfment of materials containing ssrna or cpg dna and the consecutive triggering of tlr7/9 in endosomes. however, pdcs seem to be activated for this function only in lymphoid tissues. hence, pdc might contribute to systemic ifn production during blood-borne viral infections or as a failsafe mechanism activated upon abnormal widespread dissemination of a viral infection once it has escaped local confinement at its portal of entry. cm, cell membrane; nm, nuclear membrane. particular nucleotide sequences or tertiary structures, their signaling pathways and their physiological significance have been recently reviewed (31, 32) . cytosolic rna sensors encompass dexd/h helicases among which the retinoic-acid-inducible gene (rig)-i-like receptors (rlrs) have been the most studied, namely rig-i and melanoma differentiation associated gene 5 (mda5). rig-i recognizes rna with a 5 -ppp or 5 -pp (38) (uncapped) moiety, or double-stranded rna (dsrna), both structures being present in viral, but not in cytosolic eukaryotic, rna molecules. mda5 might specifically recognize long dsrna fragments. both rig-i and mda5 contain a dexd/h box-containing rna helicase domain, and 2 caspase recruitment domains (card1/2), which bind to mitochondrial anti-viral signaling protein (mavs). rna/rlr/sting molecular complexes initiate a signaling cascade leading to irf3/7-dependent induction of ifns (figure 1 ). other dexd/h helicases can promote ifn-i production in dcs, www.frontiersin.org although their physiological roles for in vivo immune defenses against viral infections remain to be established (32) . cytosolic dna sensors able to induce ifn-i (mostly ifn-β) and ifn-iii encompass molecules belonging to different protein families, including dexd/h helicases, the inflammasome component ifn-γ-inducible protein 16 (ifi16) , the z-dna binding protein 1 (zbp1), and the cyclic gmp-amp (cgamp) synthase (cgas) (31, 32) . most of the cytosolic dna sensors activate sting and lead to irf3/7-and nfκb-dependent induction of ifn-β and ifn-iii. many cell types express zbp1 and are able to produce ifn-i upon triggering of this molecule, including macrophages, dcs, and fibroblasts following an hsv-1 infection (39, 40) . upon dna binding, cgas catalyzes the production of cgamp. cgas is critical for the detection of lentiviruses including hiv-1/2 (41, 42) and can contribute to sensing of, and protection against, other rna viruses, including in vivo in mice (43) . cgamp also acts as a secreted second messenger signal alerting uninfected cells to directly induce their expression of intrinsic immune anti-viral defenses. the cgas/sting/irf3 signaling cascade and the irf1 transcription factor are "master" inducers of cell-intrinsic immunity able to control the replication of most dna and some rna viruses at least in part independently of ifns (43) . infected cells become a factory for production of viral particles. hijacking of the translation apparatus of the host cell for massive production of viral structural proteins leads to an overload of the capacity of the er for correct folding of newly synthesized proteins. er overload induces a homeostatic response of the cell, the unfolded protein response (upr). upr aims at restoring normal er functions by inhibiting translation. upr activation in infected cells contributes to prevent viral replication, including through inhibition of the production of viral proteins, promotion of ifn-i production, and induction of cell suicide (44) . toll-like receptors (tlrs) are among the first and best characterized i 2 r 2 s. tlrs are transmembrane proteins with a leucine-rich repeat extracellular domain involved in ligand recognition and an intracellular toll/interleukin-1 receptor domain essential for signaling (45) . among the nine tlrs conserved between mouse and human, tlr3, tlr7, tlr8, and tlr9 are located in endosomes where they can detect the abnormal presence of nucleic acids such as occurs upon endocytosis of virions or of virally infected cell material. tlr3 recognizes dsrna, tlr7/8 ssrna, and tlr9 dna sequences containing unmethylated cytidinephosphate-guanosine (cpg) motifs. tlr fine specificity and signaling pathways have been reviewed recently (32) and are summarized in figure 1 . we will discuss the expression patterns and functions of endosomal tlrs with regards to ifn production in uninfected specialized immune cell types, pdcs and xcr1 + dcs. plasmacytoid dcs uniquely produce very large amounts of ifns in response to in vitro stimulation with many viruses, without being infected (46) . ifn-i mrnas represent up to 40% of all mrnas in pdcs at the peak of their activation (47) . in vitro, upon exposure to influenza virus, herpes virus type 1, cytomegaloviruses, or vesicular stomatitis virus, individual pdcs produce 100-1000 times more ifns than total pbmcs, monocytes, modcs, cdcs, neutrophils, and fibroblasts (47) (48) (49) (50) (51) (52) . however, in vitro, high molarity infection of cdcs with certain viruses unable to inhibit ifn-i production in their target cells can also induce massive ifn-β secretion (53) . pdcs produce high levels of all subtypes of ifns, contrary to many other cell types including infected cells, which often preferentially produce ifn-β (46, 47) . in vivo, pdc depletion during systemic viral infections leads to over 95% decrease of ifn-i production, while the total number of pdcs producing ifn-i (<100,000 in one mouse) is much lower than the total number of infected cells (54) (55) (56) (57) (58) (59) . this shows that in vivo also individual activated pdcs produce much more ifn-i/iii than most other cell types, including virus-infected cells. the professional ifn-producing function of pdcs largely results from their high constitutive and selective expression of irf7, tlr7, and tlr9 (figure 1) . these molecules are pre-associated in readyto-signal complexes located in specialized endosomes specific to pdcs (60, 61) . pdcs must also be equipped for efficient sensing and up-take of virions and virus-infected cells. the corresponding cell surface i 2 r 2 s remain to be identified. selective expression of tlr3 in xcr1 + dc endows them with a unique ability to produce very high amounts of ifn-β and ifn-iii upon stimulation with dsrna or hcv irrespective of their own infection. xcr1 + dcs are very potent for antigenspecific activation of cd8 + t cells, in particular through crosspresentation of exogenous antigens that they have captured from other cells and processed for association with class i major complex histocompatibility (mhc-i) molecules (62) . in mice, xcr1 + dcs are crucial for the initiation of protective adaptive immune responses against tumors and a variety of viruses (63) . mouse and human xcr1 + dcs constitutively and selectively express high levels of tlr3 (figure 1) . they produce large amounts of ifn-iii and ifn-β upon stimulation with a synthetic mimetic of dsrna, polyinosinic:polycytidylic acid (polyi:c) (64, 65) . human xcr1 + dcs uniquely respond to stimulation with hcv by producing large amounts of ifn-iii in a tlr3-dependent manner (66, 67) , irrespective of their own infection. positive feedback loops. in addition to irf7 induction, other positive feedback mechanisms exist to amplify the production of ifns rapidly after initiation of a viral infection as illustrated by the following selected examples. ifns induce the expression of many cytosolic rna/dna sensors and of tlr7. this broadens the spectrum of host's cell types able to detect endogenous viral replication for ifn induction. induction of oasl by ifns in human cells enforces rig-i signaling, counteracting viral immune frontiers in immunology | microbial immunology evasion genes interfering with this sensing pathway (68) . the ifninducible ribonuclease l (rnasel) generates viral and cellular rna degradation products, which engage rlrs for amplification of ifn production (69, 70) . the ifn-inducible protein kinase r (pkr) stabilizes ifn-i mrna (71) . to prevent unbridled responses deleterious for the host, ifn activity must be tightly controlled including during viral infections. several negative feedback loops exist to terminate ifn production, after anti-viral defenses have been activated. the isg ubiquitin specific peptidase 18 (usp18) binds to ifnar2, preventing it from recruiting signal transducer and activator of transcription 1 (stat1). ifns induce the expression of tam receptor tyrosine kinases in dcs, monocytes, and macrophages. tam receptors associate and signal in part through ifnar1. they activate the suppressors of cytokine signaling-1/3 (socs-1/3). socs inhibit tlr and rlr signaling, thereby terminating ifn production (72) . tam receptor ligands, gas6 and pros, bind phosphatidylserine on dying cells and are produced by activated dcs and monocytes/macrophages. thus, ifn induction of tam inhibitory receptors on uninfected phagocytic immune cells could limit their propensity to produce the cytokines upon engulfment of dying virally infected cells. ifns induce tetherin on most cell types. pdcs express a receptor for tetherin, leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member 4 (lilra4). lilra4 triggering on pdcs inhibits their production of ifn-i. hence, through lilra4 engagement by tetherin, pdcs can monitor their efficacy at inducing an antiviral gene expression program in neighboring cells through ifns, and timely terminate their ifn production. how positive and negative feedback loops integrate in time and space to promote optimal kinetics and intensity of ifn production in order to efficiently control viral infection without causing severe immunopathology is not completely understood. positive feedback loops may occur very rapidly after initiation of viral infection to allow rapid secretion of high levels of the cytokines for fast and strong induction of anti-viral cell-intrinsic immunity. negative feedback loops occur likely later to terminate the response and thus avoid chronicity of cytokine production and its ensuing deleterious effects. pdcs do not constitute the major source of ifn production upon local infections by several viruses in the lung or in the female reproductive tract. pdcs are dispensable for resistance against these infections (56, 73, 74) . during pulmonary infection by newcastle disease virus (ndv), ifn-i are produced locally in the lungs mainly by infected alveolar macrophages. lung pdcs do not express the cytokines (73) . selective depletion of lung alveolar macrophages leads to systemic dissemination of ndv, and to a strong activation of pdcs for ifn-i production specifically in the spleen. even in the case of systemic viral infections such as caused by intravenous injection of ndv or intraperitoneal injection of mouse cytomegalovirus (mcmv), pdc ifn production is confined to the spleen. it is not detected in other organs even those with high viral replication (59, 73) . hence, splenic pdcs are especially prone to high level ifn production upon systemic acute viral infections. pdcs located in non-lymphoid organs, in particular mucosal barrier tissues, appear to be inhibited for ifn production. thus, ifn production by infected cells serves as first line of defense to block virus replication at its portal of entry in the body. ifn production by uninfected pdcs might constitute a failsafe mechanism mainly activated in the spleen when viral infection gets systemic (75) . under these conditions, to promote health over disease, the benefits for the host of producing high circulating levels of ifns in order to induce widespread cell-intrinsic anti-viral defenses might prevail over the deleterious effects that this could cause on certain cell types or tissues. indeed, pdcs are required for protection against hsv-2 and hsv-1 in mice only in systemic but not local infections (56) . this observation is consistent with the crucial role of pdcs for protection of mice against systemic infection by mouse hepatitis virus (mhv), a fast replicating coronavirus (55) . conflicting results have been obtained on the role of pdcs during intranasal influenza infection (74, (76) (77) (78) . a possible explanation is that pdc ifn production contributes to resistance to highly pathogenic influenza strains that might systemically spread from the lung early after infection, even if at low levels. another intriguing observation is that ifns are critical for host resistance to mcmv and that pdcs are the major source of ifns in this infection model but are dispensable for virus control (54) . studies are ongoing to understand this apparent paradox. patients bearing genetic mutations disrupting endosomal tlr signaling do not appear to suffer from life-threatening viral infections (79, 80) , contrary to patients impaired in ifnar signaling (4, 81) . a notable exception is the specific susceptibility to severe herpes virus encephalitis in individuals' deficient for tlr3 signaling (82, 83) . however, contrary to extracellular tlr, endosomal tlr have evolved under strong purifying selection in human beings (84) . hence, while pdcs and endosomal tlr might have been required for protection of our species against viral infections in the past, this appears not to be the case anymore perhaps due to improved social, hygiene, and health care in modern society (75) . attesting to the importance of ifns for anti-viral defense in vertebrates, many mammalian viruses encode immune evasion genes specifically inhibiting the production of ifns in infected cells (39, 85) . pdcs or xcr1 + dcs might be essential for ifndependent host protection against these viruses, because they are spared from the intracellular functions of viral immune evasion genes (75) . to the best of our knowledge, mcmv does not encode for immune evasion genes inhibiting ifn production. however, mcmv manipulates ifn-i responses through specific inhibition of stat1 functions in infected cells. thus, pdcs might be dispensable for resistance against systemic mcmv infection due to sufficient levels of ifn production by infected cells locally in all infected tissues. hepatocyte responses to ifn-iii appear to play a www.frontiersin.org critical role in human resistance to hcv. in infected hepatocytes, hcv induces the expression of cellular micrornas binding to ifn-iii mrna and leading to its degradation. uninfected xcr1 + dcs produce high levels of ifn-iii in vitro upon hcv stimulation (66, 67) . hence, during acute hcv infection in vivo, xcr1 + dc may be a strong and early source of ifn-iii not subjected to virus immune evasion strategies, therefore, contributing to protect naturally resistant individuals. in secondary lymphoid organs, a subset of macrophages is critical for the clearance of viruses from the lymph (86) . these macrophages are located on viral entry routes, near to subcapsular sinuses where the afferent lymph drained from non-lymphoid tissues flows. contrary to other subsets of macrophages, subcapsular sinus macrophages are highly susceptible to viral infection, because they constitutively express only low levels of effector molecules of cell-intrinsic anti-viral immunity and because their responses to ifns are inhibited by their high constitutive expression of usp18. subcapsular sinus macrophages rapidly become infected by viruses incoming from the lymph and produce large amounts of ifns. this altruistic suicide prevents virus dissemination to other adjacent cell types and promotes the induction of innate and adaptive anti-viral immunity (87) . upon instruction by ifns, cells express a wide variety of viral restriction factors, whose combined action blocks pathogen invasion by interfering with the different stages of viral life cycle (figure 2a ). this has been extensively reviewed recently (88) and will only be described succinctly here. virus fusion with host cell membrane can be blocked by cholesterol-25hydrolase (ch25h) that inhibits sterol biosynthesis. some viruses enter cells by escaping from endosomes/lysosomes, which can be blocked by interferon inducible transmembrane (ifitm) proteins. virus uncoating can be blocked by tripartite motif (trim) proteins, such as trim5α, which bind to hiv-1 capsid thus promoting its degradation, and by myxoma resistance gtpases, mx1, and mx2, which efficiently trap viral structural proteins at an early stage following virus entry into the cell. mx1 inhibits a number of viruses, including influenza virus through sequestration of its nucleocapsid. mx2 associates with host cyclophilin a and hiv-1 capsid protein. virion assembly can be blocked at transcriptional, translational, and posttranslational levels. the adenosine deaminase acting on rna 1 (adar1) and the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like (apobec) deaminases induce viral rna destabilization and hypermutation (89, 90) . the sterile alpha motif and histidine-aspartic domain (hd) containing protein 1 (samhd1) blocks reverse transcription by hydrolyzing dntps (91) . adar1, apobec, and samhd1 functions have been mainly studied in infections by hiv-1 and other retroviruses. the 2 ,5 -oligoadenylate synthase (oas) proteins, the ifn-induced proteins with tetratricopeptide repeats (ifit), and pkr inhibit viral and host protein translation by using complementary mechanisms (88) . the major post-translation modification induced by ifns is the binding of the ubiquitinlike modifier isg15 to several viral and host proteins, a process called isgylation. most of the known isgylated proteins are targeted for degradation, with few exceptions that are on the contrary stabilized like irf3 (88) . finally, the egress and budding of virions of many enveloped viruses can be inhibited by tetherin or by viperin (88) . many anti-viral isgs have been functionally characterized only recently, largely thanks to large-scale screening approaches. they display a variable degree of viral specificity (43, 92) that might inversely relate to the extent of their side effects on host cells ( figure 2b ). anti-viral effectors acting on a broad spectrum of viruses often target key metabolic pathways that are also crucial for host cell functions. this is the case for the control of cholesterol metabolism by ch25h (93) or of protein translation by pkr, oas, or ifits (88) . other anti-viral restriction factors such as mx2 may specifically target one molecule of a very restricted set of viruses with no apparent side effects on host cells. some anti-viral isgs target specific functions critical for only a restricted array of viruses and might similarly exert side effects only on a subset of host cell types. for example, samhd1 inhibits retrovirus replication through dntp depletion, which might more specifically affect proliferating host cells. hence, the infected host must balance the intensity, breadth, and location of isg induction to circumvent viral replication while preventing life-threatening damages to vital cell types or tissues. one of the mechanisms contributing to this balance is translational control of the expression of isgs, especially those with pro-apoptotic or anti-proliferative functions (94) . while many anti-viral isgs are transcriptionally activated in most ifn-stimulated cells, their translation can be specifically blocked in uninfected cells by cellular microrna. this inhibition is relieved upon cell infection through negative regulation of the function of the rna-induced silencing complex. hence, ifn stimulation of uninfected cells prepares them for rapid and strong induction of cell-intrinsic anti-viral defenses upon viral infection while avoiding their unnecessary exposure to the toxic effects of certain isgs. further knowledge on the functions and the dynamic regulation of isgs is essential to develop novel therapeutic strategies against viral infections aiming at modulating ifn responses to promote their protective anti-viral cell-intrinsic functions over their deleterious toxic effects. a better understanding of the immunoregulatory effects of ifns will also help. type i interferon can modulate the functions of a broad spectrum of immune cells ( figure 3a ). we will review this knowledge, focusing on the functions of dcs, nk cells, t cells, and b cells, since they are involved in the control of most viral infections. we will discuss the hypothesis that dcs play a central role in ifn-i orchestration of innate and adaptive immunity for the induction of optimal anti-viral defenses (figure 3) . during viral infections and cancer immunosurveillance, ifn-i constitute one of the most important input signal acting on dcs to promote their delivery of appropriate output signals to t cells, b cells, and nk cells for protective immunity ( figure 3a ). dcs deliver three types of signals to activate and functionally polarize t cells. signal 1 is the triggering of the t cell receptor by viral peptide-mhc complexes. signal 2 is the triggering of activating t cell co-stimulation receptors such as cd28 or cd27 by the cd80/86 and cd70 co-stimulation molecules expressed on dcs. signal 3 corresponds to cytokines, which can promote t cell proliferation and acquisition of specific effector functions. under steady state conditions, most dcs are in an immature state characterized by low level expression of mhc-ii (signal 1) and co-stimulation molecules (signal 2) and by the lack of production of t cell-activating www.frontiersin.org cytokines (signal 3). upon activation, including early after viral infections in vivo, dcs up-regulate their expression of signal 1 and activating signal 2 and secrete t cell-activating cytokines. this process is called dc maturation. gene expression profiling of dcs stimulated by microbial stimuli identified a core set of genes upregulated in mature dcs irrespective of the stimulus they receive, irrespective of the subset they belong to, and conserved across evolution (95) . most of these genes are induced during dc maturation in part through cell-intrinsic ifn-i signaling (95) . consistently, cell-intrinsic ifnar signaling in dcs is required in many circumstances for the induction of protective immunity, including efficient cd8 t cell responses during viral infection or tumor development (96) (97) (98) , th1 responses upon polyi:c injection independently of il-12 or ifn-γ effects (99, 100) , as well as follicular helper t cell and humoral responses (101, 102) . mechanistically, ifn-i promote dc immunogenicity for efficient t cell activation through a variety of effects ( figure 3b) . it drives dc up-regulation of signal 2 in vivo during viral infections (103) and boosts their capacity to cross-present antigens for increased delivery of signal 1 to cd8 t cells (96) (97) (98) . it shapes their delivery of activating signal 3, in particular inducing il-15 and promoting or inhibiting il-12 depending on experimental conditions (58, 104) . finally, it is necessary to induce their metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, which fuels the increased needs in energy and the expansion of the intracellular organelles required for the production and proper intracellular routing of the signal 1 and 2 proteins (100, 105). selective inactivation of ifnar on cdcs compromises mouse resistance to mcmv and mhv infections (103, 106) . in contrast, ifnar expression is not required on nk cells for protection against mcmv and on pdcs, t cells, and b cells for early control of mhv replication (103, 106) . although cell-intrinsic ifn-i signaling in nk cells can promote their activation (107) (figure 3a) , ifn-i-induced il-15 trans-presentation by dcs plays a more prominent role for this function in many conditions including in vivo during mcmv infection (103, 108) ( figure 3c) . cell-intrinsic ifn-i signaling in cd4 t cells (109), cd8 t cells (110, 111) , and b cells (112) can also contribute to their efficient activation and functional polarization (figure 3 ). this depends on experimental settings. cd8 t cell-intrinsic ifn-i responses are crucial for mounting efficient cytotoxic cd8 t cell responses against lcmv but are less critical against vaccinia virus and vesicular stomatitis virus (110, 113, 114) . mechanistically, cell-intrinsic ifn-i signaling in cd8 t cells can promote their survival during their antigen-induced proliferation (110) . cell-intrinsic signaling in dcs and cd8 t cells may act in a synergistic manner. indeed, conditional inactivation of ifn-i responsiveness was required to occur simultaneously in both of these two cell types to dramatically affect cd8 t cell expansion upon vaccination with a modified ankara vaccinia virus (115) . in summary, ifn-i generally play a crucial, beneficial, role in immune defenses against viral infections, both through the induction of cell-intrinsic anti-viral defenses and through the orchestration of innate and adaptive immunity. however, if these responses are not properly regulated, they can contribute to diseases as we will now discuss. a frequent side effect of ifn-i treatment against cancer or chronic viral infections is the induction of autoimmune reactions. consistently, isg expression is a hallmark of many spontaneous systemic or tissue-specific autoimmune diseases, including systemic lupus erythematosous (sle), sjogren's syndrome, psoriasis, and other skin disorders (11) . the dysregulation of ifn-i responses observed in patients with these autoimmune diseases likely results from both genetic and environmental factors. genome-wide association studies show that polymorphisms in genes involved in ifn-i responses strongly correlate with increased susceptibility to many autoimmune diseases (11) . diverse environmental factors can also contribute to the onset of autoimmune diseases. microbial infections often precede first clinical manifestations of autoimmune diseases. whether infections (116) and/or alterations in the commensal microbiota of the affected barrier tissues (117, 118) are the cause or rather the consequence of autoimmunity is still matter of debate. infection-or dysbiosis-induced tissue damages and unbridled ifn-i responses can contribute to initiate autoimmune reactions. gender is another prominent factor affecting susceptibility to autoimmune diseases. women are more prone to autoimmunity, which may result from endocrine regulation of ifn-i responses. pdc ifn-i production is enhanced in human and mouse females, due at least in part to cell-intrinsic enhancement of tlr7/9 responses by the female hormone estradiol (119). in autoimmune diseases, different mechanisms could operate to initiate the dysregulation of immune responses leading to a vicious circle of reciprocal activation between innate ifn-i responses and adaptive self-reactive lymphocyte responses (figure 4) . adaptive immune cells are educated to spare "self." this occurs through negative selection of potentially autoimmune b and t cells during their development in the bone marrow or thymus, respectively, a process called central tolerance. self-reactive b or t cells that have escaped this pruning can be either deleted or functionally inactivated once they have egressed in secondary lymphoid organs or non-lymphoid tissues, a process called peripheral tolerance. in some individuals, polymorphisms in genes involved in the promotion of central or peripheral tolerance lead to a higher number, diversity, and/or responsiveness of self-reactive lymphocytes in the periphery, in particular of b cells secreting anti-dna or anti-rnp antibodies (120, 121) . mammalian dna or rna are poor inducers of pdc ifn-i induction under normal conditions. however, pre-existing anti-dna or anti-rnp autoantibodies can break this innate tolerance of pdc. indeed, antibodies binding to self nucleic acids can protect them from degradation and compact them into nanoparticles that are very effective for the induction of ifn-i in pdc (figure 4) . dna-containing immune complexes (ics) are frequently found in the serum of sle patients (sle-ics) and can activate pdc ifn-i production (122) . in turn, pdc ifn-i activate cdcs, monocytes (123) , and b cells, leading to a vicious circle of reciprocal activation between dcs and frontiers in immunology | microbial immunology figure 4 | a simplified model of the deleterious role of ifn-i in several autoimmune diseases. when exposed to different kinds of injuries (microbial infection, commensal microbiota dysbiosis, chemical or physical insults), healthy tissues can undergo cell damage and death. these events induce the release of apoptotic bodies encompassing self rna or dna. neutrophil recruitment and activation in inflamed tissues can also constitute a potent source of self nucleic acids, through the release of neutrophil extracellular traps (net). self rna or dna can associate with cationic peptides (e.g., ll37) as shown in psoriatic patients or with inflammatory molecules (e.g., high mobility group box 1, hmgb1) to generate nanoparticles that are extremely efficient for ifn-i production by pdc and eventually other cell types. pdc can also be efficiently activated for ifn-i production by immune complexes (ics) generated by the association between self nucleic acids and auto-antibodies as frequently found in the serum of systemic lupus erythematosus patients. ifn-i promote the differentiation and/or the maturation of antigen-presenting cells, in particular different subsets of dc. activated dc can then present self-antigens for activation of auto-reactive t cd4 + cells, including follicular helper lymphocytes, which in turn activate auto-reactive b cells for auto-antibody secretion, leading to a vicious circle of reciprocal activation between innate and auto-reactive adaptive immune cells. idc, immature dc; mdc, mature dc; mo-dc, monocyte-derived dc. see main text for further details. self-reactive lymphocytes and to the exacerbation of autoimmune responses (figure 4) . certain infections or dysbiosis of the commensal microbiota of the affected barrier tissues could promote chronic production of host amphiphatic peptides able to combine with eukaryotic dna or rna, likely released from dying cells, thus forming pdc-activating nanoparticles. indeed, in psoriatic skin, both a high expression of ll37 and a massive infiltration of pdcs is observed (124) (figure 4) . hence, to treat many autoimmune diseases, novel therapeutic strategies could be designed to target dysregulated pdc ifn-i production or b cell activation by ifn-i. one of the most common complications of primary infections by many respiratory viruses, in particular influenza virus, is a lifethreatening pneumonia due to secondary pulmonary infections by bacteria, such as streptococcus pneumoniae, staphylococcus aureus, or haemophilus influenza (125, 126) . these pathologies affect especially infants, elderly, and immunocompromised patients. retrospective studies indicate that secondary bacterial pneumonia was highly recurrent in lung tissues isolated from patients who died during last century influenza pandemics, independently of antibiotic availability (127, 128) . influenza virus induces high ifn-i responses in human beings and mice. in both hosts, secondary bacterial infections are lethal only when they occur in a limited time window following primary viral infection (3-7 days), around the peak of ifn-i responses, before complete virus clearance. mouse models of viral/bacterial coinfections are being used to dissect disease mechanisms (129) . ifnar1-deficient mice appear more resistant to secondary pulmonary bacterial infections, showing that ifn-i responsiveness contributes to disease (130) . similarly, after lymphochoriomeningitis virus (lcmv) infection, wild-type but not ifnar1-deficient mice are more susceptible to lpsinduced septic shock (131) . several mechanisms may contribute to the detrimental role of ifn-i in secondary bacterial infections ( figure 5) . early during viral infection, ifn-i decrease the host ability to control bacterial replication, by dominantly polarizing immune responses toward anti-viral functions, simultaneously inhibiting the development of the types of immune responses required for protection against most bacterial infections. ifn-i can inhibit the production of chemokines required for the recruitment to the respiratory tract of antibacterial effector innate immune cells, in particular neutrophils or monocytes/macrophages (132, 133) (figure 5) . depending on the experimental models used, ifn-i can on the contrary induce a ccr2-dependant recruitment of classical monocytes (134) . in infected tissue, ifn-i might skew the functional polarization of resident or infiltrating monocytic cells toward immunosuppression, because it does limit their antibacterial functions by inhibiting their il-1 production (135) (136) (137) while it might promote their production of il-10 and nitric oxygen intermediates. the exact nature of infiltrating monocytic cells is not clear and could correspond to activated classical monocytes, modcs, monocyte-derived macrophages, or myeloidderived suppressor cells (mdscs). the boundaries between these putatively different cell types are currently ill-defined (138) . these cells could fuel local replication of monocyte/macrophage-tropic bacteria (134) , be immunosuppressive (139) or contribute to local immunopathology (140) . the role of ifn-i on monocytes/macrophages is complex and will require further investigations to determine when it is protective versus deleterious and what the underlying mechanisms are. depending on the context, ifn-i can either promote or inhibit the induction of th1 cytokines such as il-12 and ifn-γ, and myeloid cell responses to ifn-γ (10, (141) (142) (143) . ifn-i can also polarize cd4 t cell responses toward th1 at the expense of th17, while the th17-type cytokines il-17a and il-22 are required for host defense against pulmonary www.frontiersin.org bacteria by inducing the production of anti-microbial peptides and of tissue repair molecules ( figure 5 ) (141) (142) (143) . ifn-i may not only affect host resistance to bacterial infection, but also host tolerance, i.e., the ability of the host to tolerate a given burden of pathogen without undergoing excessive tissue damages (143, 144) . hence, to counter ifn-i deleterious effects during secondary bacterial infections, it will be important to better delineate the respective contribution of lung tissue tolerance modulation and of immune-mediated resistance weakening. another well documented example of deleterious effects of ifn-i due to their inappropriate functional polarization of immune responses is the enhanced susceptibility to fungal infections of patients with genetically determined hyperactive ifn-i responses, as exemplified in the hereditary disease chronic mucocutaneous candidiadis (cmc) (figure 5 ) (145) . patients with cmc have a significant deficit in th17 cd4 t cells, at least in part as a consequence of altered responsiveness to il-6 or il-21. several stat1 mutations were identified in patients with autosomal dominant cmc. gain-of-function stat1 mutations were found to hard wire cd4 t cell responses to cytokines toward stat1 signaling, compromising their stat3-dependent ability to produce il-17 upon il-6 or il-21 stimulation. this was associated to induction of a global ifn-i transcriptomic signature in blood (145) . deleterious ifn-i effects on immunity to candida might not only occur in cmc patients but also in other types of individuals upon secondary fungal infections occurring shortly after a primary viral infection, likewise to the situation discussed above for secondary bacterial infections. indeed, polyic induced ifn-i abrogate innate immunity to systemic candidiasis in mice (146) , and ifnar-deficient mice can be more resistant to candida infection under certain experimental settings (147) . however, the role of ifn-i in the modulation of the ability of immunocompetent hosts to control fungal infection is disputed (148, 149) . the inhibition of th17 responses by ifn-i could be protective in at least one important human pathology, ms (figure 5) . ms represents a striking exception to the previously discussed detrimental role of ifn-i in autoimmune diseases. indeed, a large proportion of ms patients have low serum ifn-i activity and low isg levels. these ms patients present a significant reduction of ms relapse upon ifn-β administration (150) . the underlying mechanisms are not yet completely unraveled. however, in the experimental autoimmune encephalitis mouse model of ms, th17 responses bear a major contribution to nervous system damages and are inhibited by the il-10 and il-27 induced upon ifn-i administration (151) . in summary, ifn-i responses can be deleterious in autoimmunity by promoting a vicious circle of reciprocal activation between innate immune cells and auto-reactive cd4 t or b lymphocytes. ifn-i responses can also be deleterious upon secondary bacterial or fungal infections in the lung or the kidneys occurring shortly after a primary viral infection, by compromising the recruitment of anti-microbial innate effector cells and/or by preventing the proper functional polarization of immune responses. we will now discuss how ifn-i responses can also compromise host immune defenses against certain viruses and promote chronic infections. different lcmv strains such as armstrong and clone-13 (cl13), respectively, lead to acute versus chronic infections in mice. a hallmark of chronic lcmv infection is the loss of the proliferative potential and effector functions of anti-viral cd8 t cells, a process called exhaustion. exhausted cd8 t cells are characterized by a high expression of the inhibitory receptors pd-1, ctla4, and lag-3 (152) . in vivo blockade of these inhibitory receptors can reverse t cell exhaustion and allow resolution of the chronic infection (152) . ifn-i and isgs are induced early after infection with all strains of lcmv, albeit to lower levels with those leading to chronic infection. this early ifn-i production is critical to limit viral replication (3). in models of acute infection, ifn-i responses rapidly return to normal, undetectable, levels, before viral replication is completely controlled. in contrast, isg induction is maintained in chronic infection, including the expression of pd-1 ligands on apcs and of the immunosuppressive il-10 cytokine, consistent with a prolonged expression of ifn-i albeit at low levels (13, 14) . in vivo neutralization of ifn-i by antibody administration promoted resolution of chronic lcmv cl13 infection, allowing the frontiers in immunology | microbial immunology restoration of functional anti-viral cd8 t cell responses at least in part through cd4 t cell-and ifn-γ-dependent mechanisms (13, 14) . during persistent lcmv cl13 infection, chronic low level ifn-i production polarizes cd4 t cell responses toward t follicular helper (tfh) rather than th1 functions. thus, chronic ifn-i responses promote enhanced anti-viral b cell responses but facilitate cd8 t cell exhaustion due to deficient cd4 t cell help, therefore contributing to host failure to prevent chronic infection (153) . strikingly, establishment of chronic infection by lcmv cl13 could also be prevented by early administration of two shots of a high dose of exogenous ifn-i, at days 2 and 5 post-lcmv inoculation. this treatment allowed viral clearance by rescuing anti-viral cd8 t cell from exhaustion (154) . altogether, these studies show that the timing and duration of ifn-i production during viral infections is critical in determining how this response will impact the balance between the virus and the host. an early and robust but transient production of ifn-i promotes strong induction of cell-intrinsic viral restriction mechanisms as well as adequate polarization of adaptive anti-viral immune responses, which combined effects lead to viral clearance. in contrast, if the production of ifn-i is too low and/or too late, both viral replication and low ifn-i responses become chronic, their combined action leading to induction of immunosuppressive effects and to inadequate functional polarization of cd4 t cells. this results in cd8 t cell exhaustion and maintenance of chronic infection. chronic viral replication and cd8 t cell exhaustion is also a hallmark of hiv-1 infection. we will now discuss the complex and disputed role of ifn-i in this disease. both in hiv-1 infection and in its most relevant animal model, infection of non-human primates with simian immunodeficiency virus (siv), disease progression after the acute phase of the infection is associated with high and chronic expression of isgs while ifn-i production is inconsistently detected (155) (156) (157) . in contrast, the individuals that do not progress toward disease despite persistent high viral loads show much lower immune activation, in particular low isg expression, after the acute phase of the infection (158) (159) (160) (161) . hence, chronic low levels of ifn-i are associated to disease progression independently of the level of viral replication. therefore, an outstanding question still open for a better understanding of the physiopathology of hiv-1 infection is whether chronic ifn-i responses are merely a marker of progression, or whether they are implicated in driving disease development. in addition to mechanisms similar to those uncovered in the mouse model of chronic lcmv infection, during hiv-1 infection other effects of ifn-i could promote a vicious circle of reciprocal activation between chronic viral replication and sustained, deleterious immune responses (figure 6 ). very early after hiv-1 infection, in most individuals, ifn-i production might be too weak or too late to induce a combination of cell-intrinsic defense mechanisms and of immune responses efficient enough to prevent later establishment of chronic infection. on the contrary, as demonstrated in the case of the mouse model of lcmv infection, ifn-i responses could favor cd8 t cell exhaustion, either by direct cell-intrinsic effects on cd8 t cells (figure 6 , ) or by contributing to deprive them from cd4 t cell help (figure 6, ) . several effects of ifn-i might compromise anti-hiv-1 th1 responses or more generally contribute to the global depletion of cd4 t cells. these mechanisms include functional polarization of anti-hiv-1 cd4 t cells toward tfh rather than th1 responses, cxcl10 production leading to enhance recruitment of memory cd4 t cells to the sites of viral replication where they fuel chronic viral replication with new hiv-1 target cells (figure 6, to ) , direct pro-apoptotic and anti-proliferative effects on cd4 t cells (figure 6 , ), as well as trail induction on pdcs licensing them for killing cd4 t cells irrespective of their infection (figure 6 , ) (162, 163) . altogether, these mechanisms entertain chronic viral replication and continuous depletion of cd4 t cells, leading to the dramatically enhanced susceptibility to opportunistic infections (figure 6 , ) characteristic of the acquired immunodeficiency syndrome (aids) (figure 6, ) . other lines of evidences have been reported to support a deleterious role of pdc activation during hiv-1 infection. women undergo faster hiv-1 disease progression than men with similar viral loads, which may result in part from the highest ifn-i production of women's pdcs including in response to hiv-1 stimulation (164) . pdc recruitment and activation in the vaginal mucosa of female macaques early after local siv inoculation contribute to attract and activate cd4 t cells, which can then be infected and promote virus dissemination from its portal of entry (165) . however, in vivo blockade of pdc ifn-α production by administration of tlr7/9-antagonistic oligonucleotides early after siv infection of macaques did not decrease t lymphocyte activation, which suggests that additional sources of ifn-i likely contribute to the immune dysfunction observed in siv/hiv-1 infections. targeting dysregulated ifn-i responses during hiv-1 infection might represent an interesting adjuvant therapeutic strategy to highly active antiretroviral treatments. administration of ifn-i in the non-pathogenic siv infection model of sooty mangabeys was not sufficient to switch it into a pathogenic model. no cd4 t cell depletion ensued, no hyperactivation of immune responses were observed. viral loads were even significantly decreased. however, this could be consistent with the positive impact of early and high dose ifn-i administration in chronic lcmv infection (154) . indeed, during the review process of this manuscript, it was reported that, early during primary siv infection in the pathogenic rhesus macaque model, a high dose injection of ifn-i was protective while neutralization of endogenous ifn-i was deleterious. in contrast, in the same animal model, prolonged ifn-i administration accelerated disease development in the chronic stage of the infection (166) . in mice with a humanized immune system, pdc depletion strongly decreased isg induction and enhanced viral replication both in the acute and chronic phases of hiv-1 infection. however, pdc depletion during chronic infection decreased infection-induced t cell apoptosis and increased t cell numbers in lymphoid organs (167) . these results further emphasize the dual role of ifn-i and pdcs in the physiopathology of hiv-1 infection. a strong and transient production of ifn-i early after infection benefits the host by lowering the set-point of viral replication during chronic infection. sustained production of low levels of ifn-i during chronic infection contributes to immune dysregulation and cd4 t cell depletion. further studies will be necessary to examine whether complementing standard-of-use antiretroviral drugs with pdc www.frontiersin.org depletion, ifn-i neutralization, or selective inhibition of t cell responses to ifn-i could yield additional benefits to chemotherapy in non-human primates during chronic siv infection. ifn-i administration has been used for many years to treat another human chronic viral disease, hcv infection. roughly, half of the patients do not show sustained virological responses (svr). the treatment causes severe side effects in many individuals. new chemotherapeutic drugs very potent at blocking hcv replication in vivo have recently become available. hence, whether ifn-i administration still constitutes a viable treatment against chronic hcv infection is being questioned (168, 169) . we will now discuss this issue. chronic hcv infection is the main cause of liver cirrhosis and hepatocellular carcinoma. there is currently no vaccine against hcv. the most common therapy for chronic hcv patients is the administration of recombinant pegylated ifn-α (peg-ifn-α) combined with the anti-viral drug ribavirin. however, because of ifnar pleiotropic expression, ifn-α administration induces severe side effects including flu-like syndrome, fever, fatigue, myalgia, and nervous depression ( figure 7a ) (170) . moreover, only about half of treated patients harbor svr (171) . prior-totreatment high hepatic isg expression is a negative predictor of svr upon peg-ifn-α therapy. high isg expression in untreated patients likely results from chronic but low ifn-i production triggered by persistent hcv replication. indeed, hepatocytes from non-responder patients were found to be infected at a greater frequency and to exhibit dampened antiviral and cell death responses (172) . what the cellular sources of ifn-i production are and why they persist only in non-responder patients still remain to be established. in chronic hcv infection, cytotoxic effector lymphocytes may contribute to the development of hepatocarcinoma by causing low level but sustained hepatocyte death and renewal. in contrast, local production of ifn-γ in the liver by nk and t lymphocytes could promote resistance to disease through non-cytolytic control of viral replication. as discussed previously for lcmv and hiv-1, low chronic production of endogenous ifn-i in hcv patients could compromise both innate and adaptive anti-viral immune responses. chronic exposure to ifn-i could dampen the ability of frontiers in immunology | microbial immunology utr of ifnl3 mrna to promote their degradation. the favorable ifnl3 allele associated with responsiveness to peg-ifn-α treatment may allow endogenous expression of sufficient levels of ifnl3 for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes. this process is, however, hampered by the limited expression of the receptor for this cytokine (ifnλr1) in these patients. peg-ifn-α treatment might promote resolution of the infection by inducing ifnλr1 in these patients, potentiating their response to their endogenous production of ifnl3. in the patients that do not respond to peg-ifn-α treatment, endogenous levels of ifnl3 are insufficient for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes, due to the degradation of the corresponding mrna in infected hepatocytes. in these patient's hepatocytes, however, ifnλr1 is already expressed to high levels prior to treatment due to their high endogenous ifn-i responses. administration of exogenous ifn-λ might cure these patients. see main text for further details. nk and cd8 t cells to produce ifn-γ (173, 174) and promote cd8 t cell exhaustion (175) . it could also induce an antagonist form of cxcl10, a chemokine required for recruitment to the liver of anti-viral nk and cd8 t cell effectors (176) . it may also polarize monocytes toward immunosuppressive functions (177) . therefore, better understanding ifn-i effects in hcv infection is critical to improve care of both responders and non-responder patients to peg-ifn-α. for responder patients, the issue is to modify the treatment to favor beneficial antiviral and immunoactivating effects over side effects strongly affecting patient's quality of life (figure 7a ). this might be achieved by specific delivery of ifn-i to targeted cell types as discussed later. for non-responder www.frontiersin.org patients, the issue is to understand the mechanisms underlying treatment failure to determine whether alternative therapies could be designed (figure 7a) . genome-wide association studies identified various single nucleotide polymorphisms (snp) in the gene encoding il-28b/ifn-λ3, one of the ifn-iii, as well in its 5 and 3 non-coding regions (178) (179) (180) (181) . one snp, called rs12979860, is located 3 kb upstream of the ifnl3 gene. patients harboring the cc genotype have a favorable prognosis to ifn-i treatment. patients with the tt genotype are at high risk of treatment failure (178, 179) . in europeans, the favorable cc genotype is the major, most common, ifnl3 allele. the unfavorable tt snp is the minor allele. the frequency of these alleles is reversed in africans. the favorable allele allows escape of ifnl3 mrna from degradation by cellular microrna induced upon hcv infection (181) . until recently, ifnl3 genotypes and hepatic isg expression were considered as independent predictors of response to peg-ifn-α treatment in hcv patients (171) . here, we propose a potential explanation, which integrates both factors in a relatively simple model ( figure 7b ). our main hypothesis is that efficient control of hcv infection depends on hepatocyte response to ifn-λ rather than ifn-α. this is supported by reports that ifn-λ induces a stronger and more sustained isg expression in hepatocyte cell lines in vitro (182) , and that polyi:c-induced control of hcv replication in humanized liver in chimeric mice is correlated to the induction of ifn-λ but not ifn-i in human hepatocytes (183) . responder patients harboring the favorable ifnl3 allele preventing the degradation of the corresponding rna in infected cells might express significant levels of endogenous ifn-λ3, although this is disputed. however, they express only low levels of ifn-λr1, which limits ifnλ3 efficiency ( figure 7b ) (184) . how these patients benefit from peg-ifn-α treatment could be that it induces ifn-λr1 expression on hepatocytes thus boosting endogenous ifn-λ3 effects (184) . in contrast, high isg-expressing non-responder patients harboring the unfavorable ifnl3 allele might not express enough ifn-λ3 for virus control. however, they do express ifn-λr1 as a result of their endogenous production of ifn-i. hence, peg-ifn-α might be ineffective in these patients because they already express ifn-λr1 but fail to produce endogenous ifn-λ3 due to the degradation of its mrna in infected hepatocytes (figure 7b) . these patients may be good candidates for peg-ifn-λ therapy, currently undergoing clinical development. since the expression of ifn-λr1 is mainly restricted to epithelial cells, melanocytes, and hepatocytes, some of the side effects related to ifn-i treatment might be strongly attenuated in peg-ifn-λ therapy. however, as ifn-i are key to induce anti-viral immune responses, it will be critical to determine whether, beside viral clearance, peg-ifn-λ therapy can also induce long-term immune protection against hcv. ifn-i transduce intracellular signals through a single receptor, ifnar, but via a multitude of downstream signaling pathways. the janus activated kinase (jak)/stat pathway was the first to be identified (185) . ifnar is composed of two distinct subunits, ifnar1 and ifnar2, which are constitutively associated with members of the jak family, tyrosine kinase 2 (tyk2) and jak1, respectively (186) . the binding of ifn-i to their receptor leads to the phosphorylation of jak1 and tyk2, which in turn induce the phosphorylation and activation of the stat proteins (186) . different stat complexes can form upon triggering of ifnar (figure 8) . a transcriptional complex that forms in most conditions of ifn-i stimulation and induces the expression of many molecules of cell-intrinsic anti-viral immunity is interferonstimulated gene factor 3 (isgf3), a heterotrimer composed of pstat1, pstat2, and irf9 (187) (figure 8) . following its translocation into the nucleus, isgf3 binds to isre regulatory sequences in target genes. many molecules playing a key role in the function of innate or adaptive immune leukocytes are also induced by isgf3, including cd80, cd86, or il-15 in dc, and granzyme b in nk cells. isgf3 is generally composed of stat1 phosphorylated on tyr701 and ser727 and of stat2 phosphorylated on tyr689. however, alternative isgf3 complexes have been described in various contexts which could participate to the diversity of ifn-i effects (188) . the pstat1 homodimer also plays a prominent role in cell-intrinsic ifn-i-dependent gene induction. it binds ifnγactivated sequences (gas) and controls the expression of many pro-inflammatory molecules (187) . pstat1 homodimers can form upon stimulation with either ifn-i or ifn-γ. many gasregulated genes can be induced by either cytokines. depending on cell types, jak signaling downstream of ifnar can lead to the activation of virtually all stat proteins and to their combinatorial association into a variety of complexes with different affinities for specific gas elements (189) (190) (191) (figure 8) . this diversity contributes to ifn-i induction of different transcriptional programs in distinct cell types (39) . stat complex formation depends in part on the relative abundance of stat molecules in the cell (192) . while stat1, stat2, stat3, and stat5 can be activated in most cell populations, stat4 and stat6 are mainly activated in lymphocytes (193) . for example, quiescent nk cells express more stat4 than stat1, leading to constitutive association of ifnar to stat4 in these cells. hence, quiescent nk cells mount pstat4 homodimer-dependent responses to ifn-i stimulation, including ifn-γ production and t-bet-driven proliferation (figure 8 ) (194, 195) . changes in stat levels can also occur upon the differentiation/activation of a given cell type and lead to a shift in its functional response to the cytokines (196) . upon activation, nk cells decrease their expression of stat4 and increase that of stat1, shifting their ifn-i response from stat4-dependent in a quiescent state to stat1dependent in pre-activated cells. this translates into opposite ifn-i effects on ifn-γ production and proliferation for quiescent versus pre-activated nk cells (194) . however, this outcome can be modulated by simultaneous exposure to other cytokines such as il-15 or il-12/18. a reverse stat1-to-stat4 shift occurs in dc during their maturation, shifting their functional responses from inhibition to activation of il-12 production in response to combined stimulations with ifn-i and cd40l (197) . this frontiers in immunology | microbial immunology ifn-i binding to ifnar triggers the phosphorylation of tyk2 and jak1, which in turn phosphorylate a variety of stat proteins. activated stats are able to form complexes, as homo-or hetero-dimers. the heterodimer stat1-stat2 binds to a third partner, ifn-regulatory factor 9 (irf9), in order to form the isgf3 complex. this complex translocates into the nucleus and binds to specific regulatory sequences, ifn-stimulated response elements (isre), to activate the expression of many interferon-stimulated genes (isgs). in particular, isgf3 induces most, if not all, of the isgs encoding effector molecules of cell-intrinsic anti-viral defenses such as oas or mx1. alternative jak/stat pathways include the formation of stat1 or stat4 homodimers, which may drive different functional responses to ifn-i. stat1 homodimers bind to ifnγ-activated sequences (gas) in the promoter of certain isgs, which may promote inflammatory, anti-proliferative, and pro-apoptotic responses. stat4 homodimers also bind to gas but promote ifn-γ production and pro-proliferative responses. enables mature dc to efficiently activate cd8 t cells. other yet unknown mechanisms control the formation of different stat complexes in distinct cell types. the nature and dynamics of the signaling pathways triggered by ifn-α or -β were evaluated in bulk cultures of human blood leukocytes using flow cytometry (191) or high throughput mass cytometry (190) . a diversity of phosphorylation patterns of stat1/3/5 was observed upon ifn-i stimulation. ifn-α activation induced phosphorylation of stat1, stat3, and stat5 in most cell types, peaking at 15 min (190) . ifn-β-induced stat1 phosphorylation was found to be poor in b cells as compared to monocytes and t cells (191) . however, the underlying mechanism remains to be identified since b cells did not express lower amount of ifnar2 or stat1 or enhanced levels of the inhibitory socs1 molecule. the high stat1 activation in monocytes led to their induction of ifn-i-dependent pro-apoptotic genes while this was not the case in b cells. these results strikingly differ from those obtained in the other study upon ifn-α stimulation, where stat1 phosphorylation was on the contrary lower in cd14 + monocytes and was prolonged in b cells and nk cells (190) . the differences between these two studies might have resulted from the use of different subsets and doses of ifn-i. in any case, both studies consistently reported that cd4 t cells showed the highest activation of stat5. all cd4 t cells but not all cd8 t cells activated stat5 and for a longer time (190) . ifn-β activation of stat3 was delayed in cd4 t cells and b cells as compared to cd8 t cells and monocytes (191) . different stat complexes may lead to distinct transcriptional programs linked to different biological functions (figure 8) . more systematic studies are needed to understand this complexity. besides changing stat levels between cell types or www.frontiersin.org activation states, the processes controlling differential formation of stat complexes downstream of ifnar triggering remain to be identified. in addition to jak/stat signaling, other pathways can be activated downstream of ifnar, including those involving the phosphatidylinositol 3-kinase (pi3k), mitogen-activated protein kinases (mapk), and the crk adaptor molecules (39, 198) . this leads to the activation of other transcription factors such as irf, nf-κb, or pu.1, which contribute to orchestrate cell responses to the cytokines by regulating both distinct and overlapping sets of genes as compared to stat (199, 200) . in summary, ifnar signals through a remarkable diversity of pathways, including but not limited to diverse combinations and kinetics of stat phosphorylations. this explains at least in part the diversity of ifn-i effects, including their induction of opposite responses depending on the physiopathological contexts and/or the nature of the principal responding cell types (200, 201) . ifn-iii induce the same signaling pathways as ifn-i, although they engage a different heterodimeric receptor, composed of the il-28ra and il-10rb chains and preferentially expressed on epithelial cells including hepatocytes. in mice and human beings, numerous ifn-i subtypes exist. functional and population genetic analyses showed that these ifn-i subtypes significantly differ in their functions (202) (203) (204) (205) (206) (207) . hence, one of most extraordinary feature of ifn-i biology is how ifn-i subtypes can elicit so many pleiotropic and diverse functions by interacting with the same receptor complex (208) . both ifnar1 and ifnar2 are required for the initiation of ifn-i-dependent signals, as mice deficient in either one are highly susceptible to viral infections (3, 5) . the assembling of the ifnreceptor ternary complex is a two-step process. first, a binary complex is formed by the binding of one side of the ifn molecule to ifnar2. then, a single binary complex interacts with ifnar1 via the other side of the ifn molecule. the stability of the ternary complex will be determined in part by the association and dissociation kinetics between the cytokine and the two receptor chains, as well as by ifnar expression levels since the cell surface concentrations of the receptor subunits are relatively low. hence, both the affinity of ifn-i subsets for ifnar and the amounts of ifn-i, ifnar1, and ifnar2 will regulate their biological effects ( figure 9a ) (209, 210) . cell membrane density of ifnar1 and ifnar2 is also involved in differential ifn-β-versus ifn-α-induced functional activities, such as anti-proliferative function (211) . a variety of cell-intrinsic parameters can also impact the lifetime of the ifn-receptor ternary complex, such as the rate of endocytosis/degradation/recycling of signaling complexes, and negative isg regulators such as usp18 that decrease the affinity of ifn-ifnar1 binding (203, 212) . based on a definition of a prototypic cytokine-receptor binding module and by analogy with the epo receptor system, ifn-i subtypes were originally postulated to form ternary complexes of differing architectures, resulting in distinct geometry and assembling of intracellular signaling components (213) . experimental evidence rejected this hypothesis. rather, the differential activities of ifn-i subtypes are determined by the stability of the ligand/receptor ternary complex (207, 212) . differential affinities of the ifn-i subtypes for ifnar1 and ifnar2 extracellular domains generate subtype-specific signaling cascades and biological outcomes ( figure 9a ) (210, 214) . crystal structure of ternary ifn-i/ifnar1/ifnar2 complex illuminated the biochemical complexity of ifn-i interaction with their cognate receptors (215) . the main conformational features of ifn-i/ifnar1/ifnar2 ternary complexes are conserved among the different ifn-i, but are quite different from the other cytokine receptors (214, 215) . in the formation of the binary ifn-i/ifnar2 complex, ifn-i ligand discrimination resides on differential energetics during the interaction of anchor points with ifnar2, shared by all ifn-i, as well as on key amino acid substitution among ifn-i subtypes (215) . ifnar1 then performs major conformational changes to interact with ifn-i associated in the binary complex, thus displaying an optimized functional plasticity (215) . these differences in the chemistry of ifn-i subtype interaction with ifnar2 and ifnar1 thus explain the different affinities of ifn-α versus ifn-β within ternary complex and their differential activities (210) . the functions regulated by ifn-i strongly depend on the main responding cell types ( figure 9b ). this has been studied in vitro by examining the functional consequences of the stimulation of different cell types with ifn-i, and in vivo by determining the contribution of cell-intrinsic ifn-i responses of different cell types to resistance or susceptibility to various diseases. an emerging concept is the central role of dc responses to ifn-i for induction of protective immunity against viral infections or tumors (figure 3) . the development of mutant mice allowing conditional genetic inactivation of ifnar1 in a cell-type specific manner using the cre-lox system (216) has been instrumental in accelerating our understanding of how different cell types respond to ifn-i in vivo and what their respective contribution is to protective or deleterious ifn-i responses. this has been investigated most extensively in viral infections (106, 111, 112, 115, 217) but also in cancer (97, 98) , bacterial infections (218), autoimmunity (216, 219) , sepsis (220), or inflammatory diseases (221) . efforts are being pursued to better understand which cell types respond to ifn-i in a manner promoting protective versus deleterious effects in different physiopathological settings. that knowledge will considerably help to develop novel strategies to modulate ifn-i functions for promoting health over disease. the development of mutant mice allowing conditional genetic inactivation of stat1, stat3, and stat5 (222-226) will help better understanding how different signaling pathways in different cell types determine the outcome of ifn-i response in vivo in various conditions. this knowledge might lead to the development of strategies aiming at targeting a given cell type with a specific subset of ifn-i, or in the presence of antagonists of certain signaling pathways, to surgically tune ifn-i responses in vivo toward the most desirable outcome. frontiers in immunology | microbial immunology for example, the affinity of ifn-β for ifnar1 is 100-times higher than that of ifn-α2, and ifnβ is much more potent in inhibiting cellular proliferation or (continued ) ifn-i can also determine distinct functional outcomes. for example, during viral infections, early and transient high levels of ifn-i promote protective dc and t cell responses, while delayed, chronic and low level ifn-i production compromises host immune defenses and promotes chronic viral infections. within a given cell type, the outcome of ifn-i stimulation also depends on time of exposure to these cytokines relative to other modulatory signals (timing relative to other stimuli). for example, in naïve cd8 t cells, tcr signaling prior to ifn-i stimulation leads to increased expression of stat4 and promotes ifn-γ production and proliferation, while ifn-i stimulation prior to tcr triggering leads to stat1-dependent anti-proliferative and pro-apoptotic effects. the formation of specific stat complexes is a highly dynamic process. it depends not only on the cell type but also on its specific state at the time it sees ifn-i. hence, major parameters controlling the effects of ifn-i in a given cell type also include its microenvironment ( figure 9c ) and the timing of its exposure to the cytokines both in terms of duration of the stimulation and of previous activation history ( figure 9d) . the tam receptor ligand gas6 is expressed within tumor cells in various solid cancers (227, 228) . elevated gas6 expression is of bad prognosis in different cancers (228, 229) . in a mouse model of ovarian cancer, early during tumorigenesis tumor-infiltrating dcs were found to be immunogenic and promote antitumor immunity, but they were later altered in the course of tumor development to acquire immunosuppressive properties beneficial to the tumor (230) . one may thus hypothesize that expression of tam soluble ligands in certain tumors and of tam receptors on tumor-infiltrating dcs might contribute to dampen dc response to ifn-i and therefore facilitate their polarization by the tumor microenvironment into immunosuppressive cells (figure 9c) . acute versus chronic exposure to ifn-i can lead to strikingly opposite effects on a given cell type (13, 14, 231) . in addition to duration, the time when a cell is exposed to ifn-i can also dramatically impact its functional response, depending on its previous activation history ( figure 9d) . in vitro stimulation of dcs with ifn-β can lead to opposite outcomes depending whether it occurs simultaneously to, or after, tnfα-induced maturation. ifn-β polarizes dcs toward th1 induction in the former case, and toward il-10-secreting t cells in the latter case. these opposite effects result at least in part from the differential expression of il-12/18 by dcs (232) . similarly, ifn-i effect on the functional polarization of cd4 t cells is strongly modulated by the other cytokines present in the lymphocyte microenvironment at the same time (233) . ifn-i can also mediate opposite effects on cd8 t cells depending whether it occurs before or after cognate engagement of the t cell receptor. indeed, while cd8 t cells have the potential to respond to ifn-i by inducing both stat1-and stat4-dependent genes, this depends upon their activation history. naïve cd8 t cells respond mostly to ifn-i through stat1 signaling, leading to the inhibition of their proliferation and eventually to the induction of their apoptosis. however, cognate triggering of the t cell receptor causes a decrease in stat1 and an increase in stat4 expression in cd8 t cells. this leads to a shift of their ifn-i response from stat1-to-stat4 signaling, resulting in the promotion of their proliferation and ifn-γ production. during lcmv infection, this mechanism promotes stat4-dependant expansion of anti-viral cd8 t cells, but stat1-dependant inhibition of naïve cd8 t cell proliferation (234) . since the late 70s the clinical potential of ifn-i for the treatment of patients suffering of viral infection or cancer diseases has been widely acknowledged (235) . today, this expectation is tempered because ifn-i treatment can induce severe side effects and sufficient doses cannot be administered in patients. therefore, there is a strong need to create tuned ifn molecules devoid of side effects. based on our current understanding of ifn-i responses as reviewed above, many parameters could be tuned individually or in a combined manner to modulate ifn-i activity to promote their beneficial effects over the deleterious ones in a number of diseases. these parameters include modifying the affinity of ifn-i for its receptor, playing with the local quantity/concentration of ifn-i and with the duration of its delivery, and modulating the nature of the cells that are responding to ifn-i. we will discuss here novel strategies being developed to deliver ifn-i to, or block ifn-i responsiveness of, a specific target cell type in vivo (figure 10 ). if ifn-i-induced side effects are a consequence of the pleiotropic nature of ifn-i, and if the bioactivities mediating deleterious effects have some degree of independence from those mediating beneficial effects, one could mutate the ifn-i molecules in order to skew their activity toward a desired bioactivity. indeed, introducing key mutation in ifn-α2 allowed increasing its affinity to ifnar1 by a factor of 100. accordingly, this ifn-α2 mutant is 100times more potent in inhibiting cell proliferation, but as potent as wt ifn-α2 in inducing an anti-viral state (236) (237) (238) . hence, it is possible to tune ifn activity by modifying its binding to ifnar. however, translating such an approach for the design of molecules for clinical application is severely hampered by the poor understanding we have on the ifn-i bioactivities mediating the side effects. furthermore, we are far from having established the list of bioactivities that could be differentially modulated by changing the stability of the ifn-i/ifnar complex. we know more about the frontiers in immunology | microbial immunology cell types that mediate beneficial versus deleterious ifn responses in various diseases. hence, we will now discuss strategies aimed at focusing ifn activity to specific cell types to promote health over disease. several strategies have been developed to specifically target ifns on tumor cells, tumor-infiltrated immune cells or infected tissues. these strategies include intra-lesional injection (239, 240) , adenoviral-mediated gene transfer (241) (242) (243) , engineered tumorinfiltrating monocytes (244) , and fusion of ifns with a cleavable protecting shell (245) . another strategy to increase cytokine accumulation within the tumor or infected tissue is antibody-mediated targeting of cytokine delivery, where a cytokine moiety is fused to an antibody directed against a specific cell surface marker (figure 10) . the fusion molecule retains both antigen-binding and ifn-i bioactivities, and is enriched at the targeted site upon in vivo injection (246) (247) (248) (249) . when targeted to human cd20, ifn-i inhibited the proliferation of lymphoma cells engrafted in immunodeficient mice (250) . an ifn-i targeted to a tumor antigen can also amplify the therapeutic effect of the antibody by acting on tumor-infiltrated dcs, thus increasing antigen cross-presentation and antitumor cytotoxic t cell responses (249) . on non-targeted cells, the antibody conjugation negatively impacts ifn-i potency, but only modestly (18, 248, 251) (figure 10a) . fusion molecules generally retain full ifn-i biological activity on the cells expressing the antibody target ( figure 10b) . hence, this difference only leads to a modest ratio between the ifn-i specific activity measured on target and non-target cells (figure 10b) . such a targeting efficiency is definitely too low to reduce the toxic effect of ifn-i administration, because it will not specifically focus ifn-i activities on "beneficial cells" without stimulating "deleterious cells." the engineering of immuno-ifn-i must be improved to reach the very high targeting efficacy required to significantly diminish the treatment side effects. we recently reported an innovative strategy reaching this goal (18) . it is based on the postulate that the antibody moiety of an immuno-ifn-i stabilizes the ifn-i/receptor-complex by avidity. it also takes into account the fact that the biological potency of an ifn-i is proportional to the stability of the ifn-i/receptor complex up to a certain threshold beyond which increasing the stability does not increase its potency (238, 252) . ifn-α2 and ifn-β are used in most immuno-ifn-i studies. they have evolved to retain close to maximal potency. hence, their targeting by an antibody that only provides a modest gain in terms of biological potency. however, it is expected that decreasing the affinity of the ifn-i for its receptor, by introducing a mutation, would increase the targeting effect of the antibody (figures 10 c,d) . this is indeed the case. using an ifn-i with a single point mutation that dramatically decreases its affinity for ifnar2 ( figure 10c ) allows engineering immuno-ifns that are up to 1000-fold more potent on cells expressing the antibody target ( figure 10d) . the three log targeting efficiency of these novel types of immuno-ifns is found for various activities measured in vitro or in vivo when delivered in mice. if the toxic side effect experienced by the patients treated with ifn-i is due to systemic ifn-i activity, this targeting technology may find considerable clinical applications since such engineered immuno-ifns are virtually inactive while "en route" and are activated only after binding of the fused antibody to the desired target. it remains to define the useful targets according to pathologies, for example, tumor cells themselves and professional cross-presenting xcr1 + dcs for cancer (97, 98, 249) , or hepatocytes for chronic hcv infection. to treat autoimmune diseases, novel therapeutics targeting ifn-i have been developed, including two ifn-α-neutralizing monoclonal antibodies currently in clinical trials (sifalimumab and rontalizumab) (253, 254) . however, long-term systemic neutralization of ifn-i activity may increase susceptibility to viral infection and tumor development. alternative strategies are needed to specifically inhibit ifn-i deleterious effects in these diseases without globally compromising ifn-i anti-viral and www.frontiersin.org anti-tumoral functions. the sequential nature of the assembling of the ifn-i/receptor complex opens the possibility to design ifn-i antagonists specifically targeting the cell subsets responsible for ifn-i deleterious effects. an ifn-α2 carrying a single amino acid substitution that blocks the ifn-i/ifnar1 interaction engages ifnar2 in a complex, which cannot bind ifnar1 (255) . since the binary ifn-i/ifnar2 complex is devoid of any ifn-i activity, such mutant behaves as a potent ifn-i antagonist. when linked to an antibody specific for a cell surface marker, the antagonistic activity of the mutant ifn-i should be significantly reinforced specifically on the cells expressing the target. hence, it should be possible to design and construct targeted antagonists that inhibit responsiveness to endogenous ifn-i specifically on the cell subsets on which the cytokines act to promote autoimmunity or severe side effects, leaving the other cells fully responsive. for example, in chronic hcv patients treated with peg-ifn-α, one of the most deleterious side effects is nervous depression, which might be prevented by co-administration of an ifn-i antagonist specifically targeting neurons or other cells of the central nervous system. in the last decade, several major technological breakthroughs and the generation of novel animal models have remarkably advanced our understanding of the mode of action of ifns. in vitro high throughput screening allowed systematically studying the functions of isgs by ectopic expression or knock-down. advance biophysical investigation of the interactions between ifn-i and the ifn-i receptor allowed to rigorously investigate the mechanistic basis for the differential bioactivities of ifn-i subtypes. the analyses of the responses of different cell types to ifns or to viral infection, in vitro but also in vivo in various pathologies, demonstrated that ifn-i often mediate beneficial versus deleterious roles by acting on different cell types. from integrative analysis of these data, a picture is now emerging suggesting that it will be possible to segregate protective from deleterious ifn-i effects, based (i) on their differential induction depending on ifn-i subsets or on the magnitude/timing of ifn-i production, (ii) on their conditioning in different tissues, (iii) or on their occurrence in different cell types. hence, innovative immunotherapeutic treatments are being designed to tune ifn-i activity toward desired effects in order to promote health over disease in a manner adapted to each physiopathological condition. in particular, a proof-of-concept has been made in vitro that it will be possible to target ifn-i activity on given cell types or tissues to administer to patients sufficiently high doses of the cytokine at the site of interest while limiting unwanted effects in other tissues or cell types. the next steps will be to demonstrate efficacy of this strategy in vivo in preclinical animal models. importantly, to foster the development of these innovative immunotherapies, major efforts are still warranted to continue delineating which cell types are mainly responsible for the protective versus deleterious effects of ifn-i in different diseases. combining high throughput technologies and systems biology approaches will also advance our understanding of the molecular mechanisms dynamically controlling ifn-i responses in health and diseases, which should reveal potentially novel therapeutic targets. virus interference. i. the interferon virus interference. ii. some properties of interferon functional role of type i and type ii interferons in antiviral defense inborn errors of interferon (ifn)-mediated immunity in humans: insights into the respective roles of ifn-alpha/beta, ifn-gamma, and ifn-lambda in host defense suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type i interferon activity new insights into cancer immunoediting and its three component phases -elimination, equilibrium and escape how type i interferons work in multiple sclerosis and other diseases: some unexpected mechanisms role of type i interferons in inflammasome activation, cell death, and disease during microbial infection does type i interferon limit protective neutrophil responses during pulmonary francisella tularensis infection? front immunol type i interferons in bacterial infections: taming of myeloid cells and possible implications for autoimmunity fueling autoimmunity: type i interferon in autoimmune diseases hiv-1 immunopathogenesis: how good interferon turns bad persistent lcmv infection is controlled by blockade of type i interferon signaling blockade of chronic type i interferon signaling to control persistent lcmv infection unraveling the convoluted biological roles of type i interferons in infection and immunity: a way forward for therapeutics and vaccine design interferons, interferon-like cytokines, and their receptors dendritic cell maturation: functional specialization through signaling specificity and transcriptional programming high efficiency cell-specific targeting of cytokine activity critical role of constitutive type i interferon response in bronchial epithelial cell to influenza infection critical role of interferon-alpha constitutively produced in human hepatocytes in response to rna virus infection neutrophils responsive to endogenous ifn-beta regulate tumor angiogenesis and growth in a mouse tumor model critical role for constitutive type i interferon signaling in the prevention of cellular transformation self-priming determines high type i ifn production by plasmacytoid dendritic cells irf7-dependent ifn-beta production in response to rankl promotes medullary thymic epithelial cell development cell-intrinsic role for ifn-alpha-stat1 signals in regulating murine peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response absence of ifn-beta impairs antigen presentation capacity of splenic dendritic cells via down-regulation of heat shock protein 70 a weak signal for strong responses: interferonalpha/beta revisited novel reporter mouse reveals constitutive and inflammatory expression of ifn-beta in vivo temporal and spatial resolution of type i and iii interferon responses in vivo priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota immune sensing of dna cytosolic sensing of viruses differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor-7 positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf-7 virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting novel paradigms of innate immune sensing of viral infections breaking the barrier: membrane fusion triggers innate antiviral immunity antiviral immunity via rig-i-mediated recognition of rna bearing 5 -diphosphates interferons: signaling, antiviral and viral evasion a role for dnadependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells rice: pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity mapping the crossroads of immune activation and cellular stress response pathways tlr-mediated activation of type i ifn during antiviral immune responses: fighting the battle to win the war plasmacytoid dendritic cells and the control of herpesvirus infections specialization, kinetics, and repertoire of type 1 interferon responses by human plasmacytoid predendritic cells plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon the nature of the principal type 1 interferon-producing cells in human blood mouse type i ifn-producing cells are immature apcs with plasmacytoid morphology virus-induced interferon alpha production by a dendritic cell subset in the absence of feedback signaling in vivo tlr9-dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function viral infection switches non-plasmacytoid dendritic cells into high interferon producers plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral nk and cd8(+) t cell accrual control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon plasmacytoid dendritic cells contribute to systemic but not local antiviral responses to hsv infections dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta interferon alpha/beta and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo individual plasmacytoid dendritic cells are major contributors to the production of multiple innate cytokines in an organ-specific manner during viral infection pattern recognition receptors and inflammation intracellular toll-like receptors deciphering the role of dc subsets in mcmv infection to better understand immune protection against viral infections the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting mouse cd8alpha+ dcs and human bdca3+ dcs are major producers of ifn-lambda in response to poly ic human xcr1+ dendritic cells derived in vitro from cd34+ progenitors closely resemble blood dendritic cells, including their adjuvant responsiveness, contrary to monocyte-derived dendritic cells human type 2 myeloid dendritic cells produce interferon-lambda and amplify interferon-alpha in response to hepatitis c virus infection human blood dendritic cell antigen 3 (bdca3)(+) dendritic cells are a potent producer of interferon-lambda in response to hepatitis c virus antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor activation of ifn-&#946; expression by a viral mrna through rnase l and mda5 rnase l releases a small rna from hcv rna that refolds into a potent pamp protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity tam receptors are pleiotropic inhibitors of the innate immune response alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses differential type i interferon induction by respiratory syncytial virus and influenza a virus in vivo crosstalk between components of the innate immune system: promoting anti-microbial defenses and avoiding immunopathologies plasmacytoid dendritic cells and toll-like receptor 7-dependent signalling promote efficient protection of mice against highly virulent influenza a virus plasmacytoid dendritic cells are dispensable during primary influenza virus infection clearance of influenza virus from the lung depends on migratory langerin+cd11b− but not plasmacytoid dendritic cells human tlr-7-, -8-, and -9-mediated induction of ifn-alpha/beta and -lambda is irak-4 dependent and redundant for protective immunity to viruses pyogenic bacterial infections in humans with myd88 deficiency impaired response to interferon-alpha/beta and lethal viral disease in human stat1 deficiency herpes simplex virus encephalitis in a patient with complete tlr3 deficiency: tlr3 is otherwise redundant in protective immunity herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency evolutionary dynamics of human toll-like receptors and their different contributions to host defense type 1 interferons and the virus-host relationship: a lesson in detente subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus interferon-stimulated genes: a complex web of host defenses protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response dna deamination mediates innate immunity to retroviral infection samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection a diverse range of gene products are effectors of the type i interferon antiviral response the transcription factor stat-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation crosspriming of cd8+ t cells stimulated by virus-induced type i interferon type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd8+ t cell responses through cd8{alpha}+ dendritic cells dendritic cells require a systemic type i interferon response to mature and induce cd4+ th1 immunity with poly ic as adjuvant direct type i ifn but not mda5/tlr3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo johansson-lindbom b. type i interferon signaling in dendritic cells stimulates the development of lymph-noderesident t follicular helper cells differential responses of immune cells to type i interferon contribute to host resistance to viral infection a type i interferon autocrine-paracrine loop is involved in toll-like receptorinduced interleukin-12p70 secretion by dendritic cells tlrdriven early glycolytic reprogramming via the kinases tbk1-ikkvarepsilon supports the anabolic demands of dendritic cell activation type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells prime natural killer cells by trans-presenting interleukin 15 cutting edge: the direct action of type i ifn on cd4 t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection direct stimulation of t cells by type i ifn enhances the cd8+ t cell response during cross-priming cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn cd8 t cells specific for lymphocytic choriomeningitis virus require type i ifn receptor for clonal expansion innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd8 t cells for clonal expansion and memory formation concomitant type i ifn receptor-triggering of t cells and of dc is required to promote maximal modified vaccinia virus ankara-induced t-cell expansion antiviral immune responses: triggers of or triggered by autoimmunity? is chronic plaque psoriasis triggered by microbiota in the skin? genetic dysbiosis: the role of microbial insults in chronic inflammatory diseases the tlr-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling accumulation of peripheral autoreactive b cells in the absence of functional human regulatory t cells cvid-associated taci mutations affect autoreactive b cell selection and activation human lupus autoantibody-dna complexes activate dcs through cooperation of cd32 and tlr9 ifn priming is necessary but not sufficient to turn on a migratory dendritic cell program in lupus monocytes plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide the pathology of influenza virus infections how do viral infections predispose patients to bacterial infections? predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness pathology of human influenza revisited immune dysfunction and bacterial coinfections following influenza type i interferon: friend or foe? a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin type i ifns mediate development of postinfluenza bacterial pneumonia in mice synergistic stimulation of type i interferons during influenza virus coinfection promotes streptococcus pneumoniae colonization in mice intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population innate and adaptive interferons suppress il-1alpha and il-1beta production by distinct pulmonary myeloid subsets during mycobacterium tuberculosis infection host-directed therapy of tuberculosis based on interleukin-1 and type i interferon crosstalk inflammation. 25-hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type i interferon dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny cytomegalovirus impairs antiviral cd8+ t cell immunity by recruiting inflammatory monocytes tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice influenza a virus exacerbates staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production role of tissue protection in lethal respiratory viral-bacterial coinfection disease tolerance as a defense strategy gainof-function human stat1 mutations impair il-17 immunity and underlie chronic mucocutaneous candidiasis type i interferon inhibits interleukin-1 production and inflammasome activation type i interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during candida infections ifnalpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis interferonbeta production via dectin-1-syk-irf5 signaling in dendritic cells is crucial for immunity to c. albicans aberrant type i interferon regulation in autoimmunity: opposite directions in ms and sle, shaped by evolution and body ecology janus-like effects of type i interferon in autoimmune diseases coregulation of cd8+ t cell exhaustion by multiple inhibitory receptors during chronic viral infection type i interferon suppresses de novo virus-specific cd4 th1 immunity during an established persistent viral infection timing and magnitude of type i interferon responses by distinct sensors impact cd8 t cell exhaustion and chronic viral infection distinct transcriptional profiles in ex vivo cd4+ and cd8+ t cells are established early in human immunodeficiency virus type 1 infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd8+ t cells chronic cd4+ t-cell activation and depletion in human immunodeficiency virus type 1 infection: type i interferon-mediated disruption of t-cell dynamics host genes associated with hiv-1 replication in lymphatic tissue global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques comparative transcriptomics of extreme phenotypes of human hiv-1 infection and siv infection in sooty mangabey and rhesus macaque hiv turns plasmacytoid dendritic cells (pdc) into trail-expressing killer pdc and down-regulates hiv coreceptors by toll-like receptor 7-induced ifn-alpha plasmacytoid dendritic cells express trail and induce cd4+ t-cell apoptosis in hiv-1 viremic patients sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv-1 glycerol monolaurate prevents mucosal siv transmission type i interferon responses in rhesus macaques prevent siv infection and slow disease progression plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice chronic hepatitis c: future treatment host-targeting agents in the treatment of hepatitis c: a beginning and an end? the interferons: 50 years after their discovery, there is much more to learn immune responses to hcv and other hepatitis viruses interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis c virus infection natural killer cells are polarized toward cytotoxicity in chronic hepatitis c in an interferon-alfa-dependent manner coexpression of pd-1, 2b4, cd160 and klrg1 on exhausted hcv-specific cd8+ t cells is linked to antigen recognition and t cell differentiation evidence for an antagonist form of the chemokine cxcl10 in patients chronically infected with hcv monocyte activation by interferon alpha is associated with failure to achieve a sustained virologic response after treatment for hepatitis c virus infection genetic variation in il28b and spontaneous clearance of hepatitis c virus genetic variation in il28b predicts hepatitis c treatment-induced viral clearance a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus the favorable ifnl3 genotype escapes mrna decay mediated by aurich elements and hepatitis c virus-induced micrornas distinct and overlapping genomic profiles and antiviral effects of interferon-lambda and -alpha on hcv-infected and noninfected hepatoma cells targeted induction of interferon-lambda in humanized chimeric mouse liver abrogates hepatotropic virus infection ifn-lambda receptor 1 expression is induced in chronic hepatitis c and correlates with the ifn-lambda3 genotype and with nonresponsiveness to ifn-alpha therapies how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins regulation of type i interferon responses stat2 and irf9: beyond isgf3. jakstat (2013) single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators major differences in the responses of primary human leukocyte subsets to ifn-beta critical role for stat4 activation by type 1 interferons in the interferongamma response to viral infection immunomodulatory functions of type i interferons high basal stat4 balanced by stat1 induction to control type 1 interferon effects in natural killer cells type 1 interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection changing partners at the dance: variations in stat concentrations for shaping cytokine function and immune responses to viral infections dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of ifn-alpha/beta on antigen cross-presentation mechanisms of type-i-and type-ii-interferon-mediated signalling type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors complex modulation of cell type-specific signaling in response to type i interferons alternate interferon signaling pathways ifn-beta induces serine phosphorylation of stat-1 in ewing's sarcoma cells and mediates apoptosis via induction of irf-1 and activation of caspase-7 usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine cxcl11 expression differential activity of type i interferon subtypes for dendritic cell differentiation evolutionary genetic dissection of human interferons the receptor of the type i interferon family interferons as biomarkers and effectors: lessons learned from animal models protection against progressive leishmaniasis by ifn-beta multifaceted activities of type i interferon are revealed by a receptor antagonist receptor density is key to the alpha2/beta interferon differential activities structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation the type i interferon receptor: structure, function, and evolution of a family business differential receptor subunit affinities of type i interferons govern differential signal activation structural linkage between ligand discrimination and receptor activation by type i interferons distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system type i interferons protect t cells against nk cell attack mediated by the activating receptor ncr1 lymphadenopathy in a novel mouse model of bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/ beta cytosolic rig-i-like helicases act as negative regulators of sterile inflammation in the cns expression of type i interferon by splenic macrophages suppresses adaptive immunity during sepsis myeloid type i interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions stat3 activation is responsible for il-6-dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat3-deficient mice generation of mice with a conditional stat1 null allele conditional stat1 ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection loss of stat1 from mouse mammary epithelium results in an increased neu-induced tumor burden inactivation of stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation meta-analysis of microarray data identifies gas6 expression as an independent predictor of poor survival in ovarian cancer axl and growth arrest-specific gene 6 are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme gas6 expression identifies high-risk adult aml patients: potential implications for therapy ovarian cancer progression is controlled by phenotypic changes in dendritic cells ifnalpha activates dormant haematopoietic stem cells in vivo timing of ifn-beta exposure during human dendritic cell maturation and naive th cell stimulation has contrasting effects on th1 subset generation: a role for ifn-beta-mediated regulation of il-12 family cytokines and il-18 in naive th cell differentiation combinatorial flexibility of cytokine function during human t helper cell differentiation regulating type 1 ifn effects in cd8 t cells during viral infections: changing stat4 and stat1 expression for function interferons at age 50: past, current and future impact on biomedicine inquiring into the differential action of interferons (ifns): an ifn-alpha2 mutant with enhanced affinity to ifnar1 is functionally similar to ifn-beta an interferon alpha2 mutant optimized by phage display for ifnar1 binding confers specifically enhanced antitumor activities the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities intra-lesional low-dose interferon alpha2a therapy for primary cutaneous marginal zone b-cell lymphoma intratumoral injection of interferon-alpha and systemic delivery of agonist anti-cd137 monoclonal antibodies synergize for immunotherapy delivery of interferon alpha using a novel cox2-controlled adenovirus for pancreatic cancer therapy the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity a trial of intrapleural adenoviral-mediated interferon-alpha2b gene transfer for malignant pleural mesothelioma tumortargeted interferon-alpha delivery by tie2-expressing monocytes inhibits tumor growth and metastasis targeting cytokines to inflammation sites livertargeting of interferon-alpha with tissue-specific domain antibodies antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation targeting ifn-alpha to b cell lymphoma by a tumor-specific antibody elicits potent antitumor activities targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses targeted delivery of interferon-alpha via fusion to anti-cd20 results in potent antitumor activity against b-cell lymphoma targeted delivery of interferon-alpha to hepatitis b virus-infected cells using t-cell receptor-like antibodies variations in the unstructured c-terminal tail of interferons contribute to differential receptor binding and biological activity safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: results of a phase i, placebo-controlled, double-blind, dose-escalation study safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase i, multicentre, double-blind randomised study mutation of the ifnar-1 receptor binding site of human ifn-alpha2 generates type i ifn competitive antagonists the studies performed in the laboratories are supported by funding from inserm, cnrs, aix-marseille university, the labex mabimprove, and the european community's seventh framework programme fp7/2007-2013 (grant agreement 223608 for gilles uzé, european research council starting grant agreement number 281225 for marc dalod including salary support to emeline pollet and thien-phong vu manh). we thank past and present laboratory members for their contribution to studies on dcs or ifns. we apologize for not quoting certain studies because of space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-293646-d4qcckh1 authors: meanwell, nicholas a.; serrano-wu, michael h.; snyder, lawrence b. title: chapter 22. non-hiv antiviral agents date: 2003-12-31 journal: annual reports in medicinal chemistry doi: 10.1016/s0065-7743(03)38023-6 sha: doc_id: 293646 cord_uid: d4qcckh1 publisher summary this chapter focuses on non-hiv antiviral agents. the development of antiviral agents to treat non-hiv infections is largely focused on therapies for the treatment of chronic hepatitis infections b and c. nucleoside analog continue to be the mainstay of hepatitis b virus (hbv) therapeutics. the first small molecule inhibitor of hepatitis c virus (hcv), the ns3 protease inhibitor biln-2061, entered phase 2 clinical trials, producing a striking reduction in viral load in treated individuals. the development of the hcv replicon system and its application to screening for antiviral agents provided tangible benefit with the disclosure of mechanistically and structurally diverse hcv inhibitors. adefovir dipivoxil has been approved in the united states and the european union for the treatment of hbv, providing a second small molecule antiviral to add to lamivudine (3tc) and the injectable protein ifnα as the only approved agents for treating hbv infection. the chapter also provides details of the inhibitors of hepatitis b and c virus, the inhibitors of simplex virus and human cytomegalovirus, the inhibitors of respiratory viruses and the inhibitors of west nile virus and papilloma virus. introduction -the development of antiviral agents to treat non-hiv infections is largely focussed on therapies for the treatment of the chronic hepatitis infections b and c (1). nucleoside analogues continue to be the mainstay of hbv therapeutics and clinical development of several continued during 2002. the last year has seen the first small molecule inhibitor of hcv, the ns3 protease inhibitor biln-2061, enter phase 2 (p2) clinical trials, producing a striking reduction in viral load in treated individuals. the development of the first hcv replicon system in 1998 and its application to screening for antiviral agents is beginning to provide tangible benefit with the disclosure of mechanistically and structurally diverse hcv inhibitors. there remains considerable interest in inhibitors of herpes simplex and human cytomegalovirus viruses, particularly non-nucleoside compounds. developments in the area of respiratory virus inhibitors have focussed more on respiratory syncytial virus with a description of the first antiviral active in animal models following oral administration. the west nile virus outbreak in the us, originally confined to the east coast, broadened considerably during the summer of 2002, claiming 230 lives. in the wake of the events of september ilth, 2001, smallpox was a prominent concern as a potential agent of bioterrorism. developments in each of these areas will be reviewed. inhibitors of hepatitis b virus (hbv) -adefovir dipivoxil ihepsera'") (1) was approved in the us for the treatment of hbv on september 20', 2002 and in the european union on march 1 lth, 2003, providing a second small molecule antiviral to add to lamivudine (3tc) and the injectable protein ifna as the only approved agents for treating hbv infection. adefovir is an effective inhibitor of 3tc-resistant hbv caused by the rtm2041 and rtll8om + rtm204v mutations in the reverse transcriptase (2) and resistance to adefovir has not been seen after 48 weeks of monotherapy (3, 4) . a clinical study with entecavir (z), currently undergoing p3 trials, compared a dose of 0.1-i mg/day of 2 to 3tc (100 mg/day) for 48 days in 181 patients previously unresponsive to 3tc treatment (5). compared to 3tc, treatment with 2 resulted in lower overall viral loads, lower alt levels and a higher proportion of patients with undetectable hbv dna levels. moreover, adverse events for 2 were less than those observed with 3tc. a separate p2 trial of 2. in 177 treatment-naive patients for 22 weeks at a dose of 0.5 mglday showed that reduction in viral dna levels was independent of baseline alt levels, with treatment resulting in a 4.7-4.8 loglo reduction in hbv dna (6). l-nucleosides represent a promising area of antiviral research (7). ldt (telbivudine, 3) is currently in p3 clinical trials designed to evaluate 1200 patients for safety and efficacy compared with standard treatment in hbeag+ and . recent p2b data was released from a trial involving 104 adults randomized to receive 3tc plus 2 or 3tc monotherapy once daily for 1 year. viral load reductions of greater than 6 logto were seen for all patients in the study arms containing 3 and no treatment-limiting or dose-related adverse events were reported. the mechanism of action of l-fmau (clevudine) (fi), currently in pi/p2 trials, is not firmly understood but recent molecular dynamics simulation experiments have suggested that the triphosphate derivative of 4 may act as a competitive inhibitor rather than as a substrate of hbv polymerase (9,lo). emtricitabine (coviraciltm) (5) is also currently in p3 clinical trials for the treatment of hbv. an nda seeking approval to market 5 for the treatment of hiv was filed in september 2002 (9). results from a pi/p2 cli%zal trial with ach-126443 (elvucitabine, 5) have been released (11-13). in 36 hbv treatment-naive patients receiving single daily doses of l-100 mg of 5, mean declines in plasma hbv dna of up to 2.5 loglo were observed after 14 days of treatment. furthermore, plasma levels in excess of the i&o values for wild type and ymdd mutants were achieved in the low dose arms. it is therefore anticipated that s will be efficacious against 3tc resistant infections in an ongoing p2 trial. this mutation, which arises in response to 3tc therapy, is found in the precore region and confers hbeag negativity. the major metabolite of 7 formed in rat or human serum is the mono-ester, which is a more potent hbv inhibitor, ec% = 70 nm, than 3tc. it is postulated that the arylthio moiety is responsible for the specificity towards hbv and lower cytotoxicity than pmea, the active component of adefovir dipivoxil. additional analogs in this structural class have been prepared with the phenylthio-and 3-methoxyphenylthio ethers showing the most promise whilst other aromatic thioethers exhibited higher cytotoxicity (14, 151) . non-nucleoside inhibitors of hbv are beginning to emerge that are anticipated to show reduced cross-resistance with nucleoside analogues. at130 (b) and its close analog at61 are active against wild type hbv and the rtll80m, rtm2041, and rtll80m+rtl204v mutants (ec50 = 2-5 pm) in hepg2-derived cells (17, 18) . it has been postulated that 8 interferes with the packaging of pregenomic viral rna resulting in inhibition of viral reverse transcription. pyridinedicarboxamide s represents the first report of a non-nucleoside inhibitor of hbv reverse transcriptase enantiomer of q is active in cell culture and appears to prevent proper formation of the viral nucleocapsrd. inhibitors of hepatitis c virus (hcv) -nearly 170 million individuals are infected with hepatitis c virus (hcv) worldwide and hcv infection is responsible for 8,000-10,000 deaths annually in the united states, a burden expected to increase significantly (22, 23) . a second pegylated interferon-a (ifn), roche's pegasys, was approved in 2003, both as mono therapy and in conjunction with ribavirin (11) (copegustm) (24). however, safety concerns with combination therapy remain, as the accumulation of 11 in erythrocytes can lead to hemolytic anemia. this has prompted a search for safer interferon co-therapies which include the active enantiomer of ij, levovirin, and the prodrug viramidine (12), which improves liver at the expense of erythrocyte exposure (2526). the development of safe, efficacious, and hcv-specific antiviral agents remains an important goal and the development of subgenomic hcv replicons has dramatically enhanced the potential to identify inhibitors (27, 28) . a significant advance towards establishing a correlation between replicon inhibition and clinical efficacy was recently accomplished with the disclosure of preliminary clinical data for biln-2061, a selective inhibitor of the ns3 serine protease of hcv that is structurally related to 13. this highly modified macrocylic tripeptide derivative is extremely potent in vitro, with k, values of 0.3 nm and 0.66 nm towards hcv-la and hcv-1 b ns3 proteases, respectively (29). these figures are similar to the potency observed in cell culture in the cognate hcv replicons, ec50 = 4 nm and 3 nm, respectively (29). antiviral efficacy was established in a study conducted with 10 patients with chronic hcv and significant liver fibrosis, where all patients treated with biln-2061 (200 mg p.o. b.i.d.) displayed a decrease in serum hcv rna levels of at least 2.0 log,, copies/ml after two days of treatment (30). four of these patients recorded a reduction in viral load of more than 3 orders of magnitude and viral titers returned to baseline following cessation of therapy, with no drug-related safety issues identified (31). the intensity of effort devoted towards the discovery of inhibitors of hcv ns3 has continued, with the focus largely on peptide-based molecules that are required to effectively complement the active site and proximal regions of the protease (32-34). the crystal structure of a macrocyclic inhibitor related to 13 bound to hcv protease has been disclosed and an acyclic tripeptidic inhibitor has been used to generate resistant subgenomic replicons in which mutations mapped to the protease (3836). amongst several strategies disclosed, the c-terminal carboxylate of peptide inhibitors may be replaced with an n-acyl sulfonamide moiety, as exemplified by 14 (37). novel approaches to constrain or mimic the peptidic backbone are represented by macrocycle l5, the tetrahydroindolizine b (ic 5~ = 0.12 pm), the imidazolone 17 and the bicyclic proline derivative 18 (k, = 0.042 pm, ks0 = 0.251 pm) (38-46). these inhibitors are constructed around either an a-keto amide or a boronic acid moiety, wellprecedented as serine protease inhibitor motifs that engage the catalytic serine residue in a covalent but reversible interaction. ph 1 i however, chemical reactivity is not a prerequisite for potent inhibition in a peptidic background since phenethylamide 19 and the azapeptide 20 inhibit hcv ns3 with ki values of 0.6 and 0.2 pm, respectively (47,48). the more active diastereomer of the ahydroxy amide p3 element explored in the context of 21 was found to possess the (i?)configuration, unanticipated and explained by the presence of an intramolecular hydrogen bond that orients the lipophilic moiety of this isomer into s3, as depicted (49). non-peptidic inhibitors of ns31ns4a protease are much less common but some progress has been made in this direction. the bicyclic lactam 22 is a mechanismbased inhibitor of hcv ns3 whilst additional examples of bis-benzimidazole derivatives that rely upon zr?' to consolidate the enzyme-inhibitor complex have been described (50,51). the ns5b rna polymerase is another structurally-characterized viral protein that is an attractive target for therapeutic intervention (52-55). inhibitors of hcv polymerase can be broadly divided into nucleoside and non-nucleoside derivatives. the nucleoside analog ribavirin (ii) has been suggested to interfere with both the initiation and elongation steps of hcv rna replication (52). several hcv ns5b inhibitors incorporate modified d-ribose elements and include the 2'-me derivative 23, ec50 = 0.25 pm, the 4'-azido analog 24, ec 50 = 1.2 pm, and the 2'-deoxy-2'-fluoro cytidine derivative 25, ego = 0.74 pm (56-59). several non-nucleoside inhibitors of hcv ns5b have been reported, including a series of phenylalanine derivatives of which compound 28, k, = 2.2 fm, is representative (60,61). this compound has been co-crystallized with ns56 and appears to bind to the inactive, open conformation of the polymerase almost 35 a from the active site (62). other scaffolds with which hcv polymerase inhibitors have been discovered include an amino thiophene, represented by 27, the enolic rhodanine 28 (i&o = 1 .o pm), and structural variations of previously disclosed benzimidazole derivatives 29 (go c 0.5 fm) claimed to be active in replicons (61,63-67). mechanistic studies with the benzo[l,2,4]thiadiazine polymerase inhibitor 30 suggest interference with the initiation step of viral rna synthesis, allowing for a potential synergy with existing elongation inhibitors (68). to hcv therapy include blocking the viral rna internal ribosomal entry site (ires), binding of the viral e2 envelope glycoprotein or attachement (69-72). the highly conserved ires has been targeted by oligonucleotides and artificial ribozymes, but little progress has been made in the development of small molecule inhibitors (70,71). p2 clinical evaluation of the antisense 20-mer oligonucleotide isis-14803 revealed a l-2 logto reduction in plasma hcv rna levels in approximately 30% of the patients after 4 weeks of treatment (73). another approach, which may prove complementary to virus-specific hcv therapy, is the induction of interferon production in host cells. small molecules that act via toll-like receptor activation have been identified as activators of an immune response (74). rna interference (rnai) is a rapidly emerging technology that has proven to be a powerful means of selectively controlling protein production in cell culture. inhibition of hcv replication in replicons has been accomplished using this procedure whilst the demonstration of selective targeting of the liver protein fas in vivo using rnai holds promise for the treatment of hcv (75-78). maribavir demonstrated in viva anti-hcmv activity in all of the dosage regimens tested (100, 200, and 400 mg tid, and 600 mg bid), with mean reductions in semen hcmv titers of 2.9 to 3.7 loglo pfulml (92). the hcmv serine protease has been perceived as an attractive antiviral target. recent crystal structure data demonstrated significant conformational flexibility in the s3 binding pocket of protein complexed with two peptidomimetic inhibtors (94). a series of frans-lactams, represented by 35 and 36, 1% = 0.54 and 0.34 pm, respectively, are derived from the same structural platform as the hcv ns3 inhibitor 22 but inhibit the hcmv serine protease with excellent selectivity (9597). mechanistic studies are consistent with acylation of the active site serlz of hcmv protease in a reversible and time-dependent manner. chap. 22 non-hiv antiviral agents meanwell et al. 219 all but one of the currently licensed drugs available to treat hsv act by inhibiting the viral dna polymerase, providing a suitable backdrop for the emergence of resistant virus and a rationale for identifying inhibitors of other viral proteins (98,99). two groups have reported novel thiazole-containing inhibitors of the hsv helicase-primase. bay 57-1293 (37) is a leading pre-clinical candidate that is more potent (ec50 = 12 nm) than any anti-herpetic currently used to treat hsv infections (100,101). in a murine lethal challenge model of hsv-1 and hsv-2, 37 was protective with an edso value of 0.5 mglkg, which compares with the much higher doses of 22 and 16 mglkg for hsv-1 and hsv-2, respectively, required for acyclovir to show efficacy. additional patent applications that extend this promising chemotype have appeared (102,103). a second series of hsv helicase-primase inhibitors, of which bils-179 bs (38) is representative, has been disclosed (104). bils 179 bs inhibits viral growth with an ec50 of 27 nm, displays an excellent therapeutic index of >2000 and reduces cutaneous hsv-1 and genital hsv-2 disease in a murine model when treatment is initiated 3 hours post-infection. interestingly, when treatment was initiated 65 hours after infection, 38 reduced hsv-1 pathology by 75% and hsv-2 mortality by 75% (200 mg/kglday) when compared to acyclovir or untreated animals (104). a series of 4-oxo-dihydroquinolone derivatives that are potent and broad spectrum non-nucleoside inhibitors of dna polymerases of the herpesvirus family, including hcmv, hsv-1, hhv-8 and vzv, have been the subject of a number of recent disclosures (105). these compounds exhibit no significant inhibitory activity towards human ci-, y-, or 6-polymerases. pnu-183792 (39) is an effective antiviral in cell culture, potently inhibiting hcmv (ec 50 = 0.69 pm), vzv (ec% = 0.37 pm) and hsv (ec50 = 0.58 pm), that is active towards ganciclovir-and cidofovir-resistant hcmv and acyclovir-resistant hsv (106). excellent oral bioavailability and a protective effect in a murine cmv animal model were also reported. a series of related analogs have been reported in the recent patent literature (107,108). pyrazolopyridine derivatives have been reported to possess activity against hsv-1 in vero 76 cells with 40 having an ec50 of 0.12 fm (109-i 12). structurally related irnidazopyridine derivatives are active against hsv types l-8 with an e&o of 0.14 pm (113). cmv-423 (41) is a potent inhibitor of hcmv, ec50 = 4-7 nm, that appears to act at a step in viral replrcation preceding dna polymerization (114). overview -respiratory viruses continue to be a significant source of mortality and morbidity. the annual death rates due to influenza in the us are estimated to have doubled over the last 20 years, attributed to an aging of the population (115, 116) . this study also revealed a greater appreciation of the contribution of respiratory syncytial virus to mortality. the recently discovered human metapneumovirus (hmpv) was identified as a significant cause of wheezing in infants (117,118). the influenza inhibitor oseltamivir (tamiflutm) was approved for the treatment of influenza in adults and children and for prevention in adults and adolescents by the european community in june 2002, consolidating its position as the market leading neuraminidase (na) inhibitor. however, development of the third neuraminidase inhibitor peramivir was terminated by biocryst in june after disappointing p3 results in which the orally bioavailable compound failed to meet the key efficacy endpoint of reducing the time to onset of relief of symptoms. in march, 2003 an outbreak of severe acute respiratory syndrome (sars) emerged in southeast asia for which the culprit was quickly identified as a new coronavirus distinct from any previously identified human coronavirus. of the 1918 influenza continues and chimeric viruses containing the 1918 na or ml ion channel showed susceptibility to oseltamivir and amantadine or rimantadine, respectively, both in vitro an in viva. (119,120). the role of na inhibitors in pandemic influenza has been reviewed and the identification of structurally novel na inhibitors has continued (121). substitution of the primary amine moiety of oseltamivir with a vinyl group afforded the potent influenza b na inhibitor 42, ki = 45 nm. (122) additional sar studies around zanamivir have focussed on the c-7 hydroxyl where replacement by f or methylation affords the potent na inhibitors 43 and 44, icso values of 0.8 and 6.1 nm, respectively, which compares favorably with an i&o of 5-10 nm for the prototype (123,124). polymeric analogues of zanamivir linked to a polyglutamate backbone via the 7 position showed enhanced influenza inhibitory activity compared to monovalent analogues (125 the o-methyl analogue of zanamivir is claimed to protect mice against a lethal influenza infection following oral administration of the prodrug g whilst the bicyclic ether 48 is the first zanamivir derivative to demonstrate oral efficacy in the mouse model (127, 128) . the optimization of a screening lead into potent, cyclopentanebased inhibitors of neuraminidase using a combination of structure-based design and combinatorial chemistry has been described in detail (129). mechanism-based inhibitors of the hrv 3c cysteine protease have been probed using a combination of structure-based design principles and parallel synthesis methods in an effort to find less peptidic inhibitors (132). the chroman a emerged as an inhibitor of hrv-14 replication in cell culture, ec50 = 160 nm; however, the serotype coverage of this compound was poor with much reduced potency against other subtypes, an observation rationalized in the context of structural data (132). the hrv 2a cysteine protease releases itself from the viral p2 polyprotein, cleaves the p2 in both a cis and tram fashion to release the 2b and 2c proteins and also proteolyzes the host cap binding complex in order to compromise host cell transcription. a series of n-phenylated pyrazole derivatives have been claimed as inhibitors of this essential enzyme with 52 an effective antiviral agent in cell culture, ec50 = 1.8 pm, cc50 = 388 i.im (133). the hrv rna-dependent rna polymerase from hrv-16, a potential drug discovery target, has been cloned, expressed and purified from e. co/i and shown to be enzymatically active (134). inhibitors of resdiratotv svncvtial virus (rsv) -the role of rsv in morbidity and mortality continues to be a focus of research designed to provide a more accurate perspective of this virus as a mediator of significant disease burden, (115, 116, 135, 136) . the enhanced awareness of rsv has stimulated interest in this virus as a therapeutic target with the recent emergence of new structural classes of inhibitor. however, clinical proof-of-principle remains to be established for rsv antivirals. viropharma has suspended development of vp-14637, an rsv fusion inhibitor under examination as a topically administered agent. the benzimidazole bms-433771 (53) has been described as the first rsv inhibitor to demonstrate antiviral activity in animal models following oral administration (137, 138) . mechanism of action studies indicate that g is an inhibitor of the fusion of viral and host cell membranes and targets the rsv f (fusion) protein (137). analogues of 53 form the basis of proprietary claims (139, 140) whilst trimeris has disclosed a series of benzimidazole-based inhibitors of rsv fusion (141). representative of this series is 54, which is active in cell culture, ego = 10 ng/ml. -coronaviruses are enveloped, single-strand, positive-sense rna viruses most commonly associated with respiratory infections in man (142). two coronaviruses are the underlying cause of approximately 30% of upper respiratory tract infections, usually mild to moderate in severity and generally recognized as the common cold, although in the immunocompromised population coronavirus infections can lead to pneumonia. however, in march, 2003 an outbreak of severe acute respiratory syndrome (sars) emerged in southeast asia that very quickly spread to 27 countries, carried largely by air travelers (143-145). more than 6500 sars infections were documented worldwide within the following 2 months that were associated with substantial morbidity, including fever, non-productive cough, malaise, chills, headache and dyspnea, and significant mortality, with over 460 deaths reported (146). whilst sars appears to have originated in southern china in november, 2002, reports of major outbreaks in hong kong and toronto, canada in march 2003 brought the disease to world prominence (143-145). the attributes of modern technology, communication and international teamwork led to the rapid isolation and sequencing of the virus causing sars, identified as a novel coronavirus with little similarity to previously known human coronaviruses (147-153). final confirmation came with the demonstration that infection of monkeys with sars produced a syndrome identical to that seen in man and that virus could subsequently recovered (154). these experiments were rapidly followed by the development of diagnostic assays based on a real-time quantitative pcr method and a taqman protocol (148, 155). transmission of sars appears to be via person-person contact with infected droplets rather than airborne and, possibly, a fecal-oral route. inhibitors of coronaviruses are largely unknown although treatment with ribavirin (11) and oseltamivir have been examined in an empirical fashion (143, 156). scope of the mosquito-borne west nile virus outbreak in the us broadened considerably in the summer of 2003 extending to almost all states and causing over 2,500 cases of encephalitis and 230 deaths (157,158) . the virus presents several conventional proteins as targets suitable for intervention including the ns3 protein, which expresses serine protease, helicase and nucleoside triphosphatase activities, and the ns5 rna-dependent rna polymerase (159). moreover, screening using a cell culture assay provides a panoply of less well understood targets. however, few potent and selective wnv inhibitors have been described to date. ribavirin (ii) inhibits wnv rna production in oligodendroglial cells, a human neural cell line, with an ec50 of -60 fm (160). the nucleoside analogue hmc-ho4 (55) interferes with the helicase activity of wnv ns3 with an i& of 30 pm and is a similarly potent antiviral in cell culture (161). inhibitors of vaccinia virus (smallpox) -smallpox has aroused considerable concern with discussion prominently focussed on its potential as an agent of bioterrorism (162) (163) (164) (165) (166) (167) . many inhibitors of vaccinia virus replication interfere with host cell pathways and include 11 (inosine monophosphate dehydrogenase), the cyclopentenyl nucleoside analogue neplanocin a (s-adenosylhomocysteine hydrolase) and pyrazofurin (omp decarboxylase) (168, 169) . the @dine and 5-f cytidine analogues of neplanocin a are equally potent inhibitors of several orthopox viruses, including smallpox (170) ori-1001, which complements the el mrna start codon of hpv and has demonstrated efficacy in two animal models, is currently undergoing pi12 clinical evaluation. the fused tetracyclic amide .g is claimed to inhibit the e2-dependent binding of papilloma virus el to dna, a cntrcal step in viral replication, with an i&j of ~500 nm (175) . a vaccine derived from an hpv-16 virus-like particle, completely prevented the incidence of cervical neoplasias in hpv-16naite young women (176) . this impressive result provides strong encouragement to the development of a vaccine with a spectrum that, in addition, encompasses the broader range of hpvs (18, 31, 33 and 35) responsible for the majority of cervical cancers (177) . a combined, multivalent vaccine based on self-assembling virus-like particles and directed towards hpv-16 and hpv-18, recognized as medi-517, is entering p2 trials (178) . ----2'deoxynucleotides ";$~rn$~~ychem., 9, 899 (2002 145. nln (7~7) september section iv-cancer and infectious diseases plattner key: cord-323963-whv88ggl authors: fan, xiaofeng; xu, yanjuan; di bisceglie, adrian m. title: efficient amplification and cloning of near full-length hepatitis c virus genome from clinical samples date: 2006-08-11 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2006.06.039 sha: doc_id: 323963 cord_uid: whv88ggl abstract long rt-pcr (lrp) amplification of rna templates is sometimes difficult compared to long pcr of dna templates. among rna templates, hepatitis c virus (hcv) represents an excellent example to challenge the potential of lrp technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. the only source for viral genome amplification is clinical samples in which hcv is usually present at low titers. we have created a comprehensive optimization protocol that allows robust amplification of a 9.1kb fragment of hcv, followed by efficient cloning into a novel vector. detailed analyses indicate the lack of potential lrp-mediated recombination and the preservation of viral diversity. thus, our lrp protocol could be applied for the amplification of other difficult rna templates and may facilitate rna virus research such as linked viral mutations and reverse genetics. polymerase chain reaction (pcr) is an indispensable technique in biomedical research. with known primer sequences, it can easily amplify a dna target less than 3 kb but it has diminished power when the target is larger than 3 kb. in 1994, barnes et al. [1] first hypothesized that the inability to amplify large dna fragments was due to the misincorporation of nucleotides by most thermostable dna polymerases, which resulted in premature termination of pcr. based on this hypothesis, mixed polymerases, one of which has 3 0 -5 0 exonuclease ''proofreading'' activity to correct the misincorporation, have successfully amplified dna targets up to 42 kb [2] . however, there has been limited success in applying this concept to the amplification of large rna genomes that require the reverse transcription (rt) step prior to pcr amplification. compared to the amplification of dna targets, it is reasonable to hypothesize that the rt step is of crucial importance during long rt-pcr (lrp) performance when taking into account the following characteristics. first, in most situations, the solution buffers are not compatible between rt and pcr. only part of the rt reaction can be used for subsequent pcr and thus reduces the sensitivity dramatically. second, most rt enzymes have an inhibitory role for thermostable dna polymerases [3] . third, rt is conducted at temperatures ranging from 37°c to 50°c at which the rna template may retain its secondary structure that makes rt stop prematurely. such situations are even more challenging when trying to amplify full-length hepatitis c virus (hcv) genome, a positive sense single-strand rna virus in the family of flavirividae. there is extensive secondary structure along the whole hcv genome [4] [5] [6] . furthermore, hcv cannot be cultured in vitro. the only source of rna template for lrp is clinical samples in which hcv has a low titer. in the present study, we have investigated each step for the lrp procedure and developed a robust samples. the lrp optimization was directly conducted with serum samples collected in 2001 from two patients infected with hcv genotype 1a, referred to as jlr3037 and rj, respectively. a large volume of serum stored at à70°c was available from these two patients, which allowed repeated and detailed optimization of our lrp protocol. after the optimization, additional serum samples were used for the estimation of sensitivity, robustness, and potential recombination (see below). hcv rna levels were quantitated by bdna assay (bayer versant hcv 3.0) immediately prior to the start of this study. rna extraction. total rna was extracted from serum by using either qiaamp viral rna mini kit (qiagen, valencia, ca) or trizol ls reagent (invitrogen, carlsbad, ca) according to the instructions provided. with qiaamp viral rna mini kit, rna was extracted from 280 ll of serum and finally eluted into 60 ll of tris buffer containing 20 u/ml of rnasein ribonuclease inhibitor (promega, madison, wi). in the extraction with trizol ls reagent, 250 ll of serum was applied and the rna pellet was finally dissolved in 20 ll of nuclease-free water containing 20 u/ml of rnasein ribonuclease inhibitor (promega). additionally either glycogen (invitrogen) or transfer rna (trna) (sigma, st. louis, mo) was used for facilitating rna precipitation. for both methods, vigorous vortexing was avoided to prevent shearing of long rna templates [7] . reverse transcription. since rt is a critical step for successful lrp, we optimized this step as follows. first, we tested multiple rt enzymes alone or in combination, including amv (promega), m-mlv (promega), expand reverse transcriptase (roche applied science, indianapolis, in), transcriptor reverse transcriptase (roche applied science), superscript ii (invitrogen), superscript iii (invitrogen), and rtth dna polymerase (applied biosystems, foster city, ca) that shows reverse transcriptase activity in the presence of mncl 2 at elevated temperatures. in some experiments, we mixed a rt enzyme with pfu dna polymerase (stratagene), a similar strategy as used in long pcr, to improve full-length cdna synthesis [8] . second, previous studies showed certain chemicals might improve full-length cdna synthesis in both quantity and quality. in this study, we tried different additives at various concentrations, including dmso (5-10%) (sigma), gc-melt (0.5 m) (bd biosciences), dtt (5-10 mm), trehalose (0.6 m) (sigma) [9] , and betaine (2 m) (sigma) [9] . third, we designed a series of hcvspecific rt primers located at the 3 0 end of ns5b (table 1 ). these primers were tested for efficient priming at different concentrations. fourth, besides direct application of rt reaction in subsequent pcr, we also tried to purify rt reaction with or without rnase h digestion [11] prior to pcr, by using qiaquick pcr purification kit or qiaquick nucleotide removal kit (qiagen) or dynabeads kilobasebind-er kit (dynal) in which rt primers were biotinylated at their 5 0 ends. finally, we also investigated the role of 7-deaza-2 0 -deoxyguanosine (sigma) in the rt reaction that may improve the elongation in gc-rich domains [10] . pcr. all pcr experiments were done with dna thermal cycler 480 (perkin-elmer-cetus, norwalk, ct). the nested pcr strategy and a touchdown protocol were generally applied. at the beginning, we tested several thermostable dna polymerases for long pcr, such as expand long template pcr system (roche applied science) and elongase enzyme mix (invitrogen). we eventually focused on rtth dna polymerase, xl (applied biosystems, foster city, ca), and most of the optimization experiments were done with this enzyme. the strategy to optimize long pcr was basically similar to what we described for rt step. multiple additives were first tested, including dmso, betaine [12, 13] , and tetramethylammonium (tma) oxalate [14] . next, a series of primers were tested for their efficiency with long pcr (table 1) . meantime, since one of the mixed polymerases has 3 0 -5 0 exonuclease ''proofreading'' activity that may degrade primers, we tested phosphorothioate primers to see if the pcr amplification is improved [15] [16] [17] . to allow hot-start pcr that may diminish non-specific priming, we adopted two measures, the use of loop incorporated primers [18] and an oligonucleotide, trnc-21, which specially inhibits dna polymerase isolated from thermus thermophilus (tth pol) at low temperature [19, 20] . finally, with the dna thermal cycler 480, we empirically fixed denaturing temperature at 94°c for 30 s, elongation temperature at 72°c or 68°c for 9 min, and annealing step for 30 s. however, the annealing temperature was adjusted depending on the t m values of the primers (table 1) . molecular cloning of hcv envelope domain. hcv displays a typical quasispecies nature shared by most rna viruses. to understand if our lrp protocol conserves viral diversity, we compared the hcv quasispecies profiles derived from regular rt-pcr and lrp products in two patient samples, liv19 and liv23. the viral heterogeneity has been detailed in these two patients in our previous study based on a 1.38 kb amplicon spanning the most hypervariable region 1 (hvr1) of hcv genome [21] . in brief, serum rna was reverse transcribed with 200 u m-mlv reverse transcriptase (promega), followed by nested pcr with taq dna polymerase (applied biosystems). the pcr product was gel purified by using qiaex ii gel extraction kit (qiagen) and ligated into the ptopo-ta cloning vector (invitrogen). escherichia coli top-10 cells (invitrogen) were used for transformation and recovery of recombinant clones. approximately 15 clones for each sample were sequenced with abi prism dye terminator cycle sequencing ready reaction kit using an abi 373a automated sequencer (applied biosystems). molecular cloning of long rt-pcr product. for cloning the lrp product, we first tried several commercial cloning kits, including topo xl pcr cloning kit (invitrogen), copylight cloning kit, and clone smart blunt cloning kit (lucigen corporation, middleton, wi), without success. next, we estimated cloning efficiency of gateway technology with clonase ii (invitrogen). finally, we returned to a conventional cloning strategy in which the lrp product was digested with two restriction enzymes paci and fsei, followed by ligation into a special plasmid named pclone. the ligation product was electroporated into stbl4 cells or dh10b cells (invitrogen). the pclone vector was constructed by replacing paci-bamhi fragment of padtrack-cmv [22] with a $120 bp fragment that was assembled to include rare restriction enzymes not found in the hcv genome based on an analysis of 13 fulllength hcv genotype 1a isolates. positive recombinant clones were identified by either the digestion or partial sequencing of both ends of the insert. estimation of pcr-mediated recombination. pcr may induce a homologous recombination [23] [24] [25] . the rate of recombination is dependent on the protocol used. after the optimization of our long rt-pcr, we estimated the potential recombination related to this protocol. in doing so, sera from samples liv19 and liv23 were mixed in equal amounts, followed by the same procedures of rna extraction, rt, long pcr, and cloning. approximately 20 positive recombinant clones were sequenced at 8 domains located within 5 0 utr, core, e1, e2, ns2, ns3, ns5a, and ns5b regions, respectively. recombination would be indicated for a given clone if conflicting clusterings were noted in phylogenetic trees constructed with 1 of the 8 domains that we sequenced. genetic analysis. all sequences were aligned with clustal w (version 1.74) [26] . sequence editing and multiple sequence comparisons were performed with matched programs in the wisconsin gcg package (oxford molecular group, inc., version 10.0). the mean genetic distance (d), the number of synonymous substitutions per synonymous site (ds), and the number of nonsynonymous substitutions per nonsynonymous site (dn) were calculated with the kimura 2-parameter method (all sites) [27] in the molecular evolutionary genetics analysis software package (mega, version 3.0) [28] . all phylogenetic trees were constructed using the neighbor-joining method [29] with a bootstrap test implanted in mega. the genetic complexity at both nucleotide and amino acid levels was evaluated, respectively, for samples liv19 and liv23 by calculating normalized entropy (s n ): s n = s/lnn, where n is the total number of clones; s = à p i(pi ln pi), where pi is the frequency of each clone in the viral quasispecies population. statistical tests. student's t-test was used to analyze differences between mean values for genetic parameters when data were normally distributed. non-parametric tests were used to evaluate samples for which normal distributions were not present. the synthesis of full-length hcv cdna is a prerequisite for successful lrp. several groups have performed nested pcr of hcv 5 0 utr after rt step, assuming a positive result as the indicator of full-length hcv cdna synthesis. however, we found that multiple domains, located within 5 0 utr, core, e2, ns3, ns5a, and ns5b, respectively, could be successfully amplified after rt in which rt primers were omitted. this indicated the existence of extensive self-priming during rt presumably induced by hcv rna secondary structure or oligonucleotides in extracted rna template. using the dna thermal cycler 480, the whole procedure for lrp takes at least 2 days. we therefore amplified multiple small fragments (5 0 utr, hvr1, ns3, ns5a, and ns5b) ( table 1 ) by using the first round lrp product as the template. after the first round lrp, the effect of self-priming is reduced. the negative amplification of these small fragments indicated the absolute absence of full-length hcv cdna and thus the second round lrp is not necessary. to test all lrp conditions and parameters alone or in combination, the optimization procedure consists of hundreds of protocols. our approach that monitors the full-length cdna synthesis based on first round pcr product, although not perfect, still improves experimental progress significantly. two rna extraction procedures, based on either qiaamp viral rna mini kit (qiagen) or trizol ls reagent (invitrogen), gave similar lrp results. however, in the latter procedure, the addition of trna or glycogen during rna precipitation, even at low concentration, resulted in the failure of subsequent pcr, suggesting that these carriers had a detrimental role on rtth xl activity. the optimization of rt can be summarized as follows. 1. superscript iii outperformed all other reverse transcriptases such as amv, m-mlv, expand reverse transcriptase, transcriptor reverse transcriptase, superscript ii, and rtth dna polymerase. robust lrp results were obtained when running rt with 200 u of superscript iii at 50°c for 75 min, followed by inactivation at 70°c for 15 min. however, using a mixture of superscript iii (200 u) and amv (2.5 u) gave lrp results that were much more reproducible, with an increased yield. lrp was not successful using a mixture of rt enzyme and pfu dna polymerase, perhaps because the latter had low activity (<30%) in a nonoptimized buffer system under rt temperature (50°c) (stratagene, personal communication). 2. all additives (except dtt) that we tested in the rt reaction had an adverse role for lrp. although these additives have been reported to improve full-length cdna synthesis, they may be not compatible with superscript iii or rtth xl dna polymerase. 3. the selection of rt primers is critical for successful lrp. the most satisfactory and reproducible results were obtained only when using qr2 as the rt primer (table 1) . interestingly, lrp did not work when replac-ing qr2 with qr264 or qr274, suggesting an appropriate t m value of rt primers was required. however, lrp failed when modifying other rt primers into a similar t m value as qr2. in contrast, reproducible amplifications were obtained with other serum samples in which qr2 had one or two nucleotide substitutions. these observations suggest that efficient priming for full-length hcv cdna synthesis is domain-dependent. additionally, unlike previous reports [30] , our lrp is acceptable with qr2 in broad range of concentrations from 0.0625 lm to 1 lm. 4. there was no obvious advantage but a reduced sensitivity when purifying rt reaction by using qiagen spin columns or dynabeads. similarly, the use of 7-deaza-2 0 -deoxyguanosine in rt reaction or rnase h digestion was not advantageous. the pcr was most successful with rtth dna polymerase, xl. however, it should be noted that we did not perform detailed optimization with the another two systems, expand long template pcr system and elongase enzyme mix. the former system is the only one that uses a buffer containing mgcl 2 , which is a common component of rt buffers. this makes it potentially promising to reconcile the two buffer systems, but information about additional buffer components is not available and therefore optimization cannot be performed. there was no improvement and sometimes even an adverse effect of other measures including the addition of additives (dmso, betaine, and tma oxalate) and the use of phosphorothioate or loop incorporated primers. however, trnc-21, an oligonucleoide inhibitor to rtth dna polymerase, was required for reproducible amplification. the minimum concentration of trnc-21 is 0.4 lm. we also found no interference to lrp when increasing the concentration up to 1.2 lm. unlike rt, the requirement for primers in long pcr is less stringent as long as the t m values of primers are approximately 68°c. in the optimized protocol, rna was extracted from 280 ll of serum by using qiaamp viral rna mini kit (qiagen). 10.6 ll of rna template was mixed with 9.4 ll rt matrix consisting of 1· superscript iii buffer, 10 mm dtt, 1 lm qr2 (reverse primer), 2 mm dntps (invitrogen), 20 u of rnasein ribonuclease inhibitor, 200 u of superscript iii, and 5 u amv (promega). the reaction was performed by incubation at 50°c for 75 min, followed by heating at 70°c for 15 min. five microliters of rt reaction was applied for the first round of pcr that contained 1.25 mm mg(oac) 2 , 1· xl pcr buffer, 2 mm dntps (invitrogen), 0.4 lm trnc-21, 0.4 lm of each primer (wf33 and qr2), and 2 u rtth xl dna polymerase. cycle parameters were programmed as 94°c for 1 min fol-lowed by the first 10 cycles of 94°c for 30 s and 65°c for 9 min and final 20 cycles in which the annealing/elongation temperature was reduced to 60°c for 9 min with a 3 s autoextension at each cycle. the reaction was ended with 10-min incubation at 72°c. two microliters of the first round of pcr product was used for the second round amplification with primers wf5 and wr55. cycle parameters were the same as the first round pcr except the annealing/elongation temperature was changed to 72°c for the first 10 cycles and 68°c for the last 20 cycles, respectively. using this protocol, a 9095 bp fragment was reproducibly obtained for samples jlr3037 and rj (fig. 1) . with the optimized lrp protocol, we tested 24 hcv genotype 1a samples (serum or plasma) with various rna levels, ranging from 10 3 to 10 6 iu/ml. the predicted dna fragment of 9.1 kb was successfully amplified in all samples, even in those with low hcv rna levels. a repre-sentative result is shown in fig. 2 . these results indicate that our lrp protocol is robust and sensitive. for those samples with hcv rna levels less than 10 3 iu/ml, lrp amplification was improved by extracting total rna from 560 ll of serum instead of 280 ll of serum (data not shown). in our optimized lrp protocol, we used four primers: qr2, wf33, wf5, and wr55. sequence alignment showed that these primer domains are relatively conservative through most of hcv genotype 1a isolates, especially for 5 0 end primers wf33 and wf5. we also found that one or two nucleotide substitutions within primers qr2 and wr55 did not abrogate the lrp amplification but resulted in a diminished amount of the amplicon as determined by agarose gel electrophoresis. we encountered unexpected difficulty in cloning the lrp product that is approximately 9.1 kb in length. we repeatedly tried four commercial cloning kits: topo xl pcr cloning kit, copyright cloning kit, clone smart blunt cloning kit, and gateway technology with clonase ii. a common problem with these cloning kits was the high background of clones without the insert, which is generally assumed to be as the result of either the toxicity of foreign genes or the instability of recombinant clones. since psmart vectors from copyright and clone smart blunt cloning kits contain multiple terminators that eliminate transcription both into and out of the insert dna and therefore reduce potential toxicity of the insert, the instability of recombinant clones may be responsible for the high background with false positive clones. we therefore constructed pclone vector that contains pbr origin and restriction sites not found in hcv genome. with this conventional strategy, the lrp product was successfully cloned with dh10b e. coli cells but not stbl4 cells. positive rate for recombinant clones was about 30% which is much higher than previous reports [31, 32] . a 2 ml miniculture yielded approximately 5 lg of recombinant clones, which is a suitable amount for the performance of analysis such as sequencing. to see if our lrp protocol preserves viral diversity, we evaluated hcv quasispecies based on hvr1 domain derived either from a short amplicon (1.38 kb) or from the lrp product. we sequenced the hvr1 domain from 16 and 15 positive recombinant clones for samples liv19 and liv23, respectively. there are generally comparable levels for both genetic complexity and genetic diversity except for a significantly higher genetic complexity at the amino acid level for sample liv23 (0.829 versus 0.430, p < 0.05) ( table 2) . sample liv19 had a low genetic diversity and similar hvr1 quasispecies lineages were obtained either by short fragment amplification or by lrp ( fig. 3a) . however, when comparing hvr1 quasispecies profiles, respectively, derived from the 1.38 kb and 9.1 kb amplicons in sample liv23, only one hvr1 lineage was shared by both amplicons (fig. 3b) . lrp was performed using a mixture of equal amounts of serum from samples liv19 and liv23. twenty clones derived from the lrp product were sequenced at eight domains including 5 0 utr, core, e1, e2, ns2, ns3, ns5a, and ns5b. phylogenetic analysis showed that eight clones belonged to sample liv19 and 12 clones were from liv23. neighbor-joining trees constructed with each domain displayed consistent clustering for each clone, suggesting the absence of potential recombination induced by lrp. a representative tree constructed with the hcv e1 domain is shown in fig. 4 . although we did not sequence 20 clones in full-length, the possibility for recombination is very small, if not excluded, since the eight domains that we sequenced are evenly scattered along the entire 9.1 kb amplicon. long rt-pcr has been successfully used to amplify large or near full-length domains of rna viruses, including human coronavirus [30] , poliovirus [34] , borna disease virus [35] , porcine reproductive and respiratory syndrome virus [36] , coxsackievirus [37] , and hepatitis e virus [38] . it has also been applied to the amplification of cellular rna derived from such genes as the neurofibromatosis 1 (nf1) and polycystic kidney disease 1 (pkd1) genes [39, 40] . in these studies, a common feature was the availability of good rna templates in both quantity and quality. in contrast, hcv cannot be easily cultured in vitro although there are recent reports of the establishment of hcv cell culture by using a special hcv genotype 2a strain jfh-1 [41] [42] [43] . clinical samples from patients infected with hcv have a relatively low titer of viral rna level. in addition, hcv holds a strong structure along with the whole genome [4] [5] [6] . these features may explain the limited success of lrp with hcv. while there have been occasional reports regarding the amplification of near full-length hcv genome [44, 45] , reproducible results were only obtained with the amplification of less than 5 kb fragments in hcv [7, 33, 46, 47] . in contrast, the protocol we have described here has considerable robustness. besides the two serum samples that we used for optimizing our protocols, we successfully amplified a near full-length hcv genome from an additional 24 patient samples infected with hcv genotype 1a. we identified several critical factors for efficient amplification of a near full-length hcv genome. first, the rt step was conducted by using mixed enzymes, superscript iii and amv. superscript iii is a mutant form of superscript ii, which makes it fully active at temperature as high as 55°c. potential rna secondary structure could be melted at this temperature. however, incubation at 55°c resulted in decreased sensitivity perhaps due to the partial degradation of the rna templates. in the optimized protocol, we used 50°c for the rt reaction. it has been reported that amv especially favors the reverse transcription of genes with gc-rich domains or strong secondary structure due to its stability at higher temperatures. it is not known how these two enzymes work together, but similar cooperativity has been observed for mixed dna polymerases in long pcr [1] . in any case, we demonstrated that mixed rt enzymes improve fulllength hcv cdna synthesis in both quality and quantity. second, not all primers can effectively prime the synthesis of full-length hcv cdna. in our experiments, only one primer, qr2, met this requirement, indicating the fulllength hcv cdna synthesis is considerably dependent on the appropriate priming site. to some extent, this observation is consistent with a previous report in which differential priming of rna templates resulted in obvious differences in both accuracy and reproducibility of rt-pcr [48] . third, the use of trnc-21 in pcr steps is recommended. inclusion of trnc-21 resulted in automated hot-start pcr amplification. although there are several techniques available for the initiation of ''hot-start'' pcr, such as manual control, the use of wax, and the addition of antibodies to thermal stable dna ploymerases, none of them is as efficient and convenient as trnc-21. finally, the primers for pcr procedures should have appropriate t m values dependent on the annealing/elongation temperatures. our last optimization step for successful lrp was to raise the annealing/elongation temperature to 72°c in the second round of pcr, around 5°c above the primer t m values. the large difference between annealing/elongation temperatures and primer t m values resulted in non-specific amplification while a low annealing/elongation temperature less than 60°c always abrogated the amplification. there are two salient features for our lrp procedure: the lack of detectable recombination and the preservation of viral diversity, as estimated with samples liv19 and liv23. recombination is generally explained by template switching during pcr, particularly when the synthesis of complementary strands is stopped prematurely. the lack of detectable recombination in our lrp protocol may have contributed to the reduced cycle numbers (60 cycles versus regular 70 cycles) and vent dna polymerase that is included within rtth dna polymerase, xl, and has 3 0 -5 0 exonuclease proofreading activity. the hvr1 is located at the 5 0 end of hcv e2 domain and is the most variable region along the entire hcv genome. by comparing genetic parameters for hvr1 quasispecies profiles, our lrp protocol preserves viral heterogeneity, as also reported with a 5 kb hcv amplicon [33] . furthermore, similar hvr1 quasispecies lineages were obtained with sample liv19 while sample liv23 displayed much different hvr1 quasispecies lineages derived from either the 1.38 kb or the 9.1 kb amplicon. by using clones as direct pcr templates, we failed to amplify hvr1 domain by screening 40 clones that had no correct insert confirmed with enzyme digestion after miniculture (data not shown). this excludes the possibility for the loss of potential hvr1 quasispecies lineages during the culture due to the instability of recombinant clones. thus, these results again emphasize the bias of hvr1 quasispecies amplification when using different primer pairs as we previously reported [49] . still, defective interfering particles (dip) are another factor to be taken into account. the generation of dip, natural viral mutants with large deletions in the genome, seems to be a general phenomenon for all viruses, including hcv [50, 51] . quasispecies profiles contributed by dip could be lost in our protocol since we gel purified only the 9.1 kb fragment prior to cloning. taken together, the quantitation of viral diversity, if present at a high level within a given sample, is largely underestimated and/or biased by current protocols for pcr amplification and cloning. the technology described here should be applicable to other hcv genotypes as well as other rna viruses such as gb virus c, hiv, and dengue virus. with the amplification and efficient cloning of a near full-length viral genome, it is now possible to study linked mutations at genomewide scale. linked mutation is a common strategy exploited by viruses to counter their loss of the fitness resulting from point mutations at immune and/or drug targets. the identification of common patterns of linked mutation is helpful for the improvement of combinational antiviral strategies. in addition, our lrp protocol preserves hcv diversity and has no detectable recombination induced by pcr. these characteristics make it possible to isolate dominant, subdominant, and minor viral variants within a complex virus population, which facilitates the approach of reverse genetics. an initial step in reverse genetics is to construct vectors containing full-length viral genomes, usually assembled by overlapped pcr products that represent viral consensus sequences. however, the consensus sequence is artificial in concept and is not necessarily the dominant viral variant. as a result, replication from infectious clones with consensus viral genome may not occur. this may partially explain why the infectious hcv clone of h77 consensus did not replicate in cell culture while the one with jhf1 did [41] [42] [43] . in contrast to the existence of multiple hvr1 quasispecies lineages in the patient h77 [52] , jhf1 was derived from a patient with fulminant hepatitis. the immunocomprised status in this patient resulted in an extremely homogeneous viral population by cloning analysis of hvr1 domain [53] . in such a situation, consensus viral sequence may be equal to authentic dominant viral variant that makes an ''infectious'' clone infectious. fig. 4 . a representative neighbor-joining (nj) tree constructed based on hcv e1 domain of 20 clones derived from 9.1 kb lrp product, which was amplified using mixed serum from samples liv19 and liv23. as expected, all clones are clustered into two groups, liv19 and liv23. there is no contradictory clustering for each clone in trees constructed with other seven domains, indicating the lack of lrp-mediated recombination. pcr amplification of up to 35-kb dna with high fidelity and high yield from lambda bacteriophage templates effective amplification of long targets from cloned inserts and human genomic dna reverse transcriptase can inhibit pcr and stimulate primer-dimer formation thermodynamic and phylogenetic prediction of rna secondary structures in the coding region of hepatitis c virus detailed mapping of rna secondary structures in core and ns5b-encoding region sequences of hepatitis c virus by rnase cleavage and novel bioinformatic prediction methods detection of genome-scale ordered rna structure (gors) in genomes of positive-stranded rna viruses: implications for virus evolution and host persistence a refined long rt-pcr technique to amplify complete viral rna genome sequences from clinical samples: application to a novel hepatitis c virus variant of genotype 6 full-length cdna synthesis for longdistance rt-pcr of large mrna transcripts a highly efficient method for long-chain cdna synthesis using trehalose and betaine structure-independent dna amplification by pcr using 7-deaza-2 0 -deoxyguanosine rnase h and its effects on pcr betaine improves the pcr amplification of gc-rich dna sequences optimizing multiplex and la-pcr with betaine new specificity and yield enhancer of polymerase chain reactions phosphorothioate primers improve the amplification of dna sequences by dna polymerases with proofreading activity amplimers with 3 0 -terminal phosphorothioate linkages resist degradation by vent polymerase and reduce taq polymerase mispriming single base extension (sbe) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays controlled hot start and improved specificity in carrying out pcr utilizing touch-up and loop incorporated primers (tulips) oligonucleotide inhibitors of taq dna polymerase facilitate detection of low copy number targets by pcr inhibition of multiple thermostable dna polymerases by a heterodimeric aptamer quasispecies heterogeneity within the e1/e2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis c virus infection a simplified system for generating recombinant adenoviruses stimulation and suppression of pcr-mediated recombination factors affecting pcr-mediated recombination minimizing dna recombination during long rt-pcr clustal: a package for performing multiple sequence alignment on a microcomputer a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment the neighbor-joining method: a new method for reconstructing phylogenetic trees effective amplification of 20-kb dna by reverse transcription pcr transmission of hepatitis c by intrahepatic inoculation with transcribed rna transcripts from a single full-length cdna clone of hepatitis c virus are infectious when directly transfected into the liver of a chimpanzee cloning and characterization of a complete open reading frame of the hepatitis c virus genome in only two cdna fragments rapid rt-pcr amplification of full-length poliovirus genomes allows rapid discrimination between wild-type and recombinant vaccine-derived polioviruses amplification of a full-length borna disease virus (bdv) cdna from total rna of cells persistently infected with bdv analysis of orf 1 in european porcine reproductive and respiratory syndrome virus by long rt-pcr and restriction fragment length polymorphism (rflp) analysis the complete consensus sequence of coxsackievirus b6 and generation of infectious clones by long rt-pcr reevaluation of a north india isolate of hepatitis e virus based on the full-length genomic sequence obtained following long rt-pcr long rt-pcr of the entire 8.5-kb nf1 open reading frame and mutation detection on agarose gels yenchitsomanus, long rt-pcr amplification of the entire coding sequence of the polycystic kidney disease 1 (pkd1) gene robust hepatitis c virus infection in vitro production of infectious hepatitis c virus in tissue culture from a cloned viral genome complete replication of hepatitis c virus in cell culture amplification and fusion of long fragments of hepatitis c virus genome long pcr and its application to hepatitis viruses: amplification of hepatitis a, hepatitis b, and hepatitis c virus genomes accurate representation of the hepatitis c virus quasispecies in 5.2-kilobase amplicons a strategy for obtaining near full-length hcv cdna clones (assemblicons) by assembly pcr differential priming of rna templates during cdna synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase pcr differential amplification of hypervariable region 1 of hepatitis c virus by partially mismatched primers defective interfering particles and virus evolution visualization of hepatitis c virons and putative defective interfering particles isolated from low-density lipoproteins the outcome of acute hepatitis c predicted by the evolution of the viral quasispecies sequence analysis of hepatitis c virus isolated from a fulminant hepatitis patient we thank yunfeng feng (washington university in st. louis) and john e. tavis (saint louis university) for helpful discussions. we also thank patrick sheehy (national university of ireland cork, ireland) and rodolpho m. albano (universidade do estado do rio de janeiro, brazil) for clarifying issues regarding their work. key: cord-280878-1kt51viz authors: to, janet; surya, wahyu; torres, jaume title: targeting the channel activity of viroporins date: 2016-01-07 journal: adv protein chem struct biol doi: 10.1016/bs.apcsb.2015.12.003 sha: doc_id: 280878 cord_uid: 1kt51viz since the discovery that certain small viral membrane proteins, collectively termed as viroporins, can permeabilize host cellular membranes and also behave as ion channels, attempts have been made to link this feature to specific biological roles. in parallel, most viroporins identified so far are virulence factors, and interest has focused toward the discovery of channel inhibitors that would have a therapeutic effect, or be used as research tools to understand the biological roles of viroporin ion channel activity. however, this paradigm is being shifted by the difficulties inherent to small viral membrane proteins, and by the realization that protein–protein interactions and other diverse roles in the virus life cycle may represent an equal, if not, more important target. therefore, although targeting the channel activity of viroporins can probably be therapeutically useful in some cases, the focus may shift to their other functions in following years. small-molecule inhibitors have been mostly developed against the influenza a m2 (iav m2 or am2). this is not surprising since am2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. for other viroporins, these studies are still mostly in their infancy, and together with those for am2, are the subject of the present review. the field of viroporins was born about 20 years ago from the realization that viruses inflict injuries in the membranes of cells during infection, which results in increased membrane permeability (carrasco, 1995) . this early observation heralded the current interest in viroporins, along with the therapeutic opportunities afforded by the modulation of their channel activity using small molecules (fischer, wang, schindler, & chen, 2012; scott & griffin, 2015) , and the deeper understanding of the roles of viroporin ion channel activity on viral replication and pathogenesis (nieto-torres, verdia-baguena, castano-rodriguez, aguilella, & enjuanes, 2015) . this interest will likely increase with the discovery of new viroporins along with their ever-growing roles in the virus life cycle. indeed, the number of medically important viroporins known is expected to rise as new human viruses continuously emerge from animal hosts. it is also possible that new viroporins may have novel features and structural motifs which will contribute in updating their classifications in terms of topology and number of transmembrane α-helical domains (nieva, madan, & carrasco, 2012) . in vertebrates alone, the number of viruses was estimated to be around one million (morse, 1993) . more recent estimates quantify the viral diversity in mammals, which are the reservoir hosts of the majority of emerging zoonoses, as 320,000 (anthony et al., 2013) . this strongly contrasts with the few thousand viruses identified to date, which means that more than 99% of viruses-and their viroporins, if any-are still unknown. most viroporins have been identified as virulence factors that lead to viral attenuation when deleted. this attenuation is attributed only in part to their channel activity, but nevertheless small-molecule channel inhibitors have been sought after. the vast majority of these channel inhibitors have been developed against the influenza a virus m2 (iav m2 or am2) protein (reviewed recently in du, cross, & zhou, 2012; gu, liu, & wei, 2013) , which is the first viroporin discovered. this is not surprising since am2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. for other viroporins, these studies are still mostly in their infancy, although a highresolution structure of the hepatitis c virus (hcv) p7 protein has been recently described (ouyang et al., 2013) that may be useful for the rational design of p7 channel inhibitors in the future. however, attempts to rationally design small-molecule inhibitors against viroporins will encounter several challenges. one is the potential for sequence variability in the viroporin. indeed, it has been argued that since the number of genes susceptible to deleterious mutations increase with genome size, mutation rates are higher for small genomes (drake, 1969) . a related problem is that viral proteins, when compared to prokaryotic and eukaryotic proteins, have a higher degree of structural flexibility that may represent adaptation mechanisms to increase resistance to random mutations, especially in the case of rna viruses, which mutate and evolve faster than dna viruses. a less dense packing, and a concomitant smaller number of network interactions, translates not only to a smaller difference in energy between folded and unfolded states, but an also smaller percentual contribution of each mutation to stability (tokuriki, oldfield, uversky, berezovsky, & tawfik, 2009 ). additionally, this enhanced flexibility may have an impact on the ability of a single protein to perform a variety of tasks, consistent with the economical use of resources in viruses. adding to this intrinsic flexibility, another problem in structure-based drug discovery concerning viroporins is the strong dependence of the protein structure on environment. indeed, solution nmr can be applied to study small-membrane proteins in membrane-mimicking environments that allow rapid reorientation, but these artificial systems may also alter the intrinsic properties of the proteins (cross, dong, sharma, busath, & zhou, 2012; cross, sharma, yi, & zhou, 2011) . last, some viroporins seem to form a protein-lipid complex rather than a purely proteinaceous pore, as shown for the vp4 protein in the triatoma virus (sanchez-eugenia, goikolea, gil-carton, sanchez-magraner, & guerin, 2015) , and similar observations have also been reported for coronavirus envelope proteins (verdia-baguena et al., 2012) . in the quest to discover small molecules targeting viroporins, amantadine (amt) can be considered to be the first viroporin channel inhibitor, and one of the first antivirals licensed for use in humans. although it was known that this adamantane was active against iav (davies et al., 1964; wingfield, pollack, & grunert, 1969) , the mechanism of inhibition was described only in 1992 (duff & ashley, 1992; pinto, holsinger, & lamb, 1992) . the target itself, am2, was not known until 1985 (hay, wolstenholme, skehel, & smith, 1985) . even today, amt, together with its methylated derivative rimantadine (rim) which was licensed in the 1980s, are the only licensed antiviral drugs that target viroporins. this contrasts with the availability of hundreds of compounds that exert their effects with subnanomolar affinities on nonviral ion channels. although this phenomenon could be explained in principle by a lack of structural data for viroporins, this is not the case for am2, where precise structural data has been obtained, even for some of its amt-resistant mutants wang, qiu, soto, & degrado, 2011) . unfortunately, an effective alternative to adamantanes has not yet been licensed. with our current knowledge, the am2 channel provides yet another paradigm valid for other viroporins. it has a very simple structure compared to ion channels from higher organisms (such as k + and ca 2+ channels), but the mechanism of proton conduction and drugs binding and inhibition are not yet fully understood. indeed, the structural and functional properties of the am2 protein vary widely in changing experimental conditions, such as peptide length, drug binding, lipid composition, lipid thickness, or ph (acharya et al., 2010; cady, wang, & hong, 2011; cross et al., 2012; kovacs, denny, song, quine, & cross, 2000; thomaston et al., 2013; zhou & cross, 2013 ). an important factor in this sensitivity is the direct contact of its tm domains with the lipid molecules, in contrast to higher organism ion channels (eg, k + channels) (cuello, jogini, cortes, & perozo, 2010) , where pore-forming α-helices are surrounded and protected by additional transmembrane helices. however, while the am2 channel exists in a variety of conformational states in the apo-form, inhibitor-binding significantly reduces the conformational flexibility of the channel. the structural dynamics of the channel represents another drawback in the use of high-resolution structures for rationally designed drug discovery. amantadine is just one of the four antivirals against iav available: two neuraminidase inhibitors (oseltamivir and zanamivir) and two am2 channel blockers (amt and rim) (vanderlinden & naesens, 2014) . the seasonal flu caused by iav is associated with high medical burden (molinari et al., 2007) and sudden pandemics with high mortality rates (hay, gregory, douglas, & yi, 2001; neumann, noda, & kawaoka, 2009 ). currently, most circulating strains are amt resistant (bright, shay, shu, cox, & klimov, 2006; bright et al., 2005; deyde et al., 2007; hayden & de jong, 2011) and therefore the use of amt and rim has been discontinued for the human population (fiore et al., 2011) . the am2 channel is a homotetramer (sakaguchi, tu, pinto, & lamb, 1997) , where each monomer has 97 amino acids containing one α-helical tm domain (residues 25-46). the extramembrane c-terminal domain (residues 47-97) is exposed to the cytoplasmic side (lamb, zebedee, & richardson, 1985) and contains an amphipathic helix (residues 51-59). four of these amphipathic helices stabilize the tetrameric channel, forming a base on the membrane surface almost perpendicular to the tm bundle. the end of this c-terminal tail is disordered and interacts with the viral matrix protein 1 (m1). the m2 protein performs several critical roles for virus replication (pinto & lamb, 2006) . for instance, m2-mediated acidification of the interior of the virus is required for the uncoating of genetic material inside the cell (pinto et al., 1992) . m2 is also required to regulate the intralumenal ph of the golgi apparatus preventing the premature conformational change of hemagglutinin (takeuchi & lamb, 1994) . influenza b, the closest relative of the influenza a virus, accounts for about 50% of all influenza disease in recent years (according to the us centers for disease control and prevention website, www.cdc.gov) and has a similarly essential ph-activated proton channel m2 viroporin, the bm2 (hatta, goto, & kawaoka, 2004; pinto & lamb, 2006) . although am2 and bm2 share almost no sequence identity, both have a single tm domain and form homotetramers wang, pielak, mcclintock, & chou, 2009 ). in comparison, bm2 is completely insensitive to amt or rim (mould et al., 2003; pinto & lamb, 2006) and no bm2 inhibitors have been identified yet. the location of the binding site for the adamantanes amt and rim in am2 was initially controversial. a crystallographic structure suggested a pore-binding model in the amino-terminal part of the tm (22-46), ie, the p-binding site (stouffer et al., 2008) (fig. 1a -c). in the same and in the following year, a solution nmr-based structure of a longer construct of am2 (18-60) (pielak, schnell, & chou, 2009; schnell & chou, 2008) proposed a surface-binding site (the s-binding site) for rim, on the carboxy-terminal, lipid-facing surface of the helices (fig. 1d-g) , suggesting an alternative allosteric mechanism of inhibition. in this allosteric binding site, the adamantane group interacted favourably with the hydrophobic side chains of leu-40, leu-43, and ile-42, whereas the positively charged ammonium group interacted with the polar patch formed by figure 1 p-and s-binding sites for amt and rim to iav m2. (a-c) am2 tm domain crystallized in detergent, with the most critical residues identified by site-directed mutagenesis lining the am2 pore (a); omit map showing electron density in the amt binding region (b); structure of amantadine (nitrogen in cyan (light gray in the print version) and carbon in white) inside the binding site showing the surface associated with residues val-27 (red (light gray in the print version) surface), ala-30 (green (dark gray in the print version)), ser-31 (blue (light gray in the print version)), and gly-34 (orange (light gray in the print version)) (c); (d and e), an ensemble of 15 low-energy structures derived from nmr restraints, with tm α-helices (residues 25-46) and amphipathic helices (residues 51-59) superimposed separately (d); ribbon representation, showing the drug rim (colored in red (light gray in the print version)) (e); (f and g), surface representation of the rim-binding pocket, showing the asp-44, the indole amine of trp-41, and arg-45, which form the polar patch, as well as the hydrophobic wall composed of solution nmr structures of the (am2-bm2)tm channel in the absence and presence of rim; ensembles of 15 low-energy structures of drugbound chimera channels. rim is highlighted in red (light gray in the print version) (h); ribbon representation of drug-free (am2-bm2)tm tetramer (left), and overlay of its am2 and bm2 regions (green (dark gray in the print version)) with the corresponding regions (yellow (light gray in the print version)) of the am2 (pdb code: 2rlf) and bm2 (pdb code: 2kix) structures (right) (i); overlay of the drug-free (white) and the drugbound (cyan (light gray in the print version)) chimera structures, showing substantial differences in helical packing (j). adapted by permission from macmillan publishers ltd.: stouffer, a. l., acharya, r., salom, d., levine, a. s., di costanzo, l., soto, c. s., et al. (2008) . asp-44 and arg-45 from the adjacent subunit (fig. 1g) . a dilemmatic feature of allosteric sites is that viral mutations at these sites are easily accommodated by viruses without dramatic loss of fitness. in hcv, for example, protease inhibitor monotherapy can rapidly select resistant viral populations within a few days (schmidt et al., 2014) . however, clear support for the p-binding site was obtained from solidstate nmr in lipid bilayers (pdb id: 2kqt) (cady et al., 2010) ; the inhibitor was found in a hydrophilic pocket formed by ala-30, ser-31, and gly-34, ie, occluding the am2 channel. in this case, hydrophobic interactions were detected between the adamantane group and val-27 side chains, whereas the ammonium group was hydrogen-bonded to pore-facing residues and water molecules. the latter work revealed that the s-binding site was in fact just a low-affinity site, and only observed at high concentrations of the drug in the membrane. importantly, amt was found to undergo significant motion in the n-terminal lumen, suggesting that its structure can be improved for a better fit to the am2 channel. this pore-location (p-binding) was also further validated by chou et al. by solution nmr (pielak, oxenoid, & chou, 2011) using an m2 chimeric sample, where the n-terminal part of the m2 tm domain corresponded to that of the am2, whereas the c-terminal part was from the amt-insensitive bm2 ( fig. 1h -j). in that model, methyl groups of val-27 and ala-30 from four subunits form a hydrophobic pocket around the adamantane, with the amino group in polar contact with the backbone oxygen of ala-30. this ended the controversy, with the acceptance of the initially proposed p-binding site. there are 12 structures of the wild-type and drug-resistant mutant m2 channels available in the protein data bank solved by different techniques and conditions (see summary in gu et al., 2013) . among these, structure 2rlf solved in micelle (schnell & chou, 2008) and structure 2l0j in the bilayer environment contain, in addition to the tm domain, short c-terminal intracellular amphipathic helices. in 2rlf, the c-terminal helices were connected to the transmembrane helices via a short loop (residue 47-51) and were exposed to the solution. however, in 2l0j, the c-terminal helices were connected to the tm helices through a rigid turn (residue 47) and were positioned on the bilayer surface (sharma et al., 2010) , and this conformation was stabilized by extensive hydrophobic interactions among these helices, and polar residues asp-44 and arg-45 were buried by hydrophobic residues of the intracellular amphipathic helices, making the s-binding site more hydrophobic than that in micelles. therefore, the s-binding site was absent in 2l0j (sharma et al., 2010) . molecular dynamics simulations of the m2 proton channel with inhibitors binding at different sites have shown that the p-binding site is more stable for drug binding, but a higher energy barrier needs to be overcome for binding to occur (gu et al., 2011) . the s-binding site was less stable for drug binding but it was nearly barrier less and was easily accessed. therefore, the p-binding site represents the thermodynamic binding site where the drug molecule binds slowly and stably and dissociates even more slowly, whereas the s-binding site is a kinetic binding site where the drug molecule binds readily but less stably and dissociates easily. over the last four decades, systematic studies of amantadine analogues and library screening have elucidated structure-activity relationships (sars) and helped to identify potent wild-type (wt) am2 channel blockers (de clercq, 2006; lagoja & de clercq, 2008) . several other molecules were not based on adamantanes. for example, the spirene guanidine analogue, 2-[3-azaspiro(5,5)undecanol]-2-imidazoline (bl-1743) ( fig. 2a ) was discovered during a high-throughput screen based on the ability of inhibitors to reverse the toxicity associated with m2 channels expressed in the yeast saccharomyces cerevisiae (kurtz et al., 1995) . based on this nonadamantane, a sar study using a combination of viral plaque assays and two-electrode voltage clamp (tevc) (wang, cady, et al., 2009 ) generated 3-azaspiro[5,5]undecane hydrochloride (fig. 2b) , which lacks the (wang, cady, et al., 2009). imidazoline group of bl-1743. this compound showed an ic 50 of 0.92 ae 0.11 μm against am2, ie, more than an order of magnitude more potent than amantadine (ic 50 ¼ 16 μm) and more than 45-fold increase in potency relative to the parental bl-1743. compared to amt, this spiro-piperidine induced a more homogeneous conformation of the am2 peptide, reducing dynamic disorder near the water-filled central cavity of the helical bundle, suggesting a more extensive binding to the am2 channel, thus leading to stronger inhibitory potency. however, this compound was only effective against the wild-type am2. this is crucial because of the current prevalence of amt-resistant variants of am2, eg, l26f, v27a, and s31n, which comprise more than 99% of the reported resistance mutants (furuse, suzuki, & oshitani, 2009; suzuki et al., 2003) . of these three mutations, v27a is the only one known to originate from drug selection pressure (furuse et al., 2009) , and this mutant is the only one among the three that is completely resistant to amt and rim ). on the other hand, s31n is found in more than 98% of the mutated m2 channels in the iav subtype h3n2 viruses (bright et al., 2005) , and causes a 10-20 times decrease in the ic 50 , from low micromolar to 200 μm (amt) and more than 2 mm (rim) . the first non-adamantane inhibitor of the v27a mutant was a polycyclic pyrrolidine, a dual inhibitor of wt (ic 50 of 3.4 μm) and v27a (ic 50 of 0.29 μm) (rey-carrizo et al., 2013). later, a compound with triple inhibitor efficacy was reported by the same authors, active against the wt, v27a, and l26f mutants, with ic 50 of 18, 0.7, and 9 μm, respectively (rey-carrizo et al., 2014) . the inhibitory activity of the compounds was tested on am2 channels expressed in xenopus laevis oocytes using the two-electrode voltage clamp (tevc) technique, but most of the compounds were cytotoxic (more than amt and rim), as tested in cell-based antiviral plaque reduction assays. therefore, despite the encouraging results obtained in vitro, the toxic properties of these compounds still hinder their therapeutic potential. the spiro-piperidine inspired the generation of another compound ( fig. 3c ) that could inhibit not just the wt am2 (ic 50 ¼ 18 μm) but also mutants l26f (ic 50 ¼ 6 μm) and v27a (ic 50 ¼ 0.3 μm) (wang, ma, fiorin, et al., 2011) . direct interaction of this compound with the v27a mutant was assessed by solid-state nmr, and inhibition was further demonstrated in plaque reduction assays. this compound, however, was inactive against the s31n mutant. such observation was rationalized by the fact that the s31n mutant is more dynamic and hydrated in the pore than the corresponding wt protein , as shown by nmr studies. this lead to a disruption in the size and polarity of the pore in precisely the region that accommodates the adamantane group, thereby hampering the discovery of inhibitors of s31n. this led to attempts to increase the polarity or the dimensions of the adamantane core in order to capture more interactions with the backbone of the channel. a variety of scaffolds have been used to substitute for the hydrophobic adamantane group (hu et al., 2010; wang, ma, balannik, et al., 2011) . these compounds showed excellent activity against the wt am2, and some were highly active against v27a and l26f (wang, ma, fiorin, et al., 2011) . however, none was better than amt against s31n. attempts to introduce additional groups to the amine to enhance affinity for both s31n and wt via additional electrostatic interactions led to the discovery of benzyl-substituted amantadine derivatives ( fig. 3d ) as dual wt and s31n inhibitors with ic 50 of 35 and 59 μm for s31n and wt, respectively. the degrado group later discovered small-molecule drugs, eg, m2wj332, that locked the dynamic s31n mutant into a well-defined conformation, enabling high-resolution structure determination by solution nmr that showed the drug bound in the homotetrameric channel, threaded between the side chains of asn-31. these compounds were formed by conjugation of a-ch2-heteroaryl group to the amine of amt ( fig. 3e and f, ic 50 of 16 μm) and inhibited s31n with potencies greater than amt against am2 . the charged ammonium binds as a hydrate to one of three sites aligned along the central cavity that might stabilize hydroniumlike species formed as protons diffuse through the outer channel to the proton-shuttling residue his-37 near the cytoplasmic end of the channel. ; (e and f) compounds with ic 50 of $16 μm ; (g) compound found in wu et al. (2014) . based on structural and molecular dynamics data, the degrado group realized that inhibitors that are active against both wt and s31n channels bind in opposite orientations: the aromatic headgroup faces toward the c-terminus in wt, and toward the n-terminus in s31n. investigation of substitutions led to the identification of halide-substituted thiophene compounds. the most potent of which, n-[(5-bromothiophen-2-yl) methyl]adamantan-1-amine (fig. 3g ), inhibited both wt and s31n mutant with comparable affinities as amt against wt (wu et al., 2014) . it would be desirable to develop the same paradigm found in iav am2 and their inhibitors for other viroporins, so that their channel inhibitors can be validated. for example, many amantadine-resistant influenza viruses can be selected in cell culture (grambas, bennett, & hay, 1992; hay et al., 1985) , and some of these are also found in infected patients undergoing treatment with amantadine (shiraishi et al., 2003) . in turn, engineered viruses harboring these pore-lining mutations, while competent to replicate in mouse (abed, goyette, & boivin, 2005) , give rise to attenuated viruses that tend to revert to the wt in the absence of drug pressure suzuki et al., 2003) . another very important viroporin is p7, found in the hepatitis c virus (hcv), a member of the flaviviridae family that has six genotypes (gts). hcv is a positive-strand rna virus that causes severe liver disease and is the major cause of hepatocellular carcinomas in the developed world. hcv has chronically infected 170 million people worldwide and is responsible for more than 300,000 yearly deaths, while no prophylactic or therapeutic vaccine is available. hcv infection can be cured with drugs targeting viral enzymes and proteins, as well as host cellular proteins. one host-targeted antiviral agent (htas) is the type i ifn-α (hoofnagle et al., 1986) , which exerts antiviral activities against both rna and dna viruses through ifn-stimulated gene (isg) products. pegylated ifn has been used in combination with ribavirin (rbv), a synthetic guanosine analogue (herrmann, lee, marinos, modi, & zeuzem, 2003) , but this antiviral therapy can only last up to 1 year, associated with side effects, and is not always effective (manns, wedemeyer, & cornberg, 2006; pawlotsky, chevaliez, & mchutchison, 2007) . other drugs target hcv proteins with high potency, such as those against the hcv ns3/4a protease, the ns5a protein or the ns5b rna-dependent rna polymerase (rdrp) (sulkowski et al., 2014) [see pawlotsky, 2014; schmidt et al., 2014 , for recent reviews]. however, drug resistance creates a burden for patients, worsened by the rapid turnover of hcv replication, which produces up to 10 12 virions daily (neumann et al., 1998) . this is compounded by the error-prone activity of the hcv rna polymerase, which leads to the high genetic diversity of hcv and the formation of "quasi-species." in this complicated context, the clinical niche of p7 inhibitors is unclear. the viroporin hcv p7 is encoded within a single polyprotein precursor (simmonds, 2013) processed by host and viral proteases (moradpour & penin, 2013) . it separates the structural proteins (core and envelope glycoproteins e1 and e2) from the nonstructural proteins ns2, ns3, ns4a, ns4b, ns5a, and ns5b (lindenbach & rice, 2005) . p7 results from cleavage of e2-p7-ns2 and e2-p7 by a signal peptidase at the er (lin, lindenbach, prágai, mccourt, & rice, 1994; mizushima et al., 1994) . the p7 protein is 63 residues long with two tm domains, found mainly at the er membrane. although p7 is not necessary for rna replication (lohmann et al., 1999) , it is essential for productive hcv propagation in vivo . however, the precise role of the p7 in virus production was only determined when it was possible to replicate a genotype 2a isolate from a japanese patient suffering from fulminant hepatitis (designated jfh-1) (kato et al., 2003; wakita et al., 2005) . this isolate efficiently replicated in human hepatoma cell lines and produced infectious virus particles. this was used to demonstrate that p7 is essential for virus particle assembly and release (jones, murray, eastman, tassello, & rice, 2007; steinmann et al., 2007) . the bovine viral diarrhea virus (bvdv) (harada, tautz, & thiel, 2000) and the hepacivirus gb virus b (gbv-b) (takikawa et al., 2006) , hcv's closest relatives, also have a p7 protein crucial for virus replication. the channel activity of p7 was examined more than 10 years ago in experiments involving artificial lipid bilayers. it was suggested that p7 has preference for cations vs anions, with permeability ratios in the range 7-11 (griffin et al., 2003; montserret et al., 2010; premkumar, wilson, ewart, & gage, 2004; pavlovic et al., 2003) and more recently in xenopus oocytes (ouyang et al., 2013) . this selectivity is rather low compared to other channels, eg, for cystic fibrosis transmembrane conductance regulator (cftr) cl à channels, selectivity for anions over cations is 10-30 (anderson et al., 1991; bear et al., 1992) . for potassium channels, which conduct k + ions near the diffusion limit, the selectivity for k + over na + is about 10,000-1 (doyle et al., 1998) , and in aquaporins water to ion flux ratio is about 10 9 (pohl, 2004) . however, the biological importance of p7 cation channel activity may be relative, in the light of its recently found role to permeabilize membranes to protons, and acting to prevent acidification of intracellular vesicles (wozniak et al., 2010) . this proton channel activity was found to be crucial for the production of infectious viruses, and was observed in intracellular vesicles harboring p7. recently, this proton channel activity has been directly confirmed in vitro using purified nontagged p7 protein in a liposome-based assay (gan, surya, vararattanavech, & torres, 2014) , where the lipid composition used was the mixture paesc (pa/pe/ ps/pc 5:2:2:1 w/w) (gervais et al., 2011) . several initial papers also reported the inhibition of channel activity by some compounds. for example, his-tagged p7 from genotype 1b was inhibited by 1 μm amantadine (griffin et al., 2003) , whereas synthetic p7 from genotype 1a (strain h77) was inhibited by 100 μm hexamethyleneamiloride (hma, fig. 4a ), although this peptide was largely unpurified (premkumar et al., 2004) . another synthetic p7, also unpurified, was inhibited by 50-100 μm of the long-alkyl-chain imino sugar derivatives nn-dgj, nn-dnj (fig. 4b ) and n-7-oxanonyl-6-deoxy-dgj (pavlovic et al., 2003) . however, the channel activity of purified synthetic p7 hcv-j genotype 1b in artificial lipid bilayers was inhibited by just 50% in the presence of 200 μm hma, whereas amt did not show any effect even at 1 mm concentration (montserret et al., 2010) . in fact, the efficacy of amt in hcv culture systems is low (steinmann et al., 2007) , and no clinical benefit of amt-based combination therapy was found in comparison to standard of care (peg-ifnα plus ribavirin) (castelain et al., 2007; mihm et al., 2006) . some of these compounds have been studied in infected cells, where they reduced virus production up to 10-fold in a genotype-dependent manner (foster et al., 2014; gottwein et al., 2011; steinmann et al., 2007) , reducing secretion of hcv particles by inhibition of the acidification of viruscontaining compartments (wozniak et al., 2010) , without impairment of the infectivity of intracellular virions (foster et al., 2011; griffin et al., 2008) . to discover more p7 channel inhibitors, a liposome-based dye (carboxyfluorescein, cf) release assay has been proposed that involves the addition of p7 protein to preloaded liposomes (stgelais et al., 2007) . the method has been claimed to be sensitive to inhibitors such as amt and rim, and proposed as a high-throughput functional assay to test drug sensitivity of p7 mutants, or to discover new p7 channel activity inhibitors (foster et al., 2011; gervais et al., 2011) . these dye release assays have been previously performed with tagged p7 protein, either with flag (li et al., 2012; stgelais et al., 2009) or flu-antigen (foster et al., 2011) . however, this method has been put into question since just the c-terminal half of p7, p7(27-63) was as efficient as full-length p7 in releasing cf, while a properly reconstituted α-helical sample of p7 was not able to elicit cf release (gan et al., 2014) . nevertheless, p7 inhibitors may be able to inhibit both the proton channel activity and dye release if the mechanisms are similar, eg, via an allosteric effect that increases the rigidity of the c-terminal half (tm2) of p7. this is supported by the fact that tm2 of p7 interacts with amt and is the site of inhibition by adamantanes. indeed, amt interacts with leucines 51-57, and these leucines have been shown unequivocally to be part of a membrane inserted α-helix (cook & opella, 2009; ouyang et al., 2013) . binding of amt to p7 in dihexanoylphosphatidylcholine (dhpc) micelles led to resonance shifts in several residues, some of which are the leucines 51-57 (but not 53), ie, located in tm2 (cook & opella, 2009) . consistent with this, leu to ala mutations at this region produced an amt-resistant p7 mutant revealed using the cf release assay (stgelais et al., 2009) . the imino sugar nn-dnj and rim inhibited secreted infectivity in amt-resistant jfh-1 and con-1/jfh-1c3 viruses, whereas mutation l20f conferred resistance to rimantadine (foster et al., 2011) , consistent with rim resistance was found in gt1b patients not responding to the triple therapy ifn/rib/amt (mihm et al., 2006) . using an oligomerization assay in dhpc gels, the gt1b j4 p7 oligomerization was abolished in presence of imino-sugars, whereas rim did not affect oligomerization, suggesting two different mechanisms of action for these two compounds (foster et al., 2011) . the p7 of gt3a, but not gt1b, was found to be resistant to nn-dnj both in vitro and in culture, and this was attributed to a f25a mutation (foster et al., 2011; griffin et al., 2008) . however, direct interaction between hma or imino sugar derivatives with p7 reconstituted in a detergent or membrane has not been reported. addition of rim to gt1b j4 p7 monomer in methanol (foster et al., 2014) showed disruption at several residues, but not especially at leu-20. thus, this observation is either inconsistent with the proposed role of l20f in acquired resistance, or the methanol environment is not a good system to test this interaction. structural data for p7 was obtained initially in solvents and micelles by solution nmr. for example, solution nmr data was obtained for synthetic p7 (c27a mutant) in 50% of the helix inducer trifluoroethanol (tfe) (montserret et al., 2010) , and for recombinant p7 (c27s mutant) in dhpc micelles ). the prevalent model for the p7 monomer according to these studies was that of an α-helical hairpin with two α-helical tms kinked in the middle (cook, zhang, park, wang, & opella, 2010; montserret et al., 2010) , where the n-terminal tm helix (tm1) would face the lumen of a channel (carrere-kremer et al., 2002; chew, vijayan, chang, zitzmann, & biggin, 2009; patargias, zitzmann, dwek, & fischer, 2006 ) formed by either six or seven monomers (clarke et al., 2006; griffin et al., 2003; luik et al., 2009; montserret et al., 2010) . in a more recently published structural model of p7 (ouyang et al., 2013) , the oligomeric size is still ambiguous since this data could not be obtained in the same environment used in electron microscopy. from that model, the "old" tm1 and tm2 domains would correspond to three helical segments h1-h3, ie, h1 and first half of h2 (tm1) and second half of h2 and h3 (tm2). there are concerns, however, that this model can be present in a physiological context since the folding implied is difficult to explain by our current understanding of protein folding in the cell (madan & bartenschlager, 2015) . in the ouyang et al. paper, the authors systematically tested p7 amino acid sequences from various hcv genotypes and found that the sequence from gt5a (euh1480 strain) generated samples adequate for structure determination. although the oligomeric size of p7 in dodecyl phosphocholine (dpc) micelles was not determined, this was combined with data from negative stain electron microscopy (em), which showed hexameric, flower-shaped particles. the latter were similar to those p7 hexamers (of gt2ajfh-1) observed in em in the presence of dhpc micelles used earlier for single-particle reconstruction (luik et al., 2009) . the nmr-based model showed that the c-terminal helix, p7(27-63), is not the pore-lining sequence but instead forms a "lipid facing" part of the molecule, whereas the n-terminal half would orient lining the lumen (ouyang et al., 2013) . this is in agreement with previous models based on the partial inhibition of p7 channel activity by cu 2+ , but not by mg 2+ , which suggested exposure to a his residue located in the n-terminal half to the lumen of the channel . although this interpretation may be correct, it is unfortunate that synthetic peptides corresponding to the n-terminal half "tm1," eg, p7(1-34) or the c-terminal half "tm2," eg, p7(35-63) aggregate easily, such that individual channel activity of these peptides cannot be properly measured (montserret et al., 2010) . in fact, neither of these helices separately forms α-helical structure in micelles or membranes, and peptide p7(1-26) was totally insoluble in tfe or in detergent (gan et al., 2014) , a difficulty also encountered for the n-terminal fragment of p7(1-34) (montserret et al., 2010) . in contrast, tm domains of other viroporins are completely α-helical, eg, the iav m2 (torres, kukol, & arkin, 2000) , severe acute respiratory syndrome coronavirus (sars-cov) e , or respiratory syncytial virus (rsv) sh (gan et al., 2012) produce sharp amide i bands in the infrared spectrum consistent with $100% α-helix. nevertheless, the amt binding site in the p7 channel was determined by identifying nuclear overhauser enhancements (noes) between the protein backbone amide protons and drug protons (ouyang et al., 2013) (fig. 5) . although the full-scale structure determination of the complex could not be achieved, amt (10 mm) showed noe cross-peaks between the adamantane protons and the amide protons of val-26, leu-55, leu-56, and arg-57, ie, between the pore-forming and peripheral helices, suggesting a pocket formed by leu-52, val-53, and leu-56 from h3, and phe-20, val-25, and val-26 from h2, with the polar amino group of amt pointing to the channel lumen, with similar results also obtained for rim. amt and rim have also been reported previously to interact with some residues in the n-terminal half (cook & opella, 2009; stgelais et al., 2009) . isothermal titration calorimetry and nmr chemical shift perturbation analyses of p7(gt5a)-rim interaction produced a binding constant (kd) from 50 to 100 μm at 3 mm detergent concentration. the authors proposed that large differences in drug efficacies observed between different hcv genotypes are probably due to variations in the hydrophobicity of the binding pocket among p7 variants, and that the adamantane derivatives inhibit channel activity by restricting the structural rearrangement of the channel, ie, an allosteric mechanism. perhaps the most efficient effect for a p7 inhibitor has been reported for bit225 (n-[5-(1-methyl-1h-pyrazol-4-yl)-napthalene-2-carbonyl]guanidine) (fig. 4c ). this amiloride was identified in a bacterial assay that involved expression of p7 in e. coli; only in presence of inhibitors the normal ionic gradients are maintained and cells are able to grow despite the presence of p7. this compound had antiviral activity against the hcv model pestivirus bovine viral diarrhea virus (bvdv) with an ic 50 of 314 nm. the addition of 100 μm of bit225 blocked ion channel activity of synthetic hcv p7 protein of gt1a h77, although no data was reported in the context of hcv infection . while some docking efforts have been performed (bichmann, wang, & fischer, 2014) , direct interaction of bit225 with p7 has not been proven using biophysical or structural assays, and therefore rational optimization of the compound is still not possible. in addition to hcv p7, the human immunodeficiency virus (hiv) viroporin vpu is also targeted; bit225 was tested against the vpu tm domain using a black lipid membrane (blm) system, where the target was exposed to 40 μm of the drug (khoury, ewart, luscombe, miller, & wilkinson, 2010) . although direct interaction with vpu was not observed, this was assumed since the drug was not active against hiv-2, which has no vpu gene. one early clinical trial (phase ib/iia) involving the treatment of 24 naïve gt1 hcv patients indicated a significant reduction of viral load after 12 weeks treatment with bit225 in combination with ifnα/ribavirin (www.biotron.com.au: biotron 2011). currently, a phase 2a/2 clinical trial is in progress and the first results indicate activity against several hcv genotypes, including the difficult-to-treat gt3a and gt1a (www.biotron.com. au; may 29, 2015). since it is also active against the hiv-1 vpu, bit225 might be suitable for treatment of hcv/hiv coinfected patients. in a jul. 2015 update of a 3-month dosing trial with bit225, there was a recommendation that future trials would focus on patients infected with hcv gt3 and to further study bit225 in combination with other direct-acting antiviral drugs. in these trials, patients received 400 mg of bit225 twice daily for 3 months. a phase 2a trial on hcv demonstrated that 100% of the patients infected with hcv gt1 who received bit225 (400 mg) in combination with current standard of care therapies, ifn/rbv, had undetectable virus after 48 weeks. a phase 2 trial in hiv/hcv coinfected patients showed that all hcv gt3 patients completing 28 days of treatment with bit225 in combination with ifn/rbv have undetectable hcv load for 12 weeks (sustained viral response, svr12) after completing all therapy. bit225 is also in development for treatment of hiv, and is the pioneer in a new class of antiviral drugs that may provide a new approach to the eradication of this virus. it has shown clinical efficacy against hiv in reservoir cells, and has the potential to be combined with new or existing antiretroviral drugs to eradicate long-lived pools of virus that are not successfully eliminated with current treatments. despite these promising results, however, it remains to be demonstrated that bit225 targets p7 or vpu in infected cells. the most studied viroporin in coronaviruses is the envelope (e) protein. coronaviruses (cov) typically affect the respiratory tract and gut of mammals and birds. covs belong to the subfamily coronavirinae in the family coronaviridae, and are organized into four genera (enjuanes et al., 2000) : α, β, γ, and δ. approximately 30% of common colds are caused by two human coronaviruses-oc43 and 229e. of particular medical interest is the virus responsible for the severe acute respiratory syndrome (sars), which produced a near pandemic in 2003 (holmes, 2003) , and the recently emerged middle east respiratory syndrome coronavirus (mers-cov), which after 3 years has caused hundreds of deaths (raj, osterhaus, fouchier, & haagmans, 2014) . other coronavirus viroporins, eg, the sars-cov3a (lu et al., 2006) , a 274-amino acid protein with three putative tm domains, are also attracting increasing interest (chien et al., 2013; hsu & fischer, 2012) , but no inhibitors or structural data is available so far. currently, no effective licensed treatment exist against coronavirus infection (kilianski & baker, 2014; kilianski, mielech, deng, & baker, 2013; lou, sun, & rao, 2014) , although live vaccines consisting of attenuated viruses is a promising strategy (enjuanes, nieto-torres, jimenez-guardeno, & dediego, 2011; graham et al., 2012) , along with fusion inhibitors (reviewed in heald-sargent & gallagher, 2012) . even so, the possibility of reappearance of virulent phenotypes, drug side effects, and resistance calls for continued antiviral development. the envelope (e) proteins are 76-109 residues long with one tm domain (li, surya, claudine, & torres, 2014; parthasarathy et al., 2008 parthasarathy et al., , 2012 pervushin et al., 2009; torres et al., 2006; torres, wang, parthasarathy, & liu, 2005) , and most of them have a cytoplasmic c-terminal domain and a lumenal n-terminus (corse & machamer, 2000; nieto-torres et al., 2011; raamsman et al., 2000; ruch & machamer, 2012) . most cov e proteins are present at low concentrations in virions (corse & machamer, 2000; godet, l'haridon, vautherot, & laude, 1992; liao, yuan, torres, tam, & liu, 2006; yu, bi, weiss, & leibowitz, 1994) , but found abundantly in internal membranes of infected hosts (corse & machamer, 2003; liao et al., 2006; raamsman et al., 2000; tung et al., 1992) , eg, the er-golgi intermediate compartment (ergic), where virions assemble (lopez, riffle, pike, gardner, & hogue, 2008; nieto-torres et al., 2011) . cov e proteins have been found to be critical for viral pathogenesis, while having a protective antiapoptotic effect on infected cells which may help the virus evade premature death of host cells, thereby allowing viral replication. interestingly, deletion of e protein reduced pathogenicity and mortality in animal models through a reduced inflammation (dediego et al., 2014)-as discussed later-and this has led to the development of live attenuated vaccines based on e-deleted or e-truncated virions (almazán et al., 2013; lamirande et al., 2008; netland et al., 2010) . the only structural data available for a cov e protein is for sars-cov e, where the tm domain (e-tm) has been characterized in some detail in lipid membranes . later, solution nmr of the selectively labelled synthetic e-tm (residues 8-38) in dpc micelles produced a similar pentameric left-handed parallel bundle (pervushin et al., 2009) . in these models, asn-15 is facing the lumen of the channel whereas val-25 is involved in helix-helix interactions with other subunits (pervushin et al., 2009) . mutations at these residues, n15a and v25f, abolished channel activity in vitro (torres et al., 2007) , and introduction of these mutations in a recombinant sars-cov resulted in in vivo attenuation in a mouse model. the integrity of the tm domain, and preservation of channel activity, were shown to be important for inflammasome activation and elevated production of the proinflammatory cytokine il-1β (nieto. in the same study, revertant mutants of the channel-inactive e that regained fitness and pathogenicity also regained channel activity, as measured in black lipid membranes. both e-tm and the full-length e protein form homopentameric channels with poor ion selectivity that can be partially inhibited by the drug hma with micromolar affinity (verdia-baguena et al., 2012; wilson, gage, & ewart, 2006) . the noes between hma and e-tm suggested the presence of two binding sites at both ends of the tm domain. at the n-terminal domain, leu-19 exhibited the largest chemical shift, and the relative intensities of the hma proton cross-peaks indicated an hma:e-tm stoichiometry of 1:5 at the n-terminal binding site near asn-15, suggesting one hma molecule per e-tm pentamer. this is consistent with a pore-blocking inhibition mode similar to amt in am2, where in this case the hma molecule may be stabilized by a hydrogen bonding network to the asn-15 side chains of the sars-cov e, with the cyclohexamethylene ring pointing away from the center of the channel (fig. 6a) . at the c-terminal of the tm domain, near arg-38, the hma:e-tm stoichiometry was 1:2, suggesting for this site a rapid exchange, in the chemical shift timescale, between e-tm-bound and micelle-bound forms of hma. at this location, the amiloride group of hma is likely to be involved in interactions with the guanidinium groups of arg-38 (fig. 6b) . these results were confirmed using the extended peptide e (8-65) in mixed sds/dpc (1:4 molar ratio) micelles , where the addition of hma perturbed residues clustered near the n-terminal end of the tm domain, eg, . at the c-terminal end of the tm, leu-37 was the most affected, suggesting that the interaction of hma at the hε of arg-38 reported previously may have been an artifact due to the use of an e-tm (8-38) peptide. in any case, these results suggest a similar behaviour of hma-to-e protein and amt-to-am2 protein, ie, the presence of two binding sites, one pore-binding and one surface-binding in the tm domain. this structure provided a possible rationale for inhibition and a platform for future structure-based drug design of this potential pharmacological target. the small hydrophobic (sh) protein is found in the human respiratory syncytial virus (hrsv), an enveloped pneumovirus in the paramyxoviridae family. hrsv is the leading cause of bronchiolitis and pneumonia in infants and elderly (dowell et al., 1996) , and the most frequent cause of hospitalization of infants and young children in industrialized countries. in developing countries, hrsv is a significant cause of death, with global estimates of more than 70,000 deaths in young children. hrsv is the third most important cause of deadly childhood pneumonia after streptococcus pneumoniae and haemophilus influenzae (nair et al., 2010) . several compounds target the rsv fusion (f) protein, which facilitates viral entry through the host cell membrane (zhao, singh, malashkevich, & figure 6 binding of hma to the sars-cov e-tm pentameric channel (pervushin et al., 2009 ). (a) side view of the binding of hma to the vicinity of asn-15. the side chains of amino acids interacting with hma are shown using a stick representation; (b) binding of hma to the c-terminal binding site of the channel, in the vicinity of thr-35 and arg-38. for clarity, one of the e-tm monomers has been removed. kim, 2000) through formation of a 6-helix bundle (douglas et al., 2005; lambert et al., 1996; pastey, gower, spearman, crowe, & graham, 2000; razinkov, gazumyan, nikitenko, ellestad, & krishnamurthy, 2001; roymans et al., 2010; shepherd et al., 2006) . other approaches have involved gene transfer to expose viral proteins to cells (kumar et al., 2002) , or sirna against specific viral proteins (zhang et al., 2005) . vaccines have been recently designed based on a stabilized rsv-f form which preserves a highly antigenic site in its prefusion state, yielding rsv-specific neutralizing antibodies in mice and macaques (mclellan, chen, joyce, et al., 2013; mclellan, chen, leung, et al., 2013) . recently, a vaccine candidate based on the extracellular domain (c-terminal) of the rsv viroporin, the small hydrophobic (sh) protein, has been reported (schepens et al., 2014) . in addition, the prevention of nasopulmonary infection in mice caused by rsv has been reported using stapled peptides targeting the fusogenic f-protein 6-helix bundle . however, despite all these efforts, new fda-approved drugs have yet to emerge. the only licensed drug for use in infected individuals is ribavirin, a nucleoside analogue, but its efficacy is very limited (hall et al., 1986) . the hrsv genome transcribes 11 proteins (collins & melero, 2011) . one of these is the viroporin sh protein, which is 64-65 residues long (depending on subgroup, a or b) and has a single α-helical tm domain with n-and c-terminal extramembrane domains oriented cytoplasmically and lumenally/extracellularly, respectively (collins & mottet, 1993; gan et al., 2012) . most sh protein accumulates at the membranes of the golgi complex in infected cells, but it has also been detected in the er and plasma membranes (rixon et al., 2004) . rsv that lacks sh (rsvδsh) is still viable, and still forms syncytia (bukreyev, whitehead, murphy, & collins, 1997; karron et al., 1997; techaarpornkul, barretto, & peeples, 2001) , but is attenuated in vivo. in mouse, rsv δsh replicated 10-fold less efficiently in the upper respiratory tract (bukreyev et al., 1997) , whereas chimpanzees developed significantly less rhinorrhea than those infected with wild-type rsv (whitehead et al., 1999) . other reports have shown that the lack of sh protein leads to an attenuated phenotype in children and in rats (jin et al., 2000; karron et al., 1997) . overall, these results indicate involvement of hrsv sh protein in replication and pathogenesis. in common to the sars-cov e protein, sh protein blocks or delays apoptosis in infected cells (fuentes, tran, luthra, teng, & he, 2007) , and a similar antiapoptotic effect of sh protein has been observed for other members of the paramyxoviridae family, eg, j paramyxovirus (jpv) (jack, boyle, eaton, & wang, 2005; li et al., 2011) , mumps virus (muv), and the parainfluenza virus 5 (piv5), formerly known as simian virus 5 (sv5). in all these systems, the sh protein seems to block apoptosis during infection-at least in part-through inhibition of the tnf-α pathway (fuentes et al., 2007; li et al., 2011; lin, bright, rothermel, & he, 2003; wilson, fuentes, et al., 2006) . the mutual orientation of the tm α-helices that form the ion channel was determined in lipid bilayers using site-specific infrared dichroism (gan, ng, xin, gong, & torres, 2008) . a description of the full-length rsv sh protein monomer has been obtained by solution nmr in dpc micelles (gan et al., 2012) and later in bicelles, only for the extramembrane domains . like sars-cov e, the rsv sh protein forms homopentameric channels (gan et al., 2008 (gan et al., , 2012 that have low ion selectivity . the tm domain of sh protein has a funnel-like architecture (gan et al., 2012) , as observed in other viroporins, eg, the iav m2 (schnell & chou, 2008) , sars-cov e (pervushin et al., 2009 ) and hcv p7 (ouyang et al., 2013) . a narrower region (gan et al., 2012) in the tm domain is lined with hydrophobic side chains whereas the more open n-terminal region is lined by polar residues, ie, his-22, thr-25, and ser-29. the tm α-helix extends up to his-51 in the c-terminal region, followed by a loop, whereas the n-terminal cytoplasmic extramembrane domain forms a short α-helix (residues 5-14) present both in micelles and in bicelles . only one compound, pyronin-b, has been reported to inhibit the channel activity of the sh protein. this compound was obtained from liposome-based fluorescence assay, and was tested against purified sh protein reconstituted in planar lipid bilayers (blm). a concentration of 10 μm led to a $60% inhibition of channel activity, with a kd of $7 μm. the effect of pyronin-b was tested against rsv replication in vero cells, where the tissue culture infectivity (50% infective dose, tcid50) was zero at a drug concentration of 0.25 μm. in the nmr studies, the binding of pyronin-b to the sh protein revealed backbone resonance perturbations at both ends of the tm domain. at the n-terminal cytoplasmic end, residues more affected were ile-6 and ile-21. at the lumenal c-terminal end, the residue most affected was ala-39, and a group of nearby residues, ile-38, ile-40, leu-41, and lys-43 (fig. 7a) . interestingly, most of these juxtamembrane residues (residues 38-43) form a conserved motif in the sh protein "a 39 ilnkl 43 ," which suggests that this is a critical region for sh protein with a high barrier to resistance. determination of intermolecular nuclear overhauser effects (noes) for the drug-protein complex was not possible, probably due to weak interaction. docking studies of pyronin-b to the sh pentameric model obtained in dpc micelles (gan et al., 2012) produced two predicted binding sites at both ends of the tm domain. indeed, a majority of structures showed the drug located near residues with largest chemical shift perturbation, ie, ile-6, ile-21, and ala-39 (fig. 7) . the binding site near the n-terminus is formed by phe-14, ile-21 in one monomer (i + 1), and ile-6 of the previous (i) monomer. the one near the c-terminus is formed by residues ile-40, leu-41, and lys-43 in one monomer (i + 1) and ile-38 and ala-39 of the previous (i) monomer ( fig. 7a-c) . similar "druggable pockets" on the sh pentamer surface were identified using dogsitescorer (volkamer et al., 2012) (fig. 7d) . however, the c-terminal end of the tm domain (residues 38-41) is sufficient for inhibition, since a conservative mutation a39s almost completely prevented inhibition ($10% inhibition). only two monomers of sh protein pentamer (i and i + 1) are shown for simplicity; (d) druggable pockets (green (dark gray in the print version) mesh) predicted by dogsitescorer (volkamer, kuhn, rippmann, & rarey, 2012) . the main residues that showed largest nmr chemical shift changes are shown in red (dark gray in the print version). two monomers have been removed from the pentamer for clarity. vpu is a 81-residue small-membrane protein encoded by the singlestranded positive-sense rna human immunodeficiency virus 1 (hiv-1), and some simian immunodeficiency virus (siv) isolates. vpu is one of four hiv-1 accessory proteins involved in the release of new virions from host cell membranes (cohen, terwilliger, sodroski, & haseltine, 1988; strebel, klimkait, & martin, 1988; terwilliger, cohen, lu, sodroski, & haseltine, 1989) . without vpu, hiv particles fail to detach from the plasma membrane and accumulate in large numbers in the cell surface (klimkait, strebel, hoggan, martin, & orenstein, 1990) . the initial structural studies of vpu were performed more than 20 years ago by solution nmr in tfe/water using synthetic peptides of various overlapping lengths, which led to a model of an n-terminal hydrophobic transmembrane helix (the tm domain) and a c-terminal cytoplasmic domain with a helix-loop-helix arrangement [reviewed recently in opella, 2015] . structural similarity of vpu with am2 suggested that vpu could form pores (maldarelli, chen, willey, & strebel, 1993) and was later shown to have cation-selective channel activity (ewart, sutherland, gage, & cox, 1996; schubert et al., 1996) , but a unique structural model for the vpu oligomer cannot be defined [reviewed in radoicic, lu, & opella, 2014 ]. an analysis of the vpu tm (residues 1-40) by solid-state nmr in membranes, combined with analytical centrifugation measurements in detergent, produced data compatible with a variety of oligomers coexisting in micelles and phospholipid bilayers (lu, sharpe, ghirlando, yau, & tycko, 2010) . this led to the proposal that regulation of virus release by vpu might involve an alteration of the plasma membrane potential, and this was supported by (i) the reduced rate of hiv-1 virus release after membrane depolarization (hsu, han, shinlapawittayatorn, deschenes, & marbán, 2010) , (ii) the observed reduction in viral load and viral pathogenicity when vpu tm domain is scrambled in the context of a simian-human immunodeficiency virus (shiv) (hout et al., 2005) , and (iii) the substitution of ala-18 by a histidine (a18h) rendered hiv-1 infections susceptible to rim (hout et al., 2006) . however, recent studies challenge this model and argue against a critical involvement of vpu ion channel activity in vpu-enhanced virus release. indeed, mutations in the vpu tm domain that affect channel activity do not impair the ability to enhance virus release, whereas mutations that affect virus release retain ion channel activity (bolduan et al., 2011) . thus, the functional significance of vpu ion channel activity for hiv replication, and the role as a target for channel inhibitors, remains unclear. there are several other viroporins of which only very limited structural data is available, and no channel inhibitors have been reported. the following section will briefly describe three of such viroporins: the alphavirus 6k protein, polyomavirus agnoprotein, and the 2b protein found in picornaviruses. the 6k protein is 58-61 amino acids long and has one predicted α-helical tm domain. this viroporin is found in several members of the alphavirus genus in the positive-sense rna family togaviridae. this includes, for example, equine encephalitis viruses, chikungunya virus, sindbis virus, semliki forest viruses (sfv), ross river virus (rrv), and barmah forest virus (bfv). in these viruses, structural proteins are translated in the er from a subgenomic mrna as a single polyprotein later cleaved by proteases, in the order capsid-pe2-6k-e1 strauss & strauss, 1994) . the 6k protein was first described as early as 1980 (welch & sefton, 1980) . its expression in e. coli caused an increase in membrane permeability, whereas in eukaryotic cells it promoted viral budding (sanz, perez, & carrasco, 1994) . further, 6k proteins of rrv and bfv formed cationselective ion channels in planar lipid bilayers (melton et al., 2002) . this suggested a role for viroporins in alphavirus infection, since an increased permeability of cells to monovalent cations is followed by budding of virions. whatever the mechanism, 6k proteins have an important role in virus assembly and budding. for example, in sindbis virus, cysteine-less mutants dramatically reduced the release of virus particles, leading to aberrant enveloped particles containing multiple nucleocapsids (gaedigk-nitschko, ding, levy, & schlesinger, 1990) . other results are consistent with an indirect role in the packing of the virus spike glycoproteins; mutation c39s resulted in defective budding and a revertant contained two additional mutations at the ectodomain of the virus glycoprotein (ivanova, lustig, & schlesinger, 1995) . further, deletion of 6k in a full-length cdna clone of sfv resulted in a dramatic reduction in virus release (liljestrom, lusa, huylebroeck, & garoff, 1991) , and bhk cells expressing a δ6k strain produced thermolabile particles, suggesting a disrupted glycoprotein packing (loewy, smyth, von bonsdorff, liljestrom, & schlesinger, 1995) . despite these investigations, the specific role of 6k channel activity in these processes is still uncertain. agnoprotein is a 71-amino acids long membrane protein with one predicted α-helical tm domain required for the productive replication of the human polyomavirus jcv (john cunningham virus). jcv causes progressive multifocal leukoencephalopathy, a fatal demyelinating disease resulting from lytic infection of oligodendrocytes. agnoproteinis also found in other polyomaviruses, including bk virus, and simian virus 40 (sariyer, saribas, white, & safak, 2011) . agnoprotein was shown to play important regulatory roles in the jcv replication cycle (ellis, dang, norton, & koralnik, 2012; ellis, norton, dang, & koralnik, 2013; saribas et al., 2013) , and possesses properties commonly associated with viroporins (suzuki et al., 2013; suzuki, orba, malcino, et al., 2010; suzuki, orba, okada, et al., 2010) . for example, a deletion mutant is defective in virion release and viral propagation. also, agnoprotein localizes to the er during early stages of infection, but is also found at the plasma membrane late in infection. finally, agnoprotein, an integral membrane protein, forms homo-oligomers that enhance permeability of cells to hygromycin b and induce the influx of extracellular ca 2+ , leading to membrane dysfunction and enhancement of virus release. only a solution nmr structure of a synthetic peptide corresponding to the tm domain (thr-17 to glu-55) is available, but with an uncertain oligomeric size in lipid bilayers (coric et al., 2014) . as with the 6k protein, characterization of this viroporin is still in its infancy. protein 2b is a $100 residues long nonstructural protein found in enteroviruses [see ao, sun, & guo, 2014 for a recent review], eg, poliovirus (agirre, barco, carrasco, & nieva, 2002) . enteroviruses belong to the single-stranded positive-sense rna picornaviridae family, and include poliovirus, coxsackie virus and human enterovirus 71 (ev71). the single open reading frame encodes a peptide chain divided into three regions, p1 to p3. the 2b protein is one of the products from the polypeptides in the p2 region, and has two tm domains, one more hydrophobic than the other (de jong et al., 2003; nieva, agirre, nir, & carrasco, 2003) . the c-and n-termini of the poliovirus 2b protein are exposed to the er and golgi lumen when expressed in cells (de jong et al., 2003) , but the 2b protein in coxsackie virus adopts an opposite orientation (abed et al., 2005 , van kuppeveld, galama, zoll, van den hurk, & melchers, 1996 . poliovirus 2b protein was found to increase the concentrations of free calcium in the cytosol, which has been proposed to promote viral replication and repression of the antiviral immune response of host cells (aldabe, irurzun, & carrasco, 1997) . however, precise structural data is not yet available for this protein or its oligomers. one common, specific effect of viroporin channel activity may be associated with the observed ability of viruses to activate the host inflammasome, which activates caspase-1 to produce proinflammatory cytokines, eg, il-1β. these effects have been shown in many cases to be caused by the disruption of cellular ion homeostasis by allowing ion leakage from intracellular organelles into the cytosol, through the expression of viroporins [reviewed in guo, jin, zhi, yan, & sun, 2015] . for example, the iav activates the nlrp3 inflammasome as a result of h + or ion flux from golgi mediated by its m2 ion channel (ichinohe, pang, & iwasaki, 2010) . the 2b protein from several picornaviruses, including the encephalomyocarditis virus (emcv), poliovirus, enterovirus 71, and human rhinovirus (triantafilou, kar, van kuppeveld, & triantafilou, 2013) were shown to induce nlrp3 cytoplasmic relocalization and inflammasome activation in an intracellular ca 2+ -mediated manner (ito, yanagi, & ichinohe, 2012) . the channel activity of the sars-cov e protein is required for the processing of il-1β (nieto, which requires caspase-1 activation. channel activity of the sh protein may also result directly or indirectly in activation of the nlrp3 inflammasome (segovia et al., 2012; triantafilou, kar, vakakis, kotecha, & triantafilou, 2013) . in these cases, therefore, development of channel inhibitors should improve the pathological effects of host inflammation caused by these viruses. apart from the effects of their channel activity, viroporins are involved in many protein-protein interactions (ppis) that are susceptible for therapeutic intervention, and may be increasingly important once these interactions are properly characterized [see recent reviews fischer, li, mahato, wang, & chen, 2014; fischer et al., 2012; nieva & carrasco, 2015] . for example, the cytoplasmic regions of am2 and bm2 are important for proper virus assembly and interaction with m1 (chen, leser, jackson, & lamb, 2008; imai, kawasaki, & odagiri, 2008; imai, watanabe, ninomiya, obuchi, & odagiri, 2004; mccown & pekosz, 2006) , which can be proposed as an attractive drug target. also, the hcv p7 interacts with other viral structural and nonstructural proteins that are important to promote virus assembly and release (gentzsch et al., 2013; haqshenas, dong, ewart, bowden, & gowans, 2007; pietschmann et al., 2006; sakai et al., 2003; yi, ma, yates, & lemon, 2007) , and functions related to capsid assembly and localization of several viral proteins are not affected by either channel-inactivating mutations or treatment with rim (tedbury et al., 2011; wozniak et al., 2010) , eg, the transfer of hcv core protein from lipid droplets to the er depends on the interaction between p7 and ns2 (boson, granio, bartenschlager, & cosset, 2011) . therefore, the disruption of such interactions would require the action of ppi modulators rather than channel inhibitors. the interaction partners of e and sh proteins have been reviewed recently (torres, surya, li, & liu, 2015) . in the case of cov e proteins, the interaction with m protein has long been reported to contribute to m localization and virion formation (corse & machamer, 2003; hogue & machamer, 2008; ilk, egelseer, & sleytr, 2011; lim & liu, 2001; siu et al., 2008; vennema et al., 1996) . recent reports have highlighted interactions with protein associated with lin seven 1 (pals1) (teoh et al., 2010) and syntenin through pdz domains of e proteins, which are consistent with alterations of lung epithelia integrity and inflammation observed during cov infection. disruption of these pathways may have clear therapeutic implications, since in sars-cov-infected patients it is an exacerbated inflammatory response that leads to epithelial and endothelial damage, edema, and acute respiratory distress syndrome (smits et al., 2010) . noticeably, several other viruses, eg, human papillomavirus and influenza a, have been found to enhance pathogenesis through proteins containing pdz binding motifs (reviewed in javier & rice, 2011) , which suggests that this is a particular case of a widely used viral strategy. vpu is known for its ability to degrade the hiv-receptor cd4 (bour & strebel, 2003) , which requires interaction between vpu and cd4 cytoplasmic domains (mcnatt, zang, & bieniasz, 2013; singh et al., 2012) and probably tm domains (do et al., 2013; magadán & bonifacino, 2012) of the two proteins. this may not even require formation of oligomers, and even less channel activity. vpu also affects virus release via interaction with interferon-induced protein tetherin (bst-2) (miyagi, andrew, kao, & strebe, 2009; neil, zang, & bieniasz, 2008; skasko et al., 2012; van damme et al., 2008) , an interferon-induced host protein that inhibits the release of many enveloped viruses by tethering virions to the cell surface [reviewed in strebel, 2014] . in another case, the interaction between rsv sh and g proteins has been reported previously in infected cells (low, tan, ng, tan, & sugrue, 2008; rixon, brown, murray, & sugrue, 2005) , although the significance is not clear yet. the b-cell receptor-associated protein 31 (bap31) has been recently reported to interact with the sh protein . although this interaction has not been reported in infected cells, it could prevent the cleavage of bap31 and the formation of proapoptotic fragment p20, thus delaying apoptosis of infected cells. interaction with bap31 was also observed in the e5 viroporin from the high-risk human papillomavirus hpv-16 and hpv-31, where it is similarly thought to regulate apoptosis (regan & laimins, 2008) . jcv agnoprotein has been shown to interact with a number of cellular and viral proteins (gerits & moens, 2012; gerits et al., 2015) . however, despite all these intense research, the exact regulatory function of agnoprotein in viral replication is not fully understood. jcv agnoprotein specifically interacts with the adaptor protein complex 3 through its delta subunit. this interaction interrupts adaptor protein complex 3-mediated vesicular trafficking. the protein is then not targeted to the lysosomal degradation pathway, while allowing transport of agnoprotein to the plasma membrane. the findings suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins (suzuki et al., 2013) . these pathologically relevant interactions between viroporins and their associated viral and host proteins present an attractive therapeutic target for disruption by small molecules, as well as peptides stabilized by internal covalent linkage (stapled peptides) (walensky & bird, 2014) . in principle, both the transmembrane and extramembrane domains of viroporins can be potential templates for peptide-based modulators. for example, one could design a stapled peptide mimicking the α-helical cytoplasmic domain of the sh protein to disrupt the interactions between sh and a variant of the death effector domain (vded) of bap31, which would lead to enhanced apoptosis and cell death of infected cells. one could also mimic the transmembrane domain to disrupt monomer-monomer interactions, as shown recently for halobacterium salinarum where efflux by a small multidrug resistance protein was inhibited with peptides targeting its tm domain (bellmann-sickert, stone, poulsen, & deber, 2014) . in the case of viroporins, the disruption of the oligomeric status would abolish channel activity, a classical strategy revisited with a different approach. however, more detailed mechanistic information should be available before these conceptual strategies can be widely used. generation and characterization of recombinant influenza a (h1n1) viruses harboring amantadine resistance mutations structure and mechanism of proton transport through the transmembrane tetrameric m2 protein bundle of the influenza a virus viroporin-mediated membrane permeabilization. pore formation by nonstructural poliovirus 2b protein poliovirus protein 2bc increases cytosolic free calcium concentrations engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate demonstration that cftr is a chloride channel by alteration of its anion selectivity a strategy to estimate unknown viral diversity in mammals topology and biological function of enterovirus non-structural protein 2b as a member of the viroporin family design and pharmacological characterization of inhibitors of amantadine-resistant mutants of the m2 ion channel of influenza a virus purification and functional reconstitution of the cystic-fibrosis transmembrane conductance regulator (cftr) efflux by small multidrug resistance proteins is inhibited by membrane-interactive helix-stapled peptides docking assay of small molecule antivirals to p7 of hcv mucosal delivery of a double-stapled rsv peptide prevents nasopulmonary infection ion channel activity of hiv-1 vpu is dispensable for counteraction of cd317 a concerted action of hepatitis c virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly the hiv-1 vpu protein: a multifunctional enhancer of viral particle release incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states recombinant respiratory syncytial virus from which the entire sh gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus m2 protein modification of membrane permeability by animal viruses subcellular localization and topology of the p7 polypeptide of hepatitis c virus hepatitis c virus p7 membrane protein quasispecies variability in chronically infected patients treated with interferon and ribavirin, with or without amantadine the influenza virus m2 protein cytoplasmic tail interacts with the m1 protein and influences virus assembly at the site of virus budding determination of pore-lining residues in the hepatitis c virus p7 protein assembling an ion channel: orf 3a from sars-cov evidence for the formation of a heptameric ion channel complex by the hepatitis c virus p7 protein in vitro identification of a protein encoded by the vpu gene of hiv-1 progress in understanding and controlling respiratory syncytial virus: still crazy after all these years membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus nmr studies of p7 protein from hepatitis c virus secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis c virus by nmr spectroscopy comparative nmr studies demonstrate profound differences between two viroporins: p7 of hcv and vpu of hiv-1 nuclear magnetic resonance structure revealed that the human polyomavirus jc virus agnoprotein contains an alpha-helix encompassing the leu/ile/pherich domain infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction m2 protein from influenza a: from multiple structures to biophysical and functional insights. current opinion in virology influence of solubilizing environments on membrane protein structures structural mechanism of c-type inactivation in k+ channels antiviral activity of 1-adamantanamine (amantadine) antiviral agents active against influenza a viruses determinants for membrane association and permeabilization of the coxsackievirus 2b protein and the identification of the golgi complex as the target organelle severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis inhibition of nf-κb-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide full-length vpu and human cd4(372-433) in phospholipid bilayers as seen by magic angle spinning nmr small molecules vp-14637 and jnj-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults the structure of the potassium channel: molecular basis of k + conduction and selectivity comparative rates of spontaneous mutation recent progress in structure-based anti-influenza drug design the transmembrane domain of influenza a m2 protein forms amantadine-sensitive proton channels in planar lipid bilayers role of agnogene deletion and archetype-like regulatory region in a jcv strain isolated from the brain of a patient with jcv encephalopathy (jcve) agnogene deletion in a novel pathogenic jc virus isolate impairs vp1 expression and virion production virus taxonomy. classification and nomenclature of viruses recombinant live vaccines to protect against the severe acute respiratory syndrome coronavirus the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels antiviral agents for the treatment and chemoprophylaxis of influenza-recommendations of the advisory committee on immunization practices (acip) viral channel proteins in intracellular protein-protein communication: vpu of hiv-1, e5 of hpv16 and p7 of hcv mechanism of function of viral channel proteins and implications for drug development structure-guided design affirms inhibitors of hepatitis c virus p7 as a viable class of antivirals targeting virion release resistance mutations define specific antiviral effects for inhibitors of the hepatitis c virus p7 ion channel function of the respiratory syncytial virus small hydrophobic protein large-scale sequence analysis of m gene of influenza a viruses from different species: mechanisms for emergence and spread of amantadine resistance site-directed mutations in the sindbis virus 6k protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure structure and ion channel activity of the human respiratory syncytial virus (hrsv) small hydrophobic protein transmembrane domain two different conformations in hepatitis c virus p7 protein account for proton transport and dye release the small hydrophobic protein of the human respiratory syncytial virus forms pentameric ion channels hepatitis c virus p7 is critical for capsid assembly and envelopment agnoprotein of polyomavirus bk interacts with proliferating cell nuclear antigen and inhibits dna replication agnoprotein of mammalian polyomaviruses development and validation of a high-throughput screening assay for the hepatitis c virus p7 viroporin tgev corona virus orf4 encodes a membrane protein that is incorporated into virions development and application of hepatitis c reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (ns2) expressing fluorescent proteins or luciferase in modified jfh1 ns5a a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease influence of amantadine resistance mutations on the ph regulatory function of the m2 protein of influenza a viruses maturation of influenza a virus hemagglutinin-estimates of the ph encountered during transport and its regulation by the m2 protein the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug genotype-dependent sensitivity of hepatitis c virus to inhibitors of the p7 ion channel structural and energetic analysis of drug inhibition of the influenza a m2 proton channel free energy calculations on the two drug binding sites in the m2 proton channel nlrp3 inflammasome activation by viroporins of animal viruses respiratory syncytial viral-infection in children with compromised immune function a 2a/1b fulllength p7 inter-genotypic chimeric genome of hepatitis c virus is infectious in vitro e2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies influenza b virus requires bm2 protein for replication the evolution of human influenza viruses the molecular basis of the specific anti-influenza action of amantadine emerging influenza antiviral resistance threats ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence effect of ribavirin on hepatitis c viral kinetics in patients treated with pegylated interferon coronavirus structural proteins and virus assembly sars coronavirus: a new challenge for prevention and therapy treatment of chronic non-a non-b hepatitis with recombinant human alpha-interferon scrambling of the amino acids within the transmembrane domain of vpu results in a simian-human immunodeficiency virus (shivtm) that is less pathogenic for pig-tailed macaques a single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 vpu protein renders simian-human immunodeficiency virus (shivku-1bmc33) susceptible to rimantadine in silico investigations of possible routes of assembly of orf 3a from sars-cov membrane potential depolarization as a triggering mechanism for vpu-mediated hiv-1 release identification of hits as matrix-2 protein inhibitors through the focused screening of a small primary amine library influenza virus activates inflammasomes via its intracellular m2 ion channel s-layer fusion proteins-construction principles and applications cytoplasmic domain of influenza b virus bm2 protein plays critical roles in production of infectious virus influenza b virus bm2 protein is a crucial component for incorporation of viral ribonucleoprotein complex into virions during virus assembly encephalomyocarditis virus viroporin 2b activates nlrp3 inflammasome a pseudo-revertant of a sindbis virus 6k protein mutant, which corrects for aberrant particle formation, contains two new mutations that map to the ectodomain of the e2 glycoprotein the complete genome sequence of j virus reveals a unique genome structure in the family paramyxoviridae emerging theme: cellular pdz proteins as common targets of pathogenic viruses recombinant respiratory syncytial viruses with deletions in the ns1, ns2, sh, and m2-2 genes are attenuated in vitro and in vivo hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus respiratory syncytial virus (rsv) sh and g proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated rsv subgroup b mutant efficient replication of the genotype 2a hepatitis c virus subgenomic replicon antiviral efficacy of the novel compound bit225 against hiv-1 release from human macrophages. antimicrobial agents and cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors the human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release helix tilt of the m2 transmembrane peptide from influenza a virus: an intrinsic property intranasal gene transfer by chitosan-dna nanospheres protects balb/c mice against acute respiratory syncytial virus infection growth impairment resulting from expression of influenza-virus m2 protein in saccharomyces cerevisiae: identification of a novel inhibitor of influenza-virus anti-influenza virus agents: synthesis and mode of action influenza virus m2 protein is an integral membrane protein expressed on the infected-cell surface peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion a live attenuated severe acute respiratory syndrome coronavirus is immunogenic and efficacious in golden syrian hamsters genetic and functional heterogeneity of the hepatitis c virus p7 ion channel during natural chronic infection interaction between human bap31 and respiratory syncytial virus small hydrophobic (sh) protein structure of a conserved golgi complextargeting signal in coronavirus envelope proteins inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment function of the small hydrophobic protein of j paramyxovirus biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein internally located cleavable signal sequences direct the formation of semliki forest virus membrane proteins from a polyprotein precursor in vitro mutagenesis of a fulllength cdna clone of semliki forest virus: the small 6,000-molecular-weight membrane protein modulates virus release the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins induction of apoptosis by paramyxovirus simian virus 5 lacking a small hydrophobic gene processing in the hepatitis c virus e2-ns2 region: identification of p7 and two distinct e2-specific products with different c termini unravelling hepatitis c virus replication from genome to function the 6-kilodalton membrane protein of semliki forest virus is involved in the budding process replication of subgenomic hepatitis c virus rnas in a hepatoma cell line importance of conserved cysteine residues in the coronavirus envelope protein current progress in antiviral strategies the rsv f and g glycoproteins interact to form a complex on the surface of infected cells oligomerization state and supramolecular structure of the hiv-1 vpu protein transmembrane segment in phospholipid bilayers severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release the 3-dimensional structure of a hepatitis c virus p7 ion channel by electron microscopy a novel hepatitis c virus p7 ion channel inhibitor, bit225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-α-2b and nucleoside analogues structural and functional properties of the hepatitis c virus p7 viroporin transmembrane domain determinants of cd4 downregulation by hiv-1 vpu human immunodeficiency virus type 1 vpu protein is an oligomeric type i integral membrane protein treating viral hepatitis c: efficacy, side effects, and complications distinct domains of the influenza a virus m2 protein cytoplasmic tail mediate binding to the m1 protein and facilitate infectious virus production structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody vpu binds directly to tetherin and displaces it from nascent virions alphavirus 6k proteins form ion channels amino acid variations in hepatitis c virus p7 and sensitivity to antiviral combination therapy with amantadine in chronic hepatitis c vpu enhances hiv-1 virus release in the absence of bst-2 cell surface down-modulation and intracellular depletion two hepatitis c virus glycoprotein e2 products with different c termini the annual impact of seasonal influenza in the us: measuring disease burden and costs nmr structure and ion channel activity of the p7 protein from hepatitis c virus hepatitis c virus proteins: from structure to function influenza b virus bm2 protein has ion channel activity that conducts protons across membranes global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in e protein protects against lethal respiratory disease hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy emergence and pandemic potential of swine-origin h1n1 influenza virus subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis relevance of viroporin ion channel activity on viral replication and pathogenesis mechanisms of membrane permeabilization by picornavirus 2b viroporin viroporins: structures and functions beyond cell membrane permeabilization viroporins: structure and biological functions relating structure and function of viral membrane-spanning miniproteins unusual architecture of the p7 channel from hepatitis c virus expression and purification of coronavirus envelope proteins using a modified beta-barrel construct structural flexibility of the pentameric sars coronavirus envelope protein ion channel a rhoa-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3 protein-protein interactions: modeling the hepatitis c virus ion channel p7 influenza b virus bm2 protein is an oligomeric integral membrane protein expressed at the cell surface the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives new hepatitis c therapies: the toolbox, strategies, and challenges the hepatitis c virus life cycle as a target for new antiviral therapies structure and inhibition of the sars coronavirus envelope protein ion channel influenza m2 proton channels structural investigation of rimantadine inhibition of the am2-bm2 chimera channel of influenza viruses mechanism of drug inhibition and drug resistance of influenza a m2 channel construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras influenza virus m2 protein has ion channel activity the m2 proton channels of influenza a and b viruses combined transport of water and ions through membrane channels cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e nmr structures of membrane proteins in phospholipid bilayers mers: emergence of a novel human coronavirus. current opinion in virology rfi-641 inhibits entry of respiratory syncytial virus via interactions with fusion protein bap31 is a novel target of the human papillomavirus e5 protein easily accessible polycyclic amines that inhibit the wild-type and amantadine-resistant mutants of the m2 channel of influenza a virus 3-azatetracyclo[5.2.1.15,8.01,5]undecane derivatives: from wild-type inhibitors of the m2 ion channel of influenza a virus to derivatives with potent activity against the v27a mutant the small hydrophobic (sh) protein accumulates within lipid-raft structures of the golgi complex during respiratory syncytial virus infection the respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the rsv fusion protein a single polar residue and distinct membrane topologies impact the function of the infectious bronchitis coronavirus e protein the active oligomeric state of the minimalistic influenza virus m-2 ion channel is a tetramer the p7 polypeptide of hepatitis c virus is critical for infectivity and contains functionally important genotype-specific sequences triatoma virus recombinant vp4 protein induces membrane permeability through dynamic pores semliki forest virus 6k protein modifies membrane permeability after inducible expression in escherichia coli cells essential roles of leu/ile/phe-rich domain of jc virus agnoprotein in dimer/ oligomer formation, protein stability and splicing of viral transcripts infection by agnoproteinnegative mutants of polyomavirus jc and sv40 results in the release of virions that are mostly deficient in dna content protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein direct-acting antiviral agents and the path to interferon independence structure and mechanism of the m2 proton channel of influenza a virus identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells viroporins: structure, function and potential as antiviral targets tlr2/myd88/nf-kappa b pathway, reactive oxygen species, potassium efflux activates nlrp3/asc inflammasome during respiratory syncytial virus infection insight into the mechanism of the influenza a proton channel from a structure in a lipid bilayer modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus high frequency of resistant viruses harboring different mutations in amantadine-treated children with influenza the origin of hepatitis c virus mapping the interaction between the cytoplasmic domains of hiv-1 viral protein u and human cd4 with nmr spectroscopy the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles hiv-1 vpu protein antagonizes innate restriction factor bst-2 via lipid-embedded helix-helix interactions exacerbated innate host response to sars-cov in aged non-human primates hepatitis c virus p7 protein is crucial for assembly and release of infectious virions determinants of hepatitis c virus p7 ion channel function and drug sensitivity identified in vitro inhibition of hepatitis c virus p7 membrane channels in a liposome-based assay system structural basis for the function and inhibition of an influenza virus proton channel the alphaviruses: gene expression, replication, and evolution hiv-1 vpu-an ion channel in search of a job a novel gene of hiv-1, vpu, and its 16-kilodalton product daclatasvir plus sofosbuvir for previously treated or untreated chronic hcv infection viroporin activity of the jc polyomavirus is regulated by interactions with the adaptor protein complex 3 disruption of intracellular vesicular trafficking by agnoprotein is essential for viroporin activity and jc virus replication the human polyoma jc virus agnoprotein acts as a viroporin emergence of amantadine-resistant influenza a viruses: epidemiological study influenza virus m2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport functional analyses of gb virus b p13 protein: development of a recombinant gb virus b hepatitis virus with a p7 protein functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene the subcellular localization of the hepatitis c virus non-structural protein ns2 is regulated by an ion channel-independent function of the p7 protein the sars coronavirus e protein interacts with pals1 and alters tight junction formation and epithelial morphogenesis functional role of human immunodeficiency virus type 1 vpu detection of drug-induced conformational change of a transmembrane protein in lipid bilayers using site-directed spin labeling do viral proteins possess unique biophysical features? use of a single glycine residue to determine the tilt and orientation of a transmembrane helix. a new structural label for infrared spectroscopy conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of sars coronavirus envelope protein model of a putative pore: the pentameric α-helical bundle of sars coronavirus e protein in lipid bilayers protein-protein interactions of viroporins in coronaviruses and paramyxoviruses: new targets for antivirals? viruses the transmembrane oligomers of coronavirus protein e human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation rhinovirusinduced calcium flux triggers nlrp3 and nlrc5 activation in bronchial cells the 9-kda hydrophobic protein encoded at the 3 0 end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated the interferon-induced protein bst-2 restricts hiv-1 release and is downregulated from the cell surface by the viral vpu protein coxsackie b3 virus protein 2b contains a cationic amphipathic helix that is required for viral rna replication emerging antiviral strategies to interfere with influenza virus entry nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes coronavirus e protein forms ion channels with functionally and structurally-involved membrane lipids dogsitescorer: a web server for automatic binding site prediction, analysis and druggability assessment production of infectious hepatitis c virus in tissue culture from a cloned viral genome hydrocarbon-stapled peptides: principles, practice, and progress discovery of spiro-piperidine inhibitors and their modulation of the dynamics of the m2 proton channel from influenza a virus exploring the requirements for the hydrophobic scaffold and polar amine in inhibitors of m2 from influenza a virus molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza a virus m2 discovery of novel dual inhibitors of the wild-type and the most prevalent drug-resistant mutant, s31n, of the m2 proton channel from influenza a virus solution structure and functional analysis of the influenza b proton channel structural and dynamic mechanisms for the function and inhibition of the m2 proton channel from influenza a virus structure and inhibition of the drug-resistant s31n mutant of the m2 ion channel of influenza a virus characterization of a small, nonstructural viral polypeptide present late during infection of bhk cells by semliki forest virus recombinant respiratory syncytial virus bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees function of small hydrophobic proteins of paramyxovirus hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication therapeutic efficacy of amantadine hcl and rimantadine hcl in naturally occurring influenza a2 respiratory illness in man intracellular proton conductance of the hepatitis c virus p7 protein and its contribution to infectious virus production flipping in the pore: discovery of dual inhibitors that bind in different orientations to the wild-type versus the amantadine-resistant s31n mutant of the influenza a virus m2 proton channel compensatory mutations in e1, p7, ns2, and ns3 enhance yields of cell culture-infectious intergenotypic chirneric hepatitis c virus mouse hepatitis virus gene 5b protein is a new virion envelope protein inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns1 gene structural characterization of the human respiratory syncytial virus fusion protein core 2 modeling the membrane environment has implications for membrane protein structure and function: influenza a m2 protein this work has been funded by singapore moe tier 1 grant rg 51/13 (j.t.). key: cord-318853-mxyxwkhx authors: sallie, richard title: replicative homeostasis ii: influence of polymerase fidelity on rna virus quasispecies biology: implications for immune recognition, viral autoimmunity and other "virus receptor" diseases date: 2005-08-22 journal: virol j doi: 10.1186/1743-422x-2-70 sha: doc_id: 318853 cord_uid: mxyxwkhx much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. rna polymerases (rna(pol)) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. thus, rna(pol )causes more morbidity and premature mortality than any other molecule. the extraordinary genetic heterogeneity defining viral quasispecies results from rna(pol )infidelity causing rapid cumulative genomic rna mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking rnapol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral rna is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. this mechanism – "viral receptor disease (vrd)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type ii diabetes mellitus, and some forms of obesity. viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations. many of the world's population suffer from acute and chronic viral infection. the two common types of chronic viral hepatitis (cvh), hepatitis b (hbv) and c (hcv) are major causes of death and morbidity; conservative esti-mates suggest 400 million people are persistently infected with hbv, while hcv may infect a further 200 million. annually, in excess of two million people will die from cirrhosis or liver cancer caused by cvh, and many more suffer chronic ill health as result. during the 20 years since the human immunodeficiency virus (hiv) was identified, perhaps 40 million people have become infected worldwide and each year about a million die from resulting immunodeficiency and consequent opportunistic infections, particularly tuberculosis, and other complications. poor countries bear a disproportionate burden of disease caused by these viruses that further exacerbate poverty through pervasive economic disruption and diversion of limited resources to healthcare and disease control. emerging viral pathogens including west nile virus (wnv), the sars coronavirus, endemic viruses like murray valley, japanese, and other encephalitis viruses, dengue and yellow fever, and seasonal influenza, hepatitis a (hav) and e (hev) cause enormous further morbidity and mortality, while pandemic outbreaks of virulent influenza strains remain a constant threat. together, these viruses probably kill more people every ten days than the boxing day tsunami. rna viral infections, including foot and mouth, bovine viral diarrhea virus (bvdv) and hog cholera virus (hchv), cause similar devastation of animal populations with enormous economic consequences. rna polymerases generate massive genetic variability of rna viruses and retroviruses that circulate within infected hosts as vast populations of closely related, but genetically distinct, molecules known as quasispecies. after translation, this genetic variability causes near-infinite antigenic heterogeneity, facilitating viral evasion of host defences. tuberculosis, malaria and other cellular pathogens also express broad cell-surface antigenic heterogeneity, generated by dna-dependent rna pol . thus, rna polymerases probably cause more morbidity and premature mortality in man, and other animals, and greater economic loss, than any other molecule. despite a depressing global epidemiology that strongly suggests otherwise, the immune system is thought to "control" viruses. what practical meaning does "immune control" have for the individual? there is no argument for hbv, and other viruses, high affinity antibody, generated by prior vaccination or other exposures and directed against neutralizing epitopes, will prevent hbv infection (excepting vaccine escape mutations [1, 2] ), in part by blocking viral ligand interaction with cell receptors, or that most patients exposed to hbv develop neutralizing antibodies (hbsab), clear hbsag from serum, and will normalize liver function long term. however, even patients who develop robust immune responses to hbv, defined by high-affinity antihbsab and specific antiviral cytotoxic t cell (ctl) responses, will have both "traces of hbv [3] ... many years after recovery from acute hepatitis" [3] and transcriptionally active hbv demonstrable in peripheral blood mononuclear cells (pbmcs) [4] . furthermore, occult hbv is detected in liver tissue of patients with isolated antihbc (i.e. hbsag/hbsab negative) [5] and in patients with hbsag-negative hepatocellular carcinoma [6] suggesting, at least some patients, hbv in may persist irrespective of any immune responses, implying long term latency and low level basal replication may be a survival/reproductive strategy for hbv. for most patients, acute hcv or hiv infection results in life-long viral persistence. although many patients develop immunological responses, including specific antibody and ctl reactivity to various viral antigens, these responses have little discernible impact on either hcv or hiv replication that occurs essentially unchecked at rates estimated between 10 10 and 10 12 virions per day [7, 8] , indefinitely, while progressive destruction of liver or immune cells proceeds, commonly resulting in cirrhosis or liver cancer (for hcv) or death from immune deficiency (for hiv). evidence that prior hcv infection confers no protective immunity against heterologous hcv infection in humans [9] or chimpanzees [10] or against either homotypic [11] or heterotypic [12] human reinfection, confirmation that active hcv infection persists long after either apparent spontaneous [13] or treatmentinduced [14] viral clearance, or that vaccines causing specific antiviral b and t cell responses fail to protect against infection in animals [15] , and that antibodies to hcv envelope protein e2 are only detected in animals with persistent infection [16, 17] , further undermines the potency of "immune control" and suggests, at least for patients with hcv, the definition of "control" may need to broadened significantly. based on observations that stronger specific cd4/cd8 immune responses with t-helper (th1) cytokine profiles are found more frequently in patients with self limiting viral infections than those who develop chronic viral carriage [18, 19] it is thought ability to mount robust adaptive immune responses predicts viral clearance while failure to do so results in chronic viral carriage [20] . however, detailed and very painstaking studies, albeit in small numbers of chimpanzees [21] and patients following antiviral therapy [22] , have failed to demonstrate any relationship between t cell responses and viral clearance. although development of th1and other immune responses are certainly temporally and, probably, causally related to reduced viral replication and viral clearance the assumed direction of causality (immune response -> reduced viral replication), is not proved by the fact those responses develop, post hoc ergo propter hoc, as comforting a conclusion as it may be to reach. the first part of this paper explores the impact of rna pol fidelity on quasispecies behaviour, specifically in mediating immune avoidance during acute hcv infection. we suggest the primary event causing reduction in viral replication is inhibition of rna pol processivity by variant viral proteins, specifically envelope and envelope-related proteins. we also suggest that immune responses to viruses are thwarted initially by broad antigenic diversity generated by low rna pol fidelity but develop, when they do, after viral replication falls (because of reduced rna pol processivity) and polymerase fidelity increases -linked events that occur because of replicative homeostasisthus restricting antigenic diversity sufficiently to permit focused immune recognition. we further suggest immune responses strategically exploit replicative homeostasis to force viruses to reveal critical dominant antigenic epitopes, facilitating progressively more focused immune responses. the second part explores the ineluctable consequence of viral rna quasispecies: that is, translation of rnas into protein quasispecies with a spectrum of phenotypes and unpredictable properties, among which may be disruption of the cell surface receptors that viruses co-opt for cell entry. this innate property of viral quasispecies may explain a wide variety of diseases apart from viral autoimmunity. acute hcv and hbv infection have characteristic kinetics of viral replication, adaptive immune responses, and cause predictable tissue injury, reflected in elevated serum aminotransferases. these kinetic and transaminase responses are summarized schematically for patients with persistent infection (figure 1) [23]. initial hcv replication is very rapid and viral load increases exponentially until about week 4, at which point viraemia increases more slowly, and asymptotically, towards ~10 7 genome equivalents (geq)/ml by weeks 7-8 (these kinetics alone suggesting competitive inhibition of rna pol ). this exponential increase of viral rna in serum reflects explosive dissemination of virus in tissues, detectable by in-situ hybridisation throughout hepatocytes, including the nuclei, within days of infection [24] . viral replication declines rapidly from weeks 10-11 to weeks 14-16 falling by 10 2-3 geq/ml but lower level (~10 5 geq/ml) fluctuating replication persists, generally indefinitely, thereafter. by contrast, neither hbv dna nor hbv antigens are detectable in either viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection figure 1 viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection. data summated from figure 1 [29] and modified to represent typical patients with chronic viral persistence. note: a) high level hcv replication for 6-8 weeks prior to any immune responses, b) onset of humoral immune response well after down-regulation of viral replication [34] , and c) transaminase peaks occurs ~ 2weeks later. both hbv and hcv are non-cytolytic and viral clearance from hepatocytes, as well as hepatocyte injury, thought to be immune mediated. however, for both hbv and hcv the brisk fall in viral replication following acute infection paradoxical hcv replication kinetics figure 2 paradoxical hcv replication kinetics. if host immune clearance forces (i c , black arrows) reduce viral replication acutely (point a), then they must exceed viral expansive forces (v e , grey arrows) at that point. at equilibrium (e.g. points b through d), viral concentrations (-) and, therefore, viral forces, have fallen by 10 2-3 hence, immune forces i c must fall by >10 2-3 from a to b for equilibrium to develop. there is no evidence this happens. http://www.virologyj.com/content/2/1/70 precedes the peak of transaminase rise by at least two weeks (figure 1). if falling viral replication is due to adaptive immune responses causing hepatocyte lysis the transaminase peak should either precede or be coincident with falling replication. this temporal relationship is also inconsistent with the belief immune factors cause the falling replication seen during acute hcv or hbv, and is analagous to non-cytolytic reductions of viral replication observed for both hbv and lymphocytic choriomeningitis virus (lcv) experimentally, that suggested either [unspecified] antiviral mechanisms are operative [30,31], or that auto-inhibition of rna pol by viral mechanisms (replicative homeostasis) occurs [28] . however, if other non-cytopathic host anti-viral mechanism(s) are responsible, the kinetic paradox implies their potency falls significantly between points a and b. hepatitis c replication kinetics and their relationship to immune responses are well documented [32,33] but reveal an unexplained paradox. despite high level viral replication, adaptive cellular immune responses to hcv are completely undetectable for at least 7-10 weeks [33] after infection, while humoral responses are rarely detected before 12-14 weeks [34] , and in some patients [35] , and some chimpanzees [36] , are never detected at all. an exhaustive and very careful review of the clinical and experimental data relating adaptive immune response and hcv replication kinetics has been published recently [29] . seeking to rationalize the enigma posed by a complete lack of immune responses to hcv replication of ~10 6-7 geq/ml at week 6 but [variable] immune responses to replication at ~10 5 geq/ml after week 14, the authors conclude "..[the data]...appear[s] to be consistent with the interpretation that hbv and hcv are ignored by the adaptive immune system for about 2 months after primary infection" and "[in hcv].. the adaptive response seems to really ignore for several weeks a substantial quantity of virus (at least 10 6 copies/ml)..". this is certainly an accurate synthesis of an extensive and highly complex literature but does it make any sense? if adaptive immune responses really ignore high level hcv replication for two months, as suggested, then the following mechanism(s) are implied: a) an accurate mechanism for prompt detection of infection; b) a timing mechanism; c) a trigger mechanism for immune responses independent of any viral factor (given levels of virus are greater before immune recognition than afterwards the trigger for immune response must be either non-viral or falling (!) viraemia); and, as cytomegalovirus (cmv)-specific cd4(+) t cell responses arise within 7 days of cmv infection [37] ; d) a mechanism allowing the immune system to differentiate hcv from cmv and other viruses (and reasons to do so). while possible, this seems unusually inelegant and pointlessly counterproductive, especially as events soon after infection probably determine whether virus is cleared or chronic infection develops. it is much more likely that adaptive cellular or humoral immune responses do not develop in the first 6-7 weeks of hcv infection simply because the virus isn't "seen". why should hcv replicating at 10 6-7 geq/ml at week 6 be invisible to the immune system but visible when replicating at 10 5 geq/ml long term? dissection of this problem requires explicit analysis of what is being measured and how. assay of hcv rna and detection of hcv by immune responses measure two quite different things. quantitation of hcv is typically performed by branch-chain cdna assay (bdna) or quantitative pcr (qpcr) using probes or primers complementary to conserved 5'untranslated (5'utr) hcv rna sequences. immune responses to hcv typically "measures" envelope proteins translated from envelope-encoding rna (eerna) sequences and are directed at specific antigenic amino acid sequences and polypeptide conformations, not total viral envelope protein concentrations. while concentrations of 5'utr rna will be proportional to eerna concentrations in any given sample, they may not be identical for two reasons; i) rna transcription may prematurely terminate making 5'utr rnas relatively more prevalent than eernas and ii) hcv 5'utr is highly conserved, while eerna s are less constrained, making hybridization efficiencies of pcr primers or bdna probes greater for 5'utr rnas than for the population of eernas, causing relative under-estimation of true envelope rna concentration 1 . nonetheless, as 5'utr hcv rna concentrations will be proportional to eerna concentration, the question remains; why should envelope proteins translated from eerna sequences present at concentrations corresponding to ~10 5 5'utr geq/ml at 16 weeks be visible immunologically, but envelope proteins derived from eerna sequences corresponding to ~10 6-7 5'utr geq/ml at 4-6 weeks remain unseen? quasispecies biology, specifically variable rna pol fidelity, replicative homeostasis, and sequence-specific requirements for both genetic and immunological detection suggest an answer. rna viruses replicate by copying antigenomic templates, a process catalysed by rna pol , an enzyme lacking fidelity or proof reading function [38] [39] [40] [41] . theoretically, an rna viral genome like hcv (about 9200 bases) could assume any of 4 9200 (about 8.95 × 10 5538 ) possible sequence combinations exceeding, by some margin, population estimates of protons in the known universe (about 10 80 ), meaning the potential complexity of rna viral quasispecies is infinite, for all practical purposes. an rna pol fidelity rate of 10 -5 errors per base copied predicts at least one and as many as 10 (estimated for hiv) [39] genomic mutations will be introduced during each cycle of replication. furthermore, as hcv replication results in synthesis of ~10 12 virions per person per day [8] , on average, mutations will develop at each genomic locus ~10 7 times/day, while the probability any two genomes synthesized consecutively will be identical is about 10 -6 . the sum effect is inexorable accumulation of genomic mutationsthat, by itself, should threaten replicative fitness because of muller's ratchet [42] -and progressive dilution of wildtype genomes (figure 3), processes that make long-term stability of rna virus quasispecies highly paradoxical [43] . as argued previously, a combination of selective genomic replication and variable rna pol fidelity, both mediated by replicative homeostasis, act together to prevent rna quasispecies extinction [28] . the phenotypic consequences of viral quasispecies biology may be more important. progressive divergence of genomic rna sequences away from wild-type sequences caused by rna pol infidelity generates a massive population of closely related, but genetically distinct, rna molecules (figure 3), an effect operative at all scales from each open reading frame (orf) to whole virus species. a quasispecies of orf rnas has but one inevitable outcome; translation of a quasispecies of viral proteins with a vast and highly variable spectrum of phenotypes, some subtly nuanced, others grossly defective. furthermore, mutations simplified, two dimensional clade diagram of hyperdimensional viral rna and protein sequence-space figure 3 simplified, two dimensional clade diagram of hyperdimensional viral rna protein sequence-space. because of rna pol (p) infidelity and müller's ratchet, mutations ( ) are introduced into each rna template synthesized, and progressively accumulate, resulting in an rna quasispecies with sequence progressively divergent from consensus sequence. translation results in a spectrum of proteins ( , , , etc.) with properties that also vary progressively from wild-type sequence ( ) to highly variant proteins ( , , etc.). some rnas will be so abnormal that translation or replication fails or is truncated ( ), while others will code for grossly defective proteins ( , etc.). that create new, or obliterate pre-existing, start or stop codons in a significant proportion of rnas, will cause translation of highly unusual and heterogeneous proteins, particularly during high-level viral replication, a phenomenon that may explain hbeag. viral quasispecies cannot, and will not, produce homogeneous proteins with predictable and consistent phenotypic and antigenic properties. while rna pol infidelity will cause progressive divergence of copied sequences away from wild-type or consensus sequences, the probability of any particular sequence arising will fall dramatically with increasing genetic distance from that consensus sequence (figure 4), allowing conceptual representation of the resulting genomic (and consequent phenotypic) diversity as a frequency distribution curve, with increasingly variant sequences surrounding a 'centre of gravity of replication', formed by wild-type sequences. viral quasispecies occupy hyperdimensional sequence-spaces, hence any physical representation is necessarily simplified, but because mutation away from wildtype sequences is equally probable in all directions, variant rna and protein frequencies will be normally distributed and the standard deviation (sd, σ) -insofar as 'normal' or 'standard' can be applied to a hyperdimensional space -of that distribution will be a function of rna pol fidelity; if rna pol is completely faithful, the rnas and proteins will be monoclonal and σ = 0; if rna pol has no fidelity, rna will be synthesised randomly, and all rna and consequent protein sequences will arise with equally probability, therefore σ = ∞. while viral rna and related protein sequences are theoretically unconstrained (at least before any consideration of functionality), the sequence specificities of any reagents used in their detection (bdna probes, pcr primers, mabs etc) are not, by definition, and their specificity and the efficiency with which they detect variant molecules will fall progressively the further those variant sequences are from the consensus sequence. a zone of 'reagent specificity' may therefore be defined probably encompassing wild type and some variant sequences, but there will exist some rna sequences and corresponding proteins of any quasispecies that are undetectable with these sequence-specific reagents. a threshold of detection of any assay (including immune detection) may similarly be defined; rna or protein sequences present at concentrations below this conceptual level being undetectable by that particular assay. the hcv "early replication" paradox now partially resolves; the 5'utr sequences are both highly conserved and common to virtually all rnas in the quasispecies, therefore, the 5'utr concentration -that is, the common measure of hcv viraemia -corresponds to the area under the frequency distribution curve. by contrast, envelope rna sequences (and related envelope proteins) are not so constrained and their relevant concentrations (i.e. whether or not that rna or protein sequence is detectable) corresponds to the frequency of that specific sequence in the quasispecies and that, in turn, depends on rna pol fidelity; if rna pol fidelity is low, the frequency or concentration of any particular rna or protein sequence will also be low and may be below the detection threshold, while increasing rna pol fidelity may increase sequence frequency [i.e. the concentration of specific proteins] above detection threshold. but why should specific eerna sequence frequencies -in other words, hcv rna pol fidelity -increase after week 8, facilitating adaptive immune responses? viral autoregulation, specifically replicative homeostasis, provides an answer. interactions among species, whether between humming birds and flowering plants, primitive viroids and prokaryotic cells or hcv and man, results in an unremitting process of adaptation and responsive counter-adaptation -in effect, a molecular arms race -for each species just to maintain ecological parity. the price of survival for a species is continual evolution. survival, for viruses, requires cell entry, a precondition long antedating necessity to evade more complex host defenses, including interferons and other cytokines and adaptive immune responses, while for cells, and complex cellular organisms, cell wall defenses, including receptor polymorphisms, form a principal barrier against viral invasion. viral survival -effectively meaning rna pol survival -on an evolutionary timescale, as argued previously [28, 44] , requires control of mutation and replication rates in a manner adaptively responsive to constantly changing biota and this implies dynamic linkage of rna pol fidelity and processivity with quasispecies phenotypic and antigenic diversity, meaning an autoregulatory linkage -replicative homeostasis -between rna pol fidelity and processivity and envelope proteins, as argued previously [28] . by definition, evolutionary co-adaptation occurs in response to adaptations in locally prevalent interacting species. natural selection for beak variation(s) in darwin's finches occurs as a consequence of concrete survival benefits these variations -mediating, for example, enhanced food harvesting interactions with other variable plant or animal species -confer to individual galapagos island birds, rather than any inexorable hypothetical 'improvement' in beak function for finches in general. if a species is widely distributed in space, but population mixing is slow or incomplete, locally prevalent interactions with other species will vary and regional genetic variations will arise and be maintained, hence progressive divergence from the original genotype (speciation) may result. for viruses, and their hosts, genetic variations -reflected in viral genotype and cell surface polymorphisms and resulting disease susceptibilities -would be predicted, and are observed [45] [46] [47] [48] [49] [50] , to have frequencies that vary geographically. consider the following; an enzyme (e) functioning in a closed system synthesizes either product a or b that both interact with e to influence output such that a:e interactions cause production of b, while b:e interactions pro-duce a. irrespective of starting conditions (excluding substrate exhaustion and product inhibition), an equilibrium will eventually develop ( figure 5 ) with the relative concentrations of a:b determined by the relative association constants (k) of a:e (k a:e ) and b:e (k a:b ) and the velocity (ν) of production of a from b:e (ν a ) and b from a:e (ν b ). removal or addition of either a or b will alter equilibrium conditions but not the fact equilibrium is reached; if a is removed, for example, the increased two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent spe-cificity (red shading) and threshold of detection (tod) of any assay figure 4 two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent specificity (red shading) and threshold of detection (tod) of any assay. as mutations ( , ) accumulate and rna sequence progressively diverges from consensus sequence (0) the probability of that rna sequence and corresponding protein (e.g. envelope, env.) arising falls rapidly. standard deviation (σ) of frequency distribution is proportional to rna pol fidelity. frequency genetic distance 0 threshhold of detection (tod) reagent specificity ♦ ♦ frequency of b:e interactions will cause compensatory increased a synthesis; in this sense enzymatic autoregulation occurs. intuitive analysis suggests that enzymes acting in a milieu of increasing concentrations of inhibitory molecules become progressively less processive until reduced enzyme output is insufficient to further inhibit enzyme activity, and an equilibrium state is reached. considering viral replication, if alteration of rna pol fidelity causes synthesis of either wild-type or variant rna sequences (simplified, as a continuum between these two must exist) that are subsequently translated into either wild-type or variant polypeptides that then interact with rna pol such that wild-type: rna pol are high affinity interactions that induce rapid, low fidelity rna pol replication while variant protein: rna pol interactions are low affinity and cause high fidelity rna pol replication at low rate then an equilibrium will eventually develop. hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of rna pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. stable, highly reactive equilibria not only develop as a result of rna pol /envelope interactions and viral autoregulation, there is no option but for this to occur. generation and maintenance of polymorphisms, that is, replacement of existing genes -that, by operational dar-winian definition, have proved their functionality and evolutionary fitness by surviving to reproduce -with variant genes (polymorphisms) of uncertain functionality, fitness or overall compatibility within an organism, is an evolutionary strategy that will only be sustained on a geological timescale if new polymorphisms confer survival benefits to organisms that exceeds the risks and metabolic costs of generating and sustaining those polymorphisms. for primitive cells, lacking functional humoral, cellular or cytokine defense mechanisms, development of cell-surface protein polymorphisms is an obvious adaptive strategy to thwart invasion by primitive viruses. like other adaptive strategies, cell-surface polymorphisms are strongly selected for, and have been highly conserved over deep time, and are found in all organisms from primitive prokaryotic cells [51] and thermophilic bacteria [52] through to plants [53] as well as mammalian cells, strongly suggesting a critical evolutionary function. the lock and key hypothesis, for which there is very considerable evidence [54] [55] [56] [57] , first proposed by jbs haldane [58] , contends polymorphisms arise, and are maintained, as protection against cellular parasitism, particularly by viruses 2 . while dna-encoded protein polymorphisms form necessary defenses against viral access, they may not be sufficient; a quasispecies of cells (e.g. the liver) expressing similar and static receptor variations renders those cells vulnerable to sustained attack from any virus that successfully invades any one cell, and further dynamic modification of cell receptors, triggered by viral infection and mediated at the transcriptional level by modulation of dna dependent rna polymerase fidelity in nearby uninfected cells, by a mechanism similar to replicative homeostasis would seem possible. a clear, unambiguous band at the "c" position on a sequencing gel, causes "cytosine" to be assigned to that genetic locus. but does this certitude reflect reality, at least for viral rna quasispecies? direct pcr sequencing is an "averaging" procedure revealing the most frequent nucleotide at any particular locus. however, nucleic acids and proteins cannot express 'an average', and discrete quanta of specific nucleotides or amino acids are present at every locus. a typical clinical serum sample, containing 4 × 10 5 geq/ml hcv and mutating at 10 -5 substitutions/base, will contain examples of each possible nucleotide at every locus, but most variations will remain undetected during sequencing or any other method of quasispecies analysis. analysis of cloned dna gives cleaner data than pcr sequencing but if 100 clones (and multiple hcv quasispecies clones are highly unlikely to be identical) provides definitive sequence, would we process the 101 st to reveal different and, potentially, critical sequence variations? and if we did, how would we recognise its importance? is important sequence likely to be present at frequencies of http://www.virologyj.com/content/2/1/70 < 1%? infectious virions containing, presumably, fulllength functional genome and corresponding wild-type proteins, are often outnumbered by ~6 × 10 4 :1 in serum by defective and non-infectious particles [53] that presumably do not, suggesting that important genetic sequence and associated phenotype may occasionally be extremely rare. how the immune system recognizes uncommon, nondescript, but important protein sequences in a featureless background of similar molecules is a non-trivial problem for which replicative homeostasis may suggest a solution. replicative homeostasis, described in detail elsewhere [28, 44] , is an epicyclic mechanism of viral autoregulation that results when viral proteins, notably envelope (env), influence rna pol fidelity and processivity. the predicted consequences of replicative homeostasis for rates of intracellular viral replication and mutation, cellular expression of viral proteins and immunological responses occurring because of replicative homeostasis is represented schematically (figures 6, 7). during early viral replication in a naive cell devoid of inhibitory molecules (panel a, a), high affinity wild-type envelope:polymerase interactions predominate, causing rapid low-fidelity polymerase activity resulting in rapid synthesis of variant viral rnas and subsequently proteins, hence causing a broad spectrum of viral proteins to be expressed on the cell surface, each at concentrations below the threshold of immune detection (tod). rna pol infidelity ensures synthesis of variant viral rnas and proteins predominates early, hence variant protein molecules progressively accumulate within cells relative to wild-type viral molecules (panels b-d) and increasing the probability of variant viral envelope:rna pol interactions. variant viral envelope:rna pol interactions causing progressive inhibition of rna polymerase processivity and increasing rna pol fidelity, reducing diversity of viral rnas synthesized and progressively restricting dynamic progression of rna pol functional properties, processivity () and fidelity () predicted by replicative homeostasis figure 6 dynamic progression of rna pol functional properties, processivity ( ) and fidelity ( ) predicted by replicative homeostasis. initial state (a, corresponding to panel a, figure 7 ): in a newly infected cell, high-affinity wild-type:rna pol interactions will predominate resulting in high rna pol processivity but low fidelity causing high-level viraemia with broad virus phenotypic spectrum, maximizing cell tropism. intracellular accumulation of variant viral proteins (b, c.f. panel b, figure 7 ) reduces rna pol processivity but increases fidelity reducing viral rna synthesis and consequently, viraemia before a dynamic, fluctuating equilibrium (c, c.f. panel c or d, figure 7 ) develops in which inhibition of rna pol by variant viral proteins is balanced by increases in rna pol fidelity (with consequent synthesis of wild-type viral products tending to cause high rna pol processivity). conceptual progression of intracellular viral replication events, including variable rna pol fidelity and processivity, restriction of antigenic diversity and immune recognition under influence of replicative homeostasis env viral protein diversity expressed on the cell surface (panels b to d), increasing cell-surface concentrations of individual viral proteins above the threshold of detection (panels c, c) at which point a polyclonal immune response develops. development of low-affinity polyclonal blocking antibodies, restricting cellular egress of viral proteins, further increasing intracellular concentrations of variant envelope proteins, still further increasing the probability of variant viral envelope:rna pol interactions and inexorably further restricting antigenic diversity increasing relative expression of wild-type proteins thus further exposing these epitopes to immune surveillance and facilitating specific high-affinity immune responses, including cytotoxic t cell responses, (d,d) to wild-type proteins. thus, the immune responses can strategically utilize replicative homeostasis to force viruses to reveal important and dominant wild-type epitopes, but those responses develop initially as a consequence of restriction of rna pol fidelity that occur because of replicative homeostasis. high-affinity responses further deplete intracellular concentrations of wild-type proteins, progressively reducing wild-type envelope:rna pol interactions, greatly reducing rna pol processivity to the point of viral latency (e,e), caused by variant viral envelope:rna pol interactions. the hepatitis c "early replication" paradox now resolves completely when considered in the context of replicative homeostasis; initial high level hcv replication (due to high rna pol processivity) remains immunologically undetectable for 6-8 weeks, or more, because of low rna pol fidelity causing a broad spectrum of hcv envelope proteins each expressed on cell surfaces at concentrations below the threshold of detection even while viraemia, reflected in concentrations of 5'utr rna common to each rna species, are present at 10 6-7 geq/ml. as replication progresses, intracellular accumulation of variant viral proteins increase rna pol fidelity but decrease processivity (replicative homeostasis), downregulating hcv replication and reducing viraemia but restricting antigenic diversity and increasing expression of hcv envelope proteins to beyond the threshold of immune detection. furthermore, the temporal tissue injury (aminotransferase) paradox also resolves in this light: focussed immune recognition (including cytotoxic t cell responses) doesn't develop until after viral antigenic diversity is restricted by replictive homeostasis the transaminase peak would not be expected until after viral replication falls due to autoinhibition of rna pol processivity. varying expression of viral proteins by modulating rna pol fidelity to facilitate immune escape would seem a useful evolutionary adaptation that might be retained by more complex organisms, including cellular pathogens like tuberculosis and malaria, to optimize their stability within hosts. this mechanism of immune avoidance might also explain maternal-foetal tolerance. the human foetus maintains a stable parasitic existence during gestation (and, i expect, to university age and beyond) that is tolerated despite normal maternal immune responsiveness in general and lack of specific tolerance to paternal antigens in particular, a situation made more problematic as expressed foetal antigens are predominantly of paternal origin [54] . while immunological isolation of foetal tissue by the placental trophoblastic layer [55] , and placental display of hla-g [56] , probably contribute to foetal stability in the face of a potentially robust immune attack, neither mechanism would explain persistence of viable foetal nucleated red blood cells within the maternal circulation [57] in quantities sufficient to permit clinical prenatal diagnosis [58] . is it possible foetal tolerance is mediated by regulating the fidelity of foetal dna dependent rna transcriptases to ensure any cell-surface antigens are expressed heterogeneously and at levels below the threshold of maternal immune responsiveness? for many classical autoimmune disorders, including primary biliary cirrhosis [59] , multiple sclerosis, and rheumatoid arthritis, convincing epidemiological evidence [60] , including cases clustering [61, 62] , strongly suggests these diseases are triggered by infectious agents in genetically predisposed individuals. in others, such as diabetes mellitus, tantalizing epidemiological [63] , clinical [64] and laboratory [65] evidence has implicated enteroviruses, but has suggested viral-triggered autoimmune processes, rather than cytolytic destruction of pancreatic betacells [66] . similar circumstantial evidence exists for myocarditis, demyelinating diseases, myositis and other post infectious inflammatory disorders. when macfarlane burnet wrote autoimmunity arises from "inability to distinguish self from non-self" hbv, hcv, hiv and other viruses, now established to cause diseases with clear autoimmune features were unknown. viral infections, particularly hepatitis c -and its treatment with interferon -are associated with many varied autoimmune phenomena [67] , and thyroid disease [68] [69] [70] , diabetes mellitus [71, 72] , membranous, membranoproliferative and cryoglobulinemic glomerulonephritis, vasculitis and peripheral neuropathy [73] , and autoimmune gastritis [74] are all very well documented, although the mechanism(s) are unknown and causality is certain. classical serological markers of autoimmunity, including rheumatoid factor, antinuclear antibodies (ana), anticardiolipin, antithyroid, anti-liver/kidney/microsomal antibodies (anti-lkm), as well as hcv/anti-hcv immune complex formation and mixed essential cryoglobulinemia are common accompaniments of chronic hcv infection [73] , raising the obvious question of whether all "autoimmunity" has a viral basis. indeed, zinkernagel's pragmatic and subtly anticipatory; "if we know the infection, we call the disease immunopathologically mediated; if we do not recognize or know it, we call the disease autoimmune [75] " fully reflects recent explosive growth of information and the deeper questions this information poses. rna virus quasispecies biology, specifically the generation of rna quasispecies by rna pol , and translation of these immensely variable rnas into protein quasispecies, suggests an immediate solution to the problem of viral autoimmunity and, by extension, to autoimmunity in general, as well as suggesting a unifying hypothesis to explain other diseases known to have multi-factorial aetiologies that include inflammatory components -such as coronary artery disease -in addition to other diseasesincluding schizophrenia and some forms of depressionthat currently lack rational and coherent pathogenic explanations. viruses are known to co-opt cell surface molecules, including lectins, hormone receptors and cell signaling molecules, to access cells. receptors, and other cell surface molecules, identified as "viral receptors"or to specifically interact with viral proteins include prostaglandins, catecholamines and acetylcholine receptors [76] , serotonergic neurotransmitters (5ht) [77] , endothelial cell glycoproteins [78] , insulin-like growth factor (igf-ir) and its major signaling molecules insulin receptor substrates irs-1 [79] and irs-2 [80] , epidermal growth factor (egf) [81] , neurotrophin receptor [82] , thyroid hormone receptor tralpha1 [83] , an immunoglobulin protein superfamily [84] , low density lipoprotein (ldl) receptors [85, 86] , transferrin receptor (tfr) [87] , asialoglycoprotein receptor (asgp-r) [88, 89] , and angiotensin-converting enzyme 2 [90] , to cite biologically diverse examples. of necessity, some receptor affinity studies have used cloned viral protein ligands, an artificial situation that cannot approach the phenotypic complexity of rna viral protein quasispecies. nonetheless, variable virus receptor affinities [91, 92] , evolutionary adaptation of receptor affinity [93] , emergence of escape variants with altered receptor affinities [94] , temporal alteration of receptor usage [92] and capacity to exploit alternative entry pathways [95] have all been confirmed, suggesting viruses are capable of generating highly plastic ligands with very broad receptor affinities. if a virus co-opts a receptor for cell entry, then wild-type envelope (consensus sequence) epitopes, coded for by wild-type rna sequences, will probably form the common viral ligand. however, any viral rna quasispecies also contain a vast spectrum of rnas derived from, and similar to, envelope open reading frame (orf) consensus sequence, but variant from it. as the envelope orf quasis-pecies sequences progressively diverge from wild-type, the quasispecies of envelope proteins translated from these variant orfs will also, and inexorably, diverge in sequence, structure and biological function from wildtype envelope sequence proteins. some of these envelope proteins will be functionally identical, but others, and probably the vast majority, will range from subtly different to grossly abnormal, either due to major differences of sequence and/or chemical or steric amino acid incompatibility, or because of premature introduction of stop codons. even minor amino acid differences, as sickle cell anaemia illustrates, and has been confirmed specifically for viral receptor usage [96, 97] , may catastrophically alter a proteins' function with respect to co-opted viral receptors, with some having no binding affinity, while others will bind strongly and act as agonists, antagonists or competitive inhibitors of normal receptor function. variant and defective viruses, and their polypeptides, will be in vast molar excess compared to wild-type [53] but will exhibit similarly high antigenic variability, permitting escape from immune and other scavenger mechanisms. as many variant viral polypeptides will bind tightly to "self" receptors, but contain immunogenic non-self motifs, a polymorphic (because variant viral proteins will themselves be highly polymorphic due to the quasispecies process) immune response, apparently directed against "self" antigens, but actually targeting virus protein-receptor complexes virtually indistinguishable from normal cell receptors, will result causing apparent 'autoimmune' tissue damage. this mechanism suggests an explanation for common autoimmune phenomena. if a virus enters cells because wild-type envelope motifs interact with insulin, insulin receptor substrate [79, 80] , tsh or related molecules [83] , or acetylcholine [76] receptors, many variant envelope polypeptides, generated by envelope orf quasispecies rnas, would have similar receptor binding affinity, but may effectively disrupt receptor function, predictably causing impaired glucose tolerance or diabetes mellitus, thyroid dysfunction, or myasthenia gravis with secondary resistance to, and elevation of, the normal hormone ligand (insulin, tsh etc.). the expected consequences disruption of receptor function by variant viral proteins might explain many common biochemical pathologies; for example, what effect would chronic blockade of parathyroid (pth) receptors by viral proteins have on pth levels, the parathyroid glands, or bone? leptin is a 16kda protein hormone secreted by adipocytes and carried across the blood-brain barrier by a rate-limiting transporter to act on hypothalamic receptors [98] where, among other functions, it regulates thyrotropinreleasing hormone (trh) genes and upregulates alphamelanocyte-stimulating hormone and other anorexigenic neuropeptides [99] important to appetite-regulation and energy balance [100] . leptin also regulates a broad spectrum of other processes and behaviours including thermogenesis, blood pressure and immune function. s=serum leptin concentrations and leptin resistance, are independent markers of obesity, weight gain, systemic hypertension [101] , diabetes mellitus [102] , obstructive sleep apnoea [103] and myocardial infarction [104] , while polymorphisms of the leptin gene are associated with insulin resistance [105] and long-term risk of developing diabetes mellitus [102] . predictably, variant envelope proteins generated by envelope orf rna quasispecies from viruses utilizing leptin receptors for cell access would have similar receptor affinity, but exhibit non-physiological leptin antagonist or agonist properties, thus disrupting leptin receptor function, altering energy regulation, and causing either excess caloric intake unrestrained by satiety responses, or inappropriate satiety signals with pathologically reduced caloric intake. as clear evidence exists for viral disruption of leptin function [106] and virus-associated weight gain in humans [107] and monkeys [108] , is it possible the global epidemics of type ii diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [109] [110] [111] [112] [113] [114] [115] [116] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function? could it also be that ethnically based epidemics of obesity, diabetes mellitus, hypertension and reno-vascular disease (the 'metabolic syndrome'), as seen in pima indians, nauruans and australian aborigines [115] have developed not primarily because of exposure to "western" foods and lifestyles -that, after all, are all-pervasive without necessarily having so dramatic an effect on other groups -but because of chronic or recurrent exposure to viruses, or genotypes of viruses to which their particular repertoire of receptor polymorphisms confer no protection? or that anorexia nervosa develops, in some patients, when variant viral proteins with aberrant leptin-agonist function arise during the course of viral infection, as the temporal relationship between infection and disease onset, very clearly documented in one study [117] , suggests. cardiovascular disease, the leading cause of premature death and disability in most western countries, has a wellestablished multi-factorial basis involving a complex interplay between genetic predisposition, environmental and personal risk factors -including systemic hypertension, diabetes mellitus, hyperlipidaemia, obesity and cigarette smoking -and more recently recognized mechanisms, including endothelial dysfunction [118] , vascular inflammation [119] and leptin levels [104] . systemic hypertension, diabetes mellitus and hyperlipidaemia have long-established, but complex, patterns of inheritance, a situation further compounded by evidence receptor polymorphisms -including those of angiotensin ii type 1 receptor [120] , irs-1 gene [121] and low density lipoprotein receptor (ldlr) [122] -both confer disease susceptibility and have regionally variable prevalences [123, 124] . the flaviviradae -including hcv -as a family, and the rous sarcoma virus, utilize low density lipoprotein receptors to enter cells [85, 125] , while angiotensin ii [90] , insulin receptor substrates (irs1 and irs 2) [79] , and endothelial cell glycoproteins [78] and other receptors widely distributed in vascular tissues are known to be permissive for virus cell entry establishing, in principle and in fact, viral-protein receptor affinity relevant to cardiovascular diseases. viruses accessing cells through these receptors will generate a quasispecies of variant proteins capable of disrupting receptor function potentially causing hyperlipidaemia, hypertension, hyperglycaemia and endothelial dysfunction, as well as immune-mediated endothelial cell damage, thus establishing the necessary and sufficient conditions and a chain of events that potentially link viruses and vascular diseases, including myocardial infarction. this hypothesis exists at the confluence of established risk factors for coronary artery disease, including genetic susceptibility, polymorphisms predisposing to hypertension [126] [127] [128] , diabetes [126] and hypercholesterolaemia and substantial new data implicating vascular inflammation [119, 129] , endothelial dysfunction [119, 130] , leptin dysregulation [104] and viral infection [131, 132] in the pathogenesis of vascular disease. furthermore, this final common pathway can account for that small, but significant, group of patients with vascular diseases but no clinically identifiable risk factors, as well as the non-random co-incidence of depression and coronary artery disease [133] (as discussed below) in addition to the anti-inflammatory action of hmg-coa reductase inhibitors (statins) [134] , and their effect in lowering cardiovascular mortality independent of cholesterol reduction [135] ; if statins compete with variant viral proteins for hmg-coa reductase receptor binding, and displace immunologically attractive molecules, inflammatory responses directed at viral product, but involving endothelial cell receptors, will be ameliorated (figure 8). human immunodeficiency virus hiv-associated dementia (hivd) occurs in 15% of hiv-infected adult patients, and as a major cause of dementia in the young represents "proof of principle" of virus-caused dementia, raising the possibility other forms of virus related dementia exist. although highly active antiretroviral therapy (haart) has reduced the incidence of hiv-d by 40-50% [136] , it remains a major cause of morbidity and the pathogenesis poorly understood. direct cytopathic effects of hiv or other viruses are unlikely, while active replication of virus, high-level viral protein expression [137] , and increased viral envelope sequence-diversity in blood and brain [138] are all important, clearly indicating viral proteins are pathogenically important. the clinical features of hivd, including psychomotor slowing, apathy, and altered gait and posture, strongly suggest a subcortical dementia with involvement of the basal ganglia and striatal dopamine receptor pathways. schizophrenia, depression and bipolar affective disorder, and anorexia nervosa are highly prevalent, chronic conditions of unknown aetiology that cause enormous morbidity and generate significant health care costs. each of these disorders have well documented, albeit regionally variable, associations with receptor -including dopamine -polymorphisms [124, [139] [140] [141] [142] [143] , as well as epidemiological evidence that viral infections are aetiologically important, either directly or as precipitating events [117, [144] [145] [146] [147] , although other sero-epidemiological studies [148] and work directly seeking viral nucleic acids in patients with schizophrenia have proved negative [149] . if a virus, or viruses, use dopamine, acetylcholine [76] , neurotrophin [82] , serotonergic (5-ht) [77] , or other neuro-transmitter receptors to access cells (and, given rna virus quasispecies biology, it would be surprising if some didn't), then the rna quasispecies will generate a quasispecies of variant polypeptides potentially reactive to these receptors. while it is difficult to imagine what effect perfusing a functional human brain with a solution of antigenic, inflammatory polypeptides that bind to, and are variably disruptive of, critical neurotransmitter receptor function, might have on cognition, perception, behaviour, attention span, abstract thought, fine motor or emotional control, it is unlikely to be beneficial. in this context, the welldocumented cognitive abnormalities -unrelated to depression -found in patients with early hcv and hiv infection [150] [151] [152] are unsurprising. virus receptor disease (vrd) is quite distinct from either immune complex deposition disease due to deposition of macromolecules in tight vascular arcades, or from disease related to altered cell tropisms and is also completely independent of the primary site of viral replication; both non-inflammatory receptor blockade and immune-mediated inflammation directed at viral protein-receptor complexes could cause pathology of tissues non-permissive for and remote from the primary site(s) of viral replication with "autoimmune" damage to the liver, pancreas, brain, skin or lungs arising, for example, from chronic small intestinal virus infection. viral quasispecies biology predicts vrd will have other characteristics. first, due to replicative homeostasis, the ratio of wild type to variant viral proteins of the quasispecies will both fluctuate with time and will alter dramatically after initial infection; if wildtype proteins are dominantly agonist in function with respect to their receptor, variant proteins, most likely, will predominantly exhibit antagonist function (and vice versa). furthermore, the net effect of viral proteins (because of viral autoregulation) will fluctuate initially between receptor agonist and antagonist function, before becoming predominantly antagonistic, thus providing a possible explanation for transient thyrotoxicosis during early thyroiditis (before hypothyroidism supervenes), for hypoglycaemia seen during early insulin-receptor antibody-mediated insulin resistance [153] , and for the contradictory functions ascribed to hiv nef [154] . a corollary of fluctuating phenotypic dominance of viral protein quasispecies is that receptor affinity of these proteins will also fluctuate, and any resulting inflammation may vary in both intensity and anatomical distribution over time. second, because viruses utilize alternate receptors for cell access, apparently homogeneous disease processes could result from multiple different viruses. similarly, because cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship (1; unbound, 2; activated), receptor permis-sive for virus cell entry (3) or blocked by polymorphism (rp, 4) figure 8 cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship (1; unbound, 2; activated), receptor permissive for virus cell entry (3) or blocked by polymorphism (rp, 4). receptor blockade by variant viral envelope proteins (green e, 5), blockade by antigenic envelope proteins stimulating "autoimmune"response apparently directed against self receptors (e, 6), competitive displacement of antigenic proteins by drug (d, e.g. statin, aspirin) abrogating immune response (7). virus quasispecies produce a broad spectrum of protein phenotypes, and the receptor polymorphisms permissive for cell entry for specific viruses will be variably distributed in host populations, pathology of widely variable tissues in different individuals could result from the same virus. third, as evolutionary co-adaptation results in progressive genetic co-divergence of interacting species, the receptor polymorphisms predisposing to (or protecting against) infection by any particular virus, and resulting vrd, and the common viruses causing them, would be predicted to vary geographically, an expectation multiply confirmed for disease associated polymorphisms. as a corollary this suggests individuals migrating from regions where hosts and virus strains are stably co-adaptated to other areas, where different viruses are prevalent, might experience increased rates of vrd -beaks optimally adapted for finch survival on the galapagos may be a liability elsewhere -a prediction again amply confirmed [155] [156] [157] ;. finally, if immune mechanisms are unable to clear rna viruses like hcv and do not cause the reduced viral replication seen during acute infection, are they any more likely to be effective against other rna viruses? is it possible that self-limiting infections like influenza and sars also autoregulate their replication, and, like hcv or hbv, become partially dormant, yet remain transcriptionally active, in the face of an active and powerful immune response? pcr amplification of influenza rna from convalescent samples makes this readily testable, while the documented relationship of influenza to myocardial infarction [132] and juvenile rheumatoid arthritis [61] makes the question important. if confirmed, the well-documented seasonality of some depressive illnesses [158] and schizophrenia, [146] and increased rates of schizophrenia during influenza epidemics [144] , and the increased incidence of both depression [146] and schizophrenia [144, 145] following in-utero exposure to influenza may be more rationally explained. if quantitative pcr (qpcr) assays of both 5'utr and envelope rnas are performed serially, and data expressed as [5' utr rna]/[env rna] for each sample, then a numerical expression describing changing quasispecies complexity over time may be obtained. in case prescient genius is unappreciated, haldane formulated the "lock and key" hypothesis on the basis of protein polymorphisms, defined by gel electrophoresis, and some general musing about predation and evolutionary struggle, two decades before the nature of dna was elucidated. i have no pecuniary interests, whatever, in this work and do not stand to gain financially or otherwise from it. publish with bio med central and every scientist can read your work free of charge a new immune escape mutant of hepatitis b virus with an asp to ala substitution in aa144 of the envelope major protein emergence of vaccine-induced escape mutant of hepatitis b virus with multiple surface gene mutations in a korean child the hepatitis b virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic tlymphocyte response hepatitis b virus persistence after recovery from acute viral hepatitis hepatitis b virus occult infection in subjects with persistent isolated anti-hbc reactivity occult hepatitis b virus infection in hbs antigen-negative hepatocellular carcinoma in a japanese population: involvement of hbx and p53 hiv-1 dynamics in vivo: virion clearance rate, infected cell lifespan, and viral generation time hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy hepatitis c virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children lack of protective immunity against reinfection with hepatitis c virus superinfection by homotypic virus in hepatitis c virus carriers: studies on patients with post-transfusion hepatitis superinfection of heterologous hepatitis c virus in a patient with chronic type c hepatitis hepatitis c virus persistence after spontaneous or treatment-induced resolution of hepatitis c persistence of hepatitis c virus in patients successfully treated for chronic hepatitis c vaccina analysis of hepatitis c virusinoculated chimpanzees reveals unexpected clinical profiles development of virus-specific cd4(+) t cells during primary cytomegalovirus infection fidelity of hepatitis b virus polymerase fidelity of hiv-1 reverse transcriptase high fidelity of yellow fever virus rna polymerase lack of evidence for proofreading mechanisms associated with an rna virus polymerase rapid fitness losses in mammalian rna virus clones due to muller's ratchet eigen m: viral quasispecies replicative homeostasis: a mechanism of viral persistence the molecular epidemiology of mumps virus interleukin 6-174 g/c promoter gene polymorphism and sporadic alzheimer's disease: geographic allele and genotype variations in europe biological divergences in south-central bougainville: an analysis of blood polymorphism gene frequencies and anthropometric measurements utilizing tree models, and a comparison of these variables with linguistic, geographic, and migrational "distances classification of hepatitis c virus into six major genotypes and a series of subtypes by phylogenetic analysis of the ns-5 region a novel mechanism to explain protein abnormalities in schizophrenia based on the flavivirus resistance gene ethnic heterogeneity in allele variation in the drd4 gene in schizophrenia characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (t-rflp) structural polymorphism of the reca protein from the thermophilic bacterium thermus aquaticus single nucleotide polymorphism, haplotype diversity and recombination in the isa gene of barley polymorphism of the il-10 gene is associated with susceptibility to herpes zoster polymorphism of the interleukin-10 gene is associated with susceptibility to epstein-barr virus infection hla-dr antigen frequencies in mexican patients with dengue virus infection: hla-dr4 as a possible genetic resistance factor for dengue hemorrhagic fever polymorphism of fc receptor iia for igg in infants is associated with susceptibility to perinatal hiv-1 infection symposium sui fattori ecologi e genetici dela specilazione negli animali. supplemnto a la la ricerca scientifica high levels of hiv-1 in plasma during all stages of infection determined by competitive pcr differential imprinting and expression of maternal and paternal genomes maternal immune response to pregnancy maternal and fetal immune responses during pregnancy fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review gershwin me: lack of immunological or molecular evidence for a role of mouse mammary tumor retrovirus in primary biliary cirrhosis multiple sclerosis in australia antibodies to influenza a in a cluster of children with juvenile chronic arthritis the geographical distribution of primary biliary cirrhosis in a well-defined cohort indications that maternal coxsackie b virus infection during pregnancy is a risk factor for childhood-onset iddm. diabetologia coxsackie b virus infection and beta cell autoantibodies in newly diagnosed iddm adult patients coxsackievirus b4-induced development of antibodies to 64,000-mr islet autoantigen and hyperglycemia in mice the role of enteroviral infections in the development of iddm: limitations of current approaches hepatitis c infection and autoimmunity interferon-alpha and autoimmune thyroid disease hepatitis c and d, retroviruses and autoimmune manifestations editorial: autoimmune thyroid disease in interferon-treated patients further evidence for an association between non-insulin-dependent diabetes mellitus and chronic hepatitis c virus infection fulminant autoimmune type 1 diabetes during interferon-alpha therapy: a case of th1-mediated disease hepatitis c virus infection and autoimmunity gastric autoimmune disorders in patients with chronic hepatitis c before, during and after interferon-alpha therapy rabies virus infection selectively impairs membrane receptor functions in neuronal model cells rabies virus selectively alters 5-ht1 receptor subtypes in rat brain role of virus receptor-bearing endothelial cells of the blood-brain barrier in preventing the spread of mouse hepatitis virus-a59 into the central nervous system insulin-like growth factor i receptor signaling system in jc virus t antigen-induced primitive neuroectodermal tumors -medulloblastomas hepatitis c virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3 vaccinia virus and the egf receptor: a portal for infectivity? alterations in neurotrophin and neurotrophin receptor gene expression patterns in the rat central nervous system following perinatal borna disease virus infection direct modulation of simian virus 40 late gene expression by thyroid hormone and its receptor receptor for the group b coxsackieviruses and adenoviruses: car hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor low density lipoprotein receptor as a candidate receptor for hepatitis c virus the natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor sendai virus efficiently infects cells via the asialoglycoprotein receptor and requires the presence of cleaved f0 precursor proteins for this alternative route of cell entry role of the asialoglycoprotein receptor in binding and entry of hepatitis c virus structural proteins in cultured human hepatocytes angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus clinical coxsackievirus b isolates differ from laboratory strains in their interaction with two cell surface receptors early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals adaptation of wild-type measles virus to cd46 receptor usage evolutionary pressure of a receptor competitor selects different subgroup a avian leukosis virus escape variants with altered receptor interactions evolution of cell recognition by viruses a single amino acid alteration in the human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein confers resistance to the inhibitory effects of zanamivir on receptor binding and neuraminidase activity single amino acid substitutions in influenza haemagglutinin change receptor binding specificity the many lives of leptin leptin signaling, adiposity, and energy balance leptin signaling in the central nervous system and the periphery leptin gene polymorphism is associated with hypertension independent of obesity association between baseline plasma leptin levels and subsequent development of diabetes in japanese americans increases in leptin levels, sympathetic drive, and weight gain in obstructive sleep apnea leptin is associated with increased risk of myocardial infarction association of leptin receptor polymorphism with insulin resistance alteration of the leptin network in late morbid obesity induced in mice by brain infection with canine distemper virus human adenovirus-36 is associated with increased body weight and paradoxical reduction of serum lipids human adenovirus ad-36 promotes weight gain in male rhesus and marmoset monkeys obesity, insulin resistance and diabetes -a worldwide epidemic the worldwide epidemic of obesity in adolescents the obesity epidemic development of the obesity epidemic in denmark: cohort, time and age effects among boys born 1930-1975 the spread of the obesity epidemic in the united states global and societal implications of the diabetes epidemic etiology of the metabolic syndrome: potential role of insulin resistance, leptin resistance, and other players the obesity epidemic as harbinger of a metabolic disorder epidemic: trends in overweight, hypercholesterolemia, and diabetes treatment in geneva post-viral onset of anorexia nervosa endothelial dysfunction of the non-infarct related, angiographically normal, coronary artery in patients with an acute myocardial infarction persistent endothelial dysfunction is related to elevated c-reactive protein (crp) levels in type ii diabetic patients after acute myocardial infarction association of angiotensin ii type 1 receptor gene polymorphism with essential hypertension association between vitamin d receptor polymorphism and type 2 diabetes or metabolic syndrome in community-dwelling older adults: the rancho bernardo study normal dna polymorphism at the low density lipoprotein receptor (ldlr) locus associated with serum cholesterol level varmus he: a receptor for subgroup a rous sarcoma virus is related to the low density lipoprotein receptor a common polymorphism of uncoupling protein 2 gene is associated with hypertension epidemiology of essential hypertension: the role of genetic polymorphism genetic polymorphism of the renin-angiotensin-aldosterone system and arterial hypertension in the italian population: the geniper project low grade inflammation and coronary heart disease: prospective study and updated meta-analyses impaired endothelial function in never-treated hypertensive subjects carrying the arg972 polymorphism in the insulin receptor substrate-1 gene influenza infection exerts prominent inflammatory and thrombotic effects on the atherosclerotic plaques of apolipoprotein e-deficient mice association of influenza vaccination and reduced risk of recurrent myocardial infarction depression and cardiovascular disease: mechanisms of interaction crisby m: modulation of the inflammatory process by statins. drugs today (barc) 2003 statins and endothelial dysfunction hiv dementia: an evolving disease circular forms of unintegrated human immunodeficiency virus type 1 dna and high levels of viral protein expression: association with dementia and multinucleated giant cells in the brains of patients with aids hiv dementia patients exhibit reduced viral neutralization and increased envelope sequence diversity in blood and brain association of egf polymorphism with schizophrenia in finnish men meta-analysis identifies an association between the dopamine d2 receptor gene and schizophrenia dias neto e: association of a new polymorphism in alox12 gene with bipolar disorder anorexia nervosa (restrictive subtype) is associated with a polymorphism in the novel norepinephrine transporter gene promoter polymorphic region serologic evidence of prenatal influenza in the etiology of schizophrenia relationship between in utero exposure to influenza epidemics and risk of schizophrenia in denmark adult major affective disorder after prenatal exposure to an influenza epidemic detection of serum antibodies to borna disease virus in patients with psychiatric disorders immunoglobulin studies in patients with psychiatric diseases polymerase chain reaction (pcr) search for viral nucleic acid sequences in schizophrenia subclinical impairment of brain function in chronic hepatitis c infection hepatitis c and cognitive impairment in a cohort of patients with mild liver disease cognitive changes as early signs of hiv infection the evolving clinical course of patients with insulin receptor autoantibodies: spontaneous remission or receptor proliferation with hypoglycemia the paradox of hiv nef function: resolution of the contradictions childhood migration and cardiovascular risk migration as a risk factor for schizophrenia: a danish population-based cohort study impact of migration on coronary heart disease risk factors: comparison of gujaratis in britain and their contemporaries in villages of origin in india variability in the 5-ht(2a) receptor gene is associated with seasonal pattern in major depression i thank my wife sophie j coleman, and sons matt and tim, for everything important, my parents dick and janet for extraordinary opportunity, and some great physician-teachers -that most noble vocation -of the university of western australia medical school; professors mike mccall, dick joske, bill reed, bill musk, peter pullan, michael quinlan, dick lefroy and ted haywood. special thanks to karl ruckriegel for turning back-of-envelope sketches into first-class graphics. any remaining lack of clarity is my fault. key: cord-321230-b5a1z14w authors: samji, hasina; yu, amanda; wong, stanley; wilton, james; binka, mawuena; alvarez, maria; bartlett, sofia; pearce, margo; adu, prince; jeong, dahn; clementi, emilia; butt, zahid; buxton, jane; gilbert, mark; krajden, mel; janjua, naveed z. title: drug-related deaths in a population-level cohort of people living with and without hepatitis c virus in british columbia, canada date: 2020-10-19 journal: int j drug policy doi: 10.1016/j.drugpo.2020.102989 sha: doc_id: 321230 cord_uid: b5a1z14w background: the majority of new hcv infections in canada occur in people who inject drugs. thus, while curative direct antiviral agents (daas) herald a promising new era in hepatitis c virus (hcv) treatment, improving the lives and wellbeing of people living with hcv (plhcv) must be considered in the context of reducing overdose-related harms and with a syndemic lens. we measure drug-related deaths (drds) among hcv-negative people and plhcv in british columbia (bc), canada, and the impact of potent contaminants like fentanyl on deaths. methods: we identified drds among plhcv and hcv-negative individuals from 2010 to 2018 in the bc hepatitis testers cohort, a population-based dataset of ~1.7 million british columbians comprising comprehensive administrative and clinical data. we estimated annual standardized liverand drug-related mortality rates per 100,000 person-years (py) and described the contribution of specific drugs, including fentanyl and its analogues, implicated in drds over time. results: drds constituted 20.1% of deaths among plhcv and 4.7% of deaths among hcv-negative individuals; a 4.3-fold (95% confidence interval: 4.0-4.5) difference. drug-related mortality overtook liver-related mortality for plhcv in 2015 and hcv-negative individuals in 2016 and rose from 241.7 to 436.5 per 100,000 py from 2010 to 2018 amongplhcv and from 20.0 to 57.1 per 100,000 py for hcv-negative individuals over the same period. the proportion of deaths attributable to drugs among plhcv and hcv-negative individuals increased from 15.1% to 26.1% and 3.1% to 8.0%, in 2010 and 2018, respectively. the proportion of drds attributed solely to synthetic opioids such as fentanyl averaged across both groups increased from 2.1% in 2010 to 69.6% in 2017. conclusion: steep drug-related mortality increases among plhcv and hcv-negative individuals over the last decade highlight the urgent need to address overdose-related drivers and harms in these populations using an integrated care approach. the advent of direct acting antiviral (daa) short-course, well-tolerated therapy has dramatically altered the hepatitis c virus (hcv) treatment landscape. response rates over 95% are leading to dramatic reductions in liver-related mortality as well as new global targets to eliminate hcv as a public health threat by 2030 (n. world health organization, 2019) . however, increasing injecting drug use and the epidemic of drug overdose-related deaths in north america threaten to arrest or even reverse the progress heralded by the daa era among people who use drugs with hcv and the morbidity, mortality and well-being of all people who use drugs, regardless of hcv status. in canada, new hcv infections occur mainly among people who inject drugs (pwid) (n. z. . moreover, syndemic conditions of co-occurring mental illness, hiv co-infection and higher material deprivation are more common among people living with hcv (plhcv) compared to hcv-negative individuals (mckee et al., 2018) , prompting a need to examine data by hcv status. in british columbia (bc), an estimated 1.2 to 1.5% of the population injects drugs and more than 65% of those will have a lifetime exposure to hcv (jacka et al., 2020; public health agency of canada, 2014) . increasing rates of injecting drug use have been observed in canada and the us since 2010, likely exacerbated by policies to reduce prescription opioid overprescribing, diversion and misuse (powell et al., 2019) . other policies (e.g. prescription drug monitoring programs, more stringent guidelines) have also been implicated . a national us study observed a 76% increase in drug treatment admissions (from 13% to 22%) due to injecting drug use between 2004 and 2014; these increases were particularly pronounced among individuals 20 to 39 years of age (zibbell et al., 2018) . acute hcv infections subsequently tripled in the us between 2011 and 2015 (centers for disease control and prevention, 2017; gonzalez & trotter, 2018; zibbell et al., 2018) , representing a reversal in progress towards reducing rates of acute hcv in the us, which declined by almost 50% between 2001 and 2010. similarly, hcv incidence in bc decreased between 2001 and 2012 and then began rising thereafter (li y et al., 2019) . in both canada and the us, these "dual epidemics" of injecting drug use and hcv transmission have the potential to derail years of sustained progress in reversing the hcv epidemic, as pwid are at high risk of drug overdose-related mortality. while the rate of drug-related deaths (drds) in canada has nearly doubled from 5.8 deaths per 100,000 population in 2015 to 11.5 deaths per 100,000 population in 2017, the situation in bc is particularly concerning with 60% of canadian drds recorded in that province (statistics canada, 2019a) . in 2018, more than 1,500 people died of a drug overdose in bc -approximately 4.5 times the number of deaths due to motor vehicle accidents within the province (bc coroners service, 2020a). the proportion of drds in which synthetic opioids such as fentanyl and its analogs have been detected increased more than six fold between 2012 and 2015, prompting the bc provincial health officer to declare a state of emergency under the public health act in april, 2016 for the first time (bc gov news, 2016) . national and provincial gains in life expectancy have also been halted and reversed for the first time in four decades due to the overdose epidemic; men in bc were the most affected, losing 0.28 years between 2016 (statistics canada, 2019b ye et al., 2018) . as noted above, plhcv have a higher proportion of injecting drug use in canada and plhcv who inject drugs also have higher proportion of co-occurring mental illness, hiv co-infection, hbv co-infection and material and social deprivation (mckee et al., 2018) . it is hypothesized that as plhcv have higher drug use and are more marginalized, drds will be higher among them. however, there is no data assessing if the rate of drds is higher among plhcv compared to hcv-negative individuals. we use a population-based database of all individuals tested for hcv in bc to compare rates and trends over time in drd and liverrelated deaths (lrds) and identify whether there are differences in drds among plhcv and hcv-negative individuals. we also quantify the impact of fentanyl on drds in our study population over the last decade. the bc hepatitis testers cohort (bc-htc) includes more than 1.7 million individuals tested for hcv or hiv at the bc centre for disease control public health laboratory and all confirmed cases of hbv, hcv, hiv, and active tb reported by the public health system in bc from 1990 to 2015. individuals in bc are generally tested due to hcv risk, investigation of liver disease, routine screening in pregnancy and if they are undergoing procedures such as renal dialysis, and those tested during the mid-1990s as part of blood system lookbacks . these data were integrated with data on prescription drug dispensations, physician diagnostic and billing data, hospitalization records, emergency department visits, cancers, and deaths and are described in more detail in supplemental table s1 . additional information about the bc-htc, including methodology, data linkages, and population characteristics by hcv status has been published previously (n. z. mckee et al., 2018) . the dynamic, retrospective cohort was formed under a public health mandate of the bc centre for disease control. all residents in bc are registered in the publicly funded insurance plan that acts as a single payer system and covers services provided by fee-for-service practitioners, allowing for comprehensive population-level monitoring. eligible individuals for this study had at least one hcv test (i.e., anti-hcv or hcv rna) performed or were a reported hcv case from april 1992 to december 2015. characterization of drd was restricted to 2010 onwards to highlight the surge of drds among people with hcv over the last decade. drds were identified using bc vital statistics agency (bcvsa) death registry, which includes all deaths registered in the province of bc, underlying and/or contributing cause of death mortality data, and categorized using the international classification of diseases, tenth revision (icd-10) codes (supplemental table s2 ). any deaths not deemed "natural" (a death primarily resulting from a disease or progressive fatigue of the bodily systems) are investigated by the bc coroners service (bccs). the finalization of the medical certificates of death can take anywhere from several months to several years from date of death. bcvsa processes information received from the bccs on the finalized medical certificate of death and applies these icd-10 codes using an internationally standardized coding software provided by statistics canada. a broad definition specifying both acute and chronic poisonings was adapted from consensus recommendations for national and state poisoning surveillance developed by the safe states alliance (the safe states alliance, 2012) and which has also been used by the us centres for disease control and prevention. in addition, icd-10 multiple-cause codes provide information on the types of drugs or drug classes involved in the death ("t-codes"); of these, heroin (t40.1); natural/semisynthetic opioids (t40.2); methadone (t40.3); synthetic opioids other than methadone (including fentanyl and fentanyl analogues) (t40.4); cocaine (t40.5); other and unspecified narcotics (40.6); and psychostimulants with abuse potential (t43.6) were used. deaths involving methadone (t40.3) and natural/ semisynthetic opioids such as hydrocodone and oxycodone (t40.2) are grouped together as prescription opioid deaths (jalal et al., 2018; jones et al., 2018) , a category which includes opioids prescribed to the deceased person or prescribed to someone else (diverted). in 2016, in response to the drd public health emergency, supplementary codes to identify deaths where fentanyl ("z7221") or carfetanil ("z7222") were specifically implicated in finalized death investigations were added to the "lifestyle / environmental" field in bcvsa mortality data files; thus, we also included these codes in our categorization of "synthetic opioids other than methadone." drug-related t-codes are attached to a parent "external cause" icd code that signals the circumstances or intent under which the substance was used (accidental, intentional / suicide, homicide, undetermined intent or adverse reaction in therapeutic use) and is assigned during drds investigations. bccs investigations include toxicology results, scene evidence and death investigation information (such as existing prescription information). these investigations also help determine drug origin; for instance, whether a drug is pharmaceutically-derived (including prescribed to the deceased or diverted), non-pharmaceutical, or undetermined (e.g. fentanyl identified through toxicology but without prescription or evidence suggesting non-pharmaceutical fentanyl use) (onatrio agency for health protection and promotion (public health ontario) 2017). similarly, illicit fentanyl is differentiated from prescribed fentanyl through investigation of evidence of prescription/patch versussuggestion of a non-pharmaceutical origin (e.g., other illicit substances detected on toxicology or drug paraphernalia on the scene). heroin (6-monoacetyl morphine), which is rapidly metabolized into morphine and therefore may be underestimated, also relies on investigation to finalize classification (onatrio agency for health protection and promotion (public health ontario) 2017). most deaths involved more than one type of drug; thus, categories are not mutually exclusive. lastly, if an underlying cause of death code was recorded as icd-10 r99 (other ill-defined and unspecified causes, usually assigned when cause of death is still under investigation), and manner of death was listed as "pending," "accident," or "undetermined," we re-categorized the death as drug-related if the death met the following criteria: attributable to a person aged 20 to 64 years who also injects drugs (defined as any drug-related diagnoses, use of injection drugs, record of opioid agonist therapy dispensation or injection related infection using physician diagnostic and billing data, hospitalizations, prescription data, vital statistics and emergency room data), based on validation studies in the us and in the bc-htc (centers for disease control and prevention, 2004; to mitigate the impact of missing data on the analysis. as the majority of r99 codes are resolved within a few years as the bccs completes investigations of sudden deaths prior to death categorization, we excluded deaths occurring after december 31 st , 2018 to reduce the impact of open investigations and the need to impute missing data by re-categorizing deaths. while we were able include many drds with r99 codes in the analysis through application of our algorithm described above, it was not possible to assign t-codes to these deaths as these codes are only assigned in concluded investigations bc coroners service, 2020) . as such, analyses involving t-codes were limited to data collected on or before december 31 st , 2017. we used deidentified linked databases to identify other important variables for analyses. lrds included deaths due to decompensated liver disease, liver cancer, hcv, other types of viral hepatitis, and alcoholic or non-alcoholic liver disease. we assessed baseline participant characteristics such as age, sex and material and social deprivation (québec index of material and social deprivation) (pampalon et al., 2012) . we also evaluated concurrent conditions including hiv and hbv co-infection, major mental illness (hospitalization or at least two visits to a psychiatrist for disorders including schizophrenia, bipolar, major depressive and personality), and psychosis diagnosis as well as access to opioid agonist therapy (oat) and hcv treatment. diagnostic codes and variable definitions are presented in more detail in supplemental table s3 . we compared plhcv and hcv-negative individuals who died of drds, estimated annual age and sex-adjusted drug-related mortality rates per 100,000 person-years (py) for these groups and examined trends over time by sex. rates are age and sex-standardized to the 1991 canadian population. py were calculated from either first hcv negative or first hcv positive date, censored at death or hcv sero-conversion. secondly, we compared demographic and risk factor characteristics of people who died of liver-vs. drug-related causes and estimated annual age and sex-adjusted liver-and drug-related mortality rates per 100,000 py for these groups. lastly, we described the contribution or "co-involvement" of specific drugs, including fentanyl and its analogues, implicated in drds overall and stratified by hcv status over time. for instance, for deaths involving heroin, we identify what proportion also included fentanyl involvement each year using the t-codes described above assigned through coroner's investigation of each drug-related death. as more than one drug can contribute to a drd, categories representing specific drug involvement in a drd are not mutually exclusive. for descriptive analyses comparing groups, we used wilcoxon's rank sum test for continuous variables and the chi 2 test for categorical data. sas version 9.4 was used for all analyses. the study was approved by the university of british columbia clinical research ethics board (h14-01649). there were 1,355,952 people who were hcv tested or reported as an hcv case in the cohort as of december 31, 2015, of whom 71,715 (5.3%) were plhcv and 1,284,237 (94.7%) were hcv-negative individuals. a total of 85,899 deaths were recorded from 2000 to 2018 of which 11.7% were among plhcv. drds constituted 20.1% (2,018/ 10,024) of deaths among plhcv and 4.7% (3,598 /75,875) of deaths among hcv-negative individuals; a 4.3-fold (95% confidence interval [ci]: 4.0-4.5) difference. the majority of drds among plhcv occurred among those with a history of injecting drug use (95.4%). similarly, a majority of hcvnegative individuals individuals dying of a drd had a history of injection drug use (85.6%) (tables 1 and 2) . the population of plhcv dying of drds was also older (born prior to 1965: 52.0% vs. 27.9%), more likely to have co-infections such as hiv (13.6% vs. 1.6%), and hbv (10.5% vs. 1.1%), to have a record of oat (54.9% vs. 26.0%), and be more materially (most deprived quintile: 36.8% vs. 27.9%) and socially (most deprived quintile: 55.9% vs. 39.2%) deprived compared to the hcv-negative individuals ( table 2) . more than half of plhcv and hcv-negative groups had a major mental illness diagnosis and more than 20% in each group reported a diagnosis of psychosis. annual drug-related standardized mortality rates among plhcv and hcv-negative individuals by sex are presented in figure 1 . drugrelated mortality was 1.3 (95% ci: 1.2-1.3) to 1.5 (95% ci: 1.5-1.6) times higher in men compared to women living with hcv and from 1.8 (95% ci: 1.5-2.3) to 3.3 (95% ci: 3.2-3.5) times higher among hcv negative men compared to women. increases over time were observed for all groups, with rates among men without hcv increasing the most dramatically, from 27.1 per 100,000 py in 2010 to 96.0 per 100,000 py in 2018; a 3.5-fold increase (95% ci: 3.1-4.1). increases over the same time period were also observed for men living with hcv (283.4 to 490.0 per 100,000 py; 1.7 fold increase [95% ci: 1.7-1.8]) and women without hcv (14.8 to 29.0 per 100,000 py; 2.0-fold increase [95% ci: 1.6-2.4]) and women living with hcv (184.4 to 372.2 per 100,000 py; 2.0-fold increase [95% ci: 1.9-2.1]). the proportion of deaths attributable to drugs among plhcv increased from 15.1% (135/896) in 2010 to 26.1% (322/1274) in 2018. similarly, the proportion of deaths attributable to drugs among hcvnegative individuals more than doubled from 2010 to 2018, increasing from 3.1% (202/6540) to 8.0% (748/9361). annual liver-and drug-related standardized mortality rates among plhcv and hcv-negative individuals and overall deaths are presented in figure 2 . drug-related mortality rate rose from 241.7 per 100,000 py in 2010 to 436.5 per 100,000 py (irr: 1.8; 95% ci: 1.7-1.9) in 2018 for plhcv and from 20.0 to 57.1 per 100,000 py (irr: 2.9; 95% ci: 2.4-3.4) for hcv-negative individuals with the majority of the increase after 2014. from 2010 to 2018, liver-related mortality rates among plhcv almost halved from 313.7 to 183.2 per 100,000 py (irr: 1.7; 95% ci: 1.6-1.8); similarly, liver-related mortality rates among hcv-negative individuals decreased from 42.7 to 22.6 per 100,000 py (irr: 1.9; 95% ci: 1.6-2.2) over the same period. drug-related mortality overtook liver-related mortality for plhcv in 2015 and hcv-negative individuals in 2016. from 2010 to 2017, drds increased from 337 to 1,184, totaling 4,546 over that period. 73.7% (3,352/4,546) of drds had an assigned t-code specifying at least one drug, and 61.3% of these had more than one drug recorded. since 2010, the proportion of drds attributed solely to synthetic opioids such as fentanyl has increased in our cohort from 2.1% in 2010 to 69.6% in 2017 (figure 3) . in 2017, synthetic opioids were co-involved in 93.3% of all opioid-related deaths, 68.9% of prescription opioid deaths, 88.5% of heroin-related deaths, 79.1% of cocaine-related deaths, and 85.8% of deaths involving psychostimulants. results stratified by hcv status are included in supplemental table s4 . we examined drds in a population-based cohort of hcv testers in bc. we found that drds were higher among plhcv compared to hcvnegative individuals. drug-related causes have overtaken liver-related causes of death among plhcv, accounting for more than a quarter of deaths in this population in 2018, with similar trends observed among hcv-negative individuals. drug-related mortality has increased among men and women living with and without hcv since 2014; the steepness of the curves for each group has increased every year, indicating a worsening problem, with absolute rates several fold higher for plhcv compared to hcv-negative individuals. our data suggest that synthetic opioids such as fentanyl are ubiquitous in the drug supply, contributing to more than three quarters of deaths involving opioids in 2017. while injecting drug use was pervasive among both plhcv and hcv-negative individuals dying of drug-related causes in bc, we found differences in group characteristics. plhcv were older, had a higher proportion of co-occurring hiv, hbv infections and social and material deprivation and may therefore represent pwid with a longer duration of drug use. the median age of drd in our study among hcv-negative individuals was 37 years (vs. 49 years among plhcv); similarly, provincial data on overdose deaths illustrates that people between the ages of 30 and 39 years had the highest rate of illicit drug overdose deaths (57.4 per 100,000 person years) in 2018 (bc coroners service, 2020a). these younger hcv-negative individuals could represent pwid with shorter duration of drug use who are dying before they have a chance to access drug-related treatment services or to acquire a blood-borne pathogen. another explanation is that practices protecting people from acquiring blood-borne pathogens such as using drugs through non-injecting pathways do not protect against fentanyl exposure. more than half of overdose deaths in bc were not injecting-related in 2018 (bc coroners service, 2018). these data underscore the need to prevent initiation of injecting drug use, to promote reversal to non-injecting use, and to minimize exposure to fentanyl to reduce drds (des jarlais et al., 2018) . indeed, increasing rates of injecting drug use, acute hcv infection, and overdose deaths signal a crisis for public health in north america and elsewhere. recent outbreaks of hiv following changing drug use h. samji, et al. international journal of drug policy 86 (2020) 102989 practices and trends have been observed among pwid in both rural and urban settings. a rapid increase in new cases of hiv infection among pwid in indiana (peters et al., 2016) led the us centers for disease control to identify regions at high risk of hiv transmission due to the opioid epidemic (van handel et al., 2016) ; subsequent hiv outbreaks in washington, massachusetts and ontario among pwid have also been described (alpren et al., 2020; ball et al., 2019; golden et al., 2019) . structural inequalities such as poor economic prospects, unaddressed trauma, stigma, and unequal access to healthcare are driving this syndemic and must be addressed to reduce drug-related harms (parker et al., 2019) . overdose deaths and sequalae from drug use such as infectious disease acquisition are preventable; developing a comprehensive suite of services, such as integrating hiv pre-exposure prophylaxis services for pwid at risk of acquiring hiv as well as infectious disease treatment (antiretroviral treatment for hiv and daa therapy for hcv) and medication-assisted treatment for opioid use disorder (singer, 2020) should be explored in these contexts. our results indicate that potent synthetic opioids such as fentanyl have increasingly contaminated the illicit drug supply in bc, with the proportion of deaths with co-involvement of synthetic opioids increasing sharply across all drug categories including cocaine and stimulant-related deaths since 2015. substantiating these findings are drug testing results in bc, which found that 80% of drugs tested positive for fentanyl, including 84% of heroin samples, and 65% of nonopiate drugs such as crystal meth, ecstasy/mdma, and cocaine (vancouver coast health, 2017) . the strong association between the number of fentanyl-positive samples seized by canadian law enforcement agencies and overdose deaths in bc (karamouzian et al., 2020) also supports fentanyl's direct impact on mortality rates. while there was a decrease in overdose deaths in bc in 2019, the number of deaths and non-fatal overdoses remain at epidemic levels. moreover, the covid-19 pandemic era appears to have precipitated a striking spike in deaths, with june 2020 recording the highest number of illicit drug toxicity deaths observed in bc to date (bc coroners service, 2020b). there is thus renewed urgency to develop and implement strategies to reduce overdose deaths in bc and elsewhere. novel proximal level interventions such as take-home drug checking test strips may be explored to reduce the impact of the toxic drug supply on pwid. existing harm reduction interventions such as supervised consumption sites and take-home naloxone kits are operational in bc and have averted an estimated 3000 deaths over a 20-month period during which 2,177 deaths were observed (irvine et al., 2019) but can be expanded and implemented elsewhere. furthermore, distal interventions should address the underlying motives propelling the syndemic of mental health, blood-borne infections, and injecting drug use and other "diseases of despair" such as suicide and problematic alcohol use including unaddressed antecedent physical and psychological trauma, economic disadvantage, social isolation, and hopelessness (dasgupta et al., 2018) to create a durable impact on drug-related mortality. for instance, using "life projects" approaches that de-stigmatize and humanize pwid engaging in hcv treatment as well as culturally-safe, healing-centered approaches that acknowledge histories of trauma (e. williams et al., 2019; pearce et al., 2019) are paramount for addressing barriers to care and treatment and promoting long term wellness beyond hcv or drug treatment. notably, we found that more than half of plhcv and hcv-negative individuals were diagnosed with a major mental illness and more than a fifth of each group with psychosis. severe and persistent mental illness has been associated with an increased risk of acquiring sexually transmitted and blood-borne infections (stbbis); a recent meta-analysis observed an hcv prevalence of 17.4% among individuals with severe and persistent mental illness compared to 1% of those in the general population (hughes et al., 2016) . this increased risk is primarily due to co-morbid substance use, though a national u.s. study of veterans found a 50% increased odds of having an hcv diagnosis among veterans with severe and persistent mental illness even after controlling for substance use (himelhoch et al., 2009 ). as such, strategies to improve screening for individuals with severe and persistent mental illness for hcv, drug use and other stbbis and providing risk reduction counseling in this population are warranted. there are several limitations to consider in our study. recent deaths of undetermined intent (icd-10 code r99) pending a coroner's investigation, with manner of death reported "pending," "accident," or "undetermined," and classified as drds in our study based on age and pwid criteria may be misclassified, possibly leading to an over-estimation of drd, though age restriction and history of idu decreases figure 3 . synthetic opioid co-involvement in drug-related deaths among individuals in the bc hepatitis testers cohort, 2010-2017^(n=4546)^categories are not mutually exclusive; many deaths involve multiple drugs *proportion of total drug-related deaths with synthetic opioid co-involvement likelihood of misclassification. an estimated 1.2% (1004/85,899) of all deaths in 2018 were assigned an icd-10 r99 code. however, not including any of these deaths as drug-related would certainly under-estimate drds; we chose to be inclusive. similarly, using administrative data to identify pwid, while a useful strategy for large population-level datasets, may also lead to misclassification. another important consideration is that there may be temporal trends in the classification of deaths whereby investigation practices and therefore classification of deaths differed; for instance, as the overdose epidemic grew, it is possible that more deaths were investigated as drug-related. next, although our inclusion criteria specify that everyone is tested for hcv on entry, drds among undiagnosed hcv seroconverters who do not have a positive test will be misclassified. lastly, we see a decline in drug related mortality among men living with hcv in 2018 which preceded a decline in overall drds in bc surveillance data (bc coroners service, 2020a) in 2019. this decline in men living with hcv may be real, related to improvements in healthcare and hcv treatment access, or could be a data artifact which may become clear as additional data become available. in summary, fentanyl has significantly impacted mortality rates among pwid with and without hcv; widespread interventions to reduce harms and address the toxic drug supply are urgently needed. for plhcv, drds represent a failure of engagement in care and a missed opportunity to deliver highly effective daa treatment and resulting quality of life improvements. thus, integration of overdose prevention and hcv services is necessary to improve survivability as well as quality of life of these populations and to realize the benefit of curative hcv therapy. this work was supported by the bc centre for disease control and the canadian institutes of health research (grant numbers nhc-142832, pjt-156066, phe-141773). all inferences, opinions, and conclusions drawn in this publication are those of the authors, and do not reflect the opinions or policies of the data steward(s). mk has received research grant funding via his institution from roche molecular systems, boehringer ingelheim, merck, siemens healthcare diagnostics and hologic inc. srb has received speakers' honoraria and participated in a medical advisory board program with gilead sciences (personal payments given as unrestricted donation to bccdc foundation). all other co-authors have no potential conflict of interest to declare. opioid use fueling hiv transmission in an urban setting: an outbreak of hiv infection among people who inject drugs-massachusetts sharing of injection drug preparation equipment is associated with hiv infection: a cross-sectional study illicit drug overdose deaths in b illicit drug toxicity deaths in bc illicit drug toxicity deaths in bc excerpts from: issues to consider when analyzing icd-10 coded data on drug poisoning (overdose) deaths a surveillance data for viral hepatitis opioid crisis: no easy fix to its social and economic determinants hepatitis c virus prevalence and estimated incidence among new injectors during the opioid epidemic life projects: the transformative potential of direct-acting antiviral treatment for hepatitis c among people who inject drugs the rise of the opioid epidemic and hepatitis c-positive organs: a new era in liver transplantation understanding associations between serious mental illness and hepatitis c virus among veterans: a national multivariate analysis prevalence of hiv, hepatitis b, and hepatitis c in people with severe mental illness: a systematic review and meta-analysis. the lancet psychiatry modelling the combined impact of interventions in averting deaths during a synthetic-opioid overdose epidemic prevalence of injecting drug use and coverage of interventions to prevent hiv and hepatitis c virus infection among people who inject drugs in canada changing dynamics of the drug overdose epidemic in the united states from 1979 through identifying injection drug use and estimating population size of people who inject drugs using healthcare administrative datasets assessing hepatitis c burden and treatment effectiveness through the british columbia hepatitis testers cohort (bc-htc): design and characteristics of linked and unlinked participants twin epidemics of new and prevalent hepatitis c infections in canada: bc hepatitis testers cohort the impact of hcv svr from direct acting antiviral and interferon-based treatments on mortality in a large population based cohort study changes in synthetic opioid involvement in drug overdose deaths in the united states known fentanyl use among clients of harm reduction sites in british columbia what is killing people with hepatitis c virus infection? analysis of a population-based cohort in canada hepatitis c incidence rate among people who inject drugs (pwid) in british columbia from prescription drug monitoring programs operational characteristics and fatal heroin poisoning syndemic characterization of hcv, hbv, and hiv co-infections in a large population based cohort study. eclinicalmedicine onatrio agency for health protection and promotion (public health ontario); office of the chief coroner; ontario forensic pathology service; ontario drug policy research network an area-based material and social deprivation index for public health in québec and canada facing opioids in the shadow of the hiv epidemic another thing to live for": supporting hcv treatment and cure among indigenous people impacted by substance use in canadian cities hiv infection linked to injection use of oxymorphone in indiana a transitioning epidemic: how the opioid crisis is driving the rise in hepatitis c provincial health officer declares public health emergency |bc gov news i-track: enhanced surveillance of hiv, hepatitis c, and associated risk behaviours among people who inject drugs in canada -phase the daily -causes of death b). the daily -changes in life expectancy by selected causes of death consensus recommendations for national and state poisoning surveillance county-level vulnerability assessment for rapid dissemination of hiv or hcv infections among persons who inject drugs, united states fentanyl checking expanding to overdose prevention sites global health sector strategy on viral hepatitis impact of drug overdose-related deaths on life expectancy at birth in british columbia. health promotion and chronic disease prevention in canada increases in acute hepatitis c virus infection related to a growing opioid epidemic and associated injection drug use, united states we thank the british columbia centre for disease control (bccdc), bccdc provincial health lab, provincial health services authority, bc ministry of health, bc cancer and their respective program staff involved in data access, procurement and data management for their assistance. we also appreciate the thorough review of the paper by rosemary armour at the bc vital statistics agency and tej sidhu from the bc coroner's service. supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.drugpo.2020.102989. key: cord-341071-nwrl1qws authors: berzal-herranz, alfredo; romero-lópez, cristina title: two examples of rna aptamers with antiviral activity. are aptamers the wished antiviral drugs? date: 2020-07-22 journal: pharmaceuticals (basel) doi: 10.3390/ph13080157 sha: doc_id: 341071 cord_uid: nwrl1qws the current covid-19 pandemic has pointed out some major deficiencies of the even most advanced societies to fight against viral rna infections. once more, it has been demonstrated that there is a lack of efficient drugs to control rna viruses. aptamers are efficient ligands of a great variety of molecules including proteins and nucleic acids. their specificity and mechanism of action make them very promising molecules for interfering with the function encoded in viral rna genomes. rna viruses store essential information in conserved structural genomic rna elements that promote important steps for the consecution of the infective cycle. this work describes two well documented examples of rna aptamers with antiviral activity against highly conserved structural domains of the hiv-1 and hcv rna genome, respectively, performed in our laboratory. they are two good examples that illustrate the potential of the aptamers to fill the therapeutic gaps in the fight against rna viruses. the current global pandemic caused by sars-cov-2 has revealed the fragility of our global health security system. researchers have been warning governments about the risk of different heath threats we face today, and this outbreak has clearly shown that no country, not even the so called first world, is safe and protected against a sanitary emergency. among other deficiencies, one of the major limitations to fight against the current viral infection has been the lack of specific therapeutic agents, which is a common fact for many other infections caused by rna viruses (hiv, hcv, sars, ebola, dengue, and zika viruses are just few examples of modern viral rna outbreaks). researchers have traditionally devoted many efforts to searching for the magic molecule or therapeutic strategy that would allow fighting efficiently against these tiny organisms that so easily put the most evolved living being in check. rna viruses store all the required information for the completion of the infectious cycle in their rna genome. in addition to the information that encodes the viral proteins, the genome includes the information required to efficiently sequester and utilize the cellular machinery and all the information involved in the regulation of the viral processes. rna viruses have developed different molecular strategies to bear all the information, compacting it in different hierarchical levels of coding within the rna genome [1] [2] [3] . thus, the nucleotide sequence stores essential information in highly conserved structural domains composed of discrete structural units ( figure 1 ) [1] . this information storage system overlaps and complements the protein coding information. rna genomes are multifunctional molecules: they act as a replication template, they act as mrnas, and behave as true non-coding rnas (ncrnas), playing several essential functions for the viral cycle and the regulation of viral processes [1] . pharmaceuticals 2020, 13 elucidating the function of each rna domain and their modus operandi would provide an excellent scenario to address the control of infections caused by rna viruses, broadening beyond proteins the repertoire of potential target candidates to fight viral diseases. interfering with the structure of the functional genomic rna domains or compromising their mechanisms of action should challenge their proper functioning and therefore the completion of the viral propagation cycle. this report shows how aptamers can exploit the structural features of viral genomic rnas as therapeutic targets, while allowing the implementation of therapeutic tools, which may be developed against a variety of rna viruses. pharmaceuticals 2019, 12, x for peer review 2 of 10 and the regulation of viral processes [1] . elucidating the function of each rna domain and their modus operandi would provide an excellent scenario to address the control of infections caused by rna viruses, broadening beyond proteins the repertoire of potential target candidates to fight viral diseases. interfering with the structure of the functional genomic rna domains or compromising their mechanisms of action should challenge their proper functioning and therefore the completion of the viral propagation cycle. this report shows how aptamers can exploit the structural features of viral genomic rnas as therapeutic targets, while allowing the implementation of therapeutic tools, which may be developed against a variety of rna viruses. it was in 1990 that two independent publications from the groups of jack szostack and larry gold set the basis for the development of the currently known aptamer technology [4, 5] . the group of jack szostak described the isolation of short rna oligonucleotides that were able to efficiently bind to organic dyes. they coined the term aptamer to name these binder molecules [4] . on the other hand, the principles of the general strategy for the selection of aptamers that is known as selex were defined by the larry gold's group. selex stands for systematic evolution of ligands by exponential enrichment [5] . aptamers are defined as short oligonucleotides, dna and rna, that exhibit extraordinary binding efficiency to a specific target molecule [4] . since then, numerous reports have described the successful identification of aptamers targeting from single molecules like ions, amino acids, nucleotides, antibiotics, etc., to macromolecules like peptides, proteins, or nucleic acids, and even targeting viruses and full cells [6] [7] [8] [9] [10] [11] [12] [13] [14] . the potential of this technology was envisioned quite early, and the therapeutic application of aptamers for a wide variety of diseases was proposed. it was in 2004 that the fda approved the first aptamer to be used as a specific drug. this aptamer, which was named pegaptanib and commercialized as macugen, was meant for the treatment of the wet age-related macular degeneration [15] [16] [17] . nevertheless, the full therapeutic potential of the aptamers is still to be developed. independently of the application of the aptamers and the nature of their target, all reported aptamers have been identified following a common experimental strategy known as selex [5, 18] , which is outlined in figure 2 . briefly, a large in vitro synthesized population of variable sequence oligonucleotides, typically ranging from 10 12 to 10 16 variants, is subjected to the selex procedure, which consists of iterative cycles of exposition to the target molecule to allow their binding to the target, separation of those molecules capable of binding and their further amplification. this procedure allows the progressive enrichment of the population in winner molecules for specific binding to the desired target. those winner molecules are named aptamers. it was in 1990 that two independent publications from the groups of jack szostack and larry gold set the basis for the development of the currently known aptamer technology [4, 5] . the group of jack szostak described the isolation of short rna oligonucleotides that were able to efficiently bind to organic dyes. they coined the term aptamer to name these binder molecules [4] . on the other hand, the principles of the general strategy for the selection of aptamers that is known as selex were defined by the larry gold's group. selex stands for systematic evolution of ligands by exponential enrichment [5] . aptamers are defined as short oligonucleotides, dna and rna, that exhibit extraordinary binding efficiency to a specific target molecule [4] . since then, numerous reports have described the successful identification of aptamers targeting from single molecules like ions, amino acids, nucleotides, antibiotics, etc., to macromolecules like peptides, proteins, or nucleic acids, and even targeting viruses and full cells [6] [7] [8] [9] [10] [11] [12] [13] [14] . the potential of this technology was envisioned quite early, and the therapeutic application of aptamers for a wide variety of diseases was proposed. it was in 2004 that the fda approved the first aptamer to be used as a specific drug. this aptamer, which was named pegaptanib and commercialized as macugen, was meant for the treatment of the wet age-related macular degeneration [15] [16] [17] . nevertheless, the full therapeutic potential of the aptamers is still to be developed. independently of the application of the aptamers and the nature of their target, all reported aptamers have been identified following a common experimental strategy known as selex [5, 18] , which is outlined in figure 2 . briefly, a large in vitro synthesized population of variable sequence oligonucleotides, typically ranging from 10 12 to 10 16 variants, is subjected to the selex procedure, which consists of iterative cycles of exposition to the target molecule to allow their binding to the target, separation of those molecules capable of binding and their further amplification. this procedure allows the progressive enrichment of the population in winner molecules for specific binding to the desired target. those winner molecules are named aptamers. the researcher can control at any time the conditions of the selection and recovery steps during the process. usually, the main goal is to identify the selection to yield aptamers that exhibit high affinity pharmaceuticals 2020, 13, 157 4 of 10 by a desired target, but the researcher can also pursue the selection of aptamers that achieve stable binding by the imposed conditions. it has been shown that aptamers may bind the ligand molecule with efficiencies that even may exceed that of antibodies for their antigens [19] . aptamers show some important advantages [19] , including that the use of experimental animals is not required for their production, as opposed to antibodies. they can be chemically modified to increase their stability or resistance to nuclease degradation, by means of the addition of certain chemical groups (fluoro, methyl, or methoxy among them) at different positions of the nucleotide, or by replacing specific nucleotides by nucleotide analogs (e.g., locked nucleic acids-lnas, peptide nucleic acids-pnas). thus, in no case the specificity of the nucleotide sequence of the aptamer is altered. modifications can be also added to improve specific aptamer delivery to the target. aptamers can also be modified by incorporating different types of labeling (e.g., radioactive, fluorescent) and various functional groups, as well as to conjugate with other molecules (peg, sugar residues, sirnas, among others) to achieve many other different functionalities [20] . specificity of aptamers is determined by both their sequence and their three-dimensional folding. further, aptamers recognize specific functional groups of the target molecule in a precise spatial distribution. therefore, aptamers are good candidates to target complexly folded targets. consequently, in contrast to other nucleic acid based strategies (ribozymes, antisense oligonucleotides, sirna, among others), aptamers are postulated as excellent candidates to directly interfere with the functioning of essential structural domains of viral rna genomes. targeting viral proteins is the common strategy that has been followed in most attempts aimed to develop efficient antiviral agents regardless of the drug to be used. aptamers selected against a variety of viral proteins have also been reported in the literature. those studies account for the achievement of a range of antiviral activity levels [18] . unfortunately, up to now such a strategy has been found to be very inefficient, yielding in most cases unacceptable therapeutic results. even in the best scenarios, the achievements that have occurred over the years of enormous economic and human resources investments are unfortunately not enough to compensate for the unacceptable number of human lives and the consequences on the health and well-being of humans that viral infections take, among other consequences. this is evident in its maximum expression in a pandemic situation like the one we are currently experiencing. therefore, it is widely accepted by the scientific community the need of exploring alternative viral targets and developing of new therapeutic antiviral strategies [1] . targeting the essential information encoded in conserved structural rna domains in viral genomes represents an attractive yet unexplored strategy. nucleic acids are shown as the most likely suitable tools to interfere directly with such information, aptamers being excellent candidates for challenging the functioning of structured rna molecules. herein, we present an extension of a video we communicated at the 5th ecmc describing two examples of selection and characterization of aptamers targeting essential structural domains of respective viral rna genomes, performed in our laboratory [21] . in both cases we followed a common selex strategy ( figure 3) . briefly, the target subgenomic rna fragment was internally biotinylated during in vitro synthesis, under experimental conditions to render a theoretical incorporation of one biotinylated-utp residue per rna molecule [22, 23] . the low rate of nucleotide modification was imposed to the lowest, to achieve the minimum possible interference in the proper folding of the target viral rna by the bulky biotin residue. then, the target was immobilized to a sepharose-streptavidin column, allowing it to adopt its natural conformation. in parallel, the starting pool of rna oligonucleotides resulting from the randomization of 25-30 consecutive nucleotides flanked by fixed sequences was synthesized [24] . theoretically, each rna oligonucleotide contains a different sequence, which will determine a different molecular folding. the combination of the pharmaceuticals 2020, 13, 157 5 of 10 sequence and the structure will be responsible for the functioning of each molecule, determining whether a specific oligonucleotide is capable of binding to the target rna molecule. the pool of rna oligonucleotides was binding-challenged against the immobilized target molecule. then, only oligonucleotides capable of binding to the target rna were retained. after their selective elution and amplification by rt-pcr, the new generated pool was subsequently introduced in a new round of selection. this was repeated until rendering the most efficient binders to the viral genomic fragment [24] . theoretically, each rna oligonucleotide contains a different sequence, which will determine a different molecular folding. the combination of the sequence and the structure will be responsible for the functioning of each molecule, determining whether a specific oligonucleotide is capable of binding to the target rna molecule. the pool of rna oligonucleotides was binding-challenged against the immobilized target molecule. then, only oligonucleotides capable of binding to the target rna were retained. after their selective elution and amplification by rt-pcr, the new generated pool was subsequently introduced in a new round of selection. this was repeated until rendering the most efficient binders to the viral genomic fragment [24] . we applied the above experimental strategy to isolate aptamers against the 5′utr of the human immune deficiency type 1 virus (hiv-1) [25] . for this purpose, the target rna consisted of the genomic viral rna fragment comprising the first 308 nt of its 5′utr. this fragment is common to all genomic and subgenomic hiv-1 rnas [26] . it contains several well defined structural elements whose functioning is essential for the hiv efficient infection cycle (figure 4) . the starting rna pool consisted of theoretically 10 15 oligonucleotide variants, resulting from the randomization of 25 contiguous nucleotides. the selection cycle was repeated up to 14 times [25] . the sequence analysis of resulting molecules revealed the presence of the eight nt-long sequence, 5′-ggcaagga-3′, in the approximately 90% of the selected sequences (table 1) . interestingly, this octanucleotide is complementary to an eight nt-long sequence within the apical loop of the polya structural element of the hiv-1 5′utr. this result points to the polya domain as the target of the resulting aptamers [25] . we applied the above experimental strategy to isolate aptamers against the 5 utr of the human immune deficiency type 1 virus (hiv-1) [25] . for this purpose, the target rna consisted of the genomic viral rna fragment comprising the first 308 nt of its 5 utr. this fragment is common to all genomic and subgenomic hiv-1 rnas [26] . it contains several well defined structural elements whose functioning is essential for the hiv efficient infection cycle (figure 4) . the starting rna pool consisted of theoretically 10 15 oligonucleotide variants, resulting from the randomization of 25 contiguous nucleotides. the selection cycle was repeated up to 14 times [25] . the sequence analysis of resulting molecules revealed the presence of the eight nt-long sequence, 5 -ggcaagga-3 , in the approximately 90% of the selected sequences (table 1) . interestingly, this octanucleotide is complementary to an eight nt-long sequence within the apical loop of the polya structural element of the hiv-1 5 utr. this result points to the polya domain as the target of the resulting aptamers [25] . instead of concluding the aptamer's selection at this point, we decided to introduce an innovative further step. we complemented the selex approach with a bioinformatics analysis of the identified sequences [25] . it consisted of a thorough comparison of the sequences derived from each selection round and their respective secondary structure folding, determining the sequence and structural evolution of the resulting molecules along the process. this allowed us to identify a minimal 16 nt-long structural element common to most of the selected molecules from round 11 to 14. interestingly, this 16 nt-long structural motif contained the above mentioned octanucleotide sequence motif universally exposed in an apical loop closing a 4 bp stem of variable sequence ( figure 5 ). these results indicated that the properly folded 16 nt-long stem-loop element containing the 5′-ggcaagga-3′ sequence motif correctly exposed was responsible for the aptamers' activity. this hypothesis was confirmed by targeting the hiv-1 5′utr fragment with a synthetic 16 nt-long rna molecule (rnapt16) comprising the minimal selected structural rna. first rnapt16 showed binding affinity and efficacy in a similar extend to that shown for the parental aptamers [25] . inhibition studies of hiv-1 particles production in a cell culture analysis, by measuring the p24 viral antigen, demonstrated that rnapt16 yielded inhibition levels higher than 85%, achieving higher inhibition ratios than the two most abundant parental aptamers (xiv22 and xiv26) ( figure 5 ) [25] . instead of concluding the aptamer's selection at this point, we decided to introduce an innovative further step. we complemented the selex approach with a bioinformatics analysis of the identified sequences [25] . it consisted of a thorough comparison of the sequences derived from each selection round and their respective secondary structure folding, determining the sequence and structural evolution of the resulting molecules along the process. this allowed us to identify a minimal 16 nt-long structural element common to most of the selected molecules from round 11 to 14. interestingly, this 16 nt-long structural motif contained the above mentioned octanucleotide sequence motif universally exposed in an apical loop closing a 4 bp stem of variable sequence ( figure 5 ). these results indicated that the properly folded 16 nt-long stem-loop element containing the 5 -ggcaagga-3 sequence motif correctly exposed was responsible for the aptamers' activity. this hypothesis was confirmed by targeting the hiv-1 5 utr fragment with a synthetic 16 nt-long rna molecule (rnapt16) comprising the minimal selected structural rna. first rnapt16 showed binding affinity and efficacy in a similar extend to that shown for the parental aptamers [25] . inhibition studies of hiv-1 particles production in a cell culture analysis, by measuring the p24 viral antigen, demonstrated that rnapt16 yielded inhibition levels higher than 85%, achieving higher inhibition ratios than the two most abundant parental aptamers (xiv22 and xiv26) ( figure 5 ) [25] . particles production assays. inhibitory efficiency of rnapt16 aptamer was measured as p24 antigen production. its activity was compared with that showed by the most frequent aptamers resulting from the selex procedure lxiv22, lxiv26. data represent the mean of three independent assays. **, significant differences as compared to the control (p < 0.01). figure adapted from [27] . the innovative combination of a selex procedure with a bioinformatics analysis allowed the identification of a common 16 nt-long structural element, consisting of an octanucleotide of fixed sequence exposed in a loop motif closed by 4 bp stem of variable sequence that retained the full antiviral activity. it served as base for the design of the, to our knowledge, smallest aptamer ever described. it is worth noting that the minimum molecular size of the aptamers is an important limitation. the size is imposed by the need of carrying flanking fixed sequences required for the amplification steps of the selex procedure. the application of the bioinformatics analysis provides unique means of identifying the minimum domain responsible of the entire aptamer activity. the hepatitis c virus (hcv) has been another favorite virus for the development of nucleic acids-based antiviral strategies. the organization and structure of its rna genome have been extensively studied [28] . it contains numerous structurally conserved elements that play essential roles in the viral infection cycle (figure 1 ). among them, the so called cis-replication element (cre), located at the 3′ end of the single open reading frame of the viral genome, has been identified as a center of regulation of major viral processes (e.g., replication, translation, genome dimerization) [29] . due to its functional importance for the establishment of the viral infection, it represents a good potential antiviral target. we applied the previously described selex strategy to identify rna aptamers against a 194 nt-long subgenomic rna comprising the cre [24] . a population of theoretically more than 10 18 rna variants, resulting from the randomization of a stretch of 30 nts, was introduced in the selection process. the resulting variant molecules after six selection rounds were classified in up to five families, defined by common selected sequence motifs. as expected, each common sequence possessed its complementary counterpart within the viral cre [24] . the prevalence of each common sequence motif in the final population could be related to a direct participation in the interaction of the aptamer with the cre target or to a stabilization role of the aptamer:cre complex. interestingly, many of the selected aptamers even bore sequence motifs belonging to different families, reinforcing the idea that each sequence family accomplished different functions [30] . the antiviral potential of the selected aptamers was determined by their ability to inhibit viral replication, using a subgenomic hcv replication model in huh-7 cell line culture. results demonstrated that aptamers belonging to family 2 promoted a slightly more potent inhibitory effect on hcv replication levels ( figure 6 ). nevertheless, the inhibitory activity of the different aptamer families was quite similar, suggesting that every sequence motif must be preserved to achieve the aptamer function. importantly, two inhibition of hiv-1 particles production assays. inhibitory efficiency of rnapt16 aptamer was measured as p24 antigen production. its activity was compared with that showed by the most frequent aptamers resulting from the selex procedure lxiv22, lxiv26. data represent the mean of three independent assays. **, significant differences as compared to the control (p < 0.01). figure adapted from [27] . the innovative combination of a selex procedure with a bioinformatics analysis allowed the identification of a common 16 nt-long structural element, consisting of an octanucleotide of fixed sequence exposed in a loop motif closed by 4 bp stem of variable sequence that retained the full antiviral activity. it served as base for the design of the, to our knowledge, smallest aptamer ever described. it is worth noting that the minimum molecular size of the aptamers is an important limitation. the size is imposed by the need of carrying flanking fixed sequences required for the amplification steps of the selex procedure. the application of the bioinformatics analysis provides unique means of identifying the minimum domain responsible of the entire aptamer activity. the hepatitis c virus (hcv) has been another favorite virus for the development of nucleic acids-based antiviral strategies. the organization and structure of its rna genome have been extensively studied [28] . it contains numerous structurally conserved elements that play essential roles in the viral infection cycle (figure 1 ). among them, the so called cis-replication element (cre), located at the 3 end of the single open reading frame of the viral genome, has been identified as a center of regulation of major viral processes (e.g., replication, translation, genome dimerization) [29] . due to its functional importance for the establishment of the viral infection, it represents a good potential antiviral target. we applied the previously described selex strategy to identify rna aptamers against a 194 nt-long subgenomic rna comprising the cre [24] . a population of theoretically more than 10 18 rna variants, resulting from the randomization of a stretch of 30 nts, was introduced in the selection process. the resulting variant molecules after six selection rounds were classified in up to five families, defined by common selected sequence motifs. as expected, each common sequence possessed its complementary counterpart within the viral cre [24] . the prevalence of each common sequence motif in the final population could be related to a direct participation in the interaction of the aptamer with the cre target or to a stabilization role of the aptamer:cre complex. interestingly, many of the selected aptamers even bore sequence motifs belonging to different families, reinforcing the idea that each sequence family accomplished different functions [30] . the antiviral potential of the selected aptamers was determined by their ability to inhibit viral replication, using a subgenomic hcv replication model in huh-7 cell line culture. results demonstrated that aptamers belonging to family 2 promoted a slightly more potent inhibitory effect on hcv replication levels ( figure 6 ). nevertheless, the inhibitory activity of the different aptamer families was quite similar, suggesting that pharmaceuticals 2020, 13, 157 8 of 10 every sequence motif must be preserved to achieve the aptamer function. importantly, two aptamers, p6-96 and p6-103, significantly reduced the hcv rna levels up to 95% ( figure 6 ) [30, 31] . interestingly, the more efficient inhibitors putatively target the essential 5bsl3.2 component of the cre [30] . aptamers, p6-96 and p6-103, significantly reduced the hcv rna levels up to 95% ( figure 6 ) [30, 31] . interestingly, the more efficient inhibitors putatively target the essential 5bsl3.2 component of the cre [30] . figure 6 . inhibition of hcv replication in huh-7 cells. graph shows the hcv rna levels in a hepatoma cell line supporting the autonomous cytoplasmic replication of a hcv subgenomic construct. cells were transfected with the selected aptamers and hcv rna levels were detected by rt-qpcr 24 h after transfection. boxplot was generated with these data in order to group those aptamers with common sequence motifs, belonging to each of the identified families (1 to 5). the box reflects the 1st and 3rd quartiles, and the median is represented by a black line within the box for each aptamer family. freq, refers to the prevalence of each family within the aptamer population. in an independent series of experiments, we isolated a new collection of rna aptamers conjugated with ribozymes that specifically targeted the translation-essential hcv ires (internal ribosome entry site), which is located at the 5′ end of the rna genome [23] . those rna aptamers exhibited up to 90% viral translation inhibition rates [23, [32] [33] [34] [35] . all together the above reported results point the great antiviral potential of targeting essential structural rna domains of the hcv genome. aptamers offer a potential means for the development of efficient antiviral drugs. their mechanism resides in the recognition of functional groups in a specific structural context in the target molecule, similar to antigens recognition by antibodies. viral rna genomes store essential information for the completion of the viral propagation cycle in structural rna elements. these elements are grouped in structurally complex domains and regions that coincide with the highest conserved regions of the highly variable viral genome among different viral isolates. the preservation of their structure is essential for the proper functioning of each of these elements, offering an excellent scenario for fighting infections caused by rna viruses. aptamers have proven to be capable of interfering with the activity of these essential domains by competing with the interactions they are involved in or by modifying their structure. different groups have already reported aptamers that induce potent inhibition rates of a variety of rna viruses by targeting conserved genomic rna domains. therefore, aptamers emerge as real molecular tools to fight viral infections. figure 6 . inhibition of hcv replication in huh-7 cells. graph shows the hcv rna levels in a hepatoma cell line supporting the autonomous cytoplasmic replication of a hcv subgenomic construct. cells were transfected with the selected aptamers and hcv rna levels were detected by rt-qpcr 24 h after transfection. boxplot was generated with these data in order to group those aptamers with common sequence motifs, belonging to each of the identified families (1 to 5). the box reflects the 1st and 3rd quartiles, and the median is represented by a black line within the box for each aptamer family. freq, refers to the prevalence of each family within the aptamer population. in an independent series of experiments, we isolated a new collection of rna aptamers conjugated with ribozymes that specifically targeted the translation-essential hcv ires (internal ribosome entry site), which is located at the 5 end of the rna genome [23] . those rna aptamers exhibited up to 90% viral translation inhibition rates [23, [32] [33] [34] [35] . all together the above reported results point the great antiviral potential of targeting essential structural rna domains of the hcv genome. aptamers offer a potential means for the development of efficient antiviral drugs. their mechanism resides in the recognition of functional groups in a specific structural context in the target molecule, similar to antigens recognition by antibodies. viral rna genomes store essential information for the completion of the viral propagation cycle in structural rna elements. these elements are grouped in structurally complex domains and regions that coincide with the highest conserved regions of the highly variable viral genome among different viral isolates. the preservation of their structure is essential for the proper functioning of each of these elements, offering an excellent scenario for fighting infections caused by rna viruses. aptamers have proven to be capable of interfering with the activity of these essential domains by competing with the interactions they are involved in or by modifying their structure. different groups have already reported aptamers that induce potent inhibition rates of a variety of rna viruses by targeting conserved genomic rna domains. therefore, aptamers emerge as real molecular tools to fight viral infections. funding: this research received no external funding. unmasking the information encoded as structural motifs of viral rna genomes: a potential antiviral target unmasking the information encoded as structural motifs of viral rna genomes: a potential antiviral target structural and functional characterization of programmed ribosomal frameshift signals in west nile virus strains reveals high structural plasticity among cis-acting rna elements functional and structural characterization of the chikungunya virus translational recoding signals in vitro selection of rna molecules that bind specific ligands systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase selection in vitro of single-stranded dna molecules that fold into specific ligand-binding structures selection of high affinity rna ligands to the bacteriophage r17 coat protein in vitro selection of functional nucleic acids selection of fluorescent aptamer beacons that light up in the presence of zinc enrichment of cell-targeting and population-specific aptamers by fluorescence-activated cell sorting aptasensors for detection of microbial and viral pathogens berzal-herranz, a. in vitro and ex vivo selection procedures for identifying potentially therapeutic dna and rna molecules nucleic acid aptamers: an emerging tool for biotechnology and biomedical sensing monitoring intact viruses using aptamers anti-vegf aptamer (pegaptanib) therapy for ocular vascular diseases pegaptanib, a targeted anti-vegf aptamer for ocular vascular disease antivascular endothelial growth factor therapy for neovascular age-related macular degeneration methods developed for selex analytical applications of aptamers aptamers chemistry: chemical modifications and conjugation strategies antiviral drugs of the future degree of biotinylation in nucleic acids estimated by a gel retardation assay berzal-herranz, a. interfering with hepatitis c virus ires activity using rna molecules identified by a novel in vitro selection method anti-hcv rna aptamers targeting the genomic cis-acting replication element berzal-herranz, a. efficient hiv-1 inhibition by a 16 nt-long rna aptamer designed by combining in vitro selection and in silico optimisation strategies hiv-1 as rna evolution machine potential of the other genetic information coded by the viral rna genomes as antiviral target the role of the rna-rna interactome in the hepatitis c virus life cycle berzal-herranz, a. the 5bsl3.2 functional rna domain connects distant regions in the hepatitis c virus genome berzal-herranz, a. rna aptamers as molecular tools to study the functionality of the hepatitis c virus cre region berzal-herranz, a. rna aptamer-mediated interference of hcv replication by targeting the cre-5bsl3.2 domain inhibition of hepatitis c virus internal ribosome entry site-mediated translation by an rna targeting the conserved iiif domain berzal-herranz, a. inhibition of hepatitis c virus replication and internal ribosome entry site-dependent translation by an rna molecule berzal-herranz, a. an engineered inhibitor rna that efficiently interferes with hepatitis c virus translation and replication development of optimized inhibitor rnas allowing multisite-targeting of the hcv genome funding: this research received no external funding. the authors declare no conflict of interest. the authors declare no conflict of interest.pharmaceuticals 2020, 13, 157 key: cord-337285-t6qr41wc authors: ikeda, masanori; kato, nobuyuki title: modulation of host metabolism as a target of new antivirals() date: 2007-10-10 journal: adv drug deliv rev doi: 10.1016/j.addr.2007.03.021 sha: doc_id: 337285 cord_uid: t6qr41wc the therapy for chronic hepatitis c (ch–c) started with interferon (ifn) monotherapy in the early 1990s and this therapy was considered effective in about 10% of cases. the present standard therapy of pegylated ifn with ribavirin achieves a sustained virologic response in about 50% of patients. however, about half of the ch–c patients are still at risk of fatal liver cirrhosis and hepatocellular carcinoma. the other significant event in hepatitis c virus (hcv) research has been the development of a cell culture system. the subgenomic replicon system enables robust hcv rna replication in hepatoma cells. and recently, the complete life cycle of hcv has been achieved using a genotype 2a strain, jfh1. these hallmarks have provided much information about the mechanisms of hcv replication, including information on the host molecules required for the replication. anti-hcv reagents targeting hcv proteins have been developed, and some of them are now in clinical trials. however, the rna-dependent rna polymerase frequently causes mutations in the hcv genome, which lead to the emergence of drug-resistant hcv mutants. some of the cellular proteins essential for hcv rna replication have already been discovered using the hcv cell culture system. these host molecules are also candidate targets for antivirals. here, we describe the recent progress regarding the anti-hcv reagents targeting host metabolism. hepatitis c virus (hcv) was discovered in 1989 [1] as the causative agent of chronic hepatitis c (ch-c), liver cirrhosis and hepatocellular carcinoma (hcc) [2] . it is estimated that 170 million people worldwide are infected with hcv [3] . the ultimate goal of both clinical and basic hcv studies is the suppression of liver-related death caused by hcv infection. with respect to clinical studies, interferon (ifn) has played a major role in the treatment of patients with ch-c. ifn therapy started with ifn monotherapy in the early 1990s, and a sustained virologic response (svr) was obtained in about 10% of patients [4] . ifn therapy was developed by the hepatologists, and the current therapy of pegylated ifn (peg-ifn) with ribavirin has improved the svr to about 50% [4] . therefore, the next stage of the therapy for ch-c is to develop new anti-hcv reagents to improve the svr. during the development of ifn therapy, the most striking discovery in the basic research was the development of a cell culture system for robust hcv rna replication. in 1999, lohmann et al. [5] achieved subgenomic hcv rna replication in a human hepatoma cell line, huh-7. the advantages of this novel system (known as the replicon system) were that it provided not only a way to screen for anti-hcv reagents but also information about the mechanism of hcv rna replication. this cell culture system has been further improved, and recently the complete life cycle of hcv was achieved using a genotype 2a hcv strain, jfh1 [6] [7] [8] . this newest system has extended the targets of the anti-hcv therapy to the virus infection and release. the effects of anti-hcv reagents selected from the cell culture-based screening should be evaluated using an animal model system for hcv infection before they can be released to clinical trial. chimpanzees were the only animal model in the early hcv studies [9] . however, the use of chimpanzees is limited for ethical and financial reasons. in addition to chimpanzees, a study using tree shrews (tupaia belangeri chinensis) has been reported [10] . a different approach to the study of hcv using animal models was achieved using the related gb virus b (gbv-b). gbv-b belongs to the flaviviridae family and can be transmitted to tamarins and marmosets [11, 12] . these animal models may be valuable surrogate models for hcv study. another approach was demonstrated in a study using urokinase plasminogen activator-severe combined immunodeficiency (upa-scid) mice transplanted with human hepatocytes [13] . this chimeric mouse model can support chronic hcv viremia under the circumstance without immune system. mass screening for anti-hcv reagents using cell culture systems will become a more powerful tool when combined with small animal model systems to evaluate the antiviral effects of selected reagents before clinical trial. in considering a new strategy for ch-c to be used in place of or in combination with ifn, the main targets are hcv proteins and hcv rna. with respect to the hcv proteins, two of these, nonstructural (ns) 3-4a and ns5b, have been wellcharacterized as protease and rna-dependent rna polymerase (rdrp), respectively [14, 15] . several reagents have been reported to be inhibitors of ns3-4a serine protease, including sch6 [16, 17] , sch503034 [18] , vx-950 [19, 20] , and biln-2061 [21] . valopicitabine (nm283) was reported to inhibit ns5b rdrp [22] . hcv rna itself is also a target of antivirals, and recent rna interference technologies using sirna or shrna have targeted hcv rna [23] [24] [25] . as rdrp lacks proofreading activity, the high mutation rate of rdrp allows the virus to escape from the reagents targeting hcv proteins and hcv rna. these anti-hcv reagent-targeting viral proteins and genome will be reviewed in another section. other targets are the cellular proteins essential for hcv rna replication and infection. the expression of hcv proteins is thought to affect the host cells' gene expression profiles and vise versa [26] . the interaction of the specific cellular proteins with hcv proteins is essential for hcv replication (table 1) . cyclosporine a (csa) is one of the best characterized inhibitors targeting the cellular proteins required for hcv replication [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . the interaction of cyclophilin b (cypb) with ns5b is required for hcv rna replication [28] . csa inhibits hcv rna replication by interrupting the interaction between ns5b and cypb. heat shock protein 90 (hsp90) has also been reported to be an essential cellular protein for hcv rna replication [37] [38] [39] . knockdown or inhibition of hsp90 has been shown to result in the anti-hcv activity in cell culture and in upa-scid mouse systems [37] . fkbp8, a member of the fk506-binding protein family, specifically interacts with ns5a and forms a complex with hsp90 [38] . the la autoantigen (la) and polypyrimidine tractbinding protein (ptb) are also candidate cellular proteins for the inhibition of hcv rna replication [40] , although no inhibitors for these proteins have been reported to date. thus, inhibition of the metabolism has recently been reported as a target of the new antivirals. here, we survey the recent progress on enzyme inhibitors of the cholesterol, sphingolipid, and guanosine triphosphate (gtp) synthesis pathways, as well as other metabolic pathways. hcv was discovered to be the causative agent of non-a, non-b hepatitis by the chiron corporation in 1989 [1] . however, a treatment for patients with non-a, non-b hepatitis was established before the discovery of hcv. in 1986, hoofnagle et al. reported that ifn-α treatment normalized the serum alanine aminotransferase (alt) levels in patients with non-a, non-b hepatitis [41] . since the initial discovery of its anti-hcv activity, ifn-α has become the major reagent for ch-c treatment [4] . the replication of hcv rna itself seems to stimulate ifn production signaling, and our recent results have suggested that core and/or ns5b induce ifn-stimulated genes [42] [43] [44] . however, viral ns3-4a protease inhibits the ifn production, although it does not completely shut it off. therefore, exogenous ifn administration is needed for patients with ch-c. the svr is affected by multiple factors, such as genotype, viral load and duration of therapy. ifn-α monotherapy was begun in the early 1990s, but an svr was achieved in only about 10% of patients. in the early 2000s, ifn-α and ribavirin combination therapy was developed and the svr was improved to about 30-40%. furthermore, ifn itself has been modified by the attachment of peg, thereby enhancing its stability in the blood. the svr of the current standard therapy by peg-ifn and ribavirin is as high as 50% [4] . in the current peg-ifn and ribavirin combination therapy, the genotype of hcv is one of the major determinants of the svr. hcv genotypes are classified into 6 groups, and genotype 1 is currently considered a problem due to its ifn resistance [45] . for example, in genotype 1 hcv, 12 months of treatment resulted in an svr in 50% of patients, while in genotype 2, 6 months of treatment achieved an svr of 80-90% [46] . the precise mechanisms of the ifn resistance remain unclear. however, the recently developed ifn-resistant hcv replicon-harboring cells will be useful for studies examining ways to improve the svr [47] [48] [49] . therefore, the focus in the treatment of patients with ch-c has shifted to increasing the svr in genotype 1 hcv. before the development of an hcv replicon system, screening of anti-hcv reagents was rather difficult. the hcv replicon system developed by lohmann et al. [5] was the first milestone in hcv study using a cell culture system. the replicon system has provided a wealth of information concerning the replication machinery of hcv. we can make strategies for the achilles' heel of hcv based on the information regarding hcv rna replication. the hcv replicon has been improved to be a suitable system for the screening of anti-hcv reagents by the introduction of reporter genes such as luciferase [50] . however, this system does not contain a structural region. therefore, selectable genome-length hcv rna-replicating cell culture systems have been developed [51] [52] [53] [54] . the second milestone was the infectious virus production system established by the three groups using a genotype 2a hcv strain, jfh1 [6] [7] [8] . this system has extended the range of the hcv study to the viral entry and release. therefore, the life cycle of hcv in the cells has been reconstructed in vitro. since the development of the hcv replicon and infectious hcv production systems, many cellular proteins have been identified as essential host molecules for hcv rna replication. the hcv replicon reported by lohmann et al. contained neomycin phosphotransferase (neo) and encephalomyocarditis virus (emcv) internal ribosome entry sites (ires) instead of the hcv structural regions (fig. 1 ) [5] . this hcv replicon consists of 2 cistrons. in the first cistron, neo is translated by hcv-ires and in the second cistron ns3-ns5b is translated by emcv-ires introduced in the region upstream of the ns region ( fig. 1) . after the development of the hcv replicon system [52, 53, [55] [56] [57] [58] , genome-length hcv rna replication systems using different hcv strains (h, n, con1, and o) were developed by several groups [51] [52] [53] [54] . in these genome-length hcv rna replication systems, a complete open reading frame (orf) of hcv was introduced into the second cistron instead of the ns region (fig. 1) . for the mass screening for anti-hcv reagents, evaluation of the levels of hcv rna or hcv proteins requires time and complicated procedures. to facilitate the monitoring of the replication level of hcv rna, the reporter gene (renilla luciferase) was fused to the neo gene. in this system, anti-hcv activity was evaluated by the value of the reporter instead of the pufas [70, 103, 108] quantification of hcv rna or hcv proteins. as shown in fig. 2a , orn/c-5b/ke contains the fused renilla luciferase and neo genes in the first cistron [51] . one of the cloned cell lines, or6, was established by the g418 selection after introduction of orn/c-5b/ke rna into huh-7 cells. hcv rna and hcv proteins were stably expressed in the or6 cells, and the renilla luciferase activity was correlated well with the level of hcv rna [51] . therefore, the antiviral effect of the reagents on hcv rna replication could be monitored by the activity of renilla luciferase. the or6 assay system facilitates the mass screening for anti-hcv reagents. hcv rna replicating in or6 cells contained an adaptive mutation, k1609e, in the ns3 region. adaptive mutations have been reported to enhance the replication level of hcv rna in cell culture [59] [60] [61] . in the case of hcv-o, two adaptive mutations were required for robust replication of the genome-length hcv rna replication [60] . for example, authentic hcv-o rna with the adaptive mutations of e1202g and k1609e can robustly replicate in huh-7 cells for 9 months or more (ikeda et al., unpublished data). in 2005, three groups reported infectious hcv production systems using the jfh1 strain in cell culture [6] [7] [8] . these reports showed that the life cycle of hcv could be reconstructed in huh-7 cells, and thus became landmarks in the search for an ideal hcv cell culture system. the unique features of these systems were the origin of this strain and the cell lines. jfh1 was a genotype 2a strain derived from a patient with fulminant hepatitis and did not require any adaptive mutations for robust replication, unlike other hcv strains. the unique feature of this system was that it employed huh-7 cells such as huh-7.5 or huh-lunet cells, since the parental huh-7 cells could not support robust production of infectious hcv [6] [7] [8] 62] . recently, the genotype 1a h77-s strain was reported to produce infectious hcv in cell culture, although the production level of infectious h77-s was lower compared with that by jfh1 [63] . interestingly, five adaptive mutations were introduced into the h77-s genome in order to enhance the efficiency of infectious virus production. the presence of these adaptive mutations is the most striking and controversial characteristic regarding the production of infectious hcv described above. further study will be needed to understand the role of adaptive mutations on infectious virus production. the establishment of an infectious hcv production system gradually led to clarification of the life cycle of hcv. information regarding the hcv rna replication has been accumulated since the development of the hcv replicon system, and the infectious hcv production system [6] [7] [8] has further provided information about the step of virus entry and release. the life cycle of hcv includes the (1) receptor binding and cell entry, (2) cytoplasmic release and uncoating, (3) ires-mediated translation, (4) processing, (5) rna replication, (6) packaging and assembly, (7) virion maturation, and (8) virion release. although some of the mechanisms are still unclear, each of these steps is a target for antivirals. among the proteins involved in these steps, the protease in step (4) and polymerase in step (5) have been especially well characterized. specific inhibitors for these proteins have been developed and some of them are now in clinical trials for patients with ch-c [21, 64] . cellular proteins are required for hcv rna replication and may determine the cell tropism of hcv. as hcv is a parasite, it utilizes the cellular proteins for its replication machinery. therefore, cellular proteins essential for hcv rna replication are the targets for antivirals. using cell culture systems, several cellular proteins have been identified as effective molecules for hcv rna replication (table 1) . la and ptb were representative molecules reported as essential host factors for hcv rna replication [40] . recently, an immunosuppressant, csa, has been reported to inhibit hcv rna replication by blocking the binding of cypb to ns5b [28] . hsp90 and the fk-506binding protein 8 (fkbp8) form a complex with ns5a and geldanamycin, an inhibitor of hsp90, suppressed hcv rna replication by blocking the formation of these complex [38] . the advantage of the inhibitors targeting cellular factor is that these reagents do not affect the viral escape achieved through mutations. the high mutation rate caused by rdrp frequently produced escape mutants toward the antiviral reagents for hcv proteins. a disadvantage of the inhibitors targeting cellular factors may be that they induce side effects by inhibiting the primary roles of the cellular factors. the cellular factors are the targets of the antivirals independent of the viral escape via the genetic mutations caused by rdrp. the cellular factors were synthesized in their metabolic pathways and modified by the enzymes. these enzymes are also targets in the antiviral strategy (table 1) . furthermore, some of the reagents have already been used in the clinical treatment of the respective diseases. one of the advantages of using existing reagents is that their characterizations-including safety and side effects-have already been performed. therefore, screening of the existing reagents for anti-hcv will be a new field of antivirals. the development of a cell culture system for hcv led to the revelation that hcv incorporates many cellular factors into the replication machinery of the virus. now we have both the information of the hcv life cycle and the cell culture assay system-the input and output-that we need to develop a pool of antiviral reagents. below, we will discuss the particular host cell metabolic pathways that are currently being targeted by anti-hcv reagents including more recently found pitavastatin (ptv) (fig. 2b ). in the cholesterol-biosynthesis pathway, the region downstream of mevalonate branches into separate pathways for cholesterol and isoprenoid synthesis (fig. 3) . the attachment of the isoprenoid is called prenylation of the protein. prenylation regulates a variety of cellular functions, such as growth, differentiation, and oncogenesis. farnesyl pyrophosphate (fpp) and geranylgeranyl pyrophosphate (ggpp) are mevalonate-derived isoprenoids and are attached to the target proteins by farnesyltransferase (ftase) and geranylgeranyl transferase type i (ggtase-i), respectively. ftase and ggtase-i recognize protein substrates with a c-terminal tetrapeptide recognition motif called the caax box: in the case of ggtase-i, c is cysteine, a is an aliphatic amino acid, and x is leucine, isoleucine, valine, or phenylalanine. production of mevalonate by 3-hydroxy-3methylglutaryl coenzyme a (hmg-coa) reductase is the rate-limiting step in the cholesterol biosynthesis. statins are potent hmg-coa reductase inhibitors and are beneficial in the prevention of coronary heart disease. statins also inhibit the prenylation of the proteins. lipid metabolism is essential for the life cycle of many viruses. the cholesterol-rich lipid raft plays an important role in virus entry, replication, and assembly. hcv also forms a replication complex on the lipid raft membrane structure [65] . hcv rna replication occurs in the lipid raft and the cholesterol supply is crucial to maintain the structure of the lipid raft [65] . aizaki et al. [66] reported that lovastatin (lov), one of the hmg-coa reductase inhibitors, inhibited hcv rna replication in hcv replicon-harboring cells. statins also possess the cholesterol-independent action (pleiotropic effect) [67] . many of these pleiotropic effects are mediated by the isoprenoid. for example, inhibition of small gtp-binding proteins, ras and rho, whose proper membrane localization and function are dependent on prenylation, may play a significant role in the pleiotropic effect of statins. ras and rho are major substrates for prenylation with fpp and ggpp, respectively. gdp-bound ras and rho are localized in the cytoplasm. when fpp or ggpp is bound to the inactive ras or rho, they are translocated to the cell membrane and converted to gtp-bound active forms. recently, wang et al. [68] identified fbl2 as one of the geranylgeranylated cellular proteins required for hcv rna replication. fbl2 belongs to the fbl family of proteins, all of which contains an f box and a multiple leucine-rich repeat, with the f box binding to a multicomponent ubiquitin ligase complex. geranylgeranylated fbl2 binds to ns5a, and the resulting complex seems to be required for hcv rna replication. in hcv replicon-harboring cells, knockdown of fbl2 by sirna has been shown to reduce hcv rna by 65% [68] . depletion of the ggpp by statins may inhibit the geranylgeranylation of cellular proteins such as fbl2 and cause the anti-hcv effect in the cells. statins are among the most widely used reagents to lower cholesterol. one of the statins used clinically, lov, has been well characterized and shown anti-hcv activity in cell culture. [66, 69, 70] . however, the anti-hcv activities of other statins remain to be clarified. recently the anti-hcv activities of several statins were characterized using an or6 assay system [71] . the anti-hcv activities were tested for five statins: atorvastatin (atv), fluvastatin (flv), pravastatin (prv), simvastatin (smv), and lov. flv exhibited the strongest anti-hcv activity (50% effective concentration to inhibit hcv rna replication (ec 50 ): 0. 9 μm), while atv and smv showed moderate inhibitory effects (ec 50 : 1.39 and 1.57 μm, respectively). however, lov, which has been reported to inhibit hcv replication, was shown to possess the weakest anti-hcv activity (ec 50 : 2.16 μm). more recently, we found that ptv possessed stronger anti-hcv activity than flv (fig. 2b) . the ec 50 of ptv was calculated as 0.45 μm. the anti-hcv activities of statins were reversed by supplying mevalonate or geranylgeraniol. however, surprisingly, prv exhibited no anti-hcv activity, although it worked as an inhibitor for hmg-coa reductase. although prv is a water-soluble reagent (the others are lipophilic), prv induced the expression of hmg-coa reductase by a positive feedback mechanism. there may be another mechanism underlying the depletion of ggpp by the statins. interestingly, it has been reported that only prv has a different effect on the induction of p450 compared with the other statins [72] . ribavirin is the only reagent currently used with ifn-α to treat patients with ch-c [73] . in the previous study on anti-hcv activity using the or6 assay system, the ec 50 of ribavirin was 76 μm [74] . this concentration is much higher than the clinically achievable ribavirin concentration (10-14 μm) reported previously [75, 76] . since flvexhibited strong anti-hcvactivity, flv was examined for its anti-hcv activity in combination with ifnα in or6 cells [71] . co-treatment of ifn-α and flv exhibited synergistic inhibitory effects on hcv rna replication. for example, when administered in combination with ifn-α (2 iu/ ml) and flv (5 μm), the level of hcv rna replication was remarkably reduced to approximately 3%, compared with the effects of treatment with ifn-α alone. the combination therapy of flv may be effective for the treatment of patients with ch-c. it is not appropriate to further reduce the cholesterol level of ch-c patients who already have a normal cholesterol level. for these patients, statin-related anti-hcv reagents possessing no cholesterol-lowering activity would be good candidates for future clinical use. the specific inhibition of ggpp synthesis and prenylation will be worth testing, and ggtase-i inhibitor (ggti) is one of the candidates for this purpose. furthermore, specific inhibition of the proteins modified by ggtase-i may be more effective. fbl2 may be one of the target proteins, because its formation of a complex with ns5a is required for hcv rna replication. therefore, the reagents blocking the association of fbl2 with ns5awill be able to inhibit the hcv rna replication with fewer side effects. prenyltransferase recognizes a broad range of protein substrates with a caax motif. reid et al. [77] reported a list of hypothetical prenyltransferase substrates within the human genome. other than fbl2, the host molecules involved in hcv rna replication may be exist in this list. antiviral activity of statins has also been reported in other viruses. in the respiratory syncytial virus (rsv), lov exhibited antiviral activity via the inhibition of rhoa [78] . rhoa is activated by geranylgeranylation, and activated rhoa interacts with the f glycoprotein of rsv. flv inhibited cytomegalovirus (cmv) replication by abolishing cmv-induced nf-κb activity, which is involved in a pathway that is crucial for cmv replication [79] . in human immunodeficiency virus (hiv), lov and siv reduced hiv replication via suppression of the binding between the integrin intercellular adhesion molecule 1(icam1) and lymphocyte function associated antigen-1 (lfa-1) [80] . statins were recently shown to bind to lfa-1, and icam1-bearing viruses were reduced by statins in a dose-dependent manner. it is noteworthy that the inhibition of lfa-1 binding to icam-1 by statins is independent of the inhibition of hmg coa reductase. statins inhibited the cholesterol-biosynthesis pathway and branched prenylation pathways by depletion of mevalonate. the latter caused pleiotropic effects in growth, differentiation, and antivirals. however, an unknown function of statins may existfor example, the binding of lfa-1 is likely independent of the cholesterol-lowering and the inhibition of prenylation. furthermore, the finding that prv has a different effect on the induction of p450 than the other statins has not been clearly explained by the characterization of these mechanisms of statins. a better understanding of this finding may lead to the discovery of statin-related anti-hcv reagents that do not have exhibit any cholesterol-lowering activity or inhibition of prenylation. lipid rafts are detergent resistant membranes (drm) and are enriched in cholesterol and sphingolipids. the active replication complex of hcv is present in lipid rafts [65] . therefore, sphingolipid metabolism is also an antiviral target for hcv. serine palmitoyltransferase (spt) is the enzyme responsible for the condensation of l-serine with palmitoyl-coa to produce 3ketodihydrosphingosine in the first step of sphingolipid biosynthesis (fig. 4) . myriocin, a selective inhibitor of spt, inhibited the replication of hcv replicon [81, 82] . sakamoto et al. [81] reported that the compound na255, which is structurally similar to myriocin, also inhibited the replication of the hcv replicon. na255 has been identified as the secondary fungal metabolite derived from fusarium sp. na255 suppressed hcv replicon in a dose-dependent manner, and its ec 50 was 2 nm. they further examined the involvement of the sphingolipid synthetic pathway in hcv rna replication. fumonisin b1, an inhibitor of dihydroceramide synthase, also suppressed the replication of hcv replicon. in mammalian cells, ceramide is synthesized in the endoplasmic reticulum (er) and translocates to the golgi compartment for conversion to sphingomyelin. hpa-12, an inhibitor of ceramide trafficking from the er to the golgi apparatus, also inhibited the replication of hcv replicon. glycosphingolipids (gsls) are also a component of lipid rafts, and ppmp, an inhibitor of gsl biosynthesis, also suppressed the replication of hcv replicon. furthermore, they demonstrated that after treatment with na255, the ns5b ratio in the drm was markedly decreased. interestingly, however, the drm fraction of ns3 and ns5a were not affected. inhibition of sphingolipid biosynthesis by na255 disrupted the association of lipid rafts with ns5b, but not with ns3 or ns5a. they identified a helix-turn-helix motif (glu230-gly263) in ns5b as a sphingolipid-binding domain (sbd), which was similar in structure to the sbd of the v3 loop of hiv-1. umehara et al. [82] reported that myriocin suppressed hcv rna replication in vivo, using hcv-infected chimeric mice with humanized livers. myriocin reduced the hcv rna levels in both serum and liver to 1/10-1/100 of the levels prior to the 8 day treatment. they also demonstrated that the combined treatment of myriocin with peg-ifn reduced the hcv rna level to less than 1/1000 of the control levels. these results suggest that the sphingolipid biosynthetic pathway is also a suitable target for the development of hcv therapies. at the beginning of gtp-biosynthesis pathway, inosine monophosphate dehydrogenase (impdh) is the enzyme responsible for the conversion of inosine 5′ monophosphate (imp) into xanthosine 5′ monophosphate (xmp) (fig. 5) . ribavirin, mizoribine, mycophenolic acid (mpa), and vx-497 are impdh inhibitors and inhibit hcv rna replication. ribavirin enhanced the svr of peg-ifn therapy from 29% to 56% compared to the peg-ifn monotherapy [83] . however, the antiviral mechanisms of ribavirin remain to be clarified. four possible mechanisms have been proposed [73, 84] : (1) direct inhibition of rna replication; (2) inhibition of impdh; (3) immunomodulation; (4) mutagenesis. ribavirin is phosphorylated to mono-, di-, and triphosphate (rmp, rdp, and rtp, respectively). (1) rtp, an analog of gtp, is incorporated into replicating rna by rdrp and caused termination of the rna synthesis. (2) rmp competitively inhibits the host enzyme impdh, which is essential for the synthesis of gtp, and causes a depletion of the gtp pool. (3) rivavirin has been suggested to cause immunomodulatory effects, such as the shift of th2 to th1 in immune response, and to induce an hcvspecific t cell response. (4) ribavirin acts as an rna mutagen and causes error catastrophe. in poliovirus replication, 100 μm of ribavirin increased the mutation rate from about 1.5 mutations/genome (wild type) to about 1.9 mutations/genome and resulted in a decrease of infectivity of 70% [85] . the mutation rate increased in a ribavirin dose-dependent manner: 6.9 mutations/genome and 15.5 mutations/genome at 400 μm and 1000 μm, respectively [85] . in the clinical study of ch-c, the enhancement of svr has been observed only in combination therapy of ribavirin with ifn, but not in ribavirin monotherapy. it may be difficult to test the effect of ribavirin monotherapy, since the clinically achievable concentration of ribavirin without severe side effects such as anemia is too low (10-14 μm). however, in the cell culture model [74, 86] , a higher concentration of ribavirin suppressed hcv rna replication (ec 50 : 76 μm) [74] . mizoribine is an imidazole nucleoside that is isolated from culture medium of the mold eupenicillium brefeldianum m-2166 and is structurally similar to ribavirin. mizoribine was authorized by the japanese government as an immunosuppressive drug for renal transplantation; thereafter, lupus nephritis, rheumatoid arthritis, and nephritic syndrome were also added to the list of diseases for which this agent is indicated [87, 88] . based on the similarity of mizoribine to ribavirin, the anti-hcv activity of mizoribine has been tested using an or6 assay system. the anti-hcv activity of mizoribine (ec 50 : 99 μm) was similar to that of ribavirin [74] . furthermore, a low dose (at least 5 μm) of mizoribine was able to enhance the antiviral activity of ifn [74] . mizoribine was reported to exhibit antiviral activity on influenza virus types a and b [87] and recently on bovine viral diarrhea virus [89] and severe acute respiratory syndrome (sars)-associated coronavirus [90] . the precise antiviral mechanism of mizoribine remains unclear. however, any of the four hypothesized mechanisms of ribavirin mentioned above may be possible. since mizoribine has not been associated with severe side effects, it will be an alternative reagent for combination therapy with ifn. like mizoribine, mpa is used as an immunosuppressant and is known to inhibit impdh. it has been reported to show in vitro antiviral activity against dengue virus [91, 92] , hepatitis b virus (hbv) [93] , avian reovirus [94] , yellow fever virus [95] , and west nile virus [96] . the anti-hcv activity of mpa was reported by henry et al. [97] . at clinically relevant concentrations (1.0-6.0 μg/ml), mpa inhibited hcv rna replication to approximately 75% in a study using hcv replicon-harboring cells. furthermore, combination treatment of mpa with csa or ifn showed synergistic inhibition of hcv rna replication. we also recently confirmed that the combination of csa and mizoribine had a synergistic effect on the inhibition of hcv rna replication (yano et al., unpublished data) . these data suggest that immunosuppressive drugs possessing anti-hcv activity, such as csa, mpa, and mizoribine, may prevent not only the rejection of the graft but also the recurrence of hcv infection after liver transplantation, and that a combination of these drugs may be of additional benefit for such patients. vx-497 is a reversible uncompetitive impdh inhibitor that is structurally unrelated to other known impdh inhibitors. markland et al. [98] reported the broad-spectrum antiviral activity of vx-497.vx-497 exhibited 10-to 100-fold more potency than ribavirin against hbv, human cmv, rsv, herpes simplex virus type 1, parainfluenza 4 virus, emcv, and venezuelan equine encephalomyelitis virus in cell culture [98] . zhou et al. [99] reported that vx-497 alone had only marginal effect on hcv replicon, although combination treatment with ribavirin and vx-497 enhanced anti-hcv activity. they also reported that in their hcv replicon assay system, mpa showed only a marginal anti-hcv effect [99] . this result is different from the anti-hcv effect of mpa reported by henry et al. [97] . further study will be needed to clarify these controversial results. hcv morphogenesis is a target of antivirals in the life cycle of the virus. the hcv envelope glycoproteins e1 and e2 are highly n-glycosylated [100] . the consensus sequence for nglycosylation is asn-x-ser/thr, where x is any amino acid except for pro, and e1 and e2 contain 5-6 and 11 glycosylation sites, respectively. from the previous study using bovine viral diarrhea virus, inhibition of α-glucosidase is expected to prevent the proper folding and assembly of hcv. therefore, the n-glycosylation pathway may be a novel molecular target for antivirals. chapel et al. [101] reported an anti-hcv effect of the α-glucosidase inhibitor in the binding step using hcv virus-like particles (vlps) derived from baculovirus. the glucose analogue deoxynojirimycin derivatives, which are αglucosidase inhibitors, caused the retention of unprocessed, hyperglycosylated n-linked glycans on hcv glycoproteins and led to the reduction in binding of vlp to the cells [101] . these results will be examined using a recently developed infectious hcv production cell culture system. α-glucosidase inhibitor may be one of the candidates for an effective combination therapy. it is crucial that the svr for patients with ch-c receiving the current standard therapy of peg-ifn plus ribavirin is improved from the current value of about 50%. the anti-hcv effect of ifn-α is caused through the jak-stat signaling pathway. duong et al. [102] proposed that hypomethylation of stat1 by hcv protein caused the resistance to ifn therapy. unmethylated stat1 is less active because it can be bound and inactivated by its inhibitor, the protein inhibitor of activated stat1 (pias1). protein arginine methyltransferase 1 (prmt1) is the enzyme responsible for the methylation of stat1. hcv proteins induced the expression of the catalytic subunit of protein phosphatase 2a (pp2ac), and overexpression of pp2ac induced stat1 hypomethylation via the inhibition of prmt1. finally, pias1 interacted with and inhibited hypomethylated stat1 and resulted in the suppression of ifn signaling [102] . s-adenosyl-l-methionine (adomet) is a methyl group donor for stat1 methylation by prmt1. adomet is used for the treatment of alcoholic liver disease and is available in many countries as a nonprescription drug. betaine has been known to raise the intracellular concentration of adomet and plays the central role in the recycling of adomet. when pp2ac was overexpressed in huh-7 and uhvh 57.3 cells, ifn-α signaling was suppressed [102] . however, the co-treatment of adomet and betaine restored the ifn-α signaling. these results suggest that the addition of adomet and betaine to the current standard therapy with peg-ifn and ribavirin may enhance the svr for patients with ch-c. lipid metabolism is one of the most important pathways for hcv rna replication. other than cholesterol and sphingolipid synthesis, fatty acids are reported to be metabolites involved in hcv rna replication [70, 103] . however, the precise mechanisms of fatty acids on hcv rna replication have remained unclear. leu et al. [103] reported that polyunsaturated fatty acids (pufas) inhibited hcv replicon replication. arachidonic acid (aa), docosahexaenoic acid (dha), and eicosapentaenoic acid (epa) belong to pufas (fig. 6) and possessed anti-hcv activity. the ec 50 of aa was 4 μm. however, at 100 μm, αlinolenic acid, γ-linolenic acid (gla), and linoleic acid reduced hcv rna levels slightly, and saturated fatty acids, including oleic acid, myristic acid, palmitic acid, and steric acid, slightly enhanced hcv rna levels. similar results were also reported by kapadia et al. [70] using a genome-length hcv rnareplicating cell line. aa produces lipid mediators such as prostaglandins (pgs), thromboxanes (txs), leukotriens (lts), and lipoxins (lxs) (fig. 6) . however, the antiviral activity of these eicosanoids remains unclear. in their clinical study, hyman et al. [104] fig. 6 . fatty acid-biosynthesis pathway. the pufa metabolism from diet or membrane phospholipids. reported that oral prostaglandin e2 therapy resulted in no beneficial effect on patients with ch-c. investigation of the anti-hcv effects of the metabolites of pufas will lead to a new field of antivirals based on the host metabolism. ever since hcv was discovered to be the causative agent of non-a, non-b hepatitis virus, ifn has played the central role in treating the disease. currently ifn has been modified by peg and accompanied by the powerful partner, ribavirin, which boosts the anti-hcv activity of ifn. during the development of ifn therapy for patients with ch-c, the lack of a robust method of hcv rna replication in cell culture has hampered research into the hcv life cycle and the discovery of potent new anti-hcv reagents. it is difficult to attack the achilles' heel of hcv without information on the replication machinery of the virus. however, the development of a subgenomic replicon system by lohmann et al. [5] partially revealed the hcv life cycle. the information about hcv rna replication in the virus life cycle provided clues to the development of antivirals both from the standpoint of the virus and the host. a representative example is the discovery that ns3-4a inhibits innate immunity [105] . hcv runs through the cellular first defenses of the ifnproduction system. ns3-4a, a serine protease, cleaved the unexpected cellular target cardif and disrupted rig-i signaling [106] . hcv replicon contributed to the discovery of the viral serine protease inhibitor. surprisingly, a serine protease inhibitor, sch6, inhibited hcv rna replication not only by the inhibition of ns3-4a activity but also by the inhibition of the rig-i signaling [105] . this serine protease inhibitor possesses dual functions, inhibiting both viral (ns3-4a) and cellular (cardif) proteins involved in ifn production. viral and cellular molecules are the targets of antivirals. hcv rdrp caused a high mutation rate and the mutations accumulated in virus genome [107] . the high mutation rate enhances the viral evolution. as for the reagents targeting viral proteins, such as ns3-4a or ns5b, resistance to the therapy happens by the frequent mutations caused by rdrp. in fact, in the clinical trial of the ns3-4a protease inhibitor, vx-950, hcv rna rapidly decreased within 3 days after treatment [20] . however, hcv rna increased again at around 14 days after treatment [20] . hcv mutants may not be the problem in the anti-hcv reagent against cellular proteins, although the inhibition of the primary functions of the cellular proteins may cause side effects. in this review, host metabolic pathways are overviewed. one of the advantages of targeting host metabolism as antivirals is that multiple enzymes involved in the metabolism could become candidates for antivirals. in the strategy targeting host metabolism, we should be careful in regard to the side effects caused by inhibition of the primary function of the metabolite. to minimize these undesirable effects, pinpoint inhibition of the enzyme should be done. lipid metabolism is one of the important targets for antivirals among cellular factors. very recently, we examined the effect of ordinary nutrients on hcv rna replication [108] . using an or6 assay system, we found that linoleic acid possessed an anti-hcv effect and its combination with csa exerted synergistic inhibitory effect on hcv rna replication [108] . however, the anti-hcv mechanism of pufas remains unclear. an improved understanding of the anti-hcv effect of pufas will extend the field of host metabolism as a target of antivirals in the future. one recent striking advance is the development of a method for infectious hcv production in cell culture. this system provides information regarding the complete life cycle of hcv and will extend our understanding of the antivirals to virus entry, assembly and release. the discovery of anti-hcv reagents targeting host metabolism in the hcv life cycle will improve the svr in combination with ifn. or, the development of new anti-hcv reagents could lead to the retirement of ifn in the near future. isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome prevalence of antibody against non-a, non-b hepatitis virus in japanese patients with hepatocellular carcinoma global surveillance and control of hepatitis c, report of a who consultation organized in collaboration with the viral hepatitis prevention board antiviral therapy for chronic hepatitis c: past, present, and future replication of subgenomic hepatitis c virus rnas in a hepatoma cell line complete replication of hepatitis c virus in cell culture production of infectious hepatitis c virus in tissue culture from a cloned viral genome robust hepatitis c virus infection in vitro a critical role for the chimpanzee model in the study of hepatitis c transmission of hepatitis c virus infection to tree shrews toward a surrogate model for hepatitis c virus: an infectious molecular clone of the gb virus-b hepatitis agent development of a gb virus b marmoset model and its validation with a novel series of hepatitis c virus ns3 protease inhibitors hepatitis c virus replication in mice with chimeric human livers recent advances in the analysis of hcv ns5b rna-dependent rna polymerase structure and function of the hepatitis c virus ns3-ns4a serine proteinase discovery of sch446211 (sch6): a new ketoamide inhibitor of the hcv ns3 serine protease and hcv subgenomic rna replication in vitro antiviral activity of sch446211 (sch6), a novel inhibitor of the hepatitis c virus ns3 serine protease discovery of (1r,5s vx-950, a novel hepatitis c virus (hcv) ns3-4a protease inhibitor, exhibits potent antiviral activities in hcv replicon cells rapid decline of viral rna in hepatitis c patients treated with vx-950: a phase ib, placebo-controlled, randomized study an ns3 protease inhibitor with antiviral effects in humans infected with hepatitis c virus synthesis and pharmacokinetics of valopicitabine (nm283), an efficient prodrug of the potent anti-hcv agent 2′-cmethylcytidine clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas interference of hepatitis c virus rna replication by short interfering rnas small interfering rna targeted to hepatitis c virus 5′ nontranslated region exerts potent antiviral effect cdna microarray analysis to compare hcv subgenomic replicon cells with their cured cells nim811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis c virus alone or in combination with alpha interferon cyclophilin b is a functional regulator of hepatitis c virus rna polymerase cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes specific inhibition of hepatitis c virus replication by cyclosporin a suppression of hepatitis c virus replication by cyclosporin a is mediated by blockade of cyclophilins diverse effects of cyclosporine on hepatitis c virus strain replication evaluation of the anti-hepatitis c virus effects of cyclophilin inhibitors, cyclosporin a, and nim811 the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro combined interferon alpha2b and cyclosporin a in the treatment of chronic hepatitis c: controlled trial interferon combined with cyclosporine treatment as an effective countermeasure against hepatitis c virus recurrence in liver transplant patients with end-stage hepatitis c virus related disease hsp90 inhibitors suppress hcv replication in replicon cells and humanized liver mice hepatitis c virus rna replication is regulated by fkbp8 and hsp90 host cell factor requirement for hepatitis c virus enzyme maturation role of la autoantigen and polypyrimidine tract-binding protein in hcv replication treatment of chronic non-a, non-b hepatitis with recombinant human alpha interferon. a preliminary report differential activation of interferon-inducible genes by hepatitis c virus core protein mediated by the interferon stimulated response element hepatitis c virus proteins exhibit conflicting effects on the interferon system in human hepatocyte cells hepatitis c virus ns5b delays cell cycle progression by inducing interferon-beta via tolllike receptor 3 signaling pathway without replicating viral genomes molecular virology of hepatitis c virus ability of prolonged interferon treatment to suppress relapse after cessation of therapy in patients with chronic hepatitis c: a multicenter randomized controlled trial establishment of hepatitis c virus replicon cell lines possessing interferon-resistant phenotype epigenetic silencing of interferon-inducible genes is implicated in interferon resistance of hepatitis c virus replicon-harboring cells interferon resistance of hepatitis c virus replicon-harbouring cells is caused by functional disruption of type i interferon receptors inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas efficient replication of a full-length hepatitis c virus genome, strain o, in cell culture, and development of a luciferase reporter system efficient replication of hepatitis c virus genotype 1a rnas in cell culture selectable subgenomic and genome-length dicistronic rnas derived from an infectious molecular clone of the hcv-n strain of hepatitis c virus replicate efficiently in cultured huh7 cells persistent and transient replication of full-length hepatitis c virus genomes in cell culture effect of alpha interferon on the hepatitis c virus replicon establishment of a hepatitis c virus subgenomic replicon derived from human hepatocytes infected in vitro efficient replication of the genotype 2a hepatitis c virus subgenomic replicon subgenomic replicon derived from a cell line infected with the hepatitis c virus efficient initiation of hcv rna replication in cell culture cell culture-adaptive ns3 mutations required for the robust replication of genome-length hepatitis c virus rna mutations in hepatitis c virus rnas conferring cell culture adaptation construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras production of infectious genotype 1a hepatitis c virus (hutchinson strain) in cultured human hepatoma cells current therapy and new molecular approaches to antiviral treatment and prevention of hepatitis c hepatitis c virus rna replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2 characterization of the hepatitis c virus rna replication complex associated with lipid rafts pleiotropic effects of statins identification of fbl2 as a geranylgeranylated cellular protein required for hepatitis c virus rna replication disruption of hepatitis c virus rna replication through inhibition of host protein geranylgeranylation hepatitis c virus rna replication is regulated by host geranylgeranylation and fatty acids different anti-hcv profiles of statins and their potential for combination therapy with interferon regulation of cyp2b6 and cyp3a expression by hydroxymethylglutaryl coenzyme a inhibitors in primary cultured human hepatocytes mechanism of action of interferon and ribavirin in treatment of hepatitis c mizoribine inhibits hepatitis c virus rna replication: effect of combination with interferonalpha synergistic inhibition of intracellular hepatitis c virus replication by combination of ribavirin and interferon-alpha antiviral action of ribavirin in chronic hepatitis c crystallographic analysis of caax prenyltransferases complexed with substrates defines rules of protein substrate selectivity antiviral activity of lovastatin against respiratory syncytial virus in vivo and in vitro hydroxymethyl-glutaryl coenzyme a reductase inhibition limits cytomegalovirus infection in human endothelial cells statin compounds reduce human immunodeficiency virus type 1 replication by preventing the interaction between virion-associated host intercellular adhesion molecule 1 and its natural cell surface ligand lfa-1 host sphingolipid biosynthesis as a target for hepatitis c virus therapy serine palmitoyltransferase inhibitor suppresses hcv replication in a mouse model peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection the metabolism, pharmacokinetics and mechanisms of antiviral activity of ribavirin against hepatitis c virus rna virus error catastrophe: direct molecular test by using ribavirin antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis c virus subgenomic replicons comparative inhibitory effects of various nucleoside and nonnucleoside analogues on replication of influenza virus types a and b in vitro and in ovo mizoribine and mycophenolate mofetil inhibition of bovine viral diarrhea virus (bvdv) by mizoribine: synergistic effect of combination with interferonalpha inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus inhibition of dengue virus replication by mycophenolic acid and ribavirin mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna mycophenolic acid, an immunosuppressive agent, inhibits hbv replication in vitro mycophenolic acid inhibits avian reovirus replication the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase identification of active antiviral compounds against a new york isolate of west nile virus mycophenolic acid inhibits hepatitis c virus replication and acts in synergy with cyclosporin a and interferon-alpha broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon the effect of ribavirin and impdh inhibitors on hepatitis c virus subgenomic replicon rna glycosylation of hepatitis c virus envelope proteins antiviral effect of alphaglucosidase inhibitors on viral morphogenesis and binding properties of hepatitis c virus-like particles s-adenosylmethionine and betaine correct hepatitis c virus induced inhibition of interferon signaling in vitro anti-hcv activities of selective polyunsaturated fatty acids oral prostaglandin (pge2) therapy for chronic viral hepatitis b and c regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus genetic variation and dynamics of hepatitis c virus replicons in long-term cell culture comprehensive analysis of the effects of ordinary nutrients on hepatitis c virus rna replication in cell culture the authors thank drs. hiromichi dansako and yasuo ariumi for their stimulating discussions. key: cord-345898-a6vt8kso authors: ren, linzhu; peng, zhiyuan; chen, xinrong; ouyang, hongsheng title: live cell reporter systems for positive-sense single strand rna viruses date: 2016-01-04 journal: appl biochem biotechnol doi: 10.1007/s12010-015-1968-5 sha: doc_id: 345898 cord_uid: a6vt8kso cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. there are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand rna virus infections. the first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. during infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. using subgenomic replicon, which are genetically engineered viral rna molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. the second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. the third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. this review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades. classical reporter systems for (+) ssrna virus the first type of classical reporter systems was based on live recombinant virus carrying a reporter protein, such as enhanced green fluorescent protein (egfp), firefly luciferase (fluc), or secreted alkaline phosphatase ( fig. 1a and table 1 ). to generate a recombinant virus, a recombinant plasmid was constructed by fusing the reporter protein with the viral structural protein or by inserting the coding sequence of reporter protein behind the 3′ terminus of the viral structural protein gene [80] . subsequently, the recombinant virus was rescued in cells transfected with transcripts produced from the recombinant plasmid [80] . similarly, based on an infectious complementary dna (cdna) clone of dengue virus (denv), a recombinant denv generating luciferase was developed by engineering the luciferase gene into the capsidcoding region of an infectious cdna clone [89] . further studies indicated that the reporter system can be used to measure neutralization and antibody-dependent enhancement activity [71] . a classical swine fever virus (csfv) stably expressing luciferase was developed by inserting the luciferase gene into n pro gene of csfv [68] . the reporter virus enabled more sensitive and convenient detection of n pro protein expression and viral replication by a luciferase assay than by traditional methods [68] . the csfv n pro was detectable as early as 4.5 h post infection [68] . additionally, two semliki forest virus (sfv) constructs were developed by inserting egfp gene or renilla luciferase (rluc) gene into the virus replicase open reading frame between nsp3 and nsp4 flanked by nsp2 protease-recognition sites [60, 75] . subsequently, infectious sfv expressing detectable egfp or rluc upon infection were obtained by electroporation of in vitro transcripts of the constructs [60, 75] . another strategy of classical reporter systems utilizes a reporter virus particle (rvp) (fig. 1b) . an rvp is a type of virus-like particle (vlp) composed of viral structural proteins and a self-replicating replicon rna containing a reporter gene [17, 63] . to obtain an rvp of the west nile virus (wnv), a red fluorescent protein gene was inserted into the wnv rna genome by replacing the c, prm, and e protein genes to generate a wnv replicon rna [17, 51, 55] . subsequently, the replicon rna was transcribed in vitro and co-transfected into cells with two dna expression plasmids for viral c and prm/e proteins, and finally the rvp was generated [17, 55] . in a new protocol for the preparation of rvps, the replicon rna was stably transfected and expressed in the cells [17] . two plasmid dna expression plasmids for viral c and prm/e proteins were then co-transfected into the cells to package the replicon rnas into rvps [17] . in these rvp systems, rvps were capable of only one round of infection in susceptible cells due to the lack of structural protein genes within the replicon rnas [17, 87, 88] . however, rvps are useful tools for the study of viral genome packaging and cellular factors [87] . although recombinant viruses can be infectious, the viruses expressing fluorescent proteins tend to be attenuated due to the modification of their genomes [72] . furthermore, many viruses reporter system based on virus-like particle (vlp). the reporter gene was inserted into the viral genome by replacing the structural genes to generate a replicon. the replicon rna was transcribed in vitro and cotransfected into the cells with expression plasmids for viral structural protein, and finally the rvp was generated. then, the target cells were infected with rvp and the reporter protein can be detected in the cells. c reporter systems based on a viral subgenomic sequence. the reporter gene cassette controlled by viral promoter is constructed. the replicon rna is transcribed in vitro and transfected in to the cells. then, the reporter protein can be expressed and detected in the cells. d protease-sensor reporter systems for (+) ssrna virus. sequence of viral protease cleavage site is fused with reporter gene. the expression cassette is transfected into the cells and stably expressed in the cells. during the virus infection, the cleavage site is specifically cleaved by particular virus, and the reporter protein can be detected in cells or in cells culture medium. e replicase-sensor reporter systems for (+) ssrna virus. the reporter gene was regulated by specific viral rdrp in the defective replicon. without the virus infection, the replicon failed to express the reporter gene efficiently. however, when the cells were infected with the specific virus, the viral rdrp was provided by the virus and the defective replicon was activated, resulting in high-level expression of the reporter gene, which could be easily examined. r reporter protein, s structural protein, ns non-structural protein, rv recombinant virus, rvp reporter virus particle, pro protease, rep replicase, q quenching peptide [60] are extremely virulent pathogens, such as the middle east respiratory syndrome (mers), severe acute respiratory syndrome coronavirus (sars-cov), and csfv, which can only be handled in specialized laboratories of the highest biosafety level. to overcome these limitations, viral subgenomic replicons were developed and have been proven valuable for measuring the replication kinetics of replicon ( fig. 1c and table 2 ). self-replicating subgenomic replicons are genetically engineered viral rna molecules that are shorter than full-length viral genomes and are capable of replication but incapable of producing virions [22, 29, 62, 70] . however, they can be packaged into viral particles if viral coat proteins are provided [62] . for flaviviridae viruses, such as csfv, two self-replicating rnas (replicons) were produced by replacing the coding region for c to e2 of csfv with the rluc sequence or the coding region for c to e1 with the rluc-2a sequence [63] . subsequently, the translation and replication of the replicon rnas could be followed by the accumulation of luciferase protein as well as by detection of csfv non-structural protein (ns) 3 production within the cells [63] . similarly, two kinds of replicons were constructed for wnv and denv4 [3, 4] . one replicon encoded an rluc followed by an internal ribosome entry site (ires) element for cap-independent translation of the open reading frame encompassing the carboxy-terminal sequence of the e protein to the ns5 protein. the second replicon contained a luciferase gene, foot and mouth disease virus 2a, and a neomycin phosphotransferase gene that allows establishment of a stable mammalian cell line expressing the rluc protein in the presence of the neomycin analog, g418 [3, 4] . the stable replicon-expressing cell line has been used for cell-based screening and determination of 50 % effective concentration (ec 50 ) values for antiviral compounds that inhibited wnv replication [3, 4] . for hepatitis c virus (hcv), lohmann and his colleague established efficient cell culture systems, which were based on the self replication of engineered hcv replicons [5, 6, 48] . the first one was developed by co-transfecting in vitro-transcribed hcv bicistronic replicon rnas with the analogous mutants carrying the in-frame deletion of the ns5b polymerase active site [48] . the bicistronic replicons were composed of the hcv-ires, the neomycin phosphotransferase (neo) gene, the ires of the encephalomyocarditis virus, which directed translation of hcv sequences from ns2 or ns3 up to ns5b, and the 3′-untranslated regions (utrs) [48] . however, most of these replicons focused on hcv genotype 1b, until efficient rna replication systems for genotype 1a [7, 64] and genotype 2a [31] were established. furthermore, to construct a tricistronic hcv replicon, two sequential iress were used to initiate translation of humanized rluc and hcv nonstructural genes along with an authentic hcv ires that initiated translation of the neomycin resistance gene [12] . the results demonstrated that the tricistronic replicon was efficient to evaluate hcv infection [12] . for the chikungunya virus (chikv), the replicon was constructed by replacing the structural gene of the virus with gaussia luciferase (gluc) gene [20] . and two helper plasmids, which contain the 5′ and 3′ chikv replication signals and a subgenomic promoter followed by either the capsid gene or the remaining structural proteins, were constructed expressing either the chikv capsid protein or envelope proteins, respectively [20] . by co-transfection of the replicon and the helper plasmids, the virus replicon particles (vrps) could be efficiently produced [20] . positive-sense single strand rna virus genome encodes a large polyprotein precursor, which will be cleaved into structural and non-structural proteins by the action of cellular and viralencoded proteases [13, 14, 32, 46] . each virus with such a genome has a specific protease that along with an authentic hcv ires that initiated translation of the neomycin resistance gene. cheng et al. [12] flaviviridae pestivirus classical swine fever virus (csfv) the coding region for c to e2 of csfv was replaced with the renilla luciferase (rluc) sequence or rluc-2a sequence. the translation and replication of the replicon rnas could be followed by the accumulation of luciferase protein expression as well as by detection of csfv ns3 protein production within the cells. risager et al. [63] recognizes a specific cleavage site of the polyprotein. moreover, the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase [34] , indicating that cleavage of the polyprotein depends on the particular virus infection and that the viral protease cleavage site sequence is an ideal element to report the viral infection. during the past few decades, a large number of virus-specific proteases and their cleavage sites have been identified and used to design and engineer reporter cells for specific virus infections ( fig. 1d and table 3 ). of these viral proteases, the ns3/4a protease of hcv became an attractive target for the development of reporter systems for hcv infection. one strategy involved the fusion of egfp to secreted alkaline phosphatase (seap) through the ns3/4a protease recognition sequence, delta4ab [26, 37, 38, 41] . during hcv infection, the fused protein can be cleaved by the protease, and seap will be secreted into the extracellular culture medium. this strategy made it possible to monitor ns3/4a activity and replication of hcv genomic rna inside mammalian cells by measuring seap levels in the culture medium [26, 37, 38, 41] . in another strategy, the recombinant caspase 3 (rcasp3) was used as the specific substrate of the ns3/4a protease; the endogenous cleavage sites in the procaspase 3 molecule were substituted with decapeptides specific for the ns3/4a protease cleavage [43] . after hcv infection, the activation of rcasp3 depended on its specific cleavage by the ns3/4a protease and resulted in apoptosis of the hcv-infected cells [43] . additionally, tanaka and his colleagues established an indicator cell system in which the transcriptional factors gal4-tbp and human immunodeficiency virus type 1 (hiv-1) tat protein were utilized [77] . the chimeric tat and gal4-tbp transcription factors, both containing the hcv ns3/4a cleavage sequence of a mitochondria-resident ips-1, were manipulated to be localized in the endoplasmic reticulum. upon infection with hcv, the transcription factors were efficiently cleaved by hcv protease, migrated into the nucleus and activated the reporter gene under the tandem promoter [77] . another protease is the ns3 protease of csfv. we previously developed a dark-tobright reporter cell for csfv infection [11] . this assay was based on a novel reporter cell stably expressing egfp fused in-frame to a quenching peptide via a special recognition sequence of the csfv ns3 protease [11] . without the csfv infection, although egfp can be expressed in the reporter cell, chromophore maturation of egfp in the reporter cells was inhibited by the c-terminal quenching peptide of egfp [56] . however, when the cells were infected with csfv, the recognition sequence of the csfv ns3 protease was specifically cleaved by the protease, and the quenching peptide was released from the protein. subsequently, the egfp can undergo conformational rearrangement allowing maturation of the chromophore and gain of fluorescence, making the cell a dark-to-bright reporter of csfv infection [11, 56] . similarly, a denv-specific reporter system was created that utilized the viral protease cleavage site resulting in nuclear localization of gfp in denv-infected cells [54] . the reporter system was based on a plasmid-containing protease cleavage site of the denv-2 genome tagged with the sv40 nls (nuclear localization sequence) and egfp [54] . during denv or rvps infection, egfp is transferred from the cytoplasm to the nucleus. the reporter system was shown to be effective in detecting infection of cells by all four denv serotypes as well as by low-passaged viral strains [54] . furthermore, these systems are also valid for the detection of vlps, vrps, or rvps, and even constructs that can produce the protease rnas. positive-sense single strand rna virus genome also encodes a rna-dependent rna polymerase (rdrp), which catalyzes synthesis of viral rna and is considered as another target for virus detection and eradication [1, 2, 78] . generally, the reporter gene was regulated by specific viral rdrp in the defective replicon (fig. 1e) . without the virus infection, the replicon failed to express the reporter gene efficiently. however, when the cells were infected with the specific virus, the viral rdrp was provided by the virus and the defective replicon was activated, resulting in high-level expression of the reporter gene, which could be easily examined [44] . for alphaviruses, such as sindbis virus (sinv), a stable cell line, bhksinluc2, containing a luciferase gene under the control of the sinv subgenomic rna promoter, was established [58] . this cell line expressed high levels of luciferase activity during infection with sinv and provided a sensitive assay for titering the virus [58] . moreover, a species-specific insect cellbased system for detecting wild-type sinv infection was developed, which produced sinv minigenome containing a virus inducible promoter and a fluorescent gene [72] . during the virus infection, the fluorescent gene was specially induced and expressed in the cells [72] . in another system, replicon-defective plasmids derived from the sinv were constructed by replacing the structural genes of sinv with reporter genes and deleting 1139 bp in the ns 4 gene of the virus [44] . upon infection with the virus, the viral components induced expression of the reporter genes in the cells transfected with the replicon-defective plasmid [44] . another replicase-sensor reporter system based on a bicistronic reporter plasmid, designated as (+)fluc-(−)utr-rluc, was constructed by comprising the fluc gene and the rluc gene in reverse orientation flanked by both negative strands of the hcv 5′-and 3′-utrs, in which fluc and rluc proteins are regulated by host polymerase and functional ns5b polymerase, respectively [39] . then, the bicistronic plasmid was stably transfected into baby hamster kidney-21 (bhk-21) cells to generate the bhk-ns5b-frluc reporter cell line, which can be used to simultaneously measure cellular toxicity and intracellular rdrp activity [39] . this system is specific for the hcv rdrp activity assay and the inhibitor evaluation. cell based reporter systems, combined with other genetic and proteomic approaches including rna interference, microarray, and two-dimensional gel electrophoresis coupled with maldi-tof/tof identification, have shown promise as a strategy for research on virus infections and pathogenesis. in general, the recombinant virus or rvp can mimic the virus life cycle and the reporter protein can serve as an indirect readout, thus the functions of the viral proteins and interactions of the proteins can be elucidated by measuring the reporter protein activity. furthermore, the translation and replication of subgenomic replicon rnas could be followed by the accumulation of luciferase protein in the cells. using the reporter systems, it was demonstrated that the csfv core protein can regulate csfv rna synthesis by enhancing csfv rna-dependent rna polymerase (ns5b) activity in a virus species-specific manner [45] . further studies indicated that residues 21-99 of the core protein were required for enhancement of ns5b activity [45] . ns3, ns5a, and ns5b of csfv are replication-associated proteins. however, these proteins also play an important role in internal ribosome entry site (ires)-mediated translation of csfv. csfv ns3 is a multifunctional protein possessing serine protease, rna helicase, and nucleoside triphosphatase (ntpase) activities [81] . the rna helicase activity of ns3 could promote viral and cellular translation, whereas the protease domain of ns3 interacted with ns5b to enhance viral translation [81] . in contrast, the ns5a protein had an inhibitory effect on ires-mediated translation, while the ns5b proteins suppressed the inhibitory effect of ns5a on viral translation by binding residues 390-414 located in the cterminal half of ns5a [69] . amino acids k399, t401, e406, and l413 of ns5a and aa 63-72, aa 637-653, and the highly conserved gdd motif of ns5b were necessary for the interaction between ns5a and ns5b [69, 82] . furthermore, the 3′ terminal sequence harbored the positive and negative regulatory elements to control the ires-mediated translation of csfv [25] . the negative cis-acting element was the 3′-end hexamer cggccc sequence [25] . moreover, mutations within ires affected the translation initiation efficiency of csfv [19] . n pro of csfv was not only involved with virus rna translation in the cytoplasm [27] but also counteracted double-stranded rnamediated apoptosis and ifn-α/β induction [65] . further studies elucidated that interferon synthesis can be prevented by inhibiting transcription and promoting degradation of interferon regulatory factor 3 (irf3) via the zinc-binding ability of viral n pro [33, 73] . n pro also redistributed to mitochondria and peroxisomes to inactivate irf3 and inhibit apoptosis [28] . in addition, n pro influenced the innate immune response at local sites of virus replication in pigs and contributed to the pathogenicity and viral replication of csfv [76] . using the reporter system for denv, it was demonstrated that the 3′ utr, ns1, ns3, and ns5 were essential for viral rna replication and translation. rna replication of denv required an rna-rna-mediated circularization of the viral genome, and the 5′ and 3′ utrs formed several additional rna elements that were involved in the regulation of translation and rna replication [18] . the first 84 nt in the 3′ utr comprised a variable region (vr), which could be further divided into a 5′-terminal hypervariable region (hvr) and a 3′-terminal semi-variable region (svr) [74] . the vr was important for denv replication and was associated with the accumulation of denv rna in mammalian cells [74] . the core region of the 3′-utr of denv rna can form two dumbbell structures (5′and 3′-dbs) and four pseudoknots, which were required not only for rna replication but also for optimal translation [52] . the ns3 protein contained an n-terminal serine protease region joined to an rna helicase by an 11-amino acid linker [50] . the linker region conferred flexibility to the ns3 protein that was required both for polyprotein processing and rna replication [49, 50] . furthermore, the host factor p100 can interact with the 3′ utr to facilitate viral rna replication [42] . two nlss within the central region of ns5 ('anls' and 'bnls') can be recognized by the importin α/β and importin β1 nuclear transporters, respectively, leading to ns5 nuclear accumulation [61] . moreover, the heterogeneous ribonucleoprotein (hnrnp) c1/c2 interacted with ns1 to participate in viral rna synthesis [15, 57] . during the viral assemblying, the mature capsid protein of denv accumulated on the surface of er-derived lipid droplets in the cytoplasm [66] . in addition, denv can induce low levels of interferon regulatory factor 3 and nf-κb activation by blocking the tlr-triggered erk-nf-κb activation, thus leading to reduced cytokine production [9, 10] . additionally, the dynamics of denv infection in mice revealed that the virus localized predominantly to lymphoid and gut-associated tissues [67] . cell-based reporter systems also represent rapid, low cost, and high throughput assays for antiviral agent and antibody evaluation. replicon systems were useful for anti-viral drug development. many anti-hcv compounds targeting ns3/4a, ns5a, or ns5b have been screened by using the hcv replicon systems, which are now available with sustained virological response up to 100 % in the majority of patients [23] . recently, pan and his colleagues developed a ns3/4a protease-based reporter assay, which was not only suitable for efficiently assessing hcv infection, but was also useful for high throughput screening of anti-hcv agents [59] . in this system, virus replication/ infection in the cells could be quantitatively indicated by measuring the seap activity in the cell culture medium [59] . similarly, a dual reporter system for wnv based on a subgenomic replicon encoding rluc and neomycin phosphotransferase was generated [47] . incubation of the reporting cells with a known wnv inhibitor decreased luciferase activity as well as the replicon rna level. the efficacies of the inhibitors, as measured by the depression of luciferase activity in the reporting cells, were comparable to those derived from authentic viral infection assays, indicating that the reporting cell line can be used as a high throughput assay for anti-wnv drugs [47] . for denv, another severe mosquito-borne viral pathogen, neither a vaccine nor an antiviral therapy is currently available to treat the disease [40] . by using cell-based reporter systems, several compounds were identified as potential antiviral agents. two novel flavones, pmf and tmf, were discovered to have denv-inhibitory properties [35, 36] . one effective compound of 14,400 small-molecule chemicals was found to suppress viral rna replication [24] . a doxorubicin derivative, sa-17, can inhibit denv replication in the very early stages of the viral replication cycle with an ec 50 of 0.52 ± 0.31 μm [30] . furthermore, nucleoside inhibitors targeting viral polymerases have proved promising for the development of drugs against viruses [40] . it has been reported that a nucleoside analog, 2′-c-methylcytidine (2cmc), exerted specific anti-denv rna polymerase activity in denv subgenomic rna replicon and infectious systems as well as in suckling mice models, with a 50 % inhibitory concentration (ic 50 ) value of 11.2 ± 0.3 μm [40] . moreover, bp2109 and bp13944 were identified as small-molecule inhibitors of the denv ns2b/ns3 protease using a stable cell line harboring an efficient luciferase replicon of denv [84] [85] [86] . further studies elucidated that bp2109 inhibited replication and viral rna synthesis by interacting with the central hydrophilic portion of the ns2b cofactor with an ec 50 of 1.03 ± 0.09 μm, while bp13944 targeted the ns3 protease with an ec 50 of 0.17 ± 0.01 μm [84, 85] . in addition, 17 new compounds were discovered as ns2b-ns3 protease inhibitors with ic 50 values ranging from 7.46 ± 1.15 to 48.59 ± 3.46 μm, and 8 compounds belonging to two different scaffolds were active to some extent against denv based on luciferase reporter replicon-based assays [16] . additionally, amodiaquine (aq) inhibited denv2 infectivity with ec 50 and ec 90 values of 1.08 ± 0.09 μm and 2.69 ± 0.47 μm, respectively, and inhibited viral rna replication with an ec 50 value of 7.41 ± 1.09 μm in the replicon-expressing cells [8] . both p-hydroxyanilino and diethylaminomethyl moieties were important for aq to inhibit denv2 replication and infectivity [8] . in the context of virus disease control, a reliable, high throughput tool for antibody or vaccine evaluation is also important to allow for an understanding of the impact of neutralizing antibodies on disease progression and vaccine efficacy [53] . a virus reporter system has emerged as a promising strategy for antibody and vaccine evaluation. a reporter system using denv rvps was used to measure neutralizing antibodies in human serum samples against all • fluorescent proteins tend to be attenuated. • lower than wild-type virus yield of viral progeny • genetic modification of a viral genome may be labor intensive. • higher risk of mutation • expression vectors of structural proteins need to be co-transfected into the cells. • lower than wild-type virus yield of viral progeny • genetic modification of a viral genome may be labor intensive. four denv serotypes [53] . the results showed that denv rvps yielded serotypespecific responses and reproducible neutralization titers that were in statistical agreement with plaque reduction neutralization test (prnt) results [53] . similarly, a stable rluc reporter system for denv (luc-denv) was developed for measuring neutralization and antibody-dependent enhancement activity [71] . a luciferase value analysis using various known denv-specific monoclonal antibodies and clinical samples from infected animals and individuals showed good repeatability and a linear correlation with conventional plaque-based assays [71] . additionally, a rapid system to produce chimeric single-round infectious particles (srips) of flaviviruses was developed using a japanese encephalitis virus subgenomic replicon plasmid [83] . the srips of denv-1 were evaluated as antigens for functional antibody assays [83] . as a result, a significant correlation was shown between antibody titers obtained using srips and those obtained using denv-1 antigens, indicating that srips can be used as an alternative antigen in functional antibody assays [83] . a vrp-based neutralization assay for chikv infection was established using gluc as readout [20] . the vrps could be produced efficiently in the bhk-21 cells by co-transfecting of the chikv replicon expressing gluc and the helper rnas [20] . subsequently, the infection with vrps was measured via gluc secreted into the supernatant, and the chikv-neutralizing antibodies could be determined within a day by the assay without the need of using infectious chikv [20] . cell-based reporter systems are also well suited for use in clinical examinations and epidemiological surveillance [44] . the subgenomic reporter rna systems developed by steel and his colleagues were relatively species specific and allowed for rapid and simple visual detection of the wild-type viruses in mosquito cells [72] . moreover, the replicon-defective reporter gene assay could detect a variety of alphaviruses from tissue cultures with a limit of detection between one and ten (plaque-forming unit) pfu for sinv while other rna viruses, such as the japanese encephalitis virus and tahyna virus, displayed negative results with this system [44] . cell-based reporter systems have enhanced our understanding of the molecular mechanisms of virus replication and pathogenesis as well as virus interactions with host cells. they also provide platforms for virus detection and drug susceptibility testing (table 4 ). however, there are still several issues with these systems that need to be further explored. first, not all positive-sense single strand viruses have a successful cell-based reporter system. one future challenge will be the generation of a sensitive cell-based reporter system for detecting or tracking emerging viruses and pathogenic viruses that are currently not cultivable. second, viruses are highly variable and undergo frequent mutation, so new reporter systems for these viruses need to be developed that can identify mutations and serotypes more efficiently and accurately. additionally, future challenges will lie in optimizing cell-based reporter systems for high throughput screening of antiviral agents. rna-dependent rna polymerases, viruses, and rna silencing inhibition of rna binding to hepatitis c virus rna-dependent rna polymerase: a new mechanism for antiviral intervention construction of self-replicating subgenomic dengue virus 4 (denv4) replicon construction of self-replicating subgenomic west nile virus replicons for screening antiviral compounds the hepatitis c virus replicon system: from basic research to clinical application novel cell culture systems for the hepatitis c virus efficient replication of hepatitis c virus genotype 1a rnas in cell culture amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity dengue virus serotype 2 blocks extracellular signal-regulated kinase and nuclear factor-kappab activation to downregulate cytokine production flavivirus induces interferon-beta gene expression through a pathway involving rig-i-dependent irf-3 and pi3k-dependent nf-kappab activation a dark-to-bright reporter cell for classical swine fever virus infection tricistronic hepatitis c virus subgenomic replicon expressing double transgenes structural analysis of foot-and-mouth disease virus 3c protease: a viable target for antiviral drugs? foot-and-mouth disease virus 3c protease: recent structural and functional insights into an antiviral target role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication discovery of novel small molecule inhibitors of dengue viral ns2b-ns3 protease using virtual screening and scaffold hopping development of a novel protocol for generating flavivirus reporter particles composition of the sequence downstream of the dengue virus 5′ cyclization sequence (dcs) affects viral rna replication modulation of translation initiation efficiency in classical swine fever virus virus replicon particle based chikungunya virus neutralization assay using gaussia luciferase as readout diagnostic methods for detection of classical swine fever virus-status quo and new developments minigenomes, transcription and replication competent virus-like particles and beyond: reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses replicon cell culture system as a valuable tool in antiviral drug discovery against hepatitis c virus identification of a small-molecule inhibitor of dengue virus using a replicon system the 3′-terminal hexamer sequence of classical swine fever virus rna plays a role in negatively regulating the ires-mediated translation a reporter cell line for rapid and sensitive evaluation of hepatitis c virus infectivity and replication host factors that interact with the pestivirus n-terminal protease, npro, are components of the ribonucleoprotein complex the pestivirus n terminal protease n(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf3 by n(pro construction and applications of yellow fever virus replicons a derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro efficient replication of the genotype 2a hepatitis c virus subgenomic replicon hepatitis c virus ns3/4a protease loss of interferon regulatory factor 3 in cells infected with classical swine fever virus involves the n-terminal protease construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds development of dengue type-2 virus replicons expressing gfp reporter gene in study of viral rna replication a reporter-based assay for identifying hepatitis c virus inhibitors based on subgenomic replicon cells development of a cell-based assay for monitoring specific hepatitis c virus ns3/4a protease activity in mammalian cells a cell-based reporter assay for inhibitor screening of hepatitis c virus rna-dependent rna polymerase characterization of the activity of 2′-c-methylcytidine against dengue virus replication high-throughput cell-based screening for hepatitis c virus ns3/4a protease inhibitors functional interaction between cellular p100 and the dengue virus 3′ utr development of a cell-based assay for monitoring hepatitis c virus ns3/4a protease activity rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay the classic swine fever virus (csfv) core protein can enhance de novo-initiated rna synthesis by the csfv polymerase ns5b hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity potential high-throughput assay for screening inhibitors of west nile virus replication replication of subgenomic hepatitis c virus rnas in a hepatoma cell line flexibility between the protease and helicase domains of the dengue virus ns3 protein conferred by the linker region and its functional implications crystal structure of the ns3 protease-helicase from dengue virus a pcr-based protocol for generating west nile virus replicons identification of cis-acting elements in the 3′-untranslated region of the dengue virus type 2 rna that modulate translation and replication dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes a plasmid-based reporter system for live cell imaging of dengue virus infected cells development and application of west nile virus subgenomic replicon rna expressing secreted alkaline phosphatase structural basis of fluorescence quenching in caspase activatable identification of human hnrnp c1/c2 as a dengue virus ns1-interacting protein a cell line that expresses a reporter gene in response to infection by sindbis virus: a prototype for detection of positive strand rna viruses development of ns3/4a proteasebased reporter assay suitable for efficiently assessing hepatitis c virus infection a luciferase-based screening method for inhibitors of alphavirus replication applied to nucleoside analogues nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique analysis of classical swine fever virus rna replication determinants using replicons novel hepatitis c virus reporter replicon cell lines enable efficient antiviral screening against genotype 1a n(pro) of classical swine fever virus is an antagonist of double-stranded rna-mediated apoptosis and ifn-alpha/beta induction dengue virus capsid protein usurps lipid droplets for viral particle formation dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay classical swine fever virus ns5b protein suppresses the inhibitory effect of ns5a on viral translation by binding to ns5a construction and characterization of subgenomic replicons of new york strain of west nile virus a novel reporter system for neutralizing and enhancing antibody assay against dengue virus subgenomic reporter rna system for detection of alphavirus infection in mosquitoes zinc binding in pestivirus n(pro) is required for interferon regulatory factor 3 interaction and degradation characterization of the variable region in the 3′ nontranslated region of dengue type 1 virus insertion of egfp into the replicase gene of semliki forest virus results in a novel, genetically stable marker virus npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type i interferon induction at local replication sites establishment of an indicator cell system for hepatitis c virus an in vitro coupled transcription/ translation reporter system for hepatitis c virus rna-dependent rna polymerase comparison of six detection methods for classical swine fever virus development of egfp/gluc-tagged sindbis-like virus xj-160 effect of ns3 and ns5b proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation influence of ns5a protein of classical swine fever virus (csfv) on csfv internal ribosome entry site-dependent translation evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays novel dengue virus-specific ns2b/ns3 protease inhibitor, bp2109, discovered by a high-throughput screening assay a novel dengue virus inhibitor, bp13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral ns2b/ns3 protease characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne japanese encephalitis virus packaging the replicon rna of the far-eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system development and characterization of a stable luciferase dengue virus for high-throughput screening development of egfp/gluc-tagged sindbis-like virus xj-160 acknowledgments this work is supported by the jilin province science and technology development projects conflict of interest the authors declare that they have no competing interests. key: cord-316968-rowoylge authors: zhang, wenjuan; zhang, yuan; zhong, yang title: using maximum likelihood method to detect adaptive evolution of hcv envelope protein-coding genes date: 2006 journal: chin sci bull doi: 10.1007/s11434-006-2118-9 sha: doc_id: 316968 cord_uid: rowoylge nonsynonymous-synonymous substitution rate ratio (d (n)/d (s)) is an important measure for evaluating selective pressure based on the protein-coding sequences. maximum likelihood (ml) method with codon-substitution models is a powerful statistic tool for detecting amino acid sites under positive selection and adaptive evolution. we analyzed the hepatitis c virus (hcv) envelope protein-coding sequences from 18 general geno/subtypes worldwide, and found 4 amino acid sites under positive selection. since these sites are located in different immune epitopes, it is reasonable to anticipate that our study would have potential values in biomedicine. it also suggests that the ml method is an effective way to detect adaptive evolution in virus proteins with relatively high genetic diversity. the basic process of adaptive evolution by natural selection is the replacement of one allele gene by another with a higher fitness in a population. detecting the adaptive evolution would be helpful for better understanding of bio-evolutionary mechanism and corresponding variation in structure and function [1] . the nonsynonymous-synonymous substitution rate ratio (d n /d s ) is an important indicator of selective pressure at the protein-coding gene, with d n /d s = 1 meaning neutral mutation, d n /d s <1 purifying selection, and d n /d s >1 diversifying positive selection (i.e. adaptive evolution). in comparison with a large amount of neutral mutations and purifying selections, positive selection is rare and hardly detected effectively because it often just occurs on a few of sites or during a short period [1] . in particular, for some protein-coding sequences with high genetic diversity that might result from relatively high mutation rates or long evolutionary history, it is much difficult to infer whether positive selection exists. for example, hepatitis c virus (hcv) is a type of rna viruses with high mutation rates, and its genotypes have emerged in determining the clinic variation, main features of chronic infection and duration of antiviral therapy. although the rapid variation of hcv has attracted the attention of virologists and evolution biologists, it is still unclear how hcv evades the host immune response and the mechanism of chronic infection [2] . envelope glycoproteins e1 and e2 of hcv are involved in virus attaching to the host cell as well as in virus endocytosis and fusion with host membrane. e2 protein contains two highly variable regions called hypervariable regions 1 and 2 (hvr1 and hvr2) and two cd81-binding sites. as one of the receptors of e2 protein [3] , cd81 is a bridge between virus and host cell. hvr1 is implicated in the scarb1-mediated cell entry. it was reported that despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of hvr1 were highly conserved. the conservation of positively charged residues located at specific sequence positions of hvr1 indicates that hvr1 is involved in interactions with negatively charged molecules on host cell surface. this possible interaction probably plays a role in host cell recognition and attachment [4] . hvr2 and cd81-binding sites may be involved in sensitivity and/or resistance to ifn-alpha therapy. it is therefore considered that hcv evades the host immune response through mutation in some amino acid sites of envelope proteins, which result in recognition error during hcv contacting host cell. these mutations will be fixed under pressure driven by host immune system environment and form the adaptive evolution of hcv genomes. previous studies focused on exploration of the adaptive evolution within an individual hcv subtype. for example, positively selected amino acid sites in the entire coding sequences of hcv subtype1b were identified [5] . the increasing availability of data storing in hcv databases allows us to analyze hcv genome evolution on a large scale of genetic diversity from more quantitative frameworks based on statistical inference [6, 7] . technologically, a number of advanced methods have been proposed to reconstruct ancestral sequences or estimate the parameters under different substitution models when calculating nonsynonymous-synonymous rate ratio. for example, when all the sites evolve independently in one substitution model, a parsimony approach can be used to infer ancestral sequences and compute substitution numbers of different types. however, estimation of parameters by the parsimony approach may be biased because it does not account for multiple substitutions on one site. the maximum likelihood (ml) method is another way to estimate model parameters and more strict. it is employed not only to select the best phylogenic tree that fit the real data [1] , but also to detect select pressure on sites under different codon substitution models, especially for amino acid sites undergoing positive selection [8] . the purpose of this study is therefore to use the ml method [9] to infer adaptive evolution and positively selected amino acid sites of hcv envelope protein entire coding sequences containing all 6 hcv genotypes. the scientists that expert in the fields of hcv genetic variability and development of hcv sequence databases (such as the hepatitis virus database (japan), euhcvdb (france) [6] , and los alamos (united states) [7] ) meet to re-examine the status of hcv genotype nomenclature. hcv variants can be classified into 6 genotypes representing the 6 genetic groups defined by phylogenetic analysis [10] . the confirmed genotypes with 27 complete hcv genome sequences (18 subtypes) were defined according to the nomenclature stipulated in 2004 heidelberg conference [10, 11] . the proposal provides the framework by which the hcv databases store and provide access to data on hcv. considering the computational workload of adaptive evolution detection and statistical significance of the data analysis, this study used the sequences of these 27 complete annotated hcv genomes (table 1) [6, 10] . the average numbers of amino acid sites for e1 and e2 were 192 and 367, respectively. amino acid sequences of e1 and e2 proteins were aligned using clustal x 1.83 [12] [13] [14] . the nucleic acid sequences were aligned according to the protein alignments with tranalign program in the embosswin software package [15] . these nucleic acid sequences table 1 the entire coding sequences of hcv used in this study a) genotype genbank accession no. a) classification of genotypes is available at http://euhcvdb.ibcp. fr/euhcvdb. were retrieved from the genbank database (release 155.0). in order to infer a reliable phylogeny of hcv geno/subtypes, we used the alignment result of 27 complete hcv polyprotein coding sequences for evolution tree reconstruction. neighbor-joining method with kimura-2 parameter model implemented in mega 3.1 was used for phylogenetic analysis [16] [17] [18] . clade robustness was measured by bootstrap method with 1000 replicates. nonsynonymous-synonymous substitution rate ratio (ω = d n /d s ) was calculated by site-specific models of codon substitution models according to the results of phylogeny tree and sequences alignment. an ω significantly greater than 1 means that the nonsynonmous mutations are fixed at a higher rate than synonymous mutations and the evolution of this site is driven by positive selection. the model with maximum likelihood ratio is considered as the best model to fit the data. the likelihood-ratio test (lrt) was used to compare twice the log-likelihood differences between two nested models and with a χ 2 distribution to identify the statistics significance. the degrees of freedom (df) used in lrt were equal to the difference in the number of parameters between the two models [19] . we used three pairs of models to form three lrts: m0 (one-ratio) and m3 (discrete), m1a (nearly neutral) and m2a (positive selection), and m7 (β ) and m8 (β & ω ). the simplest model, m0, assumes one ω for all sites. model m1a (nearly neutral) allows two classes of sites with 0< ω 0 <1 and ω 1 = 1 in proportions p 0 of conserved sites and p 1 = 1−p 0 of neutral sites, respectively. based on m1a, m2a (positive selection) adds an additional class of sites with frequency p 2 = 1−p 0 −p 1 and an ω 2 estimated from the data. m3 (discrete) uses an unconstrained discrete distribution to model heterogeneous ω ratios among sites. m7 (β ) assumes a β (p,q)-distribution for 0≤ω≤1. m8 (β & ω ) adds to m7 an extra category, with proportion p 1 of sites with ω 1 , while the rest of sites (at frequency p 0 = 1−p 1 ) have ω from the β (p,q)-distribution between 0 and 1. this model can be compared with m7 to test the presence of positive sites using a likelihood-ratio test (lrt) [19] . in this study, site-specific models were used with codeml in the paml 3.14b package [9] . we tested positive selection over sites of coding sequences by comparing twice the log-likelihood differences between m1a vs. m2a and m7 vs. m8 with a χ 2 distribution in the lrt. phylogenic tree shown in fig. 1 was consistent with the phylogeny analysis based on complete genome sequences published previously [2] and the one available in http://hcv.lanl.gov/content/hcv-db/distances/hcv_vari ability.html. the results of identifying positively selected amino acid sites in the coding region of e1 are summarized in table 2 . the lrts of adaptive evolution suggested that the model of one ω ratio for all sites (m0) was rejected when compared with model m3 (2δl = 569.66, p<0.01, df = 4). the lrt statistic for comparing m1a (nearly neutral) and m2a (positive selection) showed that m2a did not have precedence over m1a (2δl = 0, df = 2). indeed, model m2a and m1a had the same likelihood value and the estimations of parameters under these models were similar. in m2a, p 1 and p 2 could be combined into one because ω 2 = 1. in this way, m2a was equivalent to m1a. therefore, we could not infer positive selection from this comparison. model m8 was significantly prior to m7 (2δl = 6.24, p<0.05, df = 2). model m3 provided three proportions of sites, p 0 , p 1 and p 2 with ω ratio of 0.0141, 0.1134, and 0.3231 respectively. it suggested a large proportion of sites (~85%) under strong purifying selection. another piece of evidence for e1 gene being negatively selected was that ω in m8 was no greater than 1. models that allow for positively selected sites are m2a and m8 in the three pairs of nested models. however, neither of these two models suggested the existence of sites of e1 protein under positive darwinian selection. the results of identifying positively selected amino acid sites in the coding region of e2 are also summarized in table 2 with ω values between 0 and 1. however, different from those in e1 protein, positively selected sites with ω ratios greater than 1 were detected in m2a (ω = 7.3185) and m8 (ω = 4.9507). these sites were 1e, 8n, 14a and 21t which all located in hvr1 of e2 protein. to assess the potential impact of the adaptive mutations, sites of e2 protein under positive selection were mapped onto immune epitope against hcv based on the epitope maps from hcv immunology database (http://hcv.lanl.gov/content/immuno/immuno-main.htm l) [20] (table 3 ). all of the amino acid sites under adaptive evolution were located in b-cell epitopes of rat. only one site (21t) was found in t-cell epitopes of human and transgenic mouse. two 14a and 21t were also located in t-helper epitopes of human. it probably suggested that humoral immune response plays a key role in the immune clearance and exert more selective pressure on hcv replication than cell mediated response. detecting adaptive evolution is a bioinformatics exploration based on the knowledge of genetics and statistics. d n and d s as well as their ratio ω which measures the selective pressure at the amino acid level provide powerful tools for better understanding of the effect of natural selection on molecular evolution. an ω significantly greater than 1 means that nonsynonymous mutations offer fitness advantages and this lineage (in lineage-specific models) or this critical amino acid site in the protein (in the site-specific models) are considered under positive selection driven by environment. though ω ratio is a sensitive measure of positive selection, both lineage-specific models and site-specific models may lack power in detecting positive selection if adaptive evolution occurs at a few time points and affects a few amino acids. we need more robust statistic tools to test the hypothesis models [19, 21] . maximum likelihood method and lrt could help to identify the best codon substitution models to fit the real data, and some models such as m2a and m8 have been successfully used for detecting positive selection. we took hcv envelope glycoprotein as an example to explore the adaptive evolution driven by immune environment pressure of coding sequences of 27 hcv containing 18 geno/subtypes and found that a number of amino acid sites were under positive selection and ml could be employed for identifying the adaptive evolution of rna virus on a large scale of genetic diversity. brown et al. [22] cloned hcv e1e2 full-length nucleotide sequences generated from serum samples of 4 chronically infected patients and identified 11 amino acid sites undergoing patient-specific adaptive evolution. in this study, we detected 4 amino acids sites of e2 protein under positive selection. two of these sites were proved in brown's work. note that a region including the n-terminal 27-31 amino acid sites in e2 is known to be the most variable and is called hypervariable region 1 (hvr1) [23, 24] . this region is surface-exposed [25] and has been proposed as a major target of the immune response probably because its hypervariable is correlated with immune evasion [26] [27] [28] . for all of the positively selected amino acid sites located in hvr1 of e2 protein and in some immune epitopes, adaptive evolution of hcv could be the consequence of the environment pressure directly driven by host immune response. recent studies revealed more information about how hcv escaping from host immune system response, but more comprehensive and careful research should be done to make clear the role of immune evasion in hcv chronically infection and explain the mechanism of hcv evolution involving immunology and virology. in other words, the positively selected sites' location indicated the immunogenicity of these sites and they might be candidate vaccination targets against hcv. the composite vaccines containing these different amino acid residues at the positively selected sites located in immune epitopes would be effective to preventing proliferation of escape mutants [5] . no amino acid sites exhibited positive selection within e1 protein in this study. it was consistent with the report from brown et al. [22] . the possible reason was that e1 was unlikely to be surface-exposed [29] and not a major target for the host antibody response. it was reported that e1 protein was a poor natural immunogen for humoral response [30] . in other words, e1 protein was not under strong selective pressure of adaptive evolution driven by immune response. suzuki and gojobori [5] identified 13 positively selected amino acid sites in the entire coding region of hcv subtype 1b by parsimony method. four of these sites were located in e1 and three located in e2. it is different from the results obtained from this study. the possible reasons may be: (1) the strategies to reconstruct ancestral sequences are different. adaptsite, the program employed in suzuki and gojobori's work, uses maximum parsimony method to perform reconstruction while codeml uses a likelihood reconstruction. thus, the reconstructed ancestral states may be different. in general, the two implementations produce similar results in dataset with high similarity among sequences. however, ml provides a more reliable result when used to analyze small-size dataset with relatively high diversity in sequence similarity [31, 32] . they focused on hcv subtype 1b [5, 33] , and deleted any sequences with gap by pairwise-alignment with reference sequence (hcv-js) to obtain dataset with highly similar sequences. our study used sequences containing 18 hcv geno/subtypes with a relatively large scale of genetic diversity; thus the ml method was adopted; (2) the methods to estimate branch length are different. adaptsite uses a neighbor-joining algorithm to estimate branch length [34] while codeml uses a codon model m0 to do it; (3) for codons that are neighbors of stop codons, adaptsite and codeml count sites differently, e.g. tac and tat, adaptsite counts s = 1 and n = 2, while codeml gives 0.429 and 2.571; (4) missing data are handled differently. adaptsite requires dataset without gaps [34] while codeml implemented in this work allows sequences data with some gaps; and (5) mutation rates in rna viruses are several orders of magnitude higher than those in dna based life-forms. by limiting genome size and content, rna virus genome can avoid deleterious mutation accumulation. it brings virus genomes to be inclined to concerted evolution or parallel evolution and own similar codon substitution patterns [35] . therefore, it is not difficult to understand why most of amino acid sites undergoing purifying selection of genetic constraints though hcv envelope proteins still possess high mutation rates. our study focused on a larger scale of genetic diversity than previous work. this study might probably miss particular positively selected amino acid sites of individual subtype but produce more general sites under positive darwinian selection to all hcv genotypes. the parsimony method of suzuki and gojobori [33] and the maximum likelihood method developed by nielsen and yang [8] are two widely used methods for detecting natural selection in homologous protein-coding sequences. however, they have their own pros and cons. the former may fail to infer positively selected sites when the branches of the phylogenetic tree are long because the maximum parsimony method is not fit for multiple substitutions. in contrast, multiple substitutions in the nielsen and yang method may be corrected by assuming the codon substitution model. suzuki also attempted to employ ml to modify previous method [36] . our study showed an application of ml method in detecting adaptive evolution in hcv envelope protein-coding sequences based on 18 geo/ subtypes. it provided an instance for similar work of high diversity homologous genes analysis. indeed, using ml method to infer adaptive evolution has been an effective strategy for some emerging viruses such as sars-cov. in this way we had successfully explored the adaptive evolution of sars-cov spike protein [37] . genetic diversity and evolution of hepatitis c virus--15 years on functional hepatitis c virus envelope glycoproteins conservation of the conformation and positive charges of hepatitis c virus e2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment positively selected amino acid sites in the entire coding region of hepatitis c virus subtype 1b hcvdb: hepatitis c virus sequences database the los alamos hcv sequence database likelihood models for detecting positively selected amino acid sites and applications to the hiv-1 envelope gene paml: a program for package for phylogenetic analysis by maximum likelihood consensus proposals for a unified system of nomenclature of hepatitis c virus genotypes chronic hepatitis c virus infection: genotyping and its clinical role a package for performing multiple sequence alignment on a microcomputer the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools multiple sequence alignment with clustal x emboss: the european molecular biology open software suite the neighbor-joining method: a new method for reconstructing phylogenetic trees mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment a simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences codon-substitution models for heterogeneous selection pressure at amino acid sites the los alamos hepatitis c immunology database statistical methods for detecting molecular adaptation evolutionary dynamics of hepatitis c virus envelope genes during chronic infection hypervariable regions in the putative glycoprotein of hepatitis c virus variable and hypervariable domains are found in the regions of hcv corresponding to the flavivirus envelope and ns1 proteins and the pestivirus envelope glycoproteins a model for the hepatitis c virus envelope glycoprotein e2 evidence for immune selection of hepatitis c virus (hcv) putative envelope glycoprotein variants: potential role in chronic hcv infections prevention of hepatitis c virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis c virus mutational evidence for an internal fusion peptide in flavivirus envelope protein e induction of hepatitis c virus e1 envelope protein-specific immune response can be enhanced by mutation of n-glycosylation sites a new method of inference of ancestral nucleotide and amino acid sequences accuracies of ancestral amino acid sequences inferred by the parsimony, likelihood, and distance methods a method for detecting positive selection at single amino acid sites adaptsite: detecting natural selection at single amino acid sites error thresholds and the constraints to rna virus evolution new methods for detecting positive selection at single amino acid sites reconstruction of the most recent common ancestor sequences of sars-cov s gene and detection of adaptive evolution in the spike protein key: cord-317595-siwzjeea authors: forni, diego; cagliani, rachele; pontremoli, chiara; pozzoli, uberto; vertemara, jacopo; de gioia, luca; clerici, mario; sironi, manuela title: evolutionary analysis provides insight into the origin and adaptation of hcv date: 2018-05-01 journal: front microbiol doi: 10.3389/fmicb.2018.00854 sha: doc_id: 317595 cord_uid: siwzjeea hepatitis c virus (hcv) belongs to the hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. hcv origin was previously dated in a range between ∼200 and 1000 years ago. hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. the closest relatives of hcv were found in horses/donkeys (equine hepaciviruses, ehv). however, the origin of hcv as a human pathogen is still an unsolved puzzle. using a selection-informed evolutionary model, we show that the common ancestor of extant hcv genotypes existed at least 3000 years ago (ci: 3192–5221 years ago), with the oldest genotypes being endemic to asia. ehv originated around 1100 ce (ci: 291–1640 ce). these time estimates exclude that ehv transmission was mainly sustained by widespread veterinary practices and suggest that hcv originated from a single zoonotic event with subsequent diversification in human populations. we also describe a number of biologically important sites in the major hcv genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. hcv exploits several cell-surface molecules for cell entry, but only two of these (cd81 and ocln) determine the species-specificity of infection. herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at cd81 were only observed in chiroptera. no evidence of selection was detected for ocln in any mammalian order. these results shed light on the origin of hcv and provide a catalog of candidate genetic modulators of hcv phenotypic diversity. hepatitis c virus (hcv) belongs to the hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. hcv origin was previously dated in a range between ∼200 and 1000 years ago. hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. the closest relatives of hcv were found in horses/donkeys (equine hepaciviruses, ehv). however, the origin of hcv as a human pathogen is still an unsolved puzzle. using a selection-informed evolutionary model, we show that the common ancestor of extant hcv genotypes existed at least 3000 years ago (ci: 3192-5221 years ago), with the oldest genotypes being endemic to asia. ehv originated around 1100 ce (ci: 291-1640 ce). these time estimates exclude that ehv transmission was mainly sustained by widespread veterinary practices and suggest that hcv originated from a single zoonotic event with subsequent diversification in human populations. we also describe a number of biologically important sites in the major hcv genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. hcv exploits several cell-surface molecules for cell entry, but only two of these (cd81 and ocln) determine the species-specificity of infection. herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at cd81 were only observed in chiroptera. no evidence of selection was detected for ocln in any mammalian order. these results shed light on the origin of hcv and provide a catalog of candidate genetic modulators of hcv phenotypic diversity. hepatitis c virus (hcv, genus hepacivirus, family flaviviridae) is a hepatotropic human pathogen with an estimated worldwide seroprevalence around 2.8% (mohd hanafiah et al., 2013) . the seven major hcv genotypes display remarkable antigenic variability and are classified into several recognized subtypes . a small number of "epidemic" subtypes (1a, 1b, 3a, and 2a) account for the overwhelming majority of infections worldwide (simmonds, 2013; messina et al., 2015) . their spread occurred recently (in the last 50-100 years), following the development of practices that cause parenteral exposure (pybus et al., 2001; simmonds, 2013; jackowiak et al., 2014) . the epidemic subtypes represent a small fraction of hcv diversity. in sub-saharan africa and south-east asia, the pattern of hcv diversity is characterized by highly divergent subtypes of the same genotype dominating transmissions across geographically contiguous areas ("endemic" transmission) (simmonds, 2013; jackowiak et al., 2014) . hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents (kapoor et al., 2011 (kapoor et al., , 2013 burbelo et al., 2012; drexler et al., 2013; quan et al., 2013; corman et al., 2015; scheel et al., 2015; walter et al., 2016; van nguyen et al., 2018) . the closest relatives of hcv were found in horses/donkeys and dogs (equine and canine hepaciviruses, ehv, and chv) (pybus and gray, 2013; pybus and theze, 2016) , but the origin of hcv as a human pathogen is still an unsolved puzzle. some authors envisaged the possibility that hcv evolved from a horse-tohuman transmission event (pfaender et al., 2014; scheel et al., 2015) , others suggested that hcv originated in relatively recent times from one or multiple cross-species transmission events from a still to be defined species (pybus and gray, 2013; scheel et al., 2015; pybus and theze, 2016) . however, its strict speciesspecificity, as well as the ability of hcv to persist lifelong in humans, led to the alternative hypothesis whereby hcvrelated viruses have been infecting humans and other primates throughout their evolutionary history (simmonds, 2013; scheel et al., 2015) . another open question is whether hcv genotypes differ in terms of transmissibility or disease outcome. conversely, the viral genotype is known to represent a predictive factor for antiviral treatment success. treatment with pegylated interferon and ribavirin (pegifn/rbv) is less likely to be successful in patients infected with genotypes 1 and 4 compared to those infected with genotypes 2 and 3 (bartenschlager et al., 2013) , and variants at the human ifnl3/ifnl4 locus were reproducibly associated with pegifn/rbv treatment outcome (o'brien et al., 2014) . interestingly, the same polymorphisms are strongly associated with spontaneous hcv clearance (o'brien et al., 2014) . recently, direct-acting antivirals (daas) have greatly improved the efficacy of treatment strategies for hcv. however, daas have limited pan-genotypic activity and tend to select for resistance-associated amino acid variants (ravs) (lontok et al., 2015) . most ravs naturally occur in treatment-naive patients and in some instances rav prevalence differs among genotypes or subtypes (rai and deval, 2011; bartenschlager et al., 2013; lontok et al., 2015; cannalire et al., 2016; chen et al., 2016; patino-galindo et al., 2016) . herein, we apply an evolutionary approach to shed light into hcv origin, to analyze its adaptation to human populations, and to verify if the emergence of drug-resistant variants is a result of positive selection. we also analyzed the most important cell-surface molecules that mediate hcv cell entry to determine whether hcv or related hepaciviruses exerted a selective pressure on these receptors and whether selection was particularly strong in specific mammalian orders or superorders. coding sequences for mammalian ocln, cd81, cldn1, and scarb1 (encoding srb1) were retrieved from the ncbi 1 database. a list of species is available in the supplementary table s1 . dna alignments were performed with the revtrans 2.0 utility 2 , mafft v6.240 as an aligner) (wernersson and pedersen, 2003) , which uses the protein sequence alignment as a scaffold for constructing the corresponding dna multiple alignment. all alignments were screened for the presence of recombination using genetic algorithm recombination detection (gard) (kosakovsky pond et al., 2006) and two methods (rdp and geneconv) (sawyer, 1989; martin and rybicki, 2000) implemented in the rdp4 program (martin et al., 2017) . rdp and geneconv were selected because they showed good power in previous simulation analyses (posada and crandall, 2001; bay and bielawski, 2011) and only breakpoints identified by both methods were accepted. the cutoff p-value was set to 0.01 in both gard and rdp4. no method detected recombination in any alignment. after running a codon model selection analysis in hyphy (delport et al., 2010) , gene trees were generated by maximumlikelihood using phyml with the approximate likelihood-ratio test (alrt) method (guindon et al., 2010) . to search for positive selection, we applied the site models implemented in paml (yang, 2007) . specifically, a model (m8, positive selection model) that allows a class of sites to evolve with dn/ds > 1 was compared to two models (m7 and m8a, neutral models) that do not allow dn/ds > 1 (yang, 2007) . positively selected sites were identified through bayes empirical bayes (beb) analysis (anisimova et al., 2002; yang et al., 2005) from model m8 (with a posterior probability cutoff of 0.90) and through mixed effects model of evolution (meme), with a default p-value cutoff of 0.10) (murrell et al., 2012) . only sites detected by both methods were considered as positively selected. very similar results were obtained using the gene tree obtained with phyml or the species tree as inputs for paml analysis. to estimate the time to the most common ancestor (tmrca) of the 7 hcv genotypes, we used alignments of 67 hcv sequences with known isolation dates (supplementary table s2 ). viral sequences were retrieved from the ncbi 1 database. we used prank (loytynoja and goldman, 2005) to generate multiple sequence alignments and guidance (sela et al., 2015) for filtering unreliably aligned codons (we masked codons with a score < 0.90), as suggested (privman et al., 2012) . the ns5b region we used for dating is non-recombining, as assessed by gard and rdp4 analyses of the entire nonstructural portion of the genome. this region was selected because it is one of the most conserved across hcv genotypes (kapoor et al., 2011) and required very minor filtering. the tmrca of ehv/chv was estimated on a phylogeny of 18 sequences with complete coding sequence information: 17 ehv isolated from horses or donkeys and 1 chv (kapoor et al., 2011; burbelo et al., 2012; walter et al., 2016) (supplementary table s3 ). guidance detected no uncertainty in the alignments (all codons had a score > 0.9) and gard detected a breakpoint at position 6729. in good agreement, rdp4 detected a breakpoint at position 6747. the terminal 2145 nucleotides were thus removed and the resulting alignment was used for tree construction and tmrca estimate. to determine whether there was sufficient temporal structure in the ns5b and ehv phylogenies to estimate divergence times, we calculate the correlation coefficients (r) of regressions of rootto-tip genetic distances against sequence sampling times (murray et al., 2016) . a method that minimizes the residual mean squares of the models, rather than one that maximizes r 2 , was applied, as suggested (murray et al., 2016) . the p-values were calculated by performing 1,000 clustered permutations of the sampling dates murray et al., 2016) . evidence for temporal structure was obtained for both phylogenies (ns5b: r = 0.25, r 2 = 0.0625, p-value = 0.024; ehv: r = 0.35, r 2 = 0.1225, p-value = 0.013). the action of saturation and purifying selection can underestimate the tmrca, and selection-informed models can improve branch length estimation (wertheim and kosakovsky pond, 2011; wertheim et al., 2013 wertheim et al., , 2014 . we thus applied a branch-site test (abs-rel, adaptive branch-site random effects likelihood) to estimate branch lengths while taking into account the effect of different selective pressures among lineages in the phylogeny. previous works showed that, in the presence of saturation and selection, abs-rel estimates branch lengths more reliably than other commonly used models such as the general time-reversible substitution model with a four-bin gamma rate distribution (gtr+ 4 ) (wertheim and kosakovsky pond, 2011; wertheim et al., 2013) . estimate of divergence times was performed by a penalizedlikelihood (pl) method implemented in r8s (sanderson, 2003) , using the abs-rel tree as the input tree. in the case of 3 extremely long (i.e., more than 50 substitution/site) terminal branches, most likely resulting from low precision in point estimates of dn/ds, mg94 branch lengths were used instead of abs-rel lengths to avoid biases in the tmrca estimates. mg94 (muse and gaut, 1994 ) is the baseline model used by abs-rel and it models synonymous and nonsynonymous substitution rate variation across branches but not across sites (muse and gaut, 1994) . the pl method in r8s employs a smoothing parameter, which represents how much the assumption of a molecular clock has been relaxed. cross-validation was run to determine the best smoothing value for the abs-rel tree (sanderson, 2003) . a latin hypercube sampling scheme (lhc) was used to sample from the abs-rel parameter distributions so as to estimate the confidence interval, as previously suggested (wertheim and kosakovsky pond, 2011; wertheim et al., 2013 wertheim et al., , 2014 . briefly, 500 samples were drawn from abs-rel analyses to estimate branch length variance, 500 trees were generated, and then used as input trees for r8s. the upper and the lower 95% bounds are used as confidence intervals. as a comparison, branch lengths were also estimated using phyml with a maximum-likelihood approach and a gtr+ 4 model (guindon et al., 2009 ). we used coding sequence information for the 67 recognized hcv subtypes (supplementary table s2 ). viral sequences were retrieved from the ncbi 1 database. as above, we used prank (loytynoja and goldman, 2005) to generate multiple sequence alignments and guidance (sela et al., 2015) for filtering unreliably aligned codons (privman et al., 2012) . gard (kosakovsky pond et al., 2006) and the above-mentioned methods implemented in rdp4 (sawyer, 1989; martin and rybicki, 2000) were used to screen for recombination. phylogenetic trees were generated by maximum-likelihood using phyml (guindon et al., 2010) after codon model selection in hyphy (delport et al., 2010) . to investigate whether episodic positive selection acted on the internal branches of hcv phylogenies, we applied the branch-site tests from paml (zhang et al., 2005) and busted (branch-site unrestricted statistical test for episodic diversification) . the p-values from both methods were fdr-corrected to account for multiple tested branches. a branch was considered positively selected when statistically significant evidence was obtained with both methods. positively selected sites were then identified through the beb analysis from model ma (with a posterior probability cutoff of 0.90) (zhang et al., 2005) and with busted (with a p-value cutoff of 0.05) . sites were called as positively selected if they were detected by both methods. beb and busted were in good general agreement. on selected branches, 63.07% of beb sites were also detected by busted and 65.68% of busted sites were also identified by beb. we used single-likelihood ancestor counting (slac) and fixed effects likelihood (fel) (kosakovsky pond and frost, 2005) to calculate the rates of nonsynonymous and synonymous changes at each site in the structural and in the non-structural region alignments (analyses were performed on alignments split on the basis of the recombination breakpoint detected by gard). both slac and fel estimate the probability of selection at each site in an alignment through the dn-ds metric (rate of nonsynonymous changes-rate of synonymous changes) (kosakovsky pond and frost, 2005) . this metric is preferred over the conventional dn/ds ratio as this latter is rendered to infinite for ds values equal to 0. although slac and fel use different methodologies to estimate substitution rates (kosakovsky pond and frost, 2005) , they yielded very similar results (for all sites in the hcv genome, spearman's correlation coefficient = 0.73, p-value < 10 −16 ). slac and fel were used to estimate the evolutionary rate at rav vs. non-rav positions. the dn-ds statistics was also exploited to provide an overall view of hcv genome evolution. in this case, only slac results are shown (those obtained with fel were very similar). a total of 127 ravs in ns3, ns4b, ns5a, and ns5b were included in the study (supplementary table s4 ). ravs were obtained by merging six recently compiled lists of naturally occurring variants associated with daa resistance (rai and deval, 2011; bartenschlager et al., 2013; lontok et al., 2015; cannalire et al., 2016; chen et al., 2016; patino-galindo et al., 2016) . specifically, a list of positions where ravs have been reported was compiled, irrespective of the viral genotype carrying the rav. for statistical analysis, six ravs that occur at codons pruned from the alignment by guidance were removed. the overall probability of rav occurrence was calculated over the number of non-pruned codons in ns3, ns4b, ns5a, and ns5b (total length = 1877 codons). for the ns5a-oas1 docking, the crystal structure of the ns5a domain i of a subtype 1b strain (pdb: 1zh1) and the structure of human oas1 (pdb: 4ig8) were obtained from the protein data bank (pdb) (tellinghuisen et al., 2005; donovan et al., 2013) . the 1b subtype was used in the original study describing interaction between oas1 and ns5a (taguchi et al., 2004) . two docking methods were applied and their results compared for consistency. specifically, we used the patchdock server (schneidman-duhovny et al., 2005) and imposed that the f37 residue in ns5a is located at the binding interface, as described (taguchi et al., 2004) . we next used cluspro (comeau et al., 2004; kozakov et al., 2017) without any assumption on the residues involved in binding. the best models from cluspro and patchdock were superimposed and revealed an almost identical binding pose. we next checked that the two oas1 regions necessary for ns5a binding (taguchi et al., 2004) were located at the interface: one of these two regions (amino acids 235-275) defines most of the contact area in the models. overall, these observations indicate that the binding interaction pose obtained with the two docking methods is reliable. the c19 sphingomyelin ligand structure was prepared for simulation using the ligprep module of maestro (schrodinger release, 2016-4: maestro schrodinger, llc, new york, ny, united states, 2016) to determine the 3d structure and ionization states at ph 7.0 ± 0.2. the ns5b structure of a subtype 1b strain (pdb id: 4mk7 chain a) was processed with protein preparation wizard module of maestro to remove crystal water molecules, add hydrogen atoms, assign bond orders, and optimize the orientation of hydroxyl groups. docking simulation was carried out with ifd protocol (sherman et al., 2006) in extra precision mode. the receptor grid was generated around the sphingomyelin binding domain (residues e230 to g263). all the simulations were performed using the opls3 force field (harder et al., 2016) . the docked ligand-receptor poses were analyzed in terms of glide score, a scoring function that estimate protein-ligand binding affinities . the 3d structures were rendered using pymol (the pymol molecular graphics system, version 1.8.4.0 schrödinger, llc). host receptors that mediate virus entry often evolve under positive selection (a situation that favors amino acid replacements over silent substitutions) to avoid viral recognition (sironi et al., 2015) . four cell-surface molecules, cd81, occludin (ocln), claudin 1 (cldn1), and scavenger receptor class b member 1 (srb1), are particularly important for hcv cell entry, although only cd81 and ocln determine the species-specificity of infection (bartenschlager et al., 2013; lindenbach and rice, 2013) . we thus reasoned that if primates experienced long-term interactions with hcv or closely related hepaciviruses, signals of positive selection should be detectable at the genes encoding hcv receptors. conversely, if selection is evident in other mammalian orders, these may represent the ancestral hcv reservoirs. we retrieved coding sequences of the four genes for mammalian species belonging to different orders, superorders or clades (supplementary table s1 ). due to the hypothesized role of bats as reservoir hosts for mammalian hepaciviruses, chiroptera were analyzed separately from other species in the laurasiatheria superorder. evidence of positive selection was searched for using models that allow dn/ds to vary among sites in the alignment (yang, 2007) . no evidence of positive selection was detected for cldn1 and srb1 (table 1) . also, selection was not detected at ocln or cd81 in primates (table 1) , an observation that does not support a long-standing selective pressure exerted by hepaciviruses on these hosts. however, positive selection was not detected for rodents, either, although these mammals are known to host a wide diversity of hepaciviruses (drexler et al., 2013; kapoor et al., 2013; scheel et al., 2015) . for laurasiatherian ocln, we detected one positively selected site located in the extracellular loop 1, which is not directly involved in hcv binding. notably, evidence of positive selection for cd81 was detected only in chiroptera (figure 1 and table 1 ). the five positively selected sites are located at the binding interface with the hcv glycoprotein. single point mutations in the corresponding region of the human receptor were shown to reduce or abolish the interaction with the hcv e2 protein and/or hcv infectivity (figure 1 and table 1 ) (drummer et al., 2002; bertaux and dragic, 2006; flint et al., 2006) . conversely, the cd81 region required for infection of hepatocytes by plasmodium yoelii (a rodent parasite) sporozoites is unaffected by selection (figure 1 ) (yalaoui et al., 2008) . the observation that the positively selected sites are involved in hcv binding and infectivity suggests that hepaciviruses contributed to shape the genetic diversity of cd81 in bats. previous studies provided estimates of the time to the most recent common ancestor (tmrca) of hcv genotypes in a range between ∼200 and 1000 years ago, with one single study indicating that hcv origin may date 2000 years back (smith et al., 1997; pybus et al., 2001; kapoor et al., 2011; simmonds, 2013; li et al., 2014; lu et al., 2014) . the tmrca of equine/canine hepaciviruses (ehv/chv) was estimated to be recent, dating around 1800 ce (pybus and theze, 2016) . it is well known that the temporal variation in rates of nucleotide substitutions often results in underestimation of the age of viral lineages (duchene et al., 2014; aiewsakun and katzourakis, 2016) . purifying selection and substitution saturation are strongly associated with temporal rate variation (duchene et al., 2014) . simulations experiments indicated that "classic" models (e.g., gtr+ 4 ) tend to underestimate branch lengths in the presence of purifying selection (wertheim and kosakovsky pond, 2011) and substitution saturation (wertheim et al., 2013) . because both phenomena are more pronounced for internal branches, length underestimation is more severe for these branches and dating inferences are consequently affected (wertheim and kosakovsky pond, 2011; wertheim et al., 2013) . the use of models that allow site-and branch-specific variation in selective pressure can improve branch length estimates in the presence of both purifying selection and substitution saturation (wertheim and kosakovsky pond, 2011; wertheim et al., 2013) . we thus applied the abs-rel model (adaptive branchsite random effects likelihood) to calculate the tmrca of relevant nodes in a phylogeny of 67 hcv strains (supplementary table s2 ). this approach was previously applied to revise the dating of other rna viruses (wertheim and kosakovsky pond, 2011; wertheim et al., 2013) . we constructed a phylogeny for the ns5b region and we found that only 0.7% of branches showed saturation of ds. as expected, branch lengths estimated with abs-rel were generally longer than those obtained with a gtr+ 4 model, and this was particularly true for internal branches (figure 2a and supplementary figure s1 ). using the abs-rel model, we estimated the tmrca of the seven hcv genotypes to be 3359 years ago (95% ci: 5221-3192) ( figure 2b) . the deepest tmrca was obtained for genotype 6 (endemic in south-east asia), with an origin dating at least 2000 years ago (figures 2b,c) . the tmrcas of genotype 3 (endemic in south asia) and of the ancestor of genotypes 1 and 4 (both widespread in central africa) were estimated to be in the 800 bce-700 ce range (figures 2b,c) . results for genotype 2 (endemic in west africa) indicated an origin around 430 years ago (figures 2b,c) . horse-to-human hepaciviral transmission was hypothesized as the origin of hcv (pfaender et al., 2014; scheel et al., 2015) . ehv isolates show limited divergence and high levels of purifying selection (simmonds, 2013; pfaender et al., 2014) . we thus used the same approach described above to obtain the tmrca of extant ehv/chv strains (figure 2a ) (supplementary table s3 ), producing an estimate of 863 years ago (95% ci: 1726-377). a number of studies have explored the recent selective events that generated intra-genotype and within-host genetic diversity (jackowiak et al., 2014) , whereas the deeper evolutionary history of hcv has remained largely unexplored. we thus used complete sequence information for the 67 recognized hcv subtypes to search for selective events that occurred during the radiation of the seven genotypes. the structural and non-structural coding regions were analyzed separately, and the core region was excluded due to the presence of a putative alternative reading frame (xu et al., 2001) . we next tested for the presence of recombination using gard and rdp4 (see the section "materials and methods"). none of the rdp4 methods detected recombination either in the structural or in the non-structural regions. conversely, gard detected a possible breakpoint at the end of ns3. the lack of consistency among recombination detection methods is somehow expected as these approaches have distinct performances depending on the amount of recombination, the genetic diversity of analyzed sequences, the tree topology, and the rate variation among sites (posada and crandall, 2001; bay and bielawski, 2011) . because our aim was to avoid the inflation of positive selection inference caused by unrecognized recombination, the alignment was split into two sub-alignments (based on the gard-detected breakpoint) with slightly different topologies (figure 3) . evidence of episodic positive selection along the internal branches of the phylogenies was searched for using two different branch-site methods, which rely on different assumptions of dn/ds variation among branches (zhang table s6 ). analysis of positively selected sites was performed by inspection of literature reports describing the effect of specific mutations, as well as by performing docking analyses. overall, positively selected sites could be categorized on the basis of the functional effect they are likely to entail, as follows: cell entry, interaction with the host immune system, and association with membrane/lipids. the details of these sites are reported below, as well as in figures 4, 5. as expected, none of the positively selected sites in e2 involved the conserved residues at the cd81 binding site (lavie et al., 2014) . interestingly though, a mutation at residue 216 in e1 was previously obtained through in vitro adaptation of hcv to mouse cells ( figure 4a ) (bitzegeio et al., 2010) . specifically, the l216f mutation (as well as two other mutations in the hypervariable region 1, hvr1) allow infection of cells expressing cd81 of rodent origin (bitzegeio et al., 2010) , suggesting that hcv adaptation to new hosts does not necessarily entail changes at the direct binding interface with cd81. several positively selected sites were detected in the transmembrane (tm) regions of both e1 and e2 (figure 4a) . a positively selected site in the e1 tm domain (residue 374) was shown to reduce the dependency on scarb1 for cell-to-cell spread in vitro, but increases viral susceptibility to antibody-mediated neutralization (catanese et al., 2013) . interestingly, a similar phenotype was described for a t563v mutant in the jfh1 viral background (corresponding to the positively selected 561 site in strain h77) ( figure 4a ) (zuiani et al., 2016) . using a site-directed saturation mutagenesis approach, the t563v mutation was shown to confer growth advantage in vitro via reduced dependency on scarb1 and probably stronger binding to cd81 (zuiani et al., 2016) . several mechanisms for hcv resistance to ifn have been proposed. the ns5a protein displays a double-stranded rnaactivated protein kinase r (pkr) binding site that overlaps with the ifn sensitivity determinant (isdr) region but includes 26 additional amino acids essential for pkr binding (gale et al., 1998) . two positively selected sites were detected within this 26 amino acid region ( figure 5a ). mutations at the second selected site (position i302 in h77, corresponding to c298 in the jfh1 sequence) arise when hcv is passaged in the presence of ifn-alpha (perales et al., 2013) . the c298i change is positively selected on the genotypes 1/4 branch and both genotypes are relatively resistant to ifn therapy (jackowiak et al., 2014) . the binding of ns5a to oas1 also contributes to inhibition of ifn antiviral activities (taguchi et al., 2004) . based on previous biochemical data (taguchi et al., 2004) , we performed protein-protein docking of ns5a domain i and human oas1. results showed that three of the positively selected sites in ns5a domain i, positions 54, 78, and 93 are at the direct binding interface with oas1 ( figure 5b) . notably, besides being described as ravs for daas, substitutions at the y93 site in the subtype 1b background occur at different frequency depending on the host's genotype at the ifnl3/ifnl4 locus (akamatsu et al., 2015; peiffer et al., 2016) . a similar trend was observed for variants at ns5a positions 31 (not included in the crystal), 37 (which modulates oas1 binding) (taguchi et al., 2004) , 52 (close to the binding interface), and 54 (a positively selected rav close to the binding interface) ( figure 5b ) (akamatsu et al., 2015) . interestingly, sites 93 and 54 are positively selected on the genotype 6 and 3 branches, respectively (supplementary table s5 ). both these figure 4 | selected sites in hcv proteins. (a) schematic representation of the hcv structural region. regions that were not analyzed (i.e., core region) or filtered due to poor alignment quality are colored in gray. the location of positively selected sites is shown and residues with known functional significance (see text and below) are underlined. sites are numbered based on the sequence of the h77 strain (af009606.1). the ectodomain region of e1 contains two short regions with sequence similarity to class ii fusion peptides (drummer et al., 2007) : in vitro mutagenesis indicated that changes at positively selected residues 285, 286, and 288 abolish or reduce viral entry (drummer et al., 2007; li et al., 2009; russell et al., 2009) . a 12 amino acid motif in e2 (the pkr-eif2α phosphorylation site homology domain, pephd) is required for pkr and perk (pkr-like er-resident kinase) inhibition (taylor et al., 1999; pavio et al., 2003) . an amino acid alignment for the pephd domain is reported (representative hcv sequences only), with positively selected sites in red. tm: transmembrane domain. (b) topological structure of the ns2 and ns4b proteins. positively selected sites are mapped (red) on the ns2 (pdb id: 2hd0) and ns4b (pdb id: 2lvg, 2jxf, 2kdr) protein structures. protein segments of unresolved structure are represented as cylinders (transmembrane domains) or dotted lines. an amino acid alignment of the second amphipathic helix of ns2 is reported for representative strains (positively selected sites in red): mutagenesis of positively charged residues at positions 131 or 134 (depending on the genotype) affect ns2 membrane association, protein stability, and efficient hcv polyprotein processing (lange et al., 2014) . the presence of at least one positively charged residue at these positions is sufficient to allow proper membrane localization (lange et al., 2014) and indeed, the two positions evolve in concert in the hcv phylogeny with a charged residue always observed at either position 131 or 134, but never at both sites. er: endoplasmic reticulum. genotypes are endemic in asia, where the frequency of the protective human ifnl3/ifnl4 variant (rs12979860) is highest ( figure 5c) . recently, a large-scale analysis of patients mostly infected with hcv genotype 3a, identified 30 sites in the viral polyprotein showing association with one or more hla alleles (ansari et al., 2017) . we found three of these sites (561 in e2, 620 in ns3, and 48 in ns4b) to be positively selected, with site 620 in ns3 (position 1646 in the polyprotein) representing one of the strongest association detected in the study (ansari et al., 2017) . all hcv proteins are tethered to intracellular membranes either via transmembrane domain or through amphipathic alpha helices or both (bartenschlager et al., 2013) . we identified several positively selected sites within amphipathic alpha helices (figure 4b ). in the case of ns4b, virtually all sites are located in these regions, where several ravs also map (supplementary table s4 ). among these, w43 (selected on the genotype 4 branch) is essential for ns4b association to the membrane and to lipid droplets (gouttenoire et al., 2009; tanaka et al., 2013) . we found positively selected sites within the sphingomyelin binding domain (sbd) of the hcv rna polymerase (ns5b). binding of sphingomyelin to ns5b allows localization of the polymerase to lipid rafts and activates the enzymatic activity in a genotype-dependent manner (sakamoto et al., 2005; weng et al., 2010) . remarkably, mutagenesis experiments indicated that the positively selected sites 244 and 238 modulate sphingomyelin binding and activation, respectively (weng et al., 2010) . we thus docked a sphingomyelin molecule onto the 3d structure of subtype 1b ns5b (figure 5d) . docking results confirmed a salt bridge interaction between residue 244 and the sphingomyelin (along with residues 241, 242, and 250) and the localization of residue 238 at the interaction surface. moreover, analysis of atomic distances indicated that two additional selected sites regions that were filtered due to poor alignment quality are colored in gray. the location of positively selected sites is shown and residues discussed in the text are underlined. positions 68 and 69 are also underlined, as a single lysine insertion between these two sites strongly increases viral replication (pflugheber et al., 2002) . (b) superimposition of ns5a-oas1 binding pose obtained using two different docking programs. for clarity, one oas1 molecule is shown (green); the binding poses of the ns5a dimer obtained with cluspro (yellow) and patchdock (light orange) are shown. the f37 residue, known to modulate ns5a binding to oas1, is marked in orange. positively selected sites are in red and labeled based on the sequence of h77. oas1 regions that are essential for ns5a binding are in dark green. (c) world map showing rs12979860 allele frequency in human populations (data from https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/ and https://alfred.med.yale.edu/alfred/high throughput.asp). (d) docking pose of ns5b (pdb id: 4mk7, white) with sphingomyelin (green). positively selected sites are colored in red and labeled when located at the ns5b-sphingomyelin binding interface. (e) ligand interaction diagram of best docked pose of ns5b-sphingomyelin. residues within a 6å distance and hydrogen bonds are shown (see legend). (231 and 73) are likely involved in the binding of sphingomyelin ( figure 5e ). a recent analysis indicated that several ravs occurred de novo on external branches of hcv phylogenies, although a minority appeared on internal branches, indicating a common origin in multiple strains (patino-galindo et al., 2016) . however, the evolutionary history of ravs has remained a poorly investigated issue. up to now, direct-acting antivirals (daas) have been developed to inhibit the function of the ns3, ns4b, ns5a, and ns5b proteins. to obtain a codon-wise measure of selective pressure acting on these proteins across the hcv phylogeny, we calculated dn-ds at each site. comparison of rav and non-rav sites revealed no statistically significant difference in dn-ds calculated with either slac (wilcoxon's rank-sum test p-value = 0.19) or fel (wilcoxon's rank-sum test p-value = 0.59) ( figure 6a ). this suggests that ravs do not originate and are not maintained in viral populations as a result of relaxed selective constraints. this overall trend does not imply that all rav sites evolve at the same rate. thus, we tested whether positions where ravs occur are more likely to diversify through the action of natural selection. nine rav positions coincided with positively selected sites, a number higher than expected by chance (binomial test, p-value = 0.012) ( supplementary table s4 ). moreover, three of these rav positions were targeted by positive selection on two distinct branches of the phylogeny, a situation observed for only 8.8% of selected sites ( figure 6a) . to gain a comprehensive view of the action of selection, we plotted dn-ds along the hcv genome and we superimposed the location of positively selected sites and of ravs ( figure 6b ). in agreement with a previous work that analyzed conservation and selection in hcv-1a and hcv-1b genomes (patino-galindo and gonzalez-candelas, 2017), dn-ds tended to be higher in the e1 and e2 regions, compared to the non-structural portions. a non-negligible fraction of codons showed almost complete conservation: about 7% displayed ds = 0 and most of these (89%) also had dn = 0. both rav positions and positively selected sites displayed variable dn-ds values. we note that this is not unexpected for positively selected sites as they were identified using branch-site tests and are thus not selected across the entire hcv phylogeny. the worldwide spread of hcv epidemic strains started recently, in the 1930s-1940s, but the existence of genetically diverse endemic hcv strains that circulate in specific geographic locations implies a long-standing association of the virus with human populations (simmonds, 2013) . using an evolutionary model that accounts for variation in the pressure of natural selection across sites and branches, we show that the common ancestor of extant hcv genotypes existed at least 3000 years ago, with a lower bound estimate of ∼5200 years before present. the same approach placed the origin of ehv/chv around 1100 ce, with a lower bound at 290 ce. although we applied a selectioninformed approach that accounts for the effect of purifying selection and is relatively robust in the presence of substitution saturation (wertheim et al., 2013) , we most likely failed to fully correct for time-dependent substitution rate variation . thus, the time of hcv and ehv/chv origin might be underestimated. however, these time estimates rule out the possibility that the use of horse serum to obtain therapeutic antitoxins originated hcv and, more generally, seem to exclude the hypothesis that the human virus derived from a cross-species transmission event from horses (pfaender et al., 2014; scheel et al., 2015) . nonetheless, the sampling of ehv is still sparse. the isolation of additional ehv sequences, possibly from a wider geographic range, may date the origin of this virus further back. horse domestication begun around 5000 years ago in central asia (outram et al., 2009) : where ehv found to be older, close contacts between horses and humans may have resulted in zoonotic cross-species transmission or in the acquisition of hcv and ehv from a common source (possibly bat or rodent). active sampling of horse and donkeys worldwide will be required to address this important point. the time frame of hcv origin and the geographic distribution of the endemic strains are not consistent with the possibility that hcv dispersed with humans following the major out-of-africa colonization routes across the old world (65000-45000 years ago) (nielsen et al., 2017) . the dating we estimated for hcv origin instead suggests that the (possible) zoonotic transmission and subsequent spread in human populations occurred in a timeframe when long-distance trade routes were being established. the deepest tmrca for hcv genotypes was obtained for genotype 6, possibly indicating an asian origin of extant strains. several historical situations may help explain the diffusion of hcv in asia and africa. maritime trading routes across the south chinese sea were already developed in the first millennium bce (hung et al., 2013) , and regular sea connections between south-east asia and india were established by 1000-500 bce (pearson, 2016) . more extensive human movements connecting asia with africa occurred starting around ∼300 bce as a result of alexander the great expansion (336-325 bce) and the full development of the silk road (since 200 bce), this latter comprising maritime and land routes that reached africa (lawler, 2014) . around this time, indian slaves, mostly women, were regularly imported to egypt (mark, 2002) . moreover, archaeological evidences suggest that trade circuits connected the east coast of africa with south asia in the first millennium ce 3 . whereas these represent possible transmission routes, it remains unclear how, in a time when parenteral exposure was limited, hcv spread and was maintained in human populations. vertical transmission of hcv is rare and sexual contact is also thought to be a scarcely efficient route for hcv dissemination. it was thus proposed that cultural practices (e.g., circumcision, tattooing, or acupunture) (shepard et al., 2005; simmonds, 2013) or natural routes such as arthropod biting (pybus et al., 2007) might account for endemic hcv transmission. this issue is not addressed by the analyses herein. however, our time estimates place the origin of ehv in a time frame when invasive veterinary procedures and parenteral exposure were most likely rare. although we cannot exclude that some forms of human intervention (e.g., the use of spurs and animal housing in crowded stables) facilitated viral transmission, natural routes are likely to account for the spread of ehv. whether the same mechanisms are responsible for the endemic transmission of hcv and of ehv remains an open question. in this respect, we note that sexually transmitted infections (stis) that disrupt mucosal integrity have been implicated in increased sexual transmission of hcv, at least in high-risk groups (chan et al., 2016) . stis have probably been common throughout human history and several such diseases are known in horses (dobson and carper, 1996; samper and tibary, 2006) . the association between hcv sexual transmission and specific stis might help explain the geographic distribution 3 http://www.sealinksproject.com/ of endemic hcv strains. for instance, lymphogranuloma venereum and granuloma inguinale are confined to tropical and subtropical regions (richens, 2006; white, 2009) and their presence may have facilitated the maintenance and diversification of endemic hcv strains in these areas. although the present-day distribution of these stis may not necessarily reflect the historical prevalence of the causative bacteria, the hypothesis of sti-facilitated hcv transmission warrants further consideration. although we found that the tmrca for hcv is greater than the previously inferred time ranges (smith et al., 1997; pybus et al., 2001; kapoor et al., 2011; simmonds, 2013; li et al., 2014; lu et al., 2014) , hcv can be thought of as a recently emerged pathogen, largely postdating the origin of our species. this observation, as well as the presently limited evidence of hepacivirus infection in non-human primates, does not support a long-standing association between these viruses and primates (simmonds, 2013; scheel et al., 2015) . the absence of selection signatures at primate cd81 and ocln, the two molecules that determine the species-specificity of hcv infection, further supports this view. however, as noted above, we also failed to detect positive selection for hcv receptors in glires, even though the wide variety of rodent hepaciviruses suggests that these mammals have been infected by hepaciviruses for a long time (drexler et al., 2013; kapoor et al., 2013; scheel et al., 2015) . thus, results obtained for the hcv receptors are clearly not conclusive and should be regarded as preliminary. indeed, the power of the positive selection tests depends not only on the strength of selection, but also on the number and divergence of the analyzed sequences (anisimova et al., 2001 (anisimova et al., , 2002 . considering these two parameters, we have most likely a power lower than 80% although accuracy is higher than 90% (anisimova et al., 2001 (anisimova et al., , 2002 . given this premise, it is nonetheless interesting that the only mammalian order showing evidence of selection at cd81 was chiroptera, which host a wide diversity of hepaciviruses (kapoor et al., 2011 (kapoor et al., , 2013 drexler et al., 2013; quan et al., 2013; corman et al., 2015; scheel et al., 2015; walter et al., 2016) . known bat hepaciviruses share very little similarity to the hcv e2 protein, and this also applies to the e2 regions involved in cd81 binding. this raises the possibility that these animal viruses engage cellular receptors distinct from cd81, although previous studies on coronaviruses showed that the same region of the cellular receptor can be bound by viruses that share little sequence or structural similarity in their glycoprotein receptor binding domains (forni et al., 2017) . also, recent studies have indicated that hcv adaptation to the usage of rodent cd81 molecules is not necessarily paralleled by changes at the direct binding interface (bitzegeio et al., 2010) . thus, the receptor preferences of non-human hepaciviruses will require experimental investigation. nonetheless, the observation that the cd81 positively selected sites in chiroptera are located in a small region that corresponds to the e2 binding site is consistent with the view that cd81-binding hepaciviruses have been infecting bats for a long time. however, we note that cd81 was also shown to interact with other pathogens. for instance, cd81 is required for human plasmodium falciparum and rodent plasmodium yoelii sporozoite hepatocyte infection (silvie et al., 2003) . even though the protein region necessary and sufficient for p. yoelii infection is distinct from that involved in hcv binding (yalaoui et al., 2008) , we cannot exclude that batinfecting plasmodium parasites (schaer et al., 2013) bind different regions of cd81. also, cd81 was shown to be important for influenza virus infection (he et al., 2013) , but the molecular details of the interaction with this virus are unknown. thus, we cannot rule out the possibility that pathogens other than hepaciviruses were responsible for the selection signatures we detected at cd81 in bats. zoonotic events are commonly associated with bursts of positive selection whereby the pathogen adapts to successfully infect and be transmitted in a new species (longdon et al., 2014) . a common trade-off of the adaptation to new hosts is the loss or reduction of infectivity in the original host (longdon et al., 2014) . indeed, hcv infection is now restricted to humans (and to experimentally infected chimpanzees). because the ancestor of hcv has not been identified, knowledge of the molecular events that allowed hcv adaptation to humans remains elusive. we identified several positively selected sites in the e1 and e2 regions and some of these were shown to modulate the viral requirement of specific cellular factors (i.e., cd81 and scarb1). whereas these changes are not expected to represent specific adaptations to the human host, they may confer a selective advantage by increasing viral titers or cell-to-cell spread, a process possibly implicated in the establishment of a persistent infection (ding et al., 2014) . an interesting possibility is that, during its spread in asia and africa, hcv has adapted in response to the genetic background of distinct human populations, as expected given the distinctive geographic localization of viral genotypes for most of their evolutionary history. a paradigmatic example of this is the distribution of the ifnl3/ifnl4 polymorphism most strongly associated with spontaneous clearance of hcv: the frequency of the protective genotype differs dramatically among populations and this variant explains part of the ethnic variance in the probability to clear hcv infection (o'brien et al., 2014) . we identified two positively selected sites (h54 and y93 in ns5a) on the branches of the asian endemic genotypes (3 and 6, respectively). analysis of patients infected with hcv subtype 1b showed that substitutions at the y93 sites and, to a lesser extent at the h54 position, vary depending on the host ifnl3/ifnl4 genotype (akamatsu et al., 2015; peiffer et al., 2016) . this observation implies viral adaptation to the host depending on ifnl3/ifnl4 allelic status. although these findings are presently limited to genotype 1b viruses (akamatsu et al., 2015; pedergnana et al., 2016; peiffer et al., 2016) , they provide a proof of principle for the hypothesis that human genetic diversity exerted a selective pressure on hcv and possibly contributed to the radiation of the seven genotypes. clearly, it remains to be evaluated whether changes at positions y93 and h54 exert their effects via modulation of oas1 binding, as the docking analysis suggests, at least for position 93. alternatively, other mechanisms may be at play: a lysine insertion between ns5a positively selected sites k68 and n69, which are not at the binding interface with oas1, regulates pkr and irf-3 activity (pflugheber et al., 2002; sumpter et al., 2004) . in general, the positively selected sites we identified herein represent excellent candidates for future functional studies as they are expected to modulate viral phenotypes. for instance, selected sites in ns5b account for genotype differences in terms of sphingomyelin-driven polymerase activation (sakamoto et al., 2005; weng et al., 2010) , and the c298i change that arose in the common ancestor of genotypes 1 and 4 may help explain the poor response of these genotypes to ifn therapy. exposure to treatment cannot be regarded as the selective force underlying the evolution of the hcv sequences analyzed herein, as all strains derive from treatment-naive subjects. it was recently reported that several ravs have a recent origin and occurred independently on distinct viral lineages (patino-galindo et al., 2016) . this observation is in line with the notion that substitutions at rav positions can reduce viral fitness (bartenschlager et al., 2013) and thus fail to be maintained in viral populations or transmission clusters. indeed, by calculating the strength of selective pressure acting on hcv proteins targeted by daas, we observed that most rav positions are subject to a degree of purifying selection similar to non-rav positions. this suggests that ravs do not arise via relaxed selection but rather that these positions are functionally constrained. however, we also found that rav positions are targeted by positive selection more frequently than expected by chance. variation at these sites must therefore be adaptive for the virus, raising the possibility that daa treatment further selects for daa-resistant viruses with high fitness. this pattern contrasts with observations in hiv-1 infection, as positive selection at codons that confer drug resistance was only observed in patients receiving antiretroviral therapy (de s leal et al., 2004) . we note, though, that most ravs investigated here were described for genotype 1 and might confer little or no drug resistance to other hcv genotypes. conversely, most rav sites were positively selected on branches different than that leading to genotype 1. their functional relevance will thus need to be analyzed further. ms conceived the study, with inputs from df and mc. ms and mc supervised the project. rc and cp performed the evolutionary analysis in mammals. df performed the molecular dating analyses. rc, cp, and df performed the evolutionary analysis of hcv phylogenies. ms and df performed the rav analyses. jv and ldg performed the docking analyses. rc and df produced the figures, with input from all authors. up provided support during the bioinformatic analyses. ms, mc, and df wrote the manuscript, with critical input from all authors. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018. 00854/full#supplementary-material time-dependent rate phenomenon in viruses association between variants in the interferon lambda 4 locus and substitutions in the hepatitis c virus non-structural protein 5a accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution accuracy and power of bayes prediction of amino acid sites under positive selection genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis c virus the molecular and structural basis of advanced antiviral therapy for hepatitis c virus infection recombination detection under evolutionary scenarios relevant to functional divergence different domains of cd81 mediate distinct stages of hepatitis c virus pseudoparticle entry adaptation of hepatitis c virus to mouse cd81 permits infection of mouse cells in the absence of human entry factors serology-enabled discovery of genetically diverse hepaciviruses in a new host a journey around the medicinal chemistry of hepatitis c virus inhibitors targeting ns4b: from target to preclinical drug candidates different requirements for scavenger receptor class b type i in hepatitis c virus cell-free versus cell-to-cell transmission sexually acquired hepatitis c virus infection: a review global prevalence of pre-existing hcv variants resistant to direct-acting antiviral agents (daas): mining the genbank hcv genome data cluspro: a fully automated algorithm for protein-protein docking highly divergent hepaciviruses from african cattle distinct patterns of natural selection in the reverse transcriptase gene of hiv-1 in the presence and absence of antiretroviral therapy codontest: modeling amino acid substitution preferences in coding sequences the impact of hepatitis c virus entry on viral tropism infectious diseased and human population history structural basis for cytosolic double-stranded rna surveillance by human oligoadenylate synthetase 1 evidence for novel hepaciviruses in rodents mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis c virus glycoprotein e1 identification of the hepatitis c virus e2 glycoprotein binding site on the large extracellular loop of cd81 the performance of the date-randomization test in phylogenetic analyses of time-structured virus data analyses of evolutionary dynamics in viruses are hindered by a time-dependent bias in rate estimates diverse cd81 proteins support hepatitis c virus infection molecular evolution of human coronavirus genomes extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes control of pkr protein kinase by hepatitis c virus nonstructural 5a protein: molecular mechanisms of kinase regulation identification of a novel determinant for membrane association in hepatitis c virus nonstructural protein 4b estimating maximum likelihood phylogenies with phyml new algorithms and methods to estimate maximumlikelihood phylogenies: assessing the performance of phyml 3.0 opls3: a force field providing broad coverage of drug-like small molecules and proteins dual function of cd81 in influenza virus uncoating and budding time-dependent estimates of molecular evolutionary rates: evidence and causes coastal connectivity: long-term trading networks across the south china sea phylogeny and molecular evolution of the hepatitis c virus characterization of a canine homolog of hepatitis c virus identification of rodent homologs of hepatitis c virus and pegiviruses not so different after all: a comparison of methods for detecting amino acid sites under selection automated phylogenetic detection of recombination using a genetic algorithm the cluspro web server for protein-protein docking determinants for membrane association of the hepatitis c virus ns2 protease domain identification of conserved residues in hepatitis c virus envelope glycoprotein e2 that modulate virus dependence on cd81 and srb1 entry factors sailing sinbad's seas origin of hepatitis c virus genotype 3 in africa as estimated through an evolutionary analysis of the full-length genomes of nine subtypes, including the newly sequenced 3d and 3e mutagenesis of the fusion peptide-like domain of hepatitis c virus e1 glycoprotein: involvement in cell fusion and virus entry the ins and outs of hepatitis c virus entry and assembly the evolution and genetics of virus host shifts hepatitis c virus drug resistance-associated substitutions: state of the art summary an algorithm for progressive multiple alignment of sequences with insertions full-length genomes of 16 hepatitis c virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes alexander the great, seafaring, and the spread of leprosy rdp: detection of recombination amongst aligned sequences detecting and analyzing genetic recombination using rdp4 global distribution and prevalence of hepatitis c virus genotypes global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence the effect of genetic structure on molecular dating and tests for temporal signal gene-wide identification of episodic selection detecting individual sites subject to episodic diversifying selection a likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome tracing the peopling of the world through genomics ifn-lambda4: the paradoxical new member of the interferon lambda family the earliest horse harnessing and milking comparative analysis of variation and selection in the hcv genome comprehensive screening for naturally occurring hepatitis c virus resistance to direct-acting antivirals in the ns3, ns5a, and ns5b genes in worldwide isolates of viral genotypes 1 to 6 protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e2 through the eukaryotic initiation factor 2alpha kinase perk trade, circulation, and flow in the indian ocean world interferon lambda 4 variant rs12979860 is not associated with rav ns5a y93h in hepatitis c virus genotype 3a interferon lambda 4 genotypes and resistance-associated variants in patients infected with hepatitis c virus genotypes 1 and 3 response of hepatitis c virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype natural reservoirs for homologs of hepatitis c virus regulation of pkr and irf-1 during hepatitis c virus rna replication evaluation of methods for detecting recombination from dna sequences: computer simulations improving the performance of positive selection inference by filtering unreliable alignment regions the epidemic behavior of the hepatitis c virus virology: the virus whose family expanded investigating the endemic transmission of the hepatitis c virus hepacivirus cross-species transmission and the origins of the hepatitis c virus bats are a major natural reservoir for hepaciviruses and pegiviruses new opportunities in anti-hepatitis c virus drug discovery: targeting ns4b donovanosis (granuloma inguinale) mutational analysis of the hepatitis c virus e1 glycoprotein in retroviral pseudoparticles and cell-culture-derived h77/jfh1 chimeric infectious virus particles host sphingolipid biosynthesis as a target for hepatitis c virus therapy disease transmission in horses r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock statistical tests for detecting gene conversion high diversity of west african bat malaria parasites and a tight link with rodent plasmodium taxa surveying the global virome: identification and characterization of hcv-related animal hepaciviruses patchdock and symmdock: servers for rigid and symmetric docking guidance2: accurate detection of unreliable alignment regions accounting for the uncertainty of multiple parameters global epidemiology of hepatitis c virus infection novel procedure for modeling ligand/receptor induced fit effects hepatocyte cd81 is required for plasmodium falciparum and plasmodium yoelii sporozoite infectivity the origin of hepatitis c virus evolutionary insights into host-pathogen interactions from mammalian sequence data expanded classification of hepatitis c virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource the origin of hepatitis c virus genotypes less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection viral evolution and interferon resistance of hepatitis c virus rna replication in a cell culture model hepatitis c virus ns5a protein interacts with 2' ,5'-oligoadenylate synthetase and inhibits antiviral activity of ifn in an ifn sensitivitydetermining region-independent manner hepatitis c virus ns4b targets lipid droplets through hydrophobic residues in the amphipathic helices inhibition of the interferon-inducible protein kinase pkr by hcv e2 protein structure of the zincbinding domain of an essential component of the hepatitis c virus replicase detection and characterization of homologues of human hepatitis viruses and pegiviruses in rodents and bats in vietnam differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys sphingomyelin activates hepatitis c virus rna polymerase in a genotypespecific manner revtrans: multiple alignment of coding dna from aligned amino acid sequences a case for the ancient origin of coronaviruses purifying selection can obscure the ancient age of viral lineages evolutionary origins of human herpes simplex viruses 1 and 2 manifestations and management of lymphogranuloma venereum synthesis of a novel hepatitis c virus protein by ribosomal frameshift hepatocyte permissiveness to plasmodium infection is conveyed by a short and structurally conserved region of the cd81 large extracellular domain paml 4: phylogenetic analysis by maximum likelihood bayes empirical bayes inference of amino acid sites under positive selection evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level a library of infectious hepatitis c viruses with engineered mutations in the e2 gene reveals growth-adaptive mutations that modulate interactions with scavenger receptor class b type i key: cord-332422-s15o0hie authors: belouzard, sandrine; cocquerel, laurence; dubuisson, jean title: hepatitis c virus entry into the hepatocyte date: 2011-11-07 journal: cent eur j biol doi: 10.2478/s11535-011-0076-y sha: doc_id: 332422 cord_uid: s15o0hie hepatitis c virus (hcv) is a small enveloped virus with a positive stranded rna genome belonging to the flaviviridae family. the virion has the unique ability of forming a complex with lipoproteins, which is known as the lipoviroparticle. lipoprotein components as well as the envelope proteins, e1 and e2, play a key role in virus entry into the hepatocyte. hcv entry is a complex multistep process involving sequential interactions with several cell surface proteins. the virus relies on glycosaminoglycans and possibly the low-density lipoprotein receptors to attach to cells. furthermore, four specific entry factors are involved in the following steps which lead to virus internalization and fusion in early endosomes. these molecules are the scavenger receptor srb1, tetraspanin cd81 and two tight junction proteins, claudin-1 and occludin. although they are essential to hcv entry, the precise role of these molecules is not completely understood. finally, hepatocytes are highly polarized cells and which likely affects the entry process. our current knowledge on hcv entry is summarized in this review. hepatitis c is a global health problem. hepatitis c virus (hcv) infects approximately 130 millions individuals worldwide with the majority remaining undiagnosed and untreated. in most infected individuals, the virus evades the immune system and establishes a chronic infection. as a consequence, hepatitis c is the leading cause of cirrhosis, end-stage liver disease, hepatocellular carcinoma and liver transplantation. to this day, there is no vaccine and the available treatment, a combination of pegylated α-interferon and ribavirin has a limited efficacy and significant side effects. however, specific direct-acting antivirals such as protease and polymerase inhibitors are under development and two ns3/4a protease inhibitors have already been approved for use in the usa. hcv belongs to the genus hepacivirus in the flaviviridae family. it is a small enveloped virus with a positive sense single stranded rna genome of 9.6 kb. the genome is translated as a polyprotein of ~3000 amino acids, which is processed during translation by cellular and viral proteases to generate the structural (capsid, e1 and e2) and non-structural proteins (p7, ns2, ns3, ns4a, ns4b, ns5a and ns5b) [1] . the structural proteins are components of the viral particle which has the peculiar feature of being associated with low-or very low-density lipoproteins (ldl or vldl) [2] . for many years, the study of the hcv life cycle has been impaired by the inability to efficiently grow hcv in cell culture. however, in recent years, several major advances have facilitated research on hcv. among them, retroviral particles pseudotyped with hcv envelope glycoproteins, e1 and e2, (hcvpp) were the first robust in vitro assay for the functional study of hcv entry [3] . following that, a major breakthrough in hcv research was the development of a cell culture system that allowed for the production of infectious particles (hcvcc) [4] . hepatocytes are major target cells of hcv infection. the liver is a complex organ, containing at least a dozen different cell types. however, 70 to 80% of the liver cell population is composed of hepatocytes that are responsible for the major liver functions such as protein synthesis, lipid and carbohydrate metabolism, bile synthesis/secretion and detoxification. to function in this capacity, hepatocytes express specific genes and their functionality requires polarization with separated apical and basolateral membrane domains, which differ in their protein and lipidic composition. it is therefore very likely that the specific architecture of the liver plays a major role in hcv propagation within this organ. the viral entry process relies on a fine interplay between the virion and the host cell. infection is initiated by the interaction of the viral particle with specific proteins on the cell surface. these proteins belong to two general categories which are classified based on the functional consequences of their interaction: attachment factors and receptors. attachment factors serve to bind and concentrate particles at the cell surface. usually, these interactions have a rather low specificity. on the other hand, receptors actively promote virus entry by inducing conformational changes of the viral glycoproteins and/or by activating signaling pathways necessary for internalization of the virion. currently, hcv entry is viewed as a complex multistep process as at least four specific cellular factors have been shown to be essential for hcv entry. these molecules are the scavenger receptor class b type i (srb1), the tetraspanin cd81 and tight junction proteins, claudin-1 (cldn1) and occludin (ocln) (reviewed in [5] ). in this review, we will first address what we know of the composition of the virion that may be of significance for viral entry, then the host entry factors required for hcv entry and we will summarize current knowledge of the mechanisms of hcv entry. the hcv rna genome, core and envelope glycoproteins, e1 and e2, are the known viral components of the virion. the hcv genome interacts with the core to form the nucleocapsid that is surrounded by a host derived lipid membrane, called the viral envelope, in which are anchored the envelope glycoproteins. hcv envelope glycoproteins are key determinants of hcv entry with a role in receptor binding and in mediating the fusion process between the viral envelope and an endosomal host cell membrane. their biogenesis has been extensively characterized in recombinant systems [6] . e1 and e2 are heavily n-glycosylated type i transmembrane proteins which form a noncovalent heterodimer. however, it has recently been shown that hcv envelope glycoproteins form large covalent complexes stabilized by disulfide bonds on secreted viral particles [7] . the presence of disulfide bridges between hcv envelope glycoproteins suggests that lateral protein-protein interactions assisted by disulfidebond formation might play an active role during the budding process of hcv particles. furthermore, functional non-infectious and capsidless structures, called subviral particles, can be produced when the hcv envelope glycoproteins are expressed alone in lipoprotein producing cell lines [8] , supporting the idea that hcv envelope glycoproteins play an active role during the budding process. the secondary and tertiary structures of envelope glycoproteins are proposed to be similar among the members of the flaviviridae family, suggesting that hcv envelope glycoproteins belong to class ii fusion proteins [9, 10] . recently, the identification of 9 intramolecular disulfide bonds in soluble e2 has shed light on its structure [11] . by compiling these results with various data relevant to e2 structure and functionality, krey et al. proposed a model for the tertiary organization of e2, which is consistent with the structure of class ii fusion protein ( figure 1 ). this model revealed the distribution of e2 amino acids among three different domains (di, dii and diii). the di domain consists of eight b-strands and is extended on the n-terminus by hypervariable region 1 (hvr1). this domain, which contains determinants for cd81 interaction, has recently been functionally confirmed [10] . the dii domain includes hypervariable region 2 (hvr2) and its most conserved part is suggested to act as a fusion loop (a.a. 502-520) that inserts into the target membrane during the first step of membrane fusion. di is connected to the diii domain by a linker region called inter-genotypic variable region (igvr). finally, diii is connected to the transmembrane domain by the flexible stem region. this latter region is potentially involved in envelope protein heterodimerization as well as in virus entry [10, 12] . an amphipathic helix, which likely folds upon membrane binding has been identified in the c-terminal part of the stem region and it has been proposed that this region might be involved in the reorganization of glycoprotein complexes taking place during the fusion process [10] . among the most variable regions, hvr1 has been shown to be essential for the interaction of hcv glycoprotein e2 with the known hcv receptor srb1 [13] . although they have an attenuated phenotype, most viruses lacking hvr1 remain infectious [14] . furthermore, it has been proposed that this region obstructs the viral cd81 binding site as well as conserved neutralizing epitopes [14, 15] , potentially playing a role in some aspects of hcv immune evasion. surprisingly, other non-conserved regions like hvr2 and igvr are essential for the assembly of functional e1e2 heterodimers [10, 16, 17] . hcv envelope glycoproteins, e1 and e2, contain up to 5 and 11 n-glycosylation sites, respectively. most hcv glycans are highly conserved [16] and some have (tm) are indicated at the c-terminus of the proteins and the position of the glycosylation sites shown in the ectodomains. for e2 glycoprotein, hypervariable region 1 (hvr1) and 2 (hvr2) and intergenotypic variable region (igvr) are indicated. furthermore di, dii and diii domains and the stem regions are shown in red, yellow, blue and grey, respectively. (b) model of e2 glycoprotein. the linear sequence of the e2 ectodomain (jfh1 strain; genbank access number ab237837) is represented as a chain of beads (colored circles) labeled with the corresponding amino acid and threaded onto a class ii fold, which is an adapted version of the model recently published by krey et al. [11] (doi: 10.1371/journal.ppat.1000762.g006). the three putative domains are presented in red (di), yellow (dii) and blue (diii), and the variable regions (hvr1, hvr2 and igvr) are indicated in brown, whereas the stem region is shown in grey. circles in pale and bright colors represent residues in the background and foreground of the domains, respectively labeled in white and black fonts. disulfide bonds are indicated by black bars. glycosylation sites are shown by green circles numbered sequentially. di domain residues that participate in cd81 binding are contoured in blue. been shown to play a role in virus assembly or entry [18] . furthermore, they form a glycan shield which protects the virus from antibody neutralization [18] . hcv virions isolated from patients display a broad range of buoyant densities and are associated with host lipoproteins and antibodies. virions and lipoproteins are believed to form complexes of very-low to low densities, called lipoviroparticles [19] . the exact nature of hcv association with lipoproteins remains elusive, yet it has been clearly demonstrated that infectivity is correlated with virus density with the highest infectivity being found in the lowest density fractions. a recent characterization of cell culture-produced particles indicates that their composition resembles the one of vldl and ldl with cholesteryl esters accounting for almost half of the total hcv lipids. thus, hcv particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far [20] . importantly, changes in the nature of hcv-associated lipoproteins by lipoprotein lipase affect hcv infectivity, suggesting that association of hcv with specific lipoproteins is important for infectivity [21, 22] . it has also been shown that the hcv assembly pathway depends on components of the vldl synthesis pathway. the contribution of different vldl pathway components is still under debate. some groups have found apob and microsomal triglyceride transfer protein as major contributors while others indicate that apoe plays a more predominant role. some explanations and models to accommodate the apparent conflicting data have recently been proposed [2] . apoe is a ligand for two molecules that are involved in hcv infection: the low-density lipoprotein receptor (ldlr) and srb1. antibodies directed against apoe inhibit hcv infection [23, 24] , thus supporting a role for apoe in hcv entry. furthermore, the apoe isoform associated with hcv particles has been shown to affect the level of virus infectivity [24] . the apoe gene is indeed polymorphic with different alleles leading to the production of 3 different isoforms, apoe2, apoe3 and apoe4. these isoforms show different affinities for ldlr, with apoe2 having a very low affinity for this receptor. infectivity of virions produced in apoe knockdown cells could be properly restored by apoe3 and apoe4 expression but only very poorly by apoe2 [24] . among other apolipoproteins, apoc1 is another factor that could also potentially play a role in hcv entry [25] . srb1 is a major receptor for high-density lipoproteins (hdl). in addition, other ligands for srb1 have been reported to include native and modified lipoproteins and modified serum proteins. this receptor plays a crucial role in selective lipid uptake and bidirectional transfer of free cholesterol. srb1 contains two membrane spanning sequences flanking a large n-glycosylated extracellular loop and two short intracellular domains ( figure 2 ). the role of srb1 in hcv entry was first suggested by its ability to mediate soluble e2 binding [13] and it was later confirmed by the inhibition of hcv infection with anti-srb1 antibodies and by silencing of the protein in hepatoma cells (reviewed in [5, 26, 27] ). srb1 residues potentially involved in e2 glycoprotein binding have recently been identified in its ectodomain [28] . indeed, amino acids 70-87 and the residue e210 are required for e2 recognition. alternative splicing of the transcript leads to the production of srb2 which differs from srb1 only in its c-terminal cytosolic domain. the srb2 c-terminal domain is reported to mediate rapid internalization and intracellular localization of the protein. the c-terminus of srb1 has recently been shown to play a role in hcv entry [29] . deletion of this domain or introduction of the endocytosis motif of srb2 into srb1 impairs hcv entry. srb1 is known to interact with the cytoplasmic adaptor molecule pdzk1 via an akl motif in its c-terminal cytosolic tail. this interaction is involved in srb1 stability in hepatocytes and it has recently been shown that pdzk1 plays an indirect role in hcv entry via its ability to interact with srb1, which leads to an enhancement of its activity as an hcv entry factor [30] . some srb1 ligands are known to affect hcv infection (reviewed in [5] ), notably hdl enhances infection by accelerating viral entry. this mechanism depends not only on the lipid transfer activity of srb1, but also on the presence of cd81 [31] , suggesting that an interplay between srb1 and cd81 occurs during hcv entry. it is also possible that lipid transfer by srb1 changes the membrane lipid composition, which in turn may affect hcv infection. in addition to the direct binding of soluble e2 to srb1, it has also been suggested that the virus can bind srb1 via its lipoprotein component [32] . together with the observation that it interacts with hcv glycoprotein e2, the role of srb1 in lipid metabolism has suggested that this entry factor may play a dual role during the early steps of the hcv life cycle [28, 31] . more recently, the role of srb1 in hcv uptake has been confirmed in vivo in a genetically humanized mouse model for hcv infection [33] . cd81 is a member of the tetraspanin family. like the other members of this family, it contains 4 transmembrane domains, a small (sel) and a large extracellular loop (lel) (figure 2 ). tetraspanins are involved in many cell functions such as adhesion, morphology, proliferation and differentiation. they are enriched in specific membrane microdomains called tetraspanin-enriched microdomains (tems), which differ from conventional raft domains, but the organization of which is dependent on cholesterol. tetraspanins interact with partner proteins to form primary complexes which can interact together to form a network of interactions within tems. the role of cd81 in hcv entry was first suggested after the demonstration that it interacts with a soluble form . the virus is internalized by clathrin-mediated endocytosis and fuses in early endosomes delivering its nucleocapsid into the cytosol. the genome of the virus is then released and translated to produce structural and non-structural proteins. the non-structural proteins are involved in viral genome replication. the mechanism of assembly is not completely understood but it is closely linked to endoplasmic reticulum (er) and lipid droplets (ld). the lipoprotein-associated virus is then released in the surrounding medium to infect new cells. the virus can also be transferred directly to the neighboring cells by cell-to-cell transmission (green arrow). bc, bile canaliculi. of e2 [34] . subsequently, it was extensively confirmed in hcvpp, hcvcc, hcv isolated from patient and in a humanized mouse model (reviewed in [5] ). the cd81 binding site for e2 has been localized within the lel [34] and characterization of chimeric proteins between cd81 and cd9, a related tetraspanin, confirmed that the lel sequence is the region of cd81 defining hcv entry [35] , with the binding site for e2 having been mapped to the variable double-helix subdomain [36] . several studies have also shown that cell susceptibility to hcv infection is closely related to cd81 expression level [37] [38] [39] . since tetraspanin molecules are known to interact with other membrane proteins, named partners, the involvement of cd81 partners in the modulation of hcv entry has been investigated. indeed, the generation of a human cell line expressing murine cd81 (mcd81) permissive to hcv infection has allowed for the analysis of the role of tem-associated cd81 in hcv infection [40] . the use of the mt81w antibody, which only recognizes temassociated mcd81 [41] and treatments with methyl-bcyclo-dextrin or sphingomyelinase showed that temassociated cd81 is not preferentially used by hcv for its entry [40] . hcv entry is a multisequential process that is highly dynamic. cd81 association with tem may lead to the sequestration of this tetraspanin which might impede its interaction with other coreceptors that are necessary for efficient hcv entry. alternatively, tetraspanins may compete with coreceptors for their interaction with cd81. in addition, the lipid composition of tems might not be suitable for hcv entry. a study of cd81-associated proteins led to the identification of ewi-2wint, a partner of cd81 that blocks e2 binding to cd81 [42] . ewi-2wint is a natural inhibitor that is produced following the cleavage of ewi-2, a major partner of cd81 that is expressed in most cells. ewi-2wint is notably absent in hepatocytes, enabling the binding of e2 to cd81 and allowing subsequent infection. in addition to the presence of specific entry factors, hepatotropism might be partly due to the absence of ewi-2wint. the regions involved in the interaction between ewi-2/ewi-2wint and cd81 have recently been identified, and it has been shown that ewi-2wint mutants, which no longer interact with cd81, are not able to exert their inhibitory effect on hcv infection [43] . tight junctions (tjs) are membrane components specialized in sealing cells. they have the double function of controlling paracellular diffusion (barrier function) and restricting the diffusion of membrane proteins and lipids of the pole (fence function) [44] . in electron microscopy, tjs form a dense structure characterized by a close apposition of membranes of neighboring cells and a cytosolic plaque. indeed, these structures are composed of transmembrane proteins and intracellular molecules. transmembrane proteins play an important role in sealing the cells by interacting with similar molecules either in cis (on the membrane of the same cell) or in trans (on the membrane of facing cells). tj functionality and architecture are regulated by signaling pathways that rely on the tj cytosolic plaque. transmembrane proteins interact with adapters of the cytosolic plaque that links the surface proteins to the actin cytoskeleton [44] . cldn1 and ocln are two transmembrane components of the tjs. cldn1 is one of the twenty four members of the cldn family [45] and evidence suggests that cldns constitute the backbone of tight junctions. these proteins consist of four transmembrane domains, two extracellular loops and two cytosolic domains. the c-terminus contains a pdz-binding motif which binds to pdz-containing plaque proteins such as the zonula occludens (zo) proteins ( figure 2 ). the first extracellular loop (ec1) contains a conserved glwxxc(8-10aa)c motif. ocln was the first integral tj protein to be identified and is a protein composed of four transmembrane domains, two extracellular loops, a short n-terminal cytoplasmic domain and a long c-terminal one ( figure 2 ). the first extracellular loop is rich (~60%) in glycine and tyrosine residues. the function of ocln is not well understood but it may participate in the assembly and barrier function of tjs [45] . recently, cldn1 and ocln have been identified as two essential entry factors for hcv [27, 46, 47] . cldn1 was the first to be identified in a complementary dna library screen for genes that confer hcvpp susceptibility to 293t cells [46] . antibodies directed against cldn1 were also shown to inhibit hcv entry [48] . two other members of the cldn family, cldn6 and cldn9, are also able to render 293t cells permissive for hcv entry [49, 50] . the first extracellular loop of cldn1 has been shown to be involved in hcv entry [46, 51] , and residues important for hcv infection have been identified in this domain ( figure 2) . indeed, mutations of the residues i32, d38 and e48 as well as in the glwxxc(8-10aa)c conserved motif affect hcv entry [46, 51] . furthermore, it has been suggested that the second extracellular loop may also modulate cldn1 function in hcv entry. the low efficacy of mouse cldn1 in mediating hcv infection is linked to three amino acid residues in the second extracellular loop (l152, i155) and the fourth transmembrane helix (v180) [52] . in contrast, the c-terminal domain of cldn1 is dispensable for hcv entry [46] . ocln was the last entry factor to be identified, and is probably involved in a late post-binding step [47, 53, 54] . by using mouse and human ocln chimeras, the species determinant for hcv entry was mapped to the second extracellular loop [55] (figure 2) . furthermore, alaninescanning mutagenesis of the two ocln extracellular loops has revealed that the second part of the second loop is required for hcv entry. this region is flanked by two cysteines for which the mutation abolishes hcv infection, suggesting that this region might be folded by a disulfide bond. ocln expression on hepatocytes as well as hcv entry is increased upon glucocorticoid treatment [56] . in contrast, ocln expression is downregulated upon hcv infection probably to prevent super-infection [54, 57] . in the liver, hepatocytes are organized in plates of single cell layers that are separated by sinusoid vessels. endothelial cells and hepatocytes are separated by the space of disse which drains lymph into the portal tract lymphatics (figure 2 ). basolateral domains of hepatocytes face the space of disse whereas apical domains form tightly closed spaces called the canaliculi in which the bile is secreted. tjs act as a barrier between the two spaces and maintain the separation between both membrane domains maintaining their distinct molecular compositions. cell polarity is achieved by a complex regulation of intracellular trafficking to deliver newly synthesized proteins to the suitable pole. it is believed that hcv enters the liver through blood flow and makes contact with hepatocytes at the basolateral membrane. in order to infect cells and propagate, the virus needs to overcome the complex organization of hepatocytes. the mechanisms leading to hcv entry into hepatocytes are still poorly understood. to date, accumulated data indicate that hcv entry is a complex multistep process involving numerous components as well as the constraints of hepatocyte polarity. in vivo, hcv enters the liver through the sinusoidal blood. before gaining access to the hepatocyte, this virus first needs to cross the sinusoidal endothelial cell barrier. c-type lectins have been proposed to play a role in assisting the virus cross the liver endothelium (reviewed in [5] ), however, this remains to be demonstrated. hcv attachment to hepatocytes is likely first mediated by heparan sulfate proteoglycans (reviewed in [58] ). due to the association of the virion with lipoproteins, the ldlr has also been proposed to play a role in the early phase of hcv entry (reviewed in [5] ). ldlr mediates internalization of low density lipoproteins that are released from the receptor upon ph decrease in endosomes. it has been shown that hcv infection is correlated with ldlr expression in hepatocytes and can be inhibited by ldlr ligands. it is therefore possible that the lipoviroparticles interact with ldlr through their apob and/or apoe components, and that these interactions are likely modulated by the processing of lvp by lpl. although recent data are in favor of the involvement of ldlr in the hcv life cycle [59, 60] , the role of this receptor in hcv entry remains unclear. after binding to the cell surface with the help of heparan sulfate proteoglycans, the virus then interacts with specific entry factors. there is evidence that the e2 glycoprotein can interact with srb1 and cd81. recently, it has been proposed that hcv virions first interact with srb1 [28] . this is in line with the observation that hcv virions can bind to cho cells expressing srb1, but not to cells expressing cd81 [46] . however, it remains to be determined whether srb1 and cd81 form a co-receptor complex or whether the virus is transferred from srb1 to cd81. currently, there is no evidence indicating that hcv particles directly interact with cldn1 or ocln. however, it has been shown that cldn1 and cd81 interact in fluorescence resonance energy transfer (fret) and fluorescence intensity ratio (fir) assays [61, 62] , suggesting that cldn1 may be a partner of cd81 in a hcv receptor complex. concordant with this hypothesis, mutation of residues 32 and 48 in the first extracellular loop of cldn1 that prevent hcv entry also abolish cldn1-cd81 interaction [62] . furthermore, antibodies directed against cldn1 or cd81, inhibit hcv entry by perturbing cldn1-cd81 interaction [62, 63] . recently, it has also been shown that epidermal growth factor receptor and ephrin receptor a2 act as host cofactors for hcv entry by regulating cldn1-cd81 co-receptor association [64] . analysis of cldn1-cd81 interaction by surface plasmon resonance demonstrated a specific interaction between cldn1 ec1 and cd81 lel [62] . finally, in polarized hepg2 cells, cd81 and cldn1 association occurs at the basolateral domain of the cells in accordance with the virus coming in contact with the hepatocyte at the sinusoidal surface of the cells [62] . although cldn1 also interacts with ocln, there is no clear relationship between cldn1-ocln association and hcv infectivity [62] . after initial binding to an attachment factor and prior uptake, viruses move laterally on the cell surface. these movements allow viruses to interact with secondary co-receptor(s) or with clathrin-coated pits. single particle imaging during viral entry in huh7.5 cells has revealed that hcv particles bind cells on filopodia and reach the cell body by a mechanism that relies on retrograde actin transport [65] . on the cell surface, the virion interacts with a receptor complex and/or co-receptors and associates with clathrin-coated pits. it has been proposed that cd81 may play a role in the transport of the virions from the basolateral face of the cells to the region of cell-cell contact and presumably the tjs where the virus could then interact with cldn1 and ocln [66] . this hypothesis is based on the observation that a soluble form of e2 glycoprotein is able to induce cd81 relocation in huh-7 cells to the cell-cell contact areas where it colocalizes with cldn1 and ocln. this transport step involves actin cytoskeleton remodeling and rho-gtpase activity. therefore, not only does cd81 mediate virus binding, but it also triggers activation of signaling pathway(s) likely required during the virus life cycle. it has been shown that hcv undergoes clathrinmediated endocytosis and fuses in early endosomes [67, 68] (figure 2 ). hcv internalization into cells is a slow process. the half-maximal internalization of hcvpp is achieved in approximately one hour. a sirna library screen led to the identification of host cofactors involved in hcv entry [65] . these proteins required for hcv infection are involved in clathrin-mediated endocytosis as well as in actin cytoskeleton dynamics or endosomal trafficking and acidification. in single particle tracking experiments, after surfing on the cell surface, virions colocalized with clathrin and the e3 ubiquitin ligase, c-cbl [65] . the exact role of c-cbl in hcv entry remains to be elucidated yet a role of this protein in clathrin-mediated endocytosis has already been shown for the bacteria listeria monocytogenes [69] . after internalization, the virus has been shown to be transported to gfp-rab5a positive early endosomes along actin stress fibers [65] . the role of microtubules in hcv infection has also been investigated [39] using pharmacological agents that are known to affect microtubules such as vinblastin, nocodazol or paclitaxel inhibit hcv infection. these molecules affect hcv entry as well as an early post-fusion step. generally, virus endocytosis is mediated by receptor(s). however, to date, the exact role of hcv co-receptors in endocytosis of the virus remains undetermined. due to the lack of a robust polarized hepatocyte in vitro model that supports hcv replication, the role of tj formation in hcv entry remains unclear. in vivo, ocln and cldn1 are concentrated in tjs, whereas cldn is also detected at the basolateral face in polarized hepg2 cells or in liver tissue. though the virus is probably internalized in the tj area in vivo, formation of tjs is dispensable for hcv entry in cell culture. some data suggest that the non-junctional pool of cldn1 is involved in hcv entry [70, 71] , whereas others have shown that cldn1 localization at cell-cell contacts is required for hcv infection. in addition, it has been shown that, in non-polarized huh7.5 cells, cell density and formation of cell-cell contacts modulate receptor expression and hcv infection [72] . more precisely, srb1 and cldn1 expression is increased in confluent cells in which they are concentrated at cell-cell contacts whereas cd81 and ocln expression levels remain unaffected by cell confluence. interestingly, infection increased in confluent cells compared to subconfluent cells, and cell-cell contacts promoted virus internalization. the relevance of cell polarity to hcv entry has only been addressed indirectly through the use of the hcvpp system. in hepg2-cd81 cells, the acquisition of polarity correlates with a reduction in hcvpp entry. hcv entry has also been studied in polarized caco-2 cells [73] . these are colorectal adenocarcinoma cells that form polarized monolayers of cells displaying a columnar morphology with an apical domain and a basolateral domain. caco-2 cells are poorly permissive to hcvpp and infection occurs at the apical surface of the cell, suggesting that tjs provide a barrier to virus diffusion. in these cells, receptor expression increased upon cell polarization and polarization induced relocalization of cldn1 from the apical domain to the tjs and the basal domain. unpolarized caco-2 cells or polarized cells are infected at similar levels, suggesting that the cldn1 receptor function is independent of tj formation. these results suggest that establishment of cell-cell contacts and polarity may be of significance in hcv entry. as a consequence, a more suitable model of polarized hepatoma cell line is much needed to study hcv entry. such a model will also be useful to understand virus spread in the liver. based on observations of hcv spread in cell culture in the presence of neutralizing antibodies, a mechanism of direct cell-to-cell hcv transfer has been proposed [74] [75] [76] [77] . the role of the different hcv co-receptors in this transfer has been investigated. both tj proteins, cldn1 and ocln, are required. however, the role of cd81 in this mechanism is more controversial. it was first proposed that hcv cell-to-cell transmission occurs independently of cd81 [74] [75] [76] . antibodies directed against cd81 are unable to inhibit hcv cellto-cell transfer, and transfer to cells expressing almost undetectable levels of cd81 was observed. in addition, in a model of upa(+/+)scid mice with humanized liver [78] , meuleman et al. showed that prophylactic treatment with anti-cd81 antibodies prevented infection, but postinfection treatment had no effect. these results suggest that virus spread may be cd81-independent. however, these data were challenged by other studies in which cell-to-cell transmission was abolished in cells lacking cd81 or treated with anti-cd81 antibodies [77, 79] . compared to cell-free infection, the role of srb1 in cellto-cell transmission is likely more important. indeed, the neutralizing effect of anti-srb1 antibodies and specific inhibitors was more pronounced on this infection route. in addition, the mutant jfh-1g451r, which displays less dependency on srb1, demonstrated a reduced cell-to-cell transmission. in the early endosome, fusion between the viral envelope and a host membrane occurs, thus allowing the nucleocapsid to be released into the cytosol. the mechanism of hcv fusion is poorly understood. fusion of the viral envelope with a host cell membrane is driven by the viral fusion protein, which undergoes major conformational changes [9] . fusion can be triggered by different mechanisms alone or in combinations of the following: receptor binding, low ph exposure and proteolytic processing. for hcv, it is believed that e2 is the protein mediating fusion. hcvpp entry is inhibited by neutralization of endosomal ph, suggesting that low ph exposure is required for hcv fusion [67, 68, 80] . by using an in vitro fusion assay between virion and fluorescently labeled liposomes, haid et al. have reported that fusion is triggered by low ph and is dependent on virus buoyant density and the lipid composition of liposomes [80] . it has been shown that e1 and e2 on the viral surface are linked by intermolecular disulfide bridges [7] . these large oligomers may form a rigid protein lattice which may have consequences for the fusion process. class ii fusion proteins undergo an oligomeric rearrangement during the fusion process, which leads to the conversion into trimers. it is likely that disulfide rearrangements are required to prime hcv fusion process. critical reshuffling of disulfide bonds has already been shown to occur for other viruses such as sindbis virus and hiv [81, 82] . for hiv, it has been proposed that protein disulfide isomerase (pdi) can act at a post-binding step to reduce two disulfide bonds in gp120 which consequently induces fusion competence. further investigations on the potential role of pdi and the disulfide-bond rearrangement of hcv envelope proteins are needed to further understand the fusion process. with the development of a cell culture system to propagate the virus, considerable progress has been made in understanding the hcv life cycle. the data accumulated on this virus indicate that hcv enters into host cells in a tightly regulated and complex multistep process involving the presence of several entry factors. another peculiarity of hcv is the hybrid nature of the lipoviroparticle. the recent progress on hcv entry has highlighted the contribution of both hcv envelope glycoproteins and lipoprotein moieties. virus-associated lipoproteins are likely playing a role in the early phase of hcv entry, whereas envelope glycoproteins are believed to take the lead after this initial step. however, the details of successive events occuring during hcv entry remain to be revealed. the recent structural model of e2 glycoproteins provides a new framework for a better understanding of the structure-function relationship of hcv envelope glycoproteins, which will lead to a better mechanistic understanding of the entry process. in spite of the major progress in the field, it is still unclear how the virus overcomes the compact and highly organized environment of liver and the role of tj formation in hcv infection remains to be elucidated. replication of hepatitis c virus assembly of infectious hepatitis c virus particles studying hcv cell entry with hcv pseudoparticles (hcvpp) hepatitis c virus molecular clones: from cdna to infectious virus particles in cell culture recent advances in hepatitis c virus entry assembly of a functional hcv glycoprotein heterodimer characterization of the envelope glycoproteins associated with infectious hepatitis c virus morphological characterization and fusion properties of triglyceride-rich lipoproteins obtained from cells transduced with hepatitis c virus glycoproteins virus membrane-fusion proteins: more than one way to make a hairpin identification of new functional regions in hepatitis c virus envelope glycoprotein e2 the disulfide bonds in glycoprotein e2 of hepatitis c virus reveal the tertiary organization of the molecule hepatitis c virus glycoprotein e2 contains a membrane-proximal heptad repeat sequence that is essential for e1e2 glycoprotein heterodimerization and viral entry the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus hypervariable region 1 differentially impacts viability of hepatitis c virus strains of genotypes 1 to 6 and impairs virus neutralization hepatitis c virus hypervariable region 1 modulates receptor interactions, conceals the cd81 binding site, and protects conserved neutralizing epitopes the neutralizing activity of anti-hepatitis c virus antibodies is modulated by specific glycans on the e2 envelope protein the variable regions of hepatitis c virus glycoprotein e2 have an essential structural role in glycoprotein assembly and virion infectivity role of n-linked glycans in the functions of hepatitis c virus envelope proteins incorporated into infectious virions hepatitis c virus particles and lipoprotein metabolism biochemical and morphological properties of hepatitis c virus particles and determination of their lipidome lipoprotein lipase mediates hepatitis c virus (hcv) cell entry and inhibits hcv infection lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis c virus (hcv) through their catalytic activities on hcv-associated lipoproteins human apolipoprotein e is required for infectivity and production of hepatitis c virus in cell culture infectivity of hepatitis c virus is influenced by association with apolipoprotein e isoforms apolipoprotein c1 association with hepatitis c virus early steps of the hepatitis c virus life cycle hepatitis c virus entry into host cells role of scavenger receptor class b type i in hepatitis c virus entry: kinetics and molecular determinants receptor complementation and mutagenesis reveal sr-bi as an essential hcv entry factor and functionally imply its intra-and extracellular domains the sr-bi partner pdzk1 facilitates hepatitis c virus entry scavenger receptor class b type i is a key host factor for hepatitis c virus infection required for an entry step closely linked to cd81 the interaction of natural hepatitis c virus with human scavenger receptor sr-bi/ cla1 is mediated by apob-containing lipoproteins a genetically humanized mouse model for hepatitis c virus infection binding of hepatitis c virus to cd81 cd81 is required for hepatitis c virus glycoprotein-mediated viral infection cd81 extracellular domain 3d structure: insight into the tetraspanin superfamily structural motifs cd81 expression is important for the permissiveness of huh7 cell clones for heterogeneous hepatitis c virus infection the level of cd81 cell surface expression is a key determinant for productive entry of hepatitis c virus into host cells initiation of hepatitis c virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein the association of cd81 with tetraspanin-enriched microdomains is not essential for hepatitis c virus entry cholesterol contributes to the organization of tetraspaninenriched microdomains and to cd81-dependent infection by malaria sporozoites the cd81 partner ewi-2wint inhibits hepatitis c virus entry interacting regions of cd81 and two of its partners, ewi-2 and ewi-2wint and their effect on hepatitis c virus infection multifunctional strands in tight junctions molecular basis of the core structure of tight junctions claudin-1 is a hepatitis c virus co-receptor required for a late step in entry human occludin is a hepatitis c virus entry factor required for infection of mouse cells monoclonal anti-claudin 1 antibodies prevent hepatitis c virus infection of primary human hepatocytes claudin-6 and claudin-9 function as additional coreceptors for hepatitis c virus the tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis c virus residues in a highly conserved claudin-1 motif are required for hepatitis c virus entry and mediate the formation of cell-cell contacts mouse-specific residues of claudin-1 limit hepatitis c virus genotype 2a infection in a human hepatocyte cell line the tight junction-associated protein occludin is required for a postbinding step in hepatitis c virus entry and infection tight junction proteins claudin-1 and occludin control hepatitis c virus entry and are downregulated during infection to prevent superinfection speciesspecific regions of occludin required by hepatitis c virus for cell entry glucocorticosteroids increase cell entry by hepatitis c virus hepatitis c virus envelope components alter localization of hepatocyte tight junctionassociated proteins and promote occludin retention in the endoplasmic reticulum hepatitis c virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies the lowdensity lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis c virus apolipoprotein e on hepatitis c virion facilitates infection through interaction with low-density lipoprotein receptor cd81 and claudin 1 coreceptor association: role in hepatitis c virus entry claudin association with cd81 defines hepatitis c virus entry inhibition of hepatitis c virus infection by anti-claudin-1 antibodies is mediated by neutralization of e2-cd81-claudin-1 associations egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy rna interference and single particle tracking analysis of hepatitis c virus endocytosis cd81 is a central regulator of cellular events required for hepatitis c virus infection of human hepatocytes hepatitis c virus entry depends on clathrin-mediated endocytosis hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrincoated vesicles listeria hijacks the clathrindependent endocytic machinery to invade mammalian cells hepatitis c virus receptor expression in normal and diseased liver tissue polarization restricts hepatitis c virus entry into hepg2 hepatoma cells hepatoma cell density promotes claudin-1 and scavenger receptor bi expression and hepatitis c virus internalization effect of cell polarization on hepatitis c virus entry hepatitis c virus cellcell transmission in hepatoma cells in the presence of neutralizing antibodies cd81 is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells real-time imaging of hepatitis c virus infection using a fluorescent cell-based reporter system neutralizing antibody-resistant hepatitis c virus cell-to-cell transmission anti-cd81 antibodies can prevent a hepatitis c virus infection in vivo advantages of a single-cycle production assay to study cell cultureadaptive mutations of hepatitis c virus low phdependent hepatitis c virus membrane fusion depends on e2 integrity, target lipid composition, and density of virus particles sindbis virus membrane fusion is mediated by reduction of glycoprotein disulfide bridges at the cell surface cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus we apologize to all authors whose work could not be cited due to space restrictions. we thank sophana ung key: cord-312080-pu6m4qad authors: he, bing; chen, guomin; zeng, yi title: three-dimensional cell culture models for investigating human viruses date: 2016-10-27 journal: virol sin doi: 10.1007/s12250-016-3889-z sha: doc_id: 312080 cord_uid: pu6m4qad three-dimensional (3d) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. moreover, these models bridge the gap between traditional two-dimensional (2d) monolayer cultures and animal models. 3d culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. in addition, 3d models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. this review summarizes and analyzes recent progress in human virological research with 3d cell culture models. we discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3d culture models. infectious diseases have been major challenges to human health and survival for centuries (morens et al., 2004) . although, over the years, significant progress has been made in the prevention and control of infectious diseases, pathogenic microbes continue to pose an enormous threat to human health (heesterbeek et al., 2015) . the level of human mortality attributed to infection is 13-15 million deaths annually (who, 2013) . in recent years, newly emerging infectious agents have represented a continuing challenge, especially those causing viral diseases, such as h7n9 influenza viruses in 2013 (lam et al., 2013) , the ebola virus outbreak in 2014 (team, 2014; heymann et al., 2015) and the zika virus outbreak in 2016 (lessler et al., 2016) . therefore, studying the mechanisms of viral diseases, antiviral agents and vaccines is critical for responding to viral diseases. our current understanding of a multitude of human viral diseases and antiviral drugs is based largely on traditional 2d cell culture, in which cells are grown on glass substrates or flat plastic, such as petri-dishes, multiwell plates and cell culture flasks baker and chen, 2012; li and cui, 2014) , or on animal model systems. indeed, 2d cell cultures have contributed tremendously to the investigation of infectious disease etiology, the immune mechanisms used to defend against human viruses barrila et al., 2010) , and especially the biochemistry and molecular biology of viral replication (andrei, 2006) . however, 2d monolayer culture has significant restrictions in mimicking the physiological complexity of the in vivo microenvironment that is encountered by pathogens; this model cannot accurately reproduce the natural infection process (barrila et al., 2010) . in addition, due to the lack of suitable cell culture models, the study of the life cycle of fastidious viruses and virus-host interactions has been hampered (lindenbach et al., 2005; sainz et al., 2009a) . furthermore, animal models are costly and their use has ethical issues (page et al., 2013) . a multitude of pathogens are species specific, and the pathogenesis mechanisms cannot be captured by time-consuming animal models (griffith and swartz, 2006) . as an example, sars (severe acute respiratory syndrome) in animal models seldom progressed to lethality, however, it may cause death in people (louz et al., 2013) . to overcome these limitations associated with 2d monolayers and animal models, various types of 3d cell culture models have been developed (edmondson et al., 2014) . 3d cell models bridge the gap between 2d cell cultures and animal models by providing an in vitro cell model system mimicking more accurately the in vivo microenvironment, which contributes to the understanding of both virus-host interaction and the fundamental mechanisms of human viruses, as well as promoting the development of antiviral drugs. cells grown on 3d culture systems can differ considerably in their morphology, viability, signaling control, gene expression, differentiation, proliferation and drug sensitivity in comparison with those grown on flat 2d tissue substrates (friedl et al., 2012; edmondson et al., 2014; antoni et al., 2015) . for example, matrigel-cultured huh7 cells assemble into 3d spheroids, whereas standard 2d-cultured cells form epithelial monolayers (molina-jimenez et al., 2012) . cells cultured in a 3d matrix can adequately reproduce the function of 3d tissues and mimic cell-cell and cellmatrix interactions in vivo. 3d culture systems have been designed to allow investigation of infectious pathogens, such as human viruses, bacteria and parasites. these models have an invaluable role in virology today. this review focuses on the recent progress of human virological research with 3d cell culture models, including human viral growth, replication, proliferation, infection, viral life cycle, virus-host interaction and the development of antiviral drugs. logical science. cells grown in a 3d environment more closely represent normal cellular characteristics and biological function that the traditional 2d monolayer culture cannot provide. for example, cells cultured in a 3d matrix can adequately reproduce the function of 3d tissues and mimic cell-cell and cell-matrix interactions that affect proliferation, differentiation, morphology and a range of cellular functions in vivo, which are lost in conventional 2d conditions (mazzoleni et al., 2009) . the classic polarized patterns of signaling that guide migration in 2d models are not essential for efficient migration in 3d models . almost all cells use lamellipodia to migrate on 2d substrates, however, multiple modes of migration are observed in three dimensions, including lamellipodial, lobopodian, amoeboid migration (madsen and sahai, 2010; and collective migration (friedl and alexander, 2011) . baker and chen (2012) further discuss examples that 3d context can provide insights in adhesion, migration and polarization which cannot be provided by the traditional 2d systems. table 1 shows the differences between 2d and 3d cell cultures. strengths and weaknesses of 3d models 3d cell cultures provide a platform for cell proliferation and differentiation. in addition, some emerging 3d culture models can allow nutrients and metabolites to be transported in and out of the 3d cells (li and cui, 2014) . although current 3d systems provide unique mechanistic insights into cell-microenvironment interaction, most 3d models do not replicate all complex physiological features of real-tissue in vivo. they often lack the normal vasculature, normal transport of small molecules, host immune responses, internal tissue tensions, tissue heterogeneity and fluid flows observed in vivo. in addition, those models cannot precisely replicate biochemical composition, gradients of soluble regulatory factors and other microenvironment factors in vivo (yamada and cukierman, 2007; grinnell and petroll, 2010; baker and chen, 2012; friedl et al., 2012) . moreover, more complex architectures are found in tissues in vivo, such as orthogonally arranged collagen sheets and collagen fibril bundles. however, the most current methods to prepare 3d fibrillar matrices result in matrices in which fibrils lack any particular organization or become aligned uniaxially (grinnell and petroll, 2010) . a brief summary of the strengths and limitations of 3d culture models is presented in table 2 . currently available and typical 3d models are spontaneous cell aggregation, liquid overlay, gyratory rotation and spinner flask spheroid cultures, scaffold-based culture systems, microcarrier beads and the rotary cell culture system (kim, 2005; page et al., 2013) , as well as 3d perfusion cell culture, microfluidic 3d cell culture and 3d cell culture by magnetic levitation (souza et al., 2010; li and cui, 2014; van duinen et al., 2015) . here, we only focus on those 3d models that have been utilized to study human viruses or will be applicable for this in the future. 3d mcs culture has become a valuable tool for mimicking the biological features and functional characteristics of native tissue. mcss are cells that aggregate and undergo the process of self-assembly on an attachment surface or scaffold. during self-assembly, mono-dispersed cells form 3d microtissues (achilli et al., 2012; . the spheroid format is particularly useful in cancer research as it enables quick discovery of morphological changes in transformed cells (antoni et al., 2015) . the most commonly used model is the multicellular tumor spheroids model, which has phenotypic characteristics close to those of human tumor tissues. consequently, it has been applied extensively in reproducing the key elements of malignant tumor behavior (hamilton, 1998) . traditional and newer techniques have been investigated for spheroid production. first, the spinner flask method, which prevents cell attachment to the vessel surface and promotes cell-cell contacts via constant stirring. this method is relatively simple and produces massive spheroids, suitable for high-throughput testing, but has a high shear force and variability in cell size/number (lin and chang, 2008; breslin and o'driscoll, 2013) . second, the hanging drop method depends on gravity forces to form spheroids on inverted substrates (kelm et al., 2003) . this method is inexpensive and suitable for highthroughput testing, and the spheroid size can be con-trolled; however, it is labor intensive and mass production is limited (lin and chang, 2008) . third, liquid overlay culture, which prevents the attachment of cells to tissue culture plates and promotes spheroid formation (achilli et al., 2012) . although this method is easy to set up, rapid and makes screening easy, the size and shape of the spheroids are heterogeneous. fourth, a rotating-wall vessel (rwv) creates a microgravity that supports cells in suspension and promotes cell aggregation into spheroids. this method allows good control of the microenvironment over time (achilli et al., 2012) . in addition, pellet culture 3d scaffolds can also form mcss, especially with micro-fluidics and the magnetic cell levitation method, which were developed in recent years to create new opportunities to form spheroids (kim et al., 2013) . mcs models have given rise to many advances in basic cell science, including understanding tumor invasion and migration, and creating models for toxicology testing and drug discovery (achilli et al., 2012; vinci et al., 2015) . the development of new technologies for analyzing spheroids has led to a rapid increase in their adoption and expansion of their applications. new technologies for analyzing spheroids have led to a rapid increase in their adoption and expansion of their applications. apart from those applications mentioned above, mcs models have also been investigated as basic units for human viruses. although the study of viruses in spheroid models is still limited so far, investigating virus-cancer interactions, the mechanism of viruses causing cancer and evaluating antiviral agents relating to tumors by those models may lead to the future direction in the field of cancer. cells cultured in 3d system can represent a more physiological microenvironment. vinci et al., 2012 as compared with 2d cultures, 3d cell cultures more accurately simulate normal cell morphology, proliferation, migration, cell-cell and cell-ecm interactions. antoni et al., 2015 edmondson et al., 2014 cell culture is flexible, cost effective and controllable, as well as a high-throughput platform. nickerson et al., 2007 several 3d models can monitor and control physiological conditions: temperature, ph, oxygen concentration, metabolites and growth factors. murakami et al., 2008; li and cui., 2014; worthington et al., 2015 limitations references in vivo complex and physiological microenvironment not to be replicated. friedl et al., 2012; van duinen et al., 2015 poor reproducibility for some biomimetic scaffolds. antoni et al., 2015 some available 3d models to be more time and expensive. vinci et al., 2012 quality of imaging interfering with 3d scaffold size, material transparency and microscope depth. antoni et al., 2015 3d cell culture using rwv or radial-flow bioreactor (rfb) the rwv is an optimized suspension culture vessel for forming 3d tissue-like assemblies (tlas). the rwv is based on a rotating cylinder that is completely full with culture medium, the sedimentation of cells in the vessel is counterbalanced by the rotating fluid, creating a constant, gentle fall of cells through the medium under conditions of physiologically relevant fluid shear (hammond and hammond, 2001; nickerson et al., 2004; barrila et al., 2010) . to generate 3d cellular aggregates, cells are first cultured in 2d monolayers. when the cells have grown to an appropriate density, they are removed from the petri dishes or flasks and re-suspended in culture medium, then they are placed within porous ecmcoated microcarrier beads for attachment (barrila et al., 2010) . lastly, the cellular aggregates are harvested and analyzed. traditional cell culture and parts of some 3d models may generate high shear forces, which may injure the cells and block proper tissue-specific differentiation (unsworth and lelkes, 1998), and inadequate nutrient and oxygenation transfer give rise to cell death, posing critical obstacles to establishing functional 3d culture systems. to overcome these problems, the nasa (national aeronautics and space administration) johnson space center developed the rwv (goodwin et al., 1992) , which provides a low fluid-shear environment and minimal turbulence that promotes cell growth and randomized gravitational vectors (goodwin et al., 1993) . the fluid dynamics of rwv bioreactors allow oxygen and nutrients to diffuse across into the cell aggregates and prevent tissue constructs from necrotic cores (goodwin et al., 2015) . studies have shown that the rwv can produce 3d models and can recreate many of the fundamental facets of the real tissue in vivo, such as 3d cellular polarity, cellular differentiation and proliferation, cell-cell interaction, multicellular complexity and functionality (rhee et al., 2001; cerwinka et al., 2012; samuelson and gerber, 2013) . rwv-derived 3d models have been applied to the investigation of infectious agents (viruses, bacteria and parasites) (barrila et al., 2010) . those models can reflect the natural infection process. the inherent flexibility of this system is an ideal platform for exploring fundamental questions in virology. a multitude of research has shown that rwv-derived models utilizing human cells are a valuable tool for investigating viral growth, replication, viral infection, viral entry, the viability of virions and virus-host interaction (margolis et al., 1997; long et al., 1998; nickerson et al., 2007; straub et al., 2007; barrila et al., 2010; berto et al., 2013; goodwin et al., 2015) . the rwv provides a useful model system for studying human viruses and has the potential for use in developing and screening antiviral drugs, as well as evaluating vaccines. the other bioreactor used for studying human viruses is the rfb, consisting of a vessel, column and pc monitoring system. it was originally designed for creating artificial liver tissues allowing human liver cells to maintain their morphological characteristics and physiological functions for a relatively long period of time (kawada et al., 1998; aizaki et al., 2003) . culture conditions can automatically be controlled. temperature, ph and oxygen concentration in the conditioning vessel are continuously monitored by pc and conditioned by mass flow controller (murakami et al., 2008) . the rfb-derived 3d models have mainly been used for hepatitis c virus (hcv). however, to our knowledge, no research associated with the rfb being used for human viruses has been reported in pubmed since 2008. the organotypic epithelial raft culture is an in vitro 3d culture system, where epithelial cells are placed on top of a dermal equivalent and then cultured at the air-liquid interface to full differentiation (meyers et al., 1992; andrei, 2006; fang et al., 2006) . raft cultures are prepared using cells or tissues derived from dispersed primary keratinocytes, explanted epithelial tissue or established cell lines (andrei et al., 2010) . organotypic raft culture was originally designed to accurately mimic the in vivo morphological and physiological characteristic of the epidermis (asselineau and prunieras, 1984; meyers et al., 1992) . this system, forming a stratified and differentiated epithelium, has provided researchers a useful means to investigate epitheliotropic viruses (chow, 2015) . over the past few years, human papillomavirus (hpv)-host interactions have been demonstrated with these raft cultures similar to those observed in vivo. this system has been a paramount milestone in the study of hpv so far. 3d scaffold materials are designed to support the attachment, proliferation and differentiation of selected cell populations grown in 3d culture models. scaffold-based 3d cultures are playing an increasing role in tissue engineering. in recent years, these models have been applied to virology. several matrices have been used to investigate human viruses. among them, the use of matrigel as a 3d scaffold has shown promise for the study of several viruses. matrigel, a gelatinous protein mixture, resembles the complex extracellular environment observed for tissues in vitro and produces a thick matrix for 3d cell culture (kleinman and martin, 2005) . hcv has been investigated by this 3d model. in addition, the use of matrigelbased systems for recombinant adenoviruses and ep-stein-barr virus has begun to capture researchers' attention (fotheringham and raab-traub, 2013; wang et al., 2014) . alginate, including sodium-alginate salt and calcium-alginate, is also used for studying hcv. mebiol gel, a thermoreversible gelation polymer (tgp), is a synthetic compound that consists of thermo-responsive and hydrophilic polymer blocks. as a 3d scaffold it was proven to be susceptible to hcv replication (rajalakshmy et al., 2015) . although the scaffold/matrix-based 3d culture models used for human viruses appear to be very limited so far, these systems may be applied in virology to define new anti-virus strategies, and may also provide a potential platform for the specific design of effective individual therapy according to patient-specific strains (aly et al., 2009 ). the multicellular complexity of tissues cannot be captured by typical 3d cell culture models, these models lack vasculature, do not provide precise control over gradients and undergo medium exchange at discrete time points instead of in a continuous manner (van duinen et al., 2015) . in recent years, several novel 3d culture models have been developed for further studying human tissue pathophysiology and physiology in vitro. microfluidic 3d cell culture allows spatial control over fluids in micrometer-sized channels. this model has become a valuable tool to further increase the physiological relevance of 3d cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over signaling gradients. van duinen et al. (2015) have reviewed the most important progress in microfluidic 3d cell culture since 2012. using hydrogels in microfluidic systems have been a recent trend, this model offers cells a more physiologically relevant 3d matrix (huang et al., 2011; chung et al., 2012) . the microfluidics 3d system will play an important role in the development of personalized medicine, especially in the field of cancer. in addition, 3d perfusion cell culture, an emerging technology, may provide a potential research avenue for commercial applications in drug discovery, regenerative medicine and tissue engineering. this model, mimicking the blood circulation in the human body, can control physiological chemostatic conditions, and create gradients of oxygen, growth factors and other biochemical signals. the 3d perfused culture model also can maintain the stability of the local microenvironment of the residing cells by continuously providing a nutrient supply and waste removal (li and cui, 2014) . souza et al. (2010) reported a novel 3d tissue culture based on magnetic cells levitation. in this method, cells bind with a magnetic iron oxide nanoparticle assembly comprising gold nanoparticles and cell-adhesive peptide sequences. by spatially controlling the magnetic field, cells are concentrated at the airliquid interface, where they aggregate to form larger 3d cultures (haisler et al., 2013) . the magnetic levitation method (mlm) and other associated techniques (cell culture, imaging and ihc) adapted for the mlm are described by haisler et al.(2013) . although there are no reports associated with studying human viruses using those models, these approaches may become valuable tools in the field of human viruses in the future. the advantages and disadvantages of different 3d culture models used for human viral growth, replication, proliferation, infection and antiviral drugs are listed in table 3 . to develop routine and advanced 3d cell culture devices, several factors must be considered, such as the cost of equipment, running cost, throughput and the simple operation. commercial development of in vitro 3d models and applications has been summarized by li and cui (2014) , including the suppliers, the core technology and 3d products. the devices, scaffolds and technical demands for different 3d cell cultures are listed in table 4 . 3d models are modular, tractable biomedical systems, which will yield great advances in our understanding of biological science. although some shortcomings have been found in the current 3d systems, 3d cell culture models hold enormous potential for the basic cell science, tissue engineering and infectious pathogens. 3d cell culture is an evolving field and requires further research for its optimization by combining a number of key areas including materials science, cell biology and bioreactor design. human papillomavirus (hpv) is small, non-enveloped viruses with a double-stranded dna genome . more than 180 types of hpv have been identified, which are classified into low-risk and high-risk hpv types (bernard et al., 2010) . the viral genome includes six early proteins e1, e2, e4, e5, e6 and e7, and the late structural proteins l1 and l2 (malik et al., 2014) . high-risk types cause cervical cancers and other anogenital carcinomas (vulvar, vaginal and anal). types 16 and 18, the two most carcinogenic hpv types, are thought to contribute to 70% of human cervical cancer (schiffman et al., 2007) . organotypic epithelial raft cultures represented a breakthrough in the study of papillomaviruses due to the strict link of hpv replication with epithelial cell differentiation (andrei et al., 2010) . so far, more than nine aspects of hpvs have been explored in these systems. hpv cannot be propagated in 2d cell monolayer cultures, so organotypic epithelial raft cultures that generate a stratified and differentiated epithelium have been applied in studying the hpv life cycle (chow, 2015) . the organotypic raft culture system has allowed the study of the en-tire differentiation-dependent life cycle of hpvs, including virion morphogenesis (mclaughlin-drubin et al., 2003; mclaughlin-drubin and meyers, 2005) . fang et al. (2006) demonstrated episomal maintenance of hpv-11 dna in n-tert cells. hpv-11 episomal dna-containbarrila et al., 2010 hjelm et al., 2010 unsworth et al., 1998 hammond and hammond, 2001 organotypic epithelial raft cultures ing cell populations grown in raft culture showed induction of a productive viral life cycle. this system has served as a faithful in vitro model for investigating propagation, infection and neutralization of hpvs, as well as producing infectious hpv virions (ozbun, 2002; mclaughlin-drubin et al., 2004; chow et al., 2009; wang et al., 2009 ). examinations of virus-host interactions have also been reported (anacker and moody, 2012) . several researchers have further used the organotypic epithelial raft cultures to study the interaction of hpv with other epitheliotropic viruses, such as herpes simplex virus (hsv) and hpvs, adeno-associated virus and hpv interaction hermonat et al., 2005) . importantly, meyers et al. (2002) demonstrated that the nonstructural genes of hpv18 functionally interact with the structural genes of hpv16, allowing the complete hpv life cycle to occur, which is the first report of the propagation of chimeric hpv by normal life cycle pathways. screening and evaluating antiviral compounds can also be carried out with raft cultures (satsuka et al., 2010) . recently, research on the relationship between tumor progression and hpv is increasing, which will hopefully lead to the development of effective treatments for hpv-associated cancer. a detailed review concerning evaluation of the efficacy of the therapeutic interventions for hpv using epithelial raft cultures has been published by andrei et al. (2010) . so far, these studies appear to be rather limited. in recent years, an increasing number of studies have addressed more specifically the role of hpv gene products and the difference in protein function between different hpv types, as well as the mechanisms of hpv carcinogenesis, especially the relationship between hpv oncoproteins (e2, e4, e5) and cervical tumors (doorbar, 2016) . the effects of hpv16 e5 deletion mutants on epithelial morphology have been reported by barbaresi et al. (2010) . mole et al. (2009) using the organotypic raft culture with epithelial cells, demonstrated that sf2/asf (splicing factor 2/alternative splicing factor) is up-regulated in response to differentiation in hpv-infected cervical epithelial raft tissue. a specific subset of sr proteins (ser-arg rich proteins) regulated by hpv16 e2, including sf2/asf, srp20 and sc35, were also overexpressed during cervical tumor progression. furthermore, organotypic raft cultures using verruciformis-derived keratinocytes could be used to reconstruct the î²-hpv life cycle and show the relationship between î²-hpv e4 expression patterns and disease severity. this finding is indicative that e4 may be a possible marker of viral expression during î²-hpv-associated skin cancer progression (borgogna et al., 2012) . a longitudinal cell culture using organotypic raft cultures has been used to investigate the immortalizing and transforming abilities of naturally occurring e6 variants in primary human foreskin keratinocytes (phfks). the observations provided insight into the mechanisms behind how phfks are immortalized and transformed into malignant tumors by the viral oncoproteins of hpv16 (richard et al., 2010) . in addition, a breakthrough in hpv chimeric genomes producing infectious virus in organotypic raft cultures was achieved. researchers constructed hpv18 chimeric genomes in which the hpv18 capsid genes were replaced with those of evolutionarily diverse pv types, including hpv45, hpv39, hpv33, hpv31, hpv11, hpv6b, hpv1a, crpv and bpv1. each of the chimeric genomes generated infectious viral particles in organotypic raft cultures (bowser et al., 2011) . human immunodeficiency virus (hiv), originally isolated from a patient with acquired immune deficiency syndrome (aids) in france in 1983 ( barre-sinoussi et al., 1983) , is a major contributor to the global burden of disease. although dramatic progress has been made in the development of novel antiviral drugs (de clercq, 2007) , an effective vaccine remains elusive despite two decades of effort (maartens et al., 2014) . the use of antiretroviral drugs has markedly reduced the mortality rate amongst aids patients. however, the effect of these drugs on oral epithelium growth and differentiation is presently unknown. a new 3d cell culture system, organotypic raft cultures of gingival keratinocytes, has been established. research demonstrated that hiv protease inhibitor amprenavir severely inhibited the growth of gingival epithelium cultured in this model. when the drug was added at day 8, amprenavir treatments altered the proliferation and differentiation of gingival keratinocytes (israr et al., 2010) . there are several other studies that have reported similar results utilizing organotypic raft cultures of gingival keratinocytes to investigate the effect of anti-hiv drugs on gingival epithelium growth and differentiation. those drugs included protease inhibitor lopinavir/ritonavir, and nucleoside reverse transcriptase inhibitors zidovudine, efavirenz and tenofovir (israr et al., 2011; mitchell et al., 2012; mitchell et al., 2014) . furthermore, balzarini et al. (2013) developed a multi-targeted drug, 6-phosphonylmethoxyethoxy-2, 4-diaminopyrimidine (pmeo-dapym), and demonstrated that this drug efficiently suppressed both hiv-1 and hsv-2 in organotypic epithelial raft cultures of primary human keratinocytes. in addition, 3d cell cultures could be developed as a potential model for studying the neuropathogenesis of hiv infection and the development of drug candidates that could effectively treat the neurological complications of hiv infection . investigating the effects of highly active antiretroviral therapy (haart) and designing multi-targeted antiviral drugs that could effectively suppress both hiv and hepatitis b virus (hbv) (or hcv) may lead to the future direction of hiv in 3d culture systems. hepatitis c virus (hcv), first identified in 1989, poses an enormous threat to public health and affects more than 170 million people around the world (choo et al., 1989; poynard et al., 2003; cox, 2015) . although the 2d cell culture model has been the standard tool for investigating hcv in cell culture, the hcv life cycle in vivo occurs in a much more complex environment compared to that in standard 2d cultures. 3d cell culture models can more closely mimic the polarized and differentiated state of hepatocytes in vivo . so far, hcv-associated research has been reported in three independent 3d cell cultures systems: 3d/rfb, 3d rwv bioreactors and the scaffold/ matrix-based 3d cultures. differentiated human hepatoma flc4 cells transfected with full-length hcv rna can produce and secrete infectious particles in the 3d rfb culture system (aizaki et al., 2003) . a similar result has been found in the rfb system following transfection of flc4 cells with a dicistronic hcv genome derived from genotype 1b, as well as in the 3d/tgp system using huh-7 cells (murakami et al., 2006) . furthermore, research has demonstrated that a long-term culture of the 3d rfb system provides a potential platform for investigating hcv dynamics, as well as examining the therapeutic effects of interferon alpha in this 3d culture model (murakami et al., 2008) . although hcv culture infection models based on the hcv jfh-1 molecule clone and huh-7 cells permit the production of virus, these recombinant hcv genomes only proliferate in sub-lines of huh-7 cells, which do not allow infection or proliferation of blood-borne hcv. a novel in vitro culture system combining 3d/tgp and immortalized human hepatocytes (hus-e/2 cells) demonstrated efficient support of the infection and replication of natural hcv (aly et al., 2009) . moreover, another system based on a 3d hollow fiber culture system and the hus-e/2 cell line was used for investigating the life cycle of blood-borne hcv, this 3d infection system allowed the reproduction of strain-dependent events reflecting virus-cell interactions and viral dynamics (aly et al., 2009) . more recently, hcv infection, replication and life cycle have been studied frequently. rwv-cultured huh-7 cells form complex, multilayered 3d aggregates, highly permitted for hcv infection, which provides a platform for studying hcv biology and the interaction between hcv infection and host cell function (sainz et al., 2009b) . cho et al. (2009) described that huh-7.5 cells cultured in a 3d peg-based hydrogel system can be efficiently infected with hcv, which was the first report of de novo infection with the virus (both replication-defective pseudovirus particles and fully infectious hcv). in addition, matrigel-embedded 3d culture of huh-7 cells or huh-7.5 cells was used as a hepatocyte-like polarized system and supported hcv infection by jfh-1 virus, as well as producing infective viral particles (molina-jimenez et al., 2012; liu et al., 2014) . researchers succeeded in reconstituting a hepatic-like structure formation from huh-7 cells using a calcium-alginate encapsulation model, which provides an opportunity for viral studies, especially for application with hcv (tran et al., 2013) . recently, mebiolgel-derived 3d culture models were shown to support cell growth as 3d spheroids for up to 63 days, this system was susceptible to hcv infection and replication; it could be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum (rajalakshmy et al., 2015) . so far, many facets of hcv have been extensively studied in 3d cell culture systems. however, knowledge of several dimensions of hcv are still limited: (i) hcv biology, (ii) anti-hcv therapeutics, (iii) hcv-hiv interaction and (iv) hcv-cancer interaction. more efforts must be taken to further research hcv. 3d culture systems will provide insight into the biophysical properties and viral morphogenesis of hcv particles and assessing anti-hcv compounds. this system may have advantages in studying aspects of hcv biology, such as viral assembly and budding, and also provides a system for screening antiviral drugs that inhibit the release or transmission of infectious hcv . hepatitis e virus (hev) was discovered during the soviet occupation of afghanistan in the 1980s. anti-hev therapy has been successfully used in chronic hepatitis e, the first vaccine available for clinical use is licensed in china (mihalcin et al., 2015) . at present, the life cycle of hev is still poorly under-stood, extensive study of the viral replication cycle has been hampered by the lack of efficient cell culture systems and animal models (osterman et al., 2015) . research demonstrated that hev can replicate efficiently in human hepatoblastoma plc/prf/5 cells cultured in a rwv for up to 5 months, hev nucleic acid was detected by reverse transcription-pcr in the supernatant of the infected cells in the 3d cell culture system. in contrast, that was not observed in the 2d monolayer system. complete virions were detection by electron microscopy. however, the study of hev using 3d systems is limited so far, and 3d cultures will offer a potential platform for in vitro cultivation of hev and investigation of the biology of hev, as well as the viability of hev in pig and other environmental samples (berto et al., 2013) . human herpes virus (hsv) is a significant pathogen and responsible for a variety of disorders (nicoll et al., 2012) . hsv-1 and hsv-2 are ubiquitous human pathogens. hsv-1 is normally associated with orofacial infections and encephalitis, whereas hsv-2 usually causes genital infections. more culture models have been applied to epithelial cells for researching hsv. the organotypic raft culture system, which accurately mimics the in vivo physiology of the epidermis, is a most powerful tool for studying infectious agents that infect the epithelium. the first report about the application of 3d organotypic raft culture for the study of hsv appeared in 1996 (syrjanen et al., 1996) . researchers demonstrated that the f strain of hsv-1 had the ability to produce lytic or nonproductive infection in hacat cells (immortalized skin keratinocytes) cultured in a 3d organotypic tissue culture. the cultures were infected with hsv-1 (5 pfu) 30 min after the lifting of the epithelial cells into the air-liquid interface and were collected 1 week after inoculation. these cultures were positive for hsv dna using pcr. one year later, another research study focused on utilizing the organotypic tissue culture methodology for the study of the infection, replication and spread of hsv-1 in fully stratified and differentiated human epithelial tissue (visalli et al., 1997) . a similar hsv-1 culture system has also been described by hukkanen et al. (1999) . they reported hsv-1 could infect immortalized hacat keratinocytes cultured in an organotypic raft culture system, the virus yield was highest when the inoculation took place 72h after seeding. in addition, 3d epithelial raft culture represents a novel model for the study of antiviral agents active against hsv. researchers have shown that specific compounds, such as foscarnet and cidofovir, can reduce the replication and spread of hsv in raft cultures of human keratinocytes (andrei et al., 2005) . so far, significant advances are still limited in our knowl-edge of hsv using 3d culture systems. anti-hsv may be the future research direction, especially for evaluation of the efficacy of new anti-hsv antivirals before clinical trials. varicella-zoster virus (vzv) is a neurotropic human alphaherpesvirus that causes varicella (chicken pox) and herpes zoster (shingles), establishes latency in multiple ganglionic neurons after primary infection and can reactivate to cause zoster (arvin, 1996; heininger and seward, 2006) . unfortunately, investigating vzv pathogenesis is challenging as vzv is strictly a human pathogen and infection is highly restricted in other species. in addition, few culture models are available for studying the interaction between vzv and human neurons, because the life span of terminally differentiated human neurons in culture is short (goodwin et al., 2013; zerboni et al., 2014) . the organotypic raft culture model has been applied in studying vzv. as keratinocytes are the main target cells for productive infection in vivo for vzv, characterization of viral replication in organotypic raft cultures of these cells represents a very relevant model for studying virus-host cell interactions and antiviral agents (andrei et al., 2005) . researchers have reported that they studied the action of antiviral compounds against vzv in organotypic epithelial raft cultures. the cultures were infected by vzv after 4 to 5 days of human keratinocyte differentiation and treated with serial dilutions of antiviral compounds. the antiviral effects were quantified by determining the viral dna load by real-time pcr for vzv. furthermore, this system could be very useful for the study of the interactions between viruses and the skin. the raft cultures have been used as a novel approach for investigating vzv replication in epidermal cells undergoing differentiation (andrei et al., 2005) . similar research has been described by mcguigan et al. (2007) . recently, a 3d model of normal human neural progenitor (nhnp) cells in tlas was used to investigate vzv infection. these cells could be effectively cultured for more than 6 months in 3d culture, and exhibited an expression profile similar to that of human trigeminal ganglia. vzv produced a persistent infection in nhnp cell tlas, which could be maintained for at least 3 months. this 3d culture system is likely to contribute to deciphering the establishment of vzv latency and reactivation in future studies (goodwin et al., 2015) . despite advances having been made in understanding vzv-host interactions, numerous questions relating to vzv pathogenesis remain unresolved (zerboni et al., 2014) . 3d culture systems have the potential to provide new approaches for preventing and treating vzv infections, in particular they may provide the cell system required for the generation of high-titer, cell-free attenuated virus for vaccine production. adenovirus (adv) is a mildly pathogenic human virus that propagates prolifically in epithelial cells. advs have been increasingly used as vectors for gene transfer in tumor cells or as oncolytic viruses (grill et al., 2002) . however, because of incomplete knowledge of the complex virus-cell interactions, the predicted replication selectivity has not been realized. in addition, rodent cells do not allow the complete lytic cycle of human adenovirus (alemany et al., 2000) . furthermore, specific constraints to adenovirus distribution and spread cannot be studied in cell cultures. mcss have been applied in studying the interaction between tumor and adenoviruses. grill et al. (2002) reported that replication-defective adenoviruses do not penetrate into spheroids; in addition, they described the propagation of replication-competent adenoviruses in spheroids. organotypic spheroids may provide a useful tool for studying the spread, oncolysis and distribution of adenoviruses. the adenovirus infection process was reproduced in organotypic raft cultures of primary human keratinocytes (noya et al., 2003) . adenovirus mutants have been found to replicate and promote the killing of cells expressing hpv e6 and e7 oncoproteins, which were present in an organotypic model of human stratified squamous epithelium derived from primary keratinocytes (balague et al., 2001) . more recently, research has demonstrated that adenoviruses could effectively deliver transgenes into the cultured 3d "mini-gut" organoids using adenoviral vectors that expressed fluorescent proteins. the transgene expression could be maintained for at least 10 days. these results were indicative that adenovirus vectors should be explored as effective gene delivery vehicles to introduce genetic manipulations in 3d organoids (wang et al., 2014) . norwalk virus was first discovered in 1972 (kapikian et al., 1972) and renamed as norovirus (nov) in 2002. nov is a human enteric pathogen causing epidemic foodborne gastroenteritis (tan and jiang, 2014) . the major barrier to the research and development of effective interventions for nov has been the lack of a robust and reproducible in vitro cultivation system (robinson and pfeiffer, 2014; ettayebi et al., 2016) . straub et al. (2007) first developed a 3d culture model with human embryonic intestinal epithelial cells cultured on collagen-i porous microcarrier beads, which were infected by genogroup i and ii human novs. the results showed that this model could support the natural growth of human novs. however, other investigators reported that using these same methods had not been suc-cessful. in 2011, straub et al. (2011) designed another 3d culture model using gastrointestinal epithelial cells (caco-2); the norovirus viral rna copy number was significantly increased (> 2 log 10 ) in this 3d caco-2 cell culture system. the results showed that this model supported norovirus replication. however, papafragkou et al. (2014) reported that using the 3d cell culture model with caco-2 cells was not suitable for the replication of norovirus. at present, there is still dispute about 3d culture models for studying novs. further research efforts need to be made in the future. apart from those viruses reviewed above, the use of 3d culture models for other viruses is currently receiving widespread attention. for example, 3d porcine epithelial cell cultures were designed to help understand the interaction between foot-and-mouth disease virus (fmdv) and porcine mucosal epithelial cells. the results demonstrated that fmdv replicated only transiently without any visible cytopathic effect, and infectious progeny virus could be recovered only from the apical side (dash et al., 2010) . researchers reported that a panel of clinical human rhinovirus (hrv) species c specimens, including hrv-c2, hrv-c7, hrv-c12, hrv-c15 and hrv-c29 types, were all capable of mediating productive infection in reconstituted 3d human primary upper airway epithelial tissues, and that the virions entered and exited preferentially through the apical surface. this model is considered as a potential tool for modeling the respiratory epithelium in the study of infections caused not only by hrvs, but also by other respiratory pathogens (tapparel et al., 2013) . in addition, 3d human intestinal enteroid cultures were designed as a novel pathophysiological model for studying human rotavirus infection, host restriction and pathophysiology (saxena et al., 2015) . furthermore, lam et al. (2012) demonstrated a method for measuring the impact of infection on the mechanics of a 3d model of connective tissue using human cytomegalovirus. table 5 shows some information on human virology in 3d cell cultures. 3d cell cultures are emerging technologies that can reproduce specific morphological and biochemical features of human cells similar to those found in vivo. recent progress in the imaging of 3d dynamics may promote the development of 3d cell culture. 3d culture models provide a potential tool for studying viral growth, infection, pathogenesis, and virus-host interaction. these culture systems have allowed a breakthrough in the cultivation of fastidious viral pathogens, which has advanced our understanding of the molecular and cellular mechanisms that underlie pathogenic processes in both the virus and host (barrila et al., 2010) . in addition, these models will play a key role in preclinical drug and therapeutic discovery. it is estimated that 10%-15% of human cancers worldwide are caused by seven human viruses (moore and chang, 2010) . as an example, high-risk hpv causes cervical cancer, kaposi's sarcoma herpesvirus leads to kaposi's sarcoma and hbv contributes to hepatocellular carcinoma. however, the understanding of the relationship between human viruses and tumors is still limited, especially the therapy strategies. 3d cell culture models, capturing tumor complexity in vitro, provide a potentially powerful physical tool for investigating virus-cancer interaction and evaluating antiviral agents. in addition, analyzing the mechanism of human tumor virology in 3d in vitro may be useful for predicting cancer progress. so far, although the development and testing of viral vaccine in 3d culture systems is limited, these models may be useful as an alternative means for evaluating preclinical vaccines in the future. moreover, 3d co-culture holds enormous potential for studying virus-virus interactions (such as hsv and hpv), and for developing novel products for the prevention, diagnosis and treatment of viral disease. in recent years, the development of multi-targeted drugs for suppressing two or more viruses has been receiving widespread attention. it might also be possible to use 3d models for the specific design of effective individual therapy according to patient-specific strains. most importantly, 3d culture systems may be used not only in future virological studies, but also in more tissue engineering. we expect that these novel, easily made and reliable models will make a contribution to the further understanding of biological science. advances in the formation, use and understanding of multi-cellular spheroids production and release of infectious hepatitis c virus from human liver cell cultures in the three-dimensional radial-flow bioreactor replicative adenoviruses for cancer therapy 3d cultured immortalized human hepatocytes useful to develop drugs for blood-borne hcv generation of organotypic raft cultures from primary human keratinocytes three-dimensional culture models for human viral diseases and antiviral drug development epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents organotypic epithelial raft cultures as a model for evaluating compounds against alphaherpesviruses three-dimensional cell culture: a breakthrough in vivo varicella-zoster virus reconstruction of 'simplified' skin: control of fabrication microtissue size and hypoxia in hts with 3d cultures skin and hair on-a-chip: in vitro skin models versus ex vivo tissue maintenance with dynamic perfusion deconstructing the third dimension: how 3d culture microenvironments alter cellular cues human papillomavirus e6e7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes a multi-targeted drug candidate with dual anti-hiv and anti-hsv activity effects of human papillomavirus type 16 e5 deletion mutants on epithelial morphology: functional characterization of each transmembrane domain isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) organotypic 3d cell culture models: using the rotating wall vessel to study host-pathogen interactions classification of papillomaviruses (pvs) based on 189 pv types and proposal of taxonomic amendments replication of hepatitis e virus in three-dimensional cell culture characterization of beta papillomavirus e4 expression in tumours from epidermodysplasia verruciformis patients and in experimental models human papillomavirus type 18 chimeras containing the l2/l1 capsid genes from evolutionarily diverse papillomavirus types generate infectious virus three-dimensional cell culture: the missing link in drug discovery differentiation of human mesenchymal stem cell spheroids under microgravity conditions viral infection of human progenitor and liverderived cells encapsulated in three-dimensional peg-based hydrogel bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds fabricated by electrospinning for 3d cell culture isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome model systems to study the life cycle of human papillomaviruses and hpv-associated cancers a highly efficient system to produce infectious human papillomavirus: elucidation of natural virus-host interactions microfluidic fabrication of microengineered hydrogels and their application in tissue engineering medicine. global control of hepatitis c virus cell interactions with three-dimensional matrices foot-andmouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells the design of drugs for hiv and hcv model systems of human papillomavirus-associated disease the biology and life-cycle of human papillomaviruses. vaccine three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors replication of human noroviruses in stem cell-derived human enteroids induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures epstein-barr virus latent membrane protein 2 effects on epithelial acinus development reveal distinct requirements for the py and yeea motifs reconfigurable microfluidic hanging drop network for multitissue interaction and analysis cancer invasion and the microenvironment: plasticity and reciprocity new dimensions in cell migration three-dimensional culture of melanoma cells profoundly affects gene expression profile: a high density oligonucleotide array study morphologic differentiation of colon carcinoma cell lines ht-29 and ht-29km in rotating-wall vessels 3d tissuelike assemblies: a novel approach to investigate virus-cell interactions three-dimensional normal human neural progenitor tissue-like assemblies: a model of persistent varicella-zoster virus infection reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity capturing complex 3d tissue physiology in vitro the organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro cell motility and mechanics in three-dimensional collagen matrices control of stem cell fate by physical interactions with the extracellular matrix biodegradable synthetic polymers for tissue engineering three-dimensional cell culturing by magnetic levitation multicellular spheroids as an in vitro tumor model optimized suspension culture: the rotating-wall vessel 3d cell culture methods and protocols modeling infectious disease dynamics in the complex landscape of global health analysis of adeno-associated virus and hpv interaction global health security: the wider lessons from the west african ebola virus disease epidemic development and characterization of a three-dimensional organotypic human vaginal epithelial cell model microfluidic hydrogels for tissue engineering herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture biomaterials offer cancer research the third dimension effect of the hiv protease inhibitor amprenavir on the growth and differentiation of primary gingival epithelium the hiv protease inhibitor lopinavir/ritonavir (kaletra) alters the growth, differentiation and proliferation of primary gingival epithelium visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis loss of cancer drug activity in colon cancer hct-116 cells during spheroid formation in a new 3-d spheroid cell culture system massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally enhanced hepatocarcinoma cell lines biomaterials for tissue engineering applications method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types high-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture three-dimensional tissue culture models in cancer biology matrigel: basement membrane matrix with biological activity the genesis and source of the h7n9 influenza viruses causing human infections in china a method for quantifying mechanical properties of tissue following viral infection three-dimensional cell culture matrices: state of the art a novel culture system to induce melanin synthesis by three-dimensional spheroid culture assessing the global threat from zika virus three-dimensional perfused cell culture recent advances in three-dimensional multicellular spheroid culture for biomedical research complete replication of hepatitis c virus in cell culture direct visualization of hepatitis c virus-infected huh7.5 cells with a high titre of infectious chimeric jfh1-egfp reporter virus in three-dimensional matrigel cell cultures bioengineered 3d platform to explore cell-ecm interactions and drug resistance of epithelial ovarian cancer cells rhinovirus replication in hela cells cultured under conditions of simulated microgravity animal models in virus research: their utility and limitations synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering hiv infection: epidemiology, pathogenesis, treatment, and prevention cancer dissemination--lessons from leukocytes human papillomavirus: current status and issues of vaccination lymphocyte trafficking and hiv infection of human lymphoid tissue in a rotating wall vessel bioreactor modelling tissues in 3d: the next future of pharmaco-toxicology and food research? preclinical development of bicyclic nucleoside analogues as potent and selective inhibitors of varicella zoster virus propagation, infection, and neutralization of authentic hpv16 virus propagation of infectious, high-risk hpv in organotypic "raft" culture human papillomavirus type 45 propagation, infection, and neutralization replication and interaction of herpes simplex virus and human papillomavirus in differentiating host epithelial tissue infectious virions produced from a human papillomavirus type 18/16 genomic dna chimera biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation hepatitis e--overview of the latest knowledge hiv nucleoside reverse transcriptase inhibitors efavirenz and tenofovir change the growth and differentiation of primary gingival epithelium effect of the hiv nucleoside reverse transcriptase inhibitor zidovudine on the growth and differentiation of primary gingival epithelium rna splicing factors regulated by hpv16 during cervical tumour progression matrigelembedded 3d culture of huh-7 cells as a hepatocyte-like polarized system to study hepatitis c virus cycle why do viruses cause cancer? highlights of the first century of human tumour virology the challenge of emerging and re-emerging infectious diseases dynamic behavior of hepatitis c virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system production of infectious hepatitis c virus particles in three-dimensional cultures of the cell line carrying the genomelength dicistronic viral rna of genotype 1b microbial responses to microgravity and other low-shear environments studying host-pathogen interactions in 3-d: organotypic models for infectious disease and drug development the molecular basis of herpes simplex virus latency activation of adenovirus early promoters and lytic phase in differentiated strata of organotypic cultures of human keratinocytes the hepatitis e virus intraviral interactome. sci rep infectious human papillomavirus type 31b: purification and infection of an immortalized human keratinocyte cell line using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses three-dimensional tissue cultures: current trends and beyond the third dimension bridges the gap between cell culture and live tissue challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models nonpolarized signaling reveals two distinct modes of 3d cell migration at the leading edge of three-dimensional cell migration viral hepatitis c. lancet mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of huh7 cell line with improved hepatocyte differentiation marker expression and hcv replication 3d cell culture systems: advantages and applications permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel the immortalizing and transforming ability of two common human papillomavirus 16 e6 variants with different prevalences in cervical cancer hydrogels for 3d mammalian cell culture: a starting guide for laboratory practice hepatitis c virus infection in phenotypically distinct huh7 cell lines three-dimensional huh7 cell culture system for the study of hepatitis c virus infection improved function and growth of pancreatic cells in a three-dimensional bioreactor environment novel human papillomavirus type 18 replicon and its application in screening the antiviral effects of cytokines human intestinal enteroids: a new model to study human rotavirus infection, host restriction, and pathophysiology human papillomavirus and cervical cancer three-dimensional tissue culture based on magnetic cell levitation human norovirus infection of caco-2 cells grown as a three-dimensional tissue structure in vitro cell culture infectivity assay for human noroviruses in vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture vaccine against norovirus growth and characterization of different human rhinovirus c types in three-dimensional human airway epithelia reconstituted in vitro ebola virus disease in west africa--the first 9 months of the epidemic and forward projections an appropriate selection of a 3d alginate culture model for hepatic huh-7 cell line encapsulation intended for viral studies microfluidic titer plate for stratified 3d cell culture assembly of a three-dimensional multitype bronchiole coculture model using magnetic levitation growing tissues in microgravity microfluidic 3d cell culture: from tools to tissue models three-dimensional (3d) tumor spheroid invasion assay advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation infection and replication of herpes simplex virus type 1 in an organotypic epithelial culture system robust production and passaging of infectious hpv in squamous epithelium of primary human keratinocytes adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids three-dimensional in vitro tumor models for cancer research and drug evaluation modeling tissue morphogenesis and cancer in 3d molecular mechanisms of varicella zoster virus pathogenesis a review of the three-dimensional cell culture technique: approaches, advantages and applications this research was supported by the national megaprojects for infectious diseases (2014zx10004002-004-001). the authors declare that they have no conflict of interests. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-314753-xflhxb13 authors: manso, carmen f.; bibby, david f.; mbisa, jean l. title: efficient and unbiased metagenomic recovery of rna virus genomes from human plasma samples date: 2017-06-23 journal: sci rep doi: 10.1038/s41598-017-02239-5 sha: doc_id: 314753 cord_uid: xflhxb13 rna viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. new sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. through selective host rna depletion and compensatory protocol adjustments for ultra-low rna inputs, we are able to detect three major blood-borne rna viruses – hiv, hcv and hev. we recovered complete genomes and up to 43% of the genome from samples with viral loads of 10(4) and 10(3) iu/ml respectively. additionally, we demonstrated the utility of this method in detecting and characterising members of diverse rna virus families within a human plasma background, some present at very low levels. by applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of hcv genotype 6, and a co-infecting human pegivirus. this method builds upon earlier rna metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses. a complex host-enriched sample was prepared by diluting in negative human plasma (nhp, negative for each hiv, hcv, and hev) stored plasmas from four samples previously characterized by routine diagnostic testing to contain hcv (x2), hiv and hev (see table 1 for details). nhp was obtained by centrifuging negative human blood for 10 minutes at 500× g to remove cell debris. the final concentration of each virus in the primary panel sample was 10 6 iu/ml (copies/ml for hiv -implied by iu henceforth for convenience), and three serial tenfold dilutions in nhp were prepared from this stock. virus multiplex reference (vmr) panel. a reagent comprising a suspension in pbs of 18 rna viruses with different genomic and structural characteristics was provided by the national institute for biological standards and controls (nibsc, potters bar, uk). each viral component and its approximate relative concentrations is given in mee et al. 42 . prior to extraction, the panel was mixed 1:1 with nhp. duplicate 400 μl extractions were performed 44 , as was genotyping of the hcv 43 and hev 68 clinical samples of indeterminate hcv genotype. four plasma samples collected from a patient between 2014 and 2016 were submitted to public health england (phe) for metagenomic analysis as previous genotyping results had been inconsistent. the most recent such test employed ns5b sequencing 43 , and reported the presence of a virus belonging to genotype 6 but was unable to resolve the subtype with any further precision. rna extraction and quantification. before extraction, all samples were centrifuged for 10 min at 2,500 × g to remove cell debris. triplicate, duplicate and single extractions were performed on the diluted vmr panel samples (referred to as 'vmr panel a/b/pbs'), the bbv panel samples ('10 6-3 -a/b'), and the patient sample series, respectively. a negative control comprising 200 μl of the same plasma used to dilute the panels was also extracted. the split rna extraction kit (lexogen) was used to extract 200 μl of each sample input, according to the manufacturer's instructions. acidic phenol was used to preferentially recover the large rna fraction, which was eluted in 12 μl of nuclease-free water. rna eluates were quantified using qubit rna hs assay kit (thermo fisher scientific), which is accurate for concentrations between 250 pg/μl and 100 ng/μl. depletion of ribosomal rna and dna digestion. ribosomal rna depletion and dna digestion was achieved using the riboerase kit (kapa biosystems). as all sample extracts were below the detection limit of the qubit quantification system, the total rna input was less than the recommended 100 ng. the manufacturer's specifications were followed with the exception of using the entire 10 μl of the extract, and after the dna digestion reaction clean up, eluting the residual rna was in 10 μl of nuclease-free water. in the case of the bbv panel, two of the three extracts of each dilution were treated with the riboerase kit before rna library preparation. the third set of extracts remained untreated and was used to monitor the effect of the rrna depletion and dna digestion upon the subsequent library preparation and sequence analysis. in the case of the vmr panel (the two duplicates) and the negative control, rrna and dna depletion was performed on all extracts. in the case of the uncharacterized hcv strain, extracts from all four samples were treated with riboerase. an additional, untreated, extract of sample 4 was included, again to monitor the process. rna library preparation with ultra-low rna input. libraries were constructed from 10 μl of extracted rna or 10 μl of rrna-depleted dnase-digested rna, using the nebnext ultra directional rna library prep kit (new england biolabs). as the protocol is designed to use a minimum rna input of 10 ng, several modifications were made to adapt it to an ultralow rna input. these are listed in table 2 . libraries were analysed for size distribution using the high sensitivity dna kit (agilent) on a 2100 bioanalyser instrument, and were quantified using the kapa sybr fast universal qpcr kit for illumina libraries (kapa biosystems) on a 7500 real-time pcr system (applied biosystems). to determine the relative abundances of viral inserts, libraries constructed from the bbv panel were analysed by qpcrs with primers and probes targeting each of the three viral components (refs 44-46 and supplementary table s1 ). reactions were performed using the quantitect virus kit (qiagen) according to the manufacturer's instructions. libraries labelled with different indexes were diluted to 2 nm and pooled. sequencing was performed on an illumina miseq instrument using the miseq reagent kit v2 (300 cycles) (illumina) according to the manufacturer's guidelines, with the following minor modifications. the library pools were denatured with 0.2 n sodium hydroxide for 2 minutes rather than 5, diluted in kit reagent ht1 to produce a 20 pm solution and then these were further diluted to 11 pm. of this library pool dilution, 600 μl were loaded onto the miseq cartridge. data analysis. all paired end fastq files were processed with trimmomatic v0.30, removing the illumina adaptor sequences, then trimming leading and trailing bases with phred scores below 20. reads were discarded where the length of either trimmed end was below 50 bases. for the determination of genome sequences of blood-borne viruses, trimmed fastq sets were normalised using the normalise-by-median.py script in the khmer package (k = 31, c = 5) 47 and submitted to the spades de novo assembler 48 without error-correction, applying the default kmer sizes of 21, 33, and 55. output contigs that matched each virus were identified with the nhmmer function of the hmmer v3.1b2 package 49 using hidden markov models (hmms) built from alignments of each virus (detailed in supplementary table s2) . where necessary, the ends of contigs were trimmed to the whole genome alignment. bwa mem (v0.7.5a, default parameters) 50 was used to map the original trimmed fastqs to the genome sequence, and the sam files were converted to bam files using samtools v0.1.19 51 while discarding reads with either 0 × 04 and/or 0 × 08 flags set (i.e. retaining only fully-mapped paired-end reads). base frequencies at each nucleotide position within each component virus sequence were obtained from bam files using quasibam v2.2, an in-house c++ program that tabulates base frequencies at each nucleotide position within a reference and generates consensus sequences based upon user-defined depth and variant percentages 52 . mapping of trimmed paired-end fastq to one or more virus reference genomes was also performed using bwa mem 0.7.5a. in each case, two independent mappings were performed, using as a reference the viral sequences, supplemented firstly by the march 2009 'grch37' release of the human genome, and secondly by a set of human rrna sequences (nr_003286.1, nr_003287.1, v00589.1, nr_003285.2, gij251831106:648-1601, and gij251831106:1671-3229, as per malboeuf et al. 38 ). the second file was used solely to derive counts for reads mapping rrna which would otherwise be subsumed into the human genome mapping results. from the filtered sam files, the numbers of reads mapping to each reference sequence were counted. counts for each of the constituent sequences of the human genome and rrna were pooled into a "human" count and an "rrna" count. quasibam was used to derive nucleotide frequencies from which depth and coverage data were calculated. a minimum depth of 10 was required for inclusion in a derived consensus sequence for the bbv panel (1 for the vmr panel). bbv and vmr panel sequences. the members of the multi-fasta reference file for the bbv panel were obtained by submitting fastq sets from the rrna-depleted sample with the highest virus concentrations to the spades-hmmer-mapping approach described in the previous paragraph. vmr panel references were derived from sequences obtained from genbank using accession numbers from mee et al. 42 . additionally, the complete genome sequence of a human pegivirus (hpgv) present in the plasma diluent was discovered in the spades contigs file. a hmm profile was constructed from an alignment of genbank sequences (supplementary table s2 ). sample with uncharacterised hcv. to obtain full-length hcv genomes, each fastq set was submitted to the spades-hmmer-mapping process. where a complete genome was not obtained, hcv-matching contigs were aligned to the full-length genomes using mega5 53 . in addition, contigs with length > 5 kb that did not align to the hmm profile were submitted to blast 54 for identification. following this analysis, an additional pegivirus genome was derived in similar fashion to the hcv genomes, using the same hmm profile as above for the nhp pegivirus. when calculating the read percentages and coverage plots, both sample-derived full-length genome sequences (hcv and hpgv) were used as the reference sequence when mapping that sample's corresponding trimmed paired-end fastqs, as well where only incomplete hcv genomes were obtained. virus (bbv) panel was prepared, comprising two strains of hcv (genotypes 1a and 1b), and one each of hiv and hev diluted in nhp to 10 6 iu/ml. three tenfold serial dilutions in plasma were made from this original panel. step manufacturer's recommendations protocol modification riboerase rna input 0. 69 . ribosomal rna depletion was performed on two of each set of triplicate extractions prior to all three being subjected to the modified library preparation protocol. data from the most concentrated rrna-depleted samples were used to generate individual virus genome sequences for use in reference mapping. during this data analysis, an unexpected human pegivirus (hpgv) was found and traced to the nhp diluent. the full genome sequence of this hpgv was determined from the 10 3 -b data and included in the mapping references. table 3 gives the read counts, genome coverages and median depths for each virus-dilution combination, across each of the three samples per dilution (10 6-3 -untreated/a/b). each test sample yielded over 800,000 reads with the exception of 10 3 -a, which gave just over 140,000 reads. with the exception of the two 10 6 samples, in which only a very small volume of nhp was added to the clinical samples, the percentage of reads mapping to the hpgv remained relatively constant at 29-39%. the exception is 10 5 -b, in which the overall viral read percentage was lower than expected, with a corresponding elevation in reads mapping to the human genome suggesting possible incomplete dnase digestion during the rrna depletion step (supplementary table s3) . with increasing dilution, the total viral read percentages (excluding hpgv) decline from over 60% to 0.23%. complete and near-complete genome coverages with depths greater than 10 were achieved at 10 6 and 10 5 iu/ml for all four viruses. a few short regions in hiv had low coverages (<10) with 10 5 -b, reflecting the reduced overall viral reads in this dataset, but at a minimum depth of 1, 99.6% coverage was achieved with this sample, with only a 32-base sequence in pol having no coverage. at 10 4 iu/ml, hcv 1a and hev continued to give 98.1-99.7% coverage with median depths over 120. hcv 1b and hiv gave 91.2-93.5% coverage (82-95 median depth) and 55.0-90.2% coverage (12-83 median depth) respectively, and in 10 3 -b, despite only 0.23% of all reads mapping to the four viruses collectively, genome coverages of 18.1-72.5% were achieved, with median depths up to 29. figure 1 illustrates coverages and depths across target genome at each dilution, showing even distributions of reads across all four target genomes and hpgv. pooling duplicates consistently improved coverages (final column, table 3 ). this is most clearly seen at the lower viral loads, where at 10 4 iu/ml, three of the four viruses achieve combined coverages of >99.4% each, and 93.5% in hiv. at 10 3 iu/ml, the combined coverages for the four viruses are effectively what would be expected were the individual coverages independent, i.e. cov aub depletion of rrna substantially enhances the recovery of blood-borne virus sequences. the percentage of reads mapping to rna virus genomes in the rrna-depleted bbv panel samples was between 40 and 150-fold higher than in corresponding untreated controls. individual target virus ratios decreased as they became more dilute, from over 100-fold for hcv in 10 5 -a to 2.9-fold for hiv at the lowest dilution. concomitantly, the ratio for hpgv rose markedly, from 4.8-fold in 10 6 -a to 175 in 10 3 -b, reflecting an effectively constant viral load against decreasing quantities of panel viruses (table 3 and fig. 1 ). genome coverage and median depth values were also much higher in the treated samples than untreated comparators. at the two highest virus concentrations, median depths were between 47-and 274-fold higher in the treated versus the untreated samples. only short fragments of hev were recovered from the untreated 10 4 dilution, and almost no hiv or hcv sequences. by contrast, near complete genomes from all four target viruses were recovered from the treated comparators, with median depths of between 83 and 457 (as noted above, hiv in 10 4 -a was an exception at 54.0% coverage and a median depth of 13). recovery of partial and complete genomes of diverse virus types from human plasma. the ability of our method to recover genome sequences from a range of rna viruses in the context of human plasma was evaluated using a virus multiplex reference (vmr) panel, putatively containing 25 genomically and physicochemically diverse viruses. two plasma-diluted panels and one pbs-diluted panel were tested (table 4 ). no reads from either of the three samples mapped to either of the two norovirus genomes, coronavirus 229e or influenza b virus. by the panel distributor's qpcr 42 , the threshold cycle (c t ) of the coronavirus was >36 and the other three were not detected, hence these four targets were excluded from further analysis. notwithstanding influenza virus a h3n2 and parainfluenza virus type 3 also not being detected by the qpcr, we recovered reads from both, with genome coverages ranging from 2.7% to 21.6%. almost no reads belonging to the panel's dna viruses were found. sixty-nine percent of all reads obtained from the pbs-diluted panel mapped to vmr panel genomes, dropping to 41-44% for the plasma-diluted samples, although the distribution of reads between targets was very uneven. parechovirus and rotavirus accounted for 78.8-87.6% and 10.6-19.5% of all viral reads respectively, with the other viruses collectively accounting for 1.7-1.9%. depths and genome coverages showed some inverse correlation with the given c q values (fig. 2) . as with the bbv panel data, coverage plots of the samples diluted in plasma were largely unbiased, giving pooled genome coverages close to those expected by independent distributions of reads between replicates ( table 4 , final column). rotavirus and coxsackievirus were exceptions, where despite large numbers of mapped reads, almost identical patterns of read coverages and gaps were observed between their replicates, with minimal additive effect. the pbs-diluted sample gave larger read numbers, but their distribution was less even throughout the genomes, resulting in relatively lower coverages. characterisation of a new subtype belonging to hcv genotype 6 and discovery of a second virus in a patient sample series. four plasma samples from a patient with hcv were used as starting material. all extracts were subjected to riboerase treatment; a second extract of sample 4 remained untreated for comparison. de novo assembly analysis of fastq sets from samples 1, 3 and 4 each gave a full-length hcv genome sequence as a single contig. for sample 2, 6 partially-overlapping contigs were obtained, covering 66% of scientific reports | 7: 4173 | doi:10.1038/s41598-017-02239-5 the hcv sequence. additionally, in all four samples, a single contig was obtained that was determined by blast and subsequent hmmer analysis to comprise an hpgv genome. the hcv and hpgv full genome sequences were combined in a single file to carry out reference mapping and nucleotide frequency determination on the four sample fastq sets (table 5 ). samples 1, 3 and 4 had hcv read percentages ranging from 1.0 to 24.3%, and gave complete genomes with median depths greater than 700. sample 2 had the lowest viral load (2,000 iu/ml), had 0.3% of reads mapping to hcv giving a genome coverage of 87% at a minimum depth of 10 (96.5% at depth ≥1) and a median depth of 43. full coverage of the hpgv genome was obtained from all samples, with median depths over 8,700, and read percentages ranging from 34.2 to 63.3%. the depth plots in fig. 3 again show unbiased and even coverages across both genomes, and the percentages of reads mapping to viral targets was again much higher in the rrna-depleted sample than in the untreated comparator (61-fold and 85-fold for hcv and hpgv respectively). table 3 . detailed sequencing data from the bbv panel. for each of the three samples (untreated, a and b) at each dilution (10 6 -10 3 ), the number and percentage of reads mapping to each virus are given, together with the genome coverages (depth ≥10) and median depths. the final column gives these last two metrics from the combined data sets of both the a and b samples. included in the analysis are data for the hpgv discovered in the sample diluent. analysis of the hcv sequence showed it to belonging to a new subtype within genotype 6 of which the details are presented in a separate manuscript (in preparation). the hpgv clustered with genotype 1 strains, and is distinct from the nhp strain. including the nhp negative control were subjected to virus-specific qpcr for the detection and quantification of hcv, hiv and hev. all were detectable in the sample libraries, but were undetectable in the riboerase-treated negative control library (supplementary table s4 ). all samples were mapped against reference sequences that included human genome and human rrna sequences to evaluate the efficiency of riboerase treatment. the average ratio of the percentages of reads mapping to rrna in the untreated versus the treated samples was 32-fold with an approximate halving of the number of reads mapping to the human genome, across all panels (fig. 4) . with the exception of the expected human pegivirus, mapping of the negative control fastq set against the reference sequences of the four bbv panel viruses, the two pegiviruses, the vmr panel and the patient hcv gave table 5 . detailed sequencing data from the patient sample series. for each of the four samples 1-4, the number and percentage of reads mapping to both the hcv and hpgv genomes are given, genome coverages (depth ≥10) and median depths. the analysis of sample 4 extracted without host rrna depletion is in the untreated column. very low numbers of reads mapped to viral genomes and no consensus sequences could be derived. further data for this section are found in supplementary tables s3 and s5 . in light of the large and ever-increasing number of human rna virus pathogens, it is perhaps unsurprising that standard serological assays and nucleic acid tests suffer from a lack of sensitivity to diverse variants of target viruses, overlook the presence of new or unexpected viruses, and provide only limited information about those targets they do successfully detect. hence the three main aims of metagenomic virology are to detect & identify known agents irrespective of their diversity, to discover novel agents of disease, and to obtain complete sequence information of detected viruses. most existing protocols achieve a maximum of two of these aims, but difficulties in selectively isolating viral rna species and short read sequences from those of the super-abundant host nucleic acid have limited the utility of metagenomic approaches in diagnostic virology. this study has addressed these limitations by establishing a novel methodology suitable for the agnostic detection and characterization of blood-borne rna viruses in plasma samples. by depleting host-derived nucleic acids and making modifications to an existing library preparation protocol to account for ultra-low rna input quantities, we have been able to reconstruct effectively full-length genomes of hcv, hev and hiv from plasma samples with viral loads of 10 4 iu/ml (copies/ml for hiv) and substantial fractions of complete genomes at 10 3 iu/ml. when applied to a series of clinical samples, we could elucidate simultaneously the full genome sequences of both a novel subtype belonging to hcv genotype 6 and a hitherto-undetected human pegivirus. additionally, our system was able to recover viral sequences from a panel of diverse rna viruses diluted in human plasma, with a broad correlation between the genomic coverage and depth metrics and approximate concentration. although full genomes were not assembled in many cases, the independence of read distribution gave sufficient genome coverage for identification. the vast majority of rna molecules in a human plasma sample are host-derived, of which up to 80% comprises the six species of human rrna. their presence in our libraries was minimised by two key protocol steps in our modified protocol. firstly, we selected an extraction method that combined a phenol/chloroform step with a column format (lexogen split rna) which increased the amount of extracted viral rna by up to one log when compared to other extraction methods (data not shown). perhaps more importantly, by controlling the final precipitation step, small rna molecules below 150 nt such as 5 s rrna and trna are excluded from the eluates, as are the majority of molecules of human genomic dna. secondly, we employed dna probes complementary to human rrna such that hybridisation and subsequent digestion by rnase h dramatically reduced their frequency in the finished libraries. whilst this methodology has been successfully used in the detection and characterisation of two haemorrhagic fever viruses, the frequency of viral reads was often below 1% and an additional hybrid-capture step was employed to elevate read numbers 36 . methods that do not deplete rrna generally give poor recovery of viral reads, yielding viral genome fragments that necessitate further work 27, 32, 55, 56 , low read numbers even at viral loads over 10 4 iu/ml [20] [21] [22] 33 , or at best, requiring dilution of both host and virus in pbs in order to recover full hiv genomes at low copy numbers 38 . the resultant rrna-depleted sample extracts typically contain quantities of nucleic acid in the low picogram range. library preparation through hexamer-mediated reverse transcription followed by multiple displacement amplification constitutes an easy and effective means of amplifying very low amounts of dna 27, 38, 57 , but in several studies (and in the authors' laboratory), significant amplification biases have been observed, leading to gaps in target genome coverage 39, [58] [59] [60] . consequently, we adopted an approach using a standard rna library preparation kit, but with substantial modification to compensate for their minimum rna input requirements of at least 10 ng and optimally 100 ng-1 µg. we made key changes to the rna fragmentation and adaptor-ligation steps of the nebnext ultra directional rna library prep kit protocol. while prior rna fragmentation with heat and divalent cations improves sequence coverage, over-fragmentation of target genomes leads to the loss of material during the library preparation process 37 . lower amounts of rna thus require shorter optimum fragmentation times and we found that 1 minute at 94 °c was optimal in terms of breadth of genome coverage. under standard kit conditions, our ultra-low rna inputs dramatically skewed the ratio of cdna to adaptor. the resulting adaptor excess led to the preferential amplification of adaptor dimers during the pcr step, and despite increasing cycle number to amplify low rna inputs, we were generally unable to generate sufficient quantities of target-specific material. accurate quantification and consequent equimolar pooling of libraries was compromised, as was the miseq clustering efficiency. we found that a reduced final adaptor concentration of 1.4 nm was crucial in reducing the amount of adaptor dimers in libraries from rrna-depleted samples whilst simultaneously extending the pcr cycle number. in the present study, serial dilutions of the blood borne virus panel were prepared in negative human plasma, reducing both the absolute quantity and relative frequency of the viral rna targets while maintaining the complexity of the sample in terms of host nucleic acid, thus mimicking that of a clinical sample. with rrna depletion, the number and diversity of viral reads was consistently high, with over 35% of all reads mapping to constituent virus genomes. throughout the three sample series, we obtained relatively high genome coverages of low-frequency viral targets. co-infections with multiple blood-borne viruses are common 61 , so whilst we speculate that the depths and coverages of target viruses would be greater yet in these samples had it not been for the confounding effect of the unexpected human pegiviruses in both the plasma diluent and the patient sample series, it was reassuring to see the method performed well under such conditions. in our experiments using negative human plasma as sample diluent, we were able to recover levels of viral genomes comparable to previous work using pbs, both for bbv panel viruses 38 and for the vmr panel 41 , and we were able to recover from a patient sample a large percentage of the genome of a previously uncharacterised subtype of hcv genotype 6 when present at 2 × 10 3 iu/ml, a diagnosis not possible using existing genotyping assays. the presence of an undiagnosed pegivirus in this sample further demonstrated the utility of the method in metagenomic analysis of blood-borne virus co-infections where the relative abundances of each virus can be highly variable 22 . furthermore, in three of the four samples, depths greater than 1,000 were routinely obtained, which are likely to be sufficient to call minority variants for clinical resistance 62 . a full description of the patient series and the new hcv strain are provided in a separate manuscript (in preparation). our approach can therefore not only accurately characterise rare or novel variants of existing viruses, but also generates the same level of information regarding unexpected viruses present in the sample. by comparison, vidisca 32, 63, 64 and other random amplification-ngs techniques 30, 31 have detected novel viruses in diverse clinical samples, but all have required further techniques to achieve full genome sequences. together with the vmr panel results, we were able to recover identifying sequence from both enveloped viruses (hcv, hiv, hev, influenza, and several paramyxoviruses), and non-enveloped viruses (several enteroviruses, astrovirus, rotavirus, and sapovirus). for the majority of viruses in the vmr panel, whilst dilution in plasma reduced the total percentage of reads recovered when compared to the panel diluted in pbs, a greater breadth of genome coverage was achieved. in the absence of any host nucleic acid background, it is possible that the pbs extracts had such ultra-low quantities of rna that despite the adjustments made to the library preparation protocol, the rna was over-fragmented, leading to a smaller number of genome fragments that were individually amplified to a greater extent than the larger array of fragments surviving the plasma extraction. in developing a similar approach, kohl et al. were only able to recover a percentage of reads exceeding 6% at a viral load over 10 7 copies/ml. at an influenza a virus concentration of over 10 5 copies/ml, this dropped to just 0.5%, and at a reovirus concentration of 10 3 -10 4 copies/ml, no viral reads were detected 24 . with our method, whole genomes were obtained for those with the highest viral loads, and for minority viral targets, there was a correlation between ostensible quantity and coverage, including for two viruses undetectable by the panel distributor 42 , a result superior to that recently obtained from influenza in clinical respiratory samples 65 . again, the presence of high quantities of one or more target is likely to have inhibited the representation of the minority species such that if tested individually, superior depths and coverages would seem likely. with further reduction to the fragmentation time, or even its abolition, it may be possible to use this method to reconstruct genomes from old, partially degraded samples such as those recently used to re-evaluate the early hiv epidemic in the americas 66 . our negative control data suggest that the level of contamination is low, with most viral reads therein belonging to the most abundant vmr panel member. nelson et al. 67 identified a second source of contamination consisting of incorrect reads from other libraries that were sequenced during the same sequencing run due to truseq index misassignment (~0.06% of reads, 0.02% here). although cross-contamination between samples during the library preparation can be another source of contamination, the qpcr results suggest no bbv panel genomes were present after library preparation in the negative control sample. to conclude, by applying the three adaptations of selective large rna extraction, rrna depletion-dnase treatment, and the extensively modified library preparation in combination, ngs data sets can be produced from plasma samples that are rich in rna virus sequence data. complex bioinformatic processing has been employed to identify viruses within a metagenomic dataset 7, 25, 26, 32, 64, 65 , but here, only simple bioinformatic processing is needed for detection and identification of known viruses, and by applying only moderately more advanced tools, an agnostic approach to virus detection can be taken, together with characterisation of the full genome even at low viral loads. the role of mutational robustness in rna virus evolution rna virus quasispecies: significance for viral disease and epidemiology ecological origins of novel human pathogens isolation of a novel coronavirus from a man with pneumonia in saudi arabia zika virus in the americas-yet another arbovirus threat reappearance of chikungunya, formerly called dengue detecting the emergence of novel, zoonotic viruses pathogenic to humans human viruses: discovery and emergence estimates of global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2015: the global burden of disease study global epidemiology and genotype distribution of the hepatitis c virus infection first genome characterization of a novel hepatitis c virus genotype 5 variant first ngs full genome characterization of a hepatitis c virus genotype 7 divergent subtype first evidence of transmission of an hiv-1 m/o intergroup recombinant virus rates of and reasons for failure of commercial human immunodeficiency virus type 1 viral load assays in brazil impact of hiv-1 genetic diversity on plasma hiv-1 rna quantification: usefulness of the agence nationale de recherches sur le sida second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test blinded, multicenter comparison of methods to detect a drug-resistant mutant of human immunodeficiency virus type 1 at low frequency global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study comparison of real-time pcr and antigen assays for detection of hepatitis e virus in blood donors an introduction to the analysis of shotgun metagenomic data metagenomic profiling of the viromes of plasma collected from blood donors with elevated serum alanine aminotransferase levels viral metagenomics applied to blood donors and recipients at high risk for blood-borne infections utility of metagenomic next-generation sequencing for characterization of hiv and human pegivirus diversity comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood protocol for metagenomic virus detection in clinical specimens host-associated metagenomics: a guide to generating infectious rna viromes virome analysis for identification of novel mammalian viruses in bat species from chinese provinces evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery two novel parvoviruses in frugivorous new and old world bats identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections using next generation sequencing to identify yellow fever virus in uganda a new phlebovirus associated with severe febrile illness in missouri a sensitive assay for virus discovery in respiratory clinical samples a viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing sequence-specific cleavage of small-subunit (ssu) rrna with oligonucleotides and rnase h: a rapid and simple approach to ssu rrna-based quantitative detection of microorganisms evaluation of convenient pretreatment protocols for rna virus metagenomics in serum and tissue samples enhanced methods for unbiased deep sequencing of lassa and ebola rna viruses from clinical and biological samples library construction for next-generation sequencing: overviews and challenges complete viral rna genome sequencing of ultra-low copy samples by sequence-independent amplification multiple displacement amplification compromises quantitative analysis of metagenomes genomic dna amplification by the multiple displacement amplification (mda) method challenges of the unknown: clinical application of microbial metagenomics development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing determining hepatitis c genotype by analyzing the sequence of the ns5b region minor groove binder modification of widely used taqman probe for hepatitis e virus reduces risk of false negative real-time pcr results fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis c virus a novel internally controlled real-time reverse transcription-pcr assay for hiv-1 rna targeting the pol integrase genomic region the khmer software package: enabling efficient nucleotide sequence analysis spades: a new genome assembly algorithm and its applications to single-cell sequencing nhmmer: dna homology search with profile hmms fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools assessment of the utility of whole genome sequencing of measles virus in the characterisation of outbreaks mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods blast+: architecture and applications comparative analysis of rna sequencing methods for degraded or low-input samples metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus whole genome amplification: abundant supplies of dna from precious samples or clinical specimens environmental whole-genome amplification to access microbial populations in contaminated sediments sequencing genomes from single cells by polymerase cloning amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded dna viruses epidemiology and impact of hiv coinfection with hepatitis b and hepatitis c viruses in sub-saharan africa highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data identification of a new human coronavirus identification of a contemporary human parechovirus type 1 by vidisca and characterisation of its full genome evaluation of unbiased next-generation sequencing of rna (rna-seq) as a diagnostic method in influenza virus-positive respiratory samples patient 0' hiv-1 genomes illuminate early hiv/aids history in north america analysis, optimization and verification of illumina-generated 16s rrna gene amplicon surveys a novel virus in swine is closely related to the human hepatitis e virus a modified rna-seq approach for whole genome sequencing of rna viruses from faecal and blood samples the authors would like to acknowledge the contribution of nibsc for kindly providing the viral multiplex reference panel and the blood borne virus unit of virus reference department, phe, for providing the hev sample. the genomic services unit at phe are acknowledged for running the miseqs. the authors are grateful to dr. brendan healy, dr. owen seddon and dr. nicola price from university hospital of wales for providing additional information regarding the patient sample series. this work was supported by funding from public health england. supplementary information accompanies this paper at doi:10.1038/s41598-017-02239-5competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-015372-76xvzvdg authors: nan title: national scientific medical meeting 1996 abstracts date: 1996 journal: ir j med sci doi: 10.1007/bf02945204 sha: doc_id: 15372 cord_uid: 76xvzvdg nan zero months months months months group a 5.53 5.09* 5.66** 6.11"* 6.26** 4 zero months group b 6.06 6.4** values are means; *p<0.05 and **p<0.0l~ most other studies show neutral effects on insulin sensitivity, with minimal incidence of glucose intolerance. this may be partly explained by the diversity of age, diagnosis (whether insufficiency or deficiency state), other pituitary deficiencies and replacement therapy, and possibly by dosage ofgh utilised in these studies. clostridium difficile-associated disease (cdad) is primarily a nosocomial condition. community-acquired disease has been described but the incidence is low "). during a recent outbreak, we prospectively reviewed all new cases of cdad to determine what proportion of cytotoxin positive cases were community or hospital acquired. during a 4 month study period, 73 cases were identified. history of diarrhoeal episodes were recorded for each case. selected isolates were typed using pyrolysis mass spectrometry (pms). community-acquired cdad was defined as diarrhoea, on or within 72 hours of admission, in association with a positive stool cytotoxin test for c.difficile and in the absence of hospitalisation within 60 days. sixty-five cases (89%) were hospital acquired. fifteen patients (20.6%) had cdad on admission; 8 (11%) were community acquired, 7 (9.6%) had been recently hospitalised (4 at st. james's hospital, 3 at other hospitals). pms typing of faecal isolates revealed that 2 predominant strains were responsible for the hospital outbreak; one of these strains was also isolated in community-acquired cases. this study suggests that the incidence of communityacquired cdad may be higher than previously reported. we suggest that all newly admitted or transferred patients with diarrhoea should be screened for this organism. several studies have confirmed that activated protein c resistance (apc-r) occurs in 20-60% of thrombosis patients and is therefore more common than congenital deficiencies in the inhibitors of coagulation such as atii1, proteins c and s. homozygosity for the factor v (fv) gene mutation is associated with a 50-100 fold increased risk of venous thrombosis while heterozygosity is associated with a 5-10 fold increased risk. the mutation, however, is highly prevalent in the general population, a prevalence of 5% has been reported in several european countries. its frequency in the population of northern ireland (ni) has not yet been reported. we screened a group of 72 generally healthy elderly individuals (av. age 81.3; range 76-97 yr on several occasions for apc-r using an assay based on the prolongation of activated partial thromboplastin time by the addition of apc. a mean ratio of 2.64 (range 1.72-3.46) was measured. seven individuals (9.8%) had ratios <2.3 (av. 2.11; range 1.72-2.28). these subjects were then analysed for the fv mutation by pcr amplification and restriction analysis. the 4 individuals with the lowest ratios (av. 2.0; range 1.72-2.15) were found to be heterozygous for the mutation. none of these 4 individuals were deficient for protein c, protein s or atiii. the frequency of apc-r (5.8%) within this ni elderly group is similar to that reported by others in the uk whose studies would have included a generally younger population. the successful ageing of these individuals perhaps underlines the low risk associated with heterozygosity. alternatively a higher prevalence of the mutation may exist in the general population of ni, where the incidence of heart disease is one of the highest worldwide. the insulin-like growth factor ii gene (igf2) is imprinted. thus, in contrast to most genes where both maternal and paternal copies (alleles) are transcribed into rna and expressed, 1gf2 is normally only expressed from the paternal copy (monoallelic expression). alterations causing biallelic expression of igf2 may lead to excess growth factor production, and thus, to tumourigenesis. this study evaluated igf2 expression in a series of childhood cancers. to date 41 tumours have been evaluated using pcr based methodology (26 wilm's tumours, 12 rhabdomyosarcomas, 3 miscellaneous). dna was extracted and a polymorphic site apal within the igf2 gene was amplified and digested. this identified 10 samples as heterozygous for igf2, meaning that separate maternal and paternal alleles were distinguishable. rna from these informative samples was extracted, pcr amplified and restriction digested, to identify monoallelic versus biallelic profiles at the expression level. samples with normal imprinting (monoallelic) displayed allele a (236bp) or allele bl/b2 (176/63 doublet). biallelic samples displayed both alleles. using this approach 3/5 informative wilm's tumours and 2/ 4 rhabdomyosarcomas demonstrated biallelic expression. in conclusion, biallelic expression of igf2 was detected in a significant number of wilm's tumours and rhabdomyosarcomas, and should be considered; with other genetic alterations, as a candidate mechanism in tumourigenesis. the proinflammatory cytokines, tnf~, il8 and il-6, may mediate host metabolic and immune responses to cancer possibly leading to paraneoplastic phenomena such as cachexia. the cellular origin of these cytokines in the cancer patient, in many cases, remains unknown. we examined proinflammatory cytokine levels intracellularly, using flow cytometry, in pbmcs from oesophageal cancer patients (n=14) and age and sex matched controls (n=10). tnf~ and il-6 levels were significantly increased (p<0.05) in pma stimulated t cells and monocytes from the cancer patients when compared to the healthy controls. these results were confirmed using standard elisa assays. following cotlagenase digestion, increased levels of tnfa and il-6,were detected in oesophageal tumour infiltrating t cells when compared to cells from normal mucosa. there was also increased production of tnf~ and il-6, but not il-ib, in malignant epithelial cells when compared to normal and halothane and maintained by 50% nitrous oxide-oxygen, 0.75-1.5% halothane, with spontaneous ventilation. tc rose marginally in group 1 and fell in group 2 (not significant, ns). tp in groups 1 and 2 at induction and 20 and 30 minutes were, respectively, (mean + sem, celsius) 31.4+0.33 vs 31.6+0.3, ns; 33.9+0.3 vs 33.4+0.3, ns and 34.5+0.4 vs 33.2+0.3, (p<0.05). overall incidence of shivering-was 14.%, but not significantly different between the groups. the data suggests that preemptive application of the space blanket increases tp in paediatric patients during general anaesthesia and tends to conserve tc. chronic actinic dermatitis (cad) an uncommon, eczematous, photosensitive eruption is diagnosed on history, clinical examination, skin biopsy and abnormal light tests. drug induced photosensitivity may look identical clinically, have a similar history and patients with cad may be treated with potentially photosensitising drugs. we therefore reviewed all patients with cad and compared their monochrumator light tests with patients who had drug induced photosensitivity. phototesting was performed on unaffected skin of the back with an irradiation monochromator; the minimal erythema dose (med) determined for a series of wavelengths between 300 and 400 nm, in 14 patients with drug induced photosensitivity and 7 patients with cad. of ten females, four males with drug induced photosensitivity, age range 40-77 (mean 61 yrs), ten (71%), were photosensitive in the uva range (320-370nm), the implicated drugs including, quinine, sparfloxacin, amiodarone, doxycycline, mefenamic acid, nalidixic acid, fenbrufen, diclofenac, enalpril and prochlorperazine maleate. three patients with rosacea were photosensitive to doxycycline. the re/nainder (29%), were tested after discontinuation of the drug and their light tests were normal. in the cad group, (four males and three females), age range 38-86 (mean 71.5 yrs), three patients (43%), were sensitive to uva, uvb and visible light, four (57%) to uva and uvb. in conclusion, uva dissociated from uvb photosensitivity seems a relative but not absolute sign of drug induced photosensitivity. this pattern of light tests should prompt a detailed drug history to elucidate the causative agent. phototesting should be performed while on the offending drug as testing days or weeks after discontinuation will give normal results. patients at risk of bone fractures by measuring bone mineral density (bmd) and markers of bone turnover and to assess the correlation of these with the severity of cld. twenty three patients with cld had bmd measured by dual-energy x-ray absorptiometry scanning of hip and lumbar spine. bone formation was assessed using serum levels of procollagen type 1 peptide, osteocalcin and bone alkaline phosphatase, and bone resorption was assessed using 2 hour urinary excretion of hydroxyproline, pyridinoline and deoxypyridinoline. 48% and 39% of patients had evidence of osteoporosis at the lumbar spine and femoral neck respectively. biochemical results showed that 74% of patients had an increase in all 3 bone resorption markers and 42% had an increase in markers for bone formation. bmd at the lumbar spine was lower in patients with cholestatic liver disease compared to patients with other types of'liver disease (p=0.004). no correlation was found between bmd and patient age, bilirubin, albumin, inr or duration of liver disease. conclusions: osteopenia occurs in up to 62% of patients with cld due to a high bone turnover state where bone resorption exceeds formation. osteoporosis is most severe in those patients with cholestasis. a detailed profile is presented of all 632 leukaemia and multiple myeloma (icd-o code 169) patients registered by the southern tumour registry during the 8-year period 1983/1990 "). annual age-adjusted rates of 13.9 and 8.7 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk of 1 in 68 for males and 1 in 116 for females. the main morphological sub-types registered were multiple myeloma (31%), cll (25%), aml (18%) cml (9%) and all (8%). one, two and five-year survival rates were examined; age at diagnosis and lesion type were extremely significant factors in relation to patient outcome. the overall incidence levels indicate that irish rates were relatively high by internatiomil standards r we assessed effects of reducing the volume of hyperbaric bupivacaine by giving half the volume as isobaric bupivacaine. when using 0.5% hyperbaric bupivacaine for spinal blockade, the segmental spread and cardiovascular effects of the block have been shown to be dependent on the volume of local anaesthetic injected. 40 patients undergoing elective surgery were studied in a prospective, randomised, double-blind trial: group 1 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine and 0.5% isobaric bupivacaine respectively; group 2 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine. there was no significant difference found between the two groups with regard to maximal height of block (group 1, mean (range), t7 (t4-ti 1); group 2, t8 (ts-t11)), rate of onset of blockade, or time to maximal blockade (group 1, mean (sem), 23.55 (2.41) rain; group 2:20.89 (2.95) min). there was no difference found between each group in either cardiovascular stability or vasopregsor usage. the administration of a mixture of 0.5% isobaric bupivacaine and 0.5% hyperbaric bupiv/~caine confers no advantages over administration of the same volume of 0.5% hyperbaric bupivacaine alone. propofolis used as a sedative during regional anaesthesia. providing titrateable sedation, it can compromise haemodynamic stability. a propofol ketamine combination provides stable haemodynamics during total intravenous anaesthesia, avoiding emergence phenomena m. we compared two sedative regimes in patients having spinal anaesthesia. following informed consent, 40 patients, asa i-ii, undergoing spinal anaesthesia were randomized to one of two groups (n=20). group i (propofol-ketamine) received loading doses of 0.4mg/kg propofol, 0.1mg/kg ketamine followed by an infusion of 1.2mg/kg/h and 0.3mg/kg/h respectively. group ii (propofol) received bolus 0.5mg/kg and infusion 1.5mg/kg/h. subsequent infusion rates were titrated to effect using a sedation score. heart rate, blood pressure, oxygen saturation, end tidal c02 and oxygen requirements were recorded. observation continued for the recovery period and patients visited the following day. data were analysed using t-test, chi 2 test and anova. groups were demographically comparable. sedative and respiratory indices were similar for both groups. there was no difference in total propofol requirements between the groups; group i -146_+194mg, group ii -137_+1:52mg (mean _+ sd). there was a large difference in mean arterial pressure, being much lower in the propofol only group. both groups had an uneventful recovery without emergence phenomena. our results do not confirm the described additive effect of andketammc . ketamine.with propofol for sedation propofol ' =~2) confers haemodynamic stability during spinal anaesthesia. we designed a controlled study to investigate whether there is a direct relationship between the degree of postoperative pain and the development of negative middle ear pressure in adults following tonsillectomy. middle ear pressure was measured by tympanometry. pressures were classified as type a (o to -99 mmh20), type b (flat) or type c (-100 to -350 mmh20) tympanograms. 30 patients with type a tympanograms, undergoing tonsillectomy were enrolled in the study. patients had daily tympanometry whilst in hospital and then weekly until amrmalisatign. a questionnaire incorporating visual analogue pain scores was filled in at the same time. a control group of 26 patients with type a tympanograms, undergoing appendicectomy and endotracheal intubation was used. follo~v up was available on 26 patients. 13 patients (50%) developed type c tympanograms, 5 patients 09%) type b and 8 (31%) patients remained unchanged. no member of the control group developed any change in middle ear pressure (chi squared = 27.5, p < vol. 165, 1996 irish journal of supplement no. 2 medical science 0.001). there was no relationship between pain scores for throat pain or otalgia and the development of negative middle ear pressure. 8 patients recorded higher pain scores for throat pain at day 7 then day 1, only 3 of this group had negative middle ear pressure. middle ear pressure reverted to normal at day 7 in 15/18 patients and in the remaining 3/18 it was normal at day 14. this study demonstrates the development of transient negative middle ear pressure following tonsillectomy in 69% of patients. this change is unrelated to the degree of postoperative pain nor is it associated with otalgia. postoperative ward analgesia remains suboptimal. this may be partially related to inadequate early use of opioids to attain minimum effective analgesic concentration (meac). we examined the incidence and predictors of severe postoperative pain on admission and discharge from our postoperative recovery room (rr). verbal pain scores were obtained in a pilot study of 202 patients on rr admission and discharge. procedures were classed as open cavitary, laparoscopic, orthopaedic, ent or body surface surgery. intraoperative use and dosage of narcotics and nonsteroidal (nsaid) analgesics, anaesthetists' experience (prefellowship or post-fellowship nchd, consultant) were noted, and rr opioid usage recorded. pain scores and analgesic use were examined using mann-whitney, ~2 analysis and logistic regression. moderate or severe pain was experienced by 25% of patients on either arrival or discharge. median intraoperative morphine dosage was 5 mg. opioid use was slightly (median morphine dosage 8 mg, p < 0.05) higher in patients undergoing cavitary surgery; these patients had the highest pain scores on rr arrival and departure. 21 patients (10%) received > 10 mg morphine intraoperatively. discharge scores of 6/10 or higher occurred in 24 patients (12%). opioid usage and pain scores were unrelated to level of training. nsaid use/nonuse was unassociated with differences in opioid use or rr pain scores. no morbidity attributable to analgesic use (desaturation, slow respiratory rate) occurred. nonattainment of meac is frequent after open cavitary surgery. conservative opioid dosages continue to be employed despite inadequate early postoperative pain relief; this does not change with increasing experience. reporting such findings in departmental audit may help to alter perioperative management; such data may serve as a baseline for future interventional studies. diclofenac is frequently used for analgesia after tonsillectomy. recently concern has been expressed about the effect of diclofenac on prolonging bleeding time. one recent retrospective study found its use in tonsillectomy was associated with an increase in reactionary haemorrhage. we designed a randomised controlled study to compare the effects of rectal diclofenac and im pethidine given at induction with pethidine alone, in children undergoing tonsillectomy. fifty nine patients were entered into the study. there were 26 males and 33 females, mean age 6 years, range (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . patients were randomised according to chart number. thirty five patients received rectal diclofenac after induction. twenty four patients acted as controls. there were no significant differences in operating time or operative blood loss between the two groups. in the recovery room the diclofenac group was significantly less restless than tl~e control group (p < 0.05, chi squared test), with less crying, movement and agitation. there was no difference in postoperative recovery and no primary or reactionary haemorrhage. one patient in the diclofenac group developed a secondary haemorrhage. this study demonstrates a significant reduction in restlessness in the recovery room in children receiving rectal diclofenac. no increase in reactionary haemorrhage was demonstrated. diclofenac remains a safe and effective analgesic agent in children undergoing tonsillectomy. elderly patients have decreased dose requirements for many drugs compared to the young. few studies have examined dose requirements of opioids in the elderly when administered via patient controlled analgesia (pca) 1. we compared the pca morphine requirements between young and elderly patients. records were retrospectively analysed from 1,000 consecutive patients receiving pca for post-operative pain. inclusion criteria (i) age less than 10 years or greater than 60 years, (ii) upper abdominal surgery, and (iii) morphine pca usage. 238 patients fulfilled the inclusion criteria. 89 patients were young and 149 were elderly. the mean age in the young was 29 years and in the elderly was 69 years. 51% were female in the younger group, 40% were female in the older group. pain scores at rest and on movement were similar in both groups 3.8 and 6.7 respectively in the young, 3.2 and 6.2 in the elderly. (p > 0.05, students t-test). morphine usage over 48 hours was 58 + 32.1mg in the young, and 37 + 20.3 in the elderly. (mean + s.d.) (p.< 0.05, students t-test). elderly patients required significantly less morphine via pca to achieve the same pain scores as the young. these findings are consistent with studies showing decreased requirements of other drugs in elderly. the erythrocyte sodium-lithium countertransport (slc) is abnormal in essential hypertension and some other forms of cardiovascular disease (cvd) but the considerable overlap in its activity in patients with these conditions and in normotensive healthy subjects remains a strong point against its possible utility as a marker for cvd. we sought to address this issue in greater detail. twenty-nine hypertensive patients (19 with family history of cvd) aged 56.4+1.9 years (mean + se) and 32 normotensive subjects (12 with family history of cvd) aged 45.9 + 1.9 years, participated after informed consent. slc were determined in 35, 50, 70, 100 and 140mm sodium chloride; and the vmax and km of the transporter determined. hypertensive and normotensive individuals with family history of cvd (n = 31) had higher slc activity (0.331 + 0.011 vs 0.238 + 0.011, p < 0.0005), greater vmax (0.473 + 0.019 vs 0.397 + 0.018 mmol/lcell.h, p < 0.006) and lower km 56.0mm (median) vs 95.5 (p < 0.001) than hypertensive and normotensive subjects without such a history (n = 30). however, none of these parameters was sufficiently discriminatory as evidenced by the considerable overlap in the scattergrams for the two groups. on the other hand not only was the median quotient vmax/km significantly different 7.89 vs 4.20 (p < 0.001), but also the scattergram separated the two groups. this may reflect an effect of hereditary factors on the identified rate-limiting step in the transport system. capsaicinadministration results in depletion of substance-p sensitive nerves. this study was carried out to observe the impact on the morphology of mitral valve endothelium. the experimental group received capsaicin i.p. on day four of life; control animals received drug vehicle only. animals were anaesthetised by chloral hydrate and hearts were removed following perfusion of 4% glutaraldehyde, and were routinely processed for scanning electron microscopy. normal endothelial morphology showed an ordered and structured pattern, with large raised nuclei covered in discrete microappendages: no zoning was observed over the valve surface. following capsaicin administration, valves were seen to be torn and possessed a denuded endothelium. nuclear bulges changed in both apparent height and area, with the surface partially denuded of microappendages. one month following systemic administration of capsaicin to neonatal rats, a serious alteration of mitral valve endothelium morphology and integrity had occurred. depletion of substance-p may have resulted in mechanical insufficiency of the mitral valve. this study was funded by the health research board. with expanding applications and increasingly aggressive stress protocols, concerns about the safety of dobutamine stress echocardiography (dse) have arisen. the purpose of this study was to analyse prospectively the safety, adverse event profile, and complication rate of dse. prospective data was recorded in a consecutive series of 309 patients undergoing dse for diagnostic evaluation of chest pain, for risk assessment following myocardial infarction or for detection of hibernating myocardium. the maximum dose of dobutamine used was 50 mcg/kg/min in 24.6% of patients and 40 mcg/kg/min in 73.1%. atropine was used in 6. 8% long-term outcome following coronary artery bypass grafting may be related to the prevalence of major risk factors and their treatment following surgery. we aimed to establish the prevalence and current management of coronary risk factors in a group of consecutive patients attending our hospital. this is a report of the first 100 patients. data was collected by a structured patient interview, chart review, physical measurements and blood sampling. there were 74 male and 26 female patients, average age 61.5 years. the mean length of time since surgery was 2.2 years. thirty-nine patients had a recurrence of angina and this occurred on average ll months after surgery. as regards risk factors, 22 were active smokers, 56 were ex-smokers and only 22 had never smoked. two thirds of the patients were taking regular exercise; only 6 took no exercise at all. seventy-two percent of patients had a cholesterol greater than 5.5mmol/l, yet only 10 of the patients were on lipid lowering drug therapy and a further 16 were on a lipid towering diet. twenty-nine patients had a systolic bp > 160 or a diastolic bp > 90 and 15 of these were on antihypertensive therapy. seventy-seven patients were overweight but most of these had received specific advice regarding weight reduction in the preceding year. our results show a high prevalence of treatable risk factors in this high-risk group with inadequate treatment in many cases. new combined primary and hospital care strategies for cardiac rehabilitation and long-term secondary prevention of coronary heart disease are required. if the goal of modern therapy of acute myocardial infarction (ami) is preservation of myocardium, the occurrence of cardiac failure could be regarded as a treatment failure. in recent studies of iv thrombolysis and primary ptca the frequency of left ventricular failure (lvf) following ami has been as low as 3.5%. this very low rate might be explained by selection bias in patients recruited to randomized trials. the purpose of this study was to examine the frequency of lvf in a consecutive alterations in nitric oxide (no) synthesis have been implicated in cardiomyopathy, ischaemic heart disease and septic shock. recent work has suggested a possible role for nitric oxide in cardiac arrhythmogenesis. the effects of inhibiting no synthesis with l-name (n c -nitro -l -arginine methyl ester) on cardiac electrophysiology have not been fully determined. the dominant frequency of electrically induced ventricular fibrillation was determined in l0 anaesthetised pigs (30-42 kg) using a fourier transform. the dominant frequency (7.8 _+ 0.5 hz in lead ii) was not altered by treatment (8.5 _+ 0.5 hz) with l-name in a group of 16 pigs (45-58 kg) the effect of l-name (10 mg/kg) was assessed in relation to energy required to defibrillate. there was no significant difference in the energies required to achieve successful defibrillation on 80% of attempts between the l-name group (104.9 _+ 8.0 j) and the control group (111.1 _ 11.9 j), and on 100% of occasions, l-name (122.5 + 11.5 j) and control (137.5 _+ 13.1j). the results show that inhibition of no synthesis has no significant effect on the dominant frequency of ventricular fibrillation or on the efficacy of defibrillation in the pig heart. received either 100mg (7pts) or 200mg (19 pts) oral flecainide followed by a maintenance dose of 100mg bd. all pts were euthyroid. none had significant hypertensive, valvular or ischaemic heart disease. 23 pts had normal echocardiograms, 3 had mildly dilated atria. of the 26 pts 17 (65%) converted to sinus rhythm, 15 within 72 hrs and 2 at 12 and 21 days respectively. 2 subsequently, he was found to have paralysis of all the muscle groups of his right upper limb apart from some flexor movements in his fingers with areflexia and sensory loss over the c5/c6 dermatomes. the findings were thought to be in keeping with a brachial plexus.lesion. an mri scan showed a "haematoma" or "fibrosis" around the brachial plexus~ emg studies revealed a complete lesion from c5/c7 with evidence of partial function at c8 and a good function at c4. despite physiotherapy, at follow up 2 months later there was no improvement in the emg findings. though brachia~ plexus injury has always been considered a complication of central venous line insertion <l), there have been no cases described, in the literature in the last 10 years. central lines are mandatory irt major surgical cases but one must always be aware of the possible complications caused by them. body weight (bw) in icu patients fluctuates due to alterations in body mass (1) and water (2) and is usually estimated rather than measured. we hypothesised that (i) estimated weights are inaccurate and (ii) changes in true body mass (tbm) are masked by changes in water content. on admission, icu patients whose expected length of stay (los) was > 5 days were placed on a bed incorporating 4 load cells (accuracy of _ 0.1 kg). bw was estimated by icu staff. mbw was then recorded 12 hourly under standard conditions. changes in mbw due to pack insertion/dressing changes were excluded. we calculated tbm as mbw minus cumulative fluid excess, corrected for insensible losses c2~. patients received standardized nutritional support from day 3 and urinary nitrogen on day 6 was calculated we studied 20 patients whose mean (sd) age, mbw, los and apache ii score were 44.3 yr (18.54), 70.5kg (14.39), 10.0 days"(6.78) and 18 (3.8) respectively. mean error in estimated weight on admission was 10.3% (nurses) and 12.2% (doctors). mean protein and calorie intake was 64g and 1670 kilocals/day. mean decrease in mbw during icu stay 0.22 kg/day (range 0.21 kg (gain) to 0.49 kg). mean reduction in tbm was 0.56 kg/day (range 0.28-0.86 kg/ day) and this correlated with urinary nitrogen loss (r=0.7). in icu, (i) estimated weight is significantly inaccurate and should not be used in physiological calculations and (ii) rapid and significant decreases in body mass occur which may be underestimated due to fluid accumulation. dopamine appears to influence pituitary function and is associated with decreased circulating human growth hormone and insulin-like growth factor i m which could exacerbate catabolism, and therefore wasting, during critical illness. at 12 hrly intervals from admission, patients whose expected length of stay exceeded 5 days, were weighed (measured body weight, mbw) under standardized conditions,-using a bed incorporating 4 electronic load cells (accuracy + o.1 kg). standard demographics, apache ii score and use of dot~amine by infusion was recorded. changes in weight due to removal./ insertion of prostheses, packs/dressings were excluded mbw was converted to true body mass (tbm) using measurements of cumulative fluid balance (1000 ml = 1 kg) and insensible lossr patients received nutritional support, starting on day 3, based on their admission mbw. there were no significant differences in mean age, weight, length of icu stay, admission apache ii scores and mean daily protein/calorie intake between 14 patients who had received dopamine by infusion (group d) and 18 who had not (group nd). mean (sd) decreases in mbw during icu stay were 0.42 (0.08) kg/day (group d) and 0.20 (0.05) kg/day (group nd) (p<0.0i). mean decrease in tbm was 0.55 (0.10) kg/day and 0.35 (0.07) kg/day respectively (p<0.05). thus group d were losing an additional 1.4 kg body mass per week relative to group nd. the use of dopamine by infusion is associated with an accelerated loss of lean body mass during critical illness. adhesion of polymorphonuclear leucocytes (pmns) to pulmonary endothelial cells is an initial step in the inflammatory process characterising the adult respiratory distress syndrome. previous studies using human umbilical vein endothelial cells (huvecs) have suggested that lipopolysaccharide (lps) is a potent stimulus for pmn adhesion to endothelial cells. the aim of this study was to investigate the effect of lps on pmn adhesion to human pulmonary artery endothelial cells (hpaecs). human pmns were coincubated with hpaecs _+ lps (0.001-10mg/ml) with 2% serum for 1 hour. the effect of phorbol myristate acetate (pma) (125 ng/ml) was also examined. percentage adhesion stimulated by pma was 66+10sd. lps did not significantly increase adhesion at any of the concentrations used. to confirm the activity of lps pmns were incubated at 37~ with 0.01-10mg/ml lps + 2% serum for 1 hour and labelled with flourescent antibodies to the mac1 adhesion molecule complex (cd 18/cd 1 lb). facs analysis indicated upregulation of both cd18 and cdllb. thus in the system used, lps did stimulate pmn adhesion molecule expression, indicating that the lack of adhesion reflects a difference in hpaec response to lps compared to that reported for huvecs. this work was supported by the health research board ireland. although inhaled corticosteroid therapy is of undoubted benefit in the management of asthma, dysphonia is a recognised sequela. this study was designed to examine longitudinally the effect of inhaled steroids on the voice and vocal cords of newly diagnosed and previously untreated asthmatics. twenty subjects were recruited and underwent voice and vocal cord assessment prior to and 3 months after starting inhaled steroid treatment. the assessment consisted of 1) rating dysphonia using a visual analogue scale, 2) acoustical analysis of the voice and 3) videostroboscopic examination of vocal cord activity. prior to commencing inhaled steroid therapy for their asthma 14 subjects had normal voices, 5 subjects were mildly hoarse and one was moderately hoarse. vocal cord pathology was noted in 12 subjects, 2 patients had vocal cord nodules and the remainder were noted to have mildly oedematous cords together with a glottic chink. at 3 month follow up, improvement in voice was noted in 2 subjects, one patient felt more dysphonic but there was no change in vocal cord appearance. one subject was noted to have developed a mid glottic chink with no associated change in voice. one subject had clearing of mild vocal cord oedema and improvement in voice. this study demonstrates that 60% of subjects commencing inhaled steroid therapy for asthma have mild vocal cord pathology. voice is more likely to be improved following use of inhaled steroids for 3 months then made worse. although the relationship between elastin degradation and emphysema is well known, recent evidence suggests that a more complex process of pulmonary remodelling occurs within the emphysematous lung. the aim of this study was to assess the extent of extracellular matrix remodelling by ultrastructural examination of its two major components, elastin and collagen. emphysema was induced in rats by the intratracheal administration of porcine pancreatic elastase (1.2u/g body weigh0 and human lungs were obtained at surgical resection for lung carcinoma. emphysema was confirmed histologically in both animal and human samples by measurement of the mean. linear intercept. matching sections were immersed in 2.5m naoh and 88% formic acid to digest elastin and collagen respectively. scanning electron microscopy with stereo-pair imaging allowed 3-d visualisation of elastin and collagen frameworks. the distribution of emphysema was primarily panacinar in rat lungs a'nd centriacinar in human lungs. as expected in both types of emphysema, elastic lamellae were disrupted and perforated with multiple fenestrations. accompanying this disintegration was a marked increase in thickness of collagen fibrils which in some cases coalesced irish journal of medical science imparting a sheet-like appearance to the airspace walls. unique to human centriacinar emphysema, collagen formed helices which spiralled around alveolar septae to form bulky walls between adjacent airspaces. in conclusion, these findings lend support to the novel concept of aberrant collagen remodelling in the pathogenesis of emphysema. small cell lung carcinoma (sclc) is the most aggressive of the four common cell types of lung carcinoma. less than 10% of patients with sclc are alive two years after diagnosis. staging procedures, treatment regimens and survival results were reviewed in a small regional centre to make a comparison with larger treatment centres. thirty-one cases of sclc seen by one physician from 1985 to 1993 were reviewed. staging was clinical. treatment was undertaken in conjunction with the local oncology and radiotherapy services. 42% of patients had limited disease where as 58% had extensive disease at diagnosis. in patients with limited disease, 20% were alive at 18 months and there was a 10% long term survival rate i.e. greater than three years. average length of survival in limited disease was 456 days. survival results were comparable with those treated with chemotherapy alone and combination chemotherapy and radiotherapy. in patients with extensive disease the best results were from those treated with a combination of chemotherapy and radiotherapy with art average survival of 353 days. these figures compare favourably with those from larger multidisciplinary centres. the factors contributing to our relative success may relate to continuity of care achieved in a smaller centre. nasal cpap is a very effective therapy for osa, but is cumbersome, and compliance varies. we prospectively evaluated 91 consecutive osa patients treated with ncpap, who were asked to complete questionnaires before, and 2 to 12 months after starting therapy. this stttdy intended to examine both the patient's subjective response to ncpap and their bed-partner's impressions also. replies were received in 84 (92%) patients. patients were divided i~to 4 groups depending on whether they had a bed-partner, and according to their response to an initiat question assessing overall improvement in sleep quality and daytime al'ertness with ncpap, ranging from 0 (minimal/none) to +4 (excellent). patients were called responders if they scored > 2. group a (45 pts, 54%) were responders with bed-partners; group b (10 pts, 12%) non-responders with bed-partners; group c (15 pts, 18%), were responders (12 pts, 14%) and nonresponders (3 pts, 4%) without bed-partners; and group d (14 pts, 17%) had stopped ncpap. nine questions were directed at the bed-partner, and assessed their perception of changes in both the patient's and their own sleep quality, daytime alertness, mood and quality of life, and also to changes in the relationship between patient and bed-partner following institution of ncpap. these questions scored from -1 (worse) to +3 (marked improvement). significant improvement in all parameters for the patient (mean + sd = 2.4 + 0.7) were noted in group a. in addition, group a bed-partners reported ~ubjective improvement in the same parameters (1.7 + 1.1). group b improvements were less, (1.8 + 1.0 in patients, and 0.6 + 1.1 in partners). overall, the data indicate a subjective success of therapy in 68% of patients, but the bed-partner's replies indicate this figure underestimates the true response rate. furthermore, the results show significant improvements in the bed-partner's sleep quality, daytime alertness, mood and quality of life, indicating that successful treatment of osa patients with ncpap also gives significant benefits to their bed-partners. vincent's hospital, dublin. neutrophil collagenase (mmp 8) is a member of the matrixmetalioproteinases (mmps), a family of highly homologous zinc endopeptidases which play a crucial role in many physiological processes. the aim of this project was to develop a purification system for mmp 8 from purulent sputum and raise polyclonal antibodies. after initial extraction, contaminating proteins were removed with a zinc chelate affinity column. mmp 8 was then separated, from another closely related mmp, gelatinase b, on a q sepharose ion exchange column using a nacl gradient. the final purification step was carried out with an orange sepharose affinity column. sds-page analysis indicated the presence of purified protein with bands corresponding to latent neutrophil collagenase (85kd) and products of coll~igenase autodegradation at lower molecular weights (55kd & 57kd). a 10fold increase in specific activity was observed, with a 20% final yield, which provided mg quantities of pure enzyme. this work is funded by forbairt and the. irish american partnership. cd30 is a protein first described as a surface marker on hodgkin's lymphoma cells. recently cd30 has been demonstrated on th2-type t lymphocytes (produce il-4, 5, 6, l0 and 13), which have a pro-inflammatory cytokine profile. but is not found on thl-type t lymphocytes (produce il-2 and ifn-gamma). its ligand, cd30l, has also been described, cd30-cd30l interaction has been shown to aid the development oft lymphocyte clones into a th2 rather than a th 1 phenotype. th2-type cytokines are inextricably linked to the aetiology of inflammatory airway disease. firstly we investigated serum cd30 levels in various patient groups. we have demonstrated significant differences in serum cd30 levels in the following groups, atopic asthmatics (mean = 149 iu/l, n = 62), non-atopic asthmatics (mean = 107 iu/l n = 13) and atopic rhinitis/dermatitis (mean = 90 iu/l n = 36) and normal controls (mean = 14 iu/l, n = 9). secondly we cultured peripheral blood mononuclear cells from allergic individuals and normals. when these cultures were stimulated with house dust mite antigen (der p 1) and il-2 or der p 1 with both il-2 and i1-4, surface expression of cd30 on t lymphocytes could be demonstrated using fluorescent staining and flow cytometric analysis, after 9 days culture in the allergic individuals but not in normals. the presence of i1-4 in the culture increased the degree of surface cd30 expression. these results are important as they show that allergic individuals have an expandable population of memory t lymphocytes which respond to allergen by expressing cd30 and developing th-2 phenotype. most work on cd30 and th-2 cytokines has hitherto been carried out an t cell clones. we have developed a relatively simple in vitro system of looking at t lymphocyte response to allergen which will allow the testing of novel therapeutic interventions with a view to modulating the immune response in allergic disease. our work also suggests that even non-allergic patients with inflammatory airway disease may have increased th2 activity, which has not been shown previously. scimitar syndrome is a rare congenital disorder consisting of a spectrum of abnormalities including hypoplasia of the right pulmonary artery, dextroposition of the heart, anomalous pulmonary venous drainage of the right lung into the inferior vena cava and anomalities of the right diaphragm. bronchiectasis and respiratory tract infections on the right side are the usual clinical presenting features. a 70 year old male patient was referred to the outpatient clinic with a history of recurrent chest infections which were slow to resolve following antibiotic therapy. physical examination revealed decreased air entry, coarse crepitations and a prolonged expiratory wheeze in the right lower lobe. the only abnormalities on routine biochemical and haematological screening were an elevated esr of 69 and a slightly raised white cell count of 15. a chest x-ray revealed hypoplasia of the right lung when compared to the left. in addition to this there was a vascular shadow present in the right lower robe representing an anomalous pulmonary vein which appeared to drain to below the diaphragm on the night side. bronchoscopy showed a normal left bronchial tree. on the right, no apical segment was detected in the right lower lobe. otherwise no endobronchial lesion was seen. a dynamic computerised axial tomographic scan of the thorax was performed. this showed a dilated anomalous right lower pulmonary vein which was clearly seen to enter the inferior vena cava below the diaphragm. in addition the right lung was again noted to be hypoplastic when compared to the left. these findings were pathognomonic of the scimitar syndrome. "the patient was treated symptomatically and is presently stable. conclusion: scimitar syndrome, with its wide spectrum of abnormalities should be considered when reviewing plain chest x-ray in patients with recurrent right lower lobe respiratory tract infection. recent studies indicate that the ability of circulating neutrophils to regulate surface levels of adhesion molecules may be altered in disease situations. the aim of this study was to determine if changes in neutrophil responsiveness accompanies chronic inflammation in cf. neutrophils in blood samples from 17 cf patients and 13 age-matched control subjects were analysed by flow cytometry for expression of l-selectin and mac-1 (cd1 lb) following stimulation by interleukin-8 (il-8) and fmlp. as expected, both il-8 and fmlp provoked a decrease in surface levels of l-selectin and an increase in cdi lb levels. however, the magnitude of these changes was significantly lower in cf patients than in control subjects (table) . these results suggest that chronic exposure to inflammatory stimulii in vivo may alter neutrophil responsiveness in cf. given the emphasis on rational prescribing, we reviewed drug use in a 208 bedded long-stay unit. prescribing patterns were analysed on an appointed day thereby obtaining a "snapshot" of prescribing practices. one hundred and ninety four long-stay residents, with a mean age of 78, were on 1188 drugs, the maximum number of drugs per patient was 12, the minimum 1 and the average 6.1. sixty percent of prescriptions fell within one of the following therapeutic categories:-central nervous system (cns) preparations (321 prescriptions), analgesics (145), gastrointestinal preparations (139) and cardiovascular preparations (i 25). there were 19 ,prescriptions for respiratory drugs and only 36 prescriptions were for antibiotics. the most commonly prescribed cns preparations were anti-psychotics (127), benzodiazepincs (1 t 3), anti-depressants (30). 43% of all analgesics prescribed (63) were nsaids. the most commonly prescribed h2 blocker was cimetidine (33). nuseals aspirin (43), digoxin (35) and captopril (24) were the most commonly prescribed cardiovascular drugs. 25% of drugs were issued on an as required basis, i.e. "prn". the most commonly prescribed prn therapeutic classes were analgesics (100 prescriptions) followed by gastrointestinal (74) and cns preparations (68). these results contrast with prescribing patterns in hospitals and general practice and may provide an insight into the challenges and realities of management in long-stay units. supported by the health research board. evaluation of physician requests to hospital based clinical pharmacist for (1) drug information, (2) possible adverse drug reaction (adr) was undertaken over a two year period from jan '94 to dec '95 (admissions -1,667, opd attendances -5,908). overall 55 requests were made. (1) drug information: advice/information on new drugs, formulation, dosage, safety consideration prior to drug prescribing was given in 23 cases. (2) suspected adr: a total of 32 suspected adverse drug reactions were investigated. in 4 cases, no adr link was established, after extensive literature/data base search. adr's were confirmed in 28 cases of which 11 were reported to n.d.a.b. regular on-going interaction between physicians and clinical pharmacy allowed critical analysis of new drugs and heightened awareness of, potential adverse drug reactions in current clinical practice. we previously demonstrated that commonly prescribed medications are not easily identified by patients, doctors or nurses in the hospital setting o,2~. we then investigated the ability of hospital pharmacists, 33 in all, to identify the same 12 commonly prescribed branded and generic drugs. correct identification as follows:-bendrofluazide k (32/33), cimetidine (33/33), diazepam 5mg (31/33), diazepam 5mg generic (30/33), digoxin (24/33), ferrous sulphate (14/33), frusemide (16/33), mefenamic acid (33/33), paracetamol (27/33), prednisolone (1 l/ 33), temezepam (33/33), theoph3/lline (25/33). pharmacists had 78% correct answers compared with 62% for nurses and 42% correct for doctors. the pharmacists had no difficulty recognising drugs with brand names written on them e.g. cimetidine, but like nurses and doctors had difficulty identifying the plain white tablets e.g. prednisolone. generic drugs were tess well recognised. a number stated that they were unwilling to definitively identify medication with no clear marking. pharmacists were also asked to list the top 5 prescribed drugs, l0 got 1/5 correct and 23 got 0/5 correct. in contrast 36 out of 80 doctors got 1/5 correct, 25 got 2 right and only 4 got 3 right. we conclude that hospital pharmacists are generally better than doctors or nurses at identifying commonly prescribed drugs but their knowledge of the top 5 prescribed drugs is not as good at that of doctors. all professionals need assistant in this important task. suggesting reduced activity or more iranians with inherited variants of cholinesterase. one iranian subject with very low activity (dibucaine number below 20, atypical) had a history of apnea. these data indicate that the frequency of atypical and heterozygote genes for cholinesterase activity leading to prolonged apnea with succinylcholine (suxamethonium) is much higher in iranian than irish populations. this study emphasises the importance of ethnic pharmacology. it is has been advocated that funding for the prescibing of methadone in general practice should be provided separate from the indicative drugs budgeting scheme on the assumption that this may act as a disincentive to g.p.s to take on care of drug addicts. the objective was to analyse the current level and cost of methadone prescribing in 550 general practices in the eastern health board over a six month period. there was a review of methadone prescriptions for gms patients from jan. to jun. 1995. 1,087 persons received prescriptions. 3,945 scripts were issued. the age-specific prescribing rate for the total population was 6/10,000 (males 10/i 0,000, females 3/10,000). males aged 25-34 years had the highest age specific rates (44/100,000). the cost of methadone prescriptions amounted to s for the six months there was a trend towards an increase in the number of g.p.s who prescribed methadone over the period. only four of the 70 g.p.s (5.7%) who prescribed methadone issued in excess of 50 scripts for the period studied. for a small number of g.p.s methadone prescribing is a significant cost item on their budget. in the light of this, government policy should be reviewed with a view to excluding methadone from the indicative drug budgeting scheme. sciences, mashhed, lran. there is increasing evidence that some of the wide variation in the response to medicines has a genetic origin which may be expressed on a racial basis. to further study inter-ethnic differences in pharmacology we compared the activity of an enzyme responsible for the breakdown of endogenous substances and drugs -serum cholinesterase (pseudocholinesterase), dibucaine and fluoride numbers -in 200 irish and 200 iranian healthy subjects. irish subjects had significantly higher serum cholinesterase activity (7.28 + 0.14 vs 5.22 + 0.09 u/ml, mean + sem, p < 0.01 drug prescribing data may reflect changes in therapy and disease pattern. we reviewed current drug use among patients (n = 578) in a dublin teaching hospital in "snapshot" fashion on a designated day, and compared it with that obtained in 1985. in 1985, patients received an average of 7.8 different drugs each, with 41% on 5 or more and 3% on none. by 1995, the average was 6.8 (range i -19), with 62% on 5 or more. the percentage of patients receiving heparin fell from 42% to 13%, due mainly to a reduction in use on the medical side. the proportion of patients prescribed hypnotics fell from 55% to 37%, while ssri's are now the most used anti-depressants. antibiotic choice changed from amp/amoxicillin to coamoxiclav and the cephalosporins. diuretics remained the most frequently used cardiovascular agents, accounting for 4% of all drugs used and prescribed for around one quarter of patients. digoxin use remained constant, and by 1995, 20% of patients were on anti-platelet doses of aspirin. at least four different agents were in use in each of calcium antagonist, beta blocker and ace inhibitor classes. some of these changes in therapy reflect therapeutic advances, changes in disease management, greater choice of therapy and amendment of less than desirable therapeutic practices. on the other hand, some may reflect fashion or pharmaceutical promotion, rather than change as a consequence of evidence-based practice. acknowledgements: pharmacy staff, st. james's hospital and the health research board. studies of in vivo endothelial function in humans have usually involved intraarterial cannulation and the subsequent administration of substances that stimulate the endothelium to produce nitric oxide (no). such techniques are invasive and potentially hazardous. an alternative non-invasive method would be of benefit. animal studies have indicated that reactive dilation of vascular beds may be at least partially endothelium dependent. this study aimed to determine whether reactive hyperaemia in the human forearm was an endothelial dependent process with the potential to be used as a non-invasive method of stimulating the endothelium. ten volunteers underwent brachial artery cannulation and randomly received either placebo or n-monomethyl-l-arginine (l-nmma) (8~mol/min), an no synthase inhibitor, for 10 minutes. following this reactive hyperaemia was induced by the inflation of an arm cuff to 200 mmhg for 5 minutes and the response to this was measured by strain-gauge plethysmography. when flows had returned to baseline the process was repeated with the remaining substance. results were analysed by repeated me~isures anova. l-nmma resulted in significant reduction of basal forearm blood flow (p<0.01). there was no significant difference in reactive hyperaemia with either l-nmma or placebo. in conclusion, no does not contribute to reactive hyperaemia in the human forearm. dublin 8. home-based infusion therapy has been widely recognised as the optimum for treatment of disease states that require daily intravenous therapy from a patient-care aspect. conditions necessitating intravenous therapies in hiv disease include: cmv retinitis, intractable cryptosporidial diarrhoea, azole-resistent candidosis, nutritional support with total parenteral nutrition, chemotherapy for aids-related malignancies and palliative care in the terminal phase of the disease. the need for such therapies is increasing as patient survival improves. in 1993, the home-infusion service was set up in recognition of the need to treat patients, requiring intravenous therapy, in the home environment. this has been brought about by the development of small, light-weight pumps suitable for ambulatory use, the development of a service for aseptic compounding and the availability of permanent in-dwelling venous catheters. we describe the impact of this service on our patient cohort. to date, sixty-five hiv positive patients have received parenteral therapy at home. patients' age, sex, risk group, cdc stage, cd4 count, indication for therapy, complication rate and response to treatment are described. the provision of this service has reduced the number and length of patient admissions with associated improvement of quality of life. in addition, it recognises that patients prefer to be treated in the home environment aided by a co-ordinated multidisciplinary approach. since blood alcohol levels over 80 mg/100 ml are now illegal for vehicle drivers we have investigated if the commonly held "safe" limit of two drinks will bring the young adult over the legal limit and if this amount of alcohol will affect their psychomotor skills. following informed consent 33 healthy volunteers, nonhabitual drinkers on no medication (16 male, 17 female), with a median age of 24 (range 20-27) years participated and refrained from alcohol for at least 2 days. each drank within 15 minutes two standard drinks (35.5 ml each ) of 37.5% vodka (2.7 units of alcohol) plus 60 ml of orange juice at about 90 minutes after a standard mid-day meal. their psychomotor performance was estimated by the number connecting technique at 45 minutes after alcohol consumption and they were also asked to rate their feelings (which included alertness, clear-headedness, competence and attentiveness) using a visual analogue scale of 1 to 10. blood samples at one and two hours later were collected from the antecubital vein and analysed on the same day for alcohol content using enzymatic methods. mean (+ sem) blood concentrations of alcohol at 1 and 2 hours respectively were 25.39 + 6.19 and 21.62 + 4.58 mg/100 ml. in males and 36.43 + 11.89 and 38.66 + 3.43 mg/l-00 ml in females. values were significantly higher in females. blood concentrations in females were also higher (p < 0.01) than in males when expressed per kg body weight. while the blood alcohol in both the genders was considerably lower than the current legal limit in ireland their psychomotor skills as estimated from their task completion time and their answers to questionnaires were indicative of an impaired cns function. thus while 2 drinks may keep many subjects below the legal limit, there is considerable inter-individual variation with females showing higher concentrations and both genders have evidence of impaired performance at these lower levels. a non-linear approach was used to develop an hrv parameter, robust to both data non'-st~itionary and missing data points. unlike the standard chaos approach, using higher dimensional embeddings and time-delays, we employed a onedimensional correlation integral plot. the parameter thus obtained, allows an estimate of the spatial spreading of the attractor (ssa) or spatial variation of rr intervals along a straight line. heart rate data from 18 volunteers (22:00 to 06:00 hr), ~ifter oral placebo or propranolol 80 mg, investigated the ability to detect drug effect. vitamin e (~-tocopherol) is the most important dietary antioxidant in lipid and cell membranes and its intake reversely relates to the incidence of coronary heart disease and certain cancers. estrogen regenerates oxidized tocopherol radical in vitro m but such interaction has not been investigated in postmenopausal women receiving estrogen containing hormone replacement therapy (hrt) although estrogen containing oral contraceptive may reduce plasma vitamin e levep. we studied 21 healthy post-menopausal women (aged 46 -56) ammenorrheic for at least one year. fifteen subjects took a combination of harmogen provera therapy and 6 acted as a control group. blood samples were taken from all subject at baseline and after 4 weeks. in the hrt group, serum fsh levels were greatly reduced (86.46 + 25.27 vs 68.67 + 11.59 iu/1, p < 0.04, mean + sd, after hrt) with an increased serum oestradiol level (< 20 -28.68 vs 603.67 + 343.56 umol/1, p < 0.0001). no change occurred in the control group. vitamin e status, measured either as plasma or red cell ~-tocopherol respectively showed no change in both groups (hrt group 26.48 + 5.42 vs 27.17 + 3.39, 23.79 + 5.19 vs 24.93 + 4.37 gmol/1, p > 0.05). we conclude that in post-menopausal women, 4 weeks estrogen containing hrt did not alter vftamin e concentrations in vivo. we assessed the clinical benefit of the newer markers of bone formation: osteocalcin (oc), procollagen 1 carboxyterminal peptide (picp), bone alkaline phosphatase (balp), and bone resorption: carboxyterminal telopeptide of type 1 collagen (ictp) and urinary deoxypyridinoline crosslinks (dpd) over traditional assays such as total alkaline phosphatase (talp) and urinary hydroxyproline (oh/pr) in 23 patients with primary hyperparathyroidism (phpt). patients were sampled basally, then at 2, 6, 24, 48 and 72 hours post surgery and again at 1.5, 3, 6 and 12 months post op. the mean basal p1cp level was 89+26 ug/l (normal: 38-202) this increased to a peak at 48 h (168+_74 ug/l), then declined to normal at 6 weeks (116+56 ug/l). mean basal urinary dpd levels were raised at 8.6+1.2 nm/mm cr. (normal 2.0-6,0), they had normalised by 6 months to 5.9-+1.4 nm/mm cr. mean balp levels were always normal, although normal the yearly mean oc level was significantly lower than the basal value. mean 1ctp, oh/pr and talp levels were always normal. therefore bone turnover in phpt is best assessed by the newer markers picp and dpd. we have previously described seasonal variation in fibrinogen with higher levels in winter. as fibrinogen is an acute phase reactant, the winter rise may be a response to seasonal infections. the present study investigates this hypothesis by examining seasonality infibrinogen and markers associated with infection: white cell count (wcc), interleukin-6 (1l-6), human herpes virus 6 (hhv6) and herpes simplex virus (hsv) antibodies. monthly blood samples from healthy volunteers age 75 and over were measured for fibrinogen, wcc, il-6, hsv and hhv6 reactivation over a 1 year time period. a rhythmometric method was used to examine the data for seasonality. statistical significance was measured using the fstatistic. a highly significant seasonal variation (sv), peaking in mid-february, was found for fibrinogen (n=24; sv=0.629 g/ 1; f=76.148; p<0.01). no significant seasonal variation was present for measures of wcc (n=24; sv=0.209 e9/l; f= 1.940; p>0.05), hhv6 (n=24; sv=0.065 au; f=0.653; p>0.05), hsv (n=7; sv=6.055 au; f=2.048; p>0.05) or il-6 (n=7; sv=1.235 pg/ml; f=0.558; p>0.05). the present investigation does not support the hypothesis that seasonal variation in fibrinogen is a direct effect of the acute phase response, initiated by a seasonal variation in level of infection. the explanation for the seasonal changes in fibrinogen remains unknown. increased plasma homocysteine and reduced plasma antioxidants are risk factors in the development of vascular disease. design: 131 subjects drawn from elderly people living in the community (median age 85 yr, range 60-102 yr; 81 female). total plasma homocysteine, vitamin c, gamma tocopherol, retinol and beta carotene were measured by high pressure liquid chromatography. homocysteine levels in elderly males [median (range) = 12.35 um (4-18.1), n=241 were significantly higher than in vol. 165, 1996 supplement no. 2 irish journal of medical science elderly females [8.45 um (4.8-24.8), n=30]. these values were also higher than in a younger (30-49 years) male cohort [mean = 7.85 um, n= 610]. no correlations to vitamin concentrations were found, nor was there a correlation to age within the elderly cohort. within the elderly females, a significant negative correlation with age was found in vitamin c, gamma tocopherol and beta carotene (p<0.05). however a significant increase in retinol was noted. a very strong correlation between vitamin c and gamma tocopherol levels was noted in the elderly population sample (p<0.001 after multiple regression). conclusion. homocysteine levels in the elderly are higher than in samples of a younger population. a gender difference is maintained in the elderly. the provision of extended care forms one part of a spectrum of health care for older people. in the eastern health board area all patients over the age of 65 must be assessed by the multidisciplinary geriatric team prior to placement. we report on the experience of the total number of referrals for assessment for extended care to one department of geriatric medicine in a 268 bed teaching hospital. ninety-eight patients listed for extended care in 1994. the mean number of days between listing for long term care and placement was 49 _+ 45 days (range 3 to 206). almost one quarter of patients died while in hospital awaiting long term care: this underlines the frailty of patients who are admitted to hospital and request long term care. two patients were transferred to other institutions and 10 patients were able to get home. of the remaining patients 35 (63%) were placed in statutory or voluntary long term care accommodation and only 37% were eligible (usually financially but in some cases due to significant disability) for nursing home care using the terms of the 1993 nursing home act. patients who are listed for long term care through a general hospital are in general very frail, they tend to have a very extended length of stay and the provisions of the nursing home act only apply to a minority. these findings underline the need for provision of adequate statutory and voluntary extended-care places within the eastern health board area. there are over 40 screening assessments for cognitive function and choosing the most appropriate may be difficult. increasingly the importance of behavioural dysfunction is recognised. can any of the cognitive assessments help to predict behavioural dysfunction.'? we compared and contrasted the folstein mini mental state examination (mmse) and the cognitive assessment schedule (cas) of the clifton assessment procedures for the elderly (cape) and compared them with the behavioural rating scale (brs) of the cape. the study was carried out on a total of 21 referrals to the occupational therapy departments by geriatricians in the meath hospital and st. james's hospital. all subjects were over 65 and medically stable. the time scale involved was may-july 1995. the mmse and the cas were administered within the one sitting and each was timed. brs was rated the same day by either a staff or family, member. the average time to complete the mmse (416 + 124 s) was longer than the cas (342 + 171 s) but this was not statistically significant. the mmse and cas were significantly correlated (r = 8208, p < 0.0001). the cas was significantly correlated with the behaviour scale (r = 0,551, p < 0.01) whereas the mmse was not. these results suggest that equivalent assessments of cognitive function may be made with the mmse or cas, but a low cas score will be a better prediction of behavioural dysfunction. a spectrum of neurological and myopathological changes are associated with patients in intensive care units. we observed several patients post discharge from icu who presented with unexplained dysphagia which we suspected may be associated with the neurological complications of sepsis. the particular complication of dysphagia as a neurological manifestation of sepsis has not been documented. our descriptive study presents a series of three patients with persistent dysphagia which may represent a similar phenomenon. we selected patients for the study ranging from 75-87 years of age and on the basis of medical history including icu stay, sepsis, and intubation. all patients presented with dysphagia as observed on videofluoroscopy. we studied the video findings in-depth in order to ascertain if similar swailow patterns were present in these patients and if this could be correlated with their medical history. each of the three patients presented with similar dysphagia signs. the oral phase of the swallow was moderately atypical but the pharyngeal phase was significantly atypical. it was felt that intubation alone was not the sole causative factor of this dysphagia. the polyneuropathy associated with sepsis in icu may explain the atypical swallow patterns observed in these patients. the severity of the persistent dysphagia can cause serious respiratory and medical consequences. there is a need for further investigation of this phenomenon to identify patients who are at risk. little attention has been paid to the prevaience and phenomenology of behavioural disturbances among medical patients despite awareness of the high prevalence of cognitive vol. 165, 1996 supplement no. 2 impairment in this patient population. we screened 79 consecutive admissions to a department of acute geriatric medicine. patients were evaluated over a 2 week period using a modified version of the brief agitation rating scale. medication use, cognitive function and impact on nursing time were also measured. the prevalence of behavioural disturbance in this population was 19/79 (24%). the most frequent behavioural abnormalities were restlessness (12), complaining (12) and screaming (6). the most common underlying disorders were dementia, stroke disease, personality disorder and paranoid psychosis. the behavioural disturbance was only documented in the medical notes in 7 patients (37%) and in only 5 cases was a psychiatric consultation sought. these findings demonstrate that behavioural disturbances are not only common but also under-documented in elderly medical patients and there is a need for training in the detection and management of behavioural symptoms in this patient group. in lower limb trauma where there is severe compound fracture, the successful treatment of this depends on adequate bone and soft tissue debridement. as a result, subsequent bone defects can lead to instability and often require large amounts of bone grafts, and major soft tissue reconstruction is reaquired to obtain skin cover. large soft defects can by reduced bv primary bone resection and shortening of the limb. this will improve the chance of bone healing if performed in the presence of an external fixator, then lengthening at a site away from the traumatised area can gradually restore limb length. two cases are presented to demonstrate .the effect of compression / distraction techniques on soft tissue and bone injuries in these difficult situations. wegener's granulomatosis -wg (15), churg strauss syndrome -css (4), polyarteritis nodosa -pan (2) and unclassified (5). using the chc definitions, the diagnoses were wg (5), microscopic polyangiitis -mpa (10), pan (1) and undefined (10). there was concordance in only 5 patients (all wg). there is significant discordance between these two criteria sets. since the acr criteria does not recognise mpa, they tend to overdiagnose wg. in addition, the chc criteria cannot be applied without a biopsy and therefore surrogate features which predict the underlying histology are required to allow more practical application of the chc definitions. the objective was to determine the value of examination of dried freshly produced saliva, under light microscopy, in patients with xerostomia related to secondary sjogrens syndrome. ten patients with known connective tissue disease or rheumatoid arthritis attending rheumatology clinic were enrolled into the study, all with symptomatic xerostomia and dry eyes. all had an abnormal schirmer's test. five normal patients were enrolled, all of whom were without clinical evidence of rheumatological disease. control patients were enrolled who had no clinical evidence of rheumatological disease, a salivary sample was collected and examined by light microscopy. serum was also examined for the presence of anti-ro/la, rheumatoid factor, and anti-nuclear factor. all ten patients demonstrated 'reindeer horn' type ferning of saliva, a pattern of shorter thicker clubbed branches of crystallised mucus, in contrast to the normal ferning pattern of the healthy subjects. conclusion: we have shown in this preliminary report that light salivary microscopy is a simple test easily performed in an outpatient setting which could be a useful diagnostic procedure in sjogrens syndrome. recently, two sets of criteria have been proposed for the nomeclature of primary vasculitides, the 1990 american college of rheumatology (acr) classification criteria and the 1992 chapel hill consensus conference (chc) definitions. the aim of this study was to determine the concordance of these two systems in a cohort of patients with primary systemic vasculitis. patients with systemic vasculitis were recruited who had a biopsy proven diagnosis or, who had typical clinical features associated with a postive antineutrophil cytoplasmic antibody (anca). the case notes were reviewed and patients were classified according to both sets of criteria. twenty-six patients were recruited, 21 of whom had a positive biopsy. applying the 1990 acr criteria, the diagnoses were, primary pulmonary hypertension (pph) typically affects young individuals, and has a high morbidity and mortality. secondary pulmonary hypertension complicating connective tissue diseases likewise carries a poor prognosis. we evaluated the acute and chronic effects of ketanserin, a selective serotonin type-2 receptor antagonist in 2 patients with pulmonary hypertension in the acute study ketanserin was administered as a peripheral venous infusion during right heart catheterisation. following encouraging results during catheterisation oral administration of ketanserin 160mg daily in divided doses was instituted. in patient 1, a 30 year old female with probable pph, serial cardiac catheterisations over a 1 year period showed a significant, sustained reduction in both mean pulmonary artery pressure from 49 mmhg at baseline to 24 mmhg at 1 year (normal 10-20 mmhg) and pulmonary vascular resistance 1118 units at baseline to 288 units at 1 year (normal <200 units). in patient 2, a 61 year old female with limited scleroderma (crest) echocardiography after 1 month's oral ketanserin showed a reduction in estimated peak right ventricular systolic pressure from 60 mmhg at baseline to 13 mmhg (normal range 15-30 mmhg). the acute and long term response to ketanserin with improyement in pulmonary haemodynamics in these patients suggests that if a beneficial effect is detected during catheterisation long term oral therapy may be worthwhile. levels were low (< 7.9 iu/1); normal though above average (>-8 -<10 iu/l) and moderate high (>10 -<15 iu/l) respectively. gonadotrophins for ovarian stimulation were commencing initially at 225 iu for group a & b and at 450 iu for group c. ivf performance was poor in most aspects (total follicles, oocytes & embryos transferred) in group b comparing with group a or c; the cumulative ongoing pregnancy rate (pr) over 2 ivf cycles in group b; was 15% comparing with 27.6% in group a (p < 0.05) however there was no significant difference in pr in group c (20%) comparing with other two groups. cycle day 3 fsh screening is predictive of follicular development in ivf. high initial dose of gonadotropins help to improve the pregnancy rate in the presence of moderate high level of fsh. the purpose of this study was to evaluate patient satisfaction with antenatal care provided in the perinatal day centre (pndc). a self administered questionnaire was administered to 61 consecutive patients. the main indications for referral were suspected small-fordates (34%), non-proteinuric hypertension (23%), glucose tolerance testing (15%), reduced fetal movements (10%) and post-term evaluation (7.5%); 51% were nulliparae. thirty-two percent of patients were reviewed in the pndc on the day of referral; the rest within 7 days. twenty eight percent of patients lived more than 10 miles from the hospital and 48% spent more than 30 minutes in travelling there. eighty five percent of patients scored their level of satisfaction with the service provided in the pndc as > 7 out of 10; only 6.5% would have preferred admission; 42% said that they would prefer to visit the pndc 5 times per week to avoid admission. the main area of dissatisfaction related to the waiting time for review prior to discharge, with 69.5% of patients waiting over 3 hours. patients attending the pndc report a high level of satisfaction; changes to reduce the visit duration have been introduced. to examine the change of taking-up the essential preconceptual measurements; rubella immune status, cervical cytology and prophylactic folic acid intake; following specific advice and publicity through 3 general public meetings with new patients prior to in vitro fertilization (ivf) programme. in 1994 we studied 281 new couples for ivf for the presence of some specific pre-conceptual data (group a). in this study we follow-up the same intake in another 229 new women interviewed to commence ivf programme from january 1995 till september 1995 (group b). in group (b) the taking-up measurements were dramatically improved. however, 30% and 18% stilldid not have rubella immunity test and cervical cytology performed; compared to 54% and 36% in group (ai respectively (p<0.001). while folic acid intake was sustained at >90% in both groups. following specific advice the rate of taking-up of preconceptual measurements prior commencing ivf programme was improved. there is a future need for continuous enhancement of the publicity and advice regarding the importance of preconceptual measurements. the aim of this study was to introduce icsi to ireland for treatment of specific cases of male factor infertility. following an introductory proving period using the bovine model, thirtyeight couples with infertility attributed to the male were selected for an icsi attempt. ovulation induction, oocyte retrieval and luteal management were as described for conventional ivf tm. the average age of patients selected for icsi were 34.5 + 3.7 years and 36.4 + 4.6 years for the female and male respectively, with an average duration of infertility of 4.8 + 2.6 years. a 64 year old woman presented with a three day history of parasthesia in her lower limbs and difficulty walking. neurological examination revealed sensory loss in her limbs and truncal ataxia. rombergs sign was positive. pelvic examination revealed a large pelvic mass that was distinct from the uterus. routine blood investigations were normal. csf culture, ct brain and serum electrophoresis were negative. anti-purkinjie cell antibodies were not present. ca-125 levels were elevated at 159 micrograms/litre. laparotomy revealed a 20 cm left ovarian tumour. a total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy was performed. histology revealed a poorly differentiated clear cell adeno-carcinoma of the left ovary. the capsule was intact and peritoneal washings were negative. she made a good postoperative recovery. she received six courses of carboplatin without ill effect. her neurological symptoms resolved. subacute cerebellar degeneration can occur as a paraneoplastic disorder in ovarian carcinoma. the mechanism by which cancer can cause neurological disorders is not fully understood. paraneoplastic cerebellar degeneration occurs with or without the presence of purkinjie cell antibodies. the aim was to review all red cell transfusions in gynaecological surgery in 1994. a retrospective review of blood bank records and individual charts was carried out. 4611 patients underwent gynaecological surgery; 97 (21%) were cross matched and 60 (1.3%) were transfused. 184 units were transfused. there were no single unit transfusions. the mean number of units transfused per patient was 3. this accounted for 33% of all units transfused this year. 92% of patients were undergoing elective surgery. the overall cross match/transfusion ratio was 2. intraoperative difficulty was recorded in 56% of cases. 64% of patients were transfused perioperatively and 36% postoperatively. the percentage of patients requiring blood transfusions in the the main individual operation categories was as follows: radical surgery: 57%; total abdominal hysterectomy and salpingo-oopherectomy, 17%; vaginal hysterectomy and repair, 16%; subtotal abdominal hysterectomy, 30%; vaginal hysterectomy alone 12.5%; and total abdominal hysterectomy alone 5%. adverse reactions to transfusions were seen in 5% of patients. conclusion: the majority of patients transfused were undergoing elective surgery. vaginal hysterectomy was associated with greater blood loss than abdominal hysterectomy. only half of all units cross matched were transfused. dilatation and curettage (d+c) is the most common operation performed in the u.k. the liberal use of d+c has been criticised. the objective of this study was to evaluate the use of outpatient endometrial pipelle biopsy and determine its safety in terms of detecting abnormalities. complications and financial costs were also evaluated. data was reviewed from an active gynaecological unit from february 1993 to january 1995, using theatre and outpatient records. a total of 303 d+cs and 104 endometrial pipelle biopsies were performed in this period. 9 malignancies were detected by d+c and 1 by pipelle biopsy. a total of 24 and 3 benign abnormalities were detected by each method respectively. there was a higher complication rate in the d+c group but the failure rate was higher in the endometrial pipelle biopsy group. the monetary savings over this period is estimated at s there were no missed malignancies to our knowledge over the 8 year period since endometrial pipelle bioposy was introduced to this hospital. our study indicates that outpatient endometrial pipelle biopsy appears to be safe, efficacious and economical. while ultrasound findings may sometimes be in conflict with clinical examination, it is the case that there are instances when ultrasound findings have, following subsequent laparotomy, been found to be wholly incorrect. it is therefore not surprising that there remain some gynaecologists who view ultrasound with scepticism, preferring to rely solely of their clinical findings. there have been few studies that directly compare clinical, ultrasound and surgical findings in the detection of pelvic masses. the objective of the study was firstly to directly compare the reliability of clinical and ultrasound examination findings in the detection of pelvic masses proven by subsequent laparotomy and secondly to determine the accuracy of ultrasound in detecting malignancy. this was as a retrospective review of 86 women who underwent a laparotomy because of a pelvic mass between january 1993 to february 1995. information was obtained from theatre and patient records. real time abdominal ultrasound was used. findings at laparotomy were correlated with clinical and ultrasound findings. the sensitivity and specificity of ultrasound in detecting a uterine mass was 94% and 99% respectively. this contrasts sharply with clinical examination (sensitivity = 74% and specificity = 94%). similar findings were obtained when ultrasound was compared to clinical examination in detecting ovarian masses. ultrasouud is capable of predicting benign disease with reasonable confidence but the prediction of malignancy is less reliable. in conclusion, ultrasound is more sensitive and specific in detecting pelvic masses compared to clinical examination. vincent's hospital, dublin. osteoporosis occurring during pregnancy or lactation is a rare event despite the homeostatic demands of the foetus for calcium. we investigated the case of a 24 year old woman who, immediately following vaginal delivery of her first child, developed severe back pain due to a vertebral compression deformity of the second lumbar vertebrae. bone mineral density (bmd) was measured by dual-energy x-ray absorptiometry. calcium metabolism and bone turnover were studied. there was a severe reduction in bmd in the spine (z-score = -4.8) and f e m o r a l neck ( z -s c o r e = -3.6); but, serial measurements showed no further reduction in bmd. indices of calcium metabolism and bone turnover were normal. pregnancy-induced osteoporosis is a severe but self-limited disorder in calcium homeostasis of unknown aetiology. women with low bmd prior to pregnancy may be at increased risk. in view of increased demand, supplemental calcium and vitamin d should be considered during pregnancy and lactation. crumlin, dublin 12. the aim of this study was to assess the clinical status on admission and the critical care m a n a g e m e n t of children p r e s e n t i n g with m e n i n g o c o c c a l i n f e c t i o n . t h i s was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+3.46). the average duration of symptoms prior to admission was 20.4 hours (+11.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a wood gas analysis on admission had a metabolic acidosis (bases excess < -5.0). the mortality rate was 10.9%. 80% of deaths were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of cardiovascular compromise, 78% were admitted to the high dependency unit, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. in this study children presenting with m e n i n g o c o c c a l infection have a high incidence of cardiovascular instability. successful management is dependent on early presentation and initiation of therapy and on aggressive intensive care monitoring and support of the cardiovascular i11 and vital organ systems. the normal crying curve and incidence of colic for term infants are well known. we studied prospectively the crying pattern and the incidence of colic in preterm'infants to determine if prematurity influenced these behaviours. the subjects were consecutive preterm infants admitted to the cork neonatal units for two and a half months from july 1995. a continuous 24 hour diary was completed on each infant by the neonatal nurses, when the babies were on full oral feeding and no longer required intensive care. the parents completed the diaries 'after discharge. colic was defined according to wessel's rule of threes. two unwell babies were excluded. the duration of follow up was from 2 to 16 weeks. fifty infants were recruited and 45 completed the study (4 lost to follow up and one withdi'awn due to sepsis). their mean (range) gestational age was 31 (26-36) weeks and birthweight was 1.52 (0.64-2.4) kg. the mean (range) age of crying onset was x(y-z) weeks; crying, peak 'was x(y-z) weeks and crying offset was x(y-z) weeks. one baby developed colic in the period of follow up. conclusions: the incidence of wessel's colic was less than expected in these preterm babies. the crying pattern according to chronological age was different from that clescribed in term babies. in general preterm babies had a delayed onset of crying, but the pattern became similar to term babies when allowance was made for gestational age. the findings'suggest that the crying patterns of early infancy have a developmental basis. we re-evaluated 18 children who had had rs (1979-86), with their closest-age siblings using the wechsler scales, coopersmith self esteem inventory and achenback child behaviour checklist (acbc) (duffy j. et al 1991). the rs patients' means were consistently lower than that of their sibs. however, comparison of mean raw data, using "t-tests", yielded significant differences only in the acbc scores, in that rs children exhibited significantly more problem behaviours than their sibs (p=0.033). after categorisation of iq data, further comparisons between the groups (using x2), found rs patients were significantly more likely to score "below average" in tests of verbal iq compared to their sibs (p=0.04). age of onset and clinical stage were also found to be more important predictors of outcome. children less than 1 year of age at onset of rs had significantly lower iq scores on all measures of cognitive ability (p=0.003) and more problem behaviours (p=0.01) than children over 1 year of age. no significant differences were found in comparison with sibs. clinical stage to which rs progressed affected only verbal iq scores. children in whom consciousness had been impaired had significantly lower iq scores than both their sibs and rs children in whom consciousness was less impaired (p=0.02). in conclusion outcome remains cautiously positive, with 13/18 rs children attending mainstream schools or in employment without apparent difficulties. a national breastfeeding policy was introduced by the department of health in 1994. factors identified for promotion of breastfeeding were based on the who/unicef "ten steps for successful breastfeeding". we present clinical cases which suggest that one step may need to be modified. the charts of breastfed babies admitted to the special care baby unit were reviewed for one year following the introduction of the national breastfeeding policy to this hospital. thirteen term breastfed babies were admitted because of fever and dehydration. none of the babies had water or bottle feed supplements. ten of the thirteen mothers were primigravida. eleven babies were admitted in the six months following the introduction of an exclusive breastfeeding policy. the nursing staff were then alerted to the risk of dehydration, but two further babies of mums committed to exclusive breastfeeding were admitted in the subsequent six months. routine biochemistry, haematology and a limited septic screen was performed in all babies. three of the thirteen babies had lumbar punctures. the mean (range) weight loss on admission was 9.5 (4.1-16)%. the mean (range) plasma sodium level was 147.6 (138-156)meq/1 and the mean (range) urea was 6.5(2.1-13.4)meq/t. there was no growth from the cultures of the blood, urine, csf and swabs. all the babies were given intravenous fluids and parenteral antibiotics for 48 hours. the outcome was satisfactory in all babies and breastfeeding was reestablished in eleven of the thirteen babies. conclusions: the common factor to these babies was inadequate fluid intake prior to admission associated with stricl~ adherence to the policy, and avoidance of all supplements including water. we conclude that the who/unicef step 6 "not to give food or drink other than breastmilk unless medically indicated" is too restrictive in the immediate postpartum period. 42.5% of children reported headache in the previous 12 months 44.8% of girls and 40.5% of boys reported headache (p < 0.0001). 0.6% of children reported daily headache 5.9% of children reported weekly headache 9.4% of children reported monthly headache 22.5% of children reported headache less often than monthly the percentage of children with headache at each frequency, other than daily, increased with increasing age. in girls headache showed a marked rise at ages 14, 15 and 16 years. reported prevalence of headache in the past year in 5 to 15 year old aberdeen children was 66% and 48% was recorded for swedish children aged 8, 11, 13 and 17 years old. this study is the first community based prevalence study of headache in irish schoolchildren. our aim was to test the hypothesis that there is no correlation between the type of feeding & swallowing disorder the child has with the neurological diagnosis or the radiological findings. a further purpose was to develop a classification of the feeding & swallowing disorders which would guide us towards a management plan. a retrospective analysis of the data collected between the years 1988-94 from the feeding & swallowing clinic at booth hall children's hospital was done. 73 children were included in our study 9 ages ranged from 4 months to 17 yrs. all the children were assessed by the members of the feeding & swallowing team and had videofluroscopic assessment by the same radiologist. neurological signs, speech therapy assessments & videofluroscopy findings were compared between children with spastic quadriparesis & those without. significant differences were noted. a clinical classification was devised using cluster analysis. we conclude that there is no causal relationship between the neurological diagnosis & the type of dysphagia. there are three distinct groups of children who require different strategies of clinical management. surveillance commenced in january 1995 to continuously monitor the incidence of surgical site infections (ssi). employing modern optical scanning technology (formic for windows version 2.0, formic limited, london) a questionnaire was designed, which required minimal completion time. the questionnaire includes relevant data based on the american national nosocomial infections surveillance system for ssi including the ssi risk index. surveillance commences at the time of surgery and continues until the patient's discharge. optical scanning technology allows rapid reading of surveillance questionnaires thereby bypassing the bottleneck of manual data entry. by october 1995, details of 2,156 procedures had been recorded. the crude ssi rate for these patients was 2.7%. the patient risk index used demonstrated that there were increased chances of developing ssi in certain patient groups. seventy-nine per cent of ssi had presented by the 10th post-operative day. the length of stay increased by an average of 4 days in patients developing ssi. regular feedback to individual surgeons, theatre and ward staff maintains awareness and highlights possible problems. we recommend optical scanning technology to all those engaged in surveillance work. this system would be especially useful were data collected is transported from outlying hospitals to a central receiving centre for collation and analysis. in 1872 francis crumpe published a paper ~ in which he described the therapeutic effect of poisonous mussels (psp) on a case of tetanus. he obtained the mussels from tralee ship canal on the occasion of its infrequent emptying, and entertained the idea of using them in tetanus after treating a young girl with psp who recovered after 24 hours. prior to the use of psp he described it's paralytic effect in two cockrels who both recovered. in concluding his successful use of psp he speculated as to the clinical nature and role of the toxin tralee ship canal was opened in 1846. the water was relatively stagnant and would have contained plenty of nutrients. as such it would have been an ideal habitat for toxic algae which may have been brought across the atlantic as spores in bilge water 2. the emptying of the canal may well have been done at times of algal blooming. this is the first irish report of psp and a most remarkable use of saxitoxin (?) in the treatment of tetanus, antedating the current management by seventy years. this study was carried out to quantify the published research on smoking in irish medical journals; to ascertain the type of research carried out; and to identify the authors of that research. during the 35 years under study, 50 papers explicitly dealing with smoking were published. only 2 papers appeared in forum. there was a decline in published papers in the eighties with a resurgence in the early nineties. of the 50 papers, a majority were observational and ten were editorials. only one paper dealt with smoking cessation, and one with preventive work. general practitioners were poorly represented as authors. one doctor (prof. r. mulcahy) published at least one paper on smoking in each quinquennium since 1960. this study underlines the relative insignificance of smoking as a topic for research in ireland. a major sea change in attitude will be required if the government's targets for smoking cessation are to be realised, particularly if they are to be achieved by relying on the medical profession. radiology represents a major cost centre within a hospital. lack of awareness of cost amongst doctors may result in the inappropriate requesting of radiology services. this study assesses doctors knowledge of the cost to a tertiary referral hospital of commonly performed radioj'ogical procedures and investigations. doctors were asked to estimate the cost of 9 items namely -chest,x-ray, arch aortogram, ultrasound abdo., lumbosacral spine, barium enema, ct brain, ct abdo., ultrasound abdomen, i.v.p. and percutaneous gastrostomy tube insertion under radiological screening. 60 doctors in st. vincent's hospital were surveyed. doctors as a group overestimated the cost of all 9 individual tests, by margins ranging from 10% (i.v.p. & gastrostomy insertion) to 100% (ct brain/ct abdo.) the total cost to the hospital of all 9 items was s consultants'overestimated this total cost by 107%, followed by registrars, interns and s.h.o's who overestimated the total cost by margins of 51%, 43% and 12% respectively . conclusion: doctors tend to overestimate what radiological procedures and investigations cost a public hospital, often by quite wide margins. thus, any excessive requesting of radiology services by docto.rs is not due to a lack of awareness of their true cost to the hospital. (ded) in dublin. secondly, to identify the major cancers contributing to years of potential life lost (ypll) for the ehb, each cca and ded in males and females. in ireland little work has been done to date on disease specific premature mortality. crude death rates weight all deaths equally; in comparison, ypll emphasise deaths among younger persons and provide a measure of the burden of premature mortality. premature mortality for the 322 deds in dublin for the years 19988 and 1989 was estimated using ypll, which was calculated by subtracting the date of death form 65. ypll due to each of the major disease groups were ranked for each ded. seventy-one thousand four hundred and sixty-eight &471468) years of potential life were lost in dublin in the 1988 and 1989. 21.9% of ypll was due to injury and poisoning, 20.64% to cancer, 15.6% to circulatory disease. however, when the major cause of ypll was established for each ded, injury and poisoning was the number one cause of death in 25.5% of the deds; cancers 24%, congenital and perinatal conditions 21.5% and circulatory disease 14%. by emphasising deaths in younger individuals ypll is a valuable tool for planning and monitoring local health promotion initiatives. serum total homocysteine (thcy) levels are inversely associated with dietary intake of folic acid and b vitamins and raised levels have been linked with chd. we have examined the association between thcy concentration and the risk of chd in middle-aged men in 18 british towns. we used a nested case control study design, within an ongoing prospective study, thcy concentration was measured in serum samples, stored at entry to the study, from 110 incident cases of myocardial infarction and 118 controls. cases and controls were frequency matched by town and age group. levels of homocysteine [geometric mean (95%c1) were significantly higher in cases than'controls: homocysteine 13.5 (12.6 -14.3)gmol/l vs 11.9 (11.3 -12.6)lamol/l; p = 0,005. there was a graded increase in the relative risk (odds ratio; or) of chd in the 2nd, 3rd and 4th quartile of thcy (or 1.4, 1.9, 2.2; trend p = 0.006) relative to the first quartile. adjustment for age, town, social class, body mass index, smoking, physical activity, alcohol intake, hypertensive status, serum cholesterol and serum creatinine did not attenuate this association, (or 2.1,2.3, 2.7; trend p = 0.04). the findings suggest that thcy is an independent risk factor for chd with no threshold level. in summer 1995, a diagnosis of cryptosporidiosis was made 25 in a child who had visited a pet farm. this child had participated in a summer project involving 161 children and nine adults. reports of a similar illness among other project members, prompted an outbreak investigation. a cohort study consisting of two phases was initiated. 95% (162/170) of project participants responded to a self-administered questionnaire in the first phase. thirteen children met the case definition, of whom seven had cryptosporidium detected in their stools. illness was significantly associated with having visited a pet farm. (p<0.000005). 75% of those ill sought medical attention, of whom two were hospitalised the second phase of the cohort study was conducted among those who had visited the pet farm. 95% (52155) were interviewed. illness was significantly associated with play in sand, to which animals had access, at a stream's edge beside a picnic area (p<0.005). contact with various animals was not statistically significantly associated with illness. however the small numbers involved may have obscured such an association. this outbreak highlights a potential hazard for children visiting pet farms and also that cryptosporidiosis is a significant but often overlooked cause of morbidity in healthy children. managers of pet farms need to be aware of the potential for transmission of disease to visiting children. strict implementation of hygiene measures is essential to minimise risk. the mrc vitamin trial highlighted the importance of folic acid in the prevention of neural tube defects(l). since 1993, the department of health has recommended periconceptional folic acid supplements. the objective of this study was to document the knowledge and behaviour of women in child bearing years to periconceptional folic acid. a cross sectional community survey was conducted using an interviewer administered questionnaire in dublin. three hundred and thirty five women took part in the study. approximately two thirds 213/335 (63.6%) had heard of folic acid. knowledge was significantly associated with higher social class and higher education (p<0.o5). few 18/335 (5.4%) had been advised to take folic acid before pregnancy. only 9/335 (2.7%) of the women in the study were currently taking folic acid supplements. three quarters of the group (75.9%) would be willing to take periconceptional folic acid supplements if they knew it would reduce the risk of malformations. the majority (77.4%) would prefer to take folic acid in tablet form. this study clearly shows that few women in childbearing years have been advised on folic acid. however, if advised appropriately the majority would be willing to take periconceptional folic acid in tablet form. future publicity campaigns involving all health professionals should address these issues. unstable intra-articular fractures with or without dislocation of the phalangeal joints often lead to joint stiffness ana loss of function. nine patients with comminuted intra-articular phalangeal fractures were treated in our unit by dynamic external fixator using "pins and rubber bands traction system". the mean age was 32.3 years, and the follow-up average was 4.6 months. five patients had full and good range of motion in the involved joints. three patients had poor results, and one patient underwent open reduction one week following the original procedure. the technique and our results are discussed. this dynamic frame is compact, comfortable for the patient, easy to apply and allows early mobilisation. careful selection of patients and close follow-up in the first few weeks are needed. this study examines how gp's store and handle vaccines. all 144 gp's in a health board region were invited to take part. gp's were interviewed in their practice premises about how they dealt with vaccines fridges were examined and temperature recorded post interview. oral polio was taken from 20 randomly selected fridges for potency testing. cold chain monitors and freeze watch indicators were used to monitor batches of vaccine stored. of the 144 gp's, 142(98.6%) agreed to participate, 140 used 111 fridges to store vaccine, 2 store vaccine at room temperature. of the 111 fridges, 6 (5.4%) had the power supply safeguarded, 9 (8.1%) had thermometers, 36 (32.4%) had vaccine only stored therein. during defrosting, vaccine was inadequately protected in 22 (19.8%). of the 138 gp's who use multi-dose vaccine vials, 133 (97.2%) keep them for further use at the end of a day/session, 3 store them at room temperature. 42 (37.8%) fridges had temperatures outside the recommended range. 13 (28.2%) coldchain monitors indicated vaccine exposed to more than 10~ 18 (90%) of the oral polio samples showed a reduction in total titre of live virus, however, none were below the minimum acceptable. this study indicates that vaccine potency could be seriously compromised due to breaks in the cold-chain and suggests the need for guidelines to be drawn up, implemented and monitored to ensure the integrity of immunisation schemes. comparison was made with a study carried out in 1983. in addition the range of antimicrobial agents tested included new oral cephalsporins and quinolones that were not then available. three hundred microorganisms isolated from mid stream urine (msu) samples were examined by standard microbiological techniques. antimicrobial susceptibility testing to 12 antimicrobial agents was performed on significant pathogens (>105 organisms per ml) by disc diffusion test, and minimum inhibitory concentrations of antibiotics was carried out by e test on organisms found resistant by disc testing. by comparison with 1983 resistance amongst e.coli, the most commonly isolated pathogen, had increased for the following: ampicillin by 6% to 61%, co amoxyclav by 4.4% from 0%, trimethoprim by 18% to 28% and nitrofuradantin by 5% from 0%. no increase in resistance occurred to cephradine (4%), or nalidixic acid (5.4%). resistance to cefixime, ofloxacin and ciprofloxacin, was 3%, no resistance was encountered to cefotaxime. for proteus species resistance to ofloxacin, ciprofloxacin and cefotaxime was 4%, and for enterobacter sp 20%. enterococci were sensitive to ampicillin and augmentin but the numbers were small. pathogens isolated from patients domiciled in the inner city were significantly more resistant to nalidixic acid (20%), cefotaxime (6%), cefixime (14%), ofloxacin and ciprofloxacin (12%) than those isolated from patients in rural areas. the purpose of this study is to examine the relative importance of obstetric complications (0c%) in the aetiology of schizophrenia and mania. using the dublin psychiatric case register, birth records of 635 patients with an icd-9 diagnosis of schizophreniaor mania were obtained. these records were evaluated, for obstetric complications using two scales, the lewis, owen and murray scale (lom) it~ and the parnas scale 121. the mothers of those going on to develop schizophrenia did not differ from those going on to mania as regards maternal age, parity, social class, or period of pregnancy. however, males who developed schizophrenia when compared to males developing mania, experienced significantly more oc's when rated by the lom scale (p=0.007) and.more frequent oc's on the parnas scale (p<0.001) of greater severity (p=0.018). no significant differences were found between females with schizophrenia and those with mania. dublin. the aim was to evaluate the diagnosis, symptomatology and level of functioning of patients presenting with a first episode of psychosis to a catchment area service and a private psychiatric hospital. all patients presenting with a first episode of psychosis were assessed using the scid-p,,the positive and negative syndrome scale (panss) and the global assessment of functioning scale (gaf). fifty-eight patients (34 male, 24 female) ranging in age from 15 to 65 years (mean + sd = 28.4 + 10.8) were included in the study. the mean total panss score was 84.8 (sd + 21.6) and was strongly correlated with the gaf score (p < 0.001) but independent of age (p = 0.16). males had a significantly lower gaf score compared to females (p = 0.04) but there was no gender difference in the total panss score (p = 0.20), twenty-five patients (43%) had a lifetime prevalence of drug abuse or dependence but only 12 patients (21%) had signs of drug abuse or dependence in the month prior to presentation. level of functioning was strongly influenced by the severity of psychopathology. substance abuse is common in individuals presenting with a first episode of psychosis. the aim was to evaluate the presence of involuntary movements in patients presenting with first episode psychosis to a catchment area service and a private psychiatric hospital. patients presenting with first episode schizophrenia and schizophreniform psychosis were assessed for involuntary movements using the involuntary movements scale (a.i.m.s.). 28 patients (17m., llf.), age range 15-67 years (mean=30.5 years.) were included in the study. one patient (3.5%) satisfied the strict criteria of schooter and kane for spontaneous dyskinesia. 7 patients (6m., if.), had minimal involuntary movements in at least 2 body areas, predominantly orofacial. the total a.i.m.s. score was positively correlated with the number of days spent in hospital per year of follow up (p=0.01). the group with involuntary movements were found to have spent more days in hospital per year of follow up, 94 v. 55 days (p=0.047). for patients with first episode schizophrenia or schizophreniform disorder spontaneous dyskinesia is not common. however involuntary movements at presentation may be a predictor of poorer outcome. psychiatry has moved from custodialcare towards care in the community. adequate reprovision will have to be made in order to discharge the remaining continuously hospitalized patients. the objectives of this study were to describe a.n entire long-stay hospital population, to examine the differences between the old and new long stay groups within this populatian and to evaluate the needs for community residential and day care facilities in order for hospital closure to take place. the study group consisted of the total long-stay population of st. davnet's hospital, monaghan. the patients were assessed using the community placement questionnaire (cpq) one hundred and twenty four patients were included in the study. fifty-six were female and 89% were single. the mean age of the total group was 68.7 years. the majority suffered from schizophrenia. the assessment revealed a globally disabled group with multiple handicaps. the new long-stay group were disabled as the old long-stay group. the patients were characterised into four groups with regard to placement recommendations. these were a specialist unit for chronically disturbed geriatrics, a geriatric unit, a high support hostel and a medium support hostel. the remaining population of this hospital were highly dependent with multiple handicaps but would live in community with adequate support. there is little difference between the needs of the old long-stay and those of the new long-stay. failure of the immune system to identify self peptides is likely to lead to the development of an autoimmune reaction. susceptibility to autoimmunity is strongly influenced by genes clustered in the hla region (chromosome 6p) particularly class i (a, b and c) and class ii (d, q and p). it has been suggested that there is an autoimmune component in the aetiology of schizophrenia. of many conflicting reports from case/control studies using hla antigens the most consistent finding has been an increased frequency of hla-a9 (now split into a23/a24). additionally, a susceptibility locus for schizophrenia has been reported near the hla locus. to attempt to confirm the hla a9 hypothesis, we have genotyped a preliminary sample of 63 familial schizophrenic probands and 77 unrelated controls at the hla-a region, using a pcr-ssop technique. the frequency of hla-a24 (the major component of a9) in patients and controls respectively was 12.5% vs 15.5%. these findings do not support the hypothesis. some of the discrepancy may be due to unspecific cross-reactions produced by commercial antisera used in the microlymphocytotoxicity method of previous studies. however it is also possible that the hla associatton with schizophrenia may reflect linkage disequilibrium with unidentified gene(s) within the hla region which is less strong in the irish population. schizophrenia is a common mental disorder affecting about 1% of the general population with a devastating disturbance of mind and personality. family, twin and adoption studies have demonstrated that the disease is largely genetic with a polygenic mode of: transmission. dopamine receptors have been implicated in the aetiology of the disease. as yet 5 dopamine genes have been identified (d1-d5). in particular the d3 receptor is expressed in the limbic regions of the brain, implicated in the control of emotions. association studies of a d3 polymorphism (glycine to serine substitution at position 9,) with schizophrenia have produced conflicting findings, many of which, however, have demonstrated a significant excess of homozygosity, or excess of the 1 -l genotype at this polymorphism. in this study, 200 familial schizophrenics and 239 irish unrelated controls were genotyped. the result show a small increase in the frequency of the 1-1 genotype which did not attain statistical significance (patients, 41.5% vs. controls, 38.5%). homozygosity (alleles 1-1 and 2-2) was also slightly increased in the patients (patients, 55.5% vs. controls, 48.9%). the small increase in the frequency of the 1-1 genotype and of homozygosity in the patients is in keeping with earlier findings but suggests that the effect, if any, of d3 sequence variation in the development of the disease is small. lack of information about general practitioners' (gp's) ability to prescribe psychotropic medication may affect patients' compliance. in this study, 73 out of 75 patients attending a psychiatry out-patient clinic completed a questionnaire which documented how many had run out of medication, the steps taken if they had and the role each patient thought their gp played in their treatment. 82% indicated that their gp knew what their current medication was but only 46% thought that their gp could provide them with a prescription if they did not have one from the clinic. this figure was similar in those who had (21%) and had not (79%) run out of medication in the past. on running out of medication, 42% of patients waited until their next appointment, 29% attended their gp and the remainder either contacted the department or went to a chemist. in conclusion, many patients do not appreciate the entitlement of their gp's to prescribe psychotropics for them. literature regarding whether or not the social class distribution of patients with psychiatric illness may differ from the general population remains controversial. we sought to clarify this by examining social class at the time of birth, to see whether patients with serious psychiatric illnesses (schizophrenia and mania) differ from the general population. paternal occupation of 450 schizophrenic patients, and 77 manic patients, from the dublin psychiatric case register, were obtained from birth registration details and categorised according to central statistics office criteria, the same-sex previous live birth was used as a matched control. there was no difference between the social class of patients with schizophrenia or mania (p=0.32). neither patients with schizophrenia (p=b.31) nor mania (p=0.153) differed from controls in social class distribution. paternal social class was found to be related to amount of time spenr in hospital (p<0.001', mean=439.62), and educational age (p<0.001, mean=15.01) and "age at onset of the illness" (p=0.03, mean=31.59). these results suggest that social class of origin may not be related to the development of either schizophrenia or mania. however, social class of origin may be relevant in terms of presentation of schizophrenia for treatment. cognitive function is widely recognised to be impaired in schizophrenia but there is an ongoing debate as to whether this impairment is generalised or localised, progressive or static, similar in both sexes, or related to symptoms. using the positive and negative syndrome scale (panss)'we measured psychopathology in 48 chronic in-patients (27m, 21 f; mean age 68.0+12.7) who satisfied feighner criteria for schizophrenia. subsequent to this, we assessed their global cognitive function using the mini-mental state examination (mmse) and their frontal cognitive function using a new instrument, the executive interview (exit). poor performance on the exit was associated strongly with increasing severity of negative (r=-0.74, p<0.001) but not positive (r=--0.09, ns) symptoms,'in both males (r=--0.73, p<0.001) and females (r=--0.76, p<0.001). overall exit performance declined modestly with increasing age (r=--0.32, p<0.05) but this phenomenon was co'nfined to females (r=--0.64, p<0.01; males: r=--0.13, ns). mmse performance was also associated with negative symptoms (r=--0.67, p<0.001) but decreased mm:e prominently with age (r=--0.59, p<0.001) and showed no gender difference. frontal dysfunction in schizophrenia appears to be intimately related to negative symptoms over the course of severe chronic illness, and may reflect among males a more static' trait deficit than is accessed by the mmse. this study was supported by the stanley foundation. while determinants of the course of schizophrenia are unclear, emerging evidence suggests that the longer psychosis proceeds unchecked before initiation of anti-psychotic therapy, the poorer may be long-term outcome. we have reported that, among older in-patients, increasing duration of initially untreated psychosis in the pre-neuroleptic era was associated with a deterioration to a state of muteness (after controlling for intervening variables). the current survivors of this population have now been examined more extensively using the positive and negative syndrome scale (panss), the mini-mental state examination (mmse) and the executive interview (exit). among these 48 patients (mean age 68.0+12.7), after controlling for age and for the duration and continuity of subsequent antipsychotic treatment, increasing duration of initially untreated psychosis was associated with greater severity of negative symptoms (p<0.005) and with lower scores on the mmse (p<0.05) but not with executive dysfunction on the exit (p=0.3). increasing duration of initially untreated psychosis appears to be associated with the evolution of more prominent negative symptoms and cognitive impairment in a manner consistent with an active, morbid process in schizophrenia that can be ameliorated by anti-psychotic drugs. this study was supported by the stanley foundation and the health research board. patients who are selected and who agree to participate in the royal college examinations play an important role. as psychiatrists and exam organisers, we should be aware of the potentially stressful experience which this might present. the purpose of our study was to elicit attitudes to the exam, and also knowledge of the examination procedure. a questionnaire comprising 10 questions was circulated to 30 patients who had participated in the royal college examinations. responses were received from 28 (93%) of the 30 patients. there were 14 males and 10 females in the responding group. none of the patients had previously participated in the examinations. all of the respondents (n=28) felt that the candidate had been polite towards them during the interview. 38% (n=9) of the patients were nervous prior to the examination, and this group was predominantly female (n=6). 21% of the patients (n=5) did not know that they would receive payment for their participation. 67% (n=l 6) did not know that they might be physically examined as part of the examination procedure. 17% (n=4) of the patients described experiences which had been upsetting for them the results of our study suggest that on the whole patients tolerate the exam procedure quite well. one of the central issues concerning physiotherapists in stroke rehabilitation is the emergence of abnormal tone. rehabilitation involves re-establishing a normal postural control mechanism (ncpm)"~. abnormal tone may develop in the presence of severe sensory and proprioceptive loss. the patients' attempts to move and find a stable base can lead to compensatory movement patterns and asymmetrical postures. positioning is used by physiotherapists to influence the distribution of muscle tone and facilitate symmetrical postures. it is essential that the patient is made aware of his 'position in space' as failure to do so presents no feedback regarding movement resulting in inertia ~2~. standardised positioning charts have been used in hospitals. the physiotherapist liaises with nursing staff regarding correct use on a 24 hour basis. this study looked at the role of a more individualised approach to positioning in the form of a photograph. patients were randomised to two positioning groups group a standard vs group b photograph, and their positioning was scored by a 'blinded' research physiotherapist over an eight week period. the results of a pilot study on a small number of patients revealed that nursing staff preferred a positioning chart individually tailored to the patient's problems. from a physiotherapy perspective, improved postural awareness correlated with better positioning scores in group b. the prevention of compensatory movements, posture is critical in stroke rehabilitation, the use of an individualised positioning chart requires further evaluation. we discuss the case of a fourteen year old boy who presented with bilateral ptosis present since birth, microcephaly and pigmentory retinopathy"!. he was found to have mild facial and proximal limb weakness. creatinine kinase and ldh were raised. muscle biopsy showed ragged red fibres consistent with a mitochondrial myopathy ~2~. electron microscopy showed abnormal mitochondria. the term mitochondrial myopathy describes a diverse range of clinical disease ~3~ and this is discussed. she developed a vasculitic skin rash with pruritis and oedema associated adenopathy, low grade fever and mouth ulcers. lab tests showed leucocytosis, eosinophilia and abnormal liver function. skin biopsy indicated an inflammatory picture without vasculitis. ct thorax confirmed axillary and para-aortic adenopathy. lymph node biopsy confirmed a reactive lymphadenopathy. the aim of. this study was to assess the characteristics of patients referred for pudendal nerve studies over a one year period. 31 consecutive patients were asked a standard questionnaire and nerve studies were performed as described by kiff and swash m and swash and snook~2k of the 31 patients, only 3 were male. the age range was from 21 to 79 (mean 50). 10 presented with constipation and 19 with faecal incontinence. two had both symptoms. bladder incontinence was presented in 10 of 31 patients. of these, faecal incontinence was the cardinal symptom in 8 patients, constipation in 2. 12 patients were nulliparous. of the remaining 17, 14 had a history of complicated births involving forceps (7), caesarean section (4), post partum haemorrhage (1), breech without forceps (2) . 14 patients had pelvic surgery and one had major trauma. 22 of 31 patients had bilaterally delayed pudental nerve terminal motor latency (pntml). 7 of 31 people had unilaterally delayed pntml, 2 were right sided, 5 were left sided. 2 had normal studi~s. the range of measurements was 1.8 -9.2 ms with a mean of 3.65 ms. in conclusionl delayed pntml was seen in 11 of 12 patients with constipation and 19 of 20 with faecal incontinence. pelvic surgery and a complicated obstetric history were significant. urological symptoms were also a common association. the p50 component of the middle latency-auditory evoked potential is attenuated in response to the second of paired clicks in a normal population. in schizophrenia, this attenuation is minimal. in alzheimer's disease (ad), the results have varied between centres depending on the frequency of the stimuli and the interclick interval. we studied 10 ad patients, 10 elderly controls (ec) and 10 young controls (yc) using a paradigm of 32 sets of paired clicks. in contrast to previous studies, our study demonstrates significantly larger absolute p50 generation and recovery amplitudes in ad patients compared to elderly controls and young controls. the purpose of study was to establish a simple screening vol. 165, 1996 irish journal of supplement no. 2 medical science test to identify asymptomatic intracranial aneurysms (icas). an association between atherosclerosis and icas is recognised. elevated serum lipoprotein (a) [lp(a)] is an independent risk factor for atherogenesis. we aimed to assess the degree of correlation between serum lp(a) and the occurrence of sporadic ruptured aneurysms and familial asymptomatic aneurysms. lp(a) levels were measured in (a) 50 patients with icas and 42 normal controls, (b) 25 first degree relatives of patients with familial subarachnoid haemorrhage (sah). icas were detected by cerebral angiography. patients with sporadic icas had significantly elevated lp(a) levels when compared with matched controls. mean level was 20.1 mg/dl in patients and 10.8mg/dl in controls. in the familial studies, 10 out of i1 subjects with asymptomatic icas had elevated lp(a) levels. one young female with elevated lp(a) had a pre-aneurysmal dilatation at operation. six out of 14 subjects without icas had elevated lp(a) levels; four of these were in the second or third decade of life and may yet develop aneurysms. conclusions: lp(a) has potential as a biological marker for icas. follow-up studies are required on angiographically negative subjects. we have begun a genetic case-control study to establish if particular apoprotein (a) gene polymorphisms can be correlated with the occurrence of icas. post mastectomy breast reconstruction has undergone several changes in the recent years. attitudes have changed towards the problem from both the patient and the reconstructive surgeon, as aesthetic outcome receives a greater emphasis than previously. there is a shift towards using autologous tissue as a means of reconstruction; these new technically difficult procedures entail a longer learning. centralization of this type of reconstruction in highly specialized centres only will serve the patient better. we share our experience of post mastectomy breast reconstruction spread out over the past five years. seventy-eight consecutive cases of breast reconstruction are included in the study. different techniques of breast reconstruction were used with a recent switch to transverse rectus abdominis myocutaneous (tram) flap; we feet that tram flap is the gold standard of breast reconstruction as far as the ultimate cosmetic result is concerned. ours is only a moderate sized study compared to some published, yet it is representative of the experience of most of the plastic surgery units in the british isles. clinically significant paraneoplastic neurologicaldisorders are rare, most are associated with small cell lung, female genital tract and breast carcinoma. the malignancy is often silent and the neurological manifestations vary from encephalomyelitis, cerebellar degeneration, sensory neuropathy to neuromuscular block. prognosis is usually poor. pathogenesis is thought to be related to cross reaction with neurons of antibody produced to tumour antigens. detection of these antineuronal antibodies in serum has assisted diagnosis of paraneoplastic encephalomyelitis in which anti-hu antibodies are present and cerebellar degeneration, in which anti-yo are found. in our lab, we used avidin-biotin-complex immunocytochemistry to detect anti-hu (cortical neuron antibodies) and anti-yo (purkinje cell antibodies) in patients' sera. tests were performed on human frontal cortex and cerebellum, at 1 in 500 and 1 in 4,000 dilutions, with positive and negative controls. of 68 sera, 6 were positive. two patients had repeat positives; in one, antibody titre rose in the second sample. subsequent patient review showed 3 positives (2 patients) had identifiable carcinoma with paraneoplastic cns signs, 2 had no identifiable malignancy but had no other cause of their cns disorder and are being followed up; details of one patient were unavailable. these results are similar to other centres. the proliferation of tumour cells despite the presence of tumouricidal mediators could be due induction of a heat shock response, a universal cellular defence mechanism in host cells and possibly tumour cells. protection may be mediated either by increasing intracellular levels or surface expression of heat shock proteins (hsp). the aim was to assess the effect of heat shock induction on tumour cell protection against host effector cells. the heat shock response was induced in sw707 colorectal cells by either sodium arsenite (0-320~tm for 6hr) or by hyperthermia (42~ for 20 rain). monocyte (m0)-mediated cytotoxicity or flow cytometry to evaluate surface expression of hsp60 and hsp70 were assessed. cytotoxicity showed a significant decrease in all treated groups (p<0.002) when compared to the control value. there was also a significant decrease in all groups (p<0.001) when compared to the 42~ value. no significant alteration in surface expression of either hsp60 or hsp70 was seen. conclusion: heat shocking tumour cells significantly protects them from m0-mediated tumour cell lysis. since the flow cytometric data indicate that there is no concomitant increase in surface expression of hsp60 and hsp70 on the tumour cell following heat shock, it can be inferred that induction of intracellular hsp levels are responsible for the protective effect on the tumour cells. a 4 week qol study in 157 consecutive advanced cancer patients was undertaken to compare the subjective 28 question fact-g with simple subjective global tools (visual analog, categorical scales: vas, cas), objective tools (spitzer qli and ecog performance status) and verbatim patient description. we anticipated the high drop out rate enrolling 157 to achieve 87 complete study patients for statistical purposes. the study sample appeared representative of the advanced cancer population in the usa. generally qol was satisfactory despite the severity of illness. there were significant differences in all measures between those who described qol in verbatim responses as positive and negative, particularly cas, vas, and qli (p<0.0001). there were significant intercorrelations between qli and ps (observer rated), vas and cas (subject rated) respectively (p<0.0001). taking patient description as the gold standard, simple, global qol measures e.g. vas or cas are as effective as multidimensional ones (fact-g and qli). irish journal of medical science males had more dysphagia. survival from diagnosis was greater for females </=65 years (female median 18 months, male median 13 months, p<0.0016). anorexia was associated with reduced survival since diagnosis in the total population and in males. anorexia anddysphagia were associated with reduced survival from referral. advanced cancer patients were polysymptomatic. symptom prevalence differed with age, gender and cancer site. the pattern of g1 symptoms was related to gender and severe weight loss. specific symptoms were associated with reduced survival. there was a gender difference in survival favoring females. the administration of recombinant interleukin-2 (rll-2) is limited by the induction of increased microvascular permeability. the in vivo antineoplastic effects of taurine in combination with rll-2 were investigated and its impact on the associated vascular leak was examined. lung metastases were established in mice via tail vein injection. ten days after injection mice were randomized into treatment groups. on day 18 the mice were sacrificed; lungs were removed, weighed and metastases counted. treatment of tumour-bearing mice with rll-2 alone resulted in a significant reduction in tumour nodule incidence compared to a control group, while the~group recei'~ing rll-2 + taurine showed an even fuither reduction in the incidence of lung nodules. animals receiving rll-2 showed a significant increase in mean wet lung weight compared to control lung weight, while mean wet lung weight of the rll-2 + taurine group was significantly less than that of the rll-2 group.and comparable to control values. animals receiving ril-2 + taurine in vivo demonstrated significantly enhanced splenocyte-mediated antimelanoma activity ex vivo compared to animals receiving rll-2 alone. thus taurine may have an important role in modulating both metastatic growth and the associated toxicity of rll-2 immunotherapy. a prospective analysis of symptoms in 1,000 patients on initial referral to the palliative care service of the cleveland clinic was made using the paradox relational database. the median number of symptoms per patient was 11 (range 1-27). the 10 most frequent symptoms were pain 82%, easy fatigue 67%, weakness 64%. anorexia 64%, >10% weight loss 60%, lack of energy 59%, dry mouth 55%,'eonstipation 51%, dyspnea 51% and early satiety 50%. patients 65 years and under had more pain, sleep problems, depression, anxiety, vomiting and headache (all p<0.01 ).'the prevalence of early satiety, nausea, vomiting and anxiety were greater in females; dysphagia 3nd hoarseness in males. patients with >/= 10% weight loss had more gi symptoms; of these females had more nausea, early satiety; the progn.o'stic significance of abnormalities in the p53 tumour suppressor gene and in the expression of its protein in colorectal carcinoma may be influenced by the method of analysis used. we studied p53 abnormalities in 66 patients with colorectal cancer followed for more than 10 years. single-strand conformation polymorphism analysis (sscp) was used to detect alterations in exons 5-8 of the p53 gene. paraffin sections were examined immunohistochemically for p53 overe,xpression with the monoclonal antibody do-7 (dako) both with and without microwave antigen retrieval. abnormalities of the p53 gene were found in 41% of cases by sscp analysis but were unrelated to age, sex, tumour size or differentiation. outcome was unrelated to sscp abnormalities (p=0.19). overexpression of p53 protein was seen in 47% of cases by immunohistochemistry without microwave antigen retrieval and in 52% of cases with microwaving. poor long-term survival was related to immunohistochemical expression of p53 protein either with (p=0.03) or without (p=0.02) microwave antigen retrieval. these results suggest that immunohistochemical detection of the p53 protein product may be more useful than sscp analysis of the encoding p53 gene in identifying those at high risk of colorectal cancer recurrence and death. dublin 9. the anti-tumour activity of tumour infiltrating lymphocytes (tils) is known to be poor and therapeutic manipulation of these cells has met with little success. suppressor macrophages (smo) influence t cell cytotoxicity and proliferation. we hypothesized that smo are a component of the lymphoreticular infiltrate and that these cells may be related to lymphocyte numbers within the tumour. tgf-b may influence macrophage phenotype. colorectal and breast tumours were obtained within an hour of resection. tumours were dissaggregated with collagenase and dnase for three hours. antibodies was used to identify smo (rfd 1 and rfd7) and t cell subsets (cd4 and cd8) by flow cytometry on the resulting cell suspension. pre-op blood was collected from patients and tgf-b levelsdetermined by elisa. conclusions: we have shown for the first time that smo, defined by the antibodies rfd1 and rfd7, are present within breast tumours. we have also shown that the balance of t cell subsets is different in these tumours and may be related to smo content. circulating tgf-b levels are increased in breast cancer and associated with greater smo numbers. this was not found to be the case in colorectal cancers. these results imply the existence of a fundamental difference in the make-up of the lymphoreticular infiltrate between these cancers. swelling of the upper limb is an uncommon but well irish journal of medical science recognised complication of breast cancer treatment. in severe cases, patients have limited arm function and feel disfigured. in a pilot study, the incidence of arm swelling following complete axillary clearance in the immediate post-operative period and at long term follow-up was investigated. arm volume measurements were performed using an opto-electronic volometer (bosl medizintechnick, hamburg). both ipsilateral and contralateral arm volumes were assessed. the expected volume of the ipsilateral arm volume was calculated using the formula vr = v1 = 31 mls for right handed people and v1 = vr = 25 mls for left handed people (vr and v1 = volume of the right and left arms respectively). the difference between the expected and actual volume of the ipsilateral arm was expressed as a percentage of the expected volume. twelve patients undergoing axillary clearance for breast cancer were prospectively evaluated pre-operatively, 48 hours and 5 days post-operatively. a second group of 48 patients who had had axillary clearance at least 3 months previously (range 3 to 116 months) were also evaluated. there was no significant change in arm volume in the immediate post operative period. clinically detectable arm welling was found on 3 patients who had undergone axillary clearance at least 3 months previously but none had any impairment of arm function. we conclude that axillary clearance can be performed safely and that arm swelling is an uncommon complication. a larger study is planned to investigate factors such as the influence of pectoralis minor division, duration of the operation and the number of axillary nodes retrieved on upper limb volume. epidermal growth factor (egf) is a potent mitogen and has been shown to accelerate healing of epithelial damage both in the skin an.d the gut. in the skin egf is not produced locally as the requisite mrna is not present but egf receptors are present on the surface of basal keratinocytes. egf is produced in various sites in the gi tract including the submandibular salivary glands. we have hypothesised that as there is upregulation of salivary egf production in some enteropathies a similar situation may occur in disorders of the skin with an associated enteropathy. using a sensitive radio-immunoassay, egf activity was estimated in stimulated saliva from 87 patients with various skin disorders, 49 patients with gastrointestinal disease, 8 patients with mixed dermatological and gut disease and 41 normal healthy volunteer controls. elevated egf activity was found in the following groups of patients : skin cancers, psoriasis, acne, oesophagitis and ulcerative colitis. the hypothesis of up-regulation of salivary egf production in skin associated enteropathy was rejected but the discovery of elevated egf activity in skin cancers and psoriasis may have aetiological and therapeutic implications. the malignant fibrous histiocytoma (mfh) is considered an uncommon malignancy. its potential for invasion, metastasis and death of patient has been reported in literature. it can be confused with other tumours including fibrosarcoma. salient histologic features include cells of both the fibrocytic and histiocytic series. mfh with its high recurrence rate and lethal potential merits an aggressive evaluation and treatment. we present unusual case of recurrent mfh treated in our unit with an open question as to what qualifies to be adequate primary surgical excision. the recommended management of localised merkel cell carcinoma has been wide surgical excision, combined with adjuvant radiotherapy in selected cases. the risk of recurrent regional disease is reported to be between 30% and 45%. a 70 year old woman with merkel cell tumour on the cheek is presented; this patient was treated exclusively with radiotherapy to a total dose of 50 gy over 18 days. the tumour regressed rapidly during the treatment, and there were no signs of local or regional recurrence. the patient is still alive and free of disease for 45 months. immune in origin with a heightened cutaneous immune response to ultraviolet light. the coexistence of cad and pbc is a new association which has not previously been documented and may not be fortuitous given the similar pathogenesis of both diseases. chronic actinic dermatitis (cad) is a rare photosensitive disorder which primarily affects elderly men resulting in an eczematous reaction to ultraviolet-radiation and sometimes visible light. the pathogenesis has been attributed to an autoimmune process, possibly in response to a photoallergen which has yet not been identified. we report a 71 year old female patient who developed cad four years after being diagnosed with primary biliary cirrhosis (pbc). abnormal monochromator irradiation tests were detected with narrow band ubv, uva and in addition visible light wavelengths. phot0provocation tests induced florid vesicular eczema and multiple patch and photo-patch tests were positive, findings typical of cad. immunoglobulin g was elevated at 2290 mg/dl and liver histology was typical of pbc with an elevated anti-mitochondrial antibody. routine biochemical and immunological tests were ~ormal and porphyrin screen was negative. azathioprine 50 mg/day induced remission of cad. pbc is an auto-immune disorder where cell-mediated immunity is impaired, suggesting that sensitized t lymphocytes may cause damage to bile ducts. the pathogenesis of cad may be autowe present two cases of cutaneous polyarteritis nodosa (pan) associated with seronegative arthritis: the first patient, a 50 year old male presented in 1993 complaining of a7 year history of pain, stiffness and swelling affecting his right ankle. he also noted intermittent tender nodules on the dorsum of his foot and over his ankle over the preceding three years. the second patient, a 51 year old male, presented .in 1994 complaining of a 5 month history of tender nodules on his shins, and pain and swelling of his right ankle. skin biopsies of the nodules in both cases showed medium vessel vasculitis consistent with polyarteritis nodosa. neither patient had any symptoms or signs to suggest systemic involvement. the only abnormality.0n laboratory investigations for vasculitis was elevated esr. x-rays showed periosteal elevation and new bone formation in case 1, and were normal in case.2. bone scan demonstrated increased uptake at the talo-navicular joint in case 1 and at the fight ankle in case 2. synovial biopsy and mri confirmed the presence of an inflammatory arthropathy in patient 1. joint involvement has been a prominent feature throughout the course in both cases requiring aggressive treatment with vol. 165, 1996 irish journal of supplement no. 2 medical science cyclophosphamide and systemic corticosteroids in case 1. cutaneous pan is a localised cutaneous vascular disorder with a benign chronic relapsing course. in one reviewl, 12 of 23 patients had arthralgias but an association with arthritis has not been emphasized in the literature to date. we conclude that this condition may present as a seronegative arthropathy in which the joint symptoms may be the most prominent feature and aggressive immunosuppresive therapy may be required for control. cardiac transplantation patients have an increased risk of skin disease. in our centre, 119 heart transplants were performed with a 5 year survival of 75%. eighty three patients are now alive and 39 have required dermatological assessment. the mean age of patient was 48.3 years, (range 11-64 years); 99 males, 20 females. skin infections were diagnosed in 13 of 39 patients. drug side effects, including sebaceous hyperplasia and steroid acne, were common. in the patients who developed skin cancer, mean time from transplant to development of lesions was 3.1 years. eleven of 36 patients had 32 non melanoma skin cancers (nmsc), 28 squamous cell carcinomas (scc), 4 basal cell carcinomas (bcc), giving scc/bcc ratio of 7:1. three of 11 patients had multiple skin cancers, one had 12 tumours. nine of 39 patients had actinic keratoses, two thirds of whom had sccs. nineteen of 39 patients had viral warts, two of whom had sccs. viral warts, premalignant and malignant lesions were located on sun exposed sites. skin complications of cardiac transplantation though mild were very common. the observed incidence of nmsc in age matched cardiac transplant recipients, appears much higher than the expected incidence of 171.38 per 100,000 population (national tumour registry 1994). regular dermatological assessment of cardiac transplant patients is necessary to detect skin disease and early skin cancer. the increased incidence of warts and skin cancer in renal transplant recipients {rtr} is well known. the oncogenic potential of unusual human papilloma virus {hpv} types has been postulated from warts and in both premalignant and nonmelanoma skin cancer (nmsc). the possible etiological role of sun-exposure in facilitating the development of hpv associated skin disorders is also suggested. a clinical study to assess the risk factors for development of these lesions in 50 rtr attending the dermatology servic& age and sex matched haemodialysis patients were similarly examined as controls. 40 male and 10 female patients with a mean duration of transplant of 10.6 years, range 1 to 25 years. a total of 191 nmsc (range 1 to 57),175 scc and 16 bcc, ratio 10.9:1, were excised from 26 rtr of which over 95% had viral warts, mosle commonly occurring on sun exposed sites and always predated the development of neoplastic lesions. both were associated with mean duration from transplantation, 4 years for warts and 10.8 for skin cancer and not the type of immunosuppressive treatment. none of the control patients had similar findings. conclusions: the close clinical association of viral wart lesions and development of skin cancer in these patients suggests a close relationship to immunosuppression, in addition to exposure to ultraviolet radiation. this study highlights the high rate of nmsc in rtr. these patients justify early and regular skin assessments soon after transplantation with advice on sun protection. sensitivity to ultraviolet (uv) light may be established by exposure to broad band uva and uvb radiation. the minimal erythema dose (med) can be determined at individual wavelengths using a monochromator. uv action spectra of photosensitive disorders may thus be constructed. we examine the value of this process in distinguishing two clinically similar photosensitive disorders. the radiation from a xenon arc is separated into component wavelengths using the monochromator. each wavelength is focused on unaffected skin, on the patient's back. the patient is exposed to a range of doses of w radiation. the med is determined for a series of wavelengths from 300 to 400 nm. chronic actinic dermatitis (cad) and drug induced photosensitivity are photosensitive disorders which may have similar clinical history and presentation. ten cad and 14 drug induced photosensitivity patients were tested. uva photosensitivity was seen in 71% of the latter group. the remaining 29% had normal mlts as the implicated drug had been discontinued prior to testing. cad patients were sensitive to both wa and wb radiation. forty-three percent of these patients were also sensitive to visible light (400 to 800 nm). monochromator light test (mlt) results show that uva photosensitivity dissociated from wb photosensitivity is indicative of a drug induced light sensitive disorder. sensitivity to both wa and wb however indicates a diagnosis of cad. mlts can therefore distinguish between clinically similar photosensitive disorders. bartholomew's hospitals, london. patients with mpd have an increased incidence of both thrombosis and haemorrhage suggesting a pivotal role for; platelets in these conditions. this study aimed to examine platelet activation antigen expression in stable patients with mpd and to examine the predictive value of these antigens prospectively. 52 patients with mpd had p selectin and gp53 measured using a refined minimally manipulative flow cytometric technique. expression of p selectin -median 5.1% (inter quartile range 2.1-10.9), control 2.0% (1.3.-4.0) and gp53 -median 4.0% (1.8-5.7), control-2.1% (1.0-2.9), were significantly elevated p<0.01. patients were followed for a median of 26 months. 15% experienced thrombosis and 6% bleeding during follow up. at entry to the study 48% of patients had previously experienced thrombosis, median disease duration 8 deaths, 3 of which were caused by thrombotic events in which the mpd was a major risk factor. increased expression of p selectin or gp53 expression failed to predict thrombosis or bleeding in this study. nor was any significant retrospective relationship demonstrated. however, previous thrombotic events were strongly associated with future events (p<0.05). this association was independent of disease, duration, age and medication. not surprisingly disease duration was also correlated with thrombotic/bleeding events. taurine levels fall in gut mucosal cells during critical illness. however, taurine transport into human intestinal cells is poorly understood. the aim was to establish the efficiency of taurine uptake by enterocytes, and to examine uptake under stressful conditions. to investigate efficiency of taurine uptake, confluent caco-2 cells were incubated for time points up to 10h. in a second study, cells were incubated for 9h with medium containing dexamethasone and / or cytokines. media for both studies was supplemented with [3h]-taurine. radioactivity was related to mg/ml protein to calculate rate of taurine uptake for each time point. study 1: uptake exhibited a steady linear response which approached saturation at 7h. maximal uptake occurred at 9h after which the rate levelled off. study 2: dexamethasone alone reduced taurine uptake by 75.4% (p<0.001) and in combination with tnf-c~ and ifn ~/ it decreased transport by 66.3%. (p<0.001). lps alone impaired uptake by 56% (p<0.001). conclusion: we have established the time course over which taurine transport reaches its maximum rate in caco-2 cells, and that corticosteroids and cytokines significantly impair uptake of taurine in these cells. elderly individuals have an increased risk of infection suggesting that immune responsiveness is altered with age. changes in the level of proinflammatory cytokine production may be an important indication of any such age related change. using flow cytometry we examined intracellular tnfet, il-lf~ and il-6 in pbmcs from normal healthy volunteers of different ages ranging from 22 up to 85 yr (n=28). tnf and il6 levels from pma stimulated cd3 positive cells (t cells) were shown, using this technique, to increase in an age dependent manner (p<0.05). no il-113 was detected in any t cell sample. no significant differences were observed between the different age groups for tnfa, il-1 g or il-6 in cd 14+ cells (monocytes). the age related changes detected by flow cytometry have been confirmed using conventional elisas. this novel method of proinflammatory cytokine detection has detected increased tnf and il6 levels in t cells from elderly healthy volunteers which may help explain some of the exaggerated inflammatory responses seen in elderly patients. detection of proinflammatory cytokines by conventional elisa or bioassay is problematic due to the presence of naturally occurring biological inhibitors. flow cytometry allows the simultaneous detection of both intra and extracellular antigens thus intracellular cytokine levels can be quantified while cell surface markers allow cell type identification. a range of monoclonal antibodies were examined for tnfcx, il-lg and il-6 using saponin permeabilisation oft cells (cd3), monocytes (cd14) and epithelial cells (ber-ep4). t cells and monocytes were grown in ~culture, t~p :to 72 hr with or without pma activation, and intracellular cytokine levels were shown to increase with time, with the stimulated samples producing more cytol<ine than the spontaneous.samples (p<0.05). elisas performed on these same samples (n--:24) correlated well with the flow cytometric results (r=0.52). tnf~x, il-113 and il-6 are detectable !n monocytes while only tnfa and il-6 have been detected in tcells and epithelial cells using this methodology. this novel technique will allow us to identify the cytokine producing cell while also avoiding the influence of the extracellular millieu, thus being useful for a number of research and diagnostic applications. all the viral hepatitides are said to be common in the middle east. hepatitis a viru s (hav) seroprevalence is said to be 100% in the adult population of gulf countries. hepatitis b, c and d seroprevalence are taken from blood banks data which are biased by selection criteria and the background of blood donors. very little is known about hev in the uae. vol. 165, 1996 supplement no. 2 irish journal of medical science the aim was to investigate the seroprevalence of hav, hbv, hcv, hdv & hev in the uae. we prospectively recruited volunteers drawn from all the uae, socioeconomic classes and from different age groups in both sexes. each donated 10ml. of venous blood and using an indirect enzyme immunoassay were tested for total ig to hav; hbsag using monoclonal antibody t.o hbsag; for hcv antibodies using recombinant antigens hc-34, hc-43, c-100 and ns-5, (3rd generations); for total ig to hdv and for total ig to hev. positive samples were retested in duplicate for confirmation. this large sero-epidemiological study clearly shows that hav is decreasing in the uae. hbv is low but significant, hcv is similar to southern european countries, hev is lower than expected and hdv does not exist in hbsag carriers in the uae. reactive oxygen and nitrogen species produced by inflammatory cells plays a role in tissue injury and inflammation. taurine, a bamino acid present in high concentrations in leukocytes functions as an anti-oxidant by scavenging the mpo derived oxidant, hypochlorous acid, to form taurinechloramine. experimental colitis was induced in male sprague-dawley rats and randomised into two groups. one group received tap water whilst the second group was supplemented with taurine (4%). these groups were sub-divided into those receiving tnbs/ ethanol (n=8) or saline (n=8). an increase in wcc count, particularly neutrophils was found in colitis. tat~rine was found to reduce the severity of the disease (ns) usin.g a colon macroscopic score. in the first group, respiratory burst activity was reduced in animals that had developed colitis (p<0.05) although mpo activity was augmented (p<0.05). nitric oxide production was 2.2 and 1.8 fold respectively higher in inflamed and non-inflamed areas of the colon than that by normal colonic mucosa. animals supplemented with taurine demonstrated attenuated m po activity (p=0.001). administration of taurine did not lead to a reduction in nitric oxide production. thus, taurine appears to have a beneficial role in reducing the severity of disease. a reduction in mpo and respiratory burst may be beneficial in reducing tissue damage. to women subsequently identified by a national screening programme which revealed 438 to be positive for rna of hepatitis c, type lb. 94 were referred to this centre for evaluation. 10 had moderate activity by modified knodell index, mean 8 (_+1.4), bridging fibrosis and a mean alt of 77 (42-176). six months dual therapy was administered. two patients withdrew due to depression (2) and hyperthyroidism (1). one patient di d not respond. seven responded with normalisation of alt and negative pcr. eight patients showed improved histology (kai 4 +_ 1.5), fibrosis was unchanged. adverse reactions in the treated group included marrow suppression (1) and thyrotoxicosis (1) . at six months follow up four remain in remission with normal alt negative pcr, three have relapsed, (pcr positive, mean alt 87(50-150). we conclude that despite adverse prognostic indicators in this homogenous group with chronic hepatitis c, lb and long disease duration, dual therapy with interferon and ribavirin achieved results comparable to prolonged high dose interferon in patients with more favourable initial prognoses. the aim of the study is to evaluate a 5 day course of azithromycin in combination with omeprazole in eradication of h.pylori. twenty-four patients with proved h.pylori infection and peptic ulcer disease were treated with omeprazole 40 mg daily for 2 months plus azithromycin 500 mg daily for 5 days. h.pylori was diagnosed on the basis of clo test and histology. this was done at the beginning and 3 months after the treatment. eradication was defined as negative clo test as well as negative histology. out of 24 patients, 2 patients were lost to follow-up. only 21 patients were analysed. sixteen patients responded to treatment and had successful eradication as well as healing of their ulcers. these included 8 patients with gastric ulcers (79.95%), 4 patients with duodenal ulcers, 2 patients with gastric and duodenal ulcers and 3 patients with non-ulcer dyspepsia. five patients did not respond including 2 with non-ulcerative dyspepsia. 2 duodenal ulcers and 1 gastric ulcer. however their ulcers have healed and symptoms relieved and they were treated with other h.pylori eradication regimes. conclusion. in this small study we have shown that azithromycin 5 mg oncedaily for 5 days is an alternative safe effective and well tolerated monotherapy for eradication of h.pylori. it has been suggested that hepatitis c virus (hcv) infection is associated with a high prevalence of autoimmune disease, but that this risk may be diminished in patients who received a low viral load at initial infection. the aim was to determine the prevalence of symptoms, signs and markers of autoimmune disease in a group of women vol. 165, 1996 irish journal of supplement no, 2 medical science infected with hcv contaminated anti-d immunoglobulin. 50 patients, mean age 44.3 years (range 32 -65 yrs) were examined. 7 patients (14%) were rheumatoid factor positive. ana was positive in 6 patients (12%) but titres were 1/10 in 4/6. thyroid globulin antibodies were found in 3 patients (6%) with 1 also being tm positive. 2 further patients were positive for thyroid microsomal ab. prevalence of apa, ara, sm were 4%, 2% and 2% respectively. one patient had cryoglobulins detected while antibodies against mitochondria and lkm-i were not found only one patient of 50 was symptomatic. these levels of antibodies are no greater than levels in the general population and similar to other groups infected with hcv contaminated anti-d. these results further support the hypothesis that either low viral innoculum or long duration of infection may in some way result in a low level of autoimmunity. a previous study (ilan et al, arch int. med., 1993; 3: 1588-1592) suggested that lga deficiency disposes towards hepatitis c virus (hcv) infection in some patients. it has also been suggested.that iga deficiency may occur as a result of the viral infection as a secondary event. the aim was to determine the prevalence of selective and partial lga deficiency in patients with hcv infection using a radio-immunodiffusion technique. serum lgg and igm levels were also examined. immunoglobulin levels were measured in 67 patients, 59 women and 8 men, mean age 45.3 years (range 29 -72 years) infected with blood between 1977 and 1990. none of the patients were found to have selective or partial iga deficiency. mean iga = 2.59 g/l (range 0.86 g.1 -5.19 g/l). furthermore no patient exhibited deficiency of igg or igm. conclusion : no evidence of iga or other immunoglobulin deficiency exists among this group of patients with hcv infection. while it may be possible that lga levels decrease following infection, no evidence exists that this phenomenon persists in the long term. hereditary non-polyposis colorectal cancer (hnpcc) is the most common form of hereditary colon cancer and accounts for up to 10% of the overall cancer incidence. the disease is inherited in an autosomal dominant manner andis characterised by predominantly right-sided tumours. hnpcc is caused by mutations in one of four mismatch repair genes. these genes are homologues of the bacterial mismatch repair systerfi and their function is to detect and correct defects in dna. the hmsh2 gene is reported to account for up to 60% of cases, hmlhi, hpms1 and hpms2 account for 30%, 5% and 5% of cases respectively. we have collected eighteen irish hnpcc families which satisfy the amsterdam criteria for the disease. we have screened the entire hmsh2 gene in eight of these families. a combination of single strand conformational analysis (sscp) followed by dideoxy sequencing was used to detect defects in the dna. sequence analysis revealed the presence of a novel polymorphismin exon 5 in one family and a possible splice site mutation in the remaining ten families, using a novel mutation detection technique. departments of medicine and immunology, university college hospital, galway. serial iga gliadin antibody (igaga) studies are used as a surrogate for repeated small intestinal biopsy to monitor dietary compliance in treated coeliac patients. we have correlated igaga levels with indices of hyposplenism (platelets and erythrocyte "pits") to determine if either index can be used to monitor adherence to a gluten free diet. the study population of 129 biopsy proven coeliacs was stratified according to dietary status into 114 gluten free diet (groups a) and 15 normal diet (group b). the iga gliadin antibody titres were significantly lower in group a than group b (44.5 : 126 units p = 0.007). in group a gliadin antibody titres showed a statistically significant correlation with both platelet (p = 0.039) and pits count (p --0.04) ; in group b gliadin antibody titres correlated with platelet counts only (p -0.003). those ona gluten free diet were further subdivided according t ~ reported degree of adherence to the diet (strict diet -sgfd, lax'diet -lgfd). significantly higher gliadin antibody titres were found in lgfd than sgfd (103.6; 27.9 p = 0.003), but the ptatelet and.pit counts were similar in the two groups. igaga is superior to platelet or pit coums in monitoring dietary'compliance in coeliac disease. hospital, galway. iga endomysial antibodies (igaea) are said to be more specific and sensitive indicators of coeliac disease than iga gliadin antibodies (igaga). initially the igaea substrate was monkey oesophagus and more recently umbilical cord. a new commercial product has becomeavailable using primate smooth muscle (accu track -diagnostic slides). we have undertaken a preliminary study to compare the performance of this lgaea assay and igaga elisa in treated and untreated coeliacs, coeliac relatives and controls. the study population consisted of 127 biopsy-proven coeliacs who were stratified according to "reported" dietary status into 89 good gluten free diet (group a), 24 poor gluten free diet (group b) 14 normal diet (group c) and 10 unaffected coeliac relatives (group d) and 5 controls (group e). elevated igaga (>25 units) were found in the following groups : a 37%, b46%, c 50%, d 20%. igaea were positive in a 27%, b 33%, c 50%. with respect to igaga positivity, there were 11 false positive igaeaa 18%, b 12.5%, c 14% and 23 false negative -a 8%, b 8%, c 14%, d 20%. in this preliminary study in untreated coeliac patients the performance of the igaea test was on a par with the igaga assay. alt and fibrosis may be associated with a non-alcohol steatohepatitis and these processes may be synergistic. finally, number and type of riba bands is not a predictor of inflammatory activity. ~stepping hill hospital, stockport, uk sk2 7je. 2royal oldham hospital, rochdale rd., oldham ol1 2jh. we investigated upper gut bleeding in patients aged 75 years and over. a proforma addressing demography, drug therapy, clinical status, timing of endoscopy / surgery, and outcome was used. 109 consecutive patients (median age 80 years, range 75-92) were studied over 6 months. 106 (97%) underwent gastroscopy with a 97% diagnostic yield. 32 patients had severe oesophagitis, 2 had oesophageal malignancy, 29 had gastric ulcers -5 of which are malignant and 23 had duodenal ulcers. ulcerogenic drugs were implicated in 52 patients. 38 patients were referred for surgery, 8 operated upon with one postoperative death. 51 had haemoglobins of 10g/dl or less. all malignant lesions were inoperable. the overall mortality was 6% reducing to 2% if neoplasla were excluded. co-morbidity influenced mortality. 92 patients were discharged with a median hospital stay of 15 days. information on cause of bleeding greatly influenced management. the prognosis of gut haemorrhage in the very old need not be so poor. a few require surgery but the majority respond to active medical resuscitation which is a key factor in determining outcome. we advocate low threshold for endoscopy, judicious use of ulcerogens and adherence to guidelines on management of upper gut haemorrhage. haemochromatosis (hh), a common recessively inherited disorder of iron metabolism is closely linked to the hla-a locus on chromosome 6. linkage studies have demonstrated a close association between (hh) and the hla alleles, a3 and b7 ("ancestral haplotype"). heterogeneity at the molecular level may account for the variance in clinical phenotypic expression. the aim was to evaluate phenotypic expression of hh in the presence/absence of the a3-b7 ancestral haplotype. 32 probands (26m:6f) from unrelated irish families were investigated. phenotypic variability was assessed with regard to l) age; 2) % trans.sat.; 3) serum ferritin; 4) liver bx iron grade; 5) body iron stores and 6)symptomatology. three males were homozygous for a3b7, 15 were heterozygous for a3b7 and 2 were non-a3b7. symptomatology, trans, sat., serum ferritin and liver bx grade were not influenced by homozygousity or heterozygousity for a3b7. conclusion: there were no significant differences in phenotypic expression on comparison of the three haplotype groups. no predominant genotype appears to be responsible for phenotypic severity in irish families indicating the possibility of multiple mutagenicity of the hh gene. of 1013 riba positive anti-d associated chronic hepatitis c patients, 438 were pcr positive but had surprisingly mild disease. the disease status of the pcr negative patients was hitherto uncertain and is the subject of this study. 20/72 riba positive patients referred to this centre were biopsied because of elevated alt (4) or florid symptoms which dated from inoculation (16). 1 2 3 4 histological activity index * 5,1,0,1 3,2,5(f), 2(f), 0,4(f), 2,1,2 0,2,1,2 2,2,3 no bile duct damage, lymphoid follicles or aggregates was observed. 3/20 had mild periportal fibrosis (f), 2 of these had steatohepatitis with obesity (1) and impaired glucose tolerance (1) suggesting dual pathology. we conclude that riba positive, pcr negative patients have minimal disease activity. elevated the association between the hla locus and haemochromatosis (hh) has allowed early identification of affected siblings. it is unclear what proportion of subjects who are predicted to be homozygous or heterozygous for the disease by hla typing develop the disease. studies correlating clinical features with hla type in families from ireland -a putative source of this celtic trait have not been described. the aim was to correlate clinical, biochemical and pathologic features of hh with hla typing in 67 first degree relatives of 12 probands. initial analyses identified 12 homozygous (hh), 40 heterozygous (hn) and 15 normal (nn) individuals. however, 11/40 hn individuals had stainable iron on liver biopsy, confirming hh. further hla analysis revealed 7 homozygous x heterozygous matings and identification of all disease haplotypes within each pedigree allowed final classification of 30 hh, 25 hn and 12 nn individuals. vol. 165. 1996 supplement no. 2 conclusion: this study demonstrates the importance of hla typing in the clinical management of families with hh, furthermore, in multiply affected families the incidertce of homozygous x heterozygous matings is high indicating the high degree of "pseudodominance" in the irish population. the degree of acute hepatic failure after severe trauma and sepsis is related to the extent of hepatocyte (hc) damage and cell death resulting from either necrosis or apoptosis. we have previously demonstrated that tnf-ct and lps can directly lead to hc necrosis, but not apoptosis. recent, studies have shown that reactive oxygen intermediates (roi) and nitric oxide (no) are capable of inducing apoptosis in eukariotic cells. however, it is unclear whether roi or no are involved in hc cell death. the aims of this study were to evaluate the role of no and roi in hc cell death (apoptosis vs necrosis). hcs were isolated from sprague-dawley rats, and cultured with the no donor, sodium nitroprusside (snp) or the roi generation system, hypoxanthine-xanthine oxidase (hx-xod) and h202. the effect of lps, tnf-t~, and ifn-y alone or in combitmtion with different antioxidants and the no synthase inhibitor, n-methyl arginine (nma) on hcs was also assessed. snp caused a dose-dependent increase in hc apoptosis. roi generated by hx-xod and h202 did not induce hc apoptosis, but were responsible for hc necrosis. tnf-ct alone failed to induce hc apoptosis, but when ~combined with antioxidants resulted in increased hc no production and apoptosis. this effect was attenuated by nma. snp also induced hc damage and hc necrosis. moreover, tnf-ct-mediated hc damage and necrosis could be further reduced by the combination of antioxidants and nma. these results indicate that roi preferentially induce hc necrosis, but not apoptosis. induction of no resulted in both hc apoptosis as well as hc necrosis, which suggest that overproduction of no may be detrimental during the sirs. irish journal of medical science intervention was not uniform. mean albumin was 34.3g/1. mean weight was 57.8kg. poor cognitive status greatly increased the requirement for dietetic consultation time. lack of dietetic resources results in inadequate monitoring of these patients following discharge. this study highlights the need for a dedicated clinical nutrition service, for medical services for older people. periconceptual consumption of folic acid has been shown to decrease the incidence of neural tube defects. the preventative strategy of universal food fortification with folic acid presents the possible risk of masking the diagnosis of cobalamin deficiency in pernicious anaemia. in addition, the ultimate longterm effect of universal exposure of adult or foetal cells to a synthetic substance, ie. folic acid, is unknown. in this study, the threshold oral dose of folic acid in a number of foods above which metabolically-unaltered vitamin appeared in serum postprandially was determined in a young and elderly population by microbiological assay of serum pre-fractionated by hplc. subjects on a five-day regime of fortified cereal and bread along with their normal unfortified diet. were shown to have a threshold level of 400~g/d, abovewhich unaltered folic acid appeared in the serum. individuals given folic acid in either isotonic saline, milkor white bread exhibited a threshold level of 200~tg per serving. from patterns of food consumption in ireland, even moderate levels of fortification are likely to lead to some population groups being exposedto excessive amounts of un-altered folic acid in serum. many older people are nutritionally compromised. there is clear evidence that: nutrition intervention reduces morbidity m and mortality in older patients. to identify the spectrum of nutritional abnormalities referred for dietetic intervention and the problems associated with nutritional assessment, 50 elderly patients were alphanumerically selected from files of the department. of nutrition and dietetics. the most common dietetj.c interventions were: use of supplements 60%; high protein high calorie diet 46%; nasogastric feeding 26%; reduction fat 8%; iron/thiamine assessment 6%; high fibre diet 6%; diabetic diet 4%; nutrition swall6w programme 2%; lipid lowering diet 2%. some 60% referred required nutritional supplements, but the profile of an increase in oxidative stress in cystic fibrosis patients has been suggested. activated neutrophjls in the presence of chronic lung inflammation in addition to increased activity of the electron transport chain in cfmay. increase free radical generation. antioxidant protection against free radical attack is likely to be compromised as a i'esult of deficiencies in fat soluble antioxidants vitamins. in the present study stimulated thiobarbituric aoid reacting substances (tbars) were measured to determine the ability of plasma to withstand lipid peroxidation. copper was used to in!tiate the breakdown of lipids to lipid hydroperoxides and eventually to aldehydes, mainly malonyldialdehyde (mda). pooled cf plasm a and pooled control plasma were incubated for 0, 30, 60, 90, 140 and 200 min. mda complexes with thiobarbituric acid which absorbs at approximately 535 rim. there is a lag phase where antioxidartts vol. 165, 1996 irish journal of supplement no. 2 medical science in the plasma or tissue protect against lipid peroxidation, then a log phase where the protective effect is overcome and finally the reaction reaches a plateau when lipid peroxidation is complete. absorbance at 532 nm was measured in all samples and zero order and first derivative spectra were obtained. the lag phase appears to be longer in the pooled cf plasma compared with controls. plasma t~-tocopherol levels were within the normal range in both groups, indicatin~ an alternative protective effect in cf. mild hyperhomocysteinaemia is an established risk factor for heart disease. a source of homocysteine in humans is the essential amino acid methionine found in protein of animal origin. in an 8-week study weekly fasting plasma homocysteine levels were examined in a group of healthy male subjects (n=6) under normal dietary conditions (weeks 1 to 4) and in response to graded increased methionine intakes (weeks 5, 6, 7). nutrient intakes, including methionine, were calculated from 4x3-day food records. under normal dietary conditions weekly mean plasma homocysteine levels were not significantly different (anova) from each other ranging from 6.82+1.77 to 9.42+2.73~tm/1. doubling daily methionine intakes (supplementing with 25mg/ kg/d) did not result in a significant increase in plasma homocysteine (8.56+3.68~m/1), however, significant increases were achieved when diets were supplemented with methionine at levels of 50 and 75mg/kg/d resulting in mean plasma homocysteine levels of 13.37+5 9 and 18.051am/1+11.08, respectively. mean plasma homocysteine levels returned to baseline (8.76 + 3.42 ~tm/l) 10 days post supplementation. we conclude that supplementary methionine results in a significant increase in plasma homocysteine only when levels of five times the normal dietary intake are reached. this study is evaluating the use of a synthetic construct which encompasses primer binding sites for lpl and a variety of cytokine and other transcripts, to quantify lpl expression in cultured human monocyte-derived macrophages. following isolation of total rna at various times during cell culture, its reverse transcription (rt) using random hexamer primers generating first strand cdna and specific amplification of lpl cdna targets by polymerase chain reaction (pcr), generates products identifiable on gel electrophoresis. quantitation of message is obtained by incorporating the pawl08 construct in the rt assay in varying quantities as an internal standard with known amounts of monocyte-macrophage rna (l~tg). pcr amplification of this construct yields size distinguishable products from that produced by the monocyte-macrophage lpl cdna transcript. pcr conditions for the assay have been optimised at 29 cycles of denaturation (tmin @94~ annealing (1.5min@55~ and extension (lmin@72~ lpl mrna has been detected in cultured monocytes and macrophages throughout their differentiation. also increased expression of monocyte lpl mrna has been observed following 15 hr incubations with chylomicrons (30t~g/6x 106 mononuclear cells/ ml)-when compared with controls. interestingly, little or no lipase mrna was detectable in circulating monocytes using identical pcr conditions to preparations of mrna from the day 4 and day 8 cultured cells. this methodology will now permit investigation of the factors controlling lpl expression in cultured human monocytic cells. replacement growth hormone (gh) therapy in adult hypopituitarism is attracting increasing interest. in 1992 markussis (l) detected premature atherosclerosis by ultrasonography in the untreated patient 9 we have shown plaque regression with patients on replacement gh (norditropin) in a 4-month trial. 11 females and 9 males were recruited, mean age 49.6 years. at each timepoint plaque characteristics were measured by duplex ultrasound. 6 patients showed a large reduction in plaque size (mean 21%) after four months treatment (p value < 0~00l). similarly, highly significant values in cholesterol, hdl and ldl and apo a1 are achieved. chol hdl ldl apo a 1 pretreatment 6.54 1.23 5.04 119 posttreatment 5.64 w 1.24 w 3.79 w 109 ~ w achieve a high degree of statistical significance (p< 0.001). the significant reductions achieved in plaque characteristics in six patients studied who showed plaque formation correlates with other parameters traditionally accepted as reducing cardiovascular risk. hypertension is found in approximately 70% of patients with cushing's syndrome, but the mechanism is poorly understood. previous studies in our unit have examined levels of exchangeable sodium, plasma renin and angiotensin ii and cardiac sensitivity to phenylephrine. one previous study has demonstrated enhanced pressor responsiveness to noradrenaline in a group of patients with cushing's syndrome due to adrenal adenoma. we have investigated the blood pressure response to noradrenaline in 7 patients with pituitary dependent cushing's syndrome and in 7 controls matched for age, sex and bmi. noradrenaline was infused for 10 minute intervals at five different concentrations between 0.01 and 0.18 mcg.kg.min -~ multiple systolic and diastolic readings were recorded and the infusion was stopped if the systolic pressure became > 200 mmhg, diastolic _> 1 l0 mmhg or the systolic pressure rose > 35 mmhg. basehne blood pressure in the patients with cushing's disease (cd) was 140/88 + 6/3 compared with 122/83 + 6/6 mmhg in the normal controls (nc). in 6 of the 7 patients with cushing's disease, the test had to be stopped before completion of the protocol, whereas this was necessary in only one control subject the change in blood pressure from basehne to the blood pressure value recorded either at the time the test was stopped or at the peak blood pressure reading during equivalent noradrenaline infusions was compared between the matched pairs. the mean change in diastolic pressure was 22 + 5 mmhg in cd compared with 7 + 2 in nc (p<0.05). there was no statistically slgmficant difference in either systolic pressure (28 + 5 vs 26 + 5 mmhg) or mean arterial pressure (23 + 5 vs 14 + 3 mmhg). these results demonstrate an increased diastolic pressor response to noradrenahne tn cushing's disease. increased pressor sensitiwty to uoradrenaline may contribute to the elevated blood pressure seen in cushing's disease. increased plasma homocysteine (thcy) is an independent risk factor for premature vascular disease. patients with insulindependent diabetes have an increased prevalence of cardiovascular disease. accordingly, we measured plasma thcy concentration in 119 such patients (15-59y), randomly selected, and in 51 control subjects. in controls, thcy was higher in males than in females (supine: geometric mean (95% ci): ,8.3 (7.2,9 6) v 5.9 (5.1,7.0) i.tmol/l, p<0.001), as previously described, but there was no gender difference in patients. male patients, without microvascular complications, had lower thcy than controls (supine: 6.5 (5.4, 7.8) v 8.3 (7.2,9 6) ~mol/l, p<0.05), but values in female patients without complications were similar to those of female controls, thcy significantly correlated with age in diabetics but not in controls, thcy increased in patients with increased severity of microvascular complications, partly due to the effect of age. thcy was higher when standing.than when supine in both controls (8 0 (7.0,9.1) v 7 1 (6.4,8.0)lalmol/l, p<0.05) and patients (6.9 (6.5,7.4) v 6.4 (6.0,6.9)l.tmol/l, p<0.02). the absence of gender difference, the association between thcy and age, and higher levels with increasing microvascular complications suggest thcy could be of pathogemc significance in iddm patients, despite unexpectedly low levels in male patients without complications. differences.between supine and erect samples may be due to haemodilution of albumin-hound thcy in the latter a review of the treatment outcome of thyrotoxicosis with standard dose/doses of radio-active iodine (sdrai) in 67 consecutive patients presented to the endocrinology department, uchg, from december 1992-december 1993 was analysed. the mean pre-treatment levels of free thyoxine (ft4) was correlated with the treatment outcome. there was statistically significant difference in the pre-treatment ft4 between responders and nonresponders to the first dose rai (p = 0.001). response with hypothyroidism and/or euthyroidlsm was considered successful treatment 43/67 (64 2%) responded to a single dose rai; 26/ 67 (38.8%) and 17/67 (25 4%) responded with euthyroldism and hypothyroidism respectively 19/67 (28.4%) and 3/67 (4.5%) responded to the second and third doses of rai respectively giving a total response rate of 92.5% and 97% respectively. interestingly, 2/67 (2.99%) patients failed to respond even to the fourth dose rai 4/67 (5.97%) patients with t3 toxicosis (3 females and 1 male). two responded to the first dose (one with hypothyroidism and the other with euthyroidism), the remaining 2 required a second dose, which produced the same results. no statistically significant difference in the response rate between t3 and t4 toxicosis (p = 0.9) was observed. inherent in the st. vincent declaration targets is the need for continuous data collection and audit. we present preliminary information from the mater database, the first prospective audit of patients from a homogenous irish population. 1,051 iddms (527m:524f) were identified with the following characteristics (mean + sd), age 37.3 + 14.4 years, duration of dm 185 + 11.2 years, bmi 24.4 + 3.0 (males) and 24.2 + 4.0 kg/m2 (females), hbalc 8.6 + 1.97% (n<6.2%). no male:females differences existed in the above nor in macrovascular complication rate 10.1% (predominantly peripheral vascular &sease and lschaemic heart disease). however, males were more likely to be current smokers (34% vs 27%, p = 0 01). hypertension rates (17.2m vs 15.4%f), cholesterol > 6.5 mmol/1 (6.2m vs 9.8%f) were similar but more males had cholesterol < 5.2 mmol/l (64.8m vs 53.8%f, p < 0.01). clinical nephropathy was present in 11.4% of males vs 8.7% in females (p<0 05) 11.9% had clinical peripheral neuropathy. retinopathy will be described elsewhere. 10.4% of females and 2.3% of males had a history of hyperthyroidism and 2.5% of females vs 1.4% of males of hypothyroidism. 7.8% had history of psychiatric disease. conclusion although not a population based study, care of iddm in ireland is almost totally hospital clinic based cigarette smoking is identified as the major problem to be addressed patients with diabetes meltitus (dm) are at a higher risk of developing vascular complications, including coronary artery disease (cad). we performed a detailed analysis of predictors of cad and its seventy in patients with dm and chest pain 19 patients in total single vessel cad (svd) in 4, double vessel (dvd) in 5, and triple vessel (tyd) in 5 on cine contrast angiography clinical, biochemical and dobutamme stress echocardiographic findings are tabulated below for patients with angiographically proven coronary artery disease. patients with tvd had a longer duration of dm (19 4 years) and were more likely to have retinopathy (100%) the sensitivity of dse was excellent for severe disease. conclusion' duration of dm, retinoapthy, and a positive dse were the best predictors of severe cad in a diabetic population with chest pain the haemodynamic hypothesis for the pathogenesis of diabetic microangiopathy argues that an initial increase in microvascular flow leads to sclerosis and disturbed microvascular autoregulation. we have recently demonstrated impairment of vasoconstrictor responses to endothelin-1, a potent endothelium-derived constrictor substance, in niddm and have suggested that this could contribute to the initiation of microangiopathy the purpose of this study was to determine whether responsiveness to endothelin-i is also impaired in iddm. non-specific vascular smooth muscle contraction was assessed using high dose serotonin eleven patients with iddm and 11 control subjects underwent forearm blood flow (fbf) measurement by venous occlusion plethysmography in response to local infusions of endothelin-1 (5 pmol/min for 60 minutes) and serotonln (30 la g/min for 2 minutes) control subjects showed slow onset vasoconstriction in response to endothehn-1 reaching maximum at 35 minutes (p<0.01) the diabetic group did not respond to endothelin-i group differences were significant (p=0.02). the two groups showed similar vasoconstriction in response to serotonln. in conclusion, vasoconstriction in response to endothelin-t is impaired m iddm non-specific vascular smooth muscle contraction is preserved. impaired vascular responsiveness to endothehn-i is a possible common mechanism for the pathogenesls of microanglopathy in 1ddm and niddm. we measured total corrected (tca) and standardised ionised calcium (lca) in a population of 76 intensive care (1cu) patients (with a mean age of 57+22 years, 59% male) to determine the prevalence of abnormalities in circulating calcium and its possible determinants severity of illness was measured by the apache ii score (acute physiological and chronic health evaluation). for comparison of ica we examined 20 subjects undergoing arterial gases which proved to be normal and 40 non-critically 111 hypoxlc subjects ica was measured on arterial gas samples and corrected for ph 84% of icu patients had a total ca (unadjusted) of < 2 2mmol/l. after adjustment for serum albumin, 55% of icu patients had an tca <2 2 mmol/1 30% of icu patients had a serum phosphate of <0.8 mmol/1 ica in controls was 1.44 + 0.017 mmol/1 and 1.38 + 0.015 mmol/1 in hypoxlc non icu patments (ns) ica in icu was lower: 1.34 +0.016 (<0.003). tca and lca were not slgmficantly related. tca and ica did not sigmficantly differ between patients who died and who survived in the icu, and they were not related to apache ii score. belfield, dublin 4. from july 1987 to june 1995 there was a 16-fold increase m the annual number of specimens submitted to the virus reference laboratory because of a perceived risk of contracting hiv through a needlestick injury, blood splash, human bite, or through occupational exposure. needlestick-associated specimens also comprise an increasing proportion of 'at-risk' specimens, rising from 0.9% m the year july 1987-june 1988 to 9.4% in the year july 1994-june 1995. between july 1992 and june 1995 a total of 1787 patients had specimens submitted for hiv antibody testing after a perceived'exposure to hiv of these only 209 patients had more than 1 specimen taken. although the time of putative exposure is rarely avadable, the median interval between 1st and 2nd postexposure specimens for these 209 patients is 3 months with 136/ 209 (65%) lying between 1 to 6 months. if the risk of hiv refection from a needlestlck injury is assessed as sufficient to warrant serological investigation, the timing and number of blood samples are important. a negative report from a single early specimen may not indicate an absence of infection a basehne specimen and follow-up specimens at 6 weeks, 3 months and at a minimum of 6 months post-exposure are recommended appropriate serology for other viral mfections (hepatitis b and hepatitis c) should also be considered. since the beginning of 1995 a significant sustained increase in the numbers of hepatitis a cases has occurred. the number of cases m 1995 was 229 against 121 in 1994. this reverses the continuous fall observed over previous years. the reason for this increase remains unidentified at present this increase has occurred following a dramatic increase in the total number of hepatitis a igm tests carried out by the vrl since february/ march 1994 the increase in the number of tests carried out since this time is primarily attributable to increased hepatitis testing following receipt of hepatitis c-contaminated rhesus anti-d immunoglobuhn. analysis of the age profiles of the positive patients and of all the referrals indicates that 95.5% of positive results were found m patients aged 50 yr or less whdst 31% of all hav igm tests were performed on individuals aged 51 yr or greater. this raises the question whether greater selectivity should be employed when requesting hepatitis a tests studies report compliance rates ranging from 30 to 50% in hiv negative patients there has been no comprehensive study of compliance in hiv positive patients. accurate measurements of compliance are not easy; easy measurements of comphance are not accurate "). to determine the compliance rate in hiv posmve patients attending st. james hospital, dublin, one hundred consecutive patients attending the service were interviewed (homosexual 46, ivdu 45, heterosexual 9). the questionnaire was divided into three sections. firstly, a medical review was completed by the clinicmn which included demographic data, cd4 count, cdc staging, karnofsky index and prescribed medication. secondly, the pharmacy detailed the medication dispensed to each patient. the third section comprised a patient interwew to determine adherence to, and understanding of prescribed drug therapy. we report an overall comphance rate of 61%. this was unevenly distrtbuted between the two main patient groups (89% in homosexuals, 24% in ivdu) the following factors were found to mfluence compliance: number of medications, cdc stage, karnofsky index, dysphagia, educational and socio economic factors. we also found that poor patient understanding of the prescribed therapy significantly affected compliance. the aim was to determine the incidence of stds in patients presenting for hiv testing at the department of genito-urinary medicine, saint james's hospital. a retrospective analysis of all patient notes who presented for hiv testing between july '94 and december '94 was undertaken. according to clinic policy all patients had been screened for the following stds; neisseria gonorrhoea, chlamydia trachomatls trlchomonas vagmalis, candida, human papilloma virus, herpes simplex virus syphilis and hepatitis b in addition intravenous drug users (ivdus) were also screened for hepatttls c all patients underwent pre test counselhng. sex, age, risk groups and diagnoses were noted. 504 patients presented for hiv testing, of whom 66% were male, 34% female, wtth an average age of 28.5 years. of the total, 8% were, or had previously been ivdus. of the total 46 ivdus, 37 were heterosexual males 2 were bisexual and 4 were females 10.2% of the male patients were homosexual and 4 4% btsexual there were 15 posmve hiv tests (3% of total); 14 males and 1 female. in this group there were 5 patients with hepatitis c, all of whom were ivdus. no other stds were detected in the hiv negative group hepatitis c was diagnosed in 20, hepatttis b in 4, anogenital warts in 58, herpes gemtalis in 10, syphilis in 3, n. gonorrhoea in 9, t. vagmalis in i, c. trachomatls m 12, g vaginalis in 5 and candldlasis in 47. this study confirms the importance of std screening in all patients requesting a hiv test. of the total testing for hiv, 30% had a concurrent std diagnosis although no stds were identified in the hiv positive group this may be more a reflection on the makeup of the irish hiv positive population, the majority being ivdus, rather than a difference m the mode of sexual toxoplasmosis is the most common opportumstlc infection of the central nervous system in aids patients. in clinical practice the diagnosis depends on clinical, radiographic and serological findings coupled to chnical response to therapy brain biopsy is not routinely performed. in this retrospective review, we describe our experience with diagnosing toxoplasmosis we examined (a) the clinical demographics and presentation, radiographic findings, response to therapy and patient outcome (b) role of polymerase chain reaction (pcr) in detecting toxoplasma gondii from blood and csf samples (c) the usefulness of serology in diagnosing acute infection. all cases diagnosed as toxoplasmosls based on the above criteria were reviewed. pcr to detect toxoplasma gondn dna used primers to the b 1 gene giving a 223bp amphficatton product serological tests used were sabln-feldmen dye test and latex agglutination. there were 25 cases diagnosed (17 m, 8f, cd4 0-300; cdc iv 19). 3 panents unknown to be hiv posmve presented with cerebral toxoplasmosis 22 patients were not receiving continuous systemic prophylaxis against pce diagnostic value oft gondii pcr in blood and csf showed a sensmvlty of 20%, specificity of 100%, ppv was 100% and npv was 40% determination of lg subtype was of limited value 25% (6) of patients were seronegatlve of whom 50% (3) had histologically proven disease 2 of these latter 3 cases were pcr negative the dye test was of poor predictive value this review confirms the need to combine all parameters in making a diagnosis of toxoplasmosis in lmmunocompromlsed hosts. the performance of the newly developed, rapid and fully automated m~croparticle enzyme immunoassay abbott imx hiv-1/hiv-2 3rd generation plus assay for the detection of antibody to hiv-1 and hiv-2 including subtype o m human serum or plasma was assessed the assay was evaluated by testmg specimens from blood donors, diagnostic populations and hospitalized patients, hiv seroconverslon panels confirmed hiv-positive specimens, and potentially interfering specimens. the abbott imx hiv-1/hiv-2 3rd generation plus assay showed an overall apparent specificity of 99.97% (lower limit of 95% ci 99.84%) in the tested blood donor populations (n=347t). this comparable to the specificity found for the abbott imx hiv-i/hiv-2 3rd generation plus eia (99 93%) and the axsym h1v-1/hiv-2 assay (99 90%). the apparent sensitivity of the abbott imx hiv-i/hiv-2 3rd. generation plus assay is at least equivalent to that of the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-i/hiv-2 assay. of 21 hiv-i seroconversion panels tested, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected seroconverslon earlier on up to 6 panels, depending on the comparison assay. among 785 specimens from asymptomatic and symptomatic hiv patients, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected 100 (785) including 7 specimens characterized as hiv-1 subtype o. the abbott imx hiv-1/hiv-2 3rd generation plus assay is an extremely sensltlve and highly specific assay for the early detection of antibody to hiv-1/hiv-2 and shows at least an equivalent performance to the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-1/hiv-2 assay. the fully automated imx instrument system offers ease of use and rapid results on a widely accepted and reliable platform. streptokinase (sk), a 47kd protein produced by group c b hemolytic streptococci, is a widely used thrombolytic agent. anti-sk antibodies arise either as a result of therapeutic administration of sk or following natural mfection with streptococci although the clinical significance of antl-sk antibodies is not clear, there is evidence that some anti-sk antibodies arising from natural infections can interfere with sk activity tn vtvo, resulting in thrombolytlc failure. to facilitate further investigations of these antibodies, we have developed and validated a highly sensitive functional assay, which measures sk neutralisatlon activity of serum independently of other circulating inhibitory factors in the sample, and a rapid and convenient enzymeimmunoassay for the detection of anti-sk antibodies. analysis of over 200 random serum samples from the local blood bank with the enzymeimmunoassay showed the prevalence of antl-sk antibodies to be approximately 13%. all the positive samples and an equal number of the negative samples randomly selected were analysed by the functional assay the agreement between the results of the two assays was excellent indicating that our enzymelmmunoassay was a convement method for detection of anti-sk antibodies which could neutrahse sk activity m vitro irish journal of medical science tuberculosis drug therapy, isolation precautions and prophylaxis. conventional methods of detection such as microscopy and culture either lack sensitivity and specificity or are timeconsuming. in this study we investigated the use of a pcr based diagnostic assay for the detection of m. tuberculosis in sputum samples this assay has been developed by bioresearch ireland (bri) and raggio italgene. sputum samples were lysed and pcr amplified using an m. tuberculosis complex-specific primers. the results obtained using the bri/c-trak tm technology were initially compared to the amplicor system (hoffman la roche). both probe detection methods represent fast and reliable methods for the detection of m. tuberculosts in clinical samples. this test is designed to eliminate the possibility of obtaining false negatives. strongyloldes stercoralis infection in humans ts endemic in the tropics. as travel is becoming more common, it will be seen more frequently. two cases of this infection in irish people are described. case i. a 21 year old women had travelled and worked in poor rural areas of mexico for one month, three years before presentation. two and a half years later she developed abdominal discomfort, anorexia and sore throat. myalgia, arthralgta and a transient skin rash began to appear in the next month. eosinophilia, mild anaemia and raised liver blood tests were noted. elisa test for strongyloides was positive but parasites were not seeri in the faeces. ivermectin was given and the patient feels better. case ii. a 31 year old nurse had arthralgia, fatigue and some weight loss for 18 months'. on two occasions in the last four years, she had been travelling extensively in s.e. asia for a total of four months. she was admitted to hospital because of acute fever and loin pain. a urinary tract infection was diagnosed. absolute eosinophil count was i'aised 0.63x10^9/1. esr was 17mm/hr. strongyloides elisa was positive and treatment administered as above. strongylotdes is the most important nematode in the returned tropical traveller. it can multiply and persist within the body for long periods of time and it can cause hypertnfection syndrome, a protean fulminating infection of bowel, lungs, blood stream and brain, in those who are lmmunocompromised. diagnosis can be difficult by stool microscopy. thlabendazole has side effects but ivermectin is safe and effective. june 1995, there was an outbreak of c.difficde-associated diarrhoea (cdad) at st. james's hospital. the aims of this study were to determine the incidence and outcome of cdad in hiv positive and negative patients we prospectively reviewed all patients with diarrhoea, a positive c.difftcile cytotoxin assay, and in whom no other infectious cause for diarrhoea was identified demographic data, history of diarrhoeal episodes, risk factors and outcome were recorded. the incidence of cdad in hiv negative patients was 1.2 per 100 hospital admissions, compared to 1 per 100 admissions m hiv positive patients. the average number of courses of antibtotlcs received, in hiv negative patients prior to the onset of symptoms was 2.3, and 76% of this group were exposed to third generation cephalosporins. hiv positive patients received an average of 3.4 courses of antibiotics and no patients received third generation cephalosporins. there were no deaths due to cdad in hiv positive patients however 4 hiv negative patients died from severe pseudomembranous colitis in conclusion we documented a unexpectedly low incidence and complication rate of cdad in hiv positive patients this is surprising considering their multiple hospital admissions and exposure to ant~microbial and chemotherapeutic agents. the number of new positive hiv specimens detected at the virus reference laboratory has risen from a cumulative total of 638 m july of 1987 to 1597 in september of 1995. we examined our data to determine the proportional make up of these positives by major risk group. in august 1987 64.7% of positive specimens were from intra venous drug abusers (ivda) by september 1995 ivda made up 49% of the total positive hiv specimens. positive specimens from homosexual individuals rose fro.m l0 9% of total positives in august 1987 to 20.6% in september 1995 there were no recorded positive specimens from heterosexual exposure in august 1987 but in september 1995 8.9% of positive specimens recorded heterosexual exposure. a further category which includes blood donors, haemophiliacs, transplant patients and organ donors made up 25% of total positives in august 1987 and in september 1995 made up 22.4% of total positives. we further examined our data in order to show when these changes occurred by ascertaining how many new positive patients have been discovered per year in each of the main risk groups. see in the united kingdom echovlrus type 22 (echo-22) is regularly isolated, with nearly 200 reports annually to the central public health laboratory. reports increased during 1986-1988 and overall it is the second most commonly reported echovlrus in the u.k. echo-22 epldemiology is different to that of other enteroviruses; over 90% of patients with echo-22 tsolated are less than 2 years of age echo-22 shows distinctive and unique cytopathogenic features in tissue culture, and based on sequence analyses, it seems to belong to a separate subgroup of picornaviruses. echo-22 has been associated with respiratory symptoms in premature infants, myocarditis and severe encephalitis. in 1989 an outbreak of acute flaccid paralysis associated with echo-22 was described in jamaica in six patients, four of whom died. we describe three cases of sudden death in infants associated with echovirus type 22 infection case i: s d. born 20/7/94; birth asphyxia and death at two days of age; echo-22 isolated on 2217194 from spleen. this study assessed the antibiotic sensitivity of organisms causing urinary tract infections (uti) among genito-urinary medicine (gum) clinic attenders in order to determine whether it is worthwhile giving tetracycline for dipstick (nitrite) positivity, even in the absence of clinical features of uti. we looked retrospectively at 100 laboratory confirmed uti's diagnosed among gum clinic attenders over a period of eight months. we assessed antibiotic sensitivities of the organisms involved, and determined how many dipstick positive urines which were left untreated turned out to be real uti's. 86% of uti's were due to coliforms and 66% of these were sensitive to tetracycline. 4% of uti's were due to staphylococcus saprophyticus, 3% due to beta haemolytic streptococcus group b, 2% due to enterococcus, 2% due to proteus species and 2% due to coagulase negative staphylococci. 25% of nephur positive urines were left untreated. 32% of these were nitrite positive. failure to treat a positive urine dipstick which turned out to be a uti necessitated a further clinic visit for adequate treatment. nitrite positive urines should be treated as a uti, even in the absence of clinical features of uti, either with trimethoprim or tetracycline. the number of untreated uti's and unnecessary extra visits to gum clinics would have been reduced with the use of judicious antibiotic therapy for nitrite positive urines. strains of enterococci resistant vancomycin have been reported with increasing frequency. in 1995, we investigated an increase in the frequency of vancomycin-resistant enterococcus faecium (vref) among patients in the haematology/oncology unit using pulse-field gel electrophoresis (pfge) to genotype these isolates and to assist in establishing the source of these vref. eighteen clinical isolates of vref from blood, urine sputum and-faeces and two environmental isolates were collected from separate patients between march and july 1995. minimum inhibitory concentrations (mics) to several antibiotics including teicoplanin and vancomycin were determined by agar dilution. pfge were performed following smal restriction endonuclease digestion. antimicrobial susceptibility testing revealed high level resistance to vancomycin and teicoplanin; mics >128 mg/ l and >32 mg/l respectively. this antibiogram is consistent with the van a phenotype. pfge of all 20 isolates revealed identical patterns indicating clonal spread of vree subsequent implementation or infection control measures reduced the frequency of vref isolation. pfge proved useful in demonstrating clonal spread of vref and.in emphasising the need for infection control measures. a prospective audit of baeteraemia in our 600 bed teaching hospital was carried out from february 1991 to march 1993. clinical and microbiological data were collected on 520 episodes of bacteraemia in 78 patients. of these 230 (62%) were hospital acquired and 200 (38%) community acquired. urinary tract and respiratory tract sources were implicated in 30% and 14% of community acquired episodes, making e. coli and s. pneumoniae the commonest community acquired isolates (41% and 16% respectively). other gram negative bacilli accounted for 13% and s. aureus for 11%. coagulase negative staphylococci were the commonest hospital acquired isolate (24%) followed by s. aureus (22%), e. coli (i5%) and enterococcus spp. (10%). enterobacter spp. were the second commonest gram negative isolate (5%). central venous cannulae were implicated in 43% of hospital acquired cases. urinary tract infections accounted for 20%. 63% of which were catheter related. invasive diagnostic procedures (angiography, prostate and liver biopsies, sinography) were implicated in t0 episodes. gentamicin resistance was found in 9% of hospital acquired aerobic gram negative bacilli and mrsa accounted for 13% of hospital acquired s. aureus. these figures are higher than expected but may be explained by outbreak of mrsa and gentamicin resistant entercobacter spp. which occurred during the study period. the past severalyears have seen a significant increase in the recognition of moraxella (branhamella) catarrhalis as a respiratory pathogen (~). the pathogenic mechanisms employed by the organism are largely unknown, but adherence may play a role ~2~. in our investigation the haemagglutinating ' activity of 40 isolates of m. catarrhalis was determined by a microtitre method. no isolate agglutinated horse, chick or sheep red blood cells (rbc). seventeen isolates agglutinated human rbc, x~hile 7 of these 17 isolates also agglutinated rabbit red. blood cells. haemagglutination of human and rabbit red blood cells was inhibited by porcine mucin. galactose inhibited the haemagglutiriating activity of the 7 isolates which agglutinate both human and rabbit rbc and yet bad no effect on the haemagglutinating activity of the isolates which haemagglutinate human rbc alone. .electron microscopy studies of the bacteria demonstrated a diffuse outer fibrillar layer on the surface of haemagglutinating positive isolates, thislayer was subsequently removed following trypsin treatment, as was the haemagglutinating activity. a 200 kda trypsin sensitive protein appears to be associated wfth haemagglutinating properties. mrsa is an increasingly important cause of morbidity, and is spreading from large hospitals to smaller community-based facilities and nursing homes. the objective of this survey was to obtain an indication of the size of the mrsa problem in ireland prior to introducing national mrsa control guidelines. a survey of all microbiology laboratories in ireland was carried out over two weeks in spring 1995. for patients from whom mrsa was isolated during the study period standard demographic and clinical data were requested and period prevalence/1000 discharges was calculated. all 45 microbiology laboratories surveyed responded. mrsa was isolated from 448 patients during the 2 week period. the period prevalence of mrsa/1,000 discharges was 16.5. males aged 65+ had the highest rate of infection (50/1000 discharges). half of all isolates were from patients in surgical or medical wards, but 4% were from community-based sources e.g. gps, nursing homes, hospices. thirty-two percent of mrsa patients were infected rather than colonised. mrsa is clearly a substantial problem in ireland. while it is largely a hospital problem at present, the increasing trend for day procedures and shorter stays means that infection will increase in the community. a survey in a university hospital in the usa revealed 41% of mrsa cases to be communityacquired. tonsil core specimens were cultured for bacteria including mycoplasma, chlamydia and ureaplasma urealyticum in 70 children undergoing tonsillectomy for recurrent acute tonsillitis. serology for chlamydia and mycoplasma pneumoniae was obtained in 55 of the children. the polymerase chain reaction (pcr) was used to investigate the presence of chlamydia pneumoniae in core tonsil tissue. ureaplasma urealyticum was cultured in three children (4.3%) and mycoplasma salivarium in two children (2.8%). culture was negative for chlamydia pneumoniae and mycoplasma pneumoniae. the complement fixation test for chlamydia species was positive in 16/55 children (29%) indicating previous infection. specific immunofluorescence testing for c. pneumoniae was positive for lgg (titre> 16) in 10/55 (18%). igm antibody to c. pneumoniae and antibodies to c. trachomatis and c. psittaci were not detected. ninechildren (16%) had titres > 32 to m. pneumoniae. pcr failed to demonstrate c. pneumoniae. aerobic and anaerobic bacteria were cultured from all specimens. the culture of ureaplasma urealyticum in 4.3% of our patients indicates a higher rate of colonisation then previously thought. this study 49 irish journal of medical science demonstrates past infection with c. pneumoniae in 18% and with m. pneumoniae in 16% of children with recurrent tonsillitis. however c. pneumoniae and m. hominis do not play a significant role in childhood recurrent tonsillitis. multiply resistant enterococci are increasingly common causes of serious infection in hospitalized patients. high level gentamicin resistance (mic > 1000 mga) in enterococci further compromises the therapy of such infections. we have identified seven clinical isolates of enterococcus hirae demonstrating high-level gentamicin resistance (hlgr: mic > i000 mg/1). to our knowledge this is the first report of hlgr for this enterococcus species. plasmid analysis has demonstrated the presence of a single, large plasmid in all seven isolates, as well as several smaller plasmids in some of the isolates. filter mating experiments have revealed that in all seven cases, hlgr was transferred to a laboratory recipient e. faecalis jh-22 by conjugation. plasmid analysis of transconjugant strains confirmed transfer of the large plasmid in all cases. based on restriction enzyme profiles, two distinct conjugative plasmids were identified for the e. hirae isolates investigated. at present we are using southern blot techniques with oligonucleotide probes designed to hybridise to the hlgr determinant found in other species of enterococcus. the results will confirm whether or not the same resistance determinant is responsible for the dissemination of hlgr in the genus enterococcus. dublin 8. aminoglycosides remain commonly used in the treatment of severe gram negative infection and have conventionally been given on a twice or thrice daily basis. single daily dosing offers advantages with respect to less nephrotoxicity, better bactericidal activity, convenience, nursing time, cost and should avoid subtherapeutic dosing which has a significant impact on outcome. we reviewed serum gentamicin assays from january to december 1995 to assess potential toxicity and subtherapeutic dosing in patients who received once daily gentamicin and those who received multiple daily dosing. 4346 assays were performed in the study period. 520 of those were random assays and not included. there was a trend towards significantly less potentially toxic levels in the once daily group compared to the multiple daily group (p<0.1). once daily dosing produced significantly less subtherapeutic dosing (p<0.001). over 98% of peak assays in the once daily group were in the recommended range. we conclude that current practice of multiple daily dosing of gentamicin leads to significant underdosing and more potentially toxic trough levels. measurement of trough assays only in patients who are treated with once daily aminoglycosides is sufficient and will have considerable cost savings. respiratory, syncytial virus (rs virus) is a major respiratory pathogen of infants less than 1 year old. it occurs in annual epidemics during the winter and early spring in temperate climates. during rs virus epidemics a significant number of infants less than 6 months old are hospitalised with symptoms of bronchiolitis and pneumonia. rs virus exists in two antigenically distinct subgroups, a and b which are known to cocirculate in the same community during the same rs virus season. there is much debate regarding the virulence of one strain over the other. using a panel of monoclonal antibodies specifically directed against the two rs virus strains, 167 rs virus isolates from specimens sent to the virus reference laboratory, university college dublin, over seven consecutive rs virus seasons (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) were typed and the rs virus subgroup predominance monitored. subgroup a was the most predominant of the rs virus isolates accounting for 67.7% of the total and was found to be the predominant rs virus strain in six out of the seven rs virus seasons studied. subgroup b predominated in a season in which the number of rs virus detections peaked much later than normal. treatment of fungal infections in patients in intensive care unit (icu.) is usually empiric. the aim of this study was to identify candida species isolated from i.c.u. patients and to test their susceptibility to antifungal agents to enable more directed therapy. forty candida sp. from 23 patients in i.c.u. were isolated from the following sites, blood culture (5), central venous catheter (7), chest drain fluid (5), wounds (8), catheter urine (8), bronchial lavage (3), sputum (4). strains were identified by standard procedures. minimum inhibitory concentrations of amphotoeracin b, 5-flucytosine and fluconazole were obtained by agar dilution test and e test. the isolates were identified as c.albicans (22), c.glabrata (8), c.krusei (5), c.tropicalis (2), c.parapsilosis (2), c.kefyr (i). all the candida species were sensitive to amphoteracin b (mic = 1-<025mg/l), and 5 flucytosine (mi c= <025mg/l). c.albicans and c.parapsilosis were fluconazole sensitive (mic = 16-15mg/l). four of eight c.glabrata were fluconazole resistant (mic = >256mg/l). c.krusei, c.tropicalis and c.kefyr were also resistant (mic = >256 mg/l). wbilst there were no major discrepancies between the agar dilution test and the e test, the agar dilution test was laborious and required a high degree of skill. the e test was easier to read and more reliable results were obtained provided the inoculum was carefully standardised. this study shows that azole antifungals should not be ganglion is probably the commonest tumour encountered in the hand and the wrist. it often arises from tendon sheath or lining of a joint capsul. the treatment can be surgical or nonsurgical, the latter includes aspiration with or without injection of steroids. surgical treatment of ganglion can pose a difficult situation to deal with. it requires hand surgeon to deal with one such problem, we present a 46 year old man with tender mass in the hypothenar eminence. during surgical exploration it was obvious that the ganglion was infiltrating the wall of the ulnar artery, and the histology proved this later. the clinical features, management arid the outcome of this unusual case are discussed. ischaemic preconditioning (ipc) of the myocardium with repeated brief periods of ischaemia and reperfusion (i-r) prior to prolonged ischaemia significantly reduces subsequent infarction. following ipc two "windows of opportunity" (early and late) exist during which prolonged ischaemia can occur with reduced myocardial infarction, we investigated if ipc of skeletal muscle prior to flap creation improved subsequent flap survival and perfusion in either early or late windows. the latissimus dorsi muscles (ldm) of sprague-dawley rats were used. group 1: (control, n=12). the ldm was elevated as a thoracodorsatly based island flap group 2: (early ipc, n=8). the ldm was preconditioned with two 30 minute episodes of normothermic global ischaemia with intervening 10 minute episodes of reperfusion prior to elevation. group 3: (late ipc, n=8). the ldm was elevated 24 hours after ipc ischaemia was created by occlusion of the thoracodorsal artery and vein and the intercostal perforators having previously isolated the muscle on these vessels. muscle perfusion was assessed by a laser doppler perfusion imager. one week after flap elevation the percentage of muscle necrosis was measured by computer-assisted planimetry ipc significantly reduced muscle flap necrosis (table) in both early and late windows. muscle flap perfusion was similar in all groups. this study compares the biochemical, serological and histopathological findings in 20 women with chronic hepatitis c virus (hcv) infection with 20 age-matched women with established autoimmune hepatitis (aih). there is increasing evidence of autoimmunity in hcv liver pathology. because of different treatment regimens for hcv (d-interferon) and aih (steroids, immunosuppression), clear distinction between the two diseases is desirable. liver enzymes (alt, ast, alkaline phosphatase), antinuclear factor (anf), anti-smooth muscle (asm) and antimitochondrial (ama) antibodies are compared for both groups of patients. liver biopsies from all women were compared using the grading and staging system of ishak et al (1995) . the results show that while some women in both groups show elevated liver enzymes, positive anf and asm autoantibodies, the values are much higher in aih. similarly aih patients show overall more severe disease on liver histology. both groups demonstrate poor correlation between histological features and autoantibody titre. we conclude that distinction between hcv and a!h is usually possible with liver histology and serology. some women with chronic hcv and positive autoantibodies however," demonstrate a histological and serological picture suggesting that chronic hcv may be mediated by an immunopathogenic mechanism. irish journal of medical science terminal changes only, 5 showed lymphocytic meningitis and 4 of these also had perivascular lymphocytic inflammation (cd3 positive) in the subependymal regions, brainstem and choroid plexus. two brains showed purulent meningitis and one case had central pontine myelinolysis probably related to profound metabolic disturbance. basal ganglia mineralization, hiv encephalitis (hive) or hiv leucoencephalopathy (hivl) were not present. these findings differ considerably from those described in us cases, in whom the majority have evidence of hiv within the cns. relatively early death from systemic infection may account for the lack of hive/hivl in these cases. the lymphocytic meningitis and perivascular inflammation may represent an immuno-allergic reaction, previously reported as "early" changes, regarded as important in inducing vascular damage which allows subsequent entry of h1v into the brain. the aim of this study is to assess apoptosis in areas of interface hepatitis and spotty necrosis in hepatitis c virus (hcv) infected liver biopsies, and to correlate the degree of apoptosis with severity of histological activity. 25 liver biopsies were randomly selected from a group of type lb hcv positive women. these patients were diagnosed by recombinant immunoblot assay (riba) test and the presence of hcv rna was confirmed using the polymerase chain reaction (pcr). apoptosis was demonstrated in the biopsies by the oncor apoptag in-situ hybridisation technique. the average number of apoptotic hepatocytes per portal tract, and within the parenchyma per 10x objective, was determined. the modified histological activity index (h.a.i.) was used to score each biopsy. comparison of the results shows that increasing numbers of apoptotic hepatocytes are consistently associated with increasing scores for interface hepatitis and spotty necrosis. it is concluded that apoptosis occurs in hcv infected livers and that it correlates with increasing histological activity .indicating a significant role for apoptosis in the pathogenesis of hcv liver disease. and university of edinburgh, scotland. paediatric aids represents only 5% of cases worldwide, in most published series parental iv drug use was the main risk factor, cns pathology was a late feature of the disease and antiretroviral treatment had been given. we studied eleven brains from romanian children with probable postnatal hiv infection using standard neuropathological stains and immunostains for lymphocyte markers and hiv p24 antigen. death was due to systemic infection, mostly pneumonia or gastroenteritis; antiretroviral treatment had not been given. three cases showed we studied the role of the p53 and bcl-2 genes in the pathogenesis of post-transplant lymphoproliferative disease (ptl). ten cases were examined by immunohistochemical and molecular methods. immunohistochemistry was performed using standard and antigen retrieval methods, with the p53 do-7 and the bcl-2 oncoprotein clone 124 antibodies. dna was extracted from paraffin blocks and subjected to pcr, and single-strand conformation polymorphism (sscp) analysis searching for mutated p53 genes. samples showing any evidence of aberrant migrations were further analysed by direct sequencing. pcr was also used to detect bcl-2 gene rearrangements. we employed a technique called representational difference analysis to search for previously undescribed translocations or deletions which may be involved in breast carcinogenesis. dna was isolated from both invasive ductal carcinoma of the breast and normal tissue from the same patient. following restriction enzyme digestion and tigation of oligonucleotide linkers, pcr was carried out on both tumour and normal dna using oligonucleotide specific primers to obtain a representation of each genome (amplicons). digestion with the same restriction enzyme removed the original linkers, and a second set of oligonucleotides were ligated to normal amplicons only. the tumour derived amplicons were subtractively hybridised to the normal and subsequent pcr was used to isolate fragments unique to the normal dna (difference products). this was possible since oligonucleotides were ligated to normal dna only. following a series of further subtractive hybridisations and subsequent amplifications, purified difference product was obtained. difference products in the size range 500-1500 bp were obtained. following further rounds of subtractive hybridisation and amplification, purified difference product will be sequenced and characterised by comparison with known gene sequences. the chromosomal location of the affected gene will be established by in-situ hybridisation and somatic ceil hybridisation, using the difference product as probe. protein s is secreted by osteoblasts and case reports of reduced bone mineral density in patients with total protein s deficiency have lead to the hypothesis that this inherited disorder is associated with generalised osteoblast dysfunction predisposing to osteoporosis ~. we have assessed bone formation in 8 patients (2 male and 6 female) with total protein s deficiency and 4 controls (2 male and 2 female) using two recently available sensitive markers; serum osteocalcin (oc) and serum procollagen 1 carboxyterminal peptide (p1cp), both secreted by the osteoblast. the mean total protein s level amongst the patients was 40 _+ 12% (ref range 62-135%), mean oc was 14.2 ng/ml (ref ~'ange 5.2-59 ng/ml) and the mean picp was 81.5 + 15 uga (ref range 38-02 ug/1). in the control group, mean oc was 12.6 _+ 5 ng/ml and the picp was 124 + 37 ug/1. there was no statistical difference between both groups using either marker. in conclusion bone formation as assessed by serum osteocalcin and p1cp appears to be normal in patients with total protein s deficiency. hereditary spastic paraparesis (hsp) is a variably expressed neurodegenerative disorder which exhibits clinical and genetic heterogeneity. hsp can be inherited in an autosomal dominant (ad), autosomal recessive (ar) or x-linked manner. ad-hsp has been linked to a number of loci. we have ruled out linkage to these loci in a large irish family affected with ad-hsp. the aim of this study was to determine whether ad-hsp is linked to spinocerebellar ataxia loci (sca), sca-i & sca-ii. ad-hsp can be clinically similar to sca. dna was extracted from blood taken from 45 co-operating family members. microsatellite markers spanning the sca-i and sca-ii loci were amplified by pcr. individuals were genotyped and linkage analysis was carried out using the linkage set of programs. significantly negative lod scores were obtained for both sca-i and sca-ii loci. d6s274 gave maximum exclusion of 18cm on either side of the sca-i locus with a lod of -2.11 at a recombination fraction of 0.18. d12s105 gave maximum exclusion of 13cm on either side of the sca-ii locus with a lod of -2.08 at a recombination fraction of0.13. other markers examined also outruled linkage to these loci. we conclude that the gene for ad-hsp is unlinked to the major sca loci. serum vitamin b12 is frequently measured in the investigation of anaemia, and in screening neurological and other disorders. frequently, patients are found with low serum vitamin b12 level with a normal hb and without clinical abnormalities relevant to vitamin b 12 deficiency. this study was carried out to determine the significance of a low serum vitamin b 12 level. 959 vitamin b12 measurements were carried out over an 8 month period using a chemiluminescence method (abbott imx). clinical data was obtained retrospectively. of the 959 samples 81 (8.4%) representing 65 patients had a low serum vitamin b12 level (>180 pg/ml) with a mean of 152 pg/ml (39-179). data was available on 44 patients. 20 (45%) had a hb below the normal range with median serum vitamin b12 level of 158 pg/ ml (75-184). 19 (45%) had a normal hb, mcv and mchc with a median serum vitamin b12 of 152 pg/ml (52-210), and 5 had a normal hb with an abnormal mcv or mch. lft's, autoantibodies, schillings test and bone marrow examination data will be presented. in conclusion in patients with a low serum vitamin b 12 level, there was no significant difference in the b12 levels in those patients with a normal or a low hb concentration. it would appear that serum vitamin b12 is a poor discriminatory test but that changing the normal range may not help in screening. low serum vitamin b12 on its own may not appear to provide adequate grounds for lifelong replacement therapy. of 1 in 133 for males and 1 in 172 for females. as expected the overall incidence of hodgkin's was lower with one third of male and one quarter of female lymphoma cases affected by the disease. a distinct age specific pattern is evident depending on lesion type. marked variation in incidence levels were noted throughout the study region. an extremely varied pattern is evident in the survival rates for lymphoma patients. the cork and kerry rates for malignant lymphoma are relatively low when compared with international levelstzl obstetric complications and schizophrenia: methodology and mechanisms perinatal complications and clinical outcome within the schizophrenia spectrum 96) negative symptoms, cognitive impairment and duration of initially untreated psychosis in schizophrenia davnet's hospital, monaghan, and royal college of surgeons in ireland retinal pathology in kearns sayre syndrome mitochondrial dna and disease mitochondrial myopathies: clinical features, investigation, treatment and genetic counselling phenytoin induced pseudolymphoma. a report of a case and review of the literature cutaneous reactions in head injured patients receiving phenytoin for seizure prophylazis hydantoin induced pseudopseudolymphoma branhamella catarrhalis: an organism gaining respect as a pathogen correlation between branhamella catarrhalis adherence to oropharyngeal cells and seasonal incidence of lower respiratory tract infections 206) epidem1ology and survival rates for all 324 lymphoma patients registered in cork and kerry over the eight year period an indepth review of all 324 lymphoma (icd -o code 196) patients registered by the sourthern tumour registry during the eight year period 1983/1990~l annual age adjusted rates of 7.6. and 5.4 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk references 1. cancer, the irish experience cancer incidence in five continents volume v immunohistochemistry for bcl-2 oncoprotein without antigen retrieval gave negative results, but with antigen retrieval, showed positive staining in 6 out of 8 cases. no bcl-2 rearrangements were detected by pcr. the combination of sscp and sequencing confirmed only wild type dna in all cases, p53 immunohistochemistry by standard methods revealed positive staining in only one out of nine samples analysed. when the antigen retrieval method was employed for this antibody, positive staining was seen in > 10% of tumour cells in four further cases.our results suggest that p53 does not play major role in ptld. bcl-2 overexpression but not rearrangement may contribute to the development of ptld. transplant arteriopathy (ta) is the major cause of death in cardiac allograft recipients. the pathogenesis is unclear. we have previously shown a plasma cell predominance in the infiltrate of ta, leading us to hypothesise a role for epstein-barr virus (ebv) infection in its pathogenesis. an association between cytomegalovirus (cmv) and ta has previously been suggested. the aim of the study was to investigate the role of epstein-barr virus (ebv) and cytomegalovirus (cmv) in the pathogenesis of ta.we performed pcr for cmv and ebv dna and protein (lmp) in seven cases of ta, involving cardiac allografts. restriction mapping was used to confirm that pcr products were either cmv or ebv dna respectively.cmv dna was found in four cases. ebv dna was found in six of the seven cases and ebv lmp staining was present in six cases. ebv was detected in all cases by either pcr or ihc.our results suggest that ebv infection may play a pathogenic role in transplant arteriopathy. the evidence for a similar role for cmv ~s less strong. st. vincent's hospital. hereditary spastic paraplegia (hsp) is a neurodegenerative disorder characterised by progressive spasticity, primarily of the lower limbs. it can be inherited in an autosomal dominant (ad), autosomal recessive or x-linked manner~ we have identified a large irish family (family a) affected with ad hsp that cosegregates with dementia. three. loci have previously been identified that are linked to ad hsp in families of different ethnic origin. the locus on chromosome 2 is reported to be the major hsp locus. the aim of the present study was to examine family a for linkage to the chromosome 2 hsp locus.dna has been extracted from blood taken from all 45 co-operating family members for genotyping. polymorphic microsatellite markers from chromosomal region 2p24-21 have been amplified by pcr, electrophoresed on a denaturing polyacrylamide gel and detected by silver staining. linkage analysis was carried out using the linkage series of programs. linkage analysis excluded the hsp gene from the chromosome 2p locusl the most significant marker was d2s367, with a lod score of-2.12 for recombination fraction 0.19, thereby excluding approximately 19cm either side of this marker. negative lod scores were also obtained for the other markers chosen (d2s391, d2s392, d2s352, d2s 174) excluding 20cm, 8cm, 4cm and 8cm respectively.the current study has therefore successfully excluded linkage of ad hsp in family a to the major locus on chromosome 2p. further studies are underway to exclude linkage of hsp in this family from other candidate loci, prior to carrying out a genome wide search. the presence of dementia in this family in association with hsp suggests that a new and as yet unidentified gene is responsible. vincent's hospital. eye and ear hospital, dublin. ched is a corneal endothelial dystrophy characterised by diffuse bilateral corneal opacities resulting in impaired vision. both autosomal dominant and autosomal recessive modes of inheritance have been described. another endothelial dystrophy, posterior polymorphous dystrophy (ppmd) has been linked to 20qll.we have used homozygosity mapping to analyse a pedigree with autosomal recessive ched for linkage to 20ql.1. all affected individuals are offspring of consanguinous matings. homozygosity mapping is based on the principle that these offspring would be homozygous for genetic markers near the disease gene. homozygous regions would be random between different offspring of these matings, except at the di-sease locus shared by affected offspring.dna was extracted from blood taken from 22 family members, 14 of which have ched. allele frequencies were determined in pooled dna from affected individuals. pooled dna from unaffected individuals was used as a control. at the disease locus, a shift in allele frequencies towards a single homozygous allele would be observed in the affected dna pool. pooled dna was genotyped by pcr for 3 polymorphic microsatellite markers in the region of20qll. pcr products were separated on a polyacrylamide gel and visualised by silver staining. similar allele frequencies were observed at these loci in both dna pools demonstrating independent assortment of alleles. in addition, affected and unaffected family members were individually genotyped at these loci and no significant loss of heterogeneity in the affected individuals at these loci was observed. these data indicate exclusion of linkage of the ched gene to 20qll. key: cord-307934-84zfabti authors: lai, chao-kuen; saxena, vikas; tseng, chung-hsin; jeng, king-song; kohara, michinori; lai, michael m. c. title: nonstructural protein 5a is incorporated into hepatitis c virus low-density particle through interaction with core protein and microtubules during intracellular transport date: 2014-06-06 journal: plos one doi: 10.1371/journal.pone.0099022 sha: doc_id: 307934 cord_uid: 84zfabti nonstructural protein 5a (ns5a) of hepatitis c virus (hcv) serves dual functions in viral rna replication and virus assembly. here, we demonstrate that hcv replication complex along with ns5a and core protein was transported to the lipid droplet (ld) through microtubules, and ns5a-core complexes were then transported from ld through early-to-late endosomes to the plasma membrane via microtubules. further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that ns5a-core complexes, but not ns4b, were present in the low-density fractions, but not in the high-density fractions, of the hcv rna-containing virions and associated with the internal virion core. furthermore, exosomal markers cd63 and cd81 were also detected in the low-density fractions, but not in the high-density fractions. overall, our results suggest that hcv ns5a is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to hcv natural infection. hepatitis c virus (hcv) is a major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. hcv is an enveloped virus with a 9.6-kb positive-strand rna genome. this genome encodes a large polyprotein, which is processed by host and viral proteases into 10 viral proteins that consist of three structural proteins, six nonstructural proteins, and a small hydrophobic peptide, p7 [1, 2] . the structural proteins, core protein and two envelope glycoproteins e1 and e2, are derived from the n terminal portion of the polyprotein and constitute physical virion components. the nonstructural (ns) proteins, ns2, ns3, ns4a, ns4b, ns5a, and ns5b, are derived from the c terminal portion of the polyprotein. most of the ns proteins (with the exception of ns2) are involved in hcv replication [3, 4] . hcv rna is synthesized in the replication complex (rc), which exists in the membranous web derived from altered er membranes [5, 6] . the hcv rc is transported on microtubules and this transport is facilitated by the interaction of ns3 and ns5a with tubulin [7] . the intact microtubule network also is directly involved in hcv rna replication [8] [9] [10] and virus release [10, 11] . following hcv rna replication, core protein and ns5a serve as central regulators of virus assembly [12] . core protein forms multimers [13] and interacts with the viral rna [14] to form the viral nucleocapsid. the core protein is localized mainly on the surface of the lipid droplets (lds) [15, 16] , which is essential for the production of infectious hcv particles [15] . further, core protein promotes the accumulation of lds to facilitate virus assembly [11, 17] and recruits viral rcs to ld-associated membranes [15] . thereby, viral rna interacts with core protein in juxtaposition to ld for virus packaging. moreover, the interaction between ns5a and core protein is essential for the recruitment of the viral rcs to lds and plays an important role in virus assembly [18, 19] . however, how viral rcs and core protein target to ld remains unclear. in addition to ns5a, other ns proteins, including ns2, ns3, and ns4b, have also been shown to influence the production of infectious virus [12] . up to now, it is not known whether the ns proteins are incorporated into infectious virions. previous studies have indicated that cell culture[20] [21] [22] [23] [24] and patients' serum-derived [25] [26] [27] [28] [29] hcv particles display heterogeneous diameters (from 35 to 145 nm) and have a broad range of buoyant density (between 1.01 g/ml and 1.17 g/ml). the main peak of both viral core protein and rna exhibited at a density of 1.15 to 1.17 g/ml in the cell culture derived-hcv (hcvcc) [30, 31] , and the highest specific infectivity of extracellular virion was observed at a density of 1.14 g/ml [20] . notably, the lowdensity fraction (density of ,1.1 g/ml) displays exosome-like structures and also contains infectivity [20] , but the nature and origin of their properties are still unknown. many types of cell continuously secrete a large number of microvesicles, called exosomes, which have a diameter of approximately 50-150 nm and have a buoyant density between 1.08 g/ml and 1.22 g/ml [32] . exosomes are released into the extracellular space from late endosomes/multivesicular bodies (mvbs) fusion with the plasma membrane [33] . more recently, the exosomes derived from cells containing hcv subgenomic replicon have been demonstrated to contain hcv rna, but not viral ns proteins [34] . our previous results [10] have shown that hcv core proteins are transported from early to late endosomes/mvb in hcv-infected cells. however, it is not known whether any hcv proteins are incorporated into the released exosomes from hcv-infected cells. in this study, the trafficking mechanism of the ns5a and core proteins is defined further. both ns5a and core proteins are found to be closely associated with and co-transported along the microtubules from the perinuclear region of cells via the lds and endosomes to the plasma membrane. this association of ns5a-core proteins implicated them in virus assembly as well as release. interestingly, we found that both ns5a and core, in addition to exosomal proteins cd63 and cd81, were detected in the lowdensity hcv particles (1.083 to 1.098 g/ml) with low-grade infectivity. ns5a appeared to be incorporated into hcv particles through interaction with core protein and microtubules during intracellular transport. our data suggest that ns5a-containing, low-density hcv particles were released in the form of exosome. huh7.5 cells, a mutant line of huh7 cells that support hcv replication at high efficiency [35] were cultured in dulbecco's modified eagle's medium (dmem) containing either 10% fetal bovine serum (fbs) or 10% dialyzed fbs [36] for a general subculturing and for preparation of purified hcv, respectively. rep 1.1 cells [7, 37] are huh7 cells that harbor a genotype 1b hcv subgenomic replicon. they were grown in the aforementioned medium containing 0.5 mg/ml of g418. plasmid puc-jc1, which encodes a chimera genome of hcv j6cf/jfh1, was constructed as previously described [38] . antibodies used in this study included anti-ns5a (austral biologicals), anti-core (affinity bioreagents inc.), anti-bromodeoxyuridine (sigma-aldrich), anti-eea1 or -cd63 or -lamp-1 (santa cruz biotechnology), mouse anti-cd81 (bd pharmingen), rabbit anti-cd81 (santa cruz biotechnology), anti-e2 (genetex, inc.), anti-calnexin (assay designs/stressgen), and anti-mouse orrabbit colloidal gold conjugates (jackson immunoresearch inc.). rabbit polyclonal abs against core (rr8) and ns4b (rr12) were described previously [15] . cy3-conjugated primary ab to btubulin and nonspecific normal mouse immunoglobulin g (igg) were obtained from sigma-aldrich. secondary abs against mouse, rabbit and goat were purchased from invitrogen molecular probes. reagents used were nocodazole (sigma-aldrich), taxol (paclitaxel; sigma-aldrich), bodipy 493/503 (invitrogen molecular probes), 4',6-diamidino-2-phenylindole (dapi; invitrogen molecular probes) and wheatgerm agglutinin (wga) alexa fluor 647 conjugate (invitrogen molecular probes). the jc1 viruses were produced and titrated in huh7.5 cells based on a previously described method [7] . the same batch of culture supernatant from virus-infected and the control cells, including hcv subgenomic replicon cells (hcv assembly-defective replicon cells) and uninfected cells, was used in all experiments. the concentrated hcv jc1 and controls were subjected to 10 to 50% sucrose gradient sedimentation centrifugation as previously described [39] . a total of 15 fractions of 0.75 ml each was collected from the bottom to the top of the sucrose gradient and monitored for hcv rna by using quantitative pcr (qpcr) [10] . fractions containing the hcv rna signal (fractions 7 to 8 and fractions 11 to 13) and their uninfected counterparts were pooled and dialyzed against tne buffer (10 mm tris, 150 mm nacl, 2 mm ethylene diamine tetraacetic acid) overnight at 4uc. the virus-containing fractions were then concentrated by 10-fold in ultracel-3k concentration devices (millipore) and further processed for electron microscopy applications or infection. infectivity of each fraction was determined by quantifying the amounts of intracellular hcv rna levels at day 3 postinfection (p.i.). huh7.5 cells in six-well plates were infected with concentrated hcv from each fraction of the sucrose gradient suspended in dmem at 37uc for 3 h. cells were washed with pbs and incubated with 2 ml of dmem containing 10% fbs. at 3 days postinfection, total rna was isolated from cell lysates using a high pure rna isolating kit (roche). viral rna was isolated from cell culture supernatants using a qiaamp viral rna kit (qiagen). qrt-pcr was performed as described previously [10, 40] . the procedure of em was carried out exactly as described previously [40] . for immuno-em, samples were first incubated with an anti-ns5a, anti-core or anti-e2 mouse mab, and followed by incubation with colloidal gold particles of 18 nm conjugated to antimouse immunoglobulin g. for immuno-em of the purified viruses, two microliters of purified virus were adsorbed onto carbon-coated grids. grids were fixed with 2% paraformaldehyde for 10 min and blocked in a solution of 0.5% bovine serum albumin (bsa) for 20 min. the grids were incubated with primary abs and normal mouse igg for overnight at 4uc. grids were then washed and incubated with goat anti-mouse and/or goat anti-rabbit colloidal gold particles (6-and 12-nm diameter) for 1 h at room temperature. after extensive washing, the grids were stained with 1% uranyl acetate for 1 min. for analysis of hcv core particle, the purified virus was treated with 0.01% saponin in pbs for 20 min before processing. all samples were analyzed under a tecnai spirit transmission electron microscope (fei co) at 120 kv. cell labeling with 5-bromouridine 5'-triphosphate (brutp) was performed according to the methods described previously [7] . hcv-infected cells (at day 10 p.i.) were grown on 4-well chamber slides. one day after seeding, cells were incubated with actinomycin d (10 mg/ml) for 30 min. then, 20 ml of brutp/ fugene 6 (roche molecular biochemicals) mixture were added to each well containing 500 ml medium with actinomycin d (10 mg/ ml). after 30 min of incubation at 37uc, cells were treated with 10 mm nocodazole or taxol for 1 h, and then fixed and processed for immunofluorescence staining as described below. cells were gown on glass chamber slides. cell fixation and immunostaining were performed by the methods described by listenberger l. l. and brown d. a. [41] . photographs of the cells were taken with a confocal microscope (zeiss confocal laser scanning microscope lsm 510meta-nlo). the procedure for quantitative colocalization analysis used in this study followed the published method [10] . the weighted colocalization coefficient is the sum of intensities of colocalization pixels relative to the overall sum of pixel intensities above the threshold. it was used to determine the relative levels of figure 1 . colocalization of ns5a with core protein and microtubules in lipid droplets. hcv-infected cells (at day 10 p.i.) were co-stained with anti-tubulin (green), -ns5a (red) (a) and/or -core (red, b; blue, c) antibodies. lipid droplets (lds) were stained with bodypi 493/503 (blue, a and b; brown, c) and nuclei with dapi (gray). stained cells were examined by confocal fluorescence microscope. merging of the images in the green, red, blue, and gray channels generated the pictures in fig. a and b. fig. c was generated by merging the images in the green, red, blue, and brown channels. yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. the upper third panels in fig. a shown for core (red) or ns5a (red) and with dapi (blue) in the lower panels of fig. a and c, respectively. colocalization efficiency between ld and core protein (b) and between ld and ns5a (d) was analyzed by using zeiss lsm zen software. (e) analysis of cellular proliferation and survival by mts assay. (f, g) quantitation of confocal microscopic fluorescent signals of core and ns5a in cells. the total fluorescence intensities of core protein and ns5a were measured using metamorph integrated morphometry analysis. a total of 20 cells were used for calculation of colocalization efficiency and total fluorescence intensity from two independent experiments and error bars represent standard deviations of the mean. noc, nocodazole. bars, 10 mm. (h) in parallel, the cell lysates were collected and then immunoblotted with antibodies against core and ns5a. results were quantified by phosphorimager counting. doi:10.1371/journal.pone.0099022.g002 were transfected with brutp in the presence of actinomycin d for 1 h and then treated with either 10 mm nocodazole or 10 mm taxol for 1 h. the cells were co-stained with mouse mab against bromodeoxyuridine (green) and rabbit polyclonal antibodies against core (rr8) (red). lds and nuclei were stained with bodypi 493/503 (blue) and dapi (cyan), respectively. enlarged views of parts of every image are shown (insets). (b) the number of brutp-labeled viral rna was counted manually using an original magnification of 6630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (c) the average distance between the center of the signal emitted by the brutp-labeled viral rna colocalization between core protein, ns5a or ns4b with the protein of interest (e.g., calnexin, ld). the shortest and longest average distances between the brutp-labeled viral rna signal center and the nearest edge of ld were calculated manually using zen software (zen 2009 light edition; carl zeiss inc). the number of brutp-labeled viral rna and antibody-labeled signals of viral proteins at the plasma membrane was manually counted in an original magnification of 630. total fluorescence intensity values of core protein and ns5a for individual cells were measured using the metamorph software (universal imaging corporation), using 8 bit images. the gray-scale of the 8-bit images ranged from 0 (black) to 256 (white). image analysis was carried out using the integrated morphometry analysis program provided by metamorph. fluorescence intensity is expressed as the integrated value of all pixels per cell that exceed the inclusive threshold value set at 40. a total of 20 cells were used in each experimental condition from two independent experiments. a celltiter96a queous one solution cell proliferation assay kit (promega) was used to evaluate cell viability, which was performed as described previously [40] . our previous studies have shown that microtubule provides the track for the movement of hcv rcs through ns5a-microtubule interaction [7] and that this transport is required for virus release [10] . we propose that microtubules also provide tracks for the transport of ns5a or the ns5a-containing rc and core protein to reach the ld, where virus assembly occurs. to test this possibility, we first investigated whether core-containing lds colocalized with ns5a and associated with microtubules in hcvinfected huh7.5 cells [at day 10 postinfection (p.i.)]. immunofluorescence staining revealed that ns5a and core protein together (fig. 1c ), or either one alone ( fig. 1a and 1b) , is colocalized on the surface of ld and is closely associated with tubulins. the microtubule network is required for the trafficking of ns5a or the ns5a-containing replication complexes and core protein to the lipid droplet we used nocodazole (which induces microtubule depolymerization) or taxol (which stabilizes tubulin polymerization) to examine whether intact microtubules are required for the transport of core protein, ns5a or viral rcs to the ld. huh7.5 cells were first infected with hcv, and treated with either nocodazole or taxol at 3 hr p.i. for 2 days. the colocalization coefficient of ld with core protein or ns5a was then calculated. under these conditions, cell viability was not affected, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2h-tetrazolium salt (mts) assay (fig. 2e) . under the control conditions [dimethyl sulfoxide (dmso)], ld colocalized with core protein throughout the entire cytoplasm, including the perinuclear region; the proportion of ld that colocalized with core protein was 69%. when the cells were treated with increasing concentrations of nocodazole, a dose-dependent decrease in the colocalization coefficient of ld with core protein was observed ( fig. 2a upper panels and 2b) . the colocalization coefficient of ld with ns5a also decreased correspondingly (fig. 2c upper panels and 2d) . taxol did not have effects on this coefficient. in order to rule out the possibility that the dose-dependent decrease in the colocalization of ld with core or ns5a by nocodazole treatments might have been the consequence of a decrease in the amounts of core and ns5a proteins, a further analysis of core protein ( fig. 2a and 3a , lower panels) and ns5a ( fig. 2c and 3c , lower panels) labeling in cells was carried out using the metamorph integrated morphometry analysis. in nocodazole-treated cells, there was no significant change on the total fluorescence intensities of core and ns5a relative to control cells ( fig. 2f and 2g ). immunoblot analysis also showed that the levels of ns5a and core proteins in the cells were not significantly affected by the low concentration (up to 4 mm) of nocodazole or taxol used (fig. 2h,) . this was in contrast to the effects of nocodazole at 10 or more mm used in most of the published studies, which inhibited hcv rna replication and thereby hcv protein level [8, 10, 42] . these data support the conclusion that the nocodazole treatment at low concentrations affected the colocalization of core and ns5a with ld ( fig. 2b and fig. 2d ). taken together, these results indicated that microtubules are involved in the transport of core protein and ns5a to the ld. newly synthesized membrane proteins generally leave the endoplasmic reticulum (er) and transported to other destinations in the cells. therefore, we next investigated whether ns5a and core proteins are transported from er to lds along the microtubules. in the presence of dmso or taxol, core protein was colocalized with the er marker protein calnexin throughout the entire cytoplasm, including the perinuclear region, with a colocalization coefficient of 32% ( fig. 3a and 3b ). in contrast, the nocodazole treatment caused a dose-dependent increase of colocalization coefficient to 75% and 84% at 2 and 4 mm nocodazole, respectively (fig. 3a, upper panels and 3b) . similarly, the colocalization coefficient of ns5a with calnexin increased from 41% to 69% and 75%, respectively (fig. 3c , upper panels and 3d). thus, the nocodazole treatment caused an increased accumulation of core protein and ns5a in the er and corresponding decrease in the lds. taken together, these results suggested that ns5a and core protein are translocated from er to lds in a microtubule-dependent manner. we further confirmed the mode of transportation of the hcv rc to the ld by immunofluorescent labeling of newly synthesized hcv rna with brutp [43] . in the dmso control and taxoltreated cells, the number of brutp-labeled speckles averaged 30 per cell, and the average distance between speckle and ld was 0.43 mm (fig. 4) . in the presence of nocodazole, the number of speckles was reduced to an average of 11 per cell (fig. 4b) , indicating that the hcv rna replication is partially suppressed by nocodazole treatments, consistent with the previous reports [8] [9] [10] . correspondingly, the average distance between the speckle center and the nearest edge of ld increased from 0.43 mm in the dmso controls and taxol-treated cells to 1.63 mm in the nocodazole-treated cells (fig. 4c) , suggesting that the hcv rcs are normally delivered to the vicinity of the lds through microtubule and that this transport is disrupted by nocodazole. taken together, these data suggest that ns5a or the ns5a-and the nearest edge of ld (hcv rna-ld distance) were analyzed by using zeiss lsm zen software. a total of 20 cells were used for quantitation and calculation of the hcv rna-ld distance and the number of brutp-labeled viral rna from two independent experiments and error bars represent standard deviations of the mean. n.s., non-significance; *, p,0.01; noc, nocodazole. bars, 10 mm. ns5a and core protein complex is cotransported from the perinuclear region to the plasma membrane via microtubule ns5a, together with core protein, is involved in virus assembly; the assembled virions are presumably transported to the plasma membrane to be released off the cell. therefore, we next analyzed whether ns5a is involved in the post-assembly processes of viral life cycle. for this purpose, we examined the colocalization of ns5a with core protein in two regions of the cytoplasm, viz. the perinuclear (region just around the nucleus) and the peripheral (region just underneath the plasma membrane) regions. hcvinfected cells were co-stained with fluorescent (alexa 647) wga (which binds glycoproteins on the cell membrane), which serves as a membrane marker, anti-core, and either anti-ns5a or -ns4b abs. as shown in fig. 5a, ns5a and core protein colocalized both in the perinuclear region and at the cell periphery. in contrast, ns4b colocalized with the core protein only in the perinuclear region of cytoplasm, but not in the peripheral region. analysis of a large number of cells indicated that core protein was colocalized with ns5a throughout the entire cytoplasm, but not with ns4b (fig. 5b, upper panels) . the total fraction of ns5a that colocalized with core protein was 76%, while the corresponding ns4b was 51% (fig. 5b, lower panel) . further, the ns5a-core complex colocalized in the peripheral region in 33% of the cells, whereas ns4b-core did not colocalize at all in the same region (fig. 5c) . we next examined the possibility that the core-ns5a complexes are also transported along microtubule to the cell periphery. in the dmso-treated cells, ns5a colocalized with core protein in both the perinuclear and peripheral regions (fig.5d ). after treatment with either nocodazole or vinblastine, the core-ns5a complexes clustered almost exclusively in the perinuclear region (fig. 5d) . these results suggested that microtubules are required for the transport of core-ns5a complexes to the cell periphery. this observation further suggests that ns5a, but not ns4b, is involved in the post-assembly transport of virus particles. to further evaluate the involvement of the ns5a-core complex in hcv assembly and release, we investigated the localization of this complex with respect to lds [15] , early and late endosomes [10, [44] [45] [46] [47] , and microtubules [10, 11] , all of which are involved in the various steps of hcv assembly and exit. the hcv-infected cells were co-stained with the ld marker (bodipy 493/503), the early endosome marker (early endosome antigen 1; eea1), the late endosome marker (lysosome-associated membrane protein-1; lamp-1), or the microtubule marker (b-tubulin). in the perinuclear region of cytoplasm, both the ns5a-core and ns4b-core complexes colocalized with the lds and the early endosomes ( fig. 5e and 5f ). in contrast, in the peripheral region, only the ns5a-core complex, but not the ns4b-core, colocalized with the late endosomes (fig. 5g) . immunoelectron microscopy (immuno-em) further showed that the late endosome/mvb contained ns5a, but not ns4b, the latter being mainly in the perinuclear regions (fig. 5h) . these results suggested strongly that ns5a-core complexes, but not ns4b, are transported through early-to-late endosomes following virus assembly at ld. furthermore, ns5a and core protein, but not ns4b, colocalized with the microtubules in the peripheral region of cytoplasm, especially at the microtubule end that is closest to the cell periphery (fig. 6a) . finally, we characterized the relationship of ns5a-core complexes with the plasma membrane. hcvinfected cells were co-stained with fluorescent (alexa 647) wga, anti-core, and anti-ns5a or -ns4b abs. as shown in fig. 6b , ns5a and core protein colocalized at the plasma membrane, particularly at the membrane curvature, which has been reported to be essential for virus exit [48] . quantitative analysis showed that the number of anti-core and anti-ns5a antibodies-labeled signals at the plasma membrane averaged 63 and 26 per cell, respectively, whereas that for anti-ns4b was less than 1. interestingly, some signals containing both ns5a and core (5 per cell on average) were also detected at the plasma membrane, whereas no ns4b-core signals were found (fig. 6c) . furthermore, immuno-em showed that ns5a (fig. 6d) , core protein (fig.6e ) and the core-ns5a complex (fig. 6f ) localized mainly to patches or clusters on the plasma membrane. taken together, these results again suggest that at least some of ns5a-core complexes (or the assembled virions) are transported from ld through early-to-late endosomes to the plasma membrane via microtubules. to confirm that ns5a is released from the plasma membrane as a component of some virion particles, we cultured hcvinfected huh7.5 cells with dialyzed serum to reduce nonspecific binding of irrelevant proteins to virus particles. the culture supernatant was subject to sucrose gradient sedimentation, and the distribution of the viral rna and infectivity of hcv were figure 5 . core-ns5a complexes are transported from perinuclear region to early and late endosomes. (a) the hcv-infected cells (at day 10 p.i.) were labeled with antibodies specific for core protein (red) and ns5a (green) (upper row) or ns4b (green) (lower row). plasma membrane and nuclei were stained with wga alexa fluor 647 conjugate (blue) and dapi (gray), respectively. in parallel with panel a, the hcv-infected cells were labeled with antibodies to core protein (green) and ns5a (red) or ns4b (red) (b, upper rows). nuclei were stained with dapi (blue). colocalization efficiency between core protein and ns5a or ns4b was analyzed by using zeiss lsm zen software (b, lower panel). error bars represent standard deviations of the mean from 20 cells in two independent experiments. the images were analyzed by using metamorph, and the proportion of cells (of 200 counted) in which the ns5a or ns4b colocalized with core protein at the cell periphery was calculated (c). (d) effects of nocodazole (noc) and vinblastine (vbl) on movement of core-ns5a protein complex to the cell periphery. the hcv-infected cells (at day 10 p.i.) were treated with nocodazole, vinblastine, or dmso for 3 h. the cells were labeled with antibodies to core protein (green), ns5a (red). nuclei were stained with dapi (blue). images with differential interference contrast (dic) and fluorescence images were then merged. an enlarged view of part of image is shown (inset). in parallel with panel a, the hcv-infected cells were labeled with antibodies to core protein (red) (e, f, g), ns5a (green, e, f, g) (upper rows), ns4b (green, e, f, g) (lower rows), eea1 (blue) (f) and lamp-1 (blue) (g). lds and nuclei were stained with bodypi 493/503 (blue) (e) and dapi (gray), respectively. the second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (a, e, f, g). colocalization of core protein with ns5a or ns4b is depicted as yellow (a, b, d, e, f, g) . colocalization of core-ns5a or -ns4b complexes with lds (e), early endosomes (f) or late endosomes (g) is depicted as white. bars, 10 mm. (h) in parallel, the hcv-infected cells was labeled with anti-ns5a or -ns4b ab. bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 18-or 12-nm gold particles, respectively. sections were visualized by em. arrows, gold-labeled ns5a (18 nm) and ns4b (12 nm) are indicated. mvb contains several intravesicular vesicles (white arrows). an enlarged view of the perinuclear region of lower left image is shown (inset). arrows, gold-labeled ns5a and ns4b. pm, plasma membrane; mvb, multivesicular bodies; n, nucleus; nm, nuclear membrane; bars, 500 nm (left panels) and 100 nm (right panels). doi:10.1371/journal.pone.0099022.g005 determined. two peaks, one from fractions 7-8, with density of 1.083 to 1.098 g/ml sucrose (low-density fractions; ldf), and another from fractions 11-13 with density of 1.145 to 1.178 g/ml sucrose (high-density fractions; hdf), were found to contain distinct hcv rna signals (fig. 7a ). fraction 12, at 1.162 g/ml sucrose, contains the largest amount of viral rna, consistent with the previously reported density of free hcv virions [27, 31] . each fraction was further analyzed for its infectivity on naive huh7.5 cells. the results showed that both the ldf and hdf from culture supernatant contained infectivity (fig. 7b) , with hdf having approximately 153-fold higher specific infectivity. thus, both the ldf and hdf are infectious, but ldf contained only 0.65% of the total infectivity; thus, the possibility that the apparent infectivity of ldf may have come from contaminations of hdf cannot be ruled out. further, we analyzed the various fractions for the possible presence of core and ns5a. as shown in fig. 7c , the hdf contained abundant core protein, but no ns5a or ns4b. in contrast, the low-density fraction 7 (1.083 g/ml) contained ns5a in addition to core protein, but no ns4b. recent studies have revealed that hcv virion release requires late endosome (or multivesicular body; mvb) [10] , the functional endosomal sorting complex required for transport (escrt) [46, 47] and hepatocyte receptor tyrosine kinase substrate (hrs) [45] , all of which are involved in the biogenesis of mvb and exosome secretion. in addition, hcv particles are associated with circulating exosomes in hepatitis patients' serum [49] . we therefore investigated whether hcv virion release from cell membrane occurs through the exosome pathway. we probed each sucrose density fraction for the presence of two exosomal markers viz. cd81 and cd63. interestingly, both cd81 and cd63 were detected in ldf (fig. 7c) , but not hdf, suggesting that the exosome is involved only in ldf virus release. since exosomes are derived from endosomes and are also secreted from normal cells to participate in a variety of cellular functions [50] , we also analyzed the sucrose gradient fractions of extracellular medium obtained from naã¯ve huh7.5 cells and cells harboring an hcv subgenomic replicon. none of the fractions obtained from these cells contained hcv ns5a protein (data not shown), consistent with the previous report [34] . these data suggest that ldf contained hcv particles released through exosome-like structure, and contained ns5a. the morphology and composition of virus particles in both the ldf and hdf were further characterized. the diameter of particles ranged from 105 to 132 nm (n = 60) and 52 to 86 nm (n = 90) in the low-and high-density pools, respectively ( fig. 7d and 7g ). the size heterogeneity of hcv particles has been described previously [20, 25, 31] . correspondingly, particles of ,130 nm and ,70 nm in diameter, probably representing the low-and high-density virus particles, respectively, were observed in the extracellular space surrounding the hcv-infected cells (fig. s1 ). after treatment with saponin (0.01%), which removed the viral envelope, the size of the particles was reduced to 35-41 nm for both the ldf and hdf viruses (fig. 7e and 7f) , consistent with its being the internal viral core [20, 51] . next, the purified virions were characterized for the presence of viral proteins and exosomal marker cd81 by immuno-em. the particles in the low-density pool could be stained with anti-cd81 and -e2 mabs, but not with a purified normal igg or an anti-ns5a mab (fig. 7d, upper panels) or an anti-ns4b ab (data not shown). in contrast, only e2 protein, but not cd81, was detected in the high-density pool (fig. 7d, lower panels) . the association of hcv envelope proteins with exosomes has previously been reported [49] . following the treatment of virus samples with detergent (0.01% saponin), the virion core of both the low-and high-density pools was stained with anti-core mab (fig. 7e) . interestingly, the virion core of ldf, but not the hdf, could be stained with anti-ns5a antibody, but neither normal igg nor anti-ns4b antibody (fig. 7e) . in parallel, the saponin-treated virus of ldf could be stained with both core and ns5a simultaneously (fig. 7f) . the specificity of staining was confirmed by staining under various treatment conditions (fig. 7h ). in all virus samples, normal igg or anti-ns4b antibody yielded only background staining. in hdf, anti-cd81 and -ns5a antibodies also yielded very little staining with or without the saponin treatment. in contrast, in ldf, anti-cd81 (40%) and -e2 (25%) stained intact virus samples strongly, while anti-core (70%) and -ns5a (40%) stained saponin-treated samples strongly. the above results prompted us to further characterize the localization of the exosomal proteins relative to ns5a and core protein on the plasma membrane. the results indicated that ns5a or core protein colocalized with cd81 at the plasma membrane by immunofluorescence staining (fig. 8a ) and immuno-em ( fig. 8b and 8c ), but not ns4b. these results again suggest that some ns5a-containing hcv particles exit the cells via fusion of mvb with the plasma membrane. taken together, these data suggest that a minor population of hcv virion exits cells via exosomes, as ns5a-containing, low-density particles, but the majority of virions do not contain ns5a and exit cells through an exosome-independent pathway. in this study, we found that in the presence of a microtubuledisrupting drug, both ns5a and core proteins failed to be transported to the ld (fig. 2) , resulting in the accumulation of ns5a and core protein in the er (fig. 3 ). in addition, ns5a and core protein colocalized with microtubule throughout the entire figure 6 . core-ns5a complexes are transported from perinuclear region to the plasma membrane via microtubules. the hcvinfected cells (at day 10 p.i.) were labeled with antibodies to core protein (red) (a, b), ns5a (blue, a; green, b) (upper rows), ns4b (blue, a; green, b) (lower row) or tubulin (green) (a). plasma membrane and nuclei were stained with wga alexa fluor 647 conjugate (blue) (b) and dapi (gray), respectively. the second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (a). colocalization of core with ns5a or ns4b is depicted as magenta (a) or yellow (b). colocalization of core-ns5a or -ns4b protein complexes with microtubules (a) or plasma membrane (b) is depicted as white. the microtubule end (white arrow) is closest to the cell periphery (a). (b) at the right is an enlarged area, marked with a white box, from the merged image. colocalization of ns5a with core protein was observed in the membrane curvature (white arrow). pm, plasma membrane; bars, 10 mm. (c) quantitation of antibody-labeled signals of viral proteins at the plasma membrane. images from 20 cells were counted manually using an original magnification of 6630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (d, e) ns5a and core are localized in clusters or patches on the plasma membrane. in parallel, the hcv-infected cells were labeled with anti-ns5a (d) or anti-core (e) antibodies. bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. sections were visualized by em. arrows, gold-labeled ns5a (d) or core protein (e). (f) immuno-em of core-ns5a complex colocalized at the plasma membrane. the hcv-infected cells were co-labeled with antibodies against ns5a (6 nm) and core (18 nm). shown is a view of the plasma membrane. arrowhead, gold-labeled ns5a. arrow, gold-labeled core. pm, plasma membrane; bars, 100 nm (d, f) and 200 nm (e). doi:10.1371/journal.pone.0099022.g006 cytoplasm, mostly also associated with lds ( fig. 1) and at the microtubule end near the plasma membrane (fig. 6a) . these results suggest that the intact microtubule network served as a transport highway for the movement of hcv ns5a (or viral rcs) and core proteins from the er towards the ld and plasma membrane for virus assembly and exit. additionally, it has been shown that the interaction between ns5a and core protein on lds plays an important role for virus assembly [18, 19] , and that core protein induces redistribution of ld to the perinuclear region for virus assembly in a microtubule-dependent manner figure 7 . association of ns5a and exosomal proteins with the low-density hcv particles. hcv virion populations were analyzed by sucrose density gradient ultracentrifugation. (a) concentrated culture medium collected from hcv jc1-infected cells was fractionated using a continuous 10-50% sucrose density gradient. hcv rna levels were determined in each fraction. (b) infectivity of each density gradient fraction. an aliquot of each fraction was used to infect naive huh7.5 cells. intracellular hcv rna copy number per mg of total rna was determined 3 days after infection by qrt-pcr. (c) western blot analysis of each density gradient fraction, shown in panel a, by using antibodies indicated at the left. (d, e and f) electron micrograph of the viral spherical structures shown by immunogold labeling. hcv particles were purified from two pooled fractions; the low-density particles (from fractions 7 to 8), and the high-density particles (from fractions 11 to 13), after dialysis and concentration. the virus samples were untreated (d) or treated with 0.01% saponin (e, f), as described in materials and methods. grids were incubated with an untreated or saponintreated purified hcv and then with antibodies against cd81, e2, or ns5a (d) or with antibodies against core protein, ns5a or ns4b (e), respectively. bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 12-nm gold particles. (f) core-ns5a complex localized in the virion core of the low-density particles. grids were incubated with a saponin-treated purified hcv and then co-labeled with antibodies against ns5a (6 nm) and core (12 nm). to determine antibody specificity, primary antibodies were replaced with nonspecific normal mouse igg. bars, 50 nm. (g) the average diameter of the low-(n = 60) and high-density (n = 90) particles was obtained from the combined analysis of two independent viral preparations and error bars represent standard deviations of the mean. (h) statistics were performed by counting hcv particles and immunogold labeled hcv particles from the combined analysis of two independent viral preparations. relative proportion of hcv particles that were labeled with antibodies (n = 60). doi:10.1371/journal.pone.0099022.g007 [11] . these studies explain previous finding that the extracellular virus production was decreased by nocodazole [10] . by immunofluorescence staining and em, we found that ns5a is closely associated with core protein in almost every step of hcv life cycle, whereas ns4b is not involved in the late steps. however, only a small number (5 signals/cell) of the ns5a-core complexes were detected at the plasma membrane of the cells, in contrast to core protein alone (,63 signals/cell) (fig. 6c) . this finding may explain why the amount of hcv rna in ldf (,1610 4 copies/ ml) was significantly lower than that of hdf (1.3610 5 copies/ml) (fig. 7a) . we suggest that ns5a-containing, low-density hcv particles constitute a minor population of the released hcv virions. nonetheless, this population was apparently infectious, though with slightly lower specific infectivity than the bulk of virus particles in hdf (fig. 7b) . the result is similar to a published report, which showed that less than 2% of all infectivity was in the low-density pool (density of ,1.1g/ml) [20] . however, we cannot exclude the possibility that ldf with very weak infectivity may have come from virus contamination from hdf. notably, we demonstrated that low-density hcv particles utilize a host exosome biogenesis pathway for the production of ns5a-containing virions. the low-density hcv particles (density of 1.08 g/ml) were also found in hepatitis c patients' serum [26, 52, 53] . most importantly, ns5a and exosomal markers coincided with core protein in the low-density fractions (fig. 7c) , which also contained hcv rna (fig. 7a ). ns5a-core complexes (fig. 7f ) and exosomal markers (fig. 7d) were detected in purified ldf virions. correspondingly, exosomal markers and ns5a or core protein were colocalized at the plasma membrane, where virus release occurs, as revealed by immunofluorescence staining and immuno-em (fig. 8) . taken together, the results indicated that the low-density, ns5a-containing virions are released off the cell via exosomes, whereas the high-density particles exit the cell via some alternative mechanism not involving the exosomal pathway. consistent with this hypothesis, hcv particles have been found to be associated with circulating exosomes in serum of hcv-infected patients [49] . combined with our previous results [10] showing that hcv particles are transported from early to late endosomes/mvb, our current studies suggest that the ns5a-containing hcv particles most likely exit the cells via fusion of mvb to the plasma membrane. exosomes have been demonstrated to facilitate the budding of human immunodeficiency virus (hiv) [54] and hepatitis a virus (hav) [55] . exosome have been known to play important roles in intercellular communications. in hiv and hav infection, exosomes are required for trans-infection of cd4 + t cells [56] and are likely to promote virus spread within the liver [55] , respectively. similarly, exosome-containing hcv virions might fuse to uninfected cell in a mechanism independent of viral envelope proteins and may contribute to hcv natural infection. interaction of ns5a with core protein plays an important role in hcv particle production [19] , but its mechanism of involvement in infectious virus production is still unclear. the association of viral ns proteins with viral genomic rna and their incorporation into viral particles is common among rna viruses, such as the ns2 of influenza virus [57] , the v protein of simian virus 5 [58] , the ns3 of coronavirus [59] , and the vpx (viral protein x) of hiv-1 [60] . however, in contrast to our observation, merz et al. [24] could not observe any ns5a in the virus preparation after affinity purification. the reason for this discrepancy may lie in the sensitivity of detection methods and the method of virus purification, as ns5a was detected only in a minor population of low-density particles in our study. it was also possible that the affinity tag used in the previous study [24] could not be detected in the low-density hcv particles. such virion-associated ''nonstructural proteins'' probably do not play structural roles in virus particles, but may participate in some yet unknown functions required at different steps of the viral life cycle. recently, an amphipathic a-helical peptide (ah) containing the n-terminal 31 amino acids of ns5a was shown to induce swelling of the vesicles leading to vesicle lysis [61] ; in addition, the infectivity of hcv particle was reduced when hcv virions were first treated with the ah peptide, causing the disruption of hcv envelope [62] . taken together, the data presented here and elsewhere show that ns5a may induce fusion and lysis of lipid vesicles as well as virus particles during the virus entry step. since ns5a is present inside the virus particles, it is likely that these membrane-altering functions of ns5a could function at a postbinding step, namely, after the virus envelope has been disrupted. indeed, a postbinding fusion step within an acidic endosomal compartment is required for hcv entry [63] . this will be an interesting topic to study in the future. in summary, our data demonstrated that a minor population of low-density hcv virions was released in the form of exosome from the infected cells, and that its virion core contains ns5a via close interaction with core protein during intracellular transport. our studies provide an additional target for designing new therapeutic approaches by possible use of exosome inhibitor to block the spread of hcv infection. figure s1 the hcv-infected cells (at day 10 p.i.) were fixed and processed for em. at the right is an enlarged area. the large-diameter (a, white arrows) and small-diameter (b, black arrows) hcv-like particles were released from plasma membrane. pm, plasma membrane; bars, 500 nm (left panels) and 100 nm (right panels). (tif) processing pathways of the hepatitis c virus proteins expression and identification of hepatitis c virus polyprotein cleavage products efficient initiation of hcv rna replication in cell culture replication of subgenomic hepatitis c virus rnas in a hepatoma cell line expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons association of hepatitis c virus replication complexes with microtubules and actin filaments is dependent on the interaction of ns3 and ns5a cytoskeletal requirements for hepatitis c virus (hcv) rna synthesis in the hcv replicon cell culture system initiation of hepatitis c virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein hepatitis c virus egress and release depend on endosomal trafficking of core protein hepatitis c virus core protein induces lipid droplet redistribution in a microtubule-and dynein-dependent manner hepatitis c virus: assembly and release of virus particles homotypic interaction and multimerization of hepatitis c virus core protein interaction of hepatitis c virus core protein with viral sense rna and suppression of its translation the lipid droplet is an important organelle for hepatitis c virus production subcellular localization of hepatitis c virus structural proteins in a cell culture system that efficiently replicates the virus an in vitro model of hepatitis c virus genotype 3a-associated triglycerides accumulation essential role of domain iii of nonstructural protein 5a for hepatitis c virus infectious particle assembly interaction of hepatitis c virus nonstructural protein 5a with core protein is critical for the production of infectious virus particles ultrastructural and biophysical characterization of hepatitis c virus particles produced in cell culture complete replication of hepatitis c virus in cell culture cell culture-grown hepatitis c virus is infectious in vivo and can be recultured in vitro identification of a residue in hepatitis c virus e2 glycoprotein that determines scavenger receptor bi and cd81 receptor dependency and sensitivity to neutralizing antibodies biochemical and morphological properties of hepatitis c virus particles and determination of their lipidome characterization of low-and very-low-density hepatitis c virus rna-containing particles hepatitis c virus: buoyant density of the factor viii-derived isolate in sucrose hepatitis c virus particle detected by immunoelectron microscopic study identification of hepatitis c virus by immunoelectron microscopy visualization of hepatitis c virions and putative defective interfering particles isolated from lowdensity lipoproteins production of infectious hepatitis c virus in tissue culture from a cloned viral genome an in vitro model of hepatitis c virion production b lymphocytes secrete antigen-presenting vesicles exosomes: endosomal-derived vesicles shipping extracellular messages shortrange exosomal transfer of viral rna from infected cells to plasmacytoid dendritic cells triggers innate immunity highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication the heat shock cognate protein 70 is associated with hepatitis c virus particles and modulates virus infectivity hepatitis c virus ns3/4a protein interacts with atm, impairs dna repair and enhances sensitivity to ionizing radiation construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras robust production of infectious hepatitis c virus (hcv) from stably hcv cdna-transfected human hepatoma cells annexin a2 is involved in the formation of hepatitis c virus replication complex on the lipid raft fluorescent detection of lipid droplets and associated proteins effect of cell growth on hepatitis c virus (hcv) replication and a mechanism of cell confluence-based inhibition of hcv rna and protein expression hepatitis c virus rna replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2 molecular determinants and dynamics of hepatitis c virus secretion regulation of hepatitis c virus secretion by the hrs-dependent exosomal pathway the escrt system is required for hepatitis c virus production vps4 and the escrt-iii complex are required for the release of infectious hepatitis c virus particles membrane fusion and fission: enveloped viruses association of hepatitis c virus envelope proteins with exosomes microvesicles and viral infection hepatitis c virus core particle detected by immunoelectron microscopy and optical rotation technique buoyant density of hepatitis c virus recovered from infected hosts: two different features in sucrose equilibrium density-gradient centrifugation related to degree of liver inflammation extraordinarily low density of hepatitis c virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction the phe105 loop of alix bro1 domain plays a key role in hiv-1 release a pathogenic picornavirus acquires an envelope by hijacking cellular membranes hiv and mature dendritic cells: trojan exosomes riding the trojan horse? ns2 protein of influenza virus is found in purified virus and phosphorylated in infected cells the paramyxovirus sv5 v protein binds two atoms of zinc and is a structural component of virions proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3 localization of the vpx packaging signal within the c terminus of the human immunodeficiency virus type 2 gag precursor protein alpha-helical peptide-induced vesicle rupture revealing new insight into the vesicle fusion process as monitored in situ by quartz crystal microbalance-dissipation and reflectometry mechanism of an amphipathic alpha-helical peptide's antiviral activity involves sizedependent virus particle lysis hepatitis c virus entry depends on clathrin-mediated endocytosis we thank dr. takaji wakita of national institute of infectious disease, tokyo, japan, for plasmid pjfh1, dr. charles m. rice from center for the study of hepatitis c, rockefeller university, new york, usa, for huh7.5 cells. key: cord-305039-grsv06j7 authors: flego, michela; ascione, alessandro; cianfriglia, maurizio; vella, stefano title: clinical development of monoclonal antibody-based drugs in hiv and hcv diseases date: 2013-01-04 journal: bmc med doi: 10.1186/1741-7015-11-4 sha: doc_id: 305039 cord_uid: grsv06j7 today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. compared to conventional antiviral drugs, monoclonal antibodies (mabs) used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. the current high cost of mabs' production, storage and administration (by injection only) and the consequent obstacles to development are outweighed by mabs' clinical advantages. these are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses. this review briefly summarizes the recent technological advances in the field of immunoglobulin research, and the current status of mab-based drugs in clinical trials for hiv and hcv diseases. for each clinical trial the available data are reported and the emerging conceptual problems of the employed mabs are highlighted. this overview helps to give a clear picture of the efficacy and challenges of the mabs in the field of these two infectious diseases which have such a global impact. the innate immune response is the first-line defense in determining the outcome of an infection. infectious agents contain conserved motifs on their surface that react with conserved pattern recognition toll-like receptors of the host. this interaction initiates a powerful innate immune response. moreover, the infectious agent's surface proteins and carbohydrates come into contact with b-cell receptors, membrane-bound immunoglobulin of isotype m (igm) or d (igd), and often induce potent antibody responses, which take some weeks to fully develop [1] . when a vertebrate organism encounters a pathogen, such as a virus or bacteria, it generates a polyclonal antibody response against numerous epitopes on different antigens during infection; therefore, polyclonal serum contains a large and diverse population of antibodies, which also include neutralizing antibodies (nabs). thus, polyclonal serum-derived biotherapeutic products can contain various nabs against multiple and distinct epitopes; these nabs provide strong protective activity due to additive or even synergistic effects on neutralization. however, in this type of product the vast majority of their constituent specific antibodies are non-neutralizing, since they are directed against misfolded protein or against epitopes on native surface proteins for which antibody binding is not protective [2, 3] . furthermore, for some viral and bacterial infections, no correlates of protection have been established; therefore, the significance of antibody titers, apart from indicating past exposure, is not clear. mechanisms of immunological escape can explain why total antibody titers are not always protective. many infectious organisms, including viruses, can constantly mutate surface proteins and exploit glycans to shield important epitopes, diverting the antibody response away from functionally important epitopes in favor of immunogenic irrelevant epitopes [4] . thanks to their protective properties, the administration of hyperimmune sera from immunized animals or immune human donors, named 'serum therapy', was the first effective treatment of infectious diseases. later, the advent of antibiotic therapy with the advances in vaccine design has meant that serum therapy was almost abandoned for many infectious diseases. nevertheless, hyperimmune human sera immunoglobulin preparations are still used to treat different bacterial toxins and virus related diseases, including those caused by cytomegalovirus (cmv), respiratory syncytial virus (rsv), hepatitis a virus (hav), hepatitis b virus (hbv), rabies, vaccinia, vesicular stomatitis virus (vsv) and measles, underscoring the fact that antibody therapy remains an effective means of treatment [5, 6] . today, the ability to rapidly generate and manipulate antibodies with a defined epitope recognition, named "monoclonal antibodies" (mabs) (figure 1 ), has opened a new window of opportunity for a rematch of antibodies in clinical practice. this achievement has been possible thanks to advances in cellular biology and biotechnology (figure 2) , and also to improved purification techniques which have made these therapeutics safer, less immunogenic and more effective. mab preparations have many advantages over immune sera-derived preparations which can vary due to both time and the source of origin, since different hosts mount different antibody responses. one advantage is that mabs, by virtue of the fact that they are chemically defined reagents, exhibit relatively low lot-tolot variability and low risk of pathogen transmission. another advantage for mab preparations is the much figure 1 schematic structure of a mab. all immunoglobulins are composed of two identical light (l) chains and two identical heavy (h) chains, linked by disulphide bonds (black dashed bars). the heavy chains contain one variable domain (vh) and three or four constant domains (ch1, ch2, ch3 and ch4) depending on antibody isotype. by contrast, the light chains contain only one variable domain (vl) and a single constant domain (cl). within the fab region, at the end of the two arms of the y-shaped molecule, the variable domain of a heavy chain pairs with the light chain variable domain to form the antigen-binding site. in more detail, within the matched v regions, three short polypeptide segments on the heavy chain and three on the light chain form the complementarity-determining regions (cdrs), which dictate the precise antigen-binding characteristics of the antibody. on the other end, the fc domain, which includes the sites for interaction with the complement system and fc receptors, mediates effector functions determining the fate of the bound antigen. greater activity per mass of protein since all the ig molecules are specific for the desired target. this phenomenon is illustrated by the report that two 0.7 mg doses of two mabs provided the same protection against tetanus toxin as 100 to 170 mg of tetanus immunoglobulins [7] . neither does mab therapy have the immunological complications associated with the use of heterologous sera in humans, such as serum sickness and immediate hypersensitivity, which significantly limited the latter's usefulness [8] . in recent years, mabs have emerged as a new class of biological drugs in oncology as well as in immune and inflammatory diseases, albeit their development in infectious diseases has been slower. to date, the only mab approved in this field is palivizumab, an anti-rsv mab licensed for prevention of severe respiratory disease in high-risk infants and immunocompromised adults. now the scenario is gradually changing and there are many antibodies against viruses and bacteria in various stages of clinical development. this trend has also been influenced by the development of different scientific disciplines, which makes it possible to study and dissect the function of individual microbial structures supporting figure 2 evolution of mabs linked to the need to decrease their immunogenicity. different methods to obtain mabs are depicted. mouse mabs, the 'hybridoma' cells derived from the stable fusion of immortalized mouse myeloma cells with lymphocytes from immunized mice, are screened to identify individual clones producing identical antibody to a single antigenic determinant [118] . chimeric mabs, the murine constant regions of both heavy and light antibody chains (mch and mcl), are replaced with human counterparts (hch1, hch2, hch3 and hcl1), leaving intact the murine variable portions (mvh and mvl) [119] . humanized mabs, only the cdrs of the murine mabs (mcdrs) from both the mvh and the mvl, are 'grafted' into a human backbone antibody [120, 121] . human mabs, 1)human memory b-cells isolated from patients are immortalized by epstein barr virus (ebv) and cpg oligodeoxynucleotide, and then screened for specific antibody production [122] . 2) transgenic mice, obtained by a genetic replacement of the mouse immunoglobulin genes with human counterparts, are used to obtain fully human mabs by traditional hybridoma technology [123] . 3) antibody libraries, constructed by in vitro combinatorial assembly of human immunoglobulin variable-region gene (v genes) and cloned to provide the display onto phage surfaces, are subjected to a panning against an antigen in order to select specific clones [124] . the first mabs of each category approved for clinical use are shown. palivizumab is the first and, so far, only mab approved for infectious diseases. the endings used to name the different types of mabs are also indicated. the development of more targeted drugs. there are excellent reviews about this topic [6, 9] . in this review we focus on the mab-therapies now underway in clinical trials (table 1 ) designed for human immunodeficiency virus (hiv) and hepatitis c virus (hcv) infectious diseases. both these worldwide epidemics require new strategies due to the lack of a definitive cure and effective vaccines, to the continuous emergence of drug resistant variants, to the toxicities of licensed drugs and to the need to ensure a treatment for all patients. in this context, antibodies represent an intriguing alternative as therapeutics; in their favor are their different resistance mechanisms and a more favorable toxicity profile when compared to other available drug classes, fitting them for use in conjunction with the current chemotherapy by slowing the onset of resistance and possibly enhancing therapeutic efficacy. the antibody structure comprises a pair of identical heavy and light chains linked by disulphide bonds held in a yshaped arrangement (figure 1 ). the fragment antigen-binding (fab) portion, the region that binds the antigen, is composed of one variable and one constant domain of both the heavy and the light chain. the remaining constant sections of the longer heavy chains form the tail of the y, termed the crystallizable fragment (fc) region, which provides the signal for effector functions. antibodies can provide protective effects through various mechanisms [10] . viral neutralization is generally meant as the ability of an antibody to provide sufficient steric interference to disrupt the interaction between a microbic antigen and its ligand in experimental conditions in vitro. this activity is clearly associated with protection, thanks to their fab domain alone, both in natural infection and after immunization. virus infection includes sequential steps beginning with attachment to cell-surface receptors and ending with delivery of the viral genetic material into the cytoplasm [11] . fusion of viral and cellular membranes is a basic entry mode for enveloped viruses, such as hiv and hcv [12] , which still differ in specific aspects of viral entry and assembly, thus offering unique therapeutic opportunities. the cell surface is certainly more directly accessible for the action of the antibodies; therefore, the phase of virus entry is one of the most important targets in preventing viral infection at the origin, and many known nabs act at this step. for the same reason, inhibition of the release of progeny virus is another possible mechanism of neutralization, as demonstrated by antibodies directed against influenza a virion surface neuraminidase [13] . in hiv and hcv fields, no virus release inhibiting antibodies have been identified to date. the interaction of hiv envelope surface protein gp120 with its host receptor, cd4, on human t cells triggers conformational changes in the envelope, resulting in exposure of a transient binding site for co-receptor ccr5 or cxcr4. this in turn promotes additional conformational changes in virus gp41 protein which allow it to insert its fusion peptide into the target cell membrane to initiate membrane fusion and viral entry into host cells. nabs can inhibit viral infection by several different mechanisms in parallel with the steps that allow the viruses to enter into cells ( figure 3 ). they can directly block virus attachment to target cells by interfering with virus-receptor interactions, as in the case of nabs against the cd4-binding site on hiv gp120 [14] . this same goal can also be achieved by directing the antibodies to the virus receptor and/or co-receptor on host cells. mabs can also block fusion at the cell membrane at the post-binding/pre-fusion stage, as exemplified by anti-cd4 [15] and/or anti-ccr5/cxcr4 (ccmotif receptor 5/cxc-motif receptor 4) mabs, under development [16] . again, mabs directed to the external proximal membrane region of hiv gp41 can interfere with conformational changes needed for membrane fusion [17] . unlike hiv, hcv entry into target cells occurs via clathrin-mediated endocytosis of the viral particle [18] . subsequent release of the viral genome into the cytosol requires the ph-dependent fusion of viral and cellular membranes. current models suggest that hcv circulate as lipoviral-particles (lvps) in the vascular system, these consisting of lipoproteins in complex with virus particles. following localization to the surface of hepatocytes through interactions of lvps with glycosaminoglycans and the low density lipoprotein receptor, specific binding of the e1 and e2 virus surface glycoproteins with the host sr-b1 scavenger receptor and cd81 occur [19] . subsequently, viral particles are translocated to regions of the membrane possessing tight junction proteins occludin and claudins; the binding to these receptors results in clathrin-mediated endocytosis. as for hiv, mabs directed against spike viral proteins, as well as against host receptors, may act at an early stage of infection by preventing the binding of the virus on the cell surface. for example, antibodies recognizing the cd81-binding site within the envelope glycoprotein e2 have been shown to block viral entry, as have a number of anti-receptor antibodies targeting cd81 [20, 21] and sr-bi [22] . some antibodies may act by blocking conformational changes and/or the requisite interactions between the viral and endosomal membranes required for fusion; although as yet no fusion determinant within the envelope glycoproteins has been defined. host protection in vivo is more complex and involves the interaction of antibodies with cells and molecules of the innate immune system. the antibody can exert protective actions through an fc region-mediated recruitment of other components of the immune system, including antibody-dependent cell-mediated cytotoxicity (adcc), complement-dependent cytotoxicity (cdc) and antibody-dependent cellular phagocytosis. receptors for the fc segment of igg (fcy receptors; fcγrs) are expressed on the surface of different types of cells, including natural killer cells (nk), monocytes, macrophages, dendritic cells and neutrophils. with the exception of γδt cells, fcγrs are not normally found on t lymphocytes. similarly, the receptor for fc segment of iga, the fcαr, involved in phagocytosis and induction of microbe killing, is expressed on monocytes, macrophages and neutrophils [23] . the adcc process is triggered by the interaction between the fc region of an antibody bound to a nonself antigen exposed on host cells, and the fc receptors on immune effector cells. the subsequent release of cytokines and cytotoxic granules containing perforins and granzymes promotes the death of the target cell. cdc is initiated by complement component c1q binding to the fc region of igg, which is in turn bound to the foreign antigen on the cell surface. this triggers a proteolytic cascade to activate the complement, so leading to the formation of a membrane attack complex that kills the target cell by disrupting its cell membrane. the fc region can also mediate complement binding to and deposition on free virions, which can cause a direct virotoxic effect or inhibit virus binding to cells. moreover, the so-called opsonization process, consisting of the binding of antibody fab portion to the antigen following by the interaction of fc domain to an fc receptor on phagocytes, is a powerful mechanism to enhance the phagocytosis [9] . with respect to hiv, a potential role for adcc in modulating the course of hiv infection was first proposed on the basis of studies showing an inverse association between adcc antibody levels and the clinical stage of disease. the strongest evidence for a role for adcc antibody in disease progression comes from a study by baum et al. of the multicenter aids cohort study [24] . in that study, rapid progressors had significantly lower adcc antibody titers against cem. nkr cells coated with gp120 than did non-rapid progressors at corresponding visits or non-progressors at any visit. morever, hiv-infected individuals with spontaneously undetectable viremia were shown to have higher adcc antibody levels than viremic subjects [25] . in the context of hcv infection, fc-mediated effector functions, although less well understood, can still have an important role. sera from both the acute and chronic phase of infection can mediate adcc via binding to viral protein e2 expressed at the cell surface [26] , while several e2-specific mabs are able to induce cdc of e2-expressing cells [27] . optimizing non-nab effector functions, such as adcc, cdc and fagogocytosis, may prove critical in the design of new effective anti-hcv therapeutic antibodies [28] . (1), as well as by binding to the viral receptor or co-receptor on host cell surface (2) . some antibodies, can neutralize viral infection through interfering with conformational changes required for membrane fusion and subsequent release of the viral core into the target-cell cytoplasm; this postbinding neutralization may occur at the cell surface (3), or inside the endosomes for the viruses (for example, hcv) whose entry into the cell requires an endocytosis step (4). antibodies recognizing viral or host proteins expressed on infected cell surface can exert protective actions through the fc-mediated effector functions (for example, cdc, adcc) (5). again, mabs may prevent the release of progeny virions (6) . at the bottom the antibody neutralizing effects on the viruses before cell binding, including the direct virolysis by cdc and the mab-mediated enhanced phagocitosis, are shown (7) . in panel b, the possible mab-mediated immunomodulary therapies are depicted. in some chronic viral infections, virus-specific immune cells may persist in a 'non-functional' state, because of an imbalance of immunoregulatory signals involving multiple inhibitory and activating receptors, triggered by soluble factors and/or cell surface ligands. therapeutic approaches using specific mabs to block host immunosuppressive molecules (antagonism) or to trigger activating receptors (agonism) may be a valid strategy to restore immune cell function and treat various chronic viral infections. system is the exhaustion of virus-specific t cells. exhaustion consists of a progressive dysfunction characterized by the inability to proliferate and to produce key antiviral and immune stimulating cytokines (for example, interleukin (il)-2, tumor necrosis factor (tnf)-a, interferon (ifn)-γ), or to lyse infected cells [29] . a feature of functional exhaustion is that it affects many antiviral properties of both mouse and human cd8+ t cells. loss of effector functions proceeds in a hierarchical manner starting with defects in il-2 production and proliferation, followed by the decrease of tnf production. cytotoxic activity is also lacking in exhausted human cd8 + t cells. at a severe stage of exhaustion, ifn-γ production is eventually compromised, with exhausted t cells ending up deleted if the high antigenic load persists [30] . exhaustion can also occur in cd4+ t cells in both mice [31] and humans [32] . probably the best explanation for this progressive dysfunction and loss of effector t cells is the continuous triggering of virus-specific t cell receptors owing to a high antigenic load in persistently infected hosts without a critical rest period. the current consensus is that functional exhaustion is a way of limiting the magnitude of effector t cell responses. although this may safeguard against autoimmune responses, it may also compromise effective immunity against persistent infectious agents and tumors [29] . exhausted t cells are subject to complex layers of negative regulation. this involves signaling through multiple inhibitory receptors that inhibit functional and proliferative responses. the cd28 family member programmed cell death 1 (pd-1) has been shown to be the most highly expressed inhibitory receptor on cd8+ t cells during chronic infection, and to have a major role in regulating t cell exhaustion during infection [33, 34] . increased expression of pd-1 by t cells also occurs during hbv and hcv infections [35] [36] [37] . several other inhibitory receptors have also been shown to induce t cell unresponsiveness during chronic infections. these receptors include cytotoxic t lymphocyte antigen 4 (ctla-4) [31, 38, 39] , t cell immunoglobulin domain and mucin domain protein 3 (tim3) [40, 41] , and lymphocyte activation gene 3 (lag-3) [38] . in addition, certain cytokines, such as il-10 and transforming growth factor-β (tgfβ) as well as regulatory t cells, may also contribute to the lack of t cell functionality during situations of high antigenic burden [42] . there is intriguing evidence that blockade of the inhibitory receptor could restore antigen t cell responses. for example, blockade of the pd-1 signaling pathway improves antigen-specific t cell proliferation and cytokine secretion in lymphocytic choriomeningitis virus (lcmv)-infected mice [31, 34] and in humans with chronic hiv [32, 43, 44] , hbv [45] and hcv [36] infections. this effect was synergistically improved in lcmv infected mouse following the simultaneous blockade of the t cell inhibitory receptors pd-1 and lag-3, thanks to which a diminished viral load in vivo was observed, although blocking lag-3 pathway alone had little effect on the severity of exhaustion [34] . moreover, mabmediated blocking of ctla-4 pathway in vitro augments hiv-specific cd4+ t-cell function suggesting that the immune modulation of this target may also provide a clinical benefit in infected individuals [39] . another example is the manipulation of signals mediated by glucocorticoid-induced tnf receptor (gitr), a recently identified member of the tnf receptor superfamily, preferentially expressed on subset cd4 +cd25+ regulatory t cells. gitr signals break the suppressive activity of this subset. in fact, an agonistic anti-gitr mab immediately injected after viral infection significantly increased the number of activated cd4+ and cd8+ t cells secreting ifn-γ [46] . one must remember that the manipulation of immunological responses could have detrimental effects on the host, as highlighted by the recent tragic human trial of tgn1412. this is a mab against human t cell costimulatory molecule cd28 developed by tegenero to treat b-cell chronic lymphocytic leukaemia, autoimmune and inflammatory diseases, on the basis of its capability of inducing preferential activation and expansion of immunosuppressive regulatory t (treg) cells, as observed in rodent models. tgn1412 has been termed a 'superagonist' because it binds to cd28 and activates t cells without the need for prior t cell antigen receptor (tcr) signaling. in a phase i clinical trial (in march 2006), following administration of tgn1412, six healthy young men suffered a life-threatening cytokine-release syndrome (crs) involving multi-organ failure, something unpredicted by the preclinical studies. it is now clear that in the presence of tgn1412, activated cd4+ effector memory t (t em ) cells were the source of the cytokines that mediated the crs observed in the volunteers. treg cells were not able to prevent systemic inflammation, probably because the balance between activated treg cell and t em cell numbers is disadvantageous for humans compared with laboratory rodents. furthermore, in macaques, but not in humans, cd4+ t cells lose cd28 expression during their differentiation into t em cells; this detail, however, had gone unnoticed despite many years of primate testing. in conclusion, this model failed to prevent the disastrous case above [47] . in view of these events, such a risk needs to be carefully assessed if the modulation of immune inhibitory or activating receptors is used for increasing the functional activity of virus-specific t cells in order to avoid nonspecific inflammation. these therapeutic approaches are being carefully evaluated for cancer as well as for hiv and hcv chronic viral disease. given the potential antiviral effect of the antibodies, viruses have evolved multiple mechanisms to protect themselves from antibody binding. one of these, the viral receptor glycosylation, is widely shared among different viruses. carbohydrates are poorly immunogenic and, therefore, do not stimulate the response of type b lymphocytes and simultaneously hide the underlying protein structures. hcv e2 protein contains up to 11 potential nlinked glycosylation sites. specific glycans mask the cd81binding site and, therefore, nab epitopes [48] . lipid shielding may represent an additional strategy used by hcv to evade the antibody response. current data suggest that key neutralizing epitopes are less accessible on lvps. more recently, hcv has been found capable of direct cellto-cell transmission, which is largely resistant to antibody neutralization [49, 50] . hiv envelope protein is also glycosilated and changes occur in the frequency and position of glycans hiv gp120; these 'evolving glycan shields' have been shown to decrease sensitivity to antibody neutralization [51] . other factors of antibody escape for hiv are: trimerization of the gp 120 and gp 41 that can shield vulnerable epitopes better exposed on the individual monomeric subunits; kinetic and spatial constraints that impede antibodies from accessing potentially vulnerable sites during receptor binding and membrane fusion process; the variable loops of gp120 that are a prime target for nabs, which usually have a very narrow breadth of reactivity [52] . finally, the high mutation rate of many viruses, including hiv and hcv, which undergo rapid antigenic variation, allows them to escape neutralization, constituting a significant hurdle for nabs development. all these problems may be counter-balanced by selecting nabs which target conserved and more accessible areas of viral particles, and/or by using mixtures of nabs which target various key epitopes. in fact, it has been demonstrated that combination therapy with mab cocktails prevents escape variants for many viruses, including influenza [53] , coronavirus [54] and lcmv [55] , and that broad neutralization in the sera of most of some individual hiv infected donors can be associated with single or four to five principal specificities [56] . recent studies have indicated that nabs play a critical role in hcv disease outcome. viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection with some evidence that these antibodies are broadly reactive [57, 58] . in contrast, chronic hcv infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection, despite the induction of crossneutralizing antibodies in the later phase of infection. current understanding of the nab response raised against hcv suggests that e2 is the major target, and that multiple epitopes within e2 may be targeted by both linear-and conformation-dependent antibodies. predominantly, these neutralization epitopes overlap with cd81binding sites and clearly demonstrate a role in inhibition of entry. currently, one of these mabs, mbl-hcv1, is being investigated in clinical trials in the prevention of liver re-infection after transplantation, for which novel antiviral preventive and therapeutic strategies are urgently needed. in fact, re-infection of the graft is universal, being characterized by accelerated progression of liver disease; ifn-based therapies exhibit enhanced adverse effects and limited efficacy in these patients [59, 60] . mbl-hcv1 is a fully human monoclonal antibody isolated from transgenic mice and directed to a highly conserved linear epitope of hcv e2 glycoprotein. it is able to neutralize pseudoviruses from multiple hcv genotypes and has demonstrated efficacy in preventing hcv genotype-1 infection in hcv naïve chimpanzees. a phase i open-labeled, dose escalation study was performed in healthy adult volunteers starting with 1 mg/kg and escalating to 3, 10, 30 and 50 mg/kg after a 10-day post-infusion safety review. mbl-hcv1 was well-tolerated without any seriously adverse effect event. based on the favorable safety, tolerability and pharmacokinetics data, a phase ii study of mbl-hcv1 in chronically infected hcv patients undergoing liver transplantation has been planned [61] . in the context of hiv disease, despite intensive study over two decades, only a small number of broadly neutralizing mabs have been identified from infected patients and little is known about their activity in vivo. these antibodies are able to inhibit viral entry of most primary hiv isolates in vitro [17, [62] [63] [64] and the exceptionally high level of mutation found in their genes may reflect chronic immune responses to hiv and persistent hypermutation and selection [65] . a number of trials evaluating different formulations of anti-hiv monoclonal antibodies are now in progress. the first trial assessed a chimeric monoclonal antibody cgp 47,439 to the v3 loop of the hiv-1 envelope gp120 over 21 weeks [66, 67] . subsequent studies evaluated the kinetics of monoclonal antibody f105 directed to the cd4-binding site of gp120 [68, 69] , a humanized antibody binding to the v3 epitope gpgraf [70] . finally, a humanized mab, kd-247 is under evaluation in clinical trials. its epitope was mapped to 6 aa, igpgra, at the tip of the v3 loop of envelope protein and demonstrates crossneutralizing activity against hiv-1 isolates in clade b [71] . a drug based on the mab cocktail mode is also currently in clinical development. in this regard it has already been observed that in hiv neutralization assays the effectiveness of a mix of broadly neutralizing antibodies increased synergistically compared to the effect of the individual antibody. the synergy effect was relatively weak, with a maximum of two-to four-fold enhancement, between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations [72] . however, the use of antibodies in the cocktail mode, as an approach to improve their effectiveness, is already recognized for other pathogens or toxins. in the case of tetanus toxin, it has been reported that combining the action of three out of four antibodies increased the neutralizing activity up to 200 times [73] . in the case of botulinum toxin, neutralizing activity has been reported up to 20,000 times higher when using a mixture of three monoclonal antibodies [74] . instead, other studies have demonstrated that the combination of two potent neutralizing mabs against hiv, vrc01 and pg9, although not synergistic, can mediate additive neutralization viral activity and provides an improved neutralization coverage of 90% to 97% of viral strains by combining independent epitope targeting [75] . in a proof-of-concept passive immunization trial with humans, it has been demonstrated that a cocktail of the three broadly neutralizing mabs -2g12, 4e10 and 2f5was able to delay viral rebound in patients whose infections were fully suppressed by antiretroviral treatment before administration of the antibodies [76] . interestingly, the main antiviral effect observed was primarily attributable to the 2g12 antibody, a mab that binds to a noncontinuous epitope composed of glycosylation residues distributed over the envelope protein gp120 [64] , whereas the other two mabs, 4e10 [77] and 2f5 [78] , recognize two adjacent highly conserved epitopes on the membrane-proximal ectodomain of the hiv-1 envelope protein gp41. in earlier phase i clinical trials, safety and tolerability were demonstrated [79, 80] . during a longterm multiple dose phase ii clinical trial, high doses of the three neutralizing antibodies were given in combination to 14 hiv-1-infected individuals at weekly intervals over three months. pharmacokinetic analysis revealed that repeated infusions at high dose levels were well tolerated by the patients and did not elicit an endogenous immune response against the monoclonal antibodies. the antibodies showed distribution and elimination kinetics similar to those seen for other human-like antibodies, though monoclonal antibody 2g12 had a significantly longer elimination half-life (21.8 +/-7.2 days) than monoclonal antibodies 4e10 (5.5 +/-2.2 days) and 2f5 (4.3 +/-1.1 days) [81] . furthermore, analyses of the emergence of mutations conferring resistance to these three mabs were performed. sequence analysis of the 2g12 epitope relevant n-glycosylation sites of viruses derived from 13 patients demonstrated that mutations in these sites are associated with resistance. in vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2g12 pressure. importantly, in vitro selection with 2f5 and 4e10 demonstrated that resistance to these nabs can be difficult to achieve and can lead to selection of variants with impaired infectivity [82] . moreover, generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication; some generated triple-resistant viruses seemed to be impaired in their replicative fitness, and none of the patients developed detectable viruses that escaped neutralization by all three mabs within the 77day observation period [83] . as is true with all mabs designed for infectious disease, the development of a successful vaccine would reduce the need for them. however, given the scarcity of drugs in the field of virology and given the slow progress on the hiv vaccine front, the development and use of microbicides, compounds that could be applied topically to prevent hiv transmission, is one of the possible strategies to counter the spread of hiv. in this regard, mabs could be proposed as suitable components of microbicides to fight hiv entry at mucosal surface. a safety study of p2g12 mab administered vaginally in healthy women has been completed. p2g12 is the broadly neutralizing 2g12 mab manufactured from tobacco plants [84] . most mabs in clinical trials have been produced using a system called chinese hamster ovary cell (cho-cell) fermentation [85] , including 2g12 used along with 2f5 and 4e10 antibodies as a cocktail. the cho-cell fermentation production method is very expensive and cannot produce enough mabs on a scale required for the global market; therefore, plant manufacture of such mabs may hopefully offer some solutions to lower production costs and improve output. the process yields five grams of purified antibody from 250 kg of tobacco and production costs could be 10 to 100 times lower than when using conventional bioreactors. this study has been designed to confirm the safety of a vaginally delivered mab p2g12 derived from plants and manufactured to good manufacturing practice (a quality standard used for the manufacture of medicinal products). the medicine is the first plant-produced antibody to be greenlit for clinical testing by britain's medicines and healthcare products agency (mhra). it took about a year to get that agency's stamp of approval because it required assurances that the drugs did not contain allergenic plant sugars or pesticides. no matter how it is produced, p2g12 antibody has not been shown to actually prevent hiv-1 infection in clinical trials; thus a version made from tobacco plants would not see approval any time soon. p2g12 would also likely be just one ingredient in a cocktail of plant-produced antibodies [84] . to eliminate or reduce the development of escape variants it has been proposed that targeting the conserved cellular receptors of the virus may open new avenues for a viable antibody therapy for hiv infection. hiv entry into cd4+ t cells requires the presence of a co-receptor, either ccr5 or cxcr4, on the target cell. thus, based on this hypothesis, mabs directed against cd4 and against the co-receptor ccr5, have been developed and are being analyzed in clinical trials. cd4 functions as a co-receptor, physically associating with the tcr during ag recognition by binding to a nonpolymorphic component of the major histocompatibility complex (mhc) class ii molecules on the surface of the antigen-presenting cell. ibalizumab, a humanized mab, binds cd4 on t cell surface away from the binding site for mhc class ii molecules. it does not inhibit gp120 binding to cd4 but appears to exert its antiviral property by post-binding conformational effects that prevent cd4bound gp120 from interacting with ccr5 or cxcr4 [19, 86] . by contrast, other monoclonal antibodies, that competitively inhibit gp120 binding, interfere with mhc class ii immune function [87, 88] . the reported human experience with ibalizumab consists of three clinical trials. during phase i study, it was observed that peak mean reductions in viral load occurred later in the higher dose cohorts, whereas the extent and duration of viral suppression correlated with the degree of cd4+ cell coating by ibalizumab, which was maintained longer in the higher dose cohorts, with a duration of 15 to 34 days. peak increases in cd4 counts at one day after infusion, well before the peak declines in viral load; this suggests that the increase may have been due to redistribution of cd4+ cells from lymphoid tissue rather than regeneration of cd4+ cells in the setting of viral suppression. a multidose study demonstrated continued safety over an extended treatment period and provided data on the development of ibalizumab resistance. resistance testing showed reduced susceptibility relative to baseline. resistant isolates remained dependent on cd4 for viral entry, suggesting that resistance did not develop through the use of alternative receptors. genotypic analysis was unable to identify mutations, diagnostic of ibalizumab resistance. consistent with the allosteric mechanism of ibalizumab's anti-hiv-1 effect, the development of resistance is associated with a reduction in the maximum percentage inhibition rather than the shift in the ic50 characteristic of competitive inhibitors [89, 90] . the half-life of iggs under normal physiological circumstances is two to three weeks [91] . in contrast, the average half-life of ibalizumab is 3 to 3.5 days [89] . this is consistent with observations of other anti-cd4 antibodies, in which internalization or shedding of the receptor results in more rapid antibody degradation. a randomized, double-blind, placebo controlled, phase iia study has evaluated the ibalizumab efficacy, the results showing a considerable viral load reduction with respect to the placebo arm [92] . ccr5 is a chemokine receptor that mediates activation and migration of t cells and other leukocytes. ccr5using (r5) viruses typically mediate transmission and then predominate through the progression to symptomatic disease. viruses can use an alternative chemokine receptor, cxcr4, either exclusively or in addition to ccr5. the cxcr4-using virus can be present initially, but tends to result in an increasing proportion of subjects in the later stages of the disease [93] . ccr5 co-receptor antagonists represent an emerging antiretroviral treatment class and the first to target a host molecule. currently, two anti-ccr5 mabs are being investigated. one of these is ccr5mab004, a fully human igg4 monoclonal antibody with robust activity against a diverse panel of hiv-1 isolates; it synergizes in vitro with other arv classes and appears safe and effective in reducing hiv viral load. high levels of receptor occupancy were observed for 14 to 28 days with the highest dose cohorts, suggesting the potential for weekly, fortnightly or even monthly dosing [94] . the other anti-ccr5 mab is pro 140, a humanized mab that also synergizes with small-molecule ccr5 antagonists in laboratory studies [95] . pro140 is being investigated in two modes of administration: the classical intravenous (iv) form, and subcutaneous (sc) form. the trial involving sc administration is the first to bear the proof of concept for a mab administered subcutaneously in hiv-1 infected subjects as a potent and longacting antiretroviral agent. an iv form of pro 140 tested as monotherapy in hiv-1 subjects with only r5 virus detectable [96] demonstrated potent and prolonged antiviral activity, with a 1.83 log10 mean reduction in hiv-1 rna and safety relative to placebo. the successive randomized, double-blind, placebo-controlled iia trial examined the antiviral activity, tolerability and pharmacokinetics of single intravenous infusions of up to 10-mg/kg of mabs. all pro 140-treated subjects treated with 10 mg/kg experienced a 1-log10-unit reduction in hiv-1 rna level, there being just one exception; a post-study analysis using the enhanced-sensitivity trofile assay determined that this subject had dual/mixed virus at screening. there was no change in co-receptor tropism or emergence of pro 140-resistant virus during the course of this study, supporting the view that pro 140 broadly inhibits r5 hiv-1 with a high barrier to resistance. the maximum tolerated dose of iv pro 140 has not been determined, suggesting a sizeable margin of safety for pro 140 sc administration study [97] . the study involving pro 140 sc administration showed virologic suppression between successive doses and no changes in r5 viral susceptibility to pro 140 following three weeks of monotherapy, indicating no adaptation of virus to use ccr5 in the presence of drug. pharmacokinetic data suggest the possibility of a drug regimen administered fortnightly for hiv infected individuals. proteins and other macromolecules drain from sc sites into both blood capillaries and the lymphatic system. in animals, proteins with molecular weights of greater than 16,000 daltons have been observed to drain primarily into the lymphatic system following sc administration [98] . such proteins transit through lymph fluid and typically are not absorbed significantly into the blood until they reach the thoracic duct. since the molecular weight of pro 140 is approximately 150,000 daltons, a substantial amount of sc pro 140 can be expected to drain into the lymphatic system and potentially encounter ccr5+ cells in lymphoid tissues prior to reaching the bloodstream. for these reasons, serum concentrations may not provide a full picture of the overall exposure following sc dosing of pro 140. sc infusion is currently used by individuals with primary immunodeficiency to self-administer at home significantly larger amounts (approximately 11 grams) and volumes (approximately 70 ml total, up to 15 ml/site) of the weekly sc-administered immunoglobulin [99] . self-administration of 324 mg sc pro 140 would be much simpler in comparison. therefore, sc pro 140 offers the potential for significant dose-dependent hiv-1 rna suppression and may offer greater convenience for many patients in terms of patient selfadministration [100] . the sc injection mode was chosen in order to evaluate pro140 safety and efficacy as an adjunct to an oral antiretroviral regimen in hiv-infected injection drug users with viral rebound and documented poor adherence to the previous antiretroviral regimen. therefore, a phase iib, national, multicenter, randomized, doubleblind, placebo-controlled study was initiated and is currently recruiting participants. given the complications that arise from the occurrence of drug resistances, the use of antibodies together with combined therapy increases the drug number and, therefore, the therapeutic opportunities. in particular, in the case of ccr5 inhibitors, one report has demonstrated that resistance to ccr5 inhibitors may increase the sensitivity of the resistant virus to certain neutralizing antibodies [101] . compared to ccr5, cxcr4-based blocking agents as therapy against hiv are less attractive due to the crucial role of cxcr4 in many biological processes, and the absence to date of known naturally occurring mutations leading to the inactivation of cxcr4 gene in humans. moreover, one major problem is linked to the fact that, whereas r5 viruses are found on their own in 50% or more of patients, viruses that using cxcr4 co-receptor (x4) usually are present mixed together with r5 viruses; therefore, the use of cxcr4 specific mabs could result in only little or transient effect on the overall viremia, also complicating the evaluation of pharmacological activity. however, antibodies against cxcr4 might still provide some benefits for some hiv positive patients when co-administrated with ccr5 antagonists, if the safety of such combinations is established [93] . there is a pressing need for antiviral agents that are effective against multiple classes of viruses. broad specificity might be achieved by targeting phospholipids that are widely expressed on infected host cells or on viral envelopes. phosphatidylserine (ps), the most abundant anionic phospholipid of the plasma membrane, is segregated at the inner leaflet of the plasma membrane of resting mammalian cells. loss of ps asymmetry occurs during apoptosis, cell injury, cell activation and malignant transformation, and results from inhibition of the translocases or activation of ps exporters, or lipid scrambling enzymes, such as scramblases. after enveloped viruses replicate within the host cell, they create their 'envelope' by carrying along part of the host cell's membrane upon exiting. as a result, the target phospholipid becomes exposed on the surface of the virus as well as on the infected host cell [102] . bavituximab is the first in a new class of patented antibody therapeutics that target and, preferentially bind, to these exposed phospholipids. it has demonstrated broad therapeutic potential across multiple oncology indications and represents a new approach to treating viral disease, too. bavituximab is currently being evaluated in randomized phase ii clinical trials for non-small cell lung cancer and pancreatic cancer, for therapy of chronic hcv infection and for hiv/hcv co-infection. the therapeutic effect of bavituximab appears to be due to adcc of tumor and virus-infected cells. since ps exposure is an early event during virus infection, adcc may limit virus spread. furthermore, in the infectious disease setting, bavituximab causes opsonization and clearance of infectious virus from the bloodstream, leaving less virus to infect other tissues. three completed phase i hcv clinical trials have shown that bavituximab is generally safe and well-tolerated. reductions in serum hcv rna levels were also observed. a randomized phase ii clinical trial with previously untreated hcv genotype-1 infected patients was designed to determine the early virologic response (evr) rate after 12 weeks of therapy with bavituximab in combination with the antiviral drug ribavirin and safety profile versus pegylated ifn-α-2a and ribavirin. the results show that the combination of bavituximab with ribavirin has a better safety profile than an ifn-containing regimen. however, the evr development in the bavituximab-containing arm was later than the ifn-containing group; therefore, a longer-term evaluation is needed to adequately compare their effectiveness. in addition, the lower dose level appears to be more active in hcv patients than the high dose does. such results suggest that future studies evaluating longer bavituximab treatment durations at or around the lower dose level in combination with ribavirin and potentially direct acting antivirals in certain patient populations may hold promise as ifn-free hcv therapeutic regimens [103] . targeting ps on cells infected with multiple different viruses and on virions themselves is a promising antiviral strategy. although resistance has developed in monotherapy trials with ibalizumab (an anti-cd4 antibody), host-derived antigen, such as anionic phospholipids, on virus-infected cells are independent of the viral genome and as a consequence the acquisition of drug resistance should be theoretically less problematic than with agents that target virus-encoded components. since the discovery of pd-1 as an inhibitory receptor associated with t-cell dysfunction, the roles of various inhibitory receptors on virus-specific cd8+ t cells have been extensively studied in human chronic viral infections, such as hcv, hbv and hiv infections. as blocking the inhibitory receptors in vitro restores the functions of virus-specific t cells, novel hiv and hcv treatments based on blockade of several immune checkpoint molecules are being investigated. in particular, mabs interfering with two major inhibitory networks of the b7:cd28 family, namely the pd-1 and ctla-4 pathways [104] , are currently being studied in clinical trials, to evaluate their safety and efficacy. these mabs recognize the pd-1 or ctla-4 receptor and neutralize the binding with their respective ligands. the pd-1:pd-l1 pathway delivers inhibitory signals which regulate t cell activation. as a result it performs a key role in various processes, namely in multiple tolerance checkpoints that prevent autoimmunity, in the suppressive tumor microenvironment, in the immune-mediated tissue damage, in host defenses aimed at eradicating microbial pathogens and tumors and finally, in t cell exhaustion that contributes to both lack of viral control during chronic infections and to t cell unresponsiveness [105] . in cancers, a strong correlation between increased pd-l1 expression on tumors and a negative survival prognosis in patients has already been observed. various studies indicate that mabs targeting the pd-1 signaling pathway reinvigorate antigen-specific t-cell responses and promote an immune response to fight tumors [106] . in hcv infection the relationship between the pd-1 expression and the outcome of the acute hcv infection was questioned; subsequently, recent studies have shown that the progression of acute hcv infection to the chronic stage is associated with a high level of pd-1 on hcv-specific cd8+ t cells, whereas the clearance of hcv infection is associated with lower levels of pd-1 expression [36] . given these premises, mdx-1106, a fully human antibody also known as ono-4538, and ct-011, a humanized antibody, both interacting with pd-1 receptor, are being developed as a treatment for cancer disease and for therapy of chronic hcv infection [107] . to date, most clinical experience with pd-1 blockade has been gained with mdx-1106 in the tumor setting. drug-related grade 3 or 4 toxic effects occurred in 14% of patients, in whom there were drug-related adverse events of special interest, those with potential immune-related causes; they included pneumonitis, vitiligo, colitis, hepatitis, hypophysitis and thyroiditis. pneumonitis (3%) ranged from isolated radiographic abnormalities to progressive, diffuse infiltrates associated with clinical symptoms in a small number of patients. although three deaths occurred, mild-to-moderate pneumonitis was managed successfully with either observation or glucocorticoids. however, objective responses were observed in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; overall, an adverse-event profile does not appear to preclude its use [108] . besides these studies, an ongoing phase i safety trial with active hepatitis c genotype 1 infected patients has been designed to assess the safety and tolerability profile of mdx-1106 [109] . clinical studies to evaluate the use of ct-011 in hcv disease have also been initiated [110] . ctla-4 is up-regulated on activated t cells and inhibits t cell activation by reducing the production of il-2 and arresting cell cycle progression. ctla-4 has also been shown to have an impact on t cell responses in animal tumor models and humans [111, 112] . human trials that used a blocking anti-ctla-4 mab demonstrated a reduction in tumor mass and clinical benefit in a substantial minority of treated subjects. studies of the role for ctla-4 in chronic infections have produced mixed results. in chronic hiv infection, many studies indicate that impaired cd4+ t cell function is associated with viral persistence [113] , although the function of ctla-4 in causing hiv persistence by suppressing t cell function remains unclear [114] . on the other hand, ctla-4's role in chronic hcv infection seems to be more defined. the hcv-specific cd8+ t cells found in the livers of chronic hcv patients overexpressed not only pd-1, but also ctla-4. co-expression of pd-1 and ctla-4 was observed in liverinfiltrating lymphocytes, but not in peripheral blood lymphocytes [36] , suggesting the phenotypic differences of virus-specific cd8+ t cells in different in vivo compartments. pd-1 and ctla-4 expressing hcv-specific t cells were profoundly dysfunctional [115] . tremelimumab is a fully human igg2 mab directed against ctla-4. while a phase ii study for hiv disease with this drug has been withdrawn prior to enrollment, clinical trials for hcv disease are still underway. tremelimumab binds to activated t lymphocytes and results in inhibition of b7-ctla-4-mediated down-regulation of tcell activation. it also acts as an il-2 stimulant. it was generated, using xenomouse technology (figure 2) , as an anticancer agent and is currently in worldwide phase iii development for malignant melanoma, phase ii development for colorectal cancer, gastrointestinal cancer, gynecological cancer and non-small cell lung cancer in the us and other countries. it is also being investigated for prostate, breast and pancreatic cancer in various countries. as for anti-pd-1 antibodies, immune-related adverse effects of tremelimumab are of special interest because of its presumed mechanism of action. most of the experience in identifying and managing ctla-4 treatment-related side effects has derived from studies in cancer, particularly in melanoma. these effects mainly include colitis/diarrhea, dermatitis, hepatitis and endocrinopathies; uveitis, nephritis and inflammatory myopathy also have been occasionally reported. these unique side effects are likely a direct result of breaking immune tolerance upon ctla-4 blockade; they are generally mild, reversible and manageable, following specific treatment guidelines that include symptomatic therapies or systemic corticosteroids [116] . in december 2008, pfizer initiated a phase ii trial in patients with latestage unresectable liver cancer who also have hepatitis c infections. the primary endpoint of this single-armed study is the ability of tremelimumab to produce tumor responses among hcv-infected patients with hepatocellular carcinoma and to produce changes in hepatitis c viral load. the first results indicate that tremelimumab demonstrated an excellent safety profile, with a promising antitumor efficacy against hcc in 17 patients, as well as an intense antiviral activity. in fact, a significant and progressive decline in serum hcv viral load was observed, this being associated with an increase in anti-hcv immune response in 76% of patients [117] . since there are multiple levels of immunoregulation, a synergistic use of antibodies against different checkpoint molecules might represent the next stage in immunotherapy for chronic infectious diseases, as evidenced from ex vivo studies about the combined pd-1/ctla-4 blockade in hcv disease [36] . furthermore, because the host mechanisms that inhibit t cell activity are common and conserved aside from specific virus-encoded immune evasion strategies, the antibodies targeting inhibitory receptors may prove extremely versatile drugs potentially effective against multiple classes of viruses. the need to treat hiv and hcv infectious diseases, two epidemics of global impact, has reawakened interest in mab-based therapy, supporting a variety of clinical studies. the results that are emerging, will help to create models for the further development of such drugs and extend their use against other viruses as well. although the mab production costs are high, increasing advances of biotechnology and production systems will make them more competitive on the market, and new approaches, such as using mab cocktails or combining mabs with available drugs, will improve effectiveness. treatment with mabs as part of a drug regimen is the most likely future for mabs that block hcv and hiv infection in order to avoid viral escape, while chronic treatment could attract further investments from pharmaceutical companies. furthermore, broad spectrum mabs, such as bavituximab and immunomodulatory mabs, could be useful against a whole range of diseases, thus extending marketability and profit margins. this review has focused on the use of intact mabs as a novel emerging and versatile class of pharmaceuticals. it is important to note, however, that biotechnology also provides the opportunity to build various antibody formats whose improved pharmacokinetics and pharmacodynamic properties could be co-opted in the fight against infectious diseases. resistance to antiretroviral drugs; the mechanisms of immune reconstitution; and, finally, operational and implementation research in resource limited settings. he chaired or participated in many international clinical studies on antiretroviral therapy, and he is currently the coordinator of the -european commission-funded hiv clinical trials network (neat). cellular and molecular immunology hemming vg: use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases passive antibody therapy for infectious diseases carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens passive immunity in prevention and treatment of infectious diseases monoclonal antibody-based therapies for microbial diseases immunotherapy with human monoclonal antibodies. fragment a specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin polyclonal immunoglobulins and hyperimmune globulins in prevention and management of infectious diseases the growth and potential of human antiviral monoclonal antibody therapeutics potent antibody therapeutics by design virus entry: molecular mechanisms and biomedical applications mechanism of membrane fusion by viral envelope proteins preparation and properties of antibody directed specifically against the neuraminidase of influenza virus the antiviral activity of antibodies in vitro and in vivo a monoclonal antibody to cd4 domain 2 blocks soluble cd4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (hiv-1) and hiv-1 infection of cd4+ cells potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the ccr5 monoclonal antibody pro 140 anti-human immunodeficiency virus type 1 (hiv-1) antibodies 2f5 and 4e10 require surprisingly few crucial residues in the membraneproximal external region of glycoprotein gp41 to neutralize hiv-1 hepatitis c virus entry depends on clathrin-mediated endocytosis hepatitis c virus cell entry: role of lipoproteins and cellular receptors anti-cd81 antibodies can prevent a hepatitis c virus infection in vivo serum-derived hepatitis c virus infection of primary human hepatocytes is tetraspanin cd81 dependent role of scavenger receptor class b type i in hepatitis c virus entry: kinetics and molecular determinants fc receptors and their role in immune regulation and autoimmunity hiv-1 gp120-specific antibody-dependent cell-mediated cytotoxicity correlates with rate of disease progression heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in hiv-1 elite controllers serum antibodies against the hepatitis c virus e2 protein mediate antibody-dependent cellular cytotoxicity (adcc) hepatitis c virus (hcv)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against hcv envelope protein advances in the assessment and control of the effector functions of therapeutic antibodies the function of programmed cell death 1 and its ligands in regulating autoimmunity and infection viral persistence alters cd8 t -cell immunodominance and tissue distribution and results in distinct stages of functional impairment t cells and viral persistence: lessons from diverse infections pd-1 expression on hivspecific t cells is associated with t -cell exhaustion and disease progression restoring function in exhausted cd8 t cells during chronic viral infection coregulation of cd8+ t cell exhaustion by multiple inhibitory receptors during chronic viral infection pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcv-specific cd8 exhaustion synergistic reversal of intrahepatic hcv-specific cd8 t cell exhaustion by combined pd-1/ ctla-4 blockade characterization of hepatitis b virus (hbv)-specific t -cell dysfunction in chronic hbv infection molecular signature of cd8+ t cell exhaustion during chronic viral infection upregulation of ctla-4 by hiv-specific cd4+ t cells correlates with disease progression and defines a reversible immune dysfunction tim-3 expression defines a novel population of dysfunctional t cells with highly elevated frequencies in progressive hiv-1 infection upregulation of tim-3 and pd-1 expression is associated with tumor antigen-specific cd8+ t cell dysfunction in melanoma patients t -cell exhaustion: characteristics, causes and conversion pd-1 is a regulator of virus-specific cd8+ t cell survival in hiv infection upregulation of pd-1 expression on hiv-specific cd8+ t cells leads to reversible immune dysfunction expression of the interleukin-7 receptor α chain (cd127) on virus-specific cd8+ t cells identifies functionally and phenotypically defined memory t cells during acute resolving hepatitis b virus infection in vivo ligation of glucocorticoid-induced tnf receptor enhances the t-cell immunity to herpes simplex virus type 1 the storm has cleared: lessons from the cd28 superagonist tgn1412 trial hepatitis c virus envelope glycoprotein e2 glycans modulate entry, cd81 binding, and neutralization hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies cd81 is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells antibody neutralization and escape by hiv-1 identifying epitopes of hiv-1 that induce protective antibodies combination therapy using chimeric monoclonal antibodies protects mice from lethal h5n1 infection and prevents formation of escape mutants human monoclonalantibody combination against sars coronavirus: synergy and coverage of escape mutants in vivo selection of neutralization-resistant virus variants but no evidence of b cell tolerance in lymphocyticchoriomeningitis virus carrier mice expressing a transgenic virus-neutralizing antibody a limited number of antibody specificities mediate broad and potent serum neutralization in selected hiv-1 infected individuals human serum facilitates hepatitis c virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis c virus infection rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis c treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus recurrence of hepatitis c virus after loss of virus-specific cd4(+) t-cell response in acute hepatitis c safety and pharmacokinetics of a novel human monoclonal antibody directed against e2 glycoprotein of hepatitis c virus (mbl-hcv1) in healthy volunteers. easl the international liver congress™ crystal structure of a neutralizing human igg against hiv-1: a template for vaccine design a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 human monoclonal antibody 2g12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals a phase i/iia clinical study with a chimeric mouse-human monoclonal antibody to the v3 loop of human immunodeficiency virus type 1 gp120 pharmacokinetics of an hiv-1 gp120-specific chimeric antibody in patients with hiv-1 disease phase i study of a human monoclonal antibody directed against the cd4-binding site of hiv type 1 glycoprotein 120 pharmacokinetics of f105, a human monoclonal antibody, in persons infected with human immunodeficiency virus type 1 monoclonal antibody hnm01 in hivinfected patients: a phase i study sequential immunization with v3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary flego et al isolates with a matching narrow-neutralization sequence motif neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody mascola neutralization coverage is improved by combining monoclonal antibodies that target independent epitopes hiv-1 delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 a broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 a phase i trial with two human monoclo-nal antibodies (hmab 2f5, 2g12) against hiv-1 long-term multiple-dose pharmacokinetics of human monoclonal antibodies (mabs) against human immunodeficiency virus type 1 envelope gp120 (mab 2g12) and gp41 (mabs 4e10 and 2f5) in vivo and in vitro escape from neutralizing antibodies 2g12, 2f5, and 4e10 hiv-1 mutants escaping neutralization by the human antibodies 2f5, 2g12, and 4e10: in vitro experiments versus clinical studies antibody production clinical trial of farmed hiv drug finally gets underway epitope mapping of ibalizumab, a humanized anti-cd4 monoclonal antibody with anti-hiv-1 activity in infected patients immunosuppression of cynomolgus renal allograft recipients with humanized okt4a monoclonal antibodies functional epitope analysis of the human cd4 molecule. the mhc class ii-dependent activation of resting t cells is inhibited by monoclonal antibodies to cd4 regardless whether or not they recognize epitopes involved in the binding of mhc class ii or hiv gp120 safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly tnx-355), an anti-cd4 monoclonal antibody, in human immuno-deficiency virus type 1-infected adults antiretroviral activity of the anti-cd4 monoclonal antibody tnx-355 in patients infected with hiv type 1 mouse/human chimeric monoclonal antibody in man: kinetics and immune response phase 2 efficacy and safety of the novel entry inhibitor, tnx-355 the biology of ccr5 and cxcr4 a phase 1, doseescalation, placebo-controlled study of a fully human monoclonal antibody (ccr5mab004) against ccr5 in patients with ccr5-tropic hiv-1 infection potent antiviral synergy between monoclonal antibody and small-molecule ccr5 inhibitors of human immunodeficiency virus type1 a phase i study to explore the activity and safety of sch532706, a small molecule chemokine receptor-5 antagonist in hiv type-1-infected patients phase 2a study of the ccr5 monoclonal antibody pro 140 administered intravenously to hiv-infected adults transport and absorption of drugs via the lymphatic system safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency diseases anti-hiv-1 activity of weekly or biweekly treatment with subcutaneous pro 140, a ccr5 monoclonal antibody neutralizing antibody and antiretroviral drug sensitivities of hiv-1 isolates resistant to small molecule ccr5 inhibitors targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases advancing clinical trials in cancer and viral infection: bavituximab antiviral the pd-1 pathway in tolerance and autoimmunity tumor b7-h1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up targeting the pd-1/b7-h1(pd-l1) pathway to activate anti tumor immunity sznol m: safety, activity, and immune correlates of anti-pd-1 antibody in cancer clinical trial.gov a service of the u.s. national institutes of health: a study of mdx-1106 to treat patients with hepatitis c infection national institutes of health: safety and tolerability study of the monoclonal antibody ct-011 in patients with chronic hepatitis c genotype 1 infection biologic activity of cytotoxic t lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients cancer regression and autoimmunity induced by cytotoxic t lymphocyteassociated antigen 4 blockade in patients with metastatic melanoma progress in defining cd4 helper cell responses in chronic viral infections pd-1 and ctla-4 inhibitory cosignaling pathways in hiv infection and the potential for therapeutic intervention functional restoration of hcv-specific cd8 t cells by pd-1 blockade is defined by pd-1 expression and compartmentalization the emerging toxicity profiles of anti-ctla-4 antibodies across clinical indications antiviral and antitumoral effects of the anti-ctla4 agent tremelimumab in patients with hepatocellular carcinoma (hcc) and chronic hepatitis c virus (hcv) infection: results from a phase ii clinical trial aacr annual meeting continuous cultures of fused cells secreting antibody of predefined specificity chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains replacing the complementarity-determining regions in a human antibody with those from a mouse humanized antibodies as potential therapeutic drugs an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus antibody engineering via genetic engineering of the mouse: xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies selecting and screening recombinant antibody libraries pre-publication history the pre-publication history for this paper can be accessed here clinical development of monoclonal antibody-based drugs in hiv and hcv diseases authors' contributions mf and aa contributed equally to writing and editing the manuscript. mc and sv critically reviewed the manuscript and made final changes. all authors have read and approved the final version of the manuscript.authors' information mf, after her doctorate (immunology, 2006, university of rome, tor vergata, italy), was enrolled as a researcher at the department of therapeutic research and medicine evaluation at the istituto superiore di sanità, rome, italy (iss). she has acquired extensive expertise in biotechnologies, such as the isolation, characterization and genetic manipulation of human mabs in single chain format for the development of biological constructs designed for clinical use. the authors declare that they have no competing interests. mc is the author of a patent about a mab against p-glycoprotein (patent number: 6063621), but this mab is not suitable for clinical applications in hiv and/or hcv diseases submit your next manuscript to biomed central and take full advantage of: key: cord-314567-purplsjn authors: fernández-ponce, cecilia; durán-ruiz, maria c.; narbona-sánchez, isaac; muñoz-miranda, juan p.; arbulo-echevarria, mikel m.; serna-sanz, antonio; baumann, christian; litrán, rocío; aguado, enrique; bloch, wilhelm; garcía-cozar, francisco title: ultrastructural localization and molecular associations of hcv capsid protein in jurkat t cells date: 2018-01-04 journal: front microbiol doi: 10.3389/fmicb.2017.02595 sha: doc_id: 314567 cord_uid: purplsjn hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd4(+) t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc1, pp1γ, ilf3, and c1qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd4(+) t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd4(+) t cells. hepatitis c virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. when expressed in cd4 + t lymphocytes, it can induce modifications in several essential cellular and biological networks. to shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed hcv-core subcellular localization and its associations with host proteins in jurkat t cells. in order to investigate the intracellular localization of hepatitis c virus core protein, we have used a lentiviral system to transduce jurkat t cells and subsequently localize the protein using immunoelectron microscopy techniques. we found that in jurkat t cells, hepatitis c virus core protein mostly localizes in the nucleus and specifically in the nucleolus. in addition, we performed pull-down assays combined with mass spectrometry analysis, to identify proteins that associate with hepatitis c virus core in jurkat t cells. we found proteins such as nolc1, pp1γ, ilf3, and c1qbp implicated in localization and/or traffic to the nucleolus. hcv-core associated proteins are implicated in rna processing and rna virus infection as well as in functions previously shown to be altered in hepatitis c virus core expressing cd4 + t cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. thus, in the current work, we show the ultrastructural localization of hepatitis c virus core and the first profile of hcv core associated proteins in t cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of hepatitis c virus core protein. thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying hcv core mediated alterations that had been described in relevant biological processes in cd4 + t cells. keywords: hepatitis c virus, immune evasion, proteomics, interactome, ultrastructure, regulatory t cells, immune tolerance introduction hepatitis c virus (hcv) infection is an important cause for chronic viral liver disease and one of the main indications for liver transplantation (anzola, 2004; dustin and rice, 2007) . hcv affects 80 million people worldwide and in more than 80% of the patients leads to chronicity (gower et al., 2014) . the high level of chronicity and the absence of a protective vaccine, makes hcv infection a significant public health problem (anzola, 2004; dustin and rice, 2007; gower et al., 2014) . the molecular mechanisms harnessed by hcv to establish a chronic infection and their implications in the innate and adaptive immune systems have not been fully elucidated. in this regard, several hcv viral proteins have been described as modulators of immunological phenomena (yao et al., 2007; krishnadas et al., 2010; tu et al., 2012; chen et al., 2015) . among them, hcv core protein has been widely associated with pathogenicity, virulence, immune evasion and immune regulation (dominguez-villar et al., 2007 , 2012a waggoner et al., 2007; tu et al., 2012; doumba et al., 2013; fernandez-ponce et al., 2014 , 2017 . however, the underlying molecular processes, as well as the behavior of hcv core protein or its interactions with host cell components, remain unclear. hcv core is a highly basic protein, which binds and protects the viral rna, multimerizing with itself to form the viral capsid (santolini et al., 1994) . in mammalian infected cells, hcv core interacts with endoplasmic reticulum membranes, lipid droplets and other cellular and viral proteins to promote assembly of new virions. however, several inhibitory molecules and the lack of some host factors can hamper viral production by disrupting the core multimerization process and the coordinated interactions with other viral proteins (mousseau et al., 2011; gawlik and gallay, 2014) . in this way, non-enveloped hcv core proteins can be directed to alternative subcellular locations and also be released into the extracellular space (maillard et al., 2001; polyak et al., 2006; tan et al., 2006) . according to these findings, in hcv chronically infected patients, hcv core protein, has been detected as non-enveloped isolated nucleocapsids, not only in hepatocytes (falcon et al., 2003) , but also in serum (maillard et al., 2001) , peripheral blood cd4 + t cells (fernandez-ponce et al., 2014) and other nonparenchymal liver cells, such as, lymphocyte, pit, endothelial, stellate, kupffer and polymorphonuclear cells (falcon et al., 2005) . in t cell lines, non-enveloped isolated nucleocapsids binding and internalization has also been described in vitro (doumba et al., 2013) . these data further support the presence of hcv core protein inside immune cells, including lymphocytes, during hcv chronic infection. interestingly, hcv core protein intracellular expression in cd4 + t lymphocytes has been shown to induce modifications in cell proliferation, cell cycle progression, expression of anergy genes, transcription of genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, transcription and translation, cytokine production, cell death and generation of a t cell regulatory phenotype with exhausted features (bergqvist and rice, 2001; bergqvist et al., 2003; dominguez-villar et al., 2007 , 2012a doumba et al., 2013; fernandez-ponce et al., 2014) , characterized by an increased expression of foxp3 (forkhead box p3) and ctla-4 (cytotoxic t-lymphocyte antigen-4) (dominguez-villar et al., 2012a) , high levels of il-10 secretion, and decreased il-2 and ifn-γ production (doumba et al., 2013; fernandez-ponce et al., 2014) . it has been described for several viruses that the specific subcellular localization of viral proteins and their interactions with host molecules can alter the spatial distribution and organization of cellular proteins, and in this way, induce diverse molecular and cellular effects (chen et al., 2002; yoo et al., 2003; ning and shih, 2004; bertrand and pearson, 2008; ponti et al., 2008; hiscox et al., 2010; zhu et al., 2013; raval et al., 2015) . as studies using the whole virus do not allow for the elucidation of the specific molecular mechanisms in which each protein is implicated, in this work, we focused on a single viral protein, showing that in cd4 + t cells, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched. in addition, we performed pull down assays, combined with mass spectrometry analysis, in order to identify host proteins associated with hcv core, which could be implicated in the functional effect previously observed to be induced by the presence of hcv-core in t cells. we found several proteins implicated in important functions that are associated with hcv core protein. thereby, our results shed light on the molecular mechanisms underlying the alterations in biological cell processes and the generation of adaptive regulatory-like cd4 + t cells in the periphery by the intracellular presence of a single hcv viral protein. human embryonic kidney (hek) lenti-x tm 293t cell line (clontech) and jurkat cell line (american type culture collection, manassas, va, usa) were maintained in dulbecco's modified eagle's medium (dmem tm ) supplemented with 10% (v/v) heat inactivated fetal bovine serum (fbs), 2 mm l-glutamine, 10 mm hepes, 1% (v/v) sodium pyruvate, 50 µm 2-mercaptoethanol, 100 u/ml penicillin and 100 µg/ml streptomycin at 37 • c, 10% co 2 . human peripheral blood samples were obtained from healthy donors upon signature of an informed consent and following approval by the ethic sub-commission of the puerta del mar university hospital (dependent from the central quality commission), in accordance to spanish and european union regulations. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphocyte separation medium (eurobiotm, montpellier, france). cells were washed three times with pbs, subsequently stimulated with 1 mg/ml phytohemagglutinin-p (pha) (sigmatm, saint louis, missouri, usa) and cultured in dmem supplemented with 1% (v/v) sodium pyruvate, non-essential aminoacids, vitamins, larginin, l-asparragin, folic acid, 10 mm hepes, 50 mm 2-mercaptoethanol, 100 mg/ml streptomycin, 100 u/ml penicillin (life technologies, carlsbad, ca, usa) and 10% heat-inactivated fbs (gibco) at 37 • c, in a 10% co2 atmosphere. 40 u/ml il-2 was added to the cultures every 48 h, for a total of 5 days to obtain blasts. hek lenti-x tm 293t cells (clontech) were used as packaging cell lines to produce lentiviral supernatant by co-transfecting plasmids pcmvdr8.91, coding for hiv-1 gag and pol proteins and pmd2.g for the vesicular stomatitis virus g protein (vsvg) together with the transfer vector phrsincppt-sewhcv-core-gfp (hcv-core) coding for the first 191 amino acids of the hcv polyprotein (serotype 1a) corresponding to hcv-core protein. transfer vector phrsincppt-sewgfp expressing only gfp (green fluorescent protein) was used as a control (dominguez-villar et al., 2007) . cells were transfected in optimem tm medium using 10 cms diameter cell-culture dishes coated with collagen (collagen i, rat tail gibco r invitrogen cell culture), and polyethylenimine (pei), branched (sigma-aldrich) as cationic polymer. transfection efficiency was evaluated by facs analysis. supernatants were collected at 48 and 72 h, centrifuged to remove cells and debris and concentrated using lenti-x tm concentrator (clontech r ) to obtain a high-titer virus-containing pellet. pellets were frozen at −80 • c and stored until use. viral titer in supernatants was determined evaluating their efficiency in infecting jurkat cells. jurkat cells were transduced using hcvcore-gfp and gfp (as control) lentiviral supernatants, previously prepared, at a multiplicity of infection (moi) of 10. aliquots from the cultures were collected after 48 h to determine the transduction efficiency. gfp expression was monitored by facs (cyanadp-mle tm ; dakocytomation tm ) presenting a transduction range >90% in all experiments. untransduced, hcvcore-gfp and gfp transduced jurkat cells were collected 48 h post-transduction, counted, washed with phosphate buffer saline (pbs) and fixed for 24 h using 4% paraformaldehyde (pfa) in pbs, ph 7.37. subsequently, cell pellets were pre-embedded in agarose 1x, taken out of the tube with a needle and sliced into approximately 1 mm 3 pieces. pre-embedding immunogold was performed. agarose pieces were washed in pbs, permeabilized using 0.05% triton x−100 in pbs and incubated in aurion blocking solution containing normal goat serum (aurion r 905.002), for 60 min at 4 • c. sections were stained immunochemically with anti-gfp antibody (abcam ab6556) diluted 1:1,000 in pbs containing 0.2% bovine serum albumin (bsa) at 4 • c, overnight, and subsequently washed with the same buffer. for gold particle staining, sections were incubated with 10 nm gold-labeled anti-rabbit igg (sigma-aldrich) diluted 1:50 in 0.2% bsa/pbs at 4 • c, overnight. agarose pieces were washed in 0.1 m caco-buffer ph 7.37 at room temperature. sections were post-fixed in 0.5% osmium tetroxide and 0.1 m caco-buffer at room temperature, for 3 h and washed with 0.1 m caco-buffer. fixed sections were dehydrated by sequential incubation in ethanol 50-100% and propylene oxide, embedded in epoxy resin and polymerized at 62 • c for 72 h. semi (500 nm) and ultra-thin (50-70 nm) sections, were obtained using a leica em uc7 ultramicrotome. uultra-thin sections were collected onto plastic-coated nickel grids and immunochemically contraststained with uranium salts (uranyl acetate) and lead citrate to reveal cell ultrastructure. finally, samples were analyzed using a transmission electron microscope em902a fa.zeiss with the item soft imaging system software (olympus). untransduced jurkat cells and pbmcs cultured as was previously described, were collected, counted, washed with pbs and 10 6 cells were incubated 30 min with 100 µl lysis buffer (50 mm tris-hcl ph 7.4, 150 mm nacl, 1% nonidetp-40, 1 mm edta, 1 mm phenylmethylsulfonyl fluoride, 1 mm na 3 vo 4 , 1 mm naf, on ice. cell debris was discarded by centrifugation at 12,000 g for 20 min, 4 • c, and soluble proteins were stored at −80 • c before further analysis. protein levels were measured using bradford protein assay kit (pierce), according to manufactures recommendations. two hundred microliter magnetic beads (dynabeads r myone tm streptavidin t1 invitrogen) were washed following manufacturer's recommendations and conjugated with hcv core genotype-1b biotin-prospec (hcv-242) by incubating 100 µl (10 mg/ml) dynabeads with 100 µl (800 pmoles) hcv core protein in pbs, for 2 h at 4 • c, with gentle rotation. hcv core protein-coated beads were washed three times by resuspension in pbs containing 0.02% twin-20 and decanting the supernatant while placed on a magnet. for mass spectrometry analysis, coated dynabeads (or uncoated as control for unspecific binding) were incubated overnight, at 4 • c, with jurkat cells protein lysates, with gentle rotation, in the following ratio: 100 µl of coated beads with 150 µg of total protein lysate. for western blot experiments, coated dynabeads were incubated overnight, at 4 • c, with protein lysates from pbmc blasts, with gentle rotation, in the following ratio: 50 µl coated beads with 3 mg protein sample. 50 µl uncoated beads were incubated with 3 mg protein lysate, in the same conditions, as a control of unspecific binding. samples were washed twice with pbs and once with ultrapure water and incubated with 150 µl of 200 mm dithiothreitol (dtt) in 50 mm ammonium bicarbonate for 5 min at 65 • c. eluated proteins were quantified and 100 µg were precipitated with acetone during 1 h at −20 • c and centrifuged 30 min at 12,000 g. subsequently, proteins were resuspended in urea 6 m, reduced with 2 mm dtt in 100 mm ammonium bicarbonate at room temperature for 45 min and alquilated with 4 mm iodacetamide 45 min, in the dark, subsequently trypsin digestion was performed with sequencing grade trypsin (promega, madison, wi, usa) at 37 • c overnight (enzyme/substrate ratio 1:50). finally, the digestion was quenched adding 5% formic acid. the resulting peptides were transferred to a clean tube, dried in speed-vac and stored at −20 • c prior to analysis by mass spectrometry (ms). peptides were resolved by reverse phase chromatography using an eksigent ultra pump fitted with a 75 µm id column 25 cm length (acclaim pepmap 100 c18 2 µm 100 a). samples were initially loaded for desalting into a 2 cm length 100 µm id precolumn packed with the same chemistry as the resolving column prior to analytical chromatography. mobile phases used were 100% water 0.1% formic acid (buffer a), 100% acetonitrile 0.1% formic acid (buffer b). gradient was developed during 95 min up to 35% b. column was equilibrated in 95% b for 10 min and 5% b for 15 min, using a constant flow of 300 nl/min. peptides eluted from the column were analyzed using a sciex 5600 tripletof tm system. data dependent acquisition was collected upon a survey scan performed in a mass range from 350 to 1,250 m/z in a scan time of 250 ms. the top 25 most intense precursors were selected for fragmentation with a transmission window width of 0.7 da. minimum accumulation time for ms/ms was set to 75 ms giving a total cycle time of 2,125 ms. fragment ions were collected in a mass range from 230 to 1,500 m/z in order to have a single q2 transmission window. precursors were excluded from further fragmentation during 15 s. raw data files were processed using proteinpilot tm 4.5 software from sciex and mgf files were loaded onto proteinscape tm 3.1 software (bruker) for data grouping and protein identification via mascot program version 2.5 (matrix science ltd, london, uk). search parameters were: homo sapiens taxonomy. trypsin specificity was used and only one missed cleavage allowed, cysteine carbamidomethylation as fixed and oxidized methionine was used as variable modification. mass tolerance of precursor ion and fragment ions were 10 ppm and 0.05 da, respectively. peptides charges of 1+, 2+, and 3+ were selected and minimum peptide length was set at five residues. peptides and proteins were considered positively identified if their mascot score was higher than 20 and 30, respectively. proteins were identified with the minimum of one peptide and a false discovery rate <1%. proteins identified with one single peptide were only included if peptide scores were significant, and peptide spectra contained most "y" and/or "b" representative ions for the corresponding peptide sequence. examples are included in supplementary table 2 . subsequently, we compared the identified proteins that bound to the dynabeads vs. the ones identified binding to hcv core protein using the "compare results" option from proteinscape software 3.1. thus, 222 proteins were found to specifically associate with hcv core protein. proteome profiling data has been deposited in the peptideatlas repository, identified as pass00982. samples obtained from the pull down assay were washed twice with pbs using the magnet, and incubated with 2x laemmli buffer at 99 • c for 5 min. subsequently, beads were removed from the proteins using a magnet and proteins resolved by sds-page and transferred to pvdf membranes (immobilon-fl). membranes were incubated with the corresponding primary antibody, followed by the appropriated secondary antibody, conjugated to hrp. primary antibodies used were pp1 gamma antibody (pa5-21671) from thermo fisher scientific r and mypt1 antibody (#2634) from cell signaling technology r . reactive proteins were visualized using an ecl system and images were acquired in a chemidoc touch imaging system (bio-rad r laboratories, hercules, ca, usa). localization and network analysis of identified candidate proteins associated with hcv core protein string database version 10 (search tool for the retrieval of interacting genes/proteins) (http://string-db.org) (szklarczyk et al., 2015) was applied to visualize detailed information about the subcellular localization and the main interactions of the identified proteins. ingenuity pathways analysis (ipa; ingenuity systems, redwood city, calif.) software was used for further protein characterization according to known and predicted associations into function canonical pathways and networks recorded in the ipa library. nuclear localization sequence (nolss) in hcv core protein prediction was obtained by entering a protein sequence in fasta format on the nod web server http://www.compbio.dundee.ac. uk/www-nod/ (scott et al., 2011) . nolss were predicted if the average score output by the artificial neural network ann of 8 consecutive windows was at least 0.8 (scott et al., 2010 (scott et al., , 2011 . to analyze the localization of hcv core protein in t cells and to circumvent the lack of optimal anti-hcv core antibodies, we took advantage of a gfp-hcv core fusion construct that has been extensively used in our laboratory (dominguez-villar et al., 2007 , 2012a fernandez-ponce et al., 2014) and an unfused gfp expressing construct as a control. jurkat cells were efficiently transduced with lentiviral vectors expressing hcvcore-gfp or gfp as a control. percentage of transduced cells analyzed by flow cytometry was >98% in all cases (data not shown). jurkat cells transduced with gfp or hcv core gfp-expressing lentiviral construct or left untransduced were subsequently immunostained with anti-gfp antibody and a 10 nm gold-labeled anti-rabbit igg and analyzed by transmission electron microscopy. as shown in figure 1 , hcv core was mostly localized in the nucleus (figures 1b,d,e) and specifically in the nucleolus where it was greatly enriched (figures 1b-f) , although some immunostaining was observed in cytoplasm ( figure 1a) . hcv-core-gfp was detected in the nucleus in 35% out of 40 hcv core gfp expressing jurkat cells analyzed, 22% showed the presence of gfp inside both nucleus and cytoplasm, 5% of the cells only in cytoplasm (figure 1a) , while 18% showed gfp immunolabelling in nucleus with enrichment in the nucleolus (figures 1b-f) . 20% of the cells were not stained. in hcv core expressing jurkat cells which showed core protein nucleolar localization, the total raw number of gold particles counted inside the nucleoli was 196, with an average of 28 gold particles per cell and a standard deviation of 13.3. regarding gfp transduced control cells, gfp was localized in the nucleus of 84% from 40 gfp-expressing jurkat cells analyzed and in both nucleus and cytoplasm in 16%. there was no recognizable co-localizing with any organelle. nucleolus localization was not visible in any cell (figures 1g,h) . no immunogold staining was observed in untransduced jurkat cells (data not shown). thus, the study revealed specific immunolabeling of hcv core protein in the nucleolus of jurkat cells. based on the findings obtained with the electron microscopy assay, we decided to identify hcv core associated proteins that could mechanistically be correlated with hcv core protein nucleolar localization. thus, we performed pull down experiments using biotinilated hcv core protein as bait. further analysis with the string platform allowed us to classify the identified proteins depending on their localization (figure 2) . thus, from the 222 associated proteins identified (supplementary table 1 ), 128 have been previously described to localize in the nucleus (figure 2a) and 43 in the nucleolus (figure 2b) , while most of the 150 proteins described in the cytoplasm (figure 2c ) have also been identified in the nucleus and/or nucleolus. string also allowed us to determine potential protein functionalities according to previously published reports. interestingly, most of the identified hcv core associated proteins were found to participate in binding processes, as indicated in figure 2 . regarding protein function, we mainly focused on the functional classification given by the ipa software, which, based on biomedical literature and integrated databases, allows to determine the most probable pathways and/or functions in which identified proteins are involved. thus, ipa core analysis of our dataset (figure 3 ) revealed that the identified hcv core associated proteins were mainly described to participate in splicing and processing of rna, cell cycle progression, cell proliferation, apoptosis and rna virus infection. our findings support the concept that the subcellular localization of hcv core protein might have an impact on cd4 + t cells functions. in order to confirm, by an alternative method, some of the associations identified by mass spectrometry analysis, and most importantly to evaluate whether such associations were also present in primary t cells (pbmc), thus enhancing the relevance of our findings. postnuclear lysates from human pbmcs blasts (see methods), were subjected to hcv core protein pull down, using a biotinilated hcv core protein (800pmoles) bound to magnetic beads as a bait. uncoated magnetic beads were used as a control. experiments were first carried out in jurkat cells and inputs from jurkat and pbmc were loaded in parallel (1.5% from the amount used per pull down), to evaluate the relative amount of each target protein (data not shown). serine/threonine-protein phosphatase was selected due to its known localization (it has a dynamic and predominantly nucleolar distribution) and to its function (it has been implicated in the regulation of several biological pathways previously described in t cells transduced with hcv core protein) (aggen et al., 2000; ceulemans and bollen, 2004; nie et al., 2013) . it was also selected based on the fact that both its catalytic subunit (pp1γ) and its regulatory subunit 12 a (mypt1), were identified by mass spectrometry, showing a despairing mascot score of 37.6 and 336.5 respectively. as shown in figure 4 , western blot analysis of (pp1γ) and (mypt1), confirmed the presence of both frontiers in microbiology | www.frontiersin.org figure 2 | "actions" view, according to the "go cellular components" distribution option, in order to identify their subcellular localization. string analysis reported that proteins were nuclear (a) (128 from 222), nucleolar (b) (43 from 222), and cytoplasmic (c) (150 from 222). colored lines between proteins indicate the type of evidence for each interaction, with a minimum required confidence score of 0.4. proteins in pbmcs lysates obtained from the pull down of hcv core protein coated magnetic beads (figure 4) . in order to identify potential nucleolar localization sequences in hcv core protein, we used the nod web server (http://www. compbio.dundee.ac.uk/www-nod/), a program that predicts the presence of nolss in eukaryotic and viral proteins, based on a database of 46 statistically analyzed human nucleolar localization sequences (scott et al., 2011) . two nolss were identified in hcv core protein ( several biological and immunological consequences of hcv core intracellular expression in cd4 + t cells have previously been described by us and others, (bergqvist and rice, 2001; bergqvist et al., 2003; dominguez-villar et al., 2007 , 2012a doumba et al., 2013; fernandez-ponce et al., 2014) . these findings suggest an important role for the intracellular presence of hcv core in hcv pathogenesis and chronification. in this study, we have analyzed the ultrastructural localization of hcv core protein in cd4 + t cells. according to studies using cell lines unrelated to the immune system, hcv core protein has been shown to localize in the endoplasmic reticulum, mitochondrial outer membrane and nucleus of human embryonic kidney 293t cells (suzuki et al., 2005) ; associated to lipid droplets in cho, hepg2 and huh7 cells lines (barba et al., 1997; boulant et al., 2006; qiang and jhaveri, 2012) , in the cytoplasm, endoplasmic reticulum, in the proximity of the nuclear membrane, in the nucleus and nucleoli of hepatocytes isolated from chronically hcv infected patients (falcon et al., 2003) ; and in the cytoplasm and nucleus of non-parenchymal liver cells such as, lymphocyte-like cells, kupffer-like cells, polymorphonuclear-like cells, pit, endothelial, stellate, and fibroblast-like cells isolated from livers of chronically hcv infected patients (falcon et al., 2005) . in the present work, we found that in cd4 + t lymphocytes, hcv core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched and mainly organized in clusters (figure 1) . regarding these findings, nuclear localization signals (nlss) described previously in hcv core protein, could be responsible for nuclear localization (chang et al., 1994; suzuki et al., 1995 suzuki et al., , 2005 , while hcv core protein traffic and residence in the nucleolus, could be explained by the presence of two nucleolar localization sequences (nolss), identified using the bioinformatic nucleolar localization sequence detector web server for eukaryotic and viral proteins (scott et al., 2011) . nolss are showed in figure 4 . the statistical variation in core protein nucleolar localization shown by hcv-core expressing cells, could be due to differences in cellular cycle stage, as it has been described for other nucleolar resident proteins (chen and huang, 2001; stoldt et al., 2007; pirlot et al., 2016) . presence of nolss in cellular or viral proteins is a key factor in their dynamic traffic and residence within the nucleolus. presumably, nolss interact with nucleolar proteins and/or rna to mediate nucleolar targeting and retention. thus, in chimeric viral proteins with mutated nolss, trafficking to the nucleolus is abrogated, and heterologous nolss insertion restores their trafficking pattern (boyne and whitehouse, 2006; emmott et al., 2008) . interestingly, the function of hcv core protein inside the virus, can partly explain its subcellular localization as hcv core protein mainly interacts with hcv genomic rna, multimerizing around it and forming the capsid shell. while multimerizing inside the virus, we have not seen any multimerization in our experiments, which is in agreement with several studies showing that mammalian cell lines fail to produce capsid assembly (bukh et al., 2002; pietschmann et al., 2002; polyak et al., 2006; rouille et al., 2006; hourioux et al., 2007) , due to the lack of host cells factors that are essential for hcv core multimerization and assembly, or to the presence of inhibitory factors that induce the majority of hcv-core to be targeted away from the er (hope and mclauchlan, 2000; mclauchlan et al., 2002; polyak et al., 2006) . thus, un-multimerized hcv core protein traffics to alternate subcellular compartments. hcv core has been shown to bind rnas in addition to hcv genomic rna (kunkel et al., 2001; cristofari et al., 2004) , including ribosomal rna (santolini et al., 1994) and trna (kunkel et al., 2001) . since the nucleolus contains several 100 copies of rrna genes and it could be a recruiting site for trnas (carmo-fonseca et al., 2000) it is likely that the nucleolar rna constitutes another important molecular target structure for hcv core protein. in agreement with our findings, several dna viruses, retroviruses and rna viruses, as well as viral proteins, have been described to traffic to the nucleolus and be associated with nucleolar proteins wurm et al., 2001; chen et al., 2002; dove et al., 2006; michienzi et al., 2006; cawood et al., 2007; hiscox, 2007; emmott et al., 2010; lam et al., 2010; jarboui et al., 2012) . the nucleolus is a dynamic nuclear organelle whose proteome is continuously changing. it seems to be a temporary storage or a sequestration site for a multiplicity of proteins (emmott and hiscox, 2009; hiscox et al., 2010) . functionally, there is extensive evidence that the nucleolus is implicated in ribosome biogenesis (stoykova et al., 1985; warner, 1990; scheer et al., 1993) , cell cycle regulation, cell growth, senescence, stress response signaling (andersen et al., 2005; emmott and hiscox, 2009; tsai and pederson, 2014; lam and trinkle-mulcahy, 2015) and the pathogenesis of several diseases such as cancer (james et al., 2014; orsolic et al., 2015; yang et al., 2015) , cardiovascular disease (hariharan and sussman, 2014) and neurodegenerative disorders (payao et al., 1994; lu et al., 1998; rieker et al., 2011; tsoi and chan, 2013; lee et al., 2014a; parlato and liss, 2014; hernandez-ortega et al., 2015) . viral protein trafficking and localization into the nucleolus has shown implications in both viral life cycle and in host cell physiology and it has been narrowly related with the loss of essential nucleolar functions (hiscox, 2002) . in addition, it has been shown that accumulation of viral proteins in the nucleolus can cause volume exclusion and crowding effects, disrupting the nucleolar architecture (hancock, 2004; hiscox, 2007) . virus infection and some viral proteins from poliovirus, avian infectious bronchitis virus (ibv), coronavirus and human immunodeficiency virus-1 (hiv-1) induce disruption of the nucleolar architecture and changes on the subcellular distribution of nucleolar proteins or proteins that traffic to the nucleolus, such as nucleolin, p53, b23.1 (waggoner and sarnow, 1998; hiscox, 2002; dove et al., 2006) . these findings are closely related to the presence of perturbations in cell cycle, cytokinesis and apoptosis in the host cells (miyazaki et al., 1996; chen et al., 2002; galati et al., 2003; hiscox, 2007) . semliki forest virus nucleocapsid migrates to the nucleolus (jakob, 1994) and porcine reproductive and respiratory syndrome virus nucleocapsid specifically interacts with the small nucleolar rna (snorna)-associated protein, fibrillarin in virus infected cells (rowland et al., 1999; yoo et al., 2003) , while hepatitis b virus (hbv) core protein usually co-localizes with the nucleolar proteins, nucleolin and b23 (ning and shih, 2004) . in addition, it has been shown that coronavirus nucleocapsid protein localization in the nucleolus of infected cells and its association to the nucleolar protein b23.1 is related to cell cycle stage and could be involved in cell cycle delay or arrest to promote virus replication wurm et al., 2001; cawood et al., 2007) . thus, trafficking to the nucleolus and nucleolar residency time of hcv core protein in cd4 + t cells could be narrowly related with previous findings from us and others showing that the intracellular presence of hcv core in cd4 + t cells induces decreasing cell proliferation, delay in cell cycle progression and a differential expression pattern of genes with relevant function, including anergy-associated genes, genes involved in cytoskeleton reorganization, vesicle trafficking, endocytosis, cytokines production, cell death, transcription, and translation (dominguez-villar et al., 2007) . in agreement with the described localization, we found hcv core protein association with several nuclear, nucleolar proteins or proteins described to traffic to the nucleolus, which showed mainly binding connections (figure 2) . the wide range of proteins associated with hcv core, correlate with the findings obtained by dolan et al. who identified two computationally predicted molecular recognition features within the n-terminal intrinsically disordered region (idr) in the hcv core sequence. the identified molecular recognition features, mediate hcv core protein binding to hcv rna and to multiple host proteins, suggesting that hcv core protein exhibits hub protein properties (dolan et al., 2015) . interestingly, pathway and network-based analysis of proteins identified to be associated with hcv core protein, indicate that a wide range of these proteins are involved in several biological signaling pathways, such as, rna processing and splicing (figure 3a) , cell cycle progression, cell proliferation, apoptosis ( figure 3b ) and infection and replication of rna viruses, including hcv ( figure 3c) . with the integrated analysis of the present data, we confer a better description of the hcv core -human t lymphocyte relationship. concerning ribosomal biogenesis, cellular proliferation, cell cycle and apoptosis; several large (rpl) and small (rps) ribosomal proteins, and proteins involved in rna processing and splicing as dead-box rna helicases, precipitated with figure 5 | predicted nucleolar localization sequences in hcv core protein. graph obtained from the nucleolar localization sequence detector web server (nod), displaying nols prediction score for each residue of hcv core protein. pink shaded regions represent the range of scores within which a 20-residues segment is predicted to be a nols. thus, pink shaded regions represent the nols candidate segment, which highlights scores above 8.0. hcv core (figures 3a,b) (rocak and linder, 2004; xu et al., 2016) . interactions between viral and ribosomal proteins, splicing factors and dead-box rna helicases including ddx5 and ddx17, have been previously described and suggested as a key mechanism for viral replication and production as well as for life cycle progression and survival (bortz et al., 2011; naji et al., 2012; yasuda-inoue et al., 2013; cervantes-salazar et al., 2015; klymenko et al., 2016; li et al., 2016) . implication of other identified proteins, such as protein red (ik) and filamin a (flna) (figure 3b ) in proliferation and cell cycle progression, have also been widely demonstrated (lee et al., 2014b; sun et al., 2014) . thus, associations of hcv core protein in t cells could alter the function of the associated proteins, explaining some of the effects shown for hcv-core expression, including cell cycle delay and inhibition of cell proliferation (dominguez-villar et al., 2007 , 2012a fernandez-ponce et al., 2014) . in addition, we found that many host proteins associated with hcv core, are involved in replication and infection of rna viruses including hcv ( figure 3c) . proteins as dexd/hbox helicases have been extensively studied in virus infection and have been described as proteins hijacked by viruses for their benefit. specifically, dhx9 is involved in virus replication, innate immunity response to viral dsrna and participation in the expression of ifn-stimulated genes (fullam and schroder, 2013) . rna binding proteins (rbps) such as the host poly(rc)-binding protein (pcbp) and different heterogeneous nuclear ribonucleoproteins (hnrps), are also interesting as they stabilize viral rnas, co-localize with the viral replicase complex and facilitate viral rna template selection (li and nagy, 2011) . in addition, interleukin enhancer-binding factor 3 (ilf3) has been shown to be recruited to hcv replication complexes (li et al., 2014) and in other infections by rna viruses, ilf3 interaction with viral proteins has been described to affect virus replication among other virus life cycle stages (patino et al., 2015) . furthermore, the subcellular localization of hcv core and its association with nuclear and nucleolar proteins found in the present study, can aid in explaining the cd4 + t cell regulatory/exhausted phenotype described by us and others, in cd4 + t cells expressing hcv core protein (dominguez-villar et al., 2012a; doumba et al., 2013; fernandez-ponce et al., 2014) . some such associations are with serine/threonine-protein phosphatase catalytic subunit (pp1γ), protein phosphatase 1 regulatory subunit 12a (mypt1) (nie et al., 2013) , interleukin enhancer-binding factor 3 (ilf3) (shi et al., 2007a,b) , complement component c1q binding protein (c1qbp) (kittlesen et al., 2000) and runt-related transcription factor 1 (runx1) (klunker et al., 2009) . in conclusion, analysis of the association of hcv core with host proteins in cd4 + t cells and the study of its ultrastructural localization, open an extensive field of study poised to understand the mechanisms underlying functional findings previously described in cd4 + t cells expressing hcv core protein, that have demonstrated to be relevant for hcv immune system evasion and thus hcv chronification. conception, design and or interpretation of the work: fg-c, ea, and rl. wb (for localization studies). cb and md-r (for association studies). performed association studies: cf-p, md-r, and as-s. performed localization studies: cf-p, jm-m, and ma-e. run statistical analyses: rl and cf-p. wrote the paper: fg-c, cf-p, ea, rl, and md-r. performed confirmation co-ip experimentos: in-s and cf-p. all authors participated in critical revision and subsequently approved the manuscript. all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. for ea. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. regulation of protein phosphatase-1 nucleolar proteome dynamics hepatocellular carcinoma: role of hepatitis b and hepatitis c viruses proteins in hepatocarcinogenesis hepatitis c virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets transcriptional activation of the interleukin-2 promoter by hepatitis c virus core protein the hepatitis c virus core protein modulates t cell responses by inducing spontaneous and altering t-cell receptor-triggered ca2 + oscillations the conserved n-terminal domain of herpes simplex virus 1 ul24 protein is sufficient to induce the spatial redistribution of nucleolin host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins structural determinants that target the hepatitis c virus core protein to lipid droplets nucleolar trafficking is essential for nuclear export of intronless herpesvirus mrna mutations that permit efficient replication of hepatitis c virus rna in huh-7 cells prevent productive replication in chimpanzees to be or not to be in the nucleolus cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein dengue virus ns1 protein interacts with the ribosomal protein rpl18: this interaction is required for viral translation and replication in huh-7 cells functional diversity of protein phosphatase-1, a cellular economizer and reset button nuclear localization signals in the core protein of hepatitis c virus nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell the hepatitis c virus protein ns3 suppresses tnf-α-stimulated activation of nf-κb by targeting lubac the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral(+) strand rna in vitro intrinsic disorder mediates hepatitis c virus core-host cell protein interactions up-regulation of foxp3 and induction of suppressive function in cd4 + jurkat t-cells expressing hepatitis c virus core protein hepatitis c virus core protein up-regulates anergyrelated genes and a new set of genes, which affects t cell homeostasis phenotypic and functional alterations of primary human pbmcs induced by hcv non-enveloped capsid-like particles uptake changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus flying under the radar: the immunobiology of hepatitis c nucleolar targeting: the hub of the matter viral nucleolar localisation signals determine dynamic trafficking within the nucleolus quantitative proteomics using silac coupled to lc-ms/ms reveals changes in the nucleolar proteome in influenza a virus-infected cells nuclear localization of nucleocapsid-like particles and hcv core protein in hepatocytes of a chronically hcv-infected patient hcv core protein localizes in the nuclei of nonparenchymal liver cells from chronically hcv-infected patients cd4 + primary t cells expressing hcv-core protein upregulate foxp3 and il-10, suppressing cd4 and cd8 t cells immune modulation by the hepatitis c virus core protein dexd/h-box rna helicases as mediators of anti-viral innate immunity and essential host factors for viral replication specific changes in the posttranslational regulation of nucleolin in lymphocytes from patients infected with human immunodeficiency virus hcv core protein and virus assembly: what we know without structures global epidemiology and genotype distribution of the hepatitis c virus infection a role for macromolecular crowding effects in the assembly and function of compartments in the nucleus stressing on the nucleolus in cardiovascular disease altered machinery of protein synthesis in alzheimer's: from the nucleolus to the ribosome the nucleolus-a gateway to viral infection? rna viruses: hijacking the dynamic nucleolus nucleolar proteomics and viral infection the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis c virus core protein core protein domains involved in hepatitis c virus-like particle assembly and budding at the endoplasmic reticulum membrane nucleolar accumulation of semliki forest virus nucleocapsid c protein: influence of metabolic status, cytoskeleton and receptors nucleolar stress with and without p53 nucleolar protein trafficking in response to hiv-1 tat: rewiring the nucleolus interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation transcription factors runx1 and runx3 in the induction and suppressive function of foxp3 + inducible regulatory t cells human papillomavirus e2 regulates srsf3 (srp20) to promote capsid protein expression in infected differentiated keratinocytes immunomodulation by hepatitis c virus-derived proteins: targeting human dendritic cells by multiple mechanisms self-assembly of nucleocapsid-like particles from recombinant hepatitis c virus core protein new insights into nucleolar structure and function. f1000prime rep proteomics analysis of the nucleolus in adenovirus-infected cells nucleolar dysfunction in huntington's disease depletion of ik causes mitotic arrest through aberrant regulation of mitotic kinases and phosphatases rabies virus phosphoprotein interacts with ribosomal protein l9 and affects rabies virus replication hnrnp l and nf90 interact with hepatitis c virus 5 ′ -terminal untranslated rna and promote efficient replication diverse roles of host rna binding proteins in rna virus replication research on nucleolar organizer regions of hippocampal neuron in alzheimer's disease nonenveloped nucleocapsids of hepatitis c virus in the serum of infected patients intramembrane proteolysis promotes trafficking of hepatitis c virus core protein to lipid droplets a nucleolar localizing rev binding element inhibits hiv replication the post-transcriptional regulator rev of hiv: implications for its interaction with the nucleolar protein b23 dimerization-driven interaction of hepatitis c virus core protein with ns3 helicase host cell interactome of hiv-1 rev includes rna helicases involved in multiple facets of virus production phosphorylation of foxp3 controls regulatory t cell function and is inhibited by tnf-α in rheumatoid arthritis nucleolar localization of human hepatitis b virus capsid protein the relationship between the nucleolus and cancer: current evidence and emerging paradigms how parkinson's disease meets nucleolar stress nf90 isoforms, a new family of cellular proteins involved in viral replication? investigation of the nucleolar organizer regions in alzheimer's disease persistent and transient replication of full-length hepatitis c virus genomes in cell culture melanoma antigen-d2: a nucleolar protein undergoing delocalization during cell cycle and after cellular stress chapter 3 assemble and interact: pleiotropic functions of the hcv core protein the hiv tat protein affects processing of ribosomal rna precursor lipid droplet binding of hepatitis c virus core protein genotype 3. isrn gastroenterol localization, quantification and interaction with host factors of endogenous htlv-1 hbz protein in infected cells and atl nucleolar disruption in dopaminergic neurons leads to oxidative damage and parkinsonism through repression of mammalian target of rapamycin signaling dead-box proteins: the driving forces behind rna metabolism subcellular localization of hepatitis c virus structural proteins in a cell culture system that efficiently replicates the virus the localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence biosynthesis and biochemical properties of the hepatitis c virus core protein characterization and prediction of protein nucleolar localization sequences nod: a nucleolar localization sequence detector for eukaryotic and viral proteins nf90 regulates inducible il-2 gene expression in t cells dynamic binding of ku80, ku70 and nf90 to the il-2 promoter in vivo in activated t-cells g1 phase-dependent nucleolar accumulation of human histone h1x ribosome biogenesis and nucleolar ultrastructure in neuronal and oligodendroglial rat brain cells interactions between filamin a and mmp-9 regulate proliferation and invasion in renal cell carcinoma. asian pac nuclear localization of the truncated hepatitis c virus core protein with its hydrophobic c terminus deleted molecular determinants for subcellular localization of hepatitis c virus core protein string v10: protein-protein interaction networks, integrated over the tree of life hepatitis c viruses genomes and molecular biology connecting the nucleolus to the cell cycle and human disease expression of expanded cag transcripts triggers nucleolar stress in huntington's disease hcv core and ns3 proteins manipulate human bloodderived dendritic cell development and promote th 17 differentiation hcv core protein interaction with gc1q receptor inhibits th1 differentiation of cd4 + t cells via suppression of dendritic cell il-12 production viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells the nucleolus and ribosome formation localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division the role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity nucleolar repression facilitates initiation and maintenance of senescence direct binding of hepatitis c virus core to gc1qr on cd4 + and cd8 + t cells leads to impaired activation of lck and akt distinct ddx deadbox rna helicases cooperate to modulate the hiv-1 rev function colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin interaction of avian influenza virus ns1 protein and nucleolar and coiledbody phosphoprotein 1 we would like to thank christian hoffmann, evelyn janssen, beatrix martiny, jessicahausmann, mojgan ghilav and consuelo rivera for their excellent technical support, the plataforma andaluza de bioinformática (centro de supercomputación y bioinformática, university of málaga) for the use of the ingenuity pathways analysis (ipa) software and the core biomedical research facility of the university of cadiz for the use of core infrastructure. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2017.02595/full#supplementary-material key: cord-334493-rt7gqiev authors: ikejiri, masahiro; ohshima, takayuki; kato, keizo; toyama, masaaki; murata, takayuki; shimotohno, kunitada; maruyama, tokumi title: 5′-o-masked 2′-deoxyadenosine analogues as lead compounds for hepatitis c virus (hcv) therapeutic agents date: 2007-11-15 journal: bioorg med chem doi: 10.1016/j.bmc.2007.08.025 sha: doc_id: 334493 cord_uid: rt7gqiev on the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (sars) coronavirus, a series of nucleoside analogues whose 5′-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate, and ether were synthesized and evaluated to develop novel anti-hepatitis c virus (hcv) agents. among these, several 5′-o-masked analogues of 6-chloropurine-2′-deoxyriboside (e.g., 5′-o-benzoyl, 5′-o-p-methoxybenzoyl, and 5′-o-benzyl analogues) were found to exhibit effective anti-hcv activity. in particular, the 5′-o-benzoyl analogue exhibited the highest potency with an ec(50) of 6.1 μm in a cell-based hcv replicon assay. since the 5′-o-unmasked analogue (i.e., 6-chloropurine-2′-deoxyriboside) was not sufficiently potent (ec(50) = 47.2 μm), masking of the 5′-hydroxyl group seems to be an effective method for the development of anti-hcv agents. presently, we hypothesize two roles for the 5′-o-masked analogues: one is the role as an anti-hcv agent by itself, and the other is as a prodrug of its 5′-o-demasked (deprotected) derivative. the hepatitis c virus (hcv), a member of the family flaviviridae, is an enveloped, positive-sense, singlestrand rna virus. 1 the virus is a major causative agent of non-a, non-b hepatitis and infects an estimated 170 million people worldwide. 2 in most cases, acute phase infection with hcv is asymptomatic; however, the virus frequently establishes chronic hepatitis in up to 80% of the infected individuals and persists for decades with a substantially high risk of developing liver cirrhosis and hepatocellular carcinoma. 3 currently, there is no vaccine available against hcv, and the approved drugs are combinations of interferon-a (ifn-a) and ribavirin (1-b-d d-ribofuranosyl-1h-1,2,4-triazole-3-carboxamide). although the use of pegylated ifn-a instead of the unmodified one has resulted in improved therapeutic effectiveness, the sustained virological response is still poor (41-55%); moreover, in some cases, significant side effects can be caused. 4 therefore, more efficacious therapies are urgently required for ensuring public health. 5 to date, a number of nucleoside analogues, including ribavirin, have been synthesized and tested for anti-hcv activity. 6, 7 among these, some 2 0 -modified ribonucleoside analogues such as 2 0 -c-methyl analogues 1, 6a 2 0 -fluoro analogues 2, 6b and 2 0 -o-methyl analogue 3 6c exhibit potent anti-hcv activities in a cell-based hcv replicon assay (fig. 1) . particularly, valopicitabine, a prodrug of 1c, is currently in phase ii clinical trials. 8 recently, the anti-hcv properties of 4 0 -substituted ribonucleosides such as 4 have also been reported by the roche group. 6d concerning the antiviral mechanism of common nucleoside analogues, it is known that most of these analogues are converted to their corresponding 5 0triphosphates by cellular and/or viral enzymes and then compete with natural triphosphates as substrates for incorporation into viral nucleic acids during viral replication. 9 therefore, the 5 0 -hydroxyl group is indispensable to the anti-hcv activity of these nucleoside analogues. 10 recently, we have reported that a carbocyclic oxetanocin analogue 5, one of whose hydroxyl groups (that corresponds to the 5 0 -hydroxyl group of the ribonucleoside) was masked by a benzoyl group, showed antiviral activity against the severe acute respiratory syndrome (sars) coronavirus (fig. 2 ). 11 interestingly, the antiviral activity of 5 (ec 50 = 14.5 lm) was much more efficacious than that of the unmasked 6 (almost no activity, ec 50 > 300 lm), which seems to indicate an inconsistency with the necessity of the 5 0 -hydroxyl group to this compound. this result attracted our interest and led us to expect that this unique trend could contribute to developing anti-hcv agents as well. thus, several nucleoside analogues, including 5 and 6, were designed as candidate compounds for the agents (fig. 3) . in this paper, we report the syntheses of these nucleoside analogues and their biological evaluations as anti-hcv agents. compounds 7-9, 11, 13, 14, 22 , and 23 were prepared based on the previous reports. 12 the syntheses of com-pounds 10, 12, and 15-19 were carried out by the treatment of diol 9 or 11 with the corresponding acyl or sulfonyl chloride (i.e., benzoyl chloride for 10 and 12; pivaloyl chloride, butyryl chloride, p-methoxybenzoyl chloride, 2,4,6-trimethylbenzoyl chloride, and benzenesulfonyl chloride for 15-19, respectively), as shown in scheme 1. in the case of compound 18, conventional conditions (trimethylbenzoyl chloride, dmap, and pyridine) required a long reaction time and led to the gradual decomposition of 9 and 18, probably because the 6-chloropurine moiety reacted with the amine bases by degrees due to its electrophilic nature. this side reaction was slightly suppressed by the use of a lownucleophilic base, n,n-diisopropylethylamine instead of pyridine and dmap. compound 10 was also prepared via an alternative route illustrated in scheme 2a. regioselective protection of the 5 0 -hydroxyl group of 2 0 -deoxyadenosine (26) yielded the benzoate 23 in 65% yield, 12c which was subsequently converted to the silyl ether 27 in 93% yield. after transformation to the 6-chloropurine derivative 28 with a 58% yield, 13 the tbs group was cleaved to afford 10 in 72% yield. compound 28 was also used as an intermediate in the synthesis of 25, as shown in scheme 2b. exposure of 28 to sodium methylthiolate resulted in the formation of the debenzoylated 6-sme analogue 29 with a 67% yield, which was reacylated into 30 in 97% yield. unfortunately, the use of thiourea in place of sodium methylthiolate led to the decomposition of the resultant products. finally, the removal of the tbs group afforded 25 in 88% yield. the synthesis of 24 is illustrated in scheme 2c. the diacetate 31, which was prepared as described in a previous report, 11 was treated with dimethylamine to furnish 32 in 62% yield. 14 subsequently, regioselective benzoylation of the 5 0 -hydroxyl group in 32 afforded 24 in 77% yield. meanwhile, the syntheses of 20 and 21 turned out to be unexpectedly difficult because of the instability of these molecules under both strongly basic and acidic conditions; for example, the exposure of 9 to bnbr-nah or bnoc(@nh)ccl 3 -cf 3 so 3 h afforded a complex mixture. thus, a stepwise protection-deprotection process was required, which is illustrated in scheme 3 (3a: synthesis of 20, 3b: synthesis of 21). the monoacetate 33, which was prepared from 26 by employing a partly modified santaniello's procedure, 15 was converted to the silyl ether 34 in 94% yield. the use of a mixed solvent (pyridine-dmf) effectively reduced the competitive silylation of the 6-amino group. protection of the amino group with pivaloyl chloride followed by the removal of the acetyl group in 35 afforded 36 with a 98% yield in two steps. the addition of dmap and meoh in the n-pivaloylation step was effective in converting the n,n-dipivaloylated by-product to the desired 35. chemoselective benzylation of the 5 0 -hydroxyl group in 36 was successfully carried out by using 2.5 equiv of potassium tert-butoxide and 1.1 equiv of benzyl bromide in thf to provide 37 in 97% yield. after the removal of the pivaloyl group (76% yield), the resultant amino group of 38 was substituted with a chloro group to provide 39, which was subsequently deprotected to afford 20 with a 34% yield in two steps. compound 21 was synthesized employing almost the same method as that for 20 described above (scheme 3b, 20% overall yield from 36). the above synthesized nucleoside analogues were assayed for their ability to inhibit hcv rna replication in a subgenomic replicon huh7 cell line (lucneo#2), 16 and the result is shown in table 1 . these cells contain a hcv subgenomic replicon rna encoding a luciferase reporter gene as a marker. the potency of the analogues against the hcv replicon is expressed as ec 50 , which was quantified by a luciferase assay after a two-day incubation period with the corresponding compound. in addition, the associated cytotoxicity, which is expressed as cc 50 in table 1 , was evaluated in a tetrazolium (xtt)-based assay according to the manufacturer's protocol. as expected, the benzoylated carbocyclic oxetanocin analogue 5 exhibited a stronger anti-hcv activity than that shown by the unprotected 6 (ec 50 = 6.4 lm and 46.1 lm, respectively). this trend seems to be consistent with that of the anti-sars-coronavirus activity, as mentioned above ( fig. 2 ; ec 50 = 14.5 lm and >300 lm, respectively). however, compound 5 displayed a cytotoxic effect at a level close to its ec 50 value (cc 50 = 20.0 lm; antiviral index = 3.1); therefore, further evaluation of this compound was not undertaken. 2.2.1. structure-activity relationship (sar) of the sugar moiety. to evaluate the effect of the sugar moiety, several 6-chloropurine derivatives that possessed the ribofuranosyl structure (7 and 8), or deoxyribofuranosyl structure (2 0 -deoxy: 9 and 10; 3 0 -deoxy: 11 and 12), or an acyclic backbone (13 and 14) were tested. among them, the 2 0 -deoxyribonucleoside derivative 10 exhibited the highest potency against the hcv replicon with an ec 50 of 6.1 lm, which is ninefold potent over that of ribavirin, and a cc 50 of 111 lm. additionally, the 5 0 -o-benzoyl analogue 10 was more potent than the corresponding unprotected analogue 9 (ec 50 : 6.1 lm (5 0 -obz, 10) vs 47.2 lm (5 0 -oh, 9)). this trend was also observed in the acyclic analogues 13 and 14 (ec 50 : 13.3 lm (àobz, 14) vs 80.8 lm (àoh, 13)). in contrast, in case of the ribofuranosyl structure, the 5 0 -hydroxyl analogue 7 was slightly more potent as compared with the 5 0 -o-benzoyl analogue 8 (ec 50 = 31.0 and 41.0 lm, respectively). these results indicate the importance of the masked 5 0 -hydroxyl group in the 2 0 -deoxyribofuranosyl structure. it is also interesting to note that the 2 0 -deoxyribonucleoside derivative 10 was more potent than the corresponding ribonucleoside derivatives such as 7 and 8 in spite of the fact that hcv is an rna virus and most of the anti-hcv agents bear ribofuranosyl structures (or their bioisosteres). 6,7 2.2.2. sar of the 5 0 -o-moiety. as a next step, we evaluated the inhibitory activities of compounds 15-21, whose 5 0 -hydroxyl groups were masked with various protective groups (i.e., pivaloyl, butyryl, trimethylbenzoyl, benzenesulfonyl, benzyl, and allyl groups) to examine the effect of the substituent group at the 5 0 -position. among these compounds, the ec 50 value of the close structural analogue 17 (p-methoxybenzoyl analogue) was comparable to that of 10 (ec 50 = 8.4 and 6.1 lm, respectively); however, 17 led to an undesir-able increase in cytotoxicity (cc 50 = 76.4 lm). notably, other types of protective groups such as ether groups (compounds 20 and 21) and a sulfonate group (compound 19) also exhibited good anti-hcv activity (ec 50 = 17.7, 24.5, and 28.8 lm, respectively). overall, it appears that protective groups containing a phenyl ring are preferable for the antiviral activity. 3. sar at position 6 of the purine base. the effect of the substituents at the purine 6-position was investigated by the evaluation of compounds 22-25. among these analogues, the 6-dimethylamino derivative 24 exhibited a good potency with an ec 50 of 22.3 lm and low cytotoxicity (cc 50 > 200 lm), while the 6-amino derivative 23 showed a weak potency (ec 50 = 105 lm). unfortunately, the others (i.e., 6-hydroxyl (hypoxanthine) analogue 22 and 6-methylthio analogue 25) did not show any significant potency (ec 50 > 100 lm). accordingly, the chloro group at the purine 6-position was considered to be important for the anti-hcv activity. 17 the luciferase assay described above revealed that compound 10 exhibited the maximum potency among the analogues. next, in order to confirm the anti-hcv activity of 10, the replicon rna levels were quantified by performing real-time rt-pcr analysis. 16 fig. 4 shows the result obtained with 10 and ribavirin (positive control). compound 10 reduced the replicon rna amount up to approximately 45% at 12.5 lm and 6% at 25 lm, which is almost consistent with the result of the luciferase assay (ec 50 = 6.1 lm). among these molecules, two compounds can be hypothesized to be species with real antiviral activity. one is the 5 0 -o-masked 2 0 -deoxyadenosine analogue itself such as 10, and the other is the deprotected 9 (or its activated form, 5 0 -triphosphate) since carboxylic ester bonds are often hydrolyzed in cultured cells, as reported earlier. 18 however, the chemically stable 5 0 -o-masked analogues 20 and 21 (bn and allyl ether, respectively) showed anti-hcv potency to some extent. moreover, the deoxyadenosine derivative 23, which would be transformed to the inactive 2 0 -deoxyadenosine after the hydrolysis, also showed potency. thus, taking these results into consideration, it would be reasonable to consider that some 5 0 -o-masked analogues themselves possess the anti-hcv potency. on the other hand, it would also be reasonable to consider that compound 9 (or its 5 0 -triphosphate) is one of the real active species since 9 also exhibited anti-hcv activity, though only moderately; in other words, compound 10 operates as a prodrug of 9. 19 therefore, at this stage, we believe that both compounds (e.g., 9 and 10) would function as the species with antiviral activity in the cells. a series of nucleoside analogues 5-25 were synthesized and their abilities to inhibit hcv rna replication were evaluated. among these, several 5 0 -o-masked analogues of 6-chloropurine-2 0 -deoxyriboside, such as 10, 17, and 20, exhibited effective anti-hcv activity. in particular, 5 0 -o-benzoyl analogue 10 exhibited the highest activity. presently, we hypothesize two roles for these 5 0 -omasked analogues: one is the role as an anti-hcv agent by itself, and the other is as a prodrug of its 5 0 -o-demasked (deprotected) derivative. there are two notable structural features of these potent compounds: one is the masked 5 0 -hydroxyl group, and the other is the 2 0 -deoxyribofuranosyl structure. in relation to the substituent group at position 6 of the purine base, the chloro group seems to be preferable. these unique features are rarely seen in common anti-hcv agents; therefore, although some issues such as improvement of the antiviral potency remain to be resolved, we hope that the present study will contribute to developing a new class of hcv therapeutic agents. (15) . to a stirred solution of 9 (63 mg, 0.23 mmol) and dmap (3 mg, 10 mol%) in pyridine (0.5 ml) was added pivaloyl chloride (43 ll, 0.35 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 30 min and then at room temperature for 15 min. the work-up process was performed in the same manner as described for 10. 6-chloro-9-(5-o-(2,4,6-trimethylbenzoyl)-b-d d-2deoxyribofuranosyl)purine (18) . to a stirred solution of 9 (100 mg, 0.37 mmol) and n,n-diisopropylethylamine (478 ll, 2.96 mmol) in acetonitrile (4.0 ml) was added 2,4,6-trimethylbenzoyl chloride (303 ll, 1.85 mmol) at ice-water temperature, and the mixture was stirred at room temperature for 3 h. the work-up process was performed in the same manner as described for 10. the crude material was purified by preparative thin layer chomatography (merck, 113895) (ethyl acetate/hexane, 2:1) to give 18 (33 mg, 21%) as a white semi-solid. 1 (19) . to a stirred solution of 9 (100 mg, 0.37 mmol) in pyridine (1.0 ml) was added benzenesulfonyl chloride (94 ll · 4 times at intervals of 30 min, 0.74 · 4 mmol) at room temperature, and the mixture was stirred at the same temperature for 3 h in total. the work-up process was performed in the same manner as described for 10. the crude material was purified by silica gel column chromatography (methanol/chloroform, 1:20) to give 19 (60 mg, 40%) as a white semi-solid. 1 (12) . to a stirred solution of 11 (600 mg, 2.22 mmol) in ch 2 cl 2 (15 ml) were added pyridine (3.5 ml) and benzoyl chloride (268 ll, 2.31 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 2 h. subsequent to the addition of saturated nahco 3 solution, the mixture was extracted with ethyl acetate. the combined organic layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1 to 2:1) to give 12 (488 mg, 59%) as a white solid. mp 160.5-161.0°c. 1 to a stirred solution of 23 (915 mg, 2.45 mmol) in pyridine (12 ml) were added imidazole (1.00 g, 14.7 mmol) and tert-butyldimethylsilyl chloride (1.11 g, 7.36 mmol) at room temperature, and the mixture was stirred overnight at the same temperature. subsequent to the addition of water, the mixture was stirred for 15 min. after concentration under reduced pressure, the residue was dissolved in ethyl acetate. the ethyl acetate solution was washed with water, 1 m aqueous hcl, saturated nahco 3 solution and brine, dried over na 2 so 4 , and concentrated under reduced pressure. were added a solution of tetraethylammonium chloride (346 mg, 2.08 mmol) in ch 2 cl 2 (2.5 ml) and then tert-butyl nitrite (313 ll, 2.60 mmol) at ice-water temperature. the mixture was stirred for 1 h at the same temperature, warmed to room temperature for 0.5 h, and stirred at 50°c for 2 h. the solvent was removed under reduced pressure, and the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:3 to 1:2) to give 28 (147 mg, 58%) as a pale yellow oil. 1 1, 810 ll, 0.81 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 5 h and then at room temperature for 1 h. the solvent was removed under reduced pressure, and the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 2:1 to 3:1) to give 10 (181 mg, 72%) as a white solid. 4.1.12. 3 0 -o-(tert-butyldimethylsilyl)-2 0 -deoxy-6-s-methyl-6-thioinosine (29). to a stirred solution of 28 (225 mg, 0.46 mmol) in dmf (3.5 ml) was added a solution of methyl mercaptan sodium salt (15% in water, 860 ll, 1.84 mmol) at ice-water temperature, and the mixture was stirred at room temperature for 3 h. after dilution of the mixture with ethyl acetate, the organic layer was washed with water, saturated nahco 3 solution and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, to a stirred solution of 31 (200 mg, 0.56 mmol) in dioxane (1 ml) was added 50% dimethylamine-water solution (8 ml) at room temperature, and the mixture was stirred overnight at the same temperature. the solvent was removed under reduced pressure, and the resultant residue was washed with ether several times, and purified by silica gel column chromatography (methanol/chloroform, 1:10) to give 32 (98 mg, 62%) as a white solid. the 1 h and 13 c nmr spectra and the mass spectrum were identical to the reported values. 14 4.1.16. 5 0 -o-benzoyl-n 6 ,n 6 -dimethyl-2 0 -deoxyadenosine (24). to a stirred solution of 32 (290 mg, 1.04 mmol) in pyridine (5 ml) was added benzoyl chloride (181 ll, 1.56 mmol) at ice-water temperature, and the mixture was stirred at room temperature for 4 h. subsequent to the addition of saturated nahco 3 solution, the mixture was extracted with ethyl acetate. the combined organic layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 3:2 to 4:1) to give 24 (306 mg, 77%) as a white semi-solid. 1 , molecular sieves 4 a (500 mg), and lipase acrylic resin from candida antarctica (300 mg) purchased from sigma were suspended in thf (20 ml), and the mixture was stirred at 60°c for 1.5 h. the enzyme was filtered off and washed with meoh, and the solvents were removed under reduced pressure. the resultant residue was purified by silica gel column chromatography (methanol/chloroform, 1:20 to 1:10) to give 33 (523 mg, 89%) as a white solid. the 1 h nmr spectrum was identical to the reported values. 15 4.1.18. 5 0 -o-acetyl-3 0 -o-(tert-butyldimethylsilyl)-2 0 -deoxyadenosine (34). to a stirred solution of 33 (670 mg, 2.29 mmol) in pyridine-dmf (1:1, 12 ml) were added tert-butyldimethylsilyl chloride (860 mg, 5.71 mmol) and dmap (140 mg, 1.15 mmol) at room temperature, and the mixture was stirred overnight at the same temperature. subsequent to the addition of water, the mixture was stirred for 20 min and then extracted with ethyl acetate. the combined organic layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate) to give 34 (940 mg, 94%) as a white solid. mp 145-146°c. 1 to a stirred solution of 34 (300 mg, 0.74 mmol) and triethylamine (408 ll, 2.94 mmol) in ch 2 cl 2 (6 ml) was added pivaloyl chloride (207 ll, 1.70 mmol) at ice-water temperature, and the mixture was stirred at room temperature for 1 h. subsequent to the addition of dmap (9 mg, 10 mol%) and methanol (2 ml), the mixture was stirred overnight at room temperature. after dilution of the mixture with ethyl acetate, the organic layer was washed with water, 5% khso 4 solution, saturated nahco 3 solution and brine, dried over na 2 so 4 , and concentrated under reduced pressure to leave the crude compound 35 (385 mg), which was employed in the next reaction without purification. to a stirred solution of 35 (385 mg) in methanol (5 ml) was added potassium carbonate (203 mg, 1.48 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 0.5 h. subsequent to the addition of acetic acid (150 ll) and water (3 ml), the organic solvent was removed under reduced pressure. the resultant mixture was extracted with ethyl acetate, and the organic layer was washed with saturated nahco 3 solution and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 3:1 to 4:1) to give 36 (324 mg, 98% from 34) as a white solid. to a stirred solution of 36 (340 mg, 0.76 mmol) in thf (7.5 ml) was added potassium tert-butoxide (210 mg, 1.89 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 1 min. subsequent to the addition of benzyl bromide (99 ll, 0.83 mmol) at ice-water temperature, the mixture was stirred at the same temperature for further 1 h. after dilution of the mixture with water, the aqueous layer was extracted with ethyl acetate. the organic layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1 to 2:1) to give 37 (397 mg, 97%) as a colorless oil. 1 to a stirred solution of 37 (390 mg, 0.72 mmol) in methanol (7 ml) was added potassium carbonate (299 mg, 2.17 mmol) at ice-water temperature, and the mixture was stirred at room temperature for 7 h. after dilution with ch 2 cl 2 , the mixture was filtrated. subsequent to the condensation of the filtrate in vacuum, the resultant residue was diluted with ethyl acetate. the ethyl acetate layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 3:2 to 3:1) to give 38 (250 mg, 76%) as a white solid. mp 134-135°c. 1 to a stirred solution of 38 (250 mg, 0.55 mmol) in ccl 4 (10 ml) were added a solution of tetraethylammonium chloride (364 mg, 2.20 mmol) in ch 2 cl 2 (2.5 ml) and then tert-butyl nitrite (326 ll, 2.75 mmol) at ice-water temperature. the mixture was stirred for 1 h at the same temperature, warmed to room temperature for 0.5 h, and stirred at 50°c for 2 h. the solvent was removed under reduced pressure, and the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:3 to 1:2) to give 39 (108 mg) with inseparable by-products, which was employed in the next reaction without further purification. to a stirred solution of 39 (108 mg) in thf (2 ml) was added 1 m thf solution of tbaf-acoh (1:1, 300 ll, 0.30 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 1 h and then at room temperature for 5 h. the solvent was removed under reduced pressure, and the resultant residue was purified by preparative thin layer chromatography (merck, 113895) (methanol/chloroform, 1:10) to give 20 (181 mg, 34% from 38) as a colorless oil. to a stirred solution of 36 (300 mg, 0.67 mmol) in thf (20 ml) was added potassium tertbutoxide (188 mg, 1.68 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 1 min. subsequent to the addition of allyl bromide (203 ll, 2.35 mmol) at ice-water temperature, the mixture was stirred at the same temperature for further 2.5 h. after dilution of the mixture with saturated nah-co 3 solution, the aqueous layer was extracted with ethyl acetate. the organic layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:2 to 1:1) to give 40 (300 mg) with inseparable by-products, which was employed in the next reaction without further purification. to a stirred solution of 40 (300 mg) in methanol (7 ml) was added potassium carbonate (253 mg, 1.84 mmol) at ice-water temperature, and the mixture was stirred at room temperature for 7 h. after dilution with ch 2 cl 2 , the mixture was filtrated. subsequent to the condensation of the filtrate in vacuum, the resultant residue was diluted with ethyl acetate. the ethyl acetate layer was washed with water and brine, dried over na 2 so 4 , and concentrated under reduced pressure. the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1 to 4:1) to give 41 (198 mg, 73% from 36) as a white solid. (11 ml) were added a solution of tetraethylammonium chloride (384 mg, 2.32 mmol) in ch 2 cl 2 (2.6 ml) and then tert-butyl nitrite (345 ll, 2.90 mmol) at ice-water temperature. the mixture was stirred for 1 h at the same temperature, warmed to room temperature for 0.5 h, and stirred at 50°c for 2 h. the solvent was removed under reduced pressure, and the resultant residue was purified by silica gel column chromatography (ethyl acetate/hexane, 1:6) to give 42 (95 mg) with inseparable by-products, which was employed in the next reaction without further purification. to a stirred solution of 42 (95 mg) in thf (1.5 ml) was added 1 m thf solution of tbaf-acoh (1:1, 330 ll, 0.33 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 1 h and then at room temperature for 6 h. the solvent was removed under reduced pressure, and the resultant residue was purified by silica gel column chromatography (methanol/chloroform, 0:1 to 1:20) to give 21 (50 mg, 28% from 41) as a colorless oil. compound 42: 1 h nmr (acetone cell culture, luciferase assay, and real-time rt-pcr analysis were performed as described previously. 16 the cytotoxicity was evaluated in a tetrazolium (xtt)-based assay according to the manufacturer's protocol (cell proliferation kit ii (xtt), roche diagnostics, cat. no. 1465015). classification and nomenclature of viruses: sixth report of the internal committee on taxonomy of viruses advances in antiviral drug design a prodrug approach involving 5 0 -monophosphate derivatives is also effective in increasing the antiviral potency a racemic mixture of 14 was employed a similar sar trend was observed in our previous study (ref. 11), and some comments are mentioned therein several 5 0 -o-acyl nucleoside analogues have been reported as prodrugs of the corresponding deacylated analogues. see ref this research was partially supported by a grant-in-aid for young scientists (b), no. 18790093, from the japan society for the promotion of science (jsps). key: cord-349358-leicos9j authors: ketzinel‐gilad, mali; shaul, yosef; galun, eithan title: rna interference for antiviral therapy date: 2006-06-16 journal: j gene med doi: 10.1002/jgm.929 sha: doc_id: 349358 cord_uid: leicos9j silencing gene expression through a process known as rna interference (rnai) has been known in the plant world for many years. in recent years, knowledge of the prevalence of rnai and the mechanism of gene silencing through rnai has started to unfold. it is now believed that rnai serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing rnai have been found in various viral pathogens. during the past few years, it has been demonstrated that rnai, induced by specifically designed double‐stranded rna (dsrna) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. however, many key questions and obstacles in the translation of rnai into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsrna that induces the system. it is expected that for the specific examples in which the delivery issue could be circumvented or resolved, rnai may hold promise for the development of gene‐specific therapeutics. copyright © 2006 john wiley & sons, ltd. the battle against viral infections is ferocious. since viruses are developing resistance to therapy, novel antiviral therapeutic modalities are in great demand. the currently approved antiviral therapies are based on the use of small molecular weight drugs, utilization of proteins simulating the innate immune response, and the adaptive immune system for both passive and active vaccination [1] . recently, an antisense drug against viral infection has also been approved, suggesting that newly developed approaches are acceptable. the first drug using antisense technology, fomivirsen (vitravene), developed by isis pharmaceuticals, was approved for the treatment of cytomegalovirus (cmv) retinitis. in general, small molecular weight drugs are mainly used today for chronic viral disease, although there are exceptions such as the anti-influenza compound, oseltamivir/tamiflu, which is used for acute infection. passive and active vaccination approaches are being developed for the prevention of severe acute diseases (in this case, there are also exceptions, such as life administration of anti-hepatitis b virus (hbv) antibodies to liver transplant patients at risk for hbv infection recurrence). the development and use of specific antiviral drugs and vaccines have been slower than expected and face major challenges. one particular example is the sluggish progress in generating effective drugs against the hepatitis c virus (hcv), even though the genomic sequence of the virus was unfolded over 15 years ago. hiv drug development also faces major drawbacks. although hiv replication is efficiently inhibited by the combination of highly active antiretroviral therapy (haart), long-term complications of haart include significant morbidity on the one hand, and the generation of multi-drug-resistant hiv strains on the other in a large proportion of patients. in spite of the comprehensive advancement in understanding the biology of the immune response, translating these findings into rational therapeutic platforms remains slower than expected. the need for preventive and effective vaccines remains as much a requisite today as it was in previous times in the history of mankind. indeed, at the beginning of the previous century, the world suffered devastating viral infection pandemics such as the one that occurred in 1918 where a quarter of the world population fell ill and the death toll reached over 25 million people from acute influenza infection. today, the achievement of peak vaccine efficacy to treat influenza stands at 75% among those under 65 years old and just 35% among the elderly. even today, the annual death toll from acute influenza infection in the usa tops 20,000 in spite of national vaccination programs. thus, different strategies are currently being suggested in an effort to be prepared for future pandemics [2] , and these include the development of escape mutants (antigenic drift) and reassortment of genetic segments of different quasispecies of the same virus or of different viruses (antigenic shift). presently, human viral pathogens are spreading worldwide, such as the much publicized menacing spread of the avian flu which is reported to have expanded to remote sites of russia and kazakhstan from the south-east, posing a major health threat to the entire world. in an effort to identify a novel class of efficient antiviral therapeutics, numerous technologies are currently being assessed for their antiviral potential. these include antisense oligonucleotides, antisense phosphorodiamidate morpholino oligonucleotides, ribozymes and, in recent times, rna interference (rnai). incidentally, we have been encouraged to learn that sirna (short interfering rna) against vascular endothelial growth factor (vegf) has recently been administered to patients in a clinical study without major side effects (asgt meeting, 2005) . in this review, we will summarize the recent developments in the use of rnai as an antiviral agent. rna interference (rnai) is a sequence-specific silencing of genes, induced by small molecules of double-stranded rna (dsrna). this phenomenon was first observed in plants in the late 1980s, but its molecular mechanism remained unclear until it was discovered in 1998 by fire et al., in the nematode c. elegans [3] . they showed that the presence of a very small quantity of dsrna led to almost a complete shut-off of the expression of the gene that was homologous to the dsrna. the interference process starts with cleavage of the dsrna that induced it into small rna duplexes, 21-23 nucleotides (nt) long, called sirnas (short interfering rnas) [4, 5] . these small dsrna duplexes have 2 nt overhang at their 3 end, a 5 -monophosphate and a 3 -hydroxyl group [6] . the enzyme responsible for that first step is dicer, a dsrna-specific nuclease that belongs to the rnaseiii family, and acts in an atp-dependent manner [7] . in the next step, the sirnas generated by the dicer are incorporated into the rna-induced silencing complex (risc), a multi-component enzymatic complex [5] . the risc unwinds the sirna in an atp-dependent manner, and uses its single-strand form to target the homologous transcript by base-pairing interactions. it then cleaves the mrna by its endonuclease component in a homology-dependent manner, only in the region corresponding to the sequence of the sirna. this process leads to degradation of the mrna [4, 8] . sirna-mediated gene silencing has also been found in lower organisms, such as plants, fungi, worms and flies [9] . it is a conserved mechanism of intracellular antiviral immunity that also protects the host genome from foreign genetic elements such as retroviruses, transposons and retrotransposons. these elements may have deleterious effects on the genomic dna of the host, and thus their mrna elimination may represent an earliest form of innate immunity. rnai was first suggested to evolve as a natural antiviral defense in plants, especially against rna viruses [10, 11] . in mammalians, rnai has also been reported to have gene-silencing properties. the rnai machinery was triggered experimentally by the introduction into the cells of artificially designed dsrna molecules, 21 nt in length, and the target gene was inhibited in a sequencespecific manner [8] . this effect has become very effective in silencing and knocking-down expression of specific genes in the cells. sirnas have become the method of choice for mammalian cell genetics as well as for potential sequence-specific therapeutic approaches. the inhibitory action of sirnas has been documented for numerous viruses. it works against rna viruses with negative-or positive-strand genome polarity, as well as against dna viruses. the sirna, as a therapeutic tool, can be targeted against the various phases of the viral life cycle of dna and rna viruses including replication, transcription, assembly of new virions, and budding out of the target cells ( figure 1 ). proteins for transcription. many rna viruses encode their own transcription factors. for example, the gag, pol and env genes of retroviruses are needed for an efficient transcription. indeed, sirnas directed against the gag and env genes of hiv-1 [12, 13] or the avian sarcoma leucosis virus [14] significantly reduce their overall transcription. another example is the dna human papilloma virus; sirna directed against the viral transcription factor e6 inhibits viral gene expression and growth in tissue culture [15, 16] . baculoviruses infect many different insect species. over many years, the autographa californica nucleopolyhedrosis virus has caused severe economic losses in the silk industry. inhibition of that virus was achieved by a pair of sirnas that target specifically the viral coded early transcription activator, and the major nucleocapsid [17] . one of the main steps in the process of viral infection is dna and rna genome replication of the virus. the genome of rna viruses, especially with plus polarity, serves both as mrna and as a replication template. many research groups applied the sirna method to inhibit replication of viruses in vitro and in vivo. in the absence of an efficient cell culture system for growing hcv, the sensitivity of hcv to rnai was shown in the replicon-based system. an hcv replicon is derived from an hcv consensus genome that was cloned from a viral rna isolated from an infected human and used to construct subgenomic selectable replicons. upon transfection of these subgenomic selectable constructs into a cell line, these rnas were found to replicate to high levels. in several studies, sirnas were directed against different targets in the virus genome [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . for example, sirna specific for the 5 untranslated region (utr) of the hcv genome, introduced into huh7 cells carrying the replicon system, inhibits hcv replication by up to 90% [20] , as measured by the expression level of the replicon luciferase reporter gene. sirnas targeting the viral polymerase ns5b region reduced expression of ns5b-luc chimera in mice [27] or in the replicon system in vitro. other studies that target other regions of the hcv genome reported a significant decline in the level of hcv proteins and the level of both the sense and antisense rna strands [25] . the sirna effect shown for hcv is ifn-and cell-cycleindependent [23] . in the hepatitis a virus (hav) replicon-based system, it has been reported that sirnas targeting the regions coding for the non-structural proteins of the virus give rise to partial inhibition of hav replication [28] . in that study, two sirnas specific for hav sequences increased rather than inhibited hav replication. this could be due to complex secondary structures of the target region that can limit and reduce the efficiency of the rnai process [29] . in another study, sirna targeted to various domains of the hav internal ribosomal entry site (ires) induced efficient and sustained suppression of viral genome translation and replication [30] . poliovirus is a highly cytopathic rna virus. sirnas specific to the poliovirus genome inhibited viral replication, as was demonstrated in a poliovirus replicon system. the sirna effect led to viral genome clearance from the infected cells, without destruction of the cells harboring the virus [31] . additional examples of inhibition of viral replication by sirna originated from the study of positive rna viruses such as dengue (denv), west nile (wnv), and severe acute respiratory syndrome (sars) [32] [33] [34] [35] [36] . sirnas targeting the 3 -utr sequence of denv, in a region that is conserved in all the dengue serotypes, reduced viral replication and infection in dendritic cells [32] . sirnas targeting the sars-cov rna polymerase gene inhibited viral rna replication, protein synthesis and reduced the viral cytopathic effects on vero cells [36] . likewise, expression vectors of sirnas specific for two different regions of the wnv genome protected 293t cells from wnv infection, and significantly reduced viral rna replication and virus production [35] . coxackievirus b3 (cvb3), a member of the picornaviridae family, is a major cause of many human diseases, such as meningioencephalitis and myocarditis. synthetic sirna targeted to the vp1 or to the viral polymerase showed antiviral effects in infected hela cells by inducing a significant reduction of viral replication [37] . the footand-mouth disease virus (fmdv) replication was inhibited in bhk-21 cells by sirnas targeting various conserved regions of the fmdv genome [38] . multiple sirnas have been used to target multiple conserved viral genes that are essential for virus replication, including a long 5non-coding region, a short 3 -non-coding region, the viral protein vpg, the viral polymerase, and the viral capsid protein vp4. the combination of those sirnas gave rise to a 10-1,000-fold inhibition in virus yield by specific inhibition of viral replication [38] . the antiviral properties of rnai have not been assessed in comparison for their effectiveness upon targeting the different intracellular stages of the viral life cycle. however, from current reports, we could surmise that targeting viral replication, similar to what has been described in several other types of antiviral methods, would probably be the suggested approach to suppress viral infection. replication of dna viruses can be inhibited by targeting their viral mrna, whereas replication of rna viruses can be inhibited by targeting either their mrnas or their viral rna, as was elegantly demonstrated for hiv [39] . in the later stages of the virus life cycle, the structural proteins are produced to assemble and form mature virions before egress. rotavirus causes severe diarrhea in infants and children worldwide. to combat this virus, dector et al., utilized sirna directed to the vp4, a viral structural protein that is essential for the attachment of the infecting virus to the cell surface. they showed a significant reduction in the number of viral particles produced in ma104, an infected monkey kidney cell line. moreover, most of the viral particles that were produced were poorly infectious [40] . however, there are only few reports assessing specifically the potential of the rnai effect on viral assembly [41] . the antiviral properties of rnai against viral assembly, a late stage in the intracellular viral life cycle, is expected to be less effective than rnai in the early steps of the viral life cycle. in many rna viruses, there is emergence of quasispecies that contain point mutations in the sirna's target sequences leading to evasion from inhibition by sirna. using a pool of sirnas to simultaneously target multiple sites in the viral genome can prevent the emergence of these resistant viruses [42, 43] . another approach that may partially solve this problem is targeting cellular factors rather than viral genes. during their life cycle, viruses apply cell membrane receptors for penetration, and cellular transcription factors for viral replication, harnessing very efficiently the cellular transcription and translation machinery for their life cycle. targeting those cellular genes may be another strategy for inhibition of viruses. and indeed, zhang et al., for example, succeeded in suppressing the replication of hcv in the replicon system by the expression of sirnas against cellular cofactors that are needed for viral replication, the polypyrimidine tract-binding protein (ptb) or eif2bg [44] . inhibition of the ptb alone by sirnas resulted in an efficient decrease in the levels of hcv proteins as well as hcv rna replication in huh7 cells harboring the hcv replicon [45] . in another study, sirna against cellular rna helicase p68 reduced hcv negative strand replication [46] . the e7 protein of human papillomaviruses (hpvs) contributes to oncogenesis. e7 was found to specifically activate the transcription of the cellular transcription factor e2f2 in an in vitro system of differentiating human keratinocytes. while suppression of e2f2 levels through the use of sirna decreased hpv replication, this loss did not affect cell proliferation. thus, e2f2 is a potential target for antiviral therapies [47] . the human cyclin t1 (hcyct1) is a cellular factor essential for transcription of messenger and genomic rnas from the long terminal repeat (ltr) promoter of the hiv-1 provirus. sirna directed against hcyct1 could effectively suppress hiv-1 replication without any induction of apoptotic cell death [48] . in previous studies, downregulation of other cellular factors, such as cd4, cxcr4, ccr5, nf-kb, p-tefb, cyclophilin a, dc-sign, spt-5 and parp-1, successfully inhibited hiv-1 replication [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] . however, since many of these molecules are essential for cellular processes (cd4, e.g., is a cell-surface molecule important for adaptive immune response), not all of them can serve as a practical target for hiv gene therapy. to conclude, sirnas can be used for inhibition of both rna and dna viral infections from the early stage of viral attachment to the cell to the late stage of viral assembly. sirnas can be targeted directly to the viral genes involved in the viral life cycle, or against cellular genes which are used by the viruses. in each case, the best strategy for viral inhibition needs to be assessed according to the virus type and its unique life cycle. interestingly, a combination therapy of two sirnas, targeting different viral sequences, each with inhibitory function, did not have an enhanced effect. this was also found by other groups, assessing the rnai effect in other human and non-human pathogens. these repeated observations could be related to the early saturation of the rnai cellular machinery. however, this issue will need further investigation, which could lead to improvement of the efficacy of rnai against viral infections. in some specific cases of acute viral infections, in particular those cases which could pose a major threat to the health care system, several hurdles remain to be overcome for the development of vaccines and specific small drugs. however, in most cases, the viral agent causing the acute severe endemic, possibly pandemic disease, could be rapidly identified and sequenced, as was the case during the recent outbreak of sars. in these cases, the development of sirnas targeted against various regions of the viral genome could lead to a quick development of a therapy against the acute viral infection. the production, delivery, dose, and modes of administration of sirnas could be tailor-designed for any group of targets. suffice to say, it is imperative that the timetable for the generation of a new sirna-based rna silencing drug could be shorter, so that a therapeutic platform against many specific infectious agents with pandemic potential could be forthcoming. obviously, it must be remembered that additional important factors need to be implemented for the development of an antiviral drug, such as in vitro and in vivo models, although these requirements are essential for any other type of therapeutic modality to be produced and tested. one recent report nicely exemplified the fact that synthetic sirna could be generated against a single (respiratory syncytial virus, rsv) or a double infection (with parainfluenza virus, piv), and rapidly tested in vitro, as a sufficiently predictive tool for an in vivo effect [65] . in this report, the sirnas against both rsv and piv were administered nasally with profound antiviral preventive and therapeutic effects without inducing interferon production. as mentioned in previous sections, sirna is very effective against other life-threatening pandemic threats such as influenza infections [66] . however, it must be remembered that we are probably just at the beginning of experimental assessments to determine the potential antiviral effects of sirna. the antiviral effects of short hairpin rnas (shrna) or sirnas were also assessed in other viral infections where there is practically no available therapy (shrnas are short hairpin rnas expressed by plasmid and viral vector systems and are subsequently processed to sirnas by the cellular machinery). sirna targeting the protease 2a region of the most common viral agent causing myocarditis, coxsackievirus b3, was found to be effective in inhibiting viral replication in vitro [67] . in addition, this same group also showed that the antisense sirna strand is critical for the rnai effect and that single nucleotide mutations at the central or 5 regions are detrimental for the antiviral effect. sirna was also effective against sars caused by the newly discovered coronavirus in a preventive model in vitro [68] . currently, since there is no available effective specific therapy against sars, rnai could be developed for this serious infection. in other cases of severe human infections by viral pathogens, there are fewer promising results than for the antiviral potential against viral replication by inducing rnai. sirna was also assessed against wnv infection. in one report, while the investigators failed to show an antiviral effect in active replicating cells [69] , they showed prevention of infection in a previous report. another group assessed the potential effect of sirna against wnv infection in vivo. again, only a preventive mode of therapy was found partially effective against viral replication and disease outcome in mice [70] . from the reports on the use of sirna against human viral pathogens causing acute disease, we could learn that for each specific pathogen infecting a specific cell lineage or tissue, we would probably need to perform an indepth assessment, with proper in vitro and in vivo models, and develop specific delivery systems. although the road towards rnai development could be visible for some of its destinations and the traveling speed could be changing, the target time remains unknown and unpredictable. an interesting new approach of preventing viral infection was reported by the group of judy lieberman [71] . in an effort to suggest a method of prevention of hsv2 sexual transmission, intravaginal installation of sirnas targeting two hsv-2 genes protected mice from a lethal hsv-2 challenge without inducing interferon-responsive genes. this encouraging result once again proves that combining a realistic method of gene delivery with a specific genetic drug payload for a specific disease could result in a beneficial gene therapy outcome. in most acute viral infections, the host overcomes the invading pathogen through a robust innate and adaptive immune response. however, in those cases where the virus causes severe disease as a result of a significant cytopathic effect, which could be related to a high multiplicity of infection (moi), low immunogenicity, high replication capacity or direct toxic effects, or a combination of all, could result in organ failure or even death. in these cases, rnai could significantly support, or even enhance, the antiviral effects for a short period of time and this could be achieved by the administration of sirnas. characterizing the specific viral pathogen, even in a pandemic situation, could enhance the rational design of a sequence-specific sirna that can be used as therapy. this situation is very different from cases of chronic viral infections of rna viruses, in that there could be quasispecies; in addition, the effect of the rnai should be prolonged and generated through an expression system rather than through a synthetic sirna administered once or only a few times. human chronic viral infections such as hbv, hcv and hiv are a worldwide threat. for hcv and hiv infections, there are no available vaccines, and, in addition, in both the prospect of vaccine development is not encouraging. furthermore, current therapeutics for both of these infections are suboptimal. for these viral infections, numerous gene-based approaches have been developed. although there are effective vaccines against hbv infection, chronic infection is still a major therapeutic challenge. the rnai was used to inhibit replication of dna viruses. hbv replication was inhibited in vitro and in vivo by rnai by us and by others [72] [73] [74] [75] [76] . sirnas targeted to different regions of the hbv surface antigen gene robustly inhibited viral gene expression and replication both in vitro and in vivo [72] . due to the overlapping gene structure of the hbv genome, targeting a region in the open reading frame (orf) of the x gene which is shared by all the viral transcripts resulted in a significant reduction of up to 90% in all viral transcripts and proteins and in a dramatic reduction of ∼95% in viral replication [75] . the x gene of hbv was also recently assessed as a target for rnai in vivo [77] . using two hbv mice models as a naked dna approach with the hydrodynamic method or expressed from an adenovector, the pol iii u6 promoter encoded short shrnas targeting conserved sequences of the hbx orf. the anti-hbv effect was significant without stimulating the interferon system. it is also known that hbx plays an important role in hepatocarcinogenesis. sirna against hbx was also used to test its effect against hepatocellular carcinoma cell lines which express hbx sequences [78] . this group demonstrated a significant reduction in cell proliferation, cell growth, anchorageindependent growth in soft agar, and tumor development in nude mice following the expression of sirna against hbx. a recent report suggested that the inhibitory effect of rnai on hbv expression is stronger than that of lamivudine in vitro [79] . we could further speculate that a combination of rnai and nucleoside analogs might encounter a synergistic effect, although this is yet to be determined. the most challenging part of rnai approaches for chronic viral infections is to design the best delivery method that would facilitate the targeting of the specific organ/cells with the appropriate expression system, for durable intracellular levels of gene-silencing effect. this also applies when designing an rnai approach for hcv infection, as well as for other chronic viral infections. the studies assessing the effect of rnai against hcv were mostly restricted to in vitro replicon systems, as discussed above. alternative in vivo systems were also adopted by some investigators, with reporting proteins used to assess the antiviral effect [80] . in early studies using the in vitro hcv replicon system, it was shown that a synthetic sirna targeting the 5 core region of hcv inhibited viral proteins and significantly suppressed viral replication for at least 8 days [21] . at about the same time, a different group showed, also in the replicon system, that sirna against the ns5b region (viral polymerase) is most effective in suppressing hcv replication [19] . this group had also transfected the huh7 replicon cells with a vector expressing complementary strands of sirna, again targeting the ns5b region, under the control of two separate h1 promoters (pcep4, invitrogen). in this case, the suppression effect on hcv replication lasted over 3 weeks. another group targeted similar hcv regions, ns3 and ns5b [26] ; they introduced shrnas targeted against these two genes into huh7-replicon cells. the delivery systems that were used in their study were plasmids or lentiviral vectors harboring shrnas against ns3 or ns5b, expressed from the u6 promoter. in both cases, they observed similar effects, i.e., suppression of hcv proteins and viral replication. however, shrna against the 5 -utr of hcv resulted in very low levels of inhibition of hcv replication. as mentioned earlier, viral replication is dependent on numerous cellular factors. targeting these viral replication/gene expression cofactors is a potential target for inhibition of viral replication. however, this approach should always be balanced against the potential of generating side effects which could overshadow the beneficial antiviral outcome. a recent study assessed this approach in vitro, targeting two hcv replication cofactors: proteasome α-subunit 7 and hu antigen r. shrna targeted against these two genes that were expressed from an expression vector transfected into huh7 hcv replicon cells showed a modest reduction of hcv transcription [81] . however, this modest inhibition on the one hand, and the potential role of both proteins in normal cellular function on the other, might suggest that it is advisable to abstain from such approaches, if possible. an interesting study in vivo targeting the hcv ires, translating a luciferase reporter protein, revealed that the in vitro synthesized shrna, administered systemically by the hydrodynamic method, encountered a sustained antiviral effect lasting over 4 days compared to synthetic sirna [80] . the use of rnai against hiv infection was reported by a number of groups (table 1) . although the results of these studies suggest that hiv could be targeted by rnai, there are major obstacles in translating this therapeutic approach into the clinical setting. most reports have used sequences from laboratory hiv strains. viruses with mutations at the rnai recognition site produced an escape mutant after a long-term rnai pressure. targeting relatively conserved hiv sequences could improve the efficacy of the rnai effect. a recent study looked at the protective effect of shrnas targeting the rev, gag, and vif sequences of a panel of hiv clades [82] . this study showed that targeting the vif hiv region had a significant inhibition effect on hiv replication. however, the long-term use of any specific sirna or shrna against hiv could probably induce the generation of escape mutants containing nucleotide substitutions at or near the target sites. furthermore, the escape from rnaimediated inhibition could also signify the emergence of mutations that change the hiv rna secondary structures [83] . all of these data emphasize the significance of hiv evolution during rnai pressure and its potential impact on the use of rnai against hiv. the virus also harbors a specific mechanism that evades the nucleicacid-based innate immunity of human cells against hiv. the genome of hiv contains a plethora of dsrna regions capable of being processed into sirnas targeting the viral genome to suppression [84] . however, the virus has evolved by a counter process, rendering itself resistant to rnai through the tat protein, altering the dicer effect on viral sequences, and abrogating the host cell innate immunity against hiv infection. interestingly, on the other hand, it is possible that hiv does apply the cellular rnai machinery for regulation of its own gene expression. the hiv nef region encodes a microrna, mir-n367, which can block nef expression [85] . later, it was shown by the same group that mir-n367 targets the hiv ltr promoter region, downregulating viral transcription; this might be a mechanism by which the virus regulates its own transcription [86] . single anti-hiv therapy is ineffective against viral replication and gene expression due to the high mutation rate of the virus. one option of overcoming this major obstacle is to generate therapeutics against highly conserved viral genomic regions. a recent report showed that it is possible to clone shrnas against the conserved regions of the hiv genome into hiv vectors, and to suppress hiv infection upon targeting the gag, pol, int and vpu sequences [87] . however, although this approach could be applied for prevention of infection, cessation of an ongoing hiv replication is prone to failure due to the high mutation rate of the virus. an alternative strategy could be to target essential cellular determinants for hiv infection. the early step of hiv infection would be attaching to the viral cellular receptor. during the progressive stage of the disease, most hiv isolates use the chemokine receptor cxcr4 for viral attachment and penetration into the host cells. patients with mutations of the cxcr4 receptor are less prone to hiv infection and are healthy by any significant measure. this finding was the rationale used to develop an anti-hiv approach by targeting cxcr4 expression and inhibiting viral fusion with the cellular membrane [54] . it is expected that such an approach will pose a major obstacle to viral evolution and prevent infection. other groups have also adopted this strategy to render cells resistant to hiv infection [52, 53, [58] [59] [60] . the hiv regulatory protein, rev, is essential for viral life cycle in a number of ways including splicing, translation and trans-activation. with regard to rev trans-activation properties, it needs to interact with the hypusine-containing protein eif-5a. the eif-5a rev cofactor is activated following a catalytic step performed by the human deoxyhypusine synthase (dhs). a recent study has suggested that rna interference inhibiting dhs blocked hiv replication [88] . again, as with other drugs in development against hiv infection, we are confronted with the following major questions: what are the potential side effects from such an approach, and what would be the therapeutic dose window? indeed, studies aimed at unfolding these issues are crucial before entering any clinical studies. other groups have also suggested the targeting of other cellular factors essential for the hiv life cycle, including parp-1 [55] , the elongation transcription factor p-tefb [56] , cyclophilin a, dc-sign [61, 63] , the human elongation transcription factor spt5 [64] , and human cyclin t1 [89] . the road towards the development of an efficient therapeutic modality using the anti-hiv rna interference strategy is bumpy, due to potential side effects; moreover, significant strides will have to be made towards harnessing clearcut approaches as described, as well as designing additional rational studies through wet and dry investigations [89] . reduction in cell-to-cell spread [170] the cellular level of dicer could be crucial for gene therapy approaches while utilizing the rnai machinery in targeted cells. recent data suggest that, although the expression of dicer and other proteins that participate in digesting long dsrnas into 21-25 nt, e.g., eif2c1 ∼ 4, decreases in differentiated cells, they retain a sufficient amount of enzymatic activity to induce rnai. however, when designing and planning any specific approach using sirna for gene therapy, it is advisable to assess the dicer activity in the targeted tissue if the expected sirna is unmet. since rna silencing acts as an antiviral defense mechanism in plants [91] , insects [92] and other eukaryotes, including mammalian cells [93] , it is not surprising that viruses have developed strategies to interfere with this effect. many plant dna and rna viruses have developed proteins that function as suppressors of rna silencing [94] [95] [96] [97] [98] . since the silencing suppression reduces the antiviral effects, the viruses can accumulate in their target cells and reach higher titers. comparing those suppressor genes did not reveal any sequence homology between them. the rna silencing suppressors can act upon the various sequences of the rnai machinery in several ways such as inhibition of sirna processing, inhibition of the incorporation step into the risc, or preventing the action of its effector molecules. although the mechanisms of inhibition of silencing are not fully understood, there are several suppressor genes that their targets have identified. the p19 protein from the tombusvirus was shown to bind specifically to the sirnas, and thus may inhibit the incorporation step of the sirnas into the silencing effector complexes [98] . the hc-pro protein, expressed by polyviruses, acts by targeting the risc [99, 100] . another example is the mosaic virus 2b protein (cmv2b). this nucleus-localized rnai suppressor protein inhibits the activity of the spreading signal of the rnai, and inhibits dna methylation processes in the nucleus that control the silencing pathways [97, 101] . the coat protein of the turnip crinkle virus (tcv) strongly suppresses the rna silencing process at an early initiation step, probably by interference with the function of the dicer cleavage reaction [96] . since the rnai machinery is conserved in mammals, it appears possible that, similar to plant viruses, viruses that infect invertebrates and vertebrates, including human viruses, have developed strategies to suppress rna silencing. and indeed, there is evidence that such inhibitors are not limited to plant viruses. in insects, the flock house virus (fhv) is a target of rna silencing. it has been shown that in drosophila and mosquito cultured cells, the fhv-encoded protein, b2, inhibited rna silencing, but the mechanism is still unknown. this protein also inhibited rnai in transgenic plants, suggesting that the rna silencing pathway is conserved in plants and animals [92, 102, 103] . recent evidence also indicates that human virus genes have the ability to inhibit the rnai pathway. both the influenza virus ns1 protein and the vaccinia virus e3l protein can inhibit rnai in drosophila s2 cultured cells [102] . the adenovirus encoded inhibitor of rnai is functional in mammalian cells. the human adenovirus inhibits rnai in the later stages of infection by suppressing the activity of dicer and the risc. the virus-associated rnas, va rnai and va rnaii, bind directly to dicer and function as competitive substrates, squelching it, and the resulting sirnas are incorporated into the active risc [104, 105] . the antiviral therapeutic potential of rnai would require identifying possible viral suppressors of the rnai machinery and designing strategies to inhibit their expression. one major drawback for most antiviral approaches is the development of resistance. this is most apparent in cases where the fidelity of the viral polymerase is low, especially in viruses with an rna genome. to overcome this hurdle, most antiviral therapeutic protocols harness a strategy that uses multiple drugs targeting different viral proteins or steps in its life cycle. one example where such resistance has been developed in vitro is in the case of rnai against the nef gene of hiv [106, 107] . to overcome this problem, it may be advisable to utilize a multi-targeted rnai approach possibly in combination with additional antiviral modalities. although targeting multiple genomic sites has probably no advantage with regard to the direct antiviral effect, it could repress the development of resistance. the initial steps towards focusing into this avenue have already been established. the group that initially described the sirna effect in vitro in the replicon system against hcv [19] have shown that, following several rounds of treatment with the same sirna against hcv, the replicon became resistant to that specific sirna, developing a point mutation at the target site. however, the replicon was still sensitive to a sirna targeting a different hcv region. in addition, the development of a resistant replicon was limited by the use of a combination of two sirnas targeting simultaneously different hcv genomic regions [108] . a similar approach has also been suggested by other groups [109] . the off-target effect of rnai is the silencing of nontargeted genes containing partial sequence identity to the sirna. in experiments conducted on specificity of sirna in cultured human cells, jackson et al., have demonstrated that sirna can cause direct downregulation of unintended targets containing as few as 11 contiguous bp complementarities [110] . to increase sirna specificity, there are many sirna design programs that employ various sequence alignment algorithms; however, maximum complementarity, by itself, is not enough for accurate prediction of off-targeting. a study done in dharmacon inc., identified a significant association between off-targeting and exact complementarity between the seed region of the sirna (bases 2-7) and their offtargeted genes. this pattern has been recognized in mirna-mediated gene silencing, thus suggesting that sirna off-targeting may operate by a mechanism similar to that of mirna targeting (a. birmingham, personal communication). until the off-target mechanism of sirna is understood, this issue can encounter deleterious side effects on the use of rnai. the role of interferon signaling in rnai has given rise to a series of conflicting reports. although most studies suggest that there is very little non-specific effect of sirna, others have shown that the jak-stat pathway is activated following sirna transfection. this effect is mediated by dsrna-dependent protein kinase (pkr) [111] . while we expect that this issue will foster additional debates, at this point, it would be important to impart cautious interpretations upon describing rnai effects. in addition, recent studies have suggested that although sirna does not activate the intracellular interferon machinery in mammalian cells upon entrance or in situ propagation inside the cells, if they are shorter than 30-nt dsrna, there is a non-specific innate immune response depicted by cytokine production [112] . furthermore, this effect is dependent on the toll-like receptor 3 (tlr3) that senses dsrna and serves as its receptor. tlr3 is located intracellularly on the endosome membrane and signals through nfκb nuclear translocation for the production of inflammatory cytokines. incubation of immune cells with sirna induces the activation of cells [113] . all of these effects could be dependent on the concentration of sirna. recent works identified putative immunostimulatory motifs within sirnas, and showed that even a slight change of these motifs did not significantly hamper the rnai process [114] . this research provides a basis for the design of synthetic sirnas that avoids activation of the innate immune response, and helps to minimize immunotoxicity. the group of reuven agami was the first to report a new vector system, called psuper, which directs the synthesis of sirnas in mammalian cells. they used the poly iii h1-rna gene promoter to express shrnas that specifically down-regulated gene expression, resulting in functional inactivation of the targeted genes [115] . recent reports showed that the expression of shrna from the h1 or the u6 pol iii promoters in a hiv-based vector induces the expression of interferon-stimulated genes (isgs). this effect is dependent on the presence of an aa dinucleotide near the transcription starting site. preserving a c/g sequence at positions −1/ + 1 prevents this effect [116] . in some cases, the expression from the u6 promoter is relatively low. the enhancer of the cmv immediateearly promoter enhances the u6 promoter activity [117] . others have also tested various promoters [118] reporting some beneficial effects on expression with the modified trna(met)-derived (mtd) promoter, upon expressing shrna against hiv-1 compared to u6 or h1 promoters [119] . it may happen that for each specific application, we would need to compare numerous regulatory elements to achieve the desired rnai effect. in some systems, it may be important to tightly control the expression of the shrna. although most controlled systems do not reach the desired stringency in vivo, some reports have suggested the use of specific systems. one such method is the tet-onoff expression technology [120] . again, each investigator should specifically assess the potential of this inducible system in a specific tissue culture or animal model. the teton-off systems were developed for naked dna transfection systems or incorporated into viral vectors like the lentiviral vector [121] . an additional long debate in the literature questions the choice of the loop structure to apply for the design of shrna. one specific strategy was to adopt the natural loop structure of microrna [118] . this was also used for shrna against hcv [80] . the major challenge in rnai gene therapy is to transform the in vitro robust effects of sirna into an in vivo genesilencing method. in other words, what would be the preferred delivery system to use in animals and later on in humans? as for gene therapy in general, and the specific aims of delivering rnai platforms, we need to tailor-design the tools to be used to the sought objective. this includes targeting the tissue, adjusting the desired level of expression (high level of sirna could induce nonspecific silencing [122] ), the longevity, and the specific maladies that we wish to treat. this is a complex situation, especially since our major barrier is the lack of a simple, non-immunogenic, targeted delivery system without side effects. however, in spite of all of these hurdles, we would like to discuss the potential available methods to deliver synthetic sirna. the most straightforward method of using sirna in vivo is by administering synthetic sirna. upon coinjecting sirna and its target being expressed from a plasmid vector, we could achieve knockdown of expression in the liver following a hydrodynamic injection. in our laboratory, we could show that hbv expression out of an hbv head-to-tail plasmid that supports viral replication in the liver of mice could effectively be silenced transiently with synthetic sirna [72] . this effect was dosedependent. however, the kinetics of this effect revealed that the silencing was transient as was also shown by other groups using a similar approach [27, [123] [124] [125] . the effect subsides after 48 to 72 h and is probably completely lost after 7 days. the silencing effect of sirna following systemic administration of duplex rna could have been hampered by various factors, thus imposing some logistic hurdles upon translating this approach into the clinical setting (additional examples of rnai against viral infections are depicted in table 1 ). although duplex rna is quite stable in serum [124] , and more stable than ssdna or ssrna, a high serum concentration could reduce stability. the introduction of phosphorothioate linkages could enhance stability in the serum [124] . others have used chemically modified sirna with the complete absence of 2 -oh residues on the sense and antisense strands of the dsrna, including 2 -fluoro, 2 -omethyl and 2 -deoxy sugars. these chemical modifications of sirna were produced in order to enhance its stability and effect [126] . not all modifications have resulted in beneficial silencing effects [127] [128] [129] ; a short review of these modifications was reported by paroo and corey [130] . one particular interesting report [131] shows that a specific sirna with a combined chemical modification had a significant and a relatively sustained effect in vitro and in vivo as compared to non-modified sirna. however, although sirna is relatively stable in the serum, there are disadvantages of using synthetic sirna: (1) the effect of synthetic sirna is transient; in order to impose a long-term effect, repeated administrations would be needed, and this might still be true for the chemically modified sirnas. (2) the production of synthetic sirna is expensive, making repeated administrations for longterm effect very costly. (3) it is very complicated to target synthetic sirna to a specific cell or tissue. however, in specific cases, the use of sirna directly administered into the target tissue could encounter a significant effect. in a recent report by dorn et al., they used sirna against the pain-related cation-channel p2x 3 , by intrathecal injection of phosphorothioated (ps) sirna in a rat model of neuropathic pain [132] . although they did not compare the non-ps-modified versus the ps-modified sirna, they have clearly shown a significant effect of sirna in relieving chronic pain. furthermore, the effect was superior to the comparable p2x 3 antisense oligonucleotides. one specific case where such an approach could be translated into an applicable clinical therapeutic modality is in post-herpetic neuralgia which could follow varicella-zoster infection. however, since chronic pain is a condition that is generally expected to last for months, this type of treatment would need to be readministered several times. a recent report took advantage of the knowledge generated regarding the stabilization of sirna by chemical modifications, and protection of the modified sirna with pegylated liposomes upon delivery. they assessed the antiviral effect of sirna against hbv in vitro and in vivo. interestingly, the modified sirna had induced less non-specific cytokine secretion combined with an effective and anti-hbv effect of up to 6 weeks after repeated weekly administrations [133] . in an effort to enhance and prolong the effect of sirna, various approaches have been undertaken using nonviral reagents. tailoring the specific delivery tactic and method is essential when designing a specific therapeutic strategy. one example is the use of sirna targeting the influenza virus genome. this malady can cause moderate to severe illness, can affect millions of people each year, and could be life-threatening. for the gene therapist, this means that the genetic therapy effect could be designed for a short window of time. in this case, the use of synthetic sirna with an enhanced transduction is an appropriate approach. in a study by ge et al., the systemic and intratracheal delivery of polyethylenimine (pei), a cationic polymer, promoting sirna delivery in mice, is beneficial for prophylaxis and therapy of the influenza virus infection [134] . pei was developed for in vitro and in vivo local and systemic gene delivery and pei-mediated gene delivery/transduction into the lung following systemic and intratracheal administrations. some investigators reported safety problems in specific cases upon the use of pei in animals. however, plasmid dna mixed with pei administered into the human bladder was safe (a. hochberg, personal communication). tompkins et al., also assessed the effect of sirna against the same pathogen, the influenza virus [135] . this group used a different therapeutic regimen. they used a preventive measure by administering naked sirna systemically, i.e., intravenously, before infection, and at the time of infection, they administered the sirna/oligofectamine  (a lipid carrier from invitrogen) intranasally. in this model, the undertaken therapeutic approach prevented death of animals. however, this study was limited to asking the general question of sirna effect against influenza virus infection rather than comparing different sirna delivery systems. thus, we cannot draw any conclusions that would suggest a preference for any specific delivery method in this disease model system. future animal studies would be required to determine the preferred delivery method. in addition to the lipid carriers, traditionally developed for in vitro and in vivo delivery of dna and now for sirna, alternative approaches have also been developed. one specific interesting report by minakuchi et al. describes the use of atelocollagen (at) [136] . at is prepared from type i collagen from calf dermis. this is a low immunogenic product that is already available in clinics for various indications like promoting wound healing. the same investigators showed that at enhanced dna delivery and supported prolonged expression. at mediated the delivery of sirna in vitro and was found in vivo to encounter a significant advantage over the sirna/liposome complex in inhibiting tumor growth in mice. recently, more sophisticated delivery methods of sirna have also been developed to treat brain tumors [137] . the obtained results could be important for those interested in developing sirna therapeutics for viral encephalitis. the model that they used in vivo to assess their delivery and rnai effects was an immunodeficient mouse with an inoculated intracranial human u87 glioma tumor that was dependent on egf signaling for growth. one of the major barriers for macromolecules to travel to diseased tissue in the brain is the blood-brain barrier (bbb). passing the bbb is a major challenge faced for the development of any pharmacological compound with high molecular weight. pegylated (polyethylene glycol) immunoliposomes (85 nm size liposomes designed with monoclonal antibodies over its outer surface) were able to support transvascular delivery of plasmid dna and to target and transduce specific cells in the brain. this group conjugated the pegylated liposomes with two monoclonal antibodies, one against the mouse transferrin receptor to enable bbb crossing, and the second against the insulin receptor to enhance cellular uptake. the generated pegylated immunoliposomes encapsidated a plasmid payload that was designed to express the sirna against the egf receptor in the transduced cell. the sirna complex of pegylated immunoliposomes showed an enhanced antitumor effect by prolonging survival of animals. the ability to treat brain tumors with a systemic approach rather than by stereotactic injection is a major achievement. recently, it has been reported that recombinant hbv capsids can be used as efficient vehicles for oligonucleotide delivery; they can encapsulate the oligonucleotides in vitro, and mediate their delivery into cells very efficiently. the process is not cell-type-specific. this method may be useful for in vivo systems for hbvinfected individuals or in other diseases provided that the immunogenicity of the viral capsids can be decreased; until then, it can be used in cell culture and in ex vivo systems [138] . the potential advantage of viral-mediated sirna delivery encouraged numerous groups to clone expression cassettes in transgenes and to encapsidate these into viral particles. each type of viral vector holds specific properties. these viral vector characteristics should be those that determine which viral delivery system needs to be applied for the specific therapeutic target. the adenovector [139] , and in particular the ad-gutless vector, hold major promise for liver-directed systemic delivery. in cases where short-term silencing effect is warranted, the non-gutless vector could then be applied; however, for prolonged silencing effects, the gutless vector might be more beneficial. in the gutless vector, there are practically no restrictions as to the size of the sequences to be incorporated and these could include marker genes, regulatory controlled cassettes or matrix-controlled regions for prolonged expression. controlling the expression of an sirna, specifically in tumor cells, could also be designed in a conditionally replicating adenovirus (crad). crads are designed to replicate and specifically kill tumor cells without harming normal cells. carette et al. [140] applied crad, which is dependent on rb deficiency for replication, to test its potential in silencing expression by shrnas, a marker gene in vitro in tumor cells. they showed that the silencing effect of a marker gene is dependent on crad replication. the combination of the crad antitumor effect with a sirna against a tumor-dependent growth gene should be assessed in the future; this possibility was not considered by this group, whether in vitro or in vivo. these issues would need further studies in an effort to assess and enhance their therapeutic potential. the major advantage of the retroviral delivery method is the potential to incorporate the payload transgene they 'carry' into the host cell genome. the integration site could not be specifically targeted and this could cause side effects. integration that occurs near cellular protooncogenes can lead to their aberrant expression from the viral ltr, or alternatively this could cause the disruption of a tumor suppresser gene expression. in the past 2 years, numerous groups have reported on various retroviral deliveries [141] , including lentiviral systems to express sirnas against viral pathogens and tumor cells. ralf bartenschlager and associates reported the use of the moloney murine leukemia virus (mo-mulv)-based vector (pbabe) as a delivery system for sirna targeting hcv [22] . in their publication, they also assessed a unique rnai approach against hcv infection. this was done in an effort to overcome the low fidelity of the viral polymerase, establishing a state of quasispecies by generating endoribonuclease-prepared sirna to simultaneously target multiple sites of the viral genome in order to prevent escape. as for the retroviral delivery approach, this group designed their sirna mainly against the viral ires sequences. their readout system to assess the silencing effect involved tissue culture cells transfected with the subgenomic hcv replicon. this replicon harbors the hcv ires upstream of the luciferase gene and the neomycin resistance region, and an additional non-hcv ires upstream of the viral non-structural sequences. the presence of the luciferase gene enables the determination of hcv ires activity in vitro. this system enables the assessment of the effect of sirna against the hcv ires as well as against the viral non-structural genome. the sirna in the retrovirus was expressed from the h1 promoter. they designed numerous sirnas in the vector and found that a specific region in the hcv ires, near the beginning of the viral coding nucleotides, was the most sensitive to rnai effect. although this is an interesting approach with clearcut results, significant developmental steps and modifications are required in order to translate this modality into the clinical setting. however, the results described in this report again suggest that the rnai encounters a therapeutic potential for chronic viral infection. similar observations were reported by other groups ( [18, 24] , see table 1 ). presently, most groups who use retroviral vectors to express sirna apply lentiviral vectors. however, it must be kept in mind that the clinical experience with this vector is very limited. numerous papers were recently published describing differently designed lentiviral vectors to meet copyright [121] or conditioning [142, 143] of sirna expression. veerle baekelandt and associates have designed a study to assess the potential use of lentiviruses in delivering sirna into brain tissue. in their recent report [144] , they constructed a lentiviral vector with sirna against the marker gene egfp. upon simultaneous administration into the brain tissue by stereotactic injection of the lentivirus expressing the egfp and the lentiviral vector expressing the sirna against the same gene, they were able to show almost complete knockdown of egfp expression. in two additional experiments in which the sirna lentivirus was administered before or after the marker gene, they were also able to show significant silencing of expression. interestingly, they claimed, albeit without showing the data, that this effect persisted for 6 months. however, the issue of delivery is again a major barrier. stereotactic administrations are possible, but alternative approaches of systemic delivery or intra-organ spreading of the vector would be beneficial. lentiviruses hold major promise in gene therapy. once the issue of integration and production is overcome, we expect that the lentiviralbased vector will be integrated into the clinical setting. the aav vector is currently perceived as a relatively safe vector since it supports long-term expression in most tissues from an episome. beverly davidson and associates reported an interesting study on the suppression of polyglutamine-induced neurodegeneration in a mouse model of spinocerebellar ataxia (sca1), a disease of the polyglutamine-expansion group which also includes huntington chorea [145] . they expressed the sirna under a modified cmv promoter due to its enhanced expression/silencing effect compared to the pol iii promoter, in their hands. in addition, they revealed that incorporating the mir23 loop (10-nt loop sequence) into the sirna expression cassette enhanced the silencing effect, resulting in improved suppression of the ataxin protein levels [118] . however, this effect was only apparent in the pol iii expression vector. how to select the best loop in any specific case is still an open question, and, presently, this is a matter of empiric assessment. in their mouse model, they injected the aav vector expressing the shrna against the mutant sca1 message directly into the brain tissue. this treatment showed longterm therapeutic effect on motor coordination as well as a histological improvement by reducing intranuclear inclusions. although this represents a step forward from previous studies with similar models that used antisense and ribozymes as therapeutic agents, we are still far from the clinic. the direct administration of a viral vector into brain tissue is a significant drawback for the current delivery systems. the potential side effects of aav administration into the brain may soon be revealed once results of clinical studies using the aav for direct brain administration in parkinson and alzheimer disease studies are available [146] . one specific point of importance should be mentioned on the issue of designing loop sequences stated previously. a number of investigators have assessed this matter as related to the effectiveness of silencing. we must bear in mind that for each specific case, there is a need to develop a specific structure to improve the silencing effect [147] . chemically modified oligonucleotides (oligos) vs. si/shrna as antiviral drugs the jury is still out as to when an antisense approach should be adopted against viral infections or when an si/shrna strategy should be applied. although antisense oligos were discovered more than 25 years ago [148] their role as antiviral tools in the clinic is still in progress. early studies with antisense oligos have shown promising antiviral potency. however, later reports determined that such approaches encounter significant problems. although comprehensive stringent comparison studies in vitro and in vivo between antisense oligos and sirna were not performed, we would like to stress a few practical points which characterize each group of compounds, in particular those which are important for those investigators interested in designing an antiviral strategy. antisense oligos with a high phosphorothioate content, which are early-generation antisense, interact directly and non-specifically with proteins, potentially interfering with their function [149] . however, recently developed 2nd and 3rd generation rnas -like oligos -have improved significantly the binding affinities of antisense oligos, as well as their nuclease and non-specific proteinbinding characteristics. the new generations include 2 -o-methyl (2om) and the additional version 2 -omethoxyethyl (2ome), both encountering improved nuclease resistance, binding affinity and reduced nonspecific binding affinity. recently, additional novel chemically modified versions of antisense oligos were developed including the locked nucleic acids (lna) [150] , anhydrohexitol nucleic acids (hnas), peptide nucleic acids (pnas), morpholino nucleic acids (mnas), and other uncharged oligos. all these novel chemicals have been tested and proven to hold significant antisense properties, with antiviral effects in models in vitro and in vivo. their effects are due to the high binding affinity and nuclease degradation resistance, without significant rnaseh activity. to overcome this hurdle, a chimeric version of oligos was generated, the gapmers, containing a core region of a phosphodiester/phosphorothioate flanked on both sides with modified oligo backbones. these gapmers also hold the rnaseh activity of traditional antisenses. when designing any specific experiment aimed to assess an antisense effect, it is important to select the best fitted oligo by testing 10 to 20 different sequences, and compare the selected oligo with appropriate controls containing scrambled, mismatched and irrelevant antisense oligos. although a significant amount of investigations would be needed to confirm which specific antiviral nucleic acid approaches would be best fitted for a specific disease therapy, by comparing antisense or ribozymes and sirna, some general guidelines can be phrased: (1) for acute viral infections, the preferred type of rna therapy could be a synthetic designed antisense or sirna. (2) for chronic viral disease targets, a preferred treatment would be such that continuously generates an antiviral drug. this could include an expression vector for any type of nucleicacid-based drug, including antisense, ribozyme or shrna. (3) for diseases of simple accessible organs, such as ocular cavity, oral cavity, vagina and epidermis, synthetic rnabased drugs might be beneficial. the need for repeated administration might be simple even in chronic disease states in cases of accessible organs. (4) for chronic multiorgan, or internal organ, involved in viral infections, e.g., chronic viral hepatitis b or c, intracellular continuous expression of high-level non-toxic, effective therapy is desirable. in this case, an expression system delivered into infected cells is warranted. designed shrna based on up-to-date criteria of shrna [151] [152] [153] might be the preferred gene therapy based treatment. (5) special attention must be given as to the specific type of virus to be targeted; whether a dna or a rna agent. furthermore, it is important to know where, inside the cellular compartments, should the rna-based drug be concentrated, e.g., cytoplasmatic vs. nuclear. for rna viruses, such as hcv, which replicate in the cytoplasm, an antisense approach could be suggested, although it should be kept in mind that the rnaseh effect is preferentially restricted to the nuclear compartment of the cell. however, for chronic hepatitis b virus infection, in which there is a nuclear reservoir of the virus in the form of super-coiled species, a vector which will express its shrna payload in the nuclear compartment is preferred. numerous new expression systems were recently suggested to encounter improved silencing properties [147] . most of these methods are waiting to be assessed as for their truly beneficial properties as antiviral reagents. in our modern world, viral infections still pose a major threat to mankind. since viruses are developing resistance to the current available therapies, there is an ongoing battle between the viruses and our ability to develop novel strategies to fight them. in vitro and in vivo experiments carried out so far conceivably demonstrate the effectiveness of rnai in inhibiting many viruses that cause severe health and economical problems. even though in vivo experiments in larger animals as well as developing efficient delivery methods have to be done before applying rnai in humans, this fascinating phenomenon will undoubtedly continue to provide new and exciting data regarding its mechanism of action and therapeutic applications in the years to come. strategies and mechanisms for host and pathogen survival in acute and persistent viral infections strategies for containing an emerging influenza pandemic in southeast asia potent and specific genetic interference by double-stranded rna in caenorhabditis elegans rnai: doublestranded rna directs the atp-dependent cleavage of mrna at 21 to 23 nucleotide intervals an rnadirected nuclease mediates post-transcriptional gene silencing in drosophila cells rna interference is mediated by 21-and 22-nucleotide rnas role for a bidentate ribonuclease in the initiation step of rna interference duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants initiation and maintenance of virus-induced gene silencing rna silencing in plants -defense and counterdefense prevention of hiv-1 infection in human peripheral blood mononuclear cells by specific rna interference inhibition of hiv-1 infection by small interfering rna-mediated rna interference rna interference against retroviruses in vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by e6 sirna selective silencing of viral gene e6 and e7 expression in hpv-positive human cervical carcinoma cells using small interfering rnas silencing structural and nonstructural genes in baculovirus by rna interference inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas rna interference blocks gene expression and rna synthesis from hepatitis c replicons propagated in human liver cells small interfering rna-mediated inhibition of hepatitis c virus replication in the human hepatoma cell line huh-7 clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas alternative approaches for efficient inhibition of hepatitis c virus rna replication by small interfering rnas interference of hepatitis c virus rna replication by short interfering rnas inhibition of hepatitis c virus protein expression by rna interference interfering with hepatitis c virus rna replication suppression of hepatitis c virus replicon by rna interference directed against the ns3 and ns5b regions of the viral genome rna interference in adult mice interference of hepatitis a virus replication by small interfering rnas local rna target structure influences sirna efficacy: systematic analysis of intentionally designed binding regions suppression of hepatitis a virus genome translation and replication by sirnas targeting the internal ribosomal entry site short interfering rna confers intracellular antiviral immunity in human cells attenuation of dengue virus infection by adeno-associated virus-mediated sirna delivery rna silencing of dengue virus type 2 replication in transformed c6/36 mosquito cells transcribing an inverted-repeat rna derived from the virus genome rna interference, arthropod-borne viruses, and mosquitoes the utility of sirna transcripts produced by rna polymerase in down-regulating viral gene expression and replication of negative-and positivestrand rna viruses inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells a small interfering rna targeting coxsackievirus b3 protects permissive hela cells from viral challenge cross-inhibition to heterologous foot-and-mouth disease virus infection induced by rna interference targeting the conserved regions of viral genome modulation of hiv-1 replication by rna interference rotavirus gene silencing by small interfering rnas rotavirus replication: plus-sense templates for double-stranded rna synthesis are made in viroplasms sequence homology required by human immunodeficiency virus type 1 to escape from short interfering rnas poliovirus escape from rna interference: short interfering rna-target recognition and implications for therapeutic approaches down-regulation of viral replication by adenoviral-mediated expression of sirna against cellular cofactors for hepatitis c virus the polypyrimidine tract-binding protein (ptb) is required for efficient replication of hepatitis c virus (hcv) rna cellular rna helicase p68 relocalization and interaction with the hepatitis c virus (hcv) ns5b protein and the potential role of p68 in hcv rna replication hpv31 e7 facilitates replication by activating e2f2 transcription through its interaction with hdacs specific inhibition of hiv-1 replication by short hairpin rnas targeting human cyclin t1 without inducing apoptosis specific inhibition of hiv-1 gene expression by double-stranded rna sirna-directed inhibition of hiv-1 infection rna interference directed against viral and cellular targets inhibits human immunodeficiency virus type 1 replication bispecific short hairpin sirna constructs targeted to cd4, cxcr4, and ccr5 confer hiv-1 resistance inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using tat-or ccr5-specific small interfering rnas expressed from a lentivirus vector inhibition of hiv-1 fusion with small interfering rnas targeting the chemokine coreceptor cxcr4 rna interference directed against poly(adp-ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells inhibition of human immunodeficiency virus type 1 replication by rna interference directed against human transcription elongation factor p-tefb (cdk9/cyclint1) suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication protection from hiv-1 infection of primary cd4 t cells by ccr5 silencing is effective for the full spectrum of ccr5 expression potent suppression of hiv type 1 infection by a short hairpin anti-cxcr4 sirna suppression of chemokine receptor expression by rna interference allows for inhibition of hiv-1 replication cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 rna interference of hiv replication lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells modulating hiv-1 replication by rna interference directed against human transcription elongation factor spt5 inhibition of respiratory viruses by nasally administered sirna the promise of sirnas for the treatment of influenza inhibition of coxsackievirus b3 replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand inhibition of sars-cov replication by sirna actively replicating west nile virus is resistant to cytoplasmic delivery of sirna use of rna interference to prevent lethal murine west nile virus infection an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection small interfering rna inhibits hepatitis b virus replication in mice short interfering rna-directed inhibition of hepatitis b virus replication inhibition of hbv replication by sirna in a stable hbv-producing cell line inhibition of hepatitis b virus expression and replication by rna interference inhibition of hepatitis b virus in mice by rna interference effective inhibition of hbv replication in vivo by anti-hbx short hairpin rnas knock-down of hepatitis b virus x protein reduces the tumorigenicity of hepatocellular carcinoma cells genomic analysis of anti-hepatitis b virus (hbv) activity by small interfering rna and lamivudine in stable hbv-producing cells small hairpin rnas efficiently inhibit hepatitis c ires-mediated gene expression in human tissue culture cells and a mouse model inhibition of hepatitis c virus translation and subgenomic replication by sirnas directed against highly conserved hcv sequence and cellular hcv cofactors lentiviral delivery of short hairpin rnas protects cd4 t cells from multiple clades and primary isolates of hiv hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome evidence that hiv-1 encodes an sirna and a suppressor of rna silencing hiv-1 nef suppression by virally encoded microrna regulation of human immunodeficiency virus 1 transcription by nef microrna lentiviral sirnas targeting multiple highly conserved rna sequences of human immunodeficiency virus type 1 identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy computational design of antiviral rna interference strategies that resist human immunodeficiency virus escape rnai induction and activation in mammalian muscle cells where dicer and eif2c translation initiation factors are barely expressed rna silencing as a plant immune system against viruses induction and suppression of rna silencing by an animal virus nucleic acid-based immune system: the antiviral potential of mammalian rna silencing adenosine kinase inhibition and suppression of rna silencing by geminivirus al2 and l2 proteins effects and side-effects of viral rna silencing suppressors on short rnas the coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step a viral protein inhibits the long range signaling activity of the gene silencing signal a viral protein suppresses rna silencing and binds silencing-generated, 21-to 25-nucleotide double-stranded rnas virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway p1/hc-pro, a viral suppressor of rna silencing, interferes with arabidopsis development and mirna unction suppression of posttranscriptional gene silencing by a plant viral protein localized in the nucleus interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing a virus-encoded inhibitor that blocks rna interference in mammalian cells adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis suppression of rna interference by adenovirus virus-associated rna human immunodeficiency virus type 1 escapes from rna interferencemediated inhibition human immunodeficiency virus type 1 escape from rna interference hepatitis c virus replicons escape rna interference induced by a short interfering rna directed against the ns5b coding region inhibition of hepatitis b virus gene expression by single and dual small interfering rna treatment expression profiling reveals off-target gene regulation by rnai activation of the interferon system by short-interfering rnas cationic liposome-mediated delivery of sirnas in adult mice small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna a system for stable expression of short interfering rnas in mammalian cells determinants of interferon-stimulated gene induction by rnai vectors an enhanced u6 promoter for synthesis of short hairpin rna short hairpin type of dsrnas that are controlled by trna(val) promoter significantly induce rnaimediated gene silencing in the cytoplasm of human cells promoter choice affects the potency of hiv-1 specific rna interference development and application of sirna expression vector conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible rna interference short-term cytotoxic effects and longterm instability of rnai delivered using lentiviral vectors efficient delivery of sirna for inhibition of gene expression in postnatal mice rna interference in mammalian cells by chemically-modified rna rna interference targeting fas protects mice from fulminant hepatitis in vivo activity of nuclease-resistant sirnas functional anatomy of sirnas for mediating efficient rnai in drosophila melanogaster embryo lysate sirna function in rnai: a chemical modification analysis functional anatomy of a dsrna trigger: differential requirement for the two trigger strands in rna interference challenges for rnai in vivo activity of stabilized short interfering rna in a mouse model of hepatitis b virus replication sirna relieves chronic neuropathic pain potent and persistent in vivo anti-hbv activity of chemically modified sirnas inhibition of influenza virus production in virus-infected mice by rna interference protection against lethal influenza virus challenge by rna interference in vivo atelocollagenmediated synthetic small interfering rna delivery for effective gene silencing in vitro and in vivo intravenous rna interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells adenovirus vectormediated doxycycline-inducible rna interference conditionally replicating adenoviruses expressing short hairpin rnas silence the expression of a target gene in cancer cells inducible, reversible, and stable rna interference in mammalian cells cre-lox-regulated conditional rna interference from transgenes cre recombinase-inducible rna interference mediated by lentiviral vectors lentiviral vector-mediated delivery of short hairpin rna results in persistent knockdown of gene expression in mouse brain rnai suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia first parkinson gene therapy trial launches optimization of an sirna-expression system with an improved hairpin and its significant suppressive effects in mammalian cells inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide antisense oligonucleotides: promise and reality lna: a versatile tool for therapeutics and genomics rational sirna design for rna interference asymmetry in the assembly of the rnai enzyme complex functional sirnas and mirnas exhibit strand bias attenuation of sars coronavirus by a short hairpin rna expression plasmid targeting rnadependent rna polymerase silencing sars-cov spike protein expression in cultured cells by rna interference using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque rna interference targeting vp1 inhibits foot-and-mouth disease virus replication in bhk-21 cells and suckling mice small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism susceptibility of human hepatitis delta virus rnas to small interfering rna action rna interference against enterovirus 71 infection inhibition of porcine endogenous retroviruses by rna interference: increasing the safety of xenotransplantation enhanced gene silencing of hiv-1 specific sirna using microrna designed hairpins expression of small hairpin rna by lentivirus-based vector confers efficient and stable gene-suppression of hiv-1 on human cells including primary non-dividing cells inhibition of hiv-1 by lentiviral vector-transduced sirnas in t lymphocytes differentiated in scid-hu mice and cd34+ progenitor cellderived macrophages efficient gene transfer of hiv-1-specific short hairpin rna into human lymphocytic cells using recombinant adeno-associated virus vectors inhibition of virus production in jc virus-infected cells by postinfection rna interference inhibition of the epstein-barr virus lytic cycle by zta-targeted rna interference selective inhibition of hepatitis b virus replication by rna interference clearance of hepatitis b virus from the liver of transgenic mice by short hairpin rnas short interfering rna-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes the investigators are supported by the israeli ministry of science, through a grant to the national gene therapy knowledge center and through the ec grant lshb-ct-2004-512034. the investigators are also supported by the blum, grinspoon, horowitz & wolfson foundations and an isf grant. we would like to thank michal gropp and hilla giladi for carefully reading the review. key: cord-347319-lcuma3eh authors: ashfaq, usman a; javed, tariq; rehman, sidra; nawaz, zafar; riazuddin, sheikh title: lysosomotropic agents as hcv entry inhibitors date: 2011-04-12 journal: virol j doi: 10.1186/1743-422x-8-163 sha: doc_id: 347319 cord_uid: lcuma3eh hcv has two envelop proteins named as e1 and e2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. fusion of the hcv envelope proteins is triggered by low ph within the endosome. lysosomotropic agents (la) such as chloroquine and ammonium chloride (nh(4)cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular ph. to investigate the antiviral effect of la (chloroquine and nh(4)cl) on ph dependent endocytosis, hcv pseudoparticles (hcvpp) of 1a and 3a genotype were produced and used to infect liver cells. the toxicological effects of chloroquine and nh(4)cl were tested in liver cells through mtt cell proliferation assay. for antiviral screening of chloroquine and nh(4)cl, liver cells were infected with hcvpp of 3a and 1a genotype in the presence or absence of different concentrations of chloroquine and nh4cl and there luciferase activity was determined by using a luminometer. the results demonstrated that chloroquine and nh(4)cl showed more than 50% reduction of virus infectivity at 50 μm and 10 mm concentrations respectively. these results suggest that inhibition of hcv at fusion step by increasing the lysosomal ph will be better option to treat chronic hcv. hepatitis c virus (hcv) is an enveloped, positive stranded rna virus classified in the family of flaviviridae. hcv causes acute and chronic hepatitis infection which can eventually lead to permanent liver damage, hepatocellular carcinoma and death. it is estimated that three to four million people are infected with hcv every year [1] . hcv genome encodes a single polyprotein precursor of just over 3000 amino acids. this polyprotein precursor is co-and posttranslationally processed by cellular (signal peptidase and signal peptide peptidase) and viral (ns2-3 and ns3-4a) proteases to yield four structural (core, e1, e2 and p7) and six non structural (ns2, ns3, ns4a, ns4b, ns5a, ns5b) proteins. hcv envelope is formed by e1 and e2 glycoprotein heterodimers which are essential for virus entry into cells [2] . hcv e1 and e2 fusion was enhanced at low ph, suggesting that hcv enters cells via the endosomal pathway and that e1 and/or e2 undergo conformational modifications that allow fusion of viral and cellular membranes [3] . there are six major and more than 80 subtypes of hcv. this classification is based on nucleotide variation among different hcv isolates. they occur in different proportion in different parts of the world. genotype 1a and 1b are the most common genotypes in the united states and europe [4, 5] . the most prevalent hcv genotype in pakistan is 3a followed by 3b and 1a [6] . presently, there is no vaccine available for prevention of hcv infection due to high degree of strain variation. current therapeutic options for hepatitis c are limited, especially for genotype 1. for genotypes 2 and 3, pegylated interferon in combination with ribavirin, can lead to a sustained virological response in up to 80% of patients [7] . however, this therapy is expensive and often associated with side effects that may lead to discontinuation of therapy [8] . hemolytic anemia, cough, shortness of breath & treatogenicity are the most common adverse effect associated with ribavirin treatment, and muscle aches, fatigue & neuropsychiatric adverse effects of ifnα lead to premature cessation of therapy in 10 to 20% of patients [9, 10] . moreover, cost of interferon for 6 month treatment ranging from pkr 50,000 to 150,000 is beyond the financial range of most patients. hence, there is a need to develop anti hcv agents, which are less toxic, more efficacious and cost-effective. the term "lysosomotropic agent" was introduced by deduve and co-workers (1974) to designate substances that are taken up selectively into lysosomes. this definition leaves open the chemical nature of a lysosomotropic substance and the mechanism of its uptake. lysosomotrpic agents such as nh 4 c1, chloroquine and methylamine penetrate acidic compartments of the cell and accumulate as protonated forms, resulting in an increase in the intravesicular ph [11] [12] [13] . chloroquine, which is widely used for the treatment of malaria, is a well-established inhibitor of autophagic proteolysis which acts by inhibiting acidification of lysosomes and endosomes [14] . it has been reported that lysosomotropic agents such as chloroquine and nh 4 cl exert direct antiviral effects on several rna viruses including coronaviruses, flaviviruses and human immunodeficiency virus (hiv) [15] [16] [17] [18] . moreover, clinical studies have demonstrated the safety, tolerability, and efficacy of lysosomotropic agents in the antiviral treatment of hiv infection [19, 20] . in the current study hcv entry is blocked by lysosomotropic agents. firstly toxicological analysis of chloroquine and nh 4 cl were done in liver cells. after toxicological analysis, antiviral effects were studied in hcvpp of 1a and 3a genotype. huh-7 and hek 293 t cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal calf serum, 100 iu/ml penicillin and 100 μg/ ml streptomycin, at 37°c in an atmosphere of 5% co 2 . huh-7 was kindly provided by dr. zafar nawaz (biochemistry and molecular biology department, university of miami, usa. the pcdna-e1e2 expression vector encoding the e1 and e2 glycoproteins (171-746) of hcv genotype 3a and 1a, was generated by inserting into a nonpackageable, cmv promoter-driven expression construct. the cmv-gag-pol murine leukemia virus (mlv) packaging construct, encoding the mlv gag and pol genes, and the ptg-luciferase plasmid provided by dr jaean dubison, france. hcvpp were produced by co-transfection of 293-t cells with equal amounts of three expression vector as described previously [21] . supernatants containing hcvpp were harvested 48 h later, filtered through 0.45 μm pore-sized membranes and stored at -80°c before use in infection of huh7 cells. mtt (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) is a rapid and sensitive in-vitro procedure for evaluating cellular toxicity of compounds. the mtt substance is reduced by mitochondrial succinic dehydrogenases in living cells to purple formazan crystals that are not soluble in aqueous water. the absorption of dissolved formazan in the visible region correlates with the number of viable cells [21] . to investigate cellular toxicity, 2 × 10 4 huh-7 cells were plated into 96-well plates. after 24 h, different concentrations of chloroquine and nh 4 cl were added and the plate was sealed and kept at 37°c in an atmosphere of 5% co 2 for 24 h. after 24 h, fresh media (100 μl) and mtt solution (5 mg/ml in pbs) were added to all wells in columns 1-11. wrapped the plate in aluminium foil and incubated for 3-4 h at 37°c. media was carefully removed and added 100 μl of dmso to dissolve the formazan crystals in columns 1-11. mtt formazan product was determined by measuring absorbance with an enzyme-linked immunosorbent assay (elisa) plate reader at a test wavelength of 570 nm and a reference wavelength of 620 nm. cell viability was obtained using the following equation. percent cell viability = test 570 nm − 650 nm/control 570 nm − 620 nm × 100 to investigate anti-fusion effect of chloroquine and nh 4 cl, huh-7 cells were incubated in the presence or absence of chloroquine and nh 4 cl at 37°c for 30 min. after 30 min huh-7 cells were infected with hcvpp of 3a and 1a genotype in the presence or absence of different concentrations of chloroquine and nh 4 cl and incubated for additional 24 h. after 24 h cells were lysed and luciferase activity was determined by using a luminometer. all statistical analysis was done using spss software (version 16.0, spss inc). data are presented as mean ± sd. numerical data were analyzed using student's t-test and anova. p value < 0.05 was considered statistically significant. mtt assay is a widely used test for evaluating cytotoxicity of compounds/herbs in cell cultures. the mtt substance is reduced by mitochondrial succinic dehydrogenases in living cells to purple formazan crystals that are not soluble in aqueous water. the absorption of dissolved formazan in the visible region correlates with the number of alive cells [21] . cytotoxic effects of chloroquine and nh 4 cl were analyzed after 24 h incubation of huh-7 cells with different concentrations of compounds. figure 1 showed that cell proliferation of liver cells is unaffected at high concentration. after toxicological analysis through mtt proliferation assay, antiviral activities of chloroquine and nh 4 cl were tested at non toxic concentrations. enveloped viruses enter cells through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. fusion of the viral envelope protein(s) is triggered by low ph within the endosome. lysosomotropic agents such as chloroquine and nh 4 cl, have been used to demonstrate the ph sensitivity of virus entry. we therefore tested the infectivity of hcvpp after treatment of target cells with different concentrations of chloroquine and nh 4 cl. hcvpp of 1a and 3a genotype demonstrated dose-dependent inhibition in the presence of chloroquine and nh 4 cl. chloroquine and nh 4 cl showed greater than 50% reduction of virus infectivity at 50 μm and 10 mm concentration respectively, suggesting a ph-sensitive route of virus entry (figure 2a and 2b ). hcv entry is a multistep process requiring four cellular receptor, fusion and endocytosis. hcv fusion depends on e1 and e2, viral dose, and occurs within a specific ph range. targeting ph dependent endocytosis is a useful tool to identify antiviral drugs against. a major advancement to look into hcv entry process was the development of hcvpp, consisting of native hcv envelope glycoproteins, e1 and e2, assembled onto [3, 22, 23] . this system is potentially very powerful tool to identify and characterize molecules that block hcv entry. in this study, hcvpp of local hcv genotype 3a and 1a were produced to study early entry steps mediated by hcv envelope glycoproteins. this assay is based on the quantification of retroviral dna synthesis, which occurs soon after the fusion of the retroviral particle with a cellular membrane. presumably, this assay is only dependent on the entry steps mediated by the heterodimer e1e2 (binding, endocytosis, and fusion) and on the activity of the reverse transcriptase of the hcvpp retroviral core. the intracellular sub-compartments such as lysosomes and endosomes have an acid nature with ph of about 5 [14] . lysosomotropic agents such as chloroquine and nh 4 cl are weak bases which have a tendency to accumulate in these compartments. lysosomotropic agents are captured by protonation inside the lysosomes and accumulate there (figure 3 ). the ratio of intra/extra lysosomal concentrations of these substances is equal to the ratio of concentration of hydrogen ions in lysosomes and in their vicinity, i.e. 1 : 100 if we suppose that ph in lysosomes is 5 and in cytoplasm 7. the amount of the permeable form of lysosomotropic agents passing through the membrane depends on the substance pka value and ph value of solution. the higher the pka value, the lower the permeable form ratio. this has led to the assumption that the specific uptake of lysosomotropic substances into lysosomes depends on the acidic ph of these compartments, ion-trapping weak bases [13] . in this model, the most suitable weak bases are those with a pka around 8. nh 4 cl and chloroquine are lysosomal weak bases that are known to affect acid vesicles leading to dysfunction of several proteins. previous studies of chloroquine and 4 cl have demonstrated that it has multiple effects on mammalian cells in addition to the elevation of endosomal ph, including the prevention of terminal glycosylation of immunoglobulin's [24] . when added to virusinfected cells, chloroquine inhibited later stages in vesicular stomatitis virus maturation by inhibiting the glycoprotein expression at the cell surface [25] , and it inhibited the production of infectious hiv-1 particles by interfering with terminal glycosylation of the glycoprotein [26, 27] . increase in ph by chloroquine and nh 4 cl, firstly inhibits low ph confirmational changes that triggers fusion, penetration and uncoating and secondly inhibits posttranslational modification of hcv enveloped proteins (e1 and e2) (figure 4 ). our results demonstrated that, chloroquine and nh 4 cl inhibit hcvpp entry in a dose-dependent manner at non toxic concentration. chloroquine and nh 4 cl resulted in greater than 50% reduction of virus infectivity at a concentration of 50 μm and 10 mm respectively, suggesting a ph-sensitive route of virus entry (figure 2a and 2b) . in conclusion, inhibition of hcv entry by increasing the ph through lysosomotropic agents is an important target to identified new drugs against hcv and combination of lysosomotropic agents with interferon will be better option to treat chronic hcv. hepatitis c virus infection the role of the hepatitis c virus glycoproteins in infection hepatitis c virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles geographical distribution of hepatitis c virus genotypes in blood donors: an international collaborative survey group atcs: hepatitis c virus type 1b (ii) infection in france and italy frequency distribution of hepatitis c virus genotypes in different geographical regions of pakistan and their possible routes of transmission chronic hepatitis c virus infection treatment of chronic hepatitis c with pegylated interferon and ribavirin ribavirin and interferon alfa-2b in chronic hepatitis c: assessment of possible pharmacokinetic and pharmacodynamic interactions side effects of therapy for chronic hepatitis c weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes van hoof f: commentary. lysosomotropic agents effect of weak bases on the intralysosomal ph in mouse peritoneal macrophages chloroquine is a potent inhibitor of sars coronavirus infection and spread effects of chloroquine on viral infections: an old drug against today's diseases? anti-hiv effects of chloroquine: mechanisms of inhibition and spectrum of activity different ph requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza a viruses comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1 hydroxychloroquine, hydroxyurea and didanosine as initial therapy for hiv-infected patients with low viral load: safety, efficacy and resistance profile after 144 weeks rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays infectious hepatitis c virus pseudoparticles containing functional e1-e2 envelope protein complexes cell surface expression of functional hepatitis c virus e1 and e2 glycoproteins chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine inhibition of human immunodeficiency virus infectivity by chloroquine anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors lysosomotropic agents as hcv entry inhibitors financial support by higher education commission pakistan is highly acknowledged. authors' contributions uaa, tj and sdr contributed equally in lab work and manuscript write up. zn and srd were the principal investigator and provide all facilities to complete this work. all the authors read and approved the final manuscript. usman ali ashfaq (phd molecular biology), imran shahid (m phil molecular biology), tariq javed (m. phil pharmaceutical chemistry, sidra rehman (msc chemistry) and sheikh riazuddin (phd molecular biology and dean post graduate study at allama iqbal medical college, lahore the authors declare that they have no competing interests. key: cord-350600-73q8mve4 authors: myint, s.; siddell, s.; tyrrell, d. title: detection of human coronavirus 229e in nasal washings using rna:rna hybridization date: 2005-12-09 journal: j med virol doi: 10.1002/jmv.1890290113 sha: doc_id: 350600 cord_uid: 73q8mve4 a method is described for the detection of human coronavirus 229e (hcv 229e) in nasal washings using rna:rna filter hybridization. volunteers were inoculated with hcv 229e, and daily nasal washings were collected. these washings were then examined for the presence of viral rna using a single‐stranded rna probe. nucleic acid hybridization is shown to be a sensitive technique for the diagnosis of hcv 229e infections. coronaviruses are a group of positive-strand rna viruses that cause a wide spectrum of disease in mammals and birds [siddell et al., 19831 . human coronaviruses are thought to cause about 15% of all common colds [monto, 19821 and have also been associated with lower respiratory tract infection [isaacs et al., 1983; mcintosh et al., 19741 . other disease associations have been suggested but are less well documented [mac-naughton and davies, 1981; riski and hovi, 19801. part of the difficulty in defining the role of hcv in disease is the difficulty in detecting the virus. currently this is dependent on culture of the virus, in either cell monolayers or organ culture, which has the disadvantage of being a lengthy procedure requiring specialist skills. immunofluorescence has been used [mcintosh et al., 19781 but has not been shown to be reliably sensitive. human coronaviruses can be divided into four serological groups, of which the oc38143 and 2293 groups cause the overwhelming majority of coronavirus-associated colds. in this paper we describe a specific and sensitive test to detect one of these major groups, hcv 2293, in nasal washings. materials t7 rna polymerase was supplied by pharmacia. boehringer mannheim supplied the restriction enzymes. 32p-labelled nucleotides were purchased from amersham international. promegaibiotec supplied the plasmid pgem-1 and rq1 dnase. vanadyl-ribonucleoside complex was purchased from bethesda research laboratories. all other chemicals were supplied by sigma. cdna cloning and subcloning the isolation of hcv-specific cdna clones will be described in detail elsewhere (myint et al., submitted) . briefly, using a method based on that of gubler and hoffmann [19831, cdnas were generated from hcv 2293 rna isolated from infected c16 cells [phillpotts, 19831. one cdna, which contained the entire open reading frame of the nucleocapsid gene, was inserted into the polylinker region of the "riboprobe vector," pgem-1. this plasmid, psmgf1, has promotor sequences for sp6 and t7 rna polymerases flanking the multiple cloning site, and thus single-stranded, hcvspecific rna transcripts can be generated. probe preparation rna probes were transcribed and labelled with 32p using the following reaction: 4 p1 5 x transcription buffer (0.2m tris hc1, ph 7.5, 30 mm mgc12, 50 mm nac1, 10 mm spermidine), 2 pl 100 mm dtt, 0.8 pl rnasin (25u/p1), 1 p1 2.5 mm atp, 1 p1 2.5 mm gtp, 1 pl 2.5 mm utp, 2.2 pl 100 pm ctp, 2 pl (1 pg) hind111 linearised psmgfl dna, 5 p1 "p-ctp (10 pci/pl), 1 pl t7 polymerase (10 u/pl). this was incubated at 37°c for 1 hr. then 1 p1 of rq1 dnase (1 pg/pl) was added and the reaction incubated again a t 37°c. after 15 min the reaction was stopped and deproteinised by phenol extraction. the aqueous phase was then precipitated overnight at -20°c by the addition of 10 pl of 7.5 m ammonium acetate and 75 pl ethanol. after centrifugation, the rna precipitate was resuspended in 100 pl te (10 mm tris hci, ph 7.5, 1 mm edta) buffer. for 6 h r at -70°c. positive signals were identified visually and by densitometry. virus in nasal washings was titrated by an end-point dilution method in flat-bottomed microtitre wells. then 5 x lo4 c16 cells were inoculated into each well of a microtitre plate and allowed to attach at 37°c for 2 hr. six 10-fold dilutions of 100 p1 nasal washing that had been stored without vrc were made in c16 growth medium. each dilution was inoculated into four wells of a row of a microtitre tray, the last two rows being used as cell controls. after 24 h r the medium was replaced with fresh c16 maintenance medium, and again at 5 days. after 10 days, the plates were fixed in formol-saline for 4 h r and stained with crystal violet. the tcidbo titre was estimated using the formula of reed and muench. the methods used have been described by callow 119851. specific igg was measured in sera collected prior to virus challenge and in sera collected 2-3 weeks after challenge. clinical score volunteers were assessed daily by a clinician who ascribed a clinical score on the basis of systemic and local symptoms and local signs. this score, along with the clinician's judgement, was used to grade the clinical illness into one of five categories: no cold, doubtful cold, mild cold, moderate cold, or severe cold (further details have been given by beare and reed [19771) . the results of virus titration and probing of nasal washings from seven volunteers are presented in table i . the elisa data are given as supportive evidence of infection. a ratio of 1.5 or greater is taken to indicate infection. figure 1 shows a typical autoradiograph of washings from three volunteers, only one of whom suffered a cold. three of the seven volunteers suffered a cold, and all three volunteers had detectable coronavirus rna in their nasal washings. none of the asymptomatic volunteers had detectable viral rna in their nasal washings. no virus was cultivated from these patients. table i1 shows a comparison of the sensitivity and specificity of virus isolation and the hybridisation method. there were no false positives or false negatives. however, there was serological evidence of infection in three volunteers who did not shed virus. the results we have obtained show that the detection of hcv 2293 infection by nucleic acid hybridisation is a reliable and specific method. it is rapid, it does not depend on having cultures of susceptible cells available, and it does not require trained personnel to recthe rna probe has been characterised regarding its sensitivity and specificity. details of this characterisation will be described elsewhere (myint et al., submitted) . hybridisation to known quantities of hcv 2293 rna showed that less than 1 ng of virus-specific rna from c16 cells infected with hcv 2293 could be detected. hybridisation to the rna of 42 common cold viruses showed that only the hcv 2293 group was detected. nasal washings nasal washings were collected from seven volunteers. details of the method of collection and design of trials at the common cold unit have been described by beare and reed [1977] . nasal washings were collected prior to challenge with hcv 2293 at a titre of 100 tcid50/ml and on the second to the sixth day thereafter. washings were collected in two aliquots. the first 1 ml of washings was collected into an empty pot, and the rest was collected directly into 500 p1 of a 200 mm stock solution of vanadyl-ribonucleoside complex (vrc). the vrc concentration was adjusted to 20 mm end-concentration once the final volume of nasal washing from each volunteer was known. nasal washings were then stored in an equal volume of nutrient broth at -70°c until required. at the end of the trial, nasal washings were thawed out and subjected to protein digestion in the following reaction: 500 p1 nasal washings, 60 pl proteinase k x 10 buffer (0.1 m tris hc1, ph 7.8,0.05 m na edta, 5% sds), and 10 pl proteinase k (20 mg/ml, stock). the reaction was allowed to proceed at 37°c for 1 hr, and then the proteins were extracted with phenol. one hundred microlitres of 3 m sodium acetate was added to 450 pl of the aqueous phase, and the nucleic acid was precipitated at -70°c for 30 min. after centrifugation at 13,ooog for 10 min, the precipitate was resuspended in 100 pl te buffer and mixed with 100 pl of 6.15 m formaldehyde/lo x ssc (1 x ssc is 0.15 m naclo.01 m sodium acetate, ph 7.0). the material was then applied directly to a nitrocellulose filter using a schleicher and schuell slot-blotting manifold. a positive control, 5 ng of poly a-selected rna from hcv 2293infected c16 cells, and a negative control, 50 ng of poly a-selected rna from uninfected c16 cells, were also applied. after baking a t 80°c for 2 hr the filters were incubated in hybridisation buffer (50% formamide, 50 mm sodium phosphate, ph 6.5, 5~ ssc, 0.1% sds, 0.05% ficoll, 0.05% pvp, 200 pg/ml denatured herring sperm dna) at 56°c for at least 4 hr. the probe was added, and hybridisation was allowed to proceed at the same temperature for 16 hr. the nitrocellulose filter was washed three times in 0 . 1~ ssc/o.l% sds at 65"c, each wash being 20 min. autoradiography was clinical scoreb ognise the rather uncharacteristic cytopathic effect of hcv infection. the results also indicate that the method is as sensitive as the procedures for virus titration used in this study. however, it is probable that virus titration may not be as sensitive as other isolation procedures, such as adaption to tissue culture by blind passage. indeed, the results of the igg immunoassay we performed suggest that three of the seven volunteers were infected, although we were not able to isolate virus. on the other hand, we have not yet tried to optimize fully the specific radioactivity of the rna probe, nor have we systematically investigated the optimal hybridisation conditions. we believe the sensitivity of the hybridisation method can also be significantly increased. despite these limitations it is clear that this nucleic acid hybridisation method is applicable to the diagnosis of coronavirus infections in the clinical setting. we intend to evaluate this method further in volunteers and in field trials, and we are sure it will prove to be a useful epidemiological tool in such studies, particularly as a large number of specimens can be simultaneously examined. it is also our intention to modify the test, in particular, by adaption to a nonradioactive labeling system. it could then be used as the primary test for the diagnosis of hcv 2293 infections. indeed, it might be the only detection method applicable in certain situasuch as in detecting virus bound to an antibody a c g fig. 1 . hybridisation analysis of hcv 2293 rna in nasal washings from three volunteers (volunteers a, c, and g in table i ). open arrowhead: 50 ng of poly a' rna from uninfected c16 cells was immobilised on the nitrocellulose filter; closed arrowhead: 5 ng of poly a ' rna from hcv 229e-infected c16 cells were immobilised. or drug. chemoprophylaxis and virus infections of the respiratory tract effect of specific humoral immunity and some non-specific factors and resistance of volunteers to repiratory coronavirus infection a simple and very efficient method for generating cdna libraries epidemiology of coronavirus respiratory infections human enteric coronaviruses. brief review coronavirus infection in acute lower respiratory tract disease of infants diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalised children viral infections of humans clones of mrc-c cells may be superior to the parent line for the culture of 229e-like strains of human respiratory coronavirus coronavirus infections of man associated with diseases other than the common cold coronaviridae-third report of the coronavirus study group we thank barbara schelle-prinz for excellent technical assistance and kerstin griebel for preparation of the manuscript. the volunteer experiment carried out as part of this work was approved by the northwick park hospital ethical committee. this work was supported by grant st25-0165-1-d from the european commission. key: cord-284376-plwyjhl8 authors: fu, xinmiao; ying, qi; zeng, tieyong; long, tao; wang, yan title: simulating and forecasting the cumulative confirmed cases of sars-cov-2 in china by boltzmann function-based regression analyses date: 2020-05-31 journal: journal of infection doi: 10.1016/j.jinf.2020.02.019 sha: doc_id: 284376 cord_uid: plwyjhl8 • cumulative confirmed cases in china were well fitted with boltzmann function. • potential total numbers of confirmed cases in different regions were estimated. • key dates indicating minimal daily number of new confirmed cases were estimated. • cumulative confirmed cases of 2003 sars-cov were well fitted to boltzmann function. • the boltzmann function was, for the first time, applied to epidemic analysis. in this journal, zhu et al. recently reported the results of their genomic analysis of multidrug-resistant klebsiella pneumoniae isolates from individual patients before and after colistin treatment highlighting the rapid emergence and multifaceted molecular mechanisms of colistin resistance in k. pneumoniae. 1 this work highlights the therapeutic and public-health challenges of colistinresistance (cr), which is increasingly used as a large resort antibiotic, despite its unattractive toxicity profile and narrow therapeutic window. 2 oral non-absorbed colistin has been proposed as a decontamination strategy in intensive care units and for patients carrying multidrug resistant enterobacterales (mdr-e). 3 , 4 the impact of decolonization strategies in terms of emergence of cr has rarely been monitored because no reliable selective medium existed and cr was not considered a public-health problem. recently, reliable universal culture media have been developed to screen for cr. 5 here, we studied the impact of non-absorbed oral colistin on the emergence of cr in the gut microbiota of patients from the rgnosis-wp3 randomized controlled trial. 6 thirty-nine subjects colonized with mdr-e were randomized to receive oral colistin sulfate 2 miu 4 times a day + neomycin sulfate 500 mg bid for 5 days followed by a fecal microbiota transplant (fmt) from healthy donors, or no intervention. stool samples were collected on visit v0 (screening sample), v2 (after 5 days of oral decontamination and before fmt for the intervention group), v3, v4 and v5, respectively 5-10 days, 30-40 days and 150-210 days later. 6 stool samples from donors and 15 subjects from the intervention group and 15 from the control group were available for this work and plated on drigalski plates (control) and superpolymyxin r plates. 5 colony forming units (cfu) counts of all gram-negative rods were determined. isolates growing on superpolymyxin r plates were identified by maldi-tof; cr was confirmed by the culture-based rapid polymyxin np test and mic determined by the microdilution method. the limit of detection was 10 2 cfu/g of stool. cr-e. coli were sequenced using the illumina hiseq technology. to determine whether cr isolates were present before the intervention, a specific mcr-1 pcr was performed on patients stool prior to intervention (v0) and on the donor's stool. 7 electroporation of plasmids was performed to localize the gene conferring resistance to colistin and neomycin and molecular typing of the electroporants was performed using pcr based replicon typing (pbrt). ✩ université de paris, iame, inserm, umr-1137 no patient or donor included in the trial carried cr isolates on v0. among the 15 patients in the intervention group two (13.3%, [ic90 −1; 3], p = 0.24) carried cr isolates at least at one visit after the intervention ( fig. 1 ) . no cr-enterobacterales was detected in the stools of subjects from the control group. among both subjects with cr-enterobacterales, one carried 10 log cfu/g of hafnia paralvei , a species which is intrinsically resistant to colistin (mic = 8 mg/l), also resistant to neomycin (mic = 32 mg/l) on visit 4 and the other carried 1 log cfu/g and 3 log cfu/g cr-e. coli at visits 4 and 5, respectively, with a colistin mic at 8 mg/l. relative abundance of cr-e. coli increased between v4 and v5 from 0.01% to 1% of the total enterobacterales population. the cr-e. coli recovered at v4 and v5 both belonged to phylogroup c st23 group and carried the serotype o176:h9. a plasmid-borne mcr-1.1 gene encoding for cr as well as a aph(3 )-ia gene conferring resistance to neomycin were identified, both being co-located on the same inchi2 plasmid. in addition, resistance genes conferring resistance to hygromycin b ( aph(4)-ia ), sulfonamides ( sul2 ), tetracyclines ( tet(a) ) and phenicols ( flor and cata1 ), all antibiotics used in veterinary medicine, were evidenced. for both subjects, cr strains could not be retrieved in the initial stool of the subject or in the donor's stool. pcr experiments performed with specific primers to detect mcr-1 gene directly on the pre-therapeutic stool were also negative. to our knowledge, this is the first report of the in-vivo selection of cr-enterobacterales in the gut microbiota of patients after oral decontamination by colistin. the selection of cr strains (a naturally-resistant h. paralvei and a mcr-1 producing e. coli ), both resistant to colistin and neomycin, may be the result either of the enrichment process by sod of preexisting cr strains that had not been initially detected because of very low abundances, or of an exogenous acquisition, either from other individuals or through fmt. 8 indeed the transmission from fmt of mdr strains from positive donors is a potential risk. 8 despite our efforts to decrease the limit of detection of mcr producers by using a pcr technique directly on the pre-therapeutic stool sample and the donors' stools, we failed to detect the parental strain, either because cr strains were in intestinal niches, the limit of detection remained too high, or the strain was acquired exogenously. however, the mcr-1 -positive e. coli is likely of animal origin according to its genetic features and its co-resistance profile. 9 indeed, phylogroup st23 is frequently encountered among avian pathogenic e. coli (apec) and co-resistances to many antibiotics used specifically in veterinary medicine is striking. 10 furthermore, the aph(3 )-ia gene confers resistance to neomycin and paromomycin, the latter commonly used in cattle and pigs. the selection of the mcr-1 producer is an illustration of the "one health" problem of resistance: a strain likely to have been selected by veterinary antibiotics among animals ended up in a patient's gut, later enriched by the use of colistin and neomycin as decontaminant. although the small number of subjects is a clear limitation, this observation is a "proof-of-concept" of the risk of selection of cr-enterobacterales after oral colistin treatment and fmt, at a time when colistin is one of the last resort antibiotics to treat mdr-enterobacterales infections. the selection of commensal cr-e. coli is especially worrying, given the pathogenic potential of e. coli and its ability to widely colonize animals and humans. given the controversial results of oral decontamination by colistin, we believe it should only be used with precautions and with thorough monitoring of cr. we read with interest a recent paper in this journal by luzatti and colleagues, 1 who explored the significance of the presence of herpes simplex virus (hsv) dna in lower respiratory tract (lrt) specimens for the diagnosis of hsv pneumonia in immunocompromised patients. the authors underlined the difficulty in gauging the clinical relevance of such a laboratory finding in the absence of histopathological data, as hsv shedding in the lrt may occur in the absence of disease. the interpretation of real-time pcr results for diagnosis of pneumocystis jirovecii (pj) pneumonia (pjp) faces an analogous challenge, since the presence of pj dna in lrt may reflect colonization (carriage) rather than infection. 2 there is limited information on the clinical value of pj real-time pcr in diagnosing pjp in patients with hematological diseases; 3-6 this is exceedingly challenging as the sensitivity of direct examination procedures is suboptimal due to low fungal burdens. 3 here, we report on our experience on this matter. a total of 219 episodes of pneumonia occurring in 192 consecutive patients with hematological disorders in which pjp was considered in the differential etiological diagnosis were included. of these, 127 episodes developed in patients undergoing either allogeneic hematopoietic stem cell transplantation-allo-hsct-( n = 86) or autologous-hsct ( n = 19), and 92 in non-transplant patients (acute leukemia, n = 61; lymphoma, n = 16; chronic leukemia, n = 8; others, n = 2). the patients were attended at the hospital clínico universitario-hcu-( n = 100) or at the hospital universitario politécnico "la fe" -hlf-( n = 92) between june 2014 and august 2019. no patients in the cohort tested positive for hiv. this study was approved by the respective hospital ethics committee and informed consent was obtained from all patients. a single specimen per episode was available for diagnosis (bal fluids, n = 179; sputa, n = 22; ta, n = 17 and bronchial biopsy, n = 1). the realcycler pjir kit r (progenie molecular, spain) was used at hcu, and the pneumocystis jirovecii real time pcr detection (certest biotech; zaragoza, spain) was employed at hlf (see footnote in table 1 ). both assays target the large sub-unit of ribosomal (mtlsu) rna gene. preliminary experiments using 5 bal specimens indicated that both assays yield comparable pcr cycle thresholds (c t s) (median, 28.2, range, 26.4-32.3 vs. median 27.5; range, 26.3-33.1, respectively; p = 0.89). all specimens tested negative by direct examination for pj, whereas 27 were positive by real-time pcr (bal, n = 18; sputa, n = 7, and ta, n = 2); following stringent clinical, microbiological and imaging criteria ( table 1 ) , pjp was deemed to be the most probable diagnosis in 12 episodes occurring in unique patients. no histopathological confirmation of pjp was available for any patient. pcr c t values inversely correlate with fungal burden in the sample. 6 which is higher in patients with pjp than in colonized individuals. 2 here, overall, pj pcr c t s in specimens from patients with pjp tended to be lower than in pj carriers ( p = 0.39); when only bal fluid specimens were considered, the difference reached statistical significance (median, 29.0; range, 26.4-34.7 vs. median 34.6; range, 30.0-41.0; p = 0.04). this finding is likely related to use of more standardized procedures for bal fluid sampling. receiver operating characteristic (roc) curve analysis showed that a threshold c t value of 30.0 in bal specimens displayed a sensitivity of 85.7% (95% ci, 45.0-100%) and a specificity of 80% (95% ci, 40.8-100%) for pjp diagnosis. a number of studies have established different c t s cut-offs for that purpose, [6] [7] [8] [9] . in our view, however, the variability in the performance of different pcr assays and sampling conditions, heterogeneity of patient populations, and in particular the lack of a pj international standard material for pcr result normalization precludes defining a consensus universal threshold nowadays. the absence of anti-pj prophylaxis, treatment with corticosteroids and serum ldh levels ≥400 u/l have been shown to be associated with pjp. 3 here, patients not undergoing anti-pj prophylaxis were more likely to display a clinically significant pj pcr result ( table 1 ). in turn, roc curve analysis indicated that a cut-off ldh value ≥400 u/l had a sensitivity of 81.8% (ci 95%, 59.0-100%) and specificity of 67% (95% ci, 34.0-99.3%) for pjp diagnosis. in univariate regression logistic models, serum ldh values ≥400 u/l were associated with a clinically significant positive pcr pj result (or, 9.0; 95% ci, 1.2-63.8; p = 0.02). in contrast, corticosteroid use within the month before sampling was not different between the probability of pneumocystis jirovecii (pj) pneumonia (pjp) for each patient was retrospectively evaluated by an expert committee including infectious diseases and microbiology specialists at both centers, on the basis of (i) documented pj presence in respiratory specimens by microscopy; (ii) compatibility of clinical signs and symptoms (at least 2 of the following: subtle onset of progressive dyspnea, pyrexia, nonproductive cough, hypoxaemia and chest pain), (iii) compatible (suggestive) radiological findings (chest radiograph and/or high-resolution computed tomographic scan detection of interstitial opacities and/or diffuse infiltration infiltrates); (iv) complete resolution of symptoms after a full course of anti-pjp treatment; (v) absence of alternative diagnosis. the efficacy of therapy was assessed on a daily basis. pjp was ruled out if real-time pcr for pj tested negative, or if clinical recovery occurred in the absence of pj-targeted antimicrobial therapy. pj colonization (carriage) was the most likely possibility when patients did not meet the above criteria and an alternate diagnosis was made. b frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. two-sided exact p values were reported and p values ≤ 0.05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. c respiratory tract specimens were obtained following conventional procedures. specimens were examined for presence of ascus or trophic forms of pj by microscopy following blue toluidine, calcofluor white or grocott's methenamine silver staining. cytospin preparations were prepared from bal specimens for direct examination. sputa and ta samples were mixed v/v with sputasol (oxoid, uk) and vortexed for 5 min. all samples were centrifuged at 30 0 0 g for 10 min, and the pellets were resuspended 1/10 in 0.9% nacl for further processing. for real-time pcr, dna was extracted from 200 μl of specimens using the qiaamp dna blood mini kit (qiagen, hilden, germany) on either qia symphony or ez-1 platforms (qiagen), following the manufacturer's instructions. at hcu, a commercially-available real-time pcr assay previously evaluated by others, the realcycler pjir kit r (progenie molecular, spain), which targets the mitochondrial large sub-unit of ribosomal (mtlsu) rna gene, was used according to the manufacturer's instructions ( http://www.progenie-molecular.com/pjir-u-in.pdf ). at hlf, the commercially-available pneumocystis jirovecii real time pcr detection. (certest biotech; zaragoza, spain), which also targets the large sub-unit of ribosomal (mtlsu) rna gene, was employed following the manufacturer instructions ( https://www.certest. es/wpontent/uploads/2019/02/viasure _ real _ time _ pcr _ pneumocystis _ jirovecii _ sp1.pdf ). at both centers pcr were performed in the applied biosystems 7500 fast real-time pcr platform (applied biosystems, ca, usa). pcr results were reported as positive or negative. for positive samples, threshold cycle (c t ) values were also recorded. no standard curve was generated with a positive control for quantitative estimations. d antimicrobial prophylaxis for pjp was performed with trimethoprim-sulfamethoxazole (tmp/smx), one double-strength tablet (160 mg tmp/800 mg smx) given 2 (in allogeneic hsct patients) or 3 times a week with oral folic acid (15, 16) . patients with suspicion of pjp according to the attending physician were treated with tmp/smx 15-20 mg/kg (tmp) 75-100 mg/kg (smx) per day for 2-3 weeks. e in all these cases, death was attributable to pjp. patients with clinically significant pj detection and pj carriers ( table 1 ) . detection or recovery of other microbial agents (one or more) was documented in 17 of the 27 specimens testing positive by pj pcr ( table 2 ). in line with a previous report, 9 this microbiological finding was significantly less frequent ( p = 0.001) in specimens from patients with pjp than in colonized patients; in fact, microbial co-detection was inversely associated with pjp in univariate logistic regression models (or, 0.024; 95% ci, 0.002-0.26; p = 0.002). strengths of the current study are the following: (i) clinical categorization of pjp was based upon stringent criteria defined by a multidisciplinary team; (ii) only hematological patients were included; (iii) a comprehensive routine investigation of microbial causes of pneumonia other than pj was conducted; (iv) the experience of two centers was collected. in addition to its retrospective nature, our study also has some limitations: (i) we cannot completely rule out that some patients categorized as being pj carriers did in fact have pjp, as most of these patients received full courses of tmp/smx in combination with antimicrobials targeting other microbial agents. the lack of standardized criteria for pjp diagnosis makes clinical misclassification of patients a potential drawback in studies such as ours, particularly when no positive microscopy or histopathology findings are available; (ii) although we evaluated bal, bronchoalveolar lavage; pjp, pneumocysis jirovecii pneumonia; ta, tracheal aspirate. a as per our routine protocol, all specimens were examined by gram and acid-fast bacilli stain. samples were also examined for presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, or the clart® pneumovir assay (genomica, coslada, spain) at both centers, as previously reported. 10 semiquantitative (sputa) and quantitative (bal and ta) cultures for bacteria were performed on conventional media: bacterial loads > 10 4 cfu/ml or > 10 5 cfu/ml were deemed to be clinically relevant on bal fluids and ta samples, respectively. specimens were cultured on bcye-alpha agar, bd (becton dickinson) mgit® ( mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and other fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. all bal fluid specimens underwent cytomegalovirus (cmv) pcr testing using the realtime cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf, as previously reported. 10 over 200 patients, only 12 presumptively had pjp; (iii) two different commercially-available pcr assays were used across centers. nevertheless, we found them to yield rather comparable c t s. in summary, we found that a positive pj pcr result in respiratory specimens from transplant and non-transplant hematological patients with pneumonia frequently reflects colonization rather than infection; pcr c t values in bal fluids, serum ldh levels and lack of co-detection of other microorganisms potentially involved may be helpful in clinical categorization in the absence of positive by pj microcopy results. we have no conflict of interest to declare. dear editor , poller et al., in this journal, provided a useful consensus for use of personal protective equipment for managing high consequence infectious disease 1 . although this was driven largely by recent ebola virus disease emergencies, we should remind your readers of the continuing problem of lassa fever (lf) in west africa. lf is a febrile infectious disease caused by lassa virus. the clinical presentation of the disease is nonspecific and includes fever, fatigue, hemorrhage, gastrointestinal symptoms, respiratory symptoms, and neurological symptoms 2 . the observed case fatality rate among patients hospitalized with severe lf is 15-20% 3 , 4 . the disease is mainly spread to humans through contamination with the urine or faeces of infected rats 2 . human-to-human transmission can occur through contact with the body fluids of infected per-sons. therefore, health care workers are at high risk for infection when the standard precautions for infection prevention and control including appropriate personal protective equipment are inadequate 5 . it is estimated that there are approximately 30 0,0 0 0 lf cases annually, resulting in approximately 50 0 0 deaths in west african countries 4 . in 2018, nigeria had a large lf outbreak, and we previously reported epidemiological characteristics of the outbreak, analyzing data collected between january and may 2018. 6 however, information on laboratory-negative suspected cases was not enough to conduct a case-control study to fully determine the risk factors and clinical characteristics of the disease. nigeria had a lf outbreak in 2019 as well. 7 here we report the epidemiological and clinical characteristics of the outbreak including case-control analysis against laboratory-negative suspected cases using data collected between 1st january 2018 and 6th october 2019. from january to december 2018, there were 3498 suspected cases, including 633 laboratory-confirmed lf cases. in 2019, there were 4019 suspected cases reported by 6th october, including 721 laboratory-confirmed lf cases. details on the case definition, laboratory test, surveillance, and data collection have been described previously. 6 of the confirmed lf cases, there were 171 fatalities (case fatality rate, 27.0%) in 2018 and 154 fatalities (case fatality rate, 21.4%) in 2019. the number of laboratory-confirmed lf cases and positivity rate peaked in the dry season (january-march) in both 2018 and 2019 ( fig. 1 (a) ). the largest number of laboratory-confirmed lf cases were reported from the neighboring edo and ondo states in both 2018 and 2019 ( fig. 1 (b) ). there were laboratory-confirmed lf cases in states such as kebbi and zamfara that had no reported cases previously, in 2019. during the study period, the detailed demographic and clinical information was collected for 1305 laboratory-confirmed lf cases (of 1354 cases, 96.4%) and 3350 laboratory-negative suspected cases (of 6163 cases, 54.4%). chi-square tests were conducted to compare the distribution of age, sex, and each symptom between the laboratory-confirmed lf cases and laboratory-negative suspected cases ( table 1 ). the proportion of children was significantly lower in laboratory-confirmed lf cases compared with that in laboratory-negative suspected cases. the proportion of males was significantly higher in laboratory-confirmed lf cases than that in laboratory-negative suspected cases. fever was the most prevalent symptom in both laboratoryconfirmed lf cases and laboratory-negative suspected cases, followed by headache ( table 1 ) . gastrointestinal symptoms, such as abdominal pain, vomiting, and diarrhea, were observed in more than 30% of laboratory-confirmed lf cases, whereas hemorrhaging was observed in 21.1% of laboratory-confirmed lf cases. while the prevalence of face/neck edema was low even in laboratoryconfirmed lf cases (6.4%), nonetheless, the odds ratio of having face/neck edema was 6.0 times high for laboratory-confirmed lf cases. we here reported the lf outbreak in 2018-2019 largest recorded in history. while previous studies have focused on laboratory-confirmed lf cases and mainly compared fatal cases and survived cases, 6 , 8 , 9 our observation revealed the difference between laboratory-confirmed lf cases and laboratory-negative suspected cases. the age and sex distribution differed significantly between laboratory-confirmed lf cases and laboratory-negative suspected cases. fever, headache, and gastrointestinal symptoms were the most common symptoms in laboratory-confirmed lf cases, which are similar to those reported previously. 6 , 8 however, these symptoms were also prevalent in laboratory-negative suspected cases. clinical guidelines for lf state that edema in the face and neck is a specific sign of the disease. 10 the present study found that the symptom had a significantly high odds ratio for confirmed lf although the prevalence of this symptom was low. unfortunately, we did not determine the differential diagnosis for the laboratory-negative suspected cases. laboratory tests for the differential diagnoses are now underway for the lf-negative samples collected during the outbreak. the results would provide us further insight for better clinical management of patients with febrile illnesses in lf-endemic areas. in addition to the standard precautions for infection prevention and control including appropriate personal protective equipment pointed out by poller et al., 1 it is important to know epidemiological and clinical characteristics of high consequence infectious diseases such as lf. that would help healthcare workers and public health officers increase an index of suspicion of the diseases, further leading to better clinical management and surveillance. the authors have declared that no conflicts of interest exist. this work was partially supported by the leading initiative for excellent young researchers from the ministry of education , culture, sport, science & technology of japan and the japan society for the promotion of science (grant number, 16809810 ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 these authors contributed equally to this article. accepted 31 december 2019 available online 15 january 2020 https://doi.org/10.1016/j.jinf.2019.12.020 © 2020 the british infection association. published by elsevier ltd. all rights reserved. recently, several studies in this journal have demonstrated the threat of animal-derived viruses to humans. [1] [2] [3] since 2018, an increase in human pseudorabies virus (prv) infection cases has been reported in china, indicating a new animal-derived virus threat to human health. porcine pseudorabies (pr), also known as aujeszky's disease, is one of the most economically important viral diseases in pigs globally. its causative agent is prv, which is classified into the genus varicellovirus of subfamily alphaherpesvirinae , family herpesviridae . prv is almost always fatal in newborn piglets, is frequently accompanied by neurological symptoms, and may cause abortions and/or stillbirths in pregnant sows. prv primarily infects members of the suidae family and can also infect other domestic and wild mammals, including horses, cattle, sheep, goats, dogs, cats, etc. currently, vaccination is the most effective strategy for pr prevention and control in pigs worldwide. in china, prv infections in pigs were first recorded in 1947. in the 1970s, an inactivated vaccine consisting of the bartha-k61 vaccine strain was imported into china. since then, this vaccine has been widely used in pig vaccination for pr prevention and control. before 2011, no large pr outbreaks were reported in pigs in china. however, after late 2011, novel prv wild-type variants emerged in nearly all regions of china and affected a number of swine herds vaccinated regularly with the bartha-k61 vaccine, resulting in significant economic losses. 4 subsequent animal experiments indicated that the bartha-k61 vaccine could not provide complete protection for pigs against a challenge with novel prv wild-type variants in china. for control and eradication of pr, the disease was listed in the "mid-and long-term animal disease prevention and control program in china (2012-2020)" by the chinese government, with the aim of eliminating pr in china by 2020 ( http://www.gov.cn/zwgk/2012-05/25/content _ 2145581.htm ). however, vaccination for prv is still voluntary and not required in china. a nationwide epidemiological investigation in 2014 demonstrated a high prevalence of 23.26% of prv among swine herds in china. 5 humans were previously regarded as refractory for prv infection, although serological prv antibody positivity was found in three cases. in 2018, the first human prv infection case with direct molecular evidence was reported in china (case 1, table 1 ). 6 in this case, the eyes of a 46-year-old woman were directly exposed to sewage on a hog farm. in the following two weeks, symptoms of fever, headache, coma, and endophthalmitis were observed in the patient. next-generation sequencing (ngs) indicated that prv dna was detected in her vitreous humor samples but not in her cerebrospinal fluid (csf). after surgery, the patient was discharged, but her vision remained impaired. in a subsequent study, zhao et al. table 1 clinical characteristics and other information on the twelve human prv infection cases in china. analyzed csf samples from four patients with encephalitis of unknown etiology using ngs (cases 2-5, table 1 ) and found molecular evidence of prv infection. 7 in addition, retinitis and blindness was observed in two cases (cases 2, 5, table 1 ), and the patient in case 3 died. the occupation of the four patients was all associated with pork production/sale/cooking. in 2019, six other human prv infection cases involving encephalitis were reported in china, and all patients were pork/pig handlers or veterinarians. [8] [9] [10] it was noted that all patients still suffered from various sequelae after discharge, except for in one case where the patient died. increasing reports on human prv infection cases in china have recently indicated that prv poses a significant threat to public health in china, especially in people in close contact with sick pigs and/or related pork products/contaminants. to reduce the risk of prv infection in susceptible workers, it is necessary to promulgate relevant policies by the chinese government to promote pr vaccine development to protect pigs from infection with novel prv wild-type variants currently circulating in china. in addition, relevant policies should be updated by the chinese government to monitor vaccination status and virus variation in pigs nationwide. moreover, it seems that prv can infect humans via injury to the skin or eyes. until now, no effective drugs to prevent the progression of the disease caused by prv infection have been reported. therefore, it is necessary to improve biosafety and self-protection awareness in susceptible populations that have contact with sick pigs and work in jobs related to handling pork products/contaminants. promoting drug development for curing prv-related disease in infected patients may also help reduce the currently increasing threat of prv to human health in china. all authors declare that they have no competing interests. dear editor, the emergence and spread of gram-negative bacteria, for example, klebsiella pneumoniae , co-producing carbapenemases and mobilized colistin resistance ( mcr ) genes limit our choice for treating multidrug-resistant infections, posing significant threats to public health. herein, we reported the discovery of mcr-9 gene in 28 k. pneumoniae strains isolated from patients in eight european countries, including belgium ( n = 2), denmark ( n = 1), montenegro ( n = 13), poland ( n = 1), romania ( n = 1), serbia ( n = 1), slovenia ( n = 1), and spain ( n = 7). notably, the co-existence of mcr-9 and the carbapenemase-encoding genes, ndm-1, vim-1, and oxa-48 were confirmed in k. pneumoniae isolates of human origin. phylogenetic analysis suggested that mcr-9 -carrying k. pneumoniae isolates, including 23 carbapenem-resistant and five susceptible k. pneumoniae strains, show a highly geographically clustered pattern. genetic environment analysis revealed the presence of insertion element is903, is1 or a cupin fold metalloprotein, wbuc, in the mcr-9 flanking. taken together, these findings indicated that mcr-9 has existed for a long time and already spread among crkp isolates of human origin in europe since 2013, further increasing the significant threat of public health through either the nosocomial spread or environmental routes. the mobilized colistin resistance ( mcr ) gene mcr-9 was detected in 28 human gut microbiomes, which has been disseminating across three continents, including asia, europe and america, recently published in the journal of infection. 1 the rapid increase in carbapenem resistance among gram-negative bacteria worldwide has greatly compromised the efficacy of carbapenem antibiotics, which has gotten renewed attention to the importance of polymyxin antibiotics for multidrug-resistant (mdr) infections. recently, sophia david and colleagues 2 reported the epidemic of carbapenem-resistant klebsiella pneumoniae (crkp) in europe, which raised concern that mobile carbapenemase resistance determinants were widely spread in european hospital settings, and inter-hospital spread is far more frequent within, rather than between, countries. however, limited information regarding the co-occurrence of carbapenemases and mcr genes in the klebsiella pneumoniae ( k. pneumoniae ) isolates have been provided. plasmid-mediated resistance genes mcr as well as tet (x3/x4) have been widespread in bacterial species of animal, human, and environment origin as well as human and animal gut microbiomes worldwide, where is a huge arg reservoir with a high horizontal gene transfer possibility. 1 , 3 -5 several studies 6 -8 illustrated that the newly described mcr-9 has spread beyond the united states into europe and asia, and into other enterobacteriaceae species. of clinical concern is the inevitable spread of a plasmid harboring the mcr-9 gene into a crkp isolate, which has been listed by the world health organization as a critical priority antibiotic-resistant bacterial pathogen for which new antibiotics are urgently needed. 9 in response to this potential clinical problem, we download > 1717 k. pneumoniae genomes isolated from 244 hospitals in 32 european countries 2 and the 14,764 complete bacterial genome sequence (accessed 26 july 2019), and explored the distribution of plasmid-mediated resistance genes mcr and tet (x3/x4). we are surprised to find that the complete mcr-9 gene (nucleotide = 100%) was present in 28 k. pneumoniae isolates of 1717 k. pneumoniae strains from belgium ( n = 2), denmark ( n = 1), montenegro ( n = 13), poland ( n = 1), romania ( n = 2), serbia ( n = 1), slovenia ( n = 1), and spain ( n = 7) (table s1 ). additionally, 23 of the 28 k. pneumoniae isolates of human origin were crkp strains and only five were carbapenem-susceptive isolates (table s1 ). as reported, these mcr-9 -harbouring strains were isolated from patients in europe between 2013 and 2014. these results suggest that mcr-9 gene might have been presented in europe for a long time and already spread to the crkp isolates, which is a major cause of both hospital-and community-acquired infections. 2 to further analyze these mcr-9 -positive k. pneumoniae isolates, the resistome of the draft genome was analyzed using the comprehensive antibiotic resistance database. 10 interestingly, the mcr-9 gene was co-existed with various carbapenemase-encoding genes: in eleven isolates with ndm-1, eight with vim-1, and two with oxa-48 ( fig. 1 (a) ). it should be noted that the mcr-9 -and ndm-1-carrying isolates were distributed in denmark ( n = 1), montenegro ( n = 8), romania ( n = 1), and serbia ( n = 1), as well as several other beta-lactam resistance determinants (for example, tem-1, cmy-16, oxa-10 and shv-11) ( fig. 1 (a) and table s1 ). moreover, the mcr-9 -and vim-1-harbouring isolates were dominant in spain ( n = 7) and slovenia ( n = 1), as well as two beta-lactamase-encoding genes (non-carbapenemases) (ctx-m-9 and shv-11) ( fig. 1 (a) and table s1 ). it is worrying that a crkp isolate from spain in 2014 was carrying mcr-9 , vim-1, and oxa-48 genes simultaneously. the presence of mcr-9 in crkp isolates from patients is of critical importance as mcr-9 could be present in hospital-borne outbreaks cre strains in the future. from whole-genome shotgun (wgs) data of the 28 mcr-9 -positive k. pneumoniae isolates, sequences types (sts) were extracted and assigned to nine different types, i.e. , 274, 461, 15, 16, 416, 1890, 37, 1942, and 147 ( fig. 1 (a) ). phylogenetic analysis suggested that mcr-9 -carrying k. pneumoniae isolates show a highly geographical clustering pattern 2 ( fig. 1 (a) ). isolates from patients in the same hospital were clustered into one clade, for example, in spain and montenegro. overall, the k. pneumoniae isolates from different countries were genetically diverse, suggesting that the mcr-9 -positive k. pneumoniae isolates were also genetically diverse and that mcr-9 could disseminate among different k. pneumoniae isolates, mainly by nosocomial transmission. 2 nowadays, all known mcr genes have been detected in various gram-negative bacterial species, whereas a small number of studies have shown the presence of mcr-1, mcr-3, mcr-7 , and mcr-8 in k. pneumoniae isolates from animal and human origin at relatively low detection rate. 11 -14 the presence of mcr-9 in the crkp isolates indicated that this novel mcr gene may already be widely spread among k. pneumoniae isolates of human origin in europe. we subsequently searched mcr-9 gene in 14,764 complete bacterial genome sequence and ncbi-nr database (14 october 2019) in the ncbi, to fully understand the prevalence of mcr-9 gene in klebsiella species isolates. interestingly, the mcr-9 gene (identity > 99% and 100% query coverage) was present in various bacterial genomes, including three klebsiella species isolates consisting of k. pneumoniae ( n = 4), k. quasipneumoniae ( n = 1), and k. oxytoca ( n = 3) (table s3) . therefore, further studies focusing on the epidemiology and transmission mechanism of mcr genes, in particular mcr-9 in klebsiella species of human origin are warranted to better understand the public health threat of emergence of antibiotic resistance among clinical k. pneumoniae . contigs carrying mcr-9 in 28 k. pneumoniae isolates could be classified into two groups (for example, gca_900513855.1 and gca_900501685.1) (table s2) . genetic environments analysis indicated that the presence of insertion element is903 and wbuc (a cupin fold metalloprotein), in the mcr-9 (gca_900513855.1, ∼28 kb) upstream and downstream flanking, respectively, similar to (identity > 99%) the plasmid sequences of pme-1a, pctxm9_020038, and pmrvim0813, and contigs from of e. coli isolate 68a and nz_naan010 0 0 063 from salmonella 15 ( fig. 1 (b) ). additionally, mcr-9 in another contig ∼8.7 kb was in the upstream of two insertion element is1 and is30 0 0, as well as a beta-lactamase-encoding resistance gene ctx-m-9, which similar to the plasmid sequence of pmrvim0813. we did not detect the downstream regulatory genes (qsec and qseb) found in the isolates that harbor mcr-9 . 8 , 16 moreover, we were unable to determine whether a complete is903 element is upstream due to a short mcr-9 -bearing contig that is available for comparison ( fig. 1 (b) ). therefore, a long-read sequencing coupled with a hybrid assembly method is needed to fully evaluate and monitor the transfer and development of args, especially mcr-9 among crkp isolates. although two unique plasmid-mediated tigecycline resistance genes firstly discovered in bacteria of animal origin in china and subsequently identified in many bacterial isolates of human, animal and environment origin, including klebsiella species, as well as human and animal gut microbiomes, 1 , 4 , 5 none of them was detected in the 1717 k. pneumoniae strains in europe. in summary, we reported the discovery of mcr-9 gene in 28 clinical k. pneumoniae strains of human origin in eight european countries. importantly, the mcr-9 gene was co-existed with different carbapenemase-encoding genes in the same strains. the spread of mcr-9 , ndm-1, vim-1, and oxa-48 and other beta-lactam resistance determinants (non-carbapenemase) carrying by crkp appears likely to be by plasmid dissemination, as the genes identified in isolates belonging to a diverse set of sts distributed in different hospitals in europe. it is noteworthy that all these mcr-9 -positive crkp strains were isolated between 2013 and 2014, highlighting an earlier presence of mcr-9 among crkp around the world than previously known. these findings raise the likelihood of ongoing undetected mcr-9 gene spread among cre strains. therefore, further study is urgently needed to understand the prevalence and dissemination of mcr-9 , especially in cre and crkp strains, and effective measures should be taken to control its spread. g.f.g. designed the study. y.n.w. and f.l. collected and downloaded the datasets. y.n.w., f.l., y.f.h., b.l.z., g.p.z., and g.f.g. analyzed and interpreted the data. y.n.w. and g.f.g. wrote the draft of the manuscript. all authors discussed, reviewed and approved the final report. supplementary information is available for this paper. correspondence and requests for materials should be addressed to g.f.g. the authors declare no competing interests. the interesting systematic review by amin-chowdhury and colleagues provides information about outbreaks of severe pneumococcal disease (spd) in closed settings that occurred in the conjugate vaccines era 1 . it shows that vaccine-type spd outbreaks are still occurring and it highlights the lack of consensus on how to manage such outbreaks. in the following, we will describe how we managed a recent outbreak of spd in norway. in march 2019, møre and romsdal hospital trust notified the norwegian institute of public health (niph) about a cluster of spd amongst men working in shipyards in møre and romsdal county. serotype data from niph were available for nine of the cases -all were serotype 4. the majority of cases had been working at one specific shipyard. municipal medical officers (mmo), the norwegian labour inspection authority (nlia), and niph formed a multidisciplinary outbreak team to investigate and control the outbreak. we formed specific case definitions: each case had to have resided in møre and romsdal county in the period from 1. january 2019 onwards and: confirmed : had invasive pneumococcal disease (ipd) with serotype 4 isolated from a normally sterile site. probable : worked at the specific shipyard and had a clinical presentation compatible with lower respiratory tract infection or ipd, but without microbiological confirmation or serotype 4 isolated from a non-sterile medium (e.g. nasopharyngeal swab or sputum culture). we identified 20 cases, ten confirmed and ten probable in the period between 28. january and 3. april ( fig. 1 ). all available isolates were serotype 4 (10 confirmed, 7 probable) and were susceptible to penicillin. fifteen isolates were sequence type (st) 801, while two were a single locus variant of 801, st 15,063. all cases were men between 20 and 60 years, with a mean age of 47 years. fifteen were hospitalized. four were norwegian citizens, the remaining came from other european countries. seven cases smoked. one case had an underlying medical risk condition. immunization history against pneumococci were unknown for all. the cases had several professions; mostly related to interior outfitting and metal welding. approximately 1800 individuals worked at the shipyard in the time period. many of them lived in temporary accommodation. at an on-site inspection of the shipyard, nlia observed a polluted atmospheric work environment and little use of personal protective equipment. several measures were put in place to control the outbreak, including information and advice to raise symptom awareness and to reinforce hand and respiratory hygiene, vaccination and occupational corrections. local medical clinics and hospital were alerted about the outbreak and advised to have a low threshold to admit and treat suspected cases. mmo held information meetings with shift leaders, and written information about spd in several languages was distributed to workers to increase spd awareness. intensified hygiene measures were implemented at the ship yard and housing quarters. nlia ordered immediate occupational corrections related to controlling the atmospheric work environment. niph recommended vaccination with the 13-valent conjugate vaccine (pcv13) to interrupt transmission and prevent disease. both the pcv13 and the 23-valent polysaccharide vaccine provide protection against serotype 4, but pcv13 was preferred as this may also affect colonization. as several work tasks were conducted in parallel process in confined spaces with suboptimal ventilation, we were unable to identify a single target group for vaccination. hence, the shipyard offered vaccination to all workers. occupational health service promptly vaccinated all workers during a four-day period. contrary to the majority of studies included in the systematic review, niph did not recommend chemoprophylaxis. as the workers were otherwise healthy (i.e. no high risk group like old age, immunocompromising conditions etc.), and since it was impossible to target a specific group of workers, niph deemed it undesirable to distribute antibiotics to 1800 asymptomatic workers, with the possibility of inducing antimicrobial resistance and possible side effects. due to high turnover of personnel it was not possible to calculate an attack rate. we did not find any new cases after control measures were implemented. no deaths have been reported in relation to the outbreak. this outbreak closely resembles one of the outbreaks described in the systematic review; between april and june 2015, an outbreak with serotype 4, st 801 occurred at a shipyard in belfast 2 . we are also aware of an outbreak this fall, 2019, at a shipyard in finland with serotype 4 (st 801), 8 and 12f 3 . although welders are a known risk group for spd, 4 in all these three outbreaks, people who worked closely alongside welders were also infected. in addition to exposure to welding fumes, the crammed and poorly ventilated working conditions, and possibly housing conditions, may have increased the risk of developing spd and facilitated the transmission of pneumococci in this closed setting. overall, this norwegian outbreak extends the knowledge about how to manage and control outbreaks of spd in closed settings. none. in this journal brunet and colleagues 1 discussed reactivation of latent infections in the context of chronic disease, solid organ transplantation or long-term immunosuppressive treatment. we recently observed the reactivation of leishmania infection in a 46-year-old patient receiving methotrexate for psoriasis, who was diagnosed with visceral leishmaniasis (vl) showing a mucocutaneous involvement. we analyzed the epidemiologic and clinical characteristics of all cases of leishmaniasis in patients with psoriasis found through a review of the literature. our patient was admitted into the infectious disease unit of paolo giaccone hospital, in palermo, with a painless and ulcerated lesion onto the oral mucosa ( fig. 1a ) , two nodular ulcerated lesions on the right knee and another one on instep of the right foot appeared one month before ( fig. 1b ) . the patient did not travel outside italy during the last year. he had been suffering from lowgrade fever in the last month. considering the above findings leishmaniasis was suspected and a needle aspiration of oral and cutaneous lesions was arranged in order to perform microscopy and leishmania-pcr, which were positive for leishmania. laboratory tests exhibited: wbc 4970/mmc, hb 13.9 g/dl, c reactive protein, 26.9 mg/l; positive serology for leishmania (igg 1/3200) and positive leishmania-pcr test on peripheral blood. abdominal us examination revealed splenomegaly (14 cm); methotrexate was suspended and liposomal amphotericin b, 4 mg/kg per day for 10 days, followed by two further administrations two weeks later was started. cutaneous and mucosal lesions improved at the end of the first 10 days of therapy and completely vanished after two further administrations, 40 days from the beginning of treatment. leishmania-pcr on peripheral blood after 10 days of therapy was negative. table 1 shows the literature data about characteristics, therapy and outcome of patients with psoriasis and leishmaniasis. leishmaniasis is a vector-born chronic infectious disease caused by protozoa of the genus leishmania and transmitted to humans by the bite of phlebotomine sandflies. in europe, the mediterranean countries are the most affected areas. leishmania parasite establishes chronic intracellular parasitism, survives for an infected person's lifetime and, in the event of major immune deficiency, may be reactivated from sites of latency. leishmaniasis can present with a spectrum of clinical manifestations and three patterns of infection are described: cutaneous (cl), mucosal or mucocutaneous (ml or mcl) and visceral leishmaniasis (vl). the infecting species of leishmania is very important in determining the clinical manifestations and the host immune response is crucial in determining the clinical outcome of infection 2 . today, non-hiv related immunosuppressive conditions are becoming increasingly prevalent, mainly because of better medical care of patients with chronic illnesses and the therapeutic use of immunosuppressive drugs. in the field of rheumatology, leishmaniasis has been reported in association with the use of various immunosuppressive drugs. 3 the introduction of tumor necrosis factor-alpha (tnf-α) antagonist drugs has received much attention recently and several cases of vl have been reported in rheumatic patients who do anti-tnf α drugs. 4 psoriasis is a chronic inflammatory autoimmune disease affecting 2-3% of the world's population and characterized by an aberrant hyper-proliferation of keratinocytes. the pathogenesis of psoriasis is complex. genetic susceptibility, environmental triggering factors and an over-reaction of local innate immune response initiate inflammation. subsequent involvement of adaptive immune response with production of th 1 cytokines, chemokines and growth factors lead to epidermal hyperplasia. 5 recently, a functional role of interleukin-17-producing t helper cells (th17) in psoriasis has been suggested by their reduction during successful anti-tnf treatment. 6 it is also known that th17 lymphocytes play an essential role in protecting against intracellular protozoa and in the successful clearance of leishmania by strengthening the th1 response. 7 in view of this, it could be argued that psoriasis may represent a protective factor for leishmania infection. indeed, in our review we did not found any case of leishmaniasis in psoriatic subjects who were not under immunosuppressive therapies. biological agents, which are powerful immunosuppressive drugs, have been more and more used in rheumatic patients and leishmania infections have been reported among anti-tnf-agents users. 8 recently maritati et al. found higher prevalence of subclinical leishmaniasis in patients with inflammatory rheumatic diseases receiving biological drugs than those treated with other immunosuppressive drugs. 9 however, leishmaniasis has also been reported in psoriatic patients not receiving biological drugs, as occurred to our patient ( table 1 ) . diagnosis of cl in psoriatic patients is challenging, as it mimics many other infections or a flare-up of psoriasis itself that can lead to ineffective and harmful changes of therapy. immunosuppressive therapies cause atypical manifestations of leishmaniasis with large lesions spread over large cutaneous areas and associated to a possible mucosal involvement. ml by l. infantum is very rare and only sporadically described in patients receiving powerful immunosuppressive therapies or in hiv-coinfected patients. mcl is mostly observed in latin america where l. braziliensis accounts for most cases, but l. panamensis, l. guyanensis, and l. amazonensis have also been implicated. only rarely cutaneous lesions extend to areas of skin distant from the mucosa involved, as in our case in which two lesions on the foot and knee were associated with the oral lesion. in the context of impaired immunity, it is also advisable to rule out vl by pcr-leismania on peripheral blood so as to establish the most appropriate therapy: intralesional or intravenous. finally, there is no agreement on appropriate screening for leishmaniasis before immunosuppressive treatments and on the strategy to be followed after the diagnosis of leishmaniasis in rheumatic patients taking immunosuppressive drugs. 10 molecular methods are highly sensitive and specific tools for the diagnosis of visceral leishmaniasis and a screening with leishmania-pcr in immunosuppressed patients living in endemic areas could be useful to identify patients at highest risk of reactivation. 9 specific leishmaniasis treatment followed by suspension of the immunosuppressive therapy was adopted by most of the authors. overall even if the treatment response is not as good as seen in the immunocompetent population, our review reports a good outcome in all cases and patients remained relapse-free without maintenance therapy and despite the ongoing use of immunosuppressive medication. in conclusion physicians must be alert to the possibility of development of leishmaniasis in immunosuppressed rheumatic patients. adequate screening for vl should be incorporated into the list of baseline studies to carry out before initiating biologic therapies, at least in endemic areas. the authors declare that there is no conflict of interest. as demonstrated in several studies in journal of infection , herpesviruses pose an increasing threat to human health. [1] [2] [3] according to international committee on taxonomy of viruses (ictv), equine herpesviruses (ehvs) belong to the family herpesviridae . until now, a total of 9 ehv species types have been determined in equines, viz. ehv1-ehv9. 4 among them, ehv1 and ehv4 are the most relevant herpesviruses affecting equines. both ehv1 and ehv4 infection are associated with upper respiratory tract disease, but only ehv1 infection could cause abortion and myeloencephalitis. ehv1 and ehv4 are prevalent in equines on all continents and have considerable economic impact on the horse industry. 5 in china, the number of equines is very large, reaching to be ∼ 6.8 million in 2018 ( http://www.stats.gov.cn/tjsj/zxfb/ ). ehv infection in equines was first reported in china in 1980, and the epidemiological investigation since then indicated ehv was prevalent in the equine population in all the studied 16 provinces in mainland china, with a seroprevalence ranging 8% −92%. [6] [7] [8] vaccination is commonly used to prevent and control ehv. however, china has not developed a commercially available ehv vaccine so far. ehv vaccine has a limited market application potential in china currently. due to the lack of relevant knowledge on ehv, most of the chinese horse owners always erroneously identified it as other common pathogen of equine respiratory diseases, and didn't realize its potential threat to equine health and reproduction. although the number of equines in china is large, most of them are labor/farming horses. to the best of our knowledge, even for racehorses, vaccination with ehv vaccine has not been performed in mainland china. considering the wide distribution and high prevalence of ehv in china, it is urgently to popularize knowledge on ehv in horse owners and promote market application prospects of ehv vaccine. in china, few veterinary researchers are currently investigating equine virus, including ehv. this is mainly caused by the change of equines' historical role. in the last century, a great number of equines were used for military in china. however, there is only one military equine farm in mainland china at present. considering a more important economic role of other domestic animals (e.g., pigs, chickens, and cattle) compared with equines, investigating equine virus (including ehv) is not a priority in the related guide policies issued by the chinese government. though epidemiological studies on ehv in china are limited, it still could be concluded that epidemic status of ehv is very complicated in china, which increases the difficulty in ehv vaccine development. in most provinces, ehv1 and ehv4 were co-circulating in equines with a high seroprevalence. 8 until now, a total of 7 ehv1 strains have been isolated from tissue samples of aborted equine fetuses (5 from farming horses in northeast china in 1980, 1 from asian wild horses in western china in 1999, 1 from farming horses in western china in 2016). 6 , 8 in addition, a novel ehv8 strain was isolated from one horse with serious respiratory disease in northern china in 2010. 9 recently, our laboratory firstly determined the molecule evinces for ehv4 and ehv5 in racehorses in sothern china (data not shown). however, a more large-scale and surveillance of ehv in equines is necessary to fully understand epidemic status of ehv in china, which could establish a foundation for updating the composition of ehv vaccine developed in china in future. in other countries, much effort has been made to develop ehv vaccine, and modified-live and inactivated virus vaccines have been registered for sale. 10 before an ehv vaccine is developed successfully in china by itself, it is necessary to vaccinate the susceptible equine population with an ehv vaccine commercially available from other countries to prevent and control ehv in china. however, a well-designed case-control animal challenge study still needs to estimate the protective efficacy of different vaccines against the field prevalent ehv strains in china. all authors declare that they have no competing interests. a recent review article on the treatment of hepatitis c with directly-acting antiviral (daa) drugs, makes numerous recommendations for baseline drug resistance testing. 1 in our local practice, we have been performing baseline drug resistance testing for some years now, prior to the publication of these guidelines. we present a recent retrospective hcv kinetics analysis of these patients' changing viral loads in response to daa therapy below. such studies have been used previously to compare viral suppressive responses in different hcv genotypes and treatment regimens. 2 , 3 the patients were a mixture of treatment-naive and treatment-experienced (including with interferon-based, ns3 protease inhibitor-based and daa-based regimens) cases. the current standard of care for hepatitis (hcv) patients is a combination of direct acting antivirals (daas), for which there are three different hcv viral protein targets (ns3, ns5a and ns5b). table 1 ns3, ns5a, ns5b resistance associated substitutions (ras), by hcv genotype, found in this patient cohort at baseline drug resistance testing (viral sequencing performed at imperial college, london, uk). the patients included a mixture of treatment-naïve and treatment experienced (i.e. interferon-based, ns3 protease inhibitor-based and more recent daa-based regimens) cases. resistance associated substitution (ras) by hcv genotype treatment with daas cure the vast majority of hcv-infected patients, with oral regimens having > 95% efficacy in most patient groups. 1 , 4 , 5 treatment failure currently affects approximately 5% of treated patients and is often associated with the selection of resistance associated substitutions (ras). 1 we performed hcv drug resistance testing both retrospectively (following treatment failures) and prospectively (prior to treatment) in our cohort of hcv genotype (g) 1-3-infected patients, during march 2015-june 2018. viral extraction, pcr and sequencing were performed at imperial college, using qiagen viral rna mini kits (qiagen pn:52906, qiagen ltd., manchester, uk), and inhouse pcr and sanger sequencing methods on an abi prism 3100-avant genetic analyser (thermo fischer scientific, loughborough, uk). the prediction of hcv genotype and drug sensitivities is derived from the geno2pheno algorithm [ www.geno2pheno.org ]. 6 treatment regimens used during this period complied with contemporaneous nhs rate cards: for non-cirrhotic or compensated cirrhotic patients: g1-treatment-naive: omb/par/rit + das + r; g1-treatment-experienced: elb/grz + /-r; g2-treatmentnaive/experienced: gle/pib; g3-treatment-naive/experienced: gle/ pib; g1-decompensated cirrhotic patients sof/led + r; g2/g3decompensated cirrhotic patients: sof/vel + r. we assessed the impact of any ras across g1-g3 on hcv rna kinetics by analysing viral load (realtime hcv viral load, abbott m20 0 0, abbott molecular uk, maidenhead, england) decline rates. we applied linear mixed regression to model the viral loads and assumed a linear decline (log 10 scale) over time, using sas statistical software (sas institute inc., nc, usa). in this cohort of 54 patients (n: g1 = 21, g2 = 6, g3 = 27), hcv ras were found as shown in table 1 . hcv rna viral load decline rates were found to be similar and not statistically different ( p = 0.760) at: −2 log 10 and −1.8 log 10 per month, respectively, for g1 and g2/g3 ( fig. 1 ). this suggests that these viral load decline rates were similar across g1-g3 infections despite baseline differences in viral load, ras profile, or a history of any previous treatment (i.e. interferon-based, older ns3 protease inhibitor-based, or more recent daa regimens). these results demonstrate similar hcv rna clearance efficacies of the various daa treatment regimens for g1-g3, in this patient cohort. although other studies on hcv kinetics have been published, they do not usually compare multiple hcv genotypes. 7 similar studies on patients infected with g4-6 viruses, and/or undergoing other daa treatment or retreatment combinations, 8 , 9 will be with great interest we have read the report of zhang et al. concerning the increased susceptibility to pertussis in chinese adults at childbearing age, as determined in a comparative seroprevalence study using samples collected from 2010 to 2016. 1 the authors describe that about 5% of the individuals had pt-igg antibodies, which is indicative of a recent infection. in the adults 20-39 years of age, 29.1% subjects had undetectable pt-igg antibodies in 2010 but 57.4% in 2015/2016. it is well known that adolescents and adults have become the reservoir of pertussis and an important source of transmission to vulnerable infants. bordetella pertussis is commonly associated with atypical pneumonia as determined in hospitalized children. 2 several seroprevalence studies conducted in different regions of china indicate that the incidence of pertussis is most likely underestimated. 3 , 4 this may be due to the use of insensitive diagnostics. at present, the diagnosis of pertussis in china is mainly based on culture. however, both the cdc and the world health organization (who) use pcr as the gold standard for diagnosis, in addition to culture. 3 oropharyngeal or nasopharyngeal swabs were obtained from 17,104 inpatients aged between 10 days and 14 years of age with clinical suspicion of pertussis, enrolled from march 2014 to february 2018 in shenzhen children's hospital. more than 50% of all patients were younger than 6 months of age. the hospitalized patients included 10,894 males and 6210 females (sex ratio, 1.75). all patients recovered after the treatment. a real time pcr assay targeting ptxa-pr was used to detect b. pertussis . of the 17,104 samples, 772 (4.51%) tested positive for b. pertussis by rt-pcr. our results indicate that despite vaccination pertussis remains a major health problem in china, since the prevalence of infection by b. pertussis in hospitalized children was high. the majority of patients were admitted because of pneumonia. the detection rate in hospitalized patients was lower than the rates reported earlier in shanghai and ji'nan. 3 , 4 this may be due to lower number of samples collected in these studies and due to the use of serology or culture methods. the overall prevalence rates were 3.97% and 5.48%, respectively. however, b. pertussis infection in female patients was significantly higher than in male patients (x 2 = 20.913, p < 0.001). this has earlier been reported by the ecdc 5 and haberling et al. 6 and may point to a genetic association with susceptibility to b. pertussis . the detection rates were dependent on age in patients (x 2 = 139.8, p < 0.001). the prevalence decreased with age: 8.71% newborns, 5.33% in infants, 2.39% in toddlers, 1.28% in (pre-) schoolers ( fig. 1 ) . the high vulnerability of newborns and young infants for b. pertussis infection may be related to a combination of insufficient herd immunity and suboptimal protection against b. pertussis infection in children too young to be fully vaccinated. since vaccination rates in infants are already at 99%, it will be difficult to improve this further. therefore, other measures must be considered, including booster vaccination at pre-school age and vaccination during pregnancy. because young infants are mainly cared for by mothers and other adults, the most important cause of infection with b. pertussis is their close contact with parents and siblings. 7 in general, b. pertussis was detected more often during seasonal changes, especially from late summer to early autumn. in hospitalized children the number of b. pertussis infections increased in march and september as compared to other months ( fig. 2 ) . the seasonal infection rates were 4.66% in spring, 4.75% in summer, 4.62% in autumn and 4.00% in winter, respectively. the prevalence in the winter season was lower but not statistically different than in other seasons (x 2 = 3.388, p = 0.336). in this study, we used real-time pcr, the most accurate method to detect b. pertussis . the detection rate may be significantly lower than the actual level, because oropharyngeal samples in most patients were collected instead of nasopharyngeal samples, and the pcr target gene was ptxa-pr instead of is4 81. is4 81 is present in high-copy numbers in b. pertussis whereas ptxa-pr is a single-copy target. however, the ptxa-pr pcr is more specific and will not detect b. parapertussis , which contributes to more than 5% of pertussis cases. 8 many studies have shown that adolescents and adults with b. pertussis infections, causing chronic cough, are an important reservoir for transmission, putting newborns at high risk. 9 maternal pertussis immunization prevents infant pertussis, as recently shown by amirthalingam et al. vaccine effectiveness against infant deaths was estimated at 95%, and disease incidence in infants < 3 months of age has remained low. 10 according to our results, vaccination of pregnant women and adults, especially those in close contact with infants and young children, may help to prevent pertussis in infants and young children in china. the authors have declared that no competing interests exist. this study was supported by the sanming project of medicine in shenzhen ( szsm201512030 ) and by the shenzhen science and technology project ( jcyj20170303155012371 ). tang and colleagues reported in this journal their experience with covid-19 disease 1 , the outbreak of which began in december 2019 in wuhan, hubei province, china 2 , 3 with spread to 33 additional countries 4-8 as of the 21st february 2020. here we report the clinical features and outcome of the first two cases of disease caused by sars-cov-2 infection in the united kingdom (uk) -the first imported and the second associated with probable person-toperson transmission within the uk. public health management will be reported separately. the index case (a) entered the uk on 23/01/20 from hubei province in china. initially asymptomatic, this individual, a 50 year-old female with no past medical history and on no regular medications, developed symptoms of fever and malaise on 26/01/20, accompanied by sore throat and dry cough. she had travelled with her partner and reported no infectious contacts prior to travel. on 28/01/20, a close household contact of the index case, a resident of the uk, developed symptoms of fever (38.5 °c), followed the next day by diffuse myalgia and a dry cough. this patient (case b) is a previously fit and well 23 year-old male. he had returned to the uk from hubei province on 06/01/20. case b promptly sought advice via the national health service (nhs) self-referral service nhs 111, and he and case a were assessed as being possibly at risk of covid-19, and were admitted to the regional infectious diseases unit at castle hill hospital, hull university teaching hospitals for isolation, assessment and diagnostic sampling. they were managed in separate negative pressure cubicles with anterooms. nursing and medical staff donned personal protective equipment (ppe) as recommended by public health england (phe). the clinical observations of each of the patients, together with their initial blood tests, are shown in table 1 . ( fig. 1 ) . clinical examination findings were unremarkable. initial investigations revealed only mild lymphopenia and elevation of crp, with mild neutrophilia in case b. periodic fever of 38-38.5 °c was observed in case b until d3 of admission. repeat blood tests in this individual on d3 demonstrated mild acute kidney injury (aki, serum creatinine 144 μmol/l). the aki was thought most likely due to dehydration, and resolved within 24 h with administration of intravenous infusion of crystalloid at 125 ml/h. cxr was normal. empirical oral antibiotic therapy (co-amoxyclav 500/125 mg p.o. t.d.s.) was administered on d3, to cover the possibility of secondary bacterial infection, but was subsequently discontinued. symptoms resolved in case a by d3 and in case b by d4 of admission. pcr testing of sars-cov-2 from nose and throat swabs taken daily was negative from d2 onwards in case a and from d5 in case b (throat swabs from this individual were negative throughout). there was no clinical indication for the use of experimental antiviral therapies. patients were deisolated according to current phe guidance, based on complete resolution of symptoms and two sequential negative respiratory pcr tests at least 24 h apart. rooms were decontaminated with 0.1% hypochlorite followed by uv light treatment. the contact of these individuals remained asymptomatic throughout the 14 days incubation period but was isolated as a precaution and to be close to family these first cases of sars-cov-2 are informative for clinicians caring for suspected and confirmed cases in the uk and elsewhere. reassuringly, illness in both individuals was relatively mild and short-lived, with no evidence of parenchymal lung disease (reflected by normal oxygenation and the absence of radiological infiltrates) or of the late-stage deterioration that has been reported in case series 9 , possibly due to the absence of comorbidities. experimental antiviral therapeutic options for severe disease were not considered necessary given the mild clinical nature of the illness. clinical illness correlated with the presence of viral rna in upper airway samples ( fig. 1 ) , with no evidence of prolonged asymptomatic shedding, although discordance between nose and throat samples in case b highlights the need to sample both areas. it was reasonably assumed that the source of infection in case b was close contact with symptomatic case a, given that the time from travel to china to onset of symptoms in case b was 22 days, although this cannot be proven. based on this assumption, the period from exposure to disease onset appeared short, at approximately 48 h, consistent with recent reports of the incubation period of sars-cov-2 6 . co-occurrence of respiratory viral infection, as we observed in case b with rhinovirus, has been described in the context of sars-cov-2 ( https://www.medrxiv.org/ content/10.1101/2020.02.12.20022327v1 ) as it has with many other respiratory viruses spread by similar routes, and may have contributed to the increased symptomatology in case b. interestingly the partner of case a, who was a close household contact, remained asymptomatic throughout and had negative tests for sars-cov-2 shedding. it will be of interest to investigate the serological responses in this individual to ascertain evidence of subclinical infection. isolation, minimisation of contacts and use of appropriate ppe is a cornerstone of management of high consequence respiratory viral infection. in the cases reported here, phe recommendations for ppe were followed 10 and there were no breeches in ppe or nosocomial transmission. this should provide reassurance to healthcare workers managing patients with suspected covid-19 in the uk that current ppe is both feasible and effective. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. dear editor , as reported in this journal 1 and elsewhere, 2 an outbreak of atypical pneumonia caused by the zoonotic 2019 novel coronavirus (sars-cov-2) is on-going in china and has spread to the world. as of feb 16, 2020 (24:00, gmt + 8), there have been 70,548 confirmed patients and more than 1700 deaths from sars-cov-2 infection in china, and 58,182 confirmed patients and 1696 deaths in the most affected province, hubei province. much research progress has been made in dissecting the evolution and origin of sars-cov-2 and characterizing its clinical features. [3] [4] [5] [6] [7] while the outbreak is on-going, people raise grave concerns about the future trajectory of the outbreak, especially given that the working and schooling time has been already dramatically postponed after the chinese lunar new year holiday was over (scheduled on jan 31). in particular, a precise estimation of the potential total number of infected cases and/or confirmed cases is highly demanding. earlier studies based on susceptible-exposed-infectious-recovered metapopulation and susceptible-infected-recovered-dead models revealed the number of potentially infected cases and the basic reproductive number of sars-cov-2. 3 , 8 , 9 these traditional epidemiological models apparently require much detailed data for analysis. 3 , 8 here we explored a simple data-driven, boltzmann functionbased approach for estimation only based on the daily cumulative number of confirmed cases of sars-cov-2 (note: the rational for boltzmann function-based regression analysis is presented in supporting information (si) file). we decided to collect data (initially from jan 21 to feb 10, 2020) in several typical regions of china, including the center of the outbreak (i.e. wuhan city and hubei province), other four most affected provinces (i.e., guangdong, zhejiang, henan, hunan) and top-4 major cities in china (i.e., beijing, shanghai, guangzhou, shenzhen). during data analysis on feb 13, 2020, the number of new confirmed cases on feb 12 in hubei province and wuhan city suddenly increased by 14,840 and 13,436, respectively, of which 13,332 and 12,364 are those confirmed by clinical features (note: all the number of confirmed cases released by feb 12 were counted according to the result of viral nucleic acid detection rather than by referring to clinical features). we thus arbitrarily distributed these suddenly added cases to the reported cumulative number of confirmed cases from jan 21 to feb 14 for hubei province by a fixed factor (refer to table s1 ), assuming that they were linearly accumulative in those days. it is the same forth with the data for wuhan city. regression analyses indicate that all sets of data were well fitted with the boltzmann function (all r 2 values being close to 0.999; figs. 1 a, b, s1 , and table 1 ). the potential total number of confirmed cases for mainland china, hubei province, wuhan city, and other provinces were estimated as 72,80 0 ±60 0, 59,30 0 ±60 0, 42,10 0 ±70 0 and 12,80 0 ±10 0; respectively; those for provinces guangdong, zhejiang, henan and hunan were 1300 ±10, 1170 ±10, 1260 ±10, 1050 ±10, 1020 ±10 and 940 ±10, respectively ( table 1 ) ; those for beijing, shanghai, guangzhou and shenzhen were 394 ±4, 328 ±3, 337 ±3 and 397 ±4, respectively. in addition, we estimated the key date, on which the number of daily new confirmed cases is lower than 0.1% of the potential total number as defined by us subjectively (refer to table 1 ). the above analyses were performed assuming that the released data on the confirmed cases are precise. however, there is a health commission, the state administration of traditional chinese medicine, the academy of chinese medical sciences, 29 provinces and cities, as well as the army ( fig. 1 ) . huoshenshan hospital is a specialized hospital established in the wuhan staff sanatorium. patients with confirmed coronavirus pneumonia have been admitted to our hospital. it has a total of 10 0 0 beds, and includes an intensive care unit, an ordinary care unit, a laboratory, and radiology and other auxiliary departments. according to the national health commission of the people's republic of china, the related design scheme of the institute was completed on january 24, 2020. construction of the hospital began on january 29th, and the hospital was completed and put into use on february 2nd. the chinese people's liberation army has transferred 1400 medical personnel to undertake the task of helping people infected with the virus. we firmly believe that chinese medical personnel and people throughout the country can work together to win this defensive battle with one heart and one mind. herpes simplex virus (hsv) pneumonia in the non-ventilated immunocompromised host: burden and predictors colonization by pneumocystis jirovecii and its role in disease ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients pcr diagnosis of pneumocystis pneumonia: a bivariate meta-analysis evaluation of pcr in bronchoalveolar lavage fluid for diagnosis of pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review molecular diagnosis of pneumocystis pneumonia in immunocompromised patients real-time pcr assay-based strategy for differentiation between active pneumocystis jirovecii pneumonia and colonization in immunocompromised patients detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients diagnosis of pneumocystis jirovecii pneumonia in immunocompromised patients by real-time pcr: a 4-year prospective study pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia a unified personal protective equipment ensemble for clinical response to possible high consequence infectious diseases: a consensus document on behalf of the hcid programme lassa fever: epidemiology, clinical features, and social consequences world health organization. lassa fever fact sheet review of cases of nosocomial lassa fever in nigeria: the high price of poor medical practice epidemiologic and clinical features of lassa fever outbreak in nigeria measures to control protracted large lassa fever outbreak in nigeria clinical and laboratory predictors of lassa fever outcome in a dedicated treatment facility in nigeria: a retrospective, observational cohort study molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation national guidelines for lassa fever case management pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds first human infection by a novel avian influenza a(h7n4) virus comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings investigation on pseudorabies prevalence in chinese swine breeding farms in 2013-2016 human endophthalmitis caused by pseudorabies virus infection clinical experience and next-generation sequencing analysis of encephalitis caused by pseudorabies characteristics of human encephalitis caused by pseudorabies virus: a case series study human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: a case report a case of human viral encephalitis caused by pseudorabies virus infection in china metagenomic data screening reveals the distribution of mobilized resistance genes tet (x), mcr and carbapenemase in animals and humans epidemic of carbapenem-resistant klebsiella pneumoniae in europe is driven by nosocomial spread antibiotic resistance gene reservoir in live poultry markets emergence of plasmidmediated high-level tigecycline resistance genes in animals and humans plasmid-encoded tet(x) genes that confer high-level tigecycline resistance in escherichia coli first report of blavim-4-and mcr-9-coharboring enterobacter species isolated from a pediatric patient a link between the newly described colistin resistance gene mcr-9 and clinical enterobacteriaceae isolates carrying blashv-12 from horses in sweden mcr-9, an inducible gene encoding an acquired phosphoethanolamine transferase in escherichia coli , and its origin discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis expansion and model-centric curation of the comprehensive antibiotic resistance database emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study novel plasmid-mediated colistin resistance gene mcr-7.1 in klebsiella pneumoniae emergence of a novel mobile colistin resistance gene, mcr-8 , in ndm-producing klebsiella pneumoniae novel plasmid-mediated colistin resistance gene mcr-3 in escherichia coli identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible salmonella enterica serotype typhimurium isolate coproduction of mcr-9 and ndm-1 by colistin-resistant enterobacter hormaechei isolated from bloodstream infection outbreaks of severe pneumococcal disease in closed settings in the conjugate vaccines era serious pneumococcal disease outbreak in men exposed to metal fume -detection, response and future prevention through pneumococcal vaccination outbreak of invasive pneumococcal disease among shipyard workers welders are at increased risk for invasive pneumococcal disease reactivation of dormant/latent fungal infection visceral leishmaniasis: host-parasite interactions and clinical presentation in the immunocompetent and in the immunocompromised host visceral leishmaniasis among immunosuppressed patients with rheumatic diseases tumor necrosis factor alpha antagonist drugs and leishmaniasis in europe psoriasis as a systemic disease the inflammatory response in psoriasis: a comprehensive review the equivocal role of th17 cells and neutrophils on immunopathogenesis of leishmaniasis leishmaniasis and biologic therapies for rheumatologic diseases subclinical leishmania infection in patients with rheumatic diseases under biological drugs appropriate screening for leishmaniasis before immunosuppressive treatments human herpesvirus 6 infection after autologous stem cell transplantation: a multicenter prospective study in adult patients next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center a different response to cytomegalovirus (cmv) and epstein-barr virus (ebv) infection in uk people with multiple sclerosis (pwms) compared to controls complete genome sequence of equine herpesvirus type 9 equine herpesviruses type 1 (ehv-1) and 4 (ehv-4)-masters of co-evolution and a constant threat to equids and beyond isolation and identification of equine herpesvirus type 1 identification of equine herpesvirus type 1 infection in a horse farm in shanghai epidemiological status of equine rhinopneumonia in china complete genomic sequence of an equine herpesvirus type 8 wh strain isolated from china equine herpesviruses 1 (ehv-1) and 4 (ehv-4)-epidemiology, disease and immunoprophylaxis: a brief review consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group differences in viral dynamics between genotypes 1 and 2 of hepatitis c virus the paradox of highly effective sofosbuvir-based combination therapy despite slow viral decline: can we still rely on viral kinetics? therapy of chronic hepatitis c virus infection in the era of direct-acting and host-targeting antiviral agents oral direct-acting agent therapy for hepatitis c virus infection: a systematic review hcv] -a web-based interpretation system to support hepatitis c treatment decisions in the era of direct-acting antiviral agents antiviral therapies for chronic hepatitis c virus infection with cirrhosis real life efficacy of ledipasvir/sofosbuvir in hepatitis c genotype 4-infected patients with advanced liver fibrosis and decompensated cirrhosis systematic review with meta-analysis: efficacy and safety of directacting antivirals for chronic hepatitis c genotypes 5 and 6 increased susceptibility to pertussis in adults at childbearing age as determined by comparative seroprevalence study ritm-tohoku collaborative research group. bordetella pertussis infection in children with severe pneumonia rapid detection of respiratory organisms with the filmarray respiratory panel in a large children's hospital in china seroprevalence of diphtheria and pertussis immunoglobulin g among children with pneumonia in ji'nan. china european centre for diseases prevention and control. annual epidemiological report infant and maternal risk factors for pertussis-related infant mortality in the united states clinical features, genotype variations of antigenic genes and macrolides resistance pertussis outbreak in a primary school in china: infection and transmission of the macrolide-resistant bordetella pertussis seroprevalence of pertussis among adults in china where whole cell vaccines have been used for 50 years sustained effectiveness of the maternal pertussis immunization program in england 3 years following introduction emergence of a novel coronavirus causing respiratory illness from wuhan the ncov outbreak joint field epidemiology investigation team an outbreak of ncip (sars-cov-2) infection in china -wuhan , hubei province a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia importation and human-to-human transmission of a novel coronavirus in vietnam transmission of sars-cov-2 infection from an asymptomatic contact in germany first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan public health england. www.gov.uk/government/publications/wuhan-novelcoronavirus-infection-prevention-and-control/wuhan-novel-coronavirus-wncov-infection-prevention-and-control-guidance accessed 3rd emergence of a novel coronavirus causing respiratory illness from wuhan a novel coronavirus outbreak of global health concern epidemiological and clinical features of the 2019 novel coronavirus outbreak in china a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical characteristics of 2019 novel coronavirus infection in china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study data-based analysis, modelling and forecasting of the novel coronavirus (2019-ncov) outbreak a data driven time-dependent transmission rate for tracking an epidemic: a case study of 2019-ncov emergence of a novel coronavirus causing respiratory illness from wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan the novel coronavirus originating in wuhan. china: challenges for global health governance emergence of sars-like coronavirus poses new challenge in china we are indebted to all colleagues who contributed to this substudy in paris and geneva. in particular, we would like to thank no public or private funds were used for the current study. eliseo albert holds a río hortega research contract from the carlos iii health institute (ref. cm18/00221 ). estela giménez holds a juan rodés research contract from the carlos iii health institute (ref. jr18/0 0 053 ). we thank all the staff of the domestic and international organizations who fought against this outbreak, including those at the various health care facilities, lassa fever diagnostic laborato-ries, nigeria centre for disease control, world health organization, african field epidemiology network, public health england, ehealth africa, pro health international, university of maryland baltimore, us centers for disease control and prevention, alliance for international medical action, médecins sans frontières, and numerous other partners. we also express our sincerest condolences to the families and friends of those who died during the outbreak. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.016 . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.01.019 . this work was supported by the national natural science foundation of china ( 31702271 ), and the guangdong provincial natural science foundation ( 2017a030310367 ). we are grateful to the patients for providing their written informed consent to publish this report. our thanks go to nursing, laboratory and medical colleagues in hull university teaching hospitals nhs trust and the newcastle upon tyne hospitals nhs foundation trust who contributed directly and indirectly to patient care, and to many colleagues in public health england and across the hcid network who contributed their time and expertise to the management of these cases. cjad is supported by a clinical research career development fellowship from the wellcome trust ( 211153/z/18/z ). we thank graduate students (boyan lv, zhongyan li, zhongyu chen, yu cheng, mengmeng bian, shuang zhang, zuqin zhang, and wei yao; all from prof. xinmiao fu's research group at fujian normal university) for data collection. this work is support by the national natural science foundation of china (no. 31972918 and 31770830 to xf). the reported cumulative number of confirmed cases may have uncertainty. assuming the relative uncertainty follows a single-sided normal distribution with a mean of 1.0 and a standard deviation of 10%, the potential total number and key dates were estimated at 95% ci. for detail, refer to the methods section and figs. 1 c, d, s2 and s3.b key date is determined when the number of daily new confirmed cases is less than 0.1% of the potential total number. tendency to miss-report some positive cases such that the reported numbers represent a lower limit. one typical example indicating this uncertainty is the sudden increase of more than 14 0 0 0 new confirmed cases in hubei province on feb 12 after clinical features were officially accepted as a standard for infection confirmation.another uncertainty might result from insufficient kits for viral nucleic acid detection at the early stage of the outbreak. we thus examined the effects of such uncertainty using a monte carlo method (for detail, refer to the methods section in si file). for simplicity, we assumed that the relative uncertainty of the reported data follows a single-sided normal distribution with a mean of 1.0 and a standard deviation of 10%. under the above conditions, the potential total numbers of confirmed cases of sars-cov-2 for different regions were estimated ( figs. 1 c, d, s2 and s3 ) and summarized in table 1 16 ,092), respectively, indicating that overall the outbreak may not be so bad as previously estimated. 9 such uncertainty analysis also allowed us to estimate the key dates at 95% ci. as summarized in table 1 , the key dates for mainland china, hubei province, wuhan city, and other provinces would fall in (2/28, 3/10), (2/27, 3/10), (2/28, 3/10) and (2/27, 3/13), respectively.finally, the ongoing sars-cov-2 outbreak has undoubtedly caused us the memories of the sars-cov outbreak in 2003. we thus collected the data from the who officiate website for analysis, and found that the cumulative numbers of confirmed cases of 2003 sars-cov both in china and worldwide were fitted well with the boltzmann function, with r 2 being 0.999 and 0.998, respectively ( figs. 1 e and f) .in summary, we found that all data sets, including both the on-going outbreak of sars-cov-2 in china and the 2003 sars-cov epidemic in china and worldwide, were well fitted to the boltzmann function ( fig. 1 and s1 ). these results strongly suggest that the boltzmann function is suitable for analyzing the epidemics of coronaviruses like sars-cov and sars-cov-2. one advantage of this model is that it only needs the cumulative number of confirmed cases, somehow as simple as the recently proposed model. 10 in addition, the estimated potential total numbers of confirmed cases and key dates may provide valuable guidance for chinese central and local governments to deal with this emerging threat at current critical stage. none. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.02.019 . we appreciate the work tang et al. have report emergence of a novel coronavirus in china. 1 the 2019-ncov broke out in wuhan, china at the end of 2019, and has attracted worldwide attention. [2] [3] [4] although the chinese government has taken active measures to control this epidemic, the virus is very infectious. according to the real-time data of the national health commission of the people's republic of china up until february 5, 2020, within a short period of half a month, the number of confirmed cases and the number of deaths were 24,443 and 493, respectively. the epidemic is progressing rapidly. 2019-ncov poses new public health challenges in china. 5 in wuhan, china, the number of local medical staff is insufficient for the demand resulting from the explosive increase in the number of infected patients. therefore, many medical personnel are needed to devote themselves to the front line of combating the virus.medical personnel throughout the country are led under the unified leadership of the chinese government. although the epidemic in wuhan is serious, a large number of medical staff rushed to wuhan to supplement the shortage of manpower in wuhan hospitals. this is a battle without smoke, the heroes of which are our medical staff. according to the national health commission of the people's republic of china, as of january 30, 2020, hubei province opened 11,0 0 0 isolated patient beds, and about 170,0 0 0 healthcare professionals from all kinds of medical institutions are working on the front lines and providing care for patients with fevers, and for suspected or confirmed patients. in this time of emergency, under the unified deployment of the chinese government, there are 52 medical teams including 6097 medical personnel from the national key: cord-332747-u46xryoo authors: mingorance, lidia; castro, victoria; ávila-pérez, ginés; calvo, gema; rodriguez, maría josefa; carrascosa, josé l.; pérez-del-pulgar, sofía; forns, xavier; gastaminza, pablo title: host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis c virus replicase complex formation date: 2018-09-18 journal: plos pathog doi: 10.1371/journal.ppat.1007284 sha: doc_id: 332747 cord_uid: u46xryoo hepatitis c virus (hcv) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. hcv replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. we focused our attention on a phosphatidate phosphate (pap) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. the best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. lipin1-deficient cell lines were generated by rnai to study the role of this protein in different steps of hcv replication cycle. using surrogate models that recapitulate different aspects of hcv infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early hcv rna replication. infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support hcv infection. finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional hcv replicase complexes. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 millions of humans are chronically infected by hepatitis c virus (hcv) worldwide [1] . chronic hcv infection is a major biomedical problem as it causes liver inflammation and fibrosis, which can lead to severe liver disease, such as cirrhosis and hepatocellular carcinoma [2, 3] . there is no vaccine against hcv and, although blood-screening tests and other prophylactic measures have reduced the dissemination of this pathogen, a number of newly acquired infections still occur associated with risk behavior or with unknown origin [4, 5] . however, chronic hcv infection can be successfully eradicated from chronically infected individuals through specific direct-acting antiviral (daa) combination therapies, virtually in all treated patients [6] . since these specific treatments have only been in place recently, there are no sufficient clinical data on the long-term benefit of these treatments in relieving the severity of advanced liver disease [7, 8] . hcv is a hepacivirus (flaviviridae) with a positive sense, single-strand rna genome that encodes a single open reading frame (orf) flanked by untranslated regions (utr), which are essential for viral polyprotein translation and viral genome replication. hcv orf is co-and post-translationally processed by cellular and viral proteases to produce ten major proteins. these have been functionally classified in a replication module, that includes the minimal viral components of the rna replicase (ns3, ns4a, ns4b, ns5a and ns5b) and an assembly module, which comprises the major structural components of enveloped hcv virions, the capsid protein (core) and the envelope glycoprotein complex formed by e1 and e2 heterodimers; as well as the polypeptides p7 and ns2, which are not structural components of virions but contribute to infectious particle assembly in a concerted action with the viral replicase [9] . hcv utilizes key aspects of cellular lipid metabolism for essentially every aspect of the virus replication cycle and strongly interferes with host cell lipid homeostasis [10] [11] [12] . in fact, chronic hcv patients display high rates of liver steatosis, severity of which inversely correlates with serum liver derived-lipoprotein [13] . thus, although host immune response remains a major component in hcv pathogenesis, direct interference of hcv infection with hepatocyte lipid metabolism may contribute to overall disease progression [13] . identified a limiting role of lipin1 pap activity in the generation of hcv replicase complexes. defective replicase assembly leads to strong inhibition of hcv propagation in lipin1-deficient cells but not that of another (+) strand rna virus, indicating a specific role for lipin1 in hcv infection. given the relevance of lipin1 for lipid metabolism and its steatogenic potential, we set out to independently verify data from published differential transcriptomic profile studies that suggested that hcv infection may alter lpin1 mrna abundance in cell culture [40, 41] . a specific and statistically significant lpin1 mrna induction (fig 1a) was observed in huh-7 cells after single cycle infection experiments [multiplicity of infection (moi) 10] with a cell cultureadapted genotype 2a hcv (d183) variant [42] at the peak of the infection (48 and 72 hours post-infection) and as compared with mock-infected cells (fig 1b) . lpin1 mrna induction was prevented when infected cells were treated with 1μm sofosbuvir (fig 1c) , an hcv rna polymerase inhibitor [43] that reduced viral rna accumulation by more than two orders of magnitude (fig 1d) , indicating that active hcv replication is required to induce lpin1 mrna accumulation. lpin1 mrna has been shown to be upregulated by mechanisms that involve induction of reactive oxygen species (ros), as treatment of the cells with ros-scavenger molecule n-acetylcysteine (nac) is capable of preventing lpin1 mrna induction under glucose deprivation [44] or during h 2 o 2 treatment [45] . given that transcriptional activation of a subset of lipogenic genes during hcv infection is also prevented by addition of antioxidants [46] , we sought to determine if lpin1 mrna induction by hcv infection is mediated by ros production and therefore dampened by nac treatment. lpin1 mrna induction was prevented in the presence of the antioxidant (fig 1e) , despite comparable hcv rna accumulation in mock-treated and nac-treated hcv-infected cells (fig 1f) , suggesting that virus replicationinduced ros production is required to induce lpin1 mrna accumulation. western-blot analysis confirmed that the observed transcriptional change leads to a correlative protein accumulation (fig 1g and 1h) ; reinforcing the notion that acute hcv infection alters lipin1 expression. in order to determine if lipin1 subcellular localization was altered during hcv infection, we performed confocal microscopy studies in control and hcv-infected huh-7 cells. lipin1 staining was observed as cytoplasmic punctated structures both in control and hcv-infected cells. to study if lipin1 signal colocalized with viral antigens, we performed double staining with antibodies against lipin1 and double-stranded rna (dsrna) or replicase subunits ns3 and ns5a. none of the viral antigens strictly colocalized with lipin1 (pearson´s<0.5) (s1a fig). however, mander´s coefficients indicate that the majority of lipin1 overlapped with a small fraction of ns3 and ns5a (s1c fig). in contrast to lipin1, lipin2 signal did not overlap with that of viral proteins ns3 and ns5a (s1b fig). our results indicate that no major lipin1 rearrangements are observed after hcv infection and that only a minor fraction of ns proteins colocalize with lipin1. to study if lipin1 plays any role in hcv infection, lipin1-deficient cells were generated by transducing human hepatoma (huh-7) cells with lentiviral vectors expressing shrnas targeting lpin1 mrna or a control vector expressing an irrelevant shrna. lipin1 expression silencing was verified by western-blot, typically 7 days post-transduction (fig 2a) . a partial (50%; shlpin1-1) and a more profound (>95%; shlpin1-2) reduction in lipin1 accumulation was observed after transduction with specific shrnas as compared with the control (fig 2b) . . rnas from mock-infected cells and hcv infected. impact of sofosbuvir (1μm; daa) (c, d) or n-acetylcysteine (10mm; nac) treatment (e, f) on hcv rna accumulation and lpin1 mrna levels. data are shown as average and standard deviation of two independent infection experiments performed in triplicate (n = 6). g-protein samples of infected and control cells collected at 48 hours post-infection were subjected to western-blot analysis to determine lipin1 and ns3 levels, using beta-actin as loading control h-quantitation of the relative lipin1 protein levels in mock and hcv-infected cells (n = 3). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). https://doi.org/10.1371/journal.ppat.1007284.g001 huh-7 cells were transduced with lentiviral vectors expressing control or lpin1-specific shrnas. seven days after transduction samples of the cells were collected for western-blot analysis using antibodies against lipin1 and lipin2 and actin as loading control. parallel cultures were subjected to an mtt assay to determine their viability as described in the methods section. (a) representative western-blot showing cellular lipin1 and lipin2 protein expression levels and a loading control (actin). (b) average expression values for lipin1 and lipin2 as determined by western-blot and cell viability as determined by an mtt assay. data are shown as average and sd of six independent transduction experiments (n = 6). (c) huh-7 cells were transduced with lentiviral vectors expressing control or lpin1-specific shrnas. at day 3 post-transduction, cells were infected at moi 0.01 with hcv d183 virus. samples of cell supernatants were collected at days 3 and 5 post-infection to determine the extracellular infectivity titer. average and sd of the infectivity titers at day 3 and 5 of two independent infections performed in triplicate (n = 6). (d) huh-7.5 cells were transduced with lentiviral vectors expressing control or lpin1-specific shrnas. silenced cells were infected 7 days after transduction at moi 0.05 with genotype 1a hcv (tncc) in the presence or absence of 2made (10μm; shcontrol+daa). intracellular hcv rna was determined by rt-qpcr 72 hours post-infection. data are shown as average and sd of three experiments performed in triplicate (n = 9). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). as expected, lipin1 and lipin2 expression is inversely correlated in lipin1 shrna-expressing cells (fig 2b) . the viability of lipin-deficient cells, as determined by mtt assay [47] was comparable to that of control cells (fig 2b) . these results illustrate that lipin1 shrna-expressing cells respond homeostatically to functionally compensate partial (shlpin1-1) and more pronounced (shlpin1-2) loss of lipin1 (fig 2b) . control and lipin1-deficient cells were infected with a genotype 2a d183 at moi 0.01 to study viral spread by determining extracellular infectivity titers at different times post infection. fig 2c shows limited propagation of this virus in lipin1-deficient cells, with a statistically significant reduction of up to two (shlpin1-1) and three orders of magnitude (shlpin1-2) in viral titer between the control and lipin1-deficient cell lines at day 5 post-infection. these results indicate that lipin1 is required for efficient hcv propagation and that homeostatic lipin2 accumulation (fig 2b) is not sufficient to support efficient hcv infection. since important differences in the interaction of different hcv genotypes with cellular lipid metabolism have previously been described [48] , we set out to determine if the observations made with a jfh1-derived virus (genotype 2a) were extensive to other hcv genotypes. we performed low multiplicity infections (moi 0.05) with genotype 1a tncc virus strain in lipin1-deficient huh-7.5 cell lines, which are susceptible to infection by this recombinant virus [49] . first, we verified that huh-7.5.1 also display a significant reduction in genotype 2a infection efficiency when lipin1 is silenced (s2b fig) . in order to determine tncc infection efficiency, intracellular hcv rna accumulation was determined 72 hours post-inoculation in control and lipin1-deficient huh-7.5 cells. viral rna detected under these conditions reflects the ability of genotype 1a to infect and replicate viral rna in the different cell lines, as treatment of control cells with an hcv polymerase inhibitor 2´-c-methyladenosine (2made; 10 μm) [50] reduced viral rna content by two orders of magnitude ( fig 2d) . lipin1 silencing consistently and significantly reduced tncc replication by approximately 3-fold both in shlpin1-1 and shlpin1-2 cell lines (fig 2d) , indicating that lipin1 is also limiting for genotype 1a hcv infection. to determine the specificity of these observations, identical lipin1-deficient and control huh-7 cultures were inoculated at moi 0.01 with a human alpha-coronavirus cov-229e bearing a gfp reporter gene (hcov-229e-gfp) [51] . inoculation of control and lipin1-deficient cells with this virus resulted in comparable progeny virus production, as determined by infectivity titration in cell supernatants 48 hours post-infection (s3 fig) . these results suggest that lipin1 is not rate limiting for cov-229e-gfp infection and that lipin1 expression is particularly limiting for hcv. to determine which aspects of the hcv replication cycle are limited by lipin1 silencing, single cycle infection experiments were conducted by inoculating control and lipin1-deficient cell cultures at moi 10 with genotype 2a d183 virus. infection efficiency was measured by titration of progeny virus infectivity present in the supernatant of infected cells and intracellular hcv rna accumulation at 48 and 72 hours post-infection. infection of lipin1-deficient cells resulted in a significant reduction of progeny infectious virus production in shlpin1-1 and shlpin1-2 cells as compared with the titers observed in the supernatants of control cells ( fig 3a) , reinforcing the notion that lipin1 silencing interferes with hcv infection. reduced virus production is likely due to parallel reduction of intracellular hcv rna levels observed in lipin1-deficient cells as compared with the control cell line (fig 3b) . this reduction was observed at all time points, except for that at 5 hours, indicating that the size of the inoculum and initial virus adsorption is comparable among the different cell lines (fig 3b) . thus, lipin1 silencing suppresses hcv infection by interfering with a step of the hcv lifecycle preceding intracellular hcv rna accumulation. next, we set out to determine if lipin1 silencing has any impact on persistent hcv infections to verify if lipin1 is also limiting for late aspects of the virus lifecycle. persistently infected cells continuously replicate viral rna, express viral antigens and secrete infectious virions. thus, it is a valuable system to measure steady-state hcv rna replication as well as infectious particle assembly and secretion. persistently infected cultures were transduced with the lentiviral vectors described above to produce persistently infected, lipin1-deficient cells (s4 fig) . shown as genome equivalents per microgram of total rna ge/μg). panels c, d-persistently infected cultures were generated by inoculation with jfh-1 virus at moi 0.01. once cultures reached >95% of hcv-positive cells, they were transduced with lentiviral vectors expressing control, hcv rna-targeting or lpin1-specific shrnas. at day 7 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine infectious virus production rate by infectivity titration hcv (c) and rna levels by rt-qpcr (d). all data are shown as mean and sd of 3 independent experiments performed in triplicate (n = 9). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). analysis of extracellular infectivity titers revealed that infectivity titers in lipin1-deficient and control cells were comparable, with the exception of a marginal reduction in shlpin1-2 expressing cells, indicating that lipin1 silencing does not strongly interfere with infectious virus production ( fig 3c) . intracellular hcv rna levels in lipin1-deficient cells were comparable to that of the control cells (fig 3d) , indicating that lipin1 expression is not rate limiting for hcv rna replication once infection has been established. taken together, the results shown above indicate that lipin1 is only limiting at early steps of hcv infection leading to viral rna accumulation. as an independent verification of the hypothesis that lipin1 is limiting for early aspects of hcv infection, we used a single cycle surrogate infection model based on the production of hcv virions bearing a defective reporter genome encapsidated by trans-complementation (hcv tcp ). hcv tcp are capable of producing abortive single-cycle infections, efficiency of which is proportional to the luciferase activity found in the target cells [52] . as expected from the results shown in fig 3, infection of lipin1-deficient cells with hcv tcp resulted in a 75% reduction in the reporter luciferase activity in shlpin1-1 cells and 90% in shlpin1-2 determined at 48 hours post-infection, proportionally to the degree of silencing in these cells ( fig 4a and 4b ). these results underscore the role that lipin1 plays at early steps of hcv infection and indicate that either viral entry or a step leading to efficient hcv rna replication is impaired in these cells. lpin-1 mrna is alternatively spliced in human liver to produce isoforms α and β, which may differ in catalytic activity, subcellular localization and gene expression regulation [53] . to determine the relative contribution of these isoforms to hcv infection, isoform-specific shrnas were generated and used to transduce huh-7 cells, as described above. lpin1α-specific shrna (shlpin1-3) reduced total lipin1 protein by 70%, while lpin1β-specific shrna (shlpin1-4) reduced total lipin1 expression by only 30% (fig 4a) . infection of control and lipin1 isoform-deficient cells with hcv tcp revealed that lipin1α silencing resulted in a strong (85%) reduction in hcv infection while lipin1β silencing resulted in a milder (55%) but significant reduction in hcv infection efficiency ( fig 4b) . these results indicate that both isoforms alpha and beta are limiting for hcv infection and suggest that the total amount of lipin1 present in the cell determines hcv infection efficiency. taken together, the results obtained with four different shrnas indicate that total lipin1 expression levels strongly correlate with hcv tcp infection efficiency (fig 4c) , underscoring a role for this host protein in early aspects of hcv infection. reduced hcv rna accumulation in a single cycle infection (fig 3) as well as reduced hcv tcp infection (fig 4) may be due to a defect in entry of incoming virions. hcv e1e2-pseudotyped retroviral vectors bearing a luciferase gene (hcv pp ) were used to measure viral entry because they constitute a sound model to study viral adsorption, receptor-mediated internalization and e1e2-mediated fusion in endosomes [54] . to assess the specificity of these observations, parallel cultures were inoculated with vsv-g pseudotyped retroviral vectors (vsv pp ). control and lipin1-deficient cell lines were infected with hcv pp (genotype 2a; jfh-1 strain) and vsvpp. as a positive control of inhibition of hcv entry, we used hydroxyzine (5 μm), which efficiently blocks hcv infection by interfering with viral entry [55] . as expected, hydroxyzine selectively inhibited hcv pp infection, as shown by reduced luciferase levels 48 hours postinoculation only in hcv pp -infected cells ( fig 5a) . interestingly, lipin1-deficient cells (shlpin1-1 and shlpin1-2) were fully susceptible to hcv pp and vsv pp infection, as comparable luciferase activity levels were found in all cell lines 48 hours post-inoculation ( fig 5a) . these results indicate that lipin1 is not rate limiting for receptor binding, particle internalization or e1e2-mediated endosomal fusion, which are steps recapitulated in this model [54] . based on the data presented thus far, we hypothesized that lipin1 is limiting for a step in the hcv lifecycle downstream of hcv entry, leading to hcv rna accumulation. to verify the hypothesis that lipin1 silencing causes strong reduction in initial hcv rna accumulation by interfering with a step downstream of viral entry, we bypassed this step of the viral replication cycle by transfecting a subgenomic hcv rna replicon bearing a reporter luciferase gene into control and lipin1-deficient cells. first, we evaluated hcv-ires driven primary translation of incoming genomes by transfection of a replication-deficient mutant replicon that bears an inactivation mutation in the catalytic site of ns5b rna polymerase [56] . luciferase activity measured at 5 hours post-transfection was not reduced in any of the cell lines, indicating that transfection efficiency and hcv ires-dependent primary translation was not significantly affected by lipin1 silencing (fig 5b) . a significant increase in renilla luciferase activity was observed in shlpin1-1 cells both when transfecting a replicon ( fig 5b) or a plasmid expressing renilla luciferase under a minimal rna polymerase ii promoter (s5a fig), suggesting that the increase in luciferase activity observed in these cells is not related with hcv. hcv rna replication was evaluated by measuring accumulation of a reporter luciferase gene at 5 and 48 hours post-transfection of a replication competent hcv subgenomic replicon. under these experimental conditions luciferase accumulation at 5 hours also represents hcv ires-driven primary translation of the input rna, while reporter luciferase activity at 48 hours depends on effective hcv rna replication. in contrast to primary translation, hcv rna replication inferred by luciferase activity at 48 hours was strongly reduced in lipin1-deficient cells (90% in shlpin1-1 and 99% in shlpin1-2 cells) (fig 5c) , indicating that initiation of hcv replication is dependent on normal lipin1 expression. this reduction was not due to a non-specific defect in luciferase expression, as co-transfection of a plasmid expressing renilla luciferase lead to comparable luciferase accumulation in all cell lines, discarding the possibility that death of transfected cells or other spurious effects are responsible for the reduced luciferase activity accumulation (s5a fig) . similar experiments were conducted in atg4b-deficient cells, as this host factor was shown to be limiting for primary hcv translation [57] . our studies confirmed that, while partial atg4b silencing (s5b fig overall, these data indicate that hcv rna replication is not initiated efficiently in lipin1-deficient cells and that blockade occurs at a step downstream translation of incoming hcv genomes. in order to determine if any of the known functions ascribed to lipin1 is required for hcv infection, we tested the ability to restore hcv infection susceptibility of silencing-resistant wild-type (wt) or mutant lipin1 versions bearing a mutation in the catalytic site responsible for its phosphatase activity (dxdxt) and a mutant in the lxxil motif, which is inactive both for transcriptional activation as well as for phosphatase activity [31] . control and lipin1-deficient cells were transfected with wt lipin1 beta cdna as well as with dxdxt and lxxil mutants. comparable overexpression levels of the wt and mutant proteins was obtained in each cell line, although overexpressed lipin1 levels were consistently higher in lipin1-deficient cells (fig 6a) . cells were subsequently inoculated with hcv d183 at moi 10 and relative infection efficiency was calculated by determining extracellular infectivity titers 48 hours post-infection. overexpression of wt lipin1 did not significantly alter susceptibility to hcv infection in control cells, although we observed a small but consistent reduction in extracellular infectivity titers when overexpressing wt and mutant lipin1 constructs (s6a fig using relative infection efficiency as readout of this set of experiments, we could clearly observe a statistically significant increase (2-fold) in the relative infection susceptibility in cells overexpressing wt lipin1 cdna as compared with mock-transfected cells or cells expressing similar or higher levels of the mutants (fig 6a) , suggesting that wt lipin1 modestly, though significantly, rescues hcv infection while phosphatase (dxdxt) and transcriptional coactivation (lxxil) mutants do not ( fig 6b) . these results suggest that lipin1 transcriptional coactivation capacity is not sufficient to support hcv infection while lipin1 phosphatase activity is essential. however, given that lxxil mutant is deficient both in transcriptional co-activation and pap activity [31], we cannot determine if transcriptional co-activation by lipin1 is also required to support hcv infection. the data described above suggest a role for lipin1 phosphatase activity in a step of hcv replication cycle between translation of the viral genome and formation of functional replicase complexes. data regarding primary translation where inferred from a surrogate model of translation based on a reporter luciferase gene ( fig 5b) . to address if indeed viral polyprotein is properly processed and inserted into detergent-resistant microdomains to form the characteristic membranous ultrastructures bearing the viral replicase, we used a replication-independent surrogate model of polyprotein expression. this system is based on a vector encoding the portion of the viral polyprotein corresponding to the replicase (ns3-ns5b) under the transcriptional control of the t7 polymerase and the translational control of encephalomyocarditis virus (emcv) ires (ptm-ns3/5b) [58] . replication-independent polyprotein overexpression control and silenced cells were inoculated with hcv e1e2 (hcv pp ) or vsv-g-pseudotyped retroviral vectors (vsv pp ) 7 days post-transduction. control cells treated with hydroxyzine (5μm) were used as positive inhibition control. luciferase activity was determined in the different cell lines at 48 hours post-inoculation. hdx, hydroxyzine pamoate (5μm). panels b, c-control and lipin1-deficient cells were transfected with a replication-deficient mutant (b) or replication competent subgenomic hcv replicon bearing a luciferase gene (c). luciferase activity was determined in the different cell lines at 5 hours post-transfection for both replicons and 48 hours post-transfection for the replication-competent replicon rna. data are expressed as average and sd of three independent experiments performed in triplicate (n = 9). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). https://doi.org/10.1371/journal.ppat.1007284.g005 systems enable assessment of polyprotein processing as well as studying the formation of virus-derived membranous structures [16, 59] . control and lipin1-deficient cells were infected with a recombinant vaccinia virus expressing t7 rna polymerase (vact7) and subsequently transfected with the plasmid ptm-ns3/5b to enable viral replicase expression. sixteen hours post-transfection, cells were processed for lipin1 is required for hcv replicase formation western-blot using anti-ns3. accumulation of ns3 is comparable in control and both lipin1deficient cells, underscoring the notion that lipin1 is not limiting for polyprotein translation and processing (fig 7a) . similarly, ns3 and ns5a expression and subcellular distribution was similar in all cell lines (fig 7b) . these results suggest that there are no major differences in accumulation of viral proteins in lipin1-deficient cells and that a step downstream is affected in these cells. transmission electron microscopy (tem) of ultrathin cell sections of cells expressing hcv replicase components shows the expected accumulation of a mixture of characteristic doublemembrane vesicles (dmv) as well as multiple membrane vesicles (mmv) (fig 7d, 7e and 7f) that were not found in mock-transfected cells (fig 7c) , as reported in previous studies using similar systems [16] . individual vesicle diameter displays heterogeneous size distribution in which the predominant population is distributed between 125-150 nm (median 150 nm; average ± sd: 167 ± 81 nm, n = 279) with larger vesicles being less predominant (fig 7g and s7 fig) . this size distribution is compatible with that observed similar replication-deficient systems and during hcv infection. treatment of these cells with 100 nm daclatasvir (dctv), resulted in a strong reduction in the number of vesicles per section area (s7b fig) , without significantly altering the size distribution of the remaining vesicles (fig 7g) , as reported by berger et al. [59] . similarly, the diameter distribution of the vesicles found in lipin1-deficient cells was comparable to that in control cells ( fig 7g) . however, hcv-induced structures were significantly less abundant 16 hours post-transfection in lipin1-deficient cells than in controls cells (s7c fig) . this reduced abundance is illustrated by a significant reduction in the fraction of cells displaying vesicular structures in lipin1-deficient cell cultures (fig 7h) despite comparable transfection efficiency and viral protein expression levels, indicating that lipin1 may be required in a critical step leading to formation of the hcv-induced vesicular compartment. to validate the tem results independently, we set out to establish a biochemical assay to evaluate replication-independent replicase complex formation. one of the characteristics of the hcv replicase complexes is that they are located in detergent-resistant membranes (drm) [19, 20] . in this sense, ns proteins are associated with replicase complexes that co-sediment with drm markers such as caveolin-2 or sigma-1 receptor (sigmar1) in low-density fractions in isopycnic gradient ultracentrifugation experiments [19, 21] . control and lipin1deficient cells were infected with vact7 and subsequently transfected with limiting doses of the plasmid ptm-ns3/5b [16] . parallel samples were treated with dctv (100 nm). sixteen hours post-transfection, cell lysates were generated and subjected to equilibrium ultracentrifugation in 10-40% sucrose gradients. gradient fractions were collected and subjected to western-blot analysis to determine the impact of lipin1 silencing on ns3, sigmar1 and actin sedimentation profiles. fig 8a shows how, as previously shown for replicon and jfh1-infected cells, ns3 can be detected in drm fractions (fractions 3-5), as determined by the presence of sigmar1 in those fractions (fractions 3-5; s8a fig), although in this experimental system, unlike during viral infection [21] , most ns3 co-sediments together with solubilized proteins, as shown for actin (fractions 9-12; s8a fig) . drm-associated ns3 is reduced in dctv-treated and lipin1-deficient cells, while total ns3 expression remains unchanged (fig 8a) . four independent experiments were performed in which relative-drm associated ns3, normalized to that found in solubilized fractions, was calculated for each experimental condition (fig 8b) . lipin1-deficient cells display a consistent and statistically significant reduction in the drmassociated, but not total ns3 abundance (fig 8a and 8b; shlpin1-1 and shlpin1-2) , similar to that observed in control cells in the presence of dctv (figs 8a and 8b; shcontrol+dctv) , consistent with the tem data (figs 7 and s7 ) and the notion that ns3 in drm fractions may reflect the abundance of replicase complexes formed in these cells. this reduction is not due to an overall reduction in cellular drm abundance in lipin1-deficient cells, as lipin1-deficient cells display similar sigmar1 distribution pattern as the control cells (s8 fig). overall, tem data and drm floatation assays strongly suggest that lipin1 is rate limiting for the generation of replicase complexes from fully processed polyprotein subunits. hepatitis c virus replication cycle is tightly linked to host cell lipid metabolism and interference with cellular lipid homeostasis contributes to viral pathogenesis [3] . one of the most evident consequences of this interference is the high prevalence of liver steatosis among chronically infected patients [13, 60] . this clinical manifestation of the infection has been linked to, among others, chronic er stress, mitochondrial dysfunction and metabolite depletion induced by hcv infection, which result in the activation of persistent homeostatic adaptation of the cellular lipid metabolism to permit cell survival, at the cost of pathogenic metabolic alterations [11, [61] [62] [63] [64] . among the different regulatory networks that have been shown to be stimulated during hcv infection, pparα [61] , pgc-1α [65] , hif-1 [66] and srebp [46, 67] have also been shown to regulate transcription of lpin1 mrna [31, 45, 68, 69] . thus, it is likely that stimulation of one or several of these regulatory networks by hcv infection results in the lpin1 mrna transcriptional activation observed in this ( fig 1a) and other studies [40, 41] . importantly, prevention of lpin1 mrna accumulation with nac ( fig 1e) did not significantly interfere with hcv rna replication (fig 1f) , suggesting that enhanced lpin1 mrna accumulation is not required for efficient hcv infection. we favor the hypothesis that ros induced by hcv protein accumulation actively participates in lpin1 induction, as treatment with the antioxidant nac prevented lpin1 mrna accumulation, similar to what has been shown for other srebp-regulated genes during hcv infection [46] . accumulation of lpin1 mrna during hcv infection results in concomitant protein accumulation (fig 1g and 1h) . however, post-translational mechanisms such as phosphorylation, acetylation or sumoylation regulate lipin1 protein stability, membrane association as well as subcellular localization thus influencing the activity of lipin1 as pa-phosphatase and as transcriptional coactivator [70] . hence, it is difficult to predict the implications of lipin1 protein accumulation during hcv infection. thus, future studies on the interference of hcv infection with cellular lipin1 functions will be required to determine its role in hcv-related pathogenesis, particularly in its contribution to steatosis. lipin1 is required for hcv replicase formation the data presented in this study provide evidence that lipin1 is rate limiting for hcv infection at an early step of the infection leading to formation of membranous hcv replicase complexes, downstream of viral polyprotein expression and processing. we provide evidence for reduced accumulation of viral rna during single cycle infection experiments (fig 2d) , that is reminiscent of a faulty initiation of viral replication, as suggested by reduced replication of a transfected subgenomic replicon in lipin1-deficient cells (fig 5d) . data obtained in replication-independent polyprotein expression models suggest that generation of the membranous compartment that contains functional replicase complexes is severely limited in lipin1-deficient cells, as suggested by a significant reduction of the fraction of cells where these structures could be visualized by tem (figs 7 and s7 ). this hypothesis is further supported by a significant reduction in drm-associated ns proteins in lipin1-deficient cells (fig 8) , which may reflect limitations in the association of viral replicase subunits with cholesterol and sphingolipid-rich membranes in lipin1-deficient cells [19, 20, 71] . four different shrnas targeting lpin1 mrna decrease susceptibility to hcv infection proportionally to their ability to reduce total lipin1 protein accumulation (fig 4) . these results, together with the cdna rescue experiments (fig 6) , strongly reduce the possibility of observing rnai-associated off-target phenomena. interestingly, homeostatic accumulation of lipin2 protein in lipin1-deficient cells (fig 2a and 2b) is not sufficient to compensate for lipin1 loss to support efficient hcv infection. the notion that, despite being capable of mutually compensating basic liver functions [36, 37], lipin1 and lipin2 play non-redundant functions in the liver has previously been proposed [36, 72, 73] . lipin1 is tightly regulated at many different levels and its activity accommodates pap activity in response to different physiological situations such as fasting and insulin signaling [70] . compelling evidence indicates that, while lipin1 and lipin2 cooperate to maintain liver lipid homeostasis, the two proteins differ in many aspects. for instance, lipin1 is transcriptionally induced by pgc-1α and it is also an inducible amplifier of this transcriptional network [31], whereas lipin2 is not [36] . lipin1 is sumoylated and sumoylation regulates its nuclear localization and function, whereas lipin2 sumoylation could not be demonstrated, despite the presence of a canonical sumoylation motif in its primary sequence [74] . lipin1 enzymatic activity is blocked by mammalian target of rapamycin (mtor)-dependent phosphorylation in response to different metabolic stimuli [30, 75], whereas lipin2 is constitutively active even when phosphorylated [76] . thus, lipin2 is considered more as a constitutive phosphatidic acid phosphatase with lower specific activity than lipin1 [76] . in addition to these differential regulatory networks, it has been shown that in vitro pap activity of purified lipin1 and lipin2 is differentially influenced by the composition of the substrates (liposomes and lipid-detergent micelles) as well as the ph at which the assay is performed [76] . this differential lipid substrate recognition may be reminiscent of the different preferential association with membranes of different subcellular compartments (s1 fig) [73, 76] . these differences suggest that, while lipin1 and lipin2 may share some common features, they are not functionally interchangeable, particularly not in the case of hcv infection [70] . our data support the notion that lipin1 silencing has a strong impact on hcv infection without affecting basic cellular functions (fig 2b) or significantly interfering with infection by an unrelated virus (s3 fig). despite great efforts and different overexpression systems, functional rescue of lipin1 functions by wt lipin1 cdna overexpression in lipin1-deficient cells only lead to a small but consistent rescue of virus infection efficiency, which was only observed when overexpressing wt lipin1 (figs 6 and s6) . given the multiple transcriptional, post-transcriptional and post-translational regulation levels existing for lipin1 expression, it is conceivable that only a fraction of the overexpressed lipin1 is fully competent to sustain hcv infection. moreover, high overexpression levels could only be achieved in lipin1-deficient cells (fig 6a) , underscoring the notion that intracellular lipin1 levels are tightly regulated by the host. nevertheless, overexpression of a mutant lipin1 lacking phosphatase activity (dxdxt) or a mutant inactive as transcriptional coactivator (lxxil) were not capable of enhancing hcv infection in lipin1-deficient cells as compared with the wt lipin1 ( fig 6b) . these data reveal that lipin1 phosphatase activity is required for lipin1 to support hcv infection and suggest that, while transcriptional co-activation by lipin1 may be important, this function is not sufficient to support hcv replication. lipins are important enzymes in the main pathway for de novo phospholipid biosynthesis by providing dag derived from the glycerol-3-phosphate pathway to produce pc and pe through the kennedy pathway [70] . production of membranous replicase compartments likely requires de novo synthesis of pc and/or pe, which are major components of biological membranes that depend on dag biosynthesis [29] . in fact, local pc biosynthesis is required for efficient replication of (+) rna viruses and certain pc species accumulate in hcv-infected cells [26, 28] . although increased lipin2 accumulation may be sufficient to compensate lipin1 silencing at the whole-cell level [37], it is possible that acute, local demand of de novo synthesized phospholipids is required at defined suborganellar compartments during early steps of hcv infection and that lipin1-deficiency shortens or alters the availability of different membrane components, demand that may not be satisfied by lipin2, given the differential regulation [76] and subcellular localization of these two proteins (s2 fig). remarkably, deletion of the yeast lipin homologs pah1/smp2 (s. cerevisae) or ned1 (s. pombe) gene, results in deregulated proliferation of the er and nuclear envelope membranes [77, 78] , with concomitant enhancement in (+) rna virus replication [79, 80] . the membranous alterations and elevation of total phospholipid content observed in pah1-deficient yeast have been ascribed to transcriptional activation of pah1-independent alternative phospholipid biosynthetic programs due to pa accumulation [77, 80] . our data in mammalian cells are more compatible with a shortage of phospholipid production, which may be at the basis of the reduced abundance viral membranous structure (figs 7 and 8) . this opposite outcomes of infection may derive from the fact that yeast use mainly pa (lipin1 pap substrate) as a precursor for pc biosynthesis, while mammals mainly use dag (lipin1 pap product) as precursor for pc biosynthesis through the kennedy pathway [77, [80] [81] [82] . moreover, in contrast to yeast and lower eukaryotes, which express only one lipin gene, three different lipin genes coordinate glycerolipid homeostasis in mammals [72] . thus, interfering with expression of one of the members of the family may not be sufficient to observe the same effects observed when deleting pah1, as transcriptional and posttranslational homeostatic compensations are in place in mammals, particularly between lipin1 and lipin2 in liver tissue [36, 37] . in this sense, deletion of either lipin1 or lipin2 in mouse models results in a relatively balanced liver phospholipid content while simultaneous deletion of both lipins is embryonically lethal [37] . accordingly, only minor alterations of er membranes [83] and no significant alterations in total pc levels [36] have been reported in lipin1-deficient mouse liver. given the fact that hcov-229e is fully capable of replicating in these cells (s3 fig), it is unlikely that a general disruption of de novo phospholipid biosynthesis occurs in lipin1-deficient cells, particularly since hcov-229e infection also induces profound er membrane rearrangements required for replication [84] , some of which are structurally similar to dmvs observed during hcv infection [85] . thus, we favor the hypothesis that a subcellular pool of glycerophospholipids is managed by lipin1 in huh-7 cells and that lipin1 silencing perturbs local levels of pa and dag, limiting local availability of precursors of structural components of virus-induced membranes. alternatively, lipin1 deficiency may alter local amounts of important signaling molecules, in particular, that of its substrate (pa) or its product (dag). deregulation of the local pa and dag pools may cause important alterations for the host cell, as both metabolites are potent chemical messengers that regulate different aspects of cellular homeostasis [86] [87] [88] . regarding pa conversion into dag by lipins, it has been shown that pah1 (yeast lipin1 homolog) phosphatase activity is critical for transforming local pools of pa into dag at the er membrane to facilitate membrane fusion events mediated by snare complexes [89, 90] . mammalian lipin1 phosphatase activity is also critical for transforming local pools of pa that accumulate at the surface of mitochondria to promote mitochondrial fission [91] or at the surface of endolysosomes to facilitate autophagy [92] . thus, lipin1 and probably other members of the lipin family modulate different aspects of intracellular membrane signaling. given that the function of host factors known to be involved in functional hcv replicase biogenesis, like vapa, vapb and osbp [11] are indirectly regulated by local pa/dag pools [93] , it is tempting to propose that lipin1 silencing interferes with the function of one or several of these, or other yet uncharacterized cellular factors. in contrast to what has been reported for other host factors required for hcv replicase complex formation [58] , we did not find evidence of lipin1 protein relocalization during hcv infection (s1 fig). thus, determining the precise mechanism by which lipin1 regulates hcv replicase formation is challenging, as association of lipin1 with different cell membranes is transient and highly regulated by posttranslational modifications [70] . moreover, some of the known lipin1 cellular functions may be compensated by other lipins, particularly lipin2 in the liver. nevertheless, our data clearly indicate that lipin1 participates at early stages of hcv replication and that the aforementioned homeostatic compensations by other lipins in regards to cellular metabolism may constitute an advantage when considering lipin1 as a host target for anti-hcv therapy. hcv antiviral compounds 2´-c-methyladenosine (2made), sofosbuvir and daclatasvir were obtained from boc sciences (ny, usa), selleckchem (texas, usa) and medchem express (new jersey, usa) respectively and dissolved in dmso to obtain 10mm stock solutions. nacetylcysteine (nac) and puromycin were obtained from sigma-aldrich (missouri, usa), dissolved in water to a final concentration of 0.5 m and 50 mg/ml respectively. hydroxyzine pamoate was purchased from sigma-aldrich (missouri, usa) and dissolved in dmso to a final 10 mm concentration. human hepatoma huh-7 and derived subclones huh-7.5, huh-7.5.1 (clone 2) have been described [94] [95] [96] and were kindly provided by dr. chisari (tsri-la jolla, ca). hek-293t cells [97] were kindly provided by dr. ortin (cnb-madrid, spain). cell cultures were maintained subconfluent in dulbecco´s modified eagle´s medium (gibco) supplemented with 10 mm hepes (gibco), 100u/ml penicillin/streptomycin (gibco), 100μm non-essential amino acids (gibco) and 10% fetal bovine serum (sigma-aldrich). genotype 2a (jfh-1 strain), d183 adapted virus (d183) and genotype 1a (tncc) virus have been described elsewhere [42, 49, 56] . tncc plasmid was kindly provided by dr. jens bukh (huidovre hospital; copenhagen, denmark). recombinant viruses were generated by electroporation of an in vitro transcribed rna as described previously [42] , using clone 2 cells (jfh-1 and d183) virus and huh-7.5 cells for tncc. supernatants containing the infectious virus were used to inoculate naive huh-7.5.1 clone 2 cells at low multiplicity of infection (moi 0.01 ffu/cell) to prepare working virus stocks. for tncc infections, supernatants of electroporated cells were directly used. lentivirus production. lentiviral vectors shrna expression were produced by co-transfection of plasmids pmdl-rre, pmd2g and prsv-rev (a gift from didier trono-addgene plasmids #12251; #12253; #12259), together with the genomic plasmid (mission plko-puro; sigma-aldrich) into hek-293t cells [21] . selected shrna target sequences are: cgagagaa agtggttgacata (shlpin1-1); (cctcagacagaaatgcagttt) shlpin1-2; cact cccagtccttccggttc (shlpin1-3); cctgttccatccttcggaaag (shlpin1-4). plasmids encoding a atg4b shrna (atg4b800) [57] , non-targeting shrna (shcontrol) as well as an shrna targeting hcv ires (shhcv)were previously described [21] . a lentiviral vector plasmid encoding lipin1-alpha cdna was purchased from genecopeia (cat nbr: ex-t7124-lv153). to generate lipin1 beta isoform, exon 7 sequence was inserted using neb-q5 mutagenesis kit (neb). similarly, silent mutations were introduced in shlpin1-2 target sequence to obtain wt lipin1beta protein expression from a cdna resistant to silencing. mutations in the conserved motifs dxdxt (didgt>eidgt) and lxxil (lghil>lghff) were also introduced in lipin1beta cdna using neb-q5 kit, based on previously described mutations [31, 98] . the sequence of the entire lpin1 cdnas was determined by sanger sequencing before use, to assess the introduction of only the desired mutations. coronavirus 229e-gfp. recombinant human coronavirus 229e-gfp [51] was kindly provided by dr. volker thiel (university of bern, switzerland). virus was propagated by infection (moi 0.01) in huh-7 cells. supernatants were collected at different time points and titrated by end-point dilution and immunofluorescence microscopy. lentiviral vectors expressing control and lpin1-specific shrnas were used to inoculate huh-7 cells. twenty-four hours later, cells were subjected to selection with 2.5μg/ml of puromycin to assess the lowest lentivirus dose capable of conferring puromycin resistance to 100% of the cell population. selected cell populations were subsequently cultured in the presence of puromycin until lpin1 silencing was ascertained by western-blot using anti-lipin1 antibodies, typically at day 6-7 post lentiviral transduction, time at which all experiments were performed in the absence of puromycin. before execution of all the experiments shown in this study, lipin1 expression was assessed by western-blot. cell viability was determined by a thiazolyl blue tetrazolium blue (mtt) formazan formation assay [47] . control and lipin1-deficient cell lines (5. 10 4 cells/well) were plated onto 12-well plates and were inoculated with d183 virus at a moi 10 ffu/cell. samples of the cells and supernatants were collected 24, 48 and 72 hours post-infection. for multiple cycle infection experiments (moi 0.01), samples of the supernatants were collected at day 3, 5 and 7 post-inoculation. cells were split 1:3 in the multiple cycle infection experiments at days 3 and 5 to maintain the cultures subconfluent. extracellular infectivity titers were determined by endpoint dilution and infection foci counting as previously described [99] . intracellular hcv rna was determined by reverse transcription and quantitative pcr (rt-qpcr) as previously described [99] . total protein samples were prepared in laemmli buffer and separated using polyacrylamide denaturing gel electrophoresis (sds-page). proteins were subsequently transferred onto pvdf membranes and incubated with 5% milk (lipins) or 3% bsa in pbs-0.25% tween20 for one hour at room temperature (rt). primary antibodies against lipin1 (clone b-12; santa cruz), lipin2 (h-160; santa cruz), ns3 (clone 2e3; biofront), beta-actin (ab8226; abcam) and tubulin (clone aa2; sigma-aldrich) were diluted in pbs-0.25% tween20 and incubated for 1 hour (four hours for lipins) at rt. membranes were subsequently washed for 20 minutes with pbs-0.25% tween20 three times. horseradish peroxidase-conjugated secondary antibodies were incubated for 1 hour at room temperature in 5% milk-pbs-0.25% tween20 and subsequently washed three times for 20 minutes at room temperature. protein bands were detected using enhanced chemoluminescence reactions and exposure to photographic films. specific bands were quantitated using the imagej software [100] on non-saturated, scanned films. confocal microscopy was performed with a leica tcs sp8 laser scanning system (leica microsystems). images of 1024 × 1024 pixels at eight bit gray scale depth were acquired sequentially every 0.13-0.3 μm through a 63x/1.40 n.a. immersion oil lens, employing las af v 2.6.0 software (leica microsystems). colocalization indexes were calculated using jacop plugin for image j [101] from a minimum of 10 regions of interest (roi). images were processed using imagej, were medians of 1 pixel were obtained for the different channels, only for illustration, not for analysis. color levels, brightness and contrast were manipulated for illustration using technical and biological controls as reference. total rna extraction was performed using the gtc extraction method [102] . purified rna (10-500 ng) was subjected to rt-qpcr using random hexamers and a reverse transcription kit (applied biosystems). quantitative pcr was performed using 2x reaction buffer from (applied biosystems) and specific oligonucleotides as previously described [99, 103] . standard curves were prepared by serial dilution of a known copy number of the corresponding amplicon cloned in a plasmid vector. control and lipin1-deficient huh-7.5 cells were inoculated with tncc virus (moi 0.05). due to the relatively low propagation levels of the tncc virus in this experimental setup, parallel cultures were infected and treated with 2´-c-methyladenosine (2made; 10μm) to determine the levels of non-replicative, background hcv rna. cells were incubated for 72 hours at 37˚c, time after which samples of the cells were collected to determine intracellular hcv rna levels by rt-qpcr. to establish persistently infected cell cultures huh-7 cells were inoculated at moi 0.01 with jfh-1 hcv strain as previously described [24] . cell cultures were maintained subconfluent for two weeks, time after which infection rates reach nearly 100% of the cells, as assessed by immunofluorescence microscopy. at this point cells were split and transduced with the corresponding lentiviral vectors in order to generate lipin1-deficient cell cultures as well as control cell lines. once silencing had been verified by western-blot, typically at day 6-8 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine intracellular hcv rna levels by rt-qpcr and extracellular infectivity titers by end-point dilution and immunofluorescence microscopy. infectious, spread-deficient hcv particles produced by trans-complementation (hcv tcp ) have previously been described [52] . briefly, huh-7.5.1 clone 2 cells expressing core-e1 and e2-ns2 regions from jfh-1 by lentiviral transduction, were electroporated with a jfh-1 subgenomic dicistronic replicon bearing a firefly luciferase gene with reagents kindly provided by dr. ralf bartenschlager (u. of heidelberg). supernatants containing hcv tcp were collected 36, 48 and 72 hours post-electroporation, pooled and assayed for viral infectivity. hcv tcp infection efficiency was determined by inoculating naïve huh-7 cells with the electroporation supernatants and measuring luciferase activity 48 hours post-infection using a commercially available kit (promega). retroviral particle production pseudotyped with different viral envelopes has previously been described [54, 55] with the materials kindly provided by dr. f. l. cosset (inserm, lyon). control and lipin-deficient cell lines were inoculated with hcv pp and vsv pp and incubated for 48 hours, time at which total cell lysates were assayed for luciferase activity using a commercially available kit (promega). a selective hcv entry inhibitor, hydroxyzine pamoate (hdx) from sigma-aldrich (missouri, usa), was used as positive control of inhibition [55] . a plasmid containing the sequence corresponding to a subgenomic jfh-1 replicon bearing a firefly luciferase reporter gene was kindly provided by dr. ralf bartenschlager (u. of heidelberg) [52] . after digestion with the restriction enzyme mlui, the linearized plasmid was transcribed in vitro using a commercial kit (megascript t7; ambion-paisley, uk). the resulting products were digested with dnase and precipitated with licl. pelleted rna was washed with 75% and 100% ethanol, and resuspended in nuclease-free water. in vitro transcribed rna was transfected into the different cell lines together with a plasmid expressing renilla luciferase under a minimal promoter (prl-null; clontech-california, usa) using lipofectamine 2000 and the manufacturer´s recommendations (life technologies-california, usa). firefly and renilla luciferase activities were measured in the sample using a commercial kit (dual luciferase assay system; promega-wisconsin, usa) at different times post-transfection. lipin1-deficient cells were generated by lentiviral transduction of shlpin1-2 shrna. at day 3 post-transduction, control and lipin1-deficient cell populations (5 x 10 4 cells/m24 well) were transfected in suspension using lipofectamine 2000 with plasmids (800 ng/m12 well) expressing wt lipin1beta isoform shlpin1_2-resistant cdna or dxdxt or lxxil motif mutants [31] . transfected cell cultures were incubated for 48 hours and subsequently inoculated at moi 10 with d183 virus. infection efficiency was determined by measuring extracellular infectivity titers 48 hours post-infection. parallel cultures were used to determine relative wt and mutant protein expression efficiency by western-blot. infectivity titers were measured as described above. the relative impact of cdna expression was estimated by determining the ratio between the infectivity found in lipin1-deficient cells and the control cells transfected with the same plasmid. in order to average experiments with different raw infection efficiency, all the experiments were referenced to the ratio in the mock-transfected cells. control and lipin1-deficient huh-7 cells were inoculated with cov-229e (moi 0.01) for 2 hours at 37˚c. cells were washed twice with warm pbs and replenished with dmem-10% fcs. extracellular infectivity titers were determined 48 hours post-infection by end-point dilution and fluorescence microscopy in huh-7 cells. huh-7 cells were inoculated at moi 10 with a recombinant vaccinia virus expressing the t7 phage rna polymerase (vact7) [104] . two hours later, cells were transfected with the plasmid ptm-ns3/5b [16, 58] (kindly provided by dr. lohmann; u. of heidelberg) and lipofectamine 2000 (thermofisher-massachussets, usa) following the manufacturer´s recommendations in terms of total dna per well (typically 4μg per 35mm dishes with 7.5 x 10 5 cells/well) and 50% of the recommended lipofectamine:dna ratio. transfected cells were cultured in the presence of the dna replication inhibitor cytosine β-d-arabinofuranoside (arac; sigma-aldrich) for 16 hours to prevent vact7 replication [105] . when indicated, media was also supplemented with 100nm daclatasvir (dctv). total cell extracts were used to determine viral protein accumulation by western-blot using anti-ns3 antibody (clone 2e3; biofront) and β-actin (abcam; ab8226) as loading control. for ultrastructural electron microscopy studies, control and lipin1-deficient cells expressing ns3-5b polyprotein (see above) were cultured on glass coverslips and fixed in situ after polyprotein expression with a mixture of 2% paraformaldehyde (taab) and 2.5% glutaraldehyde (taab) (1h at room temperature), post-fixed with 1% osmium tetroxide in pbs (45 min), treated with 1% aqueous uranyl acetate (45 min), dehydrated with increasing quantities of ethanol and embedded in epoxy resin 812 (taab). ultrathin, 70-nm-thick sections were cut in parallel to the monolayer, transferred to formvar-coated em buttonhole grids and stained with aqueous uranyl acetate (10 min) and lead citrate (3 min). sections were visualized on a jeol jem 1200 exii electron microscope (operating at 100 kv). quantitation of hcv-induced structures was performed as follows. to quantitate the differences in total vesicle abundance, tem sections were visually inspected under the microscope for the presence/absence of vesicular structures. the number of positive cells and total number of cells were inserted in a 2 x 2 contingency table to determine the statistical significance of the differences between control and lipin1-deficient cells using two-tailed fisher´s exact test or two-tailed chi square test. in addition, we determined the frequency of hcvinduced vesicles in dctv-treated control cells by dividing the number of structures per inspected area and calculating the average and sd of the frequencies found in the different images. the diameters of individual vesicles were determined manually using size-calibrated images and image j software. lipin1-deficient and control cells (7.5 x 10 5 cells) were infected with vact7 virus (moi 10) and transfected with limiting doses of ptm-ns3/5b plasmid (typically 800 ng/ well), as higher plasmid doses may difficult observing the reported differences. cells were lysed by adding 250 μl of tne (50mm tris-hcl ph 7.5, nacl 150 mm and edta 2 mm) buffer containing 0.5% triton x-114 and protease inhibitors (complete; roche-basel, sw). lysates were incubated for 30 minutes on ice before clearing them by 10-minute centrifugation at 12,000 r.p.m. clear supernatants were mixed 1:1 with 60% sucrose tne solution. this mixture was applied on top of a 40% sucrose-tne cushion and was overlaid with 20% and 10% sucrose-tne until completing the discontinuous gradient. gradients were centrifuged for 16 hours at 120,000 x g. fourteen fractions were collected from the top and analyzed by sds-page and western-blot using antibodies against ns3 (clone 2e3; biofront), sigmar1 (s-18; santa-cruz), caveolin-2 (epitomics; 3643-1) and beta actin as described previously [21] . ns3 signal was quantitated using imagej software and the fraction of drm-associated ns3 was determined as the ratio of ns3 signal in fractions 3, 4 and 5 to the total ns3 signal in the gradient. global epidemiology and burden of hcv infection and hcv-related disease immunobiology and pathogenesis of viral hepatitis liver injury and disease pathogenesis in chronic hepatitis c from non-a, non-b hepatitis to hepatitis c virus cure reversion of disease manifestations after hcv eradication risk for hepatocellular carcinoma after hepatitis c virus antiviral therapy with direct-acting antivirals: case closed? the molecular and structural basis of advanced antiviral therapy for hepatitis c virus infection unique ties between hepatitis c virus replication and intracellular lipids hepatitis c virus rna replication and assembly: living on the fat of the land the ins and outs of hepatitis c virus entry and assembly liver international: official journal of the international association for the study of the liver identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection. cellular and molecular life sciences: cmls three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication structural changes in cells imaged by soft x-ray cryo-tomography during hepatitis c virus infection hepatitis c virus rna replication occurs on a detergentresistant membrane that cofractionates with caveolin-2 characterization of the hepatitis c virus rna replication complex associated with lipid rafts replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna sigma-1 receptor regulates early steps of viral rna replication at the onset of hepatitis c virus infection hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice lpin1 rs13412852 polymorphism in pediatric nonalcoholic fatty liver disease hepatitis c virus rna functionally sequesters mir-122 an integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to hcv infection persistent hepatitis c virus infection in vitro: coevolution of virus and host sofosbuvir: a new oral once-daily agent for the treatment of hepatitis c virus infection ros-mediated p53 induction of lpin1 regulates fatty acid oxidation in response to nutritional stress induction of lipin1 by ros-dependent srebp-2 activation hepatitis c virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress a rapid colorimetric assay of fungal viability with the tetrazolium salt mtt regulation of the hepatitis c virus rna replicase by endogenous lipid peroxidation highly efficient full-length hepatitis c virus genotype 1 (strain tn) infectious culture system characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis c virus replication in vitro dendritic cell-specific antigen delivery by coronavirus vaccine vectors induces long-lasting protective antiviral and antitumor immunity efficient trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis infectious hepatitis c virus pseudo-particles containing functional e1-e2 envelope protein complexes selective inhibition of hepatitis c virus infection by hydroxyzine and benztropine. antimicrob agents chemother production of infectious hepatitis c virus in tissue culture from a cloned viral genome the autophagy machinery is required to initiate hepatitis c virus replication recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment daclatasvir-like inhibitors of ns5a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication and facts fictions of hcv and comorbidities: steatosis, diabetes mellitus, and cardiovascular diseases nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis c virus infection cellular stress responses in hepatitis c virus infection: mastering a twoedged sword entangled in a membranous web: er and lipid droplet reorganization during hepatitis c virus infection. current opinion in cell biology hepatitis c virus, cholesterol and lipoproteins-impact for the viral life cycle and pathogenesis of liver disease endoplasmic reticulum stress links hepatitis c virus rna replication to wild-type pgc-1alpha/liver-specific pgc-1alpha upregulation hepatitis c virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation the hepatitis c virus-induced nlrp3 inflammasome activates the sterol regulatory element-binding protein (srebp) and regulates lipid metabolism hypoxia causes triglyceride accumulation by hif-1-mediated stimulation of lipin 1 expression sterol-mediated regulation of human lipin 1 gene expression in hepatoblastoma cells lipin proteins and glycerolipid metabolism: roles at the er membrane and beyond morphological and biochemical characterization of the membranous hepatitis c virus replication compartment three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2 sumoylation regulates nuclear localization of lipin-1alpha in neuronal cells phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation the yeast lipin smp2 couples phospholipid biosynthesis to nuclear membrane growth an evolutionarily conserved fission yeast protein, ned1, implicated in normal nuclear morphology and chromosome stability, interacts with dis3, pim1/rcc1 and an essential nucleoporin inactivation of the host lipin gene accelerates rna virus replication through viral exploitation of the expanded endoplasmic reticulum membrane host pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis interactions among pathways for phosphatidylcholine metabolism, ctp synthesis and secretion through the golgi apparatus phospholipid biosynthesis in eukaryotes the fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins inhibition of cytosolic phospholipase a2alpha impairs an early step of coronavirus replication in cell culture membranous replication factories induced by plus-strand rna viruses shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling diacylglycerol kinases: shaping diacylglycerol and phosphatidic acid gradients to mitochondria: signaling with phosphatidic acid phosphatidic acid sequesters sec18p from cis-snare complexes to inhibit priming a common lipid links mfn-mediated mitochondrial fusion and snare-regulated exocytosis pirna-associated germline nuage formation and spermatogenesis require mitopld profusogenic mitochondrial-surface lipid signaling. developmental cell lipin-1 regulates autophagy clearance and intersects with statin drug effects in skeletal muscle pkd regulates membrane fission to generate tgn to cell surface transport carriers. cold spring harbor perspectives in biology growth of human hepatoma cells lines with differentiated functions in chemically defined medium highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication interferon modulation of cellular micrornas as an antiviral mechanism production of high-titer helper-free retroviruses by transient transfection the cellular functions of the yeast lipin homolog pah1p are dependent on its phosphatidate phosphatase activity robust hepatitis c virus infection in vitro nih image to imagej: 25 years of image analysis a guided tour into subcellular colocalization analysis in light microscopy single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction expression of the splicing factor gene sfrs10 is reduced in human obesity and contributes to enhanced lipogenesis cytoplasmic expression system based on constitutive synthesis of bacteriophage t7 rna polymerase in mammalian cells transient expression of the vaccinia virus dna polymerase is an intrinsic feature of the early phase of infection and is unlinked to dna replication and late gene expression we are grateful to drs. bartenschlager key: cord-335085-7pxkhgbq authors: dessau, r. b.; lisby, g.; frederiksen, j. l. title: coronaviruses in spinal fluid of patients with acute monosymptomatic optic neuritis date: 2009-01-29 journal: acta neurol scand doi: 10.1111/j.1600-0404.1999.tb01043.x sha: doc_id: 335085 cord_uid: 7pxkhgbq acute monosymptomatic optic neuritis (amon) may be an initial symptom of multiple sclerosis (ms). coronaviruses have been implicated in the etiology of ms. the objective of the present study was to look for coronaviral rna in amon, which could be present in the initial stages of the development of ms. material and methods ‐ spinal fluids from 37 patients with amon and 15 surgical control patients with protrusion of the intervertebral disk were assayed with a nested multiplex polymerase chain reaction with primers specific for human coronaviruses strain (hcv) 229e and oc43. results ‐ four patients and 1 control were positive for hcv‐229e. no evidence of hcv‐oc43 was found. the frequency of positive samples was low and there was no statistical difference between amon and controls. conclusion ‐ this study does not provide evidence for an etiological role of human coronaviruses in acute monosymptomatic optic neuritis. acute monosymptomatic optic neuritis (amon) may occur as an isolated event, but is often considered an initial symptom of multiple sclerosis (ms). about half of the patients will convert to clinically definite ms within 15 years (1) . amon frequently lasts from a few days to several weeks and may affect one or both eyes. the main symptoms are loss of vision, retrobulbar pain and dyschromatopsia. the presence of human coronaviral rna has been demonstrated in brain tissue and spinal fluid from patients with clinically definite ms and controls ( 2 4 ) . to investigate the possibility of an infection with human coronaviruses (hcv) in early ms and as a possible cause of amon we have analyzed cerebrospinal fluid (csf) from patients with amon using reverse transcriptase reaction and the polymerase chain reaction (rt-pcr) applying primers specific for hcv. csf was obtained by lumbar puncture from 37 untreated patients with amon. none of the patients had other symptoms of ms at the time of lumbar 88 puncture. thirty-four samples were obtained from 6 to 104 days (median 23 days) after the onset of symptoms. three patients had 2 attacks of amon and the samples were obtained in relation to the first episode 205, 182 and 71 days before their second episode of amon. in 2 patients different eyes were involved and in 1 patient the same eye. thirteen of the patients have later developed clinically definite ms. csf obtained from 15 surgical patients with protrusion of the intervertebral discs were used as controls. the median age of patients and controls with amon was 33 (range 13-57) and 51 (range 35-66) years respectively (median test p<0.001). in the amon group there were 25 female patients and among the controls there were 8 females. in the rt-pcr assay, a pool of 14 hcv negative spinal fluids, from the routine laboratory, were interspersed at every 3 to 4 patient samples and were included throughout the whole procedure starting with the rna-extraction. further negative controls, sterile water instead of sample, were added at each enzymatic reaction, starting with one in the rtreaction ending with 3 in the second pcr with the inner set of nested primers. as positive controls for the pcr, cell culture supernatant fluids containing hcv strain 229e (atcc vr-740, and hcv strain oc43 (tcidso lo4, atcc vr-759) were used. all samples were stored at -80°c until analysis. a multiplex nested rt-pcr as detailed below was used, using primers specific for both hcv-229e and hcv-oc43 (table 1) . rna extraction was performed by lysis of 100 pl spinal fluid or serum by vortexing 10 s in 400 p1 guscn-buffer (4 mm guanidiniumisothiocyanate, 1 mm dithiothreitol, 10 mm tris, ph 6). lsopropanol (500 pl) was added, vortexed briefly and spun in a microcentrifuge at 13,000 g for 10 min at room temperature. for the serum samples, complete removal of supernatant after the isopropanol precipitation was important to avoid a sticky protein precipitate following the ethanol step. to the pellet 750 pi 70% ethanol was added, the tube was vortexed and precipitated as described and the supernatant was removed. reverse transcriptase (rt) reaction was performed by mixing primers and other reagents (geneamp rna pcr kit, perkin elmer cetus) in a total volume of 20 pl. the concentration of the primers coro10 and cor012 was 0.5 pm. the other reagents were added according to the standard protocol included with the kit; 5 mm mgc12, x 1 pcr buffer 11, 1 mm of each nucleotide (dgtp, datp, dttp, dctp), rnase inhibitor 1 u/p1, reverse transcriptase 2.5 u/p1. incubation was performed at 42°c for 15 min, at 99°c for 5 rnin and 4°c for less than 1 h. a multiplex pcr using specific primersets for hcv-229e and hcv-oc43 (table 1 ) was performed in a total volume of 100 pl. the final concentration of reagents for pcr including the reagents carried over from the rt-reaction: 0.4 pm of the primers cor09 and corolo), 0.2 pm of the primers coroll and coro12, 2.5 mm mgc12, x 1 pcr buffer 11, 1 mm of each nucleotide (dgtp, dutp, dttp, dctp), dna-polymerase (amplitaq, perkin elmer cetus) 0.03 u/pl, uracil-n-glycosylase (boehringer mannheim) 0.01 u/p1, 2.5 mm mgc12. temperature cycles were 37°c (10 min), 95°c (10 rnin), then 94" -58" -72°c (1 min each, 40 cycles), 72°c (15 min) and finally soaked at -5°c. one 1. 11 was transferred to the inner pcr reactions. the inner pcr reactions were performed separately for each virus without repeating the uracil-n-glycosylase step. for hcv-oc43 primers cor05 and cor06 with an annealing temperature at 62°c and 2.25 mm mgc12 were used. for the hcv-229e primers corol and cor02 an annealing temperature of 68°c and 2.0 mm mgci2 were used. the sensitivity of the pcr assay was measured by samples containing 10 fold dilutions of the virus containing cell culture supernatants in coronavirus negative spinal fluid. the sensitivity was 10 times tcidso for both hcv-229e and hcv-oc43. the pcr products were visualized by electrophoresis at 5 v/cm on 1.5% agarose followed by southern blot on nytran filter. the blot was performed as described (5), with the exception that the setup was inverted. after overnight transfer, the nytran filter paper was rinsed briefly in 2xssc, and nucleic acids were immobilized by baking at 80°c in vacuum for 2 h. subsequently, the blots were prehybridized at 42°c for 2 h, followed by hybridization overnight with a hcv-229e or a hcv-oc43 dna fragment. these probes were produced by pcr as described above from the reference strains and labelled with biotin-7-datp by nick-translation (bionick, life technologies, rockville, usa). stringent wash was performed in 0.15 ssc for hcv-229e and in 0.2 ssc for hcv-oc43 at 70°c. probe detection was performed using the photogene detection kit according to the manufacturer's instructions (life technologies, rockville, usa). the pcr products were sequenced in both directions using primers corol and cor02 as sequencing primers, cycle sequencing reaction using the primers corol and cor02 using the taq dyedeoxy terminator cycle sequencing kit (applied biosystems, perkin elmer cetus, connecticut, usa). the sequence reactions were separated and analyzed on an applied biosystems 373a automated dna sequencer as recommended by the manufacturer. for sequence editing, the seqed software (applied biosystems) was used. alignment of the sequence results was done using the gcg software package (6). csf from 4 patients and 1 control ( table 2) were positive on nested rt-pcr using the hcv-229e primers and all samples were negative with the hcv-oc43 primers. the amplified dna fragments were only visualized after hybridization and not on the ethidium bromide stained gel, indicating that very few rna copies had been present in the original patient sample at the limit of the sensitivity of the assay. ten interspersed negative controls (pooled spinal fluids) included throughout the procedure from rna extraction to the inner pcr remained negative. water controls added for each enzymatic step also remained negative. in the amplified fragments all 261 bases between the primers (fig. 1) were sequenced. one patient (amon28) had a frameshift and a base substitution and 1 patient (amon40) had a base substitution. the control patient (pid375) had an ambiguous sequencing result. our positive control for hcv-229e and the consensus sequence has 2 base substitutions compared to the published sequence (7) used for the design of the primers, but it is however identical to another available sequence of the same gene (8) . to our knowledge the presence of human coronaviruses in csf from patients with amon has not previously been investigated. in the present study 11% of the patients with amon and 7% of the controls were positive for hcv-229e and none for hcv-oc43. we consider the possibility of false positive results due to contamination less likely, as uracil-n-glycosylase carry over prevention was used and the negative controls were consistently negative. on sequencing of the pcr products 2 base substitutions and 1 frameshift mutation were found. furthermore, 1 control patient had an ambiguous sequencing result. the error rate of pcr using the taq polymerase has been estimated to be between 1/103 and 1/104 nucleotides or less depending on the initial number of target copies (9, 10) and the reaction conditions. also the rt step may incorporate errors into the template for the pcr. in this study 6 pcr products of 261 nucleotides equal to 1566 nucleotides were sequenced (fig. 1 ) and 4 differences were found. this gives a frequency of 1 mutation per 392 bases. from these few differences cannot be distinguished between true genomic variation and experimental errors in pcr or dna-sequencing. the nucleocapsid gene of coronaviruses seems well conserved. there is only one published sequence of the atcc type strain hcv-oc43. comparison of hcv-oc43 and bovine coronavirus nucleocapsid gene (6, 11, 12) show a 96.6% homology of the coding sequences of 1347 bases, and only 6 differences over the 258 bases between the inner primers amplified in this study. thus large genomic variation may not be expected, even in strains originating from different mammal species. concerning hcv-229e there are two published sequences of the nucleocapsid gene from the same atcc type strain (7, 8) . hcv-229e is not as closely related to other known coronaviruses (7), and the natural sequence variation in clinical strains has not been studied. another study (4) has also investigated csf with pcr-technique for hcv. the csf from patients with ms and controls with other neurological disease were analyzed for the presence of human coronaviral rna sequences. the study found the csf from 10 of 20 patients with ms and 9 of 10 controls positive for hcv-oc43. the csf from 7 of 20 patients with ms and 2 of 10 controls were positive for hcv-229e. thus in total more than 80% of the tested patients were virus positive. this very high frequency of positive results also in the control is not consistent with the findings in our study. the difference could be explained by differences in sensitivity of the assays or by differences in the selection of the control group. the study by cristallo et al. (4) does not describe the level of sensitivity, and the control group in the present study consists of patients with protrusion of the intervertebral disk and not of patients with other degenerative neurological diseases. hcv-229e rna was found in the csf by rt-pcr in 4 of 37 patients with amon and in 1 of 15 controls. the frequency of positive samples was low and there was no statistical difference between amon and controls. this study does not provide evidence for an etiological role of human coronaviruses in acute monosymptomatic optic neuritis or in early ms. multiple sclerosis. clinical and pathogenetic basis human coronavirus gene expression in the brains of multiple sclerosis patients detection of coronavirus rna and antigen in multiple sclerosis brain human coronavirus polyadenylated rna sequences in cerebrospinal fluid from multiple sclerosis patients detection of specific sequences among dna fragments separated by gel electrophoresis version 9 sequence analysis of the nucleocapsid protein gene of human coronavirus 229e human coronavirus mrna for nucleocapsid protein the fidelity of dna polymerases used in the polymerase chain reactions polymerase chain reaction: replication errors and reliability of gene diagnosis sequence analysis of nucleocapsid gene and leader rna of human coronavirus oc43 sequence and analysis of bovine enteritic coronavirus finn sellebjerg, department of neurology, glostrup university hospital, denmark has provided the cerebrospinal fluids from the control patients with protrusion of the intervertebral disks. the danish multiple sclerosis society has provided financial support. key: cord-340220-raqapqg0 authors: potel, julie; rassam, patrice; montpellier, claire; kaestner, laura; werkmeister, elisabeth; tews, birke a.; couturier, cyril; popescu, costin‐ioan; baumert, thomas f.; rubinstein, eric; dubuisson, jean; milhiet, pierre‐emmanuel; cocquerel, laurence title: ewi‐2wint promotes cd81 clustering that abrogates hepatitis c virus entry date: 2013-02-16 journal: cell microbiol doi: 10.1111/cmi.12112 sha: doc_id: 340220 cord_uid: raqapqg0 cd81 is a major receptor for hepatitis c virus (hcv). it belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin‐enriched areas in the plasma membrane. in our study, we combined single‐molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of cd81 in the context of cells expressing ewi‐2wint, a natural inhibitor of hcv entry. interestingly, we found that ewi‐2wint reduces the global diffusion of cd81 molecules due to a decrease of the diffusion rate of mobile cd81molecules and an increase in the proportion of confined molecules. indeed, we demonstrated that ewi‐2wint promotes cd81 clustering and confinement in cd81‐enriched areas. in addition, we showed that ewi‐2wint influences the colocalization of cd81 with claudin‐1 – a co‐receptor required for hcv entry. together, our results indicate that a change in membrane partitioning of cd81 occurs in the presence of ewi‐2wint. this study gives new insights on the mechanism by which hcv enters into its target cells, namely by exploiting the dynamic properties of cd81. viral entry relies on a fine interplay between a viral particle and a host cell. this process is initiated by binding to an attachment factor present at the plasma membrane of target cells, which helps to concentrate virions on the cell surface. after this initial step, the viral particle interacts with one or several specific receptor(s), which actively promote virus entry by inducing conformational changes of the viral particle and/or by activating signalling pathways or internalization of the virion. a number of cell surface components exploited by viruses have now been identified (reviewed in grove and marsh, 2011) . although many viruses use a single specific receptor (i.e. poliovirus and human rhinovirus 2), several viruses exploit more than one molecular species as receptors, each with equivalent roles [i.e. angiotensin-converting enzyme (ace) or liver-sign (l-sign) for sars coronavirus]. however, other viruses require multiple cell surface components, each of which is essential. one of the best examples of this kind of viruses is the hepatitis c virus (hcv). hcv entry into hepatocytes is a multistep process involving viral envelope glycoproteins as well as several cellular attachment and entry factors. after attachment to the host cell, a complex process occurs in which the virion interacts with a series of cellular entry factors, including the tetraspanin cd81 (pileri et al., 1998) , the scavenger receptor type b class i (srb1) (scarselli et al., 2002) , two tight junction proteins, claudin-1 (cldn-1) (evans et al., 2007) and occludin (ploss et al., 2009) and additional host factors (lupberger et al., 2011; sainz et al., 2012) . hcv entry is regulated by receptor tyrosine kinases including egfr (lupberger et al., 2011) . among these entry factors, cd81 plays a major role in hcv entry (reviewed in dubuisson et al., 2008; farquhar et al., 2011) . indeed, through its large extracellular loop (lel), cd81 is involved in a direct interaction with the e2 envelope glycoprotein exposed at the surface of hcv virion. numerous studies have shown that cell susceptibility to hcv infection is closely related to the cd81 expression level. in addition, cd81 associates with cldn-1 to form co-receptor complexes that are critical for hcv entry 2010; krieger et al., 2010) . it is believed that the engagement of cd81 activates rho gtpase and mapk signalling cascades (brazzoli et al., 2008) . although mapk pathway is likely important for as yet unidentified post-entry events, cd81-triggered rho gtpase signalling probably induces actin remodelling, allowing lateral movement of cd81 necessary for hcv entry (brazzoli et al., 2008) . cd81 belongs to the tetraspanin family whose members have a strong propensity to multimerize and interact with numerous partner proteins forming scaffolds in membranes, recruiting or excluding specific proteins involved in particular cellular processes (reviewed in charrin et al., 2009a) . the precise mechanisms of interaction within and outside these clusters called tetraspanin-enriched microdomains (tems) or tetraspanin webs is still poorly understood. however, from highresolution fluorescence microscopy techniques, we now know that tetraspanins are distributed along the whole cell surface with a higher concentration in dot-like areas called tetraspanin-enriched areas (teas). these teas are stable platforms in position and shape but in permanent exchange with the rest of the membrane, indicating that inside the tetraspanin web, protein-protein interactions are transient and highly dynamic (barreiro et al., 2008; espenel et al., 2008) . in addition, cellular lipids such as cholesterol, glycosphingolipids and palmitic acid seem to play an important role in interactions between tetraspanins and their partners and therefore in building the tetraspanin web (charrin et al., 2009a) . the direct interaction between tetraspanins and their partner proteins can result in the modulation of their functions. for instance, cd81 functions in cellular processes and infectious diseases can be affected by the association with proteins of the ewi family whose members have a single transmembrane domain and several extracellular ig-domains with a conserved ewi motif, as well as a very short cytosolic tail (stipp et al., 2001) . indeed, ewi-2 modulates cellular migration (stipp et al., 2003; zhang et al., 2003; sala-valdés et al., 2006) and ewi-f/cd9p-1 inhibits plasmodium infection (charrin et al., 2009b) . a few years ago, we have identified ewi-2wint, a cleavage product of ewi-2 in which the first of the four extracellular ig-domains is cleaved off (rocha-perugini et al., 2008) . this shorter protein forming heterodimers with ewi-2 (montpellier et al., 2011) still interacts with cd81 and, can be found in most cell lines expressing ewi-2 but not in hepatocytes. importantly, in contrast to full-length ewi-2, ewi-2wint inhibits hcv infection by inhibiting viral entry (rocha-perugini et al., 2008) . in addition to the presence of specific entry factors in the hepatocytes, the lack of this specific inhibitor may contribute to the hepatotropism of hcv. although determinants for the interaction of ewi-2/ ewi-2wint and cd81 have been recently identified (montpellier et al., 2011) , the mechanism by which ewi-2wint blocks hcv entry has remained elusive. limited information is available on the exact role of each receptor in hcv entry. however, hcv entry is believed to be mediated through the formation of a tightly orchestrated hcv-entry factor complex at the plasma membrane, but for which interaction kinetics need to be defined. since the mobility is a critical determinant for the interaction capabilities and for the functions of membrane proteins, we sought to determine the impact of ewi-2wint on cd81 membrane dynamics. by combining biochemistry experiments and single-molecule microscopy experiments, we demonstrated that a change in membrane dynamics and partitioning of cd81 occurs in the presence of ewi-2wint, which affects cell-free transmission of hcv. together, our results indicate that hcv likely exploits the dynamic properties of cd81 to enter into its target cells. in a previous work, we described a human hepatoma huh-7 cell line (huh-7w7) which has lost cd81 expression and can be infected by hcv when mouse cd81 (mcd81) is ectopically expressed (rocha-perugini et al., 2009) . we took advantage of these permissive cells expressing mcd81 and the previously described mt81/mt81w monoclonal antibodies (mabs) (silvie et al., 2006) to analyse the role of tetraspanin web-associated cd81 in hcv infection. indeed, whereas mt81 mab recognizes all cd81 molecules, mt81w antibody only recognizes a fraction of mcd81 associated with the tetraspanin web or at least cd81 engaged in protein clusters enriched in cholesterol. although mt81 was fully neutralizing, hcv infection was poorly inhibited by mt81w, suggesting that only a fraction of cd81 is preferentially used for the virus entry (rocha-perugini et al., 2009) . based on these data, we made the hypothesis that ewi-2wint might inhibit hcv entry by modifying the organization of cd81 at the plasma membrane. in order to test this hypothesis, we generated huh-7w7 clones expressing mcd81 in combination with ewi-2 or dynamics and partitioning of cd81 1235 ewi-2wint and used the mt81w mab to probe modifications of cd81 organization. ewi-2 is endogenously expressed in hepatoma cells but its cleavage does not naturally occur preventing ewi-2wint production, as previously described (rocha-perugini et al., 2008) . in addition, direct expression of ewi-2wint sequence leads to the production of an unstable protein in these cells. to circumvent these technical difficulties, we used a modified ewi-2 protein that contains a furin cleavage site (ewi-2 fur ), directly upstream of the first ewi-2wint residue, leading to the production of ewi-2wint in virtually all cell lines ( fig. 1a ) (rocha-perugini et al., 2008) . we generated huh-7w7 cells stably expressing mcd81 in combination with either the parental ewi-2 protein (huh-7w7/mcd81/ ewi-2), or the cleavage-competent ewi-2 fur construct (huh-7w7/mcd81/ewi-2wint) or the empty vector (huh-7w7/mcd81/pcdna3.1) (fig. 1b) . protein expression in each cell line was controlled by flow cytometry (data not shown) and immunoprecipitation (ip) of cell surface biotinylated lysates (fig. 1b) . each of the ectopic ewi proteins was correctly expressed, as controlled by direct ip with a mab directed against a c-terminally expressed flag tag. as shown by co-ip with mcd81, in addition to endogenous ewi-2 protein (ewi-2 endo), ectopically expressed ewi-2 and ewi-2wint proteins interacted with mcd81, in mt81 and mt81w ips. the conservation of an interaction between human ewi-2/ewi-2wint proteins and murine cd81 was expected since the regions involved in protein-protein contact between these two proteins are well conserved (montpellier et al., 2011) . the cell lines were next used to generate cell clones for which expression of ectopic ewi proteins was controlled (data not shown). these cell clones were infected with infectious hcv particles produced in cell culture (hcvcc) (lindenbach et al., 2005; wakita et al., 2005; zhong et al., 2005) , which represent the most relevant tool to study hcv life cycle. we used jfh1-based renilla luciferase (r-luc) reporter hcvcc (jfh1/csn6a4/ 5′c19rluc2aubi), as described previously (delgrange et al., 2007; rocha-perugini et al., 2008; goueslain et al., 2010) . interestingly, hcv infection levels were significantly reduced in cell clones producing ewi-2wint (fig. 1c) , as compared with cells expressing the empty vector (pcdna3.1) or overexpressing ewi-2 (ewi-2). altogether these results strongly suggest that ewi-2wint interacts with mcd81 and inhibits hcv infection mediated by the murine receptor. it should be noted that ewi-2wint expression does not affect expression levels of mcd81 and other entry factors (data not shown). using the mt81w anti-mcd81 mab, we next characterized the influence of ewi-2wint on the cell surface organization of mcd81. as shown by flow cytometry analysis of huh-7w7/mcd81/pcdna3.1 and huh-7w7/mcd81/ewi-2 cells, the intensity of mt81w labelling was 15 times weaker than that of mt81 (fig. 1d) , confirming that only a fraction of cd81 molecules is recognized by mt81w. interestingly, as shown for one representative cell clone, cells expressing ewi-2wint exhibited a stronger staining with mt81w, indicating that ewi-2wint modifies cd81 membrane organization, probably by increasing the association of cd81 with the tetraspanin web. in order to carry out statistical analyses of ewi-2wint effect, we stained 14 clones expressing pcdna3.1, 12 clones expressing ewi-2 and 8 clones expressing ewi-2wint with mt81 and mt81w antibodies. for each clone, we calculated the ratio mt81w/mt81. as shown in fig. 1e , the ratio mt81w/ mt81 was significantly increased in cell clones expressing ewi-2wint indicating that ewi-2wint likely promotes a change of cd81 organization within the tetraspanin web. to strengthen the hypothesis that ewi-2wint induces a change in cd81 organization at the plasma membrane, we next analysed the membrane dynamics and partitioning of human cd81 (hcd81) molecules in huh-7 cells expressing or lacking ewi-2wint by single-molecule fluorescence microscopy, which is a method of choice to characterize the diffusion of proteins and lipids in different regions of the plasma membrane (owen et al., 2009 ). this emerging technique has been used previously to address the dynamics of the tetraspanin cd9 which has been proposed to diffuse in nanoclusters of tetraspanin/ partner, patrolling in the plasma membrane in permanent exchange with teas (espenel et al., 2008) . before carrying out single-molecule experiments, we first generated huh-7 cell clones expressing ewi-2wint. for this purpose, we used the modified ewi-2 protein that contains a furin cleavage site (ewi-2 fur ), directly upstream of the first amino acid of ewi-2wint, as described in the previous section. recently, we found that the mutation (lal) of a glycine-zipper motif in the transmembrane domain of ewi-2/ewi-2wint abolishes their interaction with cd81 and the inhibitory effect of ewi-2wint on hcv infection (montpellier et al., 2011) . moreover, we demonstrated that, if palmitoylation of two juxtamembranous cysteines is conserved, the cytosolic tail of ewi-2/ewi-2wint, which is involved in interactions with the actin cytoskeleton (sala-valdés et al., 2006) , can be replaced by that of mhc ii without affecting their biological function (qcc) (montpellier et al., 2011) . thus, in our study, we generated huh-7 cell clones stably expressing either the parental ewi-2 protein (huh-7/ewi-2), or the cleavage-competent ewi-2 fur construct (huh-7/ewi-2wint clones 1, 2 and 3), or the ewi-2 fur protein in which residues that are essential for the interaction with cd81 were mutated (huh-7/lal) (montpellier et al., 2011), or a. schematic representation of ewi-2 and ewi-2 fur proteins. the ewi-2 fur construct consists in the insertion of a cleavage sequence for furin (rgrr, see asterisk) between the first and the second immunoglobulin (ig) domains of ewi-2, so that cells containing this construct overexpress ewi-2 and express ewi-2wint. b. huh-7w7 cells were stably transfected with mcd81 and either the empty vector (pcdna3.1), ewi-2 or ewi-2 fur construct (ewi-2wint). cell surface biotinylation followed by immunoprecipitation assays were performed to control the expression of ectopic proteins. mt81 and mt81w mabs immunoprecipitate mcd81. a flag tag inserted at the c-terminus of the ewi-2 fur construct allows to immunoprecipitate ewi-2 and ewi-2wint. proteins were revealed by western blotting with hrp-conjugated streptavidin. c. several huh-7w7/mcd81 clones were tested in hcvcc infection. results are presented as related percentages to the first control clone (pcdna3.1) and reported as the mean ϯ sd of three independent experiments. ** means a p value below 0.01, as determined by unpaired t-test. d. huh-7w7/mcd81 clones expressing pcdna3.1, ewi-2 or ewi-2wint were stained with mt81 and mt81w mabs followed by pe-labelled secondary antibody and analysed by flow cytometry. cells stained with only the secondary antibody were used as controls (ctl). stainings of three representative clones are shown. e. mt81w/mt81 ratios for 14 clones expressing pcdna3.1, 12 clones expressing ewi-2 and 8 clones expressing ewi-2wint (one representative flow cytometry experiment). black bars correspond to mean ϯ sd for each group and *** means a p value below 0.001, as determined by the mann-whitney u-test. the ewi-2 fur protein in which the cytosolic tail was replaced but the two palmitoylatable cysteines conserved (huh-7/qcc) or cells expressing the empty vector (huh-7/ pcdna3.1) ( fig. 2a) . protein expression in each clone was controlled by flow cytometry (data not shown) and immunoprecipitation of cell surface biotinylated lysates (fig. 2b) . each of the ectopic ewi-2 proteins was correctly expressed, as controlled by direct immunoprecipitation with a mab directed against a c-terminally expressed flag tag (fig. 2b , left panel) or a mab directed against an n-terminally expressed ha tag (fig. 2b, right panel) . as shown by co-immunoprecipitation with cd81, in addition to endogenous ewi-2 protein (ewi-2 endo), ectopically expressed ewi-2 and ewi-2wint proteins interacted with cd81, whereas lal related proteins did not. it should be noted that, although qcc related proteins barely co-precipitate with cd81, the interaction occurs between these molecules, as previously demonstrated (montpellier et al., 2011) . cells were next infected with infectious hcvcc particles (jfh-1/ csn6a4) and infection levels were evaluated by flow cytometry using a mab directed against the non-structural protein 5 (ns5) (fig. 2c ). the proportion of infected cells was reduced in cells producing ewi-2wint (ewi-2wint 1, 2 and 3) whereas it remained unmodified in cells overexpressing ewi-2 (ewi-2) or ewi-2 proteins that no longer interact with cd81 (lal), as previously described (montpellier et al., 2011) . the proportion of infected cells was also reduced in cells expressing the qcc protein, which still interacts with cd81 (qcc). it should be noted that ewi-2wint expression does not affect expression levels of cd81 and other entry factors (rocha-perugini et al., 2008; montpellier et al., 2011) . in addition, levels of cd81 expression at the cell surface were equivalent for all the clones (data not shown). before carrying out single-molecule experiments on generated cell clones, the distribution of cd81 molecules at the basal surface of cells plated on collagen i was analysed by immunofluorescence on living cells using total internal reflection fluorescence (tirf) microscopy. as controls, we also analysed the distribution of the tetraspanin cd9 and of cd46, a type i membrane protein excluded from rafts and tems (espenel et al., 2008) . cd81, cd9 and cd46 molecules were labelled with atto647n-labelled fab fragments of ts81, syb-1 and 11c5 mabs, respectively, as previously described (espenel et al., 2008) . cd81 molecules, as well as their counterparts cd9, were distributed along the whole cell surface, with a higher concentration in dot-like areas ( fig. 3 and fig. s1 ). these structures have been described as teas in which tetraspanins are confined but from which they can also escape and freely diffuse in the plasma membrane (espenel et al., 2008) . as shown in fig. 3 and fig. s1 , no major change in the distribution of cd81, as well as that of cd9 and cd46, was observed in cell clones expressing or lacking ewi-2wint, indicating that this protein does not affect the global distribution of membrane proteins expressed at the plasma membrane. dynamics and partitioning of cd81 were next investigated using single-molecule tracking (smt), a technique based on the labelling of a low number of molecules allowing individual molecules to be optically isolated and their position accurately determined. with this technique, the position of proteins was determined frame by frame and their trajectories reconstructed. for this purpose, huh-7 cell clones were plated on collagen i-coated glass coverslips to allow attachment and cell spreading. single cd81 molecules were tracked using atto647n-labelled fab fragments of ts81 mab. for each cell clone, the apparent diffusion coefficient (adc) of at least 500 individual cd81 molecules (10-20 trajectories per cell) was calculated using a linear fit to the mean squared displacement (msd) versus time plots for the first three points of the curve (michalet, 2010) and their distribution displayed with a scatter plot. we first analysed the dynamics of cd81 in huh-7/pcdna3.1 and huh-7/ewi-2 cells for which we found no difference (data not shown). since expressing ewi-2wint in huh-7 cells required a mutated form of ewi-2 to be overexpressed, we considered that cells overexpressing the parental ewi-2 protein (huh-7/ ewi-2) were a better control than unmodified huh-7 cells (huh-7/pcdna3.1). as shown in the scatter plot in fig. 4a in which each trajectory is represented by a dot, cd81 molecules are distributed in three subpopulations (ewi-2): (i) a subpopulation characterized by the highest adc, (ii) a subpopulation characterized by the lowest adc and (iii) a major subpopulation with intermediate adc. strikingly, in cell clones expressing ewi-2wint (ewi-2wint 1 and ewi-2wint 2), a highly significant global shift of cd81 trajectories was observed with an increase of the subpopulation 2 with a low adc at the expense of the subpopulation 1 with a high adc. this effect was characterized by a decrease of the mean value of cd81 adc in ewi-2wint 1 (1 ¥ 10 -2 mm 2 s -1 ) and ewi-2wint 2 (1.1 ¥ 10 -2 mm 2 s -1 ) cells, as compared with ewi-2 cells (3.3 ¥ 10 -2 mm 2 s -1 ) ( table 1 ). these results indicate that ewi-2wint induces a global change of the behaviour of cd81 molecules in the plasma membrane. interestingly, cd81 trajectories in lal cells (in which ewi-2wint no longer interacts with cd81) were distributed similarly to those of ewi-2 cells demonstrating the specificity of the observed effect and that ewi-2wint needs to interact with cd81 to alter its diffusion. we next analysed the distribution of cd81 trajectories in cells expressing the mutant qcc which still interacts with cd81 but in which the cytosolic tail of ewi-2/ewi-2wint was replaced by that of mhc ii. as in ewi-2wint 1 and ewi-2wint 2 cells, however at a lesser extent, a global shift of cd81 trajectories a. schematic representation of ewi-2 and mutated ewi-2 fur proteins. ewi-2wint corresponds to ewi-2 without its first ig domain. in the lal mutant, mutation of a glycine zipper motif in the transmembrane domain of ewi-2 fur abolishes the interaction of ewi-2/ewi-2wint with cd81. in the qcc mutant, the cytosolic tail of ewi-2 fur was replaced by that of mhc ii but which still contains the two juxtamembranous palmitoylatable cysteines. b. huh-7 clones expressing the different flag and ha-tagged ewi constructs were cell surface biotinylated and lysed in pbs/brijo10/edta. lysates were used to carry out immunoprecipation assays with the ts81 anti-cd81 mab, the m2 anti-flag mab or the ha11 anti-ha mab. the control lines (ctl) correspond to lysates incubated with no mab. proteins are detected using hrp-streptavidin. c. each huh-7 clone was infected with hcvcc and stained using anti-ns5 mab (2f6/g11) and a secondary antibody conjugated with pe. non-infected cells served as negative controls. a representative histogram of every clone is shown. percentages corresponding to infected cells were calculated using weasel software. towards slower trajectories was seen in qcc cells. since the cytosolic tail of ewi proteins interacts with the actin cytoskeleton (sala-valdés et al., 2006) , our results indicate that differences in the diffusion of cd81 in cells expressing ewi-2wint cannot be attributed to its differential anchorage to the actin cytoskeleton. to determine whether ewi-2wint has a general effect on diffusion of membrane proteins, we also analysed the membrane behaviour of cd9 and cd46 molecules in cell clones expressing or lacking ewi-2wint. as shown in fig. 4b and fig. s2 , no significative difference was found in the distribution of cd9 and cd46 adc values, indicating that ewi-2wint does not affect their membrane diffusion. together these results demonstrate that ewi-2wint specifically affects the dynamics of cd81 molecules. since previous experiments were performed in huh-7 cell clones, we next investigated whether similar results could be obtained in another cell line. for this purpose, we generated cho cell lines expressing the human cd81 alone (cho/cd81/pcdna3.1), or in combination with either a non-cleavable ewi-2 construct (cho/cd81/ ewi-2) or a wild-type ewi-2 construct that leads to the production of ewi-2 and ewi-2wint proteins in cho cells, which both interact with cd81 (cho/cd81/ewi-2wint) (rocha-perugini et al., 2008; montpellier et al., 2011) , as shown in fig. s3 . although the distribution of cd81 trajectories in cho cells was rather different from that in huh-7 cells, we found again a statistically significant shift of cd81 trajectories towards slower trajectories (fig. 4c ) in presence of ewi-2wint. this effect was characterized by a decrease of the adc mean value of cd81 in ewi-2wint-expressing cells (1.9 ¥ 10 -2 mm 2 s -1 ), as compared with control cells (3.1 ¥ 10 -2 mm 2 s -1 ) (table 1) . altogether, our results indicate that ewi-2wint significantly restricts the diffusion of cd81 molecules at the plasma membrane. the different modes of diffusion of cd81 molecules were next evaluated by the analysis of msd versus time plots of the trajectories of individual molecules (for details see the data analysis section in experimental procedures) in each cell clone. as described for cd9 (espenel et al., 2008) three different diffusion modes were observed for cd81 trajectories in ewi-2-expressing cells ( fig. 5a and b and table 2 ): (i) pure brownian diffusion (40% of total trajectories) characterized by an adc mean value of 6.49 ¥ 10 -2 mm 2 s -1 , (ii) pure confined or restricted diffusion (50% of total trajectories) characterized by a lower adc mean value of 0.94 ¥ 10 -2 mm 2 s -1 , and (iii) diffusion with different combinations of brownian and confined modes referred to as 'mixed trajectories' (9% of total trajectories). importantly, the proportion of brownian trajectories was reduced from 40% to 23-26% of the total trajectories in ewi-2wint 1 and ewi-2wint 2 cells (fig. 5b ). this effect was characterized by an increase of confined and mixed trajectories. in addition to the increase in confinement, the adc mean value of brownian trajectories was 2.4-and 3.1-fold reduced in ewi-2wint 1 and ewi-2wint 2 cells as compared with ewi-2 cells respectively (table 2 and fig. 5c ). the effect of ewi-2wint on cd81 diffusion can be easily observed by the eye on the overlapping of all the tracked molecules with cd81 ensemble labelling as shown in fig. 5a . in addition, the analysis of the distribution of brownian and confined cd81 trajectories ( fig. 5c and d) both showed a highly significant shift of trajectories towards slower diffusion. indeed, a majority of brownian proteins diffused more slowly than 0.1 mm 2 s -1 at the expense of the highly mobile fraction in ewi-2wint-expressing cells. moreover, the emergence of a subpopulation with a very low adc (approximately 0.2 ¥ 10 -2 mm 2 s -1 ) was observed in confined trajectories in ewi-2wint cells (fig. 5d, arrows) . as shown in fig. 5b , cd81 molecules could also be transiently confined (mixed trajectories) and, in order to determine whether ewi-2wint affects the escape of cd81 molecules from confined areas, we calculated the dwell time of cd81 molecules in confined areas for each cell clone. interestingly, cd81 molecules were trapped significantly longer in confined areas in cells expressing ewi-2wint than in control cells with a dwell time of 4.8 s (ewi-2wint 1)/4.6 s (ewi-2wint 2) versus 3.4 s (ewi-2) ( table 2) . interestingly, the effect of ewi-2wint mainly depends on its interaction with cd81 since the dynamic behaviour in terms of adc mean values, diffusion modes immunofluorescence images showing the basal membrane of living huh-7 clones expressing pcdna3.1 control vector, ewi-2 or ewi-2wint 2. experiments were performed at 37°c using tirf microscopy. cells were stained with atto647n-labelled fab fragments of ts81, syb-1 and 11c5 mabs to observe cd81, cd9 and cd46 respectively. and dwell times of cd81 was not significantly modulated in cells expressing lal as compared with ewi-2 cells (fig. 5) . although less pronounced, the behaviour of cd81 trajectories in qcc-expressing cells was similar to that in ewi-2wint cells (fig. 5) , indicating that the observed effects on the diffusion of cd81 in cells expressing ewi-2wint cannot be attributed to a difference in its anchorage to the actin cytoskeleton by its cytosolic tail. together, our results demonstrate that ewi-2wint restricts the diffusion of cd81 (i) by decreasing the diffusion properties of the mobile brownian fraction and (ii) by increasing the subpopulation of cd81 molecules confined in plasma membrane areas. a previous study on the cd9 tetraspanin (espenel et al., 2008) has shown that a fraction of molecules can be purely confined in areas that are mainly enriched in cd9 and cd81 or more generally in tetraspanins. no exchange with the rest of the membrane was observed for these confinement areas. we therefore compared cd81 trajectories with the ensemble distribution of cd9 and cd46 ( fig. 6a and b) . to do so, overlapping of cd81 trajectories with ensemble cd9 and cd46 labelling was performed. in all cell clones, about 40% of cd81 confinement areas colocalized with cd9-enriched areas whereas less than 5% were colocalized with cd46. although somewhat higher, we did not find any significant increase (b) represent the distribution of all the apparent diffusion coefficients (adc) calculated for cd81 and cd9 respectively, in huh-7 clones expressing ewi-2, ewi-2wint (two clones), lal or qcc. to stain proteins of interest, cells were labelled with atto647n-labelled fab fragments of ts81 and syb-1 mabs respectively. each dot represents one trajectory, 500 trajectories are represented for each cell clone and *** indicates a p value below 0.0001 in comparison with the adc in the huh-7/ewi-2 clone, as determined by the mann-whitney u-test. c. cho cells were transfected with human cd81 in combination with either the empty vector (pcdna3.1), a non-cleavable ewi-2 construct (ewi-2) or ewi-2, which is in these cells cleaved to produce ewi-2wint (ewi-2wint). cells were used in single-molecule tracking experiments and labelled with atto647n-labelled fab fragments of ts81. * and *** indicate a p value below 0.01 and 0.0001, respectively, in comparison with the adc in cho/cd81/pcdna3.1 cells, as determined by the mann-whitney u-test. of colocalization of cd81 with cd9 in cells expressing ewi-2wint. in contrast, the proportion of cd81 trajectories overlapping cd81-enriched areas was significantly increased in cells expressing ewi-2wint, as compared with ewi-2 cells (fig. 6d) . no significant change was observed for cells expressing the lal mutant whereas an intermediate effect was observed for the qcc mutant. we also analysed the colocalization of cd81 with ewi-2/ ewi-2wint proteins (fig. 6c ). interestingly, we found an increase of the colocalization of cd81 with ewi proteins in cells expressing ewi-2wint, indicating that ewi-2wint or at least ewi-2/ewi-2wint heterodimers probably interact more strongly with cd81 than parental ewi-2 proteins. taken together, our results indicate that ewi-2wint restricts the global diffusion of cd81 and promotes its confinement in some areas of the plasma membrane that are enriched in cd81. it has been shown that cd81 and cldn1 colocalize and interact in areas of the plasma membrane 2010) , suggesting that cldn1 is likely a partner of cd81 in a hcv receptor complex. concordant with this hypothesis, mutations in the first extracellular loop of cldn1 or the use of antibodies directed against cldn1 or cd81 inhibit hcv entry by disrupting cldn1-cd81 interaction (evans et al., 2007; cukierman et al., 2009; harris et al., 2010; krieger et al., 2010) . since we demonstrated that ewi-2wint modifies the partitioning of cd81, we therefore analysed whether ewi-2wint has an impact on the colocalization of cd81 with cldn1. cell surface expression of cd81 and cldn1 on fixed pcdna3.1, ewi-2 and ewi-2wint cells was studied by confocal microscopy (fig. 7a) , and colocalization levels were quantified using the colocalizer pro software. interestingly, we found a significant increase of colocalization between cd81 and cldn1 in cells expressing ewi-2wint (fig. 7b) , indicating that in addition to the trapping of cd81, ewi-2wint likely constraints cldn1 in areas of the plasma membrane. in the first part of our results, we showed that ewi-2wint induces an increase of cd81 recognition by the mt81w antibody. since, as mentioned above, mt81w antibody only recognizes a fraction of cd81 associated with the tetraspanin web or at least cd81 engaged in protein clusters, we speculated that ewi-2wint increased the association of cd81 within membrane areas such as teas that can favour its clustering. therefore, we next tried to investigate the oligomerization of mcd81 and analyse whether its change has an influence on the recognition by the mt81w mab. in a previous study, we showed that cd81/cd82 chimeras in which the third (tm3) or the fourth (tm4) transmembrane domain of cd81 was replaced by the corresponding domain of cd82, have a tendency to oligomerize (montpellier et al., 2011) . based on this, we constructed mcd81/cd82 chimeras in which the tm3 ( fig. 8a; mtm3 ) or the tm4 (fig. 8a; mtm4 ) of mcd81 was replaced by the corresponding domain of human cd82. huh-7w7 cells that no longer express hcd81 were transfected with plasmids encoding mcd81, mtm3, mtm4 or the empty vector. the oligomerization status of mcd81 was next analysed by western blotting with the mt81 antibody. in addition to the ª 25 kda major band corresponding to the monomeric form of cd81, an additional band of ª 40 kda was seen in cells expressing the mtm3 chimera (fig. 8b) . in cells expressing the mtm4 chimera, several other additional bands (ª 60/ 70 kda and ª 90 kda) were detected (fig. 8b) . the presence of all these additional bands, which likely correspond to dimeric, trimeric and tetrameric forms of cd81 (yang et al., 2006) indicate that the replacement in mcd81 of tm3 or tm4 by the corresponding region of cd82 increases the homo-oligomerization of cd81. it has to be noted that similar results were obtained in cho cells (data not shown). we then took advantage of these chimeras to analyse whether the increase of cd81 oligomers modulates the recognition by the mt81w mab. huh-7w7 cells expressing mcd81, mtm3 or mtm4 were stained with mt81 and mt81w mabs and analysed by flow cytometry (fig. 8c) . it has to be noted that cells expressing mcd81, mtm3 or mtm4 were equally stained with mt81 mab, indicating transmembrane domain exchanges in chimeras did not affect their cell surface expression, as compared with unmodified mcd81. however, to avoid any bias of the cd81 expression level, the modulation of the recognition by mt81w was evaluated by the mt81w/mt81 ratio. interestingly, the recognition by mt81w was twice more efficient in cells expressing the mtm4 chimera for which cd81 homo-oligomerization is the strongest. we next analysed the oligomerization status of cd81 in huh-7 cell lines expressing ewi-2 or ewi-2wint 3 (see fig. 7 . ewi-2wint increases cd81-cldn1 colocalization. a. representative images from confocal microscopy experiments. fixed huh-7 cells expressing pcdna3.1 (left) ewi-2 (middle) or ewi-2wint 3 (right) were labelled with ts81 anti-cd81 and anti-cldn1 mabs followed by secondary antibodies conjugated with alexa488 (green staining) and alexa555 (red staining) respectively. b. colocalization was calculated as the number of yellow pixels (green and red pixels) over the number of yellow + red pixels, using colocalizer pro software. *** means a p value below 0.001 as determined by the mann-whitney u-test. fig. 2) . we treated cells with 2-bromopalmitate (2-bp) to expose membrane proximal cysteine residues and then cross-linked tetraspanin multimers using the homobifunctional cysteine-reactive reagent dtme. this method has previously been used to detect homo-oligomerization of tetraspanins (kovalenko et al., 2004) . the oligomerization status of cd81 in cells expressing ewi-2 or ewi-2wint 3 was compared with that of huh-7 cells and huh-7w7 cells, which no longer express cd81. although in our experimental conditions we did not detect any dimers of cd81, we found a specific additional band of ª 90 kda in cells expressing ewi-2wint (fig. 8d, arrow) . this band was seen in four independent experiments. interestingly, a band with a similar migration pattern was detected in huh-7w7 cells (fig. 8b , arrow) and cho cells (data not shown) expressing mtm4. taken together, our results indicate that cd81 tends to form cd81-enriched structures upon ewi-2wint expres-sion. in addition, these structures are preferentially recognized by the mt81w antibody. in addition to cell-free infection, hcv can also be transmitted via cell-to-cell contact for which the mechanism needs to be elucidated (timpe et al., 2008; witteveldt et al., 2009; brimacombe et al., 2011) . indeed, hcv is transmitted in the presence of mabs or patient-derived antibodies that are able to neutralize virus-free infectivity (timpe et al., 2008; brimacombe et al., 2011) . since cell-to-cell transmission has been suggested to be a major route of transmission for hcv (brimacombe et al., 2011) , we next analysed the effect of ewi-2wint on this process. briefly, hcv-infected huh-7 cells were labelled with 5-chloromethylfluorescein diacetate (cmfda) and co-cultured with the naïve target cells expressing or a. schematic representation of mtm3 and mtm4 chimeras. these chimeras were generated by replacing the third (mtm3) or the fourth (mtm4) transmembrane domain of mcd81 by the corresponding domain of cd82. b. huh-7w7 cells were transiently transfected with the empty vector (pcdna3.1), mcd81, mtm3 or mtm4, lysed in pbs/brijo10/edta and analysed by western blot with mt81 mab. c. huh-7w7 cells transiently expressing pcdna3.1, mcd81, mtm3 or mtm4 were labelled with mt81 and mt81w mabs followed by pe-conjugated secondary antibody and analysed by flow cytometry. mt81w/mt81 ratios are presented for each cell line and reported as the mean ϯ sd of three independent experiments. ** corresponds to a p value below 0.001, in comparison with cells expressing mcd81, as determined by the mann-whitney u-test. d. huh-7 clones expressing ewi-2 or ewi-2wint were used in cross-link experiments. huh-7 and huh-7w7 cells were used as controls. cells were treated with 2-bp to expose membrane proximal cysteines and cross-linked with the dtme reagent. after lysis in pbs/brijo10/cysteine, 5a6 anti-cd81 mab was used to immunoprecipitate cd81 complexes. proteins were revealed using biotinylated 1.3.3.22 anti-cd81 mab followed by hrp-streptavidin. the molecular weights of the prestained molecular ladder are indicated in kda. black arrows indicate a ª 90 kda band probably corresponding to cd81 tetramers. in (b) and (d) asterisk indicates a longer exposure of mtm4 and ewi-2wint 3 respectively. lacking ewi-2/ewi-2wint proteins. co-cultures were performed in the presence and in absence of neutralizing antibodies (3-11 anti-e2 mab) to analyse cell-to-cell and total transmission respectively. after 24 h of co-culture, de novo transmission events were determined by staining for hcv ns5 and they were quantified by flow cytometry. it is worth noting that no hcv transmission occurred in huh-7w7 cells that do not express cd81, in agreement with this entry factor being essential to both cell-free and cellto-cell transmission. as demonstrated by our data in fig. 9a , ewi-2wint expression has no significant effect on hcv cell-to-cell transmission. to support this claim, we performed another assay in which the cell seeding density was lowered or increased to reduce or increase cell-cell contacts, as compared with standard cell seeding density (medium). cells seeded at different densities were infected with hcvcc and infection levels were evaluated by flow cytometry at 48 h post infection (fig. 9b) . the more the cells were confluent at the time of infection, the less they were infected. in contrast, subconfluent cells were better infected. this is probably due to the differences in multiplicities of infection for each condition. interestingly, this effect was not observed in cells expressing ewi-2wint, it was even somewhat the opposite. when compared with control cell lines, the effect of ewi-2wint on hcv infection was less pronounced in cells seeded at high density, indicating that hcv infection was less inhibited by ewi-2wint in the presence of numerous cell-to-cell contacts. in contrast, ewi-2wint highly reduced hcv infection in subconfluent cells, as compared with control cells. together, these results demonstrate that ewi-2wint does not inhibit cell-to-cell transmission of hcv. in our study, we combined biochemistry experiments and single-molecule experiments to investigate the role of ewi-2wint in the clustering and membrane behaviour of cd81 in the context of hcv infection. we found that a change in membrane partitioning of cd81 occurs in the presence of ewi-2wint, which inhibits cell-free infection of hcv. the dynamics and partitioning of cd81 were probed using smt, a technique based on the labelling of a low number of molecules allowing individual molecules to be optically isolated and their position accurately determined. here, we especially focused on cd81 behaviour in cells expressing ewi-2, one of the primary partners of cd81 (stipp et al., 2001; charrin et al., 2003a) , or ewi-2wint, a truncated form of ewi-2 that inhibits hcv entry (rocha-perugini et al., 2008) . interestingly, membrane dynamics of cd81 was largely reduced upon ewi-2wint expression. we demonstrated that the lower adc mean value of cd81 is explained by two principal effects of ewi-2wint expression. first, the number of pure confinement largely increased, suggesting that cd81 molecules are trapped within membrane domains. ensemble labelling revealed that these domains are often enriched in cd81 and in ewi molecules, suggesting that these domains likely correspond to teas. second, the diffusion rate of cd81 brownian molecules significantly decreased when ewi-2wint was expressed. this result can be fig. 9 . ewi-2wint does not inhibit cell-to-cell transmission. a. huh-7 donor cells were infected with hcvcc and stained with cmfda. acceptor cells are huh-7 clones expressing ewi-2, ewi-2wint, lal or qcc. huh-7w7 cells serve as negative control. co-culture of donor and acceptor cells with or without neutralizing 3-11 anti-e2 mab allowed to monitor either cell-to-cell or total (cell-to-cell and cell-free) transmission of hcv. cells were labelled with anti-ns5 mab followed by pe-conjugated secondary antibody and analysed by flow cytometry. in these conditions, newly infected cells are negative for cmfda staining and positive for pe staining. for cell-free (light grey) and cell-to-cell transmission (black), results are presented as percentages relative to the total transmission. levels of infectivity of each clone are shown in dark grey. b. three conditions of cell density (high, medium and low) were used for hcvcc infection, for each huh-7 clone. after 48 h, cells were labelled with anti-ns5 mab followed by pe-labelled secondary antibody and analysed by flow cytometry. results are presented as related percentages to the infection of pcdna3.1 control cells in high-confluency condition. results are reported as the mean ϯ sd of three independent experiments (a and b). explained by the fact that cd81 diffuses in larger complexes that can be formed thanks to ewi-2wint interacting more strongly with cd81. indeed, although the diffusion of membrane proteins can be modulated by the lipidic composition of plasma membrane or the interaction with the membrane cytoskeleton, it is currently accepted that the diffusion coefficient of a molecule is dependent on its molecular weight (saffman and delbruck, 1975; gambin et al., 2006) . in our study, although we did not analyse the effect of ewi-2wint on the membrane lipid composition, our data suggest that cd81 diffusion is modulated by protein-protein interactions. indeed, we showed that the observed effects on the diffusion of cd81 molecules in cells expressing ewi-2wint were not related to anchorage to the actin cytoskeleton by its cytosolic tail. our colocalization studies indicated that ewi-2wint or at least ewi-2/ ewi-2wint heterodimers probably interact more strongly with cd81. in addition, high-molecular-weight complexes enriched in cd81 were detected in cells expressing ewi-2wint. these complexes likely correspond to the subpopulation of cd81 molecules with a very low diffusion coefficient, which have been identified in cells expressing ewi-2wint. we demonstrated that cd81 molecules display similar diffusion modes as compared with the other tetraspanin cd9, namely brownian, confined and mixed, a combination of the two first modes. however, as compared with cd9, the percentage of cd81 confined trajectories is more important, which is characterized by a low number of mixed trajectories (see espenel et al., 2008; krementsov et al., 2010) . such difference in cd81 and cd9 behaviour has also been observed in different cell lines such as cho, pc3 and hela cells (p. rassam et al., in preparation) . it has been largely demonstrated that cd81 associates with the tight junction protein cldn1 to form complexes that are essential to hcv entry (evans et al., 2007; cukierman et al., 2009; harris et al., 2010; krieger et al., 2010) . although we did not perform quantitative analyses of the interaction between cd81 and cldn1, we quantified the level of colocalization of these two hcv entry factors and found a significant increase in the presence of ewi-2wint. this suggests that ewi-2wint, by sequestering cd81 in membrane areas, might also sequester cldn1 and promote cd81-cldn1 interactions. this also suggests that cd81 and cldn1 likely interact transiently with each other in teas, then dissociate and escape from these areas. in cells expressing ewi-2wint, cd81-cldn1 complexes might be sequestered in teas. singlemolecule microscopy experiments will thus be interesting to investigate the dynamics and partitioning of cldn-1 molecules in cells expressing ewi-2wint. cd81 forms homodimers (kitadokoro et al., 2001; harris et al., 2008) , which may promote higher-order oligomeric structures. in 2006, in order to characterize tetraspanin microdomains, silvie and colleagues generated new anti-mouse cd81 antibodies among them the mt81w mab (silvie et al., 2006) . they demonstrated that mt81w immunoprecipitates cd81 only from lysates made under detergent conditions preserving tetraspanintetraspanin interactions. moreover, cd9 depletion completely removed the pool of cd81 molecules recognized by mt81w. based on this, it has been suggested that mt81w specifically recognizes cd81 associated with other tetraspanins (including other cd81 molecules) or at least cd81 proteins interacting with cholesterol that directly interact with tetraspanins (charrin et al., 2003b) . we next took advantage of this tool to analyse the role in hcv infection of cd81 molecules recognized by the mt81w antibody (rocha-perugini et al., 2009) . we showed that mt81w did not strongly affect hcv infection. furthermore, cholesterol depletion, which inhibits hcv infection and reduces total cell surface expression of cd81, did not affect recognition by mt81w. in addition, sphingomyelinase treatment, which also reduces hcv infection and cell surface expression of total cd81, raised the number of cd81 molecules recognized by mt81w. we therefore concluded that hcv does not use preferentially cd81 associated with other tetraspanins. in the present study, we used again this antibody in order to define whether ewi-2wint promotes a change in cd81 partitioning and dynamics. we found that the proportion of cd81 molecules recognized by mt81w increased upon ewi-2wint expression, suggesting that ewi-2wint promotes the association of cd81 with other tetraspanins. however, our colocalization studies showed that cd81 was trapped in areas particularly enriched in cd81 and not in cd9, in cells expressing ewi-2wint. we also demonstrated that mt81w preferentially stains cells expressing oligomeric forms of cd81 indicating that mt81w likely does not recognize cd81 associated with other tetraspanins but it is rather a low-affinity antibody that better recognizes clusters of cd81. antibodies with low monovalent affinity are indeed particularly dependent on bivalent binding, and consequently are particularly sensitive to antigen clustering. we can speculate that in depletion experiments made by silvie and co-workers, depletion of cd9, which shares the same partners as cd81, liberates partner proteins that can then interact with cd81 and thereby impede cd81 homo-oligomerization. under these conditions, the mt81w will no longer recognize cd81. our results therefore suggest that mt81w is somehow similar to c9bb, a low-affinity antibody that preferentially recognizes multimerized cd9 (yang et al., 2006) . in accordance with the claim that mt81w is a low-affinity antibody that better recognizes clusters of cd81, we showed that recognition by mt81w was increased following ceramide enrichment (rocha-perugini et al., 2009) , which is known to induce receptor clustering for many receptor molecules (reviewed in zhang et al., 2009) essential to initiation and amplification of receptor and stress-mediated signalling in almost all cell types. since receptor aggregation and trapping in ceramide-enriched microdomains may limit lateral diffusion of membrane proteins, it will be interesting to study in singlemolecule microscopy whether cd81 molecules in sphingomyelinase-treated cells behave similarly to those in cells expressing ewi-2wint. thus, our data suggest that mt81w antibody is likely a low-affinity antibody that specifically recognizes cd81-enriched structures, which might be identified as teas. taken together, our results indicate that ewi-2wint increased association of cd81 within membrane areas such as teas that favours cd81 clustering and which are specifically recognized by the mt81w antibody. ewi family members have a propensity for oligomerization. for example, it has been demonstrated that ewi-f/cd9p-1 forms oligomers at the cell surface that are directly associated with the tetraspanins cd9 and cd81 (andré et al., 2009) . ewi-2 has also been observed to form protein complexes containing at least two ewi-2 molecules (andré et al., 2009 ). in addition, ewi-2wint is associated with ewi-2 in heterodimers that are associated with cd81 and cd9 (montpellier et al., 2011) . we therefore cannot exclude the possibility that ewi-2wint needs ewi-2 to interact with cd81 and consequently to modulate its partitioning and membrane diffusion. we found that the replacement of the tm3 and tm4 of cd81 by the corresponding domains of hcd82 increases the capacity of human and murine cd81 to homooligomerize (the present work and montpellier et al., 2011) . particularly, in the context of the mtm4 chimera, cd81 formed high-molecular-weight complexes that may correspond to dimers, trimers and tetramers. interestingly, a high-molecular-weight band was seen in cells expressing ewi-2wint, suggesting that cd81 forms high-order complexes upon ewi-2wint expression. this result was in accordance with our microscopy experiments demonstrating that cd81 molecules were confined in cd81enriched areas of the plasma membrane. our previous (rocha-perugini et al., 2008; montpellier et al., 2011) and present results support the model in which cd81 clusters are deleterious for hcv entry. first, tm3 and tm4 chimeras, which form cd81 oligomers, are not functional in hcv entry. second, ewi-2wint, which promotes cd81 clustering, inhibits hcv infection. third, the mt81w mab, for which we showed a stronger affinity for cells expressing ewi-2wint or mtm4 chimera, poorly neutralizes hcv infection. in a previous work, we showed that ewi-2wint restricts hcv entry by impeding e2-cd81 binding (rocha-perugini et al., 2008) . in the present study, we demonstrated that the expression of ewi-2wint promotes the formation of cd81 oligomers that diffuse more slowly in the plasma membrane and, which are recognized by the mt81w mab. since this antibody poorly neutralizes hcv infection, we assume that the cd81 oligomers are not used during hcv entry. from all these data, we can therefore conclude that, by reorganizing cd81 molecules on the cell surface, ewi-2wint impedes the interaction between cd81 and e2 envelope glycoproteins. although additional experiments will be necessary to strengthen this hypothesis, we can assume that the effects of ewi-2wint on cd81 membrane diffusion and e2-cd81 binding are probably two inseparable functions. enveloped viruses can spread via two distinct routes, either through the cell-free aqueous environment or by remaining cell associated and being passed on by direct cell-cell contact. this latter mode of spread designated as cell-to-cell transmission not only facilitates rapid viral dissemination, but may also promote immune evasion and influence disease (reviewed in sattentau, 2008) . increasing evidences suggest that cell-to-cell transmission is a major route of transmission for hcv. indeed, hcv is transmitted in the presence of mabs and patient-derived antibodies that are able to neutralize virus-free infectivity (timpe et al., 2008; brimacombe et al., 2011) . in addition, cell-to-cell transfer of virions has been shown to be a feature common to chimeric viruses expressing the structural proteins representing the seven major hcv genotypes (brimacombe et al., 2011) . although hcv may exploit a contact structure similar to the virological synapse of hiv (brimacombe et al., 2011) or may use membrane nanotubes, the precise mechanism by which hcv is transmitted from a cell to another cell needs to be elucidated. it has been established that srb1 and the tight-junction components cldn1 and occludin are involved in hcv cell-to-cell transmission. so far, the role played by cd81 remains controversial. indeed, several studies reported hcv cell-to-cell transmission as a cd81dependent pathway (russell et al., 2008; brimacombe et al., 2011) , whereas others demonstrated a cd81independent transmission (timpe et al., 2008; witteveldt et al., 2009; jones et al., 2010) . in our study, we found that huh-7w7 cells, which do not express cd81, were resistant to both cell-free and cell-to-cell transmission indicating that cd81 is necessary for both cell-free and cellto-cell spread of hcv. we showed that ewi-2wint inhibits cell-free transmission but not cell-to-cell transmission of hcv. this finding demonstrates that cell-free and cell-tocell transmissions are driven by different mechanisms. according to the smt experiments, we could speculate that ewi-2wint inhibits cell-free transmission by modulating cd81 membrane diffusion and its clustering and that these processes are not critically involved in cell-to-cell transmission. in conclusion, by using ewi-2wint, a natural inhibitor of hcv, we defined the dynamic properties and partitioning of cd81 molecules that are necessary for productive infection. our results open the way to the understanding of the dynamics by which hcv enters into its target cells. anti-human cd81 (5a6) was kindly provided by s. levy. antihuman cd81 (ts81) and anti-mouse cd81 (mt81 and mt81w) mabs have been described previously (charrin et al., 2001; silvie et al., 2006) . 3-11 (anti-hcv e2) hybridoma was kindly provided by j. mckeating. m2 anti-flag mab was from sigma. anti-ns5 (2f6/g11) was from austral biologicals. ha11 anti-ha mab was from covance. pe-labelled goat anti-mouse and anti-rat were from bd pharmingen. peroxidase-conjugated goat antimouse and anti-rat were from sigma and jackson immunoresearch respectively. fab fragments of mabs (anti-cd81 ts81, anti-cd9 syb-1, anti-cd46 11c5) labelled with atto647n or cy3b have been described previously (espenel et al., 2008) . fab fragments of 8a12 mab [anti-ewi-2 (charrin et al., 2003a) ] were prepared and labelled with cy3b as previously described (espenel et al., 2008) . anti-cldn1 mab has been previously described (fofana et al., 2010) . plasmids pcdna3.1 plasmids expressing ewi-2, ewi-2 fur , lal, qcc, ewi-2 aga , human cd81, murine cd81 have been described previously (charrin et al., 2003a; rocha-perugini et al., 2008; montpellier et al., 2011) . the mtm3 and mtm4 chimeras were constructed by a pcr-based method. in mtm3 chimera, the amino acid (aa) sequence corresponding to the third transmembrane domain of mcd81 (aa 87-113) was replaced by the aa sequence corresponding to the third transmembrane domain of hcd82 (aa 81-108). in mtm4 chimera, the aa sequence corresponding to the fourth transmembrane domain and cytosolic tail of mcd81 (aa 204-236) was replaced by the aa sequence corresponding to the fourth transmembrane domain and cytosolic tail of hcd82 (aa 228-267). cloning details and oligonucleotide sequences are available upon request. cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 100 nm non-essential amino acids. huh-7 and cho cells were from atcc. huh-7w7 (cd81 -) cells were described previously (rocha-perugini et al., 2009) . cell transfections and generation of cell lines/clones were performed as described previously (rocha-perugini et al., 2008) . since the cleavage of ewi-2 generating ewi-2wint naturally occurs in cho cells, the mutation of two arginine residues abolishing the cleavage of ewi-2 (montpellier et al., 2011) was introduced in the coding sequence of the plasmid (pcdna3.1/ ewi-2 aga ; montpellier et al., 2011) used to generate cho/ewi-2 cells. twenty-four hours post transfection, cells were surface biotinylated as described in ref (rocha-perugini et al., 2008) and lysed in pbs 1% brij-o10 2 mm edta (pbs/brij/edta) and protease inhibitors (complete, roche applied science). lysates were pre-cleared for 2 h at 4°c with protein a-sepharose (ge healthcare), then incubated for 2 h at 4°c with specific mabs immobilized onto protein a-sepharose beads. complexes were eluted with non-reducing laemmli buffer, resolved by sds-page, transferred to a nitrocellulose membrane (ge healthcare), and immunoblotted with peroxidase-conjugated streptavidin (vector). hcvcc used in this study were based on the jfh1 strain (wakita et al., 2005) and contained cell culture-adaptive cs, n6 and a4 mutations (delgrange et al., 2007; goueslain et al., 2010) . hcvcc (jfh1/csn6a4/5′c19rluc2aubi) expressing renilla luciferase were produced as described (delgrange et al., 2007; rocha-perugini et al., 2008) . hcvcc were added to huh-7 cells [multiplicity of infection (moi) = 1] seeded the day before in 24-well plates and incubated for 2 h at 37°c. the supernatants were then removed and the cells were incubated in dmem 10% fbs at 37°c. at 40-48 h post infection, renilla luciferase assays were performed as indicated by the manufacturer (promega). hcvcc without reporter gene (jfh-1/csn6a4) stocks were produced as previously reported (delgrange et al., 2007; goueslain et al., 2010) . for the hcvcc infection assays, cells grown in 24-well plates were incubated with hcvcc (moi = 1) for 2 h at 37°c, washed, and incubated for an additional 48 h at 37°c. hcvcc infections were scored by staining for hcv non-structural protein ns5 followed by flow cytometry analyses. for ns5 staining, infected cells were permeabilized with pbs 2% bsa 0.05% saponin and washed with pbs 2% bsa. for mt81/ mt81w stainings, cells grown in 24-well plates were directly washed with pbs 2% bsa. cells were then incubated 1 h at 4°c with anti-ns5 or anti-mcd81 (mt81 and mt81w) mab. after rinsing, cells were incubated with pe-labelled goat anti-mouse or anti-rat, respectively, for 45 min at 4°c, washed, detached with pbs 2 mm edta and fixed with formalin solution (formaldehyde 4%, sigma). labelled cells were analysed using a facscalibur (becton dickinson). smt experiments were carried out as previously described (espenel et al., 2008) . briefly, cells plated on collagen-1 were incubated in dmem at 37°c for 10 min with atto647n-labelled fab fragments of mabs raised against cd81 (ts81), cd9 (syb-1) and cd46 (11c5). a homemade objective-type tirf set-up allowing multicolour single-molecule imaging and equipped with a plan fluor 100¥/1.45 na objective (zeiss, le peck, france brattleboro, vt) was used. all the experiments were performed with a 100 ms integration time. for some experiments, to achieve a better specificity in the detection of the two fluorescent signals, alternating-laser excitation (alex) was performed using an acousto-optical tunable filter and controller (aotf; aa optoelectronics) (margeat et al., 2006) . all the movies were analysed using a homemade software (named 'patrack') implemented in visual c++. trajectories were constructed using the individual diffraction limited signal of each molecule. the centre of each fluorescence peak was determined with subpixel resolution by fitting a two-dimensional elliptical gaussian function. the two-dimensional trajectories of single molecules were constructed frame per frame. only trajectories containing at least 40 points were retained. diffusion coefficient values were determined from a linear fit to the msd (mean square displacement)-t plots between the first and the fourth points (d1-4) according to the equation msd(t) = 4dt. the determination of the motional modes was performed using a homemade algorithm based on a neural network that has been trained using synthetic trajectories to detect pure brownian, confined and directed motion modes (p. dosset et al., in preparation) . due to a sliding window, the trajectory is analysed and the different modes detected within a trajectory for segments larger than 10 frames. once the motion mode is identified, the different segments are analysed by plotting the msd versus time lag. the msd curve is linearly fitted (brownian) or adjusted with a quadratic curve (4dt + v2t2) (directed diffusion) or exponential curve l2/3[1exp(-12dt/l2)] (confined diffusion), where l is the side of a square domain, the confinement diameter being related to l by dconf = (2/√l). the algorithm has been tested with simulated trajectories displaying pure brownian, confined or directed behaviour or a combination of this three modes and successfully applied to a set of single-molecule experiments previously recorded for tetraspanins diffusing into plasma membrane (espenel et al., 2008; krementsov et al., 2010) . colocalization analyses were carried out using the colocalizer pro software. briefly, images representing a single optical z-section of cells were converted to tiffs and imported into colocalizer pro software. background corrections were applied and the total number of pixels overlapping between the two different channels was calculated. colocalized pixels were divided by the total number of pixels for a chosen channel (as indicated in figure legends), yielding per cent colocalization. colocalization values from at least 10 cells were averaged to give mean per cent colocalization. cells were incubated with pbs containing 2% fatty acid-free bovine serum albumin (sigma) and 50 mm 2-bromopalmitate (2-bp, sigma) for 18 h at 37°c. then, cells were washed with pbs containing 2 mm mgcl2 (pbs/mgcl2) and treated with cysteine cross-linker dtme (0.2 mg ml -1 dithiobismaleimidoethane, pierce) in pbs/mgcl2 for 2 h at 4°c. cells were washed again with pbs/mgcl2 containing 5 mm cysteine and lysed with pbs containing 1% brijo10 (sigma), 2 mm cysteine and protease inhibitors. lysates were used for immunoprecipitation assays. huh-7 cells were infected with hcvcc (moi = 2) 24 h prior to their use in the assay. infected cells were then labelled with 5-chloromethylfluorescein diacetate (cmfda) (molecular probes, invitrogen) by incubation at 37°c during 30 min with 10 mm cmfda in medium without fbs. cells were trypsinized, washed and incubated 15 min with 3-11 mab (50 mg ml -1 ) to neutralize any remaining viral particle at the cell surface. after washing, cmfda-labelled donor cells were mixed with naïve target cell lines (ratio 1:4), seeded in 24-well plates in presence (cell-to-cell transmission) or in absence (cell-free and cell-to-cell transmission) of neutralizing mab (3-11, 50 mg ml -1 ). after 24 h, de novo transmission events were determined by staining for hcv ns5 and were quantified by flow cytometry, as described above. cell-to-cell transmission levels were defined as newly infected cells in presence of 3-11 mab. cell-free transmission levels were evaluated using the following calculation: (de novo infected cells in absence of 3-11) -(de novo infected cells in presence of 3-11). for infection with different seeding densities, 2 ¥ 10 5 , 1 ¥ 10 5 and 5 ¥ 10 4 cells per well of a 24-well plate were infected with hcvcc (moi = 1). after 48 h, hcv infection levels were determined by staining for hcv ns5 followed by flow cytometry analyses. fig. s1 . cell surface distribution of cd81 and control proteins. immunofluorescence images showing the basal membrane of living huh-7 clones expressing ewi-2wint 1, lal or qcc. experiments were performed at 37°c using tirf microscopy. cells were stained with atto647n-labelled fab fragments of ts81, syb-1 and 11c5 mabs to observe cd81, cd9 and cd46 respectively. fig. s2 . ewi-2wint has no effect on cd46 dynamics. the distribution of all the apparent diffusion coefficients (adc) was calculated for cd46 in huh-7 clones expressing ewi-2, ewi-2wint (two clones), lal or qcc. each dot represents one trajectory and 500 trajectories are represented for each cell clone. cd46 molecules were stained usingatto647n-labelled fab fragments of 11c5 mab. fig. s3 . ewi-2/ewi-2wint expression in cho cells. cho cells were stably transfected with hcd81 and either the empty vector (pcdna3.1), the uncleavable ewi-2 (ewi-2, see experimental procedures) or wild-type ewi-2 construct (ewi-2wint). it has to be noted that the cleavage of ewi-2 generating ewi-2wint naturally occurs in cho cells. cell surface biotinylation followed by immunoprecipitation assays were performed to control the expression of proteins. flag-tagged ewi-2 constructs and cd81 were immunoprecipitated with m2 and 5a6 mabs respectively. proteins were revealed by western blotting with hrpconjugated streptavidin. asterisks indicate additional cleavage products of ewi-2wint, as previously described (rocha-perugini et al., 2008) . triangles indicate an unidentified partner of cd81, which is not related to ewi-2/ewi-2wint proteins. in situ chemical cross-linking on living cells reveals cd9p-1 cis-oligomer at cell surface endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms cd81 is a central regulator of cellular events required for hepatitis c virus infection of human hepatocytes neutralizing antibodyresistant hepatitis c virus cell-to-cell transmission the major cd9 and cd81 molecular partner. identification and characterization of the complexes ewi-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells a physical and functional link between cholesterol and tetraspanins lateral organization of membrane proteins: tetraspanins spin their web the ig domain protein cd9p-1 down-regulates cd81 ability to support plasmodium yoelii infection residues in a highly conserved claudin-1 motif are required for hepatitis c virus entry and mediate the formation of cell-cell contacts robust production of infectious viral particles in huh-7 cells by introducing mutations in hepatitis c virus structural proteins early steps of the hepatitis c virus life cycle single-molecule analysis of cd9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web claudin-1 is a hepatitis c virus co-receptor required for a late step in entry hepatitis c virus entry and the tetraspanin cd81 monoclonal anti-claudin 1 antibodies prevent hepatitis c virus infection of primary human hepatocytes lateral mobility of proteins in liquid membranes revisited identification of gbf1 as a cellular factor required for hepatitis c virus rna replication the cell biology of receptormediated virus entry cd81 and claudin 1 coreceptor association: role in hepatitis c virus entry claudin association with cd81 defines hepatitis c virus entry hepatoma polarization limits cd81 and hepatitis c virus dynamics real-time imaging of hepatitis c virus infection using a fluorescent cell-based reporter system cd81 extracellular domain 3d structure: insight into the tetraspanin superfamily structural motifs evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking hiv-1 assembly differentially alters dynamics and partitioning of tetraspanins and raft components inhibition of hepatitis c virus infection by anti-claudin-1 antibodies is mediated by neutralization of e2-cd81-claudin-1 associations complete replication of hepatitis c virus in cell culture egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy direct observation of abortive initiation and promoter escape within single immobilized transcription complexes mean square displacement analysis of single-particle trajectories with localization error: brownian motion in an isotropic medium interacting regions of cd81 and two of its partners, ewi-2 and ewi-2wint, and their effect on hepatitis c virus infection quantitative microscopy: protein dynamics and membrane organisation binding of hepatitis c virus to cd81 human occludin is a hepatitis c virus entry factor required for infection of mouse cells the cd81 partner ewi-2wint inhibits hepatitis c virus entry the association of cd81 with tetraspanin-enriched microdomains is not essential for hepatitis c virus entry advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis c virus brownian motion in biological membranes identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor ewi-2 and ewi-f link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins avoiding the void: cell-to-cell spread of human viruses the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus cholesterol contributes to the organization of tetraspanin-enriched microdomains and to cd81-dependent infection by malaria sporozoites ewi-2 is a major cd9 and cd81 partner and member of a novel ig protein subfamily ewi-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5 hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies production of infectious hepatitis c virus in tissue culture from a cloned viral genome cd81 is dispensable for hepatitis c virus cell-to-cell transmission in hepatoma cells contrasting effects of ewi proteins, integrins, and protein palmitoylation on cell surface cd9 organization ewi2/pgrl associates with the metastasis suppressor kai1/cd82 and inhibits the migration of prostate cancer cells ceramide-enriched membrane domains -structure and function robust hepatitis c virus infection in vitro we thank sophana ung and andré pillez for their technical assistance. we thank yves rouillé for critical reading of the manuscript. we are grateful to s. levy, j. mckeating, t. wakita, for providing us with reagents. we thank antonino bongiovanni and frank lafont from the bioimaging center lille-nord de france for access to the instruments and technical advice. we thank nicole lautrédou for her technical advice. this work was supported by the 'institut fédératif de recherche-142' (ifr142) and by a grant from the 'agence nationale de recherches sur le sida et les hépatites virales' anrs. p.r. was supported by a fellowship from the anrs. b.a.t. was supported by a marie curie intra european fellowship within the 7th european community framework programme. c.-i.p. was supported by the postdoctoral programme posdru/89/1.5/s/60746 from european social fund. key: cord-010088-s9tfvtao authors: nan title: oral abstracts date: 2013-11-01 journal: vox sang doi: 10.1111/vox.12100_1 sha: doc_id: 10088 cord_uid: s9tfvtao nan tadokoro k and satake m japanese red cross society, tokyo, japan it is estimated that there are 2 billion hbv-infected people including 350 million hbv-carriers in the world. it is highly endemic in south africa, amazon, and southeast-central asia. the genotype of hbv is geographically characteristic, e.g. genotype b and c are the major in east asia. hbv transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. the residual risk is mainly related to donations either in the pre-sero (or pre-dna)-conversion window period or occult hbv infection (obi) where blood test is hbv-dna-positive and hbs-ag-negative. infectivity of hbv depends on the transfused blood (viral load, phase of infection, genotype, and anti-hbs in the concurrent blood) and immune status of the recipients (anti-hbs, immunocompetence). it was shown that infectivity is dependent on viral load. allain et al reported that ffps is more infectious than pcs or rbcs. the minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately 10 times higher than that of pre-acute phase. satake et al reported that transmission rate of obi-derived components with low titer anti-hbc was 1/33(3%), whereas that of anti-hbc-negative components was 11/22(50%), which was verified in the lookback programme conducted in japan. allain et al showed in the study conducted in europe that adjusted transmission rate of obi blood was 28%, and the rate was higher without anti-hbs(63.8%) and lower with anti-hbs(15.4%). discrepancy of transmission rate of obi-derived blood between above two reports might be related to the different cutoff levels of anti-hbc and presence or absence of anti-hbs. dna-positivity rate among obi-derived components is higher in those with the higher levels of anti-hbc and lower in those with the presence of anti-hbs. there has been no report of transmission by obi-derived blood with anti-hbs of 200miu/ml or more. screening test for hbv is different between countries. low endemic countries screen blood for hbs-ag, anti-hbc and mini-pooled nat, while highly endemic countries test for hbs-ag without anti-hbc, because high prevalence of anti-hbc-positive donation hamper securing necessary blood. japan as a moderately endemic country had tested for hbs-ag, mini-pool nat and anti-hbc/anti-hbs where anti-hbs of more than 200 miu/ml irrespective of anti-hbc and low anti-hbc with agglutination-inhibition titer of no more than 2 5 is qualified. transfusion-transmitted hbv cases related to window period donations declined by increasing the sensitivity of mini pool nat, whereas those related to blood with low titer anti-hbc remained stable with around 10 cases annually. in order to decrease such transmission japanese red cross implemented a novel strategy to eliminate all anti-hbc-positive donations with anti-hbs <200 miu/ml. considering the frequency of donations with low titer anti-hbc has decreased to 1.3%, loss of those donations was estimated to be covered by promoting donations, each country should establish its own hbv screening strategy considering the prevalence of hbv, residual risk of transmission, balance between safety and securing blood, and cost-effectiveness. implementation of individual donation nat and universal vaccination could further reduce further the risk of hbv transmission. our blood service is to motivate other population groups to diversify the age and gender composition of our donors. forty-two percent of the whole blood donors participate in blood donation only once a year, so we need develop programs to motivate them to re-join blood donation. also, we have to solve the annually recurring problem of blood shortages for transfusion during the winter and summer season. in the long term, our donor base is going to decrease gradually because of the low birth rate in korea and our rapidly aging population. therefore we should prepare a sustainable solution for a stable blood supply. aims: present methods to recruit blood donors based on age, gender and occupation groups for a stable blood supply. methods: to make blood donation more accessible to individual donors, fixed donation sites are continuously developed. facilities of our fixed and mobile sites are improved to provide safe and comfortable environment to donors. since 1999, we have been operating the 'registered donor system' for registered donors who agreed to donate their blood on a regular basis. to raise awareness of the importance of blood donation among youth, high-school students blood donor groups called 'red campaigners' and groups of university students called 'blood donation supporters' are actively participating in blood donation campaigns. to increase participation of the middle-aged group, agreements were made not only with enterprises and organizations but also with the government and public institutions. we have developed a computerized system for scheduling group donations according to demand and supply and for performance management. to recognize the necessity of blood donation, every 13th was designated as 'blood donation day' in 2012. to deal with donor complaints and requests, a customer relationship management center has been established. results: by increasing the number of fixed donation sites and making donation more accessible, rate of individual donations is getting increased. as of the end of june 2013, 250 agreements for blood donation had been signed with enterprises and organizations. every year more than 100 thousand donors agree to donate on a regular basis, and so far about 600 thousand donors have been registered in the 'registered donor system'. the campaign 'every 13th is blood donation day' has contributed to spread a positive perception about blood donation. conclusions: we have been very successful in engaging youth in blood donation. diversification of the donor group will take time and constant effort. however, with more participation of female donors and retention of our first-time donors, it will be possible to be self-sufficient in blood supply for transfusion and plasma fractionation. the provision of sufficient safe blood products to patients requiring transfusion is the common goal of blood transfusion services and the public expectation of absolute safety continues to be a challenge. although the focus of blood safety falls on laboratory testing, the role of pre-donation donor selection cannot be underestimated. transfusion-transmitted infections (ttis) are blood-borne microbes that can be spread from blood donor to recipient via transfusion. to prevent tti, donated blood must go through validated laboratory testing. in most countries, blood is tested for hiv, hbv, hcv, and syphilis. screening for other ttis may also be implemented in some countries after individual assessment of the prevalence of the infection in their general populations, for example, west nile virus in the united states. with the advance in testing technology, in particular the nucleic acid test, the window periods for the detection of various ttis have been significantly shortened. however, the risk of window donation still exists. furthermore, there are known or emerging ttis such as the variant creutzfeldtjakcob disease (vcjd) where there is still no suitable testing system for routine screening of donated blood. therefore, blood transfusion services have to continue practicing effective pre-donation donor selection to mitigate the tti risks. who recommends selecting voluntary, non-remunerated donors from low-risk populations for blood collection as the first step in reducing the risk of ttis. donor selection is usually conducted by a health history questionnaire to be completed by potential donors and a confidential interview. the questions asked should be effective in assessing whether the respondent's health status is suitable to donate and there is no tti risk factor. people who are deferred should be counseled and given the reason for and duration of the deferral. who has published a document titled 'blood donor selection -guidelines on assessing donor suitability for blood donation'. it recommends the following: national donor selection guidelines and criteria should be based on epidemiological and/or scientific evidence or, where evidence is limited or lacking, on best practice. donor acceptance and deferral policies for the prevention of tti should be based on up-to-date information on the local epidemiology of infections, the markers screened for, the availability of suitable blood screening and confirmatory assays, and the technologies in use. national donor selection criteria should define conditions of acceptance and deferral for each criterion. adequate resources, including a sufficient number of qualified and trained staff, should be made available for the consistent and reliable assessment of donor suitability for blood donation. quality systems should be in place for donor selection, including selection criteria, staff training and documentation. blood transfusion services should establish mechanisms for monitoring and evaluation to assess the implementation and effectiveness of donor selection criteria. in conclusion, pre-donation selection is essential to protect the safety and sufficiency of blood supply, and safeguard the health of recipients and donors. background: blood components may be contaminated by a variety of commensal, pathogenic or environmental bacteria during collection, manufacture or storage. the outcome of transfusion is dependent on the ability of the specific strain to multiply to clinically-relevant titers during storage, the pathogenicity of the strain and the patients' situation. platelets in particular are stored in conditions conducive to bacterial growth and septic reactions to these products are the most frequently documented infectious risk of transfusion. aim: the objective of this update is to review estimates of risk of bacterial sepsis and contamination of platelets, and recent findings describing interventions designed to safeguard patients. methods: this update will use recent reports and unpublished data to describe our current understanding of the role of bacteria in transfusion safety. results: historical data suggests that~1:1-3000 platelet products are contaminated with bacteria, and septic transfusion reactions occurred in 1:15-100,000 transfusions. clinical awareness of the danger and a 2004 aabb standard to 'limit and detect bacteria' in platelet products have driven the development of safety systems. the last decade has seen substantial progress in the implementation of optimal skin disinfection techniques and sample diversion strategies to reduce contamination, and many centers implemented either bacterial culture testing or pathogen inactivation processes to reduce the risk. culture testing can reduce but cannot eliminate the risk of exposure to contaminated components and sepsis. the majority of contaminated products do not cause adverse reactions, however, at the time of collection and manufacture the only means to prevent serious and fatal reactions is to ensure that that the component is functionally sterile. pathogen inactivation technologies show variable efficacy at killing bacteria, suggesting a need for strict adherence to the manufacturers suggested protocols to ensure optimum performance. alternatively, assays performed on the day of transfusion prevent the transfusion of high concentrations of bacteria that are associated with the most severe adverse reactions. conclusions: bacterial contamination and sepsis remain the greatest infectious risks of transfusion. enhanced testing or pathogen inactivation should be implemented to ensure patient safety. the australian red cross blood service, sydney, australia background: pathogen reduction technologies have been developed as a means of reducing the risk of transfusion transmission of blood-borne pathogens. published literature indicates that systems currently in use or under development effectively inactivate a range of pathogens in platelets and plasma, with the exception of some non-lipid enveloped viruses, bacterial spores and prions. development of pathogen reduction technology (prt) is ongoing, and many countries have adopted prt as part of their routine blood component processing. the aim of this update will be to review technologies currently in use or under development for treatment of labile blood components during processing. the impact of prt on blood component quality as well as ongoing challenges will be reviewed. platelets: there are currently three systems for prt treatment of platelets. these are the mirasol tm system (terumobct), the intercept blood system tm (cerus corporation) and the theraflex uvc tm system (macopharma). the intercept and mirasol systems are used widely in blood centres in europe, asia and the middle east for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the theraflex uvc system are ongoing. in vitro data suggests prt treatment leads to some loss of platelet function. however, haemovigilance reports from sev-eral countries where intercept treated platelets are transfused indicate no change in component usage, and indeed a reduction in transfusion related adverse events. similarly, there have been no reports to date of serious adverse events relating to clinical use of mirasol-treated platelets. plasma: the intercept and mirasol systems can be used to treat both plasma and platelets, and the theraflex system utilises methylene blue (mb) with visible light for prt treatment of plasma. there are some losses of coagulation factors following treatment with each of these systems. theraflex mb plasma has been transfused world-wide since 1999, and haemovigilance data indicates there is not a higher incidence of allergic reactions or other adverse events with mb-treated plasma. red cells/whole blood: commercial prt systems for red cell or whole blood components are under development. cerus corporation has continued development of a second-generation s-303 system for red cells, demonstrating in vivo recovery after 35 days storage. a mirasol prt system for treatment of whole blood is also being developed by terumobct. preliminary in vitro quality and in vivo recovery and survival data indicate that this technology may eventually become available, but further development and clinical trials are still required. challenges: concerns still exist regarding long term effects on patients receiving prt treated blood components, particularly the potential toxic effects of residual products following photochemical treatment. ongoing post-marketing surveillance and clinical trials are required to address these concerns. ideally, prt should provide proactive protection against emerging pathogens and reduce the need to introduce additional pathogen testing, minimise bacterial contamination and potentially replace processing steps such as gamma irradiation. the challenge for blood services is to understand and rationalise the costs, risks and benefits of prt compared to removing any of these tests or processes, whilst aiming to provide the highest level of blood safety. steps in getting a paper published/abstract accepted 1b-h4-01 no abstract available. 1b-h4-02 how to get your abstract accepted and how to present it daniels g many abstracts are submitted to the isbt for presentation at international and regional congresses. all abstracts are refereed by a large panel of international experts, who decide which abstracts should be accepted and which ones should be rejected. they also decide which of the accepted should be presented orally or as a poster. most of the submitted abstracts are accepted, though there is plenty of room for improvement in the quality of many of those abstracts. in this session i will describe why some abstracts are rejected and discuss how you might avoid this pitfall. i will also discuss some methods for improving your abstracts so that they represent the quality of the work you are describing and enhance the reputation of yourself and your institution. once your abstract has been accepted you will have to present it at the congress. if it is accepted for poster presentation, you will have to make a poster. the easiest way to produce a poster is to design it on a single powerpoint slide and then send the file to a company that will turn it into a poster on paper, or even on cloth for easier transportation. if your budget does not run to that, there are cheaper ways, such as printing it on single pages of a4 with the text printed in a very large font. the best posters do not have too much written information (bullet points are often best), contain diagrams and/or pictures, and are colourful and visually attractive. if, however, your abstract is accepted for oral presentation, then you will have to give a talk, usually of 10 min, and then be prepared to answer questions. this may be a daunting prospect, especially if you are not experienced in public speaking, and particularly if english is not your first language. i will discuss techniques for improving your presentation skills, both from the points of your spoken presentation and your visual aids, which will usually be a powerpoint presentation. if you are thoroughly prepared before you stand up in front of an audience, you will feel more confident and, consequently, less nervous. devine d publication of research findings and novel concepts in the biomedical literature is the mainstay of knowledge mobilization. the communication of scientific work as papers follows a well-established framework. a clear understanding of the nature of this framework and how to assess one's own work against it is critical to successful acceptance and subsequent publication of manuscripts. in this session, we will review the standard framework for scientific communication. communicating your findings is a form of scientific story telling. one must clearly explain why it was important to write the paper so that the reader will be interested and want to keep reading it. generally, an author must capture the interest of a potential reader right at the abstract which is sometimes the only way a reader sees the paper if they have discovered the work using a search engine such as medline. in the main body of the paper, the author must clearly explain how the study was designed and carried out with careful attention paid to any important details and to the statistical analysis, if appropriate. results must be presented in a manner that is clear and easily interpreted by the reader. then the work should be discussed in the context of other work in the area, emphasizing the novel findings. in this session, we will also address the following questions: how do i know if i have enough information to write a paper? where should i try to publish the paper? how should my paper be put together to give it the best chance of being accepted? what happens to my paper after i submit it? if it is returned to me with comments from the reviewers, what should i do? what is my next step if my paper is rejected, either without review or with review? we will also consider how to prepare papers in a language other than one's native language. the various sorts of scientific communications (original papers, review articles, reports, letters to the editor) will be discussed. the session is intended to provide general guidance for the publication of papers in the biomedical literature rather than be specifically focused on publication in the society's journal vox sanguinis. the membership survey commissioned by the isbt central office in 2012 gave a good insight into what members and non-members expect from the society. the main message was that members and potential members would like more educational and training resources to be available. many respondents requested that isbt should write international guidelines and develop standards. developing international guidelines when so many are already available and when countries and regions have different requirements would be a titanic task. the isbt board decided that the society should put together a repository (library) of guidelines, standards and regulatory documents that are already currently available. the repository is now ready and launched during the 24th regional congress of the isbt in kuala lumpur. for practical purposes the repository contains recent guidelines in the english language that are freely available, however following consultation with experts some manuals are included that are either relatively old or are not yet freely available on line. approximately 250 documents are in the repository from around 25 countries. the repository is a work in progress and further documents will be added in other languages. the repository is available via a link on the isbt home page www.isbtweb.org to the isbt academy e-learning portal. it can be accessed by country and by subject and a search facility is available. the guidelines are organised by six main subjects; donor, clinical, laboratory, quality/haemovigilance, processing and regulatory. there are sub topics for each main subject. isbt anticipates that you will find the repository a useful resource. background: blood services in africa operate at different levels of development from those comparable to the first world to those operating at a basic level with just hospital based blood banks and no national coordination. in recent years, countries have made efforts to improve their blood services based on voluntary non-remunerated blood donation. major challenges include low knowledge levels, inadequate funding, weak regulatory framework and poor quality systems. many international standards are too stringent for most economies in sub-saharan africa. to address these challenges, the africa society for blood transfusion (afsbt) started a program to develop blood transfusion standards relevant to africa. the afsbt step-wise accreditation standards: these standards were drafted by an afsbt task team for accreditation with guidance from the aabb and input from a team of experts. they were initially based on the who aide memoire for blood safety. an evidence-based decision-making process, where possible, was used to modify existing requirements or create new specific requirements. the goal of the standards is to provide a benchmark for accreditation of facilities and to maintain and enhance the quality and safety of blood transfusion in africa. the development process started in 2009. there is one standard with 3 progressively more rigorous steps of achievement as follows: level 1: minimum quality and operational requirements level 2: intermediate quality and operational requirements level 3: full accreditation at international standard a facility chooses the level to be assessed at. however accreditation at any grade is attained if facilities meet standards of the grade but also comply with specific requirements in section 11 which deals with legal and regulatory requirements, blood supply, equipment and supplies and clinical use of blood and blood products. application of the standards: they apply to facilities that perform any or all of the following functions: mobilization, recruitment and selection of blood donors; blood collection; components preparation; blood group and serological testing; compatibility testing; storage, handling, transportation and distribution of blood products. requirements do not apply in cases where the blood transfusion service is not responsible for an activity e.g compatibility testing. guidance document: there is a guidance document which clarifies and enhances understanding of the requirements of some of the standards. other standards are straight forward and do not need guidance. the guidance document is being updated as queries are received from the field. training: to facilitate meeting the requirements of the standards, there is a training committee, tasked with supporting training for blood services. a full training program is currently being developed. piloting: the standards were piloted in namibia and malawi. the namibia piloting was completed in 2012 while the malawi piloting will be completed in 2013. comments from these pilot sites have provided valuable input into the standards development process. conclusion: stepwise accreditation program is relevant in encouraging improvements for developing blood transfusion services. accreditation standards need to be commensurate with local needs. training is very important in helping developing blood transfusion services attain accreditation status. blood transfusion has become an integral part of modern healthcare. when it is required, it is an essential element of therapy, helps safe lives and improves quality of life. however, it is not without risk. being of human tissue origin, this risk is related to its source as well as the process involved in its provision. to ensure that blood transfusion is safe and does not cause harm to patients, these risks have to be monitored, evaluated and managed appropriately. therefore it is essential to develop system of ensuring safe blood supply and safe transfusion. quality systems should be developed covering the whole transfusion chain. policies, standards and guidelines are important tools. to ensure that the system is effective, there are various mechanisms to monitor, evaluate and analyse in order to bring about improvement. these include developing indicators, quality and clinical or transfusion audits, quality assessment programmes and haemovigilance programme. indicators can be used to monitor transfusion practice such as prevalence of transfusion transmitted disease among blood donors, crossmatch transfusion ratio, expiry fate of blood components and transfusion error. these indicators not only measure safety but also efficiency of the blood service. in countries where the blood supply is not consistent, the rate of blood requirement not met is an important indicator to monitor. performance of laboratories providing blood can be measured using quality assessment programmes. this is an important element that links the source of blood to the patient. audits covering all processes and procedures ensure that the quality and safety of blood is maintained. while these processes and procedures are controlled in ensuring safety and quality of the blood supply, the process involved in the transfusion process is sometimes not controlled by documented procedures and poorly supervised. auditing blood usage which is more complex and laborious provides an important mechanism for ensuring that this precious national resource is utilised efficiently. guidelines whether national or international are required against which these audits are performed. transfusion audits looks at the quality of care of patients besides the use of blood. it analyses the process of diagnosis, the decision making in treatment and the use of available resources. over the last two decades, haemovigilance has evolved and expanded worldwide. it focuses on adverse events that occurred throughout the transfusion chain, analysis of the facts and provide an avenue for corrective actions to be taken to prevent further recurrence. initially, its focus was more on adverse events that occurred to patients, it is now used to monitor adverse events relating to blood donors and blood donating process. in resource limited economies these tools can be used effectively when a stepwise approach is adopted. the aim is to create a culture of professionalism in delivering quality of care to patient with efficient use of available resources. however, its benefit can be extended to influence change and improvement in healthcare in general. only when deficiencies are demonstrated that request for resources can be shown to be justified. serological methods have the advantage of being fast, simple, and determining the expression of antigens directly. however, genotyping and molecular diagnostic methods have their advantages in determining subtypes and variants, as well as in detecting other rare blood groups. commercial blood group genotyping kits have been widely used, not only in clinical blood banks but also in transfusion services. these commercial kits mainly aim to analyze coding genes of abo and rh blood group systems. common alleles found in local populations are included in these kits. at present, many kinds of molecular diagnostic methods are employed in detecting blood group alleles by transfusion laboratories in china. these methods include multiplex pcr, multiplex pcr combined with pool system, sequencing, and gene chip technology. considering the genetic background of several rare phenotypes with clinical importance in chinese persons, one multiplex pcr system was developed to detect fy a , s, and ok a antigens, and the other system was developed to detect di b , k, and js b antigens. using the existing multiplex pcr-ssp assays, only one sample can be detected in a single pcr tube because the primers used in these assays are specifically devised for corresponding high-frequency alleles, and aim to screen for the negative results. therefore, the positive results would mask the negative results by adding more samples in one pcr tube. to improve the efficiency of screening, recently we established a novel method combining the multiplex pcr-ssp assays with the dna pooling strategy. the primers were designed to amplify the corresponding low-frequency snp sites. in each multiplex pcr-ssp assay, every dna pool is tested for the presence of the low-frequency snp sites. a pool is released for further processing when the positive results were found in any site. after then, each individual dna sample of the positive pool was detected respectively to determine the positive sample. finally, pcr-ssp methods were used to determine whether the positive result was caused by homozygous or heterozygous alleles. normally, the homozygous low-frequency alleles would lead to rare phenotypes. at the same time, the control system based on site-directed mutagenesis solves the problem of the lack of experiment controls due to unavailable negative or positive dna control samples. with large scale screening of population samples and diagnosis of various clinical special samples, the relationship between blood group coding genes and the expression of proteins is being clear. the frequency of multiple blood group alleles has also been investigated. the specific molecular events found in asians and chinese populations are revealing the difference among races and regions, as well as the expression diversity of blood group alleles. 1c-h6-02 diagnosis and treatment of autoimmune haemolytic anaemia autoimmune haemolytic anaemia (aiha) occurs as a result of antibodies directed against self-red blood cell (rbc) antigens, leading to the premature destruction of rbc. rbc destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses fcî¥r and c3 receptors, that bind antibody and complement coated rbc. the laboratory hallmark of aiha is a positive direct antiglobulin test (dat) although it has to be recognized that occasional cases of dat-negative aiha may occur. secondary causes for aiha such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for aiha. immunohaematology investigations for aiha should always include a dat using monospecific anti-igg and anti-c3d. on occasions, further testing with anti-iga or -igg subtypes may be necessary. while the dat provides information on bound rbc antibodies, the indirect antiglobulin test (iat) using screening and antibody identification cells will provide information on the specificity of circulating antibodies. often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. negative iat with screening and identification cells in the presence of a positive dat should raise suspicion of drug-induced aiha. in patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. elution and auto-or alloadsorbtion techniques are useful to separate allo-and autoantibodies. complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. the primary management of the patient with aiha is amelioration of the underlying condition and suppression of immune mediated haemolysis. this is usually achieved with administration of steroids or immunosuppressive agents. splenectomy may be an effective second line treatment. rituximab is increasingly becoming a promising treatment option in patients who are steroid-refractory and not suitable for splenectomy. other treatment modalities for the refractory patient include intravenous immunoglobulin and danazol. red cell transfusions in patients with aiha should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. as far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. if coincident alloantibodies are identified, antigen-negative red cells will need to be selected. clinical immunology and transfusion medicine, university & regional laboratories, lund, sweden what is the value of discussing case studies? they provide us with a forum for sharing our combined experience and permit the development of ideas and techniques as well as the possibility to see a given situation from another perspective. cases that illustrate the following will be discussed: (1) the presence of polyagglutination in a patient with a bacterial infection in europe and the usa, polyagglutination is rarely seen since monoclonal blood grouping reagents are the norm; however across asia, a broad spectrum of blood typing reagents are used and polyagglutination maybe encountered. (2) investigation of an antibody to a low-prevalence antigen in a case of haemolytic disease of the foetus and newborn antibodies to low-prevalence antigens occur not infrequently. how can they be investigated and what should be considered? (3) antibodies to a high-prevalence antigen the discussion will consider how to resolve such a case in laboratories with different levels of resources. what should be considered in different clinical situations? the goal is provide useful tips for investigation of difficult serological cases and information on how to proceed when the investigation is beyond the resources of the laboratory. haemovigilance is an important element of blood safety. it aims to identify, monitor and prevent adverse reactions, incidents and adverse events related to transfusion for both donors and patients (from 'vein to vein'). various local, regional and national haemovigilance models exist, which reflect the range of health systems and blood systems in different countries; for example, some haemovigilance systems are coordinated by professional bodies, some by blood suppliers, and some by health authorities (health departments or regulators). participation may be voluntary or mandatory, and may differ depending on whether all events or only serious ones are reportable. some systems capture events with all levels of imputability, whereas others record only those which are confirmed or highly probable. 'near miss' events are captured by some systems, and many valuable lessons can be learned from these cases. from an early stage of haemovigilance reporting it has been identified that processrelated problems are a major cause of serious transfusion complications. these include 'incorrect blood component transfused' events, where the blood component was intended for another recipient (frequently due to errors in patient identification at the time of collection of the pre-transfusion sample, or at the time of bedside administration), or did not meet the patient's special needs (such as a patient with a red cell antibody who did not receive the required antigen-negative unit). human and system factors, such as lack of awareness or training, working environment, interruptions and inadequate communications between clinical teams, or between the clinical teams and the transfusion laboratory, are very important contributors to these events. investigation of transfusion reactions, incidents and events at the hospital level is essential to identify clinical consequences, contributing factors and to develop and implement plans to prevent recurrence. reportable incidents should be notified to the haemovigilance programme. transfusion safety officers, transfusion nurses and similar roles have been introduced in many countries and they play important roles in haemovigilance, especially at the hospital level. adequate medical and transfusion laboratory support for hospital haemovigilance activities is also essential for success. hospital transfusion committees should oversee haemovigilance activities and reporting, and should ensure that hospital senior management is aware of and responds to serious reactions and events, especially where systems issues are identified to be contributory. at an international level, isbt's working party on haemovigilance brings together isbt members with an interest in haemovigilance. isbt works closely with ihn, an international collaboration of regional or national haemovigilance programmes, and other partners. ihn operates the istare database for international data sharing and benchmarking. haemovigilance reporting can identify priority areas for action (either where the events have serious clinical consequences, and/or occur frequently) and can help identify and monitor the implementation of solutions. an important feature of haemovigilance programs is the sharing of experiences and results. haemovigilance reports can both provide valuable feedback to clinical teams and hospitals locally, as well as share experiences nationally and internationally to improve patient outcomes. wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: haemovigilance systems capture data on adverse reactions and infections in recipients of blood transfusions as well as on errors and incidents in the transfusion chain. the objective is to analyse them and make recommendations for improving transfusion safety. many haemovigilance systems also collect data on complications in blood donors with a view to monitoring and improving blood donor safety. standardised definitions are necessary for classifying and comparing data in all these domains and at all levels. method: since 2004, at international meetings of blood transfusion professionals, members the international haemovigilance network (ihn) and the haemovigilance working party of the international society for blood transfusion (isbt) have collaborated in developing and validating definitions for non-infectious transfusion complications, errors and incidents in the transfusion chain as well as adverse reactions in blood donors. from 2012 contacts with other groups including the who have been put in place to ensure wide consultation as well as awareness and use of the definitions. results: standardised definitions have been published for recipient adverse reactions, for complications of blood donation and for a limited number of types of incident in the transfusion chain. the isbt haemovigilance working party and ihn are committed to ensuring that the definitions remain up-to-date and that revisions and improvements are conducted with wide consultation of professionals in relevant organisations worldwide. the haemovigilance working party and working party on transfusion-transmitted infections are collaborating on the development of definitions and criteria for assessing suspected transfusion-transmitted infections. conclusion: internationally agreed definitions are available for registration and surveillance of complications of blood donation and most types of adverse reaction in patients receiving blood transfusions. for errors and incidents in the transfusion chain, further work is necessary to improve comparability of data between hemovigilance systems. 1d-h7-03 distler pb and ashford p critical to patient safety is the capability of rapidly tracing a medical product of human origin (mpho) from donor to recipient and vice versa. traceability requires that each product be uniquely identified in order to provide a clear, unambiguous path. historically, uniqueness was defined only in the context of a single organization. for example, a blood product identifier was unique only to the blood bank that assigned it. because some medical products of human origin (mpho), especially cells and tissues, are frequently distributed across international borders, it is becoming increasingly important that identifiers of mpho need to be unique not only within an organization, but globally as well. in 2010, the world health assembly urged member states 'to encourage the implementation of globally consistent coding systems for human cells, tissues, and organs as such in order to facilitate national and international traceability of materials of human origin for transplantation.' more recently who has recognized the need for common strategies for global governance of all mhpo, including the global use of isbt 128, to ensure unique identification, optimal traceability, and interoperability between countries and across all mpho for both routine and emergency use. this requires a globally consistent coding system that can provide: a mechanism to allow distinct items to be uniquely identified and consistently characterized to all participants within the system, the means to allocate identifiers in a manner that avoids duplication, and the information infrastructure on which effective traceability can be built. isbt 128 is an international terminology, coding, and labelling system that supports the assignment of unique identifiers to support global traceability of mpho. it is currently in use by many blood banks, tissue banks, and cellular therapy facilities around the world and the who global forum on blood safety has recognized promotion of the use of isbt 128 as a priority for action in improving quality management and haemovigilance. 1d-h8-01 the university of tokyo, tokyo, japan antibodies directed to human platelet antigens (hpa) play important roles in the pathogenesis of neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). presently, six hpa biallelic systems, namely hpa-1 to -5 and -15, which are involved in immune mediated thrombocytopenia, are characterized. there are important ethnic differences in the frequency distribution of these hpa systems, the incompatibility of the hpa-1 system being the mostly involved in thrombocytopenic conditions in caucasian, whereas in japanese, the hpa-4 system is the mostly involved. the frequency distribution of hpa systems reported in other parts of asia seems to be different from caucasian as well as japanese, especially related to hpa-1 and -4, respectively. in addition to hpa, antibodies to human leukocyte antigen (hla), blood group abo, and human neutrophil antigens (hna) have also been shown to be involved in immune mediated thrombocytopenia. in fact, the majority of the cases of ptr are dependent on anti-hla antibodies, and anti-hpa antibodies comprise only a small proportion. the identification of the causative antibody is very important for the implementation of preventive/therapeutic measures for ptr, such as the selection of hla-and/or hpa-compatible platelets. on the other hand, the involvement of anti-hla antibodies in the pathogenesis of nait is questioned, but cases in which the causative antibody cannot be determined still remain relatively high. thus, for the implementation of preventive and therapeutic measures for the immune mediated thrombocytopenia, the detection and identification of the causative antibody is essential. the standard methods applied varies among the regions, the monoclonal antibody-specific immobilization of platelet antigens (maipa) and the platelet immunofluorescence test (pfit) being the preferred methods in the us and europe, whereas in japan, the mixed-passive hemagglutination is the most applied one. neither of the methods alone, however, is able to detect all the clinically significant antibodies, thus, improvement of the available methods as well as the development of new technologies is required. considering the ethnical differences of the hpa frequency distribution, we considered important to develop the research of this field also in asia, and for this purpose, the isbt platelet immunobiology working party, asia regional (isbt piwp-ar) was launched in 2010, during the xxxist international congress of the isbt in berlin, which aims the sharing of knowledge and improvement of technology in asia. a training course on platelet immunology methods and genotyping was provided in tokyo in 2010, and the first workshop of the piwp-ar was organized in taipei in 2011. in may 2013, the second training course on platelet immunology methods and genotyping was organized in guangzhou, china, and the 2nd workshop of the piwp-ar is going to be held in kuala lumpur, malaysia, during the 24th regional congress of the isbt. the presently available methods for the antigen/antibody testing, the problems related with antibody detection, and the activities of the platelet working party will be presented. nanning institute of transfusion medicine, nanning, china background: immunization against human platelet antigens (hpa) is associated with a number of clinical syndromes, including neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr), post-transfusion purpura (ptp), and other platelet immune disorders. the detection and identification of the clinical relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia. aims: the aims of this study is to investigate the characteristic of the detection of clinical relevant platelet antibodies in the asian population, and to evaluate the ability for the detection and identification of platelet antibodies in the platelet immunology laboratories in asian countries. methods: total of 378 cases that were diagnosed and studied as naitp (70), ptr (305) and ptp (3) in asian platelet immunology laboratories were reviewed. of the 378 cases, 167 were japanese in japan, 142 were chinese in china, 30 were in india, 24 were chinese in taiwan, china, 5 were in south korea, 4 were in thailand, 3 were in kuwait, and 1 case each for oman, lebanese and palestinian. the specificities of platelet antibodies in these cases were investigated and compared with the data from western country's laboratories. the methodology of detection and identification of antiplatelet antibodies in asian labs were also reviewed. results: among 378 cases, the immune thrombocytopenia associated antiplatelet antibodies were two cases of anti-hpa-1(in kuwait), 2 of anti-hpa-2 (1 in china and 1 in japan), 5 of anti-hpa-3 (three in japan, one in china and one in taiwan, china), 5 of anti-hpa-4 (all in japan), 4 of anti-hpa-5 (1 in china and 3 in japan), 1 of anti-hpa-7new(antihit a )(in japan), 1 of anti-hpa-15 (in japan), 2 of anti-hpa-21bw (in japan), 309 of anti-hla(101 in china, 147 in japan, 5 in korea,3 in thailand, 23 in taiwan, china and 30 in india), 1 of anti-a (in japan) as well as 15 cases of anti-cd36(nak a ) isoantibody (eight in china, three in japan and one case each in thailand, oman, lebanese and palestinian). thirty one cases of antibodies could not find the specificities (30 in china and 1 in kuwait). the methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (maipa), mixed passive hemagglutination (mpha) assay, platelet immunofluorescence test (pift), modified antigen capture elisa (mace), and solid phase red cell adherence (spaa) have been used in asian laboratories summary/conclusions: platelet alloantibodies are found with variable frequencies in different ethnic groups in asian population. anti-hpa-4 is mainly found in japanese individuals, while anti-cd36 (nak a ) isoantibody that occurred in cd36 type i deficient individuals is most frequent found in asian population especially in chinese population. only two cases of anti-hpa-1 antibodies were found in asian population (all in kuwait), while anti-hpa-1 is the most frequent antibody associated with severe complications in caucasian populations. however, anti-cd36 isoantibody is of a risk factor of immune thrombocytopenia in asian population, as the incidence of cd36 deficiency is 3-11%. the human platelet antigens (hpa) are a group of polymorphic antigens, expressed relatively specific on platelets, capable of eliciting an immunological response with development of alloantibodies. hpa directed alloantibodies have been implicated in neonatal alloimmune thrombocytopenia (nait), post-transfusion purpura and refractoriness to platelet transfusions. twenty-seven hpa are recognized, of which antibodies to both the given alloantigen and the antithetical alloantigen has been reported for hpa-1 to 5 and hpa-15. the remaining hpa are designated with a 'w' as an alloantibody to the antithetical antigen has yet to be reported. most hpa polymorphisms are a result of a mis-sense single nucleotide alteration and are readily detectable using standard molecular typing methods. dna based population wide genotyping have revealed considerable variation in hpa allele frequencies among different ethnic groups. the 'b' forms of hpa-1, -2, -3, and -5 are common among caucasians while hpa-4b is extremely rare. in the chinese however, hpa-1b is extremely rare while uncommon in malays. hpa-4b and hpa-21bw meanwhile are more commonly seen among asians compared to caucasians. consequent to this, alloanti-hpa-4b is the most common cause for nait in the asian population as compared to alloanti-hpa-1a among caucasians. several cases of nait due to anti-hpa-21bw have also been reported in asia. in contrast, nait due to anti-hpa-6b is rare despite the alloantigen being more common among asians. although platelet transfusion refractoriness is more commonly associated with hla antibodies, anti-hpa antibodies have been implicated in some cases. management of nait and platelet transfusion refractoriness include the supply of antigen negative platelet units. a platelet donor registry with a critical mass of hla and hpa typed blood donors is therefore necessary for effective management of these conditions. ready availability of low-cost high-throughput snp genotyping platforms allow for establishment of large hpa genotyped donor pools. knowledge of hpa genotype prevalence in the local population as well as its implications on nait and platelet refractoriness is however crucial before deciding on donor screening strategies, careful considerations would also need to be made with regards to the cost-effectiveness of such ventures in light of alternative management strategies and local incidence rates of nait. clinical -improving patient outcomes 2a-s01-01 setting up a patient blood management programme wood e 1 , engelbrecht s 1,2 and robinson k 2 1 transfusion research unit, monash university, melbourne, australia 2 australian red cross blood service, adelaide, australia patient blood management (pbm) aims to minimise unnecessary transfusions, and also to ensure that if transfusion is required it is managed appropriatelyby individualising care so that patients receive what they need when they need it. pbm is comprehensive and patient-centred, with active participation by patients and a multidisciplinary approach from the hospital team to achieving these aims. 'three pillars' of pbm have been suggested, to optimise a patient's red cell mass, reduce bleeding and improve tolerance of anaemia. in the perioperative setting, important elements of pbm include attention to medical, surgical and anaesthetic interventions and techniques to improve haemostasis and reduce blood loss. where significant intraoperative blood loss is anticipated, use of cell salvage techniques can be very valuable. pbm concepts also apply outside the perioperative setting, and the broad principles can be applied to a wide range of clinical settings, including obstetrics, trauma, critical care and haematology/oncology and other medical settings (e.g. gastroenterology). an effective hospital pbm programme requires planning and communication, with a stepby-step approach to implementation, identifying priority areas for action, engagement, education and training of staff, and on-going monitoring against plans to demonstrate progress and identify areas for improvement. feedback to staff on progress provides a sense of achievement and helps engagement. adequate resources are required, including from medical, nursing and laboratory staff from a range of disciplines who have important contributions to make in clinical practice, education and training, and audit and review. minimisation of unnecessary transfusions saves money for hospitals and the community, and other resources such as staff time, and therefore offsets the costs of establishing and maintaining a pbm programme. effective implementation requires change at individual and organizational levels, and therefore support of hospital executive management, local health authorities and the blood supplier are also very valuable. oversight of the program can be by the hospital transfusion committee or a pbm programme committee, but the particular structure and governance arrangements should be developed to suit local needs. general practitioners can play key roles in preparing patients for surgery by identification and management of anaemia, as well as other pbm activities. ultimately an effective pbm programme can optimise care and outcomes for patients, make better use of limited and precious blood supplies, and reduce risks and costs. trauma is a leading cause of death around the world, with haemorrhage accounting for more than a third of the preventable mortality, with the majority of these deaths occurring within the initial 24 h. massive blood transfusion is generally defined as the replacement bytransfusion of more than 10 units of red cells over 24 h. massive transfusionprotocols (mtps) have evolved over the past decade and are especially importantin facilitating the early delivery of copious amounts of blood products topatients who have major injuries and severe haemorrhage. various studies havealso demonstrated that with mtps, there is a more efficient use and lesswastage of blood products. however, for trauma patients, stopping thehaemorrhage and resuscitating the patient does not only involve the expedientdelivery of red cells to the injured patient. mtps have also emphasized theneed for a more balanced ratio of delivery of blood products, includingplatelets and plasma, to patients who sustain massive blood loss and havedeveloped acute traumatic coagulopathy (atc). often times, mtps also stress theimportance of the consideration of use of haemostatic adjuncts, such astranexamic acid, activated factor vii, level one transfusion units and the useof blood warmers to reverse the potential effects of hypothermia with sustainedtransfusions of large amounts of blood products. ultimately, in tandem withdamage control resuscitation, which allows permissive hypotension whilstsecuring haemostasis, mtps have been shown to also improve survival of theseseverely injured patients. a more recent paper describes the development of amassive haemorrhage protocol to aid in the identification of patients who wouldbenefit from the mtp and this may be the next step in the evolution of a workprocess by which resuscitation for severely injured patients may be optimized. background: rh blood group antigens are highly immunogenic and transfusion of rh d positive blood in rh d negative recipients is avoided. platelets do not express rh antigens, however they may contain significant amount of contaminating red cells to illicit an immune response in the patient. due to limited shelf life of platelets and inventory issues, rh d positive platelets which are not visibly contaminated with red blood cells maybe transfused to rh d negative patients including children and females of child bearing age. there has been increased focus on whether rh immunoglobulin should be routinely administered after such transfusions. in saudi arabia no clear guidelines exist on the administration of rh immunoglobulin after d positive transfusions in d negative patients. aim: the purpose of this study was to determine whether the transfusion of rh d positive platelets to patients who are rh d negative, results in d alloimmunization and whether rh immunoglobulin should be routinely administered in such patients. methods: eligibility criteria for inclusion in the study included the following: transfusion of rh d positive platelets, no anti d detectable before transfusion, no previous exposure to rh d positive blood components, and results of follow-up testing of anti-d in patients serum available. the patients blood group and antibody screening was done using liss-iat gel technology (diamed). results: one hundred rh d negative patients who received rh d positive blood components were identified. out of this 63 (63%) patients received rh d positive platelet transfusions. in 47 (74%) patients out of this, the results for post transfusion antibody screening were available. the mean age of the patients was 46.5 years and included 30 males and 17 females. average number of rh d positive platelets transfused per patient was 11.3 units and total number of platelet units transfused was 523. anti d was detectable in 4 (8.5%) patients post transfusion and included one male and three female patients. of the female patients one was a 3 year old child who received 50 units of random donor platelets and second a 21 year old women who presented in the emergency department as a case of trauma. the third female was 54 year old post-menopausal woman. conclusion: we conclude that the chances of developing d alloimmunization after receiving rh d positive platelets is generally low. however keeping in view the antenatal complications which can arise in the future in females due to this alloimmunization, it is highly recommended that rh immunoglobulin be administered to all rh d negative women in the reproductive age group and female children who receive rh d positive platelets. background: hemovigilance is a quality process which takes into account all the activities of a blood transfusion chain with the aim to improve quality and enhance safety of blood transfusion. we have implemented a transfusion feedback reporting mechanism in our hospital as a part of hemovigilance. the current study aims to collect and analyze this data to improve our transfusion system. aim: to systematically analyze the transfusion process from issue of blood components to completion of the transfusion material and methods: the transfusion feedback forms received back at the transfusion medicine department during a 3 months period were systematically analyzed for documentation related to patient identification, product identification, documentation, completeness, etc. results were analyzed statistically for specific co-relation with patient's location, time of transfusion and type of component transfused. results: of 3474 blood components were issued during the study period. transfusion feedback form were received for 2000 (57.5%) blood components, as follows: prbc-800 (40%), platelets-651 (32.5%), ffp -441 (20.7%). patient identification number /wristband check was not done in 25 (1.25%) cases. pre-transfusion verification of blood group, patients name and patient's identification number was done in 1963 (98.15%) cases. cross checking of component unit with request form was missed in 16 (0.8) cases. pre-transfusion and post-transfusion monitoring of blood pressure was documented in 698 (34.9%) episodes, monitoring of pulse in 696 (34.8%) episodes and patient's temperature was monitored in 681 (34%) episodes. signature of nurse was missing in 86 (4.3%) and that of medical officer in 11 (05%) of the form. adverse transfusion reaction was documented in 1/2000 forms, whereas the transfusion reactions notified at the blood bank during the same period were 2. of 553 (27.65%) transfusions were carried out during non routine work hours. the mean time between the issue of the components and start of transfusion was 28 min for rbc components, 21 min for platelets and 16 min for ffp. patient identification and monitoring and product identification related non-compliances significantly correlated with out of routine transfusions (p = 0.002). conclusion: though overall compliance with established procedure for transfusion was evident, activities related to unit identification, patient monitoring and identification and reporting of adverse reactions were not well documented. this emphasizes the need for ongoing training of nursing staff and medical doctors in safe blood administration practices. introduction and method: matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (maldi-tof ms) is an ideal tool for high-throughput blood group genotyping. using this technique, this swiss (bts zurich) -german (sequenom gmbh, hamburg) cooperation aims to genotype swiss blood donors for blood groups rh, kell, kidd, duffy, mnss (n = 6000), hpa and hna (n = 3000) and high frequency antigens (hfa, n = 36,000) within 3 years. specificities are grouped into single multiplex reactions (mpx, n = 10) with up to 17 snps tested simultaneously in one tube. results: using the kel-jk-fy mpx, more than 4000 donor dnas have been tested and compared to serological pre-values. concordance between geno-and phenotypes reached 100% for k/k, 99.98 for kp a 99.93% for jkand 99.23% for fy a/b/x/0 . only one discrepancy each for kp, jk and fy could be attributed to genetics, the others were erroneous serotypes revealed by genotyping. genetic discrepancies were three new variants: kel*02.03(r700g)nulla kp a relative, jk*b(mutation n.d.) and fy*b (g261r)null. serology for js a/b of some few kel*02.06 positives confirmed validity of genotyping. numbers of detected known kel*mod and null, jk*null and fy*null (gata) alleles were 3, 2 and 51, respectively. call failures (no result) were observed in less than 2% of all mpxs. genotyping for rhd, rhce (5 mpx), gypa and b (mnss, 1 mpx) on more than 4000 samples, delivered results with discrepancy rates for mnss comparable to above, and better rates for rhdce. call failure were at approximately 2% again. reproducibility, robustness and analytical accuracy of the technique allowed measurement of gene copy numbers with relevance for rhd zygosity estimation and detection of the gypb deletion in u negatives. rhd category, partial, weak, del and null alleles and the genetic correspondents of vw, mg, mi(a), he, and uvar were observed. genotyping for hpa and hna (1 mpx) showed expected results among 2300 samples with the exception of hna-1a, b and c, where frequent duplications and deletions of fcgriiib pose difficulties for all genotyping approaches in general. in hfa ('rare') genotyping (2 mpx), currently, more than 13,000 blood donors were analyzed, and delivered: 29 kk, 2 kp(a+bã�), 48 js(a+bã�), 41 kel11+kel17+, 20 lu(a+bã�), 3 lu14+lu08ã�, 52 yt(aã�b+), 17 co(aã�b+), 11 kn (aã�b+), 98 lw(a+b+), and >10 others. specificities for vel negativity and scianna have been included into hfa typing, recently. conclusion: analysis for kell, kidd and duffy showed that genotyping worked qualitatively better and to costs comparable to serology. consequently, genotyping kell, kidd and duffy instead of routinely performing a second round of serotyping as mandatory for donors in switzerland, is recommended. ahead of comparable suggestions with regard to rh and mnss, a more detailed statistical analysis of existing raw data is needed. however, genetically identified donors with rare blood phenotypes, e.g. such as yt(aã�b+), are already selected for respective transfusions and are a strong indicator for the value of the presented project. the serological data suggest that retention of the 208-210 tcc (51s in glycophorin bs) codon from the gypb pseudoexon, prior to the gene conversion insertion of gypa sequence and coding for serine in the hybrid glycophorin, is the basis of 'anek-like' activity for both hybrid glycophorins. it should be considered that these antisera react with a new kipp-related mns system antigen. genetic studies revealed the same crossover for gp.kipp and gp.yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin. background: with the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one's phenotype. there are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. however, there are several misgivings about the cost of molecular methods used to genotype antigens. aim: to evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. methods: a batch of 30 donor samples was typed using both serological and molecular methods. the test kit used for molecular methods, from gen-probe, included the typing for the following antigens -kell (k, k, kpa, kpb, kpc, jsa and jsb), kidd -(jka, jkb, jk), duffy (fya, fyb, fyx, fygatasil) , mns (m, n, s, s, s-s-uvar), rh (c, c, e, e) and dombrock (doa, dob). negatives that are obtained using this method were confirmed using serology. serological typing was performed with available antisera according to the various manufacturers' instructions. this includes kell (k, k), kidd (jka, jkb), duffy (fya, fyb), mns (m, n, s, s) and rh (c, c, e, e). the cost for the different tests were tabulated and compared. the cost includes labour, consumables and equipments. results: the results show that molecular method of typing red blood cells is more expensive than the traditional serological method. the cost of consumables is comparable for both methods. the consumables make up about 80% of the total cost for serological methods, and about 82% for molecular methods. the cost of labour is about 9% for serological methods and <1% for molecular. equipment cost contributes to <1% of the cost using serological methods and about 14% using molecular methods. conclusion: molecular methods may seem more expensive, about two times the cost of serology but results are obtained faster and are less labour intensive which could prove to be an advantage when phenotyping samples in large quantities, especially for donor phenotyping. molecular immunohaematology is a relatively new process, thus kits for these methods are expensive at the moment. as development and adoption of genotyping progresses, consumables and equipment costs are expected to become more affordable. serological methods do have their limitations even for patient testing, especially for patients with autoantibodies and patients who have had multiple transfusions. molecular methods may serve useful in overcoming these limitations. background: transfusion dependant patients often develop multiple antibodies to red cell antigens on exposure to red cells and require antigen-negative red blood cells for further transfusions. finding suitable red cell units for such patients is often difficult and time consuming, requiring phenotyping of large numbers of red cell units in inventory. the increasing cost and scarcity of anti-sera also makes this an expensive exercise. aim: in order to improve provision of phenotype-matched blood for such patients, we studied the feasibility of large-scale snp-based genotyping of common blood groups for establishment of an antigen-negative red blood cell inventory. methods: genomic dna was extracted from donor blood samples using an automated platform. samples were subjected to snp-typing for common local polymorphisms of rhce (ccee), fy (fy a /fy b ), jk (jk a /jk b ), mns (s/s) and co (co a /co b ) using taqman 㢠snp genotyping assays in 384-well plates at 5 ll final reaction volume, on a lightcy-clerii 480 real-time instrument. identification of the cc polymorphism was by probes targeted to the 48c>g and 307t>c of the rhce allele while probes targeted to 676c>g was utilised for ee detection. for the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an east-asian population. results were analysed and integrated into the blood donor software system. results: the 48c>g probe designed for identification of c was unsuccessful with poor discrimination between genotype calls. the remaining probes showed satisfactory discrimination between genotypes, with 462 of the 505 (91%) samples analysed, fully genotyped for the five alleles studied. relatively rare genotypes were successfully identified using this strategy: ccee (15/505, 3.0%), fya-/fyb+ (19/505, 3.8%), ss (5/505, 1.0%). the co2 allele responsible for co b was identified in the heterozygous state in four of 494 evaluable samples giving an allele frequency of 0.4% in our population. the estimated reagent and consumables cost for sample dna preparation and snp testing was usd12.00 compared to usd27.15 for phenotyping using commercial anti-sera. we did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. conclusions: results of this study indicate that snp genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell inventory and genotyped whole blood donor pool. the cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. in addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera. background: over the past 20 years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. since october 2012, in our transfusion center (asl caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and rh phenotype. aims: the aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. we also compared each method in regards to reliability, ease of use and reproducibility of results. methods: we selected 250 donors aged <45 years old, group o and a, rh homozygous phenotype. samples from each patient were tested by serological typing in solid phase (capture r immucor) and then dna was extracted and each sample was tested by molecular typing in microarray (bioarray solutions immucor) results: see table 1 . table 1 : summary/conclusions: the results show that the frequencies of more immunogenic antigens (k, fya, jka) reflect the specific and ethnic frequencies of the donors. we emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of 1:1000 according to american rare donor program (ardp), council of europe, international donor panel (idp), international society of blood transfusion (isbt), council of europe, japanese red cross). we only found one case of a donor expressing weak fyb which is due to mutation in fy 265c> t. the results were confirmed in 99.6% of cases by serological technique. however for the phenotype fyb weak, the result was discordant in serology, where the result was fyb-. in conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. the molecular technique is able to identify mutations in particular genes, especially in specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained. background: pathogen inactivation of blood components promises a new layer of transfusion safety, enhancing existing strategies and providing proactive protection against emerging infectious risks. processes for plasma and platelets are well established in many blood centers and those for red cells and whole blood are in development. clinical acceptance of new technologies depends on the demonstration of product safety, a minimal effect on product efficacy both in vitro and in vivo, and the range and degree of pathogens inactivated. aims: to review the major processes of pathogen inactivation of plasma and cellular blood components with a focus on the range and degree of inactivation of relevant pathogens, especially with regards to platelet pathogen inactivation. methods: this review will use recent reports and unpublished data to describe our current understanding of the potential efficacy of pathogen inactivation in improving transfusion safety. results: the major indication for platelet pathogen inactivation is bacterial contamination, a persistent problem despite multiple innovations to minimize and detect contamination. pathogen inactivation processes need to cover a wide range of possible bacterial concentrations and species. there have been few published reports using the current commercially available systems: optimized in vitro testing documents the ability of the terumo bct mirasol tm , cerus intercept tm and macopharma theraflex tm uv systems to effect a 10 2 ->10 6 log reduction of various bacterial species. most systems are less effective at inactivating bacterial spores, a particular problem as bacillus spp. is common platelet contaminant. testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. conclusions: clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection. for pathogen inactivation (pi) using amotosalen and uva light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing dna replication and rna translation. pi technology has been adopted into routine use in some european countries and is under fda review for licensure in the us. current documentation of pi efficacy relies on illuminator sensors that measure the uva light dose delivered. an indirect methodology is utilized for the validation and qc of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the compound removal device. the% amotosalen remaining is a direct measurement of the uva light delivered and photochemical conversion of amotosalen. however, a functional qc method that measures a direct target within the treated blood product has not been introduced into clinical use. residual leukocytes, platelets and potentially plasma contain mitochondrial dna (mtdna) which is a collateral target of the pi process. aims: the goal of this study was to quantify the impact of intercept treatment on platelet-derived mtdna by real-time pcr. methods: to evaluate the feasibility of detecting pi-induced mtdna modifications by real-time pcr, we spiked purified human leukocyte dna into human plasma, 35% plasma/65% intersol, or pbs. each sample (3.6 ml) was treated with 150 lm amotosalen and 3 j/cm 2 uva (n = 2). control samples were either untreated or treated in the absence of amotosalen or uva. dna was extracted from each sample (0.2 ml) in duplicate and assessed by measuring the inhibition of real-time pcr amplification in duplicate wells using mtdna-specific primers and sybr greenbased detection. amplification of sequences ranging in size from 73 to 1065 bp was evaluated over 45 cycles of amplification. subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in 35% plasma/65% inter-sol (30 ml). six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtdna sequences. results: treatment of purified dna with amotosalen plus uva resulted in 1.3 log to >6.0 log inhibition of pcr amplification (results shown in table) . the extent of pcr reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: pbs > 35% plasma > 100% plasma. exposure of platelets to amotosalen and uva showed an average of 2.5 log to 3.6 log reduction in pcr signal, with increasing inhibition observed for larger amplicons. in all cases, no pcr inhibition was observed in the absence of amotosalen and/or uva. conclusions: a quantitative real-time pcr assay specific for mtdna is capable of documenting pi-induced collateral nucleic acid modification in platelets. based on this work, this assay can be developed further for use as a quality control method for pi efficacy. background and aims: pathogen reduction technology (prt) provides a proactive approach to improving transfusion safety for platelet concentrates (pcs). however, prt treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. little is known regarding how prt-treated platelets may affect the cells of the recipient's immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient's inflammatory cells after exposure to prt-treated platelets using an in vitro whole blood model of transfusion. methods: two abo/rhd matched buffy coat derived pcs were pooled and split to form matched pairs on day-1 post-collection (n = 11). one unit was treated with the mirasol prt tm system (terumo bct), while the other unit remained as an untreated control. all units were stored at 22â°c with agitation and samples were taken on day 2 and 7 post-collection for 'in vitro transfusion' experiments. to represent a transfusion in vitro, 10% v/v platelets were incubated with abo/rhd-matched fresh whole blood, with or without lipopolysaccharide (lps; 1 mg/ml) for 6 h at 37â°c/5% co 2 . protein secretion was inhibited using brefeldin (2 lg/ml) to allow detection of intra-cellular cytokine production in monocytes (cd45 + /cd14 + ) and neutrophils (cd45 + / cd16 + ), using multi-colour flow cytometry. the fold change of cytokine production was calculated by comparison to a 'no-transfusion control' (whole blood without addition of platelets). data was analysed using a one way anova with post-hoc tests for pair-wise comparisons. results: in the absence of lps, both prt-treated and untreated platelets stored for 7 days significantly increased monocyte mip-1b expression (1.5-fold; p = 0.047), whereas exposure to day 2 platelets did not result in any significant differences in mip-1b expression. as expected, lps stimulation significantly increased monocyte production of both il-12 (5.0-to 7.4-fold) and mip-1b (1.8-to 2.4-fold). however, lps-induced monocyte il-12 production was significantly reduced by exposure to prt-treated or untreated platelets stored for 2 days (prt-treated: 3.0-fold, untreated: 2.5-fold; p < 0.0001) and 7 days (prt-treated: 4-fold, untreated: 2.1-fold; p < 0.0001). furthermore, il-12 production was significantly lower following exposure to day 7 prt-treated platelets compared to untreated platelets (p = 0.006); however there was no significant difference following exposure to day 2 platelets. lps-induced mip-1b production was not significantly differentfollowing exposure to either day 2 or day 7prt-treated or untreated platelets. exposure to platelets (untreated or prt-treated) did not significantly modulate monocyte production of ip-10, mcp-1, mip-1a, il-1a, il-1b, il-6, il-8, il-10, or tnf-a. co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. conclusion: using an in vitro whole blood transfusion model, we have demonstrated that exposure to prt-treated platelets stored for 7 days results in significant changes in the il-12 production by monocytes. these changes may reflect the way prt-treated platelets interact with immune cells upon transfusion. therefore, the effect of stored prt-treated platelets, especially in recipients with underlying inflammation, should be further examined. backgrounds: hepatitis c virus (hcv) infection is one of the major causes of chronic hepatitic disease. hcv has six genotypes and more than 80 subtypes. the epidemiology of hcv subtypes vary with different geographic distribution. understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of hcv and thus help to make effective precautionary measures. hcv subtypes are also closely related with clinical therapeutic effect. it is important for guiding clinical therapy and prognosis and predicting the possible burden of hcv infection and treatment in the future. aims: to investigate and compare the prevalence of hcv subtypes in clinical patients and blood donors in guangdong china. methods: of 191 samples ofclinical patients and 222 samples of blood donors whose hcv rna were positive were collected from guangdong province. hcv ns5b gene was amplified by rt-nested pcr and then sequenced. hcv subtypes were assigned by constructing phylogenetic trees with mega5 software. moreover, spss16.0 software was applied to compare the difference between these two groups. results: of 191 clinical patients, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 2 (1.05%), 127 (66.49%), 17 (8.90%), 5 (2.62%), 5 (2.62%), 34 (17.80%) and 1 (0.52%), respectively. of 222 blood donors, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 1 (0.45%), 92 (41.44%), 1 (6.76%), 19 (8.56%), 11 (4.95%), 83 (37.39%) and 1 (0.45%), respectively. the proportion of hcv 1b was higher in clinical patients than in blood donors(v 2 = 25.866, p = 3.66e-07), while the proportion of 3a and 6a subtypes were higher in blood donors than in clinical patients(v 2 = 6.602, p = 0.010; v 2 = 19.398, p = 1.06e-05). one possible cause was the transmission modes varied with different hcv subtypes. hcv 1b is more related with blood transfusion while 3a and 6a are more relevant to intravenous drug abuse and sexual behavior. with the anti-hcv screen implemented from 1993, the risk of hcv infection by transfusion is diminishing. the other reason was the average time from hcv infection to serious pathological lesions is about 20 years and hcv 6a was transmitted into guangdong later than 1b. conclusions: in guangdong province, hcv 1b and 6a were the predominant subtypes in clinical patients and blood donors. the proportion of hcv 1b, 3a and 6a subtypes were significantly different between clinical patients and blood donors. the reason may relate with the time of hcv transmission into guangdong area and the hcv propagation modes. blood safety and increased public health initiatives to reduce hcv infection from those in these high risk groups to the general population remain a priority. background: in japan, we routinely evaluate the presence of transmission of hbv, hcv and hiv in all transfused patients at three months after the last transfusion. although the sensitivity of detection of hepatitis b virus (hbv) in blood donation improved during recent years, transfusion transmitted hbv infection is left as a serious problem. the japanese society of transfusion medicine and cellular therapy (jstmct) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by ministry of health, labour, and welfare, every year. in this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion hbv-marker-test. aims: the aim of this study is to elucidate the cause of hbv positive in transfused patients. materials and methods: data concerning to transfused patients with positive hbvmarker were collected using results of the nationwide questionnaire survey in 2007-2011. in this questionnaire, if there is a patient showing a positive result of hbsag and/or hbvdna evaluated by post-transfusion-test, it is requested that detailed patient's characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of hbv-marker-test prior to the first transfusion, should report to the jstmct office. a number of hbsag and/or hbvdna positive patients were 234 cases in 2007-2011. among them, 19 patients were not eligible because of the absence of results of hbv-related markers. finally, a total number of 215 patients (36, 37, 41, 43, 45 patients in 2007, 2008, 2009, 2010, 2011, respectively) were enrolled for the present study. results: all the eligible patients showed positive results of hbsag and/or hbvdna in samples obtained from three months after the last transfusion. we classified the cause of these results into five categories according to results of hbv-markers tests prior to the first transfusion. (i) if a result of hbsag and/or hbvdna performed prior to transfusion is positive, a patient is categorized as the hbv carrier group. (ii) a patient showing both a negative result of hbsag and positive results of anti-hbs and/or anti-hbc are categorized as the hbv past-infection group. (iii) if a patient shows no hbv-related marker prior to the first transfusion and patient's hbvdna is identical to donor's hbvdna, we categorized as the transfusion transmitted infections (tti) group. (iv) if a patient does not show any hbv-related marker prior to the first transfusion and hbvdna is not detected in donor's blood by single nat, we categorized as the unknown cause group. results were summarized in table 1 . conclusion: although the positive result of hbsag and/or hbvdna of transfused patients has been considered the sequel of blood transfusion, we showed that hbv reactivation was also an important cause of it. to distinguish hbv reactivation from transfusion transmitted infection, we have to perform hbv-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient's serum. plenary session: it's all about red cells 2b-pl1 the english dictionary provides several definitions for the word 'myth'. one definition is, 'a widely held but false belief'. this seems suitable for the purposes of this presentation. but who decides what is false? as we shall see in the course of this presentation, some 'myths' of blood groups may not be myths at all, and some established facts may indeed be myths. perhaps a more scientific definition would be, 'a widely held but unproven belief'. abo, the original and most important blood group in transfusion and transplantation medicine, has engendered many 'myths'. these have mostly arisen through associations between abo groups and other characteristics such as personality, dis-ease, psychological traits, and ideal diet. although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. for example, the statistical associations between abo type and thrombosis, where a biochemical basis involving clearance of von willebrand factor from the blood by enzymatic cleavage appears to be affected by abh glycosylation. in rh, the first myth was that the human antibody, now called anti-d, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. hence the name rhesus, now rh, for the blood group system. in the 1940s, early serological work with rh antibodies led to two genetic theories, involving either one or three rh genes. both theories have now been rejected because molecular genetics revealed two rh genes. but was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? since the 1960s it has been commonly understood that there are two types of variant d antigens: weak d and partial d. policies for transfusing patients with these variants have often been based on this dichotomy. but is this a myth? the difficulty we have in defining the terms weak d and partial d suggests that it might be. it is always tempting to dismiss anything that we don't agree with as a myth. as wiener, one of the discoverers of the 'rhesus' antigen, wrote several papers on 'blood group mythology', doing just that. scientists should beware of falling into this trap. perhaps 'myth' is a term best avoided in science. five new blood groups -what next? the past 2 years has seen the discovery of five new blood groups. at the isbt meeting in 2012, fors, jr and lan were ratified as blood group systems and since then, the molecular basis of the vel blood group antigen has been elucidated, and the complement regulator protein, cd59 has been shown itself to be a blood group antigen. these last two discoveries will no doubt lead to their elevation to blood group systems. how has this happened? it turns out to be a mixture of old and new techniques. rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. the development of comprehensive snp arrays, exome sequencing and rapid sequencing techniques, e.g. next-generation sequencing, has provide us with tools for rapid discovery. combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens jr a and vel. however classic biochemistry and subsequent peptide and dnasequencing still play an important role and lie behind the (simultaneous) discoveries of jr a and vel, but also of lan and fors. a rare cd59-deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the cd59 protein, which was confirmed by routine dna-sequencing. the jr a and lan antigens were assigned to already well-investigated abc-transporter proteins (abcg2 and abcb6 respectively) whose presence on the red blood cell (rbc) had not been established previously and for which the function on rbcs is still not known. fors antigen was shown to result from the reactivation of the human pseudogene gbgt1. this enzyme builds the carbohydrate forssman antigen on sheep and dog rbcs but is normally inactive in humans. a mutation that reactivated the enzyme explained the unusual a pae phenotype in two families. vel was shown to be carried on a hitherto unknown protein on the rbc, smim1. the function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. absence of cd59 whose function in complement regulation is well-understood, resulted in production of an alloanti-cd59, demonstrating immunogenicity of this protein for the first time. these simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group 'home' continues at a rate unparalleled since the 1990s. as the '-omics' fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home. 2c-s04-01 teo d blood services group, health sciences authority, singapore, singapore an emerging infectious disease (eid) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. in 1992, an institute of medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. east and southeast asia, with 30% of global population, has a reputation as a hot spot for eid. within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. the emergence of severe acute respiratory syndrome (sars) exactly 10 years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional eid threats. the re-emergence of highly pathogenic avian influenza a(h5n1) virus in 2004, isolation of novel bat-associated reoviruses in 2006, emergence of artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in 2009 are some examples of eid emerging within the region since sars. at the time of writing, the situation involving human cases infected with avian influenza a(h7n9) virus is evolving, and there has been a recent report of human infection with avian influenza a(h6n1) virus as well. additionally, there are imported eids such as influenza a(h1n1) virus, west nile virus, and the present cause for concern in middle east respiratory syndrome coronavirus. climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. 40% of the world's population is now at risk of dengue, with the majority living in the asia-pacific region. the scourge of dengue is sufficiently high in the region for the association of southeast asian nations (asean) in 2011 to designate 15 june as asean dengue day. hepatitis e is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. the impact of eids on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. blood products such as hyper-immune plasma preparations may be useful treatment options in some eids. there is a need for constant surveillance and the capacity to identify, assess and manage eid risks to the blood supply. in recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. nonetheless, the volatile and ever-changing nature of eids will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community. summary: the residual transmission risk is relatively high in hbv followed by hcv and hiv-1. this finding is not new as malaysia is a country of medium seroprevalence for hbv which ranges from 1.5% to 9.8% in the general population, but is relatively low in blood donor population (0.04%). the obi nat yield was higher than acute phase wp in hbv-nat yield. these obi positivity have shown inconsistent nat results on repeat testing and also low viral loads ranging from <12 to 317 iu/ml. in contrast for hiv and hcv nat yields, their viral loads were consistently high (34,220-424,310 cp/ml and 42,780-5,980 ,350 iu/ml respectively). implementation of id-nat is probably the best option to improve blood safety especially for the detection of low viral load such as in obi and sero-negative wp donation in malaysia scenario. blood centers reported to kcdc. repository samples of these donors were tested for anti-hav igm/igg and hav-rna. if any of these test result was positive, the recipients of the blood components generated from these donations were traced. transfusion records of hospital were reviewed to identify recipients of blood suspected to be contaminated with hav. recipients were contacted by telephone. if the recipients agreed participating in the investigation, laboratory test was performed. results: from may 2007 to december 2011, 15 donors notified the blood center of having been diagnosed with hepatitis a. the median interval from donation to diagnosis in donors was 18.2 days (table) . eleven (73%) of these 15 were male donors, and the median age was 28 years (range 19-42 years). the pcr for hav rna was positive in all of 15 depository samples of these donors. none of the repository samples showed positive result of anti-hav igm. a total of 44 blood components (rbc 15 units, pc 14 units and ffp 15 units) were delivered to hospitals. twenty six products were transfused to 26 patients, and the rest 18 blood components (4 rbcs, 1 pc, and 13 ffps) were recalled immediately and discarded. twelve recipients (46%) were already expired. fourteen recipients agreed to participate in the lookback procedure through testing. twelve recipients did not showed viremic for hav, however two recipients showed positive either on the test for anti-hav igm or hav-rna. these two recipients who were 35 and 37 years old developed symptoms on 46 and 48 days after blood transfusion respectively. they were treated for hepatitis a successfully. summary/conclusions: recipients who had anti-hav igg were not infected with hav, even though they were transfused with blood suspected to be contaminated with hav. however ttha cases were those who did not have anti-hav igg and all of them were 30s. in korea, most people under 40 years old are susceptible to hav because of low immunity. there is a risk of transfusion transmitted infection, if recipients received blood contaminated with hav. 2c-s04-04 cable rt 1 , pistorius c 1 , andersson m 2 , maponga t 2 , lopez t 2 , preiser w 2 and tedder r 3 1 hav seroprevalence was highest in the black donors (86%), lower in coloured donors (62%) and lowest white donors (36%). all were hav igm negative. there was an age-related increase from 44.8% (52/116) in those 16-25 years old to 81% (47/58) in those >46 years. although no active hev infection was identified by pcr on pooled samples, 25% of donors were hev igg positive. rates were highest in whites at 33%, followed by coloured at 23% and lowest in black at 19%. again prevalence increased with age from 12.1% in those 16-25 years to 48.3% in those >46 years. discussion: the results show a lower hav seroprevalence in the white population compared to the black and coloured population groups, likely to be due to socioeconomic living conditions such a contrast is striking given the introduction of democracy in south africa almost 20 years ago. the reduction in anti-hbc (as a marker of past hbv infection) with age is reassuring, suggesting that hbv vaccination is impacting hbv population prevalence. the pattern of hev exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. given the subclinical nature of hev infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors. background: hbv demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. in addition, mutations in the polymerase region may lead to drug resistance and changes in the pres region (including deletions and mutations such as t31c and t53c) and prec/bcp region (mutations including a1762t/g1764a, g1896a, t1896a, c1766t, t1768a) are associated with higher risk of hepatocellular carcinoma (hcc). aims: to study the hbv subgenotype distribution and analyze the changes in pres region, prec/bcp and the polymerase region of hbv in chinese blood donors. methods: of 245 blood samples were selected randomly from hbsag positive blood donors from five blood centers in china. the pres plus s region or the whole genome of hbv was amplified and sequenced and hbv subgenotype was determined. the amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. the nucleotide sequences of pres region and prec/bcp region were aligned and the mutations related to hcc were determined. distribution of genotype, subgenotype, and mutations by different regions were examined using chi-square statistics. results: of 200 samples (81.6%)were subgenotyped successfully. the predominant subgenotypes were b2, c2, d1 and a1 which accounted for 50.5%, 19.2%, 5.3%, and 3.4% respectively. deletions and mutations (t31c and t53c) in pres region were found in 28 (28/200, 14.0%) samples. five of these 28 samples (2.5% of all samples) have deletions and no deletions specific to genotype d was detected. the prevalence of mutations in pres region was significantly higher in genotype c than in genotype b (p < 0.001). mutations in polymerase region were found in 14 samples (7%, 14/ 200), most of which were related to resistance to adefovir and lamivudine. mutations in prec/bcp region were found in 68 samples (29.8%, 68/228). the prevalence of hbeag was significantly lower in samples with mutations in prec/bcp region than that in the samples with no mutation (p = 0.02). more a1762t/g1764a mutations were found in c than b genotype while the opposite was observed for g1896a mutation (p's < 0.01). conclusions: subgenotype b2 was the most frequent strain circulating in hbv infected chinese blood donors, followed by c2. hcc related mutations were found less in pres region but more in prec/bcp region in blood donors. the prevalence of mutations in pres region and a1762t/g1764a mutations was higher in genotype c than in genotype b while the opposite is the case for g1896a mutations. this is consistent with the distribution of hcc related mutations in general hbv carriers in china. since all donors in this study reported not having received hbv treatment, it is not clear whether drug resistance mutations occurred spontaneously in the hbv-infected blood donors or were acquired by the donors from hbv infected patients who underwent antiviral therapy. 2c-s05-01 a fresh look at measuring quality in blood components devine d confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. the assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. however, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. this begins with the manner in which we conduct these tests. although we describe our practice as 'quality control', it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. this testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. in addition, the standards used to assess blood components often have 'wiggle room'. for example, the north american standards for the number of platelets in a whole blood-derived platelet concentrate require that at least 75% of tested products meet or exceed the required platelet count. in practice, this means that up to 25% of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. there are other examples in component quality standards of this same phenomenon. in an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. as a community, we have considerable work to do to get to this utopian ideal. first we must identify better product characteristics to use as standards for blood component production. modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some 50 years ago, yet we have not applied these advances to quality assessment for blood products. those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. production methods are only one way to impact component quality; another is the actual features of the donors themselves. this biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor viii or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. this presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality. background: blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. microparticles (mp) released in packed red cells (pc) in storage have been suggested to be mediators for transfusion-related complications. however, the underlying mechanism for mp release during storage is mostly unresolved. aims: to examine mp released in pc in storage for procoagulant and proinflammatory activity and define the role of residual platelets in pc in generation of mp during storage. methods: (i) leukoreduced (lr) and non-lr (nlr) packed cells (pc) were stored according to blood bank standards and sampled at day 0, 10, 20, 30, and 40. assay of mp was by flow cytometry using mab to label cd235a, cd45, cd41, and cd62e. thrombin generation (tg) and cd11b expression were used as measures of procoagulance and proinflammation, respectively. (ii) the impact of platelets was further evaluated by reconstituting lr pc with increasing concentrations of platelets at day 0. results: (i) multiple species of mp were released in a time-dependent manner. using nlr pc, we found that, relative to day 0, red cell mp (rmp) were increased by 2.59 at day 20, and by 8.59 by day 40. small amounts of mp from leukocytes (lmp), platelets (pmp), and endothelia (emp) were detected, generally <20% as many as rmp. levels of pmp rose rapidly from day 0 and peaked at day 20. lmp began rising at 20 days, increasing to 1.59 at day 30 and 2.49 at day 40. emp changed little over 40 days. (ii) comparing mp in nlr vs lr pc. as expected, pmp and lmp were higher in nlr pc. unexpectedly, however, the rate of rmp production was <50% as fast in lr vs nlr pc. the levels of rmp were found to be significantly associated with residual platelet counts. therefore, we investigated the possible role of platelets in rmp production. (iii) effects of residual platelets on rmp release. when lr pc were incubated with increasing numbers of platelets (0-200,000 per ll), rmp as well as pmp generation increased. rate of increase of rmp was closely associated with platelet count and storage time. this shows that residual platelets catalyze rmp generation. (iv) procoagulant and proinflammatory activities. mp-mediated thrombin generation and cd11b expression increased from day 0 to day 40 in both lr and nlr pc, but in lr pc, it was only 30-50% magnitude of nlr pc. the time course curves did not match any specific mp species. conclusions: procoagulant and pro-inflammatory mp were generated in a timedependent manner. the new finding is that residual platelets markedly augment release of rmp, which is a known indicator for storage lesion. the benefits of leukoreduction may be due to reduction of platelets and mp production in addition to reduction of wbc. reducing platelet count in pc may be beneficial in reducing storage lesion and transfusion related complications. background: it has been reported the soluble cd40 ligand (scd40l, scd154) that was accumulated during platelet storage induce polymorphonuclear leukocyte (pmn) mediated damage of human pulmonary microvascular endothelial cells(hmvecs), was a potential cofactor in developing the transfusion-related acute lung injury (trali). however the amount of scd40l was only slightly elevated in the recipient by transfused blood components as it was fully diluted in the recipient's blood circulation. the mechanism by which cd40l exert its effect is still needs to be elucidated. aim: to determine the effect of platelet derived microparticles (pmp) on pmn mediated hmvecs damage, and its correlation with pmp bounded cd40l. method: the pmps were isolated by centrifugation of the platelet-free plasma from 10 apheresis platelet concentrates (a-plts) at 20,000 g for 1 h. the pmps were counted by flow cytometric analysis, followed by western blotting that was performed on isolated pmps. the scd40l was assayed with elisa. the priming of the formyl-met-leu-phe (fmlp) activated pmn respiratory burst was measured with the hydrogen-peroxide production. a two-insult in vitro model of pmn-mediated hmvecs damage was used to investigate the effect of pmp. result: the pmp priming activity to pmn are correlative with pmp accumulations and their level of scd40l during 5 days storage (correlation was significant at the 0.05 level); pmn respiratory burst are declined by removing pmp with 0.1 lm pvdf membrane filtration or depletion pmp with cd154 monoclonal antibody combining magnetic dynabeads pan mouse igg; the lipopolysaccharides(lps) activated hmvecs were damaged more significantly by pmp isolated from 5-day stored a-plts compared with 1-day stored a-plts (p < 0.05). conclusion: platelet-derived microparticles carry concentrated cd40l signal, promote pmn mediated hmvecs damage, may be relative to developing of trali. background: continuous efforts has spared on improving the quality of platelets harvested from plateletpheresis. little is known about the contribution of donors on the variation of platelet quality, particular the effect of frequent platelet donation on donor's platelet function. aims: aim of this study is to investigate the effect of frequent platelet donation on the state of in vivo platelet activation in platelet donors. material and methods: of 107 whole blood donors and 335 platelet donors with vary donation history from 1 to 74 times (mean at 11.5 ae 12.7) were recruited. they were stratified into three subgroups according to their previous plateletpheresis history (g1: 0-1 time, g2: 2-10 times and g3: >10 times). blood sample were collected from each participant for the determination of plasma levels of soluble p-selectin (sp-selectin), marker of platelet activation and total platelet p-selectin (tp-selectin), as well as platelet count and platelet indices. results: following the increase of donation times, sp-selectin levels were steady increase (g1: 20.50 ae 5.76, g2: 23.21 ae 6.70 and g3: 25.33ae7.67 ng/ml respectly, p = 0.001) and mean platelet volume (mpv) was decrease (g1: 9.29 ae 0.89, g2: 9.17 ae 0.84 and g3: 9.02 ae 0.91 fl respectly, p = 0.039) as estimated by the analysis of covariance adjusted for sex and gender. no significant changes in tp-selectin, platelet count, platelet distribution width (pdw) were observed. further multivariate regression analysis including variables of abo blood groups as well as donation history, sex, age indicated that increased plateletpheresis donations are positively associated with the elevated sp-selectin levels in blood donors (t = 4.16, p < 0.0001). conclusion: our data suggested that frequent plateletpheresis result in the increased level of sp-selectin in platelet donors, implicating a higher state of platelet activation in vivo in frequent platelet donors. the potential effects of frequent plateletpheresis on the quality of platelet harvested and the donor complication are worthy of attention. background/aims: structural and functional changes in erythrocytes occur during ex vivo storage, including the accumulation of bioactive substances in the supernatant of red cell concentrates (rccs). many of the constituents of the supernatant fraction, which are potential mediators of transfusion-related complications, may be reduced by washing of rccs. with emerging paediatric clinical data supporting a beneficial effect of rcc washing prior to transfusion, the aim of the current study was to characterise the effects of rcc age and post-washing storage on erythrocyte structure, function and the accumulation of bioactive substances in paediatric-sized washed rccs. methods: two units of abo/rhd-and age-matched rccs (either 1-or 4-days old; n = 11 each) were pooled and equally split to obtain matched pairs (day 0). one unit was washed with 0.9% saline by repeated centrifugation then resuspended in 100 ml sag-m, while the other remained unwashed. subsequently, both rcc units were divided equally to produce 4 units of paediatric-sized washed or unwashed rccs. all units were stored at 2-8â°c and samples were taken on days 0, 1, 2, 7, and 14 of storage to measure metabolic activity and quality of rccs, as well as the concentration and activity of bioactive substances in the rcc supernatant. the overall effects of washing and storage were compared using repeated measures anova with posthoc paired t-tests as required, with p < 0.05 considered significant. results: the washing process resulted in reductions in red cell count (9.3%), haemoglobin (9.2%) and haematocrit (5.9%) compared to unwashed rccs. overall, washing and subsequent storage of 1-and 4-day old rccs significantly reduced the ph (p < 0.0001), lactate production rate (p < 0.0001), and 2,3-diphosphoglycerate concentration (p = 0.046). although the atp content of the rcc decreased during storage, it was not changed by washing (p = 0.570). haemolysis in the rccs was increased by the washing process, but remained <0.15% on day 14 for all products. extracellular potassium was significantly reduced by washing (p < 0.0001), but increased during storage in both washed and unwashed red cells (p < 0.0001 for both). washing significantly reduced the number of microparticles in the supernatant of both 1-and 4-day-old rcc compared to unwashed rccs (p = 0.01 and 0.012 respectively). however, the microparticle number in the supernatant of all rccs increased during storage. washing of both 1-and 4-day old rcc also markedly reduced the supernatant concentration of monocyte chemoattractant protein-1, scd62p, rantes, anaphylatoxins (c3a, c4a, and c5a) and iga to levels below or near the limit of detection. incubation of cultured human umbilical vein endothelial cells (huvecs) with supernatant from unwashed rcc led to endothelial cell activation, with increased cell-surface expression of e-selectin and vcam (p < 0.0001 for both). however, little or no activation was observed when huvecs were incubated with supernatant from washed rcc. conclusion: although washing affected some aspects of the in vitro quality of rccs, it effectively reduced the concentration and activity of bioactive substances in the supernatant of rccs, leading to reduced endothelial cell activation. such a reduction may be clinically beneficial in selected patient groups. blood services group, health sciences authority, singapore, singapore many of the critical issues associated withbiobanking have been effectively addressed in blood banking. blood transfusion therapy with its emphasison traceability has developed robust systems for inventory and product release. the ethos of proper quality management hasalso been an integral part of blood banking. the lessons learnt have been applied into the biobanking of cord blood, stem cells, tissue and organs. biobanking includes both banking of tissuefor research only as well as public cord blood banks that play a vital rolesupporting clinical stem cell transplantation. the growth of regenerative medicine will only increase the scope, variety and numbers in biobanking. similarly, the discovery of induced pluripotent stem cells (ips) and itspotential myriad applications has highlighted the central role of biobanking inboth diagnostics, research and therapeutics. principles and key considerations in biobanking including biospecimenprocurement, consent, processing, preservation and traceability will beaddressed. introduction: cell therapy generally includes the extraction, processing, manipulation and implantation of characterized cells effectuating specific functions in a patient. however, adjacent fields like tissue banking or tissue engineering should be incorporated. all together have donor selection and validated core procedures in common. production cycles are carried out in gmp clean rooms. furthermore, quality control includes assays which are common in transfusion medicine. it might be tempting to speculate, that cell therapy is closely related with transfusion medicine and requires minor adaptions. the big moat surrounding cell therapy: but there are huge differences: cell therapy is a domain of specialists rooted in patient care with profound knowledge about specific pathologies and how to target them. cell therapy derives from individual clinical needs and rarely is a prefabricated procedure. this is in stark contrast to transfusion medicine, which focuses on standardized products for any patient in need. today, complex regulations for cell therapy surpass those in transfusion medicine. this distracts clinicians in a cell therapy program. this may aspire transfusion medicine specialists to engage in cell therapies. however, only a small niche is left for blood centers, as two major trends arise: one is the more or less 'academic gmp' setting, utilizing the hospital exemption status. the other is the commercial arena, where companies produce standardized cell therapies to achieve maximized market shares. 'academic gmp' entities promote individualizedmainly autologoustherapies, which require a constant flow of financial assets to keep underused clean rooms running. therefore it is serious to ask why and where blood centers should engage in cell therapies. strategy first: transfusion medicine has been heavily influenced by external factors such as viral safety, blood usage, cost pressures and low resources. the term 'transfusion medicine' misleads outsiders to suppose, that it deals with transfusions, nothing else. a wise strategy must include a change in the mind-set of all. this is the most crucial issue as many clinicians are needed to collaborate and co-develop cell therapies. without deeply rooted partnerships it is impossible to establish sustainable cell therapy programs in a blood center. furthermore, a thorough evaluation includes possible products, quantities, as well reimbursement schemes. cell therapy is one of the most expensive treatments and financial assets must be secured first. second: establishing a cell therapy facility: a mock-up facility, without clean room status is highly recommended. processes will be developed, staff is enabled to learn and define procedures, before a cell therapy unit is planned. planning and establishing a cell therapy unit is very complex and specific expertise is scarce. a basic prerequisite is a project manager carrying out final decisions with a profound knowledge about processes interacting with different technologies (hvac, controls, microbiology). cell therapy units fail if project management has flaws and deep involvement of engineering with medical expertise is ignored. a cell therapy unit is an endless, stressful, path riddled with expensive failures, but rewards in the long run a blood center with exciting future prospects integrating grateful clinicians and patients. any kind of accreditation, either by regulatory authorities or any professional entity, like jacie/fact, is an important milestone in the time line of a new cell therapy and a possible showstopper of a long, expensive and enduring project. honest and thorough preparations before accreditation should start before an application may be sent. three phases are distinguishable: keeping the basics on track: running a cell therapy unit is a high wire task. fundamental knowledge about the medical background, processes, quality control, specifications is interwoven with engineering and controlling tasks. it is inevitable for anyone working in this environment to know about air quality, hygiene, staff education and additional technical features. especially controls and the design of engineering and its qualification should be documented continuously. maintenance, re-qualifications, adjustments in the control-system of a clean room facility offer chances to learn the interplay of systems. build a sufficient knowledge base and freeze the process: protocols for cell therapy are often introduced on primary events and work well in first shot experiments. as further patients are included it is very probable, that the whole process and specifications will be modified and sometimes fundamentals and documentation are out of focus. altering and scaling methods, processes and assays may or may not change the product or its intended use. slippage is often not detected and therefore first steps aim at the build-up of as much information as possible. firstly, current literature has to be collected, reanalyzed and mirrored onto the own processes. then the regulatory framework has to be analyzed. the main question is, how the product fits into the regulatory system. is it an atmp or non-manipulated cell product, is it a blood product or a pharmaceutical? pros and cons about alterations should be meticulously discussed and alternative procedures highlighted in this phaseespecially those which are already accredited. it will be very probable that the same inspectors, who have inspected similar cell therapy units, know about alternative procedures and will raise comparative questions. after building up the knowledge base it is advisable to finish a risk assessment focused on the intended use in patients and to revamp the process. after adjusting all methods, processes and assays the whole production has to be frozen. further changes are prohibited and the documentation has to be refreshed. the last test trial: in the last phase before accreditation all aspects of quality management have to be finished. risk assessments should focus on the safety and effectivity of the cell therapy. donor/patient eligibility criteria, quality control of incoming cells and tissues, production processes and their internal quality control criteria as well storage conditions have to be well documented and validated. under certain circumstances a file of clinical studies has to be prepared. all documents and clinical studies should be reviewed, regulatory aspects should be clear. a preparation project gives better chances to pass the last milestone before patients can be treated on a regular cell therapy. 2d-s07-01 burnouf t plasma fractionation is a complex biotechnological process exhibiting unique features compared to downstream technologies used for recombinant proteins, vaccines, and animal-derived antisera. in human plasma fractionation, by contrast to other biological products, multiple end-products (typically 5-10 or more) are obtained from the same manufacturing pool. some of the targeted proteins are present in plasma in large amounts, as is the case for albumin and immunoglobulin, whereas, by contrast, others, such as coagulation factors and anticoagulant proteins are in trace amounts. with the emergence of selective hemotherapy, plasma fractionation has, over the years, turned into a highly integrated protein separation process carefully designed to isolate various proteins under optimized conditions of yield and purity. current plasma fractionation methodologies combine diverse protein purification tools based on 'crude' precipitation techniques and refined chromatographic procedures. some proteins are stable and not prone to degradation, while others, with specialized functional activity, are fragile and prone to enzymatic degradation, activation, or aggregation. contamination of plasma products with harmful residual plasma protein impurities (such as activated coagulation factors or proteases) can lead to serious adverse events in some patients. in addition, while a few plasma products, like albumin and immunoglobulin, can be formulated as liquid preparations, all others products are freeze-dried to ensure long-term stability, which increases cost and technical difficulties. the diversity of protein products made from plasma explains why plasma fractionation facilities have complex design. the manufacturing lines of the various fractions should be strictly segregated from one another. in addition, the risk of viral contamination of plasma pools requires that each product be subjected to several (typically two or more) dedicated viral reduction treatments, the goal being to gradually increase the degree of viral safety along the downstream process. production zones should therefore be physically segregated to limit risks of cross-or downstream contaminations, adding to the complexity of the plant design, flows of product, personnel and wastes, and working procedures. the plasma fractionation industry has a long history from the years 1940's, when cohn and co-workers developed a sequential cold ethanol precipitation process. this method which evolved over the years, remains the core fractionation process, albeit integrated with cryoprecipitation and multiple chromatographic steps. combined with modern viral removal treatments, the current fractionation process ensures therapeutic protein products of established quality and safety, at more or less acceptable yields. the current safety record of plasma products, which contrasts with that of earlier product generations, somehow represent an impediment to the emergence of new fractionation technologies. novel plasma fractionation processes based on integrated chromatographic steps, membrane electrophoresis, aqueous two-phase system, mini-pool fractionation in disposable equipment, are being developed at pilot-scale and represent interesting alternatives. to reach the market, such technologies should be integrated with robust viral reduction steps and proven to achieve at least equal, if not superior, products yield, quality, safety, and productivity to justify the regulatory load, clinical trials, and licensing of what would be regarded by most regulatory agencies as new plasma products requiring full validation. effective specific antiviral agent is generally not available for emerging infectious disease agents such as sars-coronavirus and middle-east-respiratory-syndromecoronavirus. passive immunotherapy with plasma or plasma derived hyperimmune globulins have been used for treatment or prophylaxis against many exotoxin mediated bacterial or viral diseases such as viral hemorrhagic fever and 1918 influenza despite the lack of data from randomized control trial. antigenically shifted influ-enza a virus causes pandemics and antigenically drifted viruses are associated with seasonal outbreaks. poultry to human transmission of avian influenza a h7n9 and h5n1 virus can cause acute community acquired pneumonia with 25 to 50% mortality. risk factors including extremes of age, pregnancy, underlying medical illness and low serum igg2 are associated with severe pneumonia with delayed clearance of viral load and excessive proinflammatory response. though treatment with neuraminidase inhibitors within 48 hours after onset of symptoms should be effective, those with severe disease and respiratory failure usually present later than 5 days after symptom onset. during the 2009 pandemic of h1n1 influenza, we harvested convalescent plasma from a small percentage of recovered adults sufficient for a case control study for treating severe cases during the pandemic in hong kong. plasma supply is constrained by plasmapheresis capacity during most stages of the epidemic. between august 26 to october 31, 2009, a total of 9101 persons were successfully contacted. a total of 1309 screening and 619 whole blood donation appointments were made. in the former 786 (60.0%) attended screening but only 301 could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. for those who opted for whole blood donation, 379 (61.2%) had attended and donated. 10.5 l (21 units) and 276 l of convalescent plasma with sufficient neutralization antibodies titers was collected for passive immunotherapy as convalescent plasma or h-ivig production respectively. we recruited 93 patients with severe h1n1 2009 infection already put on neuraminidase inhibitors and requiring intensive care. twenty patients (21.5%) received convalescent plasma. mortality in the treatment group was significantly lower than the demographically matched control nontreatment group (20.0% vs 54.8%; p = 0.01). convalescent plasma treatment was associated with significantly lower day 3, 5, and 7 viral load, compared with the control group (p < 0.05) and corresponding lower serum levels of il6, il10, and tnf (p < 0.05). between 2010 and 2011, 35 patients were randomized to receive h-ivig (17 patients) or ivig (18 patients). no adverse event related to treatment was reported. serial respiratory viral load demonstrated that h-ivig treatment was associated with significantly lower day 5 and 7 posttreatment viral load when compared to the control (p = 0.04 and p = 0.02 respectively). the initial serum cytokine level was significantly higher in the h-ivig group but fell to similar level 3 days after treatment. subgroup multivariate analysis of the 22 patients who received treatment within 5 days of symptom onset demonstrated that h-ivig treatment was the only factor which independently reduced mortality [or:0.14, 95% ci, 0.02-0.92; p = 0.04]. background: in recent years, with the global spread of the west nile virus (wnv), dengue virus (denv) and chikungunya virus (chikv) that are transmissible by mosquitoes, concern has arisen regarding their entry to japan. furthermore, chikv as well as wnv and denv are potentially transfusion-transmissible, posing a serious risk for transfusion medicine. of these, wnv and denv belong to the flavivirus genus, as does the japanese encephalitis virus (jev), and they have similar antigenicity. since most japanese people are periodically vaccinated against jev, there is a possibility that anti-jev antibodies cross-react with wnv and denv and induce a protective immune response. however, because wnv and denv have similar antigenicity to jev, there is concern that the anti-jev antibodies are present in japanese plasma against wnv and denv owing to antibody-dependent enhancement (ade) in fccr-expressing cells. furthermore, if the anti-jev antibodies present in japanese plasma have high ade activity, these antibodies may act in an infection-enhancing manner rather than an infection-neutralizing manner against wnv and denv in vivo. aims: using intravenous immunoglobulin (ivig) prepared from pooled plasma from japanese donors, we evaluated its neutralizing activity and ade activity against these viruses for the purpose of estimating the potency of the japanese individuals to protect themselves against these viruses. methods: neutralizing activity (tcid 50 ) and ade activity were compared among three types of ivig, nisseki polyglobin n (made in japan), gammagard (made in germany) and sanglopor (made in the usa). tcid 50 was calculated from the results of cytopathic effect (cpe) assay using vero cells as target cells. ade activity was measured by plaque assay using bhk cells and fccr-expressing bhk cells as target cells. results: nisseki polyglobin n showed a significantly higher neutralizing activity against jev than gammagard and sanglopor. the neutralizing activity of nisseki polyglobin n against wnv was approximately a log reduction factor of 2.3 higher than that of sanglopor. furthermore, the neutralizing activity of nisseki polyglobin n showed approximately the same neutralizing activity as gammagard against wnv. none of the ivig preparations showed significant neutralizing activity against denv or chikv. nisseki polyglobin n showed only marginal ade activity against any of the viruses. conclusion: although the neutralizing activity of plasma from japanese individuals is not known, it is suggested that the japanese population as a whole has a potency to protect themselves from infection by wnv to some extent, probably due to the cross-reaction of anti-jev antibodies to wnv as a result of jev vaccination and natural infection. therefore, if wnv invades japan, a pandemic may not occur and the risk of wnv infection by blood transfusion may be low. it was suggested that plasma from the japanese individuals has almost no neutralizing activity against denv and chikv. nisseki polyglobin n showed only marginal ade activity against wnv and denv, suggesting the low possibility of the anti-jev antibodies present in japanese plasma acting as infection-enhancing agents against wnv and denv as a function of ade. background: platelet-rich-plasma (prp), platelet gel (pg), and platelet lysate (pl), are used in regenerative medicine to stimulate the healing of soft and hard tissues. in addition to their tissue regenerative properties, platelet materials are claimed, mostly through anecdotal observations, to limit post-surgical inflammation and decrease pain. the anti-inflammatory properties are thought to be due to the release of platelet components, including transforming growth factor-b1 (tgf-b1) and hepatocyte growth factor (hgf), which are known to inhibit some inflammatory pathways in vitro. however, there is a large diversity in the type of platelet fractions used in clinics. they differ significantly in protein composition and content of proinflammatory and anti-inflammatory molecules and may therefore not be equally effective in controlling inflammation. one needs to elucidate the factors responsible for the possible anti-inflammatory properties of platelet materials to standardize the preparation and clinical use of these products when an anti-inflammation effect is clinically beneficial. aims: to investigate the potential anti-inflammatory effect of various plasma/ platelet fractions using an established in vitro model of raw 264.7 mice macrophages stimulated by lipopolysaccharide (lps), and studying the production of nitric oxide (no), inducible nitric oxide synthase (inos), and cyclooxygenase-2 (cox-2). methods: apheresis platelet concentrates (pc) were obtained from three donors and separated within 3 days into pc, platelet poor plasma (ppp), platelet gel releasate (pg), frozen-thawed platelet lysate (pl), and solvent-treated platelet lysate (s/d-pl). proteins were determined, sds-page patterns obtained, and growth factors quantified by elisa. raw 264.7 cells were grown in medium supplemented with 10% of fetal bovine serum (fbs) or plasma/platelet fractions, with or without lps (500 ng/ ml added after 24 h of culture). cell growth and cytotoxicity were checked by cell count determination and mtt assay. no was determined in cell culture supernatants by colorimetric assay and inos and cox-2 in cell extracts by western blot. prp from mice and quercetin, a known anti-inflammatory compound, were used as controls. results: pc and s/d-pl had the highest mean tgf-b1 content ranging from approximately 100-200 ng/ml, and ppp the lowest (5-10 ng/ml). cell count analysis and mtt assays showed consistent cell growth and viability in all conditions evaluated but were slightly lower in the presence of lps and quercetin, as expected. there was no no, inos, cox-2 detected in absence of lps stimulation. the plasma and platelet fractions were all found to reduce no production and inos expression, when compared to fbs, after lps stimulation. interestingly, inhibition of no production and inos was generally more pronounced with s/d-pl. cox-2 synthesis was lower in the presence of s/d-pl compared to other plasma/platelet fractions and higher with pg. the mice prp did not exert any stronger anti-inflammatory action in this mice cellular model suggesting absence of species specificity. conclusions: the plasma and platelet fractions evaluated exert, to various degrees, an anti-inflammatory effect mostly revealed by inhibition of no production and inos. impact on cox-2 synthesis is less obvious. the fact that s/d-pl exhibits stronger global anti-inflammatory activity requires further studies. 2d-s08-01 tani y japanese red cross kinki block blood center, ibaraki, japan rare blood is generally defined as one that occurs at a frequency of 1:100~1000 individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. the japanese red cross (jrc) society lists 46 rare blood phenotypes that are divided in two categories as shown in table 1 . the rare blood types listed in category i occur much less frequently than those listed in category ii. we screen for rare blood cells using monoclonal antibodies (moabs). since 1987, our blood center has established 93 moabs (32 human and 61murine), and has provided them to the other blood centers. many of igg moabs are available on the machine such as beckman coulter pk-7300 blood grouping analyzer by saline or bromelin method by cross-linking with anti-human or anti-mouse igg. in addition, we routinely screen for antigen negative-cells (rhc, c, e, e, jk a , jk b , fy b , di a , m, le a , s to which antibodies are believed to be clinically significant). thus, more than 10,000 donors with rare blood phenotypes (category i, 748; category ii, 9314) are registered in japan, but the number of donors with some category i blood types are insufficient. we freeze rare blood, particularly category i types, and domestically and sometimes internationally supply these units of blood. since 1977, a total of 576 units of rare blood with phenotypes di(b-), d-, jr(a-), ko, jk(a-b-), (para-)bombay, p, en(a-), m k m k , lan-and rhnull have been supplied to 23 international countries. thus, the jrc contributes to the international panel of donors of rare blood type (idp) which is maintained by the international blood group reference laboratory (ibgrl) in bristol, uk. the idp provides information on the location of rare blood donors when they cannot be found in their respective countries. the jrc has encountered difficulties in finding rare donors with rhnull, p k , m k m k , en(a-), u-and ge-phenotypes. we also joined the isbt working party on rare donors which handles all matters related to rare blood. our rare blood donor program is successful because of international cooperation. 2d-s08-02 the china national rare blood group screening program started in 2008. the program has screened more than 1,500,000 donors in thirteen regional blood centers by using large-scale serological tests, gene diagnosis, and other different specific screening technique. now, more than 1300 donors with rare blood phenotypes have been found. including rh null, d --, wrb-, lu(a-b-), jk(a-b-), vel-, lan-, coa-, k 0 , dib-, s-as well as yta-. the primary target for the project is to screen the negative blood antigens whose conjugational antigens have high frequency, and the blood groups which is easy to produce antibodies and the negative antigen in the system is very rare in chinese population (for example, fya-, whose frequency was 1/400 in chinese han population). a professional website for rare blood groups (http://www.chinarareblood.cn)was set up and serviced in jan, 2010. the information of blood donors with rare blood types is registered into the national registering system for blood donors with rare blood types by professional technician from organizations join the program. the information includes the specificity of the rare blood types, other common blood groups, personal data of donors, and the information of contacts. except the network administrator, other visitors could only see the specificity of the rare blood type and the number of the rare blood in rare blood bank storage. application for the rare blood products should communicate with contacts through administrator. according to the standard of the pretransfusion test in china, all the donors and recipients must be identified the abo and rh systems and done the cross-match test, in addition, all the donors must pass the test for contagious marks. nowadays, more and more chinese blood centers use nucleic acid technique to detect hiv and hcv. the results of infection test must be added to the information of the blood products at every time for collecting the rare blood. in recent 2 years, 21 units (1 unit = 200 ml) blood products with different rare blood types have been used in clinical treatment. background: anti-emm is a rare specificity detecting the high-prevalence red blood cell (rbc) antigen emm (isbt 901008). five cases were reported by daniels et al, (transfusion, 1987; 27:319) and two by reid et al, (transfusion 1998; 38 (suppl) :101s). six of these were in untransfused males and anti-emm had not been implicated in a transfusion reaction, most likely because the patients were not transfused. case study: a 58-year-old, untransfused, japanese man with group a, d+, datnegative rbcs, urgently needed transfusion due to an abdominal stab wound. pretransfusion testing using gel column agglutination and peg-iat, demonstrated an antibody reactive with all panel cells, but non-reactive with autologous rbcs; anti-le a was detected by papain methods using gel column. unavoidably, one bag of crossmatch-incompatible le(a-) rbcs had to be transfused. thirty minutes after transfusion, he experienced a drop in blood pressure and hematuria. however, as his hb level was 5.5 g/dl, another two rbc bags were transfused, and his vital signs became stable. on day 3, he was transfused one rbc bag without a hemolytic transfusion reaction. on day 6, after receiving 30ml of rbcs, the patient vomited and had cola-colored urine (t-bil 6.1 mg/dl, ldh 912 u/l) and the transfusion was stopped. following transfusion, his rbcs reacted in the dat: 1+ on day 1, 2+ on day 3 and negative on day 7. thereafter his anemia improved gradually by iron medication and he was discharged on day 24. aims: to identify the antibody in the patient's plasma that caused the transfusion reaction. methods: serological testing was performed by standard methods. result: the patient's plasma reacted in saline at 4c (2+), by albumin iat (2+), peg-iat (2+), and papain-iat (3+) with all panel cells, and by peg-iat with 30 samples lacking high-prevalence antigens; the autologous rbcs were non-reactive. testing his plasma against phenotypically-similar enzyme or chemically treated rbcs showed that the antigen detected was resistant to bromelin, papain, ficin, trypsin, a-chymotrypsin, pronase, dtt, aet, and acid. two examples of emm-rbcs were non-reactive and the antibody was identified as anti-emm. the reactivity of enzyme and chemically treated rbcs is consistent with anti-emm. his rbcs reacted with antibodies to 17 high-prevalence antigens, but could not be confirmed as emmbecause anti-emm was not available. the anti-emm was igg1 and igg3 with a titer of 16 by peg-iat before transfusion rising to 128 on day 10; his serum demonstrated hemolysis after day 6. no other underlying antibodies were detected in the patient's plasma alloadsorbed to remove the anti-emm. conclusions: we report the first japanese case of anti-emm and the first to cause an acute hemolytic transfusion reaction (ahtr). in previous reports, six people with anti-emm were untransfused men and one was in a woman whose transfusion history was unknown, suggesting that the antibodies may be 'naturally-occurring'. the anti-emm reported here, also in an untransfused man, also may be considered a 'naturally-occurring' antibody. similar to the first reported example of anti-emm, the antibody reacted, albeit weakly, at 4c. our case suggests that anti-emm is clinically-significant as the patient experienced an ahtr. 2d-s09-01 background: to recruit blood donation volunteers and provide stable blood supply according to demand, it is more important to change the overall social perception than to carry out one-time event or short-term campaign. the social understanding of blood donation is formed as valuable and honorable service over certain level in korean society. nevertheless, there still are many people who don't even try to participate in blood donation because of fear, health concern, and expectation for reward. to change this culture and social awareness, it is important for the present and future blood donors to have a perception that the blood donation is the sharing one's life and a genuine service which helps other people for nothing. aim: the main purpose of this article is to introduce korean red cross' recent efforts (operation of the red campaigner and blood donation supporters, the construction of virtual blood donation experience center, the blood donation promotion program) to change the blood donation culture and widen the donor base in korea and to present their effect and improvement direction. (1) this article is comprised of the case study and analysis on the operation of red campaigner and blood donation supporters, the construction of virtual blood donation experience center and blood donation promotion program (2) this article outlines the red campaigner and the blood donation supporters and the related programs and addresses their significance in addition, describes the effect and direction for improvement, presenting research related data. (3) this article outlines virtual blood donation experience center construction and presents the exhibition in it and it suggests the effect and possibility on the authority of the research case that the education with fun has more considerable impact than learning by rote. result: to change current culture and increase positive understanding about blood donation, korean red cross is making various efforts. the red campaigner, consisting of high school students and the blood donation supporters, consisting of college students, aim to influence the youth, the potential blood donors, to have a positive understanding of blood donation and to carry out continuous and organized publicity of its importance and safety. in addition, korean red cross is making a progress in the construction of the virtual blood donation experience center which is going to be completed by the end of 2013. we expect that we can achieve the educational purpose that sends a message that the blood donation is a volunteer work to save life and make future donors to recognize the blood donation as an object of not fear, but interest. finally, 'the blood donation promotion program' which began in 2009 is designed to encourage for general groups or organizations to participate in the blood donation campaign and to create the voluntary blood donation culture. conclusion: various projects operated by korean red cross contribute to widen blood donor based by changing blood donation culture in korea and are expected to make continuous contribution. but these projects have a limitation that the partici-pant is restricted and continuous participation isn't progressing in terms of national participation. continuous and positive endeavor to foster these projects as a national campaign should be encouraged. although it is possible to increase the blood donor temporarily through one-time event when blood shortage recurs, widening the donor base by changing blood donation culture should be the fundamental solution. the changing blood donation culture and donor understanding may not be optimistic for a short-term blood shortage problem but will be useful to make conditions that expand the donor base and increase voluntary donors in the medium to longer term. understanding our future donors is of critical importance to blood collection agencies worldwide. not only do we need to know who they are, but also why they do or do not engage in the required behaviour of blood (product) donation. the surge in psychological research into blood donation in the last decade has provided significant insight into understanding the psychological factors underpinning the commencement and continuation of blood donation. this review will summarise the state of our current understanding of knowing how to effectively recruit the non-donor and the complexities that lie ahead for all involved. at the descriptive level, and stemming from the systematic application of various psychological theories and models within blood donation contexts, we now know more than ever about the key factors that non-donors report impact on their blood donation intentions and behaviour. from this, certain psychological elements such as perceived control or self-efficacythe individuals' self perception that they can cope with donationhave emerged as key determinants of donor recruitment. drawing on these results, research psychologists have worked collaboratively with blood collection agencies to develop and evaluate recruitment materials designed to target these central constructs. both laboratory and in field trials have been undertaken which have consistently shown positive effects. for example, participants receiving these materials are more likely to volunteer to donate blood than those receiving standard recruitment materials. the collaboration of researchers and blood collection agencies has furthered understanding and recruitment efficacy generating measurable, operational deliverables. however, this collaborative research has also served to highlight the challenges that lie ahead both in terms of the diversity of our non-donor population as well as the limitations of our current theoretical models. further, there is an increasing need for large scale randomized controlled field trials to systematically evaluate interventions designed on the basis of psychological research. while these needs may provide substantial challenges for researchers and blood collection agencies alike, the promise of psychology in providing the 'who' and 'how' to effectively recruit remains. background: developed countries rely solely on voluntary non-remunerated donors to ensure adequate blood supply but ageing has brought in significant pressure on the health care system including blood supply. in hong kong, blood demand has recorded a 17% increase in blood demand from year 2008 to 2012 with almost 60% blood being transfused to patients aged >60. therefore, sourcing for more blood to meet demand is one of the most urgent issues in blood service. in this report, we report how we successfully modeled donation preference into the development of a new collection site to leverage the blood demand. material and methods: demographic information of all donors with successful donations was retrieved from blood bank computer system for the years 2004-2007. statistical analyses were performed to determine how frequent they came back to donate, whether residential location affected their donation frequencies and lastly identify potential district in hong kong to build a new donor center against the donor and general population distribution. results: of 775,690 donations made by 191,302 male and 201,446 female donors were analyzed. on average 75.8% of donors donated only once during the calendar year but donors were more likely to make repeated donations at donor centers than mobile venues. significantly more donors would come back for second or more donations at donor center (36.1% vs 20.1%, p < 0.05). male were more likely to come back for repeated donations than female (43.3% vs 30.9%, p < 0.05). a total of 466,121 donations made at donor centers were further studied. upon matching with their residential address, distance from donor centers was shown to be a determining factor on their choice of donation through linear regression analysis. reduced donation frequencies were observed with increasing distance from donor centers. regression analysis then identified several districts where many donors had to travel a long distance to the nearest donor center. the district with the highest expected increase in donations, adjusted by the expected growth rate, was then chosen as the site for building the new donor center. a fixed donor center was so selected at yuen long district and open to donors by 29 july 2011. by the end of year 2011, 6160 donations (consisting of 3047 males and 3113 females) were made with more than 88.1% collection given by donors residing at yuen long and the nearby district, tuen mun. when the whole year figure was reviewed in 2012, 16,990 donations were collected which already exceeded the planned collection target of 15,000 by year 3. interestingly, same 88.1% donations were given by donors residing at these two districts. conclusion: despite being a small city, the retrospective analysis of donation behavior has provided valuable information in the service planning in hong kong. the new donor center was able to reach the planned third-year collection target by 15 months earlier. further work is being done by using the more recent data to identify the next optimal collection sites for future expansion under most best cost effective way. singapore red cross, singapore, singapore background: singapore is moving towards providing more fixed blood donation sites with the aim of enhancing donation experience and encouraging repeat donations. it was recognized that the choice of location and an understanding of the human traffic in the vicinity are elements of success. a 2-prong approach was undertaken to: collect information on the footfalls, the social profile and demographics in the designated location. collect information from potential donors on their preferences and sentiments in relation to operating hours and outreach channels for marketing communications. method: a month-long study was conducted in the vicinity by observing and counting the human traffic, the crowd density at various exit/entry points at various timings, the flow and direction of the general foot traffic, overall make up of the surrounding district such as types of corporate, civic & religious organizations operating in the vicinity and number of educational institutions. in addition, an on-site survey to determine potential donor preferences and sentiments in relation to operating hours, tactical outreach and design mechanism of the blood centre was also conducted to help develop a tactical marketing communications strategy. the results indicated that during week days, about 85% of people visiting that vicinity were youths aged 16 to 25 (50.5%) and young adults aged 26 to 39 (33.4%). the main purposes of the visit were for work related activities (22%), attending school (19.1%) and shopping (38.1%). on weekends, 76% of these age groups visited the vicinity for leisure activities or to church. another 24% who visited there were adults age 40-60 years old. most of the respondents had a specific destinations, and most of them indicated lunch time and after office hours as their preferred donation periods. a tactical marketing communications strategy targeting youth and young adults was developed to meet the behavior and preferences of the target group. social media platforms such as online mobile app advertising and locational media buys were employed. in addition, partnerships were developed with nearby educational institutions and churches to host road shows and blood donation awareness activities to engage youths and to foster fun and excitement in the social media atmosphere for the blood collection center. the strategies undertaken proofed favorable as the daily attendance at the new blood collection center has surpassed its baseline target collection of 50 units of blood a day within the 3 months of its operation (jan 2013); and now, has a daily average collection of 60 units of blood. 3a-s10-01 setting up haemovigilance programme from the very first step background: national haemovigilance programmes wherever established have yielded significant data to implement blood safety initiatives. settting up a national haemovigilance programme requires meticulous planning and the following issues need to be addressed; whether reporting will be voluntary or mandatory, what is to be reported and by whom, reporting formats and data submission, resources to sustain the programme, staff training and responsibilities. india is a country of 1.18 billion people, 2700 blood centres, one-third each in public, charitable and private healthcare sectors and annual blood collection of 9 million. given the diversity of blood centre management, setting up such a national programme is a complex task. aim: to set up a national haemovigilance programme in india. method: the ministry of health and family welfare (mohfw), govt. of india had launched the pharmacovigilance programme of india (pvpi) in july 2010, with oversight by the indian pharmacopoeia commission (ipc). adverse drug reaction (adr) monitoring centres were setup in 90 medical institutions in the country and trained staff was recruited, for data collection and submission. after the successful launch of pvpi, the mohfw, initiated the haemovigilance programme, distinct from pvpi with the co-ordinating centre at national institute of biologicals (nib), but in close collaboration with ipc. the broad organizational structure of the programme is as follows; reports will be generated in the medical institution based blood centressubmitted online to the haemovigilance national co-ordinating centre at nibreports will be reviewed by the national advisory committee which will make recommendations to the national co-ordinating centre, ipc for onward transmission to the regulatory authoritythe central drugs standards control organisation to formulate safety related regulatory changes and communicate to blood centres. the programme was formally launched in december 2012. terms of reference of the national advisory committee are: to finalize transfusion reaction reporting form (trrf) for the country. to give expert opinion for collection, collation and analysis of data and development of software for the same. to monitor the quality of data collected. to develop training modules and guidelines for implementation of haemovigilance programme. results: based on recommendations of the committee, the initial focus is on reporting serious adverse reactions as defined by the working party of the international society of blood transfusion, reporting is voluntary and a guidance document and trrf have been made available to the medical institution blood centres, which are the designated reporting centres. the reporting is online through a software -haemo-vigil accessible on the nib website. each centre has been given a unique username and password for login. the security of data submitted through the software has been validated. reporting commenced from february 2013 and till date 434 reports of adverse reactions have been submitted. the data is yet to be analysed. a series of awareness workshops are planned countrywide, five have already been organized. specific funds have been allocated by the mohfw for this programme. conclusion: a well structured programme of haemovigilance has been initiated in india and is now in a phase of development. 3a-s10-02 background: congenital haptoglobin (hp) deficiency being homozygous for a deleted allele of the hp gene, hpdel, was identified in a japanese pregnant woman who had experienced severe anaphylactic transfusion reactions (trs) after infusion of red blood cells (rbcs) and human serum albumin in 1998. in addition, the allelic frequency of hpdel was calculated to be 0.015 by a genetic study of a limited number of the japanese individuals, suggesting that hp deficiency might distribute among the japanese population as a phenotype of serum hp. aims: in this report, we present the results obtained from a hemovigilance survey carried out between 1998 and 2012, in which hp deficiency was identified among japanese patients who had experienced nonhemolytic trs (nhtrs), and those obtained from a screening of hp-deficient japanese healthy blood donors. materials and methods: patients with nhtrs: a total of 19,675 patients who had experienced nhtrs, reported by hospitals to the japanese red cross society between january 1998 and december 2012, were examined. healthy blood donors: a total of 272,068 blood donors who visited the japanese red cross blood centers in the tokyo area between june and august 2010 were examined. testing for identification of hp deficiency: (i) serum hp concentration was determined using peak-rate nephelometry with the detection limit of 6 mg/dl followed by simplified sandwich elisa with the detection limit of 3 lg/dl. individuals who showed a negative result of elisa were assessed as hp-deficient. (ii) the presence of the hpdel allele was analyzed using an allele-specific pcr method. testing for anti-hp antibody: the anti-hp antibody produced in all the hp-deficient individuals was measured by elisa and western blot analysis. it was further analyzed using ouchterlony or surface plasmon resonance technology in some cases. results: thirty-one individuals were identified as hp-deficient among the 19,675 patients who had experienced nhtrs (0.16%). all the patients, except three who could not be tested, were homozygous for the hpdel allele. they were transfused blood products [pc, 17; ffp, 1; rbcs, 9; mixed, 4] . nineteen of them (61%) experienced anaphylactic trs accompanied by severe hypotension and the other patients (39%) experienced milder nhtrs. all the patients except one had a history of transfusion. the anti-hp igg antibody was detected in 28 patients (90%). in addition to the igg antibody, the anti-hp ige antibody was detected in 20 patients (64%). hpdeficient individuals were identified among japanese blood donors with a prevalence of 105/272,068 (0.039%). all the donors were homozygous for hpdel, except one who was heterozygous for hpdel, hpdel/hp2. the anti-hp antibody was not detected in 104/105 (99%) of the hp-deficient donors. conclusions: the higher prevalence of hp deficiency caused by hpdel -homozygosity producing the anti-hp antibody among the patients with nhtrs than in the normal donors (p < 0.001), suggests a higher risk of such trs in hp-deficient patients. hp-deficient individuals are present among normal healthy donors with a prevalence that is expected from its allele frequency. they might be expected as suitable donors for hp-deficient patients to prevent hp-related anaphylaxis. hlaing aa background and objective: collection of the correct blood sample from the correct patient is a vital step in the process of safe blood transfusion. transfusion laboramethods: a study on all reports of mislabeled and miscollected specimens from january 2010 to december 2012 was undertaken and the results were analyzed. mislabeled samples were defined as samples that were incorrectly labeled at the time of collection and miscollected samples were 'wrong blood in tube' samples due to patient misidentification. errors resulting in discrepancies in blood group between the current blood sample and historical records were identified by program flagging during validation of blood group results. these discrepancies were resolved by requesting a second sample from the patient collected by another person. some errors detected at the ward level, were reported by the staff member who had sent the blood sample. workload data for group and screen samples received during 2010-2012 was collected from the annual transfusion laboratory records. using these data, ratio of errors from mislabeled and miscollected samples, to number of group and screen samples received was calculated. results: between january 2010 and december 2012 a total of 162,999 samples were received for abo grouping. of 77 incidents were recorded relating to errors in either sampling or labeling. the overall sample error rate was 0.047% or 1 in 2116 samples. of 52 cases resulted from wrong labeling during collection, 17 cases were due to patient misidentification, five were errors that had occurred during the initial request, in two incidents the cause could not be identified and one labeling error occurred in the laboratory. all errors in labeling resulted from failure to check the pre printed name label with the label on the patient's identity band. in 14 incidents, labeling was performed away from the bedside, in 11 cases name labels of a different patient were found in the correct patient's medical file, in 25 cases labels were taken from wrong patient's file and two errors were due to using prelabeled tubes. of 17 patients had been misidentified and blood taken from the wrong person. root cause for these errors was not following hospital polices in patient identification and sample collection. sixty-four percent of the errors occurred out of normal working hours, mainly during the night while the rest 36% had occurred during normal hours. conclusion: we conclude that mislabeling and miscollected sample errors represent a potential for mistransfusion in our institution. the rates of mislabeled and miscollected samples can be used as key performance indicators in this important step in the clinical transfusion process. this baseline data will be used in formulating standards of performance for sample collection and patient identification and, for implementing risk -reducing strategies. background: haemovigilance is a surveillance programme dedicated for the practice of blood transfusion. it is an important part of the quality system of the blood programme. haemovigilance programme in malaysia was initiated as a national programme in 2003 under the ministry of health (moh). since its inception in 2003 the programme has evolved and has become an integral part of our transfusion service. all adverse events and near misses were reported to the national coordinating haemovigilance unit at the national blood centre (nbc) using a standardised form and predefined criteria. these were compiled and analysed into an annual report for the national background: the process of blood transfusion from blood collection and processing to issue and bedside transfusion of blood components involves several areas of human participation. human error is therefore inevitable in this chain of events. transfusion laboratories have long focused their attention on quality control methods and quality assessment programmes dealing with analytical aspects of blood testing. however, there is enough evidence to suggest that the steps most prone to error are in fact in the pre and post analytical phases. various international accreditation bodies now require clear and effective incident reporting protocols to enhance measures for error trapping and error avoidance. aims: this study aims to quantify and characterize transfusion errors in a joint commission international (jci) accredited tertiary care centre in india. methods: all reports of transfusion related errors, registered in the blood bank or outside, between january 2008 till december 2012 were reviewed and categorised into pre-analytical, analytical and post-analytical events. the process adopted at our centre for assessing transfusion related events at the patient's side uses widely tested criteria of: (1) incident reporting (2) root cause analysis (3) identification of corrective actions results: during the study period 89,156 requests for blood and blood components were received and total of 1,98,505 blood components were issued within the hospital. a total of 16,834 reported errors were analysed. pre-analytical errors comprised a large majority (13,676; 81.24%), most of them being errors of inadequate patient information on request forms (10976; 65.2%) followed by sampling errors (1756; 10.4%). analytical errors comprised (563; 3.3%) and post analytical errors accounted for (2595; 15.4%) of the total errors reported. there was no incidence of acute haemolytic transfusion reaction or direct patient harm during the study period but on several occasions near miss events were reported which, if missed could have background: in australia, we rely on non-remunerated, voluntary donors to provide sufficient blood to meet patients' needs. for fresh components, the australian red cross blood service (blood service) is unable to import components for routine use, so is 100% self-sufficient. hospitals and pathology laboratories are under increasing pressure from government/s to improve value for money for blood and blood products, which is resulting in extra demands being placed on the blood service, especially in relation to lower age at issue and a continuing trend to hold more group o stock and less of the other groups, especially ab. with a typically seasonal inventory pattern for red cells, the blood service has focussed on closer management of blood inventory. aim: the aim of the inventory program was to improve reliability of blood coming into the supply chain and therefore improved reliability in delivery to customers. this is measured by average and variance in the number of whole blood collection packs being receipted at the processing centre. the aim was to reduce the variability in this metric, which would naturally lead to decreased inventory holding requirements, greater control and efficiency, and increased reliability and service to the customers. order fulfilment is another measure used to demonstrate improvement. methods: in order to manage blood inventory effectively, the first step was to introduce a minimum and maximum inventory level, by blood type, by state. this provided a transparent target to aim for. the bands were calculated by firstly setting a minimum inventory level using traditional supply chain safety stock calculations. the next step was to develop a 12 week inventory forecast, using a number of planning assumptions. one of the core assumptions is the number of appointments booked in the lead up to a donation. in order to improve reliability, minimum tar-gets were agreed at 3 months out (re-booking time) through to one week out. a 'traffic light' style appointments portal was developed to provide improved visibility of appointment levels for each collection mode and by state. results: results show that the quarterly standard deviation of blood coming in to inventory has improved from 1711 to 1285 in the last four financial yearsa 25% reduction. in addition, order fulfilment has improved from 82% to 95%, demonstrating that, with improved planning systems and processes, it is possible to manage inventory effectively. the results are demonstrated in the two graphs attached. summary/conclusion: the blood service in australia set a goal to improve reliability of fresh components, in particular, red cells entering finished goods inventory, to improve order fulfilment and provide service excellence to customers. by implementing robust and disciplined planning and reporting systems, reliability has improved which shows that there are methods available to improve the effectiveness of inventory management for blood components. 3a-s11-02 wooi-seong k one of the fundamentals of a blood collection center that procures, processes, stores and supplies blood and blood components to hospitals or other blood banks is an effective and sound management of blood inventory. as blood supply is dynamic, blood supply management requires continuous monitoring and interfacing between blood procurement and inventory management and with hospitals. in an effort to provide adequate, safe and equitable blood supply from voluntary non-remunerated blood donors in the face of increasing demand and decreasing donor population, blood collection centers are also challenged with blood shortages, which unless managed, could impact the healthcare delivery and negatively affect patient care. in order to provide a consistent and reliable blood supply blood centres will have to resort to creative and innovative measure. malaysia, a unique multicultural and multiethnic country celebrates significant religious and historic events as well as commemorations. as such, malaysia observes numerous national and state holidays. in fact, malaysia is ranked as the seventh country in the world in the number of observed holidays. by virtue of its geographical location, malaysia is not exempt from natural and man-made disasters, the most severe being seasonal monsoon floods and flash floods. these and the poor response to blood donation campaigns as a result of 'balik kampung' phenomenon during major holidays due to mass exodus of malaysians to their hometowns, contribute to acute seasonal blood shortages in blood collection centers around the country as well as within the region. adopting a proactive approach to blood shortages includes embracing new measures to recruit and retain blood donors, establishing a blood forecast system, developing a strong network among blood collection centers, being transparent with the blood inventory levels which will lead to greater trust and increased confidence in bts and having a contingency plan. at national blood centre (nbc), the blood action team was formed in 2010. it is a multidisciplinary team comprising of members from the donor education and publicity, donor recruitment, blood procurement, component and processing and inventory divisions as well as medical officers, transfusion medicine specialists and consultant. meetings are held regularly and this has greatly improved the communication interdepartmentally, and has fostered a team whose members are committed to improving blood supply management and preventing blood shortages through discussion and brainstorming sessions. also, blood forecasting is carried out as far ahead as months in advance. the blood stock forecasts are also communicated to blood banks from public and private hospitals which are supplied by nbc, a measure to increase transparency. since the implementation of these measures, nbc has successfully and effectively overcome the annual seasonal blood shortages for the last 3 years. evidently, blood shortages are largely preventable by adopting a proactive approach. 3a-s11-03 . the c/t ratios were calculated and analyzed for each major department. nbc and hkl had continuously introduced several interventions to reduce c/t ratios during the period of this study. results: the overall c/t ratio for hkl had been reduced from 2.2 in year 2005 to 1.9 in year 2012. the four major departments in hkl that showed high reductions in c/t ratio for the same period were obstetrics and gynecology (6.4 reduced to 2.5), surgical (2.6 reduced to 2.1), orthopedic (2.6 reduced to 2.1) and neurology (3.8 reduced to 2.2). in this study, interventions that had contributed to the drastic reduction in c/t ratio were compliance to the maximum surgical blood order schedule (msbos) which was periodically updated within each department, effective communication between clinician and blood bank staff, and continuous medical education (cme) for house officers and clinicians. the active hospital transfusion committee (htc) and hospital transfusion team (htt) also played an important role in reducing the c/t ratio by creating awareness among the paramedics and medical officers regarding the judicious use of blood and blood products, and regular monitoring and audits of the whole transfusion process starting from blood sampling to monitoring of patients during and after transfusion. summary/conclusions: this study showed that several interventions that have been introduced by hkl and nbc such as continuous medical education, compliance with updated msbos, active role of htc and htt, and effective communication between clinician and blood bank staff have successfully reduced c/t ratio in four major departments in hkl. this successful achievement needs continuous monitoring and evaluation to ensure consistency. this can also be a role model that is shared with other hospitals to ensure that the c/t ratio is within the set target. 3a-s11-04 fusion) were collected. during second step, a modeling and simulation were used to define the optimal rcl inventory for the metropolitan area. average rc cell shelflife of regional inventory as well as the number of transfused rcs were calculated. in addition it was hypothesized that an efficient turnover of rc inventory will result in inventory reduction and relatively fresh blood for the transfusion reducing the blood utilization and frequency of transfusion among non-surgical patients especially those with chronic transfusion. results: dynamic inventory management and application of inventory index at regional level (four referral hospitals providing direct health care services to 1.4 and specialized services to 2.0 million population) reduced the regional rc inventory by 32% (1100 rcs to 750 rcs; optimal hospital inventory index of 7.5). this change in inventory was accompanied by a reduction in shelf-life of transfused red cells at 36% (average shelf-life of transfused rcs reduced from 3.45 to 2.21 weeks). the total annual rc utilization and specific categorical data of patients prior to and after the implementation of bump (2010 and 2012) included in table 1 . conclusion: optimization of rc inventory by application of inventory index improved the pattern of regional blood utilization. red cell utilization among chronically transfused patients was decreased by 20% (average). chronically transfused hematology and renal patients showed the highest reduction on rcu (21-26%, p value < 0.0001). that was no change in amount of surgical transfusions. non-surgical (medical) category showed a mild reduction (4%) but statistically was not significant. the results indicate that implementation of bump could significantly improve red cell utilization among chronically transfused patients. this change may also result in reduction of transfusion associated adverse reactions and long term complications like as iron overload. 3a-s12-01 no abstract available. 3a-s12-02 burnouf t 1 , tzeng ys 2 , deng sc 2 , wang ch 2 , tsai jc 3 and chen tm 2 1 taipei medical university, taipei, taiwan 2 tri-service general hospital, taipei, taiwan 3 tatung university, taipei, taiwan background: approximately 15% of diabetic patients develop chronic ulcers, and 25% of those may undergo foot amputation. activated platelet gel, which contains growth factors, has been proposed as an adjunct to promote healing of small diabetic foot ulcers. for large un-healing diabetic ulcers, skin graft is usually needed. we have demonstrated that single-donor allogeneic platelet gel and fibrin glue improve skin grafting to achieve successful persistent healing of large ulcers [1]. however, it is not known whether autologous platelet gel can be beneficial in this application. aims: to evaluate in a prospective study the safety and efficacy of using autologous platelet gel to enhance skin graft take in non-healing diabetic lower extremity ulcers. methods: approval was obtained from the institutional review board of our hospital. eight consecutive diabetic patients aged 25-82 with nine non-healing lower extremity ulcers (median size of 50 cm 2 ; range 15-150 cm 2 ) were enrolled. their median duration of diabetes and ulcer was 10.6 years (range 5-25 years) and 6.5 months (range 3-24 months). none of the patients had received conventional skin grafting in the past. prp was prepared from 100 ml of venous blood using sep-ax-vgr protocol (biosafe sa, switzerland). autologous thrombin was prepared by activating 10 ml of plasma (tgd-001; merries international inc., taiwan). skin ulcer was debrided to remove the infected and necrotic tissues and covered with moist saline dressing. daily dressing change without additional treatment was performed. the wound was sprayed after 7 to 10 days with equal volumes (5 to 7 ml) of autologous prp and autologous thrombin to form the platelet gel within 5-10 s. thin-splitthickness skin graft with multiple slits was then applied on the wound bed and fixed with staples or cat-gut sutures. each patient was placed on antibiotics during the course according to wound cultural results. bolster dressing with sofa-tulle were used to avoid post-graft hematoma formation. negative pressure wound therapy (vac) was not used in this study. results: there was no adverse reaction during the study. eight out of nine skin grafts took well (88% healing rate). the interval between skin graft and complete wound healing ranged from 2 to 3 weeks in the eight successful cases. no ulcer recurrence was noted during the 2-19 months follow-up period. the non-successful case was an attempt to treat an ulcer that was deep to the periosteum of calcaneus bone. free tissue transfer would have been required, but the patient refused the microsurgery, due to age and medical condition, which led to skin graft loss. conclusion: this study shows for the first time, to our knowledge, the possibility to use platelet materials in combination with skin graft procedures to treat large nonhealing diabetic ulcers of lower extremity recently, human platelet lysates (pl) rich in growth factors were shown to replace fbs for ex vivo expansion of various cells, but whether they can be used for cec expansion is unknown. aims: to evaluate the possibility to isolate and propagate cecs ex vivo using a xenogeneic-free, recombinant growth factor-free medium supplemented exclusively with human pl. methods: pl was prepared by cacl 2 activation of apheresis platelet concentrates from three volunteer donors, and centrifuged to obtain a fibrin-free supernatant that was heat-treated (56â°c/30 min; hpl) or not. pl was characterized for proteins, platelet growth factors (pdgf-ab, bdnf, egf and vegf) and chemical composition. cecs were obtained from over 10 bovine corneas (bcecs) using standard procedures and grown in a dmem-f12 medium (containing sodium bicarbonate, selenium, and antibiotics) supplemented either with (i) 5% fbs, 0.5% dmso, 2 ng/ml rhu-egf, 5 lg/ml insulin, 5 lg/ml transferrin, and 1 nm cholera toxin (termed '5% fbs medium'), or with (ii) 2.5%, 5%, 7.5%, or 10% pl or hpl as the only source of protein nutrients and growth factors. cells were grown in duplicates in 6, 24 or 96-well plates at 37â°c in a controlled atmosphere containing 5% co 2 , with medium changes every two days. viable cells were counted for 7 days and cell viability was determined by mtt assay. bcec phenotype was determined by immunostaining using anti-phospho-connexin 43, anti-na/k atpase alpha-1 subunit, anti-zo-1 and purified anti-n-cadherin. anti-mouse and anti-rabbit igg fitc were used as the second fluorescent antibodies. results: pl or hpl contained 55-60 mg/ml total proteins, and a range of approximately 30-40.5, 23.1-32, 0.44-1.82, and 0.24-0.39 ng/ml of pdgf-ab, bdnf, egf, and vegf platelet growth factors, respectively. cecs could be expanded in a med-ium supplemented with 2.5-10% pl or hpl. interestingly, better cell bcec morphology and adherence was found when using hpl compared to pl. cell growth and mtt equivalent to that of the '5% fbs medium' could be achieved only using 10% hpl. in addition, bcecs could be isolated from bovine corneas and subsequently expanded using the dmem/f12 medium supplemented with 10% hpl. bcecs expanded in the hpl-medium maintained their typical morphology, adherence, transparency and phenotype. conclusion: bcecs can be isolated and expanded ex vivo in a growth medium supplemented solely with human platelet lysate material. although further studies using cec from human origin are mandatory to confirm these conclusions, such findings open a possible new paradigm for gmp-compliant, clinical-grade ex vivo propagation of cec and regenerative therapy protocols of human corneal endothelium. platelets are the smallest and second most abundant circulating cells in the blood and their primary role is to maintain the integrity of the vasculature. when blood vessel injury occurs, platelet adhesion and activation receptors recognize subendothelial matrix proteins such as collagen and this can initiate a coordinated series of reactions leading to the formation of a fibrin clot to arrest bleeding. it appears, however, that in addition to hemostasis, platelets also have important inflammatory and immunological functions. as early as the 1960's, reports began to demonstrate that platelets may play an active role in the stimulation and regulation of immune responses. for example, platelets can store and secrete several pro-and anti-inflammatory chemokines (e.g. platelet factor 4 and rantes) and cytokines (e.g. interleukin-1b and transforming growth factor-b) that can affect local immune responses such as chemoattracting neutrophils to sites of tissue damage. on the other hand, platelets may be able to directly regulate adaptive immune responses via their ability to express and secrete cd40/ cd40l co-stimulatory molecules. more recent reports have also suggested that depending on their activation state, platelets may be able to either suppress cd8+ t cell responses or under certain circumstances, present mhc class i associated peptides to activate cd8+ t cells. these studies have suggested that platelets represent a critical link between innate and adaptive immunity. platelet mhc class i expression may also have a detrimental role by conferring tumor cell resistance against immune attack. of perhaps greater interest, platelets have been shown to express the entire family of tolllike receptors (tlr) and this may allow them to act as circulating sentinel cells that first encounter bacterial products for presentation to the innate immune system. in particular, surface expression of platelet tlr4 enables platelets to present lipopolysaccharide to mononuclear cells and neutrophils which modulates their phagocytic capabilities and this has implications for the development of immune platelet disorders. furthermore, tlr9 appears to be contained within a unique platelet granule underneath the cell surface that can be expressed by platelet activation. thus, elucidating the role of platelets in sepsis and a better understanding of the apparent central role that they play as immune cells may be important for the potential development of efficient therapeutic modalities against infections. this lecture will highlight the many characteristics of platelets as immune-like cells will discuss how platelets may be the major controllers of immune responses. macquarie university, sydney, australia malaria remains a major health problem in most of the tropics, and is especially burdensome in economically underprivileged areas. our ability to reduce the high rates of morbidity and mortality due to malaria are hampered by wanning efficacy of current antimalarial drugs and the spread of insecticide-resistant mosquitoes. we desperately need a greater understanding of how the plasmodium parasite succeeds in invading and growing within red cells, how the host responds to an infection, and importantly, the protective mechanisms employed by the host to combat the infection. platelets regulate blood haemostasis, but are now also regarded as an important component of the body's early innate defense against invading microbial pathogens. recently, my laboratory discovered that platelets are able to protect against a malaria infection. in mouse models of malaria, survival to a chronic infection is reduced when platelet levels are artificially depleted. purified human platelets directly bind to p. falciparum-infected red cells in culture and kill parasite within. our current work is exploring how platelets can kill intrerythrocytic malaria parasites. i will present our current understanding of the platelet and red cell molecules involved in the killing mechanism. these include the platelet cytocidal molecule, platelet factor 4 (pf4) and the erythrocyte duffy-antigen molecule, which binds pf4 and mediates the platelet killing effect. the critical requirement of duffy has lead us to propose that platelet-mediated protection against p. falciparum infection is compromised in individuals homozygous for the common duffy-antigen negative allele. 3c-s13-01 graduate school of medicine, the university of tokyo, tokyo, japan although bleeding is a major side effect of heparin, which is used for treatment of thrombosis, heparin also causes a prothrombotic adverse drug reaction called heparininduced thrombocytopenia (hit). hit is caused by the development of platelet-activating antibodies (hit antibody), which is directed against the heparin and platelet factor 4 (pf4) complex. these reactions accelerate platelet activation and coagulation, leading to thrombosis. thus, if hit is strongly suspected clinically in cases of thrombocytopenia and thromboembolism that occur during or after heparin therapy, it is vital to stop all heparins and start administering an alternative antithrombotic drug immediately. in japan, a test to screen for hit antibody (the automated immunoassays based on two types of chemiluminescent immunoassay and a latex-enhanced immunoturbidimetric assay) was approved as a clinical laboratory test in the medical insurance system, in september 2012. only the latex agglutination test is now widely used clinically because of its simplicity, convenience and cost-effectiveness. however, these immunological methods, including enzyme immunoassays (eias), which detect binding of antibodies to immobilized pf4/heparin complexes, may not be employed suitably. the immunological hit tests are useful in diagnosing hit because of their high sensitivity; however, they also often cause overdiagnosis of hit. the value of the selected cut-offs is the key element in ruling out hit. consequently, hit should be confirmed through laboratory detection of platelet-activating antibodies by using functional assays for the hit antibody; it must also be diagnosed based on careful consideration of the clinical picture. in order to diagnose hit properly, our laboratory asks clinicians to assess the pretest probability of hit by using the scoring system (the '4t' scoring: thrombocytopenia, timing, thrombosis, and other explanations). furthermore, our expert staff ensures that the diagnosis is correctly performed, since hit has not been fully recognized in clinical practice in japan as compared to in western countries. the functional assay for hit antibody has been regarded as the gold standard for diagnosing hit in patients in spite of its disadvantages. the platelet activation test procedure is cumbersome, the tests are technically challenging, and limited to specialized laboratories. additionally, the most important requirement for the test is the selection of platelet donors with high reactivity to the platelet activation antibodies. accordingly, in japan, there are very few places where the functional assay is conducted, whereas many institutions still assess patients with only the immunological assay. in our laboratory, the heparin-induced platelet aggregation method is performed, as isotopes such as radiolabeled serotonin release assay should not be commonly used in routine laboratories. in an attempt to improve the sensitivity of the functional assay, we developed two methods for increasing hit antibody reactivity in donor platelets. one is the cooling donor platelet method, used for improving reactivity, and the other a way of donor selection by using monoclonal hit antibody. further studies are necessary to introduce a simple assay method in ordinary laboratory testing to detect platelet-activating antibodies. 3c-s13-02 autoimmune or immune thrombocytopenia (itp) is an acquired bleeding disorder with a low platelet count mediated by immune-mediated mechanisms. this condition is seen in patients with various associated diseases, such as systemic lupus erythematosus, and can also occur without an underlying disease. production of igg autoantibodies to platelet surface glycoproteins, such as gpiib/iiia and gpib, is the hallmark of the disease. it has been thought that anti-platelet autoantibodies promote platelet clearance in the reticuloendothelial system, but recent findings indicate that anti-platelet antibodies also suppress megakaryogenesis, resulting in impaired platelet production. the diagnosis of itp continues to be one of exclusion. several antigen-specific assays for detection of anti-gpiib/iiia and anti-gpib antibodies are reported to be useful in identifying itp patients, but these assays require complicated procedures such as platelet solubilization, and use of commercially unavailable monoclonal antibodies. to solve these problems, we have developed an enzyme-linked immunospot (elispot) assay for detection of circulating b cells secreting igg anti-gpiib/iiia and gpib antibodies, which is a sensitive, specific, and convenient method for evaluating the anti-platelet autoantibody response. in addition, reticulated platelets and circulating thrombopoietin (tpo) are useful in evaluating platelet production status. these findings led us to propose preliminary diagnostic criteria for itp based on a combination of itp-associated laboratory findings, including erythrocyte and leukocyte counts, anti-gpiib/iiia antibody-producing b cells, platelet-associated anti-gpiib/iiia antibodies, percentage of reticulated platelets, and plasma tpo. although the etiology of itp remains unknown, complex dysregulation of the immune system is observed in itp patients. based on a series of experiments using cd4+ t cells reactive with gpiib/iiia derived from itp patients, we have proposed a 'continuous pathogenic loop' model as a mechanism that explains ongoing antiplatelet antibody response in itp patients. this model includes b cells that produce anti-platelet antibodies, reticuloendothelial macrophages that phagocytose opsonized platelets via fcc receptors and present platelet-derived antigenic peptides, and platelet-reactive cd4+ t cells that exert their helper activity upon recognition of the antigenic peptides. once this pathogenic loop is established, anti-platelet antibody production would go on endlessly. recently, regulatory systems that control this pathogenic loop are attracting a great deal of attention. a series of studies in itp patients have found that foxp3+ regulatory t cells are reduced in circulation, bone marrow, and spleen, and are deficient in their suppressive function. in addition, a critical role of regulatory t cells in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in regulatory t cells, which spontaneously develop anti-platelet autoantibody-mediated thrombocytopenia. in addition, our recent analysis indicates that the eradication of helicobacter pylori leads to up-regulated expression of inhibitory fccgriib on macrophages, resulting in the attenuation of the pathogenic loop. therefore, therapeutic strategies aimed at interrupting this pathogenic loop would inhibit anti-platelet autoantibody production and subsequent increase in platelet count. in fact, current treatment regimens for itp, including corticosteroids, splenectomy, and rituximab, are able to suppress the pathogenic loop. interestingly, tpo mimietics have a potential to induce peripherally induced regulatory t cells, resulting in suppression of the pathogenic loop. 3c-s13-03 seguchi s 1 , maeda t 1 , kanaumi y 1 , kawamura s 1 , kodama m 1 , kawai t 1 , okazaki h 2 and miyata s 1 1 national cerebral and cardiovascular center, osaka, japan 2 the university of tokyo hospital, tokyo, japan background: heparin-induced thrombocytopenia (hit) is a devastating immunemediated thromboembolic complication of heparin therapy. heparin administration can cause conformational changes in platelet factor 4 (pf4), resulting in the production of anti-pf4/heparin antibodies. a subset of these antibodies can activate platelets and monocytes (hit antibodies), leading to thrombocytopenia and a thrombininduced hypercoagulable state. up to half of hit patients suffer from arterial or venous thrombosis. if platelet concentrates are transfused into hit patients, it is conceivable that the transfused platelets can be activated by the same mechanisms that affect the patient's own platelets and trigger the onset of new thromboembolism or exacerbate hit-associated thromboembolism. thus, platelet transfusion is thought to be contraindicated in acute hit patients. however, it remains uncertain whether platelet transfusion is a risk factor for thrombosis in hit patients since only a few studies have investigated this issue systematically. aim: the goal is to clarify whether platelet transfusion increases the risk of thrombosis in hit patients. methods: we constructed a nationwide registry for hit with the approval of the ethical review committee. between august 2008 and may 2013, 329 patients from 185 hospitals clinically suspected of having hit were retrospectively included in the registry with clinical information such as changes in platelet count, timing of heparin administration, episodes of transfusion, thromboembolic events, and the results of serological assays for hit antibodies. hit was definitely diagnosed by the detection of anti-pf4/heparin igg with platelet-activating properties at a therapeutic heparin concentration, but not at a high heparin concentration or with anti-fccriia antibodies. the assay was performed using washed platelets prepared from hit antibody-sensitive healthy volunteers at a reference laboratory. we examined patients who received transfusions of platelet concentrates after hit was suspected. results: of the 329 patients, 85 patients were ultimately diagnosed with hit (25.8%). optical density values of anti-pf4/heparin antibodies detected by elisa were significantly higher in hit patients than in non-hit patients (2.3 ae 0.95 vs 0.36 ae 0.53 for igg/a/m, p < 0.001; 1.68 ae 0.80 vs 0.25 ae 0.37 for igg, p < 0.001). the incidence of thromboembolic events was significantly higher in hit patients (51.8%) than that in non-hit patients (28.3%; p < 0.001). among the 85 hit patients, 20 patients received platelet transfusions after the onset of hit. only two of them experienced a thromboembolic event after platelet transfusion, one within 24 h and the other after 9 days. notably, neither patient was being treated with a thrombin inhibitor at the time. the incidence of thromboembolic events in hit patients who received platelet transfusions was not significantly higher compared to hit patients who did not receive platelet transfusion or non-hit patients who received platelet transfusions after the suspicion of hit arose, respectively. conclusions: to our best knowledge, this is the first systematic report that clarifies the clinical impact of platelet transfusion on the occurrence of thromboembolic events in acute hit patients whose diagnosis was confirmed by a washed plateletactivating assay. even in acute hit patients who possess platelet-activating antibodies, the transfusion of platelet concentrates does not appear to increase the risk of thromboembolism, especially while on thrombin inhibitor therapy. 3c-s13-04 lu p, ling b and li rs background: transfusion platelet matches with antigenic similarity would evoke less allorecognition and immune activation. strategies have been based on the theory that selection for hla-a and hla-b cross-reactive groups (cregs) compatible donor as well as abo/hpa-matched donor will predict good increment in platelet corrected count after platelet transfusion. aim: establish large-sized platelet donor registry with hla class i,hpa,abo-typed to meet the needs of immunized patients with platelet transfusion refractoriness. evaluate the effectiveness of platelet transfusion therapy in ptr patients. progressive management to ptr patients maintain a long-term platelet transfusion strategy. methods: to establish platelet aphaeresis donor registry in shanghai, 1931 repeat donors were typed for hla-a, -b and hpa-1,-2, -3, -4, -5, -6 and -15 using standard pcr-ssp method. eighteen patients with hematologic or oncologic diseases which refractoriness to platelet transfusions from random donors who are receiving 36 units of apheresis platelet products transfusion were studied. results: eighteen patients(eight male, 10 female)showing platelet refractoriness to random donor platelets [1 h corrected count increment (cci) <7500 ml/m 2 , percentage of platelet recovery (ppr) <20%] before. patients phenotyped for both hla-a,b and hpa-1,-2, -3, -4, -5, -6 and -15. apheresis platelets from donor registry in shanghai matched to patients abo, hpa and hla-a and hla-b cross-reactive groups (cregs) are transfused. ten patients ( show 24 h ppr >20%. the mean 1, 24 h cci and ppr values from the best donors were significantly higher than those from random donors they transfused before. conclusion: the use of hla-a,-b and hpa,abo-compatible aphaeresis platelet improves posttransfusion 1, 24 h cci values and percentage of platelet recovery in refractory patients. transfusion with hla-a,-b and hpa,abo-matched platelets is mandatory to reduce the risk of bleeding in ptr patients. refractoriness to platelet transfusions developed at least in 50% of the patients we observed and to maintain a long-term platelet transfusion strategy. establish large-sized platelet donor registry with hla class i, hpa, abo-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. the optimal strategy for platelet substitution in immunized patients remains a challenge. 3c-s13-05 xia w 1 , xu x 1 , ye x 1 , fu y 1 , deng j 1 , liu j 1 , ding h 1 , chen y 1 , shao y 1 , wang j 1 , li h 2 and santoso s 3 1 guangzhou blood center, guangzhou, china 2 department of biotechnology, guangdong food and drug vocational college, guangzhou, china 3 he institute for clinical immunology & transfusion medicine, justus-liebig univ., giessen, germany background: immunization against cd36 leads to the production of anti-nak a antibodies associated with fetal/neonatal alloimmune thrombocytopenia (fnait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). however, no data regarding the clinical relevance of cd36 immunization is currently available for chinese population. study design and methods: platelets and monocytes derived from 998 healthy blood donors were typed for cd36 deficiency using flow cytometry. in addition, four patients with suspected fnait (one case) and ptr (three cases) were investigated. nucleotide sequencing was performed to identify the mutations underlying the cd36 deficiency. transfection in mammalian cells (hek-293t) with cd36 mutated constructs was conducted to confirm these results. anti-nak a antibodies were screened by the use of platelet solid-phase kit (pak-plus, gti diagnostics). results: of 18/998 blood donors failed to express cd36 on their platelets surface. in 5/12 individuals no cd36 expression was detected both on platelets and monocytes, suggesting that the frequencies of type i cd36 deficiency (platelets and monocytes) and type ii cd36 deficiency (platelets only) were approximately 0.5% and 1.3%, respectively. nucleotide sequencing analysis of type i cd36 deficient individuals revealed eight different mutations; four of them were not described so far. however, 1228-1239del attgtgcctatt and 329-330delac appeared to be the most common mutations related to type i cd36 deficiency in south chinese population. further analysis showed that the presence of anti-nak a antibodies in one healthy donor (donor 1) as well as in three cases of ptr (patients 2-4) and one case of fnait (patient 1). these results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected cd36 cell lines. in all ptr patients, transfusion with platelets derived from cd36 negative donors resulted in good increment (24 h, ppr >20%). table 1 shows the mutations found in these five gpiv defective individuals. conclusions: more than 0.5% of cd36 type i deficient individuals are at risk to be immunized through blood transfusion or pregnancy in china. in this study, we could demonstrate that this immunization is of clinical relevance for the development of ptr and fnait. therefore, testing of anti-nak a antibodies should be considered in suspected immune mediated thrombocytopenia. a national registry of cd36 deficient blood donors should be established to maintain bleeding disorders associated with anti-nak a antibodies. since immunization against cd36 is conceivable for other asian populations an international network within laboratories in south asian region should be established in the future. 3c-s14-01 do we really need ffp? the evolving role of pf24 and pre-thawed plasma devine d fresh frozen plasma (ffp) is defined as plasma frozen within 8 hours of collection. while this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on ffp. in many blood systems, an increasing role for plasma frozen within 24 h of collection (fp24) is seen. such plasma shows little difference in functional protein levels when compared to ffp, with the exception of fviii levels which a show time related decay of activity. even factor viii loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of fp24. in addition, the activity profiles of coagulation proteins in fp24 prepared in routine production closely resemble those of commercial pooled plasma products. taken together, these observations have led many blood systems to move from the exclusive use of ffp to a mix of inventory of ffp and fp24, if not to the complete removal of ffp from their menu of offerings. the preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within 8 h of collection. since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor viii, in part owing to original role of the latter in the treatment of hemophilia a, collection of ffp has persisted as the starting material for cryoprecipitate production. in jurisdictions where hemophilia or other factor viii deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. as data began to accumulate on fp24, similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. this led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from 24 h to up to 5 days, if stored at 4â°c. such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. from the perspective of health resources management, the use of both fp24 and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. with the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as ab plasma. this presentation will focus on a review of the relevant studies of plasma quality for ffp, fp24 and pre-thawed plasma. we will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented. 3c-s14-02 background: in 2008, the australian red cross blood service (blood service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of trali (transfusion related acute lung injury). the challenge of sourcing all clinical plasma from male donors is exacerbated in australia due to its adherence to the council of europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in australia. aim: the aim of the programme was to achieve a result of 100% of clinical plasma sourced from male only donors. methods: work began in early 2008 to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that could be broken down by blood group and by day of the week. this was then formulated into a suite of reports, highlighting opportunities and variance in performance. based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. this assisted with prioritisation and workflow management. additional deliveries of blood from collection centres to processing centres. a range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation donor centre collection staff were trained to convert male ab donors over to plasmapheresis donation activity. changes to progesa to prevent manufacture of female plasma were made (after a time). results: the first report in july 2008 showed that the blood service were issuing 87.71% male clinical plasma; the group ab rate was 57.86%. the results improved dramatically by november 2010 in groups o, a and b (99.64%, 99.72% and 99.62% respectively), allowing for progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. by march 2012, the results in group ab had improved to 97.77% (chart below) and the directive was given to manufacture male clinical plasma only as routine. january 2013 was the first month ever where 100% of all clinical plasma was sourced from male donors only. in 2012/13, 99.98% of clinical plasma was male onlythe 0.02% constituted short lead time requests for iga deficient plasma. subsequent to a system change that allowed for a donor identification marker for iga deficient donors, inventory levels of this sub-product have increased by 34%, negating the need to turn female production on to accommodate sporadic demand. summary/conclusion: the multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of trali in australia. this is an achievement that many thought impossible and one that many other blood services have been unable to attain. results: quality control (qc) parameters were measured in the prepared blood components and were listed in table 1 . by standardising bc volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in sagm and plasma units while wbc and rbc contamination levels in pc and plasma were maintained low. all parameters were well within the blood component specifications set out in the council of europe guide (16th edition), the standard adopted by hkrcbts. qc parameters of the pathogen reduction-treated platelet concentrates in pas so produced were also within the hkrcbts blood component specifications. low contamination with red cells and white cells were demonstrated and the ph range was acceptable after 5 days of storage at 20-24â°c. the new t&b production method for the separation of 450-ml quadruple wb and the preparation of intercept platelet concentrates in ssp+ pas was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates. 3c-s15-01 blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. they should be managed in a way that ensures high standards of care. nevertheless, there are recognized adverse reactions that can occur during blood donation. the overall incidence of complications directly related to blood donation is 1%. they are generally more common in women, in younger and in first-time donors. although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. the majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. the incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary. iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. as each donation causes a loss of 213-236 mg of iron, repeat donation can lead to a continuous depletion of body iron stores. studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. to address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. adverse reactions such as delayed faint may occur after the donor leaves the donation venue. donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. a system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. all adverse events and reactions in donors should be identified, documented and reported. these data should be regularly analysed for possible corrective and preventive actions. the goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. for various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly. 3c-s15-02 wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. method: this 'global update' draws on work by the international haemovigilance network and international society for blood transfusion haemovigilance working party, experience in the netherlands, as well as a pubmed search using terms blood donor and adverse reaction. results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. results and discussion: the occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. a reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. fewer studies on acute complications in plasma and other types of apheresis have been published. preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. international collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. conclusion: donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. there remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation. assuming that these donors were newly infected, it is crucial for bts to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. aims: this study aimed to profile blood donors who donated during the hiv serological window period and to identify the risk factors of these blood donors. methods: past donor records of blood donors who had donated blood during the hiv serological window period (nat ultrio and discriminatory detected and negative for both anti-hiv and p24 antigen) at nbc or at blood mobiles organized by nbc from november 2007 until july 2013 were retrieved and analyzed using spss 15.0. results: a total of 18 donors were nat detected and negative for anti-hiv and p24 antigen (none in 2007, 2 in 2008, 2 in 2009, 1 in 2010, 6 in 2011, 4 in 2012 and 3 in 2013 introduction: in pakistan, the predominant reliance for blood supply is on the replacement donors, as sufficient numbers of voluntary blood donors are not available. an increase in the proportion of voluntary donors following the promotion of the concept of voluntary non-remunerated blood donation (vnrbd) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. objective: the objective of the current study was to promote vnrbd through a public awareness campaign (pac) based on a thorough analysis of the knowledge, attitudes and practices (kap) of a key segment of the society, i.e. 18-25 years old college and university students. material and methods: a cross-sectional, descriptive study was conducted over a period of three months (jan-mar 2012). multi-stage random cluster sampling approach was followed and college and university students were targeted through 20 university based blood donor organisations (bdos) out of a total 56 bdos identified. all the participants voluntarily participated in the study and informed consent was obtained orally. a pre-tested questionnaire comprising of 28 questions related to knowledge, attitudes and practices was applied. the questionnaire was kept anonymous and each question included multiple options or statements. statistical analysis was conducted by the assistance of statistical package for social sciences (spss) software version 17. results: a majority (65%) of the students had heard about blood donation through family/friends and a minority (9%) through the internet, although 45% preferred internet as spare time activity. majority (+85%) of the students had access to the internet and mobile phone. more than 50% of the respondents had donated blood: 22% donated for family, relatives or friends, 4% donated voluntarily as an act of altruism, and 25% donated voluntarily once and then stopped donating, but 55% of these respondents still considered themselves as volunteer blood donors. 35% indicated that important people in their environment had an influence on crucial decisions that they made. motivation for blood donation was a desire to help other people (73%), 28% followed friends invitations, in 21% cases respondents family or friends had received blood transfusions, 16% followed the example of fellow students. restriction for blood donation: 40% generally feared donation, 25% had a specific fear of the needle, 20% had no confidence in the public (health) sector, 25% condemned blood selling practice, 14% had no confidence in the donation procedure (hygiene), 13% experienced parental discouragement. conclusion: to overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. this strategy will help convince family replacement donors to become vnrbd and also recruit healthy individuals to become a vnrbd. the approaches and strategies for this transition can be based on the findings of the study. the reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a pac, for which detailed study is required. 3d-s16-01 murphy mf platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. there are immune and non-immune causes of platelet refractoriness. the main immune cause is hla alloimmunisation which occurs predominantly in females with a history of pregnancy. other immune causes include hpa alloimmunisation, abo incompatibility, platelet autoantibodies and drug-related platelet antibodies. the incidence of alloimmune platelet refractoriness due to hla antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. in current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, dic and splenomegaly. if there are poor responses to hla-matched platelet transfusions, the reasons should be sought including hla incompatibility which is most likely to occur in patients with unusual hla types with few well-matched donors, non-immune platelet consumption, and hpa and abo incompatibility. further serological investigations including testing for hpa antibodies may be used to differentiate between these possibilities. depending on the results, the appropriate management could be the use of abo-identical or hpa-matched platelet concentrates if the specificity of the hpa antibodies can be identified. platelet crossmatching may be helpful in some patients with non-specific hpa antibodies. the management of patients with hla and/or hpa alloimmunisation and no compatible donors may be very difficult. there is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. if bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. the management of patients with non-immune platelet consumption is similarly problematic. the usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased. 3d-s16-02 managing bleeding in cardiac surgery: despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. among all major surgical procedures, cardiac surgery with cpb still consumes a large part of the available blood supply. in england indicated that 10-15% of the blood units supplied by the national blood service is used in cardiac surgery units. in the usa, nearly 20% of blood transfusions are associated with cardiac surgery. during the early history of cardiac surgery, patients received large amounts of allogeneic blood. in the 50's, most operations were performed to correct congenital heart disease. during the 60's and 70's, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. in the 80's pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. with increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient's preoperative rbc mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. the ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. using the trac and trust scoring system predicts candidates likely for transfusion. diminished rbc mass appears to be one of the strongest predictors of transfusion. the acceptance of a lower postoperative haematocrit (in ijn the hct on bypass is 20-25% and post bypass is >25%) or haemoglobin concentration represent an important element in current blood conservation practice. the decision to transfuse a patient cannot be based only on haematocrit concentration. optimizing preoperative rbc mass involves the early detection of anaemia and its correction before surgery. preoperative autologous blood donation can be used to conserve allogeneic blood. besides economic concerns, one essential argument against pad is the lack of sufficient time because of the uncertainty of waiting list. erythropoietin has also been used to augment pad in elective cardiac surgery. acute normovolaemic haemodilution (anh) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. perioperative cell salvage (cs) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotininnow unavailable) may reduce excessive fibrinolysis and platelet dysfunction. the use of activated f vii has been reported in intractable bleeding post cardiac surgery. 3d-s17-01 the university of tokyo, tokyo, japan antibodies against human neutrophil antigens (hna) are involved in the pathogenesis of immune neutropenia, such as neonatal alloimmune neutropenia (nan), refractoriness to granulocyte transfusions, and transfusion reactions, such as febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury (trali). the hna systems are assigned to five antigen groups, namely hna-1 to 5. hna-1, -2, and -3a alloantibodies have been implicated in the pathogenesis of trali, and especially hna-3a alloantibody has been found in the severe cases requiring artificial ventilation or with fatal reactions. besides alloantibodies to hna-1, -2 and -3, those against hna-4a and hna-5a have been implicated in nan. the identification of the causative antibodies is essential for the diagnosis as well as for the prevention of these disorders. the detection of hna antibodies has been mainly dependent on cell-based assays so far. among them, the granulocyte agglutination test (gat), the granulocyte immunofluorescence test (gift) and the monoclonal antibody immobilization of granulocyte antigens (maiga) are the most commonly applied. according to the isbt working party on granulocyte immunobiology, the combination of gat and gift is presently the best means of hna antibody detection. gift is usually more sensitive than gat, however, hna-3a antibodies associated with severe cases of trali are better detectable by gat. in gat and gift, the presence of hla antibodies with broad specificities may affect the detection of hna antibodies. on the other hand, the maiga assay allows the differentiation between hna and hla antibodies. these classical methods, however, require fresh neutrophils from hna-typed donors. also, these assays are time-consuming, which makes them not appropriate for the large-scale antibody screening. in our lab, we modified the mixed-passive hemagglutination (mpha) assay, the method largely applied in japan for platelet antigen/antibody detection, for the detection of hna antibodies. recently, alternative assays have been developed, including elisa with recombinant hna (rhna), and immunofluorescence tests with transfectant cells of hna (hna-1, -2, -4, and -5). more recently, the molecular basis of hna-3 antigen has been elucidated, and stable cell lines expressing hna-3 antigens became available. these cell lines seem to have low background, and do not express detectable levels of hla antigens, which make the identification of hna antibodies easier. additionally, kits that use luminex microbeads coated with hna antigen are being developed. these kits, however, do not include hna-3 antigens. these new technologies significantly help improving the detection and identification of hna antibodies, and allow the large scale screening of hna antibodies, contributing for the reduction of the risk of the pathological conditions associated with hna antibodies, especially trali. however, these new technologies significantly increase the cost of the tests. presently, although many assays have been developed, the standard hna antisera are not necessarily available in every lab, which makes their validation difficult to be conducted. thus, the collaborative study among the various labs, by exchanging the available antisera, and comparing the test results, is essential for the improvement of this field. 3d-s17-02 one of the main sites where pmns carry out vital to surveillance functions is in the lungs. the large surface area of the lung is needed for gaseous exchange but lungs also present a vital direct mechanical barrier to the external environment. to patrol and protect this interface, about 28% of the body's total pmns are located in the pulmonary microvasculature. illness may increase the number of lung pmns as well as change their phenotype from quiescent to primed. in trali, the transfusion of blood products with either pmn reactive antibodies or biological response modifiers can activate this concentration of primed pmns to produce an augmented respiratory burst. this causes injury to the pulmonary microvasculature and consequently the symptoms of trali. circulating antibodies to pmns also can compromise their numbers and function. pmns carry human neutrophil antigens (hna) as well as class i hla, which can become targets for pmn reactive antibodies. the granulocyte immunofluorescence test (gift) and granulocyte agglutination test (gat) are primary tools for investigating these pmn reactions, as they are able to detect reaction of hna as well as some hla class i antibodies. immune neutropenias: alloimmune neonatal neutropenia (ann) occurs when a neonate's pmns are destroyed by transferred maternal antibodies developed against an inherited paternal neutrophil antigen. this is similar to haemolytic disease of the newborn, but importantly can occur with the first pregnancy. in early childhood, some children develop severe neutropenia as a result of pmn auto-antibodies. although the pathogenesis of such chronic benign autoimmune neutropenia is still not understood most of these autoantibodies demonstrate specificity for the hna system. passively acquired autoimmune neutropenia, wherein pmns are destroyed by maternal pmn auto-antibodies crossing the placenta are a rare finding. hna specificity is unlikely. autoimmune neutropenia in adults is either primary, secondary to another autoimmune disease or drug related. it can present a clinical and diagnostic challenge as many adults invariably have alloantibodies to neutrophils and the patient's neutrophil count is too low to make a definitive identification of a self reactive autoantibody. hna specificity is extremely rare. the severity of trali and immune neutropenias demand rapid and precise diagnosis with reliable neutrophil serology. the isbt granulocyte immunobiology working party maintains a list of granulocyte immunobiology reference laboratories around the world. 3d-s17-03 when seven sera from 778 donors were screened for neutrophil specific antibodies, 9% samples showed positive reaction. these results, however, could not be confirmed by gift and gat. conclusions: in this study, we found alloimmunization against hla class i and ii in 4.6% male,~4.3% nulliparous and~13.9% parous females. in contrast, alloimmunization against hna was not detectable in this cohort. these results indicate that the use of plasma containing blood products from parous females without hla antibodies pre-testing may increase the risk of trali reaction. although alloimmunization against hna seems to be a rare event in china, further observation is necessary to exclude the necessity of hna antibodies screening in our blood products. it is becoming clear that the ccn family of extracellular proteins play an important role in the health and function of several cells of the hematopoietic lineage. ccn2, also known as connective tissue growth factor, ctgf, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting a role in blood clotting and/or wound healing. on the other hand, ccn3, also known as nephroblastoma-overexpressed, nov, has been found to play an important role in hematopoietic stem cell health and function. in fact, treatment with ccn3 has recently been shown to promote hematopoietic potential, a discovery with dramatic clinical potential. initially using bioinformatics, our laboratory has discovered a signaling pathway that connects these two discoveries and appears to be a key functional node within the development of blood cells. specifically, we have found that the myeloid zinc finger protein 1, mzf1, is a transcription factor that trans-activates both ccn2 and ccn3, in distinct cell types. for example, we have shown that mzf1 can stimulate ccn2 production and secretion in stromal fibroblasts, which is then taken up by megakaryocytes and loaded into platelets. this is the first time that ccn2 loading into developing platelets has been directly achieved and observed in vitro. secondly, we have discovered that mzf1 also regulates the synthesis of ccn3 in several hematopoietic cell types. putting these results together with previous data suggests a new and immediately testable clinical treatment. it is known that both vitamin d (calcitriol) and vitamin a (all-trans retinoic acid) stimulate transcription of the mzf1 gene. (we also have new data exploring the mechanism and suggesting other pharmacological ligands). we have confirmed that treatments with either vitamin a or d activate this pathway and results in increased production of ccn2 in stromal fibroblasts, which in turn results in enhanced loading of ccn2 into developing platelets in vitro. similarly, we have observed that both vitamin a and vitamin d induce ccn3 expression, through mzf-1, and we are currently testing if this will lead to enhanced hematopoietic potential. this work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias. 3d-s18-05 background: foxp3 + t regulatory cells (tregs) consisting of natural and induced treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. while recent studies demonstrated that natural tregs are instable and dysfunctional in the inflammatory condition, induced tregs (itregs) may have a different feature. furthermore, it was reported that tolerogenic dendritic cells (tdcs) could expand itregs in vitro and this action designed to correct defects in numbers or functions of itregs may be therapeutic in the treatment of autoimmune diseases. in this study, the suppression efficacy of tgf-beta-induced tregs expanded by tdcs in vitro and in mouse model of autoimmune arthritis was determined. method: in vitro, first, cd4+ cd25ã� t cells were purified from splenocytes of d1 mice and stimulated by anti-cd3/cd28 in the presence of tgf-b1 for 5 days, which were termed 'itreg'. and tdcs derived from bone marrow of d1 mice were induced by gm-csf, il-10 and tgf-b1 and harvested after 10-day cultivation. then, itregs were expanded by tdcs at the ratio 5:1 and collected after 4 days (termed 'itreg tdc '). the phenotype, proliferation, suppression of cd4t proliferation, induction of foxp3 + tregs from foxp3ã� t cells and suppression of th17 cell differentiation were assessed. for in vivo experiments, the animal models of ra were established. in this model, arthritis was induced in d1 mice after immunized with bovine type ii collagen (cii) on day 0 and day 21, termed collagen-induced arthritis (cia). and 1 9 10 6 itregs or itreg tdc cells were transferred <iv. into cia recipient mice when cia onset. control mice were treated with pbs. clinical and histopathologic scores, cytokine and anti-cii antibody secretion in serum and cd4 + th subsets were analyzed. results: after 5-day induction in vitro, the percent of foxp3 + itregs increased from 9.8 ae 1.5% to 54.9 ae 2.5%. and these itregs could be expanded effectively by tdc in additional 5-day co-incubation, which continued to express higher levers of foxp3 (82 ae 3.6%) and increased 4.17 ae 0.17 fold. compared with itregs, the itreg tdc cells showed stronger inhibitory effect, and could prompt cd4t cells converting to foxp3 + itreg and resistant to th17 cell differentiation more effectively. during the 4-weekobservation-period, a more remarkable anti-arthritic activity, improved clinical scores and histological end-points were found in the itreg tdc -treated group than those in itreg-treated group. serological levels of tnf-a, il-6, il-17 and anti-cii antibodies were also significantly lower and tgf-b production were higher in cia mice following adoptive transfer of itreg tdc cells as compared with itregs. moreover, the itreg tdc treatment resulted in an increase in the number of tregs (cd4+ foxp3+) and a decrease in the percentage of th17 cells (cd4+ il-17+) more obviously. conclusion: these results indicated that itregs expanded by tdcs showed more effective suppression in vitro, and reduced more markedly the severity and progression of cia than itregs. this reduction was associated with modulating inflammatory cytokine and anti-cii igg secretions to lower levels and polarizing the treg/ th17 balance to treg more obviously. this study highlights the potential therapeutic utility of itregs expanded by tdcs in autoimmune arthritis and should facilitate the future design of itreg tdc immunotherapeutic strategies against ra. in may 2010 the world health assembly passed a resolution, wha 63.12, on the availability, safety and quality of blood products. this urges member states to 'take all necessary steps to establish, implement and support nationally co-ordinated bts with the aim of achieving self-sufficiency'. the resolution applies to both fresh blood components and plasma derived medicinal products (pdmps). who defines self-sufficiency as meeting national needs for six products from blood and plasma collected locally. these comprise red cells, platelet concentrates, fresh frozen plasma, and factor viii, immunoglobulin and albumin solution. the definition and overall goals were established by a who expert consensus statement on 'achieving self-sufficiency based on vnrbd' developed at a meeting of who experts in geneva switzerland in 2011. blood services develop in line with the health care system that they support. typically this involves a transition from a supply for whole blood mainly in the context of emergency management of trauma and child birth to the implementation of blood component therapy. blood component production will likely lead to an excess of recovered plasma. effective utilisation of this requires access to plasma fractionation facilities. if this can be achieved then this will reduce reliance on commercial pdmps and ensure maximal utilisation of the donated whole blood. increasing demand for pdmps will potentially lead to a requirement for introduction of plasmapheresis programmes if the goal of self-sufficiency is to be maintained. there are a number of significant barriers to both utilisation of recovered plasma and the implementation of effective plasmapheresis programmes. individual countries will need to consider these as part of their long term strategic planning process. the main barriers for low and medium hdi index countries are access to suitable plasma fractionation facilities. few countries will be able to justify creation of a national fractionation plant. regional co-operation and development of facilities will be needed. secondly plasma for fractionation is seen as a critical material in the manufacture of a medicine. quality system requirements, including full compliance with a code of good manufacturing practice will be a pre-requisite for acceptance of the plasma as suitable for fractionation. there is an urgent need to develop support programmes to raise standards to achieve this. in contrast the main barrier for most high hdi countries is cost and the easy availability of pdmps produced by commercial countries from paid plasma. the safety profile of pdmps is currently excellent since the integration of a number of specific viral inactivation steps in the manufacturing process overcomes any infectious burden associated with payment for plasma. the critical question is then how a country can assure ongoing supply at a reasonable cost. ethical principles will play an important role in defining the strategy and individual countries should be able to define their own approach without unnecessary intervention and competition from the commercial plasma industry. 4a-s19-02 background: blood transfusion service of nepal red cross society was established in the year 1966. since the beginning nrcs has been running the service exclusively with assurance of government of nepal. in 1991 the government of nepal elaborated the mandate with clear policies and declared nepal red cross society as the sole authority to conduct the national blood program through out the country. at present it consists of 86 centers in 62 districts. national leadership and oversight of management and quality standards is the responsibility of the central blood transfusion service, who in partnership with regional and district centers and aims to provide a safe and secure supply of blood/blood products to all needy in the country. in the initial years the service was made possible through collection of blood from professional donors but since 1982 collection of blood was emphasized from voluntary non-remunerated donors only. aims: blood service is provided according to the need base and with the expansion of health services, establishments of medical colleges, government hospitals, private hospitals and nursing homes the demand of blood is rapidly going up through out the country. nationwide more than 250,000 units of blood were served to the needy and around 60% of it is served through the central blood transfusion service. results: with the view of providing safe blood and bloodproducts to the needy patients all collected blood is routinely tested for hiv, hbsag, hcv, syphilis besides grouping, cross-matching and anti-d antibody identification. blood components service is limited in few centers. conclusion: blood program is run through cost recovery basis with material and nominal service charge nationwide. no defined budget is allocated for blood program. there is on and off support provided from nepal government, un agencies, national societies and international non government organizations. currently, the total revenue raised for the blood program is insufficient to meet the total cost of production of blood and blood products. 4a-s19-03 background: blood transfusion services in cambodia face a number of significant challenges in providing a supply of safe blood and blood products sufficient to meet the clinical needs of patients. these challenges include access to adequate and sustainable financing, trained staff, sufficient voluntary non-remunerated blood donors (vnrbd) and appropriate equipment and facilities. aims: a 2011 programme funded by the us presidents emergency plan for aids relief (pepfar) and managed by the us centres for disease control (cdc) was initiated by the cambodian based us cdc office, in a collaborative partnership with the nbts and ministry of health (moh). specific blood program needs were identified and presented in a logical task order framework. the task order required support at both the national and provincial level across the whole blood sector; and priorities three key deliverables: comprehensive assessment of the country's current practices and infrastructure including national policy, legislation, collection, testing and the clinical use of blood. vnrbd recruitment strategies/protocols. a new 5 year national strategic blood plan. methods: the selected technical assistance partner (ta) developed a proposal that addressed the areas of the task order, and utilised a number of strategies to deliver the technical program; including: a critical initial design phase with an introductory visit to map prior local and development partner work and draw on in-country. seeking ongoing engagement and advocacy from local thought leaders. tailoring assessment and strategy planning tools to local requirements. use of inclusive communications with all stakeholders. strengthening of local governance infrastructure (blood safety working group) . the 1 year program resulted in development and delivery of an assessment report on cambodia's blood transfusion services, a knowledge, attitudes and practices survey and a five year national strategic blood plan (nsp background: indonesia has recognized that blood transfusion plays a key role in their modern health care; yet ensuring the availability of safe blood is a challenge for a population of over 250 million living on 6000 islands with limited health care resources with a high background prevalence of hepatitis b virus (hbv) of 9.4%, human immunodeficiency virus (hiv) of 0.2%, and hepatitis c virus (hcv) 0.4%. aim: a health economic study was performed by the irc to evaluate the economic impact of individual donor (id) nat on the burden of disease (hbv, hcv, hiv) associated with transfusion transmitted infections (ttis) and understand the comprehensive costs to the ministry of health (moh) of providing id-nat-tested blood. method: a health economic analysis was performed using a markov method, which predicts the probability of an outcome based on a chain of outcomes and defined transition probabilities. the two arms of the analysis compared the testing costs and treatment costs of: (i) serology alone, and (ii) serology and nat. testing costs were defined as the total costs to the indonesian moh of purchasing the reagents used in conducting either serological testing alone or serological and id-nat testing on donor blood units. the treatment costs were defined by the outcomes associated with each of the three viruses (hbv, hiv, hcv) and the likelihood of various clinical outcomes for patients infected by transfusion. indonesia-specific data drawn from peerreviewed published sources was used to populate the model. in cases where published local data was not available, proxy published data from other geographies and data collected from interviews with local transfusion experts were used. cost information for id-nat and serology testing was sourced from test manufacturers. results: previous effectiveness of nat testingin annual collection of 2.1 million donations determined that id-nat could prevent 3187 infections missed by serology screening. if id-nat assays were not available ttis represents a calculated usd $73 million in treatment costs for the indonesian healthcare system. the introduction of id-nat could saveusd $27 million per year (or~20% savings) due to reduced treatment costs by preventing ttis. the estimated savings exclude costs associated with lost productivity and litigation. conclusion: sincethe initial analysis, additional testing has been performed in variousdemographic locations and it is now estimated that 8500 whole blood units andup to 15,000 blood components could be interdicted with id-nat. these new datarepresent even more favorable economics to the moh. while id-nat represents an additional cost tokeep blood as safe as possible, the incremental cost comparison of serology andid-nat compared to serology testing alone shows a minimum estimated savings ofusd $27 million. based on these results, id-nat blood screening appears to be aclinically and cost-effective way to prevent transmission of infection to patients. 4a-s20-01 yee-sin l tan tock seng hospital, singapore, singapore arbovirus is an acronym for a group of viruses that are transmitted by arthropod vectors, commonly mosquitoes and ticks. person-to-person transmission of arboviruses is not common. however, transmission can occur through transfusion of blood and blood products, and organ transplantation, if the virus is present in the donor's blood or organ. the majority of arboviral infections in humans are asymptomatic. if symptomatic, the symptoms generally occur 3-15 days after exposure. clinical features of arboviral infections can be classified into four groups: acute central nervous system illness, acute benign fever with or without exanthem, hemorrhagic fever, and polyarthritis. there have been published reports of transfusion-transmitted infections of a number of arboviruses, including west nile and dengue viruses, as well as colorado tick fever and tick-borne encephalitis viruses. the risk of transmission depends largely on the burden of the disease in a particular geographical location, the proportion of asymptomatic infections in which clinical diagnosis is not possible, and the availability of diagnostics to detect low levels of viruses in the blood. west nile virus (wnv) entered the united states (us) in 1999 and has spread across the entire north american continent. wnv is now the most significant arboviral infection in the us. one out of every five exposed individuals will develop illness and 1 out of 150 may suffer from severe infection with significant neurological sequelae. in 2002, 23 transfusion-transmitted wnv infections were described. since 2003, all blood donations in the us have been routinely screened for wnv rna using wnv nucleic acid test (nat). since the implementation of screening, 13 transfusion-associated transmissions of wnv have been documented (mmwr 2012) . dengue is the most common arboviral infection in the world. the world health organisation (who) estimates that 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries (who 2009 ). the ratio of asymptomatic to symptomatic infections vary widely. singapore reported three cases of transfusion-associated dengue infections involving one blood donation episode. an asymptomatic repeat blood donor informed the national blood bank that he had fever the day after blood donation. by then, packed red cells and other blood products from his donation had already been transfused to three recipients. all three recipients were infected; two of the recipients' blood and the donor's stored serum sample were tested positive for dengue serotype 2 virus by pcr. wilder-smith et al. estimated the risk for transfusion-associated dengue transmission in singapore to be 1.6-6/10,000 blood transfusions in the year 2005, assuming a ration of asymptomatic-to-symptomatic infections of 2:1 to 10:1. subsequently, teo et al. reported the practice of deferring blood donation from recently returned travellers from dengue endemic areas. the massive outbreaks of chikungunya fever in the indian ocean and southeast asia again raise concerns about blood safety. although there has yet to be a reported case of transfusion-associated chikungunya virus infection, such a transmission route remains a distinct possibility. liumbruno gm et al. reported an economic loss of approximately 2 million euros, resulting from the suspension of blood donations at the peak of the chikungunya outbreak in italy in 2008. 4a-s20-02 australian red cross blood service, perth, australia background: arthropod-borne viruses (arboviruses) are an expanding global threat to human health. among them dengue (denv) and west nile virus (wnv) have both been documented to be transfusion-transmissible and transfusion is a plausible route of infection for several others including chikungunya virus (chikv) and ross river virus (rrv). addressing the transfusion risk associated with arboviral outbreaks poses new challenges. unlike established viral threats to the blood supply (e.g. hiv, hcv and hbv) in which the risk is generalized to the population-at-large and chronic infection is common, arboviruses are characterised by focal and often seasonal outbreaks of acute, predominantly self-limiting infection. thus, unlike traditional viral threats the risk is not 'continuous' in nature complicating risk mitigation strategies. aims: to describe possible risk management strategies for arboviral transmission including; geographical donor deferral/restrictions on labile component manufacture, donation testing and pathogen inactivation (pi) which can be applied individually or in combination. methods: how strategies are applied depends on the nature of the risk i.e. whether internal (i.e. local outbreak) or an external (i.e. potentially viraemic donors returning from an outbreak affected country). geographical donor deferral can take several forms ranging from deferral of donors returning from precisely defined 'outbreak risk' areas (e.g. wnv deferral for travel to n. america) to a 'blanket' deferral of all donors returning for a defined period after return from overseas (e.g. the netherlands). while this can be an effective risk mitigation strategy it can lead to the deferral of significant numbers of donors and the impact on sufficiency needs careful consideration. in some cases (e.g. for local dengue outbreaks in australia) this can be partially addressed by restricting manufacture to 'plasma for fractionation' since pi within the fractionation process satisfactorily addresses the transmission risk. triggering implementation of deferrals for specific outbreaks is problematic because a high proportion of arboviral infections are asymptomatic so asymptomatic donor viraemia poses a risk before the first symptomatic case is apparent. instituting a deferral only during the 'transmission season' is complicated because of the difficulty in precisely defining the beginning and end donors for major transfusion-transmitted infectious diseases (ttids; hiv, hbv and hcv) has evolved from performance of serologic assays only to include nucleic acid testing (nat) for detection of window phase and occult infections in most developed and some developing countries. nat screening has also been implemented for other viruses known to be transmitted by blood components and/or plasma derivatives (parvovirus b19, hav), and for several emerging/expanding infectious disease (eid) agents that threaten blood safety in some regions (e.g., wnv in the us, hev in japan, and dengue virus in puerto rico). despite this dramatic progress, eid threats continue to be identified and cause pressure to further enhance the safety of transfusions. the concept of a 'global village' has emerged, which reflects the fact that a potential blood-borne infectious agent present in any region of the world could 'traffic' to other regions overnight. there are increasing examples of zoonotic transmissions of infectious agents, with the potential for rapid adaptation to humans and subsequent spread to blood donors and recipients. there is also intense research to identify new blood born infectious agents using novel molecular discovery strategies. although recent examples of putative new agents have proven to be non-pathogenic (hgv/gbvc, ttv and sen-v), not transmitted at significant rates by transfusions (hhv-8), or contaminants (xmrv), every newly discovered agent requires serious investigation to assess its relevance to transfusion safety. this presentation will highlight recent progress in developing approaches for systematic assessment of eid risks and for applying rational approaches to respond to new pathogens. in an effort to proactively assess transfusion safety threats in the united states and canada, the transfusion-transmitted diseases committee of the aabb has systematically reviewed relevant data and prioritized the risks of individual established and emerging disease agents; this program includes aabb website accessible fact sheets for~100 agents which are updated as new information becomes available or new agents of concern are identified. recently a research program has been started using data of id-nat users for analyses of the efficacy of various testing strategies this allows comparison of the yield and risk reduction obtained by nat and serology assays in countries with differing incidence and prevalence rates of the three major viral infections. furthermore the aabb eid sub-group and the european cdc have developed 'toolkits' for assessing the risk of eids with respect to potential impact on blood safety and availability, and for evaluating and validating the effectiveness of proposed interventions. the toolkit process, including methods to monitor eid agent emergence, identify and recognize a transfusion-transmission threat, processes to quantify risk, and the appropriate management of such threats using formal decision analysis methodologies, should be considered for implementation by all blood systems. the main challenges for hospital transfusion are patient safety, the effective use of blood, robust methods for documentation and monitoring practice, good blood stock management and low wastage, good staff training, and the rapid availability of blood for those patients who need it urgently. patient safety is paramount, but haemovigilance schemes worldwide continue to demonstrate that errors in the transfusion process result in patient morbidity and mortality. this talk will indicate where progress has been made in improving transfusion safety in hospitals in the uk, and use the implementation of an electronic transfusion management system in oxford as an example of improving patient safety. the project involved the development of an end-to-end electronic transfusion process involving barcode patient identification, handheld computers at the bedside and electronically controlled blood fridges linked to the blood transfusion laboratory information system. it was taken through pilot stages between 2001 and 2006 through to its full implementation across the acute hospitals in oxfordshire in 2006/07, and its maintenance and further development since then. there was an improvement from 11.8% to 100% of staff following the process for correct bedside patient identification; no abo incompatible red cell transfusions or serious wrong blood events in over 10years; a reduction in wrong blood in tube events; reduced nursing (one nurse rather than 2 and half the time to administer blood) and laboratory workload; and more rapid delivery of urgently required red cell units (from a median of 18 min to 45 s). the electronic system provided a simple mechanism for compliance with uk/eu regulatory requirements for the traceability of blood, and the documentation of transfusion and training. feedback from both staff and patients was positive. our group wrote a national specification and provided the material for a national recommendation for hospitals to adopt the electronic transfusion process, but wider implementation by other hospitals has been slow. an additional module into the electronic transfusion process is being implemented to provide decision support for doctors ordering blood to minimise inappropriate use of blood as part of a patient blood management programme. there are considerable barriers to improving the safety of transfusion practice in hospitals, but they can be overcome by 're-engineering' the process with the support of a multidisciplinary team with shared, achievable and measurable objectives, engaging with senior hospital management, and having a determination to see the development of a new process through to completion and its implementation as routine practice. whats new with trali? in the last 20 years there have been substantial efforts to minimise or eliminate trali. despite these activities, in 2012 the fda still reported trali as the top cause of transfusion-associated mortality. the pathophysiology of trali follows a two event mechanism. the patient's illness leads to activation of the pulmonary endothelium with the sequestration of primed neutrophilsthe first event. the transfusion is the second event, and leucocyte antibodies or biological responsive modifiers (brms), present in the blood product, activate the microbicidal arsenal of the primed neutrophils to cause injury to the pulmonary microvasculature and consequently acute lung injury (ali). hence, trali may be antibody-mediated or brm-mediated. antibodies to human neutrophil antigens (hna), hla class i and class ii have been associated and implicated with trali. interestingly, a large prospective case-controlled study of over 400,000 transfusions reported that recipients with cognate antigens to hna and hla class ii antibodies had increased risk of trali but found no statistical significance with hla class i (toy et al. 2012 blood) . that hna-3a antibodies are associated with more severe trali indicates that antibody specificity is another determinant of trali injury (reil et al. 2008 vox sang) . additionally, murine models demonstrated that antibody titre also determined the severity of injury (fung et al. 2010 blood) . while the use of male predominant plasma has significantly reduced the incidence of antibody-mediated cases, trali still occurs likely due to brms. brms are generated during the routine storage of blood products. a meta-analysis of cardiac and trauma patients concluded that older blood is associated with a significant risk of death (wang et al. et al. 2012 blood) . given the success of male predominant plasma in managing the risk of antibody-mediated trali, ongoing efforts to reduce the risk of trali are likely to focus on brm-mediated trali. developing a better understanding of the how specific brms may contribute, either individually or in combination, to the development of trali, and how brm concentrations vary between donors, will be crucial to the future development of strategies to manage the risk of brm-mediated trali. they were all thais and their forebears have lived in northeast thailand for at least two generations. hna-1,-3, and -4 were typed by the pcr-sequence specific primer (pcr-ssp). results: the gene frequencies of hna-1a, -1b, -1c were 0.697, 0.302 and 0.001, respectively. hna-1 null allele was found in one sample. the gene frequencies of hna-3a and -3b were 0.780 and 0.220, respectively. hna-4a and -4b gene frequenfrom asian populations aims: to assess whether symptoms consistent with dengue are more common in transfused patients who test positive for denv rna compared to patients who do not. methods: in 2012 and rio de janeiro, patients who were transfused were enrolled and interviews inquiring about symptoms (fever, headache, bone or joint pain, body or muscle pain, vomiting, rash, painful eyes, and bruising) were conducted between 3 to 7 days, and about 30 days following transfusion. inpatients and outpatients were included. blood samples were collected at the time of first interview. subsequently, coded samples were tested for denv rna using the hologic/novartis tma assay. we used two definitions for denv disease: the who clinical definition consisting of fever with at least two contemporaneous symptoms, and a more general definition of having â�¥3 symptoms of any kind. we calculated odds ratios (or) and 95% confidence intervals (ci) for symptomatic infection comparing transfused denv rna+ to rnaã� patients. results: of 595 patients with samples and interviews 3-7 days following transfusion 54% were inpatients while it is possible that viremia was missed in some recipients who had symptoms consistent with dengue, we did not find evidence of significantly increased rates of symptomatic infection in denv rna+ compared to rnaã� recipients. investigation into whether any of these patients acquired denv through transfusion and mie prefectures) where there are many immigrants from latin america including japanese-latin americans. methods: we conducted a questionnaire survey on chagas disease for the subjects who were borne or grew up in latin america. since jan. 8th 2013 we have performed testing for anti-t. cruzi for donors from whom informed consent for epidemiological study was obtained. tests used were ortho t. cruzi elisa assay and an immunochromatographic assay donors who were aware of chagas disease and kissing bug were 67% and 56%, respectively. nobody has ever been bitten by kissing bug. 14% of the donors had been tested for anti-t. cruzi in their country. four donors had a family member with chagas disease and they were all brazilian. four donors had been diagnosed as having a cardiac or digestive system disease before. all of the 287 blood samples obtained from the subjects were anti-t. cruzi-negative except for one false positive result. conclusions: none of the subjects surveyed were anti-t. cruzi-positive. most of latin american blood donors in tokai region of japan are brazilian and about twothirds of them were younger than 40 years. we consider that the risk of transfusion background: leukocyte antibodies presence in the blood product(s) was responsible for the development of immune mediated transfusion related acute lung injury (trali) in recipients. several studies had demonstrated that hla antibodies (class i and class ii) as well as hna antibodies (hna-1, -2, and -3) were capable to induce trali reaction. among them, hla class ii and hna-3a antibodies seemed to be the most frequent antibodies responsible for severe trali reaction. therefore, screening of hla and hna antibodies in our female blood donors may reduce the risk of trali. however, the prevalence of leukocyte antibodies among parous female blood donors in china is currently not available. study design and methods: blood samples were collected from 142 nulliparous females, 343 parous females and 453 male donors. pregnancy history was obtained from consenting donors. hla class i and ii antibodies were screened by the use of lifecodes lifescreen deluxe (lmx), and antibody specificities were determined with lifecodes lsatmclass i & ii (single antigen). antibodies against neutrophil antigens hna-1 and -2 were screened by the labscreen assay (one lambda, inc) and rapid elisa detection (bayat et al, 2009; werth et al, 2013). hna-3 antibodies were analyzed by antigen capture assay (bayat et al, 2012). the specificity of positive samples was reanalyzed by the granulocyte immunofluorescence test (gift), the granulocyte agglutination test (gat). results: screening of the total cohort of 938 donors (453 males and 485 females) showed higher prevalence of hla antibodies production in female compared with male donors (18.9% vs 4.63%). we found in 61 out of 485 females donors alloantibodies against hla class i (12.58%) and hla class ii (10.10%). analysis among female additional immunosuppression may be used in patients with low adamts13 due to autoantibodies. aims: to investigate differences between idiopathic and secondary ttp with regard to clinical presentation, management and outcomes. methods: the australian ttp registry was established in 2009, and expanded in 2012 to include all tma. of 34 hospitals currently participate in the registry. of 103 cases in 94 patients were registered from 18 sites; eight were excluded for incomplete data, leaving 95 episodes in 86 patients. results: median age was 46 years (15-83 years), 53% were female, 16% were relapses. of 51% were idiopathic and 49% had â�¥1 identified precipitant, including infection 23%, autoimmune 18%, malignancy 12%, medication 14%, pregnant/postpartum 4%, solid organ transplant 3%, and allograft 3%. adamts13 levels were available in 66 cases (37 idiopathic and 29 secondary). adamts13 was <10% in 22/37 (59%) idiopathic and 9/29 (31%) secondary cases. precipitants in cases with adamts13 <10% included infection (n = 5), autoimmune (n = 5), medication (n = 1), and pregnancy (n = 1). neurological, gastro-renal, bleeding and thrombotic presentations were similar in idiopathic and secondary cases. data on plasma therapy were available in 88 cases (table 1) . of 84/88 (95%) received pex, by centrifugation in 28/84, membrane filtration in 25/84 (30%), and unknown method in 29/84 (35%). plasma infusions were used in 11/88 cases (without pex in 4). data on additional therapy was available for 89 cases (table 2) . rituximab was given in 12/45 cases with â�¥1 precipitant, including infection (n = 5), non-haematological malignancy (n = 1), haematological malignancy (n = 3), autoimmune (n = 5), medication (n = 2), and pregnancy (n = 1). death rates were lower in idiopathic than in secondary cases (8% vs 25%, p = 0.05). conclusion: our data confirm that very low adamts13 does occur in cases with secondary causes identified. clinical presentation did not differ between idiopathic and secondary cases. plasma therapy was similar in both groups and additional immunosuppression was not only used in idiopathic cases, but also in secondary cases. mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed. background: living donor liver transplantation (ldlt) is a curative treatment for patients with end-stage liver disease. ldlt is associated with major blood loss and allogenic blood transfusions. although transfusion practices in adult liver transplant (lt) has been widely studied, the same in pediatric lt has not been well documented in india. aims: the purpose of the study was to evaluate the intraoperative transfusion requirements in pediatric ldlt recipients (<192 months) against the diagnosis, preoperative clinical and laboratory variables. methods: the data was collected retrospectively from blood bank and patient records. demographic and important preoperative variables namely clinical diagnosis, peld score, hemoglobin (hb), platelet count, inr, albumin, and fibrinogen levels were reviewed. data on intraoperative transfusion of packed red cells (prc), fresh frozen plasma (ffp), platelet concentrates (plt), cryoprecipitate was computed as ml/kg bw. massive transfusion (mt) was defined as >80 ml/kg of blood components during the surgery. analysis was done using spss software version 16. median levels were compared between mt and non-mt group using mann whitney test. results: between august 2010 to july 2013, 60 pediatric ldlt were performed in a single center in south india. thirty five (58.3%) of them were females and 25 (41.7%) males; 32 (54%) of the recipients blood group were o positive, 14 (23%) b, 9 (15%) a and 5 (8%) ab. recipient characteristics are given in table. transplant indications were biliary atresia and cirrhosis in 28, metabolic /hereditary liver disease in 24, hepatic tumors in 5 and acute liver failure in 3 recipients. of 56 (93.3%) patients received prc, 40 (70%) ffp, 17 (28.3%) plt and 34 (56.4%) cryoprecipitate. sixteen (26.7%) patients received massive transfusion (mt) with a median peld score of 22 (ã�7 to 43) compared to 9 (ã�9 to 46) recipients without mt (p < 0.005). also, mt group had significantly lowered median levels (preoperative) compared to non-mt group, viz. hb (8.6 vs 10.3 mg/dl), platelet count (96 vs 186 9 10 3 per mm 3 ), fibrinogen (137 vs 248 mg/dl), and a higher bilirubin (16.5 vs 3.7 g/dl). transfusion requirements of ffp was higher in acute liver failure (53 ae 17.3 ml/kg) compared to metabolic liver disease (11.1 ae 2.2 ml/kg) and biliary atresia (19.5 ae 4.2 ml/kg); (p = 0.001). conclusion: to conclude, massive blood transfusion requirement in pediatric recipients during ldlt was associated with higher peld score, and more deranged preoperative hematological and coagulation status. in depth analysis of recipient disease status, controlling for the effect of surgical interventional variables on larger samples are recommended to develop predictive models of transfusion therapy. conclusions: this study is the first report of hna gene frequencies in ethnic northeast thais. it could be used for the risk prediction of alloimmunization to hna and estimation of alloimmune neutropenia and trali in the ethnic northeast thai population. 3d-s18-01 distler pb 1 , slaper-cortenbach i 2 and ashford p 1 1 iccbba, san bernardino, united states of america 2 university medical center utrecht, utrecht, the netherlandsbackground: standardized isbt 128 terminology is used by cellular therapy organizations in many countries. as products evolve and new products are created, terminology is required to support the new products. changes to the terminology are managed by the cellular therapy coding and labeling advisory group (ctclag), a committee of experts representing international professional cell therapy societies, technical experts, and regulatory liaisons. since the early nomenclature was devel-oped, the ctclag has approved classes and terminology for very innovative products, including some for which therapeutic benefit has yet to be clearly demonstrated. because the term 'therapeutic cell' was used in the terminology, this became a great concern to the us fda, even to such an extent that the use of isbt 128 for cellular therapy products in the usa could be problematic. aims: the ctclag recognized the concerns raised by regulators and determined it needed to revise nomenclature to address these concerns and to be consistent with isbt 128 nomenclature used in related fields. methods: the ctclag held a face-to-face meeting and reconsidered the use of tc (therapeutic cells) terminology for products and proposed new nomenclature for the problematic terms. a draft of new nomenclature was developed and made available for public comment as well as review by the boards of ctclag sponsoring organizations (aabb, apbmt, asbmt, asfa, ebmt, fact, isbt, isct, jacie, nmdp and wmda). following this review, terminology was updated. results: major changes are:(1) the class name will comprise the type of cells and, where appropriate, the source (eg. 't cells, cord blood').(2) hyphenated class names will be replaced (3) tc and therapeutic terminology will be replaced (4) modifiers will be replaced with attributes providing the same information (5) new attributes will be added the terminology remains compatible with the single european coding system. summary/conclusions: changing terminology will create rework for facilities that have implemented isbt 128 and delay for those in the process of implementation. however, it was felt the revised terminology will provide a strong foundation for consistent nomenclature as new products are developed and address regulatory concerns. an appropriate timescale for implementation of the revised terminology in facilities already using isbt 128 will be developed. this presentation will describe the revised terminology and explain the reasoning supporting the changes.3d-s18-02 kordelas l 1 , rebmann v 2 , ludwig a-k 2 , radtke s 2 , beelen dw 1 , giebel b 2 and horn p 3 1 department of bone marrow transplantation, university hospital essen, essen, germany 2 institute for transfusion medicine, university hospital essen, essen, germany 3 university hospital essen, essen, germanybackground: graft-versus-host disease (gvhd) is a major cause of morbidity and mortality after allogeneic stem cell transplantation. a number of studies reported positive impacts of systemically applied mesenchymal stem cells (mscs) for preventing or treating acute gvhd. in contrast to the initial paradigm that mscs intercalate into injured tissues and thus reduce tissue damage, it is now widely assumed that mscs secrete a number of immune-modulatory factors, which impair inflammation and thus help to suppress gvhd. exosomes are secreted cell organelles, which exert immune-modulatory properties. these small membrane vesicles are released by a huge variety of different cell species, including mscs. methods: here, we enriched exosomes from bone-marrow derived mscs of four different unrelated stem cell donors and compared their immune modulatory properties in vitro. next, we administered immunosuppressive msc-derived exosomes in escalating doses into a 22-years female gvhd patient. this patient suffered a severe and therapy-refractory cutaneous and intestinal gvhd grade iv. we monitored the clinical effects on an in-hospital basis and correlated this with the levels of inflammatory cytokines measured in the patient's plasma. results: we show that even though all propagated msc lines released exosomes, exosome-enriched fractions differed in their potential to modulate immune responses in vitro. administration of the exosome-enriched fraction with the strongest immune suppressive in vitro effect into the gvhd patient was well tolerated and appeared to be safe. during the course of the exosome therapy a clear reduction of the proinflammatory cytokines il-6, il-8 and il-17 was observed in the patient's plasma. in line with that, the clinical cutaneous and intestinal gvhd symptoms improved significantly and the dosage of the immunosuppressive agentsparticularly of steroidscould be reduced. in total the patient was stable for 5 months. interpretation: msc exosome-enriched fractions exert immune suppressive functions in vitro and in vivo. since the in vivo administration seems to be safe, msc exosome administration appears as a promising new treatment option for steroid refractory gvhd patients. key: cord-342676-ykog278j authors: stewart, h.; bingham, r.j.; white, s. j.; dykeman, e. c.; zothner, c.; tuplin, a. k.; stockley, p. g.; twarock, r.; harris, m. title: identification of novel rna secondary structures within the hepatitis c virus genome reveals a cooperative involvement in genome packaging date: 2016-03-14 journal: sci rep doi: 10.1038/srep22952 sha: doc_id: 342676 cord_uid: ykog278j the specific packaging of the hepatitis c virus (hcv) genome is hypothesised to be driven by core-rna interactions. to identify the regions of the viral genome involved in this process, we used selex (systematic evolution of ligands by exponential enrichment) to identify rna aptamers which bind specifically to core in vitro. comparison of these aptamers to multiple hcv genomes revealed the presence of a conserved terminal loop motif within short rna stem-loop structures. we postulated that interactions of these motifs, as well as sub-motifs which were present in hcv genomes at statistically significant levels, with the core protein may drive virion assembly. we mutated 8 of these predicted motifs within the hcv infectious molecular clone jfh-1, thereby producing a range of mutant viruses predicted to possess altered rna secondary structures. rna replication and viral titre were unaltered in viruses possessing only one mutated structure. however, infectivity titres were decreased in viruses possessing a higher number of mutated regions. this work thus identified multiple novel rna motifs which appear to contribute to genome packaging. we suggest that these structures act as cooperative packaging signals to drive specific rna encapsidation during hcv assembly. hepatitis c virus (hcv) is the leading cause of chronic liver disease, hepatocellular carcinoma and liver transplants in the developed world. it is estimated that 170 million people are chronically infected worldwide, approximately 25% of whom will eventually present with liver cirrhosis. hcv therefore represents a significant health and economic burden. direct-acting antivirals (daas) targeting 3 viral non-structural proteins are now available for treatment of hcv infection. the rates of viral clearance, however, differ significantly between genotypes and a low genetic barrier to resistance for certain daas means that viral escape is highly likely. additionally, the pricing of the most recently available daa (sofosbuvir; a polymerase inhibitor) makes it unfeasible to treat the majority of patients in many countries. it is therefore apparent that further research into the basic virology of hcv is warranted. hcv is of the family flaviviridae, genus hepaciviridae and possesses a ~9.6 kb single-stranded positive-sense rna genome, encompassing highly structured untranslated flanking regions (utrs). the 5′ utr includes an internal ribosome entry site (ires), which directs cap-independent translation of a single polyprotein that is subsequently cleaved into 10 constituent proteins: core, e1, e2, p7, ns2, ns3, ns4a, ns4b, ns5a and ns5b. although the non-structural (ns) proteins contribute to some degree to the assembly and release of the virion, core, e1 and e2 play mostly structural roles 1 . the five c terminal ns proteins (ns3, ns4a, ns4b, ns5a and ns5b) were shown in 1999 to represent the minimal essential requirement for rna replication of a subgenomic replicon 2 . these proteins and the ensuing rna replication process have been available for targeted drug design 1 much longer than the processes of virion assembly, rna packaging and viral egress. the availability of a fully infectious viral molecular clone (jfh-1) means that these later stages can also be investigated in detail 3 . the role(s) of genomic rna structures during genome encapsidation has not, however, been the subject of extensive research. genomes are packaged during formation of the nucleocapsid by the core protein. core is encoded by the 5′ region of the orf and is released from the initially-translated polyprotein by host signal peptidase cleavage at the c terminus. core is then further processed by signal peptide peptidase, resulting in a mature 21 kda protein which forms homodimers and localises to cytosolic lipid droplets (for review see 1 ) . the ns proteins forming the rna replication complex remain membrane-bound within an er-derived membranous web where rna replication occurs. there is accumulating evidence that the delivery of a nascent rna by its cognate replication complex to the site of virion assembly is required for assembly initiation (for review see 4, 5) . however whether nucleocapsid formation and genome packaging occur at the lipid droplet, or whether this organelle merely acts as a storage site for core prior to its relocalisation back to the cytoplasmic face of the er (where assembly may occur), is a controversial topic 6 . following assembly the nascent nucleocapsid buds into the er, acquiring the lipid envelope and the associated e1-e2 viral glycoproteins prior to cellular egress (for review see 7) . it is apparent that the rna replication and virion assembly processes are spatially distinct within the cellular microenvironment and interactions between the replication complex and the core protein are required for efficient packaging 8 . consequently, the packaging of replication-defective genomes (which are not presented by a replication complex) is notoriously inefficient in hcv. it is this inability to study assembly without concurrent replication which has hindered the identification of rna structures contributing solely to assembly. the requirement for rna presentation by the replication complex may prevent encapsidation of defective and/or partial hcv genome fragments by core. the fact that cellular rnas are excluded from this process indicates additional selective factors must contribute. for many positive-sense rna viruses, the nucleocapsid assembly and genome packaging events occur simultaneously through recognition of a large stable rna structure (the packaging signal, ψ ) by their capsid protein. the essential role of these structural motifs within the viral rna in directing genome encapsidation has long been recognised 9 . the fact that pre-formed "empty" hcv capsid-like particles have not been identified suggests that hcv utilises a similar mechanism of rna recognition to initiate assembly 1 , although rna-free capsid-like structures have been isolated from in vitro translation systems 10 . however, unlike prototypic packaging signals such as those found in the retroviruses, alphaviruses and coronaviruses, a single rna structure which is both essential and sufficient to target non-viral rnas to a nascent hcv nucleocapsid particle has not been identified. it is possible that hcv utilises a recently discovered novel mechanism of genome encapsidation during nucleocapsid assembly, utilising multiple, relatively low-affinity structures termed packaging signals (pss) 11, 12 . we wished to investigate the possibility that similar specific secondary structures are present within hcv rnas destined for packaging, and whether their interactions with core cooperatively drive the rna encapsidation and nucleocapsid assembly processes. systematic evolution of ligands by exponential enrichment (selex) of rna aptamers against core. mature core consists of two distinct domains: the n terminal domain 1 (d1, 124 residues), which is highly basic and binds rna, and the hydrophobic c terminal domain 2 (d2, 50 residues) which possesses a membrane binding region. d2 stabilises core on the surface of lipid droplets and exposes the hydrophilic d1 to the viral rna, therefore it is essential for nucleocapsid formation in mammalian cells 13 . however, in vitro d1 is sufficient to form stable proteinase-resistant nucleocapsids when stabilised by interactions with nucleic acids [14] [15] [16] . we therefore prepared d1 as a selex target by recombinant protein expression. d1 hcv core (jfh-1) was expressed in e. coli and purified by nickel affinity chromatography against an n-terminal hexahistidine tag. the identity of the his-tagged protein was confirmed by lc-ms/ms of coomassie stained bands excised from an sds-page gel. the antigenicity of the final purified protein was also confirmed by immunoblotting for core d1 and the hexahistidine tag (fig. 1) . this protein was used for selex with a 2′ oh rna aptamer library encompassing a n35 random region. selex is an established process for the in vitro isolation of high-affinity aptamers: in vitro selected nucleic acid molecules which bind with high affinity and specificity to their target 17, 18 . each aptamer within a randomised library possesses a unique tertiary structure, depending on the series of stems, pseudoknots, kinks and/or bulges which are present in its most stable conformation. each aptamer will bind with varying affinity to the ligand of interest. selex is therefore likely to enrich for aptamers with conserved conformation rather than a unique primary sequence. locations and consensus motif of putative packaging signals (ppss). the sequences present in the final round of selex were determined by next generation sequencing (ngs) of the pool and the individual aptamer sequences were aligned to the jfh-1 genome (genbank id ab047639.1). it should be noted that known pss in other ssrna viruses are sequence/structure degenerate and their capsid/coat protein recognition sequences are discontinuous and minimal 12, 19, 20 . a consequence is that the pool of binders from selex should include a majority of oligonucleotides that, although matching essential features of hcv pss, are unlikely to match extended regions of the genome. despite this constraint, there are aptamers that match multiple sites within the genome and occur with statistical significance (a bernoulli score of 12 or more; red peaks, fig. 2 ) compared to the unselected naïve library (grey peaks, fig. 2 ). this outcome suggests that the hcv genome does contain multiple ps sites. in order to test this hypothesis, the same procedure was applied to an additional 14 genotype 2 hcv genomes, i.e. they were compared to the aptamers selected against jfh-1, and a similar picture emerged. this suggests that these ps sites are evolutionarily conserved. such peaks, within 10 nucleotides of the scientific reports | 6:22952 | doi: 10.1038/srep22952 peaks in fig. 2 , which were conserved for at least 60% of these additional strains, are highlighted by green arrows (fig. 2) . for each of these conserved peaks the 30 nucleotides 5′ and 3′ to the peak nucleotide in the jfh-1 genome were extracted, and a range of possible secondary structure folds were considered via mfold 21 . a similarity analysis revealed the potential formation of stem-loops with similar loop motifs in each of these fragments. an alignment of the loop portions of these stem-loops is displayed in fig. 3a . this analysis reveals a bias towards a grrgr loop motif, r denoting a purine, suggesting that pss should exhibit this motif or close variations thereof. in order to establish the statistical significance of this motif, we compared its occurrence in the loop portion of stem-loops in the wild-type genome with that in randomised versions of the genome, both for jfh-1 and for the other strain variants considered here. this confirmed a significant statistical bias in the wild-type genomes for this motif. we then interrogated sub-motifs of grrgr in order to identify those with an even higher statistical significance, which revealed a ggrgg motif. the positions of stem-loops with this motif across the jfh-1 genome are displayed in fig. 3b . modeling indicates that an ensemble of packaging signals with different degrees of affinity for capsid protein is required to promote efficient assembly 11, 22 . lower-affinity motifs may not necessarily be easily identifiable through a selex screen, yet their presence at statistically significant numbers within the wild-type genome statistically significant alignments of the aptamer pool with the jfh-1 genome correspond to peaks in the red curve that are above the corresponding alignment of the naïve library (grey curve). peaks occurring within 10 nucleotides of statistically significant peaks in at least 60% of the representative set of hcv genomes analysed here are marked by a green arrow. would be indicative of a potential role in virus assembly. based on previously-published models, the statistical bias observed for the ggrgg motif would be predicted to encompass at most 25% of the putative packaging signals (ppss) 19 . variations of the motif with statistical bias are the hallmark for pss with intermediate or weaker affinity for the capsid protein. the same statistical analysis was therefore used to interrogate other variations of the ggrgr motif, in an effort to identify sub-motifs that were more frequent in the wild-type genome than in the randomised version. the motif gxrxr fulfilled these criteria (see methods for details). mutagenesis to disrupt pps. since modeling suggests that ppss act collectively to promote virus assembly, an ensemble of 8 ppss was selected for mutagenesis. the region encoding ns5b was avoided as numerous studies have reported the existence of essential structures required for rna replication; mutations in this region would render the virus replication-defective 23, 24 . modeling moreover implies that a combination of high and low affinity binders is important, i.e., a combination of ppss following the ggrgg motif and those that exhibit other variations of the grrgr motif: e.g. gxrxr 19 . eight stem-loops were selected for mutagenesis, three of which contained the predicted highly conserved motif (ggrgg). the remaining five stem-loops contained the lower-affinity gxrxr motif identified above (fig. 4) . the stem-loops are numbered according to the first paired nucleotide within the context of the jfh-1 genome, according to standard nomenclature. a range of silent mutations were introduced into the orf of hcv, using the infectious molecular clone pjfh-1. these were designed to disrupt the base pairing within the stems of the ppss (fig. 4) . it was not possible in the majority of cases to completely ablate favourable folding energies for the formation of stem-loops and even mutant sites with unfavourable folding free energy might still form in the presence of a protein ligand such as core. eight mutant pss were created, each possessing between 5 and 11 silent single nucleotide polymorphisms, collectively targeting an individual pps (fig. 5) . these are referred to as the "single" pps mutants, δsl733, δsl2899, δsl3789, δsl4629, δsl4807, δsl5877, δsl6067 and δsl7580. all 57 silent mutations were also combined into a single viral genome, termed δ8xsl, in order to examine co-operative effects between the altered ppss. replication and translation are unaffected in pps mutants. rnas encoding all 8 single pps mutant viruses, the δ8xsl mutant, jfh-1 (wild-type) and jfh-1-gnd (a polymerase-deficient control that is unable to undergo viral genome replication) were electroporated into huh-7 cells. the intracellular rna was quantified by qrt-pcr, at both 4 and 48 hours post electroporation (h.p.e) (fig. 6a) . impaired replication was not observed in mutant viruses containing impaired ppss, although δsl4807 possessed a slightly decreased replication rate which was not statistically significant. this was further confirmed by detection of the viral ns5a protein compared to cellular gapdh expression via western blot (fig. 6b) . was titrated upon naïve huh-7 cells and the infectious titre calculated in infectious units per millilitre (iu/ml) as previously described 25 . this reflects the number of infectious rna-containing virions which had formed and/ or been released from within the electroporated cells. non-rna containing particles, or aberrantly-assembled defective particles, therefore do not contribute to the viral titre as they are non-transmissible. at 48 h.p.e., only the δ8xsl mutant virus displayed significantly decreased titres compared to the jfh-1 wt control, although δsl5877 and δsl7580 also appeared non-significantly decreased (fig. 7a) . the reduction in δ8xsl infectious titre was consistent across the entire 48 hours, as evidenced by time course assays during which virus was harvested every 6 hours post-electroporation to assess the rate of virion formation and release (fig. 7b) . this provides evidence that although mutating each pps independently has no apparent effect, the combined disruption of all 8 ppss affects the late stages of the viral life cycle (assembly and/or egress) whilst not affecting rna replication. rna secondary structures are altered within the packaging signal mutants. to confirm that the introduced mutations caused structural alterations, in vitro transcripts from a section of the ns4b-coding region from jfh-1 (wt) and δ8xsl mutant genomes were synthesised. rna was folded and selective 2′ -hydroxyl acylation analysed by primer extension (shape) mapping was performed as previously described 24 . briefly, folded rna was treated with a compound which reacts preferentially with single-stranded, flexible regions. this reaction forms an irreversible adduct which causes premature termination during the subsequent reverse transcription of the rna 26 . primer extensions using radiolabelled primers were conducted and the resulting fragments were resolved on a 7% polyacrylamide denaturing gel. the terminations, indicative of a flexible nucleotide, were visualised and their reactivity normalised to that of a dmso-treated control rna. the specific location of such terminations is determined by comparison to ddntp sequencing ladders. a high shape reactivity is indicative of a flexible nucleotide, available for acylation by the compound; hence only the terminal loop of a prototype stem-loop structure would appear as "reactive" nucleotides. the hcv genome is dynamic and multiple rna conformations are potentially able to form, including kissing-loops, pseudoknots, g-quadruplexes and other long-range interactions 24 . additionally the active folds of ppss may only form in the presence of an rna chaperone such as core or ns5a, which is absent during shape mapping. the shape data therefore indicates whether the engineered mutations altered the rna flexibility, rather than confirming the mfold-predicted structure of a particular motif. the reactivity profile across the region encompassing sl5877 provided evidence that the silent mutations had altered the flexibility of the mutant rna compared to wildtype (fig. 8) . together our data indicates the 8 putative packaging signals which we identified may form within the hcv genome, presumably during assembly. a mutant genome unable to form such structures displays impaired viral infectivity whilst rna replication is unaffected, indicating these motifs may interact specifically with core. the synergistic effect of these individual interactions may ensure encapsidation of a single viral genome occurs during virion assembly, thereby preventing non-specific packaging of cellular rnas. the mechanism by which the hcv core protein selectively binds and encapsidates viral genomic rna during virion assembly is currently unknown. in contrast to other positive-strand rna viruses 27, 28 there is no evidence for a single cis-acting rna structure capable of directing packaging of both viral and non-viral rnas to a nascent capsid particle. therefore it is plausible that multiple rna structures contribute to rna-core binding and act cooperatively to drive encapsidation and assembly. the presence of multiple, weak-affinity packaging signals in other rna virus genomes has recently been reported 19, 29 , which represents a novel mechanism for viral packaging in viruses which do not possess a readily identifiable prototypic packaging signal. here, we provide evidence that the abrogation of short motifs (ppss) located across the hcv genome has a significant effect on rna encapsidation, manifested by a decrease in infectious titre. this is only apparent in the multiple pps mutant (δ8xsl), consistent with rna packaging being a cooperative process in the hepaciviridae. the fact that depletion of all 8 ppss was required before a significant packaging phenotype was observed may explain why previous mutagenic screens have been unsuccessful in identifying these novel motifs 30 -if the ppss act synergistically, a threshold of ablation must be reached before significant phenotypic changes are observed. additional low-affinity ppss presumably also exist which were not mutated in this analysis. there are multiple occurrences of our described motifs within the jfh-1 genome which were not identified by our original aptamer screen; it may be that a large number of functionally redundant ppss exist and hence are able to easily compensate for many mutations which may occur during the error-prone rna replication process in vivo. in vitro transcripts of the ~1.5 kb region encompassing sl5877 and sl6067 were synthesised from both jfh-1 and mutant templates. rna was folded and subjected to structural analysis via shape mapping. following nmia (+ ) or dmso (− ) treatment and reverse transcription, a primer designed to bind approximately 20 nucleotides downstream of sl5877 was used for primer extension. products were resolved on a 7% denaturing polyacrylamide gel (upper panel) and the relative reactivity was quantified using safa 58 . sequencing ladders were included to identify individual nucleotides, based upon the wildtype genome sequence. previous models of assembly which utilise multiple contributing packaging signals predict that highly efficient capsid assembly occurs in the presence of one higher-affinity rna structure; initial binding of this motif to the structural protein then instigates additional multimerisation and concurrent interactions with the lower-affinity packaging signals 22 . the relative affinities of each of the ppss described in this study for the core protein would therefore be of significant interest. prior to the development of the infectious jfh-1 cell culture system, models of hcv virion assembly relied upon either self-assembled capsid-like particles generated from purified recombinant protein 15, 31, 32 , or by expression of core in yeast 31 , insect cells 33 or bacterial systems 34 followed by purification of the intracellular capsid-like particles. the formation of such regular capsid-like particles in these protocols requires the presence of structured rna, proving that protein-protein interactions are insufficient to drive core multimerisation. it is now recognised that these models do not reflect the assembly process in mammalian cells, therefore although investigating the binding kinetics of each pps and its respective mutant would be of significant interest, future work may have to utilise alternatives to such in vitro assembly models. additionally, the fact that intact hcv genomes cannot be packaged in in vitro assembly systems 15 supports our model wherein the structures of interest may be formed temporarily and potentially only in certain scenarios, such as during presentation of nascent rna within a replication complex. the ns5a protein (an essential component of the viral replication complex) possesses a broad rna binding spectrum 35 and interacts specifically with core protein 36 ; this combined with the fact that core exhibits extensive rna chaperone activity 37, 38 suggests a highly plausible model of rna conformational alterations occurring during rna transfer and subsequent assembly. it is well established in the literature that core binds to the viral 5′ utr 15, [39] [40] [41] [42] , although this interaction is not sufficient to drive genome packaging 43 . we did not focus upon this region as its interactions with core are already well-explored; the preferred core-rna binding motif is the iiid loop and this interaction inhibits ires-mediated translation 40, 44 . this motif is the only region of the ires which possesses similar features to the stem-loops selected for mutagenesis; specifically, the internal bulge and a g-rich stretch within the unpaired terminal loop. this suggests that multiple structures, each containing this motif in their terminal loop, may be required to interact with core during rna encapsidation. as this ires subdomain also forms long-range interactions with cres in the ns5b coding region 45, 46 , a model may be envisaged in which packaging, rna replication and translation are mutually exclusive events, partially dictated by the ligand bound to the ires iiid loop (core, cre rna or the ribosomal complex). the majority of our mutated ppss are within the non-structural protein coding regions, with the exception of sl733. trans-packaging of viral subgenomic replicons has been extensively documented (for review see) 47 and it has been noted by other research groups that the functionality of these systems reflects the lack of essential cis-acting rna elements within the core to ns2 genomic region 48 . this is supported by our data wherein only 2 of the 8 mutated ppss are located in this area (sl733 and sl2899); their removal may not reach a threshold level to affect packaging efficiency. trans-packaging studies utilising baculovirus-mediated structural protein expression also found that although the presence of the ns2 protein improves the production of replicon-containing particles, this was most apparent when ns2 is expressed in cis (within the replication-cassette), rather than in trans (within the structural protein construct) 49 . although this may be due to the protein-protein interactions required for virion assembly 50 , the fact that the pps sl2899 is present upon this particular replicon may increase the efficiency of packaging, resulting in higher titres of virus-like particles in this system. in a parallel scenario, naturally occurring mutants with deletions spanning e1-p7 have been found in multiple patient samples; as these subgenomic mutants may be packaged and released 51, 52 an essential packaging signal is definitively not located in those coding regions. given the well-recognised roles of rna structure in the hcv life cycle, it is important to highlight the novelty of the mutated structures in this report as many previously-annotated structures were not altered by our silent mutations, including the ns5b-located cres 24, 30, 53 . in addition, the genome of hcv possesses genome-scale ordered rna structure (gors); a phenomenon amongst particular rna viruses which exhibit extensive rna structure across the entire genome 54 . consequently the genome is constrained in its plasticity and evolutionary potential, in contrast to the predicted behaviour of a rna virus. it has been suggested that gors contributes to viral persistence or modulates host innate defence mechanisms. it therefore must be considered that these pps structures may additionally contribute to the overall architecture of the hcv genome and may not be solely involved in the core-rna interactions during packaging -they may have multiple functions, requiring distinct structural conformations to be adopted at precise points during the viral replication cycle. the δ8xsl mutant may represent a virus which has reached a threshold level of gors interruption. however this is unlikely; an equivalent effect upon replication and assembly would be predicted if this were the case. it is therefore apparent that our analysis does not merely reflect rna structures already annotated within the hcv genome, but rather the identification of novel structures utilised for core-rna binding. recombinant protein expression. core (d1) of jfh-1 was cloned into the pet28b(+ ) construct (novagen), in-frame with a n-terminal hexahistidine tag. recombinant protein was expressed in bl21[de3] cells (f-ompt hsdsb(rb-mb-gal dcm (de3)). luria broth media (1% glucose) was inoculated with a 1:1000 dilution of starter culture and maintained at 37 °c until an od 600 of 0.6-0.8 was reached. protein expression was then induced by the addition of 100 mm isopropyl β -d-1-thiogalactopyranoside and cultures were maintained at 20 °c for 6 hours whilst shaking, before being harvested by centrifugation. to an equilibrated ni 2+ sepharose resin column (ge healthcare). resin was washed (50 mm tris-hcl ph 7.4, 500 mm nacl, 10% glycerol, 0.06% β -mercaptoethanol, 100 mm imidazole) and the protein was eluted into wash-based buffer with 500 mm imidazole. protein was dialysed overnight into pbs and quantified by spectrometry. sds-page was conducted upon both bacterial lysates and eluted protein fractions according to previously described methods 55 using 12% gels. silver staining was conducted according to manufacturer's instructions (silverquest kit, invitrogen). immunoblots were conducted with an in-house rabbit anti-core polyclonal sera (1:2000) , commercial anti-his monoclonal antibody (1:1000, invitrogen) and hrp-labelled secondary antibodies. for protein identification, coomassie stained bands were excised, reduced, alkylated and digested with trypsin. the recovered peptides were analysed by lc-ms/ms and subsequent searching of the tandem ms data against the uniprot sequence database. recombinant d1 (1 mg) was immobilised via the his-tag to 12 mg of dynabeads his-tag isolation and pulldown magnetic beads (thermofisher scientific) following the manufacturers protocol. excess protein was removed with 3 washes of 100 mm sodium phosphate ph 8, 1 m nacl, 0.02% (v/v) tween-20. bead immobilised protein was then washed 3 times with selection buffer (pbs, 0.05% (v/v) tween-20, 1× roche complete protease inhibitor per 50 ml). a biomek 2000 laboratory automation work station (beckman coulter) was used to perform 10 rounds of in vitro selex as described previously 29 , using a n35 2′ oh rna library. this starting library was synthesised according to an experimentally and iteratively optimised protocol. it was purchased from aptait (munich, germany) who performed three rounds of synthesis, next generation sequencing (ngs) and statistical analysis of ngs data from the library using the compas software. in doing so the random region of the library has been optimised in respect of an equal distribution of nucleotide building blocks (25% of g, c, a & t, at each position), as well as in respect of a gaussian distribution of motifs of length 4-8 nucleotides. an equal distribution of nucleotides increases the sequence space on the level of shorter motifs, which finally mediate binding to the target of interest. consequently, the chance for a successful selex experiment increases with a homogeneous random region. here the library was initially transcribed with a hiscribe t7 high yield rna transcription kit (new england biolabs). negative selections were carried out at each round of selex using bare his-tag isolation and pulldown beads. the stringency of the aptamer-target interaction was raised by increasing the number of washes from 10 to 15 (slower off-rate) and by decreasing the amount of bead-immobilised protein by half (faster on-rate). selections were carried out at 37 °c. following the final round of selex, aptamers were analysed by ngs using a miseq desktop sequencer (illumina). the selex library was prepared using the miseq reagent kit following the manufacturer's protocol. raw sequencing reads were processed using in-house scripts to identify sequences that contained correct 5′ and 3′ primers. processed sequences were used for packaging signal identification (see below). potential packaging signal identification. the following hcv genotype 2 genome sequences were extracted from genbank: accession numbers ay232741; ay232730; ay746460; d00944; d50409; dq364460; ab030907.1; ab031663.1; hm777359; ab047640; af238481; af238485; af238486; ay232749; ab047640.1. the final selected aptamer library contained 112,117 unique sequences, each 35 nucleotides in length that were aligned against the jfh-1 genome as follows: each aptamer sequence was slid along the genome in increments of 1 nucleotide. for each such position of the reference frame, the subset of the aptamer sequence with the best alignment to the genome was identified according to the bernoulli score b, which benchmarks the probability of a non-contiguous alignment to that of a contiguous alignment of b nucleotides. the bernoulli scores for all reference frames of a given aptamer sequence in the library were rank-ordered, starting from the largest score, and all matches with the genome up to a bernoulli score of 12 counted. the procedure was then repeated for the other aptamer sequences, and corresponding matches added, resulting in the peaks in fig. 2. identification of a consensus motif. this analysis was repeated for all genomes listed above. peaks which occurred for at least 60% of the genomes within < 10 nucleotides of each other were marked by a green arrow to indicate their conservation. these were used as a basis to identify the highly conserved pps recognition motif. for this, sequences of 60 nucleotides, centred around the peak nucleotide, were extracted and all possible mfold predicted folds were considered. a similarity analysis of these stem-loops was performed, comparing both sequence and structure elements. the foldings of each peak area which had the highest degree of similarity with secondary structure elements in the other peaks was identified. this returned a stem-loop for each peak; an alignment of the corresponding terminal loop sequences is displayed in fig. 2 . in order to identify which features of the consensus motif are statistically significant, the number of occurrence of stem-loops with different types of sub-motifs in the genome was calculated and benchmarked against their occurrence in randomised versions of the genome. the consensus motif was grrgr; this motif was 2.05 times more likely to occur in the wildtype than randomised genomes. in order to refine this motif, we performed similar statistical tests for submotifs of this consensus motif; we identified ggrgg and gxrxr motifs as being more likely to occur in the wildtype genome than in a randomised version, by a factor of 2.36 and 1.14, respectively, suggesting that ppss with the former motif could correspond to high affinity pss, and those with the latter motif could correspond to lower affinity pss. mutagenesis of pjfh-1. the dna construct containing the jfh-1 viral genome (pjfh-1) and a replication-defective control mutant of this plasmid (termed pjfh-1-gnd, possessing a gdd > gnd mutation within the ns5b rna-dependent-rna-polymerase active site) have been described previously 3 . mutagenesis of pjfh-1 to introduce silent mutations was performed by either site-directed mutagenesis (quikchange, agilent) or through overlap pcr utilising mutagenic primers (primer sequences available upon request). the entire genome of all mutant viruses was confirmed through sanger sequencing to ensure additional mutations were not present. scientific reports | 6:22952 | doi: 10.1038/srep22952 in vitro transcription. dna constructs were linearised with xbai, briefly treated with mungbean nuclease to degrade 3′ overhangs (new england biolabs) and purified by phenol-chloroform extraction. linearised dna was used as a template for in vitro transcription to produce full-length hcv genomic rna (ribomax express; promega, as per the manufacturer's instructions). following dnase digestion, rna transcripts were purified using silica-gel columns (rneasy, qiagen) and quantified by absorbance at 260 nm prior to transfection into mammalian cells. mammalian cell culture. huh-7 cells were maintained in dulbecco's modified eagle's medium (dmem; sigma) supplemented with 10% foetal bovine serum (fbs), 100 iu/ml penicillin, 100 μg/ml streptomycin, 25 mm hepes and 1% (v/v) non-essential amino acids in a humidified incubator at 37 °c in 5% co 2 56 . infectious hcv assays. 6 .0 × 10 6 huh-7 cells were electroporated with 5 μg of viral rna at 975 μf and 260 v for 25 ms. cells were resuspended in complete medium and seeded into multiple 6 well plates (6.0 × 10 5 cells per well) (corning). 4 hours post electroporation (h.p.e.), cells were harvested with trizol (invitrogen life technologies) for rna quantification. 24 h.p.e., all remaining wells were washed with pbs to remove excessive extracellular input rna and the media replaced. 48 h.p.e., cells were harvested in trizol, protein lysis buffer or pbs, for rna quantification, protein detection and intracellular virus titration, respectively. virus supernatant was harvested at 48 h.p.e. for rna quantification and released infectious virus titrations. intracellular virus was isolated by 5 repeated freeze-thaw cycles of pbs cell lysates followed by clarification. virus titres were calculated according to previously reported methods 25 . briefly, either viral supernatant or cellular lysates were serially diluted two-fold upon naïve huh-7 cells in 96 well plate format, seeded 6 hours prior to infection. at 48 hours post-infection, cells were washed, fixed with 4% paraformaldehyde and the viral ns5a protein was detected via indirect immunofluorescence. automated counting was performed using the incucyte zoom (essen bioscience) using previously-described parameters 25 . quantitative reverse-transcriptase pcr. rna was purified by trizol extraction and diluted to 100 ng/ul. 200 ng was used in a taqman probe-based one-step qrt-pcr according to previously-reported methods 57 . to quantify rna, cycle threshold (ct) values were converted to genome copies per 200ng of input rna by comparison to a standard curve of serially-diluted pjfh-1 (10 2 to 10 9 copies). virion assembly and release replication of subgenomic hepatitis c virus rnas in a hepatoma cell line production of infectious hepatitis c virus in tissue culture from a cloned viral genome dengue virus-and hepatitis c virus-induced replication and assembly compartments: the enemy inside-caught in the web hepatitis c virus rna replication and assembly: living on the fat of the land hepatitis c virus budding at lipid droplet-associated er membrane visualized by 3d electron microscopy hepatitis c virus: assembly and release of virus particles the lipid droplet is an important organelle for hepatitis c virus production cis-acting genomic elements and trans-acting proteins involved in the assembly of rna viruses unique features of hepatitis c virus capsid formation revealed by de novo cell-free assembly solving a levinthal's paradox for virus assembly identifies a unique antiviral strategy revealing the density of encoded functions in a viral rna genetic analysis of the carboxy-terminal region of the hepatitis c virus core protein hepatitis c virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features self-assembly of nucleocapsid-like particles from recombinant hepatitis c virus core protein conformational changes accompanying self-assembly of the hepatitis c virus core protein systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase in vitro selection of rna molecules that bind specific ligands packaging signals in two single-stranded rna viruses imply a conserved assembly mechanism and geometry of the packaged genome direct evidence for packaging signal-mediated assembly of bacteriophage ms2 mfold web server for nucleic acid folding and hybridization prediction building a viral capsid in the presence of genomic rna. physical review. e, statistical, nonlinear, and soft matter physics a cis-acting replication element in the sequence encoding the ns5b rnadependent rna polymerase is required for hepatitis c virus rna replication a twist in the tail: shape mapping of long-range interactions and structural rearrangements of rna elements involved in hcv replication a novel method for the measurement of hepatitis c virus infectious titres using the incucyte zoom and its application to antiviral screening rna structure analysis at single nucleotide resolution by selective 2′ -hydroxyl acylation and primer extension (shape) functional analysis of the murine coronavirus genomic rna packaging signal conservation of a packaging signal and the viral genome rna packaging mechanism in alphavirus evolution degenerate rna packaging signals in the genome of satellite tobacco necrosis virus: implications for the assembly of a t = 1 capsid systematic analysis of enhancer and critical cis-acting rna elements in the protein-encoding region of the hepatitis c virus genome the n-terminal half of the core protein of hepatitis c virus is sufficient for nucleocapsid formation a method for in vitro assembly of hepatitis c virus core protein and for screening of inhibitors hepatitis c virus structural proteins assemble into viruslike particles in insect cells assembly of truncated hcv core antigen into virus-like particles in escherichia coli all three domains of the hepatitis c virus nonstructural ns5a protein contribute to rna binding hcv core residues critical for infectivity are also involved in core-ns5a complex formation the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+ ) strand rna in vitro rna chaperoning and intrinsic disorder in the core proteins of flaviviridae interaction of hepatitis c virus core protein with viral sense rna and suppression of its translation selective binding of hepatitis c virus core protein to synthetic oligonucleotides corresponding to the 5′ untranslated region of the viral genome specific in vitro association between the hepatitis c viral genome and core protein detection of cellular proteins and viral core protein interacting with the 5′ untranslated region of hepatitis c virus rna role of rna structures in genome terminal sequences of the hepatitis c virus for replication and assembly down-regulation of the internal ribosome entry site (ires)-mediated translation of the hepatitis c virus: critical role of binding of the stem-loop iiid domain of ires and the viral core protein hepatitis c virus rna: molecular switches mediated by long-range rna-rna interactions? a long-range rna-rna interaction between the 5′ and 3′ ends of the hcv genome are trans-complementation systems suitable for hepatitis c virus life cycle studies efficient trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles expression of hepatitis c virus (hcv) structural proteins in trans facilitates encapsidation and transmission of hcv subgenomic rna hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus genetic analysis of hepatitis c virus with defective genome and its infectivity in vitro naturally occurring hepatitis c virus subgenomic deletion mutants replicate efficiently in huh-7 cells and are trans-packaged in vitro to generate infectious defective particles detailed mapping of rna secondary structures in core and ns5b-encoding region sequences of hepatitis c virus by rnase cleavage and novel bioinformatic prediction methods detection of genome-scale ordered rna structure (gors) in genomes of positive-stranded rna viruses: implications for virus evolution and host persistence serine phosphorylation of the hepatitis c virus ns5a protein controls the establishment of replication complexes growth of human hepatoma cells lines with differentiated functions in chemically defined medium real-time detection system for quantification of hepatitis c virus genome semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments the authors would like to thank dr iain manfield (university of leeds), dr james ault (university of leeds) and dr sally harrison (leeds institute of molecular medicine next generation sequencing facility) for their help and advice regarding protein expression, mass spectrometry and sequencing, respectively. we also wish to thank dr michael blank of aptait gmbh, munich, for the provision of the n35 ssrna library used for selex and helpful discussions about the preparation of unbiased starting libraries for selex. this work was supported by a wellcome trust investigator award to mh (grant number 096670) and by financial assistance from the universities of leeds and york. rt acknowledges funding via royal society leverhulme trust senior research fellowship (lt130088) and epsrc grant ep/k028286/1 for rjb. ecd has been funded via a leverhulme trust research fellowship (ecf-2013-019). key: cord-324559-p92y5er2 authors: lillie, patrick j.; samson, anda; li, ang; adams, kate; capstick, richard; barlow, gavin d.; easom, nicholas; hamilton, eve; moss, peter j.; evans, adam; ivan, monica; phe incident team; taha, yusri; duncan, christopher j.a.; schmid, matthias l.; the airborne hcid network title: novel coronavirus disease (covid-19): the first two patients in the uk with person to person transmission date: 2020-02-28 journal: j infect doi: 10.1016/j.jinf.2020.02.020 sha: doc_id: 324559 cord_uid: p92y5er2 nan in this journal, zhu et al. recently reported the results of their genomic analysis of multidrug-resistant klebsiella pneumoniae isolates from individual patients before and after colistin treatment highlighting the rapid emergence and multifaceted molecular mechanisms of colistin resistance in k. pneumoniae. 1 this work highlights the therapeutic and public-health challenges of colistinresistance (cr), which is increasingly used as a large resort antibiotic, despite its unattractive toxicity profile and narrow therapeutic window. 2 oral non-absorbed colistin has been proposed as a decontamination strategy in intensive care units and for patients carrying multidrug resistant enterobacterales (mdr-e). 3 , 4 the impact of decolonization strategies in terms of emergence of cr has rarely been monitored because no reliable selective medium existed and cr was not considered a public-health problem. recently, reliable universal culture media have been developed to screen for cr. 5 here, we studied the impact of non-absorbed oral colistin on the emergence of cr in the gut microbiota of patients from the rgnosis-wp3 randomized controlled trial. 6 thirty-nine subjects colonized with mdr-e were randomized to receive oral colistin sulfate 2 miu 4 times a day + neomycin sulfate 500 mg bid for 5 days followed by a fecal microbiota transplant (fmt) from healthy donors, or no intervention. stool samples were collected on visit v0 (screening sample), v2 (after 5 days of oral decontamination and before fmt for the intervention group), v3, v4 and v5, respectively 5-10 days, 30-40 days and 150-210 days later. 6 stool samples from donors and 15 subjects from the intervention group and 15 from the control group were available for this work and plated on drigalski plates (control) and superpolymyxin r plates. 5 colony forming units (cfu) counts of all gram-negative rods were determined. isolates growing on superpolymyxin r plates were identified by maldi-tof; cr was confirmed by the culture-based rapid polymyxin np test and mic determined by the microdilution method. the limit of detection was 10 2 cfu/g of stool. cr-e. coli were sequenced using the illumina hiseq technology. to determine whether cr isolates were present before the intervention, a specific mcr-1 pcr was performed on patients stool prior to intervention (v0) and on the donor's stool. 7 electroporation of plasmids was performed to localize the gene conferring resistance to colistin and neomycin and molecular typing of the electroporants was performed using pcr based replicon typing (pbrt). ✩ université de paris, iame, inserm, umr-1137 no patient or donor included in the trial carried cr isolates on v0. among the 15 patients in the intervention group two (13.3%, [ic90 −1; 3], p = 0.24) carried cr isolates at least at one visit after the intervention ( fig. 1 ) . no cr-enterobacterales was detected in the stools of subjects from the control group. among both subjects with cr-enterobacterales, one carried 10 log cfu/g of hafnia paralvei , a species which is intrinsically resistant to colistin (mic = 8 mg/l), also resistant to neomycin (mic = 32 mg/l) on visit 4 and the other carried 1 log cfu/g and 3 log cfu/g cr-e. coli at visits 4 and 5, respectively, with a colistin mic at 8 mg/l. relative abundance of cr-e. coli increased between v4 and v5 from 0.01% to 1% of the total enterobacterales population. the cr-e. coli recovered at v4 and v5 both belonged to phylogroup c st23 group and carried the serotype o176:h9. a plasmid-borne mcr-1.1 gene encoding for cr as well as a aph(3 )-ia gene conferring resistance to neomycin were identified, both being co-located on the same inchi2 plasmid. in addition, resistance genes conferring resistance to hygromycin b ( aph(4)-ia ), sulfonamides ( sul2 ), tetracyclines ( tet(a) ) and phenicols ( flor and cata1 ), all antibiotics used in veterinary medicine, were evidenced. for both subjects, cr strains could not be retrieved in the initial stool of the subject or in the donor's stool. pcr experiments performed with specific primers to detect mcr-1 gene directly on the pre-therapeutic stool were also negative. to our knowledge, this is the first report of the in-vivo selection of cr-enterobacterales in the gut microbiota of patients after oral decontamination by colistin. the selection of cr strains (a naturally-resistant h. paralvei and a mcr-1 producing e. coli ), both resistant to colistin and neomycin, may be the result either of the enrichment process by sod of preexisting cr strains that had not been initially detected because of very low abundances, or of an exogenous acquisition, either from other individuals or through fmt. 8 indeed the transmission from fmt of mdr strains from positive donors is a potential risk. 8 despite our efforts to decrease the limit of detection of mcr producers by using a pcr technique directly on the pre-therapeutic stool sample and the donors' stools, we failed to detect the parental strain, either because cr strains were in intestinal niches, the limit of detection remained too high, or the strain was acquired exogenously. however, the mcr-1 -positive e. coli is likely of animal origin according to its genetic features and its co-resistance profile. 9 indeed, phylogroup st23 is frequently encountered among avian pathogenic e. coli (apec) and co-resistances to many antibiotics used specifically in veterinary medicine is striking. 10 furthermore, the aph(3 )-ia gene confers resistance to neomycin and paromomycin, the latter commonly used in cattle and pigs. the selection of the mcr-1 producer is an illustration of the "one health" problem of resistance: a strain likely to have been selected by veterinary antibiotics among animals ended up in a patient's gut, later enriched by the use of colistin and neomycin as decontaminant. although the small number of subjects is a clear limitation, this observation is a "proof-of-concept" of the risk of selection of cr-enterobacterales after oral colistin treatment and fmt, at a time when colistin is one of the last resort antibiotics to treat mdr-enterobacterales infections. the selection of commensal cr-e. coli is especially worrying, given the pathogenic potential of e. coli and its ability to widely colonize animals and humans. given the controversial results of oral decontamination by colistin, we believe it should only be used with precautions and with thorough monitoring of cr. we read with interest a recent paper in this journal by luzatti and colleagues, 1 who explored the significance of the presence of herpes simplex virus (hsv) dna in lower respiratory tract (lrt) specimens for the diagnosis of hsv pneumonia in immunocompromised patients. the authors underlined the difficulty in gauging the clinical relevance of such a laboratory finding in the absence of histopathological data, as hsv shedding in the lrt may occur in the absence of disease. the interpretation of real-time pcr results for diagnosis of pneumocystis jirovecii (pj) pneumonia (pjp) faces an analogous challenge, since the presence of pj dna in lrt may reflect colonization (carriage) rather than infection. 2 there is limited information on the clinical value of pj real-time pcr in diagnosing pjp in patients with hematological diseases; 3-6 this is exceedingly challenging as the sensitivity of direct examination procedures is suboptimal due to low fungal burdens. 3 here, we report on our experience on this matter. a total of 219 episodes of pneumonia occurring in 192 consecutive patients with hematological disorders in which pjp was considered in the differential etiological diagnosis were included. of these, 127 episodes developed in patients undergoing either allogeneic hematopoietic stem cell transplantation-allo-hsct-( n = 86) or autologous-hsct ( n = 19), and 92 in non-transplant patients (acute leukemia, n = 61; lymphoma, n = 16; chronic leukemia, n = 8; others, n = 2). the patients were attended at the hospital clínico universitario-hcu-( n = 100) or at the hospital universitario politécnico "la fe" -hlf-( n = 92) between june 2014 and august 2019. no patients in the cohort tested positive for hiv. this study was approved by the respective hospital ethics committee and informed consent was obtained from all patients. a single specimen per episode was available for diagnosis (bal fluids, n = 179; sputa, n = 22; ta, n = 17 and bronchial biopsy, n = 1). the realcycler pjir kit r (progenie molecular, spain) was used at hcu, and the pneumocystis jirovecii real time pcr detection (certest biotech; zaragoza, spain) was employed at hlf (see footnote in table 1 ). both assays target the large sub-unit of ribosomal (mtlsu) rna gene. preliminary experiments using 5 bal specimens indicated that both assays yield comparable pcr cycle thresholds (c t s) (median, 28.2, range, 26.4-32.3 vs. median 27.5; range, 26.3-33.1, respectively; p = 0.89). all specimens tested negative by direct examination for pj, whereas 27 were positive by real-time pcr (bal, n = 18; sputa, n = 7, and ta, n = 2); following stringent clinical, microbiological and imaging criteria ( table 1 ) , pjp was deemed to be the most probable diagnosis in 12 episodes occurring in unique patients. no histopathological confirmation of pjp was available for any patient. pcr c t values inversely correlate with fungal burden in the sample. 6 which is higher in patients with pjp than in colonized individuals. 2 here, overall, pj pcr c t s in specimens from patients with pjp tended to be lower than in pj carriers ( p = 0.39); when only bal fluid specimens were considered, the difference reached statistical significance (median, 29.0; range, 26.4-34.7 vs. median 34.6; range, 30.0-41.0; p = 0.04). this finding is likely related to use of more standardized procedures for bal fluid sampling. receiver operating characteristic (roc) curve analysis showed that a threshold c t value of 30.0 in bal specimens displayed a sensitivity of 85.7% (95% ci, 45.0-100%) and a specificity of 80% (95% ci, 40.8-100%) for pjp diagnosis. a number of studies have established different c t s cut-offs for that purpose, [6] [7] [8] [9] . in our view, however, the variability in the performance of different pcr assays and sampling conditions, heterogeneity of patient populations, and in particular the lack of a pj international standard material for pcr result normalization precludes defining a consensus universal threshold nowadays. the absence of anti-pj prophylaxis, treatment with corticosteroids and serum ldh levels ≥400 u/l have been shown to be associated with pjp. 3 here, patients not undergoing anti-pj prophylaxis were more likely to display a clinically significant pj pcr result ( table 1 ). in turn, roc curve analysis indicated that a cut-off ldh value ≥400 u/l had a sensitivity of 81.8% (ci 95%, 59.0-100%) and specificity of 67% (95% ci, 34.0-99.3%) for pjp diagnosis. in univariate regression logistic models, serum ldh values ≥400 u/l were associated with a clinically significant positive pcr pj result (or, 9.0; 95% ci, 1.2-63.8; p = 0.02). in contrast, corticosteroid use within the month before sampling was not different between the probability of pneumocystis jirovecii (pj) pneumonia (pjp) for each patient was retrospectively evaluated by an expert committee including infectious diseases and microbiology specialists at both centers, on the basis of (i) documented pj presence in respiratory specimens by microscopy; (ii) compatibility of clinical signs and symptoms (at least 2 of the following: subtle onset of progressive dyspnea, pyrexia, nonproductive cough, hypoxaemia and chest pain), (iii) compatible (suggestive) radiological findings (chest radiograph and/or high-resolution computed tomographic scan detection of interstitial opacities and/or diffuse infiltration infiltrates); (iv) complete resolution of symptoms after a full course of anti-pjp treatment; (v) absence of alternative diagnosis. the efficacy of therapy was assessed on a daily basis. pjp was ruled out if real-time pcr for pj tested negative, or if clinical recovery occurred in the absence of pj-targeted antimicrobial therapy. pj colonization (carriage) was the most likely possibility when patients did not meet the above criteria and an alternate diagnosis was made. b frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. two-sided exact p values were reported and p values ≤ 0.05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. c respiratory tract specimens were obtained following conventional procedures. specimens were examined for presence of ascus or trophic forms of pj by microscopy following blue toluidine, calcofluor white or grocott's methenamine silver staining. cytospin preparations were prepared from bal specimens for direct examination. sputa and ta samples were mixed v/v with sputasol (oxoid, uk) and vortexed for 5 min. all samples were centrifuged at 30 0 0 g for 10 min, and the pellets were resuspended 1/10 in 0.9% nacl for further processing. for real-time pcr, dna was extracted from 200 μl of specimens using the qiaamp dna blood mini kit (qiagen, hilden, germany) on either qia symphony or ez-1 platforms (qiagen), following the manufacturer's instructions. at hcu, a commercially-available real-time pcr assay previously evaluated by others, the realcycler pjir kit r (progenie molecular, spain), which targets the mitochondrial large sub-unit of ribosomal (mtlsu) rna gene, was used according to the manufacturer's instructions ( http://www.progenie-molecular.com/pjir-u-in.pdf ). at hlf, the commercially-available pneumocystis jirovecii real time pcr detection. (certest biotech; zaragoza, spain), which also targets the large sub-unit of ribosomal (mtlsu) rna gene, was employed following the manufacturer instructions ( https://www.certest. es/wpontent/uploads/2019/02/viasure _ real _ time _ pcr _ pneumocystis _ jirovecii _ sp1.pdf ). at both centers pcr were performed in the applied biosystems 7500 fast real-time pcr platform (applied biosystems, ca, usa). pcr results were reported as positive or negative. for positive samples, threshold cycle (c t ) values were also recorded. no standard curve was generated with a positive control for quantitative estimations. d antimicrobial prophylaxis for pjp was performed with trimethoprim-sulfamethoxazole (tmp/smx), one double-strength tablet (160 mg tmp/800 mg smx) given 2 (in allogeneic hsct patients) or 3 times a week with oral folic acid (15, 16) . patients with suspicion of pjp according to the attending physician were treated with tmp/smx 15-20 mg/kg (tmp) 75-100 mg/kg (smx) per day for 2-3 weeks. e in all these cases, death was attributable to pjp. patients with clinically significant pj detection and pj carriers ( table 1 ) . detection or recovery of other microbial agents (one or more) was documented in 17 of the 27 specimens testing positive by pj pcr ( table 2 ). in line with a previous report, 9 this microbiological finding was significantly less frequent ( p = 0.001) in specimens from patients with pjp than in colonized patients; in fact, microbial co-detection was inversely associated with pjp in univariate logistic regression models (or, 0.024; 95% ci, 0.002-0.26; p = 0.002). strengths of the current study are the following: (i) clinical categorization of pjp was based upon stringent criteria defined by a multidisciplinary team; (ii) only hematological patients were included; (iii) a comprehensive routine investigation of microbial causes of pneumonia other than pj was conducted; (iv) the experience of two centers was collected. in addition to its retrospective nature, our study also has some limitations: (i) we cannot completely rule out that some patients categorized as being pj carriers did in fact have pjp, as most of these patients received full courses of tmp/smx in combination with antimicrobials targeting other microbial agents. the lack of standardized criteria for pjp diagnosis makes clinical misclassification of patients a potential drawback in studies such as ours, particularly when no positive microscopy or histopathology findings are available; (ii) although we evaluated bal, bronchoalveolar lavage; pjp, pneumocysis jirovecii pneumonia; ta, tracheal aspirate. a as per our routine protocol, all specimens were examined by gram and acid-fast bacilli stain. samples were also examined for presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, or the clart® pneumovir assay (genomica, coslada, spain) at both centers, as previously reported. 10 semiquantitative (sputa) and quantitative (bal and ta) cultures for bacteria were performed on conventional media: bacterial loads > 10 4 cfu/ml or > 10 5 cfu/ml were deemed to be clinically relevant on bal fluids and ta samples, respectively. specimens were cultured on bcye-alpha agar, bd (becton dickinson) mgit® ( mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and other fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. all bal fluid specimens underwent cytomegalovirus (cmv) pcr testing using the realtime cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf, as previously reported. 10 over 200 patients, only 12 presumptively had pjp; (iii) two different commercially-available pcr assays were used across centers. nevertheless, we found them to yield rather comparable c t s. in summary, we found that a positive pj pcr result in respiratory specimens from transplant and non-transplant hematological patients with pneumonia frequently reflects colonization rather than infection; pcr c t values in bal fluids, serum ldh levels and lack of co-detection of other microorganisms potentially involved may be helpful in clinical categorization in the absence of positive by pj microcopy results. we have no conflict of interest to declare. dear editor , poller et al., in this journal, provided a useful consensus for use of personal protective equipment for managing high consequence infectious disease 1 . although this was driven largely by recent ebola virus disease emergencies, we should remind your readers of the continuing problem of lassa fever (lf) in west africa. lf is a febrile infectious disease caused by lassa virus. the clinical presentation of the disease is nonspecific and includes fever, fatigue, hemorrhage, gastrointestinal symptoms, respiratory symptoms, and neurological symptoms 2 . the observed case fatality rate among patients hospitalized with severe lf is 15-20% 3 , 4 . the disease is mainly spread to humans through contamination with the urine or faeces of infected rats 2 . human-to-human transmission can occur through contact with the body fluids of infected per-sons. therefore, health care workers are at high risk for infection when the standard precautions for infection prevention and control including appropriate personal protective equipment are inadequate 5 . it is estimated that there are approximately 30 0,0 0 0 lf cases annually, resulting in approximately 50 0 0 deaths in west african countries 4 . in 2018, nigeria had a large lf outbreak, and we previously reported epidemiological characteristics of the outbreak, analyzing data collected between january and may 2018. 6 however, information on laboratory-negative suspected cases was not enough to conduct a case-control study to fully determine the risk factors and clinical characteristics of the disease. nigeria had a lf outbreak in 2019 as well. 7 here we report the epidemiological and clinical characteristics of the outbreak including case-control analysis against laboratory-negative suspected cases using data collected between 1st january 2018 and 6th october 2019. from january to december 2018, there were 3498 suspected cases, including 633 laboratory-confirmed lf cases. in 2019, there were 4019 suspected cases reported by 6th october, including 721 laboratory-confirmed lf cases. details on the case definition, laboratory test, surveillance, and data collection have been described previously. 6 of the confirmed lf cases, there were 171 fatalities (case fatality rate, 27.0%) in 2018 and 154 fatalities (case fatality rate, 21.4%) in 2019. the number of laboratory-confirmed lf cases and positivity rate peaked in the dry season (january-march) in both 2018 and 2019 ( fig. 1 (a) ). the largest number of laboratory-confirmed lf cases were reported from the neighboring edo and ondo states in both 2018 and 2019 ( fig. 1 (b) ). there were laboratory-confirmed lf cases in states such as kebbi and zamfara that had no reported cases previously, in 2019. during the study period, the detailed demographic and clinical information was collected for 1305 laboratory-confirmed lf cases (of 1354 cases, 96.4%) and 3350 laboratory-negative suspected cases (of 6163 cases, 54.4%). chi-square tests were conducted to compare the distribution of age, sex, and each symptom between the laboratory-confirmed lf cases and laboratory-negative suspected cases ( table 1 ). the proportion of children was significantly lower in laboratory-confirmed lf cases compared with that in laboratory-negative suspected cases. the proportion of males was significantly higher in laboratory-confirmed lf cases than that in laboratory-negative suspected cases. fever was the most prevalent symptom in both laboratoryconfirmed lf cases and laboratory-negative suspected cases, followed by headache ( table 1 ) . gastrointestinal symptoms, such as abdominal pain, vomiting, and diarrhea, were observed in more than 30% of laboratory-confirmed lf cases, whereas hemorrhaging was observed in 21.1% of laboratory-confirmed lf cases. while the prevalence of face/neck edema was low even in laboratoryconfirmed lf cases (6.4%), nonetheless, the odds ratio of having face/neck edema was 6.0 times high for laboratory-confirmed lf cases. we here reported the lf outbreak in 2018-2019 largest recorded in history. while previous studies have focused on laboratory-confirmed lf cases and mainly compared fatal cases and survived cases, 6 , 8 , 9 our observation revealed the difference between laboratory-confirmed lf cases and laboratory-negative suspected cases. the age and sex distribution differed significantly between laboratory-confirmed lf cases and laboratory-negative suspected cases. fever, headache, and gastrointestinal symptoms were the most common symptoms in laboratory-confirmed lf cases, which are similar to those reported previously. 6 , 8 however, these symptoms were also prevalent in laboratory-negative suspected cases. clinical guidelines for lf state that edema in the face and neck is a specific sign of the disease. 10 the present study found that the symptom had a significantly high odds ratio for confirmed lf although the prevalence of this symptom was low. unfortunately, we did not determine the differential diagnosis for the laboratory-negative suspected cases. laboratory tests for the differential diagnoses are now underway for the lf-negative samples collected during the outbreak. the results would provide us further insight for better clinical management of patients with febrile illnesses in lf-endemic areas. in addition to the standard precautions for infection prevention and control including appropriate personal protective equipment pointed out by poller et al., 1 it is important to know epidemiological and clinical characteristics of high consequence infectious diseases such as lf. that would help healthcare workers and public health officers increase an index of suspicion of the diseases, further leading to better clinical management and surveillance. the authors have declared that no conflicts of interest exist. this work was partially supported by the leading initiative for excellent young researchers from the ministry of education , culture, sport, science & technology of japan and the japan society for the promotion of science (grant number, 16809810 ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 these authors contributed equally to this article. accepted 31 december 2019 available online 15 january 2020 https://doi.org/10.1016/j.jinf.2019.12.020 © 2020 the british infection association. published by elsevier ltd. all rights reserved. recently, several studies in this journal have demonstrated the threat of animal-derived viruses to humans. [1] [2] [3] since 2018, an increase in human pseudorabies virus (prv) infection cases has been reported in china, indicating a new animal-derived virus threat to human health. porcine pseudorabies (pr), also known as aujeszky's disease, is one of the most economically important viral diseases in pigs globally. its causative agent is prv, which is classified into the genus varicellovirus of subfamily alphaherpesvirinae , family herpesviridae . prv is almost always fatal in newborn piglets, is frequently accompanied by neurological symptoms, and may cause abortions and/or stillbirths in pregnant sows. prv primarily infects members of the suidae family and can also infect other domestic and wild mammals, including horses, cattle, sheep, goats, dogs, cats, etc. currently, vaccination is the most effective strategy for pr prevention and control in pigs worldwide. in china, prv infections in pigs were first recorded in 1947. in the 1970s, an inactivated vaccine consisting of the bartha-k61 vaccine strain was imported into china. since then, this vaccine has been widely used in pig vaccination for pr prevention and control. before 2011, no large pr outbreaks were reported in pigs in china. however, after late 2011, novel prv wild-type variants emerged in nearly all regions of china and affected a number of swine herds vaccinated regularly with the bartha-k61 vaccine, resulting in significant economic losses. 4 subsequent animal experiments indicated that the bartha-k61 vaccine could not provide complete protection for pigs against a challenge with novel prv wild-type variants in china. for control and eradication of pr, the disease was listed in the "mid-and long-term animal disease prevention and control program in china (2012-2020)" by the chinese government, with the aim of eliminating pr in china by 2020 ( http://www.gov.cn/zwgk/2012-05/25/content _ 2145581.htm ). however, vaccination for prv is still voluntary and not required in china. a nationwide epidemiological investigation in 2014 demonstrated a high prevalence of 23.26% of prv among swine herds in china. 5 humans were previously regarded as refractory for prv infection, although serological prv antibody positivity was found in three cases. in 2018, the first human prv infection case with direct molecular evidence was reported in china (case 1, table 1 ). 6 in this case, the eyes of a 46-year-old woman were directly exposed to sewage on a hog farm. in the following two weeks, symptoms of fever, headache, coma, and endophthalmitis were observed in the patient. next-generation sequencing (ngs) indicated that prv dna was detected in her vitreous humor samples but not in her cerebrospinal fluid (csf). after surgery, the patient was discharged, but her vision remained impaired. in a subsequent study, zhao et al. table 1 clinical characteristics and other information on the twelve human prv infection cases in china. analyzed csf samples from four patients with encephalitis of unknown etiology using ngs (cases 2-5, table 1 ) and found molecular evidence of prv infection. 7 in addition, retinitis and blindness was observed in two cases (cases 2, 5, table 1 ), and the patient in case 3 died. the occupation of the four patients was all associated with pork production/sale/cooking. in 2019, six other human prv infection cases involving encephalitis were reported in china, and all patients were pork/pig handlers or veterinarians. [8] [9] [10] it was noted that all patients still suffered from various sequelae after discharge, except for in one case where the patient died. increasing reports on human prv infection cases in china have recently indicated that prv poses a significant threat to public health in china, especially in people in close contact with sick pigs and/or related pork products/contaminants. to reduce the risk of prv infection in susceptible workers, it is necessary to promulgate relevant policies by the chinese government to promote pr vaccine development to protect pigs from infection with novel prv wild-type variants currently circulating in china. in addition, relevant policies should be updated by the chinese government to monitor vaccination status and virus variation in pigs nationwide. moreover, it seems that prv can infect humans via injury to the skin or eyes. until now, no effective drugs to prevent the progression of the disease caused by prv infection have been reported. therefore, it is necessary to improve biosafety and self-protection awareness in susceptible populations that have contact with sick pigs and work in jobs related to handling pork products/contaminants. promoting drug development for curing prv-related disease in infected patients may also help reduce the currently increasing threat of prv to human health in china. all authors declare that they have no competing interests. dear editor, the emergence and spread of gram-negative bacteria, for example, klebsiella pneumoniae , co-producing carbapenemases and mobilized colistin resistance ( mcr ) genes limit our choice for treating multidrug-resistant infections, posing significant threats to public health. herein, we reported the discovery of mcr-9 gene in 28 k. pneumoniae strains isolated from patients in eight european countries, including belgium ( n = 2), denmark ( n = 1), montenegro ( n = 13), poland ( n = 1), romania ( n = 1), serbia ( n = 1), slovenia ( n = 1), and spain ( n = 7). notably, the co-existence of mcr-9 and the carbapenemase-encoding genes, ndm-1, vim-1, and oxa-48 were confirmed in k. pneumoniae isolates of human origin. phylogenetic analysis suggested that mcr-9 -carrying k. pneumoniae isolates, including 23 carbapenem-resistant and five susceptible k. pneumoniae strains, show a highly geographically clustered pattern. genetic environment analysis revealed the presence of insertion element is903, is1 or a cupin fold metalloprotein, wbuc, in the mcr-9 flanking. taken together, these findings indicated that mcr-9 has existed for a long time and already spread among crkp isolates of human origin in europe since 2013, further increasing the significant threat of public health through either the nosocomial spread or environmental routes. the mobilized colistin resistance ( mcr ) gene mcr-9 was detected in 28 human gut microbiomes, which has been disseminating across three continents, including asia, europe and america, recently published in the journal of infection. 1 the rapid increase in carbapenem resistance among gram-negative bacteria worldwide has greatly compromised the efficacy of carbapenem antibiotics, which has gotten renewed attention to the importance of polymyxin antibiotics for multidrug-resistant (mdr) infections. recently, sophia david and colleagues 2 reported the epidemic of carbapenem-resistant klebsiella pneumoniae (crkp) in europe, which raised concern that mobile carbapenemase resistance determinants were widely spread in european hospital settings, and inter-hospital spread is far more frequent within, rather than between, countries. however, limited information regarding the co-occurrence of carbapenemases and mcr genes in the klebsiella pneumoniae ( k. pneumoniae ) isolates have been provided. plasmid-mediated resistance genes mcr as well as tet (x3/x4) have been widespread in bacterial species of animal, human, and environment origin as well as human and animal gut microbiomes worldwide, where is a huge arg reservoir with a high horizontal gene transfer possibility. 1 , 3 -5 several studies 6 -8 illustrated that the newly described mcr-9 has spread beyond the united states into europe and asia, and into other enterobacteriaceae species. of clinical concern is the inevitable spread of a plasmid harboring the mcr-9 gene into a crkp isolate, which has been listed by the world health organization as a critical priority antibiotic-resistant bacterial pathogen for which new antibiotics are urgently needed. 9 in response to this potential clinical problem, we download > 1717 k. pneumoniae genomes isolated from 244 hospitals in 32 european countries 2 and the 14,764 complete bacterial genome sequence (accessed 26 july 2019), and explored the distribution of plasmid-mediated resistance genes mcr and tet (x3/x4). we are surprised to find that the complete mcr-9 gene (nucleotide = 100%) was present in 28 k. pneumoniae isolates of 1717 k. pneumoniae strains from belgium ( n = 2), denmark ( n = 1), montenegro ( n = 13), poland ( n = 1), romania ( n = 2), serbia ( n = 1), slovenia ( n = 1), and spain ( n = 7) (table s1 ). additionally, 23 of the 28 k. pneumoniae isolates of human origin were crkp strains and only five were carbapenem-susceptive isolates (table s1 ). as reported, these mcr-9 -harbouring strains were isolated from patients in europe between 2013 and 2014. these results suggest that mcr-9 gene might have been presented in europe for a long time and already spread to the crkp isolates, which is a major cause of both hospital-and community-acquired infections. 2 to further analyze these mcr-9 -positive k. pneumoniae isolates, the resistome of the draft genome was analyzed using the comprehensive antibiotic resistance database. 10 interestingly, the mcr-9 gene was co-existed with various carbapenemase-encoding genes: in eleven isolates with ndm-1, eight with vim-1, and two with oxa-48 ( fig. 1 (a) ). it should be noted that the mcr-9 -and ndm-1-carrying isolates were distributed in denmark ( n = 1), montenegro ( n = 8), romania ( n = 1), and serbia ( n = 1), as well as several other beta-lactam resistance determinants (for example, tem-1, cmy-16, oxa-10 and shv-11) ( fig. 1 (a) and table s1 ). moreover, the mcr-9 -and vim-1-harbouring isolates were dominant in spain ( n = 7) and slovenia ( n = 1), as well as two beta-lactamase-encoding genes (non-carbapenemases) (ctx-m-9 and shv-11) ( fig. 1 (a) and table s1 ). it is worrying that a crkp isolate from spain in 2014 was carrying mcr-9 , vim-1, and oxa-48 genes simultaneously. the presence of mcr-9 in crkp isolates from patients is of critical importance as mcr-9 could be present in hospital-borne outbreaks cre strains in the future. from whole-genome shotgun (wgs) data of the 28 mcr-9 -positive k. pneumoniae isolates, sequences types (sts) were extracted and assigned to nine different types, i.e. , 274, 461, 15, 16, 416, 1890, 37, 1942, and 147 ( fig. 1 (a) ). phylogenetic analysis suggested that mcr-9 -carrying k. pneumoniae isolates show a highly geographical clustering pattern 2 ( fig. 1 (a) ). isolates from patients in the same hospital were clustered into one clade, for example, in spain and montenegro. overall, the k. pneumoniae isolates from different countries were genetically diverse, suggesting that the mcr-9 -positive k. pneumoniae isolates were also genetically diverse and that mcr-9 could disseminate among different k. pneumoniae isolates, mainly by nosocomial transmission. 2 nowadays, all known mcr genes have been detected in various gram-negative bacterial species, whereas a small number of studies have shown the presence of mcr-1, mcr-3, mcr-7 , and mcr-8 in k. pneumoniae isolates from animal and human origin at relatively low detection rate. 11 -14 the presence of mcr-9 in the crkp isolates indicated that this novel mcr gene may already be widely spread among k. pneumoniae isolates of human origin in europe. we subsequently searched mcr-9 gene in 14,764 complete bacterial genome sequence and ncbi-nr database (14 october 2019) in the ncbi, to fully understand the prevalence of mcr-9 gene in klebsiella species isolates. interestingly, the mcr-9 gene (identity > 99% and 100% query coverage) was present in various bacterial genomes, including three klebsiella species isolates consisting of k. pneumoniae ( n = 4), k. quasipneumoniae ( n = 1), and k. oxytoca ( n = 3) (table s3) . therefore, further studies focusing on the epidemiology and transmission mechanism of mcr genes, in particular mcr-9 in klebsiella species of human origin are warranted to better understand the public health threat of emergence of antibiotic resistance among clinical k. pneumoniae . contigs carrying mcr-9 in 28 k. pneumoniae isolates could be classified into two groups (for example, gca_900513855.1 and gca_900501685.1) (table s2) . genetic environments analysis indicated that the presence of insertion element is903 and wbuc (a cupin fold metalloprotein), in the mcr-9 (gca_900513855.1, ∼28 kb) upstream and downstream flanking, respectively, similar to (identity > 99%) the plasmid sequences of pme-1a, pctxm9_020038, and pmrvim0813, and contigs from of e. coli isolate 68a and nz_naan010 0 0 063 from salmonella 15 ( fig. 1 (b) ). additionally, mcr-9 in another contig ∼8.7 kb was in the upstream of two insertion element is1 and is30 0 0, as well as a beta-lactamase-encoding resistance gene ctx-m-9, which similar to the plasmid sequence of pmrvim0813. we did not detect the downstream regulatory genes (qsec and qseb) found in the isolates that harbor mcr-9 . 8 , 16 moreover, we were unable to determine whether a complete is903 element is upstream due to a short mcr-9 -bearing contig that is available for comparison ( fig. 1 (b) ). therefore, a long-read sequencing coupled with a hybrid assembly method is needed to fully evaluate and monitor the transfer and development of args, especially mcr-9 among crkp isolates. although two unique plasmid-mediated tigecycline resistance genes firstly discovered in bacteria of animal origin in china and subsequently identified in many bacterial isolates of human, animal and environment origin, including klebsiella species, as well as human and animal gut microbiomes, 1 , 4 , 5 none of them was detected in the 1717 k. pneumoniae strains in europe. in summary, we reported the discovery of mcr-9 gene in 28 clinical k. pneumoniae strains of human origin in eight european countries. importantly, the mcr-9 gene was co-existed with different carbapenemase-encoding genes in the same strains. the spread of mcr-9 , ndm-1, vim-1, and oxa-48 and other beta-lactam resistance determinants (non-carbapenemase) carrying by crkp appears likely to be by plasmid dissemination, as the genes identified in isolates belonging to a diverse set of sts distributed in different hospitals in europe. it is noteworthy that all these mcr-9 -positive crkp strains were isolated between 2013 and 2014, highlighting an earlier presence of mcr-9 among crkp around the world than previously known. these findings raise the likelihood of ongoing undetected mcr-9 gene spread among cre strains. therefore, further study is urgently needed to understand the prevalence and dissemination of mcr-9 , especially in cre and crkp strains, and effective measures should be taken to control its spread. g.f.g. designed the study. y.n.w. and f.l. collected and downloaded the datasets. y.n.w., f.l., y.f.h., b.l.z., g.p.z., and g.f.g. analyzed and interpreted the data. y.n.w. and g.f.g. wrote the draft of the manuscript. all authors discussed, reviewed and approved the final report. supplementary information is available for this paper. correspondence and requests for materials should be addressed to g.f.g. the authors declare no competing interests. the interesting systematic review by amin-chowdhury and colleagues provides information about outbreaks of severe pneumococcal disease (spd) in closed settings that occurred in the conjugate vaccines era 1 . it shows that vaccine-type spd outbreaks are still occurring and it highlights the lack of consensus on how to manage such outbreaks. in the following, we will describe how we managed a recent outbreak of spd in norway. in march 2019, møre and romsdal hospital trust notified the norwegian institute of public health (niph) about a cluster of spd amongst men working in shipyards in møre and romsdal county. serotype data from niph were available for nine of the cases -all were serotype 4. the majority of cases had been working at one specific shipyard. municipal medical officers (mmo), the norwegian labour inspection authority (nlia), and niph formed a multidisciplinary outbreak team to investigate and control the outbreak. we formed specific case definitions: each case had to have resided in møre and romsdal county in the period from 1. january 2019 onwards and: confirmed : had invasive pneumococcal disease (ipd) with serotype 4 isolated from a normally sterile site. probable : worked at the specific shipyard and had a clinical presentation compatible with lower respiratory tract infection or ipd, but without microbiological confirmation or serotype 4 isolated from a non-sterile medium (e.g. nasopharyngeal swab or sputum culture). we identified 20 cases, ten confirmed and ten probable in the period between 28. january and 3. april ( fig. 1 ). all available isolates were serotype 4 (10 confirmed, 7 probable) and were susceptible to penicillin. fifteen isolates were sequence type (st) 801, while two were a single locus variant of 801, st 15,063. all cases were men between 20 and 60 years, with a mean age of 47 years. fifteen were hospitalized. four were norwegian citizens, the remaining came from other european countries. seven cases smoked. one case had an underlying medical risk condition. immunization history against pneumococci were unknown for all. the cases had several professions; mostly related to interior outfitting and metal welding. approximately 1800 individuals worked at the shipyard in the time period. many of them lived in temporary accommodation. at an on-site inspection of the shipyard, nlia observed a polluted atmospheric work environment and little use of personal protective equipment. several measures were put in place to control the outbreak, including information and advice to raise symptom awareness and to reinforce hand and respiratory hygiene, vaccination and occupational corrections. local medical clinics and hospital were alerted about the outbreak and advised to have a low threshold to admit and treat suspected cases. mmo held information meetings with shift leaders, and written information about spd in several languages was distributed to workers to increase spd awareness. intensified hygiene measures were implemented at the ship yard and housing quarters. nlia ordered immediate occupational corrections related to controlling the atmospheric work environment. niph recommended vaccination with the 13-valent conjugate vaccine (pcv13) to interrupt transmission and prevent disease. both the pcv13 and the 23-valent polysaccharide vaccine provide protection against serotype 4, but pcv13 was preferred as this may also affect colonization. as several work tasks were conducted in parallel process in confined spaces with suboptimal ventilation, we were unable to identify a single target group for vaccination. hence, the shipyard offered vaccination to all workers. occupational health service promptly vaccinated all workers during a four-day period. contrary to the majority of studies included in the systematic review, niph did not recommend chemoprophylaxis. as the workers were otherwise healthy (i.e. no high risk group like old age, immunocompromising conditions etc.), and since it was impossible to target a specific group of workers, niph deemed it undesirable to distribute antibiotics to 1800 asymptomatic workers, with the possibility of inducing antimicrobial resistance and possible side effects. due to high turnover of personnel it was not possible to calculate an attack rate. we did not find any new cases after control measures were implemented. no deaths have been reported in relation to the outbreak. this outbreak closely resembles one of the outbreaks described in the systematic review; between april and june 2015, an outbreak with serotype 4, st 801 occurred at a shipyard in belfast 2 . we are also aware of an outbreak this fall, 2019, at a shipyard in finland with serotype 4 (st 801), 8 and 12f 3 . although welders are a known risk group for spd, 4 in all these three outbreaks, people who worked closely alongside welders were also infected. in addition to exposure to welding fumes, the crammed and poorly ventilated working conditions, and possibly housing conditions, may have increased the risk of developing spd and facilitated the transmission of pneumococci in this closed setting. overall, this norwegian outbreak extends the knowledge about how to manage and control outbreaks of spd in closed settings. none. in this journal brunet and colleagues 1 discussed reactivation of latent infections in the context of chronic disease, solid organ transplantation or long-term immunosuppressive treatment. we recently observed the reactivation of leishmania infection in a 46-year-old patient receiving methotrexate for psoriasis, who was diagnosed with visceral leishmaniasis (vl) showing a mucocutaneous involvement. we analyzed the epidemiologic and clinical characteristics of all cases of leishmaniasis in patients with psoriasis found through a review of the literature. our patient was admitted into the infectious disease unit of paolo giaccone hospital, in palermo, with a painless and ulcerated lesion onto the oral mucosa ( fig. 1a ) , two nodular ulcerated lesions on the right knee and another one on instep of the right foot appeared one month before ( fig. 1b ) . the patient did not travel outside italy during the last year. he had been suffering from lowgrade fever in the last month. considering the above findings leishmaniasis was suspected and a needle aspiration of oral and cutaneous lesions was arranged in order to perform microscopy and leishmania-pcr, which were positive for leishmania. laboratory tests exhibited: wbc 4970/mmc, hb 13.9 g/dl, c reactive protein, 26.9 mg/l; positive serology for leishmania (igg 1/3200) and positive leishmania-pcr test on peripheral blood. abdominal us examination revealed splenomegaly (14 cm); methotrexate was suspended and liposomal amphotericin b, 4 mg/kg per day for 10 days, followed by two further administrations two weeks later was started. cutaneous and mucosal lesions improved at the end of the first 10 days of therapy and completely vanished after two further administrations, 40 days from the beginning of treatment. leishmania-pcr on peripheral blood after 10 days of therapy was negative. table 1 shows the literature data about characteristics, therapy and outcome of patients with psoriasis and leishmaniasis. leishmaniasis is a vector-born chronic infectious disease caused by protozoa of the genus leishmania and transmitted to humans by the bite of phlebotomine sandflies. in europe, the mediterranean countries are the most affected areas. leishmania parasite establishes chronic intracellular parasitism, survives for an infected person's lifetime and, in the event of major immune deficiency, may be reactivated from sites of latency. leishmaniasis can present with a spectrum of clinical manifestations and three patterns of infection are described: cutaneous (cl), mucosal or mucocutaneous (ml or mcl) and visceral leishmaniasis (vl). the infecting species of leishmania is very important in determining the clinical manifestations and the host immune response is crucial in determining the clinical outcome of infection 2 . today, non-hiv related immunosuppressive conditions are becoming increasingly prevalent, mainly because of better medical care of patients with chronic illnesses and the therapeutic use of immunosuppressive drugs. in the field of rheumatology, leishmaniasis has been reported in association with the use of various immunosuppressive drugs. 3 the introduction of tumor necrosis factor-alpha (tnf-α) antagonist drugs has received much attention recently and several cases of vl have been reported in rheumatic patients who do anti-tnf α drugs. 4 psoriasis is a chronic inflammatory autoimmune disease affecting 2-3% of the world's population and characterized by an aberrant hyper-proliferation of keratinocytes. the pathogenesis of psoriasis is complex. genetic susceptibility, environmental triggering factors and an over-reaction of local innate immune response initiate inflammation. subsequent involvement of adaptive immune response with production of th 1 cytokines, chemokines and growth factors lead to epidermal hyperplasia. 5 recently, a functional role of interleukin-17-producing t helper cells (th17) in psoriasis has been suggested by their reduction during successful anti-tnf treatment. 6 it is also known that th17 lymphocytes play an essential role in protecting against intracellular protozoa and in the successful clearance of leishmania by strengthening the th1 response. 7 in view of this, it could be argued that psoriasis may represent a protective factor for leishmania infection. indeed, in our review we did not found any case of leishmaniasis in psoriatic subjects who were not under immunosuppressive therapies. biological agents, which are powerful immunosuppressive drugs, have been more and more used in rheumatic patients and leishmania infections have been reported among anti-tnf-agents users. 8 recently maritati et al. found higher prevalence of subclinical leishmaniasis in patients with inflammatory rheumatic diseases receiving biological drugs than those treated with other immunosuppressive drugs. 9 however, leishmaniasis has also been reported in psoriatic patients not receiving biological drugs, as occurred to our patient ( table 1 ) . diagnosis of cl in psoriatic patients is challenging, as it mimics many other infections or a flare-up of psoriasis itself that can lead to ineffective and harmful changes of therapy. immunosuppressive therapies cause atypical manifestations of leishmaniasis with large lesions spread over large cutaneous areas and associated to a possible mucosal involvement. ml by l. infantum is very rare and only sporadically described in patients receiving powerful immunosuppressive therapies or in hiv-coinfected patients. mcl is mostly observed in latin america where l. braziliensis accounts for most cases, but l. panamensis, l. guyanensis, and l. amazonensis have also been implicated. only rarely cutaneous lesions extend to areas of skin distant from the mucosa involved, as in our case in which two lesions on the foot and knee were associated with the oral lesion. in the context of impaired immunity, it is also advisable to rule out vl by pcr-leismania on peripheral blood so as to establish the most appropriate therapy: intralesional or intravenous. finally, there is no agreement on appropriate screening for leishmaniasis before immunosuppressive treatments and on the strategy to be followed after the diagnosis of leishmaniasis in rheumatic patients taking immunosuppressive drugs. 10 molecular methods are highly sensitive and specific tools for the diagnosis of visceral leishmaniasis and a screening with leishmania-pcr in immunosuppressed patients living in endemic areas could be useful to identify patients at highest risk of reactivation. 9 specific leishmaniasis treatment followed by suspension of the immunosuppressive therapy was adopted by most of the authors. overall even if the treatment response is not as good as seen in the immunocompetent population, our review reports a good outcome in all cases and patients remained relapse-free without maintenance therapy and despite the ongoing use of immunosuppressive medication. in conclusion physicians must be alert to the possibility of development of leishmaniasis in immunosuppressed rheumatic patients. adequate screening for vl should be incorporated into the list of baseline studies to carry out before initiating biologic therapies, at least in endemic areas. the authors declare that there is no conflict of interest. as demonstrated in several studies in journal of infection , herpesviruses pose an increasing threat to human health. [1] [2] [3] according to international committee on taxonomy of viruses (ictv), equine herpesviruses (ehvs) belong to the family herpesviridae . until now, a total of 9 ehv species types have been determined in equines, viz. ehv1-ehv9. 4 among them, ehv1 and ehv4 are the most relevant herpesviruses affecting equines. both ehv1 and ehv4 infection are associated with upper respiratory tract disease, but only ehv1 infection could cause abortion and myeloencephalitis. ehv1 and ehv4 are prevalent in equines on all continents and have considerable economic impact on the horse industry. 5 in china, the number of equines is very large, reaching to be ∼ 6.8 million in 2018 ( http://www.stats.gov.cn/tjsj/zxfb/ ). ehv infection in equines was first reported in china in 1980, and the epidemiological investigation since then indicated ehv was prevalent in the equine population in all the studied 16 provinces in mainland china, with a seroprevalence ranging 8% −92%. [6] [7] [8] vaccination is commonly used to prevent and control ehv. however, china has not developed a commercially available ehv vaccine so far. ehv vaccine has a limited market application potential in china currently. due to the lack of relevant knowledge on ehv, most of the chinese horse owners always erroneously identified it as other common pathogen of equine respiratory diseases, and didn't realize its potential threat to equine health and reproduction. although the number of equines in china is large, most of them are labor/farming horses. to the best of our knowledge, even for racehorses, vaccination with ehv vaccine has not been performed in mainland china. considering the wide distribution and high prevalence of ehv in china, it is urgently to popularize knowledge on ehv in horse owners and promote market application prospects of ehv vaccine. in china, few veterinary researchers are currently investigating equine virus, including ehv. this is mainly caused by the change of equines' historical role. in the last century, a great number of equines were used for military in china. however, there is only one military equine farm in mainland china at present. considering a more important economic role of other domestic animals (e.g., pigs, chickens, and cattle) compared with equines, investigating equine virus (including ehv) is not a priority in the related guide policies issued by the chinese government. though epidemiological studies on ehv in china are limited, it still could be concluded that epidemic status of ehv is very complicated in china, which increases the difficulty in ehv vaccine development. in most provinces, ehv1 and ehv4 were co-circulating in equines with a high seroprevalence. 8 until now, a total of 7 ehv1 strains have been isolated from tissue samples of aborted equine fetuses (5 from farming horses in northeast china in 1980, 1 from asian wild horses in western china in 1999, 1 from farming horses in western china in 2016). 6 , 8 in addition, a novel ehv8 strain was isolated from one horse with serious respiratory disease in northern china in 2010. 9 recently, our laboratory firstly determined the molecule evinces for ehv4 and ehv5 in racehorses in sothern china (data not shown). however, a more large-scale and surveillance of ehv in equines is necessary to fully understand epidemic status of ehv in china, which could establish a foundation for updating the composition of ehv vaccine developed in china in future. in other countries, much effort has been made to develop ehv vaccine, and modified-live and inactivated virus vaccines have been registered for sale. 10 before an ehv vaccine is developed successfully in china by itself, it is necessary to vaccinate the susceptible equine population with an ehv vaccine commercially available from other countries to prevent and control ehv in china. however, a well-designed case-control animal challenge study still needs to estimate the protective efficacy of different vaccines against the field prevalent ehv strains in china. all authors declare that they have no competing interests. a recent review article on the treatment of hepatitis c with directly-acting antiviral (daa) drugs, makes numerous recommendations for baseline drug resistance testing. 1 in our local practice, we have been performing baseline drug resistance testing for some years now, prior to the publication of these guidelines. we present a recent retrospective hcv kinetics analysis of these patients' changing viral loads in response to daa therapy below. such studies have been used previously to compare viral suppressive responses in different hcv genotypes and treatment regimens. 2 , 3 the patients were a mixture of treatment-naive and treatment-experienced (including with interferon-based, ns3 protease inhibitor-based and daa-based regimens) cases. the current standard of care for hepatitis (hcv) patients is a combination of direct acting antivirals (daas), for which there are three different hcv viral protein targets (ns3, ns5a and ns5b). table 1 ns3, ns5a, ns5b resistance associated substitutions (ras), by hcv genotype, found in this patient cohort at baseline drug resistance testing (viral sequencing performed at imperial college, london, uk). the patients included a mixture of treatment-naïve and treatment experienced (i.e. interferon-based, ns3 protease inhibitor-based and more recent daa-based regimens) cases. resistance associated substitution (ras) by hcv genotype treatment with daas cure the vast majority of hcv-infected patients, with oral regimens having > 95% efficacy in most patient groups. 1 , 4 , 5 treatment failure currently affects approximately 5% of treated patients and is often associated with the selection of resistance associated substitutions (ras). 1 we performed hcv drug resistance testing both retrospectively (following treatment failures) and prospectively (prior to treatment) in our cohort of hcv genotype (g) 1-3-infected patients, during march 2015-june 2018. viral extraction, pcr and sequencing were performed at imperial college, using qiagen viral rna mini kits (qiagen pn:52906, qiagen ltd., manchester, uk), and inhouse pcr and sanger sequencing methods on an abi prism 3100-avant genetic analyser (thermo fischer scientific, loughborough, uk). the prediction of hcv genotype and drug sensitivities is derived from the geno2pheno algorithm [ www.geno2pheno.org ]. 6 treatment regimens used during this period complied with contemporaneous nhs rate cards: for non-cirrhotic or compensated cirrhotic patients: g1-treatment-naive: omb/par/rit + das + r; g1-treatment-experienced: elb/grz + /-r; g2-treatmentnaive/experienced: gle/pib; g3-treatment-naive/experienced: gle/ pib; g1-decompensated cirrhotic patients sof/led + r; g2/g3decompensated cirrhotic patients: sof/vel + r. we assessed the impact of any ras across g1-g3 on hcv rna kinetics by analysing viral load (realtime hcv viral load, abbott m20 0 0, abbott molecular uk, maidenhead, england) decline rates. we applied linear mixed regression to model the viral loads and assumed a linear decline (log 10 scale) over time, using sas statistical software (sas institute inc., nc, usa). in this cohort of 54 patients (n: g1 = 21, g2 = 6, g3 = 27), hcv ras were found as shown in table 1 . hcv rna viral load decline rates were found to be similar and not statistically different ( p = 0.760) at: −2 log 10 and −1.8 log 10 per month, respectively, for g1 and g2/g3 ( fig. 1 ). this suggests that these viral load decline rates were similar across g1-g3 infections despite baseline differences in viral load, ras profile, or a history of any previous treatment (i.e. interferon-based, older ns3 protease inhibitor-based, or more recent daa regimens). these results demonstrate similar hcv rna clearance efficacies of the various daa treatment regimens for g1-g3, in this patient cohort. although other studies on hcv kinetics have been published, they do not usually compare multiple hcv genotypes. 7 similar studies on patients infected with g4-6 viruses, and/or undergoing other daa treatment or retreatment combinations, 8 , 9 will be with great interest we have read the report of zhang et al. concerning the increased susceptibility to pertussis in chinese adults at childbearing age, as determined in a comparative seroprevalence study using samples collected from 2010 to 2016. 1 the authors describe that about 5% of the individuals had pt-igg antibodies, which is indicative of a recent infection. in the adults 20-39 years of age, 29.1% subjects had undetectable pt-igg antibodies in 2010 but 57.4% in 2015/2016. it is well known that adolescents and adults have become the reservoir of pertussis and an important source of transmission to vulnerable infants. bordetella pertussis is commonly associated with atypical pneumonia as determined in hospitalized children. 2 several seroprevalence studies conducted in different regions of china indicate that the incidence of pertussis is most likely underestimated. 3 , 4 this may be due to the use of insensitive diagnostics. at present, the diagnosis of pertussis in china is mainly based on culture. however, both the cdc and the world health organization (who) use pcr as the gold standard for diagnosis, in addition to culture. 3 oropharyngeal or nasopharyngeal swabs were obtained from 17,104 inpatients aged between 10 days and 14 years of age with clinical suspicion of pertussis, enrolled from march 2014 to february 2018 in shenzhen children's hospital. more than 50% of all patients were younger than 6 months of age. the hospitalized patients included 10,894 males and 6210 females (sex ratio, 1.75). all patients recovered after the treatment. a real time pcr assay targeting ptxa-pr was used to detect b. pertussis . of the 17,104 samples, 772 (4.51%) tested positive for b. pertussis by rt-pcr. our results indicate that despite vaccination pertussis remains a major health problem in china, since the prevalence of infection by b. pertussis in hospitalized children was high. the majority of patients were admitted because of pneumonia. the detection rate in hospitalized patients was lower than the rates reported earlier in shanghai and ji'nan. 3 , 4 this may be due to lower number of samples collected in these studies and due to the use of serology or culture methods. the overall prevalence rates were 3.97% and 5.48%, respectively. however, b. pertussis infection in female patients was significantly higher than in male patients (x 2 = 20.913, p < 0.001). this has earlier been reported by the ecdc 5 and haberling et al. 6 and may point to a genetic association with susceptibility to b. pertussis . the detection rates were dependent on age in patients (x 2 = 139.8, p < 0.001). the prevalence decreased with age: 8.71% newborns, 5.33% in infants, 2.39% in toddlers, 1.28% in (pre-) schoolers ( fig. 1 ) . the high vulnerability of newborns and young infants for b. pertussis infection may be related to a combination of insufficient herd immunity and suboptimal protection against b. pertussis infection in children too young to be fully vaccinated. since vaccination rates in infants are already at 99%, it will be difficult to improve this further. therefore, other measures must be considered, including booster vaccination at pre-school age and vaccination during pregnancy. because young infants are mainly cared for by mothers and other adults, the most important cause of infection with b. pertussis is their close contact with parents and siblings. 7 in general, b. pertussis was detected more often during seasonal changes, especially from late summer to early autumn. in hospitalized children the number of b. pertussis infections increased in march and september as compared to other months ( fig. 2 ) . the seasonal infection rates were 4.66% in spring, 4.75% in summer, 4.62% in autumn and 4.00% in winter, respectively. the prevalence in the winter season was lower but not statistically different than in other seasons (x 2 = 3.388, p = 0.336). in this study, we used real-time pcr, the most accurate method to detect b. pertussis . the detection rate may be significantly lower than the actual level, because oropharyngeal samples in most patients were collected instead of nasopharyngeal samples, and the pcr target gene was ptxa-pr instead of is4 81. is4 81 is present in high-copy numbers in b. pertussis whereas ptxa-pr is a single-copy target. however, the ptxa-pr pcr is more specific and will not detect b. parapertussis , which contributes to more than 5% of pertussis cases. 8 many studies have shown that adolescents and adults with b. pertussis infections, causing chronic cough, are an important reservoir for transmission, putting newborns at high risk. 9 maternal pertussis immunization prevents infant pertussis, as recently shown by amirthalingam et al. vaccine effectiveness against infant deaths was estimated at 95%, and disease incidence in infants < 3 months of age has remained low. 10 according to our results, vaccination of pregnant women and adults, especially those in close contact with infants and young children, may help to prevent pertussis in infants and young children in china. the authors have declared that no competing interests exist. this study was supported by the sanming project of medicine in shenzhen ( szsm201512030 ) and by the shenzhen science and technology project ( jcyj20170303155012371 ). tang and colleagues reported in this journal their experience with covid-19 disease 1 , the outbreak of which began in december 2019 in wuhan, hubei province, china 2 , 3 with spread to 33 additional countries 4-8 as of the 21st february 2020. here we report the clinical features and outcome of the first two cases of disease caused by sars-cov-2 infection in the united kingdom (uk) -the first imported and the second associated with probable person-toperson transmission within the uk. public health management will be reported separately. the index case (a) entered the uk on 23/01/20 from hubei province in china. initially asymptomatic, this individual, a 50 year-old female with no past medical history and on no regular medications, developed symptoms of fever and malaise on 26/01/20, accompanied by sore throat and dry cough. she had travelled with her partner and reported no infectious contacts prior to travel. on 28/01/20, a close household contact of the index case, a resident of the uk, developed symptoms of fever (38.5 °c), followed the next day by diffuse myalgia and a dry cough. this patient (case b) is a previously fit and well 23 year-old male. he had returned to the uk from hubei province on 06/01/20. case b promptly sought advice via the national health service (nhs) self-referral service nhs 111, and he and case a were assessed as being possibly at risk of covid-19, and were admitted to the regional infectious diseases unit at castle hill hospital, hull university teaching hospitals for isolation, assessment and diagnostic sampling. they were managed in separate negative pressure cubicles with anterooms. nursing and medical staff donned personal protective equipment (ppe) as recommended by public health england (phe). the clinical observations of each of the patients, together with their initial blood tests, are shown in table 1 . ( fig. 1 ) . clinical examination findings were unremarkable. initial investigations revealed only mild lymphopenia and elevation of crp, with mild neutrophilia in case b. periodic fever of 38-38.5 °c was observed in case b until d3 of admission. repeat blood tests in this individual on d3 demonstrated mild acute kidney injury (aki, serum creatinine 144 μmol/l). the aki was thought most likely due to dehydration, and resolved within 24 h with administration of intravenous infusion of crystalloid at 125 ml/h. cxr was normal. empirical oral antibiotic therapy (co-amoxyclav 500/125 mg p.o. t.d.s.) was administered on d3, to cover the possibility of secondary bacterial infection, but was subsequently discontinued. symptoms resolved in case a by d3 and in case b by d4 of admission. pcr testing of sars-cov-2 from nose and throat swabs taken daily was negative from d2 onwards in case a and from d5 in case b (throat swabs from this individual were negative throughout). there was no clinical indication for the use of experimental antiviral therapies. patients were deisolated according to current phe guidance, based on complete resolution of symptoms and two sequential negative respiratory pcr tests at least 24 h apart. rooms were decontaminated with 0.1% hypochlorite followed by uv light treatment. the contact of these individuals remained asymptomatic throughout the 14 days incubation period but was isolated as a precaution and to be close to family these first cases of sars-cov-2 are informative for clinicians caring for suspected and confirmed cases in the uk and elsewhere. reassuringly, illness in both individuals was relatively mild and short-lived, with no evidence of parenchymal lung disease (reflected by normal oxygenation and the absence of radiological infiltrates) or of the late-stage deterioration that has been reported in case series 9 , possibly due to the absence of comorbidities. experimental antiviral therapeutic options for severe disease were not considered necessary given the mild clinical nature of the illness. clinical illness correlated with the presence of viral rna in upper airway samples ( fig. 1 ) , with no evidence of prolonged asymptomatic shedding, although discordance between nose and throat samples in case b highlights the need to sample both areas. it was reasonably assumed that the source of infection in case b was close contact with symptomatic case a, given that the time from travel to china to onset of symptoms in case b was 22 days, although this cannot be proven. based on this assumption, the period from exposure to disease onset appeared short, at approximately 48 h, consistent with recent reports of the incubation period of sars-cov-2 6 . co-occurrence of respiratory viral infection, as we observed in case b with rhinovirus, has been described in the context of sars-cov-2 ( https://www.medrxiv.org/ content/10.1101/2020.02.12.20022327v1 ) as it has with many other respiratory viruses spread by similar routes, and may have contributed to the increased symptomatology in case b. interestingly the partner of case a, who was a close household contact, remained asymptomatic throughout and had negative tests for sars-cov-2 shedding. it will be of interest to investigate the serological responses in this individual to ascertain evidence of subclinical infection. isolation, minimisation of contacts and use of appropriate ppe is a cornerstone of management of high consequence respiratory viral infection. in the cases reported here, phe recommendations for ppe were followed 10 and there were no breeches in ppe or nosocomial transmission. this should provide reassurance to healthcare workers managing patients with suspected covid-19 in the uk that current ppe is both feasible and effective. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. dear editor , as reported in this journal 1 and elsewhere, 2 an outbreak of atypical pneumonia caused by the zoonotic 2019 novel coronavirus (sars-cov-2) is on-going in china and has spread to the world. as of feb 16, 2020 (24:00, gmt + 8), there have been 70,548 confirmed patients and more than 1700 deaths from sars-cov-2 infection in china, and 58,182 confirmed patients and 1696 deaths in the most affected province, hubei province. much research progress has been made in dissecting the evolution and origin of sars-cov-2 and characterizing its clinical features. [3] [4] [5] [6] [7] while the outbreak is on-going, people raise grave concerns about the future trajectory of the outbreak, especially given that the working and schooling time has been already dramatically postponed after the chinese lunar new year holiday was over (scheduled on jan 31). in particular, a precise estimation of the potential total number of infected cases and/or confirmed cases is highly demanding. earlier studies based on susceptible-exposed-infectious-recovered metapopulation and susceptible-infected-recovered-dead models revealed the number of potentially infected cases and the basic reproductive number of sars-cov-2. 3 , 8 , 9 these traditional epidemiological models apparently require much detailed data for analysis. 3 , 8 here we explored a simple data-driven, boltzmann functionbased approach for estimation only based on the daily cumulative number of confirmed cases of sars-cov-2 (note: the rational for boltzmann function-based regression analysis is presented in supporting information (si) file). we decided to collect data (initially from jan 21 to feb 10, 2020) in several typical regions of china, including the center of the outbreak (i.e. wuhan city and hubei province), other four most affected provinces (i.e., guangdong, zhejiang, henan, hunan) and top-4 major cities in china (i.e., beijing, shanghai, guangzhou, shenzhen). during data analysis on feb 13, 2020, the number of new confirmed cases on feb 12 in hubei province and wuhan city suddenly increased by 14,840 and 13,436, respectively, of which 13,332 and 12,364 are those confirmed by clinical features (note: all the number of confirmed cases released by feb 12 were counted according to the result of viral nucleic acid detection rather than by referring to clinical features). we thus arbitrarily distributed these suddenly added cases to the reported cumulative number of confirmed cases from jan 21 to feb 14 for hubei province by a fixed factor (refer to table s1 ), assuming that they were linearly accumulative in those days. it is the same forth with the data for wuhan city. regression analyses indicate that all sets of data were well fitted with the boltzmann function (all r 2 values being close to 0.999; figs. 1 a, b, s1 , and table 1 ). the potential total number of confirmed cases for mainland china, hubei province, wuhan city, and other provinces were estimated as 72,80 0 ±60 0, 59,30 0 ±60 0, 42,10 0 ±70 0 and 12,80 0 ±10 0; respectively; those for provinces guangdong, zhejiang, henan and hunan were 1300 ±10, 1170 ±10, 1260 ±10, 1050 ±10, 1020 ±10 and 940 ±10, respectively ( table 1 ) ; those for beijing, shanghai, guangzhou and shenzhen were 394 ±4, 328 ±3, 337 ±3 and 397 ±4, respectively. in addition, we estimated the key date, on which the number of daily new confirmed cases is lower than 0.1% of the potential total number as defined by us subjectively (refer to table 1 ). the above analyses were performed assuming that the released data on the confirmed cases are precise. however, there is a health commission, the state administration of traditional chinese medicine, the academy of chinese medical sciences, 29 provinces and cities, as well as the army ( fig. 1 ) . huoshenshan hospital is a specialized hospital established in the wuhan staff sanatorium. patients with confirmed coronavirus pneumonia have been admitted to our hospital. it has a total of 10 0 0 beds, and includes an intensive care unit, an ordinary care unit, a laboratory, and radiology and other auxiliary departments. according to the national health commission of the people's republic of china, the related design scheme of the institute was completed on january 24, 2020. construction of the hospital began on january 29th, and the hospital was completed and put into use on february 2nd. the chinese people's liberation army has transferred 1400 medical personnel to undertake the task of helping people infected with the virus. we firmly believe that chinese medical personnel and people throughout the country can work together to win this defensive battle with one heart and one mind. herpes simplex virus (hsv) pneumonia in the non-ventilated immunocompromised host: burden and predictors colonization by pneumocystis jirovecii and its role in disease ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients pcr diagnosis of pneumocystis pneumonia: a bivariate meta-analysis evaluation of pcr in bronchoalveolar lavage fluid for diagnosis of pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review molecular diagnosis of pneumocystis pneumonia in immunocompromised patients real-time pcr assay-based strategy for differentiation between active pneumocystis jirovecii pneumonia and colonization in immunocompromised patients detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients diagnosis of pneumocystis jirovecii pneumonia in immunocompromised patients by real-time pcr: a 4-year prospective study pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia a unified personal protective equipment ensemble for clinical response to possible high consequence infectious diseases: a consensus document on behalf of the hcid programme lassa fever: epidemiology, clinical features, and social consequences world health organization. lassa fever fact sheet review of cases of nosocomial lassa fever in nigeria: the high price of poor medical practice epidemiologic and clinical features of lassa fever outbreak in nigeria measures to control protracted large lassa fever outbreak in nigeria clinical and laboratory predictors of lassa fever outcome in a dedicated treatment facility in nigeria: a retrospective, observational cohort study molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation national guidelines for lassa fever case management pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds first human infection by a novel avian influenza a(h7n4) virus comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings investigation on pseudorabies prevalence in chinese swine breeding farms in 2013-2016 human endophthalmitis caused by pseudorabies virus infection clinical experience and next-generation sequencing analysis of encephalitis caused by pseudorabies characteristics of human encephalitis caused by pseudorabies virus: a case series study human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: a case report a case of human viral encephalitis caused by pseudorabies virus infection in china metagenomic data screening reveals the distribution of mobilized resistance genes tet (x), mcr and carbapenemase in animals and humans epidemic of carbapenem-resistant klebsiella pneumoniae in europe is driven by nosocomial spread antibiotic resistance gene reservoir in live poultry markets emergence of plasmidmediated high-level tigecycline resistance genes in animals and humans plasmid-encoded tet(x) genes that confer high-level tigecycline resistance in escherichia coli first report of blavim-4-and mcr-9-coharboring enterobacter species isolated from a pediatric patient a link between the newly described colistin resistance gene mcr-9 and clinical enterobacteriaceae isolates carrying blashv-12 from horses in sweden mcr-9, an inducible gene encoding an acquired phosphoethanolamine transferase in escherichia coli , and its origin discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis expansion and model-centric curation of the comprehensive antibiotic resistance database emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study novel plasmid-mediated colistin resistance gene mcr-7.1 in klebsiella pneumoniae emergence of a novel mobile colistin resistance gene, mcr-8 , in ndm-producing klebsiella pneumoniae novel plasmid-mediated colistin resistance gene mcr-3 in escherichia coli identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible salmonella enterica serotype typhimurium isolate coproduction of mcr-9 and ndm-1 by colistin-resistant enterobacter hormaechei isolated from bloodstream infection outbreaks of severe pneumococcal disease in closed settings in the conjugate vaccines era serious pneumococcal disease outbreak in men exposed to metal fume -detection, response and future prevention through pneumococcal vaccination outbreak of invasive pneumococcal disease among shipyard workers welders are at increased risk for invasive pneumococcal disease reactivation of dormant/latent fungal infection visceral leishmaniasis: host-parasite interactions and clinical presentation in the immunocompetent and in the immunocompromised host visceral leishmaniasis among immunosuppressed patients with rheumatic diseases tumor necrosis factor alpha antagonist drugs and leishmaniasis in europe psoriasis as a systemic disease the inflammatory response in psoriasis: a comprehensive review the equivocal role of th17 cells and neutrophils on immunopathogenesis of leishmaniasis leishmaniasis and biologic therapies for rheumatologic diseases subclinical leishmania infection in patients with rheumatic diseases under biological drugs appropriate screening for leishmaniasis before immunosuppressive treatments human herpesvirus 6 infection after autologous stem cell transplantation: a multicenter prospective study in adult patients next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center a different response to cytomegalovirus (cmv) and epstein-barr virus (ebv) infection in uk people with multiple sclerosis (pwms) compared to controls complete genome sequence of equine herpesvirus type 9 equine herpesviruses type 1 (ehv-1) and 4 (ehv-4)-masters of co-evolution and a constant threat to equids and beyond isolation and identification of equine herpesvirus type 1 identification of equine herpesvirus type 1 infection in a horse farm in shanghai epidemiological status of equine rhinopneumonia in china complete genomic sequence of an equine herpesvirus type 8 wh strain isolated from china equine herpesviruses 1 (ehv-1) and 4 (ehv-4)-epidemiology, disease and immunoprophylaxis: a brief review consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group differences in viral dynamics between genotypes 1 and 2 of hepatitis c virus the paradox of highly effective sofosbuvir-based combination therapy despite slow viral decline: can we still rely on viral kinetics? therapy of chronic hepatitis c virus infection in the era of direct-acting and host-targeting antiviral agents oral direct-acting agent therapy for hepatitis c virus infection: a systematic review hcv] -a web-based interpretation system to support hepatitis c treatment decisions in the era of direct-acting antiviral agents antiviral therapies for chronic hepatitis c virus infection with cirrhosis real life efficacy of ledipasvir/sofosbuvir in hepatitis c genotype 4-infected patients with advanced liver fibrosis and decompensated cirrhosis systematic review with meta-analysis: efficacy and safety of directacting antivirals for chronic hepatitis c genotypes 5 and 6 increased susceptibility to pertussis in adults at childbearing age as determined by comparative seroprevalence study ritm-tohoku collaborative research group. bordetella pertussis infection in children with severe pneumonia rapid detection of respiratory organisms with the filmarray respiratory panel in a large children's hospital in china seroprevalence of diphtheria and pertussis immunoglobulin g among children with pneumonia in ji'nan. china european centre for diseases prevention and control. annual epidemiological report infant and maternal risk factors for pertussis-related infant mortality in the united states clinical features, genotype variations of antigenic genes and macrolides resistance pertussis outbreak in a primary school in china: infection and transmission of the macrolide-resistant bordetella pertussis seroprevalence of pertussis among adults in china where whole cell vaccines have been used for 50 years sustained effectiveness of the maternal pertussis immunization program in england 3 years following introduction emergence of a novel coronavirus causing respiratory illness from wuhan the ncov outbreak joint field epidemiology investigation team an outbreak of ncip (sars-cov-2) infection in china -wuhan , hubei province a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia importation and human-to-human transmission of a novel coronavirus in vietnam transmission of sars-cov-2 infection from an asymptomatic contact in germany first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan public health england. www.gov.uk/government/publications/wuhan-novelcoronavirus-infection-prevention-and-control/wuhan-novel-coronavirus-wncov-infection-prevention-and-control-guidance accessed 3rd emergence of a novel coronavirus causing respiratory illness from wuhan a novel coronavirus outbreak of global health concern epidemiological and clinical features of the 2019 novel coronavirus outbreak in china a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical characteristics of 2019 novel coronavirus infection in china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study data-based analysis, modelling and forecasting of the novel coronavirus (2019-ncov) outbreak a data driven time-dependent transmission rate for tracking an epidemic: a case study of 2019-ncov emergence of a novel coronavirus causing respiratory illness from wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan the novel coronavirus originating in wuhan. china: challenges for global health governance emergence of sars-like coronavirus poses new challenge in china we are indebted to all colleagues who contributed to this substudy in paris and geneva. in particular, we would like to thank no public or private funds were used for the current study. eliseo albert holds a río hortega research contract from the carlos iii health institute (ref. cm18/00221 ). estela giménez holds a juan rodés research contract from the carlos iii health institute (ref. jr18/0 0 053 ). we thank all the staff of the domestic and international organizations who fought against this outbreak, including those at the various health care facilities, lassa fever diagnostic laborato-ries, nigeria centre for disease control, world health organization, african field epidemiology network, public health england, ehealth africa, pro health international, university of maryland baltimore, us centers for disease control and prevention, alliance for international medical action, médecins sans frontières, and numerous other partners. we also express our sincerest condolences to the families and friends of those who died during the outbreak. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.016 . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.01.019 . this work was supported by the national natural science foundation of china ( 31702271 ), and the guangdong provincial natural science foundation ( 2017a030310367 ). we are grateful to the patients for providing their written informed consent to publish this report. our thanks go to nursing, laboratory and medical colleagues in hull university teaching hospitals nhs trust and the newcastle upon tyne hospitals nhs foundation trust who contributed directly and indirectly to patient care, and to many colleagues in public health england and across the hcid network who contributed their time and expertise to the management of these cases. cjad is supported by a clinical research career development fellowship from the wellcome trust ( 211153/z/18/z ). we thank graduate students (boyan lv, zhongyan li, zhongyu chen, yu cheng, mengmeng bian, shuang zhang, zuqin zhang, and wei yao; all from prof. xinmiao fu's research group at fujian normal university) for data collection. this work is support by the national natural science foundation of china (no. 31972918 and 31770830 to xf). the reported cumulative number of confirmed cases may have uncertainty. assuming the relative uncertainty follows a single-sided normal distribution with a mean of 1.0 and a standard deviation of 10%, the potential total number and key dates were estimated at 95% ci. for detail, refer to the methods section and figs. 1 c, d, s2 and s3.b key date is determined when the number of daily new confirmed cases is less than 0.1% of the potential total number. tendency to miss-report some positive cases such that the reported numbers represent a lower limit. one typical example indicating this uncertainty is the sudden increase of more than 14 0 0 0 new confirmed cases in hubei province on feb 12 after clinical features were officially accepted as a standard for infection confirmation.another uncertainty might result from insufficient kits for viral nucleic acid detection at the early stage of the outbreak. we thus examined the effects of such uncertainty using a monte carlo method (for detail, refer to the methods section in si file). for simplicity, we assumed that the relative uncertainty of the reported data follows a single-sided normal distribution with a mean of 1.0 and a standard deviation of 10%. under the above conditions, the potential total numbers of confirmed cases of sars-cov-2 for different regions were estimated ( figs. 1 c, d, s2 and s3 ) and summarized in table 1 16 ,092), respectively, indicating that overall the outbreak may not be so bad as previously estimated. 9 such uncertainty analysis also allowed us to estimate the key dates at 95% ci. as summarized in table 1 , the key dates for mainland china, hubei province, wuhan city, and other provinces would fall in (2/28, 3/10), (2/27, 3/10), (2/28, 3/10) and (2/27, 3/13), respectively.finally, the ongoing sars-cov-2 outbreak has undoubtedly caused us the memories of the sars-cov outbreak in 2003. we thus collected the data from the who officiate website for analysis, and found that the cumulative numbers of confirmed cases of 2003 sars-cov both in china and worldwide were fitted well with the boltzmann function, with r 2 being 0.999 and 0.998, respectively ( figs. 1 e and f) .in summary, we found that all data sets, including both the on-going outbreak of sars-cov-2 in china and the 2003 sars-cov epidemic in china and worldwide, were well fitted to the boltzmann function ( fig. 1 and s1 ). these results strongly suggest that the boltzmann function is suitable for analyzing the epidemics of coronaviruses like sars-cov and sars-cov-2. one advantage of this model is that it only needs the cumulative number of confirmed cases, somehow as simple as the recently proposed model. 10 in addition, the estimated potential total numbers of confirmed cases and key dates may provide valuable guidance for chinese central and local governments to deal with this emerging threat at current critical stage. none. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.02.019 . we appreciate the work tang et al. have report emergence of a novel coronavirus in china. 1 the 2019-ncov broke out in wuhan, china at the end of 2019, and has attracted worldwide attention. [2] [3] [4] although the chinese government has taken active measures to control this epidemic, the virus is very infectious. according to the real-time data of the national health commission of the people's republic of china up until february 5, 2020, within a short period of half a month, the number of confirmed cases and the number of deaths were 24,443 and 493, respectively. the epidemic is progressing rapidly. 2019-ncov poses new public health challenges in china. 5 in wuhan, china, the number of local medical staff is insufficient for the demand resulting from the explosive increase in the number of infected patients. therefore, many medical personnel are needed to devote themselves to the front line of combating the virus.medical personnel throughout the country are led under the unified leadership of the chinese government. although the epidemic in wuhan is serious, a large number of medical staff rushed to wuhan to supplement the shortage of manpower in wuhan hospitals. this is a battle without smoke, the heroes of which are our medical staff. according to the national health commission of the people's republic of china, as of january 30, 2020, hubei province opened 11,0 0 0 isolated patient beds, and about 170,0 0 0 healthcare professionals from all kinds of medical institutions are working on the front lines and providing care for patients with fevers, and for suspected or confirmed patients. in this time of emergency, under the unified deployment of the chinese government, there are 52 medical teams including 6097 medical personnel from the national key: cord-330110-pamxy4av authors: teissier, elodie; zandomeneghi, giorgia; loquet, antoine; lavillette, dimitri; lavergne, jean-pierre; montserret, roland; cosset, françois-loïc; böckmann, anja; meier, beat h.; penin, françois; pécheur, eve-isabelle title: mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol date: 2011-01-25 journal: plos one doi: 10.1371/journal.pone.0015874 sha: doc_id: 330110 cord_uid: pamxy4av the broad-spectrum antiviral arbidol (arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. to better understand arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. we demonstrate that arb associates with phospholipids in the micromolar range. nmr reveals that arb interacts with the polar head-group of phospholipid at the membrane interface. fluorescence studies of interactions between arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that arb interacts with tryptophan in the micromolar range. interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of arb ic50 of the hepatitis c virus (hcv) membrane fusion. since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. the resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. our findings pave the way towards the design of new drugs exhibiting arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection. distinct from specific antiviral compounds that target key viral functions are a group of broad-spectrum medicinal drugs that were originally designed for other treatments [1] [2] [3] or targeted toward a number of viruses ( [4] ; reviewed in [5] ). the advantage of this group of antivirals is that they have already met the pharmacological criteria for medicinal drugs and are already approved for clinical use in some countries. among these molecules, antiviral agents targeting viral entry of enveloped viruses are of major interest since they seize an early step in the viral life cycle, before damages have occurred to cells (recently reviewed in [6, 7] ), and since they can be incorporated into combinations of multiple drugs with different targets. one of these compounds, arbidol [arb; 1hindole-3-carboxylic acid, 6-bromo-4-[(dimethylamino)-methyl]-5hydroxy-1-methyl-2-[(phenylthio)methyl]-, ethyl ester, monohydrochloride; cas registry number 131707-23-8 ( figure 1 )], is already licensed in russia and china, and is described as an antiinfluenza drug with immunostimulant properties. arb is in use for several years as prophylaxis and treatment for influenza a and b infections. it inhibits influenza virus-induced membrane fusion and may have the capacity to induce serum interferon [8] . recent studies extended its inhibitory activity to other human viruses such as the respiratory syncytial virus, parainfluenza virus 3, rhinovirus 14 , and hepatitis b virus (reviewed in [5, 9] ). we demonstrated that it also inhibits hepatitis c virus (hcv) infection in vitro, and hcv replication [10] , hcv cell entry and membrane fusion using hcv pseudoparticles (hcvpp) and hcv grown in cell culture (hcvcc) [11, 12] . most recently, ciliberto and coworkers demonstrated the efficacy of arb derivatives at inhibiting hcv entry and replication into hepatoma cells in the low micromolar range [13] . hcv infection is a leading cause of liver diseases, including hepatocellular carcinoma, and therapeutic options are still limited (for recent reviews, see [14] and refs therein). there is thus an urgent need to develop efficient and well tolerated drugs to combat this virus. arb demonstrated a propensity to enter into hydrophobic interactions with membranes, and with membrane-like environments such as detergent micelles [12] . here we further characterize the mechanism of action of arbidol, and analyze at the molecular and atomic level the interactions of arb with membranes, tryptophan-rich derivatives and peptides. we first examined how arb inhibits hcv entry and membrane fusion using hcvpp of different genotypes, and found that arb inhibition was genotype-independent. by combining surface plasmon resonance, fluorescence and nmr spectroscopy approaches, we showed that arb directly interacts with the phospholipid membrane interface, with an affinity in the micromolar range, comparable to the concentration inhibiting hcvpp membrane fusion by 50% (ic50). arb also displayed micromolar affinity toward aromatic components of proteins such as tryptophan and derivatives, and toward peptides containing tryptophans and derived from hcv envelope glycoproteins. altogether our results demonstrate that arb interacts with the polar head of phospholipid membranes and protein motifs enriched in aromatic residues, suggesting that the inhibitory activity of arb on hcv entry and fusion could involve both types of interactions. phosphatidylcholine from egg yolk (pc, 99% pure), dimyristoylphosphatidylcholine (dmpc, 99% pure), cholesterol (chol, 99% pure), lyso-phosphatidylcholine (lysopc), dodecyl-phosphocholine (dpc), triton x-100, tryptophan octyl ester hydrochloride (toe) and n-acetyl-l-tryptophanamide (nata) were purchased from sigma. octadecyl rhodamine b chloride (r 18 ) was from molecular probes. the peptides used were part of the sequence of structural or non structural (ns) proteins of hcv and of the bovine viral diarrheal virus (bvdv). the amphiphilic helix of bvdv ns5a [15] and the transmembrane domain of hcv ns4a [16] were obtained as described previously. the peptides identified as important for hcv fusion [17] were purchased from clonstar biotech (90% purity) or sigma genosys (70% purity), respectively, and dissolved in dmso before preparation of lipid:peptide mixtures. arbidol [arb, 1h-indole-3-carboxylic acid, 6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-, ethyl ester, monohydrochloride ( figure 1 )] was a kind gift from stephen j. polyak. arb was readily soluble in ethanol, and soluble in the mm range in water. ethanol stock solutions of arb were diluted to a 1.88 mm final concentration in milliq water (the final stock solution contained 10% ethanol). for spr experiments, one mg of arb was resuspended in water, followed by centrifugation (160006g, 15 min, 4uc). arb concentration in solution was measured at 280 nm in the supernatant (arb extinction coefficient = 9510 m 21 .cm 21 ). after solvent evaporation, samples were resuspended in phosphate-buffered saline (pbs, ph 7.4) or water, and underwent 5 freeze/thaw cycles (liquid nitrogen and 37uc, respectively). liposomes were prepared by extrusion over a stack of avestin polycarbonate filters (100 nm), as described [18] . huh-7 cells [19] were maintained in dmem containing 4.5 g/ l d-glucose and 4 mm l-glutamine (invitrogen, cergy-pontoise, france), supplemented with 100 u/ml penicillin, 100 mg/ml streptomycin and 10% fcs (lonza). productions of pseudotyped viruses were obtained by the transient transfection of 293t cells by the calcium-phosphate method. for the genotype study, hcvpp of genotypes 1a (h77; af011752), 1b (con1; aj238799), 2a (jfh1; ab047639), 2b (ukn2b 2.8, ay734983), 3a (ukn3a 1.28, ay734984), 4a (ukn4 21.16, ay734987), 5a (ukn5.14.4, ay785283) and 6a (ukn6.5.340, ay736194) were produced as described previously [20] from 293t cells co-transfected with a murine leukemia virus (mlv) gag-pol packaging construct, an mlv-based transfer vector encoding gfp as a marker protein, and the e1-e2 expression constructs. supernatants were collected 48h post-transfection and filtered on 0.45 mm. for genotypes 5a and 6a, pseudoparticles were concentrated 100-fold after ultracentrifugation through a 20% sucrose cushion at 75,0006g for 2h at 4uc. pellets were resuspended in the regular medium of huh-7 cells. for infection experiments, huh-7 cells were seeded at 4000 cells/well in 96-well plates. the following day, cells were infected in the presence of increasing arb concentrations for 6 h. arbidol effect on viral infectivity was evaluated by assaying gfp activity 72 hours after infection using flow cytometry (facscalibur). pseudoparticles harbouring at their surface the influenza hemagglutinin (happ) and the envelope glycoprotein of the rd114 feline oncovirus (rd114pp) were prepared as described in [18] and [12] , respectively. lipid mixing between pseudoparticles and pc:chol:r 18 liposomes was monitored by fluorescent spectroscopy, as the dequenching of r 18 [18] . in brief, r 18 note that numbering in panel a refers to proton numbers, as identified in nmr (cf figure 5 and table 1 ). doi:10.1371/journal.pone.0015874.g001 ay734987 (4a) 9.10e 4 ; ay785283 (5a) 3.10e 4 ; ay736194 (6a) 5.10e 3 ; ha 8.10e 8 ; rd114 2.10e 6 ], and incubated 2 min. fusion was initiated by acidification to ph 5 with hcl, and recorded on an slm aminco 8000 spectrofluorimeter over a 10-min time period, at excitation and emission wavelengths of 560 nm and 590 nm, respectively. maximal r 18 dequenching was measured after the addition of 0.1% triton x-100 (final concentration) to the cuvette to lyse liposomes. the same procedure was used to follow pseudoparticle fusion in the presence of arb; in this case, after a 1-min incubation of pseudoparticles with liposomes, arb (11.3 mm final concentration) was added and incubated for 1 min, and fusion initiated by acidification. indole emission fluorescence spectra of tryptophan derivatives were recorded at excitation wavelength of 286 nm (spectral zone of lowest absorption of arb), under various conditions: nata (5 mm final) in pbs at ph 7.4 or 5.0; toe at 5 mm in lyso-pc micelles (toe:lysopc molar ratio 1:800), and in pc and pc:chol liposomes (toe:lipid molar ratio 1:20); peptides at 5 mm in pc:chol liposomes (peptide:lipid molar ratio 1:20). spectra were obtained in the absence or presence of increasing concentrations of arb (0 to 100 mm). samples were incubated 2 min at 37uc prior to recording. emission spectra were collected in the 300-400 nm region (with 2 nm-increments), with blanks substracted, using a black flat-bottom, low-binding 96-well microplate (greiner bioone). measurements were recorded on a tecan infiniteh m1000 spectrofluorimeter. k d app values were calculated from the difference between the areas under the spectra in the absence or presence of arb (da), at various arb concentrations, by nonlinear fitting using the equation da = da max c/(k d +c). fluorescence measurements were repeated three times to obtain averaged values of k d app. guvs were made by the electroformation method [21] . the flow chamber (warner instruments, connecticut, usa) used for vesicle preparation was equipped with two glass coverslips, each coated with optically transparent and electrically conductive indium tin oxide (ito) (philips, eindhoven, nl). mixtures of lipids [pc:chol:r 18 (65:30:5, molar ratio)] were prepared at 0.1 mm in chloroform. the lipid mixture (2 nmoles) was spread into a thin and uniform film on the conductive face of ito-coated slide. after chloroform evaporation, the dried lipid film was hydrated by adding water into the chamber (,400 ml) and an alternative electrical field (10 hz and 1.2v) was applied at room temperature for 3 hours. guvs in the absence or presence of increasing amounts of arb solubilized in water (0 to 40 nmoles), were observed by epifluorescence microscopy. interaction of arb with dmpc layers was investigated with a biacore 3000h using a l1 sensor chip at 30uc. the sensor chip surface was washed with a mixture of 50 mm naoh and isopropanol (6:4, v:v) , at a flow rate of 20 ml/min for 1 min. the running buffer was milliq water. the influence of liposome concentrations on the final spr signal was tested; we assayed lipid concentrations from 0.5 to 5 mm and measured the resulting resonance units (ru). we obtained a well detectable, reproducible and stable signal from 2 mm, and further increasing this concentration did not improve the signal. we therefore chose the 2 mm concentration for our experiments. dmpc liposomes were resuspended in milliq water and captured on sensor chip at 2 ml/min for 5 min. the flow rate was increased to 30 ml/min and the liposome surface was then washed with 10 mm naoh for 1 min. liposomes immobilized on the chip surface gave approx. a 5000 ru signal. to calculate arb affinity for lipids, its association to and dissociation from dmpc layers were studied at different arb concentrations in water, from 0.5 to 10 mm, at a flow rate of 20 ml/min. after each binding cycle, the sensor surface was regenerated to the original matrix by injecting 50 mm naoh/ isopropanol (6:4, v:v). the sensor surface was then coated with a fresh liposome suspension for the next binding cycle. k d values were calculated from the equilibrium resonance signal (r eq ) as a function of the analyte concentration. r eq values were estimated by extrapolation to infinite time using plots of resonance signal as a function of the reciprocal of time. apparent k d were then calculated by nonlinear fitting to the expression r eq = r max c/ (k d +c), where r max is the maximum binding capacity of the surface and c is the analyte concentration, using the sigmaplot software. for the nmr studies, the bicellar system was prepared by another sample with similar lipid concentration and higher arb content (molar ratio 1/15) was also prepared. the amount of free arb in arb:dmpc mixture was estimated after rapid separation of lipids on ultrafiltration membrane (cutoff 5000 da) and measure of arb concentration in the ultrafiltrate at 280 nm. for arb:dmpc molar ratio of 1:4 at neutral ph, free arb was found to be lower that 0.2%. we thus concluded that the amount of free arb in nmr samples was negligible. 1 h nmr experiments were performed on a bruker dmx 400 spectrometer operating at a proton frequency of 400 mhz. spectra were recorded with a 5 mm triple resonance inverse txi probe equipped with z-gradient. the p/2 pulse was 10.3 ms, the recycle delay was 3 s and solvent suppression with presaturation was used. 1d 1 h spectra were measured acquiring 120 scans. these spectra were used for the assignment of the drug signals together with 2d noesy and 1 h/ 13 c hsqc spectra (data not shown). the assignment of the lipid resonances was derived from the comparison with data in the literature [22] . to times were measured by using inversion recovery experiments with an interpulse delay ranging between 5 ms and 13 s. each measurement was repeated 3 times, adding 240 scans with a delay time between scans of 15 s. all the spectra were processed using matnmr [23] . the 1 h frequency scale is given in terms of chemical shift relative to the acetone signal used as an external reference (2.218 ppm). we have previously shown that arb could inhibit cell entry and membrane fusion of hcvpp of genotypes 1a, 1b and 2a [10, 12] , and hcvcc of genotype 2a [11] . here we sought to investigate the effect of arb on other major hcvpp genotypes as well. hcvpp infectivity toward huh-7 cells, objectifying hcvpp entry, was assayed by counting cells positive for gfp (as the marker protein), incubated with or without increasing arb concentrations for 6h (see materials and methods). a representative data set is shown in figure s1a , and inhibition obtained for the highest concentration of arb (11.3 mm) is presented in figure 2a . the inhibitory effect of arb on hcvpp cell entry depends on hcvpp genotype. indeed, within biological intrinsic variability of hcvpp preparation and samples, three cases could be distinguished: entry of hcvpp of genotypes 2a and 3a was inhibited by ca. 60%, while 1a, 1b and 2b exhibited a 40%-inhibition, but entry of genotypes 5a and 6a was weakly affected by the presence of arb ( fig. 2a) . the influence of arb on hcvpp-mediated lipid mixing was assayed by fluorescence spectroscopy using fluorescent liposomes, as previously described [18] . lipid mixing between hcvpp and liposomes was only observed at low ph and optimal at ph 5.0 [18] . in the presence of increasing arb concentrations, lipid mixing was inhibited in an arb dose-dependent manner ( figure s1b for hcvpp genotype 4a). in contrast to what was observed for hcvpp infectivity, the effect of 11.3 mm arb on hcvppmediated membrane fusion ( figure 2b ) was similar for all tested genotypes, with about 50% inhibition of membrane fusion activity. this indicates that membrane fusion inhibition by arb is not genotype-dependent. these data suggest that the differential inhibitory effect of arb on hcvpp infectivity of various genotypes is likely due to a genotype-dependent modulation of hcv glycoproteins interaction with the cellular proteins (e.g. hcv receptors) involved in hcv cell entry. conversely, arb inhibition of hcvpp membrane fusion, as assessed by a in vitro model system where the only proteins present are the viral glycoproteins, could merely reflect the interaction of arb on lipids and/or on motifs present in hcv glycoproteins of any genotype. to test these hypotheses, we further investigated arb interaction properties with lipids and protein fragments using the approaches described in the following. we previously showed that arb could interact with liposomes and membrane-like environments such as detergent micelles [12] . we further investigated this feature by studying the interactions of arb with giant unilamellar liposomes (guv) by optical microscopy ( figure 3 ). guv are pure lipid bilayers, intrinsically flexible and unstable due to their very large size (in the range of tens of mm) [24] . increasing arb concentrations were added to the chamber where guv composed of pc:chol were electroformed (see methods section), with arb-to-lipid molar ratios of 1:40, 1:20, 1:10, 1:1, 10:1 and 20:1. the guv bilayer was unaffected by the presence of arb up to a 1:20 arb-to-lipid ratio, with occasional membrane flickerings ( fig. 3c and asterisk in fig. 3e ). at higher ratios, membrane inhomogeneities and invaginations appeared (fig. 3f , asterisks in fig. 3d ), and a major overall membrane reorganization was observed at a 20:1 arb-to-lipid ratio (fig. 3g ). note that no lysis or membrane dislocation of guv was observed happ are presented as control pseudoparticles sensitive to arbidol (cf also [10] ), and rd114pp insensitive to arbidol (cf also [12] ). * 1 , the mutant hcvpp w529a (cf [17] ) are presented as a negative control of entry, displaying very low infectivity. b, membrane fusion between hcvpp and r 18 -labeled liposomes was measured by recording the kinetics of lipid mixing by fluorescence spectroscopy (excitation and emission wavelengths were 560 and 590 nm, respectively), as described in the materials and methods section. values of the last 30 s of fusion kinetics (final extent of fusion) were used to calculate the percentage of fusion in the presence of arb, relative to fusion kinetics without arb (100%). results are the mean +/2 sem of 4 separate experiments. happ and mutant hcvpp w529a were taken as controls. * 2 : no fusion was observed for rd114pp. doi:10.1371/journal.pone.0015874.g002 for any condition, even at the highest ratio (data not shown). these results reveal that only very high concentrations of arb with respect to lipids could significantly perturb the lipid organization of these bilayers. this also indicates that the direct interaction of arb to lipid bilayers at the concentrations used to inhibit hcvpp infectivity and membrane fusion (panel e) do not perturb lipid organization. in addition, hcvpp pre-incubated at neutral or acidic ph with arb, even at very high concentrations (100 mm), displayed similar morphology (visualized by transmission electron microscopy) as those observed in the absence of the drug (data not shown). indeed we counted over 160 hcvpp for each condition, and no difference in hcvpp morphology could be observed between the parameters assessed. this indicates that arb inhibition of hcvpp fusion is not due to viral particle disruption/damage. to gain insight into the molecular details of the interaction of arb with lipid membranes, we next investigated the lipid binding properties of arb by using surface plasmon resonance (spr, biacoreh technology). we used a biacore's l1 sensor chip to capture dmpc liposomes. this sensor chip displays lipophilic groups attached on the surface of a carboxymethylated dextran layer, and was shown to provide a quick and reproducible method for the preparation of bilayer-mimetic systems [25] . we first tested whether arbidol per se could bind or not to the chip. arb at 11.3 mm (the highest concentration relevant in the biological context) was injected onto the chip devoid of liposomes. this led to approx. 60 resonance units (ru, see methods section). dmpc liposomes (2 mm) captured onto the sensor chip reached about 5000 ru, and a further ,600 ru was seen when arb was pulsed onto the liposome-coated chip. the binding of arbidol alone on the l1 chip remains therefore negligible. measures of arb/dmpc association and dissociation were performed with various arb concentrations ranging from 0.5 to 11.3 mm. after passage over the surface of the sensor chip, arb bound to immobilized dmpc in a concentration-dependent manner ( figure 4 ). arb initial binding was fast, but then slowed down without reaching saturation equilibrium (from 0 to 240 s). after stopping the arb flow onto the sensor chip (from 240 s), bound arb was rapidly but incompletely dissociated from dmpc membranes. indeed, for all arb concentrations tested, about 50% of arb remained bound to dmpc. this demonstrates that arb is capable of interacting with lipid membranes, in a stable association between arb and dmpc. however the behaviour of arb binding to membranes rendered difficult the fitting of a kinetic model to the data, and hence the determination of reliable on-and off-rates. indeed using global fitting, binding curves could not be fitted properly with the different models included in the biaevalution 3.0 software (1:1 langmuir binding, bivalent analyte, heterogeneous ligand, heterogeneous analyte, conformational change), with or without mass transport. furthermore, because equilibrium was not reached during the association phase, the direct use of scatchard analysis to calculate the apparent equilibrium dissociation constant was not allowed. instead, the apparent equilibrium dissociation constant k d was calculated from the equilibrium resonance signal (r eq ) as a function of analyte concentration, r eq values being estimated by extrapolation to infinite time using plots of resonance signal as a function of the reciprocal of time [26, 27] . apparent k d was then calculated by nonlinear fitting to the expression r eq = r max c/(k d +c), where r max is the maximum binding capacity of the surface and c is the analyte concentration, using sigmaplot software. this calculation, performed on 4 separate experiments, gave an apparent k d of 6.860.4 mm (see inset to figure 4 for a representative experiment). this dissociation constant is in the same order as the ic50 of hcvpp fusion (11.3 mm) . this result indicates that the inhibitory effect of arb on hcvpp membrane fusion is at least in part deriving from arb association to lipid membranes. nmr spectroscopy was used to characterize the arb insertion in a model membrane system. the 1 h nmr spectrum of arb recorded at 305 k in deuterated water is shown in figure 5a (black spectrum), and the assignment of the proton signals deduced from 2d spectra analysis (data not shown) are reported in table 1 . in order to study arb in a membrane-mimetic environment, we used isotropic phospholipid bicelles consisting of a mixture of dmpc/dhpc in water. long-chain phospholipid molecules of dmpc self-assemble into planar bilayers, while the short-chain molecules of dhpc segregate to edge regions of high curvature [28] . bicelles with [dmpc]/[dhpc] molar ratio ,1 form fast and isotropically tumbling aggregates, amenable to solution nmr studies. still, isotropic bicelle systems are used as a phospholipid bilayer mimetic, since dmpc has been shown to form a flat bilayered surface [29] [30] [31] . the 1 h nmr spectrum of arb in this bicellar phase is shown in figure 5a (red spectrum). in this system, the spectral crowding due to the presence of phospholipid resonances allowed only the observation of protons denoted 1, 2, 3, 4, 5 and 6 of the arbidol molecule (see fig. 1a ) where only 2 signals can be distinguished for the three protons 2, 3, 4. additional 1 h resonances could be resolved from 2d 1 h noesy spectra and 1 h/ 13 c hsqc spectra ( figure s2 ) and the corresponding chemical shifts are shown in table 1 together with the assignment of the proton lines for arb in water. arbidol interaction with lipids induce chemical-shift changes in the arb resonances when compared to that observed in water. in order to investigate the immersion depth of arb in the membrane, we monitored the proton longitudinal relaxation rate of arb protons upon the addition of the soluble paramagnetic agent gadolinium-diethylenetriamine pentaacetic acid-bismethylamide gd(dtpa-bma) [32] . this paramagnetic contrast agent stays soluble in the water surrounding the membrane and induces a paramagnetic relaxation enhancement (pre) on the spin of the atoms close to the surface of the membrane. recently, pre effects due to gd(dtpa-bma) were used to probe the immersion depth and orientation of an anti-microbial peptide [33, 34] . here we measured the proton t 1 relaxation times of both arb and phospholipid protons to probe the immersion depth of arb in the membrane, using the pre values of the phospholipids as an approximated yardstick. a titration of arb with gd(dtpa-bma) was performed at increasing concentrations of the paramagnetic agent, from 2.0 to 9.7 mm. at each step of the titration, the proton t1 relaxation times for the protons 1, 2, 3, 4, 5 and 6 of arb, and for the protons alpha, beta, gamma, g1, g2, g3, c2, c3 and omega14 of the phospholipids, were measured. the plot in figure 5b shows the relaxation times as a function of the gd(dtpa-bma) concentration. this titration leads to a curve for each proton whose slope corresponds to the pre values. as shown in fig. 5c , pre measured on the arb-bicelle system range from 0.90 sec 21 to 0.03 sec 21 for the phospholipid protons and from 0.90 sec 21 to 0.20 sec 21 for arb protons. the pre observed for each spin can be described as an overall relaxation enhancement [35] , due to all the paramagnetic agents in solution. for a planar membrane surrounded by a buffer containing a non-interacting paramagnetic probe, the total pre of a nucleus with immersion depth d is given by the equation [33, 34] : pre = z/d 3 , where d is the immersion depth of a specific nucleus within the membrane plus the radius of the magnetic probe, and where the constant z is a combination of various parameters, among them a correlation time, itself a combination of the electron relaxation time, the lifetime of the intermolecular adduct bicelle-gd(dtpa-bma), and the rotational correlation time. in order to determine the immersion depth of arb, instead of determining z, we used the phospholipids as a yardstick by comparing their pre with the one of arb. the pres of the resolved signals of arb and phospholipids are reported and compared in figure 5c . this procedure is based on two assumptions: (i) the amount of free arb in solution in the presence of lipids is negligible (see nmr sample preparation in experimental procedure section), and it does therefore not affect significantly the pre of arb in the membrane ; ii) the constant z is the same for lipids and arb. figure 5c shows that methyl protons 6 of arb are at the lipid/ water interface as the gamma-proton of the hydrophilic headgroup of the lipids. protons 5 and 1 of arb are at the level of respectively protons alpha and g1 of the phospholipids. the aromatic protons 2 and 3 are the most buried protons of arb, close to the beginning of the hydrophobic chain (protons c2). the validity of assumption (ii) is supported by the noe crosspeaks detected between proton 1 of arb and the glycerol moiety of phospholipids. in addition, the maximum pre measured for arb (protons 6) and phospholipids (c protons) are about the same, suggesting that the corresponding molecule regions are the most exposed to water. this estimation of the immersion depth of arb protons relative to the phospholipids protons enables us to propose a model for the positioning of arb in the membrane, shown in figure 5d . these nmr data clearly demonstrate that arb interacts at the membrane interface, mainly at the level of phospholipid polar head. this result supports the assumption that the arb inhibitory effect on hcvpp membrane fusion is dependent, at least in part, from this interaction. a second possibility regarding arb activity is that arb might interact with key motifs present in viral proteins, thereby impeding their structural reorganization at the onset of fusion and thus leading to fusion inhibition. a first set of experiments was designed to investigate whether the order of addition of fusing partners would affect arb-induced fusion inhibition. for this purpose, we measured fusion after preincubation of hcvpp or liposomes or both in the absence or presence of 11.3 mm arb. as shown in table 2 , when arb was pre-incubated with both partners before fusion was initiated by lowering the ph, fusion inhibition was ca. 50%. in contrast, only ca. 10% fusion inhibition was observed when arb was preincubated with either hcvpp or liposomes. the greater inhibitory effect of arb when it has simultaneously access to both viral and target membranes suggests that arb could also act by interacting with selective residues of the hcv glycoprotein sequences. this assumption was tested by studying the interaction behaviour of arb with tryptophan (trp) derivatives, as tryptophan is a constituent of proteins often found in regions located close to membrane interfaces, such as stem regions in several viral fusion proteins (e.g. hiv gp41 [36] ). we also reasoned that arb, being an indole derivative, might interact with tryptophan and tyrosine residues through aromatic ring stacking. for this purpose, we tested the effect of increasing concentrations of arb on the fluorescence of n-acetyl tryptophanamide (nata) as a water-soluble trp derivative, and tryptophan octyl-ester (toe) as a membranotropic molecule ( figure 1b-c) . the fluorescence of nata and toe was recorded between 300 and 400 nm, using an excitation wavelength of 286 nm, which corresponds to an absorption minimum of arb [12] . results are presented in table 3 and figure 6 . (table 3) . similarly, the k d of arb for toe in lyso-pc micelles was in the 50 mm range at both ph. this indicates that arb is able to interact with indole rings, but with a relatively low affinity. in contrast, when arb was added to toe associated to liposomes (1:20, toe/lipid molar ratio), a marked increase in affinity was observed (table 3 , compare toe/micelles and toe/ liposomes), reaching k d values in the 10 mm range. note that toe fluorescence could not be measured in dpc micelles, due to a great intrinsic fluorescence of the dpc used for our experiments. interestingly, these k d values are comparable to arb ic50 inhibition of hcvpp fusion (see above and discussion section). indole fluorescence decreased when arb concentration increased, with virtually no measurable fluorescence for 100 mm arb ( figure 6 ). this further confirms that arb interacts with indole rings, but with a higher affinity when indole is incorporated into lipid membranes. this affinity was higher for pc than for pc:chol liposomes (table 3) , and at neutral than at acidic ph (table 3 and figure 6 ). at acidic ph, arb is most likely protonated in the lipid environment [12] , as is probably toe as well. arb affinity for toe under these conditions might then be lower than that of uncharged arb at neutral ph, because of repulsive electrostatic interactions. a third set of experiments was designed to assess the behaviour of arb in the presence of aromatic residues into protein sequences, more specifically toward trp present in peptides. for this purpose, we used synthetic membrane-binding peptides of known structure and containing only one tryptophan residue, expected to be localized at the membrane interface: the transmembrane helix of hcv ns4a protein [16] and the n-terminal amphipathic helix of bvdv ns5a protein interacting in-plane of the membrane interface [15] . intrinsic tryptophan fluorescence of both peptides was monitored in the presence of increasing concentrations of arb; this is illustrated in figure 7 for hcv ns4a peptide inserted into pc:chol liposomes (and in figure s3 for bvdv ns5a, 1:20 peptide-to-lipid molar ratio). the arb dose-dependent quenching of tryptophan fluorescence at both neutral and acidic ph clearly indicates arb interaction with both peptides (insets in figure 7 and fig. s3 ). for the ns4a peptide at both ph, a red shift of the spectral maximum, proportional to arb concentration, accompanied the fluorescence quenching; this effect was more pronounced at acidic ph (6 nm at ph 7.4 for 5 mm arb, and 12 nm at ph 5.0 for 100 mm arb). this red shift suggests that arb, when interacting with ns4a peptide, relocates its tryptophan residue to a more shallow zone of the membrane, where the trp environment would be more hydrophilic. the measure of the apparent affinity of arb for these peptides inserted into pc:chol liposomes was performed as described above. interestingly arb displayed an apparent k d toward peptide trp between 3.3 and 5.6 mm (table 4 ), twice lower than that observed for toe in pc:chol liposomes. since both peptides contain one or two tyrosine residues (hcv ns4a and bvdv ns5a, respectively) in addition to the trp, interaction of arb molecules with these aromatic residues might account for a higher affinity of arb for peptides than for a small molecule such as toe. since arb is an inhibitor of hcv membrane fusion, we reasoned that it might interact with the regions of e1 and e2 described as important for hcv fusion [11, 17] . these peptides were described as membranotropic on model membranes [37] and contain aromatic residues. we therefore analyzed the effect of increasing concentrations of arb on the fluorescence quenching of two peptide sequences derived from hcv e2 (positions 415-432 and 606-625, see aa sequences in table 5 ), and inserted into pc:chol liposomes (1:20 peptide-to-lipid molar ratio). note that we also tested a third peptide located at position 270-283 of e1, containing only one tyr; but its fluorescence quantum yield was too low to monitor any interpretable fluorescence signal (data not shown). we then calculated the k d values as described above. as shown in table 5 , e2 415-432 contains only one trp, whereas e2 606-625 contains one trp and 3 tyr. the apparent affinity was in the 15 mm range at ph 7.4 for both peptides, reminiscent to arb ic50 of hcvpp fusion. this indicates that arb is able to interact with the aromatic residues of both peptides in the membrane, and lends further support to our hypothesis that arb could interact with key residues/motifs in viral fusion proteins, which would constitute a possible (partial) explanation to its inhibition of hcvpp fusion. strikingly this affinity decreased at acidic ph for both peptides, and even drastically to 70 mm for e2 606-625. interestingly, this relatively high k d value is reminiscent of that observed for arb interaction with nata in solution (table 3) . this suggests that the interaction between the e2 peptide and the membrane would be weak at acidic ph, and that most of the peptide could be in solution. moreover an histidine residue, located in the immediate vicinity of trp in the sequence of both peptides, is expected to be charged at ph 5.0. since arb is also protonated at that ph value, this could create repulsive forces affecting the interaction between trp and arb. moreover, as protonation of the histidine cycle is expected to decrease the free energy of partition from lipids to water, the peptide could be released from the membrane at acidic ph, possibly in relation with peptide conformational change(s). this behavior in not in favor with their direct role as fusion peptides of hcv, a virus dependent on low ph for its membrane fusion activity. however, due to their membranotropism [37] , and since our and other studies showed their involvement in hcv membrane fusion [17, 38] , it is possible that the conformational changes they might undergo at low ph would lead to a proper relocation of the actual fusion peptide/loop toward the target membrane [39] (and see discussion section). this study aimed at further investigating the molecular mechanism of action by which arbidol (arb) inhibits virus cell entry and membrane fusion, using hcvpp as a model of an enveloped virus. we showed that arb displayed a dual binding capacity, on lipid membranes interface on one hand and on the aromatic residue table 4 ). doi:10.1371/journal.pone.0015874.g007 tryptophan of proteins on the other hand. it therefore appears plausible that the observed inhibitory effect of arb on viral entry and membrane fusion might result from a combined effect of binding of arb on membranes and on (fusion) proteins. from a physico-chemical point of view, arb displayed tropism for membranes or membrane-like environments such as detergent micelles, particularly prominent at low ph [12] . by combining several biochemical approaches, we show here that arb has the propensity to bind to and incorporate into lipid bilayers, with calculated apparent affinities in a similar range as the ic50 value for fusion, i.e. ca. 10 mm. our nmr studies of arb interaction with dmpc leads to a model where arb binds at the membrane interface and establishes contacts mainly with the polar heads of phospholipids (fig. 5d ). altogether these data suggest that at least part of arb inhibitory activity could be explained by its membranotropism. this physicochemical property has been further emphasized in a recent work by villalain [40] , using fourier-transform infrared spectroscopy. arb interaction with phospholipids would disturb membrane fluidity crucial to the fusion process, thereby rendering the lipid bilayer less prone to fusion. such a model is consistent with the behavior of other indole derivatives, that were shown to exhibit a preference for membrane interfaces [41, 42] , due to the flat rigid structure of these molecules and to their aromaticity, which allows them to establish cation-p interactions with the positively charged quaternary ammonium lipid headgroups [41, 43] . at low ph, the optimal ph for hcv fusion, these interactions would be favored due to the protonation of the amino groups. as was described for other substituted indoles [44] , it is possible that protonation of the carbon bearing the ester group of arb could displace this group out of the indole plane, and place it in a better position to bond with neighboring molecules. this could in turn lead to a better membrane association. arb might therefore have the propensity to intercalate into lipids of the viral and target membranes while adopting a consistent orientation by filling the gaps between lipid molecules. the interfacial region of the lipid bilayer provides a suitable environment for a wide range of chemical groups, as long as they possess a large enough hydrophobic moiety and a group capable of forming hydrogen bonds with the lipid carbonyl groups. several compounds with antiviral pharmacological properties belong to this category, in particular adamantanes active against influenza a viruses [45, 46] and against some hcv clones but not all [47, 48] , the natural triterpene glycyrrhizin efficient in the treatment of chronic viral hepatites [49] and the flavonolignan molecules composing silymarin, an herbal extract with potent anti-hcv activities [1] [2] [3] 50, 51] . in a previous study, we noticed that arb inhibition of cell entry concerned hcvpp and pseudoparticles bearing the influenza hemagglutinin (happ), but not pseudoparticles bearing the envelope glycoprotein of a feline oncogenic retrovirus (rd114pp) [12] . these data suggest that arb might display selectivity for the recognition of key motifs inside envelope proteins. this hypothesis was tested by assessing the influence of arb on the fluorescence properties of aromatic compounds derived from tryptophan (trp) and of peptides containing trp. trp is a component of proteins with interfacial properties [41, 42] , often located at the lipid/water interface and grouped into so-called tryptophan-rich motifs crucial to protein/membrane association [52] , and found in the envelope (fusion) proteins of the sars coronavirus or hiv-1 [36, 53, 54] . trp is also enriched at protein/protein binding interfaces of the small envelope protein of the hepatitis b virus [55] and of membrane proteins in general [56] . we demonstrated here that arb was able to alter/quench the fluorescence properties of small trp derivatives in solution (nata), in detergent micelles and in liposomes (toe, [57] ), in a dose-dependent manner. this occurred most likely through stacking of the aromatic rings of both molecules which is often involved in stabilization of inter-cations. interestingly the apparent affinity of the arb/trp derivative interaction was in the order: lipid bilayers.micelles.solution, indicating that arb binding strength for trp could increase in membrane environments where both molecules accomodate and get packed. indeed arb apparent affinity for toe in liposomal membranes was in the 10 mm range, a value comparable to the ic50 of fusion. arb affinity was even greater for membrane peptides containing trp and tyrosine (tyr) residues (ca. 4 mm). due to its indole group, it is conceivable that arb might display selectivity not only for indole rings (trp) but more generally for aromatic groups, as the phenol ring of tyr. a greater number of arb molecules could therefore interact with aromatic residues in peptide sequences, leading to some cooperativity in the quenching effect and to an overall larger apparent affinity. the nmr structures of synthetic peptides hcv ns4a * and bvdv ns5a peptides have been reported in references [16] and [15] , respectively. the solubilization tags kkgg and ggkk at the n-and c-terminal ends, are indicated in italic. aromatic residues trp and tyr are indicated in bold, his is underlined. b peptide-to-lipid molar ratio was 1:20. although hcv entry inhibition by arb was found genotypedependent, hcv membrane fusion was inhibited by arb in a genotype-independent manner. hcv entry and fusion are early steps in the life cycle of the virus [58, 59] . hcv first interacts through its envelope glycoproteins with a set of coreceptors at the plasma membrane level (recently reviewed in [60, 61] ) and eventually becomes endocytosed [62] [63] [64] . due to a combined action of acidification in the endosome and particular lipids like cholesterol and sphingomyelin [11, 18] , viral fusion occurs over a broad spectrum of ph's ranging from 6.3 to 5.0 [11, 18, 65] . hcv binding to the hepatocyte membrane followed by endocytosis therefore requires several cellular proteins, and most likely involves several levels of interactions (interactions between viral proteins, between cellular and viral proteins, between viral/cellular proteins and lipids). these features might explain the differential effect exerted by arb on entry of various hcv genotypes: indeed subtle differences in protein sequences could translate into modified interactions with several partners and/or at several levels. conversely some common principles of action apply to all fusion reactions, viral fusion and cellular fusion processes alike [66] . indeed all fusion processes involve two partners: lipids and the fusion protein(s). this might account for the similar inhibitory effect of arb on hcv fusion observed for all genotypes. this is in line with the observations that arb displayed potent antiviral activity against some antigenic serotypes of influenza viruses, but not against all [9] . previously we noticed that arb inhibition of primary infection of huh-7.5.1 cells with hcv (clone jfh-1) was efficient only when cells were preincubated with arb 24 or 48h before infection [10] ; in addition, inhibition of hcvpp and happ cell entry was most efficient when arb was pre-incubated with both viral and cell membranes [12] . here, using our in vitro fusion assay, we observed that arb inhibition of hcvpp fusion was maximal when both viral and target membranes were incubated with arb, before fusion was initiated. this suggests that a certain level of membrane impregnation and/or saturation with arb must be achieved to efficiently inhibit viral infection. membranes might therefore act as ''concentrators'' of arbidol, and high concentrations of the molecule might be locally achieved. this could explain why arb, exhibiting an apparent (medium to low) affinity for membranes in the mm range, exerts a relevant antiviral activity without noticeable membrane damages. along these lines, in spite of its marked membranotropism, arb displays only low toxicity [9, 10] . arb exhibited a comparable micromolar apparent affinity for aromatic residues present in membrane peptides in a membrane environment. altogether, these observations lead us to propose a mechanistic model of the way arb would inhibit hcv entry and fusion. through its membranotropism, arb is able to freely interact with viral and target membranes, and could locally get highly concentrated. arb is also able to interact with aromatic residues within viral proteins involved in membrane interactions and membrane destabilization necessary for fusion. through this dual binding capacity, arb could then locally impede the structural rearrangements required for the fusion protein to adopt its fusion conformation. the fact that arb is active in the mm range suggests that arb would act by reducing the overall speed of the fusion reaction rather than by blocking a specific protein conformation. this could therefore explain the broad antiviral spectrum of arb, and the genotype independence of its inhibitory effect on hcv fusion, since hcv envelope proteins contain well-conserved aromatic residues in all genotypes. mechanistically, the key point is the relative accessibility of these residues to arb at the membrane interface. a cooperative effect between arb and several aromatic residues might therefore occur. also the local environment of these aromatic aa is important, since the presence of residues such as histidines (his) in their vicinity could modify their accessibility with respect to ph. interestingly enough, in the sequence of both hcv e2 peptides studied here (table 5 ) and shown to be involved in hcv fusion [17] , his is contiguous to trp, and in the 606-625 peptide, his is surrounded by three tyrosines. the concept of his as a critical ph sensor at a key intramolecular domain interface in a viral fusion protein has recently emerged [67, 68] . indeed, the protonation of a sole his in the e protein of the tick-borne encephalitis flavivirus (tbev) triggers large-scale conformational changes leading to viral fusion. concerning hcv, rey and coworkers recently proposed a model of the 3d arrangement of the e2 ectodomain [39] . in this model, the fusion loop/peptide would lie within the poorly structured domain ii, and the e2 606-625 peptide would be found in the globally unstructured domain iii, where a critical his residue is disposed at the interface with domain i. the putative fusion loop contains a phenylalanine and a tyrosine [39] . at low ph, the optimal ph for hcv membrane fusion, key histidine(s) could become protonated. this could result in conformational rearrangements and, in the context of arb fusion inhibition, aromatic residues might consequently become more or less accessible to arb molecules present in their vicinity. we noted that the apparent affinity of arb for hcv peptides was weaker at ph 5.0 than at ph 7.4. at low ph, arb is also protonated, and this protonated form could exhibit a greater preference for the interfacial region of the lipid bilayer than the deprotonated form, as demonstrated for adamantanes [45] . combined with the notion that key aromatic and his residues would also display interfacial (re)localization at low ph, this would in turn explain the higher efficiency of arb at inhibiting fusion at acidic ph [12] . in conclusion our data reveal that arb directly interacts with the lipid membrane-water interface, and is able to bind to aromatic residues present in hcv glycoproteins, in their membraneassociated form. through a subtle binding interplay between arb, lipids, viral and cellular proteins, arb might efficiently block hcv entry and membrane fusion interacting with the main actors of the early steps of viral entry. most interestingly, arb inhibition of these processes demonstrated an affinity in the mm range, although the membranotropic properties of arb suggest that it could become locally more concentrated in membranes. together, these findings suggest that arb could increase the strength of viral glycoprotein's interactions with the membrane due to a dual binding mode, involving aromatic residues and phospholipids. the resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the membrane fusion process. the antiviral mechanism of arb therefore opens promising perspectives for the development of small membranotropic low affinity molecules, that would become locally concentrated in membranes and would mainly act on the kinetics of the conformational rearrangements of the viral fusion protein. figure s1 arb inhibits infectivity and membrane fusion in a dose-dependent manner. a, infectivity. results are the mean 6 sem of 5 separate experiments. black, no arb; blue, 1.9 mm; green, 5.6 mm and red, 11.3 mm arb, respectively. b, membrane fusion between hcvpp of genotype 4 (4.11.21) and r18-labeled liposomes. the lipid mixing kinetic was followed by fluorescence spectroscopy using excitation and emission at 560 and 590 nm, respectively. fluorescent liposomes (12.5 mm final lipid concentration) were added to 40 ml of hcvpp in pbs ph 7.4 at 37uc, in the absence or presence of the indicated concentrations of arb. after a 2 min-equilibration, lipid mixing was initiated by decreasing the ph to 5.0 with diluted hcl, and r18 dequenching was recorded. maximal fluorescence was obtained after addition of 0.1% final triton x-100. average value of the last 30 s of fusion (i.e. final extent of fusion) was used to calculate the percentage of fusion in the presence of arb, relative to 100% fusion without arb ( figure 2) . black, no arb; blue, 1.9 mm green, 5.6 mm and red, 11.3 mm arb, respectively. (tif) of increasing concentrations of arb (2, 5, 10, 25 and 100 mm from top to bottom). trp emission fluorescence was measured between 300 and 400 nm, with excitation at 286 nm. the apparent affinity of arb toward trp was calculated from the plot of the difference da between areas under the curve (auc) of peptide without arb (da = auc no arb 2auc with arb ) as a function of arb concentration (insert). (tif) identification of hepatoprotective flavonolignans from silymarin multiple effects of silymarin on the hepatitis c virus lifecycle silibinin and related compounds are direct inhibitors of hepatitis c virus rnadependent rna polymerase synthesis and in vitro anti-hepatitis b virus activities of some ethyl 6-bromo-5-hydroxy-1h-indole-3-carboxylates antiviral chemotherapeutic agents against respiratory viruses: where are we now and what's in the pipeline? hepatitis c virus entry: an intriguing challenge for drug discovery screening of small-molecule compounds as inhibitors of hcv entry characteristics of arbidolresistant mutants of influenza virus: implications for the mechanism of antiinfluenza action of arbidol arbidol: a broadspectrum antiviral compound that blocks viral fusion arbidol: a broad-spectrum antiviral that inhibits acute and chronic hcv infection low ph-dependent hepatitis c virus membrane fusion depends on e2 integrity, target lipid composition, and density of virus particles biochemical mechanism of hepatitis c virus inhibition by the broad-spectrum antiviral arbidol synthesis and anti-hepatitis c virus activity of novel ethyl 1h-indole-3-carboxylates in vitro hepatitis b and hepatitis c in 2009 nmr structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5a from bovine viral diarrhea virus structural determinants for membrane association and dynamic organization of the hepatitis c virus ns3-4a complex characterization of fusion determinants points to the involvement of three discrete regions of both e1 and e2 glycoproteins in the membrane fusion process of hepatitis c virus hepatitis c virus glycoproteins mediate low ph-dependent membrane fusion with liposomes growth of human hepatoma cells lines with differentiated functions in chemically defined medium characterization of host-range and cell entry properties of hepatitis c virus of major genotypes and subtypes dna-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles switchedangle spinning applied to bicelles containing phospholipid-associated peptides matnmr: a flexible toolbox for processing, analyzing and visualizing magnetic resonance data in matlab entropic stabilization and equilibrium size of lipid vesicles surface plasmon resonance in protein-membrane interactions interaction properties of the procollagen c-proteinase enhancer protein shed light on the mechanism of stimulation of bmp-1 studies of protein interactions by biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudo-first-order kinetic behavior magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules micelle-induced curvature in a water-insoluble hiv-1 env peptide revealed by nmr dipolar coupling measurement in stretched polyacrylamide gel structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution nmr studies of polypeptides identification of protein surfaces by nmr measurements with a pramagnetic gd(iii) chelate mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves positioning of micelle-bound peptides by paramagnetic relaxation enhancements nmr of paramagnetic substances membrane-induced conformational change during the activation of hiv-1 gp41 the membrane-active regions of the hepatitis c virus e1 and e2 envelope glycoproteins mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis c virus glycoprotein e1 the disulfide bonds in glycoprotein e2 of hepatitis c virus reveal the tertiary organization of the molecule membranotropic effects of arbidol, a broad anti-viral molecule, on phospholipid model membranes interfacial tryptophan residues: a role for the cation-pi effect? the preference of tryptophan for membrane interfaces cation-pi interactions in ligand recognition and catalysis the protonation of indoles. basicity studies. the dependence of acidity functions on indicator structure distribution and dynamics of adamantanes in a lipid bilayer structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug nmr structure and ion channel activity of the p7 protein from hepatitis c virus glycyrrhizin, the main active compound in liquorice, attenuates proinflammatory responses by interfering with membrane-dependent receptor signalling ground and excited state proton transfer and antioxidant activity of 7-hydroxyflavone in model membranes: absorption and fluorescence spectroscopic studies groundand excited-state proton transfer and antioxidant activity of 3-hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies the nterminal region of comparative gene identification-58 (cgi-58) is important for lipid droplet binding and activation of adipose triglyceride lipase a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry a tryptophan-rich motif in the carboxyl terminus of the small envelope protein of hepatitis b virus is central to the assembly of hepatitis delta virus particles a study of the membrane-water interface region of membrane proteins tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs replication of hepatitis c virus hepatitis c virus entry the hepatitis c virus and its hepatic environment: a toxic but finely tuned partnership virology: final entry key for hepatitis c time-and temperature-dependent activation of hepatitis c virus for low-ph-triggered entry hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrincoated vesicles rna interference and single particle tracking analysis of hepatitis c virus endocytosis functional analysis of hepatitis c virus envelope proteins, using a cell-cell fusion assay viral and developmental cell fusion mechanisms: conservation and divergence identification of specific histidines as ph sensors in flavivirus membrane fusion the ph sensor for flavivirus membrane fusion the xplor-nih nmr molecular structure determination package this work was presented at the 16 th international symposium on hepatitis c and related viruses, (nice, france, october 3-7, 2009).the authors gratefully acknowledge sylvie ricard-blum for spr analysis expertise. fluorescence experiments were performed on the platform ''production et analyse des protéines'' of the ifr 128 biosciences gerland-lyon sud. key: cord-314254-9ye8tfvz authors: pfaender, stephanie; brown, richard jp; pietschmann, thomas; steinmann, eike title: natural reservoirs for homologs of hepatitis c virus date: 2014-03-26 journal: emerg microbes infect doi: 10.1038/emi.2014.19 sha: doc_id: 314254 cord_uid: 9ye8tfvz hepatitis c virus is considered a major public health problem, infecting 2%–3% of the human population. hepatitis c virus infection causes acute and chronic liver disease, including chronic hepatitis, cirrhosis and hepatocellular carcinoma. in fact, hepatitis c virus infection is the most frequent indication for liver transplantation and a vaccine is not available. hepatitis c virus displays a narrow host species tropism, naturally infecting only humans, although chimpanzees are also susceptible to experimental infection. to date, there is no evidence for an animal reservoir of viruses closely related to hepatitis c virus which may have crossed the species barrier to cause disease in humans and resulted in the current pandemic. in fact, due to this restricted host range, a robust immunocompetent small animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control and prophylactic vaccine development. recently, several studies discovered new viruses related to hepatitis c virus, belonging to the hepaciand pegivirus genera, in small wild mammals (rodents and bats) and domesticated animals which live in close contact with humans (dogs and horses). genetic and biological characterization of these newly discovered hepatitis c virus-like viruses infecting different mammals will contribute to our understanding of the origins of hepatitis c virus in humans and enhance our ability to study pathogenesis and immune responses using tractable animal models. in this review article, we start with an introduction on the genetic diversity of hepatitis c virus and then focus on the newly discovered viruses closely related to hepatitis c virus. finally, we discuss possible theories about the origin of this important viral human pathogen. hepatitis c virus (hcv) infection is a major cause of chronic liver disease. currently, about 160 million individuals are persistently infected with hcv. 1 acute hcv infection is asymptomatic in many cases, but 50%-80% of infected individuals are unable to clear the virus and a state of persistent viral replication and hepatic inflammation, i.e., chronic hepatitis c, follows. some patients remain asymptomatic, but over years or decades others develop cirrhosis, portal hypertension, deteriorating liver function and hepatocellular carcinoma. it is due to these late complications that chronic hepatitis c is a leading cause of liver-related death and the prime indication for liver transplantation worldwide. 2 combination treatment with pegylated interferon-a and ribavirin has been the standard of care for more than a decade. 3 recently, the first directly acting antivirals targeting the viral ns3-4a protease were licensed and triple therapy has further improved treatment options for genotype 1. 4 however, resistance development, side effects and viral genotype-specific efficacy of these drugs require alternative antiviral treatment options. the hcv genome is 9.6 kb in size, encodes for a single polyprotein that is cleaved by cellular and viral proteases into at least 10 different proteins: the structural proteins core, e1, e2, the ion channel p7 and the non-structural proteins ns2, ns3, ns4a, ns4b, ns5a and ns5b. 5 these viral factors act in concert with host proteins to mediate virus entry and to coordinate rna replication and virus production which is coupled to lipoprotein biogenesis. 6 ongoing transmission of productive hcv infection is limited to human populations, although higher primates are susceptible to experimental infection. 7 due to this narrow host range, a robust immunocompetent small animal model is still lacking. however, a recent study demonstrated infection, replication and de novo virus production in an inbred mouse strain engineered to express human hcv entry factors in the liver and carrying a targeted lesion within innate immune signalling genes (stat1, irf7, irf9, ifn-abr). 8 although not fully immunocompetent, this landmark study shows that hcv can be propagated in mice and raises the hope that more robust models ultimately with fully functional innate and adaptive immune system can be developed. despite the rapid advancements in hcv research over the past decade, the origin of hcv still remains elusive, with no evidence of an animal population that might have transmitted the virus to humans. 9 nevertheless, the idea that there might be a nonhuman primate source for hcv infections, as described for hiv, 10 has engaged researchers since the discovery of hcv in 1989. 11 the recent discovery of novel hepaciviruses in different animals could now reveal new insights in the origin of hcv. several reports identified new homologs of hcv in mammals that cluster in the genus hepacivirus or the newly proposed genus pegivirus. non-primate hepaciviruses (nphv) were initially discovered in domestic dogs and subsequently in horses 12, 13 and other diverse and widespread hcv-like viruses have been reported in wild populations of rodents and bats. [14] [15] [16] tentatively assigned to this genus is gb virus b (gbv-b), a virus distantly related to hcv identified in a laboratory-housed tamarin, a new world monkey and causing hepatitis. 17 however, gbv-b infection has not been reported in any other tamarin or other new world monkey species and its origin, as well as its natural host, remains unknown. besides hepaciviruses, the flavivridae family consists of three other genera: flaviviruses including yellow fever and dengue virus; pestiviruses including bovine viral diarrhea virus and classical swine fever virus and the recently assigned pegivirus genus. pegiviruses encompass the previously unclassified gb virus a (gbv-a) which was found in primates, 18 gb virus c (gbv-c) or hepatitis g virus (hgv) which infects humans and chimpanzees 19, 20 and gb virus d (gbv-d) identified in bats. 21 of note is that in contrast to the hepaciviruses, gbv-a and some gbv-c/hgv isolates do not appear to encode a core protein. 22 25 hcv is genetically highly variable and, based on phylogenetic analyses, viral isolates are grouped into seven genotypes (1-7) (figure 1 ), which are associated with specific global regions and modes of transmission. 26 the high observed genetic diversity is a result of the error-prone replication of the virally encoded rna dependent rna polymerase, coupled with the high replication rate in vivo. 27 this genetic variability exhibited by hcv facilitates immune evasion and contributes to viral persistence. hcv genotypes differ from each other by 30%-35% at the nucleotide level. however, this genetic heterogeneity is not evenly distributed throughout the viral genome, and is concentrated in regional hot-spots. the greatest levels of diversity are observed in the e1e2 genes encoding the envelope glycoproteins e1 and e2, while other genome regions, such as those encoding the core protein or the helicase domain of the ns3 protein, exhibit higher levels of conservation. 28 despite substantial sequence variation, all genotypes share the same genome structure, encoding a single polyprotein which is postranslationally cleaved by viral and host proteases into 10 mature proteins. the single open reading frame (orf) is flanked by two highly conserved untranslated regions (utrs). hcv genotypes can be further divided into multiple distinct subtypes (a, b, c, etc.) differing by 20%-25% at the nucleotide level. 29 even within an infected patient, hcv exists as a heterogeneous population of genetically distinct yet related variants. 30 the prevalence of hcv genotypes differs significantly in different parts of the world. seventy percent of patients in north america and europe are infected with genotype 1, while hcv genotypes 2 and 3 account for about 25% of cases, but are overrepresented among patients who inject drugs. 31 in contrast, hcv genotype 3 can be detected in about 65% of patients in south east asia and genotype 4 is the major genotype in the middle east and northern africa. genotypes 5 and 7 have been detected mainly in africa, while genotype 6 has been found in china and southeast asia. 29 phylogenetic analyses, coupled with global distribution patterns of hcv genotypes, indicate that genotypes 4 and 6 emerged ca 350-700 years ago. 32 the worldwide dissemination of genotype 2 began later some 90-150 years ago, with the restricted diversity of sequences among genotype 1b indicating emergence around 60-70 years ago. 33 one has to keep in mind that this molecular clock analysis might be under-or overestimated due to mutational masking and substitutional saturation. utilizing both deep sequencing and serology-based approaches, a number of novel hepaciviruses have been identified in different mammalian host species. kapoor and colleagues 13 were the first to discover a canine homolog of hcv which was phylogenetically closely related to hcv. initially termed canine hepacivirus (chv), the virus was independently sequenced from dogs with a respiratory illness from two different outbreaks of respiratory disease in the united states, whereas no healthy animals were found to be infected. high viral loads (.10 7 viral copies/ nasal swab) were detected in nasal swabs of the infected animals. testing liver and lung samples from 19 unrelated dogs which had died of unexplained gastrointestinal illness, chv rna was found at low levels (,10 3 copies/2 ng of total rna) in liver samples, although lung tissue contained no detectable viral rna. furthermore, using in situ hybridization, kapoor et al. revealed focal and dispersed infection of canine liver and the presence of viral rna mainly in the cytoplasm of hepatocytes. strikingly, there was a high degree of genetic relatedness between viral sequences from different animals which is rather unexpected for a rna virus. the viral genome of chv encodes a 2942 amino acid (aa) polyprotein, which is predicted to be cleaved into 10 mature viral proteins (figure 2 ), in a similar fashion to that described for hcv 5 (table 1) . comparative phylogenetic analyses of conserved regions of the predicted helicase (ns3) and rna-dependent polymerase (ns5b), revealed chv to be the closest relative of hcv discovered to date, and equidistant from all seven hcv genotypes (figure 1) . the virus displayed approximately 50% nucleotide sequence divergence from hcv with a maximum aa identity to hcv in the non-structural proteins ns3 and ns5b (.55%-65%), whereas e1, the n-terminal half of e2, ns2 and the c terminus of ns5a showed the lowest aa identity (,35%-45%). surprisingly, the glycoprotein regions of chv were readily aligned with hcv, with marked similarity in the c-terminal half of e2. analyses of the e1/e2 glycosylation sites revealed 4 and 10 potential potential n-linked glycosylation (png) sites, respectively, comparable to those predicted for hcv (e1 4 or 5 and e2 up to 11). 34 png sites are essential for immune shielding, efficient entry and correct folding of the hcv envelope glycoproteins incorporated into infectious virions. 35 intriguingly, the binding site for mir-122, which is important for hcv replication, in the 59 utr could not be identified in the chv sequence. together with the absence of microrna sequences in the dog genome capable of binding to the equivalent site in chv, this finding argues that this virus might not depend on mir-122 for its replication. the mean time to the most recent common ancestor between the hcv genotypes and chv was estimated to be between 500 and 1000 years before present (ybp). in an effort to further investigate the host range of chv, the same group utilized a serology-based approach to screen for the presence of the virus in other mammalian species. 12 burbelo and colleagues used a recombinant protein expressed from the helicase domain of chv ns3 as antigen to detect chv antibodies in sera from different animals. surprisingly, they detected immunoreactivity against chv ns3 in 36 samples of 103 horses (3%), with eight horse sera also carrying viral rna, whereas 80 dogs, 14 rabbits, 81 deer and 84 cows were seronegative, with one intermediate positive sample from a cow. initial sequencing of the eight viral rna positive horse samples identified a series of genetically diverse viruses. 12 based on these findings in a different host, the authors tentatively termed these viruses nphv. 12 complete genomic sequences from these eight nphv variants were obtained ( table 2 ). the original chv variants showed a very high similarity (maximum of 0.35% divergence) to one of the eight nphv variants, whereas the eight horse-derived nphv sequences themselves had moderate genome sequence diversity from each other (6.4%-17.2%). this high degree of homology between nphv and chv combined with the lack of other hepaciviruses in dogs suggests that the chv isolate may in fact be a horse virus that was transmitted to a dog. if that was a true transmission event or simply a contamination, possibly by feeding on horse meat or veterinary products which utilized horse based components is unclear. at the aa level, the structural region showed a greater divergence than the non-structural region. of note, most sequence diversity between nphv variants was attributable to synonymous substitution (d s ), with low levels of nonsynonymous substitution (d n ) variation observed. indeed, low d n /d s ratios of between 0.03 and 0.05 indicate the action of strong purifying selection on nphv genomes when compared to hcv, which displays a higher frequency of d n variation and thus higher d n /d s ratios. in general, sequence divergence among nphv isolates was greater than subtype diversity within hcv. even though the originally predicted chv secondary structure of the 59 utr showed (table 1) . unlike hcv, which chronically infects 50%-80% of exposed humans, only 22% of horses showed co-presence of igg antibodies and viral genomes. this could be indicative for either an acute infection or would suggest that possibly the majority of equine hosts are able to clear nphv infection. to further investigate the species specificity of nphv and to address questions regarding tissue tropism or pathogenesis of this newly identified hepacivirus, the group of simmonds and colleagues analyzed nphv in domestic horses in the united kingdom. 36 screening for the presence of nphv in other mammals, the authors performed large-scale polymerase chain reaction (pcr)-based investigation of samples from dogs, cats, pigs, rodents, donkeys and horses. from this survey, three plasma samples from 142 horses (2%) were found positive for nphv rna. sequence comparison demonstrated that each of the positive horses was infected with nphv variants distinct from the eight previously identified in horses. the newly identified nphv variants displayed similar branching orders in each genome region, indicating a lack of recombination as observed previously. 12 clinical records from the time of sampling displayed no evidence for a hepatic or systemic disease. furthermore, liver function analyses revealed no indication for hepatic inflammation as c-glutamyl transferase and glutamate dehydrogenase values were within reference range, with the exception of a mildly elevated c-glutamyl transferase new hcv-like viruses in different mammalian hosts pfaender et al 4 level in one horse. moreover, with the bile acid levels within reference range, there was no sign of hepatic damage. repeated samplings from one horse four and five months after the initial sampling revealed a persistent infection. the horse remained viremic, but the viral load decreased between the fourth and fifth months from the initial 4.8310 7 copies/ml to 2.1310 5 and 7.1310 4 copies/ml, respectively. during the sampling period, the horse remained clinically unremarkable and the liver indices stayed mainly within the reference ranges, although frequently at the upper end of the reference ranges. this data indicate that hepaciviral infections in horses can be persistent. the organ tropism of nphv has yet to be elucidated and further studies on viral associated pathogenesis, the course of clinical disease and likely mode(s) of transmission are required to fully understand the nature of nphv infection in horses. the discovery of closely related hepaciviral homologs of hcv, which naturally infect equine hosts, raised the possibility that additional table 2 ). based on these sequences, the rhv genome is predicted to encode a polyprotein of 2748 aa flanked by 59 and 39 utrs (table 1 ). in one of the sequenced variants (rhv-339), a putative mir-122 seed site was apparent in the 59 utr, suggesting a possible dependence on mir-122 of these novel viruses. the polyprotein is predicted to encode 10 proteins which were similar in predicted size to those of hcv and other hepaciviruses ( figure 2 ). the rhv glycoproteins e1 and e2 contained two and four png sites, respectively, considerably less than those observed for hcv and nphv. the computed genetic distance between rhv-339 and hcv in the structural regions ranged from 67% to 77% and 65% to 70% in the non-structural regions, and was therefore substantially greater than that between hcv and nphv. additionally, another group independently described novel hepaciviruses infecting rodent hosts. 16 drexler and colleagues screened for bloodborne viruses in sera and organs from 4770 rodents and sera from 2939 bats. even though bat serum showed cross-reactivity with hcv antigens, no hepacivirus rna was detected in these samples. nevertheless, the group identified three highly divergent novel rhv clades in european bank voles (myodes glareolus) and in south african four-stripped mice (rhabdomys pumilio). to determine the genome organization and structural features of rhv, near full genomes of five representative hepaciviruses from all rodent clades were determined ( table 2 ). the five sequenced rhv genomes were predicted to encode a typical hepacivirus polyprotein comprising three structural and seven non-structural proteins of comparable size to those previously described for other hepaciviruses (figure 2 : rhv-1, 2 and 3). again, png sites in the envelope proteins, in particular in the putative e2 protein, were predicted to be fewer when compared to hcv (table 1) . the minimum aa identity of the novel rodent viruses to hcv averaged in the structural proteins between 15.1% and 31.3% and in the nonstructural proteins from 16.3% to 42.2%. based on the high degree of nucleotide sequence homology of the ns5b genes between all members of the flaviviridae family, comparative analysis was possible and revealed the grouping of rhv as a monophyletic sister-clade to hcv. interestingly, all rhv clades were slightly more related to gbv-b than to hcv (figure 1 ). further genomic analyses revealed the presence of one mir-122 binding site, again pointing to a possible hepatotropism for these viruses. indeed, the authors were able to find high viral loads in bank vole tissues infected with rhv, with the highest rna concentrations found in liver tissue: 1.8310 8 copies/g compared to significantly lower concentrations in lung, kidney, serum, spleen, heart, brain and intestine. in situ hybridization revealed foci of viral rna in the cytoplasm of m. glareolus hepatocytes, whereas no evidence of virus infection by in situ hybridization could be found in the other organs. evidence for liver inflammation was observed by histopathological examinations, which revealed low-grade focal lymphatic invasion in the rna-positive animals compared to the rna-negative animals. serological investigations of the myodes hepaciviruses revealed the presence of antibodies against the ns3 antigens of these viruses in only some of the animals (8.3% and 12.4%), and no cross-reactivity of the sera with hcv could be observed. this indicates a specific immune reaction against rhv. rna and antibodies were found in 3 of 57 (5.3%) pcr-positive bank vole sera and this low co-occurrence could indicate that bank voles might be able to clear hepacivirus infections in the majority of cases. the identification of rodent homologs of hcv could pave the way for novel surrogate animal models of hcv which may ultimately facilitate vaccine design and treatment approaches. although the group of drexler and colleagues failed to directly recover hepacivirus genomes in bats, they were able to show serological evidence for these viruses in bats, suggesting the existence of bat hepaciviruses (bhvs). indeed, the group of quan and colleagues 15 identified bats as a major natural reservoir for hepaciviruses and pegiviruses. the group utilized an unbiased high-throughput sequencing approach to enhance the knowledge of viral diversity in bats and encountered a highly diverse group of bat-derived viruses which were related to hepaciviruses and pegiviruses. the group was able to detect viral genomes in six of the eight bat families tested and in a total of 78 sera/plasma samples, one lung specimen as well as one rectal swab. the viral load was determined by quantitative pcr and ranged from 10 3 to 10 8 rna copies/ml in the sera or plasma of infected bats. taken together, the group was able to identify 83 bat-derived viruses, which potentially represent 22 novel viral species. the bhvs fell into three highly divergent clades exclusively composed of viruses from two species of african bats (hipposideros vittatus, otomops martiensseni). clade a viruses were most closely related to gbv-b whereas clade c and d viruses fell into a basal position relative to the clades containing nphv and hcv. bhv serum levels ranged from 1.07310 5 to 3310 8 rna copies/ml. five near full-length genome sequences were obtained representing all three clades of bhv (table 2) . bhv genomes encode a single positive-stranded rna genome containing a single orf, flanked at the 59 and 39 end by non-translated regions, encoding a polyprotein precursor of about 2901 to 3024 aa ( figure 2 ). consistent with other viruses in the family of flaviviridae, conserved protein domains were recognized in the predicted polyprotein. comparable to other hepaciviruses, the most variable regions of the genome encode the envelope glycoproteins, the non-structural proteins ns2 and ns5a with less than 34.5%, 33.5% and 20.4% aa sequence identity, respectively. the most conserved regions were the ns3 and ns5b proteins, with aa sequence identities of 39.2%-53.3% in the ns3 gene and 35.3%-46% in the ns5b gene. again, the translated bhv e1 and e2 protein sequence showed fewer png (6-7) sites compared to hcv (table 1) . regarding pathogenesis of these new viruses, further investigations are required. even though there were high levels of viremia detected, all bats collected were apparently healthy, suggesting that bhvs may not be pathogenic to their host. bats are probably the most abundant, diverse and geographically dispersed vertebrates worldwide. 38 the discovery of bhv and bat pegivirus (bpgv) in bats adds new members to the ever expanding list of viruses that can infect this animal reservoir. many different zoonotic viruses, including rabies virus and related lyssaviruses, nipah and hendra viruses as well as ebola and severe acute respiratory syndrome coronavirus and recently also hepadnaviruses have been found to originate in bats. 39, 40 further research might shed new light on the role of these mammals as carriers of members of the flaviviridae and may provide new insights into the evolutionary origins of hcv. pfaender et al 6 initially, novel hepaciviruses were all identified in non-primate species. recently, the group of lauck and colleagues expanded this host range further with the discovery and characterization of the first hepacivirus infecting a wild non-human primate, the black-and-white colobus (colobus guereza), an old world monkey from uganda. 37 notably, all infected animals appeared healthy at the time of sampling with no observed overt clinical symptoms. deep sequencing of rna from plasma samples of nine of these animals revealed the presence of viral rna in three animals, with the genomic architecture matching other viruses from the hepacivirus genus. the virus, named gnereza hepacivirus (ghv), was shown to share a common ancestry with gbv-b, the recently identified rhvs and one of the three recently discovered bhv clades (clade a). viral sequences covering the entire coding region as well as partial 59 and 39 utrs were obtained from all three animals. the new viruses share limited nucleotide identity across the coding region with other known hepaciviruses (hcv 43%, nphv 43%, rhv 47%, gbv-b 48% and bhv 50%). based on sequence comparison, two highly similar variants were classified as subtype ghv-1, whereas the third variant represents subtype ghv-2. interestingly, a canonical mir-122 binding site was present in both ghv subtypes. the predicted cleavage sites of the ghv polyprotein are similar to other hepaciviruses, with ten mature viral proteins predicted. the glycoproteins e1 and e2 contain four png sites each (table 1) . a striking feature of theses hepaciviruses is their unusually long ns5a gene (882-883 aa), approximately twice the length of any other known ns5a within the flaviviridae, rendering the genomic sequence over 1 kb longer than that of hcv ( figure 2 ). slidingwindow analyses of aa similarities between ghv and other members of the hepaciviruses revealed that across the region from core to ns4b ghv is most similar to bhv, whereas ghv ns5b shares the highest sequence similarity with rhv. this suggests that cross-species transmission and ancient recombination might have contributed to the evolution of ghv. phylogenetic analyses support the grouping of ghv within the hepacivirus genus (figure 1 ) and the shared common ancestry of ghv, gbv-b, rhv and bhv-112. analyses to determine the most recent common ancestor for ghv, hcv and nphv suggests an early divergence of ghv from the other hepacivirus lineages, at least 1000-1500 ybp. 37, 41 taken together, the detection of ghv represents the first documented natural infection of a non-human primate with a hepacivirus and further expands the known host range of this viral genus. as a newly proposed genus of the family of the flaviviridae, the genus pegivirus has been proposed to encompass the so far unclassified viruses gbv-a, gbv-c/hgv and gbv-d. 25 based on this new classification, different groups also identified multiple viruses falling into the pegivirus genus, which are emerging in new hosts. chandriani and colleagues 42 identified a highly divergent member of the flaviviridae family, which is likely the causative agent for an outbreak of acute hepatic disease occurring on a horse farm. the virus was present in equine serum from two overtly clinical index cases of hepatitis and from an equine plasma product which was administered to the horses before the outbreak. this new pathogen was designated theiler's disease-associated virus (tdav), presumably the causative agent of theiler's disease, an acute hepatitis in horses. viral titers in most of the infected horses ranged from 10 7 to 10 8 genomes/ml. the viral genome contained a single orf encoding a polyprotein of 3189 aa flanked by a 59 and 39 utr. based on comparison with related members of the flaviviridae, the virus is predicted to encode three structural proteins (core, e1 and e2) and seven putative non-structural proteins (a protein between e2 and ns2 as well as ns2, ns3, ns4a, ns4b, ns5a and ns5b). phylogenetic analyses grouped tdav as belonging to the newly proposed pegivirus genus (figure 1) . over the length of the entire polyprotein, the virus shares 35.5% aa identity with gbv-d, 20.5% identity with hcv (genotype 1) and 20.4% identity with nphv. the most conserved regions can be found in ns3 and ns5b, with regions exceeding 75% aa identity with gbv-d. interestingly, even though tdav is linked to an acute hepatitis, no mir-122 binding site could be identified, suggesting that, unlike hcv, the virus can replicate independently of mir-122. transmission between horses appears to be low as the virus was undetectable in horses which had contact with other infected animals. the tdav-positive horses were further ranked as clinical or subclinical, with the clinical cases displaying significantly elevated liver enzymes in serum. the subclinical cases displayed varying degrees of elevated liver enzymes in the serum, but without overt clinical manifestation of liver disease in most cases. in general, a slightly higher proportion of asymptomatic tdav-positive animals were observed. furthermore, a clear relationship between viral load and clinical hepatitis could not be observed, indicating that the viral load was not predictive of the extent of hepatic injury. further analyses of the infected horses 1 year after the outbreak provided evidence that tdav can establish a chronic infection. to further investigate the route of transmission for tdav infections, the authors performed an inoculation study in which they experimentally injected four healthy animals with a tdav-positive plasma product. even though the viral genome could be detected in all inoculated horses, only one horse showed a clear elevation of liver enzymes over the course of the study. in summary, this study provides evidence for the association of tdav with theiler's disease, identifying tdav as the sole member of the newly described genus pegivirus for which disease association has been demonstrated. kapoor and colleagues 43 independently identified another equine pegivirus (epgv) which is genetically distinct to tdav. the group screened serum samples from horses with elevated liver enzyme levels and detected viral rna in two samples, which were highly divergent from all previously known pegiviruses ( table 3) . the virus encodes a 59 utr, a single orf encoding a putative polyprotein of 3305 aa and a 39 utr. sequence alignments revealed the divergence of epgv from other pegiviruses, ranging from 62% to 77% in the structural genes and from 49% to 59% in the non-structural region. the most conserved regions were the ns3 and ns5b genes, as described for the hepaciviruses genus, whereas the highest sequence diversity can be found in the glycoproteins e1/e2 and ns4b. to analyze the prevalence and persistence of epgv, two horse cohorts were studied. in one cohort, the viremia frequencies were 25% (3/12) among horses with elevated liver enzymes and 6.4% (4/62) among healthy animals. in another herd analyzed at different time points over four years, 15%-32% of the horses were found to be positive, with two horses remaining viremic for at least three and a half years and two horses being able to clear the infection. viral genome copy numbers ranged from 10 4.5 to 10 6.5 genome equivalents/ml in the serum and viral rna could also be detected in liver and lymph node biopsy samples, as well as in peripheral blood mononuclear cells, with no major differences in viral rna between the tissues, suggesting that the virus is not strictly hepatotropic. 43 further studies are necessary to determine the prevalence, tissue tropism and possible disease association of this newly identified virus. the same group additionally identified two new species of pegiviruses in rodents (see the section on 'rodents/bats'), one from white-throated wood rats (rpgv-cc61) from which the whole genome was acquired (table 3 ) and the other from deer mice. 14 the viral genome displayed the typical flaviviridae genome organisation including a 59 utr, a polyprotein coding region (3484 aa) as well as a 39 utr. genetic analyses revealed that rpgv was substantially divergent from gbv-a, gbv-c, gbv-d and from the epgv (figure 1 ). amino-acid divergence ranged from 78% to 81% in the structural proteins and 54% to 56% in the non-structural region. apart from rodents, bats comprise the most diverse group of mammals. the pteropus giganteus bats have already been identified as carriers of gbv-d. 21 the group of quan and colleagues further enhanced our understanding of the viral diversity in bats, with the identification of bat-derived hepaciviruses and pegiviruses 15 (see the section on 'rodents/bats'). among the total of 83 bat-derived viruses uncovered, 19 potential novel viral species within the pegivirus genus were identified, which formed three distinct lineages. clade h viruses clustered with the previously identified gbv-d, whereas the clade g and k viruses formed distinct phylogenetic clusters within the genus pegivirus. interestingly, in some of the animals, coinfections of clade g and k viruses and in one case even coinfection with a hepacivirus (clade c) and a pegivirus (clade k) were observed. the genomic organization of these pegiviruses is similar to that observed for other flaviviruses. amino acid sequence identities were greatest in non-structural proteins ns3 and ns5b, whereas the envelope proteins as well as ns2 and ns5a displayed the highest variability. in summary, these studies show that also pegiviruses are widely distributed among different mammalian species and future studies are necessary to investigate disease association, transmission and tissue tropism. viruses are usually well adapted to their hosts, resulting in multiple barriers to cross-species transmission. 44 nonetheless, the majority of recent emerging infections in human populations represent zoonoses from wild animal species. 45 a recent survey, using fruit bats as a model organism, uncovered 55 novel viruses from seven viral families. 46 extrapolating this number to all mammalian species, the authors estimate a minimum of 3.2310 5 mammalian viruses awaiting discovery. although this number is likely to be an overestimation due to the tendency of bats to act as reservoirs for a broad spectrum of viruses, it is clear that many as yet undiscovered viruses circulate in wild animals, all with the potential to jump the species barrier to humans. cumulatively, these findings would support a scenario where hcv in humans is likely the result of a cross-species transmission, probably from an as yet unidentified source. current patterns of global hcv diversity could be due to a single ancestral zoonotic transmission to archaic humans prior to their global dispersal, with subsequent viral diversification in isolated populations. identification of a more closely related hepaciviral progenitor closer to the root of extant circulating hcv isolates than nphv, the current closest relative, would provide support for this scenario. 47 however, the geographical associations of different hcv genotypes, which appear to have been evolving and diverging in geographically discrete human populations over an extended time scale, could also be the result of multiple, more recent, independent cross-species transmissions to geographically separated human populations. identification of novel hepaciviruses, isolated from animal hosts, which fall within the global hcv radiation and are positioned basally to distinct hcv clades, would ultimately provide strong evidence for this alternative. 47 the .1200 bat species are known to harbour a diverse array of viruses from multiple families, many of which have crossed the species barrier to cause a variety of diseases in humans, often via an intermediate host. 48 for example, the hendra virus, an rna virus from the family paramyxoviridae, has caused multiple outbreaks in horses and four reported human fatalities. 49 interestingly, hendra virus was subsequently demonstrated to originate in fruit bats and was transmitted to humans via close contact with horses. of note, the most closely related animal hepaciviruses to hcv described to date are found in horses (nphv) 12 , and kenyan fruit bats (bhv) 15 (figure 1) . thus, if continued wild animal sampling fails to identify more likely candidates for the ancestor(s) of hcv, one could envisage an historical scenario whereby the progenitor of hcv was transmitted to humans from bats via horses, as all three species are susceptible to hepaciviral infection. however, viruses are dependent on a multitude of host factors to complete their life cycle in susceptible cells. thus, species-specific barriers to viral cross-species transmissions are likely to be proportional to the genetic relatedness of host species: one would expect viruses to jump the species barrier more easily between closely related host species. in addition to hcv infection of humans, gbv-b infection of new world monkeys and the recent discovery of distantly related hepaciviral homologs to hcv in old world monkeys 37 indicates the susceptibility of a broad array of primates to hepaciviral infection. two of the biggest infectious disease burdens afflicting humanity presently are hiv and malaria, zoonoses from chimpanzees and gorillas, respectively, 50 our closest great-ape relatives. thus, it might be also possible that great apes, or other primate species, harbor as yet undescribed hepaciviruses, which may ultimately have given rise to the current hcv pandemic. despite successful development of cell culture systems to study the complete hcv replication cycle, 51 analysis of mechanisms of virus pathogenesis and immune control as well as vaccine development are severely hampered by the lack of robust immunocompetent small animal models. furthermore, the origin of hcv has remained elusive. recently, studies combining powerful next-generation sequencing technologies with coordinated sample collection from multiple geographical localities have uncovered related hepaciviral and pegiviral homologs in diverse animal species. these research efforts have resulted in a rapid expansion of the known viral diversity in hepacivirus and pegivirus genera, and identified an increasing number of animal host species susceptible to hepacivirus and pegivirus infection. differences and similarities between these newly discovered viruses pdb-534 a only viruses listed whose near full-length genomes were generated. new hcv-like viruses in different mammalian hosts pfaender et al 8 and hcv may ultimately advance our understanding of hepacivirus biology with respect to mechanisms of hepaciviral replication, permissiveness of small animal models to productive infection, epidemiology and vaccine development, in addition to further elucidating hcv origins. future studies should address transmission and ecology of these new viruses in their natural hosts to evaluate any potential risk of trans-species transmission to humans. the generation of functional cdna clones will further advance our knowledge on hepaciviral replication strategies and could be used for the development of recombinant hcv vaccines. in conclusion, the recent discoveries of multiple novel hepaciviruses in diverse mammalian species have undoubtedly intensified research efforts to identify the true origins of hcv. ultimately, these studies will expand our knowledge of circulating mammalian viruses, in addition to further illuminating barriers/pathways to viral cross-species adaptation. evolving epidemiology of hepatitis c virus hepatitis c and liver transplantation novel therapies for hepatitis c-one pill fits all? hepatitis c virus therapy update replication of hepatitis c virus assembly of infectious hepatitis c virus particles transmission of hepatitis c by intrahepatic inoculation with transcribed rna completion of the entire hepatitis c virus life cycle in genetically humanized mice hepatitis viruses in non-human primates origin of hiv-1 in the chimpanzee pan troglodytes troglodytes isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome serology-enabled discovery of genetically diverse hepaciviruses in a new host characterization of a canine homolog of hepatitis c virus identification of rodent homologs of hepatitis c virus and pegiviruses bats are a major natural reservoir for hepaciviruses and pegiviruses evidence for novel hepaciviruses in rodents studies on the transmission of human viral hepatitis to marmoset monkeys. i. transmission of disease, serial passages, and description of liver lesions identification of two flavivirus-like genomes in the gb hepatitis agent isolation of novel virus-like sequences associated with human hepatitis molecular cloning and disease association of hepatitis g virus: a transfusion-transmissible agent identification of gbv-d, a novel gb-like flavivirus from old world frugivorous bats (pteropus giganteus) in bangladesh molecular characterization of the hepatitis g virus sequence and genomic organization of gbv-c: a novel member of the flaviviridae associated with human non-a-e hepatitis characterization of hepatitis g virus (gb-c virus) particles: evidence for a nucleocapsid and expression of sequences upstream of the e1 protein the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae expanded classification of hepatitis c virus into 7 genotypes and 67 subtypes: updated criteria and assignment web resource hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy consensus proposals for a unified system of nomenclature of hepatitis c virus genotypes genetic diversity and evolution of hepatitis c virus-15 years on new insights into the hcv quasispecies and compartmentalization epidemiology of hepatitis c: geographic differences and temporal trends the epidemic behavior of the hepatitis c virus the origin and evolution of hepatitis viruses in humans glycosylation of hepatitis c virus envelope proteins role of n-linked glycans in the functions of hepatitis c virus envelope proteins incorporated into infectious virions nonprimate hepaciviruses in domestic horses, united kingdom a novel hepacivirus with an unusually long and intrinsically disordered ns5a protein in a wild old world primate bats: important reservoir hosts of emerging viruses bats and their virome: an important source of emerging viruses capable of infecting humans bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes analysis of hepatitis c virus intrahost diversity across the coding region by ultradeep pyrosequencing identification of a previously undescribed divergent virus from the flaviviridae family in an outbreak of equine serum hepatitis identification of a pegivirus (gb virus-like virus) that infects horses a cross-species view on viruses global trends in emerging infectious diseases a strategy to estimate unknown viral diversity in mammals virology: the virus whose family expanded bats and viruses: friend or foe? emerging infectious diseases. link to mers virus underscores bats' puzzling threat evolution. great apes and zoonoses cell culture systems for hepatitis c virus this work is licensed under a creative commons attribution 3.0 unported license stephanie pfaender was funded by a stipend from the international research training group 1237 (irtg 1237) provided by the deutsche forschungsgemeinschaft. eike steinmann was supported by the deutsche forschungsgemeinschaft (ste 1954/1-1) and an intramural young investigator award of the helmholtz centre for infection research. thomas pietschmann is supported by grants from the deutsche forschungsgemeinschaft (sfb 900, project a6) and (pi 734/2-1) by grants from the helmholtz association (so-024, hai-idr) and by a grant from the european research council (erc-2011-stg_281473-virafront). we thank gisa gerold (centre of experimental and clinical infection research, hannover, germany) for critically reading the manuscript. key: cord-321505-m40s6uw9 authors: sakamoto, naoya; tanabe, yoko; yokota, takanori; satoh, kenichi; sekine‐osajima, yuko; nakagawa, mina; itsui, yasuhiro; tasaka, megumi; sakurai, yuki; cheng‐hsin, chen; yano, masahiko; ohkoshi, shogo; aoyagi, yutaka; maekawa, shinya; enomoto, nobuyuki; kohara, michinori; watanabe, mamoru title: inhibition of hepatitis c virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin rna date: 2007-08-07 journal: j gastroenterol hepatol doi: 10.1111/j.1440-1746.2007.05076.x sha: doc_id: 321505 cord_uid: m40s6uw9 background and aim: we have reported previously that synthetic small interfering rna (sirna) and dna‐based sirna expression vectors efficiently and specifically suppress hepatitis c virus (hcv) replication in vitro. in this study, we investigated the effects of the sirna targeting hcv‐rna in vivo. methods: we constructed recombinant retrovirus and adenovirus expressing short hairpin rna (shrna), and transfected into replicon‐expressing cells in vitro and transgenic mice in vivo. results: retroviral transduction of huh7 cells to express shrna and subsequent transfection of an hcv replicon into the cells showed that the cells had acquired resistance to hcv replication. infection of cells expressing the hcv replicon with an adenovirus expressing shrna resulted in efficient vector delivery and expression of shrna, leading to suppression of the replicon in the cells by ∼10(−3). intravenous delivery of the adenovirus expressing shrna into transgenic mice that can be induced to express hcv structural proteins by the cre/loxp switching system resulted in specific suppression of virus protein synthesis in the liver. conclusion: taken together, our results support the feasibility of utilizing gene targeting therapy based on sirna and/or shrna expression to counteract hcv replication, which might prove valuable in the treatment of hepatitis c. hepatitis c virus (hcv), which affects 170 million people worldwide, is one of the most important pathogens causing liver-related morbidity and mortality. 1 the difficulty in eradicating hcv is attributable to limited treatment options against the virus and their unsatisfactory efficacies. even with the most effective regimen with pegylated interferon (ifn) and ribavirin in combination, the efficacies are limited to less than half of the patients treated. 2 given this situation, the development of safe and effective anti-hcv therapies is one of our high-priority goals. rna interference (rnai) is a process of sequence-specific, post-transcriptional gene silencing that is initiated by doublestranded rna. 3, 4 because of its potency and specificity, rnai rapidly has become a powerful tool for basic research to analyze gene functions and for potential therapeutic applications. recently, successful suppression of various human pathogens by rnai have been reported, including human immunodeficiency viruses, 5, 6 poliovirus, 7 influenza virus, 8 severe acute respiratory syndrome (sars) virus 9 and hepatitis b virus (hbv). [10] [11] [12] [13] we and other researchers have reported that appropriately designed small interfering rna (sirna) targeting hcv genomic rna can efficiently and specifically suppress hcv replication in vitro. [14] [15] [16] [17] [18] [19] we have tested sirna designed to target the wellconserved 5′-untranslated region (5′-utr) of hcv-rna, and identified the most effective target, just upstream of the translation initiation codon. furthermore, transfection of dna-based vectors expressing sirna was as effective as that of synthetic sirna in suppressing hcv replication. 14 in this study, we explored the further possibility that efficient delivery and expression of sirna may be effective in suppression and elimination of hcv replication and that delivery of such hcv-directed sirna in vivo may be effective in silencing viral protein expression in the liver. here, we report that hcv replication was suppressed in vitro by recombinant retrovirus and adenovirus vectors expressing short hairpin rna (shrna) and that the delivery of the adenovirus vector to mice in vivo specifically inhibited viral protein synthesis in the liver. huh7 and retro pack pt67 cells (clontech, palo alto, ca, usa) were maintained in dulbecco's modified minimal essential medium (sigma, st. louis, mo, usa) supplemented with 10% fetal calf serum at 37°c under 5% co2. to maintain cell lines carrying the hcv replicon, g418 (wako, osaka, japan) was added to the culture medium to a final concentration of 500 mg/ml. hcv replicon plasmids, prep-feo, prep-fluc and prep-bsd were constructed from were constructed from a virus, hcv-n strain, genotype 1b. 21 the prep-feo expressed a chimeric reporter protein of firefly luciferase (fluc) and neomycin phosphotransferase. 14, 20 the prep-fluc and the prep-bsd expressed the fluc and blasticidin s (bsd) resistance genes, respectively (fig. 1) . the replicon rna synthesis and the transfection protocol have been described previously. 22 the design and construction of hcv-directed sirna vectors have been described. 14 briefly, five sirna targeting the 5′-utr of hcv rna were tested for their efficiency to inhibit hcv replication, and the most effective sequence, which targeted nucleotide position of 331 though 351, was used in the present study. to construct shrna-expressing dna cassettes, oligonucleotide inserts were synthesized that contained the loop sequence (5′-ttc aag aga-3′) flanked by sense and antisense sirna sequences (fig. 2a) . these were inserted immediately downstream of the human u6 promoter. to avoid a problem in transcribing shrna because of instability of the dna strands arising from the tight palindrome structure, several c-to-t point mutations, which retained completely the silencing activity of the shrna, were introduced into the sense strand of the shrna sequences (referred to as 'm'). 23 a control plasmid, puc19-shrna-control, expressed shrna directed towards the machado-joseph disease gene, which is a mutant of ataxin-3 gene and is not normally expressed. we have previously described the sequence specific activity of the shrna-control. 24 prior to construction of the virus vectors, we tested silencing efficiency of five shrna constructs of different lengths that covered the target sequence (fig. 2a) . the shrna-hcv-19, shrna-hcv-21 and shrna-hcv-27 had target sequences of 19, 21 and 27 nucleotides, respectively. transfection of these shrna constructs into huh7/prep-feo showed that shrna with longer target sequences had better suppressive effects (fig. 2b ). therefore, we used shrna-hcv-27m (abbreviated as shrna-hcv) in the following study. the u6-shrna expression cassettes were inserted into the stui/ hindiii site of a retrovirus vector, plncx2 (clontech) to construct plncshrna-hcv and plncshrna-control (fig. 2c) . the plasmids were transfected into the packaging cells, retro pack pt67. the culture supernatant was filtered and added onto huh7 cells with 4 mg/ml of polybrene. huh7 cell lines stably expressing shrna were established by culture in the presence of 500 mg/ml of g418. recombinant adenoviruses expressing shrna were constructed using an adenovirus expression vector kit (takara, otsu, japan). the u6-shrna expression dna cassette was inserted into the swai site of paxcw to construct paxshrna-hcv and paxshrna-control. the adenoviruses were propagated according to the manufacturer's protocol (axshrna-hcv and axshrna-control; fig. 2c ). a 'multiplicity of infection' (moi) was used to standardize infecting doses of adenovirus. the moi stands for the ratio of infectious virus particles to the number of cells being infected. an moi = 1 represents equivalent dose to introduce one infectious virus particle to every host cell that is present in the culture. pisre-ta-luc (invitrogen, carlsbad, ca, usa) contained five copies of the consensus interferon stimulated response element (isre) motifs upstream of the fluc gene. pta-luc (invitrogen), which lacks the enhancer element, was used for background determination. the pcdna3.1 (invitrogen) was used as an empty vector for mock transfection. prl-cmv (promega, madison, wi, usa), which expresses the renilla luciferase protein, was used for normalization of transfection efficiency. 25 a plasmid, pegfpneo (invitrogen), was used to monitor percentages of transduced cells. total cellular rna was extracted from cultured cells or liver tissue using isogen (nippon gene, tokyo, japan). total cellular rna (2 mg) was used to generate cdna from each sample using the superscript ii reverse-transcriptase (invitrogen). the mrna expression levels were measured using the light cycler pcr and detection system (roche, mannheim, germany) and light cycler fast start dna master sybr green 1 mix (roche). luciferase activity was measured using a luminometer, lumat lb9501 (promega) and the bright-glo luciferase assay system (promega) or the dual-luciferase reporter assay system (promega). total cellular rna was separated by denaturing agaroseformaldehyde gel electrophoresis, and transferred to a nylon membrane. the membrane was hybridized with a digoxigenin-labeled probe specific for the full-length replicon sequence, and subsequently with a probe specific for beta-actin. the signals were detected by chemiluminescence reaction using a digoxigenin luminescent detection kit (roche), and visualized by fluoro-imager (roche). for the western blotting, 10 mg of total cell lysate was separated on nupage 4.12% bis-trisgel (invitrogen), and blotted onto an immobilon pvdf membrane (roche). the membrane was incubated with monoclonal antibodies specific for hcv-ns5a (biodesign, saco, me, usa), ns4a (virogen, watertown, ma, usa), or beta-actin (sigma), and detected by a chemiluminescence reaction (bm chemiluminescence blotting substrate; pod, roche). a replicon, prep-fluc, was transfected into cells and the luciferase activities of the cell lysates were measured serially. to correct the transfection efficiency, each value was divided by the luciferase activity at 4 h after the transfection. cells were transfected with a replicon, prep-bsd, and were cultured in the presence of 150 mg/ml of bsd (invitrogen). bsdresistant cell colonies appeared after~3 weeks of culture, and were counted. transgenic mice, cn2-29, inducibly express mrna for the hcv structural proteins (genotype1b, nucleotides 294-3435) by the cre/loxp switching system. 28 the transgene does not contain fulllength hcv 5′-utr, but shares the target sequence of the shrna-hcv. although the transgenic mouse cn2 has been previously reported as expressing higher levels of the viral proteins, the expression levels of the viral core protein in the cn2-29 mice are modest and similar to that in the liver of hcv patients. thus, we chose cn2-29 mice in the present study. the mice were infected with axshrna-hcv or controls (axshrna-control or axcaw1) in combination with axcan-cre, which expressed cre recombinase. three days after the infection, the mice were killed and hcv core protein in the liver was measured as described below. the balb/c mice were maintained in the animal care facility of tokyo medial and dental university, and transgenic mice were in the tokyo metropolitan institute of medical science. animal care was in accordance with institutional guidelines. the review board of the university approved our experimental animal studies and all experiments were approved by the institutional animal study committees. the amounts of hcv core protein in the liver tissue from the mice was measured by a fluorescence enzyme immunoassay (feia) 29 with a slight modification. briefly, the 5f11 monoclonal anti-hcv-core antibody was used as the first antibody on the solid phase, and the 5e3 antibody conjugated with horseradish peroxidase was the second antibody. this feia can detect as little as 4 pg/ml of recombinant hcv-core protein. contents of the hcv core protein in the liver samples were normalized by the total protein contents and expressed as pg/mg total protein. liver tissue was frozen with optimal cutting temperature (otc) compound (tissue tek; sakura finetechnical, tokyo, japan). the sections (8 mm thick) were fixed with a 1:1 solution of acetone : methanol at -20°c for 10 min and then washed with phosphate-buffered saline (pbs). subsequently, the sections were incubated with the igg fraction of an anti-hcv core rabbit polyclonal antibody (rr8) 28 in blocking buffer or antialbumin rabbit polyclonal antibody (dako cytomation, glostrup, denmark) in pbs overnight at 4°c. the sections were incubated with secondary antibody, alexa-antirabbit igg (invitrogen) or tritic-antirabbit igg (sigma), for 2 h at room temperature. fluorescence was observed using a fluorescence microscope. statistical analyses were performed using student's t-test; p-values of less than 0.05 were considered to be statistically significant. retrovirus vectors propagated from plncshrna-hcv and plncshrna-control were used to infect huh7 cells, and cell lines were established that constitutively express shrna-hcv and shrna-control (huh7/shrna-hcv and huh7/shrna-control, respectively). there were no differences in the cell morphology or growth rate between shrna-transduced and nontransduced huh7 cells (data not shown). the hcv replicon, prep-fluc, was transfected into huh7/shrna-hcv, huh7/ shrna-control and naive huh7 cells by electroporation. in huh7/ shrna-control and naive huh7 cells, the initial luciferase activity at 4 h decreased temporarily, which represents decay of the transfected replicon rna, but increased again at 48 h and 72 h, which demonstrate de novo synthesis of the hcv replicon rna. in contrast, transfection into huh7/shrna-hcv cells resulted in a decrease in the initial luciferase activity, reaching background by 72 h (fig. 3a) . similarly, transfection of the replicon, prep-bsd, into huh7 cells and bsd selection yielded numerous bsdresistant colonies in the naive huh7 (832 colonies) and huh7/ shrna-control cell lines (740 colonies), while transfection of huh7/shrna-hcv, which expressed shrna-hcv, yielded obviously fewer colonies (five colonies), indicating reduction of colony forming units by~10 2 (fig. 3b) . there was no difference in shape, growth or viability between cells expressing the shrna or not. these results indicated that cells expressing hcv-directed shrna following retrovirus transduction acquired resistance to hcv replication. we investigated subsequently the effects of recombinant adenovirus vectors expressing shrna. axshrna-hcv and axshrna-control were used separately to infect huh7/prep-feo cells, and the internal luciferase activities were measured sequentially (fig. 4a) . axshrna-hcv caused continuous suppression of hcv rna replication. six days postinfection, the luciferase activities fell to background levels. in contrast, the luciferase activities of the huh7/prep-feo cells infected with axshrna-control did not show any significant changes compared with untreated huh7/ prep-feo cells (fig. 4a) . the dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (mts) assay showed no significant difference between cells that were infected by recombinant adenovirus and uninfected cells (fig. 4b) . in the northern blotting analysis, the cells were harvested 6 days after infection with the adenovirus at an moi of 1. feo-replicon rna of 9.6 kb, which was detectable in the untreated huh7/prep-feo cells and in the cells infected with axshrna-control, diminished substantially following infection with the axshrna-hcv (fig. 4c) . densitometries showed that the intracellular levels of the replicon rna in the huh7/prep-feo cells correlated well with the internal luciferase activities. similarly in the western blotting, cells were harvested 6 days after infection with adenovirus. levels of the hcv ns4a and ns5a proteins that were translated from the hcv replicon decreased following infection with the axshrna-hcv (fig. 4d) . these results indicated that the decrease in luciferase activities was due to specific suppressive effects of shrna on expression of hcv genomic rna and the viral proteins, and not due to non-specific effects caused by the delivery of shrna or to toxicity of the adenovirus vectors. it has been reported that double-stranded rna may induce interferon-stimulated gene (isg) responses which cause instability of mrna, translational suppression of proteins and apoptotic cell negative-or positive-control shrna plasmids was transfected. (fig. 5a) . similarly, the expression levels of an interferoninducible mxa protein did not significantly change by transfection of shrna-expression vectors (fig. 5b) . these results demonstrate that the shrna used in the present study lack induction of the isg responses both in the form of the expression plasmids and the adenovirus vectors. the effects of hcv-targeted sirna-and shrna-expressing adenoviruses were confirmed by using hcv-jfh1 virus cell culture system. transfection of the sirna #331 14 into hcvinfected huh7.5.1 cells resulted in substantial decrease of intracellular hcv rna, while a control sirna showed no effect (fig. 6a) . similarly, infection of axshrna-hcv into huh7.5.1/ hcv-jfh1 cells specifically suppressed expression of hcv rna (fig. 6b) . relative luciferase activity the effects of the shrna expression on the expression of the viral structural proteins in vivo were investigated using conditional hcv cdna-transgenic mice, cn2-29. 28 adenoviruses, axshrna-hcv, axshrna-control or axcaw1 were injected into cn2-29 mice in combination with axcancre, an adenovirus expressing cre dna recombinase. the mice were killed on the fourth day after the injection, and the hepatic expression of the hcv core protein was measured. the expressed amounts of the core protein were 143.0 ϯ 56.2 pg/mg and 108.5 ϯ 42.4 pg/mg in axcaw1 and axshrna-control-infected mice, respectively, and the expressed amount was significantly lower in mice injected with axshrna-hcv (28.7 ϯ 7.0 pg/mg, p < 0.05, fig. 7a) . similarly, the induced expression of hcv core protein was not detectable by immunohistochemistry in axshrna-hcv infected liver tissue (fig. 7c) . staining of a host cellular protein, albumin, was not obviously different between the liver infected with axcaw1, axshrna-hcv and axshrna-control (fig. 7d) . the expression levels of two isg, ifn-beta and mx1, in the liver tissue were not significantly different between individuals with and without injection of the adenovirus vectors (fig. 7b) . these results indicate specific shrna silencing of hcv structural protein expression in the liver. the requirements to achieve a high efficiency using rnai are: (i) selection of target sequences that are the most susceptible to rnai; (ii) persistence of sirna activity; and (iii) efficient in vivo delivery of sirna to cells. we have used an shrna sequence that was derived from a highly efficient sirna (sirna331), and constructed a dna-based shrna expression cassette that showed competitive effects with the synthetic sirna (fig. 2 ). 14 the shrna-expression cassette does not only allow extended half-life of the rnai, but also enables use of gene-delivery vectors, such as virus vectors. as shown in the results, a retrovirus vector expressing shrna-hcv could stably transduce cells to express hcvdirected shrna, and the cells acquired protection against hcv subgenomic replication (fig. 3 ). an adenovirus vector expressing shrna-hcv resulted in suppression of hcv subgenomic and protein expression by around three logs to almost background levels (fig. 4) . consistent results were obtained by using an hcv cell culture (fig. 6 ). more importantly, we have demonstrated in-vivo effects on viral protein expression in the liver using a conditional transgenic mouse model (fig. 7) . these results suggest that efficient delivery of sirna could be effective against hcv infection in vivo. an obstacle to applying sirna technology to treat virus infections is that viruses are prone to mutate during their replication. 32 hcv continuously produces mutated viral strains to escape immune defense mechanisms. even in a single patient, the circulating hcv population comprises a large number of closely related hcv sequence variants called quasispecies. therefore, sirna targeting the protein-coding sequence of the hcv genome, which have been reported by others, [15] [16] [17] [18] [19] may vary considerably among different hcv genotypes, and even among strains of the same genotype. 33 our shrna sequence targeted the 5′-utr of hcv rna, which is the most conserved region among various hcv isolates. 33 in addition, the structural constraints on the 5′-utr, in terms of its requirement to direct internal ribosome entry and translation of viral proteins, might not permit the evolution of escape mutations. our preliminary results have shown that the sirna-hcv suppressed replication of an hcv genotype 2a replicon 34 to the same extent as the hcv 1b replicon. although the sirna techniques rely on a high degree of specificity, several studies report sirna-induced non-specific effect that may result from induction of isg responses. 18, 31 these effects may be mediated by activation of double-strand rna-dependent protein kinase, toll-like receptor 3, 35 or possibly by a recently identified rna helicase, rig-i. 36 it remains to be determined whether these effects are generally induced by every sirna construct. sledz et al. have reported that transfection of two sirna induced cellular interferon responses, 37 while bridge et al. report that shrna-expressing plasmids induced an interferon response but transfection of synthetic sirna did not. 31 speculatively, these effects on the interferon system might be construct dependent. our shrna-expression plasmids and adenoviruses did not activate isg responses in vitro (fig. 5a,b) or in vivo (fig. 7b) . we have preliminarily detected phosphorylated pkr (p-pkr) by western blotting, and found no apparent increase of p-pkr (data not shown). these results indicate that these target sequences and structures are of sufficient specificity to silence the target gene without eliciting non-specific interferon responses. beside the canonical action of sirna, a sequence-specific cleavage of target mrna, the sirna could act as a micro-rna that suppresses translational initiation of mrna, 38 or it could mediate transcriptional gene silencing. 39 regarding our in-vivo experiments, it was difficult to differentially analyze the effect of sirna at individual sites of action because post-translational effect of sirna concomitantly destabilizes target mrna, which leads to apparent decrease of mrna transcripts. (c) immunofluorescence microscopy of hcv core protein in the liver tissue. liver sections of mice were stained using rabbit anticore polyclonal antibody and normal rabbit igg as a negative control. the upper photographs were obtained at 400¥ magnification, and the lower photographs were at 1000¥. (d) immunofluorescence microscopy of albumin in liver. liver sections from the mice were fixed and stained using rabbit antialbumin antibody and normal rabbit igg as a negative control. efficiency and safety of gene transfer methods are the key determinants of the clinical success of gene therapy and an unresolved problem. there are several reports of delivery of sirna or sirna-expression vectors to cells in vivo; 12, 40, 41 however, gene delivery methods that are safe enough to apply to clinical therapeutics are currently under development. adenovirus vectors are one of the most commonly used carriers for human gene therapies. [42] [43] [44] our present results demonstrate that the adenoviral delivery of shrna is effective in blocking hcv replication in vitro and virus protein expression in vivo. adenovirus vectors have several advantages of efficient delivery of transgene both in vitro and in vivo and natural hepatotropism when administered in vivo. the axshrna-hcv specifically blocked expression of hcv structural proteins in a conditional transgenic mouse expressing those proteins. the current adenovirus vectors may cause inflammatory reactions in the target organ, 45 however, and produce neutralizing antibodies which make repeated administration difficult. these problems may be overcome by the improved constructs of virus vectors with attenuated immunogenicity or by the development of non-viral carriers for gene delivery. 46 in conclusion, our results demonstrate the effectiveness and feasibility of the sirna expression system. the efficiency of adenovirus expressing shrna that target hcv suggests that delivery and expression of sirna in hepatocytes may eliminate the virus and that this rna-targeting approach might provide a potentially effective future therapeutic option for hcv infection. epidemiology of hepatitis c peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis c: a randomized study of treatment duration and ribavirin dose potent and specific genetic interference by double-stranded rna in caenorhabditis elegans duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells potent and specific inhibition of human immunodeficiency virus type 1 replication by rna interference modulation of hiv-1 replication by rna interference short interfering rna confers intracellular antiviral immunity in human cells inhibition of influenza virus production in virus-infected mice by rna interference alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication inhibition of hepatitis b virus replication in vivo by nucleoside analogues and sirna inhibition of hbv replication by sirna in a stable hbv-producing cell line rna interference in adult mice inhibition of hepatitis b virus expression and replication by rna interference inhibition of intracellular hepatitis c virus replication by synthetic and vector-derived small interfering rnas interference of hepatitis c virus rna replication by short interfering rnas alternative approaches for efficient inhibition of hepatitis c virus rna replication by small interfering rnas clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas letter to the editor: small interfering rna-mediated inhibition of hepatitis c virus replication in the human hepatoma cell line huh-7 rna interference blocks gene expression and rna synthesis from hepatitis c replicons propagated in human liver cells effect of alpha interferon on the hepatitis c virus replicon synergistic inhibition of intracellular hepatitis c virus replication by combination of ribavirin and interferon-alpha introduction of ns5a mutations enables subgenomic hcv-replicon derived from chimpanzee-infectious hc-j4 isolate to replicate efficiently in huh-7 cells optimization of an sirna-expression system with an improved hairpin and its significant suppressive effects in mammalian cells sequence-dependent and independent inhibition specific for mutant ataxin-3 by small interfering rna regulation of hepatitis c virus replication by interferon regulatory factor-1 production of infectious hepatitis c virus in tissue culture from a cloned viral genome robust hepatitis c virus infection in vitro efficient conditional transgene expression in hepatitis c virus cdna transgenic mice mediated by the cre/loxp system detection of hepatitis c virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (feia) mechanisms of antiviral action of interferon induction of an interferon response by rnai vectors in mammalian cells silencing viruses with rna nucleotide sequence of the genomic rna of hepatitis c virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions efficient replication of the genotype 2a hepatitis c virus subgenomic replicon recognition of double-stranded rna and activation of nf-kb by toll-like receptor 3 the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses activation of the interferon system by short-interfering rnas sirnas can function as mirnas sirna-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code sirna-mediated gene silencing in vitro and in vivo caspase 8 small interfering rna prevents acute liver failure in mice transfer of a foreign gene into the brain using adenovirus vectors direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors a model system for in vivo gene transfer into the central nervous system using an adenoviral vector clearance of adenovirus-infected hepatocytes by mhc class i-restricted cd4+ ctls in vivo helper-dependent adenovirus vectors devoid of all viral genes cause less myocardial inflammation compared with first-generation adenovirus vectors this study was supported by grants from japan society for the promotion of science, 15590629 and 16590580, and partly supported by a grant from the viral hepatitis research foundation of japan. key: cord-319754-5isw53wl authors: balgoma, david; gil-de-gómez, luis; montero, olimpio title: lipidomics issues on human positive ssrna virus infection: an update date: 2020-08-31 journal: metabolites doi: 10.3390/metabo10090356 sha: doc_id: 319754 cord_uid: 5isw53wl the pathogenic mechanisms underlying the biology and biochemistry of viral infections are known to depend on the lipid metabolism of infected cells. from a lipidomics viewpoint, there are a variety of mechanisms involving virus infection that encompass virus entry, the disturbance of host cell lipid metabolism, and the role played by diverse lipids in regard to the infection effectiveness. all these aspects have currently been tackled separately as independent issues and focused on the function of proteins. here, we review the role of cholesterol and other lipids in ssrna+ infection. the ongoing covid-19 pandemic is developing (july 2020) worldwide with devastating global consequences, both for social organization and healthcare systems. covid-19 illness is brought about by infection with the severe acute respiratory syndrome coronavirus sars-cov-2 [1, 2] , which is an enveloped positive single-stranded rna virus (ssrna+) [3] . the most abundant studies related to human diseases induced by ssrna-positive viruses refer to picornaviridae, coronaviridae, and flaviviridae [4] . this impact in a short time span has brought the biology and biochemistry of viral infection mechanisms to reach momentum. the infection mechanisms have been described for diverse unrelated viral families [5] , with the majority of them being dna viruses. within picornaviridae, coronaviridae, and flaviviridae, rhino and poliovirus (picornaviridae), sars-cov, middle east respiratory syndrome coronavirus (mers-cov), hepatitis c virus (hcv), west nile virus (wnv) and dengue virus (denv) fall within the viruses whose life cycle biology is better known. nonetheless, knowledge regarding virus entry mechanisms and other related features of the virus life cycle has been gained from the research on the influenza virus from the orthomysoviridae family and the human immunodeficiency virus from the retroviridae family. consequently, these and other unrelated viruses will be also considered in this review from the point of view of the different aspects that affect the lipidomics of the viral infection. all ssrna+ viruses initially infect mammal cells through the interaction of virus proteins with any given host cell protein. further fusion of the virus and host cell membranes is required for the viral genetic material to get into the cell. once inside the cell, the genomic and subgenomic viral rnas are translated into the virus proteins; these then lead the virus replication, which is a process that involves modulation of the host cell lipid metabolism [3, 5, 6] . consequently, along with other features, current lipid studies about the aforementioned virus infection focus their research on membrane fusion and modulation of the lipid metabolism of the host cell. these two processes are considered separated disciplines of the infection. the fight against the virus infection encompasses primarily the inhibition of the binding of the viral spike protein to the host cell's receptor protein. consequently, most of the current research focuses on the role played by viral proteins but the lipid environment, where the proteins carry out their function and regulation, is considered secondarily [7] . nevertheless, improving the knowledge on how the lipids are involved in the mechanisms of infection may provide clues to develop treatments and better counteract the virus-induced pathology [3] . to fill this gap, here, we review the main aspects regarding the lipidome regulation of the viral infection mechanism by ssrna+ viruses. the initial step in virus infection is the binding of any viral structural glycoprotein to a receptor of the host cell. the spike protein accounts for such function in coronaviruses (covs) and other enveloped viruses. after the virus is attached to the host cell protein, the process of membrane fusion starts to get the viral genome into the host cell. this process implies viral envelope and host cell membrane fusion, for which an energetically cost-effective barrier must be overcome. for example, in coronavirus, membrane fusion is driven by the fusion peptide (class i), which is localized within the spike protein (s protein) and becomes active after cleavage of the s protein at specific sites by host proteases or ph-dependent mechanisms [4, 6, 8] . a different mechanism of attachment and endocytosis drives the virus entry in the case of hcv. this mechanism is more complex than that of coronaviruses and involves interaction of the virus envelope e1 and e2 proteins (class ii fusion loop) with several host cell proteins [9] [10] [11] . however, a membrane fusion-driven pore is also required in hcv to deliver the viral genetic material into the host cell cytoplasm. two main mechanisms of membrane fusion have been described: viral endocytosis by host cell membrane (endocytic pathway), and both viral and host cell plasma membrane fusion (non-endocytic pathway). after docking of the virus to the attachment factor or the receptor on the host cell surface, the virus may internalize its genomic material or the entire particle [12] [13] [14] . the non-endocytic pathway encompasses the direct delivery of the genetic material through a pore formed in the cell membrane by the induction of viral proteins at neutral ph. this pathway is typical of non-enveloped viruses. the endocytic pathway is more complex and harnesses the host cell endocytosis machinery for the virus internalization. three main ways have been described in the endocytic pathway, namely: the clathrin-mediated endocytosis (cme), the caveolae-mediated endocytosis (cavme), and the macropycnocytosis. the best-known endocytic mechanism is the clathrin-mediated endocytosis. the cme is used by small to intermediate-sized viruses. this mechanism uses vesicles coated by the protein clathrin, which forms a polyhedral lattice that surrounds the cell membrane-derived vesicle where the virus is internalized into the cell cytoplasm through the early endosomes. clathrin coating is coordinated by the adaptor protein (ap-2) and other adaptors; it is less commonly ap-independent. the protein dynamin is involved in regulating the clathrin-coated vesicle (ccv) formation as well as its scission from the membrane. some viruses proceed to membrane fusion at this stage for releasing their genome into the cytoplasm. the early endosomes have a ph of about 6.0 to 6.5; therefore, it is considered that membrane fusion is not strictly ph-dependent. other viruses need a lower ph for the membrane fusion to be effective; thus, it is considered ph-dependent. a further step leading to endosome maturation to become late endosomes with a ph of about 5 has to proceed before the membrane fusion takes place and the genetic material is delivered to the cytoplasm. sequential acidification of the virus proteins from the early to late endosomes has also been suggested through the self-organized endosomal network. maturation of the early endosomes to late endosomes and trafficking between them is controled by the rab proteins, which are members of the ras superfamily of small g proteins. subsets of rab proteins differ between the early and late endosomes, and the rab subset change is accompanied for by formation of the phosphoinositide pi(3,5)p 2 from the precursor pi(3)p. regarding lipid composition, early endosome membrane lipids are primarily composed of unsaturated and short alkyl chains, whereas long and saturated alkyl chains, such as in gangliosides, are predominant in the membrane lipids of late endosmes. membrane fusion in some viruses requires a further step in which late endosomes are fused with lysosomes, this step giving rise to the late endosome/lysosome pathway. cholesterol depletion driven by its synthesis inhibition or extracting agents as methyl-β-cyclodextran (mβcd) is used to assess whether the virus entry takes place through the caveolae/raft endocytosis. this pathway in less known and encompasses the formation of initial endocytic vesicles enriched in cholesterol from lipid-rafts, with complex signaling routes that involve the activity of tyrosine kinases and phosphatases. thereafter, the cargo is transported to the endoplasmic reticulum (er) through early and late endosomes. most of the viruses using this endocytic pathway have different gangliosides as receptors, mainly gm1, which has a high concentration in caveolae. polyomavirus, which are non-enveloped dna viruses that replicate in the nucleus, use preferently this endocytic pathway, but picornaviruses and the coronavirus hcov-229e have also been reported to internalize through the caveolae-mediated endocytosis [15] . macropinocytosis is a phagocytic-like mechanism of virus entry that is currently utilized by the cell to internalized fluids; it is dependent on actin and implies the actin cytoskeleton rearrangement to enable internalization of the virus particle [14] . macropinocytic vacuoles (macropinosomes) are formed after membrane ruffles fold to reach at its end the membrane again, and the vacuole is closed through self-membrane fusion. these vacuoles containing the viral particle may traffick afterwards through the early and late endosome network. macropinocytosis is common to large-sized viruses. however, recent work [16] has shown that ebola virus (ebov) may use a macropinocytosis-like process to entry the host cell in a clathrin, caveolae, and dynamin-independent manner, but dependent of actin and a lipid raft. conversely, this virus may use as well an endocytic pathway that is dependent on clathrin, caveolae, and dynamin. which endocytic route is used by this virus depends on the host cell type. description of the current methodologies used to study the entry route by viruses can be found in reference [14] . some viruses may use different entry mechanisms, this feature being likely dependent upon the membrane lipid composition of the host cell they infect as well as the particular cell surface factor attachment used. cme is the entry route currently used by hcv, hiv-1, ebov, rotaviruses, and some coronaviruses, even though other routes can also be used as for ebov (see above). a reaction between clathrin and actin seems to be necessary for the effective entry of these viruses. regulation by microtubules of the cme has been reported for flaviviruses. denv, wnv, and semliki forest virus (sfv, alphavirus family, togaviridae) have been found to depend on early endosomes (rab5 protein marker) for entry but not late endosomes (rab7 protein marker), which means that they do not have strict low ph requirements or depend on different acidification mechanisms for membrane fusion. conversely, influenza avian virus (iav) needs both early and late endosomes to entry, thus reflecting low ph dependence for membrane fusion. marburg virus (marv) may use for internalization a cme through the endo/lysosomal pathway. coronaviruses differ in their internalization mechanism among strains. thus, while hcov-229e is known to use the cav-me route, sars-covs use an endocytic pathway that is clathrin-and caveolae-independent but receptor and ph-sensitive, with lipid rafts playing an essential role [17] . this endocytic mechanism implies internalization of the receptor protein angiotensin-convering enzyme 2 (ace2) along with the spike protein into the early endosomes, but the receptor is afterward recycled to the membrane via lysosomes. nonetheless, previous studies showed that sars-cov could enter through a ph-independent direct membrane fusion as it could infect cells that do not express ace2, such as enterocytes and hepatocytes [18] . recent research on the virus sars-cov-2 points to ph-independent direct cell and viral membrane fusion, which is a process that is driven by the subunit s2 of the spike protein after cleavage by the cellular serine protease tmprss2 [19] . on the contrary, the infectious bronchitis virus (ibv), a gamma-coronavirus, was reported to use the cme pathway to entry, with vesicle scission being mediated by gtpase dynamin 1, and a dependence on low ph and lipid raft localization of the receptor. tracking of the virus trip inside the cell was followed by using diverse inhibitors, cholesterol sequestering agents, and virus particles labeled with fluorescent markers. membrane fusion takes place at the late endosome/lysosome step of the endocytic pathway, with deep rearrangement of the host cell cytoskeleton being induced by the endosomal viral cargo [15] . accordingly, viruses may sequester on their own profit the diverse endocytic pathways that are currently used by the host cell, but variability of the proteins and even the general mechanisms may also exist as a consequence of virus specifity. membrane fusion has been described to proceed through the catalytic action of three different types of fusion peptides or fusion loops of class i, ii, or iii. these proteins afford the free energy necessary to overcome through conformational changes the kinetic barrier due to repulsive hydration strength. most of the knowledge on the viral and host membrane fusion has been gained from the influenza virus and its type i fusion peptide hemagglutinin. a detailed description of the three fusion peptide-guided mechanisms involved in membrane fusion has been previously reviewed in [20] [21] [22] . bringing the viral and the host membranes closer enough (c.a. 20 å) for inducing the membrane fusion is a process that entails membrane curvature and changes in the lipid bilayer phase. they are driven by the insertion of a hydrophobic region of the fusion peptide, which requires dehydration of the inter-membrane space. nonetheless, from experiments with no-protein fusogens, such as polyethilen glycol, it seems that membrane curvature stabilization is not a key player in membrane pore opening. the calculated displacement of lipids in the outer leaflet of the host membrane accounts for no more than 10% of the membrane area (about 3500 å 2 ), which does not represent a substantial energetic demand [21] . this energetic burden has been demonstrated to be afforded by the cooperation of three fusion peptides in influenza virus membrane fusion [23] , whereas two adjacent trimers of the fusion protein are required in west nile virus [24] . this result points to the fact that the viral membrane curvature may not actually impose a constraint for proceeding to the hemifusion step and the formation of a steep curvature stalk, where the outer leafleats are merged. by the mesurement of electron density profiles through x-ray reflectivity in stalks formed from bilayers in a lamellar state with different lipid compositions, aeffner et al. [25] determined that the inter-bilayer separation should attain 9.0 ± 0.5 å in order to facilitate dehydration and promote stalk formation. these authors also found that increasing the relative proportion of nonbilayer-forming, cone-shaped lipids, such as glycerophosphoethanolamine or cholesterol, favored the stalk formation by reducing the hydration energy barrier and, possibly, by contributing with their intrinsic negative curvature. as well, the energy required for dehydration was, in this study, found to decrease with the length of the acyl chains of the glycerophospholipids. however, the hemifusion stalk stage was not detected by gui et al. [26] using fluorescence and electron microscopy. the results of this study show that such a stage might be an unstable intermediate that is quickly resolved toward the postfusion stage. contrarily, localized point-like contacts were abundantly visualized in this study, where the dimples formed in the target membrane, about 5 nm wide, were drawn toward the virus surface. they were able to detect up to well-resolved four types of virus-target membrane contacts at ph 5.5 and 5.25 using liposomes of dioleylglycerophosphocholine, dopc, with 20% cholesterol. at the lowest ph, a tight contact of the two membranes through an extended length of about 100 nm (catalogued by the authors as type iii) was the predominant interaction, whose abundance was increased by about 3-fold in cholesterol-containing liposomes in comparison to only dopc liposomes. using synthetic peptides that resemble the fusion peptide hemagglutinin and electron spin resonance (esr), ge and freed [27] found that the most relevant effect of the synthetic fusion peptides was the induction of highly ordered membrane domains, which came motivated by virtue of electrostatic interactions between the peptide and negatively charged phospholipid headgroups. a similar effect was reported for two putative fusion peptides enclosed in the spike glycoprotein of sars-cov-1. it was found in this study that the inner water content in the lipid bilayer was dropped by the insertion of the fusion peptide as a consequence of increased lipid packing, but only in membranes containing negatively charged lipids, whereas the water content was only slightly altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [28] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [21, 29] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [7] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [26, 28, 30] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [22, 28] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [31] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [27, [32] [33] [34] [35] [36] . a special characteristic of the lipid rafts is the high content of cholesterol [37] [38] [39] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure 1 ), direct in vivo visualization still remains unresolved [39] . metabolites 2020, 10, x for peer review 5 of 22 altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [28] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [21, 29] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [7] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [26, 28, 30] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [22, 28] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [31] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [27, [32] [33] [34] [35] [36] . a special characteristic of the lipid rafts is the high content of cholesterol [37] [38] [39] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure 1 ), direct in vivo visualization still remains unresolved [39] . an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [25, 27] as well as the kinetics of the membrane fusion [25] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [25, 27] as well as the kinetics of the membrane fusion [25] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by statins was able to halt the viral infection at the virus entry step [26] [27] [28] 40, 41] . the effect of cholesterol on promoting membrane merging has also been observed for bis-(monoacylglycero)-phosphate (bmp) [26] . this particular phospholipid was shown to be strictly necessary for dengue virus (denv) entry even at low endosomal ph [42] . as pointed out above, the exact role played by cholesterol is not known in detail, but its intrinsic negative curvature seems to be an essential characteristic in promoting the stalk formation during virus entry. however, a recent study shows that the cholesterol action is likely to involve a direct influence on the oligomeric state of the fusion peptide after insertion into the host cell membrane, as well as on the effects of the fusion peptide on the membrane reorganization and dynamics [43] . in another recent study, a new lipid-label-free methodology was used to measure the kinetics of influenza virus infection [44] . according to the results of this study, cholesterol is able to augment the efficiency of membrane fusion in a receptor binding-independent manner. nevertheless, the rate of membrane fusion was not altered. these results led the authors to conclude that the positive effect of cholesterol in membrane lipid mixing is related to its capability to induce negative curvature. since membrane mixing was achieved in this latter study without binding of the spike protein of the influenza virus to the host cell receptor, the catalytic effect of the fusion peptide might proceed in an independent way in this virus. cleavage of the spike protein in sars-cov-1 does not seem to be also necessary for the fusion peptide to become fusogenic, but rearrangement of disulfide bridges in the s1 peptide after receptor binding are likely involved in the conformational changes driving the fusion mechanism [43, 45] . contrary to these latter results, which point to the fact that membrane fusion is independent of viral protein attachment to its receptor, guo et al. reported lipid raft-dependent viral protein binding with the suppression of viral infection if the lipid rafts were disrupted with cholesterol drug-induced depletion; lipid rafts, as recognized by the caveolin-1 marker, were the membrane domain where structural proteins of the infectious bronchitis virus (ibv) co-localized but the nonstructural proteins did not [35] . the question regarding whether the lipid-raft domains may serve as platforms to concentrate the proteins required for viral entry and, even though some evidence exists, to activate signaling pathways inside the host cell still remains unsolved. sphingomyelins (sms) are also common lipids found in lipid rafts, which contribute to make these membrane microdomains detergent-resistant [34] . the structure of a representative of this lipid class is illustrated in figure 2 . the ganglioside gm1, a sphingolipid, is used as a marker of lipid rafts [34] . sphingolipids (sls) promote to an extent higher than chol the liquid-ordered phase in the outer leaflet of the membrane bilayer because of the long saturated acyl chains they currently contain (the r group in figure 2 may extend to a length of up to 26 c), in addition to their capability to form intermolecular hydrogen bonds [46] . a relevant function of the lipid rafts has been suggested to be the connection between the events outside the cell with the pathways inside the cell, thus acting as 'signaling platforms'. with the aim of this function to be properly accomplished, the lipid rafts would act as concentrators of specific transmembrane proteins, mainly receptors, whose compatibility with the membrane phase would determine their selectivity. thus, sls would account for a role in connecting the outer leaflet with the inner leaflet through their long saturated acyl chains. regarding virus entry, research has been primarily focused toward the role played by cholesterol, but a number of studies have also enlightened the sm influence on this early step of viral infection. the displacement of cholesterol by sms and the other way round has been demonstrated, with the bilayer liquid-ordered phase being preferentially determined by the interaction between sm and cholesterol. this interaction would be controlled to a certain extent by the intracellular actin meshwork, which would also be responsible for the compartmentalization of the membrane into lipid-specific domains [47] . furthermore, the actin role is possibly extended to the routing of the viral genomic material toward the replication place inside the host cell. the hydrolysis of sm by sphingomyelinases to render the corresponding ceramide in specific membrane domains is proposed to regulate the dynamics of cholesterol in the cell membrane, the effect of such regulation being the progressive disassembly of cholesterol from the liquid-ordered phase and its displacement. since the interaction of ceramides with cholesterol has been suggested to be an apoptotic regulator, it can be expected that viral proteins would act in recruiting cholesterol to displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [48] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone ( figure 2 ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca 2+ -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e1 protein to sm/cholesterol-enriched membranes [49] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [50] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus 1 (bohv-1, herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [51] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [52] [53] [54] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [55] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [56] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. metabolites 2020, 10, x for peer review 7 of 22 displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [48] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone ( figure 2 ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca 2+ -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e1 protein to sm/cholesterol-enriched membranes [49] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [50] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus 1 (bohv-1, herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [51] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [52] [53] [54] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [55] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [56] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. some covs (hcov-oc43 and hcovhku1), as well as influenza a virus (whose fusion loop is hemagglutinin, ha) and other non-related viruses (i.e., non-enveloped simian virus 40 sv-40, of polyomavirus family), use the sialoglycan moiety (9-o-acetyl-sialic acid) of gangliosides or glycoproteins located in membrane lipid rafts as receptors for the spike protein. the amino acid trp90 in the domain a of the hcov-oc43 s protein was shown to be essential for receptor binding. however, despite the fact that binding to 9-o-acetyl-sialic acid is required for membrane fusion, further interaction of the virus protein with other host membrane sialoglycans or proteins is also necessary to induce the conformational changes leading to membrane fusion [57, 58] . conversely, formation of the complex sv40 protein with the host cell ganglioside gm1 was found to be enough to induce the membrane curvature and invaginations required for membrane fusion [59] . as already discussed above, some studies have depicted the possibility that interaction of the fusion peptide or fusion loop with negatively charged phospholipids on the host membrane might be required for an efficient membrane fusion [25] . in this regard, phosphatidylserine (ps) contained in the virus envelope has been demonstrated to serve after externalization as a virus co-receptor through the t cell immunoglobulin mucin domain 1 (tim-1) receptor in ebov and other viruses, even in an indispensable fashion [60] [61] [62] [63] . in the study of nanbo et al. [63] , flipping of ps from the inner leaflet to the outer leaflet of the cell membrane for virion adquisition and incorporation to its envelope is proposed as a previous step to tim1 binding. in herpes simplex virus (hsv), phospholipid scramblase-1 (plscr1), after activation by hsv exposure, flips both ps and akt to the outside of the membrane in a ca 2+ -dependent mechanism. ps is restored to the inner leaflet 2 to 4 h after infection to avoid apoptotic triggering [62] , suggesting a different role for ps in relation to the tim-1 ps receptor. however, the function of tim-1 as an essential receptor for hav has been disputed [64] due to the finding that quasi-enveloped ha virions (ehav) were able to infect tim1-knockout vero cells to a similar extent to naked hav. hence, the authors proposed tim1 to be an accessory attachment factor by binding ps on the hav envelope rather than an essential virus protein receptor. in spite of these contradictory data, ps seems to act in any way in virus attachment and entry in certain virus families, at least contributing to the process efficiency, but the exact role may depend on every virus or it may be complementary to other factors. a phospholipid currently associated to the inner leaflet of the lipid rafts is phosphatidylinositol (pi), which is a negatively charged phospholipid with important and versatil signaling functions ( figure 2 ) [65, 66] . abundant data suggest that a derivative of pi, the phosphatidylinositol 4,5-biphosphate (pip2), accumulates preferently in liquid-disordered phases (l d ) [7] , where the cholesterol content is presumed to be low, and interplays with ps, which is rather localized in liquid-ordered phases (l o ). pis play an essential role also in endosome maturation, which is a requisite for efficient virus infection of those using the endosomal pathway [56, 66] . during hiv infection, pip2 has been proposed to coordinate the actin cytoskeleton changes required for efficient virus entry in cd4+ t cells [67] ; after virus attachment to the host cell receptor, pip2 is recruited to the binding membrane microdomain, and in this way, pip2 controls the protein reactions, leading to actin polymerization. as well in hiv-1, the requisite of pip2 accumulation for the virus gag protein to be properly anchored and stabilized in the inner leaflet of the cell plasma membrane has been pointed out [68, 69] . two isoforms, α and γ, of the phosphatidylinositol-4-phosphate 5-kinase family type 1 (pip5k1) have recently been shown to participate in gag stabilization by pip2 through targeting the gag precursor pr55 gag to the cell plasma membrane [70] . as commented above, interaction with the headgroup of negatively charged phospholipids such as ps or pi may also contribute to the dehydration process in the formation of the hemifusion stalk, with this contribution happening by promotion of the inverted hexagonal phase in the lipid bilayer and binding of ca 2+ [25] . in in vitro experiments with cos-7 cells and multilamellar vesicles (mlvs), unspecific binding of the marburg virus (marv) mvp40 protein to pip, pip2, and even pip3 species present in the mlvs, both in the presence or absence of ps, has been reported. in this study, it was also found that with increasing ps concentration, the association of mvp40 to mlvs rose up to a threshold. furthermore, the addition of sphingosine with the aim to reduce the negative charge load in the inner leaftet of the cos-7 cells led to a decrease in the binding level. these facts suggest that the electronic density, rather than the specific lipid species, is a determinant factor for binding [70] . activation of the pi3k pathway for signaling is one of the most relevant features taking place for both entry and budding during infection by a number of viruses [58, [71] [72] [73] . pi3k converts pip2 into phosphatidylinositol 3,4,5-triphosphate (pip3). in addition to stabilizing proteins or serving as a binding factor, pip2 has been shown to collaborate with akt through the signaling pathway pi3k/akt on avoiding apoptotic events, and in this way, keeping the host cell metabolically active for virus replication and budding [71] [72] [73] . all these results clearly bring evidence that the lipid environment surrounding proteins involved in virus infection has a relevant function in the virus entry mechanism. different lipids are essential for virus docking to the cell receptor either serving directly as (co)-receptors or providing the appropriate environment (lipid rafts) for the necessary reactions (e.g., membrane curvature). in addition, the virus, through specific protein conformational changes, takes advantage of several cell signaling pathways controlled by diverse membrane lipids. this process allows the virus to govern the cell metabolism following endocytosis of the viral genetic molecules. after the virus or its genome gets inside the infected cell, ssrna+ viruses and other enveloped ones that replicate in the cytoplasm manage the cell metabolism to develop the replication scaffold, this membrane structure bolstering the so-called 'virus factory' [5, 58, [74] [75] [76] [77] [78] [79] [80] . there is consensus on that the functions of these structures are (i) to compartmentalize the diverse processes involved in viral genome replication, its envelopment, and structural protein assembly; (ii) to increase virion concentration during budding before infecting naïve cells; and (iii) to create a protected environment to escape the innate immune recognition of the viral components. virus replication imposes an extra-energetic expenditure to the cell metabolism. hence, cell central metabolism is orchestated by viral proteins to redirect toward the generation of enough energy and metabolites that are required for virus replication. in particular, building the scaffold demands a high rate of new lipid synthesis. therefore, the lipid metabolism is hijacked by the virus proteins for the de novo synthesis of fatty acids in order to generate the scaffold membranes, the replication complexes (rcs), as well as for energy production in the β-oxidation pathway in the mitochondria. concurrently, the cell metabolism needs to be kept above a threshold level to avoid exhaustion of the host cell. full understanding of the mechanisms and related factors involved in virus-host interaction is a requisite for developing efficient antiviral infection therapies. the scaffold structure raised for building the viral factory varies between different virus in their morphology and possibly lipid composition. flaviviruses develop a so-called 'membranous network' (mn) in a spherule/invagination type, while coronavirus does through a quarter-like type delimited by 'double membrane vesicles' (dmvs). nonetheless, hcv (flaviviridae) uses dmvs instead [77] ; hence, this morphological separation may have exceptions or be somewhat diffuse. an extended review of the different virus family-related morphologies of the mns as well as diverse factors influencing their formation can be found in [22, 75, 76] . it should be remarked that the exact lipid composition of the rcs' membranes is not known in detail yet, although there is evidence that their lipid profile differs from that of the organelles from which they are generated. the enrichment of typical lipids such as cholesterol, sphingomyelins, and glycosphingolipids in the lipid rafts seems to be a common feature of these mns. the rcs' membranes may be originated from the endoplasmic reticulum (er) in the perinuclear area, as for example in sars-cov and faviviridae [75, 78, 80] , from the golgi, giving rise to cytopathic vesicles (cpvs) as in togaviridae and picornaviridae [75] , from mitochondria (nodaviridae) [79] , or from the cell plasma membrane (cpvs in alphaviruses) [75] . however, vesicle trafficking between the er and the golgi organelles may contribute to an undefinition in this regard. mns, and in particular dmvs, are connected to the cytosol through a pore, which is believed to serve as the gate to the replication scaffold for the requiered metabolites, in particular nucleotides. this pore-mediated gate has not been detected up to date in sars-cov's dmvs, which raises the concern of how the required metabolites get inside the rcs. there is evidence from a number of studies that dmvs are the site of replication, but it has also been shown that dmvs can be developed irrespective of whether rna replication takes place by the sole action of the viral proteins, at least for hcv [81, 82] . viral nonstructural proteins nsp3, nsp4, and nsp6 are involved in dmv development in sars-cov-1 in a time-dependent manner and correlating with rna replication. timecourse events have been shown to run with the initial formation of single membrane vesicles (smvs) during the first 2-4 h after cell infection. these futher evolve to dmvs 16 h after infection, and they ultimately turn into multimembraneous vesicles (mmvs) close to the cis-golgi at the budding stage 36-48 h after infection, this latter transformation being coincident with the formation of vesicle packets [75, 78, 79, 83] . in hcv, ns5a seems to be enough for dmv formation, but the collaboration of ns3-5b is required for completing efficient dmvs, whereas ns4b is likely responsible for inducing the formation of smvs [77, 80, 82] . even though particular hints can be likely associated to every particular virus, there are common features shared by all ssrna+ viruses regarding rcs' structure and buildup. enveloped viruses such as ssrna+ viruses have a membrane lipid whose profile is different to that of the original organelle membrane when the envelope is created. since the viral membrane is known to be enriched in cholesterol, sphingolipids, and phospholipids with saturated acyl chains, the dmv is believed to be also primarily composed of such classes of lipids. an unusual sphingolipid, dehydrosphingomyelin, along with ps and plasmalogens of pe were reported in the hiv envelope [84] . a role for sphingomyelin-to-ceramide conversion has been proposed in wnv budding, as its envelope was found to be highly enriched in sphingomyelin [85] . more recently, using multi-color super-resolution microscopy and mass spectrometry analysis, a substantial increase in pip2 (from 11% to 51%) and pip3 (from 0.01% to 0.13%) was reported in the hiv membrane as compared with the plasma membrane of the host cell [69] ; this fact is related to the recruitment of gag protein for efficient membrane fusion as aforementioned ( figure 1) . however, the most striking and known lipid-related factor associated to the mns' development is the pi4kiii signaling pathway. the pi4pα isoform, which is mainly expressed in the er, has been shown to be a key factor for hcv replication, whereas the pi4kiiiβ is found in the golgi and is required by picornaviruses and some hcv strains [75] . this enzyme interacts with the viral protein ns5a, and disrupting this interaction prevents virus replication. the product of the pi4k enzyme is pip4; enrichment in this pi has been shown to act in different processes regarding virus replication: membrane curvature, directly or indirectly through recluting cholesterol [86] , glycosphingolipid transport to the rcs by the action of the fapp2 protein [87] , and protein concentration. however, conversely to these studies, it has been shown that currently used inhibitors of pi4kiiiα, enviroxime and bf738735, actually exert their inhibition against pi3k [88] . thus, this result points out a genomic dependence on the pi kinases in hcv; otherwise, the action on pi3k is required only at the entry stage (see above). enviroxime-like inhibitors have been shown to halt enterovirus replication through the action against pi4kβ [89] . the de novo lipid synthesis has also been evidenced for wnv, from the flaviviridae family as hcv, to proceed in a pi4p-independent fashion and, concurrently, it is not related to pi4kiii signaling [90] . there is no clear evidence on the fact that the pi4k signaling pathway has a relevant function in mns' development. hence, while pi4kiiiβ was shown to be important for sars-cov's dmv formation [91] , another study did not find its metabolite, pi4p, within the host factors involved in sars-cov replication, and the authors attibute to pi4p a function rather in virus entry. however, the authors of this latter study acknowledge that sirna methodology may provide false negatives [92, 93] . since dmvs are not common in healthy cells but they can be observed during authophagy, it has been suggested that sars-cov and other coronaviruses use the autophagy pathway for development of the dmvs; indeed, it has been shown that nsp6 in mhv or the equivalent nsp5-7 in arteriviruses, which hits the er, can activate such a pathway [79, 94] . nonetheless, dmvs are smaller than autophagosomes, and hence, they might be rather endoplasmic reticulum derived vesicles (edesomes) enriched in pi3p and not follow exactly the same synthetic route [94] . further work on coronaviruses and autopahgy found that only the lc3-i protein, the microtubule-associated proteins 1a/1b light chain 3b, is localized on the replication membranes, but the active protein lipidated with phosphatidylethanolamine lc3-ii inserted into the autophagosome membrane is absent. accordingly, present knowledge on coronaviruses in regard to autophagy suggests that they take benefit of the autophagocytic components but do not develop autophagosomes per se [95] . the autophatocytic pathway has also been associated to the start of hcv infection, but it seems not to be necessary for the infection to go on [82] . later on, it was shown that autophagy was key in rna replication at the onset of hcv infection [96] , but the virus life cycle can go ahead afterward without the autophagy system intervention. further work has shown that hcv, and possibly denv, uses the autophagy system to evade the innate immune system [97] . using immortalized human hepatocytes defective of the autophagy-related proteins either beclin (bcn1) or atg7, it was shown in the latter study that disruption of the autophagy machinery elicites activation of the interferon signaling pathway and leads to apoptosis of the infected cells. triggering of the autophagy pathways takes place after binding of the virus to the cell surface via the downregulation of mtor and inactivation of akt signaling [95] . conflicting results have been reported for the induction of autophagy by hcv in regard to the unfolded protein response (upr) [95] . recent work [98] has bound the induction of autophagy by hcv to golgi membrane fragmentation to render vesicles that colocalize with the hcv replicons. the immunity-related gtpase m protein (irgm) mediates the phosphorylation of the early autophagy initiator ulk1 as well as the golgi membrane fragmentation in response to hcv infection. the protein lc3 has also been detected in the replication membranes of the hiv-1, and the association of lc3-ii with gag-derived proteins seems to be a requisite for the efficient maturation of the gag subunit p24 [14, 99] . members of the picornaviridae family, non-enveloped viruses, have been reported to subvert the autophagosome pathway as a means to exit the infected cell without membrane lysis; support for this spreading mechanism comes from the finding of numerous extracelular vesicles that are enriched in phosphatidylserine phospholipids [14] . the best studied virus regarding autophagy is the dengue virus (denv). even though it was initially suggested that the denv replication complexes are developed from autophagosomes, further work pointed out that the replication of denv took place on invaginations arising from the endoplasmic reticulum (er), while autophagy was rather used by denv to modify the lipid metabolism in a way that is known as lipophagy [100, 101] . lipophagy was first shown to be an active way to get energy under starvation [102] through the association of autophagic components with lipid droplets (lds). recently, lipophagy has been demonstrated to regulate the fatty acid availability for the β-oxidation through contact sites between the mitochondria and the er [103] . regarding virus-associated hijacking of the cell lipid metabolism, heaton and randall [100] early showed that increased β-oxidation and the depletion of triglycerides was concurrent with and necessary for denv replication. then, these features were linked to the action of autophagy through the association with lipid droplets. a recent study by zhang et al. [104] has found that aup1, a type iii protein with signals for lds and er, plays a relevant role in lipophagy induced by denv and other flaviviruses such as wnv. unmodified aup1 is required for lipophagy triggering. a 10-fold increase in the content of diacylglycerophosphocholines (pcs) was measured in this study in infected cells containing unmodified aup1, this increase being concomitant with a depletion of triacylglycerols and cholesterol esters, whereas the contents of free fatty acids and unesterified cholesterol rose. conversely, smaller lds, but not a reduction of their abundance, were observed in aup1-knocked-out cells. thus, these data point to an augmented consumption of lds in the infected cells. this study unveils the mechanism that leads to the commented results; after the denv protein ns4a associates with aup1, the complex is relocalized from lds to autophagosomes, where the acyltransferase domain of aup1 is activated for the generation of phospholipids. this process was found to be dependent on the aup1 ubiquitylation status, with ns4a inhibiting the ubiquitylation of aup1. similar to viral entry, cholesterol has been found to be also relevant in the rcs' membranes [79, 82] . up to a c.a. 9-fold enrichment of cholesterol was found in hcv-developed dmvs as compared to its content in the er membranes from which dmvs were originated [77] . a key protein in cholesterol metabolism associated to non-vesicular transport is the oxysterol-binding protein (osbp). this protein has been described to transport cholesterol to pi4p-enriched membranes, which would agree with its collaboration in delivering cholesterol to dmvs with an abundant content of this pi [77] . the ceramide transfer protein (cert) and the four-phosphate adaptor protein 2 (fapp2) are known to undergo a similar fate in hcv infection [82] . an important protein involved in cellular lipid homeostasis is the sterol regulatory element binding protein (srebp), a bhlh-zip transcription factor with three isoforms; srebp1c regulates the expression of fatty acid (fa) biosynthesis genes [105, 106] , whereas srebp2 transactivates genes implied in cholesterol biosynthesis, intracellular lipid transport, and lipoprotein import [107] . a recent study shows that the inhibition of srebp with the retinoid derivative and rar-α agonist am580 prevents mers-cov infection by avoiding the formation of functional dmvs [105] . in this study, the lipid metabolism was the most affected pathway, with sterol biosynthesis being strengthened at expense of the glycerophospholipid metabolic pathways. fast activation of the lipid biosynthesis enzymes acetyl-coa carboxylase (acc), fatty acid synthase (fas), and hmg-coa synthase (hmgcs) was observed in such study, whose activity was partially blocked by am580 inhibition of srebp enzymes. promotion of lipid biosyntheis after infection had already been pointed out for hcv in an elegant proteomics and lipidomics study [108] . hcv infection elicites changes in the proteome of host cells that resembled the warburg effect described in cancer cells toward lactate production and the support of continuous glycolysis; concurrently, the up-regulation of citrate synthase (cs) and other lipogenic enzymes 24 h after infection was interpreted by the authors of the latter study as indicative of re-routing of the tricarboxylic acid (tca) cycle for cytosolic accumulation of citrate, which would be used in fa synthesis. the up-regulation of peroxisomal and mitochondrial fa oxidation pathways is concurrent with the other metabolic changes. an increase in pro-apoptotic ceramides was observed in the latter study as well; two possible interpretations were attributed to this finding, either a cytopathic effect after cell cycle arrest over time enough to complete virus offspring or a defense response of the host cell to avoid infection spread. blocking cholesterol suitability for the membraneous network or endosomes used for the virus replication and internalization has been demonstrated to inhibit the virus life cycle in a number of unrelated viruses. disruption of the srebp pathway restrains the andes virus (andv), an ssrnavirus, internalization, although it does not bind to the cell surface receptor [109] . in addition to srebp2, other components of this pathway were found to be necessary. the dependence of viral entry on the sterol regulatory element binding protein cleavage activating protein (scap) and the site 1 protease (s1p) was evidenced in cells null for these proteins. thus, in the study of petersen et al. [109] , the virus was not internalized in cells lacking s1p, this result pointing out that a complete cholesterol biosynthesis pathway is required. infectivity was also reduced 10-fold when the cells were treated with methyl-β-cyclodextrin (mβcd), a cholesterol sequestering agent, and comparable results were obtained after cell treatment with mevastatin or the s1p inhibitor pf-429242. however, the s1p dependence of virus infectivity does not seem to affect other viruses, thus this route being likely selective for hantaviruses [110] . in this study, the genetic or pharmacological disruption of the srebp pathway at the site of the regulatory element membrane-bound transcription factor peptidase/site 1 protesase (mbtps1/s1p) dramatically reduced viral infection, which is a feature that confirms the essential dependence of hantavirus on the high membrane cholesterol content for membrane fusion and effective infection. the down-regulation of sterol synthesis at the gene level after infection was found to be controlled by an interferon regulatory loop, in which a type i interferon-dependent mechanism down-regulates the expression of srebp2 [111] , this result showing a link between the innate immune response and cholesterol biosynthesis after viral infection. this type i interferon response toward cholesterol synthesis down-regulation was dependent on the mevalonate-isoprenoid branch as a supply of mevalonate completely blocked the cholesterol synthesis, whereas a supply of cholesterol did not. additionally, in the presence of geranylgeraniol, the type i interferon inhibition of sterol biosynthesis was severely diminished. further research has shown that interferon may regulate the sterol synthesis pathway in multiple forms through micrornas [112] . in particular, mir-342-5p was found to hit multiple srebp-independent targets of the mevalonate-sterol synthesis pathway after viral infection. the type i interferon response was also observed in regard to the impairment of the formation of double membrane structures induced by arteriviruses as replication sites [113] . host cell fight against viral infection by a reduction of cholesterol availability has been also pointed out to come from the antiviral effector protein interferon-inducible transmembrane protein 3 (ifitm3). this protein interacts with vesicle-membrane-protein-associated protein a (vapa), impeding its association with the oxysterol binding protein (osbp), and consequently, altering the normal function of osbp. as a result of the ifitm3 action, virus release into the cytosol is blocked by the accumulation of cholesterol in multivesicular bodies and endosomes. this effect restrains the membrane fusion of the intraluminal vesicles and that of the multivesicular bodies, which is a requisite for virus budding and release to the cytosol [114] . the viral accesory protein of hiv nef competes with the cholesterol transporter abca1 to prime the transport of cholesterol to lipid rafts as a viral strategy to raise the replication membranes, thus overcoming the antiviral properties of abca1 [115] . the replication of rabies virus (rabv), an ssrna virus, is halted by the action of viperin (virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible) in raw264.7 cells. this protein is induced by the rabv, ifv, hiv, or hcv infection through promotion of the innate immune response bound to the tlr4 signaling pathway. the inhibitory activity of viperin on virus budding is related to its capability to substantially drop the contents of cholesterol and sphingomyelin in the replication membranes [116] , thus pointing out the relevance of the membrane lipid composition for efficient virus replication. the induction of viperin has also been proven for hcv and ifav [111] . however, viperin does not intervene in the inhibition of arterivirus-induced double membrane formation [113] . remodeling of the lipid metabolism by virus infection may leave signals at the organism level even some years after healing. the metabolome profile of patients undergoing sars-cov-1 infection during the outbreak of 2002-2003 was assessed 12 years after overcoming the pathology [117] . an outstanding result of this study regarding disturbed lipid metabolism was the elevation of phosphatidylinositol (pi) and lysophosphatidylinositol (lpi) species concentrations in serum, which in turn correlated positively with the levels of very low-density lipoproteins (vldl); higher concentrations of products of the phospholipase a 2 (pla 2 ) such as lysophospholipids (lppls) and free arachidonic acid (aa) were also found in patients as compared to healthy voluntiers, with a correlation between the level of aa and the ratio of lpi(18:0) to total 18:0-pis being observed as well. these results show a potential high sensitivity of sars-cov patients to pla 2 activity. in the general context, the metabolome of these patients pointed to hyperlipidemia, cardiovascular abnormalities, and glucose metabolism alteration as a delayed efffect of the viral infection. nonetheless, the authors acknowledge that some of the related metabolic disturbations are likely owed to the pharmacological treatment. high levels of pla 2 group iid (pla2g2d) in lungs of middle-aged mice as compared to young mice had previously been associated to a fatal or worse outcome [118] . the authors of this study conclude that the negative influence of this enzyme in sars-cov infection was to increase the concentration of anti-inflammatory lipid mediators, mainly protaglandin d 2 (pgd 2 ), which impaired the efficient function of the immune system [119] . in the recent sars-cov-2 outbreak (covid-19), mortality has mostly affected aged people above 60 years old, thus showing an age-related fatality as for sars-cov-1 and mers-cov [120] . using a lipidomics approach, the effect of hcov-229e and mers-cov infection on the host cell lipid profile was recently investigated in cell culture [121] . the main conclusions of this study agree with the raised content of aa and lppls through plase activity, which indicates that the possible virus-induced activation of cpla 2 favors virus replication as a factor required for dmvs' formation. in this study, linoleic acid (la) or aa supplementation to the culture cells suppressed replication, which is a result that may be interpreted as a demonstration of the perturbation of the la/aa axis of the lipid metabolism. in the covid-19 outbreak, it has been suggested that increasing the levels of vitamin d could help fighting against the sars-cov infection [122] . this suggestion is based on the fact that 25-hydroxyvitamin d3 was found to protect huh7 cells against mers-cov [105] . vitamin d is a lipid-related compound belonging to the group of fat-soluble secosteroids, with the most important form in humans being vitamin d3 (cholecalciferol) [123] . in a recent study, high doses of vitamin d have shown protective effects against denv infection through regulation of the toll-like receptor expression as well as the modulation of pro-inflammatory cytokines release, which suggests that its action is focused toward the immune system modulation rather than to lipid metabolism [124] . however, evidence on the beneficial effects of vitamin d uptake is still poor, and more studies are devoted to this issue. lipids, as components of membranes, are related to viroporins, which are specific viral proteins that are known to create ion channels for ion trafficking [125] [126] [127] . the effect on cell metabolism of diverse viroporins differs among them, but there is evidence that they are closely related to viral pathogenity [125] . viroporins may play a relevant role during virus infection, as they are involved in membrane permeability and calcium homeostasis. their participation in the development of vacuoles from the er during the dmvs' formation has been suggested, but data on this issue are still scarce. the regulation of ca 2+ flux by viroporins might favor the membrane fusion through the interaction of this cation with the phospholipid headgroups and concurrently facilitate the required dehydratation reaction. viroporins are not required for virus replication with the exception of rotaviruses and picornaviruses; thus, whether this function is exerted through the ion channels or another property of viroporins remains an issue still unknown [125] . the lipid composition of the membrane may influence the viroporin activity, leading to different versions of ion channels, which depends on the electric charge that the phospholipids confer to the membrane and curvature [127] . a viroporin from rotavirus, nsp4, was shown to co-localize with the autophagy marker protein lc3 in membranes accomodating virus replication; this viroporin is implicated in the sequestering of autophagy for the transport of proteins from the er to the replication sites [128] . further research is necessary to understand the role played by viroporins in virus infection in order to consider them as potential therapeutic targets. remodeling of the virus-induced host cell lipid metabolism is a remarkable feature of the viral infection that affects viral entry, replication of the genomic material, and the releasing of progeny. a comperhensive view of the process is illustrated in figure 3 . the main actors are well known to be cholesterol, sphingolipids, and pis, but other lipid species and their related pathways such as the la/aa axis are also relevant. how to target the lipid metabolism in a safe manner to avoid virus infection or reduce its pathogenity is a promising therapeutic tool, but it demands improving the knowledge on the actual pathways that are affected over the virus life cycle. the exact mechanism through which the enzyme inhibitors act on the key enzymes of the lipid metabolism is additionally required to develop more efficient and safe therapeutic drugs. since the lipid metabolism is essential for proper cell function, selective drugs targeting the virus or exclusively the infected cells have to be used to avoid harmful side effects. a new coronavirus associated with human respiratory disease in china world health organization. who director-general's opening remarks at the media briefing on covid-19 an overview of their replication and pathogenesis the virus-host interplay: biogenesis of +rna replication complexes virus factories: associations of cell organelles for viral replication and morphogenesis virus entry: open sesame the role of cholesterol in membrane fusion world health organization. who director-general's opening remarks at the media briefing on covid-19 an overview of their replication and pathogenesis the virus-host interplay: biogenesis of +rna replication complexes virus factories: associations of cell organelles for viral replication and morphogenesis virus entry: open sesame the role of cholesterol in membrane fusion identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins structural characterization of the hcov-229e fusion core hepatitis c virus entry. in hepatitis c virus: from molecular virology to antiviral therapy how hepatitis c virus invades hepatocytes: the mystery of viral entry endocytosis of influenza viruses. microbes infect virus entry by endocytosis viral journeys on the intracellular highways infectious bronchitis virus entry mainly depends on clathrin mediated endocytosis and requires classical endosomal/lysosomal system single virus tracking of ebola virus entry through lipid rafts in living host cells sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor hepatitis c virus infection: host-virus interaction and mechanisms of viral persistence cell entry of enveloped viruses viral membrane fusion new biophysical approaches reveal the dynamics and mechanics of type i viral fusion machinery and their interplay with membranes influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates sequential conformational rearrangements in flavivirus membrane fusion energetics of stalk intermediates in membrane fusion are controlled by lipid composition visualization and sequencing of membrane remodeling leading to influenza virus fusion two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin sars-cov fusion peptides induce membrane surface ordering and curvature direct measurement of polyethylene glycol induced depletion attraction between lipid bilayers nmr structures and localization of the potential fusion peptides and the pre-transmembrane region of sars-cov: implications in membrane fusion fluorescence microscopy of the hiv-1 envelope lipid rafts are involved in sars-cov entry into vero e6 cells coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains the important role of lipid raft-mediated attachment in the infection of cultured cells by coronavirus infectious bronchitis virus beaudette strain influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion membrane lipids: where they are and how they behave the mystery of membrane organization: composition, regulation and roles of lipid rafts lipid rafts: controversies resolved, mysteries remain cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin the hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes dengue virus ensures its fusion in late endosomes using compartment-specific lipids membrane cholesterol modulates oligomeric status and peptide-membrane interaction of severe acute respiratory syndrome coronavirus fusion peptide target membrane cholesterol modulates single influenza virus membrane fusion efficiency but not rate the molecular biology of coronaviruses cholesterol interactions with ceramide and sphingomyelin sphingolipid-dependent fusion of semliki forest virus with cholesterol-containing liposomes requires both the 3-hydroxyl group and the double bond of the sphingolipid backbone both sphingomyelin and cholesterol in the host cell membrane are essential for rubella virus entry ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection role of sphingomyelin in alphaherpesvirus entry ceramide suppresses influenza a virus replication in vitro rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms differential utilisation of ceramide during replication of the flaviviruses west nile and dengue virus glucosylceramidase maintains influenza virus infection by regulating endocytosis influenza a viruses use multivalent sialic acid clusters for cell binding and receptor activation structural basis for human coronavirus attachment to sialic acid receptors lipid interactions during virus entry and infection gm1 structure determines sv40-induced membrane invagination and infection role of the phosphatidylserine receptor tim-1 in enveloped-virus entry tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and akt to the outer leaflet of the plasma membrane and promote viral entry tim1 (havcr1): an essential "receptor" or an "accessory attachment factor" for hepatitis a virus? pip5k-driven ptdins(4,5)p2 synthesis: regulation and cellular functions function and dysfunction of the pi system in membrane trafficking pip2: choreographer of actin-adaptor proteins in the hiv-1 dance synchronized hiv assembly by tunable pip2 changes reveals pip2 requirement for stable gag anchoring quantification of phosphoinositides reveals strong enrichment of pip2 in hiv-1 compared to producer cell membranes investigation of the lipid binding properties of the marburg virus matrix protein vp40 type i phosphatidylinositol-4-phosphate 5-kinase α and γ play a key role in targeting hiv-1 pr55gag to the plasma membrane make yourself at home: viral hijacking of the pi3k/akt signaling pathway hepatitis b spliced protein (hbsp) suppresses fas-mediated hepatocyte apoptosis via activation of pi3k/akt signaling multifaceted roles for lipids in viral infection ultrastructure of the replication sites of positive-strand rna viruses does form meet function in the coronavirus replicative organelle? morphological and biochemical characterization of the membranous hepatitis c virus replication compartment sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum virus factories, double membrane vesicles and viroplasm generated in animal cells three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex mechanisms of cellular membrane reorganization to support hepatitis c virus replication. viruses severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles the hiv lipidome: a raft with an unusual composition the composition of west nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for hcv replication membrane integrity and cholesterol trafficking modulation of hepatitis c virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2 pi4kiii inhibitor enviroxime impedes the replication of the hepatitis c virus by inhibiting pi3 kinases phosphatidylinositol 4-kinase iii beta is a target of enviroxime-like compounds for antipoliovirus activity west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids phosphatidylinositol 4-kinase iiiβ is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry a kinome-wide small interfering rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins host factors in coronavirus replication. in roles of host gene and non-coding rna expression in virus infection current topics in microbiology and immunology divergent roles of autophagy in virus infection the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m. proc. natl. acad. sci autophagy during viral infection-a double-edged sword dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy autophagy regulates lipid metabolism autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites flaviviruses exploit the lipid droplet protein aup1 to trigger lipophagy and drive virus production activators of the complete program of cholesterol and fatty acid synthesis in the liver common fatty markers in diseases with dysregulated lipogenesis coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target the major cellular sterol regulatory pathway is required for andes virus infection haploid genetic screen reveals a profound and direct dependence on cholesterol for hantavirus membrane fusion host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis an interferon regulated microrna provides broad cell-intrinsic antiviral immunity through multihit host-directed targeting of the sterol pathway antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry hiv-1 nef mobilizes lipid rafts in macrophages through a pathway that competes with abca1-dependent cholesterol efflux viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by tlr4 temporal proteome and lipidome profiles reveal hepatitis c virus-associated reprogramming of hepatocellular metabolism and bioenergetics critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection virus-induced inflammasome activation is suppressed by prostaglandin d2/dp1 signaling the trinity of covid-19: immunity, inflammation and intervention characterization of the lipidomic profile of human coronavirus-infected cells: implications for lipid metabolism remodeling upon coronavirus replication. viruses evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths sunlight and vitamin d for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease effect of high doses of vitamin d supplementation on dengue virus replication, toll-like receptor expression, and cytokine profiles on dendritic cells viroporins: structure and biological functions viral membrane channels: role and function in the virus life cycle autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-signaling is required for rotavirus replication this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. key: cord-298736-9bvyp21d authors: gerold, gisa; bruening, janina; pietschmann, thomas title: decoding protein networks during virus entry by quantitative proteomics date: 2016-06-15 journal: virus research doi: 10.1016/j.virusres.2015.09.006 sha: doc_id: 298736 cord_uid: 9bvyp21d abstract virus entry into host cells relies on interactions between viral and host structures including lipids, carbohydrates and proteins. particularly, protein–protein interactions between viral surface proteins and host proteins as well as secondary host protein–protein interactions play a pivotal role in coordinating virus binding and uptake. these interactions are dynamic and frequently involve multiprotein complexes. in the past decade mass spectrometry based proteomics methods have reached sensitivities and high throughput compatibilities of genomics methods and now allow the reliable quantitation of proteins in complex samples from limited material. as proteomics provides essential information on the biologically active entity namely the protein, including its posttranslational modifications and its interactions with other proteins, it is an indispensable method in the virologist's toolbox. here we review protein interactions during virus entry and compare classical biochemical methods to study entry with novel technically advanced quantitative proteomics techniques. we highlight the value of quantitative proteomics in mapping functional virus entry networks, discuss the benefits and limitations and illustrate how the methodology will help resolve unsettled questions in virus entry research in the future. viruses need to enter permissive host cells in order to propagate and spread. the penetration of cells requires an initial interaction of viral and host surface structures. typical viral attachment proteins (vap) are transmembrane glycoproteins for enveloped viruses or capsid proteins for naked viruses. alternatively, some viruses incorporate phosphatidylserine into their envelope and use this lipid for host cell interaction (amara and mercer, 2015; jemielity et al., 2013; kondratowicz et al., 2011; mercer and helenius, 2008; moller-tank et al., 2013; shimojima et al., 2012) . for the majority of viruses, however, proteins bind to cellular receptors, i.e., proteins or carbohydrates, and the expression of a particular receptor is often determining tissue and species tropism of a virus. in this review we focus on the role of protein-protein interactions (ppi) during viral penetration as protein-lipid and protein-carbohydrate interactions in cell entry of viruses have been discussed elsewhere (amara and mercer, 2015; lozach et al., 2007; stencel-baerenwald et al., 2014) . historically, the first virus receptor found was a glycan. in 1981 helenius and coworkers described sialic acid as the influenza a virus receptor (matlin et al., 1981) . in the following years it became clear that most virus receptors are proteins or protein-linked entities, such as glycosaminoglycans. as a consequence, protease treatment rendered susceptible host cells non-susceptible for many viruses. with the discovery of cr2 as epstein barr virus (ebv) and cd4 as human immunodeficiency virus type 1 (hiv-1) receptor the hunt for virus receptors began (fingeroth et al., 1984; maddon et al., 1986) . since then technological developments like antibody based affinity purification (ap), mass spectrometry (ms) of proteins, dna mediated transformation and molecular cloning led to the discovery of dozens of receptors for human pathogenic viruses (fig. 1) . nevertheless, for many viruses the cognate receptor(s) remain enigmatic and a "one fits all" method for their discovery has not yet been described. virus entry factors, which interact with vaps directly, can be differentiated into attachment factors and entry receptors. attachsurface. virus receptors not only mediate cell attachment but the ppi of vap and receptor also guides subsequent entry steps, e.g. internalization ( fig. 2 and box 1). sometimes the vap-receptor interaction unleashes the fusogenic property of viral glycoproteins by inducing conformational changes in the vap ectodomains. a well-studied example is the priming of the hepatitis c virus (hcv) glycoprotein e2 by the cd81 receptor (sharma et al., 2011) . for viruses, which fuse in intracellular vesicular compartments, a drop in ph typically acts as primer for fusion. in some cases host proteases interact with and cleave vaps rendering them fusogenic. ppis between viral and cellular factors orchestrate viral attachment, trafficking, fusion and uncoating. classical virus infection pathways are illustrated for a generic enveloped virus. note that naked virions infect cells in a similar manner with viral capsid proteins mediating critical ppis with cellular proteins (not shown). ppis facilitating virus attachment, surfing, fusion and transport are depicted. in some cases proteolytic processing mediated by host factors triggers viral membrane fusion. moreover, secondary ppis can lead to assembly of multiprotein receptor platforms that allow internalization and ultimately membrane fusion. proteolytic processing of vaps either occurs during vap biosynthesis or during virus entry. the latter applies to ebola virus, which requires processing of its envelope glycoprotein by cathepsins in endosomes (chandran et al., 2005) . moreover, interactions of vaps with secondary receptors, occurring after membrane surfing of viruses towards cellular junctions may trigger internalization (coyne and bergelson, 2006) . after endocytosis, secondary ppis with endosomal receptors can in addition to the ph drop render viral glycoproteins fusogenic (carette et al., 2011; cote et al., 2011) . subsequently viruses replicating in the nucleus are transported along the cytoskeleton towards the nuclear pore, where the genome is released into the nucleus. transport relies again on ppi of virus structural proteins and microtubular motor proteins. all viruses additionally need to undergo uncoating, i.e., the viral nucleocapsid needs to disassemble to release the genome into the cell. uncoating is poorly understood for most viruses, but as detailed below, also requires interaction of viral capsids with host proteins. fig. 2 highlights the most important steps in virus entry and critical ppi for each step. in brief, most processes during virus entry are coordinated by ppis. in fact, eighty percent of the approximately 20,000 protein coding genes of a cell are estimated to perform their function through interactions with other proteins. roughly 10,000 distinct interaction types, i.e., structurally similar interactions, and 130,000-650,000 binary protein interactions can occur in a cell (aloy and russell, 2004; stumpf et al., 2008; venkatesan et al., 2009) . thus, knowledge on protein complexes is essential for the understanding of cellular mechanisms and for the targeted interference with a protein's function. in some instances this knowledge has been successfully translated into therapeutic approaches for treatment of viral infections. the best known examples are the hiv-1 ccr5 coreceptor antagonist maraviroc and the hiv-1 gp41 fusion inhibitors (dorr et al., 2005; wild et al., 1993) . importantly, many interactions are mediated by short 10 amino acid long unstructured peptide motifs, are transient, and have relatively low affinity. classical high stringency tandem ap protocols thus often missed peptide motif based protein interactions. novel shotgun proteomics of one step affinity enriched samples provides now the opportunity to unravel a multitude of previously missed interactions. typical host peptide motifs, which coordinate ppis and occur in several hundred human proteins, are the functional unit of src-homology 2 domain (sh2-domain), sh3-domain, the post synaptic density protein (psd95), drosophila disc large tumor suppressor (dlg1), and zonula occludens-1 protein (zo-1)-domain box 1: definitions and abbreviations in virus entry. susceptible cell: cell that expresses virus receptors and entry factors and thus allows entry of a virus. permissive cell: cell that expresses all host factors required for replication, assembly and release of a virus and thus can generate infectious progeny after infection. virus entry: process of delivery of the viral genome to the viral replication compartment in the cells. depending on the replication site and the virus entry route, virus entry includes cell attachment, endocytosis, membrane penetration, trafficking in the cytoplasm, nuclear import, release of viral structural proteins like tegument and genome uncoating. virus attachment factor: cell surface protein interacting with a virus. attachment factors facilitate entry by concentrating virions at the cell surface and can promote subsequent interactions with entry receptors. virus entry receptor: cellular protein, lipid or glycan that binds the virus particle and mediates virus uptake and/or triggers critical conformational changes crucial for infection. viral receptors are often localized to the plasma membrane, but can also reside in endosomal compartments. virus entry factor: operationally defined as any cellular factor that in some way participates in the virus entry process, but not necessarily interacts with the virus. viral attachment protein (vap): viral surface protein that binds to the virus receptor. vaps of enveloped viruses are transmembrane glycoproteins, while vaps of naked viruses are viral capsid proteins. capsid (also: core): protein shell of a virus arranged around the viral genome or complexed to it; build by repeat units of single proteins or protein complexes. the capsid proteins are encoded by the viral genome. nucleocapsid: complex of viral genome and capsid, which may for enveloped viruses be surrounded by a lipid bilayer. envelope (also: peplos): host cell derived lipid bilayer surrounding the nucleocapsid of enveloped viruses. the envelope typically displays viral genome encoded transmembrane proteins. these envelope proteins are vaps and in some cases enzymes (neuraminidases) that cleave cellular carbohydrates to facilitate virus shedding or ion channel proteins that prime viral uncoating. recently, it was shown that some classical naked viruses, like hepatitis a virus, could be enveloped as well. virion surfing: directional movement of virions within the cell plasma membrane, i.e. on the cell surface or cell projections like filipodia, towards the viral entry sites virus fusion: membrane mixing of the lipid bilayer of enveloped viruses with a host cell lipid bilayer, i.e. either the plasma membrane or the endosomal membrane, resulting in the release of viral nucleocapsids into the cytoplasm. uncoating: disassembly of the viral capsid to release the viral genome into the cytosol or nucleus of a host cell. large dna viruses additionally require tegument disassembly prior to disassembly of the capsid. (pdz-domain) and the so called ww-domains which carry two tryptophan residues as signature (tompa et al., 2014) . notably, viruses and other pathogens often make use of these and other peptide motifs to hijack cellular pathways during their life cycle including virus entry hagai et al., 2014) . while transcriptomics can reveal long-term alterations of the cellular state, virus entry usually occurs within minutes and typically relies on rapid changes of protein conformation, localization, interactions and post-translational modifications (ptm). thus studying the proteome state, preferentially in a time-resolved manner, is ideally suited for understanding the mechanisms of virus entry. proteomics has already led to the characterization of global cellular alterations induced by virus infection. for instance, virus induced changes in the protein composition of subcellular hcv replication compartments revealed several host replication and assembly factors (huang et al., 2007; mannova et al., 2006; paul et al., 2013; yi et al., 2006; yi et al., 2011) . interaction proteomics at a large scale was moreover performed for all 18 hiv-1 proteins outside of the context of hiv-1 infection and revealed important virus manipulations of cellular pathways (jager et al., 2012) . another global interaction proteomics study revealed host protein interactions of viral immune modulating proteins uncovering viral immune evasion strategies (pichlmair et al., 2012) . although in principle possible, global interaction proteomics during virus attachment and entry has not yet been extensively pursued. as virus entry is the process in the viral life cycle, which can most easily be synchronized by inhibitors or temperature shifts, we believe that proteomic analyses can close many gaps of knowledge in virus entry research. below we discuss successful applications of proteomics to understand virus uptake and highlight future perspectives. the various entry routes of viruses have been extensively reviewed (grove and marsh, 2011; mercer et al., 2010; amara and mercer, 2015) . similarly, we refer the reader elsewhere for methods to define protein interactions other than ms based proteomics, i.e., native page, yeast two hybrid, protein microarrays, fluorescence resonance energy transfer, surface plasmon resonance, structurebased approaches and phage display (ngounou wetie et al., 2014) . here, we focus on the ppis and ptms that mediate virus uptake and on past and present ms proteomics based techniques to study virus invasion. to gain access to a susceptible host cell the vast majority of viruses interacts with proteinacious receptors on the cell surface. consequently, biochemical protein-based approaches successfully discovered virus receptors in the past (table 1) . typically, these approaches build on virus overlay protein blot assays (vopba). vopba relies on probing of cell lysates or cell membrane extracts, separated by gel electrophoresis and transferred onto nitrocellulose membranes, with virus particles (boyle et al., 1987) . virus bound proteins are in this case either detected by radiolabelling of the virus or by specific antibodies against the virus glycoproteins. in both cases ms fingerprinting of a corresponding polyacrylamide gel band identifies the putative receptor. a prime example for vopba approaches is the identification of alpha-dystroglycan as receptor for lymphocytic choriomenigitis virus (cao et al., 1998) . the vopba qualitative proteomics approach has the advantage that the protein is left intact until the last step of identification and thus information on the molecular weight and posttranslational modifications like glycosylation is available. however, it suffers from low sensitivity, as retrieval of proteins or peptides from polyacrylamide gels is typically low. moreover interactions could be lost under denaturing conditions. an alternative to vopba-based receptor identification is the affinity purification (ap) of virus glycoprotein -receptor complexes. this method overcomes the caveat of protein denaturation and analyzes interactions in their native state. for instance, igfusion proteins of truncated soluble viral glycoproteins were successfully used to co-purify the new world arenavirus receptor transferrin receptor 1 (radoshitzky et al., 2007) . other examples for qualitative interaction proteomics are the identification of receptors for nipah virus (ephrin b2), kshv (ephrin a2) and sars (angiotensin-converting enzyme 2) (hahn et al., 2012; li et al., 2003; negrete et al., 2005) . here, glycoprotein−fc fusion proteins served as baits for ap of the receptor post cell lysis. in a third (yan et al., 2012) . generally, biotinylation of surface proteins prior to ap or amine-reactive crosslinking are strategies to increase specificity of the ap and help avoid detection of biologically irrelevant interactions after cell lysis. a refinement of both methods is the use of biotinylated ligands equipped with hydrazine groups for crosslinking of cell surface proteins glycans. to this end, a trifunctional reagent for ligand derivatization, termed triceps, has been developed by frei et al. (2012) . critical negative controls further include, non-susceptible cell lines, which do not bind the virus, and glycoprotein mutants or peptide variants, which are deficient for receptor binding. in principle, ms fingerprinting of the purified proteins is the sole method to unambiguously identify the prey after ap. it should however be noted that analysis of the prey with respect to its sensitivity to glycosidases, proteases, heparinases and lipid raft altering agents can lead to an educated guess, which upon further validation might identify a virus receptor. a prominent example is the identification of scavenger receptor class b type i for hcv (scarselli et al., 2002) . similar to vopba approaches, receptor identification after ap relied on qualitative proteomics in the past, i.e. gel purification of the co-precipitated proteins, followed by ms fingerprinting of a defined gel band. such interaction proteomics approaches typically require a truncated and epitope tagged virus glycoprotein for efficient affinity enrichment (ae) of cellular receptors. high affinity antibodies against the viral glycoprotein may supersede epitope tagging of the bait. both vopba-based and ap-based interaction proteomics are well suited for the identification of primary receptor interactions. however, interactions with late receptors, which require a conformational change of the glycoprotein and are more difficult to synchronize, cannot be captured easily with these techniques. also virus-triggered secondary ppis are not resolved by these classical methods. the development of shotgun proteomics techniques described below thus presents a major breakthrough, which holds the promise of detecting higher order, reduced affinity and even transient virus receptor interactions. in the past decade, the development of quantitative shotgun proteomics techniques revolutionized the protein interactions field. in contrast to previous qualitative approaches, shotgun techniques avoid extensive purification from gels and instead analyze a complex mixture of proteins, thereby tremendously increasing sensitivity and throughput. technological development of high performance liquid chromatography (lc) and ultra-high resolution ion trap ms together reduced measurement time, increased peptide sensitivity and measurement reproducibility and thus made shotgun techniques possible. moreover, mass accuracy of new generation ion trap mass spectrometers increased up to 0.001 da, guaranteeing high confidence peptide identification and analysis of posttranslational modifications (ptms). lc-ms thus emerged as prime in depth proteomics method leaving behind alternative techniques like two-dimensional gel electrophoresis, low-resolution maldi and protein arrays (mann and kelleher, 2008) . shortly, in shotgun interaction proteomics affinity enriched protein mixtures are as a whole digested to peptides and analyzed by lc-ms. to separate the specific binding partners from the excess of background proteins, specific and control purifications are directly compared (vermeulen et al., 2008) . this is achieved either by stable isotope labeling of amino acids in culture (silac) or by the more recent computational based label-free quantification methods (lfq) described in the subsequent paragraph. most recent techniques use one-step ae protocols, which preserve weak and transient interactions (see below for details). fig. 1 highlights the milestones in the development of ms-based proteomics techniques and box 2 describes common proteomics terms. while quantitative shotgun proteomics has been used in a variety of fields including signal transduction, cell adhesion and even viral immune modulation, we are just starting to appreciate its power for the study of the viral replication cycle and in particular virus entry (kruger et al., 2008; pichlmair et al., 2012; weekes et al., 2014; zanivan et al., 2013) . ms based methods are commonly used to identify and quantify proteins, to profile protein-interactions or whole proteomes. therefore, proteins are isolated and proteolytically digested, the resulting peptides are separated via lc, and then analyzed by tandem mass spectrometry (ms/ms) (bottom-up approach or shotgun proteomics) (vermeulen et al., 2008) . alternatively a top-down approach can be used, where the protein is fragmented during the mass spectrometric analysis (boeri erba, 2014). for quantitative proteomics, most often bottom-up high resolution ms techniques are used in combination with either labeling or label-free methods: a powerful and the most commonly used label-based method for the quantification of proteins is silac (ong et al., 2002) . in short, an isotope label is metabolically incorporated into peptides and proteins of a cell. typically, essential amino acids with heavy isotopes (e.g., 15 n, 13 c) are fed to cells over several passages until nearly all unlabeled amino acids are replaced. as the physicochemical properties of light and heavy amino acids are nearly identical, cells labeled with the isotopic analogs behave similar or even identical to unlabeled cells. based on the isotope label silac allows the direct comparison of two cell populations (heavy and light) from two distinct experimental conditions; for instance, from cells with surface-bound virus or cells without bound virus (gerold et al., 2015) . prior to proteomic analysis, i.e., whole cell lysate analysis or ap of protein complexes, the differently labeled samples are mixed. this is advantageous as all samples are processed equally excluding sample handling biases. proteins are then digested, peptides reduced and alkylated, separated by lc and analyzed by ms. peptides derived from heavy or light cells are subsequently differentiated by their characteristic mass offset. relative abundances of proteins in each sample are calculated based on the heavy and light signal intensities in the same spectrum. typically, silac is used for relative quantification of protein levels under two different cellular conditions. of note, medium labeled amino acids are additionally available and allow expanding a silac experiment to three cellular conditions (hilger and mann, 2012) . the high accuracy of silac typically requires analysis of only two experimental replicates. in addition, two replicates of a label swap experimental condition allow assessing label-specific effects. thus, a typical dual silac experiment starts with eight experimental samples mixed into four silac pairs. those are duplicates of the forward label pair (light-condition 1; heavy-condition 2) and duplicates of the reverse label pair (light-condition 2; heavy-condition 1). fig. 3a depicts the silac workflow. silac followed by lc-ms/ms can answer various questions in virology. for example, it can be used to compare the proteome of uninfected to that of virus infected cells (e.g., zhang et al., 2013) . also cells at different time points post infection can be analyzed to understand how viruses alter host cell protein expression (weekes et al., 2014) . for instance, berard et al. performed a temporal quantification of cells infected with herpes simplex virus type 1 (hsv1) and by this showed how hsv1 modulates the metabolic function of the host cell (berard et al., 2015) . beside this, silac followed by lc-ms/ms can also be used to identify cellular targets of viral enzymes, as it has been done for the identification of the hcv protease ns3-4a targets: by comparing whole proteomes from hepatoma cells with and without the ns3-4a protease, morikawa et al. discovered that 'probable glutathione peroxidase 8' is cleaved by the viral protease (morikawa et al., 2014) . silac is also suitable to analyze how viruses alter the proteome of distinct cellular compartments. for instance, xie et al. defined the modification of lipid raft composition in hbv infection (xie et al., 2012) . in combination with ap of a protein of interest, silac followed by lc-ms/ms can also discover protein-interactions and dynamic changes in interaction complexes (reviewed e.g., in (gingras et al., 2007; trinkle-mulcahy, 2012) ). high-throughput ap-ms has led to important findings regarding virus entry, for instance the identification of receptors or receptor-complexes. a successful example is the ap-ms analysis of the hcv receptor cd81, which revealed hras as a cd81-binding partner and signaling molecule needed for hcv entry (zona et al., 2013) . a clear benefit of silac is its high reproducibility, robustness and relatively easy execution. silac is applicable to most cell culture cells and sample conditions. even silac mice have been developed in the past for ex vivo proteomics analyses (zanivan et al., 2012) . box 2: definitions and abbreviations in mass spectrometry based proteomics. proteome: entity of proteins expressed at a given cellular state or in a given tissue. the term was coined in analogy to 'genome' in 1996. ms: mass spectrometry. esi: electrospray ionization; ionization technique based on spraying of a sample solution into a strong electric field. maldi: matrix-assisted laser desorption/ionization; ionization technique based on mixing of samples with a matrix, which absorbs ultraviolet light and converts it to heat energy leading to sample vaporization. bottom up proteomics: identification of proteins based on ms peptide fingerprints after digestion of a protein into peptides using a sequence-specific enzyme such as trypsin. top down proteomics: ms analysis of entire proteins. shotgun proteomics: bottom-up proteomics technique to identify proteins in a complex mixture. proteins are digested to peptides, peptides separated by liquid chromatography and then identified by tandem ms. quantitative proteomics: shotgun proteomics with the additional dimension of measuring the quantity of a protein in a complex sample. relative quantities in two or more samples are determined either by silac or lfq (see below). absolute quantification can be achieved using spike in standards or a proteomics ruler (see paragraph on protein complex stoichiometry). interaction proteomics: unbiased identification of protein-protein interactions by affinity enrichment followed by ms. silac: stable isotope labeling by amino acids in cell culture; first quantitative method for the comparison of cellular protein amounts in two to three different cellular states. silac is based on the isotope labeling of cellular proteins by feeding cells with heavy isotope amino acids (e.g. n-15 and c-13 labeled arginine and lysine). label-free quantification (lfq): quantitative proteomics technique based on either signal intensity measurement or spectral counting followed by computational matching of peptides from multiple samples and signal normalization. ppi: protein-protein interactions. ptm: posttranslational modification. common protein modifications include among others phosphorylation, lipidation, glycosylation, methylation, acetylation, ubiquitinylation and sumoylation. ap-ms: affinity purification of proteins using antibodies against endogenous epitopes or epitope tags followed by mass spectrometric identification or quantification of the bound proteins. ae-ms: affinity enrichment of proteins using fast single step methods, which preserve transient and low affinity interactions, followed by mass spectrometric identification or quantification of bound proteins. caveats of silac are the limited sample number of maximum three and the additional labeling step requiring culturing of cells for two to three weeks prior to analysis. thus some primary human cells, which rapidly dedifferentiate ex vivo, are not suited for silac. similarly, clinical samples cannot be labeled and are thus not amenable to silac analysis. increasingly popular alternatives to silac are lfq methods. these can determine the relative or absolute abundance of a protein in complex samples. the first semi-quantitative labelfree method developed, which has since then been continuously refined, is based on the measurement of ms/ms peptide intensities (bondarenko et al., 2002) . to this end, peak areas of all identified peptides from one protein are summed up and normalized to one or several proteins with constant concentration or even to all iden-tified proteins in the sample. bondarenko et al. showed that there is a linear correlation of the abundance of a protein and its total reconstructed peak area. an alternative semi-quantitative method used is spectral sampling (liu et al., 2004) . it was shown that highly abundant proteins are sampled more frequently by lc-ms/ms and that the spectral copy number obtained increases linearly with the concentration of a protein. accordingly, the number of ms/ms spectra gained for one protein can be counted and compared between different samples measured using the same experimental conditions. several computational approaches to predict specific protein interactions in label-free datasets have been developed, among them significance analysis of interactome (saint) and mass spectrometry interaction statistics (mist) (choi et al., 2011; jager et al., 2012) . these tools compute interaction probabilities based on spectral counts of all interactions that a prey-bait pair is involved in or based on abundance, reproducibility and specificity of a preybait pair, respectively. recent developments in ms technology and analysis algorithms now allow highly sensitive, efficient and robust intensity-based label-free protein quantification (reviewed e.g., in (nahnsen et al., 2013) ). for this, samples are processed in parallel and not mixed, as in silac. thus, an unlimited number of samples can be compared. normalization is achieved post lc-ms/ms analysis by comparing all peptide signals from all ms runs and assuming that most protein signals remain unaltered in the different sample conditions. thus, no external standards are needed . however, acquisition of accurate datasets requires the analysis of at least three, typically four, biological replicates (fig. 3b ). ms combined with lfq algorithms is suitable for virological research and has been used e.g., to show that subsets of dendritic cells (dcs) differ in their ability to sense single stranded rna (ssrna) viruses (luber et al., 2010) . briefly, luber at al. compared the whole cell proteome of dc subsets by high-resolution ms combined with lfq. they found, that dc subsets differently express proteins involved in pattern recognition pathways and that this correlates with their ability to sense ssrna viruses, such as sendai virus and influenza a virus. label-free methods also allow the analysis of protein complexes. importantly, extensive purification of complexes prior to lc-ms/ms is no longer necessary for lfq. instead one ae step is sufficient for precise complex profiling, when controls are designed appropriately . false positive interaction partners, which are proteins unspecifically binding to the affinity resin, are computationally eliminated by comparing datasets to those of a control ae sample from cells without bait. fig. 3b outlines the here described lfq workflow. when compared to label-based methods, lfq techniques have several advantages: nearly all kinds of samples can be measured, including clinical material and primary cells; sample number is unlimited; no special sample processing steps are needed; slight differences in sample preparation and measurement between the different samples are tolerated. both, labeling and label-free quantification methods identify and quantify proteins with high confidence and lead to highly accurate results. each step of the proteomics workflow can be quality controlled, e.g., by immunoblotting, computational analysis of peptide intensity distributions, and clustering analysis of biological replicate datasets (fig. 3c ). to study virus entry both silac and lfq are valuable tools. lfq is ideally suited to study steady state virus receptor interactions. to this end, virus receptor-complexes are purified from cells expressing the receptor and cells lacking the receptor. comparison of both datasets then allows to define receptor interacting proteins in a given cell line. silac on the other hand can sensitively distinguish low degree quantitative changes, as they occur when virus receptor complexes change upon virus binding. briefly, heavy labeled cells are incubated with virus and light cells are left untreated (forward label); and vice versa (reverse label). after sample mixing, ap of receptor complexes and lc-ms analysis, changes in interaction strength of each protein in the receptor complex can be determined. importantly, the untreated control should ideally contain all extracellular components as the virus sample, i.e. the ideal control is incubation with virus, which had been neutralized by blocking antibodies, or incubation with a "mock" virus preparation if such antibodies are not available. using silac, we recently identified transient, virus-triggered interactions of the hcv receptor cd81. among the 26 dynamic cd81 interactors we identified serum response factor binding protein 1 as a host factor aiding hcv entry (gerold et al., 2015) . of note, similar studies will soon be possible with advanced lfq methods. fig. 3 illustrates how quantitative proteomics can identify steady state and dynamic virus-triggered receptor interactions. in summary, silac and lfq are powerful tools for analyzing virus-host interactions and global proteomic changes upon virus infection. due to the recent improvements in high resolution ms and label-free analysis tools, the highly accurate, cost effective and broadly applicable lfq methods will soon become the method of choice for profiling proteins, their interactions and whole proteomes. steady state interactions of cellular proteins, which preexist before virus attachment, can be crucial for virus infection. this has been shown in the past mainly by biochemical assays. for instance, the interaction of moesin and cd46, which is needed for measles virus entry, was demonstrated by ap followed by immunoblot analysis (schneider-schaulies et al., 1995) . since msbased proteomics became the method of choice to analyze protein interactions, several studies used mostly non-quantitative proteomic techniques to characterize interactions of virus receptors. for example chakraborty et al. (2012) analyzed immuoprecipitates of the kaposiís sarcoma-associated herpesvirus (kshv) receptor and lipid raft protein integrin ␣3␤1 via sds-page and ms of selected gel bands. they identified the association of integrin ␣3␤1 with several proteins, including epha2. the identified associations were enriched in lipid rafts of kshv infected human dermal microvascular endothelial cells, compared to uninfected cells. with virological assays chakraborty et al. then showed that this enrichment is important for effective kshv infection, especially for macropinocytosis and trafficking of the virus. also quantitative ms-based techniques in combination with traditional virological assays can be used to decipher the role of steady state protein interactions in different steps of the viral life cycle. an excellent example is the identification of a ppi network consisting of the hcv receptors cd81 and claudin1 and the membrane proteins hras, rap2b and itgb1, which are required for efficient entry of the virus into hepatocytes (zona et al., 2013) . the authors identified the hcv receptor network by silac of human hepatoma cells with and without cd81 receptor expression. the role of the identified proteins in the hcv entry process was then studied by virological assays in conjunction with rna silencing and small molecule inhibitors. using a similar workflow, host restriction factors can likewise be discovered. one example is the association of ewi-2 and ␣-actinin-4 in t-cells. gordon-alonso et al. (2012) identified the complex by analyzing ewi-2 co-precipitated proteins via non-quantitative ms, and later on showed that the complex negatively regulates hiv 1 entry. similarly, the cleavage product of ewi-2, ewi-2wint, has been shown to interact with the hcv receptor cd81 and by this inhibit hcv entry (rocha-perugini et al., 2008) . as ewi-2wint is not expressed in the target cells of hcv, which are hepatic cells, this interaction could contribute to the viral cell tropism. in summary, the analysis of steady state virus receptor interactions can reveal protein complexes critical for virus uptake and in some cases help understand host and tissue tropism. upon binding of viruses to their receptors, additional secondary ppis are triggered, most of which lead to virus entry. these interactions have at least five distinct functions depending on the virus entry strategy. first, binding of the multivalent virus particle to the cell surface can induce receptor clustering, as has been shown for phleboviruses and influenza a viruses (eierhoff et al., 2010; lozach et al., 2011) . while it is unclear whether influenza a virus triggered clustering of sialylated receptor tyrosine kinases like egfr and c-met is required for endocytic virus uptake, phlebovirus induced clustering of dc-sign seems to trigger signaling needed for uptake. other viruses, which have been suggested to induce receptor clustering, include coxsackievirus b and lassa virus (coyne and bergelson, 2006; moraz et al., 2013) . hiv-1 entry also relies on clustering of its cd4 and cxcr4 receptors on the plasma membrane. specifically, the hiv gp120 glycoprotein signals for recruitment of actin adaptor proteins filamin and debrin, which regulate actin rich cap formation at the virus entry site (gordon-alonso et al., 2013; jimenez-baranda et al., 2007) . notably, actin depolymerization is induced at later stages of the hiv entry process, highlighting that actin remodeling during virus entry is highly dynamic and tightly regulated. while proteomes of detergent resistant membrane microdomains have been determined in naïve and virus replicating cells resulting in the identification of more than 200 proteins (foster et al., 2003; xie et al., 2012) , few quantitative proteomics dataset are currently available for receptor platforms upon virus binding to the cell surface. for the hcv receptor cd81, we recently described the virus triggered receptor interactome and identified cytoskeleton regulators, cell junction proteins and a clathrin adaptor protein (gerold et al., 2015) . second, viruses can induce signaling leading to actin cortex disassembly. this is particularly important for viruses, which directly penetrate the cell at the plasma membrane and need to overcome the physical barrier of the actin cortex. viruses entering by endocytosis might similarly require local actin cortex disassembly at the site of the incoming vesicle. although the concept that the cortex can prevent or delay transit of viruses has already been suggested by colleagues in 1997 (marsh and bron, 1997) , only few studies address virus induced actin cortex remodeling. a well-studied example is hiv, which upon env binding to the cxcr4 coreceptor induces galpha i signaling and subsequent cofilin activation leading to actin depolymerization thereby facilitating nuclear translocation of hiv particles (yoder et al., 2008) . of note, not only actin cortex but also actin stress fiber disruption can aid virus entry. for instance cytomegaloviruses binding to egfr and integrin alphavbeta3, induces cofilin dependent actin stress fiber disruption thereby facilitating nuclear translocation of incoming virions (wang et al., 2005) . third, viruses, which use multiple receptors for entry, can induce their lateral translocation along the plasma membrane towards the site of endocytic uptake. a prominent example of such surfing is coxsackievirus b, which after binding to the apical decay-accelerating factor (daf) on epithelial cells induces abl kinase signaling and subsequent rac-dependent actin reorganization. this leads to the lateral translocation of the coxsackievirus b-daf complex to tight junctions, where the secondary receptor coxsackievirus-adenovirus receptor (car) is localized. after car engagement coxsackievirus b is endocytosed. this endocytosis is also triggered by ppis as initial daf binding additionally induces fyn kinase, which phosphorylates caveolin thereby facilitating uptake (coyne and bergelson, 2006) . hcv seems to use a similar multistep strategy to enter after initial binding to the basolateral side of hepatocytes, where the receptors sr-bi and cd81 reside. through a yet insufficiently described mechanism hcv also triggers lateral translocation of the cd81-virus complex to tight junctions, where the two additional entry factors cldn1 and ocln reside and endocytosis occurs (brazzoli et al., 2008) . receptor tyrosine kinases like egfr are thought to provide the trigger for lateral membrane translocation through hras signaling (zona et al., 2013) . moreover, surfing of hcv and retroviruses on filopodia has been observed and is governed by ppi with the actin cell cortex (coller et al., 2009; lehmann et al., 2005) . fourth, viruses, which enter through endocytic routes, can induce endocytic uptake mechanisms including clathrin-mediated, caveolin-dependent, macropinocytic or alternative uptake routes like the clathrin-independent carriers in parvovirus entry (nonnenmacher and weber, 2011) . on the one hand, some viruses like human rhinovirus 2 and phleboviruses hijack endogenous constitutive receptor recycling pathways. after endocytosis phleboviruses, which hijack dc-sign for cell entry into dcs, however dissociate from the receptor in the early endosome thereby avoiding antigen processing and presentation (lozach et al., 2011) . on the other hand, viruses can actively trigger their endocytosis like influenza a virus. after interaction of influenza virions with sialylated membrane receptors, the clathrin adaptor epsin-1 is recruited in a ubiquitin-dependent manner towards the receptor complex and induces clathrin-coated pits (chen and zhuang, 2008) . pi3k activation was independently reported to be required for influenza uptake, thus more than one signaling pathway might coordinate uptake of a virus. this is in line with reports, showing that influenza virus can utilize more than one endocytosis pathway to gain access to host cells (rust et al., 2004; de vries et al., 2011) . larger viruses like poxviruses enter by macropinocytosis and actively induce this uptake pathway through rac-1 and p21-activated kinase 1 (pak-1) dependent actin remodeling (mercer and helenius, 2008) . formation of plasma membrane protrusions is however not specific to poxviruses, but occurs also during entry of herpes viruses, papillomaviruses and flaviviruses (clement et al., 2006; coller et al., 2009; smith et al., 2008) . lastly, several viruses need to interact with host enzymes to trigger their uptake. a prominent example is ebola virus, which requires proteolytic processing of its envelope glycoprotein gp1 by cathepsin b and l to render it fusion competent (chandran et al., 2005) . similarly coronavirus relies on cathepsin cleavage of its spike protein to permit fusion (simmons et al., 2005) . in addition to lysosomal enzymes, cell surface proteases can prime vaps for fusion, like shown for some influenza a virus strains and sars coronavirus (bertram et al., 2010; bertram et al., 2011) . host protein disulfide isomerases similarly regulate entry by catalyzing disulfide shuffling in viral envelope proteins, as shown for dengue virus, hiv-1 and newcastle disease virus (jain et al., 2008; ryser et al., 1994; stantchev et al., 2012; vega-almeida et al., 2013) . of note, high resolution proteomics can not only reveal transient interactions of vap with enzymes, but also has the potential to identify proteolytic cleavage sites and redox modifications in vaps. it is conceivable that virus induced protein interactions during entry not only serve to promote the virus uptake pathway, but can also help cloak viruses and lead to immune evasion. for instance, the hiv capsid recruits two cofactors, cleavage and polyadenylation specificity factor subunit 6 (cpsf6) and cyclophilins (nup358 and cypa), thereby preventing antiviral ifn responses (rasaiyaah et al., 2013) . more complex viruses can further express viral gene products on the infected cell surface, which then bind receptors on bystander cells and modulate immunity. for instance, hcmv protein pul11 binds to cd45 on t cells thereby reducing t cell proliferation (gabaev et al., 2011) . while we here focus on the role of virus -host ppi in entry, it should be noted that quantitative virus entry proteomics at the same time has the potential to reveal immunomodulatory mechanisms. similarly, ppis of viral tegument and capsid proteins with host proteins occurring after membrane fusion are amenable to quantitative proteomics. we refer the reader elsewhere for the detailed description of post-fusion ppis coordinating nuclear trafficking (dohner et al., 2005; yarbrough et al., 2014) . comprehensive proteomics analyses of virus induced ppis leading to actin remodeling and endocytic uptake of virions are lacking to date. however, several studies underline the value of proteomics to analyze stimulus specific interactions in cellular signaling pathways. for instance, the ligand-induced changes of the two wnt signaling pathway members adenomatous polyposis coli protein (apc) and axin-1 identified 28 and 18 dynamic interaction partners, respectively (hilger and mann, 2012) . for other virus life cycle steps, like e.g., cmv particle assembly, interaction proteomics uncovered important mechanistic insights (moorman et al., 2010) . further, interaction proteomics of viral immunomodulatory proteins revealed virus induced alterations in human protein interaction networks and underlying viral perturbation strategies (pichlmair et al., 2012) . similar studies hold the promise of uncovering common and specific viral strategies to alter and exploit host cell functions during cell entry. post-translational modifications (ptms) play two major roles during virus entry into a host cell. first, virus receptor ptms can be critical for the localization to specific membrane microdomains. for instance, palmitoylation of the hcv receptor cd81 and its associated proteins ewi-2 and ewi-2wint regulate their interaction with each other and the localization to cholesterol rich membrane domains thereby influencing hcv entry (montpellier et al., 2011; zhu et al., 2012) . second, viruses rely on cell signaling for their uptake and such cellular signals are often transmitted via ptms like reversible protein phosphorylation. a widespread example common to viruses of diverse families including herpesviruses, papillomaviruses and flaviviruses is the triggering of receptor tyrosine kinase signaling (hahn et al., 2012; lupberger et al., 2011; surviladze et al., 2013; zheng et al., 2014) . in most cases signaling through growth factor receptors regulates actin dynamics required for virus trafficking into the cell. some respiratory viruses however use egfr signaling to suppress antiviral signaling in the airway epithelium (ueki et al., 2013) . other phosphorylation signals that coordinate virus entry are the src kinase pathway in hcmv penetration (nogalski et al., 2013) and the lassa virus induced phosphorylation of its receptor dystroglycan (moraz et al., 2013) . certain kinase activities can be a hallmark of a specific uptake pathway, as for instance pak-1 for macropinocytic virus entry (amstutz et al., 2008) . in particular for large dna viruses not only host proteins are phosphorylated early during virus infection, but also viral gene products. for instance, herpes simplex virus type 1 (hsv-1) protein pul46 is heavily phosphorylated six hours post infection and interacts with several viral and host kinases as determined by quantitative interaction proteomics (lin et al., 2013) . apart from protein phosphorylation, other ptms like ubiquitination are thought to regulate virus uptake, but are poorly characterized. ubiquitination of viral proteins seems to induce viral uncoating in at least two instances. influenza a virus m1 protein is ubiquitinated by the e3 ligase itch and this is a prerequisite for endosomal escape (su et al., 2013) . vaccinia virus on the other hand uses ubiquitinated incoming viral core proteins to trigger proteasomal degradation of its protein coat . all aforementioned studies, except for the hsv-1 study, demonstrate ptms of receptors, downstream signaling molecules or viral proteins by immunoblotting, thus requiring prior knowledge of the signaling pathways. in contrast, ms-based identification and quantification of ptms upon virus binding to a host cell is completely unbiased and thus broadly applicable. another advantage over previous immunoblot based methods is the global and quantitative measurement of ptms by ms in conjunction with the ability to pinpoint the modified amino acid. clearly, high read depth is required for ptm proteomics as the search space for the peptides explodes when taking into account all possible peptide ptms and all of their combinations. still successful large-scale measurements of protein phosphorylation (ficarro et al., 2002; olsen et al., 2006) , n-glycosylation (kaji et al., 2007) , lysine methylation (ong et al., 2004) or acetylation (kim et al., 2006) , ubiquitination (peng et al., 2003) and sumoylation (andersen et al., 2009; impens et al., 2014) have been achieved. to overcome the caveat of low abundance of modified peptides, enrichment methods for instance using anti-phosphotyrosine antibodies are available. importantly, when measuring ptms in interaction proteomics samples, which are of much lower complexity than whole cell lysates, ptm identification is facilitated. for stimuli other than viruses, ptm proteomics has yielded comprehensive datasets. for instance, phosphoproteomics of epidermal growth factor (egf) stimulated hela cells unraveled 6600 phosphorylation sites on 2244 proteins, with 14% of the interactions showing an at least 2-fold modulation by egf. the majority of proteins further showed multiple ptm sites with different kinetics highlighting how one protein can integrate numerous signals (olsen et al., 2006) . this and other studies underline the complexity of receptor signaling. how extensively virus-receptor interactions alter the phosphoproteome of a host cell, was demonstrated in a first global description for hiv-1 entry (wojcechowskyj et al., 2013) . a total of 239 phosphorylation sites in 175 proteins changed just one minute after hiv-1 binding and the study disclosed several previously unknown hiv-1 entry factors. it is conceived that most viruses will trigger ptm-dependent signaling during their entry process and this is either critical for cell penetration or for modulating cellular immunity. recent developments in ptm proteomics now allow the global mapping of virus induced changes in host proteome ptms and can thereby disclose signaling pathways active during cell entry. the stoichiometric composition of receptor complexes is critical for membrane microdomain localization, signal coordination and ligand binding. interaction proteomics can provide information on the relative abundances of a protein in a complex. typically prey protein abundance in the pull down is corrected for the prey abundance in a control pull down and subsequently normalized to the bait protein. this approach was used to estimate the stoichiometry of the yap and taz protein complexes in the human hippo pathway (kohli et al., 2014) . beyond complex stoichiometry measurements, shotgun proteomics now allows to estimate which fraction of a protein is sequestered into a protein complex. mann and colleagues recently developed a method to estimate protein copy numbers in labelfree proteomes from whole cells. this "proteomic ruler" technique determines the absolute abundance of a protein in a cell, based on the fact that the total amount of histones, which is defined by the cell ploidity and genome size, can be used for normalization of any detected protein in a proteomic dataset of sufficient depth. the minimum depth of a whole cell proteome to guarantee reliable histone based scaling is 12,000 peptides. this scaling method is similarly accurate as previous spiked-in protein epitope signature tags (prests) based methods, which required cell counting and cell labeling (wisniewski et al., 2014) . thus, whole cell proteomic datasets can provide information on total protein copy numbers in a given cell. based on this knowledge the receptor complex stoichiometry can be estimated by comparing relative abundances of co-purified proteins in an ae proteomics dataset. success-ful receptor complex stoichiometry calculation has recently been demonstrated for subcellular fractionation proteomics (borner et al., 2014) . thus, shotgun proteomics goes beyond pure identification of virus entry receptor complexes, but is also suitable to estimate complex stoichiometry and degree of sequestration of a protein into a complex. the era of systems biology and proteomics puts the virologist into the unique position of drawing a comprehensive picture of all molecular interactions during virus infection of a host cell. large scale genome and transcriptome-based methods for the characterization of virus-host interactions, including mrna microarrays, rna interference and cdna library screens, led to the discovery of a multitude of host factors. we currently lack information on the physical interconnection of these host factors and thus it is difficult to identify critical players in virus entry pathways. open access databases on ppis like the search tool for the retrieval of interacting genes/proteins (string) integrate public databases like intact and reactome and can facilitate the mapping of host factors into known networks (croft et al., 2014; orchard et al., 2014; szklarczyk et al., 2015) . however, many virus host factors do not map to previously described networks. also, virus induced changes of cellular ppis are not deposited in current databases. some repositories, however, curate host-pathogen interactions, e.g., virhostnet and the hivcentric gpsprot site (fahey et al., 2011; guirimand et al., 2015) . importantly, viruses do not play according to the rule, i.e., they may induce ppi that do not normally exist in non-infected cells. for instance, they can change the subcellular localization of a host factor and thereby connect pathways, which in an uninfected cell operate independently. thus, existing databases on host ppi may have only limited predictive value for infected cells, in particular at steps of the life cycle that heavily depend on the cell's machinery like virus entry. again this calls for repositories that either specialize in or integrate ppi from infected cells. the above described technological advances now enable rapid acquisition of large sets of protein interaction data from one-step aes. thus, proteomics is now high throughput compatible and amenable to systems biology studies (hosp et al., 2015) providing the unprecedented chance to globally define protein-protein networks during virus infection. critical for high confidence network generation and data deposition is the analysis of a large number of biological replicates, experimental conditions and controls including immunoprecipitates from bait deficient cells. furthermore, several laboratories performed control pull-downs under various experimental conditions and established a "contaminant repository for ap" (crapome) (mellacheruvu et al., 2013) . however, background binders can critically differ between experimental setups and thus a universal crapome is problematic, making internal controls more reliable . clearly, newly acquired datasets on virus infection interactomes should be integrated into established host protein-protein networks. currently, the above mentioned virhostnet is probably the most comprehensive database for virus-host interactions, which directly links interaction partners to uniprot (guirimand et al., 2015) . as many pathogens share mechanisms for exploiting host cells, a central database for host-pathogen interactions, multiplexing datasets for viruses, bacteria, fungi and parasites would allow the rapid identification of critical nodes and putative broad spectrum drug targets. clearly, publically available databases should reliably catalogue virus-host interactions and make these easily accessible to the virologist community through user-friendly web interfaces. ultimately, genomics and proteomics techniques provide essential complementary datasets, which can be integrated to reveal a box 3: network term definitions. node: component of a network, e.g., a gene or a protein. hub: node, which is highly connected and therefore a point of control of a specific subnetwork. bottleneck: node, which connects subnetworks and thus critically restricts information flow. edge: connection of two nodes in a network, e.g., representing a physical interaction of two proteins. most complete picture of molecular events during virus infection. functional genomics screens can weigh the importance of a host factor in an interaction network and reveal redundant pathways. annotation clusters can highlight the enrichment of certain cellular processes, molecular functions or protein domains . novel open access bioinformatics solutions building on available resources like string and david can help harmonizing available datasets. scripting and visualization environments like cytoscape, r and bioconductor, which were previously developed for transcriptomics and genomics data integration, are also suitable for pathway mapping of proteomics data (cline et al., 2007; gentleman et al., 2004) . proteomics-specific and publically available programs like perseus provide an additional tool for data analysis and integration (cox and mann, 2012) . meta-analysis of omics datasets together with sharing of data in a timely manner will be critical to successfully model the complexity of virus-host interactions in the future (law et al., 2013) . such networks will then allow the identification of essential nodes, bottlenecks and hubs for selection of optimal drug targets. each node in a ppi network represents a protein, while the edges indicate interactions. bottlenecks are nodes in a network, which critically connect subnetworks and thus restrict information flow, like e.g. central kinases in a signaling pathway. hubs instead are highly interlinked and therefore central points of control, but typically only control one branch of a signaling pathway. while bottlenecks are less attractive drug targets as their inhibition would shut down complete endogenous pathways, hub inhibition can lead to an efficient block of infection (box 3). furthermore, drug targeting of pathogen relevant ppis using small molecule drugs or peptidomimetics seems an attractive strategy to combat infection (de chassey et al., 2012a; law et al., 2013) . in particular, targeting of interfaces between viral and host proteins, holds the promise of minimal side effects. but even host ppis, which are triggered or exploited during virus infection, represent attractive targets. in contrast to direct acting antivirals, which often lead to rapid emergence of viral resistance mutations, host proteins and thus their interactions represent a high evolutionary barrier (gerold and pietschmann, 2014; von hahn et al., 2011) . in particular, host proteases and kinases are attractive drug targets and both protein classes can be identified by advanced interaction proteomics and ptm proteomics, respectively. taken together, increasing knowledge on interactomes during virus infection and the compilation of interactome datasets can spur the development of novel antiviral therapeutics. the experimental validation of central network hubs, also as potential drug targets, is discussed below. protein interaction network analysis allows in the next step to hypothesize, which biological pathway is critical for virus uptake and whether certain proteins are central hubs in the network. the hypothesis can then be tested by disrupting the function of one of the interaction partners and studying the resulting phenotype (fig. 3d) . to this end, blocking antibodies have been used, but the method is limited to proteins with accessible extracellular domains. alternatively, small molecule inhibitors, if available, can block the protein of interest. due to their microreversibility and rapid mode of action, such molecular probes have in the past proven indispensable to dissect the kinetics of virus-host cell interplay. the most commonly used strategy to address a protein's function was in the past the disruption or reduction of its expression by rna interference (rnai) (elbashir et al., 2001; fire et al., 1998) . here, short rna molecules sequence-specifically target complementary mrnas and induce their degradation. rnai has been used to address the role of protein interactions identified by ms in virus entry (zona et al., 2013) . however, rnai approaches can have offtarget effects (reviewed e.g., in (mohr et al., 2014) ) and the slow kinetics of action of two to three days can lead to compensatory adaptation of cells. this might be the reason why large-scale rnai screens performed poorly in studies of virus entry, which operates at a minute timescale (de chassey et al., 2012b) . since 2012, targeted genome editing became possible making use of a bacterial adaptive antiviral immune mechanism. clustered regularly interspaced short palindromic repeats (crispr) in combination with the rna guided dna endonuclease crispr associated protein 9 (cas9) is able to induce specific dna double strand breaks (gasiunas et al., 2012; jinek et al., 2012) . therefore, the system can be used to generate site-specific modifications in genomes, e.g. to knock-out specific proteins (reviewed e.g. in (doudna and charpentier, 2014) ). the crispr-cas9 technology has already been applied to analyze the function of different proteins in the viral entry process. for example crispr-cas9 knock-out of pdzd8 verified its role in hiv-1 and murine leukemia virus infections (zhang and sodroski, 2015) . in a different approach, a genome wide lentiviral crispr-cas9 library was developed to screen for cellular factors essential for entry and cell-to-cell transmission of hcv (ren et al., 2015) . in this screen, the known hcv entry factors cd81, claudin-1 and occludin were identified. while knock-out strategies are limited to host factors with non-essential endogenous function and similar to rnai compensatory effects can occur, a genetically clean knock-out system holds the promise of unambiguously defining essential virus entry factors. future studies will show whether knockout screens have higher reproducibility than rnai screens and are suitable for virus entry research. both techniques, rnai and crispr-cas9, display specific advantages and disadvantages, as reviewed in (boettcher and mcmanus, 2015) . therefore, the appropriate technique, including small molecule inhibitors, dominant negative variants and blocking antibodies, has to be chosen carefully depending on the experimental setup and the target genes. after hypothesis testing, drug targets can be defined and searches for compounds interfering with critical network nodes initiated. alternatively, novel baits can be selected and fueled into a new round of ae-ms, thereby widening the interaction network. such iterative processes will ultimately complete the picture of ppi during virus invasion (fig. 4) . proteomics is the only method to directly characterize the biologically active entity of most biological processes, the proteins. in an ae setup, it provides information on primary protein interactions and higher order complexes depending on the stringency of purification. it can further yield the order of binding as well as interaction interfaces, when crosslinkers are used (chen et al., 2010; leitner et al., 2010) . as detailed above, interaction proteomics can reveal receptor complex stoichiometry as well as ptm of the analyzed proteins. with increasing instrument sensitivity not only stable protein interactions, but also transient and virus-induced interactions are accessible. protein networks between two cellular states, e.g., with and without receptor bound virus, can be compared in a quantitative manner. in the past, detection of subtle differences in protein abundance required isotope labeling, but label-free methods are nowadays sensitive enough for most applications. a clear advantage over rna interference or image-based screens is that the host cell is in its endogenous state without the requirement for labeling of proteins or change of their expression level. recent developments moreover make proteomics high throughput screen compatible (hosp et al., 2015) , so that virus entry processes could be studied in a large panel of cell lines as well as in a time resolved manner. as every method, proteomics has its own shortcomings. certain proteins are detected as common contaminants and these false positives have to be eliminated using proper controls and by comparison to contaminant databases. when searching for interactions of endogenous proteins, antibodies of high specificity are required. in the future, crispr knockin methods might overcome this caveat and allow expression of affinity tagged proteins from their natural locus. alternatively, controlled expression systems, although not under endogenous gene control, can help express tagged proteins at endogenous levels. clearly, interaction proteomics just highlights one biological function of a given protein, i.e., its interaction network. thus the method should be considered as complimentary tool in the virologist's toolbox. to address whether an interaction is critical for virus infection, follow up analysis using established (rnai, inhibitors, dominant negative mutants) or newly developed techniques (crispr) is needed. nonetheless, we believe that interaction proteomics will prove essential to model molecular networks during virus infection, assign critical nodes, generate and test hypotheses on the molecular function of the proteins in the network and thereby reveal essential host factors, which can be targeted by antiviral drugs. genetic screens identified numerous virus receptors, but their actual binding to the respective virus has often not been formerly proven. quantitative interaction proteomics approaches hold the promise of closing this gap of knowledge. due to technological and computational developments in the past decade proteomics is now on par with genomics and allows high throughout comprehensive analyses of complex samples. recently, a 96-well compatible and streamlined interaction proteomics protocol has been developed (hosp et al., 2015) . given the fact that for many viruses the target cells are either not known or are of multiple host and tissue origin, the parallel analysis of many biological samples will in the future allow proteomics screening approaches without prior knowledge of receptor expression or susceptibility. a second consequence of the vast increase in ms sensitivity is that patient material and material from animal experiments, which is typically limited, can be analyzed in depth. transcriptomics data only accounts for a subset of pathogen induced changes in a host as it lacks information on ptms and ppi. proteomics of ex vivo samples thus holds the promise of providing a more complete picture of the virus host interplay in an infected organism. it will be interesting to demonstrate to what extend the changes in host and virus proteomes observed thus far in cell culture models, reflect the in vivo situation. notably, proteomics can not only elucidate protein networks during virus infection, but also serve to identify diagnostic and prognostic markers of virus infection and disease progression (mancone et al., 2013; rhea et al., 2010) . it also allows the identification of targets of chemical compound libraries by interaction proteomics (ong et al., 2009) . ms-based proteomics is thus valuable for virologists with a molecular biology, cell biology, and clinical focus. we believe that increasing availability of proteomics hardware, protocols and analysis software is currently spurring a shift from the largely genomics dominated field of virus infection research towards proteomic characterization of cellular perturbations by viruses. ten thousand interactions for the molecular biologist viral apoptotic mimicry subversion of ctbp1-controlled macropinocytosis by human adenovirus serotype 3 identification of sumo target proteins by quantitative proteomics quantification of the host response proteome after herpes simplex virus type 1 infection tmprss2 and tmprss4 facilitate trypsin-independent spread of influenza virus in caco-2 cells cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease investigating macromolecular complexes using top-down mass spectrometry choosing the right tool for the job: rnai, talen, or crispr identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues cd81 is a central regulator of cellular events required for hepatitis c virus infection of human hepatocytes identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus ebola virus entry requires the cholesterol transporter niemann-pick c1 kaposi's sarcoma-associated herpesvirus interacts with ephrina2 receptor to amplify signaling essential for productive infection endosomal proteolysis of the ebola virus glycoprotein is necessary for infection epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus architecture of the rna polymerase ii-tfiif complex revealed by cross-linking and mass spectrometry saint: probabilistic scoring of affinity purification-mass spectrometry data a novel role for phagocytosis-like uptake in herpes simplex virus entry integration of biological networks and gene expression data using cytoscape rna interference and single particle tracking analysis of hepatitis c virus endocytosis small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed maxlfq 1d and 2d annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions the reactome pathway knowledgebase heat shock protein 70 on neuro2a cells is a putative receptor for japanese encephalitis virus how viruses hijack cell regulation new horizons for antiviral drug discovery from virus-host protein interaction networks genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway viral stop-and-go along microtubules: taking a ride with dynein and kinesins maraviroc (uk-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor ccr5 with broad-spectrum anti-human immunodeficiency virus type 1 activity genome editing. the new frontier of genome engineering with crispr-cas9 the epidermal growth factor receptor (egfr) promotes uptake of influenza a viruses (iav) into host cells duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells gps-prot: a web-based visualization platform for integrating host-pathogen interaction data phosphoproteome analysis by mass spectrometry and its application to saccharomyces cerevisiae epstein-barr virus receptor of human b lymphocytes is the c3d receptor cr2 potent and specific genetic interference by double-stranded rna in caenorhabditis elegans unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors direct identification of ligand-receptor interactions on living cells and tissues the human cytomegalovirus ul11 protein interacts with the receptor tyrosine phosphatase cd45, resulting in functional paralysis of t cells cas9-crrna ribonucleoprotein complex mediates specific dna cleavage for adaptive immunity in bacteria bioconductor: open software development for computational biology and bioinformatics quantitative proteomics identifies serum response factor binding protein 1 as a host factor for hepatitis c virus entry the hcv life cycle: in vitro tissue culture systems and therapeutic targets analysis of protein complexes using mass spectrometry actin-binding protein drebrin regulates hiv-1-triggered actin polymerization and viral infection ewi-2 association with alpha-actinin regulates t cell immune synapses and hiv viral infection the cell biology of receptor-mediated virus entry virhostnet 2.0: surfing on the web of virus/host molecular interactions data use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions the ephrin receptor tyrosine kinase a2 is a cellular receptor for kaposi's sarcoma-associated herpesvirus triple silac to determine stimulus specific interactions in the wnt pathway a double-barrel lc-ms/ms system to quantify 96 interactomes per day hepatitis c virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins mapping of sumo sites and analysis of sumoylation changes induced by external stimuli global landscape of hiv-human protein complexes overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine filamin-a regulates actin-dependent clustering of hiv receptors a programmable dual-rna-guided dna endonuclease in adaptive bacterial immunity proteomics reveals n-linked glycoprotein diversity in caenorhabditis elegans and suggests an atypical translocation mechanism for integral membrane proteins accurate protein complex retrieval by affinity enrichment mass spectrometry (ae-ms) rather than affinity purification mass spectrometry (ap-ms) substrate and functional diversity of lysine acetylation revealed by a proteomics survey label-free quantitative proteomic analysis of the yap/taz interactome t-cell immunoglobulin and mucin domain 1 (tim-1) is a receptor for zaire ebolavirus and lake victoria marburgvirus dissection of the insulin signaling pathway via quantitative phosphoproteomics systems virology: host-directed approaches to viral pathogenesis and drug targeting actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics insulin degrading enzyme is a cellular receptor mediating varicella-zoster virus infection and cell-to-cell spread angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a proteomic perspective of inbuilt viral protein regulation: pul46 tegument protein is targeted for degradation by icp0 during herpes simplex virus type 1 infection a model for random sampling and estimation of relative protein abundance in shotgun proteomics the c type lectins dc-sign and l-sign: receptors for viral glycoproteins dc-sign as a receptor for phleboviruses quantitative proteomics reveals subset-specific viral recognition in dendritic cells egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy the t4 gene encodes the aids virus receptor and is expressed in the immune system and the brain applying proteomic technology to clinical virology precision proteomics: the case for high resolution and high mass accuracy modification of host lipid raft proteome upon hepatitis c virus replication sfv infection in cho cells: cell-type specific restrictions to productive virus entry at the cell surface infectious entry pathway of influenza virus in a canine kidney cell line the crapome: a contaminant repository for affinity purification-mass spectrometry data vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells virus entry by endocytosis rnai screening reveals proteasome-and cullin3-dependent stages in vaccinia virus infection rnai screening comes of age: improved techniques and complementary approaches role of the phosphatidylserine receptor tim-1 in enveloped-virus entry interacting regions of cd81 and two of its partners, ewi-2 and ewi-2wint, and their effect on hepatitis c virus infection a targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation cell entry of lassa virus induces tyrosine phosphorylation of dystroglycan quantitative proteomics identifies the membrane-associated peroxidase gpx8 as a cellular substrate of the hepatitis c virus ns3-4a protease tools for label-free peptide quantification ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus protein-protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches the hcmv gh/gl/ul128-131 complex triggers the specific cellular activation required for efficient viral internalization into target monocytes adeno-associated virus 2 infection requires endocytosis through the clic/geec pathway global, in vivo, and site-specific phosphorylation dynamics in signaling networks stable isotope labeling by amino acids in cell culture, silac, as a simple and accurate approach to expression proteomics identifying and quantifying in vivo methylation sites by heavy methyl silac identifying the proteins to which small-molecule probes and drugs bind in cells the mintact project-intact as a common curation platform for 11 molecular interaction databases morphological and biochemical characterization of the membranous hepatitis c virus replication compartment a proteomics approach to understanding protein ubiquitination viral immune modulators perturb the human molecular network by common and unique strategies transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses hiv-1 evades innate immune recognition through specific cofactor recruitment the sweet spot: defining virus-sialic acid interactions a dual-reporter system for real-time monitoring and high-throughput crispr/cas9 library screening of the hepatitis c virus mass spectrometry-coupled techniques for viral-related disease biomarker identification the cd81 partner ewi-2wint inhibits hepatitis c virus entry assembly of endocytic machinery around individual influenza viruses during viral entry inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus physical association of moesin and cd46 as a receptor complex for measles virus hepatitis c virus is primed by cd81 protein for low ph-dependent fusion identification of cell surface molecules involved in dystroglycan-independent lassa virus cell entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry virus activated filopodia promote human papillomavirus type 31 uptake from the extracellular matrix cell-type specific requirements for thiol/disulfide exchange during hiv-1 entry and infection estimating the size of the human interactome pooled rnai screen identifies ubiquitin ligase itch as crucial for influenza a virus release from the endosome during virus entry cellular entry of human papillomavirus type 16 involves activation of the phosphatidylinositol 3-kinase/akt/mtor pathway and inhibition of autophagy string v10: protein-protein interaction networks, integrated over the tree of life a million peptide motifs for the molecular biologist resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry respiratory virus-induced egfr activation suppresses irf1-dependent interferon lambda and antiviral defense in airway epithelium surface proteins of c6/36 cells involved in dengue virus 4 binding and entry an empirical framework for binary interactome mapping high confidence determination of specific protein-protein interactions using quantitative mass spectrometry arrest all accessories-inhibition of hepatitis c virus by compounds that target host factors integrin alphavbeta3 is a coreceptor for human cytomegalovirus quantitative temporal viromics: an approach to investigate host-pathogen interaction a synthetic peptide from hiv-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion a proteomic ruler for protein copy number and concentration estimation without spike-in standards quantitative phosphoproteomics reveals extensive cellular reprogramming during hiv-1 entry comprehensive proteomic analysis of host cell lipid rafts modified by hbv infection sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus viral subversion of nucleocytoplasmic trafficking subproteomic study of hepatitis c virus replicon reveals ras-gtpase-activating protein binding protein 1 as potential hcv rc component hepatitis c virus co-opts ras-gtpase-activating protein-binding protein 1 for its genome replication hiv envelope-cxcr4 signaling activates cofilin to overcome cortical actin restriction in resting cd4 t cells in vivo quantitative proteomics: the silac mouse silac-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers incidence of japanese encephalitis, visceral leishmaniasis and malaria before and after the wenchuan earthquake, in china efficient human immunodeficiency virus (hiv-1) infection of cells lacking pdzd8 epidermal growth factor receptor-pi3k signaling controls cofilin activity to facilitate herpes simplex virus 1 entry into neuronal cells significance of palmitoylation of cd81 on its association with tetraspanin-enriched microdomains and mediating hepatitis c virus cell entry hras signal transduction promotes hepatitis c virus cell entry by triggering assembly of the host tetraspanin receptor complex we thank stefan kunz for critical reading of the manuscript. key: cord-333331-ddcz7zck authors: yang, jin; fang, mei-xin; li, jie; lou, guo-qiang; lu, hang-jun; wu, nan-ping title: detection of hepatitis c virus by an improved loop-mediated isothermal amplification assay date: 2011-05-12 journal: arch virol doi: 10.1007/s00705-011-1001-4 sha: doc_id: 333331 cord_uid: ddcz7zck an improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (lamp) assay targeting the 5′ untranslated region (utr) was developed to detect hepatitis c virus (hcv) infection. based on an accelerating primer (ap), the present assay, named ap-lamp, has the advantages of rapidity and sensitivity over the routine lamp method. the possible ap-based amplification pathway during the reaction was revealed by restriction enzyme digestion and eletrophoresis. the detection limit of the ap-lamp assay was approximately 84 iu/ml, and no cross-detection was observed. the assay was evaluated further with 126 clinical specimens, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for detection of hcv rna. the hepatitis c virus (hcv) pandemic has become a major public health concern, with such increasing prevalence that nearly 200 million individuals are infected worldwide [1] . the majority of acute hcv infections present an asymptomatic course. many infected individuals are therefore not seen in a medical setting [28] . nearly 85% of infected people develop persistent infection and are at risk of longterm complications, ranging from mild liver damage to severe chronic hepatitis that can develop into cirrhosis, end-stage liver disease, or hepatocellular carcinomas [3] . therefore, a rapid and accurate diagnosis of hcv is important for the prevention of viral transmission and management of disease progression. screening of antibodies against hcv, however, is not a reliable method of diagnosing acute hcv infection, since the appearance of antibodies against hcv can be delayed in up to 30% of patients at the onset of symptoms [23] . moreover, the window period can be even longer in immunocompromised patients, who need to be screened routinely for hcv viremia [21] . nucleic-acid-based detection techniques are currently the most reliable methods for detecting hcv infection. a variety of molecular diagnostic assays, such as reverse transcriptase pcr [12] , nucleic-acid-sequence-based amplification [13] , transcription-mediated amplification [29] , branched-chain dna assay [26] , and in-house realtime pcr [8] , have been developed for the detection of hcv rna. these assays, whether qualitative or quantitative, are relatively time-consuming, labor-intensive, and dependent on specialized equipment. in resource-limited or point-of-care settings, the cost and technology requirement limit their universal application. since most hcv-infected individuals are asymptomatic, there are clear advantages to targeted screening for hcv in those who are at high risk. earlier detection of infection results in earlier treatment and thus earlier recovery [25] . for this reason, there is still a great need for a tool to simplify the detection of hcv rna with acceptable sensitivity and specificity, short turnaround time, and cost-effectiveness. loop-mediated isothermal amplification (lamp) is a novel rapid, accurate, and economical nucleic acid test [19] . the method is characterized by employing a dna polymerase with strand-displacement activity, along with two inner primers (fip, bip) and two outer primers (f3, b3) to form auto-cycling immediates. loop primers (lf, lb), first described by nagamine et al, could accelerate and enhance the sensitivity of the lamp assay [21] . onestep lamp assays have been successfully applied to the rapid detection of a number of rna viruses, such as influenza virus [16] , mumps virus [31] , west nile virus [24] , severe acute respiratory syndrome corona virus [15] , and hiv-1 [7] . lamp assays have also been developed to detect hepatitis viruses, such as hepatitis b virus [5] , hepatitis a virus, and hepatitis e virus [15] . at present, however, no hcv detection assay using this method has been reported. the attributes of the hcv target may interfere in complex ways with the lamp method. for instance, although the viral 5 0 untranslated region (5 0 -utr) is thought to be the most conserved portion of the hcv genome and is targeted by almost all of the commercial and in-house tests [9] , one of the most important issues may be the existence of complex secondary structure across all of this region (fig. 1b) . also, the 5 0 -utr is generally considered to be variable enough to distinguish all of the major types and many subtypes of hcv [14] . in the study described here, we have developed a modified lamp method for rapid and economical detection of hcv rna. in contrast to the routine lamp (pre-lamp) method, we designed an accelerating primer (ap) and tested the performance of the ap-based lamp assay (ap-lamp). the new assay was further evaluated using clinical samples. clinical specimens and standard twenty-five blood samples collected from patients with confirmed chronic hcv infection and 51 specimens obtained from patients suspected of having viral hepatitis who were admitted to hangzhou 2 th and infectious hospital were used for evaluation in this study. confirmed cases of hcv infection were verified by a positive result in an enzyme-linked immunosorbent assay (kehua bio-engineering, shanghai, china) for antibodies against hcv or a quantitative real-time pcr for hcv rna. a panel of 50 samples collected from healthy blood donors was also included as negative controls. in addition, five each of anti-hiv-antibody-, anti-hav-antibody-and hbv-dna-positive samples obtained from the corresponding patients were also tested. informed consent was obtained from all patients, and the study was approved by the local ethical committee, as per the declaration of helsinki (1995). five milliliters of blood was collected from each subject in a tube containing 200 ll of 0.5% edta. plasma was immediately separated after centrifugation at 1500 rpm for the in-house hcv rna standard was obtained by extracting rna from a hcv-rna-positive specimen with an hcv titer of 10 6 iu/ml. this in-house standard was anti-hcv antibody positive and calibrated in triplicate in parallel with the national hcv rna reference material (gbw09151, 2.26 9 10 3 iu/ml -4.22 9 10 7 iu/ml, genotype 1) using several commercial real-time pcr tests (kehua bio-engineering, shanghai, china; daan biotech, guangzhou, china). the gbw09151 panel was calibrated using the who hcv international standard (nibsc 96/790) [29] . hcv genotyping was performed using sequencing of the ns5b region of the hcv genome as described previously [20] . sequence analysis indicated that the hcv genotype of the in-house standard was 1b. serial dilutions of the standard sample for experimental analysis were prepared in normal human plasma and stored at -80°c until testing. viral rna was extracted from 140 ll of plasma using a tianamp virus rna kit (tiangen biotech, beijing, china) as per manufacturer's instructions. this extraction protocol used a fast spin-column procedure. rna was eluted in 60 ll of rnase-free water. the whole extraction procedure was done within an hour. to design an assay that can detect most of the genotypes of prevalent hcv strains, 338 individual sequences of the 5 0 utr region of hcv strains from the hcv database (http://hcv.lanl.gov/) were retrieved. through alignment analysis, the conserved fragments of the 5 0 utr were used to design the primer set. the hcv genotype 1b sequence (genbank accession number ay460204), chosen as a representative strain, was used as a reference for generating the set of primers. all of the primers, including two outer (f3 and b3), two inner (fip and bip) and two loop primers (lf and lb), were designed according to the guideline provided by primerexplorer v4 (http://loopamp.eiken.co.jp/). in this set, fip consisted of f1c, complementary to the f1 sequence, and f2 sequence, and bip consisted of the b1 sequence and b2c, complementary to the b2 sequence. f3 and b3 were located outside f2 and b2, while the loop primers recognized the region between f2 and f1, or b2 and b1. to strengthen the power of lamp, an additional accelerating primer (ap), located between f1 and b1, was added to the primer set. a schematic representation of the locations of the primers along with a representative alignment of the main hcv strains is shown in fig. 1 . the details of the oligonucleotide primers used for the amplification are given in table 1 . all primers were synthesized by invitrogen (invitrogen, shanghai, china). the routine one-step lamp (pre-lamp) reactions were carried out in a final volume of 20 ll containing 40 pmol each of the fip and bip inner primers, 20 pmol each of the lf and lb loop primers, 5 pmol each of the f3 and b3 outer primers, 19 thermopol buffer (new england biolabs, beverly, ma), 2 mm mgcl 2 , 1 m betaine (sigma aldrich, usa), 1.4 mm each deoxynucleotide triphosphate, 8 u of bst dna polymerase (new england biolabs, beverly, ma), 0.125 u of amv reverse transcriptase (takara, dalian, china), and 6 ll of template. the mixture was incubated at 60°c for 60 min and then heated to 85°c for 2 min to terminate the reaction. for the ap-lamp assay, the reaction mixture and the conditions were the same as those described for pre-lamp, except that 20 pmol of ap was added to the reaction mixture. a negative control was included for each lamp run. serial dilutions of the standard were used as templates for the lamp assay to evaluate its sensitivity. the specificity of the assay was tested with samples from hav-, hbv-, and hiv-infected patients and healthy donors. after amplification, the amplified dna products were analyzed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide and visualized using a bio-rad transilluminator. the restriction enzymes nhei and smai (new england biolabs, beverly, ma) were used to digest amplified products to confirm amplification specificity. digested products were analyzed by gel electrophoresis on a 1.5% agarose gel. for naked-eye visualization, one microlitre of 1000 9 diluted sybr green i (invitrogen, carlsbad, ca) was added to the reaction tube after amplification, and the reaction was observed visually. for a positive reaction, a change in the color of the reaction solution from orange to fluorescent green could be recognized. for real-time monitoring of the lamp reaction, the reaction was performed on an abi prism 7900ht sequence detection system, with 1 9 sybr-green i (invitrogen, carlsbad, ca) added to the reaction mixtures to provide the fluorescent signal. the run was set up as follows: 60 cycles of 1 min at 60°c (1 cycle corresponding to 1 min of reaction), with fluorescence reading at the end of each of these cycles. a commercial hcv rna real-time pcr detection kit (kehua bio-engineering, shanghai, china) was used according to the manufacturer's instructions. as the template, 12.5 ll of rna extract was used in a 25-ll reaction. the clinical sensitivity of this quantitation kit was 500 iu/ml [18] . to design a lamp primer set covering the main genotypes of hcv isolates, a multiple sequence alignment, including genotypes 1-6, was generated by retrieving and aligning the sequences stored in the hcv databases (http://www.hcv. lanl.gov). the primers were designed to maintain maximum conservation for annealing to the target regions. mismatches at the 5' or 3' ends of fip/bip were substituted by degenerate bases (table 1) . a database search using blast from ncbi showed that all of the primers were specific for the hcv genome. the routine lamp format (pre-lamp) for the hcvspecific assay was performed by using rna templates extracted from standard samples. the amplified dna products were subjected to electrophoresis on 1.5% agarose gels and visualized under uv light after ethidium bromide staining. as a result, a typical lamp laddering pattern was observed, indicating the different replication intermediates of the stem-loop amplification process, while no bands were obtained from the no-template control. eletrophoresis-based monitoring of the pre-lamp product showed a sensitivity of 500 iu/ml ( fig. 2a) . to test the specificity of the test, the amplified product was digested with the enzyme nhei, resulting in the detection of strong bands (fig. 3b, lanes 2) . furthermore, the possibility of crossreactivity with other viruses known to cause similar clinical signs was also investigated. no amplification of any viral rna (or dna) extracted from hbv-, hav-, or hivpositive samples was detected (fig. 3e) . to improve the efficiency of detection, an accelerating primer (ap) was designed to form an additional synthesisstarting site. different from the loop primer, which binds the single strand in the f1-f2 or b1-b2 region in the classical lamp [21, 22] , the ap designed here is complementary to one of the double-stranded regions between f1 and b1 (fig. 5a) . adding ap to the pre-lamp reaction, the assay, named ap-lamp, was carried out to evaluate its performance. serial dilutions of the templates were amplified by the ap-lamp assay. as shown in fig. 4 , the one-step ap-lamp assay had a detection limit of a 50 iu/ml of rna template. specificity tests, including restriction enzyme analysis and the use of negative samples, were also conducted, and these showed no crossreaction. since visualization of amplification products from lamp reactions without special equipment would make the assay widely applicable and available, sybr green i was added to the reaction mixtures, resulting in a color change from orange to green. the amplified products yielded a green color in positive ap-lamp reactions, demonstrating that the sensitivity of this assay is equal to that of eletrophoresis (fig. 4b ). given that the sensitivity of the ap-lamp assay for detecting hcv is higher than that of the pre-lamp method, the pathway of ap-based amplification was investigated. a dumbbell-like intermediate is the initial auto-cycling product for the subsequent amplification steps in the lamp assay. apart from the classical lamp pathway that is followed when using fip and bip, as described elsewhere [22] , the ap pathway involves the synthesis via ap priming to promote elongation followed by fip self-priming. the characteristic feature of the products of the ap pathway is that the end products are partly derived from the concatenation of ap-fip fragments (fig. 5 ). more importantly, if the ap pathway is followed in the assay, it is logical to speculate that the amplification would still occur without the outer primer (named ap-b3 lamp in this study), which is strictly required in the classical lamp [22] . three lamp formats are shown in fig. 5a . to test this hypothesis, we compared the pre-lamp, ap-lamp, and ap-b3 lamp assays using the same templates. a panel of serial log dilutions of hcv rna of known concentration was tested using the ap-lamp, pre-lamp, and ap-b3 lamp assays on the real-time 7900ht platform. the results for each dilution tested in batches of three replicates in two separate runs are given in table 2 . for the ap-lamp assay, the average threshold time (tt) required to detect a positive signal ranged from 17.99 ± 0.62 min (mean ± sd) when 5 9 10 5 iu rna was present to 31.70 ± 0.43 min when 5 9 10 2 iu rna was present, compared to 25.98 ± 0.58-42.71 ± 1.60 min in the pre-lamp assay and 16.00 ± 0.80-31.99 ± 1.06 min in the ap-b3 lamp assay. the tt value was defined as the reaction time necessary to achieve a positive signal above the baseline [10] . these results demonstrated that the ap-lamp assay was faster by 9-12 min than the pre-lamp reaction and was much faster than real-time pcr. furthermore, using the assays to analyze low-concentration standards ranging from 500 to 10 iu, no amplification was obtained when the template concentration was less than 250 iu/ml in the pre-lamp assay. the real-time ap-lamp assay consistently detected hcv rna at levels of 5 9 10 1 iu/ml, while only three of six ap-b3 lamp assays detected hcv rna. by probit regression analysis, the ap-lamp method detected 84 iu/ml with [95% probability of a positive result, as compared to 617 iu/ml and 143 iu/ml for the pre-lamp and ap-b3 lamp assay, respectively. therefore, the sensitivity of the ap-lamp assay for detecting hcv is about two times higher than that of the ap-b3 lamp assay and nine times higher than that of the pre-lamp assay. when comparing the electrophoresis bands of the products of the ap-lamp, ap-b3 lamp and pre-lamp assays, the first two showed a similar pattern. the main difference between the ap-lamp and pre-lamp assays is that there were ladder-like bands between 100 and 250 bp. the bands in this region (in rectangles with a broken line, fig. 2 ) are likely to correspond to the self-primed amplification product bounded by the 5 0 ends of the ap and fip stemloop structure (*193 bp in size), which match the expected size of the ap-pathway product. moreover, the result of the ap-b3 lamp assay provided evidence that the ap pathway is used during amplification. several reports have indicated that the lamp assay strictly requires the strand displacement function of the outer primers [22] . no lamp amplification occurs when fip, bip, f3 or b3 is absent [2] . by using the ap in the assay, this study using real-time monitoring or agarose gel electrophoresis confirmed that the outer primer . the extracted rna template was prepared from the standard plasma and was subjected to analysis by the pre-lamp and ap-b3 lamp methods. a pre-lamp assay using serial dilutions from 5 9 10 5 iu/ml to 50 iu/ml. b ap-b3 lamp assay using template concentrations from 5 9 10 5 iu to 50 iu/ml. c lowconcentration standards ranging from 500 to 50 iu/ml, detected by the pre-lamp assay. d the same standards ranging from 500 to 50 iu/ml, detected by the ap-b3 lamp reaction. the dashed box indicates the band pattern with the most significant difference between the two assays is not required. the ap-b3 lamp assay has higher sensitivity than the pre-lamp assay, but it is less sensitive than ap-lamp. this assay type also showed lower stability than ap-lamp, especially when the samples were at low concentration. in addition, taking into account the similar band patterns between the ap-lamp and ap-b3 lamp assay, these results indicate that ap-priming-based amplification is inferior to the classical pathway in the lamp process. the amplified products were digested with two restriction endonucleases to confirm the specificity and structure of the amplified products from three different lamp assays. the restriction enzyme smai recognizes the sequence between f1 and b1, while nhei cuts between b1 and b2. if the products were amplified specifically and formed the structures shown in fig. 3a , nhei digestion would yield fragments of 225, 229, 347, and 365 bp, and smai digestion would yield fragments of 106, 123 and 233 bp. since the amplified product is a concatenation of dna fragments of different sizes, the amplification kinetics probably affect the actual end products of the lamp reaction. due to cutting in the region between b1 and b2, nhei-digestion bands were observed at approximately 250-454 (225 ? 229) bp in the pre-lamp product, in contrast to 220-400 bp in the ap-lamp and ap-b3 lamp products. while cutting the region between f1 and b1, the sizes of the fragments generated by smai digestion were approximately 120 and 230 bp (fig. 3d) , which is in good agreement with the predicted sizes. the feasibility of the ap-lamp assay for detecting hcv in clinical material was assessed by using both positive and negative plasma specimens. the real-time pcr assays were performed simultaneously, and the results of both (table 3) . of the 50 samples collected from healthy volunteers, all tested negative in both ap-lamp and real-time pcr. a total of 25 samples were obtained from chronic hcv patients (genotype 1b, n = 19; genotype 2a, n = 6; all anti-hcv positive), with the viral load ranging from 1.35 9 10 3 to 3.12 9 10 6 iu/ml. none of the samples were missed by the ap-lamp assay. of 51 acute-phase samples collected from patients suspected of having viral hepatitis, three were anti-hcv positive, and these were also detected by ap-lamp. of the remaining 48 anti-hcv-negative cases, only two were positive for hcv rna by ap-lamp. these two patients later seroconverted after 1 and 3 months, respectively ( table 3 ). the ap-lamp gave a total of 5 (9.8%) positive results, and the same result was obtained by real-time pcr. in total, the ap-lamp method demonstrated 100% agreement with real-time pcr when used for analysis of clinical samples. these preliminary results suggest that the ap-lamp assay described here can be applied for the diagnosis of hcv infection in a clinical setting. among the nucleic acid amplification tests available to date, lamp method has many characteristics that make it suitable for the rapid, sensitive, and simple detection of pathogens [19] . the adaptation of the lamp technology for hcv detection in point-of-care or resource-limited setting has many potential advantages. for examples, the reaction occurs under isothermal conditions and thus does not require special equipment. the powerful amplification efficiency of the lamp assay makes it extremely rapid, and it exhibits high analytical sensitivity. furthermore, the end product can be observed immediately by visual observation, through turbidity or dye staining [27] . up to now, however, no hcv nucleic acid test has been available outside of the laboratory setting, possibly due to time, cost and technology limitations. the use of multiple primer combinations is one of the key features of the lamp method, but this can affect the primer selection for a given template, such as the hcv 5'utr, which has a complex ordered secondary structure and genotypic variation sites simultaneously. we devised an accelerating primer to improve the efficiency of amplification. just like the loop primer in lamp, the ap provides an additional starting site for dna synthesis (fig. 5 ) during the amplification, thereby reducing the overall reaction time and increasing the sensitivity. it should be mentioned, however, that in the lamp reaction, the loop region is always in a single-stranded state during the process. in contrast, the ap is located in the doublestranded region and is complementary to one of its strands. when comparing the performance of the ap-lamp and pre-lamp assays, higher amplification efficiency was found in the former. a possible explanation for this is that apart from the classical amplification process in lamp assay, there is an ap-based amplification pathway in the ap-lamp method. this notion is supported by the following evidence: first, by comparing the band patterns of ap-lamp and pre-lamp, the products of the ap pathway (ap-fip) were observed in the former. next, the ap-b3 lamp assay still amplified the target in the absence of outer primer b3. in the classical lamp format, the outer primer is important for strand displacement to form an auto-cycling intermediate product. no amplification occurs without this primer [2] . finally, by the digesting the end products with restriction enzymes that recognize different sites in the target, the most favorable structure of the amplified products was found by length polymorphism analysis, as shown in fig. 3 . it is well known that primer design in lamp is more complex and difficult than that in pcr [2] . since lamp reaction efficiency and sensitivity strongly depend on primer selection, a more flexible way for lamp primer design could promote the use of this method. summing up the above points, the ap strategy could be applied in lamp design to meet special demands under certain conditions. for instance, because both ends of the fip/bip secondary structure play a key role in amplification cycling in the lamp-based assay, using an ap could reduce the problem of selecting fip/bip. adding ap to the principle of ap in lamp amplification. a the three lamp formats in this study. the primers commonly used in the lamp assay (pre-lamp) include inner primers fip and bip, outer primers f3 and b3, and loop primers lf and lb. the loop primer anneals the partially single-stranded portion. the ap devised in this study is the additional accelerating primer located between the f1 and b1 fragments. the ap-b3 lamp assay does not use the outer primer b3. b the cyclic amplification step for the ap pathway is illustrated: the dsdna reaches a dynamic equilibrium at 60°c, and thus the ap can bind the partly free 3' end of the template to initiate strand extension. complementation of the hairpin structure (f1-f1c) induces self-primed dna synthesis pre-optimized lamp assay could avoid the need to design different primer sets for optimization. the performance of ap-lamp was investigated in this study. running the ap-lamp assay in a real-time pcr machine consistently achieved a lower limit of detection of 84 iu/ml by probit analysis. this sensitivity is comparable to a series of in-house tests published recently [4-6, 16, 30] . although this method is essentially qualitative at the outset, the sensitivity limits represent good performance, since the hcv plasma viremia in acute infections is generally higher than 10 4 copies/ml [11] . as expected, a specificity test using seronegative samples and other nontargeted virus samples demonstrated 100% exclusivity of the assay. by applying the ap-lamp assay to clinical specimens, there was 100% agreement between ap-lamp and the real-time pcr test. the ap-lamp method consistent detected hcv-infected samples with a broad range of viral loads. since the samples comprised the hcv genotypes 1b, 2a, which are prevalent in china [17] , this ap-lamp assay is expected to work for the majority of hcv-infected individuals in the local region. because of the high mutation rate of the hcv 5'utr, it is not easy to generate a single lamp primer set to detect every viral strain of an individual subtype. degenerate design is most the common way to address the issue of genotype inclusivity, but this may lower the diagnostic sensitivity due to a hybridization effect. for this reason, the ap strategy developed here would potentially be applicable in a situation like this. future evaluation of detection efficiency using an extensive collection of hcv genotypes and larger samples would be desired to validate the performance of the assay. in addition to the high sensitivity and specificity of the ap-lamp assay, its other major advantages are its rapidity and the flexibility of its detection method. the ap-lamp assay itself could be carried out in less than 45 min. only 1.5 h (including the extraction step) was needed to perform the lamp assay, compared to 2.5-3 h for the real-time pcr assay. amplification in the lamp assay can be detected with the naked eye in the form of visual fluorescence, e.g., the original orange color of the dye changes to green under natural light in the case of a positive amplification reaction [25] , thus eliminating the need for gel electrophoresis or real-time monitoring. our results, as well as the results of previous studies using a fluorescent reagent to detect the lamp product visually [24, 35] , showed a similar detection efficiency when compared to real-time pcr or electrophoresis. therefore, this hcvspecific lamp assay may be applicable under clinical or field conditions. in conclusion, as demonstrated using hcv, we have developed a lamp test using a novel principle based on an accelerating primer and have provided an alternative way to design a lamp assay for a complex target. this study presents a sensitive and specific lamp method for screening for or confirming infection with hcv in a simple, rapid, and cost-effective manner. loop-mediated isothermal amplification assay for hcv 1395 national institutes of health consensus development conference statement: management of hepatitis c real-time quantitative lamp (loop-mediated isothermal amplification of dna) as a simple method for monitoring ammonia-oxidizing bacteria natural history of chronic hepatitis c virus infection an in-house method for the detection and quantification of hcv in serum samples using a taqman assay real time pcr approach taqman amplification system with an internal positive control for hcv rna quantitation hiv-1 and hcv detection in dried blood spots by sybr green multiplex real time rt-pcr sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of hiv-1 quantitation of hepatitis c virus using an in-house real-time reverse transcriptase polymerase chain reaction in plasma samples a novel diagnostic target in the hepatitis c virus genome loop-mediated isothermal amplification integrated on microfluidic chips for pointof-care quantitative detection of pathogens dynamics of viremia in early hepatitis c virus infection design of an antisense reverse-transcriptasepolymerase chain reaction primer efficient for all hepatitis c virus genotypes: comparison of its performance vs a commercial primer evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents sequence analysis of the 5' untranslated region in isolates of at least four genotypes of hepatitis c virus in the netherlands reverse transcription-loop-mediated isothermal amplification assay for rapid detection of hepatitis e virus detection and quantification of serum or plasma hcv rna: mini review of commercially available assays hepatitis c virus genotype distribution in china: predominance of closely related subtype 1b isolates and existence of new genotype 6 variants a novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis c viral rna with armored rna as internal control loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases use of sequence analysis of the ns5b region for routine genotyping of hepatitis c virus with reference to c/e1 and 5' untranslated region sequences accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna acute hepatitis c: prevention and treatment realtime reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases comparison of conventional pcr with real-time pcr and branched dna-based assays for hepatitis c virus rna quantification and clinical significance for genotypes 1 to 5 loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products hcv screening to enable early treatment of hepatitis c: a mathematical model to analyse costs and outcomes in two populations rnase-resistant virus-like particles containing long chimeric rna sequences produced by two-plasmid coexpression system real-time quantitative assay of hcv rna using the duplex scorpion primer mumps virus reinfection is not a rare event confirmed by reverse transcription loop-mediated isothermal amplification the authors declare that they have no conflict of interest. key: cord-020010-q58x6xb0 authors: nan title: 19th icar abstracts: date: 2006-03-13 journal: antiviral res doi: 10.1016/j.antiviral.2006.02.001 sha: doc_id: 20010 cord_uid: q58x6xb0 nan the society was organized in 1987 as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its 20 th year of existence, and has about 600 members representing 30 countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, 10777 science center drive, san diego, ca 92121; phone +1 858 622 3117; fax +1 858 678 8182; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the 3 -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv-1teki (nsi phenotype) and hiv-1nl4-3 (si phenotype), and in a magi-x4 assay against hiv-1nl4-3 with hela-cd4-ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch 1 , imma clotet-codina 1 , julia blanco 1 , eduard pauls 1 , gemma coma 1 , samandhy cedeño 1 , francesc mitjans 2 , anuska llano 1 , margarita bofill 1 , bonaventura clotet 1 , jaume piulats 2 , jose este 1 1 retrovirology laboratory, fundacio irsicaixa, badalona, spain; 2 laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, 17e6, inhibited hiv-1 infection of primary macrophages. the effect of 17e6 on r5 or x4 hiv-1 replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (ec50) of 17 ± 2 g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb6 integrin (14d9.f8) also inhibited hiv-1 infection in this cell type. 17e6 reduced the detection of hiv-1 bal proviral dna in acutely infected macrophages but was completely ineffective against hiv-1 bal production in chronically infected macrophages, suggesting that 17e6 inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb6 integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant 1 , hong lu 2 , shuwen liu 2 , carol lackman-smith 3 , roger ptak 3 , jean-marc juteau 1 , shibo jiang 2 1 replicor inc., laval, que., canada; 2 f. lindsay kimball research institute, new york blood center, new york, ny, usa; 3 southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv-1 by blocking interactions between the v3 loop and cd4 has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully 2 -o-methylated derivatives) are also potent inhibitors of hiv-1-mediated membrane fusion and hiv-1 replication in a sequence-independent, sizedependent (optimal size ∼50-60 bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp41 and the hiv-1 fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp41 six-helix bundle formation, a critical step during the process of hiv-1 fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v3 loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥40 base) ps-ons may contribute to their inhibitory activity against hiv-1 fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp41 sixhelix bundle formation than shorter ps-ons (<30 bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv-1 and other enveloped viruses with type i fusion proteins. chris meier 1 , soenke jessel 1 , bastian reichardt 1 , olaf ludek 1 , jan balzarini 2 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the 3 -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv-1. additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita 1 , candace pert 2 , maria-teresa polianova 2 , alessandro ranazzi 1 , michael ruff 2 , carlo-federico perno 1 , stefano aquaro 1 1 university of rome tor vergata, italy; 2 georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv-1 commonly infected by ccr5-using (r5) strains of hiv-1. ccr5 is an attractive target for inhibition of ccr5 mediated hiv-1 entry. thus, ccr5 antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv-1 infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp120 v2 region of hiv-1, able to bind ccr5. dapta acts as selective viral entry inhibitor, displacing the binding of gp120 with ccr5. dapta anti-hiv-1 activity was evaluated in m/m infected with two different hiv-1 r5 strains, bal and 81a, in presence of several doses of the compound. dapta inhibited hiv-1 replication in m/m (>80% compared to control), measured by the p24 gag ag released in the cell culture supernatants, at concentration of 10-3 nm. pcr analysis of integrated hiv-1 proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv-1 r5 strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr5. in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of 10-3 and 10-4 nm, strongly inhibited apoptosis in sk-n-sh of 58 and 56%, respectively, compared to control. unexpectedly, tak-779 (a nonpeptidic ccr5 antagonist with potent anti-hiv-1 activity) inhibited apoptosis only of 30% compared to control. our results suggest that the development of new anti-hiv-1 compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv-1 r5 strains in the brain as direct inhibition of hiv-1 replication in m/m and gp120 related damage by ccr5 binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv-1 clades in pbmcs, in primary macrophages and several hiv-2 and siv strains in laboratory cell lines (range of 50% effective concentrations: 0.69-11 g/ml; 50% cytostatic concentration: >50 g/ml). prm-a also inhibits syncytium formation between persistently hiv-1-infected hut-78 cells and uninfected sup t1 cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp120 of hiv-1/mn in the presence of ca 2+ . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv-1. a variety of multiple mutations at n-glycosylation sites in hiv-1 gp120 are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv-1 gp120 glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/05/15), and the european commission (empro). jan muench 1 , ludger ständker 2 , knut adermann 2 , axel schulz 2 , michael schindler 2 , raghavan chinnadurai 2 , wolf-georg forssmann 2 , frank kirchhoff 2 1 department of virology university of ulm, albert-einstein allee 11, 89081 ulm, germany; 2 ipf pharmaceuticals gmbh, feodor-lynen-strasse 31, 30625 hannover, germany a variety of components in human blood might influence hiv-1 replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below 30 kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv-1 replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a 20-residue peptide corresponding to a c-terminal fragment of ␣1-antitrypsin (␣1-at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv-1 variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv-1 variants and primary isolates. virip specifically inhibited hiv-1 env function, and did not affect infection by virions containing hiv-2, siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the 20-residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv-1 gp41 fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp41 fp. our observations are evidence that a naturally occurring human substance inhibits hiv-1 infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than 350 virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv-1 entry inhibitors. stefano aquaro 1 , valentina svicher 1 , roberta d'arrigo 2 , ubaldo visco-comandini 2 , andrea antinori 2 , mario santoro 1 , giovanni di perri 3 , sergio lo caputo 4 , pasquale narciso 2 , carlo-federico perno 1,2 1 university of rome tor vergata, italy; 2 inmi l. spallanzani, italy; 3 university of turin, italy; 4 sm annunziata hospital, florence, italy to investigate gp41-variability and correlation with viroimmunological parameters in 54 hiv-infected patients (pts) receiving t20 added as a single active drug to a failing regimen. two hundred and ten hiv-gp41 sequences and clinical follow-up from 54 t20-treated patients were analyzed from baseline up to 48 weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd4 count (c/ul) was assessed by mann-whitney test. the addition of t20 to the failing antiretroviral regimen induced at 4 weeks a significant vl decrease from 5.1 log (stable in the last 12 weeks prior t20) to 4.3 log (p = .0002) and a significant cd4 increase from 48 c/ul (decreasing in the last 12 weeks prior t20) to 106 c/ul (p = .008). while vl rebounded to 4.7-5 log at 12-48 weeks, respectively, cd4 increased to 129 c/ul at 48 weeks. t20 resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v38a was the most common sign of t20 failure (27.8% of pts). the viroimmunological outcome of t20-treated pts varied according to gp41-mutations. v38a/e (38.5% of pts) was associated with a cd4 increase from bl (48 c/ul) of 4.5-fold (142 c/ul) at 24 weeks and 6.0-fold (196 c/ul) at 36 weeks (p = .004 and .02 compared without v38a/e, respectively). no significant correlation with vl was observed (from 4.9 log at bl to 4.4-4.8 at 24-36 weeks). by contrast, q40h + l45m (11.1% of pts) was associated with cd4 loss from 71 c/ul at bl to 26 c/ul at 36 weeks (p = .02), without significant changes in vl (from 5 log at bl to 5 log at 36 weeks). mutation n126k (observed in 6 pts, but never found at bl) abrogates the 4th gp41-glycosylation site and correlated with 1.7-fold cd4 increase at 24 weeks. conformational changes induced by v38a/e in the highly conserved giv motif of gp41-hr1, are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd4-recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h5n1 influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h5n1 influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as 10 2 eid 50 of a/vietnam/1203/04 (h5n1) influenza virus caused high fever (39.8-41.8 • c), weight loss (25.4% of initial), anorexia, extreme lethargy and death of animals on days 7-10 post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of 5 mg/kg/day for 5 days twice daily initiated 4 h p.i. inhibited the febrile response, reduced weight changes (14.6% of initial) and, most importantly, completely protected ferrets from lethal h5n1 infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of 2.8-6.5 log 10 eid 50 /ml on days 3, 5 and 7 p.i. systemic spread of the h5n1 virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day 5 p.i. in the brains of treated animals virus was detected in one of the two animals tested with >100-fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha1 subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h5n1 mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn 38105, usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/1203/04-like (h5n1) viruses. one virus possessed asparagine at position 31 of the m2 protein that was found in the naturally circulating virus (rgvn-1203) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn-1203sens) . balb/c mice were administered oseltamivir (1 or 10 mg/kg/day) and amantadine (1.5 or 15 mg/kg/day) twice daily for 5 days by oral gavage; the first doses were given 24 h before inoculation with 10 mld50 of h5n1 virus. combination treatment with 10 mg/kg/day oseltamivir and 15 mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h5n1 viruses (p < 0.01). treatment with oseltamivir at dosage of 10 mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days 3, 6, and 9 after inoculation (p < 0.05), but resulted in low survival rate (20%). single-drug amantadine showed dose-dependent effect only against rgvn-1203sens strain. notably, risk of death for mice that received 15 mg/kg/day of amantadine or 10 mg/kg/day of oseltamivir was similar (p < 0.05). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn-1203sens (p < 0.05) compared to singledrug usage and protected 90% of animals. importantly combination chemotherapy completely protected h5n1 virus spread to the brain of the mice: virus was not detected in brain of treated animals on days 3, 6, and 9 after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean 1 , reid asbury 2 , meera raj 1 , david lawrence 3 , martin petric 3 1 the university of british columbia, vancouver, bc, canada v6t 1z3; 2 ge healthcare bio-sciences, piscataway, nj 08855, usa; 3 british columbia center for disease control, vancouver, bc, canada v5z 4r4 in late 2002, severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the 21st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with 772 deaths reported from 29 countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh7: human liver] and primate (veroe6: monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over 50 host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard 1 , craig day 1 , robert montgomery 1 , kevin bailey 1 , matt heiner 1 , larry lauridsen 1 , robert sidwell 1 , kurt berg 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, 2005. 3, 251-262) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., 2004. apmis 112, 605-611) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning 12 h pre virus exposure at doses of 100,000 and 10,000 iu and the hydroxyethylrutoside mix (100 and 10 mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at 1.32 mg/ml (likely equivalent to an i.p. dose of 100 mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n01-ai-15435 from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk-21) cells that express the essential viral replication proteins (n, p, l and m2-1). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded 596 compounds (hit rate = 0.75%) with ec50 values ≤10 m and with selectivity index (si) values ≥10. seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a2) in a cytopathic effects inhibition assay (ec50 < 10 m; si > 10). these anti-rsv compounds represent nine different chemical classes. two compounds, a 4-aminoquinoline (ec50 = 0.25 m) and a thienopyrimidine (ec50 = 0.82 m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r44 ai047552-02. we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf1743(cf-1743) (1) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers (2) as new analogues in their own right, and 5 -phosphates (3) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides (4) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei 1 , joos van den oord 2 , pierre fiten 1 , ghislain opdenakker 1 , erik de clercq 1 , robert snoeck 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf29 gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after 4 days of differentiation and treated with serial dilutions of the test compounds. at 12 days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at 4 and 0.4 g/ml, foscarnet (pfa) at 40 g/ml and cidofovir (cdv) at 4, 0.4 and 0.04 g/ml inhibited viral dna content by more than 95%. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) 07-1 strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk-07-1 strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang 1 , oliver kregler 1 , john c. drach 2,3 , leroy b. townsend 3 , elke bogner 1 1 institut für klinische und molekulare virologie, erlangen, germany; 2 department of biologic and materials sciences, school of dentistry; 3 interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul56 and the small pul89, where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul89 is only required for duplex nicking. inhibitors targeting pul56 and/or pul89 are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul56. analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [2-bromo-4,5,6-trichloro-1-(2,3,5-tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl4rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic 50 in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv-1 infection. we report that indomethacin and aspirin inhibited hsv-1 replication at non-cytotoxic doses. in vero cells, 500 um indomethacin and 20 mm aspirin reduced hsv-1 titers 99.999 and 99.5%, respectively. electromobility shift assays revealed that hsv-1 activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv-1 titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p65, a component of the dimeric nf-kb complex, translocated to the nucleus of hsv-1 infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp4, an essential hsv immediate-early gene, 2.9and 2.5-fold, respectively, resulting in significant decreases of icp4 protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv-1 replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul97 phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp97 locus, and carrying the ul97 gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul97 by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul97-specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul97-specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic 50 of 15 m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a 3-log reduction (compared to wild-type virus) in the presence of 50 m mbv, and an ic 50 of 5 m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment (25 mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul97 in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against hsv-2 infection of human cervical cells and in a vaginal murine model. rep 9 has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). at a concentration of 100 m, rep 9 inhibited 6-logs of hsv-2 infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep 9 acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep 9 or pbs control at various times prior to vaginal challenge with a lethal dose of hsv-2 strain 186 (10 log 4 pfu). rep 9 prophylaxis provided protection to mice from hsv-2 infection and disease. protection was significant when challenged 30 min after treatment (p < 0.01). additionally, treatment with an analog of rep 9, which cannot activate tlr-9 mediated immune stimulation, was at least as active as rep 9, suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged 30 min after treatment (p < 0.001) with a trend toward protection when administered 60 min prior to challenge (p = 0.07). in summary, treatment with the rep 9 analog which has superior resistance to low ph and nuclease degradation was more effective than rep 9, in some experiments protecting 100% of mice from viral infection and disease. the testing of this ph resistant rep 9 analog in a gel formulation is currently underway. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi-4065, has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi-4065 inhibits translation initiated at the aug start site with ec50 of 308 nm (2.1 ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh-7 cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi-4065 produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi-4065 in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately 90% of mice with undetectable viral titer and the remaining mice show 1 log reduction in viral titer with 0.1 mg/mouse/day for 7 consecutive days. the fractional bioavailability of avi-4065 from a sq dose is approximately 1. the apparent elimination half life in rat, nonhuman primate and humans was 2.3, 4.5 and 11.4 h, respectively. the volume of distribution ranged from 0.6 to 1.0 l/kg and the cmax is linearly related to the dose in mg/m 2 . a phase i study in healthy volunteers in which 14 daily sq doses of 50 and 100 mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately 40-60% of the genotype-1 (gt-1) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns5b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of 4 -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a 4 -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is 4 -azido-cytidine, which shows good activity in the genotype 1b sub-genomic replicon (ic50 = 1.28 m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of 4 -azido-cytidine is a potent and highly selective inhibitor of ns5b (ic50 = 0.32 m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, 100 technology square, cambridge, ma 02139, usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns5b polymerase and ns3-4a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns5b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell 2005) . nim811, a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim811 exhibited potent anti-hcv activities in vitro. moreover, the combination of nim811 with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim811 than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim811 to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim811, with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r1479 was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r1479-tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns5b. in a transient replicon system r1479 inhibited hcv rna replication driven by genotype 1b polymerase with similar potency as compared to that driven by genotype 1a polymerase. r1479-tp inhibited native hcv replicase and recombinant ns5b from genotype 1a and 1b with similar potency. in contrast, r1479-tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and 3tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r1479-tp at concentrations up to 1 mm, suggesting high selectivity for the hcv rdrp. thus, r1479 was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype 2a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh7 cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati 1 , haitao guo 2 , gael westby 1 , anand mehta 2 , timothy block 1,2 1 institute for hepatitis and virus research; 2 drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of 80,288 drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg2.2.15, we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in 1758 initially positive hits, a hit rate of 2.2%. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to 77, currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec50 measurements below 5.0 m and cc50 measurements >50.0 m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: (1) an isobutyl-acetamide with an ec50 of 87.0 nanomolar, and a cc50 of >50 m, and (2) a carbothiamide with an ec50 of 1.6 micromolar and a cc50 of >50 m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m204v/i ± l180m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t184, s202, or m250. these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg2 cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a 3d homology model based on the hiv-1 rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m250 changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t184, s202 or m250 along with pre-existing lvdr substitutions m204v/i ± l180m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose (10 8 pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at 0, 24, or 48 h postinfection (pi). later treatment times were not evaluated. treatment at 24 or 48 h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than 10-fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by >90%; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing 10 9 -10 10 genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at 24 h pi, viral replication in spleen exceeded 10 9 genomes/g while liver and bone marrow yielded 10 8 genomes/ml. in comparison, titers in other tissues ranged between 10 5 and 10 6 genomes/g and blood yielded 10 4 genomes/ml at 24 h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately 10 10 genomes/g at day 5, then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days 7-9 at 10 8 genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv 24 h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl-4 laboratory. earl kern 1 , kathy keith 2 , robert jordan 2 , dennis hruby 2 , debra quenelle 2 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st-246, a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st-246 for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about 0.1-0.5 um of st-246. for in vivo studies, st-246 was administered once daily by oral gavage to mice using 100 mg/kg for 5, 7, 10, or 12 days beginning 4 or 24 h after intranasal inoculation with vv or cv. st-246 was highly effective (p < 0.001) in preventing mortality due to vv or cv even when treatment was delayed up to 24 h post-infection. a dosing duration of 5 days was adequate for vv infected mice, but duration of 7 days or longer was required for efficacy in cv infected mice. when st-246 was given once daily for 14 days at 100, 30, or 10 mg/kg daily at 24, 48, or 72 h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with 50 mg/kg of st-246. on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st-246 treatment successfully reduced virus titers from 3 to 5 logs 10 in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st-246 has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he16, in a mouse model when administered later in infection (oliphant et al., 2005. nat. med. 11, 522) . in this study, virus was demonstrated to be in the brains of hamsters at 5 days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. 5 dpi with 100 mg/kg of humanized monoclonal antibody, he16, survived wnv disease, whereas, 37% of placebo-treated hamsters survived ( *** p < 0.001). if administered at 2 dpi, 100% survived. we tested the hypothesis that he16 is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain 5 dpi using convectionenhanced delivery through a cannula implanted into the brain. the he16 was detected in the cns, but none was detected in the kidney. the survival of he16-treated hamsters was 88% as compared to 22% of placebo-treated animals ( *** p < 0.001). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he16 administered i.p. at 5 dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih, and grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with 50% effective concentrations (ec50s) of less than 100 nm against lassa fever virus and with ec50s of less than 2 nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein 1 , pramila walpita 1 , allison groseth 2 , heinz feldmann 2 , ramon flick 1 1 university of texas medical branch, department of pathology, galveston, tx, usa; 2 national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to 70%. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level 4 (bsl-4) agent. to avoid bsl-4 containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t7 minigenome rescue system (without the need for recombinant vaccinia virus mva-t7) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl4 agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t7) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl4 conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level 4 (bsl-4) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp30. anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire '76 strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec50 values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and 4-aminoquinoline compounds. acknowledgement: supported by nih r44 ai052917-02 and r01 ai066502-01. human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv31 episomal dna content. two of the 19 compounds caused a dose-dependent reduction in hpv31 episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼1000 copies of hpv31 per cell, compounds 2-ta and 4-ta both reduced hpv31 dna levels to below 50 copies per cell after 48 h incubation with 10 m compound. an alternative taqman tm amplicon within the hpv31 e7 gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv31 dna to the human apoe gene also demonstrated dramatic loss of hpv31 dna copies, further confirming our initial observations. finally, cells were treated with polyamides for 48 h, polyamide-containing media was removed, and episome levels were followed for 8 days. at day 6, 4 days after removal of polyamide and 3 days after sub-culturing of the cells, viral episome levels remained approximately 60% lower than control samples. by day 8, 6 days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh3 groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t7 rna polymerase from ribonucleoside 5 -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: (1) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; (2) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by 0.5-0.8 • c per modification; and (3) sirnas with borano-modifications were shown to be at least 10-fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: (1) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; (2) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; (3) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and (4) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy-2 -fluoro-2 ,3dideoxydidehydroadenosine (fd4ap, gs9148), a novel ntrti, with an excellent resistance profile toward hiv-1 variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d4) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt-2 cells. mitochondrial toxicity was assessed in hepg2 cells by measuring mitochondrial dna content. results: the d4 scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy-2 ,3dideoxydidehydroadenosine (d4ap) inhibited hiv-1 replication with a mean ec50 of 2.1 m and an 0.8-, 2.9-, and 2.9-fold change in potency against viruses containing m184v, k65r, and 6 thymidine analog mutations (tams), respectively. further exploration of d4ap was limited by its mitochondrial toxicity, which was then addressed in 2 ways: (i) preparation of l-d4ap or (ii) 2 fluorine substitution. l-d4ap exhibited an ec50 of 5.9 m but had substantially reduced potency (14-fold) toward m184v mutant viruses. fd4ap exhibited an ec50 of 12.3 m, with 0.8-, 1.2-, and 3.5-fold change in potency against viruses containing m184v, k65r, and 6 tams, respectively. no cytotoxic effects were measured up to 1 mm in mt-2 cells and no effects on mitochondrial dna were detected up to 300 m in hepg2 cells for both fd4ap and l-d4ap. conclusion: fd4ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv-1 strains. compared to d4ap, the 2 -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd4ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr-5 antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr-5 antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr-5 antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr-818, prevents the reactivation of dormant virus by inhibiting both the hiv-1 p24 and viral mrna production from latently hiv-1-infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr-818 was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr-818 was able to inhibit the ser5 phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk9 subunit of p-tefb, which is recruited to the viral promoter by the hiv-1 tat protein. since we did not find an inhibition at the level of cdk9 activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr-818 must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález 1 , sandra franco 2 , miguel a. martínez 2 , luis menéndez-arias 1 1 centro de biología molecular "severo ochoa", csic-uam, madrid, spain; 2 fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons 69-70 together with substitutions t69s and t215y in the reverse-transcriptase (rt)-coding region of hiv-1 are known to confer resistance to zidovudine (azt) and stavudine (d4t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and 10 additional mutations related to drug resistance (including t215y) showed >10-fold increased unblocking activity on azt-and d4t-terminated primers, when compared with an rt containing the insertion together with mutations t69s and t215y, in an otherwise wild-type bh10 sequence. these results suggested that other mutations associated with the complex t69sss/t215y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d4t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues 1-135 and wild-type bh10 rt residues 136-560 (l1 rt), or residues 1-135 of the bh10 rt and 136-560 of the ss rt (l3 rt), as well as an l1 rt variant with the substitution t215y (l2 rt) and an l3 rt derivative with t69sss (l4 rt). additional rts containing mutations m41l, a62v, or k70r together with the combination t69sss/t215y in the bh10 background were also obtained. atp-mediated excision activities on azt-and d4tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv-1. the l2 rt containing mutations t69sss/t215y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l1, l3, and l4 rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m41l, a62v and k70r) were responsible for the increased excision activity shown by rts bearing mutations t69sss and t215y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the 6-trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi-033, was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the 50% effective concentration (ec50) was 0.30 ± 0.18 g/ml, while the cc50 (50% cytotoxic concentration) of chi-033 for vero cells was more than 100 g/ml, thus resulting in a selectivity index of >500. detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi-033resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses 42 hiromichi tanaka 1 , kazuhiro haraguchi 1 , hiroki kumamoto 1 , takao nitanda 2 , masanori baba 2 , ginger e. dutschman 3 , yung-chi cheng 3 1 school of pharmaceutical sciences, showa university, tokyo, japan; 2 center for chronic viral diseases, kagoshima university, kagoshima, japan; 3 school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for 4 -carbon-substituted nucleosides has led to a new strategy, ring opening of 4 ,5 -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the 4 -position. as a result of this study, 4 -ethynylstavudine (4 -ed4t) was found to be more anti-hiv active than the parent compound stavudine (d4t). this compound (4 -ed4t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d4t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k103n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of 4 -ed4t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. 46 (1), 44]. in focus of the anti-hiv potency the ivs provide a 10-100-fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist 20-500-fold amplifying the anti-hiv-1 selectivity up to is∼10000. augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least 10-fold amplification of anti-hiv-1 potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as 2,6,-diamino-(9-[2-phosphonomethoxyethyl]purine (pme-dap) and 2-amino-6-cyclopropylamino-(9-[2phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv-1), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic 2,6diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv-1. the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p24 reduction assays in mt-2 cells infected with hiv-1. for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were >3 logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt-2 cells, the selectivity indexes are more than 10-fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv-1 in vitro. victor kuz'min 1 , eugene muratov 1 , anatoly artemenko 1 , ludmila koroleva 2 , vladimir silnikov 2 , v. lozitsky 3 , a. fedchuk 3 1 a.v. bogatsky physical-chemical institute, odessa, ukraine; 2 institute of chemical biology and fundamental medicine, novosibirsk, russian federation; 3 ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv-1, % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r 2 = 0.836, q 2 = 0.788). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd4 receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv-7 (vermeire et al., 2002. virology 302, 342-353) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd4 down-modulating and antiviral potencies of more than 25 cada analogs have been described (vermeire et al., 2003. mol. pharmacol. 63, 203-210) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. 1) . the anti-hiv and cd4 down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa05), an isomer of cada, was synthesized by cyclization of 1,5,7-triazabicyclo-[4.4.0]dec-5-ene (tbd) (fig. 1 ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. 1) . the anti-hiv and cd4 down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga 30602, usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code 1bl3. building of molecules and primary modeling was done with sybyl 7.1 on a silicon graphics onyx3 (r14000) workstation. the program gold 3.0 (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver 1.1, a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga 30602, usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein 1 , alexander serbin 2 , alissa bukrinskaya 1 d.i. ivanovsky institute of virology, moscow, russia; 2 health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd50). the antiviral effect of the compounds was analyzed in lymphoblastoid mt-4 cells and in hela cd4+/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv-1 replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed 1 h after infection. the anti hiv-1 effect of the compounds was gradually decreased if they were added 1 and 2 h after infection, no inhibition was observed when the compounds were added 4 h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt-2 cells or stimulated primary cd4+ t-cells were incubated with graded concentrations of tfv or cbv for 14 h (h); then extracellular drug was removed by washing. cells were further incubated without drug for 0-24 h and then infected with hiv-1 (iiib or bal). p24 was quantified on day 2; inhibition of hiv-1 replication due to intracellular drug persistence (pc50) was determined relative to a standard ec50. decay of intracellular dp/tps in cd4+ t-cells was measured using lc/ms/ms. results: in mt-2 cells, the pc50 value for tfv 12 h after drug removal remained unchanged relative to the ec50 (<3-fold shift) whereas the pc50 for cbv shifted >65-fold, indicating less persistence of cbv. in cd4+ t-cells, the pc50 value for tfv also showed a minimal shift relative to the ec50 (2.4-fold) 24 h after drug removal. cbv showed a much larger relative shift (>243-fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd4+ t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, 21 h versus cbv-tp, 5 h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt-2 cells and primary activated cd4+ t-cells, tfv had the longest persistence of antiviral activity. in cd4+ t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis 5320 is a phosphorothioate oligonucleotide with a molecular structure of t2g4t2. the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v3 loop of gp120. mapping studies with monoclonal antibodies targeting epitopes in and around the v3 loop have been used to define the binding site of isis 5320. in vitro, isis 5320 inhibits all laboratory and clinical strains of hiv-1 and hiv-2 tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr4 and ccr5 chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis 5320 resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis 5320 suggest that it may be an excellent anti-hiv topical microbicide. isis 5320 was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr5-tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd4-dependent and cd4-independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis 5320 used in combination with other microbicidal compounds. the results of these studies suggest that isis 5320 may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only 60% effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv-1 drugs, protease inhibitors (pi) are the unique to inhibit the hiv-1 production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv-1 production when pi have been removed in hiv-1 chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p24 gag protein was measured starting from 12 h after drug removal yet virus infectivity remained 1 log lower than control up to 1 week. inhibition of hiv-1 replication was still 48% and 18% upon amprenavir 20 and 4 m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd4/cd8 ratio above the levels of untreated controls until the 5th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt-2 cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt-2 cells. results: capravirine resistance selections were initiated at 1 nm (ec 50 of 1.5 nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l100i mutation emerged in rt and additional passaging led to v179d and f227c mutations at higher concentrations (100-300 nm). further increases in capravirine concentrations led to the emergence of a l100i + v179d + f227c triple mutant, which confers >1000-fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l100i, v179d and f227c were present on the same genome, with l100i as the primary mutation, and f227c and v179d were acquired sequentially at later passages. through sgs analysis, a l100i + k166r + v179d + f227c quadruple mutation on the same genome was also observed at higher capravirine concentrations (>1000 nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k103n nor y181c mutations in rt were observed. instead, the l100i mutation was initially acquired, followed by mutations f227c and v179d. addition of the k166r mutation to the triple mutant genome, l100i + v179d + f227c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi 1 , adeyemi falegan 2 1 medical microbiology, university college hospital, ibadan, nigeria; 2 dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = 0.87] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, 50,000-sample size, selected through simple random sampling technique. the mean age is 25.5 years old. relative risk [rr] calculated is 3.1, i.e. rr >1, indicating that the factor is the risk factor, and the confidential interval [ci] for rr at 95% significant level is 2.61 < 3.1 < 3.90 from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which 20% had the unprotected intercourse once or more, 25% had protected sex while 25% were not sure of using protection means. while, 20% have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu 2,3 , jacob barnor 1,6 , norio yamamoto 4 , kahoko hashimoto 1,2 , naoko miyano-kurosaki 1,2 , koichi ishikawa 5 , naoki yamamoto 5 , david ofori-adjei 6 , hiroshi takaku 1,2,7 1 department of life and environmental sciences, chiba institute of technology, chiba, japan; 2 high technology research center, chiba institute of technology, chiba, japan; 3 japan foundation of aids prevention; 4 department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; 5 aids research center, national institute of infectious disease, tokyo, japan; 6 department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; 7 bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv-1 replication in a long-term transduced pbmcs, h9, and jurkat cell culture assays (9 weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv-1 replication was similarly suppressed in the control cells expressing only vif shrna for about 3 weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of 6 avian and 12 porcine influenza a viruses isolated in germany between 1981 and 2002. virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only 3 of the 12 porzine isolates but all 6 avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between 1981 and 1987. all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g16e, s31n, and r77q in the matrix protein 2 (m2). additionally, l27a was detected in two h1n1 strains. s31n and/or l27a are well known amino acid substitutions in m2 that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin (2,3-dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin-3-thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h5n1) in mdck cells. antiviral activity was studied by virus yield assay (ec90), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h5n1) virus replication in mdck cells and compounds spiii-5h and spiii-5cl were most active (ec90 5.5 g/ml, cc50 >100 g/ml and si > 18). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai-15433 and noi-ai-30048 from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/8/34 (h1n1) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb1 segment mrnas, or the 3 -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by 1-3 orders of magnitude in a dose-dependent and sequence-specific manner over a period of 2 days. two of the p-pmo, targeting the pb1 translation start site region (pb1-aug) and the 3 terminus of np vrna (np v3 ), were evaluated by endpoint dilution (tcid50) or elisa assays against another h1n1 strain (a/wsn/33), as well as a/memphis/8/88 (h3n2) and a/thailand/1(kan-1)/04 (h5n1). the pb1-aug arp-pmo generated over 85% specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of 10-20 m. the np v3 p-pmo yielded similar results, with the exception of considerably lower efficacy against the h3n2 strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h1n1 and h5n1 fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep-2 cells were infected with hsv-1 strain us in dose 5 ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of 10 −4 m in experimental samples, or without them in control samples. then cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/1/68 (h3n2) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of 10 −3 m during the study of their anti-influenza action. diaza-18crown-6 and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza-18crown-6 derivatives that contain 2-oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv-1 by 13 and 29%, respectively. both of these compounds inhibited replication of influenza virus on 1.7 log 10 tid 50 aza-15crown-5 did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza-15crown-5 derivatives that contain 2-amino-3-phenyl-propanoyl-or 5-benzyloxy-3-oxapentyl-fragments decreased amount of cells infected by hsv-1 with virus-specific intranuclear inclusions by 41 and 47%, respectively. first compound inhibited replication of influenza virus on 1.5 log 10 tid 50 and the second one decreased virus amount on 2.0 log 10 tid 50 . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # 3147) . robert w. sidwell 1 , kevin w. bailey 1 , min-hui wong 1 , donald f. smee 1 , dale l. barnard 1 , shanta bantia 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx-1812, rwj-270201) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h1n1, h3n2) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to 6 h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n9 neuraminidase for over 24 h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to 48 h after virus exposure was protective to mice infected with influenza a (h1n1) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection 1 h pre-virus exposure in separate experiments to mice infected with an influenza a (h5n1) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of 20 and 10 mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao 2 ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to 72 h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h5n1) virus infections. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hiroshi saitoh 1 , naoko miyano-kurosaki 1,2 , hiroshi takaku 1,2 1 department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; 2 department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv2neo-u6 plasmid vectors and pvl1393based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u6 pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and 24 h later the cells were infected with either a/pr8 or b/ibaraki virus at a moi of 0.01. at 72 h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, 1992) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., 2004) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with 5-10 ld 50 of a/aichi/2/68 (h3n2) influenza virus. pre was administered by i.n. instillation 3 h before infection in the dose of 10 mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days 6 and 9 after infection their levels reached 140-160% of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h 2 o 2 , o 2 •− and no production by alveolar macrophages; the generation of ros and rni peaked on day 9. pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s11, a natural herb extract, ji-sun kwon 1 , hyun-jeong lee 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea the antiviral activity of s11, one of the traditional korean medical herb extract, against influenza virus was investigated. the 50% effective concentration (ec50) using plaque reduction assay was 31.25 ug/ml and the mean 50% cytotoxic concentration (cc50) using wst-1 assay in the mdck cells was 334 ug/ml. oral gavage treatment of the s11 to balb/c mice infected with a/pr/8/34 (h1n1) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s11 was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s11 is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku 1,2,4 , takayuki abe 3 , hitoshi takahashi 1 , naoko miyano-kurosaki 1,2 1 department life environ. sci., chiba inst. tech., chiba, japan; 2 high tech. res. center, chiba inst. tech., chiba, japan; 3 res. inst. microbial dis., osaka university, osaka, japan; 4 bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il-6, in murine raw 264.7 macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/8/34 (h1n1) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, 2 weeks apart, at a dose of 1.1 × 10 8 pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with 10 8 pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant 1 , annie lebel 2 , nathalie goyette 2 , guy boivin 2 , jean-marc juteau 1 , phil wyde 3 1 replicor inc., laval, que., canada; 2 chuq-chul and laval university, st. foy, que., canada; 3 baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥40 bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep 9 (a 40 mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep 9 was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep 9 aerosol has excellent characteristics for lung deposition and aerosol treatment with rep 9 was well tolerated and highly effective against infections with influenza a both in prophylaxis and 24 h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no1-ai-15437. irina v. alymova 1 , y. sudhakara babu 2 , allen portner 1 1 virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn 38105, usa; 2 biocryst pharmaceutical, inc., birmingham, al 35244, usa bcx 2798 is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 2798's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type 1 (rsev(hpiv-1 hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk 2 cells at moi 0.1 in the presence of increasing (from 100 to 3200 m) concentrations of compound; infected 129×1/svj mice were treated with 10 mg/kg/day of bcx 2798 twice for five days. treatment started 4 h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e527k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv-1 hn) . several mutations in hn gene of rsev (hpiv-1 hn) were selected in an animal model after the second passage of virus from mice treated with bcx 2798. two mutations, n23s and p29q, were located in the cytoplasmic domain of hn protein; mutations n173s and t553a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk 2 cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx 2798 both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv-1 hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx 2798 may not lead to appearance of clinically significant variant of viruses. kie-hoon jung 1 , michelle mendenhall 1 , lawrence m. blatt 2 , robert w. sidwell 1 , brian b. gowen 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates (30-50%) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon-1. both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level 4 containment facilities. acknowledgement: supported by contract no1-ai-30048 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding 5mono-, 5 -di-, and 5 -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers (2004) has been successfully applied to a number of different nucleosides (azt, d4t, dda, d4a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe 1b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of 4 -substituted nucleosides, including 4azido-cytidine, 4 -azido-uridine, and 2 ,3 -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (>1000-fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., 2004. mini-review. med. chem. 4, 371-382 . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns5b polymerase, an essential enzyme for viral replication. the micromolar ns5b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns5b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns5b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale 1 , dimitrios vlachakis 1 , maria chiara barbera 1 , romano silvestri 2 , colin berry 3 , johan neyts 4 1 cardiff university, the welsh school of pharmacy, cardiff cf10 3 xf, uk; 2 universita' degli studi "la sapienza", dipartimento di studi farmaceutici, 00185 roma, italy; 3 cardiff university, cardiff school of biosciences, cardiff cf10 3us, uk; 4 rega institute for medical research, k.u. leuven, b 300 leuven, belgium hepatitis c is a viral infection that affects 170 million people worldwide, including 4 million in the united states and 8 million in europe. the virus establishes a chronic infection in 55-85% of cases and 20% of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound 1 is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of 1 where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., 3650 wesbrook mall, vancouver, bc, canada v6s 2l2 hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns5b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin-4(3h)-one derivatives a series of novel 6-bromo/6,8-dibromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)-benzenesulphonamides were synthesized by condensation of 2-substituted benzo[1,3]oxazine-4-ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). 6-bromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)benzenesulphonamide (sps-ii) and 4-(4-oxo-2-phenyl-4hquinazolin-3y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv-1 in acutely infected mt-4 cells at a concentration of approximately 10 g/ml, while not being toxic to the host cell at a concentration of 60 or >125 g/ml (selectivity index: 8 and >12), respectively. in huh 5-2 cells sps-i inhibited hcv rna synthesis at ec50 of 8 g/ml, while at cc50 for cell growth 32 g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv-1, hsv-2 or vaccinia (vv) at a concentration of 50 g/ml, while not being toxic to the cells up to a concentration of 400 g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct 06511, usa hcv nonstructural protein ns5b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns5b have been used to investigate ns5b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns5b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns5b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns5b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns5b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns5b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than 170 million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than 100,000 cases of liver cancer annually worldwide and accounts for at least 40% of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p7 protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes 1a strain h77c and genotype 1b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype 2a strain jhf-1 10 um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells 1 h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after 72 h incubation (p < 0.05 and <0.01, respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype 1a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣-2b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣2b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼3-fold), or the ribavirin-interferon ␣2b combination (∼2-fold). similarly, the castanospermine-interferon ␣2b double combination was more synergistic than the castanospermine-ribavirin combination (∼5-fold), or the ribavirin-interferon ␣2b combination (∼3.3-fold). the combinations of celgosivir-interferon ␣2b or castanospermine-interferon ␣2b led to significant decreases in the ec50s of celgosivir (up to >20-fold) and castanospermine (up to >50-fold). the effective ec50s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣2b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣2b or with interferon ␣2b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about 50% of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm-107, amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (>100 m). celgosivir and castanospermine were synergistic with both amantadine and nm-107, with volumes of synergy between 60 and 150 m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm-107. mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul 120-749, korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately 9.6 kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately 3010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. the structural proteins include c, e1, e2, and p7 and the nonstructural (ns) proteins include ns2, ns3, ns4a, ns4b, ns5a, and ns5b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns5b protein, the viral rna-dependent rna polymerase (rdrp), and ns3 protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like 2 (prk2) regulates virus rna replication. hcv rna replication was inhibited when prk2 expression level was down-regulated by using a prk2-specific sirna. in this study, we investigated the anti-hcv effect of prk2 inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk2 inhibitors suppressing the endogenous prk2 activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk2 inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk2 inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk2 can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk2 activity will provide an opportunity to interfere with hcv rna replication. haitao guo 1 , tianlun zhou 2 , ju-tao guo 1 , andrea cuconati 3 , anand mehta 1 , timothy block 1 1 drexel university college of medicine, doylestown, pa, usa; 2 nucleonic inc., irvine, ca, usa; 3 hepatitis b foundation, doylestown, pa, usa more than 400 million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated 500,000 deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad38 cells, a hepg2-derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the 5 terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the 3 terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad38 produced and accumulated cccdna, which became detectable between 7 and 8 days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec50 >3 logs less than unmodified hpmpa in mt-2 cells by p24 reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc-5 human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for 6, 24 and 48 h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc-5 cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after 24 h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every 2 days for 0-10 days. levels of the diphosphates declined slowly with a t 1/2 of 5-10 days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of 15-me-hdp-cdv (1) was tested in liver s9 fractions from various species. in mouse and human liver s9 fractions, compound 1 was completely stable for 90 min while 15-20% of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound 1 retained full activity compared to hdp-cdv and compound 2 showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([1-(s)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (<5%), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs 1 that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs 2ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., 1994) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs 2 were converted to drug by cellular (caco-2, hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of 1 in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with 50% inhibitory concentrations ranging from 0.1 to 0.5 and 10 m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than 30-fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r43ai056864 and u01ai061457. lawrence trost 1 , bernhard lampert 1 , lloyd frick 2 , merrick almond 1 , george painter 1 1 chimerix, inc., durham, nc, usa; 2 dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv-1. however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx012) decreases the apparent ec 50 value against hiv-1 by up to 40-fold. cmx012 was esterified to produce cmx052 in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx052). rats were given oral doses of 10, 30 and 100 mg/kg cmx052 daily for 7 days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx012 (c max > 4 g/ml), the biologically active deesterified form of cmx052. in conclusion, cmx052 is absorbed after oral administration, converted to cmx012, and has a good preliminary toxicity profile. these results support the development of cmx052 for the treatment of drug resistant hiv infection. zhiqian wu 1 , julie breitenbach 2 , ulrika erickson 1 , john hilfinger 3 , john drach 2 , gordon amidon 1 1 department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; 2 school of dentistry, the university of michigan, ann arbor, mi, usa; 3 tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. 5 -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco-2 cell homogenate (t1/2: 2-40 min). 5 -l-isoleucyl and 5 -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept1mediated uptake as well as caco-2 permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada1. preliminary animal study shows 5 -lisoleucyl vidarabine results in >10-fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [9-␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types 1 and 2. it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼5-fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about 10-fold by combination with 1 m 2 -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on 5 -substituted vidarabine analogs (lipper et al., 1978. mol. pharmacol. 14, 366-369), we determined that minimizing metabolism of vidarabine by synthesizing 5 -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a 10-fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig 1 , anne baum 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active 3,5-bis-tertbutyl-6-fluoro-cyclosal-d4t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d4t with the antiviral active nucleoside analogues like d4a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen 1 , wolfgang fendrich 1 , tilmann schulz 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany, 2 rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d4a. for the pyrimidine-type series the fluorescent nucleoside m 5 k was synthesized as well as the dideoxy-compound dm 5 k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of 5000 compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv-1 and hiv-2 strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar 1 , richard mackman 1 , adrian ray 1 , dean boojamra 1 , lijun zhang 1 , deborah grant 1 , hon hui 1 , jennifer vela 1 , neil parkin 2 , yolanda lie 2 , kirsten white 1 , michael miller 1 , gerry rhodes 1 , manoj desai 1 1 gilead sciences, foster city, ca, usa; 2 monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine (gs9148) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs9148, resulting in the identification of gs9131 (ethylalaninyl phenyl ester of gs9148). results: gs9131 exhibits potent anti-hiv-1 activity in primary lymphocytes and t-cell lines (ec 50 < 150 nm). low cytotoxicity (cc 50 > 100 m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs9148 was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv-1 reverse transcriptase (rt) (k i = 0.8 m). unlike ddi, d4t, or d4fc, neither gs9148 nor its prodrugs inhibited mitochondrial dna replication in hepg2 cells. in a phenosense assay, gs9148 retained its full activity against hiv-1 variants with k65r, m184v or l74v mutations in rt. viruses with ≥4 thymidine analog mutations showed ≤2fold reduced susceptibility to gs9148, a shift that was smaller than that of any other tested nrti. following an oral dose of 3 mg/kg gs9131 in dogs, the bioavailability of prodrug exceeded 20%, resulting in high intracellular levels (9.0 ± 2.3 m) and prolonged retention (t 1/2 > 24 h) of gs9148 diphosphate in blood lymphocytes. conclusions: both gs9148 and its prodrug gs9131 exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs9131 possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs-7340 and gs-9131 are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; 9-[(2-phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd4ap (phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine), respectively. both gs-7340 and gs-9131 exhibit potent in vitro anti-hiv-1 activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver >10-fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs-7340 and gs-9131 is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs-7340 and gs-9131 was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was 3500-fold. sds-page analysis showed the presence of a prominent protein band at 29 kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec 3.4.16.5, cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ 14 c]gs-7340 and [ 3 h]difluorophosphonate resulted in the labeling of an identical 29 kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs-7340, approximately 6-9-fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside 5 -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to 30% by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of 0.1-0.2 m. the rate of passive drug release in physiological solution was from 33 to 50% of loaded drug during the 24-h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only 1-2 h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. 5 -triphosphate of 1-␤-d-ribofuranosyl-1h-1,2,4-triazole 3-carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin 5 -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong 1 , ebrima gibbs 2 , joel oger 2 1 department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; 2 neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the 2 (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately 60-70-folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky 1 , i. kravchenko 2 , v. larionov 2 1 research anti-plague institute, odessa, ukraine; 2 national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: (1) prolongation of the drug's action; (2) drug's concentration is maintained in therapeutic range; (3) there is no trauma to patient's skin while using td; (4) removing tts from the skin immediately stops drug's entering to the organism; (5) first-pass effect in the liver is reduced; and (6) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and 1.2-propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr (1 mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: 10 mg/mouse of dmso or 10 mg/mouse of octanol or 1 mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from 1 day before infection to 10th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/8/34 (h1n1), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of 10 −1 to 10 −7 . deaths of animals were recorded for 14 days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on 1 log 10 tid 50 . dmso and octanol did not demonstrate such activity. mikhail dobrikov 1 , serguei vinogradov 2 , barbara ramsay shaw 1 1 department of chemistry, duke university, durham, nc, usa; 2 center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv-1 reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano-2 ,3 -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r01 al52061 to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a 384 microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic50 stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, 08916 badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type 1 (hiv-1) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv-1 protease, p17-p24, was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv-1 protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv-1 repressor and hiv-1 protease, lambda phage replicated up to 1000-fold more efficiently than in cells that did not express the hiv-1 protease. this assay responds appropriately to well-known hiv-1 proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns3 protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) 3c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t20) and reverse transcription (sustiva, uc781 and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé 1 , kerstin angner 1 , anna overby 1 , david stein 2 , patrick iversen 2 , ramon flick 1 1 utmb, department of pathology, galveston, tx, usa; 2 avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about 20 subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. 1b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. 1a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic50 on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us-1 interacting with the hep-2 sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant #3147. we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve 10,000× to 100,000×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding 25 mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant #3147. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., 2002) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with 5-iodo-2 -deoxyuridine, to give intermediate 5-alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: (1) the side chain; (2) the bicyclic base; and (3) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec50 1 nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the 3 hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new 3 substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., 2001) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with 5-iodo-2 -deoxyuridine, to give intermediate 5alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis-5-nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc 50 , g/ml); (b) antiherpetic activity (hsv-1 strain kupka, ic 50 , g/ml); and (c) selectivity index (ratio of cc 50 to ic 50 ) and the structure of 48 n,n -bis-5-nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r 2 = 0.816-0.991, q 2 = 0.637-0.868). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of 5-arylethynyl-2deoxyuridines (1a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv-1. unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel 2 -deoxy (1a) and 2 -arabino (1b) uridine derivatives possessing different 5-arylethynyl substituents. dinucleosides 2 containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides 1a. these compounds as well as some of 1a and 1b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; 3,4dihydroxybenzohydroxamic acid) and trimidox (tx; 3,4,5trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a 43-and 39-fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by 50% in the resistant strain (cdv wt: 0.03 m, r: 1.28 m/gcv wt: 3.17 m, r: 122.85 m). this compared to only a moderate increase in drug dose required for dx and tx to achieve 50% inhibition in the resistant strain (dx wt: 20.71 m, r: 28.88 m/tx wt: 7.12 m, r: 11.59 m), corresponding to a 1.4-and 1.6-fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from 1-(phosphonomethoxyethyl)-cytosine (pme-c), 1-(phosphonomethoxyethyl)-5-bromo-cytosine (pme-5brc), 1-(phosphonomethoxyethyl)-5-fluoro-cytosine (pme-5fc), 9-(phosphonomethoxyethyl)-2,4-diaminopurine (pme-dap) and 9-(phosphonomethoxyethyl)-2-amino-4cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec 50 values of 0.2, 0.3, and 0.03 m in hff cells infected with hsv-1, hsv-2 or hcmv, representing a 12-26-fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec 50 values of 0.7, 0.2, and 0.6 um in hff cells infected with hsv-1, hsv-2, and hcmv, representing a 16-43-fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type 6 and 8, is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach 1 , katherine z. borysko 1 , jiri zemlicka 2 , john c. drach 1 1 biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; 2 karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., 1998. j. med. chem.) and second generation (zhou et al., 2004. j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were 10-20-fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 50 's in plaque assays = 0.35 and 21 m, respectively for wild-type (wt) and synguanol-resistant (1982r) virus]. likewise a ul97 deletion mutant (prichard et al., 1996. j. virol.) was resistant to both first and second-generation compounds (ic 50 's = 2.1 and 0.25 m in wt; 100 and 15 m in ul97 del , respectively for synguanol and cyclopropavir). ul97 from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m460i and c603y. because hcmv with either m460i or the related c607y mutation alone was sensitive to synadenol and synguanol (baldanti et al., 2002 . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad169 bac with one, the other, or both mutations in ul97. as expected, the two strains with the single mutations were 3-to 6-fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol (0.5-to 1-fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir (6to 8-fold) but less so than observed in the 1982r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul97 is required for activity against hcmv and that is likely that two mutations in ul97 are required for significant resistance. acknowledgement: supported by grants p01-ai46390 and r44-ai054135 from nih and funds from the university of michigan. svitlana zagorodnya 1 , nadiya nesterova 1 , inna alexeeva 2 , larisa palchikovskaya 2 , galina baranova 1 , alexander kobko 2 , anna golovan 1 1 zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; 2 institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. 1 and 2, as well as n-glycosidic derivatives no. 3 against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no 1, no 2, and no 3 was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc-100 r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of 1000, 500, 250, 125, 64, 32, 16, 4 , 1, 0.5 and 0.1 g/ml. the concentrations that inhibited the quantity of live cells on 50% (id50) were equal to substances no. 1-750 g/ml, no. 2-625 g/ml and no. 3-125. the minimal inhibiting concentration (mic) of nos. 1, 2, and 3 was equal to 1 g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with 28.0 up to 14. hence, the index of selectivity (is) was equal to 750 and 625 for triazine bearing tricyclic bases nos. 1 and 2, for n-glycosidic derivatives-125. in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti 1 , l. nencioni 1 , g. macrì 2 , c. nucci 2 , u. benatti 3 , m. magnani 4 , e. garaci 5 , a.t. palamara 1 1 department public health sciences, university rome "la sapienza," rome, italy; 2 department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; 3 department exp. medicine biochemistry section, university genova, genoa, italy; 4 inst. biochemistry, university urbino, urbino, italy; 5 department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c4), with increased hydrophobic properties, inhibited in vitro parainfluenza-1 and hsv-1 replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c4 administration in hsv-1-induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with 2 × 10 5 pfu/ml of hsv-1. gsh-c4, dissolved in a saline solution (150 mm, 100 ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using 0.5% fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c4 treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c4 treated animals, conjunctival hsv-1 titre, assayed by tcid50 on day 4 post-infection, was significantly reduced as compared to that of control animals (mean = 1.4 × 10 3 units/ml versus 1.1 × 10 5 units/ml, n = 10 for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c4-treated animals versus placebo animals (mean = 3.5 × 10 3 units/ml versus 8.5 × 10 5 units/ml, n = 5 per group). such results highlight the antiviral activity of gsh-c4 in vivo and suggest that topical gsh-c4 treatment could be considered as complementary therapy of hsv-1-induced keratitis. debra quenelle 1 , deborah collins 1 , latisha pettway 1 , caroll hartline 1 , james beadle 2 , w. wan 2 , karl hostetler 2 , earl kern 1 1 university of alabama school of medicine, birmingham, al, usa; 2 department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated 16-20 weeks later with 4700 pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through 21-28 days and then gradually decreased to undetectable levels by 8 weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or 10 mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated 24 h after infection and continued for 28 days. cidofovir (cdv) at 20 mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at 30 or 10 mg/kg beginning 24 h post-viral inoculation and 30 mg/kg when treatment was delayed until 48 h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep 9 has potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep 9 and compared to saline-treated infected control mice. compound was administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting at 2 days prior to infection. mice were infected with 5 × 10 5 pfu mcmv on day 0, at 3 h post-treatment. sera were collected at −22 h, at 36 hpi, and at 3 dpi for elisa analysis of ifn␥ production. spleens and livers were collected at 3 dpi for determination of virus titers. at 3 dpi, virus titers in the spleens and livers were significantly reduced by rep 9 treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep 9 but not in saline treated, infected mice or in rep 9 treated, uninfected mice. ifn␥ levels in mice treated with rep 9 peaked at 36 hpi compared to 72 hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep 9 with two oligonucleotide analogs that do not activate tlr-9 mediated immune stimulation suggests that direct antiviral activity of rep 9 and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep 9 analog exhibited even greater antiviral activity than rep 9 while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. kathy keith 1 , joseph maddry 2 , namita bansal 2 , kochurani jacob 2 , secrist john 2 , earl kern 1 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: (1) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and (2) to investigate new substituents at the purine 2-and 6-positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over 100 analogs of pmea substituted at the purine 2-or at the adenine n-6 site have been synthesized and evaluated for activity against orthopoxvirus infections. many n-6 substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine 2-position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) 2-amino-n(6)-cyclopropyl pmea had ec 50 values of 0.03-0.07 m and ode 2-hydroxylamino-n(6)-cyclopropyl pmea had ec 50 values of 0.3-1.6 m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc 50 values of 30-70 m giving selective indices of 200-1000. these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin (2,3-dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin-3thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel 4-[(1,2dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested,4-[(5-methyl-1,2-dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2-pyrimidiny)-benzene sulphonamide(spiii-5me), was the most active compound with an ec50 value of 18 mol, compared with cdv, which had an ec50 of 20 mol against vaccinia virus. all the compounds were non-toxic (>300 lm)using a neutral red uptake assay. substitution of a halogen atom in 5th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no1-ai-30049 from virology branch, niaid, nih, usa. evgeny belanov 1 , svetlana kotovskaya 2 , nikolay bormotov 1 , sergey balakhnin 1 , olga serova 1 , nataliya perova 2 , zina baskakova 2 , galina dzhumbaeva 2 , valerii charushin 3 , oleg chupakhin 2 1 state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; 2 ural state technical university, yekaterinburg, russia; 3 institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of 1,2,4-benzotriazine ( fig. 1) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk-2 cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c-3 and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project #1989. yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., 4575 sw research way-suite 230, corvallis, or 97333, usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st-246) via high throughput screening (yang et al., 2005. j. virol. 79, 13139-13149) . marker rescue of st-246 resistant variants localized compound resistance to the f13l gene that encodes a major orthopoxvirus envelope protein (p37), which is required for extracellular viral particle formation. p37 participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st-246, we examined the effects of st-246 on the production of the extracellular viral particles in bsc40 cells infected with recombinant vaccinia virus containing a gfp-tagged p37 protein. in the presence of st-246, iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st-246 prevented the intracellular localization of p37 to the late endosome compartment as measured by confocal microscopy. in contrast, st-246 did not affect localization of p37 expressed from a st-246 resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p37 in transfected cells. these results suggest that st-246 inhibits an unknown virusspecific activity that requires f13l. this work underscores the exquisite specificity of st-246 and supports continued development of st-246 as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. 4575 sw research way, suite 230 corvallis, or 97333, usa st-246 is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st-246 resistant variants was measured by fluctuation analysis and found to be 3.58 × 10 −6 . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f13l gene. the spectrum of mutations that confer st-246 resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by 100-fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f13l coding sequence by error-prone pcr and the mutated f13l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f13l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st-246 resistant, f13l proteins. sequence analysis of the compound resistant f13l alleles identified numerous silent mutations scattered throughout the f13l coding sequence and 27 point mutations leading to amino acid changes that clustered around amino acid positions 258-302 within the f13l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: (1) mutations in at least 7 positions within f13l can confer resistance to st-246 and (2) st-246 resistant mutations cluster to a 58 amino acid domain in a region of the protein of unknown function. several 5-substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec 50 values below 10 m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the 5 position had ec 50 values below 15 m against both hsv-1 and hsv-2, whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec 50 values below 10 m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the 5 position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al 35233, usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec 50 values below 1 g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec 50 values of 0.55 and 1.5 g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec 50 values 14-fold greater than a tk positive isolate. similarly, a tk negative strain of hsv-1 was 90-fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus 8 (hhv-8), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated 24 h after infection and at doses as low as 17 mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a314t and a684v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a314t and the a684v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a314t and the a684v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a314t and the a684v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a684v mutant that showed only a two to three-fold decrease in susceptibility towards the 3-hydroxy-2-phosphonomethoxypropyl (hpmp) purine derivatives, the a314t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a314t mutation appeared before the a684v substitution, and the latter mutation only occurred in conjunction with a314t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of 50 mg/kg/day for 5 days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a314t + a684v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg 1 defence science and technology laboratories (dstl), salisbury, wiltshire, uk; 6 coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor 9. there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna (7909) could provide protection from lethality against vacv in balb/c mice when administered up to 7 days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of 7909 ranging between 15 and 100 ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, 1, 2, 3, or 4 days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was 60% when treated with 30 ug 1 day post-challenge (70 mld50 challenge). a survival rate of 80% was observed when treated with 50 ug 2 days post-challenge (40mld50 challenge). the delay of treatment to either 3 or 4 days post-challenge was ineffective, indicating that the window of opportunity for delivery of 7909 is within 2 days. multiple doses of 7909 were used to attempt to extend this window of opportunity, delivering 7909 twice within a 3-day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl 2005. department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of 5 × 10 5 plaque forming units (pfu), was lethal for 50% of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c-1 cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c-1 cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of 10 7 pfu, or an intracranial injection of 5 × 10 4 pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed 24 h later by intraperitoneal treatment with (n)-mct (2x/day for 7 days) or cidofovir (1x/day for 2 days). (n)-mct treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival (placebo). statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100 mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the 30 and 100 mg/kg/day treatments significantly reduced day 5 virus titers and lung weights, and the 100 mg/kg/day treatment reduced lung consolidation. cidofovir (100 mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no-1-ai-15435 from the virology branch, niaid, nih. chelsea byrd 1 , elena sbrana 2 , shu-yuan xiao 2 , marina siirin 2 , robert tesh 2 , dennis hruby 1 , robert jordan 1 1 siga technologies, inc., corvallis, or, usa; 2 university of texas medical branch, galveston, tx, usa st-246 is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st-246 against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than 1 pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st-246 at 100 mg/kg once per day for 14 days protected ground squirrels from a lethal challenge with 100 and 1000 pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day 12 post-infection. when treatment with 48, 72, and 96 h, 100% protection was observed in the 24, 48, and 72 h groups, and 66% protection in the 96 h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st-246 at 0, 24, 48, and 72 h postinfection appeared grossly and microscopically normal. thus, st-246 appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen 1 , guang yang 2 , dennis hruby 2 , erik de clercq 1 , robert jordan 2 , johan neyts 1 1 rega institute for medical research, k.u. leuven, belgium; 2 siga technologies, inc. corvallis, or, usa st-246 is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st-246 maps to a mutation in v061, which is homologous to vaccinia virus f13l (yang et al., 2005. j. virol.) . the latter encodes the envelope protein p37 required for production of extracellular virus. deleting f13l resulted in a virus ( f13l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with 2 × 10 5 pfu of wr-vac resulted in 59 ± 11 pox tail lesions per mouse, the same inoculum of f13l-vac caused no lesions (p < 0.001). athymic nude (nu/nu) or scid mice inoculated iv with 2 × 10 5 pfu f13l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f13l-vac was 22 ± 1 days as compared to 9 ± 1 days for wr-vac-infected mice (p < 0.001); scid mice survived the infection. we next studied whether f13l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with 5 × 10 5 pfu f13l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with 5 × 10 5 pfu of wr-vac. all placebo animals, but none of the f13l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f13l-vac and were infected one week later with 2 × 10 4 pfu of wr-vac by the iv route. placebo animals developed an average of 14 ± 4 pox tail lesions; no lesions developed in the f13l-vac animals (p < 0.001). in a third set of experiments, nmri mice were inoculated iv with either 2 × 10 4 pfu of f13l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with 2 × 10 4 pfu wr-vac. the placebo group developed an average of 11 ± 8 lesions as compared to 1.2 ± 0.7 lesions in the f13l-vac mice (p < 0.05). f13l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan 1 , travis lim 1 , shuan coughlin 1 , dexin qiu 1 , zhen liu 1 , dave stein 2 , decheng yang 1 1 the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; 2 avi biopharma, inc., corvallis, or, usa background: coxsackievirus b3 (cvb3) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p007) is conjugated to the 5 end of the oligomer to improve its delivery into cells. these features make p007-conjugated pmos (p-pmos) promising candidates for the inhibition of cvb3 infection. methods: total 8 p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp1, viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the 8 p-pmos, p-pmo-6, targeting the internal ribosomal entry site in the 5 utr, showed the most potent anti-cvb3 ability in a dose-dependent manner. both infected hela and cardiomyocytes hl-1 cells treated with p-pmo-6 showed drastically reduced vp1 production and 3.0 log decreases in viral titres as compared to the controls. cell viability assay revealed that 83 and 89% of treated hela and hl-1 cells were still alive as compared to 11 and 10% of control-treated cells and 47% antiviral activity still existed after 5 days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb3 replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb3 isolate 97-927 (ic 50 , g/ml); (b) cytotoxicity in hela cells (cc 50 , g/ml); and (c) selectivity index (si = ratio of cc 50 to ic 50 ), and structure of 21 [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r 2 = 0.831-0.931, q 2 = 0.674-0.896) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb3 activity. ivanka nikolova 1 , roumena petkova 2 , boris atanassov 3 , stoyan chakarov 2 , angel s. galabov 1 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 scientific technological service, ltd., sofia, bulgaria; 3 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b1 was carried out. the wild-type disoxaril-sensitive strain (connecticut 5) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b1 and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by 9 passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b1 genome coding for the capsid protein vp1 was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp1 gene of coxsackievirus b1 was performed with the blast alignment tool. distinct alterations in the vp1 locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. 2749-2751 and an insertion of uuu at nt.2769) were observed. high-degree similarity (97%) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only 91%. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative 3-d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu-23 (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b1 infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic50 values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of 5methylthio-3-aryl-isoxazole-4-carbonitrile derivatives whose in vitro anti-coxsackievirus b1 activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds 5-methylthio-3-[4-(3-phenyl-1-propoxy)phenyl]isoxazol-4-carbonitrile (on-7) and 5-methylthio-3-[4-(4-phenoxy-1-buthoxy)phenyl]isoxazol-4-carbonitrile (on-10) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on-7 was effective when added within 1 h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on-10 when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time 0). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on-10 primarily interferes with coxsackie b1 cellular attachment. at a concentration 100 times the id 50 , inhibition of adsorption of coxsackievirus by on-10 was complete, while similar concentration of on-7 had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤1 (tgf-␤1) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in 30 non-infected c57/bl6 mice. in subsequent experiments, any animal expressing a permeability value greater than 2 sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice 8 days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of 3000, 1000, or 300 ng/kg/day of tgf-␤1 or with drug vehicle once daily via the i.p. route on 7 and 8 days post-virus inoculation (dpi), and then assayed for bbb permeability on 8 dpi. sixty-two percent (8/13) of placebo-treated animals had abnormally high permeability values, while animals treated with 3000 and 1000 ng/kg/day of tgf-␤1 had 29% (2/7) and 57% (4/7) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with 300 ng/kg of tgf-␤1 (0/8) expressed abnormally high permeability values, which was significantly lower (p < 0.01) than placebo-treated animals. these results suggest that tgf-␤1 may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. and contract no1-ai-15435 from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents 3-5 days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for 10 days beginning 4 h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine (7 mg/kg) and mk-801 (1 mg/kg), and one ampa-receptor antagonist, gyki 52466 (10 mg/kg), were administered twice daily (b.i.d.) on 4 though 9 days post-virus inoculation (dpi). gyki 52466 slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki 52466, slightly improved hamster survival (p ≤ 0.05) when treatment began on 5 dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning 5 dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using 105.3 tissue culture infectious doses of wnv. tissues of infected and control animals from 3 to 11 days post-viral injection (dpi) were fixed by cardiac perfusion using 4% paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc 1020). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as 3 and 5 dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of 7-day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly 50 million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type 2 and 3 in vero (monkey kidney cells) and hepg2 (human hepatoma cells), showing inhibitory concentration 50% (ic 50 ) values in the range 0.14-1.1 g/ml and selectivity indexes (cc 50 /ic 50 ) in the range 1000-10000. in the present work we studied the mode of action of these polysulfates against denv-2 in vero and hepg2 cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at 1 h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv-2 adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( 35 s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv-2 rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv-2 was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. 2 -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus 17d (ec 50 = 3.1 g/ml in cpe reduction assays and >98% reduction at 5 g/ml as assessed by qpcr) and dengue fever virus type 2 (ec 50 = 16.5 g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (>98% at 5 g/ml as assessed by qpcr and >98% by plaque reduction neutralization test at 50 g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin 1 , andrew shiryaev 1 , igor moiseev 1 , v sabynin 2 , larisa rustamova 2 , alexandr petkevich 2 1 state technical university, samara, russia; 2 institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an-3739 strain) on the vero cells culture. different level of antiviral activity was shown by 15 compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen 1 , donald smee 1 , min-hui wong 1 , anne pace 1 , kie-hoon jung 1 , kevin bailey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon 1) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., 2005 . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon-1 for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for 7 days, and ifn alfacon-1 was administered intraperitoneally, once per day for 10 days. treatments were initiated 1-5 days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin (5-10 mg/kg/day) with ifn alfacon-1 (5-10 mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no1-ai-15435 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler 1 , laure deflubé 1 , andrew vaillant 2 , jean-marc juteau 2 , ramon flick 1 1 department of pathology, university of texas medical branch, galveston, texas, usa; 2 replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep 9 is a 40 mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp12) as well as the wild-type rvfv (zh501), to test the ability of rep 9 to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep 9 at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep 9 analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep 9 is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep 9. adriana garozzo 1 , rossella timpanaro 1 , aldo stivala 1 , gianna tempera 1 , christian c.c. cutrì 2 , angelo castro 1 1 department of microbiological sciences, university of catania, via androne 8, 95124 catania, italy; 2 department of pharmaceutical sciences, university of catania, viale a. doria 6, 95125 catania, italy our previous studies described the synthesis and the antiviral activity of 3,4,5-trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (is-2) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is-2 addition to poliovirus type 1 infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ 3 h]uridine-labeled poliovirus to hep-2 cells in the presence or absence of is-2. no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is-2 action. justin julander 1 , aaron olsen 1 , john morrey 1 , lawrence blatt 2 , kristiina shafer 1 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c3h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc-83. virus was detected in the brain 2 days post-viral injection (dpi). brain titers increased to a peak titer of 10 9.5 50% cell culture infectious doses per gram tissue (ccid 50 /g) on 4 dpi, maintained a titer of 10 9 ccid 50 /g through 7 dpi, and dropped slightly to 10 8.6 ccid 50 /g by 8 dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in 100% survival, whereas all placebotreated animals died by 9 dpi. syrian golden hamsters were infected with 10 3 ccid 50 wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as 4 dpi. the majority of deaths occurred on 5 dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring 4 dpi. interferon alfacon 1 (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < 0.001 as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer 10 4.8 ccid 50 /g) as compared with placebo (mean titer 10 9.5 ccid 50 /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. justin julander 1 , kristiina shafer 1 , john morrey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. 183, 1431 -1436 . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a 10 −4 dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken 3 days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was 66% mortality in animals challenged with the virus up to 9 dpi. virus titers in the liver peaked 6 dpi as determined by qrt-pcr. a significant increase in serum levels of alt (6 dpi), alkaline phosphotase (6 dpi) and bilirubin (6 dpi), and a significant decrease in amylase (8 dpi), albumin (6 dpi), and glucose (6 dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon 1, a consensus interferon. ribavirin and ifn alfacon 1 both significantly (p < 0.001) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < 0.05) and ifn alfacon 1 (p < 0.01) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon 1. viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv40). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv40 has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv40 in vitro has already been demonstrated (andrei et al., 1998. antimicrob. agents chemother. 41, 587-593) . here, the activity of a new class of anp's, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4diaminopyrimidines, against polyomaviruses was assessed. confluent uc1-b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, 2pta2 or lid-1, while bsc-1 cells were infected with either the primate sv40 strain a2895, the sv40 pml-1 strain ek or the sv40 pml-2 strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after 4-6 days (murine polyoma virus) or 5-7 days (sv40). hpmpo-dapy (2,4-diamino-6-(r)-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy (2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv40 strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara 1 , sylvester mosley 1 , brian bakke 1 , joshua sadler 1 , katherine seley(radtke) 1 , sunny zhou 2 1 university of maryland-baltimore county, baltimore, md, usa; 2 washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional 5 -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv-1, thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc 27708-0346, usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv-1 reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of 2 3 -dideoxycytidine triphosphate (ddctp) resulted in a 28-fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox-2 transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr5 as a co-receptor for hiv-1 infections revealed a novel approach to hiv-1 treatments and preventions. ccr5, a member from the family of 7tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr5 antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv-1 gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p55 cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt-4 cells were labeled with [3h]-leucine or myristic acid, and 2 h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. 18-20 h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt-4 cells before infection or 1-2 h later and at concentration 2-6 ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire 1 , thomas bell 2 , sreenivasa anugu 2 , noah duffy 2 , roger le grand 3 , erik de clercq 1 , dominique schols 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 department of chemistry, university of nevada, reno, usa; 3 service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec 50 : 0.3-3.2 m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv-1, irrespective of chemokine receptor preference (r5, x4, r5/x4). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd4 receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd4 receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv-1 subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv-2 strains examined. in addition, cada potently inhibited sivmac251 infection of pbmcs isolated from macaques (ec 50 : 1.6 m). comparable results were obtained in human cells infected with sivmac251. flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd4 receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate 1,2benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus 3) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within 48 h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi (0.01) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for 48 h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole-3-carbanol [i3c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin (833), purvalanol a (570), and i3c (1000) were greater than the positive controls acv (250) and paa (60). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a 2,6,9-trisubstituted purine, primarily inhibits cdk1; and i3c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: (1) polyelectrolyte-specific interferon induction and immunomodulation; (2) electrostatic-selective prevention of viruses absorption on plasma membranes; (3) membrane-targeted blocking of post-absorption steps (fusion); (4) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as (5) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the (1) and (2) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a1/a2) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv-1 absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr5/cxcr4. as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = 20,000 (against hiv-1 strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova 1 , alexander ivanov 1,2 , georgii galegov 3 , valeria andronova 3 , maxim jasko 1 1 engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; 2 centre for medical studies, university of oslo, moscow, russia; 3 ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines, were synthesized, and their efficacies against hiv-1 and hsv-1 were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines were evaluated as substrates towards hiv-1 reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the 3 -end of the growing dna chain. available to this virus. jev genome is an approximately 11-kb single-stranded positive-sense rna that has a cap structure at its 5 terminus but lacks a poly(a) tail at its 3 -terminus. the coding region of the genome is flanked by 5 -and 3 -untranslated region (utr). the 3 -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-(2-aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the 3 -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns5 protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns5 protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand 3 -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the 3 -end 83-nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev 3 -utr 83-nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva 1 , reneta toshkova 2 , milena nikolova 3 , reneta tsvetkova 3 , stefka antonova 4 , ivana roeva 1 , munnever sokmen 5 , bektas tepe 5 , medine gulluce 6 , fikrettin sahin 6 , atalay sokmen 5 1 institute of microbiology; 2 institute of experimental pathology and parasitology; 3 institute of botany, bulgarian academy of sciences; 4 faculty of biology, department of microbiology, sofia university, sofia, bulgaria; 5 faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; 6 faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of 134 plant products, obtained from 67 bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples (11.2%) inhibited flu reproduction, and twelve samples (7.5%) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in 55.5%; the antiviral effect was connected with antioxidant activity in 100%; the anti-flu effect was associated with immunogenic properties in 100%; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy 2,6-di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose 10-3 m was studied in vitro on the model of a/hong kong/1/68 (h3n2) reproduction in tissue cultures of chorioallantoic membranes of 12 days old chick embryos. anti-hsv activity in doses 10-4 m was studied on models of reproduction of hsv-1 on cell culture hep-2. compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity (39.4 and 61% of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). 3,5-dihydrazine-carbonyl-2,6-dimethylpyridine showed high anti-hsv (52%) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # 3147 and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb 1 , angelina trifonova 1 , stephan filipov 2 , angel s. galabov 1* 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus 1 replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's 50% inhibitory concentration for poliovirus 1 (mahoney) was found to be 0.188 g/ml in the cpeinhibition test and 0.041 g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type 1 strain, lsc-2ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva-9, the six coxsackie b viruses and 6 echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by 1 lg ranged from 0.01 to 1.0 g/ml. selectivity index was greater than 100 and even greater than 1000 for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva 1 , angel s. galabov 2 1 department of medical physics and biophysics, medical university, sofia, bulgaria; 2 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/2/68 (h3n2) (2.0 of ld50) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary (5 days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee 1 , ji-sun kwon 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s22 was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s22 showed potent antiviral activities against a/pr/8/34 (h1n1) influenza virus with the 50% effective concentration (ec50) values of 62.5 g/ml and the 50% cytotoxic concentration (cc50) values of 673.86 g/ml in mdck cells. treatment with the s22 appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s22 treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s22 was extracted with chcl3, etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or 2 h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by 50, 61, 72, and 94%, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by 25, 100, 72, and 100%, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by 92, 6, 67, and 100%, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by 94, 78, and 94%, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the 20th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april 29, 2007 and will end on thursday afternoon, may 3, 2007. all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the 20 th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs 05-04-49500 and russian ministry of sciences (lot 11). key: cord-351365-dc9t3vh3 authors: todt, daniel; walter, stephanie; brown, richard j. p.; steinmann, eike title: mutagenic effects of ribavirin on hepatitis e virus—viral extinction versus selection of fitness-enhancing mutations date: 2016-10-13 journal: viruses doi: 10.3390/v8100283 sha: doc_id: 351365 cord_uid: dc9t3vh3 hepatitis e virus (hev), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. to date, hev infections can only be treated with ribavirin (rbv). major drawbacks of this therapy are that rbv is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to rbv. one of the proposed modes of action of rbv is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. these transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. in contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. emergence of these mutant viruses can lead to therapeutic failure. consequently, the onset of rbv treatment in chronically hev-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. following an overview of rna viruses treated with rbv in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of rbv on hev intrahost populations, and how hev is able to overcome lethal mutagenesis. hepatitis e virus (hev) was first described as novel agent responsible for enterically transmitted non-a, non-b hepatitis by reyes and colleagues in 1991 [1] . this was 35 years after the first documented epidemic outbreak (1955) (1956) ) of a retrospectively identified hev infection-transmitted via the fecal-oral route-in new delhi, india [2] . hev is a nonenveloped single-stranded rna virus with a 7.2 kb genome of positive orientation. three open reading frames (orfs) encode for: (1) the nonstructural proteins (orf1), comprising a methyltransferase, a papain-like cysteine protease, a helicase, and an rna-dependent rna polymerase (rdrp), connected by a y-domain and a hypervariable region (hvr); (2) the capsid protein (orf2); and (3) small proteins whose functions are not yet completely understood (orf3) [3] . the viral subgenomic rna is comparable to mammalian mrnas, flanked by a 5 -methylguanine cap and a 3 -poly(a) tail. hev has recently been taxonomically reassigned to the genus orthohepevirus in the family of hepeviridae [4] . differences in the sequences of isolates led to the current classification into seven genotypes, four of which infect humans. hev-1 and hev-2 (i.e., genotypes 1 and 2) are solely human pathogens and are mainly transmitted orally by feces-contaminated drinking water. in 1972, rbv was described as a broad-spectrum antiviral against several dna and rna viruses [27] . since then, numerous studies have reported on the in vitro antiviral properties of rbv. figure 1 provides an overview of a selection of rna viruses against which rbv was shown to be active: hepatitis c virus (hcv, flaviviridae), dengue virus (denv, flaviviridae), respiratory syncytial virus (rsv, paramyxoviridae), influenza a and b virus (orthomyxoviridae), chikungunya virus (chikv, togaviridae), poliovirus (picornaviridae), hantaan virus (bunyaviridae), and lassa virus (arenaviridae) [28, 29] ( figure 1 ). for further reading we would like to refer to other reviews like [29] [30] [31] . studying multiple viruses from the family flaviviridae, crance et al. investigated the in vitro antiviral properties of rbv against 11 flaviviruses including denv, japanese encephalitis virus (jev), and yellow fever virus (yfv). inhibition of virus replication was observed for all tested flaviviruses [32] . furthermore, effectiveness of rbv could be confirmed in vivo for yfv using a hamster model by administering early upon infection [33, 34] . however, these effects could not be confirmed in a nonhuman primate model [33] . therefore, further studies are required to evaluate the possible application of rbv as a treatment option for yfv. here, the dosage as well as the time points of treatment represent the major hurdles, which need to be overcome [33] . additionally, hemorrhagic fever-causing viruses, which are categorized into the families of arena-, bunya-, and togaviridae, were demonstrated to be susceptible to inhibition by rbv ( figure 1 ). for example, for lassa virus the antiviral efficiency of rbv was proven both in vitro and in vivo in guinea pigs and monkeys [35, 36] . hantaviruses (i.e., hantaan virus) and phleboviruses (i.e., rift valley fever virus, rvf) were also shown to be susceptible to rbv treatment [35] . in a mouse model for hantaan virus, an increase of survival and milder signs of disease were described [35] . in experimental rvf infections of mice and hamsters, rbv led to a prevention of death, delay of death, or the onset of milder symptoms, depending on the time point of administration [35] . in general, higher doses of rbv were needed to inhibit flaviviruses compared to arena-, bunya-, and hantaviruses [33] . for chikv, rbv also demonstrated antiviral effects, although to a lesser extent when compared to interferon-α (ifn-α). nevertheless, a synergistic effect of rbv and ifn-α could be demonstrated in vitro [37, 38] . moreover, the antiviral properties of rbv could be shown for members of the picornaviridae: both foot-and-mouth disease virus (fmdv) [39, 40] and poliovirus (pv) [41] were inhibited by rbv (figure 1 ). demonstrated antiviral effects, although to a lesser extent when compared to interferon-α (ifn-α). nevertheless, a synergistic effect of rbv and ifn-α could be demonstrated in vitro [37, 38] . moreover, the antiviral properties of rbv could be shown for members of the picornaviridae: both foot-and-mouth disease virus (fmdv) [39, 40] and poliovirus (pv) [41] were inhibited by rbv ( figure 1 ). figure 1 . antiviral properties of ribavirin (rbv) against rna viruses. the broad-spectrum antiviral properties of rbv have been reported for several rna viruses. depicted is a selection of the different viral families and the respective genus and species. viruses for which rbv was clinically approved are highlighted with an orange box. viruses for which lethal mutagenesis or increased mutation rate was proposed as a possible rbv mechanism are indicated in blue. while displaying broad antiviral activity against a wide range of rna viruses, clinical data on the application of rbv are still limited and restricted to only a few viruses. initially, rbv was considered as a treatment option for influenza a and b virus infections. however, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent [42, 43] . due to lack of conclusive data from clinical trials, coupled with the development of alternative antiviral therapies, rbv has never been approved for the treatment of influenza virus. nonetheless, lassa virus, hcv, and rsv are prominent examples of viruses for which rbv has received approval as an antiviral compound for clinical application [44] . rbv was shown to be effective in treating patients suffering from lassa fever [45] and can be administered orally, intravenously, or as pre-or post-exposure prophylaxis [46] ., in 1998, rbv was approved by the food and drug administration (fda) as a treatment option for hcv [47] and was, in combination with pegylated ifn-α, the standard treatment for chronic hcv infection for over two decades [48] . it has been shown that after failure of a monotherapy with ifn-α alone, a combination therapy with rbv is more effective than subsequent repetition of ifn-α monotherapy [49] . however, the sustained virological response (svr) rates varied among genotypes, and dual therapy was associated with severe side effects [48] . nowadays, rbv is no longer the standard-ofcare anti-hcv therapy, and has been replaced by direct-acting antivirals (daas). initial trials for the treatment of rsv infection showed a reduced duration of hospitalization and requirement of mechanical ventilation [50, 51] . a routine use of rbv in rsv-infected children is not recommended; however, treatment can be considered for individual cases [50] . while displaying broad antiviral activity against a wide range of rna viruses, clinical data on the application of rbv are still limited and restricted to only a few viruses. initially, rbv was considered as a treatment option for influenza a and b virus infections. however, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent [42, 43] . due to lack of conclusive data from clinical trials, coupled with the development of alternative antiviral therapies, rbv has never been approved for the treatment of influenza virus. nonetheless, lassa virus, hcv, and rsv are prominent examples of viruses for which rbv has received approval as an antiviral compound for clinical application [44] . rbv was shown to be effective in treating patients suffering from lassa fever [45] and can be administered orally, intravenously, or as pre-or post-exposure prophylaxis [46] . in 1998, rbv was approved by the food and drug administration (fda) as a treatment option for hcv [47] and was, in combination with pegylated ifn-α, the standard treatment for chronic hcv infection for over two decades [48] . it has been shown that after failure of a monotherapy with ifn-α alone, a combination therapy with rbv is more effective than subsequent repetition of ifn-α monotherapy [49] . however, the sustained virological response (svr) rates varied among genotypes, and dual therapy was associated with severe side effects [48] . nowadays, rbv is no longer the standard-of-care anti-hcv therapy, and has been replaced by direct-acting antivirals (daas). initial trials for the treatment of rsv infection showed a reduced duration of hospitalization and requirement of mechanical ventilation [50, 51] . a routine use of rbv in rsv-infected children is not recommended; however, treatment can be considered for individual cases [50] . taken together, since its first description as an antiviral in 1972, rbv has been shown to be active against a broad range of rna viruses. however, due to limited clinical trial data supporting its in vivo efficacy, clinical applications are currently limited to a minority of viruses. the broad antiviral effect of rbv against numerous rna viruses suggests different modes of action for the molecule; indeed, several antiviral mechanisms have been described in the past [29, 52] and are summarized in figure 2a . among the indirect mechanisms, a t-cell-mediated effect was described for hcv ( figure 2a) . here, the balance of t helper cells was changed by switching from a t helper type 2 phenotype to a t helper type 1 [29] . in a study by hultgren et al., an inhibition of in vitro t-cell proliferation as well as a change in secreted cytokines was observed [53] . simultaneously, alanine transaminase (alt) levels in serum were reduced with no change in hcv titers [53] . furthermore, an early switch of a t helper type 1 immune response to a t helper type 2 immune response was associated with disease progression and the development of chronicity [54] . thus, rbv restored the t helper 1 phenotype needed for balanced expression and secretion of cytokines produced from type 1 and 2 t helper cells [29] . another example where an immunomodulatory effect was described for rbv is in rsv infection. it was proposed that a t helper type 2 cytokine response initiated the cascade leading to airway hyper-reactivity, which in turn can be blocked by rbv treatment [55] (figure 2a) . another indirect mode of action for rbv is the inhibition of the cellular inosine monophosphate dehydrogenase (impdh), which was already proposed in 1973 [56] (figure 2a ). after uptake into the cell, rbv is phosphorylated to rbv mono-, di-, and triphosphate (rmp, rdp, and rtp, respectively). rmp represents a good mimic of inosine monophosphate (imp) and thereby inhibits the synthesis of imp to xanthosine monophosphate (xmp) by impdh. consequently, no guanosine monophosphate (gmp), and subsequently guanosine triphosphate (gtp), can be synthesized. in vitro, replication of measles virus in vero cells could be blocked by the addition of xmp, gmp, and to a lesser extent, also imp [56] , which underlines the mode of action of rmp. a linear correlation of the depletion of gtp pools and in vitro antiviral activity of rbv against human parainfluenza virus 3 and yfv was confirmed [57] . furthermore, the addition of guanosine to cell cultures restored the antiviral activity of rbv against gb virus b (gbv-b) [58] . in contrast, in vitro experiments with lassa virus and hantaan virus indicated that rbv did not primarily act via depletion of gtp pools for these two viruses [59, 60] . moreover, experiments with influenza a virus showed no linear correlation of intracellular gtp pools and viral replication with increasing concentrations of rbv [61] . additionally, the authors did not observe a complete restoration of influenza a virus replication after addition of guanosine [61] . no effect of guanosine or gmp on the antiviral effect of rbv against influenza a virus in mice could be demonstrated [62] . overall, these data suggest that other mechanisms for the mode of action of rbv exist. the influence of rbv on the expression of ifn-stimulated genes (isg) is controversial in the literature. most studies, both in vivo and in vitro, come from the hcv and rsv fields. rbv is able to increase the antiviral effects of an ifn-based therapy and restore ifn-responsiveness in hcv-infected livers [63] [64] [65] [66] . also, a direct, ifn-independent upregulation of isgs has been proposed [67, 68] . however, a recent study with hcv patients receiving rbv monotherapy showed a downregulation of abnormally preactivated isgs through chromatin remodeling and modulation of histone methylation, resulting in a higher liver susceptibility to ifn by lowering the baseline expression of certain isgs [69] . some rna viruses, as well as cellular mrnas, harbor a 7-methylguanosine cap structure at the 5 end [70] . the rbv-induced reduction of gtp pools within the cell was proposed to also have an effect on the capping efficiency of rna viruses (figure 2a ). for example, denv encodes for a 2 -o-methyltranferase at the n-terminus of the ns5 polymerase, termed ns5mtase dv . ns5mtase dv binds gtp and catalyzes the formation of a 5 cap structure [71] . after rbv treatment, less gtp is present and rtp was shown to compete for binding to the ns5mtase dv , thereby blocking the synthesis of the 5 cap [71] . likewise, rbv directly and strongly inhibited the viral mrna guanylyltransferase of vaccinia virus and thus prevented capping of nascent viral rna [72, 73] . however, this mechanism is controversially discussed in literature [72] [73] [74] [75] , and not all rna viruses display a 7-methylguanosine cap structure at the 5 end. therefore, this mode of action cannot account exclusively for the observed effects of rbv. another suggested mechanism is the direct impact of rbv treatment on the function of viral polymerases (figure 2a,b) . here, rtp is thought to directly inhibit viral rna replication by being recognized by the viral polymerase and thereby leading to chain termination or preventing the binding of other nucleotides important for elongation [76] . in a cell-free system, rtp was shown to inhibit the rna polymerase of influenza a virus [77] . moreover, inhibition of viral rna synthesis of vesicular stomatitis virus (vsv) in the presence of rmp, rdp, and rtp was described with the triphosphorylated form being the least active [78] . this would argue against a mode of action that is based on the incorporation of rtp in the nascent viral rna in vsv. in the same study, an inhibitory effect of rdp on la crosse virus rna synthesis was also reported [78] . interestingly, crotty et al. could demonstrate that rtp is indeed employed by pv rdrp, and that integrated rbv acts as mutagen [41] . another example of an effect of rtp on the viral polymerase is the case of reovirus. rankin et al. proposed that rtp binds close to the catalytic site of the transcriptase, thereby affecting the helicase function and subsequently lowering the binding affinity of viral rna [79] . as a consequence, elongation of the viral rna is inhibited. interestingly, no effect on the capping activity was demonstrated [79] . the nucleotide binding site of the polymerase is highly conserved among hcv genotypes, supporting this proposed mechanism [76] . indeed, in vitro analysis showed a minor decrease of hcv replication [52, 76, 80] . however, in clinical trials with rbv monotherapy, only a mild decrease of hcv replication was noticed [81, 82] . in recent years, a mutagenic effect of rbv via its incorporation into newly synthesized rna genomes, leading to viral extinction was described for several rna viruses ( figure 2b ). in contrast to dna viruses, the major characteristic of rna viruses is the occurrence of a cloud of related but genetically distinct variants in infected patients, often referred to as a quasispecies. however, the term "quasispecies" refers to a particular mutation-selection balance, with natural selection acting on the group rather than on the individual [83, 84] . it is not simply a surrogate for genetic heterogeneity [85] . while quasispecies behavior has been demonstrated experimentally in artificially expanded poliovirus populations in infected mice [86] , evidence is lacking for quasispecies' behavior in many viruses, including hev. these diverse intra-host viral populations are the result of the lack of proofreading activity of rdrp. however, due to this high variation, viral isolates are close to the error threshold, which would lead to reduction in viral fitness [87] . incorporation of rbv into newly synthesized rna genomes thereby increases the frequency of mutations in the population, pushing the virus over an error threshold and resulting in viral extinction. this mechanism of action for rbv has been described, at least in vitro, for fmdv [21] , poliovirus [28] , hcv [88] , gbv-b [58] , hantaan virus [89] , and hev [25, 26] . ever since the first reports by sidwell et al. describing rbv as a broad-spectrum antiviral [27] , there have been multiple discussions about its mechanisms of action. of course one has to always keep in mind that in vitro data, where most of the proposed models arose from, cannot just be translated into in vivo situations. remarkably enough, monotherapy with rbv is only potently effective against lassa virus [45] and hev [90, 91] . future studies should address questions regarding the biocompatibility of rbv and its availability in the targeted liver to investigate if intracellular concentrations can account for the different proposed mechanisms-for example, to outcompete cellular nucleoside triphosphates (ntps) for misincorporation. in summary, several mechanisms have been postulated for rbv activity. among these, there are indirect, immunomodulatory mechanisms and effects on impdh. furthermore, mechanisms on the virus itself were described by inhibition of the capping efficiency, the viral polymerase, and a mutagenic effect on newly synthesized rna genomes. rna viruses do not exist as a clonal population of genomes within the infected host, but rather diversify into a swarm of related but non-identical genome sequences [83] . this heterogeneous viral population-also referred to as mutant cloud, mutant swarm, or mutant spectra-is capable of better adapting to changing environmental conditions and rapidly evolving, during passage from host to host, due to its high heterogeneity. the concept of quasispecies was mainly developed by manfred eigen and peter schuster [92] . by demonstrating viral heterogeneity for fmdv [93, 94] and vsv [95, 96] domingo and colleagues and holland and colleagues were the first to extrapolate this concept to virology [84, 97] . these viral populations are the product of very high replication rates found in rna viruses, coupled with a lack of an rdrp proofreading function. for hcv, it is estimated that between 10 11 and 10 12 new virions are produced in one infected individual per day [98, 99] . estimates for hev do not currently exist, although comparably high replication rates can be assumed. there is data on 3 -end repair mechanisms identified in small rna viral polymerases [100] . in coronaviruses, for example, a 3 -to-5 exoribonuclease (exon) domain within the nonstructural protein 14 was identified as being essential for high-fidelity replication [101, 102] ; for hcv, pyrophosphorolytic and ntp-mediated nucleotide excision activity of the ns5b rdrp have been described as viral mechanisms for removing misincorporated bases [103, 104] . despite these reports, most rna viruses, and most likely also hev, do not have any real proofreading capability, causing an error-prone replication of viral genomes. together with the short generation times, this results in highly diverse intra-host populations [96] . as expected, hev also exists as a heterogeneous population within infected individuals [25, 26, [105] [106] [107] . early publications relied on the classical tools for detecting diversification of viral genomes, including restriction fragment length polymorphism (rflp) and haplotype profiling [105, 106] or clonal sequencing [107] to characterize hev intra-host diversity. recently, next-generation sequencing (ngs) methods have been utilized to study the distribution of snvs in hev genomes over time [25, 26] . hev and most other rna virus populations exist in close proximity to the so-called genomic error threshold, which defines a maximum error rate that still guarantees the maintenance and transmission of the genetic information of the master sequence [83, 84] . a replication and, most importantly, mutation rate beyond this extinction threshold causes a sharp reduction in the efficiency of transmission of the genetic information contained in the population master sequence to the next generation of viral progeny, a phenomenon sometimes referred to as error catastrophe [108] : the majority of genomes in the population are nonfunctional. broad-spectrum antiviral agents like rbv can cause increased mutation rates, and potentially can result in the extinction of the virus population in a process called lethal mutagenesis [108] . however, the mutated viral intra-host populations can acquire mutations accounting for drug resistance or decreased sensitivity to rbv as a direct consequence of the boosted complexity of the mutational spectra. this has been shown for several viruses like hcv [20] , fmdv [21] , pv [22] , sindbis virus [23] , and also for hev [24] [25] [26] . the dynamics of hev populations in patients under rbv therapy is not fully understood, but recent studies and reports from other rna viruses point to a dichotomy of opposing outcomes resulting from rbv therapy: rbv-induced lethal mutagenesis resulting in viral extinction versus the accumulation of mutations beneficial to the virus in the population, which can lead to therapeutic failure [25, 26] . as a consequence of the emergence of rbv-resistant mutations and subsequent treatment failure, clinicians could draw back on combination therapies to overcome or avoid this phenomenon. possible combinations are one mutagen and a conventional antiviral drug or using several rna mutagens in combination or sequence as proposed by perales and domingo [109] [110] [111] . hev is one of the pathogenic viruses that can currently only be treated with rbv as an off-label drug. ifn-α as an alternative therapy has been evaluated in small patient cohorts with limited success and considerable side effects [112, 113] . in addition, in vitro data suggests careful assessment of ifns when treating hev [114, 115] . considering high mortality rates of over 20% for genotype-1-infected pregnant women [116, 117] , the urgent need for extensive research in the field of novel anti-hev treatment regimens is required. patients who fail to achieve sustained virological responses after rbv therapy for hev have no further treatment options: this is particularly of importance in a solid organ transplant setting, as a reduction of immunosuppression beyond a certain level will lead to the rejection of the allograft [118, 119] , and hepatitis caused by hev cannot be impeded. recently, two independent studies were able to correlate rbv treatment failure with the emergence of novel single-nucleotide variations in the viral genome during treatment [25, 26] . both research groups identified a variant previously described, g1634r [24] , as well as other new variants, k1383n, d1384g, k1398r, v1479i, and y1587f, all in the polymerase region of orf1. in addition, todt et al. also determined nine additional snvs in orfs 2 and 3 [26] . in both studies, k1383n mutations emerged in several patients; additionally, an overall increase in viral intra-host heterogeneity could be shown [25, 26] . the authors demonstrated significant increases in the number of sites exhibiting snvs, synonymous as well as nonsynonymous, in viral populations after the first administration of rbv in nine patients. this phenomenon was observed for all orfs of the hev genome. interestingly, this increase in heterogeneity was reversible with a decline in the number of snv sites when rbv treatment was stopped. strikingly, none of the described variants that became dominant in the viral populations under treatment resulted in a decreased sensitivity to rbv when cloned into an hev subgenomic reporter replicon in tissue culture. only g1634r mutations altered the viral replication efficacy, increasing replication rates [24, 26] , while rbv sensitivity was unmodified [24, 26, 120] . why rbv treatment fails in some patients, while others are able to clear the virus under rbv monotherapy, remains an open question. rbv has been shown to block hev replication through a depletion of cellular gtp pools in cell culture model systems [121] , in addition to the strong mutagenic effect of rbv on the hev genome in vivo described above. rbv inhibits the impdh, thus causing a two-fold reduction of the intracellular gtp pools and increasing ctp and utp concentrations at the same time [122, 123] . hev genome replication is a cyclic process of alternating synthesis of negative-strand rna and positive-strand rna [124] . during the replication process, the extrinsically administered, rtp is randomly incorporated into the nascent negative-stranded rna as a result of pairing with either of the pyrimidine bases cytidine or uracil ( figure 2b, upper panel) . this negative-stranded antigenome rna then serves as a template for subsequent production of positive-stranded genomic rna. the rdrp subsequently incorporates, again randomly, a cytidine or uracil at rbv residues located in the antigenome template ( figure 2b, middle panel) . these stochastic incorporations lead to nucleotide substitutions in the newly synthesized viral genomes. additionally, rbv will also be incorporated in the positive-stranded rna genome, leading to increased amounts of replication-defective viral genomes packaged into the capsid, ultimately leading to an increase in frequency of replication-defective virions. additionally, new antigenome templates can be produced from defective positive-stranded genomes, so misincorporations are amplified in the replication process ( figure 2b, lower panel) . this results in the fixation of transitional substitutions in nascent rnas. transitional purine-to-purine (g<>a) or pyrimidine-to-pyrimidine (c<>t) nucleotide substitutions are preferentially enriched during rbv monotherapy, leading to the observed synonymous exchanges as well as to the amino acid replacements favorable for the survival of the viral population [25, 26] . whether rbv also inhibits the hev methyltransferase comparably to the direct inhibition of the vaccinia virus guanylyltransferase (see above), or if the rtp is incorporated as a cap analog [72, 73] (thus impacting correct translation) has not been investigated yet. the mutagenic effect of rbv-based therapy can have divergent effects on hev populations, which may impact the therapy success. on the one hand, rbv increases the mutation rate in the viral genome, driving the population towards its extinction threshold. in contrast, the increased variability in the viral population can result in selection of variants with improved replication fitness which become dominant in the viral population and are associated with therapeutic failure. these advantageous variants could be (i) a downregulation of the replication machinery, thus preventing the accumulation of more mutations, as shown from in vitro data when reverse engineering the k1383n variant into hev cell culture systems [25] , and (ii) an increase in viral polymerase fidelity as hypothesized by debing et al. for the k1383n variant-a mutant with a substitution in the f1-motif of the rdrp-which could hinder the incorporation of rbv into the viral genome. in fact, the lab of esteban domingo was able to dissect a multistep process of viral adaption to a mutagenic nucleoside analog in fmdv that led to an extinction escape by changing the fidelity of the polymerase [125] . hev is a life-threatening infection when immunosuppressed individuals fail to achieve an svr during rbv treatment. currently, clinicians do not have alternative therapy regimens available. recent studies have suggested that the heterogeneous viral population is able to acquire snvs that decrease rbv sensitivity [25, 26] . their data supports a conclusion whereupon the mutagenic effect of the broad-spectrum antiviral agent leads to increased heterogeneity in the intra-host viral population introducing a race between the virus trying to gain and accumulate beneficial variations and the mutagenic potential of rbv intended to drive the virus beyond an error threshold and thus into lethal mutagenesis resulting in viral extinction. hepatitis-e virus (hev)-the novel agent responsible for enterically transmitted non-a, non-b hepatitis infectious hepatitis in delhi (1955-1956): a critical study-epidemiology update on hepatitis e virology: implications for clinical practice international committee on taxonomy of viruses hepeviridae study group consensus proposals for classification of the family hepeviridae history and global burden of viral hepatitis pathogenesis and treatment of hepatitis e virus infection viral hepatitis in pregnancy-a study of its effect on maternal and foetal outcome hepatitis e virus infection in the hiv-positive patient hepatitis e virus infection in the hiv-positive patient world health organization hepatitis e (fact sheet) hepatitis e: an old infection with new implications hepatitis e in germany-an under-reported infectious disease hepatitis e virus and neurological injury guillain-barre syndrome associated with preceding hepatitis e virus infection evidence of hepatitis e virus breaking through the blood-brain barrier and replicating in the central nervous system hepatitis e virus infection as a direct cause of neuralgic amyotrophy guillain-barre syndrome following hepatitis e extra-hepatic replication and infection of hepatitis e virus in neuronal-derived cells selection of hepatitis c virus resistant to ribavirin foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe a single mutation in poliovirus rna-dependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity sindbis virus mutants resistant to mycophenolic acid and ribavirin a mutation in the hepatitis e virus rna polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients hepatitis e virus mutations associated with ribavirin treatment failure result in altered viral fitness and ribavirin sensitivity in vivo evidence for ribavirin-induced mutagenesis of the hepatitis e virus genome broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications mechanisms of action of ribavirin against distinct viruses approved antiviral drugs over the past 50 years ribavirin-current status of a broad spectrum antiviral agent interferon, ribavirin, 6-azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses treatment of yellow fever comparison of the inhibitory effects of ribavirin and interferon alfacon 1 on a yellow fever virus infection in syrian golden hamsters prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug molecular approaches for the treatment of hemorrhagic fever virus infections chikungunya: a re-emerging virus in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis action of mutagenic agents and antiviral inhibitors on foot-and-mouth disease virus the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen oral ribavirin treatment of influenza a and b ribavirin small-particle aerosol treatment of influenza intervention strategies for emerging viruses: use of antivirals effective therapy with ribavirin new opportunities for field research on the pathogenesis and treatment of lassa fever the role of ribavirin in the combination therapy of hepatitis c virus infection mechanisms of antiviral treatment efficacy and failure in chronic hepatitis c interferon and ribavirin vs. interferon alone in the re-treatment of chronic hepatitis c previously nonresponsive to interferon: a meta-analysis of randomized trials new options in the treatment of respiratory syncytial virus disease ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children ribavirin for the treatment of chronic hepatitis c virus infection: a review of the proposed mechanisms of action the antiviral compound ribavirin modulates the t helper (th) 1/th2 subset balance in hepatitis b and c virus-specific immune responses the expanding universe of t-cell subsets: th1, th2 and more reduced long-term respiratory morbidity after treatment of respiratory syncytial virus bronchiolitis with ribavirin in previously healthy infants: a preliminary report mechanism of action of 1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide (virazole), a new broad-spectrum antiviral agent the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase ribavirin induces error-prone replication of gb virus b in primary tamarin hepatocytes depletion of gtp pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on lassa virus activity of ribavirin against hantaan virus correlates with production of ribavirin-5 -triphosphate, not with inhibition of imp dehydrogenase mode of action of ribavirin: effect of nucleotide pool alterations on influenza virus ribonucleoprotein synthesis lack of interference of guanosine with ribavirin aerosol treatment of influenza a infection in mice effect of ribavirin on viral kinetics and liver gene expression in chronic hepatitis c ribavirin regulates hepatitis c virus replication through enhancing interferon-stimulated genes and interleukin 8 ribavirin improves early responses to peginterferon through improved interferon signaling hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells ribavirin enhances interferon signaling via stimulation of mtor and p53 activities ribavirin restores ifnalpha responsiveness in hcv-infected livers by epigenetic remodelling at interferon stimulated genes viral and cellular enzymes involved in synthesis of mrna cap structure a structural basis for the inhibition of the ns5 dengue virus mrna 2 -o-methyltransferase domain by ribavirin 5 -triphosphate the broad spectrum antiviral nucleoside ribavirin as a substrate for a viral rna capping enzyme the broad spectrum antiviral agent ribavirin inhibits capping of mrna ribavirin is not a functional mimic of the 7-methyl guanosine mrna cap the antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mrna cap structure in vitro the application and mechanism of action of ribavirin in therapy of hepatitis c inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate effect of phosphorylated ribavirin on vesicular stomatitis virus transcription studies on the mechanism of the antiviral activity of ribavirin against reovirus mechanism of action of ribavirin in the combination treatment of chronic hcv infection mechanisms of action of interferon and ribavirin in chronic hepatitis c: summary of a workshop antiviral action of ribavirin in chronic hepatitis c what is a quasispecies? historical origins and current scope the rna virus quasispecies: fact or fiction? quasispecies diversity determines pathogenesis through cooperative interactions in a viral population rna virus mutations and fitness for survival viral rna mutations are region specific and increased by ribavirin in a full-length hepatitis c virus replication system ribavirin causes error catastrophe during hantaan virus replication treatment of hev infection in patients with a solid-organ transplant and chronic hepatitis treatment of hepatitis e virus the hypercycle. a principle of natural self-organization. part a: emergence of the hypercycle nucleotide sequence heterogeneity of the rna from a natural population of foot-and-mouth-disease virus multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture high multiplicities of infection favor rapid and random evolution of vesicular stomatitis virus rapid evolution of rna genomes meeting on 'quasispecies: past, present and future hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy viral dynamics in hepatitis b virus infection a novel 3 -end repair mechanism in an rna virus discovery of an rna virus 3 →5 exoribonuclease that is critically involved in coronavirus rna synthesis implications of altered replication fidelity on the evolution and pathogenesis of coronaviruses ntp-mediated nucleotide excision activity of hepatitis c virus rna-dependent rna polymerase pyrophosphorolytic excision of nonobligate chain terminators by hepatitis c virus ns5b polymerase evidence for hepatitis e virus quasispecies the application of single strand conformation polymorphism (sscp) analysis in determining hepatitis e virus intra-host diversity hepatitis e virus quasispecies and the outcome of acute hepatitis e in solid-organ transplant patients theories of lethal mutagenesis: from error catastrophe to lethal defection counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment potential benefits of sequential inhibitor-mutagen treatments of rna virus infections antiviral strategies based on lethal mutagenesis and error threshold pegylated interferon-alpha for treating chronic hepatitis e virus infection after liver transplantation treatment of chronic hepatitis e in liver transplant recipients with pegylated interferon alpha-2b antiviral activities of different interferon types and subtypes against hepatitis e virus replication disparity of basal and therapeutically activated interferon signalling in constraining hepatitis e virus infection viral hepatitis during pregnancy incidence and severity of viral hepatitis in pregnancy the impact of hepatitis e in the liver transplant setting hepatitis e virus infection as a cause of graft hepatitis in liver transplant recipients mutation in the hepatitis e virus polymerase and outcome of ribavirin therapy ribavirin inhibits in vitro hepatitis e virus replication through depletion of cellular gtp pools and is moderately synergistic with alpha interferon metabolism of 5-amino-1-beta-d-ribofuranosylimidazole-4-carboxamide and related five-membered heterocycles to 5 -triphosphates in human blood and l5178y cells -beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide; a cytostatic agent hepatitis e virus replication involves alternating negative-and positive-sense rna synthesis a multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape acknowledgments: this work was supported by the german ministry for education and research (bmbf) through a ginaico grant 16gw0105. e.s. was further supported by the helmholtz centre for infection research. the authors declare no conflict of interest. key: cord-326217-ji0njeha authors: saleh, maged; rüschenbaum, sabrina; welsch, christoph; zeuzem, stefan; moradpour, darius; gouttenoire, jérôme; lange, christian m. title: glycogen synthase kinase 3β enhances hepatitis c virus replication by supporting mir-122 date: 2018-11-27 journal: front microbiol doi: 10.3389/fmicb.2018.02949 sha: doc_id: 326217 cord_uid: ji0njeha hepatitis c virus (hcv) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the hcv life cycle on key molecules in these metabolic pathways. yet, little is known on the role in the hcv life cycle of glycogen synthase kinase 3 (gsk3), one of the most important kinases in cellular metabolism. therefore, the impact of gsk3 on the hcv life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived hcv genotype 2a by exposure to synthetic gsk3 inhibitors, gsk3 gene silencing, overexpression of gsk3 constructs and immunofluorescence analyses. in addition, the role of gsk3 in hepatitis e virus (hev) replication was investigated to assess virus specificity of the observed findings. we found that both inhibition of gsk3 function by synthetic inhibitors as well as silencing of gsk3β gene expression resulted in a decrease of hcv replication and infectious particle production, whereas silencing of the gsk3α isoform had no relevant effect on the hcv life cycle. conversely, overexpression of gsk3β resulted in enhanced hcv replication. in contrast, gsk3β had no effect on replication of subgenomic hev replicon. the pro-viral effect of gsk3β on hcv replication was mediated by supporting expression of microrna-122 (mir-122), a micro-rna which is mandatory for wild-type hcv replication, as gsk3 inhibitors suppressed mir-122 levels and as inhibitors of gsk3 had no antiviral effect on a mir-122-independent hcv mutant. in conclusion, we have identified gsk3β is a novel host factor supporting hcv replication by maintaining high levels of hepatic mir-122 expression. hepatitis c virus is a member of the flaviviridae family which has a positive-sense single-stranded rna genome (lange et al., 2014b) . the hcv genome encodes the viral structural and nonstructural proteins, which are required for the hcv life cycle (lange et al., 2014b) . in addition to hcv proteins, a number of host factors have been discovered on which hcv critically depends (lange et al., 2014b) . though most of these host factors are cellular proteins, a liver-specific micro-rna (mir-122) has been identified which is mandatory for hcv replication and which is (at least partially) responsible for the hepatotropism of this virus (lange et al., 2014b) . chronic infection with hcv is not only a major cause of liver cirrhosis and hepatocellular carcinoma, but also associated with metabolic traits including insulin resistance and dyslipidemia (kawaguchi et al., 2004; shintani et al., 2004; bose et al., 2012) . while the effects of hcv infection on glucose metabolism are clearly documented in vivo and in vitro, the benefit of these alterations for the virus remains largely unclear (petta et al., 2010; negro, 2014) , although insulin resistance was identified as a negative predictor of outcome of interferon-based therapies (romero-gómez et al., 2005; dai et al., 2009; grasso et al., 2009; moucari et al., 2009; khattab et al., 2010; fattovich et al., 2011) . gglycogen synthase kinase 3 is a serine/threonine protein kinase that exists in two isoforms, gsk3α and gsk3β, which are encoded by two different genes. approximately 100 substrates of gsk3 have been proposed, highlighting a central role for gsk3 in numerous cellular processes such as proliferation, migration, apoptosis, immune modulation and -importantlyglucose metabolism (jope et al., 2007) . of note, substrates of gsk3α and gsk3β are overlapping only partially. the activity of gsk3 is modulated by inhibitory phosphorylation at ser 21 for gsk3α and ser 9 for gsk3β by upstream kinases, where the phosphorylated n-terminal tail acts as a pseudosubstrate that prevents incoming substrates from entering the catalytic center (cohen and frame, 2001) . the importance of gsk3 is further highlighted by its role in the pathogenesis of relevant diseases including diabetes, cancer and inflammation (beurel et al., 2015) . accordingly, several gsk3 inhibitors are in preclinical and clinical development, for example for the treatment of alzheimer's disease or diabetes (del ser et al., 2013; lovestone et al., 2015) . of note, gsk3 has also been identified as a host factor required for replication of influenza and sars viruses (wu et al., 2009; könig et al., 2010 ). yet, the role of gsk3 in the hcv life cycle remains to be characterized. in view of the close relationship between hcv infection and metabolic alterations, the present study aimed at investigating a possible role of gsk3 in the hcv life cycle. cell culture, subgenomic replicons, cell culture-derived hcv, plasmids huh-7.5 human hepatoma cell line was provided by charles m. rice (the rockefeller university, new york, ny) and cultured in dmem (life technologies, carlsbad, ca) containing 10% heat-inactivated fcs. hcv subgenomic replicon construct pcon1/sg-neo(i)/aflii (con1 strain, genotype 1b) (blight et al., 2000) was provided by charles m. rice. full-length hcv construct pfk-jfh1j6c-846_dg (jc1 virus, genotype 2a) (pietschmann et al., 2006) was provided by ralf bartenschlager (university of heidelberg, germany). hcv u3 virus, a genotype 2a full-length strain resistant to mir-122 inhibition was provided by jens bukh (university of copenhagen, denmark) (li et al., 2011) . huh-7.5 cells were electroporated with in vitro transcribed full-length hcv rna and 72 h later the virus infectivity in the supernatant was assessed by foci forming unit (ffu) determination using anti-hcv core monoclonal antibody (mab) c7-50 (moradpour et al., 1996) , as described (zhong et al., 2005) . the s10-3 cell line harboring a hev genotype 3 replicon derived from kernow-c1 p6 strain (provided by suzanne u. emerson (national institutes of health, md) was used as previously described (dao thi et al., 2016) . sofosbuvir (alsachim sas, illkirch-graffenstaden, france), hcv ns5b inhibitor, was resolved in dmso and used at a final concentration of 1 µm. gsk3 inhibitors lithium chloride (licl) and chir99021 (sigma-aldrich, st. louis, mo, united states) were resolved in sterile water and dmso, respectively. cytotoxicity was assessed using the wst-1 assay (sigma-aldrich). haemagglutinin-tagged gsk3β wild-type plasmid (ha-gsk3β wt pcdna3) was a gift from jim woodgett (lunenfeld-tanenbaum research institute, mount sinai hospital, toronto, on, canada) (addgene plasmid #14753) (he et al., 1995) . transfection of gsk3 constructs for overexpression was performed with a final dna concentration of 0.75 µg/ml using x-tremegene hp dna transfection reagent (roche diagnostics, mannheim, germany). immunoblotting was performed as described previously (lange et al., 2014a) . antibodies against total gsk3 α/β (5676s), phosphorylated-gsk3 α/β (ser9/21) (9331s), and β-catenin (9562s) were purchased from cell signaling technologies (danvers, ma, united states). mouse mab 9e10 against hcv ns5a was provided by charles m. rice (lindenbach, 2005) and mab 12b7 against hcv ns5b was described earlier (moradpour et al., 2002) . ecl donkey hrp-conjugated anti-rabbit (na934v) and hrp-conjugated anti-mouse (sc-2031) secondary antibodies were from ge healthcare (little chalfont, united kingdom) and santa cruz biotechnology (dallas, tx, united states), respectively. total rna extraction was done using rneasy mini kits and mirneasy mini kit for mrna and micro-rnas, respectively (qiagen, hilden, germany). reverse transcription of mrna and micro-rna was performed using the primescript rt reagent kit (takara bio, otsu, japan) and the taqman microrna reverse transcription kit (applied biosystems, foster city, ca, united states), respectively. for real-time pcr, steponeplus real-time pcr system (applied biosystems) was used. for hcv and gapdh, experiments were carried out using taqman gene expression master mix (applied biosystems). for micro-rnas, taqman universal master mix ii (applied biosystems) was used for quantification of the mirna cdnas. the following primers were used for hcv amplification: forward, 5 -acgcagaaagcgtctagccat-3 , reverse, figure 1 | gsk3 inhibition suppresses hcv replication. (a) confirmation of gsk3 inhibition by chir99021. immunoblot analysis was carried out for total and phosphorylated-gsk3α/β (ser 9/21), β-catenin, phosphorylated glycogen synthase (gs), eukaryotic initiation factor 2bε, and β-actin after treatment of huh-7.5 cells harboring con1 replicon with 5 µm chir99021 for 2, 6, 24 and 48 h, as indicated. (b) suppression of subgenomic hcv replication by gsk3 inhibition. huh-7.5 cells harboring con1 replicon were treated with different concentrations of chir99021 for 24 and 48 h. hcv rna levels normalized to gapdh mrna are expressed relative to untreated cells. (c) assessment of gsk3 inhibitors' cytotoxicity. wst-1 cytotoxicity assay was carried out for cells stimulated for 24 and 48 h with different concentrations of chir99021 at seeding densities of 10,000 and 15,000 cells per well in 96-well plates. absorbance readings (a 450 -a 690 nm) of treated cells were compared to negative control cells treated with the dimethyl sulfoxide vehicle. data are presented as mean ± sem of six independent experiments. asterisks denote statistically significant differences ( * p-value ≤ 0.05; * * p-value ≤ 0.005; * * * p-value ≤ 0.0005). frontiers in microbiology | www.frontiersin.org figure 4 | gsk3β is required for hcv replication, not gsk3α. (a) gsk3β silencing suppresses hcv replication. huh-7.5 cells harboring subgenomic con1 replicon (left panel) or replicating the full-length jc1 clone were transfected with sirnas of gsk3α (sirna #6236), β (sirna #6239) or both for 72 h. hcv rna levels normalized to gapdh mrna are expressed relative to untreated cells. data are presented as mean ± sem of two experiments performed in triplicate. asterisks denote statistically significant differences ( * p-value ≤ 0.05; * * p-value ≤ 0.005; * * * p-value ≤ 0.0005). immunoblot analysis was carried out for gsk3α/β to confirm gene silencing efficiency, and for hcv ns5b to assess suppression of hcv replication on the protein level (a, lower panel). β-actin was detected as a loading control. ns5b signal intensities were normalized to β-actin for quantification and were presented as mean ± sem of signal intensities of duplicate bands. (b) gsk3β overexpression promotes hcv expression. huh-7.5 cells harboring con1 replicon were transfected with pcdna3 plasmid expressing ha-tagged wild-type gsk3β (ha-gsk3β) for 48 and 72 h. immunoblot analysis was carried out for gsk3α/β to confirm transfection efficiency. the overexpression was confirmed by visualizing ha-gsk3β distinct bands with a slightly higher molecular weight than that of the endogenous gsk3β. hcv ns5b protein was detected to assess hcv replication level and β-actin was detected as a loading control. ns5b signal intensities were normalized to β-actin for quantification and were presented as mean ± sem of signal intensities of duplicate bands. frontiers in microbiology | www.frontiersin.org 5 -tactca ccggttccgcaga-3 , hcv taqman probe, 5 -tcctggaggctgcacgacactca-3 . taqman gene expression assay containing the pre-designed primers and taqman probe from applied biosystems was used for gapdh. for mir-122 and mir-16, pre-designed primers (assay id no. 2245 and 2420, respectively) from thermo fisher scientific were employed. for hev, the procedure and primers have been previously described (dao thi et al., 2016) . results were calculated using the 2 − ct method and shown as fold change compared to control. gene silencing was performed as described previously (lange et al., 2014a) using predesigned sirnas from life technologies. single sirnas for gsk3α (s6236 and s6237) and β (s6239 and s6240) as well as a non-targeting control sirna were transfected at a final concentration of 5 nm using lipofectamine rnaimax (life technologies). chir99021 is a highly selective small molecule inhibitor of gsk3α and gsk3β that inhibits gsk3 by competition with atp in the atp-binding site of the kinase meijer et al., 2004) . to prove efficacy of gsk3 inhibition by chir99021, immunoblot analyses of β-catenin, phosphorylated glycogen synthase (gs), and phosphorylated eif2b ε as downstream targets of gsk3 were performed. as shown in figure 1a , β-catenin was upregulated and phosphorylated gs and eif2b ε were downregulated by chir99021, as expected. we next exposed huh-7.5 cells harboring the subgenomic replicon (con1 strain) to different concentrations of chir99021 for 24 and 48 h and quantified hcv rna by pcr. as shown in figure 1b , treatment with chir99021 led to an inhibition of hcv rna replication. of note, no relevant effect on cell viability was observed as a consequence of gsk3 inhibition, as assessed by wst-1 assay ( figure 1c) . comparable results were observed for treatment of huh-7.5 cells harboring the subgenomic con1 replicon with lithium chloride, another inhibitor of gsk3 (beurel et al., 2015) (figure 2) . next, huh-7.5 cells electroporated with full-length hcv genotype 2a jc1 rna were treated with chir99021 for 72 h to assess the effects of gsk3 inhibition on infectious hcv. as shown in figure 3a , gsk3 inhibition resulted in a profound inhibition of replication of the infectious hcv clone, as well as of particle production ( figure 3b ). to assess whether both gsk3 isoforms are required for hcv replication, we silenced gsk3α and / or gsk3β gene expression by transfecting sirnas and quantified hcv rna in huh-7.5 cells harboring hcv subgenomic replicon (con1) or infectious hcv (jc1). as shown in figure 4a (upper panel), silencing of gsk3β gene expression resulted in a substantial decrease of hcv rna levels of both the subgenomic replicon and the full-length hcv construct, whereas silencing of gsk3α gene expression had only a minor effect on hcv replication. comparable results were observed for a second set of sirnas directed against gsk3α or gsk3β (supplementary figure 1) . the suppressive effect of gsk3β gene silencing on hcv replication was further confirmed on the protein level by quantifying hcv ns5b protein (figure 4a lower panel) . furthermore, the enhancing effect of gsk3β on hcv rna replication was confirmed by overexpression of a functional gsk3β construct, which increased hcv rna replication ( figure 4b ). to test whether the pro-viral effects of gsk3β are specific for hcv, we assessed the impact of gsk3 inhibition on replication of hev, another hepatotropic positive-strand rna virus. as shown in figure 5 , neither silencing of gsk3α and / or gsk3β gene figure 5 | the pro-viral effects of gskβ are limited to hcv in comparison to hev. no effects of gsk3 inhibition on hev replication. huh-7-derived cells (s10-3) harboring a selectable hev replicon construct (hev kernow-c1 strain) were treated with 5 µm chir99021 for 48 h or transfected with sirnas to silence gsk3α and β gene expression for 72 h. hev rna levels normalized to gapdh mrna are expressed relative to negative control sirna-transfected cells. data are presented as mean ± sem of two experiments performed in triplicate and compared by wilcoxon m whitney u-test (n.s., not significant) with the control group. (b) gsk3 inhibition has no effects on mir-122-resistant hcv. huh-7.5 cells, pre-treated for 24 h with 0.1% dimethyl sulfoxide (dmso), 1 µm sofosbuvir (sof) or 5 µm chir99021, were infected with either jc1 or mir-122-resistant u3 virus (moi = 0.1) and treated in the same conditions for 72 h before harvesting. hcv rna levels normalized to gapdh mrna level are expressed relative to dmso-control. data are presented as mean ± sem of two experiments performed with six samples each. (c) gsk3 inhibition has no effects on mir-122-resistant hcv infectious particles production. infectivity was determined by foci forming units (ffu) assay in cell supernatants harvested 72 h post-infection of jc1 or u3 rna and treated with either 1 µm sof or 5 µm chir99021. results were shown as infectivity level normalized to control samples cultured in presence of 0.1% dimethyl sulfoxide vehicle. for all experiments, data are presented as mean ± sem of two experiments performed with six samples each. asterisks denote statistically significant differences ( * p-value ≤ 0.05; * * p-value ≤ 0.005; * * * p-value ≤ 0.0005). expression nor treatment with the gsk3 inhibitor chir99021 had a relevant effect on hev rna levels, suggesting that the pro-viral effect of gsk3β is specific to hcv. given the well-known dependency of hcv on mir-122 (jopling et al., 2005 (jopling et al., , 2008 machlin et al., 2011) and the regulatory circuit between insulin-like growth factor 1 receptor, gsk3β, and mir-122 identified previously (zeng et al., 2010) , we assessed the effect of gsk3 inhibition on mir-122 expression in huh-7.5 cells harboring a subgenomic hcv replicon. as shown in figure 6a , treatment with chir99021 resulted in downregulation of mir-122 levels, in a similar magnitude compared to hcv replication inhibition. for further validation, huh-7.5 cells transfected with hcv jc1 or u3 full-length rna, the latter being a hcv construct replicating independently from mir-122, were treated with chir99021. as shown in figure 6b , chir99021 inhibited the replication of the full-length hcv jc1 genome, the replication of which depends on the presence of mir-122. in contrast, chir99021 had no inhibitory effect on replication, nor entry and particle production, of the hcv u3. in line with these observations, the production of jc1 infectious viral particles was strongly inhibited by chir99021 whereas this inhibitor had no effect on infectious particle production of the u3 virus ( figure 6c) , suggesting that gsk3β inhibition mainly impacts hcv rna replication. in view of remarkable associations between hcv and metabolic traits, we have investigated the role in the hcv life cycle of gsk3, a key kinase in glucose metabolism and numerous other cellular processes. our study shows that gsk3β, but not gsk3α, serves as an important host factor of viral rna replication. the proviral effects of gsk3β appeared to be mediated, at least in part, by maintaining hepatocellular levels of the key microrna mir-122. microrna-122 is an important microrna predominantly expressed in hepatocytes, where it accounts for approximately 50-70% of the entire microrna pool. the high abundancy of mir-122 in liver cells underlines key functions in liver development, growth, differentiation and homeostasis (bandiera et al., 2015) . it is not surprising, therefore, that repression of mir-122 expression promotes the development of hepatocellular carcinoma (hcc). in addition, mir-122 is involved in important metabolic functions of the liver including cholesterol and fatty acid synthesis and iron homeostasis (esau et al., 2006; castoldi et al., 2011) . a unique feature of the hcv life cycle is its dependence on the presence of mir-122, which stabilizes the hcv genome and promotes viral replication through binding to the 5 -untranslated region (5 -utr) of the viral genome (jopling et al., 2005 (jopling et al., , 2008 machlin et al., 2011) . given the liver-specific expression of mir-122, this micro-rna is also considered to be a key determinant of the hepatotropism of hcv. the strong dependence of hcv on mir-122 is evidenced by profound suppression of viral loads by therapeutic silencing of mir-122 in vivo (lanford et al., 2010; janssen et al., 2013) . given the tumor suppressor function of mir-122, these therapeutic silencing strategies may be associated with an increased risk of hcc development. in addition, a recent study has shown that hcv functionally regulates (sequesters) mir-122 within hepatocytes, which results in a de-repression of mir-122 target genes in the presence of hcv and which may partially explain the oncogenic potential of chronic hepatitis c (luna et al., 2015) . our finding that specifically gsk3β (and not gsk3α) is required to maintain mir-122 levels in hcv infected hepatocytes adds another facet to the complex reciprocal relationship between hcv, mir-122 and the implications of chronic hcv infection on metabolic and oncogenic traits. yet, it is a limitation of our study that we cannot completely exclude that targets of gsk3 other than mir-122 are additionally involved in the suppression of hcv replication by gsk3 inhibition. however, it appears plausible that depletion of mir-122 plays a key role in mediating suppression of hcv replication by gsk3-inhibition because mir-122 is one of the most important host factors on which hcv replication critically depends. a further confirmation of this notion is the important finding that the mir-122-independent hcv mutant is resistant to gsk3 inhibition. in addition, gsk3 inhibition had no suppressive effect on replication of hev, a virus which does -in contrast to hcv -not depend on mir-122. our results confirm a study by zeng et al., which has described a regulatory circuit between gsk3, mir-122 and insulin-like growth-factor 1 receptor in hepatocytes (zeng et al., 2010) . gsk3 is a multifunctional protein with an estimated 100 different substrates, thereby regulating numerous cellular processes and pathways beyond glucose metabolism. hence, inappropriate gsk3 signaling appears to be involved in diverse diseases such as diabetes mellitus, cancer or alzheimeŕs disease. though a number of gsk3 inhibitors are in preclinical and clinical development, there is a concern of serious side effects due to the pleiotropic effects of gsk3. however, some of the most significant undesirable effects of gsk3 inhibition, in particular β-catenin accumulation with potential deleterious carcinogenic effects, develop as a result of combined inhibition of gsk3α and gsk3β (rayasam et al., 2009) . in this regard, the finding that only gsk3β but not gsk3α affects mir-122 levels is important because it may serve as a proof-of-principle that selectively targeting gsk3 isoforms may be a suitable approach to manipulate specific functions of gsk3. our data further illustrate the need for dissecting the role of gsk3 isoforms separately instead of non-specifically referring to gsk3 inhibition. in view of the pleiotropic cellular functions of gsk3 it is not surprising that viruses engage this important kinase to complete their life cycle. it has been shown that replication of the sars coronavirus depends on gsk3-mediated phosphorylation of the nucleocapsid (n) protein which is a necessary process for viral replication (wu et al., 2009 ). in addition, it was shown that gsk3β promotes the life cycle of influenza virus through facilitating the viral entry step (könig et al., 2010) . the here reported role of gsk3β in the hcv life cycle adds another facet to the dependence of viruses on this central kinase. furthermore, the role of gsk3 in the hcv life cycle may be not restricted to hcv replication, as a recent report has shown that gsk3 supports hcv assembly by its involvement in lipoprotein production (sarhan et al., 2017) . while we did not make this observation in our experimental settings, our data indicate that it likely remains marginal compared to the role of gsk3 in supporting hcv rna replication. yet, the importance of these and our findings may rather consist in its implications for the understanding of the pathogenesis of chronic hepatitis c than in the identification of a novel target of antiviral therapy. gsk3 is a downstream target of insulin which is inhibited by insulin receptor signaling (welsh and proud, 1993) . reciprocally, gsk3 inhibits pivotal downstream targets of the insulin receptor under nonstimulated conditions, such as glycogen synthase (woodgett and cohen, 1984; roach, 1991) and irs-1 (eldar-finkelman and krebs, 1997; liberman and eldar-finkelman, 2005) . as a result, increased gsk3 activity has been observed in diabetic tissues (eldar-finkelman et al., 1999; nikoulina et al., 2000) and inhibition of gsk3 activity improves insulin resistance (cline et al., 2002; henriksen et al., 2003; nikoulina et al., 2002) . consequently, one may speculate that the dependence of hcv on active gsk3 could explain a benefit for hcv of insulin resistance, which is induced by hepatitis c and which had been associated with lower chances of treatment-induced viral clearance in the era of interferon therapy (romero-gómez et al., 2005) . mir-122 -a key factor and therapeutic target in liver disease glycogen synthase kinase-3 (gsk3): regulation, actions, and diseases efficient initiation of hcv rna replication in cell culture hepatitis c virus activates the mtor/s6k1 signaling pathway in inhibiting irs-1 function for insulin resistance the liver-specific microrna mir-122 controls systemic iron homeostasis in mice effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in zucker diabetic fatty (fa/fa) rats the renaissance of gsk3 insulin resistance predicts response to peginterferon-alpha/ribavirin combination therapy in chronic hepatitis c patients sofosbuvir inhibits hepatitis e virus replication in vitro and results in an additive effect when combined with ribavirin * treatment of alzheimer's disease with the gsk-3 inhibitor tideglusib: a pilot study phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action increased glycogen synthase kinase-3 activity in diabetes-and obesity-prone c57bl/6j mice mir-122 regulation of lipid metabolism revealed by in vivo antisense targeting the homeostasis model assessment of the insulin resistance score is not predictive of a sustained virological response in chronic hepatitis c patients insulin resistance predicts rapid virological response in nondiabetic, non-cirrhotic genotype 1 hcv patients treated with peginterferon alpha-2b plus ribavirin glycogen synthase kinase-3 and dorsoventral patterning in xenopus embryos modulation of muscle insulin resistance by selective inhibition of gsk-3 in zucker diabetic fatty rats treatment of hcv infection by targeting microrna glycogen synthase kinase-3 (gsk3): inflammation, diseases, and therapeutics position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome modulation of hepatitis c virus rna abundance by a liver-specific microrna hepatitis c virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3 insulin resistance predicts rapid virologic response to peginterferon/ribavirin combination therapy in hepatitis c genotype 4 patients human host factors required for influenza virus replication therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection vitamin d receptor and jak-stat signaling crosstalk results in calcitriol-mediated increase of hepatocellular response to ifn emerging therapies for the treatment of hepatitis c microrna-122 antagonism against hepatitis c virus genotypes 1-6 and reduced efficacy by host rna insertion or mutations in the hcv 5' utr serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling complete replication of hepatitis c virus in cell culture a phase ii trial of tideglusib in alzheimer's disease hepatitis c virus rna functionally sequesters mir-122 masking the 5' terminal nucleotides of the hepatitis c virus genome by an unconventional micrornatarget rna complex pharmacological inhibitors of glycogen synthase kinase 3 functional properties of a monoclonal antibody inhibiting the hepatitis c virus rna-dependent rna polymerase characterization of three novel monoclonal antibodies against hepatitis c virus core protein insulin resistance and geographical origin: major predictors of liver fibrosis and response to peginterferon and ribavirin in hcv-4 facts and fictions of hcv and comorbidities: steatosis, diabetes mellitus, and cardiovascular diseases inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes visceral adiposity index is associated with histological findings and high viral load in patients with chronic hepatitis c due to genotype 1 construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras glycogen synthase kinase 3: more than a namesake selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo multisite and hierarchal protein phosphorylation insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis c patients glycogen synthase kinase 3β inhibitors prevent hepatitis c virus release/assembly through perturbation of lipid metabolism hepatitis c virus infection and diabetes: direct involvement of the virus in the development of insulin resistance glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eif-2b multisite phosphorylation of glycogen synthase. molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-ii (glycogen synthase kinase-5) glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication a novel gsk-3 beta-c/ebp alpha-mir-122-insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellular carcinoma robust hepatitis c virus infection in vitro ms, sr, cw, jg, and cl collected the data. ms, sr, sz, dm, jg, and cl analyzed the data. all authors have contributed to the manuscript by planning the study, and preparing as well as revising the manuscript. this study was supported by the else kröner-fresenius-stiftung (2013-a165 to cl), the deutsche forschungsgemeinschaft (la 2806/2-1 and la 2806/5-1), and the swiss national science foundation (31003a_156030 and 31003a_179424 to dm). the authors express their gratitude to yolanda martinez for expert technical assistance, rolf marschalek for helpful discussions as well as ralf bartenschlager, jens bukh, suzanne u. emerson, charles m. rice, and jim woodgett for reagents. parts of this work have been presented as poster presentations at the international liver congress 2018 in paris, france, and at the annual meeting of the german association for the study of liver diseases 2018 in hamburg, germany. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02949/full#supplementary-material key: cord-344084-z4t2wkgk authors: ellwanger, joel henrique; kulmann-leal, bruna; kaminski, valéria de lima; rodrigues, andressa gonçalves; de souza bragatte, marcelo alves; chies, josé artur bogo title: beyond hiv infection: neglected and varied impacts of ccr5 and ccr5δ32 on viral diseases date: 2020-05-30 journal: virus res doi: 10.1016/j.virusres.2020.198040 sha: doc_id: 344084 cord_uid: z4t2wkgk the interactions between chemokine receptors and their ligands may affect susceptibility to infectious diseases as well as their clinical manifestations. these interactions mediate both the traffic of inflammatory cells and virus-associated immune responses. in the context of viral infections, the human c-c chemokine receptor type 5 (ccr5) receives great attention from the scientific community due to its role as an hiv-1 co-receptor. the genetic variant ccr5δ32 (32 base-pair deletion in ccr5 gene) impairs ccr5 expression on the cell surface and is associated with protection against hiv infection in homozygous individuals. also, the genetic variant ccr5δ32 modifies the ccr5-mediated inflammatory responses in various conditions, such as inflammatory and infectious diseases. ccr5 antagonists mimic, at least in part, the natural effects of the ccr5δ32 in humans, which explains the growing interest in the potential benefits of using ccr5 modulators for the treatment of different diseases. nevertheless, beyond hiv infection, understanding the effects of the ccr5δ32 variant in multiple viral infections is essential to shed light on the potential effects of the ccr5 modulators from a broader perspective. in this context, this review discusses the involvement of ccr5 and the effects of the ccr5δ32 in human infections caused by the following pathogens: west nile virus, influenza virus, human papillomavirus, hepatitis b virus, hepatitis c virus, poliovirus, dengue virus, human cytomegalovirus, crimean-congo hemorrhagic fever virus, enterovirus, japanese encephalitis virus, and hantavirus. subsequently, this review addresses the impacts of ccr5 gene editing and ccr5 modulation on health and viral diseases. also, this article connects recent findings regarding extracellular vesicles (e.g., exosomes), viruses, and ccr5. neglected and emerging topics in “ccr5 research” are briefly described, with focus on rocio virus, zika virus, epstein-barr virus, and rhinovirus. finally, the potential influence of ccr5 on the immune responses to coronaviruses is discussed. inflammatory cells play a crucial role in protecting the host from viral infections. leukocyte migration is a fundamental step of the inflammatory response to viruses, a process regulated by the interaction between chemokines and their receptors. therefore, dysregulations in the chemokine-mediated inflammatory process may contribute to viral pathogenesis (glass et al., 2003) . the c-c chemokine receptor type 5 (ccr5) interacts primarily with the chemokines ccl3 (mip-1α), ccl4 (mip-1β), and ccl5 j o u r n a l p r e -p r o o f (rantes) , which act as ccr5 agonists by stimulating cell migration and mediating inflammatory responses. on the other hand, the chemokine mcp-3/ccl7 is the main ccr5 antagonist ligand (blanpain et al., 1999; zlotnik and yoshie, 2000; glass et al., 2003; alkhatib, 2009 ). in addition to regulating the migration of non-specific leukocytes during inflammatory responses, ccr5 controls the action of specific cell types, including natural killer (nk) cells (khan et al., 2006; weiss et al., 2011) and regulatory t (treg) cells (wysocki et al., 2005; tan et al., 2009; dobaczewski et al., 2010) . ccr5 is also expressed by tissue-resident memory t cells. these ccr5 + cells support barrier immunity (davis et al., 2019) . the ccr5 is a g-protein-coupled receptor (gpcr), containing seven transmembrane α-helices, three extracellular loops, and three intracellular loops (tan et al., 2013) . figure 1 shows the structure of ccr5, highlighting its transmembrane domains and extra and intracellular loops. the specificity of interaction between ccr5 and chemokines is mediated by the second extracellular loop (samson et al., 1997) . helices 2 and 3 have a fundamental role in chemokine-induced ccr5 activation (govaerts et al., 2003) . the steps required from ligand binding culminating in cell migration encompass a series of intracellular interactions, including the g-protein heterotrimer and downstream effectors (lacalle et al., 2017) . after stimulation by chemokines or natural reactive antibodies and subsequent triggering of chemotaxis, ccr5 is phosphorylated and internalized in the cytoplasm (signoret et al., 2000; venuti et al., 2015; venuti et al., 2016; lacalle et al., 2017; venuti et al., 2017; venuti et al., 2018) . the number of available receptors on the cell surface is related to the rate of internalization and recycling of ccr5, which affects the activation of ccr5 and consequent signaling of specific pathways that culminate in chemotaxis processes (mueller et al., 2002) . of note, intracellular pools of ccr5 can be detected in the cells. these pools are probably formed by internalized or immature/precursor forms of ccr5 molecules (mirzabekov et al., 1999; kohlmeier et al., 2008; achour et al., 2009; guglielmi et al., 2011; shirvani et al., 2011) that can be rapidly expressed on the cell surface in response to viral stimuli and inflammatory responses (kohlmeier et al., 2008) . in other words, ccr5 molecules are recycled by cells. specifically, ccr5 recycling can be mediated by degradation followed by de novo synthesis (in response to stimulation by natural antibodies) or occur in the classic short-term system without de novo synthesis (in response to stimulation by ccl5, for example) (venuti et al., 2015; venuti et al., 2016) . the traffic of ccr5 between the plasma membrane and the intracellular medium is mediated by different molecules, including clathrins, β-arrestin 2, and extracellular signal-regulated kinase (erk) 1 (venuti et al., 2015; venuti et al., 2016; venuti et al., 2018) . also, intracellular cd4 regulates the expression of ccr5 on the cell surface (achour et al., 2009 ). the human ccr5 protein (352 residues) is encoded by the ccr5 gene [chromosome 3 (3p.21.31)], which is very polymorphic (blanpain et al., 2000; hoover, 2018) . among polymorphisms of the ccr5, the ccr5δ32 (rs333) has been intensively studied in different human populations. the frequency of the ccr5δ32 is quite variable. in general, the δ32 allele frequency is high in european-derived populations (for example, 16% in norway and 11% in germany) and low or absent in african and asian populations j o u r n a l p r e -p r o o f (solloch et al., 2017) . however, although the δ32 allele is more frequent in european populations, there are exceptions due to migratory events. for example, the frequency of the δ32 allele is high in south africa (13%) and chile (12%) (solloch et al., 2017) . also, the frequency of the δ32 allele can be quite variable within the same country. in brazil, the frequency of the allele in the general population is around 4-5% (silva-carvalho et al., 2016; solloch et al., 2017) . in the southern region of the country, the frequency can reach up to 9% due to the past migration of european populations to this region (boldt et al., 2009; pena et al., 2011; schauren et al., 2013) . figure 2 summarizes basic aspects of ccr5 and shows the frequency of the δ32 allele in various countries. the ccr5δ32 is the most studied genetic variant of the ccr5 gene because of its strong protective effect against hiv infection (considering susceptibility to ccr5-tropic strains). hiv entry into cd4 + t cells is mediated by the interaction of the virus with cd4 and with a co-receptor, usually ccr5. the ccr5δ32 variant is a 32 base-pair deletion in the ccr5 coding region, which causes a frameshift, resulting in a truncated protein that is not directed to the cell surface. ccr5δ32 in heterozygosis promotes a decrease in the expression of functional ccr5 on the cell surface compared to ccr5 wild-type cells. therefore, individuals with heterozygous genotype for ccr5δ32, if infected with hiv, have a small protection against disease progression due to the reduced expression of ccr5 on the surface of cd4 + t cells (reduced hiv-ccr5 interaction). in ccr5δ32 homozygous cells, no ccr5 is expressed in the plasmatic membrane. therefore, homozygous individuals for this polymorphism (δ32/δ32) show virtually total protection against hiv type 1 infection, since no ccr5 expression is verified on cell surface (no hiv-ccr5 interaction at cell surface is possible) (deng et al., 1996; dragic et al., 1996; huang et al., 1996; samson et al., 1996; wu et al., 1997; proudfoot, 2002; venkatesan et al., 2002; picton et al., 2012) . figure 3 illustrates the phenotypic effects of ccr5δ32 in human cells. the main results involving the triad "ccr5, hiv, and ccr5δ32" were published in 1996 in nature, cell and science papers by different groups (parmentier, 2015) . since then, the research involving ccr5 has explored the role of the ccr5 protein and ccr5δ32 polymorphism in different diseases, as well as the therapeutic potentials of ccr5 blockade. currently, the physical interaction of ccr5 with hiv is known in detail (shaik et al., 2019) ryst, 2015; latinovic et al., 2019) . also, a recent study has shown that molecules that inhibit ccr5 trafficking to the plasma membrane also have a therapeutic potential against hiv infection (boncompain et al., 2019) . research involving ccr5 has also brought other important advances in combating hiv infection. of note, there are already two cases of sustained remission of hiv infection following stem-cell transplantation using ccr5δ32 homozygous donor, the "berlin patient" (hütter et al., 2009) and the "london patient" (gupta et al., 2019; gupta et al., 2020) . articles that evaluated the involvement of ccr5 or ccr5δ32 in the infections caused by the mentioned viruses were analyzed. subsequently, the google scholar (https://scholar.google.com.br/) was consulted to detect relevant papers that were not indexed in pubmed, using the terms "ccr5" in association with the name of each of the viruses covered in the review. the authors of this review tried to include the largest number of original articles that addressed the topic covered in this work, intending to write a broad and complete article. however, some papers were not included because it was not possible to obtain clear conclusions from the studies. the reference lists of the consulted articles were also used to complement the selection of articles for this review. as previously mentioned in the introduction, a discussion addressing the involvement of ccr5 in tbev infection was not included in this work. articles addressing the involvement of ccr5 and ccr5δ32 in hiv infection were included only to present to the reader some basic and historical aspects related to such topics, cited mainly in the introduction section and figures, but not included as a major section. considering the importance of the coronavirus disease 19 (covid-19) pandemic, a section addressing the potential influence of ccr5 on the immune responses to coronaviruses was included in the article. review articles were also selected in pubmed and google scholar for writing the sections and paragraphs that address the basic aspects of viruses, exosomes, and diseases (e.g., epidemiological, molecular, clinical aspects). exceptionally, review articles with outstanding discussions regarding the role of ccr5 in viral infections were also cited in this work. regarding tables, it is important to note that the data available in the "population" columns are limited and often represent only general characteristics of the evaluated population. in many studies, a population can be composed of individuals from various ethnic groups. this limitation must be taken into account when evaluating the results of the studies cited in the tables. finally, also in "population" columns, "information not available" was used when this information was not clearly described in the cited article. west nile virus (wnv) is a neurotropic positive-sense single-stranded rna flavivirus endemic in various parts of the world. wnv transmission to humans occurs through the bite of infected mosquitoes, especially species of the culex genus (suthar et al., 2013) . although different animals can participate in the wnv transmission cycle, birds are the classic amplifier hosts. humans, horses and other mammals are deadend hosts (kramer et al., 2008; cdc, 2018) . among humans, blood transfusion, organ transplantation, and breast milk can also transmit the virus. vertical transmission may also occur. however, compared to transmission by mosquito bites, these routes of transmission are rare (kramer et al., 2008) . about 25% of wnv-infected individuals develop west nile fever, a clinical condition with variable symptoms and severity. in less than 1% of the infected individuals, wnv invades the central nervous system (cns), causing neurological manifestations (neuroinvasive disease), including meningitis, encephalitis, and acute flaccid paralysis. of note, west nile neuroinvasive disease shows a 10% to 20% fatality rate. severe illness is associated with older age and other factors, including genetic traces (petersen et al., 2013; sejvar, 2016) . wnv infection is considered the leading cause of arboviral encephalitis in the world (ciota, 2017) . the treatment of wnv infection is supportive (petersen et al., 2013) . it is known that the ccr5 protein interferes in the clinical course of wnv infection (glass et al., 2005; diamond, 2009; michlmayr and lim, 2014) , but the effects of ccr5δ32 on the susceptibility to this infection and disease progression are different. according to lim et al. (2010) and danial-farran et al. (2015) , ccr5δ32 has no important effect on susceptibility to wnv infection. in accordance, loeb et al. (2011) found no association between ccr5δ32 and wnv infection. conversely, there is robust populationbased data showing a strong association between the ccr5δ32 homozygous genotype and increased risk of developing symptomatic wnv infection lim et al., 2008; lim et al., 2010) . also, bigham et al. (2011) showed that the ccr5δ32 variant was associated with symptomatic wnv infection when the dominant model of inheritance was considered in the analysis. a recent meta-analysis confirmed the role of the ccr5 gene in wnv infection, specifically the association between the ccr5δ32 with severe disease (cahill et al., 2018) . table i summarizes the results of the studies that evaluated the ccr5δ32 genetic variant in the context of human wnv infection. in agreement with studies showing that ccr5δ32 homozygous genotype is a risk factor for symptomatic wnv infection in humans, ccr5-/-wnv-infected mice showed a reduced capacity of viral control, increased disease severity, impaired leukocyte trafficking towards the brain, and high mortality rates than ccr5 wild-type mice. these findings reinforce that ccr5 is a key molecule in the immune response against wnv (glass et al., 2005; durrant et al., 2015) . taking together, these pieces of evidence robustly support the role of ccr5 as a protective molecule on wnv pathogenesis. specifically, ccr5+ leukocytes play a fundamental role in combating wnv in the brain (glass et al., 2005; lim et al., 2006; michlmayr and lim, 2014; durrant et al., 2015) . in this sense, ccr5 plays a specific and non-redundant role in controlling wnv infection (lim and murphy, 2011; durrant et al., 2015; ellwanger et al., 2020a) . based on the data mentioned above, the lack of ccr5 expression linked to ccr5δ32 homozygosis is an important risk factor for increased severity of wnv-associated disease. the opposite effects of the ccr5δ32 genetic variant on both hiv and wnv infections are summarized in figure 5 . hereupon, individuals homozygous for ccr5δ32 and living in endemic areas of the wnv should take additional care to prevent wnv infection (e.g., use of repellents, mosquito nets). also, the use of ccr5 blockers to treat hiv infection may have a negative impact on populations living in wnv-endemic areas. to avoid this negative impact, hiv-infected individuals who live in such areas and who use ccr5 blockers must apply robust measures against mosquito bites lim et al., 2006; lim and murphy, 2011). j o u r n a l p r e -p r o o f influenza infection affects humans seasonally, causing recurrent epidemics and even pandemics in some years. in humans, the infection is caused basically by influenza a and influenza b, both enveloped negative-sense single-stranded rna viruses belonging to orthomyxoviridae family (krammer et al., 2018; petrova and russell, 2018) . influenza a is a zoonotic disease, and influenza b circulates primarily in humans. influenza infection affects mainly the respiratory tract, which can cause mild to severe disease depending on viral and host characteristics. secondary bacterial infection may also occur (krammer et al., 2018) . human co-infection with multiple influenza types is an important neglected problem (gregianini et al., 2019) . influenza is a prevalent infection worldwide, and new vaccines are produced annually, based on strains circulating each year in the northern and southern hemispheres (krammer et al., 2018; petrova and russell, 2018) . antivirals can be used in the treatment of influenza infection (krammer et al., 2018) . investments in new vaccines, antiviral drugs, and surveillance systems are needed to reduce the global burden associated with influenza infection (petrova and russell, 2018) . the severity of influenza infection is related to the intensity of proinflammatory responses and the predominant profile of cytokine production by the host . a body of evidence has shown that both ccl5 and ccr5 participate in the modulation of the immune response to influenza virus infection (matsukura et al., 1998; tyner et al., 2005; sládková and kostolanský, 2006; kohlmeier et al., 2008; oslund and baumgarth, 2011) . of note, ccr5 mediates the recruitment of nk cells to the lungs in influenza a infection (carlin et al., 2018) and participates in neutrophil action in the lungs during influenza pneumonia (rudd et al., 2019) . although flow cytometry data did not indicate significant changes regarding ccr5 expression on the surface of human monocytes after experimental influenza a infection (salentin et al., 2003) , various studies have shown that, in mice, the lack of ccr5 expression is associated with a higher risk of death by influenza infection (dawson et al., 2000; tyner et al., 2005; fadel et al., 2008; tavares et al., 2020) . based on these findings, it was postulated that pharmacological ccr5 blockade may have some undesirable effect on the immune response against the influenza virus in humans (fadel et al., 2008) . importantly, more research on this aspect is needed since this data suggests that, in humans, the ccr5 absence due to the ccr5δ32 polymorphism could affect pathogenesis and the lethality rate of influenza infection. falcon et al. (2015) evaluated the frequency of the ccr5δ32 in pandemic h1n1-infected spanish individuals and revealed an association between the polymorphism with fatal outcome. the ccr5δ32 was also associated with increased disease severity in other studies (keynan et al., 2010; rodriguez et al., 2013) . however, these results should be interpreted with caution since both studies were based on very small sample sizes (keynan et al., 2010; rodriguez et al., 2013) . importantly, other studies addressing humans reported no association between ccr5δ32 and severity of influenza infection (sironi et al., 2014; maestri et al., 2015; matos et al., 2019) . taking together, the body of evidence suggests that ccr5δ32 has little j o u r n a l p r e -p r o o f influence on severity of influenza infection (table 2) . results described by falcon et al. (2015) seem to be specific to that studied population, composed of individuals from 13 regions of spain. human papillomavirus (hpv) is a double-stranded dna virus belonging to the papillomavirus family. hpv is transmitted by direct contact (for example, through sexual intercourse). the hpv infection is quite common worldwide, and is usually controlled by the immune system. viral clearance occurs in most cases within 1-2 years after infection. however, if the infection is not controlled, hpv can cause noncancerous mucosal lesions or different types of malignant lesions such as anal cancer, penile cancer, vulvar cancer, head cancer, neck cancer, and especially cervical cancer. hpv is an oncogenic pathogen because it inhibits the activity of p53 and prb (retinoblastoma protein) tumor suppressor molecules through the action of e6 and e7 viral proteins, respectively. these proteins also exhibit other oncogenic mechanisms. worldwide, between 5-10% of all cancers in women are due to hpv infection (schiffman et al., 2016; sanjosé et al., 2018) . there are several hpv genotypes (>200), and those most associated with cancer are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and probably 68 (schiffman et al., 2016) . of note, the genotype 16 (hpv16) has a prominent role in cancer development. vaccination is one of the most effective ways to prevent hpv infection, having important positive impacts on multiple aspects of population health. hpv vaccine protects the vaccinated individual from infection per se and from hpv-related cancer (schiffman et al., 2016; sanjosé et al., 2018) . the expression of ccr5 is increased in cervical cancer tissues (sales et al., 2014; che et al., 2016) . also, in vitro growth, proliferation, and invasive capacity of cervical cancer cells can be inhibited through ccr5 downregulation (che et al., 2016) . these findings suggest the involvement of ccr5 in the development of cervical lesions. in a study performed with indian individuals (singh et al., 2008) , the genotype and allele frequencies of ccr5δ32 were not different between cervical cancer patients and controls. however, when the patients were stratified by cancer stage (stages 1b to 4), the ccr5 heterozygous genotype was associated with stage 1b of cervical cancer. the hpv positivity rate among the evaluated patients was not described (singh et al., 2008) . the relationship between ccr5δ32, hpv infection, and cervical lesions is addressed in other studies (table 3 ). ccr5δ32 homozygous genotype was associated with increased susceptibility to hpv infection in a study performed with swedish individuals (zheng et al., 2006) . mangieri et al. (2019) investigated the potential influence of ccr5δ32 on susceptibility to hpv infection and cervical lesions in brazilian women. however, the genetic variant was not significantly associated with susceptibility to hpv infection (considering allele frequency, codominant model and dominant model) or hpv-associated lesions (mangieri et al., 2019) . in accordance, previous studies performed with brazilian women did not find an association between ccr5δ32 and susceptibility to hpv infection (suzuki et al., 2008) or between the polymorphism and hpv-related cervical lesions (suzuki et al., 2008; santos et al., 2016) . lastly, ccr5δ32 j o u r n a l p r e -p r o o f did not affect susceptibility to hpv infection in lithuanian individuals with laryngeal cancer (stumbrytė-kaminskienė et al., 2020) . in conclusion, although tissue analysis and evidence obtained in vitro suggest that the ccr5 is potentially involved in the pathogenesis of hpv, most studies point to a lack of involvement of ccr5δ32 in susceptibility to hpv infection or hpv-associated diseases. the the ccr5 and its ligands regulate the action of t cells and other leukocytes in the liver. thus, ccr5 regulates liver inflammation and participates in the local immune response against viruses (ajuebor et al., 2006; sanchooli et al., 2014) . in mice models of hepatitis, ccr5 deficiency was associated with increased liver inflammation, tissue injury, and liver failure (ajuebor et al., 2005; moreno et al., 2005; stevens et al., 2019) . deficiency of ccr5 expression is generally associated with reduced migration of inflammatory cells, which would translate into less inflammation. this reasoning is correct and applies to different situations and tissues (braunersreuther et al., 2007; muntinghe et al., 2009; kaminski et al., 2019a) . however, ccr5 is an immunoregulatory molecule (doodes et al., 2009; dobaczewski et al., 2010; christmann et al., 2011; hütter et al., 2011) and therefore its deficiency can also cause deregulation in the action of various immune cell types (e.g., nk and treg cells), increasing the inflammatory status in some tissues. in humans, multiple evidence has shown the involvement of ccr5 (protein and gene) in distinct aspects of hbv infection (ahn et al., 2006; trehanpati et al., 2009; ahmadabadi et al., 2013; yang et al., 2018) . interestingly, ccr5δ32 and other host genetic factors can affect the immunogenicity of the hbv vaccine (ganczak et al., 2017; ellwanger and chies, 2019b) . considering these aspects, the influence of the ccr5δ32 on susceptibility/resistance to hbv and disease severity is quite plausible. several studies investigated the role of the ccr5δ32 polymorphism in hbv infection. some of them found no association between those variables (arababadi et al., 2010; khorramdelazad et al., 2013; safari et al., 2017; zhang et al., 2018; moudi et al., 2019) . in other studies, the absence of ccr5δ32 allele in the sample, due to ethnic features of the evaluated population, precluded the analysis concerning the potential impact of this genetic variant on hbv infection (ahn et al., 2006; li et al., 2011) . some authors have reported significant influences of ccr5δ32 on hbv infection. suneetha et al. (2006) reported an association between the ccr5δ32 heterozygous genotype and chronic hbv infection. in contrast, thio et al. (2007) found an association between the ccr5δ32 allele and infection recovery in a study that analyzed individuals with persistent hbv infection and individuals who recovered from the infection. subsequently, thio et al. (2008) associated the hbv infection recovery with an epistatic effect between the ccr5δ32 and the rantes-403a promoter polymorphisms. finally, abdolmohammadi et al. (2016) found a protective effect of the ccr5δ32 genetic variant against hbv infection, once the δ32 allele was more frequent in controls compared to hbv-infected individuals. recently, we investigated the frequency of ccr5δ32 in hbv mono-infected and hbv/hiv coinfected brazilian individuals (ellwanger et al., 2020b) . a control group and hiv mono-infected individuals were also evaluated in our study. a total of 1113 individuals were studied, which represents the largest study involving ccr5δ32 and hbv infection to date (see table 4 for comparisons with other studies). we found a significant protective effect of ccr5δ32 on hbv/hiv co-infection, a result probably due to the partial protective effect of ccr5δ32 against hiv infection since no important impact of ccr5δ32 on susceptibility to hbv mono-infection was observed (ellwanger et al., 2020b) . the hepatitis c virus (hcv) was formally described in 1989 (choo et al., 1989) and, currently, hcv infection is one of the most important infectious diseases in terms of global public health burden. hcv is an enveloped single-stranded positive-sense rna virus, has seven genotypes, and belongs to the flaviviridae family, hepacivirus genus (pietschmann and brown, 2019) . it is estimated that 71 million individuals are chronically infected by hcv worldwide (viganò et al., 2019) . similar to the hbv infection, hcv-infected individuals can eliminate the virus naturally or develop chronic infection, which occurs in 55-85% of the cases. chronic infection can cause liver inflammation, cirrhosis, and hepatocarcinoma (lingala and ghany, 2015) . in addition to liver damage, hcv causes a series of immune-mediated extrahepatic manifestations, including rheumatologic, dermatologic, ophthalmologic, renal, pulmonary, neuropsychiatric, cardiovascular, and hematologic manifestations, especially mixed cryoglobulinemia (romano et al., 2018) . hcv therapy using direct-acting antivirals (daas) shows cure rates over 95% (pietschmann and brown, 2019) . early treatment of infected patients decreases death rates from hcv-associated liver disease, reduces disease transmission, and alleviates extrahepatic health problems. focusing the efforts on hcv treatment is extremely important because there is no effective hcv vaccine (viganò et al., 2019) . hcv seropositivity is an important risk factor for hiv infection (zwolińska et al., 2013) . like hiv, hcv is primarily transmitted through blood transfusion and sexual intercourse, and hcv/hiv co-infection is a major problem worldwide. depression of the immune system due to uncontrolled hiv infection may contribute to hcv progression (schlabe and rockstroh, 2018) . both susceptibility to hcv infection and j o u r n a l p r e -p r o o f disease progression are affected by viral and environmental factors and physio-metabolic, immune, and genetic components of the host (ellwanger et al., 2018a) . chemokines and chemokine receptors participate in the recruitment and activity of inflammatory cells in the liver, acting on anti-hcv immune responses and ultimately modifying the rate of inflammation and other histological manifestations observed during infection. based on this rationale, the ccr5 molecule was postulated as having an impact on hcv-induced liver injury, susceptibility to hcv infection, and modulation of the possibilities of viral clearance. the downregulation of ccr5 due to ccr5δ32 may interfere in these processes (ahlenstiel et al., 2004; coenen and nattermann, 2010) . in agreement, several polymorphisms in other immune system genes [especially human leukocyte antigen (hla), mannosebinding lectin (mbl), toll-like receptor (tlr), interleukins (il), and interferon (ifn) gene families] indeed modify both susceptibility to hcv infection and disease progression (ellwanger et al., 2018a) . especially the focus of this review, clinical response to hcv therapy is influenced by ccr5 gene polymorphisms (konishi et al., 2004; omran et al., 2013) . also, there is evidence showing that ccr5 haplotypes can affect susceptibility to hcv infection (huik et al., 2013) . a recent in vitro study suggested that ccr5 blockage could have a beneficial effect on the treatment of hcv infection since ccr5 antagonists (maraviroc and cenicriviroc) inhibit hcv replication (blackard et al., 2019) . the use of ccr5 antagonists in humans is safe (fätkenheuer et al., 2010; gulick et al., 2014; giaquinto et al., 2018) and these drugs have the potential to treat a number of diseases in which ccr5 is involved, including hcv-associated liver disease. although the use of ccr5 antagonists on hcv monoinfection is not yet approved, it can be useful specifically for co-infected hiv/hcv patients, where ccr5 blocking (maraviroc) is already recommended (haïm-boukobza et al., 2013; blackard et al., 2019) . however, the detailed patterns of ccr5 expression in different tissues and at various points in the clinical course of hcv infection are still poorly understood. according to a recent study, the expression of ccr5 in cd8+ t cells is increased in the liver of chronic hcv-infected patients (pirozyan et al., 2019) , but other studies have found mixed results regarding ccr5 expression on t cells in the context of hcv infection (lichterfeld et al., 2002; vincent et al., 2005; larrubia et al., 2007; zahran et al., 2020) . therefore, considering that the role of ccr5 in hcv infection is still uncertain, the potential use of ccr5 blockers to treat hcv mono-infection should be cautious. the influence of the ccr5δ32 variant on hcv infection susceptibility was investigated by several studies. woitas et al. (2002) found a significantly higher frequency of the ccr5δ32 homozygous genotype in hcv-infected individuals compared to controls, hiv-infected and hcv/hiv co-infected individuals, suggesting the ccr5δ32 as a risk factor for hcv infection. in agreement, the δ32 allele was a significant risk factor for infection when the authors compared the hcv-infected group to both controls and hivinfected individuals. moreover, the ccr5δ32 homozygous genotype was associated with increased hcv loads. in their study, it was observed an important deviation from the hardy-weinberg equilibrium in data from hcv-infected individuals; and a high portion of the individuals included in the study was hemophiliac j o u r n a l p r e -p r o o f (woitas et al., 2002) . hemophiliac individuals were at high risk of exposure to hcv and hiv until the mid-1980s (promrat et al., 2003; zhang et al., 2003) . considering that the ccr5δ32 homozygous genotype provides protection against hiv infection, a high frequency of this genotype in an hcv-infected group may be due to hiv resistance, but not to hcv, among individuals highly exposed to both viruses. due to those and other reasons, the results of woitas et al. (2002) were criticized by different authors (klein, 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003) . in this sense, no influence of ccr5δ32 on susceptibility to hcv infection were reported in studies performed with various populations (glas et al., 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003; ruiz-ferrer et al., 2004; wald et al., 2004; wasmuth et al., 2004; thoelen et al., 2005; goyal et al., 2006) . reinforcing the observations of those different studies, our group found no association between the ccr5δ32 and susceptibility to hcv infection or hcv/hiv co-infection in a study that evaluated a large number of brazilian individuals (ellwanger et al., 2018b) . bineshian et al. (2018) did not detect the ccr5δ32 allele in any iranian hcv-infected individual and controls included in their study, preventing any conclusion in terms of susceptibility in that population. finally, it is important to mention that in addition to the study by woitas et al. (2002) , a study performed in 2013 also found an association between the ccr5δ32 homozygous genotype with chronic hcv infection in europeans, but the authors of the study mentioned that specific factors regarding selection bias (e.g., co-exposure to hiv) may have influenced their results (suppiah et al., 2013) . taken together, the above-mentioned results called attention for the importance of performing genetic variant studies in different populations, exposed to different social and environmental factors and presenting distinct ethnic backgrounds. considering multiple clinical and histological parameters, two main different results were obtained when the ccr5δ32 genetic variant was evaluated in the context of hcv-related diseases: reduced liver inflammation in δ32 allele carriers (hellier et al., 2003; wald et al., 2004; goulding et al., 2005) ; and no association between the ccr5δ32 and clinical variables (glas et al., 2003; mangia et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; ruiz-ferrer et al., 2004; morard et al., 2014; ellwanger et al., 2018b) . in the context of persistence/resolution of hcv infection and viral control, in the one hand, studies described association of the ccr5δ32 allele with reduced rates of spontaneous viral clearance (nattermann et al., 2011; morard et al, 2014) , higher viral load (yilmaz et al., 2014) , and reduced anti-hcv immune response (ahlenstiel et al., 2009 ). on the other hand, studies have reported association between the ccr5δ32 variant and increased rates of spontaneous viral clearance (goulding et al., 2005; el-moamly et al., 2013) . no significant effect of the ccr5δ32 on viral clearance was reported by other authors (mascheretti et al., 2004) . the potential impact of the ccr5δ32 polymorphism on response to hcv therapy was also evaluated by some authors. ahlenstiel et al. (2003) found an association between the ccr5δ32 allele and reduced response rates to interferon-α monotherapy, but the polymorphism did not affect the response to the combined interferon/ribavirin therapy. this finding shows that the use of more robust therapeutic regimens j o u r n a l p r e -p r o o f compensates the undesirable effects of ccr5δ32 on hcv therapy with interferon-α monotherapy. of note, the effect of ccr5δ32 may be negligible in the context of modern hcv therapies (ahlenstiel et al., 2003) . other studies did not report significant effects of the polymorphism on response to hcv therapy (glas et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; suppiah et al., 2013; morard et al., 2014) . again, it is important to mention that the ethnic distribution of the ccr5δ32 allele could interfere with study results. konishi et al. (2004) , for example, did not detect the ccr5δ32 allele in any japanese individual included in their study focused on host genetic factors involved in the response to interferon therapy. lastly, the polymorphism also does not appear to be associated with any specific hcv genotype (glas et al., 2003; wasmuth et al., 2004; goyal et al., 2006) . ahlenstiel et al. (2004) highlighted that the impact of ccr5 on hcv infection was controversial. in 2020, many controversies remain, although some points are better defined. table 5 summarizes the main findings of the studies that evaluated ccr5δ32 on hcv infection. although some studies indicate an influence of the polymorphism on susceptibility to infection, most studies indicate that the δ32 allele has little (or no) influence on hcv susceptibility. the impact of the ccr5δ32 on hcv-related liver disease is quite variable and context-dependent. finally, available data suggest some benefit of ccr5 antagonists for the treatment of hcv mono-infection. however, these data are still limited and further studies evaluating this topic are needed. poliovirus (pv) is a single-stranded rna enterovirus of the family picornaviridae. there are three types of pv: wild pv type 1 (wpv1), type 2 (wpv2), and type 3 (wpv3). the pv replicates in the tonsils and intestinal tract. in few infection cases (~1%), the virus invades the cns and can cause poliomyelitis resulting in paralysis. poliomyelitis is a condition characterized by inflammation of the gray matter of the spinal cord and muscle paralysis unleashed by pv replication in motor neurons (racaniello, 2006; kew and pallansch, 2018; keohane et al., 2020) . polioencephalitis can also occur and is characterized by the pv outbreak occurred in that country (table 6 ). the authors found no statistically significant effect of the ccr5δ32 on pv infection; only a trend of association between the δ32 allele and increased risk of pv infection was observed (rosenberg et al., 2013) . however, this study had a very small sample size (only seven cases of severe pv infection were evaluated) and therefore the results were not conclusive. in addition, due to the declining number of pv infection cases in the world, the effect of ccr5δ32 will be increasingly difficult to be assessed in population-based studies. dengue virus (denv) is a single-stranded rna virus that belongs to the flaviviridae family, flavivirus genus. there are four denv serotypes, being all transmitted to humans by aedes mosquitoes (aedes aegypti and aedes albopictus) (guzman et al., 2016) . denv infection is a global health problem with huge impacts on public health systems, especially in tropical countries (bhatt et al., 2013) , being considered the most common arbovirosis in the world (stanaway et al., 2016) . globally, the incidence of symptomatic denv infection is within the range of 50 to 100 million cases per year, resulting in ~10,000 deaths each year (stanaway et al., 2016) . clinically, dengue illness is divided into three basic phases: acute febrile phase, critical phase, and recovery (convalescent) phase. dengue disease occurs with/without warning signs or severe dengue. warning signs (suggestive signs or symptoms of important fluid loss, capillary leakage, and shock, such as severe abdominal pain and mucosal bleeding; observed at the end of the febrile phase) allow the rapid identification of patients who need more clinical attention and supportive therapy, in an attempt to avoid severe dengue. when severe disease occurs, this condition can lead to serious organ involvement, shock, and hemorrhage, among other signals and symptoms (guzman et al., 2016) . infection with a denv serotype triggers long-term immunity to that specific serotype (homotypic denv). immunity to heterotypic denv also occurs, but it is transitory. therefore, an individual can have dengue disease more than once. severe dengue occurs more frequently in recurrent infection with a different viral serotype (murphy and whitehead, 2011; st john and rathore, 2019 ). an immune response mediates denv clearance and the resolution of dengue diseases, but it is also involved in the disease pathogenesis (murphy and whitehead, 2011) . some evidence suggests the participation of ccl5/ccr5 axis in the protection against denv (sierra et al., 2014) , as well as in the pathogenesis of dengue disease (islam et al., 2019) . indeed, denv infection is associated with increased frequency of human ccr5+ t cells (de-oliveira-pinto et al., 2012; badolato-corrêa et al., 2018) . in a study performed by marques et al. (2015) , lower viral replication was found in macrophages treated with ccr5 blockers. in the same study, ccr5-/-mice were protected from denv infection. these findings suggest that ccr5δ32 could be a protective factor against denv infection. however, xavier-carvalho et al. (2013) found no statistical difference in the frequency of ccr5δ32 polymorphism between brazilian children with j o u r n a l p r e -p r o o f severe denv infection and healthy controls. in the same direction, no effect of ccr5δ32 on susceptibility to denv infection was found in a small sample-size study performed with individuals from western australia (brestovac et al., 2014) and recent data suggested no important involvement of ccr5 gene or ccr5 polymorphisms in denv infection (cahill et al., 2018; ornelas et al., 2019) . finally, the ccr5δ32 allele was not identified in a small group of indian denv-infected individuals (islam et al., 2019) . table 6 shows some details of the studies involving ccr5δ32 and denv infection. taking together, the data mentioned above indicate that the effects of ccr5 on denv infection are very different between humans and rodents. however, it should be noted that the approach of each mentioned study is quite particular, and we cannot exclude some potential effects of ccr5 and ccr5δ32 on denv infection in humans. cmv is also linked to cancer development, once several cmv proteins (e.g., pul122, pul123, pus28, pul83, pul111a) activate pro-oncogenic pathways, including angiogenesis, escape of immune control and tumor suppressors, tumoral inflammation, invasion and metastasis, genome instability, and increased cell survival and proliferation (herbein, 2018) . poor socioeconomic condition favors cmv infection. antibodies indicating past cmv infection are found in ~60% of adults from high-income countries. in low-income countries, the rate of past infections can reach 100% (griffiths et al., 2015) . cmv can manipulate the immune system producing virokines (virus-encoded cytokine/chemokine homologs) and viroceptors (virus-encoded cytokine/chemokine receptor homologs), molecules that enable the virus to evade host immune defenses. such molecules can also facilitate viral replication (lucas et al., 2001; froberg, 2004; vomaske et al., 2012) . importantly, cmv-encoded proteins can interact with ccr5, (johnson et al., 2015) . however, mixed results were reported regarding the effect of cmv on ccr5 expression since there is evidence indicating that cmv infection may reduce ccr5 expression in various cell types (lecointe at al., 2002; varani et al., 2005) . interestingly, these mixed results may not be contradictory. cmv-infected cells may indeed exhibit decreased ccr5 expression, limiting hiv infection in these cells. however, cmv-infected cells release cmv-associated soluble factors that increase ccr5 expression in non-infected bystander cells, then facilitating hiv replication in such cells and, consequently, contributing to hiv pathogenesis (king et al., 2006) . there is evidence showing that variants in the ccr5 gene can influence multiple aspects of cmv infection (loeffler et al., 2006; sezgin et al., 2011) . for example, the ccr5 promoter polymorphism rs1800023 affects cmv replication (bravo et al., 2014; corrales et al., 2015) . in a study evaluating children, kasztelewicz et al. (2017) found no influence of ccr5δ32 on susceptibility to congenital cmv infection, severity of congenital cmv disease, or cmv-related sensorineural hearing loss at birth. as an individual genetic factor, ccr5δ32 was not statistically associated with the progression of cmv retinitis, a condition that cmv can cause in immunocompromised individuals (sezgin et al., 2011) (table 6 ). bunyaviridae. cchfv circulates in africa, europe, middle east, and asia countries, and can infect a variety of domestic animals and wild species, but without causing symptomatic illness. humans are accidental hosts of cchfv, for which the virus is transmitted mainly by tick-bites (especially ticks of the genus hyalomma), although other routes of transmission also exist, such as exposure to blood of infected animals. most cchfv-infected individuals have no symptoms or have mild nonspecific febrile syndrome. however, in some individuals, the infection can cause the crimean-congo hemorrhagic fever, a severe disease characterized by fever, myalgia, hemorrhage, among other manifestations. neurological complications can also occur, being the spectrum and intensity of the disease quite variable (ergönül, 2006; bente et al., 2013; garrison et al., 2019) . hemorrhagic fever (ergönül, 2006; saksida et al., 2010; bente et al., 2013; garrison et al., 2019) . in a study performed by arasli et al. (2015) , expression of the ccr5 ligands ccl2, ccl3, and ccl4 was increased in cchfv-infected adults compared to controls. considering these same chemokines in cchfv-infected children, only ccl4 was significantly increased compared to pediatric controls (arasli et al., 2015) . engin et al. (2009) evaluated the ccr5δ32 in 15 turkish cchfv-infected individuals and observed the wild-type homozygous genotype in all cases. in a subsequent study evaluating the turkish population, rustemoglu et al. (2017) found a protective effect of ccr5δ32 heterozygous genotype and δ32 allele on cchfv infection, since the genotype and allele frequencies were higher in controls than in cchfv-infected individuals. conversely, the wild-type genotype (normal ccr5 expression) was prevalent among infected j o u r n a l p r e -p r o o f individuals. these findings suggest that ccr5 contributes to susceptibility to cchfv infection and that ccr5 down-regulation due to ccr5δ32 results in some protection against the infection. however, further studies are needed to explain the mechanisms by which ccr5 participates in cchfv infection. in the same study, the ccr5δ32 was not significantly associated with disease severity, clinical parameters, or mortality rate (rustemoglu et al., 2017) . together, these findings indicate that the effect of ccr5δ32 is given specifically on resistance against cchfv infection, without affecting the pathogenesis/outcome of crimean-congo hemorrhagic fever. the genus enterovirus is composed of non-enveloped, positive-stranded rna viruses, belonging to the picornaviridae family. enteroviruses (ev) can infect the gastrointestinal tract, cns, and other organs, including heart (tapparel et al., 2013) . cardiomyopathy is a common consequence of ev infection in the heart. ev infection is associated with myocardial inflammation (myocarditis) and other damages to heart tissues (badorff et al., 2000; cooper, 2009; sagar et al., 2012; tapparel et al., 2013; weintraub et al., 2017) . damage heart tissues. viral persistence in individuals with enteroviral cardiomyopathy is associated with an increased mortality rates (kühl et al., 2005; lassner et al., 2018) . some studies suggest that ccr5 influences different aspects of the pathogenesis of viruses belonging to the picornaviridae family, including encephalomyocarditis virus (christmann et al., 2011; shaheen et al., 2015) , coxsackievirus b3 (valaperti et al., 2013) , and rhinovirus (muehling et al., 2017) , once ccr5 participates in the regulation of the host immune response during infection by these viruses. considering the effects of the genetic variant ccr5δ32 on ccr5 expression and ccr5-related immune responses, it is possible that ccr5δ32 also shows some impact on ev-related diseases. in a german study that evaluated patients with enteroviral (chronic/inflammatory) cardiomyopathy (lassner et al., 2018) , the ccr5δ32 was strongly associated with spontaneous viral clearance and better clinical outcome (reduced mortality rate) ( table 6 ). these findings indicate a critical involvement of the ccr5 molecule in the pathogenesis of ev cardiomyopathy. it was suggested that the ccr5δ32 genotyping could be used to assist in the prediction of the clinical progression of enteroviral cardiomyopathy: the δ32 allele as a predictor of a better prognosis, without the need of antiviral interferon-β (ifn-β) therapy; and the wild-type genotype as a predictor of a worse prognosis and immediate need of antiviral ifn-β therapy (lassner et al., 2018) . the clinical use of ifn-β is effective to eliminate the virus, avoid irreversible cardiac injury, and reduce mortality rates, but it is also associated to numerous adverse effects (kühl et al., 2012; lassner et al., 2018) . considering the prognostic value of the ccr5δ32 on the clinical course of enteroviral cardiomyopathy, it is necessary to evaluate the relationship between the ccr5δ32 and the disease in different human populations, mainly through genetic association studies. if this association is confirmed in other populations, the ccr5δ32 genotyping will be a broad-spectrum clinical tool, enhancing and driving the treatment of enteroviral cardiomyopathy. jev is the etiological agent of most cases of viral encephalitis in many countries, reaching ~30% mortality rate (van den hurk et al., 2009; tiwari et al., 2012; le flohic et al., 2013) . some evidence obtained in laboratory conditions (in vitro analysis and murine models) showed that jev infection induces the up-regulation of ccr5 gene (gupta and roa, 2011; pereek et al., 2014; zhang et al., 2019) . also, increased infiltration of ccr5 + cd8 + t cells was observed in the brains of jev-infected mice (zhang et al., 2019) . in this context, using a mouse model of japanese encephalitis, larena et al. (2012) showed that ccr5 protects the host against jev infection in the cns, being essential for disease recovery. ccr5-deficient mice showed higher viral loads in the brain and spinal cord as well as increased mortality rate, as compared to control mice (larena et al., 2012) . also using a mouse model of japanese encephalitis, kim et al. (2016) demonstrated that ccr5 controls the infiltration of cd4 + foxp3 + t regulatory cells (treg) in the cns, contributing to protection against japanese encephalitis. the infiltration and action of inflammatory cells in the brain are important to limit neuroinvasive infections, processes that are regulated by multiple factors, especially cytokines, chemokines and their receptors, including ccr5. however, the uncontrolled action of inflammatory cells can cause damage to the cns. of note, cd4 + foxp3 + treg cells regulate the immune responses, avoiding undesirable or excessive action of inflammatory cells (bardina and lim, 2012; veiga-parga et al., 2013; simonetta and bourgeois, 2013; campbell, 2015; kim et al., 2016) . according to these pieces of evidence, ccr5-mediated action of treg cells is critical for the control of japanese encephalitis. however, the role of ccr5 in jev infection may be more complex than it appears at first. liu et al. (2018) demonstrated that the use of the ccr5 antagonist maraviroc reduces the jev-induced inflammation in mice brain, increasing survival rate. this result suggests that potential deleterious effects of ccr5-mediated inflammation in the brain may override the effects of ccr5-mediated action of treg cells and, therefore, the use of ccr5 antagonists to treat japanese encephalitis may be beneficial. based on these complex and contradictory results, it can be concluded that ccr5 indeed has an important influence on japanese encephalitis, but it is not yet possible to state its specific roles, once they are varied and appear to be context-dependent. also, the results obtained in animal models may not fully represent the disease course in humans. indeed, some evidences suggest the involvement of ccr5 in the pathogenesis of japanese encephalitis in humans. in indian individuals with mild cases of japanese encephalitis, a significant dowregulation of the ccr5 gene was observed as compared to healthy controls (chowdhury and khan, 2019) . however, the ccr5δ32 does not have a relevant influence on the disease. deval et al. (2019) investigated the effect of various genetic variants, including ccr5δ32, on the development of japanese encephalitis in individuals from north india (table 6 ). no statistically significant difference was found when the ccr5δ32 (as an individual factor) was compared between cases and controls (deval et al., 2019) . considering that both studies mentioned above were performed in the indian population, studies evaluating the potential effects of ccr5 and ccr5δ32 on japanese encephalitis in other populations are necessary. puumala virus (puv) infection is a zoonosis endemic in europe. puv is an enveloped singlestranded rna virus, belonging to the bunyaviridae family, genus hantaviridae. most puv infections are mild or subclinical. in some infected individuals, puv is responsible for the development of nephropathia epidemica, a milder form of hemorrhagic fever with renal syndrome, a condition typically caused by other hantaviruses (settergren, 2000; mustonen et al., 2013; lebecque and dupont, 2019 ). an in vitro study found increased ccr5 gene expression in primary monocytes infected by hantaviruses (hantaan virus, sin nombre virus and andes virus) (markotić et al., 2007) . in this sense, host genetic factors and the immune responses affect different aspects of puv infection and progression of nephropathia epidemica (mustonen et al., 2013) , although the role of ccr5 in this disease is little known. (table 6 ). of note, in their study, the authors stated hantavirus infection as the cause of nephropathia epidemica, not specifying the puv detection. no statistical difference was observed in ccr5δ32 genotype frequencies between cases and controls, indicating that ccr5δ32 does not influenced the susceptibility to hantavirus infection in the studied population. considering only nephropathia epidemica cases, the study revealed an association between the wild-type homozygous genotype (functional ccr5) and increased severity of the disease. conversely, the heterozygous genotype was considered a protective factor against increased disease severity ( in 2019, the multiple roles of ccr5 in physiological and pathological conditions gained the attention of the scientific community and lay public due to ccr5 gene editing in human embryos, the "crispr babies", performed by a chinese biophysicist. the researcher claimed to have used crispr gene editing technology to edit the ccr5 of germline cells to mimic the effects of ccr5δ32, aiming to generate babies resistant to hiv infection. the ethical, safety, and legal aspects related to this procedure have caused an intense and broad discussion in the media and scientific literature (cohen, 2019; daley et al., 2019; greely, 2019; rosenbaum, 2019) , and this case culminated in a three years prison sentence for the biophysicist responsible for the procedure (cyranoski, 2020). also, the consequences of the ccr5 absence have brought many concerns to light, once the ccr5 protein participates in tissue development processes, control of immune responses, and several other physiological processes. considering these concerns, our group and others described some undesirable effects associated to natural ccr5 absence (due to the ccr5δ32 homozygous genotype) and ccr5 editing (ellwanger et al., 2019; wang and yang, 2019; xie et al., 2019) . besides gene editing techniques, the expression of ccr5 can be artificially modulated through the use of pharmacological antagonists (e.g. maraviroc), chemokine ligands, and monoclonal antibodies (fantuzzi et al., 2019) . the use of ccr5 antagonists is well established for the treatment of hiv infection and is currently being tested for the treatment of many other diseases, such as cancer (pervaiz et al., 2019; huang et al., 2020a) , stroke (joy et al., 2019), and cocaine addiction-related disorders (hall et al., 2020; nayak et al., 2020) . taking together, it is increasingly clear that the specific inhibition of ccr5 expression through different techniques is gaining pace in different clinical contexts. the pharmacological ccr5 blockade has many benefits in different clinical situations, particularly in the treatment of hiv infection. also, the potential of gene editing (especially in somatic cells) for the treatment of genetic diseases is very promising. however, considering viral infections, two aspects must be considered, as follows: i) the non-redundant characteristics of the ccr5 should be taken into consideration when studying the ccr5 protein and the effects of ccr5δ32 on viral infections: the traditional concepts of redundancy and robustness of the chemokine system consider that the absence of a specific chemokine or chemokine receptor is adequately compensated by other molecules. although these concepts are generally correct for some chemokines/receptors and for some physiological conditions, the lack of ccr5 expression may affect the protection against some specific infectious diseases once the ccr5 absence is not perfectly compensated for by other receptors (ellwanger et al., 2020a) . the nonredundancy of ccr5 is relevant especially for infections by neuroinvasive flaviviruses (bradina and lim, 2012) . for example, the absence of ccr5 due to ccr5δ32 is considered deleterious in wnv infection lim et al., 2008) and in some aspects of tbev infection (ellwanger and chies, 2019a) . the non-redundant role of ccr5 is also likely in jev infection (larena et al., 2012) . it is possible that those individuals using ccr5 antagonists (e.g., for the treatment of hiv infection) and living in areas endemic for neuroinvasive viruses, especially wnv and tbev, may be at increased risk of j o u r n a l p r e -p r o o f developing viral encephalitis-related problems. although, it is still necessary that studies consistently evaluate this assumption, since the available evidence does not support risks for the use of ccr5 antagonists in endemic areas of flaviviruses (martin-blondel et al., 2016) . as mentioned in the topic addressing wnv in this review, it is prudent to recommend to individuals using ccr5 antagonists to adopt measures to minimize the risk of neuroinvasive infections, such as the use of mosquito repellents and mosquito nets (considering the risk of wnv infection), and to limit their exposure to tick-infested areas (considering the risk of tbev infection). concerns regarding the use of ccr5 antagonists in areas of jev circulation have also been raised by other authors (kim et al., 2016; larena et al., 2012) . if the connection between the use of ccr5 antagonists and increased risk of neuroinvasive infections is confirmed in methodologically wellcontrolled studies, these recommendations should be considered of essential importance for users of ccr5 antagonists. "extracellular vesicles" (evs) is a general term used to designate different membranous vesicles released by various cell types. evs include microparticles, microvesicles, nanovesicles, nanoparticles, ectosomes, exosomes, exovesicles, exosome-like vesicles, oncosomes, among other vesicle types. evs act in the transport of different molecules (e.g. micrornas, mrnas, proteins) between cells and tissues in a regulated and precise manner. evs promote the maintenance of physiological processes, also participating in the pathogenesis of various diseases, such as cancer and inflammatory diseases (colombo et al., 2014; théry et al., 2018) . besides, the regulation of multiple aspects of the immune system is strongly influenced by evs (o'neill and quah, 2008; colombo et al., 2014; ellwanger et al., 2016) . the multiple roles of evs in viral infections began to be studied more intensively in recent years, representing an emerging topic in the field of infectious diseases. currently, the relationship between evs and viral infections has already been explored (to a greater or lesser extent) in the context of the following . evs participate in immune evasion processes, ultimately allowing viruses to bypass host defenses (kaminski et al., 2019b) . for example, hiv is likely to usurp the exosome biogenesis pathway in such a way that enhances its infectivity, while increasing its evading strategies from the host immune defenses (ellwanger et al., 2017a) . regarding tbev, exosomes were indicated as important participants in both viral infection and pathogenesis; in this case, tick-derived exosomes facilitate tbev transmission to the host. also, exosomes derived from tbev-infected host neurons can facilitate the spread of the virus in the cns . exosomes have shown multi-dimensional roles in denv life cycle. they can modulate negatively or positively denv pathogenesis, where they are suggested as instrumental for dengue j o u r n a l p r e -p r o o f hemorrhagic fever development in reason of the transportation of specific micrornas (mishra et al., 2019) . since hcv can be found inside exosomes (liu et al., 2014) , it is not suprising that blood-derived exosomes from hcv-infected patients can transmit the virus to other cells (bukong et al., 2014) . these findings suggest that exosomes and other evs assist in the transport of hcv particles/components between cells, ultimately facilitating viral infection. of note, evs can transport multiple viral components or molecules from virus-infected cells that end up facilitating the spread of the virus in the host. on the other hand, evs can act in favor of the host, transporting molecules that assist host defenses in the elimination or control of infections (kaminski et al., 2019b) . evs can transport a range of cytokines and chemokines, such as il-1, il-2, ifn-α, ifn-β, ccl2, ccl3, ccl4, and cxcl10 (chen et al., 2011; konadu et al., 2015; hosseinkhani et al., 2018; kodidela et al., 2018; gao et al., 2019a; gao et al., 2019b; aiello et al., 2020; chiantore et al., 2020) . besides, evs and microparticles also transport chemokine receptors (shen et al., 2016; liang et al., 2018) , including ccr5 tokarz et al., 2019) . interestingly, evs from ccr5 wild type cells can deliver ccr5 molecules to ccr5 δ32/δ32 cells, making them susceptible to hiv infection . a similar phenomenon has been reported involving microparticles, hiv, and cxcr4 (rozmyslowicz et al., 2003) . ccr5 expression is also influenced by the release of evs, specifically microparticles (renovato-martins et al., 2017) . therefore, evs have the potential to attribute greater complexity to ccr5 roles in viral infections. it is possible that the presence of ccr5 in cells is not only dependent on mechanisms of membrane expression and internalization of the receptor. it can also be dependent on ccr5 delivery mediated by evs. however, the actual impact of evs on ccr5-mediated immune response in infections remains to be determined and deserves further investigation. finally, a truncated ccr5 soluble form (tsimanis et al., 2005) , as well as soluble cxcr4, have also been reported in the plasma of humans (malvoisin et al., 2011) . of note, the truncated ccr5 soluble form has ~22 kda (half the size of the ccr5 found on cell membranes) and is truncated at the end of the second extracellular loop (the third extracellular loop and the subsequent three transmembrane regions are absent) (tsimanis et al., 2005) . however, the existence of cell-free soluble ccr5 is still controversial and does not represent a consensus in the scientific community. such doubts occur mainly in consideration of the structure of the receptor, which is highly characteristic of a cell membrane-associated molecule. we believe that evscontaining ccr5 can explain potential (misleading) detections of soluble ccr5. the importance of circulating evs-containing ccr5 on viral infections represents an additional and interesting open question to be investigated. chávez et al. (2013) (ellwanger et al., 2017b) . specifically, using a mouse model of rocv-associated encephalitis, ccr5-deficient mice showed increased survival rate and reduced levels of brain inflammation compared to mice expressing ccr5, indicating the participation of ccr5 in rocv-associated encephalitis (chávez et al., 2013) . although other studies discussed in this review have shown an important involvement of ccr5 in infection by flaviviruses [especially as an important molecule for the resolution of neuroinfection, in opposition to results of chávez et al., (2013) ], the role of the ccr5 in rocv infection represents a neglected topic. this knowledge gap should be addressed in further studies since the reemergence of rocv in brazil is a concern in terms of public health (figueiredo, 2007; ellwanger et al., 2017b) . although no other rocv outbreaks have been reported in brazil after the 1980s, there is evidence of rocv circulation in animals (casseb et al., 2014; pauvolid-correa et al., 2014; silva et al., 2014) . ccr5 expression on t cells. of note, zikv is another mosquito-borne flavivirus that recently reemerged in many countries, affecting brazil in particular. zikv infection is associated with microcephaly and other human development problems (baud et al., 2017) . zachova et al. (2019) showed that epstein-barr virus (ebv)-infected b cells have increased ccr5 expression compared to ebv-non-infected cells. also, recent evidence points to a pivotal role of the ccr5 in the attenuation of rhinovirus-associated inflammation, by controlling the activity of cd4 + foxp3 + treg cells (hossain et al., 2020) . altered levels of cytokines and chemokines are associated with several aspects of zikv (barros et al., 2018; naveca et al., 2018; khaiboullina et al., 2019; lima et al., 2019) , ebv (piovan et al., 2005; ehlin-henriksson et al., 2009; cohen et al., 2017) , and rhinovirus infections (rajan et al., 2014; shelfoon et al., 2016; hansel et al., 2017) . however, the potential role of the chemokine receptor ccr5 in the pathogenesis of zikv, ebv, and rhinovirus represents open questions in the field of ccr5 research. coronaviruses are positive-sense rna viruses that belong to the coronaviridae family (li et al., 2020) . coronaviruses infect a wide range of species, including humans. until 2019, humans have faced two major outbreaks of high pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov) outbreak and the middle east respiratory syndrome coronavirus (mers-cov) outbreak (fung and liu, 2019) . in 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan (hubei province, china) and rapidly spread to more than 180 countries/regions (interactive dashboard of global covid-19 cases -johns hopkins university: https://arcg.is/0fhmtx ; dong et al., 2020) . of note, sars-cov-2 infection causes the "coronavirus disease 2019" and therefore is also called "covid-19". considering the ongoing sars-cov-2 pandemic and the clinical variability observed in the affected individuals, ranging from mild to severe respiratory disease j o u r n a l p r e -p r o o f 2020b), the following question arose: does ccr5 have any clinically relevant influence on sars-cov-2 or other coronaviruses infections? recent data indicated that sars-cov-2 binds to the ace2 receptor (andersen et al., 2020) , using this receptor for entry into human cells. therefore, it is unlikely that ccr5 or ccr5δ32 have some significant effect on the susceptibility or resistance to sars-cov-2 infection. however, ccr5 may have some impact on the clinical course of sars-cov-2 infection. the pattern and intensity of the human immune responses regulate the clinical progression and even the outcome of infections by coronaviruses (li et al., 2020) , including sars-cov-2 (qin et al., 2020; shi et al., 2020) . sars-cov-infected mice showed increased expression of ccr5 mrna in lungs along with the production of ccl2, ccl3 and ccl5 chemokines, the major ccr5 natural agonists (chen et al., 2010) . intracranial infection of mice with mouse hepatitis virus (mhv, a murine coronavirus) induces an increased ccr5 expression, which contributed to mhv-induced demyelination through ccr5-mediated macrophage recruitment to the cns (glass et al., 2001) . subsequent studies using mhv-infected mice showed that ccr5 participates in the regulation of cd4 + and cd8 + t cell activities against the virus in the cns (glass and lane, 2003a; glass and lane, 2003b) . also, sars-cov-infected human monocyte-derived dendritic cells showed increased ccr5 expression in vitro (law et al., 2009) . taking together, the findings mentioned above suggest that ccr5 could have some influence on the clinical course of sars-cov-2 infection. however, future studies addressing humans are needed to clarify this suggestion. in this sense, we emphasize that so far, there is no evidence showing a clear involvement of ccr5 in sars-cov-2 human infection. the ccr5δ32 is a highly penetrating polymorphism, and exerts a robust phenotypic effect on ccr5. however, the expression of ccr5 is regulated by other polymorphisms in addition to ccr5δ32 (mehlotra, 2019) . also, the ccr5 expression is influenced by non-coding genetic elements. the effect of the genetic backround of the host can also be exacerbated or diminished depending on the environmental conditions to which the individual is exposed (ellwanger et al., 2018c; kulkarni et al., 2019) . taking together, these factors help to explain why many of the effects exerted by ccr5 on a given disease are specific to a certain population, ethnic group, or individual. research involving ccr5 and hiv began in the mid-1990s. since then, many achievements in the field of ccr5/hiv research have been made, such as the identification of ccr5δ32 as a strong protective factor against hiv infection and the development of ccr5 antagonists for the treatment of hiv infection. currently, these drugs are being tested for the treatment of other inflammatory and infectious diseases. in this context, the use of ccr5 antagonists has raised some concerns, since the blockade of ccr5 can affect j o u r n a l p r e -p r o o f or even weaken the host defenses against certain infections, especially neuroinvasive infections by flaviviruses. however, to date, there is no strong evidence indicating that the use of ccr5 antagonists has increased the susceptibility of individuals to problematic flaviviruses infections. the frequency of ccr5δ32 is quite varied among human populations, and therefore the effects of the allele in a particular population may not apply to another population. moreover, the effects of ccr5 and ccr5δ32 are disease-specific. for instance, the ccr5δ32 does not significantly affect susceptibility to wnv infection. however, the effect of the ccr5 and ccr5δ32 on wnv disease progression is very robust. some animal-derived findings suggest the involvement of the ccr5 in the pathogenesis of denv infection. however, the effects of ccr5 on denv infection are very different between humans and rodents. of note, ccr5δ32 does not significantly affect susceptibility to denv infection or disease progression. moreover, the effect of ccr5δ32 on hbv-related disease is population-specific. interestingly, cmv release virokines, which are molecules that can manipulate the host immune response and affect the ccr5 function. the examples cited above highlight the varied impacts of ccr5 and ccr5δ32 on viral infections. the few studies involving cchfv, ev, and hantavirus infection have indicated important effects of ccr5δ32 on these conditions. based on these findings, further studies should investigate the role of ccr5δ32 and ccr5 protein in such infections, considering populations with distinct genetic backgrounds. some evidence suggested the participation of ccr5 in infections by zikv, ebv, and rhinovirus. also, a mouse model of rocv-associated encephalitis suggested an important role for ccr5 in host immune responses against the virus. however, the roles of ccr5 and ccr5δ32 in infections by zikv, ebv, rhinovirus, and rocv are still poorly understood and need to be investigated in future studies. the role of evs in the transport of ccr5 between cells indicates that the expression of ccr5 on the cell surface may also depend on the release of evs containing ccr5. also, the transport of host molecules and viruses through evs adds complexity to the topics covered in this review and should be taken into account in future studies that investigate the role of ccr5 in viral infections. gene editing technologies have the potential to be used to treat different diseases, mainly when applied to somatic cells. however, ccr5 gene editing in human embryos presents a number of ethical problems. besides, the absence of ccr5 can have deleterious effects in certain conditions, such as increased susceptibility to symptomatic wnv infection. finally, this article showed that the participation of ccr5 in different viral infections is complex and varied and, therefore, cannot be generalized. this article also pointed out neglected gaps in knowledge involving ccr5 that should be addressed in future studies. the authors declare no conflicts of interest. ccr5 polymorphism as a protective factor for hepatocellular carcinoma in hepatitis b virus-infected iranian patients cd4-ccr5 interaction in intracellular compartments contributes to receptor expression at the cell surface effects of the ccr5-δ32 mutation on antiviral treatment in chronic hepatitis c effects of the ccr5-δ32 mutation on hepatitis c virus-specific immune responses in patients with haemophilia cc-chemokine receptor 5 (ccr5) in hepatitis c--at the crossroads of the antiviral immune response? downregulation of ccr5 expression on the peripheral blood + t cells of southeastern iranian patients with chronic hepatitis b infection association of genetic variations in ccr5 and its ligand, rantes with clearance of hepatitis b virus in korea an emerging interplay between extracellular vesicles and cytokines lack of chemokine receptor ccr5 promotes murine fulminant liver failure by preventing the apoptosis of activated cd1d-restricted nkt cells ccr5 in t cell-mediated liver diseases: what's going on? the biology of ccr5 and cxcr4 gene editing of hiv-1 co-receptors to prevent and/or cure virus infection the proximal origin of sars-cov-2 host genetics, innate immune responses, and cellular death pathways in poliomyelitis patients peripheral blood cd8 + t cells ccr5 expression and its δ32 mutation in iranian patients with occult hepatitis b infections elevated chemokine levels during adult but not pediatric crimean-congo hemorrhagic fever human t cell responses to dengue and zika virus infection compared to dengue/zika coinfection enteroviral cardiomyopathy: bad news for the dystrophinglycoprotein complex ccr5 proinflammatory allele in prostate cancer risk: a pilot study in patients and centenarians from sicily the role of chemokines in the pathogenesis of neurotropic flaviviruses acute zika virus infection in an endemic area shows modest proinflammatory systemic immunoactivation and cytokine-symptom associations an update on zika virus infection crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity the global distribution and burden of dengue host genetic risk factors for west nile virus infection and disease progression a study on the association between ccrδ32 mutation and hcv infection in iranian patients ccr5 receptor antagonism inhibits hepatitis c virus (hcv) replication in vitro multiple nonfunctional alleles of ccr5 are frequent in various human populations ccr5 binds multiple cc-chemokines: mcp-3 acts as a natural antagonist analysis of the ccr5 gene coding region diversity in five south american populations reveals two new non-synonymous alleles in amerindians and high ccr5*d32 frequency in euro-brazilians targeting ccr5 trafficking to inhibit hiv-1 infection ccr5 but not ccr1 deficiency reduces development of dietinduced atherosclerosis in mice looking for biological factors to predict the risk of active cytomegalovirus infection in non-immunosuppressed critically ill patients ccr5 revisited: how mechanisms of hiv entry govern aids pathogenesis primary acute dengue and the deletion in chemokine receptor 5 (ccr5δ32) exosomes from hepatitis c infected patients transmit hcv infection and contain replication competent viral rna in complex with ago2-mir122-hsp90 identification of genetic variants associated with dengue or west nile virus disease: a systematic review and meta-analysis control of regulatory t cell migration, function, and homeostasis natural killer cell recruitment to the lung during influenza a virus infection is dependent on cxcr3, ccr5, and virus exposure dose seroprevalence of flaviviruses antibodies in water buffaloes (bubalus bubalis) in brazilian amazon division of vector-borne diseases (dvbd) mip-1 α axis in the pathogenesis of rocio virus encephalitis in a mouse model downregulation of ccr5 inhibits the proliferation and invasion of cervical cancer cells and is regulated by microrna-107 cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4 + t cells are important in control of sars-cov infection epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type i t cells infiltration into central nervous system chemokine-containing exosomes are released from heatstressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine human papillomavirus and carcinogenesis: novel mechanisms of cell communication involving extracellular vesicles sickle cell disease: a chronic inflammatory condition isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome differential expression levels of inflammatory chemokines and tlrs in patients suffering from mild and severe japanese encephalitis ccr5 regulates inflammatory gene expression in response to encephalomyocarditis virus infection west nile virus and its vectors regulation of inflammatory chemokine receptors on blood t cells associated to the circulating versus liver chemokines in dengue fever identification of a major co-receptor for primary isolates of hiv-1 association of single nucleotide polymorphisms in tnfa and ccr5 genes with japanese encephalitis: a study from an endemic region of north india virus and host determinants of west nile virus pathogenesis ccr5 signaling suppresses inflammation and reduces adverse remodeling of the infarcted heart, mediating recruitment of regulatory t cells an interactive web-based dashboard to track covid-19 in real time ccr5 is involved in resolution of inflammation in proteoglycan-induced arthritis hiv-1 entry into cd4 + cells is mediated by the chemokine receptor cc-ckr-5 ccr5 limits cortical viral loads during west nile virus infection of the central nervous system changes in chemokines and chemokine receptor expression on tonsillar b cells upon epstein-barr virus infection host immunogenetics in tick-borne encephalitis virus infection-the ccr5 crossroad host genetic factors can impact vaccine immunogenicity and effectiveness exosomes are possibly used as a tool of immune regulation during the dendritic cell-based immune therapy against hiv-i rocio virus: an overview ccr5 gene editing -revisiting pros and cons of ccr5 absence what we say and what we mean when we say redundancy and robustness of the chemokine system -how ccr5 challenges these concepts immunogenetic studies of the hepatitis c virus infection in an era of pan-genotype antiviral therapies -effective treatment is coming role of the genetic variant ccr5δ32 in hbv infection and hbv/hiv co-infection ccr5δ32 in hcv infection, hcv/hiv co-infection, and hcv-related diseases exosomes in hiv infection: a review and critical look microrna-related polymorphisms in infectious diseases-tiny changes with a huge impact on viral infections and potential clinical applications role of ccr5δ32 mutation in protecting patients with schistosoma mansoni infection against hepatitis c viral infection or progression cytochrome p450 2d6 and mdr1 gene mutation in relation to mortality in patients with crimean-congo hemorrhagic fever: a preliminary study crimean-congo haemorrhagic fever cxcr3-deficiency protects influenza-infected ccr5-deficient mice from mortality ccr5 deficiency predisposes to fatal outcome in influenza virus infection dual ccr5/ccr2 targeting: opportunities for the cure of complex disorders short-term administration of the ccr5 antagonist vicriviroc to patients with hiv and hcv coinfection is safe and tolerable emergent arboviruses in brazil human coronavirus: host-pathogen interaction possible impact of 190g > a ccr2 and δ32 ccr5 mutations on decrease of the hbv vaccine immunogenicity-a preliminary report exosomes derived from septic mouse serum modulate immune responses via exosome-associated cytokines association between cytokines and exosomes in synovial fluid of individuals with knee osteoarthritis animal models for crimean-congo hemorrhagic fever human disease pharmacokinetics, safety and efficacy of maraviroc in treatment-experienced pediatric patients infected with ccr5-tropic hiv-1 the δ32 mutation of the chemokine-receptor 5 gene neither is correlated with chronic hepatitis c nor does it predict response to therapy with interferon-α and ribavirin functional expression of chemokine receptor ccr5 on cd4 + t cells during virus-induced central nervous system disease functional analysis of the cc chemokine receptor 5 (ccr5) on virus-specific + t cells following coronavirus infection of the central nervous system chemokine receptor ccr5 promotes leukocyte trafficking to the brain and survival in west nile virus infection reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr5 following infection with a neurotropic coronavirus ccr5 deficiency increases risk of symptomatic west nile virus infection chemokine regulation of inflammation during acute viral infection the ccr5-δ32 mutation: impact on disease outcome in individuals with hepatitis c infection from a single source activation of ccr5 by chemokines involves an aromatic cluster between transmembrane helices 2 and 3 ccr5δ32 mutation does not influence the susceptibility to hcv infection, severity of liver disease and response to therapy in patients with chronic hepatitis c controls for lung dendritic cell maturation and migration during respiratory viral infection crispr'd babies: human germline genome editing in the 'he jiankui affair dual and triple infections with influenza a and b viruses: a case-control study in southern brazil the pathogenesis of human cytomegalovirus circulating human cd4 + t cells have intracellular pools of ccr5 molecules five-year safety evaluation of maraviroc in hiv-1-infected treatment-experienced patients transcriptomic profile of host response in japanese encephalitis virus infection hiv-1 remission following ccr5δ32/δ32 haematopoietic stem-cell transplantation evidence for hiv-1 cure after ccr5δ32/δ32 allogeneic haemopoietic stem-cell transplantation 30 months post analytical treatment interruption: a case report dengue infection blockade of ccr5 to protect the liver graft in hiv/hcv co-infected patients ccr5 and responses to cocaine: addiction is not just about the brain a comprehensive evaluation of nasal and bronchial cytokines and chemokines following experimental rhinovirus infection in allergic asthma: increased interferons (ifn-γ and ifn-λ) and type 2 inflammation (il-5 and il-13) association of genetic variants of the chemokine receptor ccr5 and its ligands, rantes and mcp-2, with outcome of hcv infection the human cytomegalovirus, from oncomodulation to oncogenesis intragenus (homo) variation in a chemokine receptor gene (ccr5) ccr5 attenuates neutrophilic airway inflammation exacerbated by infection with rhinovirus extracellular vesicles work as a functional inflammatory mediator between vascular endothelial cells and immune cells clinical features of patients infected with 2019 novel coronavirus in wuhan the ccr5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction the role of a mutant ccr5 allele in hiv-1 transmission and disease progression ccr5 haplotypes influence hcv serostatus in caucasian intravenous drug users the effect of the ccr5-delta32 deletion on global gene expression considering immune response and inflammation long-term control of hiv by ccr5 delta32/delta32 stemcell transplantation significance of rantes-ccr5 axis and linked downstream immunomodulation in dengue pathogenesis: a study from guwahati, india human cytomegalovirus enhances placental susceptibility and replication of human immunodeficiency virus type 1 (hiv-1), which may facilitate in utero hiv-1 transmission cytomegalovirus upregulates expression of ccr5 in central memory cord blood mononuclear cells, which may facilitate in utero hiv type 1 transmission immunological cure of hbv infection the chemokine receptor ccr5, a therapeutic target for hiv/aids antagonists, is critical for recovery in a mouse model of japanese encephalitis the role of ccr5/cxcr3 expressing cd8+ cells in liver damage and viral control during persistent hepatitis c virus infection ccr5del32 genotype in human enteroviral cardiomyopathy leads to spontaneous virus clearance and improved outcome compared to wildtype ccr5 ccr5 inhibitors and hiv-1 infection toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells puumala hantavirus: an imaging review human cytomegalovirus infection reduces surface ccr5 expression in human microglial cells, astrocytes and monocyte-derived macrophages updates on chronic hbv: current challenges and future goals review of climate, landscape, and viral genetics as drivers of the japanese encephalitis virus ecology coronavirus infections and immune responses polymorphisms of ccl3l1/ccr5 genes and recurrence of hepatitis b in liver transplant recipients horizontal transfer of exosomal cxcr4 promotes murine hepatocarcinoma cell migration, invasion and lymphangiogenesis the transcriptional and protein profile from human infected neuroprogenitor cells is strongly correlated to zika virus microcephaly cytokines phenotype evidencing a persistent inflammation in the cns the chemokine receptor ccr1 is identified in mast cell-derived exosomes reduced cc chemokine receptor (ccr) 1 and ccr5 surface expression on peripheral blood t lymphocytes from patients with chronic hepatitis c infection ccr5: no longer a 'good for nothing' gene--chemokine control of west nile virus infection genetic deficiency of chemokine receptor ccr5 is a strong risk factor for symptomatic west nile virus infection: a meta-analysis of 4 cohorts in the us epidemic ccr5 deficiency is a risk factor for early clinical manifestations of west nile virus infection but not for viral transmission chemokine control of west nile virus infection natural history of hepatitis c chemokine receptor antagonist block inflammation and therapy japanese encephalitis virus infection in mouse model the cytokine storm of severe influenza and development of immunomodulatory therapy exosome-associated hepatitis c virus in cell cultures and patient plasma genetic variants and susceptibility to neurological complications following west nile virus infection polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation viral escape mechanisms -escapology taught by viruses transfer of the chemokine receptor ccr5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection the ccr5δ32 (rs333) polymorphism is not a predisposing factor for severe pandemic influenza in the brazilian admixed population soluble chemokine receptor cxcr4 is present in human sera hcv chronic infection and ccr5-δ32/δ32 ccr5 genetic variants and epidemiological determinants for hpv infection and cervical premalignant lesions pathogenic hantaviruses elicit different immunoreactions in thp-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells dengue virus requires the ccchemokine receptor ccr5 for replication and infection development ccr5 blockade for neuroinflammatory diseases-beyond control of hiv genetic variants in the ccr gene cluster and spontaneous viral elimination in hepatitis c-infected patients expression of rantes by normal airway epithelial cells after influenza virus a infection ccr5 promoter polymorphism -2459g > a: forgotten or ignored? cells chemokine receptors as important regulators of pathogenesis during arboviral encephalitis enhanced expression, native purification, and characterization of ccr5, a principal hiv-1 coreceptor dengue haemorrhagic fever: a job done via exosomes? clinical significance of the ccr5delta32 allele in hepatitis c ccr5 deficiency exacerbates t-cell-mediated hepatitis in mice ccr5, mcp-1 and vdr gene polymorphisms are associated with the susceptibility to hbv infection pathways for internalization and recycling of the chemokine receptor ccr5 single-cell tracking reveals a role for pre-existing ccr5 + memory th1 cells in the control of rhinovirus-a39 after experimental challenge in humans ccr5δ32 genotype leads to a th2 type directed immune response in esrd patients ccr5 deletion protects against inflammation-associated mortality in dialysis patients immune response to dengue virus and prospects for a vaccine the pathogenesis of nephropathia epidemica: new knowledge and unanswered questions the predictive value of il28b gene polymorphism for spontaneous clearance in a single source outbreak cohort is limited in patients carrying the ccr5δ32 mutation analysis of the immunological biomarker profile during acute zika virus infection reveals the overexpression of cxcl10, a chemokine linked to neuronal damage chemokine ccr5 and cocaine interactions in the brain: cocaine enhances mesolimbic ccr5 mrna levels and produces place preference and locomotor activation that are reduced by a ccr5 antagonist why and where an hiv cure is needed and how it might be achieved a study of cc-chemokine receptor 5 (ccr5) polymorphism on the outcome of hcv therapy in egyptian patients exosomes secreted by bacterially infected macrophages are proinflammatory association between mbl2 haplotypes and dengue severity in children from rio de janeiro, brazil influenza-induced innate immunity: regulators of viral replication, respiratory tract pathology & adaptive immunity mir-155 induction in microglial cells suppresses japanese encephalitis virus replication and negatively modulates innate immune responses ccr5 and hiv infection serological evidence of widespread circulation of west nile virus and other flaviviruses in equines of the pantanal, brazil the genomic ancestry of individuals from different geographical regions of brazil is more uniform than expected ccr5 blockage by maraviroc: a potential therapeutic option for metastatic breast cancer west nile virus: review of the literature the evolution of seasonal influenza viruses marked differences in ccr5 expression and activation levels in two south african populations hepatitis c virus chemokine-regulated recruitment of antigen-specific t-cell subpopulations to the liver in acute and chronic hepatitis c infection chemokine receptor expression in ebv-associated lymphoproliferation in hu/scid mice: implications for cxcl12/cxcr4 axis in lymphoma generation frequency of the 32-base pair deletion in the chemokine receptor ccr5 gene is not increased in hepatitis c patients associations of chemokine system polymorphisms with clinical outcomes and treatment responses of chronic hepatitis c chemokine receptors: multifaceted therapeutic targets dysregulation of immune response in patients with covid-19 in wuhan, china one hundred years of poliovirus pathogenesis human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells metabolomics as an approach to characterise the contrasting roles of ccr5 in the presence and absence of disease microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of cd16 + , ccr5 + and tlr8 + monocytes characterization in vitro and in vivo of a pandemic h1n1 influenza virus from a fatal case uncommon immune-mediated extrahepatic manifestations of hcv infection the future of gene editing -toward scientific and social consensus ccr5 deficiency and severe polio infection in the 1984 outbreak in finland platelet-and megakaryocyte-derived microparticles transfer cxcr4 receptor to cxcr4-null cells and make them susceptible to infection by x4-hiv neutrophils induce a novel chemokine receptors repertoire during influenza pneumonia analysis of ccr5-δ32 and ccr2-v64i polymorphisms in a cohort of spanish hcv patients using real-time polymerase chain reaction and fluorescence resonance energy transfer technologies the possible role of ccr5δ32 mutation in crimean-congo hemorrhagic fever infection the ccr5-δ32 mutation: impact on disease outcome in individuals with hepatitis b infection in the southern khorasan population (east of iran) interacting roles of immune mechanisms and viral load in the pathogenesis of crimean-congo hemorrhagic fever chemokine receptor expression and chemotactic responsiveness of human monocytes after influenza a virus infection ccr5 expression is elevated in cervical cancer cells and is up-regulated by seminal plasma host genetic risk factors for community-acquired pneumonia the second extracellular loop of ccr5 is the major determinant of ligand specificity resistance to hiv-1 infection in caucasian individuals bearing mutant alleles of the ccr-5 chemokine receptor gene ccr5 plays important roles in hepatitis b infection the natural history of human papillomavirus infection ccr2 and ccr5 genes polymorphisms in women with cervical lesions from pernambuco, northeast region of brazil: a case-control study ccr5delta32 in systemic lupus erythematosus: implications for disease susceptibility and outcome in a brazilian population carcinogenic human papillomavirus infection west nile virus infection structural basis of coreceptor recognition by hiv-1 envelope spike clinical aspects of nephropathia epidemica (puumala virus infection) in europe: a review association of host genetic risk factors with the course of cytomegalovirus retinitis in patients infected with human immunodeficiency virus ccr5-dependent activation of mtorc1 regulates translation of inducible no synthase and cox-2 during encephalomyocarditis virus infection chemokine release from human rhinovirus-infected airway epithelial cells promotes fibroblast migration ccr2 positive exosome released by mesenchymal stem cells suppresses macrophage functions and alleviates ischemia/reperfusion-induced renal injury covid-19 infection: the perspectives on immune responses evidence for internal stores of ccr5 in blood cells role of cc chemokine receptor 1 and two of its ligands in human dengue infection. three approaches under the cuban situation endocytosis and recycling of the hiv coreceptor ccr5 a saint louis encephalitis and rocio virus serosurvey in brazilian horses frequency of the ccr5-delta32 allele in brazilian populations: a systematic literature review and meta-analysis cd4+foxp3+ regulatory t-cell subsets in human immunodeficiency virus infection ccr5-δ32 polymorphism and susceptibility to cervical cancer: association with early stage of cervical cancer the ccr5δ32 allele is not a major predisposing factor for severe h1n1pdm09 infection the role of cytokines in the immune response to influenza a virus infection frequencies of gene variant ccr5-δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national dkms donor centers adaptive immune responses to primary and secondary dengue virus infections the global burden of dengue: an analysis from the global burden of disease study advances in the treatment of hiv/hcv coinfection in adults ccr5 deficiency enhances hepatic innate immune cell recruitment and inflammation in a murine model of acute hepatitis b infection combined effect of hpv and several gene snps in laryngeal cancer association between vitamin d receptor, ccr5, tnf-α and tnf-β gene polymorphisms and hbv infection and severity of liver disease interaction between rantes promoter variant and ccr5δ32 favors recovery from hepatitis b frequency of the ccr5-δ32 mutant allele is not increased in belgian hepatitis c virus-infected patients japanese encephalitis: a review of the indian perspective retinopathy severity correlates with rantes concentrations and ccr 5-positive microvesicles in diabetes soluble chemokine ccr5 receptor is present in human plasma ccl5-ccr5 interaction provides antiapoptotic signals for macrophage survival during viral infection ccr5δ32 mutation, mycoplasma pneumoniae infection, and asthma innate immune interleukin-1 receptor-associated kinase 4 exacerbates viral myocarditis by reducing ccr5 + cd11b + monocyte migration and impairing interferon production ecology and geographical expansion of japanese encephalitis virus maraviroc -a ccr5 antagonist for the treatment of hiv-1 infection human cytomegalovirus inhibits the migration of immature dendritic cells by down-regulating cell-surface ccr1 and ccr5 role of regulatory t cells during virus infection reduced cell surface expression of ccr5 in ccr5δ32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration the role of natural antibodies to cc chemokine receptor 5 in hiv infection class b β-arrestin2-dependent ccr5 signalosome retention with natural antibodies to ccr5 the abrogation of phosphorylation plays a relevant role in the ccr5 signalosome formation with natural antibodies to ccr5 erk1-based pathway as a new selective mechanism to modulate ccr5 with natural antibodies real life experiences in hcv management t-cell surface ccr5 density is not correlated with hepatitis severity in hepatitis c virus/hivcoinfected individuals: implications for the therapeutic use of ccr5 antagonists cytomegalovirus cc chemokine promotes immune cell migration the ccr5δ32 allele is associated with reduced liver inflammation in hepatitis c virus infection gene-edited babies: what went wrong and what could go wrong cc chemokine receptor 5 δ32 polymorphism in two independent cohorts of hepatitis c virus infected patients without hemophilia dilated cardiomyopathy ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions frequency of the hiv-protective cc chemokine receptor 5-δ32/δ32 genotype is increased in hepatitis c a new coronavirus associated with human respiratory disease in china ccr5 levels and expression pattern correlate with infectability by macrophagetropic hiv-1, in vitro critical role for ccr5 in the function of donor cd4 + cd25 + regulatory t cells during acute graftversus-host disease single nucleotide polymorphisms in candidate genes and dengue severity in children: a case-control, functional and meta-analysis study the potential risks of c-c chemokine receptor 5-edited babies in bone development bioinformatic identification of key genes and pathways that may be involved in the pathogenesis of hbv-associated acute liver failure effects of the chemokine receptor 5 (ccr5)-delta32 mutation on hepatitis c virus-specific immune responses and liver tissue pathology in hcv infected patients hepatitis b virus infection multiparametric flow cytometry analysis of peripheral blood b cell trafficking differences among epstein-barr virus infected and uninfected subpopulations differential expression of tim-3, pd-1, and ccr5 on peripheral t and b lymphocytes in hepatitis c virus-related hepatocellular carcinoma and their impact on treatment outcomes correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1a and c-c motif chemokine receptor 5 genes and infection with the hepatitis b virus in three ethnic groups in china pd1 + ccr2 + cd8 + t cells infiltrate the central nervous system during acute japanese encephalitis virus infection high frequency of ccr5-δ32 homozygosity in hcv-infected, hiv-1-uninfected hemophiliacs results from resistance to hiv-1 genetic polymorphism of chemokine receptors ccr2 and ccr5 in swedish cervical cancer patients host micrornas and exosomes that modulate influenza virus infection structure of cc chemokine receptor 5 with a potent chemokine antagonist reveals mechanisms of chemokine recognition and molecular mimicry by hiv exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral rna and proteins to the vertebrate neuronal cells chemokines: a new classification system and their role in immunity protective effect of ccr5-δ32 against hiv infection by the heterosexual mode of transmission in a polish population american 395 symptomatic wnv+ individuals; 145 symptomatic but noninfected individuals; 1318 controls (blood donors)the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection glass et al. (2006) american 224 symptomatic wnv+ individuals; 1318 controls (blood donors)corroborating data from glass et al. (2006) , the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection lim et al. (2008) american 634 wnv+ individuals; 422 non-infected individuals ccr5δ32 homozygous genotype was associated with clinical symptoms of wnv infection; no association between ccr5δ32 and susceptibility to wnv infection lim et al. (2010) american, canadian 385 symptomatic wnv+ individuals; 328 asymptomatic wnv+ individuals; 1318 controls [blood donors from glass et al. (2006)] no association of ccr5δ32 and wnv infection considering symptomatic and asymptomatic wnv+ individuals; considering a dominant model of inheritance of ccr5δ32 and using controls from glass et al. (2006) , the ccr5δ32 was associated with symptomatic wnv infection and infection risk bigham et al. (2011) key: cord-327135-4c2flue4 authors: chinnaswamy, s title: gene–disease association with human ifnl locus polymorphisms extends beyond hepatitis c virus infections date: 2016-06-09 journal: genes immun doi: 10.1038/gene.2016.24 sha: doc_id: 327135 cord_uid: 4c2flue4 interferon (ifn) lambda (ifn-λ or type iii ifn) gene polymorphisms were discovered in the year 2009 to have a strong association with spontaneous and treatment-induced clearance of hepatitis c virus (hcv) infection in human hosts. this landmark discovery also brought renewed interest in type iii ifn biology. after more than half a decade since this discovery, we now have reports that show that genetic association of ifnl gene polymorphisms in humans is not limited only to hcv infections but extends beyond, to include varied diseases such as non-alcoholic fatty liver disease, allergy and several other viral diseases including that caused by the human immunodeficiency virus. notably, all these conditions have strong involvement of host innate immune responses. after the discovery of a deletion polymorphism that leads to the expression of a functional ifn-λ4 as the prime ‘functional’ variant, the relevance of other polymorphisms regulating the expression of ifn-λ3 is in doubt. herein, i seek to critically address these issues and review the current literature to provide a framework to help further understanding of ifn-λ biology. supplementary information: the online version of this article (doi:10.1038/gene.2016.24) contains supplementary material, which is available to authorized users. in the year 2003, two different groups reported on the presence of three novel genes closely placed to each other, on human chromosome 19 that coded for interferons (ifns) with potent antiviral properties. 1, 2 these genes due to their relatedness to the interleukin 10 (il-10) family were initially christened il-28a, il-28b and il-29, and subsequently changed to ifnl2, ifnl3 and ifnl1. 3 the genes encode ifnλ-2, ifnl-λ3 and ifnl-λ1, respectively; together with the newly discovered ifnl-λ4 (or ifnl4) they constitute the type iii ifns or the lambda ifns (ifnl-λs). ifnl-λ1, 2 and 3 activate antiviral responses through the jak-stat (janus kinase-signal transducer and activator of transcription) pathway by utilizing a distinct receptor complex made of a heterodimer formed between ifn-λr1 and il-10r2. 2, 3 subsequent studies showed that unlike the receptors that bind to type i ifns, the ifn-λ receptors were expressed on selective cell types mainly of epithelial origin, hepatocytes and some immune cells. [3] [4] [5] [6] the discovery of ifn-λs was seminal in the sense that it showed the presence of an alternate system to the well-known type i ifns (ifn-α and ifn-β) that the different nucleated cells in the body, especially the ones on epithelial surfaces, can utilize to combat viral infections. later studies in mice have shown that type iii ifns form a strong barrier at the host-environment interface, which encompasses large regions of epithelial lining to the respiratory, gastrointestinal and urogenital tracts of mammals. [7] [8] [9] [10] a major boost in the area of ifn-λ research came after another discovery in the year 2009. three independent groups conducted genome-wide association studies (gwass) involving treatment response to chronic hepatitis c virus (hcv) infections, in three different geographical regions of the world, and reported that single-nucleotide polymorphisms (snps) in the ifnl locus (figure 1 ), had strong association with treatment-induced hcv clearance irrespective of ethnicity and geographical location of the hosts. [11] [12] [13] the search for a 'causative' or a 'functional' snp at the ifnl locus that could give a biological explanation for the hcv-gwas results was taken up rigorously by several groups, but none seem to have given a better explanation to the hcv-ifn-λ 'puzzle' than the group from the national institutes of health, usa, that discovered the presence of another ifnl upstream to ifnl3, named as ifnl4 (refs 14,15; figure 1 ; in figure 1b , the alleles for the respective snps are shown as beneficial (b) or non-beneficial (nb) with respect to the studies on hcv (reviewed in ref. 15 ; the major allele for each of the snps depicted in figure 1b has been shown to be the beneficial one in hcv infections). functional ifn-λ4 is expressed only in a subset of individuals, due to a frameshiftcausing deletion polymorphism (ifnl4-δg; rs368234815) in the first exon of ifnl4 (figure 1 ). 14 the presence of the alternate allele (ifnl4-tt) renders ifnl4 a pseudogene and this allele is seen iñ 50% of the european population and in most of the east asian population, but ifnl4 is a functional gene (ifnl4-δg allele) in majority (~95%) of the african population, 16 suggesting that human evolution has played an active role in elimination of a functional ifn-λ4 in the human species. 17 in fact, the pseudogene shows strong positive selection in human evolution, whereas the functional gene is conserved in other mammalian species except in mice and rats where the gene is completely absent. 17 in this article, i review the literature on genetic association studies that have shown the involvement of the hcv-gwas snps in non-hcv disorders that involve both viral diseases and some non-infectious conditions. i also update the progress on transcriptional studies of the ifnl4 gene and examine whether the functional ifn-λ4-generating snp is sufficient to explain the molecular mechanism of causality in the diseases it is associated with, and whether the other ifnl locus snps (mainly the ones regulating ifn-λ3 expression) may have any functional roles to play in the observed phenotypes. ifnl-λs and innate immunity against viruses innate and adaptive immunity are the two indispensable arms of the mammalian immune system. although we had a clearer understanding of the principles of functioning of the adaptive immunity arm, a lack of advanced molecular techniques and incomplete understanding of molecular mechanisms made us remain unaware of the intricacies of functioning of the innate immunity arm, for a long time. 18 with the advent of superior molecular biology techniques and the discovery of the pathogenassociated molecular pattern (pamp) or pattern recognition receptors, 19 we now have better understanding of how nucleated cells can differentially recognize different classes of pathogens and propagate signals to their surroundings, in the process raising the immediate alarm in the host. 19 large strides were made in the area of molecular recognition of viral pamps and signal transduction that leads to raising of antiviral states within virusinfected cells. 10, 20 the epithelial cells, being at the interface between the host and the environment in the respiratory, gastrointestinal and the urogenital tract, are not only prone to a variety of viral infections but are strategically located to respond and propagate alarm signals to the underlying immune cells ( figure 2) . however, the primary function of the epithelium is to provide a physical barrier between the underlying lamina propria and the lumen of the cavity or the exterior. even though they can sense and respond to pamps and damage-associated molecular patterns, 21 the epithelial cells are not professional immune cells and due to their high level of differentiation, may lack the plasticity required to send out amplified and prolonged signals to the lamina propria. therefore, a crucial link still remained missing about how an adaptive immune response is shaped within distantly located lymph nodes that have obligatory 'immune-rich environments', by taking cues from signals generated by viral infections at the epithelium ( figure 2 ). the discovery of the new class of effector immune cells called the innate lymphoid cells (ilcs) may seem to have provided the answer to this puzzle ( figure 2 ). ilcs are derivatives of common lymphoid precursors along with t and b cells. 22 these cells are stationed near the epithelial surfaces in larger numbers and respond to signals from the surrounding cells by secreting cytokines and chemokines in large quantities, thereby acting as signal amplifiers in both health and disease. 23 the ilcs are considered the innate immunity counterparts to the various th (t helper) cell (cd4+ (cluster of differentiation)) subsets of the adaptive immune system (figure 2 ; ref. 24) . for example, ilc2s respond to il-33 generated from influenza virus-infected epithelial cells by secreting il-5 and il-13, both known inducers of th2 immunity. 25 the natural killer cells, now classified as ilc1 cells, are considered the tc (t cytotoxic) cell (cd8+) counterparts. 24 even though most studies so far on ilcs have been on mice, ilcs have also been characterized in a variety of human tissues 26 and are deregulated in many human diseases. 27 with emerging roles of type iii ifns at the epithelium, 8, 10, 28 ilcs and ifn-λs now seem to be the major players in innate immunity at barrier surfaces. 10 15 ). the minor allele (mi) is the non-beneficial allele (that leads to expression of a functional ifn-λ4 and also lowers expression of ifn-λ3) and the major allele (ma) is the beneficial allele (that does not produce a functional ifn-λ4 and is associated with higher levels of expression of ifn-λ3) for all snps, again with respect to hcv studies. 15 the information on ld values has been obtained from 1000 genomes project reference panel (http://www.1000genomes.org), october 2014 release; some ld (r 2 ) values for yri (marked with *) were obtained from ref. 14. the maf values were obtained from dbsnp (national center for biotechnology information). in asian population, mafs of rs12979860, rs8099917 and rs4803217 are from jpt population; and mafs of rs368234815, rs8103142 and rs28416813 are from chb-jpt populations. yri, yoruba in ibadan, nigeria; ceu, residents with ancestry from northern and western europe; chb/jpt, han chinese in beijing, china/japanese in tokyo, japan; -, maf information not available for the ta repeat polymorphism rs59702201. how ilcs respond and interact with the epithelial cells during viral infections and how they cross-talk with other immune cells at the barrier surfaces in shaping and maintaining the optimal th responses, will form the next exciting wave in innate immunity research. similarly, research on the production, regulation and functions of ifn-λs in viral infections has also been exciting in the last decade. human ifn-λs are secreted (except ifn-λ4 that is poorly secreted 30 ) in response to the detection of viral rna intermediates from the cytoplasm of epithelial cells via the toll-like receptor and retinoic acid inducible gene i-like receptor) pathways. 4, 7, 31 ifn-λs are also known to be secreted by macrophages, plasmacytoid dendritic cells, monocyte-derived dendritic cells and hepatocytes. 4, 6, [32] [33] [34] the ifn-λr1 receptor is expressed on limited cell types including epithelial cells, hepatocytes, b cells and monocytes. 5, 35 there is currently no information on whether the newly discovered ilcs secrete any of the ifn-λs and whether they express ifn-λr1. ifn-λs act in a paracrine and/or autocrine manner to raise an antiviral state in the infected and to-be-infected cells by reprogramming the target cell gene expression patterns. 28, 36 the ifnl snps would have functional roles if they can affect the diversification of innate and adaptive immune cell subsets. diversification of ilc subsets will lead to the polarization of dendritic cells and macrophages and will eventually influence the th1/th2 balance by favoring either a th1 or a th2 response ( figure 3 ). 37 although there is no evidence for this belief, it is known that the ifnl snps do affect the expression of ifn-λ3 (ref. 15 ) and that they give rise to a new ifn (ifn-λ4). 14 ifn-λ1, 2 and 3 are known to deflect the th1/th2 balance to a th1 predominant mode in vitro and also in vivo in humans and mice 38, 39 (reviewed in ref. 37) . no such studies are reported for ifn-λ4. one hypothesis is that different levels of ifn-λ3 expression dictated by the underlying genetic polymorphisms are responsible for eliciting a th1 or a th2 response. 37 the role played by ifn-λ4 in this process is only speculative at this stage ( figure 3 ). how ifn-λs interact with the newly discovered ilcs is also unknown. a recent report showed that rotavirus infection in mice is controlled by il-22 produced by ilc3s for which the presence of an intact ifn-λ signaling pathway is required. 40 an even more important question in humans will be on what role does the ifnl snps, and therefore ifn-λ4, play in the diversification and functioning of ilcs. ifn-λ4 apart from being antiviral to hcv 14 is also known to inhibit other flaviviruses such as dengue virus, yellow fever virus 41 and human corona viruses. 30 some of the other ifn-λs are known to be induced by several human pathogens including m. tuberculosis, 42 human papilloma virus, 43 influenza virus 44 and human metapneumovirus (also induces ifn-λ4), 45 and have clear antiviral activities against some but not other viruses in mice (reviewed in ref. 46) . in a recent finding, murine ifn-λ3 but not ifn-α/ifn-β was responsible for protecting mice against norovirus persistence in mice colon, strikingly, even in the absence of t and b lymphocytes. 9 similar results were seen in reovirus infections of mice colon. 8 in conclusion, ifn-λs are potent antiviral molecules and their cross-talk with innate immune cells can potentially figure 2 . immune responses to viral infections of the epithelia. resident macrophages and dendritic cells (dcs) form the first line of cellular resistance to invading viruses. the newly discovered innate lymphoid cells (ilcs) 22 may have important roles due to their ability to respond to signaling molecules/alarmins secreted by the epithelial cells. ilcs diversify and respond by secreting large amounts of effector cytokines and chemokines locally. these effector molecules can potentially lead to the polarization of dcs and macrophages, and therefore may be critical in shaping the adaptive immunity mediated by t-helper (th) and t-cytotoxic (tc) cells that develop in the nearby lymphatic tissue (green lobed structure). mhc, major histocompatability complex. orchestrate innate immunity against several viruses and they may be particularly important at the barrier surfaces. ifn-λs also modulate adaptive immunity by affecting th1/th2 balance, tilting it to a th1-favoring response that is required for clearing viral infections. the function of the newly discovered ifn-λ4 in these immune processes remains to be determined. in cognizance of the potential that ifn-λs may hold in innate immunity, researchers across the world have got intrigued with the ifnl snps and have started to test them in candidate gene case-control studies in both infectious and non-infectious diseases. so far, association has been reported with nonalcoholic fatty liver disease (nafld), allergy and infections with several viruses. causing chronic infections and is especially a problem in immunosuppresed individuals. egli et al. 47 tested the association of the ifnl snp rs8099917 with cmv replication in a small number of solid organ transplant patients (n = 38) who were seronegative for cmv (but received an organ transplant from a cmvseropositive donor) and who had stopped receiving antiviral prophylaxis. they found that the minor allele at rs8099917 (g) was associated with decreased cmv replication (p = 0.036) in a dominant model of inheritance. further, their in vitro studies provided evidence for a beneficial effect of the minor allele against cmv replication. 47 a study in cmv-seropositive kidney transplant patients 48 also found that the minor allele (t) at rs12979860 had a dominant beneficial effect against cmv replication. although in another study in allogenic stem cell transplant recipients, 49 the minor allele t at rs12979860 protected recipients against cmv infection in a recessive model of inheritance. two more studies have also reported on the association of ifnl snps with cmv. 50, 51 the first report by bibert et al. 50 tested for the occurrence of cmv retinitis among those human immunodeficiency virus (hiv)-infected patients who were at risk of developing cmv retinitis due to low cd4 counts and cmv seropositivity. they found that carriers of two copies of the minor allele (δg/δg), increased the risk of getting cmv retinitis (p = 0.007) in multivariate regression models. in the second report by manuel et al., 51 solid organ transplant patients were tested for the association of rs368234815 with cumulative incidence of cmv replication. the results showed that the minor allele homozygotes (δg/δg), only in the pre-emptive antiviral therapy group but not among those receiving antiviral prophylaxis, had higher incidence of cmv replication. these results are indeed very interesting and sound, but the paradoxical findings of the five groups in terms of the model of inheritance of ifnl snps raise some questions. although two groups showed that their results fit best with a recessive model of inheritance of ifnl snps 50,51 in affecting cmv replication/retinitis wherein minor homozygosity was non-beneficial, the other three studies show an opposite trend where the minor allele had a beneficial effect against cmv replication in both patients and cell culture experiments, involving either dominant 47, 48 or recessive models of inheritance. 49 in stark contrast, a dominant model of inheritance (of the non-beneficial ifnl snp minor allele) has consistently given the best explanation on the observed phenotypes in association studies with both spontaneous clearance and ifn-based treatment response in chronic hcv infections. [11] [12] [13] 15 this discrepancy within the cmv studies may be partly due to statistical fluctuations owing to low sample sizes, 47, 50 and high heterogeneity among patient groups, end points and antiviral regimens that are followed in the different studies. the discrepancy needs be resolved by well-designed replication studies to gain a proper understanding of the role of ifnl snps in cmv replication and disease. human t-lymphotrophic virus. human t-lymphotrophic virus (htlv)-1 is an ancient retrovirus causing chronic infections in humans and is associated with adult t-cell leukemia/lymphoma. it is also associated with inflammatory disorders such as htlv-1associated myelopathy/tropical spastic papaparesis (ham/tsp), htlv-associated arthropathy and several other related disorders including rheumatoid arthritis. a spanish study reported for the first time that an ifnl snp (rs12979860) was associated with ham/ tsp in a small number of patients (n = 41) who had htlv-1 proviral dna in their blood cells. 52 they showed that the presence of the minor allele made 12/41 patients to have a sixfold higher risk (p = 0.03) of having symptoms of ham/tsp compared with asymptomatic carriers (n = 29) in a dominant model of inheritance. however, proviral dna load was a confounder in this association; covariate analysis suggested that both the snp and proviral dna load were linked in their association with ham/tsp. the minor allele-carrying genotypes (ct and tt) were indeed having higher proviral dna in their blood cells compared with the major homozygotes (p = 0.01). the major drawback of this study seems to be the low sample number; although the strength of the study was that there was significant effect of the minor allele on proviral dna copy number, a functional association that could be directly linked with the antiviral role of ifn-λ3. it is known that the ifnl snp minor alleles are associated with decreased expression of ifn-λ3 (reviewed in ref. 15 , although an opposite effect is evident in chronic hcv infections, where the minor allele carriers have low baseline virus levels 11 ). in a later study from brazil, 53 both rs8099917 and rs12979860 were tested in a cohort consisting of 229 htlv-positive subjects (93 ham/tsp patient and 136 asymptomatic carriers). the minor allele g of rs8099917 was significantly associated with ham/tsp in both univariate and multivariate analysis with a recessive model of inheritance (p o 0.001). in this study, the proviral dna load as a covariate did not seem to interfere with the association of rs8099917 with ham/tsp unlike the previous spanish study. 52 with respect to rs12979860, the minor allele t was associated with ham/tsp only as a heterozygote in univariate analysis (p = 0.01) and weakly in multivariate analysis (p = 0.06). however, a series of studies have also reported conflicting results to the above two reports on the association of ifnl snps with htlv-1-associated diseases. first, sanabani et al. 54 show clearly a lack of association of ifnl snp rs12979860 with ham/tsp and/or adult t-cell leukemia/lymphoma. this brazilian study had more number of samples (n = 112) than the spanish study of trevino et al. (n = 41). 52 they also did not find any correlation of proviral dna load with rs12979860 genotypes. another report from brazil with a sample size of 79 also reflected similar findings, where they found no association of rs12979860 with ham/tsp and proviral dna load. 55 this study also compared 300 healthy controls with 79 htlv-1-positive subjects and found no association with rs12979860 and htlv-1 infection. yet another recent study from brazil analyzed the genotypes at rs8099917, rs12979860 and rs8103142 in 300 healthy controls and 96 htlv-1-infected individuals, and found no association with htlv-associated arthropathy and any of the three snps when tested individually. 56 when they carried out haplotype analysis, they found some association (p = 0.01) with htlv-1 infection and one of the seven haplotypes (cct) involving the three snps (rs8099917, rs12979860 and rs8103142); with htlv-associated arthropathy and another haplotype (ttg; p = 0.05). they also found an association of the three snps individually with levels of some cytokines (such as ifn-γ) and proviral dna load (p o 0.05). 56 however, no multiple testing corrections seem to have been carried out in their analysis, thus severely undermining the results. 56 further, a japanese study also failed to see any association with adult t-cell leukemia/lymphoma and rs8099917 and also with htlv-1 and hcv mono-or co-infections. 57 last, a study from france on 95 htlv-1-positive subjects of afro-carribean lineage compared the distribution of genotypes of rs12979860 and the ifn-λ4-generating snp rs368234815, and found no association with ham/tsp. 58 in summary, the results so far are not entirely convincing on a true association of the ifnl snps with htlv infection-related diseases. further, in the two reports 52,53 that did see an association, the models of inheritance used to fit the phenotype data do not agree with each other, raising doubts on the underlying functionality of the observed genetic associations. therefore, more functional characterization of the observed association may be needed to rule out false positivity. if at all there is another human disease where ifnl snps were expected to have associations as strong as that of hcv infections, it was the case of hepatitis b virus (hbv). this expectation is because both viruses are hepatotropic and cause chronic infections; both diseases can be effectively treated using ifn-α even though they differ in their pamp ligands recognized by innate immune receptors. 59, 60 mixed results have been obtained about the role of ifnl snps in both spontaneous clearance and ifn-α-induced clearance of hbv and the literature until the year 2013-2014 has been reviewed elsewhere. [61] [62] [63] more studies have also been conducted since then (results from 10 of them are summarized in supplementary table 1 ), but have largely failed to resolve the conflict. two studies have also reported on a lack of association of ifnl snps on spontaneous as well as ifn-induced clearance of hepatitis d virus, a co-infecting satellite virus that requires hbv for replication and assembly. 64, 65 although consistent results were obtained across numerous studies with hcv-ifnl snp association mainly because they had only virological end point phenotypes 15 that correlated well with serological and biochemical parameters of the infection, several drawbacks in case of hbv studies may be responsible for the inconsistent results. some of them are: presence of different hbv genotypes as mixed infections, with some genotypes (genotype d 62 ) showing better association than others; variation in treatment regimens (ifn alone or with nucleotide analogs); end point phenotypes varying from serology to viral load, to biochemical parameters without a common quantitative parameter to assess the phenotype; prolonged clinical course of the disease with fluctuating virological, serological and biochemical markers; and prevalence rates of disease differing in different ethnicities/populations, to name a few. the reports that have shown an association of ifnl snps with hbv spontaneous or treatment-induced clearance are largely in agreement with the results from hcv studies in terms of the model of inheritance (supplementary table 1 ). although the conflicting data so far on hbv-ifnl snp association do lead to doubts about its true positive nature, the data are also not fully supportive of the notion that the association may be false positive. in fact it has been argued that if properly assessed, the association between hbv disease progression and ifnl snps could have clinical value. 62 but it appears that the effect of the ifnl snps on hbv persistence and/or progression within the human host is highly variable; it may involve more complex interactions with other variables and genes than was observed in chronic hcv infections. human immunodeficiency virus. another important viral disease that has been tested for the influence of ifnl snps is acquired immunodeficiency syndrome (aids) caused by hiv. particularly, it was of interest to see how the ifnl snp beneficial alleles are distributed in a unique group of hiv-infected individuals that can suppress the virus from replicating to high levels (defined as elite controllers/suppressors or natural viral suppressors) and defer the progression to aids (called as long-term non-progressors) without any antiretroviral therapy; and another unique group that remains hiv-seronegative despite being at high risk for infection due to intravenous drug use (highly exposed seronegative or exposed seronegative)). more interestingly, the natural viral suppressor patients have also been known to efficiently clear hcv in hcv-hiv co-infections, compared with controls in both african americans 66 and caucasians. 67 a few reports have come up in this area, some showing no correlation of hiv infection/disease progression to the ifnl snps, whereas others see clear association. [68] [69] [70] [71] [72] [73] [74] one of the earlier reports tested the association of rs12979860 in 291 high-risk seronegative and 1221 hiv-positive subjects comprising both white and black subjects in the usa. 68 no association was evident for either hiv positivity or for disease progression within the infected subjects. in another study reported by rallon et al., 69 the association of rs12979860 was tested in two different cohorts. the first comprised of 30 long-term non-progressors and 38 typical progressors to aids; the second included 29 exposed seronegative and 29 hiv-positive partners. thus, the study tested both hiv progression and protection, although in a small number of patients. no significant difference in distribution of genotypes between cases and controls was evident in both cohorts, although the beneficial cc genotype was more frequent in the exposed seronegative patients compared with hiv-positive patients (62% vs 45%), suggesting that there may be a protective role for the cc genotype against hiv that was not detected in the study, likely due to inadequate power. another study from the usa tested whether the beneficial cc genotype at rs12979860 was overrepresented in 25 african-american elite controllers/suppressors compared with hiv-infected patients with high viral loads, and found no statistically significant difference. 70 confirming this report, sajadi et al. 71 also found that cc genotype (for rs12979860) was not significantly over-represented in 48 natural viral suppressors of african-american origin compared with hivpositive (n = 124) and -negative controls (n = 173). three reports published subsequently have seen association with rs12979860 and rs368234815, and spontaneous control of hiv and/or aids progression. [72] [73] [74] interestingly, all three studies were carried out on whites/caucasians, whereas the reports that failed to see an association described above were mostly carried out on african americans (except that by rallon et al 69 that used white subjects) or mixed group of blacks and whites. 68 first, machmach et al. 72 found an association of rs12979860 with spontaneous hiv control when tested on 53 white natural viral suppressors and 389 matched non-controllers at p = 0.02 after correcting for occurrence of hla-b57 (human leukocyte antigen) protective alleles (which also have independent association with hiv and hcv spontaneous clearance) and gender, in multivariate analysis. second, machmach et al. 73 confirmed and extended their results in another report, where they found the association of rs368234815 with aids progression. last, a recent study carried out on a well-characterized spanish white cohort of hcvseropositive men exposed to hiv infection through shared needles shows a clear association of ifn-λ4-generating rs368234815 with hiv positivity. 74 the study had 213 men who were hiv seropositive and 188 were highly exposed seronegative. they found that the protective tt/tt genotype was overrepresented in the highly exposed seronegative group (0.49 vs 0.41; p = 0.006). further, this association had no interaction with the hiv-protective ccr5 (c-c chemokine receptor type 5) deletion (p = 0.7). interestingly, all three studies that found an association show data that the non-beneficial minor alleles are following a recessive model of inheritance, 72-74 suggesting a common underlying functionality in the observed genetic association between the three studies. this is however different from the dominant model of inheritance seen in hcv studies, [11] [12] [13] 15 suggesting that the two viruses may have different interactions with ifn-λ-driven immune responses. however, unlike in the case of cmv studies discussed above, [47] [48] [49] [50] [51] all three hiv studies [72] [73] [74] show that the minor alleles are non-beneficial, similar to the observations in chronic hcv infections. 15 in summary, it is evident that ifnl snps do associate with hiv replication and disease progression, even though in an ethnicity-specific manner. it appears that the beneficial alleles of ifnl snps have protective roles in whites/caucasians. in african americans, more studies with the functional ifn-λ4-generating snp rs368234815 rather than rs12979860 will reveal any true association. this is especially true as the two snps are not in strong linkage disequilibrium (ld) in this population (figure 1b) . herpes simplex virus. herpes simplex virus resides inside the human host latently in sensory neurons, but gets reactivated due to the altered host immunity and replicates efficiently in epithelial cells causing genital or oral lesions (the latter is referred to as 'cold sores'). an association of rs12979860 with the recurrence and severity of herpes simplex virus-1-induced 'cold sores' was reported from a study on a small number of individuals (n = 57). 75 a recent report carried out on a large number of individuals (n = 2192 for genital herpes; n = 1511 for oral herpes) clearly found no association of rs368234815 with recurrence of genital or oral herpes episodes. 76 among other phenotypes, the latter report ruled out an effect of the snp on 'frequency of recurrence' of oral herpes, it however did not rule out an effect of the snp on severity of infection. non-infectious diseases and miscellaneous conditions. ifnl snps have also been tried as candidate gene snps in some noninfectious inflammatory diseases. the association of ifnl snps with nafld was earlier reported by petta et al. 77 this study with 160 subjects identified an independent association with the cc genotype at rs12979860 and the severity of lobular inflammation (cc genotype positively correlated with severity of inflammation) in a cohort of patients with nafld (p = o 0.001). the study was conducted taking lead from previous reports of increased hepatic necroinflammation in hcv patients with the cc genotype at rs12979860. 78 another report showed no such association in 195 caucasian biopsy-confirmed nafld patients. 79 however, all the patients in this cohort were obese (with body mass index 430), whereas in the previous study only 40% were obese. after the latter report, petta et al. 80 revisited their data and indeed found an association with the ifnl snp only with their non-obese patients (n = 94; p = 0o0.001) but not with the obese patients (n = 66; p = 0.13). these initial doubts seem to have been resolved with a recent report by eslam et al. 81 who worked on a relatively large number of nafld patients (n = 488) and confirmed the association of the cc genotype at rs12979860 with increased severity of liver inflammation and fibrosis (p = o 0.0001). they also found a similar strong association with hepatic inflammation and fibrosis in separate cohorts of viral hepatitis c (n = 3129) and b (n = 555), strongly suggesting that even though the major allele genotype cc is beneficial against hcv and hbv, it is also responsible for excess inflammation of the liver in viral and non-viral hepatitis. all four studies 77-81 report on dominant models of inheritance for the minor alleles; but unlike in the context of chronic hcv infections, [11] [12] [13] the minor alleles in nafld are beneficial rather than being non-beneficial, as they are responsible for less severe hepatic inflammation. extending the potential role for ifnl snps in inflammatory diseases, there is also a recent report that showed association of rs12979860 and allergy in children aged o5 years. 82 although the study involved a small sample size (non-allergic, n = 35; allergic cohort 1, n = 35; food allergy cohort 2, n = 30), a large effect size (odds ratio = 4.56, p = 0.004, for cohort 1) was seen wherein the non-beneficial t allele at rs12979860 was over-represented in the allergy group in a dominant model of inheritance. another interesting finding in this study was that the effect of the ifnl snp was more pronounced in females than males. this report suggests that the non-beneficial ifnl snp minor allele-carrying genotypes may predispose children to an allergic phenotype by skewing the th1/th2 balance to a th2 predominant one. this extrapolation is also based on the findings from a recent study on mice model of allergic asthma, which showed that ifn-λ2 was able to rescue mice from allergy by suppressing th2 cytokines. 39 these conclusions would also suggest a role for ifnl snps in immune response to respiratory viral infections, which account for disease exacerbations in conditions such as asthma. 83 a study carried out on infants with respiratory syncytial virus-induced bronchiolitis shows no association of r12979860 and rs8099917 with viral load or other clinical features. 84 interestingly, the non-beneficial tt genotype of rs12979860 was associated with early age of hospitalization in the infants (p = 0.005). more studies are awaited on the association of ifnl snps with allergy and allergy-related diseases. in the autoimmune disorder multiple sclerosis, rs8099917 and rs12979860 did not show any association with ifn-β treatment. 85 as evidence for a potential role of ifn-λs and ifnl snps in adaptive immune responses, a study has linked ifnl snps with development of effective vaccine response against human influenza virus in immunosuppressed individuals. 86 the study shows that minor allele (g)-carrying individuals at rs8099917 show better vaccine responses (odds ratio = 1.99; p = 0.038) in a dominant model of inheritance, and that t cells from minor allele-carrying individuals produce more il-4 (a th2 cytokine). this study also showed that recombinant ifn-λ3 increased th1 responses from human peripheral blood mononuclear cells while inhibiting th2 responses (th2 cytokines are needed for effective seroconversion). 86 studies have accumulated evidence on the different transcription factors (tfs) that bind and drive transcription from all four ifnl genes. roles for several virus-inducible tfs such as nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), ifn regulatory factor-3 and ifn regulatory factor-7 have been defined for ifnl1, 2 and 3 genes, and have been reviewed elsewhere. 28, 87, 88 we recently showed that apart from these three tfs, specificity protein 1 ( a gc-rich dna-binding tf) also has a role in driving transcription from the ifnl4 promoter in a549 cells 89 (figure 4a) . a cpg island of~1.5 kb is located upstream of the ifnl3 gene and overlapping the ifnl4 gene, and also includes two of the most important ifnl snps rs12979860 and rs368234815. epigenetic regulation of the transcriptional activity at the ifnl locus is likely to involve this cpg island and needs further exploration. 90, 91 even though ifnl4 messenger rna (mrna) was shown to be highly expressed from stimulated primary hepatocytes in the original report that described the discovery of ifnl4, 14 subsequent studies have not seen robust expression levels of ifnl4 gene in human samples. for example, amanzada and others found ifnl4 transcripts expressed at 4.3-5.6-fold lower levels than ifnl2/3 mrna in liver biopsies of hcv-infected patients. 92 in another report, ifnl4 mrna was detectable in only in 7/23 and 8/23 patient-derived peripheral blood mononuclear cells that were stimulated or not with ifn-poly(i:c), respectively. 93 all 23 patients carried at least one copy of the functional ifn-λ4-generating allele, δg. in the report by liang and colleagues also, ifnl4 transcripts were seen in only 33/70 liver biopsies from hcv-infected patients. 94 furthermore, lu et al. 41 found that only 2% of the transcripts generated from poly(i:c)-stimulated primary hepatocytes derived from two heterozygous (tt/δg at rs368234815) individuals, represented functional ifn-λ4 transcripts that originated from the δg allele. similarly, they also found no expression of the full-length functional ifn-λ4 mrna transcripts from poly(i: c)-stimulated a549 cells. it is possible that the qpcr assays utilized in these studies may be suffering from technical difficulties in dealing with several different mrna isoforms of ifnl4 gene. 14 all the four studies described above have used primer sets where at least one of the primers binds to the exon/intron/exon-intron junctions (figure 4b) . using a primer set that uniquely binds to the 3′-untranslated region of ifnl4 mrna, ifn-λ4 induction was seen at levels similar to that of ifn-λ2/3, upon human metapneumovirus infection of a549 cells by banos-lara et al. 45 we used the same primer set and found similar high expression levels upon poly(i:c)/hcv rna transfection in a549 cells. 89 two other studies have reported no such problems in amplifying ifnl4 transcripts from liver biopsies. 95, 96 although honda et al. 96 used the allelespecific taqman assay described earlier, 14 konishi et al. do not provide the primer sequence information that they used in their taqman assays. 95 the inconsistency in the above reports in detection of ifnl4 transcripts (either the full-length functional ifn-λ4 isoform or the other isoforms) suggests that rna-sequencing may be the optimal approach to measure ifnl4 transcripts. furthermore, the pre-mrna splicing mechanism that leads to the expression of different ifn-λ4 isoforms 14 under different stimulation and cell-type conditions needs to be examined. the relevance of other 'ifn-λ3 functional snps' after the discovery of functional ifn-λ4-generating snp till date, three functional snps have been identified that regulate ifnl3 gene transcription/translation (referred to as 'ifn-λ3 functional snps' in this section). although rs28416813 is a snp present at~37 nucleotides upstream of the start codon of ifnl3 and is known to affect downstream gene expression by differentially binding to nf-κb, 97 rs4803217 was identified in the 3′-untranslated region region of ifnl3 (figure 1a ) that affects stability of the mrna by interfering with au-rich element decay (amd). 98 both these snps are in high ld with each other and with rs12979860 and were predicted to be two of the four potential causal snps from among the hcv-gwas hits. 15, 99 a recent study defines another mechanism by which rs4803217 affects the stability of rna by remodeling its secondary structure. 100 furthermore, a third functional snp is the ta repeat polymorphism rs59702201, originally reported by the mizokami group. 101 this snp located within the proximal promoter affects transcription of ifnl3, depending on the number of repeats present. 101 recent reports have confirmed the significance of this snp in association studies involving chronic hcv infections. [102] [103] [104] apart from several reports that showed genotype-dependent differences in expression levels of ifn-λ3 (reviewed in ref. 15 ) recent reports also have confirmed this finding in ex vivo and in vivo conditions. 94, 105 these results suggest that ifn-λ3 expression levels dictated by the alleles present at the three functional snps may have a role in the observed phenotype in health and disease. however, the discovery of the ifn-λ4-generating snp (rs368234815, referred to as 'ifn-λ4 functional snp' in this section) has overshadowed the importance of the 'ifn-λ3 functional snps', as most of the new reports have chosen to test for the 'ifn-λ4 functional snp' rather than rs12979860, which is the tag-snp for the 'ifn-λ3 functional snps'. 15 another snp within the coding region of ifnl4 that leads to a non-synonymous mutation in the functional ifn-λ4 protein is thought to be a better marker of association along with rs368234815, in chronic hcv infections. 106 so the question arises: is the ifn-λ4-generating snp the sole functional snp or the other snps regulating the expression of ifn-λ3 also have independent roles? the answer to this question will be difficult to obtain under in vivo conditions due to high ld between these snps in most populations that precludes any attempts to assess their independent effects (figure 1b) . a recent report compared the strength of association of rs4803217 and rs368234815 in ifn treatment response in hcv-infected african americans, who have the lowest reported ld values among all ethnicities between rs368234815 and rs12979860 snps (figure 1b) . 107 they found that rs368234815 was more strongly associated with the outcome than rs4803217. 107 in another independent report, lu et al. applied multivariate regression to test the independence of rs368234815 from rs4803217 in predicting response to ifn treatment in hcv patients of african-american decent. 100 they found that correcting for the effect of s368234815 on rs4803217 abolished the latter snp's association with treatment response, whereas correcting for the effect of the latter snp reduced the strength of association of the former snp (from p = 0.004 to 0.065). 100 this could have resulted due to the low sample size (n = 169); nevertheless, more results are awaited to conclude that ifn-λ4-generating snp is the sole functional snp and that the observations giving credence to the functionality of the other 'ifn-λ3 functional snps may only be artifacts. studies so far point to the functional ifn-λ4-generating snp rs368234815 as the prime causal variant at the human ifnl locus. therefore, a detailed investigation is needed on the function of ifn-λ4 as both an antiviral cytokine in different viral diseases and as a potential player in diversification and maintenance of innate and adaptive immune cell subsets. in case of ifn treatment response in chronic hcv infections, even though the ifn-λ4 -generating snp can explain a large amount of variance in the observed phenotypes, 107 questions still remain on its role in spontaneous clearance of hcv. it is known that expression of functional ifn-λ4 is associated with high levels of ifn-stimulated gene (isg) expression in non-responders who further fail to upregulate their isg expression upon ifn-α treatment. 15 however, such an elegant explanation is lacking in the case of spontaneous clearance of hcv ( figure 5 ). although the majority of studies on spontaneous hcv clearance are with rs12979860 and not rs368234815, the fact that these two snps are in high ld in most populations 14 allows us to extrapolate the results. so, how does the expression of a functional ifn-λ4 in patients who get an acute hcv infection will lead them to become chronically infected? one explanation could be that similar to the ifn treatment nonresponders group, the subjects expressing functional ifn-λ4 also have higher baseline levels of isgs, which do not get further upregulated upon acute hcv infection. having a higher basal innate immune response may have helped those populations with higher frequency of the functional ifn-λ4-generating allele in dealing with prevalent viral infections. higher baseline levels of isgs in people who can express a functional ifn-λ4 may be offering protection against excess inflammation of the liver in hepatitis of non-viral origin such as nafld. [78] [79] [80] [81] however, as is evident from studies on spontaneous and treatment-induced clearance of hcv, such a pre-emptive state may become nonbeneficial in dealing with hepatitis of viral origin. further, there may be other conditions that we do not yet know in which expression of a functional ifn-λ4 may be beneficial. therefore, an epidemiological investigation to assess basal isg expression levels, in humans both in health and disease, and their correlation with expression of ifn-λ4, is needed. further, studies are also needed to examine why hepatic ifn-λ3 levels are lower in the beneficial allele/genotype-carrying individuals during chronic hcv infection and how this phenomenon is reversed once treatment is initiated (figure 5c ; ref. 94) . although ifn-λ1, 2 and 3 are known to associate with a th1 response, 38, 39, 86 no information is available in this regard for ifn-λ4. if the hypothesis that higher levels of ifn-λ3 expression (due to presence of major alleles) will lead to a th1 predominant response is to be believed, then by extrapolation, ifn-λ4 should promote th2 responses (figure 3 ; as 'ifn-λ3 functional snps' and 'ifn-λ4 functional snp' are in strong ld and the minor allele will give rise to ifn-λ4). this explanation is difficult to reconcile with the observations that ifn-λ4, except for being a poorly secreted ifn, requires the same dimeric receptor for signaling and can stimulate not only similar set of isgs but also at similar levels when compared with ifn-λ3. 14, 30, 36 besides, both ifn-λ4 and ifn-λ3 have similar antiviral potencies in vitro. 14,41 therefore, expression of ifn-λ4 and its associated isgs in a subset of individuals carrying minor alleles is less likely to favor a completely opposite phenotype to that seen in those carrying major alleles that induce higher levels of ifn-λ3 expression. human ifnl snps came to the limelight after genetic studies showed their relevance in chronic hcv infections in the year 2009. since then, case-control studies in humans have been reported involving ifnl snps and several other viral diseases, nafld, allergy and even in vaccine responses. although it appears that the associations are well replicated (such as in nafld and aids) and strong in some diseases (such as in pediatric allergy), conflicting reports weaken the association in some (such as those involving htlv and hbv), whereas further studies are needed in others (such as those involving cmv and herpes simplex virus) to clear discrepancies. the ifnl-λs may have critical roles in shaping innate and adaptive immunity in general and in viral infections in particular. however, unlike their effect in hcv infections, the ifnl snps may involve interactions with variables other than just standard end point phenotypes in many of the reported diseases/ conditions. therefore, future studies should aim at dissecting these interactions to arrive at meaningful results. well-designed replication studies and functional studies to strengthen the genetic association results are required to arrive at firm conclusions. importantly, ifn-λ4 has emerged as the key causal link to the genetic associations, but many questions remain on its functional role in the diversification and shaping of innate and adaptive immune responses in viral infections and other inflammatory conditions. 14) and therefore are expected to express ifn-λ4. 94 a recent study has documented that type iii ifns including ifn-λ2/3 and ifn-λ4 expression levels correlate with isg expression in the liver of patients undergoing anti-hcv therapy. 94 the ifn-λ2/3 and ifn-λ4 levels and their associated isg levels are higher in the liver of patients who will eventually not respond to the therapy 94 compared with those who will respond, suggesting that an unknown effect inhibits the expression of ifn-λ3 in the latter group of patients (shown as an ellipse in c). it seems that ifn-α-ribavirin treatment induces the expression of ifn-λ3 once treatment begins (depicted as a circle in c) preferably in those patients who will eventually respond to the treatment 94 (these patients have lower frequency of the functional ifn-λ4-generating alleles 14 and hence are shown without ifn-λ4 expression (c)). this induction is further dependent on whether the patients carry beneficial alleles at rs12979860. the patients who have the beneficial alleles show higher increase in their hepatic ifn-λ3 levels than those who do not, once treatment is initiated. 94 a previous report also had seen similar changes in serum ifn-λ3 levels. 109 the isg levels are shown in correlation with ifn-λ3 expression. ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28 r il-28a, il-28b, and il-29: promising cytokines with type i interferon-like properties viral infection and toll like receptor agonists induce a differential expression of type i andλ interferons in human plasmacytoid and monocyte-derived dendritic cells ifn-lambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo interferon type i gene expression in chronic hepatitis c an important role for type iii interferon (ifn-lambda/il-28) in tlr-induced antiviral activity leukocytederived ifn-α/β and epithelial ifn-λ constitute a compartmentalized mucosal defense system that restricts enteric virus infections interferon-λ cures persistent murine norovirus infection in the absence of adaptive immunity guarding the frontiers: the biology of type iii interferons genetic variation in il28b predicts hepatitis c treatment-induced viral clearance il28b is associated with response to chronic hepatitis c interferon-alpha and ribavirin therapy genome-wide association of il28b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c avariant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus genetic variants at the ifnl3 locus and their association with hepatitis c virus infections reveal novel insights into host-virus interactions ifn-λ4: the paradoxical new member of the interferon lambda family selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) innate immunity: an overview pathogen recognition and inflammatory signaling in innate immune defenses innate immunity to virus infection intestinal epithelial cells: regulators of barrier function and immune homeostasis innate lymphoid cells in the initiation, regulation and resolution of inflammation the biology of innate lymphoid cells the 3 major types of innate and adaptive cell-mediated effector immunity il-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo human innate lymphoid cells regulation of the adaptive immune system by innate lymphoid cells interferon-λ: immune functions at barrier surfaces and beyond innate lymphoid cells: balancing immunity, inflammation and tissue repair in the intestine interferon lambda 4 signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia ifn-alpha regulates tlr-dependent gene expression of ifn-alpha, ifn-beta, il-28 and il-29 type iii ifns are produced by and stimulate human plasmacytoid dendritic cells expression profiles of human interferon-alpha and interferon-lambda subtypes are ligandand cell-dependent despite ifn-lambda receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type iii interferons: implications for therapeutic applications of these cytokines transcriptome analysis reveals a classical interferon signature induced by ifnλ4 in human primary cells the impact of the interferon-lambda family on the innate and adaptive immune response to viral infections human interferon lambda-1 (ifn-lambda1/il-29) modulates the th1/th2 response il-28 a (ifn-λ2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease interferon-l and interleukin 22act synergistically for the induction of interferonstimulated genes and control of rotavirus infection interferon-λ4 is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden interferon lambda-2 levels in sputum of patients with pulmonary mycobacterium tuberculosis infection interferon lambda 1 expression in cervical cells differs between low-risk and high-risk human papillomavirus-positive women influenza virus activation of the interferon system impact and regulation of lambda interferon response in human metapneumovirus infection interferon-λ in the context of viral infections: production, response and therapeutic implications immunomodulatory function of interleukin 28b during primary infection with cytomegalovirus association between individual and combined snps in genes related to innate immunity and incidence of cmv infection in seropositive kidney transplant patients effect of the il28b rs12979860 c/t polymorphism on the incidence and features of active cytomegalovirus infection in allogeneic stem cell transplant patients the ifnl3/4 δg variant increases susceptibility to cytomegalovirus retinitis among hiv-infected patients influence of ifnl3/4 polymorphisms on the incidence of cytomegalovirus infection after solid-organ transplantation development of tropical spastic paraparesis in human t-lymphotropic virus type 1 carriers is influenced by interleukin 28b gene polymorphisms il28b gene polymorphism snp rs8099917 genotype gg is associated with htlv-1 carriers lack of evidence to support the association of a single il28b genotype snp rs12979860 with the htlv-1 clinical outcomes and proviral load htlv-1-associated myelopathy/ tropical spastic paraparesis is not associated with snp rs12979860 of the il-28b gene il28b gene polymorphisms and th1/th2 cytokine levels might be associated with htlv-associated arthropathy paradoxical expression of il-28b mrna in peripheral blood in human t-cell leukemia virus type-1 mono-infection and co-infection with hepatitis c virus absence of association of ifnl3/il28b rs 12979860 and ifnl4 ss 469415590 polymorphisms with the neurological status of htlv-1 afro-caribbean subjects in martinique nucleic acid sensors involved in the recognition of hbv in the liver-specific in vivo transfection mouse models-pattern recognition receptors and sensors for hbv regulation of hepatic innate immunity by hepatitis c virus systematic review with meta-analysis: do interferon lambda 3 polymorphisms predict the outcome of interferon-therapy in hepatitis b infection? one more piece in the interleukin 28b gene puzzle? the case of hepatitis b interleukin 28b genetic polymorphism and hepatitis b virus infection no impact of interleukin-28b polymorphisms on spontaneous or drug induced hepatitis delta virus clearance effects of polymorphisms in interferon λ3 (interleukin 28b) on sustained virologic response to therapy in patients with chronic hepatitis d virus infection hepatitis c infection in hiv-1 viral suppressors hepatitis c virus replication in caucasian hiv controllers il28b polymorphism does not determine outcomes of hepatitis b virus or hiv infection interleukin-28b gene polymorphisms do not influence the susceptibility to hiv-infection or cd4 cell decline protective interleukin-28b genotype affects hepatitis c virus clearance, but does not contribute to hiv-1 control in a cohort of african-american elite controllers/ suppressors il28b genotype does not correlate with hiv control in african americans il28b single-nucleotide polymorphism rs12979860 is associated with spontaneous hiv control in white subjects ifnl4 ss469415590 polymorphism is associated with unfavourable clinical and immunological status inhiv-infected individuals ifnl4 rs368234815 polymorphism is associated with innate resistance to hiv-1 infection a systematic analysis of host factors reveals a med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication interferon lambda 4 genotype is not associated with recurrence of oral or genital herpes il28b and pnpla3 polymorphisms affect histological liver damage in patients with nonalcoholic fatty liver disease genome wideassociation study identifies il28b polymorphism to be associated with baseline alt and hepatic necro-inflammatory activity in chronic hepatitis c patients enrolled in the ideal study il28b rs12979860 is not associated with histologic features of nafld in a cohort of caucasian north american patients reply to: "il28b rs12979860 is not associated with histologic features of nafld in a cohort of caucasian north american patients interferon-λ rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease genetic variations in il28b and allergic disease in children anti-viral agents: potential utility in exacerbations of asthma evaluation of interleukin 28b single nucleotide polymorphisms in infants suffering from bronchiolitis il28b polymorphisms are not associated with the response to interferon-β in multiple sclerosis il-28b is a regulator of b-and t-cell vaccine responses against influenza mechanisms of type iii interferon expression interferon induction and function at the mucosal surface roles for transcription factors sp1, nf-ĸb, irf3, and irf7 in expression of the human ifnl4 gene il28b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction identification of improved il28b snps and haplotypes for prediction of drug response inn treatment of hepatitis c using massively parallel sequencing in a crosssectional european cohort interferon-λ4 (ifnl4) transcript expression in human liver tissue samples impaired induction of interleukin 28b and expression of interferon λ4 associated with nonresponse to interferon-based therapy in chronic hepatitis c hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis c: a phenotype-genotype correlation study interferon-lambda4 genetic polymorphism is associated with the therapy response for hepatitis c virus recurrence after a living donor liver transplant hepatic interferon-stimulated genes are differentially regulated in the liver of chronic hepatitis c patients with different interleukin-28b genotypes a single nucleotide polymorphism associated with hepatitis c virus infections located in the distal region of the il28b promoter influences nf-ĸb mediated gene transcription the favourable ifnl3 genotype escapes mrna decay mediated by au-rich elements and hepatitis c virus-induced micrornas estimating the net contribution of interleukin-28b variation to spontaneous hepatitis c virus clearance ifnl3 mrna structure is remodelled by a functional non-coding polymorphism associated with hepatitis c virus clearance genetic variation of the il-28b promoter affecting gene expression prevalence of thymine-adenine dinucleotide repeat, il28b and ifnl4 in thai population and correlation with spontaneous clearance and treatment outcome of hepatitis c infection pretreatment prediction of the outcome of response-guided peginterferon-α and ribavirin therapy for chronic hepatitis c a thymineadenine dinucleotide repeat polymorphism near il28b is associated with spontaneous clearance of hepatitis c virus ifnl3 genotype is associated with differential induction of ifnl3 in primary human hepatocytes reduced ifnλ4 activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes comparison of functional variants in ifnl4 and ifnl3 for association with hcv clearance il-28b genetic variation is associated with spontaneous clearance of hepatitis c virus, treatment response, serum il-28b levels in chinese population impact of il28b gene polymorphisms on interferon-λ3 plasma levels during pegylated interferon-α/ribavirin therapy for chronic hepatitis c in patients coinfected with hiv the author declare no conflict of interest. key: cord-023017-k6edtg58 authors: nan title: aasld abstracts (pp. 282a–382a) date: 2006-02-10 journal: hepatology doi: 10.1002/hep.1840380505 sha: doc_id: 23017 cord_uid: k6edtg58 nan of well-characterized human hepatocyte cell lines could facilitate cell therapies such as htx and bioartificial livers. toward this goal, we have developed a tightly regulated human hepatocyte line. methods human hepatocytes were immortalized with a retroviral vector encoding human telomerase reverse transcriptase (htert) and green fluorescent protein (gfp) cdnas flanked by a pair of loxps. after retroviral transduction, a single cell clone was obtained using flow cytometric cell sorting followed by limiting dilution method. the resultant gfp-positive clones were supertransduced with tamoxifen-inducible cre recombinase expression cassette, mercremer. recovery of the reverted form of htert-immortalized cells was conducted by tamoxifen treatment and subsequent gfp-negative cell sorting with moflo. the expression of hepatocyte markers and albumin secretion was compared before and after tamoxifen treatment. transplantation effect of the reverted cells (lx lo9) into the liver were evaluated in a pig model of liver failure induce by an intravenous administration of d-galactosamine (d-gal). resu1ts:a non-tumorigenic human hepatocyte cell line ttnt-16-t3 was established. albumin secretion and gene expression of liver markers were increased in the reverted ttnt-16-t3 cells. transplantation of the reverted cells via the portal vein of pigs treated with d-gal was effective to prolong the survival by providing liver support until spontaneous hepatic regeneration occurred. conclusion: we here demonstrate reversible immortalization of human hepatocytes using tamoxifen-induced crellox self-excision. the resulting cell line would be highly desirable for research and therapeutic applications. acknowledgmenk the work was supported in part by life science project of 2lst century, japan. background and aim: stem cells play a key-role in tissue homeostasis. haematopoietic stem cells (hscs) could migrate into the liver and transdifferentiate, becoming a "liver stem cell reserve''. we aimed to analyse: human hsc (h-hsc) ability to engraft into the mouse liver and to contribute to its regeneration after a toxic damage. materials and methods: nodiscid mice were so divided: a) chimeric not-damaged mice: mice were submitted to irradiation followed by h-hsc intraperitoneally (i.p.) injection; finally they were killed 22, 27 and 31 days after; 8) chimeric damaged mice: mice were irradiated, injected with h-hscs and, 21 days after, damaged using ally1 alcohol (aa), i.p.; killing was performed 1 , 5 and 10 days after the damage; c) damaged not-chimeric mice: mice were damaged (with aa injection) and killed 1, 5 and 10 days after; d) not-chimeric not-damaged mice: mice were killed without any treatment. spleens (s), bone marrows (bm) and livers (l) were tested by flow-cytometry to detect h-hscs and immunohistochemistry (l only) to localize h-hscs and their hepatic derivatives. results: flow-cytometry: group a) presence of low percentage of human cells; groups c) and d) human cells undetectable; group b) presence of low percentage of human cells in bm and s while in the l they represented up to 20% of whole cellular population (the difference respect to the group a has high statistical significance, being alpha<0.001%). immunohistochemistry showed the human cell differentiation into parenchymal cells. conclusions: our results suggest that hscs could be an option to improve thie liver regeneration after a toxic injury, supporting the feasibility of bm-derived liver stem cell hypothesis. we have previously demonstrated that cd8-mediated immune damage of allogeneic liver parenchymal cells is difficult to regulate due to the activity of "immunoresistant" cd8+ t cells. in prior studies we demonstrated that short-term interference with cd401 cd40l (but not cd281b7) costimulation transiently suppressed cdb-dependent hepatocyte rejection. recently, we have determined that targetintg lfa-1 alone preferentially suppresses cd8dependent versus cd4-dependent hepatocyte rejection. in addition, short-term immunotherapy targeting both lfa-1 and cd40lcd40l costimulation produced synergistic effects and resulted in longterm suppression of cd8-dependent rejection such that hepatocyte survival up to 90 days was achieved in the majority of recipient mice. the purpose of this study was to determine whether targeting both cd40lcd40l and lfa-1 mediated signals results in an immunoregulatory state which controls naive cd8+ t cells. methods: 2 x lo6 fvbln (halat, hepatocytes, were transplanted into cd4 ko or c57bl16.scid (all h-2b) mice, and recipients were treated with anti-lfa-1 mab (0.3mg d0-6) and anti-cd4ol mab (lmg, do, 2,4,7) . hepatocyte survival was monitored by detection of serum halat reporter product by elisa. hepatocyte recipients with longterm survival (>60 days) were challenged by adoptive transfer of 2 million naive cdb' t cells (isolated from cd4 ko mice) which are sufficient to initiate rejection of functioning hepatocellular allografts in scid recipients. results: combined treatment with anti-lfa-1 mab and anti-cd40l mab significantly prolonged hepatocyte allograft survival in cd4 ko mice such that 95% achieved hepatocyte survival >60 days posttransplant (mst>105 days, n= 17) in comparison to either agent alone (anti-cd40l mab mst=35 days, n=4, p<o.ool; anti-lfa-1 mab mst=77 days, n=9, p<o.ool). recipient mice induced to achieve hepatocyte survival >60 days (by short-term treatment with anti-lfa-1 and anti-cd40l mab) received adoptive transfer of 2 million nayve purified cd8+ t cells. continued survival of longterm hepatocellular allografts despite adoptive transfer of cd8+ t cells was observed in 60% (3 of 5) recipients. in 2 recipients, adoptive transfer of cd8+ t cells was associated with loss of hepatocellular allograft function by 31 and 56 days following cell transfer. in contrast, control scid mice with functioning hepatocellular allografts which were reconstituted with 2 million naive cd8+ t cells rapidly rejected hepatocytes with mst of 17 days (n=5). conclusion: targeting of both cd40l cd4ol and lfa-1 not only prevents immunoresistant cdb-dependent immune damage of liver parenchymal cells, but also x lo3 vs. 1.321.3 x lo3 and 3.920.4 x lo3 vs. 0.321.0 x 103, respectively) while at 72 hrs it was 2.4t0.8 x lo3 vs. 2.122.0 x 103 (ns) . in the spleen, significantly higher heparanase treated cells were located within the tissue, showing proliferative activity at 48 and 72 hrs post transplantation. already by 24 hrs after transplantation the proliferating index of sec increased from 150.5 in controls to 2256 in heparanase treated rats (i'<o.o2). conclusions: heparanase increases the long-term presence of transplanted cells within portal radicles, thereby facilitating their engraftment. it has also a significant effect on ec proliferation. disc 1 o s u r e s : yaacov baruch -no relationships to disclose orit goldshmit -no relationships to disclose neta ilan -no relationships to disclose gideon shoshany -no relationships to disclose vladislav tsiperson -no relationships to disclose ella veitzman -no relationships to disclose israel vlodavsky -no relationships to disclose 264 cell activation. arunzazu sanchez, peter n u n , insa s schroeder, valentina m factor, snorri s thorgeirsson, national cancer institute, bethesda, m d substantial evidence suggests that proliferation of hepatic stem cell progenies (referred to as oval cells) is responsible for liver regeneration when the replicative capacity of hepatocytes is impaired. it is well established that activity of the tnf-a , nf-kb, il-6, and stat3 pathways is necessary for the regenerative response of hepatocytes following partial hepatectomy. however, much less is known about the signals mediating oval cell proliferation and differentiation. the aim of the present study was to evaluate the involvement of nf-kbistats signaling pathways in regulation of oval cell activation. high activity of nf-kb was found in regenerating liver after aaflph protocol coinciding with the proliferation of oval cells. urification of the oval cell population by magnetic beads sorting using ov1 antibody confirmed that the activation of both nf-kb and stat3 is specific for this cellular compartment. three distinct subpopulations of oval cells were identified based on intensity of ov1 staining by facs and named as ov1 dim, medium and bright. real time pcr analysis showed that they represent oval cells at different stages of differentiation along the hepatocytic lineage. ov1 dim subpopulation was the closest to the hepatic stem cell, as judged by high expression of c-kit. on the contrary, ov1 bright cells displayed the highest expression of hepatic differentiation markers, ttr, albumin, connexin 32 and hnf-4. nf-kb was uniformly activated in the entire oval cell compartment, whereas stat3 was phosphorylated only in the most differentiated ov1 bright subpopulation. furthermore, activation of stat3 tightly correlated with a high proliferative activity in these cells. together, these data provide the first evidence that the transcriptional activity supported by nf-kb and stat3 is required for oval cell activation. the differential induction of these two transcription factors suggests that they may control different stages of oval cell activation. disclosures: introduction: haematopoietic stem cells (hscs) have been shown to have greater plasticity than previously thought, with studies demonstrating that both human and rodent hscs can differentiate into hepatocytes. our group has also confirmed that human stem cell derived hepatocytes are the result of true differentiation and not the result of cellular fusion (newsome et al, gastroenterology 2003) . the extent to which endogenous hscs are naturally mobilized and contribute to liver repair is however uncertain. aims 1) to establish whether liver injury mobilizes endogenous hscs into the circulation & into the liver. 2) to establish if circulating stem cells in liver injury have altered stem cell potential. patients and methods 4omls peripheral blood was analysed from the following groups (n=8): normal adult controls, patients with alcoholic hepatitis, abstinent chronic alcoholic liver disease and paracetamol-induced liver injury. using a flow cytometry sorting machine, circulating cd34 positive hscs were quantified as a percentage of the number of mononuclear cells according to the ishage flow cytometry protocol. equal numbers of cd34 positive cells from each gmup were collected and cultured, to assess their in vitro stem cell properties, as judged by number of colony forming units (cf'us) produced. for patients with alcoholic hepatitis, age, sex, biochemical parameters, maddrey score and outcome (mortality) was recorded. liver biopsies from paracetamol induced liver injury and alcoholic hepatitis patients were stained for cd34 and c-kit markers to quantitate stem cell numbers (number/ portal triad) and compared with normal adult controls. results 1.levels of circulating cd34 positive hscs were significantly elevated in patients with alcoholic hepatitis (0.19% 20.06; p<0 .05) when compared with all other groups. the corresponding cd34 positive values for the other groups are as follows: healthy controls (0.05% 20.01); paracetamol liver injury (0.08% 20.04) and abstinent alcoholic cirrhotics (0.04% 20.002).there was no significant difference in mean mononuclear cell count between these groups. 2.patients surviving their alcoholic hepatitis had significantly higher levels of circulating cd34 positive stem cells (0.26% 20.09; p<o.o5) when compared with those who had died (0.057% 20.03). the surviving alcoholic hepatitis patients had significantly lower maddrey scores (22.1 92.1; p=o.oos) than those who had died (91.67 228) whilst no significant difference in sex or age distribution was demonstrated between these two groups. the total mononuclear cell counts were lower in the deceased alcoholic hepatih group (0.85xlox9l1 2 0.5) when compared with those that had survived (2.7xlox911 2 0.26). if cd34 numbers are adjusted for the total mononuclear cell counts then the increase in circulating stem cells between these two groups became even more apparent (0.44 ? 0.34 and 4.98xlox6 cellsll 24.8 ; p<0 .05). 3.hscs from alcoholic hepatitis patients produced greater numbers of cfus compared with adult control hscs (245 vs.65). 4.liver biopsies from paracetamol and alcoholic hepatitis patients contained greater numbers of stem cells compared with healthy controls (p<0.05). control (0.65 cellsltriad ?o.l) , alcoholic hepatitis (1 cellltriad 20.17) and paracetamol liver injury (1.9 cells/ triad 2 0.35). stem cells were only identifiedwithin the portal triads and not within the hepatic parenchyma in all of these groups. conclusions 1. alcoholic hepatitis is associated with increased levels of circulating hscs both in the blood and the liver. 2. these mobilized cells display in vifro stem cell potential at a level equal to or higher than control hscs. 3. increased levels of stem cell mobilization are associated with improved clinical outcome. disclosures: background klf6 is a ubiquitously expressed kruppel like zinc finger transcription factor identified as a tumor suppressor gene in prostate cancer (narla et al, science 2001) . in addition, klf6 is frequently lost and/or mutated in hepatocellular carcinomas (tal-kremer, aasld 2002-#924) . klf6 is also an immediate early gene that is rapidly upregulated in hepatocytes following partial hepatectomy and in hepatic stellate cells after injury. its expression is regulated in a tissuerestricted manner during development (laub et al, mech dev 2001) . these broad roles yet defined sites of expression suggested a potentially important role of klfg in development. the aim of this project was to examine the role of klf6 in hepatic differentiation using an embryonic stem (es) cell differentiation system in which undifferentiated cells can be driven along tissue specific lineages under defined stimuli. materials: two targeting constructs were generated in which the second exons of klf6 were replaced by a neomycinllac z cassette or hygromycin cassette. klf6 (+/-) es cells were generated by homologous recombination through selection with a klf6 targeting vector containing the neomy&/lac z cassette. to obtain klf6 (-/-) es cells, klf6 (+/-) clones were subjected to the second round of homologous recombination. a l l es cell dones were confirmed to have a normal karyotype after selection. methods and resulk in the undifferentiated state, klfg (-/-)null es cells grew more slowly than wild type es cells, with doubling times up to 50% longer. following differentiation into embryoid bodies (eb) over 6 days in culture, the number of ebs was reduced by > 80% compared with wild type ebs. the disparity in growth rate between wt and (-/-) es cells was greater in later stages than in the initial 2 days of differentiation. this was accompanied by prolongation of the epiblast-like stage, associated with markedly delayed expression of endodermal mesodermal and ectodermal markers. next, we examined the capacity of klfg (-/-) ebs to differentiate into hepatocyte like cells by withdrawing leukemia inhibitory factor after two days, then culturing ebs under serum free conditions for 8 days, followed by growth on gelatinized dishes. basic fibroblast growth factor and dexamethasone were added into the medium starting day 6 and day 10. whereas expression in klfg (+/+) ebs of alpha-fetoprotein and albumin mrna occurred after 10 days of differentiation, their expression was almost absent and significantly delayed in klf6 (-/-) ebs. an impaired ability of klf6 (-/-) cells to differentiate along hematopoietic and neuronal lineages was also observed. in conclusion, these data indicate a broad and proximal role of klfg in tissue specific development with specific role in hepatocellular development. cell transplantation has been recognized as a possible future treatment for liver cirrhosis. one of the major questions is which cell will be of greatest usespleen might be a major source of stem cell, if exist, since splenomegaly is a common in cirrhotic patients and even splenectomy is performed for treatment. recent advance in the isolation technique of stem cells based on the efflux of fluorescent dyes hoechst 33342 has turned out to be an efficient method to purify stem cells called side population (sp) cells. we utilized this method to isolate stem cells from spleen. we also transfused these sp cells into rat liver treated with carbon tetrachloride (cc14) to see whether these sp cells proliferate and differentiate into both hepatocytes or cholangiocytes. methods. c57/6j male mice and female nodlscid mice were obtained from nihon crea. egfp transgenic (tg) rat were obtained from nihon slc. splenocytes were prepared by digesting in 0.5% collagenase solution and forcing tissue through sterile mesh. splenocytes were then resuspended at lo6 cellslml in hanks balanced salt solution (hbss) containing 2% fbs, 1mm hepes (hbss+), and ug/ml hoechst 33342 w or w/o verapamil and were incubated at 37 degrees centigrade for 90min. splenocytes were then stained for 30min. on ice with fitc or pe conjugated various antibodies against sca-1, c-kit, thyl.1, cd45 and cd34. splenocytes were washed in hbss+ alone 3times and then in hbss+ with 2ug/ml propidium iodide. splenocytes were analyzed and sorted on a dual-laser facstar plus flow cytometer. ( excitation:36nm, emission:424/bp44 & 660/bp20 filter & 640-nm short-pass dichroic mirror). we also prepared spcells from donor egfp tg rats' spleen as described above. these spleen egfp positive sp cells were directly injected into liver of anesthetized recipient nodlscid mice. the recipient nodlscid mice were injected with 20 ug. cc14 intrapentoneally every once a week before and after transplantation. immunohistochemical staining were performed to identify engrafted egfp positive cells, liver specific proteins such as cytokeratinl8j9, afp and albumin. results. initial hst333422 staining of low density splenocytes showed the sp fraction to be readily detectable ( 0.3 -1%). these cells were verapamil sensitive and could be stained with hst33342 after verapamil treatment. the frequency of each surface markers positive cells were as follows (table 1 and 2). two weeks after cell transplantation, donor derived egfp positive cells were engrafted. the some engrafted cells distributed in a cluster indicating donor cell proliferation. hepatic differentiation of engrafted cells were shown by double staining of egfp and rat albumin or cytokeratinld. we observed some of afp positive cells in egfp positive cells, but very few of cytokeratinl9 positive cells. conclusions. the adult rodent spleen had consistently detectable fraction of sp cells. the surface marker profiles were different from those of bone marrow sp cells. the splenic sp cells could be engrafted in liver and differentiate into hepatocyte like cells. our results suggest that splenic sp cells could be exploited for the repair of damaged liver. backgroundlaim : it has been reported that bone marrow cells (bmcs) can replace liver in a murine model of tyrosinaemia and correct this metabolic disease through the fusion of donor bmcs to recipient hepatocytes. the aim of this study was to test whether or not this replacement is a general phenomenon in other liver injury models. the following three models were tested; (1) hepatitis b transgenic mouse (tgn(alblhbv)), (2) albumin-urokinase transgenic mouse (tgn(alblp1au)) and (3) carbon tetrachloride (cc14) treatment model. as the selective liver injury models, irradiated tgn(alb1hbv) and tgn(alblp1au) were transplanted with lx106-lx107 bmcs from green fluorescent protein (gfp) transgenic mice (tgn(actbegfp)) or from @-galactosidase transgenic mice (tgn(mtnlacz)). as a non-selective liver injury model, irradiated c57bl/6 mice were transplanted with 1x106 bmcs from tgn(actbegfp) or tgn(mtnlacz) followed by the administration of cc14 (0.02mllkg animal weight, twice a week for 4 weeks). a cci4 protocol without preparative irradiation was also tested, in which c57bl16 mice were first administered ccl, 8 times, then followed by transplantation of tgn(actbegfp) or tgn(mtnlacz) bmcs. after the analysis of the donor cell engraftment in the peripheral blood, these recipient mice were sacrificed and checked for donor-derived hepatocytes at 2-47 weeks post-transplantation. sixty liver sections for each animal (containing approximately 1 . 5~1 0~ hepatocytes), were analyzed for gfp positive cells and the whole livers were inspected for @-galactosidase expression with x-gal cytohistochemistry. however, there were no gfp positive hepatocytes and no gross blue staining of the livers with x-gal in any of the 18 recipient mice. gfp positive cells were only located in sinusoids or associated with larger vessels. they were readily distinguishable from hepatocytes through their morphology and were negative for albumin by immunostaining. in addition, the livers from 4 female animals with gender mismatched bm transplantation were also tested with y chromosome fish analysis, which might be a more sensitive method to detect donor derived cells than transgenic epitope tagging. total of 5 isolated hepatocytes were positive for y chromosome in 4 . 1~1 0~ hepatocytes analyzed, although it still remains to be elucidated whether these cells arise from spontaneous fusion events or from transdifferentiation. conclusions: these results demonstrate that there is little or no contribution of bmcs to the replacement of injured livers in these models. thus, we do not believe that bm derived cells can generally lead to a cure of liver damage. background &aims: mouse embryonic stem (es) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. we previously reported that es cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (hnf-3b). in the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugtlal) in undifferentiated and differentiating hnf-3btransfected es (hnf-3 b-es) cells. materials & methods: hnf3b-es cells were established from a mouse es cell line. undifferentiated hnf-3b-es cells were main-tained in gelatin-coated dishes without feeder cells in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), 0.1 mm of z-mercaptoethanol(2me), 0.1 mm of non-essential amino acids (neaa), 1 mm of sodium pyruvate and 1000 ulml of leukemia inhibitory factor (lif). undifferentiated hnf-3b-es cells were allowed to differentiate in dmem supplemented with 10% fbs, 0.1 mm 2-me, 0.1 mm neaa, 1 mm sodium pyruvate, and 20 nglml of fibroblast growth factor 2 (fgfz) in the absence of leukemia inhibitory factor (lif). differentiating hnf-3 b-es cells were collected for rt-pcr analysis on day 14, and for western blotting on days 14 and 28. results: the expression of organic anion transporting polypeptide 1 (oatpl), multidrug resistance-associated protein 1 (mrpl), mrp2, mrp3, and ugtlal was not seen in the undifferentiated hnf-3 b-es cells by rt-pcr, whereas all were expressed in differentiating hnf-3 b-es cells. protein expression for oatpl, mrpl, mrp2, mrp3, and ugtlal was also observed in the differentiating hnf-3 b-es cells by western blotting. an immunofluorescence examination revealed that oatpl was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with cd26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes. we have used cdna microarray analysis of clonal murine hepatoblasts to identify genes that are significantly and preferentially expressed in undifferentiated hepatoblasts compared to adult mouse liver. unique stem cell genes were identified by overexpression in undifferentiated hepatoblasts compared to adult liver. 61 genes were identified by these criteria and were designated candidate stem cell genes (cscg). 54 cscg were distributed on all chromosomes of the mouse except chromosomes 12,14 and the y chromosome. however, chromosomes 2, 3 and 19 showed a preferential distribution of cscg compared to the total number of genes mapped on these chromosomes. approximately half of our cscg are commonly expressed in other murine stem cell populations including embryonic stem cells, neural stem cells, mesenchymal stem cells and hematopoetic stem cells, suggesting that hepatoblasts share a molecular signature with other stem cell populations. some of the commonly identified stem cell genes include: sc14a2, c-myc, nek2, phosphodiesterase 7, nicastrin, smarca 3 and smarca 5 and pias3. these genes function in a variety of pathways including ion transport, anion exchange, cell cycle control, chromatin organization, dna binding activity and signal transduction. approximately 25 % of cscg are expressed in early endoderm, fetal liver and/or the pancreatic bud. a search for homology to known proteins indicated that some of these genes contain homology to membrane transporters, zinc finger proteins and spindle apparatus components. this class of genes is of particular interest because they may represent new hepatic lineage markers. the results of this analysis leads us to conclude that a network of pathways some of which are in common with other stem cell populations function together to maintain the liver stem cell phenotype. valentina m factor, aranzazu sanchez, ju-seog lee, tanya n hoang, snom' s thorgeirsson, national cancer institute, bethesda, m d rat liver epithelial cells (rle) were isolated from normal adult rat livers and established as a cell line . rle cells were found to express undifferentiated characteristics resembling adult liver stem cells. we have addressed cellular and molecular mechanisms controlling lineage commitment of hepatic stem cells using rle-13. to induce rle differentiation along the hepatocytic lineage, differentiation protocols were developed consisting of a sequential treatment with the demethylating agent 5-aza-cytidine (5ac), fibroblast growth factors 1 and 2 (fgf), oncostatin m (osm) and hepatocyte growth factor (hgf) in the continuous presence of the synthetic glucocorticoid dexamethasone (dex). these treatments resulted in a remarkable enlargement in cell size and organelle complexity as shown by facs and confocal microscopy. morphological maturation of rle was paralleled by a decrease in cell proliferation. more significantly, rt and real time pcr analysis revealed an induction of hepatocyte specific markers such as tat, ttr, glucose-6-phosphatase, and connexin 32. in addition mrnas encoding liver enriched transcription factors hnf lalp, hnf 3ru, hnf 4, and clebp p were notably induced along with hepatocytic differentiation. the pcr results were supported by a cdna microarray analysis. comparison of the gene expression profiles following the treatment protocols reflected an activation of the hepatocytic differentiation program as judged by increased expression of a differentiation-associated gene set (phosphofructokinase, glutathione-s-transferase) and decreased expression of a cell cycle regulated gene set. currently, transplantation studies are performed to identify the potential of the differentiated rle to repopulate and restore the diseased livers. the results indicate that cell lines derived from adult liver stem cells may provide a renewable resource for transplantation. adult liver stem cells (adulis) can be isolated from rodent bone marrow. when cultured under specific conditions in co-culture with isogeneic hepatocytes, adulis fiom 14 days bile duct ligated (bdl) rats are transdifferentiating into a hepatocyte-like lineage and are able to produce urea from ammonia. the aim of the study presented is to describe the hepatocyte specific metabolic capacity of cultured adulis from normal or bdl rats in single or co-culture with isogeneic hepatocytes, with or without interleukin-3 (il-3). methods: adulis were isolated by a two-step immunoisolation procedure (i.e. beta-2-microglobulin negativity and thy-1 positivity) from rat femoral bone marrow (male wistar rats, 250g) and cultured on a matrigel layer in 24-well polystyrene dishes at a cell density of 50'000 cells/cm* in small hepatocyte media. isogeneic hepatocytes were isolated by a two-step collagenase perfusion technique and seeded (100,000 cells/cm2) on an inlay with a collagen-coated ptfe membrane (poresize: 0.4pm) for co-culture experiments. of note, the culture media was supplemented with 5% isogeneic serum and dexamethason m) was administered after culture day 3. il-3 was added in the corresponding experimental groups (10nglml). after removal of the hepatocyte-inlay (in the co-culture groups) adulis cultures were exposed to 1.5mmol nh4ci for 5 hours in dmem and urea formation determined thereafter with a colorimetric assay. cells were harvested in trizol, total rna extracted and after reverse transcription cdna used for real-time pcr determination of 18s(rm~) content to standardize the metabolic signal for cell number. relative ureagenesis values were compared statistically using jandel scientific. the significance level was set at pc0.05. results: the amount of adulis isolated from normal rat femoral bone marrow (n=lo) was 1.4620.75 x lo6 and 1.8321.97 x lo6 in animals (n=6) after seven days of bdl (p=n.s.). relative urea synthesis in cultures from normal animals was 1.0320.42 in single and 1.3820.41 in co-culture at culture days 3, 6, 9, and 12. with addition of il-3 to the culture media urea genesis was determined to be 1.6820.6 in single and 2.6521.0'2 in co-culture. in cell cultures from seven days bdl rats relative urea formation was 1.5821.43 in single and 2.6351.32 in co-culture. with the addition of il-3 to the culture media values were 2.3920.51 in single and 3.1620.81 in co-culture. addition of il-3 to the culture media increased the metabolic signal in all cell cultures from normal animals (anova on ranks, p<0.05) but not in cultures with cells isolated from bdl animals (p=n.s.). co-culture induced stronger ureagenesis under all culture conditions examined (paired t-test: p<0.05). conclusions: to exclude immunological interference from adult immunocompetent bone marrow cells cultures were strictly isogeneic (i.e. adulis, hepatocytes, and serum from the same animal). co-culturing adulis with isogeneic hepatocytes increased ureagenesis in all paired culture experiments. as there is no direct cell contact between hepatocytes and adulis paracrine soluble, so far undetermined, factors must be involved. interestingly by the addition of il-3 transdifferentiation was inducible in cultures of adulis from normal animals but no additional effect was detectable in the cell cultures from bdl animals. in further studies it will be necessairy to determine if cholestatic serum also induces accelerated and more pronounced hepatocyte specific metabolic capacity in adulis from normal animals. factors responsible for this transdifferentiation should then be isolated from cholestatic serum and might be used to support the failing liver in vivo potentially by activation of the adulis pool in the bone marrow and the liver. disclosures: saji, shinji tamura, yuichi yoshida, shinichi kiso, ayuko iizuka, hitoshi matsumoto, takako kawasaki, yoshihiro kamada, yuji matsuzawa, graduate school of medicine, osaka university, suita, osaka, japan background and aims: recently, the evidence that bone marrow cells (bm cells) have trans-differentiating potential into hepatic lineage cells has been described in animal transplantation experiments and human pathology of bone marrow transplantation recipients. however, the molecular mechanism underlying this phenomenon has remained unclear because of lack of effective culture system. to address this issue, we developed a novel in vitro culture system in which murine bm cells are cultured with growth factors and investigated factors required for the transdifferentiation of em cells into hepatic linage cells. methods and results: (1) bm cells were isolated from the femora of 6-to 10-week-old male c57bl/6] mice. they were cultured in three-dimensional culture system using type i collagen gel and stimulated without or with the growth factors (egf, hgf, hb-egf, osm, bmp-4, bfgf, fgf-4; 2ongiml) for 12 day. to investigate the effect of these growth factors, the gene expression of albumin was examined by quantitative rt-pcr. when bm cells were cultured with hgf, osm, bfgf and fgf-4, albumin was induced effectively. among these growth factors, bfgf was found to induce albumin the most effectively in the cultured bm cells. (2) bm cells were cultured in collagen gel with bfgf (2onglml) for 6, 12, 18 days. the gene expression of hepatocyte-specific markers (albumin, cytokeratin 18, alphal-antitrypsin, glucose 6 phosphates and tyrosine aminotransferase), cholangiocyte-specific marker (cytokeratin 19) and liver-enriched transcription factors (hnflalpha, hnf3alpha, hnf3beta, hnf4alpha, gata4, gata6, clebpalpha, and ciebpbeta) were examined by rt-pcr. upon the stimulation of bfgf, bm cells were found to express cytokeratin 18, alphal-anitripsin and glucose 6 phosphates, cytokeratin 19 and liver-enriched transcription factors including hnflalpha, hnfjalpha, hnf3beta, gata4 and gata6. (3) bm cells were cultured in collagen gel with bfgf (20ngiml) for 6 days. we assessed expressions of albumin and ck18 proteins of cultured bm cells by immunohistochemistry. about 5% cells of them were positively stained for both albumin and cytokeratin 18. conclusions: we established a novel in vitro culture system of bm cells and demonstrated that basic fibroblast growth factor could induce the trans-differentiation of bm cells into hepatic lineage cells the most effectively. furthermore, this conversion was associated with induction of liver-enriched transcription factors including hepatocyte nuclear factors and gata family proteins. these results indicate these liver-enriched transcription factors are the major components, which induce this trans-differentiation. disclosures: ayuko iizuka -no relationships to disclose yoshihiro kamada -no relationships to disclose takako kawasaki -no relationships to disclose shinichi kiso -no relationships to disclose hitoshi matsumoto -no relationships to disclose yuji matsuzawa -no relationships to disclose yukiko saji -no relationships to disclose shinji tamura -no relationships to disclose yuichi yoshida -no relationships to disclose crtteil, france; nadine martin, hbpital henri mondor, criteil, france; dominique couchie, anne m preaux, yannick laperche, elie 5; zafrani, inserm u581, cre'teiz, france liver can regenerate from oval precursor cells when the replication of hepatocytes is inhibited. these cells proliferate in the periportal region, migrate in the lobule and differentiate into hepatocytes. stromal-derived factor-1 (sdf-1) is a chemokine which plays a major role during embryogenesis. since sdf-1 and its sole receptor cxcr4 are involved in the differentiation of progenitor cells (e.g. b lymphopoiesis, digestive epithelial cell renewal), the aim of our work was to study the expression of sdf-1 and cxcr4 in a model of hepatic regeneration from precursor cells. hepatic regeneration from oval cells was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. rats were sacrificed the day of surgery (day 0) and at days 1,5,9,14 and 21 after partial hepatectomy. rare oval cells were observed at day 1. their number was moderate at day 5, marked at day 9 and 14 and declined thereafter. oval cells predominated in the periportal region and usually formed canalar structures resembling cholangioles. individual or cholangiole-forming oval cells strongly expressed sdf-i, as demonstrated at the mrna level by in situ hybridization (ish) and at the protein level by irnmunohistochemistry. interlobular bile duct epithelial cells were also positive for sdf-1 protein but negative for its mrna. cxcr4 mrna hepatic levels, as assessed by quantitative rt-pcr, paralleled the degree of oval cell proliferation. ish showed marked cxcr4 mrna expression in individual as well as in cholangiole-forming oval cells. in order to determine which role the sdf-1icxcr4 couple could play in hepatic regeneration from oval cells, rats were treated or not with fucoidan (25 mglkg ip, twice daily), a sulfated polysaccharide known to bind to sdf-1 and to block its biological effects. as compared to untreated animals, oval cell proliferation was markedly decreased in five of the seven fucoidan-treated rats. in conclusion, oval cells express sdf-1 and its receptor cxcr4 during hepatic regeneration from precursor cells. our results strongly suggest that the sdf-1lcxcr4 couple acts in an autocrinelparacrine way to stimulate the proliferation of these precursor cells. disclosures: dominique couchie -no relationships to disclose background: bone marrow cells and embryonic stem cells are being used for cell transplantation. although these cells are known to be multipotent and capable to become hepatocyte, several problems have been pointed out, such as cell fusion and teratoma generation. thus we paid attention to side population (sp) cells, those with a verapamil-sensitive ability to efflux hoechst 33342. sp cells have been identified in several tissues. the bone marrow sp phenotype appears to be a common feature of stem cells, but hepatic and splenic sp cells have been less well characterized. aim: we tried to separate and culture sp cells from non-parenchymal liver cells (npcs) and splenocytes, and examined whether they would obtain hepatic phenotype. methods: 8-12-week-old female transgenic rats that ubiquitously express egfp were used to isolate non-parencymal liver cells by the standard collagenase two-step perfusion method, and splenocytes were isolated by forcing the tissue through sterile mesh, then mononuclear cells were separated by ficoll density gradient centrifugation. they were resuspended at lo6 cellslml in hanks balanced salt solution (hbss) containing 2% fetal calf serum, 1mm hepes (hbss+), and 5uglml hoechst 33342 (hst) w or wlo verapamil and were incubated at 37 degrees centigrade for 90min. cells were washed twice in hbss+ and then resuspended in hbss+ with 2uglml propidium iodide. cells were analyzed and sorted on a dual-laser facstar plus flow cytometer. rt-pcr was performed with rna extracted from freshly isolated sp cells. isolated sp cells were cultured with male rat primary cultured hepatocytes in collagen gel sandwich. cell morphology was monitored under a phase-contrast microscope and a fluorescence microscope. the expression of albumin, cytokeratin 18, and cytokeratin 19 was analyzed by immunohistochemistry. fluorescence in situ hybridization (fish) for the sry gene was performed to exclude ceil fusion. results: the rate of npc-sp was 0.05% of isolated npcs. 89% of npc-sp expressed cd45, while 55% of npc main population (mp) did. freshly isolated sp cells were smaller than mature hepatocytes. they looked like monocytes or lymphocytes. these cells did not express liver specific markers, such as albumin, alpha-fetoprotein, cytokeratin 18, or cytokeratin 19. during one-week culture with hepatocytes using collagen gel sandwich method, we could find no change in gfp-positive sp cells, but after further culture, we found several colonies consisting of gfp-positive sp cells, whose shape became polygonal. after 2-week-culture, most of gfp-positive cells were stained positive for albumin. npc-sp cells died within one week without hepatocyte co-culture. npc-mp cells formed colonies after 2 weeks culture with hepatocytes, but unlike sp cells, they looked like fibroblasts and were negative for albumin. freshly isolated sp cells from splenocytes were also small and monocyte-like, and expressed no liver specific markers. spleen sp cells could culture for more than 2 weeks in the same way as npc-sp cells, and looked like hepatocytes, but did not proliferate. after having been cultured, most of gfp-positive cells were stained positive for albumin and cytekeartin 18, and negative for cytokeartin 19; on the other hand splenic main population cells after 2 weeks' culture expressed those very little. conclusion: we succeeded in culturing liver and spleen sp cells. sp cells which did not express mrna of liver specific markers, such as albumin or cytokeratin 18, became to express those after the culture with hepatocytes. these results suggest that these sp cells have plasticity and obtain hepatic function. thus, tissue stem cells, especially sp cells, are useful for cell transplantation. spleen sp cell might be a candidate for source of cell transplantation for treatment of liver cirrhosis. miura, takashi goto, ken-ichiro mikami, kunio nakane, kazuo yoneyama, hirokazu nagai, kunihiko terada, toshihiro sugiyama, katsuyuki imai, haruki senoo, sumio watanabe, akita university, akita, japan background epimorphin, a mesenchymal cell surface-associated molecule identified as an epithelial morphogen, is detected on stellate cells in the liver. we have reported that a contact with hepatic stellate cells (hscs) promotes differentiation of hepatic stem-like cells (hslcs). here, we show the involvement of epimorphin in that process.materials and methods: hslcs and hscs were isolated from healthy adult rats using two-step collagenase perfusion and percoll gradient centrifugation, and maintained standard medium, dulbecco's modified eagle's medium with 10% fetal bovine serum. hslcs were cultured in stellate cell-conditioned medium, co-cultured with hscs to maintain cellcell contact or in the presence of epimorphin. phenotypical and morphologic changes were investigated by rt-pcr and with an electron microscopy, respectively. results: hslcs are polygonal in shape and assume a cobblestone appearance when cultured in standard medium. transmission electron microscopy showed that hslcs , 20 to 25 micro-m in diameter, have a round to ovalshaped nucleus, a few vacuoles, scant organelles and a high nucleuslcytoplasm ratio compared with normal hepatocytes. the phenotypic properties of hslcs did not change as well as their size and shape during this experiment. hslcs characterized by rt-pcr are follows: positive, c-kit, musashi-1 (a neural stem cell marker), alpha-fetoprotein, cytokeratinl9, connexin43; negative, albumin, transferrin, tyrosine aminotransferase, gamma-glutamyl transpeptidase. hslcs cultured in stellate cell-conditioned medium had no phenotypical and morphological changes. hslcs co-cultured with hscs expressed albumin, transferrin, and tyrosine aminotransferase, which were inhibited by an anti-epimorphin antibody. furthermore, epimorphin induced the markers not only for hepatocytes including albumin, transferrin and tyrosine aminotransferase, but also for cholangiocytes including gammaglutamyl transpeptidase in addition to increased expression of connexin43 and cytokeratinl9, with decreased expression of c-kit and musashi-1. in addition, clebp beta was enhanced, which has been reported to mediate through morphogenesis by epimorphin. hslcs co-cultured with hscs piled up and subsequent development of bile-canalicui-like structures, which was dramatically inhibited by an anti-epimoirphin antibody. hslcs, close to epimorphin, stated piling up, changed their shape from flat to cuboidal, became rich in mitochondria and rough endoplasmic reticulum and formed bile-canalicui-like structures. conclusions: hslcs have a self-renewing capacity and multilineage differentiation potential. epimorphin is involved in differentiation of hslcs though a contact with hscs. disclosures: takashi goto -no relationships to disclose n order to identify new and differentially expressed genes in fetal rat liver that are specific for epithelial stemlprogenitor cells and genes involved in liver progenitor cell differentiation, we used murine cdna microarrays containing 8,976 cdnas, available at the functional genomic facility, aecom. the expression pattern of fetal liver stemlprogenitor cells was studied from embryonic day 13 through birth, 7 days after birth and in adult liver. the driver rnas were isolated from cells adhered to the dish after plating the cell suspension in order to remove the blood cells. reference rna was isolated from the livers of newborn rats. we found that 511 genes present on the cdna microarrays were developmentally regulated. these genes fall in two major hierarchical clusters, according to their pattern of expression. the 281 genes that are down-regulated during fetal liver development were distributed in functional groups and further analyzed. in this study, special attention was paid to genes that were induced in fetal liver but were not expressed (or expressed at a very low level) in adult liver. these genes are of special interest because they can serve as specific markers for identification and for isolation of liver stemlprogenitor cells. in addition, these genes represent links to understanding the fetal liver specific molecular pathways that govern cell proliferation, survival, apoptosis and differentiation. to determine which of the 281 over-expressed genes in 13-14 day fetal liver that are down-regulated in adult liver are progenitor cells specific, we searched in the available databases whether the expression of these genes in adult liver was previously reported. seventy genes were further analyzed: the clones of interest were hybridized to radioactive labeled 32p cdna synthesized from fetal and adult liver rnas. for 48 of the clones, we found that there was little or no expression in adult liver. the expression level of 25 selected clones was analyzed further by quantitative pcr, and they were confirmed as highly induced in fetal hepatoblasts compared to adult liver. half of the 48 clones are ests. the known genes fall in different categories, the major four being: genes related to transcription; signal transduction; morphogenesis, histogenesis and organogenesis; cell adhesion, de-adhesion and migration. some of the known genes over-expressed in fetal liver that are not expressed or expressed at very low level in adult liver are: grblo (aa260248), fh12 (aa023645), tnc (aa270625), peg3 (aa003064), hey1 (aa049474), enah ((aa217593), pkcd (aa276844), lox (w96914), shcbpl (aa265225), magoh (aa254528), manba (aa200473), klf5 (aa432818), gpc3 (aa274932), pcolce2 (aa153907), ppap2c (aa220316), nfkb2 (aa060802), adam19 (aa051790), akapl2 (aa387076), tagln (bc003795. it should be noted, that most of the 48 clones and all those listed here that we have identified as liver progenitor cells specific, are expressed in stem cells of embryonic, hematopoietic, or mesenchymal origin. two of the presented genes encode cell surface proteins: a disintegrin and metalloproteinase domain 19 (adaml9) (meltrin beta), glypican 3. using in situ hybridization, we are currently verifying whether our putative liver progenitor cell specific genes are expressed in hepatoblasts and in rare progenitor cells that remain in the adult liver. identifying and cloning new genes that are expressed uniquely in liver stemlprogenitor cells will allow us to design a method for isolation of these cells and to study their role in liver development, growth control and regeneration. hepatocyte transplantation has been shown to be an effective treatment for liver diseases including fulminant hepatic failure and metabolic defects in liver function. however, the availability of sufficient numbers of hepatocytes with which to conduct clinical studies limits the wide-spread availability of this therapy. previously, we have shown that human placenta derived stem cells (pdsc) differentiate along neural or hepatic lineages depending on the culture conditions and suggested that these stem cells could be a source of cells for clinical transplantation. here we report on a protocol to further optimize hepatic differentiation of pdsc. a three stage culture system was applied to induce hepatic differentiation. ae cells were propagated in dmem based standard media which contains egf 10 nglml for a week (stage i) and subsequently with growth factors such as fgf-1, 2, 4, 7, 8, oncostatin m, hgf, andlor dexamethasone (dex) and insulin-transferrin-selenium (its) for 2 weeks (stage 11). the cells were then changed to media supplemented with different nuclear hormone receptor agonists for 1 week to stimulate hepatic maturation (stage 111). total rna samples were examined for hepatocyte specific gene expressions with real-time quantitative pcr (rtq-pcr). additional studies examined hepatic differentiation on different culture substrates including collagen, gelatin, fibronectin, arg-gly-asp-ser, and matrigel 1%, 20%, 100% were also examined. in the three stage differentiation system, 20% (vlv) matrigel coated plates and dexlits containing media, and phenobarbital (pb) were identified as the best conditions to upregulate liver specific gene expression. the principal human drug-metabolizing enzymes (cytochrome p-450, cyp) were also examined by rtq-pcr. cypla1, 2c8,2d6, and 3a4 were induced with either rifampicin or pb. in parallel, expression of the relevant liver-enriched transcription factors, hnf-4, clebp-a and ciebp-b mrnas were increased under conditions which induced hepatic differentiation. under slightly different conditions, differentiation of pdsc into cells which expressed pax6, pdxl and nkx2.2, insulin and glucagon was observed. flow cytometric analysis showed isolated nayve ae cells contains a population of cells which express the embryonic stem (es) cell markers, ssea-3, 4, tra 1-60, and tra 1-81. furthermore, the placental stem cells formed embryonic body (eb)-like structure when cells were cultured on matrigel. the eb-like structures retained the expression of ssea-3, 4, tra 1-60, and tra 1-81. these data indicate that cells can be isolated from term placenta which express markers of es cells. under appropriate conditions pdsc expand, and differentiate into cells with characteristics of hepatocytes, neurons, or pancreatic cells. we propose that pdsc could provide a new source of cells for clinical transplantation and regenerative medicine. unlike with es cells, there should be no social, ethical or religious objections to the isolation and use of placental-derived stem cells. using the ssh technology, we have identified previously 643 induced clones in 13 day fetal compared to adult liver; 202 of these clones were independent transcripts, 64 of them represented ests and4 appeared to be new sequences (petkov et al., genomics 6 8 197-209,2000) . applying the rapid amplification of 5' and 3' cdna ends to subtracted genes (generace kit, invitrogen), we have obtained the full-length cdnas for two of the subtracted clones. one of them (clone 2g8) encodes a novel putative serine proteasel subtilase highly expressed in fetal liver and comparatively low in adult. the expression of 2c8 mrna was analyzed by northern blots with rna isolated from different rat tissues: fetal and adult liver, isolated 14 day fetal hepatoblasts, brain, bone marrow, kidney, spleen, intestine, colon, stomach and muscle. on northern blot with total rna, a strong signal was detected with fetal liver/ hepatoblasts rna and a weak signal with adult liver rna, which showed that the expression of this mrna is developmentally regulated. to confirm the liver specific expression of 2g8, quantitative rt-pcr was camed out. the results of this analysis confirmed our previous results and revealed also a very low expression of 2g8 mrna in the colon. the 2g8 cdna is 3345 nucleotides in length and codes for a novel protein of 691 amino acids. we were able to identify in the data bases the sequences of a rat contig which contains the complete gene for 2g8. the gene coding for 2g8 comprises 21,920 bp of the rat genome and includes 12 exons and a long 3'-utr segment of 1,000 nucleotides. the initiation of the translation begins with an aug codon, 188 nucleotides downstream from the 5'-end of mrna. using specific primers designed from the contig sequences, we obtained clones corresponding to the upstream promoter region and downstream of the gene in the pcr4 top0 vector (invitrogen). these two upstream and downstream clones were ligated to the 2c8 cdna upstream and downstream sequences, respectively, so that the whole gene with the 5' and 3' regulatory sequences was obtained in the pcr4-top0 vector. analysis of the structure of 2g8 showed that this protein resembles 8 others mammalian subtilases, named convertases described during the last decade: furin, pc1/3, pc2, pc4, pace4, pc516 and pc7ilpcipc8 and ski-1isip. these proteases are synthesized as proproteins and secreted from the cell. 2g8 contains a putative signal sequence for release from the er located between amino acid 30 and 31 (alanine 4 glutamine). however, it does not share the consensus cleavage sequences characteristic for the other convertases: (k,r)-(x)n-(k,r) for processing, release and secretion of the active enzyme form. at present, we do not know the processing site of the proprotein. in situ hybridization experiments showed that this serine protease is expressed in fetal liver hepatoblasts. convertases are secretory proproteins implicated in tissue specific processing of hormones, growth factors, metalloproteinase, extracellular matrix proteins, viral proteins etc. they show tissue specificity and some are implicated in development and differentiation. 2g8 is a novel convertase, it is developmentally regulated and most likely functions in processing and activation of growth factorslcytokines or extracellular matrix proteins implicated in morphogenesis. further studies of its promoter region will reveal the control sequences and transcriptional activators and repressors regulating its expression during development. college, london, uk introductioniaims: bone marrow cells (bmcs) can contribute to regeneration of the chronically damaged liver but in human studies and animal models the magnitude of this axis is highly variable. in a murine model of hepatitis b we examined whether this pathway of regeneration is enhanced by inhibiting endogenous hepatocyte regeneration. methods: 2 month old female mice transgenic for hepatitis b surface antigen (hbs-tg) received lethal irradiation and were then transplanted with c57b1/6j male bmcs by tail vein injection. 6 weeks later half the mice were treated with retrorsine, a pyrrolizidine alkaloid, to irreversibly block proliferation of endogenous hepatocytes. mice were sacrificed at 3 and 6 months following retrorsine injections. y chromosome containing hepatocytes were identified using in situ hybridisation and phenotype markers (positivity for cytokeratins 8/18, cytochrome p450 and glycogen, negative for cd45). results: in the control mice with chronic liver damage there was an increase in y chromosome positive hepatocytes over time, but the proportion remained <5% of the total number of hepatocytes. however, 19.3% (corrected count) of y chromosome containing hepatocytes could be found repopulating the livers of the mice that had received retrorsine. conclusions: bmcs contribute to the regeneration of the chronically damaged liver, but under conditions where regeneration of endogenous hepatocytes is possible this is minor. however, when chronic liver damage occurs and regeneration of the endogenous hepatocytes is inhibited, the contribution to liver regeneration from the bone marrow is significantly enhanced. beaujon, paris, france; thierry poynard, groupe hospitalier pitie-salpetriere, paris, france background portal hypertension depends in part on the development of hepatic fibrosis. thus, since fibrotest (ft) is a potential biochemical marker of fibrosis, this test was prospectively used to evaluate the presence and severity of portal hypertension in patients with different liver diseases. methods: 89 consecutive patients (56 males; 32% chronic viral hepatitis c, 9% hepatitis 8, 33% alcoholic liver disease, 7% transplanted, 19% miscellaneous) with transjugular liver biopsy had same day measurements done for hemodynamic parameters (gradient, free hepatic, wedged pressures), histological parameters (fibrosis scoring system in 4 stages, necroinflammatory activity grades and modification of architecture in 3 classes), and biochemical parameters via blood sample (ft for fibrosis and actitest for activity). the measurements of histological and biochemical parameters were done blind to any other characteristics. sensitivity (se), specificity (sp), predictive values (npv and ppv), spearman correlation (sc), accuracy (ac), kappa (k) and the area under the roc curves (auroc) were assessed. the main endpoint was the prediction of elevated portal pressure (epp), defined by a gradient of 2 5 mm hg. the secondary endpoint was the prediction of highly elevated portal pressure (hepp) of 212 mm hg (hepp). results: the mean ft value was 0.47 (se 0.05) for 15 patients without epp, 0.73 (0.04) in 23 patients with moderate epp and 0.87 (0.03) in 49 patients with hepp (p<o.ol between all groups, dunnett's multicomparison test) (figure 1 ). high aurocs of ft were obtained for the prediction of epp, 0.86 (0.04) and hepp 0.78 (0.05); a cutoff at 0.70 had a 95% ppv for the presence of epp (se=76%, sp=80%) and a 0.45 cutoff had a 100% npv for the exclusion of hepp (se=100%, sp=28%). there was a significant correlation between ft and gradient (sc= 0.50, p<o.ool). the auroc of ft for the histological architectural disturbance (fibrosis or cirrhosis) was very high 0.94, (0.03). there were also excellent ft diagnostic values for stages f2f3f4 ac=87%; k=0.54 (0.11); sc =0.57, p<o.ool; auroc=0.87 (0.04). therefore histological staging did not give higher diagnostic values than ft for epp, auroc=0.91, (0.03) introduction: the prevalence of gastric varices ranges from 20-25% in cirrhotic patients and is associated with high morbidity and mortality rates. conventional endoscopic hemostatic treatment is not effective for gastric variceal bleeding. tips can achieve initial control of bleeding but it is associated with high cost, shunt dysfunction, and increased encephalopathy. cyanoacrylate injection has been shown to be very effective with 90% hemostasis and low rebleeding rates of 0-28%. in our current series of gastic varix patients at the university of virginia, injection with n-butyl-2-cyanoacrylate (histoacryl) has yielded similar results (scaldwell, fda-ide). however, distal embolization after n-butyl-2-cyanoaaylate injection has been reported and probably results from "beading" upon polymerization. whether other cyanoacrylates with different physical-chemical properties have less embolic risk with equal or greater efficacy remains to be determined. &to investigate and compare the efficacy and safety of two different cyanoacrylates in the management of gastric varices. meth-ods:polymerization properties of two cyanoacrylates, n-butyl-2cyanoacrylate (histoacryl) and octyl-cyanoacrylate (dermabond), were compared in vitro by adding a drop of blood to different concentrations of n-butyl-2-cyanoacry1atel:ethiodol and octyl-cyanoacry-1ate:ethiodol (ethiodol, iodinated poppy seed oil, is radio opaque and delays polymerization). the two cyanoacrylates were then compared for "beading" by injecting each cyanoacrylate solution into a flowing circular system of fresh frozen plasma and then collecting the resultant polymer. lastly, cyanoacrylate injection was tested in an animal model using the femoral vein of a pig to simulate a gastric varix. each femoral vein was injected with n-butyl-2-cyanoacrylate and octyl-cyanoacrylate, respectively, and then both veins were ligated and examined for histologic change. results n-butyl-2-cyanoacry1ate:ethiodol (1:l) and octyl-cyanoacrylate alone had similar polymerization properties as far as initiation of polymerization to formation of a "hard substance"these concentrations were used for subsequent testing. preliminary results from the circular flowing system show that n-butyl-2-cyanoacrylate forms a "beaded" and "crystallized" polymer whereas the octyl-cyanoacrylate polymer is more "stringy" and "rubbery". in the animal model, n-butyl-2-cyanoacrylate had formed a large, extended thrombus in the vein with an irregular surface that appeared granular. in contrast, octyl-cyanoacrylate appeared uniform throughout and occluded the vein wall with only a s m d thrombus in the center. conclusion octyl-cyanoacrylate appears to be a viable alternative with possible lower embolic risk. vessel occlusion appears to involve a combination of polymer and thrombus formation, although interaction with the dotting cascade has not been adequately investigated. further studies in an animal model of gastric varices are in development to assess the relative safety and efficacy of these and other cyanoacrylates. disclosures: stephen evgeny farber, doron fisher, rami eliakim, nira beck-razi, ahuva angel, ella veikman, irit chermesh, camal yassin, diana gaitini, michael libas, rambam medical center, ha+, israel; soboh soboh, porria hospital, tiberia, israel; yaacov baruch, rambam medical center, haqa, israel background: current guidelines recommend that patients with liver cirrhosis, undergo egd screening every 1-2 years for the presence of ev. patients with large ev should be treated with beta-blockers and those with small ev should be rescreened. platelet count and us correlate with high risk patients but are not accurate enough to avoid endoscopy in low risk patients. barium swallow is preferred by most patients but data concerning its sensitivity is not sufficient. to evaluate the accuracy of be to diagnose esophageal varices in patients with compensated cirrhosis and portal hypertension at an early stage. patients and methods: 45 patients with liver cirrhosis where evaluated by egd (japanese general criteria), as a gold standard and be for the presence and size (small, large) of varices. x-rays were taken within 3 weeks of endoscopy, with 98% barium sulfate (e-z-hd), as suspension adjusted for double contrast studies and barium paste (e-z, 60%, esophageal cream) for mucous coating. results: 45 patients diagnosed by liver biopsy; 60% postnecrotic, 20% cryptogenic, 10% nash, 10% alcohol. 90% were child-pugh's score a and 10% were child-pugh's r. the correlation between egd and be was very high; r=0.84, p<o.oool. there were only 4 false negative results with be. overall sensitivity of re was 89%. all large varices (f2-f3) were diagnosed by be. sensitivity declined with f1 varices to 73%. the positive predictive value was 86%. the cost of x-rays was one fourth that of egd. conclusions: re can be used as a non invasive procedure for the screening of patients with varices and compensated liver disease at a minimal cost. using be will allow to avoid universal prophylaxis with beta-blockers. studies dealing with the prevention of rebleeding have shown that hemodynamic response to pharmacological treatment of portal hypertension is adequate when the hepatic venous pressure gradient (hvpg) decreases to ?12mmhg or by ?20% from baseline. with beta-blockers these targets are achieved in only around 30% of cases. however, even with such a high rate of poor hemodynamic response, variceal bleeding occurs in few patients when beta-blockers are used for primary prophylaxis (around 15%). the aim of this study was to assess whether the cut-off value to define response in primary prophylaxis may be defined more accurately and to investigate whether the acute response to beta-blockers may predict evolution (avoiding a second study). methods: a total of 101 cirrhotic patients with large varices and without pre-vious bleeding were investigated. a baseline hemodynamic study was performed in all patients. after initial measurements, propranolol was administered in 55 patients (0.15mg/kg i.v.) and measurements were repeated 20 minutes later. subsequently, nadolol was chronically administered to prevent variceal bleeding. a second hemodynamic study was performed 1 to 3 months later in 72 patients. results: during a mean follow-up of 37 months, 17% of patients had a bleeding episode and 19% died. there was a significant correlation between acute and chronic hemodynamic response to beta-blockers (r=0.6, p=o.ool). using roc curve a decrease of hvpg ?lo% from baseline was the better cut-off point to predict bleeding, both for acute and chronic studies. hemodynamic responders (defining response as a decrease of hvpg to ?12mmhg or ?lo%), had a significantly lower probability of bleeding (p?o.ol) and of requiring hospital admission because decompensations of cirrhosis, while survival probability was higher, both after acute and chronic beta-blocker administration. the hvpg decreased ?20% in 38% of cases and decreased ?lo% in 74% (p?o.ool) after acute beta-blocker. these figures were 31% vs 68% (p?o.ool) after a chronic administration. conclusions: our results suggest that: 1) a decrease of hvfg ?lo% from baseline may be the target to identify hemodynamic responders in primary prophylaxis of variceal bleeding; 2) ,4cute hemodynamic response to beta-blockers may provide an accurate prognostic information on long-term risk of variceal bleeding. ut; william m grosse, corneliu sanda, milton taylor, lndiana university, bloomington, in treatment of chronic hepatitis c patients with type 1 interferon (ifn-alpha) and ribavirin leads to a sustained virologic response in -50% of patients. the elimination of serum hcv rna in an individual patient displays biphasic kinetics with the first order attributed to the direct antiviral effects of ifn-alpha and the second order attributed to an immune mediated clearance of infected cells by induction of th1 cytokines, primarily ifngamma (type 2 ifn). we have postulated that the direct antiviral effects as well as the th1 response of current therapies may be enhanced by the addition of type 2 ifn (ifn-gamma). to study this hypothesis, we examined the activity of a type 1 ifn, infergen; (ifn-alfaconl) in combination with a type 2 ifn, actimmune; (ifn-gamma l b ) in preclinical models of hcv. these included an infectious flavivirus system (west nile virus) and the hcv replicon. in addition, global transcriptional profiling was examined utilizing the hg-u133a genechip; (affymeirix, santa clara, california) system. effective concentration (ec) studies of the l effects of ifn-alfaconl, ifn-gamma l b and the combination against the west nile virus and the hcv replicon are shown below: analysis of synergy for the combination of ifn-alfaconl and ifngamma l b in the hcv replicon using the chau-talalay methodology demonstrated very strong synergistic effects for a range of doses (c1<0.1 for all observations). initial statistical analysis (pv<o.ool; fold change 2 1.2) of global transcriptional profiling revealed that treating hcv replicon containing cells with ifnalfaconl up-regulated 156 transcripts, while 61 transcripts were significantly down-regulated. ifn-gamma l b treated cells had 109 transcripts up-regulated and 32 down-regulated transcripts. most significant was the finding that several transcripts were up-regu-lated by the combination of ifn-alfaconl and ifn-gamma l b that were not up-regulated by either ifn alone. these transcripts were involved in cell-cycle regulation, antigen presentation, apoptosis and immuno-activation. in addition, several known interferon stimulated genes were highly elevated by the combination when compared to the monotherapy treatments. these results demonstrate synergistic effects of the combination of type 1 and type 2 ifns for the treatment of chronic hepatitis c both on a cellular and molecular level. further study in hcv infected patients is warranted. hcv is leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. clearance of hcv is associated with strong t cell immunity to hcv immunogens including the non-structural ns3 protein (ns3). dendritic cells (dcs) play a pivotal role in priming rare antigen specific t cells to initiate protective hcv immunity. therefore, dcs are an important target to elicit broader and more robust immune responses against hcv. to target immunogens to dcs, 12-mer peptides were screened from a phage display peptide library for specific binding to human dcs. a candidate 12-mer peptide (dc-peptide# 3) was identified that bound human monocyte-derived dcs. dc-peptides bind to ligands that are strongly involved in receptor mediated endocytosis. dc-peptides could be linked to hcv ns3 biochemically or fused genetically with preservation of dc targeting specificity and immunogenicity. dcs pulsed with ns3 that has been genetically fused to dc peptide augmented t ceil proliferation >3 fold better thanns3 alone, and skewed t cells towards thl polarization resulting in elevated levels of ifn-.)i (higher than with ns3 alone) but not il-4 or il-10. t cells also acquired an activated phenotype (cd69+). to test in viva efficacy of ns3-dc peptide fusion protein, nod-scid mice were xenotransplanted with hcv-nake t cells, and vaccinated with three separate weekly injections of autolo-gous dcs (106) pulsed with nothing, ns3 alone, or the ns3-dc pep-tide#3 fusion construct. vaccination using dcs pulsed with ns3-dc peptide# fusion protein significantly increased ns3-specific t cell proliferationlactivation and enhanced the expression of ifn-y, and tnf-a compared to ns3 alone but no detectable levels of il-10 or il-4 were observed. these data indicate that targeting hcv subunits to dcs using specific dc-peptides represents a novel vaccine approach that may facilitate targeting of immunogenic antigens to dcs to elicit specific t cell immune responses against ns3 of hcv. this immunotherapeutic strategy can be implemented against the broad range of immunogenic antigens of various pathogens. it has recently been reported that the administration of eta in the setting of chronic hcv infection enhances ifn-induced viral clearance (1). the mechanism of these synergistic effects remains to be understood. our hypothesis was that eta enhances antiviral effects of ifn by increasing t cell reactivity to antigens. methods: peripheral blood mononuclear cells (pbmcs) were isolated in cpt tubes from blood of two healthy volunteers. cells were washed and then cultured in complete media 24-well plates in the absence of any antigen (negative control) and in the presence of immobilized anti-cd3 antibody (i-cd3, 150 nglml, positive control) as a nonspecific t cell stimulant. i-cd3 stimulated cells were treated immediately with peg ifn alpha-2a at two different concentrations consistent with physiologic doses in humans (10 nglml or 100 nglml) with or without the addition of eta at two different concentrations that were also consistent with physiologic doses in humans (1.65 pglml or 3.30 pglml). supernatants from each of the previous conditions were collected and assessed by elisa for ifn-7 secretion as a marker of t cell activation. results: our findings are displayed in the figure below. there was no spontaneous ifn-7 detected in cells that were not stimulated. ifn-y was detected at a modest level after exposure to i-cd3 alone, a response that became greater after exposure of cells to peg ifn alone or eta alone. production of ifn-7 was substantially enhanced in cells that were exposed to a combination of peg ifn and eta compared to those exposed to peg ifn alone or eta alone. conclusion: our findings suggest that eta has a synergistic effect to that of peg ifn in promoting in vitro activation of pbmcs and ifn-7 production in healthy individuals. increased t cell activation with eta may also suggest that tnfa suppress t cell function and may contribute to refractoriness to inf therapy in hcv patients. the influence of absolute dose and dose in mg per kg body weight of ribavirin on sustained virologic response (svr) in interferonbased combination treatment of chronic hepatitis c as well as the influence of dose reduction is still controversial. here, we address this problem by reanalyzing data of 343 previously untreated patients with chronic hepatitis c from a multicenter trial who were treated with interferon alfa-2a plus ribavirin and amantadine or interferon alfa-2a plus ribavirin (berg et al, hepatology 2003 ,37,1359 -1367 and completed therapy. thereby, we used a multivariate approach to account for correlations of the dose of ribavirin with body weight and body mass index (bmi) and known predictor variables from this data set which are low baseline hcv rna, high platelet counts, high pretreatment alt, and low y glutamyl transpeptidase (ggt) as well as hcv genotype non-1. because per protocol dosage of ribavirin was weight-adjusted (1000 mg for body weight below 75 kg and 1200 mg for body weight 75 kg or more) and body weight was positively correlated with ggt and negatively correlated with platelet counts (spearman rank correlation, p<o.ol% and p=0.2'x), respectively, we used a stratification with respect to genotype (gt 1 vs. non-1), ggt, and platelet counts in the analysis of ribavirin dose. when comparing body weight, bmi, the absolute intention to treat (itt) ribavirin dose as well as the itt dose of ribavirin measured in mg per kg body weight and the respective ribavirin doses at the end of treatment (eot), a significant association with svr was found for the eot ribavirin dosage (mg per kg body weight) and bmi (p=1.8% and p=3.0%, respectively). for predicting svr, a weight-based ribavirin dosage threshold of 13.75 mg/kg was calculated using receiver operating characteristics (roc) curves. the svr rate was 66% (1121169) in patients with a ribarivin dose of more than 13.75 mglkg as compared with 46% (79l172) in patients receiving equal or less than 13.75 mglkg ribavirin at eot (odds ratio 2.3; 95% ci 1.5-3.6; p=o.l%). in conclusion, the analysis of ribavirin dosage motivates new considerations of weight-adjustments of the ribavirin dosage to further increase svr in hcv-infected patients treated with combination therapy. effective and approved for treatment of patients with chronic hepatitis c virus (hcv) infection, the medications have many side effects and frequently require dose reduction or discontinuation. improved adherence to the medical regimen increases sustained response rates. data currently available regarding dose reduction, discontinuation and svr are largely derived from registration trials with experienced hepatologists at academic centers. anecdotal information suggests that dose reduction and discontinuation rates are higher and svr lower in community practice, where the majority of hcv patients are treated. actual dose reduction and discontinuation rates and svr in community practice are unknown. aim: we sought to determine dose reduction and discontinuation rates and svr in community practice in comparison with academic centers within the context of a prospective, randomized, controlled, multi-center trial. methods: patients with chronic hcv (+hcv rna) were evaluated at academic centers and community sites within the context of a randomized trial comparing peg ifn a (1.0 pglkglwk) + ribavirin (800-1400 mgld) vs. peg ifn a (1.5 pglkglwk) + ribavirin (800-1400 mg/d). demographic, serological (alt), virological (hcv rna level and genotype) and histological data were obtained. eligible patients were randomized to one treatment group. medications were administered for 24 weeks (hcv gt non-1) or 48 weeks (hcv gt 1). tolerability (dose reduction rates, adverse events and discontinuation rates) through week 24 of therapy was determined and comparisons were made between academic (ac) and community-based centers (cc). results: 294 patients have been enrolled, 74 in ac and 220 in cc. 6 patients in ac and 80 in cc remain on therapy within the initial 24 weeks and are not included in this report. genotype, histology, gender and age profiles were similar in the 2 groups. * designates a significant difference p<0.05. by week 24,7168 (10 %) in ac discontinued medications in comparison to 29/140 (21%) in cc". 3/68 (4%) discontinuations were due to aefsae in ac in comparison to 101140 (7%) in cc. 2155 (4%) patients in ac who did not discontinue by wk 24 received peg ifn dose reduction in comparison to 23/108 (21%) in cc". 14/55 (25%) patients in ac who did not discontinue by week 24 received ribavirin dose reduction in comparison to 31/108 ( the clinical outcome in response to combination therapy for treatment of chronic hepatitis c virus (hcv) infection appears to be different for caucasian versus african american patients. combination therapy of ribavirin and interferon alpha-2b has generally been found to be more effective for eliminating hcv in caucasians. the present study has been undertaken to further define this difference in response to therapy between caucasians and african americans and to determine whether the difference may be related to a difference in t cell-dependent immune responses to hcv in the two patient populations. to this end peripheral blood mononuclear cells (pbmc) were collected from patients prior to the initiation of combination therapy with ribavirin and pegylated interferon alpha-2b, week 0, and at 28 weeks and 44 weeks after initiation of therapy. the pbmc were stimulated in vitro with recombinant hcv-superoxide dismutase (sod) fusion proteins for ns3 (sod-c33c), core (sod-c22-3), ns4 (sod-c100-3), ns5 (sod-nss), and the relevant recombinant sod controls from yeast or bacteria (all generous gifts from dr. michael houghton, chiron). after four days, 3h-tymidine was added to the cultures and incubation continued for 16 hours to measure proliferation. the cultures were harvested on day five. supernatants from identical cultures were collected for assay of th1, il-2 and interferon gamma (infy), and t h~, il-10 and il-4, cytokine production. t cell responses to tetanus toxoid and the mitogen phytohemagglutinin (pha) were used as positive controls for t cell response potential. to date 68 patients have entered the study. week 0 data has been collected from 64 patients and 9 normal controls. data have been collected from only 25 patients that have completed the study through week 44. the results indicate that generally all patients' t cells were stimulated by pha and to lesser extent tetanus toxoid. the major cytokine stimulated by both pha and tetanus toxoid was infy. few of the patients had significant t cell responses to hcv proteins at time 0 measured either as proliferation or cytokine production. over the period of combination therapy, most patients in which serum virus titers were reduced to non detectable levels had significant increases in t cell responses to hcv proteins. although t cell proliferation was varied among the patients, there was generally a marked increase in infy production but little production of other cytokines. responses to hcv proteins were less consistent in those patients that either did not clear the virus or in which there was resurgence of virus after initial clearance. hepatitis c virus (hcv) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer (hcc). the high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for hcv. the hcv ns3 serine protease is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. intracellular biological processes can be specifically manipulated by anti-viral antibodies. we have recently presented the modulation of hcv ns3 serine protease transforming activity by non-neutralizing recombinant intracellular antibodies (intrabodies). we herein report the development of a novel bacterial genetic screen for inhibitors of ns3 serine protease catalysis and its application for the isolation of single-chain antibody-inhibitors. our screen is based on the concerted co-expression of a reporter gene, of recombinant ns3 and of stabilized single-chain antibodies (scfvs) in e. coli. the reporter system had been constructed by inserting a short peptide corresponding to the ns5aib cleavage site of ns3 into a permissive site of the enzyme p-galactosidase. the resulting cleavable lac2 gene is carried on a low copy plasmid that also carries the ns3 coding sequence. the resultant p-galactosidase enzyme when active, conferring a lac' phenotype (blue colonies on indicative x-gal plates) while co-expression of active ns3 results in loss of / 3 -galactosidase activity (transparent colonies on x-gal plates). the identification of inhibitors, as shown here by isolating ns3 inhibiting single-chain antibodies is based on the appearance of blue colonies (ns3 inhibited) on the background of colorless colonies (ns3 active). our source of inhibitory scfvs was a scfv library we prepared from spleens of ns3-immunized mice. once isolated, the inhibitors were validated as genuine and specific ns3 binders by an elisa and as bone fide ns3 serine protease inhibitors by an in vitro catalysis assay. these antibodies are also analyzed for intracellular immunization against hcv ns3 serine protease, as inhibiting intracellular antibodies. single chain ns3 neutralizing antibodies may emerge as useful clinical reagents for the treatment of infectious diseases and cancer. our genetic screen, although applied here for the isolation of antibody-based inhibitors, should be applicable for the identification of candidate inhibitors from other sources. 1ntroduction:genetic heterogeneity is an important feature of hepatitis c virus (hcv) genomes with six known genotypes and more than 50 subtypes. genotypes 1,2 and 3 are broadly distributed in patients around the world, whereas the other types are more geographically restricted. the peptidomimetic inhibitor biln 2061, optimized to have a high affinity for genotype 1 ns3 protease, has been reported to reduce viral load in genotype 1 hcv infected individuals. in this study, the ability of biln 2061 to inhibit the ns3 proteases from the other widespread genotypes 2 and 3 was assessed. methods: the ns3 protease domains and the ns3-ns4a proteins of genotypes la, lb, 2ac, 2b and 3a were cloned, expressed in e. coli and purified. protease activity was evaluated using fluorogenic depsipeptide substrates derived from the amino acid sequence at the ns4a-ns4b and ns5a-ns5b junctions. results: the activity of the ns3 protease domains revealed no major differences among the various genotypes. for the ns3-ns4a proteins, the catalytic efficiencies of the non-genotype 1 enzymes, although higher than the ones observed for the corresponding protease domains, were similar to that observed for genotype 1 (within 3-fold). differences in activity observed among genotypes were mainly related to changes in k,,, values. binding constant (k,) values for biln 2061 were similar among non-genotype 1 proteases with k,'s ranging from 80-90 nm for the ns3-ns4a proteins, up to a 60-fold reduction in affinity when compared to genotype 1. hepatitis-c-infection (hcv) is associated with an increased incidence of the chronic fatigue syndrome and depressive mood changes. beside immunological changes the modulation of the serotonergic system might be related to these psychiatric syndromes. serotonin concentration, 5-ht uptake and the activity of the enzyme mao-b in platelets are useful peripheral parameters to monitor serotonergic activity and function in patients with a chronic hepatitis c (chc). method in a prospective controlled study biochemical measures included assays of 5-ht concentration and 5-ht uptake activity at a low physiological substrate concentration in platelets as well as mao-b activity of 64 patients with a chronic hepatitis c were compared to 21 age and gender-matched healthy subjects. furthermore the effect of interferon-alpha treatment on serotonergic parameters was evaluated in 30 patients before and during treatment. results: the mean serotonin platelet concentration did not differ between controls and hcv-infected patients. however, 5-htuptake and the mao-b activity were significant lower in patients with hcv-infection (p<o.ool respectively, t-test, two-tailed). interferon-treatment led to an increase in 53it reuptake (p=o.ol), to an increase of mao-b activity (p<o.ool) and to a decrease of serotonin concentration (p<o.ol). thus, during hcv-infection serotonin concentration was stabilized by a decrease in re-uptake and reduced metabolism by lowering mao-b activity. treatment with interferon-alpha led to a decompensation of those mechanisms by increasing the mao-b activity and 5-ht-reuptake, followed by a decreased serotonin concentration. conclusion: our data support the hypothesis of a tryptophan depletion and serotonergic deficit in patients with chronic hcvinfection and interferon-alpha treatment. furthermore the results give evidence for a useful role of serotonin reuptake inhibitors for the treatment of the serotonergic deficiency during chronic hcvinfection and interferon-alpha treatment. chronic background: recent large prospective trials demonstrated that the combination therapy of interferon (1fn)-alphalribavirin significantly increased the ratio of a sustained virological response in patients with chronic hepatitis c in comparison with ifn monotherapy, especially in patients with high hcv-rna titer and genotype lb. however, the mechanism of beneficial effect of ribavirin is still unknown. on the other hand, interleukin 18 (il-m), originally termed ifn-y inducing factor, is one of proinflammatory cytokines, which acts on th-1 cells and strongly induces intrahepatic nk or nkt cells as well as activated t cells to produce ifn-y. to clarify the effect of ribavirin in combination with ifn, special emphasis on il-18, we examined the correlation between serum il-18 level and anti-viral effect, and compared it with ifn monotherapy in patients with chronic hepatitis c. patients and methods: all patients in this study are biopsy proven chronic hepatitis c with high hcv-rna titer (hcv-rna>loo kcopieslml before therapy) and serotype 1 (genotype l a or lb). forty-two patients were treated with ifn alpha-2b (6 mu) in combination with ribavirin (600-800 mg) daily for 2 weeks, following 22 weeks with ifn alpha-2b (6 mu) 3 times a week (the combination group). another 40 patients who were treated with ifn alpha-2b alone between 1998 and 1999 (before introduction of ribavirin in japan) with the same schedule are used as control (the monotherapy group). serum samples were taken before, 2 weeks after administration and 12 weeks after cessation of therapy. serum il-18 are examined by enzyme linked immuno-sorbent assay (elisa), using human il-18 elisa kit (mbl, nagoya, japan). the il-18 ratio is defined as serum il-18 level before administration divided by serum il-18 level 2 weeks after administration. hcv-rnas are quantitatively examined before and 2 weeks after administration. sustained viral responses are confirmed 12 weeks after cessation of administration by rt-pcr. results: there are no differences in the background of patients between the combination group and the monotherapy group. in the combination group, the decline of hcv-rna level highly correlates with the il-18 ratio in patients with higher viral titer (hcv-rna>500 kcopieslml before therapy) despite no correlation is observed in patients with lower viral titer (hcv-rna<soo before therapy). similarly, the hcv-rna level 2 weeks after administration controversially correlates with the il-18 ratio in the higher titer group despite no correlation in the lower titer group. interestingly, serum il-18 level itself does not correlate with the decline of hcv-rna level in both the higher and lower hcv-rna titer group. on the contrary, in the monotherapy group, the level of up-regulation of serum il-18 is lower than that of the combination group. furthermore, although the decline of hcv-rna also correlates with the il-18 ratio in the higher titer group, the level of a correlation coefficient is lower than that of the combination group. however, the sustained viral clearance 12 weeks after cessation of treatment is not correlated with the il-18 ratio. it suggests that ribavirin enhances the up-regulation of serum il-18 level in combination with ifn therapy and the effect of ribavirin is critical for the early anti-viral response during therapy, not for sustained viral response. objective: treatment with pegylated interferon alfa and ribavirin has an efficacy in chronic hepatitis c patients with sustained response rates of about 50%. however, response in genotype 1 patients with high viral load is considerably lower and is not significantly improved by pegylation of interferon. methods: in the present study, the efficacy of cifn daily dosing and induction therapy followed by combination with ribavirin in 200 naive patients with chronic hepatitis c and genotype 1 was evaluated. au patients had histologically proven hepatitis, elevated alt values and were viremic, the average weight was 80 kg. patients were treated with cifn dosages of 27 or 18 ug qd for 4 weeks, followed by 18 or 9 ug qd for 8 weeks. treatment was continued with cifn at 9 ug qd with ribavirin (7.5 or 15 mglkgld) for another 36 weeks. results: at 48 weeks therapy an undetectable hcv-rna was observed in 79 % and 72 % in the cifn 27/18 and 18/9 ug groups, respectively. data regarding sustained response rates showed 64 '10 and 58 % for cifn 27/18 and 18/9 ug groups, respectively. due to side effects cifn had to be dose reduced in 11% and discontinued in 6% of patients. addition of ribavirin potentiated the effect of consensus interferon in a dose-dependent manner as has been shown for other interferons previously. when viral response rates were related to the initial viral load, in the genotype 1 i low viral load group, sr rates of 71 and 74% were obtained for the cifn 27/18 and 18/9 ug groups (diff. n.s.). in contrast, genotype 1 / high viral load patients showed sr rates for the cifn 27/18 and cifn 18/9 ug groups of 41 and 49% (diff. sig.), respectively. the viral response rates obtained with daily dosing of cifn were significantly higher than an independently run control arm using cifn at 9 ug tiw with ribavirin irrespective of the initial viral load. four patients (2%) experienced grade i11 thrombocytopenias, while no grade iv neutropenias or thrombocytopenias were observed. the overall tolerability in the cifn 18/9 ug qd arm was comparable to standard therapy with pegylated interferon a2b and ribavirin, while the cifn 2711819 ug arm was less tolerable during the high dosing period. however, the withdrawal rates were not sigruficantly affected by the higher dose of cifn. conclusions: cifn daily dosing l induction therapy in combination with ribavirin thus shows significant response rates with the highest response rates in genotype 1 patients seen to-date. the sustained response rates for genotype 1 i high viral load patients are higher than the ones demonstrated in the peg interferon and ribavirin registration trials. these data suggest that for difficultto-treat genotype 1 / high viral load patients cifn with daily and higher initial dosing may be a worthwhile alternative to standard combination therapy with pegylated interferons. introduction: hcv-induced cirrhosis or hepatocellular carcinoma account for at least a quarter of all patients undergoing liver transplantation. almost 100% of hcv related liver transplant patients are re-infected with hcv after liver transplantation, often within days. in most of these cases, the recurrent infection leads to chronic hepatitis. these patients are in need of preventive therapy against re-infection of the transplanted liver. antibodies may be used as a stand-alone prophylactic therapy to prevent re-infection following liver transplantation or in cases of accidental exposure (e.g. needle stick injury). in addition, it may be feasible to use monoclonal antibodies in combination with other antivirals to treat chronic hcv patients. purpose: to develop an effective therapy to prevent hcv infection in liver transplant patients. methods: several human monoclonal antibodies were generated from peripheral blood b cells isolated from individuals infected with hcv genotype lb. these antibodies are directed against the hcv envelope protein (e2) of the virus and have high affinity and broad reactivity against different genotypes. the antibodies were shown to neutralize hcv in vitro in a cell-based infection assay and in vivo using the hcv-trimera mouse model. hepex-c, a single human monoclonal antibody, was further developed and studied clinically for safety, tolerability, and potential antiviral activity in phase l a and l b clinical studies in chronic hcv patients. results: in a phase l a study, single doses of 0.25, 1.0, 2.5, 10, and 40 mg of hepex-c were administered to a total of 15 chronic hcv patients. these patients had varying levels of hcv-rna (2.6 x lo3 to 1 x lo6 iulml) and endogenous anti-e2 antibodies (5 to 550 pglml). doses of hepex-c were well tolerated and no moderate or serious adverse events (sae) were reported. in 8 out of 15 patients, hcv-rna levels were reduced at least by half immediately following infusion and then gradually returned to initial levels. a multidose phase l b study of 10,20,40,80, and 120 mg of hepex-c in 25 hcv chronic patients was conducted. doses were administered weekly for 3 weeks and then 3 times a week during the fourth week. the patients were followed for an additional 3 months. multiple doses were well tolerated; no drug related saes were reported and no specific pattern of ae noted. 9 out of 20 patients from the 10 through 80 mg cohorts had at least 1 log infusion related reduction in hcv-rna from pre-treatment levels following administration of hepex-c. data from the 120 mg cohort are pending. conclusions: the good safety and efficacy data from this trial provided a basis for a phase 2a pilot study of the safety and efficacy of hepex-c for the prevention of recurrence in hcvinfected patients who have received a hepatic allograft for endstage liver disease. a multicenter, blinded, placebo controlled study is currently underway in 19 liver transplant patients receiving 20, 40, 80, and 120 mg doses of hepex-c. logic response rates to interferon (ifn) therapy in aa patients w i t h chronic hcv infection have been reported to be lower than that for caucasians (ca), but the effect of therapy on hepatic histology in aa patients remains to be assessed. objective: to evaluate histologic progression of disease in conjunction with anti-viral efficacy and safety in non-hispanic aa hcv genotype 1 patients and non-hispanic ca, hcv genotype 1 patients treated with peginterferon alfa-2a (40kd) in combination with ribavirin (rbv). methods an open-label, multicenter study was conducted to evaluate the efficacy and safety of the combination of peginterferon alfa-2a (4okd) and rbv in aa patients and in ca patients. all patients were required to have previously untreated chronic hcv genotype 1 infection, elevated alt levels and. a liver biopsy obtained within the 24 months prior to enrollment demonstrating chronic hepatitis but without cirrhosis. patients received peginterferon alfa-2a (4okd) 180 pg sc once weekly plus rbv 1ooo or 1200 mg orally based on body weight (<75 kg or =75 kg) for 48 weeks, with 24 weeks of treatment-free follow-up. histologic responses based on the knodell hai score were determined from liver biopsies obtained prior to treatment and within 4 weeks of completion of the 24-week untreated follow-up period. hai activity scores range from 0-18 and fibrosis scores range from 0-4. the histology outcome was evaluated for patients with paired biopsies only. improvement in necrointlanunatory activity was defined as 2 2 point decrease, and improvement in fibrosis as 2 1 point decrease. the primary efficacy endpoint was sustained virologic response (svr) with undetectable hcv rna (<50 iulml) at 72 weeks. results: the intent-to-treat population included 106 patients. paired biopsies were available for 53 of the 78 aa patients and 16 of the 28 ca patients. these patients were representative of all patients with respect to knodell hai scores. mean baseline hai activity scores were 7.2 0.32 for aa and 6.9 & 0.69 for ca patients; mean baseline fibrosis scores were 1.8 + 0.14 and 1.9 + 0.3, respectively. the svr rate for all aa patients was 20l78 (26%) and for all ca patients, 11/28 (39%). for those patients with paired biopsies, svr rates were 17/53 (32%) in aa patients and loll6 (63%) in ca patients. the majority of patients in both groups exhibited improvement in activity scores: 64% for the aa group and 69% for the ca group. more importantly, the table beiow shows that the proportion of patients with worsening fibrosis was similar in both groups, while 13/53 (25%) of aa patients and only 1/ 16 (6%) of ca patients showed fibrosis improvement. mean changes in fibrosis scores were -0.4 +-0.14 for aa and -0.1 t 0.14 for ca patients. among aa patients who had paired biopsies, 5/17 (29%) patients who achieved svr had fibrosis improvement; and 8/36 (22.2%) viral nonresponders exhibited fibrosis improvement. the only patient who had fibrosis improvement among ca was a viral non-responder. conclusions: therapy with peginterferon alfa-2a (4okd) plus rbv produced improvement in necroinflanunation and fibrosis in a substantial group of aa patients in this study. although the svr of 26% in aa with genotype 1 hcv is the highest response to combination therapy yet reported in this population, even patients without an svr achieved a benefit with regard to hepatic histology. higher sustained response rates (svr). however, modification of interferon-alfa by pegylation also reduces biologic potency. delivery of a bio-optimized alpha interferon in an unmodified form (consensus interferon, infergen(r)) by continuous infusion can be expected to provide sustained and constant levels of a fuuy potent protein. we hypothesize that such an approach may potentidy result in greater antiviral activity and improved tolerability by avoiding wide swings in interferon levels. methods:we conducted a phase 2 pilot study to assess the safety, tolerability and viral kinetics of mergen (12 fg/day) by continuous infusion using a subcutaneous infusion device (medtronic minimed(r) pump) in combination with ribavirin (loo0 mg or 1200 mg daily). the minimed model 508 micro infusion pump is cleared for use in the treatment of diabetes. hcv rna viral levels were measured at days 1, 3,7,10,14,21,28 and then at weeks 6 and 12. results: among 10 nonresponders treated to date with infergen by continuous infusion with available early viral kinetics data, 7 showed a substantial reduction in hcv viral levels (at least 1 loglo reduction in 4, and 2 loglo reduction in 3). all 7 of these patients were genotype 1 and 6 had less than 1.0 loglo decrease with prior treatment while 1 had a 1.5 loglo decrease at similar time-points with other combination therapy [either pegylated interferonlrbv (4 pts) or interferon-alfa/rbv (3 pts)]. no serious adverse events were reported and 3 patients discontinued (2 due to moderate interferon-related side effects and 1 due to inability to acclimate to the pump). other patients continuing on study experienced mild adverse events that have been tolerable. conclusions: consensus interferon administered by continuous delivery via the medtronic minimed pump shows early substantial reduction of hcv rna levels among nonresponder patients and shows a good safety and tolerability profile. updated data on a total of 22 patients (including 10 na'ive genotype 1 patients) will be presented. background side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in a given hcv patient population. furthermore, it has been suggested that higher ribavirin doses and longer treatment duration are required to achieve a sustained response rate in patients with genotype 1. coupling of ribavirin to hemoglobin offers the potential of a therapeutic with improved safety and efficacy by targeting the delivery of ribavirin to key tissues infected by viral infections such as hcv. these tissues include liver parenchymal cells and macrophages, both of which possess receptors for binding and internalization of hemoglobin-haptoglobin complexes as part of the natural clearance pathway for cell-free hemoglobin. hemoglobin thus serves as a natural drug carrier for targeted therapy of such receptor-bearing tissues. aim: to evaluate the effect of a ribavirinhemoelobin coniueate (rbv-hb. hrc 203) in a mouse model of viral hepatitis induced by the coronavirus, mhv-3. methods: ribavirin was covalently coupled to highly purified hemoglobin hbao at a mean molar drug ratio of 8 1 (ribavirin:hemoglobin). the rbv-hb was evaluated for retention of haptoglobin binding and cellular uptake in vitro, as well as the ability to recover bioactive ribavirin following enzymatic cleavage from rbv-hb. the rbv-hb was complexed to haptoglobin prior to administration to mhv-3 infected balblc mice, and carried one-third of the drug dose that was used for the ribavirin group. the rbv-hbtreated group was compared to control infected untreated and ribavirin-treated groups for survival, clinical behavior, viral titer, biochemistry and liver histopathology. results: rbv-hb was specifically internalized by cultured human and mouse hepatic cells but not by control non-hepatic cells. ribavirin enzymatically cleaved from rbv-hb retained in vitro bioactivity similar to unmodified ribavirin. untreated and mhv-3 infected mice all developed clinical and biochemical signs of acute viral hepatitis and died by day 4 post infection (altmax 7000 iuil). livers recovered from untreated and infected mice showed greater than 90% necrosis. in contrast, survival was enhanced in both ribavirin and rbv-hb treated and mhv-3 infected mice with a marked reduction in biochemical (alt 1000 iuil) and histologic evidence of hepatic necrosis ( 4 0 % ) . clinically, rbv-hb treated mice behaved normally at all time points, in contrast to ribavirin treated mice which developed lethargy and abnormal fur texture compatible with drug toxicity or ongoing viral infection. conclusions: in this study, a beneficial effect of rbv-hb was shown on the course of mhv-3 infection as demonstrated by prolonged sunival, improved clinical behavior, and a reduction in biochemical and histologic disease. the study provides a rationale for additional studies to examine the mechanism for the beneficial effect. ( introduction: the teravic-4 study targets patients with chronic hepatitis c who do not show a very early viral response at week 4 of therapy, as defined by detectable serum hcv rna (>50 kjlml, pcr methodology). after 48 weeks of therapy, these patients are expected to achieve a lower rate of sustained viral response (svr) than patients with undetectable hcv rna at week 4 (vr4). models based on viral dynamics and data from pilot studies with conventional interferon (ifn) suggest that a treatment period longer than 48 weeks may lead to a higher svr rate in these difficult-to-treat patients. this trial compares 72 versus 48 weeks of treatment with pegasys(') plus copegus('i in treatmentna'ive patients without vr4. methods: this phase 111, randomized, parallel group, multicenter study is being conducted in spain in accordance with the principles of good clinical practice.(l) after signing the informed consent form, all patients received the study treatment (peginterferon alfa-2a [40kd] [pegasys(']] 180 pg once weekly and ribavirin [copegus(')] 800 mg daily 1400 mg bid]) for 4 weeks. at this time, viral response (vr) was assessed using the cobas amplicor hcv(') test v2.0 (detection limit hcv rna 50 iulml). patients achieving vr4 continued therapy for an additional 20-or 44-week period, according to their hcv genotype and viral load. patients without vr4 were randomized 1:l to continue the study treatment for either an additional 44 weeks (total active treatment duration 48 weeks or an additional 68 weeks (total active treatment duration 72 weeks ). a 24-week follow-up period is ongoing to determine the svr rate. results: the mean age of the patients was 42 years, the mean weight was 73.5 kg, and 37% were female. the prevalence of hcv genotype 1 or 4 was 78%. vr4 occurred in 36% out of 511 patients, (23% of those with hcv genotype 1 or 4,83% of those with other hcv genotypes). hcv genotype distribution, viral load, age, and body weight were similar in the randomized study groups (group-48 and group-72). among patients without vr4, hcv rna was undetectable in 62"h in group-48 and in 69% in group-72 at week 24. the biochemical response rate (br) at week 24, defined as normal alt levels, was similar as well: 81% in group-48 and 78% in group-72. the dropout rate after 24 weeks was 9% in group-48 and 6% in group-72. at the end of therapy (eot), hcv rna was undetectable in 93% of patients with vr4. the eot vr rate in group-48 was 74% versus 81y0 in group-72. the rate of br in patients with vr at eot was also higher in group-72 (85%) than in group-48 (77%). dropout rates and reasons for dropping out by the eot visit were different between groups: 17% in group-48 and 36% in group-72. therapy was well-tolerated and no unexpected adverse events were observed. the prolongation of therapy from 48 to 72 weeks did not cause an increase in the frequency of neutropenia or thrombocytopenia. conclusions: pegasys(') 3 80 p g weekly plus copegus(') 400 mg bid administered for 72 weeks offers higher rates of vr and br at eot than 48 weeks of treatment in patients with detectable hcv rna four weeks after treatment initiation. the 800 mglday dose of ribavirin used in the trial has been proven to be optimal in patients infected with hcv genotype 2 or 3, but not in those with genotype 1 who require 100011200 mglday. this evidence was not established when the trial was initiated. "the teravic-4 study group also included v. ripollks background isis 14803 is an antisense phosphorothioate oligodeoxynucleotide inhibitor of hepatitis c virus (hcv). in a previous phase i study, plasma hcv rna reductions 2 1.0 log were observed in 3 chronic hcv patients treated with isis 14803 for 4 weeks. aims: the goals for this phase i1 study were to evaluate the safety, antiviral efficacy, and pharmacokinetics of isis 14803 given for 12 weeks. methods: the study was conducted in noncirrhotic chronic hcv patients. following 2 weeks of thrice weekly intravenous dosing at 2.5 mglkg ideal body weight (ibw), patients were treated for an additional 10 weeks at higher doses given either once (group a) or twice weekly (group b). the two doses studied were 4 (initial 3 patientslgroup) and 6 mglkg ibw. results: of 43 patients enrolled into the study, 41 had genotype 1 hcv, 39 had plasma hcv rna levels 2 2 x lo6 copieslml, and all but two had previously failed interferon-based therapies. two of the 16 group a and 5 of the 18 group b patients treated at 6 mglkg ibw had plasma hcv rna reductions 2 1.0 log. three in group b had reductions of 3.2-3.8 log. hcv levels remained 2.5 log reduced at 110 days after the last isis 14803 dose in one of these patients. seventeen patients had transient elevations in alt including all those with hcv rna reductions. peak alt levels ranged 5-30 times the upper limit of the normal range (x uln). for those with hcv rna reductions, the alt flares were temporally associated with the reductions. the alt flares were not associated with any clinical signs, symptoms, or sequelae. there were no changes in prothrombin time or albumin level. minor alkaline phosphatase (5 2.2 x uln) and bilirubin (5 1.5 x uln) elevations occurred in 2 patients. dosing with isis 14803 was continued during 7 alt flares, all of which resolved to baseline levels despite continued dosing. three flares, ranging from 9-29 x uln, occurred 3-8 weeks after the last dose of isis 14803, when presumably little or no drug remained in the liver. similar alt elevations have not been observed in the clinical studies of other phosphorothioate oligodeoxynucleotides. aside from the alt flares, common adverse events in this study were mild to moderate headache, fever, chills, fatigue, nausea, myalgia and other flu-like symptoms. the fever and chills usually occurred several hours after the end of the 6 mglkg infusions and resolved within 24 hours. two serious adverse events that were considered possibly related to isis 14803 occurred in the study: one patient had cryoglobulinemic glomerulonephritis and one was hospitalized for precautionary monitoring of an altlastlbilirubin flare. the former event was possibly due to an exacerbation of a pre-existing condition. the isis 14803 plasma pharmacokinetics observed in this study were consistent with those seen in the previous phase i study. conclusions: isis 14803 appears to have significant antiviral activity in some chronic hcv patients. the plasma hcv rna reductions (up to 3.8 log) achieved in this study were in difficult-to-treat patients (i.e., genotype 1, high virus level, andlor nonresponders to interferon-based therapy). although, alt flares were observed in some patients, the results suggest the flares may not be due to a direct hepatotoxic effect of the drug. other than the alt flares, isis 14803 treatment was generally well tolerated. this study established the dose of 6 mglkg ibw twice weekly for further clinical evaluation. a study of isis 14803 in combination with peginterferon and ribavirin for patients not achieving an early virologic response during standard peginterferon and ribavirin therapy is in progress. with the long serum half-life of albumin. the objectives of this phase 1/2, open-label, dose escalation study were to evaluate the pharmacokinetics, safety, tolerability, immunogenicity, and pharmacodynamics of albuferon in subjects with hepatitis c virus (hcv) who had previously failed ifna containing regimens. methods: subjects were initially enrolled in 4 sequential dose groups (10-12 subjects per each group at 7 pg, 20 pg, 40 pg, and 80 pg). within each dose group a minimum of 5 and a maximum of 6 subjects per cohort were enrolled sequentially to receive 1 or 2 subcutaneous (sc) doses of albuferon administered 14 days apart. dose escalation beyond 80 pg included single injections of 120 pg, 180 pg, 240 p g 320 pg and 400 pg. plasma albuferon concentrations and antibody to albuferon were measured by elisa. hcv rna was measured using the amplicor hcv monitor kit (roche). 2' 5' oligoadenylate synthetase (oas1) mrna levels in whole blood was measured by a research-based taqman pcr assay. results: of the 63 subjects currently enrolled, 96% were infected with hcv genotype 1 with a mean baseline viral burden of 2 million copies/ml. 78% of subjects enrolled in these cohorts had previously failed pegylated ifna containing regimens. albuferon pharmacokinetics showed linear increases in auco.8 and c, , , with mean terminal half-lives of 139-158 hours with doses of 80 pg and higher. t, , , occurred between days 4 and 5. there is an approximately 40% increase in auc after the second injection in the double injection cohort at 80 pg. albuferon was well tolerated and there were no discontinuations. no subjects developed detectable anti-albuferon antibodies. adverse events were transient and most were mild to moderate. the most common adverse events were injection site erythema (39%), headache (30%), fatigue (26%), mylagia (21%) and arthralgia (19%). the mean reductions in the nadir neutrophil counts in the higher single dose cohorts ranged from 40-60%. there was induction of oasl mrna expression in all cohorts, with approximately 9-fold increase in median values at day 7. in the single dose cohorts (=80 pg), 52% of subjects showed a maximal 0.55 log or greater reduction in hcv viral load during the first two weeks. also, 20% or greater reductions in alt levels were observed in 36% of subjects in these single dose cohorts. conclusions: in the ongoing phase 112 study, albuferon demonstrated a favorable safety and immunogenicity profile. the pharmacokinetic profile supports dosing every 2-4 wks given its reduced clearance and extended half-life of up to 158 hours. all cohorts showed evidence of biological activity, as demonstrated by oasl induction, with anti-viral activity evident in the higher single dose cohorts. disclosures: v balan -human genome sciences, inc.: investigator t bambury -human genome sciences, inc.: investigator g everson -human genome sciences, inc.: investigator w freimuth -no relationships to disclose h mesghali -no relationships to disclose j murray -no relationships to disclose d nelson -human genome sciences, inc.: investigator l novello -no relationships to disclose b osborn -no relationships to disclose j recta -no relationships to disclose g subramanian -no relationships to disclose m sulkowski -human genome sciences, inc.: investigator j zhong -no relationships to disclose introduction pirfenidone (pfd) is an orally bioavailable pyridone derivative that affects a variety of cytokines, including inhibition of tgfbeta, tnf-alpha, pdgf, and egf. pfd has been shown in clinical studies to improve physiologic parameters in patients with pulmonary fibrosis. we have previously shown that pfd decreased hepatic fibrosis in two different rat models of cirrhosis of hepatology 37: [797] [798] [799] [800] [801] [802] [803] [804] [805] 2002) . our objective in this pilot clinical study was to evaluate the safety and preliminary activity of pfd in patients with cirrhosis of varying etiologies. methods all patients had histologic andlor clinical evidence of cirrhosis. pfd was given orally at a dose of 400 mg tid for 12 months. physical examination and labs including alt, ast, bilirubin, albumin and prothrombin time, platelet count were assessed at baseline and on monthly basis. hcv rna levels were measured in patients with chronic hepatitis c (cobas amplicor hcv monitor v2.0). liver biopsies were obtained at baseline and after 12 months of treatment and were read independently by two hepatopathologists who were blinded to the biopsy sequence. modified histological activity (hai) index of knodell and ishak fibrosis stage were used to assess changes in necroinflammatory scores and fibrosis stage, respectively. change in steatosis was also assessed. results a total of 26 patients with cirrhosis due to hepatitis c (15), ethanol (s), amyloidosis (l), autoimmune disease (1) and budd-chiari syndrome (1) were included. the mean age was 57 years (range, 29-75) with 13 males. liver biopsies at end of therapy showed a 2-point or greater reduction in the ha1 necroinflammatory score in 41% of the patients. steatosis decreased in 33% of the patients, was unchanged in 42% and worsened in 25%. while there was no significant reduction in the ishak fibrosis stage, improvement in interstitial fibrosis was noted. evidence of cell regeneration was seen in some patients. hcv rna levels were measured in 15 patients with chronic hepatitis c. at 6 months, 9 patients had a decrease in viral load, 2 patients remained unchanged and 4 patients displayed an increase in viral load compared to baseline. no patient had a sustained virologic response. 4 out of 15 (27%) hcv patients had normalization of alt, 7 out of 15 (47%) had decreased alt values, 1 did not change (7%) and 3 patients showed a modest increase in alt (20%). pfd was well tolerated with the predominant drugrelated adverse events being nausea, photosensitivity rash, and itching occurring in 15% of the patients and which improved after 2 to 3 months of therapy. conclusions: in this pilot study, treatment of cirrhotic patients with pirfenidone for one year was well tolerated. a significant reduction in necroinflammation ( 2 2-point reduction in ha1 grade) and steatosis was observed in a substantial proportion of patients. a subset of patients with chronic hcv infection showed on-treatment reduction in hcv rna levels. longer treatment duration or a less cirrhotic patient population may be needed to demonstrate effects on fibrosis. these data support conducting clinical studies to evaluate a potential role of i'fd in the treatment of steatosis, or in combination therapy for chronic hepatitis c. this work was supported by grants of marnac, inc and intermune, inc. disclosures: arnulfo alvarez -no relationships to disclose gil arechiga -no relationships to disclose juan armendariz-borunda -no relationships to disclose background: viral kinetic modeling of hcv response to interferon-based therapy provides important insights into factors associated with treatment outcomes. hcv/hiv coinfected patients appear to have lower overall response rates than that observed in monoinfected subjects, but the reason for this is not clear. we speculated that kinetic responses in key parameters would be decreased in coinfected subjects. methods: hcvlhiv coinfected patients and hcv monoinfected patients prospectively matched for treatment, genotype, age (k 5 yrs), gender, race and histology were evaluated. coinfected patients were randomized within the context of a large us. multicenter trial (actg 5071) to receive pegylated interferon alfa-2a + ribavirin vs. interferon alfa-2a + ribavirin. quantitative hcv rna (roche cobas amplicor) kinetic testing was performed at 0, 6, 12, 24, 48, and 72 hours and at days 7, 14. non-linear regression and linear models were evaluated in an effort to best predict response and identify prognostic factors. results: twenty-seven subjects underwent viral kinetic sampling and evaluation. these included twelve hcvlhiv case subjects (10 men, 2 women) and 15 matched hcv controls (some patients double-matched). the mean age of coinfected subjects was 46.1 years (range, 38-60). among hiv+ subjects the mean cd4+ count was 325 cells/mm3 (range, 175-855). 11/12 had baseline hiv rna 1400 copieslml. mean hcv viral load was 6.6 log iulml among coinfected vs. 6.2 log iulml in controls. 75% of coinfected subjects had hcv genotype 1. the remainder were genotype 2.25% of coinfected patients had no detectable virus 24 weeks after completion of 48 weeks of therapy (svr). overall svr in control subjects was 46.6%. efficiency ( e ) of phase 1 response (a) slope at 72 hours, lambda2 (slope of phase 2 decline) were calculated. efficiency of clearance ( e ) at 72 hours was highly associated with actual viral clearance across all groups (p=o.ool) but a2 was not. regression analysis failed to demonstrate a relationship between e and baseline cd4+ count, hcv viral load or genotype. in contrast, #955;2 was significantly associated with genotype. viral kinetic parameters were not predictive of svr. conclusion: viral kinetic modeling demonstrated that the efficiency of clearance at 72 hours was significantly associated with viral clearance during treatment. coinfection status did not affect key kinetic parameters. peg-ifn was superior to standard interferon in terms of viral clearance efficiency, particularly in the coinfected group. diminished svr in coinfected patients may be related to immune factors that are operative after reduction of viral loads to undetectable levels. the prevalence of chronic hepatitis c (hcv) infection is greater in african-americans (aa) than in caucasian-americans (ca). however, small studies and post-hoc analyses of larger clinical trials have not found racial differences in disease severity at study entry, though indicate that interferon and ribavirin combination therapy is not as efficacious in aa as in ca. it is important to identify disease and patient characteristics that differ between aa and ca in a large study designed to ascertain whether the apparent racial difference in response to therapy can be explained. aim: the study of viral resistance to antiviral therapy in chronic hepatitis c (virahep-c) is investigating reasons for the lack of sustained viral response (svr) to pegylated interferon and ribavirin therapy in previously untreated patients chronically infected with hcv genotype 1. a key objective is to determine whether previously reported response rate differences between aa and ca exist in a large multi-center cohort. methods: virahep-c will recruit 200 aa and 200 ca from 8 clinical centers in the united states. hypothesizing that aa and ca patients differ by factors that influence svr which could explain racial differences in svr, we compare demographic, clinical, histological, virological and health-related quality of life variables (hrqol) before treatment begins. liver biopsies performed within 18 months of study entry were assessed without knowledge of patient race by a central pathologist. results: based on the first 155 participants recruited, age and sex distributions are similar in the two racial groups with patients averaging 48 years of age and 2/3 being male more than 80% of the cohort has at least a high school education, but aa are more likely to have less than a high school education. body mass indices tend to be higher among aa than among ca (p=.06), though waistto-hip ratios are similar (mean 0.9 in each group). there are no racial differences in mode of hcv transmission, estimated duration of hcv infection, or baseline serum hcv rna levels. aa are more likely to have sub-genotype l b (p=.004).the ishak fibrosis score does not differ by race, but severe lobular inflammation is greater in aa patients (p=.03). diabetes and hypertension are more common in aa. the sf-36 physical component score in ca is similar to the general population and higher than among aa (p=.002). the sf-36 mental component score is similar in the two racial groups and comparable to the general population. summa-wlconclusion: with few exceptions, pretreatment virological, clinical and liver biopsy findings were generally similar in the two racial groups. differences in response are unlikely to be explained by these factors. lshak flbrosls score 58 (%) severa lobular influnmation (%) hypertension (%) diabetes ( bar-llan university, ramat-gan, israel background: combination therapy with peginterferon alfa and ribavirin for 48 weeks achieves sustained virologic response rates (svr) of 54-61%0 in patients with chronic hepatitis c. however, the svr rates are highly variable according to baseline (hcv genotype) and on-treatment parameters (initial viral decline). thus, it must be anticipated that current standard therapy recommendations lead to under-treatment in some and over-treatment in other individual patients. objectives: comparison between a dynamically individualized treatment schedule according to the early virologic response versus the standard of care combination therapy with peginterferon alfa-2a (40kd) (pegasys @) (peg-ifn) (180 pg qw) plus ribavirin (copegus @) (rbv) (1000-1200 mg qd) for 48 weeks. the primary aim of the study was to increase svr, while optimizing the available drugs, treatment duration, quality of life and the socio-economical burden of therapy. the secondary aim of the study was to enable a comprehensive analysis of virallhostlimmune correlates of response to treatment (ongoing). methods: all patients (n=273) were initially treated with peg-ifnlrbv for 6 weeks and initial viral kinetics were defined according to centralized serum hcv rna quantifications (cobas amplicor@ hcv monitor v2, roche molecular systems) on baseline and days 0,1,4,7,8,15,22 and 29 . after classification into viral response categories at 6 weeks, patients were randomized (n=270) to individualized therapy (arms al, a2, b1, b2, c, d) or continuation of standard therapy (std arm). treatment tailoring included: in rapid viral responders (rvr)discontinuation of rbv (al) or shortening of treatment duration to 24 weeks (a2); in slow partial responders (spr)addition of histamine (bl) or prolongation of treatment to 72 weeks (b2); in flat partial responders (fpr)addition of histamine (c); in null responders (nur)retreatment with high-dose of peg-ifn (360 pg qw) plus rbv (d). svr was defined as undetectable serum hcv rna (< 50 iulml) at the end of 24 weeks of untreated follow-up. results: demographic and baseline virologic parameters were similar in the standard and the individualized treatment groups. according to the initial viral decline patients were categorized as rvr (51% for genotype 1 and 94% for genotype 2-3), spr (35% and 5% accordingly), fpr (5% and 0% accordingly), nur (10% and 1% accordingly) or unclassified (1%). the overall svr rates for genotype 1 patients were 49% for the individualized and 56% for the standard treatment arm (ns), and for genotype 2-3 patients 90% and 87% respectively (ns). svr rates (by itt analysis) according to hcv genotype and within each initial viral response category and arm are given in the table. conclusion: the overall sustained virologic response rates of 53% in genotype 1 and 88% in genotype 2-3 patients with chronic hepatitis c treated with peginterferon alfa-2a (40kd) in combination with ribavirin support previously presented data. individualized treatment according to initial viral kinetics appears to be clinically feasible, but did not improve the sustained virologic response rate with the drugs and dosages usable at the time of this study. nevertheless, the possibility that in rapid viral responders discontinuation of ribavirin does not decrease the sustained virologic response rate warrants future prospective trials. supported by the european community (qlk2-2000-00836), hoffmann la-roche and maxim pharmaceuticals. 10% 10% (hepatology 2000; 32(suppl) :844a). the rectally administered ifn is transferred not into blood but into regional lymphatic system and reaches to the thoracic duct via intraperitoneal lymphatics (pharmaceut. res. 1986; 3,116) . therefore, the ifn suppository appears to have different antiviral mechanism from the conventional ifn injection. in this study, we performed a combination of the ifn suppository and ifn injection. the antiviral effect and immune-related markers were examined comparing with the controls (ifn injection therapy). (patients and methods) fourteen patients with chronic hepatitis c were given an ifn suppository and ifn alpha (6-10m units) injection daily for 4 weeks. after that, only ifn injection was continued three times a week for 20 weeks. the control group contains 9 patients with chronic hepatitis c, who received ifn alpha (6-10m units) injection daily for 4 weeks and were followed by the ifn injection three times a week for 20 weeks. the viral loads were 1.52-2400kiulml (median 280kiulml) in the ifn suppository group and 18.7-616kiulml (median 97kiulml) in the ifn injection group. the genotype population (lb/2a,zb) was 5/9 and 3l6, respectively. there is no significant difference in the clinical background in the two groups. serum hcv rna was tested every 4 weeks. peripheral blood nk cell activity and cd4lcd8 ratio were investigated before and 4 weeks after the beginning of the therapy. the ifn suppository contains low dose ifn alpha (ball-1). (results) serum hcv rna turned negative in 73.3% of the ifn suppository group and in 66.7% of the ifn injection group (n.s.) after 4 weeks (end point of ifn suppository administration). the hcv rna seronegative rates of the two groups were 80.0% and 77.6% (n.s.) after 24 weeks, respectively. however, while the nk cell activity was decreased in 78% of the ifn injection-treated patients after 4 weeks, only 36% of the ifn suppository patients had decreased nk cell activity (p=0.049, chi-square test). furthermore, whereas serum alt levels were significantly decreased after 4 weeks in the ifn injection group (83.5+47.9 iull vs. 53.3+30.1 iull, p=0.026, paired t test), no change was seen in the ifn suppository group (80.1iu/1+63.9 vs. 80.5+53.7, n.s.). (con-clusion) the conventional ifn therapy decreased nk cell activity and serum alt levels. however, the ifn suppository combination therapy maintained augmented nk cell activity and elevated alt levels. these findings suggest that the ifn suppository continuously activates the hosts' immunity during ifn treatment. we used the ifn suppository only for 4 weeks; however, longer administration may lead to more frequent elimination of hcv the aim of the study was to evaluate the efficacy and tolerability of induction dose pegylated-interferon and ribavirin vs pegylatedinterferon and ribavirin as treatment strategies in relapsers to standard interferon + ribavirin in chronic hepatitis c patients. methods: 110 patients, virological relapsers after a first treatment with standard interferon and ribavirin for at least 6 months, were randomized to receive either pegylayed-interferon alpha-2b 2 fglkg qw plus ribavirin 800-1000mg qd during 8 weeks then peg-interferon alpha-2b 1 pglkg qw plus ribavirin 800-1000mg qd during 40 weeks (n= 52 pts) or peg-interferon alpha-2b 1.5 pglkg qw plus ribavirin 800 -1000mg qd during 48 weeks (n= 58 pts). efficacy assessments consisted of serum hcv rna level by pcr and serum alt at the end of treatment and after 24 weeks of follow up (week 72) and liver fibrosis with metavir score before treatment and at week 72. safety evaluations included adverse events and laboratory tests. results: patients were not different for baseline characteristics in induction and non induction groups including sex (male 58 % and 71% ), mean age ( . liver fibrosis metavir score decreased in 41% of patients in non-induced group and in 19% of patients in induced group (p=0,16) whatever the response (34% in sustained responders and 25% in non responders or relapsers). conclusion 1) combination therapy with pegylated-interferon is efficient in half of relapsers to standard combination therapy. 2) the response is similar to the response observed in naive patients. 3) 8 weeks induction dose does not increase the virological response. 4) we observed a regression of liver fibrosis in 30% of patients whatever treatment regimen and virological response. backgroundanemia has been shown to be an important factor in impairing hrql in cancer patients receiving chemotherapy, with changes in hb directly correlated with changes in hrql (gabrilove et al., j clin oncol2001). since 29-36% of patients with hcv develop anemia as a side effect of combination ifnlrbv therapy (rebetron@ package insert), it is important to assess the relationship between hb and hrql in this population. the objectives of this analysis were to (1) descriptively correlate changes in hb to changes in hrql in an anemic hcv-infected population; and (2) analyze the independent relationship of hb to hrql. methods: hrql scores and hb values were obtained from clinical trial data of 185 anemic hcv-infected patients (hb 10.8 t-0.9 gldl) who had been receiving ifnlrbv therapy for 12-14 weeks (afdhal et al., ddw 2003) . during the 8-week double-blind phase, patients were randomized to receive either epoetin alfa 40,000 u once weekly or placebo. hrql was measured using the short form-36 health survey (sf-36), an accepted and validated tool that measures 8 domains of hrql (table l) , and the linear analog scale assessment (lasa), which measures constructs of overall quality of life, energy, and activity. patients were categorized into the following groups based on their change in hb levels between randomization and week 9: (1) decrease in hb; (2) hb increase from 0 to <2 gldl; and (3) hb increase 2 2 gldl. changes in hrql (by domain of each instrument) corresponding to the aforementioned hb categories were summarized (table 1) . regression analyses were conducted to evaluate the independent impact of hb change on hrql improvement. the factors included in the regression model (in addition to hb change) were: age, gender, baseline hb, baseline hrql domain, fibrosis status, rbv dose change, duration of hcv therapy, and hcv rna. results: changes in hb values were directly related to changes in hrql for all 8 domains of the sf-36 and the lasa (table 1) . hrql changes were seen in an hb-dependent manner; patients whose hb increased 2 2 g/dl had higher improvements than patients whose hb increased between 0 to <2 gldl. similarly, patients whose hb decreased from randomization to week 9 had mean decreases in hrql for most of the domains (table 1) and minimal increases for others. regression analysis substantiated that the hb change was a significant independent predictor (p=.006 to r.001) of hrql change in all subscales of the lasa and the sf-36, with the exception of bodily pain and general health. conclusions: similar to cancer patients receiving chemotherapy, improvement in hb is a strong independent predictor of improvement in the hrql of hcv-infected patients. patients on combination ifnlrbv therapy should be monitored and considered for anemia treatment to improve hrql and potentially enhance their adherence to hcv therapy. virological response. the current trial was designed to see if better results could be achieved by retreating with higher doses of peginterferon alfa-2b (peg2b) + weight-based ribavirin. aim to compare the efficacy, safety and tolerability of three different doses of peg2b + weight-based ribavirin among interferonlribavirin non-responders. methods: patients were randomized to 1 yr of treatment with peg2b 0.5, 1.5 or 3.0 mcglkglwk, plus ribavirin 12-15 mglkglday. treatment assignment was stratified for sex, race, hcv genotype and histologic fibrosis. treatment was stopped at 24 wk if pcr(+). doses were reduced by 33% for toxicity; growth factors were not allowed. results: patients: enrollment took place between february 2001 and november 2002, with 963 patients recruited from 100 centers; data forms have been received on 794 thus far. enrollment was stopped in the low-dose group after fda approval of higher doses of peg2b. the study population is 31% female, 16% african-american, 93% genotype l, and 63% f2/3/4. efficacy: on-treatment virological response rates were dose-related at 24 wk but less so at 48 wk (see table) . this was partly due to a higher rate of discontinuation after a satisfactory response at 24 wk on the higher dose. on-treatment response rates were lower among african-americans and patients with more advanced fibrosis. sustained response data will be available for most patients by october 2003. 'tolerability: the rate of dose reduction was 33% on peg2b 1.5 mcglkglwk, vs. 45% on 3.0. rates of discontinuation were the same for peg2b 1.5 and 3.0 mcglkglwk: 25% vs. 24% overall, and 13% vs. 14% for adverse events. the frequencies of subjective adverse events were similar between the two groups. some degree of iieutropenia was observed in 27% on 1.5 mcglkglwk, vs. 32% on 3.0; however, only 14 patients in the study discontinued treatment for neutropenia overall. conclusions: 1) thirty to 40 percent of interferon/ of high-dose peginterferon alfa-pb + ribavirin m s l d abstracts 313a ribavirin non-responders achieved initial clearance of viremia on peginterferon alfa-2b + ribavirin. 2) doubling the peg2b dose to 3.0 mcglkglwk resulted in a higher viral clearance rate at 24 wk but a similar rate at 48 wk. 3) the higher dose of peg2b 3.0 mcglkglwk was well tolerated, with a higher rate of dose reduction but an identical rate of discontinuation. beliefs about therapy and psychosocial factors may influence the course of treatment and therefore could be important for developing comprehensive medical care plans that improve treatment adherence and optimize response to therapy. objectives: (1) to describe baseline indices of social support, depression, and self-efficacy (i.e., perceived confidence in the ability to perform health behaviors necessary for successful treatment) of patients with hcv genotype 1 participating in a treatment trial of combination peginterferon and ribavirin therapy. (2) to assess the degree to which baseline psychosocial measures are interrelated. methods: the sample consisted of the first 155 patients enrolled in the virahep-c study, a multicenter, collaborative clinical trial, designed to examine reasons for non-response to hcv therapy in african american and caucasian patients. using a touch-screen computer, patients completed the 21-item medical outcomes study social support survey (mos-sss); the 20-item centers for epidemiological studies depression scale (cesd); 24 questions about self-efficacy, and the sf-36 quality of life instrument. questionnaires were completed during an 8-week period prior to initiation of therapy. mean scores were calculated for each of the 4 subscales of the mos-sss (l=supported none of the time, 5=all the time): a) tangible support (material aid, behavioral assistance); b) affectionate (expressions of lovelaffection); c) emotional-informational (expressions of understandinglencouragement); and d) social interaction (others to have an enjoyable time with). similarly, mean scores were calculated for 5 subscales of the selfefficacy instrument (o=no confidence, 10= high): a) obtaining help; b) communicating with physicians; and managing c) symptoms, d) depression, e) medications. correlation analysis was used to compare each subscale with the cesd and sf-36. results: of the participants in the analysis, 43% were african american and 33% were female. on average, perceived social support and self-efficacy beliefs were high at the initiation of treatment. mean scores for the tangible, affectionate, emotional, and social interaction subscales were 4.2 (sd=0.9), 4.4 (sd=0.8), 4.4 (sd=0.7) and 4.2 (sd=0.8), respectively. for self-efficacy, the mean scores were 8.0 (sd=2.0) for obtaining help; 9.2 (sd=1.5) for communicating with physicians; 7.3 (sd=2.2) for managing symptoms; 8.3 (sd=1.6) for managing depression; and 9.4 (sd=1.3) for managing medications. the distribution of social support and self-efficacy scores were similar for both racial groups and genders. depressive symptoms were common in the sample at baseline with 55% having a score of at least 16 on the cesd and 7% having a score of at least 28. in general, the social support and self-efficacy subscales were unrelated to the cesd and to the subscales of the sf-36. conclusion: african american and caucasian study participants rate their social support and self-efficacy beliefs high in the weeks prior to initiating combination antiviral therapy for hcv, although depressive symptoms are common. social support and self-efficacy instruments may measure different psychosocial constructs as compared with other instruments used in hcv research like the sf-36 and cesd. as a result, social support and selfefficacy may represent new and important predictors of adherence and sustained virologic response (svr) in hcv treatment. baseline data from virahep-c will be used to evaluate factors associated with adherence and svr to determine if racial differences exist among these measures. if so, this information can be used to develop new initiatives for educating patients and families prior to initiating hcv therapy. background and aims: interferon(1fn) a-2blribavirin therapy is an effective anti-viral therapy for the patients with hepatitis c virus (hcv) genotype 1. however, the response to this therapy is not satisfactory because only about 40-50% of the patients become sustained responder(sr)s. further, the patients receiving ifn a-2blribavirin therapy have been frequently withdrawn because of severe adverse effects. the efficacy of ifn therapy is mediated by both genomic type and viral load and to immunological response of the hosts. reportedly, some amino acids were identified to modulate host's immunological response. in order to achieve more higher sr ratio after ifn a -2blribavirin therapy and to decrease the number of withdrawn patients, it seems important to improve the nutritional condition and to upregurate potential immunological response by modifying the balance of amino acid. in the present study, we investigated the dynamics of a panel of 41 amino acids during ifna-2blribavirin therapy, then analyzed the effects of nutritional condition on the clearance of hcv. materials and methods: seventy patients with chronic hcv infection were included after informed consent. six-million unit of ifna-2b in combination with ribavirin (600-800mglday) were given during 6 months. during the first 2 weeks of the therapy, ifn a -2b was given daily. blood samples were obtained after overnight fasting at zweeks, 8weeks, and 24weeks of the therapy and served for the analysis of amino acids. at the same time, peripheral blood mononuclear cells (pbmc) were collected and the number of ifny-and il4-producing cells were measured by facs analysis. thllth2 ratio was calculated as following; thll results: hcv rna was undetectable in 22%, 52%, and 78% of the patients at 2, 8, and 24 week of the therapy, respectively. thllth2 ratio decreased gradually during the therapy. thllth2 ratio at the end of therapy (24 week of the therapy) was significantly smaller than that at the beginning of the therapy (17.8210.9 vs 13.558.0, p=o.o11). however, there was no significant correlation of thll th2 level with the rate of hcv eradication. total amino acids (taas) and branched chain amino acids (bcaas) decreased gradually during the therapy. even at 8week of the therapy, there was a significant decrease in taa and bcaa (p=0.017 and p<o.oool, respectively). fischer ratio (fr) also showed drastic change. it changed from 2.83 (at the beginning of the therapy) to 2.19 (at 8 week of the therapy), indicating that the balance of amino acid rapidly altered to the similar condition to the patients with liver cirrhosis. of interest, the levels of taa and bcaa at the beginning of the therapy were significantly higher in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (taa: 3243+.275 vs 28772252 nmol/ml; p=0.004, bcaa: 470292 vs 377265 nmollml; p=0.024). although we could not find a significant difference, fr at the beginning of the therapy was larger in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (2.8120.51 vs 2.4750.68, p=0.15). analysis of each amino acid showed that there were significant differences in the concentration of citrulline and ornithine, both amino acid are involved in urea cycle, between hcv rna-negative and hcv rna-positive group at the end of the therapy. conclusions: interferon a-zblribavirin therapy induces malnutrition at early stage of the therapy. successful elimination of hcv rna seemed to depend much on nutrition rather than the ratio of thl/th2 during the therapy, suggesting that better response in interferon a-zblribavirin therapy might be possible by improving the condition of nutrition at the beginning of the therapy. in addition, adequate nutrition support would contribute to the improvement of the prevalence of hcv infection in egypt varies between 9% and 24% and approximately 90% are infected with hcv genotype 4.to date limited data exist on the effect of antiviral therapy on genotype 4-infected subjects. the purpose of this study was to assess the effect of interferon (standard or pegylated) in combination with ribavirin in na'ive-to-treatment subjects with chronic hepatitis c who reside in egypt. methods: in a prospective, open-label, randomized clinical trial, 200 egyptian subjects with chronic hepatitis c documented by liver biopsy and detectable hcv rna in serum received interferon alfa 2b (ifn-a2b) 3 mu tiw or pegylated ifn-a 2b (100 mcglweek) in combination with weight-based oral ribavirin (800 or 1000 mg for both groups). the study design was that if hcv rna was detectable at week 24, the treatment was stopped but if hcv rna was undetectable at week 24, treatment was extended for a total of 48 weeks. subjects were observed for an additional 24 weeks and hcv rna status at week 72 determined the response to antiviral therapy. safety laboratory tests were done at regular intervals. the study received irb approval. the hcv rna was done by a pcr technique with a detectability cutoff of 50-copieslmb results: both randomized groups were similar at baseline without statistical significant difference, 190 (90%) were infected with genotype 4, mean age was 39.5 years ? 8.7 years, 158 were men (79%), mean bmi was 27.8 2 4 kglm2, mean inflammatory ha1 score was 7/18 2 2 and fibrosis stage 2.716 t 1.3. of the 190 genotype-4 infected individuals, 156 have finished therapy (week 48) and 78 had completed the 24week follow-up (week 72). the study will be completed by october 2003. the table shows the intention to treat rates of undetectable hcv rna in genotype-4 infected subjects. serious adverse events that led to discontinuation of medications were observed in 6 subjects in the standard ifni'rbv group and in llsubjects in the peg ifnirbv, one death was observed in the latter group. expected adverse events were observed in similar rates in both groups. laboratory adverse events were observed between 2"h and 10% of subjects receiving either treatment regimen depending on the week of therapy. no growth factors were used in this population. in summary: subjects with chronic hepatitis c and genotype-4 infection have a similar antiviral response to standard or pegylated interferon in combination with ribavirin. the safety and tolerability of the medications is similar to what has been observed for other hcv genotypes. patients infected with hcv genotype 4 respond effectively to therapy and should be encouraged to undergo antiviral treatment. daily for 4 weeks, followed by 3 times a week for 20 weeks (week 24) and followed up for 24 weeks after cessation of therapy (week 48). the expression of socs-1 protein in the liver specimen obtained before the treatment was investigated by immunohistochemistry for socs-1. results: patients with the expression of socs-1 protein in the liver were defined as "socs-1 positive group". all other patients without staining were classified as "socs-1 negative group". sixteen patients were socs-1 negative and 61 patients were socs-1 positive. we compared baseline characteristics among two groups. socs-1 positive group had a significant lower rate of svr to ifn therapy than socs-1 negative group (p=0.0014). socs-1 positive group had significantly higher hcv-rna titer than socs-1 negative group (p=0.015). we analyzed factors with ifn svr in univariate logistic regression analysis. not only hcv genotype (p=o.oool) and hcv-rna titer (p<0.0001), but also the expression of socs-1 were found to be significant predictors of ifn svr (p =0.004 background: interferon(1fn) and ribavirin combination therapy is now a standard regimen for chronic hepatitis c infection and achieves a higher sustained virological response than interferon monotherapy worldwide. but the response rate to patient with genotype l b and high viral load is still not satisfactory. some virological factors have been shown to influence the effect of interferon monotherapy in previous studies. however little is known about those influencing the outcome of combination therapy. to examine the genetic basis of the genotype l b virus resistant to combination therapy, we analyzed full-length nucleotide and deduced amino acid sequences of hcv rna. method: among patients infected with genotype l b and high viral load, 6 patients treated with monotherapy : (group a-3 nullresponders, 3 relapsers) and 10 patients treated with combination therapy: (group b-3 nullresponders, 7 relapsers) were studied. the group a consists of 6 naive patients and group b consists of 5 naive patients and 5 patients who failed to previous ifn monotherapy. the group a patients were treated with 6-18miu of ifn, there times a week for 6 months. the group b patients were treated with 6miu-1omiu of ifn three times a week and ribavirin 600mg-800mg per day for 6 months.sera were obtained from the patients at three points: 6 months before the treatment, just before the treatment, and after or during the treatment. complementary dna was reverse-transcribed from total rna extracted from these sera, amplified by polymerase chain reaction and directly sequenced. the mutation rates during natural course of infection and during the treatment were calculated by comparing the nucleotide sequences obtained from the samples just before the treatment and 6 months before the treatment, and just before the treatment and after or during the treatment. the deduced amino acid sequences in different region were also analyzed in each case. result: the mutation rates of natural evolution analyzed in 14 patients were 1.89~10-~nt isitelyear. the mutation rates of hcv rna from nullresponders during the treatment were almost identical with those before the treatment in both groups a and bcu (3.3052.06 vs 3.0421.22, p=ns and 0.681-0.69 vs 0.6420.78, p=ns, respectively).in contrast, those from relapsers during the treatment were higher than those before the treatment in both groups ( table. no major differences were observed in the frequency or intensity of saes and aes. conclusion: the combination of peginterferon alfa-2a (40kd) (pe-gasysb) and ribavirin (copegusb) was safe and effective in patients with hcv genotype 1. despite the use of a dose of ribavirin (800 mglday) that is lower than the recommended dose for patients infected with g1 (100011200 mglday), approximately half of the patients in the study achieved an svr. because the study could not be blinded, a bias might have influenced the outcome of the study as suggested by the unequal discontinuation rate. these preliminary data do not support the hypothesis that higher svr rates can be achieved in patients with hcv g l by extending treatment duration. final data will be presented. *itt population, defined as all patients who took at least one dose of study medication and had at least one hcv rna evaluation postbaseline. **safety population, defined as all patients who took at least one dose of the study medication elusive goal, particularly in previously treated patients who failed or relapsed following prior therapy. we hypothesize that response rates in these patients who are re-treated with pegylated interferon and ribavirin will be improved compared to standard interferon preparations with or without ribavirin. the present prospective study is designed to determine the efficacy and safety of treatment with peg-intron (pegylated interferon alfa-2b) and ribavirin in a group of patients who failed prior therapy with interferon with or without ribavirin. methods: 462 aatients are uresentlv enrolled in this multi-center study. the first250 enrolled patie& were treated with 1.5 mg/kg o~p e g -intron sc q week, and ribavirin 800 mg po qd, which was standard at the time. the intended duration of treatment is 48 weeks. 225 patients have been trcated for at least 48 weeks and week 72 data is available in 141 patients. adverse events: dose reduction was required in 22% of pts most commonly due to leukopenia and anemia. permanent discontinuation of therapy was required in 8% most often due to: depression or anxiety serious adverse events: (1.8% of pts) included one patient each with neutropenialmrsa sepsis, optic neuritis with monocular blindness, perirectal abscess, cellulitis and multi-organ failure, cutaneous and pulmonary sarcoidosis, pneumonia, homicidal ideation, and suicide. conclusions: 1) overall etr was 50% and svr was 41%. 2) svr was significant in genotype non-1 patients and patients who failed or ;elapsed after previous monotherapy 3) 18% a multi-centre, randomised controlled study comparing the combination of interferon and ribavirin with no treatment was undertaken. patients were eligible if they had biopsy proven mild hcv (defined as necro inflammatory scores less than 4 and fibrosis scores less than 3 using the ishak scoring system. interferon was administered at a dose of 3 mu thrice weekly for 48 weeks regardless of viral genotype. ribavirin was administered according to the patient's body weight ( 5 75kg 1000mg/day, > 75kg 1200mgl day in two divided doses). randomisation was stratified according to viral genotype (1 and non-1). the primary end point was the virological outcome of treatment with sustained response defined as an undetectable hcv rna pcr 6 months post cessation of therapy. in addition there were a number of secondary aims: 1. viral kinetics. serum was collected at baseline, days 3,7,10 14 and week 12 for quantitative rt pcr to determine hcv viral load. the ability of early phase kinetics and the presence or absence of a 2 log drop at week 12 to predict eventual outcome is assessed in the context of mild disease. 2. health economics. the collection of resource use data in hcv infected patients who have mild, moderate and cirrhotic disease was performed as an integral part of this study. this data has been incorporated into a markov chain based model to assess likely future costs of the disease and their possible avoidance by treating mild cases. response rates from this trial will form the basis of the model. 3. quality of life assessments. sf36 and euroquol data were collected at baseline, weeks 1 2 2 4 and 48 during treatment and at post treatment weeks 12 and 24 in order to compare quality of life between treated patients and controls before during and after therapy. results. 98 patients received treatment and were matched with 98 controls (no treatment). sustained viral response rates will be available at the end of july when the follow up period of the trial is complete and will be included in the presented results. end of treatment results are shown in table 1. 30% of patients infected with genotype 1 and 61% of patients infected with genotype non-1 achieved end of treatment responses. sustained viral response rates to date are shown in table 2 .20% of patients infected with genotype 1 and 53% of patients infected with genotype non-1 have achieved sustained viral responses so far. 25% of patients in the treatment group were unable to complete at least 6 months of therapy due to side effects of medications. there were 4 hospitalisations in the treatment group, 3 of which were related to adverse events. patients with mild hcv have lower response rates to those with more severe histological change on liver biopsy. side effects are common and frequently result in treatment discontinuation, lowering response rates. the decision to treat patients with mild histological change must be weighed against the side effects. deferring treatment until later in the disease process does not prejudice response to therapy and may increase likelihood of success. the hcv ns5b polymerase is essential for hcv viral replication and infectivity as demonstrated in a chimpanzee model. the enzyme adopts a unique molecular structure that resembles a "thumb-palm-finger" which has some features different from other known dna and rna polymerases, highlighting the attractiveness for this enzyme as a target of antiviral therapy. from an in-house high throughput screening campaign and the subsequent lead optimisation, we have identified a novel chemical series of substituted thiophene-2-carboxylic acids that have good potency against hcv polymerase in vitro. x-ray crystallography studies revealed that these compounds bind to an allosteric site that is -35 angstrom away from the active site. further studies on selected members of the series confirmed the ability of these compounds to inhibit the sub-genomic replication of hcv in huh-7 cells. moreover, a representative compound was also found to have good in vitro safety index and favourable in vitro and in vivo metabolic and pharmacokinetic properties. in an open and prospective trial we investigated, if a pre-treatment with citalopram as an ssri can reduce the frequency of ifn-associated depression in hepatitis-c-infected psychiatric risk patients during methadone substitution. 36 patients with a chronic hepatitis c were treated with pegylated interferonalpha 2b and ribavirin according to body weight. 11 patients without any psychiatric history (group a) were compared to 25 patients during methadone substitution. the methadone substituted patients were separated into two groups: the first group (group b, n = l l ) were only treated with ifn-alpha and ribavirin and the second group (group c, n=14) received a two week pre-treatment with citalopram (20mglday) before combination treatment with ifn-alpa and ribavirin was started. antidepressant treatment was continued over the study period of four months. the hamilton depression rating scale (hamd, 17-item) was used and major depression defined as >20 points. patients were followed over the first 4 treatment months. group differences were calculated with anova for parametric and chi2-test for non-parametric scales. results: hamd scores at baseline were significantly higher in the psychiatric groups as compared to the controls (p=o.olo dallas, t x background: peg plus rbv is the mainstay of chronic hepatitis c treatment. however, an upper limit in terms of safety and efficacy for dosing with peg has not been evaluated. methods: we studied a group of nake patients using either conventional weight-based dosing of peg; (1.5 pglkg) or double the dose (3.0 pglkg) plus rbv 13+2mg/kg/day for 48 weeks. genotype 1 patients were randomized 1:l and to receive medication accordingly in unblinded fashion, with intent to enroll a total of 1,100 nayve patients n, an additional 300 previous nr or r to any type of interferon i rbv in a non randomized arm, using only the 3.0 pglkg peg dose. values for hcv rna at week 12 were compared to those obtained at baseline. results: to date, 573 patients 306 n, 193 nr, 74 r have enrolled in the study. 175 patients 106 n, 48 nr, 21 r have reached week 12. hcv rna results at week 12 are shown in the table below. treatment was well tolerated in most patients. dose reductions were required in 20% of n 1.5pglkg peg and 29% of n 3.0pglkg peg patients. side effects were equivalent between the two groups as shown in the table below. serious adverse events (sae's) were less than 5% and equally observed in both arms of the n patients. none were directly attributed to study drug. there were 25 patients in the wk 12 analysis that discontinued therapy. the reasons these patients were discontinued after reaching wk 12 were side effects (36%), +hcv rna (32%). it is interesting to note that 40% of the patients that were discontinued had 2 2 log drop or negative hcv-rna at week 12 and a positive hcv rna was not a reason for their discontinuation. conclusion: 3.op.glkg peg dosing does not appear to improve 12 wk viral response, but we await 24 wk results and sustained viral response rates. increased side effects as a result of doubling the interferon dose were not observed. more data is needed to verify the long-term efficacy and safety of this dosing regimen for patients with chronic hepatitis c . this study was supported by a grant from integrated therapeutics group a subsidiary of schering plough. background and aim: one of the major adverse effects of the combination therapy for chronic hepatitis c is ribavirin-induced hemolyhc anemia. however, little is known about the mechanism of this anemia. oxidative stress has been suggested as potentially important pathological mechanism in hepatitis c. this burden may cause peroxidation of erythrocyte membrane phospholipid in conspiracy with the accumulation of ribavirin triphosphate in the erythrocyte, which potentially attenuates the mobility of erythrocyte membrane. the aim of this study was to examine the change of fatty acid composition in erythrocyte membrane and the effects of vitamin e and vitamin c on fatty acid composition in combination therapy. methods: thirty-nine patients with chronic hepatitis c were enrolled in this prospective study. they were randomized to receive daily oral vitamin (500 mg of vitamin e plus 750 mg of vitamin c) (vitamin group) or none (control group), in addition to injections of 6 million units of interferon-a-2b daily for two weeks, followed by thrice-weekly for 24 additional weeks, plus daily oral ribavirin (600 or 800 mg) for 26 weeks. blood samples were obtained at 0, 2, 4, 8, and 26 weeks after initiation of therapy. phospholipid was separated by one-dimensional thin-layer chromatography after extraction of total lipid from the erythrocyte ghosts. following transmethylation, fatty acid methyl esters were quantified by gas chromatograph. a-tocophenol concentrations in erythrocyte were determined by high performance liquid chromatography. plasma thiobarbituric acid reactive substances (tbars) were measured by spectrofluorometer. results seven patients were obliged to suspend or cease receiving therapy because of adverse effects including anemia by 8 weeks after initiation of therapy. twenty-one patients have completed the assigned therapy up to now. among the 26 kinds of fatty acid analyzed, the mean content of 205n-3 polyunsaturated fatty acid (pufa) significantly decreased at 8 (0.96 5 0.12 mol% vs. in a cross-sectional study we investigated mxa expression in patients with chronic hcv infection(n = 60 ) and in patients with chronic hcv infection receiving ifn-alpha therapy (n = 23) as well as in healthy controls (n = 22). in a prospective study with 16 chronically infected patients (hcv genotype 1) known to be ifn non-responders, we followed mxa gene expression during combination therapy with pegylated ifn-alpha2b and ribavirin. results: patients with chronic hcv infection showed higher mxa gene expression levels than healthy controls, indicating that hepatitis c virus induces ifn production. however there was no correlation between mxa gene expression and clinical parameters (viral load, alt, liver histology). patients with chronic hcv infection receiving ifn-alpha had significantly higher mxa levels than patients without treatment or healthy controls. further we addressed the question whether mxa expression in pbmcs during therapy in previous non-responders could be a predictive marker of therapy outcome. mxa expression was clearly induced in all but one patient compared to pretreatment values (median: 10.2 fold). importantly, the level and kinetics of mxa expression did not correlate with the response to antiviral therapy. surprisingly, one patient showed an unusually low pre-treatment level of mxa expression and a total lack of mxa induction during ifn therapy, which was associated with complete non-response to therapy. in a neutralization assay, we could detect neutralizing antibodies to ifn-alpha2b in the serum of this patient whereas no neutralizing antibodies were found in all others patients. hepatitis c virus (hcv) infection is a common cause of transfusion acquired hepatitis and is today a major health problem throughout the world. the present study reports the prevalence of hepatitis c virus (hcv) infection in general population and in various high-risk groups from the city of hyderabad in south india. a total of 308 out of 3589 (8.6%) people (both general and risk groups) tested positive for hcv rna by rt-pcr, while anti-hcv antibody positivity, as determined by third generation eia, was found to be 5.8% (209/3589). this suggests that a number of cases go unreported, as screening of blood and blood products is done primarily by elisa. among 124 chronic renal failure (crf) patients with history of either renal transplant or hemodialysis, 46% were infected with hcv alone, 6.5% hbv alone while 37.1% were found to be co-infected with bothe hcv and hbv. our findings implicate these viruses as the major cause of post transplant hepatitis in indian patients with crf and indicate the necessity for immediate implementation of stringent screening procedures for these viral infections. additionally we report here that tattooing and slashing (a cultural practice among one sect of muslims) increase the mode of transmission of hcv infection. in addition, we also report a high incidence of hcv infection ( background in japan, long-term treatment with glycyrrhizin, given as snmc, is used to reduce alt and the risk of hepatocel-glycyrrhizin given as stronger neo-lular carcinoma in patients with chronic hepatitis c. phase 1/11 studies confirmed the alt lowering effect of glycyrrhizin therapy in western patients with chronic hepatitis c. the treatment duration in these studies was limited to 4 weeks. alt response was lost during follow-up. aim 1) to evaluate which dose frequency is required beyond week 4 to maintain the initial alt response. 2) to evaluate the effect of snmc treatment on liver histology. methods: hcv-rna positive patients with alt >2x uln, fibrosis stage 2 3 or necro-inflammation score 2 6 (ishak's score) who were not eligible for interferon therapy (prior non response, absolute contraindications) could enter this multi-center, randomized, open phase i1 clinical trial. all patients were treated 4 weeks with six infusions weekly of 100 ml snmc (minophagen pharmaceutical co. ltd., japan) containing 200 mg glycyrrhizin. patients with an alt response at week 4 (defined by a decrease of more than 50 percent of the baseline value or alt 51.5 uln) were randomized to continue treatment in three dose frequency groups for a total of 26 weeks. snmc was given 6 times weekly (group l), objectives: it is important to maintain reduced serum alanine aminotransferase (alt) levels in cases with chronic hepatitis c (ch-c) that do not respond to interferon (ifn) and in those with no indication of ifn therapy. we reported previously that dietary restriction of iron intake reduces serum alt levels in such patients. we evaluated ch-c patients treated with iron-restricted diet for two or more consecutive years, mainly focusing on the balance of energy intake, physical examination, and changes in hematological indices of nutrition. methods: twenty-two patients with ch-c (males, 18; females, 4; mean age, 56 year-old) that consulted our outpatient department were enrolled in this study. the inclusion criteria were as follows: 1) elevation of alt levels above the upper normal limit for 3 months or more; 2) positive tests for hcv-antibody and hcv-rna; 3) absence of other causes of ch (alcoholic liver disease, drug-induced liver injury, hemochromatosis) and negativity for hepatitis b surface antigen and for serum anti-nuclear and anti-mitochondria1 autoantibodies. twenty cases had received ifn therapy for more than 12 months before the beginning of the study; none of them responded to ifn therapy. dietary prescriptions included iron intake 7 mglday or less, energy intake 30 kcallkglday, protein intake 1.1-1.2 glkglday, and a fat energy fraction of 20%. nutritional balance was evaluated based on meal records, and instructions was given when necessary. results: the average energy intake before dietary prescription was 2184 kcal(36.7 kcallkg)lday, and it was significantly reduced to 1655 kcal(28.5 kca1lkg)lday (p < om), and then maintained stable at 30 kcallkglday. the average protein intake before dietary prescription was 85.7 g (1.45 glkg)lday and it was reduced to 1.1-1.2 glkglday after the prescription. the average fat intake of 66.5 g (1.1 glkg)lday and the average fat energy fraction of 27% before the dietary prescription were significantly decreased to 30.8 g (0.52 glkg)lday; p < 0.01 and 16% (p < 0.001), respectively, after dietary instructions. the fat energy fraction was maintained at a level of 20% or less. carbohydrate intake did not change remarkably during the observation period, although the carbohydrate energy fraction significantly (p < 0.001) increased. the average iron intake decreased significantly (p < 0.001) from 9.6 (before) to 6.1,5.2, 5.1,5.2, and 5.1 mglday 6, 12, 18, and 24 months after die* prescription, respectively. body mass index (bh4i) before diet prescription was 23.9 on average; bmi had no significant change throughout the course. the body fat percentage was 24.6% on average before the diet instructions, and it significantly decreased after the diet. the average values of aspartate aminotransferase and alt before diet prescription were 65 iull and 66 lull, respectively, and they were significantly reduced to 48 iull and 49 iull, respectively, after 24 months (p < 0.01). serum iron levels significantly decreased after 18 (p < 0.01) and 24 (p < 0.05) months, while unsaturated iron binding capacity tended to increase. the average serum ferritin levels were 376, 210, 189, 189, 141 nglml before and 6,12, 38, and 24 months after diet, respectively; there was a significant reduction (p < 0.01) in the values measured before and after the diet instructions. the average levels of hemoglobin, albumin and cholinesterase did not change significantly during the follow-up period. conclusions: restriction of iron intake is safe and well tolerated for a long period. the results of our present study suggest that decreased dietary intake of iron may constitute an important adjuvant therapy in patients with ch-c. chru, nice, france; c henquell, chru, clermont ferrand, france; c dizrcha, s ughetto, p dechelotte, hotel-dieu, clemont ferrand, france; h lafeuille, chru, clermont fewand, france; g bommelaer, hotel-dieu, clemont fewand, france peginterferon (pegifn) background: hcv co-infection is common among patients with hiv disease. it has been documented that hiv co-infection accelerates the course of liver disease in patients with hcv, and that liver failure is higher in co-infected patients. at this moment, there is no effective treatment for hcv non-responders to interferon therapy. mono-therapy and interferon-ribavirin combination is only effective in 2-3% and 5-10% interferon resistant patients respectively. treatment strategies to slow the progression to cirrhosis and to prevent liver failure in this population are needed. objective: this is a report of a study that examines the efficacy of peg ifn alfa 2-a (pegasys) vs pegasyslrbv 800mg in co-infected hcvlhiv patients that have not responded to a previous 6-12 months course ifn-alfa. the study also examines the histological benefit of treatment in this population. patients and methods: 76 patients were randomized 1:l to receive either pegasys 180mcg weekly x 24 weeks (group 1) or pegasys 180mcg weekly plus 800mg rbv for 24 weeks (group 2). patients that have hcv non-detectable or 210g decrease from baseline at week 24 (groupl), were added rbv 800mg. all similar responders (group 2) continue treatment for a total of 48 weeks. baseline demographics were similar between both groups. more than 70% of patients were nonresponders to ifn monotherapy.most patients in both groups (80%) were genotype l. the mean hiv baseline log was 2.92 (sd.64) group 1, vs 2.85 (sd.57) group 2 and most patients had baseline cd4 levels>400 cells, and were in stable antiretroviral therapy. the majority of patients (81%) are non cirrhotic, with mean grading group 1 6.63 (sd 3.24), group 2 7 (sd 3.64), and mean staging, group 1 3.51 (sd 2.2), and group 2 3.48 (sd 1.70 conclusions: this study is completed and pending some results, that will be available for presentation. sustained virological response(svr) (intention to treat), will be of the order of 5-20%. (11 group 2 results are pending). in this study, 10 patients were discontinued because adverse events and 10 were lost to follow up, before week 24th.svr in patients that completed at least 24 weeks of therapy will be of the order of 7-26%.a significant number of end of treatment responders are relapsing at week 72th. histology analysis show improvement in both groups of the mean grading and staging after treatment. in responders and relapsers the fpr becomes static or regressive.these results show that pegasys lrbv therapy is effective in this population.the study also suggests that longer duration of therapy ,and higher doses of rbv should be studied in coinfected nonresponders. * -pending results, will be available for presentation. disclosures: josi. rodriguez-orengo -no relationships to disclose maribel rodriguez-torres -roche laboratories: investigator; other: grant to perform study. adverse events by week 12 of therapy were grade 2 to 3 in severity and were therapy related. see table 2 . sixteen patients who continued therapy to week 24, three dropped out due to adverse events (1 anemia, 1 hyperbilirubinemia and 1 related death due to self-overdose). this patient had a history of hypothyroidism and mild untreated depression. at 48 weeks of therapy, 1 patient discontinued treatment due to suicidal thoughts. there was no difference between the ethnic groups regarding the drop out rate due to adverse events. hivfhcv coinfected population had a poor tolerance to pegylated interferon and ribaiirin. this was markedly increased in the first 12 weeks of therapy. the use of high dose pegylated interferon and ribavirin led to a significant drop out rate in comparison to the low dose pegylated interferon. background during liver injury, poorly characterized factors activate quiescent hepatic stellate cells (hsc) to become proliferative, matrix-synthesizing myofibroblasts. activated hsc are the major source of liver collagen and thus, play a key role in the fibrotic response to liver injury.the sns appears to promote fibrosis in injured livers because hepatic fibrosis is increased in the spontaneously hypertensive rat, which has an overactive sns. conversely, prazosin, an adrenoceptor antagonist, inhibits fibrosis in toxin-damaged rat livers. hsc express several neuronal proteins, including glial fibrillary acidic protein (gfap). they also contain synaptic vesicles and receive autonomic fibers. therefore, our hypothesis is that hsc are hepatic neuroglial cells that produce and respond to neurotransmitters in order to become activated during liver injury. methods: hsc were isolated from normal mice and dbh-lmice that cannot produce norepinephrine (ne) due to targeted disruption of the dopamine p-hydroxylase (dbh) gene. lysates of culture-activated hsc were analysed by rt-pcr, immunoblot and hplc to determine if they express adrenoceptors, catecholamine biosynthetic enzymes andlor produce ne. the effect of adrenoceptor antagonists and ne on hsc growth in vitro was also assessed. then in vivo activation of hsc by hepatotoxic diets was evaluated in control and dbh-lmice by comparing numbers of a-smooth muscle actin (asma)+ cells with immunohistochemistry and the induction of tgfp-1 and collagen a-1 gene expression by ribonuclease protection analysis of whole liver rna. results: hsc express a-and p-adrenoceptors, tyrosine hydroxylase and dbh. hsc from control, but not dbh-i-, mice release ne. endogenous ne is an autocrine growth factor for hsc because a-and p-adrenoceptor antagonists inhibit proliferation of hsc cultured from control mice. moreover, hsc from dbh-lmice, which cannot make ne, grow poorly in culture and are rescued by addition of ne. exogenous ne also promotes hsc proliferation. inhibitor studies demonstrate that the latter effect is mediated via g-protein coupled adrenoceptors that activate mitogen activated kinases and phosphatidylinositol 3 kinase pathways. hsc activation in response to diet-induced liver injury is inhibited in dbh-imice, as evidenced by reduced hepatic accumulation of asma (+) hsc and inhibited hepatic induction of background & aims: hepatic cirrhosis is six times more prevalent in obese individuals than in the general population, and obesity is one of the risk factors for liver fibrosis in which plasma adiponectin levels are decreased. adiponectin is an adipocytokine, which we previously identified by screening adipose-specific genes in the human cdna project. hepatic stellate cells (hscs) play central roles in liver fibrosis. when they are activated, they undergo transformation to myofibroblast-like cells, then proliferate, migrate, produce transforming growth factor-pl (tgf-pl), and various extracellular matrix proteins, and express a-smooth muscle actin (a-sma). we previously reported that adiponectin suppresses not only the proliferation and migration of hscs, but also the tgf-pl-induced fibrogenic gene expression in hscs. adiponectin could have biological significances in liver fibrosis. in this study, in order to clarify the effect of adiponectin on liver fibrosis in vivo, we tested the role of adiponectin on liver fibrosis using adiponectin-knockout (ko) mice and adenovirus mediated adiponectin expression system. methods: (1) to investigate the anti-fibrogenic effects of physiological concentrations of adiponectin, male wild type (wt) mice and ko mice were used. mice were each injected with a dose of carbon-tetrachloride (ccl4) (300 pllkglbw) intraperitoneally twice a week for 12 weeks to induce liver fibrosis. (2) to investigate the anti-fibrogenic effects of excessive concentrations of adiponectin, male wt mice were used. mice were injected cc14 (1000 pl/kg/bw) intraperitoneally twice a week for 12 weeks. mice were divided into 6 groups. control, received an injection of corn oil only; gr.1, mice treated with cc14 for 12 weeks; gr.2, mice treated with cc4 for 12 weeks after infusion of adenovirus producing adiponectin (adadn); gr.3, mice treated with cc4 for 12 weeks after infusion of adenovirus producing p-galactosidase (adlacz); gr.4, mice treated with cc14 for 12 weeks with adadn infusion at 6 week; gr.5, mice treated with cclb for 12 weeks with adlacz infusion at 6 week. results: (1) ko mice showed extensive liver fibrosis with an enhanced expression of tgf-p1 and connective tissue growth factor (ctgf) compared to wild type (wt) mice (p<0.05). the hydroxyproline content and the numbers of a-sma positive cells in mice liver significantly increased in ko mice. (2) the fibrosis areas were significantly decreased in gr.2 and gr.4 compared to those in gr.3 and gr.5, respectively. the hydroxyproline content in mice liver significantly decreased in gr.2 and gr.4 compared to that in gr.3 and gr.5, respectively. moreover, in gr.4, the hydroxyproline content was significantly decreased compared to that in 6-weeks of c c 4 treatment even though ccl, was given for an additional 6-weeks (total 12 weeks). conclusions: the findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention. disclosures: (ttg) is observed in mature scars and might promote wound repair by protecting the neomatrix from degradation by matrix metalloproteinases (mmps).however, such ecm crosslinking has the potential to hinder the matrix remodelling required for resolution of liver fibrosis. we have explored this hypothesis by examining expression of ttg and its crosslink product epsilon-(gamma-glutamyl) lysine in livers of: a) rats administered cc14 for 6 weeks (6wk c c 4 cohort)after which liver fibrosis resolves spontaneously after 28 days; b) rats administered cc14 for 12 weeks (12wk cc14 cohort) which develop micronodular cirrhosis; c) 12 wk ccl4 treated rats allowed to recover for 365 days after last cc14 dose (12 wk+365d cohort) which show partial resolution of fibrosis but with persistence of a macronodular cirrhosis. although ttg was identified by immunohistochemistry within and around fibrotic bands in 6 wk ccld cohort, no crosslinks were detectable. however, both ttg and crosslinks were detected in and around mature fibrotic bands in the 12wk cc14 cohort and in persisting fibrotic bands in the 12wk+365d cohort. western blotting of liver homogenates from these two cohorts using antibody against the crosslink revealed a major product of 160 kd which was degradable to lower molecular weight products following incubation of homogenates with bacterial collagenase but was refractory to mmp-2. to evaluate if ecm crosslinking in livers correlated with its resistance to mmps, 10 micron cryostat sections of livers from the three cohorts were incubated with purified active forms of mmp-1 or mmp-2 for 8 hr at 37 c and any residual ecm was stained with sirius red. ecm of 6 wk cc14 cohort was effectively degraded by these mmps but that of 12wk c c 4 and 12 wk+365d cohorts was only minimally degraded. hsc freshly isolated from normal rat or human liver showed no ttg expression whilst hsc activated for 7-15 days on plastic substrate expressed ttg protein (by western blotting) and mrna (by rt-pcr). there was ttg in cell lysates and in conditioned media of activated hsc. pure rat tail type i collagen incubated with conditioned media of activated hsc incorporated crosslink antigen, confirming that hsc secreted functional enzyme. however, no crosslink was found in type i collagen incubated with media from hsc which had ttg gene silenced by cell transfection with small inhibitory rna. cultured hsc which had ttg gene silenced had enhanced apoptosis (by acridine orange staining) following serum deprivation in culture. this suggests ttg or ttg activity promotes survival of these cells. we conclude that in persistent, incompletely resolving liver fibrosis there is evidence of ttg mediated crosslinking of the liver ecm and resistance to exogenous mmps. both features are lacking in ecm of fully resolving fibrosis. ecm crosslinking by ttg might limit resolution of cirrhosis. activated hsc are a potential source of ttg following liver injury and, as this protein supports their survival, it might contribute to their persistence in cirrhotic liver. we recently defined multifunctional biochemical properties of the long and frizzled variants and hypothesized that they may be inherent in the 192-aa-long-specific and the 235-aa-fz-specific modules, respectively. we thus transfected pcdnas.l-v5-his vectors containing variant-specific sequencer3 in mhat3fls315 hepatoma cells. this cell line stably expresses a truncated hnf3 protein turning off endogenous expression of liver-specific genes, such as albumin or c18. consistently, mhat3fls315 hepatoma cells transfected with an empty vector showed no endogenous c18 expression. by immunohistochemistry, using variant-specific and tag-specific antibodies and after analysis by confocal microscopy, long was localized in large supranuclear vacuolae suggesting protein storage/maturation in golgi structures or in beaded perinuclear vacuolae suggesting rer cysternae. in contrast, fz was strongly detected in close proximity to the plasma membrane of single cells. clusters of fz-transfected cells showed a strong and dense signal at sites of cell-cell contact, outlining single cells. these findings were confirmed by transfection of prep-7 vectors containing full-length long or fz forms of human c18 and confocal microscopy analysis after immunohistochemistry using an antibody directed against an epitope common to all c18 forms. immunoblot analysis of conditioned media showed that the fulllength long form was secreted into the medium but not fz. the latter was detected as a heavily glycosylated protein (> 300 kd) in the cell layers, predominantly in the triton-x-100 insoluble pellet after sonication and 10% sds solubilization, suggesting binding to extracellular matrix proteins. finally, glycosydase analysis of fz and long n-terminal modules showed an important increase in mobility after pngase f plus sialidase a digestion. these data show that, in addition to the previously described plasma (long) and tissue (short) forms of cl8, the fz form constitutes the pericellular matrix form, suggesting that the specific modules of c18 regulate tissue targetting through protein-protein interactions. backgr0und:during liver fibrosis, hepatic stellate cells (hsc) acquire an activated phenotype, proliferate and produce an excess of collagens. mycophenolic acid (mpa) is a known immunosuppressive drug. which inhibits the proliferation of b-and t-lymphocytes by inosine mono phosphate dehydrogenase (impdh) inhibition, causing an intracellular guanosine depletion. in addition, mpa has shown to inhibit growth of mesangium cells in the kidney and of skin-and tenon fibroblasts. therefore, we hypothesize that the proliferation of hsc may also be influenced by mpa. in this study we explored whether mpa is able to inhibit the proliferation of primary isolated rat hsc in vitro. furthermore, we studied the in vitro effects, mechanism of cellular uptake, and in vivo pharmacokinetics of mpa coupled to mannose-6-phosphate modified human serum albumin (m6p-hsa). m6p-hsa is a newly developed, hsc-specific drug carrier, homing towards the upregulated m6pligf-i1 receptor on activated hsc. in this way we hope to achieve cell-specific delivery of mpa to the hsc avoiding undesired effects of mpa on the immune system. meth-odslresults: in primary cultures of hsc a dose dependent reduction in the number of brdu-positive nuclei was observed after 24h incubation with 750,3000 and 6000 nm mpa, as assessed immunohistochemically. coupling of mpa to m6p-hsa via a biodegradable ester linker resulted in a conjugate with a maximum drug: carrier ratio of 1.2:l. analysis of the synthesized constructs was performed by hplc, fplc and ms. this conjugate was able to inhibit 3t3-fibroblast growth, as detemined by brdu-incorporation (elisa) in a dose dependent manner, reducing proliferation to 38.0 2 14.5%, 30.0 t 18.0% and 18.0 2 16.8% of control at 120, 240 and 480 uglml of conjugate, respectively. when cells were co-incubated with an excess of m6p-hsa, a competitor for receptor mediated uptake of the conjugate, the anti-proliferative effect was reduced by 69 yo. the organ distribution of the conjugate, 1251 labeled, was evaluated by iv injection of a tracer dose in rats 3 weeks after bile duct ligation (bdl-3). 47.3 t-4.1 % of the injected dose accumulated in the liver after 10 min of injection. in contrast, spleen and thymic gland accumulated 1.47 f 2.65% of the injected dose. intra-hepatic distribution was assessed in bdl-3 rats by immuno-histochemical double staining for hsa and specific markers for kupffer cells (ed-2), hsc (desminelgfap) or endothelial cells (his52). m6p-hsa-mpa showed a non-parenchymal distribution and co-localization with hsc and kupffer cells. conclusions: mpa is able to inhibit the proliferation of primary isolated rat hsc. this suggests that stellate cells are dependent on intracellular impdh activity to proliferate. coupling of mpa to mcip-hsa, a stellate cell specific drug carrier, resulted in a pharmacologically active construct, able to inhibit fibroblast proliferation in vitro after specific uptake via the m6p/igf-ii receptor. the major part of the injected conjugate accumulates in the hsc and kupffer cells of the liver, and avoiding uptake in the major resident organs for band t-lymphocytes. future studies will assess the advantage of this first hsc-selective compound in animals with liver fibrosis. background: platelet-derived growth factor (pdgf) is the most potent stimulator of migration and proliferation of mesenchymal cells. the expression of pdgf-p-receptor is increased during liver fibrosis. our aim was to investigate the effect of p3-integrin subunit blockade using the specific non-peptidic inhibitor emd409915 on migration and proliferation of hepatic stellate cells (hsc) and human foreskin fibroblast (hff). materials and methods: cell migration of human hsc and hff was assessed using a scratch assay. scratches of 600-700 pm width were made in confluent cell monolayers of cells following 24 h starvation in serum-free medium. after wounding. cells were stimulated with pdgf-bb (10 nglml) with or without emd409915 (merck, darmstadt, germany) at increasing concentrations (10-10m -10-6m). cell proliferation was measured as dna synthesis by brdu-incorporation. mitogen-activated protein kinase (erk112, p38, sapkljnk and akt) phosphorylation was evaluated by phospho-mapk-specific western blotting of cell lysates after 10 min of stimulation. results: stimulation of human hsc cells with pdgf-bb at 10 nglml in the absence of other growth factors resulted in pronounced stimulation of cell migration. pdgf-stimulated migration of human hsc was inhibited dose-dependently between 10-9 and 10-6 m by emd409915, with complete abrogation of migration at 10-6m. surprisingly, human skin fibroblast migration was not affected by b3-blockage. there was no effect of p3 integrin inhibition on cell proliferation as measured by brdu-incorporation, neither in human hsc-nor in hff cells. pre-incubation of hsc cells with the p3 integrin inhibitor at 10-6m did not affect the activation of p38 mapk, p44l42 mapk or sapkljnk. conclusions: 1. pdgf-bbinduced migration is strongly b3integrin dependent in human hsc, but not in human skin fibroblasts. 2. in contrast to cell migration, hsc and hff proliferation is p3-integrin-independent. 3. p38, p44l42, sapkljnk and akt mitogen-activated protein kinase signalling pathways are not modified by p3-integrin inhibition. 4. p3-integrin antagonist may be an adjuvant approach to treat hepatic fibrosis. epithelial to mesenchymal transition (emt) is defined as a process, in which epithelial cells loose their epithelial characteristics and acquire typical characteristics of fibroblasts. epithelial cells are tightly attached to their neighboring cells via cell adhesion molecules and they adhere with their basal side to their underlying basement membrane matrices, whereas the apical side faces a lumen. in contrast to immobile epithelium, mesenchymal fibroblasts are specifically designed to invade extracellular matrix (ecm). this is reflected by their prominent mesenchymal cytoskeleton and by their lack of a typical polarity. in the adult organism, emt occurs in epithelia in response to injury, potentially as a means to replenish fibroblasts, which are required for repair of injury. in organ fibrosis however, enhanced conversion of epithelium into fibroblasts is considered to contribute to progression of disease, as parenchymal epithelial cells acquire phenotypic and functional properties of activated fibroblasts. recent studies provided increasing evidence that parenchymal epithelium can potentially contribute to activated fibroblasts by emt, as in mouse models of kidney fibrosis 36% of activated fibroblasts were found to be of tubular epithelium origin. in liver fibrosis, stellate cells are considered the principal source of activated fibroblasts, whereas the role of hepatocytes is considered minor, even though they constitute for more than 80% of the liver mass. here we provide evidence that the pro-fibrotic growth factors tgf-beta1 and egf induce expression of fibroblast-markers fibroblast specific protein-1 (fspl), alpha-smooth muscle actin (alpha-sma) and type i collagen in primary mouse hepatocytes in vitro. furthermore, tgf-beta1 and egf induce acquisition of fibroblast characteristics, migratory capacity and release of mmp-2, suggesting emt of hepatocytes in vitro. additionally, progression of liver fibrosis in a mouse model of tetracarbon chloride-induced liver disease is associated with appearance of fspl positive hepatocytes, indicating emt, which suggests a role of emt in liver fibrosis. in conclusion, our results for the first time provide evidence for an active role of hepatocytes in liver fibrosis by undergoing emt. human studies -serum mcp-1 levels were significantly elevated in both cfld (929t67 pglml; p<o.oool) and ba (1,2902220 pglml; p<o.oool) vs controls (602240 pglml). in celd subjects serum mcp-1 was negatively correlated with the stage of fibrosis (r= conclusion: these results provide strong evidence for a role for mcp-1 in early fibrogenic events in cholestatic liver injury, ie., ductular proliferation and procollagen q(i) mrna expression, and suggest that serum mcp-1 may be a very good marker of the onset of cholestatic liver disease. this is the first study to clearly demonstrate elevated excretion of mcp-1 into bile in obstructive cholestasis, implying a potential role for mcp-1 in hsc and inflammatory cell recruitment to the biliary tree in cholestatic liver injury. [background and aim] angiotensin i1 (ang 11) is considered to play an important role in hepatic fibrogenesis. however, the molecular mechanism of renin-angiotensin system in hepatic fibrogenesis has not been clarified. we previously reported that ang i1 up-regulated mrna expression of pro a (i) collagen and tgf-p in isolated rat hepatic stellate cells and an angiotensin converting enzyme inhibitor, lisinopril, reduced carbontetrachroride-induced rat hepatic fibrosis. ang i1 type i receptor blocker (arb) have shown a good clinical response in patients with hypertension, and a promising preventive effect has been described in rat hepatic fibrosis. recently, the rholrho-kinase has been demonstrated to be involved in the signal transduction pathway of the ang i1 type i receptor signaling downstream. thus,the aim of this study was to evaluate the role of rholrho-kinase pathway in hepatic fibrogenesis in rat. to that effect, a rat hepatic fibrosis model was produced by chronic administration with cholinedeficient, l-amino acid-defined (cdaa) diet and the effect of an arb and a rho-kinase inhibitor (y27632) in this model was investigated. methods: wistar rats (male, 6 weeks-old) were continuously fed the cdaa diet or the control choline supplemented, l-amino-defined (csaa) diet. the rats were treated with 1 mglkg candesartan cilexetil, an arb (a kind gift by takeda chemical induustries, ltd., osaka, japan), or 1 mglkg y27632, a rho-kinase inhibitor (a generous gift by mitsubishi welpharma co., tokyo, japan), once daily through stomach tube. twelve weeks after the start of administration, the liver was excised and subjected to histological examination for fatty change and fibrosis by hematoxylin and eosin staining. serum alanine aminotransferase (alt) values and hyaluronic acid (ha) levels were also measured. since cdaa diet causes hepatic steatosis and fibrosis via production of free radicals, we also studied the oxidative membrane damage by immunostaining of 4-hydroxynoneal (hne) adduct. results: histologic examinations showed severe steatosis and fibrosis in the liver of cdaa-fed rats and no significant change in csaa-fed rats. the liver weight of cdaa-fed rats was significantly increased than that of csaa-fed rats. both candesartan cilexetil and y27632 remarkably reduced not only fibrosis, but also severe steatosis and the liver weight of the treated rats was significantly reduced in both treated groups. serum ha levels but not alt values significantly elevated in cdaa-fed rats compared to csaa diet fed group. the increased levels of serum ha were significantly reduced by the treatment with candesartan cilexetil or y27632. the number of hne-positive hepatocytes increased by cdaa diet and this increase was attenuated in the candesartan cilexetil-or y27632-treated groups. conclusion: these results suggests that an arb, candesartan cilexetil, and a rho-kinase inhibitor, y27632, show the same inhibitory effect on hepatic steatosis and fibrosis induced by chronic cdaa diet, and this free radicalinduced hepatic injury is a good therapeutic target of arbs or rho-kinase inhibitors. the rholrho-kinase may be a key enzyme system in the ang i1 signaling involved in the hepatic fibrosis. in liver cirrhosis, malnutrion and portal congestive enteropathy facilitate bacterial translocation from the intestine to mesenteric lymph nodes and the development of endotoxemia and spontaneous bacterial peritonitis in ascitic patients. igf-i is an anabolic hormone synthesized in the liver, whose levels are reduced in hepatic cirrhosis. igf-i possesses hepatoprotective properties and displays trophic effects on the intestine. here we investigated whether igf-i therapy could in-fluence portal pressure and bacterial translocation in experimental cirrhosis. methods: liver cirrhosis was induced in sprague-dawley rats (n=30) by oral administration of carbon tetrachloride during 8 weeks. after this time, rats were randomized in two groups to receive subcutaneous injection of recombinant human igf-i (2 microgllo0 g body weight in two separated doses per day) (ci-igf, n=15) or vehicle (ci, n=15), for 21 days. eight healthy rats, receiving vehicle were studied in parallel. at the end of treatment period, animals underwent laparotomy to measure portal pressure and to collect blood and tissue samples. endotoxin quantification in blood samples was carried out with a colorimetric limulus amebocyte lysate test. liver and ileum histopathological changes were assessed in tissue sections stained with masson's trichrome, using a numerical scoring system. bacterial translocation was evaluated by culturing blood samples and mesenteric lymph nodes in appropriated culture media. results: treatment with igf-i significantly improved biochemical markers of liver damage, being the alt values significantly lower in the ci-igf group than in ci rats (p<0.05). in addition we found that igf-i administration to cirrhotic rats reduced both the hepatic histopathological score ( the rate of progression to cirrhosis varies among individuals infected with hepatitis c virus (hcv) cholesteryl ester transfer protein (cetp) is a plasma glycoprotein involved in lipid metabolism which transfers cholesterol esters from hdl to vldl and ldl. obesity and steatosis are emerging as key factors in the pathogenesis of liver fibrosis in hcv. variability in function or levels of cetp may affect lipid transport and therefore steatosis. the uncommon vallval genotype results in decreased cetp plasma activity and may protect against myocardial infarction. we tested for a relationship between possession of cetp ile405val polymorphisms and rate of fibrosis in 327 white european patients chronically infected with hcv. rate of fibrosis was calculated by dividing the fibrosis score (ishak modification of knodell) by infection duration. genotyping was by reverse line blot hybridisation. a significant association was seen between rate of fibrosis and the ile405val polymorphism in cetp (p =0.002 (anova)) (figure 1) . disease association showed patients possessing the val allele (which is associated with lower cetp activity) were more likely to be slow fibrosers (no cirrhosis for >30 years) genotype frequency p=0.0017, allele frequency p=0.0007, odds ratio=1.8. (table 1) . possession of the val allele is associated with slow rate of disease progression in hcv. the functionally less active forms of cetp may result in decreased liver steatosis as a result of altered composition or increased levels of hdl, and therefore less fibrosis when a second insult such as infection with hcv is sustained. backgroudslaims: hepatitis c virus (hcv) is a major cause of chronic liver disease worldwide. however, little is known about exact mechanism of fibrogenesis by specific hcv gene or protein because the study of hcv-induced liver fibrosis has been mainly studied in human or animal model. moreover, the dynamically molecular study of hcv role in relation to liver fibrogenesis or immune response has been hampered due to lack of efficient in vitro hcv culture system. in the present study, we investigated whether hcv core protein directly influence on the liver fibrogenesis through stimulation of hepatic stellate cell(hsc) in in vitro or not. methods human and rat hscs were isolated using collagenase perfusion system and density gradient method, and other human hsc line (l190) was purchased from atcc. we established co-culture system of primary hsc and hepg2-core stable cell line which hcv-core gene was transfected into hepg2 cell. co-culture system was divided into two way, mixed co-culture and separated co-culture system. immunocytochemical staining was performed to identify the cytokines such as transfoming growth factor pl (tgf-p1) and a-smooth muscle actin(a-sma) overexpressed from hsc in the liver fibrogenesis. a-sma, tgfpl, transforming growth factorp receptor i1 (tgforii), collagen type i were also quantitated by western blot analysis. the expression of mmp-2 and collagen type i in the culture media was measured and analyzed by each zymogram and elisa. results the expression of tgf-p1 and a-sma was significantly higher in mixed co-culture of hepg2-core plus hsc than in hepg2 plus hsc as negative control. also the markers related fibrosis such as a-sma, tgf-pl, collagen type i and tgfrii, mmp-2 and collagen type i were highly expressed in separated co-culture of hepg2core and hsc conclusions in conclusion, hcv core protein may play a direct role in the fibrogenesis via upregulation of a-sma, tgf-pl, collagen type i and tgfrii, mmp-2 and collagen type i. further study is needed to clarify exact signal transduction of fibrogenesis by hcv core protein in the co-culture system. background: to current knowledge, transforming growth factor beta (tgf-beta) signaling is mandatory to produce liver fibrosis. various molecular interventions designed to affect the tgf-beta system were successfully used to inhibit fibrogenesis. activated hepatic stellate cells have been considered as a major producer of extracellular matrix proteins in liver injury and fibrosis. in the present study, we wondered whether follistatin, activin inhibitory protein, reduce apoptosis and prevent liver fibrosis and whether activin plays a key role in liver fibrogenesis. methods: wistar male rats weighing around 380g were injected intraperitoneally with dimethylnitrosamine (dmn) (lopg/g body weight) three times a week for three weeks. either follistatin or saline were also injected intravenously three times a week. on the 22nd day, blood was collected and biochemical parameters were measured using the standard methods. the liver was either fixed with 4% bufferedparaformaldehyde for histological examination, or frozen immediately in liquid nitrogen for the rna analysis. tissue sections were either stained with hematoxyline-eosin, masson-trichrome, or subjected to immunohistostaining using antibodies against collagen type iv, alpha-smooth muscle actin (sma), fibronectin, tgf-beta. the mrna expression of activin, tgf-beta, timp were measured by rt-pcr. apoptosis was analyzed by tunel methods. wistar male rats were injected with dmn (10kglg body weight) once and liver tissues were obtained and fixed with 4% buffered-paraformaldehyde 0, 12, 24, 36, 48, 60, 72 hours and 7 days after injection for immunohistochemical staining. hepatocytes and hepatic stellate cells were isolated by collagenase perfusion method and by collagenase-pronase perfusion method and analyzed activin and tgf-beta mrna expression by rt-pcr. results: 50% of control rats died, whereas none of follistatintreated rats died within 22 days. the serum level of hyaluronic acid in follistatin-treated rats were significantly reduced. ast and alp levels were also decreased significantly. apoptosis was reduced by follistatin dose-dependently. the expression of tgfbeta, timp, collagen lv and alpha-sma expression were also decreased in follistatin treated rats. we then examined activin expression in liver after dmn treatment. activin expression was observed at the maximum level in hepatocytes 12 hours after dmn treatment. tgf-beta expression was not detectable 12 hours after dmn administration, and it was sfxikingly increased in stellate cells 48 hours after dmn administration but it was not detectable in hepatocytes. activin appeared in hepatic stellate cells as well as in hepatocytes 72 hours after dh4n administration. inducible nitric oxide synthase (inos) has been reported to play pivotal roles in the development of various types of liver injury and inos expression may be important in the process of fibrogenesis of nonalcoholic steatohepatitis in obesity. no-induced active substance was shown to activate matrix metalloproteinase. the inos mrna and protein activity have been found to be induce in rats on a high-fat diet. objectives we investigated whether 1) induction of nos occurs in liver of mice fed high-fat diet, and 2) nos increases matrix metalloproteinase activity and reduces collagen content, thus attenuates liver fibrosis. we compared fatty and fibrotic changes of hepatic tissues in inos-knockout (inos-'-) and wild-type (inos+/+) mice which were fed with high-fat diet for 12 weeks starting from 8 weeks old. marked induction of inos mrna occurred in inos+/' mice, but not in inos-/-mice. immunohistochemically, nitrotyrosine staining which is a footprint of no-induced active substance, showed positive in inos+'+ mice, and negative in inos-'-mice. in histopathologically showed fatty metamorphosis, but did not have any distinction between groups inos+'+ and inos-/-. the extracellular collagen content with azan staining in inos+/+ mice was markedly decreased compared with that in inos-'-mice. in gelatin zymography of matrix metalloproteinases we found that prommp-2 and prommp-9 were increased both in inos+'+ and inos-'-mice, but their active form was found only in inos+'+ mice. similar results were obtained from in situ zymography of hepatic tissue. the stronger gelatinolytic activity was found diffusely in hepatic tissues of inos+/+ mice than inos-/-mice. (129xc57bl16) were subjected to either sham operation or bile-duct ligation. animals were sacrified two weeks after sur-gery and blood and liver samples obtained. bile duct ligationinduced elevation of serum liver enzymes was similar between wt and atla (-/-) mice. however, the liverlbody weight ratio was greater in wt than in atla (-/-) mice. bile duct ligated wt mice showed severe septa1 fibrosis, as assessed by trichromic masson and sirius red staining. in contrast, atla (-/-) mice showed minor fibrotic lesions, which were mainly located in peribiliary areas. collagen accumulation, as assessed by morphometric analysis of sirius red staining and hepatic hydroxyproline content, was markedly lower in atla (-/-) mice compared to wt mice. positive sirius red stained area in bile duct ligated wt and atla (-/-) mice were 8.150.9 and 4.651.0%, respectively (p<0.05). the increase in hepatic concentration of bioactive tgfb and proinflammatory cytokines (tnfa and illb) was attenuated in atla (-/-) mice compared to wt mice, as assessed by elisa. moreover, immunohistochemistry analysis revealed decreased lipid peroxidation products as well as decreased phosphorylation of c-jun and p42-44 mapk in atla (-/-) mice compared to at1 (+/+) mice. chronic liver failure stimulates the onset of interstitial liver fibrosis, which eventually results in "capillarization" of the sinusoid, impeding clearance of toxic substances by hepatocyte cells. the goal of this work was to search for the therapeutical effect of adjuvant gene therapy using adenoviral vectors containing cd-nas for human urokinase plasminogen activator and human matrix metalloproteinase-8 (ad-hupa plus ad mmp-8) on cirrhosis and its relationship with manganese brain accumulation, striatum dopamine and its metabolites in the rat. mn+2 is a wellknown neurotoxic metal which has been found accumulated in brain and blood of cirrhotic patients with hepatic encephalopathy. mnt2 elimination takes place via the hepatobiliary route. methods 200 gr wistar rats underwent bile-duct ligation (bdl) for 4 weeks and concomitantly treated with 1 mglml of mncl2 in drinking water (bdl/md2). after this point, five animals were sacrificed and serum, liver and brain tissue (striatum) were obtained. of the remaining bdl/mn+2-cirrhotic animals (n=10), 5 were injected with ad-hupa plus ad-mmp-8 (3x10" + 1.5~10~' vp/kg respectively) and 5 rats injected with (4.5~10" vp/kg of ad-p-gal). this treatment lasted 10 days. then, biological samples were recollected as before. an additional experimental model represented by cirrhotic rats injured chronically for 7 weeks with cclb were also monitored for their response to this therapy. results seven wk-cc4-cirrhotic animals treated with ad-hupa plus ad-mmp-8 (total 4.5x101'vp/kg) were monitored at 2,4,6,8, 10 and 12 days after combined gene therapy treatment (n=18). these animals showed improvement in liver fibrosis of up to 55% as compared with their ad-p-gal (n=18) treated cirrhotic counterparts. these results correlated with hydroxyproline detemiinations. furthermore, survival was determinated in 28 additional cirrhotic animals. 14 cirrhotic rats treated with ad-hupa plus ad-mmp-8 had a significantly higher probability of survival at 60 days after beginning of treatment as compared with 14 ad-@-gal cirrhotic rats. bdl/mn+2 injured rats displayed tremors, rigidity, and gait abnormalities. ten days after treatment with combined therapeutic gene therapy, these symptoms decreased. also, liver fibrosis was evidently less (25%) as compared with ad-p-gal treatment rats. brain tissue (striatum) was recollected 10 days after bdl/mnf2 animals were injected with either ad-hupa plus ad-mmp-8 or ad-p-gal alone. dopamine (3.04 mglgr) was decreased in 30% in ad-p-gal treated animals, as compared with 4.79 mglgr of dopamine found in ad-hupa plus ad-mmp-%treated animals. dyhydroxyphenylacetic acid (dopac a main dopamine metabolite) was as high as in ad-hupa plus ad-mmp-%treated animals (13.56 mglgr striatum) indicating a higher dopamine turnover in nontherapeutically treated cirrhotic animals. moreover, animals treated with ad-hupa+ad-mmp-8 showed a 50% decrease in ascitis and gastric varices as compared with ad-p-gal-treated animals. (gastroenterology 1221924,2002) . while ppar-a was reported to be involved in induction of antioxidant enzymes expression and activities (life sci 63:135,1998). furthermore, in respect of hepatic fibrosis, oxidative stress induces hepatic fibrosis and many reports have demonstrated that several antioxidants can inhibit hepatic fibrosis. therefore, in the present study, we examined whether ppar-a ligands, i.e. wy-14,643 and fenofibrates, can suppress hepatic fibrosis by attenuating oxidative stress in experimental rat model and possess a possibility of therapeutic candidate for hepatic fibrosis. methods: fibrosis was induced in male wistar rats by intraperitoneal administration of thioacetamide (taa) (200 mglkg twice weekly for 6 weeks). in the treated groups, ppar-a ligands, wy-14,643 (wy) and fenofibrates, were fed a diet containing 0.1% (wlw), and ppar-y ligands, pioglitazone (pio), were fed containing 0.01% (wlw) all through the experiment. control cirrhotic rats received saline injections for 6 weeks. after killing all rats, histological examinations (he, azan staining, immunohistochemistry of a-sma), serum value of alt and hyaluronic acid, mrna expression of ppars, acyl-coa oxidase (aco), a1 (i) procollagen, and antioxidant enzymes, such as superoxide dismutase (sod) and catalase. we also determined lipid peroxidation (lpo), glutathione levels, and activities of sod and catalase in the perfused liver. results: semi-quantitative analyses of fibrotic area revealed that wy co-administration with taa reduced to only 19% of the area of taa-treated rats. there was no significant difference in azan staining of the liver between taatreated rats and taa-treated rats. wy administration could not lower serum alt value in chronic and acute taa injury. these observations suggest that wy fails to modulate the hepatotoxicity of taa. an increased expression of ppar-cy in taatreated rats were observed, but the expression of ppar-a was abolished in taa-and taa-treated rats. wy intensively enhanced mrna levels of aco which was regulated by ppar-a, increase nearly 50-fold than controls, but the expression of aco diminished in taa-and taa-ppar-a activators, wy-14,643 and fenofibrates, treated rats as well as ppar-a. the mrna levels of a1 (i) procollagen and tgf-/3l were strongly increased in taa-and taa-administrated rats than in controls, while they were dramatically suppressed in taa-treated rats. the catalase mrna was reduced to 15% of the controls in taa-and taa-treated rats, however, taatreatment prevented this decrease to 70% of the control levels. catalase activity increased 2-fold in taatreated rats than in controls, while it decreased 60% of the controls in taa-and taa-treated rats. in taa-and taa-treated rats an increase of lpo was observed, but not in ta-atreated rats. we c o n h e d that fenofibrates treatment could reduce hepatic fibrosis to approximately 16% of taa treatment for 6 weeks by semi-quantitative analysis of fibrotic area. conclusion: our data indicate that war-a activators, not ppar-y, can markedly inhibit hepatic fibrosis in the experimental taa-induced rat cirrhotic model. we suggest that ppar-a and catalase are important in the development of hepatic fibrosis. therefore, we conclude that suppression of hepatic fibrosis by ppar-a activators is probably due to their antioxidant effects via increased activitiy of catalase which reduces hydrogen peroxide, and that fibrates, such as fenofibrates and bezafibrates, might be new candidates for the treatment of hepatic fibrosis. fibrosis is a common end-point in clinical trials of chronic hepatitis c. liver biopsy remains the gold standard for fibrosis evaluation. however, variability in fibrosis distribution within the liver (sampling variability) is a potential limit to liver biopsy. in order to assess the influence of sampling variability on the accuracy of liver fibrosis assessment with biopsy, we measured liver fibrosis on virtual biopsies of increasing length reconstituted from digitalized images of large liver sections using two different methods of fibrosis assessment. method large sections (3cmz) were performed from 17 surgical liver samples with chronic hepatitis c and various degree of fibrosis. measurement of fibrosis on the whole section was considered as the reference value. from the digitalized image of the whole section, virtual biopsies of increasing length (2.5 to 200mm) were reconstituted. fibrosis was assessed independently on each individual virtual biopsy using both image analysis and meta-vir score. results were compared to the reference value. results: a total of 10.659 virtual biopsies were studied. using image analysis, a strong dispersion of area of fibrosis was observed for virtual biopsies smaller than 4omm. only 22% of 1,5 mm length virtual biopsies had a measured of area of fibrosis equal to reference value 2 10%. this percentage increased progressively with increasing size (30%, 50%, 69% for biopsies of 25mm, 4omm, loomm length, respectively). using metavir scoring system, 65% of biopsies of 1,5 cm length were correctly categorized according to the score assessed on the whole large section. it increases to 75% for a 2,5 cm size without any substantial benefit for longer biopsies. a same trend was observed whatever the stage of fibrosis. conclusion: sampling variability of fibrosis is a significant limit for fibrosis assessment with liver biopsy. this study suggests that a length of at least 25mm is necessary to valuably evaluate fibrosis with semiquantitative score. sampling variability become a major limitation for more accurate method such as automated image analysis. (msi-1) , a rna-binding protein, is highly observed in developing central nervous system and thought to be a mammalian neural stemlprogenitor cell marker. we have reported that cells positive for musashi-1 (msi-1) protein appeared during spontaneous resolution of rat liver cirrhosis and that some msi-1-postive cells were also positive for matrix metalloproteinase (mmp)-13, possibly implicating active participation of stem cells in the resolution of collagen. though msi-1 antigen is expected to be expressed during very early stage of differentiation in the cellular lineage of liver, the type of cells appeared in the damaged liver and expressed msi-1 remain to be elucidated. the present study is designed to characterize the msi-1-positive cells in cirrhotic liver from the aspect of differentiation, significance and expected function of stemlprogenitor cells in the liver, especially fibrolytic function. methods: rat liver fibrosislcirrhosis was established by intraperioneal injection of cc14 twice a week. liver samples were obtained 2, 5 and 7 days after the last injection of 12-week cc14 intoxication. liver tissue samples were immunohistochemically stained for msi-1, c-kit, nestin, a-smooth muscle actin (a-sma), ck-19 and mmp-13. gene expression of msi-1 was also observed by reverse-transcription polymerase chain reaction (rt-pcr). results: neural stemlprogenitor cells, as defined by their expression of msi-1 andlor nestin, were not observed either in normal rat liver or after 4 weeks of c c 4 administration. rt-pcr analysis revealed very slight signals of msi-1 mrna at 2 days after discontinuance of 8-week ccld treatment. on the contrary, remarkable increase of msi-1 mrna was observed at 2 days after the 12 weeks ccl4 treatment, then the signals decreased gradually. immunohistochemical analysis showed that a considerable number of cells positive for msi-1 appeared in the early recovery stage from advanced liver fibrosis, especially at day 5 after the last injection of 12 weeks ccl4 treatment. expression of msi-1 in liver tissue was proceeded by and partly overlapped with that of c-kit, a marker of hematopoietic stem cells. some msi-1-positive cells observed around perivenular regions were as very small as oval cells, while the size of msi-1-positive cells present along the resolving fibrous bands varied and some of them expressed mmp-13. some ductal cells were positive for msi-1 but not for ck-19 examined in serial sections. msi-1 positive cells did not express hepatocytes markers such as albumin or afp. cells positive for a-sma and msi-1 were observed in some portions, but most of msi-1 expressing cells did not show positive staining for a-sma or desmin. at day 7, a few msi-1-positive cells were observed around the remaining fibrous bands. s-adenosyl-l-methionine represses the fibrosis, constitute a good model for studying the mechanisms responsible for antifibrotic therapy. to test whether administration of s-adenosyl-l-methionine, a precursor of glutathione and an agent shown to prevent liver toxicity in a variety of settings, could repress the activity of the mouse pro-alpha 2(i) collagen gene in hepatic stellate cells, homozygous transgenic mice harboring the -17 kb to +54 bp of the proximal promoter of the mouse alpha 2(i) collagen gene cloned upstream of the escherichia coli beta-galactosidase reporter gene (lacz) were used. chronic liver injury was induced by injecting intraperitoneally 5 mllkg of body weight of cc4 (25% vlv in mineral oil) three times per week for 4 weeks. s-adenosyl-l-methionine was administered intraperitoneally at a dose of 10 mglkg of body weight every day for 4 weeks. control groups were given either mineral oil or s-adenosyl-lmethionine alone. hepatocellular damage and protection by sadenosyl-l-methionine was confirmed by measuring serum levels of transaminases; s-adenosyl-l-methionine lowered alt levels in the ccl4-treated mice from 143 to 28 ull and ast from 171 to 66 ull (control values in the absence of cc14 were 26 and 58 ull for alt and ast, respectively). hematoxylin and eosin staining in the cc14-treated mice revealed the presence of mallory bodies, lymphocyte infiltration, centrilobular steatosis, and perivenular and pericellular fibrosis, and s-adenosyl-l-methionine minimized the pathology score. masson's trichrome staining showed less collagen deposition in mice treated with cc14 plus s-adenosyl-lmethionine than in the cc4-treated mice. histochemical analysis using x-gal staining allowed for the precise identification of the cell type in which the beta-galactosidase gene was active as driven by the pro alpha 2(i) collagen promoter. results indicated activation of the pro alpha 2(i) collagen promoter in mice treated with ccl, and repression of such activation in mice co-treated with s-adenosyl-l-methionine. immunofluorescence analysis of mice injected with cc14 revealed colocaliiation of alpha-smooth muscle and beta-galactosidase positive cells, suggesting that the activation of the promoter occurred only in hepatic stellate cells. these results suggest that one mechanism by which administration of s-adenosyl-l-methionine, a precursor of glutathione synthesis, could ameliorate liver fibrosis is by decreasing the responsiveness of the promoter of the alpha 2(i) collagen gene to a profibrogenic stimuli such as ccb. these transgenic mice should prove to be useful in further studies on how s-adenosyl-l-methionine exerts this repression of the alpha 2(i) collagen gene and perhaps to studies with other liver toxins such as alcohol. background endothelin-1 (et-i), the most powerful constrictor of the liver vasculature and stimulator of the synthesis, by kupffer cells, of a potent systemic vasodilator, platelet-activating factor, is also implicated in the fibrosis of lung, kidney and liver. thus increased hepatic concentrations of et-1 and its receptors in human and experimental cirrhosis suggest its major role in the pathology of chronic liver disease and its complications (fibrosis, portal hypertension and systemic hypotension). we investigated whether et-1 receptor antagonism, after the development of fibrosis and cirrhosis, arrestslreverses the progression of chronic liver disease. methods: chronic liver injury was induced in rats by cc14 treatment ( of the development of cirrhosis in group 1 (histopathology score of 3.7 ? 0.41 vs 4.8 2 0.16, p< 0.01) and reversal of cirrhosis in group 2 (histopathology score of 3.6 2 0.41 vs 5 2 0, p<o.ol) rats. this was accompanied by reduced a-smooth muscle actin-positive cells (activated stellate cellslmyofibroblasts) in the tak-044treated versus saline-treated rats receiving cc4. tak-044 treatment caused significant amelioration of portal hypertension (p<0.05 in both groups), systemic hypotension (p<0.05 in both groups) and liver injury (reduced activities of serum ast, alt and ldh), and improved hepatic synthetic capacity (increased serum albumin concentration) in both groups of rats relative to the vehicle-treated rats. tak-044 treatment reduced collagen synthesis as evidenced by decreased hepatic hydroxyproline, collagen-a type i mrna expression, and mrna and protein expression of a potent fibrogenic cytokine tgf-p1. conclusions: the results emphasize the role of et-1 in the development of cirrhosis and strongly suggest that blockade of its actions can be a rational therapy for chronic liver disease and its complications. subunits which are reported to be stimulatory. however p50 and p65 can form homodimers which have been described as inhibitors and stimulators of gene expression respectively. the aim of the study was to determine the role of the p50 subunit of nfkb in the progression of fibrosis. methods -chronic liver fibrosis was induced in p50 knockout and wild type control mice by administering twice weekly ccl4 injections for 12 weeks. livers and blood samples were harvested at day 1, 3, 5,7 and 14 after the last ccl4 injection. results -p50 knockout mice developed more severe liver disease as measured by serum alt's and peak fibrosis scores. the increased liver injury in p50 knockout mice was associated with a massive infiltrate of inflammatory cells into the liver compared to the control wild type mice. the majority of the inflammation was neutrophilic as measured by immunohistochemistry, however we also observed an increase in cd3 positive t cells. taqman quantitative rt-pcr analysis was used to measure collagen i, alpha-smooth muscle actin (asma) and timpl gene expression in the p50 knockout and wild type mice. all three genes were elevated in the mice laking p50 subunit of nfkb. immunostaining confirmed an increase in asma positive cells in the livers of p50 knockout mice compared to wild type controls. conclusion: the p50 subunit of nfkb may play an important role in protecting against the development of liver fibrosis and reducing liver inflammation in chronic liver injury. gnainsky, volcani center, bet dagan, israel; orna halevy, hebrew university of jerusalem, rehovot, israel; gadi spira, technion -israel institute of technology, haifa, israel; rafael bruck, wolfson medical center, holon, israel; arnon nagler, sheba medical center, tel hashomer, israel; mark pines, volcani center, bet dagan, lsrael introduction halofuginone is an inhibitor of hepatic fibrosis as demonstrated by prevention of dimethylnitrosamine (dmn) and thioacetamide (taa)-induced fibrosis. halofuginone is also responsible for the resolution of advanced fibrosis and increase in cirrhotic liver regeneration. methodslaims in this study atlas microarray technique was applied in vivo in an effort to identify genes involved in the halofuginone-dependent prevention of liver fibrosis. to that end we compared cdna array hybridization of taa-treated rat liver biopsies to biopsies of livers from rats treated with both taa and halofuginone. results from the 588 genes of the array, 13 were differentially expressed. one of the genes up-regulated by halofuginone was identified as protein tyrosine phosphatase of regeneration liver -1 (prl-l), an immediate early gene known to be induced during liver regeneration. the atlas microarray results were confirmed by northern blot analysis and by in situ hybridization. hepg2 cells in culture expressed high levels of prl-1 gene that was down-regulated upon serum starvation. halofuginone increased the prl-1 gene expression in the serum-starved cells in a dose and time-dependent manner. on the protein level, halofuginone caused translocation of prl-1 from hepg2 and huh-7 nucleus outer membranes to the cytosol as demonstrated by immunofluorescence using prl-1 antibodies. prl-1 translocation is known to be associated with alterations in cell cycle. halofuginone caused accumulation of hepg2 cells in the gym phase as analyzed by flow cytometry suggesting a putative g2im arrest. conclusions these results demonstrate the involvement of halofuginone in the hepatocytes cell cycle through prl-1 gene expression and its cellular translocation. nathalie senant, universitk victor segalen bordeaux 2, bordeaux, france; genevieve freyburger, hdpital pellegrin, bordeaux, france; toulouse, france; alexis desmouliire, universitk victor segalen bordeaux 2, bordeaux, france several lines of evidence incriminate the hemostasis system in liver fibrogenesis. first, experimental and human pathological data suggest a role for micro-thrombosis in fibrosis progression. secondly, several hemostasis proteins, such as thrombin, can regulate the expression of fibrogenic mediators through signaling by cell-surface receptors. the aim of this study was thus to evaluate the effect of direct thrombin inhibition on experimental liver fibrosis. fibrosis was induced in rats by administration of cc14 in olive oil orally 3 times a week for either 3 or 7 weeks. the thrombin antagonist ssr 182289 (sanofi-synthelabo research) was given daily orally at 30 mglkg (a dose that has been shown to significantly inhibit thrombin in vivo) starting 1 week after the beginning of cc14 intoxication. control groups received either the respective solvents or ssr 182289 alone. nine animals were used in each group. fibrosis was quantified with histomorphometrical analysis of sirius red-stained liver sections and expressed as a percentage of the surface of the section. we also measured the level of fibrogenic cell activation with quantitation of the area occupied by alpha-smooth muscle actin (asma)-positive cells. the levels of tissue inhibitor of matrix metalloproteinase-1 (timp-i) transcripts were analyzed by northern blot on total liver rna and expressed as timp-l/gapdh ratios following quantitative measurement with an instant imager. after 3 weeks of cc14, the area of fibrosis increased from 0.285 % 0.08 in controls to 2.62 i 0.68 % and was slightly but non significantly decreased by ssr 182289 (2.26 lr 0.62 %, ns). the area of asma-positive cells was however significantly decreased by ssr 182289 from 3.83 ? 1.04 to 2.86 i 0.79 % (p =0.03). in animals receiving cc14 for 7 weeks, treatment with ssr 182289 resulted in a decrease in fibrosis area from 7.01 t 0.24 to 4.62 t 0.77 % (p =0.05). similarly, the asma-positive area decreased from 11.57 5 5.57 to 7.71 i 3.89 % (p =0.05). the timp-i/gapdh ratio was decreased by ssr 182289 at 3 weeks (from 0.118 2 0.018 to 0.087 2 0.014, p =om). ssr 182289 alone had no effect on the measured parameters at both time points. additionally, it did not alleviate the acute toxicity of cc14 as shown by the levels of serum transaminases and the area of necrosis that were not significantly modified. altogether, these data provide evidence that thrombin antagonism can reduce the extent of liver fibrosis in this model. ongoing experiments should clarify whether this results from an anticoagulant effect or an inhibition of thrombin signaling effects. center, tel aviv, israel background and aims: proliferation and "activation" of hepatic stellate cells (hscs) and production of collagen are involved in the pathogenesis of liver inflammation and fibrogenesis. ligands of nuclear hormone receptors, such as triiodothyronine (t3) and peroxisome proliferator activator receptor y (ppary) ligands have been shown to affect fibrogenesis. levels of ppary are reduced in activated stellate cells and addition of the iigand increases ppary receptor expression and ameliorates fibrosis. in contrast, t3 has the opposite effect on liver fibrosis. low circulating t3 levels reduces liver fibrosis and cirrhosis. in this study, we investigated the in vitro effects of t3, ppary ligand and their combination on proliferation and activation of a rat hsc cell line. methods: transforming growth factor (tgf-pi) was used to induce myofibroblast-like phenotype in rat hscs (hsc-t6). activation of thyroid hormone and ppary receptors was induced with t3 and 15-deoxy-prostaglandinj(2)(pj2), respectively. signal transduction markers and activation marker a-smooth muscle actin were determined using western blotting. collagen i expression was measured by northern blotting. results: both t3 and pj (2) inhibit hsc proliferation induced by platelet-derived growth factor and by tgf-p. t3 inhibits cell proliferation by inducing apoptosis, while pj(2) reduces expression of proliferation marker cyclin d1. both "i3 and pj(2) decrease expression of activation marker a-smooth muscle actin. in addition, t3 strongly increases both basal and tgfeinduced collagen i expression. by contrast, pj(2) decreases collagen expression and inhibits t3-induced collagen production in tgfp-activated hscs. conclusion: both t3 and pj(2) reduce mitogen-induced proliferation of rat hscs, but have different effects on collagen i expression. these results suggest that prolierationlactivation and collagen i expression are two differently regulated processes in kato, sadakazu aiso, hiromasa lshii, keio university, tokyo, japan connective tissue growth factor (ctgf) is a profibrogenic molecule involved in several human fibrotic disorders including liver fibrosis. in human as well as experimental liver fibrosis, ctgf overexpression, through upregulation of ctgf mrna in hepatic stellate cells (hscs), is associated with the degree of fibrosis. it has been known ctgf is produced in many cell types in the live and exogenous ctgf modulates even the activation of hscs. although alcohol is the major cause of liver fibrosis, the role of ctgf in alcoholic liver fibrogenesis, has been poorly understood. cytochrome p-4502e1 (cyp2el)-mediated oxidative stress, as a result of chronic and excessive ethanol consumption, has been implicated as a crucial factor in alcoholic liver fibrogenesis. the goal of a series of our studies was to clarify the effect of cyp2e1mediated ethanol oxidation on ctgf expression. we previously reported the results of the assay using the well-established hepg2 cell line constitutively expressing cypzei, which was kindly provided by dr. a. cederbaum ( mt. sinai school of medicine, ny). ctgf mrna quantitation assay by a real-time reverse polymerase chain reaction (rt-pcr) showed a significant increase in ctgf mrna levels in ethanol-treated e9 cells in a time-dependent manner up to 36 hours after initial ethanol treatment. there was nearly a 2-fold increase in ctgf mrna levels when ethanoltreated e9 cells were coincubated with phorbol myristate acetate (pma), a reagent which enhances expression of cyp2e1. ctgf mrna levels were significantly reduced when ethanol-treated e9 cells were co-incubated with 4-methylpyrazole(4mp), a cyp2el inhibitor, or antioxidants, n-acetylcysteine and trolox. in the present study, we visualized the state of oxidative stress in ethanol-treated e9 cells by using mab5f6, a newly-established marker for oxidative stress. the mab5f6, which was generously provided by dr. k uchida (nagoya university, japan), was raised against the acrolein-modified keyhole limpet hemocyanin to verify the protein-bound acrolein in vivo. the staining, procedure was detailed elsewhere. immunohistochemical detection of proteinbound acrolein revealed e9 cells were stained when treated with ethanol. without ethanol treatment, no staining was observed in ethanol upregulates pro-fibrotic connective cells via cytochrome p-4502e1-mediated e9 cells even at a 24 -hour incubation period. moderate or no staining was observed when ethanol-treated e9 cells were coincubated with antioxidants. staining intensity by mab5f6 is associated with ctgf mrna levels in this cell line. collectively, these data suggest ctgf mrna upregulation in e9 cells seems to be mediated by the state of oxidative stress. our present study first linked cyp2el-mediated oxidative stress with ctgf gene regulation, thus suggesting a possible involvement of ctgf in alcoholic liver fiberogenesis. disclosures: sadakazu aiso -no relationships to disclose hiromasa ishii -no relationships to disclose mikio kajihara -no relationships to disclose (2) human livers with biliary fibrosis. methods: changes in ntp-dase2 expression in rat liver were determined in normal and 2-wk bile duct ligated (bdl) or 6-wk c c 4 rat livers and pflpm using confocal immunofluorescence, immunoblot, and northern blot. changes in ntpdase2 expression in human liver were determined in de-paraffinized liver biopsy specimens using confocal immunofluorescence. results: as was previously reported, ntp-dase2 was expressed in pf surrounding intrahepatic bile ducts in normal and sham bdl controls. in bdl rat livers, however, nt-pdase2 fluorescence was completely lost. no decrease in ntp-dase2 fluorescence was noted in cc14 rat livers within portal areas, although an increase in ntpdase2 fluorescence was seen in central areas. pf from sham bdl control animals expressed nt-pdase2 and the intermediate filament vimentin. "pf" from bdl animals did not express vimentin but were positive for a-smooth muscle actin (sma), suggesting that they were in fact pmf. most bdl pmf did not express ntpdase2; however, occasional cells were seen with sma and ntpdase2 in a pen-nuclear compartment. immunoblot analysis of pf revealed a marked decrease in ntpdase2 detection in bdl cells relative to sham bdl. no change was noted in ccll or c c 4 control cells. northern blot analysis revealed no differences in ntpdase2 mrna levels in sham bdl, bdl, ccl, control, or cc14 pf. confocal micrographs of normal human biopsy specimens revealed ntpdase2 co-localization with vimentin in pf surrounding intrahepatic bile ducts. however, ntpdase2 fluorescence was either markedly decreased or lost in specimens from patients with primary biliary cirrhosis. no loss of ntpdase2 fluorescence was seen in biopsy specimens from patients with hepatitis c. conclusions: ntpdase2 expression is specifically decreased in biliary fibrosis, largely through a posttranslational mechanism. "portal fibroblasts" from bdl rats are in fact portal myofibroblasts, which lose expression of ntpdase2. consistent with findings in rats, ntpdase2 expression is also specifically lost in human biliary fibrosis. work is now underway to determine whether loss of ntpdase2 in the peri-biliary compartment alters nucleotide signaling in bile duct epithelia. myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. we hypothesised that marrow derived myofibroblasts contribute to liver cirrhosis. methods. we sought to analyse the origin of myofibroblasts within cirrhotic liver in two scenarios: three male recipients of female liver transplants who have subsequently developed post transplant hepatitis c cirrhosis and a female recipient of a male bone marrow transplant who later developed hepatitis c induced cirrhosis. through the use of in situ hybridisation for the y chromosome we have been able to track cells of male origin. results. we have detected significant numbers of y chromosome positive cells in fibrotic areas. by combining the in situ hybridisation with immunocytochemistry these cells were found to be positive for vimentin, alpha-smooth muscle actin and negative for cd45. these cells were therefore determined to be of a myofibroblast phenotype. in the liver transplant recipients, 15.4% (corrected count 33.6%) of the myofibroblasts were y chromosome positive. in the female recipient of the male bone marrow transplant, 12.5% (corrected count 27.3%) of the myofibroblasts were y chromosome positive. conclusions. within all the livers analysed we found frequent myofibroblasts within and adjacent to the fibrotic bands that are of bone marrow origin indicating an extrahepatic cell contribution to the pathogenesis of human liver cirrhosis. (hsc) is the major fibrogenic cell of the liver and a key target of potential antifibrotic therapies. hepatocyte growth factor (hgf) is antifibrotic in several models of experimental liver fibrosis, but its mechanism of action is uncertain. potential use of hgf as a therapeutic peptide is limited by its size and need for parenteral administration. to overcome these potential limitations, refanlin has been developed as a synthetic small molecule sflhgf mimetic, that has demonstrated antifibrotic properties in renal fibrosis. aim: to study the in vitro and in vivo antifibrotic efficacy of refanlin. methods: serum starved lx2 cells, an immortalized human hsc line, were treated with 12 mglml of refanlin for 24 hours. proliferation studies using tritiated thymidine incorporation were performed. rna was isolated, and a panel of fibrosis markers (al-smooth muscle actin (asma), collagen i, bpdgf-receptor, 9, 14 (mmp2, 9, 14) , tissue inhibitor of metalloproteinase-1,2 (timp1,2), were assessed by real time rt-pcr analysis. cirrhosis was induced in sprague-dawley rats by if thioacetamide (taa) (200 mg/kg tiw) for six weeks. in the refanlin treated group, 1 mglkg of refanlin was administered five days per week for six weeks, concurrently with the taa. control rats received ip pbs five days per week for six weeks, along with taa. liver tissue was formalin-fixed and paraffin-embedded, then stained with 1% picrosirius red and computer morphometric analysis was performed for collagen quantitation using the bio-quanttm software. results: in cultured human stellate cells, there was a 60% decrease in asma, a 70% decrease in the expression of tgfb-receptor and mmp2, and a 50% decrease in timp2. there was no effect on proliferation. in vivo, fibrosis decreased by one metavir stage in rats treated with c6 and thioacetamide for 6 weeks. furthermore, c6 led to a significant decrease in portal pressure compared with non-treated rats with fibrosis. conclusions: refanlin may have antifibrotic efficacy through its inhibition or downregulation of tgfb receptor as well as mmp2. in vivo antifibrotic activity of refanlin is associated with reduced portal pressure. these experiments better define the potential targets of hgf's antifibrotic activity. ongoing studies in mechanistically distinct models of fibrosis will broaden our understanding of this agent's effects and point towards therapeutic trials in humans. kanto, aki sato, michiyo inoue, hideki miyatake, mitsuru sakakibara, takayuki yakushijin, chika oki, tetsuo takehara, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim hepatitis c virus (hcv) infection induces a wide range of chronic liver injuries. impaired thl response against hcv may be involved in hcv persistence, however, its precise mechanism is largely unknown. dendritic cells (dc) are the most potent antigen-presenting cells that play essential roles in initiating as well as regulating anti-virus immune responses. blood dc are grouped as myeloid (mdc) and plasmacytoid dc (pdc). with respect to their functions, mdc produces il-12 and tnf-a upon stimulation and drives thl response, while pdc releases considerable amount of type-i ifn upon virus infection and mainly skews th2. we previously reported that monocytederived dc is functionally impaired (kanto t. et al. j lmrnunol 1998) , however, the roles of blood dc subsets in the direction of thllth2 in hcv infection are still elusive. to address these issues, we compared the numbers and functions of mdc and pdc between chronically hcv-infected patients and uninfected donors. subjects and methods sixty-six hcv-positive patients (43 chronic hepatitis and 23 asymptomatic carriers) and 19 agematched uninfected donors were enrolled. mdc or pdc was determined by the patterns of lineage markers, hla-dr, cdllc and cd123. each type of dc was magnetically separated or sorted under facs vantage and was subjected to the functional analyses. the ability of dc to polarize naive cd4 t-cells towards thl or th2 was estimated by the pattern of ifn-ylil.-4/il-10 production from mdc-or pdc-primed cd4. results absolute numbers of mdc, pdc and their cd34+ progenitors are lower in hcv-infected patients than in control subjects, most significantly in those with chronic active hepatitis (mdc, pdc, cd34, median, patients vs. controls; 4.2,1.0,0.511*.1 vs. 6.9,3.0,1.9/pl, p<0.05). both types of dc from patients are impaired in the stimulation of allogeneic cd4 t-cells and the production of il-12 p70 or ifn-a compared with healthy donors. on encounter with nayve cd4 t-cells, mdc from the patients are less able to drive thl response, while pdc from them prime more il-10 producing t-cells than those from donors. exogenous il-12 significantly improved the patient mdcdriven thl response, however, it simultaneously increased il-10 producers both in mdc and pdc. in contrast, ifn-y or il-15 stimulated the patient mdc to drive thl without increasing mdc-or pdc-primed il-10 producing cells. conclusion both types of blood dc in chronic hcv infection are reduced and play decisive roles in the development of an impaired thl and an il-10-rich environment, thus favoring hcv persistence. ifn-y or il-15 is feasible to restore the altered t-cell polarizing ability of blood dc in chronic hepatitis c. hepatitis c virus (hcv) replicates its genome in a membraneassociated complex composed of viral nonstructural proteins and rna as well as altered cytoplasmic membranes and other as yet unidentified host cell components. a specific membrane alteration, designated membranous web, was recently identified as the site of hcv rna replication in huh-7 cells harboring subgenomic replicons (gosert et al. j virol 2003; 77:5487-5492) . here, we describe hcv replicons that allow the direct visualization of hcv replication complexes in living cells. methods: a transposon-mediated random insertion screen followed by selection of viable replicons was performed to identify sites that tolerate insertions in nonstructural protein 5a (ns5a). the green fluorescent protein (gfp) coding sequence was subsequently inserted into selected sites. rna was in vitro transcribed from subgenomic replicon constructs harboring these insertions, followed by electroporation into huh-7 cells and selection in the presence of g418. results: two sites in the c-terminal domain of ns5a were found to tolerate the gfp insertion and allowed efficient initiation and maintenance of hcv rna replication. expression of a ns5a-gfp fusion protein of the expected molecular mass could be demonstrated by immunoblot, indicating that the gfp was stably retained and that processing of the viral protein occurred normally. more important, expression levels were robust enough to allow direct visualization of this fusion protein by fluorescence microscopy. gfp fluorescence was found in a cytoplasmic reticular pattern and in brightly fluorescing dots, the typical distribution of hcv nonstructural proteins in replicon cells. by confocal laser scanning microscopy, gfp fluorescence was found to colocalize with other hcv nonstructural proteins in the brightly fluorescing dots, suggesting that these represent hcv replication complexes, previously identified as membranous webs. metabolic labeling of the nascent viral rna with brutp and (immuno)electron microscopy studies are underway to further validate these findings. moreover, live cell imaging studies using these replicons have successfully been initiated. conclusion: we have identified sites in ns5a that allow insertion of gfp in the context of functional hcv replicons and direct visualization of hcv replication complexes in living cells. these findings have implications for the structure and function of the hcv ns5a protein as well as for the architecture of the hcv replication complex. this system will allow to gain a dynamic view of the formation and turnover of hcv replication complexes and may be useful to select for cell populations permissive for hcv rna replication. infection. masahisa jinushi, tetsuo takehara, tomohide tatsumi, takuya miyagi, takahiro suzuki, tatsuya kanto, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim it has been generally accepted that the pertubation of cellular immunity plays an important role in establishment and persistence of chronicity in hcv infection as well as resistance to ifn therapy. accumulating lines of evidence have unveiled that dendritic cells (dcs), most potent antigen presenting cells, are impaired in patients with hcv infection, which may be involved in disturbance of anti-hcv immune responses. we recently reported that mhc class i-related chain a and b(mical b),ligands of nk activating receptor nkgzd, are induced on dcs upon interferonaand gain their ability to antivate nk cells; their expression is severely impaired in dcs derived from hcv-infected individuals (hcv-dc) (3. immunol. 170 1249 immunol. 170 -1256 immunol. 170 ,2003 . in the present study, we evaluated whether various types of cytokines have an effect for inducing micalb expression on hcv-dcs, thus resulting in triggering nk cell activation by dcs. methods dcs from healthy volunteers (n-dcs; n=15) or hcv-dcs (n=20) were generated from monocyte treated with gm-csf (1000 ulml) and il-4 (500 ulml) for 6 days. after stimulated with various cytokines (ifnalp, ifny, tnfa, il-12, il-15 or il-18) for 24 h, dcs were subjected to flow cytometric analysis of micalb expression. to investigate the involvement of each cytokine in mica/b expression on ifnalp-stimulated dcs, we employed elisa and rt-pcr to examine the cytokine expression (tnfa, il-lp, il-6, il-12~70, il-15 and il-18) in ifnalp-stimulated dcs, and also assessed micalb expression on dcs in the presence of neutralizing antibodies for these cytokines. to further address the stimulatory capacity of dc, allogeneic nk cells were cocultured with n-dcs or hcv-dcs at a ratio of 5:l for 24 h, and applied for flow cytometry of intracellular ifny expression, as well as 51cr release assay for the cytolysis of k562 target cells. in some experiments, masking antibody for nkg2d or micalb was added during nkldc cocultures, and then subjected to nk cell function assays described above. results micalb were induced on n-dcs, but not on hcv-dcs, upon iifnalp(% mic positive: n-dc 42.1% vs hcv-dc 3.4%; p<o.ol). in sharp contrast, micalb expression was increased on hcv-dcs at levels similar to n-dcs in the presence of of 5onglml il-15 (but not other cytokines) (%mic: n-dcs 49.4% vs hcv-dcs 47.6%). moreover, n-dcs, but not hcv-dcs, was capable of producing il-15 in response to ifnalp. little differences were detected in n-dcs and hcv-dcs for the production of tnfa, il-lp, 1l-6, il-12~70, or il-18. in addition, ifnalp-mediated micalb induction in n-dcs were completely inhibited in the presence of neutralizing antibody of anti-il15l il-15 receptorcu. even much lower doses (1nglml) of il-15 restored the ability of hcv-dcs to induce micalb and to activate nk cells in response to ifnalp. the ability of hcv-dcs for nk cell activation was largely diminished by adding the masking antibody of nkg2d or micalb. conclusion the current study demonstrated that il-15 acts as an indispensable mediator to induce micalb expression on ifnalp-stimulated dcs, and that hcv-dcs lack the ability to produce il-15 and to induce micalb expression in response to ifnalp. since defect in il-15 production is responsible for impaired nk cell activities by hcv-dcs, these findings shed light on the possibility that il-15 could improve the ifn-mediated anti-viral immune responses in chronic hcv-infected patients. the cellular immune response contributes to viral clearance as well as liver injury in acute and chronic hepatitis c virus (hcv) infection. understanding the relative immunodominance of hcvencoded peptides is important for understanding pathogenesis as well as potential vaccine candidates. we have developed a comprehensive and sensitive method to screen immune responses to all potential hcv epitopes in hcv-exposed individuals. methods: subjects with evidence of spontaneously resolved infection (eia+/hcv rna-neg, n = 15) and patients with evidence of chronic hcv infection (mean hcv rna level = 23.6 million copieslml; n = 15, all genotype la) had either leukophoresis or one-unit blood draw. we synthesized 750 peptides (15 mer-peptides overlapping by 11 amino acids) that spanned the entire hcv genotype l a polyprotein; responses to 33 pools of peptides were screened directly ex vivo by an ifn-gamma elispot assay. briefly, autologous dendritic cells (dcs) were generated from peripheral blood mononuclear cells selected by plastic adherence then cultured with gmcsf, il-4 and 10% hs for 5 d. dcs were pulsed with peptide pools to stimulate bead-purified cd8+ t cells. the remaining cd4+ t cells (5.0 x lo5 cells/well) were stimulated the same day with the peptide pools; 10 wells with no peptide (or siv peptides) were used as controls. only responses greater mean + 3 sds above controls were considered positive. the frequency and vigor of cd4+ t cell responses were statistically greater in resolved vs. chronic infection (mean 9.4 pools out of 33; median 8 in resolved vs. mean 3.86 pools; median 3.0 in chronics; p =.005). there was an inverse trend between # peptide pools with cd4+ t cell responses and hcv rna level. as shown in the figure, the hcv peptide pools differentially elicited responses; specifically, resolved infection was associated with greater cd4+ responses to pools core b, ela, ns3 protease-2, ns3 helicase-3, ns3 helicase-4, ns3 helicase-5, ns3 helicase-6, ns4b-2, ns5a-2, ns5a-3, ns5b-2. on the average, cd8+ t cells responded to 4.8 pools in resolved infection vs. 1.3 pools in chronics (p =.as). cd8+ t cell responses specifically directed against core 8, ns3 helicase-5, ns3-helicase 6, ns4b-2, ns5a-2 were more frequent in resolved as compared to chronic infection. conclusions: comprehensive mapping of hcv-specific immune responses by overlapping pools reveals significant differences in immunogenicity in resolved vs. chronic infection. in particular, regions core b (amino acids 96-203), ns3 helicase-5 (aa 1369-1479) and helicase-6 (1469-1579), ns4b-2 (1801-1899), ns5a-2 (2076-2191) induce both cd4+ and cd8+ t cell immunity and thus represent potential vaccine candidates. supported by nih r01 dk60590. sud, geoffiey c farrell, priyanka bandara, james g kench, storr liver unit, westmead hospital, westmead, nsw, australia; geoffiey w mccaughan, aw marrow gastroenterology (and liver centre, camperdown ns w, australia; jacob george, storr liver unit, westmead hospital, westmead, ns w, australia introduction: chronic hepatitis c virus (hcv) infection is associated with an increased prevalence of type 2 diabetes mellitus, which is known to accelerate fibrosis in nash. we hypothesized that viral-induced insulin resistance (ir) may be a mechanism for fibrogenesis in chronic hcv infection. methods and results: 1. to assess the influence of hcv infection on ir independent of any effect of hepatic fibrosis, 121 hcv subjects with minimal (stage 1) or no (stage 0) fibrosis were compared with 137 healthy volunteers matched by gender, body mass index (bmi) and waistlhip ratio. although the hcv subjects were younger than the healthy volunteers (p=0.004), they had significantly higher levels of markers of ir including fasting glucose (p=o.ol), insulin (p=o.o02), cpeptide (p<o.ool) and homa-ir (p=o.o02). 2. in 260 hcv patients (fibrosis stage 0 to 4), e examined the viral and diseaserelated factors (portallperiportal & lobular inflammation, viral genotype) which may be involved in the pathogenesis of ir in a multivariate model, controlling for other demographic and biochemical variables. by multiple linear regression analysis, independent predictors of homa-ir included bmi (p<o.ool), failed previous antiviral treatment (p<o.ool), portal inflammatory grade (p<o.ool) and genotype 3 status (p=o.ol). genotype 3 cases had significantly lower homa-ir than other genotypes at each stage of hepatic fibrosis. the adjusted difference in homa-ir between genotype 3 and non-genotype 3 was -0.58 (95% ci: -0.13 to -1.02; p=o.ol). 3. we then assessed if ir was associated with increased fibrotic severity. by multiple ordinal regression analysis, independent predictors for the stage of fibrosis in the 260 hcv subjects were homa-ir (or 1.3, p<o.ool), portal inflammatory grade (or: 5.3, p<o.ool), past alcohol intake (or 1.7, p<o.ool), age (or 1.1, p <0.001), alt (or 1.0, p=0.04), platelet count (or 0.93, p<o.ool), and serum cholesterol level (or 0.65, p=o.ool). as cirrhosis is known to cause ir, a separate multivariate analysis was performed for the 236 non-cirrhotic subjects. the independent predictors for fibrosis were unaltered and homa-ir remained in the model. in a subgroup of 117 patients with estimated duration of infection, multiple linear regression analysis showed that homa-ir was independently associated with an increased rate of fibrosis progression (p=0.03). conclusion: hcv infection induces insulin resistance irrespective of the severity of liver disease, and this effect appears to be genotype-specific. further, increased insulin resistance contributes to fibrotic progression. disclosures: priyanka bandara -no relationships to disclose geoffrey c farrell -no relationships to disclose jacob george -no relationships to disclose jason m hui -no relationships to disclose james g kench -no relationships to disclose geoffrey w mccaughan -no relationships to disclose background: approximately one half of hepatitis c virus (hcv) infected patients will fail to respond to current treatment regimens, pegylated interferon alfa and ribavirin. modeling of hcv rna decay in response to antiviral therapy can provide insight into viral and host determinants of viral response. aims: pharmacokinetic, pharmacodynamic, and immunologic outcomes were evaluated on 24 co-infected patients: 1) to evaluate the relationship between hcv rna decay and peg-ifn alfa2b serum concentrations in hivlhcv co-infected patients and 2) to evaluate association between hcv-specific immune responses and virologic outcome. methods: 24 co-infected, therapy-nake patients were treated with peg-ifn alfa2b 1.5pglkglwk and rbv 13 2 2 mglkglday. serum was obtained for hcv rna, hiv-1 rna and serum peg-ifn alfa2b levels at baseline, after the first dose (6,12, 24 hours) and on days 2, 3, 5, 6; after the second dose (6, 12, 24 hours) and on days 8,9; and after the third dose on days 14,15, and 16. peripheral blood mononuclear cells were obtained at baseline, weekly for the first month, and monthly thereafter. hcv and hiv-1 rna levels were determined by reverse-transcriptase polymerase chain reaction and peg-ifn alfa2b levels were measured by elisa. cd4+ cell proliferative responses to hcv antigens (core, ns3, ns4, ns5) and hiv-1 gag were performed at baseline, day 7 and 14, and weeks 4, 8, 12, and 24. among 24 co-infected patients, 21 are males, the mean age was 46 2 5.8 years, 8 are caucasian, 12 are african-american and 4 are hispanic. 20l24 patients were infected with genotype 1, 3/24 patients with genotype 2, and 1/24 with genotype 3a. results: pharmacokinetics: to date, 21 patients have been evaluated. 10/21 patients were end of treatment responders, but 2 of these were "late responders" (i.e. hcv rna declines after week 8 and is undetectable by week 24). data fitting during the first seven days revealed a free virion clearance rate c of 13.3 (day-l) and an infected cell clearance rate 6 of 0.21 (day-'). the half-lives were 2.7 hours and 4.0 days, respectively. pharmacodvnamics: viron production was blocked after the first dose of peg-ifn alfa with an average maximum effectiveness (emax ) of 89%. we estimated that the fifty percent effective concentration (ec50) of peg-ifn alfa2b, the theoretical in v i m drug concentration at which the drug efficacy is 5070, was five-fold lower in responders compared with non-responders while the maximum (cmax) and minimum (c,,,in) concentrations did not differ significantly ( table i ). the calculated minimum ( € , , , i n ) and maximum (emax) efficacy were also significantly increased in responders. immunolonic: the mean number of cd4+, cd8+ and cd56+ cells did not differ significantly at baseline or during treatment between responders and nonresponders. peripheral blood cd4+ hcv-specific proliferative responses did not differ significantly between responders and nonresponders except that the response to core antigen was increased in non-responders compared with responders at weeks 4 and 12. hiv gag cd4+ proliferative responses were stronger than hcv-specific responses at all time points evaluated. conclusions: the serum peg-ifn alfa2b concentration necessary for an end of treatment response is fivefold lower in responders compared with non-responders while there are no significant differences in the maximum and minimum peg-ifn alfa2b concentrations in responders and nonresponders. these data suggest that host factors responsible for a viral response may be different in responders compared with nonresponders, independent of serum peg-ifn alfa2b concentrations. is a significant predictor of liver disease severity in patients who are coinfected with hepatitis c virus (hcv). this accelerated liver disease is presumed to be due, in part, to hn-related immune suppression. hcv quasispecies variability is likely a marker of immune pressure on the virus. hyuotheses: 1. hcv quasispecies variability is decreased in those with hiv coinfection, and 2. treatment with highly active antiretroviral therapy (haart) is associated with increased variability, methods: hcv-infected patients with or without hiv were enrolled in a cross-sectional study. patients with genotype 1 infection and a parenteral risk factor for hcv were included. those with injection drug use (idu) were estimated to have acquired hcv 2 years after the onset of idu. alcohol use was determined using standard questionnaires. haart data were collected from the hiv clinic's database. only those with complete records were included. hcvquasispecies variability was determined using clonal frequency analysis of the second envelope gene hypervariable region. quasispecies heterogeneity was characterized according to complexity (the normalized number of unique quasispecies variants within a sample) and diversity (the average genetic distance between individual variants within the same sample). groups were compared using the student t-test for unequal variances, anova and the wilcoxon rank-sum test where appropriate. linear regression was used to assess the independent effects of hiv infection and haart on complexity and diversity. results: to date quasispecies data are available on 43 patients, 22 of whom are hivpositive. as expected, hiv+ subjects had significantly lower cd4 counts (mean 508 vs 956 cellslul, p<.oool). mean age and hcv duration were significantly lower in the hiv+ group (40.3 vs 45.5, p=.0017, and 15.7 vs 22.2 years, p=.oo18, respectively). the groups did not differ with respect to alcohol use over their lifetime or in the 6 months prior to enrollment. quasispecies complexity and diversity are detailed in the table below. while there was no significant difference among groups by anova (p=.16), the hiv+ subjects not on haart had less complexity and diversity than the other two groups, and differences between groups 1 and 2 and groups 2 and 3 were significant (table) . both hiv infection and haart were significantly and independently associated with complexity in a linear regression model; hiv infection predicted decreased complexity (p= .001), while haart predicted increased complexity (p=.007). in addition, higher alcohol use in the prior 6 months was independently associated with decreased complexity (p=.007). hiv infection and haart also were independently associated with decreased and increased diversity, respectively (p=.ool and.034). the cd4 count did not predict complexity or diversity, and the effect of haart appeared to be independent of the cd4 count. conclusion: hcv coinfection with hiv is associated with decreased hcv quasispecies complexity and diversity. while this effect is not completely reversed by haart, treatment of hiv is associated with significantly increased hcv complexity and diversity in our sample. furthermore, the effect of haart on hcv quasispecies variability appears to be independent of its effect on the cd4 count. these findings suggest that the immune pressure on hcv is enhanced in hcvlhn coinfected individuals who are on haart when compared to those who are not on haart. diversity" group (mean .t sd) (mean f sd) in the majority of cases, hepatitis c virus (hcv) becomes chronic and is associated with impaired viral-specific t cell immune responses; the mechanisms underlying viral persistence and lack of protective immunity are poorly understood. considering dendritic cells (dcs) play critical roles in initiating and modulating immune responses, we explored the effect of hcv proteins on dc genetic expression, phenotype, and function. methods: human dendritic cells (dcs) were generated following plastic adherence of monocytes from normal subjects and culture with gm-csf and il-4. autologous non-adherent cells were infected with vaccinia constructs expressing various hcv proteins (core, nssa, ns5b) or an irrelevant protein /3-galactosidase @-gal) as the control. following induction of apoptosis with uv irradiation, these vaccinia-infected cells were co-cultured with dcs. the phenotype, gene expression profiles and functions of the dcs were analyzed. dcs were evaluated after 8, 30, and 48 hrs of co-culture as well as after maturation with a cytokine cocktail (tnf-a, il-lp, pge2, il-6) for an additional 48 hrs. results: by facs analysis, we found no alteration in expression of co-stimulatory markers (cdso, cd86, cd40,) or mhc class i or i1 molecules. by elispot assay, we found that the recall response of cd4t t cells for cmv following pulsing of hcv-dc or control (p-gal)-dc was also not altered. however, between 2-10% of the genes probed in the bd clontech nylon array were differentially regulated. real time pcr has confirmed increased expression of scyc 1 (lymphotactin) in the presence of ns5a in 2 normals as well as 2 separate biological replicates. both clontech and affymetrix gene arrays revealed ns5a-induced increases in expression of il-8, a chemokine with pro-inflammatory properties. functional assays reveal that dcs which had engulfed apoptotic cells containing hcv core, ns5a, or ns5b demonstrated comparable ability to macropinocytose as measured by uptake of dextran-fitc or albumin-fitc when compared to p-gal-dcs. however, following uptake, the hcv-dcs died at a significantly greater rate than p-gal-dcs (-30%). conclusions: taken together, these results demonstrate that following engulfment of apoptotic cells containing hcv proteins, differential gene expression occurs in human dendritic cells, although the expression of mhc and costimulatory molecules and ability to stimulate memory (cmv) responses remain intact. the mechanisms underlying the surprising induction of death of dc-hcv in the macropinocytosis assay is currently under further investigation. institutes of health, bethesdu, md steatosis is a common histological feature of chronic hepatitis c virus (hcv) infection. the cause of hepatic steatosis in hepatitis c remains to be elucidated, but its association with obesity, diabetes and hyperlipidemia suggests an underlying metabolic dysfunction similar to that found in patients with nonalcoholic steatohepatitis (nash). additionally, recent data suggest an association between body mass index (bmi) and the degree of hepatic steatosis and fibrosis in hcv infection. insulin resistance (ir) is a key factor in the pathogenesis of nash and may play a similar role in the steatosis seen in chronic hepatitis c &: to compare the prevalence and severity of hepatic steatosis, visceral adiposity, j r and their relation to disease severity in african americans (aa) vs caucasians (ca) with genotype 1 hcv infection being considered for anti-viral therapy. methods: virahep-c is a prospective study of response rates to peginterferon and ribavirin in treatment-na'ive aa vs ca patients with genotype 1 hcv infection. all patients underwent liver biopsy within 18 months of enrollment. biopsies were read blindly by a central pathologist and scored for inflammation and fibrosis using the histologic activity index [i-iaii and for steatosis (0-4+) and features of steatohepatitis (zone 3 ballooning, zone 3 perisinusoidal fibrosis and mallory bodies). waist circumference and waist-hip ratio were measured as determinants of visceral adiposity. ir was determined using fasting glucose and insulin levels based upon the homa method. results: to date, 177 patients have been enrolled and 166 have complete liver biopsy information (75 aa and 91 ca). steatosis was present in 66 patients (40%) with similar rates among aa (39%) and ca (41% ca). grades of steatosis were also similar between aa and ca 1+ (38% vs 41%), 2+ (45% vs 27%), 3+ (14% vs 27%) and 4+ (3% vs 5%) (p = ns). patients with steatosis had significantly higher values for homa index (4.0 vs 2.4, p<o.ol), total hai score (10.3 vs 8.7, p<o.oool), hai inflammation score (8.5 vs 7.1, p<o.oool), waist circumference (40.2 in vs 36.5 inches, p<o.oool) and bmi (30.6 vs 26.7, p<o.oool). there were no significant differences in amount of alcohol consumption between patients with and without steatosis. spearman's non-parametric correlations were performed to assess associations between the ordinal variables (scores for fat, steatohepatitis ballooning degeneration + perisinusoidal fibrosis + mallory bodies], total hai, inflammation and fibrosis) and one continuous variable (homa index). waist circumference, waist-hip ratio, bmi, homa index, total hai, hai inflammation and hai fibrosis all correlated with both the fat score and steatohepatitis score (p<o.ol). median homa index levels were significantly greater among aa than ca (3.1 vs 2.7, p< 0.05), but step-wise logistic regression showed that race was not an independent predictor of steatosis after controlling for alt, ast, waist circumference, waist-hip ratio, bmi and homa index. conclusions: in patients with chronic hepatitis c and genotype 1, steatosis and steatohepatitis correlate strongly with the presence of insulin resistance and visceral adiposity. race is not an independent predictor of steatosis after controlling for other factors. bulk populations of antigen-presenting dendritic cells (dcs) contain myeloid dcs and plasmacytoid dcs. myeloid dcs polarize t lymphocytes to thl phenotype that produce interferon (1fn)-7. plasmacytoid dcs produce abundant amounts of interferon (1fn)-alpha. both ifn-?-producing t cells and adequate amounts of ifn-alpha are essential for viral clearance. in patients with chronic hepatitis c virus (hcv) infection, dcs are infected with hcv, which is presumed to have a role in viral persistence. however the functional implication of this has not been addressed. the aim of this study is to evaluate the functional capabilities of both myeloid and plasmacytoid dcs in patients with chronic hcv infection. myeloid dcs were defined as linage-, cdllc+, hla dr+ and cd123-cells, and plasmacytoid dcs as lineage-, hla dr+, cdllcand cd123+ cells among the peripheral blood mononuclear cells (pbmcs). the frequencies of myeloid and plasmacytoid dcs were measured in 64 patients with chronic hcv infection and 34 agematched control subjects by four-color flow cytometry. to assess the production of ifn-alpha by plasmacytoid dcs, these were cultured with graded doses of herpes simplex virus-1 (10-100 plaque-forming unitsldc). the frequencies of plasmacytoid dcs expressing intracellular ifn-alpha among total plasmacytoid dcs were counted by two-color flow cytometry. to evaluate the capacity of myeloid dcs to induce th polarization, dcs were cultured with autologous naive t cells for 7 days. polarization of t cells to thl phenotype was assumed when na'ive t cells expressed intracellular ifn-y. steatosis has been reported to occur in up to 50% of liver biopsies in patients with chronic hepatitis c virus (hcv) infection.risk factors for steatosis include alcohol abuse and those found in nonalcoholic steatohepatitis: obesity, increasing age, insulin resistance and hypertriglyceridemia. studies have shown an association between genotype 3 and hepatic steatosis in chronic hcv infection, and others have found an association between steatosis and fibrosis stage. previously published studies have been based primarily on cohorts of clinic referral patients and often have not analyzed for confounding variables. we herein report a population-based study of hepatic steatosis in hepatitis c. methods: in a study of 924 alaska nativestamerican indians with chronic hcv infection, 185 patients underwent at least one percutaneous liver biopsy as part of evaluation for treatment. all patients had a positive hcv rna by pcr and were negative for hbsag and hiv. all biopsy slides were reviewed by a pathologist blinded to patient identity, demographic, clinical and biological data. histologic activity was scored using the knodell system and fibrosis using the ishak system. steatosis was graded as 0, 1 (4 so%), 2 (30-65%), and 3 (> 65%). patients were analyzed for a) gender, age, estimated length of infection and bmi at the time of biopsy, b) genotype, current ethanol use, hcv rna within 1 year of biopsy, c) alt within 30 days of biopsy, and d) histologic activity and fibrosis found on biopsy, using univariable and multivariable analysis. results: steatosis was found in 79/185(43%) of liver biopsies; 62 (33%) had grade 1; 13 (7%) grade 2, and 4 (2%) grade 3. genotype distribution was as follows: 114 genotype 1 (62%), 44 genotype 2 (24%), and 27 genotype 3 (14%). steatosis was found in 39% (441114) of genotype 1,48% (21/44) of genotype 2, and 52% (14/27) of genotype 3 patients (p = 0.34). grade 2 or 3 was found in 6% (7/114) of genotype 1, 11% (5/44) of genotype 2, and 19% (5/27) of genotype 3 patients (p = 0.09). there was no significant association found between steatosis and gender (p = 0.93); age at biopsy date (p = 0.09); alt (p = 0.58); histologic activity (p = 0.27), viral load (p = 0.27) and estimated length of infection (p = 0.22). there was a significant association of steatosis with bmi (< 0.01), fibrosis score (p < 0.01) and current ethanol use (p = 0.03). bmi 2 30 occurred in 52% (42/81) of genotype 1, 39% (11/28) of genotype 2, and 27% (6/22) of genotype 3 patients (p = 0.10). after controlling for bmi, there was no significant relationship found between steatosis and genotype. for bmi < 30, 23% (9/39) of genotype 1, 47% (8/17) of genotype 2, and 50%(8/16) of genotype 3 patients had steatosis (p = 0.08). for bmi 2 30,60% (25142) of genotype 1,55% (6111) of genotype 2, and 67% (4/6) of genotype 3 patients had steatosis (p = 1.00). multivariable analysis was performed on 5 variables (p 0.25 in univariable analysis) and genotype. these included age (categories < 30,30-40,40-50, so+), bmi (< 30,30+), current ethanol use (yes, no), ishak fibrosis score (0-1, 2 21, and estimated length of infection (5 10, 11-15, 16-25, 25+ years) . a significant association with steatosis was found in 3 variables: ishak fibrosis score 2 2 (or 4.2, 95% ci 1.5-11.6, p = 0.005), bmi -> 30 (or 3.7, 95% ci 1.6-8.8, p = 0.003), and current ethanol use (or 2.5, 95% ci 1.0-5.8, p = 0.04) after adjustment for hcv infection length, which itself was not statistically significant (p = 0.82). conclusion: contrary to a number of previously reported studies, this population-based study did not find an association between steatosis and genotype, including genotype 3 in chronic hcv infection after multivariable analysis. we did find that ishak fi-brosis score, bmi and current ethanol use were associated with steatosis after adjustment for hcv infection length, but age and histologic activity were not associated. infection results in necroinflammatory liver disease characterized by the insidious progression of hepatic fibrosis and loss of functioning hepatocyte mass. a cell-mediated immune response with prominent lymphocytic infiltration of liver is thought to play a major role in pathogenesis, although little is known about the molecular mechanism underlying the liver injury associated with this viral infection. on the other hand, non-immune mechanisms have also been proposed as another mechanistic candidate for such sophisticated processes. the recent intensive interest is that the expression of hcv proteins seemingly alters lipid metabolism and transport in the liver in association with the reactive oxygen species (ros) mediated carcinogenesis, although the molecular basis of underlying mechanisms as well as responsible elements of hcv gene for this is yet to be established. thus, the aim of this study was to clarify whether hcv core protein expression directly alters lipid metabolism-relating enzymes to (cause steatosis, especially focusing on nuclear receptors and atp binding cassette transporter proteins expression. methods: 1. the complementary dna clone of full length hcv core (aa 1-191) was derived from serum of hcv i b patient by reverse transcription and nested polymerase chain reaction, and hcv-core expression plasmid (pcag-hcvcore) was prepared by a standard procedure. control plasmid was also prepared with p-galactosidase (pcag-lacz). 2. hepg2 cells were transfected with pcag-hcvcore or pcag-lacz, thereafter harvested for enzymatic triglycerides (tg) assay, hcv core antigen rneasurement by elisa at 24 h or 48 h. also, lipid metabolism-related enzymes mrna expression was estimated by semi-quantified rt-pcr; mitochondrial and peroxisomal fatty acids ,&oxidation, o-oxidation, abc transporters (mdr3, mrp2, bsep), microsomal tg transfer protein (mtp), ldl receptor, and nuclear receptors. resultss: 1. hcv core expression was evidenced at 24 and 48 h at the similar level. 2. hepatic tg was increased in hcv core expressing cells, along with down-regulation of carnitine palmitoyl transferase 1 a(cptla), a precious protein in liver mitochondrial fatty acid@-oxidation (50% at 24 h, 85% at 48) and up-regulation of acyl-coa oxidase 1 (acol), a precious protein in peroxisomal fatty acidso-oxidation (130% at 48 h). cyp4a11, a precious protein in microsomal o-oxidation, and mtp, a precious protein in vldl assembly, were not detected or unchanged. superfamily 2 (sf2) helicases and helicase-like proteins share 6 conserved motifs. alignments reveal that several additional conserved motifs are present in the sf2 helicase encoded by the hepatitis c virus (hcv). the roles of two such motifs are examined here using structure-based site-directed mutagenesis. the first motif (yrgxdv) forms a loop that connects sf2 helicase motifs 4 and 5, at the tip of which is arg393. when arg393 is changed to ala, the resulting protein retains a nucleic acid stimulated atpase but cannot unwind rna, unwinds dna poorly, and does not translocate on ssdna. dna and rna stimulate atp hydrolysis catalyzed by r393a like the wildtype but the mutant protein binds dna more weakly than wildtype both in the presence and absence of a non-hydrolysable nucleotide analogue. thus, this "arg-clamp" motif anchors the protein on nucleic acid enabling processive unwinding. the second motif (dfsldptf) forms a beta-loop between sf2 motifs 5 and 6 that connects domains 2 and 3. when f444 in this "beta-arm" is changed to ala, the resulting protein is devoid of all activities. when f438 is changed to ala, the protein retains nucleic acid stimulated at-pase, but unwinds dna and rna poorly. in this case, uncoupling of atp hydrolysis and unwinding is due to the fact that the f438a mutant does not release dna upon atp binding like the wildtype. the f438a mutant also has a lower melting temperature than the wildtype indicating the hydrophobic pocket formed by the beta-arm and residues in domain 3 stabilizes the protein. data support an inchworm model for helicase action and identify two new potential sites for rational hcv drug design. this work was supported by the aasld liver scholar award from the american liver foundation. tat is an early gene product of hiv-1 that is essential for replication and viral gene expression. this transactivating protein is secreted by hiv-infected cells and taken up by neighboring cells. tat modulates expression of specific cellular genes and may be a key player in the interactions between hiv and other infections. one of the most common coinfections observed among hiv patients is hepatitis c virus (hcv). if hiv tat has an effect on hcv replication, this could help explain the rapid development of liver disease and cancer among hiv patients. aim this study was designed to test the hypothesis that tat alters hcv replication. methods: soluble tat was added to the media of huh-7 cells containing a hcv replicon. replicon replication was quantitated using a ribonuclease protection assay. using an ltr-luciferase plasmid to transfect hela cells, we developed a series of controls outlining the relative oxidative state of the tat protein. results exposure to tat protein in the reduced state substantially increased hcv replicon replication. oxidized tat was found to have no significant effect on the replication of the replicon. the increase was dependant on length of exposure to tat and the concentration of tat. present work: further assays seek to define the nature of the effect by tat on hcv replication. conclusion: tat may upregulate hcv replication directly or indirectly, and thereby play a role in modulation of hcv infection and pathogenesis. elucidating the complex interactions between hiv and hcv will be critical in evaluating and developing workable treatments for the increasing number of coinfected hiv/hcv persons with liver disease. acknowledgements: this research was supported by nih grants ai34764, ai54626, ai54238, ca54576 and ca89121. we have found previously that expression of hepatitis c virus proteins in cell lines or in the liver of transgenic mice inhibits interferon alpha induced signaling through the jak-stat pathway (heim et al., j virol, 1999, 73:8469 and blindenbacher et al., gastroenterology, 2003, 1241465) . the aim of the present study was to investigate if the same inhibition takes place in livers cells of patients with chronic hepatitis c. from february 2001 to april 2002, all patients with chronic hepatitis c referred to the outpatient liver clinic of the university hospital basel were asked for their permission to use part of the liver biopsy for this study. for non-hcv controls, patients that underwent ultrasoundguided liver biopsies of focal lesions (mostly metastasis of carcinomas) were asked for their permission to obtain a biopsy from the normal liver tissue outside the focal lesion. the protocol was approved by the ethical commission of basel. written informed consent was obtained from all patients that agreed to participate in the study. after removal of a 20 to 25mm long biopsy specimen for routine histopathological workup for grading and staging of the liver disease, the remaining 5 to 20 mm long biopsy cylinders were immediately incubated in pbs or pbs with human interferon alpha (lo00 iulml) for 10 to 60 minutes at 37°c. the samples were then used for the preparation of cytoplasmic and nuclear extracts and in some cases for immunofluorescence studies. in a first part, the ex vivo stimulation of biopsy tissue was validated. biopsy samples of patient with different degrees of fibrosis were incubated for 0, 10, 20,30 and 60 minutes, and the nuclear translocation (a surrogate marker for activation) of statl was visualized by immunofluorescence. we found a transient activation of stat1, with a peak after 20 minutes of stimulation with interferon alpha. the signal was shut down in all biopsies after 60 minutes. interferon alpha diffused readily in the biopsy cylinders regardless of the degree of fibrosis. in the second part, 44 consecutive biopsies of patients with chronic hepatitis c and 12 consecutive control biopsies of patients who underwent ultrasound-guided biopsy of focal lesions in otherwise healthy livers (mainly liver metastasis of carcinomas) were used for semiquantitative assessment of interferon alpha induced statl activation using gel shift assays. we found a signhcant inhibition of statl dna binding in patients with chronic hepatitis c compared to controls. as in our previous work with hcv protein expression systems, statl phosphorylation was not impaired in human liver biopsies from patients with hepatitis c. we conclude that interferon induced intracellular signaling can be analyzed semi-quantitatively in human liver biopsies ex vivo by gel shift assays. using this newly validated method, we found that signaling is impaired in liver biopsies from patients with chronic hepatitis c. the block in stat signaling is at the level of dna binding in the nucleus, whereas stat activation at the interferon receptor functions normally. methods we studied the intrahepatic and peripheral hcv-specific cds+ t cell response in a cohort of 15 hla-a2 positive chronically hcv infected patients by tetramer staining, intracel-m a r ifngamma staining and cfse labeling. in addition, viral amino acid sequences corresponding to the four ctl epitopes used in the study were deduced by nucleotide sequence analysis to assess the potential role of viral escape variants. results (1) substantial higher numbers of hcv specific cd8+ t cell responses were detectable in the liver compared to the peripheral blood, suggesting compartmentalization at the site of infection. in addition, intrahepatic cds+ t cells were multispecific whereas peripheral hcv specific cds+ 'i' cell responses were primarily monospecific. these results suggest, e.g., the persistence of hcv-specific t cells at the site of disease or the direct priming of t cells in the liver. (2) epitope-specific cd8+ t cells that were detectable in peripheral blood as well as liver were characterized by a good peripheral proliferative capacity after peptide specific stimulation suggesting that proliferation is a prerequisite of peripheral virus-specific cd8+ t cells to accumulate in the liver. this is further supported by the observation in one patient that the lack of proliferation of virus-specific cd8+ t cells in the peripheral blood was associated with the absence of the same cds+ t cell response in the liver. (3) despite the strong accumulation of hcv specific cd8+ t cells in the liver, the majority of those cells was unable to secrete cytokines after peptide specific stimulation indicating that dysfunction of intrahepatic hcv specific cd8+ ' i cells might contribute to viral persistence. importantly, ifn gamma producing tetramer positive cd8+ t cells were only detectable in patients with low viral titers or in patients with high viral titers but with sequence variations in the corresponding viral epitope, suggesting viral escape. the relative contribution of t cell dysfunction versus viral escape to viral persistence is not known. it is important to note, however, that both mechanisms can operate within the same patient. insulin resistance (ir) is a frequent feature in chronic hepatitis c while risk factors of steatosis are body mass index in patients infected with genotype 1 and viral load in those infected with genotype 3. in patients with chronic hepatitis c, we adressed the following issues: 1) is ir the cause or consequence of steatosis and fibrosis ? 2) what are the risk factors of ir ; 3) does ir play a role (and to what extent) in the occurrence of steatosis ? 4) is ir involved in the progression of fibrosis ? therefore, this study was designed to assess relationships between ir, steatosis and fibrosis according to hcv genotypes in non diabetic patients. methods: 152 non-diabetic patients with biopsy proven non-cirrhotic chronic hepatitis c had fasting serum glycemia and insulinemia measurements. ir was evaluated by using homa model assessement. 4 groups of patients were defined according to hcv genotypes (1 or 3) and degree of steatosis (absent or mild vs moderate to severe). results: the 4 groups were similar in terms of age, sex ratio, bmi and disease duration. prevalence of ir (homa higher than1.64) was significantly higher in genotype 1 patients with steatosis than that of other patients (77% vs 27,23 and 21% respectively, p=o.oool). ir was significantly associated with extensive fibrosis in genotype 1 patients (p=0.008) but not in genotype 3 patients. among genotype 1 patients, independent parameters associated with ir were age (p=0.002) and steatosis (p=o.o3) but not fibrosis. independent risk factors for steatosis in genotype 1 and genotype 3 patients were dr (p=o.o4) and viral load (p=0.02) respectively. steatosis was associated with significantly higher fibrosis score whatever the genotype (p=o.o1). among genotype 1 patients, the median progression rate of fibrosis was significantly higher in those having steatosis and ir together than in other patients (0.1 vs 0.05, p =0.02). conclusions : in non-diabetic patients with non-cirrhotic chronic hepatitis c 1) steatosis and fibrosis are associated with ir in genotype 1 patients but not in genotype 3 patients, 2) among genotype 1 patients, ir mainly depends on age but not fibrosis 3) ir is a major risk factor of steatosis in genotype 1 patients, 4) the combination of steatosis and insulin resistance is a risk factor of fibrosis progression. these results suggest that ir is not the consequence but rather the cause of steatosis and fibrosis progression. . however, in the first month of life, in 4 of these 13 newborns, hcv rna was only detected in pbmcs and not plasma. in 2 neonates, the sscp band patterns of pbmc-derived viral sequences were different from maternal sequences in serum or pbmc; however, they were identical to hcv rna negative strand amplified from mothers' pbmc. the latter strongly suggests that the infection was transmitted through maternal infected pbmcs. another infant harbored different hcv strains in serum and pbmc; both strains were present in the mother's serum. only 7 of 13 hcv-rna positive children developed hcv antibody at one year. hiv infection was found in 2 out of 13 hcv rna-positive and in 6 out the 35 hcv rna-negative infants (ns). conclusions: we have found that 1) hcv strains infecting neonates may be derived from strains residing in maternal pb-mcs. 2) hiv+ pregnant women who were hcv rna positive in pbmcs were more likely to transmit hcv to their infants. these data suggest that one mechanism for perinatal transmission of hcv is maternal-fetal transfusion of hcv-infected pbmcs late in pregnancy or during labor and delivery. additionally, hcv infected neonates may have a limited ability to develop hcv antibodies in the first year of life and thus there may be an underestimation of the prevalence of hcv infection by anti-hcv determination among children born to hcv rna positive mothers. (hcc) in this clinical setting is very high. however, the molecular mechanisms of how hcv related proteins may contribute to hcc is not well defined. hcv core protein, a viral structural protein, has been implicated in tumor development. the goal of this study was to characterize the transcriptional regulation induced by core protein expression with respect to generations of a malignant phenotype. meth0ds:a cdna fragment encoding hcv core protein of l b genotype was subcloned into the ptre2hyg vector and co-transfected with ptet-on vector (clontech) into a human huh 7 hepatoma derived cell line using polyamine (mirus) as a carrier. stably transformed clones were selected by growth in culture medium containing g418 and hygromycin. clones positive for hcv core protein expression were screened by immunoblotting from cells grown in the presence and absence of tetracycline. one clone with core expression tightly regulated by tetracycline was chosen for further analysis. cell proliferation was evaluated by a modified mtt assay, at 6,12,24, 36 and 48 hours after induction of core protein expression. twenty-four hrs after hcv core expression, the cellular transcriptional changes were analyzed by microarray analysis using human genome u133 array sets (affymetrix). the microarray data were validated by real-time pcr on selected genes. results hcv core protein expression was tightly regulated by the presence of tetracycline and the protein levels were dependent on the amount of the tetracycline present in the culture medium. core protein expressing cells showed significant increase in growth compared to non-expressing cells at 6,12, 24,36 and 48 hours after addition of tetracycline indicating a proliferation stimulus provided by core protein. microarray analysis using human gene chip u133 revealed that 1072 of 39,000 genes were significantly changed (> 3 fold), with 626 up-and 446 down-regulated. this screen was quite informative since 52 genes involved in regulation of cell proliferation (38 genes) and apoptosis (14 genes) were changed respectively. conclusions: enhanced growth of liver-derived cells by hcv core protein is due to cellular transcriptional changes induced upon core expression; two major molecular pathway(s) are involved 1) those involved in celi growth control and 2) those involved in cell survival. such gene regulation by hcv core protein is important to the molecular pathogenesis of hcc daudi-cd4 cells were infected with pnl4-3, a t-tropic hiv molecular clone. 24 hours later, uninfected and hiv-infected cells were incubated with either 10% high titer hcv-positive patient serum, 10% hcv-negative patient serum, or 10% fbs (no human serum). infections were continued for 3,4,5, and 6 days; cells were harvested, stained with annexin v and propidium iodide, fixed, and apoptosis was measured by flow cytometry. annexin v is a marker of early apoptosis, and propidium iodide indicates cell death either by apoptosis or necrosis. results: six high titer hcv-positive sera and six hcv-negative sera were tested in five separate experiments, and in each case, the percentage of viable cells was higher in the hivihcv-coinfected cells compared to those infected with hiv only. to further elucidate dynamics of the course of coinfection, we did a time course consisting of harvests every eight hours beginning at day 3 (the earliest point at which apoptosis had been observed in prior experiments) and continuing until day 6 plus 16 hours. the results of the five harvests from day 5 plus 8 hours to day 6 plus 16 hours are shown in the figure below. the percentages of viable cells and those undergoing apoptosis are shown as measured by flow cytometry. during this 32-hour interval the percentage of viable (negative for apoptosis) cells fluctuated very little in untreated cells, or in cells incubated with hcv-positive serum, or even in hn-infected cells incubated with hcv-positive serum. in striking contrast, hiv-infected cells that were incubated with hcv-negative patient serum dropped to only 16% viability by the end of the 32 hours, compared to 64% viability in the hiv-infected cells incubated with hcv-positive patient serum. this effect was not observed when apoptosis was induced by either of two chemical inducers of apoptosis in daudi cells: peroxisome proliferator-activated receptor gamma ligand and calpain inhibitor 11. next, we sought to determine if the effect of apoptosis inhibition was dependent on hcv particles. when h ninfected cells were kept separate in culture from hcv-infected cells by a membrane that prevented virus passage, apoptosis in the hn-infected cells was still inhibited. conclusions: collectively, these results suggest that in our experimental conditions: 1) hcv-positive sera, but not hcv-negative sera provide a protective effect to hiv-induced apoptosis in daudi-cd4 cells, and 2) a cellular factor induced by the presence of hcv, rather than hcv particles themselves, is responsible for the inhibition. methods: serum and pbmc samples from an individual with acute hcv-infection (genotype l a ) were collected every two to three months over a time period of 20 months beginning 2 months after infection. pcr amplification of vrna using a set of overlapping primers for the entire hcv genome was performed and pcr-products population sequenced using an abi automated sequencer. cd8+ t cell responses were defined using an ifngamma elispot assay using an overlapping synthetic peptide set spanning the whole hcv-genome (genotype la). based on the hla-type the complete viral genome was also screened for putative cds+ epitopes using a web based epitope prediction program (syfpeithi). results: elispot screening with the whole hcv-genome peptide set detected only two ex-vivo ifn-gamma responses (ns3 1073-1081 and ns5b 2594-2602), although neither epitope was associated with the development of mutations. however, over the 20 month period of observation we detected a total of 25 mutations throughout the genome (2 in el, 4 in e2,3 in ns2,5 in ns3, 2 in 353a ns4 and 9 in ns5), in addition to multiple changes in the hypervariable regions. interestingly, only five of these mutations resided within known hla-restricted optimal epitopes. however, 17/25 mutations (68%) were located within predicted epitopes based on known hla-binding motifs of the subject. in order to begin to define the rate at which these mutations develop an intermediate time point of 12 months was also sequenced. interestingly, 19/25 (76%) of the mutations had already occurred by this time. it is likely that at least some of these mutations are the direct result of immune pressure exerted through cd8+ t lymphocytes. we are currently generating peptide-specific cds+ t cellines against these regions exhibiting viral evolution, providing an opportunity to determine which of these regions are associated with previously undescribed cd8+ responses. conclusions: hypothesizing that evolving mutations in hcv might be the result of immune pressure by ctl, longitudinal full length sequencing of hcv may represent a powerful tool to define additional hcv-specific cd8+ t cell responses, especially those exerting substantial selective pressure. using this approach we were able to identify a number of candidate regions where cd8+ t cell responses may be present and driving viral evolution. determining the extent to which viral escape from cd8+ responses occurs during hcv infection will elucidate its overall impact in preventing proper control of hcv. indicate that the main open reading frame of hcv contains rna structures in the corelarfp (alternate reading frame protein) and nssb (polymerase) genes. we hypothesized that one or more of these structures are cis-acting replication elements (cre)s. methods. we used custom software, thermodynamic rna folding programs, and classical comparative phylogenetic analysis to build secondary structural models. we then used directed mutagenesis in the subgenomic replicon system to seek stem-loop elements in ns5b that are required for viability. the mutations we introduced maintained the sequence of the polymerase protein, i.e., they were silent, synonymous codon substitutions. structural probing was carried out on replicon rna. rnase t1 and lead (ii), and nuclease v1, were used to identify loops and helices, respectively. results. mutations in ns5bsl3.2 blocked replication, indicating that this novel structure is an essential cis-acting replication element (cre the aims of this study were: 1) to correlate mean alt level with hcv rna by pcr positivity or negativity, 2) to compare clinical, demographic and risk factor data as well as viral load and genotype between persons with pnalt, persistently elevated alanine transaminase (pealt) and fluxuating alanine transaminase (fluxalt) who had at least 3 years of data available. methods: for each person in the cohort from whom 2 2 pcr results were available (n=278), we calculated the mean level of all alts measured over a 3-year period after enrollment. for a subset of the cohort, we selected persons who met the following criteria: 2 positive pcr tests 2 1 year apart with the first positive pcr occurring prior to date of the first alt and a minimum of 6 alt levels measured over the subsequent 3 years with a minimum interval of 1 month between alt measurements (n=129). we defined a person as having pnalt or pealt when 2 5 of 6 alt levels were normal or elevated, respectively, during the 3-year follow-up period. persons who did not fit into either of the above 2 categories were defined as fluxalt. we reviewed clinical data via chart review, demographic and risk factor data via interviews, as well as data on viral load (performed by branched dna assay version 2.0) and genotype (restriction polymorphism analysis background: hcv infection rarely presents acutely, but when it does it offers a potential early "window" for effective therapy. it has been suggested that such therapy is efficacious due to enhanced activity of t cell responses, which are most active during acute disease. methods: 8 subjects with acute hcv infection were comprehensively mapped for cd8+ t cell responses with an interferongamma elispot assay using 301 overlapping peptides, spanning the entire hcv polyprotein. responses were confirmed by establishing peptide-specific t-cell lines and intracellular cytokine staining. the screening for hcv-specific cd8+ t cell responses was repeated during and after therapy. responses detected in the screening assay were longitudinally studied before, during and after therapy using single peptide elispot ,as well as tetramer assays. we also studied cd4 proliferative responses over time in some subjects using recombinant hcv proteins. results: cd8+ t cell responses were detected in 6/8 subjects before therapy was initiated and were typically multispecific, with up to 5 epitopes targeted. after the start of therapy, frequencies of hcv-specific cd8+ t-cells declined following suppression of hcv viremia. therapy also did not increase the breadth of the hcv-specific response as no additional specificities were detected at later timepoints, when elispot screening was repeated. in 5/8 subjects viral relapse occurred after therapy was stopped, with 2 subjects being retreated successfully. relapse was not predicted by the presence or absence of hcv-specific cd8+ t-cell responses. during viral relapse, a vigorous expansion of pre-existing hcv-specific cd8+ t-cells was observed for most specificities, with single responses reaching almost 3% of cd8+ t cells as measured by tetramer staining. these responses were unsuccessful in containing hcv. in the two subjects who were retreated after viral relapse, we observed the identical pattern of declining cd8+ t cell responses as in the first treatment course. in contrast to cd8+ t cell responses, cd4 proliferative responses usually became more vigorous after virus was suppressed on therapy. however, such responses also did not protect from viral relapse. conclusions: these data suggest that although multispecific functional cd8+ t cell responses may be present during acute disease, this does not predict a successful outcome of antiviral therapy. rather than boosting cd8+ t cell responses, therapy and viral suppression appear to lead to their attenuation, even though cd4+ t cell responses may be restored. figure) in the fibrous septum of portal tract in cirrhotic liver. significantly less fltl immunopositivity occurred in nd liver. hepatocytes were negative for fltl. conclusion: the greater de of genes involved in immune activation, fibrosis, cellular proliferation and cell signalling indicated that these processes are more active in the cirrhotic liver that has developed hcc than the cirrhotic liver without hcc development. plgflfltl signalling has an important role in neo-vascular development within organs. the decreased expression of plgf and its receptor flt 1 in cirrhosis with hcc implies that vascular proliferative signals although normally active in cirrhosis itself may be decreased in cirrhotic livers with hcc. prospective analysis of cirrhosis prior to hcc development is needed to indicate whether these findings represent a premalignant phenotype. background: hepatitis c virus (hcv) infection often leads to chronic liver diseases including liver cirrhosis and hepatocellular carcinoma (hcc). at least 10 hcv proteins have been identified, which serve as viral structural and non-structural proteins required for viral replication and virion formation. several viral proteins have been implicated in hcv persistence and the development of hcc. we have previously reported that the ns2 protein inhibits gene expression f+om various cellular and viral promoters in liver and non-liver derived cells. thus, expression of endogenous cellular proteins was significantly reduced in ns2 expressing cells. in this regard, the ns2 protein was recently found to be an inhibitor of the pro-apoptotic cide-b protein and therefore may contribute to hepatic oncogenesis. to understand the molecular basis of repressed gene expression, we constructed successive deletion mutants of the ns2 protein and tested their effect on luciferase expression driven by cmv promoter. meth-ods: the cdnas encoding the full-length (1-217), n-terminal part (1-91) and internal parts of ns2 were generated by pcr and cloned into the pcr3.1 vector for expression under control of the cmv promoter. a liver-derived huh-7 cell line was co-transfected with a plasmid encoding the luciferase reporter gene and plasmid encoding various deletion mutants of the ns2 gene. inhibition of gene expression was measured by luciferase activity assay at day two after transfection. cell viability was also evaluated as well. results: deletion constructs 61-121, 61-111, 71-121 and 71-111 exhibited a significant inhibitory effect on luciferase activity comparable to the fulllength ns2 protein, whereas the deletion constructs 81-111 and 81-121 lost the inhibitory effect. therefore, the minimal amino acid residues required for the inhibition of gene expression by this viral non-structural protein was mapped to residues 71-111. interestingly, this region partially overlaps with the binding site of the ns2 to a newly identified cide-b pro-apoptotic protein. il12 plays an essential role in the antagonism of t helper 2 differentiation and in the induction of antiviral host defence. we postulated that genetically determined attenuation of il12 production could provide a plausible immunological mechanism able to determine outcome of hcv infection and have investigated a recently described polymorphism in the il12b gene. methods: we have extracted genomic dna from whole blood taken from 196 hcv antibody positive patients. 124 patients had chronic hcv with detectable hcv rna and 72 had spontaneously resolved infection, testing hcv rna negative on several occasions. genotyping for a single nucleotide polymorphism (snp) at position (a16974c) on the ill2b gene was performed using polymerase chain reaction and restriction digest. maximal in-vitro il-12 production by cultured mononuclear cells in response to sac (staph aureus cowan) stimulation was determined in 24 cases by elisa. results: of the hcv rna positive cases 81 (65%) were homozygous for the 'a' allele, 39 (31%) heterozygous 'ac'; and 4 (4%) were homozygous 'cc' whereas of the hcv rna negative cases 36 (50%) were 'aa', 33 (46%) were 'ac'; and 3 (4%) were 'cc'. hcv rna negative cases were significantly more likely to be heterozygous for lac' than hcv rna positive cases (p=0.04). of the 24 patients studied for il-12 production, 13 were genotype 'aa' and 11 'ac'. 13 were hcv rna positive (8 genotype lac' and 3 'aa') and 11 hcv rna negative (3 genotype 'aa' and 3 'ac'). maximal il-12 production with sac stimulation was lower in the 11 genotype 'aa' cases (mean 104 units) than in the 13 genotype 'ac' cases (mean 228 units) (p= 0.08). hcv rna positive cases produced significantly less il-12 than did hcv rna negative cases (mean 84 units compared to 266 units, p= 0.016). comparison of hcv rna positive or negative cases only, revealed a trend for higher maximal il-12 production with genotype 'ac' regardless of hcv outcome. conclusion: cases heterozygous for the variant 'c' allele at position 16974 of the il-12 p40 gene are significantly more likely to be hcv rna negative than those homozygous for the 'a' allele. recent data found carriage of the variant 'c' allele to be associated with greater il-12 production capacity and our data from a small number of cases supports this. genetically influenced enhancement of il-12 production appears to be a factor influencing the outcome of hcv infection. we have previously demonstrated that hepatitis c virus (hcv) core protein expression in huh-7 hepatoma cells increased reactive oxygen species (ros) derived from mitochondria without inducing apoptosis. the aim of this study was to investigate whether hcv core protein inhibits the rosassociated apoptosis induced by deoxycholic acid (dca). methods:: we measured ros level and 8-hydroxy-2'-deoxyguanosine (8-ohdg) content, and evaluated apoptosis in the presence or absence of dca (500 pm), using a human hepatoma-derived cell line with tightly regulated hcv core protein expression under the control of a tet-offrm promoter. cells were incubated with 5pm of chloromethyl2',7'-dichlorodihydrofluorescein diacetate for 30 min for measurement of ros. cellular 8-ohdg content was quantified with enzyme-linked immunosorbent assay. fragmented nuclei were assessed with 4',6-diamidino-2-phenylindiole staining and dna fragmentation was evaluated by genomic dna laddering. the degree of apoptosis was quantified with enzyme-linked immunosorbent assay. we also examined whether the general caspase inhibitor (zvad-fmk) inhibited the ros-associated apoptosis induced by dca. in some experiments, cells were incubated with 500wm of ursodeoxycholic acid (udca) in addition to dca. the experiments were repeated 3 or 4 times. results:: strong core expression was detected 72 hours after withdrawal of tetracycline from the culture medium. the expression of core protein increased the basal ros level (1.6 2 0.17-fold, p<0.05) and 8-ohdg content (1.13 i 0.005-f0ld, p<0.05) of huh-71191-20 cells without inducing apoptosis. dca stimulation produced a 4.0-fold increase in ros content in the presence of core expression and a 2.5-fold increase in the absence of core expression. similarly, the 8-ohdg content was significantly increased by dca regardless of hcv core expression (1.13-fold, p=0.02 for core expression, 1.15-fold, p=o.o1 for core non-expression). nevertheless, hcv core protein significantly suppressed the ros-associated apoptosis induced by dca (w0.05). also apoptosis induced by dca was almost completely inhibited by zvad-fmk. udca significantly decreased the ros (p<0.05) and the 8-ohdg content (p=o.oos) in the core-expressing cells and attenuated dca-induced apoptosis. on-treatment virological response (otr) was defined as complete loss or greater than 100-fold drop in hcv viremia within 3-6 months of therapy by quantitative or qualitative rt-pcr (roche cobas amplicor v2.0), based on recommended early virological testing for continued therapy. forty-six patients with at least 3 months of continued antiviral therapy were examined thus far, including 30 with genotype 1 (cl) and 16 with either genotypes 2 or 3 infection (c2). on-treatment response (otr) was achieved in 29/46 (63%) patients overall, including 47% among c1 and 94% in c2 groups. positive and negative control groups included 19 healthy hcv seropositive but nonviremic subjects without history of antiviral therapy ("recovered") and 23 healthy hcv seronegative volunteers, respectively. hcv-specific cd4 t cell response was examined using recombinant hcv core, ns3l4, ns5 and control proteins in standard proliferation and ifn-gamma (ifng) elispot assay at baseline (pre-treatment) and at 1, 3 andlor 6 months during antiviral therapy. results: hcv-specific cd4 t cell response was significantly greater in the recovered compared to the chronic patients, consistent with its expected role in natural hcv clearance. among the chronics, c2 patients (genotype non-1) displayed a baseline t cell response to hcv ns3/4 that was modestly but significantly greater than c1 patients ( the progression of fibrosis in chronic hepatitis c virus (hcv) infection differs among individuals and determines the ultimate prognosis and thus the need for therapy. the molecular mechanisms associated with the progression of fibrosis are poorly understood. gene expression profiling technologies allow the analysis of gene networks whose expression is associated with specific pathological conditions. we used real-time quantitative rt-pcr assays to compare the mrna expression of 148 selected genes in liver biopsies of controls (n=10 normal liver samples) and of 57 untreated patients with chronic hcv infection and different stages of fibrosis according to metavir, i.e. stage flal (n=11), f1a2 (n=9), f2a1 (n=10), f2a2 (n=lo), f3a2 ( n = l l ) and f4a2 (n=6). in order to limit the number of pcr experiments, we first studied total mrna pools which were prepared by mixing amounts of individual liver biopsies mrna of each group. this pooled sample analysis allowed the selection of genes displaying significant different expression (> 2-fold variation) in comparison to "normal livers". the selected genes were then studied at the individual level and their diagnostic performance was assessed using roc curves. the most informative genes were used to construct specific gene expression signatures. we identified several genes specifically involved in various stages of fibrosis. the dysregulated genes mainly encoded extracellular matrix proteases, growth factors, cytokineslchemokines, and ifncu-induced genes. we also observed a statistically significant association between hcv rna amount (as determined with the same real-time quantitative rt-pcr technology) and expression of several genes, mainly ifn-a-induced genes including stat1, ifi27, mix1, oas2 and gip2. understanding the correlates of protective immunity in this setting is an important first step in the development of potential vaccine candidates. methods: two recipients of frozen patellar allograft tissue procured from the same donor developed evidence of acute hcv within 6 months of surgery. in retrospect, the donor was confirmed to be hcv antibody negative but hcv rna positive (genotype la). both patients were enrolled in a prospective study of t cell immunity requiring whole unit blood draw at baseline, 2, 4, 6 and 12 months. comprehensive hcv-genomewide analyses were determined by ifn-y elispot responses to 33 overlapping peptide pools that spanned the entire hcv polypeptide (genotype la, 3010 aa, total 750 peptides). peptide responses were defined as greater than mean plus 3 sd compared to 10 control wells. results: patient 1 (51 yo wf) cleared the virus spontaneously within 6 months; when first evaluated, the pt. demonstrated cd4+ t cell responses to 8 of 33 (24%) peptide pools, with effector frequencies as high as 1 in 5,000 (to ns3helicase-7, amino acids 1569-1667). ifn-y cd8+ t cells were detected following stimulation with 6 of 33 (18%) pools (highest frequency to pool ns5b-5, aa 2829-2927). remarkably, 6 months later, the repertoire of the hcv-specific cd4 t t cell response had expanded further, and patient 1 demonstrated responses to 13 of 33 (39%) pools. in contrast, patient 2 (49 yo wm) demonstrated persistent viremia (4.4 million copieslml, bayer assay) and lacked cd4+ t cell responses to any peptide pools when first evaluated; only one peptide pool (ns3-helicase-5, aa 1369 -1478) elicited responses in cd8+ t cells. despite virologic clearance with antiviral treatment (pegylated interferon and ribavirin), elispot screening failed to reveal emergence of new cd4+ or cd8+ t cell responses (6 months later). conclusions: comprehensive analyses of hcv-genome-wide cd4+ and cd8+ t cell responses reveal significant differences in patients exposed to the same hcv innoculum and correlate with spontaneous clearance versus chronicity. in hcv infection that resolves spontaneously, the repertoire and strength of the hcv-specific immune response may continue to expand in the first year after infection (and may target more than one-third of the hcv polypeptide). in contrast, the profile of t cell responses remains narrow, weak, and constant in the acute infection that becomes chronic, even after successful antiviral therapy. alcohol consumption exacerbates liver injury in chronic hepatitis c and enhanced oxidative stress is one possible pathophysiological mechanism. we have previously developed a hepatoma cell line with conditional, stable expression of hcv core protein and constitutive expression of cyp2e1. these cells demonstrate dosedependent ethanol toxicity when core protein is expressed. the aims of this study were to determine whether reactive oxygen species (ros) production and mitochondrial dysfunction are responsible for ethanol-induced cytoxicity. methods: huh-7 cells with conditional expression of core protein and constitutive expression of cyp2e1 (l14 subclone) were exposed to 0.1 micromolar tbooh and/or ethanol (25 mm) for up to 24 hrs. cytotoxicity was measured by mtt assay or trypan blue exclusion. ros production was assayed with the oxidation sensitive fluorescent dye dcfda. mitochondria1 membrane potential was analyzed by flow cytometry using the dye jc-1 as a probe. apoptosis was detected by cellular dna content analysis with flow cytometry. results: in the presence of 0.1 pm tbooh, there was a progressive increase in cell death in huh-7 cells expressing no heterologous proteins (2 2 2%), cypzel only (12 2 l%), core only(l5 2 5%), or core plus cypzel (3927 x). addition of 25 mm ethanol to core/ cyp2e1 expressing cells further increased cell death to 5326%. dna content analysis and nuclear morphology showed that this type of cell death represented necrosis and not apoptosis. effects on mitochondrial membrane potential were also examined. under control conditions, only 250.5% of cells had depolarized mitochondria. expression of corelcyp2el and exposure to tbooh depolarized mitochondria in 47211% of cells. similar to cell death, ethanol (25 mm) addition increased depolarization to 7027% of cells. compared to control cells, core protein increased ros by a factor of 1.220.3, and the combination corelcyp2el by 2.150.3. furthermore, cells expressing corelcyp2el ampiified the effect of exogenous tbooh. incubation with tbooh (0.1 pm) had no effect on ros content of control cells. it approximately doubled the ros content of cells expressing either core or cyp2e1 and it increased ros content of cells expressing both corelcyp2el by 4.120.5 fold. in all cases the increase in ros was completely blocked by the antioxidant n-acetyl cysteine (20 mm). the antioxidant also completely blocked both mitochondrial depolarization and cytotoxicity. conclusions: hcv core protein and cyp2el synergistically enhance ros production in hepatoma cells, amplify the oxidative stress produced by extracellular ros, and sensitize cells to mitochondrial depolarization and necrotic cell death caused by alcohol. since all these effects are blocked by antioxidants, the formation of ros is likely to be the primary event which subsequently produces mitochondrial dysfunction and cell death. sponse associated with different outcomes of hcv infection may provide important insights into our understanding of hcv pathogenesis. for this purpose, we compared the functional features of hcv-specific cd8 cells in patients with chronic hepatitis c (23 patients, ch), chronic asymptomatic carriers of hcv with persistently normal alt and positive serum hcv-rna (12 patients, as) and in subjects with resolved hcv infection, either spontaneously (8 subjects, sp) or following anti-viral treatment (12 subjects, rt). functional analysis was carried out with 5 highly immunogenic peptides corresponding to ns31073-1081 , ns31406-1415 , ns41812-1821 , ns41992-2000 , ns52627-2635 , containing previously identified hla-a2 restricted epitopes. since the different hcv genotypes were represented in the different groups of patients in different proportions, different sets of peptides designed on genotype 1, genotype 2 and genotype 3 sequences were synthesized and used to reproduce more closely the sequence of the viruses infecting each individual patient and responsible for in vivo priming of the cds response. the immunological parameters tested were: a) frequency of hcv specific, cd8+ t cells by tetramer staining, both ex-vivo and after in vitro stimulation with synthetic peptides; b) ifn-7 production by intracellular cytokine staining; c) cytolytic activity by a standard %hromium release assay. the results of our study indicate that subjects recovered from hcv infection following treatment show lower ex-vivo frequencies of circulating hcv-specific cd8 cells compared to ch patients but their cells are able to expand and to produce ifn-7 more efficiently after in-vitro stimulation. indeed, hcv-specific t cell lines were induced in 83% of rt subjects and in 74% of ch patients; moreover, ifn-7 production was induced in 100% of rt subjects compared to 67% of ch patients (p<0.05). in the group of subjects spontaneously recovered from infection, ex vivo cd8 frequencies were below the threshold of tetramer detection; however, peptide stimulation in vitro was able to expand tetramer+ cd8 cells and to induce ifn-7 production in 37% and 63% of the subjects, respectively. these lower levels of reactivity of sr subjects compared to tr patients are likely due to the different time elapsed from recovery, which was approximately 3 years in tr subjects but much longer in sp subjects, who had recovered even decades before the time of immunological analysis. finally, the group of as patients showed the lowest levels of response to hcv, in terms of both frequencies of hcv-specific circulating cd8 cells and cytokine secretion. in conclusion, the hcv-specific, cd8-mediated response has different features in patients with different outcomes of infection, showing a hierarchy of efficiency declining from subjects recovered after treatment who displayed the best responses, to chronic hepatitis patients, subjects recovered spontaneously and asymptomatic hcv-carriers, who appear to be the least responsive as a likely result of a deeper condition of tolerance to hcv. hepatitis c virus (hcv) infection is the most frequent cause of chronic liver disease in western countries and it has been involved in the development of cirrhosis with the risk of hepatocellular carcinoma. cyclooxygenases (cox) are crucial enzymes in the biosynthesis of prostaglandins and cox-2, the inducible isoform, has been implicated in inflammation, fibrogenesis and carcinogenesis. to address whether cox-2 may play a role in hcvrelated hepatic inflammation and fibrosis, we determined the cox-2 expression pattern in the liver tissue from 27 patients with hcv-induced chronic hepatitis (low histological activity=22, high histological activity=5) and 5 with end-stage cirrhosis, searching for correlations between the expression level of this enzyme and the histological activity of liver disease as well as with the intrahepatic expression and activity of metalloproteinases (mmps). we also investigated whether structural and non-structural hcv proteins may promote cox-2 expression in the human hepatocytederived cell line ccl13. western-blot and rt-pcr analysis for cox-2 demonstrated that cox-2 expression levels were higher in mild chronic hepatitis (mch, 2.6 fold), severe chronic hepatitis (sch, 3 fold) and cirrhosis (2.4 fold) than in normal liver. moreover, pge2 levels were also higher in liver samples from patients with sch (1.9 fold) and in those with cirrhosis (3.3 fold) than in normal liver. besides other cell types, hepatocellular cox-2 immunoreactivity was markedly observed in hcv cirrhosis, and although some cox-2 positive hepatocytes were observed at the edge of hepatic lobules, the majority of them were mainly restricted to the regenerative nodules. in contrast, none or few cox-2 expressing hepatocytes located in periportal areas were observed in patients with mch and sch, respectively. interestingly, we found a significant correlation between the intrahepatic expression and activity of cox-2 and mmp-2 and -9 in all the histological groups of patients analyzed. cox-2 mrna, protein and activity was de novo induced in resting ccl13 cells stably transfected with hcv core and ns5a, and this effect was higher (1 fold and 2.5 fold) when activated with cytokines and phorbol esters, respectively. in conclusion, our results provide evidence that a virus-induced hepatocellular cox-2 upregulation could mediate important pathogenic events in the course of chronic hcv infection, suggesting that specific cox-2 inhibitors might be useful for chemoprevention and therapy of hcv-related liver disease. were nahe to hbv. thl responses to hcv proteins ns3, ns4 and core were sought by elispot. as a control thl responses were also sought to hbsag, where results were analysed according to recovery from hbv infection or nayve to hbv. thl responses to each of the 5 proteins was performed before and after depletion of t-regs using anti-cd25 (>80% depletion). results: following treg depletion thl responses were revealed de novo in 3/12 and enhanced in 8112 patients with ns3,2 and 9 with ns4 respectively and 4 and 7 with hcv core respectively. the overall increase in the thl responses with t-reg depletion was significant for both hcv core (median increase 13-fold p = 0.004) and ns3 (5backaround: in both chronic viral hepatitis b and c viral persistence is thought to be due to an inadequate antiviral t cell response, which in turn may be caused by inappropriate priming of t cells by dendritic cells. an important subset of peripheral blood dendritic cells, the plasmacytoid dendritic cell, has been identified to be the major interferon-alpha producing cell in humans. since both hepatitis b and hepatitis c infection can be successfully treated by exogenous interferon-alpha, an impairment of endogenous interferon-alpha producing cells is an attractive hypothesis for the pathogenesis of chronic viral persistence. methods: patients with acute symptomatic ( n = l l ) and chronic hepatitis c (n=25), sustained responders to interferon-alpha therapy (n=22) and patients with acute hepatitis b (n=lo) as well as healthy controls (n=20) were included in this study. plasmacytoid dendritic cells were stained for facs-analysis in peripheral blood mononuclear cells with antibodies against bdca-2 and cd4 and by exclusion of lineage marker positive cells (cd3, cd14, cd16, cd19). in addition pdc were stained with various activation and maturation markers (e.g. cdso, cd83, cd86). production of interferon-alpha was measured by elisa after stimulation with cpg. results: in acute hepatitis c, ifn-alpha production was about 45-fold reduced as compared to healthy controls (15 pg/50000 pbmc vs. 701 pg150000 pbmc). this reduction was caused by both a significant reduction in absolute numbers of pdc (3.8/~1 vs. 8.4/pl, p=0.0032) as well as by a reduction of ifn-alpha production per cell (0.19 pg/pdc vs. 3.45 pg/pdc; p=0.0004). in chronic hepatitis c, ifn-alpha production was still reduced by over 50%, although absolute numbers of pdc were similar to healthy controls. following spontaneous or treatment induced viral clearance both number and ifn-alpha secretion of pdc were not different from healthy controls. importantly, in acute hepatitis b, which runs a self-limited course in the majority of cases, also the ifn-alpha production was reduced (21 pg150000pbmc). this reduction was also caused by both the reduction of absolute pdc count (5.291~1, p=o.l) as well as by the ifn-alpha secretion per pdc (0.32 pg1pdc; p=0.0007). using a panel of dc activation and maturation markers we did not find any evidence of a more immature or more activated phenotype of pdc in acute hepatitis c. exogenous ifn-alpha administration during acute hepatitis c led to a further reduction of ifn-production by pdc. conclusions: acute viral hepatitis has a dramatic impact on frequency and function of plasmacytoid dendritic cells in the peripheral blood, indicating that pdc play an important role during this phase of viral hepatitis. however, no differences were found between patients with self-limited vs. chronically evolving acute hepatitis c or patients with acute self-limited hepatitis b. future studies have to clarify whether the reduced ifn-alpha secretion by pdc contributes to viral persistence or whether this is rather part of a physiological response to acute viral disease. it has been suggested that hepatocellular steatosis, a frequent histological feature of chronic hepatitis c, is more frequent in hcv genotype 3 infection, and disappearance of steatosis in patients who clear hcv genotype 3 infection after antiviral therapy suggests a possible direct role of this hcv genotype. in order to assess the direct causal role of hcv in steatosis, we studied the relationship between hcv rna load and steatosis according to the hcv genotype. methods: we studied 176 patients with chronic hepatitis c (genotype 1, n = 83 ; genotype 3, n = 93). the serum hcv rna level was measured at the time of liver biopsy by means of a third-generation "branched dna"-based assay (versant hcv rna 3.0 quantitative assay, bayer diagnostics). steatosis was graded as absent, mild (< 10% of hepatocytes), moderate (10 to 30% of hepatocytes) or marked (> 30% of hepatocytes). daily alcohol intake during the 6 months prior to liver biopsy, and the body mass index (bmi), were recorded. results: steatosis was more frequent in patients with genotype 3 infection than in those with genotype 1 infection (84.9% vs 74.7%, ns). steatosis was significantly more severe in hcy genotype 3 than in genotype 1 infection (moderate or marked in 54.8% vs 25.3%, p=o.oool). in patients infected by genotype 3, the severity of steatosis was significantly related to hcv rna load but not to alcohol intake or bmi. in contrast, in patients infected by hcv genotype 1, the severity of steatosis was not related to the hcv rna level but was significantly influenced by alcohol intake and bmi. bivariate analysis demonstrated the effect of the hcv genotype on the association between the severity of steatosis and viral load in genotype 3-infected patients, and between the severity of steatosis and exogenous metabolic factors in genotype 1-infected patients. multivariate analysis showed that : (i, in patients infected by hcv genotype 3, only hcv rna load was independently related to the severity of steatosis (odds ratio (or) = 2.5, 95% confidence interval (ci): 1.4-4.4; p = 0.001), (ii) in patients infected by hcv genotype 1, bmi higher than 28.0 kglm2 (or = 5.8, 95% ci: 1.3-26.9; p = 0.016), alcohol intake exceeding 30 glday (or = 29.6, 95% ci: 3.2-272.0; p = 0.009), and histological grade (or = 20.0, 95% ci: 2.6-152.0; p = 0.001) were independently related to the severity of steatosis. conclusion: our results suggest : (i) steatosis is a cytopathic lesion induced by hcv genotype 3 ; (ii) hcv genotype 1 is not steatogenic per se or at the usual in vivo expression levels, and liver steatosis is a feature of associated steatohepatitis in these patients. the effect of hcv genotype 3 sequences and the role of their expression levc4s must be tested in vitro, in order to better reflect the in vivo situation. in animal models, steatosis is associated with oxidative stress and lipid peroxidation which is known to promote fibrogenesis. this study was aimed to assess in patients with chronic hepatitis c whether steatosis contributes to fibrosis through oxidative stress. methods: markers of lipid peroxidation and antioxidant status were measured in blood from 192 chronic hepatitis c patients and age and sex matched healthy controls. intrahepatic levels of these markers were also assessed in a subgroup of 23 patients and controls. results: the lipid peroxidation marker malondialdehyde was significantly higher in the blood and in the liver of patients compared to controls (p=0.05 and p=o.o01 respectively) while the antioxidant glutathione peroxidase was significantly lower (p=0.05 and p =0.0001 respectively). the antioxidant superoxide dismutase was significantly higher in the blood and lower in the liver compared to controls (p=o.o01 and p=0.002 respectively). autoantibodies to soluble liver antigen (sla) are specific for autoimmune hepatitis. the molecular characterization of the sla antigen has allowed the establishment of highly sensitive and specific radioligand assays. to determine serum anti-sla a specific radioligand assay was used. total rna was isolated and reverse transcribed from hepg2 cells. the cdna encoding sla was amplified by pcr and used as a template to express sla protein eukaryotically in a tnt coupled reticulocyte lysate system (promega corporation, southampton, uk). anti-sla was measured retrospectively in 16 consecutive patients (10 girls, median age at transplant 52 months; range 8-160) with dn-aih, in whom sera were available before transplant, 1,2,6,12 and 24 months post-transplant and at the time of the diagnosis of dn-aih. eight patients had biliary atresia, 2 alagille syndrome, 2 cryptogenic cirrhosis, 2 alpha-1-antitrypsin deficiency, 1 druginduced acute liver failure and 1 bsep deficiency. the patient with acute liver failure did not have pre-transplant serum specimen stored. twelve patients had developed smooth muscle andlor antinuclear antibodies, two anti-liver kidney microsomal (one atypical) and two anti-mitochondria1 antibody. before transplantation, anti-sla was negative in 13/15 cases, the two positive patients having cryptogenic cirrhosis and biliary atresia. post transplant, anti-sla remained positive in these two and became positive in further 9 patients. of these, in 7 patients anti-sla became positive at a median of 17 months (range: 1-115) before the clinical diagnosis of dn-aih. in 5 children anti-sla became detectable as early as 2 months post-surgery. in 6 patients anti-sla levels were highest at the time of the clinical diagnosis of dn-aih. the presence of anti-sla in dn-aih supports the autoimmune nature of this condition; the frequent appearance of anti-sla before the clinical manifestation of the disease makes it a potential predictive marker for development of dn-aih. all 38 patients showed positive response to the therapy, with respect to remarkable release of severe meteorism, active diet, and significant improvement of liver and kidney functions. however, no difference was presented in the markers of electrolytes, blood routine and blood gas analysis before and after the mars, while the effects on serum levels of alanine aminotransferase, aspartate aminotransferase and y-gt were remained uncertain since the alt decreased from 941 ull to 690 ullduring a in conjunction with observations from other centres, our data show that mars treatments resulted in a ramarkable removal of bilirubin, uric acid, bun, cr and ammonia, which could therefore decrease the toxic effects that higher concentrations of these compounds exert on liver and kidney function and could thus contribute to improvements in multiple organ dysfunctions. we found additionally that the higher concentration of serum toxin before detoxication therapy, the more efticacy removal could be achieved which presents mars could be {of therapeutic results even in the very serious cases. it remains questionable whether some of useful substances characted in low molecular weight such as trhlthyrotrophin-releasing factor), gnrh), adh(anti-diuretic hormone and calcitonin will be also removed by albumin dialysis, whether the added synthesis function is necessary for an artificial liver support, and whether this encouraging survival rate applies to long term outcome remains open for further investigation. scoring system has recently been introduced to determine priority for organ allocation in liver transplantation (lt). there is limited available data on resource utilization in the post-meld era.aims: 1. evaluate the effect of the meld system on lt-associated resource utilization and post-lt survival. 2. compare the costs of lt in the periods before and after the implementation of meld. methods : patients undergoing lt at our center in the 6 months following meld implementation(cases) were compared to those undergoing lt in the immediately preceding 9-month period (controzs). the outcome parameters studied were: 1) resource utilization: defined as ( there were no significant differences between the two groups in terms of age, sex distribution or presence of hepatocellular carcinoma (hcc). the average meld among cases was 25.9, compared to 24.1 in controls (p =0.19, ns). overall los was similar in the two groups. however, pre-lt los, especially in the intensive care unit (icu), was significantly higher in the pre-meld or control group. the pre-lt cost was also higher in this group(p=0.05), translating into significantly higher total costs of lt in the pre-meld period (p=o.ol). the need for post-lt hd, pmv and pressors was no different between the two groups. this data is summarized in table 1. the meld score was found to be significantly correlated with bile d u d damage is a maior feature in liver graft rejection. ductopenia (dp), defined as loss of more than 50%-of bile ducts, is a major hallmark of chronic liver graft rejection (cr), which usually leads to graft failure within the first post transplant year. in a previous study we have shown that in patients who developed dp and cr a deficient proliferative response of canals of hering (coh) was present in the preceding biopsies showing acute rejection (ar) when compared with a control group who experienced ar but did not progress to cr. these findings support the postulation that coh represent a regenerative compartment of the biliary unit in the liver. aim of the present studv: to analyze whether preservation of coh might contribute to the reversibility of dp. patients and methods we studied 2 groups of patients. the index group (ig, n=5) developed loss of bile ducts without progression to graft failure due to cr during a follow up of 5 years after transplantation. the second group (crg, n=12) were all cr patients, retransplanted at a median time of 7 months (range 2-19 months). reperfusion (rb), 1 week (1-w), 1 month (1-m) biopsies were studied in both groups. in the crg the last biopsies (lb) before retransplantation taken at a median time of 160 days (range 42-329 days) were also studied. additionally, in the ig the 1& (1-y), 2nd (2-y) and 5* (5-y) annual biopsies were also included. bile ducts and coh were identified by immunohistology using cytokeratin 7. ki67 (mib) was applied to investigate the proliferative activity. the number of bile ducts and coh were counted per portal tract and the number of k67+ cells was counted as ratio of the total number of cells in these structures. clinical follow-up of the ig group was studied based on liver function tests at similar time points as the biopsies. the ig group showed damaged bile ducts and decreasing numbers of bile ducts in the course of time, leading to dp at a median of 3 years. contrastingly, the crg developed dp at a median time of 160 days, observed in the last biopsies taken before retransplantation. when compared with the crg, the ig showed significantly less proliferative activity in bile ducts at 1-w (p=o.o43) but not in rb, 1-m and 1-y, the latter was compared with the lb of the crg. coh showed an initial increase at 1-w in the ig, but a progressive decrease in the subsequent biopsies, reaching significant loss at 5-y (p=0.043). in the crg, progressive loss of coh started at 1-w, leading to a significant loss within 1 year. numbers of coh were consistently lower in the crg at all time points except rb, although not statistically significant. there were also no significant differences in proliferative activity both within the ig in the course of time and when compared with crg. all ig patients continuously showed abnormal liver function tests apart from the bilirubin levels, which were elevated during the first month after transplantation but were normal after 1-y. at 5-y, median serum level of alkaline phosphatase was 228 u/l, gamma glutamyl transpherase was 312 ull, bilirubin was 26 micromoll1 and asatlalat were 96193 ull. conclusion: the ig showed a much slower course of loss of bile ducts when compared with crg, leading to graft survival of more than 5 years. a higher proliferative activity in bile ducts at 1 week in the crg did not prevent the progressive development of dplcr in crg. the initial increase of coh at 1 week in the ig might be responsible for the prolonged preservation of coh in the ig. as coh are believed to represent a regenerative compartment of the biliary unit, our findings indicated that prolonged preservation of coh might contribute to the delayed occurrence of dp but apparently not to reversibility of dp. absence of reversibility of dp is supported by the abnormal liver function tests. (ltx) . recurrent hcv poses a significant and possibly increasing threat to graft and patient survival. diagnosis of acute rejection and alterations in immunosuppression (is) may significantly impact the tempo of post-tx hcv. these associations motivated us to re-examine risk factors for early acute rejection (ear) in a large and contemporary cohort of ltx recipients. methods: the study cohort comprised of 282 consecutive adults undergoing primary ltx between 1/1/99 and 10/31/2002 for chronic liver disease with a minimum of 7 day graft and patient survival. recipients received triple is, typically with steroids, tacrolimus, and mycophenolate mofetil. ear was defined as biopsyproven rejection or treatment for presumed rejection within 6 months of ltx. risk factors for ear were determined by cox proportional hazard methods. spearman rank and phi correlations were used to determine associations between factors. results: 177 men (63%) and 105 women (37%) underwent deceased (n = 236; 84%) or living donor (n = 46; 16%) ltx. the etiologies of chronic liver disease were hcv (n=138; 49%), aih i pbc / psc (n=40; 14%), hbv (n= 34; 12%), cryptogenic (n=27; lo%), alcohol (n=24; 9%), and miscellaneous (n=19; 7%). our overall incidence of ear was 45% (126 i282 recipients); 118 recipients (94%) had biopsy-proven rejection while 8 recipients (6%) were treated for rejection without histologic confirmation. cox univariate models showed that hcv, female gender, meld 2 28, year 2000 compared to year 1999, and lower day 5-7 tacrolimus (tac d5-7) level were positively associated with ear while asian compared to caucasian ethnicity was negatively associated with ear (table la) . factors such as recipient and donor age, recipient african american or hispanic ethnicities, donor type (deceased or living), other years (2001 and 2002) , child's score and class, pre-ltx icu location, and cold and warm ischemia times were not significant risk factors. cox multivariate models showed that hcv diagnosis and female gender remained independent and significant risk factors for ear (table lb) . hbv etiology and asian ethnicity were significantly correlated (phi coefficient 0.53; p = <0.0001) and thus, did not remain independent risk factors in multivariate analysis. while lower tacrolimus level tended to predispose to ear, meld 2 28 and year 2000 were no longer associated with ear. lower tacrolimus level was significantly correlated with both higher meld score (spearman rank correlation -0.34; p = 0.0000) and later transplant year (spearman rank correlation -0.17; p = 0.019). conclusions: hcv etiology of liver disease is strongly associated with ear after ltx. risk of ear is higher for hcv than etoh, cryptogenic, and hbv etiologies and comparable to aih / psc i pbc etiologies; this effect is independent of gender, ethnicity, year, meld, and post-ltx is. recipients with high meld scores are also at increased risk for ear, at least partly because of lower post-ltx is. it is unclear whether the strong association of hcv etiology with ear is because hcv infection results in an immunologic environment predisposing to ear or whether we are unable to accurately diagnose rejection in the setting of hcv recurrence. aim: although the donor pool has expanded in response to increasing waiting lists the quality of donor livers has suffered as a result. it remains unclear whether such marginal organs can be safely used in high-risk recipients or whether they should be implanted solely into good recipients. we aimed to define recipient selection criteria for implantation of these grafts. methods: a prospectively collected database containing 397 patients who underwent orthotopic liver transplantation between 1994 and 2002 was analysed. donors were scored using a formula derived by logistic regression that identified 3 covariates (graft steatosis, donor age and cold ischaemia time) that independently correlated with primary graft dysfunction. this enabled them to be stratified into either marginal or non-marginal groups. using logistic regression analysis, recipient factors independently correlated with 1-year post transplant survival in the marginal group (recipient age, plasma albumin and urea) were built into a mathematical model and each patient was given a score accordingly. patients with scores higher than the defined optimum cut-off value were considered as high-risk and the others with lower scores as low-risk recipients. outcomes were then evaluated in these subpopulations results: marginal and non-marginal donor groups consisted of 65 and 332 patients respectively. the recipient score derived from the multivariate analysis was as follows : score = 1.989 x plasma albumin+ 1.22 x age group + 2.076 x urea, with the recipient albumin level coded as 1 for < 3.1 gldl and 0 for 2 3.1 gldl, the recipient age group coded as 2 for > 55 years, as 1 for 43-55 years, and as 0 for 22.4 mgldl, and as 0 for 5 22.4 mgldl. the roc curve analysis showed that the score had a good discriminating power (area under the curve 0.78, p =.001) and the ideal cut-off value demarcating high-risk from low-risk recipients was 3.25. therefore the recipients were classed as high-risk if they had a plasma albumin level below 3.1 gldl with either an age of over 55 or a urea level of above 22.4 mgldl or when they had a urea level over 22.4 mgldl with an age of over 42. of the 65 recipients in marginal group, 28 (43%) were classified as high-risk and 37 (57%) were classified as low-risk. there was a huge 1-year survival difference between high-risk and low-risk recipients (60.7% and 91.9%, respectively, p <.0001 background single-center experience with pretransplant use of rabbit anti-human-thymocyte globulin suggests that despite early depletion of lymphocytes, a third of all pediatric liver recipients develop early rejection, while the remainder demonstrate clinical graft adaptation. purposelmethods: to identify potential mechanisms, 31 pediatric liver recipients, median age 7.8 years, median followup 8 months, received pre-and post-transplant measurements of 1. whole blood concentrations of tacrolimus (tac), 2. interdose changes in mitogen-stimulated t-and b-cell responses (slr), 3. dendritic cell (dc),and peripheral blood mononuclear cell subsets, and 4. cd8+28-suppressor effect on donor antigenpresenting cell types (cd34fmonocytes and cd19+ b-cells). all patients received ratg preconditioning with a total dose of 5 mglkg in two divided doses. the first dose was given before liver transplantation (ltx). maintenance agent was tac without steroids. in 9 patients this was replaced with sirolimus (srl). mitogen-stimulated t and b-cell responses were compared with those seen in a historical population, who had not received ratg pretreatment. results: all measurements were performed at a median interval of 47 days (22-57 days) after ltx. 1. in slr, the frequency of t-cells expressing the cytokines ifn-g, tnf-a, and il-2 decreased with increasing total exposure (auc) to tac in historical controls (n=6). this relationship was markedly dampened in ratg patients (n=15), as suggested by slopes of 0.0023, 0.032, and 0.0006 relating ifn-g, tnf-a and il-2 on the y-axis to tac auc. 2. significant decrease in cd4 absolute counts at 1 week and partial reconstitution at 2 months after ratg pretreatment (mean pretx-3120 vs 1 week-1920 vs month 2-2117 celllmm3). during this period, monocytes and b-cells remained stable. 3. dc2 remained unchanged, while dc1 frequency increased significantly, from 0.15 to 0.23, p=0.05. 3. in coculture experiments, purified cd8+28-subpopulations from two recipients receiving minimal tac doses induced decreased cd86 expression in donor apc, but increased its expression in hla-mismatched apc. eleven of 31 subjects experienced rejection, while 18 subjects are being maintained on daily (n=13) or every other day (n=5) doses of tac or srl. conclusions: despite lymphocyte depletion, rejection in the setting of t-cell anergy can be explained by relative sparing of antigen-presenting cells, which can recruit alternative effector mediators. the appearance of donor-specific cd8 + suppressor cells in recipients on low immunosuppression suggests that these conditions may also foster a favorable immunomodulatory response toward the liver allografts. (dropout) . the objective of this study was to evaluate the impact of the hcc-adjusted model for end-stage liver disease (meld) organ allocation scheme on the intention-to-treat outcome. under the meld scheme, patients with hcc meeting the united network for organ sharing (unos) t1 (single lesion under 2 cm) and t2 (single lesion between 2 to 5 cm, or 2 to 3 lesions none exceeding 3 cm) criteria were eligible for an initial meld priority score of 24 and 29, respectively. they were also entitled to an increase in meld score, corresponding to a 10% increase in mortality, for every 3 months on the waiting list. methods: excluding patients undergoing living-donor liver transplantation, we prospectively evaluated 102 consecutive patients with hcc listed for olt since january 1998. the kaplan-meier probabilities of olt and dropout among 44 patients with hcc listed under the meld system between february 2002 and january 2003 were compared with 58 patients listed between january 1998 and january 2002 under the previous system of organ allocation. follow-up in the pre-meld group was censored on february 27,2002, when the meld system for organ allocation was implemented by unos. for patients under meld, follow-up was censored on february 27,2003, when further refinements of the meld policy for hcc were made. results: the baseline characteristics were not significantly different between the two groups. eighteen of 44 patients (41%) in the meld group and 26 of 58 patients (45%) in the pre-meld group received chemoembolization or various ablation treatments before olt (p=0.89). all patients in the meld group met t1 (5 patients) or t2 criteria (39 patients). in the pre-meld group, 6 patients had hcc stage exceeding t2 but meeting our proposed expanded criteria (single lesion not exceeding 6.5 cm or no more than 3 lesions none greater than 4.5 cm with total tumor diameter not exceeding 8 cm) by the time of olt. the kaplan-meier cumulative probabilities for olt at 3, 6, and 8.5 months of longest followup were 22.5%, 64.0% and 88.0%, respectively, in the meld group, versus 17.2%, 24.7%, 35.8%, and 47.2% at 3, 6, 9, and 12 months, respectively, in the pre-meld group (p=0.0006). under meld, none of the 5 patients with t1 lesion had received olt. the cumulative probabilities for olt for the 39 patients with t2 hcc in the meld group were 25.5%, 69.2%, and 89.2%, respectively at 3, 6, and 8.5 months (p=o.oool versus the pre-meld group). the cumulative probability of dropout was 5.6% at 8.5 months of longest followup without olt under meld, whereas the cumulative probability of dropout increased from 7.2% at 6 months to 37.8% at 12 months in the pre-meld era. the difference did not reach statistical significance (p=0.74) largely due to low dropout rates in the first 6 months for both groups. among the patients who received olt, 3 of 22 patients in the meld group versus 9 of 25 patients in the pre-meld group had pathologic hcc stage in the explant exceeding t2 criteria (p=0.78). unfavorable histologic tumor features in the explant, including either poorly differentiated grade or microvascular invasion or both, were observed in 4 of 22 patients in the meld group versus 5 of 25 patients in the pre-meld group ( i " 1.0). the short duration of follow-up under meld precluded comparison of intention-totreat survival or hcc recurrence between the two groups. con-clusion: the hcc-adjusted meld system significantly improved the probability of timely olt, and was not associated with selection of a greater proportion of hcc with unfavorable explant tumor histology. given the low probabilities for dropout in the first 6 months following listing for olt even in the pre-meld era, patients with hcc might have indeed received too high a priority score in the first year under meld. disclosures: nancy l ascher -no relationships to disclose nathan m bass -no relationships to disclose randomized at d7 to receive maintenance is regimen with ciclosporine microemulsion + prednisone (group 1) or without steroids (ciclosporine microemulsion + placebo, group 2) after a 7 days blinded oral steroid tapering period. results : 193 patients were recruited and a total of 174 were randomized at d7 (group 1 = 90, group = m). there was no difference between the 2 groups for baseline characteristics, proportion of patients with hepatitis c (18.9% and 20.2%), or cyclosporine blood levels. the incidence of treated biopsy confirmed acute rejection at 6 months was 24.4% in group 1 and 38.1% in group 2 (p= 0.03), with a trend for higher incidence of grade 213 rejection ( 18.9% vs 28.6% ; p=0.12). this difference was maintained in hcv pos and hcv neg patients. no difference was observed between the 2 groups in terms of adverse events, infections, incidence of hypertension and renal dysfunction. changes from baseline were similar with regards to metabolic parameters. a trend towards a better glucose tolerability was observed, less patients receiving an antidiabetic treatment in the placebo group ( 2 vs 10). conclusion : peritransplant immunosuppression with basiliximab, cyclosporine microemulsion, and steroids resulted in excellent low rejection rates in lt patients. early steroid withdrawl strategy at day 14 is not supported by the results of this study, which showed an higher incidence of acute rejection and a trend to more severe acute rejection, only balanced by a trend to a lower need of antidiabetic treatment. to determine if an association exists between meld score and post transplant graft loss within 3 months. methods from 2/28/02 through 10/31/02,2,745 patients underwent liver transplantation, 92% of whom were followed for a minimum of three months. cox regression analysis was utilized to assess the relationship between meld score and post transplant outcome. transplantation. seventy one percent of patients were positive for autoantibodies: 52.9 % were positive for anti-nuclear antibody, 23.5% for anti-smooth muscle antibody and 0% for anti-liver kidney microsomal antibody. the average alt elevation was 224 (range 61-486) and ast elevation 166 (range 52-346). an elevated ggtp was seen in 70.6% of patients. all patients were hepatitis c pcr negative. at the time of diagnosis, 12 patients were being treated with cyclosporine, 5 with f'k506, and 7 with steroids in addition to calcineurin inhibitors. after diagnosis, all patients received standard therapy with l-zmg/kg of steroids. in addition, 89% (15117) began hthioprine and 24% had their calcineurin inhibitor dose decreased. after a mean of 2.6 years of follow-up (range 0.53-5.93), 59% remain steroid dependent and 12% are off steroids. one patient was re-transplanted for biliary complications and 1 patient required conversion to sirohus. conclusion: diagnosis of de novo am requires a high index of suspicion as auto-antibodies are often negative. steroid therapy, often with the addition of azathioprine, is effective in controlling disease. many patients, however, become steroid dependent in order to remain in remission. background. liver allografts have an improved outcome compared with other solid organ grafts, and rodent studies have suggested that activation-associated lymphocyte death may play a key role. studies from our laboratory have shown increased levels of apoptotic lymphocytes in human liver grafts compared with renal grafts and native liver. although the levels of apoptosis did not differ significantly between those who developed acute rejection (rej) and those who did not (nr), the earliest timepoint studied was 3 days post-olt. we hypothesized that the very early postoperative period (within 48 hours) would reveal a relationship between leukocyte apoptosis and rejection. aims. the aim of this study was to determine the amount of early leukocyte apoptosis and its relationship with the degree of lymphocyte activation, subsequent rejection status and degree of donor cell chimerism. methods. peripheral blood mononuclear cells were isolated from 76 patients undergoing olt and were collected on the day prior, 5 hrs after reperfusion (day 0) and 24 hrs post-olt (day 1). dna and rna were prepared and real-time pcr used to quantify apoptosis (ligation-mediated pcr), lymphocyte activation (il-2, ifn-gamma, il-10, cd40 ligand, using gapdh as an internal standard) and donor cell chimerism (y chromosome dyz3 or donor-specific drb1). results. the mean level of circulating apoptotic cells in day 1 recipient pbmc was higher than healthy controls (0.9% t-0.2 vs 0.2% ? 0.1, ~~0 . 0 1 3 ) . apoptosis was greater in nr (1.1% 2 0.3) compared with rej (0.3% t-0.1, p=o.o21). on day 1 the pbmc from nr had increased expression of ifn-gamma (p=0.006), il-10 (p=0.016), cd40 ligand (p=0.02) and il-2 (trend) compared with rej, despite no difference in lymphocyte counts. donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased leukocyte apoptosis in the nr group. interestingly, the level of chimerism 5 hrs postreperfusion (day 0) was significantly higher in nr (3.8% f 0.6) compared with rej (1.2% t-0.4, p=0.004) and there was a close correlation between chimerism on day 0 and pbmc cytokine expression on day 1 (r=0.58, p=0.006). conclusions. patients who did not experience rejection had a paradoxical increase in markers of lymphocyte activation at day 1, in association with higher levels of leukocyte apoptosis. this suggests that recipient cell death may have a role in graft acceptance and reduced acute rejection in human olt. the higher donor cell chimerism seen in non-rejectors implicates the passenger leukocytes in the process of heightened cell death. disclosures: andrew clouston -no relationships to disclose wenyi gu -no relationships to disclose julie r jonsson -no relationships to disclose elizabeth e powell -no relationships to disclose daina m vanags -no relationships to disclose amadeo marcos, bridget flynn, paulo fontes, thomas cacciarelli, wallis marsh, michael devera, obaid shakil, noriko murase, anthony demetris, john fun% thomas e stanl, university of pittsburgh, pittsburgh, pa the seminal mechanism of organ engraftment is thought to be immune activation-dependent clonal exhaustion-deletion. the conventional use of heavy immunosuppression may depress the initial step of donor-specific activation to the extent that the treatment is anti-tolerogenic. to avoid this pitfall, we have applied 2 therapeutic principles in management of 78 adults cadaveric liver recipients transplanted between 9/2002-5/2003. the treatment principles were, first, host conditioning prior to transplantation, and second, minimum post-transplant immunosuppression. the host conditioning was done with one gram of methylprednisolon and a single infusion of 30 mg of alemtuzumab, completed before liver revascularization. there were 49 males and 29 females in the group with mean age 52.3110.3 years (32-73). main causes of liver disease were hepatitis c (40%), cholestatic liver disease (18%), alcoholic liver disease (22%). daily post-transplant monotherapy with tacrolimus (trough target 10 ng/ml) was started 12-24 hours postoperatively (starting tacrolimus dose: 10.5+5 mg, median 10). because of suspected neurotoxicity, 5 patients were switched to cyclosporin. in addition sirolimus monotherapy was substituted for tacrolimus in one patient for management of nephrotoxicity. immune activation diagnosed by liver function tests or by mild or equivocal rejection in liver biopsies frequently was not treated, and tended to resolve spontaneously. clinically and pathologically significant rejection was treated with a bolus of methylprednisolone and/or a 30 mg dose of alemtuzumab. nine patients (11.5%) died 5 sepsis; 2 pnf; one coagulation disorder (hypercoagulable state), and one congestive heart failure. of the 69 surviving recipients four experienced rejection during the first two months which was readily reversed with a bolus of methylprednisolon. after demonstrating the absence of immune activation in liver biopsies at 120 days post-transplantation, weaning from monotherapy was initiated in 49 patients (with the aim of completely stopping treatment in selected cases). seven patients experienced one episode of rejection that was reversed with single dose of methylprednisolone and/or infusion of 30 mg of alemtuzumab. presently, 14 patients are on once a day monotherapy, 14 patients on every-other-day, 10 patients on three timedweek, 10 patients on twicelweek and one patient on once-a-week immunosuppressive regimen (latest tacrolimus dose 5.7t3.5 mg, median 5.5). the patients on cyclosporin and sirolimus are also following the same stepwise weaning pattern. no cases of new onset diabetes, renal failure or hypertension was found in these patients. mean serum creatinine level in the study group at the time of transplantation was 1.020.4 mgldl and at the latest follow up 1.220.3 mgldl. cmv infection was seen in 22% of the patient population but was easily treatable with antivirals. no ptld was seen during the follow-up period. hepatitis c recurrence was seen in over 70% of the patients. response to anti-hcv therapy in this group, after reduction of immunosuppression and especially during the weaning process was promissing. conclusion: by applying the principles of immunosuppression outlined above, it has been possible to drastically reduce the amount of total immunosuppression relative to any (of our) pre-vious experience with liver transplantation. to see less side effects of chronic and high dose immunosuppressive therapy, and probably to have a chance to get a better response to treatment in our hepatitis c population. the results suggest the possibility of systematically achieving drug-free tolerance after liver transplantation. background:end stage liver disease as a consequence of hepatic sarcoidosis is an uncommon indication for liver transplantation (lt). consequently, there is a paucity of information on the pre-lt findings and postoperative course of individuals transplanted for hepatic sarcoidosis. the purpose of this study was to evaluate our experience with lt for sarcoidosis. methods: cases were identified by review of the mount sinai hospital lt database. patient records were reviewed; including the pathologic analysis of the hepatic explant, to confirm that sarcoidosis was responsible for liver failure. for each case, two control patients with other causes of liver failure matched for age, gender and date of transplant were selected. data collected encompassed a mean follow up period of 5 years (range 1-8 years) post-lt. two-tailed student's t-test was used for comparison of continuous data, two-tailed fisher's exact test for comparison of categorical data and log rank test for comparison of survival d,ita. results: hepatic sarcoidosis was the indication for lt in 7 of 2016 adult-lt (0.3%) performed from september 1988 -june 200.3. the mean age at transplant was 56 years and 4/7 (57%) were males. the diagnosis of sarcoidosis was established by findings of extensive, non-caseating granulomas in pre-lt biopsy specimens or in the native liver explant. in 2/7 cases, sarcoid had been previously diagnosed by biopsies of lung parenchyma or mediastinal lymph node. 6 of 7 patients were ama negative. the sole exception was a patient with an ama titer of 1:320 in whom sarcoid was still considered the most likely diagnosis based on liver biopsy findings of granulomas present in both portal and lobular areas and granulomas in a lung biopsy performed 10-years prior to lt. extrahepatic disease was limited to pulmonary involvement in 4 patients with radiographic findings of either interstitial infiltrates (2) or interstitial infiltrates and hilar adenopathy (2). the mean age at transplant and gender distribution were identical in cases and controls. the indications for lt in the control group were: hepatitis c (7), pbc (2) and one patient each with hereditary hemochromatosis, cryptogenic cirrhosis, hepatitis b and laennec cirrhosis. no statistically significant differences between the groups were present with respect to pre-lt levels of ast, alt, alkaline phosphatase, total bilirubin, serum creatinine or prothrombin time. cases and controls had a similar prevalence of ascites and anti-hepatitis b core antibodies. in contrast, sarcoid cases were much more likely to have a diagnosis of diabetes mellitus (100% vs. 21%, p=.002) and less likely to have antibodies to hepatitis c (0% vs. 50%, p=.05). standard orthotopic lt was performed (two control patients received right trisegment grafts). rates of acute cellular rejection were 71% in cases and 43% in controls (p=o.44) with cases experiencing a greater number of acute rejection episodes per patient (1.9 vs. 0.6 episodeslpatient). de novo autoimmune hepatitis developed in one case and one control patient. both de novo autoimmune hepatitis and chronic rejection developed in one case patient. recurrence of hepatic sarcoidosis was diagnosed in two patients at 1.5 and 5.6 years of follow-up. among cases, the one-year graft and patient survival rates were 100% and five-year graft and patient survival rates were 86%. there was no statistically significant difference in five-year graft or patient survival between cases and controls. conclusions: end-stage liver disease as a consequence of sarcoidosis is a rare indication for lt. these patients share many features in common with those transplanted for other indications. despite the small number of patients, a relatively high rate of acute rejection and de novo autoimmune hepatitis was observed in patients with sarcoidosis. recurrence of hepatic sarcoidosis was observed. five-year graft and patient survival rates were comparable to those of patients transplanted for other indications. disclosures: sander florman -no relationships to disclose leona kim schluger -no relationships to disclose kevin m korenblat -no relationships to disclose evan j lipson -no relationships to disclose 440 transplantation. james d eason, ari j cohen, safheesh nair, george loss, ochsner clinic foundation, new orleans, la sirolimus has been used successfully in improving renal function in olt recipients previously treated with tacrolimus and steroids. we report our experience with early sirolimus conversion in steroid-free olt recipients to determine safety and efficacy in patients never treated with steroids. methods: we performed 220 olt over a threeyear period. steroid-free immunosuppression with rabbit atg induction and tacrolimus and mmf was used in 140 of these patients. twenty-five of these steroid-free recipients were converted from tacrolimus to sirolimus within one week to ll months of transplant. results of these patients receiving sirolimus conversion were reviewed. results: renal insufficiency was the reason for conversion in18 patients, while seven patients were converted because of neurotoxicity. six patients were dialysis-dependent at the time of conversion. one-year patient survival in this high-risk group was 80% (20/25) compared to 88% in patients who remained on tacrolimus. the four deaths were in patients converted because of renal failure, three of whom were on dialysis. the incidence of rejection was 31% in sirolimus patients compared to 24% in patients who remained on tacrolimus. rejection was treated by the addition of mmf or reintroduction of low-dose tacrolimus. only one patient required steroids to reverse rejection. conclusion: early sirolimus conversion is safe and effective lawal, sandy florman, isabel fiel, ronald gordon, myron schwartz, charles miller, thomas d schiano, the mount sinai medical center, new york, ny introduction: primary non-function (pnf) fter liver transplantation occurs in approximately 5% of cases and is fatal without timely retransplantation. pnf has been associated with many risk factors, however the etiology remains unknown. some evidence suggests that ultrastructural changes in the liver may be causative. aim: we sought to examine the hepatic ultrastructure of donor allografts in patients with and without pnf using electron microscopy. medical center, over 2000 adult orthotopic liver transplants were performed. patients with the classic clinical presentation of pnf requiring retransplant were identified. archived pre-and postreperfusion donor liver biopsies were examined by electron microscopy in 9 patients with pnf and in 9 matched controls. each pnf case was matched by donor age t5years, gender, cold ischemic time klhour and the donor's cause of death. all biopsies were blindly reviewed by the same pathologist. particular attention was paid to abnormalities of the mitochondria, endoplasmic reticulum and sinusoidal endothelial cells. in addition, the glycogen content of the cells was also assessed. the recipient age and creatinine, as well as the donor serum peak transaminases and bilirubin were compared.non parametric tests with p values <0.05 were regarded as significant using the spss 11.5 program. results: overall, patients with pnf were older (55.7 t 10 vs. 48.99 years, p=0.03) and had higher peak alt levels (141 ? 185 vs. 40 2 32 u/l, p=0.04). there was no significant difference in recipient peak serum creatinine, donor peak serum ast, sodium or donor peak serum bilirubin. in all cases, the endoplasmic reticulum and sinusoidal endothelial cells were ultrastructurally normal. the hepatocytes had variable degrees of glycogen pooling and moderate to severe fatty infiltration. in 7/9 (78%) pnf cases vs. 2ly (22%) control had intramitochondrial crystalline inclusions on pre-perfusion biopsy. conclusion: liver allografts from patients with primary non-function have significant mitochondria1 ultrastructural changes on pre-perfusion biopsies, and may thus have some intrinsic mitochondria abnormalities. introduction: the ideal liver allocation system would allocate donated livers to patients who are most likeiy to die without a transplant, but who have the lowest predicted mortality once they have been transplanted. this mode of allocation assesses the condition of both the recipient and the donor at the time of transplantation. in this study we have constructed a self-organising map (som: this is a neural network or non-linear mode of decision analysis suitable for modelling complex multidimensional relationships) and then validated it by using the som to classify survival in an unrelated population. patients and methods: we have previously described a som consisting of 72 (50 recipient and 22 donor) factors (inputs) constructed from 827 (449 male; median age 52 years; median meld score 16) consecutive primary liver transplants undertaken between 01/01/1993 and 31/07/2002 in the queen elizabeth hospital, birmingham, uk. using a 3 neuron version of this som with three output functions (patient survival at 3 months; 1 year and 5 years) we used the som to classify the post-transplant survival of all primary graft recipients (n=200 patients) over a 5 year period from a north american centre (125 male; median age 48 years; median meld score 16). the birmingham (uk) patients were first classified by the som and then the probabilities of survival (p,) at 3 months, 1 year and 5 years calculated by dividing the number of surviving patients by the total number of patients in neuron i. using this som, each of the wisconsin (us) patients was classified to each of the neurons and the distribution of survival probes for each neuron compared between the two populations using the chi squared test. results: there was no significant difference in the survival probabilities of patients in each neuron when the wisconsin population was compared with the birmingham population. thus, the birmingham som was able to classify successfully the survival of the wisconsin patient population at 3 months, 1 year and 5 years following transplantation by using the same donor and recipient factors ( table 1) . analysis of the inter-neuronal survival probabilities demonstrated that for 3 month and 1 year survival, neuron 3 was associated with a significantly lower survival probability than neurons 1 and 2 (p=0.006 for 3 months; p= 0.006 for 12 months). further, if recipients from neuron 1 received livers from patients classified to neuron 3 by "computer simulated transplantation", a proportion of patients then had a lower predicted survival probability (by som re-distribution to neuron 3). this analysis indicates that patient survival post-transplantation is significantly infuenced by both the condition of the recipient and donor factors at the time of transplantation. conclusions: (1)som analysis provides an efficient and automated method for assessing the probability of survival of individual patients in an unrelated population at 3 months, 1 year and 5 years following liver transplantation. (2)the model not only assesses the pre-transplant condition of the patient, but also considers a wide range of donor factors when predicting the probability of survival post-transplant. (3)this unique resource can match a single liver to a population of recipients most likely to die without a transplant whilst ensuring the best possible post-transplant survival for the most suitable recipient (4)som analysis can also assess the probability of survival of individual recipents matched to a range of possible donated livers when they are being considered for transplant programmes. purpose of study: to assess the efficacy of targeted prophylaxis in patients identified to be at highest risk of if1 after orthotopic liver transplantation. introduction: historically, invasive fungal infection (in) has complicated up to 20% of cases of orthotopic liver transplantation (olt). retrospective analysis has identified a variety of risk factors associated with a high risk for the subsequent development of ifi. these include patients undergoing retransplantation, transplantation for fulminant hepatic failure (fhf), requiring haemodialysis and prolonged intensive care unit (icu) stay. prophylactic anti-fungal therapy is safe and can reduce the incidence of ifl in olt recipientri. in recent years our unit has moved towards targeting prophylaxis to higher risk transplants. we assessed the efficacy of our policy in reducing the incidence of invasive fungal infection. methods: a retrospective audit was conducted comparing two groups of adult olt recipients over two 5 year time periods, 1990-1994 when targeted prophylaxis was not used (group 1) and 1997-2001 when targeted prophylaxis was used (group 2). data were collected with respect to risk factors for ifi, anti-fungal prophylaxis use and development of ifi. results: there was no difference in the overall number of risk factors for if1 associated with olt between the two groups. however, a higher proportion of patients in group 2 had high risk factors for if1 compared to group 1 (p=0.07). there was a significant difference in the use of targeted anti-fungal prophylaxis given to high risk patients in group 2 compared to group 1 (52.3% cf 23.8%, p=0.03), and there was a significant reduction in the number of ifis in group2 compared with group 1 (4.5% cf 28.6%, conclusion: a policy of targeting anti-fungal prophylaxis to highest risk liver transplant recipients leads to a significant reduction in if1 this group. there remains a background incidence of infection in low risk or long term recipients in whom prophylaxis is not given. background: only few studies have described risk factors associated with cellular rejection. the diagnosis of cellular rejection requires histology. there are no data evaluating the absence of rejection in a protocol biopsy population. aim: to assess predictive factors associated for the absence of cellular rejection in a protocol liver biopsy population and to evaluate the usefulness of protocol liver biopsies. method 449 consecutive patients transplanted on a data-base at our centre. protocol liver biopsies were performed between 5 and 10 days post transplantation and then when clinically indicated, during the first 3 months. rejection was scored prospectively. the following variables were examined with respect to absence of rejection over 3 months and in the first protocol liver biopsy: a) donor factors: age, gender, race, donorlrecipient gender match, abo match. b) pre-transplantation recipient factors: age, sex, aetiology, ascites, oesophageal varices, tips, encephalopathy grade, renal support, ventilation, total bilirubin, albumin, inr, ast, alt, creatinine, urea, c) graft and surgical factors: surgeon's visual assessment of graft, cold ischaemic time, use of venovenous by-pass, intraoperative blood transfusion and initial maintenance immunosuppression of either cyclosporin or tacrolimus in triple-dual or monotherapy. standard treatment of rejection was 1 g iv daily of methylprednisolone x 3 days. results: absence of rejection over 3 months was in 69 (15%), mild in 103 (21%) and moderatelsevere in 277 (62%). in the first biopsy: absence of rejection in 92 (21%), mild in 178 (39%), moderate in 159 (35%) and severe in 20 (4.5%). univariate analysis showed for both first protocol biopsies and 3 months, that 4 variables were significantly correlated with non rejection: pre-transplantation use of renal support (haemodialysis or hemofiltration), (p=0.042), cold ischaemic time >15 hours (p=0.030), suboptimal graft visually (p=0.042) and blood transfusion <= 7 units cp=0.008). no correlation was found between aetiology of liver diseases, initial maintenance immunosuppression. conclusion: a suboptimal graft with prolonged cold ischaemic time in recipients with previous need of renal support seems to be associated with absence of rejection. these data are in contrast with previous reports. these factors may be helpful in identifying patients who do not need to be submitted to protocol liver biopsy. tables 1 and 2 1 child developed abnormal transaminases and was found to have chronic hepatitis on liver biopsy and was treated with steroids. 2 required adjusting cyclosporine dosage to maintain trough levels in the identified range (range 65-90 microgramll as per protocol). summary: there was good correlation between the trough levels taken on 2 occasions in the stable paediatric post liver transplant group of patients. the range of c2 peak levels were also similar suggesting good bioavailablity conclusion: this preliminary study on long term post transplant recipients suggests that a peak c2 level is within the range of 280-460 microgramll. background hepatocytes and cholangiocytes release substantial amounts of adenosine triphosphate into bile, where it is rapidly degraded by membrane-bound ecto-atpase and 5'-nucleotidase. this degradation process leads to the generation of adenosine and inorganic phosphate (i?#. whereas adenosine is reabsorbed in a sodium-dependent manner back into hepatocytes, little is known about the fate of biliary pi. in rat, biliary pi concentration is 0.01 mm, which is about 100 fold lower than in hepatocytes (1 mm) and 200 fold lower than in plasma (2.3 mm)9 indicating active reabsorption of pi from bile canaliculi andlor from the biliary tree. aim: the purpose of the present study was to functionally characterize canalicular p, reabsorption in rat liver and to identify the involved p, transport system(s). methods: p, transport was determined in isolated canalicular liver plasma membrane (clpm) vesicles using a rapid filtration technique. identification of putative p, transporters was performed with reverse transcriptase-polymerase chain reaction (rt-pcr) from rat liver total mrna using sodiumlphosphate cotransporter specific primers for napi-iib (gggattgggaaattcatccti ttccaacacaaggttggtca), napi-111 subtype pit-1 (catctcggtgggatgtgcitgttgctctctcctccttca) and napi-i11 subtype pit-2 (gctctaccattggcttctcglaca-gaggaagtgcctggaga)3. on the protein level, phosphate transporter expression was confirmed by western blot analysis in isolated basolateral (blpm) and canalicular (clpm) rat liver plasma membrane vesicles. specific polyclonal antibodies were raised in rabbits against antigenic peptides from mouse napi-iib and human napi-iiilpit-2 showing >88% identity with rat homologues. results: transport studies in isolated clpm vesicles demonstrated sodium-dependent p, uptake (najut > nag 3877 pmollmin; k&t > kg 107 pmollmin). initial na+-dependent p, uptake was linear for at least 6 sec and exhibited a clear overshoot indicating transient intravesicular concentration of p, (secondary active p, transport). initial p, uptake rates (4 sec) were saturable with increasing p, concentrations and exhibited an apparent k,,, value of -11 km. furthermore, sodium-dependent p, transport was higher at an acidic (phout = 6.5; ph,, = 7.4) as compared to an alkaline extravesicular ph (8.0). in addition, an intravesicular negative membrane potential stimulated sodium-dependent p, transport indicating a na:p, stoichiometry of > 1. these data are comparable with the transport characteristics of sodiumlphosphate cotransporters napi-iib, napi-iiilpit-1 and napi-iiilpit-23. mrnas of all these three napi's were found to be expressed in rat liver by rt-pcr. however, on the protein level only napi-iib was found to be expressed selectively in clpm, whereas napi-iiilpit-2 was detected in blpm. no clearcut localization of napi-iiilpit-1 protein could be obtained. conclusions: the canalicular membrane of rat hepatocytes localizes the sodiumlphosphate cotransporter napi-iib, which can reabsorb p, from primary hepatic bile back into hepatocytes. in contrast, napi-iiilpit-2 was found to be expressed at the basolat-era1 hepatocyte plasma membrane. the results indicate that napi-iib regulates p, concentration in bile and may play an important role in the overall p, homeostasis in rat liver backgroundlaims. organic anion transporting polypeptides (oatps) are a family of transport proteins of the basolateral hepatocyte membrane. human oatp8 (slc21a8) is predominantly expressed in hepatocytes and is an uptake system for xenobiotics such as digoxin. the oatp8 gene promoter possesses an inverted repeat (ir1) element at nt -82l-70 relative to the transcription start site, that binds and is activated by the farnesoid x receptorlretinoid x receptor (fxrlrxr). ligands of fxr include bile salts such as chenodeoxycholic acid (cdca), previously shown to activate the oatp8 gene. however, because oatp8 is only a poor bile salt but an efficient xenobiotic transporter, we investigated whether xenobiotics such as rifampicin regulate the oatp8 gene. rifampicin is a prototypic ligand of the xenobiotic receptor pxr (pregnane x receptor). methods. endogenous oatp8 mrna levels in caco2 cells were quantified by real-time pcr. an oatp8 gene promoter construct containing nucleotides -120l +38 relative to the transcription start site (luc-120) was assayed for reporter activity in transiently transfected cells. the fxr binding site in the oatp8 gene (ir1 element) was characterized using the luciferase construct ir1-tk-luc that contained a thymidine kinase promoter under the control of the irl element. results. endogenous oatp8 mrna levels in caco2 cells were induced 7fold by cdca (100 pmolll). interestingly, incubation of cells with rifampicin (10 pmolll) resulted in a 4.5fold increase in oatp8 mrna levels. to study the mechanism of rifampicinmediated induction of oatp8, the promoter sequence was searched for potential pxr binding sites. because the ir1 element, previously shown to bind the fxrlrxr heterodimer, was the only nuclear receptor binding site found, we hypothesized that the induction by rifampicin could be mediated through the fxr element. we, therefore, studied the effect of cdca and rifampicin on the ir1 element. in cells cotransfected with the ir1-tk-luc construct and expression plasmids coding for fxrlrxr, pxrlrxr or car (constitutive androstane receptor)lrxr, fxrlrxr induced the irl element llfold in the presence of cdca and 2.5fold in the presence of rifampicin, indicating that rifampicin is capable of activating fxr. in contrast, pxrlrxr or carlrxr did not activate the ir1 element in the presence of cdca or rifampicin. to confirm that an oatp8 promoter construct is also activated by rifampicin, huh7 cells cotransfected with the luc-120 construct and fxrlrxr expression plasmids were incubated with cdca or rifampicin. cdca induced oatp8 promoter activity -zfold, rifampicin -15fold, confirming that rifampicin transactivates the oatp8 promoter. to investigate whether other xenobiotics also activate the fxr element, huh7 cells cotransfected with the ir1-tk-luc construct and fxrlrxr expression plasmids were incubated with rifampicin, rifamycin, ru486, clotrimazol, phenobarbital or pcn. a 1.4fold (rifamycin) to 2.5fold (clotrima-201) induction of the ir1 element was seen in the presence of these xenobiotics. conclusions. the human oatp8 gene is induced transcriptionally by xenobiotics that represent prototypic ligands of the xenobiotic receptor pxr. however, activation does not occur through pxr but through activation of fxr bound to the ir1 element in the oatp8 gene promoter. the data thus indicate that pxr ligands such as rifampicin and clotrimazol can also activate fxr and thereby induce the fxr-regulated transporter gene oatp8. disclosures: may-britt becker -no relationships to disclose michael fried -no relationships to disclose diana jung -no relationships to disclose gerd a kullak-ublick -no relationships to disclose peter j meier -no relationships to disclose epidemiologie de population epi 106 seema sonnad -no relationships to disclose 428 preservation of canals of hering mitigates bile duct loss in liver graft rejection. ivlarius c van den michael angelis -no relationships to disclose jeffery cooper -no relationships to disclose erick edwards -no relationships to disclose richard b freeman jr -no relationships to disclose ann harper -no relationships to disclose abigail mithoefer -no relationships to disclose no relationships to disclose the data was not available. the ca 19-9, cea, and afp levels were obtained using standard commercially available assays results: out of a total of 108 patients with esld fifty-eight patients fulfilled the inclusion and exclusion criteria. of these, thirty-three patients had evidence of ascites and twenty-five did not. the etiology of liver disease in the two groups was similar. the mean levels of ca 19-9, cea and afp levels in patients with ascites were not significantly different from those without ascites (ca 19-9 48.92 % 65 furthermore, 15 out of these 58 patients with esld had levels -> 37 ulml, the upper limit of normal in this assay unitslml] vs 6.53 5 6.13 conclusions: ascites does not seem to make an impact on the serum levels of ca 19-9 chhaya hasyagar -no relationships to disclose savant mehta -no relationships to disclose 445 severe mitochondrial toxicity after liver transplantation in hiv-hcv coinfected patients liver transplantation (lt) may be the only potentially curative treatment available to these patients at this stage. however its feasibility and benefit has still to be established. severe recurrence of hepatitis c on the liver graft may complicate the post operative course as recently suggested by us and others. mitochondrial toxicity of highly active antiretroviral therapy (haart) may also play an active role on the liver graft of htv-hcv co-infected patients. we aimed to study this complication on the liver graft of hiv-hcv coinfected patients. patients and methods: between he had an history of episode of pancreatitis and haart therapy was azt-3tc-nelfinavir. at m10 post lt, microvesicular steatosis was noted. at m18 a low content of liver mtdna was found (mtdnalnuclear dna = 0.28). 3 other patients had microvesicular steatosis respectively at m2, m4 and m6 post lt. one of these 3 patients had low content of liver mtdna (mtdnalnuclear dna = 0.20). severe defect of complex iv of the respiratory chain was noted in 1 of these 3 patients. no deletion of mtdna was observed by southern blot or long range pcr conclusion: mitochondrial toxicity on the liver graft may become a major problem during post lt c o m e and could worsen graft lesions related to hcv recurrence on the liver graft in hiv patients daniel azoulay -no relationships to disclose henri bismuth -no relationships to disclose denis castaing -no relationships to disclose duclos-vallee -no relationships to disclose cyrille feray -no relationships to disclose michelle gigou -no relationships to disclose catherine guettier -no relationships to disclose philippe ichai -no relationships to disclose claude jardel -no relationships to disclose anne lombes -no relationships to disclose bruno roche -no relationships to disclose faouzi saliba -no relationships to disclose didier samuel -no relationships to disclose elina teicher -no relationships to disclose daniel vittecoq -no relationships to disclose 381a 457 identification of sodiumlphosphate cotransporter type iib marek nowicki, usc, los angeks, ca; jorge rakela, tomasz laskus, there is growing evidence that patients with chronic hepatitis c are more likely to have significant changes in their physical and mental well being, commonly manifested as fatigue and depression, than patients with liver disease of other etiology. recently published studies demonstrated also that hcv infection is associated with cognitive dysfunction. hepatitis c virus (hcv) was reported to replicate in monocyteslmacrophages and lymphoid cells. we have recently demonstrated that leukocytes carry hcv across the blood-brain barrier and we found hcv rna in the central nervous system (cns) in some infected patients (j virol 2002, 76, 10064-10068; 76, 600-608) . however, biological basis for neurocognitive abnormalities observed in hcv-infected patients remains unclear. aim: to determine the pattern of gene expression in cns in hcv-positive patients as compared to hcv-negative controls. material and methods we analyzed samples of brain tissue obtained at autopsy from 3 hcv-positive patients and 3 hcv-negative control patients. all were men of similar age, none was infected with hiv. all 3 hcv+ patients and 2 out of 3 controls had liver cirrhosis. only 2 deaths (one in each group) were liverrelated. the analysis of gene expression was conducted using three different techniques: differential display (genhunter inc), reverse northern, and microarray analysis (bd atlas plastic miroarray). reverse northern analysis was used for confirmation of differential display findings. analysis of microarray data was done using atlas image 2.01 (bd) and cluster 2.20 and treeview 1.60 (m.eisen; ucb). only those genes that were up or downregulated 1.8 times in reverse northern and/or microarray analysis were considered differentially expressed. results: the most striking finding was downregulation of mitochondria] oxidative phosphorylation genes in all hcv-infected patients as compared to controls; impairment of brain oxidative/ energy metabolism has been previously suggested to be the proximate cause of many disorders that impair mentation. another consistent finding in differential display and microarray analysis was downregulation of multiple ribosomal proteins genes and several genes involved in transcription regulation. these could indicate reduced metabolic activities perhaps secondary to deficiencies in oxidative phosphorylation. we also observed upregulation of several genes involved in immune response. there was upregulation of mhc class i and class i1 and several lymphokine receptors like interferon (alpha, beta), il-6, il-9, il-17 as well as lfa-1 ligand intracellular adhesion molecule icam-2 and ceacam1. furthermore, upregulation of cd8o and allograft inflammatory factor-1 gene (aif-1) suggested the presence of activated microglia cells and/or activated macrophages. conclusions: we found downregulation of oxidative phosphorylation genes and upregulation of genes involved in immune response in brains from hcv-positive patients when compared to hcv negative patients. our findings provide the likely substrate for neuropsychiatric symptoms and cognitive impairment associated with hcv infection. medicine, ehime, japan; emmett v schmidt, raymond t chung, massachusetts general hospital, boston , m a background/aims: we previously reported cell-based hcv replication using a novel binary expression system in which cells were transfected with a t7 polymerase-driven full length hcv cdna plasmid (pt7-flhcv-rz) and infected with vaccinia-t7 (pnas 989847). to circumvent vaccinia-induced cytotoxicity, we sought to determine whether replication-defective adenoviral vectors expressing t7 or cell lines stably transfected with t7 could support hcv replication. because vaccinia interferes with pkr function, we further sought to define the antiviral activity of interferon-ar (ifn) in these models. methods: 24 hours after transient transfection of cv-1 and huh7 cells with pt7-flhcv-rz, cells were treated with recombinant replication-defective adenovirus vectors expressing t7 polymerase (ad-t7pol). medium with or without 1000 iulml of ifn was changed at day 1 post-infection and every 2 days thereafter. cells were harvested on day 1, 2, 3, 5, 7 and 9 post-infection. for t7-stably transfected cell lines (huh-t7), we transiently transfected with pt7-flhcv-rz, then added ifn to selected cells in analogous manner. hcv positive and negative strand were measured by strand specific real-time rt-pcr. hcv core protein expression was assessed by western blotting. results: in the hcv replication system with recombinant adenovirus vectors and with t7 stable cell lines, no cytotoxicity was observed at 9 days. with pt7-flhcv-rz and ad-t'lpol, hcv positive and negative strand rna expression was strongest in the first 3 days post-infection and diminished thereafter, but were expressed throughout the 9 days. in the 2 days after adding ifn, hcv positive strand was significantly decreased (0.48520.213 vs. 0.162+0.017 in cv1, 0.123t0.051 vs. 0.035?0.012 in huh7, hcv/ gapdh copy ratio, pc0.05) than the samples without ifn. sustained expression of hcv rna and ifn inhibitory effect was also observed in t7-stable huh-t7 cell lines. pkr expression was strongly induced by ifn, suggesting that pkr is appropriately induced by ifn. conclusions: we have successfully substituted recombinant adenovirus vectors or t7 stable cell lines in our cell-based binary hcv replication system. in these systems, ifn inhibits hcv replication, and upregulates the pkr pathway. these improved binary systems are a durable and more authentic model for identification of host cellular processes critical to hcv replication. disclosures:jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose norio horiike -no relationships to disclose yoshitaka kamegaya -no relationships to disclose morikazu onji -no relationships to disclose emmett v schmidt -no relationships to disclose the meld scoring system for the allocation of cadaveric donor liver for transplantation gives significant priority to cirrhotic patients with a small, t1 or t2, hepatocellular carcinoma. patients on the wait list were given adjusted meld scores equivalent to 3-month mortality rates of 15% and 40% for t1 and t2 lesions, respectively based on theoretical tumor doubling times. frequently these patients have compensated cirrhosis and hence their intrinsic meld score is lower, yet they are not candidates for surgical resection secondary to the underlying cirrhosis. to assess whether the additional meld points for hcc is valid, we sought to examine the impact of the meld scoring system on a single center's waiting list one year after implementation.methods: records of all patients undergoing liver transplantation (oltx) at uthsc-san antonio from feb 27,2002 through feb 27,2003 were retrospectively reviewed. pathology and radiology reports as well as data from unet were collated for analysis and review. results over a 1-year time period following implementation of the meld system, 126 patients underwent oltx at our center. of these 24 were excluded from analysis because of the following reasons: status 1 (lo), living-related recipient (6), pediatric non-tumor (8). the table below summarizes the pertinent findings. of the 102 remaining patients, 36 (35%) underwent oltx with suspected hcc (4 tl, 32 t2).none of the hcc patients had their meld score downgraded because of tumor progression (t2-t3). at transplant, 2 patients were found to have extra hepatic disease(one with known hcc and the other unknown) and the cases were aborted. sensitivity of radiological evaluation for the presence of hcc was 92.5% and specificity 94%. the calculated meld score at the time of listing for oltx and at the time of oltx was significantly lower for those patients thought to have an hcc. exception points for hcc gave those patients a significantly higher average meld score at the time of oltx than patients without hcc. patients wlo hcc had a aupper; true meld of +3.1 at the time of oltx compared to listing as opposed to hcc patients whose a-upper; true meld was -0.3. during the study period 34 patients (none with suspected hcc) died on the waiting list or were too sick to transplant while 215 new patients were listed compared to 24 dying the prior year with 163 new registrants (15.8% vs 14.7%). mean meld score of those dying during the study period was 22.6 8 (median 22.5) and mean time on the list was 284 421 days (median 99 days). of those patients dying. 16 had a meld score of 1 2 4 . conclusion: meld has effectively prioritized cadaveric donor livers to the sicker patients. however, patients with hcc are given too much priority as evidenced by all of the wait list deaths occurring in patients without hcc and lack of tumor progression (to t3) sufficient to loose meld exception points in those with hcc. likewise, the mean meld score of those dying was less than that given to patients with t1 lesions(22.6 vs. 24). the recent downward adjustments of meld exceptions to 20 and 24 for ti and t2 lesions, respectively, are reasonable. ongoing analysis will be needed to accurately place hcc patients in the current allocation system. there was no significant difference (p>0.05) in weight gain between the sexes, those with a bm1>30 pre-transplant and those with a bmi<30 pre-transplant or those on prolonged courses (>3 months) of steroids. weight gain was significantly greater (p<0.05) in patients aged over 50 compared to those under 50 and those transplanted for chronic liver disease compared with fulminant liver failure. a pre-transplant bmi>30 was a strong indicator that the patient would still have a bmi>30 at 3 years. there was no effect of immunosuppression on weight gain: corticosteroids were discontinued in 59.3% by 3 months and long term use of steroids was not associated with weight gain. weight gain was similar in those on cyclosporine and on tacrolimus.conclusions: a bm1>3o is common in the liver transplant population, but seems to unrelated to specific immunosuppression and may be more closely linked to lifestyle. most weight gain occurs after the first 6 months, and intervention with dietary and lifestyle advice at this point could be implemented to minimise the long-term morbidity and mortality risks associated with obesity. serum tumor marker estimations are often performed as a part of evaluation for liver transplantation. there is a paucity of information on the effect of esld on the levels of these tumor markers making interpretation difficult one tumor marker, ca-125, has been consistently reported to be elevated in patients with ascites from liver disease. purpose:-to compare the levels of serum ca 19-9, cea and afp in patients with esld with or without ascites.-to compare the serum ca 19-9 levels in patients with esld vs normal controls methods: all patients referred for liver transplantation between jan 2000 and dec 2002 at our center were included in the study. men with end stage liver disease (esld) develop a plethora of debilitating symptoms, including severe muscle wasting, that render many of these patients non-suitable for liver transplantation. esld-associated hypogonadism, i.e. testosterone deficiency, may, in part, be responsible for the development and progression of many of these incapacitating symptoms. in general, hypogonadism can be effectively treated with topical testosterone replacement (ttr), which avoids the first pass effect via the hepatic system in contrast to oral anabolic steroids. ttr has been shown to effectively improve muscle mass and overall well being in patients with hiv. however, little is known about the effects of ttr in men with esld. the aim of this study was to determine the potential benefits and safety of ttr in patients with esld and muscle wasting. method:the medical records of liver transplant patients treated for at least 3 months with testosterone gel 1% (5 grams per day) therapy for muscle wasting (mw) from january 2002 to march 2003 were reviewed. information collected included; demographics, albumin, pre-albumin, transferring, testosterone, estrogen, lh and fsh. drug safety results were also collected and included, acute cellular rejection, cholestasis, malignancy, and patient or graft loss. tumor markers for afp, ca19-9, cea, psa, prolactin levels and radiographic imaging was reviewed to rule out and potential malignancy. patients not treated with testosterone but with a similar clinical setting were compared (group 2). results: thirteen patients were identified with esld and mw, group 1 (n=8), group 2 (n=5). demographics were as follows: (group 1) 6 males, 2 females, and mean age 56 years; (group 2) 4 males, 1 female, mean age 53 years. disease etiology for group 1: (hcv (n=5), hcv/hiv (n=l), aih (n=l), cryptogenic (n=l)) group 2: hcv (n=4), hcvilaennec's (n=l). most patients in group 1 stated a subjective improvement in muscle strength, and overall wellbeing. in group 1, serum albumin levels increased from day 1, 30, 90 (2.18/2.7875/3.45 g/dl, respectively) while those in group 2 did not (2.30/2.16/2.0 g/dl) (p= 0.0002). similar results were seen for prealbumin (days 1, 30, 90) group 1 (10/18/22 mgldl) versus group 2 (10/8/6 mg/dl). in group 1, 7/8 patients received a liver transplant, while 1 remains listed for olt. in contrast, 3/5 patients in group 2 were transplanted while 2/5 died. two patients in each group were not listed at the time of evaluation due to extreme deconditioning. of those in group 1, one patient was transplanted and another patient remains listed for olt with an albumin greater than 3.5 gldl. this patient presently has no radiographical evidence of ascites. the two patients of group 2, who were not listed for lt due to extreme deconditioning, died on day 28 and 35, respectively. there was no rise in the tumor marker elevations in any of the patients. after transplantation, no patients in either group suffered from rejection or graft failure. ascites accumulation, as assessed by abdominal ultrasound imaging, appeared to be less in patients receiving ttr (group 1) compared to all 3 living patients in group 2, who had persistent ascites. one patient of the treatment group developed marked cholestasis 10 days after starting ttr and received a liver transplant 14 days later. prior to starting ttr, this patient underwent weekly large volume paracentesis and suffered from marked deconditioning and recurrent bouts of hepatic encephalopathy while maintaining a meld score of 8-9 points over a 6-month period. conclusion: our preliminary data suggest that ttr increases muscle strength (subjective measures), stimulates albumin synthesis and improves the outcome of olt. thus, ttr (testosterone gel 1%) appears to be of great benefit in patients with esld and muscle wasting. further studies are needed to determine the efficacy and safety of ttr in patients with esld. percutaneous ethanol injection (pei), radiofrequency tumor ablation (rf), and laser thermal ablation (lta) are percutaneous techniques used in the treatment of unresectable hcc. percutaneous seeding of neoplastic cells along the needle track is a rare complication of these tecniques, and it has been recently suggested to consider with caution these techniques as a "bridge" treatment in cirrhotic patients with hcc candidates to lt. the aim of this study was to verify whether percutaneous ablation treatments represent a risk factor for hcc recurrence in these patients. during the period 1990 -2002,32 patients (mean age 53 yrs) with hcc complicating liver cirrhosis previously treated with percutaneous ablation treatments underwent lt in 3 italian centers. among the 32 patients, 13 had monofocal hcc, 19 multifocal hcc. in the latter group, 5 had monolobar disease, 14 had bilobar disease. considering the 13 patients with monofocal hcc, 7 were treated with rf (associated with transarterial chemoembolization [tace] in one case), 4 with pei (associated with tace in one case), 2 with rf and pei (associated with tace in one case). among the 19 patients with multifocal hcc, 9 were treated with rf (associated with tace in four cases), 9 with pei (associated with tace in four cases), 1 with lta associated with tace. on the whole, 38 nodules were treated in the 32 patients. furthermore, 31 additional untreated hcc nodules were found at pathological examination of the explanted liver. among the 19 rf-treated nodules, 6 showed total necrosis, 12 partial necrosis, 1 absence of necrosis. among the 15 pei-treated nodules 4 showed total necrosis, 5 partial necrosis, 6 absence of necrosis. the 2 nodules treated with rf and pei showed total necrosis in one case and partial necrosis in the other one while the 2 nodules treated with lta, showed partial necrosis. the mean follow-up period was 35 months (range 3-130 months). five patients died during the follow up and the actuarial survival rate was 94% at one year, 85% at 3 and 5 years. three patients (9.4%) had post-transplant recurrence of hcc which was the cause of death in all cases; the diagnosis of tumor recurrence was made at 2 months from lt (peritoneal carcinomatosis), at 8 months from lt (bone), and at 9 months from lt (abdominal liymphnodes and bone), respectively. the 3 patients had all been treated with pei before lt and no one of them showed recurrence of hcc at the abdominal wall level. according to our results, percutaneous ablation techniques performed before lt do not seem significantly affect both survival and tumor recurrence rate in patients transplanted because of hcc complicating liver cirrhosis. in particular, no cases of recurrence at the abdominal wall level were observed in the overall series and no one case of hcc recurrence was observed in patients treated with rf alone or in combination. background: transjugular intrahepatic portosystemic shunt (tips) is valuable in the management of portal hypertension (phtn) in patients awaiting liver transplantation (olt). recurrent phtn after olt can be refractory to medical management and portosystemic shunting may be considered in rare situations, either as a bridge to retransplantation or as definitive therapy. in this report we review our experience and outcomes with tips after olt. methods: records of 309 primary adult olt recipients, between january 1995 and december 2003, were retrospectively reviewed. evidence of refratory post-olt phtn was noted. those who required tips were the subject of this review. demoraphics, indications for olt and tips, evidence of allograft dysfunction and outcomes after tips were described. results: during the study period 6 tips were placed in 6 patients at a mean of 13.5 months after olt (range 2-36 months). there were 3 males and 3 females, age 53.0l7.8 years. hepatitis c was the primary indication for olt in 5 and primary biliary cirrhosis in 1. indications for tips included refractory ascites (6), variceal bleeding (2) and various degree of associated hepatic vein outflow stenosis (3). five patients had resolution of phtn and 1 patient with refractory ascites had severe hepatic vein outflow stenosis and associated hepatitis c in the allograft. two patients required re-olt for recurrent hepatitis c. there were 3 deaths: liver failure 1 month after tips done in the setting of allograft dysfunction, 3 months after tips with subsequent re-olt and organ failure, and lung cancer 5 months after tips. bridging fibrosis was present in 3 patients, 2 needed re-olt and 1 died from liver failure while waiting for olt. currently, 3 patients are alive without evidence of phtn 3, 9 and 58 months after tips. conclusions: 1-tips effectively and safely controls phtn after olt. 2-tips in setting of a moderate to severe allograft dysfunction does not abrogate the need for re-olt and can be associated with a high mortality. 3-timely re-olt should be considered in the presence of fibrosis from recurrent hcv and phtn. 4-tips has been successful in the setting of mild-moderate hepatic vein outflow stenosis and phtn. introduction: biliary tract complications after liver transplantation (lt) are reported to occur in 13% to 35% of patients. the frequency of biliary infections is not well documented. therefore, we prospectivly obtained bile samples during diagnostic and or therapeutic endoscopic retrograde cholangiography (ercp) in all liver transplant patients from 0112001 to 1112002. methods: in 67 patients (mean age 53.6 years, range 24-73 years) a total of 172 ercp's were performed (2.57 ercflpatient). all patients had a lumen adapted, end-to-end biliary anastomosis. lt was performed between 3.2 months and 2.8 years before the intervention. cholangitis was defined as the presence of cholestasis with clinical and biochemical signs of infection not explained otherwise. only in 19 cases of 172 interventions, positive culture results were combined with clinical signs of cholangitis. the follwoing risk factors were identified stenosis of any type, plastic endoprothesis, choledocholithiasis and previous papillotomy. patients with these risk faktors had significantly higher incidence of positive bile cultures (77.6% vs 40%, p<o,ool; chi-square). in addition, they were more often infected with multiresistant bacteria (p<0.05). 49.3% were resistant to gentamycin, 44.0% to ceftriaxone. vancomycin (1.7% resistance) was the most effective antibiotic. imipenem (18.2%), ciprofloxacin (27.5%) and amoxicillin (28.8%) had intermediate incidence of resistant bacteria. conclusion: in patients with biliary tract complications after lt, biliary infection is common. clinical cholangitis, however, occurs predominantly in those with risk factors. gram-positive bacteria were more commonly demonstrated than gram-negative. cocolonisation with anaerobes andlor candida can occur. ciprofloxacin and amoxicillin seem to be appropriate antibiotics for prophylaxis. selection of an empiric antibiotic therapy for patient after liver transplantation with sings of cholangitis should be directed toward agents with a low incidence of resistance or a combination therapy. disclosures: thomas buratti -no relationships to disclose adolescent health related quality of life after liver transplantation. shikha s sundaram, katie neighbors, lisa amaruso, james sinacore, estella m alonso, northwestern university, chicago, il background: adolescence is an important developmental stage, during which children with chronic diseases begin to assume some of the responsibility for their own medical care. many children who received liver transplants as infants are now reaching adolescence and their unique health perceptions should be considered in providing appropriate healthcare. the aims of this study were to 1) quantify the health related quality of life (hrqol) in adolescent liver transplant recipients and compare this to a published healthy population, 2) compare parental ratings of adolescent hrqol to adolescent self-reports of hrqol, and 3) assess adolescent hrqol and it's relationship to disease specific disability. methods: a cross sectional mailing survey of liver transplant recipients between ages 11-18 years, surviving transplant by at least 1 year and receiving care at our center was conducted. primary caregivers of these patients were also recruited. adolescents completed the child health questionnaire, child form 87 (chq-cf87) and primary caregivers the child health questionnaire, parent form 50 (chq-pf50). demographic and medical data was collected using an institutional transplant database and standard demographic survey. a disease specific disability score was calculated using the liver transplant disability scale (ltds). mean scores for 12 chq-cf87 subscales, including general and mental health, physical and social functioning and self-esteem were calculated. these were compared to published normative values from a healthy population and mean chq-pf50 subscale scores. results: thirty-seven of 53 surveys mailed were received for a response rate of 69.8%. adolescent respondents had a mean age of 14.122.13 years, were 65% female and 81% caucasian. cadaveric donors accounted for 67.6% of transplants and 38% of patients were transplanted for biliary atresia. a majority (86.5%) of respondents had private insurance and 86.5% indicated mothers were their primary caregivers. after transplantation, adolescents had a mean global health score of 59.28518.47 as compared to mean normative score of 66.38214.62, p=0.031. they had higher scores than the normative population for role behavioral, 94.89211.29 vs. 86.542 21.45, p=o.ool and mental health, 77.53z10.81 vs. 72.65215.95, p=0.021. all other scores were similar between the 2 groups. significant positive correlations between adolescent and parental scores were observed in relation to physical functioning, (r =0.64, p<0.0005), self-esteem (r = 0.72, p < 0.0005) and mental health (r = 0.59, p<0.0005). the remaining sub-scales lacked statistically significant correlation. an inverse relationship between inpatient hospitalization during the previous 6 months and mean global health score was observed 0 inpatient days had a mean of 76214.77,l-4 inpatient nights had a mean of 65235.5 and greater than 4 inpatient nights 38.3243.1. there were no other significant correlations between subscale scores and ltds scores. conclusion: adolescent liver transplant recipients report their hrqol as equal or better than the normative population. parents may be adequate proxies for some, but not all domains of adolescent hrqol in this population. were quantitated from wild type and fxrnull mice. results:transfection of the moatp4-pgl3 into hepg2 cells led to a 5.5fold ( 2 1.0 ) increase in reporter gene activity over vector controls. co-transfection with the bile acid receptor fxr and addition of its ligand, chenodeoxyacholic acid (cdca), resulted in a further 6.25 ( t 0.35 ) -fold increase suggesting fxr-mediated transactivation.fxrlrxr cotransfection and addition of ligands, cdca and 9-cis retinoic acid decreased transactivation suggesting rxr-mediated antagonism ( jbc, 278: 10028,'03 ). hnf-1 a also transactivated moatp4 promoter as shown previously. mutation of ir-1 sequence in moatp4 promoter abolished fxr-transactivation. emsa analysis using rat liver nuclear extracts showed specific binding of nuclear proteins to moatp4 fxre which was supershifted by fxr and rxr antibodies. mrna levels for moatp4 in fxrnull mice were reduced by 48.3 % compared to wild-type mice ( 0.612 in wild-type vs 0.296 infxr-null mice). conclusion: based on the above data, we suggest that moatp4 is regulated by bile acids by binding to fxr via ir-1 in its promoter. , and g1249a encoded for amino acid changes i1173f, a1450t, and v4171, in the third membrane spanning domain, the second nucleotide binding domain, and the second membrane spanning domain, respectively. the full-length coding regions of the reference mrp2 and three mutants sequences were inserted into the baculovirus genome. sf9 insect cells were infected with the baculovirus stocks, and membranes were isolated from the infected cells expressing mrp2 or the mutants. atpase activity was measured by determining release of inorganic phosphate by a colorimetric assay. atp-dependent transport of 3h-e23g (estradiol-p-(3p-d-glucuronide)) or 3h-e217g (estradiol-p-(17p-d-glucuronide)) was determined by uptake into inside out membrane vesicles collected on filters and counted by liquid scintillation. results: the total expression levels of mrp2 and the three mutants in the infected sf9 cells were comparable by densitometry of western blots of three levels of each protein, indicating total expression in sf9 cells was not influenced by the mutations. mrp2 atpase activity was significantly stimulated by the uricosuric probenecid lmm, the cholestatic 250pm, and the mild choleretic e23g 1mm. although the control and stimulated at-pase activities of the v417i mutant were not different from mrpz, the i1173f mutant had significantly less baseline and less stimulated atpase activities, indicating impaired function. a1450t had less probenecid-stimulated atpase activity, but activity stimulated by e217g and e23g was not different from mrp2. ursodiol saturably stimulated atpase activity of mrpz by 520% with km =72138pm. although the three mutants transported 3h-e23g with affinities not significantly different from mrp2 (km=122223 pm), the vmax of i1173f was only 5.1% (15414 pmollmglmin) and the vmax of v417i was 157% (4701251 pmollmglmin) compared to the vmax of mrpz (29912186 pmollmglmin); the vmax of a1450t was not different (2967287 pmollmglmin). ursodiol activated transport of 3h-e23g (60nm) with a peak effect of 950% at 100pm; higher ursodiol concentrations (up to 1mm) returned transport to near control values. mrpz-mediated atp-dependent transport of 3h-e217g (44nm) in the presence of ursodiol (1oopm) for the mutants differed from mrp2 (lo%, 67%, and 140% for i1173f, a1450t, and v4171, respectively). conclusions: ursodiol markedly activated mrp2 transport of e23g. although ursodiol has several anticholestatic effects, these data show its direct effect on transport of mrpz substrates, and may suggest activation of mrp2 as a target in the treatment of cholestatic liver disease. the mrp2 mutants showed several important differences in activity, including changes in transport vmax, and transport in the presence of ursodiol, and activation of atpase activity by ursodiol, probenecid, and the mrp2 substrates e217g and e23g. further characterization of these mrp2 mutants and others will lead to a better understanding of mrp2 structure and function and may yield new therapeutic approaches. also, although e217g is an established mrp2 substrate, these data show that its noncholestatic isomer e23g is also an mrpz substrate, implicating mrp2 in the disposition of other estrogen metabolites, which can be interconverted to cholestatic compounds like e217g. argentina; nicholas f larusso, mayo medical school, clinic and foundation, rochester, m n previous work from our laboratory is consistent with an important role for aquaporins (aqps), a family of water channel proteins, in canalicular bile secretion principally via agonist-induced exocytic insertion of aqps into the hepatocyte canalicular membrane. to further define the pathways and molecular mechanisms for water movement across hepatocytes, our aim here was to directly assess osmotic water permeability (pf) and activation energy (ea) in basolateral (blmv) and canalicular membrane (cmv) vesicles using stopped-flow spectrophotometry. scattered light intensity was used to follow changes in vesicle volume in response to gradients induced by sucrose solutions of different osmolarities. results: the time course of scattered light for blmv and cmv fit a single-exponential function. in blmv, pf was 10824 pm.sec-' (25 oc) with an ea of 7.7 kcallmol; in cmv, pf was 8615 pm.sec-' (25 oc) with an ea of 8.0 kcallmol. the aqp blocker, dimethylsulfoxide, significantly inhibited the pf of both basolateral (8124 pm.sec-l; -25%) and canalicular (59?4 pmsec-'; -30 %) vesicles consistent with the presence of aqps in both vesicle populations. experimental data for cmv from dibutyryl camp-treated hepatocytes fit best to a double-exponential curve implying the presence of two functionally distinct cmv populations; one population had pf and ea values similar to those of cmv from untreated hepatocytes and the other population had a very high pf (6552135 pm.sec-l, 25 oc) and very low ea (2.8 kcallmol). dimethylsulfoxide completely inhibited the high pf value in this second vesicle population. in contrast, the pf of blmv was unaltered by camp treatment of hepatocytes. conclusions: our data are consistent with the notion that the canalicular but not the basolateral membrane becomes more permeable to water after a secretory stimulus due to the insertion of aqp molecules. the data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation. disclosures: ariel j caride -no relationships to disclose sergio a gradilone -no relationships to disclose bing q huang -no relationships to disclose key: cord-350640-sz6xj5o3 authors: menzel, nicolas; fischl, wolfgang; hueging, kathrin; bankwitz, dorothea; frentzen, anne; haid, sibylle; gentzsch, juliane; kaderali, lars; bartenschlager, ralf; pietschmann, thomas title: map-kinase regulated cytosolic phospholipase a2 activity is essential for production of infectious hepatitis c virus particles date: 2012-07-26 journal: plos pathog doi: 10.1371/journal.ppat.1002829 sha: doc_id: 350640 cord_uid: sz6xj5o3 hepatitis c virus (hcv) has infected around 160 million individuals. current therapies have limited efficacy and are fraught with side effects. to identify cellular hcv dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. using this approach we identified the mapk/erk regulated, cytosolic, calcium-dependent, group iva phospholipase a2 (pla2g4a) as a novel hcv dependency factor. inhibition of pla2g4a activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. exogenous addition of arachidonic acid, the cleavage product of pla2g4a-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. strikingly, production of infectious dengue virus, a relative of hcv, was also dependent on pla2g4a. these results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define pla2g4a-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny. approximately 160 million people are chronically infected with hepatitis c virus (hcv) [1] . without treatment, at least 20% of patients will develop liver cirrhosis and of these, approximately 15% will progress to liver cancer within ten years [2] . hcv is the sole member of the genus hepacivirus within the family of flaviviridae. its plus strand rna genome encodes a polyprotein that is flanked by non-translated regions. proteolytic processing releases ten viral proteins which coordinate viral rna replication and particle assembly. the non-structural proteins ns3, ns4a, ns4b, ns5a and ns5b in conjunction with cellular co-factors are both necessary and sufficient to catalyze genome replication [3] . core protein, envelope protein 1 and 2 (e1, e2) reside in the very n-terminal portion of the viral polyprotein and compose the virus particle encasing the rna genome. these proteins are essential for virus assembly. interestingly, the ion channel protein p7, and the ns2 protease also contribute functions to the production of infectious viral progeny [4, 5] . lipid droplets have been recognized as an essential cellular organelle for production of infectious hcv progeny [6] . during virus production core protein resident on lipid droplets recruits viral proteins and rna, which is an essential prerequisite for virus production [6] . in turn, core protein is loaded onto these cellular organelles through an interaction with diacylglycerol acyltransferase-1 (dgat-1) [7] , an enzyme which catalyzes the final step in the biosynthesis of triglycerides that is essential for lipid droplet biogenesis [8] . in addition, host factors involved in the biosynthesis and secretion of human lipoproteins have emerged as essential cofactors for virus production. specifically, apolipoprotein b (apob), apolipoprotein e (apoe) and microsomal triglyceride transfer protein (mttp) were shown to contribute to virus production [9, 10, 11] . likely as a consequence, infectious hcv is a ''lipo-viro particle'' rich in cholesteryl esters and comprising viral proteins, apob and apoe [12, 13] . cells constantly respond to environmental changes by sensing these alterations through dedicated receptors and associated signaling cascades that reprogram cellular processes. such signaling-dependent modifications may also influence important cellular hcv dependency factors regulated by these pathways thus providing a lead for identification of novel and possibly druggable host factors crucial for hcv. using this approach we show that mitogen-activated protein kinase (map kinase) regulated enzymatic activity of pla2g4a is crucial for production of infectious hcv progeny highlighting the intricate involvement of host cell lipids and lipid-modifying enzymes in the replication of this virus. the mapk/erk signaling pathway is involved in hcv assembly/release cultured cells respond to multiple stimuli by growth factors and hormones present in serum-containing culture media. therefore, to reduce the complexity of our experimental system we developed a transient virus replication assay and cultured cells in serum-free conditions. when transfecting our infectious firefly luciferase reporter virus genome luc-jc1 [14] into highly permissive huh-7.5 human hepatoma cells [15] cultured in the presence or absence of serum, we measured comparable levels of luciferase activity 4 to 48 h after transfection ( figure s1a ). likewise, transduction of luciferase activity upon inoculation of naïve cells with culture fluid from the transfected cells was similar ( figure s1b ). therefore, in this transient time course viral rna replication and production of infectious progeny particles was comparable in both serum-free and serum-containing conditions. to identify new host factors involved in hcv replication and/ or virus production we used pathway-specific inhibitors of central signal transduction cascades including akt/pkb, mtor, wnt, jnk and mapk/erk (figure 1 and data not shown). to reveal rapid, signal-mediated changes of rna replication and virus production we added the inhibitors 41 h post transfection during the logarithmic growth phase of cells. one hour later culture medium was replaced with fresh medium with or without inhibitors and virus production as well as rna replication was assessed 6 h later. this procedure which is summarized in figure 1a ensures that specifically infectivity of particles produced and released during inhibitor treatment -and thus blockade of the respective signaling cascade -is evaluated. among the inhibitors tested, u0126 a selective inhibitor of the mapk/erk pathway, substantially decreased the production of infectious virus particles as is evident from the dose-dependent reduction of luciferase activity in the inoculated cells ( figure 1b) . interestingly, u0126 did not measurably impede rna replication and only affected hcv particle production when added in serum-free medium. this latter finding may be related to a much more efficient blockade of the mapk/erk pathway in serum-free conditions compared to serum-containing medium that is evident from a lower level of phosphorylated erk1 and erk2 in the presence of the drug when serum was absent ( figure 1c ). collectively, these results suggested that the applied doses of u0126 efficiently prevented phosphorylation of erk under serum-free conditions thus impeding production of infectious hcv progeny. interestingly, pd98059 and sorafenib which inhibit the mapk/erk pathway upstream of u0126 [16] [17] also reduced production of infectious hcv particles ( figure s2 ) further confirming the role of mapk/ erk signaling in hcv morphogenesis. notably, at least under serum free conditions, these inhibitors also reduced rna replication. this may either be linked to a more potent suppression of mapk/erk signaling or due to inhibition of additional signaling events. to confirm these findings, we analyzed the impact of u0126 on the production of infectious wildtype jc1 particles [18] in transfected cells ( figure 1d and e). congruent to our findings with reporter viruses, treatment of cells with u0126 reduced both extracellular and intracellular levels of infectious hcv particles ( figure 1d ). although intracellular levels of core and ns5a were moderately reduced in the absence of serum ( figure 1e ), addition of u0126 did not further reduce abundance of viral proteins suggesting that the inhibitor did not prevent rna translation or rna replication. we also tested if presence of u0126 interferes with hcv cell entry by adding the drug to infectious reporter virus particles that had been produced in the absence of the inhibitor ( figure s3 ). since hcv infection was not decreased, we concluded that addition of u0126 does not prevent hcv cell entry but interferes with production of infectious progeny particles under serum-free conditions. the mapk/erk regulated pla2g4a is involved in production of infectious hcv besides activating transcription factors in the nucleus, erk1/2 also directly regulate the activity of cytoplasmic enzymes. therefore, we searched for cellular factors that are regulated by the mapk/erk pathway and operate at the er, the presumed site of hcv particle production. based on these criteria we focused on pla2g4a, which is activated by mapk/erkdependent phosphorylation [19] and recruited to the er by ca 2+ ions [20, 21] , as a possible candidate host factor that may be responsible for the observed u0126-dependent blockade of hcv particle production. in line with our finding that u0126 only inhibited hcv in the absence of serum, phosphorylation of pla2g4a was selectively inhibited under these conditions and not affected when serum was present (figure 2a ). to investigate if pla2g4a activity is relevant for the production of infectious hcv particles we treated luc-jc1 transfected cells with pyrrolidine-2 (py-2), a specific inhibitor of this type of phospholipase [22, 23] . irrespective of culturing these cells in the presence or absence of serum, we observed a strong and dose-dependent inhibition of production of infectious particles resulting in a more than 100-fold reduction of luciferase transduction at 5 and 20 mm of py-2, respectively ( figure 2b ). similar to u0126, rna replication and cell entry were not affected by py-2 ( figure 2b and data not shown). moreover, accumulation of hcv proteins in cells transfected with authentic hcv was not changed by addition of py-2 ( figure 2c ). however, the human genome encodes more than 30 phospholipase a2s. these enzymes cleave fatty acids at the c2 atom of phosphoglycerides and thus modulate membrane properties. among all pla2s only pla2g4a, which is recruited to perinuclear membranes by ca 2+ and activated by extracellular stimuli via the mitogen activated protein kinase pathway, specifically cleaves lipids with arachidonic acid. metabolism of arachidonic acid yields prostaglandins and leukotriens, important lipid mediators of inflammation. we show that inhibition of pla2g4a produces aberrant hcv particles and that infectivity is rescued by addition of arachidonic acid. our results suggest that a specific lipid (arachidonic acid) is essential for production of highly infectious hcv progeny, likely by creating a membrane environment conducive for efficient incorporation of crucial host and viral factors into the lipid envelope of nascent particles. strikingly, pla2g4a is also essential for production of highly infectious dengue virus (denv) particles but not for vesicular stomatitis virus (vsv). these observations argue that hcv and denv which unlike vsv produce particles at intracellular membranes usurp a common host factor (pla2g4a) for assembly of highly infectious progeny. these findings open new perspectives for antiviral intervention and highlight thus far unrecognized parallels in the assembly pathway of hcv and denv. [14] and seeded into replicate tissue culture plates. at 4 h post transfection (hpt), medium was changed to culture conditions with or without fcs. inhibitors (e.g. u0126) were added into the medium at 41 hpt. one hour later, medium was replaced by medium containing given doses of the inhibitor. finally, at 48 hpt cells and culture fluids were analyzed. (b) hcv rna replication in cells prepared as in (a) was measured by using a luciferase reporter assays (top panel). the release of infectious particles was titers of both extracellular as well as intracellular infectivity were strongly impaired by 50-and 10-fold, respectively ( figure 2d ). interestingly, py-2 also inhibited production of infectious genotype 1a, 3a and 5a hcvcc particles, indicating that pla2g4a activity is important for hcv virus production across different hcv genotypes ( figure s4 ). intracellular phospholipase a2 enzymes comprise cytosolic, ca 2+ -dependent enzymes (cpla2) as well as the structurally similar ca 2+ -independent lipases (ipla2) [24] . to investigate if ipla2 activity contributes to hcv particle production we treated luc-jc1 transfected cells with bromenol lactone (bel) a specific inhibitor of ipla2 [25, 26] . however, bel neither affected hcv rna replication nor production of infectious particles ( figure 2e ), supporting the notion that specifically the pla2g4a is involved in the hcv life cycle. to determine whether utilization of pla2g4a activity is unique to hcv or common to other enveloped viruses, we analyzed production of infectious vsv and denv in the presence of py-2. in case of vsv, a member of the family rhabdoviridae which assembles infectious virus particles at the plasma membrane [27], we used a replication competent reporter virus (designated vsv*m q ) that expresses a gfp transgene from an additional transcriptional unit placed between the g and l genes [28] . interestingly, py-2 treatment of vsv*m q infected huh-7.5 cells did neither affect intracellular level of gfp ( figure 3a ) nor production of infectious vsv progeny particles ( figure 3b ) indicating that unlike for hcv, production of infectious vsv particles did not rely on pla2g4a activity. in contrast, production of infectious denv, a relative of hcv from the genus flavivirus that is thought to assemble at intracellular membranes [29] , was heavily impaired by py-2 treatment ( figure 3 ). strikingly, like for hcv, rna replication was not affected ( figure 3c ) and release of particles was only moderately reduced as is evident from ca. 10-fold lower copy numbers of viral rna in the culture fluid of py-2 treated cells compared to mock treated denv infected cells ( figure 3d ). importantly, when we quantified the infectivity of released particles using a limiting dilution assay we noted an approximately 1,000-folder lower infectivity titer for particles produced in the presence of py-2 as compared to particles produced in the absence of the compound ( figure 3e ). since py-2 did not grossly inhibit cell entry ( figure 3f ) we concluded that inhibition of pla2g4a activity via py-2 primarily impairs infectivity of released particles ( figure 3e ). in summary these results indicate that pla2g4a is a key host enzyme required for efficient release and high infectivity of hcv and denv, but not vsv particles. to corroborate our finding that pla2g4a is involved in production of infectious hcv particles we knocked down expression of the enzyme in hcv transfected cells using rna interference ( figure 4) . surprisingly, despite decreased abundance of pla2g4a in sirna-transfected cells ( figure 4a ), we observed at best a moderate reduction of the total cellular pla2g4a activity as determined by a commercial enzymatic test ( figure 4b ). in line with the result of the enzymatic test, knock down of pla2g4a did not impede production of infectious hcv particles ( figure 4c ). in contrast, reduction of virus titer correlated again with pla2g4a inhibition upon treatment with py-2 ( figures 4b and c) . to confirm that indeed pla2g4a was directly contributing to hcv particle production rather than alternative enzymes which may share a similar enzymatic activity, we combined sirna treatment with application of py-2. under these conditions the reduction of pla2g4a within cells should increase the susceptibility of hcv to treatment with py-2 because due to lower abundance of the host factor a lower level of the drug should be sufficient to prevent hcv particle production. as expected, sirna and py-2 treatment did not decrease hcv rna replication ( figure 4d and e). in fact rna-replication moderately increased in cells that were treated this way compared to cells receiving a scrambled sirna and no py-2 ( figure 4e ). despite of this we found significantly lower levels of infectious virus particles secreted from cells receiving the pla2g4a-specific sirna and 5 or 10 mm py-2 as compared to cells treated with the scrambled sirna and these drug doses ( figure 4e ). collectively, these data indicate that sirna treatment does not sufficiently suppress pla2g4a enzyme activity to limit hcv production in huh-7.5 cells. however, when adding the pla2g4a-specific inhibitor to these cells, knock down of pla2g4a increased the susceptibility to the drug, arguing that the abundance of enzymatically active pla2g4a is important for production of infectious particles. arachidonic acid (aa) restores efficient production of infectious hcv in the absence of pla2g4a activity mammals encode genes for more than 30 phospholipase a 2 s (pla 2 -s) and related enzymes which are further divided into several classes [24] . these enzymes share the property of hydrolyzing the sn-2 position of membrane glycerophospholipids to release free fatty acids and lysophospholipids. among pla 2 -s only the pla2g4a displays selectivity for cleaving phospholipids carrying aa at the sn-2 position [30, 31] . while local release of aa modifies membrane properties including curvature and fluidity [32, 33, 34] , this lipid is also a precursor for production of bioactive prostaglandins (pgs) and leukotriens (lts) which play essential roles in inflammatory reactions. in fact, production of pgs and lts is reduced by ca. 90% in pla2g4a deficient mice highlighting the dominant role of this phospholipase for generation of these lipid mediators [35, 36] . given these circumstances we wanted to distinguish if properties of the pla2g4a-derived cleavage products (aa and/or lysophosphatidic acid) directly contribute to hcv particle production, or if these molecules may indirectly promote virus production through activating inflammatory reactions. since aa is further metabolized by lipoxygenases and cyclooxygenases to yield prostaglandins and leukotrienes we determined by inoculation of naïve cells with culture fluids collected at 48 hpt and determination of luciferase activity in cells 72 h after inoculation (bottom panel). data are shown as means +/2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (c) analysis of erk1/2 expression and phosphorylation in luc-jc1 transfected and u0126-treated cells. cell lysates were collected 48 hpt. erk proteins were detected using erk-and phospho-erk-specific antibodies (bottom and top panel, respectively). (d) cells were transfected with jc1 rna and subjected to the assay described in (a). culture fluids and cells were harvested 48 hpt and extracellular and intracellular infectivity was determined using a limiting dilution assay. intracellular infectious particles were collected by three repetitive cycles of freeze and thaw. (e) aliquots of cell lysates were analyzed for expression of ns5a, core and actin using mono-specific antibodies. doi:10.1371/journal.ppat.1002829.g001 assessed whether inhibition of aa metabolism by application of (s)-flurbiprofen or nordihydroguaiaretic acid (ndga), inhibitors of cyclooxgygenases and lipoxygenases, respectively, prevents efficient hcv particle production. however, neither drug modulated hcv rna replication or decreased production of hcv particles ( figure s5 ). in fact high doses of (s)-flurbiprofen even slightly increased levels of infectious hcv ( figure s5a ). these results argue against the notion that aa metabolites and their biological activities are crucial for production of infectious hcv. next, we analyzed if application of aa or related fatty acids restores production of hcv particles in the presence of the pla2g4a inhibitor py-2. remarkably, we observed a pronounced and dose-dependent restoration of infectious particle production in the presence of aa ( figure 5a ). importantly, 5,8,11,14-eicosatetraynoic acid (etya) a derivative of aa with four triple bonds at positions 5, 8, 11, and 14 of the acyl backbone did not restore virus production ( figure 5b ). likewise further natural fatty acids with 1, 2, 3 or 4 double bonds at various positions did not relieve the blockade of virus production caused by addition of the pla2g4a inhibitor ( figure s6 ). among all tested lipids only aa itself and to a moderate level 5,6-dehydro aa (5,6-dha) restored virus production ( figure 5a and c). notably, aa did not increase hcv cell entry since the infectivity of particles produced in the absence of the drug was not stimulated by addition of aa during cell entry ( figure s7 ). next, we investigated if repression of denv infectious particle production is also relieved by addition of aa. as is depicted in figure s8 , we noted a trend that high doses of aa partially restore production of infectious denv progeny in the presence of py-2. however, the rescue of infectious virus production was moderate and not statistically significant. to investigate if in the context of hcv, addition of aa truly rescues the blockade of pla2g4a enzymatic activity and not simply over-stimulates production of hcv particles, we applied aa to hcv transfected cells in the absence of py-2. interestingly, under these conditions we observed a moderate increase of hcv infectivity ( figure 5d ). this finding mirrors the moderate gain of infectivity when cells were treated with flurbiprofen that prevents aa metabolism. since both treatments are expected to increase availability of aa, these data suggesting that availability of aa may be sub-saturating in non-treated hcv transfected cells. collectively, these results indicate that specific properties of aa, which is created by cleavage of glycerophospholipids carrying this lipid at the sn-2 position by pla2g4a, are important for production of highly infectious hcv progeny particles. pla2g4a contributes to association of core with lipid droplets and to core protein envelopment pyrrolidine has been described as precursor for compounds against the ns3-protease and rna-polymerase of hcv [37] . as the pla2g4a-specific inhibitor py-2 shares a heterocyclic ring with pyrrolidine we wanted to exclude that py-2 may prevent virus production indirectly by inhibiting the viral protease or polymerase. both enzymes contribute to active rna replication complexes and may be required to feed in newly synthesized viral rna into assembling virus particles. to address this, we treated jc1transfected cells with a polymerase inhibitor (29-c-methyladenosine; 29cma), a protease inhibitor (boceprevir), with py-2, or with quinidine, the latter being a class i antiarrhythmic drug recently found to inhibit production of infectious hcv [38] . as expected, only the rna-replication inhibitors (29cma and boceprevir) reduced abundance of hcv rna in transfected cells ( figure s9a ) dose dependently. while at the used doses (well beyond the ic 90 for all compounds) all drugs moderately impaired release of hcv core protein (2-5-fold), only py-2 and quinidine strongly reduced infectivity of particles ( figure s9c ) to levels more than 20-fold lower compared to the dmso control. thus, it is unlikely that indirect effects of py-2 on rna-replication are responsible for the reduced amount of secreted particle and their impaired infectivity. rather these findings argue that py-2 directly interferes with hcv assembly and the infectivity of released particles. to investigate how py-2 impairs hcv assembly, we investigated the subcellular localization of core, and pla2g4a in the presence or absence of py-2. adipose differentiation-related protein (adrp), a host protein interacting with the surface of lds was stained in parallel. since we were unable to detect endogenous pla2g4a with commercial antibodies, we created a stable huh-7.5 cell line ectopically expressing a gfp-tagged pla2g4a protein. as is shown in figure s10 we did not see gross changes of the distribution of these proteins during the short term py-2 treatment. moreover, localization of gfp-pla2g4a did not differ between cells expressing hcv proteins and those cells that were not positive for hcv. these findings provide preliminary evidence that localization of epitope tagged pla2g4a is not influenced by hcv. more work, ideally with untagged pla2g4a will be needed to fully resolve the localization and trafficking of this protein in the presence or absence of hcv and py-2. next we assessed the amount of intracellular core protein that is resistant to proteolysis by proteinase k. reasoning that core protein that is surrounded by a membrane should be protected from digestion by the protease, this assay estimates the number of core protein that has acquired a lipid envelope. since among members of the family flaviviridae virus particle envelopment depends on expression of functional glycoproteins and as for hcv deletion of e1-e2 abrogates production of infectious progeny [39] , we used a jc1 mutant carrying a deletion of e1-e2 genes as a control and reference. as expected, when the protease was added to cell lysates prepared by repetitive cycles of freeze together with detergent (triton x-100), the viral protein was completely degraded ( figure 6a ). however, when cell lysates were incubated with the protease in the absence of detergent, a substantial amount of core protein resisted digestion indicating protection by a membrane envelope. notably, the amount of protected core protein was approximately 3-fold lower in py-2 treated compared to dmso treated jc1 transfected cells, resembling the phenotype figure 2 . inhibition of pla2g4a by py-2 impairs infectivity of hcv. (a) u0126 prevents phosphorylation of pla2g4a in the absence of serum. cells were cultured in presence or absence of serum and the u0126-assay was carried out as described in figure 1a . cell lysates were analyzed using antibodies specific for pla2g4a or the s505-phosphorylated enzyme. (b) luc-jc1-transfected cells were treated with indicated doses of py-2 as outlined in figure 1a . the influence on rna replication (left panel) and production of infectious particles (right panel) was determined as described in the legend to figure 1 . data are shown as means +/2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (c) analysis of ns5a and core protein levels in jc1-transfected cells in the presence or absence of py-2. (d) secreted and intracellular infectious hcv was quantified using a limiting dilution assay. (e) an ipla 2 -specific inhibitor does not impede hcv rna replication or virus production. huh-7.5 cells were transfected with luc-jc1 and treated with given doses of bel, an ipla2-specific inhibitor, using the procedure outlined in figure 1a . luciferase activity was measured in transfected (left panel) and in the inoculated cells (right panel). data are shown as means +/2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). doi:10.1371/journal.ppat.1002829.g002 of cells transfected with jc1de1e2 ( figure 6a and b), arguing that py-2 had decreased the amount of enveloped core protein structures. since trafficking of core protein to lipid droplets (lds) [6] is crucial for assembly of infectious progeny particles we analyzed the influence of pla2g4a on the accumulation of core protein on lds. to this end, we prepared lds from jc1-transfected and py-2 treated cells and analyzed the abundance of core protein on the surface of this cellular organelles. the quality of our ld preparation was monitored by detection of actin (cytosol), adrp, calreticulin (er) and golgi matrix protein (golgi) in total lysates and the ld fraction ( figure 6c ). importantly, calreticulin and golgi matrix protein were below the detection limit of our assay in the ld fraction whereas adrp was readily detected thus confirming that our procedure successfully enriched cellular lds. notably, py-2 moderately increased the total cellular level of core protein but at the same time reduced the abundance of core in the ld fraction of the cell lysate. this difference evident by western blot was further confirmed using a core-specific elisa demonstrating a ca. 2-fold higher core protein amount in the lysate of py-2-treated cells and about 3-fold lower levels in the ld fraction. collectively, these data indicate that py-2-dependent inhibition of pla2g4a impedes association of core with lds which in turn may limit core protein envelopment and particle release. in principle inhibition of pla2g4a by py-2 may reduce infectivity by preventing assembly/release of particles and/or by altering particle properties including the association with lipoproteins. therefore, we used ultracentrifugation of hcv particles through density gradients to analyze the amount and density of virus particles released from cells treated in the presence or absence of py-2 ( figure 7 ). using this approach we noted that the distribution of hcv core protein-containing structures throughout the density gradient was essentially unchanged with peak core protein levels in fractions with a density of 1.11-1.18 g/ml irrespective of py-2 treatment ( figure 7a) . notably, the overall amount of released core protein was moderately reduced (ca. 2-3fold; figure 7a and c) when particles were produced in the presence of the pla2g4a inhibitor. most strikingly, inhibitor treatment heavily decreased the infectivity of released hcv particles by 50-to 100-fold ( figure 7a ). to elucidate, why particles produced in the presence of py-2 are less infectious, we analyzed their protein composition. to this end we transfected jc1 carrying a flag-epitope tag at the n-terminus of e2 [13] into huh-7.5 cells where endogenous apoe was silenced by a specific shrna and replaced by ectopic expression of an ha-tagged, shrna resistant, human apoe gene ( figure s11 ). this approach enabled us to monitor co-precipitation of viral and host factors with an apoe-specific or a virus envelope-specific antibody. notably, treatment of transfected cells with py-2 reduced the amount of secreted apoe and core protein by ca. 25% and 65%, respectively ( figure 7b and 7c, respectively) . therefore, we normalized the culture fluids to equal quantities of apoe or core protein before the apoe-specific or flag-specific co-precipitation. remarkably, py-2 treatment reduced the level of core protein co-precipitating with apoe by 5-fold ( figure 7b) . likewise, inhibition of pla2g4 lowered the association of apoe and core with the flag-tagged e2 by ca. 5-fold ( figure 7c ). in summary, inhibition of mapk-dependent pla2g4a activity by py-2 moderately decreased the titer of released hcv particles but heavily impaired infectivity of both intracellular and extracellular particles likely through gross changes of their protein composition. in this study we manipulated key cellular signaling cascades to identify host cellular hcv dependency factors. we report that blockade of the mapk/erk cascade by a well-established specific inhibitor (u0126) potently repressed production of infectious hcv progeny ( figure 1 ). making reasonable assumptions (activation by erk, function at the er membrane), we focused on pla2g4a, an erk-regulated host enzyme that is recruited to the er-membrane by ca 2+ , as possible new hcv dependency factor for hcv assembly. our further experiments provide three lines of evidence supporting our conclusion that the erk-dependent activation of pla2g4a is crucial for production of infectious progeny: first, we show that py-2 impedes production of infectious hcv in a dose-dependent fashion ( figure 2) . notably, phospholipase a 2 enzymes are subdivided into several classes including secreted pla 2 s (spla 2 s), ca 2+ -dependent cytosolic (cpla 2 s), ca 2+ -independent cytosolic (ipla 2 s), platelet-activating factor acetylhydrolases (paf-ahs), lysosomal pla 2 s and the most recently identified adipose-specific pla [24] . importantly, py-2 potently inhibits pla2g4a (the a isoform of the ca 2+ -dependent cytosolic pla 2 s, also named cpla 2 a) in various in vitro assays [22] . in contrast it interferes with pla2g4b (cpla 2 b) and pla2g4c (cpla 2 c) only at very high doses, probably by a non-specific mechanism, and it does not inhibit the secreted spla 2 s [22] . congruently, bel, a ''suicide substrate'' and specific inhibitor of ipla 2 s with a .1,000fold selectivity for ipla 2 s over cpla 2 s [26] , did not impede hcv particle production ( figure 2) . second, using rna interference we show that reduction of the abundance of pla2g4a enzyme increased susceptibility of hcv to inhibition by py-2 ( figure 4) . surprisingly, we did not observe an influence of the knock down of pla2g4a on hcv particle production ( figure 4) . however, we note that in spite of clearly reduced abundance of the enzyme in sirna-treated cells we measured only a small decline of pla2g4a enzyme activity. it is possible that the active, phosphorylated pla2g4a enzyme has a relatively long half-life which may preclude reduction of the active enzyme to a level that limits hcv infectious particle production under these experimental conditions. third, we observed an almost complete restoration of hcv infectivity upon supplementing py-2 treated cells with aa ( figure 5) . importantly, among all pla 2 enzymes, only the pla2g4a displays a preference for cleaving glycerophospholipids carrying the polyunsaturated aa at the sn2-position [24] . moreover, related fatty acids including etya which differs from aa only by triple-bonded c-atoms in place of the double-bonded c-atoms in aa, were unable to restore virus production ( figure 5 and s7). even 596-dha (with a single triple-bonded c atom) only partially compensated production of infectious hcv in the presence of py-2 indicating that highly specific properties of aa, the cleavage product of pla2g4a, are crucial for production of infectious hcv progeny. aa is the precursor for biologically active lipid mediators including prostaglandins, thromboxane and leukotrienes collectively termed eicosanoids. these molecules are synthesized from aa through the cyclooxygenase and lipoxygenase pathways and play important roles during inflammation. however, since inhibitors of both pathways of aa metabolism did not impede production of infectious hcv particles ( figure s6 ), we believe that the properties of aa itself rather than indirect effects caused by aa-metabolites are important for hcv. our data support the conclusion that pla2g4a activity is relevant for hcv assembly in two principal ways. first, inhibition of pla2g4a decreased the amount of core protein associated with lipid droplets and reduced the level of core protein that is protected from proteolytic digestion ( figure 6 ). the latter finding may indicate a lower level of intracellular core protein that is encased in membranes -possibly within virus particles -and therefore protected from proteolysis. moreover, we observed reduced levels of extracellular hcv particles (2-3-fold; figure 7 ). it is currently unclear why inhibition of pla2g4a reduces the level of core protein at lds. in principal several mechanisms account for this including an increase of core assembly and subsequent unloading from lds or a reduced trafficking of core to lds possibly due to aberrant processing of the protein by signal peptide peptidase cleavage. however, since we observed lower levels of secreted virus particles we consider it unlikely that increased assembly and protein unloading from lds is responsible. moreover we did not detect an overt processing defect of core in the presence of py-2 (figure 2 and 6) . notably, gubern et al. have shown that mapk-dependent phosphorylation of pla2g4a at ser 505 is necessary for biogenesis of lipid droplets [40, 41] . therefore, it is tempting to speculate that blockade of pla2g4a by py-2 reduces lipid droplet biogenesis, thus limiting abundance of core protein at these organelles which are essential for hcv particle production [6] . assuming that core protein has to be unloaded from lds to drive budding and virus production which is consistent with the recent findings of counihan et al. [42] , it is reasonable to suggest that reduced core protein levels at lds may decrease membrane envelopment and particle release. while aa (the product of the pla2g4a-catalzed triglyceride cleavage) is apparently not required for lipid droplet biogenesis [40] , it nevertheless seems to be essential for the second prominent influence of pla2g4a on hcv, i.e. the production of highly infectious hcv progeny. remarkably, both hcv and denv produced and released in the presence of py-2 were approximately 100-fold less infectious as compared to viruses assembled in the absence of the drug (figures 2 and 3 ). since addition of py-2 to particles generated in the absence of the drug, did not impede cell entry (data not shown and figure 3 ), we exclude that py-2 interferes with cell entry of hcv or denv. rather, our results indicate that particle properties are altered when py-2 is present. while the density spectrum of released hcv particles was unchanged, coprecipitation with apoe-or envelope-specific antibodies provide firm evidence that blockade of pla2g4a disturbs the composition/ structure of released hcv particles. specifically, we noted 5-fold reduced levels of core co-precipitating with anti-apoe and likewise 5-fold lower amounts of apoe and core co-precipitating with the envelope protein-specific pull down. these results argue that either less particles are directly associated with apoe or that particles contain lower levels of apoe. since apoe is important for infectivity of hcv particles [43, 44] , a defect in the loading of this protein onto hcv particles may explain their reduced infectivity. in the envelope-specific pull down mediated by the flag-tagged viral e2 protein, we observed both 5-fold lower apoe and also core protein. on one hand this may indicate lower abundance of both proteins in secreted enveloped hcv particles. on the other hand, this may reflect incorporation of lower numbers of glycoprotein complexes into the viral envelope and in turn reduced precipitation efficiency. unfortunately, due to insufficient sensitivity of currently available envelope protein detection systems, we are currently unable to distinguish between these two possibilities. nevertheless, these results argue that blockade of pla2g4a by py-2 prevents normal loading of viral (core and envelope proteins e1 e2) and host proteins (apoe) onto nascent hcv particles. as a consequence, virus attachment or the interaction with entry factors or membrane fusion might be inefficient. notably, recent evidence suggests that aa and other poly-unsaturated fatty acids increase membrane fluidity [32, 33, 34] . thus, pla2g4a activity in the vicinity of particle production may modify membrane characteristics including curvature and fluidity. these altered properties may disturb virus budding and/or the trafficking of viral and host-derived components to the site of virus assembly and thus result in the production of particles with disturbed stoichiometry, aberrant envelope composition, and poor infectivity. a more detailed proteomic and lipidomic comparison between particles produced in the presence or absence of py-2 should help to clarify this in the future. our finding that hcv and denv particle assembly depend on pla2g4a activity while vsv apparently does not rely on this host factor indicates fundamental differences between the assembly of enveloped vsv particles compared to hcv and denv. it remains to be shown how exactly pla2g4a contributes to production and release of infectious denv particles. while it has been reported that denv may also usurp lds for its assembly [45] , welsch and colleagues showed that denv assembly sites are physically linked to rna replication sites on modified er figure 1a . (d) knock down of pla2g4a was monitored by western blotting. (e) hcv rna replication was measured in cell lysates (top panel) and release of infectious particles was determined by inoculation of naïve huh-7.5 cells (bottom panel) and luciferase assays. the box plots in panels (b) and (e) as well as in the following figures visualize the full distribution of the data; the central horizontal line in each box indicates the value of the median, whereas the box represents the range between the lower and upper quartile of the data, i.e. the area in which the central 50% of the measurements lie. the whiskers extend from the quartiles to the minimum and maximum measurements, respectively. statistical significance of difference between means is indicated using asterisks (*): n.s -not significant, * marginally significant (p#0.1), ** significant (p#0.05), *** highly significant (p#0.01). doi:10.1371/journal.ppat.1002829.g004 structures [29] . unlike for hcv, aa did not consistently restore virus production of denv supporting the notion that pla2g4a may participate in hcv and denv morphogenesis through different mechanisms. more work will be needed to fully understand the role of this host factor for these two viruses. along these lines it will be interesting to analyze if other enveloped viruses (e.g. coronaviruses) that like hcv and denv assemble progeny particles at intracellular membranes depend on pla2g4a as well. such studies could in the future reveal common replication mechanisms between hcv and denv (and figure 5 . arachidonic acid restores production of infectious hcv particles in py-2-treated huh-7.5 cells. at 32 hpt given doses of (a) arachidonic acid (aa), (b) 5,8,11,14-eicosatetraynoic acid (etya) or (c) 5,6-dehydro arachidonic acid (5,6-dha) were added to the medium of luc-jc1 transfected cells. rna replication and virus production in the presence or absence of py-2 was determined as described in figure 1a . data are shown as means +/2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (d) rna replication and virus production in cells treated as above except that medium was only supplemented with aa (and not with py-2). doi:10.1371/journal.ppat.1002829.g005 figure 6 . pla2g4a activity contributes to the association of core with lipid droplets and the membrane envelopment of core. cells were treated with py-2 as outlined in figure 1a . (a) freeze and thaw lysates of these cells were prepared as described in materials and methods and were left untreated or were incubated with proteinase k in the presence or absence of triton x-100. the abundance of core protein was determined by western blot. cells transfected with a jc1 mutant lacking the coding region of e1 and e2 served as control. (b) the % of core protein protected from proteolytic digestion was determined by chemiluminescence imaging and evaluated by using the imagej software. mean values of two independent experiments are shown. (c) total cell lysates were subjected to western blots for detection of core, adrp, calreticulin and golgi matrix protein (left panel). in parallel equal amounts of total cell lysates were used for preparation of lipid droplets by ultracentrifugation. lipid droplet associated proteins were analyzed by western blotting (right panel). (d) the amount of core protein in the total lysates and the ld fractions was determined by using a core-specific elisa. statistical significance of differences of means: n.s -not significant, * marginally significant (p#0.1), ** significant (p#0.05), *** highly significant (p#0.01). doi:10.1371/journal.ppat.1002829.g006 possibly other viruses) that may provide valuable insights into conserved assembly pathways of enveloped viruses. finally, inhibitors of the pla2g4a which have been pursued and brought into clinical development for treating inflammatory diseases may prove useful as antiviral therapeutics for the treatment of chronic hcv infection and possibly other viral diseases. antibodies were obtained from the following companies: apolipoprotein b (millipore); pla2g4a (abcam); p(s505)-pla2g4a, erk1/2, p-erk1/2 (cell signaling); actin (sigma. hcv particles produced in the presence of py-2 display aberrant protein composition. (a) virus particles produced in the presence or absence of py-2 were separated by using ultracentrifugation and an iodixanol step gradient. ten fractions were collected from the bottom, and core protein abundance (left panel) and infectivity titers (right panel) were determined. one example of two independent experiments is given. (b) flag-jc1 rna [13] was transfected into huh-7.5-ha-apoe cells ( figure s11 ) and cells were treated with py-2 as outlined in figure 1a . the total amount of apoe secreted from transfected cells was determined using an elisa (top panel). virus containing culture fluid of py-2 and untreated cells were normalized for equal quantities of apoe and precipitated with apoe-specific antibodies. the amount of co-precipitated core protein was determined by elisa and is expressed relative to the untreated control. data are shown as means +/2 sd of three independent experiments (c) flag-jc1 transfected huh-7.5-ha-apoe cells were treated as in (b). the amount of secreted hcv core protein was determined by elisa (left panel) and normalized to equal amounts of core prior to precipitation with flag-specific antibodies. the amount of co-precipitated apoe and core was determined by western blot and elisa, respectively and is expressed relative to the untreated control. data are shown as means +/2 sd of three independent experiments. doi:10.1371/journal.ppat.1002829.g007 pd98059 (cell signaling); sorafenib (bay 43-9006, alexa biochemicals); proteinase k (roche); cpla2a-sirna (thermo); etya; 5,6dehydro arachidonic acid; (s)-bromoenol lactone; ndga (cayman); quinidine; arachidonic acid (sigma); flag-agarose (sigma). huh-7.5 cells were grown in dulbecco's modified minimal essential medium (dmem; life technologies) supplemented with 2 mm l-glutamine, nonessential amino acids, 100 u/ml of penicillin, 100 mg/ml of streptomycin, and 10% fetal calf serum (dmem w/fcs). the plasmids pfk-luc-jc1 and pfk-jc1, encoding the genotype 2a/2a chimera jc1 with or without the firefly luciferase reporter gene have been described [14, 18] . chimeric hcv constructs pfk-h77/c3 encoding the genotype 1a/2a chimera [18] , ps52/jfh1 (a4550c) encoding the genotype 3a/2a chimera [46] , and psa13/jfh1 (c3405g-a3696g) encoding the genotype 5a/2a chimera have been described [47] . for the shrna-mediated knockdown of apoe expression, the vector plenti-39-u6-ec-ep7 [48] which contains a blasticidine resistance gene was modified to harbor an shrna specific to the 39untranslated region of human apoe (59-gccgaagcctg-cagccatgcg-39). as control, a construct containing a nontargeting shrna was used. the lentiviral plasmid pwpi hapoe-linker-ha-gun encodes the human apoe3 variant with an ha tag added to the 39-end via a glycine-glycine-serine-glycine linker in the self-inactivating pwpi vector [49] which comprises a gfp-ubiquitin-neomycinphosphotransferase fusion protein as selectable marker. the gene encoding pla2g4a (thermo scientific, cdna clone mhs4426) was n-terminally fused to a gfp-tag and subcloned into the pwpi vector. the creation of a huh-7.5-cell line expressing gfp-pla2g4a fusion protein is described below. finally, pfk-jc1-de1-e2 was created by a pcr-based strategy. in this construct the entire e1 and e2 coding region is deleted and the core coding region is directly fused in frame to the coding region of p7. sequence information is available upon request. hcvcc particles and firefly luciferase hcv reporter virus were generated as reported previously [50] . luciferase reporter virus infection assays were carried out as described [50] . for inhibitor assays, cells were pre-treated with cellular or viral inhibitors to completely abolish enzyme activity 41 h after transfection of hcv-rna. one hour later, supernatants were removed and fresh medium with inhibitors was added to harvest newly synthesized virus in a period of six hours. at 48 hpt, supernatants and cell lysates were used for the infection of naïve huh-7.5 cells or subjected to assays as described below. lipid droplets were isolated according to a published protocol [51] with minor modifications. briefly, jc1-transfected cells of 106100-mm plates were scraped into 50 ml pbs and pelleted by centrifugation at 10006g. cells were resuspended in ice-cold hlm buffer (20 mm tris-hcl (ph 7.4); 1 mm edta) with protease inhibitors (complete mini; roche) and incubated for 10 min on ice. cells were homogenized by eight gentle strokes with a potter-elvehjem tissue homogenizer and nuclei were removed from lysates by low-speed centrifugation (10006g). density of the post-nuclear supernatant was adjusted with sucrose (20% final) and samples were loaded below a discontinuous sucrose gradient (0, 5, 20%). flotation of lipid droplets through the gradient was achieved by centrifugation at 28,0006g for 30 min, 4uc. the white band containing lipid droplets at the top of gradient was collected and proteins were characterized by immunoblotting or core elisa. confluent jc1-transfected cells were suspended in 170 ml pk buffer (50 mm tris-hcl (ph 8.0); 10 mm cacl 2 ; 1 mm dtt) and homogenized by five freeze-and-thaw cycles. the lysate was divided into three 50 ml fractions and treated with or without 50 mg/ml proteinase k for one hour on ice. as control, the third sample additionally containted 5% (v/v) triton x-100 during protease digestion. the reactions were stopped by 5 mm pmsf for 10 min on ice and addition of laemmli buffer. samples were analysed by immunoblotting. huh-7.5 cells were transfected with sirnas specific to pla2g4a (d-009886-01, -02; thermo) or scramble sirna (4390846; ambion) in a forward transfection procedure according to the manufacture's protocol (rnaimax; life technologies). to achieve an efficient knock-down, cells were transfected twice within 96 h and re-seeded in 6-wells at a density of 2.5610 5 cells per well. cells were infected with a 20-fold concentrated stock of luc-jc1 virus and 41 h later the inhibitor assay was performed as described above. the efficiency of pla2g4a knock-down was verified by immunoblotting. viral rna was prepared from infected cells using a nucleo spin rnaii kit (macherey-nagel) according to the manual's instructions. 5 ml of the rna sample was used for hcv-specific quantitative reverse transcription-pcr (qrt-pcr) analysis using a lightcycler 480 device (roche). hcv-specific qrt-pcrs were conducted in duplicate measurements as published [52] to normalize for equal quantities of total rna in the samples, the gapdh-specific mrna was detected in parallel employing gapdh-specific oligonucleotides (s-gapdh, 59-gaaggt-gaaggtcggagtc-39; a-gapdh, 59-gaagatggtgat-ggg atttc-39) and a gapdh-specific probe (tib molbiol), 640-gapdh-bbq probe (59-lc640-caagcttcccgttct-cagcct-bbq-39). reactions were performed in three stages by using the following conditions: stage 1 (rt), 3 min at 63uc; stage 2 (initial denaturation), 30 s at 95uc; stage 3 (amplification), 45 cycles of 10 s at 94uc and 20 s at 58uc. the amount of hcv rna was calculated by comparison to serially diluted in vitro transcripts and normalized to the amount of gapdh, which served as a housekeeping gene. hcv core protein within cell lysates and culture fluids was quantified with a commercially available diagnostic kit (architect anti-hcv; abbott). one mg of total rna or 1/5 of rna extracted from 100 ml cell culture supernatant was reverse transcribed into cdna using the high capacity cdna reverse transcription kit (applied biosys-tems) following the manufacturer's protocol. quantitative rt-pcr was performed on an abi prism 7000 sequence detection system (applied biosystems). the reaction was carried out in a final volume of 15 ml, including 7.5 ml 26 green dye master mix (p.j.k., kleinblittersdorf), 2 ml cdna template, 1.5 ml primer mix (5 mm each), 4 ml rnase-free sterile water. reactions were carried out using the following settings: 95uc: 10 minr406 [95uc: 30 secr55uc: 60 secr72uc: 60 sec]. the amounts of denv rna were calculated from a standard curve derived from serially diluted in vitro transcripts of known concentration. primers used for the amplification were: sdv2-9687, 59-gcccttctgttcacaccatt-39 and asdv2-9855, 59-ccacatttgggcgtaagact-39. to quantify core protein, cell culture supernatants or immunoprecipitated flag-jc1 particles were diluted in pbs/1% triton in a ratio of 1:30. the core elisa was performed with a commercially available diagnostic kit (architect anti-hcv, abbott). apoe was quantified according to the manufacturer's instructions (mabtech). density gradient centrifugation was performed as described recently [53] . briefly, viruses were separated by overnight centrifugation through a 0% to 40% iodixanol step gradient at 154,0006g. ten fractions of 1 ml were collected from the bottom and analyzed for virus infectivity, core protein levels, and viral rna copies. following py-2 treatment, aliquots of cell culture supernatants were subjected to core or apoe elisa in order to equilibrate the volumes for immunoprecipitations. to capture flag-jc1 particles or ha-apoe, equilibrated supernatants were mixed with either 20 ml flag-agarose or 3 mg anti-ha antibody and 30 ml g-protein agarose (roche) overnight at 4uc in gentle rotation. immunoprecipitated proteins were washed three times in pbs, eluted with 50 ml pbs/1% triton for 10 min at 50uc and analyzed by core elisa or immunoblotting. phospholipase a2 activity was measured according to the manufacture's protocol (enzchek phospholipase a2 kit; life technologies). in brief, huh-7.5 cells were harvested from 35-mm wells, resuspended in 200 ml enzchek pla2 reaction buffer and disrupted by sonication. to avoid any measurement of ipla2 activity, bromenol lactone was added to all samples at a concentration of 5 mm. liposomes were prepared with the enzchek phospholipase a2 substrate and mixed with lysates at a ratio of 1:1 to give a total volume of 100 ml. samples were transferred in 96-wells and pla2g4a activity was monitored by the intensity increase of a single wavelength at 515 nm in a fluorescence microplate reader (flx800; biotek). for the generation of stable huh-7.5-ha-apoe cells, lentiviral gene transfer was used as described before [54] . endogenous apoe expression in huh-7.5 cells was silenced using plenti-39-u6-ec-ep7 [48] which contains a blasticidine resistance gene and an shrna specific to the 39-untranslated region of human apoe (59-gccgaagcctgcagccatgcg-39). subsequently, apoe expression was restored by transduction with pwpi hapoe-linker-ha-gun described above. lentiviral particles were generated by transfection of pcmv dr.74, pcz vsv-g and a derivative of either plenti-39-u6-ec-ep7 or pwpi at a ratio of 3:1:3 into 293t cells. lentiviral particles were collected 48 h post transfection and used to transduce target cells. selection was carried out in the presence of 5 mg/ml blasticidine or 0.75 mg/ml g418. for generation of huh-7.5-gfp-pla2ga4 cells, huh-7.5 cells were transduced with lentiviruses carrying the pwpi-gfp_pla2g4a vector. transduced cells were selected in the presence of 5 mg/ml blasticidine. the protocol for immunostaining was carried out as described previously (frenzen, hueging, steinmann, plos pathogens april 2011 volume 7 issue 4 e1002029). immunostainings of core and adrp proteins were performed at dilutions of 1:7000 respectively 1:500. texas red and cy-5 secondary antibodies were used at dilutions of 1:1000. statistical data analysis was performed using the free statistical environment r. data were initially visualized using histograms, boxplots and qq-plots, and normality of the distributions was assessed. statistical significance of differences was then calculated using welch's t-test if data were sufficiently well approximated by a normal distribution, or using the wilcoxon rank sum test as a non-parametric alternative for non-normal data. p-values were calculated and statistical significance reported as highly significant (***) if p#0.01, significant (**) if p#0.05, and marginally significant (*) if p#0.1. differences were considered not significant (n.s.) for p.0.1. figure 1a . hcv rna replication in cells was measured by using a luciferase reporter assays (top panels). the release of infectious particles was determined by inoculation of naïve cells with culture fluids collected at 48 hpt and determination of luciferase activity in cells 72 h after inoculation (middle panels). data are shown as means +/2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). the bottom panels display erk1/2 expression and phosphorylation in luc-jc1 transfected and inhibitor treated cells. erk proteins were detected as described in figure 1 . (tif) figure s3 influence of mapk/erk inhibitor u0126 on hcv cell entry. luc-jc1 particles prepared in the presence or absence of fcs were supplemented with the given dose of u0126 or left untreated. virus suspensions were incubated with huh-7.5 cells for 4 h at 37uc. subsequently, unbound particles as well as the inhibitors were removed and cells were cultured in fcs-containing culture fluid until the analysis of hcv infection 72 h later. data are shown as means +/2 sd of three independent experiments. (tif) figure s4 py-2 impedes production of infectious hcv across different hcv genotypes. cells were transfected with indicated chimeric hcv genomes encoding structural proteins of genotype 1a, 3a or 5a, and subsequently treated with py-2 as described in figure 1a . production of infectious progeny was quantified using a limiting dilution assay. two independent experiments are shown in the two panels. mean values of six replicates +/2 sd of the replicates are given. (tif) figure s5 blockade of arachidonic acid metabolism by inhibition of cyclooxygenases and lipoxygenases does not impede production of infectious hcv. luc-jc1 transfected huh-7.5 cells were treated with given doses of (s)-flurbiprofen (a) or ndga (b) as outlined in figure 1a . rna replication in transfected cells and release of infectious particles was determined by luciferase asssays. data are shown as means +/ 2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (tif) figure s6 fatty acids with varying degree of unsaturation are unable to restore virus production in py-2treated huh-7.5 cells. luc-jc1-transfected cells were loaded with given lipids 32 hpt and subsequently subjected to the py-2 inhibition assay outlined in figure 1a . hcv rna replication and virus production was determined by luciferase assays in cells treated with different fatty acids data are shown as means +/2 sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (tif) figure s7 arachidonic acid does not increase hcv cell entry. luc-jc1 particles were supplemented with aa or left untreated. virus suspensions were incubated with huh-7.5 cells for 4 h at 37uc. subsequently, unbound particles as well as the inhibitors were removed and cells were cultured in fcscontaining culture fluid until the analysis of hcv infection 72 h later. data are shown as means +/2 sd of three independent experiments. (tif) figure s8 influence of aa production of infectious denv particles in the presence or absence of py-2. cells were transfected with a denv rna and treated as described in figure 1a . infectivity of released particles was determined by inoculation of naïve huh-7.5 cells. statistical significance of differences of means: n.s -not significant, * marginally significant (p#0.1), ** significant (p#0.05), *** highly significant (p#0.01). (tif) figure s9 hcv protease or polymerase inhibitors do not impede production of infectious particles in the transient assay. given drugs were applied to jc1-transfected huh-7.5 cells as outlined in figure 1a [13] . rna replication (left) and production of infectious particles (right) was monitored using luciferase assays (means +/2 sem are shown). (tif) evolving epidemiology of hepatitis c virus aging of hepatitis c virus (hcv)-infected persons in the united states: a multiple cohort model of hcv prevalence and disease progression replication of subgenomic hepatitis c virus rnas in a hepatoma cell line hepatitis c virus p7 protein is crucial for assembly and release of infectious virions hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus the lipid droplet is an important organelle for hepatitis c virus production efficient hepatitis c virus particle formation requires diacylglycerol acyltransferase-1 thematic review series: glycerolipids. dgat enzymes and triacylglycerol biosynthesis hepatitis c virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins cellular determinants of hepatitis c virus assembly, maturation, degradation, and secretion human apolipoprotein e is required for infectivity and production of hepatitis c virus in cell culture characterization of low-and very-low-density hepatitis c virus rna-containing particles biochemical and morphological properties of hepatitis c virus particles and determination of their lipidome characterization of the early steps of hepatitis c virus infection by using luciferase reporter viruses highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication targeting the raf-mek-erk mitogen-activated protein kinase cascade for the treatment of cancer sorafenib blocks the raf/mek/erk pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model plc/prf/5 construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras ) cpla2 is phosphorylated and activated by map kinase a novel arachidonic acid-selective cytosolic pla2 contains a ca(2+)-dependent translocation domain with homology to pkc and gap a calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase a2 with membrane in the macrophage cell line raw 264.7 a pyrrolidine-based specific inhibitor of cytosolic phospholipase a(2)alpha blocks arachidonic acid release in a variety of mammalian cells pyrrolidine inhibitors of human cytosolic phospholipase a(2) recent progress in phospholipase a research: from cells to animals to humans regulation and inhibition of phospholipase a2 suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase a2. mechanism-based discrimination between calcium-dependent andindependent phospholipases a2 fusion-active glycoprotein g mediates the cytotoxicity of vesicular stomatitis virus m mutants lacking host shut-off activity composition and three-dimensional architecture of the dengue virus replication and assembly sites processive interfacial catalysis by mammalian 85-kilodalton phospholipase a2 enzymes on product-containing vesicles: application to the determination of substrate preferences cytosolic phospholipase a2 effects of fatty acid unsaturation numbers on membrane fluidity and alpha-secretase-dependent amyloid precursor protein processing the modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function eicosatetraynoic and arachidonic acid-induced changes in cell membrane fluidity consonant with differences in computer-aided design-structures role of cytosolic phospholipase a2 in allergic response and parturition reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase a2 optimization of novel acyl pyrrolidine inhibitors of hepatitis c virus rnadependent rna polymerase leading to a development candidate a cell protection screen reveals potent inhibitors of multiple stages of the hepatitis c virus life cycle production of infectious hepatitis c virus in tissue culture from a cloned viral genome group iva phospholipase a2 is necessary for the biogenesis of lipid droplets ccaat/enhancer binding proteins are not required for hiv-1 entry but regulate proviral transcription by recruiting coactivators to the long-terminal repeat in monocytic cells trafficking of hepatitis c virus core protein during virus particle assembly apolipoprotein e on hepatitis c virion facilitates infection through interaction with low-density lipoprotein receptor infectivity of hepatitis c virus is influenced by association with apolipoprotein e isoforms dengue virus capsid protein usurps lipid droplets for viral particle formation robust hepatitis c genotype 3a cell culture releasing adapted intergenotypic 3a/2a (s52/jfh1) viruses highly efficient jfh1-based cell-culture system for hepatitis c virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection the zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses lentiviral vectors interfering with virus-induced cd4 down-modulation potently block human immunodeficiency virus type 1 replication in primary lymphocytes hepatitis c virus hypervariable region 1 modulates receptor interactions, conceals the cd81 binding site, and protects conserved neutralizing epitopes isolation of lipid droplets from cells by density gradient centrifugation a novel diagnostic target in the hepatitis c virus genome low ph-dependent hepatitis c virus membrane fusion depends on e2 integrity, target lipid composition and density of virus particles a thirdgeneration lentivirus vector with a conditional packaging system we are grateful to takaji wakita for jfh1 and to jens bukh for the j6cf strain, to marc p. windisch for boceprevir, to timothey tellinghuisen for 29cma, to charles rice for the ns5a-specific antibody 9e10 and huh-7.5 cells, to darius moradpour for the core-specific antibody c7-50, to john maclauchlan for the adrp-specific antibody and to gert zimmer for vsv*m q . we would also like to thank all members of the department of experimental virology for helpful suggestions. key: cord-347992-coby2m6e authors: marton, soledad; reyes-darias, josé a.; sánchez-luque, francisco j.; romero-lópez, cristina; berzal-herranz, alfredo title: in vitro and ex vivo selection procedures for identifying potentially therapeutic dna and rna molecules date: 2010-06-28 journal: molecules doi: 10.3390/molecules15074610 sha: doc_id: 347992 cord_uid: coby2m6e it was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. in vitro selection and evolution strategies have been extremely useful in the analysis of functional rna and dna molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize dna and rna molecules with potential therapeutic and diagnostic applications. the great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. this review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids. nucleic acids, particularly rna, are extremely versatile molecules. apart from their role as carriers of genetic information they can also express a phenotype, e.g., they may show a catalytic activity, have a specific binding function, or have the capacity to recruit specific molecules. the appearance of sequence variation in a nucleic acid can, in some cases, provide an advantage to an organism: a key feature in natural evolution. over the last 20 years, advances in molecular biology and biotechnology have seen the development of methods that allow the effect of such naturally arising variation to be mimicked in the laboratory. it was in the 1960s when spiegelman's group first observed evolution in vitro [1] . these authors reported that changes in the rna genome of the qβ bacteriophage during replication led to the formation of rna molecules that were more efficiently copied by the viral replicase. these genomes lacked unnecessary sequences and were synthesized at a greater rate. however, these finding fell into oblivion until 1990, when the great potential of in vitro selection and evolution techniques was reported by three independent groups [2] [3] [4] [5] . since then, numerous authors have used these technologies to study the chemical and catalytic properties of nucleic acids (for further information see [6] [7] [8] [9] [10] [11] [12] [13] . they have also helped uphold the rna world hypothesis, and it seems likely that they may soon have applications in biomedicine. indeed, new selection methods -known as ex vivo selection procedures -are now being used to identify molecules that target viruses, subcellular fractions and even whole cells. these techniques overcome some of the limits imposed by in vitro technology and provide new environments and conditions in which to explore the properties and functions of nucleic acids. this review highlights the most recent advances in in vitro and ex vivo selection procedures for nucleic acids, and discusses their potential application in biomedicine. in vitro selection strategies have been used to select nucleic acids with a large variety of properties. although each strategy differs according to the feature or phenotype sought, all in vitro selection methods follow the same three-step pattern ( figure 1 ). genetic variability is introduced into the system (generally by chemical synthesis) to yield nucleic acid populations, the heterogeneity of which is determined by fixing the number of nucleotides to be mutagenized and the mutation rate per nucleotide. in most cases, completely random synthesis involving a fixed number of nucleotides yields a starting population of variant molecules that, a priori, differ only in the sequence and the structure of the variable region. constant sequences, or primer binding sites (pbs), flanking the variable region are incorporated during the design of the starting population to facilitate the amplification of desired molecules, although this limits the structural diversity of the rna populations to specific conformations [14] . different approaches have been tried to minimize the effect of pbss, e.g., the addition of customized primers or adapters by ligation before amplification, and their removal prior to the selection step [15] [16] [17] . the selection strategy needs to be specifically designed according to the phenotype sought. the initial pool of variants usually contains very few active molecules corresponding to the desired phenotype, and their enrichment can only be made possible by properly designing the selection step. the selection of inactive molecules may also be important since the analysis of these molecules can provide very valuable information on the sequence and structural requirements of active molecules [18] . in vitro selection strategies have been widely used for the selection of nucleic acids capable of catalyzing specific chemical reactions, i.e., dnazymes and ribozymes [19] . these strategies have also been successfully used to identify dna and rna molecules with affinity for a specific ligand (i.e., aptamers) [3] , being known as selex, which stands for systematic evolution of ligands by exponential enrichment [5] . sequence variants are separated into active, those that satisfy selection criteria, and inactive molecules. active molecules are selectively amplified by pcr resulting in the production of a new pool of molecules that can be the input of a new selection cycle. an additional reverse transcription and in vitro transcription steps are required, before and after pcr, respectively, when the selection is performed on rna molecules. alternatively, active and inactive resulting pools can be cloned and analyzed. variants that show the required phenotype need to be replicated to ensure their passage into the next generation and therefore their persistence in the population. specific primer binding sites are used to amplify the selected molecules. when necessary a rna polymerase promoter is incorporated at the 5' end of the pbs during the amplification step. in addition to these general steps, the amplified molecules may require additional manipulations prior to their introduction into the next round of selection when the selected molecule is ssdna, both dna strands must be separated and the positive one isolated, e.g., by incorporating a biotinylated residue into the unwanted strand [20, 21] via asymmetric pcr [22] . as a result of the described selection cycle, a population enriched in the sought-after molecules (but not composed of them entirely) is produced; a new selection cycle can then be undertaken. by iteratively executing the process of selection and amplification the complexity of the original population is reduced and enriched with candidates of interest. during this process, the stringency of selection can be increased to achieve the isolation of the molecules with the desired phenotype. the great advances made in in vitro selection procedures over recent decades have helped us improve our knowledge of the plasticity of nucleic acids and their potential applications as therapeutic tools. indeed, such have been the advances that selection strategies within living cells are now contemplated. these new methodologies are named according to the specific procedure employed in each method, e.g., ex vivo, in vivo, in cell selection, or cell-selex etc. these are here all referred to as ex vivo selection strategies. ex vivo selection methodologies follow the general pattern described above for in vitro techniques. these systems have mainly been used in the isolation of dna and rna molecules that interfere with the activity of a target molecule. when the expression of an oligonucleotide leads to a desired cell phenotype, it allows for the selection of these cells and the subsequent isolation of the oligonucleotide itself. following these principles a strategy was used to identify a transcriptional activator regulated by tmr (tetramethylrosamine) [23] . for this purpose, a chimera was constructed by tethering a tmr aptamer to the transcriptional activator for ms2 protein (also known as n40-26) [24] . variability was introduced by randomizing seven nucleotides within the linker region of the two rna domains. selection was performed in yeast cells containing a construct coding for the his3 and lacz genes under the control of the lexa operator, and expressing a lexa-ms2 coat protein fusion. this is a hybrid protein that binds to the operator and to the n40-26 ms2 rna hairpin present in the rna construct. only cells expressing an active transcriptional activator were capable of growth in the absence of histidine, and were the only ones capable of expressing β-galactosidase. the stringency of the selection process was increased by the presence of varying amounts of a competitive inhibitor of the his3p activity. analysis of the selected yeast clones revealed specific rna sequences that responded to tmr enhancing the transcription activator effect [23] . the selection of an artificial ribosome switched on or off via the external addition of a small molecule to the growth medium deserves special mention. the escherichia coli 16s ribosomal rna was fused to an aptazyme, a ribozyme capable of binding thiamine pyrophosphate (ttp) through an aptamer domain. the binding of ttp activates the ribozyme, reducing gene expression by cleaving the 16s ribosomal rna and thus working as a riboswitch. this allows for the identification and selection of specific e. coli colonies according to the phenotype expressed [25] . the ttp riboswitch was also included in the 5`utr region of a reporter gene, 30 nt upstream of the shine-dalgarno sequence. this linker between these elements was randomized and e. coli colonies selected depending on the expression of the reporter gene in response to ttp [26] . ex vivo selection methods in cells are limited by the number of sequences that can be studied, this number being determined by the number of available cells. another problem is the possibility of there being more than one sequence variant per cell, which could lead to many false positive. ellington's group developed a very interesting strategy that allowed the direct selection of active molecules instead of selecting cell clones [27] . the cleavage products of the autocatalytic ribozymes synthesized in the cell nucleus were extracted via hybridization to a biotinylated oligonucleotide, allowing the direct identification of active molecules. the selection of nucleic acids against whole cell targets has been successfully used to select nucleic acids that target cell surface receptors. this technology also known as cell-selex could have a role to play in the treatment of cancer. for most types of cancer cell there is a shortage of highly specific surface markers that can be used with diagnostic and therapeutic intent. aptamers generated from whole living cells are the optimal molecular probe for characterizing target cells at the molecular level. when bound to the membrane receptors of cell lines they provide an effective means of identifying disease markers. the pharmacokinetic and pharmacodynamic properties of a nucleic acid, and its resistance to nucleases, all condition its effectiveness as a therapeutic molecule. after selection, the most effective molecules can be modified to improve their nuclease resistance as well as their affinity for their targets, their cellular uptake and selectivity. a great diversity of post-selection modifications has been described. only those of interest for therapeutic purpose are discussed here. these include modifications of oligonucleotide size and sequence, and mainly certain chemical modifications (for a review see [28, 29] . advances in chemical synthesis have allowed the production of conjugates that combine an oligonucleotide sequence with compounds such as fluorophores, peptides, carbohydrates and lipids ( figure 2 ). these modified oligonucleotides show advantageous properties with respect to the native form. the ligands are usually linked to the 5' or 3' termini, which are the most accessible regions for chemical conjugation reactions; in addition, any disruption of the nucleic acid's folding and functional properties are minimized [30] . conjugations at the 2' position of ribose or involving the internucleotidic phosphodiester bonds are also possible. fluorophore conjugation is already used in clinical diagnosis, e.g., in fluorescence in situ hybridization (fish) and molecular beacons. fish can detect specific genes in cells, while molecular beacons act like switches, emitting fluorescent light when bound to their target sequence. cell uptake has been improved by the conjugation of oligonucleotides to peptides capable of translocating them across the cell membrane by a non-receptormediated endocytotic mechanism. some of the most frequently used peptides are residues 43-58 of the third helix of the antennapedia homeodomain (penetratin), the highly arginine/lysine rich region of the hiv-1 tat protein, the hydrophobic signal peptide, the nuclear localization sequence (nls), and transportan [31] . besides improving cellular uptake, peptide-oligonucleotide conjugates show increased stability to nucleases degradation and enhanced binding [32] . carbohydrate-oligonucleotide conjugates (cocs) have similar applications. in addition they confer cell or tissue specificity by their binding to sugar receptors (i.e., lectins) present at the cell surface capable of recognizing and internalizing (by endocytosis) glycoproteins bearing specific carbohydrates moieties [33] . lipophilic oligonucleotide conjugates (locs), such as cholesterol, reduce the hydrophobic character of the oligonucleotide, and some bind to blood lipoprotein carriers. lipophilic oligonucleotides are also used for designing supramolecular assemblages such as micelles, vesicles and liposome networks [34] . the ability of certain rna polymerases to incorporate modified nucleotides to the growing chain, such as the 2'-modified ribonucleosides, makes it possible to use the selection procedure with populations of chemically modified oligonucleotides [35, 36] . chemical modifications of the 2' hydroxyl group of rna, such as 2' fluoro, amino, methoxy and amido modifications, are noteworthy for their potential therapeutic applications since they increase rna stability, conferring greater resistance to nucleases. another important modification for therapeutic purposes is the use of locked nucleotides (lnas) in the nucleotidic chain. lnas contain a methylene link between the 2'-o and 4'-c of the ribose ring which locks the sugar moiety in the 3' endo conformation [37] . this generates the most stable hybrids ever characterized, with a δt m of +3 to +10 per lna residue upon binding to dna and rna respectively, thus conferring nuclease resistance [38, 39] . the introduction of lna modifications into in vitro selection techniques has so far been restricted to post selection incorporation (for a review see [40] ). nevertheless, it has recently been shown that locked nucleotides can be incorporated enzymatically into both dna and rna [41] [42] [43] . spiegelmers (spiegel = mirror in german), unnatural but biostable l-forms of d-aptamers, were developed to allow oligonucleotides to escape nuclease attack. naturally occurring proteins are lchiral, therefore natural nucleases digest d-oligonucleotides while l-nucleosides escape this fate. to obtain spiegelmers, natural oligonucleotides are used during the selection cycle against unnatural dproteins that mirror the natural structure of the l-target [44, 45] . the selection process yields l-form aptamer sequences that by virtue of the law of symmetry recognize their natural target. spiegelmers have been selected against peptide hormones such as gonadotropin-releasing hormone (gnrh) [46] , and ghrelin, an endogenous ligand for growth hormone secretagogue receptor 1a [47] . both have a neutralizing effect in vivo against these hormones after systemic administration. in vitro selection strategies have been extensively and successfully used to characterize known ribozymes and dnazymes, and to isolate new catalytic nucleic acids with unsuspected activities. in fact, the first observation of a dna molecule catalyzing a chemical reaction (dnazyme) was made when using an in vitro selection strategy [48] . although ribozymes and dnazymes have been extensively assayed as potential therapeutic agents, and different clinical trials have already tested their efficiency against various diseases [49] [50] [51] [52] , very few reports have described the direct application of in vitro selection strategies in the development of potentially therapeutic catalytic nucleic acids. ellington's group recently described a procedure aimed at identifying cleaving ribozymes active within the cell milieu, but this has not yet been used with therapeutic intent [27] . most of the work referred to herein describes the use of in vitro and ex vivo selection strategies for the identification of aptamers of therapeutic potential. assays with catalytic nucleic acids engineered by so-called 'rational design' are beyond the scope of this review. the idea that aptamers can modulate the activity of target proteins emerged from basic studies of viruses. in the 1980s, research on hiv and adenovirus led to the discovery that these viruses contain several structural rna domains that bind to viral or cellular proteins with high affinity and specificity. not surprisingly, functional analyses of these viral rna ligands demonstrated that the viruses had evolved these aptamers either to modulate the activity of proteins essential for their replication [53] or to inhibit the activity of proteins involved in cellular antiviral responses [54] . the first study performed to determine whether an rna aptamer could be used to inhibit the activity of a pathogenic protein was published in 1990. this work reported that the tar aptamer, evolved by hiv to recruit viral and cellular proteins to viral transcripts, could be turned against the virus to inhibit its replication [55] . the in vitro aptamer selection strategies developed during the 1990s prompted the idea of sullenger's group that therapeutic aptamers might be possible. several such aptamers have now completed various stages of preclinical development, and a number of others are currently being tested clinically (table 1) . indeed, one aptamer is already on the market as a therapeutic drug several modifications of the general selection process scheme have been described in order to achieve different goals. for example, the toggle-selex strategy is used for the selection of potentially therapeutic nucleic acids [56] . toggle-selex was designed for the isolation of aptamers with a broad range of specificities for closely related targets, such as the homologous proteins of different species. these aptamers were obtained by performing the selection procedure for related targets (i.e., homologous proteins) in alternating cycles. such alternation ensures that the rna or dna variants resulting from selection will bind to both proteins, most likely to domains conserved between them. sullenger's group described an in vitro selection strategy in which rna aptamers that bind both human and porcine thrombin were selected by "toggling" the protein target between the human and porcine forms in successive rounds of selection [56] . this yielded a family of aptamers, all of which bound both thrombin types with high affinity. toggle-25, a characteristic member, inhibited two of thrombin's most important functions: plasma clot formation and platelet activation [56] . this strategy could facilitate the isolation of ligands with properties required for gene therapy or other therapeutic or diagnostic applications. to date, the only aptamer approved by the fda [73] , known as pegaptanib or macugen, was approved in december 2004 for the treatment of wet type age-related macular degeneration (amd). this aptamer binds to vascular endothelial growth factor, vegf 165 [57, 74] , the main isoform of a family of growth factors involved in promoting blood vessel development and maintenance via tyrosine kinase receptor signaling. vegf 165 is also involved in several pathological processes such as amd, diabetic retinopathy and cancer [75] . a phase ii clinical trial to evaluate the use of this aptamer in the fight against diabetic retinopathy is currently underway [76] . the selection procedure involved a 2'-fluoro-pyrimidine (2'-fy)-modified rna pool. additional modifications were made after selection by adding 2'-o-methyl (2'-mr) to all purine residues of the aptamer except two, by adding a 3' cap, and by adding polyethylene glycol (peg; 40 kd) to the 5' end ( figure 3 ). the macugen aptamer binds to the heparin-binding domain of vegf 165 [75, 77] and efficiently inhibits the growth of blood vessels [57, 74] . this agent is of particular interest with respect to preventing tumor angiogenesis. different aptamers are currently being tested for the treatment of other degenerative diseases. niu's group attempted to interfere with the function of the glur2 ampa receptor associated with cerebral ischemia and amylotrophic lateral sclerosis [78] . a selected aptamer known as an58 acts as a glutamate antagonist preventing glutamate-induced activation of the cationic channel. interestingly, the aptamer adopts two mutually exclusive non-interchangeable isoforms that are both necessary for proper inhibition to occur [79] . recently, new rna aptamers have been isolated that bind to the nogo-66 neural receptor as antagonists of myelin-derived ligands. nogo-66 signaling blocks neurite outgrowth, but the binding of the aptamers allow axon growth in rat ganglion cells in vitro [80] . this aptamer is of special interest in the search for agents that aid neural repair, e.g., after spinal cord trauma. aptamers can also be targeted against disease-causing proteins such the scrapie isoform of the prion protein (prp sc ). a 2'-fy rna aptamer known as saf-93 [81] has been shown to prevent its aggregation in cell free systems, while a 2'-amino-2'-deoxypyrimidine-modified aptamer known as dp7 slows down its aggregation in neuroblastoma cells [35] . both aptamers target the same conserved region involved in prion interactions. neutrophil elastase (hne) is involved in the pathogenesis of inflammatory diseases such as acute respiratory distress syndrome (ards), septic shock, emphysema and arthritis, as well as ischemiareperfusion injuries [82] . a covalent inhibitor of hne, a diphenyl phosphate derivative of valine, has been coupled to an rna library to enhance its binding to hne [83] . after in vitro selection, an rna aptamer conjugated with dna:valp (rna10.11:dna:valp) was isolated that binds hne with high affinity. the bound molecule, unlike the aptamer rna 10.11 or dna:valp alone, also inhibits lung inflammation in an ex vivo rat model of ards [83] . more efficient inhibitors of hne were obtained from a valyl phosphonate:dna pool [84] . after selection, aptamer inhibitor ed45 inhibited hne formation two orders of magnitude greater than rna.10.11:dna:valp [83] . a truncated dna aptamer version named nx21909, composed of two annealed dna oligonucleotides, was tested in a rat model of lung inflammation and was found to inhibit neutrophil infiltration by 53% at a dose of 40 nmol [85] . immunoglobulin e (ige) plays an important role in protecting mammals from parasites [86] . however, its overproduction due to exposure to environmental antigens can result in allergies, atopic dermatitis and allergic asthma [87] . dna selection was performed against human ige to produce aptamers that bind it with high affinity [88] . these aptamers inhibited the binding of ige to its receptor fcεri, and also prevented ige-mediated cellular degranulation in the serum of patients with allergy to grass pollen. in patients exposed to grass pollen extract, the ic50s for the dna aptamers were 2-6 µm, but when triggered by anti-ige antibodies they reached 200-300 nm. these dna aptamers represent a novel class of ige inhibitors that may prove useful in the fight against allergic diseases. cancer has been one of the diseases most targeted by aptamers. alteration of the signaling pathways results in the escape of tumor cells from the control of cell division and apoptosis. the formation of metastases is also promoted. all of these processes have been the target of aptamers. the tyrosine kinase receptors (tkr) has been targeted by two aptamers: ret (rearranged during transfection) and her 3 (human epidermal growth factor receptor). the ret aptamer, d4, a 2'-fy aptamer, was isolated by ex vivo selection against the extracellular mutant ret c634y receptor [89] . briefly, an rna population was incubated against a parental pc12 cell line and variants expressing different ret mutants as a negative selection step. the unbound fraction of rna molecules was recovered and incubated against pc12 cells expressing the recombinant ret c634y receptor. the selected d4 aptamer inhibits neurite outgrowth and reverts the neoplastic phenotype of nih/men2a cells in vitro [89] and in three dimensional collagen gel matrix cultures [90] . the her-3 aptamer, a 2'-fy aptamer, known as a30, was isolated against the her-3 monomeric extracellular domain; this prevents her-2 signaling via her-3 heterodimerization in cell culture [91] . cell adhesion misregulation involved in metastasis has also been prevented by aptamers. the plasminogen activator inhibitor-1 (pai-1) is overexpressed in breast cancer cells and binds to vitronectin, leading to the loss of adhesion. a 2'-fy aptamer known as sm-30, specific for plasminogen activator inhibitor-1 (pai-1), restores cell adhesion to vitronectin-coated plates in vitro [92, 93] . another type of therapeutic signaling modulation is the targeting of nuclear factor κb (nf-κb) inside cells. maher's group isolated two rna aptamers, α-p50 and r1, by two independent in vitro selection procedures. these bind nf-κb p50 and p65 isoforms in vitro respectively [94, 95] . these aptamers underwent further ex vivo selection using the yeast three-hybrid system [96, 97] . the inhibition of nf-κb might be of therapeutic interest in different types of cancer, hiv-1 infection and inflammatory diseases. in fact, gene therapy with different anti-nf-κb aptamers administered via an adenoviral vector suppresses doxorubicin resistance in vivo in a lung tumor xenograft mouse model [98, 99] . the inhibition of nucleophosmin oligomerization by an aptamer promotes higher p53 levels and, consistently, sensitizes cells to dna-damaging-agent-induced apoptosis in cell culture [100] . the stimulation of the immune system can also be used in anti-cancer therapy. the activation of cd8 + t cells within a tumor would promote its cytotoxic involution. in this respect, the modulation of cytotoxic t-cell antigen-4 (ctla-4), 4-1bb and ox40 receptors by aptamers has been explored. multimeric aptamer forms frequently improve aptamer signaling properties and have proven especially importance in this area. it has been shown that an antagonist rna aptamer against ctla-4, a negative regulator of t-cell activation, inhibits its function [101] , while an agonistic aptamer against 4-1bb, a major co-stimulatory receptor, leads to the activation of t cells [102] . immunity against the tumor is induced in vivo in both cases. a specific aptamer for the dimeric murine ox40 combined with a dendritic cell-based tumor vaccine promotes tumor immunity in a xenograft melanoma model in mice [103] . aptamers have also been shown able to promote tumor cell death. when expressed in cells, an aptamer selected against nucleophosmin was shown to prevent the latter's oligomerization. higher p53 levels were therefore promoted that led to apoptosis. in addition, the sensitivity of cells to dnadamaging agents in cell culture was increased [100] . research into anti-angiogenesis aptamers has provided some very interesting results. sullenger's group reported the selection of specific rna aptamers against the ang1 and ang2 genes [36, 104] . aptamer ang9-4 binds to ang1 and inhibits its signaling pathway, leading to the reduced survival of huvec cells in vitro [36] . similarly, intraocularly administered aptamer 11-1.41 binds to ang2 and inhibits angiogenesis in rat corneal micropockets [104] . a 3' deoxythymidine cap protects this aptamer from rnases. the pegylated version of this molecule was shown to inhibit tumor angiogenesis and growth in an in vivo murine metastatic colon cancer model following systemic administration [105] . while the large majority of aptamers have been isolated by selex [106] , the anti-proliferative dna aptamer as-1411 was developed based on observations that guanosine-rich oligonucleotides have antiproliferative effects in tumor cells [107] . molecular studies have shown that this aptamer binds to the cell surface protein nucleolin and inhibits the activity of nf-kb, a ubiquitous transcription factor, through intracellular complex formation [108] . clinical studies of as-1411 have focused on patients with renal, pancreatic and other solid tumors. the aptamer was administered to patients as a continuous infusion for 4 or 7 days. selectins are a family of cell adhesion molecules expressed by leukocytes, endothelial cells and platelets [109] . they are involved in a number of inflammatory diseases as well as tissue injury and infection. dna selection against the l-selectin/igg fusion protein (ls-rg) was performed to find aptamers that could be tested in vivo [110] . aptamers ld201, ld174 and ld196 all bound with a kd of 1.8 nm at 37 ºc. truncated versions of these aptamers inhibited sl-rg binding to its ligand sialyl lewis x (slex) with an ic 50 of 3 nm. aptamer ld201t1 blocked l-selectin-mediated adhesion of human lymphocytes and neutrophils and inhibited human cell trafficking to peripheral and mesenteric lymph nodes in scid mice. platelet-derived growth factor (pdgf) is a ubiquitous mitogen and chemotactic growth factor in the form of three disulphide-linked dimers made of two homologous chains, a and b. it is involved in wound healing and is linked to the progression of numerous diseases, including atherosclerosis and glomerulonephritis [111, 112] . a hallmark of malignant transformation is the loss of dependence on exogenous mitogenic stimulation; many tumor cell lines are thought to produce and secrete pdgf for this reason [113] . dna selection against recombinant human pdgf-ab yielded dna specific aptamers of the pdgf b-chain that bound with subnanomolar affinity [114] . the consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a threenucleotide loop at the branch point. the pdgf aptamers inhibited the mitogenic effects of pdgf-bb in cells that expressed pdgf β receptors [114, 115] . peg-modified aptamers in a rat model of mesangioproliferative glomerulonephritis led to a 64% reduction in mitoses by day 6, and 78% by day 9. there was also a 95% reduction of proliferating mesangial cells by day 9 and a markedly reduced glomerular expression of the endogenous pdgf b-chain. aptamer-treated animals also showed a reduced influx of monocytes/macrophages and the overproduction of glomerular extracellular matrix on day 6 [109] . further studies revealed the inhibition of other disease mechanisms by the above aptamer in experimental glomerulonephritis [116, 117] . the expression of pdgf β receptors in tumors is associated with increased interstitial fluid pressure (ifp) in the dermis. this reduces the gradient between capillaries and the interstitium and impedes the exchange of solutes, such as anticancer agents, over the capillary membrane [118] . increasing this gradient may facilitate the transport of anticancer drugs to tumors [119] . to reduce the ifp, the pdgf-b aptamers [109] were tested in a rat tumor model. the treated animals had an ifp of 9.7 mm hg compared to 14.6 mm hg in scrambled-rna-treated animals [120] . another dna aptamer known as e1-0030 that targets the pdgf-b subtype is currently undergoing phase i clinical trials as an antivascular endothelial growth factor compound [121] . blood fluidity and blood vessel resistance are involved in numerous vascular diseases such as coronary and thrombotic syndromes and myocardial infarction. certainly, these factors must be carefully controlled during coronary surgery. the proliferation of cardiac and vascular cells is key in the development of vessel resistance in diseases such as cardiac intimal hyperplasia, cardiac hypertrophy and atherosclerosis, as well as in the development of malignancies [122, 123] . a recent study has reported the development of an rna aptamer able to specifically recognize members of the e2f transcription factors involved in cell proliferation. the binding of the 2'-fy aptamer 8-2 mainly to e2f3, reduces intimal hyperplasia and the pathological proliferation and migration of vascular smooth muscle cells (vsmcs) after bypass surgery in a mouse model [124] . this aptamer avoids the side effects derived from cross reactivity with other members of the e2f family. in a different approach, selex has been performed with the e2f1 protein to find in vitro selected rna aptamers that bind to and inhibit e2f activity. clone e1 rna was found to bind to e2f1 and blocked the latter's attachment to its dna binding site [125] . by impeding e2f activity, the e2f rna aptamer inhibited s-phase induction by 90% compared to controls. thus, both natural and in vitro selected aptamers appear able to limit cell proliferation. the coagulation signaling cascade offers several targets for the modulation of blood fluidity. the conversion of pro-thrombin to thrombin is delayed by a long half-life (15 h) 2'-fy aptamer targeting the catalyst factor viia in a dose dependent manner [126] . nevertheless, the rapid restoration of cascade integrity is needed to prevent the harmful effects of coagulation deficiency. with this aim, an rna-based aptamer-antidote system has been developed [127, 128] . the reg-1 rna aptamer targets factor xia. an antisense rna molecule was designed to specifically bind the 5'-half of the aptamer (the antidote). binding of the antidote abolishes the aptamer's binding to its target, thereby reversing the anticoagulant effect ( figure 4 ). neither antidote nor aptamer have been seen to cause any adverse effect in phase i clinical trials (ia and ib) when given to healthy people and patients with stable cardiovascular disease receiving antiplatelet therapy [61, 63] . the results of another phase i clinical trial (ic) indicate that the anticoagulation effect can be modulated by varying the dose of antidote rna [62] . in a recent phase iia trial, percutaneous coronary injection of the aptamer increased the activated clotting time (act) in patients immediately after its administration, reaching values close to those obtained with heparin. act values were restored 15 min after the administration of the antidote [129] . thrombin is a natural target for anticoagulation therapy and numerous aptamers have been generated with different capacities to inhibit its activity in vitro [56, 70] . the archemix corp., in collaboration with arca biopharma inc. (formerly nuvelo inc.), has tested nu-172, a dna aptamer against thrombin. in phase i clinical trials, nu-172 was administered intravenously as a continuous infusion to healthy volunteers. the results showed an increase in activated clotting time with a return to baseline when administration ceased (see archemix corp. website). a phase ii clinical study is currently underway with the goal of using nu-172 in coronary artery bypass graft surgery and percutaneous coronary intervention. figure 4 . mechanism of the apatamer-antidote pair for anticoagulant therapy.the intrinsic pathway of the blood coagulation cascade involves the activation of factor x. anticoagulation system reg1 consists of rb006 (drug), an injectable rna aptamer that specifically binds to activated factor ix (ixa) and prevents the proteolytic cleavage of factor x; and rb007 (antidote), a rna antisense oligonucleotide that neutralizes the anticoagulating effect of the aptamer rb006. in the presence of the antidote, the aptamer is released from factor ixa and clotting parameters return to normal. together with activated factor viii (viiia), factor ixa catalyzes the cleavage of factor x (pro-enzyme) to yield activated factor x (xa), which is required for the blood clotting cascade. a dna/rna aptamer conjugated to peg, known as arc-1779, generated against vwf (von willebrand factor), a central factor in the adhesion of platelets to the endothelial surface at vascular injury sites [130] , has been examined in a phase i trial in healthy volunteers. the aptamer increased platelet function in a whole-blood assay sensitive to vwf-mediated platelet inhibition. moreover, a slow intravenous bolus followed by 4 h of continuous infusion inhibited more than 95% of vwf function, which returned to baseline over 12-16 h after administration was suspended. nf-κb is involved in inflammation responses and modulates the synthesis of chemokines, interferons, major histocompatibility complex (mhc) proteins, growth factors, and the cell adhesion molecules that play a role in ischemia-reperfusion injuries seen in most myocardial infarctions [131] . a natural double stranded dna aptamer was found that binds to nf-κb with high affinity. in a rat cardiac ischemia-reperfusion model, this aptamer significantly reduced the expected injury [115] . in a rat cardioplegic arrest model, animals transfected with the nf-κb dna aptamer showed improved recovery of left ventricular function as well as coronary flow three days post-transfection compared to scrambled-dna controls (97% vs. 61%) [132] . the aptamer-treated group also showed a lower percentage of neutrophil adhesion to endothelial cells (38% vs. 81%) and a lower level of interleukin il-8 (109 vs. 210 ng/mg). the same aptamer was also studied in a murine model of nephritis, in which it abolished glomerular inflammation and the expression of inflammatory markers il-1α, il-1β, il-6, icam-2 (intracellular adhesion molecule 2), and vcam-1 (vascular cell adhesion molecule 1) [133] . a rat global brain ischemia model showed inhibition of tnf-α, il-1β and icam-1 expression in nf-κb aptamer-treated animals after 1 h of ischemia. moreover, 7 days after ischemia, neuronal damage was significantly attenuated in the nf-κb-aptamer-treated group compared to controls [134] . the proteins of different pathogens have also attracted the attention of researchers as targets for inhibitory nucleic acids. a recent study reported the use of a 2'-fy aptamer against the extracellular domain of the erythrocyte membrane protein 1 (pfemp1) of the parasitic protozoan plasmodium falciparum, achieving the efficient inhibition of erythrocyte rosseting in blood cultures [135] . rna viruses, especially hiv-1 and hcv, have been the main targets for therapeutic nucleic acids with catalytic activity. rossi's group designed a very interesting ex vivo selection procedure to identify anti-hiv hammerhead ribozymes. a pool of hammerhead catalytic domains containing randomized binding arm sequences was assayed against an hiv-1 chimera containing the thymidine kinase gene. after cell transfection of the ribozyme population expressed under the control of the u16snorna promoter, gancyclovir-resistant cells were selected. such antibiotic resistance suggests the presence of an active anti-hiv ribozyme [136] . banerjea's group designed a strategy to identify accessible cleavage sites within the hiv-1 gag rna and to pick out dnazymes effective against this target [137] . two dnazyme variant populations were synthesized. the specificity of the first was limited to all possible augs in the target rna, whereas the second population was designed to cleave any potential target site. these options were made possible by either fixing the nucleotides immediately preceding the catalytic motif to ca (for aug cleavage) or totally randomizing the seven bases on either side of the catalytic motif. dnazymes selected from both populations showed target-specific cleavage activities in the presence of mg 2+ , and significantly interfered with hiv-1-specific gene expression. this strategy could be used for the selection of effective target sites in any target rna [137] . viral proteins are favorite targets for the development of therapeutic aptamers. rna aptamers have been selected against different viral enzymes and proteins involved in host-cell interactions, such as hiv-1 reverse transcriptase (rt), hiv-1 glycoprotein 120, hcv rna-dependent rna polymerase (rdrp), sars coronavirus ntpase/helicase, and the hemagglutinin protein of human influenza virus b, all of which show efficient viral inhibition [138] [139] [140] [141] [142] [143] . human influenza b virus hemagglutinin protein has been inhibited in vitro by the rna pool resulting from 15 rounds of in vitro selection [143] . further studies are required for the identification of independent aptamers. recently, a dna aptamer (termed 93del) has been described that adopts a novel type of dimeric quadruplex folding and shows anti-hiv1 integrase activity in the nanomolar range in vitro by blocking several catalytic amino acid residues essential for integrase function. several other g-rich dna aptamers have been identified as remarkable hiv1-in inhibitors with ic50 values in the nanomolar range. t30695 is one such aptamer, and has been well studied in recent years, [144, 145] . astier-gi's group described the characterization of two dna aptamers (27v and 127v) that specifically bind to hepatitis c virus (hcv) rna polymerase (ns5b), inhibiting its activity in vitro [146] . aptamer 27v competed with the rna template for binding to the enzyme and blocked both the initiation and elongation phases of rna synthesis, whereas aptamer 127v exclusively inhibited initiation events. the authors also determined that, in addition to an in vitro inhibitory effect on rna synthesis, aptamer 27v interfered with the multiplication of hcv jfh1 in huh7 cells. the efficient cellular entry of these short dnas, and the inhibitory effect observed in human cells infected with hcv, indicate that aptamers are useful tools for the study of hcv rna synthesis; their therapeutic against hcv infection is also attractive [146] . an alternative and innovative potential therapeutic approach that has attracted much hope is the targeting of the structural domains of viral genomes which are frequently concentrated within untranslated terminal regions (utrs). the hiv-1 trans-activation response (tar) element is a polyfunctional rna domain mainly involved in the activation of transcription by its binding to the viral protein tat. aptamer r06 binds the tar element in vitro by a loop-loop interaction [147] . the intracellular expression of the aptamer by the nucleolar u16 rna promoter inhibits hiv-1 infection by more than 90%. this strategy takes advantage of hiv-1 rna nucleolar-trafficking to efficiently colocalize the aptamer and target [148] . the same r06 aptamer has been improved by the inclusion of extra binding rna domains targeting additional 5'-utr sites [149] . our own results show that presynthesized aptamers, or aptamers produced intracellularly via u6 rna promoter-driven expression, both with multiple binding sites targeting the whole hiv-1 5'-utr, efficiently inhibit hiv-1 replication in cell culture (sánchez-luque et al., unpublished). there have been different attempts to isolate rna aptamers against different domains of the hcv internal ribosome entry site (ires) located within the 5'-utr [150] [151] [152] . aptamer 3-07 binds domain iii-iv of hcv ires inhibiting its activity in cell-free systems [151] . this inhibition was further improved by conjugation of 3-07 with 2-02 in a chimeric molecule that targets domain ii [153] . rna aptamer ap30 isolated against the hcv (-) strand ires domain i partially inhibits hcv rna replicase-mediated (+) strand synthesis, probably by interfering with (-) strand binding [154] . a step forward was the design of an innovative selection approach to identify chimeric ribozyme/aptamer rna molecules against the entire hcv-ires. each selection cycle includes two consecutive selection steps for binding and cleavage of the viral rna [155] . after six selection rounds, seven families of inhibitor rnas were identified simultaneously targeting two sites within the hcv-ires, one for each inhibitory domain. these chimeric rna inhibitors promoted ires inhibition by up to 95% in cell extracts, identifying new targets for the development of anti-hcv agents. further characterization revealed up to 50% inhibition of viral translation and replication in a human liver cell line [156, 157] . the ability of selex to generate aptamers against any kind of target provides the possibility of their being used as therapeutic antidotes or palliatives. ricin is a toxic heterodimeric lectin from the castor bean plant ricinus communis. it disrupts protein synthesis by inactivation of the ribosomes. an aptamer isolated against the a chain partially restores protein synthesis levels in cell free translation systems and cell cultures [158] . a different approach has been explored for cocaine and anticonvulsant mk-801 alleviation. both compounds preferentially bind the open isoform of nicotine acetylcholine receptors (nachrs) found at neuromuscular junctions, autonomic ganglia and in the central nervous system. rna aptamers developed against nachrs with equal binding affinity for both open and closed sodium-potasium channel isoforms partially restore isoform equilibrium, alleviating drug channel inhibition in cells [159] [160] [161] . aptamers can distinguish between different isoforms of the same protein or different members of the same family. aptamer-sirna/toxin conjugates have been developed to deliver therapeutic agents within a specific target cell. ex vivo selection procedures performed against a specific cell type have yielded aptamers that are very efficiently taken up by those cells; they could therefore be used as a specific delivery tool. the tta1 aptamer of tenascin c, selected against the human glioma u251 cell line [110, 162] , was efficiently taken up by human tumor cells in a xenograft glioblastoma and breast tumor model [163] . aptamers targeting the prostate-specific membrane antigen (psma) are those most used as delivery tools. coffey's group reported two 2'-fy rna aptamers, a9 and a10, that bind specifically recombinant psma [164] . since psma is continually recycled from the cell surface, these aptamers are appropriate vehicles for delivering therapeutic compounds via the endosomal pathway. small interfering rnas against pro-survival factors over-expressed in prostate cancer cells, such as plk-1 and bcl-2 and eukaryotic elongation factor 2, have been delivered by the a10 aptamer [165] [166]. the aptamer-driven uptake of plk-1 bcl-2 sirnas leads to the death of psma-positive cells and tumor regression following intra-tumoral injection in mice xenograft models [165] . the improvement of the delivery vehicles were achieved in several ways and tumor regression through the almost complete loss of cell viability was observed when the eukaryotic elongation factor 2 sirna was delivered by a dimeric aptamer [166] . the delivery of pharmacological compounds by aptamers has also been studied by different approaches. doxorubicin (dox) is a chemotherapeutic intercalating agent used against cancer. dox intercalates between the aromatic rings of the gc pairs of the a10 aptamer, enhancing its cytotoxicity against psma-expressing cells and reducing the same in non-expressing cells [167] . the same results have been reported when using the a9-genolin conjugate, a toxin that blocks protein synthesis [168] . side toxicity of the chemotherapeutic compound can be further prevented by encapsulating it within nanoparticles or nanoconjugates [169] [170] [171] . aptamer b40, specific for hiv-1 gp120 [140] , has also been used to deliver sirna against infected cells. hiv-1 gp120 is found on the surface of infected cells and promotes cell fusion. consequently, besides neutralizing infective particles, the b40 aptamer can be used for anti-hiv-1 sirna delivery to infected cells. the first attempt involved a chimera of the b40 aptamer and sirna targeting the overlapping rev tat region [172] . the chimera was internalized in an aptamer-dependent manner, with inhibition dependent on the interference machinery. the aptamer-sirna linkage was further improved for the easy combination of sirnas to the aptamer and better sirna processing by the cellular machinery. this improved chimera resulted in the specific inhibition of hiv-1 replication and infectivity in pbmc and cultured cem t cells [173] . finally, aptamers can also be used to colocalize rna inhibitors with their specific target molecules at the subcellular level. aptamers can target rna domains, e.g., in viral rna genomes; they can therefore improve trans-cleaving ribozymes by anchoring them to their target. chimeric molecules composed of a hammerhead ribozyme and an aptamer both targeting the hcv ires have efficiently inhibited ires activity in human hepatocyte cell cultures [156, 157] . the procedures used to identify dna and rna molecules of interest in large populations of variant nucleic acid molecules have contributed significantly to the development of nucleic acid-based therapeutic drugs. aptamers show high specificity for their targets and have low toxicity and immunogenicity profiles. since the 1990s, the design and isolation of specific aptamers using selection and evolution techniques has been optimized and even automated. this has lead to great advances in our knowledge of aptamers as therapeutic agents and has expanded our bank of inhibitory nucleic acids and their possible targets, which now include cytokines, cell receptors, viruses and even whole cells. an aptamer drug is now on the market, and several mid and late clinical trials in progress that appears to confirm the great potential of these tools. with the improvement and optimization of selection strategies, and the ongoing discoveries made in this field, success in the development of nucleic acidbased therapeutics protocols might be predicted. an extracellular darwinian experiment with a selfduplicating nucleic acid molecule in vitro genetic analysis of the tetrahymena selfsplicing intron in vitro selection of rna molecules that bind specific ligands selection in vitro of an rna enzyme that specifically cleaves singlestranded dna systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase in vitro selection and evolution of rna: applications for catalytic rna, molecular recognition, and drug discovery in vitro selection of hairpin ribozymes in vitro selection of catalytic polynucleotides in vitro selection of functional nucleic acids in vitro selection of functional nucleic acid sequences in vitro selection procedures for identifying dna and rna aptamers targeted to nucleic acids and proteins berzal-herranz, a. rna selection and evolution in vitro: powerful techniques for the analysis and identification of new molecular tools aptamers: a new class of oligonucleotides in the drug discovery pipeline? in vitro rna random pools are not structurally diverse: a computational analysis in vitro selection using a dual rna library that allows primerless selection short bioactive spiegelmers to migraineassociated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-selex minimal primer and primer-free selex protocols for selection of aptamers from random dna libraries in vitro selection of active hairpin ribozymes by sequential rna-catalyzed cleavage and ligation reactions ribozyme catalysis of metabolism in the rna world selection-by-function: efficient enrichment of cathepsin e inhibitors from a dna library an allosteric synthetic dna engineering a ligand-dependent rna transcriptional activator in vivo evolution of an rna-based transcriptional activator aptazyme-mediated regulation of 16s ribosomal rna reengineering a natural riboswitch by dual genetic selection direct selection for ribozyme cleavage activity in cells chemical modification of sirnas for in vivo use therapeutic applications of dna and rna aptamers recent developments in oligonucleotide conjugation cell penetrating peptides: overview and applications to the delivery of oligonucleotides sequence-specific activity of antisense oligonucleotides conjugated to poly (l-lysine) carriers the cluster glycoside effect nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids prion-proteinspecific aptamer reduces prpsc formation a nuclease-resistant rna aptamer specifically inhibits angiopoietin-1-mediated tie2 activation and function the first analogues of lna (locked nucleic acids): phosphorothioate-lna and 2'-thio-lna structural studies of lna:rna duplexes by nmr: conformations and implications for rnase h activity the conformations of locked nucleic acids (lna) locked nucleic acids: promising nucleic acid analogs for therapeutic applications in vitro incorporation of lna nucleotides polymerase chain reaction and transcription using locked nucleic acid nucleotide triphosphates efficient enzymatic synthesis of lna-modified dna duplexes using kod dna polymerase mirror-image rna that binds dadenosine mirror-design of loligonucleotide ligands binding to l-arginine in vivo properties of an anti-gnrh spiegelmer: an example of an oligonucleotide-based therapeutic substance class inhibition of ghrelin action in vitro and in vivo by an rna-spiegelmer a general purpose rna-cleaving dna enzyme ribozyme structures and mechanisms gaining target access for deoxyribozymes dnazymes as potential therapeutic molecules inhibition of hiv-1 replication by rna-based strategies regulatory pathways governing hiv-1 replication identification and characterization of a hela nuclear protein that specifically binds to the trans-activation-response (tar) element of human immunodeficiency virus overexpression of tar sequences renders cells resistant to human immunodeficiency virus replication generation of species cross-reactive aptamers using "toggle pegaptanib, a targeted anti-vegf aptamer for ocular vascular disease antivascular endothelial growth factor therapy for neovascular agerelated macular degeneration inhibition of von willebrand factor-mediated platelet activation and thrombosis by the anti-von willebrand factor a1-domain aptamer arc1779 first-in-human evaluation of anti von willebrand factor therapeutic aptamer arc1779 in healthy volunteers first-in-human experience of an antidote-controlled anticoagulant using rna aptamer technology: a phase 1a pharmacodynamic evaluation of a drug-antidote pair for the controlled regulation of factor ixa activity a randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor ixa inhibitor phase 1b randomized study of antidote-controlled modulation of factor ixa activity in patients with stable coronary artery disease discovery and development of the g-rich oligonucleotide as1411 as a novel treatment for cancer the nucleolin targeting aptamer as1411 destabilizes bcl-2 messenger rna in human breast cancer cells discovery and development of anticancer aptamers inhibition of platelet-derived growth factor b signaling enhances the efficacy of antivascular endothelial growth factor therapy in multiple models of ocular neovascularization sequential loss of tumor vessel pericytes and endothelial cells after inhibition of platelet-derived growth factor b by selective aptamer ax102 derivation of rna aptamer inhibitors of human complement c5 selection of single-stranded dna molecules that bind and inhibit human thrombin in vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits pilot study of the efficacy of a thrombin inhibitor for use during cardiopulmonary bypass pegaptanib sodium for ocular vascular disease 2'-fluoropyrimidine rna-based aptamers to the 165-amino acid form of vascular endothelial growth factor (vegf165). inhibition of receptor binding and vegf-induced vascular permeability through interactions requiring the exon 7-encoded domain the biology of vegf and its receptors a phase ii randomized doublemasked trial of pegaptanib, an anti-vascular endothelial growth factor aptamer, for diabetic macular edema inhibition of vegf blocks tgf-beta1 production through a pi3k/akt signalling pathway rna aptamers selected against the glur2 glutamate receptor channel one rna aptamer sequence, two structures: a collaborating pair that inhibits ampa receptors aptamer antagonists of myelin-derived inhibitors promote axon growth characterization of 2'-fluoro-rna aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion the role of neutrophil elastase in chronic inflammation in vitro selection of rna-based irreversible inhibitors of human neutrophil elastase highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized dna-valine phosphonate library protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury high-affinity ige receptor on eosinophils is involved in defence against parasites the human ige network high-affinity oligonucleotide ligands to human ige inhibit binding to fc epsilon receptor i neutralizing aptamers from whole-cell selex inhibit the ret receptor tyrosine kinase distribution and bioactivity of the ret-specific d4 aptamer in three-dimensional collagen gel cultures inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3 antimetastatic potential of pai-1-specific rna aptamers rna aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1 selection and characterization of an rna decoy for transcription factor nf-kappa b selection and characterization of anti-nf-kappab p65 rna aptamers yeast genetic selections to optimize rna decoys for transcription factor nf-kappa b characterization of anti-nf-kappab rna aptamer-binding specificity in vitro and in the yeast three-hybrid system h1 rna polymerase iii promoter-driven expression of an rna aptamer leads to high-level inhibition of intracellular protein activity rna aptamertargeted inhibition of nf-kappa b suppresses non-small cell lung cancer resistance to doxorubicin rna aptamers interfering with nucleophosmin oligomerization induce apoptosis of cancer cells multivalent rna aptamers that inhibit ctla-4 and enhance tumor immunity multivalent 4-1bb binding aptamers costimulate cd8+ t cells and inhibit tumor growth in mice assembling ox40 aptamers on a molecular scaffold to create a receptor-activating aptamer inhibition of rat corneal angiogenesis by a nuclease-resistant rna aptamer specific for angiopoietin-2 inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin 2-specific rna aptamer aptamers: an emerging class of therapeutics antiproliferative activity of grich oligonucleotides correlates with protein binding agro100 inhibits activation of nuclear factor-kappab (nf-kappab) by forming a complex with nf-kappab essential modulator (nemo) and nucleolin novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers tenascin-c aptamers are generated using tumor cells and purified protein a subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-b chain mrna platelet-derived growth factor (pdgf) and pdgf receptor are induced in mesangial proliferative nephritis in the rat structural and functional studies on platelet-derived growth factor inhibitory dna ligands to platelet-derived growth factor b-chain in vivo transfection of cis element "decoy" against nuclear factor-kappab binding site prevents myocardial infarction specific antagonism of pdgf prevents renal scarring in experimental glomerulonephritis the effects of platelet-derived growth factor antagonism in experimental glomerulonephritis are independent of the transforming growth factor-beta system transport of molecules in the tumor interstitium: a review delivery of molecular medicine to solid tumors inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors emerging pharmacologic therapies for wet age-related macular degeneration braking the cycle e2f: a link between the rb tumor suppressor protein and viral oncoproteins distinct roles of e2f proteins in vascular smooth muscle cell proliferation and intimal hyperplasia inhibition of cell proliferation by an rna ligand that selectively blocks e2f function blocking the initiation of coagulation by rna aptamers to factor viia rna aptamers as reversible antagonists of coagulation factor ixa antidote-mediated control of an anticoagulant aptamer in vivo nucleic acid aptamers as antithrombotic agents: opportunities in extracellular therapeutics von willebrand factor and the endothelium in vascular disease rel/nf-kappa b/i kappa b family: intimate tales of association and dissociation a novel strategy for myocardial protection using in vivo transfection of cis element 'decoy' against nfkappab binding site: evidence for a role of nfkappab in ischemia-reperfusion injury transcription factor decoy for nfkappab inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis nuclear factor-kappa b decoy attenuates neuronal damage after global brain ischemia: a future strategy for brain protection during circulatory arrest in vitro selection of rna aptamers against a conserved region of the plasmodium falciparum erythrocyte membrane protein 1 use of a u16 snorna-containing ribozyme library to identify ribozyme targets in hiv-1 in vitro-selected rna cleaving dna enzymes from a combinatorial library are potent inhibitors of hiv-1 gene expression rna pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase bent pseudoknots and novel rna inhibitors of type 1 human immunodeficiency virus (hiv-1) reverse transcriptase neutralization of infectivity of diverse r5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'f-rna aptamers in vitro selection of rna aptamers that bind the rna-dependent rna polymerase of hepatitis c virus: a possible role of gc-rich rna motifs in ns5b binding isolation of inhibitory rna aptamers against severe acute respiratory syndrome (sars) coronavirus ntpase/helicase an efficient rna aptamer against human influenza b virus hemagglutinin mechanism of inhibition of hiv-1 integrase by g-tetrad-forming oligonucleotides in vitro structure-activity of tetrad-forming oligonucleotides as a potent anti-hiv therapeutic drug astier-gin, t. inhibition of hepatitis c virus (hcv) rna polymerase by dna aptamers: mechanism of inhibition of in vitro rna synthesis and effect on hcv-infected cells in vitro selection identifies key determinants for loop-loop interactions: rna aptamers selective for the tar rna element of hiv-1 endogenous expression of an anti-tar aptamer reduces hiv-1 replication bimodal loop-loop interactions increase the affinity of rna aptamers for hiv-1 rna structures apical loop-internal loop interactions: a new rna-rna recognition motif identified through in vitro selection against rna hairpins of the hepatitis c virus mrna rna aptamers targeted to domain ii of hepatitis c virus ires that bind to its apical loop region a hepatitis c virus (hcv) internal ribosome entry site (ires) domain iii-iv-targeted aptamer inhibits translation by binding to an apical loop of domain iiid increased inhibitory ability of conjugated rna aptamers against the hcv ires isolation and characterization of rna aptamers specific for the hcv minus-ires domain i berzal-herranz, a. interfering with hepatitis c virus ires activity using rna molecules identified by a novel in vitro selection method berzal-herranz, a. inhibition of hepatitis c virus internal ribosome entry site-mediated translation by an rna targeting the conserved iiif domain berzal-herranz, a. inhibition of hepatitis c virus replication and internal ribosome entry site-dependent translation by an rna molecule protective effects of anti-ricin a-chain rna aptamer against ricin toxicity in vitro selection of rna molecules that displace cocaine from the membranebound nicotinic acetylcholine receptor mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and mk-801 minimal rna aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor a tenascin-c aptamer identified by tumor cell selex: systematic evolution of ligands by exponential enrichment tumor targeting by an aptamer identification and characterization of nucleasestabilized rna molecules that bind human prostate cancer cells via the prostate-specific membrane antigen cell type-specific delivery of sirnas with aptamer-sirna chimeras cell-specific induction of apoptosis by rationally designed bivalent aptamer-sirna transcripts silencing eukaryotic elongation factor 2. curr. cancer drug targets an aptamer-doxorubicin physical conjugate as a novel targeted drug-delivery platform aptamer:toxin conjugates that specifically target prostate tumor cells targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo formulation of functionalized plga-peg nanoparticles for in vivo targeted drug delivery targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized pt(iv) prodrug-plga-peg nanoparticles novel dual inhibitory function aptamer-sirna delivery system for hiv-1 therapy selection, characterization and application of new rna hiv gp 120 aptamers for facile delivery of dicer substrate sirnas into hiv infected cells the work of berzal-herranz's group is funded by grant bfu2009-08137 from the spanish ministry of science and innovation, grant cts-5077 from the junta de andalucía, and by feder funds from the e.u. key: cord-343963-99rd3o79 authors: wong, mun-teng; chen, steve s-l title: emerging roles of interferon-stimulated genes in the innate immune response to hepatitis c virus infection date: 2014-12-29 journal: cell mol immunol doi: 10.1038/cmi.2014.127 sha: doc_id: 343963 cord_uid: 99rd3o79 infection with hepatitis c virus (hcv), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. hcv infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. upon hcv infection, the host induces the interferon (ifn)-mediated frontline defense to limit virus replication. conversely, hcv employs diverse strategies to escape host innate immune surveillance. type i ifn elicits its antiviral actions by inducing a wide array of ifn-stimulated genes (isgs). nevertheless, the mechanisms by which these isgs participate in ifn-mediated anti-hcv actions remain largely unknown. in this review, we first outline the signaling pathways known to be involved in the production of type i ifn and isgs and the tactics that hcv uses to subvert innate immunity. then, we summarize the effector mechanisms of scaffold isgs known to modulate ifn function in hcv replication. we also highlight the potential functions of emerging isgs, which were identified from genome-wide sirna screens, in hcv replication. finally, we discuss the functions of several cellular determinants critical for regulating host immunity in hcv replication. this review will provide a basis for understanding the complexity and functionality of the pleiotropic ifn system in hcv infection. elucidation of the specificity and the mode of action of these emerging isgs will also help to identify novel cellular targets against which effective hcv therapeutics can be developed. hepatitis c virus (hcv) infects more than 170 million people worldwide and represents a heavy burden to global health, with the highest prevalence rates found in africa and the eastern mediterranean. 1 acute hcv infection is asymptomatic, and in 70% of infected individuals, the virus persists and progresses to chronic liver diseases, including fibrosis, steatosis, cirrhosis and hepatocellular carcinoma. 2, 3 furthermore, hcv is a major cause of type i mixed cryoglobulinemia, which occurs in 10% of patients. 4 using the hcv genotype 2a isolate japanese fulminant hepatitis-1 genome-based cell culture-derived infectious hcv (hcvcc), 5 zhong et al. 6 demonstrated that hcv and cells coevolve in vitro during chronically persistent infection, which involves the selection of viral mutants with increased infectivity and cells with resistance to viral entry and/or rna replication. in this coevolution process, hcv exhibits multifaceted interactions with the host cells, and these cellular stress responses subsequently affect virus replication. for instance, infection with hcvcc or expression of the japanese fulminant hepatitis-1 genome has been shown to trigger cytopathic effects, endoplasmic reticulum (er) stress, the unfolded protein response (upr), autophagy and the innate immune response. [7] [8] [9] [10] [11] [12] [13] [14] in the competition between this virus and host cells, viral infection often triggers a first-line host defense through the production of type i interferon (ifn), which is a broadly acting antiviral cytokine, and inflammatory cytokines. these cytokines confer an antiviral state on the host cells, thereby interfering with viral replication. 15, 16 with the ability to enhance the immune response for virus clearance or to inhibit viral replication, ifn-based therapies have been used to treat hcvinfected patients for over two decades. 17 to guard against viral infection, the host cell has developed multiple restriction strategies to limit viral infection. the expression of many of these restriction factors is subject to transcriptional regulation by ifn. 13, 14 upon infection by viruses such as hcv, viral rna is first sensed by cellular pattern recognition receptors (prrs), and the prr-mediated recruitment of adaptor proteins and the activation of downstream signaling lead to ifn production. 16, 18, 19 after binding to its receptor (ifnar) complex present on the cell surface, ifn triggers the janus kinase (jak)/signal transducer and activator of transcription (stat) pathway to drive the synthesis of over 300 ifn-stimulated genes (isgs), which block virus replication at different phases of the virus replication cycle. [19] [20] [21] [22] these isgs are usually not synthesized at the basal state, but are induced to express and mediate the antiviral effector functions of ifn upon viral infection or ifn treatment. in response, viruses have developed elaborate strategies to escape the ifn antiviral system by blocking the expression or antiviral functions of ifn. 19, 23, 24 therefore, the host and hcv maintain a homeostatic state, allowing tightly restricted viral replication without killing the host. 13, [25] [26] [27] recent studies on genome-wide sirna screens have also added new candidates to this growing list of anti-hcv isgs. these findings highlight the complexity and pleiotropic roles of ifn and its induced isgs in modulating innate immunity and virus replication. nevertheless, the complete spectrum of isgs and their functionality in suppressing hcv replication have yet to be defined. 28, 29 in this review, we focus on the molecular aspects of the type i ifn system and its effector mechanisms in modulating hcv replication. first, we briefly discuss the signaling triggered by the retinoic acid-inducible gene 1-like receptor (rlr) and the toll-like receptor (tlr), which leads to type i ifn synthesis and ifn-mediated signaling pathway activation, resulting in the expression of a variety of effector isgs. we also summarize the strategies that hcv uses to escape ifn antiviral surveillance. additionally, we highlight what is currently known regarding the pivotal isgs in viral infections, with an emphasis on their anti-hcv activities, and the emerging isgs identified from recent genome-wide sirna screens in relation to anti-hcv activities. finally, we discuss the potential functions of several critical cellular factors, such as high-mobility group box 1 (hmgb1) and immunity-related gtpase family m (irgm), and cellular pathways, such as upr and autophagy, during hcv infection. although these cellular determinants are not stimulated by ifn, these factors critically control the host immune response. therefore, these determinants may also play crucial roles in modifying hcv replication. this review provides a perspective for a better understanding of the anti-hcv mechanisms of ifn, isgs and several critical cellular determinants known to contribute to the regulation of innate immunity. the gathered information not only provides a clearer picture for the specificity, functionality and complexity of the ifn system and its effector mechanisms in the control of hcv infection, but also helps to identify novel cellular targets against which efficacious therapeutic strategies can be developed. clinically, the identification of new isgs will also help to optimize the current ifn-based therapy and to provide a basis for more accurate predictions of ifn treatment outcomes. hcv is an enveloped, positive-sense, single-stranded rna virus classified within the genus hepacivirus in the flaviviridae family. 3 currently, hcv isolates are classified into seven major genotypes, i.e., genotypes 1 through 7, and an array of subtypes. 30 hcv genotypes differ by 20%-35% in genome sequence, 31 whereas subtypes within each genotype can differ by least 15%. 30 genotype 1 is the most prevalent (46%), followed by genotype 3 (30%); genotypes 2, 4 and 6 (cumulatively approximately 22%); and genotype 5 (less than 1%). 32 different genotypes exhibit distinct geographic distributions. genotype 1 predominates in america and europe, genotype 2 in japan, genotype 3 in asia, genotype 4 in africa and middle east and genotype 6 in southeast asia. 32 hcv is transmitted via blood transfusion, intravenous drug abuse, unsafe therapeutic injection, liver transplantation and other risk factors. 33 the combination of pegylated ifn-a and ribavirin is the standard therapy for hcv infection. however, this treatment is associated with side effects, and the efficacy of this regimen varies among genotypes, limiting the success rate of this treatment. 34 compared with genotype 2, infection with genotypes 1a and 1b results in more severe liver disease and low responsiveness to ifn therapy. seventy-one percent of patients with genotype 2 infection respond to ifn therapy, whereas only 28% of genotype 1a and 26% of genotype 1b show a response. 35 patients infected with genotype 6 generally show higher sustained virological responses to ifn therapy than genotype 1infected patients, 36 whereas genotype 3-infected patients show a lower sustained virological response compared with genotype 1-infected patients. 37 the heterogeneity of hcv genotypes also translates to differences in the manifestation of liver disease. 38 for example, hepatic steatosis is most common in patients infected with genotype 3 and is attributed to its core protein. 37 recently, the use of active direct-acting antiviral molecules to block hcv infection has led to substantial improvements in sustained virological response rates in genotype 1-infected patients. however, the use of these drugs may allow selection of resistant variants if direct-acting antiviral monotherapy is adopted, and a high relapse rate occurs after direct-acting antiviral treatment is discontinued. 39 the approximately 9.6-kb hcv genome contains a single open reading frame flanked by untranslated regions (utrs) at its 59 and 39 ends (figure 1 ). 3 the internal ribosome entry site (ires) located in the 59-utr directs cap-independent translation, whereas the 39-utr contains sequences critical for viral replication and translation. 40 the 39-utr (positioned at nucleotides 9389-9679 of the hcv genome) contains a poly(u/uc) (pu/uc) tract located at nucleotide positions 9436-9600, which was identified as an hcv pathogen-associated molecular pattern (pamp) that triggers rlr-mediated type i ifn production ( figure 1 ). 22 translation of the hcv genomic rna produces a single polyprotein of approximately 3000 amino acids, which is further processed by cellular and viral proteases to yield the structural proteins core, e1 and e2 and the non-structural (ns) proteins p7, ns2, ns3, ns4a, ns4b, ns5a and ns5b (figure 1 ). 3 ns proteins participate in different phases of the viral replication cycle (figure 1 ). for example, ns3 to ns5b proteins are important for rna replication. 3 jones et al. 41 showed that p7 and ns2 are required for entry and assembly of the virus. other researchers have also reported that ns2, 42-45 ns3/4a 46 and ns5a 47, 48 are involved in virion assembly and production. hepatocytes are the primary target cells for hcv replication. upon infection, the virus particle circulating in the blood biochemically resembles the very low-density lipoprotein, which is rich in apolipoprotein (apo) e and apob. 3, 49 first, the apolipoprotein-associated lipoviral particle (lvp) attaches to glycosaminoglycan and low-density lipoprotein receptor and then interacts with cluster of differentiation 81 (cd81) and scavenger receptor class b number 1 (figure 2 ). 50 the lvp is subsequently translocated to the tight junction of hepatocytes where the lvp binds to the tight junction proteins claudin-1 and occludin followed by internalization of the hcv particle via ph-dependent endocytosis, which occurs on the plasma membrane ( figure 2 ). 50 in addition to these receptors, cell surface molecules, such as epidermal growth factor receptor, ephrin receptor a2 and niemann-pick c1-like l1 cholesterol uptake receptor, are also essential for virus internalization. 50 subsequent to internalization, the acidic ph in the endosome triggers fusion of the viral envelope with the endosomal membrane, allowing the release of the viral genome into the cytoplasm ( figure 2 ). 50 hcv genomic translation occurs at the rough er, and hcv rna replicates in an er-derived or er-associated lipid-rich environment termed the membranous web ( figure 2 ). 51 all hcv ns proteins except for ns2 are involved in viral rna replication. the ns proteins are colocalized with the replicating viral rna on a light density, detergent-resistant cytoplasmic membrane structure termed a 'lipid raft'. 52 lipid droplets (lds), which comprise a neutral lipid core with a single phospholipid layer, serve as energy storage sites and reservoirs of neutral lipids in adipose tissue and hepatocytes. 53 lds are indispensable for viral rna replication and infectious virus formation. 54 during the initial stage of virus assembly, hcv core protein interacts with lds, 55 and the viral replication complex is also directed to lds in an ns5a-and core-dependent manner, allowing encapsidation of the viral rna by the core protein and assembly of the nucleocapsid ( figure 2 ). 47, 54 additionally, the interaction of ns3 and ns5a with actin and tubulin in the microtubule network mediates translocation of the hcv replication complex to lds. 56 the late stage of virus assembly, which occurs in the lumen of the er, involves the acquisition of a lipid envelope, the embed assembly and budding liver cell figure 2 hcv replication cycle. as shown, the hcv lvp is coated with apob and apoe, which are marked by light green and light blue stripes, respectively, on its surface. the lvp attaches to srb1 and to cd81 and further interacts with the tight junction protein claudin-1 and with occludin. virus entry into cell proceeds through receptor-mediated endocytosis at the cell surface. subsequent to internalization, the viral envelope fuses with the endosomal membrane under acidic ph, and the viral genome is uncoated and released into the cytoplasm. to dissect these two events, internalization and fusion are conventionally depicted as two seemingly separate steps in the cytoplasm. viral rna is translated at the er to produce the polyprotein, which is subsequently processed into mature structural and non-structural proteins. viral non-structural proteins, in conjunction with host factors, form a membranous web where viral rna replication occurs. hcv particle assembly most likely initiates near the er and ld where core protein and viral rna accumulate. finally, hcv particles are secreted into the extracellular milieu via the secretory pathway. viral replication and assembly occur in the proximity of lds and in lipid raft microdomains, which are shown in the inserted dashed rectangle. apo, apolipoprotein; cd81, cluster of differentiation 81; er, endoplasmic reticulum; hcv, hepatitis c virus; ld, lipid droplet; lvp, lipoviral particle. ding of e1 and e2 into the envelope and the formation of the nascent virion ( figure 2 ). then, the nascent virus particles associate with apob, apoe and other very low-density lipoprotein lipids to form lvps. 50 finally, lvps are released from cells through the very low-density lipoprotein secretion pathway or the endosome secretory pathway ( figure 2 ). 50 similar to the ns proteins, p7 plays numerous crucial functions in virion assembly and egress. 57 p7, an integral, oligomeric membrane protein consisting of 63 amino acids, is grouped into the family of viroporins that form membrane pores or channels. 58 in functioning as an ion channel, p7 modulates membrane permeability to facilitate virus entry by promoting virus uncoating and to enhance assembly or release. 58 p7 conducts ions across the membrane, and this channel activity can be abrogated by the drug amantadine and iminosugar derivatives. 59, 60 during maturation and egress, the ion channel activity of p7 maintains the ph gradients within the secretory pathway and thereby stabilizes the hcv particle. 50 in addition, p7 has been shown to be necessary for capsid assembly and envelopment because mutations in p7 result in the accumulation of incompletely assembled capsids that are unable to encapsidate viral rna. 61 the ifn systems constitute the first-line defense mechanism against viral infection in humans. 62 based on their antiviral properties, ifns are grouped into three classes: type i, ii and iii ifns. 63 in humans, type i ifns comprise a large group of molecules encoded by multiple genes, mainly ifn-a and ifn-b, and other genes such as ifn-e, ifn-k and ifn-v. 64 ifn-a and ifn-b combat viruses directly by inhibiting virus replication or indirectly by inducing the innate immune response. 63 most cell types can elicit a type i ifn response by activating the tlr, rlr and jak-stat pathways. 64, 65 type ii ifn contains only one member, ifn-c. unlike type i ifns, which are elicited as a direct response to viral infection, ifn-c is secreted by natural killer cells and mitogenically activated t cells. 63 ifn-c exerts potent anti-hcv activity in vitro and mediates antiviral t-cell responses. it has also been reported that ifn-c inhibits hcv infection by downregulating claudin-1 and cd81. 66 type iii ifns consist of three members, termed ifn-l1, ifn-l2 and ifn-l3 or il-29 (l1) and il-28a/b (l2/3). as with type i ifns, viral infection also directly activates type iii ifns. however, the antiviral properties and the mechanisms of action of type iii ifns remain unknown. type iii ifns can be secreted by many cell types, but their receptors show a limited tissue distribution. 63 hcv infection results in type iii ifn induction predominantly in the human liver. despite modulation by the irf3 and nf-kb pathways for induction of type iii and type i ifns, these two systems upregulate distinct subsets of isgs with different kinetics of induction. 67 during hcv infection, cells produce type i ifn to counteract viral infection, to modulate viral replication and to activate natural killer cells, dendritic cells and kupffer cells. 68 recognition of pamps by prrs, including tlrs and rlrs, triggers ifn synthesis and ifn-mediated cascade signaling pathways, leading to the production of type i ifn and a wide range of isgs to mediate ifn antiviral activity ( figure 3 ). 13, [20] [21] [22] upon virus infection, tlrs and rlrs operate through different signaling pathways, depending on the nature of the viral signals. tlrs are expressed and localized in the intracellular compartment, similar to endosomes, or on the cell surface. 69, 70 unlike rlrs, tlrs potentially detect viral double-stranded rna (dsrna) released by cells into the extracellular milieu ( figure 3 ). 69 three types of tlrs, i.e., tlr3, tlr7 and tlr9, are involved in the recognition of virus infections. tlr3 detects the dsrna formed during the replication of positive-stranded rna viruses, whereas tlr7 recognizes the urine-rich ribonucleotide region of rna, and tlr9 senses dna pamp motifs encoding cpg dinucleotides. 69 upon binding to a pamp, tlr3 dimerizes and initiates the binding of its cytosolic toll-il-1 receptor to the adaptor protein toll-il-1 receptor domain-containing adaptor inducing ifn-b (trif), resulting in the association of tlr3 with trif ( figure 3 ). 13, 14, 19 this interaction leads to the recruitment of tumor necrosis factor (tnf) receptor-associated factor (traf) 6, traf3 and the traf family member nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) activator-binding kinase 1 (tbk1), resulting in the phosphorylation and activation of ifn regulatory factor (irf) 3 by tbk1 and by inhibitor of kappa b (ikb) kinase-related kinase (ikk)e. 19, 70 after phosphorylation, the irf3 protein dimerizes and is translocated into the nucleus to form an enhanceosome complex with nf-kb and other transcription factors, thereby inducing the expression of target genes, such as ifns. 70 moreover, the binding of viral dsrna to tlrs also activates nf-kb activity and pro-inflammatory cytokine synthesis through the interaction of trif with receptor-interacting protein-1. tlr7 and tlr9 bind to myeloid differentiation pro-inflammatory response 88 (myd88) and activate il-1 receptor-associated kinase (irak) and traf6, followed by the activation of ikka/b, which in turn activates nf-kb through phosphorylation, polyubiquitination and proteasomal degradation of its associated inhibitor ikba. migration of nf-kb into the nucleus results in ifn production ( figure 3 ). 70 the rlr receptors consist of rig-i, melanoma differentiation-associated protein 5 (mda5) and laboratory of genetics and physiology-2. 13,16,18 rig-i recognizes the hcv replication intermediate dsrna within hours of infection, which triggers the downstream signaling before the viral protein is extensively synthesized (figure 3 ). 71 rig-i senses the short, non-self dsrnas with 59-triphosphates, whereas rnas lacking 59-ppp, such as picornaviruses, are recognized by mda5. 72 both rig-i and mda5 contain two n-terminal caspase activator and recruitment domains (cards). 24 the recognition of dsrna by rig-i is dependent upon the atp-driven translocase activity of cards and helicases, and binding to dsrna induces conformational changes in rig-i that facilitate its oligomerization and translocation from the cytosol to the mitochondrial surface. 73, 74 on the mitochondria, rig-i engages via its tandem cards with the card of its downstream effector, mitochondria antiviral signaling protein (mavs), which is also termed ifn-b promoter stimulator-1, virus-induced signaling adaptor or card adaptor inducing ifn-b ( figure 3 ). 16, 18 the chaperone protein 14-3-3e and the ring finger domaincontaining e3 ubiquitin (ub) ligase triple motif-containing protein (trim) 25 also participate in this process. trim25 mediates the ubiquitination of rig-i at position lys-63, which is important for mavs binding and for ifn production. 74 the interaction between rig-i and mavs promotes the formation of a signaling complex on the mitochondrial surface that recruits and activates the downstream classical ikk complex, ikka/ikkb, and two non-classical ikk-related kinases, tbk1 and ikke. 75, 76 activation of tbk1 and ikke leads to the phosphorylation, dimerization and nuclear translocation of the transcription factor irf3 ( figure 3 ). 14 traf3, traf6 and mitogen-activator protein kinase/extracellular signal-regulated kinase (erk) kinase 1 (mekk1) are also recruited to mavs to activate nf-kb. 24 the canonical ikka and ikkb induce nf-kb-dependent gene transcription via phosphorylation, polyubiquitination and proteasomal degradation of ikba, thereby resulting in the release and nuclear migration of nf-kb. nf-kb activation involves the interaction of card9 with b-cell lymphoma/leukemia 10 protein. activated nf-kb and irf3 are translocated into the nucleus to form an enhanceosome, thereby stimulating the expression of ifn and inflammatory cytokines with the help of other cellular factors, such as activating transcription protein 2 and c-jun. 63 then, secreted ifn binds to ifnar on the cell surface and triggers the jak-stat signaling pathway ( figure 3 ). 75, 77 following ifnar receptor binding, tyrosine kinase-2 and jak1 are activated and phosphorylate stat1 and stat2 to form a heterodimer, which subsequently recruits irf9 to form the transcription factor ifn-stimulated gene factor 3 (isgf3). 75 then, isgf3 is translocated into the nucleus, binds to ifnsensitive responsive element (isre) and transactivates the expression of various isgs, such as 29-59 oligoadenylatesynthetase (2-5oas)/29,59-linked oligoadenylate (2-5a)-dependent, latent endoribonuclease (rnase l), dsrna-dependent protein kinase r (pkr), and irf7 ( figure 3 ). 78 acute hcv infections can be spontaneously cleared in some infected individuals, suggesting that the innate immunity induced by hcv pamp sensing can control acute viral infection. 19, 23 however, 80% of acutely infected people are not effectively cleared of hcv infection, and these patients may further develop chronic infection, suggesting that hcv has developed strategies to escape or to counteract the host immune response, leading to the emergence of resistance to ifn therapy. in this regard, several hcv proteins have been shown to block host antiviral responses, resulting in progression to chronic hcv infection ( figure 4 ). 13, 23, 24 obtaining further data regarding hcv evasion of host innate immunity will certainly improve ifn-based therapy outcomes. core protein hcv core protein is involved in the formation of the viral nucleocapsid and modulates many cellular functions, including transcription and signal transduction. expression of the full-length hcv genome or core protein downregulates ifn signaling by depressing stat1 tyrosine phosphorylation, which then blocks stat1 heterodimerization with stat2 and inhibits ifn signal transduction and isg expression ( figure 4) . 79 in addition, expression of core protein induces synthesis of suppressor of cytokine signaling 3 (socs3) in hepg2 cells. 80 socs3 is an important repressor of the jak-stat pathway due to its ability to inhibit stat1 phosphorylation ( figure 4 ). 77, 80 thus, hcv core protein induces socs3 and suppresses ifn-mediated isg expression. 79-81 socs3 expression is upregulated in chronically hcv-infected patients who are ifn non-responders compared with responders. 80 core protein expression has also been demonstrated to inhibit irf1 synthesis, transcriptionally repressing several isgs, such as il-15, il-12 and pkr. 82 many viruses use molecular mimicry as an important immune evasion strategy to promote virus survival and persistence. 83 viruses express proteins that are structurally similar to host defense proteins, and these viral proteins can act as immune modulators. 83 hcv employs this molecular mimicry strategy to resist type i ifn through its e2 envelope protein. 84 ,85 e2 comprises a 12-amino acid sequence identical to eukaryotic initiation factor 2a (eif2a) and pkr. 84 this domain operates to prevent pkr-dependent phosphorylation of eif2a and repression of protein synthesis, thus possessing an ability to resist type i ifn treatment ( figure 4 ). 84, 85 ns3/4a the hcv ns3/4a protease is not only responsible for the maturation of ns proteins, viral rna replication and virion morphogenesis but is also important for suppressing the host antiviral system. 13, 19, 23, 24, 86 the ns3/4a complex is anchored to the intracellular membrane through the ns4a transmembrane domain and the amphipathic a-helix at the ns3 n-terminus. 87 all these domains facilitate cleavage of their two cellular targets, mavs and trif, which act as key players in type i ifn production ( figure 4 ). 88, 89 mavs is an essential antiviral signaling protein in the rlr system and, therefore, is an ideal target for viral immune evasion. ns4a serves as the primary membrane subcellular targeting subunit to escort ns3/4a to mavs. 86 ns3/4a binds to mavs on mitochondria 88 and cleaves mavs at cys-508, resulting in the dislocation of the n-terminal portion of mavs from mitochondria and the suppression of ifn production ( figure 4 ). 71 the hydrophobic amino acid stretch in the ns3 amphipathic a-helix is also required for controlling rig-i signaling. 86 cleavage of mavs and reduction of ifn levels have been observed in chronically hcv-infected patients. 90 thus, ns3/4a-mediated cleavage of mavs rig-i signaling impairs ifn synthesis. 90 additionally, the ns3/4a protease also cleaves trif, an adaptor protein linking tlr3 to kinases responsible for activating irf3 and nf-kb ( figure 4 ). 89, 91 cleavage of trif interferes with poly(i:c)-activated tlr signaling and irf3 and nf-kb activation, thereby limiting the expression of multiple host defense genes and enhancing hcv persistence. 89 stimulator of interferon gene (sting), which is also known as mediator of irf3 activation (mita), is a 42-kda protein mainly localized to the er. 92 in response to dsdna transfection or dna virus infection, sting plays a crucial role in the activation of transcription pathways, essential for effective innate immune signaling. 92 upon dsdna stimulation, sting polymerizes and translocates from the er to a cytoplasmic punctate structure where the sting polymer provides a platform to connect tbk1 with irf3, which phosphorylates irf3, thereby triggering downstream signaling. 93 in viral infection, ns4b from yellow fever virus (yfv) blocks the induction of the ifn production pathway through an interaction with sting. 94 ns2b3 from dengue virus (denv) acts as a protease to cleave sting, thereby shutting down ifn signaling. 95 in hcv infection, ns4b interacts with and sequesters sting on the er to inhibit the association of sting with tbk, suppressing ifn signaling ( figure 4 ). 96, 97 therefore, targeting sting to inhibit innate immunity might be beneficial for virus survival. the mature hcv ns5a is present as two phosphoproteins, the hypophosphorylated p56 and hyperphosphorylated p58. 98, 99 ns5a phosphorylation occurs at multiple serine residues, such as serine 225, 229 and 232 upstream of the ifn sensitivity determining region (isdr) of ns5a, which spans residues 237-276 (based on genotype 1b hcv-j strain), and these serine residues are important for hyperphosphorylation. 98, 100 the functional and locational significance of ns5a p56 and p58 remains unclear; however, maintenance of these two forms at a specific ratio is critical for hcv replication. 100 functions as a pleiotropic protein that modulates the host environment to favor virus replication and persistence. 102 additionally, ns5a binds to myd88, which is a major adaptor molecule in the tlr pathway, and inhibits the recruitment of irak1 to myd88, attenuating tlr signaling and impairing cytokine production. 103 a sequence within the ns5a isdr, which spans residues 237-302, was shown to be responsible for interaction with the death domain of myd88 in macrophage cells. 103 pkr is an ifn-induced gene product that is activated by binding to dsrnas commonly produced during viral replication. ns5a rescues hcv replication in ifn-treated cells and inhibits ifn antiviral activity by binding to pkr and blocking pkr autophosphorylation and eif2a phosphorylation. 23, 104 ns5a expression is sufficient to rescue the replication of an ifn-sensitive virus. 102 the interaction of pkr with ns5a requires the isdr that overlaps a broader pkr-binding region, residues 234-366, and results in the inhibition of pkr activation and resistance to ifn in hcv-expressing cells. 104, 105 consistent with this mechanism, mutations in or deletion of isdr correlate with sensitivity to ifn-a-mediated antiviral activity. 102, 105, 106 moreover, meta-analysis and long-term follow-up support the association of this isdr region with the outcome of ifn therapy. this region, which encompasses a genetically flexible domain that allows mutations to occur, is the key site of adaptation to ifn therapy and influences the fitness of hcv rna replication. 23 in contrast, other studies suggest that the inhibitory effect of ns5a on ifn may be independent of pkr. ns5a increases expression of il-8, also known as chemokine cxcl8, by upregulating the il-8 promoter, which in turn, inhibits ifn antiviral activity and facilitates virus infection. 107, 108 in a cell culture model, il-8-positive cells are associated with chronic hcv infection, and il-8 removal mitigates hcv replication. 108 importantly, the serum level of il-8 is elevated in chronic hepatitis c patients compared with control individuals or is higher in ifn non-responders relative to responders. 23 these observations suggest that ns5a expression increases il-8 production, which somehow perturbs the ifn antiviral pathway. moreover, ns5a impedes the 2-5oas/rnase l system to inhibit ifn signaling. 109 the 2-5oas/rnase l antiviral pathway is present in virtually every cell. 110 this pathway involves the activation of a latent endoribonuclease and degrades hcv mrna with a dsrna structure during replication. 110 ns5a physically binds to 2-5oas through amino acid residues 1-40 of ns5a. 109 a single point mutation at amino acid 37 of ns5a affects the ns5a and 2-5oas binding and the antiviral activity of the 2-5oas/rnase l system. 109 thus, ns5a inhibits ifn antiviral activity in an isdr-independent manner. moreover, ifn-resistant strains, such as genotypes 1a and 1b, have fewer rnase l recognition sites in their genomes than the ifn-sensitive strains, such as genotypes 2 and 3, providing a means for ifn-resistant strains to escape from nucleotylic cleavage. 110 apoptosis also plays a key role in the host defense system by restricting viral spread and persistence. blocking apoptosis could be critical for the establishment of life-long persistence in the host organism. ns5a was shown to block the activation of caspase 3 and to inhibit proteolytic cleavage of the death substrate poly(adp-ribose) polymerase in tnf-a-induced cells. 111 adenovirus infection in ns5a-transgenic mice downregulates and upregulates the expression of t-box transcription factor 21 and trans-acting t cell-specific transcription factor 3, respectively, resulting in lower ifn-c expression and a delay in virus clearance. 112 furthermore, stable expression of ns5a in the human hepatoma cell line huh7 decreases sensitivity to tnf-a-mediated apoptosis, and activation of caspase-3, 8 and 9 by tnf-a is inhibited in ns5a-expressing cells. 113 thus, ns5a protects cells from tnf-a-mediated apoptotic death. hcv-induced er stress hcv protein expression can induce an er stress response and lead to calcium release from the er, which in turn activates the cyclic amp responsive element-binding protein that binds to the cyclic amp responsive element in the promoter of protein phosphatase 2a (pp2a), resulting in upregulation of pp2a. 114 expressed in essentially all cell types, pp2a is a serine/threonine phosphatase that is involved in multiple cellular processes, such as the cell cycle, signal transduction and stress response. 115, 116 increased expression of pp2a has been observed in a cell line inducibly overexpressing hcv protein, in liver extracts from hcv transgenic mice and in liver biopsies from patients with chronic hepatitis c. 117 duong et al. 117 showed that upregulation of pp2a by hcv can inhibit the enzymatic activity of protein arginine methyltransferase 1 (prmt1), which leads to decreased methylation of stat1. hypomethylated stat1 is more prone to bind to protein inhibitor of activated stat1 and inhibits stat1 dimerization, resulting in impaired nuclear translocation into the nucleus, binding of stat1 to the isre, and isg production. 117 thus, hcv-induced pp2a activation disrupts the ifna-induced antiviral state, leading to hcv evasion of innate immunity. these authors also showed that prmt1 can methylate hcv ns3 at arginine 467, resulting in the inhibition of ns3 helicase activity. 118 therefore, hcv-mediated pp2a upregulation enhances ns3 helicase activity by inhibiting prmt1 enzymatic activity, which in turn facilitates virus replication. 118 human genomes encode hundreds of isgs, 23 and the first isg, 54k, was discovered more than 25 years ago. 119 synthesis of some isgs can be triggered by viral infection without ifn production. some isg products can directly regulate cellular processes, such as protein synthesis and cell growth, survival and apoptosis, whereas others may modify the ifn antiviral activity against invading viruses. 120 the gene products of isgs can target many steps in the hcv replication cycle to limit viral replication. 23 many pamp receptors and their subsequent sig-naling partners are also isgs. 23 isgs expressed at the basal level provide antiviral surveillance before ifn activation or therapy; however, their levels markedly increase after ifn production. 23 in the innate immune response to virus infection, viral rna acts not only as a inducer of the production of ifn and its effector functions but also as a substrate and product for cellular enzymes, such as pkr and 2-5oas/rnase l. 121 the inverse correlation between the upregulated expression of isgs, such as oas-like (oasl), isg15 and viperin, in liver biopsies from hcv-infected patients and infected hepatocytes and decreased viral rna levels suggest the anti-hcv activities of these isgs. 122 in this section, we will highlight the involvement of isgs that are critical for modulating innate immunity in hcv replication, and the potential functions of these isgs, as outlined in table 1 . rig-i rig-i, which is encoded by the ddx58 gene and which belongs to the dexd/h box rna helicase (ddx) family, is a key player in the defense against invasion by many rna viruses. 72 rig-i senses the intracellular viral components and initiates antiviral responses to stimulate ifn production (table 1 ). in turn, ifn activates the transcription of rig-i, hence forming a positive feedback loop for amplifying antiviral signals. 123 studies have shown that rig-i is essential for eliciting an immune response against vesicular stomatitis virus (vsv), sendai virus, newcastle disease virus (ndv) and hepatitis c virus. 72, 124, 125 knockout of the rig-i gene in mouse embryonic fibroblasts severely limits type i ifn production and isg activation, thereby potentiating viral replication; conversely, overexpression of rig-i restricts viral replication. 72 in addition, upregulation of rig-i gene expression has been observed in ifn-treated human dendritic cells, suggesting that rig-i serves as an isg. 126 rig-i contains two tandem cards at its n-terminal region, with a repressor domain in its c-terminal region. the card is responsible for downstream signaling and activation of type i ifn after recognition of non-self rna, whereas the repressor domain is essential for the autoregulation and recognition of viral rnas. 127 without viral stimulation, the card interacts with the helicase domain, placing rig-i in an auto-inhibitory state and disabling signal transduction. 127 upon binding to viral rna, rig-i undergoes conformational changes that expose the card, allowing rig-i to be ubiquitinated. 127 rig-i is ubiquitinated by two different ligases, trim25 and ring finger protein 125. trim25 ubiquitinates rig-i at lysine 172 to mediate the antiviral response, whereas ubiquitination by ring finger protein 125 regulates the degradation of rig-i by the proteosome, thereby downregulating rig-i-mediated signaling. 123 ubiquitination by trim25 induces rig-i to form a tetramer, promoting the cards of rig-i to engage with the cards of mavs. this results in the accumulation of mavs on the mitochondrial membrane and the activation of ikk and tbk1, which, in turn, activates the transcription of nf-kb, irf3 and irf7 to promote ifn production. 123 moreover, ubiquitination by trim25 also prevents cards from interacting with the helicase domain to reinstate the auto-inhibitory state. 127 ubiquitination at lysine 172 is crucial for rig-i function because a mutation at this residue renders rig-i unable to bind to mavs, thus abrogating downstream signaling. 128 ddx60 is also a dexd/h box helicase whose function remains unclear. 129 ddx60 slightly resembles the yeast ski protein, which is a cofactor of the rna exosome required for controlling host rna quality. 129 ddx60, which is the human homolog of yeast ski, and the rna exosome exhibit antiviral activity against monkey leukemia virus and sindbis virus (sinv). 129 ddx60 expression is upregulated during infections of measles virus (mev) and hcv. 28, 129 the ddx60 mrna level is robustly upregulated in human fetal liver cells within 24-48 h after hcv infection, providing a means to initiate the antiviral mechanism. 130 unlike rig-i, ddx60 does not contain the cards to interact with mavs. 129 after viral infection, ddx60 is induced and binds to rig-i as well as mda5 and laboratory of genetics and physiology-2 and promotes the binding of rig-i to dsrna. 28,129 ddx60 is essential for type i ifn expression during dna virus infection and is induced to suppress viral replication in a rlr-dependent manner (table 1 ). 129 ddx60 knockdown reduces the expression of type i ifn after hcv, hiv and yfv infections. 28 irf1 irf1 was first identified as a transcriptional activator of the ifn-a/b gene. in unstimulated cells, irf1 is expressed at a low level; however, its expression is increased by the induction of ifn-a/b, tnf-a, il-1 and viral infection. 131 nevertheless, the precise pathway leading to irf1 activation by virus infection remains elusive. irf1 activation may proceed through a pkrdependent pathway after virus infection. pkr indirectly phosphorylates irf1 and activates its dna-binding properties. thus, activated irf1 regulates the promoter function of ifna/b promoter and acts as a modulator of many isgs by binding to the isre in the promoter region, thereby regulating viral replication (table 1) . 82, 132 irf1 controls the ifn antiviral response by affecting a set of isgs, such as irf7 and irf3. 131 irf1 cooperates with irf3 and irf7 to regulate cellular antiviral genes, such as ifn-a/b. hcv infection increases the level of irf1, which may affect other irf pathways and isg expression, thereby leading to a reduction in viral replication. 132 irf1 overexpression induces an antiviral state that affects various viruses, including ndv, vsv and hcv. 133, 134 the expression level of irf1 is reduced in cells harboring hcv subgenomic replicons (sgrs), whereas irf1 overexpression in these cells increases the isre activity and attenuates hcv replication. 134 additionally, hcv infection mediates irf1 expression, thus affecting the intracellular level of hcv rna. 134 however, hcv may evade the irf1 anti-hcv effect through core-mediated suppression of irf1 synthesis. 82 irf7 is an essential transcription factor for the induction of ifn-a/b and isgs. all of the elements of ifn responses, either innate or adaptive immunity, are regulated by irf7. 135 irf7 is constitutively expressed in certain cells, such as macrophage and plasmacytoid dendritic cells, priming these cells for rapid ifn production. 23 during infection, ifn-a/b binds to its receptor and activates the jak-stat pathway, resulting in irf7 expression. 23 then, irf7 is phosphorylated, forms a heterodimer with irf3 and is translocated into the nucleus. 136 in the nucleus, the irf7-irf3 heterodimer binds to the irf elements in the promoter region of ifn-a genes, leading to enhanced expression of the ifn-a subtype and a diverse range of isgs (table 1) . 23, 135 in turn, these events increase the abundance of rig-i and viral pamp signaling components, whereas sustained signaling serves to amplify ifn production. 23, 135 moreover, irf7 induces expression of other isgs without activating ifn signaling. 137 thus, irf7-mediated transcriptional cascades serve as an intrinsic antiviral mechanism allowing rapid isg expression before ifn production. irf7 plays an important role in eliminating hcv infection. sirna knockdown of irf7 decreases ifn-a production and increases the hcv titer. 138, 139 mice lacking irf7 show rapidly lethal infection by west nile virus (wnv) and high virus burdens. 140 irf7 deficiency represses the induction and accumulation of ifn-a, thus favoring wnv replication. 140 although hcv seems to suppress the basal expression of irf7, tlr7 stimulation activates irf7 and suppresses hcv replication. 138 this observation suggests that hcv may only partially inhibit irf7 activity in hcv-expressing cells. 138 pkr pkr, which is also known as eif2ak2, is a serine/threonine kinase that phosphorylates eif2a in response to virus infection. this ifn-inducible kinase has two distinct activities: autophosphorylation, resulting in its activation, and phosphorylation and inactivation of eif2a. through phosphorylation events, pkr mediates the inhibition of translation initiation of both cellular and viral mrna. [141] [142] [143] it is well documented that the anti-hcv activity of pkr occurs through its translational control (table 1) . [144] [145] [146] however, viruses have evolved elaborate strategies to counteract the detrimental effects of pkr. hcv ires activity has been shown to be resistant to pkr activation in cells harboring hcv sgr and in the hcv infection model (table 1) . [147] [148] [149] mechanistically, viruses may use their proteins to impede the dsrna-dependent pathway in various ways, such as sequestering dsrna, inhibiting pkr activation, producing pkr pseudosubstrates, activating antagonist phosphatases and degrading pkr. 142 as indicated above, hcv employs ns5a and e2 to antagonize pkr function, resulting in resistance to ifn and a blockade of the pkr-mediated inhibition of viral protein synthesis (table 1) . analogous to alphaviruses sinv and semliki forest virus, 150 hcv can activate pkr and eif2a phosphorylation to enhance its own viral protein translation (table 1) . 151, 152 compared with other previously studied dsrnas, domains iii-iv of the hcv ires were shown to bind to the n-terminal dsrnabinding domain of pkr, leading to increased pkr autophosphorylation and activation. 152 additionally, cap-dependent but not hcv ires-mediated translation is inhibited by pkr and eif2a phosphorylation. 152 these results indicate that while escaping the deleterious effects of pkr activation, hcv can employ its structured ires to direct its own protein translation. karamichali et al. 153 demonstrated that activated pkr or silencing pkr upregulates or downregulates hcv ires activity (table 1) . these authors further showed that the inhibitory effect of ns5a on ires-dependent translation occurs through pkr inactivation. 153 in contrast, hcv can translate its viral protein via a bacterial-like pathway that uses eif5b, which is an analog of bacterial if2, and eif3, instead of eif2a and its gtpase-activating protein eif5, as the initiation factor (table 1) . 154 the use of eif2a-independent translation initiation provides an alternative tactic for hcv translation when eif2a is inactivated by phosphorylation under stress conditions. many lines of evidence have revealed that hcv-mediated phosphorylation and activation of pkr, in turn, inhibit its downstream target, eif2a, and attenuate the expression of host cellular proteins, including isgs, without any inhibitory effects on viral ires-mediated viral protein translation (table 1) . 143, 155, 156 pkr knockdown in hcv-infected cells restores isg expression and enhances the antiviral effect of ifn. 155 these results demonstrate that hcv escapes ifn antiviral activity by promoting the phosphorylation of pkr and inhibiting the production of antiviral isg proteins, thus providing an interesting pathway for the virus to evade the ifn antiviral response. furthermore, accumulating evidence has revealed that pkr and eif2a participate in the formation of stress granules (sgs). sgs are large, dynamic structures between 50 to 200 nm in size, that form in the cytoplasm when cells undergo extracellular stresses, including viral infections. 157 sg formation is important for the posttranscriptional regulation of gene expression. 157 sgs contain stalled translation pre-initiation complexes, including cellular mrnas, translational initiation factors, the small subunit of the ribosome and many cellular rna binding proteins, such as t-cell-restricted intracellular antigen 1 (tia-1), the homologous tia-1-related protein tiar and rasgap-sh3 domain binding protein 1 (g3bp1), involved in regulating mrna functions. [158] [159] [160] [161] many viruses, including hcv, can modulate sg assembly and co-opt sgs to promote their own protein synthesis. 162, 163 hcv induces sg formation via eif2a phosphorylation (table 1) . 156, 164 consistent with this notion, upregulation of the regulatory subunit of protein phosphatase 1 that dephosphorylates eif2a and growth arrest dna damage-inducible protein 34, inhibits sg formation. 164 these results indicate the importance of eif2a phosphorylation in hcv-induced sg formation. moreover, garaigorta et al. 156 demonstrated that hcvinduced sg formation is ifn-and pkr-dependent and is inversely correlated with the induction of isg proteins, such as myxovirus resistance gene a (mxa) and ub-like (ubl)specific protease 18 (usp18), in hcv-infected cells without affecting the mrna levels of these isgs. furthermore, the sg proteins tia-1, tiar and g3bp1 have been shown to play a critical role in hcv replication and infectious virus production. 156 in support of this finding, g3bp1 was also reported to be essential for hcv rna replication, presumably through its relocalization to lds or its interaction with ns5b. 165, 166 the results of garaigorta et al. 156 demonstrated that hcv hijacks pkr phosphorylation-triggered sg formation to downregulate the translation of antiviral isgs, thereby promoting viral rna replication, virus assembly and egression. oas and rnase l upon sensing and activation by the pamp of viral dsrna, certain ifn-stimulated 2-5oas proteins can synthesize 2-5a from atp. after binding to 2-5a short oligoadenylates, a ubiquitous, latent endonuclease, rnase l, is activated through dimerization and degrades either cellular or viral rnas, resulting in the inhibition of protein synthesis, cellular apoptosis and impaired virus propagation. 121, 167, 168 therefore, the oas/ rnase l pathway represents a critical arm of ifn's antiviral effector mechanism against many viruses, including hcv. 121 depending on the specific rna substrates and the extent of enzymatic activity, rnase l can block different types of viruses through different mechanisms, such as apoptosis, or through the 'suppressor of virus rna' derived from cellular or viral rna. 168 nevertheless, some members of the oas family can exert antiviral activity independent of rnase l. 169 the oas system has been reported to exert anti-hcv effects through the rnase l pathway. the ua and uu dinucleotides within loops of predicted stem-loop structures in the viral rna is prone to cleavage by rnase l (table 1) . 110 additionally, the sensitivity of hcv infection to ifn therapy correlates with the efficiency of rnase l-mediated viral rna cleavage. 110 the anti-hcv activity of oas1 p46 and oas3 p100 in the oas family occurs in an rnase l-dependent fashion (table 1) . 170 hcv replication is suppressed in hcvcc-infected huh7 cells co-cultured with hepatic stellate cells (lx-2) treated with 59ppp-dsrna or incubated with conditioned medium from lx2 cells stimulated with 59ppp-dsrna. 125 in these hcvccinfected cells, the expression of oas-1 and mxa is upregulated. 125 the two different domains in oas-like a (oasla), a major isoform in human liver that is induced by hcv, contribute to the antiviral activity. the n-terminal oas homology domain, which lacks the cleavage activity, impairs cell proliferation as well as viral replication, whereas the c-terminal ublike domain impedes hcv replication without affecting cell growth (table 1 ). 171 the ifn-stimulated gene 20 kda protein (isg20) has emerged as a second ifn-regulated rnase that inhibits rna virus replication. 172,173 isg20, along with the closely related isg20l1 and isg20l2, belongs to the yeast rna exonuclease 4 homolog subfamily within the deddh exonuclease family and members of this superfamily possess both rnase and dnase activities. 173 the 39-59 exonuclease activity of isg20 demonstrates a greater preference for single-stranded rna than for single-stranded dna. 173 isg20 overexpression restricts infection by encephalomyocarditis virus, vsv, influenza virus (infv), human immunodeficiency virus (hiv), yfv, picornavirus and hcv. 172,173 isg20 has been reported to impair hcv genotype 1b sgr replication in hek293 cells (table 1) . 146 in addition, isg20 can hinder genotype 2a viral rna replication either in sgr or hcvcc infection, and its anti-hcv effect is not shared with isg20l1 and isg20l2 (table 1) . 172 apart from degrading viral rna through its 39-59 exonuclease activity, 146,172 the anti-hcv mechanism of isg20 in hcv replication remains poorly understood despite its possible action on cellular factors. 173 adar rna-specific adenosine deaminase (adar) is constitutively expressed in normal cells as an inactive form. 174 however, viral infection triggers the two mammalian adar genes, adar1 and adar2, to express two active proteins, adar1 and adar2. 174 adar catalyzes adenosine to inosine editing in rnas that possess double-stranded structures. 174, 175 because i is recognized as guanosine by rna polymerase, a to i editing causes nucleotide substitution as well as dsrna destabilization because of the reduced stability of i:u mismatch base pair compared with the normal base pair. 174, 175 the rna editing ability of adar affects many biological processes, including viral replication and persistence, apoptosis, ion channel function and the posttranscriptional modification of genes. 174, 176 only the adar1 transcription level is induced by ifn treatment and by pathogen infections. 177 in addition, adar1, but not adar2, affects the stability of hcv replicon rna (table 1) . 148 in hcv sgr replication, ifn-a treatment decreases viral rna replication and concomitantly increases adar1 expression, suggesting that adar1 possesses an antiviral activity in the hcv rna replicon. 148 adar1 knockdown conversely increases the hcv replicon rna. 148 loss of hcv rna by adar1 may be due to several reasons. 148 first, an i base-specific rnase might target mutated viral rna. second, the mutated rna might lead to insufficient replication and genome instability. 148 third, the cellular mrna involved in viral replication may also be targeted by adar1. thus, the rna editing ability of adar1 negatively affects hcv rna replication, representing a potent strategy in anti-hcv therapy. in sharp contrast, the replication of hepatitis delta virus (hdv) benefits from adar1 editing. 178 the editing of hdv rna by adar1 converts the uag stop codon to a uig tryptophan codon, allowing the synthesis of a larger hdv antigen. 179 without viral rna editing, the hdv genome cannot be packaged into a virion. 179 nonetheless, adar overexpression increases rna editing but decreases hdv replication. 180 ifn-inducible transmembrane protein (ifitm) family ifitm family members, including ifitm1, ifitm2 and ifitm3, inhibit, in an ifitm-specific manner, the replication of diverse pathogenic membrane-enveloped viruses, including marburg virus and ebola (ebov) filoviruses; severe acute respiratory coronavirus; hiv; rift valley fever virus (rvfv); respiratory syncytial virus; reovirus; flaviviruses, including denv and wnv; and hcv. [181] [182] [183] [184] [185] [186] in contrast, ifitms show no inhibitory effects on entry of amphotropic mouse leukemia virus, machupo virus, lassa virus and lymphocytic choriomeningitis virus. 183 ifitms are topologically located at different intracellular membrane compartments. ifitm2 and ifitm3, which are type ii transmembrane proteins, are primarily localized to endosomes and lysosomes, 187, 188 whereas ifitm1 also localizes to the cell periphery. 189, 190 ifit3 interacts with tbk1, irf3 and other ifitm members and enhances ifn signaling. 191, 192 lipid raft membranes, which are enriched in cholesterol and sphingolipids, play vital roles in cellular pathways and in virus entry, assembly and budding. 193, 194 vesicle-associated membrane protein-associated protein a (vapa) and oxysterolbinding protein (osbp) modulate the intracellular trafficking and de novo synthesis of cholesterol. 195 vapa interacts with osbp to regulate the transfer of cholesterol from the er to other organelles. 196, 197 the regulation of intracellular cholesterol homeostasis, particularly in the endosomal compartment, is critical for the entry of viruses such as ebov and marburg viruses. 198 ifitms have been demonstrated to interfere with virus infection by blocking virus-endosome fusion (table 1) , 183, 184 presumably through the modification of cellular membrane properties, such as fluidity and spontaneous curvature. 184, 188, 199 amini-bavil-olyaee et al. 188 demonstrated that the interaction of ifitm3 with vapa antagonizes the association of vapa with osbp, thereby inducing the accumulation of cholesterol in multivesicular bodies and in late endosomes. the disruption of intracellular cholesterol homeostasis subsequently impairs the membrane fusion of intraluminal virion-containing vesicles and endosomes, resulting in a block of vsv release into the cytosol. 188 using immortalized human hepatocytes and huh7 infection models, raychoudhuri demonstrated that ifitm1 expression inhibits hcv replication but not at virus entry. 200 later, wilkins et al. 186 identified that ifitm1 is a hepatocyte tight junction protein whose antiviral action occurs through modification of the interactions of the hcv coreceptors cd81 and occludin, thereby inhibiting hcv entry (table 1) . 186 this study represents an interesting mode of antiviral innate immunity; an isg can exert its anti-hcv action by disrupting viral coreceptor associations. the ifn-induced protein with tetratricopeptide repeats (ifits) family represents a class of isgs featured by their unique helix-turn-helix motifs, known as tetratricopeptide repeats. ifits mediate a broad range of protein-protein interactions; in particular, the tetratricopeptide repeat motif is critical for modulating protein translational initiation and transport, cell proliferation and migration, virus replication, and antiviral signaling. [201] [202] [203] [204] proteins in the ifit family have been linked to ifn antiviral functions, including those against wnv and lymphocytic choriomeningitis virus. 205 ifit3 plays an important role in modulating innate immunity by bridging tbk1 to mavs on mitochondria as ifit3 expression facilitates the association of its tetratricopeptide repeat motif with the n terminus of tbk1, thereby enhancing irf3-mediated gene expression. 192 ifit1, which is also known as isg56, belongs to a family that also contains other stress-induced, structurally related proteins, p60, p58 and p54, in humans. ifit1 acts as a negativefeedback regulator for sendai virus-triggered induction of type 1 ifn antiviral signaling transduction, presumably through its interaction with the adapter protein sting and through disruption of the normal association between sting/mita and mavs or tbk1. 203 moreover, ifit1/2 preferentially targets mutants of poxvirus, coronavirus, and wnv that lack 29-o methylation in their viral rna cap, thereby rendering these mutant viruses unable to replicate. 204 this study addresses the mechanism by which 29-o methylation of the 59 cap of viral rna renders viruses insensitive to ifit-mediated host innate antiviral activity. wang et al. 147 demonstrated that ifit1 mediates its ifn antiviral activity and blocks hcv rna replication, presumably by targeting an eif3-dependent step in viral ires-mediated translation (table 1) . 147 in immortalized human hepatocyte and huh7 infection models, raychoudhuri documented that ifit1 expression inhibits hcv replication by suppressing hcv ires-mediated transcription. 200 conversely, ifit1 knockdown facilitates hcv replication. 200 these results suggest that ifit1 restricts hcv infection primarily at the viral translation/replication site. protein posttranslational modifications by ub and ubl modifiers not only play important roles in numerous cellular processes, such as protein localization, interaction, activity and degradation, signal transduction, vesicular trafficking and dna damage repair, [206] [207] [208] but also modulate pathogen-host interactions, such as the viral replication cycle and the host antiviral response. 208-210 isg15, which was the first ubl protein modifier identified, 211 is post-translationally attached via its c terminus to the lysine residues of isgs and to hundreds of target proteins involved in different pathways. 212, 213 similar to its ub homolog, isg15 is linked to proteins via a tightly regulated process known as 'isgylation', and the activating e1 (ube1l), conjugating e2 (ubch8), and ligating e3 (ceb1) enzymes catalyze these sequential events. 211, 214 isg15, together with its conjugation e3 ligase (ceb1) and its deconjugation enzyme usp18, are in the same isg15/usp18 ubl pathway. isgylation modulates signal transduction pathways and host antiviral responses. isg15 exerts its modulatory roles by inhibiting virus release, isoylating viral proteins, or modifying host proteins. 214 isg15 targets many cellular proteins, including jak1, stat1 and many isgs. three antiviral effector molecules, irf3, rig-i and pkr, are also modified by isgylation. 214 activated irf3 is stabilized by isgylation and therefore, positively regulates type i ifn signaling. 215, 216 the isg15 conjugation-mediated reduction of the non-isgylated rig-i correlates with the reduced ndv-triggered ifn response. 123 additionally, viral rna-independent pkr activation requires the isgylation of pkr. 217 isg15 expression enhances ifn-mediated antiviral activity against many viruses, including hiv and sinv. 214 overexpressing isg15 in ifn-a/b receptor knockout mice decreases sinv replication and protects the mice from sinv-induced lethality. 218 isg15 2/2 mice are more susceptible to infection by many rna and dna viruses, such as infv and herpes simplex virus (hsv) type 1, and the protection effect of isg15 from sinv infection is dependent on isgylation. 219 lu et al. 215 demonstrated that induction of isg15 expression in ndv-infected cells counteracts the ub-mediated degradation of irf3 and enhances the ndv-mediated host innate antiviral response. their findings revealed a feedback mechanism of isg15 in enhanced antiviral immunity. despite functioning as an antiviral molecule, isgylation of the antiviral rig-i enzyme inhibits ifn signaling in mouse embryonic fibroblast cells. 123 using the genotype 2a j6/japanese fulminant hepatitis-1 chimeric hcv infectious model, chen et al. 220 unexpectedly found that isg15 acts as a pro-hcv regulator because increased isg15/isgylation facilitates hcv production, whereas blocking isgylation decreases virus production (table 1) . moreover, knockdown of ube1l, the e1 activating enzyme, inhibits hcv replication, particularly hcv egress, without affecting ifn-mediated isg expression in hcv-infected cells. 220 using the hcv-huh7. 25 .cd81 infection system, arnaud et al. dissected the acute ifn response to hcv infection into early, pkr, and late, rig-i, phases. 221 hcv infection rapidly induces the expression of many irf3-dependent genes, including isg15, through a pkr-dependent mechanism before the rig-i phase, which recruits mavs. 221 then, isg15 induction blocks hcv rna-mediated rig-i activation by inhibiting rig-i ubiquitination, thereby negatively controlling the rig-i/mavs pathway. 221 these studies illustrate that hcv may exploit isg15 to antagonize host innate immunity and to promote viral replication. the deconjugation of usp15 from its target proteins is catalyzed by usp18 (mouse ortholog ubp43). 222 usp18 can function in both isg15-dependent and isg15-independent modes. usp18 was shown to bind to ifnar2 and attenuate the jak-stat pathway, thereby negatively regulating ifn signaling (table 1) . 223 reduced usp18 expression results in increased antiviral activity against many viruses, such as sinv, hepatitis b virus and vsv, in usp18 knockout mice. 218, [223] [224] [225] usp18 knockdown is concomitant with increased cellular protein isgylation, prolonged stat1 tyrosine phosphorylation and enhanced isg expression, thus greatly enhancing the anti-hcv potency of ifn. 226 all these studies suggest that usp18 disruption can impede its negative regulatory effect on ifn signaling, resulting in sustained jak-stat activity and antiviral activity. consistent with these observations, murray et al. 227 demonstrated that ifn-a signaling and isg induction were greatly increased when ups18 was knocked down in both hcv sgr-and hcvcc-infected huh7 cells. however, usp18 knockdown did not have a significant effect on anti-hcv activity. 227 these observations suggest a slight dependency of ifn-mediated antiviral activity on usp18 activity. 227 additionally, usp18 upregulation is predictive of a nonsustainable viral response to ifn treatment. 34, 228, 229 the expression levels of ups18 and isg15 increase in liver biopsy specimens from chronically hcv-infected patients who do not respond to ifn-based therapy, inferring that hcv hijacks the isg15/usp15 pathway to evade the antiviral immune response and to facilitate its replication (table 1) . 34, 230 this observation also explains, at least partially, the failure of ifn-based treatments in non-responders, although non-responders express higher levels of isgs, particularly isg15, compared with ifn responders. 34, 230 taken together, these findings demonstrate that usp18 is an attractive target for the development of anti-hcv therapeutics. viperin, which stands for virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible, plays crucial roles in virus replication, signaling and the immune response. 231, 232 the viperin protein sequence is highly conserved, and all viperin homologs contain three functional domains: the amphipathic, n-terminal domain, which mediates er and ld association; the central cxxxcxxc motif, which is functionally important for fe-s cluster formation; and the highly conserved c-terminal domain, which is essential for antiviral activity. [231] [232] [233] in addition to type i, type ii and type iii ifns, dsdna and dsrna analogs, bacteria, lipopolysaccharide, poly(i:c) and a broad spectrum of dna and rna viruses can induce viperin expression. 231, 232, 234 viperin expression regulates many cellular functions, such as forming lds and reducing membrane fluidity. 231 viperin possesses antiviral activity against diverse families of dna and rna viruses, including infv, hiv, sinv, the flaviviruses japanese encephalitis virus, denv and wnv, and the hepacivirus hcv (table 1) . 231, 232, 234 viperin functions in different ways to defend against virus infections. for instance, viperin alters membrane fluidity by interacting with farnesyl diphosphate synthase, which is an enzyme essential for isoprenoid biosynthesis, thus disrupting the formation of lipid rafts, the sites of infv budding, leading to interference with virus release from the cell surface. 235 the induction of viperin into hiv-1-infected cells disrupts lipid rafts, causing viperin redistribution to cd81 compartments, where hiv-1 buds in human macrophages. 236 the radical s-adenosyl-methionine enzymatic activity of viperin is required for the inhibition of hiv production. 236 in cells infected with japanese encephalitis virus, the antiviral function of viperin is attenuated due to its degradation by the proteasome-mediated protein degradation system. 237 in contrast, viperin enhances human cytomegalovirus infection through its interaction with the viral mitochondrial inhibitor of apoptosis vmia protein, resulting in viperin relocalization from the er to mitochondria. 238 in mitochondria, viperin interacts with the mitochondrial trifunctional protein and reduces cellular atp generation, resulting in the disruption of the actin cytoskeleton and enhancement of the virus infection. 238 viperin is upregulated in huh7 cells transfected with either poly(i:c) or hcv rna, 239 and transient expression of viperin in hcv sgr replicating cells significantly decreases hcv replication. 239 the putative radical s-adenosyl-methionine enzymatic activity of viperin is required for this anti-hcv activity. 146 helbig et al. 240 further demonstrated that the restriction of hcvcc replication by viperin depends on both the n-terminal amphipathic a-helix and the c-terminal domain. the anti-hcv function of viperin coincides with its binding to ns5a at the ld interface, whereas ns5a normally associates with the human homolog of the 33-kda vesicleassociated membrane protein-associated protein (hvap-33), which is a pro-viral cellular factor, at the viral replication complex. 239 the interaction of hcv ns5a with hvap-33 was previously shown to be critical for the formation of the viral replication complex. 52 therefore, the association between viperin and hvap-33 requires both of their c-terminal domains, which then disturbs the interaction of hvap-33 with ns5a and inhibits hcv replication (table 1) . 241 together, these findings imply that viperin hinders viral rna replication by perturbing the interaction between hvap-33 and ns5a. by conducting bioinformatic analyses of murine bone marrowderived macrophages, liu et al. showed that cholesterol-25-hydroxylase (ch25h), which is an ifn-a-and ifn-c-stimulated isg, can mitigate the replication of many membraneenveloped viruses, including hiv, vsv, hsv and murine c-herpesvirus, and many pathogenic viruses, such as rvfv, ebov, russian spring-summer encephalitis virus and nipah virus in vitro and in vivo. 242 these viruses contain different structural characteristics in their fusion proteins. for instance, hiv and ebov contain class i fusion peptides, rvfv and russian spring-summer encephalitis virus harbor class ii peptides, and vsv and hsv belong to class iii fusion proteins. 243, 244 the broadly antiviral action of the ch25h gene product is mediated by the ability of its enzymatic product, 25hydroxycholesterol, to inhibit ph-dependent and ph-independent membrane fusion between cells and viruses, as typified by vsv and hiv, respectively (table 1) . 242 this study not only demonstrates that ifn can confer an antiviral state to host and/ or target cells by inducing a natural oxysterol inhibitor but also suggests that modification of membrane oxysterols can be used as a potential antiviral approach. 242 determining whether this broad antiviral isg can block hcv-mediated membrane fusion would be interesting. several genome-wide sirna screens were recently performed to identify isgs or ifn-mediated effector genes (iegs) that mediate ifn antiviral functions. these studies have identified many new isgs or iegs and have revealed interesting features of the actions of isgs. using an overexpression screen approach, schoggins et al. 28 demonstrated that each virus exhibits a unique but partially overlapping profile of antiviral isg expression. the expression levels of isgs may vary depending on viral infection or on the time, dose, or cell type used for ifn treatment. 28 in hcv infection, higher expression levels of 36 unique isgs were found to correlate with a reduction in the hcv viral load. 245 schoggins et al. 28 further observed that multiple isg genes could target each viral species with a range of inhibitory activities. a set of effectors, including irf1, c6orf150 (also known as mb21d1), heparanase, rig-i, mda5 and ifitm3, exert broad antiviral activities against different viruses, including hcv, yfv, wnv, chikungunya virus, venezuelan equine encephalitis virus and hiv-1. 28 however, other effectors, such as ddx60, ifn-inducible proteins 44l and 6, ifitm2, mapk kinasekinase 14, moloney leukemia virus 10, nicotinamidephosphoribosyltransferase, oasl, receptor transporter protein 4, three prime repair exonuclease 1 and protein unc-84 homolog b, display species-specific antiviral effector functions. 28 these results also demonstrated that different isgs can exert additive antiviral effects on virus replication. 28 remarkably, several isgs, such as adar, family with sequence similarity 46, member c, lymphocyte antigen 6e and mucolipin-2, can enhance the replication of certain viruses. 28 certainly, further characterizing how these isgs antagonize ifn-mediated antiviral functions and determining which steps of virus replication are targeted by these isgs are important. these findings indicate the complexity of the type i ifn-mediated innate immune response in virus replication. performing a sirna-based 'gain of function' screen, metz et al. 246 identified several new anti-hcv isgs in addition to those previously reported anti-hcv isgs. this study demonstrated that both ifn-a and ifn-c can upregulate the expression of several isgs, including ifit3, trim14, phospholipid scramblase 1 and inducible nitric oxide synthase 2. these isgs possess anti-hcv activity, although the precise roles of these isgs in hcv replication are not understood. 246 this study also reported a substantial overlap in antiviral innate immune responses triggered by either cytokine. 246 however, some isgs are more specifically induced by ifn-a or by ifn-c. for instance, phospholipid scramblase 1 and nitric oxide synthase 2 primarily function as ifn-c-mediated anti-hcv effectors. 246 moreover, different isgs function additively or synergistically to interfere with hcv infection, 246 indicating that the combinatorial and concerted actions of multiple effectors mediate repression of hcv replication. in addition to the signaling molecules involved in the ifn/ jak-stat/isgpathway, the majority of genes identified by fusco et al. are not transcriptionally activated by ifn. 247 in contrast to the notion that isgs target specific virus replication steps, some of these genes can exert ifn-mediated antiviral effects at multiple steps of the hcv replication cycle. 247 for instance, dipeptidyl-peptidase 4/cd26/adenosine deaminase complexing protein 2 blocks virus entry, initial rna replication, and amplified translation. myst1 histone acetyltransferase inhibits hcv entry, translation, rna replication and virion release, and protein phosphatase 3, catalytic subunit, b isoform (ppp3cb) impairs virus entry, initial rna replication and subsequent translation. taken together, these findings reveal that these ifn-insensitive iegs, together with isgs, constitute the host cellular genes mediating the antiviral activity of ifn against viral replication. a functional genomic screen has shown that several new genes comprising the u4/u6.u5 tri-small nuclear ribonucleoprotein (snrnp) possess the ability to mediate ifn antiviral activity. 248 u4/u6.u5 tri-snrnp is the major component of human spliceosome complexes involved in mrna processing. 249 this genomic screen demonstrated that squamous cell carcinoma antigen recognized by t cells (sart1) is a u4/ u6.u5 tri-snrnp-specific factor required for ifn-a-mediated anti-hcv activity, although sart1 is not induced by ifna. 248 the anti-hcv activity of sart1 acts by regulating the expression of isgs, such as mxa, oas and pkr, either in the presence or absence of exogenous ifn-a. 248 this genetic screen links an unappreciated role of rna processing to the control of antiviral immunity. in this section, we discuss recent findings regarding the roles of several cellular factors and/or machinery involved in the immune response in modulating hcv replication. although these determinants are not directly induced or activated by ifn, knowledge of their interplay with the host immune response will help to elucidate their effects on hcv infection. the functions of these cellular determinants in hcv infection are summarized in table 2 . ikka hcv can co-opt an intrinsic innate pathway and hijack cellular lipid metabolism to facilitate its assembly. ikka was initially identified as a critical factor for hcv replication in a genomewide rna interference screen. 250 subsequently, hcv infection was shown to activate ikka through the interaction of the viral genome 39-utr with dead box polypeptide 3, x-linked (ddx3x). 251 ikka translocates into the nucleus and induces the cbp/p300-mediated expression of lipogenic genes, including sterol regulatory element-binding proteins, followed by the promotion of core-mediated ld formation and the enhancement of hcv assembly ( table 2 ). 251 dansako demonstrated that upon hcv expression, class a scavenger receptor type 1 (msr1) expressed on the plasma membrane of infected and adjacent uninfected cells can bind to dsrna released from infected cells and mediate its endocytosis and transport to endosomes where the dsrna is sensed by tlr3 and initiates a local antiviral ifn response to restrict hcv replication. 252 the msr1-mediated binding, transport, and release of dsrna at the acidified endosome requires a stretch of conserved basic residues within the c terminus of the collagen superfamily domain of msr1. 252 therefore, msr1 acts as a key element for the tlr3-mediated prr, thereby rendering both infected and uninfected hepatocytes refractory to hcv replication (table 2) . hmgb1, which is an abundant nuclear protein that mediates activation of host immune responses and inflammation, represents a prototype damage-associated molecular pattern that participates in the pathogenesis of diverse pathogens. 253, 254 hmgb1 is passively released by cell injury or ischemia without pathogen invasion, but is actively secreted from stimulated immune cells, such as natural killer cells, macrophages and mature dendritic cells. 253 many types of tlrs, such as tlr2, tlr4 and tlr9, can act as receptors for hmgb1. 253 the production of reactive oxygen species can mediate translocation from the nucleus to the cytoplasm and the subsequent release of hmgb1. 253, 255 interestingly, it has been shown that hcv core and ns5a can trigger oxidative stress in infected cells. [256] [257] [258] jung et al. 259 demonstrated that hcv infection causes the nuclear-to-cytoplasmic translocation of hmgb1 and its release into the extracellular milieu. tlr4 acts as a major component of the receptor complex that recognizes lipopolysaccharide lps and plays a role in the production of pro-inflammatory cytokines and antiviral ifns via signaling myd88 and the tlr adapter protein trif. 260 jung et al. 259 also demonstrated that hmgb1 interacts with tlr4 to activate ifn signaling (table 2 ). because hmgb1 is present at higher levels in the sera of patients with chronic hepatitis and cirrhosis compared with those detected in control individuals, 261 the results of jung et al. may help to elucidate the potential inhibitory action of hmgb1 in hcv propagation in chronically hcv-infected patients. 259 autophagy autophagy is a conserved 'self-eating' process that engulfs and delivers cytoplasmic cargos and invading pathogens within double-or multiple-membrane autophagosomal structures to lysosomes for degradation. [262] [263] [264] [265] the purpose of autophagic induction is to maintain cellular homeostasis in the host when the host undergoes extracellular or intracellular stresses. autophagy plays pivotal roles in the stress response, nutrient deprivation, damaged organelles, unfolded protein aggregation, intracellular quality control and cell death. [262] [263] [264] [265] the autophagic process requires two ubl conjugation complexes: autophagy-related gene (atg) 12-atg5-atg16l and microtubule-associated protein 1 light chain 3-phosphatidylethanolamine. 262, 266 autophagy has emerged as an immune regulator that commands the innate and adaptive immune responses against intracellular viruses. [267] [268] [269] [270] [271] autophagy also participates in the modulation of virus-host interactions. in contrast, viruses can subvert the host autophagic pathway to potentiate their own growth. 272, 273 analogously, hcv is able to subvert the host autophagic machinery and enhance viral growth, including rna replication, 274 translation of the incoming viral rna genome 275 and the release of infectious viruses ( table 2) . 276 two laboratories have independently demonstrated that hcv can activate autophagy via er stress-mediated induction of the upr and that upr-autophagy is required for hcv replication. 7,277 hcv ns3, ns4b, ns5a and ns5b have also been implicated in the induction of autophagy. 12, 27 huang et al. 278 showed that hcv induces er stress and inhibits the akttuberous sclerosis-mtor complex 1 signaling pathway, resulting in autophagy activation. in contrast, shrivastava et al. 279 demonstrated that hcv induces autophagy by stimulating beclin mrna expression and by activating mtor signaling, which may enhance hepatocyte growth. ke and chen 277 demonstrated that in the context of hcv infection or without hcv infection, activation of the upr and autophagy downregulates innate immunity; in contrast, disruption of the upr and autophagy upregulates innate immunity. these results demonstrate that hcv hijacks upr and autophagy to stimulate viral rna replication by suppressing immune antiviral immunity. the upr-autophagy pathway represents a unique mode of reversible control in the innate immunity capacity in target cells. 10, 27 subsequently, shrivastava et al. 280 found that beclin or atg7 gene silencing in genotype 1a h77 strain hcv-infected immortalized human hepatocyte upregulates ifn signaling and isg expression, which are concurrent with apoptotic cell death. together, the results from these two groups suggest that autophagy may protect hcv-infected cells from the damage caused by excessive ifn antiviral stimulation, thereby promoting hcv rna replication. furthermore, a specific mode of autophagy, termed 'mitophagy', was recently reported to play a critical role in hcv replication and in the elimination of damaged mitochondria in infected cells in a parkin-dependent manner. 281 knockdown of parkin and pink gene expression suppresses viral rna replication (table 2 ). 281 these results suggest a critical role for mitophagy in hcv replication. nevertheless, the molecular basis for the roles of autophagy and mitophagy in suppressing innate antiviral immunity in hcv infection has yet to be investigated. a recent study has demonstrated that many different families of rna viruses can target the autophagy network to promote viral growth. 282 among these targets is irgm, which modulates autophagy by interacting with many autophagy-associated proteins, such as atg5, atg10 and light chain 3c. strikingly, irgm knockdown impairs autophagy induced by many viruses, such as hcv, mev and hiv-1, resulting in mitigated viral replication (table 2 ). 282 moreover, the c protein of mev, ns3 of hcv, and nef of hiv-1 were shown to induce autophagy by interacting with irgm. these results suggest that rna viruses have evolved to use a common strategy to target a critical molecule in autophagy to benefit their growth. microrna is a class of endogenous small non-coding rnas that bind to the 39-utr of target mrnas to control gene expression. 283 micrornas also participate in innate and adaptive immunity response by binding to their complementarily mrnas and regulating the expression and translation of their target genes. 284 for example, mir-155 regulates the host antiviral immune response by promoting type i ifn, whereas mir-16 enhances mrna degradation. 285 mir-21 was shown to be upregulated in liver samples from hepatocellular carcinoma patients and in hcv-infected cells. 286 during hcv infection, mir-21 expression is activated by the pkce/jnk/cjun and pkca/erk/cfos pathways. 287 cjun and cfos form the ap-1 protein, which binds to the mir-21 promoter and activates mir-21 expression. 287 mir-21 upregulation was shown to suppress the expression of myd88 and irak1, which are two genes involved in the tlr signaling cascade, thereby repressing the production of type i ifn and isg and promoting hcv replication ( table 2) . 287 these results indicate that hcv usurps mir-21 to enhance its replication. likewise, mir-21 also increases the production of hiv, vsv and enterovirus 71 by suppressing type i ifn production. 287 the mechanisms by which viruses and cells coevolve and the tactics each party employs to establish the dynamic equilibrium are emerging as a fascinating area in hcv-host interaction research. previous studies that aimed to understand the hcv cell coevolution process have revealed several interesting aspects of virus-host cell interactions, such as er stress, upr, autophagy and innate antiviral immunity responses in hcv replication. 11, 12, 27 further determining how the virus-cell interplay subsequently reshapes the host defense mechanisms and how virus replication is modulated in response to these cellular stresses will be important for gaining a complete understanding of the molecular basis of the hcv-host interaction in the pathogenesis of hcv infection. viral infection can trigger the ifn-mediated frontline host defense mechanism, including the production of a wide range of isgs to limit virus replication. many studies have also hitherto demonstrated that some of the identified isgs can exert broad antiviral activities against a diverse spectrum of viruses, whereas other isgs may have virus type-specific functions. the majority of studied isgs mediate ifn antiviral activities, acting as negative regulators in virus replication. strikingly, some isgs function as negative modifiers in the innate immune response, thereby promoting virus replication. nevertheless, the modes of action of most of the isgs remain unclear. although most identified isgs target individual steps of virus replication, some isgs seem to act at multiple stages of the virus replication cycle. determining the mechanisms by which these isgs function at different steps of the virus replication cycle would be interesting. current studies have indicated that different types of ifns may substantially overlap in mediating their innate immune response by activating the same set of isgs. however, the induction of some isgs may be unique to only one type of ifn, indicating the specificity in the induction of these isgs by ifns. clearly, different isgs can additively or synergistically suppress hcv replication, suggesting that inhibiting hcv replication depends on the combinatorial effects of individual isgs induced by ifn under the specific context of hcv infection. therefore, ifn-mediated suppression of hcv replication is not caused by a single isg but rather by the concerted actions of multiple isgs. moreover, gene expression profiling of hepatocytes from chronically hcv-infected patients treated with ifn has consistently shown differences between ifn-responders and ifnnon-responders. for instance, the levels of specific isgs, such as isg15 and usp18, and viral sensors, such as rig-i, mda5 and laboratory of genetics and physiology-2, are upregulated in patients with non-sustained virological responses compared with patients with sustained virological responses. 34, 228 therefore, profiling gene expression for cytoplasmic viral sensors and related regulators involved in the innate antiviral immune response can identify new isgs that can be used as markers for predicting the clinical outcome of ifn therapy. in conclusion, the emergence of complexity in the highly pleiotropic type i ifn system in hcv infection reveals that the host has evolved to erect multiple checkpoints for anti-hcv innate immune surveillance to ensure that hcv is under tight control at all times, even when a single effector fails to confer antiviral activity, without drastically downgrading the overall efficacy of the ifn treatment. therefore, further deciphering which isgs and/or iegs are induced by ifns upon hcv infection and the specificity and action of these isgs and other cellular immune regulators on hcv replication will not only provide insights into how ifn functions to obstruct hcv replication but will also reveal novel cellular targets against which effective and efficacious anti-hcv therapeutics can be developed. unscrambling hepatitis c virus-host interactions replication of hepatitis c virus hepatitis c virus infection and mixed cryoglobulinemia production of infectious hepatitis c virus in tissue culture from a cloned viral genome persistent hepatitis c virus infection in vitro: coevolution of virus and host perturbation of autophagic pathway by hepatitis c virus viruses and the autophagy machinery impact of the autophagy machinery on hepatitis c virus infection autophagy: a novel guardian of hcv against innate immune response hepatitis c virus and cellular stress response: implications to molecular pathogenesis of liver diseases autophagy in hepatitis c virus-host interactions: potential roles and therapeutic targets for liver-associated diseases regulation of hepatic innate immunity by hepatitis c virus immune responses to hcv and other hepatitis viruses toll-like receptor and rig-i-like receptor signaling innate immune modulation by rna viruses: emerging insights from functional genomics treatment of chronic non-a, non-b hepatitis with recombinant human alpha interferon. a preliminary report innate immune recognition of viral infection activation and evasion of antiviral innate immunity by hepatitis c virus regulation of innate immunity against hepatitis c virus infection induction and evasion of innate antiviral responses by hepatitis c virus innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna evasion of intracellular host defence by hepatitis c virus hepatitis c virus evasion from rig-i-dependent hepatic innate immunity viral determinants of resistance to treatment in patients with hepatitis c innate immune responses in hepatitis c virus infection suppression of innate antiviral immunity after hepatitis c virus infection: role of the unfolded protein response and autophagy a diverse range of gene products are effectors of the type i interferon antiviral response intrinsic antiviral immunity expanded classification of hepatitis c virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource consensus proposals for a unified system of nomenclature of hepatitis c virus genotypes global distribution and prevalence of hepatitis c virus genotypes global burden of hepatitis c: considerations for healthcare providers in the united states hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection hepatitis c virus genotypes in the united states: epidemiology, pathogenicity, and response to interferon therapy. collaborative study group systematic review: epidemiology of hepatitis c genotype 6 and its management hepatitis c virus infection and liver steatosis clinical significance of hepatitis c virus genotypes treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus characterization of the terminal regions of hepatitis c viral rna: identification of conserved sequences in the 59 untranslated region and poly(a) tails at the 39 end hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus structural and functional characterization of nonstructural protein 2 for its role in hepatitis c virus assembly hepatitis c virus ns2 protein contributes to virus particle assembly via opposing epistatic interactions with the e1-e2 glycoprotein and ns3-ns4a enzyme complexes trans-complementation of an ns2 defect in a late step in hepatitis c virus (hcv) particle assembly and maturation determinants of the hepatitis c virus nonstructural protein 2 protease domain required for production of infectious virus ns3 helicase domains involved in infectious intracellular hepatitis c virus particle assembly essential role of domain iii of nonstructural protein 5a for hepatitis c virus infectious particle assembly interaction of hepatitis c virus nonstructural protein 5a with core protein is critical for the production of infectious virus particles hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor the ins and outs of hepatitis c virus entry and assembly replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft lipid droplets: a unified view of a dynamic organelle the lipid droplet is an important organelle for hepatitis c virus production structural determinants that target the hepatitis c virus core protein to lipid droplets association of hepatitis c virus replication complexes with microtubules and actin filaments is dependent on the interaction of ns3 and ns5a hepatitis c virus p7 protein is crucial for assembly and release of infectious virions the elusive function of the hepatitis c virus p7 protein the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug antiviral effects of amantadine and iminosugar derivatives against hepatitis c virus hepatitis c virus p7 is critical for capsid assembly and envelopment viruses and interferons interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures induction and function of type i and iii interferon in response to viral infection viruses and interferon: a fight for supremacy inhibition of hepatitis c virus infection by interferon-gamma through downregulating claudin-1 hcv infection induces a unique hepatic innate immune response associated with robust production of type iii interferons pathogenesis of chronic viral hepatitis: differential roles of t cells and nk cells innate immunity to virus infection toll-like receptors and viruses: induction of innate antiviral immune responses viral and therapeutic control of ifn-beta promoter stimulator 1 during hepatitis c virus infection differential roles of mda5 and rig-i helicases in the recognition of rna viruses cytosolic viral sensor rig-i is a 59-triphosphate-dependent translocase on double-stranded rna the mitochondrial targeting chaperone 14-3-3 regulates a rig-i translocon that mediates membrane association and innate antiviral immunity hcv innate immune responses cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus regulation of jak-stat signalling in the immune system down-regulation of the internal ribosome entry site (ires)-mediated translation of the hepatitis c virus: critical role of binding of the stem-loop iiid domain of ires and the viral core protein hepatitis c virus core protein blocks interferon signaling by interaction with the stat1 sh2 domain ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling-3 analysis of interferon signaling by infectious hepatitis c virus clones with substitutions of core amino acids 70 and 91 repression of interferon regulatory factor 1 by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes viral immune evasion: a masterpiece of evolution inhibition of the interferon-inducible protein kinase pkr by hcv e2 protein hepatitis c virus and interferon resistance control of innate immune signaling and membrane targeting by the hepatitis c virus ns3/4a protease are governed by the ns3 helix a 0 structural determinants for membrane association and dynamic organization of the hepatitis c virus ns3-4a complex hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system toll-like receptor 3-mediated activation of nf-kb and irf3 diverges at toll-il-1 receptor domain-containing adapter inducing ifn-b sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway sting regulates intracellular dnamediated, type i interferon-dependent innate immunity denv inhibits type i ifn production in infected cells by cleaving human sting hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity phosphorylation of hepatitis c virus-encoded nonstructural protein ns5a phosphorylation of the hepatitis c virus ns5a protein in vitro and in vivo: properties of the ns5a-associated kinase phosphorylation of hepatitis c virus ns5a nonstructural protein: a new paradigm for phosphorylation-dependent viral rna replication? reduction of hepatitis c virus ns5a hyperphosphorylation by selective inhibition of cellular kinases activates viral rna replication in cell culture hepatitis c virus ns5a: tales of a promiscuous protein hepatitis c virus nonstructural protein 5a modulates the toll-like receptor-myd88-dependent signaling pathway in macrophage cell lines evidence that hepatitis c virus resistance to interferon is mediated through repression of the pkr protein kinase by the nonstructural 5a protein how hepatitis c virus counteracts the interferon response: the jury is still out on ns5a control of pkr protein kinase by hepatitis c virus nonstructural 5a protein: molecular mechanisms of kinase regulation hepatitis c virus nonstructural 5a protein induces interleukin-8, leading to partial inhibition of the interferoninduced antiviral response relationships between hepatitis c virus replication and cxcl-8 production in vitro hepatitis c virus ns5a protein interacts with 29,59-oligoadenylate synthetase and inhibits antiviral activity of ifn in an ifn sensitivity-determining region-independent manner activation and evasion of the antiviral 29-59 oligoadenylate synthetase/ribonuclease l pathway by hepatitis c virus mrna hepatitis c virus ns5a protein protects against tnf-alpha mediated apoptotic cell death inhibition of intrahepatic gamma interferon production by hepatitis c virus nonstructural protein 5a in transgenic mice hepatitis c virus nonstructural protein 5a inhibits tumor necrosis factor-a-mediated apoptosis in huh7 cells activation of endoplasmic reticulum stress response by hepatitis viruses upregulates protein phosphatase 2a protein phosphatase 2a: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling pp2a targeting by viral proteins: a widespread biological strategy from dna/rna tumor viruses to hiv-1 hepatitis c virus inhibits interferon signaling through upregulation of protein phosphatase 2a upregulation of protein phosphatase 2ac by hepatitis c virus modulates ns3 helicase activity through inhibition of protein arginine methyltransferase 1 transcriptional induction of two genes in human cells by b interferon functional classification of interferon-stimulated genes identified using microarrays a central role for rna in the induction and biological activities of type 1 interferons targeted impairment of innate antiviral responses in the liver of chronic hepatitis c patients negative feedback regulation of rig-i-mediated antiviral signaling by interferoninduced isg15 conjugation immune signaling by rig-i-like receptors retinoic acid inducible gene-i (rig-i) signaling of hepatic stellate cells inhibits hepatitis c virus replication in hepatocytes interferon-b pretreatment of conventional and plasmacytoid human dendritic cells enhances their activation by influenza virus a structure-based model of rig-i activation emerging role of ubiquitination in antiviral rig-i signaling ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling hepatitis c virus induces interferon-and interferon-stimulated genes in primary liver cultures irf family of transcription factors as regulators of host defense regulation of pkr and irf-1 during hepatitis c virus rna replication constitutive expression of an isgf2/irf1 transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection regulation of hepatitis c virus replication by interferon regulatory factor 1 irf-7 is the master regulator of type-i interferon-dependent immune responses hepatitis c virus infection impairs irf-7 translocation and alpha interferon synthesis in immortalized human hepatocytes irf-3, irf-5, and irf-7 coordinately regulate the type i ifn response in myeloid dendritic cells downstream of mavs signaling hepatitis c virus inhibits intracellular interferon alpha expression in human hepatic cell lines serum-derived hepatitis c virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes interferon regulatory factor irf-7 induces the antiviral alpha interferon response and protects against lethal west nile virus infection antiviral actions of interferons impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection pkr protein kinase is activated by hepatitis c virus and inhibits viral replication through translational control pkr-dependent mechanisms of gene expression from a subgenomic hepatitis c virus clone identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication new antiviral pathway that mediates hepatitis c virus replicon interferon sensitivity through adar1 hepatitis c virus controls interferon production through pkr activation translational resistance of late alphavirus mrna to eif2a phosphorylation: a strategy to overcome the antiviral effect of protein kinase pkr initiation of protein synthesis by hepatitis c virus is refractory to reduced eif2.gtp.met-trna(i)(met) ternary complex availability translational insensitivity to potent activation of pkr by hcv ires rna hcv ns5a co-operates with pkr in modulating hcv ires-dependent translation eukaryotic translation initiation machinery can operate in a bacterial-like mode without eif2 hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress p bodies, stress granules, and viral life cycles stress granules: sites of mrna triage that regulate mrna stability and translatability stress granules eukaryotic stress granules: the ins and outs of translation regulation of stress granules and p-bodies during rna virus infection diversion of stress granules and p-bodies during viral infection dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection hepatitis c virus hijacks p-body and stress granule components around lipid droplets hepatitis c virus coopts ras-gtpase-activating protein-binding protein 1 for its genome replication viral encounters with 29,59-oligoadenylate synthetase and rnase l during the interferon antiviral response new insights into the role of rnase l in innate immunity the oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities the ribonuclease ldependent antiviral roles of human 29,59-oligoadenylate synthetase family members against hepatitis c virus 59-oligoadenylate synthetaselike gene highly induced by hepatitis c virus infection in human liver is inhibitory to viral replication in vitro antiviral activities of isg20 in positive-strand rna virus infections isg20, an actor of the innate immune response adenosine deaminases acting on rna (adars) are both antiviral and proviral adenosine deaminases acting on rna, rna editing, and interferon action rna-specific adenosine deaminase adar1 suppresses measles virus-induced apoptosis and activation of protein kinase pkr expression of interferoninducible rna adenosine deaminase adar1 during pathogen infection and mouse embryo development involves tissueselective promoter utilization and alternative splicing rna editing of hepatitis delta virus antigenome by dsrna-adenosine deaminase a specific base transition occurs on replicating hepatitis delta virus rna rna editing in hepatitis delta virus the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 limits the severity of acute influenza in mice the broad-spectrum antiviral functions of ifit and ifitm proteins ifitms restrict the replication of multiple pathogenic viruses distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm1 is a tight junction protein that inhibits hepatitis c virus entry ifitm3 inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication ifit1 is an antiviral protein that recognizes 59-triphosphate rna ifn-induced tpr protein ifit3 potentiates antiviral signaling by bridging mavs and tbk1 pathogens: raft hijackers cholesterol-binding viral proteins in virus entry and morphogenesis the diverse functions of oxysterolbinding proteins lipid traffic: floppy drives and a superhighway short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions ebola virus entry requires the cholesterol transporter niemann-pick c1 ifitm proteins restrict viral membrane hemifusion isg56 and ifitm1 proteins inhibit hepatitis c virus replication tpr proteins: the versatile helix the isg56/ifit1 gene family isg56 is a negativefeedback regulator of virus-triggered signaling and cellular antiviral response 29-o methylation of the viral mrna cap evades host restriction by ifit family members coordinated regulation and widespread cellular expression of interferon-stimulated genes (isg) isg-49, isg-54, and isg-56 in the central nervous system after infection with distinct viruses tracing the history of the ubiquitin proteolytic system: the pioneering article the ubiquitin code interplay between viruses and host sumoylation pathways viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways ubiquitin-like protein modifiers and their potential for antiviral and anti-hcv therapy isg15 and immune diseases human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways proteomic identification of proteins conjugated to isg15 in mouse and human cells the antiviral activities of isg15 isg15 enhances the innate antiviral response by inhibition of irf-3 degradation positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification activation of double-stranded rna-activated protein kinase (pkr) by interferon-stimulated gene 15 (isg15) modification down-regulates protein translation identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment hepatitis c virus reveals a novel early control in acute immune response ubp43 (usp18) specifically removes isg15 from conjugated proteins ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity the level of hepatitis b virus replication is not affected by protein isg15 modification but is reduced by inhibition of ubp43 (usp18) expression isg15 inhibits nedd4 ubiquitin e3 activity and enhances the innate antiviral response silencing of usp18 potentiates the antiviral activity of interferon against hepatitis c virus infection knockdown of usp18 increases a 2a interferon signaling and induction of interferon-stimulating genes but does not increase antiviral activity in huh7 cells potential relevance of cytoplasmic viral sensors and related regulators involving innate immunity in antiviral response interferon signaling and treatment outcome in chronic hepatitis c the isg15/usp18 ubiquitin-like pathway (isgylation system) in hepatitis c virus infection and resistance to interferon therapy viperin: a multifunctional, interferon-inducible protein that regulates virus replication the role of viperin in the innate antiviral response the interferon inducible gene: viperin viperin, a key player in the antiviral response the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts hiv-1 infection of human macrophages directly induces viperin which inhibits viral production the cellular antiviral protein viperin is attenuated by proteasome-mediated protein degradation in japanese encephalitis virus-infected cells human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity analysis of isg expression in chronic hepatitis c identifies viperin as a potential antiviral effector the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein 5a viperin inhibits hepatitis c virus replication by interfering with binding of ns5a to host protein hvap-33 interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol virus membrane-fusion proteins: more than one way to make a hairpin class ii enveloped viruses a novel unsupervised method to identify genes important in the antiviral response: application to interferon/ribavirin in hepatitis c patients identification of type i and type ii interferon-induced effectors controlling hepatitis c virus replication a genetic screen identifies interferon-a effector genes required to suppress hepatitis c virus replication a functional genomic screen reveals novel host genes that mediate interferon-alpha's effects against hepatitis c virus pre-mrna splicing: awash in a sea of proteins a genome-wide genetic screen for host factors required for hepatitis c virus propagation hepatitis c virus infection activates an innate pathway involving ikk-a in lipogenesis and viral assembly class a scavenger receptor 1 (msr1) restricts hepatitis c virus replication by mediating toll-like receptor 3 recognition of viral rnas produced in neighboring cells hmgb1 is a therapeutic target for sterile inflammation and infection release of chromatin protein hmgb1 by necrotic cells triggers inflammation high-mobility group box 1, oxidative stress, and disease human hepatitis c virus ns5a protein alters intracellular calcium levels, induces oxidative stress, and activates stat-3 and nf-kb hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production hepatitis c virus protein expression causes calcium-mediated mitochondrial bioenergetic dysfunction and nitro-oxidative stress hepatitis c virus infection is blocked by hmgb1 released from virus-infected cells pattern recognition receptors and inflammation serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma autophagy fights disease through cellular self-digestion self-eating and selfkilling: crosstalk between autophagy and apoptosis autophagy: process and function autophagy: from phenomenology to molecular understanding in less than a decade autophagy and the integrated stress response autophagy protects against sindbis virus infection of the central nervous system autophagy is an essential component of drosophila immunity against vesicular stomatitis virus enhancing immunity through autophagy eating the enemy within: autophagy in infectious diseases autophagy genes in immunity autophagy, immunity, and microbial adaptations autophagy in immunity and inflammation induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro hepatitis c virus inhibits akt-tuberous sclerosis complex (tsc), the mechanistic target of rapamycin (mtor) pathway, through endoplasmic reticulum stress to induce autophagy hepatitis c virus upregulates beclin1 for induction of autophagy and activates mtor signaling knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy autophagy and rna virus interactomes reveal irgm as a common target micrornas and immunity: novel players in the regulation of normal immune function and inflammation how do micrornas regulate gene expression? microrna regulation of innate immune responses in epithelial cells regulation of gene expression by microrna in hcv infection and hcv-mediated hepatocellular carcinoma hcv-induced mir-21 contributes to evasion of host immune system by targeting myd88 and irak1 this study was supported by research grants from the ministry of science and technology (101-2320-b-001-022-my3) and academia sinica, taipei. this manuscript was edited for the english language by american journal experts (aje). attribution-noncommercial-noderivs 3.0 unported license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ key: cord-256036-gd53s4dv authors: sandmann, lisa; ploss, alexander title: barriers of hepatitis c virus interspecies transmission date: 2013-01-01 journal: virology doi: 10.1016/j.virol.2012.09.044 sha: doc_id: 256036 cord_uid: gd53s4dv hepatitis c virus (hcv) is a major causative agent of severe liver disease including fibrosis, cirrhosis and liver cancer. therapy has improved over the years, but continues to be associated with adverse side effects and variable success rates. furthermore, a vaccine protecting against hcv infection remains elusive. development of more effective intervention measures has been delayed by the lack of a suitable animal model. naturally, hcv infects only humans and chimpanzees. the determinants of this limited host range are poorly understood in part due to difficulties of studying hcv in cell culture. some progress has been made elucidating the barriers for the hcv lifecycle in non-permissive species which will help in the future to construct animal models for hcv infection, immunity and pathogenesis. hepatitis c virus (hcv) is a positive-sense, single stranded rna virus classified within hepacivirus genus of the flaviviridae family. hcv has a high propensity for establishing lifelong persistent infections, which are associated with a significant risk of progressive liver fibrosis and hepatocellular carcinoma. worldwide at least 130 million people are chronically infected resulting in an estimated 366,000 deaths due to cirrhosis and cancer annually (perz et al., 2006) . epidemiological data is incomplete and in the united states alone the frequency of chronic carriers may be twice as high (edlin, 2011) . thus, hcv poses a major public health threat. treatment options have improved but are still limited. furthermore, the current standard of care, consisting of a combination of pegylated interferon (ifn) alpha, the nucleoside analog, ribavirin (rbv), and one of two hcv ns3-4a protease inhibitors, boceprevir or telaprevir, is not well tolerated. prior to the approval of these directly acting antivirals (daas) roughly 50% of genotype 1 infected patients -clinically, the hardest hcv substrain to treat -who underwent treatment, cleared the infection. inclusion of a hcv protease inhibitor to the peg-ifn/rbv regiment has increased sustained virological response (svr) rates in certain clinical trial cohorts to 60-70% kwo et al., 2010; mchutchison et al., 2009; poordad et al., 2011) . currently, numerous viral and host proteins are being pursued as antiviral drug targets. second-generation protease inhibitors and several compounds interfering with the functions of the ns5a phosphoprotein and the rna-dependent rna polymerase, ns5b, are already being tested in clinical trials (yang et al., 2011) . a combination of orally administered daas with distinct mechanisms of action holds promise to boost svr rates across all hcv patient cohorts. despite these successes unexpected toxicity in late-stage clinical trials has led to the discontinuation or delays in development of some very potent compounds, including the protease inhibitor biln2061 (lamarre et al., 2003) , the nucleotide polymerase inhibitor bms-986094 (vernachio et al., 2011) , and the cyclophilin a antagonist, alisporivir (coelmont et al., 2009) . these challenges highlight the need for thorough pre-clinical testing in predictive animal models. likewise, the development of pan-genotypic prophylactic or therapeutic vaccines instrumental in containing the hcv epidemic in resourcepoor communities would be greatly accelerated by a tractable animal model. chimpanzees are the only available immunocompetent in vivo experimental system, but their use is limited by ethical concerns, restricted availability and prohibitively high costs. undoubtedly, more tangible animal models are needed not only to prioritize clinical development of vaccine and drug candidates, but also to gain deeper insights into virus-host biology. the study of hcv in conventional cell culture systems, i.e. human hepatoma cells, may not accurately reflect host responses to infection. with the advent of more sensitive detection methods for viral infection and improved technical ability to culture primary hepatocytes it is now possible to dissect hcv infection in a physiological-relevant environment (jones et al., 2010a; ploss et al., 2010) . however, even in the most advanced tissue culture platforms, including micropatterned primary hepatocyte co-cultures (mpccs; jones et al., 2010a; khetani and bhatia, 2008; ploss et al., 2010) , cultures of human fetal hepatocytes marukian et al., 2011) , or hepatocyte-like cells derived from induced pluripotentstem cells (ipscs; roelandt et al., 2012; schwartz et al., 2012; wu et al., 2012) , important features of liver biology as yet cannot be adequately recapitulated. within the liver, numerous cell types, including hepatocytes and various non-parenchymal cell subsets (i.e. liver sinusoidal endothelial cells, stellate cells, and oval cells), are arranged in an intricate three-dimensional architecture. nutrient and oxygen gradients within the liver result in a compartmentalization of the liver, referred to as zonation, affecting metabolism, detoxification and response to injury. whether those specific microenvironments impact hcv infection is not understood and may only be adequately modeled within the three-dimensional context of the liver. furthermore, a tractable animal model may be suitable to decipher mechanisms of viral persistence and pathogenesis and assess disease states and comorbidities, such as hiv, alcohol or nutritionally exacerbated viral hepatitis, or extrahepatic manifestations associated with hcv infection. the only hosts known to be naturally permissive to hcv infection are humans and chimpanzees (table 1 ). the basis for this limited species tropism is incompletely understood and the topic of this review. the chimpanzee model played an instrumental role in early characterization of non-a, non-b hepatitis (houghton, 2009) which ultimately led to the discovery of hcv as the etiologic agent for the classically defined non-a non-b hepatitis by michael houghton and his team in 1989 (choo et al., 1989) . subsequently, many important discoveries were facilitated by their use; for example it was first demonstrated in chimpanzees that in vitro transcribed rna from a hcv cdna clone was infectious (kolykhalov et al., 1997) . furthermore, experimental infection of chimpanzees with hcv helped to define the nature of protective immunity (farci et al., 1992; prince et al., 1992) and to demonstrate the significance of cellular subsets, most stringently the role of cd4 and cd8 t cells in controlling chronic hcv infection shoukry et al., 2003) . chimpanzees remain the only fully immunocompetent animal model for hcv infection and consequently, have and continue to play an important role in evaluating the preclinical efficacy of vaccine candidates (reviewed in houghton, 2011) . precedence for the efficacy of new treatment modalities, such as the ifn-free control of hcv infection with daas (olsen et al., 2011) or interference with the liver specific microrna (mir) 122 (lanford et al., 2010) was first shown in chimpanzees. despite their utility, the use of large apes in biomedical research has raised ethical concerns, which culminated in the ban of experiments conducted in chimpanzees in many countries. an nih moratorium on 'non-essential' chimpanzee research (nih, 2011) is likely to constrain hcv research in the future and has made the need for alternative animal models even more pressing. the natural host reservoir of hcv remains poorly defined. while chimpanzees can be experimentally infected with hcv, the prevalence in chimpanzees or other great apes, gorillas and orangutans in the wild is not known. in search of alternative, more readily accessible experimental models for hcv numerous species have been tested for their susceptibility to hcv. woodchucks, old-and new-world monkeys, including cynomolgus, rhesus, japanese, green monkeys, doguera (abe et al., 1993) , chacma baboons (sithebe et al., 2002) , cottontop tamarins (garson et al., 1997) and marmosets appear to be mostly resistant to hcv infection. surprisingly, tree shrews (tupaia belangeri) which are distantly related to primates (schmitz et al., 2000; xu et al., 2012) appear to support hcv infection to some level (xie et al., 1998; xu et al., 2007) (table 1 ) and were recently shown to develop signs of severe liver disease, including steatosis, fibrosis and cirrhosis (amako et al., 2010) . while these data are encouraging additional studies with larger cohorts of this outbred and thus, genetically heterogeneous species, are needed to define more clearly the natural course of hcv infection in tree shrews. genetically related viruses, which replicate more readily in non-human species have been considered as potential surrogates for the study of hcv in vivo. most prominently, gb viruses (gbv), named after the surgeon george baber (gb) who presented with acute viral hepatitis in the 1960s (deinhardt et al., 1967) , specifically gbv-b, can be transmitted to new world monkeys (bukh et al., 2001; karayiannis et al., 1989; lanford et al., 2003; schaluder et al., 1995; simons et al., 1995) . gbv-b and hcv both belong to the flaviviridae family but share only approximately 28% amino acid identity within their polyprotein, which constrains the utility of this model for testing of drugs specific to hcv or vaccine candidates formulated with hcv specific antigens. another caveat is the different course of gbv-b infection in tamarins and marmoset, which results in acute hepatitis butin contrast to hcv -does not establish chronicity. more recently, other viruses that are genetically more related to hcv than gbv-b have been detected in other species. non-primate hepaci-virus (nphv), also known as canine hepacivirus (chv) has been identified in dogs (kapoor et al., 2011) and horses (burbelo et al., 2012) . intriguingly, chv/nphv appears to have a broader tissue tropism in these species but has yet to be shown to cause hepatitis. comparative analysis of chv/nphv and hcv may help to shed light on potentially common viral and host factors determining viral host and tissue tropism. since hcv does not readily replicate in non-human species conceptually the most straightforward strategy would be to introduce the relevant tissue compartment -a human liverinto mice. to this end various approaches have been taken including transplantation of human liver pieces under the kidney capsule (ilan et al., 2002) , engraftment of ''human ectopic artificial livers'' (heals) in the peritoneal cavity (chen et al., 2011) or actual expansion of human hepatocytes within the liver parenchyma (reviewed in de jong et al. (2010) and meuleman and leroux-roels (2008b) of immunocompromised mice. for the latter approach, suitable xenorecipients combine two features; immunodeficiency to prevent graft rejection and liver injury to provide a growth stimulus for the normally quiescent hepatocytes as well as yield the transplanted human cells a competitive growth advantage over endogenous murine cells. the best-characterized liver injury mouse strains are urokinase-type plasminogen activator (upa) transgenic mice (heckel et al., 1990) and mice deficient in fumaryl acetoacetate hydrolase (fah; grompe et al., 1993) . when crossed to an immunodeficient background the livers of either line can be highly repopulated with human hepatocytes, rendering the human liver chimeric mice susceptible to hcv infection (bissig et al., 2010; mercer et al., 2001; meuleman et al., 2005) . such humanized mice have been invaluable to study basic aspects of hcv biology within the three-dimensional context of the liver and assessing preclinically the efficacy of antiviral compounds and biologics (reviewed in meuleman and leroux-roels (2008a) ). however, presumably due to their immunocompromised status hcv infected human liver chimeric mice generally do not exhibit signs of progressive liver disease, a process that is thought to be immune-mediated in humans. to overcome this caveat, engrafting components of a human immune system alongside human hepatocytes in a single recipient has been proposed (legrand et al., 2009 ). proof-of-concept for this strategy was provided in a recent study showing that mice co-injected with a mixture of human hematopoietic stem cells, fetal hepatoblasts and other nonparenchymal cells could be infected with hcv and develop signs of liver fibrosis (washburn et al., 2011) . while intriguing, human immune responses are generally weak in these reconstituted mice and additional modifications will be needed to improve both cellular complexity and functionality of the engrafted human immune system (reviewed in shultz et al. (2007) and willinger et al. (2011) ). the utility of these xenotransplantation models is hampered by low through-put, intricate logistics, high costs and technical difficulties in their production but also the inherent donor-to-donor variability and differences in the human chimerism between animals. an inbred mouse model for hcv would overcome the technical difficulties of the xenotransplantation model. mice with inheritable susceptibility to hcv could be produced fairly easily in large quantities, would be amendable to genetic manipulations on defined genetic backgrounds and viral infection could be analyzed with a vast amount of existing tools and reagents. the challenge is to elucidate and overcome restrictions for hcv to complete its lifecycle in mouse cells and mice ( fig. 1) . in recent years progress has been made in explaining aspects of the restricted host range of hcv. in the following sections, we will highlight barriers in the hcv lifecycle in non-permissive species, focusing on mice, and discuss putative strategies to overcome them. a large number of cellular factors has been implicated in facilitating hcv entry into human cells. in a coordinated multistep process (table 2 , reviewed in ploss and evans (2012) ) hcv, complexed with host lipoproteins in so called lipo-viroparticles, is thought to initially attach to glycosaminoglycans, then binds to low-density lipoprotein receptor (ldlr), the scavenger receptor class b type i (scarb1; scarselli et al., 2002) and the tetraspanin cd81 (pileri et al., 1998) on the hepatocyte surface, before engaging the tight junction proteins, claudin-1 (cldn1; evans et al., 2007) and occludin (ocln; liu et al., 2009; ploss et al., 2009) , ultimately resulting in endocytotic uptake (fig. 1 ). more recently, the receptor tyrosine kinases epidermal growth factor receptor (egfr) and ephrin receptor a2 (epha2; lupberger et al., 2011) and the cholesterol uptake receptor niemann-pick c1 like 1 (npc1l1; sainz et al., 2012) have been implicated in the viral entry process by indirectly influencing the composition of membrane microdomains. the c-type lectins liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (l-sign, cd209l; cormier et al., 2004) , which is expressed on liver sinusoidal endothelial cells (lsecs) and the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign, cd209), expressed by dendritic and kupffer cells have been shown to bind to hcv e2 (gardner et al., 2003; lozach et al., 2003; pohlmann et al., 2003) . c-type lectins on sinusoidal cells may be able to trap hcv in the liver, but it is not known whether this can lead to productive infection of hepatocytes in trans or whether sequestered hcv is subjected to lysosomal degradation. other molecules such as cldn6, cldn9 (meertens et al., 2008; zheng et al., 2007) , transferrin receptor (tfr; martin and uprichard, 2010) and protocadherin 5 (pcdhb5; wong-staal et al., 2008) have been proposed to be involved in influencing hcv entry. however, cldn6, cldn9 nor pcdhb5 are expressed at appreciable levels in the liver (table 2) . thus, strong experimental evidence supporting their suggested relevance for viral entry and independent confirmation of these results still remain pending. in contrast to human hepatocytes, murine cells do not support hcv entry thereby creating a first and important barrier for a broader host tropism of the virus. in an attempt to define the determinants restricting hcv entry into non-human cells a cdna complementation screen was conducted in mouse cells ectopically expressing human cd81, scarb1 and cldn1 . human ocln was identified as the missing factor needed to facilitate viral entry into human and mouse cells. while cd81, scarb1, cldn1 and ocln are all required, only cd81 and ocln need to be of human origin to promote viral entry into mouse lymph node, liver,placenta lymph node, spleen, pancreas ã¾ ã¾ 54.5 gardner et al. (2003) cells ). these observations are in line with previous reports demonstrating that in contrast to cd81 of various non-human primate species, mouse cd81 does not support hcv pseudoparticle entry into human hepg2 hepatoma cells, which lack endogenous expression of cd81 (flint et al., 2006) . similarly, in human cells lacking endogenous ocln expression, primate sequences function equivalently to human ocln, whereas canine, hamster, mouse and rat ocln had intermediate to low activities, and guinea pig ocln was completely nonfunctional (michta et al., 2010) . for both cd81 and ocln, differences in the second extracellular loops are in part responsible for this apparent functional activity. in contrast, the residues (i32 and e48) within the first extracellular loop of cldn1 required for hcv entry (evans et al., 2007) are conserved between mice and man. thus, it is not surprising that mouse cldn1 supports hcv uptake. originally, it was shown that human but not mouse scarb1 can bind to soluble hcv e2 (scarselli et al., 2002) . nonetheless, in both in vitro ) and in vivo (dorner et al., 2011) infection assays mouse and human scarb1 were equally functional. these apparently conflicting data are likely attributable to the read-out system and highlight that hcv e2 binding may not be the most reliable proxy for functional entry assays. to date any differences in the susceptibility of cells of non-human origin to hcv entry are largely accounted for by differences in critical positions within respective orthologues of cd81, scarb1, cldn1 and ocln or their limited or absent expression. the observation that cd81 and ocln comprise the minimal set of human factors required to render mouse cells permissive to hcv entry in vitro provided the blue print for engineering a mouse that at least supports viral uptake in vivo. genetically humanized mice have been successfully constructed by expression of receptors and/or coreceptors in mice for other human pathogens, such as poliovirus (hcd155; racaniello and ren, 1994) , measles virus (hcd46/cd150; sellin and horvat, 2009), human coronavirus (hcd13; lassnig et al., 2005) , human immunodeficiency virus (hcd4/ccr5/cxcr4; klotman and notkins, 1996) and listeria monocytogenes (lecuit et al., 2001) . earlier attempts to render mice susceptible to hcv infection by transgenic expression of cd81 were not successful (masciopinto et al., 2002) . expression of human cd81 in a wide variety of tissues increased the binding of recombinant e2 to liver, thymocytes and splenic lymphocytes in comparison to non-transgenic controls, but due to the lack of human ocln human cd81-transgenic mice were resistant to hcv infection. recently, it was demonstrated that adenoviral delivery of human cd81 and ocln is sufficient to allow hcv infection of fully-immunocompetent inbred mice (dorner et al., 2011) . using intergenotypic virus chimeras expressing cre recombinase to activate a cellular reporter, in vivo uptake of diverse hcv genotypes could be conveniently quantified by bioluminescent imaging. utilizing blocking antibodies specific to cd81 or the viral envelope protein e2, expression of entry factor mutants and mice with a targeted disruption of the scarb1 gene validated uptake of hcv into murine hepatocytes in an hcv glycoprotein-mediated fashion. furthermore, it established a precedent for applying mouse genetics to dissect the viral entry process, in addition to validating the role of scarb1 for hcv uptake in vivo for the first time. beyond studying hcv entry, this model can be employed to evaluated passive and active immunization strategies (dorner et al., 2011, in press; giang et al., 2012) . the transient adenoviral delivery approach is high-throughput and allows rapid evaluation of mutant genes. however, in order to accurately reproduce the complex process of hcv entry in vivo, it will be important to achieve native expression patterns of the human hcv entry factor orthologues by using transgenic and/or knock-in approaches. recently, transgenic mice expressing human cd81, scarb1, cldn1 and ocln were generated, but did not appear to be permissive to hcv infection in vivo (hikosaka et al., 2011) . this apparent discrepancy between transient adenoviral and stable transgenic expression approaches can be in part explained by the lower level of entry factor expression in the transgenic mice and the requirement for a very sensitive reporter system to quantify viral entry (dorner et al., 2011) . as an alternative to this host adaptation approach to overcome species barriers it may be possible to adapt hcv to usage of entry factor orthologs from other species. using an unbiased selection approach, a laboratory strain of hcv was adapted to utilize mouse cd81 (bitzegeio et al., 2010) . taking advantage of the high mutational plasticity of hcv, three adaptive mutations in the viral glycoproteins e1 and e2 were identified that allowed the virus to enter cells expressing human scarb1, cldn1, ocln and mouse cd81. interestingly, the resulting murine-tropic (mt) hcv was also capable of efficiently utilizing mouse ocln and had a lower dependency on scarb1. whether mthcv is capable of infecting mouse hepatocytes in vitro or in vivo has yet to be shown. clearly, this study provides an important proof-of-concept for the validity of the viral adaptation approach but also raises a few potential caveats. the adaptive mutations within the viral envelope appear to have resulted in conformational changes. consequently, it remains to be tested whether mthcv faithfully recapitulates viral entry and raises concerns of whether the neutralizing capacity of antibodies or human specific therapeutics to block hcv entry can be adequately tested. the contribution of other host factors implicated in viral entry to the species tropism of hcv is not well understood. most human and murine orthologues share a high degree of amino acid sequence similarity (table 2 ) but, as discussed above, differences in individual residues can drastically affect viral uptake efficiency. the exact roles of egf receptor and ephrin receptor a2, which are both expressed in mouse and human hepatocytes, in this process are incompletely defined. both receptor tyrosine kinases (rtks) appear to mediate entry by regulating cd81-cldn1 co-receptor associations and viral glycoprotein-dependent membrane fusion (lupberger et al., 2011) . recently, it was shown that egfr activation is dependent on interactions between hcv and cd81 but not cldn1 (diao et al., 2012) . although, it is conceivable that expression of human egfr and/or ephrin receptor a2 could increase hcv entry efficiency in vivo their murine orthologues appear to exert sufficient functionality in genetically humanized mice. interestingly, although npc1l1 has been shown to play a role in the viral entry in human cells in vitro and in vivo (sainz et al., 2012) it appears to be dispensable for entry into mouse cells. in contrast to humans, the murine ortholog of npc1l1 has approximately 85% sequence similarity to the human protein and is abundantly expressed in the gut but not in the liver (altmann et al., 2004) . nonetheless, hcv is still capable of entering into hepatocytes of mice modified to express a combination of human cd81 and ocln (dorner et al., 2011) suggesting some level of redundancy for npc1l1 in this process. it is conceivable that hcv entry would be affected, perhaps becoming more efficient, if npc1l1 along with human cd81 and ocln was ectopically co-expressed in the liver, which remains to be experimentally tested. it was recently demonstrated that hepatic overexpression of human npc1l1 in transgenic mice (temel et al., 2007) or via adenoviral delivery modulates cellular and plasma lipid metabolism (kurano et al., 2012) . altered cellular lipid profiles can arguably affect the composition of membrane microdomains and consequently, the trafficking of membrane proteins including hcv entry factors. following receptor mediated endocytosis the viral membrane has to fuse with the endosome to eventually release the hcv rna genome from the nucleocapsid into the cytoplasm. it has been known that viral rna translation occurs in vivo, when the rna is directly introduced into mouse hepatocytes, e.g. by hydrodynamic delivery (mccaffrey et al., 2002) . this suggests that the hcv internal ribosome entry site (ires) within the 5 0 untranslated region is functional in mouse cells. recently, a genetically humanized mouse model was constructed utilizing cell culture produced recombinant hepatitis c virus to activate a cellular encoded reporter (dorner et al., 2011, in press ). in this model hcv is taken up into murine hepatocytes in a viral glycoprotein-dependent fashion providing further evidence that fusion and uncoating work efficiently in this background. the advent of hcv replicons, i.e. subgenomic or full-length genomes containing a dominant selectable marker such as neomycin amino transferase (blight et al., 2000; lohmann et al., 1999) , allowed for assessing whether hcv is capable of replicating in non-human cells. indeed, when these replicons were introduced into mouse hepatoma cell lines and embryonic fibroblasts, cell clones harboring replicating hcv rna could be identified at very low frequency (uprichard et al., 2006; zhu et al., 2003) . these data suggest that all necessary host factors are present in mouse cells and sufficiently similar to support hcv rna replication. however, it is conceivable that the viral rna replication machinery cooperates more robustly with essential human host factors than their murine counterparts. targeted and genome wide loss of function screens led to the discovery of numerous cellular factors putatively promoting or restricting hcv replication in human cells borawski et al., 2009; coller et al., 2009; jones et al., 2010b; li et al., 2009; ng et al., 2007; randall et al., 2007; reiss et al., 2011; supekova et al., 2008; tai et al., 2009; trotard et al., 2009; vaillancourt et al., 2009) . unfortunately, independent studies are often minimally overlapping and the relevance of many of these interactions to hcv biology remains to be demonstrated. strong experimental evidence has been provided for the critical role of cyclophilin a (cypa) in rna replication and virion assembly (kaul et al., 2009; yang et al., 2008) . another essential co-factor for rna replication independently identified by several groups borawski et al., 2009; reiss et al., 2011; tai et al., 2009; trotard et al., 2009) , is phosphatidylinositol 4 kinase iii (pi4kiii). pi4kiii is recruited to the membranous web via interaction with ns5a. the human and murine orthologues of cypa and pi4kiii share a high degree of sequence similarity at 98.2 and 98.6%, respectively. it is yet to be shown whether ectopic expression of human cypa and pi4kiii augments hcv rna replication in mouse cells. likewise, there is little evidence for the accumulation of adaptive mutations boosting viral rna replication in mouse cells by fostering more efficient interactions with mouse orthologues of host factors. cellular micrornas can drastically influence virus replication and pathogenesis (gottwein and cullen, 2008) . a liver specific microrna, mir-122, was shown to be critical for efficient hcv rna replication (jopling et al., 2005) . mir-122 is derived from a liver-specific noncoding polyadenylated rna transcribed from the gene hcr and interacts with two sites in the viral 5 0 non-translated region (ntr) (jopling et al., 2008 (jopling et al., , 2005 . those interactions presumably protect the 5 0 terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the rna terminus (machlin et al., 2011) , resulting in greater viral rna abundance in both infected cultured cells and in the liver of infected chimpanzees (lanford et al., 2010) . the exact sequence of mir-122 is highly conserved in vertebrate species; comparatively, mouse mir-122 is sequence-wise identical and with 50,000 copies per hepatocytes similarly expressed as the human counterpart (chang et al., 2004) . consequently, mir-122 is not likely to contribute to the restricted host tropism in non-permissive species. it is possible that cell-intrinsic, species-specific restriction factors, similar to those that control retroviral infection, such as fv1, trim5 or apobec3 cytidine deaminases (reviewed in bieniasz (2004) ), interfere with hcv rna replication in murine cells. however, heterokaryons of mouse and human cells capable of supporting the hcv lifecycle suggest that dominant negative inhibitors of hcv replication do not exist (frentzen et al., 2011) . differences in the magnitude, nature and kinetics of an antiviral response between hosts are known to restrict tropism of certain viruses, such as myxoma virus, which is only permissive in mouse cells that have impaired ifn responses . the interferon system also plays a pivotal role in limiting hcv both under physiological conditions and when triggered during therapeutic intervention. hundreds of ifn stimulated genes (isgs) are induced in the liver during hcv infection (reviewed in ) and isg products can potently inhibit hcv infection fig. 2) . pattern recognition receptors (prrs) such as toll-like receptors (tlrs)-3 and 7 in endosomes as well as a family of rig-i like receptors (rlrs), including the retinoic-acid-inducible gene i (rig-i) and the melanoma-differentiation-associated gene 5 (mda5) sense the presence of viral rnas. subsequently, host antiviral responses are activated resulting in the production of cytokines, such as type i and iii interferons (yoneyama et al., 2004) . when ifn signaling is blunted, hcv replicates more efficiently in both human hepatoma cells (blight et al., 2002) and primary human hepatocytes marukian et al., 2011) . similarly, in mouse embryonic fibroblasts with targeted disruptions in protein kinase r (pkr; chang et al., 2006) or interferon regulatory factor 3 (irf3; lin et al., 2010) rna replication is enhanced. whether disruption of these or other innate signaling pathways are sufficient to render mice expressing human hcv entry factors permissive to hcv infections has yet to be shown. hcv has devised mechanisms to counteract innate defenses but those may not work equally efficient in cells from different species. for example, ns3-4a serine protease is not only essential for processing of the hcv polyprotein but has been shown to cleave the mitochondrial associated antiviral signaling protein (mavs; meylan et al., 2005) , the t-cell protein tyrosine phosphatase (tc-ptp; brenndorfer et al., 2009 ) and tir domain-containing adapter inducing ifn (trif; li et al., 2005) thereby modulating antiviral signaling. the cleavage motifs of mavs and trif are partially conserved between mice and man/chimpanzees (fig. 2) and it remains to be demonstrated whether these antiviral evasion strategies are functional in murine cells. contrarily, mavs from certain new world monkey species, including rhesus macaques and baboons, both of which are resistant to hcv infection, is functional for interferon signaling even in the presence of heterologously expressed ns3/4a protease in human epithelial cells. thus, escape from hcv antagonism of innate immune sensing may explain at least part of hcv species restriction (patel et al., 2012) . furthermore, other aspects of hcvs antiviral evasion strategies may work less efficiently in mouse cells. for example, ns4b was shown to inhibit rlr-mediated ifn signaling through association with the stimulator of interferon genes (sting; nitta et al., in press) . similarly, overexpression data suggests that the hcv core protein interferes with ifn signaling downstream of the ifn receptor, presumably through direct interaction with stat1, leading to reduced phospho-stat1. the protein sequences of mouse and human stat1 are highly conserved ( 490% sequence identity), mouse and human sting are only ca. 60% identical (ca. 80% similar) which may result in a weaker interaction of ns4b with the murine orthologue and consequently, more exacerbated rlr signaling. the development of the infectious cell culture system, which recapitulates all parts of the viral lifecycle paved the way to study hcv assembly, egress and release. increasing evidence points to a connection between the viral lifecycle and the biogenesis of host very low-density lipoproteins (vldl) (huang et al., 2007; merz et al., 2011; ye, 2007) . very low-density viral particles can be detected in sera from hcvã¾ patients using antibodies targeting lipoproteinassociated proteins, notably apolipoproteins (apo) b and e (nielsen et al., 2006) . the role of specific apolipoproteins in virion assembly is incompletely understood. loss of function experiments have pointed towards apoa1 (mancone et al., 2011) , apob (huang et al., 2007) , and apoe (reviewed in bartenschlager et al. (2011) ) as potential players in this process. while human cells with a functional vldl pathway are capable of producing infectious hcv it was unclear whether other species would also support late stages of the hcv lifecycle. to investigate this process mouse hep56.1d cells harboring a subgenomic hcv replicon were transcomplemented with hcv core, e1, e2, p7 and ns2 proteins but only marginal amounts of infectious hcv were released . a comparative transcriptome analysis between naã¯ve mouse cells and those containing hcv replicons hinted that low levels of apoe in the replicon-containing cells might limit hcv assembly. indeed, complementation with human or mouse apoe was sufficient to rescue infectious hcv production from these cells, yielding infectious titers similar to those observed in the widely used human hepatoma cell line, huh-7.5. these data confirm the critical importance of apoe in hcv assembly and provide evidence that mouse cells can support the late stages of hcv lifecycle, if critical components of the vldl pathway are present. however, it remains unclear whether hcv can assemble in more physiologically relevant systems and importantly if mice are capable of producing infectious particles in vivo. substantial progress has been made in our understanding of the hcv lifecycle which is being translated into improved therapies. experimental systems devised to study hcv in cell culture have enabled comparative studies to elucidate the barriers that restrict hcv infection to humans and chimpanzees. while murine cells can be engineered to express the human orthologs of the entry molecules cd81 and occludin to allow entry, viral rna replication is still low in these cells. this is at least in part due to varying antiviral defenses. murine cells with an intact vldl pathway can produce infectious virions. taken together these observations raise the hope that an inbred mouse model for hcv infection can be achieved. recapitulating the entire viral lifecycle in a mouse would certainly be of great importance but it remains to be seen whether such a model would also mimic clinical relevant disease aspects. in humans pathogenesis in the context of chronic hcv infection is thought to be driven by persistent liver inflammation ultimately resulting in fibrosis, cirrhosis and liver cancer. thus, establishing hcv innate immune response to hcv infection. ch; chimpanzee, dsrna, double-stranded rna; er, endoplasmatic reticulum; hu, human; ifn, interferon; ifnar, interferon-alpha receptor; il, interleukin; irf, interferon regulatory factor; jak, janus kinase; ms, mouse; mavs, mitochondrial associated antiviral signaling protein; mda5, melanoma-differentiation-associated gene 5; pkr, protein kinase r; rh, rhesus macaque; rig-i, retinoic-acid-inducible gene i; ssrna, single-stranded rna; stat1, signal transducer and activator of transcription protein-1; stat2, signal transducer and activator of transcription protein-2; sting, stimulator of interferon genes; tlr, toll-like receptor; trif, tir-domain-containing adapter-inducing interferon; tyk, tyrosine kinase. inlet: sequence alignment of the ns3-4a cleavage motif within mavs from different permissive and non-permissive species. persistence in the presence of a functional immune system is likely necessary to model adequately hcv pathogenesis. studies characterizing chronicity in humanized mice engrafted with both human hepatocytes and components of a human immune system (washburn et al., 2011) as well as long-term follow up studies in tree shrews (amako et al., 2010 ) hint that it may become possible to dissect liver disease progression in small animal models. the course of hcv infection has been studied in great detail in chimpanzees and by and large mirrors adequately acute and chronic hcv infection in humans. whether hcv infection in other species such as inbred mice will follow a similar clinical course is unclear. however, even if hcv infections were to cause e.g. mostly acute hepatitis in mice inheritable traits rendering animals permissive to hcv could be combined with genetic backgrounds that are more prone for chronicity and liver disease progression. different approaches, including both viral and host adaptations are likely needed to construct a selection of hcv permissive animal models, which in combination may be suitable to replace the chimpanzee. a cost-effective animal model that is amendable to genetic manipulations and can be reproducibly produced at higher throughput would undoubtedly lend itself to address currently intractable problems in hcv research. lack of susceptibility of various primates and woodchucks to hepatitis c virus niemann-pick c1 like 1 protein is critical for intestinal cholesterol absorption pathogenesis of hepatitis c virus infection in tupaia belangeri expression of paramyxovirus v proteins promotes replication and spread of hepatitis c virus in cultures of primary human fetal liver cells assembly of infectious hepatitis c virus particles roles for endocytic trafficking and phosphatidylinositol 4-kinase iii alpha in hepatitis c virus replication intrinsic immunity: a front-line defense against viral attack human liver chimeric mice provide a model for hepatitis b and c virus infection and treatment adaptation of hepatitis c virus to mouse cd81 permits infection of mouse cells in the absence of human entry factors efficient initiation of hcv rna replication in cell culture highly permissive cell lines for hepatitis c virus genomic and subgenomic rna replication class iii phosphatidylinositol 4-kinase alpha and beta are novel host factor regulators of hepatitis c virus replication nonstructural 3/4a protease of hepatitis c virus activates epithelial growth factor-induced signal transduction by cleavage of the t-cell protein tyrosine phosphatase host range studies of gb virus-b hepatitis agent, the closest relative of hepatitis c virus, in new world monkeys and chimpanzees serology-enabled discovery of genetically diverse hepaciviruses in a new host mir-122, a mammalian liverspecific microrna, is processed from hcr mrna and may downregulate the high affinity cationic amino acid transporter cat-1 replication of hepatitis c virus (hcv) rna in mouse embryonic fibroblasts: protein kinase r (pkr)-dependent and pkr-independent mechanisms for controlling hcv rna replication and mediating interferon activities humanized mice with ectopic artificial liver tissues isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis c virus (hcv) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for hcv (stat-c) inhibitors rna interference and single particle tracking analysis of hepatitis c virus endocytosis l-sign (cd209l) and dc-sign (cd209) mediate transinfection of liver cells by hepatitis c virus new horizons for studying human hepatotropic infections studies on the transmission of human viral hepatitis to marmoset monkeys. i. transmission of disease, serial passages, and description of liver lesions hepatitis c virus (hcv) induces epidermal growth factor receptor (egfr) activation via cd81 binding for viral internalization and entry a genetically humanized mouse model for hepatitis c virus infection study of hepatitis c virusentry in genetically humanized mice perspective: test and treat this silent killer claudin-1 is a hepatitis c virus co-receptor required for a late step in entry lack of protective immunity against reinfection with hepatitis c virus diverse cd81 proteins support hepatitis c virus infection completion of hepatitis c virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines l-sign (cd 209l) is a liver-specific capture receptor for hepatitis c virus lack of susceptibility of the cottontop tamarin to hepatitis c infection human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis c virus viral and cellular micrornas as determinants of viral pathogenesis and immunity hcv persistence and immune evasion in the absence of memory t cell help loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator expression of human factors cd81, claudin-1, scavenger receptor, and occludin in mouse hepatocytes does not confer susceptibility to hcv entry the long and winding road leading to the identification of the hepatitis c virus prospects for prophylactic and therapeutic vaccines against the hepatitis c viruses hepatitis c virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins the hepatitis c virus (hcv)-trimera mouse: a model for evaluation of agents against hcv telaprevir for previously untreated chronic hepatitis c virus infection real-time imaging of hepatitis c virus infection using a fluorescent cell-based reporter system comparison of u2os and huh-7 cells for identifying host factors that affect hepatitis c virus rna replication position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome modulation of hepatitis c virus rna abundance by a liver-specific microrna characterization of a canine homolog of hepatitis c virus studies of gb hepatitis agent in tamarins essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics microscale culture of human liver cells for drug development transgenic models of human immunodeficiency virus type-1 transmission of hepatitis c by intrahepatic inoculation with transcribed rna modulation of lipid metabolism with the overexpression of npc1l1 in mice liver efficacy of boceprevir, an ns3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis c infection (sprint-1): an open-label an ns3 protease inhibitor with antiviral effects in humans infected with hepatitis c virus comparison of tamarins and marmosets as hosts for gbv-b infections and the effect of immunosuppression on duration of viremia therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection studying human pathogens in animal models: fine tuning the humanized mouse a transgenic model for listeriosis: role of internalin in crossing the intestinal barrier humanized mice for modeling human infectious disease: challenges, progress, and outlook immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif a genomewide genetic screen for host factors required for hepatitis c virus propagation replication of subgenomic hepatitis c virus replicons in mouse fibroblasts is facilitated by deletion of interferon regulatory factor 3 and expression of liver-specific microrna 122 tight junction proteins claudin-1 and occludin control hepatitis c virus entry and are downregulated during infection to prevent superinfection replication of subgenomic hepatitis c virus rnas in a hepatoma cell line mouse hepatic cells support assembly of infectious hepatitis c virus particles dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e2 egfr and epha2 are host factors for hepatitis c virus entry and possible targets for antiviral therapy masking the 5 0 terminal nucleotides of the hepatitis c virus genome by an unconventional microrna-target rna complex hepatitis c virus production requires apolipoprotein a-i and affects its association with nascent low-density lipoproteins the role of transferrin receptor in hepatitis c virus entry hepatitis c virus induces interferon-lambda and interferonstimulated genes in primary liver cultures expression of human cd81 in transgenic mice does not confer susceptibility to hepatitis c virus infection determinants of hepatitis c translational initiation in vitro, in cultured cells and mice telaprevir with peginterferon and ribavirin for chronic hcv genotype 1 infection the tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis c virus hepatitis c virus replication in mice with chimeric human livers biochemical and morphological properties of hepatitis c virus particles and determination of their lipidome the human liver-upa-scid mouse: a model for the evaluation of antiviral compounds against hbv and hcv the human liver-upa-scid mouse: a model for the evaluation of antiviral compounds against hbv and hcv morphological and biochemical characterization of a human liver in a upa-scid mouse chimera cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus species-specific regions of occludin required by hepatitis c virus for cell entry identification of host genes involved in hepatitis c virus replication by small interfering rna technology association between hepatitis c virus and very-lowdensity lipoprotein (vldl)/ldl analyzed in iodixanol density gradients hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type-i interferon-dependent innate immunity sustained viral response in a hepatitis c virus-infected chimpanzee via a combination of direct-acting antiviral agents convergent evolution of escape from hepaciviral antagonism in primates the contributions of hepatitis b virus and hepatitis c virus infections to cirrhosis and primary liver cancer worldwide binding of hepatitis c virus to cd81 hepatitis c virus host cell entry human occludin is a hepatitis c virus entry factor required for infection of mouse cells persistent hepatitis c virus infection in microscale primary human hepatocyte cultures hepatitis c virus glycoproteins interact with dc-sign and dc-signr boceprevir for untreated chronic hcv genotype 1 infection immunity in hepatitis c infection transgenic mice and the pathogenesis of poliomyelitis cellular cofactors affecting hepatitis c virus infection and replication recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis c virus identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor the human scavenger receptor class b type i is a novel candidate receptor for the hepatitis c virus molecular and serologic analysis in the transmission of the gb hepatitis agents the complete mitochondrial genome of tupaia belangeri and the phylogenetic affiliation of scandentia to other eutherian orders interferon-stimulated genes and their antiviral effector functions a diverse range of gene products are effectors of the type i interferon antiviral response modeling hepatitis c virus infection using human induced pluripotent stem cells current animal models: transgenic animal models for the study of measles pathogenesis memory cd8 ã¾ t cells are required for protection from persistent hepatitis c virus infection humanized mice in translational biomedical research isolation of novel virus-like sequences associated with human hepatitis lack of susceptibility of chacma baboons (papio ursinus orientalis) to hepatitis c virus infection identification of human kinases involved in hepatitis c virus replication by small interference rna library screening a functional genomic screen identifies cellular cofactors of hepatitis c virus replication hepatic niemann-pick c1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe kinases required in hepatitis c virus entry and replication highlighted by small interference rna screening replication of a hepatitis c virus replicon clone in mouse cells identification of a lipid kinase as a host factor involved in hepatitis c virus rna replication inx-08189, a phosphoramidate prodrug of 6-o-methyl-2 0 -c-methyl guanosine, is a potent inhibitor of hepatitis c virus replication with excellent pharmacokinetic and pharmacodynamic properties disruption of erk-dependent type i interferon induction breaks the myxoma virus species barrier a humanized mouse model to study hepatitis c virus infection, immune response, and liver disease improving human hemato-lymphoid-system mice by cytokine knock-in gene replacement characterization of the role of the protocadherin b famility in hcv entry productive hepatitis c virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation transmission of hepatitis c virus infection to tree shrews evaluating the phylogenetic position of chinese tree shrew (tupaia belangeri chinensis) based on complete mitochondrial genome: implication for using tree shrew as an alternative experimental animal to primates in biomedical research efficient infection of tree shrew (tupaia belangeri) with hepatitis c virus grown in cell culture or from patient plasma cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro anti-hcv drugs in the pipeline reliance of host cholesterol metabolic pathways for the life cycle of hepatitis c virus the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses claudin-6 and claudin-9 function as additional coreceptors for hepatitis c virus replication of hepatitis c virus subgenomes in nonhepatic epithelial and mouse hepatoma cells the authors thank dr. cynthia de la fuente for critical discussions and editing. l.s. is a fellow of the german national merit foundation and is supported by a stipend from the organization of supporters of the hannover medical school (gesellschaft der freunde der medizinischen hochschule hannover e.v.). a.p. is a recipient of the astellas young investigator award of the infectious disease society of america and a liver scholar award from the american liver foundation. work in the laboratory is supported in part by the starr foundation, the greenberg medical institute, the national institutes of health and the bill and melinda gates foundation. the funding sources were not involved in the writing of the report. key: cord-324984-ojrpsdt9 authors: ji, xingyue; li, zhuorong title: medicinal chemistry strategies toward host targeting antiviral agents date: 2020-02-14 journal: med res rev doi: 10.1002/med.21664 sha: doc_id: 324984 cord_uid: ojrpsdt9 direct‐acting antiviral agents (daas) represent a class of drugs targeting viral proteins and have been demonstrated to be very successful in combating viral infections in clinic. however, daas suffer from several inherent limitations, including narrow‐spectrum antiviral profiles and liability to drug resistance, and hence there are still unmet needs in the treatment of viral infections. in comparison, host targeting antivirals (htas) target host factors for antiviral treatment. since host proteins are probably broadly required for various viral infections, htas are not only perceived, but also demonstrated to exhibit broad‐spectrum antiviral activities. in addition, host proteins are not under the genetic control of viral genome, and hence htas possess much higher genetic barrier to drug resistance as compared with daas. in recent years, much progress has been made to the development of htas with the approval of chemokine receptor type 5 antagonist maraviroc for human immunodeficiency virus treatment and more in the pipeline for other viral infections. in this review, we summarize various host proteins as antiviral targets from a medicinal chemistry prospective. challenges and issues associated with htas are also discussed. viral infections still represent a major global public health problem. although hundreds of viruses are known to be pathogenic, only less than 10 of them can be treated in clinic with available antiviral drugs. for some highly pathogenic virus such as zika (zikv), ebola (ebov), severe acute respiratory syndrome (sars), as well as many others, there is still no effective drugs on market against them. most of the antiviral drugs approved are designed to target viral proteins to inhibit viral infections, and they are named as direct-acting antiviral agents (daas). 1 daas have been demonstrated to be very successful in clinic to combat viral infections, and are generally considered as very safe for human use because most of the targeted viral proteins have no human homologs. however, viral polymerases, one of the hottest targets for antiviral design, do share some structural similarity with their human counterparts, especially at the active sites, which is the major underlying reason for the toxicity observed with nucleoside-based antivirals. in addition, viral proteins do not normally share structural similarity among different species or even genotypes of virus, and hence one antiviral agent targeting a specific viral protein can hardly impart the same inhibitory effects against the other virus. consequently, the antiviral drugs available on market can barely be employed to treat newly emerging virus, and the magnitude of this problem is further exacerbated by the lack of broad-spectrum antiviral drugs on market. the other inherent limitation of daas is that daas, in most cases, have low genetic barrier to drug resistance because they act directly on viral proteins, and the resulted selective pressure will facilitate the mutations of virus during replication, which will make the virus refractory to the treatment of daas. 2 altogether, there still exist great unmet needs for the treatment of viral infections, and new antivirals with broad-spectrum antiviral activities as well as new mechanism of action are highly demanded. it is widely known that viruses are unable to complete their replication without the help of the host. they will hijack various host proteins or pathways throughout their replication cycle to facilitate their replication, and hence the inhibition or knockdown of such host proteins will block viral replication. therefore, host proteins as such are potential antiviral targets for drug development. the host targeting antivirals (htas) are complementary to the daas, and are superior to daas from several aspects. chiefly, a specific host protein might play crucial roles in the replication of several types of viruses, and thereby its inhibition will yield broad-spectrum antiviral activities. htas as such are likely to be effective against newly emerging virus, because they may also leverage on the same host protein for replication. for example, heat shock protein 90 (hsp90), a host chaperone responsible for the folding, assembly, and maturation of endogenous proteins, is also found to be crucial for maturation of many viral proteins, and therefore, hsp90 inhibitors are endowed with broadspectrum antiviral activities. 3 altogether, targeting host factors is a very promising strategy with possibility to address the critical challenges faced with the daas. in this review, we summarize the recent advances made in htas from a medicinal chemistry standpoint, and the host targets are generally classified into three different categories based on the development stage of their corresponding inhibitors/modulators, namely the ones which reached food and drug administration (fda) approval, that have entered clinical trials and those in preclinical studies. the focus is mainly placed on the smallmolecule based htas, and the antibody or small interfering rna (sirna)-based strategy will not be included. for several well-studied host targets with quite a lot of known inhibitors, because each one can stand alone as an independent review, and this review is not intended to be comprehensive, so for such targets, only a selection of representative inhibitors will be presented to discuss related issues. it should be noted that chemokine receptor type 5 (ccr5) antagonist but not chemokine receptor type 4 (cxcr4) antagonist has reached fda approval. however, cxcr4 is discussed in this section because it is closely related to ccr5. the chemokine receptors cxcr4 and ccr5 belong to the superfamily of g-protein-coupled receptors with seven transmembrane domains, and both are involved in the entrance of human immunodeficiency virus (hiv) virus. 4 the glycoprotein 120 (gp120) on the surface of hiv envelop will bind to the cd4 receptor on the surface of t lymphocytes, leading to the conformation change of gp120. the gp120 will then bind to coreceptor cxcr4, ccr5, or both to facilitate the entrance of hiv virus. 5 hiv-1 virus can thus be broadly classified into three types based on the coreceptor tropism, namely the x4-, r5-, and dual-tropic viruses. the feasibility of targeting cxcr4 or ccr5 for hiv therapy is supported by the fact that ccr5 mutations confer the host with resistance to hiv infection. 6 ample studies have demonstrated that cxcr4 antagonists can block hiv-1 infection through cxcr4. 7, 8 generally, cxcr4 antagonist can be divided into two types: peptide and nonpeptide based. the peptide-based antagonists were designed using the natural chemokines of cxcr4 as template, such as stromal cell-derived factor 1α (sdf-1α) and viral macrophage inflammatory protein ii (vmip-ii), both of which can bind to and then activate cxcr4 to initiate downstream signaling pathway. for example, peptide v1 (1; figure 1 ) was derived from the first 1 to 21 residues of vmip-ii, and it can block the interaction between cxcr4 and hiv-1, and thereby inhibit the entry of both x4-and dual-tropic hiv-1 virus. interestingly, replacement of all the residues in 1 with all d-amino acid lead to much more potent antiviral activities. 9 due to the inherent limitation with the peptide-based antagonist, several nonpeptide based antagonists have been developed. amd3100 (2) was discovered by random screening, and it inhibited hiv-1 infection by disrupting the interaction between cxcr4 and hiv-1. 10 encouragingly, it showed antiviral efficacy in x4 hiv-1-infected patients in a phase ii clinical trial. however, compound 2 lacks oral bioavailability due to its high positive charge, and cardiotoxicity was also reported in the clinical trial. 11 the structural optimization identified amd3465 (3) with just one macrocyclic structure retained. this compound retained all the biological profiles of 2, and it showed improved antiviral activity (ic 50 : 1-10 nm). it also inhibited the replication of drug-resistant virus strain (zidovudine). 12 amd070 (4), a noncyclam derivative, was discovered by anormed as a new cxcr4 antagonist. it showed much improved oral bioavailability (rat: 20%; dog: 80%), but hepatotoxicity was observed in preclinical studies. 13 further structural optimization led to gsk812397 (5) without the basic sidechain. it showed potent anti-hiv-1 activity with an ic 50 value of 1.5 nm, and it did not show any detectable toxicity (>1000 nm) in in vitro assay. in addition, 5 also presented acceptable pharmacokinetic profiles with the oral bioavailability in the range between 10% and 21% among different species. 14 further structural modification with constraining conformation (6) and/or opening up the tetrahydroquinoline ring (7) resulted in a series of new derivatives with retained anti-hiv activities. 15, 16 however, none of these compounds have been advanced into clinical trials. although compound 2, also known as plerixafor, was approved by the fda for autologous transplantation in patients with non-hodgkin's lymphoma or multiple myeloma, 17 no cxcr4 antagonist has been approved for the treatment of hiv infections. the underlying reasons are at least twofold: one major concern with the cxcr4 antagonist is the toxicity issue, especially that cxcr4 18, 19 or sdf-1α 20 knockout mice die prenatally with multiple neurological, cardiac/vascular, and hematopoietic defects. such potential adverse outcomes are exacerbated in the case of hiv treatment, wherein a life-long treatment is required; the other minor issue with these cxcr4 antagonists is the lack of oral bioavailability. iv injection is not practical in the case of hiv treatment, because this will definitely hurt the compliance of the patients. in comparison, the development of ccr5 antagonists for hiv treatment is much more successful. one ccr5 antagonist maraviroc (8; figure 2 ) has been approved by fda as hiv entry inhibitor in 2007. 21 with a long plasma half-life (15-23 hours), 22 8 is administrated orally once daily. the clinical data showed that 8 is well tolerated in patients and it can significantly repress the viral load in r5-infected haart-treatment experienced patients. moreover, it is active against 200 clinically derived hiv-1 envelope-recombinant pseudoviruses, with 100 of them being resistant to existing drug classes, demonstrating the merits of htas. 23 in the clinic, it is primarily recommended for the patients infected with r5-tropic hiv virus, and a tropism test (although not approved by fda) is highly required to determine the viral tropism population within the patient before the treatment to ensure treatment success. the first generation of the test (known as trofile) failed to distinguish r5 from r5/x4 mixed tropism, resulting in significant drug failure in clinic in r5/x4-infected patients. 24 therefore, 8 is just designated as a backup regimen, despite that a new reliable and cost-efficient tropism test is available in clinic now. interestingly, the combination of 8 with raltegravir/tenofovir/emtricitabine led to faster reduction of 2-long term repeat (2-ltr + ) newly infected cells and recovery of cd4 + t-cell counts after 48 weeks of treatment, 25 and 8 in combination with reverse transcriptase inhibitors is being tested in clinic for preexposure prophylaxis. 26 however, the use of 8 is accompanied with severe side effects, and in some cases even life-threatening conditions such as hepatotoxicity and heart attack were observed. to mitigate these limitations, the second generation of ccr5 antagonists have been developed. two clinical candidates vicriviroc (9) 27 and aplaviroc (10) 28 have made to phase ii trials. however, the studies were halted f i g u r e 2 the chemical structures of representative chemokine receptor type 5 antagonists due to insufficient efficacy and observed hepatotoxicity for 9 and 10, respectively. much of the subsequent medicinal chemistry effort is devoted to the structural modification toward 8 to 10, aiming to improve either efficacy and/or adme profiles. for example, pfizer discovered compound 8's structural analogue pf-232798 (11), 29 which showed not only potent anti-hiv activity (ic 50 : 2.0 nm) and moderate herg inhibition (ic 50 : 12 μm), but also superior oral bioavailability as compared to 8. in addition, compound 11 is also active against maraviroc-resistant hiv-1 isolate strain cc185. the data from phase-ii trials demonstrated its superior safety in patients with no adverse effects observed at a dosage up to 250 mg. however, no further data were disclosed after that. 30 gsk 163929 (12) with a 4,4-disubstituted piperidine scaffold exhibited potent anti-hiv activity and excellent pharmacokinetics (pk) profiles, but it did not progress to clinic trials due to toxicity concerns. 31 the other two representative new scaffolds derived from compound 8 are monocyclic piperidine amides and cyclic and acyclic urea-piperidines. some representative candidate compounds from these two series are shown in figure 2 . among these compounds, only tak-220 (13) was progressed to phase-i clinical trials. 32 however, no further update about the status of compound 13 has been reported. inspired by the scaffold of 9 and 10, several new ccr5 antagonists have been devised as depicted in figure 2 . 33 the most promising antagonist among this series is the one developed by incyte based on the structure of 10. incb9471 (16) showed potent antiviral activity against r5 hiv-1 strains at ic 50 values in sub-nanomolar range. in addition, it presented excellent pk profiles with oral bioavailability being 100% and 95% in rat and dog, respectively. 34 as such, 16 has been advanced to phase ii trials, and the data showed that it was well-tolerated in humans. however, its clinical studies were halted due to business issues. besides the chemotypes described above, ccr5 antagonists with miscellaneous scaffolds have also been discovered, yet none of them have made to clinical trials. 35 targeting cxcr4 or ccr5 alone for hiv treatment possesses several downsides. chiefly, a tropism test is required before the treatment, and tropism shifts are frequently observed in both treatment-experienced and treatment-naive hiv-infected patients. therefore, a dual antagonist against both cxcr4 and ccr5 would be perfect to mitigate these limitations. the first dual-tropic inhibitor amd3451 (17; figure 3 ) with n-pyridinylmethyl cyclam scaffold was discovered in 2004. it was moderately active against not only x4-and r5-tropic hiv strains but also dual -tropic hiv strains with ic 50 in the range of 1 to 30 μm. 36 although 17 is not potent enough for further development, it established the proof of concept of targeting cxcr4 and ccr5 simultaneously for hiv-1 treatment. ribavirin (18; figure 4 ) is an approved antiviral drug used in clinic to treat respiratory syncytial virus (rsv), hepatitis c virus (hcv) infection and viral hemorrhagic fever. although its specific antiviral mechanism of action f i g u r e 3 the chemical structure of a dual-tropic inhibitor amd3451 the chemical structures of 18 and 19 ji and li | 5 remains largely uncertain, it is widely accepted that it elicits its antiviral efficacy via modulating host pathways. for example, 18 showed moderate inhibitory potency against inosine-5′-monophosphate dehydrogenase (impdh) with a k i value of around 250 nm, which is believed to be highly involved in the replication of various viruses (see below). in addition, several other mechanism of actions have also been proposed and evidenced: (a) direct inhibition of rna polymerase by converting 18 to its triphosphate form to competitively bind to the nucleotides binding site in rna polymerase 37, 38 ; (b) 18 can act as a mutagen by inserting into the viral rna to push the virus beyond the threshold of error catastrophe 39, 40 ; (c) 18 shows immunomodulatory effect of shifting a th2 response in favor of a th1 phenotype, which helps to clear virus infections. 41 although the combination of 18 and interferon 2α (ifn-2α) used to be the soc for hcv treatment, it is notorious for several severe side effects. one major adverse effect associated with the use of 18 is hemolytic anemia. 42 to alleviate this unwanted effect, taribavirin (19) was developed as the prodrug of 18. ideally, 19 can be metabolized to 18 mainly in the liver to target hcv-infected hepatocytes, and hence the distribution of 18 within red blood cells will be significantly decreased, and thereby the development of hemolytic anemia will be subsequently eliminated. indeed, the clinical data from phase iii trials revealed that patients receiving 19 (fixed dosage 600 mg, bid) and ifn showed significantly lower rates of anemia as compared to the ones in 18 (1000-1200 mg) and ifn group (5.3% vs 24%). 43 however, the sustained virologic response (svr) rates for 19 and 18 group are 38% and 52%, respectively, failing to demonstrate the noninferiority end point for efficacy. the viser-2 trials also failed to meet the noninferiority end points. 44 with the approval of several daas such as sofosbuvir and ledipasvir, the treatment of hcv in clinic has been significantly revolutionized, and several both ribavirin (rbv)-and ifn-free regimens have been approved with better efficacy and safety profiles as compared to the old soc. initially, it was expected that the hcv treatment would not benefit much from the combination of rbv with other daas due to the safety concern associated with rbv. however, very interestingly, it has been found recently that rbv remained an indispensable component for the optimal treatment for some difficult-to-cure subgroups of hcv patients. 45 for example, in a phase 3 c-edge studies, the combination of rbv with elbasvir/grazoprevir achieved much higher svr ( [3/5] , respectively) as compared to the group without rbv in patients infected with genotype 4 hcv. 46 in addition, rbv increased the barrier to resistance, especially in patients receiving daas with low barriers to resistance. importantly, the combination of rbv with daas showed much improved safety and tolerability as compared with the combination with ifn, and the frequency and severity of anemia is significantly reduced with an adverse effect-induced discontinuation rate of less than 3%. 45 nevertheless, the last generations hcv daas (including glecaprevir/pibrentasvir, sofosbuvir/velpatasvir, sofosbuvir/velpatasvir/ voxilaprevir) are highly effective in most cases without any need to use rbv. cyclophilins are a family of cellular peptidyl-prolyl cis-trans isomerases (ppiase) and are involved in many cellular processes. the important roles cyclophilin a (cypa) plays during hcv replication were found by an unexpected clinical finding. cyclosporin a (csa; 20; figure 5 ), which shows high binding affinity to cypa, is an approved immunosuppressive drug. in a clinical trial for csa's therapeutic potential against hepatitis-associated inflammation, it was unexpectedly found that a significantly more potent antiviral response was observed in the combination group of csa and ifn-α2b as compared with the ifn-α2b alone group. 47 the function of cypa in the replication of hcv was subsequently confirmed in vitro. 48 the main cellular function of cyps is to convert the conformation (trans-to cis-form) of prolines in the protein, which is essential for trafficking of many proteins and forming protein complexes, and these functions are also indispensable for the replication of hcv. therefore, it is anticipated that the cypa inhibitors will show anti-hcv activity. although 20 is an approved drug, its main indication is suppressing immunoreaction after organ transplantation, which is an unwanted "side-effect" for antivirals. fortunately, the immnosuppressive effect of 20 is attributed to the inhibition of calcineurin (cn) but not cypa, and hence it is feasible to eliminate the immnosuppressive effect while retaining the anti-hcv activity by making new csa analogues. nim811 (21) is one of such analogues with a methyl-isoleucine at position 4 of csa. it showed anti-hcv activity in vitro with an ic 50 value of 0.12 μm, whereas its immunosuppressive effect was completely eliminated. 49 the phase i clinical trial showed that 21 is well-tolerated with no obvious adverse effects observed. however, in a subsequent double-blind, placebo-controlled study, the monotherapy with 21 failed to yield significant viral load reduction in genotype 1 hcv-infected patients. the underlying reason is attributed to a relatively low trough concentration (0.47 μm) at a dosage of 600 mg (bid), which is lower than the ic 50 value (1.5 μm) of 21 against hcv in the presence of serum, and the dosage elevation did not result in proportional exposure. 50 therefore, the clinical trial with 21 is not continued. alisporivir (22) is a more potent nonimmunosuppressive csa analogue with an anti-hcv ic 50 values of 0.045 and 0.33 μm in the absence and presence of serum, respectively. in addition, it is much more difficult to develop resistance against 22 both in vitro and in patients, as compared to other daas. 51 in phase ii trial, 22 was studied in combination with rbv as an ifn-free regimen in genotype 2 and 3 patients, and about half of the patients receiving 22 (800 mg, qd) plus rbv, and one-third of those receiving 22 only (1000 mg qd) were cured with a svr. 52 however, unfortunately, in a pivotal phase iii trial designed to study the combination of 22 with rbv and peg-ifn-α2a in treatment-naive, genotype 1 hcv patients, six cases of severe pancreatitis along with one death were reported, and the clinical trial with 22 was then put on hold. it is worth noting that all the severe side effects were observed in the triple-therapy aim including rbv and peg-ifn-α2a, both of which are notorious for their adverse effects, while the monotherapy with 22 or the ifn-free regimen did not result in the same side effects. 53 to mitigate the observed side effects with 21 and 22, the third generation of csa derivatives have been potent anti-hcv activity in vitro with an ec 50 and ec 90 value of 0.1 and 0.3 μm, respectively, and exhibited synergistic effect with ifn-α and additive effect with rbv. it also showed low potential for drug-drug interaction with no obvious induction on the major cytochrome p450 enzymes 1a2, 2b6, and 3a4. in addition, 23 also showed acceptable pk profiles with an oral bioavailability of around 20% in rat and monkey. 54 as such, 23 has been advanced into clinical trials. in patients with genotype 1 hcv infection, 900 mg/day of 23 achieved a decline in plasma viremia by 2.2log 10 after 15 days. 55 it is interesting to note that 23 showed totally different side effects from those of 21 and 22, indicating that the adverse effects may not be associated with the inhibition of cyps, but are likely resulted from the off-target effects of individual inhibitors. non-csa based cyps inhibitors have also been discovered. sanlifehins (sfa) including sanlifehin a (24), b (25), c (26) , and d (27) are a class of natural occurring polyketides isolated from the soil bacterium streptomyces sp. strain a92-308110. sfas were identified as cyps inhibitors with stronger potency as compared to csa derivatives, particular 25, of which the inhibitory potency against all cyps was 30-to 50-fold more potent than 20. it also showed much more potent antiviral activity in vitro with an ec 50 value of 70 nm against hcv genotype 1b. interestingly, albeit slightly less potent as compared to against the wild type, 24, 26, and 27 retained inhibitory effect against csa-resistant huh 9 to 13 subgenomic replicon with ec 50 values ranging from 3.3 to 6.8 μm. 56 however, the pk studies revealed that sfa suffered from poor water solubility (<25 μm) and poor oral bioavailability (<4%). moreover, sfa possessed undesirable immunosuppressive activity via an unknown mechanism. 57 structural modifications have been made to 24, and it was revealed that only the macrocyclic moiety was essential for the cyps inhibition, and modification on the sidechain had little effect on the binding affinity. 58 removal of the spirolactam moiety on the sidechain of 24 only led to the loss of immunosuppressive activity but not the cyps inhibition. such structure-activity relationships are very important for further optimization of sfa as anti-hcv agents ( figure 6 ). both csa and sfa derivatives are macrocyclic molecules with large molecular size, and as such both suffered from some limitations, including poor cell membrane permeability, high risk of drug-drug interactions and off-target toxicity, and synthetic inaccessibility for structural optimization and manufacturing. in 2016, a new family of nonpeptide based small-molecule cyps inhibitors have been designed using fragment-based strategy. 59 the crystal structure of cypa indicated that its ppiase catalytic site consisted of hydrophobic, aromatic, and polar residues, next to the catalytic site is a deep pocket called gatekeeper site, which might contribute to the substrate binding specificity ( figure 7) . a total of 34 409 fragments were docked into these two sites, and several fragments were identified to bind to these two sites separately. eventually, fragment 28 and 29 from each binding site were it has been well-established that cyps are involved in a broad range of viral infections, including hiv-1, 60 influenza virus, 61 hbv, 62 sars coronavirus, 63 human cytomegalovirus (hcmv), 64 papillomavirus, 65 and nidovirus, 66 among others. therefore, it can be envisioned that cyps inhibitors should exhibit broad-spectrum antiviral activity against these viruses. indeed, cyps inhibitors were reported to show broad-spectrum antiviral activities. 67, 68 consequently, the development of cyps inhibitors could benefit the treatment of a variety of viral infections, possibly including the newly emerging unidentified viral infections. it is widely known that virus will hijack a wide range of host factors to facilitate its replication. meanwhile, the host has also evolved an innate immune system to counteract viral infections. compounds with the capacity to mediate the host antiviral pathways are expected to confer broad-spectrum antiviral activity, and nitazoxanide (32; figure 9 ) is such a compound that regulates several host antiviral pathways to convey broad-spectrum antiviral profiles. 32 is originally an fda approved drug for the treatment of diarrhea caused by cryptosporidium parvum and gastrointestinal disorders as the most frequently observed side effects. it also showed no effect on cardiac repolarization in a clinical trial for cardiac safety. 69 in recent studies, 32 was revealed as a promising antiviral agent against a wide range of pathogenic viruses including influenza virus, 70 hbv, 71 hcv, 71 ebov, 72 denv, 73 jev, 74 hiv, 75 and zikv, 76 among others with ic 50 values ranging from 0.06 to 1.0 μg/ml 77, 78 its mechanism studies revealed that multiple host antiviral pathways were involved, and as such 32 is widely known as a polypharmacology antiviral agent. 32 activated protein kinase r (pkr), which plays vital roles in innate immune system. the activation of pkr leads to the phosphorylation of eukaryotic initiation factor 2α, an important host restriction factor against viral replications. 79 hbv viral protein hbx was found to interact with host protein damage-specific dna-binding protein 1 (ddb1) to promote transcription of covalently closed circular dna (cccdna) and degradation of a host restriction factor chromosomes 5/6 (smc5/6). 80 32 was reported to disrupt the interaction between hbx and ddb1 to block the transcription from cccdna. 81 in addition, 32 was also able to activate cellular antiviral response and induce the expression of a subset of ifn-stimulated genes, especially interferon regulatory factor 1, 82 which is known to block the replication of a wide range of viruses. 83 other host targeting mechanisms are also involved in the broad-spectrum antiviral profiles of 32. 72 since 32 has been safely used in clinic for many years, it has been advanced into clinical trials for the treatment of several viral infections. the most advanced one is the treatment of acute uncomplicated influenza with 32. in a phase 2b/3 trial, significant reductions in tci50 viral titer and alleviation in symptoms were observed in 32 (600 mg, twice daily) group as compared to the placebo. no resistance was identified in the influenza virus collected from the patients receiving 32, and no adverse effect on humoral immune response was reported. 84 a large global phase 3 trial is being conducted. several clinical trials of 32 for the treatment of other viral infections are also ongoing. in a pilot clinical trial of 32 for the treatment of chronic hbv, the serum hbv dna of eight of nine patients became undetectable after 4 to 20 weeks of treatment with 32, and more importantly, three out of nine subjects became hbeag negative, which is a rare outcome for current standard of care. this proof-of-concept study presented 32 as a very promising drug to achieve a hbv cure. 85 32 was also tested in clinical trials against hcv infections, and it showed very pronounced efficacy either as monotherapy or in combination with ifn-α and rbv. for example, administration of 500 mg 32 twice a day for 48 weeks with 180 μg of ifn-α once weekly from week 13 to 48 achieved a svr of 79% in patients infected with chronic hepatitis c (chc) genotype 4, as compared to 50% for ifn-α and rbv. however, the development of 32 as an anti-hcv drug was not further pursued due to the approval of several new daas, despite that 32 has much higher genetic barrier to resistance as compared to daas. interestingly, 32 is considered as a prodrug because it is rapidly hydrolyzed to tizoxanide (33) in the plasma with a half-life of only 6 minutes. compound 33 showed broad-spectrum antiviral activity against a panel of viruses both in vitro and in vivo. to obtain new candidate compounds with improved potency and safety profiles, structural modifications were made to 32 primarily on the phenyl ring a and the thiazole moiety. it was revealed that electron-withdrawing group such as nitro and chloro group at c-5 position was favorable for the antiviral activity, and replacement of the thiazole ring with phenyl ring retained the activity. the structural optimization led to the f i g u r e 9 the chemical structures of nitazoxanide analogues identification of a candidate compound rm-5038 (34) with a chloro group at the c-5 position, which showed comparable activity to that of 32 with ec 50 values in the submicromolar range. 34 was considered to be superior as compared to 32 due to the replacement of the potential cytotoxicity nitro group in 32. 32 also suffers from poor systemic exposure after oral administration, and it is primarily biodistributed in the gastric intestinal tract, and excreted via urine and faeces. for antiviral therapy, it is preferred to increase the systemic exposure of the active drug. for such purpose, amino acid based prodrugs for 33 were devised, 86 and they showed much improved aqueous solubility as compared to the parent drug. as such, prodrug 35 exhibited significantly improved pk profiles with oral bioavailability being around 20% in rats, as compared to 2.8% and 0 for 32 and 33, respectively. in addition, 35 also showed preferable safety profiles in laboratory animals, with a no observed adverse effect level being 25 mg/kg/day for a consecutive 28 days in beagle dogs, and it did not present any obvious toxicity in rats after a single oral dosage of 300 mg/kg, and only minor toxicity on central nervous system and respiratory system was observed at a single dosage of 1000 mg/kg. all these pk results present 35 as a very promising candidate compound for further development, and it is now undergoing phase i clinical trial. α-glucosidase is an enzyme removing glucose units from n-linked glycans attached to a nascent glycoprotein, which is essential for proper folding and functions of many glycoproteins. most viral envelope glycoproteins contain n-linked glycans, and α-glucosidase (especially endoplasmic reticulum [er] α-glucosidase) is highly involved in their proper folding and maturation. therefore, the inhibition of er α-glucosidase would yield broad-spectrum antiviral activity. indeed, er α-glucosidase inhibitors showed pronounced antiviral activity against a series of enveloped viruses both in vitro and in vivo including hiv, 87 hcv, 88 human coronavirus, 89 influenza a virus, 90 and denv. 91 although α-glucosidase is critical in the proper folding of viral envelop glycoproteins, it is less important to host cells, and the host cells can well-tolerate the complete shutdown of these er α-glucosidase. 92 moreover, several glucosidase inhibitors are being used in clinic for treating type ii diabetes and gaucher disease. consequently, targeting α-glucosidase for antiviral therapy would not raise a red flag on toxicity issues. to date, a wide range of α-glucosidase inhibitors have been discovered, 93 among which the iminosugars are the most promising inhibitors as antiviral agents. it is widely accepted that the antiviral profiles of iminosugars is attributed to the inhibition of er α-glucosidases (i and ii). the natural occurring iminosugars 1-deoxynojirimycin (dnj; 36; figure 10 ) and castanospermine (37; figure 10 ) has been used as starting points for further modifications. the modifications to 36 were primarily made at the amino position by introducing an alkyl chain. the n-butylated dnj 38 has been advanced to clinical trials for hiv treatment. in a phase ii study, although 38 showed the chemical structures of iminosugars ji and li | 11 some efficacy on viraemia, it failed to maintain a serum concentration needed to inhibit hiv replication in vitro, 87 so the clinical trials of 38 for hiv treatment have been discontinued. the other dnj analogue mon-dnj (39) also showed broad-spectrum antiviral activity, and it has recently been tested in human against denv infection. in the phase i trial, 39 has been demonstrated to be well-tolerated in human with no severe side effects observed after a single dosage of 1000 mg, and the pk data indicated a low interindividual variability and good linearity over a wide range of dosage (nct02061358). in a recent study, 39 has been tested in a proof-of-concept non-human primate trial against ebov infections. however, 39 failed to yield any survival benefit to macaques infected with ebov-makona, despite that 39 showed definitive antiviral activity against ebov in vitro. 94 celgosivir (40), a prodrug of 37, has also been tested in human against dengue fever. in a phase ib, placebo-controlled study, 40 failed to meet the primary end point in patients infected with uncomplicated dengue fever. however, in a mouse model study, it was confirmed that the dosing regime was crucial for the efficacy, 95 and therefore, a four-time daily dosing regime is planned for a phase ii trial (nct02569827). 40 was also studied for the treatment of patients infected with genotype i hcv. in a phase ii trial, 40 only showed a moderate antiviral effect as a monotherapy, but exhibited synergistic effects with ifn-based therapies. 88 however, further development of 40 as anti-hcv agents were discontinued due to inferior efficacy as compared with other approved daas. although the results from the clinical trials of 39 and 40 demonstrated the safety of iminosugars, they are all limited by poor pk profiles and insufficient efficacy. to address these limitations, further structural modifications were made to 36 by introducing various substituted alkyl sidechain, and several analogues were identified as potent antiviral activity against bovine viral diarrhea virus (bvdv), tacaribe virus, and denv with ec 50 values in the submicromolar range, which is hundreds fold more potent than 38. in addition, these compounds showed much superior pk profiles, especially compound 42, which had an oral bioavailability of 94%. most importantly, they all demonstrated significant protective effects in both ebov and bvdv infected mice models, and the in vivo glycan analysis also indicated significant er α-glucosidase suppression in the compounds treatment group. 96 it should be noted that the inhibition of other glucosidase such as intestinal glycosidase will lead to unwanted side effects. to avoid such side effects, a prodrug of compound 42 was designed by masking the free hydroxyl group by acylation. the tetrabutyrate prodrug 43 exhibited preferred stability toward simulated gastric and intestinal fluid, and yet was readily converted to the parent drug in the plasma and liver of mice. in a cell-based assay, compound 43 showed inhibitory activity against ebov with ec 50 value of 15.6 μm, which is slightly less potent as compared with the parent compound (4.1 μm). compound 43 also showed much improved overall drug exposure after either oral or intravenous administration to mice. 97 another strategy to alleviate the side effects of iminosugars is to design more specific inhibitors toward er α-glucosidase. in 2016, the crystal structure of er α-glucosidase ii in complex with 36 and 39 has been successfully resolved, which provided significant insights into the interactions between inhibitors and the active site of the enzyme, laying a firm foundation for the structure-based drug design of more specific inhibitors. 98 3.4 | inosine-5′-monophosphate dehydrogenase impdh is an enzyme catalyzing the conversion of inosine monophosphate (imp) to xanthosine monophosphate, which is a critical step in the de novo biosynthesis of guanine nucleotides. 99 inhibition of impdh will lead to the decrease in the intracellular guanosine-5'-triphosphate (gtp) level, which will disrupt the gene synthesis of both dna and rna virus, and thereby inhibit viral replication. therefore, impdh inhibitors are expected to show broadspectrum antiviral activities. indeed, impdh inhibitors exhibited broad-spectrum antiviral activities against both dna and rna virus in vitro. 100 the approved non-nucleoside-based impdh inhibitors include mycophenolic acid (44) , its prodrug mycophenolate mofetil (45) and mizoribine (46; figure 11 ), and all of them were approved for prevention of organ transplant rejection, but not for antiviral therapy, nevertheless, all these inhibitors were reported to inhibit the replication of a wide range of virus in vitro and even in vivo. for example, 44, an uncompetitive impdh inhibitor with respect to imp and nad+, is active against flaviviruses, paramyxoviruses, orthopoxviruses, avian reoviruses, and denvs with ec 50 values in low micromolar range, and its antiviral potency is closely correlated to the intracellular gtp levels, indicating the involvement of impdh inhibition in its antiviral mechanism. 101,102 however, one major limitation with the application of 44 is the presence of a phenolic hydroxyl group, which is prone to glycosylation for excretion, and thereby limits its efficacy. to overcome this limitation, a series of phenyloxazoles were developed by the vertex group and others. the representative compound from this series is vx-497 (47) , which shows high affinity to impdh with a k i value of 10 nm. it shows potent antiviral activity against a wide range of dna and rna virus including hbv, hcmv, rsv, and murine encephalomyocarditis virus, hcv, zikv, and ebov, 103 among others with ic 50 values ranging from low micromolar to submicromolar levels, and its antiviral activity can be reversed by the addition of guanosine, indicating that its antiviral mechanism is resulted from impdh inhibition. some other vx-497 derivatives also exhibited similar antiviral profiles. 104, 105 in light of the successful application of rbv in clinic for hcv treatment, as a more potent impdh inhibitor and antiviral agent as compared to rbv, 100 other mechanisms are involved in the antiviral action of rbv. in addition, rbv has to be used in combination with ifn-alpha or other anti-hcv agents in clinic, and the monotherapy with rbv failed to yield any antiviral efficacy in patients. 108 although quite a few other impdh inhibitors are under development, they are all intended for other indications. consequently, special attentions should be paid to this issue in pursuit of impdh inhibitors as antiviral agents. kinase represents a huge family of host proteins, and have been successfully targeted by a myriad of smallmolecule inhibitors for the treatment of cancers and inflammatory diseases in clinic. mounting evidence have shown that viruses hijack a variety of human kinases throughout the entire viral life cycle to facilitate their replications, and some kinases are broadly required. [109] [110] [111] therefore, it can be anticipated that the inhibition of such kinases would lead to the disruption of a broad spectrum of viral replications. since tremendous kinase inhibitors have been approved in clinic for treating cancer and inflammatory diseases, and more are under development in the pipelines, increasing efforts have been devoted to repurposing approved kinase inhibitors as broad-spectrum antiviral agents. in this section, only one type of kinases along with their respective inhibitors are picked for the discussion of related issues, and the selection of these examples is not based on the "importance" of the articles, but rather whether there are appropriate issues to discuss. the numb-associated kinases (naks) constitute a diverse family of ser/thr kinases with a broad range of cellular functions. naks have been found to play key roles in a diverse range of human diseases ranging from parkinson's and prostate cancer to viral infections. host kinases adaptor protein 2 (ap2)-associated protein kinase 1 (aak1) and cyclin g-associated kinase (gak), two kinases from this family, are found to regulate intracellular trafficking of a variety of viruses including denv and ebov. two approved kinase inhibitors sunitinib (49) and erlotinib (50) were found to potently inhibit aak1 and gak, respectively, and both are reported to exhibit potent broad-spectrum antiviral activity in vitro. in addition, the combination of 49 and 50 showed very pronounced protective effects against morbidity and mortality in denv and ebov infection mouse models. 112 a cocktail treatment containing 49 and 50 against ebov infection is being investigated in clinic trial, but no clinical data have been disclosed yet (nct02380625). gak and aak1 were also reported to regulate the binding of ap2m1 to hcv core protein, which is essential to the hcv assembly. inhibitors of gak and aak1 disrupted the interaction between ap2m1 and core, and thus inhibited hcv replication with ec 50 values ranging from 0.15 to 1.8 μm. 113 although 49 and 50 exhibited potent inhibitory activity against gak and aak1, both of which are known as pankinase inhibitors, and thus raise off-target toxicity concerns when used as antiviral agents. in 2015, herdewijn et al developed a series of highly selective inhibitors against gak. the crystal structure of gak in complex with one of these inhibitors has been resolved, showing that inhibitors as such behaved as classic type i adenosine triphosphate (atp)-competitive kinase inhibitors. these compounds showed pronounced anti-hcv activity in vitro with ec 50 in the low micromolar range. 114 further structure optimization lead to the identification of compound 51 with a k d value of 8.9 nm, which is endowed with broad-spectrum antiviral activity against a panel of viruses including denv, ebov, chikv with ec 50 values in the low micromolar range. the mechanism of action studies confirmed that the inhibition of gak is an important target underlying the broad-spectrum antiviral activity of compound 51. 115, 116 the other chemotype of gak inhibitor is quin(az)oline, 117 ,118 yet their antiviral activity was not probed. many other kinases are also involved and play essential roles in different life cycle of various viruses, and their corresponding kinase inhibitors show broad-spectrum antiviral activities against a wide range of viruses both in vitro and in vivo. [109] [110] [111] since host kinase is not under the genetic control of virus, kinase inhibitors for antiviral treatment have much higher genetic barrier to resistance. for example, denv developed resistance against the viral ns4b inhibitor sdm25n after eight passages, yet it remained sensitive to the treatment of 49 and 50 under the same conditions. 119 despite with advantages of broad-spectrum antiviral profiles and higher genetic barrier to resistance, there still remain concerns or limitations for repurposing kinase inhibitors as antiviral agents. chiefly, toxicity is the major concern associated with kinase inhibitor, because most of kinases (if not all) play essential roles to regulate the cellular functions. however, it should be noted that, in most cases, the duration of antiviral therapy can be as short as several weeks or even several days, and hence the incidence of severe side effects can be significantly decreased. in addition, the potential toxicity can be further diminished by operating in a well-defined therapeutic window. since most of the kinase inhibitors are designed to target the atp binding site, which is highly conserved among different kinase families, so nearly all kinase inhibitors possess cross inhibitory activity against other kinases, which could be problematic. first, targeting multiple kinase may result in off-target side effects; second, it is challenging to understand the mechanism underlying the antiviral action of these inhibitors because multiple kinases are involved. however, one could also argue that pan-kinase inhibition is favorable for antiviral therapy in that the off-target kinase(s) may also contribute to the viral replication, and targeting multiple kinases simultaneously may result in not only synergetic antiviral activity but also high genetic barrier to resistance as well. altogether, targeting kinases represent a promising strategy for the development of antiviral agents, especially the ones with broad-spectrum antiviral profiles and high genetic barrier to resistance. sodium taurocholate cotransporting polypeptide (ntcp) is expressed on the hepatic basolateral membranes specifically and functions as a cotransporter for bile acids and sodium ions. recently, ntcp has been identified as hbv/hdv infection receptor via interacting with hbv large surface protein, and the silence of ntcp inhibited hbv and hdv infections. 120 therefore, the inhibition of ntcp would block the entry of hbv/hdv virus, and the subsequent formation of the persistent viral reservoir: cccdna will also be halted, which cannot be achieved by current therapy. 121 were also significantly declined in all myrb groups but not the control tdf group, 122 demonstrating superiority by targeting ntcp (figure 12 ). the other discovered ntcp inhibitors include fda approved drugs (ie, csa, 123 ezetimibe, and ritonavir, 124 etc), fasiglifam, 125 oxysterol, 126 vanitaracin a, 127 nti007, 128 and among others. 129 the inhibition of ntcp by these inhibitors normally will result in the loss of transporter function of ntcp, leading to the inhibition of bile acid uptake, which might cause unwanted adverse effects. interestingly, shimura et al 130 identified several csa f i g u r e 1 2 the chemical structures of a subset of gak and aak1 inhibitors. aaak1, ap2-associated protein kinase 1; gak, cyclin g-associated kinase derivatives with anti-hbv activity in vitro via direct interaction with ntcp to inhibit viral attachment. these compounds inhibited multiple hbv genotypes including one clinically relevant nucleoside analog-resistant hbv isolate. importantly, they did not compromise the transporter function of ntcp. two analogs scy446 (56) and scy450 (57; figure 13 ) also did not show meaningful inhibition against cn, and therefore, these two compounds did not show any unwanted immunosuppressive effects. taken together, these results showed that the inhibition of viral attachment via ntcp can be functionally separated from the bile acid uptake, and future efforts should be dedicated to new ntcp inhibitors with transporter function retained to eliminate unwanted adverse effects. farnesoid x receptor (fxr) belongs to the nuclear receptor superfamily and plays regulatory roles in the metabolism of bile acid, lipid, and glucose. recently, it was identified as a proviral host factor for hbv virus. 131 silence of fxr by small hairpin rna resulted in significant decrease in the levels of cccdna, pregenomic-and precore-rnas, secreted relaxed circular dna (rcdna), and hbsag by 40%-70%, and the same effect was observed with the treatment of a fxr agonist gw4064 (58; figure 14 ). importantly, in c3h/hen adult mice infected with a recombinant aav2/8-hbv vector, gw4064 (50 mg/kg/d) significantly suppressed the rcdna and hbsag titers (mean rcdna variation: −0.52 and −0.93log10; mean hbsag variation −0.89 vs −0.73log10, at day 21 and 28, respectively). 132 interestingly, 58 together with other fxr agonists such as way362450 (59), fexaramine (60), and chenodeoxycholic acid (61) also showed inhibitory effect against hcv replications in huh7.5 cells with ic 50 values ranging from 0.75 to 7.03 μm. the mechanism of action studies showed that compound 58's anti-hcv activity is fxr-dependent. it has been reported that fxr agonist downregulated the expression of srib, which is a coreceptor for hcv entrance. as expected, the treatment of 92 also dose-dependently decreased the level of sr-bi. in addition, 58 also presented synergistic anti-hcv activity with other approved daas, and it remained sensitive against other daa-resistant hcv mutations, suggesting the advantages of targeting host factors. 133 encouragingly, one specific fxr agonist eyp001 (structure not disclosed) has successfully entered clinical trial for hbv treatment, and the data from phase i trial showed that eyp001 is very well-tolerated in humans with no treatment-related discontinuations or severe side effects. the pk of eyp001 was linear up to 500 mg with t max and t 1/2 being 2.3-2.5 and 1.4-2.4 hours, respectively. 134 the phase ii trial of eyp001 is ongoing. f i g u r e 1 3 the chemical structures of a subset of sodium taurocholate cotransporting polypeptide inhibitors. the r group in 56 and 57 was not specified in the original paper 130 diacylglycerol acyltransferases (dgats) are the key enzymes catalyzing the biosynthesis of endogenous triglycerides, which are necessary for the biogenesis of lipid droplets in the liver. it has been reported that lipid droplets are the major site for hcv particle assembly and production, and dgats, especially dgat-1, play vital roles during hcv infection. dgat-1 forms a complex with nonstructural protein 5a (ns5a) and core protein to enhance the interaction of the latter two, and the trafficking of ns5a to lipid droplets is also highly dependent on dgat-1's activity. 135 the silence of dgat-1 significantly impaired hcv entry. 136 therefore, dgat-1 could serve as a viable host target for anti-hcv agents development. one specific dgat-1 inhibitor pradigastat (62; figure 15 ) developed by novartis significantly decreased the level of hcv rna in cell supernatant at a concentration of 10 μm, indicating that compound 62 inhibited the assembly or release of virions. since compound 62 is in clinical development for the treatment of dyslipidemia, and it has demonstrated convinced efficacy and safety profiles. 137 therefore, it was directly tested in patients with genotype 1 or 3 hcv infections for safety and efficacy. however, disappointedly, 14 days of treatment with compound 62 failed to afford any significant deduction in serum hcv rna levels in either gt1 or gt3 patients, and thus the trial was terminated. 138 since the pk studies showed that the predicted concentration of compound 62 in liver is approximately 10 to 20 μm based on the plasma concentration on day 8 and 14, which is much higher than the concentration needed for dgat-1 inhibition (ic 50 : 66 nm), so the lack of efficacy is not due to pk problem. the other possibility is that hcv virus hijacked other compensate pathway to facilitate the assembly and release while dgat-1 activity is inhibited in vivo, or dgat-1 plays a nonenzymatic roles during hcv replication, so the inhibition of its enzymatic activity would not yield any inhibitory activity against hcv replication. hsp90 is a highly conserved chaperone protein that assists the maturation of its clientele proteins. there are four the viral entry and intracellular trafficking stage, hsp90 is reported to be critical for the intracellular translocation of viral proteins as well as other host factors critical for viral replications. for example, hsp90 is required for the nuclear translocation of ebv and hsv-1 dna polymerase and 139,140 rna-dependent rna polymerase of influenza virus. 141 to facilitate viral gene expression, hsp90 also activate several host signaling pathways. for instance, hsp90 is upregulated to activate akt and nuclear factor κb for viral gene expression upon hcmv infection. 142 as a chaperone protein, hsp90 is indispensable for the maturation, accurate folding, and maintenance of stability of various proteins including viral proteins. for example, hsp90 facilitates the accurate folding of ns5a of hcv virus in the replication complex to promote viral replication. 143 it can also help to maintain the stability of various viral proteins, including polymerases of vsv, 144 chikv, 145 and rsv, 146 and ribonucleoprotein complex 147 to facilitate viral genome replications. in addition, hsp90 is also involved in the formation of viral capsid. hsp90 can maintain the stability of capsid precursor p1 protein of poliovirus, 147 and it can also increase affinity between core protein dimers to facilitate hbv capsid formation. 148 market for unknown reasons. 64 was shown to inhibit rsv replication in an in vivo model of well-differentiated primary human airway epithelial cells at concentration as low as 1.9 nm. moreover, despite extensive replication in the presence of 64, no resistance against 64 was observed even after 17 passages, which is in direct contrast to previously reported rsv inhibitor. 155 similarly, 63 was reported to inhibit the replication of poliovirus both in vitro and in vivo, and no resistance against 63 was detected after 10 passages, despite that poliovirus is feature with rapid replication rate and high mutation frequency. 156 and inhibited nucleocapsid egress from the nucleus. 159 many other hsp90 inhibitors have also shown definitive antiviral activities against a panel of virus strains. 160, 161 although around 17 hsp90 inhibitors are under development in clinical trials, they are all for cancer indication and none is being tested for antiviral purpose albeit with well-established preclinical results. the major concern associated with the application of hsp90 inhibitors for antiviral treatment is the toxicity. most of the hsp90 inhibitors in clinical trials are accompanied with some severe side effects including cardiotoxicity, gastrointestinal toxicity, and/or ocular toxicity amongst other side effects. [162] [163] [164] it is now widely accepted that these unwanted toxicities are mainly attributed to paninhibition of hsp90 isoforms. for example, the observed cardiotoxicity mainly resulted from the inhibition hsp90α, which is responsible for the maturation of herg channel. 165 therefore, hsp90 isoform-selective inhibitors are expected to side-step some detrimental toxicity observed with pan-inhibitors. indeed, several hsp90 isoform-selective inhibitors against grp94 166, 167 (72) (73) and hsp90β 168 (71) have been successfully developed with much better safety profiles. these inhibitors were devised for cancer treatment, and their antiviral profiles were not investigated. very recently, two grp94 selective inhibitors 72 and 73 were found to inhibit the replication of denv and zikv with ic 50 values in the low nanomolar range, and it was further confirmed that grp94 was essential for the replication of denv and zikv. 169 therefore, it can be deduced that grp94 might be indispensable for the replications of other viruses as well. it can be envisioned that antiviral therapy with hsp90 inhibitors can benefit from hsp90 isoform inhibition, and isoform-selective show broad-spectrum antiviral activities. as compared to hsp90 inhibitors, hsp70 inhibitors are under developed, and the development of hsp70 inhibitors is faced with several challenges: hsp70 has a high affinity toward adp, and the conformation state of hsp70 makes the atp binding site less accessible. 178 therefore, it is very hard to design inhibitors targeting the atp binding site. 179 due to high sequence similarity between hsp70i and hsc70, most of the available inhibitors are unable to discriminate between those two isoforms. shown in figure 10 are a selected subset of hsp70 inhibitors, and all those inhibitors are intended for cancer indication. mkt-077 (76; figure 17 ) is the most advanced one, which has entered phase i clinical trial for cancer treatment. however, its clinical trial was halted due to severe renal dysfunction observed in patients. 180 albeit with highly structural similarity toward 80. 182 in addition, 80 suppressed the inflammation response associated with dengue fever. more importantly, no resistance to 80 was detected even after 10 passages, while significant resistance was observed with a viral ns5 inhibitor under the same conditions, highlighting the advantages of htas over daas in terms of resistance selection. 80 and its analogues have also exhibited broadspectrum antiviral profiles with inhibitory activity against hcv, 183 zikv, 184 west nile, and japanese encephalitis viruses. 182 the hsp70i is at relatively low level in unstressed cell, while its expression is significantly induced upon f i g u r e 1 7 the chemical structures of hsp70 inhibitors and downregulators, hsp, heat shock protein viral infections, indicating an integral role of hsp70i in viral infections. to achieve hsp70 isoform inhibition, several inhibitors targeting an allosteric site on hsp70 were elegantly designed with selectivity toward hsp70i. 185, 186 hs-72 (81) is one of such inhibitors, and it was shown to inhibit the entry of denv mainly by disrupting the association of hsp70i with the denv receptor complex. 187 in comparison, hsc70 selective inhibitors have not been precedented. however, in contrast to direct inhibition, hsc70 downregulators have been reported with broadspectrum antiviral activities. for example, imb-dm122 (82), an analogue derived from natural compound oxymatrine, was discovered as a hsc70 downregulator. the half-life of hsc70 messenger rna (mrna) was reduced by 78% followed by the treatment of 82 (500 μg/ml) in huh7.5 cells. as such, 82 is effective to inhibit hcv replication at a concentration of 125 μg/ml. in addition, 82 is well-tolerated in mice with no obvious toxicity at a single dosage of 1000 mg/kg (intraperitoneal [ip]). 188 intensive structural modifications were further made to the sidechain and/or substituent at the nitrogen position of 82, yielding several analogues with ic 50 s against hcv in the low micromolar range (ie, 83). [189] [190] [191] the oxymatrine analogues were also shown to inhibit both wild-type and lamivudine-resistant hbv infection via downregulating hsc70 expression with excellent safety profile in mice (ld 50 = 750 mg/kg, oral administration). 192 the activity against other virus was also reported with oxymatrine analogues. 193, 194 the other naturally occurring hsc70 downregulator is lycorine (84) . 121 it was reported to decrease the hsc70 mrna level does-dependently with definitive anti-hcv activity in vitro. the structural optimization led to several derivatives with ic 50 values ranging from low micromolar to submicromolar levels. the sar study showed that the double bond between c3 and c4 and the basic nitrogen at n-5 position are crucial to the anti-hcv activity. 195 interestingly, its naturally occurring cousin lycoricidine (85) possessed much more potent inhibitory effect against hcv with an ec 50 value of 0.55 nm, and the mechanism of action studies revealed that downregulation of hsc70 expression at least partially account for the observed antiviral activity. 196 although these compounds are confirmed to downregulate hsc70 to exert their antiviral efficacy, their respective physical binding protein(s) remain to be clarified, but it can be deduced that their physical binding partner(s) must be host factor(s). host cells have developed an innate immune system to act as the first-line defense against invading virus. in addition, a series of intracellular restriction factors are also expressed endogenously to counteract viral infections, 197 and apolipoprotein b mrna-editing enzyme catalytic polypeptide-like 3g (apobec3g, a3g) is one such factor, which possesses the capacity to restrict the replication of a panel of viruses including hiv-1, hbv, hcv, and ev71 among others via different mechanisms. a3g is known as a cytidine deaminase, catalyzing the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. its anti-hiv-1 activity is closely associated with its deaminase function, by which consistent mutation from dg to da in the positive strand of viral dna is frequently observed. 198 the high level of g to a mutation is attributed to the c to u transitions occurred on the complementary negative-strand dna by the cytidine deaminase activity of a3g. introduction of deoxyuracils in the proviral dna can ultimately lead to its cleavage and degradation by specific ap endonucleases. additionally, high percentages of g to a mutation in the hiv-1 genome will result in the loss of functions of viral proteins. 199 interestingly, the antiviral activity of a3g against other virus is independent of its deaminase activity. for example, a3g is reported to inhibit hcv replications by binding to the c-terminus of hcv ns3 protein to reduce its helicase activity, which is essential for hcv replication. 200, 201 a3g was also reported to inhibit hbv replication both in vitro and in vivo, but its anti-hbv mechanism is not related to its deaminase activity, because a3g did not yield any g to a hypermutation within the hbv genome, and the catalytically inactive a3g derivatives were able to result in the same magnitude of hbv inhibition as compared with its wild-type counterpart. 202, 203 although host cells are endowed with antiviral restriction factors such as a3g, viruses are so cunning that they developed their own mechanism to evade host innate immune system. for example, hiv-1 expresses a viral protein ji and li vif, which binds to a3g and form an ubiquitin ligase complex with cullin 5 (cul5), elongin b/c (elob/c), and cbfβ, leading to the ubiquitination and subsequent proteasomal degradation of a3g. 204 in the cases of hbv and hcv infections, endogenous a3g is also eliminated by some unknown mechanisms. therefore, agents either disrupting the formation of ubiquitin complex, stabilizing a3g or inducing the expression of a3g are expected to demonstrate antiviral activity. in 2008, chen et al identified two compounds imb-26 (86; figure 18 ) and imb-35 (87) , which directly binds to a3g and disrupted its interaction with vif, and therefore rescue a3g from vif-mediated degradation. both compounds showed a3g-dependent anti-hiv-1 activity in nonpermissive h9 cells with ec 50 values in the low nanomolar range. moreover, no cytotoxicity was observed at a concentration of around 4 μm, indicating a therapeutic index of greater than 200, and the ld 50 value for 87 is as high as greater than 1000 mg/kg (ip). 205 86 also showed strong antiviral activity against hcv in vitro via stabilizing intracellular a3g. 206 due to the presence of a bromo substituent at the α position of amide, which is highly reactive as a alkylation agent, structural modifications were made to 86, and it turned out that the bromo is not essential for the antiviral activities. most of the synthesized derivatives showed potent antiviral activity against hcv and ev71 virus with ic 50 values ranging from 0.57 to 80 μm. 207, 208 since the amide linkage is liable to hydrolysis, a methylene group was inserted between the carbonyl and amino group. however, such modification resulted in complete loss of activity. 207 to further address this issue, a ring formation strategy was employed to generate compound with 2-aryl-isoindolin-1-ones scaffold (88) . these analogues showed much-enhanced stability toward hydrolysis, and most importantly, the anti-ev71 activity was retained with ic 50 in the low micromolar range. 209 interestingly, the derivatives of 86 also showed definitive antiviral activity against both wild-type and tamiflu resistant influenza virus in vitro with ic 50 values in the low micromolar range, 210 despite the fact that a3g does not yield any inhibitory activity against influenza virus replication, 211 indicating that other a3g-independent antiviral mechanism must be involved. vif, and both inhibited viral replication via a3g pathway. the immunoprecipitation experiment confirmed that neither compound disrupted the interaction between vif and a3g, and thus inhibited the ubiquitination of a3g. in addition, neither compound impaired 20s proteasome activity, suggesting that recovery of a3g is not simply caused by inhibiting the general proteasome activities. the specific mechanism(s) underlying the recovery of a3g need further clarification. 215 both compounds also showed high cytotoxicity toward 293t cells with ic 50 values being 30 and 50 μm, respectively, necessitating further structural optimization. benzimidazole derivatives were also identified as potent anti-hiv-1 agents via stabilizing a3g. 216 the structural modifications led to the identification of compound 93 with an anti-hiv-1 ic 50 value of 3.4 nm in h9 cells, and no toxicity in mice was observed in 2 weeks after the ip injection of 1000 mg/kg of compound 93. the mechanism of action studies showed that this type of compounds stabilized a3g via disrupting the interaction between vif and eloc, which is essential for the vif-mediated a3g degradation. 217 in 2012, zuo et al 218 confirming its a3g-dependent antiviral mechanism. the coimmunoprecipitation and computational analysis suggested that 94 directly bound to eloc at the interface of eloc-vif interaction to suppress vif activity. the sar studies concluded that both the naphthoyl and benzoyl group were essential for the activity, and modifications made to the ester group were well-tolerated. 219 the abovementioned antiviral strategy is based on the recovery of a3g to inhibit viral infection. very interestingly, it was reasonably argued that hiv-1 virus might actually benefit from sub-lethal levels of a3g-induced mutation, which might contribute to the high mutation rate of hiv-1 virus and the capacity of the virus to escape innate immune system and evolve resistance against antiretroviral drugs. therefore, current antiretroviral treatment regime may benefit from the inhibition of a3g deaminase activity. 220 however, a proof of concept study needs to be established to support such hypothesis. bone marrow stromal cell antigen 2 (bst-2), also known as tetherin or cd317, is a potent ifn-induced antiviral molecule inhibiting the release of various enveloped virus particles from infected cells, including filoviruses, 221, 222 arenaviruses, 223 paramyxovirus, 223 γ-herpesviruses, 224 and among others. 225 the broad-spectrum antiviral profiles of bst-2 is attributed to its ability to target a common feature shared by these viruses: host cell-derived lipid bilayer. since the target of the bst-2 is not encoded by viral genome, so the virus cannot mutate the viral protein to evade the antiviral activity of bst-2. however, viruses have evolved other mechanisms to counteract the action of bst-2 by expressing different viral proteins to physically bind to bst-2, and thereby block the interaction between bst-2 and its target. such proteins include hiv-1 vpu, hiv-2 env, siv env, siv nef, kshv k5, and the ebov glycoprotein. for example, hiv-1 vpu, a type i integral membrane protein, can either induce the ubiquitination of bst-2 for degradation or downregulate the cell-surface bst-2 level by sequestering the de novo synthesized bst-2 away from the plasma membrane to counteract its antiviral activity. 226 therefore, it can be envisioned that disruption of the interaction between bst-2 and these viral proteins or stabilization of bst-2 could yield antiviral agents. zhang et al 227 developed a cell-based high-throughput enzyme-linked immunosorbent assay for the quantification of cell-surface bst-2 levels, 227 and a lead compound imb-la (95; figure 19 ) was identified to inhibit vpu-mediated bst-2 degradation and recover the expression of bst-2 at the cell surface. 228 human dead-box polypeptide 3, an atpase/rna helicase, was founded to be involved in a variety of cellular biogenesis process, including cellular differentiation, cell-cycle regulation, apoptosis, and cell survival. recently, it has also been identified as an essential host factors for the replication of both dna and rna virus, including hiv, 231 hcv, 232 denv, 233 and wnv, 234 among others. although the exact mechanism(s) ddx-3 employed to facilitate viral replication is yet to be elucidated, ddx-3 has been proposed as a very promising host target for broad-spectrum antiviral agent development. the crystal structure of ddx-3 in complex with amp has been resolved, 235 and a pharmacophore has been proposed for virtual screening, 236 value of 10 μm. 241 in a follow-up study, the structure-based drug design strategy was employed to devise new inhibitors with more potent activity. the structural modification was mainly made to the two phenyl rings to form more interactions with the other two unexplored binding pockets within the rna-binding site, and one inhibitor which showed much more potent antibudding against filoviruses, arenavirus, and rhabdovirus vlps. for example, both compounds inhibited egress of lfv-z vlps by more than 10-fold at a concentration of 1 μm, and as expected, showed no inhibitory activity against a ppxy mutant and budding defective virus, confirming the antibudding mechanism via inhibition of ppxy and nedd4 interaction. in addition, no cytotoxicity toward hek293t cells was observed for both compounds at concentrations tested (1 μm). 242 in a follow-up study, an intensive structural modifications were made to the lead compound 102, and several more potent analogues were identified with nanomolar activity in the antibudding assay. for example, compound 103 can achieve more than 90% inhibition of li et al identified a natural compound cajanine (105; figure 22 ) as a potent hcv inhibitor via a cell-based phenotype-screening assay with an ic 50 value of 3.12 μm. 244, 245 the structural-activity relationships study revealed that both the hydrophobic benzene ring (106) and prenyl group (107) were not essential for the anti-hcv activity, and several derivatives with more potent anti-hcv activity (ic 50 < 1 μm) were also obtained. the mechanism of action study demonstrated that 105 and its derivatives showed no inhibitory effect against hcv viral proteins, and yet led to the downregulation of a host target chondroitin sulfate n-acetylgalactosaminyltransferase 1 (csgalnact-1), which is a key enzyme responsible for the initiation of chondroitin sulfate chain. csgalnact-1 was shown to play a key role during the replication of hcv virus, and its expression was elevated upon hcv infection. knockdown of csgalnact-1 by sirna led to significant suppression of hcv replications. it is interesting to note that 105 showed no effect on the mrna level of csgalnact-1, and the downregulation of csgalnact-1 protein level was recovered by a general protease inhibitor mg132, indicating that cajanine promoted the degradation of csgalnact-1 by proteasome pathway. in consistency with such host targeting mechanism, 105 showed the same magnitude of inhibitory effect against both wild-type and drug resistant hcv virus strains, and it also showed very pronounced synergistic effects with other daas to inhibit hcv replications, indicating that 105 and its derivatives are worthy of further studies. although it is still unclear whether csgalnact-1 is broadly required for the replication of other viruses, our unpublished data showed that 105 and its derivatives also possessed inhibitory effect against other virus such as influenza virus, hiv-1, hbv, and coxsackie b virus. although it is ascertained solidly that 105 inhibits hcv replication via downregulation of csgalnact-1 protein, it remains unclear how 105 leads to the csgalnact-1 degradation, and which protein(s) 105 binds to physically. answering these types of questions would definitely provide new host targets for the development of new antiviral agents. throughout a drug repurposing campaign, estrogen receptor α (erα) inhibitor tamoxifen (108; figure 23 ) was identified as an hcv inhibitor. the known targets of 108 include erα, p-glycoprotein, calmodulin, and protein kinase c, and so forth. the inhibitors against p-glycoprotein, calmodulin and protein kinase c failed to inhibit hcv f i g u r e 2 2 the chemical structure of cajanine along with its synthesized analogues replication, excluding the possibility of these proteins as the anti-hcv targets for 108. a specific sirna against erα significantly suppressed hcv rna in replicon-containing cells, and transient transfection with erα (but not erβ) expression plasmids also augmented hcv replications. altogether, these results confirmed the important roles erα played during hcv replication. the subsequent binding assay showed that domain c of erα physically bound to ns5b but not ns3, ns4b, and ns5a, and such interaction is essential for the hcv genome replications. the selective estrogen receptor modulators (serms) toremifene (109) and clomiphene (110) were also reported to inhibited several other viruses such as hiv-1, 246 ebov, 247 and hsv. 248 however, the mechanism studies have excluded erα as potential antiviral targets, and the observed broad-spectrum antiviral property for serms resulted from the inhibition of other host factors, including protein kinase c (hiv-1) 249 and chloride channel (hsv-1). 248 these host proteins together with erα represent promising targets for the development of novel antiviral agents to combat against drug resistance and newly emerging unknown viruses. quite a few other host proteins or pathways have also been validated as feasible antiviral targets in vitro and even in laboratory animals, such as lipid biosynthesis and metabolism pathways, 250, 251 pparα, 252 and hnf4α, 253 among others. 121, [254] [255] [256] table 1 summarized the host targets included in this review along with the development stages of their respective inhibitors/modulators. it should be noted that the focus of this review is just on small-molecule based htas, and other validated antiviral host targets with only antibody or sirna-based modulators were not included. it is reasonable to anticipate that such host proteins can also be targeted by small-molecule modulators to convey antiviral activity. notably, the antiviral host targets studied only account for a very small portion of the host factors confirmed to be essential for viral replications. in 2016, ammari et al 257 released a database for hostpathogen interactions, with which one can search the known host-pathogen interactions by inputting either the genes, proteins or the viruses. it can be speculated that most of the recorded host-virus interactions must play critical roles during viral replications, and thus could serve as new targets for the development of htas. daas have shown great success in combating viral infections in clinic, and they are generally safe to use because they directly target viral proteins, which lack homologs in human. however, daas also suffer from several inherent limitations due to its viral protein targeting nature: viral proteins varied among different species and even different genotypes, and thus daa targeting one specific viral protein is unlikely to exhibit inhibitory effect against viral proteins from other viral species or variants. therefore, daas are normally narrow-spectrum antiviral agents, and this is also the underlying reason for the lack of effective antiviral drugs against newly emerging viruses; the other major downside for daas is they are prone to cause drug resistance, particular among rna viruses with very high frequency of replication errors. hta agents perfectly complement to daas in regard to narrow-spectrum activity although a large number of host factors are known to play vital roles throughout the whole life cycle of virus, only very few of them were explored as antiviral targets. this is primarily attributed to several concerns raised by htas. chiefly, targeting a host protein may potentially lead to unwanted toxicity issues, because the target protein may be indispensable for some cellular functions. however, this may not be intrinsically true for all the host proteins, and many host genes are known to be nonessential for cellular functions. therefore, the inhibition of such host proteins will be well-tolerated, and the on-target based toxicity can be minimized. even though the target host protein is essential for some cellular functions, the on-target based toxicity can be mitigated in many ways. first, it csgalnact-1 cajanine hcv, influenza, and hiv preclinical ns5b-erα tamoxifen hcv preclinical is known that the expression level or the activity of many cellular proteins are elevated to facilitate viral replications. therefore, it is possible to knockdown the target protein to a level, at which the viral replication can be blocked while the normal cellular functions can still be maintained. second, most of the time, alternative pathways or proteins exist for a cellular function, and hence the inhibition of one of these proteins or pathways may lead to the blockage of viral replication, but not the cellular function. third, the on-target based toxicity is closely associated with the duration of treatment, and the treatment of most of viral infections only lasts for several weeks or even days. therefore, the toxicity concern raised by targeting a host factor for virus treatment can be further alleviated. it is worth noting that quite a few approved drugs targeting host factors for other indications are considered to be generally safe to use in clinic for years. consequently, toxicity issue is something that one should pay attention to, but not something used to bias against host target antiviral agents. the other major challenge faced with the development of htas is that the antiviral phenotype observed with htas is merely in vitro artifact in some cases, and the correlation between in vitro and in vivo or clinical efficacy is very poor as in the case of development of impdh inhibitors as anti-hcv agents. the other example is the repurposement of statins as anti-hcv drugs. statins showed very pronounced inhibitory effects against hcv replication in vitro, but yield unsatisfactory outcomes in clinic trials. the lack of reliable predictive in vitro models indeed increased the attrition rates in the development of htas. however, despite with these challenges, development of htas can be highly rewarding because htas can potentially address the unmet needs in the treatment of viral infections. the work from the ji lab is financially supported by the national natural science foundation of china (81773608) recent advancement of direct-acting antiviral agents (daas) in hepatitis c therapy the competitive binding between inhibitors and substrates of hcv ns3/4a protease: a general mechanism of drug resistance hsp90: a promising broad-spectrum antiviral drug target the hiv coreceptors cxcr4 and ccr5 are differentially expressed and regulated on human t lymphocytes hiv: cell binding and entry. cold spring harb perspect med the geographic spread of the ccr5 delta32 hiv-resistance allele ccr5 receptor antagonists in preclinical to phase ii clinical development for treatment of hiv targeting chemokine receptor cxcr4 for treatment of hiv-1 infection, tumor progression, and metastasis exploring the stereochemistry of cxcr4-peptide recognition and inhibiting hiv-1 entry with d-peptides derived from chemokines amd3100, a small molecule inhibitor of hiv-1 entry via the cxcr4 co-receptor the bicyclam amd3100 story amd3465, a monomacrocyclic cxcr4 antagonist and potent hiv entry inhibitor multiple-dose escalation study of the safety, pharmacokinetics, and biologic activity of oral amd070, a selective cxcr4 receptor inhibitor, in human subjects blockade of x4-tropic hiv-1 cellular entry by gsk812397, a potent noncompetitive cxcr4 receptor antagonist synthesis of a novel tricyclic 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system and cxcr4 antagonists with potent activity against hiv-1 design of novel cxcr4 antagonists that are potent inhibitors of t-tropic (x4) hiv-1 replication plerixafor: in patients with non-hodgkin's lymphoma or multiple myeloma function of the chemokine receptor cxcr4 in haematopoiesis and in cerebellar development the chemokine receptor cxcr4 is essential for vascularization of the gastrointestinal tract defects of b-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the cxc chemokine pbsf/sdf-1 ccr5 inhibitors: emerging promising hiv therapeutic strategy efficacy of short-term monotherapy with maraviroc, a new ccr5 antagonist, in patients infected with hiv-1 chemokine control of west nile virus infection virological and immunological response to antiretroviral regimens containing maraviroc in hiv type 1-infected patients in clinical practice: role of different tropism testing results and of concomitant treatments intensification of a raltegravir-based regimen with maraviroc in early hiv-1 infection maraviroc and reverse transcriptase inhibitors combinations as potential preexposure prophylaxis candidates (s)-methyl-1-piperazinyl]-4-methylpiperidine (sch-417690/sch-d), a potent, highly selective, and orally bioavailable ccr5 antagonist pharmacokinetics and short-term safety of 873140, a novel ccr5 antagonist, in healthy adult subjects an imidazopiperidine series of ccr5 antagonists for the treatment of hiv: the discovery of n-{(1s)-1-(3-fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-1h-imidazo[4, 5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]propyl}acetamide (pf-232798) is pf-232798 a possible successor to maraviroc? novel 4,4-disubstituted piperidine-based c-c chemokine receptor-5 inhibitors with high potency against human immunodeficiency virus-1 and an improved human ether-a-go-go related gene (herg) profile discovery of a piperidine-4-carboxamide ccr5 antagonist (tak-220) with highly potent anti-hiv-1 activity design, synthesis, and biological evaluation of novel 2-methylpiperazine derivatives as potent ccr5 antagonists discovery of incb9471, a potent, selective, and orally bioavailable ccr5 antagonist with potent anti-hiv-1 activity ccr5 receptor antagonists in preclinical to phase ii clinical development for treatment of hiv inhibition of human immunodeficiency virus replication by a dual ccr5/cxcr4 antagonist inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction rna virus error catastrophe: direct molecular test by using ribavirin the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen the antiviral compound ribavirin modulates the t helper (th) 1/th2 subset balance in hepatitis b and c virus-specific immune responses ribavirin-induced anemia in hepatitis c virus patients undergoing combination therapy a phase iii study of the safety and efficacy of viramidine versus ribavirin in treatment-naive patients with chronic hepatitis c: viser1 results safety and efficacy of viramidine versus ribavirin in viser2: randomized, doubleblind study in therapy-naive hepatitis c patients ribavirin revisited in the era of direct-acting antiviral therapy for hepatitis c virus infection efficacy and safety of grazoprevir/elbasvir+/− rbv for 12 or 16 weeks in patients with hcv g1, g4 or g6 infection who previously failed peginterferon/rbv: c-edge treatment-experienced combined interferon alpha2b and cyclosporin a in the treatment of chronic hepatitis c: controlled trial specific inhibition of hepatitis c virus replication by cyclosporin a nim811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis c virus alone or in combination with alpha interferon safety, pharmacokinetics, and antiviral activity of the cyclophilin inhibitor nim811 alone or in combination with pegylated interferon in hcv-infected patients receiving 14 days of therapy alisporivir-a host-targeting antiviral, provides low viral breakthrough rate and high barrier to resistance in hcv genotype 1 treatment-naïve patients in the phase iib essential study alisporivir plus ribavirin is highly effective as interferon-free or interferon-add-on regimen in previously untreated hcv-g2 or g3 patients: svr12 results from vital-1 phase 2b study interferon (ifn)-free alisporivir (deb025) treatment in the vital-1 study has a more beneficial overall safety profile vs ifn-containing treatment scy-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis c virus rna replication in vitro the cyclophilin inhibitor scy-635 suppresses viral replication and induces endogenous interferons in patients with chronic hcv genotype 1 infection preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family sanglifehrin a, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action sanglifehrin−cyclophilin interaction: degradation work, synthetic macrocyclic analogues, x-ray crystal structure, and binding data fragment-based discovery of a new family of non-peptidic smallmolecule cyclophilin inhibitors with potent antiviral activities catalysis of cis/trans isomerization in native hiv-1 capsid by human cyclophilin a cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the hbx protein involved in hepatitis b virus replication nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a cyclophilin a is required for efficient human cytomegalovirus dna replication and reactivation target cell cyclophilins facilitate human papillomavirus type 16 infection cyclophilins and cyclophilin inhibitors in nidovirus replication p0890: novel cyclophilin inhibitor cpi-431-32 shows broad spectrum antiviral activity by blocking replication of hcv, hbv and hiv-1 viruses discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance analyzing the relationship of qt interval and exposure to nitazoxanide, a prospective candidate for influenza antiviral therapy-a formal tqt study synergistic effect of nitazoxanide with neuraminidase inhibitors against influenza a viruses in vitro tizoxanide and other thiazolides are potent inhibitors of hepatitis b virus and hepatitis c virus replication the fda-approved oral drug nitazoxanide amplifies host antiviral responses and inhibits ebola virus nitazoxanide: a first-in-class broad-spectrum antiviral agent comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens systematic identification of synergistic drug pairs targeting hiv pediatric drug nitazoxanide: a potential choice for control of zika nitazoxanide inhibits paramyxovirus replication by targeting the fusion protein folding: role of glycoprotein-specific thiol oxidoreductase erp57 nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus the anti-hepatitis c agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2α via protein kinase activated by double-stranded rna activation hepatitis b virus x protein identifies the smc5/6 complex as a host restriction factor inhibition of hbv transcription from cccdna with nitazoxanide by targeting the hbx-ddb1 interaction nitazoxanide inhibits human norovirus replication and synergizes with ribavirin by activation of cellular antiviral response interferon regulatory factor-1 (irf-1) is involved in the induction of phosphatidylserine receptor (psr) in response to dsrna virus infection and contributes to apoptotic cell clearance in chse-214 cell effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial a pilot clinical trial of nitazoxanide in the treatment of chronic hepatitis b synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs the safety and efficacy of combination n-butyl-deoxynojirimycin (sc-48334) and zidovudine in patients with hiv-1 infection and 200-500 cd4 cells/mm3 an alpha-glucosidase i inhibitor for the potential treatment of hcv infection inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus nl63 spike protein-mediated entry by altering the glycan processing of angiotensin i-converting enzyme 2 in vivo therapeutic protection against influenza a (h1n1) oseltamivir-sensitive and resistant viruses by the iminosugar uv-4 dengue virus evolution under a host-targeted antiviral glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum synthetic heterocyclic candidates as promising α-glucosidase inhibitors: an overview assessment of the potential for host-targeted iminosugars uv-4 and uv-5 activity against filovirus infections in vitro and in vivo dose-and schedule-dependent protective efficacy of celgosivir in a lethal mouse model for dengue virus infection informs dosing regimen for a proof of concept clinical trial small molecule inhibitors of er α-glucosidases are active against multiple hemorrhagic fever viruses ester prodrugs of ihvr-19029 with enhanced oral exposure and prevention of gastrointestinal glucosidase interaction structures of mammalian er α-glucosidase ii capture the binding modes of broadspectrum iminosugar antivirals impdh as a biological probe for rna antiviral drug discovery: synthesis, enzymology, molecular docking, and antiviral activity of new ribonucleosides with surrogate bases. nucleosides nucleotides nucleic acids broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro an impdh inhibitor, suppresses replication of zika virus and other emerging viral pathogens synthesis and antiviral activity of a novel class of (5-oxazolyl)phenyl amines synthesis and broad-spectrum antiviral activity of some novel benzoheterocyclic amine compounds a randomized, double-blind, placebo-controlled dose-escalation trial of merimepodib (vx-497) and interferon-alpha in previously untreated patients with chronic hepatitis c merimepodib, pegylated interferon, and ribavirin in genotype 1 chronic hepatitis c pegylated interferon and ribavirin nonresponders ribavirin as therapy for chronic hepatitis c. a randomized, doubleblind, placebo-controlled trial repurposing of kinase inhibitors as broad-spectrum antiviral drugs repurposing kinase inhibitors as antiviral agents to control influenza a virus replication new connections: kinase inhibitors as antivirals anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap2m1 essential for viral assembly selective inhibitors of cyclin g associated kinase (gak) as anti-hepatitis c agents optimization of isothiazolo[4,3-b]pyridine-based inhibitors of cyclin g associated kinase (gak) with broad-spectrum antiviral activity 3-b]pyridines as inhibitors of cyclin g associated kinase: synthesis, structure-activity relationship studies and antiviral activity identification and optimization of 4-anilinoquinolines as inhibitors of cyclin g associated kinase sgc-gak-1: a chemical probe for cyclin g associated kinase (gak) anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus targeting hepatitis b virus cccdna by crispr/cas9 nuclease efficiently inhibits viral replication final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of myrcludex b in combination with tenofovir in patients with chronic hbv/hdv co-infection cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilinindependent interference with the ntcp receptor use of fda approved therapeutics with hntcp metabolic inhibitory properties to impair the hdv lifecycle inhibitory effect of fasiglifam on hepatitis b virus infections through suppression of the sodium taurocholate cotransporting polypeptide evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells overexpressing a membrane transporter ntcp isolation and structure of vanitaracin a, a novel anti-hepatitis b virus compound from talaromyces sp design and synthesis of a novel candidate compound nti-007 targeting sodium taurocholate cotransporting polypeptide [ntcp]-apoa1-hbx-beclin1-mediated autophagic pathway in hbv therapy concept of viral inhibitors via ntcp cyclosporin derivatives inhibit hepatitis b virus entry without interfering with ntcp transporter activity epigallocatechin gallate inhibits hepatitis b virus via farnesoid x receptor alpha farnesoid x receptor-alpha is a proviral host factor for hepatitis b virus that is inhibited by ligands in vitro and in vivo farnesoid x receptor agonist gw4064 indirectly inhibits hcv entry into cells via down-regulating scavenger receptor class b type i the selective fxr agonist eyp001 is well tolerated in healthy subjects and has additive anti-hbv effect with nucleoside analogues in heparg cells diacylglycerol acyltransferase-1 localizes hepatitis c virus ns5a protein to lipid droplets and enhances ns5a interaction with the viral capsid core hepatitis c virus entry is impaired by claudin-1 downregulation in diacylglycerol acyltransferase-1-deficient cells effect of the dgat1 inhibitor pradigastat on triglyceride and apob48 levels in patients with familial chylomicronemia syndrome a diacylglycerol transferase 1 inhibitor is a potent hepatitis c antiviral in vitro but not in patients in a randomized clinical trial herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus human butyrate-induced transcript 1 interacts with hepatitis c virus ns5a and regulates viral replication antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus molecular chaperone hsp90 is a therapeutic target for noroviruses heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers hsp90, an unlikely ally in the war on cancer geldanamycin, a ligand of heat shock protein 90, inhibits herpes simplex virus type 2 replication both in vitro and in vivo geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro inhibition of heat-shock protein 90 reduces ebola virus replication heat shock protein 90 controls hiv-1 reactivation from latency hsp90 inhibitors reduce influenza virus replication in cell culture hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance a novel class of geldanamycin derivatives as hcv replication inhibitors targeting on hsp90: synthesis, structure-activity relationships and anti-hcv activity in gs4.3 replicon cells synthesis and biological evaluation of heat-shock protein 90 inhibitors: geldanamycin derivatives with broad antiviral activities hsp90 inhibitor at-533 blocks hsv-1 nuclear egress and assembly inhibition of heat-shock protein 90 reduces ebola virus replication inhibition of hsp90 attenuates porcine reproductive and respiratory syndrome virus production in vitro hsp90 molecular chaperone inhibitors: are we there yet? heat shock protein 90: inhibitors in clinical trials targeting the molecular chaperone heat shock protein 90 (hsp90): lessons learned and future directions the herg channel is dependent upon the hsp90alpha isoform for maturation and trafficking paralog-selective hsp90 inhibitors define tumor-specific regulation of her2 development of a grp94 inhibitor structure-guided design of an hsp90beta n-terminal isoform-selective inhibitor small molecule grp94 inhibitors block dengue and zika virus replication involvement of endoplasmic reticulum chaperones in the folding of hepatitis c virus glycoproteins molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo posttranslational folding of vesicular stomatitis virus g protein in the er: involvement of noncovalent and covalent complexes folding, interaction with grp78-bip, assembly, and transport of the human immunodeficiency virus type 1 envelope protein heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo heat shock cognate protein 70 is involved in rotavirus cell entry in vivo and in vitro association of hsc70 with polyomavirus capsid proteins the heat shock cognate protein 70 is associated with hepatitis c virus particles and modulates virus infectivity allosteric opening of the polypeptide-binding site when an hsp70 binds atp atpases as drug targets: insights from heat shock proteins 70 and 90 a phase i and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog mkt 077 phase i trial of the selective mitochondrial toxin mkt077 in chemoresistant solid tumours defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection allosteric heat shock protein 70 inhibitors block hepatitis c virus assembly heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70 heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70 an inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating hsp70 as a host antiviral target small molecular compounds that inhibit hepatitis c virus replication through destabilizing heat shock cognate 70 messenger rna evolution of matrinic ethanol derivatives as anti-hcv agents from matrine skeleton synthesis and biological evaluation of sophocarpinic acid derivatives as anti-hcv agents synthesis, structure−activity relationship and biological evaluation of novel n-substituted matrinic acid derivatives as host heat-stress cognate 70 (hsc70) down-regulators design and synthesis of oxymatrine analogues overcoming drug resistance in hepatitis b virus through targeting host heat stress cognate 70 antiviral effect of matrine against human enterovirus 71 sar evolution and discovery of benzenesulfonyl matrinanes as a novel class of potential coxsakievirus inhibitors design, synthesis and structure-activity relationship optimization of lycorine derivatives for hcv inhibition evaluation of anti-hcv activity and sar study of (+)-lycoricidine through targeting of host heat-stress cognate 70 (hsc70) zinc finger antiviral protein inhibits coxsackievirus b3 virus replication and protects against viral myocarditis hypermutation of hiv-1 dna in the absence of the vif protein single-strand specificity of apobec3g accounts for minus-strand deamination of the hiv genome host apobec3g protein inhibits hcv replication through direct binding at ns3 host apolipoprotein b messenger rna-editing enzyme catalytic polypeptide-like 3g is an innate defensive factor and drug target against hepatitis c virus inhibition of hepatitis b virus replication by apobec3g inhibition of hepatitis b virus replication by apobec3g in vitro and in vivo ubiquitination of apobec3 proteins by the vif-cullin5-elonginb-elonginc complex small molecular compounds inhibit hiv-1 replication through specifically stabilizing apobec3g host apolipoprotein b messenger rna-editing enzyme catalytic polypeptide-like 3g is an innate defensive factor and drug target against hepatitis c virus synthesis and antiviral activity of a series of novel n-phenylbenzamide and n-phenylacetophenone compounds as anti-hcv and anti-ev71 agents synthesis and antiviral activity of n-phenylbenzamide derivatives, a novel class of enterovirus 71 inhibitors synthesis and broad antiviral activity of novel 2-aryl-isoindolin-1-ones towards diverse enterovirus a71 clinical isolates synthesis and antiviral activity of substituted bisaryl amide compounds as novel influenza virus inhibitors high level expression of the anti-retroviral protein apobec3g is induced by influenza a virus but does not confer antiviral activity small-molecule inhibition of hiv-1 vif synthesis and structure-activity relationship studies of hiv-1 virion infectivity factor (vif) inhibitors that block viral replication design, synthesis, and biological evaluation of 2-amino-n-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent hiv-1 vif inhibitors small molecules that inhibit vif-induced degradation of apobec3g development of benzimidazole derivatives to inhibit hiv-1 replication through protecting apobec3g protein identification of an hiv-1 replication inhibitor which rescues host restriction factor apobec3g in vif-apobec3g complex small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between vif and elonginc indolizine derivatives as hiv-1 vif-elonginc interaction inhibitors first-in-class small molecule inhibitors of the single-strand dna cytosine deaminase apobec3g broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein infectious lassa virus, but not filoviruses, is restricted by bst-2/tetherin molecular mechanism of bst2/tetherin downregulation by k5/mir2 of kaposi's sarcoma-associated herpesvirus is tetherin a true antiviral: the influenza a virus controversy antagonism of cd317 restriction of human immunodeficiency virus type 1 (hiv-1) particle release and depletion of cd317 are separable activities of hiv-1 vpu high-throughput assay to identify inhibitors of vpu-mediated down-regulation of cell surface bst-2 a small molecule compound imb-la inhibits hiv-1 infection by preventing viral vpu from antagonizing the host restriction factor bst-2 2-thio-6-azauridine inhibits vpu mediated bst-2 degradation a novel peptide to disrupt the interaction of bst-2 and vpu requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function ddx3 dead-box rna helicase is required for hepatitis c virus rna replication strategies for development of dengue virus inhibitors p-body components lsm1, gw182, ddx3, ddx6 and xrn1 are recruited to wnv replication sites and positively regulate viral replication crystal structure of conserved domains 1 and 2 of the human dead-box helicase ddx3x in complex with the mononucleotide amp pharmacophore modeling and molecular docking led to the discovery of inhibitors of human immunodeficiency virus-1 replication targeting the human cellular aspartic acid−glutamic acid−alanine−aspartic acid box polypeptide 3 toward the discovery of novel anti-hiv drugs. second-generation inhibitors of the cellular atpase ddx3 with improved anti-hiv activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation targeting ddx3 with a small molecule inhibitor for lung cancer therapy ketorolac salt is a newly discovered ddx3 inhibitor to treat oral cancer targeting the human dead-box polypeptide 3 (ddx3) rna helicase as a novel strategy to inhibit viral replication discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors small-molecule probes targeting the viral ppxy-host nedd4 interface block egress of a broad range of rna viruses quinoxaline-based inhibitors of ebola and marburg vp40 egress design and synthesis of cajanine analogues against hepatitis c virus through downregulating host chondroitin sulfate n-acetylgalactosaminyltransferase 1 total synthesis of cajanine and its antiproliferative activity against human hepatoma cells ligands of the antiestrogen-binding site are able to inhibit virion production of human immunodeficiency virus 1-infected lymphocytes fda-approved selective estrogen receptor modulators inhibit ebola virus infection inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and nppb effect of tamoxifen on regulation of viral replication and human immunodeficiency virus (hiv) long terminal repeat-directed transcription in cells chronically infected with hiv-1 different anti-hcv profiles of statins and their potential for combination therapy with interferon identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor naringenin inhibits the assembly and long-term production of infectious hepatitis c virus particles through a ppar-mediated mechanism effects of bezafibrate in patients with chronic hepatitis c virus infection: combination with interferon and ribavirin herpes simplex virus type 1 abrogates the antiviral activity of ch25h via its virion host shutoff protein ubiquitin c-terminal hydrolase-l3 promotes interferon antiviral activity by stabilizing type i-interferon receptor dauricine combined with clindamycin inhibits severe pneumonia co-infected by influenza virus h5n1 and streptococcus pneumoniae in vitro and in vivo through nf-kappab signaling pathway hpidb 2.0: a curated database for host-pathogen interactions he got both his bachelor's and master's degrees in chemistry from the university of science and technology of beijing in 2006 and 2008, respectively. thereafter, he moved to peking union medical college institute of medicinal biotechnology, and received his phd degree in medicinal chemistry under the supervision of professor zhuorong li in 2011. after that, he took an assistant professor position in the same institution, and he worked as a postdoctoral fellow in dr binghe wang's lab from chinese academy of medical sciences and peking union medical college. she got her bachelor's degree at peking medical school in 1984, then she moved to peking union medical college and received her master's degree in medicinal chemistry in 1988. her research interests include the development of anti-infection, antiosteoporosis, and anticancer drugs. she has published over 150 peer-reviewed scientific papers and over 30 patents medicinal chemistry strategies toward host targeting antiviral agents key: cord-305085-bv7udg9k authors: lawrence, robert m. title: chapter 13 transmission of infectious diseases through breast milk and breastfeeding date: 2011-12-31 journal: breastfeeding doi: 10.1016/b978-1-4377-0788-5.10013-6 sha: doc_id: 305085 cord_uid: bv7udg9k nan a large body of evidence clearly demonstrates the protective effects of breastfeeding and documents the transmission of specific infections to infants through breast milk. the fear and anxiety that arise with the occurrence of any infectious disease are even greater in the situation of the breastfeeding mother-infant dyad. uncertainty and lack of knowledge often lead to proscribing against breastfeeding out of fear, which then deprives the infant of the potential protective, nutritional, and emotional benefits of breastfeeding exactly at the time when they are most needed (see the discussion of immunologic benefits of human milk in chapter 5). decisions concerning breastfeeding in a mother with an infectious illness should balance the potential benefits of breastfeeding versus the known or estimated risk for the infant acquiring a clinically significant infection via breastfeeding and the potential severity of the infection. documenting transmission of infection from mother to infant by breastfeeding requires not only the exclusion of other possible mechanisms of transmission but also the demonstration of the infectious agent in the breast milk and a subsequent clinically significant infection in an infant that was caused by a plausible infectious process. the first step is to establish the occurrence of a specific infection (clinically or immunologically evident) in a mother and demonstrate the persistence of the infectious agent such that it could be transmitted to the infant. isolation or identification of the infectious agent from the colostrum, breast milk, or an infectious lesion of the breast is important but not necessarily proof of transmission to an infant. epidemiologic evidence of transmission must be considered, including identifying characteristics of the organism that relate an isolate from an infant to the maternal isolate. infectious organisms can reach the breast milk either by secretion in the fluid or cellular components of breast milk or by contamination of the milk at the time of or after expression. a reasonable mechanism of infection via breast milk should be evident and proved through either animal or human studies. demonstration of a subclinical or clinically evident infection in an infant should follow these outlined steps. exclusion of other possible mechanisms of transmission (exposure to mother or other persons/ animals via airborne, droplet, arthropod, or vector modes of transmission or through direct contact with other infectious fluids) would complete the confirmation of transmission of infection via breastfeeding. it is essential to exclude prenatal or perinatal transmission of infection to a fetus/infant, but doing this can often be difficult. clinical case reports or studies confirming the isolation of an infectious agent from the milk are important. to determine a reasonable estimate of the risk for infection via breast milk, larger epidemiologic studies are needed that compare infection rates in breastfed infants versus formula-fed infants, robert m. lawrence addressing the issues just identified. timing of breastfeeding is important relative to the timing of maternal infection and to the presence of a pathogen in colostrum or breast milk. the duration of breastfeeding is another important variable to consider in the estimate of risk because shedding of a pathogen in breast milk may be intermittent. these considerations are only some of the variables to be taken into account, in general, to assess the risk for transmission of an infectious agent from mother to infant via breast milk or breastfeeding. efforts to prove transmission of infection in a particular maternal-infant dyad can be just as difficult and must consider many of the same factors. this chapter focuses on a discussion of specific, clinically relevant, infectious agents and diseases, with reasonable estimates of the risk for infection to infants from breastfeeding. the basic tenet concerning breastfeeding and infection is that breastfeeding is rarely contraindicated in maternal infection. 243 the few exceptions relate to specific infectious agents with strong evidence of transmission and to the association of an infant' s illness with significant morbidity and mortality. the risk or benefit of breastfeeding relative to immunization of a mother or infant is discussed for certain microorganisms. appendix d addresses drugs in breast milk and includes table d-1, on antiinfective agents, and chapter 5 reviews how breastfeeding may protect against infection. chapter 21 addresses specific concerns relating to banked breast milk and includes standards developed by the human milk banking association of north america to guide the appropriate handling of banked human milk relative to possible infectious agents. isolation precautions have undergone some revisions in terminology and conceptualization. 143 understanding that the transmission of microorganisms can occur with a known infection and with unrecognized sources of infection, recommendations have been made for standard precautions to be applied to all patients to protect health care workers from potentially infectious body fluids. additionally, precautions based on the predominant modes of transmission have been recommended to protect against infection through the airborne route, direct contact, or contact with droplets. although these precautions are intended to be used in clinical situations to protect health care workers, they may be applied in certain situations to the mother-infant dyad to prevent transmission of infectious agents from one to the other or to other hospitalized mothers and infants. these precautions are useful most often when a mother and infant are still hospitalized. the use of such precautions within the home is not meant to limit breastfeeding. they are intended to allow breastfeeding in the majority of cases and to facilitate the continuation of breastfeeding with some additional safeguards in certain situations, after short temporary periods of stopping breastfeeding, and when to safely use expressed breast milk (see appendix f). standard precautions include preventing contact with blood, all body fluids, secretions and excretions, nonintact skin, and mucous membranes by (1) careful handwashing before and after every patient contact; (2) use of gloves when touching body fluids, nonintact skin, or mucous membranes or any items contaminated with body fluids (linens, equipment, devices, etc.); (3) use of nonsterile gowns to prevent contact of clothing with body fluids; (4) use of masks, eye protection, or face shields when splashing with body fluids is possible; and (5) appropriate disposal of these materials. standard precautions should be applied to all patients regardless of actual or perceived risks. the centers for disease control and prevention (cdc) does not consider breast milk a body fluid with infectious risks and thus these policies do not apply to breast milk. (see section on misadministration of breast milk later in this chapter as a possible exception to this concept.) in considering breastfeeding infant-mother dyads and standard precautions, body fluids other than breast milk should be avoided, and only in specified situations should breast milk also be avoided. in general, clothing or a gown for the mother and bandages, if necessary, should prevent direct contact with nonintact skin or secretions. avoiding infant contact with maternal mucous membranes requires mothers to be aware of and understand the risks and to make a conscious effort to avoid this type of contact. the use of gloves, gowns, and masks on infants for protection is neither practical nor appropriate. the recommendations concerning the appropriateness of breastfeeding and breast milk are addressed for specific infectious agents throughout this chapter. human immunodeficiency virus (hiv) infection is an example of one infection that can be prevented by the use of standard precautions, including avoiding breast milk and breastfeeding. the recommendations concerning breastfeeding and hiv and the various variables and considerations involved are discussed later. airborne precautions are intended to prevent transmission via droplet nuclei (dried respiratory particles smaller than 5 mcm that contain microorganisms and can remain suspended in the air for long periods) or dust particles containing microorganisms. airborne precautions include the use of a private room with negative-air-pressure ventilation and masks at all times. in the case of pulmonary tuberculosis (tb), respiratory protective devices (requiring personal fitting and seal testing before use) should be worn. airborne precautions are recommended with measles, varicella or disseminated zoster, and tb. breastfeeding in the presence of these maternal infections is prohibited for the infectious period. this is to protect against airborne transmission of the infection from the mother and to allow the infant to be fed the mother' s expressed breast milk by another individual. the exception to allowing breast milk would be local involvement of the breast by varicella-zoster lesions or mycobacterium tuberculosis, such that the milk becomes contaminated by the infectious agent. transmission via droplets occurs when an individual produces droplets that travel only a short distance in the air and then contact a new host's eyes, nose, mouth, or skin. the common mechanisms for producing droplets include coughing, sneezing, talking (singing or yelling), suctioning, intubation, nasogastric tube placement, and bronchoscopy. in addition to standard precautions applied to all patients, droplet precautions include the use of a private room (preferred) and a mask if within 3 feet (0.9 m) of the patient. droplet precautions are recommended for adenovirus, diphtheria, respiratory infections, haemophilus influenzae, neisseria meningitidis or invasive infection, influenza, mumps, mycoplasma, parvovirus, pertussis, plague (pneumonic), rubella, and streptococcal pharyngitis, pneumonia, or scarlet fever. the institution of droplet precautions with a breastfeeding mother who has these infections should be specified for each particular infection. this may require some period of separation for the infant and mother (for duration of the illness, for short-term or complete treatment of the mother, for the infectious period) with use of expressed breast milk for nutrition in the interim. prophylactic treatment of the infant, maternal use of a mask during breastfeeding or close contact combined with meticulous handwashing, and the mother's avoidance of touching her mucous membranes may be adequate and reasonable for certain infections. contact precautions are meant to prevent transmission of infection via direct contact (contact between the body surfaces of one individual with another) and indirect contact (contact of a susceptible host with an object contaminated with microorganisms from another individual). contact precautions include cohorting or a private room, gloves and gowns at all times, and handwashing after removal of gown and gloves. contact precautions are recommended for a long list of infections, such as diarrhea in diapered or incontinent patients with clostridium difficile infection, escherichia coli o157:h7, shigella, rotavirus, hepatitis a, respiratory illness with parainfluenza virus or respiratory syncytial virus (rsv), multidrug-resistant (mdr) bacteria (e.g., enterococci, staphylococci, gramnegative organisms), enteroviral infections, cutaneous diphtheria, impetigo, herpes simplex virus (hsv) infection, herpes zoster (disseminated or in immunocompromised individuals), pediculosis, scabies, staphylococcus aureus skin infection, viral hemorrhagic fevers (e.g., ebola, lassa), conjunctivitis and abscesses, cellulitis, or decubitus that cannot be contained by dressings. 94 for a breastfeeding infant-mother dyad, implementation of precautions for each of these infections in a mother requires meticulous attention to gowning and handwashing by the mother and a specialized plan for each situation. each of these transmission-based precautions can be used together for organisms or illnesses that can be transmitted by more than one route. they should always be used in conjunction with standard precautions, which are recommended for all patients. the red book: report of the committee on infectious diseases by the american academy of pediatrics (aap) 96 remains an excellent resource for infection control guidelines and recommendations to prevent transmission in specific situations and infections. routine culturing of breast milk or culturing breast milk to screen for infectious agents is not recommended except when the milk is intended as donor milk to another mother' s child directly or through human milk banks. see chapter 21 for specific bacterial count standards for raw donor milk and for pasteurization of donor milk. breastfeeding and the expression of or pumping of breast milk (referred to as expressed breast milk) for later use are not sterile activities. in general expressed breast milk should not contain large numbers of microorganisms (less than 10 4 for raw milk and less than 10 6 for milk to be pasteurized), nor should it contain potential pathogens such as s. aureus, β-hemolytic streptococci, pseudomonas species, proteus species, or streptococcus faecalis or faecium. few studies have examined "routine" culturing of milk and the significance of specific bacterial colony counts relative to illness in infants. the studies have been primarily concerned with premature or low-birth-weight (lbw) infants who remain hospitalized and are commonly fed via enteral tubes. a study from canada tested 7610 samples of milk for use in 98 preterm infants. 242 the study did not identify any adverse events in the infants attributed to organisms growing in the milk samples, and routine bacteriological testing of expressed breast milk was not recommended. a study from chicago examined gram-negative bacilli in the milk used in premature infants. 48 samples were tested before feeding and from the nasogastric tubes during feeding. milk samples from before feeding were less likely to contain gram-negative bacilli (36%) than milk samples from the nasogastric tubing (60%). feeding intolerance was observed when there were more than 10 3 colony-forming units per milliliter (cfu/ml), and episodes of sepsis were identified when the bacterial counts in the milk were greater than or equal to 10 6 cfu/ml. this study recommended the routine bacteriologic testing of expressed breast milk. another study from arkansas focused on contamination of feeding tubes during administration of expressed breast milk or formula. 277 ten infants in the neonatal intensive care unit (nicu) were exposed to greater than 10 5 gram-negative bacteria in their feeding tubes. the three infants who were fed expressed breast milk with contamination at greater than 10 5 organisms remained well, but the seven formula-fed infants with high levels of bacterial contamination in the feeding tubes developed necrotizing enterocolitis. the gram-negative bacteria with high level contamination in the feeding tubes were either enterobacter or klebsiella in all cases. many nicus consider 10 5 to 10 6 cfu/ml as the significant bacterial count for gram-negative bacilli in breast milk that places premature and lbw infants at greater risk for infection. even less data are available concerning specific bacterial colony counts for gram-positive organisms and the risk to the infant. generally less than 10 3 gram-positive organisms per ml of milk is considered acceptable, with only case reports and no controlled trials to support this cutoff. when the presence of an infectious illness in an infant and/or the breastfeeding mother' s breast when breast milk is seriously considered as a possible mechanism of transmission to the infant, culturing breast milk to identify the organism may be warranted and useful. more important than hurrying to culture breast milk is the careful instruction of mothers on the proper technique for collecting expressed breast milk, storing it, and cleaning the collection unit. the reinforcement of proper technique from time to time, especially when a question of contamination arises, is equally important. many small reports comment on the contamination of breast milk with different collection methods. relative comparisons suggest decreasing contamination of expressed breast milk when collected by the following methods; drip milk, hand pumped milk, manual expression, modern electric pumped milk. one group from malaysia published results showing no difference in contamination between milk collected by electric pump versus manual expression when collected in the hospital. expressed breast milk collected at home by breast pump had higher rates of contamination with staphylococci and gram-negative bacteria. 46 discussion continues about the need to discard the first few milliliters of milk to lower bacteria numbers in expressed breast milk without any evidence to suggest if this is truly necessary. 62, 337 no evidence shows that cleansing the breast with anything other than tap water decreases the bacterial counts in cultured expressed breast milk. 414 if an infant is directly breastfeeding, collecting milk for culture by manual expression and trying to obtain a "midstream" sample (as is done with "midstream" urine collection for culture) is appropriate. if an infant is being fed expressed breast milk, collecting and culturing the milk at different points during collection (utilizing the same technique the mother uses [manual expression, hand pump, or electric pump]) and administration is appropriate. this might include a sample from immediately after collection, another of stored expressed breast milk, and a sample of milk from the most recent infant feeding at the time the decision to culture is made. please see box 13-1 for the basic steps in culturing expressed breast milk. interpretation of such culture results can be difficult and should involve a pediatric infectious disease expert, a microbiologist, and hospital epidemiologist. additional organism identification is often required, utilizing antibiogram patterns or molecular fingerprinting by various techniques to correlate a bacterial isolate from breast milk with an isolate causing disease in infant or mother. misadministration of breast milk, also known as misappropriation, breast milk exposure, and accidental ingestion of breast milk, and other terms, is a medical-legal issue when it occurs in a hospital. this scenario occurs when one infant receives breast milk from another mother by mistake. this occurrence can be very distressing to the families (recipient patient, recipient parent, and donor mother) and medical staff involved. the actual risk for transmission of an infectious agent to an infant via a single ingestion of expressed breast milk (the most common occurrence) from another mother is exceedingly low. in this scenario, the cdc recommends treating this as an accidental exposure to a body fluid, which could be infectious. 84 bacterial, fungal, or parasitic infection from the one exposure is highly unlikely. the concern is about viral pathogens, known to be blood-borne pathogens, which have been identified in breast milk and include but are not limited to hepatitis b virus (hbv), hepatitis c virus (hcv), cytomegalovirus (cmv), west nile virus, human t-cell lymphotropic virus (htlv), and hiv. most hospitals have protocols for managing the situation from both the infection control/prevention and the medical-legal perspectives. these protocols advise informing both families about what occurred, discussing the theoretical risks of harm from the exposure, and reviewing test results and/ or recommending testing to determine the infectious status of each mother relative to the above mentioned viruses. hcv is not a contraindication to breastfeeding and west nile virus infection in lactating women is rare. 74, 177 neither infection has a documented effective form of prevention or acute treatment. testing either mother (donor or of recipient infant) for these agents is not warranted. prenatal testing for hiv is more commonplace throughout the world. the incidence of hiv among women of childbearing age is low, although it varies significantly by geographic location, and the hospital or locale-specific incidence would be important to know to estimate risk. most women and medical staff are aware that hiv can be transmitted by breastfeeding; therefore breast milk from hiv-positive women is rarely if ever stored in hospitals. the risk for transmission of hiv via breastfeeding is due to the volume of feedings over months (estimated at 400 to 500 feedings in the first 2 months of life) compared with the small "dose of exposure" from one or two "accidental feedings." transmission of hiv from a single breast milk exposure has never been documented. immunologic components in breast milk, along with time and cold of storage, inactivate the hiv in expressed breast milk. for these reasons, the risk for transmission of hiv via expressed breast milk consumed by another child is thought to be extremely low. htlv-i/ii infection in childbearing women is uncommon except in certain geographic regions (japan, africa, the caribbean, and south america). transmission of htlv via breast milk does occur and, like hiv, appears to be related to the volume and duration of breastfeeding. limiting the duration of breastfeeding is effective in decreasing transmission. 407, 409, 446 freezing and thawing expressed breast milk decreases the infectivity of htlv-i. 11 in areas of low prevalence, a positive test in a mother should be suspected to be a false positive test, and retesting with both antibody and polymerase chain reaction (pcr) testing should be performed. for these reasons the transmission of htlv-i/ii via accidental expressed breast milk exposure is thought to be extremely low. although the majority of women are cmv positive by childbearing age and cmv transmission occurs via breastfeeding, the risk for cmv in a full-term infant is low. premature or lbw infants are at greater risk for developing disease with cmv infection. freezing expressed breast milk (at −20° c) for 3 to 5 days significantly decreases the infectivity of cmv. here again the risk for cmv transmission from a single accidental exposure to cmv-positive expressed breast milk is extremely low. with a discussion of theoretical risk should be a discussion of possible preventive interventions, such as vaccination or antimicrobial postexposure prophylaxis. if donor mothers are positive for hbv, it is appropriate to give recipient infants hepatitis b virus immunoglobulin (hbig) and hbv vaccines if they have not already received them. if a box 13-1. culturing breast milk 1. wash hands as per routine. 2. wash breast with warm tap water and a clean washcloth. 3. manually express breast milk ("midstream" collection is not required) or attach breast pump flange (previously cleaned as per routine) for collection and collect milk. 4. place a 3 to 5 ml sample of expressed breast milk in a sterile container with a nonleakable top. 5. deliver to the labatory in less than 1 hour or refrigerate at 4° c until delivery. before sending samples to the viral lab or for nucleic acid/ po lymerase chain reaction (pcr) testing, confirm that the laboratory will accept and process the sample as requested and that the appropriate collection container and prelaboratory management of the specimen are utilized. 6 324 it may also be appropriate to consult a pediatric infectious disease specialist. additional important components of the hospital-based protocols for managing accidental expressed breast milk exposure include ongoing psychosocial support for the families and staff, documentation of medical discussions with the families, investigative steps, consents and interventions, and the demonstration of ongoing infection control efforts to prevent additional events of misadministration of breast milk. microorganisms produce a whole spectrum of clinical illnesses affecting mothers and infants. many situations carry the risk for transmission of the involved organism from a mother to the infant, or vice versa; in general, however, infants are at greater risk because of such factors as inoculum size and immature immune response. as always, an infection must be accurately diagnosed in a timely manner. empiric therapy and initial infection control precautions should begin promptly based on the clinical symptoms and the most likely etiologic agents. when dealing with a maternal infection, clarifying the possible modes of transmission and estimating the relative risk for transmission to the infant are essential first steps to decision-making about isolating a mother from her infant and the appropriateness of continuing breastfeeding or providing expressed breast milk. breastfeeding infrequently is contraindicated in specific maternal infections. 243 often the question of isolation and interruption of breastfeeding arises when symptoms of fever, pain, inflammation, or other manifestations of illness first develop in a mother and the diagnosis is still in doubt. a clinical judgment must be made based on the site of infection, probable organisms involved, possible or actual mechanisms of transmission of these organisms to the infant, estimated virulence of the organism, and likely susceptibility of the infant. additionally, by the time the illness is clearly recognized or diagnosed in a mother, the infant has already been exposed. given the dynamic nature of the immunologic benefits of breast milk, continuation of breastfeeding at the time of diagnosis or illness in a mother can provide the infant protection rather than continued exposure in most illnesses. stopping breastfeeding is rarely necessary. many situations associated with maternal fever do not require separation of mother and infant, such as engorgement of the breasts, atelectasis, localized nonsuppurative phlebitis, or urinary tract infections. appendix f lists a number of clinical syndromes, conditions, and organisms that require infection control precautions in hospitals. this appendix also includes short lists of possible etiologic agents for these conditions and appropriate precautions and recommendations concerning breastfeeding for different scenarios or organisms. this chapter considers specific infectious agents that are common, clinically significant, or of particular interest. bacillus anthracis, a gram-positive, spore-forming rod, causes zoonotic disease worldwide. human infection typically occurs due to contact with animals or their products. three forms of human disease occur: cutaneous anthrax (the most common), inhalation anthrax, and gastrointestinal (gi) disease (rare). person-to-person transmission can occur as a result of discharge from cutaneous lesions, but no evidence of human-to-human transmission of inhalational anthrax is available. no evidence of transmission of anthrax via breast milk exists. standard contact isolation is appropriate for hospitalized patients or patients with draining skin lesions. the issue of anthrax as a biologic weapon has exaggerated its importance as a cause of human disease. the primary concerns regarding anthrax and breastfeeding are antimicrobial therapy or prophylaxis in breastfeeding mothers and the possibility that infant and mother were exposed by intentional aerosolization of anthrax spores. the cdc published recommendations for treatment and prophylaxis in infants, children, and breastfeeding mothers. 72 the recommendations include the use of ciprofloxacin, doxycycline, amoxicillin, and several other agents without discontinuing breastfeeding. little available is information on ciprofloxacin and doxycycline in breast milk for prolonged periods of therapy or prophylaxis (60 days) and possible effects on infants' teeth and bone/cartilage growth during that time period. depending on the clinical situation and sensitivity testing of the identified anthrax strain, other agents can be substituted to complete the 60-day course. the cdc has approved the use of ciprofloxacin and doxycycline for breastfeeding women for short courses of therapy (less than several weeks). simultaneous exposure of infant and mother could occur from primary aerosolization or from spores "contaminating" the local environment. in either case decontamination of the mother-infant dyad' s environment should be considered. breastfeeding can continue during a mother' s therapy for anthrax as long as she is physically well. open cutaneous lesions should be carefully covered and, depending on the situation, simultaneous prophylaxis for the infant may be appropriate. considerable justifiable concern has been expressed because of the reports of sudden infant death from botulism. infant botulism is distinguished from food-borne botulism from improperly preserved food containing the toxin and from wound botulism from spores entering the wound. infant botulism occurs when the spores of clostridium botulinum germinate and multiply in the gut and produce the botulinal toxin in the gi tract. 17 the toxin binds presynaptically at the neuromuscular junction, preventing acetylcholine release. the clinical picture is a descending, symmetric flaccid paralysis. not every individual who has c. botulinum identified in the stool experiences a clinical illness. the age of infants seems to relate to their susceptibility to illness. the illness is mainly in children younger than 12 months of age; the youngest patient described in the literature was 6 days old. 17 most children become ill between 6 weeks and 6 months of age. the onset of illness seems to occur earlier in formula-fed infants compared with breastfed infants. when a previously healthy infant younger than 6 months of age develops constipation, then weakness and difficulty sucking, swallowing, crying, or breathing, botulism is a likely diagnosis. the organisms should be looked for in the stools, and electromyography may or may not be helpful. in a group reviewed by arnon et al, 19 33 of 50 patients hospitalized in california were still being nursed at onset of the illness. a beneficial effect of human milk was observed in the difference in the mean age at onset, with breastfed infants being twice as old as formula-fed infants with the disease. the breastfed infants' symptoms were milder. breastfed infants receiving iron supplements developed the disease earlier than those who were breastfed but unsupplemented. of the cases of sudden infant death from botulism, no infants were breastfed within 10 weeks of death. all were receiving iron-fortified formulas. in most cases, no specific food source of c. botulinum can be identified, but honey is the food most often implicated, and corn syrup has been implicated in infants older than 2 months of age. honey may contain botulism spores, which can germinate in the infant gut. however, botulin toxin has not been identified in honey. it has been recommended that honey not be given to infants younger than 12 months of age. this includes putting honey on a mother' s nipples to initiate an infant' s interest in suckling. arnon 18 reviewed the first 10 years of infant botulism monitoring worldwide. the disease has been reported from 41 of the 50 states in the united states and from eight countries on four continents. the relationship to breastfeeding and human milk is unclear. in general the acid stools (ph 5.1 to 5.4) of human milk fed infants encourage bifidobacterium species. few facultative anaerobic bacteria, or clostridia, existing as spores, are present in breastfed infants. in contrast, formula-fed infants have stool phs ranging from 5.9 to 8.0, with few bifidobacteria, primarily gram-negative bacteria, especially coliforms and bacteroides species. c. botulinum growth and toxin production decrease with declining ph and usually stops below ph 4.6. breast milk also contains additional protective immunologic components, which purportedly have activity against botulinum toxin. 269 the relationship between the introduction of solid foods or weaning in both formula-fed and breastfed infants and the onset of botulism remains unclear. for a breastfed infant, the introduction of solid food may cause a major change in the gut with a rapid rise in the growth of enterobacteria and enterococci followed by progressive colonization by bacteroides species, clostridia, and anaerobic streptococci. feeding solids to formula-fed infants minimally changes the gut flora as these organisms already predominate. although more hospitalized infants have been breastfed, sudden-death victims are younger and have been formula fed, which supports the concept of immunologic protection in the gut of a breastfed infant. much work remains to understand this disease. clinically, constipation, weakness, and hypotonicity in a previously healthy child constitute botulism until ruled out, especially with recent dietary changes. at this time, no reason exists to suspect breastfeeding as a risk for infant botulism, and some evidence suggests a possible protective effect from breastfeeding. breastfeeding should continue if botulism is suspected in mother or infant. brucella melitensis has been isolated in the milk of animals. foods and animals represent the primary sources of infection in humans. brucellosis demonstrates a broad spectrum of illness in humans, from subclinical to subacute to chronic illness with nonspecific signs of weakness, fever, malaise, body aches, fatigue, sweats, arthralgia, and lymphadenitis. in areas where the disease is enzootic, childhood illness has been described more frequently. the clinical manifestations in children are similar to those in adults. 259 infection can occur during pregnancy, leading to abortion (infrequently), and can produce transplacental spread, causing neonatal infection (rarely). the transmission of b. melitensis through breast milk has been implicated in neonatal infection. 259, 260 there have been eight cases of brucellosis in infants that were possibly associated with breastfeeding, but brucella was not isolated from the breast milk in any of those cases.* one case of brucellosis in an infant caused by breast milk transmission, with b. melitensis isolated from the breast milk, before antibiotic treatment was given to the mother has been documented. 415 additionally, brucella melitensis has been cultured from women with breast lumps and abscesses. 295 only one of six women described in this report was lactating at the time of diagnosis, and no information about the infant was given. brucellosis mastitis or abscess should be considered in women presenting with appropriate symptoms and occupational exposure to animals, contact with domestic animals in their environment, or exposure to animal milk or milk products (especially unpasteurized products). the breast inflammation tends to be granulomatous in nature (without caseation) and is often associated with axillary adenopathy; occasionally systemic illness in the woman is evident. treatment of brucellosis mastitis or abscess should be treated with surgery or fine needle aspiration as indicated and 4 to 6 weeks of combination antibiotic therapy with two or three medications. temporary interruption of breastfeeding with breast pumping and discarding the milk to continue stimulation of milk production is appropriate. breastfeeding should then continue after an initial period of 48 to 96 hours of therapy in the mother. acceptable medications for treating the mother while continuing breastfeeding include gentamicin, streptomycin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and rifampin (see appendix d). chlamydial infection is the most frequent sexually transmitted disease (std) in the united states and is a frequent cause of conjunctivitis and pneumonitis in an infant from perinatal infection. the major determinant of whether chlamydial infection occurs in a newborn is the prevalence rate of chlamydial infection of the cervix. 364 chlamydial immunoglobulin a (iga) has been found in colostrum and breast milk in a small number of postpartum women who were seropositive for chlamydia. no information is available on the role of milk antibodies in protection against infection in infants. 389 it is not believed that chlamydia is transmitted via breast milk. use of erythromycin or tetracycline to treat mothers and oral erythromycin and ophthalmic preparations of tetracyclines, erythromycin, or sulfonamides to treat suspected infection in infants are appropriate during continued breastfeeding. separating infants from mothers with chlamydial infections or stopping breastfeeding is not indicated. simultaneous treatment of mothers and infants may be appropriate in some situations. corynebacterium diphtheriae causes several forms of clinical disease, including membranous nasopharyngitis, obstructive laryngotracheitis, and cutaneous infection. complications can include airway obstruction from membrane formation and toxinmediated central nervous system (cns) disease or myocarditis. the overall incidence of diphtheria has declined even though immunization does not prevent infection but does prevent severe disease from toxin production. fewer than five cases are reported annually in the united states. transmission occurs via droplets or direct contact with contaminated secretions from the nose, throat, eye, or skin. infection occurs in individuals whether they have been immunized or not, but infection in those not immunized is more severe and prolonged. as long as the skin of the breast is not involved, no risk for transmission exists via breast milk. no toxin-mediated disease from toxin transmitted through breast milk has been reported in an infant. breastfeeding, along with chemoprophylaxis and immunization of affected infants, is appropriate in the absence of cutaneous breast involvement (see appendix f). maternal infection with neisseria gonorrhoeae can produce a large spectrum of illness ranging from uncomplicated vulvovaginitis, proctitis, pharyngitis, conjunctivitis, or more severe and invasive disease, including pelvic inflammatory disease, meningitis, endocarditis, or disseminated gonococcal infection. the risk for transmission from mother to infant occurs mainly during delivery in the passage through the infected birth canal and occasionally from postpartum contact with the mother (or her partner). risk for transmission from breast milk is negligible, and n. gonorrhoeae does not seem to cause local infection of the breasts. infection in neonates is most often ophthalmia neonatorum and less often a scalp abscess or disseminated infection. mothers with presumed or documented gonorrhea should be reevaluated for other stds, especially chlamydia trachomatis and syphilis, because some therapies for gonorrhea are not adequate for either of these infections. with the definitive identification of gonorrhea in a mother, empiric therapy should begin immediately, and the mother should be separated from the infant until completion of 24 hours of adequate therapy. treatment of the mother with ceftriaxone, cefixime, penicillin, or erythromycin is without significant risk to the infant. single-dose treatment with spectinomycin, ciprofloxacin, ofloxacin, or azithromycin has not been adequately studied but presumably would be safe for the infant given the 24-hour separation and a delay in breastfeeding without giving the infant the expressed breast milk (pump and discard). doxycycline use in a nursing mother is not routinely recommended. careful preventive therapy for ophthalmia neonatorum should be provided, and close observation of the infant should continue for 2 to 7 days, the usual incubation period. empiric or definitive therapy against n. gonorrhoeae may be necessary depending on an infant' s clinical status and should be chosen on the basis of the maternal isolate' s sensitivity pattern. the mother should not handle other infants until after 24 hours of adequate therapy, and the infant should be separated from the rest of the nursery population, with or without breastfeeding. haemophilus influenzae type b can cause severe invasive disease such as meningitis, sinusitis, pneumonia, epiglottitis, septic arthritis, pericarditis, and bacteremia. shock can also occur. because the increased utilization of the h. influenzae type b conjugate vaccines, invasive disease caused by haemophilus has decreased dramatically, more than 95%, in the united states. most invasive disease occurs in children 3 months to 3 years of age. older children and adults rarely experience severe disease but do serve as sources of infection for young children. children younger than 3 months of age seem to be protected because of passively acquired antibodies from the mothers, and some additional benefits may be received from breast milk. transmission occurs through contact with respiratory secretions, and droplet precautions are protective. no evidence suggests transmission through breast milk or breastfeeding. evidence supports that breast milk limits the colonization of h. influenzae in the throat. 185 in the rare case of maternal infection, an inadequately immunized infant in a household is an indication to provide rifampin prophylaxis and close observation for all household contacts, including the breastfeeding infant. expressed breast milk can be given to an infant during the 24-hour separation after the mother' s initiation of antimicrobial therapy, or if the mother' s illness prevents breastfeeding, it can be reinitiated when the mother is able (see appendix f). although uncommon in the united states, leprosy occurs throughout the world. this chronic disease presents with a spectrum of symptoms depending on the tissues involved (typically the skin, peripheral nerves, and mucous membranes of the upper respiratory tract) and the cellular immune response to the causative organism, mycobacterium leprae. transmission occurs through long-term contact with individuals with untreated or multibacillary (large numbers of organisms in the tissues) disease. leprosy is not a contraindication to breastfeeding, according to jeliffe and jeliffe. 202 the importance of breastfeeding and urgency of treatment are recognized by experts who treat infants and mothers early and simultaneously. no mother-infant contact is permitted except to breastfeed. dapsone, rifampin, and clofazimine are typically and safely used for infant and mother regardless of the method of feeding (see appendix d). listeriosis is a relatively uncommon infection that can have a broad range of manifestations. in immunocompetent individuals, including pregnant women, the infection can vary from being asymptomatic to presenting as an influenza-like illness, occasionally with gi symptoms or back pain. severe disease occurs more frequently in immunodeficient individuals or infants infected in the perinatal period (pneumonia, sepsis, meningitis, granulomatosis infantisepticum). although listeriosis during pregnancy may manifest as mild disease in a mother and is often difficult to recognize and diagnose, it is typically associated with stillbirth, abortion, and premature delivery. it is thought that transmission occurs through the transplacental hematogenous route, infecting the amniotic fluid, although ascending infection from the genital tract may occur. 122 early and effective treatment of a woman can prevent fetal infection and sequelae. 206, 257 neonatal infection occurs as either early-or late-onset infection from transplacental spread late in pregnancy, ascending infection during labor and delivery, infection during passage through the birth canal, or, rarely, during postnatal exposure. no evidence in the literature suggests that listeria is transmitted through breast milk. treatment of the mother with ampicillin, penicillin, or trimethoprim-sulfamethoxazole is not a contraindication to breastfeeding as long as the mother is well enough. expressed colostrum or breast milk also can be given if the infant is able to feed orally. the management of lactation and feeding in neonatal listeriosis is conducted supportively, as it is in any situation in which an infant is extremely ill, beginning feeding with expressed breast milk or directly breastfeeding as soon as reasonable. n. meningitidis most often causes severe invasive infections, including meningococcemia or meningitis often associated with fever and a rash and progressing to purpura, disseminated intravascular coagulation, shock, coma, and death. transmission occurs via respiratory droplets. spread can occur from an infected, ill individual or from an asymptomatic carrier. droplet precautions are recommended until 24 hours after initiation of effective therapy. despite the frequent occurrence of bacteremia, no evidence indicates breast involvement or transmission through breast milk. the risk for maternal infection to an infant after birth is from droplet exposure and exists whether the infant is breastfeeding or bottle feeding. in either case the exposed infant should receive chemoprophylaxis with rifampin, 10 mg/kg/dose every 12 hours for 2 days (5 mg/kg/dose for infants younger than 1 month of age), or ceftriaxone, 125 mg intramuscularly (im) once, for children younger than 15 years of age. close observation of the infant should continue for 7 days, and breastfeeding during and after prophylaxis is appropriate. the severity of maternal illness may prevent breastfeeding, but it can continue if the mother is able, after the mother and infant have been receiving antibiotics for 24 hours. a period of separation from the index case for the first 24 hours of effective therapy is recommended; expressed breast milk can be given during this period. respiratory illness caused by bordetella pertussis evolves in three stages: catarrhal (nasal discharge, congestion, increasing cough), paroxysmal (severe paroxysms of cough sometimes ending in an inspiratory whoop, i.e., whooping cough), and convalescent (gradual improvement in symptoms). transmission is via respiratory droplets. the greatest risk for transmission occurs in the catarrhal phase, often before the diagnosis of pertussis. the nasopharyngeal culture usually becomes negative after 5 days of antibiotic therapy. chemoprophylaxis for all household contacts is routinely recommended. no evidence indicates transmission through breast milk, with similar risk to breastfed and bottle-fed infants. in the case of maternal infection with pertussis, chemoprophylaxis for all household contacts, regardless of age or immunization status, is indicated. in addition to chemoprophylaxis of the infant, close observation and subsequent immunization (in infants older than 6 weeks of age) are appropriate. despite chemoprophylaxis, droplet precautions and separation of mother and infant during the first 5 days of effective maternal antibiotic therapy are recommended. expressed breast milk can be provided to the infant during this period. staphylococcal infection in neonates can be caused by either s. aureus or coagulase-negative staphylococci (most often s. epidermidis) and can manifest in a wide range of illnesses. localized infection can be impetigo, pustulosis in neonates, cellulitis, or wound infection, and invasive or suppurative disease includes sepsis, pneumonia, osteomyelitis, arthritis, and endocarditis. s. aureus requires only a small inoculum (10 to 250 organisms) to produce colonization in newborns, most often of the nasal mucosa and umbilicus. 193 by the fifth day of life, 40% to 90% of the infants in the nursery will be colonized with s. aureus. 126 the organism is easily transmitted to others from mother, infant, family, or health care personnel through direct contact. outbreaks in nurseries were common in the past. mothers, infants, health care workers, and even contaminated, unpasteurized, banked breast milk were sources of infection. 298, 326 careful use of antibiotics, changes in nursery layout and procedures, standard precautions, and cohorting as needed decreased the spread of s. aureus in nurseries. now the occurrence of methicillin-resistant s. aureus (mrsa) is again a common problem, requiring cohorting, occasionally epidemiologic investigation, and careful infection control intervention. there are numerous reports of mrsa outbreaks in nicus.* the significance of colonization with staphylococcus and the factors leading to development of disease in individual patients are not clear. the morbidity and mortality related to s. aureus infection in neonates is well described. 192, 195, 219 management of such outbreaks has been reviewed. 147, 250 little has been written about the role of breastfeeding in colonization with s. aureus in nicus, wellbaby nurseries, or at home. mrsa is an important pathogen worldwide. community-acquired mrsa is different from hospital-acquired mrsa. community-acquired mrsa is usually defined as occurring in an individual without the common predisposing variables associated with hospital-acquired mrsa, lacking a mdr phenotype (common with hospital-acquired mrsa), frequently carrying multiple exotoxin virulence factors (such as panton-valentine leukocidin toxin), as well as carrying the smaller type iv staphylococcal cassette cartridge for the meca gene on a chromosome (hospital-acquired mrsa carries types i-iii staphylococcal cassette cartridge) and as being molecularly distinct from the common nosocomial strains of hospital-acquired mrsa. community-acquired mrsa is most commonly associated with skin and soft tissue infections and necrotizing pneumonia and less frequently associated with endocarditis, bacteremia, necrotizing fasciitis, myositis, osteomyelitis, or parapneumonic effusions. community-acquired mrsa is so common, it is now being observed in hospital outbreaks. 24, 144, 164, 358 community-acquired mrsa transmission to infants via breast milk has been reported. 34, 144, 210, 253, 286 premature or small-forgestational-age infants are more susceptible to and at increased risk for significant morbidity and mortality due to mrsa due in part to prolonged hospitalization, multiple courses of antibiotics, invasive procedures, and intravenous (iv) lines, their relative immune deficiency due to prematurity and illness, and altered gi tract due to different flora and decreased gastric acidity. therefore colonization with mrsa may pose a greater risk to infants in nicus in the long run. full-term infants develop pustulosis, cellulitis, and soft tissue infections, but rarely has invasive disease been reported. 82, 132, 298 fortunov et al 132 from texas reported 126 infections in term or late-preterm previously well infants including 43 with pustulosis, 68 with celluliltis or abscesses, and 15 invasive infections. family history of soft tissue skin infections and male sex were the only variables associated with risk for infection; cesarean delivery, breastfeeding, and circumcision were not. 132 nguyen et al 298 reported mrsa infections in a well-infant nursery from california. the eleven cases were all in full-term boys with pustularvesicular lesions in the groin. the infections were associated with longer length of stay, lidocaine injection use in infants, maternal age older than 30 years, and circumcision. breastfeeding was not an associated risk factor for mrsa infection. 298 the question of the role of circumcision in mrsa outbreaks was addressed by van howe and robson. 426 they reported that circumcised boys are at greater risk for staphylococcal colonization and infection. 426 others report that s. aureus carriage in infants (and subsequent infection) is most likely affected by multiple variables including infant factors (antibiotics, surgical procedures [circumcision being the most common], duration of hospital stay as a newborn), maternal factors (previous colonization, previous antibiotic usage, mode of delivery, length of stay), and environmental factors (mrsa in the family or hospital, nursery stay versus rooming-in, hand hygiene).* gerber et al 147 from the chicago area published a consensus statement for the management of mrsa outbreaks in the nicu. the recommendations, which were strongly supported by experimental, clinical, and epidemiologic data, included using a waterless, alcohol-based hand hygiene product, monitoring and enforcing hand hygiene, placing mrsa-positive infants in contact precautions with cohorting if possible, using gloves and gowns for direct contact and masks for aerosolgenerating procedures, cohorting nurses for care of mrsa-positive infants when possible, periodic screening of infants for mrsa using nares or nasopharyngeal cultures, clarifying the mrsa status of infants being transferred into the nicu, limiting overcrowding, and maintaining ongoing instruction and monitoring of health care workers in their compliance with infection control and hand hygiene procedures. evaluation of the outbreak could include screening of health care workers and environmental surfaces to corroborate epidemiologic data and laboratory molecular analysis of the mrsa strains if indicated epidemiologically. the use of mupirocin or other decolonizing procedures should be determined on an individual basis for each nicu. s. aureus is the most common cause of mastitis in lactating women. 317, 394, 395, 436 recurrence or persistence of symptoms of mastitis is a well described occurrence and an important issue in the management of mastitis. communityacquired mrsa has been associated with mastitis as well. 342 pasteurization, s. aureus was not detected in any of the 6820 samples of expressed breast milk. colonization of one infant with mrsa was identified, but no mrsa infections were identified in any of the hospitalized infants in the nicu during the 18 months of the study. 26 novak et al 300 identified mrsa in 57 of 500 samples (11%) of expressed fresh-frozen milk from 500 different donors from five brazilian milk banks. only 3 of the 57 samples were positive with high-level bacterial counts of mrsa: greater than 10,000 cfu/ml. these were the only samples that would not have been acceptable by bacteriological criteria according to brazilian or american criteria for raw milk use. they did not investigate other epidemiologic data to identify possible variables associated with low or high level contamination of expressed breast milk with mrsa. 300 management of an infant and/or mother with mrsa infection relative to breastfeeding or use of breast milk should be based on the severity of disease and whether the infant is premature, lbw, very-lowbirth-weight (vlbw), previously ill, or full term. full-term infants who themselves or their mothers develop mild to moderate infections (impetigo, pustulosis, cellulitis/abscess, mastitis/breast abscess, or soft tissue infection) can continue breast feeding after a short period of interruption (24 to 48 hours). during this time, pumping to maintain the milk supply should be supported, an initial evaluation for other evidence of infection should be done in the maternalinfant dyad, the infected child and/or mother should be placed on "commonly" effective therapy for the mrsa infection, and ongoing observation for clinical disease should continue. the mother and infant can "room-in" together in the hospital, if necessary, with standard and contact precautions. culturing the breast milk is not necessary. empiric therapy for the infant may be chosen based on medical concerns for the infant and the known sensitivity testing of the mrsa isolate. appropriate antibiotic choices include short-term use of azithromycin (erythromycin use during infancy [less than 6 weeks of age], or breastfeeding associated with an increased risk for hypertrophic pyloric stenosis), sulfamethoxazoletrimethoprim (in the absence of g6pd deficiency and older than 30 days of age), clindamycin, and perhaps linezolid for mild to moderate infections. infants in nicus (premature, lbw, vlbw, and/ or previously ill), who themselves or their mothers have a mrsa infection, should have the breast milk cultured and suspend breastfeeding or receiving breast milk from their mother until the breast milk is shown to be culture negative for mrsa. the infant should be treated as indicated for their infection or empirically treated if symptomatic (with pending culture results) and closely observed for development of new signs or symptoms of infection. pumping to maintain the milk supply and the use of banked breast milk are appropriate. the infant should be placed on contact precautions, in addition to the routine standard precautions. the infant can be cohorted with other mrsa-positive infants with nursing care cohorted as well. for the mother with mrsa infection, she should be instructed concerning hand hygiene, the careful collection, handling, and storage of breast milk, contact precautions to be used with her infant, and the avoidance of contact with any other infants. the mother can receive several possible antibiotics for mrsa that are compatible with breastfeeding when used for a short period. if the mother remains clinically well, including without evidence of mastitis, but her breast milk is positive for mrsa greater than 10 4 cfu/ml, empiric therapy to diminish or eradicate colonization would be appropriate. various regimens have been proposed to "eradicate" mrsa colonization, but none have been proven to be highly efficacious. these regimens usually include systemic antibiotics with one or two medications (rifampin added as the second medication), nasal mupirocin to the nares twice daily for 1 to 2 weeks with routine hygiene, with or without the usage of hexachlorophene (or similar topical agent or cleanser) for bathing during the 1 to 2 week treatment period. there is no clear information concerning the efficacy of using similar colonization eradication regimens for other household members or pets in preventing recolonization of the mother or infant. before reintroducing the use of the mother' s breast milk to the infant at least two to three negative breast milk cultures should be obtained after completion of therapy. routine screening of breast milk provided by mothers for their infants in nicus for the presence of mrsa is not indicated in the absence of mrsa illness in the maternal-infant dyad, an mrsa outbreak in nicus, or a high frequency of mrsa infection in a specific nicu. one case of staphylococcal scalded skin syndrome was reported by katzman and wald 208 in an infant breastfed by a mother with a lesion on her areola that did not respond to ampicillin therapy for 14 days. subsequently the infant developed conjunctivitis with s. aureus, which produced an exfoliative toxin, and a confluent erythematous rash without mucous membrane involvement or nikolsky sign. no attempt to identify the exfoliative toxin in the breast milk was made, and the breast milk was not cultured for s. aureus. the child responded to iv therapy with nafcillin. this emphasizes the importance of evaluating mother and infant at the time of a suspected infection and the need for continued observation of the infant for evidence of a pyogenic infection or toxin-mediated disease, especially with maternal mastitis or breast lesions. this case also raises the issue of when and how infants and their mothers become colonized with s. aureus and what factors lead to infection and illness in each. the concern is that staphylococcus can be easily transmitted through skin to skin contact, colonization readily occurs, and potentially serious illness can occur later, long after colonization. in the case of staphylococcal scalded skin syndrome or toxic shock syndrome (tss), the primary site of infection can be insignificant (e.g., conjunctivitis, infection of a circumcision, or simple pustulosis), but a clinically significant amount of toxin can be produced and lead to serious disease. toxic shock syndrome can result from s. aureus or streptococcus pyogenes infection and probably from a variety of antigens produced by other organisms. tss-1 has been identified as a "superantigen" that affects the t lymphocytes and other components of the immune response, producing an unregulated and excessive immune response and resulting in an overwhelming systemic clinical response. tss has been reported in association with vaginal delivery, cesarean delivery, mastitis, and other local infections in mothers. mortality rate in the mother may be as high as 5%. the case definition of staphylococcal tss includes meeting all four major criteria: fever greater than 38.9° c, rash (diffuse macular erythroderma), hypotension, and desquamation (associated with subepidermal separation seen on skin biopsy). the definition also includes involvement of three or more organ systems (gi, muscular, mucous membrane, renal, hepatic, hematologic, or central nervous system); negative titers for rocky mountain spotted fever, leptospirosis, and rubeola; and lack of isolation of s. pyogenes from any source or s. aureus from the cerebrospinal fluid (csf). 368 a similar case definition has been proposed for streptococcal tss. 451 aggressive empiric antibiotic therapy against staphylococci and streptococci and careful supportive therapy are essential to decreasing illness and death. oxacillin, nafcillin, first-generation cephalosporins, clindamycin, erythromycin, and vancomycin are acceptable antibiotics, even for a breastfeeding mother. the severity of illness in the mother may preclude breastfeeding, but it can be reinitiated when the mother is improving and wants to restart. standard precautions, but allowing breastfeeding, are recommended. staphylococcal enterotoxin f has been identified in breast milk specimens collected on days 5, 8, and 11 from a mother who developed tss at 22 hours postpartum. 428 s. aureus that produced staphylococcal enterotoxin f was isolated from the mother' s vagina but not from breast milk. infant and mother lacked significant antibody against staphylococcal enterotoxin f in their sera. the infant remained healthy after 60 days of follow-up. staphylococcal enterotoxin f is pepsin inactivated at ph 4.5 and therefore is probably destroyed in the stomach environment, presenting little or no risk to the breastfeeding infant. 35 breastfeeding can continue if the mother is able. coagulase-negative staphylococcal infection (the predominant isolate is staphylococcus epidermidis) produces minimal disease in healthy, full-term infants but is a significant problem in hospitalized or premature infants. factors associated with increased risk for this infection include prematurity, high colonization rates in specific nurseries, invasive therapies (e.g., iv lines, chest tubes, intubation), and antibiotic use. illness produced by coagulasenegative staphylococci can be invasive and severe in high-risk neonates, but rarely in mothers. there are reports of necrotizing enterocolitis associated with coagulase-negative staphylococcus. at 2 weeks of age, for infants still in the nursery, s. epidermidis is a frequent colonizing organism at multiple sites, with colonization rates as high as 75% to 100%. serious infections with coagulase-negative staphylococci (e.g., abscesses, iv line infection, bacteremia/sepsis, endocarditis, osteomyelitis) require effective iv therapy. many strains are resistant to penicillin and the semisynthetic penicillins, so sensitivity testing is essential. empiric or definitive therapy may require treatment with vancomycin, gentamicin, rifampin, teicoplanin, linezolid, or combinations of these for synergistic activity. transmission of infection in association with breastfeeding appears to be no more common than with bottle feeding. as with s. aureus infection control includes contact and standard precautions. occasionally, during presumed outbreaks, careful epidemiologic surveillance may be required, including cohorting, limiting overcrowding and understaffing, surveillance cultures of infants and nursery personnel, reemphasis of meticulous infection control techniques for all individuals entering the nursery, and, rarely, removal of colonized personnel from direct infant contact. s. epidermidis has been identified as part of fecal microbiota of breastfed infants. 203 s. epidermidis has also been identified in the breast milk of women with clinical evidence of mastitis. 107 nevertheless, s. epidermidis is rarely associated with infection in full-term infants. conceivably breast milk for premature infants could be a source of s. epidermidis colonization in the nicus. the other factors associated with hospitalization in a nicu noted previously presumably play a significant role in both colonization and infection in premature infants. the benefits of early full human milk feeding potentially outweigh the risk for colonization with s. epidermidis via breast milk. 348 ongoing education and assistance should be provided to mothers about the careful collection, storage, and delivery of human breast milk for their premature infants. 353 s. pyogenes (β-hemolytic group a streptococcus [gas]) is a common cause of skin and throat infections in children, producing pharyngitis, cellulitis, and impetigo. illnesses produced by gas can be classified in three categories: (1) impetigo, cellulitis, or pharyngitis without invasion or complication; (2) severe invasive infection with bacteremia, necrotizing fasciitis, myositis, or systemic illness (e.g., streptococcal tss); and (3) autoimmune-mediated phenomena, including acute rheumatic fever and acute glomerulonephritis. gas can also cause puerperal sepsis, endometritis, and neonatal omphalitis. significant morbidity and mortality rates are associated with invasive gas infection; mortality rate is 20% to 50%, with almost half the survivors requiring extensive tissue débridement or amputation. 347 infants are not at risk for the autoimmune sequelae of gas (rheumatic fever or poststreptococcal glomerulonephritis). transmission is through direct contact (rarely indirect contact) and droplet spread. outbreaks of gas in the nursery are rare, unlike with staphylococcal infections. either mother or infant can be initially colonized with gas and transmit it to the other. in the situation of maternal illness (extensive cellulitis, necrotizing fasciitis, myositis, pneumonia, tss, mastitis), it is appropriate to separate mother and infant until effective therapy (penicillin, ampicillin, cephalosporins, erythromycin) has been given for at least 24 hours. breastfeeding should also be suspended and may resume after 24 hours of therapy for the mother. group b streptococcus (gbs, streptococcus agalactiae) is a significant cause of perinatal bacterial infection. in parturient women, infection can lead to asymptomatic bacteriuria, urinary tract infection (often associated with premature birth), endometritis, or amnionitis. in infants, infection usually occurs between birth and 3 months of age (1 to 4 cases per 1000 live births). it is routinely classified by the time of onset of illness in the infant: early onset (0 to 7 days, majority less than 24 hours) and late onset (7 to 90 days, generally less than 4 weeks). infants may develop sepsis, pneumonia, meningitis, osteomyelitis, arthritis, or cellulitis. early-onset gbs disease is often fulminant, presenting as sepsis or pneumonia with respiratory failure; three quarters of neonatal disease is early onset. type iii is the most common serotype causing disease. transmission is believed to occur in utero and during delivery. colonization rates of mothers and infants vary between 5% and 35%. postpartum transmission is thought to be uncommon, although it has been documented. risk factors for early-onset gbs disease include delivery before 37 weeks' gestation, rupture of membranes for longer than 18 hours before delivery, intrapartum fever, heavy maternal colonization with gbs, or low concentrations of anti-gbs capsular antibody in maternal sera. 95 the common occurrence of severe gbs disease before 24 hours of age in neonates has lead to prevention strategies. revised guidelines developed by the aap committees on infectious diseases and on the fetus and newborn 95 have tried to combine various variables for increased risk for gbs infection (prenatal colonization with gbs, obstetric and neonatal risk factors for early-onset disease) and provide intrapartum prophylaxis to those at high risk ( figure 13 -1) the utilization of these guidelines and intrapartum prophylaxis across the united states has decreased the incidence of early-onset disease by approximately 80%. in 2005, the incidence of early-onset disease was 0.35 cases per 1000 live births. 95 late-onset gbs disease is thought to be the result of transmission during delivery or in the postnatal period from maternal, hospital, or community sources. dillon et al 112 demonstrated that 10 of 21 infants with late-onset disease were colonized at birth, but the source of colonization was unidentified in the others. gardner et al 141 showed that only 4.3% of 46 children who were culture negative for gbs at discharge from the hospital had acquired gbs by 2 months of age. anthony et al 15 noted that many infants are colonized with gbs, but the actual attack rate for gbs disease is low and difficult to predict. acquisition of gbs through breast milk or breastfeeding is uncommon. cases of late-onset gbs disease associated with gbs in the maternal milk have been reported. 58, 214, 313, 366, 438 some of the mothers had bilateral mastitis, at least one had delayed evidence of unilateral mastitis, and the others were asymptomatic. it was not clear when colonization of the infants occurred or when infection or disease began in the infants. the authors discussed the possibility that the infants were originally colonized during delivery, subsequently colonized the mothers' breasts during breastfeeding, and then became reinfected at a later time. butter and demoor 56 showed that infants initially colonized on their heads at birth had gbs cultured from their throat, nose, or umbilicus 8 days later. whenever they cultured gbs from the nipples of mothers, the authors also found it in the nose or throat of the infants. byrne et al 58 presented a review of gbs disease associated with breastfeeding and made recommendations to decrease the risk for transmission of gbs to infants via breastfeeding or breast milk. some of their recommendations included confirming appropriate collection and processing procedures for gbs cultures 370 in medical facilities to decrease false-negative cultures, reviewing proper hygiene for pumping, collection, and storage of expressed breast milk with mothers, reviewing the signs and symptoms of mastitis with mothers, and utilizing banked human milk as needed instead of mother' s milk. when a breastfed infant develops late-onset gbs disease, it is appropriate to culture the milk. (see discussion of culturing breast milk earlier in this chapter.) consider treatment of the mother to prevent reinfection if the milk is culture positive for gbs (greater than 10 4 cfu/ml), with or without clinical evidence of mastitis in the mother. withholding the mother' s milk until it is confirmed to be culture negative for a pathogen is appropriate and should be accompanied by providing ongoing support and instruction to the mother concerning pumping and maintaining her milk supply. serial culturing of expressed breast milk after treatment of the mother for gbs disease or colonization would be appropriate to insure the ongoing absence of a pathogen in the expressed breast milk. there are reports of reinfection of the infant from breast milk. 23, 225 eradication of gbs mucosal colonization in the infant or the mother may be difficult. some authors have recommended using rifampin prophylactically in both the mother and infant at the end of treatment to eradicate mucosal colonization. 23 (see chapter 16 for management of mastitis in the mother.) a mother or infant colonized or infected with gbs should be managed with standard precautions 94 while in the hospital. ongoing close evaluation of the infant for infection or illness and empiric therapy for gbs in the infant are appropriate until the child has remained well and cultures are subsequently negative at 72 hours. occasionally, epidemiologic investigation in the hospital will utilize culturing medical staff and family members to detect a source of late-onset gbs disease in the nursery. this can be useful when more than one case of late-onset disease is detected with the same serotype. cohorting in such a situation may be appropriate. selective prophylactic therapy for colonized infants to eradicate colonization may be considered, but unlike gas or staphylococcus infection, gbs infection in nurseries has not been reported to cause outbreaks. no data support screening all breastfeeding mothers and their expressed breast 4 cbc including wbc count with differential and blood culture. 5 applies only to penicillin, ampicillin, or cefazolin and assumes recommended dosing regimens. 6 a healthy-appearing infant who was ≥ 38 weeks' gestation at delivery and whose mother received ≥ 4 hours of iap before delivery may be discharged home after 24 hours if other discharge criteria have been met and a person able to comply fully with instructions for home observation will be present. if any one of these conditions is not met, the infant should be observed in the hospital for at least 48 hours and until criteria for discharge are achieved. milk for gbs as a reasonable method for protecting against spread of gbs infection via expressed breast milk. selective culturing of expressed breast milk may be appropriate in certain situations. the face of tuberculosis (tb) is changing throughout the world. in the united states the incidence of tb rose during 1986 through 1993 and has been declining since then. 60 increased rates of tb were noted in adults between 25 and 45 years of age, and because these are the primary childbearing years, the risk for transmission to children increased. tb during pregnancy has always been a significant concern for patients and physicians alike. 340 it is now clear that the course and prognosis of tb in pregnancy are less affected by the pregnancy and more determined by the location and extent of disease, as defined primarily by chest radiograph, and by the susceptibility of the individual patient. untreated tb in pregnancy is associated with maternal and infant mortality rates of 30% to 40%. 365 effective therapy is crucial to the clinical outcome in both pregnant and nonpregnant women. tb during pregnancy rarely results in congenital tb. any individual in a high-risk group for tb should be screened with a tuberculin skin test (tst). no contraindication or altered responsiveness to the tst exists during pregnancy or breastfeeding. interpretation of the tst should follow the most recent guidelines, using different sizes of induration in different-risk populations as cutoffs for a positive test, as proposed by the cdc. 68 figure 13 -2 outlines the evaluation and treatment of a pregnant woman with a positive tst. 398 treatment of active tb should begin as soon as the diagnosis is made, regardless of the fetus' gestational age, because the risk for disease to mother and fetus clearly outweighs the risks of treatment. isoniazid, rifampin, and ethambutol have been used safely in all three trimesters. isoniazid and pyridoxine therapy during breastfeeding is safe, although the risk for hepatotoxicity in the mother may be a concern during the first 2 months postpartum. 391 congenital tb is extremely rare if one considers that 7 to 8 million cases of tb occur each year worldwide and that less than 300 cases of congenital tb have been reported in the literature. as with other infectious diseases presenting in the perinatal period, distinguishing congenital infection from perinatal or postnatal tb in infants can be difficult. postnatal tb infection in infancy typically presents with severe disease and extrapulmonary extension (meningitis, lymphadenopathy, and bone, liver, spleen involvement). airborne transmission of tb to infants is the major mode of postnatal infection because of close and prolonged exposure in enclosed spaces, especially in their own household, to any adult with infectious pulmonary tb. potential infectious sources could be the mother or any adult caregiver, such as babysitters, day care workers, relatives, friends, neighbors, and even health care workers. the suspicion of tb infection or disease in a household with possible exposure of an infant is a highly anxiety-provoking situation ( figure 13 -3). although protection of an infant from infection is foremost in everyone' s mind, separation of the infant from the mother should be avoided when reasonable. every situation is unique, and the best approach will vary according to the specifics of the case and accepted principles of tb management. the first step in caring for the potentially exposed infant is to determine accurately the true tb status of the suspected case (mother or household contact). this prompt evaluation should include a complete history (previous tb infection or disease, previous or ongoing tb treatment, tst status, symptoms suggestive of active tb, results of most recent chest radiograph, sputum smears, or cultures), physical examination, a tst if indicated, a new chest radiograph, and mycobacterial cultures and smears of any suspected sites of infection. all household contacts should be evaluated promptly, including history and tst with further evaluation as indicated. 68 continued risk to the infant can occur from infectious household contacts who have not been effectively evaluated and treated. an infant should be separated temporarily from the suspected source if symptoms suggest active disease or a recent tst documents conversion, and separation should continue until the results of the chest radiograph are seen. because of considerable variability in the course of illness and the concomitant infectious period, debate continues without adequate data about the appropriate period of separation. 278 this should be individualized given the specific situation. hiv testing and assessment of the risk for mdr tb should be done in every case of active tb. sensitivity testing should be done on every mycobacterium tuberculosis isolate. table 13 -1 summarizes the management of the newborn infant whose mother (or other household contact) has tb. initiation of prophylactic isoniazid therapy in the infant has been demonstrated to be effective in preventing tb infection and disease in the infant. therefore continued separation of infant and mother is unnecessary after therapy in both mother and child has begun. 114 the real risk to an infant requiring separation is from airborne transmission. separation of the infant from a mother with active pulmonary tb is appropriate, regardless of the method of feeding. however, in many parts of the world, after therapy in the mother and prophylaxis with isoniazid in the infant has begun, the infant and mother are not separated. with or without separation, the mother and infant should continue to be closely observed throughout the course of maternal therapy to ensure good compliance with medication by both mother and infant and to identify, early on, any symptoms in the infant suggestive of tb. tuberculous mastitis occurs rarely in the united states but does occur in other parts of the world* and can lead to infection in infants, frequently involving the tonsils. a mother usually has a single breast mass and associated axillary lymph node swelling and infrequently develops a draining sinus. tb of the breast can also present as a painless mass or edema. involvement of the breast can occur with or without evidence of disease at other sites. evaluation of extent of disease is appropriate, including lesion cultures by needle aspiration, biopsy, or wedge resection and milk cultures. therapy should be with multiple anti-tb medications, but surgery should supplement this, as needed, to remove extensive necrotic tissue or a persistently draining sinus. 16 neither breastfeeding nor breast milk feeding should be done until the lesion is healed, usually 2 weeks or more. continued anti-tb therapy for 6 months in the mother and isoniazid for the infant for 3 to 6 months is indicated. in the absence of tuberculous breast infection in the mother, transmission of tb through breast milk has not been documented. thus even though temporary separation of infant and mother may occur pending complete evaluation and initiation of adequate therapy in the mother and prophylactic isoniazid therapy (10 mg/kg/day as a single daily dose) in the infant, breast milk can be expressed and given to the infant during the short separation. breastfeeding can safely continue whether the mother, infant, or both are receiving anti-tb therapy. anti-tb medications (isoniazid, rifampin, pyrazinamide, aminoglycosides, ethambutol, ethionamide, p-aminosalicylic acid) have been safely used in infancy, and therefore the presence of these medications in smaller amounts in breast milk is not a contraindication to breastfeeding. although conflicting, reports indicate that breastfeeding by tst-positive mothers does influence infants' responses to bacille calmette-guérin notes: 1 further workup should always include evaluation of tb status of all other household (or close) contacts by tuberculin skin testing (tst), review of symptoms, physical examination, and chest x-ray (cxr). sputum smears and cultures should be done as indicated. 2 separation should occur until interpretation of cxr film confirms absence of active disease, or, with active disease, separation should continue until individual is no longer considered infectious: three negative consecutive sputum smears, adequate ongoing empiric therapy, and decreased fever, cough, and sputum production. separation means in a different house or location, not simply separate rooms in a household. duration of separation should be individualized for each case in consultation with tb specialist. 3 this assumes no evidence of breast involvement, suspected tb mastitis, or lesion (except in status 5, when breast involvement is considered). risk to infant is via aerosolized bacteria in sputum from the lung. expressed breast milk can be given even if separation of mother and infant is advised. 4 tst positive, no symptoms or physical findings suggestive of tb, negative cxr film. 5 prophylactic therapy: isoniazid 10 mg/kg/day, maximum 300 mg for 6 months; pyridoxine 25 to 50 mg/day for 6 months. empiric therapy: standard three-or four-drug regimens for 2 months, and treatment should continue for total of 6 months with isoniazid and rifampin when organism is shown to be sensitive. suspected multidrug-resistant (mdr) tb requires consultation with tb specialist to select optimum empiric regimen and for ongoing monitoring of therapy and clinical response. vaccine, the tst, and perhaps the m. tuberculosis bacillus. despite efforts to identify either a soluble substance or specific cell fractions (gamma/delta t cells) in colostrum and breast milk that affect infants' immune responsiveness, no unified theory explains the various reported changes and no evidence has identified a consistent, clinically significant effect. 39, 213, 319, 367 viral infections arboviruses arboviruses were originally a large collection of viruses grouped together because of the common mode of transmission through arthropods. they have now been reclassified into several different families: bunyaviridae, togaviridae, flaviviridae, reoviridae, and others. they include more than 30 human pathogens. these organisms primarily produce either cns infections (encephalitis, meningoencephalitis) or undifferentiated illnesses associated with fever and rash, severe hemorrhagic manifestations, and involvement of other organs (hepatitis, myalgia, polyarthritis). infection with this array of viruses may also be asymptomatic and subclinical, although how often this occurs is uncertain. some of the notable human pathogens include bunyaviridae (california serogroup viruses), hantavirus, hantaan virus, phlebovirus (rift valley fever), nairovirus (crimean-congo hemorrhagic fever), alphavirus (western, eastern, and venezuelan equine encephalomyelitis viruses, chikungunya virus), flavivirus (st. louis encephalitis virus, japanese encephalitis virus, dengue viruses, yellow fever virus, tick-borne encephalitis viruses), and orbivirus (colorado tick fever). other than for crimean-congo hemorrhagic fever and for reported cases of colorado tick fever associated with transfusion, direct person-to-person spread has rarely been described. recent outbreaks of chikungunya virus infection in reunion island and in india described infection in young infants probably secondary to vertical spread from mother to infant transplacentally. 146, 339, 422 a few cases of early fetal deaths were associated with infection in pregnant women. the cases of vertical transmission occurred with near-term infection in the mothers, and the infants developed illness within 3 to 7 days of delivery. 146, 339 no evidence for transmission via breast milk or breastfeeding is available. little evidence indicates that these organisms can be transmitted through breast milk. the exceptions to this include evidence of transmission of two flaviviruses via breast milk, west nile virus, and yellow fever vaccine virus. standard precautions are generally sufficient. with any of these infections in a breastfeeding mother, the severity of the illness may determine the mother' s ability to continue breastfeeding. providing the infant with expressed breast milk is acceptable. (see the discussion of west nile virus and yellow fever vaccine virus later in this chapter.) in general, treatment for these illnesses is supportive. however, ribavirin appears to decrease the severity of and mortality from hantavirus pulmonary syndrome, hemorrhagic fever with renal failure, and crimean-congo hemorrhagic fever. ribavirin has been described as teratogenic in various animal species and is contraindicated in pregnant women. no information is available concerning ribavirin in breast milk, with little information available on the use of iv or oral ribavirin in infants. arenaviruses are single-stranded ribonucleic acid (rna) viruses that infect rodents and are acquired by humans through the rodents. the six major human pathogens in this group are (1) lymphocytic 6 sensitivity testing should be done on any positive culture. 7 isoniazid 10 mg/kg/day for 3 to 9 months depending on mother's or contact's status; repeat tst at 3 months and obtain normal cxr in infant before stopping isoniazid. before beginning therapy, workup of infant for congenital or active tb may be appropriate. this workup should be determined by clinical status of infant and suspected potential risk, and may include tst after 4 weeks of age, with cxr, complete blood count, and erythrocyte sedimentation rate, liver function tests, cerebrospinal fluid analysis, gastric aspirates, sonography/computed tomography of liver/spleen, and chest if congenital tb is suspected. 8 breastfeeding is proscribed when separation of mother and infant is indicated because of risk for aerosolized transmission of bacteria. expressed breast milk given to infant via bottle is acceptable in absence of mastitis or breast lesions. 9 consult with tb specialist about mdr tb. empiric therapy will be chosen based on the most recent culture sensitivities of index patient or perhaps suspected source case, if known, as well as medication toxicities and other factors. 10 tb mastitis usually involves a single breast with associated axillary lymph node swelling and, infrequently, a draining sinus tract. it can also present as a painless mass or edema of breast. 11 with suspected mastitis or breast lesion caused by tb, even breast milk is contraindicated until lesion or mastitis heals, usually 2 weeks or more. 12 patient has a documented, recent tst conversion but has not been completely evaluated. evaluation should begin and cxr done and evaluated in less than 24 hours to minimize separation of this person from infant. further workup should proceed as indicated by symptoms, physical findings, and cxr results. choriomeningitis virus, (2) lassa fever virus, (3) junin virus (argentine hemorrhagic fever), (4) machupo virus (bolivian hemorrhagic fever), (5) guanarito virus (venezuelan hemorrhagic fever), and (6) sabia virus. the geographic distribution of these viruses and the illness they cause are determined by the living range of the host rodent (reservoir). the exact mechanism of transmission to humans is unknown and hotly debated. 25, 69, 131 direct contact and aerosolization of rodent excretions and secretions are probable mechanisms. lymphocytic choriomeningitis virus is well recognized in europe, the americas, and other areas. perinatal maternal infection can lead to severe disease in the newborn, but no evidence suggests transmission through breast milk. 28, 224 standard precautions with breastfeeding are appropriate. lassa fever (west africa) and argentine hemorrhagic fever (argentine pampas) are usually more severe illnesses with dramatic bleeding and involvement of other organs, including the brain. these fevers more frequently lead to shock and death than do the forms of hemorrhagic fever caused by the other viruses in this group. person-to-person spread of lassa fever is believed to be common, and transmission within households does occur. 212 this may relate to prolonged viremia and excretion of the virus in the urine of humans for up to 30 days. 330 the possibility of persistent virus in human urine, semen, and blood after infection exists for each of the arenaviruses. the possibility of airborne transmission is undecided. current recommendations by the cdc 69 are to use contact precautions for the duration of the illness in situations of suspected viral hemorrhagic fever. no substantial information describes the infectivity of various body fluids, including breast milk, for these different viral hemorrhagic fevers. considering the severity of the illness in mothers and the risk to the infants, it is reasonable to avoid breastfeeding in these situations if alternative forms of infant nutrition can be provided. as more information becomes available, reassessment of these recommendations is advisable. a vaccine is in clinical trials in endemic areas for junin virus and argentine hemorrhagic fever. preliminary studies suggest it is effective, but data are still being accumulated concerning the vaccine' s use in children and pregnant or breastfeeding women. cytomegalovirus (cmv) is one of the human herpesviruses. congenital infection of infants, postnatal infection of premature infants, and infection of immunodeficient individuals represent the most serious forms of this infection in children. the time at which the virus infects the fetus or infant and the presence or absence of antibodies against cmv from the mother are important determinants of the severity of infection and the likelihood of significant sequelae (congenital infection syndrome, deafness, chorioretinitis, abnormal neurodevelopment, learning disabilities). 234 about 1% of all infants are born excreting cmv at birth, and approximately 5% of these congenitally infected infants will demonstrate evidence of infection at birth (approximately five symptomatic cases per 10,000 live births). approximately 15% of infants born after primary infection in a pregnant woman will manifest at least one sequela of prenatal infection. 96 various studies have detected that 3% to 28% of pregnant women have cmv in cervical cultures and that 4% to 5% of pregnant women have cmv in their urine. 120, 172 perinatal infection certainly occurs through contact with virus in these fluids but usually is not associated with clinical illness in fullterm infants. the lack of illness is thought to result from transplacental passive transfer of protective antibodies from the mother. postnatal infection later in infancy occurs via breastfeeding or contact with infected fluids (e.g., saliva, urine) but, again, rarely causes clinical illness in full-term infants. seroepidemiologic studies have documented transmission of infection in infancy, with higher rates of transmission occurring in daycare centers, especially when the prevalence of cmv in the urine and saliva is high. cmv has been identified in the milk of cmv-seropositive women at varying rates (10% to 85%) using viral cultures or cmv deoxyribonucleic acid (dna) pcr. 172, 301, 397, 430 cmv is more often identified in the breast milk of seropositive mothers than in vaginal fluids, urine, and saliva. the cmv isolation rate from colostrum is lower than that from mature milk. 172, 396 the reason for the large degree of variability in identification of cmv in breast milk in these studies probably relates to the intermittent nature of reactivation and excretion of the virus in addition to the variability, frequency, and duration of sampling of breast milk in the different studies. some authors have hypothesized that the difference in isolation rates between breast milk and other fluids is caused by viral reactivation in cells (leukocytes or monocytes) in the breast leading to "selective" excretion in breast milk. 301 vochem et al 430 reported that the rate of virolactia was greatest at 3 to 4 weeks postpartum, and yeager et al 455 reported significant virolactia between 2 and 12 weeks postpartum. antibodies (e.g., secretory iga) to cmv are present in breast milk, along with various cytokines and other proteins (e.g., lactoferrin). these may influence virus binding to cells, but they do not prevent transmission of infection.* several studies have documented increased rates of postnatal cmv infection in breastfed infants (50% to 69%) compared with bottle-fed infants (12% to 27%) observed through the first year of life 120, 281, 397, 430 in these same studies, full-term infants who acquired cmv infection postnatally were only rarely mildly symptomatic at the time of seroconversion or documented viral excretion. also, no evidence of late sequelae from cmv was found in these infants. postnatal exposure of susceptible infants to cmv, including premature infants without passively acquired maternal antibodies against cmv, infants born to cmv-seronegative mothers, and immunodeficient infants, can cause significant clinical illness (pneumonitis, hepatitis, thrombocytopenia).* in one study of premature infants followed up to 12 months, vochem et al 430 found cmv transmission in 17 of 29 infants (59%) exposed to cmv virolactia and breastfed compared with no infants infected of 27 exposed to breast milk without cmv. no infant was given cmv-seropositive donor milk or blood. five of the 12 infants who developed cmv infection after 2 months of age had mild signs of illness, including transient neutropenia, and only one infant had a short increase in episodes of apnea and a period of thrombocytopenia. five other premature infants with cmv infection before 2 months of age had acute illness, including sepsis-like symptoms, apnea with bradycardia, hepatitis, leukopenia, and prolonged thrombocytopenia. 430 vollmer et al 431 followed premature infants with early postnatal cmv infection acquired through breast milk for 2 to 4.5 years to assess neurodevelopment and hearing function. none of the children had sensorineural hearing loss. there was no difference between the 22 cmv-infected children and 22 matched premature control cmv-negative infants in terms of neurologic, speech and language, or motor development. 431 neuberger et al 296 examined the symptoms and neonatal outcome of cmv infection transmitted via human milk in premature infants in a case-control fashion; 40 cmv-infected premature infants were compared with 40 cmv-negative matched premature infants. neutropenia, thrombocytopenia, and cholestasis were associated with cmv infection in these infants. no other serious effects or illnesses were found directly associated with the infection including intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, duration of mechanical ventilation or oxygen therapy, duration of hospital stay or weight, gestational age, or head circumference at the time of discharge. exposure of cmv-seronegative or premature infants to cmv-positive milk (donor or natural mother' s) should be avoided. 379 various methods of inactivating cmv in breast milk have been reported, including holder pasteurization, freezing (−20° c for 3 days), and brief high temperature (72° c for 10 seconds). 120, 135, 155, 393, 455 one small, prospective study suggests that freezing breast milk at −20°c for 72 hours protects premature infants from cmv infection via breast milk. sharland et al 379 reported on 18 premature infants (less than 32 weeks) who were uninfected at birth and exposed to breast milk from their cmv seropositive mothers. only one of 18 (5%) infants became positive for cmv at 62 days of life, and this infant was clinically asymptomatic. this transmission rate is considerably lower than others reported in the literature. cmvseronegative and leukocyte-depleted blood products were used routinely. banked breast milk was pasteurized and stored at −20° c for various time periods and maternal expressed breast milk was frozen at −20° c before use whenever possible. the infants received breast milk for a median of 34 days (range 11 to 74 days) and they were observed for a median of 67 days (range 30 to 192 days). breast milk samples pre-or postfreezing were not analyzed by pcr or culture for the presence of cytomegalovirus. 379 buxmann et al 57 demonstrated no transmission of cmv in 23 premature infants receiving thawed frozen breast milk until 33 weeks (gestational age + postnatal age) (less than or equal to 31 weeks gestational age) born to 19 mothers who were cmv-igg negative. cmv infection was found in five premature infants of 35 infants born to 29 mothers who were cmv-igg positive and who provided breast milk for their infants. three of the five children remained asymptomatic. one child development a respirator-dependent pneumonia and the second developed an upper respiratory tract infection and thrombocytopenia in association with their cmv infections. 57 yasuda et al 454 reported on 43 preterm infants (median gestational age 31 weeks) demonstrating a peak in cmv dna copies, detected by a real-time pcr assay, in breast milk at 4 to 6 weeks postpartum. thirty of the 43 infants received cmv dna-positive breast milk. three of the 30 had cmv dna detected in their sera, but none of the three had symptoms suggestive of cmv infection. much of the breast milk had been stored at −20° c before feeding, which the authors propose is the probable reason for less transmission in this cohort. 454 lee et al 248 reported on the use of maternal milk frozen at −20°c for a minimum of 24 hours before feeding to premature infants in a nicu; 23 infants had cmv-seropositive mothers and 39 infants had cmv-seronegative mothers. two infants developed cmv infection, which was symptomatic. they were both fed frozen thawed milk from cmv-seropositive mothers. 248 others have reported individual cases of cmv infection in premature infants despite freezing and thawing breast milk. 268, 314 simple freezing and thawing of breast milk does not completely prevent transmission of cmv to premature infants. the efficacy of freezing and thawing breast milk for varying lengths of time to prevent cmv infection in premature infants has not been studied prospectively in a randomized controlled trial. eleven of 36 neonatal units in sweden (27 of which have their own milk banks) freeze maternal milk to reduce the risk for cmv transmission to premature infants. 314 a prominent group of neonatologists and pediatric infectious disease experts in california who recognize the significant benefits of providing human milk to premature and lbw infants recommend screening mothers of premature infants for cmv igg at delivery and, when an infant' s mother is cmv igg positive at delivery, using either pasteurized banked human milk or frozen then thawed maternal breast milk for premature infants until they reach the age of 32 weeks. 445 in consideration of the low rates of cmv virolactia in colostrum 169, 397 and the predominant occurrence of virolactia between 2 and 12 weeks (peak at 3 to 4 weeks) postpartum, 430,455 they reasonably propose beginning colostrum and breast milk feedings for all infants until the maternal cmv serologic screening is complete. they appropriately recommend close observation and follow-up of premature infants older than 3 weeks of age for signs, symptoms, and laboratory changes of cmv infection until discharge from the hospital. 445 cmv-seropositive mothers can safely breastfeed their full-term infants because, despite a higher rate of cmv infection than in formula-fed infants observed through the first year of life, infection in this situation is not associated with significant clinical illness or sequelae. dengue viruses (serotypes dengue 1 to 4) are flaviviruses associated primarily with febrile illnesses and rash; dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. the mosquito aedes aegypti is the main vector of transmission of dengue virus in countries lying between latitudes 35 degrees north and 35 degrees south. more than 2.5 billion people live in areas where transmission occurs; dengue virus infects over 100 million individuals a year and casuses approximately 24,000 deaths a year. 159, 163 although dengue hemorrhagic fever and dengue shock syndrome occur frequently in children younger than 1 year of age, they are infrequently described in infants younger than 3 months of age. 167 there are also differences in the clinical and laboratory findings of dengue virus infection in children compared with adults. 222 boussemart et al 49 reported on two cases of perinatal/prenatal transmission of dengue and discussed eight additional cases in neonates from the literature. prenatal or intrapartum transmission of the same type of dengue as the mother was confirmed by serology, culture, or pcr. phongsamart et al 333 described three additional cases of dengue virus infection late in pregnancy and apparent transmission to two of the three infants with passive acquisition of antibody in the third infant. sirinavin et al 386 reported on 17 cases in the literature of vertical dengue infection, all presenting at less than 2 weeks of age, but no observations or discussion of breast milk or breastfeeding as a potential source of infection were published. watanaveeradej et al 439 presented an additional three cases of dengue infection in infants documenting normal growth and development at follow-up at 12 months of age. it has been postulated that more severe disease associated with dengue disease occurs when an individual has specific igg against the same serotype as the infecting strain in a set concentration, leading to antibody-dependent enhancement of infection. the presence of preexisting dengue serotype specific igg in an infant implies either previous primary infection with the same serotype, passive acquisition of igg from the mother (who had a previous primary infection with the same serotype), or perhaps acquisition of specific igg from breast milk. watanaveeradej et al 439 documented transplacentally transferred antibodies against all four serotypes of dengue virus in 97% of 2000 cord sera at delivery. follow-up of 100 infants documented the loss of antibodies to dengue virus over time with losses of 3%, 19%, 72%, 99%, and 100% at 2, 4, 6, 9, and 12 months of age, respectively. no evidence is available in the literature about more severe disease in breastfed infants compared with formula-fed infants. no interhuman transmission of dengue virus in the absence of the mosquito vector and no evidence of transmission via breast milk are known. only one report of a factor in the lipid portion of breast milk, which inhibits the dengue virus, is available, and no evidence for antibody activity against dengue virus in human breast milk is known. 127 breastfeeding during maternal or infant dengue disease should continue as determined by the mother' s or infant' s severity of illness. epstein-barr virus (ebv) is a common infection in children, adolescents, and young adults. it is usually asymptomatic but most notably causes infectious mononucleosis and has been associated with chronic fatigue syndrome, burkitt lymphoma, and nasopharyngeal carcinoma. because ebv is one of the human herpesviruses, concern has been raised about lifelong latent infection and the potential risk for infection to a fetus and neonate from the mother. primary ebv infection during pregnancy is unusual because few pregnant women are susceptible. 149, 189 although abortion, premature birth, and congenital infection from ebv are suspected, no distinct group of anomalies is linked to ebv infection in fetus or neonate. also, no virologic evidence of ebv as the cause of abnormalities was found in association with suspected ebv infection. culturing of ebv from various fluids or sites is difficult. the virus is detected by its capacity to transform b lymphocytes into persistent lymphoblastoid cell lines. pcr and dna hybridization studies have detected ebv in the cervix and in breast milk. one study, which identified ebv dna in breast milk cells in more than 40% of women donating milk to a breast milk bank, demonstrated that only 17% had antibody to ebv (only igg, no igm). 204 another study examining serologic specimens from breastfed and bottle-fed infants showed similar seroprevalence of ebv at 12 to 23 months of age (36/66 [54.5%] and 24/43 [55.8%]) in the breastfed and bottle-fed children, respectively. 236 the question of the timing of ebv infection and the subsequent immune response and clinical disease produced requires continued study. differences exist among the clinical syndromes that manifest at different ages. infants and young children are asymptomatic, have illness not recognized as related to ebv, or have mild episodes of illness, including fever, lymphadenopathy, rhinitis and cough, hepatosplenomegaly, or rash. adolescents or young adults who experience primary ebv infection more often demonstrate infectious mononucleosis syndrome or are asymptomatic. chronic fatigue syndrome is more common in adolescents and young adults. burkitt lymphoma, observed primarily in africa, and nasopharyngeal carcinoma, seen in southeast asia, where primary ebv infection usually occurs in young children, are tumors associated with early ebv infection. 246 these tumors are related to "chronic" ebv infection and tend to occur in individuals with persistently high antibody titers to ebv viral capsid antigen and early antigen. the questions of why these tumors occur with much greater frequency in these geographic areas and what cofactors (including altered immune response to infection associated with coinfections, immune escape by ebv leading to malignancy, or increased resistance to apoptosis secondary to ebv gene mutations) may contribute to their development remain unanswered. 21, 289 it also remains unknown to what degree breast milk could be a source of early ebv infection compared with other sources of ebv infection in an infant' s environment. similar to the situation of postnatal transmission of cmv in immunocompetent infants, clinically significant illness rarely is associated with primary ebv infection in infants. more data concerning the pathogenesis of ebv-associated tumors should be obtained before proscribing against breastfeeding is warranted, especially in areas where these tumors are common but the protective benefits of breastfeeding are high. in areas where burkitt lymphoma and nasopharyngeal carcinoma are uncommon, ebv infection in mother or infant is certainly not a contraindication to breastfeeding. marburg and ebola viruses cause severe and highly fatal hemorrhagic fevers. the illness often presents with nonspecific symptoms (conjunctivitis, frontal headache, malaise, myalgia, bradycardia) and progresses with worsening hemorrhage to shock and subsequent death in 50% to 90% of patients. personto-person transmission through direct contact, droplet spread, or airborne spread is the common mode of transmission. however, the animal reservoir or source of these viruses in nature for human infection has not been identified. attack rates in families are 5% to 16%. 330 no postexposure interventions have proved useful in preventing spread, and no treatment other than supportive is currently available. a recent report documented the presence of ebola virus in numerous body fluids including in breast milk. one acute breast milk sample on day 7 after the onset of illness and a "convalescent" breast milk sample on day 15 from the same woman were positive for ebola virus by both culture and pcr testing. 30 in the same study, saliva remained virus positive for a mean of 16 days after disease onset, urine was positive for a mean of 28 days, and semen for a mean of 43 days after the onset of disease. no information is available concerning the risk for transmission of these viruses in breast milk or additional risks or benefits from breastfeeding. contact precautions are recommended for marburg virus infections and contact and airborne precautions for ebola virus infection. given the high attack and mortality rates, these precautions should be carefully instituted and breastfeeding not allowed. if any other suitable source of nutrition can be found for an infant, expressed breast milk should also be proscribed for the infant of a mother with either of these infections for at least 3 weeks postrecovery. the diagnosis of hepatitis in a pregnant woman or nursing mother causes significant anxiety. the first issue is determining the etiology of the hepatitis, which then allows for an informed discussion of risk to the fetus/infant. the differential diagnosis of acute hepatitis includes (1) common causes of hepatitis, such as hepatitis a, b, c, and d; (2) , igm anti-hbcag, anti-hcv) as the initial diagnostic tests. simultaneous consideration of other etiologies of acute liver dysfunction is appropriate depending on a patient' s history. if the initial diagnostic tests are all negative, subsequent additional testing for anti-hepatitis d virus (hdv), hcv rna, hepatis g virus (hgv) rna, anti-hepatis e virus (hev), or hev rna may be necessary. if initial testing reveals positive hbsag, testing for anti-hdv, hbeag, and hbv dna is appropriate. these additional tests are useful in defining the prognosis for a mother and the risk for infection to an infant. during the diagnostic evaluation, it is appropriate to discuss with the mother or parents the theoretic risk for transmitting infectious agents that cause hepatitis via breastfeeding. the discussion should include an evaluation of the positive and negative effects of suspending or continuing breastfeeding until the exact etiologic diagnosis is determined. the relative risk for transmission of infection to an infant can be estimated and specific preventive measures provided for the infant (table 13-2) . hepatitis a virus (hav) is usually an acute selflimited infection. the illness is typically mild, and generally subclinical in infants. occasionally, hav infection is prolonged or relapsing, extending 3 to 6 months, and rarely it is fulminant, but hav infection does not lead to chronic infection. the incidence of prematurity after maternal hav infection is increased, but no evidence to date indicates obvious birth defects or a congenital syndrome. 372, 464 hav infection in premature infants may lead to prolonged viral shedding. 349 transmission is most often person to person (fecal-oral), and transmission in food-borne or water-borne epidemics has been described. transmission via blood products and vertical transmission (mother to infant) are rare. 440 transmission in daycare settings has been clearly described. infection with hav in newborns is uncommon and does not seem to be a significant problem. the usual period of viral shedding and presumed contagiousness lasts 1 to 3 weeks. acute maternal hav infection in the last trimester or in the postpartum period could lead to infection in an infant. symptomatic infection can be prevented by immunoglobulin (ig) administration, and 80% to 90% of disease can be prevented by ig administration immune serum globulin within 2 weeks of exposure. hav vaccine can be administered simultaneously with ig without affecting the seroconversion rate to produce rapid and prolonged hav serum antibody levels. transmission of hav via breast milk has been implicated in one case report, but no data exist on the frequency of isolating hav from breast milk. 440 because hav infection in infancy is rare and usually subclinical without chronic disease and because exposure has already occurred by the time the etiologic diagnosis of hepatitis in a mother is made, no reason exists to interrupt breastfeeding with maternal hav infection. the infant should receive ig and hav vaccine, administered simultaneously. hepatitis b virus (hbv) infection leads to a broad spectrum of illness, including asymptomatic seroconversion, nonspecific symptoms (fever, malaise, fatigue), clinical hepatitis with or without jaundice, extrahepatic manifestations (arthritis, rash, renal involvement), fulminant hepatitis, and chronic hbv infection. chronic hbv infection occurs in up to 90% of infants infected via perinatal and vertical transmission and in 30% of children infected between 1 to 5 years of age. given the increased risk for significant sequelae from chronic infection (chronic active hepatitis, chronic persistent hepatitis, cirrhosis, primary hepatocellular carcinoma), prevention of hbv infection in infancy is crucial. transmission of hbv is usually through blood or body fluids (stool, semen, saliva, urine, cervical secretions). 94 vertical transmission either transplacentally or perinatally during delivery has been well described throughout the world. vertical transmission rates in areas where hbv is endemic (taiwan and japan) are high, whereas transmission to infants from hbv carrier mothers in other areas where hbv carrier rates are low is uncommon. 399 transmission of hbv to infants occurs in up to 50% of infants when the mothers are acutely infected immediately before, during, or soon after pregnancy. 462 hbsag is found in breast milk, but transmission by this route is not well documented. beasley 31 and beasley et al 32 demonstrated that although breast milk transmission is possible, seroconversion rates are no different between breastfed and nonbreastfed infants in a long-term follow-up study of 147 hbsag-positive mothers. hill et al 176 followed 101 breastfed infants and 268 formula-fed infants born to women who were chronically hbsag positive. all infants received hepatitis b immunoglobulin at birth and a full series of hepatitis b vaccine. none of the breastfed infants and nine of the formulafed infants were positive for hbsag after completion of the hbv vaccine series. breastfeeding had occurred for a mean of 4.9 months (range 2 weeks to 1 year). transmission, when it does happen, probably occurs during labor and delivery. another report from china followed 230 infants born to hbsag-positive women. the infants received appropriate dosing and timing of hbig and hbv vaccine. at 1 year of age, anti-hbs antibody was present in 90.9% of the breastfed infants and 90.3% of the bottle-fed infants. 437 risk factors associated with immunoprophylaxis failure against vertical transmission of hbv include hbeag-seropositive mothers and elevated hbv dna "viral loads" in the mothers. 392 in 2009 the aap committee on infectious diseases stated that "that breastfeeding of the infant by a hbsag-positive mother poses no additional risk for acquisition of hbv infection by the infant with appropriate administration of hepatitis b vaccine and hbig." 96 screening of all pregnant women for hbv infection is an essential first step to preventing vertical transmission. universal hbv vaccination at birth and during infancy, with administration of hepatitis b immunoglobulin (hbig) immediately after birth to infants of hbsag-positive mothers, prevents hbv transmission in more than 95% of cases. breastfeeding by hbsag-positive women is not contraindicated, but immediate administration of hbig and hbv vaccine should occur. two subsequent doses of vaccine should be given at appropriate intervals and dosages for the specific hbv vaccine product. this decreases the small theoretic risk for hbv transmission from breastfeeding to almost zero. when acute peripartum or postpartum hepatitis occurs in a mother and hbv infection is a possibility, with its associated increased risk for transmission to the infant, a discussion with the mother or parents should identify the potential risks and benefits of continuing breastfeeding until the etiology of the hepatitis can be determined. if an appropriate alternative source of nutrition is available for the infant, breast milk should be withheld until the etiology of the hepatitis is identified. hbig and hbv vaccine can be administered to the infant who has not already been immunized or has no documented immunity against hbv. 400 if acute hbv infection is documented in a mother, breastfeeding can continue after immunization has begun. acute infection with hcv can be indistinguishable from hepatitis a or b infection; however, it is typically asymptomatic or mild. hcv infection is the major cause of blood-borne non-a, non-b hepatitis (nanbh). chronic hcv infection is reported to occur 70% to 85% of the time regardless of age at time of infection. sequelae of chronic hcv infection are similar to those associated with chronic hbv infection. bortolotti et al 47 the two commonly identified mechanisms of transmission of hcv are transfusions of blood or blood products and iv drug use. however, other routes of transmission exist because hcv infection occurs even in the absence of obvious direct contact with significant amounts of blood. other body fluids contaminated with blood probably serve as sources of infection. transmission through sexual contact occurs infrequently and probably requires additional contributing factors, such as coinfection with other sexually transmitted agents or high viral loads in serum and other body fluids. studies of transmission in households without other risk factors have demonstrated either low rates of transmission or no transmission. the reported rates of vertical transmission vary widely. in mothers with unknown hiv status or known hiv infection, the rates of vertical transmission were 4% to 100%, whereas the rates varied between 0% and 42% in known hiv-negative mothers. 113 these same studies suggest that maternal coinfection with hiv, hcv genotype, active maternal liver disease, and the serum titer of maternal hcv rna may be associated with increased rates of vertical transmission. 263, 307, 461 the correlation between hcv viremia, the hcv viral load in a mother, and vertical transmission of hcv is well documented. 288, 355, 406, 456 the clinical significance and risk for liver disease after vertical transmission of hcv are still unknown. the timing of hcv infection in vertical transmission is also unknown. in utero transmission has been suggested by some studies, 125 whereas intrapartum or postpartum transmission was proposed by ohto et al 308 when they documented the absence of hcv rna in the cord blood of neonates who later became hcv rna positive at 1 to 2 months of age. more recently, gibb et al 150 reported two pieces of data supporting the likelihood of intrapartum transmission as the predominant time of vertical transmission: (a) low sensitivity of pcr for hcv rna testing in the first month of life with a marked increase in sensitivity after that for diagnosing hcv infection in infants and (b) a lower transmission risk for elective cesarean delivery (without prolonged rupture of membranes) compared with vaginal or emergency cesarean delivery. 150 another group, mcmenamin et al, 275 analyzed vertical transmission in 559 mother-infant pairs. the overall vertical transmission rate was 4.1% (18/441), with another 118 infants not tested or lost to follow-up. comparison of the vertical transmission rate was no different for vaginal delivery or emergency cesarean in labor versus planned cesarean (4.2% vs. 3.0%). this held true even when mothers had hepatitis c rna detected antenatally (7.2% vs. 5.3%). the authors did not support planned cesarean delivery to decrease vertical transmission of hepatitis c infection. no prospective, controlled trials of cesarean versus vaginal delivery and the occurrence of vertical hepatitis c transmission are available. the risk for hcv transmission via breast milk is uncertain. anti-hcv antibody and hcv rna has been demonstrated in colostrum and breast milk, although the levels of hcv rna in milk did not correlate with the titers of hcv rna in serum. 36, 162, 256, 355 nevertheless, transmission of hcv via breastfeeding (and not in utero, intrapartum, or from other postpartum sources) has not been proven in the small number infants studied. transmission rates in breastfed and nonbreastfed infants appear to be similar, but various important factors have not been controlled, such as hcv rna titers in mothers, examination of the milk for hcv rna, exclusive breastfeeding versus exclusive formula feeding versus partial breastfeeding, and duration of breastfeeding.* zanetti et al 461 including 23 infants whose mothers were seropositive for hcv rna. eight infants in that study were infected with hcv, their mothers had both hiv and hcv, and three of these eight infants were infected with both hiv and hcv. the hcv rna levels were significantly higher in the mothers coinfected with hiv compared with those mothers with hcv alone. overall, the risk for hcv infection via breastfeeding is low, the risk for hcv infection appears to be more frequent in association with hiv infection and higher levels of hcv rna in maternal serum, no effective preventive therapies (ig or vaccine) exist, and the risk for chronic hcv infection and subsequent sequelae with any infection is high. it is therefore appropriate to discuss the theoretic risk for breastfeeding in hcv-positive mothers with the mother or parents and to consider proscribing breast milk when appropriate alternative sources of nutrition are available for the infants. hiv infection is a separate contraindication to breastfeeding. additional study is necessary to determine the exact role of breastfeeding in the transmission of hcv, including the quantitative measurement of hcv rna in colostrum and breast milk, the relative risk for hcv transmission in exclusively or partially breastfed infants versus the risk in formulafed infants, and the effect of duration of breastfeeding on transmission. the current position of the cdc is that no data indicate that hcv virus is transmitted through breast milk. 83 therefore breastfeeding by a hcvpositive, hiv-negative mother is not contraindicated. infants born to hcv rna-positive mothers require follow-up through 18 to 24 months of age to determine infants' hcv status, regardless of the mode of infant feeding. infants should be tested for alanine aminotransferase and hcv rna at 3 months and 12 to 15 months of age. alanine aminotransferase and anti-hcv antibody should be tested at 18 to 24 months of age to confirm an infant' s status: uninfected, ongoing hepatitis c infection, or past hcv infection. hepatitis d virus (hdv) is a defective rna virus that causes hepatitis only in persons also infected with hbv. the infection occurs as either an acute coinfection of hbv and hdv or a superinfection of hbv carriers. this "double" infection results in more frequent fulminant hepatitis and chronic hepatitis, which can progress to cirrhosis. the virus uses its own hbv rna (circular, negative-strand rna) with an antigen, hdag, surrounded by the surface antigen of hbv, hbsag. hdv is transmitted in the same way as hbv, especially through the exchange of blood and body fluids. hdv infection is uncommon where the prevalence of hbv is low. in areas where hbv is endemic, the prevalence of hdv is highly variable. hdv is common in tropical africa and south america as well as in greece and italy but is uncommon in the far east and in alaskan inuit despite the endemic occurrence of hbv in these areas. 390 transmission of hdv has been reported to occur from household contacts and, rarely, through vertical transmission. no data are available on transmission of hdv by breastfeeding. hdv infection can be prevented by blocking infection with hbv; therefore hbig and hbv vaccine are the best protection. in addition to hbig and hbv vaccine administration to the infant of a mother infected with both hbv and hdv, discussion with the mother or parents should include the theoretic risk for hbv and hdv transmission through breastfeeding. as with hbv, once hbig and hbv vaccine have been given to the infant, the risk for hbv or hdv infection from breastfeeding is negligible. therefore breastfeeding after an informed discussion with the parents is acceptable. hepatitis e virus (hev) is a cause of sporadic and epidemic, enterically transmitted nanbh, which is typically self-limited and without chronic sequelae. hev is notable for causing high mortality rate in pregnant women. transmission is primarily via the fecal-oral route, commonly via contaminated water or food. high infection rates have been reported in adolescents and young adults (ages 15 to 40 years). tomar 416 reported that 70% of cases of hev infections in the pediatric population in india manifest as acute hepatitis. maternal-neonatal transmission was documented when the mother developed hepatitis e infection in the third trimester. although hev was demonstrated in breast milk, no transmission via breast milk was confirmed in the report. five cases of transfusion-associated hepatitis e were reported. 416 epidemics are usually related to contamination of water. person-to-person spread is minimal, even in households and day care settings. although ig may be protective, no controlled trials have been done. animal studies suggest that a recombinant subunit vaccine may be feasible. 344 hev infection in infancy is rare, and no data exist on transmission of hev by breastfeeding. no evidence of clinically significant postnatal hev infection in infants or of chronic sequelae in association with hev infection and no documented hev transmission through breast milk is available. currently no contraindication exists to breastfeeding with maternal hev infection. ig has not been shown to be effective in preventing infection, and no vaccine is available for hev. hepatitis g virus (hgv) has recently been confirmed as a cause of nanbh distinct from hepatitis viruses a through e. several closely related genomes of hgv, currently named gbv-a, -b, and -c, appear to be related to hcv, the pestiviruses, and the flaviviruses. epidemiologically, hgv is most often associated with transfusion of blood, although studies have identified nontransfusion-related cases. hgv genomic rna has been detected in some patients with acute and chronic hepatitis and a small number of patients with fulminant hepatitis. gbv-c/hgv has also been found in some patients with inflammatory bile duct lesions, but the pathogenicity of this virus is unconfirmed. hgv rna has been detected in 1% to 3% of healthy blood donors in the united states. 8 feucht et al 128 described maternal-to-infant transmission of hgv in three of nine children. two of the three mothers were coinfected with hiv and the third with hcv. none of these infants developed signs of liver disease. neither the timing nor the mode of transmission was clarified. lin et al 255 reported no hgv transmission in three mother-infant pairs after cesarean delivery and discussed transplacental spread via blood as the most likely mode of hgv infection in vertical transmission. wejstal et al 442 reported on perinatal transmission of hgv to 12 of 16 infants born to hgv viremic mothers, identified by pcr. hgv did not appear to cause hepatitis in the children. 442 fischler et al 130 followed eight children born to hgv-positive mothers and found only one to be infected with hgv. that child remained clinically well, while his twin, also born by cesarean delivery and breastfed, remained hgv negative for 3 years of observation. five of the other six children were breastfed for variable periods without evidence of hgv infection. ohto et al 309 examined hgv mother-to-infant transmission. of 2979 pregnant japanese women who were screened, 32 were identified as positive for gbv-c/hgv rna by pcr; 26 of 34 infants born to the 32 hgv positive women were shown to be hgv rna positive. reportedly, none of the infants demonstrated a clinical picture of hepatitis, although two infants had persistent mild elevations (less than two times normal) of alanine aminotransferase. the viral load in mothers, who transmitted hgv to their infants, was significantly higher than in nontransmitting mothers. infants born by elective cesarean delivery had a lower rate of infection (3 in 7) compared with infants born by emergency cesarean delivery (2 of 2) or born vaginally (21 of 25) . in this study, hgv infection in breastfed infants was four times more common than in formula-fed infants, but this difference was not statistically significant because only four infants were formula fed. the authors report no correlation between infection rate and duration of breastfeeding was seen. testing of the infants was not done frequently and early enough routinely through the first year of life to determine the timing of infection in these infants. 309 schröter et al 371 reported transmission of hgv to 3 of 15 infants born to hgv rna positive mothers at 1 week of age. none of 15 breast milk samples were positive for gbv-c/hgv rna, and all of the children who were initially negative for hgv rna in serum remained negative at follow-up between 1 to 28 months of age. 371 the foregoing data suggest that transmission is more likely to be vertical, before, or at delivery rather than via breastfeeding. the pathogenicity and the possibility of chronic disease due to hgv infection remain uncertain at this time. insufficient data are available to make a recommendation concerning breastfeeding by hgv-infected mothers. herpes simplex virus types 1 and 2 (hsv-1, hsv-2) can cause prenatal, perinatal, and postnatal infections in fetuses and infants. prenatal infection can lead to abortion, prematurity, or a recognized congenital syndrome. perinatal infection is the most common form of infection (1 in 2000 to 5000 live births, 700 to 1500 cases per year in the united states) and is often fatal or severely debilitating. the factors that facilitate intrapartum infection and predict the severity of disease have been extensively investigated. postnatal infection is uncommon but can occur from a variety of sources, including oral or genital lesions and secretions in mothers or fathers, hospital workers and home caregivers, and breast lesions in breastfeeding mothers. a number of case reports have documented severe hsv-1 or hsv-2 infections in infants associated with hsvpositive breast lesions in the mothers. 116, 161, 338, 403 cases of infants with hsv gingivostomatitis inoculating the mothers' breasts have also been reported. in the absence of breast lesions breastfeeding in hsv-seropositive or culture-positive women is reasonable when accompanied by careful handwashing, covering the lesions, and avoiding fondling or kissing with oral lesions until all lesions are crusted. breastfeeding during maternal therapy with oral or iv acyclovir can continue safely as well. inadequate information exists concerning valacyclovir, famciclovir, ganciclovir, and foscarnet in breast milk to make a recommendation at this time. breastfeeding by women with active herpetic lesions on their breasts should be proscribed until the lesions are dried. treatment of the mothers' breast lesions with topical, oral, and/or iv antiviral preparations may hasten recovery and decrease the length of viral shedding. human herpesvirus 6 (hhv-6) is a cause of exanthema subitum (roseola, roseola infantum) and is associated with febrile seizures. hhv-6 appears to be most similar to cmv based on genetic analysis. no obvious congenital syndrome of hhv-6 infection has been identified, although prenatal infection has been reported. 118 seroepidemiologic studies show that most adults have already been infected by hhv-6. therefore primary infection during pregnancy is unlikely, but reactivation of latent hhv-6 infection may be more common. no case of symptomatic hhv-6 prenatal infection has been reported. the significance of reactivation of hhv-6 in a pregnant woman and the production of infection and disease in the fetus and infant remains to be determined. primary infection in children occurs most often between 6 and 12 months of age, when maternally acquired passive antibodies against hhv-6 are waning. febrile illnesses in infants younger than 3 months of age have been described with hhv-6 infection, but infection before 3 months or after 3 years is uncommon. various studies involving serology and restriction enzyme analysis of hhv-6 isolates from mother/infant pairs support the idea that postnatal transmission and perhaps perinatal transmission from the mothers are common sources of infection. one study was unable to detect hhv-6 in breast milk by pcr analysis in 120 samples, although positive control samples seeded with hhv-6-infected cells did test positive. 119 given the limited occurrence of clinically significant disease and the absence of sequelae of hhv-6 infection in infants and children, the almost universal acquisition of infection in early childhood (with or without breastfeeding) and the absence of evidence that breast milk is a source of hhv-6 infection, breastfeeding can continue in women known to be seropositive for hhv-6. human herpesvirus 7 (hhv-7) is closely related to hhv-6 biologically. primary infection with hhv-7 occurs primarily in childhood, usually later in life than hhv-6 infection. the median age of infection is 26 months, with 75% of children becoming hhv-7 positive by 5 years of age. 63 seroprevalence of hhv-7 antibody has been reported to be 80% to 98% in adults, and passive antibody is present in almost all newborns. 306, 408 like hhv-6, hhv-7 infection can be associated with acute febrile illness, febrile seizures, and irritability, but in general it is a milder illness than with hhv-6 with fewer hospitalizations. virus excretion of hhv-7 occurs in saliva, and pcr testing of blood cells and saliva are frequently positive in individuals with past infection. 463 congenital infection of hhv-6 was detected via dna pcr testing in 57 of 5638 of cord blood samples (1%), but hhv-7 was not detected in any of 2129 cord blood specimens. 165 hhv-7 dna was detected by pcr in 3 of 29 breast milk mononuclear cell samples from 24 women who were serum positive for hhv-7 antibody. 137 in the same study, small differences were seen in the hhv-7 seropositive rates between breastfed infants and bottle-fed infants at 12 months of age (21.7% versus 20%), at 18 months of age (60% versus 48.1%), and at 24 months of age (77.3% versus 58.3%, respectively,). none of these differences were statistically significant. given that, in general, hhv-6 infection occurs earlier than hhv-7 infection in most infants and that hhv-6 is rarely found in breast milk, it seems unlikely that hhv-7 in breast milk is a common source of infection in infants and children. the infrequent occurrence of significant illness with hhv-7 infection, with the absence of sequelae except in patients who had transplantation surgery at older ages and the common occurrence of infection in childhood argue, that no reason to proscribe against breastfeeding for hhv-7 positive women exists. human papillomavirus (hpv) is a dna virus with at least 100 different types. these viruses cause warts, genital dysplasia, cervical carcinoma (types 6 and 11), and laryngeal papillomatosis. transmission occurs through direct contact and sexual contact. laryngeal papillomas are thought to result from acquiring the virus in passage through the birth canal. infection in pregnant women or during pregnancy does not lead to an increase in abortions or the risk for prematurity, and no evidence indicates intrauterine infection. hpv is one of the most common viruses in adults and one of the most commonly sexually transmitted infections. diagnosis is usually by histologic examination or dna detection. spontaneous resolution does occur, but therapy for persistent lesions or growths in anatomically problematic locations is appropriate. therapy can be with podophyllum preparations, trichloroacetic acid, cryotherapy, electrocautery, and laser surgery. interferon is being tested in the treatment of laryngeal papillomas, with mixed results. 109 prevention against transmission means limiting direct or sexual contact, but this may not be sufficient because lesions may not be evident and transmission may still occur. rintala et al 346 examined the occurrence of hpv dna in the oral and genital mucosa of infants during the first 3 years of life. hpv dna was identified in 12% to 21% of the oral scrape samples and in 4% to 15% of the genital scrape samples by pcr. oral hpv infection was acquired by 42% of children, cleared by 11%, and persisted in 10% of children; 37% of the children were never infected. they did not report on breast milk or breastfeeding in that study. the question of the source of the infection remains undetermined. the breast is a rare site of involvement. 110 hpv types 16 and 18 can immortalize normal breast epithelium in vitro. 441 hpv dna has been detected in breast milk in 10 of 223 (4.5%) of milk samples from 223 mothers, collected 3 days postpartum. 361 no attempt was made to correlate the presence of hpv dna in breast milk with the hpv status of an infant or to assess the "viral load" of hpv in breast milk or its presence over the course of lactation. a second study found dna of cutaneous and mucosal hpv types in 2 of 25 human milk samples and 1 of 10 colostrum samples. 64 no reports of hpv lesions of the breast or nipple and documented transmission to an infant secondary to breastfeeding are available. no increased risk for acquiring hpv from breast milk is apparent, and breastfeeding is acceptable. even in the rare occurrence of an hpv lesion of the nipple or breast, no data suggest that breastfeeding or the use of expressed breast milk is contraindicated. measles is another highly communicable childhood illness that can be more severe in neonates and adults. measles is an exanthematous febrile illness following a prodrome of malaise, coryza, conjunctivitis, cough, and often koplik spots in the mouth. the rash usually appears 10 to 14 days after exposure. complications can include pneumonitis, encephalitis, and bacterial superinfection. with the availability of vaccination, measles in pregnancy is rare (0.4 in 10,000 pregnancies), 148 although respiratory complications (primary viral pneumonitis, secondary bacterial pneumonia), hepatitis, or other secondary bacterial infections often lead to more severe disease in these situations. prenatal infection with measles may cause premature delivery without disrupting normal uterine development. no specific group of congenital malformations have been described in association with in utero measles infection, although teratogenic effects of measles infection in pregnant women may rarely manifest in the infants. perinatal measles includes transplacental infection when measles occurs in an infant in the first 10 days of life. infection from extrauterine exposure usually develops after 14 days of life. the severity of illness after suspected transplacental spread of virus to an infant varies from mild to severe and does not seem to vary with the antepartum or postpartum onset of rash in the mother. it is uncertain what role maternal antibodies play in the severity of an infant's disease. more severe disease seems to be associated with severe respiratory illness and bacterial infection. postnatal exposure leading to measles after 14 days of life is generally mild, probably because of passively acquired antibodies from the mother. severe measles in children younger than 1 year of age may occur because of declining passively acquired antibodies and complications of respiratory illness and rare cases of encephalitis. measles virus has not been identified in breast milk, whereas measles-specific antibodies have been documented. 1 infants exposed to mothers with documented measles while breastfeeding should be given immunoglobulin (ig) and isolated from the mother until 72 hours after the onset of rash, which is often only a short period after diagnosis of measles in the mother. the breast milk can be pumped and given to the infant because secretory iga begins to be secreted in breast milk within 48 hours of onset of the exanthem in the mother. table 13 -3 summarizes management of the hospitalized mother and infant with measles exposure or infection. 148 mumps is an acute transient benign illness with inflammation of the parotid gland and other salivary glands and often involves the pancreas, testicles, and meninges. mumps occurs infrequently in pregnant women (1 to 10 cases in 10,000 pregnancies) and is generally benign. mumps virus has been isolated from saliva, respiratory secretions, blood, testicular tissue, urine, csf in cases of meningeal involvement, and breast milk. the period of infectivity is believed to be between 7 days before and 9 days after the onset of parotitis, with the usual incubation period being 14 to 18 days. prenatal infection with the mumps virus causes an increase in the number of abortions when infection occurs in the first trimester. a small increase in the number of premature births was noted in one prospective study of maternal mumps infection. 383 no conclusive evidence suggests congenital malformations are associated with prenatal infection, not even with endocardial fibroelastosis, as originally reported in the 1960s. perinatal mumps (transplacentally or postnatally acquired) has rarely if ever been documented. natural mumps virus has been demonstrated to infect the placenta and infect the fetus, and live attenuated vaccine virus has been isolated from the placenta but not from fetal tissue in women vaccinated 10 days before induced abortion. antibodies to mumps do cross the placenta. postnatal mumps in the first year of life is typically benign. no epidemiologic data suggest that mumps infection is more or less common or severe in breastfed infants compared with formula-fed infants. although mumps virus has been identified in breast milk and mastitis is a rare complication of mumps in mature women, no evidence indicates that breast involvement occurs more frequently in lactating women. if mumps occurs in the mother breastfeeding can continue because exposure has already occurred throughout the 7 days before the development of symptoms in the mother and secretory iga in the milk may help to mitigate the symptoms in the infant. human parvovirus b19 causes a broad range of clinical manifestations, including asymptomatic infection (most frequent manifestation in all ages), erythema infectiosum (fifth disease), arthralgia and arthritis, red blood cell (rbc) aplasia (less often decreased white blood cells or platelets), chronic infection in immunodeficient individuals, and rarely myocarditis, vasculitis, or hemophagocytic syndrome. vertical transmission can lead to severe anemia and immune-mediated hydrops fetalis, which can be treated, if accurately diagnosed, by intrauterine transfusion. inflammation of the liver or cns can be seen in the infant, along with vasculitis. if the child is clinically well at birth, hidden or persistent abnormalities are rarely identified. no evidence indicates that parvovirus b19 causes an identified pattern of birth defects. postnatal transmission usually occurs person to person via contact with respiratory secretions, saliva, and rarely blood or urine. seroprevalence in children at 5 years of age is less than 5%, with the peak age of infection occurring during the schoolage years (5% to 40% of children infected). the majority of infections are asymptomatic or undiagnosed seroconversions. 417 severe disease, such as prolonged aplastic anemia, occurs in individuals with hemoglobinopathies or abnormal rbc maturation. attack rates have been estimated to be 17% to 30% in casual contacts but up to 50% among household contacts. in one study of 235 susceptible pregnant women, the annual seroconversion rate was 1.4%. 223 no reports of transmission to an infant through breastfeeding are available. excretion in breast milk has not been studied because of limitations in culturing techniques. rat parvovirus has been demonstrated in rat milk. the very low seroconversion rate in young children and the absence of chronic or frequent severe disease suggest that the risk for parvovirus infection via breast milk is not significant. the possibility of antibodies against parvovirus or other protective constituents in breast milk has not been studied. breastfeeding by a mother with parvovirus infection is acceptable. poliovirus infections (types 1, 2, and 3) cause a range of illness, with 90% to 95% subclinical, 4% to 8% abortive, and 1% to 2% manifest as paralytic poliomyelitis. a review by bates 29 from 1955 of 58 cases of poliomyelitis in infants younger than 1 month of age demonstrated paralysis or death in more than 70% and only one child without evidence of even transient paralysis. more than half the cases were ascribed to transmission from the mothers, although no mention was made of breastfeeding. breastfeeding rates at the time were approximately 25%. prenatal infection with polioviruses does cause an increased incidence of abortion. prematurity and stillbirth apparently occur more frequently in mothers who developed paralytic disease versus inapparent infection. 188 although individual reports of congenital malformations in association with maternal poliomyelitis exist, no epidemiologic data suggest that polioviruses are teratogenic. also, no evidence indicates that live attenuated vaccine poliovirus given during pregnancy is associated with congenital malformations. 89, 170 perinatal infection has been noted in several case reports of infants infected in utero several days before birth who had severe disease manifesting with neurologic manifestations (paralysis) but without fever, irritability, or vomiting. additional case reports of infection acquired postnatally demonstrate illness more consistent with poliomyelitis of childhood. these cases were more severe and involved paralysis, which may represent reporting bias. 89 no data are available concerning the presence of poliovirus in breast milk, although antibodies to poliovirus types 1, 2, and 3 have been documented. 270 in this era of increasing worldwide poliovirus vaccination, the likelihood of prenatal or perinatal poliovirus infection is decreasing. maternal susceptibility to poliovirus should be determined before conception and poliovirus vaccine offered to susceptible women. an analysis of the last great epidemic in italy in 1958 was done using a population-based case-control study. 336 in 114,000 births, 942 infants were reported with paralytic poliomyelitis. a group of matched control subjects was selected from infants admitted to the hospital at the same time. using the dichotomous variable of never breastfed and partially breastfed, 75 never-breastfed infants were among the cases and 88 among the control group. the authors determined an odds ratio of 4.2, with 95% confidence interval of 1.4 to 14, demonstrating that the risk for paralytic poliomyelitis was higher in infants never breastfed and lowest among those exclusively breastfed. because by the time the diagnosis of poliomyelitis is made in a breastfeeding mother, the exposure of the infant to poliovirus from maternal secretions has already occurred, and because the breast milk already contains antibodies that may be protective, no reason exists to interrupt breastfeeding. breastfeeding also does not interfere with successful immunization against poliomyelitis with oral or inactivated poliovirus vaccine. 71 the occurrence of human t-cell leukemia virus type i (htlv-i) is endemic in parts of southwestern japan, 66, 105, 207, 450 the caribbean, south america, 156 and sub-saharan africa. htlv-i is associated with adult t-cell leukemia/lymphoma and a chronic condition with progressive neuropathy. the progressive neuropathy is called htlv-i associated myelopathy or tropical spastic paraparesis. 136 other illnesses have been reported in association with htlv-i infection including dermatitis, uveitis, arthritis, sjögren syndrome in adults, and infective dermatitis and persistent lymphadenitis in children. transmission of htlv-i occurs most often through sexual contact, via blood or blood products, and via breast milk. infrequent transmission does occur in utero or at delivery and with casual or household contact. 291 seroprevalence generally increases with age and varies widely in different regions and in populations of different backgrounds. in some areas of japan, seropositivity can be as high as 12% to 16%, but in south america, africa, and some caribbean countries the rates are 2% to 6%. in latin america seropositive rates can be as high as 10% to 25% among female sex workers or attendees to std clinics. 156 in blood donors in europe, the seroprevalence of htlv-i has been reported at 0.001% to 0.03%. the seroprevalence in pregnant women in endemic areas of japan is as high as 4% to 5% and in nonendemic areas as low as 0.1% to 1.0%. htlv-1 is not a major disease in the united states. in studies from europe the seroprevalence in pregnant women has been noted to be up to 0.6%. these pregnant women were primarily of african or caribbean descent. 138 htlv-i antigen has been identified in breast milk of htlv-i positive mothers. 220 another report shows that basal mammary epithelial cells can be infected with htlv-i and can transfer infection to peripheral blood monocytes. 254 human milk from htlv-i positive mothers caused infection in marmosets. 221, 453 htlv-i infection clearly occurs via breastfeeding and a number of reports document an increased rate of transmission of htlv-i to breastfed infants compared with formula-fed infants.* ando et al 12, 13 in two separate reports demonstrated a parallel decline in antibodies against htlv-i in both formula-fed and breastfed infants to a nadir at approximately 1 year of age and a subsequent increase in antibodies from 1 to 2 years of age. the percentage of children seropositive at 1 year of age in the breastfed and formula-fed groups, respectively, was 3.0% and 0.6%, at 1.5 years of age it was 15.2% and 3.9%, and at 2 years of age it was 41.9% and 4.6%. a smaller group of children followed through 11 to 12 years of age demonstrated no newly infected children after 2 years of age and *references 9, 10, 12, 13, 178-180, 407. no loss of antibody in any child who was seropositive at 2 years of age. 12, 13 transmission of htlv-i infection via breastfeeding is also clearly associated with the duration of breastfeeding. 407, 409, 446, 447 it has been postulated that the persistence of passively acquired antibodies against htlv-i offers some protection through 6 months of life (table 13-4) . other factors relating to htlv-i transmission via breast milk have been proposed. yoshinaga et al 460 presented data on the htlv-i antigen producing capacity of peripheral blood and breast milk cells and showed an increased mother-to-child transmission rate when the mother' s blood and breast milk produced large numbers of antigen-producing cells in culture. 460 hisada et al 183 reported on 150 mothers and infants in jamaica, demonstrating that a higher maternal provirus level and a higher htlv-i antibody titer were independently associated with htlv-i transmission to the infant. ureta-vidal et al 421 reported an increased seropositivity rate in children of mothers with a high proviral load and elevated maternal htlv-i antibody titers. various interventions have been proposed to decrease htlv-i transmission via breastfeeding. complete avoidance of breastfeeding was shown to be an effective intervention by hino et al 180, 181 in large population of japanese in nagasaki. avoiding breastfeeding led to an 80% decrease in transmission. breastfeeding for a shorter duration is another effective alternative. ando et al 11 showed that freezing and thawing breast milk decreased the infectivity of htlv-i. sawada et al 363 demonstrated in a rabbit model that htlv-i immunoglobulin protected against htlv-i transmission via milk. it is reasonable to postulate that any measure that would decrease the maternal provirus load or increase the anti-htlv-i antibodies available to infants might decrease the risk for transmission. the overall prevalence of htlv-i infection during childhood is unknown because the majority of individuals do not manifest illness until much later in life. the timing of htlv-i infection in a breastfeeding population has been difficult to assess because of passively acquired antibodies from the mother and issues related to testing. furnia et al 139 in areas where the prevalence of htlv-i infection (in the united states, canada, or europe) is rare, the likelihood that a single test for antibody against htlv-i would be a false positive test is high compared with the number of true positive tests. repeat testing is warranted in many situations. 66 quantification of the antibody titer and the proviral load is appropriate in a situation when mother-to-child transmission is a concern. a greater risk for progression to disease in later life has not been shown for htlv-i infection through breast milk, but early-life infections are associated with the greatest risk for adult t-cell leukemia. 402 the mother and family should be informed about all these issues. if the risk for lack of breast milk is not too great and formula is readily available and culturally acceptable, then the proscription of breastfeeding, or at least a recommendation to limit the duration of breastfeeding to 6 months or less, is appropriate to limit the risk for htlv-i transmission to the infant. freezing and thawing breast milk before giving it to an infant might be another reasonable intervention to decrease the risk for transmission, although no controlled trials document the efficacy of such an intervention. neither ig nor antiviral agents against htlv-i are available at this time. human t-cell leukemia virus type ii (htlv-ii) is endemic in specific geographic locations, including africa, the americas, the caribbean, and japan. transmission is primarily through intravenous drug use, contaminated blood products, and breastfeeding. sexual transmission occurs but its overall contribution to the prevalence of htlv-ii in different populations remains uncertain. many studies have examined the presence of htlv-i and ii in blood products. pcr testing and selective antibody tests suggest that about half of the htlv seropositivity in blood donors is caused by htlv-ii. htlv-ii has been associated with two chronic neurologic disorders similar to those caused by htlv-i, tropical or spastic ataxia. 258 a connection between htlv-ii and glomerulonephritis, myelopathy, arthritis, t-hairy cell leukemia, and large granulocytic leukemia has been reported. mother-to-child transmission has been demonstrated in both breastfed and formula-fed infants. it appears that the rate of transmission is greater in breastfed infants.* htlv-ii has been detected in breast milk. 174 nyambi et al 304 reported that htlv-ii transmission did correlate with the duration of breastfeeding. the estimated rate of transmission was 20%. the time to seroconversion (after the initial loss of passively acquired maternal antibodies) for infected infants seemed to range between 1 and 3 years of age. 304 at this time avoidance of breastfeeding and limiting the duration of breastfeeding are the only two possible interventions with evidence of effectiveness for preventing htlv-ii mother-to-child transmission. 207 with the current understanding of retroviruses, it is appropriate in cases of documented htlv-ii maternal infection to recommend avoiding or limiting the duration of breastfeeding and provide alternative nutrition when financially practical and culturally acceptable. mothers should have confirmatory testing for htlv-ii and measurement of the proviral load. infants should be serially tested for antibodies to htlv-ii and have confirmatory testing if seropositive after 12 to 18 months of age. further investigation into the mechanisms of transmission via breast milk and possible interventions to prevent transmission should occur as they have for hiv-1 and htlv-i. human immunodeficiency virus type 1 (hiv-1) is transmitted through human milk. refraining from breastfeeding is a crucial aspect of preventing perinatal hiv infection in the united states and many other countries. the dilemma is the use of replacement feeding versus breastfeeding in countries where breastfeeding provides infants with significant protection from illness and death due to malnutrition or other infections. the question of the contribution of breastfeeding in mother-to-child hiv-1 transmission is not a trivial one when one considers the following: 3. the who estimates that 2.7 million people were newly infected with hiv-1 in 2007, with children younger than 14 years old making up 370,000 of that 2.7 million. (this number has declined due to increasing access to interventions to prevent mother-to-infant transmission. availability of antiretroviral therapy for prevention of mother-to-child hiv transmission in developing countries in 2007 was estimated to reach 33% of the mothers who needed it.) 419 4. breastfeeding contributes an estimated 10% to 20% increase in the overall mother-to-child transmission rates, over and above intrauterine and intrapartum transmission, when no specific interventions to prevent transmission via breastfeeding are utilized. 5. despite a dramatic increase in the number of people receiving antiretroviral therapy in developing countries (3 million), this represented only 31% of the individuals who needed treatment. 419 the evidence of hiv transmission via breastfeeding is irrefutable. multiple publications summarize the current evidence for hiv transmission via breastfeeding in the literature. 232, 341, 420 since 1985, case reports have documented hiv transmission via breast milk to children around the world. 182, 198, 249, 465 primary hiv infection in breastfeeding mothers, with the concomitant high viral load, is associated with a particularly high rate of hiv transmission via breast milk. palasanthiran et al 322 estimated that risk at 27%.large observational studies have demonstrated higher rates of hiv transmission in breastfed infants of mothers with chronic hiv infection compared with formula-fed infants. 43, 108, 124 a systematic analysis of published reports estimated the additional risk for perinatal hiv transmission due to breastfeeding to be 14% (95% confidence interval 7% to 22%). 117 more recently published cohort studies similarly attributed additional risk for hiv transmission due to breastfeeding at 4% to 22% over and above the risk from prenatal and intrapartum transmission. 38, 104, 121 laboratory reports demonstrate the presence of cell-free virus and cell-associated virus in breast milk as well as various immunologic factors that could block or limit infection.* a dose-response relationship has been observed, correlating the hiv viral load in human milk as well as a mother' s plasma viral load with an increased transmission risk for the breastfed infant. 335, 345, 351, 373 many of the potential risk factors associated with human milk transmission of hiv is higher the longer the duration of breastfeeding. 108, 251, 282, 290, 424 maternal characteristics related to transmission of hiv via human milk include younger maternal age, higher parity, lower cd4+ counts, higher plasma viral loads, and breast abnormalities (mastitis, abscess, or nipple lesions). characteristics of human milk that relate to a higher risk for transmission include higher viral load in the milk, lower concentrations of antiviral substances (lactoferrin, lysozyme), and lower concentrations of virus-specific cytotoxic t-lymphocytes, levels of various interleukins (il-7, il-15), 434, 435 secretory iga, and igm. mixed breastfeeding is also associated with a higher risk for hiv transmission compared with exclusive breastfeeding. 99, 100, 410 the measurable benefits of breast milk versus the relative risk for hiv transmission to the infant due to exclusive breastfeeding (with optimization of other factors to decrease hiv transmission) have been reported in a couple of studies. 97, 229 the measurable benefits of receiving breast milk versus the relative increased risk for hiv transmission will need to be determined in a prospective fashion in different locales. 247 a number of potential interventions to prevent breastfeeding transmission of hiv-1 can be utilized (box 13-3) . the simplest and most effective is the compete avoidance of human milk. this is a practical solution in places like the united states and other countries where replacement feeding as well as other strictly medical interventions are feasible and reasonable, and the risk for not providing breast milk to the infant is negligible. in resourcepoor situations, where the risk for other infections is high without the benefits of breast milk, exclusive breastfeeding is appropriate, with any other reasonable and culturally acceptable interventions to decrease hiv transmission via breast milk. potentially effective interventions include exclusive breastfeeding, early weaning versus breastfeeding for longer durations, education, and support to decrease the likelihood of mastitis or nipple lesions. 191 other possible interventions include treating a mother with antiretroviral therapy for her own health (cd4 counts less than 350) or prophylactically to decrease the human milk viral load, treating an infant prophylactically for a prolonged period of time (6 weeks to 6 months) to protect against transmission via breastfeeding, treating the milk itself to decrease the viral load (by pasteurization or other methods), 316, 318 treating acute conditions in mothers and infants (e.g., mastitis, breast lesions, infant candidiasis), and enhancing an infant' s own defenses via vitamins, immunization, or antiretroviral therapy. some of these may not be feasible in certain settings such as pasteurization or maternal antiretroviral therapy. others may not be culturally acceptable, such as treating expressed breast milk before giving it to an infant or even exclusive breastfeeding. significant data demonstrate the advantage of breastfeeding, even for hiv-infected or hivexposed infants. the complete avoidance of breastfeeding in certain situations may lead to increased risk for illness and death due to other reasons besides hiv transmission. 106 a study from kenya showed improved hiv-1-free survival rates in a formula-fed group of children born to hiv-positive mothers, but the breastfed and formula groups had similar mortality rates (24.4% versus 20.0%, respectively) and similar incidences of diarrhea and pneumonia in the first 2 years of life. 272 no difference in the two groups was seen in the prevalence of malnutrition, but the breastfed infants had better nutritional status in the first 6 months of life. arpadi et al 20 recommend additional nutritional interventions to complement breastfeeding in this population after 6 months of age. two reports from zambia document the benefit of exclusive breastfeeding for decreasing late hiv transmission and the lower mortality at 12 months in infants who had continued breastfeeding rather than had discontinued breastfeeding at 4 months of age. 229, 385 in malawi, hiv-infected and hiv-exposed infants who were breastfed (exclusive breastfeeding for 2 months and mixed feeding after that) had lower mortality at 24 months than those who were not breastfed. 405 a report from botswana examined breastfeeding plus infant zidovudine prophylaxis for 6 months versus formula feeding plus infant zidovudine for 1 month; this study showed a decreased risk for vertical transmission with formula feeding, but also increased cumulative mortality for the hiv-infected infants at 7 months of age who were in the formula-fed group. 411 a study from south africa examining the use of vitamin a also demonstrated less morbidity in hiv-infected children who were breastfed than not breastfed. 102 other abstract reports have shown increased morbidity in hiv-infected children due to diarrhea, gastroenteritis, and hospitalization after weaning from breastfeeding. 205, 226, 315, 413 exclusive breastfeeding in most areas of the world is essential to infant health and survival, even in the situation of maternal hiv infection. 97, 99, 100, 229 the duration of exclusive breastfeeding is crucial to decreasing the risk for hiv infection in infants versus the risk for malnutrition and other infections with early weaning. in the mashi study in botswana, thior et al 411 evaluated infants randomized to breastfeeding plus infant zidovudine for 6 months or formula feeding plus 1 month of infant zidovudine. the cumulative infant mortality was significantly higher at 7 months for the formula-fed group but at 18 months it was similar between the two groups. the breastfed infants were more likely to become hiv infected despite the 6 months of zidovudine prophylaxis. 411 becquet et al 33 analyzed data from cote d'ivoire for 2001 to 2005; 47% of the hivexposed infants were breastfed for a median of 4 months, and 53% were formula fed and observed for 2 years. no significant difference in the rate of hiv infection was seen in the two groups, and no significant difference between the two groups was seen for morbid events (diarrhea, acute respiratory infections or malnutrition) or hospitalization or death. the authors attributed these good outcomes to effective nutritional counseling and care, access to clean water, and the provision of a safe and continuous supply of breast milk substitute. 33 coovadia et al 97 studied exclusive breastfeeding in the first 6 months of life as an intervention in south africa. of the exclusively breastfed infants, 14.1% at 6 weeks of age and 19.5% at 6 months of age were hiv infected. breastfed infants who also were fed solids or formula milk were more likely to acquire infection than exclusively breastfed infants. the cumulative mortality at 3 months of age was markedly lower for exclusively breastfed infants (6.1%) versus 15.1% in the infants receiving mixed feedings. kuhn et al 230 examined the effects of early, abrupt weaning on hiv-free survival of 958 children in zambia. infants were randomly assigned to two different counseling programs that advised either abrupt weaning at 4 months or prolonged breastfeeding. in the weaning intervention group, 69% of mothers stopped breastfeeding by 5 months compared with a median duration of breastfeeding of 16 months in the control group. the study found no significant difference in hiv-free survival at • women and their health care providers need to be aware of the potential risk for transmission of hiv infection to infants during pregnancy and in the peripartum period and through breast milk. • documented, routine hiv education and routine testing with the consent of women seeking prenatal care are strongly recommended so that each woman knows her hiv status and the methods available both to prevent the acquisition and transmission of hiv and to determine whether breastfeeding is appropriate. • at delivery, education about hiv and testing with the consent of women whose hiv status during pregnancy is unknown are strongly recommended. knowledge of a woman' s hiv status assists in counseling on breastfeeding and helps each woman understand the benefits to herself and her infant of knowing her serostatus and the behaviors that would decrease the likelihood of acquisition and transmission of hiv. • women who are known to have hiv infections must be counseled not to breastfeed or provide their milk for the nutrition of their own or other' s infants. • in general, women who are known to be hiv seronegative should be encouraged to breastfeed. however, women who are hiv seronegative but at particularly high risk for seroconversion (e.g., injection drug users and sexual partners of known hiv-positive persons or active drug users) should be educated about hiv with an individualized recommendation concerning the appropriateness of breastfeeding. in addition, during the perinatal period, information should be provided on the potential risk for transmitting hiv through breast milk and about methods to reduce the risk for acquiring hiv infection. • each woman whose hiv status is unknown should be informed of the potential for hiv-infected women to transmit hiv during the peripartum period and through breast milk and the potential benefits to her and her infant of knowing her hiv status and how hiv is acquired and transmitted. the health care provider needs to make an individualized recommendation to assist the woman in deciding whether to breastfeed. 24 months in the two groups (83.9% versus 80.7%). children already infected by 4 months of age had a higher mortality if they were assigned to the early weaning group (73.6% versus 54.8%). additional analysis showed that in mothers with less severe hiv disease early weaning was clearly harmful to the infant. 231 arpadi et al 20 studied the growth of hiv-exposed, uninfected children who were exclusively breastfed for 4 months with rapid weaning to replacement foods or exclusively breastfed until 6 months and then continued breastfeeding with complementary foods. weight-for-age z scores dropped markedly in both groups from 4 to 15 months of age but less so in the continued breastfeeding group. length-for-age z score also dropped dramatically, but was not influenced by continued breastfeeding. even in this hiv-exposed, uninfected group of children, additional nutritional interventions are essential to complement breastfeeding beyond 6 months of age. 20 in recent years the discussion around preventing hiv transmission via breastfeeding and in the number of studies examining the important issues have increased. 98, 233, 283 the fact that intrapartum and perinatal transmission of hiv from mothers to infants has decreased markedly due to the increased utilization of antiretroviral therapy during pregnancy, delivery, and postnatally for prevention emphasizes the importance of now working harder to decrease breast milk transmission of hiv. in considering different possible interventions to decrease mother-infant hiv transmission, it is crucial to reemphasize the goals of optimizing maternal health and survival and optimizing infant health and survival at the same time. a laboratory report shows that mothers receiving highly active antiretroviral therapy (haart) while breastfeeding do have decreased whole breast milk hiv-1 viral loads (23/26 mothers had less than 50 copies/ml) compared with mothers who did not receive haart (9/25 with less than 50 copies/ml). however, the whole milk hiv-1 dna load (measured as "undetectable" at less than 10 copies/10 6 cells) was not significantly different in the haart (13 of 26 mothers)] and non-haart (15 of 23) groups. 378 hiv-1 dna is incorporated into cells found in breast milk. another group showed significantly lower hiv rna levels in the breast milk of women treated with nevirapine, zidovudine, and lamivudine compared with women not receiving antiretroviral therapy. 152 the use of maternal haart seems to decrease hiv-1 transmission via breastfeeding. one group working in mozambique, malawi, and tanzania working with mother-infants pairs receiving haart as prevention during pregnancy compared one cohort (809 mother-infant pairs) who received supplementary formula and water filters for the first 6 months of life with a second cohort (251 motherinfant pairs) breastfeeding exclusively and the mothers receiving haart for the first 6 months. the cumulative incidence rate of hiv infection at 6 months of age was 2.7% for the formula-fed infants and 2.2% for breastfed infants. through 6 months of age no apparent additional risk for late postnatal transmission of hiv was observed. 323 the petra study team working in tanzania, south africa, and uganda examined the efficacy of three shortcourse regimens of zidovudine and lamivudine in preventing early and late hiv transmission in this predominantly breastfeeding population. 332 there were four regimens: a, zidovudine and lamivudine starting at 36 weeks' gestation plus intrapartum medication and 7-days' postpartum treatment; b, same as a without the prepartum component; c, intrapartum zidovudine and lamivudine only; d, placebo. at week 6 the hiv transmission rates were 5.7% in group a, 8.9% in group b, 14.2% in group c, and 15.3% in group d. at 18 months the hiv infection rates were 15% in group a, 18% in group b, 20% in group c, and 22% in group d. although a measurable decrease in transmission at 6 weeks of age was observed, limited protection was seen at 18 months of age. an observational study from tanzania compared maternal haart for 6 months with exclusive breastfeeding and abrupt weaning at 5 to 6 months of age with a historical control of the same feeding schedule without the postnatal maternal haart. in the treatment group the cumulative hiv transmission was 4.1% at 6 weeks, 5.0% at 6 months, and 6.0% at 18 months of age. the cumulative hiv infection or death rate was 8.6% at 6 months and 13.6% at 18 months of age. the cumulative risk for hiv transmission was 1.1% between 6 and 18 months. the hiv transmission in this treatment group was half the transmission rate in the historical control group. 218 another study in sub-saharan africa with 6 months of maternal haart and exclusive breastfeeding for 6 months demonstrated 94% hiv-free survival at 12 months of age; the maternal and infant mortality rates for the treated mother-infant pairs were significantly lower than the country' s maternal and infant mortality rates. 264 antiretroviral therapy prophylaxis for infants is another investigated intervention to decrease hiv transmission via breastfeeding. in a study from cote d'ivoire comparing different groups over time, infants received zidovudine (zdv) alone as zdv prophylaxis, a single dose of nevirapine (nvp), and 7 days of zidovudine (zdv) as nvp/ zdv prophylaxis, or a single dose of nevirapine plus zidovudine and lamivudine (3tc) for 7 days as nvp/zdv/3tc prophylaxis. formula feeding (ff) was compared with exclusive shortened breastfeeding (esb) upto 4 months of age and prolonged breastfeeding (pb). the cumulative transmission rates at 18 months were 22.3% in 238 infants in the zdv + pb group, 15.9% in 169 infants in the nvp/zdv + esb group, 9.4%, in the 195 infants in the nvp/zdv +ff group, 6.8% in the 198 infants in the nvp/zdv/3tc + esb group, and 5.6% in the 126 infants in the nvp/zdv/3tc + ff group. 252 kumwenda et al 235 working in malawi demonstrated decreased hiv transmission with breastfeeding and two different infant prophylaxis regimens. at 9 months of age, they observed a 10.6% occurrence of hiv transmission for infants receiving a single dose of nevirapine plus 1 week of zidovudine compared with 5.2% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of daily nevirapine, and 6.4% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of nevirapine and zidovudine. 235 in the mitra study in tanzania in which the median time of breastfeeding was 18 weeks, the hiv transmission rate at 6 months in the infants who received zidovudine plus 3tc for 1 week plus 3tc alone for breastfeeding through 6 months of age was less than 50% of the transmission rate for those infants receiving only 1 week of zidovudine plus 3tc. 217 a summary of three trials in ethiopa, india, and uganda compared a single dose of nevirapine at birth for infants with 6 weeks of daily nevirapine in predominantly breastfed infants whose mothers were counselled regarding feeding per the who/ unicef guidelines. at 6 months 87 of 986 infants in the single-dose group and 62 of 901 in the extended-dose group were hiv infected, which was not statistically significant. the authors suggested that a longer course of infant antiretroviral prophylaxis might be more effective. 388 the potential effect of breastfeeding on the hivpositive mother needs to be adequately assessed in relation to the mother's health status. from uganda and zimbabwe mbizvo et al 271 reported no difference in the number of hospital admissions or mortality between hiv-positive and hiv-negative women during pregnancy. in the 2 years after delivery the hiv-positive women had higher hospital admission (approximately two times increased risk) and death rates (relative risk greater than 10) than hiv-negative women. 271 chilongozi et al 90 reported on 2292 hiv-positive mothers from four sub-saharan sites followed for 112 months. serious adverse events occurred in 166 women (7.2%); 42 deaths occurred in the hiv-positive women, and no deaths occurred in 331 hiv-negative women. 90 several studies have examined breastfeeding relative to mothers' health and reported conflicting results. the first study from kenya demonstrated a significantly higher mortality rate in breastfeeding mothers compared with a formula-feeding group in the 2 years after delivery. the hypothesized explanation offered by the authors for this difference was increased metabolic demands, greater weight loss, and nutritional depletion. 294 a second study from south africa showed an overall lower mortality rate in the two groups with no significant difference in mortality rate in the 10 months of observation. 101 kuhn et al 227 reported no difference in mortality at 12 months after delivery between 653 women randomly assigned to a short breastfeeding group (326 women, median breastfeeding duration 4 months, 21% still breastfeeding at 12 months) and a long breastfeeding group (327 women, 90% breastfeeding at 5 months, 72% breastfeeding at 12 months, median 15 months). the hiv-related mortality rates were high (4.9%), but not associated with prolonged lactation. 227 walson et al 433 followed 535 hiv-positive women for 1 to 2 years in kenya. the mortality risk was 1.9% at 1 year and 4.8% at 2 years of follow-up. although less than 10% of women reported a hospitalization during the 2 years, they experienced various common infections (pneumonia, diarrhea, tb, malaria, stds, urinary tract infections, mastitis). breastfeeding was a significant cofactor for diarrhea and mastitis but not for pneumonia, tb, or hospitalization. 433 in summary, breastfeeding of infants by hiv-positive mothers does lead to an increased risk for hiv infection in the infants. much remains to be understood about the mechanisms of hiv transmission via breast milk and the action and efficacy of different interventions to prevent such transmission. the complete avoidance of breastfeeding is a crucial component for the prevention of perinatal hiv infection in the united states and many other countries. in resource-poor settings, where breastfeeding is the norm and where it provides vital nutritional and infection protective benefits, the who, unicef, and the joint united nations programme on hiv/ aids (unaids) recommend education, counseling, and support for hiv-infected mothers so they can make an informed choice concerning infant feeding. mothers choosing to breastfeed should receive additional education, support, and medical care to minimize the risk for hiv transmission and to optimize their own health status during and after breastfeeding. mothers choosing to use replacement feedings should receive parallel education, support, and medical care for themselves and their infants to minimize the effect of the lack of breastfeeding. good evidence now shows that antiretroviral prophylactic regimens for mothers or infants while continuing breastfeeding does decrease postnatal hiv transmission. early weaning is associated with increased morbidity and mortality. further carefully controlled research is indicated to adequately assess the risks and benefits to infants and mothers of prolonged breastfeeding with antiretroviral prophylaxis for either or both mothers and infants. along with this, hiv testing rates must be improved at the same time as increased availability and access to antenatal care, hiv prevention services, and hiv medical care for everyone must be increased. the availability and free access to antiretroviral medications must also improve. the decision about infant feeding for hivpositive mothers remains a difficult one, but this is slowly changing with increasing options. the goals remain 100% hiv transmission prevention, optimal maternal health and survival, and long-term infant health and survival. human immunodeficiency virus type 2 (hiv-2) is an rna virus in the nononcogenic, cytopathic lentivirus genus of retroviruses. it is genetically closer to simian immunodeficiency virus than to hiv-1. the clinical disease associated with hiv-2 has similar symptoms to hiv-1 infection but progresses at a slower rate to severe immunosuppression. hiv-2 is endemic in western africa and parts of the caribbean and found infrequently in europe and north and south america. 190, 305 it is transmitted via sexual contact, blood, or blood products and from mother to child. routine testing for hiv-2 is recommended in blood banks. antibody tests used for hiv-1 are only 50% to 90% sensitive for detecting hiv-2. 65 specific testing for hiv-2 is appropriate whenever clinically or epidemiologically indicated. vertical transmission occurs infrequently. ekpini et al 121 followed a large cohort of west african mothers and infants: 138 hiv-1 positive women, 132 hiv-2 positive women, 69 women seropositive for both hiv-1 and 2, and 274 hiv seronegative women. a few cases of perinatal hiv-2 transmission occurred, but no case of late postnatal transmission was observed. 121 it is probable that hiv-2 transmission via breast milk is less common than with hiv-1, but insufficient data support that the risk for transmission is zero. mothers who test positive for hiv-2 should be tested for hiv-1, and guidelines for breastfeeding should follow those for hiv-1 until additional information is available. rabies virus produces a severe infection with progressive cns symptoms (anxiety, seizures, altered mental status) that ultimately proceeds to death; few reports of survival exist. rabies occurs worldwide except in australia, antarctica, and several island groups. in 1992 more than 36,000 cases of rabies were reported to the who, a number that is probably a marked underestimate of the actual cases. 67 between 1990 and 2003, 37 cases of human rabies were reported in the united states. 70, 78 postexposure prophylaxis is given to thousands of patients each year. rabies virus is endemic in various animal populations, including raccoons, skunks, foxes, and bats. because of aggressive immunization programs, rabies in domesticated dogs and cats in the united states is uncommon. the virus is found in the saliva and tears and nervous tissue of infected animals. transmission occurs by bites, licking, or simply contact of oral secretions with mucous membranes or nonintact skin. many cases of rabies in humans now lack a history of some obvious contact with a rabid animal. this may be a result of the long incubation period (generally 4 to 6 weeks, but can be up to 1 year, with reports of incubation periods of several years), a lack of symptoms early in an infectious animal, or airborne transmission from bats in enclosed environments (caves, laboratories, houses). person-to-person transmission via bites has not been documented, although it has occurred in corneal transplants. 44 rabies viremia has not been observed in the spread of the virus. no evidence exists indicating transmission through breast milk. in the case of maternal infection with rabies, many scenarios can occur before the onset of progressive, severe cns symptoms. the progression and severity of maternal illness can preclude breastfeeding, but separation of an infant from the mother is appropriate regardless of the mother' s status and method of infant feeding (especially to avoid contact with saliva and tears). breastfeeding should not continue when the mother has symptoms of rabies, and the infant should receive postexposure immunization and close observation. an infant may received expressed breast milk, but the expression must occur without possible contamination with saliva or tears from the mother. depending on the scenario, the nature of a mother' s illness, the possible exposure of an infant to the same source as the mother, and the exposure of a child to the mother, postexposure immunization of an infant may be appropriate. a more common scenario is a mother' s apparent exposure to rabies (without exposure for the infant), necessitating postexposure immunization of the mother with rabies vaccine. in the majority of cases, in the absence of maternal illness, breastfeeding can reasonably continue during the mother' s five-dose immunization series in 28 days. in a rare situation in which apparent exposure of mother and infant to rabies occurs together, postexposure treatment of both mother and infant should be instituted, and breastfeeding can continue. respiratory syncytial virus (rsv) is a common cause of respiratory illness in children and is relatively common in adults, usually producing milder upper respiratory tract infection in adults. no evidence indicates that rsv causes intrauterine infection, adversely affects the fetus, or causes abortion or prematurity. rsv does produce infection in neonates, causing asymptomatic infection, afebrile upper respiratory tract infection, bronchiolitis, pneumonia, and apnea. mortality rate can be high in neonates, especially in premature infants and ill full-term infants, particularly those with preexisting respiratory disease (hyaline membrane disease, bronchopulmonary dysplasia) or cardiac disease associated with pulmonary hypertension. rsv is believed to be transmitted via droplets or direct contact of the conjunctiva, nasal mucosa, or oropharynx with infected respiratory secretions. documentation of rsv infection is rarely made in adults, and spread from a mother or other household contacts probably occurs before a diagnosis can be made. therefore risk for rsv transmission from breast milk is probably insignificant compared with transmission via direct or droplet contact in families. in nurseries, however, it is appropriate to make a timely diagnosis of rsv infection in neonates to isolate infants from the others and prevent spread in the nursery. ribavirin is not recommended for routine use. it is infrequently used in patients with potentially life-threatening rsv infection. rsv infection should be suspected in any infant with rhinorrhea, nasal congestion, or unexplained apnea, especially in october through march in temperate climates. prophylaxis against rsv with rsv-specific immunoglobulin iv (rsv-igiv) during this season for infants at highest risk for severe disease is appropriate. debate surrounds the topic of the effect of passively acquired antibodies (in infants from mothers before birth) against rsv on the occurrence and severity of illness in neonates and infants. it appears that a higher level of neutralizing antibody against rsv in neonates decreases the risk for severe rsv disease. 153, 239 some controversy remains concerning the measurable benefit of breastfeeding for preventing serious rsv disease. 3, 54, 115 some studies have shown benefit and others no effect. controlling for possible confounding factors (e.g., smoking, crowded living conditions) in these studies has been difficult. at this point, no reason exists to stop breastfeeding with maternal rsv infection; a potential exists for benefit from nonspecific factors in breast milk against the rsv. infants with rsv infection should breastfeed unless their respiratory status precludes it. rotavirus infections usually result in diarrhea, accompanied by emesis and low-grade fever. in severe infections the clinical course can include dehydration, electrolyte abnormalities, and acidosis and can contribute to malnutrition in developing countries. generally, every child will have at least one episode of rotavirus infection by 5 years of age. 157 in developed countries, rotavirus is often associated with diarrhea requiring hospitalization in children younger than 2 years of age, but rarely associated with death. worldwide rotavirus is the leading cause of diarrhea-related deaths in children younger than 5 years old. estimates suggest that in children younger than 5 years old rotavirus infection leads to more than 100 million occurrences of diarrhea, 2 million hospital admissions, and 500,000 deaths each year. 157 fecal-oral transmission is the most common route, but fomites and respiratory spread may also occur. spread of infection occurs most often in homes with young children or in daycare centers and institutions. in hospitalized infants or mothers with rotavirus infection, contact precautions are indicated for the duration of the illness. no evidence indicates prenatal infection from rotavirus, but perinatal or postnatal infection from contact with the mother or others can occur. no case of transmission of rotavirus via breast milk has been documented. breast milk does contain antibodies to rotavirus for up to 2 years. human milk mucin has been demonstrated to inhibit rotavirus replication and prevent experimental gastroenteritis. 457 the mechanisms of rotavirus immunity are not well understood. they are thought to be multifactorial with cell-mediated immunity limiting severity and the course of infection, while humoral immunity protects against subsequent infections. innate and adaptive responses at the level of the mucosa are probably the most important. 134 exclusive breastfeeding may decrease the likelihood of severe rotavirus-related diarrhea by as much as 90%. 93, 377 although breastfeeding does not prevent infection with rotavirus, it seems to decrease the severity of rotavirus-induced illness in children younger than 2 years old. 93, 123, 184 at least one study suggested that this may represent simply the postponement of severe rotavirus infection until an older age. 93 one study suggested that protection against rotavirus rapidly declines upon discontinuation of breastfeeding. 356 this delay in rotavirus infection until the child is older may be beneficial in that the older child may be able to tolerate the infection or illness with a lower likelihood of becoming dehydrated or malnourished. continuing breastfeeding during an episode of rotavirus illness with or without vomiting is appropriate and often helpful to the infant. no reason to suspend breastfeeding by a mother infected with rotavirus is apparent. two rotavirus vaccines (rotateq and rotarix) have been licensed for use in more than 90 countries, but less than 20 countries have routine immunization programs. additional types of rotavirus vaccines are undergoing study in various countries, specifically examining the efficacy of the vaccines in low and medium income countries. 444 some of the explanations for the slow implementation of an effective vaccine globally include differences in protection with specific vaccines in high income countries compared with low or medium income countries, the unfortunate association with intussusception in the united states, the delayed recognition of the significant rotavirus-related morbidity and mortality, and the cost of the new vaccines. the question of variable efficacy of the specific rotavirus vaccines in developed and developing countries remains an important one. several trials are examining this issue and attempting to address factors such as maternal transplacentally transferred antibodies, breastfeeding practices (especially immediately before immunization with a live oral rotavirus vaccine), stomach acid, micronutrient malnutrition, interfering gut flora, and differences in the epidemiology of rotavirus in different locations. 327 evidence indicates that maternal immunization with rotavirus vaccine can increase both transplacental acquisition of antibodies and secretory iga in breast milk. 334 additionally, oral rotavirus vaccines have been able to stimulate a good serologic response in both formula-fed and breastfed infants, although the antigen titers may need to be modified to create an optimal response in all infants. 86 the actual protective effect of these vaccines in different situations and strategies will require measurement in ongoing prospective studies. congenital rubella infection has been well described, and the contributing variables to infection and severe disease have been elucidated. the primary intervention to prevent congenital rubella has been to establish the existence of maternal immunity to rubella before conception, including immunization with rubella vaccine and reimmunization if indicated. perinatal infection is not clinically significant. postnatal infection occurs infrequently in children younger than 1 year of age because of passively acquired maternal antibodies. the predominant age of infection is 5 to 14 years old, and more than half of those with infections are asymptomatic. postnatal rubella is a self-limited, mild viral infection associated with an evanescent rash, shotty adenopathy, and low-grade transient fever. it most often occurs in the late winter and spring. infants with congenital infection shed the virus for prolonged periods from various sites and may serve as a source of infection throughout the year. contact isolation is appropriate for suspected and proved congenital infection for at least 1 year, including exclusion from day care and avoidance of pregnant women, whereas postnatal rubella infection requires droplet precautions for 7 days after the onset of rash. rubella virus has been isolated from breast milk after natural infection (congenital or postnatal) and after immunization with live attenuated vaccine virus. both iga antibodies and immunoreactive cells against rubella have been identified in breast milk. breastfed infants can acquire vaccine virus infection via milk but are asymptomatic. because postpartum infection with this virus (natural or vaccine) is not associated with clinically significant illness, no reason exists to prevent breastfeeding after congenital infection, postpartum infection with this virus, or maternal immunization with rubella vaccine. severe acute respiratory syndrome (sars) is a term that could be applied to any acute serious respiratory illness caused by or associated with a variety of infections agents; since 2003, however, it has been linked with sars-associated coronavirus (sars-cov). in the global outbreak of 2002 to 2003, more than 8400 probable cases of sars and more than 800 deaths occurred. more than the actual number of affected individuals or its associated mortality rate (approximately 10% overall, and closer to 50% in persons older than 65 years of age), it was what we did not know about this new unusual illness, and the tremendous publicity surrounding it, that made sars such a sensation. we now know the cause of this illness, known as the sars-cov. sars-cov was shown not to be closely related to the previously characterized coronavirus groups. 265, 350 despite intense international collaboration to study the illness and the virus, many things are not known, such as the degree of infectiousness, the actual period of transmissibility, all the modes of transmission, how many people have an asymptomatic infection compared with those with symptoms or severe illnesses, how to make a rapid diagnosis of confirmed cases, and where it originated. at least 21 cases of probable sars in children have been described in the literature. 42, 187, 380, 387 in general, the illness in children is a mild, nonspecific respiratory illness, but in adolescents and adults it is more likely to progress to severe respiratory distress. it has been reported that children are less likely to transmit sars than adults. 187 the overall clinical course, the radiologic evolution, and the histologic findings of these illness are consistent with the host' s immune response playing a significant role in disease production. five infants were born to mothers with confirmed sars. the infants were born prematurely (26 to 37 weeks), presumably due to maternal illness. although two of the five infants had serious abdominal illnesses (other coronaviruses have been associated with reported outbreaks of necrotizing enterocolitis), the presence of sars-cov could not be demonstrated in any of these infants. 380 no evidence of vertical transmission of sars is available. the mode of feeding for any of the reported cases of young children with sars or the infants born to mothers with sars was not mentioned. as with other respiratory viruses predominantly transmitted by droplets, transmission via breast milk is an insignificant mode of transmission, if it occurs at all. the benefits of breastfeeding being what they are, mothers with sars should continue breastfeeding if they are able, or expressed breast milk can be given to an infant until the mother is able to breastfeed. in this era of worry about biologic terrorism, smallpox is an important concern. the concern for infants (breastfed or formula-fed) is direct contact with mothers or household members with smallpox. smallpox is highly contagious in the household setting due to person-to-person spread via droplet nuclei or aerosolization from the oropharynx and direct contact with the rash. additional potential exposures for infants include the release of a smallpox aerosol into the environment by terrorists, contact with a smallpox-contaminated space or the clothes of household members exposed to an aerosol, and infection via contact with a mother' s or a household member' s smallpox vaccination site. these risks are the same for breastfed and formulafed infants. no evidence for transmission of the smallpox virus via breast milk exists. a contact is defined as a person who has been in the same household or had face-to-face contact with a patient with smallpox after the onset of fever. patients do not transmit infection until after progression from the fever stage to the development of the rash. an exposed contact does not need to be isolated from others during the postcontact observation period (usually 17 days) until the person develops fever. temperature should be monitored daily in the exposed contact. personal contact and breastfeeding between mother and infant can continue until the onset of fever, when immediate isolation (at home) should begin. providing expressed breast milk for the infant of a mother with smallpox should be avoided because of the extensive nature of the smallpox rash and the possibility of contamination (from the rash) of the milk during the expression process. no literature documents transmission of the smallpox virus via expressed breast milk. the other issue for breastfeeding infants is the question of maternal vaccination with smallpox in a preexposure event vaccination program. children older than 1 year of age can be safely and reasonably vaccinated with smallpox in the face of a probable smallpox exposure. smallpox vaccination of infants younger than 1 year of age is contraindicated. breastfeeding is listed as a contraindication to vaccination in the preevent vaccination program. it is unknown whether vaccine virus or antibodies are present in breast milk. the risk for infection due to contact or aerosolization of virus from a mother' s smallpox vaccination site is the same for breastfed and formula-fed infants. the advisory committee on immunization practices also does not recommend preevent smallpox vaccination of children younger than 18 years old. 443 a report documents tertiary contact vaccinia in a breastfeeding infant. 140 a united states military person received a primary smallpox vaccination and developed a local reaction at the inoculation site. despite reportedly observing appropriate precautions, the individual' s wife developed vesicles on both areolae (secondary contact vaccinia). subsequently, the breastfeeding infant developed lesions on her philtrum, cheek, and tongue. both the mother and infant remained well and the infections resolved without therapy. culture and pcr testing confirmed vaccinia in both the mother' s and the infant' s lesions. the breast milk was not tested. 140 in a review from 1931 to 1981, sepkowitz 375 reported on 27 cases of secondary vaccinia in households. the cdc reported 30 suspected cases of secondary/tertiary vaccinia with 18 of those cases confirmed by culture or pcr. the 30 cases were related to 578,286 vaccinated military personnel. this is an incidence of 5.2 cases per 100,000 vac cinees and 7.4 cases per 100,000 primary vaccinees. 79 in a separate report on the civilian preevent smallpox vaccination program, 37,802 individuals were vaccinated between january and june 2003, and no cases of contact vaccinia were reported. 77 the risk for contact vaccinia is low. the risk is from close or intimate contact. in the above-mentioned case, the risk for the infant was contact with the mother' s breasts, the inadvertent site of her contact vaccinia. breastfed and formulafed infants are equally at risk from close contact in the household of a smallpox vaccinee or a case of secondary vaccinia, and separation from the individual is appropriate in both situations. if the breast of the nursing mother is not involved, expressed breast milk can be given to the infant. tt virus (ttv) is a recently identified virus found in a patient (tt) with posttransfusion hepatitis not associated with the other hepatitis-related viruses a through g. ttv has been described as an unenveloped, circular, single-stranded dna virus. 311 this virus is prevalent in healthy individuals, including healthy blood donors, and has been identified in patients with hepatitis. ttv dna has been detected in infants of ttv-positive and ttvnegative mothers. ohto et al 310 reported no ttv dna was detected in cord blood from 38 infants, and it was detected in only 1 of 14 samples taken at 1 month of age. they noted an increasing prevalence from 6 months (22%) to 2 years (33%), which they ascribed to acquisition via nonparenteral routes. in comparisons of the ttv dna in ttvpositive mothers and their ttv-positive infants, 6 of 13 showed high level nucleotide sequence similarity, and 7 of 13 differed by greater than 10%. 310 schröter et al 371 reported on ttv dna in breast milk examined retrospectively. notably, ttv dna was detected in 22 of 23 serum samples of infants at 1 week of age, who were born to 22 women viremic for ttv dna. twenty-four women who were negative for ttv dna gave birth to 24 children who were initially negative for ttv dna and remained negative throughout the observation period (mean 7.5 months, range 1 to 28 months). ttv dna was detected in 77% of breast milk samples from ttv viremic women and in none of the breast milk samples from ttv-negative women. no clinical or laboratory evidence of hepatitis was found in the 22 children who were observed to be ttv dna positive during the period of the study. 371 other authors have reported ttv in breast milk detected by pcr. they describe the absence of ttv dna in infants at 5 days and 3 months of age, and 4 of 10 infants were positive for ttv dna at 6 months of age, suggesting the late acquisition of infection via breastfeeding. 197 tt virus is transmitted in utero and is found in breast milk. no evidence of clinical hepatitis in infants related to ttv infection and no evidence for a late chronic hepatitis exist. given the current available information, no reason to proscribe breastfeeding by ttv-positive mothers is compelling. certainly more needs to be understood concerning the chronic nature of this infection and the possible pathogenesis of liver disease. no documented evidence indicates that women with breast cancer have rna of tumor virus in their milk. no correlation between rna-directed dna polymerase activity has been found in women with a family history of breast cancer. rna-directed dna polymerase activity, a reserve transcriptase, is a normal feature of the lactating breast. 91, 129, 352 epidemiologic data conflict with the suggestion that the tumor agent is transmitted through the breast milk. the incidence of breast cancer is low among groups who had nursed their infants, including lower economic groups, foreign-born groups, and those in sparsely populated areas. 262 the frequency of breast cancer in mothers and sisters of a woman with breast cancer is two to three times that expected by chance. this could be genetic or environmental. cancer actually is equally common on both sides of the family of an affected woman. if breast milk were the cause, it should be transmitted from mother to daughter. when mother-daughter incidence of cancer was studied, no relationship was found to breastfeeding. sarkar et al 360 reported that human milk, when incubated with mouse mammary tumor virus, caused degradation of the particular morphology and decreased infectivity and reverse transcriptase activity of the virions. they suggest that the significance of this destructive effect of human milk on mouse mammary tumor virus may account for the difficulty in isolating the putative human mammary tumor agent. sanner 359 showed that the inhibitory enzymes in milk can be removed by special sedimentation technique. he ascribes the discrepancies in isolating virus particles in human milk to these factors, which inhibit rna-directed dna po lymerase. the fear of cancer in breastfed female offspring of a woman with breast cancer does not justify avoiding breastfeeding. breastfed women have the same breast cancer experience as nonbreastfed women, and no increase is seen in benign tumors. daughters of breast cancer patients have an increased risk for developing benign and malignant tumors because of their heredity, not because of their breastfeeding history. 280, 287 unilateral breastfeeding (limited to the right breast) is a custom of tanka women of the fishing villages of hong kong. ing et al 194 investigated the question, "does the unsuckled breast have an altered risk for cancer?" they studied breast cancer data from 1958 to 1975. breast cancer occurred equally in the left and the right breasts. comparison of patients who had nursed unilaterally with nulliparous patients and with patients who had borne children but not breastfed indicated a highly significantly increased risk for cancer in the unsuckled breast. the authors conclude that in postmenopausal women who have breastfed unilaterally, the risk for cancer is significantly higher in the unsuckled breast. they thought that breastfeeding may help protect the suckled breast against cancer. 194 others 274 have suggested that tanka women are ethnically a separate people and that left-sided breast cancer may be related to their genetic pool and not to their breastfeeding habits. no mention has been made of other possible influences, such as the impact of their role as "fishermen" or any inherent trauma to the left breast. 274 in 1926, lane-claypon 240 stated that a breast that had never lactated was more liable to become cancerous. nulliparity and absence of breastfeeding had been considered important risk factors for breast cancer. macmahon et al 262 reported in 1970 that age at first full-term pregnancy was the compelling factor, and the younger the mother, the less the risk. in a collective review of the etiologic factors in cancer of the breast in humans, papaioannou 325 concludes, "genetic factors, viruses, hormones, psychogenic stress, diet, and other possible factors, probably in that order of importance, contribute to some extent to the development of cancer of the breast." 325 wing 449 concluded in her 1977 review on human milk and health that "in view of the complete absence of any studies showing a relationship between breastfeeding and increased risk of breast cancer, the presence of virus-like particles in breast milk should not be a contraindication to breastfeeding." henderson et al 173 gradually, studies have appeared challenging the dogma. brinton et al, 52 mctiernan and thomas, 276 and layde et al 245 showed the clearly protective effects of breastfeeding. another example is a study conducted to clarify whether lactation has a protective role against breast cancer in an asian people, regardless of confounding effects of age at first pregnancy, parity, and closely related factors. 458 in a hospital-based case-control study of 521 women without breast cancer, statistical adjustment for potential confounders and a likelihood ratio test for linear trend were done by unconditional logistic regression. total months of lactation regardless of parity was the discriminator. regardless of age of first pregnancy and parity, lactation had an independent protective effect against breast cancer in japanese women. 458 although breast cancer incidence is influenced by genetics, stress, hormones, and pregnancy, breastfeeding clearly has a protective effect. "there is a reduction in the risk of breast cancer among premenopausal women who have lactated. no reduction in the risk of breast cancer occurred among postmenopausal women with a history of lactation," according to newcombe et al, 299 reporting a multicenter study in 1993. varicella-zoster virus infection (varicella/chickenpox, zoster/shingles) is one of the most communicable diseases of humans, in a class with measles and smallpox. transmission is thought to occur via respiratory droplets and virus from vesicles. varicella in pregnancy is a rare event, although disease can be more severe with varicella pneumonia, and can be fatal. congenital varicella-zoster virus infection occurs infrequently, causing abortion, prematurity, and congenital malformations. a syndrome of malformations has been carefully described with congenital varicella-zoster virus infection, typically involving limb deformity, skin scarring, and nerve damage, including to the eye and brain. 148 perinatal infection can lead to severe infection in infants if maternal rash develops 5 days or less before delivery and within 2 days after delivery. illness in infants usually develops before 10 days of age and is believed to be more severe because of the lack of adequate transfer of antibody from the mother during this period and transplacental spread of virus to the fetus and infant during viremia in the mother. varicella in a mother occurring before 5 days before delivery allows sufficient formation and transplacental transfer of antibodies to the infant to ameliorate disease even if the infant is infected with varicella-zoster virus. mothers who develop varicella rash more than 2 days after delivery are less likely to transfer virus to the infant transplacentally; they pose a risk to the infants from postnatal exposure, which can be diminished by the administration of varicellazoster ig to the infant. postnatal transmission is believed to occur through aerosolized virus from skin lesions or the respiratory tract entering the susceptible infant's respiratory tract. airborne precautions are therefore appropriate in the hospital setting. infants infected with varicella-zoster virus in utero or in the perinatal period (younger than 1 month of age) are more likely to develop zoster (reactivation of latent varicella-zoster virus) during childhood or as young adults. postnatal varicella from nonmaternal exposure can occur but is generally mild when it develops after 3 weeks of age or when a mother has passed on antibodies against varicella-zoster virus via the placenta. severe postnatal varicella does occur in premature infants or infants of varicella-susceptible mothers. when a mother' s immune status relative to varicella-zoster virus is uncertain and measurement of antibodies to varicella-zoster virus in mother or infant cannot be performed promptly (less than 72 hours), administration of vzig 81 or ivig to the infant exposed to varicella or zoster in the postnatal period is indicated. ideally a mother' s varicella status should be known before pregnancy, when varicella virus vaccine could be given if indicated. varicella-zoster virus virus has not been cultured from milk, but varicella-zoster virus dna has been identified in breast milk. 459 antibody against varicella-zoster virus has also been found in breast milk. 270 breast milk from mothers who had received the varicella vaccine in the postpartum period was tested for varicella-zoster virus dna. varicella dna was not detected in any of the 217 breast milk samples from the 12 women, all of whom seroconverted after vaccination. 45 one case of suspected transfer of varicella-zoster virus to an infant via breastfeeding has been reported, but virus may have been transmitted by respiratory droplet or exposure to rash before the mother began antiviral therapy. 459 isolation of an infant from the mother with varicella and interruption of breastfeeding should occur only while the mother remains clinically infectious, regardless of the method of feeding. as soon as the infant has received varicella-zoster ig, expressed breast milk can be given to an infant if no skin lesions involve the breasts. persons with varicella rash are considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted, usually in 6 to 10 days. immunocompetent mothers who develop zoster can continue to breastfeed if the lesions do not involve the breast and can be covered because antibodies against varicella-zoster virus are provided to the infant via the placenta and breast milk and will diminish the severity of disease, even if not preventing it. conservative management in this scenario would include giving an infant varicella-zoster ig as well (see table 13 -5). 331 it is estimated that 150 to 300 asymptomatic cases of west nile infection occur for every 20 febrile illnesses and for every one case of meningoencephalitis associated with west nile virus. west nile fever is usually a mild illness of 3 to 6 days' duration. the symptoms are relatively nonspecific, including malaise, nausea, vomiting, headache, myalgia, lymphadenopathy, and rash. west nile disease is characterized by severe neurologic symptoms (e.g., meningitis, encephalitis, or acute flaccid paralysis, and occasionally optic neuritis, cranial nerve abnormalities, and seizures). children are infrequently sick with west nile virus infection and infants younger than 1 year of age have rarely been reported. 331 the case-fatality rate for 2003 in the united states was approximately 2.5%, but has been reported as high as 4% to 18% in hospitalized patients. the case-fatality rate for persons older than 70 years of age is considered to be higher, 15% to 29% among hospitalized patients in outbreaks in romania and israel. 331 the primary mechanism of transmission is via a mosquito bite. mosquitoes from the genus culex are primary vectors. the bird-mosquito-bird cycle serves to maintain and amplify the virus in the environment. humans and horses are incidental hosts. the pathogenesis of the infection is believed to occur via replication of the virus in the skin and lymph nodes, leading to a primary viremia that seeds secondary sites before a second viremia causes the infection of the cns and other affected organs. 59, 111 transmission has been reported in rare instances during pregnancy 7,73 via organ transplant 199 and percutaneously in laboratory workers. 75 a study of west nile virus infection in pregnancy documented four miscarriages, two elective abortions, and 72 live births. cord blood samples were tested in 55 infants and 54 of 55 were negative for anti-west nile virus igm. three infants had west nile virus infection, which could have been acquired congenitally. three of 7 infants who had congenital malformations might have been caused by maternal west nile virus infection based on timing in pregnancy, but no evidence of west nile virus etiology is conclusive. 312 west nile virus transmission occurs via blood and blood product transfusion, 186 and the incidence has been estimated to be as high as 21 per 10,000 donations during epidemics in specific cities. 40 no evidence of direct person-to-person transmission without the mosquito vector has been found. one case of possible west nile virus transmission via breastfeeding has been documented. 74 the mother acquired the virus via packed rbc transfusions after delivery. the second unit of blood she received was associated with other blood products from the same donation causing west nile infection in another transfusion recipient. eight days later the mother had a severe headache and was hospitalized with fever and a csf pleocytosis on day 12 after delivery. the mother' s csf was positive for west nile virus-specific igm antibody. the infant had been breastfed from birth through the second day of hospitalization of the mother. samples of breast milk were west nile virus-specific igg and igm positive on day 16 after delivery and west nile virus-specific igm positive on day 24. the same milk was west nile virus rna positive by pcr testing on day 16, but not on day 24 after delivery. the infant tested positive for west nile virus-specific igm in serum at day 25 of age, but remained well without fever. no clear-cut exposure to mosquitoes for the infant were reported. the cord blood and placenta were not available to be tested. igm antibodies can be found in low concentrations in breast milk, but this is not common or as efficient as the transfer of iga, secretory iga, or igg into breast milk. a review of west nile virus illness during the breastfeeding identified six occurrences of breastfeeding during maternal west nile virus illness. 177 five of the six infants had no illness or detectable antibodies to west nile virus in their blood. one infant developed a rash and was otherwise well after maternal west nile virus illness, but was not tested for west nile virus infection. two infants were identified who developed west nile virus illness while breastfeeding, but no preceding west nile virus infection was demonstrated in their mothers. two other breastfeeding infants developed west nile virus-specific antibodies after their mothers acquired west nile virus illness in the last week of pregnancy, but congenital infection could not be ruled out. live virus was not cultured from 45 samples of breast milk from mothers infected with west nile virus during pregnancy, but west nile virus rna was detected in two samples and 14 samples had igm antibodies to west nile virus. 177 the above data suggest that west nile virus infection through breastfeeding is rare. to date evidence of significant disease due to west nile virus infection in young breastfeeding children is lacking. at this time, no reason exists to proscribe breastfeeding in the case of maternal west nile virus infection if a mother is well enough to breastfeed. as with many other maternal viral illnesses, by the time the diagnosis is made in a mother, the infant may have already been exposed during maternal viremia and possible virolactia. the infant can and should continue to receive breast milk for the potential specific and nonspecific antiviral immunologic benefits. yellow fever virus is a flavivirus which is transmitted to humans by infected aedes and haemogogus mosquitos in tropical areas of south america and africa. large outbreaks occur when mosquitos in a populated area become infected from biting viremic humans infected with yellow fever virus. transmission from the mosquitos to other humans occurs after an incubation period in the mosquito of 8 days. direct person-to-person spread has not been reported. illness due to yellow fever virus usually begins after an incubation period of 3 to 6 days, with acute onset of headache, fever, chills, and myalgia. photophobia, back pain, anorexia, vomiting, and restlessness are other common symptoms. the individual is usually viremic for the first 4 days of illness until the fever and other symptoms diminish. liver dysfunction and even failure can develop as can myocardial dysfunction. cns infection is uncommon but symptoms can include seizures and coma. medical care should include intensive supportive care and fluid management. one case of congenital infection after immunization of a pregnant woman with the attenuated vaccine strain has been reported. one of 41 infants whose mothers had inadvertently received the yellow fever virus vaccine during pregnancy developed igm and elevated neutralizing antibodies against the yellow fever virus without any evidence of illness or abnormalities. 418 a more recent study 404 from brazil examined inadvertent yellow fever virus immunization during pregnancy during a mass vaccination campaign in 2000; 480 pregnant women received the yellow fever virus at a mean of 5.7 weeks' gestation, the majority of whom did not know their pregnancy status at the time. seroconversion occurred in 98.2% of the women after at least 6 weeks after vaccination. mild postvaccination illness (headache, fever, or myalgia) was reported by 19.6% of the 480 women. the frequency of malformations, miscarriages, stillbirths, and premature deliveries was similar to that found in the general population. at the 12-month follow-up point, 7% of the infants still demonstrated neutralizing antibodies against yellow fever virus, but after 12 months only one child was still seropositive. 404 transmission of the yellow fever vaccine virus through breastfeeding was recently reported from brazil. 85 the mother was immunized during a yellow fever epidemic in a nonendemic area in brazil; 15 days after delivering a healthy female infant (39 weeks' gestational age) the mother received the 17dd yellow fever vaccine, and 5 days later the mother reported headache, malaise, and low-grade fever that persisted for 2 days. the mother continued breastfeeding and did not seek medical care for herself. at 23 days of age the infant became irritable, developed fever, and refused to nurse. the infant developed seizures and subsequent evaluation of the infant demonstrated an abnormal csf and ct of the brain showed bilateral areas of diffuse low density suggestive of inflammation and consistent with encephalitis. yellow fever-specific igm antibodies were identified in the infant' s serum and csf. reverse-transcriptase polymerase chain reaction (rt-pcr) testing of the csf also demonstrated yellow fever virus rna identical to the 17dd yellow fever vaccine virus. breast milk and maternal serum were not tested for yellow fever virus. 85 yellow fever virus, wild or vaccine type, has not been identified in human breast milk, although another flavivirus, west nile virus, has been detected in milk from a few lactating women with west nile virus infection. 177 (see the section on west nile virus.) yellow fever vaccine-associated neurologic disease occurs at different rates in different age-groups, including 0.5 to 4.0 cases per 1000 infants younger than 6 months of age. 285 the 17d-derived yellow fever vaccines are contraindicated in infants younger than 6 months of age. since 2002, the advisory committee on immunization practices has recommended, based on theoretical risk, that yellow fever vaccine be avoided in nursing mothers, except when exposure in highrisk yellow fever endemic areas is likely to occur. 76 no case of transmission of yellow fever virus from an infected mother to her infant via breastfeeding or breast milk has been reported. published information on the severity of yellow fever virus infection in infants younger than 1 year of age, potential protection from passively acquired antibodies, or protection from breast milk is limited. no information on a differential risk in breastfed versus formula-fed infants is available. given the well documented method of transmission of yellow fever virus via mosquitos, and the lack of evidence of transmission via breast milk, it makes more sense to protect all infants against mosquito bites than to proscribe breastfeeding, even in the mother infected with yellow fever virus. continued breastfeeding or use of expressed breast milk will depend on a mother's health status and ability to maintain the milk supply while acutely ill. if another source of feeding is readily available then temporarily discarding expressed breast milk for at least 4 days of acute illness in the mother is a reasonable precaution. lyme disease, as with other human illnesses caused by spirochetes, especially syphilis, is characterized by a protean course and distinct phases (stages) of disease. lyme borreliosis was described in europe in the early twentieth century. since the 1970s, tremendous recognition, description, and investigation of lyme disease have occurred in the united states and europe. public concern surrounding this illness is dramatic. lyme disease is a multisystem disease characterized by involvement of the skin, heart, joints, and nervous system (peripheral and central). stages of disease are identified as early localized (erythema migrans, often accompanied by arthralgia, neck stiffness, fever, malaise, and headache), early disseminated (multiple erythema migrans lesions, cranial nerve palsies, meningitis, conjunctivitis, arthralgia, myalgia, headache, fatigue, and, rarely, myocarditis), and late disease (recurrent arthritis, encephalopathy, and neuropathy). the varied manifestations of disease may relate to the degree of spirochetemia, the extent of dissemination to specific tissues, and the host' s immunologic response. the diagnosis of lyme disease is often difficult in part because of the broad spectrum of presentations, inapparent exposure to the tick, and the lack of adequately standardized serologic tests. culture of the spirochete, borrelia burgdorferi, is not readily available. enzyme-linked immunosorbent assay (elisa), immunofluorescent assay, and immunoblot assay are the usual tests. pcr detection of spirochetal dna requires additional testing in clinical situations to clarify and standardize its utility. gardner 142 reviewed infection during pregnancy, summarizing a total of 46 adverse outcomes from 161 cases reported in the literature. the adverse outcomes included miscarriage and stillbirth (11% of cases), perinatal death (3%), congenital anomalies (15%), and both early-and late-onset progressive infection in the infants. silver 384 reviewed 11 published reports and concluded that lyme disease during pregnancy is uncommon, even in endemic areas. although the spirochete can be transmitted transplacentally, a significant immune response in the fetus is often lacking, and the association of lyme infection with congenital abnormalities is weak. 401, 448 little published information exists on whether b. burgdorferi can be transmitted via breast milk. one report showed the detection of b. burgdorferi dna by pcr in the breast milk of two lactating women with untreated erythema migrans, but no evidence of lyme disease or transmission of the spirochete in the one infant followed for 1 year. 369 no attempt to culture the spirochete was made, so it is not possible to determine if the detectable dna was from viable spirochetes or noninfectious fragments. in that same study of 56 women with untreated erythema migrans who had detectable b. burgdorferi dna in the urine, 32 still had detectable dna in the urine 15 to 30 days after starting treatment, but none had it 6 months after initiating therapy. ziska et al 466 reported on the management of nine cases of lyme disease in women associated with pregnancy; seven of the nine women were symptomatic at conception and six received antibiotics throughout pregnancy. follow-up of the infants showed no transmission of lyme disease, even in the seven infants who had been breastfed. 466 the lack of adequate information on transmission of b. burgdorferi via breast milk cannot be taken as proof that it is not occurring. if one extrapolates from data on syphilis and the treponema pallidum spirochete, it would be prudent to discuss the lack of information on the transmission of b. burgdorferi via breast milk with the mother or parents and to consider withholding breast milk at least until therapy for lyme disease has begun or been completed. if the infection occurred during pregnancy and treatment has already been completed, an infant can breastfeed. if infection occurs postpartum or the diagnosis is made postpartum, infant exposure may have already occurred. again, discussion with the mother or parents about withholding versus continuing breastfeeding is appropriate. after prenatal or postnatal exposure, an infant should be closely observed and empiric therapy considered if the infant develops a rash or symptoms suggestive of lyme borreliosis. treatment of mother and infant with ceftriaxone, penicillin, or amoxicillin is acceptable during breastfeeding relative to the infant' s exposure to these medications. doxycycline should not be administered for more than 14 days while continuing breastfeeding because of possible dental staining in the neonate. continued surveillance for viable organisms in breast milk and evidence of transmission through breastfeeding is recommended. a large body of information is available on various "lyme vaccines" used in dogs, but these vaccines are only partially protective and must be repeated yearly. preliminary information suggests that a vaccine for use in humans safely produces good serologic responses, but protective efficacy has not been demonstrated, and no information exists on its use during pregnancy or breastfeeding. syphilis is the classic example of a spirochetal infection that causes multisystem disease in various stages. both acquired syphilis and congenital syphilis are well-described entities. acquired syphilis is almost always transmitted through direct sexual contact with open lesions of the skin or mucous membranes of individuals infected with the spirochete, treponema pallidum. congenital syphilis occurs by infection across the placenta (placentitis) at any time during the pregnancy or by contact with the spirochete during passage through the birth canal. any stage of disease (primary, secondary, tertiary) in a mother can lead to infection of the fetus, but transmission in association with secondary syphilis approaches 100%. infection with primary syphilis during pregnancy, without treatment, leads to spontaneous abortion, stillbirth, or perinatal death in 40% of cases. similar to acquired syphilis, congenital syphilis manifests with moist lesions or secretions from rhinitis (snuffles), condylomata lata, or bullous lesions. these lesions and secretions contain numerous spirochetes and are therefore highly infectious. postnatal infection of an infant can occur through contact with open, moist lesions of the skin or mucous membranes of the mother or other infected individuals. if the mother or infant has potentially infectious lesions, isolation from each other and from other infants and mothers is recommended. if lesions are on the breasts or nipples, breastfeeding or using expressed milk is contraindicated until treatment is complete and the lesions have cleared. spirochetes are rarely identified in open lesions after more than 24 hours of appropriate treatment. penicillin remains the best therapy. evaluation of an infant with suspected syphilis should be based on the mother' s clinical and serologic status, history of adequate therapy in the mother, and the infant' s clinical status. histologic examination of the placenta and umbilical cord, serologic testing of the infant' s blood and csf, complete analysis of the csf, long bone and chest radiographs, liver function tests, and a complete blood cell count are all appropriate given the specific clinical situation. treatment of the infant should follow recommended protocols for suspected, probable, or proven syphilitic infection. 96 no evidence indicates transmission of syphilis via breast milk in the absence of a breast or nipple lesion. when a mother has no suspicious breast lesions, breastfeeding is acceptable as long as appropriate therapy for suspected or proven syphilis is begun in the mother and infant. giardiasis is a localized infection limited to the intestinal tract, causing diarrhea and malabsorption. immunocompetent individuals show no evidence of invasive infection, and no evidence exists that documents fetal infection from maternal infection during pregnancy. giardiasis is rare in children younger than 6 months of age, although neonatal infection from fecal contamination at birth has been described. 22 human milk has an in vivo protective effect against giardia lamblia infection, as documented by work from central africa, where the end of breastfeeding heralds the onset of giardia infection. 145 this has been reaffirmed in undeveloped countries around the world. the protective effect of breast milk has been identified in the milk of noninfected donors. 151 the antiparasitic effect does not result from specific antibodies but rather from lipase enzymatic activity. the lipase acts in the presence of bile salts to destroy the trophozoites as they emerge from their cysts in the gi tract. hernell et al 175 demonstrated that free fatty acids have a marked giardiacidal effect, which supports the conclusion that lipase activity releasing fatty acids is responsible for killing g. lamblia. g. lamblia have also been reported to appear in the mother' s milk, and the parasite has been transmitted to newborns via that route. the exact relationship of breastfeeding to transmission of g. lamblia and the effect on infants continue to be studied, even though symptomatic infection in breastfed infants is rare. 151 one report from the middle east suggests that even partial breastfeeding is protective against infection with intestinal parasites, including cryptosporidium and giardia lamblia. 41 breastfeeding by mothers with giardiasis is problematic mainly because of the medications used for therapy. metronidazole' s safety in infants has not been established, and little information is available on quinacrine hydrochloride and furazolidone in breast milk. paromomycin, a nonabsorbable aminoglycoside, is a reasonable alternative recommended for treatment of pregnant women. breastfeeding by a mother with symptomatic giardiasis is acceptable when consideration is given to the presence of the therapeutic agents in the breast milk. hookworm infection, most often caused by ancylostoma duodenale and necator americanus, is common in children younger than the age of 4 years, and there is at least one report on infantile hookworm disease from china. 374 this publication from the chinese literature reports hundreds of cases of infantile hookworm disease that include the common symptoms of bloody stools, melena, anorexia, listlessness, and edema. anemia, eosinophilia and even leukemoid reactions occur as part of the clinical pictures in young children. they also note at least 20 cases of hookworm diseases in newborn infants younger than 1 month of age. in the discussion of infantile hookworm infection, they note four routes of infection: direct contact with contaminated soil, "sand-stuffed" diapers, contaminated "washed/wet" diapers, and vertical equal to transmammary transmission or transplacental transmission. they postulated that infection of infants before 40 to 50 days of age would most likely be due to transplacental transmission and infection before environmental contact would most likely be due to transmammary transmission. ample evidence is available in veterinary medicine of transmammary spread of helminths. 302, 382 at least two reports suggest the possibility of transmammary transmission of hookworms in humans. setasuban et al 376 described the prevalence of necator americanus in 128 nursing mothers as 61% and identified n. americanus in breast milk in one case. nwosu 303 documented positive stool samples for hookworms in 33 of 316 neonates (10%) at 4 to 5 weeks of age in southern nigeria. the majority of neonatal infections were due to ancylostoma duodenale although necator americanus is more prevalent in that area of nigeria. examination of colostral milk did not demonstrate any hookworm larvae. 303 additional epidemiologic work is necessary to determine the potential significance of transmammary spread of helminths in humans, and more careful examination of breast milk as a source of hookworm infection is required before reasonable recommendation are possible. malaria is recognized as a major health problem in many countries. the effect of malaria infection on pregnant and lactating women and thus on the developing fetus, neonate, and growing infant can be significant. the four species of malaria, plasmodium vivax, p. ovale, p. malariae, and p. falciparum, vary in the specific aspects of the disease they produce. p. vivax exists throughout the world, but p. falciparum predominates in the tropics and is most problematic in its chloroquine-resistant form. malaria in the united states is most often seen in individuals traveling from areas where malaria is endemic. the parasite can exist in the blood for weeks, and infection with p. vivax and p. malariae can lead to relapses years later. transmission occurs through the bite of the anopheline mosquito and can occur via transfusion of blood products and transplacentally. congenital malaria is rare but seems to occur more often with p. vivax and p. falciparum. it usually presents in the first 7 days of life (range 1 day to 2 months). it may resemble neonatal sepsis, with fever, anemia, and splenomegaly occurring in the most neonates and hyperbilirubinemia and hepatomegaly in less than half. malaria in infants younger than 3 months of age generally manifests with less severe disease and death than in older children. possible explanations include the effect of less exposure to mosquitoes, passive antibody acquired from the mother, and the high level of fetal hemoglobin in infants at this age. 22 the variations in the infection rates in children younger 3 months of age during the wet and dry seasons support the idea that postnatal infection is more common than congenital infection. no evidence indicates that malaria is transmitted through breast milk. the greatest risk to infants is exposure to the anopheline mosquito infected with malaria. the main issues relative to malaria and breastfeeding are how to protect both mothers and infants effectively from mosquitoes and what drugs for treating malaria in mothers are appropriate during lactation. protection from mosquito bites includes screened-in living areas, mosquito nets while sleeping, protective clothing with or without repellents on the clothes, and community efforts to eradicate the mosquitoes. chloroquine, quinine, and tetracycline are acceptable during breastfeeding. sulfonamides should be avoided in the first month of an infant' s life, but pyrimethamine-sulfadoxine (fansidar) can be used later. mefloquine is not approved for infants or pregnant women. however, the milk/plasma ratio for mefloquine is less than 0.25, there is a large volume of distribution of the drug, high protein binding of the drug limits its presence in breast milk, and the relative importance of breastfeeding in areas where malaria is prevalent shifts the risk/benefit ratio in favor of treatment with mefloquine. the single dose recommended for treatment or the onceweekly dose for prevention allows for continued breastfeeding with discarding of the milk for short periods after a dose (1 to 6 hours). maternal plasma levels of primaquine range from 53 to 107 ng/ml, but no information is available on levels in human milk. primaquine is used in children, and once daily dosing in the mother would allow discarding milk with peak levels of drug. therefore breastfeeding during maternal malaria even with treatment is appropriate with specific medications. strongyloides stercoralis is a nematode (roundworm). most infections are asymptomatic, but clinically significant infection in humans can include larval skin invasion, tissue migration, intestinal invasion with abdominal pain and gi symptoms, and a loeffler-like syndrome due to migration to the lungs. immune-compromised individuals can develop dissemination of larvae systemically, causing various clinical symptoms. humans are the principal hosts, but other mammals can serve as reservoirs. infection via the skin by filariform larvae is the most common form of transmission; ingestion is an uncommon occurrence. transmammary transmission of strongyloides species has been described in dogs, ewes, and rats. 211, 302, 382 only one report of transmammary passage of strongyloides larvae in humans is available. in 76 infants younger than 200 days of age, 34% demonstrated the presence of strongyloides fuelleborni on stool examination. the clinical significance of this was not elucidated. strongyloides larvae was identified in only one sample of milk from 25 nursing mothers. 53 in the absence of an understanding of the clinical significance of strongyloides in the stools of young infants, given the lack of exclusion of the most common mechanism of transmission (through the skin) in the single report and the apparent infrequent evidence of these larvae in human milk, it is difficult to make any recommendations concerning breastfeeding and strongyloides. toxoplasmosis is one of the most common infections of humans throughout the world. the infective organism, toxoplasma gondii, is ubiquitous in nature. the prevalence of positive serologic test titers increases with age, indicating past exposure and infection. the cat is the definitive host, although infection occurs in most species of warmblooded animals. postnatal infection with toxoplasmosis is usually asymptomatic. symptomatic infection typically manifests with nonspecific symptoms, including fever, malaise, myalgia, sore throat, lymphadenopathy, rash, hepatosplenomegaly, and occasionally a mononucleosis-like illness. the illness usually resolves without treatment or significant complications. congenital infection or infection in an immunodeficient individual can be persistent and severe, causing significant morbidity and even death. although most infants with congenital infection are asymptomatic at birth, visual abnormalities, learning disabilities, and mental retardation can occur months or years later. the syndrome of congenital toxoplasmosis is clearly defined, with the most severe manifestations involving the cns, including hydrocephalus, cerebral calcifications, microcephaly, chorioretinitis, seizures, or simply isolated ocular involvement. the risk for fetal infection is related to the timing of primary maternal infection, although transmission can occur with preexisting maternal toxoplasmosis. 241 in the last months of pregnancy the protozoan is more readily transmitted to the fetus, but the infection is more likely to be subclinical. early in pregnancy the transmission to a fetus occurs less frequently but does result in severe disease. treatment of documented congenital infection is currently recommended, although duration and optimal regimen have not been determined, and reversal of preexisting sequelae generally does not occur. 343 prevention of infection in susceptible pregnant women is possible by avoiding exposure to cat feces or the organism in the soil. pregnant or lactating women should not change cat litter boxes, but if they must, it should be done daily and while wearing gloves. the oocyst is not infective for the first 24 to 48 hours after passage. mothers can avoid ingestion of the organism by fully cooking meats and carefully washing fruits, vegetables, and food preparation surfaces. 94 in various animal models, t. gondii has been transmitted through the milk to the suckling young. the organism has been isolated from colostrum as well. the newborn animals became asymptomatically infected when nursed by an infected mother whose colostrum contained t. gondii. only one report has identified t. gondii in human milk, and some question surrounds the reliability of that report. 241 transmission during breastfeeding in humans has not been demonstrated. breast milk may contain appropriate antibodies against t. gondii. given the benign nature of postnatal infection, the absence of documented transmission in human breast milk, and the potential antibodies in breast milk, no reason exists to proscribe breastfeeding by a mother known to be infected with toxoplasmosis. trichomonas vaginalis is a flagellated protozoan that can produce vaginitis (see chapter 16 for a discussion of vaginitis) but frequently causes asymptomatic infection in both men and women. the parasite is found in 10% to 25% of women in the childbearing years. it is transmitted predominantly by sexual intercourse, but it can be transmitted to the neonate by passage through the birth canal. this parasite often coexists with other stds, especially gonorrhea. infection during pregnancy or while taking oral contraceptives is more difficult to treat. some evidence suggests that infection with and growth of the parasite are enhanced by estrogens or their effect on the vaginal epithelium. no evidence indicates adverse effects on the fetus in association with maternal infection during pregnancy. occasionally female newborns have vaginal discharge during the first weeks of life caused by t. vaginalis. this is thought to be influenced by the effect of maternal estrogen on the infant' s vaginal epithelium and acquisition of the organism during passage through the birth canal. the organism does not seem to cause significant disease in a healthy infant. no documentation exists on transmission of t. vaginalis via breast milk. the difficulty encountered with maternal infection during lactation stems from metronidazole (flagyl), the drug of choice, being contraindicated for infants. case reports describe treatment of neonates with metronidazole without adverse effect. although topical agents containing povidone-iodine (betadine) or sodium lauryl sulfate (trichotine) can be effective when given as douches, creams, or suppositories, metronidazole remains the treatment of choice. the aap advises using metronidazole only with a physician' s discretion and considers its effect on a nursing infant unknown but possibly a concern. the potential concerns are metronidazole' s disulfiram-like effect in association with alcohol, tumorigenicity in animal studies, and leukopenia and neurologic side effects described in adults. on the other hand, metronidazole is given to children beyond the neonatal period to treat serious infections with various other parasites, such as entamoeba histolytica. the current recommendation for lactating women is to try local treatment first, and if these fail, then to try metronidazole. a 2-g single-dose treatment produces peak levels after 1 hour, and discarding expressed breast milk for the next 12 to 24 hours is recommended. if this treatment also fails, a 1-g twice-daily regimen for 7 days or a 2-g single daily dose for 3 to 5 days is recommended, with discarding of breast milk close to the dose and timing of feedings distant from the dose. infants who exclusively breastfeed are presumed at greater risk from exposure to metronidazole than those who are only partially breastfed. candida consists of multiple species. the most common species affecting humans include c. albicans as the dominant agent and c. tropicalis, c. krusei, and c. parapsilosis, as well as many other uncommon species. in general, candida exists as a commensal organism colonizing the oropharynx, gi tract, vagina, and skin without causing disease until some change disrupts the balance between the organism and the host. mild mucocutaneous infection is the most common illness, which can lead to vulvovaginitis, mastitis, or, uncommonly, oral mucositis in a mother, and thrush (oral candidiasis) and candidal diaper rash in an infant. invasive candidal infection occurs infrequently, usually when a person has other illness, impaired resistance to infection (hiv, diabetes mellitus, neutropenia; decreased cell-mediated immunity in premature infants or lbw or vlbw infants), or disrupted normal mucosal and skin barriers and has received antibiotics or corticosteroids. invasive disease can occur through local spread, and may occur more often in the genitourinary tract (urethra, bladder, ureters, kidneys), but usually develops in association with candidemia. the bladder and kidney are more frequently involved, but when dissemination occurs via candidemia, a careful search for other sites of infection should be made (e.g., retina, liver, spleen, lung, meninges). 279 transmission usually occurs from healthy individuals colonized with candida through direct contact with them or through contact with their oral or vaginal secretions. intrauterine infection can occur through ascending infection through the birth canal but is rare. no distinct syndrome of congenital candidal infection exists. most often an infant is infected in passing through the birth canal and remains colonized. postnatal transmission can occur through direct contact with caregivers. the mother and infant serve as an immediate source of recolonization for each other, especially during the direct contact of breastfeeding. for this reason, an infant and breastfeeding mother should be treated simultaneously when treating thrush, vulvovaginitis, diaper candidiasis, or mastitis. colonization with this organism usually occurs in the absence of any clinical evidence of infection. simultaneous treatment should occur even in the absence of any clinical evidence of candida infection or colonization in the apparently uninvolved individual of the breastfeeding dyad. no well-controlled clinical trials define the most appropriate or most effective method(s) of treatment for candidal infection in breastfeeding mother-infant pairs. the list of possible treatment products is extensive and includes many anecdotal and empirical regimens. in the face of this absence of data, brent 51 conducted a survey of members of the academy of breastfeeding medicine concerning the respondents' approach to diagnosis and treatment of thrush in the breastfeeding dyad. most of the respondents relied on the history and physical examination of the infant, but only a third rated the examination of the mother as very important in making a diagnosis. only 7% reported using laboratory testing to make the diagnosis. twentyone percent of the respondents reported using only oral nystatin for the infant when the mother was asymptomatic. almost half treated the infant and the mother with topical nystatin, and 13% used oral nystatin for the infant and oral fluconazole for the mother when the mother had breast pain. less than 5% used oral fluconazole for both infant and mother, and other therapies were used by about 15% of the respondents. for recurrence of persistence of the thrush, more respondents reported treating the mother or both the infant and mother with fluconazole, and almost a quarter reported using other therapies. considerable discussion of mammary candidosis/candidiasis, the clinical diagnosis of candidal involvement of the breast, the significance of pain with breastfeeding, and the presence or absence of candida albicans in milk samples is ongoing. 14, 133, 166 this topic will continue to be debated because additional prospective studies are necessary to clarify specific issues. data are inadequate to make specific recommendations about various clinical situations regarding candida and breastfeeding. clinical practice will vary with experience, especially for the more problematic clinical situations. some general guidelines follow. (see chapter 16 for a discussion of mastitis.) the treatment of mucocutaneous candidiasis should probably begin with a topical agent, such as nystatin, clotrimazole, miconazole, econazole, butaconazole, terconazole, or ciclopirox. treatment should continue for at least 2 weeks, even with obvious improvement in 1 or 2 days. failures most often result from inadequate therapy involving the frequency of application, careful washing and drying before application, or, in the case of diaper candidiasis, decreasing the contact of the skin with moisture. nystatin oral suspension is less effective for the treatment of oral candidiasis in infants, now compared with the past, supposedly due to increasing resistance. 154 gentian violet (diluted to 0.25% to 1.0%) applied to the breast or painted onto an infant' s mouth is being recommended more frequently. other topical preparations have been recommended for the mother' s breast including mupirocin, grapefruit seed extract, or mixtures of mupirocin, betamethasone ointments, and miconazole powder. controlled clinical trials for efficacy and toxicity are not available. when good adherence to the proposed regimen with topical agents fails, or when infant or mother are severely affected by pain and decreased breastfeeding, systemic therapy is appropriate. fluconazole and ketoconazole are the most commonly used systemic agents for oral or diaper candidiasis and vulvovaginitis or mastitis. fluconazole has a better side effect profile than ketoconazole, and more data are available concerning its safe use in children younger than 6 months of age and even neonates and premature infants. 87, 154, 209 fluconazole is not currently approved for use in infants younger than 6 months of age. for severe invasive infections in infants, amphotericin b with or without oral flucytosine, iv fluconazole, voriconazole or caspofungin are reasonable choices in different situations. use of itraconazole in infants has not been adequately studied to date. maternal use of fluconazole during breastfeeding is not contraindicated because only a small amount of medicine compared with the usual infant dose reaches the infant through breast milk. amphotericin or caspofungin therapy in mothers is also not contraindicated because these are both poorly absorbed from the gi tract. whenever a mother is treated for candidal mastitis or vulvovaginitis, the infant should be treated simultaneously, at least with nystatin oral suspension as the first choice of medication. any predisposing risk factors for candidal infection in mothers and infants should be reduced or eliminated to improve the chance of rapid, successful treatment and to decrease the likelihood of chronic or recurrent disease. for mothers, such interventions might include decreasing sugar consumption, stopping antibiotic use as soon as possible, and consuming some form of probiotic bacteria, such as acidophilus (in yogurt, milk, or pill form), to reestablish a normal colonizing bacterial flora. for infants, breastfeeding can enhance the growth of specific colonizing bacterial flora such as lactobacillus, which can successfully limit fungal growth. breastfeeding should continue with appropriate support and problem-solving with a professional who is knowledgeable about breastfeeding. hiv-1, hiv-2, htlv-i, and htlv-ii are the only infectious diseases that are considered absolute contraindications to breastfeeding in developed countries. when the primary route of transmission is via direct contact or respiratory droplets/particles, temporary separation of mother and infant may be appropriate (whether the infant is breastfed or formula fed), but expressed breast milk should be given to the infant for the organism-specific immunologic benefits in the mother' s milk. in most instances, by the time a specific diagnosis of infection is made for a mother, the infant has already been exposed to the organism and providing expressed breast milk to the infant should continue. (refer to appendix f for specific exceptions, such as lassa fever.) regarding antimicrobial therapy for mothers and continued breastfeeding, the majority of the medications commonly used in adults can be used to treat the same infection in infants. the additional amount of medication received by infants via breast milk is usually insignificant. in almost all instances, an appropriate antimicrobial agent for treating mothers that is also compatible with breastfeeding can be chosen. unless the risk to infants for transmission of an infectious agent via breast milk that leads to a clinically significant illness in the infants is documented, breastfeeding should continue. measles antibodies in the breast milk of nursing mothers spectrum of breast tuberculosis respiratory syncytial virus infection among young children with acute respiratory tract infection in iraq probable breast milk borne brucellosis in a young infant breast milk transmission of cytomegalovirus (cmv) infection congenital and perinatal cytomegalovirus infections intrauterine west nile virus: ocular and systemic findings the cloning and clinical implications of hgv and hgbv-c bottle feeding can prevent transmission of htlv-i from mothers to their babies transmission of adult t-cell leukemia retrovirus (htlv-i) from mother to child: comparison of bottle-with breastfed babies effect of freezethawing breast milk on vertical htlv-i transmission from seropositive mothers to children long-term follow up study of vertical htlv-i infection in children breastfed by seropositive mothers long-term followup study of htlv-i infection in bottle-fed children born to seropositive mothers the yeast connection: is candida linked to breastfeeding associated pain? epidemiology of group b streptococcus: maternal and nosocomial sources for acquisition tuberculosis and pregnancy and tuberculous mastitis infant botulism infant botulism: anticipating the second decade protective role of human milk against sudden death from infant botulism growth faltering due to breastfeeding cessation in uninfected children born to hiv-infected mothers in zambia other viral infections of the fetus and newborn protozoan and helminth infections (including pneumocystis carinii) recurrent group b streptococcal disease in infants: who should receive rifampin? methicillin-resistant staphylococcus aureus sccmec type iv: nosocomial transmission and colonisation of healthcare workers in a neonatal intensive care unit stringent precautions are advisable when caring for patients with viral hemorrhagic fevers prevalence of methicillin-resistant staphylococcus aureus in expressed breast milk in a neonatal intensive care unit transmision de brucelosis por lactancia materna: presentacion de dos casos congenital lymphocytic choriomeningitis virus infection in twins poliomyelitis in pregnancy, fetus and newborn assessment of the risk of ebola virus transmission from bodily fluids and fomites transmission of hepatitis by breastfeeding evidence against breast feeding as a mechanism for vertical transmission of hepatitis b two-year morbidity-mortality and alternatives to prolonged breast feeding among children born to hiv infected mothers in cote d'ivorie transmission of methicillin-resistant staphylococcus aureus to preterm infants through breast milk a new staphylococcal enterotoxin, enterotoxin f, associated with tss staphylococcus aureus isolate mother-to-infant transmission of hepatitis c outbreak of methicillin-resistant staphylococcus aureus colonization and infection in a neonatal intensive care unit epidemiologically linked to a healthcare worker with chronic otitis estimating the timing of mother-to-child transmission of human immunodeficiency virus in a breast-feeding population in kinshasa mycobacteriareactive t cells are present in human colostrum from tuberculin-positive, but not tuberculin-negative nursing mothers estimated risk of transmission of the west nile virus through blood transfusion in the us partial breastfeeding protects bedouin infants from infection morbidity: prospective cohort study children hospitalized with severe acute respiratory syndrome-related illness in toronto a prospective study of infants born to women seropositive for human immunodeficiency virus type 1. hiv infection in newborns french collaborative study group clinical virology postpartum varicella vaccination: is the vaccine virus excreted in breast milk? contamination of breast milk obtained by manual expression and breast pumps in mothers of very low birth weight hepatitis c virus infection and related liver disease in children of mothers with antibodies to the virus clinical and laboratory observations, gram-negative bacilli in human milk feedings: quantitation and clinical consequences for premature infants prenatal transmission of dengue: two new cases community associated methicillin-resistant staphylococcus aureus in hospital nursery and maternity units thrush in the breastfeeding dyad: results of a survey on diagnosis and treatment reproductive factors in the aetiology of breast cancer transmammary passage of strongyloides sp. larvae in the human host alaska rsv study group: risk factors for severe respiratory syncytial virus infection among alaska native children detection of human immunodeficiency virus type 1 (hiv-1) proviral dna in breast milk and colostrum of seropositive mothers streptococcus agalactiae as a cause of meningitis in the newborn and bacteraemia in adults incidence and clinical outcome of cytomegalovirus transmission via breast milk in preterm infants </=31 weeks neonatal group b streptococcal infection related to breast milk epidemiology of tuberculosis in the united states probable transmission of brucellosis by breast milk does discarding the first few milliliters of breast milk improve the bacteriological quality of bank breast milk? primary human herpes virus 7 infection: a comparison of human herpes virus 7 and human herpes virus 6 infections in children analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis centers for disease control and prevention: testing for antibodies to hiv-2 in the united states public health service working group: recommendations for counseling persons infected with human t-lymphotropic virus, types i and ii centers for disease control and prevention: screening for tuberculosis and tuberculosis infection in high-risk populations update: management of patients with suspected viral hemorrhagic fever-united states poliomyelitis prevention in the united states: introduction of a sequential vaccination schedule of inactivated poliovirus vaccine followed by oral poliovirus vaccine-united states prevention: recommendations for antimicrobial prophylaxis for children and breastfeeding mothers and treatment of children with anthrax prevention: possible west nile virus transmission to an infant through breastfeeding-michigan prevention: yellow fever vaccine; recommendations of the advisory committee on immunization practices (acip) update: cardiac and other adverse events following civilian smallpox vaccination-united states centers for disease control and prevention: secondary and tertiary transfer of vaccinia virus among u.s. military personnel-united states and worldwide centers for disease control and prevention: a new product (varizig) for post exposure prophylaxis of vericells available under an investigational new drug application expanded access protocol centers for disease control and prevention: communityassociated methicillin-resistant staphylococcus aureus infection among healthy newborns: chicago and los angeles county centers for disease control and prevention: what to do if an infant or child is mistakenly fed another woman' s expressed breast milk prevention: transmission of yellow fever vaccine through breast feeding: brazil take of rhesushuman reassortment tetravalent rotavirus vaccine in breastfed infants candida infections in the neonate prevalence of methicillin-sensitive and methicillin-resistant staphylococcus aureus in pregnant women coxsackieviruses, and enteroviruses morbidity and mortality among a cohort of human immunodeficiency virus type 1 infected and uninfected pregnant women and their infants from malawi, zambia, and tanzania electron microscopic detection of simian-type virus particles in human milk staphylococcus aureus outbreaks among newborns: new frontiers in an old dilemma breastfeeding and the risk of life-threatening rotavirus diarrhea: prevention or postponement? committee on infectious diseases: american academy of pediatrics: red book report of the committee on infectious disease committee on infectious diseases: american academy of pediatrics: red book report of the committee on infectious disease committee on infectious disease: american academy of pediatrics: red book report of the committee on infectious disease mother-tochild transmission of hiv-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort breastfeeding, hiv transmission and infant survival: balancing pros and cons influence of infantfeeding patterns on early mother-to-child transmission of hiv-1 in durban, south africa: a prospective cohort study. south african vitamin a study group method of feeding and transmission of hiv-1 from mothers to children by 15 months of age: prospective cohort study from durban are hivinfected women who breastfeed at increased risk of mortality? morbidity in children born to women infected with human immunodeficiency virus in south africa: does mode of feeding matter? pilot epidemiologic study of transmission of cytomegalovirus from mother to preterm infant by breastfeeding mother-to-child transmission of human immunodeficiency virus type: report from the nairobi study human milk and hiv infection: epidemiologic and laboratory data hiv, breastfeeding and under 5 mortality: modelling the impact of policy decisions for or against breastfeeding staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics hiv-1 transmission through breast-milk: appraisal of risk according to duration of feeding task force on recurrent respiratory papillomatosis presence of papillomavirus in condylomatous lesions of the mamillae and in invasive carcinoma of the breast variations in biological features of west nile viruses group b streptococcal carriage and disease: a 6 year prospective study hepatitis in the obstetric patient prophylactic isoniazid protection of infants in a tuberculosis hospital breast-feeding protects against respiratory syncytial virus infections neonatal herpes simplex infection possibly acquired via maternal breast milk risk of human immunodeficiency virus type 1 transmission through breastfeeding serologic evidence for congenital transmission of human herpesvirus 6 examination of human breast milk for evidence of human herpesvirus 6 by pcr cytomegalovirus infection of breast milk and transmission in infancy late postnatal mother-to-child transmission of hiv-1 in abidjan listeriosis during pregnancy and in neonates rotavirus infections in young nicaraguan children european collaborative study: children born to women with hiv-1 infection: natural history and risk of transmission european paediatric hepatitis c virus network: effects of mode of delivery and infant feeding on the risk for motherto-child transmission of hepatitis c virus flora of the umbilical stump: 2479 cultures a lipid inhibitor of dengue virus in human colostrum and milk, with a note on the absence of anti-dengue secretory antibody vertical transmission of hepatitis g nonspecific antiviral substances in human milk against arbovirus and murine leukemia virus genetic evidence for mother-to-infant transmission of hepatitis g virus stringent precautions are not advisable when caring for patients with viral hemorrhagic fevers evalution and treatment of community acquired staphylococcus aureus infections in term and late-preterm previously healthy neonates diagnostic value of signs and symptoms of mammary candidosis among lactating women immunity and correlates of protection for rotavirus vaccines rate of inactivation of cytomegalovirus in raw banked milk during storage at 20 degrees c and pasteurisation htlv-i transmission from mother to child detection of human herpesvirus 7 (hhv-7) dna in breast milk by polym erase chain reaction and prevalence of hhv-7 antibody in breast-fed and bottle-fed children a new endemic focus of human t-lymphotropic virus type ii carriers among orinoco natives in colombia estimating the time of htlv-i infection following mother-to-child transmission in a breast-feeding population in jamaica tertiary contact vaccinia in a breastfeeding infant community acquisition of group b streptococcus by infants of colonized mothers infectious diseases of the fetus and newborn infant hospital infection control practices advisory committee: guidelines for isolation precautions in hospitals transmission of community-associated methicillian-resistant staphylococcus aureus from breast milk in the neonatal intensive care unit giardiasis and breastfeeding in urban africa multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of la reunion management of outbreaks of methicillin-resistant staphylococcus aureus infection in the neonatal intensive care unit: a consensus statement chickenpox, measles and mumps seroepidemiology in various population groups of the greater montreal area mother-to-child transmission of hepatitis c virus: evidence for preventable peripartum transmission cholate-dependent killing of giardia lamblia by human milk triple antiretroviral prophylaxis administered during pregnancy and after delivery significantly reduces breast milk viral load: a study within the drug resource enhancement against aids and malnutrition program risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level comparison of fluconazole and nystatin oral suspensions for treatment of oral candidiasis in infants rapid high temperature treatment of human milk htlv-i: a new problem for latin america rotavirus vaccines opportunities and challenges detection of human immunodeficiency virus type 1 (hiv-1) dna and p24 antigen in breast milk of hiv-1-infected ugandan women and vertical transmission denuge and denuge hemorrhagic fever tubercular mastitis child to mother transmission of herpes simples virus-1 infection at an unusal site vertical transmission of hepatitis c virus dengue: an update epidemiology and diagnosis of hospital acquired conjunctivitis among neonatal intensive care unit patients congenital infections with human herpes virus 6 (hhv6) and human herpes virus 7 (hhv7) the absence of candida albicans in milk samples of women with clinical symptoms of ductal candidiasis dengue hemorrhagic fever in infants: research opportunities ignored epidemiology of transmission of cytomegalovirus from mother to preterm infant by breastfeeding cytomegalovirus transmission to preterm infants during lactation congenital malformations and oral poliovirus vaccination during pregnancy mammary tuberculosis: analysis of thirty-eight patient cytomegalovirus in human milk an epidemiologic study of breast cancer detection of htlv-ii in breast milk of htlv-ii infected mothers killing of giardia lamblia by human milk lipases: an effect mediated by lipolysis of milk lipids risk of hepatitis b transmission in breast-fed infants of chronic hepatitis b carriers transmission of west nile virus through human breast milk seems to be rare milk-borne transmission of htlv-i as a major route in the endemic cycle primary prevention of htlv-i in japan primary prevention of htlv-1 in japan apparent vertical transmission of human immunodeficiency virus type 1 by breast-feeding in zambia virus markers associated with vertical transmission of human t lymphotropic virus type 1 in jamaica rotavirus antibodies in the mother and her breastfed infant incidence of haemophilus influenzae in the throats of healthy infants with different feeding methods transfusion transmission of west nile virus: a merging of historical and contemporary perspectives clinical presentations and outcomes of severe acute respiratory syndrome in children poliomyelitis in pregnancy: a twenty-year report from long-term serologic follow-up of patients for epstein-barr virus after recovery from infectious mononucleosis the global distribution of human immunodeficiency virus type 2 (hiv-2) infection interventions for preventing late postnatal mother-to-child transmission of hiv methicillin-resistant staphylococcus aureus colonization and its association with infection among infants hospitalized in neonatal intensive car units staphylococcus aureus in the infant upper respiratory tract. i. observations on hospital-born babies unilateral breast feeding and breast cancer staphylococcus aureus in australasian neonatal nurseries identification of human t-cell lymphotropic virus type iia infection in the kayapo, an indigenous population of brazil mother-to-infant transmission of tt virus in japan italian register for hiv infection in children: hiv-1 infection and breast milk transmission of west nile virus from an organ donor to four transplant recipients tuberculosis mastitis methicillin-resistant staphylococcus aureus infections among healthy full-term newborns human milk in the modern world staphylococcus epidermidis: a differential trait of the fecal microbiota of breastfed infants epstein-barr virus shedding in breast milk post weaning gastroenteritis and mortality in hiv-1 uninfected african infants receiving antiretroviral prophylaxis to prevent mtct of hiv-1 successful antepartum treatment of listeriosis low risk for motherto-child transmission of human t lymphotropic virus type ii in non-breastfed infants staphylococcal scalded skin syndrome in a breastfed infant fluconazole prophylaxis against fungal colonization and infection in preterm infants transmission of staphylococus aureus between healthy lactating mothers and their infants by breastfeeding transmammary transmission of strongyloides ratti case-control study of mastomys natalensis and humans in lassa virusinfected households in sierra leone transfer of tuberculin immunity from mother to infant recurrent group b streptococcal disease in an infant associated with the ingestion of infected mother' s milk mammary tuberculosis: report on 52 cases eradication of methicillin-resistant staphylococcus aureus from a neonatal intensive care unit by active surveillance and aggressive infection control measures prevention of mother-to-child transmission of hiv-1 through breast feeding by treating infants prophylactically with lamivudine in dar es salaam prevention of mother-to-child transmission of hiv through breastfeeding by treating mothers with triple antiretroviral therapy in dar es salaam, tanzania: the mitra plus study clinical outcomes in methicillin-resistant staphylococcus aureus colonized neonates in the neonatal intensive care unit demonstration of adult t-cell leukemia virus antigen in milk from three seropositive mothers oral infection of a common marmoset with human t-cell leukemia virus type i (htlv-i by fresh human milk of htlv-i carrier mothers the differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection human parvovirus b19 infection in women of childbearing age and within families lymphocytic choriomeningitis in the newborn late-onset and recurrent neonatal group b streptococcal disease associated with breast-milk transmission diarrhoea in uninfected infants of hiv-infected mothers who stop breastfeeding at 6 months: the ban study experience accessed 12/19 prolonged breastfeeding and mortality up to two years postpartum among hiv positive women in zambia hiv specific secretory iga in breast milk of hiv-positive mothers is not associated with protection against hiv transmission among breast-fed infants high uptake of exclusive breastfeeding and reduced postnatal hiv transmission; prospective results from the zambia exclusive breastfeeding study accessed 12/19 effect of early, abrupt weaning on hiv-free survival of children in zambia differential effects of early weaning for hiv-free survival of children born to hiv-infected mothers by severity of maternal disease breastfeeding and aids in the developing world hiv prevention is not enough: child survival in the context of prevention of mother to child hi transmission congenital and postnatally acquired cytomegalovirus infections: long-term follow-up extended antiretroviral prophylaxis to reduce breast milk hiv-1 transmission breast milk is not a significant source for early epstein-barr virus or human herpes virus 6 infection in infants: a seroepidemiologic study in 2 endemic areas of human t-cell lymph tropic virus type i in japan evidence for mother-to-child transmission of human t-lymphotropic virus type ii mother-to-child transmission of human t-lymphotropic virus type ii (htlv-ii) role of maternal antibody in pneumonia and bronchiolitis due to respiratory syncytial virus a further report on cancer of the breast repeated congenital infection with toxoplasma gondii is ingestion of milk associated bacteria by premature infants fed raw human milk controlled by routine bacteriologic screening? maternal and child health technical information bulletin cytomegalovirus in human breast milk: risk to the premature infant the independent associations of parity, age at first full term pregnancy, and duration of breastfeeding with risk for breast cancer epstein-barr virus estimating infant mortality from hiv and other causes in breastfeeding and bottle-feeding populations postnatal cytomegalovirus infection from frozen breast milk in preterm low birth weight infants postntal transmission of hiv from mother to child eradication of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit: which measures for which success? international multicentre pooled analysis of late postnatal mother-to-child transmission of hiv-1 infection. ghent international working group on mother-to-child transmission of hiv 18 month effectiveness of short-course antiretroviral regimens combined with alternatives to breastfeeding to prevent hiv mother-tochild transmission breast milk transmission of a panton-valentine leukocidin-producing staphylococcus aureus strain causing infantile pneumonia mammary epithelial cells support and transfer productive human t cell lymphotropic virus infections mechanism of vertical transmission of hepatitis g absence of infection in breast fed infants born to hepatitis c virus-infected mothers intrauterine listeria infection: prenatal diagnosis by biophysical assessment and amniocentesis ejpidemiologic features of htlv-ii: serologic and molecular evidence neonatal brucellosis probable transmission of brucellosis from breast milk to a newborn a multicenter therapeutic study of 1100 children with brucellosis lactation and cancer of the breast: a summary of an international study human immunodeficiency virus infection as risk factor for mother-to-child hepatitis c virus transmission: persistence of anti-hepatitis c virus in children is associated with the mother' s antihepatitis c virus immunoblotting pattern increased infant human immunodeficiency virus type one free survival at one year of age in sub-saharan africa with maternal use of highly active antiretroviral therapy during breast feeding the genome sequence of the sars-associated coronavirus diagnostic approach cytomegalovirus infection of extremely low-birth weight infants via breast milk freezethawing of breast milk does not prevent cytomegalovirus transmission to a preterm infant human milk siga binds to botulinum type b 16 s toxin and limits toxin adherence on t84 cells antimicrobial factors and microbial contaminants in human milk: recent studies morbidity and mortality patterns in hiv-1 seropositive/seronegative women in kampal and harare during pregnancy and in the subsequent two years morbidity and mortality in breastfed and formula-fed infants of hiv-1-infected women: a randomized clinical trial predominance of left-sided breast tumors obstetric management of hepatitis c-positive mothers: analysis of vertical transmission in 559 mother-infant pairs evidence for a protective effect of lactation on risk of breast cancer in young women: results from a case-control study prospective study of the incidence and complications of bacterial contamination of enternal feeding in neonates effect of treatment on the contagiousness of patients with active pulmonary tuberculosis infectious diseases of the fetus and newborn infant does breast feeding increase the child' s risk of breast cancer? role of breast milk in acquisition of cytomegalovirus infection hiv transmission through breastfeeding: a study in malawi prevention of breast milk tranmission of hiv: the time is now mother-to-infant transmission of hepatitis c virus: rate of infection and assessment of viral load and igm anti-hcv as risk factors yellow fever vaccine nosocomial transmission of methicillin-resistant staphylococus aureus from a mother to her preterm quadruplet infants breastfeeding family history and breast disease transmission of hepatitis c virus from mothers to infants: its frequency and risk factors revisited immune escape by epstein-barr virus associated malignancies the duration of breastfeeding by hiv-1 infected mothers in developing countries: balancing benefits and risks search for possible routes of vertical and horizontal transmission of adult t-cell leukemia virus outbreak of invasive disease caused by methicillin-resistant staphylococcus aureus in neonates and prevalence in the neonatal intensive care unit human immunodeficiency virus type 1-infected cells in breast milk: association with immunosuppression and and vitamin a deficiency effect of breastfeeding on mortality among hiv-1 infected women: a randomised trial breast brucellosis in taif, saudi arabia: cluster of six cases with emphasis on fna evaluation case control study of symptoms and neonatal outcome of human milktransmitted cytomegalovirus infection in premature infants human milk glycosaminoglycans inhibit hiv glycoprotein gp 120 binding to its host cell cd4 receptor risk factors for neonatal methicillin-resistant staphylococcus aureus infection in a well-infant nursery lactation and a reduced risk of premenopausal breast cancer contamination of expressed human breast milk with an epidemic multiresistant staphylococus aureus clone human cytomegalovirus infection of breast milk strongyloides papillosus: prenatal and transmammary infection in ewes human neonatal infections with hookworms in an endemic area of southern nigeria. a possible transmammary route mother-to-child transmission of human t-cell lymphotoropic virus types i and ii (htlv-i/ii) in gabon: a prospective follow-up of 4 years transfer of human herpes virus 6 and 7 antibodies from mother to their offspring vertical transmission of hepatitis c virus transmission of hepatitis c virus from mothers to infants for the vertical transmission of hepatitis viruses collaborative study group, motherto-infant transmission of gb virus type c/hgv for the vertical transmission of hepatitis viruses collaborative study group: tt virus infection during childhood tt virus: virological and genomic characteristics and disease associations birth outcomes following west nile virus infection of pregnant women in the united states neonatal group b streptococcal disease associated with infected breast milk transmission of cytomegalovirus to extremely preterm infants through breast milk early breastfeeding cessation among hiv-exposed negative infants and risk of serious gastroenteritis: findings from a perinatal prevention trial in kampala, uganda inactivation of human immunodeficiency virus type i in human milk: effects of intrinsic factors in human milk and of pasteurization lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome human immunodeficiency virus and other viruses in human milk: placing the issues in broader prospective effect of breast-feeding on immune response to bcg vaccination brucellosis in a mother and her young infant: probable transmission by breast milk brucellar arthritis of knee in a child breast-feeding during primary maternal human immunodeficiency virus infection and risk of transmission from mother to infant treatment acceleration program and the experience of the dream program in prevention of mother-to-child transmission of hiv updated u.s. public health service guidelines for the management of occupational exposures to hiv and recommendations for post exposure prophylaxis etiologic factors in cancer of the breast in humans: collective review methicillin-resistant staphylococcus aureus in milk oral rotavirus vaccines: how well will they work were they are needed most? determinants of acquisition and carriage of staphylococcus aureus in infancy early acquisition of cytomegalovirus infection clinical virology west nile virus: a primer for the clinician petra study team: efficancy of three short course regimens of zidovudine and lamivudine in preventing early and late transmission of hiv-1 from mother to child in tanzania, south africa, and uganda (petra study): a randomized, double-blind, placebo-controlled trial dengue virus infection in late pregnancy and transmission to the infants effect of maternal rotavirus immunization on milk and serum antibody titers cell-free virus in breast milk of hiv-1-seropositive women role of breastfeeding in paralytic poliomyelitis bacterial contamination of human milk: container type and method of expression maternal herpetic breast infection: another hazard of neonatal herpes simplex mother-to-child transmission of chikungunya virus infection care of the pregnant woman with tuberculosis and her newborn infant: a pediatrician' s perspective read jsand the committee on pediatric aids: human milk, breastfeeding, and transmission of human immunodeficiency virus type 1 in the united states postpartum mastitis and community-acquired methicillin-resistant staphylococcus aureus infectious diseases of the fetus and newborn infant hepatitis e virus breastmilk infectivity in human immunodeficiency virus type 1-infected mothers high risk types of human papillomavirus (hpv) dna in oral and genital mucosa of infants during their first 3 years of life: experience from the finnish hpv family study streptococcal toxic shock syndrome, including necrotizing fasciitis and myositis late-onset septicemia in a norwegian national cohort of extremely premature infants receiving very early full human milk feeding hepatitis a outbreak in a neonatal intensive care unit: risk factors for transmission and evidence of prolonged viral excretion among preterm infants characterization of a novel corona virus associated with severe acute respiratory syndrome longitudinal analysis of human immunodeficiency virus type 1 rna in breast milk and of its relationship to infant infection and maternal disease attempts to detect rna tumour virus in human milk is breast milk collected at home suitable for raw consumption by neonates in brizilian public neonatal intensive care units? prevalence of hiv-1 dna and p24 antigen in breast milk and correlation with maternal factors follow-up of transmission of hepatitis c to babies of human immunodeficiency virus-negative women: the role of breastfeeding in transmission occurrence of acute diarrhea in atopic and nonatopic infants: role of prolonged breast-feeding an outbreak of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit hospital transmission of community acquired methicillin-resistant staphylococcus aureus among postpartum women removal of inhibitors against rna-directed dna polymerase activity in human milk effect of human milk on mouse mammary tumor virus human papillomavirus dna detected in breast milk control of a cluster of a community-associated methicillin-resistant stahpylococcus aureus in neonatology immunoglobulin prophylaxis against milkborne transmission of human t cell leukemia virus type 1 in rabbits chlamydial infections pregnancy and pulmonary tuberculosis possible breast milk transmission of group b streptococcal infection evidence for transmission of lymphocyte responses to tuberculin by breast-feeding, lancet i:529 toxic shock syndrome detection of borrelia burgdorferi dna by pcr in the urine and breast milk of patients with lyme borreliosis prevention of perinatal group b streptococcal disease. revised guidelines from cdc detection of tt virus dna and gb virus type c/hepatitis g virus rna in serum and breast milk: determination of mother-to-child transmission effects of hepatitis a and b in pregnancy on the mother and fetus human immunodeficiency virus load in breast milk, mastitis and mother-to-child transmission of human immunodeficiency virus type 1 how contagious is vaccinia? transmammary transmission of necator americanus larva in the human host a study of infectious intestinal disease in england: risk factors associated with group a rotavirus in children highly active antiretroviral therapy started during pregnancy or postpartum suppresses hiv-1 rna, but not dna, in breast milk prevention of postnatal cytomegalovirus infection in preterm infants infants born to mothers with severe acute respiratory syndrome tuberculosis of the breast masquerading as carcinoma: a study of 100 patients transmammary transmission of strongyloides stercolis in dogs low birth weight and maternal virus diseases: a prospective study of rubella, measles, mumps, chickenpox, and hepatitis lyme disease during pregnancy no benefit of early cessation of breastfeeding at 4 months on hiv-free survival of infants born to hiv-infected mothers in zambia: the zambia exclusive breastfeeding study vertical dengue infection: case reports and reviews a young infant with severe acute respiratory syndrome six week extended-dose neviapine (swen) study team: extended-dose nevirapine to 6 weeks of age for infants to prevent hiv transmission via breastfeeding in ethiopia, india, and uganda: an analysis of three randomized controlled trials chlamydial secretory iga antibodies in human milk hepatitis d virus factors associated with immunoprophylaxis failure against vertical transmission of hepatitis b virus phagocytosis-associated oxidative metabolism in human milk macrophages management of mastitis in breastfeeding women communityacquired methicillin-resistant staphylococcus aureus among patients with puerperal mastitis requiring hospitalization working parents: the impact of day care and breast-feeding on cytomegalovirus infection in offspring breast milk and the risk of cytomegalovirus infection tuberculosis, an old disease but a new threat to mother, fetus, and neonate vertical transmission of hepatitis b antigen in taiwan yeast-recombinant hepatitis b vaccine: efficacy with hepatitis b immune globulin in prevention of perinatal hepatitis b virus transmission maternal lyme disease and congenital heart disease: a case-control study in an endemic area significance of postnatal mother-to-child transmission of htlv-i on the development of adult t-cell leukemia/lymphoma disseminated neonatal herpes simplex virus type i from a maternal breast lesion the effects of yellow fever immunization (17dd) inadvertently used in early pregnancy during a mass campaign in brazil the impact of breastfeeding on the health of hiv positive mothers and their children in sub-saharan africa prospective study of mother-to-infant transmission of hepatitis c virus inhibitory effect of maternal antibody on mother-to-child transmission of human t-lymphotropic virus type i seroepidemiological study of human herpes virus 6 and 7 in children of different ages and detection of these two viruses throat swabs by polymerase chain reaction short-term breast-feeding may reduce the risk of vertical transmission of htlv-i. the tsushima atl study group infant feeding and risk of mother-to-child transmission of hiv-1 in sao paulo state, brazil. sao paulo collaborative study for vertical transmission of hiv-1 breast feeding plus infant zidovudine prophylaxis for 6 months vs. formula feeding plus infant zidovudine for 1 month to reduce mother-to-child hiv transmission in botswana: a randomized trial: the mashi study isolation of the aids virus from cell-free breast milk of three healthy virus carriers rates of diarrhoea associated with early weaning among infants in kisumu, kenya. in program and abstracts of the 14th conference on retroviruses and opportunistic infections contamination in expressed breast milk following breast cleansing brucellar meningitis in an infant-evidence for human breast milk transmission hepatitis e in india human parvovirus b19 congential yellow fever virus infection after immunization in pregnancy unaids/who: report on the global hiv/aids epidemic a review of hiv transmission through breastfeeding mother-to-child transmission of human t-cell leukemia/ lymphoma virus type i: implication of high antiviral antibody titer and high proviral load in carrier mothers clinical profile of chikungunya in infants infective and antiinfective properties of breast milk from hiv-1 infected women postnatal transmission of the human immunodeficiency virus type 1 from mother to infant: a prospective cohort study in kigali, rwanda mother-tochild transmission of human t-lymphotropic virus type ii the possible role of circumcision in newborn outbreaks of community associated methicillin-resistant staphylococcus aureus brucellosis chez un nourrisson de 3 mois recovery of staphylococcal enterotoxin f from the breast milk of a woman with toxic-shock syndrome evidence for sexual and mother-to-child transmission of human t lymphotrophic virus type ii among guaymi indians transmission of cytomegalovirus to preterm infants through breast milk postnatally acquired cytomegalovirus infection via breast milk: effects on hearing and development in preterm infants pregnancy and lactation in relation to breast cancer risk morbidity among hiv-1-infected mothers in kenya: prevalence and correlates of illness during 2-year postpartum follow-up high concentrations of interleukin 15 in breast milk are associated with protection against postnatal hiv transmission low and undectable breast milk interleukin-7 concentrations are associated with reduced risk of postnatal hiv transmission lactation mastitis: a descriptive study of the experience breastfeeding does not pose any additional risk of immunoprophylaxis failure on infants of hbv carrier mothers recurrent neonatal group b streptococcal disease associated with infected breast milk transplacentally transferred maternal-infant antibodies to dengue virus vertical transmission of hepatitis a resulting in an outbreak in a neonatal intensive care unit immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 e6 or e7 perinatal transmission of hepatitis g virus (gb virus type c) and hepatitis c virus infections: a comparison advisory committee on immunization practices; healthcare infection control practices advisory committee: recommendations for using smallpox vaccine in a pre-event vaccination program global rotavirus surveillance: determining the need and measuring the impact of rotavirus vaccines recommendations for minimizing cmv exposure in breast milk fed very low birth weight (vlbw) preterm infants. available at www.breastfeeding. org/articals/cmvpremi.pdf maternal-infant transmission of htlv-i: frequency and time course of seroconversion. abstract w-53 mother-to-child transmission of human t-cell lymphotropic virus type i associated with prolonged breast-feeding maternal lyme disease and congenital malformations: a cord blood serosurvey in endemic and control areas human versus cow' s milk in infant nutrition and health human t-lymphocyte retroviruses working group on severe streptococcal infections: defining gas streptococcal toxic shock syndrome: rationale and consensus definition acquired cytomegalovirus infection of breast milk in infancy oral transmission of human t-cell leukemia virus type 1 into a common marmoset as an experimental model for milk-borne transmission evaluation of cytomegalovirus infections transmitted via breast milk in preterm infants with a real-time polymerase chain reaction assay sequelae of maternally derived cytomegalovirus infections in premature infants mother-to-infant transmission of hepatitis c virus human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis independent protective effect of lactation against breast cancer: a case-control study in japan a maternal factor for mother-to-child transmission: viral antigen-producing capacities in culture of peripheral blood and breast milk cells mother-to-infant transmission of hepatitis c virus infectious diseases of the fetus and newborn infant sensitive method for detection of human herpes viruses 6 and 7 in saliva collected in field studies survey of 34 pregnant women with hepatitis a and their neonates postnatal transmission of aids-associated retrovirus from mother to infant disseminated lyme disease and pregnancy. 9th annual international scientific conference on lyme disease and other tick-borne disorders a. siblings at home have active chickenpox when neonate and mother are ready for discharge from hospital.no no 1. mother: if she has a history of chickenpox, she may return home. without a history, she should be tested for varicellazoster virus antibody titer.* if test is positive, she may return home. if test is positive, she may return home. if test is negative, varicella-zoster ig † is administered and she is discharged home. 2. neonate: may be discharged home with mother if mother has history of varicella or is varicella-zoster virus-antibody positive. if mother is susceptible, administer varicella-zoster ig to infant and discharge home or place in protective isolation. b. mother has no history of chickenpox; exposed during period 6-20 days antepartum. ‡ ‡if exposure occurred less than 6 days antepartum, mother would not be potentially infectious until at least 72 hours postpartum. §considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted. ¶dosage of acyclovir for pregnant woman is 30 mg/kg/day; for seriously ill infant with varicella, 750 to 1500 mg/m 2 /day. elisa, enzyme-linked immunosorbent assay; fama, fluorescent antibody to membrane antigen; la, latex agglutination. key: cord-347710-ff64y6ef authors: wan, qianya; song, dan; li, huangcan; he, ming-liang title: stress proteins: the biological functions in virus infection, present and challenges for target-based antiviral drug development date: 2020-07-13 journal: signal transduct target ther doi: 10.1038/s41392-020-00233-4 sha: doc_id: 347710 cord_uid: ff64y6ef stress proteins (sps) including heat-shock proteins (hsps), rna chaperones, and er associated stress proteins are molecular chaperones essential for cellular homeostasis. the major functions of hsps include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. regarded as a double-edged sword, hsps also cooperate with numerous viruses and cancer cells to promote their survival. rna chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnrnps), which are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. hnrnps involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., parkinson’s diseases, alzheimer disease), stroke and infectious diseases. in this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. as sps also attract a great interest as potential antiviral targets (e.g., covid-19), we also discuss the present progress and challenges in this area of hsp-based drug development, as well as with compounds already under clinical evaluation. stress proteins (sps) are a diverse group of proteins that are synthesized at increased levels when cells are exposed to either intracellular or extracellular stressful stimuli. they exhibit protective effects against stresses. stress proteins include heat shock proteins (hsps), rna chaperone protein (rnps), and proteins mainly function in the endoplasmic reticulum (er): peptidyl-propyl isomerases, protein disulfide isomerases (pdis) and the lectin-binding chaperone system. 1 sps are ubiquitously expressed in all kind of cells, triggering signal cascades for neutralizing and eradicating the stresses occurring both intracellularly (e.g., pathogen invasion) and extracellularly (e.g., starvation, stimulation by cytokines/chemokines or hormones). responses triggered by sps can either activate pathways to promote cell survival or initiate cell death (i.e., apoptosis, necrosis, pyroptosis or autophagic cell death) for eliminating the damaged cells to protect a particular organ/ tissue under given conditions. it is widely noted that the dysregulation of stress proteins is associated with a variety of human diseases, including cardiovascular diseases, neurodegenerative diseases (e.g., parkinson's diseases, alzheimer disease), stroke, human cancers and infectious diseases. in this review, we focus on their functions and update findings involved in infectious diseases, particularly, the diseases caused by viral infections. heat shock proteins in 1962, an italian geneticist ritossa inadvertently elevated the incubation temperature of drosophila larvae and discovered an increased gene transcription of unknown proteins. he nominated these protein as hsps. 2 further studies have revealed a large number of hsps, which form a big family and are ubiquitously expressed in cells. based on the molecular weight, hsps are classified into different families, including hsp100s, hsp90s, hsp70s, hsp60s, hsp40s, and some small hsps . [3] [4] [5] hsps belong to the largest family of chaperones. the hsp expression is rapidly induced when cells meet physiological or environmental attacks such as starvation, high temperature, hypoxia or hyperoxia, pathogen invasion, malnutrition and exposure to chemicals or uv, etc. they form a network to promote or stabilize the correct folding of substrate protein to gain its functional/active conformation (fig. 1 ), although they may not associate with the substrate protein in the final structure. 3, 6 hsps are important factors in regulating cell survival, differentiation and cell death. accumulating evidence shows that some hsps participate in not only innate cellular immunity but also antigen presentation in adaptive immune response. 7, 8 hsps also serve as potential biomarkers for some diseases. it has been shown that the increase of hsp70 in plasma is associated with heart failure, 9 and the elevated hsp27 level in human peripheral blood mononuclear cells is related with coronary artery disease (cad). 10 hsp90s. hsp90, an abundant chaperone in all eukaryotic cells, controls a variety of critical signalling pathways in eukaryotic cells. 5, 6, 11 hsp90 is an atp-dependent chaperone with different isoforms, including 1) hsp90α (hsp90aa1, or hspc1), hsp90α-a2 (hspaa2, or hspc2) and hsp90β (hspab1, or hspc3) locate in cytoplasm. 2) glucose regulated protein grp94 (hspc4 or gp96) locates in the er. 3) trap1 (hspc5) locates in mitochondria. 12 among them, hsp90α and hsp90β account for the greatest proportion in humans. hsp90 contains three regions: an atpasedependent hydrolytic domain in the n-terminal region, a middle linker region, and a dimerization domain in the c-terminal region. 5 like other hsps, hsp90 binds non-native substrate peptide to prevent its aggregation and degradation. when hsp90 binds atp, a transient dimerization of the n-terminal domain allows the substrate peptide binding to hsp90. then the atp hydrolysis and energy release lead to a conformational change of the n-terminal domain that facilitates the correct folding of the substrate petite ( fig. 1) . 5, 6 besides, hsp90 is also involved in telomere maintenance, apoptosis, and cell cycle progression, etc. 6, 13 it is well known that hsp90 not only interacts and contributes to rna polymerase assembly and nuclear import of some (−) ssrna viruses (e.g., pb2 of influenza virus), but plays crucial roles in the folding process of viral capsid proteins and virion assemblies as well. 14 cochaperones of hsp90. cochaperones of hsp90 regulate hsp90 functions at many aspects. cdc37 (also called p50) delivers kinase to hsp90 and inhibits its atpase activity; carboxyl terminus of hsp70-interacting protein (chip) functions as e3 ubiquitin ligase; hsc70/hsp90-organizing protein (hop, also called sti1) inhibits dimerization of the n-terminal domain; and the activator of hsp90 atpase 1 (aha) and p23 participates the maturation of substrate peptides. 11, 15 hsp70s. hsp70 is a subfamily of hsps' superfamily with~70 kda molecular weight. it accounts for the majority of molecular chaperones in cells. 11 the members of the hsp70 family mainly include: (1) hsp72 (hspa1a), hsp70-2 (hspa2), hsp70b' (hspa6) and hsc70 (hspa8) are commonly located in the cytosol; (2) grp75 (hspa9) is located in mitochondria; (3) grp78 (hspa5) is associated with the er. 16 hsp70 consists of two domains: a 44-kda nucleotide-binding domain (nbd) which can be divided into four subdomains (ia, ib, iia, and iib) in the n-terminal region and a 28-kda substrate-binding domain (sbd) composed of c-terminal α-helical (sbdα) and n-terminal β-sheet (sbdβ) subdomains in the c-terminal region. 17, 18 as a critical component of cellular protein surveillance, the atp-dependent molecular chaperone protects cells from damage caused by stress and takes part in a number of folding processes, including folding of newly synthesized polypeptides, recognition and refolding of misfolded or aggregated proteins, solubilization or degradation of proteins, transporting proteins, assembly or disassembly of oligomeric protein complexes, and the regulation of certain natively folded proteins. 5, 13, 19, 20 the functions of hsp70 are not limited to host protein folding. its functions are considerable during viral infection. the members of hsp70s exhibit quite different roles in the course of virus life cycle. for example, hsp72, hsp70b' and hsc70 participate in the hcv viral entry, virion assembly and translation of the viral genome. grp78 in er is associated with the homeostasis of viral proteins and prevents the overload of viral proteins in host cells. in hepatocytes, the elevated grp78 stimulates innate immunity to restrict or eliminate hepatitis b virus (b) replication. 21 grp75 interacts with the ns5a protein of hcv in mitochondria. 22 accumulating evidence shows that hsp70 interacts with viral components of human cytomegalovirus, rabies virus, respiratory syncytial virus, human papillomavirus, herpes simplex virus. chaperone cycle of hsp70. the chaperone cycle is mediated by the n-terminal nbd, which regulates the binding of hsp70 with substrates through switch of two states. the first state is the atpbound state with low affinity for substrate binding, i.e., a high association and dissociation rate of the substrate peptides to the sbd. the second state is atp hydrolysis that switches to the adpbound and nucleotide-free state. at this state, the substrate exchange rates are low while the affinity to substrates is high. the chaperone activity of hsp70 mostly relies on atp hydrolysis. the basal atpase of hsp70 is normally low unless it is stimulated by the substrate peptide itself. it takes 20-30 min for a molecule of atp to be hydrolysed per hsp70 molecule at 30°c. as a result, it is necessary for some cochaperones to encounter with hsp70 atp to induce atp hydrolysis and help the increase of the affinity for substrate peptides. 19, 23 cochaperones of hsp70. the most crucial cochaperones of hsp70 are members of the j-domain proteins (jdps) family and nucleotide exchange factors (nefs) family. previous studies focused on the function of hsp70 machinery and led to the development of a "canonical model" for its mode of action. the model contains two steps. first, the unfolded peptide substrates bind jdps of the hsp40 family; then the substrates are delivered to hsp70 that stimulate the hsp70's atpase activity. simultaneously, jdps prevent the aggregation of unfolded proteins. second, nefs work as substrate release factors that assure the substrate to fold into the correct and active conformation. in this way, the cochaperones strongly facilitate the function of hsp70. therefore, hsp70 generally does not work individually but cooperates with cochaperones. 23, 24 hsp60s. hsp60s are large cylindrical oligomers with two back-toback rings. 19 the non-native proteins of the central cavity in each ring are folded into the native protein through an atp-dependent process. 25, 26 hsp60s are classified into two subfamilies. group i is mainly in prokaryotes, while group ii appears in eukaryotic cytosol and some archaea. 27, 28 the most well-studied one in group i is the groel-groes chaperonin system in the cytosol of bacteria. groel is an about 57 kda protein with two rings arranged back-to-back; and 7 subunits form a tetradecamer structure. groes is the cochaperone of groel. 11 the two rings-tetradecamer structure appears in two fig. 1 the general chaperone cycle of heat shock proteins. initially, unfolded client protein bound to the hsp70-hsp40 chaperones interacts with the hsp90. atp binding to hsp90 induces the client proteins transfer from hsp70 to hsp90. later, the conformation of hsp70-hsp40 chaperones will be released. finally, the hydrolysis of atp induces additional conformation changes leading to the client protein release forms include asymmetric (1 groel: 1 groes) and symmetric (1 groel: 2 groes) complexes, which are described as "bullet" 14, 29, 30 and "american football" 31 shaped respectively. the groel-groes chaperonin undergoes allosteric regulation dependent on atp and which completes the protein folding function. the polypeptide binds to the hydrophobic sites of one of the seven subunits of groel and changes conformation upon atp binging and hydrolysis. with the help of cochaperone groes, the polypeptide finishes its folding process. 32 in contrast to the groel-groes system, the mammalian homolog hsp60/hsp10 system is less studied. hsp60 is thought to be imported into the mitochondria and converted into its mature form with a molecular size of 58 kda. [33] [34] [35] hsp60 exhibits important roles in facilitating protein folding, transportation and proteostasis in mitochondria. 36, 37 group ii chaperonins include the archaeal thermosome and eukaryotic cct (chaperonin-containing tcp1, or called tric), which are oligomers with eight to nine subunits with molecular weight 57-61 kda in each ring. compared to group i, group ii chaperonins show different allosteric movements by atp binding. 11, 19 hsp60 family is known to participate in viral life cycle at various stages from viral attachment to the replication of the viral genome. hsp60s are essential for host cell immunity regulation. some viral proteins require hsp60 for its translocation within host cells. pb2 is a subunit of influenza a viral rna polymerase, which mostly locates in the nucleus but also appears in mitochondria. 38 when the virus infects the host cells, pb2 is responsible for maintaining the function of mitochondria. pb2 interacts with mitochondrial antiviral signaling protein (mavs) to downregulate intracellular immune response by decreasing the level of ifnβ so that the invading virus can easily escape from the defence of host cells. 39 hsp60 takes the role of transporting pb2 from cytosol to mitochondria in the host cells. besides, hsp60 shows great regulatory functions on innate immunity by inducing proinflammatory cytokine release, such as tnf-a, il-6, and il-1b, etc. 40 small heat shock proteins. small heat shock proteins (shsps) are a group of small proteins with a low molecular weight ranging from 15 to 40 kda. 4 there are 10 members in the shsp family and some are ubiquitous including hsp27 (hspb1), hspb5 (αb-crystallin, or αbc), hsp20 (hspb6), and hsp22 (hspb8), while the others are tissue-specific including hspb2 (myotonic dystrophy protein kinase, or mkbp), hspb3, hspb4 (αa-crystallin, or αac), hspb7 (cvhsp), hspb9, and hspb10 (sperm outer dense fiber protein, or odf). 41 shsps can exist as monomers, dimers or even large multimeric complexes in the cells. 42 the structure of shsps is different from the other hsps due to their less conserved sequences. the basic structure of shsps is a conserved α-crystallin domain (acd) flanked by two non-conserved domains including the n-terminal sequence (nts) and the c-terminal sequence (cts). among these domains, acd becomes the characteristic of different shsps. 43, 44 shsps play crucial roles in several physiological processes regarding stress tolerance, apoptosis, aging, and longevity. [45] [46] [47] [48] the phosphorylation together with the n-terminal wdpf motif helps shsps to form homo-or hetero-oligomers. 49, 50 the oligomerization is the hallmark of shsps for supporting their quite different activities. phosphorylation favors small oligomer formation while dephosphorylation provokes a shift toward large oligomer formation. 51 oligomerization dynamics is crucial for chaperone activity because it gives rise to the possibility to format different homo-and hetero-oligomers, each with different binding properties to chaperone a broad range of substrates. 41, 52 for instance, the phosphorylated species are required for actin dynamics. small phosphorylated dimers/tetramers bind f-actin to regulate actin polymerization. 53 among different shsps, hsp27 has been broadly studied. hsp27 exists in all tissues though it is known to mainly express in cardiac, skeletal and smooth muscles. 54 its importance has been demonstrated in cell differentiation, cell survival, cellular innate immunity, viral protein translation, and intracellular virus transport, etc. [55] [56] [57] same as the other shsps, hsp27 shares a highly conserved α-crystallin domain. 41, 58, 59 hsp27 is capable of oligomerization and phosphorylation. there are three serine residues 15, 78, and 82 can be phosphorylated by different kinases including p90rsk, pkg, mapkap kinases, etc. 55 the phosphorylation of hsp27 is a reversible process. the dephosphorylation contributes to the formation of large size oligomers. 60, 61 hsp27 can not only form large homo oligomers up to 800 kda; but it can also cooperate with other shsps (e.g., hsp20) to form heteromeric structures. 56, 58 hsp27 is upregulated and activated upon infections. 62, 63 the elevated hsp27 activity promotes either cytoskeletal stability or cell motility, 64, 65 and prevents apoptosis. 66 hsp27 is required for il-1-induced expression of the pro-inflammatory mediators il-6, il-8, and cyclooxygenase-2. [67] [68] [69] hsp27 is also linked to various signalling pathways involved in the development, differentiation, and cell growth. 70, 71 the long-term and high-level expression of hsp27 stimulated by variety of stresses (such as hbv or ebv infections) enhances carcinogenesis, cell survival, stemness of cancer cells, cancer metastasis, tumour formation and drug resistance. transcriptional regulation of the hsps. heat shock factors (hsfs) display great contributions in regulating the transcriptional activation of hsp genes. in all invertebrate animals, only hsf1 is responsible for the transcriptional activation. in vertebrates, four members of hsf family (hsf1-4) regulate hsp expression. 72 among them, hsf1 is the most critical one. the fibroblasts from hsf1 −/− mice undergo apoptosis upon heat stress because of no hsp transcription. 73 upon stress conditions, the originally monomeric hsf1 in the cytoplasm could trimerize and translocate into the nuclei to promote the hsp expression by binding on the heat shock elements (hse) in the promoter region. 74 protein disulfide isomerase protein disulfide isomerase (pdi) is a multifunctional oxidoreductase and chaperone that catalyses the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (er). during disulfide bond formation, cysteine residues at the cghc active site of pdi accept two electrons from the cysteine residues in polypeptide substrates, leading to the reduction of pdi and oxidation of the substrate. then pdi transfers the electrons to an acceptor to start another cycle of disulfide bond formation. 75 in addition to pdi's catalytic function as a thiol-disulfide isomerase, it also exhibits molecular chaperone properties for glycosylated protein quality control. 76 erp57 (pdia3, grp58) is possibly the most thoroughly studied pdi family member that shares a similar structure consisting of four domains (namely a-b-b'-a') and possesses two localization sequence-an er retention signal (qdel), and a nuclear localization signal (kpkkkkk). unlike other pdi family members that directly bind the substrates for their reductase or isomerase activities, the b domains of erp57 have a high affinity to associate with calreticulin (crt) and calnexin (cnx), which would help to recognize and recruit polypeptide segments of the glycoproteins. 77 if the protein is not correctly folded, udpglucose:glycoprotein glucosyltransferase (uggt) would be recruited to reglycosylate the proteins, allowing them to be recognized and re-associated by erp57/crt/cnx complex. 76, 78, 79 considering the essential roles of pdis in the oxidative folding and chaperone-mediated protein quality control, they are now linked to a growing range of diseases including those are caused by virus infection. proteins that interact non-specifically with rna and resolve the non-functional inhibitory structures are usually referred to as rna chaperones, which have distinct roles without common sequences or motifs. 80, 81 they participate in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, histone-like nucleoid structuring, intracellular immunity, and viral rna replication and translation. rna molecules mostly rely on welldefined 3d structures to fulfill their functions. however, the process of rna folding is very complicated. 82 the multitude of possible rna base-pairings together with the high stability of rna duplexes would give rise to a large number of alternative secondary and tertiary structures that are thermodynamically as stable as the functional, native structure. 83 rna chaperones promote rna folding by accelerating the escape from kinetic folding traps and prevent rnas from being trapped in nonfunctional conformations. [84] [85] [86] so far, no protein has been characterized whose primary function is to resolve nonspecifically misfolded rnas in cells. 80, 81 hnrnps are a group of heterogeneous nuclear ribonucleoproteins. they are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. more than 20 hnrnps have been identified to date. hnrnps contain common rna binding motifs like arginine glycine boxes (rgg boxes), rna recognition motifs (rrms), hnrnp k homology (kh)-domains and zinc finger (zf)-domains (khzf domain). 87 well-defined functions of this family include transcription regulation, pre-mrna splicing, 3′-end formation, mrna packaging, rna transport, translational regulation, rna silencing, dna repair, and telomere biogenesis. they also have the ability to shuttle between nucleus and cytoplasm, therefore could transiently help to form rnp complexes in nucleus and also participate in rna metabolism in cytoplasm. 88 a large collection of hnrnps are involved in virus activities, most of which were first identified using viral rna-protein binding assays, followed by functional assays. 89 the importance of stress proteins one of the main functions of stress proteins is to maintain cellular homeostasis. under pressure, stress proteins are hyperactive to release the pressure. hsp27, hsp70 and hsp90 accumulate to a very high level in quite a few types of cancer cells. [90] [91] [92] although the mechanism underlying the increase has not been fully understood, it suggests that the fast increased hsps respond to the folding stresses. with tumor progression, the accumulating oncogenic proteins need powerful protein folding ability. under long-term stresses, hsps participate or promote carcinogenesis, cell survival, anti-apoptosis, angiogenesis, cancer cell stemness, invasion and metastasis. 93 however, hsps and rna chaperones are downregulated in nearly all age-related neurodegenerative diseases including alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, and several polyglutamine diseases such as huntington's disease and different forms of spinocerebellar ataxias 94, 95 (fig. 2) . downregulated rna chaperones lead to disorder of rna metabolism; 94 while the attenuated hsps result in a failure of the protein quality control (pqc) system to adequately handle the folding or timely degradation of proteins in neurologic disease. 95 both mechanisms cause protein aggregates, the hallmark of age-related neurodegenerative diseases. in this review, instead of paying much attention to these topics, we would only focus on the main biological functions and target values of stress proteins in human diseases caused by pathogen infections, particularly by virus infections because the welldeveloped antibiotics have already controlled the bacterial infection very well since 1950s. viruses infection causes a variety of diseases that are highly associated with dysregulation of stress proteins (fig. 2 ), e.g., respiratory symptoms, 96, 97 gastroenteritis, [98] [99] [100] [101] hemorrhagic fevers. 102, 103 critically, it has been demonstrated that neurodegenerative diseases as well as neurobehavioral disorders are the consequences of loss of neurons and axons in the central nervous system with ageing. it is evidenced that these diseases are possibly caused by chronic neuropathic viral infections. 104 stress proteins are involved in many steps of virus infection process, including virus entry, uncoating, replication, gene expression, as well as virus assembly and releasing as steps 1-5 shown (fig. 3 ) some viruses replicate in the nucleus, while stress proteins take part in the virus protein and/or rna nuclear import/export processes as setp a-c shown (fig. 3 ). the summary of the relationship between stress proteins, virus infection as well as host cell response is listed in table 1 . in this section, we would review and present new findings on hsp90 family proteins in the life cycles of different viruses including rna virus, dna virus and retrovirus. in addition, we also discuss their functions in virus-induced cellular response. the function of hsp90 family in rna virus infection virus entry. in the case of rna virus, hsp90 is critical for the entry of enterovirus a71 (ev-a71), 107 japanese encephalitis virus (jev) and dengue virus (denv). 108 ev-a71 entry is significantly blocked when cells are pre-treated with hsp90 inhibitors or hsp90-specific sirnas. 107, 109 since both denv virus and jev belong to the flavivirus, the entry of the these two viruses differently utilize hsp90 with the support of hsp70s in both neuroblastoma and microglial cells. 95, 108 denv depends much more on hsp90 to enter into cells as compared with jev since anti-hsp90 antibodies or hsp90 inhibitors block the majority of denv entry 110 but only a small portion of jev entry. 111 additionally, both hsp90 and hsp70 are associated with membrane lipid rafts in response to denv infection. 110 however, in denv infected liver cells (hepg2), neither hsp90 nor hsp70 works as the receptor to enable denv internalization, 110 indicating alternative entry mechanisms in different cell types. it is likely that the receptor functions of hsp90 and hsp70 are replaced by other unknow molecules. virus replication. hsp90 protein facilitates virus replication in several aspects. first, hsp90 works as a classic chaperone protein to stabilize virus proteins. hsp90 stabilizes paramyxoviruses polymerase and l protein, as well as assists virus replication. inhibition of hsp90 could hamper virus replication and shorten the half-life of l protein in vesicular stomatitis virus (vsv), human parainfluenza viruses-2 (hpiv-2), human parainfluenza viruses-3 (hpiv-3), simian virus 41 (sv41), or respiratory syncytial virus (rsv) infection cells. 112, 113 similarly, hsp90 is shown to maintain the stability of chikungunya virus (chikv) non-structural proteins (nsps) including nsp3 (a protein essential for rna synthesis) and nsp4 (rna-dependent rna polymerase, rdrp); 114 and the protease of hcv nonstructural protein ns3. 115 second, hsp90 modulates virus polymerase activity to enhance virus replication. taking hcv as an example, hsp90 indirectly modulates the hcv polymerase ns5 activity by maintaining the stability of kinase phosphoinositide dependent kinase l (pdk1), an upstream kinase of ns5 phosphorylation kinase prk2. 116 besides, mediated by fkbp8, hsp90 forms a complex with ns5 and directly regulates ns5 activity. 117 hsp90 inhibitors suppress viral replication by disrupting the hsp90-ns5 complex formation. 117 third, hsp90 manipulates the proper location of virus polymerases. during influenza virus infection, hsp90 interacts with the viral rdrp subunits polymerase basic protein-1 (pb1) and -2 (pb2) to form a complex, and then co-translocates into nucleus. 14, 118 in this process, hsp90 maintains the stability of pb1 and pb2. after entry into nucleus, hsp90 dissociates from the hsp90/pb1/pb2 complex and forms a new functional complex with polymerase acidic protein (pa). 119 extending study shows that the state of hsp90 acetylation is strictly regulated by histone deacetylases 6/8 while some viruses (such as influenza, sv40, hbv etc) also enter into nucleus of host cells. they may undergo other steps in their life cycle which are labelled as a-c: virus nucleus import, nucleus export, and virus rna processing. during the process of virus infection, hsp90, hsp70, hsp60, hsp40, hsp27, and pdis participate in the virus entry and uncoating steps. hsp90, hsp70, hsp60, hsp40, hsp27 and rna chaperones take part in the virus replication step. hsp70, hsp40 and rna chaperones are required in virus gene expression step. hsp90, hsp70 and hsp40 assist virus assembly. hsp70 and rna chaperones contribute to the virus release. while hsp90 is also important in virus nucleus import and export. hsp70 plays a role in the virus nucleus import. and rna chaperones play a major role in virus rna processing, including replication, initiation of translation, stabilization and decay 485, 486 (hdac6/8) in the influenza rdrp nuclear import stage. hdac6/8 inhibitors efficiently limit the polymerase nuclear import and suppress virus replication. 120 in the course of influenza infections, hsp90 expression is stimulated through mtor/p70s6k signalling. 121 our recent studies show that hsp90 also exhibits significant importance on ev-a71 replication through pim1 signalling (unpublished). it has been shown that ev-a71 infection elevated both the mrna and protein levels of pim1. 122 the elevated pim promoted ev-a71 replication while knockdown of pim1 decreased ev-a71 replication. knockdown of hsp90β decreases 60% of virus replication 12h post infection (p.i.), while the secreted virions decrease by approximately 80%, indicating the crucial roles of hsp90β in both virus replication and secretion (fig. 4a-c) . other researchers reported that hsp90β is responsible for ev-a71 assembly which may be the reason that hsp90β attenuates the secretion of ev-a71 virions in our study. 107, 109, 122 notably, hsp90β contributes to virus replication more than that of secretion. our data also shows that knockdown or knockout of hsp90β decreased the proteins expression level of ev-a71 structure protein (fig. 4d , e). and hsp90 inhibitors 17-aag, geldanamycin (ga) and ver50588 all dramatically inhibit ev-a71 protein expression (fig. 4f ). among them, ver50588 display the strongest inhibitory effect which has not been reported before. we predicted that hsp90β is a potential target of pim1 (fig. 4g ). to address this hypothesis, we conducted experiments by overexpression pim1 and knockdown of pim1. accordingly, the phosphorylated status of hsp90β is increased and decreased (fig. 4h,i) . more importantly, knockout of hsp90β by crispr/cas9mediated gene editing almost completely abolishes the effects of pim1 signaling on ev-a71 replication (fig. 4j, k) . viral protein maturation, virion assembly, and release. during the viral protein expression and maturation, hsp90 works as a classic chaperone to monitor the proper folding of viral proteins. hsp90 modulates the maturation of hcv nonstructural protein 2/3 (ns2/ 3) kinase. 123 hcv ns2/3 is cleaved into two separate proteins right after translation, a key step of ns2/3 protein maturation. hsp90 strictly regulates the proper folding of newly synthesized ns2/3 protein. 123 hsp90 and its co-chaperone p23 form a complex to assist the proper folding of capsid precursor polyprotein p1 of poliovirus, rhinovirus, and coxsackievirus; 124 while the inhibitor ga reduces the maturation of p1, leading to immature p1 degradation in proteasome. 124 during the virion assembly, hsp90 interacts with capsid vp1 protein of noroviruses and the termini of the murine norovirus 1 genome. 124, 125 this interaction not only stabilizes vp1, but ensures the viral genome to be encapsulated into capsids as well. 124 hsp90 interacts with and stabilizes influenza neuraminidase (na), a major surface glycoprotein involving in virion release. 126 more importantly, it emphasizes the hsp90-na complex formation on promoting cell survival, leading to more virus production. 126 the function of hsp90 family in dna virus infection virus entry. the entry of dna viruses includes steps of crossing over cell membrane and nuclear import. hsp90 is mainly shown the ability to assist the nuclear import of virus. the nuclear transport of many viruses depends on the microtubules (mt) and mt-dependent molecular motor dynein/dynactin complex. 127 virus strictly modulates the status of tubulin acetylation, a critical event for the transportation of viral components. [128] [129] [130] in hsvinfected cells, hsp90 co-localizes with acetylated tubulin and capsid protein vp5. 131 hsp90 inhibitors disrupt its binding to the acetylated tubulin, thereby inhibiting the nuclear transport of hsv capsid protein. 131 during hbv infection, the glucocorticoid receptor shows a strong possibility to enhance the nuclear import of hbv. 132 hsp90 facilitates glucocorticoid receptor redistribution from the cytoplasm to the nucleus. 133 virus replication. during dna retroviral replication, hsp90 mainly contributes to regulating and maintaining the reversetranscriptase (rt) activity. taking hepatitis b virus (hbv) as an example, the reverse transcription is an essential step to generate viral genomic dna in type vii viruses. the beginning of reverse transcription is the recognition and interaction of rt with an rna signal (the packaging signal, ε) on the pre-genomic rna. 134 it was identified that hsp90 is an essential host factor that facilitates duck hepatitis b virus (dhbv) replication by interacting with viral rt. 135 treating with hsp90 inhibitors or monoclonal antibodies (mab) sufficiently block rt-ε binding. 135 hu et al. demonstrated that rt-ε interaction depends on hsp90's atp hydrolysis activity. 136 two independent regions in the terminal protein (tp) and the rt domains of polymerase separately bind with hsp90 at the n-terminal and c-terminal fragments, and both domains are essential for ribonucleoprotein (rnp) and protein priming. 137, 138 although a model is established to show how hsp90 bridges the two separate rt domains of polymerase together to enable the formation of an rnp complex with the hbv rna; 137 there are still some fundamental questions to be addressed. firstly, whether the hsp90 chaperones or hsp70 chaperones are essential for the rt-ε interaction. stahl et al. believed that hsp70 chaperones are much more important for the rt-ε interaction. they proposed that hsp90β another hsp90 family member, is shown a critical regulator in stabilizing and activating rt, allowing its preferential binding to the pregenomic rna during hbv replication. 143 the replication of most dna viruses occurs in the nucleus, where virions form in replication centres. therefore, the proper location of viral proteins is quite important for virus replication. hsp90 is also found to regulate the location of virus dna polymerase in virus-infected cells. after treated with hsp90 inhibitors, hsv polymerases is mislocalized from the nucleus to the cytoplasm and subsequently degraded in a proteasomedependent manner. 144, 145 similar to hsv, the nuclear translocation of dna polymerase of ebv also requires hsp90. 146, 147 during the polymerase transportation, the polymerase catalytic subunit balf5 forms a complex with bmrf1 in the assistance of hsp90β. hsp90 inhibitors effectively block the translocation of viral dna polymerase. 146, 147 virus gene expression. hsp90 is important for virus gene expression both at the transcription and translation levels. the transcription of hsv immediate-early α (ieα) genes is initiated by the transcription factor complex, which is composed of octamerbinding transcription factor 1 (oct-1), host cell factor 1 (hcf-1) and viral protein 16 (vp16). 148, 149 in the complex, vp16 is the major virus-encoded transcriptional activator that controls the efficiency and level of viral gene transcription. in the transcription process, hsp90α is shown to maintain the stability of vp16 by keeping it from degradation in a macroautophagy-mediated manner. 150 similarly, hsp90 also regulates the transcription of human cytomegalovirus (hcmv) immediate-early genes through activating akt and nf-κb signalling pathways, which are critical for major immediate early genes transcription. 151, 152 at the translation level, hsp90 promotes the translation of conserved herpesvirus protein kinases (chpks), including herpes simplex virus type 1 and 2 (hsv-1, hsv-2), varicella-zoster virus (vzv), ebv, kshv. 153 chpks play important roles in multiple processes, including gene expression, 154-156 viral dna replication, [156] [157] [158] capsid nuclear egress, 159, 160 and dna damage responses. 161, 162 the translation of ebv nuclear antigen 1 (ebna1) protein is also manipulated by hsp90. 163, 164 ebna1 is critical for cellular transformation, tumorigenesis, and the maintenance of viral episomes. [165] [166] [167] the ebna1 translation is strictly regulated by hsp90 through the gly-ala repeat domain to keep ebna1 at a relatively low level. 168 it has been demonstrated that hsp90 inhibitors block the translation of ebna1; and mutation of gly-ala repeat domain abrogates the inhibition of ebna1 translation. 163, 164 hsp90 does not interact with ebna1directly, a bridge protein may be involved in this process. inhibiting ebna1 expression strongly suppresses both ebv-induced primary b cell transformation in vitro and lymphoproliferative disease in scid mice in vivo. 163, 164 virus assembly. only a few papers reported the function of hsp90 in dna virus assembly. the activated hsp90 is needed for hbv assembly. 169, 170 hsp90 enhances the affinity of core protein dimers for capsid formation and prevents capsid dissociation. 169 besides, the reactive oxygen species (ros)-promoted hbv capsid assembly also requires an active form of hsp90. 170 virus-induced tumorigenesis. as described before, ebv is the causative regent of several tumors, including burkitt's lymphoma 171 and nasopharyngeal carcinoma (npc). 172 the latent membrane protein 1 (lmp1) is regarded as an oncoprotein that promotes tumor metastasis and invasiveness through inducing the expression of matrix metalloproteinase 9 (mmp9), mimicking the tumor necrosis factor receptor (tnfr) superfamily proteins, and activating the nf-kb, mapk, pi3k/akt and jak/stat signal transduction pathways. 173, 174 hsp90 seems positively promoting cell growth in ebv-positive nasopharyngeal carcinoma cells and ebv-infected t and nk cells. 175, 176 hsp90 inhibitors, at13387 and biib021, potently inhibit cell growth and induce apoptosis by impeding lmp1 function through activating its downstream signaling pathways described above. 175, 176 immunity modulation. we have discussed how hsp90 is hijacked by dna viruses to promote viral replication above. under certain conditions, hsp90 exhibits antiviral activities by promoting cell immunity. in the acute infection stage, hsp90 is induced to express in the cell surface of ebv-infected b cells. it has a strong ability to expand gamma delta t cells (γδ t cells) population. 177 the γδ t cells have potent antiviral ability in the acute phage when a host is infected by hiv, influenza, sendai, coxsackie, vaccinia, vsv, or hsv-1. the γδ t cells work as both early sentinels of the immune system by providing immediate protection and as bridging elements between innate immunity and adaptive immunity. 178 since hsp90 can work as an immune sensor and assist antigen presentation, it may function in the same way in ebv infection. 177, [179] [180] [181] [182] the function of hsp90 family on cell transformation during retrovirus infection several hsps functions as oncoproteins to promote cellular transformation. hsp90 participates in the htlv-1-induced cellular transformation. the tax protein of htlv-1 controls viral replication and induces t lymphocyte transformation. 183 nf-κb pathway is one of the main targets essential for this process; 184 while hsp90 acts as an important partner of tax by binding with and maintaining its stability in nucleus. 153, 185 hsp90 inhibitors (e.g.,17-dmag) or hsp90-depletion by sirnas cause tax degradation in proteasome, inhibition of nf-κb signalling, and activation of the long terminal repeat (ltr) of htlv-1. 153, 185 oral treatment with fig. 4 pim1 signaling promotes ev-a71 replication through hsp90β phosphorylation(unpublished data). a rd cells were treated with scramble/hsp90β sirna for 24h, the effects of hsp90β knockdown were determined by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. b, c rd cells were treated with scramble /hsp90β sirna for 4h, then infected with ev-a71 at moi of 1 for indicated time. the intracellular (b) and extracellular (c) viral rna levels were detected by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. d, e knockdown of hsp90β by sirna or knockout by cripsr/cas9 mediated gene editing in rd cells, and then cells were infected with ev-a71 at moi of 1 for indicated time. the protein level of ev-a71 was determined by western blot. f rd cells were treated with hsp90 inhibitors at different concentrations and infected with ev-a71 at moi of 0.01 for 48h. the protein level of ev-a71 was determined by western blot. g pim1protein interaction network was predicted using online genemania program. h pim 1 was overexpressed in 293t cells and cell lysate was collected for immunoprecipitation assay. the phosphorylation status of hsp90 was detected by western blot. i pim1 was overexpressed/ knocked down in 293t cells. and cell lysate was collected for native page analysis. j wt rd cells and hsp90β knockout (hsp90β-ko) cells were pretreated with 2 μm pim1 inhibitor sgi-1776 for 2 h, then the cells were infected cells with ev-a71 at moi of 0.01 for 48h. intracellular viral rna level was determined by rt-qpcr. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. k pim1 was overexpressed in wt/hsp90β -knockout rd cells, and infected with ev-a71 at moi of 1 for 12 h. the protein level of ev-a71 was determined by western blot hsp90 inhibitor 17-dmag significantly suppresses the aggressive infiltration into multiple organs in atl mice. 153, 185 functions of hsp70 family in rna virus infection virus entry. viruses in different families (e.g., picornaviridae, flaviviridae, and reoviridae) take advantage of hsp70 family proteins for their entry into host cells. in the case of picornaviridae, for example, the coxsackievirus a9 (cav-9) uses hsp70 homolog grp78 for its entry. 186 it shows that antibodies against grp78 block 50% of virus binding. integrin αvβ3 is another famous virus receptor. 187 when cells are simultaneously treated with grp78 and integrin αvβ3 antibodies, virus binding is blocked completely. therefore, grp78 functions as a co-receptor of cav-9. besides, grp78 can interact with major histocompatibility complex (mhc) i molecules on the host cell membrane after infection of cav-9. mhc i molecules help virus internalization into mammalian cells. 186 in the course of ev-a71 infection, hsp70 is dramatically upregulated and interacts with ev-a71 on the cell surface. hsp70 antibody significantly inhibits virus binding to the cell surface. 188 besides enteroviruses, many viruses of flaviviridae family also require hsp70 to entry into host cells. by affinity chromatography assay, hsp70 is discovered to form a complex with hsp90 and denv receptor that facilitates viral entry. 110, 189 hsp70 interacts with dnev envelope protein (e protein) and plays a significant role in virus attachment. 190 similarly, antibodies against hsp70 and hsp90 significantly inhibit denv infection. 110, 189 the same mechanism is also observed in jev infection. 191 hsp70 is enriched in the lipid raft and colocalized with the e protein in jev-infected huh7 cells. 192 the depletion of cholesterol disrupts the enrichment and colocalization of the e protein and hsp70 to a raft membrane. eventually, it decreases jev entry without any effects on virus attachment. 192 these results suggest that hsp70 works as a receptor of jev; and lipid rafts serve as an organizing centre to facilitate jev entry. at the late stage of jev entry, hsc70 (isoform d) is upregulated in c6/36 cells upon jev infection. however, it seems that hsc70 is not required for virus attachment to the cell membrane but needed for virus penetration into the host cells. it is suggested that hsc70 holds an intense involvement in clathrinmediated endocytosis at the late stage of viral entry, which helps jev to penetrate into host cells. 193 recently, it is reported that grp78 is also required for jev both in the attachment and entry steps. 194 antibody targeting the n-terminal of grp78 significantly prevents virus attaching to host cells whereas antibody targeting the c-terminus fails to block the attachment. knockdown of grp78 also inhibits jev internalization. the colocalization and interaction between grp78 and jev envelope protein provide solid evidence to show the importance of grp78 in the process of virus attachment and entry. 194 interestingly, grp78 is secreted out of host cells after jev infection, and the secreted grp78 cooperates with jev to promote virus infection. 195 recent studies show that grp78 is a receptor of sars-cov, mers-cov, and sars-cov-2 viruses. 196 zikv infection is positively regulated by hsp70 at multiple stages. 197 hsp70 inhibitors impair virus entry, rna replication, and capsid assembly of different zikv strains in diverse cell lines. 190, 197 rotavirus infection also needs the assistance of membrane-resident hsc70. 198 hsc70-specific monoclonal antibodies inhibit virus internalization and infection without effect on virus attachment. 198 further evidence shows that the whole virus particle and a short domain (or a peptide) in the cterminal region of vp5 is sufficient to bind to hsc70. 199 the atpase domain of hsc70 is proved to be necessary for its interaction with vp5 and induction of virion conformation change for the entry. 200 virus replication. hsp70 family proteins participate viral replication by employing different mechanisms. first, hsp70 family proteins facilitate the formation of virus replication complex and/ or maintain the stability of complex proteins. in some cases, hsp70 family proteins directly interact with viral polymerase to enhance viral replication. for example, during the mumps virus (muv) infection, the expression level of hsp72 is increased. the c-terminal region of hsp72 interacts with the n-terminal region of p protein, which is an essential component of rdrp complex. knockdown of hsp72 results in accumulated ubiquitinated p protein as well as increased cell apoptosis. 201 besides, hsp70 is also reported to regulate l protein, another muv polymerase component. hsp70 cooperates with hsp90 to regulate l protein levels. hsp90 inhibitor, 17-aag, reduces the l protein level through promoting degradation via the c terminus of hsp70interacting protein (chip) -mediated proteasomal pathway. hsp70 inhibitor ver155008 together with 17-aag enhances l protein degradation. therefore, hsp90 and hsp70 together regulate the stability of l protein and ensure the proper virus replication complex (vrc) formation. 202 in the case of canine distemper virus (cdv) infection, the increased hsp70 results in an elevated expression of light nucleocapsid (nc-l) variant, which displays polymerase activity. 203, 204 a more direct evidence is that hsp70 facilitates viral rna production in cell-free transcriptional assays. 204 furthermore, it is demonstrated that hsp70 interacts with and regulates nc polymerase activity dependent on the hsp70 atp activity; because hsp70 antibody significantly inhibits nc polymerase activity and supplementation of purified recombinant hsp70 enhances both the basal and stress-induced nc polymerase activity. 205 the other members of hsp70s also regulate vrc formation. hsp72 physically interacts with several replication proteins of flavivirus including ns5a, ns3, and ns5b (rdrp). for example, hsp72 participates the vrc formation of hcv. 206 downregulating hsp72 leads to a decreased number of vrc in hcv-infected cells, while overexpression of hsp72 raises the number of vrc. 206 hsc70 is associated with vrc by binding on the 3' polyu/uc motif of hcv rna genome. 207 hcv accumulation and virion production are significantly suppressed when cells are treated with hsp70 or hsc70 inhibitors. 208 similar result is reported in the case of rsv infection. ectopic expression of rsv nucleocapsid protein (n protein) and phosphoprotein (p protein) are detected to interact with hsp70 in 293t cells. 209 the n protein is responsible for interacting with the viral rna, and p protein interacts with n protein and with the rdrp l to form the nucleocapsid. in rsvinfected cells, hsp70 redistributes into lipid-raft membranes and colocalizes with virus n protein and lipid raft marker gm1. 210 although hsp70 inhibitors suppress rsv polymerase activity; 210 it only disrupts viral gene expression but do not affect rna polymerization. 211 therefore, more detailed studies are needed to understand the functions of hsp70 in modulating rsv gene expression and replication. in term of ebola virus (ebov) replication, the mechanism of hsp70 involved is much more complicated. by using immunoprecipitation and mass spectrometry assays, it has been identified that the n protein interacts with hsp70, nef, bag2, and the hsp70 co-chaperone dnaja2. 212 the n protein recognizes and binds to the viral rna genome to establish a steady n protein-rna complex structure (rnp). this complex further interacts with viral proteins vp30, vp35 and rdrp l, to finally form vrc. here, hsp70 functions to maintain the stability of n protein and helps to facilitate vrc formation. 212, 213 in addition, hsp70 is also copurified with l polymerase in insect cells. 214 besides, hsc70 interacts with the terminal non-coding regions of the ebov genome. disruption of the interaction by mutating the binding site potently inhibits the minigenome replication of ebov. 215 another mechanism that hsp70 employs to support virus replication is to modulate nuclear import of polymerase or nuclear capsid. some rna viruses also replicate in the nuclear such as cdv and influenza virus. therefore, nuclear transportation becomes a critical step for their replication. upon cdv infection, hsp70 is shown a strong contribution to viral replication by interacting with and promoting the translocation of the nucleocapsid particles from the cytosol to nucleus. 216 similarly, during influenza infection, hsp70 interacts with pb2 or pb1 monomers and pb2/ pb1 heterodimer in hela and hek293t cells, and sequentially translocates into the nucleus with pb2 monomers or pb2/pb1 heterodimers. 217 if hsp70 and pb2/pb1 polymerases are retained in the cytosol, the polymerase activity reduces dramatically. the shuttling of hsp70 between nuclear and cytoplasmic compartments underlies the modulatory effect of hsp70 on influenza virus replication. 217 virus gene expression. besides viral entry and replication, hsp70 also contributes to viral protein translation. positive singlestranded rna viruses (e.g., sars-cov-2, hcv, zika, ev-a71, etc) use the internal ribosome entry site (ires) to initiate the translation of their own proteins but inhibit the host cellular cap-dependent translation through regulation or cleavage of eukaryotic translation initiation /elongation factors (eifs/eefs). in the case of coxsackievirus b3 (cva b3) infection, hsp70 is upregulated to enhance the initiation and elongation of viral translation. 218 in the translation initiation step, hsp70 upregulates ires-acting factor lupus autoantigen protein expression and activates eif4e binding protein 1 (eif4ebp1), a cap-dependent translation suppressor. in the elongation step, hsp70 activates the akt-mammalian target of rapamycin complex 1 (mtorc1) signal cascade, leading to activation of eef2 via kinase p70s6k-and cdc2-mediated phosphorylation and inactivation of eef2 kinase (ef2k). 218 hsc70 enhances the ires activity in ev-a71 infected cells. hsc70 interacts with 2a protease of ev-a71 to enhance eif4g cleavage that impairs host cell cap-dependent translation but enhances viral ires-mediated translation. 219 therefore, hsc70 may serve as an antiviral target against ev-a71 and hcv infections. some viruses do not have ires sequence, and virus replication produces plenty of dsrnas which trigger the activation of protein kinase-rna-activated (pkr)-eif2a signalling cascade that shuts down global translation in cells and releases stresses. 220, 221 to circumvent the pkr-mediated block to viral proliferation, influenza a virus induces the cellular tetratricopeptide repeat (tpr)domains containing jdp protein, p58ipk. influenza virus downregulates pkr in an hsp70-dependent way. [222] [223] [224] [225] [226] in uninfected and unstressed cells, p58ipk activity is clogged with hdj1 by forming a complex. 225, 226 during influenza a infection, the amount of hdj1 in p58ipk-hdj1 complex decreases to an undetectable level. these findings suggest that the activation of p58ipk appears to be a sequel to the hsp70-mediated release of hdj1 from the p58ipk-hdj1 complex, which allows the monomeric p58ipk to inhibit pkr. 225 in the hsp40 chaperone part, we would discuss more details about the regulation of pkr signaling by influenza virus. virion assembly. hsp70 is reported to assist some viruses' assembly. during morphogenesis of the double-stranded rna reovirus, hsp70 contributes to the assembly of trimeric sigma 1 protein, which is responsible for the interaction with host cell receptor. 227 while the n-terminal segment of the sigma 1 protein folds and trimerizes cotranslationally in an hsp70-independent manner, a post-translational fold of the c-terminal globular domain is dependent on hsp70. in this process, hsp70 binds cotranslationally to the region downstream the n-terminal αhelical coiled-coil, which presumably helps to inhibit unwanted interaction and misfolding. trimerization of the c-terminal domain of the sigma 1 protein is coupled to the atp-dependent release of hsp70 from the ribosome. 227 besides, in hela cells infected by poliovirus or coxsackievirus b1, hsp70 is detected to interact with the capsid precursor p1. 228 in the complex, p1 is mainly newly synthesized and has a longer half-life than that of total p1. the hsp70-p1 complex is regarded as an assembly intermediate of picornaviruses. 228 interestingly, some researchers demonstrate that hsp70 inhibits influenza virus replication by blocking nuclear export of viral ribonucleoprotein complex (vrnp), and subsequent viral morphogenesis via disassociating m1 from vrnp. 229, 230 virus release. the evidence of hsps on virus releasing is limited. both hsp70 and hsc70 can interact with ns5a protein; although they play different roles in hcv infection. 231 silencing of hsp70 decreases viral protein expression, but the virus protein level is not affected. 232, 233 instead, interfering hsc70 reduces extracellular virion production. 232, 233 moreover, hsc70 is embedded in the viral capsid. and co-localization between hsc70 and core and e2 structural proteins of hcv has been found in lipid droplets. therefore, hsp70 and hsc70 may regulate hcv infection release at different steps. the function of hsp70 family proteins in dna virus infection virus entry and genome releasing. instead of virus attachment and entry into the host cells, here we talked about virus entry into the cytosol from er. the cytosol entry from er is a key step in sv40 infections. hsc70 is reported to be essential in this step. hsc70 interacts with and is regulated by sgta. 234 further studies show that hsp70 superfamily member hsp105 forms a subunit in the hsc70-sgta complex to facilitate sv40 cytosol entry. 235 in addition, grp78/bip also plays a role in sv40 cytosol entry. grp78 interacts with sv40 capsid protein in a dnajb11-dependent manner to help sv40 disassemble and enter into the cytosol. 236 hsp70 is also needed for the genome release of some dna viruses. such a process is described in adenovirus infection. after the release of the virion from endocytic vesicles into the cytoplasm, hsp70 and hsc70 immediately attach to the hexon protein, one of the major adenovirus coat proteins. 237 hsp70/ hsc70 and its co-chaperone bag3 interact with the penton base protein, the viral capsid constituent responsible for virus internalization. 238, 239 the intact nucleocapsid is transported to nucleus through the typical nls-dependent nuclear import machinery. 240 the nucleocapsid anchors to the nuclear pore through its hexon protein by interacting with components of the pore complex. then viral dna is transferred into the nucleus in a hsp70-dependent manner but leaving hexon outside the nucleus because the purified hexon, instead of viral dna, enter the nucleus in a hsp70-independent manner. 240 a possible explanation is that the intact nucleocapsid is too large to pass through the nuclear pore complex, while the disassembly of nucleocapsid facilitates the entry of viral dna into nucleus. however, more solid evidence is needed for such an explanation. 238 other examples for the contribution of hsc70 in viral genome release in host nucleus are hsv and polyomavirus. in hsv-infected cells, the translocation of hsc70 from the cytosol to nucleus is triggered by the immediate-early viral protein icp0. hsc70 is colocalized with the components of the 26s proteasome and virus ul6 portal protein, which provides the conduit for dna entry and exit from the capsid. ul6 is highly ubiquitinated in the nucleus, indicating that hsc70 may be responsible for the correct folding and degradation of ul6 in the ubiquitin-proteasome pathway, though there was no direct evidence that ul6 is a substrate of hsc70. 241 it is also believed that hsc70 contributes to polyomavirus genome nuclear import through its interaction with all viral capsid proteins vp1, vp2 and vp3 both in vitro and in vivo. hsc70 translocates from the cytosol to the nucleus accompanied by the translocation of capsid proteins upon infection with polyomavirus. 254 gene expression and protein maturation. most viruses manipulate the cellular transcription and translation machinery and shut off host protein synthesis, so that they can take advantage of these machineries and recruit initiation and elongation factors for the expression of viral proteins. some host factors exploited by viruses interact closely with components of hsp70 complex. therefore, the chaperone system is highly important for viral gene expression. several transcription initiation factors are well known to physically interact with the hsp70 co-chaperone bag1 in vitro. bag1 stimulates general transcription activity in an hsp70dependent manner. [242] [243] [244] the stimulation of global transcription is detected in cells upon infections by either human polyomavirus, john cunningham virus (jcv) or hcmv. 245, 246 however, the detailed molecular mechanism of the general transcriptional activation by viruses is to be further investigated. the typical example of hsp70 system in regulating the maturation of viral proteins is shown in hbv large envelope protein (lhbsag). [247] [248] [249] the mature lhbsag has a unique dual transmembrane topology. initially, the c-terminal of lhbsag cotranslationally resides in er, while the n-terminus is resident in the cytosol and later is translocated into er for post-tranreported that the expression of grp78 is stimulatedslation. to ensure the correct topology, hsp70 system strictly regulates the post translocation of n-terminal of lhbsag. during the translation, hsc70 interacts with the n-terminal of lhbsag at amino acids 63 to 107 and suppresses lhbsag translocation into er. 248, 249 hsc70 cochaperones hip and bag1 also regulate the activity of hsc70 in an antagonistic way. 250, 251 overexpressed hip promotes hsc70 activity resulting in more cytosol retention of lhbsag n-terminal; 250 while bag1 overexpression could inhibit hsc70 activity to promote nuclear translocation. 251 in the post-translation process, grp78 binds with lhbsag and hsc70 to facilitate the er translocation of hbv large surface antigen. 247, 248, 252, 253 the function of grp78 is regulated by both positive regulator er-localized dnaj-domain-containing protein 4 (erdj4) and negative regulator bip-associated protein (bap). 253 increased bap destabilizes lhbsag/bip complex. 253 together, hsp70 chaperone system is crucial in modulating the sophisticated topogenesis of hbv envelope protein. 247 virus assembly. a lot of dna viruses normally assemble in the nuclei of infected cells. taking polyomavirus as an example, all capsid proteins are synthesized in the cytosol, whereas subsequent assembly of virions only takes place in the nucleus. during polyomavirus infection, the constitutive form of hsp70 and hsc70 are coimmunoprecipitated with all three viral capsid proteins (vp1, vp2, and vp3). hsp70 is translocated from cytoplasm to the nucleus in the late stage of infection, coincident with localization change of the viral capsid proteins. 254 in vitro studies show that hsp70 functions to keep proper assembly of polyomavirus. 255 the prokaryotic hsp70 chaperone dnak also interacts with recombinant vp1 at the c-terminal domain where it links pentamers in an assembled capsid. 255 when dnak binds to vp1, it inhibits vp1 assembly, which is induced by calcium in vitro. however, combining the hsp70 chaperone system including dnak, dnaj and grpe with vp1 together is sufficient to assemble vp1 into uniform capsids in the presence of atp alone without calcium. 255 the function of hsp70 family in retrovirus infection human t lymphotropic virus type 1 (htlv-1) is a well-investigated example of retrovirus that interacts with hsp70 proteins. during htlv-1 infection, syncytium formation is a key factor for cell-tocell virus transfer. the syncytium formation is subject to close cellto-cell interactions. 256, 257 cell membrane-resident hsc70 is necessary in this process as hsc70-specific monoclonal antibodies eliminates the syncytium formation and htlv-1 infectivity. the same outcome presents when cell is treated with a peptide derived from the htlv-1 glycoprotein gp46, which binds to hsc70. 258, 259 interestingly, although hsc70 enhances the syncytium formation, it has no effect on virus entry. 259 hsp70 also plays an important role in the post-entry steps. during human immunodeficiency virus type 1 (hiv-1) infection, viral protein r (vpr) stimulates interaction between the viral preintegration complex and karyopherin-alpha to facilitate viral nuclear import. hsp70 functionally overlaps with vpr in this process. 260 when vpr is deficient, hsp70 could rescue virus nuclear import by interacting with karyopherin-alpha at the n-terminal that also binds vpr. interestingly, some researchers argue for the antiviral role of hsp70 because hsp70 and vpr share the same substrate. it seems that hsp70 would compete and inhibit vpr function. since hiv-1 needs vpr to manipulate cell cycle and apoptosis, hsp70 neutralizes the function of vpr in hiv-1 infection. 261, 262 recently, hiv is observed to package hsp70 as part of virion core. the virion-incorporated hsp70 atpase activity and correct conformation of hsp70-virion are essential for hiv infection, since inhibition of hsp70 atpase activity interrupts the hsp70-virion core association and diminishes virus infectivity. 263 the effects of hsp70s on host cells upon dna virus infection cellular transformation. dna viruses those do not encode polymerase in their genome are dependent on host dna replication machinery. to replicate in quiescent cells, the virus has to reinitiate cell cycle thereby transforming the host cells. some mechanisms have evolved in enabling viruses to overcome the restriction points of cell cycle. the best-investigated example is sv40. the large and small t antigen (tag) are central for the sv40 transformation ability. at their n terminus, both types of tag contain the j-domain, the signature motif of an hsp70 cochaperone. mutation or deletion of the j-domain disrupts the functional association of tag with hsp70s. the ability of tag to transform mammalian cells is subsequently obliterated. 264 also, tag sequesters the retinoblastoma family proteins (prb, p107, and p130) and liberates members of the e2f family of transcription factors in a hsc70-atp hydrolysis-dependent manner. 265, 266 the free e2f family proteins subsequently trigger the expression of the s-phase genes leading to dna replication. 267 the above observations are interpreted in the following four steps. firstly, the large tag combines with prb-e2f complex. subsequently, the prb-e2f-tag complex associates with hsc70 in the presence of atp as hsc70 atp-bound form presents high substrate association rate to facilitate tag-hsc70 complex formation. the third step is that the j-domain of tag in the tag-hsc70 complex stimulates atp hydrolysis. this process is dependent on the active site of hsc70 and does not occur at the t-antigen binding site. meanwhile, hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. in the last step, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f. after adp dissociation and rebinding of atp to hsc70, e2f and the prb-tag complex is released from the hsc70-substrate complex. [268] [269] [270] [271] a second strategy of tag to transactivate e2f transcription is independent of the disruption of the prb-e2f complex that also involves the jdomain and hsp70 protein. [271] [272] [273] [274] tag functions like bag1 to assiste the assembly of a transcription initiation complex on the respective promoters in the presence of hsc70. alternatively, tag could induce hsc70 to disassemble an inhibitory silencer complex fig. 5 model for the participation of hsc70 in the tag induced disassembling of prband e2f. firstly, tag combines with prb-e2f complex to facilitate t-antigen-hsc70 complex formation. then tag-hsc70 complex stimulates atp hydrolysis, and hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. finally, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f to initiate cell cycle reprogram or to assist with remodelling the chromatin (fig. 5) . hpv and adenovirus have similar transforming activities by disrupting prb-e2f complexes. although neither e7 (hpv) nor e1a (adenovirus) protein contains a j-domain, both proteins could transform cells in a way similar to that described for sv40 tag. e7 interacts with tumor suppressor htid-1. 275 the c terminus of e7, which mediates the interaction with htid-1, is essential for the physical disruption of the prb-e2f complex though it is not necessary for direct interaction with prb. 276, 277 these observations suggest that the interaction with htid-1 is involved in the disruption of the prb-e2f complex, providing e7 with the jdomain necessary to recruit hsc70 for the complex dissociation in analogy to the function of sv40 tag. alternatively, the binding of e7 to htid-1 could transform cells through inhibition of the assumed tumor suppressor function of htid-1. e1a directly interacts with hsc70 to disrupt the prb-e2f complex. 278 in conclusion, most double-stranded dna viruses depend on hsp70 chaperones for the reprogramming of the host cells to reenter the cell cycle. cell survival and apoptosis. since hsp70 systems are essential modulators for cell survival under stress conditions, the induction of hsp70 protein facilitates virus infection by keeping the cell alive until mature viruses are ready to leave. this is the main reason why the disruption of apoptotic pathway is a quite common phenomenon in viral infections. [279] [280] [281] in the early stage of viral life cycle, viral reproduction is simply vulnerable to cell death. naturally, viruses are evolved in manipulating cellular apoptosis. in ebv-infected cells, nuclear oncoprotein ebna3a helps hsp70 nuclear translocation and hsp70 chaperone complex formation to immortalize b cells through inhibiting apoptosis. 282 in contrast, apoptosis is beneficial for virus spreading when virions are finally assembled. [283] [284] [285] [286] [287] [288] [289] [290] virions are found in apoptotic bodies and subsequently engulfed by phagocytic cells. it has been suggested that virus can infect neighbour cells without being detected by the host immune system. 291, 292 on the other hand, the decrease of hsp70 mrna level may lead to the timed induction of apoptosis at the late stage of adenovirus infections. innate immunity. hsp70 has been previously described to influence innate immunity and inflammatory responses. 293 it has been believed that hepatocyte is devoid of innate immunities. ma et al. reported that the expression of grp78 is stimulated by hbv replication. the elevated grp78 protein in turn activated innate immune response by induction of ifnβ expression. 294 further studies showed that hsp70 greatly contributes to cellular innate immunity in response to either virus or bacterial infections. in the course of bacterial infections, the elevated intracellular levels of hsp70 protect cells from lps-mediated inflammation. through its interaction with tnf receptor associated factor 6 (traf6), hsp70 can inhibit its ubiquitination and thereby block the activation of transcription factor nf-kb. 295, 296 weiss et al. has presented more details on this mechanism. hsp70 binds with ikk, leading to disturbing the function and stability of nf-κb/ikbα/ikk complex and further impairing ikbα phosphorylation. 297 the disturbance of this complex also affects ikbα proteasomal degradation and nuclear translocation of nf-κb complex. 298 the inhibition of nf-κb signalling has great therapeutic significance because it can prevent massive tissue damage medicated by the excessive inflammation response. a more recent study provides another approach for hsp70 to regulate inflammation. pierre et al. described an nf-κb independent pathway that hsp70 could impact nlrp3/asc inflammasome formation through its association with nlrp3. 299 aside from the anti-inflammation function of intracellular hsp70, the secreted extracellular hsp70 binds to dendritic cells and macrophages before being recognized by its binding elements, most of which are innate immune receptors. 293, 300 toll-like receptors (tlrs) detect virus invasion and immediately trigger intracellular innate antiviral response. 301 they belong to type i integral membrane glycoproteins of il-1 receptor (il-1r) superfamily. 302 it has been suggested that tlr2/tlr4 involved in the initiation of innate immunity by extracellular hsp70. hsp70 utilizes both tlr2 and tlr4 to transduce its proinflammatory signal in a cd14-dependent manner to promote proinflammatory cytokine production via myd88/irak/nf-κb axis signalling cascade. 8, 301 another study clearly demonstrated the function of tlr4 and its direct interaction with hsp70. 303 after ligand binding, tlrs dimerize and undergo a conformational change required for recruiting downstream signaling molecules, including the adaptor molecule myeloid differentiation primary-response protein 88 (myd88), il-1r-associated kinases (iraks), tgfβ-activated kinase (tak1), tak1-binding protein 1 (tab1), tab2 and tnf-receptor-associated factor 6 (traf6). [304] [305] [306] [307] many other innate immunity-related signaling pathways would then be activated, for example, phosphorylation of nf-κb via tak1/ikk activation. mapks p38, jnk, and erk pathway are also activated, then subsequently activate creb and ap-1 transcription factors. both ap-1 and nf-κb activate proinflammatory cytokine expression, including tnfα, il-6, il-1β, and a number of other cytokines and chemokines. 308-311 the effects of hsp60s on host cells upon rna virus infection immunity modulation. few studies show the function of hsp60s in the life cycle of rna virus; however, the function of hsp60 in regulating host immunity has been widely studied. [312] [313] [314] [315] [316] the majority of studies show that hsp60 works as an activator of immune response. in the case of jev infection, hsp60 facilitates virus-induced inflammation by promoting il-1β production via increasing nlrp3 inflammasome activity and nfκb phosphorylation. 317 however, under certain conditions, viruses also utilize hsp60 to evade host cell immune response. the genome-wide rna interference (rnai) screen identifies the interaction of hsp60 with pb2 of influenza virus. 318 hsp60 helps pb2 translocation from the cytosol into mitochondria. 38, 39 then the mitochondrial pb2 interacts with and modulates the activity of mitochondrial antiviral signaling protein (mavs) to suppress interferon β (ifnβ) production. 38 therefore, hsp60 determines the effect of pb2 on both mitochondrial stability and the level of ifn-β production. 38, 39 besides, denv infection also elevates hsp60 expression. silencing of hsp60 results in an increase of ifn-α production and decrease of virus reproduction in macrophages. 319 however, the detailed mechanism remains elusive. apoptosis regulation. viruses use different mechanisms to modulate apoptosis. in the case of hcv infection, ros production is regarded as the major contributor of hcc although it also promotes cell apoptosis. 320 study shows that the n-terminal domain of hcv core protein can induce ros production by interacting with hsp60 and inhibiting the normal function of hsp60 in releasing protein stress. 321, 322 while another rna virus, rotavirus sa11, tries its best to delay the early apoptosis through modulating hsp60 stability. 323 hsp60 helps to translocate nsp4 protein of rotavirus into mitochondria from cytosol and induces apoptosis. 323 to yield more viruses, rotavirus infection increases the hsp60 phosphorylation at tyr 228 by activated src kinase that leads to the ubiquitin-mediated proteasomal degradation. 323 even though virus postpones apoptosis via hsp60, the main function of hsp60 is to refold proteins in mitochondria. 323, 324 the function of hsp60 family in dna virus infection virus replication. in the previous sections, we have discussed that hsp90 is important for rt-ε rna complex formation in hbv infection. however, it has been shown that hsp60 directly regulates rt activity before the rt-ε rna complex formation. 325, 326 hbv replication is markedly suppressed when hsp60 is knocked down by specific sirnas. 325 hsp60 transiently interacts with rt to activate rt in an atp-dependent manner. 326 upon rt activation, hsp60 immediately dissociates from the hsp60/rt complex without being encapsidated into viral nucleocapsid. 326 more detailed research shows that at least one of two rt fragments, residues 1-199 of terminal protein (tp) domain and 680-842 of rnase h (rh), is necessary for hsp60 binding. 327 the tp domain is also responsible for the binding of hsp90 in the rt-ε rna priming step. 138 apoptosis regulation. similar to hcv infection, hbv infection also induces strong apoptosis which is thought mainly contributed by hbx protein. 328 studies show that overexpression of hsp60 facilitates hbx-induced apoptosis. 329, 330 the interaction of hbx and hsp60 has been observed and confirmed by different methods including affinity purification, mass spectrometry and co-immunoprecipitation. 329 hsp60 binds on a small domain (residues 88-117) of hbx. 329 furthermore, hsp60 also forms a complex with hbx and hsp70 in the mitochondria. 330 however, the mechanism of how hsp60 enhances the apoptosis remains unclear during hbv infections. immunity modulation. hsp60 is involved in both the innate and adaptive immune response. 314 here, we focus on how hbv harnesses hsp60 to evade host immune response. numerous studies show that hbv employs active means to escape innate immune response and induce immunosuppression. 331 among these strategies, hbv infection increases the population of cd4 + cd25 + t regulatory cells (tregs) which can produce an amount of il-10 and tgf-β. 332 il-10 is also called cytokine synthesis inhibitory factor (csif) displaying anti-inflammatory properties. 333 it influences both the first and the second line of immune defence. 334 hbv infection increases serum shsp60 level and makes use of hsp60 to activate cd4 + cd25 + regulatory t via tlr2/myd88/il10 signaling. 335 the function of hsp60 family in retrovirus infection although the role of hsp60 in dna/rna virus infection process has been widely studied; only limited knowledge has been obtained in retrovirus infection. hsp60 is encapsidated into hiv particles, 336 but we have no idea about its function. the integrase catalyzes the integration of hiv-1 pro-viral dna in the host genome. 337 a small portion of hsp60 colocalizes and interacts with the viral integrase (in). 338 hsp60-hsp10 complex maintains the integrase at an active form and stimulates its activity in an atp-dependent manner. 338 the integration is a critical step for successful infection of hiv-1. the function of hsp40 family in rna virus infection virus replication. hsp40s regulate rna viral replication by modulating polymerase activity, replication complex and nuclear transportation. in jev-infected cells, hsp40/dnaj homolog hdj2 interacts and colocalizes with ns5 protein, an rdrp essential for viral rna genome replication. 339 overexpressed hdj2 promotes jev replication significantly. however, how hdj2 modulates ns5 activity remains elusive. 339 influenza virus also takes advantage of hsp40 to promote replication by assisting vrc to relocate into nucleus. the replication of influenza virus occurs in the nucleus of host cells; therefore, nuclear trafficking of viral ribonucleoprotein (vrnp) complex is required. the vrnp is composed of viral rna (vrna), polymerase heterotrimer (pa, pb1, pb2) and nucleoprotein (np). 340 np has a key function of interacting with importins through its nuclear localization signals. 341, 342 hsp40s have two strategies to help vrnp transport into nucleus. first, hsp40/ dnajb1 interacts with np at the early stage of infection and ensures efficient association between np and importin alpha. 343 this interaction is mediated by the j domain of hsp40 and the nterminal region of np. 343 another strategy is that hsp40/dnaja1 binds with the pb2 and pa polymerase subunits, then cotranslocates into nucleus with pb1-pa complex. 344 besides, hsp40/dnaja1 enhances viral rna synthesis both in vivo and in vitro. 344 different from dnajb1, dnaja1 mainly depends on its c-terminal substrate-binding domain instead of typical j domain to manage viral rna synthesis. 344 in the replication process, hsp70 is reported to enhance polymerase nuclear translocation. 217 thus it is proposed that dnaja1 cooperates with hsp70 and assist rna polymerase nuclear import. 217, 343 virus gene expression. viral rna replication produces plenty of double-stranded rna (dsrna) molecules. host cell detects these dsrna and activates interferon-induced protein kinase (pkr) to restrict viral replication by phosphorylating eukaryotic initiation factor eif2α and preventing protein synthesis. 220, 221, 345 however, in order to escape from the antiviral response of host cells, the influenza virus smartly blocks the activation of pkr/eif-2α. influenza virus ns1 directly binds the n-terminal of pkr and inhibits pkr activation. 225 besides, np protein also interacts with hsp40, leading to dissociation of hsp40 and p58ipk. 346 it is reported that type iii hsp40/p58ipk is an inhibitor of pkr, [222] [223] [224] 226, 347 while hsp40 is the inhibitor of p58ipk. 225, 348 therefore, the dissociation of hsp40 results in activation of p58ipk. subsequently, p58ipk inhibits the activity of pkr/eif-2α. as a result, influenza virus releases the inhibition of protein synthesis. however, in the late stage of infection, influenza virus m2, hsp40 and p58ipk form a stable complex that would lead to pkr activation, er-stress-induced cell death and virion release. 349 protein maturation. flavivirus genome encodes a large polyprotein which is later cleaved into several mature structures and nonstructure proteins. the mature proteins then form vrcs. at the beginning, hsp40 family protein dnajc14 participates in the vrc formation of flavivirus. during yellow fever virus (yfv) infection, dnajc14 is recruited to non-structural protein clustering sites with ns3 and ns5 to form vrc. 350 however, either knockdown or overexpression of dnajc14 inhibits yfv and hcv replication. 350, 351 later, it has been demonstrated that dnajc14 overexpression affects yfv polyprotein processing and alters vrc assembly. overexpression of dnajc14 alters the cleavage sites of ns3/4a and ns4a/2k and gives rise to abnormal ns3 to ns3-4a ratios, suggesting that the chaperone activity of dnajc14 modulates ns3/ 4a/2k cleavage that ensures appropriate expression level of ns3 and ns4a. the inhibition of vrc formation upon ectopic expressing dnajc14 is caused by chaperone dysregulation. 351, 352 immunity modulation. hsp40s sometimes act to help the virus evade host immunity, while sometimes it exhibits antiviral activity by increasing host immune response under certain conditions. it is reported that the expression of dnajb1/hsp40 and hsp70 is induced by polyi:c stimulation. 353 hsp40 cooperates with hsp70 to suppress the mda5/mavs pathway though interacting with mda5 and inhibiting mda5 multimer formation. 353 however, dnaja3 shows its ability to suppress virus replication. during hfdv infection, vp1 is able to suppress the type i interferon signaling via suppression of phosphorylation, dimerization, and nuclear translocation of irf3. 354 however, dnaja3 induces lysosomal degradation of vp1 protein. therefore, dnaja3 indirectly stimulates the immune response of host cells. 354 the function of hsp40 family in dna virus infection virus replication. evidence suggests that hsp40s regulate the initiation of dna virus replication. in the case of hpv replication, it starts with the recognition of protein e2 on the origin (ori) sequence and recruitments of replication initiator e1, which displays atpase and helicase activities. [355] [356] [357] hsp40 (hdj1 and hdj2) and hsp70 enhance e1 binding on the ori independently and additively. hsp40 directly binds with e1 and remains in the e1ori complex, whereas hsp70 transiently interacts with e1 in an atp-dependent manner. 355 subsequent study reveals an additional role of hdj2 in facilitating e1 helicase function by replacing e2 in the e1/e2/ori complex. 358 the stable association of e2 to ori flanking the e1 binding site may act as a dna clamp to prevent dna unwinding. similarly, hsp40 (htid1) also has similar functions to hdj1 and hdj2 with independent chaperone activity. hsp40 interacts with hsv-1 replication initiator protein and helicase protein ul9, thereby promotes their binding to the replication origin. 359, 360 this provides another example of the involvement of j-proteins in the replication process of eukaryotic dna viruses. except for enhancing hbv replication, a possible negative role of hsp40 has also been reported. the core protein is a key component of viral capsid and essential for virion assembly, while hbx is a multifunctional virulence factor implicated in viral replication and hepatocarcinogenesis in human. a yeast 2-hybrid approach is used to identify interactions between the core protein and two hsp40s, hdj1 and htid1. 361, 362 individual expression of each hsp40 in hepatocytes transfected with a replicationcompetent hbv construct shows an inhibitory effect on both viral replication and capsid formation. further studies reveal that both core and hbx proteins are destabilized by co-expression with hdj1 or htid1; because they are targeted for enhanced proteolytic degradation. except inhibiting hbv replication, hdj1 also facilitates proteasome-mediated degradation of hbx. 361 virus induced cellular transformation. as mentioned above, some viruses can induce cell transformation and tumorigenesis. here, we discuss the role of hsp40 proteins on virus-induced cellular transformation. hsp40 may have a negative role in hbv-induced cell transformation. it has been demonstrated that hbx is the major factor for induction of hepatocyte transformation. [363] [364] [365] [366] nevertheless, overexpression of hsp40s (hdj1 and htid1) significantly enhance the proteasome-mediated degradation of hbx. another study suggests that htid1 interacts with e7 oncoprotein and promotes the cellular transformation by hpv16; 275 because the binding sequence of e7 shares high similarity to the tag oncoproteins of sv40. nuclear entry of viral pre-integration complex. hiv can efficiently infect nondividing cells. 367 this requires active transport of the viral pre-integration complex (pic) into the nucleus without breaking down nuclear membrane. some components of pic implicated in regulating nuclear import include the central dna flap and viral proteins in, ma, and vpr of hiv-1 (or vpx of hiv-2). 288,368-372 hsp40/dnajb6 interacts and enhances the nuclear localization of vpx as well as promotes the nuclear import of viral pic. 373 similarly, dnajb6 also promotes vpr nuclear localization during hiv-1 infection; 374, 375 particularly, the long isoform of dnajb6 is extremely important in this process. 375 the expression level of dnajb6 s/l isoform is regulated by the polyadenylation factor cstf64. high level of cstf64 favors dnajb6-s isoform production, whereas a low level of cstf64 enhances dnajb6-l isoform production. 374 regulation of viral gene expression. a notable example of hsp40 involvement in regulating retrovirus gene expression is its importance in enhancing the gene expression during hiv-1 infection. 376 nef protein, an important viral protein associated with pathogenesis and disease progression, stimulates hsp40 expression by enhancing hsp40 promoter activity via hsf1 transcription factor. [377] [378] [379] hsp40 and nef co-translocate into nucleus where they become a part of cdk9-associated transcription complex to enhance long terminal repeat (ltr) mediated gene expression. 376, 380 the binding of hsf1 on hiv-1 ltr promoter induces viral gene expression directly. 380 interestingly, hsp70 seems to act contrary to hsp40, which also presents in the nef-hsp40 complex. different from hsp40, hsp70 suppresses viral replication and gene expression; while hsp40 rescues the hsp70downreguled viral gene expression. hsp70 inhibits cdk9 phosphorylation, an essential event for high-affinity binding of hiv-1 transactivator of transcription-positive transcription elongation factor b complex for transactivating response rna. 381 it is also reported that some other hsp40 family proteins negatively regulate hiv replication. hsp40a1, b1, b6 and c5 (but not c3) are able to limit hiv-1 production, while they have no effect on viral gene expression upon infection by adenovirus, hsv-1 or vaccinia virus. the conserved dnaj domain is suggested to be responsible for the inhibiting hiv-1 reproduction. the hsp70/ hsp40 complex specifically recognizes and inhibits the rev translation or accelerates its degradation, leading to inhibiting viral gene expression. 382 small heat shock proteins are the most upregulated proteins identified in host cells under stress conditions, for example, when cells are exposed to elevated ros level, abnormally high temperature, or pathogen invasions. 383 in most cases, shsps are responsible for recognizing misfolded proteins and transferring them to other atp-dependent chaperones for proper folding, or proteasomes or autophagosomes for degradation. 49, 384 hsp27 is one of the most ubiquitously expressed shsps with the highest level in skeletal, smooth, and cardiac muscles. 59, 385 like all other shsps, hsp27 shares a highly conserved α-crystallin domain, or socalled c-terminal domain. it contains 6-8 β-strands, forming 2 β-sheets as intermolecular interaction sites. 41, 59 because of the importance of hsp27, in this section, we mainly focus on the function of hsp27 in virus infection. hsp27 has been shown particular importance in viral infections. rajaiya et al. suggested that the association of hsp27 with p38 or nfκb/p65 plays key roles in controlling the expression of proinflammatory mediators in virus-infected cells. 64 fukagawa et al. suggested that the phosphorylated hsp27 is upregulated through the pi3k/akt pathway upon ebv infection. 386 we would discuss the special roles of hsp27 in different viral infections in the following sections. the function of hsp27 in rna virus infection hsp27 is upregulated during ev-a71 infection by proteomics analysis. knockout of hsp27 results in the suppression of virus replication and protein expression level, while their restoration appears after hsp27 is restored. also, hsp27 enhances viral ires activity by promoting 2a pro -mediated eif4g cleavage. 57 interestingly, the nuclear translocation of hsp27 from cytosol is reversely correlated with the relocalization of rna chaperone hnrnpa1 from nucleus to the cytosol for initiating viral protein translation upon ev-a71 infection. however, knockout of hsp27 blocks hnrnpa1 cytosol relocolization, indicating a fundamental role of hsp27 in regulating the import/export of nuclear proteins during virus infection (fig. 6) . 383 hsp27 is rapid upregulated at the early stage (4 hours post infection) of coronavirus infection, 387 suggesting an important role in virus early replication and possibly a good target for treating sars-cov-2 infection. swine fever virus (csfv) is a member of the family flaviviridae. hsp27 is found to bind with ns5a, which is a non-structural protein in response to viral replication and assembly. hsp27 depletion enhances the virus replication while the replication is suppressed by ectopic expression of hsp27 through activating nf-κb signaling in pk-15 cells. 388 porcine epidemic diarrhoea virus (pedv) infection causes high fatality in swine. the hsp27 is obviously upregulated in pedv infected marc-145 cells. 389 however, the virus titer is declined by ectopic expression of hsp27. hsp27 could activate nf-κb signalling and enhance the transcription of ifnβ and downstream interferon stimulated genes (isgs). 389 the function of hsp27 in dna virus infection pro-inflammatory cytokines play crucial roles in early antiviral infections. adenovirus infection causes a wide variety of diseases. 390 the internalization of adenovirus leads to the activation of erk1/2, which could in turn trans-activate nf-κb and ap-1 to induce pro-inflammatory cytokine il-8 expression in different experimental systems. [391] [392] [393] a study shows that downregulation of hsp27 leads to an increased release of il-8 from keratinocytes stimulated with uv or tnfα. the increase of il-8 is suppressed by nf-κb inhibitor and correlated with an enhanced iκb-α degradation and phosphorylated iκb-α accumulation in hsp27-depleted cells. in addition, hsp27 is shown to be associated with the iκb kinase (ikk) complex. because the synthesis of prostanoid pge2 and il-8 is regulated by nf-κb, it could be a likely mechanism of hsp27 in modulating inflammatory cytokine production. 70 further studies also show that hsp27 is of particular importance in the cyclooxygenase-2 and il-6 expression via activating p38 mapk/mk2 signalling and the consequential stabilization of cyclooxygenase-2 and il-6 mrnas. 68 aside from its involvement in pro-inflammatory response regulation, researches have also found the antioxidant role of hsp27 in regulating stress caused by ros. 49 it is commonly agreed that hsp27 regulates enzyme activities by upholding glutathione in reduced form, such as glutathione reductase and glucose-6phosphate dehydrogenase. 394 more recent evidence correlates the level of shsps with the intracellular content of iron, a catalyzer of hydroxyl radical generation. hsp27 also exhibits an important role in oxidized protein degradation machinery. 395, 396 however, there is also controversial evidence indicating the involvement of hsp27 in accumulation of oxidized proteins that benefits herpesvirus replication. experimental models are set up using two distantly related herpesviruses rhesus rhadinovirus (rrv) and hsv-1. they are close relative of kaposi's sarcoma-associated herpesvirus (kshv). the oxidized proteins are accumulated during these viral infections. results show the removal of only a part of oxidized proteins in a proteasome-dependent manner, while some others resisting degradation. 397 oxidized proteins resisting proteolysis become sequestered in foci within the nucleus and coincided with hsp27-enriched foci; although they are not associated with virus-induced chaperone enriched domains (vice). furthermore, the accumulation of oxidized proteins is more pronounced in hsp27-depleted cells. 398 one possible explanation is that hsp27 buffers the toxic effects of those defective proteins undergoing proteolysis through aggregation in the nucleus. the roles of hsp27 are most likely not mutually exclusive during virus infection. hsp27 also contributes to virus replication. porcine circovirus type 2 (pcv2) is a single-stranded dna virus that causes the postweaning multisystemic wasting syndrome (pmws) in pigs. the phosphorylated hsp27 is upregulated in the nucleus in pcv2infected pk-15 cells. hsp27 inhibitors such as sb203580 suppress pcv2 replication. the same result appears upon hsp27 knockdown. in contrast, ectopic expression of hsp27 promotes viral replication. 399 moreover, the phosphorylation of hsp27 is also upregulated in ebv-positive cells, as well as the phosphorylated (activated) akt levels. when ebv-positive cells are treated with pi3k inhibitors, the phosphorylated hsp27 level is decreased, suggesting that the phosphorylation of hsp27 is upregulated through the pi3k/akt signalling pathway upon ebv infection. 386 however, studies also show that hsp27 can both positively and negatively regulate the virus replication depending on the virus/ cell types. tong et al. reported that hsp27 works as an antiviral protein against hbv replication through enhancing ifn production in hepatocytes. the hsp27 expression level is increased in both hbv-infected human liver tissues and hbv-producing hepg2.2.15 cells. 400 the function of pdis on virus entry and uncoating the entry of some viruses into eukaryotic cells is governed by redox-regulated processes. one example is newcastle disease virus (ndv), a bird virus in the family of paramyxoviruses. this negative-sense, single-stranded rna paramyxovirus gains entry to its host cell through large conformational changes in its fusogenic f-protein, which involves thiol/disulfide exchange. 401 overexpression of pdi and erdj5 (a pdi family reductase with an extra j domain) leads to an increase of viral membrane fusion, indicating a route whereby virus can take advantage of the pdi family to gain access to host cells. 402 fig. 6 the controversial function of hsp27 during virus infection. hsp27 would enhance 2a protein-mediated eif4g cleavage, which would in turn promote ires function and viral genome replication. it correlates with hnrnpa1 translocation from host cell nuclei to cytosol, and the consequential virus protein translation. hsp27 is also able to suppress apoptosis via caspase 3 inhibition. on the other hand, hsp27 could help activating ikk complex, modulating nf-κb and ap-1 to induce the expression of pro-inflammatory cytokines reduces β1 and β3 integrins allowing denv entry. 403, 404 also, thiol blockers and pdi inhibitors decrease the entry of rotavirus in ma104 cells, indicating the involvement of thiols for infectivity. 405 the entry of hiv is regulated by pdis. the envelope of hiv becomes unhinged by pdi for entry. ryser et al. firstly reported that cleavage of two disulfide bonds in the gp120 surface component of the hiv-1 envelope is required for virion entry into cd4 + cells. in this process, the pdi on cell surface is responsible for this effect. 406 pdi inhibitors sufficiently prevent the reduction and block the cleavage of surface-bound disulfide conjugate, thereby prevent infection at the level of hiv-1 entry. 407 now, there are numerous studies on how envelope binds on host cells. results from different groups have demonstrated that gp120 moves laterally along the membrane surface until it collides with a patch of pdi in a domain of the membrane that distinguishes from a typical lipid raft. pdi reduces two disulfide bonds in gp120, producing conformation changes that likely stabilize the binding of gp120 to cd4 and expose the v3 loop for subsequent binding to the chemokine coreceptor. following this, gp41 undergoes rearrangement into its fusogenic intermediates and entry occurs. 408, 409 during the entry, galectin-9 binds pdi to regulate the redox environment on the cell surface and enhance hiv entry. 410 taken together, the reports from these three groups have profound implications for our understanding of the hiv virion surface structure and viral entry. the other examples are the nonenveloped polyomavirus (py). py particles contain a layer of coat protein vp1. this single protein, arranged as 72 pentamers, forms the shell surrounding the viral genome. 411 after internalization, py penetrates the er membrane to gain access to the cytosol and then the nucleus for viral genome transcription and replication. pdis isomerize or reduce the virus disulfide bonds to generate a membrane transportcompetent intermediate for host cell membrane penetration. 412 for example, sv40 entry involves caveolar/lipid raft-mediated endocytosis, vesicular transport to er and translocation into the cytosol. erp57 isomerizes the interchain disulfides connecting vp1 for virus uncoating. 413 the further study demonstrates that erp57 and pdi operate in concert with erp29 to unfold the vp1 c-terminal arm. pdi and erp72 reduce py, while erp57 principally isomerizes the virus in vitro. mutagenesis study subsequently identified that the residues c 11 and c 15 of vp1 are important for infection, suggesting a role for these residues during isomerization. 414 pdis regulate viral protein translation a little evidence shows that pdis are involved in viral protein translation. some positive single-stranded rna (+ssrna) virus depends on ires-mediated translation for viral protein synthesis. ev-a71 infection is potently inhibited by an active compound oblongifolin m (om), which is isolated from herb garcinia oblongifolia. further studies show that om suppresses the viral ires-mediated translation of polypeptide via suppressing erp57, and ectopic expression of erp57 increases the ires activity and partially rescues the decreased viral replication caused by om treatment. 415 the detailed mechanism how erp57 downregulates ires activity would be further investigated. several studies have demonstrated the implication of redox balance disruption in the establishment of viral infection and the progression of virus-induced diseases. and accumulated ros in turn may modulate the viral replication and cellular response that also contribute to viral pathogenesis. 416, 417 virus-induced oxidative stress has been reported during hiv, 418 influenza virus, 419 hbv, 420 hcv, 421 encephalomyocarditis virus (emcv), 422 respiratory syncytial virus (rsv), 423 and jev 424 infections. considering its thioredoxin-like sites, erp57 has been thought a main player in the mechanisms of cell protection against oxidative stress, regardless of its subcellular location. redox proteomics analysis of hpv positive tissues shows that the expression level of erp57 and gst is positively correlated with tissue redox status, suggesting its potential association with viral-induced oxidative dna and protein damages. 425 er stress is another consequence caused by virus activities. viral infection would lead to exploitation of the er membrane, accumulation of misfolded proteins, and imbalance of calcium concentration. influenza a virus (iav), hbv, jev, denv, and zika virus all hijack host cell process to enhance viral pathogenesis, such as facilitating viral folding and trafficking, affecting receptor interaction, and modulating host immune responses. [426] [427] [428] therefore, as a major factor in er stress response, erp57 would be critical for viral protein glycosylation and maturation, which may in turn affect virus release and infection (fig. 7) . the life cycle of most rna viruses is completed in the cytoplasm of host cells. to complete the life cycle, virus is often able to induce redistribution of many host cell proteins; particularly, those proteins involved in rna metabolism and functional regulation of viral rnas. hnrnps, such as hnrnp a1, 429 hnrnp c1/c2, 430 hnrnp d 431 and hnrnp i, 432 are mainly resident in the nuclear but quite often shuttle to cytoplasm for stabilizing the viral rna and initiating cap-independent translation. viruses employ various means to redistribute hnrnps. for instance, the ebov inhibits the nuclear import of hnrnp c1/c2. vp24 binds to npi-1 subfamily karyopherin-alpha nuclear import proteins at the c-terminal region (amino acids and prevents their interaction with tyrosine-phosphorylated stat1 (pstat1) and hnrnp c1/c2. the inhibition results in cytoplasm retention of pstat1 and hnrnp c1/c2. 430 435 however, the detailed mechanism of how 2a pro and core protein induce the redistribution remains unknown. virus replication. it has been well documented that hnrnps greatly contribute to the virus replication. for example, hnrnp i/ ptb and hnrnp c participate replication of different viruses, e.g., coronavirus, 436 hcv 437 and poliovirus. 438 the role of hnrnp i/ptbs in viral rna synthesis seems to vary among different viruses. ptbs bind ucuaa pentanucleotide repeats at ires and 3′-utr. ptbs modulate coronavirus rna synthesis. 436 however, the function of hnrnp i/ptb is to some degree complicated in hcv rna replication. it selectively interacts with the 3′ end of the hcv genomic rna. the upstream sl2 and sl3 stem-loop structures are essential for hnrnp-i/ptb i binding whereas the most 3′-terminal stem-loop sl1 is not needed. hnrnp i/ptb i and hnrnp c specifically bind the pyrimidine-rich region of in the 3′ntr of hcv rna genome. 437 possibly, hnrnp i/ptb i is recruited for helping to initiate viral negative-strand (−) rna genome replication, to stabilize rna genome, and/or to regulate the encapsidation of genomic rna. 439, 440 up to date, the detailed function of hnrnp i/ptb i binding on 3′utr has not been elucidated. some studies showed that ptbs have an inhibitory effect on the synthesis of hcv rna genome, 441 while aizaki et al. reported that ptbs are required for efficient hcv rna replication. 442 it has been postulated that they suppress the initiation of viral genome rna replication but enhance ires-mediated translation or facilitate the replication-translation switch. it is shown that hur can compete hnrnp i/ptb i binding on 3′ utr of the viral rna to facilitate la binding to the 3′ utr; while la protein is critical for hcv genome replication. [443] [444] [445] although hnrnp c has many similarities with hnrnp i and both bind with the 3′utr of the hcv rna genome to facilitate viral rna replication; 437 more details demonstrate that only hnrnp c binds the 3′-ends of viral rnas with both negative and positive polarities. 446 besides, hnrnp c also binds at the 5′-end on negative-strand rna of poliovirus to facilitate the synthesis of positive-strand rna; 438 while mir-555 decreases the expression of hnrnp c thereby inhibits poliovirus replication. 447, 448 virus rna splicing. the main function of hnrnps is modulating rna process and metabolism, including splicing, stabilization and transportation. it has been documented that hnrnp k helps the rna splicing of iav. iav is a major human pathogen with a genome comprised of eight negative-stranded rna segments. two viral rna segments, ns1 and m, undergo alternative splicing and yield several proteins including ns1, ns2, m1, and m2 proteins. two of the influenza virus rna segments generate spliced products: ns segment codes for non-structural protein ns1 and nuclear export protein nep/ns2; m segment encodes the matrix protein (m1) and ion channel (m2). ns1-bp properly splices the viral m1 mrna segment to yield the m2 mrna without affecting the splicing of m4 mrna or ns mrna segments. in this process, hnrnp k works as a mediator to bridge the interaction of ns1-bp (binding protein) and m mrna. lack of neither ns1-bp nor hnrnp k ensures the proper splicing of m mrna. 449, 450 further studies show that ns1-bp and hnrnp k bind m mrna downstream of the m2 5′ splice site (5′ ss). ns1-bp binds most proximal to the 5′ss, partially overlapping the u1 snrnp binding site, while hnrnp k binds further downstream and promotes u1 snrnp recruitment. mutation of either or both the hnrnp k and ns1-bp-binding sites results in m segment mis-splicing and attenuated iav replication. 451 virus translation. virus rna translation often harnesses host cellular proteins. most hnrnps positively regulate virus translation. positive-sense single-stranded rna viruses normally contain an ires sequence that drives viral protein translation independence of the cellular cap-dependent translation. hnrnps bind with the ires of different viruses to assist their translation. during hcv virus infection, hnrnp i, hnrnp l, hnrnp d, [452] [453] [454] hnrnp a1 and hnrnp k 455 all participate the translation of hcv viral protein by binding with 5′utr sequence. hnrnp i (ptb3), is one of the polypyrimidine tract-binding proteins (ptbs) that has been reported to bind viral rna recurrently. they could bind the ires of picornaviruses, including cardio-aphthovirus group (including emcv, ev-a71) and entero-rhinovirus group (poliovirus and hrv-2). 89, 456 ptbs also bind hcv utr regions, 441 calicivirus rna 457 and coronavirus rnas. 436 for picornavirus and hcv, it is proposed that ptbs could help ires folding into a translation-competent structure. 458 although unlike canonical transient interactions shared by rna chaperones, it is not clear whether ptbs are eliminated after the ires has folded properly. 459 ptb binds the 5' utr of hcv rna genome to initiate translation. in translation assays, ptb antibody efficiently blocks the ires-mediated translation in vitro. 460 three rrm motifs of ptb monomer directly bind with ires of fmdv to stabilize ires structure and enhance eif4g entry to ires. 459 hnrnp l is capable of binding the 3′ border of the ires. [461] [462] [463] hnrnp l binds with single stranded-hcv rna when preannealing singlestranded rna with mir-122. 463 hnrnp d, also referred to as aurich element rna-binding protein 1 (auf1), shuttles between the cytosol and nucleus. it is found that hnrnp d could also interact with the stem-loop ii of hcv 5′ utr, and its overexpression enhances hcv ires-dependent translation. 452 pim1 inhibitors induce hnrnp d relocalization from nucleus to cytosol so that it binds the ires and inhibit ev-a71 protein translation. 122 similarly, hnrnp k interacts with sl1 located in the 5′ -utr of hcv genome where is bound with mir-122 binding site. 464, 465 mir-122 is required for hcv replication. it binds at a conserved sequence in the 5′ -utr and increases the stability of hcv rna. [466] [467] [468] it is also reported that residues 25-91, a hydrophilic region near the n terminus of hcv core protein, binds to proline-rich domains of hnrnp k and negatively regulates the effect on human thymidine kinase transcription. 469 however, its function on viral replication is not well addressed. while hnrnp a1 binds with both 5′-and 3′-utr of hcv rna, they form a complex with ns5b and septin 6 to assist viral protein translation. the c-terminal of hnrnp a1 and n-terminal of septin-6 are required in the translation process. since hnrnp a1 has many homologous such as hnrnp a/b, hnrnp a2/b1, and hnrnp a2; all of them may substitute for hnrnp a1 in regulating ires-mediated translation. 470 the enteroviruses' ires sequence also interacts with hnrnps. hnrnp a1 specifically binds on the 5′-utr of ev-a71 and enhances ires-dependent translation. 471 apigenin, a dietary flavonoid, interacts with hnrnps and interferes with their rna editing activity. 472 the binding of hnrnp a1 with viral rna is significantly blocked when the cells are infected with ev-a71 upon treating with apigenin, leading to marked suppression of iresmediated translation. it is noted that hnrnp a1 redistribution is not affected in this experiment. recent studies show that hnrnp a1 cytoplasmic translocation is strictly regulated by some signalling or stress proteins, such as mink/p38 mapk pathway 473 and hsp27. 57 p38 inhibitors or hsp27 knockout dramatically block hnrnp a1 translocating from nucleus into cytosol, leading to inhibition of virus replication. 57, 473 except for promoting viral protein translation by binding with the viral ires sequence, hnrnps can directly bind with virus proteins to facilitate virus replication. upon cvb3 infection, 3c pro binds and cleaves hnrnp m to facilitate virus replication. 474 however, hnrnp m does not affect ires activity and rna stability. 475 hnrnp k is required for denv replication. 476 the core protein of dnev interacts with hnrnp k to release the inhibitory effects of hnrnp k on transcriptional activator c/ebpb. 477, 478 other studies demonstrate that hnrnp c1/c2 interact with viral rna and ns1 protein of dnev and facilitate virus reproduction. [479] [480] [481] [482] hnrnp d binds on both 5′-and 3′-utr of enteroviruses and inhibits translation without affecting rna decay. 434 on the other hand, virus also applies strategy to release the inhibitory effects of hnrnp d via protease 3cd cleavage. 434 the cytoplasm translocation of hnrnp d is dependent on the expression of 2a pro and viral rna replication. 433, 434 hnrnp d also inhibits nucleocapsid expression by interacting with an au-rich sequence (nt 41 to 60) in the 3′ utr of niv. 483 virus rna export. hnrnp a2/b1 interacts with ns1 of influenza virus, leading to decrease of the viral ns1 rna/protein levels as well as ns1 rna nuclear export. 484 rna polyadenylation. the gag gene of rous sarcoma virus contains a cis-acting negative regulator of splicing (nrs) element. the general function of nrs is usually manifested by binding serine/arginine-rich (sr) protein hnrnp h and u1/u11 snrnps, resulting in inhibition of splicing by acting as a decoy 5′ splice site. evidenced by in vitro polyadenylation analysis, a new function of nrs element is revealed that it is required for 3′ ltr polyadenylation. in this process, hnrnp h binds on nrs element and promotes polyadenylation; however, mutation of the binding sites of u1 and u11 snrnps to the nrs does not affect polyadenylation. 485, 486 an opposite result shows that polyadenylation stimulatory activity of nrs is dependent of u1 and/or u11 snrnp as well as sr proteins; while hnrnp h seems not participating in the splicing control or rous sarcoma virus polyadenylation. 487 the functions of rna chaperones in regulating viral activities of dna viruses virus replication. the transient lytic dna replication of hcmv relies on the cis-acting element orilyt, six viral-encoded core proteins, the proposed dna replication initiator protein ul84, ie2, irs1 and the gene products from the ul112/113 loci. here it is reported that hnrnp k is sufficient to interact with ul84 protein of hcmv thereby promotes viral replication. the interaction is further enhanced by another two virus proteins ul44 and ie2. 488 gene expression. hnrnps regulate dna virus gene expression at both transcriptional and post-transcription levels either positively or negatively. at the transcription level, hnrnp d0b and hnrnp a/b are capable of binding with cis-acting aagtatgca core element of hpv18 late promoter to suppress the late genes l1 and l2, which are components of virus capsid proteins. 425, 489 hnrnp k binds enhancer ii (enii) to promote hbv replication. ectopic expression of hnrnp k augments hbv replication; while hnrnp k knockdown significantly decreases hbv viral load. 490 further study reveals that apobec3b forms a super complex with hnrnp k that alters the enii binding activity via conformational change, therefore suppresses the s gene promoter activity. 491 in the course of hsv infection, the immediate early protein ie63 (icp27), hnrnp k and casein kinase 2 (ck2) together form a big complex. ck2 is capable of phosphorylating hnrnp k and icp27. the phosphorylated icp27 is responsible for its nucleocytoplasmic translocation and interaction with hnrnp k. up to date, the function of the complex formation is not well elucidated. 492, 493 it may affect icp27 to recruit the cellular rna polymerase ii for the transcription of certain late genes. [494] [495] [496] at the post-transcription level, hnrnps regulate the polyadenylation, splicing, and translation during dna virus infections. hpv genome can be divided into an early region and a late region, followed by the proximal early (pae) and the distal late (pal) polyadenylation signals, respectively. 497, 498 the virion production mainly depends on the differentiation-dependent induction of l1 and l2 late genes. it has been shown that hnrnp h downregulates the late gene l2 at an early stage by interacting with the multiple ggg motifs located 174 nucleotides downstream of the early polyadenylation signal pae. the hnrnp h binding promotes polyadenylation at early polyadenylation signal and inhibits the l2 mrna production, since l2 mrna production need read-through into the late region and polyadenylation of the late transcripts at the pal. 499 while at the late infection stage, hnrnp h is captured by l1 to release the inhibitory effect on l2. 499, 500 this process is called late gene autoregulation which enables rapid viral capsid protein production. 500 another example for hnrnps modulating the polyadenylation is that sm polymerase (pol) mrna of ebv early protein is cleaved and polyadenylated inefficiently. 501 under certain conditions, ebv early protein sm may harness hnrnp a1 and hnrnp c to help the processing of polymerase mrna. viral rna splicing is also modulated by hnrnps. hnrnp a1 negatively regulates the expression of hpv late genes by affecting rna splicing. it directly binds with the late regulatory element (lre) in differentiated hpv16-infected cells. 502 hnrnp a1 binds with splicing silencer element to suppress the use of the 3′ splice site located immediately upstream of aug at late 1 (l1) mrna. hnrnp a1 inhibits the splicing of late mrnas through the splicing silencer sequence and prevents the premature expression of l1 gene. [503] [504] [505] on the other hand, there is another 17 nucleotides resident immediately downstream of the splice site which counteracts the effect of hnrnp a1-binding splicing silencers. 506 hnrnp i also interferes with the splicing inhibitory elements locating at the upstream and downstream of major late 5′ splice site sd3632, thereby activates the late gene expressions. 507 at the translation level, hnrnp i and hnrnp k inhibit the translation of hpv late gene l2 via binding on a specific cis-acting element in the 3′ end of l2 mrna; 508 whereas the inhibition of l2 translation can be disrupted by c-src-mediated phosphorylation of hnrnp k at multiple tyrosine residues. 509 cellular transformation. hnrnps also exhibit some other functions during virus infections. auf1 works as a major component of c promoter binding factor 2 (cbf2) to interact with ebna2 and mediate ebna2 targeting to the latency c promoter (cp) of ebv, thereby inducing b-cell immortalization and viral latency in humans. 510 auf1 also binds with the eber1 noncoding rna of ebv. in ebv-positive cells, eber1 is abundant; therefore it may compete with auf1-interacting targets in the host cells. 511 both ebna2 and eber1 are proposed to facilitate cell transformation. immunity modulation. aside from affecting virus life cycle directly, hnrnps are able to regulate viral activities through modulating immune response. during hsv-1 infection, hnrnp a/b form a complex with viral dna, followed by homodimerization and demethylation. these events result in translocating the complex to cytosol and activating natural immunity through type i interferon signalling. moreover, the complex promotes n6methyladenosine modification and cgas-sting-related mrnas translocation upon infection by dna viruses, further enhancing the immune response for virus elimination. 512 the function of rna chaperones in regulating the viral activities of retroviruses as we have mentioned above, hnrnp a1 can bind to rna/proteins and participate intracellular nucleo-cytoplasmic transportation, 513 as well as alternative splicing of mrna in eukaryotes. during retrovirus infection, hnrnps participate in viral rna transcription, 514 splicing, 515-517 translocation 516 and translation. transcription. in hiv-infected cells, viral nef protein is required for high-level viral replication. 518 it is reported that nef, eed, kinase lck and npkc subfamily (pkcδ/θ) form a complex nakc (nef-associated kinase complex) responsible for promoting viral replication. 519 later on, it is found that hnrnp k is also a partner of the complex. it bridges the interaction of nef, eed and the kinases. the hnrnp k-nucleated complex activates erk1/2 and results in suppressing hiv promoter, enabling suboptimal amounts of tat and transcription factors (e.g., nf-kb) for initiation of transcription. 514 post-transcription. hiv-1 takes advantage of alternative splicing to generate doses of messenger rnas to encode the various viral proteins. it is known that over 40 message rnas are created by alternative splicing from a single pre-mrna. 520,521 alteration of splicing patterns dramatically affects the infectivity and pathogenesis of hiv-1. 521, 522 the splicing of hiv mrna is mainly controlled at the early stages of spliceosome assembly on pre-mrna by a stepwise association of the small nuclear ribonucleoprotein particles (snrnps) u1, u2, and u5·u4/u6. the early steps include the recognition of the 5′ splice site and the branch point sequence by u1 and u2 snrnps, respectively. 523 rs splicing factors, which contain a domain rich in arginines and serines (rs domain), assist these steps. u2af, one of the splicing factors which consists of two subunits u2af65 and u2af35, interacts with the polypyrimidine tract and the 3′ splice site, respectively. 524 then the interaction mediates the association of u2 snrnp with the branch point sequence. [525] [526] [527] sr splicing factors are essential for virtually every step of spliceosome assembly, including early recognition of splice sites, recruitment of basic splicing factors to the pre-mrna, and formation of bridging contacts with other rs domain-containing splicing factors. 528 prior to forming spliceosomes, the pre-mrna is packed with hnrnps. 529 it has been documented that pre-mrnas bind with different subsets of hnrnps, indicating sequence specificity at some degrees. a direct role of hnrnps in constitutive splicing has not been observed; although it seems that the binding of hnrnps exhibits an unspecific nature. it is widely believed that hnrnps employ crucial functions of modelling pre-mrna structure and initiating recognition of splice sites. 530 the cis-acting sequence elements of cellular and viral pre-mrnas undergoing alternative splicing regulate the process either positively or negatively. they bind trans-splicing factor machinery together and form splicing complex. the majority of trans-acting factors are either hnrnps or sr family proteins. these proteins also regulate alternative rna splicing either positively or negatively. sr proteins often regulate splicing in a positive way while some hnrnps mainly do the job in a negative way. 528, 530 alternative incomplete splicing of hiv-1 genomic mrna produces more than 40 unique viral mrna species within an hiv-1-infected cell through highly accurate regulation by cis exon splicing silence elements (ess) and trans hnrnps. the production of tat, rev and vpr proteins is highly controlled because they play key roles in hiv-1 multiplication. ess2 is the first identified ess in the hiv-1 genome that locates downstream of 3′ ss a3 within exon 4 and specifically represses tat mrna splicing. 531 ess2 is mapped to a 10 nt core sequence cuagacuaga. 532 ess3, which locates at exon 7, represses splicing at 3′ ss a7. 533 fine structure mutagenesis indicated that ess3 is bipartite; and each sub-elements [agaucc (ess3a) and uuag (ess3b)] inhibits splicing independently. 534 a third ess (auaguuaguccuagg, essv), which locates downstream of 3' ss a2 in exon 3 within the vif coding sequence, represses splicing at 3′ ss a2. 535 to modulate the expression of tat protein, several hnrnps participate the splicing process by interacting with these ess, intron splicing silencer (iss) elements directly or interfering enhancer splicing element (ese) activity. for example, hnrnp a1 and hnrnp k synergistically bind on ess2 and inhibit the utilization of a3 splicing site. 536, 537 the uag triplets in ess2 is required for hnrnp a1 binding, and several hnrnp a1-binding sites are also found at sls3. the c-terminal gly domain of hnrnp a1 is important for the binding. sr proteins sc35 and srp40, the strong activators of site a3, have similar binding sites to hnrnp a1. hence, they may compete with each other to bind ess2 and ese2. 536 another study shows that hnrnp h displays an inhibitory effect on splicing. hnrnp h binds to ess2 and competes with u2af35 for binding to the exon sequence flanking 3′-splice site a3. this binding results in the inhibition of splicing at the 3′-splice site a3. 538 hnrnp a1 also inhibit tat splicing by binding with iss, which is independent of exon splicing silencer (ess2) and blocks the entry of u2 snrnp but not u2af65. [539] [540] [541] the up1 domain of hnrnp a1 is responsible for the iss binding, while the rgg domain of hnrnp a1 is not needed for the alternative splicing activity. 542, 543 in addition to ess2 and iss elements that are regulated by hnrnp a1, blocking the binding of sr protein sc35 on ese is another strategy for the virus to inhibit the tat expression at the early infection stage. hnrnp a/b proteins bind the ese locating at tat exon 2 to repress splicing, whereas sc35 binds the ese to activate splicing. the binding sites of hnrnp a1 and sc35 are overlapping within the juxtaposed ese/ess9. it seems that hnrnp a1 inhibits the splicing of the upstream intron by binding to the ess and directly masking the sc35 binding site. 544, 545 similarly, hnrnp a1 is also reported to compete with asf/sf2 at ese3/(gaa)3. therefore, the ratio of asf/sf2 to hnrnp a1 determines the utilization of ese3/(gaa)3 for activation or repression at site a7. 515, 541, 546 the expression of other hiv proteins is also regulated by hnrnps via modulating splicing. hnrnp a/b proteins inhibit the splicing at 3′ splice site a2 which is used to generate vpr mrna. hnrnp a/b proteins bind with essv with a consensus sequence pyuag. the splicing at splice site a2 is increased when the three pyuag motifs within essv are mutated, leading to increased vpr mrna levels and reduced skipping of the noncoding exon flanked by a2 and d3. 547 others reported that hnrnp d also binds at essv and functions as an inhibitor of splicing. 548 the binding of hnrnp a/b proteins at essv blocks u2af65 binding to the ppt of the repressed 3′ splice site and inhibits the splicing efficiency of 3′ splice site. 549 interestingly, hnrnp h is found to positively regulate the exon 6d splicing of hiv mrna. it interacts with the sequence cgga and enables u1 snrnp assembly onto exon 6d. 550, 551 further study shows that hnrnp h family proteins function as a splicing enhancer through enhancing the atp-dependent spliceosomal complex formation. 552 rna translocation. after transcription and splicing, lots of spliced and unspliced rna molecules of hiv exist in the nucleus. translocation of rna into the cytoplasm is dependent on the rev protein. on one hand, some hnrnps facilitate rna translocation. in hiv-1 genome, two sequences similar to the hnrnp a2 response element (a2re) function as cis-acting rna trafficking sequence that binds to the trans-acting trafficking factor hnrnp a2. the binding mediates a specific rna trafficking pathway characterized extensively in oligodendrocytes. a2re-1 locates within the major homology region of gag gene; while a2re-2 locates at a region overlapped between vpr and tat coding sequence. hnrnp a2 binds to both a2res in vitro to form a complex, which is necessary for rna transportation in oligodendrocytes in vivo. a2re-mediated rna transport requires both microtubule and hnrnp a2. if gag and vpr rnas containing a2re-1 and a2re-2 respectively are differentially labelled, it is observed that they are co-assembled into the same rna trafficking granules and cotransported to the periphery of the cell. although the tat rna contains a2re-2, it is not transported as efficiently as vpr rna. [553] [554] [555] hnrnp d also has a similar function in assisting rna translocation. 556 on the other hand, hnrnp a2b1, hnrnp c and hnrnp u can retain the hiv-1 genomic rna in nucleus. depletion of hnrnp a2b1 results in cytoplasmic redistribution of the virus rna genome in the absence of rev. 557, 558 an n-terminal fragment of the hnrnp u specifically targets the 3′ long terminal repeat (3′ltr) and blocks the cytoplasmic accumulation of mrnas, thereby affecting hiv gene expression. 559 besides hiv, the regulatory protein orf57 of kaposi's sarcomaassociated herpesvirus (kshv) also interacts with hnrnp k. ck2 can phosphorylate orf57 and promote orf57-hnrnp k interaction. the orf57-hnrnpk-ck2 complex may be important for rna export of kshv since orf57 is responsible for the nuclear export of viral mrnas. 560,561 hnrnp a1 interferes with the binding of rex to rexresponse element (xre). the rex protein of htlv-1 mediates the nuclear export of unspliced and incompletely spliced viral mrnas. this process partly depends on the binding of rex to rex-regulatory sequences xre. 562 viral protein translation. hnrnp a1 is induced to redistribute into the cytoplasm in the late infection stage of hiv and enhances the ires-mediated translation. 563 hnrnp d helps the redistribution of hiv mrna into the cytoplasm, and p45 and p42 isoforms increase viral gag protein synthesis while p40 and p37 suppressed this process. 556 hnrnp i interacts with a novel ires within a latently expressed gene (vcyclin) of kshv and enhances vflip expression. 564 the network of hsps and their co-chaperones is essential for cells to maintain protein homeostasis, including nascent protein folding, protein translocation across membranes, and protein complex formation. many stress stimuli can disrupt protein homeostasis such as thermal stress, nutrient starvation, chemical toxicity, oxidative stress, hypoxia, inflammation, and virus infection. [565] [566] [567] [568] stresses can lead to protein misfolding and aggregation that need to be resolved quickly to prevent cell and tissue damage. 568 hsps and their partners may facilitate protein degradation when cells cannot cope with massive damaged proteins. many lines of evidence suggest hsps as major factors for protein surveillance to protect host cells against virus infections. it was observed that hsp70/hsp90 expression increased dramatically in hsv-infected cells. 569 overexpression of hsp70 inhibits the translocation of viral capsid into nucleoli during flavivirus infections. although the interaction has not yet been investigated in natural infection, the in vitro findings have suggested that hsp70 may act as a negative regulator of viral capsid protein to protect host cells against wnv infection by abolishing cytotoxic effects induced by the viral capsid. 570 studies from ours and other groups show that almost all hsp subfamily members (hsp90s, hsc70, hsp70, grp78, erp57, hsp27) are highly responsible to enterovirus infection and play key roles in all stages of virus life cycle. not surprisingly, demonstrated that all most all hsps (hsp90s, hsp70s, hsp60s, hsp40s and hsp27) participate coronavirus infection, 145, 196, 387, 571 suggesting good target for anti-covid-19 drug development. as discussed in above sections, hsps exhibit immunomodulatory effects on both innate and adaptive immune responses. [572] [573] [574] hsps are capable of regulating not only intracellular innate immunity, but extracellular innate and adaptive immunity as well. hsps released from tumor cells can bind to surface receptors on antigen presenting cells (apcs) and elicit tumor-specific killers by means of antigen crosspresentation. 575, 576 for example, hsp27 positively modulates nf-κb phosphorylation, increases ifnβ transcription and downstream antiviral interferon-induced genes (isgs). pedv infection downregulates hsp27 expression to escape host antiviral surveillance. 389 additionally, extracellular hsps can also regulate cytokine production by dendritic cells (dcs), underscoring a connection between innate and adaptive immune responses modulated by hsps. 577 the purified or recombinant hsp70 can stimulate the production of pro-inflammatory cytokines and c-c chemokines in monocytes, macrophages and dcs, and upregulate mhc and costimulatory molecules in dcs. 578 binding of extracellular hsp72 to human monocytes and dendritic cells can induce the production of the pro-inflammatory cytokines including tnf-α, il-1β, il-6 and il-12 and ifn-γ. 579 in the process of above hspinduced cytokine production, hsps act as internal stimulus of the cd14/tlr (tlr2 and tlr4) complex signal transduction pathways that further activates nf-κb and mapks signalling. 580, 581 although the main functions of hsps are to protect cells upon stresses, they are often hijacked by many viruses to achieve successful infections. recent study has demonstrated that denv ns3 protein acts as a viral suppressor of rna silencing by interacting with hsc70, thereby interrupting host antiviral system by rnai pathway and subsequently enhancing denv replication. 582 ev-a71 takes advantage of hsps (hsp90, hsc70, erp57, and hsp27) to enhance 2a pro -mediated eif4g cleavage that is favour viral protein translation and blocks host protein translation. 57, 219, 415 viruses also initiate er stress in host cells after infection. they have to manipulate upr activation leading to cell survival, rather than inflammation induction, autophagy and apoptosis. denv modulates upr activation in a sequential manner to prolong the viral life cycle by allowing cellular adaptation to cope with the infection-induced er stress. upr is transiently induced by perk pathway resulting in phosphorylation of eif2α and subsequently translational attenuation in the early phase of infection. this transient event allows viral protein synthesis and accumulation that finally trigger upr by ire1-xbp1 (x-box binding protein 1) axis in the mid-phase of infection. this results in the increased expression of grp78 to facilitate protein folding and also the increased expression of gadd34 (growth arrest and dna damage 34), which dephosphorylates eif2ɑ, and thus allowing protein translation to be continued. the increased grp78 also prevents cellular apoptosis-mediated by chop (pro-apoptotic protein during stress persistence). finally, the increased viral proteins transiently trigger atf6 arm of upr to provide the active spliced xbp1 for sustaining upr activation in the late phase. 583 jev infection requires hsp70s in particular stages of its life cycle for survival and establishment of infection, including viral entry, replication, 194 and maturation. 195 cell-surface hsp70 directly interacts with jev envelope protein. 192 antibodies against hsp70 or 90 significantly block virus entry. 111 it is also observed that hsp70 colocalizes and directly interacts with jev replicase complex. in addition, hsp70 also interacts with ns3, ns5 and viral dsrna that stabilizes vrc formation during jev infection. 194, 584 er stress and antiviral in mammalian cells, the er stress is sensed and mediated by three er transmembrane receptors: pancreatic er kinase (pkr)-like er kinase (perk), inositol-requiring enzyme 1 (ire1) and activating transcription factor 6 (atf6). in resting cells, these three sensors are maintained in inactive states via interactions with the er resident chaperone grp78. when unfolded or misfolded proteins accumulate in the er lumen, grp78 dissociates from these three transducers, resulting in activation and initiation of the upr. viruses also take advantage of or revolt er stress response by different means for favour its life cycle at specific stage(s) of infection. the er stress response constitutes a cellular process triggered by a variety of conditions disturbing protein folding in the er. eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, i.e., the er stress and upr, aiming to clear unfolded/misfolded, and excessive amount of protein production; thereby restoring er homeostasis. in cases er stress cannot be resolved, upr would be triggered and lead to cell death. er stress and upr could be observed in a large amount of virus infections, as most of viruses require host er machinery for viral protein synthesis and modification. virus infection usually causes er stress. when large amount of misfolded/unfolded proteins or excessively expressed viral proteins accumulate inside er lumen, er stress response is triggered as indicated by fast elevated expression of er chaperone proteins, including grp78/ bip, grp94, calnexin and calreticulin. it is well documented that er stress response is triggered by a number in some cases, er stress plays a role in virus pathogenesis. for instance, jev, bvdv, tulv, severe acute respiratory syndrome coronavirus (sars-cov) and wnv have all been shown to induce apoptosis through upr. [585] [586] [587] [588] [589] oxidative stress is mediated by er stress during hcv infection. 21 certain viruses modulate er stress response or preferentially activate different pathways of upr. hcv infection activates the atf6 pathway while blocks the ire1 pathway. instead, hbv infection stimulates both atf6 and ire1 signalling but has no effects on perk signalling although both virus infect the same kind of hepatocytes. 590-592 hsv-1 develops a virulence factor enabling dephosphorylation of eif2α, one of the downstream effectors of the perk pathway. 593 some consequences of upr are beneficial for viral life cycle. for example, atf6-induced expression of chaperone proteins may help viral protein folding and prevent protein aggregation. the perk-eif2α-activated atf4 may help re-establish cell metabolism and resume protein translation. the ire1-xbp1 pathway may facilitate virus replication by enhancing er protein-folding ability and er membrane biosynthesis. 594 the activated atf6 promotes the replication of asfv and lymphocytic choromeningitis virus (lcmv). 595,596 denv envelope protein directly interacts with grp78, which provides a scaffold for its association with two other chaperones calnexin and calreticulin. this complex significantly improves the proper folding and stability of denv e protein, thereby leading to increase of virus production. 597 other than denv, grp78 is also demonstrated to promote hcmv, jev and rgnnv infections. 195, 598, 599 the ire1-xbp1 pathway is activated by iav to facilitate its replication. 600 however, other upr outcomes are detrimental for virus replication. the perk-eif2α-mediated global translation attenuation is known as an antiviral response to restrict viral replication, such as infection by denv or wnv. 583, 601 the activated pkr phosphorylates eif2α at the ribosomal interface, which in turn causes a general inhibition of protein synthesis and blockage of vsv replication. 602 the ire1-xbp1(s)-mediated er-associated protein degradation (erad) pathway reduces intracellular hbv particles by degrading its envelope proteins. 603 another approach of er stress contributing to virus pathogenesis goes through modulating host cell immune responses. vsv, hcv and sars-cov are able to inhibit the type i ifn signaling pathway by activating the perk signalling, which leads to the phosphorylation-dependent ubiquitination and subsequent degradation of ifnar1, thereby promoting immune evasion and virus pathogenesis. 604, 605 wnv has also been reported to induce er stress and inhibit type i ifn signaling pathway for escaping from the host immune response. 601 us11 protein of hcmv activates upr to facilitate the degradation of class i major histocompatibility complex (mhc1), leading to immune evasion. 606 moreover, er stress is responsible for viral pathogenesis by interconnecting with the inflammatory responses. for example, hcv induces inflammatory responses by activating ire1, which interacts with traf2 to phosphorylate jnk, leading to activation of inflammation mediators. 607 ns4b and ns5a proteins of hcv activate nf-κb via er stress-elicited calcium depletion and ros production. 604 the x protein (hbx) of hbv induces the expression of cyclo-oxygenase 2 (cos2), a key mediator of inflammation, through perk-eif2α-activated atf4. 608 rna chaperons and human diseases caused by viral infections hnrnps impact mrna metabolism including transcript synthesis, processing and degradation as well as translation. 432 the function of hnrnps is determined or modulated by cellular localization. the mechanisms that regulate the nucleo-cytoplasmic shuttling are, therefore, of extreme importance. most hnrnps possess a conventional nuclear localization signal (nls) and are predominantly present in the nucleus during steady state. they are able to translocate in the cytosol upon post-translational stimulation or by the recruitment of other hnrnps. 609 post-translational modifications like methylation, phosphorylation, ubiquitination and sumoylation are reported to affect biological activity and subcellular localization of hnrnps. 610 as stated in the above sections, virus always employs different strategies to redistribute hnrnps in host cells. 430, 431, 433, 434 how dysregulation of hnrnps causing human diseases has gained an increasing interest. the expression level of hnrnps is altered in many types of diseases, including varieties of human cancers and neurodegenerative diseases (e.g., spinal muscular atrophy (sma), amyotrophic lateral sclerosis (als), alzheimer's disease (ad), and fronto-temporal lobe dementia). in this section, we mainly focus on the relationship between hnrnps and diseases caused by virus infections. enteroviruses are common pathogens that cause human diseases worldwide. although most enteroviral infections are subclinical, they may cause a wide spectrum of diseases including mild upper respiratory illness (common cold), febrile rash (hand, foot, and mouth disease and herpangina), aseptic meningitis, heart failture, pleurodynia, encephalitis, acute flaccid paralysis (paralytic poliomyelitis) and neonatal sepsis-like disease. 293, 611 besides, several studies showed that enterovirus sequences could be detected in neuronal cell bodies of the spinal cord of 60-88% amyotrophic lateral sclerosis (als) patients, suggesting that persistent enterovirus infection may have a relationship with als. 295, 297, 299 enterovirus infections are supposed to cause or increase the risk of als; because the replication and translation of poliovirus, ev-a71, and coxsackievirus redistribute numerous hnrnps (such as hnrnp a1) into the cytoplasm, the same localization during als pathogenesis. for example, hnrnp k, hnrnp a1, hnrnp m and hnrnp d shuttle into cytoplasm to assist virus replication or translation. 300, 455, 472, [612] [613] [614] dislocated hnrnp a1 and loss of splicing function have been regarded as a toxic mechanism in als pathogenesis. 301 hsv infection is one of the most common causes of infectious disease in humans. 302 hsv infection often causes watery blisters in the skin or mucous membranes of the mouth, lips, nose, or genitals. 615 as neurotropic and neuroinvasive viruses, hsv-1 and -2 persist in the infected individuals through hiding in the neuron cells from immune surveillance. when the carrier's immunity compromised, hsv is reactivated that causes new sores. more seriously, accumulating evidence shows that hsv infection is associated with the pathogenesis of alzheimer's disease. 105, 616, 617 during hsv infection, hnrnp k positively promotes hsv replication; 618 while hnrnp a2/b1 negatively regulates hsv replication by triggering immunity response. 512 hbv, hcv, ebv, hpv and hiv are oncogenic viruses that account for over 20% of human cancers. hcv or hbv infection leads to a wide spectrum of liver disease ranging from acute hepatitis (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (hcc). 619,620 hdv can only infect people who are already infected with hbv. the coinfection of hdv and hbv increases the risk of liver cirrhosis and liver cancer. 621, 622 except for damaging the liver, 10% of hbv infected patients also have other symptoms outside the liver, such as serumsickness-like syndrome, membranous glomerulonephritis, acute necrotizing vasculitis (polyarteritis nodosa) and papular acrodermatitis of childhood (gianotti-crosti syndrome). 623 multiple steps of hcv reproduction need the presence of hnrnps. the rna replication of hcv needs the presence of hnrnp i, hnrnp c; 437, 442, 446 while virus rna translation requires hnrnp a1, hnrnp d, hnrnp i, hnrnp k and hnrnp l. 452, 460, [462] [463] [464] [465] besides, hnrnp a2, hnrnp l, hnrnp u and hnrnp k are highly expressed in hcc tissue. they promote liver cancer progression. 8, 304, 305, 624 ebv has been implicated in several diseases, including infectious mononucleosis, 625 burkitt's lymphoma, 626 hodgkin's lymphoma, 627 nasopharyngeal carcinoma, 628 multiple sclerosis 629 and lymphomatoid granulomatosis. 630 other diseases associated with ebv infections include gianotti-crosti syndrome, erythema multiforme, acute genital ulcers, oral hairy leukoplakia, 631 and disorders related to α-synuclein aggregation (e.g. multiple system atrophy, dementia with lewy bodies, and parkinson's disease). 632 during ebv infection, hnrnp a1 and hnrnp c assist the polyadenylation of ebv rna. hnrnp d takes part in cellular transformation-induced by ebv. 510 hpvs are a group of dna viruses with more than 150 members that infect cutaneous and mucosal epithelia. the acute infection of hpv causes benign cutaneous lesions such as non-genital and genital warts, or flat cervical condylomas. 633 about 15 hpv subtypes that infect genital tract are capable of inducing malignant tumors, most commonly in the cervix. 308,309 these cancer-associated hpvs are grouped into high-risk types, while those not associated with cervical cancer are regarded as low-risk types. 307 most infections by low-risk type hpvs are asymptomatic, except for infection by hpv6 and hpv11 that cause most cases of genital warts (condyloma acuminatum). 309 hpv-related cancers are the result of long-term persistent infection with high-risk types. hpv16 and hpv18 are the most common carcinogenic types which account for a larger proportion of cervical cancers, squamous cell cancers and adenocarcinomas in all regions. 311 hpvs are also implicated in the development of a variable proportion of vulvar, vaginal, penile, and oropharyngeal cancers, anal cancer, 634 head and neck cancers, 635-637 lung cancer and skin cancers. hnrnp a1 is involved in splicing of hpv rna; while hnrnp h plays a role in the polyadenylation of hpv. hnrnp k and hnrnp i are required for viral protein translation. besides acquired immunodeficiency syndrome (aids), hiv infection is also able to cause human cancers because hiv infection impairs immunity system. people with aids are not only more easily infected with bacteria, viruses, fungi, and parasites; 638 but at high risk for developing various viral-induced cancers as well, including burkitt's lymphoma, kaposi's sarcoma, primary central nervous system lymphoma, and cervical cancer. 639 515, [532] [533] [534] [535] [536] [537] [544] [545] [546] [547] [548] [549] [550] [551] [552] [553] [554] [555] [556] [557] [558] [559] 564 potential therapeutic value by targeting stress proteins interest in targeting stress proteins in various diseases is increasing overtime. in this part, we will discuss the potential therapeutic value by targeting stress proteins. first, targeting stress proteins have a wide spectrum of antiviral ability. for example, hsp90 inhibitors, which are originally developed for anticancer, have been demonstrated to possess antiviral activity in cultured cells against poliovirus, rhinovirus, ev-a71, 107,109 coxsackievirus, 124 rsv, 112, 113 vsv, paramyxoviruses (hpiv2, hpiv3, sv5, sv41), influenza virus, 126 chikv, 114 hcv, 115, 124 and hsv, 131, 144, 145, 148, 149 hbv, 135, 169 ebv, 146, 147, 163, 164 hcmv 151, 152 and htlv. 153, 185 depleting hsp60 by sirna functionally suppresses infections by influenza virus, 38,39 denv, 319 hbv, 325, 329, 331 hcv, 321, 322 rotavirus, 323 and hiv. 338 targeting hnrnp a1 is able to inhibit the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c could be a strategy to combat viral infections by hcv, 438 [461] [462] [463] 475 therefore, stress proteins are particularly attractive as antivirals targets for those lacking therapies and newly emerging viral diseases. second, under disease situations, the cellular need of stress proteins is usually stronger than that in normal conditions which enable specific selectivity of those drugs targeting stress proteins. for example, mutant p53 relies much more on hsp90 function than wild type p53. hsp90 inhibitor gm can easily disturb the association of mutant p53 with hsp90, resulting in mutant p53 degradation while not affecting wild type p53. 310 additionally, stress proteins derived from stressed cells display higher affinity to clients and inhibitors. tumor cell-derived hsp90 exhibits a 100fold higher binding affinity for 17-aag than that from normal cells, since tumor cell-derived hsp90 complexes with activating cochaperones p23 and hop exhibit increased atpase activity and possess higher affinity to hsp90 inhibitors. in contrast, hsp90 in normal cells exists as an uncomplexed species with low atpase activity and low affinity to hsp90 inhibitors. 294 further study demonstrates that the difference is caused by a distinctive portion of hsp90 forming complexes in cancer cells with oncogenic partners, such as bcr-abl-hsp90 complex in mutant b-raf-hsp90 in skmel28 melanoma cells, k562 chronic myeloid leukemia cells, her3-hsp90 and raf1-hsp90 complex in mda-mb-468 breast cancer cells. hsp90 inhibitor pu-h71 selectively binds to specific hsp90-oncoprotein networks in these cancer cells. 570 these characters of cancer cells enable stress proteins to exhibit stronger biological activity and to be discriminated by their inhibition under stress as compared with normal conditions. similarly, inhibitors of stress proteins show high prospect for antiviral. hsp70 inhibitor jc40 inhibits pan-flavivirus (denv2 and denv4) replication in mddcs, with negligible toxicity to host cells. 640 these experiments highlight the feasibility of using hsp inhibitors therapeutically in humans. third, it is not observed drug resistance using hsp inhibitors for antiviral. for instance, hsp90 inhibitors are refractory to develop drug resistance. this is clearly demonstrated in rsv infection. 113 when rsv is repeatedly treated with hsp90 inhibitors, no drug resistance was observed either in extensive passaging of the virus in cultured cells or in mice undergoing long-term treatment with hsp90 inhibitors. similar result is also observed in denv infections. 640 treatment of denv up to 10 passages with hsp70 inhibitor jc40 does not cause any drug resistance. the lack of viral drug resistance to hsp90 and hsp70 inhibitors suggests that such an antiviral approach may be particularly useful for treating chronic viral infections in which drug resistance is most frequently observed. the three features of stress proteins inhibitors make them extremely powerful antiviral agents, suggesting great application potential for treating the diseases caused by virus infections, such as covid-19. challenges and perspectives: target-based drug development targeting hsp90 for antivirus drug development. hsp90 is thought to be the most abundant and evolutionarily conserved heat shock protein. a unique pocket in n-terminal region of hsp90 is required for binding with atp and co-chaperones. 641 hsp90 forms a flexible dimer by interaction of c-terminal domains. the formation and dissociation of compact dimers involving nterminal domains are important for the molecular chaperone activity. 642 the middle domain of hsp90 tends to recruit and facilitate unfolded client proteins to assemble. the c-terminal domain of hsp90 possesses a highly conserved peptide sequence for interacting with co-chaperones. 643 over 20 co-chaperones selectively interact with hsp90 to regulate atpase activity and recruit client proteins to assemble a big complex under certain conditions. [642] [643] [644] [645] it has been shown that more than 300 client proteins require hsp90 and co-chaperones for folding and maturation. 646 the mechanism of selectivity may rely on the direct interaction of co-chaperones with specific clients. therefore, most hsp90 inhibitors achieve their inhibitory effects by suppressing the atpase activity or disrupting the interaction between hsp90 and its co-chaperones. expression of hsp90 and its client proteins are increased during viral infection and in most cancer cells. hsp90 as an effective anticancer drug target has already grabbed attention; and a series of hsp90 inhibitors as potential drugs have been intensively investigated in the laboratories, preclinical and clinical scenarios. the successful use of hsp90 inhibitors in cancer therapy makes it much easier to apply them for treating virus infections. as described above, we have comprehensively summarized the functions of hsp90 and its client proteins in a diversity of virus reproduction processes. the potential of hsp90 inhibitors has been well demonstrated to protect cultured cells against infections by ev-a71, 107,109 poliovirus, rhinovirus, coxsackievirus, 124 paramyxoviruses (hpiv2, hpiv3, sv5, sv41),vsv, rsv, 112,113 influenza virus, 126 chikv, 114 hcv, 115,124 hsv, 131,144,145,148,149 hbv, 135,169 ebv, 146, 147, 163, 164 hcmv, 151, 152 and htlv. 153, 185 notably, administration of hsp90 inhibitors to infected animals exhibits little toxicity while potently suppresses the replication of poliovirus, 107,109 ebv, 163, 164 hbv, 135 chikv 114 and hcv. 647 these experiments highlight the feasibility of using these inhibitors therapeutically in clinic. here we would briefly enumerate the present hsp90 inhibitors and their potential for antiviral therapies. most hsp90 inhibitors hamper hsp90 function by competitively binding to the atp binding site of hsp90, blocking the interaction with co-chaperones, or modulating acetylation. by doing so, we also try to address the possibility of repurposing hsp90 inhibitors as candidate antiviral drugs ( table 2) . inhibitors targeting hsp90 atpase activity. some hsp90 inhibitors competitively bind to the atp pocket in the n-terminus of hsp90, leading to blocking of atp hydrolysis and the closure of nterminus of hsp90 dimer. in addition, another atp binding site has been found in the c-terminus of hsp90. 648 recently, some natural products and their derivatives have been reported to competitively bind to the atp pocket in the c-terminus of hsp90. generally, inhibitors of hsp90 atpase are classified into three types: (i) ansamycins, (ii) non-ansamycins and (iii) those block hsp90 c-terminal atpase activity. ansamycins are antibiotics that possess the benzoquinone as structure core. these antibiotics include geldanamycin (ga), herbimycin a, and the macbecins. they inhibit hsp90 activity and degrade its client proteins. 649 ga competitively binds to the atp pocket in the n-terminus and inhibits the atpase activity. 649 it has shown potent antiviral activity against coronavirus infection in the culture cells, 145 indicating a good potential for treating covid-19. tanespimycin (17-allylamino-17-demethoxygeldanamycin, 17-aag) is an analogue of ga. 650, 651 alvespimycin (17-dimethylaminoethylamino-17demethoxygeldanamycin, 17-dmag) is an analogue of 17-aag, with high solubility in water. in addition, retaspimycin hydrochloride (ipi-504) is a more water-soluble derivative of 17-aag. 652 non-ansamycin inhibitors of hsp90 include luminespib, 653 biib021, 654 ganetespib, 655 onalespib, 656 snx-5422, 657 etc. another type of hsp90 inhibitors binds to the c-terminal atp pocket rather than n-terminal atp pocket. novobiocin blocks the c-terminal atpase activity. 658, 659 in in vitro and in vivo assays, treatment with novobiocin strongly reduces the expression of hsp90-dependent client proteins, such as raf-1 and p60v-src. a natural rotenoid, deguelin, is suggested to bind to the atp pocket in the c-terminus without affecting the atp pocket in the nterminus. 660 treating with deguelin leads to reduced hsp90 clients, such as cdk4, akt and mek1/2. 661 like deguelin, epigallocatechin gallate (ecgc) is reported to bind to the atp pocket in the c-terminus of hsp90 without affecting the nterminal atp pocket. 662 compared with inhibition of the nterminal atp pocket of hsp90, inhibitors targeting the c-terminal atp pocket lead to a stability of hsp70 and a negative instability of client proteins of hsp90. however, the potential of these cterminal inhibitors and their molecular mechanisms remain elusive. the structural features of the c-terminal domain of hsp90 need to be further analysed. such analysis may provide important hints on how to design effective hsp90 inhibitors without hsr induction. inhibitors of hsp90's co-chaperones. as the functions of the hsp90 chaperone machinery are highly dependent on the associated co-chaperones, it is possible to selectively inhibit downstream signaling of hsp90 by disrupting certain protein-protein interactions. inhibitors targeting the interactions may present an alternative approach to prevent the toxicity induced by other hsp90 inhibitors. as a result, great efforts are put into disrupting the interaction between hsp90 and its cochaperones and are rewarded recently in this area. here we briefly present the advance in hsp90-cdc37 complex and hsp90-hop-hsp70 ternary complex. human cdc37 is a well-studied co-chaperone of hsp90. the middle domain and c-terminus of cdc37 are critical for the interaction with hsp90. currently, most of the reported agents for manipulating the hsp90-cdc37 interaction are natural products and their derivatives. these agents include celastrol, aferin a, sulforaphane, kongensin a and platycodin d. they are capable of dissociating the hsp90-cdc37 complex. [663] [664] [665] [666] [667] in addition, a peptide (pep-1, ac-khfgmlrrwdd-nh2) is developed and shows strong inhibitory effects on the formation of hsp90-cdc37 complex. 668 another well-known hsp90/co-chaperone complex is hsp90-hop-hsp70 ternary complex, which facilitates the transfer of unfolded client proteins from hsp70 to hsp90. notably, six active compounds have been identified after screening the ncgc compound library. 669 these compounds possess similar structural cores and have no effect on hsp70 expression. studies on hsp90-co-chaperone inhibitors are limited. great effort should be paid on the efficacy and toxicity for further drug design and clinical studies. hsp90 inhibitors blocking deacetylation of hsp90. the chaperone function of hsp90 is also controlled by posttranslational modification (ptm). the well-studied ptms in hsp90 are acetylation and deacetylation in the m-domain of hsp90. vorinostat (suberoylanilide hydroxamic acid, saha) dissociates her2/erbb2 from hsp90 via acetylation of hsp90 that leads to degradation of her2/erbb2. 670 laq824 induces hsp90 acetylation that reduces hsp90 client protein levels. 671 romidepsin is involved in the dissociation of mutant p53 and raf-1 from hsp90 via acetylation of hsp90. 672 in addition, the nuclear import of iav polymerase needs the deacetylation of hsp90 which is strictly regulated by histone deacetylases 6/8 (hdac6/8). 120 hdac6/8 inhibitors efficiently limit polymerase nuclear import and suppress virus replication. 120 therefore, hsp90 inhibitors that block hsp90 deacetylation would potentially be used to treat influenza a virus infection. their potential usage for combating other viruses (such as sars-cov-2) needs to be extensively explored. novel class of hsp90 inhibitors for hsp90 cleavage. recently, hsp90 cleavage has been observed under various stimuli such as uv irradiation, 673 ascorbate/menadione, 674 hdac inhibitors, 675 proteasome inhibitors 676 etc. therefore, hsp90 cleavage is considered as another mechanism. hsp90 cleavage induced by these inhibitors can be classified into two types: enzymatic cleavage and non-enzymatic cleavage. 674, 676 the enzymatic cleavage produces a 55 kda fragment of hsp90 via caspase 10 activation, while the non-enzymatic cleavage utilizes reactive oxygen species (ros) to chemically degrade hsp90 to an~70 kda fragment. additionally, some substances have been reported to present hsp90 cleavage activity, but the mechanism remains unclear. targeting hsp70s for drug development: one kind of antiviral drugs are currently designed based on different properties of hsps. considering hsp70 and hsc70 are potential targets for hcv, they load the dcs with hsp70 for a prolonged potent antigen-and tumor-specific t cell responses directed against multiple epitopes. 677 hsp70 inhibitors (such as quercetin, ver155008 and jc40) also show great potential for treating flavivirus and enterovirus infections. 202, 208, 640 inhibitors blocking the interaction between grp78 and spike protein of coronavirus would be a useful approach for combating the infection by sars-cov, mers-cov, and sars-cov-2 infections. 196, 387, 571 however, the efficacy and toxicity of these inhibitors are not yet well investigated in clinic studies. targeting hsp60s for drug development: as described above, hsp60s play critical roles in different diseases including autoimmune diseases, human cancers and virus infection-induced diseases. studies show that silencing of hsp60s by sirna significantly decrease influenza virus, 38,39 denv 319 and hbv 325, 331 reproduction by activating immunity response; and is capable of releasing hcv 321,322 , rotavirus, 323 hbv 329 and hiv 338 infectioninduced pathogenesis. therefore, small molecule regents targeting hsp60s are potentially useful as therapeutics in these diseases. 678, 679 currently, the known hsp60 inhibitors are either from natural products or synthetic compounds (table 3) . mechanistically, they can be grouped as two types. type i inhibitors block atp binding and hydrolysis. type ii inhibitors covalently interact with certain cysteine residues of hsp60. however, the detailed binding sites have not been identified. the natural products of hsp60 inhibitors include mizoribine, epolactaene, myrtucommulone a, stephacidin b. mizoribine is an imidazole nucleoside antibiotics isolated from eupenicillium brefeldianum. 680 it directly binds to hsp60 and inhibits the chaperone activity of the hsp60-hsp10 complex. 33 epolactaene is isolated from the fungal strain penicillium sp. bm 1689-p. 681 its derivate tert-butyl ester etb has similar activity as epolactaene. 682 they inhibit hsp60-hsp10's chaperoning activity by covalent and selective bound to hsp60 at residue of cys442, close to the atp binding pocket. 683, 684 interestingly, etb does not inhibit the atpase activity of hsp60, 685 suggesting that the covalent interaction between etb and cys442 may allosterically modulate hsp60-hsp10's chaperoning activity without interfering with its atpase activity. myrtucommulone a (mc) binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. 686 mc is a non-prenylated acylphloroglucinol with multiple reported bioactivities, including antibacterial, 687,688 antioxidant, 689 antiinflammatory, 690, 691 and anti-tumor properties. 692, 693 in addition to these natural products, several other natural products are also reported to interact with hsp60 without direct proof. stephacidin b is isolated from aspergillus ochraceus wc76466 while avrainvillamide is isolated from aspergillus sp. cnc358. 694, 695 interestingly, dimeric stephacidin b is converted into monomeric avrainvillamide in tissue culture media which implies avrainvillamide is the actual active species. 696 pull down assay only shows hsp60 may be a putative target. 696 further validation studies have yet to be performed. besides the natural products identified above as potential hsp60 inhibitors, quite a few synthetic molecules have also been discovered to be able to modulate hsp60 activity. o-carboranylphenoxyacetanilide is firstly identified as an hif-1α inhibitor. 697 later it is found that o-carboranylphenoxyacetanilide selectively and directly binds to hsp60 and inhibits hsp60-hsp10's atpase activity and refolding activity. 685 hsp60 can interact with hif-1α, suggesting that inhibition of hif-1α activity by carboranylphenoxyacetanilide is possibly through targeting hsp60. 685 the other class of synthetic compounds identified to inhibit hsp60 is gold porphyrins. 698 it directly binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. although several hsp60 inhibitors potently suppress virus reproduction, a lot of work remains to be done to verify if they can serve as drugs for treating diseases caused by virus infections. targeting hnrnps for antivirus drug development. drugs targeting rna chaperones may have a wide spectrum of antivirus capability. hnrnp a1 participates the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c suppresses the replication of hcv, 438 514, 536, 537 however, the available regents developed targeting hnrnps as antiviral drugs are limited. to date, only three agents targeting hnrnps have been reported. apigenin is a flavonoid abundant in fruits and vegetables. it targets hnrnp a2 and blocks hnrnp a2 dimerization thereafter affects the alternative splicing of key mrnas. 699 apigenin efficiently inhibits the interaction of hnrnp a1 and a2 with ev-a71 rna thereby inhibits ev-a71 rna translation. 472 quercetin is also a flavonoid abundantly present in plants. it binds to the c-terminal region of hnrnp a1, impairing the ability of hnrnp a1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. 700 in addition, quercetin down-regulates hnrnp a1 expression. 701 the antiviral ability of quercetin has been verified in some viruses such as inhibiting influenza entry, 702 ev-a71, 703 and hcv, 208 jev and denv reproduction. 704 however, in these researches, they explain the antivirus mechanism of quercetin is by inhibiting hsp70 expression 208 or inhibiting virus proteases 703 without mentioning the function of hnrnp a1. another compound vpc-80051 targets rna binding motif of hnrnp a1 and alters hnrnp a1 splicing activity. 705 its antiviral ability remains to be further investigated. targeting er stress pathways for drug development: during the past few years, the enlarging role of er stress-mediated pathways is becoming quite an attractive topic for broad-spectrum antiviral therapy, especially in the field of virus reproduction and pathogenesis, considerable progress has also been made for potential antiviral agents development. among pathways involved in er stress, the three upr pathways are the most thoroughly studied, thus antiviral targets related with these pathways are considered as the first class. extensive investigation for antiviral development has been put int to the perk-eif2α pathway. a small chemical compound named salubrinal, identified by boyce and his colleagues, specifically inhibits the formation of pp1/gadd34 complex as well as suppresses hsv γ134.5-mediated eif2α dephosphorylation. it could also significantly reduce hsv replication. 706 their research suggested that salubrinal was able to attenuate pp1/gadd34-dependent eif2α dephosphorylation, which would in turn inhibit denv replication. 707 a glucose analog 2-deoxy-d-glucose is responsible for the activation and phosphorylation of eif2α, leading to suppression of herpesvirus (kshv) replication. this agent is a potential candidate for anti-herpesvirus, and hopefully is tested in clinical therapies. 708 other than eif2α, tremendous efforts have also been made in developing drugs to target ire1-xbp1 pathway. 3,5-dibromosalicyladehyde is an endoribonuclease that specifically interacts with ire1 and blocks the downstream iav replication. 600 wp1130 is another ire1-xbp1 inhibitor with a broad-spectrum antiviral effects against multiple viruses, namely mnv-1, lcv and so on. 709 er stress-mediated apoptosis signaling is another noteworthy pathway for drug development and selection. rana catesbeiana ribonuclease (rc-rnase) is reported to be efficient in suppression of jev replication via activation of caspase-3, caspase-8 and caspase-9 pathways. 710 dubble-stranded rna activated caspase oligomerizer (draco) is reported to have broad anti-viral effects in clinical therapy. draco selectively activates apoptosis in cells infected with dsdna viruses but has no harmful effect on normal healthy cells. over 15 different kind of viruses can be eradicated by draco, including hiv and dengue virus. 711 drugs targeting other signal transducers are also investigated, for example jnk, jak, bcl-2 and chop. a promising agent vaticanol b can protect host cells from er stress-induced apoptosis. 712, 713 some researchers choose to focus on viral proteins that could trigger er-stress, including non-envelope glycoproteins of sfv, precursor glycoprotein of lcmv, glycoproteins tulv, several nonstructural protein of sars-cov, multiple non-structural proteins of flavivirus family, envelope protein of pedv, e1, e2 and nonstructral protein of hcv, surface protein of mhv, icp0 glycoprotein b of hsv1, structural protein 4 of chikv. viral proteins that are responsible for upr activation and apoptosis induction are given special attentions such as hcv (e1, e2, core), hcmv (pul37x1, pul38) and sv5 v protein. great advances have been made in this area, current progress includes, clemizol for hcv ns4b, vaccine vectors for hsv-1 γ134, and norakin for iav hemagglutinin a. 714, 715 due to their potential side effects on normal cell functions, the usage of inhibitors targeting stress proteins are very cautious in clinic settings; and the gone with wind infection manner of some virus (e.g., sars-cov) limits the opportunity of clinic studies, giving rise to additional challenges for developing stress protein-based drugs against the common and special infectious diseases such as severe acute respiratory symptom (sars) caused by coronavirus. we thank prof. robert weiss at cornell university college of veterinary medicine for critical reading and comments in preparation of this manuscript. the work was partially supported by grants from the science technology and innovation committee of shenzhen municipality [jcyj20170818100531426, jcyj20180507181627057]; grants from national science foundation of china in and out of the er: protein folding, quality control, degradation, and related human diseases a. new puffing pattern induced by temperature shock and dnp in drosophila molecular chaperone functions of heat-shock proteins the growing world of small heat shock proteins: from structure to functions 70 and 90, anti-apoptotic proteins, in clinical cancer therapy molecular chaperones in protein folding and proteostasis the role of heat shock proteins in antigen cross presentation novel signal transduction pathway utilized by extracellular hsp70: role of toll-like receptor (tlr) 2 and tlr4 heat shock protein 70 acts as a potential biomarker for early diagnosis of heart failure hsp27 expression in the human peripheral blood mononuclear cells as an early prognostic biomarker in coronary artery disease patients chaperone machines for protein folding, unfolding and disaggregation the hsp90 family: structure,regulation, function, and implications in health and disease molecular chaperones and protein quality control involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits the hsp90 chaperone machinery heat shock proteins: cellular and molecular mechanisms in the central nervous system structural analysis of substrate binding by the molecular chaperone dnak the human hsp70 family of chaperones: where do we stand? gymnastics of molecular chaperones the kinetic parameters and energy cost of the hsp70 chaperone as a polypeptide unfoldase hepatitis c virus, er stress, and oxidative stress chaperones in hepatitis c virus infection hsp70 chaperone dynamics and molecular mechanism the hsp70 chaperone machinery: j-proteins as drivers of functional specificity two families of chaperonin: physiology and mechanism folding of newly translated proteins in vivo: the role of molecular chaperones mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets group ii chaperonins: new tric(k)s and turns of a protein folding machine structure of holo-chaperonin studied with electron microscopy oligomeric opal0 on top of two layers of cpn60 rings with two stripes each a chaperonin from a thermophilic bacterium, thermus thermophilus. in molecular chaperones review: a structural view of the groe chaperone cycle groel and the groel-groes complex mammalian hsp60 is a major target for an immunosuppressant mizoribine mammalian hsp60 is quickly sorted into the mitochondria under conditions of dehydration mammalian 60-kda stress protein (chaperonin homolog) physicochemical properties of the mammalian molecular chaperone hsp60 down-regulation of hsp60 suppresses the proliferation of glioblastoma cells via the ros/ampk/mtor pathway the pb2 subunit of the influenza virus rna polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon association of the influenza virus rna polymerase subunit pb2 with the host chaperonin cct heat shock protein 60: regulatory role on innate immune cells the small heat shock proteins family: the long forgotten chaperones crystal structure and assembly of a eukaryotic small heat shock protein a first line of stress defense: small heat shock proteins and their function in protein homeostasis the small heat shock protein hsp27: present understanding and future prospects structure and function of small heat shock/α-crystallin proteins: established concepts and emerging ideas mechanism of chaperone function in small heat shock proteins regulation of aging and age-related disease by daf-16 and heatshock factor hsp27 negatively regulates cell death by interacting with cytochrome c some like it hot: the structure and function of small heat-shock proteins small heat shock protein activity is regulated by variable oligomeric substructure small heat shock proteins hsp27 and αb-crystallin: cytoprotective and oncogenic functions heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27 muscle develops a specific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology heat shock protein 27: its potential role in vascular disease hsp27 responds to and facilitates enterovirus a71 replication by enhancing viral internal ribosome entry site-mediated translation heterooligomeric complexes formed by human small heat shock proteins hspb1 (hsp27) and hspb6 (hsp20) large potentials of small heat shock proteins blocking tumor cell migration and invasion with biphenyl isoxazole derivative kribb3, a synthetic molecule that inhibits hsp27 phosphorylation quantification of hsp27 and hsp70 molecular chaperone activities human adenovirus type 19 infection of corneal cells induces p38 mapk-dependent interleukin-8 expression mapk and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption heat shock protein 27 mediated signaling in viral infection hsp27 expression regulates cck-induced changes of the actin cytoskeleton in cho-cck-a cells increased hsp27 after androgen ablation facilitates androgenindependent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of hsp27 heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (tak1)-mediated signaling hsp27 regulates pro-inflammatory mediator release in keratinocytes by modulating nf-κb signaling increased expression of the m (r) 27,000 heat shock protein (hsp27) in in vitro differentiated normal human keratinocytes 28-kda mammalian heat shock protein, a novel substrate of a growth regulatory protease involved in differentiation of human leukemia cells regulation of hsf 1 function in the heat stress response: implications in aging and disease targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis novel aspects of heat shock factors: dna recognition, chromatin modulation and gene expression proteins of the pdi family: unpredicted non-er locations and functions the protein disulfide isomerase family: key players in health and disease erp57 functions as a subunit of specific complexes formed with the er lectins calreticulin and calnexin protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and erp57 rna chaperone stpa loosens interactions of the tertiary structure in the td group i intron in vivo rna chaperones and the rna folding problem probing the folding landscape of the tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway exposing the kinetic traps in rna folding strategies for rna folding and assembly protein enhancement of hammerhead ribozyme catalysis a. chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis domain analysis of the saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, nab2p. dissecting the requirements for nab2p-facilitated poly(a) rna export role of rna chaperones in virus replication heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications altered states: selectively drugging the hsp90 cancer chaperone heat shock proteins promote cancer: it's a protection racket heat shock proteins and cancer: intracellular chaperones or extracellular signalling ligands? disruption of rna metabolism in neurological diseases and emerging therapeutic interventions heat shock proteins as potential targets for protective strategies in neurodegeneration severe respiratory viral infections: new evidence and changing paradigms pathogenesis of viral respiratory infection. respiratory disease and infection-a new insight, intechopen lvd viruses causing gastroenteritis: the known, the new and those beyond identification of mw polyomavirus, a novel polyomavirus in human stool discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin a highly prevalent and genetically diversified picornaviridae genus in south asian children immunological features underlying viral hemorrhagic fevers pathogenesis of the viral hemorrhagic fevers the role of viruses in neurodegenerative and neurobehavioral diseases herpes viruses and alzheimer's disease: new evidence in the debate virus infection and human cancer: an overview. recent results cancer res heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for therapy dengue virus entry into liver (hepg2) cells is independent of hsp90 and hsp70 heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells characterization of putative japanese encephalitis virus receptor molecules on microglial cells antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo heat-shock protein 90 is essential for stabilization of the hepatitis c virus nonstructural protein ns3 destabilization of pdk1 by hsp90 inactivation suppresses hepatitis c virus replication through inhibition of prk2-mediated viral rna polymerase phosphorylation hepatitis c virus rna replication is regulated by fkbp8 and hsp90 identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis hsp90 inhibitors reduce influenza virus replication in cell culture mc1568 inhibits hdac6/8 activity and influenza a virus replication in lung epithelial cells: role of hsp90 acetylation autophagy is involved in regulating influenza a virus rna and protein synthesis associated with both modulation of hsp90 induction and mtor/p70s6k signaling pathway pim1 impacts enterovirus a71 replication and represents a potential target in antiviral therapy host cell factor requirement for hepatitis c virus enzyme maturation evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp90 is a therapeutic target for noroviruses influenza a virus neuraminidase protein interacts with hsp90, to stabilize itself and enhance cell survival viral transport and the cytoskeleton the hepatitis e virus open reading frame 3 product interacts with microtubules and interferes with their dynamics the ins and outs of tubulin acetylation: more than just a post-translational modification? regulation of microtubule assembly and stability by the transactivator of transcription protein of jembrana disease virus heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin glucocorticoid stimulates hepatitis b viral gene expression in cultured human hepatoma cells geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus hepatitis b virus replication hsp90 is required for the activity of a hepatitis b virus reverse transcriptase requirement of heat shock protein 90 for human hepatitis b virus reverse transcriptase function in vitro reconstitution of a functional duck hepatitis b virus reverse transcriptase: posttranslational activation by hsp90 hbv polymerase interacts independently with nterminal and c-terminal fragments of hsp90beta chaperone activation of the hepadnaviral reverse transcriptase for template rna binding is established by the hsp70 and stimulated by the hsp90 system efficient hsp90-independent in vitro activation by hsc70 and hsp40 of duck hepatitis b virus reverse transcriptase, an assumed hsp90 client protein hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids hsp90 makes the human hbv pol competent for in vitro priming rather than maintaining the human hbv pol/ pregenomic rna complex expression of stable hepatitis b viral polymerase associated with grp94 in e. coli herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro hsp90 inhibitors: a potential treatment for latent ebv infection? nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 the herpes simplex virus vp16-induced complex: the makings of a regulatory switch herpes simplex virus disrupts nf-kappab regulation by blocking its recruitment on the ikappabalpha promoter and directing the factor on viral genes heat-shock protein 90α is involved in maintaining the stability of vp16 and vp16-mediated transactivation of α genes from herpes simplex virus-1 geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus curcumin inhibits human cytomegalovirus by downregulating heat shock protein 90 a novel hsp90 inhibitor, 17-dmag, induces tax down-regulation and its oral administration to atl-model mice intervenes against the infiltration property of the atl-like lymphocytes and provides extended survival period processing of the herpes simplex virus regulatory protein alpha 22 mediated by the ul13 protein kinase determines the accumulation of a subset of alpha and gamma mrnas and proteins in infected cells icp22 and the ul13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of rna polymerase ii epstein-barr virus-encoded protein kinase (bglf4) is involved in production of infectious virus the human cytomegalovirus ul44 protein is a substrate for the ul97 protein kinase distinct and separate roles for herpesvirus-conserved ul97 kinase in cytomegalovirus dna synthesis and encapsidation the human cytomegalovirus ul97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress viral mimicry of cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress conserved herpesvirus kinases target the dna damage response pathway and tip60 histone acetyltransferase to promote virus replication sumo binding by the epstein-barr virus protein kinase bglf4 is crucial for bglf4 function hsp90 inhibitors block outgrowth of ebv-infected malignant cells in vitro and in vivo through an ebna1-dependent mechanism ganetespib, an hsp90 inhibitor, kills epstein-barr virus (ebv)-infected b and t cells and reduces the percentage of ebv-infected cells in the blood ebp2 plays a key role in epstein-barr virus mitotic segregation and is regulated by aurora family kinases epstein-barr virus provides a survival factor to burkitt's lymphomas structure of the p53 binding domain of hausp/usp7 bound to epstein-barr nuclear antigen 1 implications for ebv-mediated immortalization self-inhibition of synthesis and antigen presentation by epstein-barr virus-encoded ebna1 heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers reactive oxygen species promote heat shock protein 90-mediated hbv capsid assembly virus particles in cultured lymphoblasts from burkitt's lymphoma epstein-barr virus in the pathogenesis of npc all three domains of the epstein-barr virus-encoded latent membrane protein lmp-1 are required for transformation of rat-1 fibroblasts epstein-barr virus induces invasion and metastasis factors a novel hsp90 inhibitor at13387 induces senescence in ebvpositive nasopharyngeal carcinoma cells and suppresses tumor formation the heat shock protein 90 inhibitor biib021 suppresses the growth of t and natural killer cell lymphomas heat shock protein 90 expression in epstein-barr virus-infected b cells promotes gammadelta t-cell proliferation in vitro tcrgammadelta cells and viruses heat shock protein 90 (hsp90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway nlr, the nucleotide-binding domain leucine-rich repeat containing gene family the hsp90-sgt1 chaperone complex for nlr immune sensors molecular mechanisms of cellular transformation by htlv-1 tax activation of nf-kappab by htlv-i and implications for cell transformation hsp90 protects the human t-cell leukemia virus type 1 (htlv-1) tax oncoprotein from proteasomal degradation to support nf-κb activation and htlv-1 replication grp78, a coreceptor for coxsackievirus a9, interacts with major histocompatibility complex class i molecules which mediate virus internalization integrin alpha v beta 6 is an rgd-dependent receptor for coxsackievirus a9 heat shock protein 70 as a supplementary receptor facilitates enterovirus 71 infections in vitro surface proteins of c6/36 cells involved in dengue virus 4 binding and entry zika virus dependence on host hsp70 provides a protective strategy against infection and disease heat shock protein 70 on neuro2a cells is a putative receptor for japanese encephalitis virus association of heat-shock protein 70 with lipid rafts is required for japanese encephalitis virus infection in huh7 cells heat shock cognate protein 70 isoform d is required for clathrin-dependent endocytosis of japanese encephalitis virus in c6/36 cells grp78 is an important host factor for japanese encephalitis virus entry and replication in mammalian cells japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity natural products may interfere with sars-cov-2 attachment to the host cell heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells heat shock cognate protein 70 is involved in rotavirus cell entry interaction of rotaviruses with hsc70 during cell entry is mediated by vp5 the peptide-binding and atpase domains of recombinant hsc70 are required to interact with rotavirus and reduce its infectivity heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway heat shock protein 90 ensures efficient mumps virus replication by assisting withfang viral polymerase complex formation constitutive overexpression of the major inducible 70 kda heat shock protein mediates large plaque formation by measles virus enhanced production of morbillivirus gene-specific rnas following induction of the cellular stress response in stable persistent infection the highly inducible member of the 70 kda family of heat shock proteins increases canine distemper virus polymerase activity heat shock protein 72 is associated with the hepatitis c virus replicase complex and enhances viral rna replication tylophorine analogs allosterically regulates heat shock cognate protein 70 and inhibits hepatitis c virus replication the heat shock protein inhibitor quercetin attenuates hepatitis c virus production human respiratory syncytial virus n, p and m protein interactions in hek-293t cells evidence for an association between heat shock protein 70 and the respiratory syncytial virus polymerase complex within lipid-raft membranes during virus infection interactome analysis of the human respiratory syncytial virus rna polymerase complex identifies protein chaperones as important cofactors that promote l-protein stability and rna synthesis elucidation of the cellular interactome of ebola virus nucleoprotein and identification of therapeutic targets an rna polymerase ii-driven ebola virus minigenome system as an advanced tool for antiviral drug screening recombinant rna-dependent rna polymerase complex of ebola virus a small stem-loop structure of the ebola virus trailer is essential for replication and interacts with heat-shock protein a8 cellular stress response induces selective intranuclear trafficking and accumulation of morbillivirus major core protein heat shock protein 70 modulates influenza a virus polymerase activity heat shock protein 70 promotes coxsackievirus b3 translation initiation and elongation via akt-mtorc1 pathway depending on activation of p70s6k and cdc2 hsc70 regulates the ires activity and serves as an antiviral target of enterovirus a71 infection the double-stranded rna-dependent protein kinase is also activated by heparin repression of the pkr protein kinase by the hepatitis c virus ns5a protein: a potential mechanism of interferon resistance the 58,000-dalton cellular inhibitor of the interferon-induced double-stranded rna-activated protein kinase (pkr) is a member of the tetratricopeptide repeat family of proteins purification and partial characterization of a cellular inhibitor of the interferon-induced protein kinase of mr 68,000 from influenza virus-infected cells characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsrna-activated protein kinase the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr the cellular inhibitor of the pkr protein kinase, p58(ipk), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity c-terminal trimerization, but not n-terminal trimerization, of the reovirus cell attachment protein is a posttranslational and hsp70/atp-dependent process association of heat shock protein 70 with enterovirus capsid precursor p1 in infected human cells heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo polo-like kinase 1 is involved in hepatitis c virus replication by hyperphosphorylating ns5a the ns5a-binding heat shock proteins hsc70 and hsp70 play distinct roles in the hepatitis c viral life cycle structural characterization of the hsp70 interaction domain of the hepatitis c viral protein ns5a sgta-dependent regulation of hsc70 promotes cytosol entry of simian virus 40 from the endoplasmic reticulum a non-enveloped virus hijacks host disaggregation machinery to translocate across the endoplasmic reticulum membrane bip and multiple dnaj molecular chaperones in the endoplasmic reticulum are required for efficient simian virus 40 infection adenovirus capsid proteins interact with hsp70 proteins after penetration in human or rodent cells novel partner proteins of adenovirus penton co-chaperone bag3 and adenovirus penton base protein partnership nuclear import of adenovirus dna in vitro involves the nuclear protein import pathway and hsc70 nuclear sequestration of cellular chaperone and proteasomal machinery during herpes simplex virus type 1 infection transcriptional stimulation by the dna binding protein hap46/bag-1m involves hsp70/hsc70 molecular chaperones transcriptional activation by the human hsp70-associating protein hap50 the hsp70-associating protein hap46 binds to dna and stimulates transcription bag-1, a novel bcl-2-interacting protein, activates expression of human jc virus bag-1m, an isoform of bcl-2-interacting protein bag-1, enhances gene expression driven by cmv promoter chaperone action in the posttranslational topological reorientation of the hepatitis b virus large envelope protein: implications for translocational regulation sequence-specific repression of cotranslational translocation of the hepatitis b virus envelope proteins coincides with binding of heat shock protein hsc70 chaperones involved in hepatitis b virus morphogenesis bag-1m accelerates nucleotide release for human hsc70 and hsp70 and can act concentration-dependent as positive and negative cofactor hsp70 interacting protein hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of bag-1 molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo mammalian bip controls posttranslational er translocation of the hepatitis b virus large envelope protein in vivo and in vitro association of hsc70 with polyomavirus capsid proteins chaperone-mediated in vitro assembly of polyomavirus capsids productive infection and cell-free transmission of human t-cell leukemia virus in a nonlymphoid cell line detection of lymphocytes producing a human retrovirus associated with adult t-cell leukemia by syncytia induction assay 71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human t-cell lymphotropic virus type 1 heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human t-cell lymphotropic virus type i heat-shock protein 70 can replace viral protein r of hiv-1 during nuclear import of the viral preintegration complex heat-shock proteins reverse the g2 arrest caused by hiv-1 viral protein r heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein r atpgammas disrupts human immunodeficiency virus type 1 virion core integrity the amino-terminal transforming region of simian virus 40 large t and small t antigens functions as a j domain the molecular chaperone activity of simian virus 40 large t antigen is required to disrupt rb-e2f family complexes by an atp-dependent mechanism atp-dependent simian virus 40 tantigen-hsc70 complex formation the regulation of e2f by prb-family proteins mechanism of regulation of hsp70 chaperones by dnaj cochaperones investigation of the interaction between dnak and dnaj by surface plasmon resonance spectroscopy priming polyvalent immunity by dna vaccines expressing chimeric antigens with a stress protein-capturing, viral j-domain loss of p19(arf) eliminates the requirement for the prb-binding motif in simian virus 40 large t antigenmediated transformation novel mechanisms of e2f induction by bk virus large-t antigen: requirement of both the prbbinding and the j domains j domain-independent regulation of the rb family by polyomavirus large t antigen the j domain of simian virus 40 large t antigen is required to functionally inactivate rb family proteins a novel human dnaj protein, htid-1, a homolog of the drosophila tumor suppressor protein tid56, can interact with the human papillomavirus type 16 e7 oncoprotein identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 e7 protein the human papillomavirus e7 oncoprotein and the cellular transcription factor e2f bind to separate sites on the retinoblastoma tumor suppressor protein differential distribution of the adenovirus e1a proteins and colocalization of e1a with the 70-kilodalton cellular heat shock protein in infected cells to kill or be killed: viral evasion of apoptosis apoptosis in viral pathogenesis viruses and apoptosis epstein-barr virus nuclear antigen (ebna) 3a induces the expression of and interacts with a subset of chaperones and co-chaperones caspase activation is required for permissive replication of aleutian mink disease parvovirus in vitro the mechanisms of direct, virus-induced destruction of neurons the hepatitis c virus core protein interacts with ns5a and activates its caspase-mediated proteolytic cleavage apoptosis in coxsackievirus b3-caused diseases: interaction between the capsid protein vp2 and the proapoptotic protein siva the apoptotic capability of coxsackievirus b3 is influenced by the efficient interaction between the capsid protein vp2 and the proapoptotic host protein siva hiv-1 vpr induces apoptosis through caspase 9 in t cells and peripheral blood mononuclear cells adenovirus encoding hiv-1 vpr activates caspase 9 and induces apoptotic cell death in both p53 positive and negative human tumor cell lines influenza virus ns1 protein induces apoptosis in cultured cells neurovirology and developmental neurobiology programmed cell death in virus infections of the nervous system ts-hsp70 induces protective immunity against trichinella spiralis infection in mouse by activating dendritic cells through tlr2 and tlr4 multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma hsp70 inhibits lipopolysaccharide-induced nf-κb activation by interacting with traf6 and inhibiting its ubiquitination hsp70/dnaja3 chaperone/cochaperone regulates nf-κb activity in immune responses the role of the membrane-initiated heat shock response in cancer enhanced heat shock protein 70 expression alters proteasomal degradation of ikappab kinase in experimental acute respiratory distress syndrome hsp70 is a negative regulator of nlrp3 inflammasome activation functional domains of hsp70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity hsp70 peptidembearing and peptide-negative preparations act as chaperokines toll-like receptor signalling toll-like receptor 4 (tlr4) is essential for hsp70-like protein 1 (hsp70l1) to activate dendritic cells and induce th1 response toll-like receptor signaling and its inducible proteins inflammation in gastric cancer: interplay of the cox-2/prostaglandin e2 and toll-like receptor/myd88 pathways tak1-ecsit-traf6 complex plays a key role in the tlr4 signal to activate nf-kappab cutting edge: traf6 mediates tlr/il-1r signaling-induced nontranscriptional priming of the nlrp3 inflammasome heme oxygenase-1 regulates dendritic cell function through modulation of p38 mapk-creb/atf1 signaling activated apoptotic cells induce dendritic cell maturation via engagement of toll-like receptor 4 (tlr4), dendritic cell-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin (dc-sign), and beta2 integrins il-1β induction of muc5ac gene expression is mediated by creb and nf-κb and repressed by dexamethasone induction of proinflammatory response in prostate cancer epithelial cells by activated macrophages autoimmune and inflammatory mechanisms in atherosclerosis heat shock proteins as ligands of toll-like receptors the hsp60 immune system network heat shock protein and innate immunity heat shock proteins: mediators of atherosclerotic development hsp60 critically regulates endogenous il-1β production in activated microglia by stimulating nlrp3 inflammasome pathway genome-wide rnai screen identifies human host factors crucial for influenza virus replication rna interference mediated silencing of hsp60 gene in human monocytic myeloma cell line u937 revealed decreased dengue virus multiplication hepatitis c virus: from oxygen free radicals to hepatocellular carcinoma mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein interaction of hepatitis c virus core protein with hsp60 triggers the production of reactive oxygen species and enhances tnf-alpha-mediated apoptosis tyrosine phosphorylation modulates mitochondrial chaperonin hsp60 and delays rotavirus nsp4-mediated apoptotic signaling in host cells cooperation between heat shock proteins in organizing of proteins spatial structure antisense oligodeoxynucleotides targeted against molecular chaperonin hsp60 block human hepatitis b virus replication human hepatitis b virus polymerase interacts with the molecular chaperonin hsp60 binding site analysis of human hbv pol for molecular chaperonin, hsp60 hepatitis b virus x protein: a multifunctional viral regulator interaction of the hepatitis b virus x protein (hbx) with heat shock protein 60 enhances hbx-mediated apoptosis hbx protein of hepatitis b virus (hbv) can form complex with mitochondrial hsp60 and hsp70 innate immune responses in hepatitis b virus (hbv) infection circulating and liver resident cd4 + cd25 + regulatory t cells actively influence the antiviral immune response and disease progression in patients with hepatitis b interferon effects on interleukin-10 secretion mononuclear cell response to interleukin-10 is normal in multiple sclerosis patients il-10-producing regulatory b cells (b10 cells) in autoimmune disease hepatitis b virus replication could enhance regulatory t cell activity by producing soluble heat shock protein 60 from hepatocytes an hsp60 related protein is associated with purified hiv and siv molecular mechanisms in retrovirus dna integration functional interactions of human immunodeficiency virus type 1 integrase with human and yeast hsp60 dnaj homolog hdj2 facilitates japanese encephalitis virus replication structure of the ribonucleoprotein of influenza virus a classical bipartite nuclear localization signal on thogoto and influenza a virus nucleoproteins an unconventional nls is critical for the nuclear import of the influenza a virus nucleoprotein and ribonucleoprotein human heat shock protein 40 (hsp40/dnajb1) promotes influenza a virus replication by assisting nuclear import of viral ribonucleoproteins dnaja1/hsp40 is co-opted by influenza a virus to enhance its viral rna polymerase activity molecular cloning and characterization of the human doublestranded rna-activated protein kinase induced by interferon influenza a virus nucleoprotein exploits hsp40 to inhibit pkr activation p58ipk, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eif2alpha signaling biochemical and genetic evidence for complex formation between the influenza a virus ns1 protein and the interferon-induced pkr protein kinase interaction of hsp40 with influenza virus m2 protein: implications for pkr signaling pathway flavivirus replication complex assembly revealed by dnajc14 functional mapping identification and characterization of the host protein dnajc14 as a broadly active flavivirus replication modulator chaperone-assisted protein folding is critical for yellow fever virus ns3/4a cleavage and replication dnajb1/hsp40 suppresses melanoma differentiation-associated gene 5-mitochondrial antiviral signaling protein function in conjunction with hsp70 cellular dnaja3, a novel vp1-interacting protein, inhibits footand-mouth disease virus replication by inducing lysosomal degradation of vp1 and attenuating its antagonistic role in the beta interferon signaling pathway human hsp70 and hsp40 chaperone proteins facilitate human papillomavirus-11 e1 protein binding to the origin and stimulate cell-free dna replication targeting the e1 replication protein to the papillomavirus origin of replication by complex formation with the e2 transactivator cell-free replication of the human papillomavirus dna with homologous viral e1 and e2 proteins and human cell extracts chaperone proteins abrogate inhibition of the human papillomavirus (hpv) e1 replicative helicase by the hpv e2 protein the human dnaj protein, htid-1, enhances binding of a multimer of the herpes simplex virus type 1 ul9 protein to oris, an origin of viral dna replication activation of the herpes simplex virus type-1 origin-binding protein (ul9) by heat shock proteins turnover of hepatitis b virus x protein is facilitated by hdj1, a human hsp40/dnaj protein negative regulation of hepatitis b virus replication by cellular hsp40/dnaj proteins through destabilization of viral core and x proteins hepatitis b virus x protein in the pathogenesis of hepatitis b virusinduced hepatocellular carcinoma high-level expression of hepatitis b virus hbx gene and hepatocarcinogenesis in transgenic mice x protein of hepatitis b virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis hepatitis b. x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma retroviral infection of non-dividing cells: old and new perspectives hiv-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway a nuclear localization signal within hiv-1 matrix protein that governs infection of non-dividing cells hiv-1 genome nuclear import is mediated by a central dna flap functional analysis of the simian immunodeficiency virus vpx protein: identification of packaging determinants and a novel nuclear targeting domain simian immunodeficiency virus vpx is imported into the nucleus via importin alphadependent and -independent pathways hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 vpx-mediated preintegration complex interference of dnajb6/mrj isoform switch by morpholino inhibits replication of hiv-1 and rsv large isoform of mammalian relative of dnaj is a major determinant of human susceptibility to hiv-1 infection heat shock protein 40 is necessary for human immunodeficiency virus-1 nef-mediated enhancement of viral gene expression and replication hiv-1 nef and host cell protein kinases nef triggers a transcriptional program in t cells imitating single-signal t cell activation and inducing hiv virulence mediators the plasma membrane as a combat zone in the hiv battlefield cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways reciprocal regulation of human immunodeficiency virus-1 gene expression and replication by heat shock proteins 40 and 70 novel role of hsp40/dnaj in the regulation of hiv-1 replication heat shock proteins as cellular lifeguards structural and functional diversities between members of the human hspb, hsph, hspa, and dnaj chaperone families muscle develops a dpecific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation epstein-barr virus upregulates phosphorylated heat shock protein 27 kda in carcinoma cells using the phosphoinositide 3-kinase/akt pathway proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo cellular hsp27 interacts with classical swine fever virus ns5a protein and negatively regulates viral replication by the nf-κb signaling pathway down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism the heat-shock response adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2 nf-κb and the map kinases/ap-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein i/ii, a modulin from oral streptococci signaling pathways for glycated human serum albumin-induced il-8 and mcp-1 secretion in human rpe cells mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery heat shock proteins: essential proteins for apoptosis regulation apoptosis versus cell differentiation: role of heat shock proteins hsp90, hsp70 and hsp27 small heat shock proteins hsp27 (hspb1), αb-crystallin (hspb5) and hsp22 (hspb8) as regulators of cell death modification and reorganization of the cytoprotective cellular chaperone hsp27 during herpes simplex virus type 1 infection heat shock protein 27 is involved in pcv2 infection in pk-15 cells hspb1 is an intracellular antiviral factor against hepatitis b virus role of thiol/disulfide exchange in newcastle disease virus entry overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein endothelial cell surface expression of protein disulfide isomerase activates beta1 and beta3 integrins and facilitates dengue virus infection protein disulfide isomerase mediates dengue virus entry in association with lipid rafts inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry the catalytic activity of protein disulfide isomerase is involved in human immunodeficiency virus envelope-mediated membrane fusion after cd4 cell binding protein-disulfide isomerase-mediated reduction of two disulfide bonds of hiv envelope glycoprotein 120 occurs post-cxcr4 binding and is required for fusion galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance t-cell migration and hiv entry structure of murine polyomavirus complexed with an oligosaccharide receptor fragment how viruses use the endoplasmic reticulum for entry, replication, and assembly simian virus 40 depends on er protein folding and quality control factors for entry into host cells a pdi family network acts distinctly and coordinately with erp29 to facilitate polyomavirus infection oblongifolin m, an active compound isolated from a chinese medical herb garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of erp57 cellular self-defense: how cellautonomous immunity protects against pathogens role of free radicals in viral pathogenesis and mutation ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: role for lymphocyte loss in hiv infection. free radic role of oxidants in influenza virus-induced gene expression investigation of oxidative stress and antioxidant defense in patients with hepatitis b virus infection and the effect of interferon-alpha plus lamivudine combination therapy on oxidative stress hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production oxidative damage to neurons caused by the induction of microglial nadph oxidase in encephalomyocarditis virus infection cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures oxidative stress in hpv-driven viral carcinogenesis: redox proteomics analysis of hpv-16 dysplastic and neoplastic tissues folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment n-glycosylation of the premembrane protein of japanese encephalitis virus is critical for folding of the envelope protein and assembly of viruslike particles hepatitis c virus glycoprotein folding: disulfide bond formation and association with calnexin an rna chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis the ebola virus vp24 protein prevents hnrnp c1/c2 binding to karyopherin α1 and partially alters its nuclear import hnrnps relocalize to the cytoplasm following infection with vesicular stomatitis virus hnrnp proteins and the biogenesis of mrna direct and indirect effects on viral translation and rna replication are required for auf1 restriction of enterovirus infections in human cells cellular mrna decay protein auf1 negatively regulates enterovirus and human rhinovirus infections heterogeneous nuclear ribonucleoprotein a2 participates in the replication of japanese encephalitis virus through an interaction with viral proteins and rna viral and cellular proteins involved in coronavirus replication hnrnp c and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'ntr of the hcv rna genome mechanistic consequences of hnrnp c binding to both rna termini of poliovirus negative-strand rna intermediates characterization of multimeric complexes formed by the human ptb1 protein on rna heterogeneous nuclear ribonucleoprotein i (hnrnp-i/ptb) selectively binds the conserved 3′ terminus of hepatitis c viral rna role of la autoantigen and polypyrimidine tract-binding protein in hcv replication polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis hur displaces polypyrimidine tract binding protein to facilitate la binding to the 3′ untranslated region and enhances hepatitis c virus replication the la antigen binds 5′ noncoding region of the hepatitis c virus rna in the context of the initiator aug codon and stimulates internal ribosome entry site-mediated translation hepatitis c virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human la autoantigen hur, a protein implicated in oncogene and growth factor mrna decay, binds to the 3′ ends of hepatitis c virus rna of both polarities delayed kinetics of poliovirus rna synthesis in a human cell line with reduced levels of hnrnp c proteins microrna-555 has potent antiviral properties against poliovirus cellular rna binding proteins ns1-bp and hnrnp k regulate influenza a virus rna splicing ns1-binding protein (ns1-bp): a novel human protein that interacts with the influenza a virus nonstructural ns1 protein is relocalized in the nuclei of infected cells co-regulatory activity of hnrnp k and ns1-bp in influenza and human mrna splicing rna-binding protein hnrnp d modulates internal ribosome entry site-dependent translation of hepatitis c virus rna two distinct binding modes of a protein cofactor with its target rna mechanism for nucleic acid chaperone activity of hiv-1 nucleocapsid protein revealed by single molecule stretching heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 5′ untranslated region and participates in virus replication the polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation feline calicivirus replication: requirement for polypyrimidine tract-binding protein is temperature-dependent internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of unr to two distinct sites on the 5′ untranslated region evidence for an rna chaperone function of polypyrimidine tractbinding protein in picornavirus translation interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis c virus rna genome and its functional requirement in internal initiation of translation hnrnp l is required for the translation mediated by hcv ires heterogeneous nuclear ribonucleoprotein l interacts with the 3 border of the internal ribosomal entry site of hepatitis c virus hnrnp l and nf90 interact with hepatitis c virus 5′-terminal untranslated rna and promote efficient replication a human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein k (hnrnp k) recognizes the 5′ terminal sequence of the hepatitis c virus rna heterogeneous ribonucleoprotein k (hnrnp k) binds mir-122, a mature liver-specific microrna required for hepatitis c virus replication modulation of hepatitis c virus rna abundance by a liver-specific microrna global epidemiology and genotype distribution of the hepatitis c virus infection position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome hepatitis c virus core protein interacts with heterogeneous nuclear ribonucleoprotein k an rna-binding protein, hnrnp a1, and a scaffold protein, septin 6, facilitate hepatitis c virus replication hnrnp a1 interacts with the 5' untranslated regions of enterovirus 71 and sindbis virus rna and is required for viral replication apigenin inhibits enterovirus-71 infection by disrupting viral rna association with trans-acting factors the role of misshapen nck-related kinase (mink), a novel ste20 family kinase, in the ires-mediated protein translation of human enterovirus 71 n-terminomics tails identifies host cell substrates of poliovirus and coxsackievirus b3 3c proteinases that modulate virus infection heterogeneous nuclear ribonucleoprotein m facilitates enterovirus infection the heterogeneous nuclear ribonucleoprotein k (hnrnp k) is a host factor required for dengue virus and junín virus multiplication the heterogeneous nuclear ribonucleoprotein k (hnrnp k) interacts with dengue virus core protein identification of heterogeneous nuclear ribonucleoprotein k (hnrnp k) as a repressor of c/ebpmediated gene activation role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release identification of human hnrnp c1/c2 as a dengue virus ns1-interacting protein heterogeneous nuclear ribonucleoprotein k supports vesicular stomatitis virus replication by regulating cell survival and cellular gene expression downregulation of nipah virus n mrna occurs through interaction between its 3′ untranslated region and hnrnp d hnrnp a2/b1 interacts with influenza a viral protein ns1 and inhibits virus replication potentially through suppressing ns1 rna/ protein levels and ns1 mrna nuclear export the negative regulator of splicing element of rous sarcoma virus promotes polyadenylation a cellular protein, hnrnp h, binds to the negative regulator of splicing element from rous sarcoma virus efficient polyadenylation of rous sarcoma virus rna requires the negative regulator of splicing element analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication viral dna replication orientation and hnrnps regulate transcription of the human papillomavirus 18 late promoter host heterogeneous ribonucleoprotein k (hnrnp k) as a potential target to suppress hepatitis b virus replication cytidine deaminase apobec3b interacts with heterogeneous nuclear ribonucleoprotein k and suppresses hepatitis b virus expression the multifunctional herpes simplex virus ie63 protein interacts with heterogeneous ribonucleoprotein k and with casein kinase 2 ck2 protein kinase is stimulated and redistributed by functional herpes simplex virus icp27 protein rna polymerase ii holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells association of herpes simplex virus type 1 icp8 and icp27 proteins with cellular rna polymerase ii holoenzyme herpes simplex virus 1 icp27 is required for transcription of two viral late (gamma 2) genes in infected cells human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences a downstream polyadenylation element in human papillomavirus type 16 l2 encodes multiple ggg motifs and interacts with hnrnp h specific interaction between hnrnp h and hpv16 l1 proteins: implications for late gene auto-regulation enabling rapid viral capsid protein production the epstein-barr virus (ebv) sm protein enhances pre-mrna processing of the ebv dna polymerase transcript the alternative splicing factor hnrnp a1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element a 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hfip1, cstf-64, hnrnp c1/c2, and polypyrimidine tract binding protein identification of an hnrnp a1-dependent splicing silencer in the human papillomavirus type 16 l1 coding region that prevents premature expression of the late l1 gene inhibition of hpv-16 l1 expression from l1 cdnas correlates with the presence of hnrnp a1 binding sites in the l1 coding region identification of a 17-nucleotide splicing enhancer in hpv-16 l1 that counteracts the effect of multiple hnrnp a1-binding splicing silencers polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, sd3632 translational inhibition in vitro of human papillomavirus type 16 l2 mrna mediated through interaction with heterogenous ribonucleoprotein k and poly(rc)-binding proteins 1 and 2 c-src-mediated phosphorylation of hnrnp k drives translational activation of specifically silenced mrnas regulation of the epstein-barr virus c promoter by auf1 and the cyclic amp/protein kinase a signaling pathway auf1/hnrnp d is a novel protein partner of the eber1 noncoding rna of epstein-barr virus nuclear hnrnpa2b1 initiates and amplifies the innate immune response to dna viruses hnrnp a1 selectively interacts through its gly-rich domain with different rna-binding proteins hiv nef enhances tat-mediated viral transcription through a hnrnp-k-nucleated signaling complex cooperative-binding and splicing-repressive properties of hnrnp a1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly actin-associated hnrnp proteins as transacting factors in the control of mrna transport and localization genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients novel (n)pkc kinases phosphorylate nef for increased hiv transcription, replication and perinuclear targeting cloning and functional analysis of multiply spliced mrna species of human immunodeficiency virus type 1 alternative splicing of human immunodeficiency virus type 1 mrna modulates viral protein expression, replication, and infectivity a naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production exon recognition in vertebrate splicing intron recognition comes of age identification, purification, and biochemical characterization of u2 small nuclear ribonucleoprotein auxiliary factor interaction of u2af65 rs region with pre-mrna of branch point and promotion base pairing with u2 snrna a potential role for u2af-sap 155 interactions in recruiting u2 snrnp to the branch site sorting out the complexity of sr protein functions hnrnp complexes: composition, structure, and function alternative pre-mrna splicing: the logic of combinatorial control balanced splicing at the tat-specific hiv-1 3'ss a3 is critical for hiv-1 replication splicing efficiency of human immunodeficiency virus type 1 tat rna is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2 the tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3′ splice site behind the scenes of hiv-1 replication: alternative splicing as the dependency factor on the quiet an exonic splicing silencer downstream of the 3' splice site a2 is required for efficient human immunodeficiency virus type 1 replication biochemical and nmr study on the competition between proteins sc35, srp40, and heterogeneous nuclear ribonucleoprotein a1 at the hiv tat exon 2 splicing site rna biology identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new hiv-1 splicing regulator, protein hnrnp k a second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of tat mrna and binds protein hnrnp h control of hiv-1 env rna splicing and transport: investigating the role of hnrnp a1 in exon splicing silencer (ess3a) function the hnrnp a1 protein regulates hiv-1 tat splicing via a novel intron silencer element hnrnp a1 controls hiv-1 mrna splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure solution structure of the hiv-1 intron splicing silencer and its interactions with the up1 domain of heterogeneous nuclear ribonucleoprotein (hnrnp) a1 a truncated hnrnp a1 isoform, lacking the rgg-box rna binding domain, can efficiently regulate hiv-1 splicing and replication sc35 and heterogeneous nuclear ribonucleoprotein a/b proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate hiv-1 tat exon 2 splicing hnrnp a1 recruited to an exon in vivo can function as an exon splicing silencer a janus splicing regulatory element modulates hiv-1 tat and rev mrna production by coordination of hnrnp a1 cooperative binding rna splicing at human immunodeficiency virus type 1 3' splice site a2 is regulated by binding of hnrnp a/b proteins to an exonic splicing silencer element differential hnrnp d isoform incorporation may confer plasticity to the essv-mediated repressive state across hiv-1 exon 3 human immunodeficiency virus type 1 hnrnp a/b-dependent exonic splicing silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts sr proteins and hnrnp h regulate the splicing of the hiv tev-specific exon 6d the secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity members of the heterogeneous nuclear ribonucleoprotein h family activate splicing of an hiv-1 splicing substrate by promoting formation of atp-dependent spliceosomal complexes rna trafficking signals in human immunodeficiency virus type 1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly a late role for the association of hnrnp a2 with the hiv-1 hnrnp a2 response elements in genomic rna, gag, and vpr localization differential effects of hnrnp d/auf1 isoforms on hiv-1 gene expression depletion of hnrnp a2/b1 overrides the nuclear retention of the hiv-1 genomic rna mapping of determinants required for the function of the hiv-1 env nuclear retention sequence inhibition of hiv-1 gene expression by a fragment of hnrnp u nuclear mrna export: insights from virology protein kinase ck2 phosphorylation regulates the interaction of kaposi's sarcoma-associated herpesvirus regulatory protein orf57 with its multifunctional partner hnrnp k heterogeneous nuclear ribonucleoprotein a1 interferes with the binding of the human t cell leukemia virus type 1 rex regulatory protein to its response element human immunodeficiency virus type 1 (hiv-1) induces the cytoplasmic retention of heterogeneous nuclear ribonucleoprotein a1 by disrupting nuclear import: implications for hiv-1 gene expression a polypyrimidine tract facilitates the expression of kaposi's sarcoma-associated herpesvirus vflip through an internal ribosome entry site heat shock protein hsp72 plays an essential role in her2-induced mammary tumorigenesis. oncogene the role of heat shock protein 70 in mediating agedependent mortality in sepsis peptides and aptamers targeting hsp70: a novel approach for anticancer chemotherapy activation of hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation monitering heat shock protein 70 and heat shock protein 90 during herpes simplex virus type 1 infection hsp70 functions as a negative regulator of west nile virus capsid protein through direct interaction mitochondrial hsp70, hsp40, and hsp60 bind to the 3′ untranslated region of the murine hepatitis virus genome immunomodulatory activity of extracellular hsp70 mediated via paired receptors siglec-5 and siglec-14 extracellular cell stress proteins as biomarkers of human disease molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation cross-presentation of the oncofetal tumor antigen 5t4 from irradiated prostate cancer cells-a key role for heat-shock protein 70 and receptor cd91 membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen stress for maintaining memory: hsp70 as a mobile messenger for innate and adaptive immunity novel heat shock protein hsp70l1 activates dendritic cells and acts as a th1 polarizing adjuvant chaperokine activity of heat shock proteins hsp70 as endogenous stimulus of the toll/interleukin-1 receptor signal pathway chlamydial heat shock protein 60 activates macrophages and endothelial cells through toll-like receptor 4 and md2 in a myd88-dependent pathway association of hadha with human rna silencing machinery dengue virus modulates the unfolded protein response in a time-dependent manner heat shock protein 70 is associated with replicase complex of japanese encephalitis virus and positively regulates viral genome replication japanese encephalitis virus activates autophagy through xbp1 and atf6 er stress sensors in neuronal cells bovine viral diarrhea virus infection induces autophagy in mdbk cells the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis the emerging roles of viroporins in er stress response and autophagy induction during virus infection stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis b virus hepatitis b virus x protein (hbx) activates atf6 and ire1-xbp1 pathways of unfolded protein response herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perk and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein membrane biogenesis and the unfolded protein response the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection role of the host cell's unfolded protein response in arenavirus infection interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone bip/grp78, which is required for virion assembly betanodavirus up-regulates chaperone grp78 via er stress: roles of grp78 in viral replication and host mitochondria-mediated cell death influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ire1) stress pathway west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2α kinases perk and pkr activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production hepatitis c virus ns4b induces unfolded protein response and endoplasmic reticulum overload response-dependent nf-κb activation the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor human cytomegalovirus protein us11 provokes an unfolded protein response that may facilitate the degradation of class i major histocompatibility complex products hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response endoplasmic reticulum stress induced by hepatitis b virus x protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4 functional diversity of the hnrnps: past, present and perspectives heterogeneous nuclear ribonucleoproteins (hnrnps) in cellular processes: focus on hnrnp e1's multifunctional regulatory roles acute and chronic disease caused by enteroviruses hnrnp a1 alters the structure of a conserved enterovirus ires domain to stimulate viral translation heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 59 untranslated region and participates in virus replication molecular mechanism of action of hnrnp k and rtn3 in the replication of enterovirus 71. chin sherris medical microbiology, 6e anti-herpetic medications and reduced risk of dementia in patients with herpes simplex virus infections-a nationwide, population-based cohort study in taiwan antivirals reduce the formation of key alzheimer's disease molecules in cell cultures acutely infected with herpes simplex virus type 1 the heterogeneous nuclear ribonucleoprotein k is important for herpes simplex virus-1 propagation hepatitis b virus epidemiology and natural history of hcv infection pathobiology of chronic hepatitis virus infection and hepatocellular carcinoma (hcc). tal hepatitis delta virus polyarteritis nodosa and extrahepatic manifestations of hbv infection: the case against autoimmune intervention in pathogenesis autoantibody recognition of an n-terminal epitope of hnrnp l marks the risk for developing hbv-related hepatocellular carcinoma benign lymphadenopathies epstein-barr virus-associated lymphoproliferative disorders: experimental and clinical developments epstein-barr virus-associated hodgkin's lymphoma human papillomavirus and epstein-barr virus in nasopharyngeal carcinoma in a low-incidence population epstein-barr virus infection and multiple sclerosis: a review lymphomatoid granulomatosis: a practical review for pathologists dealing with this rare pulmonary lymphoproliferative process epstein-barr virus and skin manifestations in childhood monoclonal antibodies against epstein-barr virus cross-react with alpha-synuclein in human brain human papillomavirus (hpv) type distribution and serological response to hpv type 6 virus-like particles in patients with genital warts the global health burden of infection-associated cancers in the year 2002 trends in human papillomavirus-associated cancers-united states epidemiology and natural history of human papillomavirus infections in the female genital tract case-control study of human papillomavirus and oropharyngeal cancer review of human immunodeficiency virus type 1-related opportunistic infections in sub-saharan africa the treatment of patients with hiv defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection the hsp90 mosaic: a picture emerges the chaperone hsp90: changing partners for demanding clients structure of tpr domain-peptide complexes: critical elements in the assembly of the hsp70-hsp90 multichaperone machine hsp90 and co-chaperones twist the functions of diverse client proteins the 'active life' of hsp90 complexes structure and functional relationships of hsp90 cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp90 inhibitor as a selective inhibitor of hepatitis c virus novobiocin and additional inhibitors of the hsp90 cterminal nucleotide-binding pocket the amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an atp/adp switch domain that regulates hsp90 conformation 17-allylamino-17-demethoxygeldanamycin induces downregulation of critical hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells inhibition of signal transduction by the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis antitumor efficacy of ipi-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib nvp-auy922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis biib021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein hsp90 ganetespib, a unique triazolone-containing hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy the heat shock protein 90 inhibitor, at13387, displays a long duration of action in vitro and in vivo in non-small cell lung cancer snx2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against her kinase-dependent cancers novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins the heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized atpbinding domain in the carboxyl terminus of the chaperone design, synthesis, and biological evaluation of novel deguelinbased heat shock protein 90 (hsp90) inhibitors targeting proliferation and angiogenesis structural basis for depletion of heat shock protein 90 client proteins by deguelin epigallocatechin-3-gallate is a novel hsp90 inhibitor novel hsp90 inhibitor platycodin d disrupts hsp90/cdc37 complex and enhances the anticancer effect of mtor inhibitor sulforaphane inhibits pancreatic cancer through disrupting hsp90-p50cdc37 complex and direct interactions with amino acids residues of hsp90 withaferin a targets heat shock protein 90 in pancreatic cancer cells a novel hsp90 inhibitor to disrupt hsp90/cdc37 complex against pancreatic cancer cells natural product kongensin a is a non-canonical hsp90 inhibitor that blocks rip3-dependent necroptosis discovery and identification of cdc37-derived peptides targeting the hsp90-cdc37 protein-protein interaction targeting the hsp90-cdc37-client protein interaction to disrupt hsp90 chaperone machinery activity of suberoylanilide hydroxamic acid against human breast cancer cells with amplification of her-2 chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor laq824 modulation of p53, erbb1, erbb2, and raf-1 expression in lung cancer cells by depsipeptide fr901228 caspase-10-mediated heat shock protein 90β cleavage promotes uvb irradiation-induced cell apoptosis hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death suberoylanilide hydroxamic acid induces ros-mediated cleavage of hsp90 in leukemia cells proteasome inhibitor-induced cleavage of hsp90 is mediated by ros generation and caspase 10-activation in human leukemic cells dendritic cells transfected with heat-shock protein 70 messenger rna for patients with hepatitis c virus-related hepatocellular carcinoma: a phase 1 dose escalation clinical trial hsp60 chaperonopathies and chaperonotherapy: targets and agents hsp60 as a drug target studies on bredinin. i. isolation,characterization and biological properties hiroyuki epolactaene, a novel neuritogenic compound in human neuroblastoma cells, produced by a marine fungus structure-activity relationships of epolactaene derivatives: structural requirements for inhibition of hsp60 chaperone activity epolactaene binds human hsp60 cys442 resulting in the inhibition of chaperone activity crystal structure of the human mitochondrial chaperonin symmetrical football complex identification of hsp60 as a primary target of o-carboranylphenoxyacetanilide, an hif-1α inhibitor mitochondrial chaperonin hsp60 is the apoptosis-related target for myrtucommulone isolation and antibacterial activity of acylphloroglucinols from myrtus communis oligomeric acylphloroglucinols from myrtle (myrtus communis) antioxidant activity of oligomeric acylphloroglucinols from myrtus communis l myrtucommulone from myrtus communis, exhibits potent antiinflammatory effectiveness in vivo identification of molecular targets of the oligomeric nonprenylated acylphloroglucinols from and myrtle (myrtus communis) and their implication as anti-inflammatory compounds myrtucommulone from myrtus communis induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9 dual induction of mitochondrial apoptosis and senescence in chronic myelogenous leukemia by myrtucommulone a stephacidin a and b: two structurally novel, selective inhibitors of the testosterone-dependent prostate lncap cells avrainvillamide, a cytotoxic marine natural product, and derivatives thereof evidence for the rapid conversion of stephacidin b into the electrophilic monomer avrainvillamide in cell culture boron-containing phenoxyacetanilide derivatives as hypoxiainducible factor (hif)-1α inhibitors anticancer gold(iii) porphyrins target mitochondrial chaperone hsp60 molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnrnp) a1 as the molecular target of quercetin in its anti-cancer effects in pc-3 cells quercetin targets hnrnpa1 to overcome enzalutamide resistance in prostate cancer cells quercetin as an antiviral agent inhibits influenza a virus (iav) entry. viruses inhibition of enterovirus 71 replication and viral 3c protease by quercetin antiviral activity of baicalein and quercetin against the japanese encephalitis virus computer-aided discovery of small molecules targeting the rna splicing activity of hnrnp a1 in castration-resistant prostate cancer a selective inhibitor of eif2alpha dephosphorylation protects cells from er stress dengue virus serotype infection specifies the activation of the unfolded protein response activation of the unfolded protein response by 2-deoxy-dglucose inhibits kaposi's sarcoma-associated herpesvirus replication and gene expression antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response rana catesbeiana ribonuclease inhibits japanese encephalitis virus (jev) replication and enhances apoptosis of jev-infected bhk-21 cells broad-spectrum antiviral therapeutics vaticanol b, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress the hepatitis c virus (hcv) ns4b rna binding inhibitor clemizole is highly synergistic with hcv protease inhibitors spontaneous and engineered compensatory hsv mutants that counteract the host antiviral pkr response open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source [81671995]; the general research grant of hong kong [11100215] ; and strategic funds from city university of hong kong. competing interests: the authors declare no competing interests.commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-002774-tpqsjjet authors: nan title: section ii: poster sessions date: 2017-12-01 journal: j urban health doi: 10.1093/jurban/jti137 sha: doc_id: 2774 cord_uid: tpqsjjet nan food and nutrition programs in large urban areas have not traditionally followed a systems approach towards mitigating food related health issues, and instead have relied upon specific issue interventions char deal with downstream indicators of illness and disease. in june of 2004, the san francisco food alliance, a group of city agencies, community based organizations and residents, initiated a collahorarive indicator project called rhe san francisco food and agriculture assessment. in order to attend to root causes of food related illnesses and diseases, the purpose of the assessment is to provide a holistic, systemic view of san francisco\'s food system with a focus on three main areas that have a profound affect on urban public health: food assistance, urban agriculture, and food retailing. using participatory, consensus methods, the san francisco food alliance jointly developed a sec of indicators to assess the state of the local food system and co set benchmarks for future analysis. members collected data from various city and stare departments as well as community based organizations. through the use of geographic information systems software, a series of maps were created to illustrate the assets and limitations within the food system in different neighborhoods and throughout the city as a whole. this participatory assessment process illustrates how to more effectively attend to structural food systems issues in large urban areas by ( t) focusing on prevention rather than crisis management, (2) emphasizing collaboration to ensure institutional and structural changes, and (3) aptly translating data into meaningful community driven prevention activities. to ~xplore the strategies to overcome barriers to population sample, we examined the data from three rapid surveys conducted at los angeles county (lac). the surveys were community-based partic· 1patory surveys utilizing a modified two-stage cluster survey method. the field modifications of the method resulted in better design effect than conventional cluster sample survey (design effect dose to that if the survey was done as simple random sample survey of the same size). the surveys were con· ducte~ among parents of hispanic and african american children in lac. geographic area was selected and d1.v1ded int.o small c~usters. in the first stage, 30 clusters were selected with probability proportionate to estimated size of children from the census data. these clusters were enumerated to identify and develop a list of households with eligible children from where a random sample was withdrawn. data collectmn for consented respondents involved 10-15 minutes in-home interview and abstraction of infor· ma~ion from vaccine record card. the survey staff had implemented community outreach activities designed to fost~r an~ maintain community trust and cooperation. the successful strategies included: developing re.lat1on .w.1th local community organizations; recruitment of community personnel and pro· vide them with training to conduct the enumeration and interview; teaming the trained community introduction: though much research has been done on the health and social benefits of pet ownership for many groups, there have been no explorations of what pet ownership can mean to adults who are marginalized, living on fixed incomes or on the street in canada. we are a community group of researchers from downtown toronto. made up of front line staff and community members, we believe that community research is important so that our concerns, visions, views and values are presented by us. we also believe that research can and should lead to social change. method: using qualitative and exploratory methods, we have investigated how pet ownership enriched and challenged the lives of homeless and transitionally housed people. our research team photographed and conducted one-on-one interviews with 11 pet owners who have experienced home· lessncss and live on fixed incomes. we had community participation in the research through a partnership with the fred vicror centre camera club. many of the fred victor centre camera club members have experienced homelessness and being marginalized because of poverty. the members of the dub took the photos and assisted in developing the photos. they also participated in the presenta· tion of our project. results: we found that pet ownership brings important health and social benefits to our partici· pants. in one of the most poignant statements, one participant said that pet ownership " ... stops you from being invisible." another commented that "well, he taught me to slow down, cut down the heavy drugs .. " we also found that pet ownership brings challenges that can at times be difficult when one is liv· ing on a fixed income. we found that the most difficult thing for most of the pet owners was finding affordable vet care for their animals. conclusion: as a group, we decided that research should only be done if we try to make some cha.nges about what we have learned. we continue the project through exploring means of affecting social change--for example, ~eti.tions and informing others about the result of our project. we would like to present our ~mdmgs and experience with community-based participatory action resea.rch m an oral. presentarton at yo~r conference in october. our presentation will include com· mumty representation ~f. both front-hne staff and people with lived experience of marginalization and homdessness. if this is not accepted as an oral presentation, we are willing to present the project m poster format. introduction the concept of a healthy city was adopted by the world health organization some time ago and it includes strong support for local involvement in problem solving and implementation of solutions. while aimed at improving social, economic or environmental conditions in a given community, more significantly the process is considered to be a building block for poliq reform and larger scale 'hange, i.e. "acting locally while thinking globally." neighbourhood planning can he the entry point for citizens to hegin engaging with neighbours on issues of the greater common good. methods: this presentation will outline how two community driven projects have unfolded to address air pollution. the first was an uphill push to create bike lanes where car lanes previously existed and the second is an ongoing, multi-sectoral round table focused on pollution and planning. both dt•monstrate the importance of having support with the process and a health focus. borrowing from traditions of "technical aid"• and community development the health promoter /planner has incorporated a range of "determinants of health" into neighbourhood planning discussions. as in most urban conditions the physical environment is linked to a range of health stressors such as social isolation, crowding, noise, lack of open space /recreation, mobility and safety. however typical planning processes do not hring in a health perspective. health as a focus for neighbourhood planning is a powerful starti_ng point when discussing transportation planning or changing land-uses. by raising awareness on determmants of health, citizens can begin to better understand how to engage in a process and affect change. often local level politics are involved and citizens witness policy change in action. the environmental liaison committee and the dundas east hike lanes project resulted from local level initiatives aimed at finding solutions to air pollution -a priority identified hy the community. srchc supported the process with facilitation and technical aid. _the processs had tangible results that ultimately improve living conditions and health. •tn the united kmgdom plannm in the 60's established "technical aid" offices much like our present day legal aid system to provide professional support and advocacy for communities undergoing change. p2-15 (c) integrating community based research: the experience of street health, a community service agency i.aura cowan and jacqueline wood street health began offering services to homeless men and women in east downtown ~oronto in 1986. nursing stations at drop-in centres and shelters were fo~lowed by hiv/aids prevent10~, harm reduction and mental health outreach, hepatitis c support, sleeping bag exchange, and personal tdennfication replacement and storage programs. as street health's progi;ams expanded, so to~ did the agency:s recognition that more nee~ed t~ be done to. address the underl~ing causes of, th~ soct~l and economic exclusion experienced by its clients. knowing t.h~t. a~voca~y ts. helped by . evtd~nce , street he.alt~ embarked on a community-based research (cbr) initiative to 1dent1fy commumty-dnven research priorities within the homeless and underhoused population. methods: five focus groups were conducted with 46 homeless people, asking participants to identify positive and negative forces in their lives, and which topics were important to take action on and learn more about. findings were validated through a validation meeting with participants. results: participants identified several important positive and negative forces in their lives. key positive forces included caring and respectful service delivery, hopefulness and peer networks. key negative forces included lack of access to adequate housing and income security, poor service delivery and negative perceptions of homeless people. five topics for future research emerged from the process, focusing on funding to address homelessness and housing; use of community services for homeless people; the daily survival needs of homeless people and barriers to transitioning out of homelessness; new approaches to service delivery that foster empowerment; and policy makers' understanding of poverty and homelessness. conclusions: although participants expressed numerous issues and provided much valuable insight, definitive research ideas and action areas were not clearly identified by participants. however, engagement in a cbr process led to some important lessons and benefits for street health. we learned that the community involvement of homeless people and front-line staff is critical to ensuring relevance and validity for a research project; that existing strong relationships with community parmers are essential to the successful implementation of a project involving marginalized groups; and that an action approach focusing on positive change can make research relevant to directly affected people and community agency staff. street health benefited from using a cbr approach, as the research process facilitated capacity building among staff and within the organization as a whole. p2-16 (c) a collaborative process to achieve access to primary health care for black women and women of colour: a model of community based participartory research notisha massaquoi, charmaine williams, amoaba gooden, and tulika agerwal in the current healthcare environment, a significant number of black women and women of color face barriers to accessing effective, high quality services. research has identified several issues that contribute to decreased access to primary health care for this population however racism has emerged as an overarching determinant of health and healthcare access. this is further amplified by simultaneous membership in multiple groups that experience discrimination and barriers to healthcare for example those affected by sexism, homelessness, poverty, homophobia and heterosexism, disability and hiv infection. the collaborative process to achieve access to primary health care for black women and women of colour project was developed with the university of toronto faculty of social work and five community partners using a collaborative methodology to address a pressing need within the community ro increase access to primary health care for black women and women of colour. women's health in women's hands community health centre, sistering, parkdale community health centre, rexdale community health centre and planed parenthood of toronto developed this community-based participatory-action research project to collaboratively barriers affecting these women, and to develop a model of care that will increase their access to health services. this framework was developed using a process which ensured that community members from the target population and service providers working in multiculrural clinical settings, were a part of the research process. they were given the opportunity to shape the course of action, from the design of the project to the evaluation and dissemination phase. empowerment is a goal of the participatory action process, therefore, the research process has deliberately prioritized _ro enabling women to increase control over their health and well-being. in this session, the presenters will explore community-based participatory research and how such a model can be useful for understanding and contextualizing the experiences of black women and women of colour. they will address. the development and use of community parmerships, design and implementation of the research prorect, challenges encountered, lessons learnt and action outcomes. they will examine how the results from a collaborative community-based research project can be used as an action strategy to poster sessions v61 address che social determinants of women health. finally the session will provide tools for service providers and researchers to explore ways to increase partnerships and to integrate strategies to meet the needs of che target population who face multiple barriers to accessing services. lynn scruby and rachel rapaport beck the purpose of this project was ro bring traditionally disenfranchised winnipeg and surrounding area women into decision-making roles. the researchers have built upon the relationships and information gachered from a pilot project and enhanced the role of input from participants on their policy prioriries. the project is guided by an advisory committee consisting of program providers and community representatives, as well as the researchers. participants included program users at four family resource cencres, two in winnipeg and two located rurally, where they participated in focus groups. the participants answered a series of questions relating to their contact with government services and then provided inpuc as to their perceptions for needed changes within government policy. following data analysis, the researchers will return to the four centres to share the information and continue che discussion on methods for advocating for change. recommendations for program planning and policy development and implementation will be discussed and have relevance to all participants in the research program. women's health vera lefranc, louise hara, denise darrell, sonya boyce, and colleen reid women's experiences with paid and unpaid work, and with the formal and informal economies, have shifted over the last 20 years. in british columbia, women's employability is affected by government legislation, federal and provincial policy changes, and local practices. two years ago we formed the coalition ior women's economic advancement to explore ways of dealing with women's worsening economic situations. since the formation of the coalition we have discussed the need for research into women's employabilicy and how women were coping and surviving. we also identified how the need to document the nature of women's employability and reliance on the informal economy bore significanc mechodological and ethical challenges. inherent in our approach is a social model of women's health that recognizes health as containing social, economic, and environmental determinants. we aim to examine the social contexc of women's healch by exploring and legitimizing women's own experiences, challenging medical dominance in understandings of health, and explaining women's health in terms of their subordination and marginalizacion. through using a feminist action research (far) methodology we will explore the relationship between women's employability and health in 4 communities that represent bricish columbia's social, economic, cultural/ethnic, and geographic diversities: skidegate, fort st . .john, lumhy, and surrey. over the course of our 2 year project, in each community we will establish and work with advisory committees, hire and train local researchers, conduct far (including a range of qualitative methods), and support action and advocacy. since the selected communities are diverse, the ways that the research unfolds will 1·ary between communities. expected outcomes, such as the provision of a written report and resources, the establishment of a website for networking among the communities, and a video do.:umentary, are aimed at supporting the research participants, coalition members, and advisory conuniuces in their action efforts. p2t 9 (c) health & housing: assessing the impact of transitional housing for people living with hiv i aids currently, there is a dearth of available literature which examines supporrive housing for phas in the canadian context. using qualitative, one-on-one interviews we investigace the impact of transitional housing for phaswho have lived in the up to nine month long hastings program. our post<'r pr<·senta-t1on will highlight research findings, as well as an examination of transitional housing and th<· imp;kt it has on the everyday lives of phas in canada. this research is one of two ground breaking undertakings within the province of ontario in which fife house is involved. p2-20 (c) eating our way to justice: widening grassroots approaches to food security, the stop community food centre as a working model charles l.evkoe food hanks in north america have come co play a central role as the widespread response to growing rates of hunger. originally thought to be a short term-solution, over the last 25 years, they have v62 poster sessions be · · · 1· d wi'thi'n society by filling the gaps in the social safety net while relieving govemcome mst1tut1ona 1ze . . . t f the ir responsibilities. dependent on corporate donations and sngmauzmg to users, food banks men so th' . ·11 i i . are incapable of addressing the structural cause~ of ~u~ger. 1s pres~ntation w1 e~~ ore a ternanve approaches to addressing urban food security while bmldmg more sustamabl.e c~mmumt1es. i:nrough the f t h st p community food centre, a toronto-based grassroots orgamzanon, a model is presented case 0 e 0 h'l k' b 'id · b that both responds to the emergency food needs of communities w 1 e wor mg to. u1 ~ sustama le and just food system. termed, the community food centre model. (cfc), ~he s~op is worki?g to widen its approach to issues of food insecurity by combining respectful ~1rect service wit~ com~~mty ~evelop ment, social justice and environmental sustainability. through this approach, various critical discourses around hunger converge with different strategic and varied implications for a~ion. as a plac~-based organization, the stop is rooted within a geographical space and connected directly to a neighbourhood. through working to increase access to healthy food, it is active in maintaining people's dignity, building a strong and democratic community and educating for social change. connected to coalitions and alliances, the stop is also active in organizing across scales in connection with the global food justice movement. inner city shelter vicky stergiopoulos, carolyn dewa, katherine rouleau, shawn yoder, and lorne tugg introduction: in the city of toronto there are more than 32,000 hostel users each year, many with mental health and addiction issues. although shelters have responded in various ways to the health needs of their clients, evidence on the effectiveness of programs delivering mental health services to the home· less in canada has been scant. the objective of this community based research was to provide a forma· tive evaluation of a multi-agency collaborative care team providing comprehensive care for high needs clients at toronto's largest shelter for homeless men. methods: a logic model provided the framework for analysis. a chart review of 56 clients referred over a nine month period was completed. demographic data were collected, and process and outcome indicators were identified for which data was obtained and analyzed. the two main outcome measures were mental status and housing status 6 months after referral to the program. improvement or lack of improvement in mental status was established by chart review and team consensus. housing outcomes were determined by chart review and the hostel databases. results: of the clients referred 75% were single and 98% were unemployed. forty four percent had a psychiatric hospitalization within the previous two years. the prevalence of severe and persistent men· tal illness, alcohol and substance use disorders were 60%, 26% and 37% respectively. six months after referral to the program 37% of clients had improved mental status and 41 % were housed. logistic regression controlling for the number of general practitioner and psychiatrist visits, presence of person· ality or substance use disorder and treatment non adherence identified two variables significantly associ· ated mental status improvement: the number of psychiatric visits (or, 1.92; 95% ci, 1.29-2.84) and treatment non adherence (or, 0.086; 95% ci, 0.01-0.78). the same two variables were associated with housing outcomes. history of forensic involvement, the presence of a personality or substance use disorder and the number of visits with a family physician were not significantly associated with either outcome. conclusions: despite the limitations in sample sire and study design, this study can yield useful informa· tion to program planners. our results suggest that strategies to improve treatment adherence and access to mental health specialists can improve outcomes for this population. although within primary care teams the appropriate collaborative care model for this population remains to be established, access to psychiatric follow up, in addition to psychiatric assessment services, may be an important component of a successful program. mount sinai hospital (msh) has become one of the pre-eminent hospitals in the world by contributing to the development of innovative approaches to effective health care and disease prevention. recently, the hospital has dedicated resources towards the development of a strategy aimed at enhancing the hospital's integration with its community partners. this approach will better serve the hospital in the current health care environment where local health integration networks have been struck to enhance and support local capacity to plan, coordinate and integrate service delivery. msh has had early success with developing partnerships. these alliances have been linked to programs serving key target populations with _estabhshe~. points of access to msh. recognizing the need to build upon these achievements to remain compe~mve, the hospital has developed a community integration strategy. at the forefront of this strategy is c.a.r.e (community advisory reference engine): the hospital's compendium of poster sessions v63 community partners. as a single point of access to community partner information, c.a.r.e. is more than a database. c.a.r.e. serves as the foundation for community-focused forecasting and a vehicle for inter and intra-organizational knowledge transfer. information gleaned from the catalog of community parmers can be used to prepare strategic, long-term partnership plans aimed at ensuring that a comprehensive array of services can be provided to the hospital community. c.a.r.e. also houses a permanent record of the hospital's alliances. this prevents administrative duplication and facilitates the formation of new alliances that best serve both the patient and the hospital. c.a.r.e. is not a stand-alone tool and is most powerful when combined with other aspects of the hospital's community integration strategy. it iscxpected that data from the hospital's community advisory committees and performance measurement department will also be stored alongside stakeholder details. this information can then be used to drive discussions at senior management and the board, ensuring congruence between stakeholder, patient and hospital objectives. the patient stands to benefit from this strategy. the unique, distinct point of reference to a wide array of community services provides case managers and discharge planners with the information they need to connect patients with appropriate community services. creating these linkages enhances the patient's capacity to convalesce in their homes or places of residence and fosters long-term connections to neighborhood supports. these connections can be used to assist with identifying patients' ongoing health care needs and potentially prevent readmission to hospital. introduction: recruiting high-risk drug users and sex workers for hiv-prevention research has often been hampered by limited access to hard to reach, socially stigmatized individuals. our recruitment effom have deployed ethnographic methodology to identify and target risk pockets. in particular, ethnographers have modeled their research on a street-outreach model, walking around with hiv-prevention materials and engaging in informal and structured conversations with local residents, and service providers, as well as self-identified drug users and sex workers. while such a methodology identifies people who feel comfortable engaging with outreach workers, it risks missing key connections with those who occupy the margins of even this marginal culture. methods: ethnographers formed a women's laundry group at a laundromat that had a central role as community switchboard and had previously functioned as a party location for the target population. the new manager helped the ethnographers invite women at high risk for hiv back into the space, this time as customers. during weekly laundry sessions, women initiated discussions about hiv-prevention, sexual health, and eventually, the vaccine research for which the center would be recruiting women. ra.its: the benefits of the group included reintroducing women to a familiar locale, this time as customers rather than unwelcome intruders; creating a span of time (wash and dry) to discuss issues important to me women and to gather data for future recruitment efforts; creating a location to meet women encountered during more traditional outreach research; establishing the site as a place for potential retention efforts; and supporting a local business. women who participated in the group completed a necessary household task while learning information that they could then bring back to the community, empowering them to be experts on hiv-prevention and vaccine research. some of these women now assist recruitment efforts. the challenges included keeping the group women-only, especially after lunch was provided, keeping the membership of the group focused on women at risk for hiv, and keeping the women in the group while they did their laundry. conclusion: public health educators and researchers can benefit from identifying alternate congregation sites within risk pockets to provide a comfortable space to discuss hiv prevention issues with high-risk community members. in our presentation, we will describe the context necessary for similar research, document the method's pitfalls and successes, and argue that the laundry group constitutes an ethical, respectful, community-based method for recruitment in an hiv-prevention vaccine trial. p3-0t (c) upgrading inner city infrastructure and services for improved environmental hygiene and health: a case of mirzapur in u.p. india madhusree mazumdar in urgency for agricultural and industrial progress to promote economic d.evelopment follo_wing independence, the government of india had neglected health promotion and given less emphasis on infrastructure to promote public health for enhanced human pro uct1v_ity. ong wit r~p1 m astrucrure development, which has become essential if citie~ are to. act ~s harbmger.s of econ~nuc ~owth, especially after the adoption of the economic liberalization policy, importance _is a_lso ~emg g1ve.n to foster environmental hygiene for preventive healthcare. the world health orga~1sat10~ is also trj:'1!1g to help the government to build a lobby at the local level for the purpose by off~rmg to mrroduce_ its heal.thy city concept to improve public health conditions, so as to reduce th_e disease burden. this pape~ 1s a report of the efforts being made towards such a goal: the paper descr~bes ~ c~se study ?f ~ small city of india called mirzapur, located on the banks of the nver ganga, a ma1or lifeline of india, m the eastern part of the state of uttar pradesh, where action for improvement began by building better sanitation and environmental infrastructure as per the ganga action plan, but continued with an effort to promote pre· ventive healthcare for overall social development through community participation in and around the city. asthma physician visits in toronto, canada tara burra, rahim moineddin, mohammad agha, and richard glazier introduction: air pollution and socio-economic status are both known to be associated with asthma in concentrated urban settings but little is known about the relationship between these factors. this study investigates socio-economic variation in ambulatory physician consultations for asthma and assesses possible effect modification of socio-economic status on the association between physician visits and ambient air pollution levels for children aged 1 to 17 and adults aged 18 to 64 in toronto, canada between 1992 and 2001. methods: generalized additive models were used to estimate the adjusted relative risk of asthma physician visits associated with an interquartile range increase in sulphur dioxide, nitrogen dioxide, pm2.5, and ozone, respectively. results: a consistent socio-economic gradient in the number of physician visits was observed among children and adults and both sexes. positive associations between ambient concentrations of sul· phur dioxide, nitrogen dioxide and pm2.5 and physician visits were observed across age and sex strata, whereas the associations with ozone were negative. the relative risk estimates for the low socio-«onomic group were not significantly greater than those for the high socio-economic group. conclusions: these findings suggest that increased ambulatory physician visits represent another component of the public health impact of exposure to urban air pollution. further, these results did not identify an age, sex, or socio-economic subgroup in which the association between physician visits and air pollution was significantly stronger than in any other population subgroup. eco-life-center (ela) in albania supports a holistic approach to justice, recognizing the environ· mental justice, social justice and economic justice depend upon and support each other. low income cit· izens and minorities suffer disproportionately from environmental hazards in the workplace, at home, and in their communities. inadequate laws, lax enforcement of existing environmental regulations, and ~ea.k penalties for infractions undermine environmental protection. in the last decade, the environmental 1ust1ce m~ve~ent in tirana metropolis has provided a framework for identifying and exposing the links ~tween irrational development practices, disproportionate siting of toxic facilities, economic depres· s1on, and a diminished quality of life in low-income communities and communities of color. the envi· ~onmental justice agenda has always been rooted in economic, racial, and social justice. tirana and the issues su.rroun~ing brow~fields redevelopment are crucial points of advocacy and activism for creating ~ubstantia~ social chan~~ m low-income communities and communities of color. we engaging intensively m prevcnnng co'.'1mumnes, especially low income or minority communities, from being coerced by gov· ernmenta~ age_nc1es or companies into siting hazardous materials, or accepting environmentally hazard· ous_ practices m order to create jobs. although environmental regulations do now exist to address the environmental, health, and social impacts of undesirable land uses, these regulations are difficult to poster sessions v65 enforce because many of these sites have been toxic-ridden for many years and investigation and cleanup of these sites can be expensive. removing health risks must be the main priority of all brown fields action plans. environmental health hazards are disproportionately concentrated in low-income communities of color. policy requirements and enforcement mechanisms to safeguard environmental health should be strengthened for all brownfields projects located in these communities. if sites are potentially endangering the health of the community, all efforts should be made for site remediation to be carried out to the highest cleanup standards possible towards the removal of this risk. the assurance of the health of the community should take precedence over any other benefits, economic or otherwise, expected to result from brownfields redevelopment. it's important to require from companies to observe a "good neighbor" policy that includes on-site visitations by a community watchdog committee, and the appointment of a neighborhood environmentalist to their board of directors in accordance with the environmental principles. vancouver 1976-2001 michael buzzelli, jason su, and nhu le this is the second paper of research programme concerned with the geographical patterning of environmental and population health at the urban neighbourhood scale. based on the vancouver metropolitan region, the aim is to better understand the role of neighbourhoods as epidemiological spaces where environmental and social characteristics combine as health processes and outcomes at the community and individual levels. this paper builds a cohort of commensurate neighbourhoods across all six censuses periods from 1976 to 2001, assembles neighbourhood air pollution data (several criterion/health effects pollutants), and providing an analysis to demonstrate how air pollution systematically and consistently maps onto neighbourhood socioeconomic markers, in this case low education and lone-parent families. we conclude with a discussion of how the neighbourhood cohort can be further developed to address emergent priorities in the population and environmental health literatures, namely the need for temporally matched data, a lifecourse approach, and analyses that control for spatial scale effects. solid waste management and environment in mumbai (india) by uttam jakoji sonkamble and bairam paswan abstract: mumbi is the individual financial capital of india. the population of greater mumbai is 3,326,837 and 437 sq. km. area. the density of population 21, 190 per sq. km. the dayto-day administration and rendering of public services within gr. mumbai is provided by the brihan mumbai mahanagar palika (mumbai corporation of gr. mumbai) that is a body of 221 elected councilors on a 5-year team. mumcial corporation provides varies conservancy services such as street sweeping, collection of solid waste, removal and transportation, disposal of solid waste, disposal of dead bodies of animals, construction, maintance and cleaning of urinals and public sanitary conveniences. the solid waste becoming complicated due to increase in unplanned urbanization and industrialization, the environment has deteriorated significantly due to inter, intra and international migration stream to mumbai. the volume of inter state migration to mumbai is considerably high i.e. 20.89 lakh and international migrant 0.77 lakh have migrated to mumbai. present paper gives the view on solid waste management and its implications to environment and health. pollution from a wide varity of emission, such as from automobiles and industrial activities, has reached critical level in mumbai, causing respiratory, ocular, water born diseases and other health problems. sources of generation of waste are -household waste, commercial waste, institutional waste, street sweeping, silt removed from drain/nallah/cleanings. disposal of solid waste in gr. mumbai done under 1 incineration 2. processing to produce organic manure. 3. vermi-composting 4. landfill the study shows that the quantity of waste disposal of through processing and conversion to organic ~anure is about 200-240 m.t. per day. the processing is done by a private agency m/s excel industries ltd. who had set up a plant at the chincholi dumping ground in western mumbai for this purpose. the corporation is also disposal a plant of its waste mainly market waste through the environment friendly, natural pro-ces~ known as vermi-composing about 100 m.t. of market waste is disposed of in this manner at the various sites. there are four land fill sites are available and 95 percent of the waste matter generated m mumbai is disposed of through landfill. continuous flow of migrant and increa~e in slum population is a complex barrier in the solid waste management whenever community pamc1pat1on work strongly than only we can achieved eco-friendly environment in mumbai. persons exposed to residential craffic have elevated races of respiratory morbidity an~ ~ortality. since poverty is an important determinant of ill-health, some h~ve argued that t~es~ assoc1at1ons may relate to che lower socioeconomic status of those living along ma1or roads. our ob1ect1ve was to evaluate the association between traffic intensity at home and hospital admissions for respiratory diagnoses among montreal residents older than 60 years. morning peak traffic estimates from the emmej2 montreal traffic model (motrem98) were used as an indicator of exposure to road traffic outside the homes of those hospitalised. the influence of socioeconomic status on the relationship between traffic intensity and hospital admissions for respiratory diagnoses was explored through assessment of confounding by lodging value, expressed as the dollar average over road segments. this indicator of socioeconomic status, as calculated from the montreal property assessment database, is available at a finer geographic scale than socioeconomic information accessible from the canadian census. there was an inverse relationship between traffic intensity estimates and lodging values for those hospitalised (rho -0.23, p 3160 vehicles during che 3 hour morning peak), even after adjustment for lodging value (crude or 1.35, cl95% 1.22-1.49; adjusted or 1.13, cl95% 1.02-1.25). in montreal, elderly persons living along major roads are at higher risk of being hospitalised for respiratory illnesses, which appears not simply to reflect the fact that those living along major roads are at relative economic disadvantage. the paper argues that human beings ought to be at the centre of the concern for sustainable development. while acknowledging the importance of protecting natural resources and the ecosystem in order to secure long term global sustainability, the paper maintain that the proper starting point in the quest for urban sustainability in africa is the 'brown agenda' to improve che living and working environment of che people, especially che urban poor who face a more immediate environmental threat to their health and well-being. as the un-habitat has rightly observed, it is absolutely essential "to ensure that all people have a sufficient stake in the present to motivate them to take part in the struggle to secure the future for humanity.~ the human development approach calls for rethinking and broadening the narrow technical focus of conventional town planning and urban management in order to incorporate the emerging new ideas and principles of urban health and sustainability. i will examine how cities in sub-saharan africa have developed over the last fifty years; the extent to which government policies and programmes have facilitated or constrained urban growth, and the strategies needed to achieve better functioning, safer and more inclusive cities. in this regard i will explore insights from the united nations conferences of the 1990s, especially local agenda 21 of the rio summit, and the istanbul declaration/habitat agenda, paying particular attention to the principles of enablement, decentralization and partnership canvassed by these movements. also, i will consider the contributions of the various global initiatives especially the cities alliance for cities without slums sponsored by the world bank and other partners; che sustainable cities programme, the global campaigns for good governance and for secure tenure canvassed by unhabit at, the healthy cities programme promoted by who, and so on. the concluding section will reflect on the future of the african city; what form it will take, and how to bring about the changes needed to make the cities healthier, more productive and equitable, and better able to meet people's needs. heather jones-otazo, john clarke, donald cole, and miriam diamond urban areas, as centers of population and resource consumption, have elevated emissions and concentrations of a wide range of chemical contaminants. we have developed a modeling framework in which we first ~stimate the emissions and transport of contaminants in a city and second, use these estimates along with measured contaminant concentrations in food, to estimate the potential health risk posed by these che.micals. the latter is accomplished using risk assessment. we applied our modeling framework to consider two groups of chemical contaminants, polycyclic aromatic hydrocarbons (pah) a.nd the flame re~ardants polybrominated diphenyl ethers (pbde). pah originate from vehicles and stationary combustion sources. ~veral pah are potent carcinogens and some compounds also cause noncancer effects. pbdes are additive flame retardants used in polyurethane foams (e.g., car seats, furniture) 105fer sessions v67 and cl~ equipm~nt (e.g., compute~~· televisio~s). two out of three pbdes formulations are being voluntarily phased by mdustry due to rmng levels m human tissues and their world-wide distribution. pbdes have been .related to adv.erse neurological, developmental and reproductive effects in laboratory ijlimals. we apphed our modelmg framework to the city of toronto where we considered the southcattral area of 21 by 21 km that has a population of 1.3 million. for pah, local vehicle traffic and area sources contribute at least half of total pah in toronto. local contributions to pbdes range from 57-85%, depending on the assumptions made. air concentrations of both compounds are about 10 times higher downtown than 80 km north of toronto. although measured pah concentrations in food date to the 1980s, we estimate that the greatest exposure and contribution to lifetime cancer risk comes from ingestion of infant formula, which is consistent with toxicological evidence. the next greatest exposure and cancer risk are attributable to eating animal products (e.g. milk, eggs, fish). breathing downtown air contributes an additional 10 percent to one's lifetime cancer risk. eating vegetables from a home garden localed downtown contributes negligibly to exposure and risk. for pbdes, the greatest lifetime exposure comes through breast milk (we did not have data for infant formula), followed by ingestion of dust by the toddler and infant. these results suggest strategies to mitigate exposure and health risk. p4-01 (a) immigration and socioeconomic inequalities in cervical cancer screening in toronto, canada aisha lofters, rahim moineddin, maria creatore, mohammad agha, and richard glazier llltroduction: pap smears are recommended for cervical cancer screening from the onset of sexual activity to age 69. socioeconomic and ethnoracial gradients in self-reported cervical cancer screening have been documented in north america but there have been few direct measures of pap smear use among immigrants or other socially disadvantaged groups. our purpose was to investigate whether immigration and socioeconomic factors are related to cervical cancer screening in toronto, canada. methods: pap smears were identified using fee codes and laboratory codes in ontario physician service claims (ohip) for three years starting in 1999 for women age 18-41 and 42-66. all women with any health system contact during the three years were used as the denominator. social and economic factors were derived from the 2001 canadian census for census tracts and divided into quintiles of roughly equal population. recent registrants, over 80% of whom are expected to be recent immigrants to canada, were identified as women who first registered for health coverage in ontario after january 1, 1993. results: among 397,967 women age 18-41 and 328,885 women age 42-66, 55.3% and 55.5%, rtspcctively, had pap smears within three years. low income, low education, recent immigration, visible minority and non-english language were all associated with lower rates (least advantaged quintile:most advantaged quintile rate ratios were 0.84, 0.90, 0.81, 0.85, 0.83, respectively, p < 0.05 for all). similar gradients were found in both age groups. recent registrants comprised 22.5% of women and had mm;h lower pap smear rates than non-recent registrants (37.2 % versus 63. 7% for women age 18-41 and 35.9% versus 58.2% for women age 42-66). conclnsions: pap smear rates in toronto fall well below those dictated by evidence-based practice. at the area level, immigration, visible minority, language and socioeconomic characteristics are associated with pap smear rates. recent registrants, representing a largely immigrant group, have particularly low rates. efforts to improve coverage of cervical cancer screening need to be directed to all ~omen, their providers and the health system but with special emphasis on women who recently arrived m ontario and those with social and economic disadvantage. challeges faced: a) most of the resources are now being ~pent in ~reventing the sprea.d of hiv/ aids and maintaining the lives of those already affected. b) skilled medical ~rs~nal are dymg under· mining the capacity to provide the required health care services. ~) th.e comphcat1o~s of hiv/aids has complicated the treatment of other diseases e.g. tbs d) the ep1dem1c has led. to mcrease number of h n requiring care and support. this has further stretched the resources available for health care. orp a s d db . . i methods used on our research: 1. a simple community survey con ucte y our orgamzat1on vo · unteers in three urban centres members of the community, workers and health care prov~ders were interviewed ... 2. meeting/discussions were organized in hospitals, commun.ity centre a~d with government officials ... 3. written questionnaires to health workers, doctors and pohcy makers m th.e health sectors. lessors learning: • the biggest-health bigger-go towards hiv/aids prevention • aids are spreading faster in those families which are poor and without education. •women are the most affected. •all health facilities are usually overcrowded with hiv/aids patients. actions needed:• community education oh how to prevent the spread of hiv/aids • hiv/aids testing need to be encouraged to detect early infections for proper medical cover. • people to eat healthy • people should avoid drugs. implications of our research: community members and civic society-introduction of home based care programs to take care of the sick who cannot get a space in the overcrowded public hospitals. prl-v a te sector private sector has established programs to support and care for the staff already affected. government provision of support to care-givers, in terms of resources and finances. training more health workers. introduction: australian prisons contain in excess of 23,000 prisoners. as in most other western countries, reliance on 'deprivation of liberty' is increasing. prisoner numbers are increasing at 7% per annum; incarceration of women has doubled in the last ten years. the impacts on the community are great -4% of children have a parent in custody before their 16th birthday. for aboriginal communities, the harm is greater -aboriginal and/or torres strait islanders are incarcerated at a rate ten times higher than other australians. 25% of their children have a parent in custody before their 16th birthday. australian prisons operate under state and territory jurisdictions, there being no federal prison system. eight independent health systems, supporting the eight custodial systems, have evolved. this variability provides an unique opportunity to assess the capacity of these health providers in addressing the very high service needs of prisoners. results: five models of health service provision are identified -four of which operate in one form or another in australia: • provided by the custodial authority (queensland and western australia)• pro· vided by the health ministry through a secondary agent (south australia, the australian capital territory and tasmania) • provided through tendered contract by a private organization (victoria and northern territory) • provided by an independent health authority (new south wales) • (provided by medics as an integral component of the custodial enterprise) since 1991 the model of the independent health authority has developed in new south wales. the health needs of the prisoner population have been quantified, and attempts are being made to quantify specific health risks /benefits of incarceration. specific enquiry has been conducted into prisoner attitudes to their health care, including issues such as client information confidentiality and access to health services. specific reference will be made to: • two inmate health surveys • two inmate access surveys, and • two service demand studies. conclusions: the model of care provision, with legislative, ethical, funding and operational independence would seem provide the best opportunity to define and then respond to the health needs of prisoners. this model is being adopted in the united kingdom. better health outcomes in this high-risk group, could translate into healthier families and their communities. p4-04 (a) lnregrated ethnic-specific health care systems: their development and role in increasing access to and quality of care for marginalized ethnic minorities joshua yang introduction: changing demographics in urban areas globally have resulted in urban health systems that are racially and ethnically homogenous relative to the patient populations they aim to serve. the resultant disparities in access to and quality of health care experienced by ethnic minority groups have been addressed by short-term, instirutional level strategies. noticeably absent, however, have been structural approaches to reducing culturally-rooted disparities in health care. the development of ethnic-specific h~alth car~ systems i~ a structural, long-term approach to reducing barriers to quality health care for eth· me mmonty populations. methods: this work is based on a qualitative study on the health care experiences of san francisco chinatown in the united states, an ethnic community with a model ethnic-specific health care infrastrucrure. using snowball sampling, interviews were conducted with key stakeholders and archival research was conducted to trace and model the developmental process that led to the current ethnic-specific health care system available to the chinese in san francisco. grounded theory was the methodology ijltd to analysis of qualitative data. the result of the study is four-stage developmental model of ethnic-specific health infrastrueture development that emerged from the data. the first stage of development is the creation of the human capital resources needed for an ethnic-specific health infrastructure, with emphasis on a bilingual and bicultural health care workforce. the second stage is the effective organization of health care resources for maximal access by constituents. the third is the strengthening and stability of those institutional forms through increased organizational capacity. integration of the ethnic-specific health care system into the mainstream health care infrastructure is the final stage of development for an ethnic-specific infrastructure. conclusion: integrated ethnic-specific health care systems are an effective, long-term strategy to address the linguistic and cultural barriers that are being faced by the spectrum of ethnic populations in urban areas, acting as culturally appropriate points of access to the mainstream health care system. the model presented is a roadmap to empower ethnic communities to act on the constraints of their health and political environments to improve their health care experiences. at a policy level, ethnic-specific health care organizations are an effective long-term strategy to increase access to care and improve qualiiy of care for marginalized ethnic groups. each stage of the model serves as a target area for policy interventions to address the access and care issues faced by culturally and linguistically diverse populations. users in baltimore md: 1989-2004 noya galai, gregory lucas, peter o'driscoll, david celentano, david vlahov, gregory kirk, and shruti mehta introduction: frequent use of emergency rooms (er) and hospitalizations among injection drug users (idus) has been reported and has often been attributed to lack of access to primary health care. however, there is little longitudinal data which examine health care utilization over individual drug use careers. we examined factors associated with hospitalizations, er and outpatient (op) visits among idus over 14 years of follow-up. methods: idus were recruited through community outreach into the aids link to lntravenous experience (alive) study and followed semi-annually. 2,551 who had at least 2 follow-up visits were included in this analysis. outcomes were self-reported episodes of hospitalizations and er/op visits in the prior six months. poisson regression was used accounting for intra-person correlation with generalized estimation equations. hits: at enrollment, 73% were male, 95% were african-american, 33% were hiv positive, median age was 35 years, and median duration of drug use was 15 years. over a total of 37,512 visits, mean individual rates of utilization were 11 per 100 person years (py) for hospitalizations and 123 per 100 py for er/op visits. adjusting for age and duration of drug use, factors significantly associated with higher rates of hospitalization included hiv infection (relative incidence [ri(, 1.4), female gender (ri, 1.2), homelessness (ri, 1.6), as well as not being employed, injecting at least daily, snorting heroin, havmg a regular source of health care, having health insurance and being in methadone mainte.nance treatment (mmt). similar associations were observed for er/op visits except for mmt which was not associated with er/op visits. additional factors associated with lower er/op visits were use of alcohol, crack, injecting at least daily and trading sex for drugs. 10% of the cohort accounted for 45% of total er/op visits, while 11 % of the cohort never reported an op visit during follow-up. . . . lgbt) populations. we hypothesized that prov1dmg .appomtments .for p~t1~nts w1thm 24 hours would ensure timely care, increase patient satisfaction, and improve practice eff1c1ency. further, we anticipated that the greatest change would occur amongst our homeless patients.. . methods: we tested an experimental introduction of advanced access scheduling (usmg a 24 hour rule) in the primary care medical clinic. we tracked variables inclu~ing waiting ti~e fo~ next available appointment; number of patients seen; and no-show rates, for an eight week penod pnor to and post introduction of the new scheduling system. both patient and provider satisfaction were assessed using a brief survey (2 questions rated on a 5-pt scale). results and conclusion: preliminary analyses demonstrated shorter waiting times for appointments across the clinic, decreased no-show rates, and increased clinic capacity. introduction of the advanced access scheduling also increased both patient and provider satisfaction. the new scheduling was initiated in july 2005. quantitative analyses to measure initial and sustained changes, and to look at differential responses across populations within our clinic, are currently underway. introduction: there are three recognized approaches to linking socio-economic factors and health: use of census data, gis-based measures of accessibility/availability, and resident self-reported opinion on neighborhood conditions. this research project is primarily concerned with residents' views about their neighborhoods, identifying problems, and proposing policy changes to address them. the other two techniques will be used in future research to build a more comprehensive image of neighborhood depri· vation and health. methods: a telephone survey of 658 london, ontario residents is currently being conducted to assess: a) community resource availability, quality, access and use, b) participation in neighborhood activities, c) perceived quality of neighborhood, d) neighborhood problems, and e) neighborhood cohesion. the survey instrument is composed of indices and scales previously validated and adapted to reflect london specifically. thirty city planning districts are used to define neighborhoods. the sample size for each neighborhood reflects the size of the planning district. responses will be compared within and across neighborhoods. data will be linked with census information to study variation across socio-eco· nomic and demographic groups. linear and gis-based methods will be used for analysis. preliminary results: the survey follows a qualitative study providing a first look at how experts involved in community resource planning and administration and city residents perceive the availability, accessibility, and quality of community resources linked to neighborhood health and wellbeing, and what are the most immediate needs that should be addressed. key-informant interviews and focus groups were used. the survey was pre-tested to ensure that the language and content reflects real experiences of city residents. the qualitative research confirmed our hypothesis that planning districts are an acceptable surrogate for neighborhood, and that the language and content of the survey is appropriate for imple· mentation in london. scales and indices showed good to excellent reliability and validity during the pre· test (cronbach's alpha from 0.57-0.96). preliminary results of the survey will be detailed at the conference. conclusions: this study will help assess where community resources are lacking or need improve· ment, thus contributing to a more effective allocation of public funds. it is also hypothesized that engaged neighborhoods with a well-developed sense of community are more likely to respond to health programs and interventions. it is hoped that this study will allow london residents to better understand the needs and problems of their neighborhoods and provide a research foundation to support local understandmg of community improvement with the goal of promoting healthy neighborhoods. p4-08 (a) hiv positive in new york city and no outpatient care: who and why? hannah wolfe and victoria sharp introduction: there are approximately 1 million hiv positive individuals living in the united sta!es. about. 50% of these know their hiy status and are enrolled in outpatient care. of the remaining 50 yo, approx~mately half do not know their status; the other group frequently know their status but are not enrolled m any .sys~em of outpatient care. this group primarily accesses care through emergency departments. when md1cated, they are admitted to hospitals, receive acute care services and then, upon poster sessions v71 di5'harge, disappear from the health care system until a new crisis occurs, when they return to the emergency department. as a large urban hiv center, caring for over 3000 individuals with hiv we have an active inpatient service ".'ith appr~xi~.ately 1800 discharges annually. we decided to survey our inpatients to better charactenze those md1v1duals who were not enrolled in any system of outpatient care. results: 18% of inpatients were not enrolled in regular outpatient care: 2% at roosevelt hospital and 35% at st.luke\'s hospital. substance abuse and homelessness were highly prevalent in the cohort of patients not enrolled in regular outpatient care. 84% of patients not in care (vs. 33% of those in care) were deemed in need of substance use treatment by the inpatient social worker. 74% of those not in care were homeless (vs. 15% of those in care.) patients not in care did not differ significantly from those in me in terms of age, race, or gender. patients not in care were asked "why not:" the two most frequent responses were: "i haven't really been sick before" and "i'd rather not think about my health. conclusions: this study suggests that there is an opportunity to engage these patients during their stay on the inpatient units and attempt to enroll them in outpatient care. simple referral to an hiv clinic is insufficient, particularly given the burden of homelessness and substance use in this population. efforts are currently underway to design an intervention to focus efforts on this group of patients. p4.q9 (a) healthcare availability and accessibility in an urban area: the case of ibadan city, nigeria in oder to cater for the healthcare need of the populace, for many years after nigeria's politicl independence, empphasis was laid on the construction of teaching, general, and specialist hospital all of which were located in the urban centres. the realisation of the inadequacies of this approach in adequately meeting the healthcare needs of the people made the country to change and adopt the primary health care (phc) system in 1986. the primary health care system which is in line with the alma ata declaration of of 1978, wsa aimed at making health care available to as many people as possible on the basis of of equity and social justice. thus, close to two decades, nigeria has operated primary health care system as a strategy for providing health care for rural and urban dwellers. this study focusing on urban area, examimes the availabilty and accessibility of health care in one of nigeria's urban centre, ibadan city to be specific. this is done within the contest of the country's national heath policy of which pimary health care is the main thrust. the study also offers necessary suggestion for policy consideration. in spite of the accessibility to services provided by educated and trained midwifes in many parts of fars province (iran) there are still some deliveries conducted by untrained traditional birth attendants in rural parts of the province. as a result, a considerable proportion of deliveries are conducted under a higher risk due to unauthorised and uneducated attendants. this study has conducted to reveal the pro· portion of deliveries with un-authorized attendants and some spatial and social factors affecting the selection of delivery attendants. method: this study using a case control design compared some potentially effective parameters indud· ing: spatial, social and educational factors of mothers with deliveries attended by traditional midwifes (n=244) with those assisted by educated and trained midwifes (n=258). the mothers interviewed in our study were selected from rural areas using a cluster sampling method considering each village as a cluster. results: more than 11 % of deliveries in the rural area were assisted by traditional midwifes. there are significant direct relationship between asking a traditional birth attendant for delivery and mother age, the number of previous deliveries and distance to a health facility provided for delivery. significant inverse relationships were found between mother's education and ability to use a vehicle to get to the facilities. conclusion: despite the accessibility of mothers to educated birth attendants and health facilities (according to the government health standards), some mothers still tend to ask traditional birth attendants for help. this is partly because of unrealistic definition of accessibility. the other considerable point is the preference of the traditional attendants for older and less educated mothers showing the necessity of changing theirs knowledge and attitude to understand the risks of deliveries attended by traditional and un-educated midwifes. p4-12 (a) identification and optimization of service patterns provided by assertive community treatment teams in a major urban setting: preliminary findings &om toronto, canada jonathan weyman, peter gozdyra, margaret gehrs, daniela sota, and richard glazier objective: assertive community treatment (act) teams are financed by the ontario ministry of health and long-term care (mohltc) and are mandated to provide treatment, rehabilitation and support services in the community to people with severe and persistent mental illness. there are 13 such teams located in various regions across the city of toronto conducting home visits 1-5 times per week to each of their approximately 80 respective clients. each team consists of multidisciplinary health professionals who assist clients to identify their needs, establish goals and work toward them. due to complex referral patterns, the need for service continuity and the locations of supportive housing, clients of any one team are often found scattered across the city which increases home visit travel times and decreases efficiency of service provision. this project examines the locations of clients in relation to the home bases of all 13 act teams and identifies options for overcoming the geographical challenges which arise in a large urban setting. methods: using geographic information systems (gis) we geocoded all client and act agency addresses and depicted them on location maps. at a later stage using spatial methods of network analysis we plan to calculate average travel rimes for each act team, propose optimization of catchment areas and assess potential travel time savings. resnlts: initial results show a substantial scattering of clients from several act teams and substan· rial overlap of visit travel routes for most teams. conclusions: reallocation of catchment areas and optimization of act teams' travel patterns should lead to substantial savings in travel times, increased service efficiency and better utilization of resourc_~· ~e l'<!tenri~i modifications to catchment areas must be conducted with appropriate attention to specific clients servtce needs, service continuity and applicable regulations by the mohl tc. venice lido is a popular island within and outside italy because it hosts the international cinema festival yearly. only few people remember the island for its hospital, which, in the fifties, was one of the most important hospitals in italy and in europe, being a modern center for wind-, sun-and thermal-therapy. since the sixties, its fame became to drop away, since its structure was no longer adequate for the evolving medicine science. at the end of the seventies all the wards were moved into a new big building in the same area. at present the hospital is made up by about forty 'pavilions' (mostly partially used or empty) and a big building, disseminated in a 10 hectares area, on the sea front, on the 500 m long beach. the aim of my research is to suggest a realistic solution, sustainable on the functional use of the area according to basic local needs and economically realizable. i used a philological and multi-disciplinary method, more in detail: -from historical documents, i found what was the very first call of the property and how it adapted to the needs of the demographic development and the sanitary supply over time; -by contacting some operators from different fields, i realized which functions were suitable to the buildings in existence, to the population's needs and to the present sanitary supply. moreover the planning idea considers: -the debate going on between local people who strongly want to preserve the property, and die ones who want to renew it by different new opportunities; and -the regional sanitary politics, aiming at rationalizing the regional hospitals dislocation, and the budget needs. this plan analyzes and proposes some new alternative solutions to satisfy different needs. the different activities will be dealt out and the environmental fall will be limited as much as possible. after having analyzed its basic call, considered its environmental peculiarities and its position, verified the emerging needs of the local population, the plan proposes 5 different activities: the hospital; the social-rehabilitation center; the elderly house; the thermal center; the residential center; and other activities. such a plan wants to share in the solution of a basic problem by giving architectonic and functional dignity to an historical completely degraded area and by starting again a social-economical broken off process, by bringing in venice lido some fresh necessary elements for its so wished new throw. p4-14 (c) dilemma of free health care in spokane, wa david bunting introduction: the paper reports on the activities of project access spokane [pas], a physician sponsored initiative to provide uncompensated health care to low income, uninsured residents of spokane county wa. program providers included all county hospitals, over 600 physicians, other medical professionals, and specialized clinics. each prescription requires only a $4 co-payment. sponsored and administered by the county medical society, pas is funded by local and national foundations, private corporations, municipalities, civic organizations and individuals. method: the paper is based on a first year assessment report funded by pas, using hcfa [health care financing administration] insurance claim data documenting utilization rates, disease categories and donated charge information and patient cares [centralized applications, referrals and enrollment status) data. results: while essentially free for enrollees, the program involved real costs. an administrative organization to refer over 700 patients to any of over 800 providers had to be developed and staffed, using both paid and unpaid personnel. methods to identify potential patients and veri.fy eligibility. had to ~devised. patient appointments and transportation had to be scheduled and monitored. pu~hc relanons, provider solicitations and fundraising activities were continuous. information requirements regarding program operation were also significant. data reporting the volume. ~nd value of dona~ed medical services were necessary to demonstrate accomplishments and attr~ct addmon~l support. providers required assurances their contributions were both significant and eqmtable. funding sources sought evidence that their support had important consequences. however, generating this ~~ta proved difficult. many providers failed to submit appropriate insurance claims forms because .of a~dmonal donated effort and administrative cost, thereby not only reducing their own apparent conmbut1ons but also the overall significance of the program. conclusions: during its initial year, the estimated cost to deliver free health care was ~bout 5500 per enrollee. the lesson is that without an elaborate administrative structure and record keeping s.yst~~· efforts to provide free health care probably will fail. eligible enrollees c~n not be ~parated from in~hgi ble ones, care will not be accessed or delivered in an efficient manner, neither fundmg nor ~upport "". 1 11 be offered without evidence of tangible benefits, and providers will withdraw if they perceive mequ1table tteannent. v74 p4-1s (c) mobility in prostitution and the impact of health therese van der helm and henk sulman poster sessions introduction: many of the estimated 8.000 commer~ial ~ex wor~ers (c~w)in a'.'1~terdam ~~me from developing countries and eastern europe. to gain ins~ght mto their workmg and_ hvmg condinons the intermediary project (ip) at the municipal health serv1~e _contacts these women via ?utreach work on regular basis. since the new brothel law in october 2000 1s 1~troduc~d, ~sw ~rom outside the eu and who have no dutch residence permit are not allowed to work m prost1tut10n. smee then, many of these csw therefore have gone "underground." the consequences of the new law in the netherlands will be discussed with regard to the accessibility of csw and their risk for stl/hiv acquisition and unplanned pregnancies. methods: topics during outreach work are the interventions to increase the knowledge of safe sex and birth control for csw. written information is handed out in relevant languages and with addresses of health and social services. in conversations with the women, it appeared that many are not aware of these services. to provide easier access to health care for women, staff of the ip carries out free sti control csw working places, in windows brothels and sex houses. also, women are offered vaccination against hepatitis b (hbv). results: from january 2004 till july 2005 we approached 900 csw for first contact. one third was dutch; others were from developing countries, other eu countries and eastern europe. 350 sti consul· rations were done among non-iv drug using sex workers, of whom 50 % was dutch. of these, 2 % was diagnosed with syphilis, 1 % with gonorrhea, and 9% with chlamydia. of 234 women tested for hiv, none were infected. due to the high turnover of csw in .brothels, most wnmen were tested only once. among 845 csw tested for hbv -of whom one third is dutch -22 % had antibodies for hbv, 12 were carrier of hbv, most of whom came from hbv endemic areas. conclusions: sti control and hbv vaccination in brothels is an important support in health care. our findings suggest that csw do not play an important role in the transmission of sti. many csw are highly mobile and often not aware of the existing health and social services. continuous outreach is important to remain in contact with this mobile population. regulations in the new brothel law should not hamper contacts with (illegal) csw. heart disease (cho) is projected to become the leading cause of death. studies conducted in europe and the united states have proven a higher rate of cho in south asians who have migrated from south asia, most notably india, pakistan and bangladesh. approximately 50% of all heart attacks among south asian men occur under the age of 55; 25% of them occur under the age of 40-unheard of in any other population. associated risk factors such as diabetes, high blood pressure and cholesterol are also com· mon in this group. the population of south asians residing in nyc has more than doubled over the past decade to over 300,000, the majority being young, working-class and with limited health care access. many south asian men are employed as taxi drivers (4 out of 10 nyc taxi drivers are from south asial, working daily12 hour plus shifts. methods: acknowledging that this vulnerable, at-risk group was unavailable for outreach through conventional venues, three of the hospitals of the new york city health and hospital corporation, (elmhurst, queens and bellevue) conducted a one day outreach effort at nyc's jfk international air· ~rt's taxi holdin~ ar~a, ~here over one thousand taxis regularly assemble. bringing an outreach event directly to the taxi dnvers workplace, a tent was set up where cardiovascular-related health screenings, in~luding bl~ pressure, sugar, cholesterol, body mass index (bmi), were provided. results were shared with and explamed to the drivers and each participant received a south asian health education brochure. a total of 84 taxi drivers who identified as south asian were involved. rau/ts: participants were all male, ranging from 20 to 61 years; mean age, 41.5. screenings revealed 57% hypertensive; 46% had cholesterols over 200; 26% blood glucose over 160; 91 % had a bmi greater than 25. conc~on: a unique outreach activity, adapting to logistical, occupational limitations, is pre· se~ted. on-site f~back and explanation of results, reiterating and reinforcing the concern that south asia~. men are at mcreased danger for early coronary heart disease, and the dissemination of a culturally sensmve and r~levant brochure, may heighten awareness of prevailing cardiac risk factors in this immi· grant community. p4-17 (c) the community-hospital integration program framework: community-hospital panoenhips to improve the population's health john stevenson, richard blickstead, ann-marie marcolin, and sandi kendal v75 introduction: st. josephs health centre is a catholic community teaching hospital located in the heart of southwest toronto. st. joseph\'s was founded from a community-expressed need for hospital services, and since then has stayed committed to fostering a healthy community through collaboration and parmership. st. joseph's has developed an innovative model, the community-hospital integration program (chip), to strengthen st. joseph's partnerships with community stakeholders, and facilitates the building of links between community needs, service provision, and public policy outcomes to improve the health and wellness of the diverse neighbourhoods they together serve. methods: development of the chip framework st. joseph's health centre developed the chip framework through a multidisciplinary approach that included extensive international literature reviews, consultations, and key informant interviews with community service agencies and academics. to ensure active community voice, st. joseph's has hosted a number of community consultations including a community outreach forum attended by over ninety representatives from 68 community partners. results: the chip framework the chip framework aims to improve the health and wellness of the urban communities served by st. josephs health centre through four intersecting pillars: • raising community voices provides an infrastructure and process that supports community stakeholder input into health care service planning, decision-making, and delivery by the hospital and across the continuum of care; • sharing reciprocal capacity promotes healthy communities through the sharing of our intellectual and physical capacity with our community partners; • cultivating integration initiatives facilitates vertical, horizontal, and intersectoral integration initiatives in support of community-identified needs and gaps; and • facilitating healthy exchange develops best practices in community integration through community-based research, and facilitates community voice in informing public policy. the chip framework drives the complex inter-relationships between community-hospital engagement, reciprocal capacity-building, integration initiatives, and community-based research and evaluation, to create an interconnected network of health care services. the fluidity and simplicity of this framework, and its dedication to balancing community needs and hospital mandate, posits the st. josephs health centre's chip model as an integral component in building healthy and thriving communities. p4-18 (c) home based care promotion: improving acess to quality services and livelihood in the face of aids home based care is a service provided to persons affected/living with hiv/aids, a menu of care/support services at home/community. it is a prominent alternative approach. in uganda, hospital bed ocupacny is high, patient health care worker ratio deteriorating. empowering communities offers solutions to the problems; adressing cost, effectiveness of care. hiv/aids demands changes in attitudes, behaviour, approaches which is enhanced in home/community settings. for example art requires high levels of adherecnce, which medical workers have vety little opportunity to enforce. it also demands other support mechanisims in terms of nutrition, personal displine, etc which work best in stigma free environments. hence the relevance of home based care. this paper highlights the gaps that call for increased action in terms of home based care, examples of whre it has worked key challenges and recommendations for the future. p4-19 (c) health care for one ... health care for alli katharina kovacs burns introduction: at the surface, it appears that every person in canada _has. ~ccess to a publicly funded health care system. those living in urban centers should have the best ava1l~b1hty, chmce, and access to a variety of health care services because of the distribution of health care services, fac1lmes, and health professionals in concentrated in urban centers. this is not true for everyone -people with low income or who are homeless experience the challenges of social exclusion and being marginalized: there are many factors at play making it difficult for the latter group of people to access health care. service they need,.when they need them. health care is not as accessible or inclusive as intended. the quesuon to be explore.cl 1~: if health care is available and accessible to one person, what makes it not available to all people? the ~1gmfic~nce of having marginalized people accessing health care and other services includes e~hanced quality of hfe and decreased risks associated with being homeless, and cost savings in ion~ ter~ w1rh acute health care. methodology: community-based participatory research is applied m a case study on health care access and challenges experienced by low income and homeless people in one urban centre, edmonton, edmonton, and their challenges. the data is being gathered and ana_lyzed usmg basic m1x~d methods. results: the results will focus on how services can be more mtegrated and accessible to all people with low income and who are homeless. there will also be discussion about specific perceptions of not only the service providers but also of people with low income an~ who are homeless~ and vario~s decision makers. the results will be compared to the literature regardmg health care services access mother urban centers in canada and elsewhere. conclusions: recommendations will need to address social exclusion and other factors which can make health care and other services more available to all people in the community. the implications for health care will also be discussed. additional information will be gathered in the next phases of the study to develop a community services access model. p4-20 (c) availability and access exemplified: a case study beth hayhoe and ruth ewert introduction: access to health care in canada requires either the correct documentation or money. street youth have neither. in addition, health services in canada are designed by adults for adults. youth in general are often wary of having trust in adults with respect to their health concerns and frequently feel misunderstood. street youth are even more mistrustful of adults and public organizations, making their contact with health care professionals minimal. this combined with their risky behaviours and dangerous environment creates a population at risk for numerous health issues but with no place to have them investi· gated. at our non-government, not for profit health service designed specifically to provide a broad range of health care to street youth, professional services are provided almost entirely by volunteers. methods: using a retrospective analysis of the 11 years of data gathered from this organization and reviewing the initial reasons gathered from youth about their needs, the success of the health centre was examined. results: all the things youth had listed as being important in a health centre have been implemented, and even more things have been added to improve the breadth and quality of services offered. the use of the health centre has increased by more than six times since its opening in 1994. in addition, tens of thousands of visits have been paid to the health centre, costing the government and taxpayers nothing. as far as we know there is nothing else in canada that offers so many diverse and innovative services to youth in need. conclusion: it is clear from this case, that health care targeted at the needs of specific disadvan· taged populations can successfully provide them with appropriate health care. a model of accessible health care for marginalized populations can easily be developed for high impact and low cost from this successful case. toronto is one of the most ethno-racially diverse cities in the world of the estimated 2,529,280 toronto residents, 24% (597,218) live in scarborough population growth in scarborough is faster than toronto. scarborough's population increased by 6% from 1996 to 2001 while toronto increased only by 4%. toronto's population is projected to increase by 10% (252 000) in 2011 while scarbor· ough's will increase by 12%. (census canada 2001) low income, pove;ty, seniors, hidden homeless, new~omers to canada, the uninsured, are just a few of the issues concerning health care in our community. health care needs in scarborough are greatly impacted by diversity of ethnic and racial groups, poverty and settlement issues. this paper will share how the scarborough hospital (tsh) an urb~n communir_y hospital cares f~~ it~ community. tsh recognizes: •the changing community• b_arriers t~ accessing care • lnequalmes m health care the hospital in caring for its community pro· v1~es services that '!'e~e cu~turally, racially, and linguistically sensitive to our community. the paper will share the hospitals unique program on access and equity. the paper will share the needs in scar· borough and tsh's response to these needs: working in partnership with the local health committee, i:ne scarborough homeless co.mminee, the scarborough network of immigrant services organiza· nons a~d others. 1:'1e paper will also share the many initiatives this urban community hospital has taken viz. community and home programs in service areas such as mental health, haemodialysis day care, t~~ home oxyg~n program, the palliative at-home program, and above all support for the volunteer clinic for the uninsured. j"'"1tllu:lion: in canada's universal health care system it is generally assumed that everyone has equal access to health care. however, some immigrant groups in canada have historically been uninswed. these groups include failed refugee claimants, those overstaying their visas, and new immigrants in transition who are waiting to become eligible for health insurance. some estimates have suggested that at the present time there may be as many as one hundred thousand undocumented and thus uninsured immigrants in toronto alone. understandably, information on the characteristics of this marginalized population is very limited, since undocumented immigrants tend to have little contact with formal institutions. knowledge of the demographic characteristics of this population may aid in the development of health and social programs to better identify and meet the needs of undocumented immigrants in toronto. we conducted a review of all undocumented, uninsured patients, 15 years of age or older at access alliance multicultural community health centre (aamchc) between 1994 and 2004. demographic information such as age, gender, preferred language, length of time living in canada, selfreported education level and household income were recorded. rmdts: 72% of all adult patients at access alliance were undocumented and uninsured. 80% were under40years of age (with a median age of 30 years), 72% were women and only 19% spoke english. this group lived in canada for an average period of 2.2 years before they were first seen at aamchc. 74% of undocumented clients reported having completed at least a secondary or post-secondary education. 81% had self-reported household incomes of less than 20,000$/year, while 97% of households reported earning less than $30,000/year. the mean income per person in an average household was 425$/month. conchuions: uninsured individuals at aamchc were predominantly young females with limited knowledge of the english language. despite having attained a high level of education most were living well below the poverty line. recognition of these characteristics may assist in the development of health and social programs that are better adapted to meet the needs of undocumented immigrants. p4-23 (c) sharing expertise: a role for the hospital lactation consultant in the community. badrgrmuul: st. michael's hospital is a tertiary care hospital that serves a large inner city population in downtown toronto. in order to provide care to this population, the hospital has developed a number of unique outreach roles. one such role was developed to work with pregnant and breastfeeding women living in regent park, a social housing complex located near the hospital. the population of regent park includes new immigrants, refugees and the socially disadvantaged. approach: the outreach lactation consultant provides care, as part of the canada prenatal nutrition program, in the community prenatally, in the hospital during their inpatient stay and postnatally when they return to the community. the continuity of care enhances the probability of long term successful breastfeeding. aside from the health benefits of breastmilk, breastfeeding is especially important for women who are financially needy and must depend on food banks for formula. . . lason learned: aher two years of partnership, the relationship between the an-hospital ~stpar tum experience for breastfeeding mothers and the community postpartum support, the breastfeeding rate ~thin this community is high. the professional support and visibility of the 1:8ctation consultant, both m the hospital and in the community, contributes to the support of breastfee~m~.. . in light of ongoing funding constraints, this ha1son between hospital and community could be at risk and discontinued. in light of the benefits to the mother/baby dyad, the lactation consultant as a community partner in the canada prenatal nutrition program should be safeguarded. . lldpodiu:tion: although the importance of adequate health care in pregna~cy is well recognised, ethnic disparities in utilization of prenatal care still exist in many western coun~~es. a fun~amental factor in these disparities may be language proficiency, as it influences women's ab1hty. to ob~m and understand health information, to find the way in the health care system and to communicate with health care v78 poster sessions providers. we investigated the role of language proficiency in use and knowledge of folic acid as aspect of prenatal care in an urban multi-ethnic pregnancy c?hort. . . design: prospective cohort study. setting and parnc1pants: amsterdam ~regnant women attending obstetric care providers for their first antenatal visit (n= 8050). country of birth defined ethnicity: the netherlands, surinam, antilles, turkey, morocco, ghana, other non-~est~m (nw) and other western (w) country. main outcome measures: knowledge about and use of.fohc a~d (fa) supplements in pregnancy, and determinants of these in diffe~~t ethnic ~~ups. deterrrunants mcl~ded age, ed~ tion, parity, pregnancy intention and dutch prof1c1ency. stansncs: ~2 to co~p~re e~c groups, stranfied logistic regression (forward stepwise method) to explore determmants "'.'thm ethmc groups .. use of fa supplements was significantly lower among ghanaian, moroccan, turkish and other nw women (21%to41 %) than among dutch (86%) or other w women (78%). use amongsurinames and antillean women was intermediate (51%and60%). ethnic differences in fa knowledge were similar. knowledge was the strongest determinant of use in all ethnic groups, with odds ratios (ors) ranging from 10.9 to 42.0. language proficiency was the strongest determi~a~t of kno~ledge in ethnic groups with a mother tongue different from dutch (ors good vs. low proficiency ranging from 3.2 ro 16.0). educational level had a modifying role, as shown by an interaction effect between education and language proficiency in the nw group. here, the odds of having fa knowledge given a high education and good dutch proficiency was 20 times the odds given a good proficiency but low education. conclusions: appropriate periconceptional use of folic acid supplements in non-western ethnic groups is low, reflecting an absence of knowledge that is largely determined by the inability to speak and understand the language of the habitual country. tailored interventions using communication channels most likely to address (pregnant) women from ethnic minority groups are necessary. apan from specific interventions, our results are in support of strong incentives on language education despite current political debate. introduction: recent research suggests living in an economically disadvantaged neighborhood is asso· ciated with decreased likelihood of undergoing mammography and increased risk of late-stage breast can· cer diagnosis. long distances and travel times to facilities offering low-or no-cost mammography may be imponant barriers to adherence to mammography screening recommendations for residents of economically disadvantaged neighborhoods. the purpose of this study was to examine whether facilities providing low-and no-cost screening mammography were less spatially accessible in low-income neighborhoods in chicago, and the extent to which the relationship between neighborhood income and the spatial accessibility of facilities varied by the proponion of african-american residents in the neighborhood. methods: the sample consisted of 343 chicago neighborhoods. for each neighborhood, we con· structed three measures of the spatial accessibility of facilities: street network distance to the nearest facility, public transportation travel time to the nearest facility, and shortest automobile travel time to a facility. using 2000 decennial census data, we characterized the neighborhoods according to proportion of residents with incomes below the poverty line, proportion of residents in each of four raciavethnic groups (african-american, latino, white, and other), and population density. we used ordinary least squares (ols) and spatial exogenous lag regression to examine relationships. model 1 estimated the relationship between neighborhood poverty and the spatial accessibility of facilities, adjusting for raciaveth· nic composition and population density. model 2 added a multiplicative interaction term between neighborhood poverty and african-american. restllts: we identified deven facilities that provided low-or no-cost screening mammography to chicago residents in 2004. we found that the distance and travel times via automobile and public trans· portation to facilities generally decreased as neighborhood poveny increased. however, we also found that the ~egative associations between neighborhood poverty and two of the spatial accessibility mea· sures -distance and public transportation travel time -were less strong in neighborhoods with the highest proponions of african-american residents. among neighborhoods in the highest tertile for poveny, th~ mean distance and mean public transportation travel time to facilities were over twice as long in neighborhoods in the highest tertile than in those in the lowest tenile of african-american residents. . ~ persistent socioeconomic and racial/ethnic disparities in breast cancer stage at diagno-s11 ~nd su~1val suggest that ensuring an equitable distribution of affordable mammography is a worth-w~e policy .goal. the ~~dy sugges~ that ~ture investigations should consider both neighborhood soc1oecono1d1c charactensbes and rac1avethmc composition when examining the spatial distribution of health resources. , 2004) . epidemiological data suggest tha~ hiv prevalence among msm is over 1 % in some metropolitan cities, such .as beijing (ibid.). due to widespread homophobia in chinese society, however, this population remains invisible within current health/social services. lack of research on sexuality, specifically homosexuality, has impaired our understanding of health and health practices of chinese msm. focusing on the illness experiences of chinese msm with hiv/aids, this paper explores the socio-cultural impacts of hiv/ aids on this group. the data used for this paper were collected through semi-constructed in-depth face-toiace interviews with 21 adult plwhas (including 11 msm) in beijing, china. with the permission of the participants, the interviews were audio-taped or recorded in notes. the transcribed interviews and interview notes were analyzed by using n-vivo, a software program for qualitative data analysis. this phenomenological study aimed to understand the experiences chinese plwhas (including msm) from their own perspectives. results: it is found that the illness experiences of chinese msm with hiv/aids are profoundly shaped by the socio-cultural meanings of homosexuality, which are further complicated by the dominant discourses on hiv/aids in china. at a macro level, homophobia in the larger society has decreased the capability of this group to respond to this epidemic in a more efficient way. at a meso level, aids-phobia within the chinese msm circuits has prevented this group from openly confronting this disease and offering support to those who are already affected by it. at a micro level, however, these hiv-infected/ affected msm have tried their best to locate spaces to live and to construct hope for their future lives despite various barriers within family, community, and the larger society. this study suggests that facilitating chinese msm's response to the aids crisis urgently requires bridging awareness, commitment, knowledge and resources both within and without this group. it also illustrates the importance of understanding homosexuality in the local context of hiv i aids, which will be helpful for developing culturally sensitive hiv/aids programs for this population on the community, national and international levels. introduction: having a regular family doctor (rfd) is known to be positively correlated with a good medical follow up, including access to preventive and/or chronic care. inversely, a lack of primary care may lead to high rates of avoidable hospitalizations, especially among poor people and/or in underserved neighbourhoods. in such a matter, a recent comparative study showed that paris was better ranged than other cities, such as nyc, tokyo or london. nevertheless, despite a quasi universal health insurance and a high density of general practitioners, a noticeable fraction of the paris population does not have any regular physician and we aimed to understand who they are and for what reasons rhey don't have any rfd. mdbods: cross sectional population survey among a random sample of households in 2 ~nderpriv ileged neighbourhoods in paris inner city, performed in 2003, using a face-to-face quest10nna1re collecr-1ng more than 400 social and health characteristics. ruults: a quarter (26.3 %) of rhe study popularion did not have any rfd. this proporrion was iignificantly higher among male (or= 2.00, 95%ci = [ 1.41-2.781), younger (e.g. or 18 -29/> .s l _= 4._oo, 95"1.ci = (2.13-7.69)), and/or unemployed (or =2.01, 95%ci = ij .21-3.3411_ people. in multtvanate analysis, after a full adjustment on gender, age, health status, health insura~ce, income, occupat10n and tducation level, we observed significant associations between having no rfd and: ~arrtal and_ pare~t hood status (e.g. or single no kids/in couple+kids = 2.12, 95%ci = ( 1.26-3.59()~ quality of relattonsh1ps with neighbours (or bad/good= 3 .82, 95%ci = [ 1.84-7.94)), and length of residence m the neighbourhood (with a dose/effect statistical relationship). . co11clusion: gender, age, employment status, mariral and parenthood stat~s as well as ~e1gh bourhood anchorage seem to be major predictors of having a rfd, even when um.versa! health i~sur ance has reduced most of financial barriers. in urban contexts, where residential migrattons and single lift (or family ruptures) are frequent, specific information may be conducted to encourage people to ket rfd. :tu~y tries to assess the health effects and costs and also analyse the availability and accessibility to health care for poor. . methods: data for this study was collected by a survey on 300 households of the local community living near the factories and 100 households where radiation hazard w~s n?~ present. ~~art from mor· bidity status and health expenditure, data was collected ~n access, a~ail~b1.hty and eff1c1ency of healrh care. a discriminant analysis was done to identify the vanables that d1scnmmate between the study and control group households in terms of health care pattern. a contingent valuation survey was also undertaken among the study group to find out the factors affecting their willingness to pay for health insurance and was analysed using logit model. results: the health costs and indebtedness in families of the study group was high as compared to control group households and this was mainly due to high health expenditure. the discriminant analysis showed that expenditure incurred by private hospital inpatient and outpatient expenditure were significant variables, which discriminated between the two types of households. the logit analysis showed !hat variables like indebtedness of households, better health care and presence of radiation induced illnesses were significant factors influencing willingness to pay for health insurance. the study showed that study group households were dependent on private sector to get better health care and there were problems with access and availability at the public sector. conclusion: the study found out that the quality of life of the local community is poor due to health effects of radiation and the burden of radiation induced illnesses are so high for them. there is an urgent need for government intervention in this matter. there is also a need for the public sector to be efficient to cater to the needs of the poor. a health insurance or other forms of support to these households will improve the quality for health care services, better and fast access to health care facilities and reduces the financial burden of the local fishing community. the prevalence of substance abuse is an increasing problem among low-income urban women in puerto rico. latina access to treatment may play an important role in remission from substance abuse. little is known, however, about latinas' access to drug treatment. further, the role of social capital in substance abuse treatment utilization is unknown. this study examines the relative roles of social capital and other factors in obtaining substance abuse treatment, in a three-wave longitudinal study of women ages 18-35 living in high-risk urban areas of puerto rico, the inner city latina drug using study (icldus). social capital is measured at the individual level and includes variables from social support and networks, familism, physical environment, and religion instruments of the icdus. the study also elucidates the role of treatment received during the study in bringing about changes in social capital. the theoretical framework used in exploring the utilization of substance abuse treatment is the social support approach to social capital. the research addresses three main questions: ( t) does social capital predict parti~ipating in treatment programs? (2) does participation in drug treatment programs increase social capital?, and (3) is there a significant difference among treatment modalities in affecting change in ~ial capital? the findings revealed no significant association between levels of social capital and gettmg treatment. also, women who received drug treatment did not increase their levels of social capital. the findings, however, revealed a number of significant predictors of social capital and receiving drug ~buse treatment. predictors of social capital at wave iii include employment status, total monthly mcoi:rie, and baseline social capital. predictors of receiving drug abuse treatment include perception of physical health and total amount of money spent on drugs. other different variables were associated to treatment receipt prior to the icldus study. no significant difference in changes of social capital was found among users of different treatment modalities. this research represents an initial attempt to elucidate the two-way relationship between social capital and substance abuse treatment. more work is necessary to unden~nd. ~e role of political forces that promote social inequalities in creating drug abuse problems and ava1lab1hty of treatment; the relationship between the benefits provided by current treatment poster sessions v81 sctrings and treatment-seeking behaviors; the paths of recovery; and the efficacy and effectiveness of the trtaanent. and alejandro jadad health professionals in urban centres must meet the challenge of providing equitable care to a population with diverse needs and abilities to access and use available services. within the canadian health care system, providers are time-pressured and ill-equipped to deal with patients who face barriers of poverty, literacy, language, culture and social isolation. directing patients to needed supportive care services is even more difficult than providing them with appropriate technical care. a large proportion of the population do not have equitable access to services and face major problems navigating complex systems. new approaches are needed to bridge across diverse populations and reach out to underserved patients most in need. the objective of this project was to develop an innovative program to help underserved cancer patients access, understand and use needed health and social services. it implemented and evaluated, a pilot intervention employing trained 'personal health coaches' to assist underserved patients from a variety of ethno-linguistic, socio economic and educational backgrounds to meet their supportive cancer care needs. the intervention was tested with a group of 46 underserved cancer patients at the princess margaret hospital, toronto. personal coaches helped patients identify needs, access information, and use supportive care services. triangulation was used to compare and contrast multiple sources of quantitative and qualitative evaluation data provided by patients, personal health coaches, and health care providers to assess needs, barriers and the effectiveness of the coach program. many patients faced multiple barriers and had complex unmet needs. barriers of poverty and language were the easiest to detect. a formal, systematic method to identify and meet supportive care needs was not in place at the hospital. however, when patients were referred to the program, an overwhelming majority of participants were highly satisfied with the intervention. the service also appeared to have important implications for improved technical health care by ensuring attendance at appointments, arranging transportation and translation services, encouraging adherence to therapy and mitigating financial hardship -using existing community services. this intervention identified a new approach that was effective in helping very needy patients navigate health and social services systems. such programs hold potential to improve both emotional and physical health out· comes. since assistance from a coach at the right time can prevent crises, it can create efficiencies in the health system. the successful use of individuals who were not licensed health professionals for this purpose has implications for health manpower planning. needle exchange programs (neps) have been distributing harm reduction materials in toronto since 1990. counterfit harm reduction program is a small project operated out of a community health centre in south-east toronto. the project is operated by a single full-time coordinator, one pan-rime mobile outreach worker and two peers who work a few hours each week. all of counterfit's staff, peers, and volunteers identify themselves as active illicit drug users. yet the program dis~rib utes more needles and safer crack using kits and serves more illicit drug u~rs t~an the comb1~e~ number of all neps in toronto. this presentation will discuss the reasons behind this success, .s~1f1cally the extended hours of operation, delivery models, and the inclusion of an. extremely marg1~ahzed community in all aspects of program design, implementation and eva.luat1?n. ~ounterfit was recently evaluated by drs. peggy milson and carol strike, two leading ep1dem1olog1st and researchers in the hiv and nep fields in toronto and below are some of their findings: "the program has experienced considerable success in delivering a high quality, accessible and well-used program .... the pro· gram has allowed (service users) to become active participants in providing. services to others and has resulted in true community development in the best sense. " ... counterf1t has ~~n verr succe~sful attracting and retaining clients, developing an effective peer-based model an.d assisting chen~s ~1th a vast range of issues .... the program has become a model for harm red~ctmn progr~ms withm the province of ontario and beyond." in june 2004, the association of ?ntano co~mumty heal~~ <:en· ires recognized counterfit's acheivements with the excellence m community health initiatives award. in kenya, health outcomes and the performance of government health service~ have det~riorated since the late 1980s, trends which coincide with a period of severe resource constramts necessitated by macro-economic stabilization measures after the extreme neo-liberalism of the 1980s. when the govern· ment withdrew from direct service provision as reform trends and donor advocacy suggested, how does it perform its new indirect role of managing relations with new direct health services providers in terms of regulating, enabling, and managing relations with these health services providers? in this paper therefore, we seek to investigate how healthcare access and availability in the slums of nairobi has been impacted upon by the government's withdrawal from direct health care provision. the methodology involved col· leering primary data by conducting field visits to 8 health institutions located in the slum areas of kibera and korogocho in nairobi. purposive random sampling was utilized in this study because this sampling technique allowed the researcher(s) to select those health care seekers and providers who had the required information with respect to the objectives of the study. in-depth interviews using a semi-structured ques· tionnaire were administered ro key informants in health care institutions. this sought to explore ways in which the government and the private sector had responded and addressed in practical terms to new demands of health care provision following the structural adjustment programmes of the 1990s. this was complemented by secondary literature review of publications and records of key governmental, bilateral and multilateral development partners in nairobi. the study notes a number of weaknesses especially of kenya's ministry of health to perform its expected roles such as managing user fee revenue and financial sustainability of health insurance systems. this changing face of health services provision in kenya there· fore creates a complex situation, which demands greater understanding of the roles of competition and choice, regulatory structures and models of financing in shaving the evolution of health services. we rec· ommend that the introduction of user fees, decentralization of service provision and contracting-out of non-clinical to private and voluntary agencies require a new management culture, and new and clear insri· tutional relationships. experience with private sector involvement in health projects underlines the need not only for innovative financial structures to deal with a multitude of contractual, political, market and risks, but also building credible structures to ensure that health services projects are environmentally responsive, socially sensitive, economically viable, and politically feasible. purpose: the purpose of this study is to examine the status of mammography screening utilization and its predictors among muslim women living in southern california. methods: we conducted a cross-sectional study that included 202 women aged ::!: 40 years. we col· leered data using a questionnaire in the primary language of the subjects. the questionnaire included questions on demography; practices of breast self-examination (bse) and clinical breast examination (cbe); utilization of mammography; and family history of breast cancer. bivariate and multiple logistic regression analyses were performed to estimate the odds ratios of mammography use as a function of demographic and other predictor variables. . results: among the 202 women, 78% were married, 68% were 40-50 years old, and 20% had family h1story of breast cancer. thirty-two percent of the participating women never practiced bse and 32% had not undergone cbe during the past two years. the data indicated that 46% of the women did not have mammography in the last two years. logistic regression analysis showed that age (0r=5.1, 95% confi· dcnc~ interval (cl)=l.8-14.2), having clinical breast examination (0r=24.9, 95% cl=8.4-73.7), and practtce of self-breast examination (0r=2.6, 95% cl= 1.1-6.2), were strong predictors of mammography use . . conclusions: the data point to the need for intervention targeting muslim women to inform and motivate th.cm a~ut practices for early detection of breast cancer and screening. further studies are needed to investigate the factors associated with low utilization of mammography among muslim women population in california. we conducted a review of the scientific literature and° government documents to describe ditnational health care program "barrio adentro" (inside the neighborhood). we also conducted qualiurivt interviews with members of the local health committees in urban settings to descrihe the comm unity participation component of the program. rtsmlts: until recently, the venezuelan public health system was characterized by a lack or limited access w health care (70% of the population) and long waiting lists that amounted to denial of service. moit than half of the mds worked in the five wealthiest metropolitan areas of the country. jn the spring oi2003, a pilot program hired 50 cuban mds to live in the slums of caracas to provide health care to piople who had previously been marginalized from social programs. the program underwent a massive expansion and in only two years 20,000 cuban and 6,500 venezuelan health care providers were working acmss the country. they provide a daily average of 20-40 medical consultations and home visits, c1lly out neighborhood rounds, and deliver health prevention initiatives, including immunization programs. they also provide generic medicines at no cost to patients, which treat 80% of presenting ill-ij!m, barrio adentro aims to build 8,000 clinics (primary care), 1,200 diagnostic and rehabilitation ctnrres (secondary care), and upgrade the current hospital infrastructure (tertiary care). local health committees survey the community to identify needs and organize a variety of lobby groups to improve dit material conditions of the community. last year, barrio adentro conducted 3.5 times the medical visits conducted by the ministry of health. the philosophy of care follows an integrated approach where btalrh is related to housing, education, employment, sports, environment, and food security. conclusions: barrio adentro is a unique collaboration between low-middle income countries to provide health care to people who have been traditionally excluded from social programs. this program shows that it is possible to develop an effective international collaboration based on participatory democracy. low-income americans are at the greatest risk of being uninsured and often face multiple health concerns. this evaluation of the neighborhood health initiative (nh!), an organization which uses multiple programmatic approaches to meet the multiple health needs of clients, reflected the program's many activities and the clients' many service needs. nh! serves low-income, underserved, and hard-to-reach residents in the des moines enterprise community. multiple approaches (fourth-generation evaluation, grounded theory, strengths-and needs-based) and methods (staff and client interviews, concept mapping, observations, qualitative and quantitative analysis) were used to achieve that reflection. results indicate good targeting of residents in the 50314 zip code and positive findings in the way of health insurance coverage and reported unmet health needs of clients. program activities were found to match client nttds, validating the organization\'s assessment of clients. important components of nhi were the staff composition and that the organization had become part of both the formal and informal networks. nhi 1 1 positioned as a link between the target population and local health and social sc:rvice agencies, working to connect residents with services and information as well as aid local agencies in reaching this underserved population. p4-36 (c) welfare: definition by new york city maribeth gregory for an individual who resides in new york city, to obtain health insurance under the medicaid policy one must fall under certain criteria .. (new york city's welfare programs 2003) if the individual _is on ssi or earns equal to or less than $934 per month, he is entitled to receive no more than $5,600 m resources. a family the size of two would need to earn less than $942 per month to qualify for no greater than ss,650 worth of medicaid benefits. a family of three would qualify for $5,650 is they earned less than $942 per month and so on. introduction: the vancouver gay communiry has a significant number of asian descendan!l. because of their double minority status of being gay and asian, many asian men who have sex with men (msm) are struggling with unique issues. dealing with racism in both mainstream society and the gay communiry, cultural differences, traditional family relations, and language challenges can be some of their everyday srruggles. however, culturally, sexually, and linguistically specific services for asian msm are very limited. a lack of availability and accessibiliry of culturally appropriate sexual health services isolates asian msm from mainstream society, the gay community, and their own cultural communities, deprives them of self-esteem, and endangers their sexual well-being. this research focuses on the qualita· tive narrative voices of asian msm who express their issues related to their sexualiry and the challenges of asking for help. by listening to their voices, practitioners can get ideas of what we are missing and how we need to intervene in order to reach asian msm and ensure their sexual health. methods: since many asian msm are very discreet, it is crucial to build up trust relationships between the researcher and asian msm in order to collect qualitative data. for this reason, a community based participatory research model was adopted by forming a six week discussion group for asian msm. in each group session, the researcher tape recorded the discussion, observed interactions among the participants, and analyzed the data by focusing on participants' personal thoughts, experiences, and emotions for given discussion topics. ra11lts: many asian msm share challenges such as coping with a language barrier, cultural differ· ences for interpreting issues and problems, and westerncentrism when they approach existing sexual health services. moreover, because of their fear of being disclosed in their small ethnic communities, a lot of asian msm feel insecure about seeking sexual health services when their issues are related to their sexual orientation. conclflsion: sexual health services should contain multilingual and multicultural capacities to meet minority clients' needs. for asian msm, outreach may be a more effective way to provide them with accessible sexual health services since many asian msm are closeted and are therefore reluctant to approach the services. building a communiry for asian msm is also a significant step toward including them in healthcare services. a communiry-based panicipatory approach can help to build a community for asian msm since it creates a rrust relationship between a worker and clients. p4-38 (c) identifying key techniques to sustain interpretation services for assisting newcomers isolated by linguistic and cultural barriers from accessing health services s. gopi krishna lntrodaetion: the greater toronto area (gta) is home to many newcomer immigrants and other vulnerable groups who can't access health resources due to linguistic, cultural and systemic barriers. linguistic and cultural issues are of special concern to suburbs like scarborough, which is home to thousands of newcomer immigrants and refugees lacking fluency in english. multilingual community ~nterpreter. service~ (mcis) is a non-profit social service organization mandated to provide high quality mterpretanon services. to help newcomers access health services, mcis partnered with the scarborough network of immigrant serving organizations (sniso) to recruit and train volunteer interpreters to accompany clienrs lacking fluency in english and interpret for them to access health services at various locati?ns, incl~~ing communiry ~c:-lth centres/social service agencies and hospitals. the model envisioned agencies recruin~ and mcis ~.mm.g and creating an online database of pooled interpreter resources. this da.tabase, acces&1bl~ to all pama~~g ?rganization is to be maintained through administrative/member · ship fees to. be ~1d by each parnapanng organization. this paper analyzes the results of the project, defines and identifies suc:cases before providing a detailed analysis for the reasons for the success . . methods:. this ~per~ q~ntitative (i.e. client numben) and qualitative analysis (i.e. results of key •~ormant m~rv1ews with semce ~sers and interpreters) to analyze the project development, training and 1mplementanon phases of the project. it then identifies the successes and failures through the afore· mentioned analysis. poster sessions vss resljts: the results of the analysis can be summarized as: • the program saw modest success both ia l?lllls of numbers of clients served as well as sustainability at various locations, except in the hospital iririog. o the success of the program rests strongly on the commitment of not just the volunteer interprmr, but on service users acknowledgments through providing transponation allowance, small honororia, letter of reference etc. • the hospital sustained the program better at the hospital due to the iolume and nature of the need, as well as innate capacity for managing and acknowledging volunceers. collc/llsion: it is possible to facilitate and sustain vulnerable newcomer immigrants access to health !ul'ices through the training and commitment of an interpreter volunteer core. acknowledging volunteer commitment is key to the sustenance of the project. this finding is important to immigration and health policy given the significant numbers of newcomer immigrants arriving in canada's urban communities. nity program was established in 1993 to provide support to people dying at home, especially those who were waiting for admission to the resi<lential hospice. with the advent of haart and corresponding challenges for people living with hiv/aids in the community, the community program has developed a unique case management approach. this model features an interdisciplinary team providing a clientdrivcn service. the case manager provides continuity of care to clients in a variety of settings both within and outside of the health care system. a case study is used to demonstrate how formal and informal networks of support facilitate efficient and appropriate resource utilization to promote client health. this case study demonstrates the value of consistent coordination with a client who has complex physical, spiritual, substance use and mental health needs. the authors will show how they liaised with the housing authority, police, social services as well as the family physician, clinic staff and a hospital emergtncy room to ensure this client did not fall between the cracks. tarek hussain recognition of the importance of community involvement and sectoral cooperation in health and !ocial services, formalized in alma-ata declaration in 1978, was reinforced at riga meeting held at the mid·point between alma-ata and the year 2000. then at the 20th anniversary of alma-ata declaration, ir was declared that 'primary health care is everybody's business'. health does not exist in isolation. health cannot be defined only as an outcome of 'medical care' but also as one of 'social action'; the responsibility of disease prevention and health promotion rests not only on governments, but also with don·govemmentorgan izations, the community, and individuals. the major accomplishment to date may be that rhere is growing realization in the health sector that community involvement is more than a political right-it is absolute necessary. lessons have learned during the implementation of disease-specific l'cltical programmes, such as cdd, ari, and epi; first, integrated and holistic approaches to childcare art needed, and second, the need for community involvement has become evident. imc! is a broad inte-gra1ed strategy with an overall objective contributing to reducing child morbidity and mortality in developing countries. and one of the imci components, 'community !mci', which is now broadening ~s an entry point for child-focused community development initiatives. community jmcl/c~1ld health is ~n mtcgrated approach to the promotion of key family and community practices that have impact on: child growth and development, disease prevention, home care for sick, malnourished child~en, a.nd care-seekmg behavior and compliance with advice and treatment. the presentation addres~es, m brief, the ~evel opinent of community !mci, operational aspect of community/im cl, key strategies and tools ava1la~le for implementation of c/imci. the paper draws some successful programme examples on working logether for imc! with the community in various countries which had substantial benefns on programme outcomes. p4-4 t (c) healthy child screening: an innovative service initiative ann-marie marcolin and joyce allen . introduction: with mounting attention for creative and integrated models of c~re .that are populallon and community focused, the healthy child screening initiative achiev.es t~ese pnncipl~s and more! . • parkdale high park rotary (sponsorship) the heal~h ~h1ld scree?1?g is a ~opl~-centred model of care. agency and professional sector-specific silos are ehmmated, ~rov1dmg f~~1hes wit~ one-sto~ access to primary prevention services in a local school gym! children at nsk are rece1vmg early mtervent10n and appropriate referrals to the right care provider, at the right time a~d in t~e right place. further, with a complimentary focus on prevention, the program serves to keep at nsk children healthy. healthy child screening results: the service model is developed around four distinct phases: 1) planned outreach; 2) coordinated intake and registration; 3) interdisciplinary screening; and 4) targeted referral. since the fall 2003 and through six collaborative healthy child screenings 158 children ranging in age from 2 to 6 have benefited from this unique service model of care. conclusion: the presentation will provide practical information on the development and implementation of the model. there will also be a focus on lessons learned in building community service provider-hospital relationships. according to statistics canada (2001) 49% of the toronto population was born outside of canada and 21 % were new immigrants. immigrants often arrive in canada in superior health compared to the canadian born population, but they lose this health advantage over time. changes in immigrant health status over time may be attributed to a variety of factors including barriers to accessing and receiving care as well as limitations in the mainstream health service organizations and their approaches to health care delivery (hyman, 2001 ) . for example, immigrant women are less likely to receive pap tests, which places them at a greater risk for developing cervical cancer. similarly, immigrant women receive less mammography screening than their canadian counterparts. the immigrant women's health centre mobile health unit (mhu) was created as an alternative care delivery model to address the need for increased accessibility as well as culturally and linguistically appropriate reproductive health care. toronto western hospital and the immigrant women's health centre have formed a collaborative partnership which incorporates a primary care nurse practitioner and lay ethnocultural counselors providing care on the mhu. currently, a qualitative ethnonursing study is underway to understand the unique experiences of women using the mhu for their reproductive health care as well as the perspectives of the mhu health care providers. based on a literature review and preliminary findings from provider and participant interviews, the proposed presentation will address the themes of health status for immigrant and refugee women, accessibility issues as well strategies to improve their reproductive health care. we will discuss benefits as well as limitations to health care provision using this alternative service delivery model. michael carden, brian edlin, andrew talal, elizabeth getter, and marla shu lntrod.ction: to date most active drug users have not had access to treatment for hepatitis c virus (hcv) infection and substantial barriers continue to exist that interfere with treatment availability for this population. methods: active illicit drug users interested in pursuing hcv care and treatment were recruited in partnership w~th co~munity-based organizations (cbo's) in new york city. baseline data were collected on quahty of hfe, substance use patterns, depression, health care utilization, hcv health beliefs and other relev.ant variables. medical evaluations were conducted, including liver biopsy when indicated, an~ treatment ts ~ffered when no contraindications are present. participants also receive psychiatric evaluahons to determme the appropriateness of interferon treatment. renlts: fifteen individuals have been recruited to date and received an initial medical evaluation. the mean age was 40 years and _the average age of initial heroin or cocaine use was 16.8. eight (53%) ha~ been homeless at least once m the last 6 months and s (33%) were homeless for most or all of this penod. fourteen (93%) had a history of injection drug use with the mean age at first injection being 21 111s1frsewons v87 ,an. elrven persc.!ns (79%) reported inje~~ng .heroin or cocaine within the last 30 days while the .wing four (21 l'o) reported regular non-m1ect1on use of cocaine and street-obtained benzodiazepines. sijiy seven percent of the sample (n= 10) reported ever being referred to hcv treatment while 2 ( t 3 % ) ftponeddiattreann~nt had been offered by~ ~e.dical provider. none had ever received a liver biopsy or araanent. most believed that hcv was a s1gmf1cant threat to their health (80%) and that there was a pm)dchance they would eventually die if their hepatitis c was not treated (67%). though 7 individuals 147%1 believed that there was no cure for hcv, 13 (87%) reported that they would initiate treatment if i was recommended by their doctor. thirteen (87%) had a current psychiatric diagnosis including «pmsion, anxiety disorder, schizophrenia, schizoaffective disorder, and borderline and antisocial persooaliry disorders. among eight individuals who had the beck depression inventory administered, the mean score was 29.75. attendance rates were 83% (83/100) at scheduled medical and psychiatric 1ppointments and 50% (2/4) at liver biopsy. <andtuions: active drug users, despite experiencing multiple psycho-social problems, can be mgaged in hcv care using a multidisciplinary approach, but require intensive follow-up support. hcv aunnent of active drug users should not be dismissed. n-44 (c) antiretroviral therapy in mv infected infants: when to initiate therapy an african experience kingsley okonkwo, ademola adeyemo, israel sokeye, and nwaife umeike /""°""ction: as technology for early diagnosis of human immunodeficiency virus [hiv] improves, m to commence highly active antiretroviral therapy [haarn is yet to be fully evaluated. this prospective study describes our initial experience with early haart in hiv infected nigerian infants and rqions the outcome. we also analyzed the socioeconomic implications of early haart therapy in infants in the african setting. method: hiv infected infants were started on haart before 6 months and followed up at 4 mkly intervals.we monitored baseline and 3 monthly cd4 . conclusion: initial results suggest early haart before 6 months resulted. in improv~d out~ome in african children. treatment appears to be as effective as in developed counmes. in. a~nca as. m most developing countries with limited resources it is possible and effective to use haart m 1~~ants if p~oper llloniroring facilities are available. however the occurrence of long-term drug related tox1c1ty and mk of emergence of resistant viral strains create need for further evaluation of haart in infants. background: most risk factors for cardiovascular disease (cvd) can be mitigated through ~th clinical interventions and lifestyle change. we used data from a telephone survey of new york city (nyc) adults to characterize health care access and healthy behavior among those with three or more cvd risk factors. methods: the study population included respondents to the nyc community health survey 2002 (n= 9,674) self-reporting~ 3 of the following: smoking, diabetes, high cholesterol level, high blood pres· sure, body mass index >25, and age >45 (males) or >55 (females) (n=2,439). results: based on self-report, an estimated 1.447,000 (24%) of nyc adults have~ 3 or more cvd risk factors. this population is 51 % male, 47% white, 25% black, and 53% with s 12 years of education. most report good access to health care, indicated by having health insurance (95%), regular doctor (89%), their blood pressure checked within last 6 months (91 %), and their choles· terol checked within the past year (90% ). only 29% reported getting at least 20 minutes of exercise ~ 4 times per week and only 9% eating ~ 5 servings of fruits and vegetables the previous day. among current smokers, 59% attempted to quit in past 12 months, but only 32% used medication or counseling. implications: these data suggest that most nyc adults known to be at high risk for cvd have access to regular health care, but most do not engage in healthy lifestyle or, if they smoke, attempt effective quit strategies. more clinic-based and population-level interventions are needed to support lifestyle change among those at high risk of cvd. introduction: recently, much interest has been directed at "obesogenic" (obesity-promoting) (swinburn, egger & raza, 1998) built environments, and at geographic information systems (gis) as a tool for their exploration. a major geographical concept is accessibility, or the ease of moving from an origin to a destination point, which has been recently explored in several health promotion-related stud· ies. there are several methods of calculating accessibility to an urban feature, each with its own strengths, drawbacks and level of precision that can be applied to various health promotion research issues. the purpose of this paper is to describe, compare and contrast four common methods of calculating accessibility to urban amenities in terms of their utility to obesity-related health promotion research. practical and conceptual issues surrounding these methods are introduced and discussed with the intent of providing health promotion researchers with information useful for selecting the most appropria1e accessibility method for their research goal~ ~ethod: this paper describes methodological insights from two studies, both of which assessed the neighbourhood-level accessibility of fast-food establishments in edmonton, canada -one which used a relatively simple coverage method and one which used a more complex minimum cos1 method. res.its: both methods of calculating accessibility revealed similar patterns of high and low access to fast-food outlets. however, a major drawback of both methods is that they assume the characteristics of the a~e~ities and of the populations using them are all the same, and are static. the gravity potential method is introduced as an alternative, since it is ·capable of factoring in measures of quality and choice. a n~mber of conceptual and pr~ctical iss~es, illustrated by the example of situational influences on food choice, make the use of the gravity potential model unwieldy for health promotion research into sociallydetermined conditions such as obesity. co.nclusions: i~ ~ommended that geographical approaches be used in partnership with, or as a foun~ation for, ~admonal exploratory methodologies such as group interviews or other forms of commumty consultation that are more inclusive and representative of the populations of interest. qilhl in los angeles county ,,..ia shaheen, richard casey, fernando cardenas, holman arthurs, and richard baker ~the retinomax autorefractor has been used for vision screening of preschool age childien. ir bas been suggested to be used and test school age children but not been validated in this age poup. ob;taiw: to compare the results of retinomax autorefractor with findings from a comprehensive i!' examination using wet retinoscopy for refractive error. mllhods: children 5-12 years old recruited from elementary schools at los angeles county were iaml with snellen's chart and the retinomax autorefractor and bad comprehensive eye examination with dilation. the proportion of children with abnormal eye examination as well as diesensitiviry and specificity of the screening tools using retinomax autorefractor alone and in combinalion wirh snellen's chart. results of the 258 children enrolled in the study (average age= 8.5± 2.1 years; age range, 5-12 years), 6?% had abnormal eye examination using retinoscopy with dilation. for the lerinomax, the sensitivity was 85% (95% confidence interval [ci] 78%-90%), and the specificity was 31% (95% ci, 22%-41 o/o). simultaneous testing using snellen's chart and retinomax resulted in gain in 111sitiviry (94%, 95% cl= 89, 97), and loss in specificity (28%, 95% cl= 19%-38%). the study showed that screening school age children with retinomax autorefractor could identify most cases with abnormal vision but would be associated with many false-positive results. simuhaneous resting using snellen's chart and retinomax maximize the case finding but with very low specificiry. mdhotjs: a language-stratified, random sample of 2366 members of the college of family physicians of canada received a confidential survey. the questionnaire collected data on socio-demographic characteristics, medical training, practice type, setting and hcv-related care practices. the self-adminisratd questionnaire was also made available to participants for completion on the internet. batdti: response proportion was 33%. median age was 41 years (47% female) and the proporlionoffrench questionnaires was 26%. approximately 88% had completed family medicine residency lllining in canada; median year of training completion was 1995. sixty-seven percent, 38% and 29% work in private offices/clinics, community hospitals and emergency departments, respectively. regarding ~practices, 94% had ever requested a hcv test and 87% of physicians had screened for hcv iafrction in rhe past 12 months· median number of tests was 10. while 17% reported having no hcv-uaed patients in their practic~, 44% had 1-5 hcv-infected patients. regarding the level of hcv care provided, 4.3% provide ongoing advanced hcv care including treatment and dose monitoring for ctmduions: in this sample of canadian family physicians, most had pro~ided hcv screening. to •least one patient in the past year. less than half had 1-5 hcv-infected patients and 41 % provide ~:relared care the role of socio-demographic factors, medical training as wel_i as hcv ca~e percep-lldas 10 rhe provision of appropriate hcv screening will be examined and described at the time of the canference. '4-50 (c) healthcare services: the context of nepal meen poudyal chhetri """1tl.ction healthcare service is related with the human rights and fundamental righ~ of the ci~ ciaaiuntry. however, the growing demand foi health care services, quality heal~care service, accessib1b~ id die mass population and paucity of funds are the different but interrelated issues to .be ~ddressed. m nepat.1n view of this context, public health sector in nepal is among other sectors, which is struggling -.i for scarce resources. . . . nepal, the problems in the field of healthcare servic~s do not bnut ~o the. paucity of faads and resources only, but there are other problems like: rural -urban imbalance, regional unbalance, poster sessio~ f the ll ·m1 ·ted resources poor healthcare services, inequity and inaccessibility of the poor management o , . poor people of the rural, remote and hilly areas for the healthcare services and so on.. . . . · . i f ct the best resource allocation is the one that max1m1zes t e sum o m ivi ua s u11 · ea t services. n a , · h d' ·b · · · h . ·t effi.ciency and efficient management are correlated. it might be t e re istn utmn of mes. ence, equi y, . . . . 1 . income or redistribution of services. moreover, maximizanon of available resources, qua tty healthcare services and efficient management of them are the very important and necessary tools and techmques to meet the growing demand and quality healthcare services in nepal. p4-51 (a) an jn-depth analysis of medical detox clients to assist in evidence based decision making xin li, huiying sun, ajay puri, david marsh, and aslam anis introduction: problematic substance use represents an ever-increasing public health challenge. in the vancouver coastal health (vch) region, there are more than 100,000 individuals having some probability of drug or alcohol dependence. to accommodate this potential demand for addiction related services, vch provides various services and treatment, including four levels of withdrawal management services (wms). clients seeking wms are screened and referred to appropriate services through a central telephone intake service (access i). the present study seeks to rigorously evaluate one of the services, vancouver detox, a medically monitored 24-bed residential detox facility, and its clients. doing so will allow decision makers to utilize evidence based decision-making in order to improve the accessibility and efficiency of wms, and therefore, the health of these clients. methods: we extract one-year data (october 1, 2003 -september 30, 2004 from an efficient and comprehensive database. the occupancy rate of the detox centre along with the clients' wait time for service and length of stay (los) are calculated. in addition, the effect of seasonality on these variables and the impact of the once per month welfare check issuance on the occupancy rate are also evaluated. results: among the 2411 clients (median age 40, 65% male) who were referred by access! to vancouver detox over the one-year period, 1448 were admitted. the majority (81 %) of those who are not admitted are either lost to follow up (i.e., clients not having a fixed address or telephone) or declined service at time of callback. the median wait time was 1 day [q3-ql: 3-1], the median los was 5 days iq3-qt: 6-3], and the average bed occupancy rate was 83%. however, during the threeday welfare check issue period the occupancy rate was lower compared to the other days of the year 175% vs. 84%, p conclusion: our analysis indicates that there was a relatively short wait time at vancouver detox, however 40% of the potential clients were not served. in addition, the occupancy rate declined during the welfare check issuance period and during the summer. this suggests that accessibility and efficiency at vancouver detox could be improved by specifically addressing these factors. background: intimate partner violence (ipv) is associated with acute and chronic physical and men· tal health outcomes for women resulting in greater use of health services. yet, a vast literature attests to cultural variations in perceptions of health and help-seeking behaviour. fewer studies have examined differences in perceptions of ipv among women from ethnocultural communities. the recognition, definition, and understanding of ipv, as well as the language used to describe these experiences, may be different in these communities. as such, a woman's response, including whether or not to disclose or seek help, may vary according to her understanding of the problem. methods: this pilot study explores the influence of cultural factors on perceptions of and responses to ipv among canadian born and immigrant young women. in-depth focus group interviews were con· ducted with women, aged 18 to 24 years, living in toronto. open-ended and semi-structured interview questions were designed to elicit information regarding how young women socially construct jpv and where they would go to receive help. interviews were transcribed, then read and independently coded by the research team. codes were compared and disagreements resolved. qualitative software qsr n6 was used to assist with data management. . ruu~ts_: res~nses_abo~t what constitutes ipv were similar across the study groups. when considering specific ab.us1ve ~1tuanons and types of relationships, participants held fairly relativistic views about ipv, especially with regard to help-seeking behaviour. cultural differences in beliefs about normaive m;ile/femal~ relations. familial.roles, and customs governing acceptable behaviours influenced partictpants perceptions about what 1n1ght be helpful to abused women. interview data highlight the social l05ter srnfons v91 11111 suucrural _impact these factors ha:e on you?g women and provide details regarding the dynamics of cibnocultur~ m~uences on help-~eekmg behav10ur: t~e ro~e of such factors such as gender inequality within rtlaoo?sh1ps and t_he ~erce1ved degree of ~oc1al 1solat1on and support nerworks are highlighted. collc~ the~ findmgs unde~score the _1mporta_nc_e of understanding cultural variations in percrprions of ipv ~ relanon to ~elp-seekmg beha~1':'ur. th1s_mformation is critical for health professionals iodiey may connnue developmg culturally sensmve practices, including screening guidelines and protorol s. ln addition, _this study demonstr~tes that focus group interviews are valuable for engaging young romen in discussions about ipv, helpmg them to 'name' their experiences, and consider sources of help when warranted. p4-s3 (a) health problems and health care use of young drug users in amsterdam .wieke krol, evelien van geffen, angela buchholz, esther welp, erik van ameijden, and maria prins /11trod11ction: recent advances in health care and drug treatment have improved the health of populations with special social and health care needs, such as drug users. however, still a substantial number dots not have access to the type of services required to improve their health status. in the netherlands, tspccially young adult drug users (yad) whose primary drug is cocaine might have limited access to drugrreatment services. in this study we examined the history and current use of (drug associated) treatmmt services, the determinants for loss of contact, and the current health care needs in the young drug mm amsterdam study (yodam). methods: yodam started in 2000 and is embedded in the amsterdam cohort study among drug mm. data were derived from y ad aged < 30 years who had used cocaine, heroin, ampheramines and i or methadone at least 3 days a week during the 2 months prior to enrolment. res11lts:of 195 yao, median age was 27 years (range: 18-30 years), 72% was male and 83% had 1dutch nationality at enrolment. nearly all participants (97%) reported a history of contact with drug llt.lnnent services (methadone maintenance, rehabilitation clinics and judicial treatment), mental health car? (ambulant mental care and psychiatric hospital) or general treatment services (day-care, night-care, hdp for living arrangements, work and finance). however, only 61 % reported contact in the past six l!xlllths. this figure was similar in the first and second follow-up visit. among y ad who reported no current contact with the health care system, 87% would like to have contact with general treatment serl' icts. among participants who have never had contact with drug treatment services, 67% used primarily cocaine compared with 22% and 8% among those who reported past or current contact, respectively. saied on the addiction severity index, 70% reported at least one mental health problem in the past 30 days, but only 11 % had current contact with mental health services. concl11sion: results from this study among young adult drug users show that despite a high contact rm with health care providers, the health care system seems to lose contact with yao. since 87% indicatt the need of general treatment services, especially for arranging house and living conditions, health m services that effectively integrate general health care with drug treatment services and mental health care might be more successful to keep contact with young cocaine users. mtthods: respondents included adults aged 18 and over who met dsm-iv diagn?snc criteria for an anxiety or depressive disorder in the past 12 months. we performed two sets of logisnc regressmns. thtdichotomous dependent variables for each of the regressions indicated whether rhe respondenr_vis-ud a psychiatrist, psychologist, family physician or social worker in the _past_ 12 months. no relationship for income. there was no significant interaction between educatmn an mco~:· r: ::or respondents with at least a high school education to seek help ~rom any of the four servic p were almost twice that for respondents who had not completed high school. th . d ec of analyses found che associacion becween educacion and use of md-provided care e secon s · · be d · · ·f· ly 1 ·n che low income group for non-md care, the assoc1anon cween e ucatlon and was s1gm icant on -· . . . . use of social workers was significant in both income groups, but significant only for use of psychologists in che high-income group. . . . conclusion: we found differences in healch service use by education level. ind1v1duals who have nor compleced high school appeared co use less mental he~lt~ servi~es provided ~y psyc~iatrists, psycholo· gists, family physicians and social workers. we found limited e.v1dence _suggesting the influence of educa· tion on service use varies according to income and type of service provider. results suggesc there may be a need to develop and evaluate progr~ms.designe~ to deliver targeted services to consumers who have noc completed high school. further quahtanve studies about the expen· ence of individuals with low education are needed to clarify whether education's relationship with ser· vice use is provider or consumer driven, and to disentangle the interrelated influences of income and education. system for homeless, hiv-infected patients in nyc? nancy sahler, chinazo cunningham, and kathryn anastos introduction: racial/ethnic disparities in access to health care have been consistently documented. one potential reason for disparities is that the cultural distance between minority patients and their providers discourage chese patients from seeking and continuing care. many institucions have incorporated cultural compecency craining and culturally sensicive models of health care delivery, hoping co encourage better relacionships becween patients and providers, more posicive views about the health care system, and, ulcimacely, improved health outcomes for minority patients. the current scudy tests whether cultural distance between physicians and patients, measured by racial discordance, predicts poorer patient attitudes about their providers and the health care system in a severely disadvantaged hiv-infected population in new york city that typically reports inconsistent patterns of health care. methods: we collected data from 396 unscably housed black and latino/a people with hiv who reported having a regular health care provider. we asked them to report on their attitudes about their provider and the health care system using validated instruments. subjects were categorized as being racially "concordant" or "discordant" with their providers, and attitudes of these two groups were compared. results: the sample consisted of 256 (65%) black and 140 (35%) latino/a people, who reported having 80 (20%) black physicians, 49 (12%) latino/a physicians, 167 (42%) white physicians, and 100 (25%) physicians of another/unknown race/ethnicity. overall, 260 (75%) subjects had physicians of a different race/ethnicity than their own. racial discordance did not predict negative attitudes about rela· tionship with providers: the mean rating of a i-item trust in provider scale (lo=high and o=low) was 8.0 for both concordant and discordant groups, and the mean score in 13-icem relationship with provider scale (4=high and !=low) was 3.5 for both groups. however discordance was significantly associated with distrust in che health care syscem: che mean score on a 7-icem scale (5=high discrust and l=low distrust) was 3.4 for discordant group and 3.0 for che concordant group (t= 2.66, p= 0.008). we further explored these patterns separacely in black and lacino/a subgroups, and using different strategies ro conceptualize racial/ethic discordance. conclusions: in this sample of unscably housed black and latino/a people who receive hiv care in new york city, having a physician from the same racial/ethnic background may be less important for developing a positive doctor-patient relationship than for helping the patients to dispel fear and distrust about the health care system as a whole. we discuss the policy implications of these findings. ilene hyman and samuel noh . .abstract objectiw: this study examines patterns of mental healthcare utilization among ethiopian 1mm1grants living in toronto. methods: a probability sample of 342 ethiopian adults ( 18 years and older) completed structured face-to-face interviews. variables ... define, especially who are non-health care providers. plan of analysis. results: approximately 5% of respondents received memal health services from mainstream healthcare providers and 8% consulted non-healthcare professionals. of those who sought mental health services from mainstream healthcare providers, 3.1 % saw family physicians, 2.1 % visited a psychiatrist. and 0.6% consulted other healthcare providers. compared with males, a significantly higher proportion 1gsfer sessions v93 ri ftlnales consulted non-healthcare_ professionals for emotional or mental health problems (p< 0.01 ). tlbile ethiopian's overall use of mamstream healthcare services for emotional problems (5%) did not prlydiffer from the rate (6%) of the general population of ontario, only a small proportion ( 12.5%) rjerhiopians with mental health needs used services from mainstream healthcare providers. of these, !oj% received family physicians' services, 4.3 % visited a psychiatrist, and 2.2% consulted other healthll/c providers. our data also suggested that ethiopian immigrants were more likely to consult tradioooal healers than health professionals for emotional or mental health problems ( 18.8% vs. 12.5% ). our bivariate analyses found the number of somatic symptoms and stressful life events to be associated with an increased use of medical services and the presence of a mental disorder to be associated with a dfcreased use of medical services for emotional problems. however, using multivariate methods, only die number of somatic symptoms remained significantly associated with use of medical services for emooonal problems. diu#ssion: study findings suggest that there is a need for ethnic-specific and culturally-appropriate mrcrvention programs to help ethiopian immigrants and refugees with mental health needs. since there ~a strong association between somatic symptoms and the use family physicians' services, there appears robe a critical role for community-based family physicians to detect potential mental health problems among their ethiopian patients, and to provide appropriate treatment and/or referral. the authors acknowledge the centre of excellence for research in immigration and settlement (ceris) in toronto and canadian heritage who provided funding for the study. we also acknowledge linn clark whose editorial work has improved significantly the quality of this manuscript. we want to thank all the participants of the study, and the ethiopian community leaders without whose honest contributions the present study would have not been possible. this paper addresses the impact of the rationalization of health-care services on the clinical decision-making of emergency physicians in two urban hospital emergency departments in atlantic canada. using the combined strategies of observational analysis and in-depth interviewing, this study provides a qualitative understanding of how physicians and, by extension, patients are impacred by the increasing ancmpts to make health-care both more efficient and cost-effective. such attempts have resulted in significantly compromised access to primary care within the community. as a consequence, patients are, out of necessity, inappropriately relying upon emergency departments for primary care services as well as access to specialty services. within the hospital, rationalization has resulted in bed closures and severely rmricted access to in-patient services. emergency physicians and their patients are in a tenuous position having many needs but few resources. furthermore, in response to demands for greater accountability, physicians have also adopted rationality in the form of evidence-based medicine. ultimately, ho~ever, rationality whether imposed upon, or adopted by, the profession significantly undermines physu.:1ans' ability to make decisions in the best interests of their patients. johnjasek, gretchen van wye, and bonnie kerker introduction: hispanics comprise an increasing proportion of th.e new york city (nyc) populanon !currently about 25%). like males in the general population, h1spamc males (hm) have a lower prrval,nce of healthcare utilization than females. however, they face additional access barriers such as bnguage differences and high rates of uninsurance. they also bear a heavy burden of health problems lllehasobesity and hiv/aids. this paper examines patterns of healthcare access and ut1hzat1on by hm compared to other nyc adults and identifies key areas for intervention. . . . 148 9 01 5 8 9 01 and older are significantly lower than the nhm popu anon . 10 v. . 10, p<.05), though hi\' screening and immunizations are comparable between the two groups. conclusion: findings suggest that hm have less access t? healthcare than hf or nhm. hown1r, hm ble to obtain certain discrete medical services as easily as other groups, perhapsdueto!rtor are a hm. i i . subsidized programs. for other services, utilization among 1s ower. mprovmg acc~tocareinthis group will help ensure routine, quality care, which can lead to a greater use of prevennve services iii! thus bener health outcomes. introduction: cancer registry is considered as one of the most important issues in cancer epidemiology and prevention. bias or under-reporting of cancer cases can affect the accuracy of the results of epidemiological studies and control programs. the aim of this study was to assess the reliability of the regional cancer report in a relatively small province (yasuj) with almost all facilities needed for c3llcll diagnosis and treatment. methods: finding the total number of cancer cases we reviewed records of all patients diagnoicd with cancer (icd 140-239) and registered in any hospital or pathology centre from1999 until 2001 i n yasuj and all (5) surrounding provinces. results: of 504 patients who were originally residents of yasui province, 43.7% wereaccoulll!d for yasuj province. the proportion varies according to the type of cancer, for exarnplecancetsofdiglstive system, skin and breast were more frequently reported by yasuj's health facilities whereas cancmoi blood, brain and bone were mostly reported by neighbouring provinces. the remaining cases (56.3%1 were diagnosed, treated and recorded by neighbouring provinces as their incident cases. this is partly because of the fact that patients seek medical services from other provinces as they believed that the facil. ities are offered by more experienced and higher quality stuffs and their relative's or temporary acooiii' modation addresses were reported as their place of residence. conclusion: measuring the spatial incidence of cancer according to the location of report ortht current address affected the spatial statistics of cancer. to correct this problem recording the permanm! address of diagnosed cases is important. p4-60 (c). providing primary healthcare to a disadvantaged population at a university-run commumty healthcare facility tracey rickards the. c:ommuni~y .h~alth ~linic (chc) is a university sponsored nurse-managed primary bealthwt (p~c:l clime. the clm1c is an innovative model of healthcare delivery in canada that has integrated tht principles of phc ser · · h' . vices wit ma community development framework. it serves to provide access to phc services for members of th · · illi · dru is ii be . . e community, particularly the poor and those who use or gs, we mg a service-learning facil'ty f d · · · · · · d rionll h . . .,m.:. · t · . meet c ient nee s. chmc nursing and social work staff and srudents r·--· ipa em various phc activities and h .l.hont" less i . f . outreac services in the local shelters and on the streels to'"" popu auon o fredericton as well th chc · model iii fosterin an on oi : . • e promotes and supports a harm reduction . · local d!or an~ h ng ~art:ersh1p with aids new brunswick and their needle exchange program, w1tha ing condoms and :xu:t h:~~~e e~aint~nance therapy clients, and with the clie~ts themselves ~_r; benefits of receiving health f ucation, a place to shower, and a small clothing and food oai~· care rom a nurse p · · d d · --""~'i"· are evidenced in th r research that involved needsaans mvo ves clients, staff, and students. to date the chc has unacn-1 · sessment/enviro i . d ; •• '""""1ll eva uanon. the clinic has also e . d nmenta scan, cost-benefit analysis, an on-go...,,1"".'i'~ facility and compassionate lea x~mme the model of care delivery' focusing on nursing roles wi~ cj rmng among students. finally, the clinic strives to share the resu•p v95 . -arch with the community in which it provides service by distributing a bi-monthly newsletter, and plllicipating in in-services and educational sessions in a variety of situations. the plan for the future is coolinued research and the use of evidence-based practice in order to guide the staff in choosing how much n~ primary healthcare services to marginalized populations will be provided. n-61 (c) tuming up the volume: marginalized women's health concerns tckla hendrickson and betty jane richmond bdrotbu:tion: the marginalization of urban women due to socio-economic status and other determinants negatively affects their health and that of their families. this undermines the overall vitaliry of urban communities. for example, regarding access to primary health care, women of lower economic surus and education levels are less likely to be screened for breast and cervical cancer. what is not as widely reported is how marginalized urban women in ontario understand and articulate their lack of access to health care, how they attribute this, and the solutions that they offer. this paper reports on the rnults of the ontario women's health network (owhn) focus group project highlighting urban women's concerns and suggestions regarding access to health care. it also raises larger issues about urban health, dual-purpose focus group design, community-based research and health planning processes. mdhods: focus group methodology was used to facilitate a total of 30 discussions with 55 urban and 54 rural women across ontario from 2003 to 2005. the women were invited to participate by local women's and health agencies and represented a range of ages, incomes, and access issues. discussions focussed on women's current health concerns, access to health care, and information needs. results were analyzed using grounded theory. the focus groups departed from traditional focus group research goals and had two purposes: 1) data collection and dissemination (representation of women's voices), and 2) fostering closer social ties between women, local agencies, and owhn. the paper provides a discussion and rationale for a dual approach. rax/ts: the results confirm current research on women's health access in women's own voices: urban women report difficulty finding responsive doctors, accessing helpful information such as visual aids in doctors' offices, and prohibitive prescription costs, in contrast with rural women's key concern of finding a family doctor. the research suggests that women's health focus groups can address access issues by helping women to network and initiate collective solutions. the study shows that marginalized urban women are articulate about their health conctrns and those of their families, often understanding them in larger socio-economic frameworks; howtver, women need greater access to primary care and women-friendly information in more languages and in places that they go for other purposes. it is crucial that urban health planning processes consult directly with women as key health care managers, and turn up the volume on marginalized women's voices. women: an evaluation of awareness, attitudes and beliefs introduction: nigeria has one of the highest rates of human immunodeficiency virus ihivi seroprrvalence in the world. as in most developing countries vertical transmission from mother to child account for most hiv infection in nigerian children. the purpose of this study was ro. determine the awareness, attitudes and beliefs of pregnant nigerian women towards voluntary counseling and testing ivct! for hiv. mnbod: a pre-tested questionnaire was used to survey a cross section '.>f.240 pregnant women ~t 2 (lrlleral antenatal clinics in awka, nigeria. data was reviewed based on willingness to ~c~ept or re1ect vct and the reasons for disapproval. knowledge of hiv infection, routes of hiv transm1ssmn and ant1rnroviral therapy iart) was evaluated. hsults: 72% of the women had good knowledge of hiv, i 5% had fair knowledge while 1.1% had poor knowledge of hiv infection.48% of the women were not aware of the association of hreast milk feeding and transmission of hiv to their babies. majority of the women 87% approved v~t while 13% disapproved vct, 93% of those who approved said it was because vct could ~educe risk of rransmission of hiv to their babies. all respondents, 100% who accepted vc.i ~ere willing to be tnted if results are kept confidential only 23% accepted to be tested if vc.t results w.111 be s~ared w1.th pinner and relatives 31 % attributed their refusal to the effect it may have on their marriage whale 69 '-gave the social 'and cultural stigmatization associated with hiv infection for their r~fusal.s 9 % wall accept vct if they will be tested at the same time with their partners.81 ~0 of ~omen wall pref~r to breast feed even if they tested positive to hiv. women with a higher education diploma were 3 times v96 more likely to accept vct. knowledge of art for hiv infected pregnant women as a means of pre. vention of maternal to child transmission [pmtct) was generally poor, 37% of respondents wm aware of art in pregnancy. conclusion: the acceptance of vct by pregnant women seems to depend on their understanding that vct has proven benefits for their unborn child. socio-cultur al factors such as stigmatizationof hiv positive individuals appears to be the maj_or impedi~ent towards widespread acceptanee of ycr in nigeria. involvemen t of male partners may 1mpro~e attitudes t~wa~ds vct:the developmentofm novative health education strategies is essential to provide women with mformanon as regards the benefits of vct and other means of pmtct. p4-63 (c) ethnic health care advisors in information centers on health care and welfare in four districts of amsterdam arlette hesselink, karien stronks, and arnoud verhoeff introduction : in amsterdam, migrants report a "worse actual health and a lower use of health care services than the native dutch population. this difference might be partly caused by problems migrants have with the dutch language and health care and welfare system. to support migrants finding their way through this system, in four districts in amsterdam information centers on health care and welfare were developed in which ethnic health care advisors were employed. their main task is to provide infor· mation to individuals or groups in order to bridge the gap between migrants and health care providers. methods: the implementat ion of the centers is evaluated using a process evaluation in order to give inside in the factors hampering and promoting the implementat ion. information is gathered using reports, attending meetings of local steering groups, and by semi-structu red interviews with persons (in)directly involved in the implementat ion of the centers. in addition, all individual and groupcontaets of the health care advisors are registered extensively. results: since 2003 four information centers, employing 12 ethnic health care advisors, are implemented. the ethnicity of the health care advisors corresponds to the main migrant groups in the different districts (e.g. moroccan, turkeys, surinamese and african). depending on the local steering groups, the focus of the activities of the health care advisors in the centers varies. in total, around 2000 individual and 225 group educational sessions have been registered since the start. most participants were positive about the individual and group sessions. the number of clients and type of questions asked depend highly on the location of the centers (e.g. as part of a welfare centre or as part of a housing corporation). in all districts implementa tion was hampered by lack of ongoing commitment of parties involved (e.g. health care providers, migrant organization s) and lack of integration with existing health care and welfare facilities. discussion: the migrant health advisors seem to have an important role in providing information on health and welfare to migrant clients, and therefore contribute in bridging the gap between migrants and professionals in health care and welfare. however, the lack of integration of the centers with the existing health care and welfare facilities in the different districts hampers further implementation . therefore, in most districts the information centres will be closed down as independent facilicities in the near future, and efforts are made to better connect the position of migrant health advisor in existing facilities. the 2005 who report ranks the philippines as ninth among 22 countries with a high tb prevalence. about a fourth of the country's population is infected, with majority of cases coming from the lower socioeconomic segments of the community. metro manila is not only the economic and political capital of the philippines but also the site of major universities and educational institutions. initial interviews with the school's clinicians have established the need to come up with treatment guidelines and protocols for students and personnel when tb is diagnosed. these cases are often identified during annual physical examinations as part of the school's requirements. in many instances, students and personnel diagnosed with tb are referred to private physicians where they are often lost to follow-up and may have failure of treatment due to un monitored self-administered therapy. this practice ignores the school clinic's great potential as a tb treatment partner. through its single practice network (spn) initiative, the philippine tuberculosis initiatives for the private sector (philippine tips), has established a model wherein school clinics serve as satellite treatment partners of larger clinics in the delivery of the directly observed treatment, short course (dots) protocol. this "treatment at the source" allows school-based patients to get their free government-suppl ied tb medicines from the clinic each day. it also cancels out the difficulty in accessing medicines through the old model where the patient has to go to the larger clinic outside his/her school to get treatment. the model also enables the clinic to monitor the treatment progress of the student and assumes more responsibility over their health. this experience illustrates how social justice in health could be achieved from means other than fund generation. the harnessing of existing health service providers in urban communities through standardized models of treatment delivery increases the probability of treatment success, not only for tb but for other conditions as well. p4-66 (c) voices for vulnerable populations: communalities across cbpr using qualitative methods martha ann carey, aja lesh, jo-ellen asbury, and mickey smith introduction: providing an opportunity to include, in all stages of health studies, the perspectives and experiences of vulnerable and marginalized populations is increasingly being recognized as a necessary component in uncovering new solutions to issues in health care. qualitative methods, especially focus groups, have been used to understand the perspectives and needs of community members and clinical staff in the development of program theory, process evaluation and refinement of interventions, and for understanding and interpreting results. however, little guidance is available for the optimal use of such information. methods: this presentation will draw on diverse experiences with children and their families in an asthma program in california, a preschool latino population in southern california, a small city afterschool prevention program for children in ohio, hiv/aids military personnel across all branches of the service in the united states, and methadone clinic clients in the south bronx in new york city. focus groups were used to elicit information from community members who would not usually have input into problem definitions and solutions. using a fairly common approach, thematic analysis as adapted from grounded theory, was used to identify concerns in each study. next we looked across these studies, in a meta-synthesis approach, to examine communalities in what was learned and in how information was used in program development and refinement. results: while the purposes and populations were diverse, and the type of concerns and the reporting of results varied, the conceptual framework that guided the planning and implementation of each study was similar, which led to a similar data analysis approach. we will briefly present the results of each study, and in more depth we will describe the communalities and how they were generated. conclusions: while some useful guidance for planning future studies of community based research was gained by looking across these diverse studies, it would be useful to pursue a broader examination of the range of populations and purposes to more fully develop guidance. background: the majority of studies examining the relationship between residential environments and cardiovascular disease have used census derived measures of neighborhood ses. there is a need to identify specific features of neighborhoods relevant to cardiovascular disease risk. we aim to 1) develop methods· data on neighborhood conditions were collected from a telephone survey of s,988 fesi· dents in balth:.ore, md; forsyth county, nc; and new york, ny. a sample of 120 of the i.ni~~l l'elpondents was re-interviewed 2-3 weeks after the initial interview t~ measure the tes~-~etest rebab1~1ty of ~e neighborhood scales. information was collected across seven ~e1ghborho~ cond1~ons (aesth~~ ~uah~, walking environment, availability of healthy foods, safety, violence, social cohesion, and acnvmes with neighbors). neighborhoods were defined as census tracts or homogen~us census tra~ clusters. ~sycho metric properties.of the neighborhood scales were accessed by ca~cu~~.ng chronba~h s alpha~ (mtemal consistency) and intraclass correlation coefficients (test-r~test reliabilmes) .. pear~n s .corre~anons were calculated to test for associations between indicators of neighborhood ses (tncludmg d1mens1ons of race/ ethnic composition, family structure, housing, area crowding, residential stability, education, employment, occupation, and income/wealth) and our seven neighborhood scales. . chronbach's alphas ranged from .73 (walking environment) to .83 (violence). intraclass correlations ranged from .60 (waling environment) to .88 (safety) and wer~ high~~~ .7~ for ~urout of the seven neighborhood dimensions. our neighborhood scales (excluding achv1hes with neighbors) were consistently correlated with commonly used census derived indicators of neighborhood ses. the results suggest that neighborhood attributes can be reliably measured. further development of such scales will improve our understanding of neighborhood conditions and their importance to health. childhood to young adulthood in a national u.s. sample jen jen chang lntrodfldion: prior studies indicate higher risk of substance use in children of depressed mothers, but no prior studies have followed up the offspring from childhood into adulthood to obtain more precise estimates of risk. this study aimed to examine the association between early exposure to maternal depl'elsive symptoms (mds) and offspring substance use across time in childhood, adolescence, and young adulthood. methods: data were obtained from the national longitudinal survey of youth. the study sample includes 4,898 mother-child/young adult dyads interviewed biennially between 1992 and 2002 with children aged 4 to 16 years old at baseline. data were gathered using a computer-assisted personal interview method. mds were measured in 1992 using the center for epidemiologic studies depression scale. offspring substance use was assessed biennially between 1994 and 2002. logistic and passion regression models with generalized estimation equation approach was used for parameter estimates to account for possible correlations among repeated measures in a longitudinal study. rnlllta: most mothers in the study sample were whites (42%), urban residents (79%), had a mean age of 31 years with at least a high school degree (82%). the mean child age at baseline was 9 years old. offspring cigarette and alcohol use increased monotonically across childhood, adolescence, and young adulthood. differential risk of substance use by gender was observed. early exposure to mds was associated with increased risk of cigarette (adjusted odds ratio (aor) = 1.52, 95% confidence interval (0): 1.12, 2.08) and marijuana use (aor = 1.46, 95% ci: 1.02, 2.08), but not with alcohol use across childhood, adolescence, and young adulthood, controlling for a child's characteristics, socioeconomic status, ~ligiosity, maternal drug use, and father's involvement. among the covariates, higher levels of father's mvolvement condluion: results from this study confirm previous suggestions that maternal depressive symptoms are associated with adverse child development. findings from the present study on early life experi-e~ce have the potential to inform valuable prevention programs for problem substance use before disturbances become severe and therefore, typically, much more difficult to ameliorate effectively. the ~act (~r-city men~ health study predicting filv/aids, club and other drug transi-b~) study 15 a multi-level study aimed at determining the association between features of the urban enyjronment mental health, drug use, and risky sexual behaviors. the study is randomly sampling foster sessions v99 neighborhood residents and assessing the relations between characteristics of 36 ethnographically defined urban neighborhoods and the health outcomes of interest. a limitation of existing systematic methods for evaluating the physical and social environments of urban neighborhoods is that they are expensive and time-consuming, therefore limiting the number of times such assessments can be conducted. this is particularly problematic for multi-year studies, where neighborhoods may change as a result of seasonality, gentrification, municipal projects, immigration and the like. therefore, we developed a simpler neighborhood assessment scale that systematically assessed the physical and social environment of urban neighborhoods. the impact neighborhood evaluation scale was developed based on existing and validated instruments, including the new york city housing and vacancy survey which is performed by the u.s. census bureau, and the nyc mayor's office of operations scorecard cleanliness program, and modified through pilot testing and cognitive testing with neighborhood residents. aspects of the physical environment assessed in the scale included physical decay, vacancy and construction, municipal investment and green space. aspects of the social environment measured include social disorder, social trust, affluence and formal and informal street economy. the scale assesses features of the neighborhood environment that are determined by personal (e.g., presence of dog feces), community (e.g., presence of a community garden), and municipal (e.g., street cleanliness) factors. the scale is administered systematically block-by-block in a neighborhood. trained research staff start at the northeast corner of an intersection and walk around the blocks in a clockwise direction. staff complete the scale for each street of the block, only evaluating the right side of the street. thus for each block, three or more assessments are completed. we are in the process of assessing psychometric properties of the instrument, including inter-rater reliability and internal consistency, and determining the minimum number of blocks or street segments that need to be assessed in order to provide an accurate estimate of the neighborhood environment. these data will be presented at the conference. obj«tive: to describe and analyze the perceptions of longterm injection drug users (idus) about their initiation into injecting. toronto. purposive sampling was used to seek out an ethnoculturally diverse sample of idus of both genders and from all areas of the city, through recruitment from harm reduction services and from referral by other study participants. interviews asked about drug use history including first use and first injecting, as well as questions about health issues, service utilization and needs. thematic analysis was used to examine initiation of drug use and of injection. results: two conditions appeared necessary for initiation of injection. one was a developed conception of drugs and their (desirable) effects, as suggested by the work of becker for marijuana. thus virtually all panicipants had used drugs by other routes prior to injecting, and had developed expectations about effects they considered pleasureable or beneficial. the second condition was a group and social context in which such use arose. no participants perceived their initiation to injecting as involving peer pressure. rather they suggested that they sought out peers with a similar social situation and interest in using drugs. observing injection by others often served as a means to initiate injection. injection served symbolic purposes for some participants, enhancing their status in their group and marking a transition to a different social world. concl111ion: better understanding of social and contextual factors motivating drug users who initiate injection can assist in prevention efforts. 10 ma!onty of them had higher educational level (57%-highschool or higher).about 20.2 yo adffiltted to have history of alcohol & another 12.4% had history of smoking. only 3.2% people were on hrt & 3.1 % were receiving steroid. majority of them (81.2) did not have history of osteoporosis. 13.6% have difficulty in ambulating. only 8.8% had family history of osteoporosis. bmd measurements as me~sured by dual xray absorptiometry (dexa) were used for the analysis. bmd results were compare~ w1~ rbc folate & serum vitamin b12 levels. no statistical significance found between bmd & serum v1taffiln b12 level but high levels of folate level is associated with normal bmd in bivariate and multivariate analysis. conclusion: in the studied elderly population, there was no relationship between bmd and vitamin b12; but there was a significant association between folate levels & bmd. introduction: adolescence is a critical period for identity formation. western studies have investigated the relationship of identity to adolescent well-being. special emphasis has been placed on the influence of ethnic identity on health, especially among forced migrants in different foreign countries. methodology: this study asses by the means of an open ended question identity categorization among youth in three economically disadvantaged urban communities in beirut, the capital of lebanon. these three communities have different histories of displacement and different socio-demographic makeup. however, they share a history of displacement due to war. results and conclusion: the results indicated that nationality was the major category of identification in all three communities followed by origin and religion. however, the percentages that self-identify by particular identity categories were significantly different among youth in the three communities, perhaps reflecting different context in which they have grown up. mechanical heart valve replacement amanda hu, chi-ming chow, diem dao, lee errett, and mary keith introduction: patients with mechanical heart valves must follow lifelong warfarin therapy. war· farin, however, is a difficult drug to take because it has a narrow therapeutic window with potential seri· ous side effects. successful anticoagulation therapy is dependent upon the patient's knowledge of this drug; however, little is known regarding the determinants of such knowledge. the purpose of this study was to determine the influence of socioeconomic status on patients' knowledge of warfarin therapy. methods: a telephone survey was conducted among 100 patients 3 to 6 months following mechan· ical heart valve replacement. a previously validated 20-item questionnaire was used to measure the patient's knowledge of warfarin, its side effects, and vitamin k food sources. demographic information, socioeconomic status data, and medical education information were also collected. results: sixty-one percent of participants had scores indicative of insufficient knowledge of warfarin therapy (score :s; 80%). age was negatively related to warfarin knowledge scores (r= 0.27, p = 0.007). in univariate analysis, patients with family incomes greater than $25,000, who had greater. than a grade 8 education and who were employed or self employed had significantly higher warfarm knowledge scores (p= 0.007, p= 0.002 and p= 0.001 respectively). gender, ethnicity, and warfar~n therapy prior to surgery were not related to warfarin knowledge scores. furthermore, none of t~e. m-hospital tea~hing practices significantly influenced warfarin knowledge scores. however, panic1~ants who _rece1v~d post discharge co~unity counseling had significantly higher knowledge scores tn comp~r1son with those who did not (p= 0.001 ). multivariate regression analysis revealed that und~r~tandmg the ~oncept of 1?ternational normalized ratio (inr), knowing the acronym, age and receiving ~ommum1!' counseling after discharge were the strongest predictors of warfarin kn~wledge. s~1oeconom1c status was not an important predictor of knowledge scores on the multivanate analysis. poster sessions v101 ~the majority of patients at our institution have insufficient knowledge of warfarin therapy.post-discharge counseling, not socioeconomic status, was found to be an important predictor of warfarin knowledge. since improved knowledge has been associated with improved compliance and control, our findings support the need to develop a comprehensive post-discharge education program or, at least, to ensure that patients have access to a community counselor to compliment the in-hospital educatiop program. brenda stade, tony barozzino, lorna bartholomew, and michael sgro lnttotl#ction: due to the paucity of prospective studies conducted and the inconsistency of results, the effects of prenatal cocaine exposure on functional abilities during childhood remain unclear. unlike the diagnosis of fetal alcohol spectrum disorder, a presentation of prenatal cocaine exposure and developmental and cognitive disabilities does not meet the criteria for specialized services. implications for public policy and services are substantial. objective: to describe the characteristics of children exposed to cocaine during gestation who present to an inner city specialty clinic. mnbods: prospective cohort research design. sample and setting: children ages 5 to 15 years old, referred to an inner city prenatal substance exposure clinic since november, 2003. data collection: data on consecutive children seen in the clinic were collected over an 18 month period. instrument: a thirteen (13) page intake and diagnostic form, and a detailed physical examination were used to collect data on prenatal substance history, school history, behavioral problems, neuro-psychological profile, growth and physical health of each of the participants. data analysis: content analysis of the data obtained was conducted. results: twenty children aged 6 to 14 years (mean= 9.8 years) participated in the study. all participants had a significant history of cocaine exposure and none had maternal history or laboratory (urine, meconium or hair) exposure to alcohol or other substances. none met the criteria of fetal alcohol spectrum disorder. all were greater than the tenth percentile on height, weight, and head circumference, and were physically healthy. twelve of the children had iqs at the 19th percentile or less. for all of the children, keeping up with age appropriate peers was an ongoing challenge because of problems in attention, motivation, motor control, sensory integration and expressive language. seventy-four percent of participants had significant behavioral and/or psychological problems including aggressiveness, hyperactivity, lying, poor peer relationships, extreme anxiety, phobias, and poor self-esteem. conclusion: pilot study results demonstrated that children prenatally exposed to cocaine have significant learning, behavioural, and social problems. further research focusing on the characteristics of children prenatally exposed to cocaine has the potential for changing policy and improving services for this population. methods: trained interviewers conducted anonymous quantitative surveys with a random sample (n= 148) of female detainees upon providing informed consent. the survey focused on: sociodemographic background; health status; housing and neighborhood stability and social resource availability upon release. results: participants were 70% african-american, 16% white, 9% mixed race and 5% native american. participants' median age was 37, the reported median income was <s2,000 year, and 53% reported receiving a high school diploma or equivalent. women reported a number of health conditions rypical of underserved populations, including asthma (42%), sexually transmitted infections (39%), high blood pressure (19%), hcv (14%), diabetes (5%) and hiv (4%). nearly half (46%) did not anticipate having a place to stay for at least 30 days upon release. factors significantly associated housing stability upon release were: high family support score (adjusted odds ratio [aor) 6.15; 95% confidence interval (95% cl) 1.76, 21.61), high neighborhood stability score (aor 4.41; 95% cl 1.25, 15.52), wanting a commercial sex worker (csw) support group (aor 0.25; 95% cl 0.10, 0.62); and identifying as bisexual (aor 0.24; 95% cl 0.07, 0.75). women desired employment (95%), housing (84%), education (77%), drug treattnent (74%), help with custody of children (31 %), childcare (28%), and domestic violence suppon (20%) services. conclusions: female detainees represent one of urban centers' most marginalized pcjpwatioos. short periods of detention represent a unique opportunity for _interven?~ns bri~ co~s and public health institutions. service providers should collaborate ~1th local ~ails to .p~de a con1111uwnof care for female detainees upon release that includes transportation , housing, residential drug treallllcnt, employment, education, family reunification, childcare and domestic viol~nce su~rt. ~pecial attenlion is warranted to lesbian and bisexual women and csws, who may be especially margmalized &om family and service-based support networks. introduction: "health care and ethnic minority immigrants" examines how health care policy dil· ferences between canada and the united states impact the health-related hardships, access and use of preventative care, and health outcomes of urban ethnic minority immigrants in both countries. methods: the findings of this paper build on the results of my qualitative comparative study of hotel workers in vancouver, bc and seattle, wa that revealed greater health related hardships for similarly matched employees in seattle compared to vancouver. in this paper, i examine the generalizability of these findings at the cross-national level for similar ethnic minority immigrant groups. i compare the impact of health care policy differences on specific groups that have emigrated to cities in both canada and the united states. these include immigrants from china, viemam, philippines, west indies, africa, sri lanka, pakistan, and latin america. comparative statistical analysis -utilizing rel· evant data from statistics canada and the u.s. census -reveal the extent to which health policy differ· ences help explain how current health policy in the united states disadvantages urban ethnic minority immigrants. results: many ethnic minority immigrants are a part of the working poor, a group which disproportionately lacks health insurance coverage in the united states. their position in the labour market makes them most vulnerable to exclusion from health care services. the data reveals how current u.s. health policy creates barriers for recently arrived ethnic minority immigrants to access health insurance and care. it remains difficult to ascribe health outcome differences directly to policy differences because of the challenge in disentangling the complex interacting interventing variables, including income inequality, that determine health outcomes. yer suggestive evidence suggests that health policy in the united states is harming the health of urban ethnic minority immigrants. the current health policy regime in the united states harms the health access, care, and outcomes of urban ethnic minority immigrants. comparing the fortunes of similar immigrants in cana· dian and u.s. cities reveals these patterns of disadvantage and some of their consequences. these barriers are an understudied factor in the sociological literature on "segmented assimilation" and social exclu· sion. the paper ends with policy recommendatio ns to improve access to health care among ethnic minor· ity immigrants in both countries, with the goal of reducing health related hardships, and improving health outcomes. mass index deyu pan, richard baker, and keith norris badtgrou~ over the past 3 decades, there has been dramatic increase in prevalence of obesity in the us population. however, the relationship between obesity and the prevalence of cardiovascular dis· ease (cv~) risk factors in different race/ethnic groups over time is not well known. . ik~rgn: _we use 2 cross-sectional , nationally representative surveys: national health and nutri· t10nal examination survey (nhanes iii 1988 -1994 (n= 14 029) and nhanes 1999 nhanes -2002 , including white, black, and hispa~ic races who were no~preg~ant, aged 20 to 74 years. res11lt1: the preval~nce of high cholesterol level (~ 240 mg/dl), untreated high blood pressure (2: l40/90 mm hg) an~ diabetes were calculated according to bmi group (lean, <25; overweight, 25-29; and obese, 2: 30) for different race/ethnicity groups. over the approximately 10 years period (from 1988l ~94 ~o [1999] [2000] [2001] [2002] , reducrion in 3 cvd risk factors in non-hispanic white has been observed. for mmonty race/~hnicity groups, black and hispanic, there had been increases in prevalence in high blood fa pressure and diabetes. the lean group experienced larger increase in prevalence of those cvd risk ctors. . fo~~ the prevalence in cvd risk factors over time had reduced in most of the bmi ca1egoes r t e w ~~e population. however, for black and hispanic, prevalence of 2 of 3 cvd risk factors ave generauy uncreased, especially in overweight and obese groups. methods: strategies identified to achieve these goals include: developing meaningful neighbourhood and district boundaries for comparing health information, providing free access to neighbourhood health profiles (including relevant data at the smallest level for which valid rates could be calculated); mapping relationships of inequality at a variety of nested levels; providing a range of formats (maps, tables) to meet the needs of users; providing technical support; seeking user input to advance and improve the site; and conducting workshops to foster access and use of data for decision-making , advocacy and collaboration. an evaluation strategy is being developed to assess the efforts and impact of these strategies. a preliminary assessment of the results of the first six months of activity will be completed in october 2005. results: significant differences in health are shown at all the geographic levels indicating success in developing the boundaries. results tabulated for the first six months use of the site include: site visits; media use; reference/acknow ledgement of the site in other documents; number of contacts through presentations and workshops; feedback from users identifying strengths and limitations; and, response from health decision makers. members of the partnership are identifying additional tools and services to further support actions that reduce health inequalities. the assessment will be used to develop more specific goals, targets and monitoring mechanisms. conclusions: our experience with paper-based health profiles indicates that they have been used extensively for program planning and advocacy. the greater availability of information in a publiclyavailable web-based format is expected to translate into even greater usefulness and wider application. the web site has had considerable usage to date, extensive media coverage. site users and workshop participants have provided positive feedback and suggestions for next steps. the six month review will be available in october 2005. in india, more than 4 and a half people are hiv-positive and half a million have aids. india currently has the largest number of positive people in the world, oumumbering south africa and accounting for nearly one tenth of the global hiv/aids prevalence. the world bank predicts that by 2015, there will be 35 million hiv/aids cases in india. the central challenge facing hiv prevention efforts in south asia today is learning how to respond to the societal determinants of vulnerability to hiv. hiv/aids is an issue largely engulfed by social stigma. stigma and discrimination are often considered the foremost barriers to effective poster sess10ns v104 prevention and care initiatives. stigma can make a person afraid of disclosing their status~ ~yone el se, and may make them feel depressed or alone. stigma can .also le~d to poor adherence to medicanon, ~ likelihood to access health and social services, and less desire to hve. hiv+ men and women may beafraidtotd! their co-workers that they have hiv for fear of their reactions or for fear of losing their jobs. in a study 1 conducted of people living with hiv/aids in delhi, india ov~r. 2003-~, many respo~ts shared their experiences of discrimination in their families, in their communmes, an~ m health ~e settmgs. these findings will be shared in the workshop, and will be compa~ed to m~ ex~nences workmg as an ~ co~l~ at the center for aids prevention studies in san francisco, cahforma and congreso de lannos unidos m philadelphia, pennsylvania. the workshop will include an overview of hiv/aids in urban contexts in tht united states (los angeles, san francisco, new york, philadelphia) and south asia (delhi, hyderabad, chennai). differences in methods for outreach, prevention and treatment efforts between devdoped world and developing world contexts will be discussed. a large portion of the workshop will be centered on discussion. participants will engage in an exercise to categorize their ideas of stigma. the workshop will also include a powerpoint presentation on the quantitative data gathered from the survey. the number of homeless and runaway youth is increasing in toronto, which is currently home to 3 out of 4 youth in the gt a who live alone, and it is the preferred locale for the street-involved and homeless youth. the city's youth population is expected to grow by nearly 20% by 2011, the school dropout rate is rising, and there are is a paucity of empirical data on what works with these at-risk youth. over a two-year period, (2002) (2003) (2004) , youthlink-inner city, conducted an evaluation of the impact of a harm reduction program aimed at reducing street youth's risk of contracting/infecting others with the hep c virus, which has both short-term and long-term deleterious effects. demographic data from 102 street-youth, along with resources/services used, employment methods, type and frequency of drug use, health status, police contacts, and their views of program impact were analyzed using a standardized sur· vey. additionally, focus groups with youth, agency staff and community key informants were conducted, as well as in-depth interviews with three youth, over a sixth-month period. this paper details study results, which both inform both practice and future study about what works, why it works, and how best to work with these vulnerable youth. sho~ed .that the wasteland in this area was contaminated by as cd zn pb cu simultaneously. the con-ta~matton of as ~d was quit significant. the contamination problems and environmental issues in this region are very. serious. the awareness on environmental and health issues was investigated by spot survey and analysis. overall awareness among the residents in the region is very poor. the measures must be taken to assu~e t~e .sustainable economy development by local and central gorvernments. keywords nandan guangx1; mmmg area; environmental awareness. concern like, epileptic fits, vision problems, earache, cough of more than 3 weeks and urinary problems. ninety-five subjects did not take bath regularly and 42 were exposed to sex. the health problems identified are only of a particular point of time and it may be difficult to interpret the depth of "iceberg" of the street children's world also draw the health-illness continuum. periodic interaction and follow-up is very difficult as they generally disappear from the research setting due to their frequent mobility for survival measures. as per the prediction of the iceberg theory, only some problems may have been identified, larger problems in respect to their health may not have been identified. in spite of various limitations, this attempt had proven that, it is possible to enter the street children world, explore the iceberg of illnesses, and establish data of health-illness continuum through an effective nursing agency. it is recommended to deworm these children, provide nutritional education, establish "condom corners" and "street children help line" in the city and urban slum areas and undertake action research on their health. malnutrition is a serious problem in developing countries like bangladesh. malnutrition causes a great deal of child suffering. malnutrition is one of the major causes of morbidity and mortality among the child. the aim of present study was to investigate the nutritional status and its relation to socioeconomic conditions of urban disadvantaged child of under five years age. nutritional status was determined by anthropoemetric measurement(heig ht, weight, mid-arm circumference etc.). determination of total protein and serum albumin were done for biochemical examination. haematological examination was done by blood haemoglobin profile estimation by cynmethhaemoglob in method and determination of haematocrit value or packed cell volume (pcv). stool examination for ova of hook worm, round worm, trichuris species and other species were also done. relevant information on socioeconomic characteristics was recorded by interviewing the house hold head using pretested questionnaire. our study demonstrated that nutritional status of the children are positively correlated with family income, parents level of formal education and negatively related to the family size. the effect of family income on the nutritional status, haematological and biochemical indices of urban disadvantaged child under five will be discussed in detail which will help us to determine proper way of utilization of health care facilities exists in developing countries. introduction: with the increase in morbidity and reduction in mortality and with the introduction of highly active antiretroviral therapy (haart), there is increasing focus on the determinants of healthrelated quality of life (hrqol) in hiv-infection. the focus on the present study is to examine the relationship between social support, depression, and hiv disease severity, and the extent to which these variables impact on hrqol in adult men with hiv-infection. methods: as part of a larger ongoing natural history study focusing on the neurobehavioural complications of hiv and aids, we administered questionnaires to assess depression (beck depression inventory), social support, and hrqol (mos-hiv) to 366 adult men with hiv-infection. a structured interview was used to collect health information and data on hiv disease severity. physical and mental health summary scores (phs and mhs) were derived through principal components analysis. participants were grouped according to level of social support: "well supported" (n= 180), "moderately supported" (n=90), "little or ineffective support" (n=96). analysis of variance was conducted to assess the effects of three levels of perceived social support, depression status (present vs absent), and hiv disease severity (aids vs non-aids) on mental and physical health dimensions of hrqol. potential interaction effects were also evaluated. results: social support was found to have a significant effect on both mhs and phs. presence of depression was found to have a significant effect on mhs but not phs. hiv disease severity (presence of aids diagnosis) was found to have a significant effect on phs but not mhs. manov a analyses revealed no significant interactions effects. conclusions and implications: level of social support, presence of depression and hiv disease severity were shown to have independent effects on hrqol. feeling "well supported" appears to have significant benefits for mental and physical health. biological indicators of hiv disease appear to have a selective impact on physical health while presence of depression is related to significant reductions in mental health. the development and evaluation of behavioural interventions to improve perceived social support network and depression will likely result in significant improvements in health outcomes of adults with hiv and aids. introduction: ongoing medical advances have greatly enhanced the longevity of penons living with aids (plwa). however, for certain subgroups living with hiv/aids, including, members of minority groups, women and the economically underprivilege d, aids outcomes have not changed so favorably. one obvious factor to consider when explaining disparities in aids outcomes is the environ· ment within which an individual resides. few studies of aids outcomes have focused on how commu· nity setting influences the effectiveness of care delivery. the principle research questions were: 1) what is the relationship between the locations of areas with concentrations of pl wa and the distribution of both primary and ancillary service providers? 2) how has the widespread availability of haart (after 1996) impacted aids outcomes at the community level? 3) how do aids mortality and fatality rates vary when related community characteristics, such as household income, ethnic mix, and geographic access to primary and ancillary hiv/aids services are considered? we examine this issue across residential communities in the los angeles area. methods: los angeles county's comprehensive aids service providers database (compiled and maintained by aids project los angeles) was geo-referenced and distances between weighted mean population centers and the nearest hiv/aids service providers were calculated to provide a measure of access which was then merged with aids diagnosis and mortality data along with relevant socioeconomic and population indicators and for analysis using bivariate and multivariate statistics at the zip code level. results: there is a growing disparity in hiv/aids outcomes between low-income minority areas and affluent non-minority areas in the post-haart era (p = 0.002). ancillary aids services such as case management and counseling and testing are located near the places where low income hiv/aids service consumers are likely to live (p =.000). in spite of having more access to ancillary services low income areas continue to be associated with smaller decreases post-haart aids mortality rates (p=0.301). conclusions: given the increasing disparities in aids outcomes between lower and upper income communities in the post -haart era, more specific research into the capacities of aids services providers and the efficiency of their interventions is required. although it is clear that ancillary services are operating in the areas of greatest need, their effectiveness remains limited. understanding the reasons for t~is, be they lack of adequate resources for inner city service providers, failure to reach target popula· tmns or other causes, requires additional research. in 20?3, y.out~ who were able to speak either french or english and had been absent from their parent's/ c~regivers ~es.1dence for at least three consecutive nights took part in the survey which consisted of inter· viewer-adm1ms tered question~aires. p.arti~i~an~s were recruited from drop in centres in 7 cities across <?-nada.y~uth self-report as either using in1ect1on or non-injection drugs. statistical analyses were car· r1ed out using sas version 8. the 'home' is a contested site for many women living in urban areas. women living in poverty and using illicit drugs are largely excluded from participation in dominant 'home-base' gender roles of wife and mother because of economic disadvantage. they thus 'make home' in ways that are specific to their everyday experiences. while research has established links between housing, health and drug use, this literature does not tend to consider the meanings that home may have within the lives of women who lack access to adequate tenured living spaces. this presentation reports the findings of my master's thesis work. using grounded theory methodology within a poststructuralist feminist conceptual framework, i conducted in-depth, open-ended interviews with 11 women living in the toronto area. the women were recruited from within the community through posters at different social service agencies (e.g., drop-in centres, community health centres). they were asked to describe their housing experiences and what home meant to them over the course of their lives. through the data analysis process, a substantive theory of 'making home' for women living in poverty and using illicit drugs emerged. the central concept of making home is a continual process of learning about and interacting with the environment. the process of making home entails different subprocesses: finding home (knowing what is available and accessible, and accessing home), adaptation, and leaving home. the different meanings of home that women developed from their experiences and knowledge emerged as critical components for the process of making home. the women i interviewed were very mobile and experienced constant change with regards to home. home was not considered only in relation to physical living space. home could be made within a relationship or with regards to a possession. while dominant meanings of home relation to tenured living spaces are generally positive, my research indicated that home could also be a negative context. the presentation will describe my research process and findings as well as the theoretical and policy implications of my results. in the western democracies, social citizenship bundles the right to social provision (defined as a share of the social good, not necessarily proportionate to the market value of a citizen's contribution) with participation in the paid labour force. for this reason, social citizenship rights, which usually involve some kind of redistributive measures, are a key mechanism by which states ensure economic equality, or at least, economic equality of opportunity, but are also a mechanism of social exclusion. social rights are inscribed around a single kind of relationship, that which exists between a male family breadwinner and the paid labor market; those who make economic contributions that fall outside this relationship -such as unpaid care-work, or, work that occurs in shadow or underground economiesare to a greater or lesser degree barred from accessing citizen-based social provisions. furthermore, social provision that occurs outside of the breadwinner/market-eco nomy dyad is very often meanstested and is accompanied by regulatory state apparatuses (this includes a range of social services, from welfare payments, to child-care subsidies, to disability pensions). social citizenship is thus stratified; the legal relationship that is at the heart of social citizenship acts as a form of social closure against claims for a share of the social good made by those who are 'lesser' citizens than others. of particular interest in this paper is how citizenship inequalities translate into inequalities in the social provisions that emerge from economic participation: the right to safety on the job, protection from employer harassment, protection from the vagaries of the market as well the right to adequate, comprehensive, and appropriate health services. drawing on mixed methods, longitudinal data from adult sex workers (n= 170) located in two research sites with different social welfare regimes -one in canada and one in the u.s. -we examine the consequences of working in a "shadow" or "underground" economy on various facets of health and access to health services, taking into consideration the mediating role that citizenship constructs and social welfare regimes play in accessing other key social and economic determinants of health. methods: the study population included methadone patients (re)admitted at the mhs in the period from 1/1/1996 to 31/1/2002, born in the netherlands, morocco, surinam, dutch antilles or turkye, and officially registered in the population register of amsterdam. the population register was used to ascer· rain the vital status. causes of death were provided by the central bureau of statistics. mortality was categorised as hiv related, directly drug related (overdose) and other.mortality. results: the methadone patients had the following characteristics: 38 years of age at entry; 81% male; 37% ethnic minority; 37% ever injected. in total, 9558 personyear (py) of observation time and 173 deaths (18% hiv related, 14% overdose; 57% other) were observed. hence, the crude mortality rate was 18/1000 py (95% ci 16-21/1000 py). the percentage of (ever) injectors was higher among the native dutch than among the ethnic minorities, 51 % and 14 % respectively. higher rates were observed among native dutch drug users compared to those belonging to an ethnic minority (age adjusted hazard ratio (hr) 1.9 (95% ci 1.4-2.7). the age adjusted hr of (ever) injecting drug users versus non injecting users was 3.0 (95% cl 2.2-4.1 ). after additional adjustment for route of administration a non significant difference between the dutch and ethnic minorities remained (hr 1.3, 95% cl 0.9-1.9, p-value 0.151. conclusion: the mortality rate among the heroin users in amsterdam is strongly affected by the high prevalence of non-injecting drug use. moreover, this study confirms that differences in mortality between the native dutch and the ethnic minorities is related to differences in route of administration. the ongoing reduction of injecting drug use (due tot selctieve mortality, and switching route ofadministration and popularity of crack cocaine) may lead to a further reduction of mortality rates among heroin users in amsterdam. interventions preventing the initiation of injecting should be encouraged. . introduction: food insecurity is an inequality that is central in the lives of many low-income cana· d1ans. people lack food security when regular access to nutritious food is limited or variable due to high prices'. low income, lack of transportation or inadequate food distribution, or they become disadvan· taged m other w~ys through the acquisition of food. a group comprising academics, health profession· als, and commumty workers who have experience in food insecurity, developed a pilot study in ottawa that sought to und~rstand the lived experience of food insecurity. the study also used the united states department of agriculture (usda) community food security module. methods: a series of group meetings determined the best research approaches; questionnaires and consent forms were developed by a working group. twenty-three self-identified food insecure respon· dents were interviewed. the data were analysed using frequency distributions· the food security module was coded according to the usda coding guide, and open-ended responses w~re coded using a grounded approach. renlts: preliminary results from our pilot study have given the research team cause for considerable concern. out of 14 households without children, 11 experienced food insecurity with hunger; 5 ~ere at the severe l~vel. out of 9 households with children, 7 experienced food insecurity with hunger. ~•rst person reflecnons commonly featured feelings of depression low self-esteem and despair. participants' account tha d . an 1 cu ty gettmg to the store, severely limited putting knowledge and skills mto practice. l'oster sessions v109 conclusion: based on these early findings, our research team is looking to challenge what appear to be assumptions about poverty and food insecurity and for best ways to use this information. some policy approaches and interventions that encourage people to eat better and exercise more, may be missing the mark for low-income individuals, and may serve to maintain food insecurity. future research will build on this pilot. with methods augmentation, and a comprehensive knowledge dissemination plan, we hope co contribute to research and policy frameworks at all levels. ps-25 (a) mental health and the corrections system: population-based analyses in urban, semi-urban, and rural settings julian somers, robert watts, and michelle patterson introduction: according to recent epidemiological research, rates of mental disorders vary considerably across countries and across regions within countries. nevertheless, rates of mental disorders (including substance use disorders) are consistently higher in populations involved in the corrections sys-1em. courts have long recognized the heavy burden of mental illness within their purview, and have developed innovative programs to better manage the needs of such individuals. the majority of these innovations (e.g., specialized courts) exist in urban settings. however, few studies have examined the degree of variability in rates of different mental disorders between courts in urban and other settings (e.g., semi-urban, rural). variability between courts in different settings, if present, holds implications for needs-based resource planning. the present study was conducted as part of a long-term inter-ministerial collaboration in british columbia (bc). analyses were conducted on linked administrative data regarding population health and correctional services. a database was constructed including all individuals sentenced in bc in a single year (n=49,142). corrections records were matched to records of health services utilization (hospital and outpatient services) for mental disorders and substance use disorders in the years preceding sentencing. services for mental health and substance-related problems were aggregated into three groups: severe mental illness (smi); less-severe mental illness (lsmi); alcohol/other drug (ad). courts were grouped, based on their annual volumes, inro three categories: "urban"; "semi-urban" and "rural." rates of service use for mental disorders were compared between groups for 1-year and 5-years preceding sentencing. results: there were significant differences in the rates of mental disorders in courts of different size (urban, semi-urban, rural) . however, differences between the rates of disorders within each of these groups exceeded the discrepancies between groups. comparatively high and low rates of disorders were observed in courts of each size. moreover, the courts with the highest rates of certain disorders (e.g., ad) did not have the highest rates of other types of mental disorders (e.g., smi). conclusions: rates of mental disorders differed significantly among the annual populations sentenced across courts in bc. urban courts (which process comparatively large numbers of people) have implemented services to address the needs of the mentally ill. the present analyses strongly suggest that program development should be closely linked to the demonstration of need. the results caution that a uniform approach to court programs for the mentally ill is likely to be inefficient, over-supplying services in some settings and underestimating need in others. results: public settings used for injection are characterized by highly unhygienic conditions, the presence of unsafely disposed syringes, limited availability of sterile syringes and a lack of sterile water for injecting. a number of unsafe injection practices observed were common among public injectors. the presence of police officers nearby and the potential of assault by street predators fostered rushed injecting among those consuming drugs in public spaces. the ecological features of these environments encourage unhygienic and unsafe injecting practices, heighten risk for overdose and the transmission of blood borne viruses. introduction: approximately 13% of women experience depression in the first weeks or months after giving birth. although it has been argued that postpartum depression (ppd) is a culture-bound p~ nomenon, experienced only in western, industrialized countries, recent international research studies have confirmed that the prevalence of ppd is similar around the world. however, little is known about variables that may influence the experience of ppd in women from diverse cultures and immigrant women. research is needed to identify the role culture may play in the presentation of ppd (i.e., types of symptoms most commonly reported), how women from various racial, ethnic and cultural backgrounds perceive and attribute their symptoms of ppd, and finally, how culture can be appropriately addressed in ppd treatment programs. method: to examine the role of culture in ppd, semi-structured interviews and self-report question· naires were administered to clients of the women's health centre of st. joseph's health centre, tor· onto. participants were identified as either first generation canadians (relative newcomen) (n=8) or second generation canadian (parents were immigrants to canada) (n=6). data were collected qd these two groups of women based on: severity of depression, symptom presentation, perceived role of social supports and culturally-specific traditions, and barriers and responses to treatment. the interview data were analyzed using qualitative methods (thematic content analysis), and the following themes were identified: 1) stress about passing on their culture to their children, 2) lack of social support and feelings of isolation, 3) perceived importance or lack of importance of cultural based tra· ditions, 4) conflicts with family members. about cultural traditions and beliefs, 5) mental health stigma within ethnic communities and beyond, 6) race-and sex-based discrimination, and 7) language barriers. conclrllion: identifying the role culture plays in the presentation of ppd, and how women from diverse backgrounds perceive and attribute their symptoms of ppd, is critical in order to establishcultur· ally appropriate guidelines for treatment options. furthermore, information gathered from this study can be used to develop policies and resources for delivering culturally competent care for newcomer wo~ who are dealing with ppd as well as the issues around acculturation (i.e., language barriers, racism). llus is of particular importance for urban health centers which provide postpartum care to diverse groups of women, and particularly recent immigrants or newcomers. ps-~8 (al. contaminated 'therapeutic landscape': perceptions of the aamjiwnaang fint nation keyln smith and isaac luginaah . in~: this. paper pres;ents the findings of an ongoing study among the aamjiwnaang f!rst nanon. aam11wnaang is located m the heart of samia's 'chemical valley', surrounded by cherrucal ~(ants such as esso, imperial oil, shell, suncor energy, and canada's largest hazardous waste disposal sate. safety kleen. this fint nation community is located within the st. clair river •area of concem'. as destgna~ by health canada for the population of samia and the surrounding region. although this comm_umty, by way of. its proximity to the numerous chemical plants is already exposed to high levels pollu~on, recent che?ucal dumping in ~mjiwnaang, as well as a failed proposal for another ethanol plant m the community, only helped to mtensify community concerns about potential health impacts of exposure. research on the cultural beliefs and traditions of first nation communities has established that th~ ~isting rel~tionship between first nations people and 'mother earth' is not only physical, but also_ spmtual. in this study we explore how the complex relationship between the aamjiwnaang first naaon and 'mother earth' be ha · · · i .. may c ngmg 111 a contammated 'therapeutic' landscape. the study a so ~lores h?w aam11wnaang residents are coping with these changes and with the probable conramina· boa of their community. ra!jts: aamjiwnaang residents acknowledge that they are living in a contaminated environment and are being impacted by emissions from the surrounding 'chemical valley' especially for future generations, and have expressed concerns that members of the community and mother earth are 'sick' as a result of this exposure. while moving from that 'place' has been discussed, residents have strong personal and generational connections to the land and insist however, that aamjiwnaang is their 'home' and will remain that way despite growing community concerns about the impact from industry. the contribution of this study lies in the direct involvement of community leadership in designing and conducting this research and distributing the results to further local policy development regarding community concerns on the reserve. background: truck driver are at very high risk for drug abuse because the factor contributing to drug abuse among driver are multiple. it is important to gain an understanding of the key factors that contributing to drug abuse among truck drivers. methods: seventy-truck driver aged 25-35 years were conveniently selected from the population. all the participants were male. an interview schedule was developed in the light of literature data and data were collected by personal interview with informed consent in selected urban area of eastern part of nepal. results: a considerable percentage of truck driver have less knowledge about drug abuse, its effect on body and their complication. majority of the participants (90%) explained that lack of education, financial crisis were leading factors of drug abuse. the average numbers of respondents were believed that lack of meaning in life, marital disharmony. friendship with drug abuser, stress, tiredness, modernization of life pattern and freedom from the home pressure were the influencing risk factors to drug abuse. conchuion: this study reveals those truck drivers are at risk in urban area for developing drugs abuse, mostly influenced by social and environmental factors. hence, these groups should undergo certain educational programme to prevent not only drug abuse, but also prevent transmission of infectious disease among these groups. introduction: food insecurity is an inequality that is central in the lives of many low-income canadians. our group of university researchers, health professionals, and community developers conducted a study in ottawa with community workers who work with people identified as food insecure. the purpose of the study was to understand the nature of their work, and their perspectives, perceptions and experiences of the causes and consequences of food insecurity. (a parallel study was conducted with food insecure participants). methods: sampling was through local knowledge of organizations involved in the provision of food to community members. written permission for the researchers to contact workers in confidence was obtained from each organization. thirteen in-depth interviews were conducted. the interviews consisted of a number of open-ended questions. the data were collated, and analysed using a grounded approach. results: workers interviewed represented organizations that offered diverse programming and methods of food distribution intended to address the needs of the community. the provision of food was seen as a necessary response to address hunger that arose for the majority from poverty caused by low income, or levels of government income support and safety nets that were insufficient to support food requirements. workers reported that food requirements varied according to different. ethnic backgrounds, ages of recipients and family configurations, and with food preferences, and d'.etary (health) requirements. workers' observations of the effects of insufficient food on adults and children such as depression, low energy and non-participation or social disengagement matched those of food insecure respondents. food insecurity was cited as "another srressor among the mountain of problems." overall, workers presented a picture of a silent, stigmatized, poor population including children, and a lack of advocacy to address the issues. be tha th · ·ty the results suggest that workers and organizations, like the commuruty mem rs t ey msecun • · · · · f ilab'l' f food · t d ·n thei·r ab1'l1'ty to address food insecurity. l1m1tanons m terms o ava 11ty o , serve, are restnc e 1 . . funding, and donations appeared to set the para?1eters of pr~g:am r.espon~ to commuruty food msecurity. workers were concerned with program des1g": and a~m1mstranon that m general addr~d hunger on an emergency short-term basis, although food msecunty was long-ter_m for ~any. sustainable solutions that include knowledge translation strategies, and health and social pohcy responses were suggested and will be explored in future research that hopes to build on this pilot. background: socioeconomic deprivation as well as low levels of soc!al s.uppoi:r have .been associated with poor outcomes following cardiac surgery. pre-operative depression m ~anents with coron~ry artery disease awaiting cabg ranges from 27-47% and is an independent predictor of post-operanve mortality. since st. michael's hospital has a large inner city population, this study was designed to determine the both the prevalence of depressive symptomatology (ds) in patients awaiting cabg as well as its relationship with socioeconomic status (ses) and social support. methods: consecutive patients referred for isolated cabg were invited to complete the centre for epidemiological studies depression scale (ces-d) as well as a social support scale and a life stressors inventory. a ces-d score of 2: 16 represents mild ds and a score of 2:27 represents moderate to severe ds. participants also recorded information on functional status, perceived progression of heart failure, demographic and ses variables. all participants with ces-d scores 2: 16 were considered to have ds. results: of the 75 patients (75%) who returned the survey, 22 (29.3%) of participants had a ces-d score 2: 16 with 8 (10.7%) of those having scores suggestive of severe depression. females and those with ongoing medical concerns tended to have more ds (p= 0.06 and p= 0.07 respectively). age was not related to ds. perceived worsening of symptoms and increased ccs angina class were significantly related to the presence of ds (p= 0.03 and p= 0.03 respectively) but not the number of diseased heart vessels. satisfaction with personal relationships including having someone to confide in (p=0.01), feeling lonely (p= 0.04) and not having a partner (p= 0.007) were significantly related to ds. being very upset by a recent breakup in the immediate family was also significantly related to ds (p= 0.02). having greater than a grade 9 education was the only ses variable related to ds (p=0.06). multivariate logistic regression suggested that not being partnered and having low education were the most significant predictors of ds. conclnsion: pre-operative depression is present in at least one quarter of patients referred for cabg. since ds is related to poor outcomes following cabg, single patients with few social supports and low education should be considered at high risk for ds. these findings support the need for the development of supportive interventions in patients at risk in order to decrease post-operative morbidity. this study examines the relationship between childhood sexual abuse, sexual assault during adolescence, and hiv-risk-related behaviours in a sample of 919 homeless youths. over the past decade, there has been increasing research devoted to the impact of childhood sexual abuse. some studies have suggested that the experience of childhood sexual abuse is associated with substance use and abuse, at· risk sexual behaviour and subsequent higher rates of hiv infection (ompad et al., 2005; cinq-mars et al., 2003; brown et al., 1997) . other studies have found that childhood abuse, especially sexual abuse, is c_o~n among stree~ youth (noell et al. 1999 ). this study compares sexually abused males and females livtng on the street ~th non-sexually abused street youth, with regards to unsafe sexual behaviours and e~ures to potmnal t:dv sources (e.g., tattooing, body piercing and injection drug use). the hypothe-sis oft~ ~nt study is that the prevalence of hn-risk-related behaviours will be higher among street youth vtctuns of sexual abuse than those who have not been sexually abused. the sample includes 919 youths aged 13 to 25 who met specific criteria's for itinerancy (649 male and 270 female, with a mean ~of 19.4 y· and a standard deviation of 2.9). they were recruited through five organisations working ~th~ you~ and were~ of an ongoing cohort study. all youth completed a 45 to 60 minute ques· ~onnaue on their sexual behaviours, use of drugs and alcohol and other hiv risk behaviours. oral spec· unens for hiv testing were. also collected. a total of 357 (38.9%) youth reported at least one incident of ~i •~use/assault (1~1 girls (67.0% of all the girls) and 176 boys (27.2% of all the boys]). preliminary descnpnve analyses pomt to sexually abused/assaulted youth showing the highest prevalence of sexual poster sessions v113 at-risk behaviours. for example, 43.3% of sexual abuse/assault victims have engaged in prostitution in their lives compared with 13.4% in the non-sexually abused/assaulted group. in addition, 42.6% of sexual abuse/assault victims reported having injected drugs, compared with 32.0% in the non-sexually abused/assaulted group. more statistical analyses will bring other significant factors to light, especially when we analyze separately those victim of childhood sexual abuse as compare to those victim of sexual assault after childhood. elucidating the risk factors for getting involved in hiv-risk-related behaviours is important for the development of comprehensive and appropriate prevention and treatment intervention strategies. introduction: new immigrants and refugees to canada frequently emigrate from regions of the world where intestinal parasitic infections with worms are endemic. of note, some of these parasites area capable of surviving for years to decades within a given host and can have life threatening consequences many years after initial infection. thus, early diagnosis and treatment of intestinal parasitic worms is considered important in high risk immigrant populations, however there remains a lack of consensus regarding the best method of accomplishing this. diagnosing worm infections through stool examinations is costly and has limited sensitivity, while presumptive treatment of all immigrants, although advocated by some, is expensive and results in healthy individuals being unnecessarily treated. herein, we examine the utility of a hematologic marker (i.e. peripheral blood eosinophilia) as a screening instrument to identify parasitic worm infections in new immigrants to toronto. we identified 192 adults, 15 years of age or older, who were screened for intestinal parasitic infections by stool examination as part of a recently developed screening protocol at a downtown toronto community health centre. we examined the prevalence of infections with worms and subsequently evaluated the impact of several demographic factors on the presence of worm infections. we also determined the sensitivity and specificity of peripheral blood eosinophilia (defined as greater than 500 eosinophils per ml) as a marker for intestinal parasitic worm infections. results: overall, 21 % of the 192 immigrants tested had evidence of at least one worm infection on a single stool examination, while 4% of such individuals bad infections with multiple worms concurrently. age did not appear to be associated with the presence of worms, however 29% of males had evidence of worm infections compared with 16% of females (p= 0.03). immigrants from asia, sub-saharan africa, and central america had the highest burden of overall infection. the sensitivity and specificity of peripheral blood eosinophilia for worm infections was 24% and 92% respectively. conclusions: one in five recent immigrants presenting to a community health centre in downtown toronto had evidence of at least one intestinal parasitic worm infection. the highest burden of illness was in men and in those emigrating from less developed regions of the world. peripheral blood eosinophilia is a poor screening test for worm infections however its presence is highly suggestive of the existence of worms in new immigrants. more than a decade ago, the journal of american college health recommended that a national study be conducted to measure the health status of african american college students, 'both health disparities and [their] positive healthful behaviors' (fennell, 1994) . it was later decided that when exploring the level of health risk behaviors for african american students, gender is an important variable and should be included in the research (fennell, 1997) . at historically black college and universities (hbcus) college health researchers must tackle gender disparities through highly-effective health promotion and disease prevention programs. the purpose of this review is to examine the literature that addresses the overarching health behaviors (i.e. risky sexual behavior, poor diet, smoking, physical inactivity, etc.) of african american men who attend hbcus. despite the various social, cultural, and academic benefits of african american students who attend hbcus, the number of research studies that adequately address their health and health behaviors is limited. studies indicate that african american men who attend hbcus are actively engaged in poor health behaviors. compared to females at hbcus, african american men are more likely to behave in ways that could result in injuries and the use tobacco, alcohol, and ~ther dru~. the city of toronto has recently proposed to conduct a street count of the homeless. like proposals that have come before, this most recent proposal is controversial among many homeless advocates and service providers because it is perceived as intrusive, likely to undercount the target population, and unnecessary for addressing the problem. advocates of the count view it as necessary to gauge the scope of the problem and advise city leaders about how to most effectively direct resources ro.the various homeless services. this paper first reviews the history of efforts to count homeless populat10ns m toronto, describing the motivations and arguments of people on both sides of the issue. second, the paper reviews the methodological complexities of counting homeless populations, describing approaches that use shelter-counts, administrative data and street observations. those interested in counts for toronto and elsewhere can benefit from this review of approaches that have been proposed and carried out in other municipalities. third, the paper proposes a new community-base d strategy that joins the skills of methodologists, local experts, and service providers to ensure a fair and accurate count. the method of systematic social observation avoids some of the problems of early counts by utilizing a repeated identification approach to minimize undercount bias. the method also has the advantage of involving and compensating homeless individuals for assisting with the street count estimates. the estimation approach can be implemented on a rolling basis throughout the year. drug transitions) is a multi-level study aimed at examining the associations between features of the urban environment and mental health, drug use, and risky sexual behaviors. research participants are being recruited in 36 new york city (nyc) neighborhoods. an essential first step in the implementation of this study was to delineate boundaries for each target neighborhood that could then be used to establish sampling frames and as key units of analytic interest. although many neighborhood studies rely on pre-established definitions (e.g. those used for the census or administrative purposes), such definitions do not always reflect contemporary settlement patterns. we felt that street level observations were a neces· sary component of the definition process. methods: the procedures used to delineate neighborhoods were: (1) development of census block maps of targeted areas; (2) review of land use maps and census tract data to ensure, prior to going into the field, tha.t particular blocks are residential and likely to have sufficient population for recruitment purposes; and (3) field vislts and observation in each of the targeted areas. field observations focused on: housing characteristics, including housing type and condition; characteristics of commercial areas, such as likely customer base (e.g. local or no~, socio-economic status and ethnicity of customers); pedestrian volume (including con· gregauons of people m parks and outside stores or residences); obstructions to pedestrian traffic (e.g. ma1or thoroughfares, multi-block industry or institutions); and maintenance and use of open spaces. results: in making the final decisions regarding neighborhood boundaries and the inclusion or exclusion of particular. blocks, we considered a broad range of factors including evidence of homogeneity an~ heterogeneity (socmeconomic, ethnic, housing type, environmental) across blocks within and blocks ad1acent to a nei.ghborhood, and across the 36 sample neighborhoods; the potential for efficient recruit· ment of appropriate particip.ants (i.e. sufficient local pedestrian traffic); and boundaries used in reporting census data (s? that population data can be used in our analysis). . altho~gh utilization of field observations for neighborhood definitions is relatively time consuming (ap~~ox1mately 2 hours for a 12 block area, with more diverse neighborhoods taking even longer), we annc1p~t~ that it will substantially improve the quality and consistency of our data gath· ~rmg ~nd analyse~. spec1f1c e?'amples from neighborhoods defined through this process will be given dur· m~ this presentatmn. we will also discuss the merits and limitations of characterizing neighborhoods usmg street level observations. has been no consideration for how social networks are associated with sse. the objective of this study was to identify personal and social network characteristics associated with being a recipient of sse. in this cross-sectional study, idus who injected in the past 6 months were recruited from syringe exchange programs in montreal, canada, between april 2004 and january 2005. information on each participant and on the persons with whom contact had occurred in the past month were elicited using a structured, interviewer-administe red questionnaire. participants provided detailed demographic and drug use information on up to 5 idu members with whom drugs or injecting materials were shared. using logistic regression, personal and network characteristics were examined in relation to receipt of sterile syringes. res.its: of 277 idus, 39% reported receiving sterile syringes in the past month from another idu. the sample mean age was 33 years (range 18-55), 73% male, 91 % caucasian, and 85% cocaine injectors. in multivariate analyses adjusted for age and gender, recipients of sterile syringes were more likely than non-recipients to share drugs after preparation (0r=2.39(1.06-5.42 )), to require help or to have helped inject another idu (or= 3.48(1.60-7.60)), to have asked someone to get sterile syringes from a syringe exchange program (or=2.54[1.20-5.40 )), to lend syringes (or=2.44[1.16-5.16) ) and to use drug preparation water that was previously used by another iou (0r=2.46(1.42-4.28) ) during the past 6 months. recipients also had a higher rate of change (p=0.01) of idus in their drug injecting network during the past month. with respect to social networks, 45% ( 171/348) of idu network members were providers of sterile syringes and were more likely than non-providers to inject everyday (or= 2.50( 1.51-4.15)). when stratified by their role as a sexual partner or as someone who provides support (e.g. friend, family member), further risk behaviours were observed in relation to sharing injecting materials with the participant. conclusion: recipients of sterile syringes and their idu network members had several high-risk behaviours for infection with bloodborne diseases. sse may afford an opportunity for the dissemination of other salutary behaviours such as information sharing on safe injecting practices between recipients and distributors. furthermore, drug injecting networks may be an avenue to reach idus who may not otherwise be exposed to preventive measures. there are numerous sources of stigma and labelling of mental and physical illness. first, the sociocultural dimension to stigma and labelling clearly influences patients, families, health care professionals and society's perception and treatment of illness. this creates multiple challenges related to illness identification and recovery, particularly in culturally diverse societies. second, the media is a major source of stigma and stereotyping with regard to a variety of mental and physical illnesses. despite increased public knowledge of many illnesses, studies have shown that stigma has intensified over the years in certain cases. a third element of stigma and labels appears with the use of diagnosis as a solution to human problems with the result that difficult to treat patients are repudiated and social issues are unaddressed. this presentation will serve to outline the development of stigma, various expressions of stigma, and consequences of stigma. it will provide some practical recommendations for the reduction of stigma related to mental and physical illness. purpose: to assess the knowledge, attitude, and practice about immunization for parent of 2-3 years old hispanic and african american children in los angeles county (lac). methods: we conducted two cluster surveys in lac. the sample was selected with probability proportionate to estimated size at the first stage and simple random sample of children at the second stage. data were collected through interview. we absuacted vaccine coverage data from immunization record card (irc) at home or from clinic records. the participation was 81 % in hispanics and 83% in african americans populations. irc was not available at home for 26% of african american and 16% of hispanic children. results: all parents perceived their children got all necessary vaccine~. fa~orable attitudes to.w~rd immunization were reported by 82 % of african american and 90% of h1s?amc parents. '.he m~1ority of the parents (90% african american and 95% hispanic) agreed that vaccines prevent senous d1se~ses among children. regarding vaccine safety, significantly more hispanic parents (91 %) than african american parents (75%) mentioned that vaccines are safe. introduction: cervical cancer has been shown to be preventable by papanicolau tests in heterosexual women; however, the utility of papanicolau test in lesbians is controversial. cervical cancer is caused by the human papillomavirus (hpv) which is transmitted by sexual _intercourse; howev_er, its' transmission by homosexual intercourse is also controversial. the goal of this study was to review the evidence for papanicolau tests in lesbians. methods: a pub med, ovid ( 1996 ovid ( to 2005 , and cochrane library search was performed using the mesh headings "homosexuality , female," "vaginal smears," and "papillomavirus, human." in pub med, the clinical queries feature was used with "diagnosis" and "broad, sensitive" filters. a google advanced search of the internet was conducted with the terms "lesbian," "cervical cancer," and "pap rest." the url was limited to ".ca," ".edu," or ".gov." results: the search yielded 8 original articles and 4 reviews, but no cochrane reviews. none of the publications were canadian. 111 website hits were found and selected websites were reviewed. studies showed that lesbians have fewer papanicolau tests than heterosexual women; however, many reported risk factors for cervical cancer, including multiple past or current sexual partners (male and female), early age of first coitus, history of sexually transmitted diseases, and cigarette smoking. hpv has been detected in 13-30% of lesbians and 6-19% of lesbians who have never had sex with men. case studies of abnormal papanicolau tests in lesbians who have never had sex with men have also been reponed. sexual transmission of hpv among lesbians can be explained by several factors: 1) 53-99% of lesbians have had sex with men and 21-30% of lesbians continue to have sex with men. 2) hpv may be transmitted by sexual behaviours among women, like digital-vaginal sex, digital-anal sex, oral sex, and the use of insertive sex toys. 3) hpv transmission requires only skin-to-skin contact. genital hpv has been identified on human fingers. some professional organizations, like the society of obstetricians and gynecologists of canada and the american cancer society, recognize the need for papanicolau tests in lesbians. other organizations, like the canadian cancer society, do not have official policy statements. the current ontario cervical screening program does not specifically address lesbians. conclusion: although the data was limited, most studies recommended that lesbians should receive papanicolau tests. the frequency of these tests was controversial. policy makers and professional organizations should address the needs of this special population and make specific recommendations . monique chaaya, ahia sibai, hiam chemaitelly, and zana el roueiheb . literature concerning the mental and physical effect of work at an old age is contradictory. in addinon, urban environments are physically and socially harsh with reduced potential for income generation and employment and increased potential for depression. the present paper is an attempt to study how do work, or lack of work, link to the experience of depression among older adults in underprivileged lebanese urban communities. the data comes from a cross-sectional survey, where 740 elderly residing in 3 underprivileged communities in beirut, including a palestinian refugee camp, were successfully inter-v1ewed through face-to-face interviews. logistic regression was performed to assess the effect of work on depression ad_justing for many other covariates. data showed that 17.5% of people aged 60 years and more were still workmg. there was a highly protective effect of work on depression in elderly males (or= 0.342, 95% cl= 0.128-0.909). the current study is the first of its kind in the arab region and more work shou_ld be _don_e in this area. it is important for the lebanese government to consider elderly rights for social mclus10n m terms of employment opportunities. amam nuru-jeter, nancy adler, and burton singer . l~u~on:_ the socioeconomic gradient is perhaps the most persistent and significant finding in social epidem1ological research. the insistent nature of the ses effect is evidenced by its significance after acc?untmg for ~umerous confounds. the significance and magnitude of the effect, however, varies by ::•al group. evidence_ o_f the relative effect of race and ses is inconclusive. further, less attention has f ~ ptid to the specific interactions of race and ses than to examinations of which is a better predictor 0 d ~ th. ~sues are further complicated by the explanatory complexity of the various measures of ses anl . owht ey relate to the health of various social groups. understanding the specific nature of these re a11ons ips 1s necessary for guiding he ith d · i 1· · h a an soc1a po mes and programs aimed at improving t e poster sessions v117 health of our most disadvantaged groups, and therefore population health, overall. this study examines the synergistic effects of ses with both race and gender in predicting group differences in mortality. methods: analyses use data from the national longitudinal mortality survey (n= 559, 715) for the years 1979, 1980, and 1981-85; and were linked to the national death index for deaths occurring through 1985. results were confirmed using data from the national mortality followback survey for people who died in 1986 (n= 13,491) and the 1986 national health interview survey (denominator). we used correlation analysis and the standardized mortality ratio to examine ses (education and income), race, and gender as predictors of group differences in changes in mortality. results: for the total population, analyses support the income gradient showing significant declines in mortality ratios from low to high income along the income strata. education shows a significant drop in mortality with completion of high school and completion of college, exhibiting a threshold rather than a gradient effect. compared to whites, blacks receive diminishing health returns for each subsequent level of income and education; with whites closely mirroring the total population in ses thresholds. similarly, compared to men, women do not experience the same health gains at comparable ses levels. for education, black men and women only show significant mortality declines upon completion of high school. the ses gradient operates differently among race and gender subgroups. threshold effects suggest resource gains in life opportunities at particular milestones along the ses gradient. further, ses matters less for blacks and women compared to whites and men suggesting differing implications for health and social policy aimed at reducing disparities. objective: we assessed the impact of diabetes in a large puerto rican community of chicago by measuring the prevalence of diagnosed diabetes and calculating the diabetes mortality rate. methods: we analyzed data from a comprehensive health survey conducted in randomly selected households in community areas. questions on diagnosed diabetes and selected risk factors were asked. in addition, mortality data were analyzed in order to calculate the age-adjusted diabetes mortality rate. when possible, rates were compared to those found in other studies. results: the diabetes prevalence located in this community (20.8%: 95% cl= 10.1 %-38.0%) is one of the highest ever reported for puerto ricans. for instance, it is more than three times higher than the prevalence for the us (6.1 %) and twice the prevalence for puerto ricans in new york ( 11.3%) and puerto rico (9.3%-9.6%). diagnosed diabetes was found to be significantly associated with obesity (p=0.023). the prevalence was particularly high among older people, females, those horn in the us, and those with a family history. the diabetes mortality rate (67.6 per 100,000 population) was more than twice the rate for all of chicago (31.2) and the us (25.4). conclusions: understanding why the diabetes and mortality rates for puerto ricans in this commu· nity are so much higher than those of other communities is imperative. collaboration between researchers, service providers and community members can help address the issues of diabetes education, early screening and diagnosis and effective treatment needed in this community. introduction: sars is a respiratory illness that is spread from person to person through dose contact. this infectious disease outbreak was unusual due to the high rate of infection among health care workers. the aim of the study is to explore the demographic and attitudinal determinants of psychological distress due to sars among health care workers of a toronto hospital. methods: a total of 997 adult participants completed questionnaires co assess psychological distress (impact of event scale -jes) and attitudes to sars. of these, 845 participants completed the questionnaire during the sars i outbreak, and 152 participants completed the questionnaire during the sars ii outbreak. participants also provided information on demographic variables, including occupation and exposure to sars. principal components analysis was used to derive eight attitudinal scales: fear: system, protection, prevention, job stress, stigma, instrumental coping, and psychological copm~. l111ear regression analyses were applied to evaluate the associations of the demographic and amtudmal variables, as well as time, with psychological distress. . . regression analyses showed that time and higher scores on fear, 1ob stress, stigma'. and instrumental coping were associated with a higher total ies score: (r2 =.30). contrary to expectations, none of the demographic variables was associated with total ies score. conclusion: the presence of psychological distress in health care workers is indicative of intrusive or avoidant responses to thoughts, feelings, and memories associated with the sars outbreak. this study shows that ( 1) fear for one's health and the he~lth of.others, (2) ex~erien~in~ job stress, (3)_tbe per· ception of stigma, (4) usage of instrumental copmg skills, ~n~ (5) t1m_e s1grufi~an~ly c~nmb~te to increased psychological distress in health care workers. potential mterventmns during mfecttous diseaie outbreaks in health care settings should be targeted to minimize the impact of these factors. purpose: the purpose of this study was to expand knowledge regarding feeding practices, know!· edge and nutritional beliefs of mothers currently residing in the dominican republic (dr). the rising incidence of obesity in children is a major national health concern and obesity in first and second-generation immigrant children also continues to rise. we know that parents (particularly mothers) shape children's early diet patterns and that immigrant mothers experience additional challenges related to acculturation and assimilation into a new culture, however there is a paucity of literature concerning the effect of immigrant adaptation to american culture on the nutrition of children. in addition, new immigrant knowledge about the causes of obesity and the resulting health implications for childhood obesity has not been assessed and reported. this qualitative study used focus group methodology to discuss views and practices related to the introduction of food for infants and feeding practices for young children, along with a discussion on knowledge and beliefs related to the causes and health implications of child· hood obesity. ten dominican mothers' of young children (birth to 6 years old) who reside in a small town in the western frontier area of the dr were participants in the focus group. results from this srudy will be compared to results from an identical study that will be conducted in fall 2005 with new immi· grant mothers from the dr to the boston area. we will compare feeding practices, beliefs and knowledge about nutrition for young children between these two groups. methods: using participatory principles, a focus group design was used to interview a group of mothers in the dr. the investigator along with a bilingual translator conducted the focus group. the session was tape recorded and is currently in the process of being transcribed and translated. transcribed data will be systematically analyzed themes will be identified. a qualitative data analysis software will be used to organize and code the data according to the specific aims of the study. supporting quotations will be selected to substrate each theme. lmpli~tions for urban health practice: an ultimate goal of this study is to lay a foundation for understanding the process of acculturation on feeding practices of mothers when they immigrate to the us. understanding beliefs, knowledge and feeding practices that mothers use with young children will help m ~he ~evelopment of culturally and linguistically appropriate educational strategies and materials for use m primary prevention of pediatric obesity. in canada more than seventy-five percent of immigrants choose to stay in three major urban centers of toronto, montreal and vancouver. approximately fifty percent of the immigrants eventually set· tie m ~~e greater tor<_>nto area. there is mounting evidence that the increasing immigrant population has a_ sigmfic~nt health disadvantage over canadian-born residents. this health disadvantage manifests particularly m the ma1"ority of 1"mm1"gr t h h d be · · h . . . . an s w o a en m canada for longer than ten years. this group as ~n associ~te~ with higher risk of chronic disease such as cardiovascular diseases. this disparity twccb n ma1onty of the immigrant population and the canadian-born population is of great importance to ur an health providers d" · i i · b as isproporttonate y arge immigrant population has settled in the ma1or ur an centers. generally the health stat f · · · · · · h h been . us 0 most 1mm1grants 1s dynamic. recent 1mm1grants w o av_e ant •;ffca~ada _for less ~han ~en years are known to have a health advantage known as 'healthy imm1• ~ants r::r · ~:s eff~ 1~ defined by the observed superior health of both male and female recent immiimmigrant participation in canadian society particularly the labour market. a new explanation of the loss of 'healthy immigrant effect' is given with the help of additional factors. lt appears that the effects of social exclusion from the labour market leading to social inequalities first experienced by recent immigrant has been responsible for the loss of healthy immigrant effect. this loss results in the subsequent health disadvantage observed in the older immigrant population. a study on patients perspectives regarding tuberculosis treatment by s.j.chander, community health cell, bangalore, india. introduction: the national tuberculosis control programme was in place over three decades; still tuberculosis control remains a challenge unmet. every day about 1440 people die of tuberculosis in india. tuberculosis affects the poor more and the poor seek help from more than one place due to various reasons. this adversely affects the treatment outcome and the patient's pocket. many tuberculosis patients become non-adherence to treatment due to many reasons. the goal of the study was to understand the patient's perspective regarding tuberculois treatment provided by the bangalore city corporation. (bmc) under the rntcp (revised national tuberculosis control programme) using dots (directly observed treatment, short course) approach. bmc were identified. the information was collected using an in-depth interview technique. they were both male and female aged between 4-70 years suffering from pulmonary and extra pulmonary tuberculosis. all patients were from the poor socio economic background. results: most patients who first sought help from private practitioners were not diagnosed and treated correctly. they sought help form them as they were easily accessible and available but they. most patients sought help later than four weeks as they lacked awareness. a few of patients sought help from traditional healers and magicians, as it did not help they turned to allopathic practitioners. the patients interviewed were inadequately informed about various aspect of the disease due to fear of stigma. the patient's family members were generally supportive during the treatment period there was no report of negative attitude of neighbours who were aware of tuberculosis patients instead sympathetic attitude was reported. there exists many myth and misconception associated with marriage and sexual relationship while one suffers from tuberculosis. patients who visited referral hospitals reported that money was demanded for providing services. most patients had to borrow money for treatment. patients want health centres to be clean and be opened on time. they don't like the staff shouting at them to cover their mouth while coughing. conclusion: community education would lead to seek help early and to take preventive measures. adequate patient education would remove all myth and conception and help the patients adhere to treatment. since tb thrives among the poor, poverty eradiation measures need to be given more emphasis. mere treatment approach would not help control tuberculosis. lntrod#ction: the main causative factor in cervical cancer is the presence of oncogenic human papillomavitus (hpv). several factors have been identified in the acquisition of hpv infection and cervical cancer and include early coitarche, large number of lifetime sexual partners, tobacco smoking, poor diet, and concomitant sexually transmitted diseases. it is known that street youth are at much higher risk for these factors and are therefore at higher risk of acquiring hpv infection and cervical cancer. thus, we endeavoured to determine the prevalence of oncogenic hpv infection, and pap test abnormalities, in street youth. ~tbods: this quantitative study uses data collected from a non governmental, not for profit dropin centre for street youth in canada. over one hundred females between the ages of sixteen and twentyfour were enrolled in the study. of these females, all underwent pap testing about those with a previous history of an abnormal pap test, or an abnormal-appearing cervix on clinical examination, underwent hpv-deoxyribonucleic (dna) testing with the digene hybrid capture ii. results: data analysis is underway. the following results will be presented: 1) number of positive hpv-dna results, 2) pap test results in this group, 3) recommended follow-up. . the results of this study will provide information about the prevalence of oncogemc hpv-dna infection and pap test abnormalities in a population of street youth. the practice implic~ tions related to our research include the potential for improved gynecologic care of street youth. in addition, our recommendations on the usefulness of hpv testing in this population will be addressed. methods: a health promotion and disease prevention tool was developed over a period of several years to meet the health needs of recent immigrants and refugees seen at access alliance multicultural community health centre (aamchc), an inner city community health centre in downtown toronto. this instrument was derived from the anecdotal experience of health care providers, a review of medical literature, and con· sultations with experts in migration health. herein we present the individual components of this instrument, aimed at promoting health and preventing disease in new immigrants and refugees to toronto. results: the health promotion and disease prevention tool for immigrants focuses on three primary health related areas: 1) globally important infectious diseases including tuberculosis (tb), hiv/aids, syphilis, viral hepatitis, intestinal parasites, and vaccine preventable diseases (vpd), 2) cancers caused by infectious diseases or those endemic to developing regions of the world, and 3) mental illnesses includiog those developing among survivors of torture. the health needs of new immigrants and refugees are complex, heterogeneous, and ohen reflect conditions found in the immigrant's country of origin. ideally, the management of all new immigrants should be adapted to their experiences prior to migration, however the scale and complexity of this strategy prohibits its general use by healthcare providers in industrialized countries. an immigrant specific disease prevention instrument could help quickly identify and potentially prevent the spread of dangerous infectious diseases, detect cancers at earlier stages of development, and inform health care providers and decision makers about the most effective and efficient strategies to prevent serious illness in new immigrants and refugees. lntrodmction: as poverty continues to grip pakistan, the number of urban street children grows and has now reached alarming proportions, demanding far greater action than presently offered. urbanization, natural catastrophe, drought, disease, war and internal conflict, economic breakdown causing unemployment, and homelessness have forced families and children in search of a "better life," often putting children at risk of abuse and exploitation. objectives: to reduce drug use on the streets in particular injectable drug use and to prevent the transmission of stds/hiv/aids among vulnerable youth. methodology: baseline study and situation assessment of health problems particularly hiv and stds among street children of quetta, pakistan. the program launched a peer education program, including: awareness o_f self and body protection focusing on child sexual abuse, stds/hiv/aids , life skills, gender and sexual rights awareness, preventive health measures, and care at work. it also opened care and counseling center for these working and street children ar.d handed these centers over to local communities. relationships among aids-related knowledge and bt:liefs and sexual behavior of young adults were determined. rea.sons for unsafe sex included: misconception about disease etiology, conflicting cultural values, risk demal, partner pressur~, trust and partner significance, accusation of promiscuity, lack of community endorsement of protecnve measures, and barriers to condom access. in addition socio-economic pressure, physiological issues, poor community participation and anitudes and low ~ducation level limited the effectiveness of existing aids prevention education. according to 'the baseline study the male children are ex~ to ~owledge of safe sex through peers, hakims, and blue films. working children found sexual mfor~anon through older children and their teachers (ustad). recommendation s: it was found that working children are highly vulnerable to stds/hiv/aids, as they lack protective meas":res in sexual abuse and are unaware of safe sexual practices. conclusion: non-fatal overdose was a common occurrence for idu in vancouver, and was associated with several factors considered including crystal methamphetamine use. these findings indicate a need for structural interventions that seek to modify the social and contextual risks for overdose, increased access to treatment programs, and trials of novel interventions such as take-home naloxone programs. background: injection drug users (idus) are at elevated risk for involvement in the criminal justice system due to possession of illicit drugs and participation in drug sales or markets. the criminalization of drug use may produce significant social, economic and health consequences for urban poor drug users. injection-related risks have also been associated with criminal justice involvement or risk of such involvement. previous research has identified racial differences in drug-related arrests and incarceration in the general population. we assess whether criminal justice system involvement differs by race/ethnicity among a community sample of idus. we analyzed data collected from idus (n = 1,084) who were recruited in san francisco, and interviewed and tested for hiv. criminal justice system involvement was measured by arrest, incarceration, drug felony, and loss/denial of social services associated with the possession of a drug felony. multivariate analyses compared measures of criminal justice involvement and race/ethnicity after adjusting for socio-demographic and drug-use behaviors including drug preference, years of injection drug use, injection frequency, age, housing status, and gender. the six-month prevalence of arrest was highest for whites (32%), compared to african americans (25%) and latinos (27% ), in addition to the mean number of weeks spent in jail in the past 6 months (7.0 vs. 5.8 and 4.2 weeks). these differences did not remain statistically significant in multivariate analyses. latinos reported the highest prevalence of a lifetime drug felony conviction (48%) and mean years of lifetime incarceration in prison (13.3 years), compared to african americans (48%, 10.7 years) and whites (34%, 6.9 years). being african american was independently associated with having a felony conviction and years of incarceration in prison as compared to whites. the history of involvement in the criminal justice system is widespread in this sample. when looking at racial/ethnic differences over a lifetime including total years of incarceration and drug felony conviction, the involvement of african americans in the criminal justice system is higher as compared to whites. more rigorous examination of these data and others on how criminal justice involvement varies by race, as well as the implications for the health and well-being of idus, is warranted. homelessness is a major social concern that has great im~act on th~se living.in urban commu?ities. metro manila, the capital of the philippines is a highly urbanized ar~ w.1t~ the h1gh~st concentration of urban poor population-an estimated 752,229 families or 3,005,857 md1v1duals. this exploratory study v122 is the first definitive study done in manila that explores the needs and concerns of street dwdlent\omc. less. it aims to establish the demographic profile, lifestyle patterns and needs of the streetdwdlersindit six districts city of manila to establish a database for planning health and other related interventions. based on protocol-guid ed field interviews of 462 street dwellers, the data is useful as a template for ref!!. ence in analyzing urban homelessness in asian developing country contexts. results of the study show that generally, the state of homelessness reflects a feeling of discontent, disenfranchisem ent and pow!!· lessness that contribute to their difficulty in getting out of the streets. the perceived problems andlar dangers in living on the streets are generally associated with their exposure to extreme weather condirioll! and their status of being vagrants making them prone to harassment by the police. the health needs of the street dweller respondents established in this study indicate that the existing health related servias for the homeless poor is ineffective. the street dweller respondents have little or no access to social and health services, if any. some respondents claimed that although they were able to get service from heallh centers or government hospitals, the medicines required for treatment are not usually free and are beyond their means. this group of homeless people needs well-planned interventions to hdp them improve their current situations and support their daily living. the expressed social needs of the sucet dweller respondents were significantly concentrated on the economic aspect, which is, having a perma· nent source of income to afford food, shelter, clothing and education. these reflect the street dweller' s need for personal upliftment and safety. in short, most of their expressed need is a combination of socioeconomic resources that would provide long-term options that are better than the choice of living on the streets. the suggested interventions based on the findings will be discussed. . methods: idu~ aged i 8 and older who injected drugs within the prior month were recruited in 2005 usmg rds which relies on referral networks to generate unbiased prevalence estimates. a diverse and mon· vated g~o~p of idu "seeds." were given three uniquely coded coupons and encouraged to refer up to three other ehgibl~ idu~, for which they received $5 usd per recruit. all subjects provided informed consent, an anonymous 1~t erv1ew and a venous blood sample for serologic testing of hiv, hcv and syphilis anti~!· results. a total of 213 idus were recruited in tijuana and 206 in juarez, of whom the maion!)' were .male < 9 .l.4% and 92.2%) and median age was 34. melhotls: using the data from a multi-site survey on health and well being of a random sample of older chinese in seven canadian cities, this paper examined the effects of size of the chinese community and the health status of the aging chinese. the sample (n=2,272) consisted of aging chinese aged 55 years and older. physical and mental status of the participants was measured by a chinese version medical outcome study short form sf-36. one-way analysis of variance and post-hoc scheffe test were used to test the differences in health status between the participants residing in cities representing three different sizes of the chinese community. regression analysis was also used to examine the contribution of size of the chinese community to physical and mental health status. rmdts: in general, aging chinese who resided in cities with a smaller chinese population were healthier than those who resided in cities with a larger chinese population. the size of the chinese community was significant in predicting both physical and mental health status of the participants. the findings also indicated the potential underlying effects of the variations in country of origin, access barriers, and socio-economic status of the aging chinese in communities with different chinese population size. the study concluded that size of an ethnic community affected the health status of the aging population from the same ethnic community. the intra-group diversity within the aging chinese identified in this study helped to demonstrate the different socio-cultural and structural challenges facing the aging population in different urban settings. urban health and demographic surveillance system, which is implemented by the african population & health research center (aphrc) in two slum settlements of nairobi city. this study focuses on common child illnesses including diarrhea, fever, cough, common cold and malaria, as well as on curative health care service utilization. measures of ses were created using information collected at the household level. other variables of interest included are maternal demographic and cultural factors, and child characteristics. statistical methods appropriate for clustered data were used to identify correlates of child morbidity. preliminary ratdts: morbidity was reported for 1,087 (16.1 %) out of 6,756 children accounting for a total of 2,691 illness episodes. cough, diarrhoea, runny nose/common cold, abdominal pains, malaria and fever made up the top six forms of morbidity. the only factors that had a significant associ· ation with morbidity were the child's age, ethnicity and type of toilet facility. however, all measures of socioeconomic status (mother's education, socioeconomic status, and mother's work status) had a significant effect on seeking outside care. age of child, severity of illness, type of illness and survival of father and mother were also significantly associated with seeking health care outside home. the results of this study have highlighted the need to address environmental conditions, basic amenities, and livelihood circumstances to improve child health in poor communities. the fact that socioeconomic indicators did not have a significant effect on prevalence of morbidity but were significant for health seeking behavior, indicate that while economic resources may have limited effect in preventing child illnesses when children are living in poor environmental conditions, being enlightened and having greater economic resources would mitigate the impact of the poor environmental conditions and reduce child mortality through better treatment of sick children. inequality in human life chances is about the most visible character of the third world urban space. f.conomic variability and social efficiency have often been fingered to justify such inequalities. within this separation households exist that share similar characteristics and are found to inhabit a given spatial unit of the 'city. the residential geography of cities in the third world is thus characterized by native areas whose core is made up of deteriorated slum property, poor living conditions and a decayed environment; features which personify deprivation in its unimaginable ma~t~de. there are .eviden~es that these conditions are manifested through disturbingly high levels of morbidity and mortality. ban · h h d-and a host of other factors (corrupt10n, msens1t1ve leaders 1p, poor ur 1ty on t e one an , . · 1 f · 1 · · th t ) that suggest cracks in the levels and adherence to the prmc1p es o socta 1usnce. ese governance, e c . . . . . ps £factors combine to reinforce the impacts of depnvat10n and perpetuate these unpacts. by 1den· grou o . ·1 "id . . bothh tifying health problems that are caused or driven by either matena _or soc1a e~nvanon or , t e paper concludes that deprivation need not be accepted as a way. of hfe a~d a deliberate effon must be made to stem the tide of the on going levels of abject poverty m the third world. to the extent that income related poverty is about the most important of all ramifications of po~erty, efforts n_iu_st include fiscal empowerment of the poor in deprived areas like the inner c~ty. this will ~p~ove ~he willingness of such people to use facilities of care because they are able to effectively demand 1t, smce m real sense there is no such thing as free medical services. ). there were 322 men with hiv-infection included in the present study (mean age and education of 41.8 (sd=8.4) and 13.9 (sd=2.7), respectively). a series of multiple regressions were used to examine the unique contributions of symptom burden (depression, cognitive, pain, fatigue), neuropsychologic al impairment (psychomotor efficiency), demographics (age and education) and hiv disease (cdc-93 staging) on iirs total score and jirs subscores: ( 1) activities of daily living (work, recreation, diet, health, finances); (2) psychosocial functioning (e.g., self-expression, community involvement); and (3) intimacy (sex life and relationship with partner). resnlts: total iirs score (r 2 "0.43) was associated with aids diagnosis (ii= 0.11, p <0.01) and symptoms of pain (ii= -0.14, p < 0.01 ), fatigue (ji = -0.34, p < 0.001) and cognitive difficulties (p =0.30, p < 0.001 ). for the three dimensions of the iirs, multiple regression results revealed: ( 1) activities of daily living (r2=0.42) were associated with aids diagnosis (ii =0.17, p < 0.01) and symptoms of pain <p =-0j6, p < 0.01 ), fatigue (ji = -0.31, p < 0.001) and cognitive difficulties (ji =0.32, p < 0.001 ); (2) psychosocial functioning (r2=0.31) was associated with was associated with symptoms of fatigue (ii= -0.25, p <0.001) and cognitive difficulties <ii =0.19, p <0.001); and (3) intimacy (r2=0.17) was related to was associated with symptoms of fatigue (ij = -0.17, p < 0.01) and cognitive difficulties (ii= 0.19, p < 0.001 ). methods: the sif evaluation methodology involves a comprehensive database located at the sif, a randomly selected p~o.spective coh~rt of sif users, and two pre-existing external control cohorts. . . ~ts: in addmon to reporting process data and descriptions of the sif users, we report on data indicating that the establishment of the sif has been independently associated with reductions in public ~':°g ~ (p <?.001), and syringe sharing (aor =0.3[95%ci: 0.1 _ 0.7); p =0.013) and other unsafe m1ecnon pract1_ces_ (p ~ 0.010), and increased uptake of detox services (log-rank p =0.017). as well, we report on da~ md1cat1ng that the sif has not prompted adverse changes in community drug use patterns. the vancouver sif has been well accepted by the target population, and while adve~ events s~c~ as overdoses have occurred, these events have been successfully managed. externally compiled data indicate that the sif has been associated with substantial declines in public disorder !'oster sessions v125 associated with injection drug use, syringe sharing, and increased uptake of detox services. as well, the establishment of the sif has not exacerbated community drug use patterns. ongoing evaluation activities wiu involve assessing the impact of the sif on a variety of outcomes, including infectious disease transmission, fatal overdose, and utilization of health and social services. city, brazil, 1996 -2002 maria cristina almeida, waleska caiaffa, renato assum;iio, and fernando proietti dengue cases were described according to time, space, intensity, age, gender, onset of symptoms, residence address and census tract. spatial distribution of two groups (children and women over 64 years old and men aged 20-59) were compared, under assumption that they have distinct behaviors regarding their displacement around the city. analysis included local moran index, ripley\\'s k function and recurrence of census areas over time. about 99,559 cases were identified in seven epidemic waves. concentration of cases in small areas, followed by a spread either in space or time suggested endemic patterns. regarding age-gender groups, distinct patterns suggested that residence might not be a good proxy in determining the local of infection for men aged 20-59. dengue is shifting to endemic pattern in this city and transmission may not be sole related to home environment, suggesting that additional spatial information must be couected aiming efficient control measures. murukan kandamuthan and subodh kandamuthan lnl7uduction: illness or disablement of a child may affect the economic functioning of a family as it alters the employment pattern and earnings of parents this paper tries to assess the health status and the quality of life of disabled children, and how generally, severe disablement in a child affected their fami-lies\' finances, and to quantify any such effects the problems faced by children in their home and to provide information relevant to the formation of criteria for new or increased cash support. methods: a case-control study was done in the thiruvananthapuram district, capital city of kerala by selecting a random sample of 300 families with severely disabled children below 15 years and a random sample of 300 normal children matched for age and occupation of the parents. comparative and subjective data were collected. money spent on children\'s medical care, loss of earnings of the parents, and other economic burden to the family due to the disability of the child in the family. a discriminant analysis along with univariate analysis was done to assess the factors that mostly differentiate the disabled and normal children. the mean expenditure of the families with a disabled child was rs. 852/-per month, which is significantly higher than the corresponding expenditure of rs. 389/-per month of families with normal child, (t= 16.86, p <.00001). of the disabled children, 81 % were not getting any social security payments and 90% had no special concessions for medical and other educational purposes. the analysis of income and expenditure pattern indicated that financial impact of disablement in a child on the family is significant. the discriminant model results revealed that medical expenditure was a significant variable that differentiated the disabled and normal child. the study found that the health status and quality of life of the disabled children were far from satisfactory. this in fact affected the economic status of the disabled children. in addition to reduced earnings, there are extra costs for disabled children for travel, domestic help, medical care, and health care expenditures (hospital care, physician services, dentistry, drugs and others) for disabled individuals. a strong case can be made for their long-term care by improving the financial support to such families possibly through the introduction of an allowance specially to compensate for the earnings lost by parents due to the disability of their children. widows in india are considered disadvantage group of population. because they are characterized by pangs of separation from spouse, and consequently they go through mental agony, social ~s?lation, and economic dislocation. in addition to these, poverty, landlessness, homelessness, malnutr1non etc. endure serious deterioration of their health. according to census 2001, there are 44 million women are widowed and one fourth live in urban areas. there is a noticeable increase of urban widows from the previous census (almost half). the paper examines the type of disability and chronic ailment borne among elderly widows (60 years and above). fifty second round of national sample survey aske~ the individuals three sets of questions regarding their health: their status of health, whether they are physically poster sessions v126 immobile, and whether or not they have had any specific chronic illnesses. although health infrastruc-1 1 ·1 ble 1 ·n urban setting in india as compared to rural counterpart, prevalence of tures are arge y ava1 a . . . . . h · ·11 · h h"gher 1 "n urban areas analysis shows 1omt problems m old age qmte common c romc 1 ness is muc 1 • . . and nearly 44 percent elderly widows are suffering from this ail'.11ent in urban areas. one-fifth of widows are suffering from high/low bp. heart disease, diabetes and urinary problems are much ~ore pre~alent in urban areas though disabilities is comparatively lower in urban areas. however, the d~fference is not statistically significant. disability with respect to vision is more pronounced and one third_ of the total respondents have problems regarding eyesight. one fifths ofdderly widows are _ha.rd of hearing. though disability increases with age, similar trend is not observed with respect to chr~mc illness. the percepnon of self-health among elderly becomes poorer as their age increases. the perceived health status need not be always corrected to objective indicators of health. it is noted that around 27 per~en~ of those perceived their health as "excellenr" or "very good" are found to be suffering from chrome disease of1omts and 17 percent have visual disability. to conclude an elderly widow living in urban areas have similar health problems as she jives in rural areas. this can be easily explained, as they were not made aware to seek treatment from health facility. in addition, the study shows that the economic conditions are deterrent factors for their illness. it is essential that intervention should be taken up improving their economic conditions so that wellbeing of this most vulnerable group of the society can be taken care of. heart failure is a disease that affects nearly 15 million people world wide. the united states alone accounts for one third of this population. blacks are stricken with heart failure twice as often as whites. men are more likely to develop chf than women, however since the majority of the chf population are seniors, there are actually more women than men. congestive heart failure (chf) has become a pandemic. as the baby boomer generation ages, the number of chf cases will rise dramatically. (young, j. & mills, r.,2004). after the age of 45, each proceeding decade doubles the incidence level of chf. the average person has a 1 in 5 chance of developing heart failure within their lifespan according to the framingham study (nih.gov). individuals aging 65 or older compose over half of the entire documented heart failure population. in this age group, chf is the leading cause of hospitalization. many patients are continuously readmitted. it is estimated that the chf population will increase by one million within the next 25 years. mortality was at 50% for a two year period and 70% for five years (young, j. & mills, r.,2004 ). data from: aha as technology evolves, the understanding of the nature of heart failure has become clearer. in the beginning of the 20th century heart failure was diagnosed as a condition that presented with edema (swelling) in the extremities caused by water retention. treatment in the beginning of the past cenrury was limited to abdominal drainage and diuretics. the discovery of the heart's insufficient pumping ability in heart failure patients lead to new surgeries and devices such as heart transplants and ventricular assist devices (bridge until transplant is available). ventricular hypertrophy often occurs before the development of heart failure. hypertrophy is the term given for cardiac remodeling. cardiac remodeling also includes chamber dilation (young, j. & mills, r.,2004) . chf financial burden the us healthcare financing administration has ranked chf as the highest cost illness in the diagnose-related area. direct costs related to chf in the us for 2004 were nearly $26 billion. these costs included hospital admissions, physician fees, home health aids and medications. indirect costs were over $2 billion in 2004. loss of jobs or death due to chf was the criteria for this category (young, j. & mills, r., 2004). . introduction: the injection drug user quality of life scale (iduqol) is a relatively new scale ~es1gned to capture the unique and individual circumstances that determine quality of life among injection drug users (idus). the 20 life areas comprising the instrument were based on the research literature an~ ~ocus group discussions with idus. the purpose of the present study was to evaluate the content validity of t.h~ iduqol using judgmental methods based on subject matter experts' (smes) ratings. content vah~1ty refers to the de~ee to which elements of an assessment tool are representative of the construct of interest and appropnate for a given population. methods: data were obtained from six smes, from the field of idu research and practice, who ~ere. asked t.o evaluate various aspects of the iduqol, including the title of the instrument, administraon instruchons, clarity of the names and descriptions of each life area, response format, scoring instruc-no~s and examples, record form and whether each life area should be included in the instrument. sme ratings were made using either 3 or 4 point scales. sm es were also given the opportunity to comment on poster sessions v127 each section and to suggest whether important life areas had been overlooked, were redundant, not relevant, or in need of revision. (cvi) and the ~v.erage ~eviation index (ad) were used to evaluate smes' agreement on ratings of the content vahd1ty vanables. both measures provided support for the content validity of various aspects of the iduqol, although results from the stricter ad index noted some areas of disagreement, including the description of particular life areas (e.g., being useful, drugs, sex), the inclusion of some life areas (e.g., drug treatment, education, independence and free choice), and the ease of the scoring instructions. the findings from this study provide (a) content validity evidence to support the use of the iduqol as well as (b) recommendations to suengthen both the instrument (e.g., improved descriptions for some items) and the accompanying administration and scoring manual (e.g., an easier scoring template). changes made to the instrument and its manual make the iduqol even easier for researchers, practitioners, and program evaluators to use as a way of assessing, and tracking changes over time or as a result of interventions in, quality of life in injection drug users. introduction: strict adherence to prescribed regimens is required to derive maximal benefit from highly active antireuoviral therapy (haart) in persons living with hiv/aids (phas). adherence is a key determinant of the degree and durability of viral suppression. adherence is also essential in reducing the spread of hiv and ensuring that today's treatments remain effective for as long as possible. the objective of this study is to conduct a systematic review of the research literature on the effectiveness of education and patient support strategies for improving adherence to haart. methods: a systematic search of the core databases was performed from january 1996 until may 2005. randomized controlled trials examining the effectiveness of education and patient support interventions to improve the adherence to haart were considered for inclusion. only those studies that measured adherence at a minimum of six weeks were included. study selection, quality assessments, and data abstraction were performed independently by two reviewers. results: study heterogeneity with respect to differing populations, interventions, outcomes, and length of follow-up did not allow for meta-analysis. we included 19 studies involving 2, 159 ph as. sample sizes ranged from 22 to 367. study duration ranged from a single session to a variable number of sessions delivered over one year. many of the interventions involved the provision of an educational component to improve adherence and often addressed strategies to overcome barriers to adherence and the development of problem-solving skills. they ranged from simple interventions (e.g., the provision of reminder devices) to complex interventions (e.g., cognitive-behavioural therapy delivered by licensed psychologists). eleven of the 19 studies demonstrated a statistically significant advantage associated with the described adherence intervention. the advantage associated with adherence outcomes does not seem to translate into the more clinically relevant outcome of viral suppression. only 4 out of 12 studies that reported virological or immunological results found a significant effect associated with the intervention. the studies had several methodological shortcomings. conclusions: there is a need for standardization and methodological rigour in the conduct of adherence trials. some interventions may have a significant impact on improving adherence. further research is required before any specific adherence improving strategy can confidently be incorporated into standard clinical practice. focus groups were used to collect data from a purposive sample of treatmentexperienced participants. focus group data were analyzed using a grou~ded t~eory appr~ach. i:ne themes identified from the interviews were used to identify the effects of ant1rerrov1rals on quality of hfe. the content of the mos-hiv was appraised against the themes identified from our analysis. participants also completed the mos-hiv survey and were asked whether the survey covered all important aspects of quality of life on antiretroviral medications. results: five key informant interviews and five focus groups with 38 participants were used to identify effects on quality of life that are not directly ~aptu~ed by_ the mos-hiv health survey. our~~ showed that our participants described quality of hfe as mvol~mg_ a set of ~rsonal trad~ffs ~stay alive. in most cases, worsened quality of life was traded-off for gams 1~ longevlty from ~~canon use. these eff~'ts or tradeoffs included: ( 1) downstream consequences of side effects and toxlanes; (2) loss of lrufe. pende~t decision-making privileges, (3) having to choose drugs over ~areer; (~)burden of medication-taking responsibilities; and (5) living life under a pretense and havmg to hide-the net effect of the tradeoffs, or "prices" to pay led to what was described as "longevity with hn without hope and future~. conclusion: our study highlights five major themes summarizing the impact of haart on quality on life, and suggests that currently used scales for measuring quality of life do not a~atd~ cap~e these findings and the associated consequences. the conceptual framework for measuring quality of bfe in hiv should be reconsidered in order to better capture the important effects of the medications on quality of life. this may involve widening the boundaries of the definition of health-related quality oflife to include aspects such as finances, employment, and housing. we should further consider the development of a haart adverse effect specific instrument, using a broad definition of adverse effect in order to capture all types of adverse impacts of hiv treatments on quality of life. introduction: measles (rubeola) is the most contagious vaccine preventable disease of humans. while cases of measles are uncommon in canada today, the disease continues to be a leading cause of morbidity and death in the developing world. as a result of human migration, measles cases still occur in canada, primari~ among immigrants, returning travelers, and other susceptible groups. canada's national advisory council on immunizations (naci) recommends that immigrants without records of measles immunization be considered for vac:cination since these individuals may not have been appropriately vaccinated in their native country. on rhe other hand, natural immunity to measles in immigrants is likely to be high given the high incidence of disease in developing parts of rhe world. thus it remains unclear if the most efficient strategy to achieve high levels of measles immunity in immigrants should entail initial serologic screening followed by vaccination of susceptible individuals or preemptive vaccination of all persons lacking immunization records. understanding the epidemiology of measles immunity in immigrant populations could help guide such decisions. . methods: we identified 527 adults, 15 years of age or older, who were screened for serologtc immunity to measles as part of a recently developed screening protocol at a downtown toronto community health centre. we subsequently determined if these individuals had any immunization records from their native country or from within canada. we then examined the relationship between several demographic factors and immunity to measles. results: 93% of immigrants had no prior immunization records. overall, 93% of the 527 immigrants rested for immunity to measles were found to be immune; 88% of those under the age of 20, 93% of those between 20 and 39 years of age, 99% of those between 40 and 59 years of age, and 100% of those 60 years of age or older. gender, education status, household income, and immigration status were nor ~ssoc1ated wirh immunity to measles. immunity to measles was lowest among immigrants from eastern europe (8~%), whil~ immigrants from other regions of the globe had greater than 90% immunity. c~lusrons: immigrants and refugees appear to have reasonably high levels of immunity to mea· sics, possibly related to natural infection wirh the measles virus. immigrants from eastern europe appear to have slightly. lower l~vel~ of immunity to the measles virus. universal screening for immunity to meales or preemptive vac:cmauon may both be inefficient public health strategies, however further research is needed to corroborate these findings. nancy r~ss, stephane tremblay, saeeda khan, daniel crouse, mark tremblay, and jean-marie berrhelor o~ve: the speed of the rise in obesity in places like canada suggests that rather than a shift in the gcncnc_ com~sirion of the population, the root of the obesity pandemic is an environment that suprrs ~besity. this paper examines the influence of neighbourhood and metropolitan characteristics. bour~lts:. while accounting. for individual socio-demographics and behaviours, residents of neighs with a large proportmn of poorly educated individuals had higher bmis than those living in poster sessions v129 neighbourhoods with more highly educated individuals (p <.01 ). residing in a neighbourhood with a high proportion of recent immigrants was associated with lower bmi for men (p<.01), but not women. neighbourhood dwelling density, a proxy for walkability, was not associated with bm! for either sex. metropolitan sprawl was associated with higher male bmi (p=.02) but the effect was negligible for women (p=.09). bmi was significantly lower for women (p<.01) living in a city in the province of quebec, even after accounting for the influence of individual and neighbourhood covariates. conclusions: bm! was strongly patterned by individual-level social position in urban canada. the incremental influence on bmi of neighbourhood related to the neighbourhood's social conditions and not to their physical form. metropolitan sprawl was associated with higher bm! for men, a group with longer car-dependent commutes than women. the findings suggest that both individuals and their environments (both social and physical) influence the distribution of bmi in urban populations. introduction: urban environments have been linked to a range of human health issues. in singapore, prevalence of end stage renal disease (esrd) is predicted to rise (2633 in 1999 to 6000 in 2010 , kidney international, vol.67, 2005 . the clinical and economic burden of esrd has promoted development of strategies aimed at preventing the development and progression of chronic kidney disease. these include population based screening programs such as the national kidney foundation, sin-gapore\'s (nkfs) "partnership for prevention."the program, aims to reduce incidence of esrd through comprehensive intervention and screening for early detection of urinary abnormalities, blood pressure (bp), and random blood glucose (rbg). objective of this study is to examine gender, race and age wise prevalence of elevated bp, rbg and proteinuria. methodology: this current analysis includes 575,438 subjects, age 18 to 86 years, who underwent screening during september 1999 and december 2004. participants were asked to give demographic information and the history of diseases. tests were given to get clinical information such as proteinuria, blood glucose, sbp, and dbp. blood pressure abnormalities were defined according to ]nc vi criteria. proteinuria was defined as the presence of 1 plus or greater protein (equivalent to> 30 mg/di) on dipstick analysis. results: there were 296, 116 (51.5%) males. racial distribution was chinese (78.8% ), malay (8.8% ), indians (8. 7%) and others (3. 7% ).among participants, who were apparently "healthy" (asymptomatic and without history of dm, ht, or kd), gender and race wise % prevalence of elevated (bp> 140/90), rbg (> 140 mg/di) and positive urine dipstick for protein was as follows male: (20.5;6.9; 3.5) female:(13.6;5.0;3. 2) chinese:( 17.1;6.0;3) malay: (19.4;7.3;5.6) indian:( 15.9;7.5;3.0) others: (15.4;4.5;2.9) total:(l 7.1, 6.1,3.2). percentage of participants with more than one abnormality were as follows. those with bp> 140/90mmhg, 14% also had rbg> 140mg/dl and 6.4% had proteinuria> i. those with rbg> 140mgldl, 11 % also had proteinuria> 1 and 35% had bp> 140/90mmhg. those with proteinuria> 1, 18% also had rbg> 140mg/dl, and 38% had bp> 140/90mmhg. conclusion: we conclude that sub clinical abnormalities in urinalysis, bp and rbg readings are prevalent across all genders and racial groups in the adult population. the overlap of abnormalities, point towards the high risk for esrd as well as cardiovascular disease. this indicates the urgent need for population based programs aimed at creating awareness, and initiatives to control and retard progression of disease. introduction: various theories have been proposed that link differential psychological vulnerability to health outcomes, including developmental theories about attachment, separation, and the formation of psychopathology. research in the area of psychosomatic medicine suggests an association between attachment style and physical illness, with stress as a mediator. there are two main hypotheses explored in the present study: ( t) that individuals living with hiv who were upsychologically vulne~able" at study entry would be more likely to experience symptoms of depression, anxiety and phys1ca! illness over. the course of the 9-month study period; and (2) life stressors and social support would mediate the relat10nship between psychological vulnerability and the psychological ~nd physical outcomes. . (rsles), state-trait anxiety inventory (stai), beck depr~ssi~n lnvento~ (bdi), and~ _21-item pbys~i symptoms inventory. we characterized participants as havmg psychological vulnerability and low resilience" as scoring above 35 on the raas (insecure attachment) or above 120 on the das (negative expectations about oneself). . . . . . " . . ,, . results: at baseline, 55% of parnc1pants were classified as havmg low resilience. focusmg on anxiety, the average cumulative stai score of the low-resilience group was significandy hi~e~ than that of the high-resilience group ( 18.45 sd= 10.6 versus 9.57 sd= 8.6; f(l,80)= 16.74, p <.001). similar results were obtained for bdi and physical symptoms (f( 1,80)= 14.65, p<.001 and f( 1,80)= 5.50, p<.05, respec· tively). after controlling for resilience, the effects of variance in life stres".°rs averaged over time wa~ a_sig· nificant predictor of depressive and physical symptoms, but not of anxiety. ho~e_ver, these assooan~s became non-significant when four participants with high values were removed. s1id1larly, after controlling for resilience, the effects of variance in social support averaged over time became insignificant. conclusion: not only did "low resilience" predict poor psychological and physical outcomes, it was also predictive of life events and social support; that is, individuals who were low in resilience were more likely to experience more life events and poorer social support than individuals who were resilient. for individuals with vulnerability to physical, psychological, and social outcomes, there is need to develop and test interventions to improve health outcomes in this group. rajat kapoor, ruby gupta, and jugal kishore introduction: young people in india represent almost one-fourth of the total population. they face significant risks related to sexual and reproductive health. many lack the information and skills neces· sary to make informed sexual and reproductive health choices. objective: to study the level of awareness about contraceptives among youth residing in urban and rural areas of delhi. method: a sample of 211 youths was selected from barwala (rural; n= 112) and balmiki basti (urban slums; n= 99) the field practice areas of the department of community medicine, maulana azad medical college, in delhi. a pre-tested questionnaire was used to collect the information. when/(calen· dar time), by 2, fisher exact and t were appliedxwhom (authors?). statistical tests such as as appropriate. result: nearly 9 out of 10 (89.1 %) youth had heard of at least one type of contraceptive and majority (81.5%) had heard about condoms. however, awareness regarding usage of contraceptives was as low as 9.4% for terminal methods to 39.3% for condom. condom was the best technique before and after marriage and also after childbirth. the difference in rural and urban groups was statistically signif· icant (p=.0001, give confidence interval too, if you provide the exact p value). youth knew that contra· ceptives were easily available (81 %), mainly at dispensary (68.7%) and chemist shops (65.4%). only 6.6% knew about emergency contraception. only advantage of contraceptives cited was population con· trol (42.6%); however, 3.8% believed that they could also control hiv transmission. awareness of side effects was poor among both the groups but the differences were statistically significant for pills (p=0.003). media was the main source of information (65%). majority of youth was willing to discuss a~ut contraceptive with their spouse (83.4%), but not with others. 51.2% youth believed that people in their age group use contraceptives. 35% of youth accepted that they had used contraceptives at least once. 81 % felt 2 children in family is appropriate, but only 59.7% believed in 3 year spacing. . conclusion: awareness about contraceptives is vital for youth to protect their sexual and reproduc· tive health .. knowledge about terminal methods, emergency contraception, and side effects of various contraceptives need to be strengthened in mass media and contraceptive awareness campaigns. mdbot:ls: 740 elderly aged 60+ were interviewed in 3 poor communities in beirut the capital of f:ebanon, ~e of which is a palestinia~. refugee camp. depression was assessed using the i 5-item geriat· nc depressi~n score (~l?s-15). specific q~estions relating to the 3 aspects of religiosity were asked as well as questions perta1rung to demographic, psychosocial and health-related variables. results: depression was prevalent in 24% of the interviewed elderly with the highest proportion being in the palestinian refugee camp (31 %). mosque attendance significantly reduced the odds of being depressed only for the palestinian respondents. depression was further associated, in particular communities, with low satisfaction with income, functional disability, and illness during last year. condiuion: religious practice, which was only related to depression among the refugee population, is discussed as more of an indicator of social cohesion, solidarity than an aspect of religiosity. furthermore, it has been suggested that minority groups rely on religious stratagems to cope with their pain more than other groups. implications of findings are discussed with particular relevance to the populations studied. nearly thirty percent of india's population lives in urban areas. the outcome of urbanization has resulted in rapid growth of urban slums. in a mega-city chennai, the slum populations (25.6 percent) face greater health hazards due to overcrowding, poor sanitation, lack of access to safe drinking water and environmental pollution. amongst the slum population the health of women and children are most neglected, resulting in burden of both communicable and non-communicable diseases. the focus of the paper is to present the epidemiology profile of children (below 14 years) in slums of chennai, their health status, hygiene and nutritional factors, the social response to health, the trends in child health and urbanization over a decade, the health accessibility factors, the role of gender in health care and assessment impact of health education to children. the available data prove that child health in slums is worse than rural areas. though the slum population is decreasing there is a need to explore the program intervention and carry out surveys for collecting data on some specific health implications of the slum children. objective: during the summer of 2003 there was a heat wave in central europe, producing an excess number of deaths in many countries including spain. the city of barcelona was one of the places in spain where temperatures often surpassed the excess heat threshold related with an increase in mortality. the objective of the study was to determine whether the excess of mortality which occurred in barcelona was dependent on age, gender or educational level, important but often neglected dimensions of heat wave-related studies. methods: barcelona, the second largest city in spain (1,582,738 inhabitants in 2003) , is located on the north eastern coast. we included all deaths of residents of barcelona older than 20 years that occurred in the city during the months of june, july and august of 2003 and also during the same months during the 5 preceding years. all the analyses were performed for each sex separately. the daily number of deaths in the year 2003 was compared with the mean daily number of deaths for the period 1998-2002 for each educational level. poisson regression models were fitted to obtain the rr of death in 2003 with respect to the period 1998-2002 for each educational level and age group. results: the excess of mortality during that summer was more important for women than for men and among older ages. although the increase was observed in all educational groups, in some age-groups the increase was larger for people with less than primary education. for example, for women in the group aged 65-74, the rr of dying for 2003 compared to 1998-2002 for women with no education was 1.30 (95%ci: 1.04-1-63) and for women with primary education or higher was 1.19 (95%ci: 0.90-1.56). when we consider the number of excess deaths, for total mortality (>=20 years) the excess numbers were higher for those with no education ( 17 5. 7 for women and 46. 7 for men) and those with less than primary education (112.5 for women and 11-2 for men) than those with more than primary edm:ation (75.0 for women and -10.3 for men). conclusion: age, gender and educational level were important in the 2003 barcelona heat wave. it is necessary to implement response plans to reduce heat morbidity and mortality. policies should he addressed to all population but also focusing particularly to the oldest population of low educational level. introduction: recently there has been much public discourse on homelessness and its imp~ct on health. measures have intensified to get people off the street into permanent housing. for maximum v132 poster sessions success it is important to first determine the needs of those to be housed. their views on housing and support requirements have to be considered, as th~y ar~ the ones affected. as few res.earch studies mclude the perspectives of homeless people themselves, httle is known on ho~ they e~penence the 1mpacrs on their health and what kinds of supports they believe they need to obtain housing and stay housed. the purpose of this study was to add the perspectives of homeless people to the discourse, based in the assumption that they are the experts on their own situations and needs. housing is seen as a major deter· minant of health. the research questions were: what are the effects of homelessness on health? what kind of supports are needed for homeless people to get off the street? both questions sought the views of homeless individuals on these issues. methods: this study is qualitative, descriptive, exploratory. semi-structured interviews were conducted with homeless persons on street corners, in parks and drop-ins. subsequently a thematic analysis was carried out on the data. results: the findings show that individuals' experiences of homelessness deeply affect their health. apart from physical impacts all talked about how their emotional health and self-esteem are affected. the system itself, rather than being useful, was often perceived as disabling and dehumanizing, resulting in hopelessness and resignation to life on the street. neither welfare nor minimum wage jobs are sufficient to live and pay rent. educational upgrading and job training, rather than enforced idleness, are desired by most initially. in general, the longer persons were homeless, the more they fell into patterned cycles of shelter /street life, temporary employment /unemployment, sometimes addictions and often unsuccessful housing episodes. conclusions: participants believe that resources should be put into training and education for acquisition of job skills and confidence to avoid homelessness or minimize its duration. to afford housing low-income people and welfare recipients need subsidies. early interventions, 'housing first', more humane and efficient processes for negotiating the welfare system, respectful treatment by service providers and some extra financial support in crisis initially, were suggested as helpful for avoiding homelessness altogether or helping most homeless people to leave the street. this study is a national homelessness initiative funded analysis examining the experiences and perceptions of street youth vis-a-vis their health/wellness status. through in-depth interviews with 140 street youth in halifax, montreal, toronto, calgary, ottawa and vancouver, this paper explores healthy and not-so healthy practices of young people living on the street. qualitative interviews with 45 health/ social service providers complement the analysis. more specifically, the investigation uncovers how street youth understand health and wellness; how they define good and bad health; and their experiences in accessing diverse health services. findings suggest that living on the street impacts physical, emotional and spiritual well·being, leading to cycles of despair, anger and helplessness. the majority of street youth services act as "brokers" for young people who desire health care services yet refuse to approach formal heal~h care organizational structures. as such, this study also provides case examples of promising youth services across canada who are emerging as critical spaces for street youth to heal from the ravages of ~treet cultur~. as young people increasingly make up a substantial proportion of the homeless population in canada, it becomes urgent to explore the multiple ways in which we can support them to regain a sense of wellbeing and "citizenship." p5-77 (c) health and livelihood implications of marginalization of slum dwellers in provision of water and sanitation services in nairobi city elizabeth kimani, eliya zulu, and chi-chi undie . ~ntrodfldion: un-habitat estimates that 70% of urban residents in kenya live in slums; yet due to their illegal status, they are not provided with basic services such as water sanitation and health care. ~nseque~tly, the services are provided by vendors who typically provide' poor services at exorbitant prices .. this paper investigates how the inequality in provision of basic services affects health and livelihood circumstances of the poor residents of nairobi slums . . methods: this study uses qualitative and quantitative data collected through the ongoing longitudmal .health and demographic study conducted by the african population and health research center m slum communities in n ·rob" w d · · · · ai 1. e use escnpnve analytical and qualitative techmques to assess h~w concerns relating to water supply and environmental sanitation services rank among the c~mmumty's general and health needs/concerns, and how this context affect their health and livelihood circumstances. results: water (32%) and sanitation (20%) were the most commonly reported health needs and also key among general needs (after unemployment) among slum dwellers. water and sanitation services are mainly provided by exploitative vendors who operate without any regulatory mechanism and charge exorbitantly for their poor services. for instance slum residents pay about 8 times more for water than non-slum households. water supply is irregular and residents often go for a week without water; prices are hiked and hygiene is compromised during such shortages. most houses do not have toilets and residents have to use commercial toilets or adopt unorthodox means such as disposing of their excreta in the nearby bushes or plastic bags that they throw in the open. as a direct result of the poor environmental conditions and inaccessible health services, slum residents are not only sicker, they are also less likely to utilise health services and consequently, more likely to die than non-slum residents. for instance, the prevalence of diarrhoea among children in the slums was 31 % compared to 13 % in nairobi as a whole and 17% in rural areas, while under-five mortality rates were 151/1000, 62/1000 and 113/1000 respectively. the results demonstrate the need for change in governments' policies that deprive the rapidly expanding urban poor population of basic services and regulatory mechanisms that would protect them from exploitation. the poor environmental sanitation and lack of basic services compound slum residents' poverty since they pay much more for the relatively poor services than their non-slum counterparts, and also increase their vulnerability to infectious diseases and mortality. since 1991 iepas've been working in harm reduction becoming the pioneer in latin america that brought this methodology for brazil. nowadays the main goal is to expand this strategy in the region and strive to change the drug policy in brazil. in this way harm reduction: health and citizenship program work in two areas to promote the citizenship of !du and for people living with hiv/aids offering law assistance for this population and outreach work for needle exchange to reduce damages and dissemination of hiv/aids/hepatit is. the methodology used in outreach work is peer education, needle exchange, condoms and folders distribution to reduce damages and the dissemination of diseases like hiv/aids/hepatitis besides counseling to search for basic health and rights are activities in this program. law attendance for the target population at iepas headquarters every week in order to provide law assistance that includes only supply people with correct law information or file a lawsuit. presentations in harm reduction and drug policy to expand these subjects for police chiefs and governmental in the last year attended 150 !du and 403 nidu reached and 26.364 needles and syringes exchanged. in law assistance 740 (420 people living with aids, 247 drug users, 43 inject drug users, 30 were not in profile) people attended. 492 lawsuits filed 218 lawsuits in current activity. broadcasting of the harm reduction strategies by the press helps to move the public opinion, gather supporters and diminish controversies regarding such actions. a majority number of police officer doesn't know the existence of this policy. it's still polemic discuss this subject in this part of population. women remain one of the most under seviced segments of the nigerian populationand a focus on their health and other needs is of special importance.the singular focus of the nigerian family welfare program is mostly on demographic targets by seeking to increase contraceptive prevalence.this has meant the neglect of many areas of of women's reproductive health. reproductive health is affected by a variety of socio-cultural and biological factors on on e hand and the quality of the service delivery system and its responsiveness on the other.a woman's based approach is one which responds to the needs of the adult woman and adolescent girls in a culturally sensitive manner.women's unequal access to resources including health care is well known in nigeria in which stark gender disparities are a reality .maternal health activities are unbalanced,focusi ng on immunisation and provision of iron and folic acid,rather than on sustained care of women or on the detection and referral of high risk cases. a cross-sectional study of a municipal government -owned hospitalfrom each of the 6 geo-political regions in igeria was carried out (atotal of 6 ce~ters) .. as _part ~f t~e re.search, the h~spital records were uesd as a background in addition to a 3-week mtens1ve mvesuganon m the obstemc and gynecology departments. . . . : little is known for example of the extent of gynecological morbtdtty among women; the little known suggest that teh majority suffer from one or more reproductive tr~ct infect~ons. although abortion is widespread, it continues to be performed under ilegal and unsafe condmons. with the growing v134 poster sessions hiv pandemic, while high riskgroups such ascomn;iercial sex workers and their clients have been studied, little has been accomplished in the large populat10ns, and particularly among women, regardmgstd an hiv education. . . conclusions: programs of various governmentalor non-governmental agen,c1es to mvolve strategies to broaden the narrow focus of services, and more importan~, to put wo~en s reproducnve health services and information needs in the forefront are urgently required. there is a n~d to reonent commuication and education activities to incorprate a wider interpretation of reproducnve health, to focus on the varying information needs of women, men, and youth and to the media most suitable to convey information to these diverse groups on reproductive health. introduction: it is estimated that there are 250-300 youths living on the streets, on their own with the assistance of social services or in poverty with a parent in ottawa. this population is under-serviced in many areas including health care. many of these adolescents are uncomfortable or unable to access the health care system through conventional methods and have been treated in walk-in clinics and emergency rooms without ongoing follow up. in march 2004, the ontario government provided the ct lamont institute with a grant to open an interdisciplinary and teaching medical/dental clinic for street youth in a drop-in center in downtown ottawa. bringing 5 community organizations together to provide primary medical care and dental hygiene to the streetyouths of ottawa ages 12-20, it is staffed by a family physician, family medicine residents, a nurse practitioner, 2 public health nurses, a dental hygienist, dental hygiene students and a chiropodist who link to social services already provided at the centre including housing, life skills programs and counselling. project objectives: 1. to improve the health of high risk youth by providing accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. 2. to model and teach interdisciplinary adolescent care to undergraduate medical students, family medicine residents and dental hygiene students. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative:a) semi-structured interviews b) focus groups with youth quantitative:a) electronic medical records for 12 months b) records (budget, photos, project information). results: in progress-results from first 12 months available in august 2005. early results suggest that locating the clinic in a safe and familiar environment is a key factor in attracting the over 130 youths the clinic has seen to date. other findings include the prevalence of preventative interventions including vaccinations, std testing and prenatal care. the poster presentation will present these and other impacts that the clinic has had on the health of the youth in the first year of the study. conclusions: 1) the clinic has improved the health of ottawa streetyouth and will continue beyond the initial pilot project phase. 2) this project demonstrates that with strong community partnerships, it is possible meet make healthcare more accessible for urban youth. right to health care campaign by s.j.chander, community health cell, bangalore, india. introduction: the people's health movement in india launched a campaign known as 'right to health care' during the silver jubilee year of the alma ata declaration of 'health for all' by 2000 ad in collahoration with the national human rights commission (nhrc). the aim of the campaign was to establish the 'right to health care' as a basic human right and to address structural deficiencies in the pubic health care system and unregulated private sector . . methods: as part of the campaign a public hearing was organized in a slum in bangalore. former chairman of the nhrc chaired the hearing panel, consisting of a senior health official and other eminent people in the city. detailed documentation of individual case studies on 'denial of access to health care' in different parts of the city was carried out using a specific format. the focus was on cases where denial of health services has led to loss of life, physical damage or severe financial losses to the patient. results: _fourte_en people, except one who had accessed a private clinic, presented their testimonies of their experiences m accessing the public health care services in government health centres. all the people, e_xcept_ one person who spontaneously shared her testimony, were identified by the organizations worki_ng with the slum dwellers. corruption and ill treatment were the main issues of concern to the people. five of the fourteen testimonies presented resulted in death due to negligence. the public health cen· n:s not only demand money for the supposedly free services but also ill-treats them with verbal abuse. five of these fourteen case studies were presented before the national human right commission. the poster sessions v135 nhrc has asked the government health officials to look into the cases that were presented and to rectify the anomalies in the system. as a result of the public hearing held in the slum, the nhrc identified urban health as one of key areas for focus during the national public hearing. cond#sion: a campaign is necessary to check the corrupted public health care system and a covetous private health care system. it helps people to understand the structure and functioning of public health care system and to assert their right to assess heath care. the public hearings or people's tribunals held during the campaign are an instrument in making the public health system accountable. ps-82 (a) violence among women who inject drugs nadia fairbairn, jo-anne stoltz, evan wood, kathy li, julio montaner, and thomas kerr background/object ives: violence is a major cause of morbidity and mortality among women living in urban settings. though it is widely recognized that violence is endemic to inner-city illicit drug markets, little is known about violence experienced by women injection drug users (!du). therefore, the present analyses were conducted to evaluate the prevalence of, and characteristics associated with, experiencing violence among a cohort of female idu in vancouver. methods: we evaluated factors associated with violence among female participants enrolled in the vancouver injection drug user study (vidus) using univariate analyses. we also examined self-reported relationships with the perpetrator of the attack and the nature of the violent attack. results: of the 346 active iou followed between december 1, 2003 and may 6, 2005, 73 (21.1 %) had experienced violence during the last six months. variables positively associated with experiencing violence included: homelessness (or= 3.46, 95% ci: 1.66-7.21, p < 0.01), public injecting (or= 3.45, 95% ci: 1.43 -8.35, p < 0.01 ), frequent crack use (or= 2.99, 95% ci: 1. 72 -5.17, p < 0.01 ), recent incarceration (or =2.81, 95% cl: 1.38 -5.72, p < 0.01), receiving help injecting (or =2.77, 95% cl: 1.54-5.00, p < 0.01 ), shooting gallery attendance (or =2.46, 95% ci: 1.22 -4.93, p < 0.01 ), sex trade work (or =2.30, 95% cl: 1.35 -3.93, p < 0.01 ), frequent heroin injection (or= 1.96, 95% cl: 1.13 -3.40, p < 0.02), and residence in the downtown eastside (odds ratio [or] = 1.85, 95% ci: 1.09 -3.13, p < 0.02). variables negatively associated with experiencing violence included: being married or common-law (or =0.47. 95% ci: 0.25 -0.87, p < 0.02) and being in methadone treatment (or =0.53, 95% ci: 0.31 -0.91, p < 0.02). the most common perpetrators of the attack were acquaintances (48.0%), strangers (27.4%), police (9.6%), or dealers (8.2%). attacks were most frequently in the form of beatings (65.8%), robberies (21.9%), and assault with a weapon (13.7%). conclusion: violence was a common experience among women !du in this cohort. being the victim of violence was associated with various factors, including homelessness and public injecting. these findings indicate the need for targeted prevention and support services, such as supportive housing programs and safer injection facilities, for women iou. introduction: although research on determinants of tobacco use among arab youth has been carried out at several ecologic levels, such research has included conceptual models and has compared the two different types of tobacco that are most commonly used among the lebanese youth, namely cigarette and argileh. this study uses the ecological model to investigate differences between the genders as related to the determinants of both cigarette and argileh use among youth. methodology: quantitative data was collected from youth in economically disadvantaged urban communities in beirut, the capital of lebanon. results: the results indicated that there are differences by gender at a variety of ecological levels of influence on smoking behavior. for cigarettes, gender differences were found in knowledge, peer, family, and community influences. for argileh, gender differences were found at the peer, family, and community l.evels. the differential prevalence of cigarette and argileh smoking between boys and girls 1s therefore understandable and partially explained by the variation in the interpersonal and community envi.ronment which surrounds them. interventions therefore need to be tailored to the specific needs of boys and girls. introduction: the objective of this study was to assess the relationship between parents' employment status and children' health among professional immigrant families in vancouver. our target communmes v136 poster sessions included immigrants from five ethnicity groups: south korean, indian, chine~e, ~ussian, and irani~ with professional degrees (i.e., mds, lawyers, engineers, ma?~ger~, and uru~ers1ty professors) w11h no relevant job to their professions and those who had been hvmg m the studied area at least for 36 months. methodology: the participants were recruited by collaboration from three local community agencies and were interviewed individually during the fall of 2004. ra#lts: totally, 109 complete interviews were analyzed: 33 from south-east asia, 59 from south asia, 17 from russia and other eastern europe. overall, 14.5% were employed, 38.5% were underemployed, 46% indicated they were unemployed. overall, 58.5% were not satisfied with their current job. russians and other eastern europeans were most likely satisfied with their current job, while south-east asians were most satisfied from their life in canada. about 53% indicated that their spouses were not satisfied with their life in canada, while 55% believed that their children are very satisfied from their life in canada. in addition, around 30% said they were not satisfied from their family relationship in canada. while most of the responders ranked their own and their spouses' health status as either poor or very poor, jut 3% indicated that their first child's health was very poor. in most cases they ranked their children's health as excellent or very good. the results of this pilot study show that there is a need to create culturally specific child health and behavioral scales when conducting research in immigrant communities. for instance, in many asian cultures, it is customary for a parent either to praise their children profusely, or to condemn them. this cultural practice, called "saving face," can affect research results, as it might have affected the present study. necessary steps, therefore, are needed to revise the current standard health and behavioral scales for further studies by developing a new scale that is more relevant and culturally sensitive to the targeted immigrant families. metboda: database: 2003 national health survey (ministry of health www.msc.es). two thousand interviews were performed among madrid population (0.04% of the whole); 593 corresponded to older adults (0.04% of the 1. 7 million aged 50 years and over). study sample constitutes 95.3% (565 out of 593) of those older adults, who live in urban areas. demographic structure (by age and gender) of this population in relation to health services use (medical consultations, dentist visits, emergence services, hospitalisation) was studied using general linear model univariate procedure. a p0.005), while age was associated with emergence services use (26% of the population: 21 %, 28% and 45% of each age group) and hos~italisation (17% .oft~~ population: 13%, 20% and 31%, of each age ~oup) (p0.005) was fou~d with respect to dennst v1s1ts (18% vs 20%), medical consultations (29% vs 36%), and emergence services use (26% vs 26%), while an association (p= 0.005) was found according to hospitalisation (20% vs 16%). age. an~ g~der interaction effect on health services use was not found (p> 0.005), but a trend towards bosp1tal1sanon (p=0.04) could be considered. concl.uions: demographic structure of urban older adults is associated with two of the four health se~ices use studi~. a relation.ship ber_ween age. and hospital services use (emergence units and hospitalisanon), but not with ~ut-hosp1tal sei:vices (medical and dentist consultations), was found. in addition ro age, gender also contnbutes to explam hospitalisation. . sexua experiences. we exammed the prevalence expenences 10 relation to ethnic origin and other sociodemographic variables as wc1i as y1j7 die relation between unwanted sexual experiences, depression and agreuion. we did so for boys and prts separately. mdhods: data on unwanted sexual expcric:nces, depressive symptoms (ce.s-d), aggrc:uion (bohi-di and sociodemographic facron were collected by self-report quescionnairc:s administettd to 35 31 students in the: 2nd grade (aged 12-16) of secondary schools in amsterdam, the netherlands. data on the nature ol unwanted sexual experiences were collected during penonal interviews by trained schoolnursn. ltaijtj: overall prevalences of unwanted sexual experiences for boys and girls were 6.5% and 5.7% respectively. unwanted sexual experiences were more often ttported by turkish ( 17.1 %), moroc· an (10.4%) and surinamese/anrillian boys (7.4%) than by dutch boys (2.2%). moroccan and turkish girls, however, reported fewer unwanted sexual experiences (respectively 2.3 and 2.7%) than durch girls did (6.9%). depressive symptoms(or=4.6, cl=3.1-7.0) covert agression (0r•4.9, cl•3.2-7.7) and cmrt aggression (or= 2.6, cl• 1.6-4.4) were more common in girls with an unwanted sexual experi· met. boys with an unwanted sexual experience reported more depressive symptoms (or= 2.2; cl• i . .l· 3.9) and oven agression (or= 1.5, cl= 1.0-2.4) . of the reported unwanted sexual experiences rnpec· timy 17.5% and 73.5% were confirmed by male and female adolescents during a personal interview. cond11sion: we ..:an conclude that the prevalence of unwanted sexual experiences among turkish and moroccan boys is disturbing. it is possible that unwanted sexual experiences are more reported hy boys who belong to a religion or culture where the virginity of girls is a maner of family honour and talking about sexuality is taboo. more boys than girls did not confirm their initial disdosurc of an lllwalltc:d sexual experience. the low rates of disclosure among boys suggcsu a necd to educ.:atc hcahh care providen and others who work with migrant boys in the recognition and repomng of 1exu.il ... iction. viramin a aupplc:tmntation i1 at .h'yo, 1till far from tafl'eted 100%. feedinit pracn~:n panku· lerty for new born earn demand lot of educatton ernpha111 a• cxdu11ve hrealt fecdtnit for dnared rcnoj of 6 months was observtd in only 6.s% of childrrn thoulh colckturm w.11 givm 1n rn% of mwly horn ct.ildrm. the proportion of children hclow-2 waz (malnounshrdl .con" a• h!jh •• 42.6% anj "rt'i· acimy tc.. 11 compared to 1998 data. mother's ~alth: from all is 10 womm in ttprod~uvr •ill' poup, 83% were married and among marned w~ .\9% only w\"rt' u1mic wmr cnntr.-:cruve mt1h· odl 44% were married bdorc thc •ar of 18 yean and 27% had thnr ftnc prcicnancy hcftitt dlt' •icr nf 21 yean. the lt'f'vicn are not uutfactory or they arc adequate but nae unh1ed opumally. of thote' l'h mothen who had deliverrd in last one year, 80% had nailed 11ntmaral eum1nat1on 11 ira" oncc, .~o-... bad matt rhan four ttmn and ma1ortty had 1heir tetanus toxotd tnin,"t1or"'" nlht "'"'"· ljn1r11ned rn· win ronductrd 12.4% dchvcnn and 26% had home deh\'t'oc'i. ~md~: the tervtcn unbud or u111led are !tu than dnarame. the wr· l'kft provided are inadequate and on dechm reprcwnttng a looun1t ~p of h11hnto good coytti\#' ol wr· ncn. l!.ckground chanpng pnoriry cannoc be ruled out u °"" of thc coatnbutory bc10f. ps-ii ia) dcpn:wioa aad anuccy ia mip'mu ia awccr._ many de wn, witco tui~bmjer. jack dekker, aart·jan lttkman, wim gonmc:n. and amoud verhoeff ~ a dutch commumry-bucd icudy thawed 12-moarh•·prc:yalm«i al 17 .44'1. kw anx1· ay daorden and 13. 7% foi' dqrasion m anmttdam. nm .. 11p1tficantly hlllhn than dwwhrft .. dw ~thew diffamca m pttyalcnca att probably rdarcd to tlk' largr populanoa of napaan 111 ..\mturdam. <turkish 5%, moroccan 9%, and surinam 10%1. lndttd. ic'wt'll ltudla hatt ~ ~ high prevalena rata among migrant poupi in rbe ncdwrlands. howc'ytt au ttlluchn iuffenod from wry low raporne rara, or med screnung talel thac lack a ~y yabdarcd ~ in order to study the prevalence of depression and anxiety, and the (barriers to) use of mental health care among the different ethnic groups the following study was recently conducted: . the study consisted of a two-step approach. the first step was mcl~ded m the ~ h ith survey of 2004 (ahs), and consisted of three screening scales for depression and aruaety (klo, g::q-12 and mhl-5). in this survey all respondents were asked_ permission for~ second app~ 1:' 18t consisted of a structured interview containing the cidi for aruaety and depression, and questtollllalres on health care seeking, amongst others. all respondents who gave permission for a second approachwere invited by jetter for a suuctured interview with multilingual interviewers at home at a preset date and time in the following week. . in total, 439 dutch, 317 turkish, 322 moroccan and 124 surinam respondents took part in the ahs and gave permission for a second approach. in the second step, fo~ 21~ tur~h, 184m~roc can 87 surinamese and 320 dutch respondents, information from the extensive interview was available (res~nse rates between 60 and 70% ). since the data collection was completed only recently (june 2005), prevalence estimates can be presented at the conference for the first time. we have shown that with this approach it is feasible to achieve an acceptable response rate in a study on mental health among migrants. therefore we expect that this study will provide reli· able estimates of depression and anxiety in the turkish, moroccan and surinam migrants in amstw!3~· for the first time. in addition, insight into mechanisms underlying the differences between and within ethnic groups and into barriers to mental health care will provide pretexts for the improvement of pre· vention and mental health care for migrant groups. this study aimed to determine spatial patterns of mortality and morbidity of five major health problems in an urban environment: homicides, pregnancy among adolescents ((<20 years old), asthma hospitalization among young children(< 5 years old) and two mosquito-borne diseasesdengue and visceral leishmaniasis, during 1999-2003. all events were obtained through the city health database and, subsequently, geoproccsscd using the address of residence and the geographical and administrative division of the municipality, composed by 80 unit of planning (up), which in tum were formed, each one, by census tract units. two research questions were investigated: are there spatial patterns to the events dis· tribution, and moreover, are these patterns overlapping across space? we use thematic maps, index of comparative mortality/morbidity by up and the overlapped rank of the 20th worse up rates for each event. a spatial pattern of high rates of homicides, proportion of young mothers and hospitalization of asthma were overlapping in areas social and economically disadvantaged. for mosquito-borne diseases, high rates with great dispersion were found in unprivileged areas in contrast with very low rares among privileged ones. these results pointed toward a coexistence of heavier burden of diseases for those living in areas of the city where misery, poverty, lack of political public health may be modulating social health problems. a possible environmental intervention in one mosquito•bome disease might be playing a role in the occurrence of other. although with limitations, this study may provide useful information for a joint urban planning under the public perspective, articulated for use in health impact assessment. jalil safaei bdrod#aion: the association of socioeconomic status (ses), usually measured by income, and health status is an established result in bcalth studies. the poor and low income individuals have lower health sta· tus then_ those with higher incomes. in general. such finding has been usually obtained from sample surveys on speafic populations. a problem with many sample survey$ is that their results are not comparable aaoss ~ndaries as they may use different sampling methods, ask different questions, categorize the answers differently, or define groups differently. moreover, the sample data need to be standardized using the demographic: katurea of a ~ population. this study, however, uses the national population health survey (nphs) cycle 3 public use mictodata files which is based on a common survey template ·~~three~ area in canada, namely montreal, toronto, and vancouver. it also utili7.es the built-m stanclatdizaaon of the nphs data which accounts for its complex survey design. . ~:the stu~ usc:s two well-known measures of health inequalitythe relative index of meqaality and die concentranon indexto capture the extent of income related health inequality among poster sessions v139 urban female and male populations in each of the three metropolitan areas. personal income is classified into ten groups by the nphs with "no income" as the first group and "more than $60,000" as the last. health status is measured by three variables -having a chronic condition (chc), self assessed health (sah), and health utility index (hui) -using appropriate indices. alternative formulations are used to calculate the standard deviations of the estimated or calculated measures. the findings of the study suggest that the measured health inequality depends on the health measure used. for chc and hui the measured inequality indices do indicate poorer health for lower income individulas, however, they are not statistically insignificant. health inequalities are more pronounced when health is measured by sah. the results do not support any systematic ranking of the three metropolitan areas in terms of health inequalities. they do not reveal any systematic pattern of inequality between men and women, either. conclusions: despite the use of highly aggregated nphs data, income related health disparities are observed in the three metropolitan areas in canada. this is more the case with self perceived (sah) health. such disparities are preventable and call for broader health policies that address poverty and low income among other social determinants of health. introduction: the analysis of health indicators under the spatial perspective is configured as an important instrument in the detection of intra-urban differentials. this study aimed to examine the spatial distribution of the births occurred in belo horizonte, in 2001, analyzing the presence of spatial clusters of health indicators for newborns (rn) and their mothers, using data from the information system on live birth database (sinasc). method: for each covered area of the basic units of health (ubs), we calculated the proportion of: adolescent mothers, mothers with less than 8 years of schooling, first pregnancy, mothers with four or more pregnancies, dead babies born on previous pregnancies, stillbirths, cesarean section, less than four prenatal care attendance, moderate and severe hypoxemia in the 1st and 5th minutes of life, low or very low birth weight. we used empiric bayesian methods for smoothing the estimates. for spatial analysis, the indicators obtained from the global moran (i) index and local indicators of spatial association (lisa) were used. maps were built to allow the visualization of the spatial clusters. in all analysis, significance statistics was considered p~ 0.05. results: a total of 36, 12 7 births of residents in belo horizonte were registered in 200 i. the estimated bayesian proportions for the entire municipality was close to the one observed for all variables. analysis using lisa showed the presence of relevant spatial clusters for adolescent and low educated mothers, dead babies form previous pregnancies, cesarean section, low attendance to the prenatal care especially in area with low socio-demographic characteristics. three areas presented consistent clusters, with important spatial auto-correlation for almost all studied indicators. conclusion: the used methodology was configured as a great instrument of detection risk areas where clustering occurs. it can easily be incorporated in any surveillance system as a mechanism for controlling events related to births in the municipality. moreover, it can be applied to discriminate target areas for prompt public health interventions, such as improving health services access and the consolidation of better obstetric practices. introduction: gentrification, the displacement of low income and non-majority people out of longtime neighborhoods and residences is a global phenomenon. it has been identified as occurring in both developed and less developed countries and as inequality persists or increases, many communities are vulnerable to disruption and loss. while there may be some benefits to gentrification, these benefits are less likely to impact those who have been forced to move out of a gentrifying community or those who remain but economically stressed. . . . . this paper lays out a theoretical framework for 1dent1fymg and ~ddressm~. the_ health consequences of gentrification on residents living in a community prior .to o.r durmg gent~1~1cat.10n. by drawing on the literature of housing, sociology, health and urban plannmg, 1t places genmf1catt~n and neighborhood change into the context of other forces affecting th~ .hea.lth of vulnerable populations. it then proposes solutions to prevent or mitigate the impacts of genmf1cat1on. results: four main categories of consequences potentiallr re~ult from ~entr~fica~~n: !'°°r housing· related issues, spatial effects, mental health impacts and contr1bunons to racial ~spanttes m heal~. the housing issues include increased susceptibility to asthma, lead exposure, allergiesl~topy an~. acetdents/ injuries. spatial effects include reduced access to health ~are, reduce~ access to 1obslttadinonal food sources, decreased physical activity, and increased obesity. the __ pnmary mental health effects_ :ire increased stress increased risk of depression and disruption of trad1t1onal support networks. in addinon to the above he~lth issues, gentrification has the potential to increase racial disparities in health by reduc· ing access to long term and multi-culturally experienced health care provide~s. conclusions: public health practice provides a framework for addressmg the health consequences of gentrification. in this context primary prevention would involve preventing gentrification in the first place along with a renewed commitment to helping distressed communities a~~ im_proving the quality ~f life for long term residents. the next layer of meeting the challenges of gentrification would be to assjst long rime residents to stay in a community and thus potentially allowing them to participate in the bene~ts of gentrification (such as better public services). only if all these efforts fail -and these other prevennon strategies must be a priority, public policy should then work to help people and instirutions move to other communities through grants and the provision of alternative safe affordable housing and neighborhoods. sarah romans, marsha cohen, and tonia forte lnh'odlletion: there has been sustained interest in urban rural (ur) differences in mental health over the last 50 years of epidemiological research, with most studies reporting higher urban rates of mor· bidity, particularly when psychotic disorders have been studied. most recently, in britain, urban rates of non-psychotic disorder were greater than rural and semi-rural rates, both before and after the more adverse circumstances of urban dwellers were considered (paykel et al 2003) . surprisingly, there were no ur differences in help seeking. in canada, wang (2004) found greater urban rates for major depression only after he controlled for the confounding effects of race, immigration, employment and marital status, a result reminiscent of work from the us (blazer et 1985) . rural participants were less likely to have sought help for their mental health problems. in this study, we sought to examine urban/rural differences in rates of depression and help seeking in canada. method; data came from the 2000 canadian community health survey 1.2, a community survey conducted by statistics canada of people aged 15 and older, with the total sample size of 36,984 respon· dents, 77% response. analyses used weighted data; differences were assessed by chi-squared. ra.its: bivariate cross-tabulations showed a modest increase in 12 month depression rates for urban (5.4%) over rural (4.3%, p= 0.03) dwellers; there were no ur differences when the sample was examined separately by gender and age group ( 15-29, 30-44, 45-69) . in general, more urban (10.6%) than rural people (8. 7%, p= 0.002) had sought mental health treatment in the past year. there was no ur difference in helping seeking amongst those with depression (u 58.9%, r 50.1 % ns). amongst the whole population, helping seeking was gendered with more urban men accessing help (7.4%) than rural men (5.4%, p=0.004); this did not apply for women (13.7% vs 12.1% p=0.1). ~ons: the urban-rural demographic continues to generate intriguing findings, even recently when internal migration is common and people can seek out the environment most conducive to their health, ~th physical and mental. people with depression are a disadvantaged, frequently stigmatized group, with ~r quality of life a_nd often impaired function. unlike many other factors associated wi~h urban or rural hfe per se, depression can be treated. the low help seeking rates is a cause for concern, m both the ur~n and_ rural populations. it behooves researchers, policy makers and service planners to ensutt effective services are available for both humane and economic reasons. methods: using united states 2000 census income data, we assigned the 12 manhattan community districts to two major socioeconomic groups, manhattan i and ii. manhattan i is composed of community districts with average household incomes above the median for new york city; manhattan ii is those below. with public new york city department of health 1999 death records and 2000 us census data, we calculated a "standard" new york city population and estimated mortality rate distributions for manhattan i and ii. we then compared these age-adjusted actual mortality distributions with a standard t-test. results: we explored premature deaths using several cut-off points (before the ages of 65, 70, and 75), and with subcomparisons for males and females. every comparison showed the wealthier area, manhattan i, to have significantly lower premature mortality rates than the lower income manhattan ii. further, we compared age-adjusted mortality rates among the city's five boroughs, and found no significant differences based on local geography alone. conclusions: these findings suggest that in manhattan, characterized by its extremes of wealth and poverty, health status, as measured by premature mortality, does vary significantly with the local income level. in manhattan, the relative average income by community district varies as much as 4: 1. this ratio is greater than that found in the other cities we have studied: for example, the range among tokyo's kus is half that in manhattan, or about 2: 1. paris has shown the longest life expectancy and changing premature mortality rates. from our preliminary work, we expect to find less variation in premature mortality rates in comparable cities. this study will continue to explore the relationship of premature mortality rates and life expectancy to income levels and different health systems among wcp cities with a view to measuring health policy options. introduction: according to the world health organization (who), smoking-related illness is currently the world's second major cause of death, currently responsible for one out of ten adult deaths worldwide. the who's framework convention on tobacco control (fctc) aims to reduce tobaccorelated disease and deaths by changing national public policies. mexico, which, according to the who, had smoking prevalences of 32% among adults in 1998 and 23% among health care providers (hcps) in 1997, ratified the fctc in 2004. objective: to examine knowledge of and attitudes towards cigarette smoking and smoking cessation among hcps, and clinical practices regarding smoking cessation. methods: in june 2005, a convenience sample of hcps from one public clinic in urban oaxaca, mexico, part of mexico's national network of public health care clinics, were interviewed in spanish using a verbally administered questionnaire that was standard among all participants. during primary care outpatient visits, hcps were observed treating patients to assess the relative importance of smoking cessation as a health care priority. results: of the 18 hcps interviewed, including ten physicians, three nurses, and five medical students, 67% reported smoking regularly. all hcps reported assessing patients' history of smoking, knowledge of the health risks associated with smoking, and feeling qualified to discuss these risks with patients. all hcps reported counseling patients who currently smoked to quit. all hcps reported recommending nicotine replacement therapy (eg., patch and/or gum) and/or behavioral therapy (eg., psychologist, support groups) to patients who smoked. all hcps reported that there was no government funding for smoking cessation, and that all costs associated with smoking cessation were patients' out-of-pocket expenses. observation of hcp interaction with patients revealed that hcps always inquired about smoking history with new patients and rarely inquired about smoking history with returning patients. hcps were not observed counseling patients who reported current smoking on smoking cessation methods and its benefits. observation of the clinical environment suggested a focus on preventative child health (eg., immunization, water-borne illnesses), family planning, and chronic diseases (eg., diabetes, hypertension). conclusions: while the mexican government has made smoking cessation a public health priority and hcps report addressing smoking cessation, observation of hcps suggests: 1) smoking status is assessed only in new patients; and 2) smoking cessation methods are not addresse~. observ_atio~ of hcps and the clinical environment in a public clinic in oaxaca suggests that smoking cessation 1s of lower priority than other heath care issues. p6-04 (a) urban agriculture and food and nutrition security in kampala, uganda fiona yeudall, renee sebastian, abdelrahman lubowa, selahadin ibrahim, and donald cole introduction: urban agriculture is an important livelihood strategy contributing to household food security by increasing access and availability to food in urban settings (koc et al., 1999) . the purpose of poster sessions v142 rhis study was to examine relationships between child nutritional securiry outcomes, household food security, and urban agriculture activiries in kampala, uga~da. . . questionnaires assessing socio-demographic, farmmg and household food secunry (hfsi characreristics were administered to 270 households. food diversiry was ~lculate~ from ~e number of foods consumed over 24 hours for one child (2-5 years) per household. heigh~ weight,. nud-upper·ann· circumference (muac) and tricep skinfolds (tsf) were measured for the mdex child. z-sc.ores for height-for·age (haz), weighr-for-age (waz) and body mass index (zbmi) were ~~ulated usmg cen· rres for disease control and prevenrion reference data. z-scores for body composition measures were calculated using nhanes i and ii data for african americans. the lms method was used to c~.t for skewed z·score indices. all dara was checked for normaliry and univariable and backward mulnvanablc linear regression analysis conducted. ruwlts: household food securiry was significantly associated with wealth (b= 0. 71, p< 'a acre were more dependenr on assets for hfs. although sex of head of household was ~ot related to j-:ifs, it modi· fied relationships in rhar female headed households had greater food securtry when fai:mmg less than 1,4 acre compared ro male headed households, and vice versa. hfs was significantly associated with food diversiry (s=0.15, p). urbanization, especially in developing countries, has substantially increased the vulnerability of rhe mass of low-income urban dwellers. the livelihoods and quality of life for many of the poor espe· cially in larin america, asia, and africa, have deteriorated significantly over the years. urban areas are increasingly unable to provide for their populations, resulting in poverry, massive unemploymenr, job cuts, poor housing, lack of or poor public services, and a compromised health status. botswana, a country rhat lies in the sourhern parr of africa, has had its share of urban problems such as rhe mush· rooming of squarters and low-income urban sertlements. despite efforts to address the substandard living conditions in low-income urban areas, these problems have continued to grow. these living con· diuons are porenrially stressful to rhe residenrs and likely affects their health. the primary aim of the 11udy wa1 to examine the complex relationship between communiry-level stressors and interveners and health-related quality of life among residents of low-income neighborhoods in francistown, botswana. u1i"" a croa1-icctional quantitative design (both descriptive and explanatory) and using primarily cloae-mded interview• with a random sample of 388 residents, this study examined the role of chrome life 1trnson and environmental quality on overall health status qualiry of life) and the physical, psy· chological and level of independence domains of health. the major hypothesis of the study was that community-levrl stre1sor1 would influence health-related quality of life and that social capital would moderate these relationships. findings indicate that neighborhood quality is a powerful predictor of healrh 1tatu1 rhan socioeconomic status and individual life stressors. social capital was also found to he a 11111ificant positive predictor of healrh and also moderator of structural factors. social capital moderated the effects of low environmental quality on level of indrpendence and on physical health outcomn, but nor on psychological and global health outcomes. these findings suggest that as the environment get1 betttr. stresson are reduced, hence promoting berter health outcomes. the study ends with implication• for social justice, public health and social work practice, and research, focusing mostly on the ~ole of social capital and the environmental quality in predicting health outcomes. spt· cafically • anenhon should be focused on political and civic society's commitment for social justice and poveny alleviation, ~uction of the threat of insecuriry and violence; cultivating social capital and good governance; and improving the health and social environments, especially housing and environ· mental 1aiutanon to name a few. urban and other uprisings by non-elites that lead to transitions, can disrupt sexual and injection "risk" networks that transmit infectious diseases by changing mixing patterns. they can also weaken urban social networks, their associated protective norms and their informal social control mechanisms which can lead to increased sexual and drug risk behaviors and violence (fullilove 2004; wallace & wallace1998, aral 2002 , friedman & reid 2000 hankins et al 2002) . for example, the 1979 revolution and transition to theocratic rule, and subsequent urban and national conflicts, have been followed by many urban youth in iran engaging in clandestine high-risk sexual and drug behaviors. in palestine, conflicts and restrictions on movement have led to increased fatalities from chronic diseases due to limited access to hospitals and modern medical facilities (union of palestinian medical relief committees); and heroin use and violence-related blood exposure have also increased. social movements "from below" that are rooted both in urban social network dynamics and in underlying patterns of injustice can have both protective and risky effects. for example, the social movements that led to the collapse of the ussr and its dependent governments in other countries-with subsequent increases in sex trade, alcoholism, drug use, tuberculosis, hiv, stis, and mortality in many localities-were based originally on such city-based movements in eastern europe. their impacts on health were mediated by urban social dynamics and structures. some urban social movements have improved health by prevent· ing the spread of hiv, hepatitis and other diseases. examples include movements of (a) gays and lesbians and (b) drug users. these have been rooted in social networks that overlap with risk networks. theories of urban health should include social conflict as a core concept. mechanisms that generate conflicts and the pathways by which conflicts affect health should be a major part of urban health research agendas. p6-07 (c) demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital (lasuth) chest clinic john bako, adewale akeredolu, and wale alabi tuberculosis has become a resurgent public health problem in recent times in the world. because resources are limited, control programmes frequently must target population at greatest risk. the purpose of this study was to study the demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital. a total of 76 patients who have been both radiologically and bacteriologically confirmed tuberculosis patients were used for this study between january and march. 57 (75%) were males, while 19 (25%) were females. notable risk factors among patients with tuberculosis were overcrowding (46.1 % ), homelessness ( 11.8%) cigarette smoking (22.4% ), alcoholism (30.3%), non vaccination (25%), secondary contact (36.8%) and poor knowledge (92. l 'x,). man· agement history patterns among the patients were herbs ( 13.2 'yo), orthodox medicine ( .h . .l'x.). intervention targeting early-identified groups may be an effective way to reduce the incidence of tuber· culosis. such intervention should focus on health education, modification of life style and improvement of standard of living. p6-08 (c) profiles in urban health in 9 cities of the americas in the countries of the americas, there is an increasing migration of national and international populations to urban centers. this presentation will focus on some of the issues created by this influx of populations, the ways that 9 cities in 8 countries are dealing with them, and the efforts to promote local participation and solutions in management and decision-making. the methodology used to collect this information has been to draw upon and analyze information produced by the mayor's and ministry of health and development offices in each of the cities under consideration. an analysis of the impact of urbanization on health and health determinants will be presented. the information covers issues such as health status by geographic areas within the cities, highlighting issues such as marginality, barriers and physical, economic and cultural constructs and inequities in the delivery of and access to essential services (such as health, education, water, and basic sanitation). issues of governance and citizen participation will be highlighted to provide insight in~o the balance of power and mechanisms for decision-making at the local level. part of the analysis will show the relationship between democratic processes and social participation. public ~olicies will be reviewed to indicate those that are most beneficial in promoting health and overcoming barriers to it, as well as ways of capitalizing on local assets and resources. final~y, evidence of effectiveness of various strategies will be indicated. the presentation will conclude wuh suggesuons for future strategic directions to improve the quality of life and the promotion of health in large urban centers of the americas. introduction: much of the illicit drug in mexico is concentrated in northern border areas. the 2000 mile border between the u.s. and mexico is also characterized by migration; the border crossing between tijuana, baja california, mexico and san diego, california, u.s.a. is rep~rt~dl~ the busiest land border crossing in the world. we attempt to describe the migration scene among m1ect1on drug users (idus) m tijuana and investigate service needs. methods: migration trends were investigated in a cross-sectional study conducted from february to june 2005 among idus in tijuana. enrollment criteria included informed consent, being 18 years or older, and having injected drugs within the prior month. subjects were recruited by respondent-dnven sampling and were administered a quantitative survey and serology for hiv, hcv, and syphilis. logistic regression was used to compare idus who had migrated to the u.s. versus those who had not. results: of 222 idus enrolled, 91 % were male and median age and age at first injection were 35 and 20, respectively. drug combinations injected most frequently in the past 6 months were heroin (35%1 and crystal methamphetamine mixed with heroin (53%). of those enrolled, 212 responded to quesnons on migration and drug treatment and were included in this analysis. although 76% had resided in tijuana for at least 5 years, 70% of users were born outside of baja california. working outside of mexico was common, with 38% working abroad in the last 10 years (94% in u.s.), and 17% in the past year. in the last 6 months, 10% of idus had crossed the border to the u.s., with 57% crossing at least once per month. those working outside of mexico in the past year were less likely to have ever received substance abuse treatment, [29% vs. 53%, or 0.38, ] and were marginally less likely to have received drug treatment in the past 6 months [6% vs. 18 %, or 0.28, 95% ci (0.06-1.2)]. drug use patterns, age and gender did not differ between migrants and non-migrants, although migrants were more likely to report being in need of substance abuse treatment (68% vs. 57%, respectively). conclusions: migration is common among idus in tijuana. maintaining drug treatment regimens and determining how to best target education and services to a highly mobile population pose challenges to officials on both sides of the border. these data underscore the need to develop coordinated binational prevention and treatment efforts. introduction: despite the expanding literature on the important role public policy plays in influencing the broader determinants of the public\'s health, profound differences exist among jurisdictions in rhe attention placed upon such activities. we take an international perspective on health by examining rhe d?minant public _health paradigms of canada, usa, uk, and sweden and exploring how these paradigms shape public health practice. _method: we carefully analyze governmental and public health agencies documents to discern ~he dommant para~igms driving public health approaches and practices. we also consider the unique paht1· cal and economic contexts within each nation and consider how these contexts drive public health pahcy and approaches. . . ~u~: the canadian and usa public health communities -with some exceptions --focus upon m~ividuahzed approaches to risk management. in contrast, the uk and swedish public health scenes are oriented toward broader approaches to health determinants. we find that the extent to which govern· ments, public health agencies and public health workers concern themselves with public policy approaches £<_> ~ddress ~ro.ad~r .determinants of health depends upon the particular health parad'.~ adhered. ~o withm each 1urisd1ctton. and whether a paradigm is adopted depends upon the ideologi~a and pol~ncal context of each nation. nations such as sweden that have a long tradition of public policies promonng social jus~ce an~ equity are naturally receptive to evolving population health concepts. '[he usa represen~ a ~bey en~ro~~t where such is~ues are clear!~ subordinate. ., our findings mdicate that there 1s a strong political component that influences pubh ~ealth a~proaches and practi~ within the jurisdictions examined. the implications are that those seek· m~ to raise the broader detennmants of the public's health should work in coalition to raise these issues with non-health organizations and age · ca d d th · badrgrollnd: in developed countries, social inequalities in health have endured or even worsened comparatively throughout different social groups since the 1990s. in france, a country where access to medical and surgical care is theoretically affordable for everyone, health inequalities are among the high· est in western europe. in developing countries, health and access to care have remained critical issues. in madagascar, poverty has even increased in recent years, since the country wenr through political crisis and structural adjustment policies. objectives. we aimed to estimate and compare the impact of socio· economic status but also psychosocial characteristics (social integration, health beliefs, expectations and representation, and psychological characteristics) on the risk of having forgone healthcare in these 2 dif· fercnt contexts. methods: population surveys conducted among random samples of households in some under· served paris neighbourhoods (n= 889) and in the whole antananarivo city (n= 2807) in 2003, using a common individual questionnaire in french and malagasy. reslllts: as expected, the impact of socioeconomic status is stronger in antananarivo than in paris. but, after making adjustments for numerous individual socio-economic and health characteristics, we observed in both cities a higher (and statically significant) occurrence of reponed forgone healthcare among people who have experienced childhood and/or adulthood difficulties (with relative risks up to 2 and 3.s respectively in paris and antananarivo) and who complained about unhealthy living conditions. in paris, it is also correlated with a lack of trust in health services. coneluions: aside from purely financial hurdles, other individual factors play a role in the non-use of healthcare services. health insurance or free healthcare seems to be necessary hut not sufficienr to achieve an equitable access to care. therefore, health policies must not only focus on the reduction of the financial barriers to healthcare, but also must be supplemented by programmes (e.g. outreach care ser· vices, health education, health promotion programmes) and discretionary local policies tailored to the needs of those with poor health concern .. acknowledgments. this project was supported by the mal>io project and the national institute of statistics (instat) in madagascar, and hy the development research institute (ird) and the avenir programme of the national institute of health and medical research (inserm) in france. for the cities of developing countries, poverty is often described in terms of the living standard~ of slum populations, and there is good reason to believe that the health risks facing these populations are even greater, in some instances, than those facing rural villagers. yet much remains to be learned ahour the connections between urban poverty and health. it is not known what percentage of all urban poor live in slums, that is, in communities of concentrated poverty; neither is it known what proportion of slum residents are, in fact, poor. funhermore, no quantitative accounting is yet available that would sep· arare the health risks of slum life into those due to a househoid•s own poverty and those stemminic from poveny in the surrounding neighborhood. if urban health interventions are to be effectively targeted in developing countries, substantial progress must be made in addressing these cenrral issues. this paper examines poverty and children's health and survival using two large surveys, one a demographic and health survey fielded in urban egypt (with an oversampling of slums) and the other a survey of the slums of allahabad, india. using multivariate statistical methods. we find, in both settings: ( 11 substan· rial evidence of living standards heterogeneity within the slums; (21 strong evidence indicating that household-level poverty is an imponant influence on health; and (3) staristically significant (though less strong) evidence that with household living standards held constant, neighborhood levels of poverty adversely affect health. the paper doses with a discussion of the implications of these findings for the targeting of health and poverty program interventions. p6-13 (a) urban environment and the changing epidemiological surfacr. the cardiovascular ~ &om dorin, nigeria the emergence of cardiovascular diseases had been explained through the concomitants o_f the demographic transition wherein the prevalent causes of morbidity and monality ~hangr pr~mmant infectious diseases to diseases of lifestyle or chronic disease (see deck, 1979) . a ma1or frustration m the v146 poster sessions case of cvd is its multifactural nature. it is acknowledged that the environment, however defined is the d · f · t' b tween agents and hosts such that chronic disease pathogenesis also reqmre a me 1an o mterac ion e . spatio-temporal coincidence of these two parties. what is not clear is which among ~ever~( potennal fac· · h b pace exacerbate cvd risk more· and to what extent does the ep1dem1olog1cal trans1· tors m t e ur an s ' . . . . tion h othesis relevant in the explanation of urban disease outlook even the developmg cities like nigeri~: thesis paper explorer these within a traditional city in nigeria. . . . the data for the study were obtained from two tertiary level hospitals m the metropolis for 10 years (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) . the data contain reported cases of cvd in the two facilities for the period. adopting a series of parametric and non-parametric statistics, we draw inferences between the observed cases of cvds and various demographic and locational variables of the patients. findings: about 28% of rhe cases occurred in 3 years (1997) (1998) (1999) coinciding with the last year of military rule with great instability. 55.3% occurred among male. 78.8% also occurred among people aged 31-70 years. these are groups who are also likely to engage in most stressful life patterns. ~e study also shows that 63% of all cases occurred in the frontier wards with minor city areas also havmg their •fair' share. our result conformed with many empirical observation on the elusive nature of causation of cvd. this multifactoral nature had precluded the production of a map of hypertension that would be consistent with ideas of spatial prediction. cvd -cardiovascular diseases. mumbai is the commercial capital of india. as the hub of a rapidly transiting economy, mumbai provides an interesting case study into the health of urban populations in a developing country. with high-rise multimillion-dollar construction projects and crowded slums next to each other, mumbai presents a con· trast in development. there are a host of hi-tech hospitals which provide high quality care to the many who can afford it (including many westerners eager to jump the queue in their healthcare systems-'medical tour· ism'), at the same time there is a overcrowded and strained public healthcare system for those who cannot afford to pay. voluntary organizations are engaged in service provision as well as advocacy. the paper will outline role of the voluntary sector in the context of the development of the healthcare system in mumbai. mumbai has distinct upper, middle and lower economic classes, and the health needs and problems of all three have similarities and differences. these will be showcased, and the response of the healthcare system to these will be documented. a rising hiv prevalence rate, among the highest in india, is a challenge to the mumbai public healthcare system. the role of the voluntary sector in service provision, advocacy, and empowerment of local populations with regards to urban health has been paramount. the emergence of the voluntary sector as a major player in the puzzle of urban mumbai health, and it being visualized as voices of civil society or communiry representatives has advantages as well as pitfalls. this paper will be a unique attempt at examining urban health in india as a complex web of players. the influence of everyday socio·polirical-cultural and economic reality of the urban mumbai population will be a cross cutting theme in the analysis. the paper will thus help in filling a critical void in this context. the paper will thus map out issues of social justice, gender, equiry, effect of environment, through the lens of the role of the voluntary sector to construct a quilt of the realiry of healthcare in mumbai. the successes and failures of a long tradi· tion of the active advocacy and participation of the voluntary sector in trying to achieve social justice in the urban mumbai community will be analyzed. this will help in a better understanding of global urban health, and m how the voluntary sector/ngos fir into the larger picture. ba~und: o~er. half _of n~irobi's 2.5 million inhabitants live in illegal informal settlements that compose 5 yo of the city s res1dent1al land area. the majority of slum residents lack access to proper san· iranon and a clean and adequate water supply. this research was designed to gain a clearer understand· mg of what kappr · · · h f . . opnate samtanon means or the urban poor, to determine the linkages between gender, hvehhoods, and access to water and sanitation, and to assess the ability of community sanitation blocks to meet water and sanitation needs in urban areas. m~tbojs_: _a household survey, gender specific focus groups and key informant interviews were conducted m maih saba, a peri-urban informal settlement. qualitative and quantitative research tools were u~ to asses~ the impact and effectiveness of community sanitation blocks in two informal settlements. results ropna e samtarmn me u es not only safe and clean latrines, but also provision ° adequate drainage and access to water supply for cleaning of clothes and homes. safety and cleanliness poster sessions v147 were priorities for women in latrines. levels of poverty within the informal settlements were identified and access to water and sanitation services improved with increased income. environmental health problems related to inadequate water and sanitation remain a problem for all residents. community sanitation blocks have improved the overall local environment and usage is far greater than envisioned in the design phase. women and children use the blocks less than men. this is a result of financial, social, and safety constraints. the results highlight the importance a need to expand participatory approaches for the design of water and sanitation interventions for the urban poor. plans need to recognize "appropriate sanitation" goes beyond provision of latrines and gender and socioeconomic differences must be taken into account. lessons and resources from pilot projects must be learned from, shared and leveraged so that solutions can be scaled up. underlying all the challenges facing improving water and sanitation for the urban poor are issues of land tenure. p6-16 (c) integrating tqm (total quality management), good governance and social mobilization principles in health promotion leadership training programmes for new urban settings in 12 countries/ areas: the prolead experience susan mercado, faren abdelaziz, and dorjursen bayarsaikhan introduction: globalization and urbanization have resulted in "new urban settings" characterized by a radical process of change with positive and negative effects, increased inequities, greater environmental impacts, expanding metropolitan areas and fast-growing slums and vulnerable populations. the key role of municipal health governance in mitigating and modulating these processes cannot be overemphasized. new and more effective ways of working with a wide variety of stakeholders is an underpinning theme for good governance in new urban settings. in relation to this, organizing and sustaining infrastructure and financing to promote health in cities through better governance is of paramount importance. there is a wealth of information on how health promotion can be enhanced in cities. despite this, appropriate capacity building programmes to enable municipal players to effectively respond to the challenges and impacts on health of globalization, urbanization and increasing inequity in new urban settings are deficient. the who kobe centre, (funded by the kobe group( and in collaboration with 3 regional offices (emro, searo, wpro) with initial support from the japan voluntary contribution, developed a health promotion leadership training programme called "prolead" that focuses on new and autonomous structures and sustainable financing for health promotion in the context of new urban settings. methodology: country and/or city-level teams from 12 areas, (china, fiji, india, japan, lebanon, malaysia, mongolia, oman, philippines, republic of korea, tonga and viet nam) worked on projects to advance health promotion infrastructure and financing in their areas over a 9 month period. tools were provided to integrate principles of total quality management, good governance and social mobili1.ation. results: six countries/areas have commenced projects on earmarking of tobacco and alcohol taxes for health, moblization of sports and arts organizations, integration of health promotion and social health insurance, organizational reforms, training in advocacy and lobbying, private sector and corporate mobilization and community mobilization. results from the other six areas will be reported in 01..;obcr. conclusions: total quality management, good governance and social mobilization principles and skills are useful and relevant for helping municipal teams focus on strategic interventions to address complex and overwhelming determinants of health at the municipal level. the prolead training programmes hopes to inform other processes for building health promotion leadership capacity for new urban settings. the impact of city living and urbanization on the health of citizens in developing countries has received increasing attention in recent years. urban areas contribute largely to national economies. however, rapid and unplanned urban growth is often associated with poverty, environmental degradation and population demands that outstrip service capacity which conditions place human health at risk. local and national governments as well as multi national organizations are all grappling with the challenges of urbanization. with limited data and information available, urban health characteristics, including the types, quantities, locations and sources in kampala, are largely unknown. moreover, there is n? basis for assessing the impact of the resultant initiatives to improve health ~onditions amo~g ~o":1":1um ties settled in unplanned areas. since urban areas are more than the aggregation ?f ~?pie w~th md_1v1dual risk factors and health care needs, this paper argues that factors beyond the md1v1dual, mcludmg the poster sessions v148 · i d h · i · ment and systems of health and social services are determinants of the health soc1a an p ys1ca environ . of urban populations. however, as part of an ongoing study? ~s pape~ .addresses the basic concerns of urban health in kampala city. while applying the "urban hvmg conditions and the urban heal~ pen· alty" frameworks, this paper use aggregated urban health d~ta t~ explore the role of place an~ 111st1tu· tions in shaping health and well-being of the population m kampala by understanding how characteristics of the urban environment and specific features of the city are causally related to health of invisible and forgotten urban poor population: results i~dica~e that a .range o~ urb~n he~l~h hazards m the city of kampala include substandard housing, crowdmg, mdoor air poll.ut1on, msuff1c1ent a~d con· taminated water, inadequate sanitation and solid waste management services, vector borne .diseases, industrial waste increased motor vehicle traffic among others. the impact of these on the envtronment and community.health are mutually reinforcing. arising out of the withdra"'.al of city pl~nning systems and service delivery systems or just planning failure, thousands of people part1cularl~ low-mc~me groups have been pushed to the most undesirable sections of the city where they are faced with ~ va_r1ety ~f envj· ronmental insults. the number of initiatives to improve urban health is, however, growing mvolvjng the interaction of many sectors (health, environment, housing, energy, transportation and urban planning) and stakeholders (local government, non governmental organizations, aid donors and local community groups). key words: urban health governance, health risks, kampala. introduction: the viability of urban communities is dependent upon reliable and affordable mass transit. in particular, subway systems play an especially important role in the mass transit network, since they provide service to vast numbers of ridersseven of the 95 subway systems worldwide report over one billion passenger rides each year. surprisingly, given the large number of people potentially affected, very little is known about the health and safety hazards that could affect both passengers and transit workers; these include physical (e.g., noise, vibration, accidents, electrified sources, temperature extremes), biological (e.g., transmission of infectious diseases, either through person-to-person spread or vector-borne, for example, through rodents), chemical (e.g., exposure to toxic and irritant chemicals and metals, gas emissions, fumes), electro-magnetic radiation, and psychosocial (e.g., violence, workstress). more recently, we need to consider the threat of terrorism, which could take the form of a mass casualty event (e.g., resulting from conventional incendiary devices), radiological attack (e.g., "dirty bomb"), chemical terrorist attack (e.g., sarin gas), or bioterrorist attack (e.g., weapons grade anthrax). given the large number of riders and workers potentially at risk, the public health implications are considerable. methods: to assess the hazards associated with subways, a structured review of the (english) litera· ture was conducted. ruults: based on our review, non-violent crime, followed by accidents, and violent crimes are most prevalent. compared to all other forms of mass transit, subways present greater health and safety risks. however, the rate of subway associated fatalities is much lower than the fatality rate associated with automobile travel (0.15 vs. 0.87 per 100 million passenger miles), and cities with high subway ridership rates have a 36% lower per capita rate of transportation related fatalities than low ridership cities (7.5 versus 11.7 annual deaths per 100,000 residents). available data also suggest that subway noise levels and levels of air pollutants may exceed recommended levels. . ~: there is a paucity of published research examining the health and safety hazards associated with subways. most of the available data came from government agencies, who rely on passively reported data. research is warranted on this topic for a number of reasons, not only to address important knowled~ gaps, but also because the population at potential risk is large. importantly, from an urban perspecnve, the benefits of mass transit are optimized by high ridership ratesand these could be adversely affu:ted by unsafe conditions and health and safety concerns. veena joshi, jeremy lim. and benjamin chua ~ ~rban health issues have moved beyond infectious diseases and now centre largely on chrome diseases. diabetes is one of the most prevalent non-communicable diseases globally. 9 % of adult ¥151 benefit in providing splash pads in more parks. given the high temperature and humidity of london summers, this is an important aspect and asset of parks. interviewed parents claimed to visit city parks anywhere between 1 to 6 days per week. corrduion: given that the vast majority of canadian children are insufficiently active to gain health benefits, identifying effective qualities of local parks, that may support and foster physical activity is essential. strategies to promote activity within children's environments are an important health initiative. the results from this study have implications for city planners and policy makers; parents' opinions of, and use of city parks provides feedback as to the state current local parks, and modifications that should be made for new ones being developed. this study may also provide important feedback for health promoters trying to advocate for physical activity among children. introdt1clion: a rapidly increasing proportion of urban dwellers in africa live below the poverty line in overcrowded slums characterized by uncollected garbage, unsafe water, and deficient sanitation and overflowing sewers. this growth of urban poverty challenges the commonly held assumption that urban populations enjoy better health than their rural counterparts. the objectives of this study are (i) to compare the vaccination status, and morbidity and mortality outcomes among children in the slums of nairobi with rural kenya, and (ii) to examine the factors associated with poor child health in the slums. we use data from demographic and health survey representative of all slum settlements in nairobi city carried out in 2000 by the african population & health research center. a total of 3,256 women aged 15-49 from 4,564 households were interviewed. our sample consists of 1,210 children aged 0-35 months. the comparison data are from the 1998 kenya demographic and health survey. the outcomes of interest include child vaccination status, morbidity (diarrhea, fever and cough) and mortality, all dichotomized. socioeconomic, environmental, demographic, and behavioral factors, as well as child and mother characteristics, are included in the multivariate analyses. multilevel logistic regression models are used. l'nlimin11ry rest1lts: about 32 % of children in the slums had diarrhea in the two weeks prior to the survey, compared to 16% of rural children. these disparities between the urban poor anj the rural residents are also observed for fever (64% against 42%), cough (46% versus 20%), infant mortality (91/ 1000 against 76/1000), and complete vaccination (48% against 64%). preliminary multivariate results indicate that health service utilization and maternal education have the strongest predictive power on child morbidity and mortality in the slums, and that household wealth has only minor, statistically insignificant effects. conclruion: the superiority of health of urban children, compared with their rural counterparts, masks significant disparities within urban areas. compared to rural residents, children of slum dwellers in nairobi are sicker, are less likely to utilize health services when sick, and stand greater risk to die. our results suggest policies and programs contributing to the attainment of the millennium development goal on child health should pay particular attention to the urban poor. the insignificance of socioeconomic status suggests that poor health outcomes in these communities are compounded by poor environmental sanitation and behavioral factors that could partly be improved through female education and behavior change communication. introduction: historic trade city surat with its industrial and political peace has remained a center of attraction for people from all the comers of india resulting in to pop.ulatio~ explosio~ a~d stressed social and service infrastructure. the topography,dimate and demographic profile of the city 1s threat to the healthy environment. aim of this analysis is to review the impact of managemt'nt reform on health indicators. method: this paper is an analysis of the changing profile of population, sanitary infr~s~rucrure, local self government management and public health service reform, secondary health stat1st1cs data, health indicator and process monitoring of 25 years. . . health of entire city and challenge to the management system. plague outbr~ak (1994) was the turning point in the history of civic service management including p~blic ~e~lth service management. ~ocal self government management system was revitalized by reg~lar_ field v1s1ts o~ al~ cadre~, _decentraltzanon of power and responsibility, equity, regular vigilant momtormg, commumcanon facility, ream_approach and people participation. reform in public health service management was throu_gh stan~~rd1zed intervention protocol, innovative intervention, public private partnership, community part1c1panon, academic and service institute collaboration and research. sanitation service coverage have reached nearer to universal. area covered by safe water supply reached to 98%(2004) from 40% (1991) and underground drainage to 97% (2004) from 17% ( 1991) the overhauling of the system have reflected on health indicators of vector and water born disease. malaria spr declined to 1.23 (2004) from 23.06'yo(!991) and diarrhea case report declined to 1963(2004) from 3431 (2004). except dengue fever in 2002 no major disease outbreaks are reported after 1991. city is recipient of international/national awards/ranking for these achievements. the health department have developed an evidence and experience based intervention and monitoring system and protocol for routine as well as disaster situation. the health service and management structure of surat city have emerged as an urban health model for the country. introduction: the center for healthy communities (chc) in the department of family and com· munity medicine at the medical college of wisconsin developed a pilot project to: 1) assess the know· ledge, attitudes, and behaviors of female milwaukee public housing residents related to breast cancer; 21 develop culturally and literacy appropriate education and screening modules; 3) implement the developed modules; 4) evaluate the modules; and 5) provide follow-up services. using a community-based participatory research model the chc worked collaboratively with on-site nurse case management to meet these objectives. methods: a "breast health kick off event" was held at four separate milwaukee public housing sites for elderly and disabled adults. female residents were invited to complete a 21-item breast health survey, designed to accommodate various literacy levels. responses were anonymous and voluntary. the survey asked women about their previous physical exams for breast health, and then presented a series of state· ments about breast cancer to determine any existing myths. the final part gathered information about personal risk for breast cancer, the highest level of education completed, and whether the respondents h;td ever used hormone replacement therapy and/or consumed alcohol. responses were collected for descriptive analysis. results: a total of 45 surveys (representing 18% of the total female population in the four sites) were completed and analyzed. 89% reported that they had a physical exam in the previous rwo years. 96% of respondents indicated they never had been diagnosed with breast cancer. 85% reported having had a mammogram and 87% having had a clinical breast exam. those that never had a mammogram reported a fear of what the provider would discover or there were not any current breast problems ro warrant an exam. 80% agreed that finding breast cancer early could lower the chance of dying of cancer. over 92% reported that mammograms were helpful in finding cancer. however, 27% believed that hav· ing a mammogram actually prevents breast cancer. 14% indicated that mammograms actually cause cancer and 16% reported that a woman should get a mammogram only if there is breast cancer in her family. conclusion: this survey indicates that current information about the importance of mammograms and clinical breast exams is reaching traditionally underserved women. yet there are still critical oppor· tunities to provide valuable education on breast health. this pilot study can serve as a tool for shaping future studies of health education messages for underserved populations. located in a yourh serv· ~ng agency m downtow~ ottawa, the clinic brings together community partners to provide primary medical care. and dent~i hygiene t? the street youths of ottawa aged 12-20. the primary goal of the project is to provide accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. these efforts respond to the pre-existing body of evidence suggesting that the principle barrier in accessing such care for these youths are feelings of intimidation and vulnerability in the face of a complex healthcare system. the bruyere fhn satellite clinic is located in the basement of a downtown drop-in and brings together a family medicine physician and her residents, a dental hygienist and her 2nd year students, a nurse practitioner, a chiropodist and 2 public health nurses to provide primary care. the clinic has been extremely busy and well received by the youth. this workshop will demonstrate how five community organizations have come together to meet the needs of high risk youths in ottawa. this presentation will showcase the development of the clinic from its inception through its first year including reaction of the youths, partnerships and lessons learned. it will also focus on its sustainability without continued funding. we hope to have developed a model of service delivery that could be reproduced and sustained in other large cities with faculties of medicine. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative-a) semi structured interviews with providers & partners b)focus groups with youth quantitative a)electronic medical records for 12 months records (budget, photos, project information). results: 1) successfully built and opened a medicaudental clinic which will celebrate its 1 year anniversary in august. 2) over 140 youths have been seen, and we have had over 300 visits. conclusion: 1) the clinic will continue to operate beyond the 18 month project funding. 2) the health of high risk youth in ottawa will continue to improve due to increased access to medical services. p7-11 (a) health services -for the citizens of bangalore -past, present and future savita sathyagala, girish rao, thandavamurthy shetty, and subhash chandra bangalore city, the capital of karnataka with 6.5 million is the 6th most populous city in india; supporting 30% of the urban population of karnataka, it is considered as one of the fastest growing cities in india. known as the 'silicon valley of india', bangalore is nearly 500 years old. bangalore city corporation (bmp), is a local self government and has the statutory commitment to provide to the citizens of bangalore: good roads, sanitation, street lighting, safe drinking water apart from other social obligations, cultural development and poverty alleviation activities. providing preventive and promotive heahh services is also a specific component. the objective of this study was to review the planning process with respect to health care services in the period since india independence; the specific research questions being what has been the strategies adopted by the city planners to address to the growing needs of the population amidst the background of the different strategies adopted by the country as a whole. three broad rime ranges have been considered for analysis: the 1950s, 1970s and the 1990s. the salient results are: major area of focus has been on the maternal and child care with activities ranging from day-care to in-patient-care; though the number of institutions have grown from 5 to the current day 79, their distribution has been far from satisfactory; obtaining support from the india population projects 3 and 8 major upgradarions have been undertaken in terms of infrastructure; over the years, in addition to the dispensaries of modern system of medicine, local traditional systems have also been initiated; the city has partnered with the healthy cities campaign with mixed success; disease surveillance, addressing the problems related to the emerging non-communicable diseases including mental health and road traffic injuries are still in its infancy. isolated attempts have been made to address the risks groups of elderly care and adolescent care. what stands out remarkably amongst the cities achievements is its ability to elicit participation from ngos, cbos and neighbourhood groups. however, the harnessing of this ability into the health sector cannot be said totally successful. the moot question in all the above observed development are: has the city rationally addressed it planning needs? the progress made so far can be considered as stuttered. the analysis and its presentation would identify the key posirive elements in the growth of banglore city and spell a framework for the new public health. introduction: anaemia associated with pregnancy is a major public health problem all over the world. different studies in different parts of india shown prevalence of anaemia between 60-90%. anaemia remains a serious health problem in pregnancy despite of strong action taken by the government of india through national programmes. in the present study we identified th~ social beha~iors, responsible for low compliance of if a tablets consumption in pregnancy at community level and intervention was given with new modified behaviors on trial bases. . in vadodara urban. 60 anganwadies out of 289 were selected from the list by random sampling for tips (trials of improved practices) study. . . participants: 266 pregnant women (132, intervention group+ 134, control. group) registered m the above 60 anganwadies. study was conducted in to three phases: phase: 1. formative research and baseline survey (frbs). data was collected from all 266 pregnant women to identify behaviors that are responsible for low compliance of ifa tablets. both qualitative and quantitative data were collected. haemoglobin was estimated of all pregnant women by haemo-cue. phase: 2. phase of tips. behaviors were identified both social & clinical for low compliance of ifa tablets consumption in pregnancy from frbs and against those, modified behaviors were proposed to pregnant women in the intervention group on trial bases by health education. trial period of 6 weeks was given for trial of new behaviors to pregnant women in the interven· tion group. phase: 3. in this phase, feedbacks on behaviors tried or not tried were taken from pregnant women in intervention group. haemoglobin estimation was carried out again in all 266 pregnant women. at the end of the study, messages were formulated on the bases of feedbacks from the pregnant women. results: all pregnant women in the intervention group had given positive feedback on new modified behaviors after intervention. mean haemoglobin concentration was higher in intervention group (10.04±0.11 gm%) than control group (9.60±0.14 gm%). ifa tablets compliance was improved in intervention group (95.6%) than control group (78.6%). conclusion: all pregnant women got benefits after trial of new modified behaviors in the intervention group. messages were formulated from the new modified behaviors, which can be used for longterm strategies for anaemia control in the community. introduction: in order to develop a comprehensive mch handbook for pregnant women and to assess its effect among them, a pilot study was carried out at the maternal and child health training institute (mchti), in dhaka, bangladesh. methods: from mchti a sample of 600 pregnant women was selected and all subjects were women who were attending the first visit of their current pregnancy by using a random sampling method. of the 600 subjects, 240 women were given the mch handbook as case and 360 women were not given the handbook as control. data on pre and post intervention of the handbook from the 240 cases and 360 controls were taken from data recording forms between the 1st of november 2002 and 31st of october, 2003 and data was analysed by using a multilevel analysis approach. this was a hospital-based action (case-control) research, and was applied in order to measure the outcome of pre and post intervention following the introduction of the handbook. data was used to assess the effects of utilisation of the handbook on women's knowledge, practice and utilisation of mch services. results: this study showed that the change of knowledge about antenatal care visits was 77.1% among case mothers. knowledge of danger signs improved 49.2 %, breast feeding results 31.5%, vaccination 32.0% and family planning results improved 60.3% among case. results showed some positive changes in women's attitudes among case mothers and study showed the change of practice in antenatal care visits was .u.5% in the case. other notable changes were: change of practice in case mother's tetanus toxoid (ti), 55.2%; and family planning 41.2%. in addition, handbook assessment study indicated that most women brought the handbook on subsequent visits (83.3%), the handbook was highly utilised (i.e. it was read by 84.2%, filled-in by 76.1 %, and was used as a health education tool by 80.4%). most women kept the handbook (99.5%) and found it highly useful (78.0%) with a high client satisfaction rate of 88.0%. conclusion: pregnant women in the case group had higher knowledge, better practices, and higher utilisation of mch services than mothers in the control groups who used alternative health cards. if the handbook is developed with a focus on utilising a problem-oriented approach and involving the recomendations .of end~users, it is anticipated that the mch handbook will contribute significantly to ensuring the quahry of hfe of women and their children in bangladesh. after several meetmgs to identify the needs of the community, a faso clinic was opened at ncfs. health care professionals from smh joined with developmental and social service workers from ncfs to implement the faso diagnostic process and to provide culturally appropriate after-care. the clinic is unique in that its focus is the high risk urban aboriginal population of toronto. it accepts referrals of not only children and youth, but also of adults. lessons learned: response to the faso clinic at native child and family services has been overwhelming. aboriginal children with f asd are receiving timely diagnosis and interventions. aboriginal youth and adults who have been struggling with poveny, substance abuse, and homelessness are more willing to enter the ncfs centre for diagnosis and treatment. aboriginal infants prenatally exposed to alcohol born at st. michael's hospital or referred by other centres have access to the developmental programs located in both of the partnering agencies. the presentation will describe the clinic's development, and will detail the outcomes described, including interventions unique to the aboriginal culture. p7-15 (c) seeds, soil, and stories: an exploration of community gardening in southeast toronto carolin taran, sarah wakefield, jennifer reynolds, and fiona yeudall introduction: community gardens are increasingly seen as a mechanism for improving nutrition and increasing food security in urban neighbourhoods, but the evidence available to support these claims is limited. in order to begin to address this gap in a way that is respectful of community knowledge and needs, the urban gardening research opportunities workgroup (ugrow) project explored the benefits and potential risks of community gardening in southeast toronto. the project used a community-based research (cbr) model to assess community gardens as a means of improving local health. the research process included interviews, focus groups, and participant observation (documented in field notes). we also directly engaged the community in the research process, through co-learning activities and community events which allowed participants to express their views and comment on emerging results. most of the research was conducted by a community-based research associate, herself a community gardener. key results were derived from these various sources through line-by-line coding of interview transcripts and field note review, an interactive and iterative process which involved both academic and community partners. results: these various data sources all suggest that enhanced health and access to fresh produce are important components of the gardening experience. they also highlight the central importance of empowering and community-building aspects of gardening to gardeners. community gardens were thought to play a role in developing friendships and social support, sharing food and other resources, appreciating cultural diversity, learning together, enhancing local place attachment and stewardship, and mobilizing to solve local problems (both inside and outside the garden). potential challenges to community gardens as a mechanism for communiry development include bureaucratic resistance to gardens, insecure land tenure and access, concerns about soil contamination, and a lack of awareness and under· standing by community members and decision-makers of all kinds. conclusion: the results highlight many health and broader social benefits experienced by commu· nity gardeners. they also point to the need for greater support for community gardening programs, par· ticularly ongoing the ongoing provision of resources and education programs to support gardens in their many roles. this research project is supported by the wellesley central health corporation and the centre for urban health initiatives, a cihr funded centre for research development hased at the univer· sity of toronto. p7-16 (c) developing resiliency in children living in disadvantaged neighbourhoods sarah farrell, lorna weigand, and wayne hammond the traditional idea of targeting risk reduction by focusing on the development of eff~ctive coping strategies and educational programs has merit in light of the research reportmg_ that_ ~10lupl.e forms of problem behaviour consistently appear to be predicted by increasing exposure to 1den_uf1able risk factors. as a result, many of the disadvantaged child and youth studies have focused on trymg to better _unde.r· stand the multiple risk factors that increase the likelihood of the development of at nsk behaviour m ch1ldren/youth and the potential implications for prevention. this in turn has led t_o. the conclus1on that community and health programs need to focus on risk reduction by helpm~ md1v1duals develop more effective coping strategies and a better understanding of the limitations of cenam pathologies, problematic v156 poster sessions coping behaviours and risk factors potentially inheren~ in high needs co~unities. ~owever, another ai:ea of research has proposed that preventative interventions should cons1de~ .~rotecnve fa~ors alo~~ with reducing risk factors. as opposed to just emphasizing problems, vulnerab1ht1es, and deficits, a res1liencybased perspective holds the belief that children, youth and their families. have strengths, reso~ce.s and the ability to cope with significant adversity in ways that are not only effective, but tend to result m mcreased ability to constructively respond to future adversity. with this in mind, a participatory research project sponsored by the united way of greater toronto was initiated to evaluate and determine the resiliency profiles of children 8 -12 years (n = 500) of recent immigrant families living in significantly disadvantaged communities in the toronto area. the presentation will provide an overview of the identified protective factors (both intrinsic and extrinsic) and resiliency profiles in an aggregated format as well as a summary of how the children and their parents interpreted and explained these strength-based results. as part of the focus groups, current community programs and services were examined by the participants as to what might be best practices for supporting the development and maintaining of resiliency in children, families and communities. it was proposed that the community model of assessing resiliency and protective factors as well as proposed best strength-based practice could serve as a guide for all in the community sector who provide services and programs to those in disadvantaged neighbourhoods. p7-17 (c) naloxone by prescription in san francisco, ca and new york, ny emalie huriaux the harm reduction coalition's overdose project works to reduce the number of fatal overdoses to zero. located in new york, ny and san francisco, ca, the overdose project provides overdose education for social service providers, single-room occupancy hotel (sro) residents, and syringe exchange participants. the project also conducts an innovative naloxone prescription program, providing naloxone, an opiate antagonist traditionally administered by paramedics to temporarily reverse the effects of opiate overdose, to injection drug users (idus). we will describe how naloxone distribution became a reality in new york and san francisco, how the project works, and our results. the naloxone prescription program utilizes multiple models to reach idus, including sro-and street-based trainings, and office-based trainings at syringe exchange sites. trainings include information on overdose prevention, recognition, and response. a clinician conducts a medical intake with participants and provides them with pre-filled units of naloxone. in new york, funding was initially provided by tides foundation. new york city council provides current funding. new york department of mental health and hygiene provides program oversight. while the new york project was initiated in june 2004, over half the trainings have been since march 2005. in san francisco, california endowment, tides foundation, and san francisco department of public health (sfdph) provide funding. in addition, sfdph purchases naloxone and provides clinicians who conduct medical intakes with participants. trainings have been conducted since november 2003. to date, nearly 1000 individuals have been trained and provided with naloxone. approximately 130 of them have returned for refills and reported that they used naloxone to reverse an opiate-related overdose. limited episodes of adverse effects have been reported, including vomiting, seizure, and "loss of friendship." in new york, 400 individuals have been trained and provided with naloxone. over 30 overdose reversals have been reported. over half of the participants in new york have been trained in the south bronx, the area of new york with the highest rate of overdose fatalities. in san francisco, 570 individuals have been trained and provided with naloxone. over 96 overdose reversals have been reported. the majority of the participants in san francisco have been trained in the tenderloin, 6th street corridor, and mission, areas with the highest rates of overdose fatalities. the experience of the overdose project in both cities indicates that providing idus low-threshold access to naloxone and overdose information is a cost-effective, efficient, and safe intervention to prevent accidental death in this population. p7-18 (c) successful strategies to regulate nuisance liquor stores using community mobilization, law enforcement, city council, merchants and researchers tahra goraya presenta~ion _will discuss ~uccessful environmental and public policy strategies employed in one southen:1 cahf?rmna commumty to remedy problems associated with nuisance liquor stores. participants ~111 be given tools to understand the importance of utilizing various substance abuse prevention str~tegi~ to change local policies and the importance of involving various sectors in the community to a~_1st with and advocate for community-wide policy changes. recent policy successes from the commultles of pa~ad~na and altad~na will highlight the collaborative process by which the community mobilized resulnng m several ordmances, how local law enforcement was given more authority to monitor poster sessions v157 nonconforming liquor stores, how collaborative efforts with liquor store owners helped to remove high alcohol content alcohol products from their establishments and how a community-based organiz,uion worked with local legislators to introduce statewide legislation regarding the regulation of nuisance liquor outlets. p7-19 (c) "dialogue on sex and life": a reliable health promotion tool among street-involved youth beth hayhoe and tracey methven introduction: street involved youth are a marginalized population that participate in extremely risky behaviours and have multiple health issues. unfortunately, because of previous abuses and negative experiences, they also have an extreme distrust of the adults who could help them. in 1999, toronto public health granted funding to a non governmental, nor for profit drop-in centre for street youth aged 16-24, to educate them about how to decrease rhe risk of acquiring hiv. since then the funding has been renewed yearly and the program has evolved as needed in order to target the maximum number of youth and provide them with vital information in a candid and enjoyable atmosphere. methods: using a retrospective analysis of the six years of data gathered from the "dialogue on sex and life" program, the researchers examined the number of youth involved, the kinds of things discussed, and the number of youth trained as peer leaders. also reviewed, was written feedback from the weekly logs, and anecdotal outcomes noted by the facilitators and other staff in the organization. results: over the five year period of this program, many of youth have participated in one hour sessions of candid discussion regarding a wide range of topics including sexual health, drug use, harm reduction, relationship issues, parenting, street culture, safety and life skills. many were new youth who had not participated in the program before and were often new to the street. some of the youth were given specific training regarding facilitation skills, sexual anatomy and physiology, birth control, sexually transmitted infections, hiv, substance use/abuse, harm reduction, relationships and discussion of their next steps/future plans following completion of the training. feedback has been overwhelmingly positive and stories of life changing decisions have been reported. conclusion: clearly, this program is a successful tool to reach street involved youth who may otherwise be wary of adults and their beliefs. based on data from the evaluation, recommendations have been made to public health to expand the funding and the training for peer leaders in order ro target between 100-200 new youth per year, increase the total numbers of youth reached and to increase the level of knowledge among the peer leaders. p7-20 (c) access to identification and services jane kali replacing identification has become increasingly more complex as rhe government identification issuing offices introduce new requirements rhar create significant barriers for homeless people to replace their id. new forms of identification have also been introduced that art' not accessible to homekss peoplt-(e.g. the permanent resident card). ar rhe same time, many service providers continue to require identifi· cation ro access supports such as income, housing, food, health care, employment and employmt·nt training programs. street health, as well as a number of other agencies and community health centres, h,1, been assisting with identification replacement for homeless peoplt· for a number of years. the rnrrt·nr challenges inherent within new replacement requirements, as well as the introduction of new forn1' of identification, have resulted in further barriers homeless people encounter when rrring to access t:ssential services. street health has been highlighting these issues to government identification issuing offices, as well as policy makers, in an effort to ensure rhar people who are homeless and marginalized have ac'ess to needed essential services. bandar is a somali word for •·a safe place." the bandar research project is the product of the regent park community health centre. the research looks ar the increasing number of somali and afri· can men in the homeless and precariously house population in the inner city core of down~own toronto. in the first phase of the pilot project, a needs assessment was conducted to 1dennfy barners and issues faced by rhe somali and other african men who are homeless and have add1cr10ns issues. th_e second phase of rhe research project was to identify long rerm resources and service delivery mechamsms that v158 poster sessions would enhance the abiity of this population to better access detox, treatment, and post treatment ser· vices. the final phase of the project was to facilitate the development of a conceptual model of seamless continual services and supports from the streets to detox to treatment to long term rehabilitation to housing. "between the pestle and mortar" -safe place. p7-22 (c) successful methods for studying transient populations while improving public health beth hayhoe, ruth ewert, eileen mcmahon, and dan jang introduction: street youth are a group that do not regularly access healthcare because of their mis· trust of adults. when they do access health care, it is usually for issues severe enough for hospitalization or for episodic care in community clinics. health promotion and illness prevention is rarely a part of their thinking. thus, standard public health measures implemented in a more stable population do not work in this group. for example, pap tests, which have dearly been shown to decrease prevalence of cer· vical cancer, are rarely done and when they are, rarely followed up. methods to meet the health care needs and increase the health of this population are frequently being sought. methods: a drop-in centre for street youth in canada has participated in several studies investigating sexual health in both men and women. we required the sponsoring agencies to pay the youth for their rime, even though the testing they were undergoing was necessary according to public health stan· dards. we surmised that this would increase both initial participation and return. results: many results requiring intervention have been detected. given the transient nature of this population, return rates have been encouraging so far. conclusion: it seems evident that even a small incentive for this population increases participation in needed health examinations and studies. it is possible that matching the initial and follow-up incentives would increase the return rate even further. the fact that the youth were recruited on site, and not from any external advertising, indicates that studies done where youth trust the staff, are more likely to be successful. the presentation will share the results of the "empowering stroke prevention project" which incor· porated self-help mutual aids strategies as a health promotion methodology. the presentation will include project's theoretical basis, methodology, outcomes and evaluation results. self-help methodology has proven successful in consumer involvement and behaviour modification in "at risk," "marginalized" settings. self-help is a process of learning with and from each other which provides participants oppor· tunities for support in dealing with a problem, issue, condition or need. self-help groups are mechanisms for the participants to investigate existing solutions and discover alternatives, empowering themselves in this process. learning dynamic in self-help groups is similar to that of cooperative learning and peertraining, has proven successful, effective and efficient (haller et al, 2000) . the mutual support provided by participation in these groups is documented as contributory factor in the improved health of those involved. cognizant of the above theoretical basis, in 2004 the self-help resource centre initiated the "empowering stroke prevention project." the project was implemented after the input from 32 health organizations, a scan of more than 300 resources and an in-depth analysis of 52 risk-factor-specific stroke prevention materials indicated the need for such a program. the project objectives were:• to develop a holistic and empowering health promotion model for stroke prevention that incorporates selfhelp and peer support strategies. • to develop educational materials that place modifiable risk factors and lifestyle information in a relevant context that validates project participants' life experiences and perspectives.• to educate members of at-risk communities about the modifiable risk factors associated with stroke, and promote healthy living. to achieve the above, a diverse group of community members were engaged as "co-editors" in the development of stroke prevention education materials which reflected and validated their life experiences. these community members received training to become lay health promoters (trained volunteer peer facilitators). in collaboration with local health organizations, these trained lay health promoters were then supported in organizing their own community-based stroke prevention activities. in addition, an educational booklet written in plain language, entitled healthy ways to prevent stroke: a guide for you, and a companion guide called healthy ways to pre· vent stroke: a facilitator's guide were produced. the presentation will include the results of a tw<>tiered evaluation of the program methodology, educational materials and the use of the materials beyond the life of the project. this poster presentation will focus on the development and structure of an innovative street outreach service that assists individuals who struggle mental illness/addictions and are experiencing homelessness. the mental health/outreach team at public health and community services (phcs) of hamilton, ontario assists individuals in reconnecting with health and social services. each worker brings to the ream his or her own skills-set, rendering it extremely effective at addressing the multidimensional and complex needs of clients. using a capacity building framework, each ream member is employed under a service contract between public health and community services and a local grassroots agency. there are public health nurses (phn), two of whom run a street health centre and one of canada's oldest and most successful needle exchange programs, mental health workers, housing specialists, a harm reduction worker, youth workers, and a united church minister, to name a few. a community advisory board, composed of consumers and professionals, advises the program quarterly. the program is featured on raising the roors 'shared learnings on homelessness' website at www.sharedlearnings.ca. through our poster presentation participants will learn how to create effective partnerships between government and grassroots agencies using a capacity building model that builds on existing programs. this study aims to assess the effects of broadcasting a series of documentary and drama videos, intended to provide information about the bc healthguide program in farsi, on the awareness about and the patterns of the service usage among farsi-speaking communities in the greater vancouver area. the major goals of the present study were twofold; ( 1) to compare two methods of communications (direct vs. indirect messages) on the attitudes and perceptions of the viewers regarding the credibility of messengers and the relevance of the information provided in the videos, and (2) to compare and contrast the impact of providing health information (i.e., the produced videos) via local tvs with the same materials when presented in group sessions (using vcr) on participants' attitudes and perceptions cowards the bc healrhguide services. results: through a telephone survey, 545 farsi-speaking adults were interviewed in november and december 2004. the preliminary findings show that 53% of the participants had seen the aired videos, from which, 51 % watched at least one of the 'drama' clips, 8% watched only 'documentary' clip, and 41% watched both types of video. in addition, 27% of the respondents claimed that they were aware about the program before watching the aired videos, while 73% said they leaned about the services only after watching the videos. from this group, 14% said they called the bchg for their own or their "hildren's health problems in the past month. 86% also indicated that they would use the services in the future whenever it would be needed. 48% considered the videos as "very good" and thought they rnuld deliver relevant messages and 21 % expressed their wish to increase the variety of subjects (produ\:e more videos) and increase the frequency of video dips. conclusion: the results of this study will assist public health specialists in bc who want to choose the best medium for disseminating information and apply communication interventions in multi\:ultural communities. introduction: many theorists and practitioners in community-based research (cbr) and knowledge transfer (kt) strongly advocate for involvement of potential users of research in the development of research projects, yet few examples of such involvement exist for urban workplace health interventions. we describe the process of developing a collaborative research program. methods: four different sets of stakeholders were identified as potential contributors to and users of the research: workplace health policy makers, employers, trade unions, and health and safety associations. representatives of these stakeholders formed an advisory committee which met quarterly. over the 13 month research development period, an additional 21 meetings were held between resc:ar~h~rs and stakeholders. in keeping with participant observation approaches, field notes of group and md1v1~ ual meetings were kept by the two co-authors. emails and telephone calls were also documented. qu~h tative approaches to textual analysis were used, with particular attention paid to collaborattve v160 poster sessions relationships established (as per cbr), indicators of stakeholders' knowledge utilization (as per kt), and transformations of the proposed research (as per cbr). results: despite initial strong differences of opinion both among stakeho~ders .an~ between stakeholders and researchers, goodwill was noted among all involved. acts of rec~proc1ty included mu.rual sharing of assessment tools, guidance on data utilization to stakeho~der orga~1zat10ns, and suggestions on workplace recruitment to researchers. stakeholders demonstrated mcreases m concep~ual. un~erstand ing of workplace health e.g. they more commonly discussed more complex,. psychosocial md1cators of organizational health. stakeholders made instrumental use of shared materials based on research e.g. adapting their consulting model to more sophisticated dat~ analysis. sta~ehol?~rs recogni_zed the strategic use of their alliance with researchers e.g., transformational leadership trainmg as a~ inducement to improve health and safety among small service franchises. stakeholders helped re-define the research questions, dramatically changed the method of recruitment from researcher cold call to stakeholderbased recruitment, and strongly influenced pilot research designs. owing a great deal to the elaborate joint development process, the four collaboratively developed pilot project submissions which were all successfully funded. conclusion: the intensive process of collaborative development of a research program among stakeholders and researchers was not a smooth process and was time consuming. nevertheless, the result of the collaborative process was a set of projects that were more responsive to stakeholder needs, more feasible for implementation, and more broadly applicable to relevant workplace health problems. introduction: environmental groups, municipal public health authorities and, increasingly, the general public are advocating for reductions in pesticide use in urban areas, primarily because of concern around potential adverse health impacts in vulnerable populations. however, limited evidence of the relative merits of different intervention strategies in different contexts exists. in a pilot research project, we sought to explore the options for evaluating pesticide reduction interventions across ontario municipalities. methods: the project team and a multi-stakeholder project advisory committee (pac), generated a list of potential key informants (kl) and an open ended interview guide. thirteen ki from municipal government, industry, health care, and environmental organizations completed face to face or telephone interviews lasting 30-40 minutes. in a parallel process, a workshop involving similar representatives and health researchers was held to discuss the role of pesticide exposure monitoring. minutes from pac meetings, field notes taken during ki interviews, and workshop proceedings were synthesized to generate potential evaluation methods and indicators. results: current evaluation activities were limited but all kls supported greater evaluation effons beginning with fuller indicator monitoring. indicators of education and outreach services were imponant for industry representatives changing applicator practices as well as most public health units and environmental organizations. lndictors based on bylaw enforcement were only applicable in the two cities with bylaws, though changing attitudes toward legal approaches were being assessed in many communities. the public health rapid risk factor surveillance system could use historical baseline data to assess changes in community behaviour through reported pesticide uses and practices, though it had limited penetration in immigrant communities not comfortable in english. pesticide sales (economic) data were only available in regional aggregates not useful for city specific change documentation. testing for watercourse or environmental contamination might be helpful, but it is sporadic and expensive. human exposure monitoring was fraught with ethical issues, floor effects from low levels of exposure, and prohibitive costs. clinical episodes of pesticide exposure reported to the regional poison centre (all ages) or the mother risk program (pregnant or breastfeeding women) are likely substantial underestimates that would be need to be supplemented with sentinel practice surveillance. focus on special clinical populations e.g., multiple chemical sensitivity would require additional data collection efforts . . conc~ons: broad support for evaluation and multiple indicators were proposed, though con-s~raints associate~ with access, coverage, sensitivity and feasibility were all raised, demonstrating the difficulty of evaluating such urban primary prevention initiatives. interventionists. an important aim of the youth monitor is to learn more about the health development of children and adolescents and the factors that can influence this development. special attention is paid to emo· tional and behavioural problems. the youth monitor identifies high-risk groups and factors that are associated with health problems. at various stages, the youth monitor chancrs the course of life of a child. the sources of informa· tion and methods of research are different for each age group. the results arc used to generate various kinds of repons: for children and young persons, parents, schools, neighbourhoods, boroughs and the municipality of rotterdam and its environs. any problems can be spotted early, at borough and neigh· bourhood level, based on the type of school or among the young persons and children themselves. together with schools, parents, youngsters and various organisations in the area, the municipal health service aims to really address these problems. on request, an overview is offered of potentially suitable interventions. the authors will present the philosophy, working method, preliminary effects and future developments of this instrument, which serves as the backbone for the rotterdam local youth policy. social workers to be leaders in response to aging urban populations: the practicum partnership program sarah sisco, alissa yarkony, and patricia volland 1"'"1tliu:tion: across the us, 77.5% of those over 65 live in urban areas. these aging urban popu· lations, including the baby boomers, have already begun encounter a range of heahh and mental hcahh conditions. to compound these effects, health and social service delivery fluciuates in cities, whit:h arc increasingly diverse both in their recipients and their systems. common to other disciplines (medicine, nursing, psychology, etc.) the social work profession faces a shortage of workers who are well-equipped to navigate the many systems, services, and requisite care that this vast population requires. in the next two decades, it is projected that nearly 70,000 social workers will be required to provide suppon to our older urban populations. social workers must be prepared to be aging-savvy leaders in their field, whether they specialize in gerontology or work across the life span. mllhotu: in 2000, a study conducted at the new york academy of medicine d<>1:umcntcd the need for improved synchroniciry in two aspects of social work education, classroom instruction and the field experience. with suppon from the john a. hanford foundation, our team created a pilot proj~"t entitled the practicum pannership program (ppp) in 11 master's level schools of social work, to improvt" aginr exposure in field and classroom content through use of the following: i) community-university partnrr· ships, 2) increased, diverse student field rotations, ll infusion of competcn1."}'·drivm coursework, 41 enhancement of field instructors' roles, and 5) concentrated student recruitment. we conductt"d a prr· and post-test survey into students' knowledge, skills. and satisfaction. icarlja: surveys of over 400 graduates and field inltnk."tors rcflected increased numlk-n of .1rrm:y· univmity panncrships, as well as in students placed in aging agencin for field placements. there wa1 11 marked increase in student commitments to an aging specialization. onr year por.t·gradu:nion rcvealrd that 93% of those surveyed were gainfully employed, with 80% employed in the field of aginic. by com· bining curricular enhancement with real-world experiences the ppp instilled a broad exposurr for llu· dents who worked with aging populations in multiple urban settings. coltdtuion: increased exposure to a range of levels of practicr, including clinical, policy/ajvocaq, and community-based can potentially improve service delivery for older adulh who live in elfin, and potentially improve national policy. the hanford foundation has now elected to 1uppon cxpantion of the ppp to 60 schools nationwide (urban and rural) to complement other domntic initiatives to cnhalk"c" holistic services for older adults across the aging spectrum. bodrgnn.ntl: we arc a team of rcscarcbcn and community panncn working tcj8c(her to develop an in"itepth understanding of the mental health needs of homeless youth ~ages 16 to 24) (using qualiutivc and quantitative methods 8' panicipatory rncarch methods). it is readily apparmt that '-neless youth cxpcricnce a range of mental health problems. for youth living on the street, menul illnew may be either a major risk factor for homelessnal or may frequently emcsge in response to coping with rhe multitudinous stressors associated with homclcslllcsi including exposure to violence, prasutt to pamaplte in v162 poster sessions survival sex and/or drug use. the most frequent psychiatric diagnoses amongst the homeless gencrally include: depression, anxiety and psychosis. . . . the ultimate ob1ective of the pr~am of rei:e~ is to ~evelop a plan for intervention to meet the mental health needs of street youth. prior t_o pl~nnmg mtervenbons, .it is necessary to undertake a comprehensive assessment ~f mental health needs m this ~lnerable populanon. thus, the immediate objective of this research study is to undertake a comprehensive assessment of men· tal health needs. . . melbotlology: a mixed methodology triangulating qualitative, participatory acnon and quantitative methods will capture the data related to mental health needs of homeless youth. a purposive sample of approximately 60-80 subjecrs. ages 16 to 24, is currently being ~ted ~participate from the commu.nity agencies covenant house, evergreen centre fo~ srrc;et youth, turning p?1?t and street ~ serv~. youth living on the street or in short -term residennal programs for a mmimum of 1 month pnor to their participation; ages 16 to 24 and able to give infonned consent will be invited to participate in the study. o..tcomes: the expected outcome of this initial survey will be an increased understanding of mental health needs of street youth that will be used to develop effective interventions. it is anticipated that results from this study will contribute to the development of mental health policy, as well as future programs that are relevant to the mental health needs of street youth. note: it is anticipated that preliminary quantitative data (25 subjects) and qualitative data will be available for the conference. the authors intend to present the identification of the research focus, the formation of our community-based team, relevance for policy, as well as preliminary results. p7-31 (a) the need for developing a firm health policy for urban informal worken: the case of despite their critical role in producing food for urban in kenya, urban farmers have largely been ignored by government planners and policymakers. their activity is at best dismissed as peripheral eveo, inappropriate retention of peasant culture in cities and at worst illegal and often some-times criminal· ized. urban agriculture is also condemned for its presumed negative health impact. a myth that contin· ues despite proof to the contrary is that malarial mosquitoes breed in maize grown in east african towns. however, potential health risks are insignificant compared with the benefits of urban food production. recent studies too rightly do point to the commercial value of food produced in the urban area while underscoring the importance of urban farming as a survival strategy among the urban poor, especially women-headed households. since the millennium declaration, health has emerged as one of the most serious casualties consequent on the poverty, social exclusion, marginalisation and lack of sustain· able development in africa. hiv/aids epidemic poses an unprecedented challenge, while malaria, tuber· culosis, communicable diseases of childhood all add to the untenable burden. malnutrition underpins much ill-health and is linked to more than 50 per cent of all childhood deaths. kenya's urban poor people ~ace ~ h~ge burde~ of preventable and treatable health problems, measured by any social and bi~ medical md1cator, which not only cause unnecessary death and suffering, but also undermine econonuc development and damage the country's social fabric. the burden is in spite of the availability of suitable tools and re:c=hnology for prevention and treatment and is largely rooted in poverty and in weak healah •rstems. this pa~ therefore challenges development planners who perceive a dichotomy instead of con· tmuum between informal and formal urban wage earners in so far as access to health services is con· cemed. it i~ this gap that calls for a need to developing and building sustainable health systems among the urban mformal ~wellers. we recommend a focus on an urban health policy that can build and strengthen the capacity of urban dwellers to access health services that is cost-effective and sustainable. such ~ health poli<=>: must strive for equity for the urban poor, displaced or marginalized; mobilise and effect1~ely use sufficient sustainable resources in order to build secure health systems and services. special anenti_on. should ~ afforded hiv/aids in view of the unprecedented challenge that this epidemic poses to africa s economic and social development and to health services on the continent. methods: a review of the literature led us to construct three simple models and a composite model of exposure to traffic. the data were collected with the help of a daily diary of travel activities using a sample of cyclists who went to or come back from work or study. to calculate the distance, the length of journey, and the number of intersections crossed by a cyclist different geographic information systems (gis) were operated. statistical analysis was used to determine the significance between a measure of exposure on the one hand, and the sociodemographic characteristics of the panicipants or their geographic location on the other hand. restlltj: our results indicate that cyclists were significantly exposed to road accidents, no matter of where they live or what are their sociodemographic characteristics. we also stress the point that the fact of having been involved in a road accident was significantly related to the helmet use, but did not reduce the propensity of the cyclists to expose themselves to the road hazards. condlllion: the efforts of the various authorities as regards road safety should not be directed towards the reduction of the exposure of the vulnerable users, but rather towards the reduction of the dangers to which they could face. keywords: cyclist, daily diary of activities, measures of exposure to traffic, island of montreal. p7-33 (a) intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods olumuyiwa akinbamijo intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods abstract urbanization panicularly in nigerian cities, ponends unprecedented crises of grave dimensions. from physical and demographic viewpoints, city growth rates are staggering coupled with gross inabilities to cope with the consequences. environmental and social ills associated with unguarded rapid urbanization characterize nigerian cities and threaten urban existence. this paper repons the findings of a recent study of the relationship between environmental health across inrraurban residential communities of akure, south west nigeria. it discuses the typical urbanization process of nigerian cities and its dynamic spatial-temporal characteristics. physical and socio-demographic attributes as well as the levels and effectiveness of urban infrastructural services are examined across the core residential districts and the elite residential layouts in the town. the incidence rate of cenain environmentally induced tropical diseases across residential neighborhoods and communes is examined. salient environmental variables that are germane to health procurement in the residential districts, incidence of diseases and diseases parasitology, diseases prevention and control were studied. field data were subjected to analysis ranging from the univariate and bivariate analysis. inferential statistics using the chi-square test were done to establish the truthfulness of the guiding hypothesis. given the above, the study affirms that there is strong independence in the studied communities, between the environment and incidence of diseases hence health of residents of the town. this assertion, tested statistically at the district levels revealed that residents of the core districts have very strong independence between the environment and incidences of diseases. the strength of this relationship however thins out towards the city peripheral districts. the study therefore concludes that since most of the city dwellers live in urban deprivation, urban health sensitive policies must be evolved. this is to cater for the urban dwellers who occupy fringe peripheral sites where the extension of facilities often times are illegally done. urban infrastructural facilities and services need be provided as a matter of public good for which there is no exclusive consumption or access even for the poorest of the urban poor. many suffer from low-self esteem, shame and guilt about their drug use. in addition, they often lack suppon or encounter opposition from their panners, family and friends in seeking treatment. these personal barriers are compounded by fragmented addiction, prenatal and social care services, inflexible intake systems and poor communication among sectors. the experience of accessing adequate care between services can be overwhelming and too demanding. the toronto centre for substance use in pregnancy (t-cup) is a unique program developed to minimize barriers by providing kone-stop" comprehensive healthcare. t-cup is a primary care based program located in the department of family medicine at st. joseph\'s health centre, a community teaching hospital in toronto. the interdisciplinary staff provides prenatal and addiction services, case management, as well as care of newborns affected by substance use. regular care plan meetings are held between t-cup, labour and delivery nurses and social workers in the y164 poster sessions maternity and child care program. t-cup also connects "'.omen with. inpatient treatment programs and community agencies such as breaking the cycle, an on-site counselmg group for pregnant substance users. · f · d d h ith method: retrospective chart review, qualitative patient ~ans action stu ~· an ea care provider surveys are used to determine outcomes. primary outcomes mclude changes m maternal su~tance use, psychosocial status and obstetrical complications (e.g. pre-rupture of membrane, pre-eclampsia, placen· ral abruption and hemorrhage). neonatal measures ~~nsisted of .bir~h pa_rame~ers, length of h~spital st.ay and complications (e.g. feral distress, meconium stammg, resuscitation, 1aund1ce, hypoglycemia, seventy of withdrawal and treatment length). chart review consisted of all t-cup patients who met clinical cri· reria for alcohol or drug dependence and received prenatal and intra-partum care at st. joseph's from october 2003 to june 2005. participants in the qualitative study included former and current t-cup patients. provider surveys were distributed on-site and to a local community hospital. raulb: preliminary evaluation has demonstrated positive results. treatment retention and satisfaction rates were high, maternal substance use was markedly reduced and neonatal outcomes have shown to be above those reported in literature. conclusion: this comprehensive, primary care model has shown to be optimal in the management of substance use in pregnancy and for improving neonatal outcomes. future research will focus on how this inexpensive program can be replicated in other health care settings. t-cup may prove to be the optimal model for providing care to pregnant substance users in canada. lntrod11ction: cigarette smoking is one of the most serious health problems in taiwan. the prevalence of smoking in 2002 is 48.1 % in males 5.9% in females aged 18 years and older. although the government of taiwan passed a tobacco hazards control act in 1997, it has not been strongly enforced in many places. therefore, community residents have often reported exposure of second hand smoke. the purpose of the study was to establish a device to build up more smoke-free environments in the city of tainan. methods: unique from traditional intervention studies, the study used a healthy city approach to help build up smoke-free environments. the major concept of the approach is to build up a healthy city platform, including organizing a steering committee, setting up policies and indicators, creating intersectoral collaboration, and increasing community participation. first, more than 80 enthusiastic researchers, experts, governmental officers, city counselors and community leaders in tainan were invited in the healthy city committee. second, smoke-free policies, indicators for smoke-free environments, and mechanisms for inter-departmen· tal inspections were set up. third, community volunteers were recruited and trained for persuading related stakeholders. lastly, both penalties and rewards were used for help build up the environments. raults: aher two-year (2003 aher two-year ( -2005 execution of the project, the results qualitatively showed that smoke-free environments in tainan were widely accepted and established, including smoke-free schools, smoke-free workpla~es, smoke-free households, smoke-free internet shops, and smoke-free restaurants. smoke~s were. effectively educated not to smoke in public places. community residents including adults and children m the smoke-free communities clearly understand the adverse effects of environmental tobacco smoke and actively participated anti-smoking activities. conclruions: healthy city platform is effective to conquer the barrier of limited anti-smoking rc:sources. nor. only can it enlar:ge community actions for anti-smoking campaigns, but also it can provide par_merships for collaboratjon. by establishing related policies and indicators the effects of smoke· free environments can be susta1·ned a d th · · · ' · n e progression can be monitored m a commuruty. these issues are used ~· oi::c it~ goals, weuha identifies issues that put people's health at risk. presently, team com~u:c: ran ee~tion !earns. (iats) that design integrative solutions ~tesj'°~ g om six to fifteen members. methods in order to establish wo-poster sessions v165 projects for weuha, the following approach was undertaken: i. a project-polling template was created and sent to all members of the alliance for their input. each member was asked to identify thdr top two population groups, and to suggest a project on which to focus over a 12-18 month period for each identified population. 2. there was a 47% response to the poll and the top three population groups were identified. data from the toronto community health profile database were utilized to contextualize the information supplied for these populations. a presentation was made to the steering committee and three population-based projects were selected, leaders identified and iats formed. three population-based projects: the population-based projects and health care issues identified are: newcomer prenatal uninsured women; this project will address the challenges faced by providers to a growing number of non-insured prenatal women seeking care. a service model where the barrier of "catchments" is removed to allow enhanced access and improved and co-ordinated service delivery will be pilot-tested. children/obesity/diab etes: using a health promotion model this team will focus on screening, intervention, and promoting healthy lifestyles (physical activity and nutrition) for families as well as for overweight and obese children. seniors health promotion and circle of discharge: this team will develop an early intervention model to assist seniors/family unit/caregivers in accessing information and receiving treatment/care in the community. the circle of discharge initiative will address ways of utilizing community supports to keep seniors in the community and minimize readmissions to acute care facilities. results/expected outcomes: coordinated and enhanced service delivery to identified populations, leading to improved access, improved quality of life, and health care for these targeted populations. introduction: basic human rights are often denied to high-risk populations and people living with hiv/aids. their rights to work and social security, health, privacy, non discrimination, liberty and freedom of movement, marriage and having a family have been compromised due to their sero-positive status and risk of being positive. the spread of hiv/aids has been accelerating due to the lack of general human rights among vulnerable groups. to formulate and implement effective responses needs dialogue and to prevent the epidemic to go underground barriers like stigma need to be overcome. objective: how to reduce the situation of stigma, discrimination and human rights violations experienced by people living with hiv/aids and those who are vulnerable to hiv/aids. methodology and findings: consultation meetings were strm.-rured around presentations, field visits, community meetings and group work to formulate recommendations on how govt and ngos/cbos should move forward based on objective. pakistan being a low prevalence country, the whole sense of compl;u:enc.:y that individuals are not subject to situations of vulnerable to hiv is the major threat to an explosion in th•· epidemic, therefore urgent measures are needed to integrate human rights issues from the very start of the response. the protection and promotion of human rights in an integral component of ;tll responses to the hiv/aids epidemic. it has been recognized that the response to hiv/aios must he multi sectoral and multi faceted, with each group contributing its particular expertise. for this to occur along with other knowlcdg<" more information is required in human rights abuses related to hiv/ aids in a particular scenario. the ~·on sultarion meetings on hiv/aids and human rights were an exemplary effort to achieve the same ohj<..:tivc. recommendations: the need for a comprehensive, integrated and a multi-sectoral appro;u.:h in addressing the issue of hiv/aids was highlighted. the need social, cultural and religious asp•·ct' to he: prominently addressed were identified. it was thought imperative measures even in low prevalence countries. education has a key role to play, there is a need for a code of ethics for media people and h<"alth care providers and violations should be closely monitored and follow up action taken. p7-38 (c) how can community-based funding programs contribute to building community capacity and how can we measure this elusive goal? mary frances maclellan-wright, brenda cantin, mary jane buchanan, and tammy simpson community capacity building is recognized by the public health agency of canada (phac) as an important strategy for improving the overall health of communities by enabling communities to addre~s priority issues such as social and economic determinants of health. in 2004/2005 phac.:, alberta/nwf region's population health fund (phf) supported 12 community-based projects to build community capacity on or across the determinants of health. specifically, this included creating accessible and sup· portive social and physical environments as well as creating tools and processes necessary for healthy policy development and implementation. the objective of this presentation is to highlight how the community capacity building tool, developed by phac ab/nwf region, can demonstrate gains in v166 poster sessions · · the course of a pror· ect and be used as a reflective tool for project planning and community capacity over . . . . i · a art of their reporting requirements, 12 pro1ect sites completed the community caparny eva uanon. s p . . th t i ii i'd d . building tool at the beginning and end of their ~ne-year prorect. e oo ~o ects va 1 an reliable data in the context of community-based health prorects. developed through a vigorous ~nd collabora11ve research process, the tool uses plain languag~ to expl~re nine key f~atures o~ commuruty cap~city with 35 't ch with a section for contextual information, 26 of which also mdude a four-pomt raong 1 ems, ea f fu d · scale. results show an increase in community capacity over the course o the nde prorects. pre and post aggregate data from the one-year projects measure~ statistic.ally si~n~ficant changes for 17 of the 26 scaled items. projects identified key areas of commumty capacity bmldmg that needed strengthemng, such as increasing participation, particularly among people with low incomes; engaging community members in identifying root causes; and linking with community groups. in completing the tool, projects examined root causes of the social and economic determinants of health, thereby exploring social justice issues related to the health of their community. results of the tool also served as a reflec· cion on the process of community capacity building; that is, how the project outcomes were achieved. projects also reported that the tool helped identify gaps and future directions, and was useful as a project planning, needs assessment and evaluation tool. community capacity building is a strategy that can be measured. the community capacity building tool provides a practical means to demonstrate gains in community capacity building. strengthening the elements of community capacity building through community-based funding can serve as building blocks for addressing other community issues. needs of marginalized crack users lorraine barnaby, victoria okazawa, barb panter, alan simpson, and bo yee thom background: the safer crack use coalition of toronto (scuc) was formed in 2000 in response to the growing concern for the health and well-being of marginalized crack users. a central concern was the alarm· ing hepatitis c rate ( 40%) amongst crack smokers and the lack of connection to prevention and health ser· vices. scuc is an innovative grassroots coalition comprised of front-line workers, crack users, researcher! and advocates. despite opposition and without funding, scuc has grown into the largest crack specific harm reduction coalition in canada and developed a nationally recognized sarer crack kit distribution program (involving 16 community-based agencies that provide outreach to users). the success of our coalition derives from our dedication to the issue and from the involvement of those directly affected by crack use. setting: scuc's primary service region is greater toronto, a diverse, large urban centre. much ofour work is done in areas where homeless people, sex trade workers and drug users tend to congregate. recently, scuc has reached out to regional and national stakeholders to provide leadership and education. mandate: our mandate is to advocate for marginalized crack users and support the devdopmentof a com.p.rehensive harm reduction model that addresses the health and social needs facing crack users; and to fac1htare the exchange of information between crack users, service providers, researchers, and policy developers across canada. owrview: the proposed workshop will provide participants with an overview of the devdopment of scuc, our current projects (including research, education, direct intervention and consultation), our challenge~ and s~ccesses and the role of community development and advocacy within the coalition. pre-senter~ will consist of community members who have personal crack use experience and front-line work· ers-, sc.uc conducted a community-based research project (toronto crack users perspectives, 2005) , in w~ich 1 s focus groups with marginalized crack users across toronto were conducted. participants iden· t1f1ed health and social issues affecti h b · · · d " red . . ng t em, arrsers to needed services, personal strategies, an oue recommendations for improved services. presenters will share the methodology, results and recommen· datmns resulting from the research project. conc/usio": research, field observations and consultations with stakeholders have shown that cradck shmoke~s are at an. increased risk for sexually transmitted infections hiv/aids hepatitis c, tb an ot er serious health issues health · ff, · ' ' · · . · issues a ectmg crack users are due to high risk behavmurs, socio· economic factors, such as homeless d. · · · · d · 1 . 1 . ness, 1scrsmmat1on, unemployment, violence incarceraoons, an soc1a 1so at1on, and a lack of comprehe · h i h · ' ns1ve ea t and social services targeting crack users. · · sinct · s, owever arge remains a gross underesurnaoon. poster sessions v167 these are hospital-based reports and many known cases go unreported. however teh case, young age at first intercourse, inconsistent condom use and multiple partnersplace adolescents at high risks for a diverse array of stls, including hiv. about 19% of female nigerian secondary school students report initiating sexual intercourse before age 13 years. 39% of nigerian female secondary school students report not using a condom the last time they had sexual intercourse. more than 60% of urban nigerian teens report inconsistent condom use. methods: 371 adolescents were studied, ages 12 to 19, from benin city in edo state. the models used were mother-daughter(119), mother -son(99), father -son (87), and father-daughter(66). the effect of parent-child sexual communicationat baseline on child\'s report of sexual behavior, 6 to 12 months later were studied. greater amounts of sexual risk communication were asociated with markedly fewer episodes of unprotected sexual intercourse, reduced number of sexual partners and fewer episodes of unprotected sexual intercourse. results: this study proved that parents can exert more influence on the sexual knowledge attitudes and practise of their adolescent children through desired practises or rolemodeling, reiterating their values and appropriate monitoring of the adolescents\' behavior. they also stand to provide information about sexuality and various sexual topics. parental-child sexual communication has been found to be particularly influential and has been associated with later onset of sexual initiation among adolescents, less sexual activity, more responsible sexual attitudes including greater condom use, self efficacy and lower self -reported incidence of stis. conclusions: parents need to be trained to relate more effectively with their children/wards about issues related to sex and sexuality. family -based programs to reduce sexual risk-taking need to be developed. there is also the need to carry out cross-ethnicaland cross-cultural studies to identify how parent-child influences on adolescent sexual risk behavior may vary in different regions or countries, especially inthis era of the hiv pandemic. introduction: public health interventions to identify and eliminate health disparities require evidence-based policy and adequate model specification, which includes individuals within a socioecological context, and requires the integration of biosociomedical information. multiple public and private data sources need to be linked to apportion variation in health disparities ro individual risk factors, the health delivery system, and the geosocial environment. multilevel mapping of health disparities furthers the development of evidence-based interventions through the growth of the public health information network (phin-cdc) by linking clinical and population health data. clinical encounter data, administrative hospital data, population socioenvironmental data, and local health policy were examined in a three-level geocoded multilevel model to establish a tracking system for health disparities. nj has a long established political tradition of "home rule" based in 566 elected municipal governments, which are responsible for the well-being of their populations. municipalities are contained within counties as defined by the us census, and health data are linked mostly at the municipality level. marika schwandt community organizers from the ontario coaliti~n again~t pove~, .along ":ith ~edical practitioners who have endorsed the campaign and have been mvolved m prescnbmg special diet needs for ow and odsp recipients, will discuss the raise the rates campaign. the organizati~n has used a special diet needs supplement as a political tool, meeting the urgent needs o.f .poor ~ople m toront~ while raising the issues of poverty as a primary determinant of health and nutrtnous diet as a preventative health mea· sure. health professionals carry the responsibility to ensure that they use all means available to them to improve the health of the individuals that they serve, and to prevent future disease and health conditions. most health practitioners know that those on social assistance are not able to afford nutritious foods or even sufficient amounts of food, but many are not aware of the extra dietary funds that are available aher consideration by a health practitioner. responsible nurse practitioners and physicians cannot, in good conscience, ignore the special needs diet supplement that is available to all recipients of welfare and disabiliry (ow and odsp). a number of toronto physicians have taken the position that all clients can justifiably benefit from vitamins, organic foods and high fiber diets as a preventative health measure. we know that income is one of the greatest predictors of poor health. the special needs diet is a health promotion intervention which will prevent numerous future health conditions, including chronic conditions such as cardiovascular disease, cancer, diabetes and osteoporosis. many communiry health centres and other providers have chosen to hold clinics to allow many patients to get signed up for the supplement at one time. initiated by the ontario coalition against poverty, these clinics have brought together commu· niry organizers, community health centers, health practitioners, and individuals, who believe that poverty is the primary determinant of poor health. we believe that rates must be increased to address the health problems of all people on social assistance, kids, elders, people with hiv/aids -everyone. even in the context of understaffing, it could be considered a priority activity that has potentially important health promotion benefits. many clients can be processed in a two hour clinic. most providers find it a very interesting, rewarding undertaking. in 2004 the ontario coalition for social justice found that a toronto family with two adults and two kids receives $14,316. this is $21,115 below the poverty line. p7-43 (c) the health of street youth compared to similar aged youth beth hayhoe and ruth ewert . lntrod~on: street youth are at an age normally associated with good health, but due to their risky ~hav1ours and th~ conditions in which they live, they experience health conditions unlike their peer~ an more stable env1r~nments. in addition, the majority of street youth have experienced significant physical, sexual ~nd em.ot1onal abuse as younger children, directly impacting many of the choices they make around their physical and emotional health. we examined how different their health really is. . , methodl: using a retrospective analysis of the 11 years of data gathered from yonge street mis· 510~ 5 • evergreen health centre, the top 10 conditions of youth were examined and compared with national tren~s for similar aged youth. based on knowledge of the risk factors present in the group, rea· sons for the difference were examined. d' ~its: street youth experience more illness than other youth their age and their illnesses can bt . irect t ·~kc~ to the. conditions in which they live. long-term impacts of abus~ contribute to such signif· ~~nt t e t 0 d~slpl air that youth may voluntarily engage in behaviours or lack of self care in the hope at t cir 1ve~ w1 perhaps come to a quicker end. concl11non: although it has ion b k h th' dy clearly shows 3 d'fi . h g ee~ no~n t at poverty negatively affects health, ~siu be used to make ; erence m t .e health of this particular marginalized population. the infonnanon can relates to th . ecommendatio.ns around public policy that affects children and youth, especially as it e1r access to appropriate health care and follow up. p7-44 (cl why do urban children · b gt . tarek hussain 10 an adesh die: how to save our children? the traditional belief that urban child alid. a recent study (dhs d fr 17 r~n are better off than rural children might be no longer v urban migrants are highata th om h c~untn~s i demonstrates that the child survival prospects of rural· er an t ose m their r j · · ·grants. in bangladesh, currently 30 million 0 ~r~ 0~1gm and lower than those of urban non-idi million. health of the urban 1 ~ p~e are hvmg m urban area and by the year 2025, it would be so the popu at1on 1s a key a eals that urban poor have the worse h 1 h . concern. recent study on the urban poor rev ea t situation than the nation as a whole. this study shows that infant poster sessions v169 mortality among the urban poor as 120 per thousand, which are above the rural and national level estimates. the mortality levels of the dhaka poor are well above those of the rest of the city's population but much of the difference in death rates is explained by the experience of children, especially infants. analyzing demographic surveillance data from a large zone of the city containing all sectors of the population, research showed that the one-fifth of the households with the least possessions exhibited u5 child mortality almost three times as high as that recorded by the rest of the population. why children die in bangladesh? because their parents are too poor to provide them with enough food, clean water and other basic needs to help them avoid infection and recover from illness. researchers believed that girls are more at risk than boys, as mothers regularly feed boys first. this reflects the different value placed on girls and boys, as well as resources which may not stretch far enough to provide for everyone. many studies show that housing conditions such as household construction materials and access to safe drinking water and hygienic toilet facilities are the most critical determinants of child survival in urban areas of developing countries. the present situation stressed on the need for renewed emphasis on maternal and child healthcare and child nutrition programs. mapping path for progress to save our children would need be done strategically. we have the policies on hand, we have the means, to change the world so that every child will survive and has the opportunity to develop himself fully as a healthy human being. we need the political will--courage and determination to make that a reality. p7-45 (c) sherbourne health centre: innovation in healthcare for the transgendered community james read introduction: sherbourne health centre (shc), a primary health care centre located in downtown toronto, was established to address health service gaps in the local community. its mission is to reduce barriers to health by working with the people of its diverse urban communities to promote wellness and provide innovative primary health services. in addition to the local communities there are three populations of focus: the lesbian, gay, bisexual, transgendered and transexual communities (lgbtt); people who are homeless or underhoused; and newcomers to canada. shc is dedicated to providing health services in an interdisciplinary manner and its health providers include nurses, a nurse practitioner, mental health counsellors, health promoters, client-resource workers, and physicians. in january 2003 shc began offering medical care. among the challenges faced was how to provide responsive, respectful services to the trans community. providers had considerable expertise in the area of counselling and community work, but little in the area of hormone therapy -a key health service for those who want to transition from one gender to another. method: in preparing to offer community-based health care to the trans community it was clear that shc was being welcomed but also being watched with a critical eye. trans people have traditionally experienced significant barriers in accessing medical care. to respond to this challenge a working group of members of the trans community and health providers was created to develop an overall approach to care and specific protocols for hormone therapy. the group met over a one year period and their work culminated in the development of medical protocols for the provision of hormone therapy to trans individuals. results: shc is currently providing health care to 281 registered clients who identify as trans individuals (march 31 2005) through primary care and mental health programs. in an audit of shc medical charts (january 2003 to september 2004) 55 female-to-male (ftm) and 82 male-to-female (mtf) clients were identified. less than half of the ftm group and just over two-thirds of the mtf group presented specifically for the provision of hormones. based on this chart audit and ongoing experience shc continues to update and refine these protocols to ensure delivery of quality care. conclusion: this program is an example of innovative community-based health delivery to a population who have traditionally faced barriers. shc services also include counselling, health promotion, outreach and education. p7-46 (c) healthy cities for canadian women: a national consultation sandra kerr, kimberly walker, and gail lush on march 4 2005, the national network on environment and women's health held a pan-canadian consultation to identify opportunities for health research, policy change, and action. this consultation also worked to facilitate information sharing and networking between canadian women working as urban planners, policy makers, researchers, and service workers on issues pertaining to the health of women living in canadian cities. methods: for this research project, participants included front-line service workers, policy workers, researchers, and advocates from coast to coast, including francophone women, women with disabilities, racialized women, and other marginalized groups. the following key areas were selected as topics for du.bnes i1 alto kading .:auk of end·sugr ieaal clileue ia singapore, accounting for more than so% of new can singapore (nkfs) to embark on a prevention program (pp) 10 empo~r d1ahc 1j1u1f dieir condition bttter, emphasizing education and disease sdf·managemen1 lkilla a. essennal camponenn of good glycaemic control. we sought 10 explore the effects of a 1pecialijed edu.:a11on pro· pun od glycacmic conuol, as indicated by, serum hba ic values budine serum hba ic values were determined before un<k-rgmng the education progr ;am, which couisted of comprehensive dietary advice, ideal weight goals, and improving lipid profiln. serum hbaic values were obtained ar 3,6,9 months later to determine impact of the program. analy11• wu done uling paired !·tests significant reduction in hba le levels from 0 ro 9 month• was observed in females, chinese race, older age group(> so yean). ohew-ibmi ~ 27.nwm2, wai11 hip ratio> l),up to primary and above secondary level education and those having om urine iclt showed that increasing hbalc levels (9) had increasing urmary protein (38.± 117; .18 ±i ih so± 136) and crearinine (s2.s ± 64 7s ± 71; ioi± 7s) levels fbg rnults showed that the management nf d1abetn m the nkfs preven· tion programme is effec;rive. results also indicated 1har hba le leve11 have a linnr trend wnh unnary protein and creatinine which are imponant determinants of renal diseate tal family-focused cinical palbway1 promoce politivc outcollln for ua inner city canu allicy ipmai jerrnjm1 care llctivirits in preparation for an infanr'' dilchargr honlr, and art m1endnl lo improve effi.:k'fl.:tn of c.are. 11lere i11 paucity of tttran:h, and inconsi1trncy of rnulta on 1ht-•m!*-1 of f1m1ly·fc"-'uw d1nm 1a: to determinr whrthrr implrmentation of family.focuted c:pt 1n 1 ntnn.tt.tl unit w"n mg an inner city 1;ommunity drcl't'aki leftarh of lf•y (i.osi and rromclll'i family uo•fkllon and rt.1j1 nest for dikhargr. md6odt: family-focuk"d cpi 1041 data wm coll«ted for all infant• horn btrwttn 29 and 36 wft"k• 1t"lal111mi atr who wrtt .1dm111ed to the ntonatal unit lmgdl of -.y 111. 9 n. 14.8 day'o p c o.osi ind pma .11 d•mr., ho.nr 137.3 t 1.3 n. 36.4 ± i. i wb, p < o.os) wett n«01fiamly f.lfrt 1n the pre.(]' poup. ~11.fxtmon icofn for famihn wrre high. and families noctd thc:y wnr mott prepued to ah thrar t..lby "'-· thett was .a cosi uving of s 1,814 (cdn) per patient d1teharpd home 1n the pmi-cp poap c.-pated 10 the p"''lfoup· cortclaion· lmplrmrnr.rion of family·foanrd c:p. in a nrona1.1i umt tc"fyidi an 1nnn an com· muniry decre.ned length of'"'" mft with a high dcgrft of family uujamon, and wrre coll~nt at least 35% percent of the kathmandu population lives in slum like conditions with poor access to basic health services. in these disadvantaged areas, a large proportion of children do not receive treatment due to inaccessibility to medical services. in these areas, diarrhea, pneumonia, and measles, are the key determinants of infant mortality. protein energy malnutrition and vitamin a deficiency persists and communicable diseases are compounded by the emergence of diseases like hiv/aids. while the health challenges for disadvantaged populations in kathmandu are substantial, the city has also experienced various forms of innovative and effective community development health programs. for example, there are community primary health centers established by the kathmandu municipality to deliver essential health services to targeted communities. these centers not only provide equal access to health services to the people through an effective management system but also educate them hy organizing health related awareness programs. this program is considered one of the most effective urban health programs. the paper/presentation this paper will review large, innovative, and effective urhan health programs that are operating in kathmandu. most of these programs are currently run by international and national ngos a) early detection of emerging diseases in urban settings through syndromic surveillance: 911 data pilot study kate bassil of community resources, and without adequate follow-up. in november 2003 shelter pr.oviders ~et with hospital social workers and ccac to strike a working group to address some of th~ issues by mcre.asing knowledge among hospital staff of issues surrounding homelessness, and to build a stro?g workmg relationship between both systems in hamilton. to date the hswg has conducted four w~lkmg to~ of downtown shelters for hospital staff and local politicians. recently the hswg launched its ·~ool.k1t for staff working with patients who are homeless', which contains community resources and gu1dehnes to help with effective discharge plans. a scpi proposal has been submitted to incre~se the capacity of the hswg to address education gaps and opportunities with both shelters and hospitals around homelessness and healthcare. the purpose of this poster presentation is to share hamilton's experience and learnings with communities who are experiencing similar issues. it will provide for intera~tion around shared experiences and a chance to network with practitioners across canada re: best practices. introduction and objectives: canadians view health as the biggest priority for the federal government, where health policies are often based on models that rely on abstract definitions of health that provide little assistance in the policy and analytical arena. the main objectives of this paper are to provide a functional definition of health, to create a didactic model for devising policies and determining forms of intervention, to aid health professionals and analysts to strategize and prioritize policy objectives via cost benefit analysis, and to prompt readers to view health in terms of capacity measures as opposed to status measures. this paper provides a different perspective on health, which can be applied to various applications of health such as strategies of aid and poverty reduction, and measuring the health of an individual/ community/country. this paper aims to discuss theoretical, conceptual, methodological, and applied implications associated with different health policies and strategies, which can be extended to urban communities. essentially, our paper touches on the following two main themes of this conference: •health status of disadvantaged populations; and •interventions to improve the health of urban communities.methodology: we initially surveyed other models on this topic, and extrapolated key aspects into our conceptual framework. we then devised a theoretical framework that parallels simple theories of physkal energy, where health is viewed in terms of personal/societal health capacities and effort components.after establishing a theoretical model, we constructed a graphical representation of our model using selfrated health status and life expectancy measures. ultimately, we formulated a new definition of health, and a rudimentary method of conducting cost benefit analysis on policy initiatives. we end the paper with an application example discussing the issues surrounding the introduction of a seniors program.results: this paper provides both a conceptual and theoretical model that outlines how one can go about conducting a cost-benefit analysis when implementing a program. it also devises a new definition and model for health barred on our concept of individual and societal capacities. by devising a definition for health that links with a conceptual and theoretical framework, strategies can be more logically constructed where the repercussions on the general population are minimized. equally important, our model also sets itself up nicely for future microsimulation modeling and analysis.implications: this research enhances one's ability to conduct community-based cost-benefit analysis, and acts as a pedagogical tool when identifying which strategies provide the best outcome. p7-06 (a) good playgrounds are hard to find: parents' perceptions of neighbourhood parks patricia tucker, martin holmes, jennifer irwin, and jason gilliland introduction: neighbourhood opportunities, including public parks and physical activity or sports fields hav~ been. iden.tified as correlates to physical activity among youth. increasingly, physical activity among children 1s bemg acknowledged as a vital component of children's lives as it is a modifiable determinant of childh~d obesity. children's use of parks is mainly under the influence of parents; therefore, the purpose of this study was to assess parents' perspectives of city parks, using london ontario as a case study.m~~: this qualitative study targeted a heterogeneous sample of parents of children using local parks w1thm london. parents with children using the parks were asked for 5 minutes of their time and if willing, a s.hort interview was conducted. the interview guide asked parents for their opinion 'of city parks, particularly the one they were currently using. a sample size of 50 parents is expected by the end of the summer.results: preliminary findings are identifying parents concern with the current jack of shade in local parks. most parents have identified this as a limitation of existing parks, and when asked what would make the parks better, parents agree that shade is vital. additionally, some parents are recognizing the v170 poster sessions focused discussions during the consultation: 1. women in _poverty 2. women with disability 3. immi· grant and racialized women 4. the built and _physica_l environment. . . . . r its· participants voiced the need for integration of the following issues withm the research and policy :::na; t) the intersectional nature of urban women's health i~sues wh~ch reflects the reality of women's complex lives 2) the multisectional aspect of urban wo_m~n s health, 1ss~es, which reflects the diversity within women's lives 3) the interse~roral _dynamics within _womens hves and urban health issues. these concepts span multiple sectors -mdudmg health, educat10n, and economics -when leveraging community, research, and policy support, and engaging all levels of government.policy jmplicatiom: jn order to work towards health equity for women, plans for gender equity must be incorporated nationally and internationally within urban development initiatives: • reintroduce "women" and "gender" as distinct sectors for research, analysis, advocacy, and action. •integrate the multisectional, intersectional, and intersecroral aspects of women's lives within the framework of research and policy development, as well as in the development of action strategies. • develop a strategic framework to house the consultation priorities for future health research and policy development (for example, advocacy, relationship building, evidence-based policy-relevant research, priority initiatives}.note: research conducted by nnewh has been made possible through a financial contribution from health canada. the views expressed herein do not necessarily represent the views of health canada.p7-47 (c) drugs, culture and disadvantaged populations leticia folgar and cecilia rado lntroducci6n: a partir de un proyecto de reducci6n de daiios en una comunidad urbana en situ· aci6n de extrema vulnerahilidad surge la reflexion sobre el lugar prioritario de los elementos sociocuhurales en el acceso a los servicios de salud de diferentes colectivos urbanos. las "formas de hacer, pensar y sentir" orientan las acciones y delimitan las posibilidades que tienen los individuos de definir que algo es o no problema, asf como tambien los mecanismos de pedido de ayuda. el analisis permanenre del campo de "las culturas cotidianas" de los llamados "usuarios de drogas" aporta a la comprension de la complejidad del tema en sus escenarios reales, y colabora en los diseiios contextualizados de politicas y propuestas socio-sanitarias de intervenci6n, tornandolas mas efectivas.mitodos: esta experiencia de investigaci6n-acci6n que utiliza el merodo emografico identifica elementos socio estructurales, patrones de consumo y profundiza en los elementos socio-simb61icos que estructuran los discursos de los usuarios, caracterizandolos y diferenciandolos en tanto constitutivos de identidades socia les que condicionan la implementaci6n del programas de reduccion de daiios.resultados: los resultados que presentaremos dan cuenta de las caracteristicas diferenciadas v relaciones particulares ~ntre los consumidores de drogas en este contexto espedfico. a partir de este e~tudio de caso se mtentara co1?1enzar a responder preguntas que entendemos significativas a la hora de pensar intcrvcnciones a la med1da de poblaciones que comparten ciertas caracteristicas socio-culturales. (cuales serian las .motivaciones para el cambio en estas comunidades?, cque elementos comunitarios nos ayudan a i:nnstnur dema~~a? • cque tenemos para aprender de las "soluciones" que ellos mismos encuenrran a los usos problemat1cos? methods: our study was conducted by a team of two researchers at three different sites. the mapping consisted of filling in a chart of observable neighbourhood features such as graffiti, litter, and boarded housing, and the presence or absence of each feature was noted for each city block. qualitative observations were also recorded throughout the process. researchers analyzed the compiled quantitative and qualitative neighbourhood data and then analyzed the process of data collection itself.results: this study reveals the need for further research into the effects of physical environments on individual health and sense of well-being, and perception of investment in neighbourhoods. the process reveals that perceptions of health and safety are not easily quantified. we make specific recommendations about the mapping methodology including the importance of considering how factors such as researcher social location may impact the experience of neighbourhoods and how similar neighbourhood characteristics are experienced differently in various spaces. further, we discuss some of the practical considerations around the mapping exercise such as recording of findings, time of day, temperature, and researcher safety.conclusion: this study revealed the importance of exploring conceptions of health and well-being beyond basic physical wellness. it suggests the importance of considering one's environment and one's own perception of health, safety, and well-being in determining health. this conclusion suggests that attention needs to be paid to the connection between the workplace and the external environment it is situated in. the individual's workday experience does not start and stop at the front door of their workplace, but rather extends into the neighbourhood and environment around them. our procedural observations and recommendations will allow other researchers interested in the effect of urban environments on health to consider using this innovative methodology. introduction: responding ro protests against poor medical attention for sexually assaulted women and deplorable conviction rates for sex offenders, in the late 1970s, the ontario government established what would become over 30 hospital-based sexual assault care and treatment centres (sactcs) across the province. these centres, staffed around the clock with specially trained heath care providers, have become the centralized locations for the simultaneous health care treatment of and forensic evidence collection from sexually assaulted women for the purpose of facilitating positive social and legal outcomes. since the introduction of these centres, very little evaluative research has been conducted to determine the impact of this intervention. the purpose of our study was to investigate it from the perspectives of sexually assaulted women who have undergone forensic medical examinations at these centres.method: women were referred to our study by sactc coordinators across ontario. we developed an interview schedule composed of both closed and open-ended questions. twenty-two women were interviewed, face-to-face. these interviews were approximately one-to-two hours in length, and were transcribed verbatim. to date, 19 have been analyzed for key themes.results: preliminary findings indicate that most women interviewed were canadian born (79'yo), and ranged in age from 17 to 46 years. a substantial proportion self-identified as a visible minority ( 37'x.). approximately half were single or never married (47%) and living with a spouse or family of origin (53%). most were either students or not employed (68%). two-thirds (68%) had completed high school and onethird (37%) was from a lower socio-economic stratum. almost two-fifths (37%) of women perceived the medical forensic examination as revictimizing citing, for example, the internal examination and having blood drawn. the other two-thirds (63%) indicated that it was an empowering experience, as it gave them a sense of control at a time when they described feeling otherwise powerless. most (68%) women stated that they had presented to a centre due to health care concerns and were very satisfied ( 84 % ) with their experiences and interactions with staff. almost all (89%) women felt supported and understood.conclusions: this research has important implications for clinical practice and for appropriately addressing the needs of sexually assaulted women. what is apparent is that continued high-quality medical attention administered in the milieu of specialized hospital-based services is essential. at the same time, we would suggest that some forensic evidence collection procedures warrant reevaluation. the study will take an experiential, approach by chroruclmg the impa~ of the transition f m the streets to stabilization in a managed alcohol program through the techruque of narrative i~:uiry. in keeping with the shift in thinking in the mental health fie!~ ~his stu~y is based on a paradigm of recovery rather than one of pathology. the "inner views of part1c1pants hves as they portray their worlds, experiences and observations" will be presented (charm~z, 1991, ~· 38~)-"i?e p~ of the study is to: identify barriers to recovery. it will explore the exj?cnence of ~n~t1zanon pnor to entry into the program; and following entry will: explore the meanmg ~nd defirutto~s of r~overy ~~d the impact of the new environment and highlight what supports were instrumental m movmg pan1apants along the recovery paradigm.p7-st (a) treating the "untreatable": the politics of public health in vancouver's inner city introdudion: this paper explores the everyday practices of therapeutic programs in the treadnent of hiv in vancouver's inner city. as anthropologists have shown elsewhere, therapeutic programs do not siinply treat physical ailments but they shape, regulate and manage social lives. in vancouver's inner city, there are few therapeutic options available for the treatment of 1-ilv. public health initiatives in the inner city have instead largely focused on prevention and harm reduction strategies such as needle exchange programs, safe injection sites, and safer-sex education. epidemiological reports suggest that less than a quarter of those living with hiv in the downtown eastside (dtes) are taking antiretroviral therapies raising critical questions regarding the therapeutic economy of antiretrovirals and rights to health care for the urban poor.methods: this paper is drawn from ethnographic fieldwork in vancouver's otes neighborhood focusing on therapeutic programs for hiv treatment among "hard-to-reach" populations. the research includes participant-observation at inner city health clinics specializing in the treatment of hiv; semi· structured interviews with hiv positive participants, health care professionals providing hiv treatment, and administraton working in the field of inner city public health; and, lastly, observation at public meetings and conferences surrounding hiv treatment.r.awlts: hiv prevention and treatment is a central concern in the lives of many residents living in the inner city -although it is just one of many health priorities afflicting the community. concerns about drug resistance, cost of antiretrovirals, and illicit drug use means that hiv therapy for most is characterized by the daily observation of their medicine ingestion at health clinics or pharmacies. daily observed treatment (dot) is increasingly being adopted as a strategy in the therapeutic management of "untreatable" populations. dot programs demand a particular type of subject -one who is "compliant" to the rules and regimes of public health. over emphasis on "risky practices," "chaotic lives," and "~dh~rence" preve~ts the public health system from meaningful engagement with the health of the marginalized who continue to suffer from multiple and serious health conditions and who continue to experience considerable disparities in health.~ the ~ffec~s of hiv in the inner city are compounded by poverty, laclc of safe and affordable houamg, vanous 1llegal underground economies increased rates of violence and outbreaks of ~~~·~ly tr~nsmitted infections, hepatitis, and tuberculosi: but this research suggests 'that public health uunauves aimed at reducing health disparities may be failing the most vulnerable and marginal of citiztl1s. margaret malone 1~ vi~lence that occurs in families and in intimate relationships is a significant urban, ~unity, and pu~hc health problem. it has major consequences and far-reaching effects for women, ~~--renho, you~ sen1on, and families. violence also has significant effects for those who provide and ukllc w receive health care violence · · i · · . all lasses, · is a soc1a act mvolvmg a senous abuse of power. it crosses : ' : ' ~ 11 s;nden, ag~ ~ti~, cultures, sexualities, abilities, and religions. societal responseshali ra y oc:used on identificatton, crisis intervention and services for families and individuajs.promoten are only "-"--:-g to add h · ' · i in intimate relationshi with"-~"'.". ress t e issues of violence against women and vjoence lenga to consider i~ m families. in thi_s p~per, i analyze issues, propose strategies, and note c~· cannot be full -...l'-~ whork towards erad1canng violence, while arguing that social justice and equity y -.1ucvcu w en thett are people wh mnhod: critical social theory, an analysis that addresses culturally and ethnically diverse communities, together with a population health promotion perspective frame this analysis. social determinants of health are used to highlight the extent of the problem of violence and the social and health care costs.the ottawa charter is integrated to focus on strategies for developing personal skills, strengthening community action, creating supportive environments, devdoping healthy public policies, and re-orientating health and social services. attention is directed to approaches for working with individuals, families, groups, communities, populations, and society.ratdts: this analysis demonstrates that a comprehensive interdisciplinary, multisectoral, and multifaceted approach within an overall health promoting perspective helps to focus on the relevant issues, aitical analysis, and strategies required for action. it also illuminates a number of interacting, intersecting, and interconnecting factors related to violence. attention, which is often focused on individuals who are blamed for the problem of violence, is redirected to the expertise of non-health professionals and to community-based solutions. the challenge for health promoters working in the area of violence in families and in intimate relationships is to work to empower ourselves and the communities with whom we work to create health-promoting urban environments. social justice, equiry, and emancipatory possibilities are positioned in relation to recommendations for future community-based participatory research, pedagogical practices for health care practitioners, and policy development in relation to violence and urban health. the mid-main community health center, located in vancouver british columbia (bc), has a diverse patient base reflecting various cultures, languages, abilities, and socio-economic statuses. due to these differences, some mid-main patients experience greater digital divide barriers in accessing computers and reputable, government produced consumer health information (chi) websites, such as the bc healthguide and canadian health network. inequitable access is problematic because patient empowerment is the basis of many government produced chi websites. an internet terminal was introduced at mid-main in the summer of 2005, as part of an action research project to attempt to bridge the digital divide and make government produced chi resources useful to a broad array of patients. multi-level interventions in co-operation with patients, with the clinic and eventually government ministries were envisioned to meet this goal. the idea of implementing multi-level interventions was adopted to counter the tendency in interactive design to implement a universal solution for the 'ideal' end-user [ 1 ), which discounts diversity. to design and execute the interventions, various action-oriented and ethnographic methods were employed before and during the implementation of the internet terminal. upon the introduction of the internet terminal, participant observation and interviews were conducted using a motion capture software program to record a digital video and audio track of patients' internet sessions. this research provided insight into the spectrum of patients' capacities to use technology to fulfil their health information needs and become empowered. at the mid-main clinic it is noteworthy that the most significant intervention to enhance the usefulness of chi websites for patients appeared to be a human rather than a technological presence. as demonstrated in other ethnographic research of community internet access, technical support and capacity building is a significant component of empowerment (2). the mid-main wired waiting room project indicates that medical practitioners, medical administrators, and human intermediaries remain integral to making chi websites useful to patients and their potential empowerment. (1) over the past 5 years the environmental yo~th alliance has been of~ering a.youth as~t. mappin~ program which trains young people in community research and evaluation. wh1~st the positive expenenc~ and relationships that have developed over this time attest to the success of this program, no evaluations has yet been undertaken to find out what works for t.he youth, what ~ould be changed, and what long term outcomes this approach offers for the youth, their local community, and urban governance. these topics will be shared and discussed to help other community disorganizing and uncials governments build better, youth-driven structures in the places they live.p7-55 (a) the world trade center health registry: a unique resource for urban health researchers deborah walker, lorna thorpe, mark farfel, erin gregg, and robert brackbill introduction: the world trade center health registry (wfchr) was developed as a public health response to document and evaluate the long-term physical and mental health effects of the 9/11 disaster on a large, diverse population. over 71,000 people completed a wfchr enrollment baseline survey, creating the largest u.s. health registry. while studies have begun to characterize 9/11 bealth impacts, questions on long-term impacts remain that require additional studies involving carefully selected populations, long-term follow-up and appropriate physical exams and laboratory tests. wtchr provides an exposed population directory valuable for such studies with features that make ita unique resource: (a) a large diverse population of residents, school children/staff, people in lower man· hattan on 9/11 including occupants of damaged/destroyed buildings, and rescue/recovery/cleanup work· ers; (b) consent by 91 % of enrollees to receive information about 9/11-related health studies; (c) represenration of many groups not well-studied by other researchers; (c) email addresses of 62% of enrollees; (d) 30% of enrollees recruited from lists with denominator estimates; and (e) available com· parison data for nyc residents. wfchr strives to maintain up-to-date contact information for all enrollees, an interested pool of potential study participants. follow-up surveys are planned.methods: to promote the wtchr as a public health resource, guidelines for external researcher.; were developed and posted on (www.wtcregistry.org) which include a short application form, a twopage proposal and documentation of irb approval. proposals are limited to medical, public health, or other scientific research. researchers can request de-identified baseline data or have dohmh send information about their studies to selected wfchr enrollees via mail or email. applications are scored by the wtchr review committee, comprised of representatives from dohmh, the agency for toxic subst~nces and disease registry, and wtchr's scientific, community and labor advisory committees. a data file users manual will be available in early fall 2005.~suits: three external applications have been approved in 2005, including one &om a non-u.s. ~esearcher, all requesting information to be sent to selected wtchr enrollees. the one completed mail· mg~~ wtchr enrollee~ (o 3,700 wfc tower evacuees) generated a positive survey response rate. three additional researchers mtend to submit applications in 2005. wfchr encourages collaborations between researchers and labor and community leaders.conclusion: studies involving wtchr enrollees will provide vital information about the long· term health consequences of 9/11. wtchr-related research can inform communities, researcher.;, policy makers, health care providers and public health officials examining and reacting to 9111 and other disasters. t .,. dp'"f'osed: thi is presentation will discuss the findings of attitudes toward the repeat male client iden· 1 ie as su1e1 a and substance us'n p · · · · i · 'd . . -1 g. articipants will learn about some identified effective strategies or service prov1 ers to assist this group of i · f men are oft · d bl men. n emergency care settings, studies show that this group 0 en viewe as pro emaric patient d i r for mental health p bl h h 5 an are more ikely to be discharged without an assessmen 200!) ea 1 1 rofr ems t. an or er, more cooperative patients (forster and wu 2002· hickey er al., · r y resu ts om this study suggest th · · ' ' l · d tel' mining how best to h 1 . d 1 at negative amtudes towards patients, difficu nes e · as well pathways l_e_ p patientsblan ~ck of conrinuity of care influence pathways to mental health care. • uc\:ome pro emat1c when p ti k · che system. m a ems present repeatedly and become "get stuc id methods: semi-structured intervie d . · (n=5), ed nurses (n=5) other ed ;s were con ucted with male ed patients (n=25), ed phys1oans ' sta (n= 7) and family physicians (n= 7). patients also completed a poster sessions v175 diagnostic interview. interviews were tape-recorded, transcribed verbatim and managed using n6. transcripts were coded using an iterative process and memos prepared capture emergent themes. ethics approval was obtained and all participants signed a detailed informed consent form.introduction: urban settings are particularly susceptible to the emergence and rapid spread of nt•w or rare diseases. the emergence of infectious diseases such as sars and increasing concerns over the next influenza pandemic has heightened interest in developing and using a surveillance systt·m which detects emerging public health problems early. syndromic surveillance systems, which use data b,1scd on symptoms rather than disease, offer substantial potential for this by providing near-real-rime data which are linked to an automated warning system. in toronto, we are piloting syndromic data from the 911 · emergency medical services (ems) database to examine how this information can be used on an ongoing basis for the early detection of syndromes including heat-related illness (hri), and influenza-like-illness (ill). this presentation will provide an outline of the planned desi_gn of this system and proposed evaluation. for one year, 911 call codes which reflect heat-related illness or influenza-like-illness will be selected and searched for daily using software with a multifactorial algorithm. calls will he stratified by call code, extracted from the 911-ems database and transferred electronically to toronto public health. the data will be analyzed for clusters and aberrations from the expected with the realtime outbreak and disease surveillance (rods) system, a computer-based public health tool for the early detection of disease outbreaks. this 911-ems surveillance system will be assessed in terms of its specificity and sensitivity through comparisons with the well-established tracking systems already in place for hr! and ill. others sources of data including paramedic ambulance call reports of signs and . this study will introduce complementary data sources t~ the ed ch1e~ complamt an~ o~~rthe-counter pharmacy sales syndromic surveillance data currently bemg evaluated m ~ther ontar~o cltles. . syndromic surveillance is a unique approach to proactive(~ dete~tmg early c.hangesm the health status of urban communities. the proposed study aims to provide evidence of differential effectiveness through investigating the use of 911-ems call data as a source of syndromic surveillance information for hr! and ili in toronto. introduction: there is strong evidence that primary care interventions, including screening, brief advi<:e, treatment referral and pharmacotherapy are effective in reducing morbidity and mortality caused by substance abuse. yet physicians are poor at intervening with substance users, in part because of lack of time, training and support. this study examines the hypothesis that shared care in addictions will result in decreased substance use and improved health status of patients, as well as increased use of primary care interventions by primary care practitioners (pcps). methods: the addiction medicine service (ams) at st. joseph's health centre's family medicine department is in the process of being transformed from its current structure as a traditional consult service into a shared care model called addiction shared care (asc). the program will have three components: education, office systems and clinical shared care. as opposed to a traditional consult service, the patient will be booked with both a primary care liaison worker (pcl) and addictions physician. patients referred from community physicians, the emergency department and inpatient medical and psychiatric wards will be recruited for the study as well as pcps from the surrounding community. the target sample size is 100-150 physicians and a similar number of patients. after initial consult, patient will be recruited into the study with their consent. the shared-case model underlines the interaction and collaboration with the patient's main pcp. asc will provide them with telephone consults, advice, support and re-assessment for their patients, as well as educational sessions and materials such as newsletters and informational kits.results: the impact of this transition on our patient care and on pcp's satisfaction with the asc model is currently being evaluated through a grant provided by the ministry of health & long term care. a retrospective chart review will be conducted using information on the patient's substance use, er/clinic visits, and their health/mood status. pcp satisfaction with the program will be measured through surveys and focus groups. our cost-effectiveness analysis will calculate the overall cost of the program per patient..conclusion: this low-cost service holds promise to serve as an optimal model and strategy to improve outcomes and reduce health care utilization in addict patients. the inner city public health project introduction the inner city public health project (icphp) was desi.gned to explore new an~ innovative ways to reach marginalized inner city populations that par-t1c1pate m high health-nsk beha~1ors. much of this population struggles with poverty, addictions, mental illness and homelessness, creatmg barriers to accessing health services and receiving follow-up. this pro1ect was de~igned to evaluat~ .~e success of offering clinics in the community for testing and followup of communicable diseases uuhzmg an aboriginal outreach worker to build relationships with individuals and agencies. v1n(demographics~ history ~f testi~g ~nd immunization and participation in various health-risk behaviors), records of tesnng and 1mmumzat1ons, and mterviews with partner agency and project staff after one year.. results: t~e chr ~as i~strumental in building relationships with individuals and partner agencies ' .° the c~mmun_1~ re_sultmg m req~ests for on-site outreach clinics from many of the agencies. the increase m parnc1pat10n, the chr mvolvement in the community, and the positive feedback from the agen? staff de~onstrated that.the project was successfully creating partnerships and becoming increasingly integrated m the community. data collected from clients at the initial visit indicated that the project was reaching its target populations and highlighted the unique health needs of clients, the large unmet need for health services and the barriers that exist to accessing those services. ~usion: the outreach clinics were successful at providing services to target populations of high health-nsk groups and had great support from the community agencies. the role of the chr was critical to the success of the project and proved the value of this category of health care worker in an urban aboriginal population. the unmet health needs of this disadvantaged population support the need for more dedicated resources with an emphasis on reducing access barriers. building a caring community old strathcona's whyte avenue, a district in edmonton, brought concern about increases in the population of panhandlers, street people and homeless persons to the attention of all levels of government. the issue was not only the problems of homelessness and related issues, but feelings of insecurity and disempowerment by the neighbourhood residents and businesses. their concerns were acknowledged, and civic support was offered, but it was up to the community itself to solve the problem. within a year of those meetings, an adult outreach worker program was created. the outreach worker, meets people in their own environments, including river valley camps. she provides wrap-around services rooted in harm reduction and health promotion principles. her relationship-based practice establishes the trust for helping clients with appropriate housing, physical and/or mental health issues, who have little or no income and family support to transition from homelessness. the program is an excellent example of collaboration that has been established with the businesses, community residents, community associations, churches, municiple services, and inner-city agencies such as boyle street community services. statistics are tracked using the canadian outcomes research institute homes database, and feedback from participants, including people who are street involved. this includes an annual general meeting for community and people who are homeless. the program's holistic approach to serving the homeless population has been integral, both in creating community awareness and equipping residents and businesses to effectively interact with people who are homeless. through this community development work, the outreach worker engages old strathcona in meeting the financial and material needs of the marginalized community. the success of this program has been surprising -the fact is that homeless people's lives are being changed; one person at a time and the community has been changed in how they view and treat those without homes. over two years, the program has successfully connected with approximately seventy-five individuals who call old strathcona home, but are homeless. thirty-six individuals are now in homes, while numerous others have been assisted in obtaining a healthier and safer lifestyles by becoming connected with other social/health agencies. the program highlights the roots of homelessness, barriers to change and requirements for success. it has been a thriving program and a model that works by showing how a caring community has rallied together to achieve prosperous outcomes. the spn has created models of tb service delivery to be used m part~ers~1p with phannaceunca compa-. · · -. t' ns cooperatives and health maintenance orgamzanons (hmos). for example, the mes, c1v1c orgamza 10 , . · b tb d' · spn has established a system with pharmaceutical companies that help patients to uy me 1cmes at a special discounted rate. this scheme also allows patients to get a free one-_month's worth of~ dru_g supply if they purchase the first 5 months of their regimen. the sy_s~e~s were ~es1gned to be cm~pattble with existing policies for recording and documentation of the ph1hppme national tuberculosis program (ntp). aside from that, stakeholders were also encouraged to be dots-enabled through the use of m~nual~ and on-line training courses. the spn initiative offers an alternative in easing the burden of tb sc:rv1ce delivery from rhe public sector through the harnessing of existing private-sector (dsos). the learnmgs from the spn experience would benefit groups from other locales that _work no~ only on ~ but other health concerns as well. the spn experience showcases how well-coordinated private sector involvements help promote social justice in health delivery in urban communities.p7-63 (c) young people in control; doing it safe. the safe sex comedy juan walter and pepijn v. empelen introduction: high prevalence of chlamydia and gonorrhoea have been reported among migrants youth in amsterdam, originating from the dutch antilles, suriname and sub-sahara africa. in addition, these groups also have high rates of teenage-pregnancy (stuart, 2002) and abortions (rademakers 1995), indicating unsafe sexual behaviour of these young people. young people (aged 12 -30) from the so· called urban scene (young trendsetters in r&b/hip hop music and lifestyle) in amsterdam have been approached by the municipal health service (mhs) to collaborate on a safe sex project. their input was to use comedy as vehicle to get the message a cross. for the mhs this collaboration was a valuable opportunity to reach a hard-to-reach group.mdhods: first we conducted a need assessment by means of a online survey to assess basic knowledge and to similtaneously examine issues of interest concerning sex, sexuality, safer sex and the opposite sex. second, a small literature study was conducted about elements and essential conditions for succesful entertainment & education (e&e) (bouman 1999), with as most important condition to ensure that the message is realistic (buckingham & bragg, 2003) . third a program plan was developed aiming at enhancing the stl/hiv and sexuality knowledge of the young people and addressing communication and educational skills, by means of drama. subsequently a safe sex comedy show was developed, with as main topics: being in love, sexuality, empowerment, stigma, sti, hivand safer sex. the messages where carried by a mix of video presentation, stand up comedy, spoken word, rap and dance.results: there have been two safe sex comedy shows. the attendance was good; the group was divers' with an age range between 14 and 50 year, with the majority being younger than 25 year. more women than men attended the show. the story lines were considered realistic and most of the audients recognised the situations displayed. eighty percent of the audients found the show entertaining and 60% found it edm:arional. from this 60%, one third considers the information as new. almost all respondents pointed our that they would promote this show to their friends.con.clusion: the s.h<_>w reached the hard-to-reach group of young people out of the urban scene and was cons1d_ered entert~mmg, educational and realistic. in addition, the program was able in addressing important issues, and impacted on the percieved personal risk of acquiring an sti when not using condoms, as well as on basic knowledge about stl's. introduction: modernity has contributed mightily to the marginality of adults who live with mental illnesses and the subsequent denial of opportunities to them. limited access to social, vocational, educational, and residential opportunities exacerbates their disenfranchisement, strengthening the stigma that has been associated with mental illness in western society, and resulting in the denial of their basic human rights and their exclusion from active participation in civil society. the clubhouse approach tn recovery has led to the reduction of both marginality and stigma in every locale in which it has been implemented judiciously. its elucidation via the prism of social justice principles will lead to a deeper appreciation of its efficacy and relevance to an array of settings. methods: a review of the literature on social justice and mental health was conducted to determine core principles and relationships between the concepts. in particular, fondacaro and weinberg's (2002) conceptualization of social justice in community psychology suggests the desirability of the clubhouse approach in community mental health practice. a review of clubhouse philosophy and practice has led to the inescapable conclusion that there is a strong connection between clubhouse philosophy-which represents a unique approach co recovery--and social justice principles. placing this highly effective model of community mental health practice within the context of these principles is long overdue. via textual analysis, we will glean the principles of social justice inherent in the rich trove of clubhouse literature, particularly the international standards of clubhouse development.results: fondacarao and weinberg highlight three primary social justice themes within their community psychology framework: prevention and health promotion; empowerment, and a critical pnsp<"<·tive. utilizing the prescriptive principles that inform every detail of clubhouse development and th<" movement toward recovery for individuals at a fully-realized clubhouse, this presentation asserts that both clubhouse philosophy and practice embody these social justice themes, promote human rights, and empower clubhouse members, individuals who live with mental illnesses, to achieve a level of wdl-heing and productivity previously unimagined.conclusion: a social justice framework is critical to and enhances an understanding of the clubhouse model. this model creates inclusive communities that lead to opportunities for full partic1pil!ion 111 civil society of a previously marginalized group. the implication is that clubhouses that an· based on the international standards for clubhouse programs offer an effective intervention strategy to guarantee the human rights of a sizable, worthy, and earnest group of citizens. to a drastic increase in school enrollment from 5.9 million in 2002 to 7.5 million in 200.s. however, while gross enrollment rates increased to 104°/., in the whole country after the introduction of fpe, it remained conspicuously low at 62% in the capital city, nairobi. nairobi city's enrollment rate is lower .than thatof all regions in the country except the nomadic north-eastern province. !h.e.d1sadvantage of children bas_ed in the capital city was also noted in uganda after the introduction of fpe m the late 1990s_-many_ education experts in kenya attribute the city's poor performance to the high propornon of children hvmg m slums, which are grossly underserved as far as social services are concerned. this paper ~xammes the impact of fpe and explores reasons for poor enrollment in informal settlements m na1rob1 city. methods: the study utilizes quantitative and qualitative schooling data from the longitudinal health and demographic study being implemented by the african population and health research center in two informal settlements in nairobi. descriptive statistics are used to depict trends in enrollment rates for children aged 5-19 years in slum settlements for the period 2000-2005. results: the results show that school enrollment has surprisingly steadily declined for children aged 15-19 while it increased marginally for those aged 6-14. the number of new enrollments (among those aged 5 years) did not change much between 2001 and 2004 while it declined consistently among those aged 6-9 since 2002. these results show that the underlying reasons for poor school attendance in poverty-stricken populations go far beyond the lack of school fees. indeed, the results show that lack of finances (for uniform, transportation, and scholastic materials) has continued to be a key barrier to schooling for many children in slums. furthermore, slum children have not benefited from fpe because they mostly attend informal schools since they do not have access to government schools where the policy is being implemented.conclusion: the results show the need for equity considerations in the design and implementation of the fpe program in kenya. without paying particular attention to the schooling needs of the urban poor children, the millennium development goal aimed at achieving universal primary education will remain but a pipe dream for the rapidly increasing number of children living in poor urban neighborhoods.ps-04 (c) programing for hiv/aids in the urban workplace: issues and insights joseph kamoga hiv/aids has had a major effect on the workforce. according to !lo 35million persons who are engaged in some form of production are affectefd by hiv/aids. the working class mises out on programs that take place in communities, yet in a number of jobs, there are high risks to hiv infection. working persons sopend much of their active life time in workplaces and that is where they start getting involved in risky behaviour putting entire families at risk. and when they are infected with hiv, working people face high levels of seclusion, stigmatisation and some miss out on benefits especially in countries where there are no strong workplace programs. adressing hiv/aids in the workplace is key for sucessfull responses. this paper presents a case for workplace programing; the needs, issues and recommendations especially for urban places in developing countries where the private sector workers face major challenges. key: cord-342901-ca2xxkb2 authors: lloyd, richard e. title: nuclear proteins hijacked by mammalian cytoplasmic plus strand rna viruses date: 2015-05-31 journal: virology doi: 10.1016/j.virol.2015.03.001 sha: doc_id: 342901 cord_uid: ca2xxkb2 abstract plus strand rna viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular rna-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building rna replication complexes. in the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. many mammalian viruses hijack a common group of the same factors. this review summarizes recent gains in our knowledge of how cytoplasmic rna viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus rna replication and common themes employed between different virus groups. viral spread and ultimately pathogenesis require efficient replication in key host cells that aid spread of the virus within hosts and throughout host populations. rna viruses are typically small, encoding as little at three genes, and thus must rely on many host factors interacting with viral rnas to assist with essential replication functions, and control many interaction points within host cells to promote replication. this often results in redirecting host metabolism on several levels to support the infection and at the same time suppress innate host defense systems that are triggered. comparing plus and minus stranded rna viruses, there are stark differences at the time of uncoating of genomic viral rna in the cytoplasm. the plus strand rna virus genome that is released is naked, however the minus strand rna virus genome is completely enclosed in a functional nucleocapsid with rna replicase poised ready to produce transcript mrnas. thus, the plus strand virus rna can, and does, interact with many host rna binding proteins (rbps), whereas there is a little opportunity for minus strand virus genomic rna to interact directly with host rbps. most rna-binding proteins are nuclear shuttling proteins and many more nuclear rbps have been reported to play roles in replication of plus strand rna viruses than minus strand rna viruses. accordingly, this review focuses heavily on plus stranded rna viruses, particularly mammalian viruses. rna viruses interact with a multitude of host factors during the course of infection. several screening approaches have been employed to identify which of the 15-20,000 proteins that may be expressed in a given cell are host factors required for rna virus replication. these include genetic screens in yeast that implicated 130 proteins that could affect plant virus replication (tomato bushy stunt virus) (jiang et al., 2006) and about 100 genes that affect brome mosaic virus (kushner et al., 2003; panavas et al., 2005) . rnai knockdown studies in mammalian cells with hepatitis c virus (hcv), dengue virus (denv) and west nile virus (wnv) have identified several hundred other genes that affect virus replication. however, many or most of these may function quite indirectly, affecting pathways that produce metabolites or products the virus needs, movement or trafficking of constituents that are directly required, factors that control divalent cation fluxes and atpase pumps, the stress or innate immune activation levels that counteract general cellular biosynthetic potential, or include general off-target effects from the silencing step. it is likely that the spectrum of factors that directly interact in meaningful ways with virus rna and proteins will be larger than that known today, but also smaller than the first lists that have emerged from such screenings (box 1). recently the novel approach of thiouracil cross-linking mass spectroscopy (tux-ms) was used to more precisely identify host proteins bound to poliovirus rna during replication. this procedure identified all proteins known to interact with enterovirus rna, plus 66 additional factors previously unidentified (lenarcic et al., 2013) . eight of the new proteins were chosen and validated as playing roles in replication, indicating this new method is powerful and should be applied to other virus systems. however, standard molecular biology and biochemical approaches will still be required to tease out the functions and impact of each of these factors on virus replication. proteins that interact with viral rna do not present interesting targets for antiviral development unless it is determined that they play critical roles in virus replication. plus strand rna viruses must translate incoming viral genomic rna as the first biosynthetic step in replication cycles, thus, control of translation becomes the first battleground with the host that involves co-opted nuclear factors. it makes sense for the virus to utilize the host factors it commonly encounters at sites of replication. thus, translation regulation involves virus co-opting of cellular translation factors. these are mostly cytoplasmic resident proteins since translation is a cytoplasmic process. however, translation does not occur on transcripts that are naked and devoid of rna-binding proteins, rather, cellular transcripts are continually bound to a host of rna binding proteins from the instant they emerge from rna polymerase during their synthesis. in mammalian cells, rna binding proteins control most aspects of rna biology and the rna cycle; from splicing, transport out of the nucleus, cellular function, transcript-specific translation control, and cytoplasmic localization and mrna half-life. mammalian cells encode hundreds of rbps ( $ 860), most with several splice variants (castelló et al., 2012) . the cytoplasmic milieu encountered by plus strand rna virus genomes as they are released from capsids is poised to greet the interloper as any other mrna, with a ready store of rna binding proteins ready to interact and impart functions. no wonder viruses have evolved to interact with rbp in diverse ways to promote replication. recent research indicates that interactions of viruses with rbp can both benefit and inhibit virus replication, depending on stages of the replicative cycle. rbp interaction with complexes may afford molecular switching events that promote rna replication over translation as one example. the complete virus "interactome" will contain proteins that provide a diverse range of functions during virus replication; including protein and rna chaperone functions, transport functions, helicases, factors and enzymes that regulate vesicular traffic, membrane functions and lipid metabolism. notably, many of these are cytoplasmic proteins, thus beyond the purview of this review. most of the nuclear rbps are constantly shuttling between the nucleus and cytoplasm, thus, the definition of nuclear factor in this context refers solely to shuttling proteins that are more concentrated in the nucleus under normal physiological conditions. nuclear pore disruption: how cytoplasmic viruses get their stuff an infecting virus may encounter sufficient nuclear shuttling proteins to initiate translation or the first rounds of rna replication. however, rapidly expanding rna virus replication requires a growing storehouse of supplies and will quickly expend the limited cytoplasmic supply of many nuclear factors. it was noticed early during studies of poliovirus-infected cells that many nuclear resident proteins showed improper cytoplasmic localization, including ptb, la autoantigen, sam68, nucleolin and others (back et al., 2002; mcbride et al., 1996; meerovitch et al., 1993; waggoner and sarnow, 1998) . in fact, many viruses disrupt trafficking of proteins and mrnps at the nuclear pore as a mechanism to restock the cytoplasmic storehouse with needed supplies. this dysregulation is usually required for efficient viral replication and can involve increased export of outbound cargo from the nucleus and/or blockage of inbound cargo from the cytoplasm. detailed descriptions of the mechanisms involved are beyond the scope of this article, but recent reviews cover advances in virus disruption of nuclear pores (le sage and mouland, 2013; yarbrough et al., 2014) . the range of viruses known to disrupt nuclear-cytoplasmic traffic is diverse and includes viruses that replicate in the cytoplasm (enterovirus, cardiovirus, coronavirus, rhabdovirus) and viruses that replicate in the nucleus (influenza, hiv-1, herpes simplex virus 1, adenovirus). the mechanisms that disrupt nuclear transport are also diverse. just two examples are enteroviruses that employ viral 2a proteinase to cleave at least three nuclear pore proteins (nup 62, nup 98, and nup 153) (park et al., 2008 (park et al., , 2010 watters and palmenberg, 2011) and cardiovirus that employs l protein to induce a phosphorylation cascade that results in hyperphosphorylation of nup 98, nup62, nup153 and nup 214 as well as dysregulation of the pore complex (bacotdavis and palmenberg, 2013; basta et al., 2014; porter and palmenberg, 2009; ricour et al., 2009; watters and palmenberg, 2011) . in addition, l protein directly binds and inhibits the active cellular transport protein ran gtpase (bacotdavis and palmenberg, 2013) . notably some of the nup targets are the same for both picornavirus genera, an example of convergent evolution providing two different approaches to accomplish similar functional goals. picornaviruses such as poliovirus (pv) and encephalomyocarditis virus (emcv) do not contain m7gtp cap structures on genomic rna to recruit ribosomes, instead they use an internal cap-independent mode of translation that requires a large folded rna structure to recruit ribosomes called an internal ribosome entry site (ires). ires elements have complex rna folds and are only active when bound with specific proteins called ires transactivating factors (itafs) that are thought to provide rna chaperone functions (table 1) . itafs are not canonical translation initiation factors that function in cap-dependent translation but are proteins known to play primary roles in other aspects of rna biology in the cell. itafs are all of cellular, not viral origin, which makes sense for plus strand viruses because the ires must function before any viral proteins can be synthesized. the ires plus the required itafs and canonical translation factors that make up a functional unit are referred to as iresomes because they function together as a complex. the functional relationships between itafs, canonical translation factors and ribosome recruitment are still unclear but significant strides have been made in understanding them in three virus systems. although the first itafs were discovered in the context of viral ires translation, many of the same itafs are thought to play similar roles promoting cap-independent translation for cellular ires elements. for example, ptb is an itaf for bip, bag1, apaf-1, unr, p53 iress; hnrnp a1 is an itaf for cyclin d1 and c-myc iress; and hnrnp c1/ c2 is an itaf for xiap and c-myc iress (reviewed in ). there are distinct classes of virus iress that are referred to by a somewhat inconsistent and evolving nomenclature. picornavirus ires elements can be classified into at least five types based on structure, limited sequence homology and phylogeny. type 1 iress are large $ 450 nucleotide segments that are encoded by poliovirus and other enteroviruses; type 2 iress are similarly large and occur in cardioviruses (emcv), aphthoviruses (fmdv) and parechoviruses; and type 3 ires occurs only in hepatitis a virus (hav). type 4 iress (sometimes called class 2 or type 3) are smaller $ 330 nucleotide segments and occur in non-picornaviruses (hepatitis c virus and pestiviruses) but are also found among the newer picornavirus families teschovirus, sapelovirus, senecavirus, tremovirus. type 5 iress are encoded in the newly defined picornavirus genera kobuvirus, salivirus and paraturdivirus (sweeney et al., 2012) . one of the first host proteins ever shown to interact with picornavirus rna by uv-crosslinking was polypyrimidine tract binding protein 1 (ptb1, shortened to ptb) . ptb is a shuttling, but mostly nuclear resident protein that associates with pre-mrnas and plays roles in pre-mrna processing, alternative splicing, mrna metabolism and transport (kafasla et al., 2010; sawicka et al., 2008) . ptb binds polypyrimidine tracts in pre-mrna introns to repress exon inclusion but can actually bind quite varied rna structures. thus, ptb can also stabilize certain mrnas against degradation by binding to the 3 0 untranslated regions. in virus replication schemes ptb plays roles that support and stimulate cap-independent translation driven by picornavirus and hcv rna. ptb exists as one of three alternatively spliced isoforms (ptb1, ptb2, ptb4) and contains four-rna binding domains (rbds) of the rnp1/rnp2 class (kafasla et al., 2010; sawicka et al., 2008) . though ptb was the first, a wide spectrum of factors, largely nuclear rbps, have also been proposed as poliovirus itafs, including lupus autoantigen (la) (meerovitch et al., 1993 (meerovitch et al., , 1989 , poly(rc)binding proteins (pcbps) (blyn et al., 1996; gamarnik and andino, 1997) , upstream of n-ras (unr) (anderson et al., 2007; boussadia et al., 2003; hunt et al., 1999) , srp20 (bedard et al., 2007) and glycyl-trna synthetase (gars) (andreev et al., 2012) (table 1 ). in order to make sense of the relative impact of this range of factors on basic translation, pestova and hellen have used in vitro translation initiation experiments reconstituted with a complete set of purified factors to define the minimal set of factors absolutely required for ires translation. in these reconstituted translation systems, the hcv class of ires minimally requires no itafs and only translation factor eif3 to bind ribosomes (hellen, 2009; pestova et al., 1998) . for both picornaviruses emcv and pv, several canonical translation factors and only one itaf is minimally required. emcv requires only ptb as an itaf and poliovirus requires only pcbp2 as an itaf to initiate translation (pestova et al., 1996; sweeney et al., 2014) . however, minimal requirements in vitro likely do not reflect the conditions of severe competition among mrnas for ribosomes in cells, suggesting the other factors proposed as itafs may play less fundamental, but still critical roles during infection and can contribute to pathogenesis. in addition, certain itafs play crucial roles in regulating the conversion of translation-competent genomic rnas into replicationcompetent rnas. ptb was known as a nuclear splicing factor when its role as an itaf of poliovirus and emcv was discovered; a classic hijacked nuclear protein pressed into a new role required for the virus. however, ptb is now known to also support cap-independent translation of cellular ires-containing mrnas that were discovered after the viral iress. these include apaf-1, bag-1 and mutant forms of c-myc transcripts associated with more aggressive tumor growth sawicka et al., 2008) . thus ptb can interact with a wide range of rnas to promote cap-independent translation and may be considered a pro-translation, general itaf. continuous investigations of ptb have revealed biochemical details about how it supports iresome function in virus translation. the rna binding function of ptb is modular and split between four rna-binding domains (rbds) that are distributed along an overall extended and flexible structure. this extended flexible nature of ptb is important for ires function. the ptb rbds recognize short pyrimidine-rich sequences but have distinct rna structural preferences. the two n-terminal rbds (rbd1 and rbd2) recognize short pyrimidine tracts contained in loops, while the rbds3-4 preferentially bind to larger flexible rna sequences. these features give ptb the ability to bind to a variety of rna structures and enables ptb to function as a versatile adapter protein that facilitates formation of many rna-protein regulatory complexes (clerte and hall, 2009) . recently, emcv and poliovirus ires structures have been used most extensively as models to study the itaf functions of ptb. mapping studies indicated that two ptb moieties bind the emcv ires, one upstream in a non-essential region of the ires and another in the ires core. in the core, the orientation of a single ptb binding to ires was determined by binding of ptb mutants and hydroxyl radical probing. rbd1 and rbd2 bind near the 3 0 end of the core ires sequence, and rbd3 binds near the 5 0 end. binding of ptb to multiple regions on the ires simultaneously enables stabilization and constraint of the ires tertiary structure and is consistent with multiple rbd-rna interactions proposed in the splicing functions of ptb (oberstrass et al., 2005; kafasla et al., 2009) . this illuminates how an itaf mechanistically provides an rna-chaperone function. ptb is thought to promote rna looping also, partly because rbds 3 and 4 bind rna in anti-parallel directions (oberstrass et al., 2005; lamichhane et al., 2010) . further work established which of the multiple rbd-ires interactions are critical for itaf function. using ptb with point mutations in various rbds, it was shown that both poliovirus and emcv ires rnas required simultaneous interaction with three of the four ptb rbds, however the list of most critical rbds differed strand shown in red) breathes allowing cloverleaf and anti-cloverleaf to form, stabilized by binding pcbp and hnrnp c respectively. polymerase replicase complex also builds on 5 0 cl and initiates replication on negative strand template that is properly positioned for precise-end initiation. hnrnp c can also interact with sl on the 5 0 end of the negative strand that also requires breathing to form. in this scenario pabp may be able to rebind to poly(a) tail and hnrnp oligomerization and pabp interaction with pcbp may facilitate genome circularization in double-stranded rf intermediate. between the viruses (kafasla et al., 2011) . this indicates that the modular and elongated nature of ptb can adapt to disparate rna structures using different sets of rbds to provide similar chaperone function. besides providing an rna chaperone function, ptb also provides a second critical function as it helps recruit or position the canonical translation initiation factor eif4gi on the ires (fig. 1a ). in the case of pv, both ptb and eif4g bind the same rna stem loop structure (stem loop v) comprising much of the ires core. the orientation of ptb binding is such that rbds 3 and 4 bind the base of stem loop v, whereas rbds 1 and 2 bind the stem loop itself in the same region proposed to bind the central domain of eif4gi. the two proteins do not compete for binding, rather ptb is thought to stimulate ires activity by ensuring that eif4gi binds in the correct orientation and position to function properly for ribosome entry at a nearby aug (kafasla et al., 2010) . other picornaviruses also utilize ptb as an itaf. coxsackievirus b3 is another enterovirus closely related to poliovirus with a type 1 ires, thus there is no surprise that ptb interacts with cvb3 rna and promotes translation in a similar fashion as an itaf. however, ptb also interacts with the cvb3 3 0 utr and this interaction may stimulate ires translation through long-range looping or genome circularization bridged by ptb (verma et al., 2010) . foot-andmouth disease virus ires is a type 2 ires structure distantly related to emcv, sharing little sequence homology but some secondary structural similarity. however, fmdv requires both ptb and itaf 45 (also known as ebp1) to minimally support translation, whereas emcv requires only ptb. the fmdv ires also binds three of four ptb rbds, where rbds 3 and 4 bind distally located regions of the ires and can function as a minimal ires when supplied as a truncated form of ptb (song et al., 2005) . changes in ires structure were analyzed to compare effects of itaf binding on rna conformation shifts in emcv and fmdv iress. despite the different itaf requirements, in both cases when itafs interacted with their cognate iress, similar conformation changes occurred where two domains were brought into closer compacted proximity (yu et al., 2011a) . thus, ptb provides the same structural role building the functional iresome, whether alone or in conjunction with other itafs. poly(rc)-binding protein (pcbp) is the only obligate itaf for enteroviruses using type 1 iress (sweeney et al., 2014) . unlike ptb, that binds widely separated sites on the emcv ires, pcbp binds to a restricted area of stem loop iv of the pv ires (fig. 1a) . this binding region of sliv is distant from the ires core in the two dimensional secondary structure (blyn et al., 1996) . but pcbp also binds stem loop b of the cloverleaf structure (cl) at the 5 0 terminus of enterovirus rna (fig. 1) andino, 2000, 1997) . there are four pcbp isoforms, however pcbp1 and pcbp2 are expressed in more abundance and both bind pv ires. pcbp1/2 isoforms can form dimers or heterodimers and each can interact with pv ires rna, but pcbp2 plays a more dominant role. pcbp binds rna through three kh domains and interacts 6-fold more strongly with the ires than cloverleaf rnas in isolation (gamarnik and andino, 2000) . pcbp2 is the isoform required for both translation initiation and also for rna replication. kh1 is the primary domain that interacts with pv ires domain iv (silvera et al., 1999; walter et al., 2002) . for closely related cvb3, individual kh1 and kh3 domains of pcbp bind to ires stem loop iv, kh1 interacts with subdomain iv/c rna, whereas kh3 interacts with subdomain iv/b (zell et al., 2008a) . another host factor, srp20, interacts with pcbp2 through its kh3 domain and was found to be critical for itaf function of pcbp2 in cells. in vitro translation in hela cell extracts depleted of srp20 are deficient in supporting poliovirus translation initiation and srp20 sirna knockdown in hela cells restricted poliovirus translation. (bedard et al., 2007) . like other itafs, srp20 is strongly relocalized from the nucleus to cytoplasm during poliovirus infection. srp20 may bind pcbp2 rather than viral rna directly since deletion of its rrm-rna interaction motif does not alter its localization during infection, however, this truncated form strongly repressed virus translation, likely via a dominant negative process (fitzgerald and semler, 2011) . many other factors have been shown to promote virus translation and have considered itafs or itaf ancillary factors, but compared to pcbp and ptb much less is known about their function at the biochemical level in promoting ires translation. the binding sites of some of these other itafs are poorly characterized. la enhances and corrects aberrant translation of pv in reticulocyte lysates that lack other itafs (meerovitch et al., 1993) and 40s ribosome subunit binding to pv ires is inhibited by a dominant negative la protein (costa-mattioli et al., 2004) . nucleolin interacts with poliovirus ires and enhances ires-dependent translation (izumi et al., 2001) . unr has also been reported to interact with ires elements (pv and rhinovirus) acting as an rna chaperone and also binds pabp that is bound to poly(a) tails in cellular mrnas (anderson et al., 2007; chang et al., 2004; hunt et al., 1999) . presumably this interaction may also occur with viral polysomes and can contribute to genome looping or circularization that makes continuous translation more efficient. that unr is important in vivo was demonstrated by gene knockout of both unr alleles in mouse cells that reduced translation of pv and human rhinovirus (hrv) ires's by 90% and could be rescued by introduction of unr expression plasmid (boussadia et al., 2003) . rna affinity pulldown of cellular proteins using ev71 5 0 utr bait revealed ptb, unr, pcbp1/2, hnrnp a1 and 10 other proteins. hnrnp a1 interacts with stem-loops ii and vi of the ev71 5 0 utr, and is proposed to be another enterovirus itaf. the roles of this interaction in replication are unclear since knockdown of hnrnp a1 had no effect on viral replication. in contrast, knockdown of both hnrnps a1 and a2 reduced viral rna synthesis and virus output, suggesting that hnrnp a2 can substitute for hnrnp a1 (lin et al., 2009) , however the relative importance of hnrnp a1 versus pcbp2 or ptb in ev71 replication was not evaluated. gars is perhaps the most unusual proposed itaf; a trna synthetase house-keeping enzyme that binds the ires core in the apical portion of domain v that mimics the anticodon-stem-loop of trna gly (andreev et al., 2012) . so what are we to make of all the proposed itafs for pv and other closely related enteroviruses? recently, in vitro reconstitution of translation with purified factors has been accomplished on the pv ires and other type 1 iress and those experiments indicate that pcbp2 is the only mandatory itaf required to accomplish 48s ribosome assembly and initiation. ptb showed minor stimulation of translation, and other proposed itafs (la, unr, srp20, gars) produced no measureable effect on 43s or 48s translation complex formation in vitro. pcbp2 may also promote recruitment of 43s complexes by direct interaction with eif3 (sweeney et al., 2014) . the weak stimulation of pcbp2-dependent 48s complex formation by ptb in vitro is likely due to its imposed reorientation of eif4g binding to the ires (kafasla et al., 2010) . but many itafs promote translation in cells by providing functions that promote efficiency, beyond the minimal requirements for ires translation initiation. intracellular conditions encountered by viruses include intense competition among mrnas for ribosomes in cells, forcing viruses to utilize multiple mechanisms to seize translation control. thus, the other itafs that aid ires translation efficiency in vivo play less fundamental, but still critical roles during infection and contribute to pathogenesis. future work will discern new molecular details how this happens. comparatively little is known about factor requirements for the type 3 ires of hav. unlike most of its picornavirus cousins, this virus replicates very slowly, it does not cause host translation shutoff and is relatively non-cytopathic in tissue culture cells. the ires is unique in that it requires intact eif4g1, whereas the pv ires functions better with only the cleaved central domain of eif4gi (borman and kean, 1997; hambidge and sarnow, 1992) . both ptb and pcbp have been implicated as itafs for hav (yi et al., 2000; zhang et al., 2007) . hav 3cpro inhibits hav ires-dependent translation and cleaves ptb. this finding suggests ptb cleavage regulates the switch from viral translation to rna replication and strengthens a role of ptb as an itaf for this ires (kanda et al., 2010) . the smaller type 4 ires elements, ( $ 300 nt) hepatitis c virus (hcv) and pestiviruses such as bovine viral diarrheal virus (bvdv) and classical swine fever virus (csfv) have been extensively studied. the hcv ires has simpler factor requirements than type 1 or 2 iress. it requires only one canonical initiation factor, eif2, and no itafs to form 48s translation initiation complexes in vitro with reconstituted systems, it interacts directly with ribosomes and ribosomal 18s rna (malygin et al., 2013; pestova et al., 1998) and can recruit translation factor eif3 (ji et al., 2004; siridechadilok et al., 2005) . despite its simpler minimal requirements in vitro, like poliovirus, several factors have been described that enhance hcv translation in lysate systems and cells, including la protein. ptb also weakly interacts with the ires, but appears to attenuate translation instead of stimulating it and its putative role as a functional itaf involved in ribosome recruitment has been challenged (brocard et al., 2007; domitrovich et al., 2005; ito and lai, 1999) , several lines of evidence indicate that la is an itaf. la enhances translation in the context of the competitive translation environment during infection in cells with replicons or virus. sirna depletion of la inhibits hcv translation and a dominant negative form of la inhibits translation (costa-mattioli et al., 2004) in vivo. further, the fact that la protein can be exploited as a therapeutic target for hcv suggests a significant role in hcv replication. a cell permeable peptide corresponding to the n-terminal 18 amino acids of la inhibits hcv ires-mediated translation and also inhibits hcv replication in cells (fontanes et al., 2009 ). lapeptide-mediated inhibition of hcv ires-translation blocked interaction of la and other itafs (ptb and pcbp2) with the ires, but could be rescued with exogenous ptb and pcbp2. this implied that la peptide sequesters ptb and pcbp2 as its mode of action (fontanes et al., 2009) . other knockdown experiments implicated several host factors in hcv translation or hcv virus replication in cells, including la, ptb, pcbp2 and proteasome alpha-subunit 7 (psma7) (kruger et al., 2001; shirasaki et al., 2010) . all four factors are induced in huh-7.5 cells during hcv replication, suggesting the virus has coopted factors that are naturally up-regulated during infection. shrna knockdown of la repressed production of hcv core protein 70%, further supporting a role as an itaf in vivo. la has been reported to be a telomerase component, thus telomerase activity and expression of other telomerase subunits increased coordinately with la induction during chronic hcv infection (shirasaki et al., 2010) . the linkage of hcv-induced la expression to increased telomerase activity in chronically-infected liver may be important in development of hepatocellular carcinoma. evidence from in vitro translation experiments in somewhat artificial rabbit reticulocyte lysates suggest that ptb is not a true hcv itaf that facilitates 40s ribosome subunit binding to the hcv ires. rather, ptb may stimulate translation indirectly through bringing other factors bound to the x region of the 3 0 utr into proximity with the ires (brocard et al., 2007) . ptb is proposed to loop hcv rna by binding the 3 0 utr x region and 5 0 utr ires region simultaneously. another factor, nsap1 (also called hnrnp q) stimulates hcv ires translation by binding an a-rich region downstream of the initiator aug and facilitating formation of 48s ribosome initiation complex by also binding the 40s ribosome proteins. this is the first reported interaction of an itaf directly with a ribosome, thus nsap1 provides more of a translation-factor role than other canonical itafs that play "chaperone-like" roles (kim et al., 2004; park et al., 2011) . ptb also supports translation initiation of aichivirus (av) that contains a novel type of picornavirus ires that differs structurally from type 1 and 2 iress. in reconstitution experiments this ires requires interaction with both eif4g and ptb. unlike other iress, the av ires has a strong requirement for a novel nuclear factor dhx29, that is also a ribosome-associated helicase. dhx29 releases the av ires initiation codon from a strong stable hairpin to aid anticodon base pairing by the ribosome (yu et al., 2011b) (table 1) . various itafs are either essential for virus translation or play less critical roles that enhance efficiency of translation. high translation efficiency is critical for most viruses to produce sufficient proteases or other factors to control activation of innate immune pathways that would block further replication. therefore, itafs have long been considered host range factors that determine tissue and host tropism and pathogenesis. the attenuated sabin vaccine strains of poliovirus lost neurovirulence partly through nucleotide changes in the ires that alter ptb binding . restricted ires function will result from a lack of the proper itaf in certain cells. for instance, a neuronal variant of ptb (nptb) is required for the ) with sl-i through iv, the 3 0 db region containing duplicate dumbbell structures (db1 and db2) (light blue) and the 3 0 cs/sl region (red) containing the conserved 3 0 sl. all structures from sl-1 through db-2 participate in pseudoknots (not depicted). to facilitate translation poly(a)-binding protein binds a-rich sequences in the db region allowing protein-bridge looping to translation factor eif4g associated with the 5 0 cap structure. yb-1 binds the 3 0 sl and represses translation and replication. ptb and la bind 3 0 utr may also facilitate translation. (b) long range interaction between complimentary sequences in 5 0 and 3 0 regions (5 0 cs, 3 0 cs; 5 0 uar, 3 0 uar) facilitate genomic looping associated with rna replication. this looping remodels rna into new conformations that may promote binding of additional host rbps not associated with translating denv rna such as eef1, and the dsrna binding proteins nf90, nf45 and rha that bind the 3 0 sl. in hcv, nf90 binds both 5 0 and 3 0 utr of hcv rna and rha is involved in bridging 5 0 and 3 0 utrs (not depicted). a similar arrangement may exist in flaviviruses where nf90 and rha may also bind 5 0 utr stem loops and stabilize the looped structure to promote negative strand rna replication. the ns5 rdrp binds to the 5 0 sl1 which due to looping helps position the polymerase near the 3 0 terminus . ns5 may also be recruited by la, which binds the 5 0 utr. neurovirulence of the gdvii strain of tmev (pilipenko et al., 2001) . ptb1 expression patterns have a powerful tropic effect on a chimeric poliovirus containing the hrv2 ires element (called pv1(ripo)). this virus has a growth defect in mouse cells because it cannot interact with endogenous murine ptb (jahan et al., 2013 (jahan et al., , 2011 . this is proposed to interfere with the thermodynamics of folding of the iresome into a functional structure. the virus also has a severe neuro-attenuation defect in humans but retains highly specific virulence against glioblastoma cells and hela cells that express high levels of ptb (gromeier et al., 2000) . modern proteomic approaches are now being applied to identify the full complement of host factors that interact with viral rnas, and have identified over 30 proteins that interact with fmdv ires directly or indirectly (pacheco et al., 2008) . of these gemin5 was found to bind to the ires at a domain 5 hairpin flanked by a/u/c-rich sequences via its c-terminal domain. gemin5 is the rna-binding component of the survival of motor neuron (smn) complex that assembles sm proteins onto spliceosomal snrnas. gemin5 binding did not enhance fmdv translation, but rather inhibited it (table 1 ) (box 1). rna structure analysis using 2 0 hydroxyl acylation analyzed by primer extension (shape) revealed gemin5 induced conformation changes that out-competed shape changes induced by ptb. thus gemin5 may competitively inhibit ptb itaf binding (piñeiro et al., 2013) . gemin5 also interacts with the hcv ires and may provide similar functions (piñeiro et al., 2013) . gemin5 is cleaved in fmdv infection by l protease (piñeiro et al., 2012) similar to gemin3 cleavage by poliovirus that blocks assembly of the smn complex (almstead and sarnow, 2007) . gemin5 cleavage alleviates translational repression of the ires. the similarity of gemin 5 and gemin 3 cleavages suggests that inactivation of the smn complex may be required by a wide range of picornaviruses. rna decay regulator auf1 (which has 4 isoforms) is cleaved by poliovirus and rhinovirus 3cd proteinase. auf1 is a factor that binds au-rich elements in mrna 3 0 utrs and promotes rapid mrna decay and turnover. auf1 follows the familiar pattern of strong relocalization from nucleus to cytoplasm during poliovirus infection . surprisingly though, auf1 does not bind pv 3 0 utr but rather binds the ires at stem loop iv and negatively regulates rhinovirus and poliovirus infections via translation inhibition. however, discordant findings with closely related cvb3 indicated that auf1 was bound to the 3 0 utr (wong et al., 2013) . 3cpro cleavage inactivates rna binding function of auf1 and relieves translation restriction (cathcart et al., 2013) . thus, both auf1 and gemin5 act as a "hijacked" restriction factor in translation and must be remedied by inactivation by a viral proteinase. however, auf1 may not be a restriction factor for all picornaviruses. emcv infection induces strong cytoplasmic relocalization of auf1, but was not cleaved by emcv 3cpro, even at late times after infection (cathcart and semler, 2014) . caliciviruses such as feline calicivirus and human norovirus share a non-structural protein coding region that is distantly related to picornaviruses, yet translate by an unusual capindependent mechanism that does not involve an ires. rather, translation of norovirus involves interaction of initiation factor eif3 with the viral vpg protein covalently linked to the 5 0 end (daughenbaugh et al., 2003 (daughenbaugh et al., , 2006 . ptb interacts with both 5 0 and 3 0 ends of feline calicivirus genomic rna, and also subgenomic rna. fcv infection induces nuclear to cytoplasmic relocalization of ptb that is coincident with the switch from early translation to late rna replication. ptb inhibited fcv translation in vitro, thus ptb is proposed to be a negative regulator that may aid the switch from translation to rna replication . flavivirus rna is unique in containing a 5 0 cap structure, but no poly(a) tail to facilitate typical pabp binding that promotes 5 0 -3 0 looping. thus, flaviviruses use cap-dependent translation machinery but it is unknown how viral translation is promoted over cellular translation. dengue virus promotes both cap-dependent translation and a form of non-canonical translation that is not ires dependent and does not require a functional m7g cap structure (edgil et al., 2006) . surprisingly, denv can bind pabp in an a-rich region upstream of the 3 0 sl, resulting in promotion of translation (polacek et al., 2009a (polacek et al., , 2009b likely by binding cap-binding translation factor eif4g and promoting 5 0 -3 0 interactions similar to host mrna (fig. 2) . denv rna is also known to bind several host factors that could play roles in translation or rna replication, including ptb, la, y box-binding protein1 (yb-1), translation elongation factor eef-1a and p100/tudor-sn (table 1 ) (de nova-ocampo et al., 2002; garcía-montalvo et al., 2004; lei et al., 2011; paranjape and harris, 2007; polacek et al., 2009a polacek et al., , 2009b yocupicio-monroy et al., 2007; yocupicio-monroy et al., 2003) . yb-1 binds the 3 0 sl of denv and represses translation, possibly in a role that regulates the switch from translation to rna replication discussed below (paranjape and harris, 2007) , and binding of eef-1a to the 3 0 sl has no effect on translation (davis et al., 2007) (fig. 2) . la binds to both 5 0 and 3 0 utrs (garcíamontalvo et al., 2004) and could play a role of stabilizing looped rna structures via protein bridging and indirectly support translation through ribosome recycling. ptb has been proposed to play positive roles in virus replication but the mechanism(s) remains elusive. ptb relocalizes to the cytoplasm with variable completeness in infections in different cell types. ptb cytoplasmic localization is weak in huh7 cells, however ptb binds the 3 0 stem-loop region of denv rna in these infections (fig. 2) and could play roles in rna looping to promote translation and rna replication. nuclear to cytoplasmic relocalization of ptb was associated with increased denv translation and replication and sirna knockdown of ptb inhibited replication and overexpression increased replication (agis-juárez et al., 2009). however, in huh7 cells knockdown studies indicated that ptb did not have an effect on denv rna translation, but promoted negative strand rna synthesis and interacted with viral protein ns4a (jiang et al., 2009 ). thus, additional work is required to pin down the specific mechanisms of ptb-specific stimulation of denv replication. in theory, nuclear factors may not be necessary for support of viral rna synthesis. each virus synthesizes its own complete rnadependent rna replicase, an enzyme function that is uniquely not present in host cells. further, in the cytoplasm where virus replication occurs, viruses duplicate functions such as mrna capping and polyadenylation that are carried out in the nucleus for host transcripts. thus, one may not expect many dependent interactions between virus rna replicases and host factors to have evolved to support viral rna replication. for negative strand viruses that contain rna covered in nucleocapsid proteins, this may be more expected. however, plus strand rna viruses make extensive use of host rbps for important roles in the replicative process, not in rna polymerase enzymatic activity per se, but in promotion of its temporal and spatial regulation. a key feature for host factors is helping to organize complex 5 0 -3 0 genome interactions in alternate configurations that sequentially promote translation, then rna synthesis. all plus strand rna viruses are faced with the problem that the same genomic template is used for both 5 0 -3 0 transit by ribosomes and 3 0 -5 0 transit by the viral rna polymerase. since translation and rna replication machinery proceed in opposite directions there must be a regulated switch from translation to rna replication that clears the template of all elongating ribosomes before negative strand rna synthesis can take place. for plus strand rna viruses the details of this regulation are emerging. a common theme is that sufficient translation/production of key virus proteins is required before modifications are triggered in host factor structure/protein interactions that shift host factor roles from promoting translation to promoting rna synthesis. accumulating evidence indicates that both translation and negative strand rna replication occur on templates circularized via complex rna-rna, protein-rna and protein-protein interactions. further, the switch in template use involves complex shifts in these interactions allowing host factors to change roles simultaneously. pcbp2 helps mediate a switch from poliovirus translation to rna replication due to changes in its rna binding properties. pcbp binds both the ires and 5 0 cloverleaf (cl) stem b, and its binding the cloverleaf stimulates translation early during infection (gamarnik and andino, 1998) . the pcbp complexed on the 5 0 cloverleaf promotes translation in conjunction with the 3 0 poly (a) tail in a circularization model based on protein-protein interactions. the c-terminal kh3 domain of pcbp is required to stimulate translation and can be tethered directly to pv rna and still promote translation. in the current model the 5 0 cl-pcbp complex interacts with the 3 0 poly(a)-pabp complex to form a 5 0 -3 0 circular structure that enhances translation by facilitating ribosome reloading as ribosomes recycle from the stop codon ( fig. 1b) (ogram et al., 2010) . this is consistent with the finding that pcbp2 and 5 0 cloverleaf function during de novo assembly of polysomes (kempf and barton, 2008) . thus, pcbp strongly promotes translation by interaction with the 5 0 cl in the phase before viral proteins accumulate. but once virus proteins accumulate from sustained translation, pcbp bound to the 5 0 cl also promotes binding of viral polymerase precursor 3cd to the adjacent cloverleaf stem loop d (sld). the recruited 3cd promotes translation inhibition (gamarnik and andino, 1998) . further, cleavage of pcbp1 and 2 mediated by 3cpro occurs during this phase of the replication cycle (fig. 1b) . cleavage occurs between the kh2 and kh3 domains, resulting in truncated pcbp2 lacking the kh3 domain that cannot support translation but binds the cl and supports rna replication (perera et al., 2007) . in fact, several itafs for pv are cleaved by 3cpro in midphase of the replication cycle. in addition to pcbp, ptb is cleaved by 3cpro in a reaction that was shown to inhibit pv translation (back et al., 2002) and la is similarly cleaved (shiroki et al., 1999) . even though cleavage of pcbp2 and ptb inhibits poliovirus translation and contributes to a switch in rna template usage, for related rhinoviruses things may be different. pcbp2 and ptb are differentially cleaved during infection with three hrv serotypes in hela cells but are not cleaved in human lung epithelial cells that sustain productive infections. this suggests that some itaf cleavages may not be required by hrv to mediate a switch in template usage (chase and semler, 2014) . like picornaviruses, hcv must clear genomic rna templates of ribosomes to allow use by rna replicase complexes. several insights have emerged about how hcv regulates the switch and mechanisms involve both host factors and accumulation of nascent hcv proteins. early studies showed hcv core protein binds to the ires and linked translation production of hcv core protein to translation repression in a direct feedback loop (zhang et al., 2002) . expression of hcv ns3 protease also has the effect of blocking ires translation and increasing replicon replication. ns3 protease and la protein compete for binding the ires in the same sliv region, thus ns3 was proposed to directly inhibit itaf binding to the ires, thereby reducing translation activity (ray and das, 2011) . additional work indicated that la protein binds to a gcac sequence near the initiator aug in sliv of the ires, but this enhances rna replication, not translation, by promoting linkage between 5 0 and 3 0 utrs through la (kumar et al., 2013) . since both la and ns3 have similar dissociation constants for binding hcv ires rna, ns3 may not actually evict all the ires-bound la, but competitively reach a binding equilibrium that promotes replication by recruiting ns5b to a replication complex involving la, ns3 and both ends of the viral rna (kumar et al., 2013) . another class of nuclear factors called nf/nfar proteins nf90/nfar (nuclear factor associated with dsrna, also called ilf3) bind to both ends of the genome of hcv and the pestivirus bvdv (isken et al., 2007 (isken et al., , 2003 (isken et al., , 2004 . nf90/nfar is a part of group of interacting nuclear factors containing dsrna-binding motifs and multiple isoforms that include nf45 and rna helicase a (rha). nf90/nfar is associated with hcv translation inhibition and increased rna replication (isken et al., 2007 (isken et al., , 2004 (isken et al., , 2003 . another group used sirna approaches to show that efficient hcv translation was dependent upon several members of the cellular 5 0 -3 0 deadenylation-dependent mrna decay pathway, rck/p54, lsm1, and patl1. the requirement of these factors for efficient translation was linked to the 5 0 and 3 0 -utrs. the 3 0 utr also interacted with lsm1-7 complexes. all of these factors are core components of p-bodies where non-translating silenced mrnas are stored before undergoing decay. this raises the possibility that the decay factors could play roles in the switch to replication, but the proposed role from experiments for hcv interaction with reconstituted lsm1-7 complexes was support of translation (scheller et al., 2009) . it is possible that co-opting p-body components downregulates functions of p-bodies that repress hcv translation. hcv infection results in significant reductions in the number of p-bodies in cells both in vitro and also in hepatocytes from patients infected with hcv (pérez-vilaró et al., 2014 . rna synthesis of viral negative strands initiates replication of genomic templates from the 3 0 -terminus. thus, it was a surprise when rna polymerase complexes for poliovirus were found to bind near the 5 0 terminus of the plus strand genome template on the cloverleaf (andino et al., 1993) . this suggested that the template is circularized rather than linear to allow close proximity of the replicase complex and 3 0 end of the template where the polymerase must initiate. cellular mrnas and viral genomes are thought to translate optimally on rnas circularized by interactions between translation factors eif4g, eif4b and pabp that promote ribosome recycling (kuyumcu-martinez et al., 2004) . thus, rna replication may first begin on templates transitioning from a circular configuration. indeed, this paradigm seems to be conserved among many plus strand rna viruses. in viruses, the long-range interactions that circularize templates are mediated by either protein-protein interactions or rna-rna interactions or both. for pv, circularization is also promoted by a complex of pcbp bound to the cloverleaf that interacts with pabp bound to poly (a) tail forming a protein-protein bridge (herold and andino, 2001 ). an analogous arrangement occurs in coronavirus mouse hepatitis virus (mhv) where interaction between 5 0 and 3 0 ends is promoted by another protein bridge involving ptb bound to 5 0 utr and hnrnp a1 bound to the 3 0 utr (huang and lai, 2001) . for dengue virus cyclization occurs via long range rna-rna interactions of complementary sequences in the corresponding 5 0 and 3 0 utrs (filomatori et al., 2011; lodeiro et al., 2009; polacek et al., 2009a polacek et al., , 2009b sztuba-solinska et al., 2013; villordo et al., 2010) (fig. 2) . in hcv an rna sequence in the ns5b gene region interacts with the 3 0 utr via a kissing-loop to facilitate placement of the polymerase at the exact 3 0 end of the genome (friebe et al., 2005) . the latter two examples above emphasize rna-rna interactions without factors, but it is possible that host factors could promote or stabilize these long-range interactions. for instance, hcv interactions may be aided by hnrnp a1, which binds ns5b and both ends of the genome (kim et al., 2007) . finally, the host rbp nf90/nfar circularize the hcv genome by binding both ends of the genome. these interactions may occur sequentially after kissing loop and hnrnp a1 interactions form as nf90 binding blocks translation and stimulates rna replication (isken et al., 2007 (isken et al., , 2004 (isken et al., , 2003 . rha can also play a role in the complex with nf90/nfar-1 as a bridging factor between the 5 0 and 3 utrs of hcv that promotes genome circularization (isken et al., 2007) . several of these factors also bind to denv2 3 0 utr (nf90, nf45, rha) and nf90 is critical for replication (gomila et al., 2011) . together, these findings suggest that conformations of circularized genomic rna can differ between translation and replication. accordingly, switching between translation and replication can be facilitated by virus recruitment of host factors that provide conformational remodeling of rna structures. for the coronavirus mouse hepatitis virus (mhv), crosstalk between the viral utrs relies on interactions between ptb and hnrnp a1, which bind the 5 0 utr and 3 0 utr, respectively. mutations that block interaction of these factors with mhv rna reduce replication and transcription (huang and lai, 2001) . mhv replication was not restricted in cells that do not express hnrnp a1, since the viral rna can also bind several other hnrnp proteins in replacement roles. these were identified by rna affinity with negative strand utr to be hnrnp a2/b1, hnrnp a/b and hnrnp a3, all which are closely related to hnrnp a1 (shi et al., 2003) . for enteroviruses there is a clear dependence on co-opted pcbp2 to promote negative strand rna synthesis built on the platform of the extended 5 0 cloverleaf. only intact pcbp functions in translation but either intact or cleaved (by 3cpro) pcbp2 can bind the 5 0 cloverleaf (cl) on stem-loop b(slb) (perera et al., 2007) and promote genome circularization and recruitment of 3cd to the cloverleaf on the adjacent loop, positioning near the 3 0 poly (a) template due to the protein-bridge circularization (herold and andino, 2001) . initial reports indicated the pcbp binding region was cl slb, however this was later shown to be insufficient, that an additional c-rich spacer region located just downstream of the cl is also required for pcbp binding. mutation of six c residues in this region to a abolished replication in hela cells but had no effect on translation. it was found that pcbp did not bind to 88 nt cl alone and but did bind 142 nt extended cl including this region (toyoda et al., 2007) . point mutations in this region strongly affect pv neurovirulent phenotypes (de jesus et al., 2005) . similarly, pcbp2 was shown to bind the analogous c-rich spacer region in cvb3 rna (fig. 1c) . pcbp2 did not bind cvb3 cl alone but 3cpro binding to sld allowed recruitment of pcbp2 to the minimal cl (zell et al., 2008b) . also, pcbp kh domains 1 and 3 interact with the extended cloverleaf rna of cvb3 (zell et al., 2008a) . taken together these reports make it clear that pv co-opts pcbp to provide multiple functions for virus replication, including: promoting genome circularization, as an itaf, switching from translation to rna synthesis, and properly assembling the replicase on the 5 0 cl. the cl is a required partner in several of these steps and can be seen as a general promoter for rna synthesis that provides a platform for binding host factors and polymerase (vogt and andino, 2010) . nucleolin is a shuttling rna helicase that is largely concentrated in the nucleolus and binds pre-rrnas. nucleolin was initially reported to bind poliovirus 3 0 utr and enhance replication and may play roles in negative strand rna replication (waggoner and sarnow, 1998) . subsequently another report indicated nucleolin interacts with poliovirus ires and enhances ires-dependent translation (izumi et al., 2001) . whether interactions at two sites on opposite ends of the viral genome require one nucleolin moiety or looped circularized rna has not been determined and more precise functional roles of nucleolin have not been identified. fmdv interacts with rna helicase a (rha) and alters its distribution in the cell from mostly nuclear to cytoplasmic. rha is involved in diverse cellular functions as it enhances gene expression by interacting with cbp/p300 and rna polymerase ii and responds to ifn alpha signaling to increase its activity (aratani et al., 2001; fuchsová et al., 2002) . during fmdv infection rha can bind the s domain of the virus 5 0 utr and co-precipitates with fmdv 2c and 3a proteins that function in the replicase complex. though the precise function of rha remains undetermined, knockdown of rha reduces virus titers, indicating that rha plays roles in supporting replication (lawrence and rieder, 2009 ). fmdv-induced movement of rha out of the nucleus is associated with demethylation or arginine residues at the c terminus and did not require activity of fmdv leader protein. rather nuclear egress involves demethylation activity provided by jumonji c-domain containing protein 6 (jmjc) that is stimulated by fmdv (lawrence et al., 2014) . flavivirus 3 0 utrs are complex structures that contain cisacting elements required for translation, cyclization and replication. accordingly, this regulatory rna region binds several host factors that may play roles in rna synthesis, including la, ptb, yb-1, eef1a, nf90, rha, and nf45 (fig. 2) . flavivirus rna replication is dependent on many of these co-opted host factors, however not all are thought of as nuclear factors. translation elongation factor 1a (eef1a), which is abundant in both the cytoplasm and nucleus, binds to three sites within the 3 0 utr of west nile virus and other flaviviruses. this interaction supports negative strand rna synthesis but not translation (blackwell and brinton, 1997; davis et al., 2007) . eef1a immunoprecipitates with the ns5 replicase complex proteins of wnv and colocalizes with replication complexes, further supporting a role for this factor in minus strand rna synthesis (davis et al., 2007) . flavivirus rna shifts to an alternate conformation based on complex long-range binding interactions that form a new panhandle structure. this panhandle structure positions 5 0 and 3 0 termini close together and promotes rna replication (fig. 2) . binding of a complex of double-strand binding factors nf90, rha and nf45 to the 3 0 sl likely occurs in concert with the formation of the panhandle and promotes rna replication (gomila et al., 2011) . the possibility exists that these factors may also bind the 5 0 utr and stabilize the long-range looped structure similar to their role with hcv rna (isken et al., 2007) . further, la binds to both 5 0 and 3 0 utrs and could play a role stabilizing this structure as well as recruiting ns5 and ns3 (garcía-montalvo et al., 2004) . the ns5 rdrp binds to the 5 0 sl1 which due to looping helps position the polymerase near the 3 0 terminus . similar to picornaviruses and hcv, ptb also binds the 5 0 utr of dengue virus (denv) and japanese encephalitis virus (jev) (anwar et al., 2009; kim and jeong, 2006) . although the 5 0 utr binding site suggested a possible role of ptb in flavivirus translation, ptb knockdown inhibits denv negative strand rna replication and does not affect virus translation or viral entry. ptb also interacts with ns4a protein, a component of the replicase, further suggesting that ptb plays a role in viral rna replication (anwar et al., 2009; jiang et al., 2009) . surprisingly, ptb knockdown did not inhibit jev replication so its requirement is not conserved. ptb also binds 3 0 utrs of both viruses. though the 5 0 utr of both viruses are conserved, the differential requirement for ptb may stem from the fact that the 3 0 utrs are divergent. in addition, ptb translocates to the cytoplasm in denv-infected cells and is found colocalized with calnexin endoplasmic reticulum marker, ns1 and ns3 (agis-juárez et al., 2009) . further work will be required to elucidate precise functional roles of ptb in flavivirus replication. for hcv, as discussed above, the switch from translation to rna replication is stimulated by binding of core and ns3 to the ires. however, some translation may still occur and be coupled to rna replication at the same subcellular membrane compartment termed a replicasome. pulse chase and other analyses in infected cells supported this conclusion since restriction of rna synthesis with an ns5b polymerase inhibitor reduced translation even when levels of hcv rna were unaltered (liu et al., 2012) . as with any rna virus, the overlapping, ongoing replication and translation processes make separation of the roles of host factors very difficult to tease apart. even though ptb may not be a bona fide itaf for hcv, it interacts with the poly(u/c) tract in the hcv 3 0 utr and may promote replication. during infection in huh7 cells a phosphorylated form of ptb not found in uninfected cells associates with the membrane-bound hcv replication complex (chang and luo, 2006) . synaptotagmin-binding cytoplasmic rna-interacting protein (syn-crip)(nsap1)(hnrnp q) is another member of the heterogenous nuclear ribonucleoprotein (hnrnp) family that plays roles in mrna maturation and also binds hcv rna and enhances ires-dependent translation. syncrip/hnrnp q also plays a role in rna replication as it associates with the replication complex in membrane fractions and colocalizes with nascent virus rna. immunodepletion or sirna knockdown of nsap1 decreases rna replication, indicating dual functions of nsap1 in the hcv replicative cycle . finally, the rna chaperone nucleolin has also been proposed to play a role in hcv replication, as it binds the virus ns5b rna polymerase via its glycine-arginine-rich domain. transient expression of ns5b recruits or sequesters nucleolin from the nucleus to cytoplasm and may modulate the oligomerization of ns5b that is required for rnrp activity (hirano et al., 2003; kusakawa et al., 2007) . after sindbis virus (sinv) infection numerous nuclear proteins shift to the cytoplasm including hnrnp k, hnrnp a1, and hur. hnrnp a1 interacts with the viral 5 0 utr, and with the genomic (g) and subgenomic (sg) rna promoters. knockdown of hnrnp a1 resulted in markedly decreased synthesis of g and sg rna both in infected cells and in vitro (gui et al., 2010; lin et al., 2009) . a recent report also indicates that sinv infection results in cytoplasmic relocation of ptb and tia1, but no specific function of these factors in replication is known (sanz et al., 2015) . several reports indicate that sinv nonstructural proteins interact with stress granule nucleating proteins g3bp1 and g3bp2, including nsp4 polymerase (cristea et al., 2010) , nsp3 and nsp2 (cristea et al., 2006) (gorchakov et al., 2008) . the interaction of g3bp1 with nsp3 has been shown to have the proviral effect of inhibiting stress granule formation (panas et al., 2012) . tia1 and ptb also enter the cytoplasm during sinv infection but not during ectopic expression of nsp proteins, indicating replicative processes may be required (sanz et al., 2015) . recently the combined approach of isotope labeling of purified semliki forest virus replicase/ modified lysosome complexes with proteomics analysis identified 78 host proteins associated with the functional replicase. several familiar proteins colocalized with replicase including pcbp1, hnrnp m, hnrnp c, and hnrnp k. silencing experiments indicated that hnrnp m and c are antiviral for sfv, chikungunya and sinv. differential silencing results with hnrnp k indicated opposite roles of this rbp in chikv and sinv versus sfv. this suggests that interactions of host factors with replicase complexes are not always proviral and that the roles of hnrnps may be interchangeable or more nuanced in various cells/ virus combinations (varjak et al., 2013) . nuclear factors modulate astrovirus and norovirus rna replication ptb also binds the 3 0 utr of astrovirus. seven host proteins from caco2 cell lysates could be cross-linked to 3 0 -utr rna probes in vitro and mobility shift assays indicated that two complexes of host factors form on 3 0 utr probes and that ptb is part of one of the complexes. sirna knockdown of ptb reduced virus replication, suggesting that ptb is required for replication (espinosa-hernández et al., 2014) . it is unclear if negative strand or plus strand rna replication is affected by ptb. human norovirus was shown to bind la and ptb to the 3 0 utr that contains a small stem-loop structure (gutiérrez-escolano et al., 2003) , however, since human norovirus cannot be cultivated in vitro, most recent work has focused on murine norovirus that replicates in macrophage cell lines. murine norovirus rna contains three stable stem loop structures and a single stranded polypyrimidine (py) tract within the 3 0 terminal stem loop. both ptb and pcbp bind the py tract, but this is not essential since viruses lacking this region are viable in cells and in mice. however, py-deleted virus suffer a fitness cost and is less virulent in mice . rna affinity chromatography-mass spectroscopy identified over 50 host factors that bound to discreet structures in murine norovirus rna. many were common to other rna viruses discussed above, including ptb, la, ddx3, nucleolin, hnrnpk, pcbp1/2, eef1a and hnrnp a species. sirna knockdown of ptb, la and ddx3 in raw264.7 cells resulted in deficient virus replication (vashist et al., 2012) suggesting these play roles in replication, but much more work will be required to elucidate how these factors function in norovirus infections. a long range 5 0 -3 0 rna-rna looping interaction was described in murine norovirus rna that is stabilized by binding pcbp2 and hnrnp a1. mutations in the rna-rna complimentary binding motif reduced binding of both host factors to rna and also inhibited rna replication; and pcbp2 and hnrnp a1 colocalized to virus replication complexes in cells. all these results indicate the looping interaction stabilized by the host factors plays an important roles in replication (lópez-manríquez et al., 2013) . feline calicivirus also binds nucleolin on the 3 0 utr together in a complex with viral protein ns6/7 and infection of feline kidney cells results in nuclear-cytoplasmic relocalization of nucleolin to sites enriched for ns6/7 (cancio-lonches et al., 2011) . finally, ptb plays a negative role in calicivirus translation and therefore may promote the switch from translation to replication . several host proteins interact with the 3 0 end of the transmissible gastroenteritis coronavirus (tgev) genome. genomic rna ends were used as bait in rna affinity pulldowns to identify host factors. the only protein to bind the 5 0 end was ptb, whereas nine other proteins bound the 3 0 terminus rna, including several hnrnps, pabp and two amino-acyl trna synthetases. knockdown of pabp, hnrnp q and glutamyl-prolyl-trna synthetase inhibited replication of a replicon, indicating that they may play functional roles in replication (galán et al., 2009) . since hnrnps oligomerize, they may participate in rna-protein complexes by bringing transcription-regulating sequences (trs) trs-l and trs-b into close proximity to facilitate coronavirus discontinuous transcription (sola et al., 2011b) . further investigation of the role of ptb in tgev replication indicated that ptb affects virus rna accumulation and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites. interestingly, sirna knockdown of ptb in two cell lines increased virus mrna levels and virus titer, suggesting that ptb interaction inhibits virus replication, the opposite of most other rna viruses. ptb relocalized from the nucleus to discreet structures that contained tia1 and tiar and may be reminiscent of stress granules. these foci also contained viral genomic and subgenomic rna and ptb could play a role in sequestration of some virus rna into novel foci involved in posttranscriptional regulation of virus gene expression (sola et al., 2011a) . there is not substantial evidence that the tia1-foci are bonafide stress granules but may represent replication complexes. nuclear factors regulate enterovirus plus strand rna synthesis compared to minus strand synthesis, less is known about the synthesis of plus strands from the minus strand anti-genomic template in rna virus systems such as poliovirus. the template for plus strand synthesis is different, being mostly double-stranded rna instead of single stranded rna, and this may have profound influence on the host factors that interact with virus rna. this double stranded structure is called replicative form (rf) in picornaviruses. hnrnp c supports virus positive strand replication by binding to the anti-cloverleaf that forms on the 3 0 end of the minus strand of rf (fig. 1d ). since the rf template is completely double stranded, the anti-cloverleaf can only form by dsrna breathing allowing strand separation at the terminus. the strand separation is aided progressively by host factors that recognize the cognate plus strand stem-loop b and anti-stem-loop b, which are pcbp2 and hnrnp c1/c2, respectively and prevent renewed basepairing that would zip the rf back to completely double-stranded structure. in this situation there is opportunity for the factors pcbp and hnrnp c to interact to support positive strand rna synthesis, yet this has not been reported. hnrnp c also is relocalized from the nucleus to the cytoplasm during infection and like pcbp2, can interact with pv 3cd polymerase precursor. a current model proposes that hnrnp c maintains the 3 0 end of the template in a single strand form via its chaperone function and then recruits the polymerase (brunner et al., 2005) . using the hela in vitro replication system, depletion of hnrnp c inhibited production of plus strand rna product, and replenishment with full length but not truncated hnrnp c restored rna synthesis (brunner et al., 2005) . hnrnp c also interacts with the 5 0 end of the negative strand rna on a stem loop structure that can only form through breathing of the full length double strand rf replication intermediate (ertel et al., 2010) . although rf is typically depicted in cartoons as a linear structure, this finding raises the possibility that oligomerization of hnrnp c could maintain a loop with both ends of the double-stranded template in close proximity to promote rna strand synthesis (fig. 1d ). hnrnp c functions in pre-mrna processing as a tetramer that forms via a coil-coil interactions involving its oligomerization domain (whitson et al., 2005) . an engineered tandem cloverleaf approach was used to separate functions required for pv minus strand rna synthesis from plus strand rna synthesis. this approach revealed several requirements for plus strand synthesis that were surprising. first, the short stem a is essential for plus strand synthesis but not minus strand synthesis, suggesting the stem-loop structure is functional on the anti-cloverleaf. second, the complete plus strand version of the cl is required, including binding sites for pcbp and 3cd polymerase precursor. a trans-initiation model was proposed where replicase builds on the plus strand cl to prime plus strand synthesis the same way it does for negative strand rna synthesis (vogt and andino, 2010) . however, hnrnp c binding to the anti-cl will also help position the 3 0 end of the negative strand to allow precise rna replication initiation by the replicase. after synthesis of pv plus strand transcripts an rna processing step removes vpg at the 5 0 end of a portion of transcripts. all pv rna associated with polysomes lacks vpg, having been removed by a host unlinkase enzyme, whereas all encapsidated rna retains vpg. cellular 5 0 tyrosyl-dna phosphodiesterase-2 (tdp2) is a hijacked cellular dna repair enzyme that performs the vpg unlinkase function for enteroviruses. pv infection relocalizes the bulk of tdp2 from the nucleus to cytoplasm. a hypothesis predicts that vpg is a capsid packaging signal that must be removed so that a pool of nascent transcripts can continue to engage ribosomes and produce additional virus proteins (virgen-slane et al., 2012) . the effect of preventing vpg unlinking by using click chemistry to form an uncleavable bond for tdp2 was tested and found to not affect translation or replication efficiency, indicating that vpg does not inhibit initial steps in either process (langereis et al., 2013) . in flaviviruses, plus strand rna synthesis is initiated by a promoter-protein complex that builds on a conserved stem-loop structure on the 3 0 terminal 96 nt of minus strand rna. this structure is specifically bound by four host proteins, one of which is la autoantigen (yocupicio-monroy et al., 2003) and another is tiar. tiar and its closely related paralog tia1 are nuclear proteins that shuttle into the cytoplasm during environmental stress and help nucleate formation of rna stress granules. tia1 also binds wnv ( à ) rna and both proteins bind wnv rna through their conserved rrm2 motifs (li et al., 2002) . wnv replication was repressed in tiar knockout cells and mutations of the uaauu tiar recognition motif in the 3 0 sl affected replication but not translation. several mutant viral rnas that only weakly bound tiar rapidly reverted to wild type phenotypes in vivo, suggested that tiar interaction was not required for low level minus strand replication but allowed efficient high level plus strand rna synthesis from the minus strand template (emara et al., 2008) . the ability of wnv and denv to hijack tiar and tia1 from the nucleus and place it into new roles in rna synthesis provides another advantage; hijacking also sequesters these proteins from their role in forming stress granules (sg) (emara and brinton, 2007) . many viruses induce sgs by interrupting cellular pathways and homeostasis, particularly protein synthesis, and by triggering activation of pkr. these virus-derived perturbations drive sg formation through phosphorylation of eif2 à or other mechanisms (kedersha et al., 2013 (kedersha et al., , 1999 reineke and lloyd, 2013) . accumulating evidence suggests that sgs may provide platforms to activate innate immune functions and they are seen as antiviral (lloyd, 2013) . indeed, many viruses have evolved mechanisms to suppress sg formation, and co-opting key sg nucleating factors such as tiar and g3bp1 is one mechanism employed by flaviviruses and hcv (ariumi et al., 2011; emara and brinton, 2007; ruggieri et al., 2012) . further experiments with chimeras of wnv that had different rna replication efficiencies and different abilities to induce or control sgs indicated that early viral rna synthesis cannot exceed the ability to protect product dsrna with virusinduced membranes. virus-induced membranes block access of pkr to the dsrna intermediates. viruses that replicate too quickly overwhelm the limited virus-induced membranes that protect dsrna, allowing pkr activation and sgs formation that inhibits replication (courtney et al., 2012) . several plus strand viruses have multiple open reading frames and use subgenomic plus strand transcripts (sgrna) to express structural proteins. the synthesis of subgenomic plus strand rna is regulated differently than genomic rna, however is still controlled by transcription elements that function in cis or trans. this provides the opportunity for subgenomic transcription elements to assemble alternate replicase complexes containing different host factors than those modulating genomic rna replication. coronaviruses promote sgrna transcription using interactions between 5 0 terminal leader sequences and intergenic (ig) sequences just upstream of each orf. one host factor, hnrnp a1 binds to both leader and ig sequences in mhv negative strand template rna (li et al., 1997) . overexpression of hnrnp a1 promotes mhv rna replication while a dominant negative hnrnp a1 reduces replication (shi et al., 2000) and the interaction of hnrnp a1 with the leader and ig sequences is critical for in vitro sgrna transcription . hnrnp a1 could function by recruiting virus n nucleocapsid protein (wang and zhang, 1999) . ptb is another prominent nuclear factor that may regulate sgrna transcription as it binds the plus strand leader sequence and also the 5 0 utr in minus strand rna in reactions that promote sgrna replication specifically li et al., 1999) . the alphavirus sindbis virus induces a shift of hnrnp k from the nucleus to cytoplasm where it colocalizes with viral nonstructural protein 2 and co-immunoprecipitates with subgenomic but not genomic rna, nsp1 and nsp2. sirna knockdown of hnrnp k reduced gene expression from a subgenomic promoter. these findings indicate that hnrnp k has a role, details yet to be determined, in sgrna synthesis (burnham et al., 2007) . viruses must maintain the integrity of their viral rnas and suppress rna decay pathways. alphavirus and coronavirus genomes are capped and poly-adenylated, retaining two modifications that stabilize mrnas against rna decay. however, binding of certain host factors to virus rna can also protect against rna decay machinery. hur is associated with prolonged mrna stability in cells and binds to u-rich elements (ures) in alphavirus 3 0 utrs (garneau et al., 2008; sokoloski et al., 2010) . as with many other factors, hur is selectively relocalized to the cytoplasm during sindbis virus infection. two other alphaviruses, ross river virus and chikungunya virus, lack the conserved 3 0 ures but still tightly bind hur via alternative binding elements (dickson et al., 2012) . knockdown of hur increased decay of sindbis virus rnas and reduced virus replication yields in both human and mosquito cells, indicating that hur binding to sinv rna blocks components of the cellular rna decay pathway (sokoloski et al., 2010) . other viruses lack either 5 0 m7gtp caps or poly(a) tails and may specifically co-opt or target host factors to protect against decay. many of these are resident cytoplasmic factors but some are nuclear shuttling proteins. the pv 5 0 cl binds pcbp to support rna replication as discussed above, but this also stabilizes pv plus strand rna in hela lysates. a mutant pcbp protein that cannot bind the cl was associated with increased decay of pv rna in the hela in vitro replication system and it was proposed that pcbp binding blocks xrn1 exonuclease activity. this was supported by showing that capped pv rnas bypassed the requirement for pcbp2 in stability assays (murray et al., 2001) . poliovirus also achieves greater rna stability through cleavage and proteolytic degradation of rna decay factors xrn1, dcp1a and pan3 by poliovirus proteinases and virus-activated proteasomal decay (dougherty et al., 2011) . further, 3cpro cleavage of the rna destabilizing factor auf1 likely contributes to poliovirus rna stability wong et al., 2013) . flaviviruses generate short non-coding rnas (sfrna) from the 3 0 utr due to the ability of this structure to stall and sequester xrn1. denv2 sfrna accumulates during infection and acts a sponge and binds host factors g3bp1, g3bp2 and caprin1. sequestration of g3bp was linked to dysregulation of translation of several mrnas of specific interferon stimulated genes, thus interfering in innate immunity . importantly sfrna also sequesters xrn1, and results in dysregulation of host mrna stability and accumulation of uncapped mrna decay intermediates in cells (moon et al., 2012) . the stem-loops and u-rich tract contained in the 3 0 utr of hcv bind la protein and inhibit decay of hcv rna in hela extracts (spångberg et al., 2001) . similar to alphaviruses, the hcv 3 0 ure can bind hur and knockdown of hur expression in cells resulted in reduced hcv translation and rna replication using replicon models (korf et al., 2005; spångberg et al., 2000) . the stimulatory effects of hur on ires translation were confirmed using dicistronic vectors in rabbit reticulocyte lysates, xenopus laevis oocytes and hela cell extracts but may not involve direct interaction of hur with the ires (rivas-aravena et al., 2009) . though hur is associated with rna stability in many systems, its importance for hcv rna remains unclear. proteomics analysis indicates more than 70 host proteins are enriched from cells with hcv 3 0 utr probes and several of these are associated with regulation of rna stability in cells, including yb-1, nf90, hnrnp c and hnrnp d . the discussion herein mostly focused on nuclear shuttling rbps and their roles in regulating mammalian plus strand rna virus translation and rna replication. however, cytoplasmic viruses exploit many other types of cytoplasmic factors involved in translation regulation, membrane formation and remodeling, and virus assembly and egress that were not discussed. as research on rna virus replication has continued, several common themes have emerged. first, the array of host proteins that plays significant roles in replication will continue to grow as the newly expanded list of interacting factors identified from proteomics approaches undergo analysis. second, although many host proteins are hijacked by viruses, several ubiquitous host proteins, such as ptb, pcbp and hnrnp a1 are used by several classes of viruses, often in different ways. third, the production of key virus proteins shifts the function of some host factors from support of translation to rna replication. fourth, the rna chaperone functions described for host factors in supporting iresome activity often provide similar or analogous chaperone functions supporting rna structures and long range interactions in rna synthesis. fifth, the new concept of host factors being sequestered or sponged away from normal host roles may be common, and may be a variant form of hijacking. sixth, some interactions of host factors with virus rna inhibit replication or translation and must be opposed by viruses. many nuclear host factors discussed are highly conserved, which can enable cross-species infection in the case of alphavirus and flaviviruses, but also restrict virus tissue tropism and control pathogenesis in mammals where gene expression patterns are so variable. also, the complete lists of factors used by any one virus are unique evidence that viruses evolve many different ways to take over cell functions. however, many of the diverse host factors that are hijacked perform related functions for the virus in replication. our understanding of co-opted factors, though expanding rapidly, is far from complete. we certainly do not have a comprehensive list of factors utilized by any single virus. future expansion of proteomics research and new approaches such as tux-ms will fill in the catalog, but substantial traditional biochemical wet bench science will be required to tease apart functions and impact of newly discovered factors, many of which may be novel to science as a whole. adaptation of in vitro replication systems used for poliovirus to study other virus families will help dissect the roles of factors in viral replication complexes, particularly where the same factor may play roles in sequential replicative processes. finally, the increased use of super-resolution fluorescence microscopy and cryo-electron microscopy of whole cells will also be important to finely localize factors and virus-induced structures in cells. polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in vero cells inhibition of u snrnp assembly by a virus-encoded proteinase internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of unr to two distinct sites on the 5 0 untranslated region poliovirus rna synthesis utilizes an rnp complex formed around the 5 0 -end of viral rna glycyl-trna synthetase specifically binds to the poliovirus ires to activate translation initiation the polypyrimidine tract-binding protein is required for efficient dengue virus propagation and associates with the viral replication machinery dual roles of rna helicase a in crebdependent transcription hepatitis c virus hijacks p-body and stress granule components around lipid droplets translation of polioviral mrna is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3c(pro) encephalomyocarditis virus leader protein hinge domain is responsible for interactions with ran gtpase functional analysis of rna structures present at the 3 0 extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence encephalomyocarditis virus leader is phosphorylated by ck2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins a nucleo-cytoplasmic sr protein functions in viral ires-mediated translation initiation g3bp1, g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna flaviviral rnas: weapons and targets in the war between virus and host translation elongation factor-1 alpha interacts with the 3 0 stem-loop region of west nile virus genomic rna poly(rc) binding protein 2 binds to stem-loop iv of the poliovirus rna 5 0 noncoding region: identification by automated liquid chromatography-tandem mass spectrometry intact eukaryotic initiation factor 4g is required for hepatitis a virus internal initiation of translation unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus evidence that ptb does not stimulate hcv ires-driven translation functional interaction of heterogeneous nuclear ribonucleoprotein c with poliovirus rna synthesis initiation complexes heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna nucleolin interacts with the feline calicivirus 3 0 untranslated region and the protease-polymerase ns6 and ns7 proteins, playing a role in virus replication insights into rna biology from an atlas of mammalian mrna-binding proteins cellular mrna decay protein auf1 negatively regulates enterovirus and human rhinovirus infections differential restriction patterns of mrna decay factor auf1 during picornavirus infections the polypyrimidine tract-binding protein (ptb) is required for efficient replication of hepatitis c virus (hcv) rna unr, a new partner of poly (a)-binding protein, plays a key role in translationally coupled mrna turnover mediated by the c-fos major coding-region determinant differential cleavage of ires trans-acting factors (itafs) in cells infected by human rhinovirus the domains of polypyrimidine tract binding protein have distinct rna structural preferences upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased interactions with and expression of ptb-1 and yb-1 la autoantigen is necessary for optimal function of the poliovirus and hepatitis c virus internal ribosome entry site in vivo and in vitro west nile virus infections suppress early viral rna synthesis and avoid inducing the cell stress granule response tracking and elucidating alphavirus-host protein interactions host factors associated with the sindbis virus rna-dependent rna polymerase: role for g3bp1 and g3bp2 in virus replication the genomelinked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment 22 vpg of murine norovirus binds translation initiation factors in infected cells interaction between the cellular protein eef1a and the 3 0 -terminal stem-loop of west nile virus genomic rna facilitates viral minus-strand rna synthesis mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence translation elongation factor-1alpha, la, and ptb interact with the 3 0 untranslated region of dengue 4 virus rna dephosphorylation of hur protein during alphavirus infection is associated with hur relocalization to the cytoplasm role of la autoantigen and polypyrimidine tract-binding protein in hcv replication poliovirus-mediated disruption of cytoplasmic processing bodies dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly mutation of mapped tia-1/ tiar binding sites in the 3 0 terminal stem-loop of west nile virus minus-strand rna in an infectious clone negatively affects genomic rna amplification mechanistic consequences of hnrnp c binding to both rna termini of poliovirus negative-strand rna intermediates ptb binds to the 3 0 untranslated region of the human astrovirus type 8: a possible role in viral replication rna sequences and structures required for the recruitment and activity of the dengue virus polymerase re-localization of cellular protein srp20 during poliovirus infection: bridging a viral ires to the host cell translation apparatus a cell-permeable peptide inhibits hepatitis c virus replication by sequestering ires transacting factors kissing-loop interaction in the 3 0 end of the hepatitis c virus genome essential for rna replication nuclear dna helicase ii is recruited to ifn-alpha-activated transcription sites at pml nuclear bodies host cell proteins interacting with the 3 0 end of tgev coronavirus genome influence virus replication two functional complexes formed by kh domain containing proteins with the 5 0 noncoding region of poliovirus rna switch from translation to rna replication in a positive-stranded rna virus interactions of viral protein 3cd and poly(rc) binding protein with the 5 0 untranslated region of the poliovirus genome la protein binds to ns5 and ns3 and to the 5 0 and 3 0 ends of dengue 4 virus rna the 3 0 untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and mammalian cells nf90 binds the dengue virus rna 3 0 terminus and is a positive regulator of dengue virus replication different types of nsp3-containing protein complexes in sindbis virus-infected cells intergeneric poliovirus recombinants for the treatment of malignant glioma hnrnp a1 interacts with the genomic and subgenomic rna promoters of sindbis virus and is required for the synthesis of g and sg rna la, ptb, and pab proteins bind to the 3( 0 ) untranslated region of norwalk virus genomic rna translational enhancement of the poliovirus 5 0 noncoding region mediated by virus-encoded polypeptide 2a identification of cellular factors associated with the 3 0 -nontranslated region of the hepatitis c virus genome a cytoplasmic 57-kda protein that is required for translation of picornavirus rna by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein ires-induced conformational changes in the ribosome and the mechanism of translation initiation by internal ribosomal entry poliovirus rna replication requires genome circularization through a protein-protein bridge direct interaction between nucleolin and hepatitis c virus ns5b polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3 0 untranslated region, thereby altering rna conformation heterogeneous nuclear ribonucleoprotein a1 binds to the 3 0 -untranslated region and mediates potential 5 0 -3 0 -end cross talks of mouse hepatitis virus rna unr, a cellular cytoplasmic rnabinding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus rna nuclear factors are involved in hepatitis c virus rna replication members of the nf90/nfar protein group are involved in the life cycle of a positive-strand rna virus complex signals in the genomic 3 0 nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral rna an internal polypyrimidine-tract-binding protein-binding site in the hepatitis c virus rna attenuates translation, which is relieved by the 3 0 -untranslated sequence nucleolin stimulates viral internal ribosome entry site-mediated translation a host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, pv1(ripo) polypyrimidine tract binding protein-1 (ptb1) is a determinant of the tissue and host tropism of a human rhinovirus/ poliovirus chimera pv1(ripo) coordinated assembly of human translation initiation complexes by the hepatitis c virus internal ribosome entry site rna polypyrimidine tract-binding protein influences negative strand rna synthesis of dengue virus identification of essential host factors affecting tombusvirus rna replication based on the yeast tet promoters hughes collection activation of picornaviral iress by ptb shows differential dependence on each ptb rna-binding domain polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus ires structure via binding multiple sites in a unique orientation polypyrimidine tractbinding protein stimulates the poliovirus ires by modulating eif4g binding hepatitis a virus (hav) proteinase 3c inhibits hav ires-dependent translation and cleaves the polypyrimidine tract-binding protein polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation stress granules and cell signaling: more than just a passing phase? rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress granules poly(rc) binding proteins and the 5 0 cloverleaf of uncapped poliovirus mrna function during de novo assembly of polysomes an rna-binding protein, hnrnp a1, and a scaffold protein, septin 6, facilitate hepatitis c virus replication a cellular rna-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis c virus polyprotein initiation codon polypyrimidine tract-binding protein interacts with the 3 0 stem-loop region of japanese encephalitis virus negative-strand rna the role of ires trans-acting factors in regulating translation initiation inhibition of hepatitis c virus translation and subgenomic replication by sirnas directed against highly conserved hcv sequence and cellular hcv cofactors involvement of proteasome alpha-subunit psma7 in hepatitis c virus internal ribosome entry site-mediated translation human la protein interaction with gcac near the initiator aug enhances hepatitis c virus rna replication by promoting linkage between 5 0 and 3 0 untranslated regions functional interaction of hepatitis c virus ns5b with nucleolin gar domain systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus cleavage of poly(a)-binding protein by poliovirus 3c protease inhibits host cell translation: a novel mechanism for host translation shutoff rna looping by ptb: evidence using fret and nmr spectroscopy for a role in splicing repression modification of picornavirus genomic rna using "click" chemistry shows that unlinking of the vpg peptide is dispensable for translation and replication of the incoming viral rna redistribution of demethylated rna helicase a during foot-and-mouth disease virus infection: role of jumonji cdomain containing protein 6 in rha demethylation identification of rna helicase a as a new host factor in the replication cycle of foot-and-mouth disease virus functional interaction between cellular p100 and the dengue virus 3 0 utr viral subversion of the nuclear pore complex thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification polypyrimidine tract-binding protein binds to the leader rna of mouse hepatitis virus and serves as a regulator of viral transcription heterogeneous nuclear ribonucleoprotein a1 binds to the transcription-regulatory region of mouse hepatitis virus rna cell proteins tia-1 and tiar interact with the 3 0 stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication hnrnp a1 interacts with the 5 0 untranslated regions of enterovirus 71 and sindbis virus rna and is required for viral replication syncrip (synaptotagmin-binding, cytoplasmic rna-interacting protein) is a host factor involved in hepatitis c virus rna replication hepatitis c virus translation preferentially depends on active rna replication regulation of stress granules and p-bodies during rna virus infection structural and functional studies of the promoter element for dengue virus rna replication norovirus genome circularization and efficient replication are facilitated by binding of pcbp2 and hnrnp a1 hcv ires interacts with the 18s rrna to activate the 40s ribosome for subsequent steps of translation initiation human protein sam68 relocalization and interaction with poliovirus rna polymerase in infected cells a cellular protein that binds to the 5 0 -noncoding region of poliovirus rna: implications for internal translation initiation la autoantigen enhances and corrects aberrant translation of poliovirus rna in reticulocyte lysate a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability poly(rc) binding proteins mediate poliovirus mrna stability structure of ptb bound to rna: specific binding and implications for splicing regulation the 5 0 cl-pcbp rnp complex, 3 0 poly(a) tail and 2a(pro) are required for optimal translation of poliovirus rna riboproteomic analysis of polypeptides interacting with the internal ribosome-entry site element of foot-and-mouth disease viral rna sequestration of g3bp coupled with efficient translation inhibits stress granules in semliki forest virus infection yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of rna viruses y box-binding protein-1 binds to the dengue virus 3 0 -untranslated region and mediates antiviral effects differential targeting of nuclear pore complex proteins in poliovirus-infected cells specific cleavage of the nuclear pore complex protein nup62 by a viral protease translation-competent 48s complex formation on hcv ires requires the rna-binding protein nsap1 cellular protein modification by poliovirus: the two faces of poly(rc)-binding protein canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry a prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis c and classical swine fever virus rnas hepatitis c virus infection inhibits p-body granule formation in human livers hepatitis c virus infection alters p-body composition but is independent of p-body granules cell-specific proteins regulate viral rna translation and virus-induced disease gemin5 promotes ires interaction and translation control through its c-terminal region gemin5 proteolysis reveals a novel motif to identify l protease targets conformational changes in the solution structure of the dengue virus 5 0 end in the presence and absence of the 3 0 untranslated region poly(a)-binding protein binds to the nonpolyadenylated 3 0 untranslated region of dengue virus and modulates translation efficiency leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses interplay between ns3 protease and human la protein regulates translation-replication switch of hepatitis c virus diversion of stress granules and p-bodies during viral infection inhibition of mrna export and dimerization of interferon regulatory factor 3 by theiler's virus leader protein the elav-like protein hur exerts translational control of viral internal ribosome entry sites picornavirus modification of a host mrna decay protein dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection inhibition of host protein synthesis by sindbis virus: correlation with viral rna replication and release of nuclear proteins to the cytoplasm polypyrimidine-tractbinding protein: a multifunctional rna-binding protein translation and replication of hepatitis c virus genomic rna depends on ancient cellular proteins that control mrna fates heterogeneous nuclear ribonucleoprotein a1 regulates rna synthesis of a cytoplasmic virus la protein required for internal ribosome entry sitedirected translation is a potential therapeutic target for hepatitis c virus replication intracellular redistribution of truncated la protein produced by poliovirus 3cpro-mediated cleavage the n-terminal k homology domain of the poly(rc)-binding protein is a major determinant for binding to the poliovirus 5 0 -untranslated region and acts as an inhibitor of viral translation structural roles for human translation factor eif3 in initiation of protein synthesis sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells the polypyrimidine tract-binding protein affects coronavirus rna accumulation levels and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites rna-rna and rna-protein interactions in coronavirus replication and transcription evidence for an rna chaperone function of polypyrimidine tract-binding protein in picornavirus translation hur, a protein implicated in oncogene and growth factor mrna decay, binds to the 3 0 ends of hepatitis c virus rna of both polarities binding of the la autoantigen to the hepatitis c virus 3 0 untranslated region protects the rna from rapid degradation in vitro the mechanism of translation initiation on type 1 picornavirus iress a distinct class of internal ribosomal entry site in members of the kobuvirus and proposed salivirus and paraturdivirus genera of the picornaviridae structural complexity of dengue virus untranslated regions: cis-acting rna motifs and pseudoknot interactions modulating functionality of the viral genome replication of poliovirus requires binding of the poly(rc) binding protein to the cloverleaf as well as to the adjacent c-rich spacer sequence between the cloverleaf and the internal ribosomal entry site magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of semliki forest virus identification of rnaprotein interaction networks involved in the norovirus life cycle polypyrimidine tract-binding protein interacts with coxsackievirus b3 rna and influences its translation a balance between circular and linear forms of the dengue virus genome is crucial for viral replication an rna virus hijacks an incognito function of a dna repair enzyme an rna element at the 5 0 -end of the poliovirus genome functions as a general promoter for rna synthesis viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells distinct poly(rc) binding protein kh domain determinants for poliovirus translation initiation and viral rna replication the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo differential processing of nuclear pore complex proteins by rhinovirus 2a proteases from different species and serotypes solution structure of the symmetric coiled coil tetramer formed by the oligomerization domain of hnrnp c: implications for biological function genetics of poliovirus cytoplasmic redistribution and cleavage of auf1 during coxsackievirus infection enhance the stability of its viral genome viral subversion of nucleocytoplasmic trafficking functional significance of the interaction of hepatitis a virus rna with glyceraldehyde 3-phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function mosquito la protein binds to the 3 0 untranslated region of the positive and negative polarity dengue virus rnas and relocates to the cytoplasm of infected cells cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3 0 untranslated region rna common conformational changes induced in type 2 picornavirus iress by cognate transacting factors the mechanism of translation initiation on aichivirus rna mediated by a novel type of picornavirus ires interaction of poly(rc)-binding protein 2 domains kh1 and kh3 with coxsackievirus rna poly(rc)-binding protein 2 interacts with the oligo(rc) tract of coxsackievirus b3 rna interaction and cleavage of poly(c)-binding protein 2 by hepatitis a virus protease autogenous translational inhibition of core protein: implication for switch from translation to rna replication in hepatitis c virus formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein a1 and transcription-regulatory elements of viral rna research in the author's laboratory is supported by nih public health service grant ai50237, by nci, national institutes of health cancer center support grant p30ca1251230; and by national institutes of health grants hd007495, dk56338, and ca125123 (to the integrated microscopy core at baylor college of medicine). key: cord-007890-bie1veti authors: nan title: ecc-4 abstracts date: 2002-04-16 journal: int j antimicrob agents doi: 10.1016/s0924-8579(02)00033-x sha: doc_id: 7890 cord_uid: bie1veti nan f spain introduction: the prevalence of erythromycin resistance (er-r) in group a streptococci (gas) has increased in spain since early 90s with current rates exceeding 40% in some regions. this study determined the emm -types associated to erythromycin resistance in spain. material and methods: isolates belonged to the sauce* surveillance collection. rapid sequence analysis of specific pcr products was used to deduce emm -types corresponding to the majority of the known gas m serotypes. pcr primers used: gasm1 (5?-tattgcgct-tagaaaattaa-3?) and gasm2 (5?-gcaagttctt-cagcttgttt-3?). sequencing was done with the big dye terminator mix and autosequenator (applied biosystems). dna sequences were subjected to homology searches against the bacterial dna database. results: overall, 670 gas isolates (345 er-r) were analysed. three m-types (m4, st1812 and m12) accounted for 66.4% of the er-r isolates, whereas they just represented a 16.7% of the ery-s. for er-r isolates the strongest association was seen with m4 (or0/12; 95% ci 6.7 á/22.1), and m75 was second after m4 only in the last temporal period of the study (1998 á/1999) . no homogeneous distribution of er-r m-types by centres was seen. conclusions: few m-types (leading by m4) are responsible for the er-r in spain. but for m4, the remaining er-r m types (st1815, m12 and m75) did not show a temporally nor geographically homogeneous distribution. *sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en espana' (susceptibility to the antimicrobials commonly used in the community in spain ) and is the spanish word for the willow tree. significant increase in the prevalence of erythromycin-resistant, clindamycin and miocamycin-susceptible (m-phenotype) streptococcus pyogenes in spain (1998 ( á/2001 purpose: a variety of methods is used for a molecular typing of enterococcus spp. and related gram-positive bacteria. these include dna-based methods such as macrorestriction analysis using pulsedfield gel electrophoresis (pfge), ribotyping, and amplification-based methods such as rapid amplification of polymorphic dna (rapd) and amplified fragment length polymorphism (aflp). we used a homogeneous strain collection of 24 transconjugants resulting from filter-matings with different antibiotic-resistant e. faecium and a recipient isolate from our lab. the influence of transferred antibiotic-resistance determinants on the outcome of different typing methods was investigated. results: fragment patterns resulting from pfge indicated minor differences between the transconjugants and the recipient. in respect to different primers used for rapd, none or only a single fragment shift was detected in the resulting fragment patterns. aflp clusters all transconjugants into a group of major relatedness, but the result was strongly dependent on the mathematical method used for cluster analysis. fragment patterns of digested plasmids showed the possession of different or only widely related plasmids in the transconjugants. conclusions: the results of this study clearly show that under certain situations typing methods commonly used for enterococci and related gram-positive bacteria come to their limits. the sasss network aims to set up a national surveillance study to obtain standardized information on antimicrobial susceptibility to various bacterial pathogens. currently, 25 hospitals are participating in the project from different geographical regions in saudi arabia. during the 1st year (2001), the sasss focused on setting up this network. overall, high frequencies of resistance to antibiotics to different bacterial pathogens in saudi arabia were seen. geographical variations of resistance were noticed, which could be related to different prescribing practices. approximately, 9 and 32% of escherichia coli and k. pneumoniae , respectively, were extended spectrum â-lactamases (ebls) producers. resistance of enterobacteriacae group to carbapenem and pipracillin/tazobactam is low. resistance of pseudomonas aeurginosa to various anti-pseudomonal antibiotics including carbapenem is high and alarming. methicillin resistant staphylococcus aureus (mrsa) comprised 36% of s. aureus isolates. no vancomycin intermediate s. aureus (visa) was detected. high level resistance to gentamicin in enterococcus were seen in 12% of the isolates and only 2% of enterococcus facieum were glycopeptide resistant. resistance of streptococcus pneumoniae to penicillin ranged between 2% to almost 52%. surveillance of antibiotic resistance on a national level is necessarily to give guidance to practicing physicians on the best agents to use. the world-wide problem of betalactam resistance (r) in streptococcus pneumoniae (sp) has been complicated by increasing r to macrolides and some older fluoroquinolones (fq) (ciprofloxacin cip). aim of our study was to evaluate rate of acquisition of resistance to different fq: cip, sparfloxacin (spx) and levofloxacin (lev) of sp strains with different levels of susceptibility to penicillin (p). fifteen strains were serially and daily passaged in subinhibitory concentrations of these four antibiotics by a gradient plate method until acquisition of resistance. clinical strains isolated from children in day-care centers were used. five strains were susceptible (s) to penicillin (p) (one reference strain, four clinical isolates: p micsb/0.064 mg/l): five were intermediate (i) to p (one reference strain: p mic 0.19 mg/l, four clinical strains p mics: 0.5 á/1 mg/l), five were resistant (r) to p (p mics 1.5 mg/l). mean of number of passages (n ) necessary to reach i or r level with each fq as selecting agent are in the following table: spx and lev induced resistance but more slowly than cip. our results show that rate of acquisition of resistance to fq is strongly related to alteration of susceptibility to p, probably by modification of cell wall. these results are concordant with clinical results. clinical relevance of phase variation in pneumococcal opacity: nasopharyngeal (np) colonization in children from day care centers (dcc) soa8.11 carsenti h, mancini g, bensoussan m, dunais b, pradier ch, dellamonica p. archet hospital, infectious disease, nice, france streptococcus pneumoniae (sp) adherence to nasopharyngeal (np) epithelium is a prerequisite for induction of otitis transparent sp (t) have been shown to colonize the np of infant rats better than opaque (o) sp. opaque sp has proven more virulent than the t form during systemic infection in a mouse model. aim of this study was to evaluate phase variation in the nasopharynx of children. sp strains were isolated during a winter epidemiology study of np samples in children from family dcc. mics determinations were performed by e -test for penicillin (p), amoxicilline (amx) and ceftriaxone (cro). serotypes were performed using the quellung reaction. upon oil immersion microscopic examination short chains of six to eight cocci were noted as 0, '/, '/'/, '/'/'/ for absence, 1, 2, !/3 chains by field, respectively. phase variation was detected on catalase trypticase soja plates, amx and cro mics and bactericidal activity was determined for 10 pairs of o and t variants with different serotypes and susceptibility to penicillin. seventy strains of sp were screened for phase variation. nine out of 42 with chain length 0, '/ had o variants while 23 out of 28 strains with chain length '/'/ or '/'/'/ showed o variants. proportion of o variants was predominant when chain length increased. serotype 23f was prevalent. bactericidal activity of o variants showed a four-to eightfold increase of mbc. o variants may be present in np of children while t are predominant form for colonization. these virulent variants with lower level of autolysis showed less susceptibility to killing by antibiotics. they may persist in np and explain the absence of eradication by active molecules. antimicrobial resistance among clinical strains of s. pneumoniae isolated from patients with community-acquired respiratory tract infections (carti) in russia soa8.12 kozlov rs a , bogdanovitch tm a , sivaya ov a , agapova ed b , ahmetova li b , furletova b , gudkova lv b , gugutsidge b , ilyina vn b , marusina b , multich ig b , ortenberg ea b , schetinin ev b , shturmina purpose: to determine the antimicrobial resistance of pneumococci causing carti in different russian cities. methods: a total of 142 non-duplicate strains isolated in 14 russian cities in 2001 were studied. antimicrobials tested included penicillin (pen), amoxicillin (amo), erythromycin (ery), azithromycin (azi), clarithromycin (cla), midecamycin (mid), spiramycin (spi), clindamycin (cli), levofloxacin (lev), vancomycin (van), rifampicin (rif), tetracycline (tet) and co-trimoxazole (sxt). susceptibility testing was performed by broth microdilution with interpretation of the results according to nccls guidelines (2001) a map of bacterial resistance in a hungarian region soa8.16 farkas a a , juhasz a b , orosi p a , miszti c c , balogh m d . a kenezy teaching hospital, hygienie, debrecen, hungary , b kenezy teaching hospital, laboratory, debrecen, hungary , c university of debrecen, microbiology lab, debrecen, hungary , d regional hospital berettyóújfalu, laboratory, debrecen, hungary background: regional trends of microbiological resistance pattern constitute basic data and qualifying criteria for effective infection control. purpose: the aim of our study was to establish an internationally compatible regional database in a hungarian county hajdú -bihar. methods: our model is the national nosocomial infections society publications' format from the u.s. published in 2001. it contains data regarding various icu types, ambulatory patients and hospitalised patients. the same format is used for antibiotic utilisation data and device related infections' rates as well. we collected cleaned data of years 2000 á/2001 from all the microbiological laboratories of our county. results: ciprofloxacin p e. coli 6.8 4.5 9.6 75 á/above 90 susceptibilities not significantly different from u.s. data were as follows: mr cns, streptococcus pneumoniae /penicillin and 3rd generation cephalosporin, pseudomonas aeruginosa /piperacillin and enterobacter spp. and escherichia coli /ceftriaxon. conclusions: this database proved to be a very useful tool for choosing primary wards of active surveillance including places for infectious disease physician's visit (icu, rehabilitation unit). additional analysis is needed at an individual institution's level for other heavily used (or useable) antibiotics and bacteria as well (aminoglycosides, beta-lactam á/beta lactamase inhibitor combinations, 2nd generation cephalosporins, corynebacteria . to compare epidemiological, clinical, and immunological features of add before and after haart introduction, between the 436 patients (p) diagnosed in 1985 á/1995, and the 103 p detected since 1997, in a case-control study. though the mean number of newly diagnosed aids p had a sharp drop in the haart era, from 65 p/year in 1991 á/ 1995 to 22 p/year since 1997 (p b/0.001), the distribution of add and underlying immunodeficiency showed limited changes. when excluding a greater frequency of tuberculosis (tb) (p b/0.001) and wasting syndrome (p b/0.04), all other add did not show a different frequency before and after 1997. a tendency towards a higher mean cd4 count at aids disease was noticed: 78 vs 61 cells/ml (p b/0.003), with a significant difference for candida esophagitis , toxoplasmosis, kaposi sarcoma and tb (p b/0.002 á/b/0.03). the limited variation of clinical and immunological presentation are attributable to the poor impact of haart before aids recognition: 88.4% of p detected since 1997 did not receive haart or had insufficient compliance to antiretrovirals, so that 58.2% of p were aids presenters. during the haart era, an increase of mean age and sexual transmission was found (p b/0.001). notwithstanding the effects of haart on the natural history of hiv disease, the consequences on add distribution and related immunodeficiency were negligible, since most p could not benefit from haart before aids onset. a high clinical suspicion for add should be maintained when facing p with missed or undertreated hiv disease. radata */communication internet platform management of resistance analysis guided haart switch for implementation in clinical practice of hiv-infected individuals soa9.3 paech v, lorenzen t, stoehr a, plettenberg a. ifi, interdisciplinary infectiology and immunology, hamburg, germany purpose: hiv-resistance analyses are indicated to prepare switch of haart in hiv-infected individuals with failure to ongoing haart regimen. specialists at several responsible sites often feel lack of complementary informations if interpretation of resistance analyses is done independent from each other. clinical benefits from resistance analysis assays are sigfnificantly higher for those physicians, who can access external advice from hiv-experts for possible treatment options. the database concept 'radata' (www.radata.de) was developed in germany to generate expert advice for implementation in haart switch. results: fifteen hiv-treatment centres, seven laboratories and 15 high ranked authorities in hiv-medicine contribute to radata database since it is started in january 2002 in germany. hiv-infected subjects are eligible to participate at the project after presentation of failure to haart (viral load !/1000 c/ml). expert advice is generated after all data are evaluated and based on recommendations of 2 á/4 external hiv-experts. observation after therapy switch is scheduled for a period of 12 months. conclusions: radata is a novel database concept with features for evaluation of data and availability of complementary information to participating sites. the project is designed to provide its proficiency to patients and centres from germany and foreign countries. further information will be provided after the number enclosed subjects have enlarged. in vitro effects of hiv infection on abc transporter expression and antiretroviral drug efficacy soa9.4 therefore, we evaluate in primary cultures of human monocytederived macrophages (mdm) and lymphocytes, effects: (1) of retroviral infection and haart on the expression and activity of p-gp and mrp; and (2) of specific inhibitors of these host proteins on antiretroviral activities of nrti, non-nrti and ip. results: on the one hand, we evidenced a transitory increase of p-gp mrna expression in lymphocytes and mdm in response to in vitro hiv infection. this was correlated to an increased p-gp cell surface expression and activity, and an increased tnf-alpha production and mrna. in contrast, no significant modulation of mrp was observed. on the other hand, psc833 and probenecid potentiated in vitro the anti-hiv activity of azt and indinavir. these effects were accentuated when psc833 and probenecid were combined. conclusion: these results showed that: (1) hiv infection by increasing abc transporter expression could favorise the efflux of antiretroviral drugs and decrease their pharmacological effects; and (2) specific inhibitors of these transporters could reverse these deleterious effects. effects of interferon alpha plus ribavirine therapy on frequencies of hcv, hiv and cmv specific cd4-t-cell responses in peripheral blood of hiv/hcv coinfected patients after 6 months of treatment soa9.5 methods: two groups of patients with chronic hcv infection were studied: 26 hiv coinfected progressors with antiretroviral therapy and 13 hiv-negative controls. twelve hcv/hiv and 9 hcv patients have already reached 6 months of ifn-alpha'/ribavirine therapy. virusspecific cd4-t-cells in peripheral blood were analyzed by ifngamma-elispot-assays using hiv-p24, one cmv and three hcv (core, ns3, ns4) antigens. results: (1) at baseline, hcv-specific cd4-th1-cells frequencies were significantly lower than hiv-and cmv-specific ones; (2) frequencies of cd4-th1-cells against hcv as well as against cmv were similar in the two groups; (3) in hcv'//hiv'/, hcv specific cd4-t-cell frequencies did not change between baseline and 6th month of anti-hcv treatment, decreased in three and increased in only one case. hiv-and cmv-specific frequencies were decreased in seven patients. similar results were observed in hiv-negative group. conclusion: (1) hcv-specific immune responses might be more prone to tissue compartmentalization than hiv-specific ones; (2) immune defects induced by hiv infection might not be responsible for the low level of hcv-specific responses observed in hiv-progressors; (3) ifn-alpha'/ribavirine therapy influence on hcv-and hivspecific cd4-t-cell frequencies after 6 months of treatment will be discussed. frequencies of hiv-1-p24 specific th1 cells (elispot) are correlated with plasma hiv-1 viral load in a cohort of lt-np and slow progressors soa9.6 martinez v a , alatrakchi n a , costagliola d b , bonduelle o a , agut h c , autran b a , alt study group a . a hopital pitié-salpêtrière, laboratoire d'immunologie cellulaire, paris, france , b faculté de medecine saint-antoine, inserm sc4, paris, france , c hopital pitié-salpêtrière, laboratoire de virologie, paris, france background: hiv-1-specific t helper-1 cell responses have been associated with long-term-non-progression (lt-np) in hiv infection but the correlation between frequencies of hiv-1-p24-specific th1 cells and viral load has not yet been studied. we prospectively quantified these frequencies by using an ifn-gamma elispot assay in a cohort of lt-np. methods: a cohort of 62 lt-np and slow progressors (infection !/ 8 years and cd4 counts !/600/mm 3 ) was analysable. hiv-1-p24specific t cells were analyzed using: proliferation, ifn-gamma eli-spot assays and ifn-gamma production in cell supernatants. results: wide ranges were observed in the frequencies of hiv-1-p24-specific cd4 th1 cells as assessed by elispot (0-3770 sfc/106 pbmc) with a median of 70 sfc/106 pbmc. these frequencies were negatively correlated with viral load (r0/(/0.304, p 0/0.026) but not with cd4 counts and associated with a low level of t cell activation assessed by cd71 on cd4 cells (r0/(/0.266, p 0/0.035). similar results were obtained with t cell proliferation and ifn-gamma production. conclusion: interestingly, the numbers of hiv-1-p24-specific th1 cells correlate with plasma viral load, independently of cd4 counts indicating that: (1) the defect in hiv-1-specific cd4 th1 cells does not reflect the global cd4 depletion; and (2) these responses are strongly correlated to the control of virus replication. impact of drug á/drug interactions on therapeutical management of active tuberculosis in hiv infected patients soa9.7 martinez v a , truffot c b , caumes e c , katlama c c , jarlier v b , bricaire f c , jouan m d . a department of infectious and tropical diseases, pitié salpêtrière hospital, institut pasteur, unité de génétique mycobactérienne, paris, france , b department of bacteriology, pitié salpêtrière hospital, paris, france , c department of infectious and tropical diseases, pitié salpêtrière hospital, paris, france , d institut pasteur, unité de génétique mycobactérienne, paris, france since 1996 and the use of haart, management of hiv patients with active tuberculosis raised the question of drug á/drug interactions and therapeutical management of both infections. retrospective cohort study: follow-up of 43 hiv patients with active tuberculosis diagnosed between 1996 and 2000. studied data included evolution of tuberculosis and hiv, cd4 cell counts, plasma hiv viral loads, antituberculosis and antiretroviral regimens. ninety-four percent of patients were treated by quadruple combination antituberculosis drug with rifabutin for five patients. fourteen patients were treated by double antiretroviral therapy of nucleoside reverse transcriptase inhibitors (nrtis) and 29 by triple or more drugs (nrtis and/or nonnucleoside reverse transcriptase inhibitors nnrtis and/or protease inhibitors pis). the median follow-up was 24 months. there was no difference on cd4 cell counts and viral loads in the two groups and between the patients treated by nnrtis and pis at the diagnosis of tuberculosis, at the time of antituberculosis drug discontinuation and concerning cure rates of tuberculosis. on the other hand, the plasma hiv viral load was significantly better controlled in patients with nnrtis than with pis (p b/0.005). in hiv patients with active tuberculosis receiving haart, antiretroviral combination including nnrtis allows a better control of viral replication than regimen including pis without impact of the use of rifampin or rifabutin. adherence is essential to the effectiveness of antiretroviral therapy. a pharmacy visit would improve the patient advisement. a survey was carried out over a period of 3 months in the u.m.i.t. a self-report was distributed to 150 patients. ninety were evaluated. for 66%, information's given by the clinician were sufficient and for 76% the treatment advice cards were useful. however, 72% of them would like to attend a pharmacy visit. the topics they would prefer to be tackled were drug interactions (51%), side effects (49%) and effect of forgetting (44%). the treatment was well accepted and tolerated for, respectively 91 and 65% of the patients. the viral load and the cd4 count were well known by, respectively 52 and 46%. however, inaccurate pattern of treatment was frequent ( !/50%) and bad adherence was observed: treatment forgotten occasionally (60%), regularly (30%) or inadequate attitude when the treatment was forgotten (76%). number of pills, dose frequency, length of the treatment would be risk factors of nonadherence. for the majority of patients, a pharmacy visit is necessary and beneficial. the first result shows a better understanding of the treatment, an improvement of the adherence and an enhancement of plasma concentration of antiretroviral drugs. in vivo activity of glycopeptides against s. aureus infection in a rabbit endocarditis model: is mic predictive for in vivo efficacy? soa10.3 asseray n, caillon j, lemabecque v, jacqueline c, batard e, potel g, bugnon d. laboratoire antibiologie, faculte de medecine, nantes, france we have studied the in vivo efficacy of vancomycin (v) and teicoplanin (t) against five staphylococcus aureus (sa) strains: two methicillin-susceptible (mssa 1 and 2), two methicillin-resistant (mrsa 3 and 4) and one glycopeptide-intermediate (gisa 5) strain, in a rabbit endocarditis model. mics of v and t (v/t) were 0.5/0.25, 1/0.5, 1/1, 0.5/0.5, and 4/8, for mssa 1, mssa 2, mrsa 3, mrsa 4, and gisa 5, respectively. the animals were randomly infected with one of these strains, then treated for 2 days by v or t. a continuous infusion of v, simulating a 30 mg/kg/24 h human dose was used. t was infused as a continuous infusion allowing simulating a 6 mg/kg human dose, following an initial bolus. these regimens achieved clinically relevant serum steady-state concentrations of glycopeptides ( !/20 mg/ l). results were as follows: expressed in log cfu/g of vegetations (mean9/sd, followed in parenthesis by the number of rabbits used). purpose: to determine the prevalence of the decreased glycopeptides susceptibility among 187 clinical isolates of staphylococcus aureus collected from patients hospitalized in strasbourg university hospital between 15/01/2000 and 01/03/2000. the susceptibility to glycopeptides of 38 s. aureus isolates collected from hospital environment during approximately the same period was also investigated. methods: the susceptibility to glycopeptides was studied among the mrsa isolates, using: á/ detection of the decreased susceptibility to glycopeptides using agar plates containing 6 mg/l teicoplanin, á/ detection of hetero-visa strains using agar plates containing 4 mg/ l vancomycin, á/ determination of the mics of vancomycin and teicoplanin using the agar dilution and the e -test strips methods. results: thirty-nine percent of s. aureus clinical isolates (74 out of 187 strains) are mrsa. no visa or hetero-visa strain was detected. six percent mrsa isolates are teicoplanin intermediate s. aureus strains. in the environment, 13% s. aureus isolates are methicillinresistant (five out of 38). the five strains are all susceptible to glycopeptides. conclusion: the results regarding vancomycin are reassuring. however, the high rate of mrsa and the presence of teicoplanin intermediate s. aureus isolates prove that prevention and control measures need to be improved. comparative investigation of polymerase chain reaction and a conventional methods for detection of methicilin resistant staphylococcus amont clinical isolates soa10.5 kantardjiev tv a , vacheva-dobrevski rs b , panajotov sv a , bachvarova am a , velinov ti a , levterova vs a . a national center of infectious and parasitic diseases, microbiology, sofia, bulgaria , b military medical academy, clinical microbiology, sofia, bulgaria purpose: identification on methicillin resistant staphylococci has a great clinical implication and significant impact of antibiotic therapy. the aim of this study is to compare the disc-diffusion test (ddt), oxacillin agar screen test (oast) and pcr for detection of mec a gene. fifty selective clinical isolates (41 staphylococcus aureus and nine s. epidermidis ) determined as methicillin resistant by ddt were enrolled in the study. ddt was performed with oxacillin disk (1 mkg/ml) on mueller-hinton agar (mha) without nacl (nccls, 2000) . oast was performed on mha with 4% nacl, oxacillin 6 mkg/ ml, t 35 8c. these strains was genotypically characterized for the mec a gene presence by pcr method using the mec a 1-5?-aaa atc cat ggt aaa ggt tgg c-3? and the mec a 2 á/5?-agt tct gca gta ccg gat ttg c-3? primers (gibco, brl) . results: in the group of 50 mrs isolates, detected by pcr, positive results were as follow: 23 s. aureus and six s. epidermidis . for six s. aureus isolates ddt and oast were positive; pcr-negative. for two s. aureus and two s. epidermidis isolates pcr was positive; phenotypic methods-negative. conclusions: accurate and rapid detection of mrs is a constant challenge for laboratories. the pcr assay (first time in our country) appears to be more reliable than routine susceptibility testing for the rapid diagnosis of mrsa infections at hospitals, particularly due to the heterogeneous resistance of many strains. 7.39/1.7 (6) 9.79/0.9 (5) 8.79/0.9 (5) 8.49/1.3 (8) 8.29/1.1 (4) v 3 . 0 9/1.3* (7) 8.39/1.5 (5) 3.09/0.9* (4) 6.69/1.6 (6) 6.79/1.9 (6) t 3 . 0 9/0.6* (4) 7.59/0.4 (4) 4.59/1.6* (6) 8.29/0.6 (4) 5.79/1.9 (6) distribution and antibiotic susceptibilities of bacteria isolated from suspected urinary tract infections of inpatients in hungary soa13.2 rozgonyi f, csukás z, kamotsay k, szabó d, ostorházi e, berek z, maródi c. institute of medical microbiology, semmelweis university, budapest, hungary between l january 1997 and 31 december 2000, a total of 12, 143 urine samples were cultured 40% as native urine (nu) and 60% as uricult-plus (up) (orion diagnostica, finland) . cultivations were negative in 39% of nu and 42% of up specimens, while contamination was revealed in 20% of nu and 30% of up. in the clinical bacteriologically evaluable positive 1726 nu and 1656 up cultures, the distribution of the gram-negatives was very similar with the predominance of escherichia coli (43 and 41%) followed by enterobacter spp. the distribution of gram-positives differed significantly according to the types of specimens. nu resulted in 13% group-d and 6% group-b streptococcus while up did 22 and 1.5%, respectively. third generation cephalosporins and fluoroquinolons were equally very effective against e. coli strains, while ampicillin inhibited growth of 42% only. carbapenems, cefepime and the fluoroquinolons were the most active against enterobacter strains. interestingly, trimethoprim'/ sulfarmetoxazole combination could inhibit more than 75% of enterobacteriaceae strains. piperacillin'/tazobactam (89%), imipenem (83%), and ciprofloxacin (82%) could be the drog of choice against pseudomonas aeruginosa . enterococcus strains were highly sensitive to glycopeptides (100%), nitrofurantoin (99%), imipenem (95%) and amoxicillin'/clavulanic acid (94%). antimicrobial susceptibility levels of escherichia coli isolates cultured from urine at a tertiary care teaching hospital. temporal trend and comparison between community-acquired and nosocomial urinary tract infection soa13.3 nanetti a, manfredi r, valentini r, calza l, chiodo f. uni versity of bologna, infectious diseases, bologna, italy in order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). when evaluating sensitivity levels of 2070 community-acquired pathogens (1999 á/2001), a significant resistance rise was limited to cotrimoxazole (p b/0.01) and nalidixic acid (p b/0.02), while a tendency towards increased resistance regarded norfloxacin (p 0/ 0.05) (fig. 1) . when 1570 community-acquired e. coli isolates were compared with 2687 nosocomial strains (tested in the years 2000 á/ 2001), a greater susceptibility of community-acquired e. coli isolates was limited to cotrimoxazole versus all other compounds in the year 2000 (p b/0.03), while it was extended to amoxicillin, cephalotin, nitrofurantoin and piperacillin in the year 2001 (p b/0.0001) (fig. 2) . on the whole, e. coli showed an elevated sensitivity rate ( !/90% of tested strains) to nitrofurantoin, gentamicin, amikacin, and 2nd-and 3rd-generation cephalosporins, while only amoxicillin and piperacillin had a mean resistance rate !/30%, regardless of the community or nosocomial origin. a permanent surveillance of sensitivity levels of the most common pathogens responsible for infectious diseases enables to identify local antimicrobial activity and its temporal variations, and plays a key role in starting empiric therapy, pending bacterial identification and in vitro assays. conclusion: in uti, the antimicrobial agents such as 1st cg combined with aminoglycosides are recommended as initial treatment as well as the 3rd cephalosporin generation at monotherapy. in addition, the fluoroquinolones and aminoglycosides are effective in uti. prevalence of resistance mutations to antirretrovirals and relation to virological failure s3.4 garcia f a , suarez s a , alvarez m a , martinez nm a , valera b b , pasquau j b , hernandez quero j a , maroto mc a . a hospital san cecilio, microbiology, granada, spain , b hospital virgen nieves, microbiology, granada, spain purpose: to investigate the prevalence of resistance mutations in the reverse transcriptase (rt) and protease (p) genes of hiv and to relate it with the type of virological failure (vf), we have studied 88 patients (11% naïve or pregnant women, 31% were first vf, 23% were second vf, 35% more than two vf. resistance mutations were investigated using trugene hiv-1 genotyping kit (visible genetics). results: global prevalence of resistance mutations for rt inhibitors (rti) has been !/20% for m41l, d67n, k103n, m184v, l210w, t215yf, and for l10i, m36i, l63p, a71vt, l90m for p inhibitors (pi). the prevalence of resistance mutations for the naïve patients studied was very low (a98g, v118i for rti and l10i, m36i, m46i */all n0/1 */and l63p n0/4 for pi). for patients on first vf only k103n, m184v, t215yf (rti) were !/20%, as well as l10i, d30n, l63p (pi); when patients on second vf were studied, then m41l, e44d, k103n, m184v, g190a, l210w, t215yf, k219qe (rti) and m36i, l63p, a71t (pi) were !/20% prevalence; finally, when patients with more than two vf were studied, the following resistance mutations were !/20%: m41l, d67n, k70r, k103n, v118i, y181c, m184v, g190a, l210w, t215yf (rti), and l10i, m36i, m46il, l63p, a71t, l90m (pi). conclusions: the prevalence of primary resistance in the population studied is very low; the prevalence of mutations in the reverse transcriptase and protease genes increase in parallel to the type of virological failure. genotypic resistance in hiv-1 rna from patient plasma compared with rapid virus isolation and phenotypic resistance in patient pbmcs s3.5 stuermer m, groeschel b, cinatl j, doerr hw. institute for medical virology, university clinic frankfurt, frankfurt, germany objective: to compare hiv-1 virus isolation in the presence of antiretroviral drugs with plasma hiv-1 genotyping. materials and methods: hiv-1 genotyping was performed using the viroseqtm vers. 2 from applied biosystems. interpretation of genotype was done according to international standards. cd4-cells were purified from patient plasma and cultivated in microtiter plates coated with anti-cd3 and anti-cd28 antibodies in the presence of different concentrations of antiretroviral drugs. virus production was measured using a p24 antigen assay. phenotypic activity was expressed as 50% reduction of p24 concentrations. results: seventeen samples were analyzed. for 11 samples results were obtained from both methods, two samples could not be analysed by phenotyping and four samples not by genotyping. only 3/11 samples showed total and 2/11 samples partial concordance, 6/11 samples showed discordance between the two assays. in discordant samples the genotype gave a definite interpretation. conclusion: hiv-1 virus isolation and phenotyping from pbmcs may overcome the problem of currently used resistance assays, which analyse only the reverse transcriptase and the protease gene of hiv-1. possible mutations in other regions may influence viral fitness and therefore contribute to the growth of the virus population present. the lack of concordance between the two assays is related to the different blood compartments used. the clinical value of resistance tests using pbmcs is under investigation. interleukin-6 co-operates with a new type i ifn, ifn-tau to inhibit early steps of hiv-i biological cycle s3.6 rogez c a , clayette p a , martin m a , dereuddre-bosquet n a , martal j b , dormont d c . a cea, drm, fontenay-aux-roses, france , b inra, physiologie animale, jouy-en-josas, france , c cea, crssa, ephe, drm, fontenay-aux-roses, france background: type i interferons (ifn) exhibit efficient antiviral activities notably against hiv, but severe side effects restrict their clinical uses. ifn-tau is an ovine or bovine non-cytotoxic type i ifn which displays higher inhibitory effects towards hiv replication than ifn-alpha, particularly in human monocyte-derived macrophages (mdm). the antiretroviral activity of ifn-tau seems to involve antiviral and immunomodulatory mechanisms: il-6 synthesis is increased in dose-dependent manner in mdm treated with ifn-tau and a specific inhibition of il-6 biological activity decreases its antiretroviral efficiency. results: after a 1-h infection, a significant decrease of intracellular hiv rna amount was found in mdm treated with ifn-tau. in parallel, no additive inhibition was observed with ifn-tau during the elongation of proviral dna. these results suggest either an inhibition of hiv nucleocapsid uptake or an immediate hiv rna degradation, and the expression of 2?,5?-oas, mxa protein and pkr was then measured. ifn-tau induced the expression of these three host cell factors. the role of il-6 on these different steps was evaluated and we showed that il-6 co-operates with ifn-tau during the very early step of hiv biological cycle. conclusion: altogether, these results evidence that ifn-tau uses the same antiretroviral pathway as others type i ifn in mdm, and that il-6 takes part to its inhibition of early steps of hiv biological cycle. actinomycin-d as a modulator of resistances due to cell-wall active agents like bacitracin (bc) and lysozyme (lz) s4.7 chakrabarty an a , dastidar sg b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india it was observed that development of lzr in the lzr mutants took placed at three different levels and was accompanied with unselected, distinctive and elevated levels of bcr. similarly, bcr in bcr-mutants were also detected at three different levels. although the levels of bcr (100/200 mg/ml) in the bcr mutants could be raised only by persistent efforts, an increase in the levels of lzr (as cross-resistance) in the same mutants could be easily achieved. a correlation of actinomycin-d resistance with lzr and bcr of the mutant bacteria and the effects of lipase treatment on the same showed a 4 á/20-fold rise in actinomycin-d resistance of the lzr and bcr mutants of gram-positive bacteria compared with their correspondence wild-types. these findings suggest that lzr and bcr are controlled by several genes accounting for reduced cell-wall and cell-membrane permeability and indirectly, by phenotypic alteration of the lipid content of the cell-wall. thus, the alteration of cell-walls and membranes and a phenotypic extra lipid layer can work in conjunction with the efflux pump mechanisms finally determining the levels of drug-resistance. experimental development of drug resistance to non-antibiotics: a role of alteration of membrane fluidity and efflux systems s4.8 dastidar sg a , mazumdar k a , asok kumar k a , chakrabarty an b . a jadavpur university, pharmaceutical technology, calcutta, india , b department of medical microbiology, calcutta university, calcutta, india drug resistance among clinical strains was studied by selecting mutants resistant to promazine (pr) and methdilazine (md). the results showed that successive step-up mutants of pr and md developed cross-resistance to several unrelated drugs, which in subsequent steps had broader resistance spectra with higher levels of resistance. experiments on the membrane fluidity or permeability of bacterial cells using diphenyl hexatrine (dph), a fluorescent probe on md-mutants showed that three was marked reduction in the membrane fluidity and permeability. when several analogues of the basic phenothiazine structure, e.g. 2-chlor-methyl-n -methyl-pyrrolidine (cmp), methyl-1-methyl-2-pyrrolidone-4-carboxylate (mmpc), 3 hydroxymethyl-n -methyl-pyrrolidine (hmp) and md with final substitution were tested for antibacterial function on different strains, highest activity was observed with respect to md. with anaerobic bacteria the resistance(s) dependent on efflux pumps showed higher levels of resistance even to md. we have found the non-antibiotic agents triflupromazine, trimeprazine and diclofenac sodium have high degree of activity against vibrios, staphylococci and pseudomonads. the explanation of such a phenomenon in terms of possible efflux pumps will be discussed. csf, plasma and urine pcr in lyme neuroborreliosis s7.3 pícha d a , moravcová l a , lásiková š a , marešová v a , ž ïárský e b . a charles university, 2nd medical school, 1st clinic for infectious diseases, prague, czech republic , b department of cellular and molecular biology, charles university, 3rd medical school, 1st clinic for infectious diseases, prague, czech republic the main reason for high diagnostic value of pcr in neuroborreliosis (nb) is the direct way of spirochete detection. two sets of primers in nested pcr were used: one for plasmide gene encoding ospc protein and second for chromosomal gene 16s rdna. so far 25 patients with clinically manifested involvement in nb were enrolled into the prospective designed study (being continued). the main including criterion was positive prove of intrathecal specific antibody secretion (in 22 patients) and pcr positivity in csf (in 3). all patients were repeatedly examined by neurologist and samples of csf, plasma and urine were taken: (1) before treatment; (2) after treatment; (3) after 3 months. before treatment were 12 patients pcr positive in csf, six in plasma, and 10 in urine. five were parallel positive in csf and plasma and four in all three body fluids. urine after treatment was positive in seven (28%) cases and completely negative after 3 months. the pcr has had relative high sensitivity (44 %), but does not rich the sensitivity of antibody index (88%). supported by grant mzcr 6244; 111300003. consumption of imipenem correlates with b-lactam resistance in pseudomonas aeruginosa s12.5 lepper pm a , hö gel j b , trautmann m a , grusa e c . a department of medical microbiology and hygiene, university of ulm, ulm, germany , b department of biostatistics, university of ulm, ulm, germany , c hospital memmingen, central pharmacy, memmingen, germany purpose: in the present study we investigated the monthly consume of three anti-pseudomonas-active antibiotics, namely imipenem, piperacillin/tazobactam (pt) and ceftazidime during a period of 3 years (1997 á/2000) . the use of these antibiotics was correlated to the rate of resistance in pseudomonas aeruginosa . results: inspection of the time series for use of imipenem, ceftazidime, and pt, and the corresponding time series for resistance (each available from july 1997 to july 2000) indicates a remarkable coincidence between use of imipenem and resistance against the three antibiotics mentioned. pearsons's coefficient of correlation for the use of imipenem and the resistance against imipenem was 0.62 (p b/0.001), between imipenem use and pt resistance was 0.57 (p b/0.005), and between imipenem use and ceftazidim resistance 0.56 (p b/0.005). we found positive regression coefficients quantifying an association with imipenem use in the same month (p b/0.01) and with the use during the preceding month (p b/0.05). the same was true when checking dependence of ceftadizime resistance (p b/0.05) and pt resistance (p b/0.01) on imipenem use observed during the same month. neither the use of ceftadizime nor of pt could be identified as factors creating resistance to one of the three antibiotics under consideration within a reasonable period of time. conclusion: there might be a strong pressure towards resistance created by carbapenems. this could limit the use of carbapenems for initial empiric therapy. treat hard and fast: short course antibiotic treatment and its relation with patient compliance and effectiveness s12.6 perez-gorricho bpg a , ripoll m b , pechere jc c . a niño jesus hospital, infectious diseases, madrid, spain , b insalud, outpatient consult, madrid, spain , c university of geneve, microbiology, geneve, switzerland 'treat hard and fast ': short course antibiotic treatment and its relation with patient compliance and effectiveness. finding the important implications for the way in which physicians manage patients with mild á/moderate respiratory tract infections, and the relation of this management with the perception of antibiotic effectiveness, and the compliance with the antibiotic regimen has been the main purpose of the research. in a pan-european market research study of more than 3000 patients, designed to determine behaviour to the antibiotic management of mild-moderate respiratory tract infections, patient expectations of antibiotic therapy were identified, particularly those aspects that relate to efficacy and compliance. the study identifies three key drivers of patients perceived antibiotic efficacy: length of antibiotic course, time to onset of symptom relief and time to complete resolution of symptoms. the results demonstrate that once daily treatment for short periods is perceived by patients to be significantly more effective than longer antibiotic courses and thus better meets patient expectations of therapy. in this study, a macrolide, azithromycin, was selected as the drug therapy of shortest course, being the antibiotic with the shortest dosage schedule for common outpatient infections. the perception of efficacy with short course therapy also correlates with overall satisfaction with management by the physician and with patient compliance with antibiotic therapy. purpose of the study: group b streptococci (gbs) remain a major cause of neonatal infections. consensus guidelines have recommended an intrapartum antibioprophylaxis by amoxicillin, which has reduced the incidence of early-onset neonatal gbs infections. however, an increased incidence of beta-lactam-resistant gram-negative neonatal sepsis has been reported. the aim of our study was to analyse the consequences of this antibioprophylaxis on the intestinal microbial colonization of newborns. a study of the fecal flora was carried out on 50 stools samples from 3 days-old newborns divided into groups: group a intrapartum treated mothers (n 0/25); and group b untreated mothers (n0/25). both groups were matched with regards to known factors affecting intestinal microbial colonization: gestational age, type of delivery and feeding. results: colonization by enterobacteria and enterococci was not significantly different and occurrence of amoxicillin-resistant enterobacteria was similar (11/13 and 13/16 in groups a and b, respectively). however, the colonization by clostridia was modified: the number of newborns colonized was significantly less important in group a than in group b (group a: 3/25 and group b: 10/25 p b/0.05). conclusion: in our study, intrapartum antibioprophylaxis did not affect intestinal colonization by aerobes but reduced significantly colonization by clostridia, potentially anaerobic pathogens. impact of an antibiotic policy restricting the use of b-lactams and macrolides on the incidence of clostridium difficile associated diarrhoea in general medical, renal and elderly patients s12.8 boswell tc a , pacey s b , broomfield s c , westmoreland d c , yates c c . a nottingham city hospital, microbiology, nottingham, uk , b nottingham city hospital, pharmacy, nottingham, uk , c nottingham city hospital, infection control, nottingham, uk the purpose of the study: to investigate the short-term impact of a new antibiotic policy for the treatment of urinary and respiratory infections on the incidence of clostridium difficile associated diarrhoea (cdad) in hospitalised medical, elderly care and renal patients. the results obtained: a policy restricting the use of b-lactams (except parenteral penicillin), and promoting alternative antibiotics including levofloxacin for pneumonia, and doxycycline for non-pneumonic respiratory infections, was launched in july 2000. as a result there was a significant and sustained reduction in use of aminopenicillins, cefuroxime and macrolides, with a corresponding increase in doxycycline and levofloxacin. the incidence of cdad was determined during the 1st 12 months of the new policy and compared to the last 12 months of the old policy. the incidence of cdad fell from 10.02 to 4.91 per 1000 patients, and from 1.2 to 0.59 per 1000 in-patient days (p b/0.00001). in contrast, there was no change in the incidence of cdad in other specialties (surgery, oncology etc.) that had not introduced the new policy. there was no change in the incidence of nosocomial bacteraemia with quinolone-resistant coliforms or mrsa, despite the increased use of levofloxacin. conclusions: hospital-wide reduction of b-lactam and macrolide use in medical patients can result in a significant and immediate reduction in cdad. longer follow-up will determine if this effect is sustained. use of imipenem/cilastatin i.v. (tienam i.v.) for the treatment of low respiratory tract infections in intensive care units s12.10 izzo l a , orsetti r b , boschetto a a , binda b a , della casa u a , caramanico l a , la mazza a a . a department of surgery, universitá degli studi di roma 'la sapienza','p. valdoni', rome, italy , b s. camillo-forlanini, intensive care unit, rome, italy ventilator associated pneumonia (vap) is considered the most frequent infection in the intensive care unit (icu), occurring in 9 á/24% of patients intubated for longer than 48 h besides nosocomial pneumonia is a common complication in the critically ill surgical or trauma patient. inadequate treatment can lead to the complications of acute respiratory distress syndrome (ards), empyema, and lung abscess. the most important aetiological agents both in vap and in pneumonia which arise as complication in surgical or trauma patients are bacteria, whit a marked predominance of staphylococcus aureus and pseudomonas aeruginosa . the authors present their experience (30 cases) on the employment of imipenem/cilastatin i.v. (tienam i. v.) as initial empirical monotherapy at the dose of 500 mg)/3/day or 1 g)/3/ day for the treatment of the serious lower respiratory tract infection in an icu. tienam is a well tolerated broad spectrum antibacterial agent that is effective against the majority of gram-positive and gram negative aerobic and anaerobic bacteria including most pseudomonas species. except one patient deceased for causes related to his very poor general conditions and three cases in which has been necessary the addition of an aminoglycoside, in all the other patients the imipenem/ cilastatin (tienam) monotherapy has shown satisfactory clinical and bacteriological responses. clinical auditing of the impact of recommendations on antibiotic treatment s12.11 kinoo j a , david-ouaknine f a , hacquard b a , echard y a , decazes jm b . a centre hospitalier lagny marne la vallée, lagny sur marne, france , b hospital saint louis, paris, france the aim of this study was to assess the impact of curative antibiotic recommendations on suitable prescriptions at lagny-marne la vallée hospital (general hospital, 714 beds). two prospective exhaustive audits were made (all complete hospitalizations, excluding psychiatry, february á/may 1997 and 2000) of the detailed curative antibiotic prescriptions, before and after distribution of internal recommendations. the same methodology, designed by a multidisciplinary team, was used for both periods. the same antibiotics were available at the pharmacy. the prescriptions were assessed by an infectious diseases specialist and a pharmacist using pre-established criteria: literature recommendations (1997 audit), internal recommendations (2000 audit). six hundred and fifty-six prescriptions for 498 patients were collected and analysed in 1997, 738 for 497 patients in 2000. exhaustivity of the recovered prescriptions was over 95%. patient characteristics, infection sites and microbiological findings were similar for both groups. suitable prescriptions were significantly increased (47 á/59%, p b/0.001). unsuitable prescriptions (economic reasons, too short or too long course, incorrect administration, or underdosage) were significantly reduced. prescriptions for incorrect indications were unchanged and necessary combined treatment not being prescribed, increased. local recommendations improved prescriptions, but efforts have to be done in order to go on the improvement of the practice behaviour. cost-effectiveness analysis of antibiotic therapy in hospitalized patients with copd exacerbations (ae-copd) s12.12 beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy antibiotic costs represent a high burden of total drug costs for hospital administrations. a scientific approach considering also the economic aspects of each therapeutic decision may gain optimal treatment objectives at pondered costs. in our study we retrospectively evaluated the clinical effectiveness and costs of antibiotic therapy in patients with ae-copd. from 1997 to 2001 , 1058 our retrospective study results support previous pharmaco-economic considerations according which in choosing an antibiotic regimen for ae-copd we must take into consideration the expected clinical and microbiological results without forgetting to consider the economic burden of our decisions. significant increase in fungaemia due to non-albicans candida species s20.4 shah pm. klinikum der j.w. goethe universitaet, schwerpunkt infektiologie, frankfurt am main, germany until 1996, predominant candida species in blood cultures was candida albicans . it accounted for 72.8% of all candida species cultured from blood. since then we have observed a gradual increase in number of non-albicans candida species. from 1999, onwards nonalbicans candida species out-number c. albicans . this observation is especially important as non-albicans candida species are generally non-susceptible to azole derivatives and empirical use of azoles in suspected candidaemia should not be recommended. amphotericin b is uniformally active against almost all candida species. echinocandin may be an alternative. see figure below. a search for newer antifungal chemotherapeutics s20.5 chakrabarty an a , dastidar sg b , saha b b , basu l b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india fungal infections due to the mucor-rhizopus (m-z) group present formidable problems due to lack of appropriate and effective drugs against them, as seen in increasing number of clinical situations; death due to mycoromycosis is nearly inevitable. we analysed the biological 'weak-spots' of the mucor-rhizopus group and attempted to devise suitable drugs using their weak-spots. we have noted that like many free-living fungi, the m-z fungi are facultatively chemoautotrophic (can grow on simple sources of carbon and nitrogen and a solution of mineral salts), like the human pathogenic chemoautotrophic nocardioform bacteria. we devised a minimal medium based on that of davis and mingioli, supplemented with simple chemical compounds as sole sources of carbon and nitrogen. the key chemical here was diphenylamine with trypan blue (dpa á/tb) and other similar sources of c and n. we found that while media free of these chemicals (controls) allowed good growth of different strains of m-z fungi, a mixture of dpa á/tb completely prevented their growth over a wide concentration range. experiments with immunocompromised mice showed that these drugs at the concentrations used are well tolerated; mice experimentally infected with several clinical isolates of m-z fungi and receiving these chemicals showed that these fungi could not grow in vivo. in vitro activity of newer fluoroquinolones against multi-drug resistant salmonella typhimurium s33.4 nolones resistance is being also reported. we have studied the in vitro activity of b-lactams and fluoroquinolones against multi-drug resistant s. typhimurium from human sources. material and methods: fifty multi-drug resistant s. typhimurium were tested against cefazolin, cefuroxime, cefotaxime, cefepime, ofloxacin, levofloxacin, and moxifloxacin, by the agar dilution method according nccls guidelines. results and conclusions: all the strains were resistant to four or more of the following antibiotics: ampicillin, tetracyclines, chloramphenicol, streptomycin, sulphonamides and nalidixic acid. a high proportion of strains were intermediate or resistant to amoxicillin/clavulanate. we found no resistance to cephalosporins. nevertheless, 26% were intermediate to first and/or second gen. cephalosporins. cefotaxime and cefepime were the most active cephalosporins (mic50: 0.1 mg/l). though increasing fluoroquinolones resistance has been described among this kind of strains, no resistance to fluoroquinolones was found here. levofloxacin was the most active fluoroquinolone (mic90: 0.06 mg/l), followed by ofloxacin (mic90: 0.1 mg/l) and moxifloxacin (mic90: 0.2 mg/l). high rates of resistance to antibiotics by salmonellae from diarrhoeic children in zliten-libya s33.5 ghenghesh ks a , ben ali m b , abuhelfaia a b , dufani ma a . a faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya , b faculty of arts and sciences, el-ghomes university, biology, el-ghomes, libyan arab jamahiriya salmonellae are major bacterial cause of diarrhoea in libya particularly in children. included in the present study 23 salmonella species isolated from 169 children with diarrhoea in zliten city-libya. the children aged between a few days to 10 years. the organisms were tested for their susceptibility to antibacterial agents using the disc diffusion method. of the isolates examined, 23 (100%) were resistant to ampicillin, 22 (95.7%) to amoxicillin á/clavulanic acid combination, 20 (87%) to cefoxitin, 22 (95.7%) to chloramphenicol, 21 (91.3%) to doxycycline, 18 (78.3%) to gentamicin, 1 (4.3%) to nalidixic acid, 1 (4.3%) to trimethoprim á/sulphamethoxazole and none (0.0%) were resistant to norfloxacin. a strong relationship was observed between the availability of antibiotics in the pharmacies of the city and resistance of the isolated salmonellae to these drugs. the misuse of the antibiotics by the community may be an important factor (among others) in the emergence of these high rates of resistance by the salmonellae examined. effect of ceftriaxone along with probiotics administration on intestinal ecosystem and betalactamase activity s34.6 bertazzoni minelli e a , benini a a , zoppi g b . a department of medicine and public health-pharmacology section, university of verona, verona, italy , b department of paediatrics, university of verona, verona, italy oral bacteriotherapy during antibiotic treatment is a much debated topic. aim: to study whether different probiotics can prevent imbalance of the intestinal ecosystem (dysbiosis) in children during therapy with ceftriaxone (cx). methods: fifty-one children (mean age 5.1 years) with febrile respiratory tract infections were treated with cx 50 mg/kg/day iv, alone (therapy 1) and along with the following preparations: saccharomyces boulardii (2); enterococcus spp. (3); lactulose (4); l. casei gg (5); l. rhamnosus , l. bifidus and l. acidophilus (6); b. bifidum and l. acidophilus (7); and a mixture of various lactobacilli and bifidobacteria at high concentrations (8). faecal samples, collected before and after treatment, were analysed for microflora composition, cx concentration, and beta-lactamase (bl) activity. results: cx causes intestinal dysbiosis. no c. difficile was found. faecal bl increased after therapy in all treated groups. cx alone increased bl activity in 60% (3/5) of children (no activity before treatment); a higher incidence (75 á/80%) was found in groups 2 and 5. after therapies 3, 4, 6, 7, and 8, bl activity was found in 1 or 2 more children. cx was detected in 36% of faecal samples. conclusions: the probiotics administration seems to protect against dysbiosis caused by cx and to contain the increase in faecal bl activity. the effects differ according to the probiotic administered and are peculiar to certain bacterial species. these preliminary data need further studies. comparative study of initial and acquired drug resistance in pulmonary tuberculosis in iran s37.4 mansoori d, arami s, mirabolhasani z. nritld, infectious disease, tehran, islamic republic of iran purpose: resistant to anti-tuberculosis agents particularly multiple drug resistant (mdr) is a major obstacle in treatment tuberculosis in the world. between september 1996 and march 2000 for 273 smear and culture positive pulmonary tuberculosis patients (old 0/86, new0/ 187) pretreatment susceptibility tests of isolated bacilli to inh, rif, emb and stm were performed by standard proportional method and the results were attributed to three groups: (i) newly diagnosed without any history of treatment; (ii) patients with history of treatment for one course; (iii) patients with history of treatment for two or more courses supposed to be mdr cases. the results were collected for each drug individually and different combinations of two, three and four medications. results: resistance to one, two, three and four drugs was significantly increased in group iii comparing to groups ii and i, also in group ii compared to group i. we observed a high rate of primary resistance to inh and stm in groups i and ii and a high rate of mdr (inh and rif resistance) in groups ii and iii. conclusion: the duration of bacilli exposure to antituberculosis agents in the past is a major factor in developing resistance. in contrast to who's guideline, due to high rate of primary resistance especially to stm in our area, we do not recommend addition of stm for treatment of patients whose initial four-drug regimens have been failed (group ii). donors, to understand host interactions with this bacteria, to develop new methods of diagnosis and define new vaccine candidates. nineteen tb patients and seven healthy donors were enrolled in french hospitals. cellular immune responses were evaluated by lymphoproliferation and ex-vivo quantification of specific th1 cells by elispot-ifn-gamma assays. four recombinant proteins of m. tuberculosis were tested: esat-6, 85b, erp and tb b1.3 and compared with tuberculin. we confirmed that 85b (but not esat-6 in our study) gives higher responses in tb patients compared to donors according to the results in proliferation assay (p 0/0.04). in addition, frequencies of th1 cd4 specific cells for esat-6 and tuberculin were statistical different between the two groups (p0/0.04 and 0.028, respectively). 41.1% for 85b and 70.5% for esat-6 of the patients tested were responders in elispot-assay versus 57.9% for both in proliferation assay. for the new antigens erp and tb b1.3, no difference was observed between the two groups. in conclusion, 85b and esat-6 are recognised by a large number of our patients. they seem to be promising antigens for the development of new methods of diagnosis or for the development of new vaccines. erp and tb b1.3 are not preferentially recognised by tb patients. other exported antigens will be tested. the incidence of tuberculosis (tb) is increasing worldwide. in recent some years, geographical differences in the incidence of tb in former yugoslavia have been observed. an important rise in tb cases was registered in the bordering region of bosnia. it is likely that poorer living conditions, influenced by war and emotional stress, may promote such rising incidence of tb. renal tuberculosis was diagnosed in 25 patients (female 21, male 4) from district brcko in bosnia, during the period of 2 years, 2000 á/2001. at the same time none patient had active pulmonary tb lesions, fibrous lesions were noticed in 18 patients, but we did not diagnose any signs of previous pulmonary tb in seven patients. seven patients developed relapse of renal tb after 1 á/5 years of previous treatment. guided by clinical parameters, precisely done renal echosonography enabled early suspicion and searching for renal tb, by radiological and other methods. bacteriological diagnosis was performed by detection mycobacterium tuberculosis on loewenstein á/jensen medium in 18 patients. pcr as simple, fast and highly sensitive method enabled diagnosis in incipient stadium of disease, so antituberculous therapy could be instituted some months earlier. prompt diagnosis of renal tuberculosis (using pcr, besides standard methods) is necessary, otherwise delayed diagnosis may be dangerous. a study on resistance to first generation anti-tuberculosis drugs in mycobacterium kansasii s37.7 mirsaeidi sm, farnia p, mohammadi f, mansoori sd, jabbari r, taghizadeh r, masjedi mr, velayati aa. national research inistitute of tuberculosis and lung diseases, infectious diseases and immunology (nritld ), tehran, islamic republic of iran purpose: this research has been performed to determine antibiotic resistance of atypical mycobacteria especially mycobacterium kansasi . results: twenty-three pigmented colonies which indicated atypical agents from nritld's mycobacterium culturebank were selected, they then underwent type identification and antibiogram for inh, rif, etb, stm. nine samples were m. kansasi and 14 were other non-mtb, 4 was m. gordonei , 2 m. xenopi , 3 mac, 1 m. bovis , 2 m. tererra , 1 m. asiatioum , 1 m. marinum and 1. mean age in m. kansasi cases was 34'/ 8.1 year and in non-kansasi cases 52'/ 15.6 year and in whole society ntm was 44.9'/16.4. frequency of resistance in kansasi group were 4 to inh (44%), 4 to rif (44%) 4 to etb (44%), 5 to stm (55%) and prevalence of mdr was 4 (44%) and in non-kansasi group frequency of resistance were 13 (92%) to inh, to rif 9 (64%), to etm 9 (64%) and to stm was 10 (71%), to mdr was 9 (64%). conclusion: a significant difference was seen between the age groups of patients who are affected with m. kansasi and non-kansasi (p b/ 0.01), also in frequency of resistance to first generation anti-tb drugs. m. kansasi is detected as the most common atypical mycobacterium agent in pulmonary infections and attention to antibiogram is recommended before treatment. buruli ulcer caused by mycobacterium ulcerans , is the third most common mycobacterial after tuberculosis and leprosy in west africa. nowadays, the only effective treatment is surgery. it consists in a large excision of the lesions, often followed by a skin transplant. in this study, the effectiveness of rifampin, amikacin and their combination were estimated in the treatment of mice, which were infected experimentally by m. ulcerans . after 15 weeks of treatment with rifampin, amikacin or their combination, no more viable bacilli were found in infected tissues. the animals were kept for 3 other months. among the mice treated with rifampin alone, two mice out of 30 relapsed. the minimal inhibitory concentration of these isolated strains went from 0.5 to 8 mg/ml. the dna sequence, obtained from a 93-pb of the rpob gene from these strains, showed a missense mutations, which affect a ser-415 replaced by a phenylalanine. this modification on the gene leads to an important inefficacy of treatment when rifampin was used alone. this study showed that rifampin and amikacin have a bactericidal action on m. ulcerans and that a combination of these antibiotics is necessary to avoid the selection of resistant mutants. histopathologic and electron microscopy studies of a severe isolated hiv enteropathy detected in an aids presenter. favorable response to haart introduction or1.1 manfredi r, calza l, chiodo f. university of bologna, infectious diseases, bologna, italy advanced hiv infection was detected in a heterosexual female with a 1-year history of chronic diarrhoea and severe wasting, as expressed by a body weight of 39 kg, a cd4'/ count of 64 cells/ml, and a plasma viraemia of 2.5 million copies/ml. a malabsorption syndrome was confirmed by d-xylose test, but repeated pathogen search tested negative at stool examination and light microscopy, scanning electron microscopy (em), and transmission em study of enteric mucosa. em assays detected an ultrastructural modification of duodenal mucosa never reported to date: an extensive thinning of enterocytic microvilli, disappearance of glycocalix, and large vacuolization of the enterocyte cytoplasm. two weeks after starting an indinavir-based haart, diarrhoea disappeared and our patient significantly gained body weight: 6 kg after 4 months,15 kg after 8, and 18 kg after 1 year, paralleling a cd4'/ increase to 299 cells/ml, and undetectable hiv viraemia. the subsequent 3-year follow-up confirmed absence of gut disturbances, a stable body weight, a cd4'/ count of 350 á/480 cells/ml, and hiv viraemia persistently b/50 copies/ml. repeated endoscopy and related histopathologic and em assays documented a notable improvement of mucosal damage, with complete cure reached after 2 years of haart. a direct intestinal localization of hiv may be responsible for severe diarrhoea, malabsorption, and wasting, though the morphological features of hiv enteropathy are still unclear. haart acts favourably also against isolated hiv-related enteropathy. kaposi's sarcoma in a non-hiv immunocompetent adult: relapsing due to the development of a squamus cell carcinoma or1.2 sioula e a , magira ee a , georgopoulou c a , rontogiani d b , gounaris t a . a evagelismos, internal medicine and infectious diseases, athens, greece , b evagelismos, pathology, athens, greece a 26-year-old heterosexual hiv negative girl was diagnosed with cutaneous kaposi's sarcoma. the disease was started 2 years earlier with the appearance of lesions on the left feet and on the right knee. absolute number of cd4 and cd8 were 1200 and 486 cells/dl, respectively with a decreased lymphocyte proliferation. human herpesvirus type 8 had been detected in biopsy specimens and she placed on recombinant interferon alpha-2b. follow up few months later the lesions decreased in size. two years after the onset of the disease the patient readmitted because of a mass on the left paratracheal region along with mediastinitis. her body temperature was increased. the patient underwent thoracic ct scan, which demonstrated mediastinal well defined soft tissue infiltration associated with mediastinitis and a well-defined mass in the left paratracheal region. the mass biopsy revealed squamous cell carcinoma. several violaceus lesions were observed on the arms, hands and face. severe bilateral lymphedema of the legs with a reddish papules nodules and tumors from 0.5 to 7 cm in diameter on the soles, toes and calves were present. this case illustrates the significant relapsing of the cutaneous kaposi's sarcoma soon after the appearance of and the carcinoma and the mediastinitis. a 58-year-old male patient with insulin-dependent diabetes underwent cardiac surgery for aortocoronary bypass 2 years ago. two weeks after surgery he developed mediastinitis and sternal osteomyelitis caused by methicillin-resistant staphylococcus aureus (mrsa). twice, revisions and plastic surgery for sternal osteomyelitis were performed. the patient received initially treatment with vancomycin. then the patient received intravenous outpatient treatment with teicoplanin for 6 weeks followed by by treatment with fusidic acid and then trimethoprim/sulfametrol. the fistula was closed. four months later he presented again with substernal pain and purulent discharge. the culture revealed the growth of staphylococci which were first mistaken for coagulase-negative staphylococci. after closer investigation these staphylococci were identified as small variant mrsa. computer tomography (ct) revealed multiple mediastinal abscesses. the patient was treated with intravenous linezolid 600 mg bid for 10 days and then switched to oral linezolid 600 mg bid. the oral therapy was pursued for 16 weeks under close surveillance. the patient improved substantially, the purulent discharge disappeared. the mediastinal abscesses were not detected any longer by ct at the end of treatment. the treatment with linezolid was well tolerated. platelets decreased initially but rose to normal values without treatment modification. nosocomial pneumonia due to stenotrophomonas maltophilia in a profound granulocytopenic patient hospitalized for community-acquired staphylococcus aureus severe sepsis or1.5 radulescu a a , sasca n b , lupse m c , tatulescu d c . a university of medicine and pharmac, epidemiology, cluj-napoca, romania , b the teaching hospital of infectious diseases, laboratory, cluj-napoca, romania , c university of medicine and pharmacy, infectious diseases, cluj-napoca, romania objective: to present the diverse opportunistic infections in immunocompromised patients and treatment difficulties. findings: a 35-year-old women was admitted to the teaching hospital of infectious diseases cluj with a 2-day history of fever, myalgia, lumbar pain, hemorrhage syndrome. severe sepsis was diagnosed and the conditions that evolved in it were paronychia in a patient with chronic leukemia having prolonged and profound granulocytopenia due to aggressive treatment with ifn. the condition at admission was critical due to trombocytopenia ( b/2000 platelets/ml) and hemorrhage syndrome. the evolution was favorable under antimicrobial treatment (imipenem), blood and platelet transfusion, intravenous immunoglobulins, granulocyte colony-stimulating factor, antifungal prophylaxis and supportive care. blood and pus cultures revealed mssa. in the 6th day of hospitalization she developed bronchopneumonia and respiratory failure. the sputum culture was positive for stenotrophomonas maltophilia susceptible to ceftazidime, fluoroquinolones. treatment was unsatisfactory until introducing ticarcilline á/clavulanate and ciprofloxacine. she had uneventful recovery despite remaining granulocyto-trombocytopenic. conclusions: treatment of infections with emerging agents in immunocompromised patients is difficult, guidance by results of susceptibility testing being misleading with a poor correlation between the tests and treatment outcome. early disseminated listeriosis in a liver transplant recipient (ltr): a rare case due to an in vitro multiresistant strain or1.6 manfredi r, de ruvo n, vivarelli m, bellusci r, montalti r, la barba g, abtueli aden a, cucchetti a, attard l, calza l, cavallari a. university of bologna, infectious diseases, bologna, italy a ltr receiving cyclosporin, azathioprine and steroids, developed an extraordinary episode of sepsis and pleural effusion due to a multiresistant listeria monocytogenes (lm) isolate. a lm strain serov. 4 showing the same, extensive resistance pattern (all penicillins and 1stand 2nd-generation cephalosporins), was isolated from multiple blood cultures and pleural fluid 2 weeks after surgery, while stool exam was negative. our p spent her life in countryside and bred some animals, but denied consumption of uncontrolled food. iv cotrimoxazole administration achieved a complete clinical and microbiological cure in 10 days. underlying immunodeficiency may prompt unusual/severe lm infection, but because of its usual community-acquired origin, lm disease remains infrequent in hospitalized p. only seven anecdotal reports of lm infection were described in ltr, 1986 á/2000, all but 1 occurring months á/years after surgery. an early respiratory and systemic infection caused by a community-acquired lm strain which proved resistant to first-choice antibiotics, but had a favorable response to cotrimoxazole (used only once in a ltr with lm sepsis), characterized our episode. an epidemiological survey retrieved the possible source of this usually community-acquired infection. lm should be regarded as an emerging opportunistic pathogen in ltr, and specific risk factors should be seeked. when immunodeficiency is of concern, the unpredictable sensitivity of lm should prompt in vitro assays to adjust antimicrobial therapy problems for discussion hbv á/hcv and liver carcinogenesis: where does the viral influence end? or2.2 pappas ga. university hospital, internal medicine, ioannina, greece hepatocellular carcinoma (hcc) is a major clinical problem worldwide, usually evolving over a long-standing liver pathology, in the latter stages in the form of cirrhosis. hbv and hcv chronic infection is a common etiology of cirrhosis, and hence, hcc. a number of studies have attempted to clarify the role of these viruses into the progression towards hcc. does their end in the stage of cirrhosis? is progression towards hcc independent of the etiology of cirrhosis (since alcoholic cirrhosis also proceeds to hcc)? do the trials with interferon alfa for patients with hbv or hcv cirrhosis exhibit a favorable result due to the antiviral properties of interferon, or is interferon exhibiting anti-oncogenic potential?, and is hcc cytokine and hormone sensitive (view ongoing trials with somatostatine analogues versus hcc)? (hence, if we treat alcoholic cirrhosis patients with interferon could we have a favorable response?). which patients with hbv or hcv cirrhosis are eligible for interferon treatment?: interferon treatment is a potential hazard for those with thrombocytopenia. how ethical is it to conduct a randomised trial where one leg of cirrhotic patients is left without antiviral therapy? and on the basis of which classification system should the two legs of such a trial be separated? moreover, do viral proteins with oncogenic potential exist (the controversy over the recently discovered hbv protein is still, unresolved)? a major topic awaiting for a major debate. to assess the role of hiv-associated campylobacteriosis (c) according to haart availability, 17 patients with positive culture were identified since 1991. compared with the â/1000 hiv-infected p followed in the last decade, no epidemiological differences were shown, save a greater sexual exposure to hiv (p b/0.005). the introduction of haart caused a drop of frequency of c (from 1.8 to 0.9 episodes per 1000 p-year; p b/0.0001), and modified clinical features, with disappearance of dissemination and mortality, reported in 7 and 2 patients before 1996 (p b/0.03). hiv-related immunodeficiency and disease stage were significantly related to c features before and after haart availability: p b/0.0001 for cd4 and neutrophil count, p b/0.007 for aids diagnosis. most cases (15) were community-acquired, but alimentary or environmental risk factors were never found. ten patients received cotrimoxazole prophylaxis (nine before 1996; p b/ 0.03), while no relationship occurred with steroid or antibiotic use, caused 14 cases out of 17. a 100% sensitivity was found to quinolones, followed by cephalosporins (82.3%), gentamicin (76.5%), macrolides (64.7%), and cotrimoxazole (47.1%). a 8 á/18-day antimicrobial therapy cured 16 p , but relapses caused by similar strains occurred in 6 patients within 2 á/8 weeks, all in the pre-haart era (p b/0.05). c still occurs in the haart era, probably due to its varied mode of transmission. the frequency of c is greater in hiv-infected patients, but less frequent visceralization, recurrences, and mortality characterized the haart era. objective: to determine the incidence and risk factors for nosocomial viral respiratory infections (nrvi) and involvement of human coronaviruses (hcov) in a neonatal and pediatric intensive care unit. methods: prospective observational study. nasal samples were obtained by cytological brush at admission and weekly thereafter for all hospitalized infants. nasal samples were taken monthly from staff. virological studies were performed, using immunofluorescence for respiratory syncitial virus (rsv), influenza viruses, paramyxoviruses, and adenoviruses; both immunofluorescence and rt-pcr were used for hcov detection. results: during 1998, 42 hcov related nrvi were detected in 152 nn and six in 92 children. three hcov-related outbreaks were observed (february, august and december), associated with a high prevalence of infection in staff. during august outbreak, 18 hcovinfected nrvi were detected over 23 hospitalized infants. seventy-five of hospitalized preterm nn with gestational age under 32 weeks and 52.4% of staff members were infected. risk factors for nrvi in nn were birth weight, gestational age, ventilation, oxygenation and hospitalization length. ninety-two percent of infected preterm nn were symptomatic, mainly with bradycardia and respiratory worsening. conclusions: these data provide additional evidence for a significant role of hcov in nrvi occurring in hospitalized preterm nn. strain typing and screening of 8 dna targets to assess echinococcus sp transmission in new and old geographic endemic foci or2.5 bart jm a , piarroux r a , dia l b , benchikh-elfegoun mc c , vuitton da a , bardonnet k a . a serf, parasitology, besancon, france , b national centre of veterinary study, parasitology, nouakchott, mauritania , c university of mentouri, parasitology, constantine, algeria purpose: cystic echinococcosis is due to echinococcus granulosus. parasite cycles depending on the main intermediate host species involved in different foci have been described promoting mixed infection in the same definitive host. strain typing is a tool to identify the main intermediate host involved via the dogs in the human infection route and to focus the control measures. many dna targets have been used to compare samples and to access the parasite cycle in different countries. but no study has compared the value of each of these targets. eight targets have been tested in mauritania where echinococcosis is an emergent disease, and in algeria where strain typing has never been done. thirty-five cyst samples from human, ovine, camel and bovine have been tested with six nuclear and two mitochondrial targets. results: the two mitochondrial targets and four out the six nuclear targets have allowed to discriminate the different foci. two strains have been found infectious to human : the 'sheep' strain in algeria and the 'camel' strain in mauritania. conclusion: although overlapping geographically sometimes, this raises the question of the respective genetic evolution of the different strains and of their involving in human infection. alveolar echinococcosis in france: an update or2.6 bardonnet k, bresson-hadni s, bartholomot b, gérard a, watelet j, beytout j, saurin jc, piarroux r, vuitton da, who centre collaborating for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the highest prevalence rate for alveolar echinococcosis (ae) in europe has been found in france. in 1997, a french observatory of human ae was done in order to get data that could be used to evaluate presentation, evolution and management of ae. material-methods: french cases were collected for the period 1982 á/ 2002. registration of every case was performed with the subject's agreement. a questionnaire was filled in by referring to the patients' medical files or to practitioners or to patients themselves. completeness of the collection of cases was ensured by multiplying the sources of information. results: two hundred and sixty nine french patients were registered. sex ratio averaged 1. mean age at diagnostic was 54.8 years. 12.5% of diagnosis was performed in 'echinococcosis free' french areas. symptoms, but not always specific liver symptoms, were present at diagnosis in 76.4% of cases. the liver was the main location of lesions in 93.6% of cases. a wide spectrum of management of the patients was observed, accounting for regional differences. conclusion: this french observatory of human ea will facilitate a better management of the disease at the national level. it shows new epidemiological trends, and especially an extension of the endemic area. can coins and paper currency transmit bacillus anthracis ? or2.7 ghenghesh ks. faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya anthrax is an often fatal bacterial infection caused by bacillus anthracis . recent events that began in september 2001 in us has gained the organism worldwide attention and heightened awareness of and concern about anthrax. many cases of anthrax with a number of deaths have been reported as a result of contact with envelopes, sent through postal mail, containing b. anthracis endospores. a number of studies have shown that currency is colonized with bacterial organisms, that include enteropathogens (e.g. shigella sp.), other enteric flora (e.g. escherichia coli ) and potential pathogens (e.g. staphylcoccus sp. , pseudomonas sp. and bacillus sp.). furthermore, methicillin-resistant s. aureus (mrsa) isolates that produced enterotoxin (seb) and toxic shock syndrome toxin-1 also been reported. all of these studies do agree on that currency may be considered as a method of spreading potentially pathogenic and pathogenic bacteria in the community. therefore, currency could also be a vehicle for spreading other highly pathogenic organisms that include b. anthracis . in addition, the introduction of the 'euro' could also allow such bacteria greater freedom to travel across the euro zone. the threat of using currency, particularly paper notes, in spreading lethal organisms should be investigated and proper measures to prevent the use of such a method by terrorists should be implemented. salvage of temporary femoral catheters for haemodialysis using antibiotics in ambulatory patients or2.8 gerasimovska v, oncevski a, dejanov p. department of nephrology, clinical centre, skopje, the former yugoslav republic, macedonia the stay of femoral catheters (fc) for haemodialysis is typically short-term for several days. we used fc as a temporary vascular access (va) for a longer period of time in outpatients going on regular ambulatory haemodialysis, who had a problem with their permanent access. we analysed 43 patients who were discharged from hosptal with fc. duration time of fc was between 13 and 183 days (average 44.2 days) with cummulative total of 1989 days. the incidence of bacteriaemia was 2.51 episodes/1000 catheter days. in six patients we had signs of infection, so according to our protocol we took blood cultures from peripheral vein, and from catheter (at same time) and started with antibiotic therapy (ab) systemically and locally (ab was 'locked' in catheter) with different duration of time. dominant microorganism was staphylococcus coagulasa negative, and much less staphylococcus aureus , and enterococcus.ab that were frequently used were: cefotaxim, vancomycin and ciprofloxacin. at one of six patients we removed catheter at once without trying to save the catheter. catheter tip was sent for microbiological analysis too. criteria for catheter-related bacteriemia (crb) was found in only one patient, and for possible crb in five patients after we removed the catheters. in the absence of clinical signs of infection, ab treatment was not provided for positive tip culture alone or for positive blood culture of the catheter with negative blood culture from peripheral vein. advances in meningitis education or2.9 holt de a , tait mi b , cavanna al b , worgan-brown s a , hart b a . a the meningitis trust, stroud, uk , b the computer-aided learning unit, school of health science, university of wales, swansea, uk background: meningitis remains an important cause of death worldwide despite improvements in diagnosis, treatment and prevention. clinical and lay awareness of the disease relies on education, however educational delivery has changed and the introduction of material suitable for computer and internet application is now necessary. we have developed educational material on cdrom and on the internet applicable both at tertiary university and secondary school level. application: a computer-aided learning program on cdrom, covering all aspects of meningitis has been produced. it is suitable for undergraduate teaching of healthcare professionals from student nurse and doctors to pharmacists. in order to reach school children in a form acceptable to both pupils and teachers, we have developed a curriculum-linked website. these applications are simple to use and can be incorporated into existing courses of study, so that issues raised can be discussed with tutors and group peers. comment: the introduction of new methods of teaching and learning mean that compatible educational material must be produced. we believe that these applications, focusing on meningitis, are the first of their kind and that they offer tutors the opportunity to progress their teaching of the disease both in methodology and content. brivudin compared to famciclovir for improved therapy of herpes zoster: effects on acute disease and postherpetic neuralgia or3. potentially treatment-related adverse events occurred in 11.8% of the brivudin recipients and in 10.1% of the famciclovir recipients (p 0/ 0.26). conclusions: in zoster patients ]/50 years, brivudin 1)/125 mg and famciclovir 3)/250 mg showed equivalent effects on prevalence and duration of phn. brivudin is as effective as famciclovir in stopping viral replication in acute herpes zoster. brivudin offers the advantage of a once daily dosage regimen while being as well tolerated as famciclovir. activity of complexes of pt(ii) and pd(ii) with pyridine-2-carbaldehyde thiosemicarbazone (hfotsc) (acta virol., 2001, 45, 87) with selectivity index (si) 1.5 times higher than that of acyclovir (acv). in order to evaluate virus specific response and structure á/activity relationships we continue our investigations with three pt(ii) and three pd(ii) complexes. the activity was evaluated against sensitive to acv hsv 2 (strain bja) and resistant strains r-100 (hsv 1) and pu (hsv 2) and compared to that obtained against strain victoria (hsv 1) infection. si was indicative for activity. the virus specific response was demonstrated by the fact that viruses sensitive to acv were also sensitive to pt(hfotsc) 2 ]cl 2 , while acv resistant viruses were sensitive to [ptcl(fotsc) ]. the structure á/activity relationship was proved by the fact that the less active against hsv infection was [pd(fotsc)]. influenza diagnosis, treatment, and the impact of new antivirals on current treatment behaviours during influenza outbreaks or3.3 schaetz l a , sessa a b , a hoffman-la roche f. basel, switzerland , b italian college of general practitioners, italy introduction: annual influenza epidemics severely affect individuals, families, health care systems and society. the availability of new and specific antivirals provides an opportunity for better management of influenza. methods: during the 1999/2000 and 2000/2001 influenza seasons, physicians ( â/100/country) and public ( â/1000/country) in the usa and europe were interviewed to determine perceptions of influenza and behaviours for its treatment. results: patients recognise influenza illness as severe and identify it by symptoms of fever, muscle aches/pains and cough. physicians use these symptoms to diagnose influenza clinically (93% fever, 78% muscle aches/pains, 47% cough); their main treatment objective being to reduce complications. antibiotics for influenza treatment are broadly recommended/prescribed by about 30% of european physicians, whereas currently available antivirals are only recommended by 10%. the recommendation of antivirals by us physicians increased from 47% (season 99/00) to 62% (00/01) and markedly decreased antibiotic use (from 25 to 11%). experience from the two influenza seasons shows that influenza antivirals are only used while the virus is circulating and that the volume of use is proportional to the size of the outbreaks. conclusions: experiences in the usa show that with prompt outbreak information antivirals can be used appropriately in times of influenza activity. influenza treatment with oseltamivir: costs and benefits for the individual as well as for society or3.4 objective: to evaluate the effects of treatment of influenza with antivirals (oseltamivir) on health outcomes and costs to patients and society. methods: based on clinical trial data and data from the literature a simulation model has been developed. the underlying clinical pathway covers morbidity and mortality due to influenza and its specified complications. health outcome data and costs were attached to events in the model. the model compares various scenarios, which are defined by treatment schemes within defined populations and other parameters. application of the model is shown using uk unit cost data simulating an otherwise healthy adult population comparing oseltamivir with usual care. results: early treatment results in reduced morbidity, which translates into faster recovery and return to normal activities (1.93 days). lower morbidity and mortality make this a cost-effective intervention from a societal perspective. the analysis covers more than 10 different scenarios and the incremental cost effectiveness ratios will be discussed. conclusion: antiviral treatment appears to be effective in terms of health outcome and cost for otherwise healthy adults from the perspectives of both the individual patient and society. however, this effect is very sensitive to time when treatment is started and the accuracy of the diagnosis of influenza. oseltamivir is well tolerated by all patient groups or3.5 thakar b a , dutkowski r b , froelich e c , gilbride j a , ward p a . a roche global development, welwyn, uk , b f hoffman-la roche, nutley, usa , c f hoffman-la roche, basel, switzerland introduction: oral oseltamivir, the ethyl ester pro-drug of a potent inhibitor of influenza virus neuraminidase, is licensed for the treatment and prophylaxis of influenza in the usa. patients and methods: safety data [adverse events, laboratory safety evaluations] derived from clinical trials involving !/12 000 subjects (including â/1000 children and â/1200 high-risk adults) and 400 healthy volunteers in a large study investigating ecg parameters. spontaneous event reports from medwatch or yellow-card reports following use by â/2 000 000 individuals worldwide. an observational case-control study of !/10 000 subjects with influenza-like illness treated with oseltamivir. results: oseltamivir was well tolerated in clinical trials; drug-related side-effects were limited to transient gi effects occurring in 5/1:10 exposed individuals. these resolved spontaneously and caused drop out in b/1% of treated subjects. no effects on ecg parameters were noted at doses ]/sixfold above the licensed regimen. oseltamivir had no adverse effects on pulmonary function. no additional effects were identified among high-risk adults or children, or following prolonged dosing for prophylaxis. occasional reports of liver dysfunction have been documented post-marketing but causal association has not been established. conclusions: oral oseltamivir is an effective and safe antiviral suitable for influenza management in all patient groups. the decision to stop the vaccination against smallpox and the loss of specific immunity of a high proportion of the population made apocalyptic the perspective of a natural or provoked re-emergence of smallpox. therefore, it is important to improve the current capacities to prevent or to treat the orthopoxvirus infections. uracil dna glycosylase (udg) is one viral enzyme indispensable to the replication of poxviruses. udg of the copenhagen strain of vaccinia virus (vv) was characterized with the aim of defining specific inhibitors susceptible to be used as a new class of active antiviral substances on the viruses of the orthopoxvirus genus. the activity of this enzyme was analysed in real time, in an original method, on a pcr quantitative instrument by digestion of amplified dna revealed by fluorescent intercaled molecules. this technique was used to screen and select several active antiviral substances on udg. moreover, the antiviral activity was estimated by the cytopathic effect of the vv on infected vero cells. the cytotoxicity was determined by inhibition of trypan blue exclusion. the specificity of action of each tested compound was estimated by the selective index (50% cytotoxic dose/50% effective dose). two antiviral compounds were selected for their inhibitory effect on udg activity and on vv replication in vero cell culture : ('/)-5-iodo-2?-deoxyuridine and 4-chlorouracil. these compounds are candidates for the chemotherapy of poxvirus infections. objective: to study the efficacy and tolerance of russian antiviral drugs produced from dna in a limited resources context. results: the drug derinat was produced from salmons' milt. mm of dna was 270 á/500 kda, hyperchrome effect !/37%, protein content b/0.5%. the conjugation of the dna with fe3'/ resulted in a new drug named ferrovir which influences dna and rna synthesis during early stages of hiv-1 replication by blocking the virus's action on cells' metabolism and reduces cytomegalovirus titre in fibroblast cells for 1.5 á/2.0 ig tcid50. a protective effect of ferrovir against fatal herpes encephalitis mice was found. the drugs are not toxic. ic50!/ 4000 mg/ml. ec90 of ferrovir against hiv-1 was 800 mg/ml. in limited clinical trials patients received 75 mg of drugs twice daily (7 á/14 days). administration was well tolerated and no side effects were observed. derinat in 88.1% cases of herpesvirus infection (42 patients) improved the healing and shortened duration of illness. hiv-infected patients (34) treated with ferrovir showed sustained, elevated cd4'/ counts and a significant reduction in hiv-1 viral load (median 1.8 ig). the apparent remission was found in patients with concomitant hiv and herpes virus infection. conclusions: antivirals show good antiviral potency against rnaand dna-viruses; are well tolerated by patients and are useful in case of mixed infections; low price makes them accessible to populations with low financial resources. ortho total hcv core antigen assay can aid early prediction of response in patients treated with interferon/ribavirin or3.8 lunel f a , veillon p b , payan c b . a ahu angers, laboratoire de bacterio-virologie, angers, france , b chu angers, laboratoire de bacterio-virologie, angers, france aim: evaluate the predictive value of total hcv core antigen assay and viral kinetics in patients with chronic hcv. methods: one hundred and twenty two patients infected by genotype 1, 4, 5 or pretreatment viral load (bdna 2.0, chiron) !/3 meq/ml, with no previous treatment, received 6 mu interferon (ifn) during 12 months (m). ribavirin was given with ifn after 3 months therapy, for 9 months in patients with detectable rna. viral load was expressed as log (ui/ml) and hcv ag as log (pg/ml)/10 000). results: pretreatment ag values were correlated with viral load (r 2 0/0.779). we observed a rapid decrease of ag (5.2 log pg/ml) and viral load (5.1 log ui/ml) after m1 in sustained responders (sr). in patients who relapsed (rr) after ifn alone, the fall was less important (2.6 log pg/ml, 3.6 log ui/ml) during m1. in sr and rr to combination therapy, the decrease of ag and viral load at m1 was, respectively, (ag: 1.2 and 1.4 log pg/ml; rna: 2.4 and 1.5 log ui/ml). we did not observed significant variation of ag and viral load in nonresponders. the negative predictive value of hcv rna and ag after m1 of treatment were 100%, and positive predictive values were 81 and 82%. after 1 month of ifn alone, the hcv ag decrease was highly predictive of sr, correlated with rna negativation and early reduction of hcv rna ( !/2 log). conclusion: early measurements of total hcv core antigen are useful to predict long-term response to treatment. lamivudine in the treatment of acute hepatitis b or3.9 vincenti a, meini m, luchi s, de gennaro m, ricciardi l, moneta s, scasso a. infectious diseases department, infectious diseases, lucca, italy acute hepatitis b is a self-limiting infection, but in some cases its course may be particularly severe. we report a case of a 77-years-old woman affected by acute hepatitis b treated with lamivudine. on admission in the hospital the alanino-aminotransferase was1060 u/l, the aspartate-aminotransferase 1017 u/l, bilirubin 10,2 mg/dl, hbsag, hbcigm and hbeag were positive, hbv dna was 300.000 copies/ml. during the following days, the levels of ast and alt gradually rose; on the 14th day prothrombine time was 39%, bilirubin 30 mg/dl and the patient developed signs of encephalopathy. four plasmapheresis were practiced without benefit, so the patient was treated with lamivudine, 100 mg/day. after 24 days of therapy, lamivudine was discontinued because of the appearance of diffuse maculopapular rash. at this time the results of liver function tests were normal; after four months hbsag and hbv dna were no longer detectable. in our patient lamivudine prevented an acute hepatic failure. our experience suggests a promising role of lamivudine in the treatment of acute hepatitis b, but how long such therapy have to be practiced and in which patients? prospective, controlled, clinical studies using lamivudine in patients with acute-hepatitis b are necessary. the cost-effectiveness of amantadine versus symptomatic care in the treatment of influenza or3.10 morris s a , carman wf b , barber j c . a city university, london, uk , b west of scotland specialist virology centre, glasgow, uk , c alliance pharmaceuticals, chippenham, uk aim: to assess the cost-effectiveness of amantadine versus best symptomatic care in the treatment of influenza in the uk. methods: we constructed an economic model populated with parameters from the published literature. the model structure is the same as that used in the economic evaluation of zanamivir published by the national institute for clinical excellence in the uk. we conducted a cost-utility analysis (incremental cost per qaly gained) of amantadine versus best symptomatic care. the analyses are conducted for all adults (average-risk group) and the at-risk population (high-risk group), based on the prevalence of influenza over an average season and when the virus is circulating. the perspective is that of the nhs. results: in the average-risk group the incremental cost per qaly gained of amantadine relative to best symptomatic care is uk£30,488 during an average influenza season and uk£12,538 when the virus is circulating. for high-risk individuals the figures are uk£35,278 and uk£14,526, respectively. the results are sensitive to the hospitalisation rate. conclusions : if the threshold for cost-effectiveness is £20,000 per qaly gained amantadine represents value for money in the treatment of influenza in a variety of scenarios, including the baseline for both average-risk and high-risk groups when the virus is circulating. background: surveillance studies all over the world have revealed an extraordinary increase in the prevalence of penicillin resistant streptococcus pneumoniae . the newer quinolones are believed to have broad activity against s. pneumoniae . methods: a total of 87 penicillin resistant clinical strains isolated from patients at hacettepe children's hospital, ankara, turkey between 1999 and 2001 were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections. the minimum inhibitory concentrations (mics) of the penicillin, amoxicillin/clavulanic acid, doxycycline, azithromycin, clarithromycin, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin were determined using the nccls recommended procedure for e -test. results: the range of mics, mic50 and mic90 values for all agents tested against the strains are shown in the table. gemifloxacin and moxifloxacin had the highest in-vitro activity among the quinolones tested. all strains tested were susceptible to b/0.2 mg/ml gemifloxacin, b/1 mg/ml moxifloxacin and 2 mg/ml levofloxacin. conclusions: there is some degree of resistance to all the drugs except the newer quinolones which were active against all isolates studied. purpose: stenotrophomonas maltophilia prevalence is growing, mainly in some hospital areas. s. maltophilia is frequently multidrug resistant. fluoroquinolone (fq) resistance varies from one to another study, but in whole resistance is moderate to high. gyra and parc qrdr partial codes have been recently described. we have studied correlations between fq-resistance and mutations in these sequences in s. maltophilia clinical strains. material and methods: gyra and parc qrdr regions from six fqresistant and two fq-susceptible s. maltophilia clinical strains were amplified and sequenced. mics of ciprofloxacin (cfx), gatifloxacin (gfx) and clinafloxacin (cnfx) were determined by the agar dilution method, according guidelines defined by nccls for p. aeruginosa . results and conclusions: mics ranges of cfx, gfx and cnfx for resistant strains were 8 á/32, 1 á/32 and 0.5 á/8 mg/l. susceptible strains had mics of cfx, gfx and cnfx of 1, 0.2 and 0.06 á/0.1 mg/l, respectively. most susceptible and resistant strains had no significant mutations in the fragments sequenced. only one resistant strain (mic of cfx 16 mg/l) and one susceptible strain (mic of cfx1 mg/l) had a significant gyra mutation, the same in both strains (ile112 á/val). thus, fq resistance in s. maltophilia shall derive from changes in other areas in the topoisomerases or probably from other mechanisms of resistance, such as efflux pumps. purpose: corynebacterium urealyticum is the cause of encrusted cystitis and other inespecific utis and systemic infections. it is frequently multi-drug resistant, with a high rate of resistance to fluoroquinolones (fq). the mechanisms of resistance to fqs have not been described in c. urealyticum . we describe the c. urealyticum parc gene qrdr region and its relationship with quinolone resistance. materials and methods: the activity of ciprofloxacin (cfx), levofloxacin (lfx), gatifloxacin (gfx), clinafloxacin (cnfx) and moxifloxacin (mfx) against 30 c. urealyticum clinical strains was determined following nccls guidelines for enterococci. we amplified and sequenced their parc qrdr by standard methods. results and conclusions: five strains (16.6%) were cfx-susceptible (mic 0.1 á/0.5 mg/l), 5 had mics 1 á/2 mg/l and 20 (66.7%) were highlevel cfx-resistant (mic 32 á/128 mg/l). cnfx was 64-fold more active than cfx. mfx and gfx had mics90 of 4 and 8 mg/l. all the strains, including the type strain, showed a c to t change at the 2614 position referred to wild type s. aureus parc gene, leading to a ser-80-phe change, described as the main parc change in fq-resistant s. aureus . this finding suggest that this mutant sequence, as compared with parc sequences from other grampositives, might be the wild-type for this species, and might explain in part its high resistance rate, and its apparent lightness to develop high-level resistance. purpose: during routine surveillance, we identified 32 ciprofloxacinresistant (mic!/8 mg/l) pneumococcal isolates and compared clinical details and resistance patterns. results: they were isolated from sputa (29) and blood cultures (3) from adults, most with heart or lung disease. hospital admissions were common; half had been inpatients in the previous 3 months. nineteen patients received quinolones in the preceding 3 months, in part reflecting the local policy (introduced in 1997) of penicillin and ofloxacin for first line treatment of pneumonia. thirteen patients had radiological signs of pneumonia and 10 were pyrexial with raised inflammatory markers. agar dilution mics for quinolones, including norfloxacin with and without reserpine, penicillin and erythromycin were performed. an increase in norfloxacin mics was noted over the period 1997 (16 á/64 mg/l) to 2001 (256 mg/l). fluoroquinolone efflux was suggested in three isolates. resistance to moxifloxacin (mic 8 á/16 mg/l) was noted from 1998 onwards. all isolates were serotype 9v and resistant to penicillin (mic!/0.5 mg/l). thirty-one were resistant to erythromycin (mic!/1 mg/l). conclusion: the 1997 policy of using quinolones may have contributed to the development of quinolone resistance and this cluster of isolates. the increasing levels of quinolone resistance observed raise concerns about the future use of newer quinolones for the treatment of respiratory infections. s. maltophilia has emerged in the last years as an important nosocomial pathogen, inherently resistant to most of the antimicrobial agents. new quinolones has been proposed as a treatment of choice because their enhanced activity, but several parameters (t a , atmosphere, method) can affect the results of mics. methods: we have performed mics using two different methods (agar dilution and microdilution) and different conditions: 35 and 30 8c of temperature; atmosphere of o 2 and co 2 , and incubation times of 18, 24 and 48 h. a total of 78 strains were assayed with nine quinolones following standard nccls. comparisons were made between results with 18 á/24 and 24 á/48 h using the x 2 -test (a 0/0.05). results: no differences were found between 18 á/24 and 24 á/48 h results with agar dilution, except with 3 atbs in the case of mics at 30 8c co 2 . on the contrary, almost all the atbs showed significant differences in the results with 24 and 48 h using microdilution method, at any condition of ta or atmosphere. comparison of mics (p values, significance level 0/95%) with incubation times of 24 and 48 h at different procedure conditions. the incubation time is a parameter that seems to affect significantly the results of mics of quinolones when microdilution method is used, whereas only few differences can be encountered with the agar dilution method. results: breakpoints were used as proposed by nccls 1999. during the study period the pneumococci resistance was noted as follows: 65% to pc, 39% to em and 73% to sxt. the rank order of activity of the five fqs against multi-drug resistant pneumococci was: cip (mic 90:2 mg/l), ofx (mic 90:2 mg/l), lvx (mic 90:1 mg/l), grx (mic 90:0.25 mg/l), tvx (mic 90:0.25 mg/l). conclusions: in romania, fluoroquinolones represent alternative treatment to beta-lactams and macrolides for first-line empirical treatment for respiratory tract infections caused by pneumococci but, continued vigilance for emerging resistance to fqs is further indicated. introduction and material/methods: susceptibility testing (semiautomated broth microdilution method, sensititre, trek diagnostics, usa, following nccls recommendations) was performed with six different quinolones to 817 streptococcus pneumoniae isolates with a ciprofloxacin-cip */mic ]/2 mg/l collected in two consecutive sauce$ surveillances in spain (1996 á/97/1998 á/99). nccls resistance (r) breakpoints were used ( ]/8 for ofloxacin-ofl and levofloxacin-lev; ]/2 for sparfloxacin-spa; ]/4 for gatifloxacin-gat */and moxifloxacin-mox), but for gemifloxacin-gem */where ]/1 was used. results were as follows. conclusions: for cip-r isolates gem and mox were the most active agents. gem was the only agent not influenced by cip mic increase regarding prevalence of r , with 0% resistance for strains with cip mic ]/8 mg/l. $sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en españ a' (susceptibility to the antimicrobials commonly used in the community in spain) and is the spanish word for the willow tree. in vitro activity of gatifloxacin and seven other antibiotics against respiratory and urinary tract pathogens from the community. first results of the basic */study ps110 grimm h, on behalf of a european multicenter study group, institute for med. microbiology, 88250 weingarten, germany a total of 24 centers in austria, belgium, france, germany, italy, portugal, spain and switzerland are involved in the basic study (bacterial annual susceptibility information collection). the mics of gatifloxacin (gati), ciprofloxacin (cipro), clarithromycin (clari), benzylpenicillin g (pen), amoxicillin (amox), amoxicillin/clavulanic acid (augm), cefuroxime (cur) and cefixime (cix) were determined using the microdilution method. each center is requested to investigate 30 strains each of the following species: s. pneumoniae (spn), s. pyogenes (spy), s. aureus (sau), e. faecalis (efa), m. catarrhalis (mca), h. influenzae (hin), e. coli (eco), k. pneumoniae (kpn), p. mirabilis (pmi) and p. aeruginosa (pae). so far approximately 1400 strains are enrolled. some important mic90%/percentage resistance were as follows: from the oral antibiotics tested gatifloxacin has the highest activity and broadest spectrum against all relevant respiratory and urinary tract pathogens. gatifloxacin is a promising alternative for therapy of respiratory tract bacterial infections. in vitro activity of gatifloxacin against bordetella pertussis in comparison with erythromycin, ciprofloxacin and levofloxacin ps111 bourgeois n, pangon b, ghnassia jc, doucet-populaire f, de versailles ch. microbiologie, le chesnay, france purpose of the study: bordetella pertussis infections are far more common in adults and adolescents than is generally estimated. however, they are often not recognised. infected or colonised adults can act as a reservoir of infection, passing it to children. fluoroquinolones are currently recommended for the treatment of respiratory tract infection in adult patients, which is usually empirical. gatifloxacin is a novel 8-methoxyquinolone, with a potent activity against both gram-negative and -positive bacteria. the in vitro activity of gatifloxacin was compared with those of erythromycin, the drug of choice for both treatment and prophylaxis of pertussis, ciprofloxacin and levofloxacin, against 52 clinical isolates strains of b. pertussis including 12 erythromycin resistant strains. results: we used the agar dilution method on mueller á/hinton medium supplemented with 10% horse blood to determine the mic of each antibiotic. gatifloxacin (mic90, 0.125mg/l) was as active as ciprofloxacin and levofloxacin (mic90, 0.06mg/l) against both sensitive erythromycin (mic90, 0.015 mg/l) and resistant erythromycin (mic90, !/512 mg/l) strains. conclusion: gatifloxacin may be an effective drug in the treatment or prophylaxis of adults with suspected or confirmed pertussis. ex vivo serum activity (killing rates) after gemifloxacin 320 mg versus trovafloxacin 200 mg single doses against ciprofloxacin-susceptible andresistant streptococcus pneumoniae ps112 calvo a a , giménez mj b , alou l a , gó mez-lus ml a , aguilar l b , prieto j a . a microbiology department, universidad complutense, madrid, spain , b glaxosmithkline, medical department, tres cantos, madrid, spain serum bactericidal activity was measured ex vivo after single dose administration of gemifloxacin (gem) 320 mg and trovafloxacin (tro) 200 mg to 12 healthy volunteers in a randomized, cross-over phase i trial. blood samples were collected 1 h (cmax) after dosing and serum killing rates were determined against a serotype 3 penicillin (pen) á/ciprofloxacin (cip) susceptible strain (s3) (mics of 0.03, 1, 0.015 and 0.06 mg/l for pen, cip, gem and tro) and a serotype 9 pen á/cip resistant strain (s9) (mics of 2, 4, 0.03 and 0.25 mg/l for pen, cip, gem and tro). tubes with 1.6 ml of serum sample and 0.4 ml broth (50% todd á/hewitt'/50% hbss) were incubated over 3 h at 35 8c. final inocula was 10 7 cfu/ml. mean colony counts for samples and controls (k ) are shown in the figure: gem exhibited higher colony counting decrease of the initial inocula, versus tro, for both strains. after 3 h incubation, the initial inocula decrease obtained with tro and the cip susceptible strain was similar to that obtained with gem and the resistant strain, showing a lower influence of cip mic increase in the ex vivo bactericidal activity of gem versus tro. urine bactericidal activity after administration of gemifloxacin and trovafloxacin single doses in a phase i study ps113 garcía-calvo g a , parra a a , giménez mj b , ponte c a , aguilar l b , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain urine bactericidal activity after o.d. administration of gemifloxacin (gem) 320 mg and trovafloxacin (tro) 200 mg, was assessed in six adult males in a cross-over phase i trial. urine killing rates (ukr) against escherichia coli atcc 25922 (mic mg/l of 0.008 and 0.015 for gem and tro) and s. saprophyticus atcc 1970 (mic mg/l 0.015 and 0.06 for gem and tro) were performed with samples collected at 0, 2 á/4, 16 á/24 and 60 á/72 h. a 1.6 ml of iso-sensitest broth and 0.4 ml of bacterial logarithmic growth were added to 2 ml sample, giving a final inoculum of 107 cfu/ml. colony counting was performed after 1, 2, 3 and 4 h incubation. percentages of initial inocula reduction (iir) were calculated. mean urine concentrations measured by bioassay were (mg/l): 28.4, 14.7 and 0.5 for gem, and 3.6, 3.0 and 0.1 for tro. against e. coli , an iir of 99.9% was obtained after 2 h incubation with all samples except with tro at 60 á/72 h. against s. saprophyticus an iir of 90% was obtained after 3 h incubation with all samples except with tro at 60 á/72 h, where bacterial regrowth was found. the maintenance over 72 h of gem urine antibacterial activity suggests its efficacy in the treatment of uncomplicated cystitis. influence of the decreased susceptibility to ciprofloxacin on gemifloxacin versus levofloxacin efficacy in experimental pneumococcal pneumonia in guinea pigs ps114 garcia-olmos m a , parra a a , gimenez mj b , garcia-calvo g a , ponte c a , aguilar l b , soriano f a . a fundacion jimenez diaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain the efficacy of ciprofloxacin (cip), levofloxacin (lev) and gemifloxacin (gem) in the treatment of pneumococcal pneumonia was assessed in a guinea pig model using three strains (s) with mics (mg/l) of 2, 2 and 0.03 (s1), 32, 4 and 0.25 (s2) and 64, 32 and 1 (s3) for cip, lev and gem, respectively. intraperitoneal treatments started 1 h after s. pneumoniae intratracheal inoculation, and continued t.i.d up to four doses. ten animals were included in each group. doses (mg/kg) used were 20, 16 and 6 for cip, lev and gem, respectively, in order to mimic auc0-24 h and cmax obtained in humans after standard doses. animals that survived 48 h after inoculation were sacrificed and colony counts were performed in lungs ( the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of nosocomial pneumonia. our aim was to study the pharmacokinetic á/pharmacodynamic appropriateness of lfx 500mg iv bid in the treatment of six inpatients with ventilator-associated pneumonia (vap) (459/25 years; 4m á/2f; 749/9 kg). blood and urine samples were collected in steadystate conditions at appropriate intervals. lfx concentrations were analysed by hplc. the aetiological agent was identified in all the cases and its in vitro sensitivity to lfx was always assessed. the results obtained mean values (9/sd) of the major pharmacokinetic parameters were: cmax, 8.789/2.05 mg/ml; vdss, 1.259/0.36 l/kg; t1/2b, 4.699/0.87 h; cl, 3.659/1.01 ml/min/kg; auc0-t, 32.479/12.80 mg/ml h. cumulative urinary excretion was 82.899/10.23%, confirming that lfx clearance is mainly renal. clinical cure and microbiological eradication were obtained in all the patients after a 7 á/13 day therapy. a suprainfection due to acinetobacter anitratus insensitive to lfx occured in 1 case. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/10; auc/mic !/125) in all the cases. the conclusion reached the findings suggest that lfx 500 mg bid iv may be considered effective in the treatment of vap caused by sensitive bacteria. comparative pharmacokinetics of levofloxacin in patients with lower respiratory tract infections (lrti) being treated with sequential therapy ps116 pea f a , brollo l a , lugatti e b , di qual e a , dolcet f b , talmassons g b , furlanut m a . a institute of clinical pharmacology and toxicology, dpmsc, university of udine, udine, italy , b division of pneumology, sm misericordia hospital, udine, italy the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of lrti. our aim was to study the pharmacokinetic á/pharmacodynamic appropriateness of a standard switch lfx iv/os regimen (500 mg iv od for 5 á/7 days followed by 500 mg os od for 4 á/10 days) in the treatment of seven inpatients with lrti (709/17 years; 6m á/1f; 769/15 kg). blood samples were collected in steady-state conditions at appropriate intervals. lfx plasma concentrations were analysed by hplc. the aetiological agent was identified in 2/7 cases and its in vitro sensitivity to lfx was assessed. the results obtained absolute oral bioavailability was 1.079/0.19, with a cmax of 10.899/3.39 vs 8.369/3.90 mg/ml after iv and oral administration, respectively. no significant difference in the main pharmacokinetic parameters was observed between the two routes. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/10; auc/mic !/125) in the two assessable cases. all the patients were clinically cured after a 9 á/15 day therapy. the conclusion reached the ad interim findings show that lfx 500 mg od may guarantee per os an exposure similar to that achievable after iv administration, suggesting that sequential therapy may be considered effective in the treatment of lrti. levofloxacine in the exacerbations of copd due to pseudomonas ae ps117 micheletto c, tognella s, pomari c, dal negro r, ospedale orlandi, div.pneumologia, bussolengo, italy development of antibiotic resistance in bacteria is a problem of great concern. gram-negative bacteria, including multidrug-resistant (mdr) pseudomonas aeruginosa (ps), are responsible for a significant proportion of episodes of copd exacerbations, particularly in elderly (1). aim was to check the susceptibility to common antimicrobial treatments against ps strains isolated from bronchial secretions in patients with severe exacerbations of copd. methods: microbial investigations were conducted on 290 specimens: spontaneous purulent sputum (88.4%), and tracheobronchial aspirates (11.6%, collected with a protected specimen brush). results: fifty-seven ps pathogen strains (106 cfu) were identified (19.6%) and tested over a 6-month period: ps. aeruginosa 91.2%; ps. putida 3.5%; ps. fluorescens 3.5%, and burkholderia cepacia 1.8%. the assessed susceptibility to most common antibiotics was: levofloxacine (90%), ciprofloxacine (84%), ipenem cil. (88%), ceftazidime (84%); amikacin (84%), and piperacillin'/tazobactam (74%). a much lower susceptibility was found for ticarcillin á/clavulanic acid (58%), gentamicin (48%), and netilmicin (40%). (1) at present, levofloxacine proves the most effective antimicrobic option for treating copd exacerbations due to ps infection; (2) a much more efficient policy of antibiotic prescribing should be promoted in order to prevent the selection of resistant strains in these cases. these results confirm the excellent 'in vitro' activity of levofloxacin against nosocomial gram-negative pathogens, including the esbl producing strains (90% of escherichia coli , e. cloacae and k. pneumoniae were inhibited at 0.5 mg/l). levofloxacin was more rapid than ciprofloxacin to determine a bactericidal effect particularly against s. maltophilia . moreover, considering the favourable pk/pd profile, levofloxacin can represent a valid therapeutic option for the treatment of severe gram-negative nosocomial infections. moretti f, quiros-roldan e, casari s, chiodera a, viale p, carosi g. university of brescia, institute of infectious and tropical diseases, brescia, italy a 34-year-old man, ivdu, hiv positive was attended in aur hospital for fever and toracic pain. a x-chest radiography revealed a round lesion of 5 cm near the lingula with central hyper-diaphan area. lymphocytes cd4'/ count was 21 cells/mm 3 and viral load 91.900 cp/ ml. hospital stay rhodococcus equi was found in cultures of peripheral blood, faecal and sputum specimens. antibiotic treatment with oral rifampin (600 mg/qd) and with intravenous imipenem (500 mg tid) was started. due to the persisting fever, immodificated radiography and negativity for p. carinii , mycobacteria and bacteria in bal coltures, imipenem was substituted by parenteral vancomycin (400 mg bid). after 10 days, because of persisting fever and increase of the diameter of the lung lesion (6 cm) vancomycin was sustituted by oral levofloxacyn (500 mg bid), continuing rifampin. after a 4 days course of levofloxacyn therapy the fever remitted. the patient was discharged with levofloxacyn (500 mg bid) and rifampin and, after 2 months of follow-up, a radiological control pointed-out a remarkable resolution in the lung lesion. we may suppose that levofloxacyn can be effective for the treatment of r. equi infection, even if more studies (particularly controlled studies) are necessary. liberti a a , izzo b a , loiacono l b , calabria g a , patarino t a , izzo e a . a ii department, naples, d. cotugno hospital, italy , b iii department, naples, d. cotugno hospital, italy the increasing prevalence of salmonella typhi strains with reduced susceptibility of chloramphenicol had prompted the search for other antibiotics with the same efficacy.quinolones are a class of antibiotic with an activity in vitro and in vivo against enteropathogenes. we inwestigated the use of levoxacin in two regimens of treatment of typhoid and paratyphoid infection. patients and methods: thirty-two adult patients were incluted in this study from september 1999 to april 2001; 26 patients had positive culture for s. typhi and six had positive cultures for s. paratyphi . all isolated were fully susceptible to levoxacin. we compared treatment with levoxacin for 7 days, 500 mgr b.i.d. (group 1, 16 patients), with treatment for 10 days, 500 mgr b.i.d. (group 2, 16 patients). clinical cure was defined as defervescenze of fever by day 3 of treatment, with an absence of complications and no clinical relapse during the followup. results and conclusion: the clinical cure rate was 87% (14 patients) for group 1 and 94% (15 patients) for group 2; the difference in these rates was not statistically significant. the blood cultures of all patients were sterile by day 2 of treatment and remained so until the 6th month of follow-up, no subjects had clinical or microbiological relapse and all stool cultures remained negative, also. the two regimens of treatment was good tollerated and no adverse event was registered; it was concluded that levoxacin treatment for 10 days in enteric fever is not necessary the mulidrug-resistence of s. typhi led to the use of quinolones as the first-line drug in the treatment of enteric fever. pefloxacin in the treatment of patient with acute infectious diarrhoea ps121 troselj-vukiae tvb a , strahinja v b , poljak i a , stojanoviae d c , nikoliae n d . a department of infectious disease, university hospital center, rijeka, croatia , b glaxosmithkline, marketing, rijeka, croatia , c dept. rijeka, institute of public health, rijeka, croatia , d dept. rijeka, maritime academy, rijeka, croatia the purpose of the study was to investigate clinical and bacteriological efficiency in 5 and 7 days pefloxacin treatment and to compare it with symptomatic therapy. the results obtained in 47 patients treated with pefloxacin the therapy was clinically effective already in the third day while in the control group this happend in the 7th day. bacteriological eradication was noted in 18 patients (95%) of the first and 25 patients (93%) of second group in the 5th days of the treatment. they all had negative cultures 1 and 4 weeks after pefloxacin protocols were completed. only 22 patients (63%) in control group had negative stool cultures in the 7th day of the treatment and all of them 4 weeks after it ended. there was no statistically significant difference in clinical (p 0/0.232) and bacteriological (p 0/0.972) efficiency between 5 and 7 days pefloxacin treatment protocols. both protocols significantly differed in clinical (p b/0.001) and bacteriological (p0/0.017) eradication from the control group. the conclusion reached is that the efficiency of pefloxacin (quinolones) in the treatment of acute infectiuos diarrhoea and justifies their use in the more severe forms of the disease. dalhoff a a , ullmann u b , schubert s b . a bayer ag, wuppertal, germany , b university of kiel, institute of med. microbiology, kiel, germany background: the antibacterial activity of moxifloxacin (mxf) was compared to levofloxacin (lev), amoxicillin (amx), clarithromycin (cla) and erythromycin (ery) in an in vitro model. method: pharmacokinetics in bronchial mucosa (bm) and serum (s) following single oral doses of 400 mg mxf or cla and 500 mg lev, amx or ery were simulated using a one compartment model. bacteria tested staphylococcus aureus (nos. 133, 25895), streptococcus pneumoniae (nos. 4241, 672). aliquots were taken (0 á/8 h) and plated on to brain heart infusion agar for enumeration. results: s. pneumoniae 4241 was eliminated by all agents studied. significant differences were apparent with s. pneumoniae 672 and the s. aureus strains: objective: to compare the safety and efficacy of once-daily moxifloxacin with once-daily ceftriaxone in the treatment of cap in hiv-infected patients (pts). methods and results: in a retrospective survey, oral moxifloxacin (400 mg daily)/12 á/15 days) was compared to standard regimen of i.v. ceftriaxone (2 g daily)/10 á/12 days) for treatment of cap in hiv'/ pts. adults pts with clinical signs and symptoms of cap and consistent chest x-ray findings were included. pts had a median age of 39 years (range 29 á/529 and 70% were male). demographic characteristics were similar in both treatment groups; 15 pts received mxifloxacin and 25 pts ceftriaxone. clinical success rates were 94% for moxifloxacin and 96% for ceftriaxone. at a post-study evaluation approximately 8 weeks later, 2 moxifloxacin-treated pts and 3 ceftriaxone-treated pts had relapsed. the adverse events reported were comparable for both treatment groups. there were four-related adverse events (3 gi, 1 headache) for moxifloxacin-treated and 7 (4gi, 3 skin) for ceftriaxonetreated pts. conclusion: the results of this study show that moxifloxacin as oral therapy is as effective and well tolerated as i.v. ceftriaxone in the treatment of hiv'/ pts with cap. therapy with moxifloxacin was not associated with any significant clinical or laboratory abnormalities. these data suggest that once-daily oral administration of moxifloxacin is potentially convenient and cost-effective alternative therapy for cap in pts with hiv infection. moxifloxacin in the treatment of acute maxillary sinusitis after first-line therapy failure or acute sinusitis with high risk of complications ps124 gehanno p a , berche p b , perrin a b , arvis p c . a ent department, bichat claude bernard hospital, paris, france , b microbiology department, necker enfants malades hospital, paris, france , c bayer pharma, medical affairs department, paris, france the efficacy and safety of moxifloxacin (mxf) 400 mg once daily for 7 days was evaluated in the treatment of acute maxillary sinusitis after first-line therapy failure, or acute sinusitis with high risk of complications. in this prospective, multicenter study, a total of 216 patients with acute bacterial sinusitis confirmed by sinus x-ray were valid for efficacy analysis: one hundred and seventy five patients (81.0%) had an acute maxillary sinusitis which failed to respond to a previous antibiotic therapy given for a mean duration of 7.2 days, and 41 (19.0%) had an acute sinusitis with high risk of complications (frontal: 24, pan-sinusitis: 15 and sphenoid: 2). ninety two patients (42.6%) were microbiologically valid. clinical cure and continued clinical cure rates at 7 á/10 and 28 á/35 days post-therapy were 92.6 and 99.0%, respectively. clinical cure rates at 7 á/10 days post-therapy were 94.9 and 82.9% in sinusitis after first-line therapy failure and in sinusitis with high risk of complications, respectively. bacteriological eradication rate during therapy (day 3 á/4) was 95.7%. at 7 á/10 days post-therapy. bacteriological success rates were 97.1% in patients who failed to respond to a previous antibiotic and 95.4% of patients who had sinusitis with high risk of complications. 12.2% of patients experienced drug-related adverse events, abdominal pain (2.4%), nausea (2.4%) being the most frequently reported events. mxf was rapidly effective and a well-tolerated treatment for this kind of infection. neisseria gonorrhoeae with decreased susceptibility to penicillin and ciprofloxacin: novel mutation patterns in the gyr a and par c genes of ciprofloxacin resistant isolates and plasmid profile of penicillin resistant isolates of n. gonorrhoeae in india (delhi) ps125 chaudhry u, saluja d. dr. b. r. ambedkar center for biomedical research, university of delhi, delhi, india commercial sex workers (csws) serve as the most important reservoir of gonorrhoea. periodic monitoring of the antimicrobial susceptibility profile of neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. in india, such a surveillance of the in vitro antimicrobial susceptibility of n. gonorrhoeae was established in 1997. significant increasing trend of penicillin and ciprofloxacin resistance with high mic of 2 á/16 and 1 á/32 mg/ml, respectively were found over the years (1997 á/2001) . the molecular basis of ciprofloxacin resistance, i.e. mutations in the gyr a and the par c genes of 170 isolates, were analyzed. four isolates (with an mic of 32 mg/ml for ciprofloxacin) harbored triple mutations (ser-91 to phe, asp-95 to asn and val-120 to leu) in the gyr a gene. the third mutation of val-120 to leu, lies downstream of the quinolone resistance determining region of the gyr a and has not been described before in gonococcus. in addition, these isolates had a phe-100 to tyr substitution in the par c, a hitherto unknown mutation. the alterations in the par c gene were seen in these isolates only in the presence of changes in the gyr a gene and comprised amino acid changes at codons 91, 100, 104, 109, and 131. the presence of b-lactamase plasmid among the penicillinresistant isolates was determined by their plasmid profiles and further confirmation was carried out by a pcr based protocol. our findings suggest that emergence of penicillin and ciprofloxacin resistance in n. gonorrhoeae isolates from a major std center in india, indicates the need for the increased awareness and prudent use of antimicrobials. in vitro activity of newer antibiotics against methicillin-resistant staphylococcus aureus ps126 gutierrez zufiaurre mn, sanchez hernandez j, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: mrsa are frequently co-resistant to a number of structurally unrelated antibiotics. more than 70% mrsa are resistant to gentamycin, ciprofloxacin, macrolides and clindamycin. newer antibiotics active against multi-drug resistant grampositives have been developed. we have tested the in vitro activity of newer antibiotics against genetically-characterized, high level ciprofloxacin resistant mrsa. material and methods: thirty-six ciprofloxacin-resistant, gyra/grla mutant mrsa clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), gatifloxacin (gfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method, according nccls guidelines. results and conclusions: all the strains were resistant to cfx, 55,5% were lfx-susceptible and 63.9% were gfx-susceptible. nevertheless, mics of lfx and gfx for all susceptible strains was in the highest extreme of the susceptibility range. mfx was the most active quinolone. almost all the strains were high-level er-resistant with constitutive mlsb phenotype. tl did not improve significantly its behaviour, although it was 10-fold as active as er against the only er-susceptible strain. va, lin and syn had excellent activity against all the strains. they showed a very homogeneous behaviour against all the strains that were included in a range of 1 á/4 mg/l of lin and va and 0.5 á/1 mg/l. sanchez hernandez j, gutierrez zufiaurre mn, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: corynebacterium urealyticum is the etiologic agent of encrusted cystitis and inespecific utis, and can be also involved in systemic infections. c. urealyticum is frequently multi-drug resistant, so only glycopeptide antibiotics and tetracyclines have high susceptibility rates, while fluoroquinolones resistance rates vary significantly. we have tested the in vitro activity of linezolid, telithromycin, synercid and newer fluoroquinolones against multi-drug resistant c. urealyticum clinical strains. material and methods: sixty-four c. urealyticum clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method according guidelines defined by the nccls for enterococci. results and conclusions: results confirm the high resistance rate to older fluoroquinolones and macrolides, with !/50% cfx resistance and 100% er-resistance. lfx was more active (mic90 32 mg/ml). mfx was the most active fluoroquinolones (mic90 4 mg/ml). tl improve its behaviour in respect to er (range 0.5 á/1 mg/ml). va, lin and syn had excellent antimicrobial activity. no resistant strains were found. mic90 were 1, 0.5 and 1 mg/ml, respectively. mics were similar for all the strains independently of their resistance to other antibiotics plasma concentrations, urinary excretion and bactericidal activity of linezolid (600 mg) versus ciprofloxacin (500 mg) in healthy volunteers after a single oral dose ps128 wagenlehner fme a , wydra s a , onda h a , kinzig-schippers m b , sö rgel f b , naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, (ibmp), nürnberg-heroldsberg, germany purpose of the study: in a randomized cross-over study 12 volunteers received a single oral dose of 600 mg linezolid versus 500 mg ciprofloxacin to assess plasma concentrations (up to 24 h), urinary excretion (by hplc), and urinary bactericidal titers (ubt) up to 120 h. the mean maximum plasma concentration of linezolid was 12.1 mg/ ml and of ciprofloxacin 2.4 mg/ml. the cumulative renal excretion (mean) of parent drug was 40%/41% for linezolid/ciprofloxacin. ubts were determined for a reference strain and five gram-positive clinical uropathogens with the following mics (mg/ml) for linezolid/ciprofloxacin: s. aureus atcc 27278 (4/0.25), s. aureus (mssa) (2/32), s. aureus (mrsa) (2/128), s. saprophyticus (msse) (2/0.5), e. faecalis (2/1), e. faecium (2/2). results: median ubts measured within the first 6 h for linezolid were 1:96 for enterococcal strains and 1:128 to 1:256 for the four staphylococal strains. median ubts for ciprofloxacin were 1:64 for the two enterococcal strains, 1:384 to 1:512 for the two ciprofloxacin susceptible, 1:1.5 for the two resistant staphylococcal strains. areas under the ubt á/time-curve showed statistically significant differences only for the two ciprofloxacin resistant staphylococcal strains in favour of linezolid. conclusion: linezolid exhibited the same bactericidal activity against ciprofloxacin resistant as susceptible strains. linezolid should be tested for treatment of complicated uti due to gram-positive uropathogens in a clinical trial. whitehouse t a , cepeda ja a , tobin purpose: we performed pharmacokinetics within a double-blind, randomised trial comparing linezolid and teicoplanin in intensive care patients with known or suspected gram-positive infection. they received either 600 mg linezolid intravenously 12-hourly, or 400 mg teicoplanin 12-hourly for the first three doses and once daily thereafter (or every 3rd day if renally impaired). blood samples were collected to create serum pharmacokinetic profiles. linezolid was quantitated by hplc and teicoplanin by fluorescence polarization immunoassay. results: twenty two patients were studied in the linezolid group (m á/f 13:9, mean age 54 years [range 19 á/90 years]). median treatment duration was 8.5 days (range 4 á/18). eighteen patients were treated with teicoplanin (m á/f 13:5, mean age 57 years [range 17 á/86]) for median 9 days (range 4 á/17). steady state peak concentrations (mean9/ sd) for linezolid and teicoplanin were 12.89/5.0 and 10.59/4.7 mg/l, respectively. trough concentrations at day 4 were 4.79/4.3 mg/l for linezolid and 7.99/2.5 mg/l for teicoplanin. recommended breakpoints of staphylococcus aureus are 4 mg/l for linezolid and 8 mg/l for teicoplanin. accumulation occurred in one 90-year-old linezolidtreated patient with impaired renal function. conclusion: current recommended dosing regimens for linezolid and teicoplanin are generally appropriate in the critically ill, though a more detailed analysis is required. stamos g, lebessi e, ioannidou s, paleologou n, kallergi k, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of the study was to investigate the susceptibility of methicillin resistant staphylococcus aureus (mrsa) isolates from a 500-bed paediatric hospital to quinupristin/dalfopristin (q/d, streptogramin combination) and linezolid (lzd, oxazolidinone). material: we performed a retrospective analysis of 85 mrsa strains, isolated from patients hospitalized in miscellaneous medical departments [neonatal unit (35) , surgical wards (15), orthopaedic wards (9), oncology unit (7), other wards (8) and outpatient clinic (11)], during a 3-year period (1998 á/2000) . the sources of isolation were pus (23), throat (22), nasal (8), bronchial (5), skin (6), stool (6) ear (4) and other (11) specimens. all isolates were sensitive to glycopeptides, while 22.4% were resistant to gentamicin and 7.1% to erythromycin. methods: the sensitivity testing was performed by a disk diffusion method (bbl sensitivity disks, becton dickinson), according to nccls guidelines. the breakpoint zone diameters for lzd and q/ d were ]/21 and ]/19 mm for susceptibility and 5/17 and 5/15 mm for resistance, respectively. results: all isolates were proved to be susceptible to both antibiotics. the mean inhibition zones were 29.1 mm for lzd and 26 mm for q/d. conclusions: lzd and q/d are very promising antimicrobial agents, showing excellent activity against mrsa clinical isolates. the prudent therapeutic use is strongly recommended to avoid the emergence of resistance. in vitro activity of streptogramins and oxazolidinones against streptococcus pneumoniae clinical isolates ps131 stamos g, lebessi e, paleologou n, psatha m, sanida p, zaphiropoulou a, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of this study was to evaluate the in vitro activity of linezolid (lzd), a member of oxazolidinones and the streptogramin combination quinupristin/dalfopristin (q/d) against clinical isolates of streptococcus pneumoniae from a tertiary care paediatric hospital. material: a total of 53 pneumococcal isolates exhibiting reduced susceptibility to common antibiotics were included in the study. the strains were isolated from middle ear fluid (34), eye (4), nasal (5) blood (6) and other (4) cultures during the last 4 years. the percentages of the isolates that were resistant to penicillin, erythromycin, cotrimoxazole and clindamycin were 67.9, 67.9, 67.9 and 26.4%, respectively. methods: the susceptibility testing was performed by a standard disk diffusion method (bbl sensitivity disks, becton dickinson). in case of marginal results or intermediate sensitivity to quinupristin/ dalfopristin, the mic was determined using the e -test method (ab biodisk). results: all isolates were found to be sensitive to lzd. q/d was very active as well, except for two isolates that exhibited intermediate susceptibility, showing cross-resistance to macrolides and clindamycin, as well. conclusions: the new antimicrobial agents show excellent activity against resistant to common antimicrobials pneumococcal isolates, but the clinical use is not suggested, unless no other therapeutic solutions are available. linezolid is a new oxazolidinone with excellent activity against gram-positive organisms including glycopeptide-resistant strains of staphylococci and enterococci. in icu linezolid has been used for the treatment of severe gram-positive infections in a control trial. the susceptibility pattern of all gram-positive isolates from icu patients has been studied. methods: a total of 2427 specimens from icu patients were processed, 767 were from patients enrolled in the antibiotic trial. all methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci (cons), enterococcus sp and methicillin-sensitive staphylococcus aureus (mssa) were tested. the break point for linezolid was 4 mg/l and for teicoplanin 8 mg/l. isolates were tested for susceptibility by e -test. results: linezolid (1063 isolates) mic 50/90 (mg/l) were as follows: mrsa (n 0/541) 0.6/2, cons (n0/143) 0.9/1.4, enterococcus sp (n0/39) 0.7/0.8, mssa (n0/340) 0.8/1. teicoplanin (782 isolates) mic 50/90(mg/l) mrsa (n0/415) 1.9/3.5, cons (n 0/100) 4/7.4, enterococcus sp (n0/24) 0.9/1.4, mssa (n0/243) 2.2/2.6. all grampositive isolates were inhibited by concentrations of linezolid below the breakpoint including eight strains of staphylococci resistant to teicoplanin. conclusions: linezolid was highly active against grampositive isolates. resistance to teicoplanin was similar to other reported series. there was no emergence of resistance to linezolid. mrsa colonization is often a limiting factor for discharge from icu. clearance of mrsa is seldom achieved with conventional glycopeptide treatment. the oxazolidinone, linezolid, has excellent soft tissue and respiratory tract penetration and might be expected to eradicate carriage in some patients. we recently performed a doubleblind randomized trial in 204 icu patients with known/suspected gram positive infection. 100 received intravenous linezolid, 600 mg b.d., and 104 patients received teicoplanin 400 mg o.d. mrsa clearance was assessed at end of treatment (eot), at 7-and 21days follow-up. results: in the linezolid and teicoplanin groups, 45 and 43 were known to be colonized with mrsa at study entry, respectively, while 39 and 40 were not. detection of clearance of mrsa colonization at eot was 51% for linezolid vs 19% for teicoplanin group (x b p b/ 0.005), at 7 days it was 35% vs 14% (x b p b/0.1), and at 21 days 19% vs 33% (ns). conclusion: short-term mrsa clearance can be achieved in significantly more patients treated with linezolid however this was not maintained at 21 days, either because of incomplete initial eradication or recolonization. further analysis will follow when molecular typing of the isolates is completed. penetration of linezolid into bone, fat and muscle during hip arthroplasty ps134 lovering am, bannister gc, zhang j, macgowan ap, southmead hospital, bcare, bristol, uk there are limited data describing the concentrations and penetration of linezolid (lzd) into tissues, such as bone, that can be used to guide therapy for non-vascular infections. here we report the concentrations and penetration of lzd for bone, fat and muscle in comparison with cefamandole (cmd). twelve patients received 600 mg lzd as a 20 min infusion and 1000 mg cmd as a bolus injection immediately before hip arthroplasty. bone, fat, muscle and blood samples were collected at timed intervals after the infusion and assayed by a validated hplc method. for bone, peak levels of both agents occurred 10 min after administration with mean levels of lzd 9.1 mg/kg versus cmd 17.5 mg/kg, decreasing to lzd 5.7 mg/kg versus cmd 9.7 mg/kg at 30 min. correction for blood concentrations gave penetration of lzd 51% versus cmd 25% at 10 min and lzd 46% versus cmd 23% at 30 min. for fat and muscle, peak levels occurred 20 min after infusion. mean levels were lzd 5.2 mg/kg versus cmd 10.9 mg/kg (fat) and lzd 13.4 mg/kg versus cmd 10.9 mg/kg (muscle). correction for blood concentrations gave penetration of lzd 37% versus cmd 19% (fat) and lzd 95% versus cmd 31% (muscle). we conclude that linezolid exhibits rapid penetration into bone and associated soft tissues achieving levels in excess of the mic for sensitive organisms; with a similar distribution and penetration profile to agents currently used for treatment of infections in these tissues. bacqué e a , barrière jc a , berthaud n b , desmazeau p b , dutruc-rosset g b , dutka-malen s b , ronan b b . a aventis pharma, chemistry, paris, france , b aventis pharma, disease group, paris, france xrp2868 is a new oral streptogramin composed of 2 semi-synthetic synergistic components in a 30/70 (w/w) association: rpr202868 (5d-(1-morpholino)methyl-5d,5g-dehydro pristinamycin i e ) from pi a and rpr132552 [(16r )-16-deoxy-16-fluoro pristinamycin ii b ] from pii b by original synthetic routes. the association has antibacterial activity against staphylococci including methicillin */mls b -resistant strains (mic90 range: 0.12 */1 mg/ml), streptococci (mic90: 0.25 mg/ml), pneumococci including multidrug resistant strains (mic90: 0.50 mg/ ml), enterococci including vancomycin-resistant strains (mic90: 4 mg/ ml), m. catarrhalis and neisseria spp. (mic90: 0.12 mg/ml), h. influenzae (mic90: 1 mg/ml), legionella spp. (mic90: 0.06 mg/ml) and anaerobes (mic range: 0.06 á/4 mg/ml). xrp2868 is generally bactericidal at the concentration of 2 á/4)/the mic against s. aureus , s. pneumoniae , h. influenzae and demonstrates consequent pae (2.1 á/ !/5.7 h at 2 á/4)/mic, following an exposure of 0.25 á/2 h). mutants of s. aureus to xrp2868 were isolated at low frequencies (3.6)/ 10 (9 á/9.7)/10 (10 ) at 2)/ and 4)/mic while no mutant could be isolated at 8)/mic. these results suggest that xrp2868 (30/70 w/w) is a promising compound for the treatment of community-acquired infections. ex vivo evaluation of rpr202868/rpr132552 (xrp2868), a new oral streptogramin ps136 berthaud n, diallo n. aventis pharma sa, infectious disease group, paris, france the intra cellular activity of xrp2868, was assessed in j774 murine macrophages containing ingested staphylococcus aureus (three strains) or l. pneumophila (one strain). at the concentration of 8 )/ the mic, growth of s. aureus was strongly inhibited after a 3-h period of incubation (d log 10 cfu/ml vs t 0 controls range: (/2.16 á/(/1.24, according to the strain tested). at the concentration of 8)/ the mic, growth of intracellular l. pneumophila was inhibited after a 48 á/ 72-h period of incubation (d log 10 cfu/ml vs t controls about (/1.30 and (/1.48 at 48 and 72 h, respectively). rpr202868 and rpr132552 alone had also inhibiting effect on bacterial growth (d log 10 cfu/ml vs t 0 controls after 72 h of incubation about (/1.54 and (/0.87, respectively). the bactericidal activity of xrp2868 was also assessed against slowly growing s. aureus , under experimental conditions mimicking those observed in patients with an infected indwelling device (first step of infection: adherence to inert support; declared infection: biofilm model). under the experimental conditions, xrp2868 demonstrated a rapid and potent bactericidal effect against s. aureus adherent to an inert support or included in biofilm. this effect was observed at each of the three concentrations tested (5, 10, 50 and 8, 100 and 200)/ mic, respectively). in vivo evaluation of xrp2868 (rpr202868/rpr132552), a new oral streptogramin ps137 berthaud n, huet y, aventis pharma sa, infectious disease group, paris, france the oral efficacy of xrp2868, was assessed in staphylococcus and pneumococcal murine infections. mice were challenged ip ( )/10, and )/100 times ld 50 ). abscesses were established by intramuscular injection of about 10 7 bacteria into the right thigh of mice. pneumonia was established by intranasal injection of about 10 7 bacteria. mice were treated twice a day for 1 (staphylococcus aureus septicaemia and abscesses), 2 (s. pneunomoniae septicaemia) or 3 days (s. pneumoniae pneumonia). six to 15 days post infection, for septicaemia and abscesses the results were expressed as ed 50 , whereas for pneumonia they were expressed as the dose yielding an average survival time (ast) significantly longer than that of the untreated infected controls. xrp2868 was efficacious in the treatment of experimental infections in mice caused by mls b -sensitive and -constitutively resistant s. aureus (ed 50 range: 20 á/44 and 24 á/60 mg/kg/administration in septicaemia and abscess models, respectively). it was also efficacious in the treatment of infections caused by s. pneumoniae whatever the serotype and the resistance profile of the strains tested (ed 50 range: 28 á/55 mg/ kg/administration in septicaemia, ast: 50 mg/kg/administration). these results suggest that xrp2868 might be effective for the treatment of staphylococcal and pneumococcal community-acquired infections. xrp2868 (rpr202868/rpr132552), a new oral streptogramin: bactericidal activity and pharmacokinetics in a model of streptococcus pneumoniae mouse pneumonia ps138 berthaud n, huet y, diallo n. aventis pharma sa, infectious disease group, paris, france the bactericidal activity against streptococcus pneumoniae of xrp2868, a new oral streptogramin composed of two semi-synthetic synergistic components in a 30/70 (w/w) association (rpr202868, pristinamycin i derivative and rpr132552, pristinamycin ii derivative), was assessed in lungs of mice with pneumonia. mice were inoculated intranasally with about 10 7 cfu of strain 6254-01 (mls bresistant). eighteen hours later (t 0 ), animals received xrp2868 (120 mg/kg p.o). the administration was repeated 6, 24, 30, 48 and 54 h afterwards. to study the influence of varying the ratio of pi to pii component administered on activity and pk parameters, ratios of rpr202868 to rpr132552 ranging from 0/100 to 100/0 were also administered under the same conditions. after an oral unitary administration at 120 mg/kg, xrp2868, as well as ratios of rpr202868 to rpr132552 ranging from 50/50 to 90/10, demonstrated strong and quick bactericidal activity in lungs. lung levels of rpr202868 and rpr132552 were generally equal or two times higher than blood levels. resulting rpr202868/rpr132552 ratios in blood and lung, although not in accordance with the initial ratio administered, were synergistic for 3 á/4 h in blood and for 1 á/3 h in lungs explaining the activity observed. conclusion: based on in vitro data, telithromycin is a good candidate for the treatment of rti. in vitro evaluation of abt-773, telithromycin and azithromycin against streptococcus pneumoniae , moraxella catarrhalis , haemophilis influenzae and methicillin-resistant staphylococcus aureus ps140 steele-moore l, berg d, barnes i, couch k, klein j, holloway w. christiana care, infectious disease, wilmington, us macrolide resistant streptococcus pneumoniae (sp) is a worldwide concern predominantly because these isolates tend to be multiply drug resistant. new agents with increased activity against these pathogens are clinically important. the ketolide class of antimicrobial agents demonstrate excellent in vitro activity against sp, even those that are macrolide resistant. the in vitro activities of the ketolides abt-773 (a) and telithromycin (t) were compared to azithromycin (az) against clinical isolates of sp, moraxella catarrhalis (m.cat), haemophilis influenzae (h.flu) and mrsa. organisms tested: 51 strains of sp (including 14 az resistant), 25 h.flu, 25 mrsa and 10 m.cat. microdilution mic tests were performed following nccls recommendations using freshly prepared plates containing haemophilis test medium for h.flu, cation adjusted mueller-hinton broth (camhb) with laked horse blood for sp and camhb for m.cat and mrsa. the new ketolides, a and t had superior activity against sp including the az resistant strains (mic90s: a0/0.12 mcg/ml, t 0/0.25, az0/4). all compounds had excellent activity against m.cat while none demonstrated activity against mrsa. h.flu activity was comparable among a, t and az. these new ketolides are not currently approved by the fda; however t has been approved in europe. ló pez h, vidal gi, salomó n jm, scaglione m, zitto tr. centro de infectología, infectious diseases, buenos aires, argentina objective: we evaluated the impact of the initial treatment failure rate, hospitalisation and costs in outpatient treatment of adult cap in argentina comparing amoxicillin, clarithromycin and telithromycin. method: a probabilistic model was implemented in outpatient treatment of cap. we estimated an initial treatment with amoxicillin, clarithromycin or telithromycin. we assumed expected clinical cure at 90.1, 88.5 and 94.6%, respectively. for those patients with failure treatment we evaluated a second-line of antibiotics (amoxicillin followed by clarithromycin and clarithromycin followed by new fluorquinolone) or hospitalisation. patients with telithromycin and failure treatment must be hospitalised without a second line of outpatient treatment. costs of cap included drug's costs by 10 days, medical visits, chest radiographies, analysis and hospitalisation. results: we estimated treatments in 100 patients and first-line drug failure in 10, 11 and 5 patients with amoxicillin, clarithromycin and telithromycin, respectively. costs in outpatient treatment were: hospitalisation $12,224 and $13,489.4; second-line drug $1039 and $1045; second-line hospitalised $1320.4 and $1232 with amoxicillin and clarithromycin, respectively and hospitalisation with telithromycin $6137. conclusions: telithromycin showed lower clinical failure, hospitalisation and costs in cap. some studies suggest shortening cap telithromycin treatment to 7 days helping adherence to treatment and decreasing costs even more. the new macrolides */a good alternative of tetracycline in the treatment of mediterranean spotted fever ps142 popivanova nip a , petrov aip a , boykinova obb a , kazakova zkk a , baltadjiev agb b . a medical university, infectious disease, plovdiv, bulgaria , b department of anatomy, medical university, plovdiv, bulgaria mediterranean spotted fever (msf) caused by rickettsia conorii appears an endemic disease for some regions in bulgaria. frequently the disease has a severe course with multiple organ lesions. the early and adequate treatment is of extreme importance for the outcome of the disease. searching an alternative antibiotic treatment of this disease we considered macrolides for their good cell and tissue penetration and dose-dependent bacteriostatic and bactericide effect. we treated 27 msf patients with doxycycline 2)/100 mg/day, 25 patients with clarithromycin 2)/250 mg/day, as well as 25 patients with midecamycin 2)/400 mg/day and midecamycin acetate 30 mg/kg/day. as a surrogate marker for treatment evaluation the effect on the febrile syndrome was used. our findings showed that by the 5th day of treatment the fever normalized in 89.47, 88.24 and 76.47% of the patients treated with doxycycline, clarithromycin and midecamycin, respectively. for the same period the patient fever decreased below 38 8c in 100, 100 and 94.12%, respectively. the intoxication symptoms were influenced within the same period equally in all treated patients. conclusions: we suggest that the new macrolides appear a good alternative of tetracycline on patients with msf. erythromycin resistance of gram /'// cocci in bulgaria. benefits of the new macrolides in the treatment of respiratory infections ps143 popivanova ni a , yovtchev ip b , dobreva md c , argirova ta c . a medical university, infectious disease, plovdiv, bulgaria , b medical university, ear-nose-throat disease, plovdiv, bulgaria , c medical university, microbiology, plovdiv, bulgaria in the recent 2 years we tested erythromycin sensitivity of 474 species of gram /'// cocci isolated from throat and nose secretions, ear and eye effusions, sputa, cerebrospinal fluid and blood cultures, vaginal and urethral secretions, urine and fecal samples from patients with inflammatory diseases of the listed organs and systems. staphylococcus aureus , staphylococcus coagulase /(//, streptococcus â-haemolyticus , enterococcus were isolated. of these microorganisms s. aureus was the most abundant. our resistograms revealed sensitivity of the gram /'// cocci in 76.97 and 68.60% for 2000 and 2001, respectively, and resistance of 23.03 and 31.40%, respectively. in the tests with midecamycin and midecamycin acetat the same microorganisms showed sensitivity of 85.38% and resistance of 14.62%. the clinical findings showed excellent effect of the new macrolides including clarithromycin and azalides */azithromycin. we conclude the resistance of gram /'// cocci and especially of s. aureus to erythromycin increases very quickly and has reached dramatical extent. by now, the new 14, 15, and 16-membered ring macrolides and azalides show high antibacterial activity and good clinical effect. pappas ga, liberopoulos e, tsiara s, elisaf m, tsianos e. university hospital, internal medicine, ioannina, greece quinupristin/dalfopristin (q/d) is a novel injectable streptogramin antibiotic which initiation in therapeutics was hailed as an important step towards treatment of vancomycin-resistant enterococcus faecium (vref) species. initial reports concluded in an excellent response of vref to q/d. reports of q/d-resistant strains of e. faecium have emerged, both in usa and europe. we report two cases of e. faecium bacteremia in which the responsible isolate was not sensitive to q/d. the first patient was a woman with acute leukemia and septicemia. e. faecium was cultured from blood samples: the species was resistant to almost all antibiotics, exhibiting sensitivity only to tetracycline (!), while its sensitivity to q/d was indermediate. the second patient was a man with endocarditis, in whom blood cultures isolated e. faecium sensitive to a number of antibiotics, including ciprofloxacin and vancomycin; still the sensitivity to q/d was indermediate. q/d has not been officially introduced to the antibiotic arsenal of greek medicine. moreover, the drug has never been used in our hospital, not even experimentally. other e. faecium species isolated in our hospital have been sensitive to q/d. how much hope can be put on a drug that seems to be partly useless before its initiation? increasing reports of v/ q-resistant strains of e. faecium from all over europe raises fears that the v/d story might well end before it begins. varadinova t a , diakov t a , karagiozova d a , genova p a , pardon p b , baudry c b , quideau s b . a sofia university, virology, sofia, bulgaria , b et institut du pin, centre de recherche en chimie moleculaire, universite bordeaux 1, bordeaux, france vegetable tannins posses a wide range of biological activities. the aim of the present study was to evaluate the cytotoxicity of five purified vegetable tannins against mdbk cells. the maximal nontoxic concentrations (mnc) and the concentrations required to inhibit cell growth by 50% (cc 50 ) were evaluated after 24, 48 and 72 h periods of action. mnc values after 48 h indicated that compounds stimulated cell surveillance when applied in concentrations lower than 0.01 mm. cc 50 values indicated: (i) a decrease in cytotoxicity after 48 h as cc 50 were up to 70 times lower than those observed after the 24 h period, and (ii) a re-increase in cytotoxicity when the period of action was prolonged up to 72 h as cc 50 were 2500 times lower than those observed after the 48 h period. these data thus appear to reveal the capability of the investigated natural polyphenolic products to stimulate cell surveillance in a time-dependent manner. antibacterial effect survey of enoxolone on periodontopathogenic and capnophilic bacteria isolated from specimens of patients with periodontitis ps146 salari mh a , kadkhoda z b , sohrabi n a . a tehran university of medical sciences, pathobiology, tehran, islamic republic of iran , b tehran university of medical sciences, dentistry, tehran, islamic republic of iran objectives: most of the microorganisms associated with periodontitis are capnophilic and anaerobic bacteria. purpose of this study was to detection of antibacterial effect of enoxolone on periodontopathogenic and capnophilic bacteria. methods: in this study 136 periodontopathogenic and capnophilic bacteria were isolated from 400 specimens of patients with periodontitis by culture method. an anti-bacterial activity of enoxolone against these microorganisms was investigated by minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) methods. results: based on our findings the mic, mbc and lethal does 50 (ld50) of enoxolone for actinobacillus actinomycetemcomitans , eikenella corrodens and capnocytophaga were '8, 16, 64', '16, 16, 32', and '8, 16, 32' mg/ml, respectively. conclusion: our results show enoxolone has antibacterial effect on a. actinomycetemcomitans , e. corrodens and capnocytophaga spp. sakalova stn. medical university, microbiology, grodno, belarus we have synthesized diamides of dicarboxylic acids, with components such as 5-nitrothiazole benzolsulphamides and triazol. all the above compounds exhibited bacteriostatic activity towards some microorganisms. for further studies of bacteriostatic activity of amides and diamides of dicarboxylic acids, as well as for determination of 'structure á/activity' relationship, we have synthesized a range of monoamides. antibacterial activity of the synthesized compounds was studied in vitro by agar dilution methods. for this purpose, approximately 50 various gram-positive and -negative microorganisms including clinical strains of staphylococcus aureus , bacillus subtilis , serratia marcescens , escherichia coli , proteus morganii , micrococcus lisodeicticus , staphylococcus epidermidis , shigella sonnei , salmonella typhimurium , yersinia enterocolitica . minimum inhibitory concentration was expressed in mg/ml. nitazole was used as a comparison substance. analysis that new derivatives of 5-nitrotiasole have high antibacterial activity relative towards certain microorganisms included strains obtained from infection department's patients. results will be shown. microbial susceptibility to the essential oil of ziziphora clinopodiodes lam. ps148 purpose of the study: antimicrobial activities of essential oils vary from oil to oil and from one micro organism to another. the antimicrobial and chemical properties of essential oil from ziziphora clinopodiodes lam. has not been studied and hence the present study was planned to evaluate those properties against a series of micro organisms viz, escherichia coli , staphylococcus aureus , pseudomonas aeroginosa , klebsiella pneumonia , bacillus subtilis , bacillus licheniformis , streptococcus faecalis , candida albicans and saccharomyces cerevisiae . results: z. clinopodiodes lam. essential oil was found to have remarkable antimicrobial property against all the microorganisms but p. aeroginosa . the oil exhibited its best antimicrobial activity within a maximum of 45 min. seventeen components were identified by gas chromatography and mass spectrometry (gc and gc/ms) analysis of the oil, out of which pulegone (24.5%), neomenthol (12.8%), cyclohexene,5-methyl-3-(1-methenyl)trans (12.2%), 2,4-cycloheptadien-1-one,3,6,6-trimethyl (9.2%), piperitone (4.2%), and limonene'/ 1,8-cineole (4.1%) constitute major parts of the oil. conclusion: monoterpenes seem to have antimicrobial role. it seems necessary to explore antimicrobial properties of new harmless antimicrobial agents from natural sources as substitutes for common chemical drugs. methanol extract of carpobrotus edulis enhances killing of methicillin resistant staphylococcus aureus phagocytosed by thp-1 human monocyte derived macrophages and promotes the release of modulators of cellular immunity ps149 although alkaloids from the family mesembryanthemaceae have anti-cancer activity, species of this family have received little attention. because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, we have studied a crude methanol extract of carpabrotus edulis , a common plant found along the portuguese coast, for properties normally associated with plasma membrane active compounds. the results of this preliminary study show that the extract is non-toxic at concentrations that prime thp-1 human monocytederived macrophages to kill ingested methicillin resistant staphylococcus aureus and promote the release of lymphokines associated with cellular immune functions. the extract also induces the proliferation of thp-1 cells within 1 day of exposure and 2 days earlier than that induced by phytohemagglutinin. similar results were obtained with monocyte-derived macrophages isolated from human peripheral blood. the active component or components of the plant extract may be exploited as intracellular active anti-bacterials as well as modulators of cellular immunity. enhancing of erythromycin production by saccharopolyspora erythraea nur001 with common and uncommon oils ps150 hamedi j a , malekzadeh f a,b saghafi-nia ae b . a department of biology, faculty of science, university of tehran, tehran, islamic republic of iran , b shafe-e-sari co., antibiotic production co., teheran, iran enhancing effect of various oils on the erythromycin production by saccharopolyspora erythraea nur001 were evaluated in a complex medium consisting soybean flour and dextrin as the main substrates. the biomass, erythromycin, dextrin and oil concentrations, and ph value were measured on a daily basis. also, the kinds and frequencies of fatty acids of the oils used were determined. saturated fatty acids in the shark oil were higher than that of vegetable oils used. erythromycin concentration in the melon (cucumis melo var. inderus cultivar mashhad ) seed oil containing medium was 4.56 times higher than that of the control medium (without oil) and 1.18 times higher than that of rapeseed oil containing medium. erythromycin concentration in the other oil containing media, including rapeseed, soybean, shark (carcharhinus dussumieri ), and safflower oils was 3.88, 3.03, 2.59, 2.44 time higher than that of control medium, respectively. the melon seed oil had the least enhancing effect on the biomass production, and thus decreasing the cost of the biomass separation. can varicella be eliminated by universal childhood vaccination ? */ epidemiological and economic data from germany ps151 wutzler p a , banz k b , neiss a c , goertz a d , bisanz h d . a institute for antiviral chemotherapy, university of jena, jena, germany , b outcomes international, basle, switzerland , c institute of medical statistics and epidemiology, technical university, munich, germany , d glaxosmithkline, pharma, munich, germany purpose: universal varicella vaccination in childhood is expected to reduce substantially the number of uncomplicated cases of chickenpox and to decrease the number of complicated cases requiring hospitalization. to generate fundamental data for decisions of the health authorities epidemiological and health-economic data were collected in two large studies. using an age-structured decision analytic model the benefits, costs and cost-effectiveness of a varicella immunization program over a period of 30 years were assessed. results: it could be shown, that the vast majority of varicella cases occur in children aged less than 8 years. in 16.3% of the cases a severe course was assessed. overall incidence of complications was estimated to be 5.7%. a routine varicella vaccination program targeting healthy children could prevent 82.6% of varicella cases and over 4700 major complications per year provided the coverage rate would be 85%. under these conditions the elimination of varicella is predicted to be achievable within 15 á/20 years. a combined measles, mumps, rubella and varicella vaccine is expected to provide the required coverage. conclusions: routine childhood varicella vaccination appears to be a highly efficient strategy to significantly reduce the sizeable burden of varicella and leads to significant savings from both societal and payer's perspective. bulgakova va, balabolkin ii, sentsova tb. scientific center of child health, russian academy of medical sciences, scientific research institute of pediatrics, moscow, russian federation objective: to estimate efficacy of vaccine influvac at children with allergic diseases. methods: twenty children aged 3 á/18 years with allergic diseases received vaccine influvac (solvay pharma). for the control group of children, with allergic pathology did not receive this vaccine because of an intolerance of chicken protein (10 children). results: all vaccinated children for the observable season of 6 months did not get influenza. general and aboriginal reactions to a vaccine did not occur. in control group for the observable season two children were ill with influenza and four children with acute respiratory virus infection (60%). among vaccinated children there was an increase in titre to a protective level (1:40 and above) to all to three strains of influenza 30 days after injection. vaccine influvac can be recommended for an immunisation against influenza of children with an allergic pathology because of efficacy and absence of side effects. ló pez h, zitto tr, vidal gi, cánepa mc, salomó n jm, scaglione m. centro de infectología, infectious diseases, buenos aires, argentina objectives: our study examines the possible economic impact of the influenza on health working adults in argentina, and the intervention cost saving with immunization. methods: this is a theoretical study based on a mathematical model. population data was published in 2001s national statistics. the global incidence of influenza infection was estimated at 5%. we have estimated the direct cost on influenza infection (outpatient visits, drugs and hospitalization) and indirect cost (work absenteeism and productivity loss) and projected net saving for the 20 á/64 year-old vaccinated group. the vaccine effectiveness was estimated at 70 and 90%. the price of vaccine was $8 each. results conclusion: influenza vaccination is effective in diminishing cases of flu and reducing working-day loss. its a safe and cost-effective vaccine. ló pez h, zitto tr, cánepa mc. centro de infectología, infectious diseases, buenos aires, argentina flu infection is a major cause of illness and one of the most common cause of work absenteeism, increasing institution costs, healthcare provider visits, use of drugs, and decreasing work productivity. vaccine against flu has an effectiveness between 70 and 90%, in health adults. objective: evaluate the impact of flu-like respiratory tract infections in a health institution staff during 1 year, comparing vaccinated with not-vaccinated groups. methods: we evaluated all causes of absenteeism along 1 year (2001), based on the written note made by the professional who has evaluated ill people, selecting flu-like respiratory infection causes. we evaluated age, working days loss related to illness, and cost on vaccinated and not-vaccinated groups. results: one hundred and sixty eight of the total staff (479 people) were vaccinated, 21 of them had flu-like infection, resulting in 40 working days lost. for not-vaccinated group, 33 people had flu and 56 lost days. lost cost for vaccinated group was of $1267, and for notvaccinated group, $1874. conclusion: we observed a decrease in working days loss and money waste related to flu-like infections on the vaccinated group. because of safety and effectiveness of vaccine against flu, the implementation of vaccination will be cost-effective for all institution staff. we studied functioning of the interferon system in 108 children with atopic bronchial asthma (ba) at the age of 2 á/14 years. the control group included 10 healthy children. we investigated the interferon status (method of grigoryan s.) and serum concentrations of ifngamma (ifng) (elisa). there was a decrease in the ifn-producing ability of leukocytes to the synthesis of ifna at 50% and ifng at 84% of children with ba. serum level of ifn of children with ba during all period of illness is compared to the children without predisposition to atopy (94.99/10.3 and 192.39/7.8 pg/ml accordingly) was significantly decreased. production of ifna increased after using viferon (recombinant a2b ifn and antioxidants). decreased ability of gammainterferonogenesis in the most children was not affected by the action of immunomodulators. there was shown interferon system's dysfunction in the development of atopy and increasing predisposition to respiratory infections and to persistent of atypical infections in children with ba. harxhi a, pilaca a, pano k. university hospital center of tirana, infectious diseases, tirana, albania congo-crimean haemorrhagic fever is viral disease with a high rate of mortality that is caused by a nairovirus, bunyaviride species. this is a zoonotic disease, which affects sporadically humans and is geographically distributed even in eastern europe and balkan. during the months of may and june 2001, in northeast of albania were reported eight cases of haemorrhagic fever. serologic tests performed in the laboratory of reference in thesaloniki, greece confirmed the diagnosis of congo-crimean haemorrhagic fever. in the mean time, who reported the outbreak in southwest kosovo of 69 cases suspected for haemorrhagic fever from which 18 were confirmed laboratory as congo-crimean haemorrhagic fever. we are describing here the clinical history of one of eight cases with cchf in albania. from the epidemiological point of view this case was considered peculiar, as it was the only one hospitaly acquired, and due to the gravity of the haemorrhagic syndrome was admitted at the intensive care unit at infectious diseases service, university hospital center of tirana. results: in the study were included 150 patients ]/18 years of age with documented hbsag-carrier ]/6 months (average age */39.4 years, male */58%, female */42%). hbv dna in serum was tested by qualitative and quantitative pcr (commercial test-system ampli-sens hbv). hbeag, hbeab, hbsag were detected by elisa (hoffmann la roche). hbv dna by qualitative pcr was detected in 41% patients, by qualitative pcr was detected in 13% patients in the concentration ]/10 5 copies/ml, in 4.3% ]/10 6 copies/ml, in 4.3% ]/10 7 copies/ml, in 2.1% ]/10 8 copies/ml ( fig. 1 ). hbv dna level distribution among the hbsag carriers. elevated (1.5 the upper limit of normal) alt level was determined in 3.8% of the hbv dna negative and 25.4% of the hbv dna positive patients. hbeag was detected in 10.2% of the hbv dna positive patients and had not been determined in the hbv dna negative patient. eleven percent of patient had the combination of the biochemical, serological and virology criteria, which are typical for active chronic hepatitis b (hbsag-carrier !/6 months, hbv dna ]/ 10 )/5 copies/ml, elevated alt). conclusion: in smolensk 41% of the hbsag-carriers have viral replication confirmed by qualitative pcr. eleven percent of them have active chronic hepatitis b. kandemir o a , polat a b , kaya a a . a medicine of faculty, mersin university, clinical microbiology and infectious disease, mersin, turkey , b medicine of faculty, mersin university, pathology, mersin, turkey the exact potential of nitric oxide in the pathogenesis of chronic viral hepatitis is not known. the elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions, mainly via peroxynitrite, a potential oxidant that is produced by the reduction of superoxyde anion with nitric oxide. the intensity and the distribution of the immunohistochemical staining of intrahepatic inducible nitric oxide synthase were studied in the biopsy specimens obtained from 63 patients with viral hepatitis and 13 patients with elevated transaminase levels from other etiology. hepatic inducible nitric oxide synthase staining was significantly more intense in the viral hepatitis group (p 0/0.000). inducible nitric oxide synthase staining levels correlated well with the severity of the viral hepatitis using the knodell's liver histological activity index (r0/0.393, p 0/0.002). among the viral hepatitis group, the pathological distribution of the inducible nitric oxide synthase staining favored the periportal regions whereas less staining was observed in the bile duct and parenchyma regions. as nitric oxide mediated nitration of hepatocellular proteins is found to be elevated in the inflamed hepatic tissues and it well correlated with the severity of the disease, we suggest that inducible nitric oxide synthase can possibly have a critical role in the pathogenesis of chronic viral hepatitis. there were 36 patients under observation who were divided into two groups, the first of 18 patients (eight chronic virus hepatitis; three chronic virus hepatitis'/steatosis; four steatohepatitis; three chronic cryptogenic hepatitis; there were 14 men and four women aged from 35 to 74. the second group consisted of 18 patients, eight with chronic virus hepatitis; four with steatohepatitis; six with chronic cryptogenic hepatitis. there were 15 men and three women aged from 40 to 80. diagnosis was confirmed with the help of clinical data, biochemical tests, serological markers, psr-diagnostics and ultrasound examination and computer tomography of the abdomen. in the first group of patients the treatment with ursofalk was administered at the dosage of 10 mg/kg of body mass from 1 month to 3 years with improvement in general condition of the patients: heaviness, pain under the right rib, nausea and skin itch have disappeared. in all the cases, improvement in the biochemical blood analysis took place during treatment. the average index of alt activity was before */105.88 u/l, after */62.06 u/l and ast */156.44 and 95.05 u/l; alp */196.39 and 171.87 u/l; ggt */163.2 and 82.86 u/l; chol */5.38 and 4.8 mmol/l; tg */2.15 and 1.75 mmol/l. in the second group of patients the treatment was carried out with various hepatoprotectors during the courses from 1 to 6 months. before the average index of alt activity was 181.3 u/l, after */165.5 u/l; ast */126.9 and 129.0 u/l; alp */229.8 and 213.5 u/l; ggt */204.2 and 238.0 u/l; chol */5.85 and 5.67 mmol/l; tg */1.72 and 1.8 mmol/l. treatment of patients suffering from hepatitis of viral and other aetiologies with ursofalk produces a positive effect on both clinical symptomatic and biochemical indices. remission was more stable during a long period of taking the preparation. the hepatoprotective effect of ursofalk during the 3 years was sustained for the whole of the period of the treatment. after stopping, an acute attack of cytolytic syndrome was observed. with other hepatoprotectors we did not get any improvement in clinical scene of the disease or in biochemical indices. shavlov nm, kletsky sk. minsk, belarus hsv-1 and -11 have possibility to damage different organs and systems. sometimes they cause damage of the liver, which resemble viral hepatitis. the etiology of such hepatitis may be confirmed only by results of liver biopsy. we have diagnosed 12 cases of herpetic hepatitis: eight children and four adults. clinical course was different. in five cases the acute beginning took place: high temperature, the jaundice at the 2 á/3 day (the level of bilirubin was 200 á/350 mkmol, especially direct), cholestasis, the pain in upper right part of abdomen. the ascites was found in three patients with acute hepatitis during 1st week from the beginning of disease. in seven cases the beginning was gradual. the temperature was subfebrile, prolonged; malaise and moderate pain in upper part of abdomen were constant complaint. the jaundice was moderate; bilirubin increased until 120 mkmol. the level of alt was moderately increased (5 á/7 times). the blood analysis showed moderate leukocytosis with neutrophilia, and increased sre. the serological markers of hepatitis a, b, c, d were negative in all cases. hsv-1 and -2 were found in the blood. the diagnosis was defined by the results of hystochemical investigations, when the viruses were found in liver bioptat, and confirmed with the results of specific treatment. specific damage of liver cells was found: protein dystrophy and specific inclusions in cell nucleus. in all cases the treatment with acyclovir were given. the results we have observed during 1st week: the temperature became normal, the jaundice decreased and bilirubin was normal during 5 á/10 days. in one case the recidive took place 2 weeks later after treatment. the second course of acyclovir with intron a gave good results. nesic z a , delic d b , prostran m a , vuckovic s a , stojanovic r a . a department of pharmacology, school of medicine, university of belgrade, belgrade, yugoslavia , b clinical center of serbia, institute for infection and tropical disease, belgrade, yugoslavia the large number of unsolved cases of acute and chronic hepatitis has most probably the viral etiology. in mid 1990s, two independent groups of authors reported a new human hepatotropic virus, with flavivirus like genomes, hepatitis g virus (hgv). the aim of this pilot study was to determine the prevalence of hepatitis g viral infection among patients at high risk of exposure to blood and blood products, as well as to evaluate if the risk of hgv infection was higher among them than in the general population. immunoenzyme test on microtitration plate for detection antibodies against hgv e2 antigen in plasma or sera (r&d systems, minneapolis, usa) was used for evidencing anti-hgv igg antibodies in sera. anti-hgv antibodies were detected in the control group (blood donors) in 8.3% (2/24) patients. prevalence of anti-hgv antibodies among i.v. drug users was evidenced in 42.8% (9//21), in hemophiliacs 41.2% (7/17), in patients acquiring multiple blood transfusion 42.8% (3/7), in hemodialyzed patients 33.3% (4/12) and in patients with transplanted organs 57.14% (4/7). our results suggest that patients exposed to blood or blood products have a higher risk of hgv infection than general population. evaluation of ortho total hcv core antigen assay in assessment and follow-up of patients treated for chronic hcv ps162 lunel f, veillon p, payan c. chu angers, laboratoire de bactério-virologie, angers, france an assay to quantitate 'total' hvc core antigen (hcv ag) in serum or plasma, may reflect viral load, has been developed by ortho-clinical diagnostics. methods: we evaluated hcv ag with two quantitative assays for hcv rna: bdna 3.0 (bayer) and monitor 2.0 (roche). we studied 191 samples from untreated patients and 237 from 144 patients with chronic hcv treated with ifn or ifn/ribavirin. results: correlation of ag and quantitative assays was high (r0/ 0.85 for bdna 3.0 and 0.83 for monitor 2.0). no difference between the levels of rna and ag among hcv genotypes (r0/0.82 á/0.96) was found. ag values, before treatment, were significantly lower in sustained responders (sr) than in other groups (5.4 log versus 6 log, p b/0.001). in patients treated with ifn or combination therapy, we found very good correlation between decrease and negativation of ag and viral load: 2 log iu/ml decline after m1 of interferon was significantly correlated with the negativation of hcv ag and sr. thirty-eight/41 of sr had a rna load decrease !/2 log iu/ml and 39/ 41 had a negativation of hcv ag after m1. conclusion: total hcv core ag appears to be a new tool for monitoring patients with hcv infection. hepatitis c virus rna and hcv core antigen kinetics predict the efficiency of interferon-alfa and ribavirin therapy in naive patients infected by hcv genotype 2 or 3 ps163 fifty-five patients infected by genotype 2 or 3 were treated with a primary dose of 3 (if hepatitis c virus (hcv) rna b/3 meq/ml) or 6 million units of interferon alfa-2b (ifn) thrice weekly for 12 months. ribavirin was added at month 3 (m), until m12 if hcv rna was found positive after m2 of ifn. the viral kinetic was assessed during the follow up by serial measurements of hcv rna (bdna 3.0 and monitor 2.0) and using a new assay from ortho-clinical diagnostics which is able to quantify total hcv core antigen. sustained virologic response was observed in 65% of the patients (36/55). after 1 month of ifn treatment, sustained responders had a fall of hcv rna and hcv core antigen higher than non-responders (4.339/1.84 log ui/ml versus 0.159/0.31 log ui/ml, p b/0.001, for hcv rna) and (1.689/ 0.89 log (pg/ml)/10 000) versus 0.179/0.18 log (pg/ml)/10 000) p b/ 0.001, for hcv core antigen). after 1 month of ifn, the positive and negative predictive values of sustained response were, respectively, 100 and 17.4% for hcv rna negativation and 97.5 and 37.5% for hcv antigenemia negativation. these results suggest that both kinetics of viral load and antigenemia are highly predictive of sustained response. theodorou m, petinelli i, pontikaki d, mela c, blana a, papanastasiou a, toliopoulos a, stavrakaki m, sagkana e. microbiology department, western attika general hospital, greece, egaleo, greece greece has accepted a big number of economic immigrants lately. we investigate the prevalence of hepatitis b/c as well as the epidemiological features that might influence the public health. 1.235 economic immigrants from: albania 630, eastern europe 411 and from asiatic-african countries 194, visited our hospital to be checked in order to get a health certificate to obtain the green card. they where tested for hepatitis b/c. the serological markers were determined by immunoenzymatic method. all hbsag('/) and anti-hcv were further tested for hbv dna and hcv rna by competitive rt pcr. hcv rna('/) were genotyped by strip hybridization immunoassay. in 630 albanians 17.5% were hbsag('/), 14.6% hbv dna('/), 1.1% anti-hcv('/) and 0.8% hcv rna('/). in 411 east europeans 5.1% were hbsag('/), 4.4% hbv dna('/), 2.43% anti-hcv('/) and 1.9% hcv rna. in 194 asians-africans, 2.6% were hbsag('/) and 2% hbv dna('/). in 101 pakistanis, 26.7% were anti-hcv('/) and 22.0% hcv rna('/). of the rest of asians-africans, 11.82% were anti-hcv('/) and 9.3% hcv rna('/). albanians: higher prevalence of hbv infection (14.6%). greek blood donors: 1% pakistanis: hcv infection is 22% (predominance of 3a type), general greek population: 2%. public health services in greece and europe must take appropriate measures. el zawawy la a , mohamed on b , ali sm a , eissa me a , allam sr a . a faculty of medicine, parasitology, alexandria, egypt , b high institute of public health, microbiology, alexandria, egypt the purpose of the study was to investigate the influence of schistosomal suppression on the antibody response to hepatitis-b vaccine (hbv) and to study if the vaccine has any protective effect on experimental () infection. the results obtained revealed that infection reduced the serum antibody level against hbv. parasitological and histopathological findings showed significant protection against infection. the conclusion reached was; in order to reduce the incidence of virus-b infection especially in schistosomiasis endemic areas, public health officials should evaluate a policy for regulation of hbv booster vaccination to enhance the population immunity against hepatitis-b infection. cooper e, fisher t, shingadia d. newham general hospital, family clinic, london, uk sustained anti-retroviral combination chemotherapy requires excellent adherence to the regimen so as to suppress viral replication sufficiently to delay the emergence of resistance. if chemotherapy were taken to scale, e.g. in africa, erratic adherence might soon lead to multi-resistant circulating virus. we reviewed our experience in a well established london family clinic with a team including community nurses. we reviewed the records of the 23 african immigrant children, aged 2 á/14, treated with anti-retrovirals exclusively at our centre throughout 2001. whereas 14 had undetectable hiv rna within the year, only four had undetectable rna throughout the year. four failed therapy through proven resistance mutations, but nine were considered through circumstantial evidence to have rising viral loads primarily because of poor adherence. three were known to have stopped taking drugs for extended periods. the three boys over 11 years were unreliable in adherence, but the one girl in this age-group was fully adherent. our preliminary assessment is that for the children in our families, despite a team approach and home visits, nonadherence to haart may be twice as common as selection of a dominant viral mutant as a primary cause of failure to sustain viral suppression. quiros-roldan e, moretti f, castelli f, el-hamad i, carosi g. the prevalence of hiv related lipodystrophy-syndrome depending on the definition and severity of lipodystrophy ranges from 5 to 80%. we have retrospectively reviewed the medical records of 58 african patients followed. the characteristics are shown in the 3.4% of africans had triglycerides !/200 mg/ml and 1.7% had cholesterol !/250 mg/ml, none had both metabolic alterations. glycemia !/120 mg/ml was observed in 8.6% patients. it is interesting highlight that in any the africans morphological changes were noted and all of them showed weight stable. although the low prevalence of metabolic alterations may be attributed to the different ethnic alimentary behavior if self-body perception by african is not as accurate as by caucasian on the estimation of the body changes have to be investigated. alabaz d a , alhan e a , yaman a b , evliyaoglu n a , kocabas e a , aksaray n a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey hepatitis a virus (hav) infection is usually asymptomatic in children. however, it may occasionally cause a severe disease with high morbidity and mortality, and loss of school or business days. in a previous study, we have shown that every one of two to three school children from upper social classes living in adana carries high risk of hav infection. it is well known that maternally transmitted anti-hav antibodies interfere with hav vaccination. in an effort to determine the optimal age for hav vaccination, 122 babies (52% girls and 48.5% boys) born in our hospital were prospectively followed up at least 24 months for the presence of maternal antibodies to hepatitis a (anti-hav igg). anti-hav igg titers were measured from the blood specimens obtained at birth from the mothers and from the offsprings at months, 0, 3, 6, 9, 12, 15, 18, 21 and 24. the prevalence of positive anti-hav at birth (95%) was similar to those of hav seroprevalance studies carried out in adults in our area. the disappearance of antibodies occurred between the 1st and 21st month of life. the prevalence of anti-hav igg among children aged 0, 9, 12, 15, 18 and 21 months were 95, 60, 32, 9, 4 and 0%, respectively. in light of these findings, we suggest that hepatitis a vaccination be given after 21 months of age. earlier vaccination may be ineffective due to interference with maternally transmitted anti-hav antibodies. ghaderi b, alaghebandan r, rastegar lari a. department of microbiology, iran university, tehran, islamic republic of iran diarrhoea is a major public health problem in developing countries. amoebiasis is one of the most common causes of diarrhoea in iran as an endemic area for amoebiasis. little, however, is known about the extent of the condition in our society. the aim of this study is to determine socio-demographic and clinical characteristics of patients with intestinal amoebiasis. during july and august 2001, we collected 90 patients with diarrhoea among 5000 patients who visited at a referral hospital in shahriar area (in countryside of tehran), iran. thirty out of 90 patients (33%) had intestinal amoebiasis and were followed up prospectively until the resolution of the illness. nineteen of 30 (63.3%) patients were male and the remaining of 36.7% was female. the patients were aged 1 á/76 with mean of 28.4 years. most of the patients (70%) were below 30 years of age and the peak of occurrence was between the age of 17 and 26 years. watery diarrhoea with abdominal cramps was the main clinical feature. seventy percent of patients were resident in urban area and the remaining (30%) in rural area. average family income was low and all patients were in low socioeconomic level. water supplying system for all patients was pipeline water. low socioeconomic level associated with poor personal hygiene was the most important factor for highly prevalence of this problem in our society. also it seems that food plays important role in transmission of protozoa then water. the new strategy for allele identification of the genes coding for pertactin and pertussis toxin subunit s1 in bordetella pertussis ps170 bordetella pertussis strains demonstrate a significant polymorphism in toxin s1 subunit and pertactin, which are major protective antigens of the organism. monitoring the changes in prevalence of particular alleles of genes coding for these proteins in local b. pertussis populations is an essential issue in cases of the observed decrease of vaccination effectiveness. we have developed a new method for allele identification of these genes, which eliminates the necessity of dna sequencing. the approach is based on the identification of the number of repeats or the presence of specific nucleotides in the polymorphic regions or residues, respectively, of the genes and utilises products of their full or partial pcr amplification. the nucleotide heterogeneity in each polymorphic site is analysed either by the differential digestion of the amplicons or by the arms (amplification-refractory mutation system) methodology. numbers of repeats in particular regions of the genes are revealed by the size analysis of the adequate pcr products or their restriction fragments. in all cases the presence, size or pattern of dna molecules obtained is visualised by the agarose gel electrophoresis. the preliminary analysis of the recent and archival b. pertussis strains identified in poland was performed using the described approach. the presented strategy provides a much easier, faster and more cost-effective than dna sequencing mean to study the polymorphism of the major b. pertussis antigens. vaccination coverage and history of vaccine preventable infectious diseases among students in second year of medicine and pharmacy of tours university ps171 borderon jc a , hamed a b , ragot s b . a centre hospitalier universitaire, tours, france , b médecine préventive universitaire, tours, france the purpose of this study was to determine the level of infectious risk in students who will be exposed to patients. information was obtained by a questionnaire for each student, and by checking medical records for immunization coverage and vaccine preventable infectious diseases. answers could be specified for 138 students, of whom 97 females (f) and 41 males (m). the number of non-immunized students was against diphtheria: two, tetanus: three, pertussis: four, poliomyelitis: two, hepatitis b: six, and hepatitis a: 127, respectively. among the 52 students non-vaccinated against measles, 14 (nine f and five m) had no history of that disease. among 56 (31 f and 25 m) non-vaccinated against rubella, 17 (10 f and seven m) had no history of that disease, uncertain in seven others (six f and one m). the date of vaccination was often late regarding recommendations. fifteen students had no history of varicella. one student had not received bcg vaccination. fifty-eight students had received two, and 14 three bcg vaccines. post-bcg tuberculin skin testing was missing after 28 first bcg, 14 second and 3 third bcg. the date of the first tuberculin test was often one or several years after bcg vaccination. adverse effects of vaccination were rarely reported: two cases of fever (dt polio, measles); three cases of local reaction (dt polio, dtp polio). one case of contraindication for influenza vaccine: egg allergy. the survey shows failure in immunization coverage actually recommended in health care students. objectives: to present the morbidity of rabies and evaluate the efficiency of our prophylaxis scheme in lasi county. material and method: we made a retrospective study of the rabies cases in the patients admitted in our unit in a 13th-year period. we have analysed all the clinical, epidemiological and biological aspects. results: in a 30-year period, 22 cases of rabies were admitted in the clinical infectious diseases hospital lasi. the highest incidence was for 1986 á/1990 */eight cases (36%); the highest yearly cases were three cases in 1974 and 1986. most of the patients were male (59%), came from suburban areas (21 patients). eight cases occurred in may á/june, wild animals were involved in half the cases (fox, wolf). for 18 patients, no prophylaxis was performed and an incomplete course in four cases. the period of time to the appearance of the first symptom was 18 á/120 days. the prophylaxis scheme led to a good protection. conclusions: in lasi county, rabies is a problem with a prevalence of 0.73%/year. trends in the use of antimicrobials in riyadh in 2000 were analyzed. data was obtained from a survey of 652 randomly selected families of school children aged 6 á/8 years in a 3-month period in 2000. one hundred and ninety-nine (33.8%) students were on antibiotics in the month preceding the study; 108 (56%) received antibiotics for the diagnosis of pharyngitis; 177 (90%) students antibiotics were prescribed by a physician; and in 161 (83%) the duration of antibiotics was less than 1 week. this study shows a major problem in antibiotics prescription in our community and also the need to establish effective antibiotics policy in general practice to limit the potential emergence of drug resistance bacteria in the community. mir s a , cura a a , erdogan h a , guler s a , sengul gn a , koyu a a , ozinel ma b . a department of pediatrics, ege university medical faculty, izmir, turkey , b department of microbiology, ege university medical faculty, izmir, turkey antibiotic susceptibility spectrum of childhood urinary tract infection agents are geographical variation. the current antibiotic regimens and the selection of antibiotics for prophylaxis should be re-evaluated periodically. the objective of our study was to determine the local resistance rates to antibiotics and to give a direction for the selection of antibiotics in uti treatment. we evaluated 4124 urinary culture assays retrospectively, sent from pediatrics and pediatric surgery inpatient and outpatient clinics of our hospital during the last 6 months, and investigated the isolated pathogens and the resistance rates to antibiotics. in addition, the data obtained were compared with of 5 years ago. with respect to the resistance rates to antibiotics of uti pathogens, the resistance rates of e. coli for carbapenems, aminoglycosides and third generation cephalosporins were 1, 10 and 10%, respectively as before, but the rates for ampicillin increased from 67 to 75% and for tmp-smx it increased from 49 to 61%. we concluded that the resistance profiles to antibiotics should be reviewed every 5 years at least and thus the selection of proper antibiotics would lessen the morbidity as well as the medical expenses. chanturidze tk, tsiklauri r. ministry of labor, health and social affairs, public health, tbilisi, georgia purpose: since 1991 infectious diseases (id) are increasing in georgia. this study is aimed to reveal economic barriers of effective id control by assessing financial contribution to id from public and private sources, household's total spending on health and their capacity to pay. sources: 1) national household expenditure and revenue survey. 2) who fair financial contribution methodology. 3) meta-analysis. results: 51.8% of population leaves under the poverty level; 70% out of total household expenditure (average 270 gel; us$ 130) 30% is spent on food */non-subsistence income covers expenditures on goods and services including health; 15% of population refuses health services because of inability to pay; public spending on health comprises 33% of total health expenditures; public spending on id control is below 1 gel per capita; almost all private spending goes to id treatment and equals to 28.6 gel per patient. conclusions: insufficient public spending on id control transfers the burden to the population with extremely low capacity to cover health expenses. refusal to utilize health services, and incomplete treatment and increases the threat of id spread and drug resistance. government should increase the allocations to id from public sources for effective id control in georgia. antimicrobial consumption trends in children's university hospital ps177 ratchina s a , averchenkova l b , jarkova l a . local surveillance of antimicrobial (am) consumption is essential to promote the rational use of this group of drugs. the purpose of this study was to analyze the trends of am use in the children university hospital in 1998 and 2000. data on am usage were obtained from the hospital drugstore requests in the 250-beds multi-ward children university hospital. consumption was expressed as the number of ddd per 100 bed-days (b á/d). the total am consumption figures were similar in 1998 and 2000 (7.7 and 8.2 ddds/100 b á/d, respectively) with notable differences in am prescribing patterns. penicillin consumption increased from 1.8 to 3.8 ddds/100 b á/d mostly due to amoxicillin. the overall aminoglycoside usage remained comparable (0.95 vs. 0.82 ddds/100 b á/d) though amikacin has considerably replaced gentamicin. there was a sevenfold increase of ciprofloxacin (0.03 vs. 0.2 ddds/100 b á/d) along with the evident decrease of tetracycline and co-trimoxazole consumption found (1.5 vs. 0.9 ddds/ 100 b á/d and 0.8 vs. 0.1 ddds/100 b á/d, respectively). the tendency to prescribe more effective in respect of the local resistance data and/or more safe am was detected in 2000 comparing with 1998 that can be explained by the introduction of the local guidelines for infectious diseases management in 1999. clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis */a pilot study ps178 the aim of this pilot study was to determine the efficacy and tolerability of clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis (ct). fifty-two patients older than 18 years of age with diagnosed chronic chlamydial prostatitis were enrolled. the presence of ct in expressed prostatic secretion or urine specimen voided immediately after prostatic massage was confirmed by isolation on mccoy cells and lugol staining. the majority of patients suffered from suprapubic pain and pain in the groin. twelve patients had no clinical symptoms. according to rectal palpation, prostate gland was normal in 35 patients, tender and soft in 12 and firm in five patients. clarithromycin was administered orally 500 mg twice daily for 14 days. simultaneously the patients' partners received 500 mg orally twice daily for 7 days. clinical efficacy and tolerability of administered clarithromycin were evaluated 1 á/7 days and 4 á/6 weeks after the end of treatment. bactericidal efficacy of administered drug was evaluated 4 á/6 weeks after the end of treatment. the eradication of ct was achieved in 30 out of 40 patients, while 28 patients were clinically cured. two patients had nausea and elevated serum transaminases. in asymptomatic patients, the eradication of ct was achieved in 10 of 12 patients who reported no side effects. this pilot study has shown an excellent efficacy and tolerability of clarithromycin in the treatment of patients with chronic chlamydial prostatitis. women with diagnosis of urinary tract infections (uti) often demonstrate vaginal colonisation with alpha-haemolytic escherichia coli strains. in the present study we decided to evaluate a distribution of virulence genes encoding for cytotoxic necrotizing factor type 1 (cnf1), p-fimbriae, s/f1c-fimbriae aerobactin (aer), and afa genes in alpha-haemolytic e. coli strains isolated from gynaecological material in our region and to compare the detected sequences in clinical isolates of other diagnostic groups. of 127 alpha-haemolytic e. coli strains, 41 were isolated from urine, 44 from gynaecological specimen, and 42 were faecal strains. e. coli strains were tested for the production of haemolytic phenotype on blood agar plates. the amplification of virulence factors was performed by pcr according to previously described protocols (le bouguenec et al., 1992; blanco et al., 1996; and yamamoto et al., 1995) . we found that all gynaecological alphahaemolytic strains were positive for cnf1, (p b/0.001 compared to 78% of urine strains, and p b/0.0001 compared to 61% of faecal strains). similarly, sfa/foc specific dna sequences were found in 100% of gynaecological isolates (p0/0.01 compared to 85% of urine strains and p 0/0.005 compared to 83% of faecal strains). from this point of view, the female genital tract seems to be a potential reservoir of these uropathogenic e. coli strains. azithromycin in the treatment of pelvic inflammatory disease caused by chlamydia trachomatis ps180 administered in 35 hospitalized patients with chlamydial pid. the diagnosis was made prior to hospitalization. microbiological analysis of urine, blood and swab specimens collected from endocervix, vagina and urethra confirmed c. trachomatis to be the single suspected causative pathogen of pid. the presence of c. trachomatis in swab specimens from endocervix was examined by dnk/rnk hybridization. azithromycin was administered 5 á/7 days after samples for microbiological analysis were collected in dose of 1)/500 mg iv for 5 days. clinical efficacy and tolerability of therapy were assessed 1 á/7 days after the end of therapy and clinical and microbiological analysis 3 á/4 weeks after completion of therapy. the eradication of c. trachomatis and normalization of gynecological findings were achieved in 33 and disappearance of subjective symptoms in 30 patients. no side effects and deviations from normal values in hematologic and biochemical blood parameters were recorded. this study showed high bactericidal efficacy, rapid clinical effect and good tolerability of once-daily administration of 500 mg azithromycin for 5 days in the treatment of patients with pid caused by c. trachomatis . altindis m a , cevrioglu s b , aktepe oc a , cetinkaya a . a kocatepe university school of medicine, microbiology, afyon, turkey , b kocatepe university school of medicine, obstetrics and gynecology, afyon, turkey diagnosis of the causative organism of the vaginitis is usually based on clinical criteria. a standardized, laboratory based and rapid diagnostic test for the identification of these organisms is desirable. to determine the laboratory method that best predicted the causative organism, we calculated the sensitivity, specificity and predictive value of positive and negative test for clinical criteria, an oligonucleotide probe test (affirm vpiii-bd usa) and compared them with the combination of positive vaginal culture and gram-stained vaginal smear. we evaluated 40 consecutive women aged 21 á/48 years, attending for vaginal discharge. vaginal swab specimens were used for culture of gardnerella vaginalis , trichomonas vaginalis and candida sp, preparation of a vaginal smear for gram-stain interpretation and wet mount evaluation and affirm test. affirm detected g. vaginalis in 10 (25%), candida sp in three (7.5%) women and no trichomoniasis case found by any methods. the sensitivity and negative predictive values of affirm test and clinical signs were same (100%) in identifying of bacterial vaginosis. however, affirm test was more specific (94 vs 72%) and also has higher positive predictive value (80 vs 53%) than clinical signs. we did not evaluate the results for patients with candidiasis because of less number. according to these results affirm-microbial identification tests are objective and specific for the rapid diagnosis of the bacterial vaginosis. comparison of efficacy of single dose of tinidazole with standard dose of metronidazole in giardia lamblia infection (preliminary report) ps182 fallah m a , moshtaghi aa b . a university of medical sciences, parasitology, hamadan, islamic republic of iran , b university of medical sciences, pediatrics, hamadan, islamic republic of iran objectives: giardia lamblia is the most common intestinal protozoa in developing countries. treatment of the infection with metronidazole, the drug of choice, requires a long course of therapy and produced some side effects. the object of this study is to determine efficacy and side effects of tinidazole in g. lamblia infection. this is a preliminary report of an ongoing trial. methods: a randomized controlled clinical trial was carried out and 47 subjects with g. lamblia infection were treated with tinidazole or metronidazole. tinidazole 50 mg/kg single dose and metronidazole 25 mg/kg three times a day for 7 days were given orally to 24 and 23 children, respectively. parasitological cure was documented when there were consecutive negative stool examinations at 1 á/2 weeks after therapy. results: twenty-one of 23 individuals treated with tinidazole and 20 of 24 children treated with metronidazole had parasitological cure. cure rates between two groups were not significant statistically. no major side effects were observed except one case in metronidazole group who had mild headache and abdominal pain for 2 days. conclusions: we concluded, tinidazole at the dose has efficacy equal of metronidazole in the treatment of g. lamblia infection. because of single dose prescription, short course of therapy and good compliance of patients, this preparation is preferred to metronidazole in giardial infection. ebeid fa, seif el-din sh. theodor bilharz research institute, pharmacology, cairo, egypt b-carotein was given in different doses starting from 2.5 to 10 mg/kg body weight (b.w.) for different groups of albino mice 4 days before infection with s. mansoni . infection of animals was done by body immersion using 80 egyptian strain of s. mansoni cercariae/mouse. forty-nine days after infection the animals were sacrificed and hepatic and mesenteric worms were extracted and determined. ova count in liver and intestinal tissue and the total number of worms/animals were also determined in experimental groups comparing with infected control animals. the results indicated marked decrease in number of worms and ova count in both liver and intestine comparing with control ones. this reduction increased significantly with increasing dose. it was concluded that b-carotein could be used as a prophylactic agent against s. mansoni infection. barisic z, babic-erceg a, borzic e, zoranic v, carev m, kaliterna v. department of microbiology, public health institute, split, croatia the aim of this study is to determine frequency of pseudomonas aeruginosa urinary tract infection (uti) in outpatient's population in south croatia and to suggest optimal antimicrobial treatment for these patients. during 3 months long observation period, from total number of 13 158 examined urine specimens, significant bacteriuria was found in 2341 specimens. p. aeruginosa was the sixth most common isolate, it was isolated from 94 specimens (4.02%). these 94 specimens were taken from 57 different patients. susceptibility testing was performed by disk diffusion method, and the following results were obtained: resistance to cefibuten occurred in 94.74% patients, to norfoxacin in 49.12%, to ciprofloxacin in 47.37%, to gentamicin in 42.11%, to netilmicin in 35.09%, to amikacin in 24.56%, to ceftazidime in 3.51% and to imipenem in 1.75% patients. p. aeruginosa strains showed better susceptibility to tested parenteral antibiotics than to antibiotics for oral use which complicated treatment in outpatients. the best susceptibility was shown to imipenem, but this drug is inappropriate for use in outpatients setting, so the best choice for treatment p. aeruginosa uti in our outpatients is treatment with ceftazidime, and the second choices are aminoglycoside drugs amikacin and netilmicin. silan l a , breza j b , krcmery v jr. c . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school, bratislava, slovakia , c department of pharmacology, st. elisabeth cancer institute, bratislava, slovakia we studied the clinical efficacy of oral treatment with ciprofloxacin/ cpf/ alone and combined with clarithromycin in patients with complicated urinary tract infection/cuti/ with or without an indwelling catherer. patients were randomly allocated to 600 mg cpf/cpf group/ or to 600 mg cpf plus 600 mg cam/combination group/ for 14 days. evaluation was done on day 14 according to the criteria advocated by the japanese urinary tract infection committee. in patients with a urinary catherer, the combination achieved a higher complete bacterial elimination rate /50%/ and clinical efficacy rate / 84%/ than cpf alone /30 and 61.5%, respectively/. while no significant difference was found in the bacterial elimination rate between the two groups, the clinical effect of the combination /40%/ was superior to that of cpf alone /23%/ in patients with an indwelling catherer. the better clinical efficacy of the combination may partly be attributed to the antibiofilm effect of cam in the clinical setting. the results also indicated that difficulties still remain in the treatment of cuti in patients with an indwelling catherer. in conclusion, clinical study suggested that cam might have an inhibitory action on biofilm formation in the clinical setting. combination of cam with other appropriate antimicrobial agents may have a favorable effect on the treatment of cuti. vesicoureteral reflux and urinary tract infections */the management of primary vesico-eureteral reflux in children ps186 the children studied presented with primary vesicoureteral reflux at derer s universitz hospital in bratislava between 1974 and 1994. seven hundred and sixty patients, 179 boys and 581 girls, suffering from primary vesicoureteral reflux in age from 6 months to 18 years were tested and systematically analyzed outcomes data for seven treatment alternatives. key outcomes identified were probability of reflux resolution, likelihood of developing pyelonephritis and scarring, and possibility of complications of medical and surgical treatment. available outcomes data on the various treatment alternatives were summarized and the relative probabilities of possible outcomes were compared for each alternative. conclusions: increased of urinary tract infection, vesicoureteral reflux nephropathy includes early diagnosis, appropriate evaluation, effective atb therapy, and surgery indicated. the main determinants of renal damage are bstruction, age, sex, predisposition on renal scarring, reflux grade and laterality, therapeutic delay, individual susceptibility, bacterial virulence and immunogenetic status. 3) the one and only absolute indication for surgical management is failure of medical therapy to prevent chronic recurrent pyelonephritis, renal injury or other reflux complications and eliminations of the reflux condition will minimize their likelihood. genetically conditioned immunopathogenic mechanisms are involved in the pathogenesis of the chronic recurrent pyelonephritis in patient suffering from vur. for most children we recommended continuous antibiotic prophylaxis as initial treatment-medical therapy is based on the principle that reflux often resolves with time, and antibiotics maintain urine sterility and prevent infections while the patients awaits spontaneous resolution. 6) vur predispose an individual to renal infection, the immunological and inflammatory reaction caused by a pyelonephritic infection may result in renal injury or scarring. silan l a , breza j b . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school of medicine, bratislava, slovakia elderly patients with uti are believed less likely to be cured by antimicrobial therapy than younger patients. the reasons for this poorer outcome have not yet been clarified. we have investigated the efficacy of antimicrobial therapy in elderly patients with complicated uti. five hundred patients, 260 men and 240 women, who had complicated uti/268 symptomatic and 240 symptomatic and were 21 á/80 years of age, were treated with one of three different drugs, one was a never quinolone and two were oral cephems. multivariate logistic regression analysis of treatment outcome revealed that the clinical response was significantly related to general underlying diseases and diseases of the urinary tract, but not to age, symptomatic or asymptomatic uti, or infection site such as the kidney or bladder. we concluded that the clinical effectiveness of an antimicrobial agent was not directly related to age, and that urological examination for underlying disease and control of them is quite important for effective treatment and control of complicated utis, especially in elderly patients. the study on the frequency and antimicrobial resistance of escherichia (e ) coli in urine isolates of patients admitted to maribor teaching hospital in 1998 and 2000 ps188 rebersek gorisek j a , baklan z a , unuk s a , novak d b . a department for infectious diseases, teaching hospital maribor, maribor, slovenia , b department for microbiology, regional institute of public health maribor, maribor, slovenia purpose: the aim of the study was to determine the frequency and antimicrobial resistance of escherichia coli isolated from urine samples of patients admitted to maribor teaching hospital in 1998 and 2000. the frequency and the antimicrobial resistance were compared between years 1998 and 2000. methods: in the prospective study going on between 1998 and 2000, all urine isolates from patients at maribor teaching hospital were collected and analysed. urine cultures were done using the modified sanford method. the susceptibility testing was performed by disk diffusion method according to nccls. results: in the year 1998, 2563 urine isolates and in the year 2000, 2325 urine isolates were analysed. e. coli represented 39.1% of urine isolates in 1998 and 39.4% of urine isolates in 2000. e. coli resistance rates (%) to amoxycillin was 39.5 in the year 1998 and 38.4 in the year 2000; to amoxycillin/clavulate was 15.4 and 10.5; to cefalotin was 7.8 and 5.7; to cefaclor 2.8 and 2.18; to trimethoprim sulfamethoxazole was 15.0 and 18.4; to ciprofloxacin was 4.9 and 6.9; to gentamicin was 2.3 and 2.2. conclusion: compared to 1998, the frequency of e. coli isolated from urine samples is similar to that in the year 2000. the resistance to amoxycillin, cefaclor and gentamicin is stable. the resistance to trimethoprim sulfamethoxazole and ciprofloxacin is increased and the resistance to amoxycillin/clavulate and cefalotin is decreased. prevalence of the resistance to metronidazole, furazolidone and nitrofurantoin in helicobacter pylori clinical strains ps189 de la obra sanz p a , roman jl a , lomas e a , villar h b , lopez-brea m a . a hospital de la princesa, microbiology, madrid, spain , b hospital de san agustin, microbiology, aviles, spain the objective of this study was to determine the prevalence of metronidazole, furazolidone and nitrofurantoin resistance in helicobacter pylori clinical isolates. methods: a total of 286 strains of h. pylori were included in this study. all these were tested against metronidazole, and 164 against furazolidone and nitrofurantoin by an agar dilution method. resistance was defined as: metronidazole, mic !/8 mg/l; and mic !/4 mg/l for furazolidone and nitrofurantoin. results: sixty-eight strains were resistant to metronidazole (23.8%). the mic50 and mic90 values were 1 and 32 mg/l, respectively. three of 164 strains (1.8%) were furazolidone resistant (mic 0/4 mg/l), two of these strains were metronidazole resistant (mic0/16 mg/l) and they had mic of 2 mg/l for nitrofurantoin. the mic50 and the mic90 were 0.125 and 0.5 mg/l, respectively for furazolidone. only one of the 164 strains (0.6%) was nitrofurantoin resistant (mic 4 mg/l), this strain was metronidazole resistant (mic 128 mg/l) and had a mic0/1 mg/l for furazolidone. the mic50 and the mic90 were 0.5 and 1 mg/l, respectively for nitrofurantoin. conclusion: the low frequency of furazolidone and nitrofurantoin resistance, compared to metronidazole suggests that the furazolidone and the nitrofurantoin may be good alternatives to metronidazole for treatment of h. pylori infections. antimicrobial resistance of campylobacter isolated from human origins ps190 zhukhovitsky vg, drabkina iv. department of bacteriology, botkin hospital, moscow, russian federation the purpose of the study was to determine the antimicrobial resistance of 50 thermophilic enteropathogenic campylobacter spp. (tec) isolated from human under acute diarrhoea in 2001 in moscow. among 50 tec strains 45 c. jejuni and five c. coli were identified. the antibiotic tested by disk diffusion test on mueller-hinton agar with sheep blood were ampicillin (a), amoxycillin/clavulanate (ac), imipenem (i), meropenem (m), erythromycin (e), clarithromycin (cl), tetracycline (t), doxycycline (d), gentamicin (g), azithromycin (az), chloramphenicol (ch), lincomycin (l), ciprofloxacin (c), nalidixic acid (na). the resistant rate of the tec isolates was highest for na (42%) followed by a (38%), t (36%), d (20%), n (20%) and cl (16%). a moderate resistance rate was obtained for a (8%), ch (6%), az (6%), ac (2%). none of the isolates demonstrated resistance to i, m and g and four of 50 isolates (8%) were sensitive for all the antibiotics tested. mic90 to na was estimated as 64 mg/l. among 21 na resistant tec strains 20 (40%) were identified as c. jejuni and one (2%) as c. coli . among c. jejuni and c. coli na resistant rate was 44 and 20%, respectively. one na resistant c. coli and nine na resistant c. jejuni were resistant to ciprofloxacin. ring c, atanassova v. department of food hygiene and microbiology, school of veterinary medicine, hannover, germany aim of the study: poultry meat is known to be often contaminated with salmonella and other foodborne pathogens and thus has to be considered as a possible source for human infections. the aim of the study was to monitor the resistance of salmonella isolates from poultry meat of different european countries to various antibiotics. material and methods: from september 2001 to december 2001 a total of 422 samples of frozen poultry meat from france, germany, italy, spain, the netherlands and portugal were examined for the prevalence of salmonella using classical cultural detection as well as rflp-pcr. all isolates were tested for their sensitivity towards ampicilline, kanamycine, ciprofloxacine, tetracycline, trimethoprim, sulfamethoxazole, nalidixic acid and erythromycine using standard procedures. results: from 18.01% of all examined samples salmonella spp. were isolated. of these isolates 32.9% were characterized as salmonella , 42.1% as s. hadar and 17.1% as s. typhimurium . nearly 100% of all isolates were resistant to erythromycin. resistance towards four or more isolates was observed in several cases. discussion: the consumption of poultry meat, if insufficiently prepared, has still to be considered as a major source for human infection with salmonella spp. the question arises whether the resistance of the isolates to various antibiotics is of clinical importance in the treatment of the patients. objective: to provide insight into the epidemiologic situation of salmonellosis for the nis area (the largest area in serbia, with inhabitants */648,000). methods: the material was processed at the institute for public health (epidemiology and microbiology divisions). isolation of microorganisms was performed on apparatus for rapid identification (vitek-biomerieux) and by applying elisa tests and classical microbiological methods. results: in the period 1992 á/2000, 2857 salmonella laboratory confirmed cases were reported. the greatest number of diseased in the 0 á/4 years group. the most frequent isolated salmonellae were: s. enteritidis (83.7%) and s. typhimurium (11.2%), s. hadar , s. agona , s. virchow , s. infantis , s. derby , s. enteritidis showed the greatest sensitivity to antibiotics with the infrequent resistance to ampicillin and trimethoprim-sulfamethoxazole. s. typhimurium showed the greater resistance to the wide spectrum of antibiotics and some isolates were resistant to all antibiotics tested. the less common types of salmonella were sensitive to all antibiotics except trimethoprimsulfamethoxazole and ampicillin. conclusion: specific resistance to some antibiotics was related to serotypes. typhoid fever */retrospective study of 52 cases in lebanon ps193 tohme a, abboud j, ghayad e. hôtel-dieu hospital, internal medicine, beirut, lebanon objectives: to present epidemiological and clinical features of typhoid fever in lebanon. methods: fifty-two patients were seen at hotel-dieu hospital of beirut between 1995 and 1999. diagnostic criteria were positive blood culture for s. typhi or paratyphi and/or a somatic o agglutinin titer ]/ 1/160 as determined by the widal test with symptoms suggestive of typhoid fever. we also present an epidemiological study of 3864 cases registered by the ministry of health during the same period. results: among the 3864 cases, 40% of the patients' ages were between 15 and 40 years and 33% were less than 14 years. the overall male to female ratio was 0.98 and 24% of cases were seen on january, february and 11% on august. among the 52 patients, young adults were the most affected. average duration of symptoms before the diagnosis was 109/8 days. the main presenting symptoms were: fever (96%), diarrhoea (37%), abdominal pain (31%) and headache (29%). complications were noted in 33% of cases and digestive complications were the most prevalent. leucopenia was not a helpful diagnostic marker. s. typhi was the most frequent (84%) serotype identified. resistance to ampicilline was 13%, to cotrimoxazole and chloramphenicol 10% for each. the mortality rate was 2%. conclusion: typhoid fever is still an endemic disease in our country and the occurrence of resistant strains of s. typhi will favor ceftriaxone or fluoroquinolones in the treatment. maaloul i a , hammami b a , zambaa f a , elleuch r a , hammami a b , -ben jemaa m a . a chu hedi chaker, service des maladies infectieuses, sfax, tunisia , b chu habib bourguiba, laboratoire de microbiologie, sfax, tunisia although its not very frequent, the psoas abscess is not an exceptional entity. in order to specify its clinical, biological, radiological and evolutionary features, a retrospective study has been led in our service, on a period of 14 years (january 1988 á/december 2001). on the whole, 25 cases have been listed. they were 16 men and 9 women. the age average was 40 years (extreme 14 á/74 years). the study did not find any underlying diseases, except diabetes mellitus for three patients. the clinical symptoms were dominated by fever with abdomino-lumbar aches (20 cases), and psoitis (eight cases). biology showed an inflammatory syndrome in all cases and a hyperleucocytosis in 17 cases. the diagnosis of psoas abscess, evoked on clinical data, has been confirmed by the imagery data: ultra-songraphy (16 cases), ct scanning (six cases), magnetic resonance imaging (three cases). the tubercular etiology has been confirmed in six cases, among which two were associated to escherichia coli (one case) and to brucella melitensis (one case). the other etiologic agents were dominated by staphylococcus aureus (eight cases), b. melitensis (two cases), e. coli (one case), bacteroides fragilis (one case), streptococcus anginosus (one case), fusobacterium nucleaticum (one case) and candida glabrata (one case). all patients received an anti-infectious treatment adapted to the micro organism in question. a drainage of the abscess has been realized for 15 patients (percutaneous: nine cases, surgical: six cases). the evolution was favourable for 23 patients. however, two patients had a relapse after stopping the treatment. conclusion: the diagnosis of the psoas abscess, difficult on the clinical data, is based on the imagery techniques (us, ct, rmi). the percutaneous drainage guided by the imagery is recommended (in an etiological and therapeutic aim). associated to an adapted antibiotherapy, it allows to defer a surgical drainage. zezoski mbz, nikolova o, gavriloski pmg. medical center, infectious diseases, prilep, the former yugoslav republic, macedonia purpose: to make a list of the most frequent abdominal changes in patients with human brucellosis. materials and methods: there were 769 new patients with human brucellosis, between 01 1991 and 12 2001. diagnosis was made using standard clinical, biochemical and serological investigations (bab, wright, coomb's, rvk, 2-mercaptoethanol, elisa igm and igg), and specially ultrasound examination of the abdomen and retro peritoneum. results: weight loss is the most frequent change, presented in 665 (86.5%) patients. follow atypical abdominal pain in 89 (5.3%), vomiting in 59 (7.7%), diarrhea in 44 (5.3%), enlarged liver in 594 (77.2%), enlarged spleen in 573 (74.5%) and hepatic lesion with increased ast and alt in 174 (22.6%). conclusion: although frequent, abdominal changes seldom could be missed in patients with human brucellosis. we recommend routine ultrasound examination with standard biochemical test for liver function, due to avoid unnecessary complications. osteoarticular complications are common in brucellosis. the most common site of involvement is the sacroiliac joint. the osteoarticular complications such as, sacroiliitis and spondylitis are diagnosed with radiologically. in the present study, we aimed to determine the severity (grade) of sacroiliitis by using some laboratory parameters such as esr, crp and tube agg test. seventy-two (34 male, 38 female) patients with brucellosis were included in the study. osteoarticular involvement was present in 44 patients. the most common osteoarticular finding was sacroiliitis in the patients (86%). twenty (20) healthy subjects were formed the control group. there was statistically significant difference between patients and controls regarding esr, crp, and tube agg test (p 0/0.000, 0.0017, 0.000, respectively). in addition, sacroiliitis has an effect on esr and crp. there was a positive correlation between the grade of sacroiliitis and the value of crp (p0/0.006, r 0/0.414). in conclusion, it has been suggested that, crp may be used as an auxiliary or a secondary parameter in grading sacroiliac joint involvement in brucellosis. magira ee a , papandreoy s a , gounaris t a , spirelis ma a , tasopoulos g a , anagnostopoulou m b , paniara o b , gounari p a , sioulal e a . a evagelismos, internal medicine, athens, greece , b evagelismos, microa 65-year-old greek farmer was admitted to the hospital because of painful scrotal swelling, hepatosplenomegaly, lumbar pain and lowgrade fever accompanied by profuse sweating. his life style included occupational animal exposure ingestion of raw milk and dairy products. the laboratory data were within the normal ranges. focal hypoechoic right testicular lesions, swelling of the concurrent epididimis along with an increase in the vascularity of the right testis were seen on an echo examination. these findings were consisting in unilateral epididimo-orchitis. a ct scan of the lumbar spine area showed a decrease of the signal intensity localized in the anterior aspect of l5 vertebral body at the diskovertebral junction involving the subchondrial parts of the l5 and s1 vertebrae standard tube agglutination test was positive for antibodies to brucella melitensis (titer !/1/1280). cultures of blood specimens were positive for b. melitensis . the patient had been given to a combination of antibiotics with doxycycline, streptomycin and rifampin. a remarkable improvement of his clinical condition was showed 2 weeks later. this case illustrates the following point: in areas in which brucellosis is endemic when scrotal abnormalities are seen the possibility of genitourinary tract complications of brucella should be considered. pappas ga a , akritidis nk b , mastora m b , tsianos e a . a university hospital, internal medicine, ioannina, greece , b 'g. hatzikosta ' hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and forms of complications associated with brucella infection. patients and methods: we studied the 100 most recent patients, in all larger series approaching 1000, diagnosed as suffering from brucellosis, and assessed the presence of signs and symptoms of arthritis and spondylitis, or other forms of bone involvement. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures or cultures from other media. results: osteoarticular complications were noted in 23 patients, 13 presenting with arthritis, and 10 presenting with spondylitis. eight patients presented with genitourinary complications, either orcheoepididymitis (four patients), or hematuria resolving with treatment (four patients). meningitis was present in two patients. gastrointestinal complications (vomit and diarrhea) were present in three patients, while one patient presented with ascites. respiratory tract complications, in the form of pneumonia (four patients) or bronchitis (three patients) were noted in seven patients, while one patient with pneumonia exhibited pleural fluid. skin rashes, of macular type, were present in three patients. no patient presented with complications from the heart. hematologic complications were frequent, in the form of severe (one patient) or moderate (two patients) pancytopenia, isolated thrombocytopenia (three patients), or lymphocytosis (eight patients). akritidis nk a , pappas ga b , mastora m a . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and modes of bone and joint involvement in the course of brucellosis. patients and methods: we studied the 100 most recent patients, in all larger series approaching 1000, diagnosed with brucellosis, and assessed the presence of arthritis and spondylitis. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures. results: twenty-three patients exhibited a form of osteoarticular involvement. arthritis was present in 13 patients, most often involving the knees, but also the hips, elbows, even smaller joints as intephalangeal joints of the hand. synovial fluid, when aspirated, was often characterised by an intense mononuclear infiltrate. spondylitis was present in 10 patients, most often involving the lumbar spine, but also the thoracic spine. the characteristic erosion on the upper anterior crest of the vertebral body was visible in plain x-rays in three patients, while mri and bone scan were helpful in other cases. discussion: osteoarticular involvement in the course of brucellosis is the most common focal presentation of the disease. acute brucellosis is often accompanied by bone and joint ache, especially of the lumbar spine, still frank involvement in the form of arthritis and spondylitis is not rare. arthritis usually presents in the acute form of the disease, while spondylitis tends to be characteristic of a chronic form of the disease, often necessitating prolonged use of antibiotics. bosilkovski m, krteva l, caparoska s, grozdanovski k, sajn b. clinic for infectious diseases and febrile conditions, medical faculty, skopje, the former yugoslav republic, macedonia one hundred and twenty-six patients with brucellosis were studied prospectively. seventy-eight (61%) of them had osteoarticular involvement. peripheral arthritis in 46 (59%) patients was the most frequent, followed by spondilitis in 27 (35%), sacroiliitis in 15 (19%), rarely bursitis, tendinitis and osteomyelitis. the overall male to female ratio was 2:1. their average age was 39 (sd 20) years. direct contact with animals was the reason for acquisition of the illness in 59% of patients, in 29% alimentary or aerogenous route was incriminated, and in 12% the route of aqisition was unknown. the average duration of the symptoms from the onset to establishing the diagnosis was 68 (sd 78) days. the main presenting symptoms were joint pain (100%), sweating (77%), fatigue (58%) and fever (57%). hepatomegaly was present in 58%. in 49% of patients, involvement of some other system was evident. comparison with patients, who did not have osteoarticular illness, showed that patients with osteoarticular involvement had significantly more often joint pain, fatigue, weight loss and more prolonged duration of symptoms before the diagnosis was established. doxycycline and chloroquine as combination therapy for chronic q fever endocarditis ps201 calza l, attard l, manfredi r, chiodo f. division of infectious diseases, university of bologna, s. orsola hospital, bologna, italy introduction: endocarditis is the main clinical manifestation of chronic q fever, occurring in about 60 á/70% of all reported cases, and it is diagnosed almost exclusively in patients with either cardiovascular abnormalities or an immunocompromised condition. case report: a 62-year-old caucasian male patient with biological prosthetic aortic valve was first hospitalized because of an interstitial pneumonia. six months later, our patient was re-admitted owing to intermittent fever, chills and weight loss. echocardiographic study showed a small vegetation of 12 mm in diameter on left cusp of aortic valve. serology for coxiella burnetii revealed a complement-fixing igg antibody titer to phase i antigen of more than 1:2048, consistent with chronic q fever endocarditis. antimicrobial therapy with i.v. doxycycline and oral chloroquine was started, leading to a clinical and echocardiographical recovery. therapy was continued by oral doxycycline and chloroquine, and the patient remained asymptomatic during a 2-year follow up. conclusion: the optimal treatment of q fever endocarditis has not been well established: the most effective antimicrobials are fluoroquinolones and rifampin, but chloramphenicol, doxycycline and trimethoprim are also useful. the role of chloroquine in combination with doxycycline seems to be promising, because chloroquine may increase the lysosomal ph, enhancing the doxycycline bactericidal activity. akritidis nk a , pappas ga b , mastora m a , liappis e a , tsianos e b . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scopes: to review the incidence and the forms of lower respiratory tract infection in patients suffering from q fever, and their clinical and radiological characteristics. patients and methods: twenty-seven patients diagnosed as suffering from q fever, were assessed for the presence of lower respiratory tract infection. the diagnosis was confirmed serologically. results: thirteen patients expressed lower respiratory tract pathology, as confirmed by clinical examination and chest x-ray. in 11 of these patients the main cause of admission was respiratory tract symptoms, ranging from dry cough to hemoptysis. chest x-ray was pathological in 13 patients: 10 patients had lobar pneumonia, two of them multiple nodular opacities, and one of them bronchopneumonia. hepatitis was a common finding. all patients were treated with tetracycline. discussion: although coxiella burnetii infection is acquired via the respiratory tract, it is paradoxical that symptoms attributed to the lung are not invariably positive. q fever pneumonia is an atypical pneumonia that usually follows a benign course. diangostic suspicion is usually raised by the epidemiologic pattern and the accompanying mild hepatitis. pleural effusion is not a common finding. the usual radiologic appearance of q fever pneumonia is that of a lobar or segmental pneumonia. one important aspect of q fever pneumonia is its common presentation in the form of multiple nodular opacities often necessitating the exclusion of malignancy. (16), culture (4), and serology'/culture (2). there were 0 á/4 cases per year, mainly in october (73%). a history of exposition to hare was present in 24/25 (96%) and to marmot in 1/25 (4%). skinning (18/25 72%), animal contact (5/25 20%), bite (1/25) and wound during bait preparation with frozen meat for hunting (1/25) were noted. the initial clinical presentation was ulceroglandular (77%), glandular (19%) and pneumonic (4%). the involved nodes distribution was axillary (12/24), cubital/axillary (10/24) and cubital (2/24). median incubation period was 3 days (range 1 á/6); time to consultation 3 days (range 0 á/7), and time for effective treatment 7 days (range 0 á/23). an initial diagnosis of tularemia was made presumptively in 42%. effective antibiotic regimen used was aminoglycosides in 73% (19/26), and tetracyclines in 27% (7/26). note that intravenous netilmicin was used in 6 cases. complication rate was 35% (9/26) with one death (4%), and was associated with delay in effective treatment ( !/8 days of illness) (p b/0.05). conclusion: in our area tularemia occured mainly in male population, during autumn, with a short incubation period and history of hare contact. delay before appropriate treatment increased the complication rate. bompolaki i, doukakis s, triantafillidou d, polimili g, kastanakis m, nikiforakis k, vittorakis e, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece a severe frontal and/or retroorbital headache represents the most common neurologic manifestation of murine typhus. other neurologic manifestations as confusion, stupor, nuchal rigidity and in severe cases delirium, extreme agitation or coma appear less commonly. eightyfour patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi, were studied from our team. seventy-four patients (88%) presented headache and nine patients (11%) presented confusion. one patient (1.1%) presented nuchal rigidity in combination with severe headache and confusion giving us the suspicion of meningitis. in this case a lumbar puncture was performed emergently and the cerebrospinal fluid (csf) was examined. the findings of csf were proteins: 9 mg/dl, wbc: 16/ml and glucose: 73 mg/dl and its culture was negative. all patients were treated with a specific anti-rickettsial treatment. the outcome of murine typhus was favorable for all 84 patients (100%). no one patient presented neurologic sequelae. conjunctivitis usually accompany rickettsial diseases such as rocky mountain spotted fever, epidemic typhus and murine typhus. eightythree patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january 1993 and the first semester of 1998. the clinical examination of these patients revealed the presence of conjunctivitis in 21/83 patients (25.6%). in the same time these patients referred retroocular pain and mild photophobia. this study showed that in murine typhus the injection of conjunctivae is rather common. almost a quarter of the patients presented conjunctivitis despite the fact, that this ocular manifestation is less severe than in other typhus and spotted fevers. eighty-three patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january 1993 and the first semester of 1998. three blood samples were obtained from each patient for the study of their hematological abnormalities. the first sample was obtained on admission, the second sample 2 weeks after the first, the third sample, 1 month after the second. on admission 26/83 patients (31%) presented anemia, 6/83 patients (7%) presented leukopenia and 42/83 patients (51%) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was 12.4 g/dl, 6.1)/10 3 and 147)/10 3 /ml, respectively. two weeks later anemia was presented in 45/83 patients (54%), 3/83 patients (4%) presented leukopenia, 3/83 patients (4%) presented leucocytosis and 15/83 patients (18%) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was 11.3 g/dl, 7.0)/10 3 and 248)/10 3 /ml, respectively. one month later 11/42 patients (26%) had anemia and 2/42 patients (5%) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was 12.4 g/dl, 6.4)/10 3 and 242)/10 3 /ml, respectively. our study showed that early thrombocytopenia and anemia are frequent in murine typhus and that white blood cells count is usually normal. renal function in murine typhus: a study of 83 cases ps207 doukakis s, polimili g, triantafillidou d, kastanakis m, vittorakis e, palla k, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece the clinical course of murine typhus is usually uncomplicated and the mortality rate is low ( b/1%). advanced age and prolonged interval before administration of a specific anti-rickettsial treatment are correlated with severity of the disease. renal function in murine typhus is usually unaltered except in elderly patients with prolonged hypotension. eighty-three patients with compatible clinical status of murine typhus and high serological titres of antibodies against rickettsia typhi, were studied from our team, during a period of time between january 1993 and the first semester of 1998. three blood samples were obtained from each patient for the study of their renal function. the first sample was obtained on admission, the second sample approximately 2 weeks after the first, the third sample, taken from the half of the patients, was obtained one month after the second. on admission 4/83 patients (5.0%) presented acute renal failure. the outcome of murine typhus was favourable for all 83 patients (100%). the four patients who presented acute renal failure reversed after the administration of anti-rickettsial treatment and careful administration of fluids. in murine typhus the induction of hypovolaemia insufficiently corrected by normal homeostatic mechanisms may lead to prerenal azotaemia. in these cases the immediate onset of an antirickettsial treatment and the correction of hypovolaemia are essential for the rapid clinical improvement of the patient. experimental ocular toxoplasmosis: clinical, histopathological, immunological and therapeutic studies ps208 el zawawy lae a , hammoda na a , allam sr a , ali sm a , galal as b . a faculty of medicine, parasitology, alexandria, egypt , b faculty of medicine, ophthalmology, alexandria, egypt the purpose of the study was to investigate clinical, histopathological, immunological and therapeutic features of an experimental model of ocular toxoplasmosis in sensitized and non sensitized rabbits and to assess the influence of treatment by interleukin2 (il-2) on ocular lesions. the results obtained was that 'toxoplasma' retnochoroiditis developed in both groups of rabbits with more pronounced effect in non sensitized animals. administration of il-2 improved ocular lesions in both groups with more evident effect in sensitized rabbits. immunological findings were consistent with clinical and histopathological observations. the conclusion reached was that; ocular lesions were manifested in non sensitized rabbits more than in sensitized ones. il-2 revealed a significant impact on improving the host defense against toxoplasm infection in eye. immunotherapy with il-2 would open the way for a new range of treatment based on immunomodulation. express-diagnostic of streptococcus antigen for the adequate antibiotic therapy in patients with pharyngitis ps209 pertseva to, konopkina li, kireeva tv. dsma, internal medicine , dniepropetrovsk, ukraine the purpose of the study: evaluation of effectivness of streptococcus express-diagnostic for the adequate antibiotic therapy in patients with pharyngitis. results: we deal with clinical and microbiological comparison in 53 patients with pharyngitis. using of streptococcus antigen express diagnostic in swabs from the backside of pharynx allowed to get positive results in the seven cases (13.2%). following cultural study has confirmed these results. positive test was more probable in patients with pronounced fever (more than 38 8c), headache, weakness and in cases associated with chronic tonsillitis. isolated streptococcus pyogenes was susceptible to ampicillin, claritromicin, erytromicin, azytromicin, , clindamicin, ceftriaxon, levafloxacin, oxacillin, cefuroxim, roxytromicin. conclusion: using of the express diagnostic of streptococcus antigen allows to restrict groundless prescription of antibiotic therapy in patients with other types pharyngitis (i.e. viral, candidal etc.). alabaz d a , turgut m a , kocabas e a , tumgor g a , yaman a b , alhan e a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey chickenpox is a common viral infection that usually follows a benign, self limited course in healthy children. the most common complication in children with varicella is superimposed cutaneous infections with pyogenic bacteria (streptococcus pyogenes and staphylococcus aureus ). varicella gangrenosum, a necrotising soft tissue infection complicating the vesicular eruption of chickenpox, is rare. here we present three cases with necrotising soft tissue infections following chicken pox. these children were admitted because of common crusted lesions and necrotising soft tissue infection over the neck, the back, and the inguinal area. they all had the contact history and ensuing vesiculopapular rush. these infections were caused by group a streptococci in two cases, and s. aureus in one case. after instituting of appropriate antibiotic therapy, each patient underwent a surgical exploration with fasciotomies and debridement. widespread use of varicella vaccine may decrease invasive infections in children, adolescents, and adults, thus decreasing the burden the disease with its complications impose up on the family and the society . cefprozil is a second generation cephalosporin. the aim of this open, multicentre, non-comparative study was to investigate the efficacy and safety of cefprozil in the treatment of streptococcal tonsillopharyngitis. fifty-eight patients were clinically assessed for signs and symptoms of streptococcal infection. laboratory confirma-tion was sought using three tests; culture, rapid strepto test and estimation of antistreptolysin (aso). one or more tests were done in 47 of the 58 patients. treatment was for 5 days, 25 mg/kg per day in children, 500 mg per day in adolescents. patients were again clinically assessed on the 5th á/6th day. the results showed clinical success in 56 patients (96.5%) and in 46 of the 47 who had laboratory tests (97.8%). two patients had treatment withdrawn because of nausea and abdominal pain (3.5%). of the 47 patients with laboratory tests at least one test was ositive. the most helpful was the strepto test giving the quickest positive result in 92% of those tested (38/41). culture was positive in 58% of those tested (24/41). the aso test was of limited value. in conclusion, cefprozil showed high clinical efficacy and safety in the treatment of streptococcal tonsillopharyngitis. tsiara s, militadou g, milionis c, elisaf m. internal medicine department, university of ioannina, ioannina, greece streptococcus group b agalactiae (gbs) is a rare pathogen for healthy male adults. we present an old man in whom (gbs) was isolated in blood cultures. case report: a 79-year-old man was admitted to the hospital in order to investigate osteolytic lesions in the lumbar spine. two weeks before, he experienced severe low back pain radiating to the right leg and fever arising to 39.50 8c with chills and rigors. a gbs was isolated from blood cultures and treatment with penicillin was initiated. a spine ct scan revealed osteolysis in the t12 and s1 vertebrae and the patient transferred to us. he was a previously healthy man. he received only antihypertensive therapy. on admission he was afebrile with arthralgias and myalgias. on clinical examination there was tenderness on the right pleurospondylic angle radiating to the right leg. laboratory data on admission: hb: 11.2 g/dl, wbc: 7.4)/10 9 /l, esr: 77 mm/h. biochemical values, serum protein electrophoresis, rectal examination and a prostatic specific antigen (psa) were normal. a bone marrow aspiration and biopsy were negative. a transoesophageal ultrasound revealed vegetation on the right cusp of the aortic valve, with low grade regurgitation. an mri of the lumbar spine revealed infectious myositis with concomitant osteomyelitis involving the l4, l5 vertebrae without any evidence of osteolytic lesions. a thorough investigation did not revealed any underlying immunosuppressive disease. treatment with vancomycin and gentamycin iv was initiated and the patient discharged from the hospital in excellent health after 6 weeks. discussion: gbs infections usually occur in neonates and pregnant, or in adults with underlying disease as diabetes mellitus or immunosuppression. the most common site of infection is soft tissue. we present this case because gbs infections are rare in the elderly in the absence of underlying disease. common sites of involvement are soft tissues, while bone, joints, and heart valves account to 1 á/2% of the involved organs. although our patient had more than one site of involvement he responded well to medical treatment without surgical debriment or heart valve replacement. objective: to evaluate the safety and efficacy of rhugm-csf in combination with broad-spectrum antibiotics for the treatment of ccnf. methods and results: a retrospective review of all patients (pts) with ccnf treated with antibiotics'/rhugm-csf at our hospital from january 1997 to december 2001 was performed. five patients were identified with the diagnosis of ccnf. ages ranged from 33 to 71 years; there were three women and two men. dental infection was the most common source of ccnf in 90%. one patient had acute tonsillitis leading to ccnf. all cases studied experienced infection of the neck with spread into the submandibular, submental, sublingual, retropharyngeal, and parapharyngeal spaces. all infections were polymicrobial. diabetes mellitus was a co-morbidity in one case. all pts were treated with dual antibiotic coverage (vancomycin'/meropenem), rhugm-csf (10 mcg/kg/daily given s.c.) and aggressive wound care. rhugm-csf was given for 7 á/10 days. in spite of the severity of the infection all pts recovered and do not experienced local or systemic complications. discussion: ccnf is a severe bacterial infection of the cervical fascia resulting in extensive fascial necrosis with widespread undermining of the surrounding tissues. prompt antibiotic therapy combined with rhugm-csf resulted in a 100% overall survival in our experience. to our knowledge, this is the first report describing the successful treatment of ccnf with use of broad-spectrum antibiotics combined with rhugm-csf. the following case adds to the clinical manifestations and course of meningococcal disease. a previously healthy 3-year-old girl presented acutely with high fever purpuric rash including conjuctival haemorrhages and hypotension. the child had also neck stiffness. a presumptive diagnosis of meningococcal septicaemia was made and treatment with penicilline, chloramphenicole, fluids and inotropes was initiated. laboratory investigations showed wbc: 4300/ml, hb: 12.2 g/dl, hct: 39.5%, esr: 1 mm in the first hour, pt: 26 s, aptt: 90 s. neisseria meningitidis group b was isolated from the blood cultures. csf obtained after antibiotics were started did not grow n. meningitidis . the patient had an adverse outcome. she died after 12 h of hospitalization. this patient developed a fulminating meningococcal septicaemia. shock is a clinical diagnosis arising from the failure of compensatory mechanisms that maintain perfusion of vital organs at the expense of non vital. septic shock results from loss of circulating plasma volume due to increased vascular permeability and maldistribution of intravascular volume. in young children there is a prevalence of serogroup b meningococcal disease which can be explained by the immaturity of the immune system and by the fact the group b capsule synthesis is known to inhibit alternative complement pathway activation. this case emphasizes the need for further protection against n. meningitidis group b. . results: forty-eight patients were included in this study. the etiological agents were: viral (n 0/26, 54.2%, mean ('x ) age0/28 years), streptococcus pneumoniae (n 0/8, 16.6%,'x age0/53), neisseria meningitidis (n0/7, 14.6%, 'x age0/28), s. viridans (n0/6, 12%, 'x age0/34), p. multocida (n0/1, age 75). the peak incidence of bacterial meningitis was in winter (pneumococcal 73%, meningococcal 86%, s. viridans 83%) .the cerebrospinal fluid (csf) findings in viral meningitis were 'x white cells 0/340/mm 3 , 'x pmn0/27%, 'x glucose csf/ serum 0.55, 'x protein 39 mg/dl and in bacterial meningitis were 'x white cells0/5800/mm 3 , 'x pmn0/80%, 'x glucose csf/ serum0/ 0.2, 'x protein0/350 mg/dl, gram stain was positive in 55%, culture was positive in 45%. all pneumococcal and meningococcal strains were susceptible to penicillin. the case fatality rates for pneumococcal and meningococcal meningitis were 25 and 14.3%, respectively. conclusions: the cases of bacterial meningitis were according to typical epidemiological features of age and season. the case fatality rate of pneumococcal meningitis appear to be high regardless of susceptibility to penicillin. none had received pneumococcal vaccine prior to becoming ill. diagnosis and therapy of meningococcal meningitis */trend and particularities of a 'romanian model' ps216 lintmaer i, moroti r, popescu a, popescu c. institute of infectious diseases matei bals , unit 5 , bucharest, romania background: newer diagnosis methods and antimicrobials are expected to change the management of menigococcal meningitis (mm) and to improve its prognosis. objectives: to determine the changes in the diagnosis methods and therapy of mm patients in a infectious diseases hospital. to compare mm management in bucharest with literature data. methods: retrospective rewiew of mm in adult patients hospitalized over a 6-year period. our results were compared with other studies made in the 90s, taken from medline. results: there were 97 episodes of mm during the study period (52 episodes in 1995 á/1997 and 45 episodes in 1998 á/2000) . we noticed a defined diagnosis increase and increased blood culture specificity. the antimicrobial monotherapy was maintained but penicilin was replaced by ceftriaxone. hhc was replaced by dexamethasone in pathogenic therapy. we noticed a shorter length of treatment and a reduced lethality. the most important differences between our results and other studies are: monotherapy regimens are less frequent and therapy lengths are longer; however, prognosis is similar. conclusions: the mm management has been modified in the last 3 á/ 4 years: prognosis is improved, but the changes do not bring clear cost/ effective benefits. tiouiri th, kilani b, amari l, zouiten f, kanoun kf, ghbontini a, ben chaabene t. rabta hospital, infectious diseases, tunis, tunisia objectives: in order to study epidemiological, clinical and therapeutical characteristics of the infective endocarditis (ie). methods: we reviewed all the cases of ie fulfilling the duke criteria. data were collected during a 19-year period (1980 á/1998) in the unit of infectious diseases. results: one hundred and eight cases were identified. the mean age was 37.5 years. sex ratio was 0.8. eighty-five ie (78.6%) occured in patients with native valve, and 23 ie (21.4%) with prosthetic valve. fever was the most common sign, 12% had a congestive heart failure, 26.9% had cutaneous signs. the most common primary focus of ie was orthodontic. blood cultures were positive in 63% of cases. in one case, serological test identified rickettsia conori . streptococci and staphylococci were isolated in 27.8 and 25.9%, respectively. echocardiography detected abnormalities in 63.4% of cases. rheumatic heart disease was the most predisposing condition. empirical therapy was based on combination of b lactam with aminoglycoside. recovery was obtained for 60 patients. cardiac surgery was performed in 26 cases. overall mortality rate was 19.4%. conclusion: ie affects young persons. prevention needs eradication of acute rheumatic arthritis. a major outbreak of legionnaires' disease in spain: diagnostics aspects ps218 guerrero c a , toldos cm a , yagü e g b , ramírez c a , rodríguez t a , -segovia m a . a hospital morales meseguer, servicio de microbiología, murcia, spain , b departamento de microbiología, facultad de medicina, universidad de murcia, murcia, spain objective: to evaluate the value of different methods (serological tests, culture of respiratory secretions, blood cultures and urinary antigen testing) for the diagnosis of legionella pneumophila pneumonia during an outbreak in spain. results: we have studied 542 patients from a recent outbreak of legionellosis in murcia (spain). the diagnosis was achieved in 305 patients. urinary antigens were positive in 187 patients. in the 355 patients with urinary antigen negative the serological response was demonstrated by indirect immunofluorescence (ifa) in 118 patients. all blood cultures processed were negative. sputum samples were obtained from 154 patients, of these l. pneumophila was isolated only in six patients. in all of them direct immunofluorescence test (dfa) was positive. conclusions: although the serological diagnosis was the most sensitive method the urinary antigen testing was of great value in the rapid diagnosis of the legionella's outbreak in murcia. the isolation of l. pneumophila by culture showed a poor sensitivity probably because of the low severity of the illness. purpose: to evaluate chloramphenicol for an initial empiric antibiotic treatment of purulent meningitis in adults. study group: one hundred and twenty patients hospitalized for the diagnosis purulent meningitis in the department in years 1997 á/2000, 67 males and 53 females, age range 15 á/81 years, mean age 52.6 years. children up to 15 years were not included. method: a retrospective analysis of the study group focused on antibiotic treatment both initial and changes during treatment. results: chloramphenicol was used as an initial antibiotic in 59 (49%), 3rd generation cephalosporin in 30 (25%), penicillin in 21 (17%), ampicillin in five (4%) and other antibiotic in five (4%), respectively. during treatment chloramphenicol was switched for 3rd gen cephalosporin in seven of 23 patients with streptococcus pneumoniae meningitis and in five of 28 patients with meningitis of unknown etiology. the reason for the change was non-improving csf formula in three, persisting csf culture positivity in two and persisting coma in seven patients. conclusion: because of repeated necessity to switch chloramphenicol for 3rd gen cephalosporin during treatment of purulent meningitis of pneumococcal and unknown etiology the initial treatment strategy was changed in 2001. third gen cephalosporin is now used as a first choice antibiotic, what is in consent with international recommendation of treatment. to evaluate and compare groups treated initially with chloramphenicol and with 3rd gen cephalosporin will need several more years. low prevalence of multi-drug resistant mycobacterium tuberculosis in jerez de la frontera-cadiz (spain) ps220 alados jc, aller ai, de miguel c, de francisco jl, calbo l. hospital del sas-jerez , microbiologia, jerez de la frontera, cadiz, spain introduction and aim: previous reports indicate that multi-drug resistance mycobacterium tuberculosis (mtb) is an worldwide problem. the aim of this study was to review the resistance of mtb to the first-line antimycobacterial agents in our area. material and methods: over a period of 4 years (1998 á/2001), 151 strains of mtb isolated from non-treated patients with tuberculosis (38 strain in 1998, 48 in 1999, 37 in 2000 and 28 in 2001) were studied. these isolates were tested for in vitro drugs susceptibility to isoniacid-i, rifampicin-r, streptomycin-s and ethambutol-e using the bact/ alert method (organon teknica) as described by the manufacturer. results: our results showed that 10.6% (16/151) strains were resistant to one or more drugs. single drug resistances were detected on nine strains to i (6.3%), one to r (0.66%), two to s (1.3%), one to e (0.66%). three mtb strains were resistant to more than one drug but only one was multi-drug resistant (i'/r).the incidence of i-resistant strains over the period fell from 13% in 1998 to 3.6% in 2001. conclusions: (1) multi-drug resistance is not an important problem in our area. (2) isoniacid resistance was declined to an admissible level. to investigate the anti-tuberculosis drug resistance pattern of pulmonary tuberculosis isolates in southern taiwan, an area with higher tuberculosis incidence and mortality than other regions of the island, we performed a hospital-based surveillance at a southern taiwan medical center from 1996 to 2000. the combined drug resistance rates to at least one of five first-line agents was 84.8%, and to both isoniazid and rifampin (multi-drug resistance, mdr) was 11.4%, indicating high resistance rates compared with those reported in the who/iuatld global project and in northern taiwan. the resistance rates to two second-line drugs, cycloserine, and kanamycin, were 75.7 and 23.7%, respectively. a significant decreasing trend in resistance rates to all drugs except streptomycin was observed during the 5-year period. though combined drug resistance rate may not be the most accurate tool as it includes previously treated cases which inflates the resistance rate, the observation of trends in the susceptibility of pulmonary tuberculosis in accompany with the increasing percentages of tuberculosis patients receiving complete treatment course and the decreasing percentages of cases lost of follow-up in kaohsiung after the institution of new governmental regulations for case management in 1997 suggest the usefulness of intervention programs. lipid profile in patients with multidrug resistant pulmonary tuberculosis ps223 extrapulmonary tuberculosis may sometimes present with confusing clinical manifestations. a 77-year-old female patient was admitted with a history of recurrent supra-sternal abscess for 1 year. mri confirmed the presence of sternal osteomyelitis and an anterior mediastinal mass. the diagnosis of tuberculosis was proved by histologic examination and acid-fast stain. the patient was treated with first-line agents, which isoniazid, rifampin, pyrazinamide, and ethambutol. tobacco smoking as a factor of the decrease of chemotherapy effectiveness and of the development of the drug resistance in patients with pulmonary tuberculosis ps225 shprykov as a , zhadnov vz a , shprykova on b . a medical academy, department of tuberculosis and lung diseases, nyzhny novgorod, russian federation , b medical clinic for infectious #2, laboratory of bacteriology, nyzhny novgorod, russian federation studies of the effect of smoking on the results of chemotherapy of 798 patients with tuberculosis of lungs. intensive tobacco smoking slowed down clearance of positive sputum and of lung tissue destruction (in smokers 92.1% and 70.1 vs. 98.0% and 90.6% in nonsmokers, p b/0.01). drug-resistant mtb strains have been found to be isolated more often in smokers */43.4 vs. 19.4% in non-smokers, p b/ 0.01. resistance to streptomycin and isoniazid prevailed, reaching in heavy smokers 50.0 and 30.5%, respectively. resistance to rifampicin increased 1.5 times. the concentration of rifampicin in the blood serum of heavy smokers decreased in 1.8 times. clinical data are in complete correlation with the findings of our experiments: 67% of experimental cultures developed resistance to streptomycin, isoniazid and less to rifampicin in the study of drug sensitivity under the effect of tobacco smoke condensate. thus, our findings show the development of drug resistance in mtb under the effect of components of tobacco smoke and also showed less effectiveness in therapy. kilani kb, ammari la, tiouiri ht , ben chaabène tbc. rabta hospital, infectious diseases, tunis, tunisia guerrero c a actinomycosis is a chronic disease characterized by abcess formation, tissue fibrosis and draining sinuses. it is caused by anaerobic bacteria belonging to the genus actinomyces. thoracic actinomycosis may involve the lungs, pleura, mediastinum or chest wall. the authors present a case of pulmonary actinomycosis complicating a cervicofacial site. a 32-year-old man with a history of cervicofacial actinomycosis treated by penicillin g 2 years ago was admitted because of right-sided chest pain for 2 months before presentation, cough and fever. physical examination shows a painless indurated mass in the neck with multiple fistula of the sternum. chest radiograph and ct scan revealed a mass in the upper lobe of the right lung with an infiltrate of the upper lobe of the left one. magnetic resonance imaging confirms the previous lesions, with extending process to the sternum and right collar bone. bronchoscopy was performed while patient was on antimicrobial therapy. culture of bronchoalveolar lavage fluid was negative. transbronchial biopsy was not conclusive. fungal serologies were negative for aspergillosis, histoplasmosis, blastomycosis. bacterial examination of purulent drainage from sternal wound shows inclusion bodies identified as actinomyces. he was treated then with penicillin iv for 2 months, than switched to doxycycline. after 8 months of treatment, he is asymptomatic with radiological improvement. kanellopoulou m a , skarmoutsou n a , iglezos i b , mylona e a , martsoukou m a , apostolopoulou f b , papafrangas e a . a laboratory of clinical microbiology, sismanoglio general district hospital of attica, athens, greece , b 2nd department of pneumology, sismanoglio general district hospital of attica, athens, greece introduction: achromobacter xylosoxidans is a rare human pathogen. it is an important cause of bacteremia in patients with cardiac diseases, malignancies and immunosuppression. it has been recently recognized as an emerging microorganism in cystic fibrosis (cf), whose its pathogenic role is unknown. aim: to investigate the sensitivity to eleven different antibiotics of 27 a. xylosoxidans strains isolated from adults with cf, during 2001. methods: the susceptibility was tested by kirby bauer and microdilution methods (wider i, fransisco soria melguizo, s.a.), according to nccls recomendations. results: the resistance to antibiotics was as follows : gentamicin, tobramycin, aztreonam 100%, amikacin 96%, ceftazidime 85%, ticarcillin 66%, carbapenems, cotrimoxazole 59%, colistin 51% and piperacillin 22%. conclusions: (1) a. xylosoxidans isolated from cf patients appeared resistant to the most usually tested antibacterial agents. (2) colistin which is used as aerolized antibiotic for cf patients seems to be effective in the half of the isolated strains. (3) piperacillin was the most active antibiotic against a. xylosoxidans . morris ka, perry jd, jain s, gould fk. microbiology department, freeman hospital, newcastle upon tyne, uk alafosfalin, l-alanyl-l-1-aminoethylphosphonic acid, is an antibacterial peptide mimetic which inhibits peptidoglycan biosynthesis. we report the in-vitro activity of this compound in combination with ceftazidime, cefsulodin, fosfomycin, piperacillin/tazobactam, aztreonam, ciprofloxacin and timentin. drug combinations were evaluated against 20 burkholderia cepacia strains, and 20 pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. for this purpose a chequerboard technique was adopted using doubling dilutions of each antibiotic incorporated into a highly defined agar medium free of antagonists. the minimum inhibitory concentrations (mics) and fractional inhibitory concentrations (fics) of all the antibiotic combinations were determined which revealed the antibiotic interaction occurring. alafosfalin in combination with ceftazidime, meropenem, piperacillin/tazobactam and timentin demonstrated the highest percentages of synergy in both b. cepacia and p. aeruginosa . synergy was shown to occur in 15, 15, 10 and 10% of b. cepacia strains respectively, and in 10, 5, 5 and 5% of p. aeruginosa strains. these four combinations were re-tested with all 40 isolates and the results were shown to be reproducible. alafosfalin shows potential as a treatment for cystic fibrosis patients colonised with p. aeruginosa and/or b. cepacia , when applied in combination with these agents. community-acquired pneumonia */does its aetiology matter? ps229 lintmaer i, popescu a, popescu c. institute of infectious diseases matei bals, unit 5, bucharest, romania the aetiology of a pneumonia is not one of the criteria used to determine pneumonia's severity. however, it is accepted that identified based á/based therapy is less expensive (and possibly more effective). objectives: our study aims were to: (1) to evaluate the role of aetiology identification in pneumonia; (2) to evaluate the first-line therapy in pneumonia. methods: we conducted a retrospective study in an infectious diseases hospital on 1126 patients with pneumonia. we excluded all the cases with nosocomial pneumonia. primary end-point was the 28day clinical failure (deaths, icu admission), secondary end-points were the average time of fever and length of stay and the antimicrobial regimen changes. results: causative agent identification rate was 24.6%. the evolution was different for patients with identified aetiology compared with other patients in terms of: 28-day failures, length-of-stay and changes of the antimicrobial regimen. the 61 patients with inadequate first-line therapy had a more severe course of illness with a greater rate of 28day clinical failure, longer fever and length-of-stay. conclusions: pneumonia's treatment was better for the patients with identified causative agent. that is why we should include aetiology among the pneumonia severity criteria, especially at an 'after 3-day therapy' re-evaluation. results: pneumomococcal aom was detected in 94 children (41.0%) and s.pn. was the only pathogen in 80.9%. the resistance rates of the organism to antibiotics were as follows: penicillin 48.9% (micb/1 mg/ml; intermediately resistant 31.9%, mic 1.5 mg/ml 8.5%, and mic!/2 mg/ml; highly resistant 8.5%), erythromycin 55.3%, clindamycin 11.7%, cotrimoxazole 41.5% and chloramphenicol 6.4%. all isolates were uniformly susceptible to rifambicin and vancomycin. the large majority of pneumococcal isolates (80.8%) had the mphenotype and the remaining strains (19.2%) the constitutive mls phenotype. a various of serogroups were detected; the serogroup 19 was the most predominant one (50.0%), followed by serogroups 14 (14.9%), 23 (10.6%) and 6 (6.4%). the non-typable s.pn. strains compromised the 8.5% of the strains. conclusions: high prevalence of resistance to penicillin, macrolides and cotrimoxazole in pneumococcal aom of childhood was recognized. a strategy for preventing aom caused by drug-resistant pneumococci is mandatory to start. material and methods: a total number of 82 strains were examined. the sensitivity test was performed by kirby bauer, microdilution method (pasco, difco) according to nccls guidelines and by e -test. results: a percentage of 5.5 % of s. pneumoniae strains revealed high level resistance to penicillin (mic]/2 mg/ml), while the 28% showed intermediate resistance (mic 0.12 á/1 mg/ml). the resistance to erythromycin and cotrimoxazole was 8.3 % (mic ]/1 mg/ml) and 5.5 % (mic]/4/76 mg/ml), respectively. all strains were sensitive to cefotaxime (mic 0.05 mg/ml), vancomycin (mic5/0.5 mg/ml), meropenem (mic 5/0.25 mg/ml) and levofloxacin (mic5/2 mg/ml). conclusions: (1) the prevalence of high resistance s. pneumoniae to penicillin seems to be low in examined strains (5.5%). (2) intermediate resistance to penicillin of s. pneumoniae isolates was high as expected (28%). (3) most of the strains were sensitive to erythromycin (91.7%) and cotrimozaxole (94.5%). (4) s. pneumoniae isolates were completely (100%) sensitive to levofloxacin, vancomycin and meropenem. beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy aim: of this study was a retrospective (1997 á/2001) evaluation of the more effective and practical antibiotic treatment in 1058 ae-copd patients (pts) admitted to our unit. methods: before introducing any antimocrobial drug, sputum specimens were collected for microbiological purposes, while blood analysis, to monitor adverse systemic effects, were performed at the beginning and the end of treatment. antibiotic treatment ranged from 8 to 15 days according to four regimens: regimen a (832 pts)0/oral therapy only: 30.9% with amc 1 g b.i.d.; 24.4% with cip 500 mg b.i.d.; 17.4% with dox 100 mg u.i.d.; 10.2% with lev 750 mg u.i.d.; 8.5% with cla 500 mg b.i.d.; 7.8% miscellaneous. regimen b (94 pts)0/sequential therapy (e.v. for 3 days0/oral): 56.4% with amc 1 g b.i.d.; 37.2% with cla 500 mg b.i.d. regimen c (68 pts)0/e.v. therapy only: same drugs. regimen d (64 pts)0/an association of two antibiotics. results: of 1058 evaluated pts, only 199 (19.6%) required a second regimen of treatment because of failure of the previous one: 19.7% of regimen a; 12.7% of regimen b; 23.5% of regimen c, and 14% of regimen d. mild adverse effects were detected only in four pts. our results confirm that oral antibiotic treatment is practical, safe, and effective, and can be considered as the first line regimen also in hospitalized patients with ae-copd. becher g a , gillissen a a , rothe m b . a st. george medical center, robert-koch-hospital, leipzig, germany , b filt, lung and chest diagnostics ltd., berlin, germany patients with severe form of chronic obstructive pulmonary disease (copd) are prone by frequent exacerbations. bacterial infections are judged to cause at least half of exacerbations. haemophilus influenzae and streptococcus pneumoniae are the most frequent isolates, gramnegative bacilli account for the severe cases, aggravating the inflammatory process in the airways eventually leading to respiratory insufficiency. the aim of this ongoing placebo controlled, parallel group, mono center study trial is to evaluate beneficial effect of cefixim to reduce bacterial load and pulmonary inflammation in patients (n0/ 30) with acute bacterial exacerbation of severe copd. thus, 30 patients received in randomized fashion either cefixim (400 mg/day) or placebo (5 days). on days 1, 2, 5 and 8 breath condensate is collected using 'ecoscreen' (jaeger germany) for ltb4-, il-8-, nitrite-and ph-analysis. in parallel sputum gathered for detection of bacterial strains, and for ltb4-, and il-8 quantification purposes. these data are compared to clinical outcome parameters such as lung function tests, radiographic findings, serum inflammatory markers and length of hospital stay. the preliminary data obtained confirm successful antibiotic therapy with oral cefixim in bacterial related acute exacerbations of copd is a useful approach to reduce bacterial load, and concomitantly lower inflammatory indices of the central and peripheral airways leading to clinical improvement of the patients. soriano garcia f a , fenoll a b , fernandez-roblas r a , coronel p c , gimeno m c , rodenas e c , garcia m a , granizo jj d . a fundacion jimenez diaz, microbiology, madrid, spain , b instituto de salud carlos iii, centro nacional microbiologia, majadahonda, spain , c tedec meiji farma, scientific, alcala de henares, spain , d fundacion jimenez diaz, epidemiology, madrid, spain purpose: to describe the susceptibility of streptococcus pneumoniae against cefditoren and 10 other antimicrobials by serotype a multicenter study in south europe was carried out. a total of 877 strains were collected between september 2000 and march 2001 from adult patients (more than 16 y.o.) with respiratory tract infection (respiratory tract samples and blood cultures). all the isolates were sent to a central laboratory (fundació n jiménez díaz, madrid, spain) where susceptibility test was performed by broth microdilution (sensititre) following nccls recommendations. serotype was determined by quellung reaction and dot assay in carlos iii institute in 806 strains. results: a total of 25 strains (3.1%) were not typable. the most prevalent serotypes were 23 (15.4%), 6 (11.4%), 19 (10.8%), 3 (9.6%), 14 (8.9%) and 9 (8.1%). two hundred and sixty-four strains were grouped in 27 different serotypes. the proportion of susceptible strains by serotype to penicillin, erythromycin and levofloxacin were: serotype 3 (90.9, 93.5, 100%); 6 (50.0, 34.8, 96.7%); 9 (36.9, 86.2, 93.8%); 14 (38.9, 48.6, 94.4%); 19 (60.9, 44.8, 97.7%); 23 (58.9, 49.2, 99.2%). the mic 90 to cefditoren was 5/0.03 (serotype 3); 0.5 (serotype 6, 9 and 19) and 1 mg/l (serotype 14 and 23). conclusions: the most prevalent serotype was 23. the susceptibility was higher in serotype 3 than in serotypes 14 and 23. community acquired pneumonia */a study among closed military community of young people ps235 martynova av, turkutyukov vb, vostrikova aa, andryukov bg. vladivostok state medical university, epidemiology, vladivostok, russian federation purpose: the etiology of pneumonia is still partly unknown. we should like to clear up an etiological role of respiratory pathogens in community-acquired pneumonia among youth. and we had chosen for it a model of a closed community both investigation of etiology of disease and for further investigation of mechanisms of transmission drug-resistant mechanisms. methods: we studied 300 adults in age of 17 á/25 from closed military collectives who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during winter 2000 á/2001. we did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. results: streptococcus pneumoniae is recognized as an important cause of community-acquired pneumonia, it probably accounts for 65% of cases of community-acquired pneumonia among youth. chlamydia pneumoniae and mycoplasma pneumoniae responsible for approximately 20% of cases. haemophilus influenzae caused 7.5% sever cases of disease, 3% of all cases were due to moraxella catharralis . conclusion: pneumococcial infection accounted for 65% of the cases diagnosed. s. pneumoniae was the most common bacterial infective agent, with a low incidence of both m. pneumoniae and s. pneumoniae . other causative pathogens occurred only within groups of individuals with deficiency of immunological status. berezin en a , cardenuto md a , nobuko e b , guerra ml c , brandileone mc d . a santa casa, pediatrics, s. paulo, brazil , b santa casa, microbiology, s. paulo, brazil , c adolfo lutz, microbiology, s. paulo, brazil , d adolfo lutz, bacteriology, s. paulo, brazil to determine antimicrobial susceptibility of sp isolated from the upper respiratory tract, we collected np swab specimens from 80 children, between 3 months and 5 years old. those children attended the outpatient clinic in s. paulo city, with diagnosis of bacterial infection requiring antibiotic therapy between march 15, 2000 to may 20, 2001. penicillin susceptibility of isolates was determined by screening with oxacillin 1 mcg disk and performing the minimal inhibitory concentration by the e -test. we performed also susceptibility test for amoxicillin and cefaclor. results: sp was recovered from np of 42 children (52.5%). the carriage of sp was more prevalent in children attending day care centers, children with young siblings at home, and children with tobacco users at home. the prevalence of penicillin non-susceptible strains was 40.4% all of them with intermediate resistance. all the strains were susceptible to amoxicillin and 19.2% were resistant to cefaclor. serotypes 14.6b, 19f, 9n, 4, 6a and 5 were the most common. these findings suggest that nasopharyngeal isolates of streptococcus pneumoniae from children with upper respiratory infections can be used to conduct surveillance for antimicrobial resistance in a defined geographic area. we were able to conclude also that penicillin intermediate resistent strains can be susceptible to amoxicillin. hinojosa rm a , saenz a a , collazo m a , echaniz g b . a universidad autonoma de nuevo leon, infectologia, monterrey, mexico , b instituto nacional de salud publica, epidemiologia, cuernavaca, mexico the emergence of penicillin-and multidrug-resistant pneumococcal strains has become a global concern, necessitating the identification of the epidemiological spread of such strains. material: ninety streptococcus pneumoniae clinical isolates were collected from march 1997 through march 1999. typing was done by the capsular reaction with pooled, type, or group antisera. susceptibility testing to 11 antimicrobials was done by the e -test and the disk diffusion method. results: only 46 (51%) s. pneumoniae strains were classified by serotyping; the most frequent types were 6a/b, 23f, 9v, 19f and 4. the oxacillin screening test detected 37.2% penicillin-resistant s. pneumoniae strains isolated from children and 44.6% from adults. the susceptibility percentage of s. pneumoniae to ceftriaxone was 93% in both children and adults. s. pneumoniae isolates from children exhibit a susceptibility of 88% to azithromycin, while in adults 91% of the isolates were susceptible. for the rest of the antimicrobial agents, the susceptibility ranged from 69 to 89%. s. pneumoniae had a lower susceptibility to ceftazidime and ciprofloxacin. conclusions: ceftriaxone and azithromycin had a good in-vitro activity against s. pneumoniae strains. but the percentage of penicillinresistant s. pneumoniae detected in this study is alarming, therefore we conclude that a continuous surveillance system is necessary in mexico. vertkine al, prokhorovitch ea, alexanyan la. a retrospective analyses of 221 cases of ambulant pneumonia with fatal outcome was made. among the patients who died from ambulant pneumonia the prevailing age was over 60 (63.8%) and the prevailing sex was male (68.8%). 95.9% had pneumonia accompanied with some pathology: chronic lung disease (43.9%), alcoholism (26.2%), diabetes mellitus (10.4%). 62.5% of the patients had a big volume of lungs lesion */55.7% of the patients suffered from bilobular pneumonia and 6.8% */from trilobate pneumonia. in 57.0% of the cases pneumonia was complicated with abscess formation and/or exudative pleurisy. we studied the antibiotics therapy used for the patients treatment. the change of antibiotics was made only in 53 cases (24.0%) whereas in the other cases no change of preparations was made though the signs of the therapy non-effectiveness were obvious. thus, the rational antibiotics therapy with the timely change of non-effective antibacterial drug is significantly important. while choosing the antibiotics, the patient's age, the accompanying diseases, the volume of the lungs lesion and complications which define pneumonia seriousness are to be taken into consideration. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to describe the management of lower respiratory tract infections (lrti) in healthy adults, by general practitioners (gp). methods: a questionnaire was sent to a representative national sample of 4092 gps. this questionnaire assessed their perception and management of lrti, the indication for antibiotics (ab) in a case of lrti in a healthy adult with no focal signs and no signs of severity, knowledge of the micro-organisms responsible for acute bronchitis and knowledge of the afssaps (french agency for the safety of health products) recommendations. results: three thousand seven hundred and thirty-eight gps, who reported seeing an average of 131 patients per week, including 10.39/ 9.6 patients with lrti, returned the questionnaire. the main results are presented in the following for the majority of gps, the main objective of the visit is to determine the indication for antibiotics. according to gps, alp and whooping cough are rare, while atypical pneumonia is frequent. gps also declare that the diagnosis of alp is often easy right from the first visit, in contrast with that of atypical pneumonia. complementary investigations are not often requested. gps consider that they often delay prescription of antibiotics (41%) and declare that they tend to prescribe a macrolide as first-line treatment. finally, gps have a poor knowledge concerning the micro-organisms responsible for acute bronchitis and the majority of gps declare to be familiar with afssaps recommendations. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to study the management of one case of lower respiratory tract infection (lrti) in adults by general practitioners (gps). methods: prospective study conducted on a representative national sample of 4092 gps. each gp had to include the first healthy adult patient seen during the 3-week data collection period, either on a home visit or in the office for recent cough and acute fever !/37.8 8c. clinical data, the diagnostic perception and the therapeutic approach to the patient were collected by means of a standardised questionnaire, distributed by abbott laboratories. results: three thousand seven hundred and thirty-eight general practitioners included 3738 patients. conclusions: during lrti in adults, gps observe few focal signs, confirming the marked predominance of bronchitis compared to pneumonia. in view of the frequency of the signs, the diagnosis of pneumonia appears to be overestimated. one-third of clinical situations were diagnosed as 'bacterial superinfection of acute bronchitis', despite it is not a recognised diagnostic entity. antibiotic prescription was immediate in 87% of cases and delayed in 10% of cases. this last point shows that clinical practice differs from the gp's perception of their described prescribing practice shown in a simultaneous survey (56% of gps declared that they prescribed antibiotics immediately, while 41% delayed this prescription). results: thirty-three patients (20 men, mean age 49 b 19 years, mean apache ii score 15.3 &bdqup;b 7.3) during the years 1998 á/ 2001 were prospectively studied. thirteen patients (39%) had no identifiable risk factor for severe cap. an etiologic factor was revealed in 15 patients (45%). in 14 of them this was achieved with noninvasive methods. psb cultures were taken from eight patients and were positive in only 1. the offending organisms included: streptococcus pneumoniae in six cases, gnb as the sole pathogen in six cases, haemophilus influenzae (with s. pneumoniae or klebsialla pneumoniae ) in two cases, s. aureus in two and legionella pneumophila in one patient. initial antibiotic regimen a combination of marcodile '/3rd gen cephalosporin9/aminoglycoside was successful in 12 patients who all survived and had to be changed empirically or according to culture results in 17 patients who had a mortality of 65%. the overall mortality rate was 42%. the identification of the causative factor did not seem to have any impact on survival. conclusion: severe cap in our icu was most often caused by s. pneumoniae and gbn. the high mortality of this entity seems to be influenced by the immediate use of the appropriate antibiotic combination and not by the identification of the causative organism. this underscores the need for knowledge of topical microbiology which helps in designing an effective empirical initial antibiotic regimen. mily mn a , golubev sa b , lugovoy vy a , voronov gg b . a vitebsk emergency hospital, pharmacotherapy unit, vitebsk, belarus , b basic and clinical pharmacology department, vitebsk state medical university, vitebsk, belarus purpose: the study aims were to assess the spectrum and predictors of the antibiotic use during pneumonia management in a regional emergency hospital in belarus. patients, treatment and physicians characteristics of 195 cases (2000 á/2001) were collected and possible associations were examined with using defined daily doses methodology (ddd) and american thoracic society (ats) guidelines. results: the mean treatment duration was 13.99/7.4 days, the total antibiotic ddd/100 bed-days was 130.4. the ddd/100 bed-days of the most used antibiotics were: penicillins 54.0; aminoglycosides 29.5; macrolides 21.9; cephalosporins 9.8; tetracyclines 8.2. in manova certain ats categories were associated with ddd/day, but not with frequency of definite antibiotic use. ddd/day was higher in case of multilobar infiltrates ( conclusion: our study indicated the low rate of macrolides and cephalosporins using, and the high one of aminoglycosides. antibiotic prescriptions were associated with disease severity and physician personality rather then with empirical choice rules recommended. acute exacerbation of copd: most frequent infecting agents and their suspectability to the different types of penicillins. analysis of medical documentation ps243 pertseva to, bogatska ke, gashynova ky. dsma, internal medicine , dniepropetrovsk, ukraine number of copd cases has been increased in ukraine. treatment of acute exacerbation (ae) of copd is not always successful because of inadequate antibiotic therapy. the aim of study was to reveal most frequent infecting agents and their susceptibility to the different types of penicillins in patients with ae of copd. medical documentation of 48 patients with ae of copd (type i) was studied. data of sputum analysis and susceptibility of isolated agents to penicillin, ampicillin, oxacillin and amoxicillin/clavulone acid were evaluated. there were patients with haemophilus influencae/parainfluencae (43.8%), klebsiella pneumoniae (25%), staphylococcus aureus (12.5%), pseudomonas fluorescens , pseudomonas putida , serratia marcescens , serratia liquefaciens (6.8% each), streptococcus agalactiae , acinetobacter baumanii (4.1% each) in samples of sputum. in 31.3% cases there was mixt infection. 6.8% had no any bacterial agents. only 10% of agents were susceptible to penicillin, 35% */to ampicillin, 15% */to oxacillin. however, 70% of microorganisms were susceptible to amoxicillin combined with clavulone acid. this study has shown that most frequent infecting agents caused ae of copd were gram-negative microorganisms and s. aureus . according to antibiogram the prescription of amoxicillin/clavulone acid is most expedient in this case. pertseva to, bogatska ke, konopkina li, kireeva tv, gashynova ky. dsma, internal medicine department, dniepropetrovsk, ukraine we examined 32 patients (22 men, mean age 52.39/4.7 years) with acute exacerbation of cob (type i). the most frequently isolated agents were haemophilus influenzae and parainfluenzae */eight patients, gram-negative rods in 8, staphylococcus aureus in two and mixed in six and one patient had no bacterial agents isolated in their sputum. high susceptibility to azithromycin was found in all cases of gram-positive agents and in h. influenzae and parainfluenzae . other gram-negative agents were resistant to this drug in vitro. however, treatment with 500 mg/day during 3 days was clinically effective in 93.7% of cases. only 25.0% of patients had a further acute exacerbation of cob. there were peculiarities of the infecting agents causing acute exacerbation of cob in this study: klebsiella pneumoniae and s. aureus were found more frequently than in other studies. high efficacy of surnamed in the treatment of acute exacerbation of cob was established in 68.7%. 25.0% had partial positive clinical effect after this therapy. there were no patients with adverse events. efficacy and tolerance of amoxicillin 30 mg/kg bid versus amoxicillin 15 mg/kg tid in the treatment of acute otitis media (aom) in children 5/2 years ps245 borek m a , guggenbichler jp b . a biochemie gmbh, international medical department, kundl, austria , b department of pediatrics, university of erlangen, erlangen, germany five hundred and sixteen patients (mean age 49/2.6 years) with clinical and otoscopic diagnosis of aom were included in a randomized, double blind, multicentre study, and were treated 10 days either with amox 30 mg/kg bid or amox 15 mg/kg tid. assessments were made during therapy (day 3 á/5), after end of therapy (eot, day 12 á/14) and follow up (fu, day 38 á/46). the primary efficacy endpoint was the clinical response at eot defined as success (cure/improvement) or failure. both regimens were well tolerated; one or more drug-related adverse events (aes) were reported in 17.2% (11/64) of bid patients and in 21.7% (13/60) of tid patients. the most frequently reported drugrelated aes in each group were gastrointestinal symptoms (bid 11.5% vs. tid 13.5%), which were mainly of mild or moderate severity. both regimens were clinically equivalent. the higher cure rates in the bid group suggest a possible higher benefit from bid therapy in children 5/ 2 years. children attending family day-care (fdc) should be less exposed to upper respiratory tract infections than those in group day-care (gdc) and therefore to antibiotic treatment; fewer should thus carry resistant bacteria. to test this hypothesis, np carriage of sp and hi with reduced susceptibility to penicillin (pdsp and hi bl'/, respectively) was investigated among children in fdc (maximum three children) and in gdc (25 á/100 children) in the alpes maritimes (france) between november 1999 and march 2000. a two stage cluster sample of children attending gdc or fdc was selected. np samples were cultured for sp and hi. penicillin susceptibility was tested by disk diffusion and e -test, and b-lactamase production by api-nh † tests (biomerieux, lyon). two hundred and thirty-five children in fdc and 298 in gdc aged 6 á/36 months were sampled. age and sex distribution were similar in both groups. sp was isolated in 80 children in fdc (34%), and in 163 (54.7%) children in gdc (p b/10 á/6 ). proportions of pdsp were 52.5 and 55.8%, respectively (p0/0.6). hi was present in 37.6% of children in gdc vs. 23.8% in fdc (p b/ 0.001). proportions of hi bl'/ were 40.2% vs. 46.4%, respectively (p0/0.4). antibiotic exposure during the previous 3 months concerned 46.2% of children in gdc vs. 48.7% in fdc (p 0/0.6). there was no correlation between antibiotic use and carriage of pdsp or hi b'/ strains. sp and hi carriage rates are significantly lower among children in fdc than in gdc. advising alternative types of daycare for children attending gdc should reduce exposure and thus limit the spread of resistant bacteria. however, the proportion of pdsp and hi bl'/ is similar in both groups and comparable patterns of antibiotic use are observed. continued efforts must concentrate on parental education and enforcement of recommendations for management of pediatric upper respiratory tract infections. during a period between january 1999 and august 2001, 43 invasive haemophilus influenzae (hi) isolates had been collected at the children's hospital of tunis. we used haemophilus test medium to test antibiotic susceptibility. the mic of beta-lactams was measured by e -test. beta-lactamase production was determined by using the cefinase test and biotyping by apinh. presence of capsular antigen was determined by using hi typing anti sera. hi strains were isolated from meningitidis (35), bacteremia (5) and arthritis (3). all strains were serotype b and 62.2% of them belonged to biotypes i and ii. amoxicillin resistance with beta-lactamase producing mechanism occured in 34.8%. mic90 of beta-lactamase producing strains was 32 vs 0.5 mg/l in non-producing one. there is no betalactamasenegative amoxicillin resistant among these invasive isolates. antibiotic resistance concerned chloramphenicol: 9.3%, trimethoprim-sulfamethoxazole: 11.6%, tetracycline: 4.6% and kanamycin: 11.6%. invasive hi infections in tunisian children's were always associated with type b strains. introduction of a hib vaccine programme in tunisia is recommended. the aim of this study was to analyse the clinical picture and treatment of neurological manifestations of neuroborreliosis in children. the study included 22 children (2 á/16 years) with neuroborreliosis diagnosed on the basis of the clinical and serologic criteria. symptoms of facial palsy occurred in six children symptoms of iii á/vi cranial nerves palsy in three children, meningitis in four and paresthesias in three. symptoms of v or viii nerve palsy, mental disturbances, radiculoneuritis or cerebellitis were found in singular cases. all children received ceftriaxone intravenously 3 á/4 weeks. total recovery was obtained in 18 children following the first course of therapy. recovery following the second course of therapy (amoxicillin) occurred in one child with mental disorders and one with vi nerve palsy. improvement was achieved after the second course in the patient with radiculitis, however, muscular atrophy persisted. irreversible, unilateral deafness was found in a child with viii nerve palsy in spite of three courses of therapy applied. infection with borrelia burgdorferi in children causes a wide spectrum of neurological manifestations. facial palsy was the most common sign in our study. applying ceftriaxone in the treatment of neuroborreliosis is characterised by a good effectiveness. double infection by c. pneumoniae and m. pneumoniae as a cause of cystic changes in the lungs ps249 streharova a, moravcikova d. department of infectious diseases, university of trnava, trnava, slovakia chlamydia pneumoniae and mycoplasma pneumoniae are human respiratory pathogens manifested in early childhood. immunological disbalance could trigger autoaggressive diseases. the authors describe the case of a15-year-old girl with development of multiorgan failure and septic state, which followed multiple cystic changes in the lungs. the girl did not have a diagnosis of cystic fibrosis. the authors consider that cystic changes are a consequence of double infection by c. and m. pneumoniae. dzarlieva m, momeva l, temelkovska g, balevska p, pejkovska m. medical centre, neonatology, bitola, the former yugoslav republic, macedonia unicef skopje has supported a nationwide safe motherhood needs assessment in representative samples of hospitals. eighteen of 28 maternity wards and facilities renovated and certified as 'babyfriendly'. all mature newborns with successful adaptation to extra uterine life and satisfactory vital parameters are 24 h during the day with their mothers at rooming-in system. aim: with rooming-in system we reached decreasing of incidence of neonatal infections. material and methods: history records of newborns from our department. for the period of 9 months, 907 neonates have been borne. with suspection of infection there */49 babies (5.4%). newborns borne through meconium stained liquid */40 (4.4%). results: microbiological findings: from blood culture */staphylococcus coagulaza negative from swabs */staphylococcus aureus , escherichia coli , staphylococcus epidermidis. conclusion: in 1999, from all babies who had risk factors for infection in 56 newborns (5%) we had positive findings and in year 2000 (before rooming-in sistem), in 44 (3.9%). after that period (with rooming-in) 15 newborns (1.5%). with rooming-in system we reached decreasing in incidence of neonatal infections by breaking the chain of infection */only mothers take care of their babies with help of the staff. newborns are in their micro environment, the same they will have at their home. with this practice we have also reduction of nosocomial infections. a study on antibiotic susceptibility and resistance factor transmissibility among antibiotic resistant salmonellae isolated from children affected to diarrhea ps251 sharifzadeh a. azad university of shahrekord, microbiology, shahrekord, islamic republic of iran in spite of happened drug resistance, antibacterial therapy still is the best route of treatment of salmonellosis in man and animals. in order to detection of dominants serotypes of salmonellae in children and detection of antibiotic susceptibility and r-factor transmissibility among those isolated salmonellae. this study was conducted on 400 diarrheic stool samples were collected from children affected by diarrhea in ayatollah kashany hospital of shahrekord, during spring of 1999 to autumn of 2000. after isolation and identification of salmonellae, seven serotypes were detected. one of those was s. typhi and another six serotypes were s. paratyphi b . in order to detection of antibiotic different antibiotic disks were used in disk diffusion method. best results were taken from ceftizoxim, cephtriaxon, cephazolin and chloramphenichol. the r-factor were transferred from isolated salmonellae to escherichia coli k12 in all of cases of resistance to penicillin and ampicillin. pharyngeal colonization by streptococcus pneumoniae and group a bhs were evaluated in 652 randomly selected school children aged 6 á/ 8 years in riyadh, saudi arabia. fifty-six children (8.6%) had positive culture for either organisms of the 56 isolates from school children, 17 (30%) were s. pneumoniae , of them 14 (82%) were penicillin-sensitive, three (18%) were penicillin-resistant, and two (11.7%) were resistant to two antimicrobials. forty isolates of bhs (71%) were group a bhs. all isolates were penicillin and erythromycin sensitive. the carrier rate among school children for penicillin-resistance s. pneumoniae and resistance to two antimicrobials were (4.6%) and (3%), respectively. the carrier rate of group a bhs was (6.1%). riyadh has a low rate of antibiotic-resistant s. pneumoniae and a similar rate of group a bhs carriers among school children as that seen in temperate areas. boukadida j a , boukadida n b , hannechi n a , said h b , erraii s a , elmhabrech h a . a chu f. hached, laboratoire de microbiologie, sousse, tunisia , b c s base, sousse, tunisia the acute pharyngitis is a very frequent pathology in which group a streptococcus is the most incriminated bacteria. however, other non a b-haemolytic streptococcus (sbna) could be responsible. the aim of this work is to determine the part of each non a b hemolytic streptococci (sbna) in acute pharyngitis and the related antibiotics susceptibility pattern. the study was realized in sousse-tunisia (north africa) during 5 months from may 2001. the origin materials of isolates are throat swab (transystem venturi, copan, bovezzo). the mean age of patients is 23 years with extremes 3 á/72 years. the samples are cultured on blood agar plates in a delay of 3 h maximum. identification was done to samples that have over than 20 b-hemolytic colonies, groupage with pastorex strep. sanofi pasteur france, susceptibility pattern according to nccls norms, mic is determined by e -test. the control strain is s. pneumoniae atcc 49619. twentyone clinical isolates of sbna are distinguished from 80 clinical isolates of b-hemolytic streptococci recovered from 143 patients with acute pharyngitis without symptoms of viruses' infections (tearing, corysa, sneeze). all b-hemolytic streptococcus represents 55.94% of all collected samples. sbna were 26.25% of the isolates. sbna were 11 strains group g streptococci, seven strains group c streptococci and three strains group f streptococci. susceptibility pattern of each sbna to antimicrobial agents is as follow: group g streptococci: peni g 0/100%, amoxicillin 0/100%, pristinamycin 100%, clindamycin and erythromycin0/45.46%, tetracyclin 0/9.1%, telithromycin 63.63% and levofloxacin0/45.45%. group c streptococci: peni g0/ 100%, amoxicillin 0/100%, pristinamycin 100%, clindamycin0/ 57.15%, erythromycin0/42.86%, tetracyclin0/71.42%, tel-ithromycin0/85.72%, levofloxacin0/100%. group f streptococci-peni g0/100%, amoxicillin0/100%, clindamycin0/66.66%, erythro0/33.34%, tetracyclin 0/66.66%, telithromycin 0/100%, levofloxacin0/66.66%. all sbna have mic to penicillin g under 0.1 mg/l. according to available data, penicillin g and amoxicillin still the reference treatment of acute bacterial pharyngitis in spite of the new antibiotics introduction. berezin en a , quevedo sg b , nicolla l b , viegas d c , eizenberg b d , pedrosa f b , santos ag a . a santa casa, pediatrics, s. paulo, brazil , b elly lilly, scientific, s. paulo, brazil , c fac. abc, pediatrics, santo andre, brazil , d university hospital, pediatrics, s. paulo, brazil a multicenter, open label, prospective, randomized trial in which patients 2 á/16 years of age with proven gabhs pharyngitis were randomized to receive either 10-day course of the broad spectrum oral cephalosporin, cefaclor or a 10-day course of amoxicillin. patients were included if they have signs and symptoms of streptococcal tonsillopharyngitis and a rapid streptococcal rapid test positive . patients were evaluated at days 3 á/5, 13 á/16 and 24 á/35 posttherapy. pharyngeal cultures were conducted at baseline and at follow-up visit (13 á/16 days). we considered for bacteriologic eradication analysis only patients with positive culture to gabhs. there were 148 patients with a rapid streptococcal rapid test. clinical success were achieved in around 95% of the patients. for evaluate eradication of the initial pathogen we considered 42 patients of the amoxil arm and 40 patients of the cefaclor arm. thirty-four of 42 patients of the amoxil arm (80.9%) and 36 of 40 (90%) patients of the cefaclor arm were considered bacteriological cured at the second culture performed at day 13 á/16. ten days of a penicillin or amoxicillin therapy may not be the best therapeutic choice for all pediatric patients. in developing countries where rheumatic fever is still an important problem to evaluate the bacterial eradication achieved with the different antibiotics may be important. prophylactic affect of saccharomyces boulardii for antibiotic-associated diarrhea in a paediatric age group ps256 erdeve o, tiras u, camurdan mo, tanyer g, dallar y. ankara education and research hospital, pediatrics, ankara, turkey the process resulting in antibiotic associated diarrhea is the alteration of enteric flora due to bacteria. saccharomyces boulardii is a yeast, isolated from cover of a kind of hazelnut and its usage became widespread recently. there is no enough study about s. boulardii activity at pediatric ages. we aimed to define s. boulardii activity on azithromycin and sulbactam-ampiciline, and associated diarrhea at pediatric age group. the 18.9% of cases only with antibiotic usage developed diarrhea, whereas rate was 5.7% for probiotic using group (p b/0.05). all of the clostridium difficile toxin a defined cases were from sulbactam-ampiciline using group. the rate of diarrhea for sulbactam-ampiciline using group was 25.6%, while it was 5.9% for group used s. boulardii as probiotic beside sulbactam-ampiciline (p b/ 0.05). it was observed that probiotic usage decreases diarrhea rate four times (p b/0. 05). when age groups considered, the rate of sulbactamampiciline associated diarrhea increased at 1 á/5 ages and s. boulardii effect on preventing diarrhea was significant at 1 á/12 ages (p b/0.05). antibiotic associated diarrhea is a common clinical problem at pediatric age group. s. boulardii is a hope giving probiotic especially for sulbactam-ampiciline associated diarrhea. grey e a bilikova e a , hafed bm a , kovacicova g a , chovancova d b , huttova m b , krcmery v a . a school of health, trnava university, trnava, slovakia , b postgraduate academy of medicine, neonatal clinic, bratislava, slovakia the purpose of the study: the aim of the study was to find out, whether artificial abortion of a mother has impact on neonatal infection of her future baby. the results obtained: therefore, we compared 39 neonates with infection, who were born to mothers with past history of abortion (1 á/7 artificial abortions within 10 years), with the babies of mothers, who have not experienced abortion before. control group consisted of 207 neonates hospitalized at the same clinic, at the same time period. according to the analysis of risk factors for neonatal infections, it was found, that prematurity (28 á/32 weeks of gestation) (30.77 vs. 14.01%, p 0.019) and low birth weight-1500 á/2500 g (58.97 vs. 39.13%, p 0.03) were significantly more frequently observed in babies of mothers with past history of abortion. drug abuse (heroin) (12.82 vs. 1.45%, p 0.003) and nicotine use (15.38 vs. 2.42%, p 0.0027), were more significantly related to neonatal infections in babies of mothers with past history of abortion. etiological analysis showed that only candida albicans (5.13 vs. 0%, p 0.024) was significantly related to neonatal infections in mothers with past history of abortion. the conclusion reached: in conclusion, artificial abortion has not only direct impact to the health of the mother, but also on her next pregnancy. bacteraemia and patient mortality in a peadiatric intensive care unit ps258 armenian sh, singh j, arrieta ac. children's hospital of orange county, infectious disease, orange, usa purpose: this study will identify the factors that significantly contribute to mortality in patients with bloodstream infections (bsi) at a pediatric intensive care unit (picu). results: medical records of 63 patients who were admitted to the picu and had a documented bsi were reviewed. there were 74 separate episodes of bsi's, with nine patients having multiple bsi's during their hospital stay. a casecontrol model was used. cases were bsi's with eventual mortality (n0/27; 36.5%) and controls were those who survived bsi (n0/47; 63.5%). patients who died were older (5.7 vs. 2.4 years; pb/ 0.01), more likely to have a nosocomial bsi (42.9 vs. 16.7%; pb/ 0.05), longer hospitalization prior to bsi (30.5 vs. 9.8 days; pb/ 0.05), and have a polymicrobial bsi (66.7 vs. 30.6%; pb/ 0.05). infection related mortality (irm)-defined as death within 7 days of bsi */was significantly higher in those receiving inadequate antibiotic treatment at the time of diagnosis of bsi (54.5 vs. 12.7%; p b/ 0.01), as well as in those with gram negative bacteremia and/or fungemia (35 vs. 13%; p b/ 0.05). logistic regression was used to adjust for potential confounding variables. conclusions: we found that being older, multiple organisms, and a longer hospitalization prior to the bsi were significantly associated with overall patient mortality. irm was significantly higher for those with inadequate initial antibiotic coverage and in those with gram negative bacteremia and/or fungemia. somer a a , gü r d a , diri s a , yalcýn i a , salman n a , ongen b b , gü rler n b . a department of pediatric infectious diseases, istanbul university istanbul medical faculty, istanbul, turkey , b department of microbiology and clinical microbiology, istanbul university istanbul medical faculty, istanbul, turkey teicoplanin is a glycopeptide antibiotic active against a broad range of gram-positive pathogens including methicillin-resistant staphylococci and offers the advantages of once daily administration, choice of administration route, lack of requirement for routine therapeutic drug monitoring and lower propensity to cause nephrotoxiticy and anaphylactoid-like reactions. in this study the efficacy and safety of teicoplanin were evaluated retrospectively in children with serious bacterial infection. sixty-three children (18 girls, 45 boys) aged between 1 month and 15 years were treated with teicoplanin (three loading dosages of 10 mg/kg at 12 h intervals, followed by a maintenance dosage of 10 mg/kg/day). the infections treated were pleural empyema (n0/25), joint and bone infections (n 0/22), septicemia (n0/9), skin and soft tissue infections (n0/4), and lung abscess (n0/3). the pathogens isolated were staphylococcus aureus (n 0/30, 16 of which were methicillin resistant), coagulase-negative staphylococci (n0/4), s. pneumoniae (n0/4), and group a hemolytic streptococci (n 0/2). the duration of therapy ranged from 14 to 81 days (median 28 days). clinical success (cure plus improvement) was achieved in 96.8% of cases. no side effects attributable to teicoplanin therapy were encountered. teicoplanin appears to be an effective and well-tolerated treatment for serious gram-positive infections in children. the risk of infection is present in all children with acute leukemia. two hundred and sixty episodes of febrile neutropenia were analyzed in 149 children (aged 1.5 á/16 years) with aml */43 cases and all */ 106 cases over 10 years during cytostatic therapy (protocols: mbfm */ 87 aml and mbfm */90 all). a degree of fn occurred in 90% of cases in aml, in 53% */in all. the sites of infection were: blood'/ central venous catheter (32%) and respiratory tract (49%). pathogens isolated from blood were: gram-positive */cns */70%, streptococcus spp. */18.7%, gram-negative */enterobacteria */41%, pseudomonas aeruginosa */23%, candida spp. */23.5%, aspergillus spp. */5.9%, other */5.9%. for the treatment of fn we used empirical antibiotics regimens. clinical response was noticed: in 1st line of therapy */ cephalosporins 3 á/4 generation used */45%, carbapenems used */60% as 2nd á/3rd lines */vancomycin */70%, amphotericin b */80%. thirtytwo percent of children with aml and 5.2% children with all died because of sepsis. conclusion: carbapenems are more active in the 1st line of antibiotics therapy both for patients with aml and all. vancomycin is useful in the 2nd line for patients with all. amphotericin b and vancomycin are useful in combination in the 2nd line for patients with aml. pavlenishvili ivp. medical academy, pediatrics, tbilisi, georgia our drug of choice in cases of complicated neonatal sepsis i. pavlenishvili, g, iobashvili tbilisi, medical academy georgian society of pediatrics chemotherapy (gspc) with collaborations of drug and therapeutic committee (dtc) of childers hospital 'republic' finished the observation study about nosocomial infections of neonatal icu in above mentioned hospital. study begins from november 2000. during this period we observe 45 cases of neonatal sepsis, most of them were due gram-negative bacteria. thirty-four cases of neonatal sepsis were cause different stains of escherichia coli , proteus spp. and acinetobacter . we notice that during last 2 years the role of acinetobacter in etiology of neonatal sepsis and nosocomial complications of neonatal sepsis in our hospital is gradually increased. as our observation reveals most optimal in the case of complicated neonatal sepsis was (according criteria of dts) use of ceftasidime. almost in all cases we used ceftasidime with success. we have only one case of mortality. since the macrolide resistance (mr) in streptococcus pyogenes has been increasing in europe, we studied the incidence and genetic basis of mr in s. pyogenes in russia. s. pyogenes isolated at baseline and during follow-up visits from children with acute pharyngitis receiving penicillin or midecamycin (16-membered macrolide) were studied. mr was evaluated by agar dilution (nccls), resistance mechanisms */by pcr. a total of 149 s. pyogenes were obtained at the baseline, 21 (14%) of which were erythromycin-resistant: 19 strains (90%) were erm (a)-positive, one */mef (a)-positive, and one was negative for all primers used. all erm (a)-strains were inducibly resistant to clindamycin and represented seven pfge profiles with one profile found in 11/ 19 strains. in three midecamycin treated patients mr was selected during the therapy (one strain had erm (a), one */mef (a), one */ unknown resistance determinant). mef (a)-strain obtained during follow-up visit had different pfge pattern with mef (a) strain isolated at baseline, while both the baseline and follow-up strains with unknown mechanism of resistance had unique pfge pattern. these data showed moderate incidence of erythromycin-resistant s. pyogenes in smolensk. ribosomal methylation (erm (a)) was the most common mechanism and though the polyclonal nature of mr was established, most erm (a)-strains belonged to only a few clones. bacillus cereus infections in traumatology-orthopaedy department: retrospective study and re-evaluation of healthcare practices ps263 bacillus cereus was cultured for 30 patients from traumatology department who had developed postoperative wound infections between august 1997 and march 2000. all patients presented inferior members open fractures, frequently contaminated with telluric material and requiring external fixators. genomic study of clinical isolates by pulsed field gel electrophoresis and random analysis polymorphic dna, allowed us to eliminate an outbreak. furthermore, the reduced delay in which patients developed the infection (6 days'//-2) led us to re-evaluate the procotols used in our institution. indeed, all patients had received amoxycillin '/ clavulanate iv 2g for antibioprophylaxis during anesthetical induction then relayed per os for 48 hours. because of the production of a potent beta-lactamase by the bacteria, this association could not be efficient. furthermore, accordingly to the afnor en 1040 norm, we have tested clinical isolates' sensitivity to the principal antiseptics used for antisepsis and disinfection (iodophors, chloride derivated and biguanidines) and observed a major resistance of all strains tested. even if these postoperative wound infections are considered as nosocomial because of the delay in appearance, we actually think that bacillus cereus were initially present in telluric material. this fact led us to propose a systematic screening for bacillus at admission for this type of wound and to administrate quinolones such as ciprofloxacin for prophylaxis. internal thiols and reactive oxygen species in the candidacidal activity exerted by a n-terminal peptide of human lactoferrin ps 264 the purpose of the study: the emergence of candida albicans strains resistant to current antifungals points to the need for new antifungal agents, e. g. antimicrobial peptides. the results obtained : we report that hlf(1-11), a synthetic nterminal peptide of human lactoferrin, displays excellent killing effect against fluconazole-resistant c. albicans and that sub-optimal concentrations of this peptide combined with fluconazole act synergistically. previous investigations revealed that hlf(1-11) required an energized mitochondrion, atp release by candida , and ligation of atp receptors for its killing effect. we now report that reactive oxygen species (ros) are involved in the killing effect of hlf(1-11). since internal thiols protect cells from oxidative damage, our observation that hlf(1-11) caused a 20% reduction of internal thiols in candida is of interest. as expected, n-acetyl-l-cysteine (nac), which is a precursor of glutathione and a ros scavenger, inhibited the killing effect of hlf(1-11). diamide, which oxidizes internal thiols, was candidacidal and hlf(1-11) and diamide acted synergistically in killing c. albicans and ros production. moreover, the hlf(1-11)-induced activation of mitochondria was inhibited by nac, indicating that internal thiols/ros affect mitochondrial activity. the conclusion reached : the candidacidal activity of hlf(1-11) involves ros production and reduction of internal thiols. objective : an audit was conducted to explore the observation of increasing resistance to ciprofloxacin in enterobacteriacea isolated from specimens from such patients in the hematology unit. results : enterobacteriacea isolates in specimens from such patients processed over the years 1997 to 2001 were examined. number of specimens per year was */109, 152, 132, 160 and 170 respectively and the annual percent ciprofloxacin resistant enterobacteriacea from these was 2%, 9%, 12%, 14% and 21%. conclusion : conclusion drawn from the audit was that rapid rise in ciprofloxacin resistance may possibly be attributed to the use of this fluoroquinolone as a single agent in dose of 250/500 mg [cut off patient weight 40 kg] twice a day in neutropenic patients till neutrophils exceed 500/c.mm. subsequent to the audit, prophylaxis protocol was modified to use of oral colistin 3-mega units/twice a day in combination with ciprofloxacin 250/500 mg twice a day. a prospective audit is proposed to test the benefits of this combination. rhinocerebral zygomycosis: diagnostic dilemma for emergency physician: can the associated morbidity and mortality in this rare but deadly disease be reduced? ps 266 samavedam s, guleri as. western infirmary, clinical microbiology, glasgow, united kingdom rhinocerebral zygomycosis [rcz] is a rare, invasive, rapidly progressive opportunistic infection caused by ubiquitous fungi of the order mucorales. it usually occurs in diabetics or immunocompromised patients. the emergency physician will typically see patients with rcz in its earliest stages masquerading as a variety of other less serious diseases. the key markers like necrotic patch on hard palate, nasal septum or turbinate, marked facial pain, and cellulites with marked eye and neurological signs may present late in disease. we report a case of rcz caused by rhizopus arrhizus [oryzae] in an 84 year old woman with poorly controlled diabetes. she presented with a right-sided facial droop of short origin and being generally unwell. ct scan was non-conclusive and delayed presentation of key markers of rcz permitted disease to rapidly progress. despite an intensive antifungal therapy with ambisome and insulin sliding scale, patient rapidly succumbed within 4 days. fine needle aspiration cytology is less invasive, easier and equally effective alternative to pre op biopsy. the key to successful reduction in morbidity and mortality associated with this rapidly fatal disease is -increasing awareness of the disease, an early diagnosis, correction of underlying metabolic derangement, prompt intensive antifungal therapy with amphotericin b and radical surgical debridement of the necrotic tissue. an 'optimal dosage' of ambisome requires discussion. clinical audit in the haematology ward of a tertiary care hospital: study of degree of correlation between bacteraemia and oro-pharyngeal screens in immunocompromised patients over five years and role of antibiotic prophylaxis ps 267 guleri as, butcher i. western infirmary, clinical microbiology, glasgow, united kingdom introduction : a clinical audit was carried out over 5-years [1997] [1998] [1999] [2000] [2001] in the immunocompromised patients including neutropenic patients and bone marrow [autograft] transplant recipients in hematology ward of gartnaval general hospital, glasgow, a tertiary care center. objective : it was aimed to establish the degree of correlation between bacterial isolates in oro-pharyngeal screen during bacteraemia episodes and role of antibiotic prophylaxis. methods purpose : to assess whether antiretroviral therapy (art) intensification, gm-csf use and remune initiation before stopping art lead to viremia containment, and long periods off art. methods : ten adults with chronic hiv disease, hiv-1 rna levels (vl) b/1.7 log copies/ml and median cd4'/ -t cell count of 385/ml were enrolled. after art intensification with ddi (6 months) '/ hydroxyurea [hu](5 mo.) '/ gm-csf (3 mo.) and a remune dose, art was stopped but remune continued. art was resumed if rebound vl did not decrease to b/4.7 log in 3 months or if cd4'/ counts decreased to b/200. results : vl rebounded in all patients after stopping art, and 7 developed an acute retroviral syndrome (ars). cd4'/-t cells decreased, and cd8'/38'/ increased (5-fold). after a median stoppage of 16 weeks, art was resumed in 9 patients and vl decreased to b/ 1.7 log, cd4'/ counts were regained, il-2 and il-15 levels rose. at the 2nd interruption, 9/9 patients had a rebound, and 3/9 had a 2nd ars, but peak vl and loss of cd4'/ were lower (p 0/0.003). after 21.3 weeks off art, 8 patients resumed therapy. the breadth and magnitude of hiv-specific activity increased and thymus size grew. the patients were off art for a median of 49.5 out of 80 weeks. two of them are off art for 80 and 44 weeks respectively ( vlb/4 log). conclusions : this approach led to a high ars incidence, long periods off art, increases in hiv-specific responses, il-2 and il-15 levels, and thymus size. urinary tract infection in hospitalized population is a significant problem. this is a study of catherized patients urinary infection in corfu hospital. in 2000 á/2001, 3500 urine cultures were sent to our bacteriology laboratory. 650 of them were collected from the catheters. clinical data of age and type of disease were analysed. the samples were cultured on mc conkey agar-urotube and vitek cards. the organisms identified with vitek automatic system. the sensitivity was tested with vitek and kirby-bauer method. results: no growth of organisms in 46.5%. positive cultures 35%. 62.2% of the bacteria were gram((/) rods (45.6% e. coli , pseudomonas spp. 0%), 16% were gram('/) cocci (enterococcus spp.) and 22% was candida spp. the resistance of e. coli was: 37% to ampicillin , 17% to co-trimoxazol and 2% to quinolons. e. faecalis was resistant 10% to vancomycin . conclusions : (1) the possible interference of acetaminophen in the amoxicillin/ clavulanic acid (a/c) or erythromycin (ery) efficacy in the treatment of acute otitis media (aom), and its possible role in the evolution to otitis media with effusion (ome), were determined in a gerbil model. a 23f streptococcus pneumoniae strain exhibiting a mic of a/c and ery of 1/0.5 and 0.12 mg/l, respectively, was used. both antibiotics were tested at 2.5 and 10 mg/kg. acetaminophen at 50 mg/kg was administered 30 min before each antibiotic dose. antibiotic concentrations in serum and middle ear exudate were determined. both antibiotics significantly reduced the number of culture-positive ears and colony counts, with serum concentrations over the mic of the microorganism for ]/15% of the dosing interval. antibiotic concentrations in middle ear exudate were almost identical in animals receiving and not receiving acetaminophen. clinical and microbiological efficacy was correlated with antibiotic concentrations in middle ear exudate ]/1.7 times the mic of the microorganism, for both antibiotics. both antibiotics demonstrated efficacy in the treatment of pneumococcal aom, with the same rate of ome. acetaminophen, concomitantly administered, did not interfere the efficacy of the two antibiotics tested and did not prevent the evolution of aom to ome. parra a a , ponte c a , cenjor c b , garcía-olmos m a , giménez mj c , aguilar l c , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b fundación jiménez díaz, otorrinolaryngology, madrid, spain , c glaxosmithkline, medical department, madrid, spain a gerbil model of otitis media with effusion (ome) induced by haemophilus influenzae (amoxicillin/clavulanate-a/c-and erythromycin-ery-mics of 1/0.5 and 4 mg/l, respectively) was used to evaluate the efficacy of a/c (10/2 and 15/3 mg/kg) and ery (20 and 50 mg/kg). antibiotics were administered subcutaneously 2 h post-middle ear inoculation, and continued t.i.d for 24 h, with or without acetaminophen (ap), at 50 mg/kg, administered 30 min before each antibiotic dose. antibiotic concentrations in serum and middle ear (me) were measured by bioassay. me samples for colony counting were collected on day 2. a/c reduced (p 5/0.05) positive me samples and colony counts versus untreated controls or ery: me positive cultures of 90% for controls, 0% for a/c 15, 7% for a/c 10, 17% for a/c 10'/ap, 90% for ery 20, 57% for ery 50 and 87% for ery 50'/ap. this was due to a/c (but not ery) concentrations in me exceeding 1.8 times the mic despite the higher percentage of antibiotic penetration of ery versus a/c (43 versus 8/14%). animals receiving ap showed less polymorphonuclear cells and more bacteria in me than those receiving only antibiotics, suggesting that the anti-inflamatory drug diminish the phagocytes and therefore, the efficiency in bacterial clearance. amoxycillin treatment for acute otitis media caused by penicillinresistant streptococcus pneumoniae . a pharmacodynamic analysis pm103 parra a a , ponte c a , cenjor c b , garcía-calvo g a , giménez mj c , aguilar l c , soriano methods: a serotype 23f streptococcus pneumoniae strain exhibiting a mic of amoxycillin of 1 mg/l was used in an experimental model performed in gerbils (meriones unguiculatus ) following previously described procedures. amoxycillin was tested at the following doses: 0.2, 0.4, 0.8, 1.25, 2.5 and 5 mg/kg. amoxycillin concentrations in serum and middle ear exudate were determined after drug administration. results: doses of ]/2.5 mg/kg significantly reduced the number of culture-positive ears, colony counts and otorrhoea (p 5/0.05) as compared with untreated controls or animals treated with doses lower than 1.25 mg/kg. doses of ]/2.5 mg/kg achieved antibiotic concentrations in the middle ear 1.4 á/2.4 times higher than the mic of the infecting strain and serum concentrations over the mic for 14 á/19% of the dosing interval. conclusions: amoxycillin at doses achieving serum concentrations similar to those obtained in children after standard doses, obtained therapeutic and microbiological efficacy regardless the susceptibility of the infecting strain. better correlation was found between antibiotic efficacy and antibiotic concentrations in middle ear exudate than between efficacy and serum concentrations, which were suboptimal from the pharmacodynamic perspective. increasing prevalence of amoxycillin á/clavulanate-resistance among e. coli strains in a hungarian university hospital pm104 veréb i, vígh a, urbán e, hajdú e, nagy e. department of clinical microbiology, university of szeged, szeged, hungary background: amoxicillin á/clavulanate resistance (acr) is an emerging problem in escherichia coli as reported from different parts of europe. the aims of the present study were to evaluate statistically the prevalence of acr among e. coli isolates and to investigate the genetic background of the resistance. methods: all e. coli strains isolated between 2000 1 1 and 2001 9 1 were screened for acr by kirby á/bauer disc diffusion method. the resistance to other beta-lactam á/beta-lactamase inhibitor combinations and to different beta-lactam antibiotics were also tested. selected strains underwent determination of beta-lactamase activity. confirmatory tests for suspected extended spectrum beta-lactamase were performed. pcr testing for tem and shv genes were carried out on plasmids isolated from selected strains. results: in 2000 out of 1109 e. coli strains 102 (9.2%) were found to be resistant to amoxicillin clavulanate (amc). most of the resistant strains (95%) were obtained from the genitourinary tract and no acr isolate was found in blood cultures. in 2001 out of 2167 isolates 239 (11%) proved to be acr and 2.5% were isolated from blood cultures and 66.9% from the genitourinary tract. thirty-five selected strains were further analysed. thirty-two were also resistant to (sam) and six were further resistant to tzp. quantitative beta-lactamase determination showed increased activity in strains which were partially susceptible to amc. the presence of esbl could be proved only in three acr isolates. this open parallel-group study compared the efficacy and tolerability of cef with cfix 400 mg once daily in the treatment of community acquired uncomplicated uti. seventy-eight female patients were randomized to receive either oral cef or cfix for 5 or 10 days. the efficacy of treatment was evaluated by clinical response (by symptoms of uti dysuria frequency urgency suprapubian pain and by clinical signs) by bacteriologic response and health status measures at baseline and posttherapy. results: the clinical cure (complete resolution of symptoms and signs) rate for patients receiving cef was 95.12% of the 41 evaluable patients and 89.18% of the 37 patients receiving cfix. bacteriologic response (based on the results of urine cultures obtained posttherapy) the pathogen was eradicated in 82.3% for cef 73.3% for cfix. no drug related side effects have been reported in cef and side effects were experienced by 5.40% of the patients receiving cfix. improvement in health status comparing visual scale scores baseline and poststudy to have detected a higher change in average score from 17 to 94 in cef, from 20 to 82 in cfix. wilcoxon improvement value was significant on the 3rd day of therapy in case of cef and on the 5th day of therapy in cfix group. in conclusion, the results of this study indicate that cef course is more effective than cfix in producing a favourable clinical outcome and achieving higher bacteriologic eradication rate, furthermore cef was better tolerated. cefepime is a fourth generation cephalosporine that has a broader spectrum of antibacterial activity than the third generation cepfalosporines and is more active in vitro against gram-positive aerobic bacteria. the purpose of this study was to measure cefepime concentrations in plasma, bile fluid and gall bladder tissue in patients undergoing cholecystectomy. thirty patients 12 male, 18 female, mean age: 48 years had data acceptable for analysis and were included in this study. all patients received iv 2 g of cefepime. several hours after administration and at different time intervals, during surgery, samples were obtained from plasma, bile fluid and gall bladder tissue concomitantly. antibiotic levels were measured by an agar diffusion method. the mean delta time was 2849/255 min. the values for plasma, bile fluid and gall bladder tissue, were 33.69/28.5, 6.69/4.3 and 16.19/12.9 mg/ml, respectively. the plasma/bile fluid ratio was 2.69/ 2.4. there was a significant correlation between plasma and gall bladder tissue concentration (r0/0.771, p0/ b/0.0001). a correlation between bile fluid and plasma cefepime concentration was not observed. the minimum inhibitory concentration (mic) data from previous in vitro studies indicate that the cefepime concentration observed in plasma bile and tissue samples of this study would be adequate against typical biliary tract pathogens. furthermore, these cefepime concentrations correlated well with the favorable clinical outcome reported in previous clinical studies in biliary tract infections. there was also good correlation between delta time and plasma and tissue concentrations and if the dose were given closer to the time of surgery, cefepime concentration would be higher reducing the possibility of an infection. objectives: the use of antibiotics may lead to decreased colonization resistance and increased formation of resistant bacteria. present concept was developed to overcome these untoward effects. methods: b-lactamase of bacillus licheniformis was overproduced in bacillus subtilis . this targeted recombinant b-lactamase enzyme (trbl) was released in the small bowel from a controlled-release formulation. beagles (n0/6) were treated bid with either 20 mg/kg ampicillin (i.v.)'/placebo (p.o.), 20 mg/kg ampicillin (i.v.)'/trbl (p.o.) or only placebo (i.v.'/p.o.). stool was collected at days 4 and 10. samples were cultured for total and main groups of aerobic and anaerobic bacteria and yeast. temperature gradient gel electrophoresis (tgge) was used to separate the ribosomal rna genes. results: ampicillin'/placebo group had clearly decreased counts of both aerobic and anaerobic bacteria during the treatment, whereas those receiving trbl had only minor overall changes and some occasional changes by single species. intravenous ampicillin decreased the fecal similarity percentage to 60%. the similarity percentage during treatment with ampicillin'/trbl did not differ from that of placebo (86 vs. 81%). conclusions: according to our results the trbl can maintain the large intestinal microflora almost unchanged. these results indicate that trbl is a promising novel approach for overcoming the ecological adverse effects on gut flora caused by b-lactam antibiotic agents. adamis since broad-spectrum â-lactams combined with amikacin are often applied for nosocomial infections, their pharmacokinetic interactions might be interesting. one gram of aztreonam and 0.5 g of amikacin were administered intravenously single and in combination in six healthy volunteers. blood samples were collected at regular time intervals and concentrations of antimicrobials were determined by a microbiological assay applying a strain developing resistance to single agent after serial passages. mean concentrations of amikacin in serum when administered alone and in combination with aztreonam were 26.2 and 20.3, 11.8 and 12.3, 8.4 and 12.2, 6 .3 and 9.7, 1.8 and 4.3 and 0 and 1.3 mg/ml immediately after and 0.5, 1, 2, 4 and 8 h after infusion of antimicrobials. respective concentrations of aztreonam were 63.5 and 25.3, 27.5 and 21.6, 24.2 and 10.5, 19.5 and 13.6, 5.8 and 4.4 and 1.8 and 4 .0 mg/ml. aucs for amikacin when administered alone and in combination with aztreonam were 35.59/10.9 and 50.59/7.8 mg h/l, respectively. respective auc for aztreonam were 99.79/35.2 and 76.49/31.5 mg h/l. it is concluded that the co-administration of aztreonam and amikacin results in earlier clearance of aztreonam and in higher levels of amikacin compared to the administration of each single antimicrobial. molecular modelling of b-lactams reveals the structural basis for their inhibition of penicillin-binding proteins, susceptibility to b-lactamases and oral bioavailability pm110 grail bm, gupta s, payne jw. school of biological sciences, university of wales, bangor, uk b-lactam antibiotics are peptide mimetics that act as suicide substrates for transpeptidase enzymes that cross link bacterial cellwall peptides. for the first time, the structural and electronic features needed for their recognition by transpeptidase have been fully described, using innovative molecular modelling techniques to compare the conformational forms adopted by cell-wall peptides and blactams. comparison of features in the backbone and c-terminal regions of conformers of active b-lactam antibiotics and model cellwall peptides, has allowed definition of the molecular recognition template required for substrate recognition by transpeptidase. these shared structural features allow both to act as substrates and to acylate the active-site serine. however, a significant difference in a critical backbone torsion between the two substrates, provides an explanation for the inability of the enzyme á/antibiotic complex to undergo the deacylation step that causes inhibition of transpeptidase. on the other hand, b-lactamases appear to have evolved molecular mechanisms that facilitate the deacylation reaction through compensating for the altered structural orientations in b-lactams caused by the different backbone torsion. finally, analysis of the conformer repertoires of blactams for structural features required for substrate uptake by peptide transporters, provides insights into how their structures can be tailored for optimal oral absorption. antimicrobial susceptibility of proteus mirabilis clinical isolates producing extended spectrum beta-lactamases (esbls) pm111 objectives: the aim of the present study was to determine in vitro susceptibility to antimicrobials of proteus mirabilis isolated from urinary tract infections. methods: we studied the susceptibility profile of esbl positive p. mirabilis strains in three adopted children from india with age range from 20 months to 2 years. esbl was identified using the synergic effect of clavulanate with betalactams (ceftazidime and cefotaxime the in vivo efficacy of amoxicillin (amx) sub-therapeutic doses (3.12 mg/kg, t.i.d for 48 h, achieving serum levels over the mic of only 3% of the dosing interval) and concomitant specific serotherapy (single intraperitoneal dose of 1/4 diluted hyperimmune serum (hs) obtained from mice immunized with the heat-inactivated strain) was assessed in a pneumococcal sepsis balb/c mouse model. mice (five mice/ treatment group) were intraperitoneally infected with 1.5 )/10 8 cfu/ ml of a serotype 6b penicillin-resistant strain (mic of 2 and 4 mg/l for penicillin and amx, respectively). treatments started 1 h after bacterial inoculation. study groups were: control (k; receiving nonimmune serum (nhs)), amx'/nhs, hs, and amx'/hs. survival rates (%) over time were: purpose of the study: the study was performed to determine the consumption of imipenem and resistance of gram-negative pathogens (pseudomonas aeruginosa , acinetobacter sp., klebsiella sp., escherichia coli , proteus mirabilis , serratia marcescens , enterobacter sp. ) to imipenem. gram-negative pathogens were isolated at the sestre milosrdnice university hospital from zagreb, croatia, in 1999 and 2000. the imipenem sensitivity testing was performed by disk diffusion and e -test methods. the consumption of imipenem was expressed in ddd/100 hospital days in the same periods. results obtained: imipenem resistance of acinetobacter sp. decreased significantly in the year 2000 (p0/0.0052), especially in the first 6 months (p 0/0.021) when the lowest consumption of imipenem was recorded. imipenem resistance of other gram-negative pathogens did not decrease significantly. conclusion reached: comsumption of imipenem might lead to changes in resistance to imipenem among acinetobacter strains. vacheva-dobrevski rs, savov ez. military medical academy, clinical microbiology, sofia, bulgaria purpose: acinetobacter baumanii is becoming increasingly frequent nosocomial pathogen at our hospital, and beta-lactam resistant strains are on the increase, especially among icu isolates. to study the susceptibility of a. baumanii clinical isolates to beta-lactams and to determine the esbl-producing strains during 2001, year. a total 438 gram-negative nonfermenters (gnnf) isolates was investigated by semiautomated mini api system (bio merieux, france). eighty-four a. baumanii non-repeated isolates was studied for esbl-producing by double-disk synergy test (ddt) and atb-blse test (bio merieux, france). mics for beta-lactams were determined by e -test (ab biodisk, sweden). results: a. baumanii (n0/84) showed a multidrug resistance. the isolates were resistant to cefotaxime (79%), cefoxitin (96%), ceftazidime (49%), amoxicillin/clavulanate (76%), piperacillin (72%), aztreonam (84%), imipenem (12%). the 32 (38%) of investigated a. baumanii expressed esbl activity and originated more frequently from icu (78%). esbls producing strains were isolated from endotracheal aspirate (52%), surgery wounds (29%), blood culture (5.7%). conclusions: in general resistance levels were higher in clinical isolates a. baumanii to beta-lactams. the ddt seems to be a practical method for esbl-screening; atb-blse method is more sensitive. our study display to be the first report of esbl-producing a. baumanii strains from our country. carbapenems seems to be the most active agents against a. baumanii . salmonella infantis , strain 111, was isolated from a newborn baby at wassila bourguiba maternity in tunis. it exhibited high resistance to penicillins, extended-spectrum cephalosporines (cefotaxime, ceftriaxone, ceftazidime, cefpirome) and aztreonam but remained susceptible to cefoxitine and imipenem. involvement and characterization of enzymatic mechanism in b-lactam resistance were investigated in strain 111. isoelectricfocusing revealed that this strain produced a b-lactamase of pi 6. this enzyme had a broad-substrate profile, hydrolyzing amoxicillin, ampicillin, ticarcillin, cephaloridine, cefuroxime, cefotaxime, ceftriaxone, cefpirome and ceftazidime. the highest specific activity was observed with ampicillin. cefotaxime was hydrolyzed the most efficiently of the extended-spectum cephalosporines. the pi 6 extended-spectrum b-lactamase (esbl) was inhibited by clavulanic acid and sulbactam. no inhibition of the esbl was observed with 1 mm edta. thus, no metal ion is involved in hydrolysis for this b-lactamase. resistance due to the production of the pi 6 esbl was transferred with dna plasmid into escherichia coli . on the basis of substrate and inhibition profiles and isoelectric point, the pi 6 esbl was not previously described in s. infantis in tunisia. the presence of such a resistance on a plasmid raises concer for rapid dissemination among bacteria and loss of effectiveness of blactams. poizot-martin i a , enel p b , benhaïm s c , vion-dury f c , dinh t c , drogoul mp c , gastaut ja c . a assistance publique hôpitaux de marseille, cisih sud, pr ja gastaut, marseille, france , b assistance publique hôpitaux de marseille, cellule santé publique dmi2, marseille, france , c assistance publique hôpitaux de marseille, cisih sud, marseille, france objective: to assess liver biopsy (lb) practices in a cohort of 255 co-infected hcv and hiv patients followed up in an hiv specialized medical unit. method: transversal study with questionnaire among patients in pre-therapeutic's evaluation with pcr'/ and without lb at 6 months. results: among the 255 patients, 28 (11%) are lost of follow up, 159 (62.3%) have had lb, 68 (26.2%) have no lb. the characteristics of these 68 patients are: median age 0/38.59/6 years; sex ratio 0/2.14, cdc-stage a0/33.3% b 0/47.0% c 0/19.7%, undetectable viral load0/33.3%, median cd40/3269/204, anti-retroviral therapy0/ 95.5%, hcv-genotype 10/46.7%; 3a0/31.7%; 40/21.7%. causes of non-made lb are: (1) refusal from patients because of biopsy's fear0/ 35.3%; (2) contraindications because of hiv infection0/33.8% (clinical events0/16.2% which contraindicate anti-hcv treatment, grade iii thrombocytopenia0/8.8% which contraindicate biopsy, non-adherence to previous hiv follow up0/8.8%); (3) other0/30.9% (alcoholism0/13.2%, psychiatric/depressive disorders0/11.8%, decompensated cirrhosis0/5.9%). drug use or methadone/buprenorphine treatment are not considered as contraindication. conclusion: one-third of patients are afraid of lb. alcoholism and psychiatric/depressive disorders are the principal contraindications to anti-hcv treatment. it seems important to improve information of patients about lb and to focus on alcohol and psychiatric/depressive disorders management in such population. kashiwagi kk a , furusyo nf a , nakashima hn a , kashiwagi sk b , hayashi jh a . a department of environmental medicine and infectious disease, kyushu university, fukuoka, japan , b national kyushu medical center, fukuoka, japan the purpose of the study: the aim of this prospective study was to explore the effect of htlv-i co-infection on the development of hcc among patients with chronic hcv viremia. a total of 764 consecutive patients with chronic hcv viremia were studied and followed-up over a mean period of 5.1 years: 172 (22.5%) were infected with htlv-i infection and 592 (77.5%) were not. the results obtained the annual hcc development rate was 3.4% in patients co-infected with hcv and htlv-i and 1.4% in patients infected with hcv alone. hcc was significantly higher in 33 (19.2%) of the 172 patients co-infected patients than in 39 (6.6%) of the 592 patients infected with hcv alone (p b/ 0.001, logrank test). in patients under the age of 55 years, hcc development was significantly higher in seven (17.1%) of 19 patients co-infected with hcv and htlv-i than in eight (2.9%) of 220 patients with hcv alone (pb/ 0.05, logrank test), whereas there was no significant difference in hcc development between patients over age 55 with or without htlv-i infection (26 (2.0%) of 131 and 31 (9.9%) of 312, respectively). the conclusion reached htlv-i infection accelerates the development of hcc in chronic hcv patients, especially among patients under the age of 55 years. to analyze hbv genotype-related clinical differences among patients with chronic hbv infection, all 158 patients were serially tested for serum alanine aminotransferase (alt) and hepatitis b e antigen (hbeag) and followed up for a mean 10.8 (6.4) year period. genotypes b and c were found in 58 (36.7%) and 100 (63.3%) of the patients, respectively. hbeag positivity and alt abnormality rates at the start of the observation period were significantly higher in genotype c patients (66.0 and 84.0%) than in genotype b patients (34.5 and 22.4%). the annual rate of spontaneous hbeag disappearance in genotype b patients was much higher than in genotype c patients (8.38 versus 2.34%, respectively). patients with genotype c who were continuously hbeag negative from entry had significantly higher alt abnormality (58.8%) than those with genotype b (19.2%). interestingly, patients with genotype c who became hbeag negative by interferon treatment had high alt abnormality (58.8%). all patients with alt abnormality were serum hbv dna positive. these findings indicate that hbv genotype c patients are more severe liver deterioration because of the delay of hbeag disappearance and continued hbv replication after hbeag disappearance. nossik nn a , nebolsin ve b , zheltukhina ga c , yevstigneeva rp c . a the d.i. ivanovsky institute of virology, viral reproduction, moscow, russian federation , b pparminterprisis co., chemistry, mocow, russian federation , c moscow state academy of fine chemical technology, piptide chemistry, moscow, russian federation objective: to study the effects of 'gamma'-l-glutamylhistamine (glu-ha) derivates on non-specific immunity ('alfa'-ifn, 'gamma'-ifn and nk cell activity) and antiviral activity on the experimental influenza and herpes virus infections in mice. the glu-ya and its derivate glu-ii were synthesized by peptide chemistry techniques. the glu-ha and glu-ii was administered i.p. 0.05 and 0.5 mg/kg before and after influenza virus (type a/aichi) and showed a protective effect even at the high infective dose (100ld50) */the rate of protection0/ 42 á/51/60% in the positive/control group. they were not very effective in the protection of herpes simplex virus encephalitis in mice. the model of the physico-emotional stress in mice was used to investigate the ifn system and nk cell activity. the production of ifns and nk cell activity of splenocytes decreased in 2 h after the stress and back to normal level in 7 á/10 days. it was shown that glu-ha and glu-ii can protect or substantially prevent the decrease in nk cell activity and ifns synthesis in post-stress period (so normally did not induce the ifns' synthesis). conclusions: the glu-ha and glu-ii showed antiviral effect against influenza virus infection in mice. the immunomodulating activity and ability to normalize the ifn synthesis and nk cell activity depressed the post-stress period and probably play an essential role in the antiviral activity. no dose adjustment of an anti-influenza prodrug oseltamivir is required in patients with hepatic impairment pm120 oo c a , snell pr b , liu b a , martin d b , simkins t b , small i b , ward p b . a hoffmann-la roche inc., global development, nutley, usa , b roche products ltd., global development, welwyn garden city, uk background: oseltamivir (ose; ro 64-0796, tamiflu † ) is an oral ethyl ester prodrug of its active metabolite oseltamivir carboxylate (oc: ro 64-0802), a potent and selective neuraminidase inhibitor of the influenza virus. the purpose of the study is to evaluate the need for ose dosage adjustment in hepatic impaired patients (hi). method: healthy volunteers (hv) versus hi (child-pugh score 7 á/ 9) [matched on the basis of age (9/10 years), gender and weight (9/ 20%)] were compared. each subject received 75 mg ose. results: based on c max (ng/ml) and auc inf (ng h/ml) analysed using nominal times, ls mean ratios and 90% ci between hi and hv were similar. ose* values in hi were marginally elevated but not sufficiently to require dose adjustment. the aim of this study is to investigate the influence of molecular structure of macrocyclic pyridinophanes and their analogs on antiinfluenza and antiherpetic activity of these compounds. we used 4d-qsar approaches on the basis of simple representation of molecular structure. such representation for biologically active substances allows the description of the spatial structure of compounds with the complete stereochemical information. it determines spatial structures either promoting or interfering of the concrete biological activity. it is easy to realize the molecular design of compounds with the given level of activity with the help of the combinations of simplexes. statistic characteristics for qsar of partial least-squares models are satisfactory (r0/0.92 á/0.97; cvr0/0.76 á/0.86). the molecular fragments that increase the antiviral activity were defined and will be demonstrated. this information was used for design and directed synthesis of several novel antiviral agents with predicted high anti-influenza or antiherpetic activities. predicted activities were confirmed experimentally. 4d-qsar approaches are useful for development of antiviral compounds. this work was partially supported by intas foundation (grant intas 97-31528). lozitsky vp. ukrainian mechnikov research anti-plague institute, chemotherapy, odessa, ukraine the purpose of this study was to research the anti-influenza activity of proteolytic inhibitor e-aca. it prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into cells. e-aca brings down proteolytic cleavage of ha-precursor to ha-1 and ha-polypeptides and reduces the infectious virus harvest. it shows the prophylactic and therapeutic action during the experimental influenza reducing the enhancement of alkaline proteases activity in lungs after infection. e-aca promotes the intensification of specific antibodies production and cell immunity, prevents vessels' permeability and hemorrhagic phenomena, decreases the destruction of bronchi's epithelium. it reduces the duration of intoxication, catarrhal instances and hyperthermia in sick children. e-aca improves the indexes of immunity, non-specific resistance and decreases the rate of bacterial complications. application of e-aca for treatment influenza and other arvi in children is recommended in ukraine on the base of results of our researches. the higher effects demonstrated as a result of combine usage of e-aca with specific ig, or deitiforin, or unithyol, or ribavirin. in our opinion, the study of effectiveness of e-aca combine application with inhibitors of influenza na is the perspective direction of anti-influenza researches development. we should we treat immediately all varicella patients with acyclovir if the patient is older than 20 years pm123 during past 2 years in institute for infectious and tropical diseases in belgrade, 89 immunocompetent varicella patients were treated and cured. among them 32 were older than 20 years (35.95%). x-rays were performed in all patients. diagnosis of pneumonia was made in 32 patients (35.95%), but in 26 (81.25%) patients older than 20 years. varicella is a benign, self-limited disease, if it strikes early, i.e. preschool, school children and teenagers. at that time there is no need for specific therapy. but in neonates, immunocompetent adults and in all immunocompromised patients it can be difficult and life-threatening disease. in immunocompetent adult population pneumonia is a very serious, sometimes fatal complication. knowing the pathophysiology of primary varicella á/zoster infection, specific therapy with acyclovir should be started immediately after making the diagnosis in patients older than 20 years, without waiting for x-ray proof of pneumonia. brivudin compared to acyclovir for the treatment of herpes zoster: effects on acute disease and posttherapeutic pain pm124 objective: comparison of efficacy and safety of brivudin 1)/125 mg and acyclovir 5)/800 mg, both for 7 days, in the treatment of herpes zoster. methods: randomised, double-blind study on 1227 immunocompetent patients ]/18 years (brivudin: n 0/613, acyclovir: n 0/614). a subgroup of patients ]/50 years (brivudin: n0/309, acyclovir: n0/299) was examined for the occurrence of posttherapeutic pain in a poststudy survey. posttherapeutic pain was defined as any zoster-associated pain, regardless of intensity, after the end of acute zoster. results: brivudin was superior to acyclovir in reducing time to last occurrence of new vesicles (rr(itt): 1.13 [1.01 á/1.27], p 0/0.01). the advantage of brivudin was more pronounced in patients ]/50 years (rr(itt): 1.16, [1.01 á/1.34], p0/0.02). incidence of posttherapeutic pain was significantly lower with brivudin (32.7%) than with acyclovir (43.5%, p0/0.006). duration of pain was comparable in both treatment groups (rr: 1.11, [0.93 á/1.32], p 0/0.27). potentially treatment-related adverse events occurred in 7.7% of the brivudin recipients and in 10% of the acyclovir recipients. conclusions: brivudin 125 mg once daily for 7 days is superior to standard acyclovir in stopping viral replication in acute herpes zoster. in patients ]/50 years, brivudin is more effective than acyclovir in reducing the risk of developing posttherapeutic pain. brivudin is as well tolerated as acyclovir. varadinova t a , genova p a , garcia-raso a b , terron a b , fiol j b , badenas f b . a laboratory of virology, sofia university, sofia, bulgaria , b chemistry, universitat de les balears, palma de mallorca, spain we have published that cu(ii) complexes of acyclovir (acv) are active against hsv infection (mbd, 1996) . here we present data on the activity of acv complexes of ni(ii), cd(ii), co(ii) and ag(i) against resistant to acv hsv 1 strain r-100 in comparison with the effect against acv sensitive strain victoria. selectivity indexes (si) compared to that of acv were indicative for activity. the following data were obtained: (i) 1 was 10 times less selective inhibitor of strain r-100 than of strain victoria; (ii) under the action of [cd(acv)cl 2 ], [ni(acv) 2 (h 2 o) 4 ]cl 2 ×/2acv and [ni(acv)(no 3 )] 3 ×/5h 2 o was up to 90% higher than that in the control; (ii) 2 was 28 times less selective inhibitor than acv; (iv) si of 3 was two times higher for strain r-100 and five times lower for strain victoria then that of acv. these data show that the selectivity of acv against resistant hsv 1 strains can increase when acv is bond to a proper metal ion. methods: an antiviral activity against herpes simplex virus of the type i (hsv-i/leningrad/248/88) and variant hsv-1 (vvt/4/00r) resistant to acyclovir was determined using commonly accepted method. viruses were grown on a continuous culture. maximal toxic dose was determined by the administration of compounds orally (300 mg/kg) or intraabdominally (100 mg/kg) to white mice that had mass 18 á/20 g. condition of the animals was controlled during 72 h. mice pneumonia model was used for the testing activity in vivo. results: derivatives tested have activity against hsv-1 and hsv-1 resistant to acyclovir. maximum protection of the cells up to 80% was reached at concentration of compounds 100 á/10 mg/kg. tested compounds have low toxicity and animals did not die after intraabdominal and after per oral administration of the substances. using these compounds led to essential relief of diseases in animals. the number and square of virus specific areas of inflammation in lung was decreased to compare with control untreated group. tested compounds protected animals similar to acyclovir that was used as control. conclusion: derivatives of carboalkoxysulfanilic acids are active against hsv in vitro and in vivo and act on the acyclovir resistant variant viruses. markiewicz r a , szepietowski jc b . a jelfa s.a., medical department, jelenia gora, poland , b department of dermatology, university of medicine, wroclaw, poland background: denotivir is a 5-benzoamino-4?-chloro-3-methyl-4isothiazolecarboxanilide anti-inflammatory agent with antiviral and immunomodulatory activities. it possesses also mild antibacterial and antifungal action. the purpose of the study: the aim of this presentation is to give an overview of recent studies demonstrating denotivir efficacy in herpetic infections. the results obtained: in vitro studies revealed that denotivir in the doses below its cytotoxicity (about 25 um) significantly inhibited (by 90 á/99%). herpes simplex virus (hsv)-1 and hsv-2 replication in fibroblast and kidney cell cultures. moreover, it was showed that denotivir in the dose of 37 mg/ml markedly inactivated hsv-2 after 30 min incubation in 37 8c. in giunea pigs research, 2% denotivir in 90% dmso appeared to be superior to 90% dmso alone and untreated groups in the therapy of animal skin infected by hsv-2. there was no huge difference in eythema and oedema scorings between studied groups, however in the group treated with denotivir, in contrast to others, no vesicles developed. several clinical studies showed usefulness of denotivir in controlling herpetic infections in dermatology, ophthalmology and otolaryngology. in the majority of studies within few hours after the drug application itch and pain relief was noted and within 1 á/2 days the vesicular lesions were dried up. the conclusion reached: in conclusion, denotivir is an effective antiherpetic agent. this study was conducted on 12 cows affected with teat papillomatosis. in the first step, each cow was located on one of three groups. the first group contained four cows from 1 to 4 years that were treated with fig tree (ficus carica) latex. the second group contained four cows from 1.5 to 4 years that were treated with a 10% solution of salicylic acid, and the third group contained four cows as control. in group one and two following treatment with fig tree latex and salicylic acid, superficial necrosis begun from day 5 and all of the warts disappeared by day 30. in the control group, after day 20, there were no changes in number of lesions, but some of them were larger than first observation. on day 25, one of the marked warts disappeared and on day 35 another wart was disappeared but six were present until day 45. comparison of effects of salicylic acid and fig latex showed similar effects in treatment of udder papillomatosis in cow. laboratory markers of skeletal muscle toxicity in hiv-infected patients: a cross-sectional case-control survey of frequency, potential correlation with antiretroviral therapy, clinical significance, and outcome pm130 manfredi r, motta r, patrono d, calza l, chiodo f, boni p. to assess skeletal muscle toxicity among â/1000 hiv-infected outpatients (p), the 129 p who had ]/1 altered cpk assay ( !/195 u/l) between may and november 2001, were compared with 387 p randomly selected among those who had ]/2 laboratory exams in this 6-month interval, in a 1:3 case-control study. among the 129 p with altered cpk levels only six were females, and 110 received antiretrovirals. the overall frequency of altered cpk among all p who underwent ]/2 laboratory workouts in 6 months was 14.4%. cpk alteration was transient in 98 p, with values ranging from 196 to 3463 (mean 256.29/62.3) u/l, but was recognized ]/2 times in 2 á/6 months in 31 p (24%), 24 of them showing concomitant high aldolase levels (3.1 á/10.8 u/l). a myopathy or a rhabdomyolisis were recognized in four p only; a myositis was confirmed in one p by histopathology. in a multivariate logistic regression analysis, when excluding the unexpected prevalence of the male gender (p b/0.0001), no significant difference emerged between p and controls as to age, risk for hiv infection, iv drug use, duration of hiv infection, prior anti-hiv therapy and its length, selected drug combinations, administered nucleoside analogues,hiv disease stage, mean cd4'/ count and hiv viremia, signs and duration of lipodystrophy, increased glucose, triglyceride and cholesterol levels, and other therapies. muscle abnormalities, though frequently asymptomatic, are underestimated hiv disease complications, and the role of metabolic (i.e. mitochondrial) alterations, deserves investigation. poor efficacy of non-nucleoside reverse transcriptase inhibitor (nnrti)based salvage haart in hiv-infected patients heavily pre-treated with all other classes of antiretroviral compounds pm131 manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy poorly comparable literature series show conflicting results of nnrti-based rescue haart: 20 á/70% rate of virologic success. to assess the response to a 4 á/5-drug rescue haart including a nnrti, 72 patients (p) treated with nucleoside analogues (na) and protease inhibitors (pi) for !/18 and !/15 months, respectively but naïve to nnrti, with a viremia !/10 000 copies/ml, were prospectively followed during 1 á/2 years, provided that they ensured a !/90% adherence. efavirenz was used in 41 p, nevirapine in 29, and delavirdine in two. most p (88.9%) had an early laboratory improvement, but mean peak viral load decrease was 0.4 log, and a significant reduction vs baseline (p b/0.02) lasted 3 months only. a mean 17% increase of zenith cd4 count was obtained (p b/0.001), but only 18.1% of p remained !/200 cells/ml 1 year after switch to nnrti. in a multivariate analysis, the concurrent introduction of novel pi(s) (32 p) and/or different na(s) (19 p) acted favorably until the 9th month of follow-up (p b/0.04), while genotypic mutations conferring nnrti cross-resistance, usually associated with a broad resistance profile, predicted failure in all p (p b/0.001), and the response did not vary according to duration and type of prior therapy, and selected nnrti. a deep salvage nnrtibased haart has a poor and transient virologic outcome also in nnrti-naïve p, while a more evident and sustained immunologic response is expected. p who can introduce novel pi/na and have no mutations impairing nnrti activity are entitled to a better outcome. fatal lactic acidosis without elevation of liver-enzymes during the treatment with stavudine, didanosine and efavirenz: a case report pm132 winzer r, langmann p, väth t, zilly m, klinker h. medizinische poliklinik der universität würzburg, schwerpunkt hepatologie/infektiologie, ürzburg, germany nucleoside reverse transcriptase inhibitors (nrtis) cause various side effects, many of which are thought to be due to their effects on mitochondria. a 36-year-old hiv positive (hiv rna: 6000 copies/ml, cd4 cell count: 359/ml), obese (body-mass-index: 40.9), therapy-naïve female patient, who after 7 months of well tolerated and effective antiretroviral therapy (stavudine, didanosine, efavirenz), had slight gastrointestinal discomfort and suddenly developed a lactic acidosis (arterial-ph 7.03 [7.36 á/7.44 she died 4 days later despite intensive care (continuous venovenous haemodiafiltration, sodium-bicarbonate infusion, high doses of vitamins, respiration). the pathologic examination showed an enlarged liver (2370 g) with yellowish appearance and pasty consistency, which microscopically appeared as a massive macro-and microvesicular fatty degeneration, and only slight signs of terminal pancreatitis. this reported case gives evidence that a massive lactate acidosis may develop without previously disarranged laboratory parameters for liver or pancreatic function. a fatal outcome may evolve without further accompanying-illnesses. efficacy and tolerability of atorvastatin in the treatment of hypercholesterolemia in hiv-infected patients receiving haart pm133 calza l, manfredi r, chiodo f. division of infectious diseases, university of bologna, bologna, italy introduction: significant increases in plasma triglyceride and cholesterol levels have been reported in patients treated with haart, and prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of hiv-infected persons. patients and methods: fourteen hiv-infected patients on pi-based haart since at least 12 months and presenting hypercholesterolemia ( !/290 mg/dl) of at least 6-month duration and unresponsive to a hypolipidemic diet and physical exercise, have been treated with a single daily dose of atorvastatin (20 mg) for 18 months. results: one patient was ecluded from evaluation due to early dropout. ongoing antiretroviral treatment included ritonavir in four cases, indinavir in four, nelfinavir in three, and saquinavir hard-gel in two. at the close of 18-month follow-up of atorvastatin therapy, a decrease of total cholesterol level of 21.9% versus respective baseline value was observed; eight out of 13 patients reached normal values for cholesterol. mild gastroenteric symptoms were found in only one of the 13 treated patients, while no skeletal muscle and liver toxicity has been observed. discussion: in our study, pharmacological treatment with atorvastatin proved certainly effective in the management of diet-resistant hypercholesterolemia, and was associated with a favourable tolerability and adherence profile. the effect of combination antiretroviral therapy regimens on hiv-1 proviral dna level in peripheral blood mononuclear cells (pbmcs) was examined in 12 hiv-1-positive patients, using endpoint dilution pcr and serially cloning and sequencing of the gag region of hiv-1. the major clone was defined as the most numerous of 10 analyzed clones, and observation periods ranged from 8 to 32 months (mean, 19.89/10.2 months). in five patients (one with primary-stage hiv-1 infection) receiving three antiretroviral drugs, hiv-1 rna levels reduced to undetectable (i.e. b/100 copies/ml). hiv-1 proviral dna levels and the number of major clones reduced in four of these patients. hiv-1 rna levels reduced, but remained detectable, in five other patients. in the two remaining patients (both receiving two rather than three antiretroviral drugs) hiv-1 rna levels increased. these results suggested that the population of the major clones may be affected when hiv-1 rna levels reduce following combination regimens of antiretroviral therapy. saquinavir hard gel (shg) as a part of a spontaneous 12 á/18-month deintensification anti-hiv regimen following successful highly active antiretroviral therapy (haart) pm135 manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy the induction-maintenance concept was poorly studied in hiv'/ patients (p), and shg was never assessed after prolonged response to potent protease inhibitors (pi)-based haart. shg-naïve p who refused indinavir, ritonavir, or nelfinavir-based haart after achieving long-term viral suppression, and resorted to shg'/2 nucleoside analogues (na), were followed prospectively. in 60.7% of the 61 p assessed for 12 á/18 months, ]/1 na was changed. prior haart was interrupted after 8.49/2.9 months, due to adverse events (46 p), or p's request (15 p), while a viremia b/50 copies/ml was present since 4.79/ 1.5 months. a viremia of 50 á/1000 hiv-rna copies/ml was maintained in 49 p (80.3%), while a higher viral load occurred in 12 p after 5.99/0.6 months, and was related to a pre-haart viremia !/100 000/ ml, a more frequent !/100% recovery of cd4 count, mutations of codons 48 á/90, and failure to change na (p b/0.05 á/0.001). a cd4 drop !/20%/150 cells/ml was found after 7.19/0.5 months in only eight p, who also had virologic failure: immunologic deterioration was earlier and deeper when na were not changed (p b/0.05). all the 37 p who introduced shg'/novel na after a successful !/6-month induction with a potent pi-based haart had a stable 12 á/18-month outcome. a suboptimal haart including the less effective but better tolerated shg may be effective for !/1 year, especially when novel na are introduced, and specific mutations are absent. despite a lower potency, drugs with a good safety and compliance profile may be recovered for simplified regimens. objective: to evaluate efficacy of antiretroviral therapy (art) with two or three drugs in the nervous 'reservoir'. patients: thirteen acute neurological and art naive aids patients underwent a paired and simultaneous sample from plasma and cerebrospinal fluid (csf) for a quantitative detection of hiv-1 rna (amplicor roche) before art. all patients underwent a ct and/or mr of the brain to perform a diagnosis. all of them had an hiv related neurological acute inflammatory disease. after diagnosis all patients received art: 7/13 received two nrti and 6/13 received haart including two nrti and one protease inhibitor. all patients underwent a paired and simultaneous follow-up from plasma and csf during the 2nd month of treatment. results: in all patients baseline levels of hiv-rna were higher (p b/ 0.05) in the plasma (log 10 5.37'/0.93) than in the csf (log 10 4.33'/1.379). the 7/13 patients who received dual therapy had undetectable levels (cut-off 200 copies/ml) of viral rna at the follow-up in csf, but not in plasma: three of these seven patients had a detectable plasma hiv-1 rna. all 6/13 patients with haart had undetectable hiv-1 rna both in plasma and in csf at the follow up. conclusions: dual nrti therapy is rapidly effective in csf (because of an high penetration of drugs through a more permeable blood á/brain barrier and lower hiv rna baseline levels) but not in plasma. haart is rapidly and equally effective both in csf and plasma. there are no reports of fulminant and fatal hepatic failure after the start of highly active antiretroviral therapy (haart) in an hiv subject without chronic viral hepatitis. case report: a 30-year-old naive aids woman with clinical symptoms due by a pcp was observed. baseline alt was increased (0.5 m.n.v.) because of a mild hepatosteatosis and a silent cholelithiasis. serology for hbv, hdv and hcv was negative; igg anti-hav, anti-ebv, anti-cmv and anti-hsv were present. hiv-1 rna was 5.7 log 10, cd4'/ count was 26/ml. during the pcp treatment with cotrimoxazole alt values increased ( !/5 m.n.v.); nevertheless, she completed the treatment. liver enzymes returned to the pre-treatment values over several days. then she started haart with stavudine, lamivudine and efavirenz. after 10 days the patient showed an efavirenz-related skin rush that resolved within 5 days, without treatment discontinuation. fourteen days after the start of haart jaundice appeared. laboratory revealed severe alt increase ( !/8 m.n.v.) and hyperbilirubinemia (17 mg/dl) and she died because of an acute liver failure syndrome within few days. an haart efavirenzbased regimen can result highly hepatotoxic when given in presence of a hepatosteatosis, of a recent hepatotoxicity caused by a nonantiretroviral treatment and of a previous idiosyncratic reaction to efavirenz. our experience with bulgarian herbal extracts for improving the general condition of hiv-positive patients pm138 methods: we used a combination of 19 bulgarian herbal extracts and treated six patients, divided in two groups: three with symptomatic and three with asymptomatic hiv-infection. the all three patients with asymptomatic hiv-infection were treated only with herbal extracts, another three patients with symptomatic hiv-infection were treated with combination of herbal extracts and anti-retroviral therapy. the general status of patients has been evaluated by both subjective and objective surveillance. the immunologic monitoring has been performed by absolute count of cd4'/ lymphocytes. results: all patients have shown an obvious improvement in their general condition: high spirit and working capacity, good appetite and sleep, a restoration of body weight. the number of cd4'/ lymphocytes has been lightly increased or constant. conclusion: the combination of bulgarian herbal extracts has shown significant positive effect on the general condition and improve the quality of life. antifungal activity of in vitro and in vivo combinations of voriconazole with 5-fluorocytosine and amphotericin b against candida and cryptococcus spp pm139 hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: the present study was designed to determine whether the activity of voriconazole (vor), a novel triazole, was reduced against candidal and cryptococcal infections by the addition of standard antifungal agents, amphotericin b (amb) and 5-fluorocytosine (5-fc). vor was tested in combination with standard antifungal agents both in vitro, using a checkerboard mic determination test, and in vivo in immune normal guinea pig models of fungal infections. the results indicate that the efficacy of vor against candida albicans and c. neoformans was not antagonised by amb or 5-fc in vitro. furthermore, in guinea pig models of systemic candidiasis and intracranial cryptococcosis, no antagonism was observed between the lower doses of vor and either amb or 5-fc on the basis of reductions in tissue fungal loads. at the highest doses of vor, both amb and 5-fc showed some antagonism, but the combinations were still effective in significantly reducing fungal tissue loads compared with vehicle-treated control animals. conclusion: these results suggest that vor may be used in combination with standard antifungal agents, and future studies to elucidate the clinical potential of vor combination therapies in the management of candida and cryptococcus infections are warranted. itraconazole in the treatment of pityriasis versicolor pm140 tiodorovic j, jovanovic d, binic i, nikolic lj. faculty of medicine, clinic of dermatovenerology, nis, serbia, yugoslavia a comparison of two short-term dose schedules with itraconazole was carried out in 50 patients with pityriasis versicolor. the patients were divided in two groups. each group consisted of 25 patients who completed the therapy and controls. the clinical diagnosis was confirmed mycologically, by direct microscopic examination. the first group received 400 mg of itraconazole daily for 3 days. the second group received 200 mg daily for 5 days. the patients were controlled clinically and mycologically 15 and 30 days after the initiation of treatment. erythema, scaling and pruritus was evaluated clinically. clinically and mycologically cured patients accepted as cured. the cure rate were 76% in the first group and 72% in the second group at day 30. the effects of these two groups are similar. none of the patients reported side-effects. fungal urinary infections: emerging species, antifungal susceptibility trends and antibody response pm141 badawi he a , kamel ai b , fam ns a , el-sayed me a , elian sae c . a theodor bilharz medical research institute (tbmri ), microbiology, giza, egypt , b theodor bilharz medical research institute (tbmri ), urosurgery, giza, egypt , c faculty of medicine, medical microbiology and immunology, cairo university, cairo, egypt objectives: to assess the role of candida species in 120 patients with urinary tract infections (utis) with or without schistosomiasis and/or cancer bladder, to compare chromogenic; chromagar (cma), biggy agar; morphologic (corn meal, rice agar-tween 80) media and biochemical candifast test for identification of candida species. susceptibility to antifungal agents using e -test and candifast and the performance of elisa test for detection of anticandida antibodies (igm and igg) in serum were evaluated. results: c. albicans was the most frequent (43.4%) species responsible for fungal utis. however, non-albicans species, c. glabrata (23.3%), c. tropicalis (20%) and c. krusei (13.3%) were also isolated. rice agar-tween 80 was found to be cheap, available and sufficient to make a final identification (100%). cma could not identify c. glabrata . biggy agar could not adequately differentiate candida species. candifast biochemical identification showed low sensitivity of 83.3%. e -test on sabouraud dextrose agar (sda) is simple method for mics determination and could detect s-dd strains in case of azoles. conclusion: the emergence of non-albicans species such as c. glabrata , c. tropicalis and c. krusei have contributed to complicated utis. this necessitates accurate isolation and identification of candida to the species level. morphology on rice agar-tween 80 and antifungal susceptibility using e -test on sda is a simle rapid scheme for routine identification of clinically important yeasts. purpose: classification of allergic fungal rhinosinusitis (afr) is based on the immunologic relationship of the host to the fungus. afr must be differentiated from other fungal rhinosinusitis infections, which include acute invasive, chronic invasive, fungal balls and saprophytic colonization. although many cases of fungal rhinosinusitis is caused by species of aspergillis , dermatiaceous moulds have become an emerging pathogen in immunocompetent individuals. results: our case study involved a 36 year male suffering from facial pain, headache, postnasal drip and loss of smell. he was hiv negative and a nonsmoker. the following laboratory tests were performed: ige-2331.40 (0.00 á/25.00) iu/ml iga-2.16 (0.70 á/4.00) g/l igm-0.61 (0.40 á/ 2.30) g/l allergen specific ige for alternaria */20.00 (0.00 á/0.5) ku/l fbc-normal except eosinophils slightly raised 0.60 (0.00 á/0.50))/10 9 / l. ct scans indicated fungus proliferation, bone erosion and extension of disease into adjacent anatomic area. sinus tissue following debridement was sent for microscopy and culture. hyphae was microscopically observed and cultures yielded two dermatiaceous fungi, bipolaris spp and alternaria spp . conclusion: it is important to differentiate these two species from curvularia , helminthosporum , drechelria and exserohilum . knowledge of these dermatiaceous fungi is important in directing appropriate antifungal therapy and selecting the correct antigens for postsurgical immunotherapy after initial debridement and irrigation. antifungal activity of in vitro and in vivo combinations of voriconazole with 5-fluorocytosine and amphotericin b against aspergillus fumigatus pm143 hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: a key requisite for a new antifungal drug is to demonstrate that it is devoid of significant antagonism in combination with other agents. combinations of the new triazole, voriconazole (vor), and standard antifungal agents (5-fluorocytosine or amphotericin b; 5-fc or amb) were tested against aspergillus fumigatus in vitro and in guinea pig models of infections to confirm that antifungal activity was not antagonised by using combination therapies. vor was studied in combination with amb or 5-fc in vitro, using a checkerboard mic determination test, and in vivo, using immune normal and immunocompromised guinea pig models of systemic aspergillosis. results: the results indicate that the potency of vor was not antagonised by amb or 5-fc in vivo; indeed, at lower concentrations of vor, significant improvements in reducing fungal burden in both in vivo models were achieved by the addition of amb. in vitro, no antagonism was found between vor and amb, although 5-fc had a significant antagonistic effect on vor activity. conclusion: these results from in vitro and in vivo models of aspergillosis suggest that vor may be used in combination with standard antifungal agents and, therefore, justify further examinations of vor combination therapies in a clinical setting. in vitro activity of caspofungin compared to that of amphotericin b, fluconazole, and itraconazole against candida species pm144 arikan s, sancak b, hascelik g. department of microbiology and clinical microbiology, hacettepe university medical school, ankara, turkey purpose: to evaluate the in vitro activity of caspofungin against various candida spp. and particularly against isolates with decreased amphotericin b, fluconazole, and itraconazole susceptibilities. methods: susceptibility tests were done by nccls m27a microdilution guidelines for 239 clinical candida strains. the mics (mg/ml) were read at 24 and 48 h. results: caspofungin mics at 24 h are shown in the table. mics at 48 h were similar to 24 h readings. expectedly, no evidence of crossresistance was detected between caspofungin and other drugs tested. caspofungin was similarly active against fluconazole-or itraconazolesusceptible and resistant isolates. conclusions: (1) caspofungin is active in vitro against all candida spp. tested. (2) caspofungin mics are slightly higher for c. parapsilosis compared to other species. (3) its activity against fluconazole-and itraconazole-resistant isolates is noteworthy. (4) validation of these data require clinical investigations. oropharyngeal microbiological samples of 80 bmt patients were evaluated. weekly cultures (days (/7, 0 and '/7) revealed presence of fungi in 27 patients (33.7%): in four (5%) patients before bmt only, in 12 (15%) after bmt only, and in 11 (13.8%) both before and after bmt. in one patient candida norvegensis was isolated from the throat, buccal and palatal surfaces. three c. albicans , two c. krusei , and three c. norvegensis from four patients were chosen to comper their antifungal sensitivities and extracellular virulence factors. using fungitest method we determined the sensitivities of these isolates to flucytosine, amphotericin b, miconazole, ketoconazole and fluconazole. the fluconazole sensitivities were also determined by the e -test. on the basis of the fungitest data the three c. norvegensis isolates were sensitive to flucytosine, amphotericin b, and also to ketoconazole. in case of fluconazole and miconazole they proved to be susceptible dependent upon dose. the mic fluconazole values determined by the e -test for the three c. norvegensis isolates were 96, ]/256 and ]/256 mg/ml, respectively. the oropharyngeal isolates of c. norvegensis produced high amounts of extracellular aspartic protease and phospholipase similarly to c. albicans strains. these enzymes may contribute to the pathogenesis of this new emerging candida species. in vitro activities of antifungal and antiseptic agents against rhodotorula sp pm148 preney l, théraud m, guiguen c, gangneux jp. laboratory parasitologie-mycologie, chu de rennes, france purpose of the study: rhodotorula species are common saprophyte yeasts widespread in nature. since the last 10 years, they have been implicated in several severe infections, especially in immunocompromised patients, and various antifungal therapies were used. however, only limited data are available on the susceptibility of rhodotorula sp. to antifungal and antiseptic agents. material and methods: in this work, we evaluated the in vitro activities of eight antifungal agents against 30 strains of rhodotorula (21 strains of r. rubra and nine strains of r. glutinis using atb fungus system (biomerieux) and etest strips (ab-biodisk). beside, the effect of eight antiseptic agents was assessed on a suspension of r. rubra . the quantification of yeasts after exposure to antiseptic agents was performed by subculturings using a microtitration method in 96well plates. results and discussion: all strains tested were susceptible to amphotericin b, 5fc, and nystatin. twenty-nine out of 30 strains were susceptible to ketoconazole, 21 out of 30 were intermediate to econazole. all strains were resistant to fluconazole (cmi!/64 mg/ml) and itraconazole (cmi!/1 mg/ml), and 29 out of 30 were resistant to miconazole, suggesting that antifungal therapy must be adapted when rhodotorula yeasts are implicated in invasive infection. beside, 5 min exposure to sodium hypochlorite 128, chlorhexidine 0.5% or ecodiol (isopropyl alcohol'/alkylamin) showed fungicidal activities. susceptibility testing of aspergillus fumigatus and emerging aspergillus pathogens by a modification of the nccls m38-p method pm149 logotheti m a , kapsanaki-gotsi e b , velegraki a a , zagoura d b . a department of microbiology, mycology reference laboratory, medical school, university of athens, athens, greece , b biology department, section ecology and systematics, university of athens, athens, greece aspergillosis in high risk groups of patients is still associated with high mortality rate (30 á/90%). aspergillus fumigatus is the primary pathogen, while other opportunistic aspergillus species are emerging. amphotericin b (ab), itraconazole (it), voriconazole (vo) and terbinafine (te) minimum inhibitory concentrations (mic) were determined by modifying the nccls m38-p microdilution method. stock drug solutions were prepared in rpmi 1640, dimethyl sulfoxide (dmso), and polyethylene glycol (peg 400). inocula, of the a. fumigatus group (7), a. flavus group (6) (2) and the m38-p quality control strains were prepared according to, and by modifying, the nccls guidelines. plates were incubated at 30 and 35 8c and read at 24 and 48 h. peg 400 effectively dissolved it and vo, while either dmso or peg dissolved te. low 30 and 35 8c á/48 h ab, it, vo and te mics (0.062 á/0.5 mg/l) were recorded. a. terreus (1) and a. parasiticus (2) were resistant to ab. certain clinical isolates demonstrate clinical and in vitro resistance. standardization of susceptibility testing would offer reliable assistance in selecting and monitoring antifungal therapy. otag f, aslan, g, ozturk c. microbiology department, faculty of medicine, mersin university, mersin, turkey rates of opportunistic fungal infections have risen markedly. because some of these species have potential resistance to antifungal agents, rapid presumptive species level identification is crucial in allowing for directed antifungal therapy. in this study, 55 isolated yeasts from the clinical specimens were identified by atb id 32 c (biomerieux, france). the number of identified yeasts were, respectively; 33 (60%) candida albicans , six (10%) c. glabrata , five (9.1%) c. tropicalis , three (5.4%) c. parapsilosis , two (3.6%) c. krusei , two (3.6%) c. kefyr , one (1.8%) c. guillermondii , one (1.8%) c. dubliniensis . twenty-one of 55 strains were investigated for antifungal sensitivities by atb fungus kit (biomerieux, france). the results are as follows: 100% sensitivity was detected to myconasol, 94% to flusitozin, nystatin and econasol, 95% to amphtericin b and ketokonazol. it is important to achieve empirik treatment of the opportunistic candida infections and the following of resistance to antifungals. shakhmatov da, strelchenco, ov. novosibirsk state medical academy, dermatovenerology, novosibirsk, russian federation at the present stage in russia with a background of a high case rate of syphilis, it becomes necessary to exclude biological false positive serological tests. because the serodiagnosis of syphilis has significant limitations, the direct detection of t. pallidum in suspect blood may serve as an alternate diagnostic strategy. polymerase chain reaction (pcr) has been the most widely used amplification method. the study of 56 patients receiving examination related and treatment for syphilis in std clinic and persons directed from other hospitals where routine serologic examination revealed doubtful results. pcr reaction was carried out with nested primer pairs based on the dna sequence of the 47 á/1 and 47 á/2 kda gene of t. pallidum . pcr was utilized with whole blood. a complex of serological tests: fta-abs and tit was used as the &rdqup; gold standard''. as a result the sensitivity of pcr was 91.2% and specificity was 90.9%. selective comparison of pcr results with vdrl, the fta-abs and treponemal immobilisation test (tit) has shown concurrence 96.8%. in conclusion, the preliminary results of pcr in whole blood in syphilis detection revealed its high sensitivity and specificity; possibility to obtain rapid results in unclear cases. chlamydia pneumoniae (cp) is an atypical pathogen whit intracellular location, whose eradication is very difficult. in the past years it has been objects of many studies that lead to the demonstration of a relationship between its presence and the development of widespread multifactorial pathologies such as atherosclerosis and asthma. the lack of its eradication can become an important clinical and social problem. the study objective is the comprehension of pathogen á/host interaction mechanism, to characterize therapeutics protocols that cold lead to complete eradication of cp from organism. the research had been principally made on the studying the molecular mechanisms that are at the root of pathogen permanence inside host cell. using proliferation and apoptosis tests we underlined a different behaviour of infected cells towards control cells. in presence of p1 (20 mg/ml), i.e. a peptide that can inhibit the proliferation and induce apoptosis in vitro inhibiting nf-kb, uninfected cells proliferation decreased of 35% in comparison whit the controls, while the one of infected decreased only of about 15%. moreover, using various apoptosis-inducers, the infected cells showing apoptosis were about 10% while the uninfected were about 40%. the caspace iii activity increased significantly in uninfected cells. in conclusion, cp could delay its elimination from the host inhibiting the apoptosis via nf-kb activation. hryniewiecki t a , gzyl a b , rawczynska-englert i a , a department of acquired valvular heart disease, national institute of cardiology, warsaw, poland , b department of sera and vaccines, national institute of hygiene, warsaw, poland infective endocarditis (ie) frequently causes problems in diagnosis, especially where blood cultures are negative and with fungal etiology (also as a fungal superinfection in bacterial ie). the purpose of the study: the purpose of the study was to evaluate the usefulness of broad-range fungal pcr in diagnosis of fungal superinfection of bacterial ie. twenty-five blood samples were taken for analysis from patients with infective endocarditis. ie was diagnosed according to duke criteria including positive blood cultures. suspicion of fungal superinfection was established on serological investigation in five patients, confirmed by blood culture in two patients. control group consisted of 15 patients without infection. dna was isolated using the commercially available s.n.a.p. kit. amplification products were analyzed by gel electrophoresis stained with ethidium bromide. the results obtained: fungal dna was found in two patients with fungal superinfection of bacterial ie confirmed by culture. in the remaining patients with ie and controls no fungal dna was found. the conclusion reached: broad-range fungal pcr is a fast and inexpensive tool for the detection of fungal dna, but it is more prone to contamination than species-specific pcr. the method may be valuable in the identification of fungal superinfection of bacterial ie or diagnosis of fungal ie. rivanera d, lilli d, lozzi ma, piunno m, mancini c. microbiology, science and public health, rome, italy aim: the aim of this study was to evaluate the eia method for detection of antibody to ttv virus (ttv) and to investigate the anti-tt virus prevalence in patients with hepatitis b (hbv) virus, hepatitis c (hcv) virus, in group of 'high risk'subjects to hepatitis and in healthy subjects. the elisa methods (nuclear laser vienna lab) using ttv s and ns antigens: orf1 (770 aa) and orf2 (202 aa) was applied to detect anti-ttv; the serological screening was performed from 250 samples to italian subjects. results: the positive rates of anti-ttv antibodies were 11.29% in 62 patients with hepatitis b á/c and 14.06% in 119 'high risk' hepatitis patients. the anti-ttv was also found in 7.56% in 69 healthy people. conclusions: the anti-ttv were detected in all groups studied, however, its positive rate was similar in patients with hepatitis b á/c and in 'high risk' hepatitis respect to heathly people. our results shown that tt virus is frequent in italy both in patients infected by others transmitted viruses and in general population. the positivity found in healthy adults included in our studies suggests that the virus might be transmitted non-parenterally. the study of pattern of antibody to ttv may be an infectious marker of ttv similar to that of anti-hcv. a stress test on a miniaturized identification system designed for neisseria and haemophilus pm155 rich m a , bannatyne rm a , memish za b . a king fahad national guard hospital, division of microbiology, riyadh, saudi arabia , b king fahad national guard hospital, infection prevention and control, riyadh, saudi arabia we report an incident that occurred in our laboratory when the bbl crystal identification system for neisseria and haemophilus was used to identify a haemophilus-like-organism. the numerical profile generated was not in the system database. conventional biochemical tests subsequently revealed an identification of brucella melitensis , a common isolate in our area. as a result of this revelation we subjected this system to a mini 'stress-test' with a collection of 20 isolates of b. melitensis . two numerical profiles were obtained, 1740616606 and 1740616607, neither of which are listed in the system database. brucella species have been misidentified as moraxella species, moraxella phenylpyruvica , and as haemophilus influenzae biotype iv in various identification systems. two cases of laboratory-acquired brucellosis have been attributed to misidentification. to its credit the bbl crystal identification system for neisseria and haemophilus neither generates a profile number with a misidentified organism nor assigns a confidence level. instead it properly directs the user to resort to conventional methods to secure an identification. if further studies on additional brucella isolates and strains from different geographical sources confirm the unique biochemical profiles identified here, it may be worthwhile to incorporate these into the database where they would be of considerable assistance in areas where brucellosis is widespread. cloning and characterization of aflmp1 in aspergillus flavus pm156 chong tk, woo pcy, leung asp, yuen ky. the university of hong kong, microbiology, hong kong, hong kong purpose of the study: to clone and characterize an antigenic protein for serodiagnosis of infection caused by aspergillus flavus which is the commonest aspergillus species causing aspergilloma (ao) and invasive aspergillosis (ia) in asia. result obtained: we cloned the aflmp1 gene, which encodes the first antigenic cell wall protein in a. flavus . aflmp1 codes for a protein, aflmp1p, of 273 amino acid residues, with sequence features that are present in mp1p and afmp1p, the antigenic cell wall mannoprotein in penicillium marneffei and aspergillus fumigatus that we described previously. it contains a serine-and threonine-rich region for o glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. specific anti-aflmp1p antibody was generated with recombinant aflmp1p protein purified from escherichia coli to allow further characterization of aflmp1p. indirect immunofluorescent staining indicated that aflmp1p is present in the cell walls of the hyphae and conidia of a. flavus . furthermore, it was observed that patients with ao and ia due to a. flavus develop a specific antibody response against aflmp1p. conclusion reached: this suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with ao or ia, and the protein may represent a good cell surface target for host humoral immunitiy. grape purpose: to investigate the basis for increasing resistance to trimethoprim and sulphamethoxazole. methods: pcr screening for integrons of 105 clinical urinary tract isolates was performed. isolates were tested for resistance to 12 antibiotics. integrons in 14 isolates were sequenced. results: integrons of class 1 were found in 43 isolates and class 2 integrons were found in 10. eight isolates in the study were resistant to five antibiotics or more and not shown to carry any integron. nineteen of 69 isolates resistant to trimethoprim did not carry integrons. only one of these isolates was shown to carry sul1 and is thus probably also carrying an integron. none of the 19 isolates were shown to carry dfr8 , one of five trimethoprim resistance genes known to exist outside integrons. three isolates were resistant to sulphonamides but were not shown to carry neither sul1 nor sul2 . only dfr and aad gene cassettes were found in the sequenced integrons. conclusions: resistance to trimethoprim in 19 of 69 trimethoprim resistant isolates is mediated by genes not detectable, as in the case with three sulphonamide resistant isolates. sequenced integrons that contain dfr genes do not carry any gene cassettes mediating resistance to modern antibiotics. unusual diagnosis tool for an unusual presentation of alveolar echinococcosis: report of two cases of local progression after an animal bite pm158 bardonnet k, bart jm, loiseau j, gérard a, estavoyer jm, heyd b, badet jm, dubiez a, piarroux r, bresson-hadni s. who collaborating centre for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the classical human contamination route for alveolar echinococcosis (ae) is ingestion of eggs. two exceptional human cases are reported with extensive local evolution of ae after a bite. case no. 1: between 1954 and 1972, a patient underwent surgery seven times for a muscle growing tumour which developed after a bite. the diagnosis of muscle ae was assessed on histopathological examination. in 1980, serological tests were in accordance with echinococcus sp infection. case no. 2: in 1985, a man presented 'cat-scratch fever' with a right supraclavicular tumefaction following a cat bite. between 1986 and 2000, five recurrences occurred. different surgical explorations indicated multiple abscesses of the cervical muscles. in 2000, serological tests were in favour of echinococcus sp infection and the pathologist described a parasitic wall suggesting hydatidosis, but specific pcr from histological samples prompted the diagnosis of ae. conclusion: in these exceptional observations, the liver which is the most usual location of ae was lesion-free. the chronic inflammatory ae lesions have developed in the local lymphatic chain area of the bite site. to perform diagnosis in these very unusual forms of ae, it is necessary to add unusual tests such as specific pcr to classical tests. antibiotic resistance in foodborne salmonella is an emerging public health concern. integrons are now recognized as the main genetic vehicles of antibiotic resistance in gram-negative bacteria, including in salmonella . the purpose of the present study was to investigate the presence of class i integrons in resistant isolates of several serotypes of salmonella isolated from poultry products and to determine their association with multidrug-resistance phenotypes. a total of 20 isolates of salmonella belonging to seven different serotypes were tested. the most frequent multiresistant phenotype, found alone or together with other resistances, was to streptomycin and tetracycline. all but seven were resistant to three or more antimicrobial agents, including quinolones and amoxicillin. pcr analysis with the 5?cs and 3?cs primers detected the presence of class i integrons of 1.5 kb in one isolate, with the multiresistant phenotype: amoxicillin, chloramphenicol, streptomycin, trimethoprim-sulphametoxazol and tetracycline. our findings suggest that the uncontrolled use of the antimicrobial agents in food animals may have contributed to the development of the pattern of resistance observed in salmonella isolates. also the presence of integrons in low prevalent human salmonella serotypes but associated with food animals underscores the public health problem of antibiotic resistance acquisition and spread. prevalence and antimicrobial resistance of campylobacter jejuni and c. coli isolated from broilers and pigs in france pm161 avrain l a , humbert f b , sanders p c , kempf i a . a afssa, umb, ploufragan, france , b afssa, hqpap, ploufragan, france , c afssa, lermvd, fougères, france in 1999, 620 caeca from standard, export or free-range broilers and in 2000, 600 fecal samples from pigs, were collected in french slaughterhouses. prevalence of campylobacter jejuni and c. coli strains was 56.6% in standard, 51.3% in export and 80% in free-range broilers. in standard and export productions, the most often isolated species was c. jejuni , whereas c. coli was predominant in free-range production. 53.8% samples collected from pigs contained c. coli . the sensitivity of strains to ampicillin, nalidixic acid, enrofloxacin (broilers) or ciprofloxacin (pigs), tetracycline, erythromycin and gentamicin was tested by an agar dilution method. in broilers, the percentages of resistant strains were, respectively 22, 25, 17, 57, 0.3 and 0% for c. jejuni and 29, 43, 40, 70, 31 and 0% for c. coli . in pigs the percentages of resistant c. coli were, respectively 12, 21, 12, 83, 65 and 0%. in broiler production, significant differences between distributions of species or percentages of resistant strains were observed according to type of production or administrated antimicrobials. the enzyme dhps (dihydropteroate synthase) participates in the folate synthesis pathway, and is well recognized as the target for sulphonamides. the enzyme preceding dhps in this pathway, pppk (dihydropterin pyrophosphokinase), is another interesting candidate drug target. the metabolic role of pppk is to provide one of the substrates for dhps. earlier studies have suggested that pppk and dhps enzymes need to have physical contact with each other for full enzyme activity. studies of potential interactions between the enzymes have been initiated. so far, indication of a weak interaction has been detected in gelfiltration experiments and the two-hybrid system. to confirm these results, we are currently developing a method to study substrate channeling, as interfering with such interactions could lead to impaired growth and thus be used as inhibitory drugs. we have also cloned and sequenced the operons coding for the enzymes in the folate biosynthesis from different clinical isolates of streptococcus pyogenes . comparisons revealed some isolates with a mosaic structure in the operon, suggesting that horizontal transfer of genetic material has occurred. multi-resistance gene cluster on a plasmid in a clinical isolate of e. faecium pm163 werner g, hildebrandt b, klare i, witte w. department of nosocomial infections, robert koch institute, wernigerode branch, germany purpose: strain uw786 was isolated from an urine sample of a patient with a permanent catheter. the purpose of our study was to identify and localize the resistance determinants in this isolate. results: isolate uw786 was resistant to the following antibiotics: penicillin, ampicillin, gentamicin (high-level), streptomycin (highlevel), erythromycin, clindamycin, vancomycin, teicoplanin, ciprofloxacin, moxifloxacin, nourseothricin, rifampicin, and fusidic acid (lowlevel, mic0/4 mg/l); but showed susceptibilities to oxytetracycline, phosphomycin, chloramphenicol, trimethoprim/sulfamethoxazol, linezolid, and quinupristin/dalfopristin. hybridization, pcr and sequencing experiments localized a cluster consisting of several resistance genes in a composite element on a plasmid. the cluster included genes and transposons tn1546 (vana) á/tn917 (ermb) á/tn1505 (aade á/ sat4 á/apha-3). the plasmid itself was not transferable in filter-matings into a fusidic acid high-level resistant enterococcus faecium recipient while selecting either for erythromycin or vancomycin resistances. however, after transposing a tn916-related determinant into uw786, determinants became mobilizable with the help of the conjugative transposon. transconjugants were, besides others, high-level resistant to fusidic acid, but susceptible to penicillin and ampicillin. pfge of transconjugants demonstrated a pattern almost identical to the recipient but clearly different from the donor. conclusion: resistance genes in e. faecium could be arranged in a cluster and are mobile via mobilizable/transferable plasmids. lilli d, rivanera d, barbacini ig, lozzi ma, mancini c. department of science and public health, university la sapienza, microbiology, rome, italy aim: hepatitis g virus (hgv), a new rna virus that is parenterally trasmitted has frequentley been found in patients with chronic hepatitis c infection but its role in chronic liver desease is unknown. the aim of this study was to determine the prevalence of hgv infection in patients infected with hcv. ninety-eight patients infected with hcv were evaluated for the presence of hgv rna. the hcv genotypes distribution was 30 genotype 1b, 10 genotype 1a, 54 genotype 3a and four genotype 4c/4d. hcv rna and hgv rna were detected by rt-nested pcr. results: infection with hepatitis g virus was detected in 21 (21.4%) patients and 77 (78.6%) were hgv rna negative. none of our patients with genotypes 1a and 4c/4d results hgv rna positive. prevalence of hgv infection was 10% in patients infected with hcv genotype 1b and 33.3% with genotype 3a. conclusions: infection with hgv occurred frequently (21.4%) in this sample of patients with chronic hepatitis c. we observed a height prevalence of hcv/hgv coinfection in patients infected with hcv genotype 3a. this association with hcv genotype 3a was indipendent of the source of infection, infact some of our patients have not history of intravenous drug use. characterization of extended-spectrum beta-lactamase (esbl)mediated resistance in salmonella spp. from durban, south africa pm165 moodley p a , essack s b , gajee k a , sturm w a . a department of medical microbiology, nelson r. mandela school of medicine, school of infection, university of natal, durban, south africa , b school of pharmacy and pharmacology, university of durban westville, durban, south africa background: gastroenteritis is a common condition among the paediatric population presenting to king edward viii hospital in durban, south africa. from july 2001, we noticed that the susceptibility of the salmonella spp. isolated from stool samples among these children were resistant to multiple antibiotics. aim: to characterize the phenotype of the resistance mechanisms involved. methods: minimum inhibitory concentrations (mics) of ampicillin, azithromycin, ciprofloxacin, cefepime, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, chloramphenicol, cotrimoxazole and gentamicin were determined by means of the agar dilution method. isolates were subjected to the e -test for extended-spectrum betalactamase (esbl) production. isoelectric focusing was performed as a preliminary step in enzyme characterization. results and conclusion: thirty isolates of multiresistant salmonella spp. were obtained. antibiogram typing revealed six different resistance phenotypes. all isolates depicted ceftazidime/ceftazidime á/clavulanate ratio of !/8 and were considered putative esbl-producers. isolates expressed 1 á/3 beta-lactamases each with pi values ranging between 5 and 8.2 indicative of tem-, shv-and/or ctx-m-related esbls. nine isolates expressed two beta-lactamases each and two isolates expressed three beta-lactamases each. there was evidence of the simultaneous expression of both tem-and shv-derived esbls as well as the simultaneous expression of multiple tem-or shv-derived esbls in single isolates, a phenomenon reported in esbl-positive klebsiella pneumoniae isolated at the same hospital. neutrophils exhibit reduced chemiluminescence response to serum opsonized klebsiella pneumoniae producing extended spectrum b-lactamases (esbl) pm166 objective: to investigate the ability of esbl and non-esblproducing klebsiella pneumoniae isolates treated with human serum to induce a chemiluminescence response in neutrophils. methods: oxidative burst induced by the interaction of esbl (n0/ 81) and non-esbl-producing (n0/152) klebsiella pneumoniae isolates with neutrophils from healthy individuals was monitored by measuring the chemiluminescence response (cl). pooled sera from healthy individuals served as source of complement for pretreatment of the bacteria. cl responses triggered by serum treated zymosan served as positive control. the serum opsonized klebsiella strains were arbitrarily graded as high (h) and low (l) inducers of cl when the cl response induced by the bacteria was cl b/60 and !/60%, respectively, of that induced by opsonized zymosan. results: out of 152 non-esbl-producing klebsiella isolates, 61.8% induced high cl response in neutrophils whereas only 42% of 81 esbl-producing klebsiella isolates did so (pb/ 0.02). conclusions: strains harboring the esbl plasmid were more virulent than non-esbl-producing strains by virtue of their higher tendency to escape serum-dependent recognition by neutrophils. osiris */an automated system for susceptibility testing in agar diffusion technique pm167 chegrani f, kolbert m, shah pm. universitätsklinik frankfurt, zentrum der inneren medizin med iii schwerpunkt infektiologie, frankfurt am main, germany objective: osiris measured zone sizes were compared to manually measured inhibition zones using round 90 (rp) and 120 mm mueller hinton square agarplates (sqp). variations of 9/3 mm in zone size measurements were defined as tolerable. 'very major errors' (vme) were defined as classification of a resistant organism as sensitive by osiris. thirty thousand two hundred and ninety-eight single measurements testing 42 antibiotics on 352 staphylococci and 113 enterobacteriaceae were done according to the din 58940 recommendations. results: vancomycin, rifampicin, gentamicin gave the best results on rp with a concordance of 96, 88 and 86%. vancomycin, rifampicin, teicoplanin performed best with 97, 94, 93% on sqp testing staphylococci . worst results on rp gave cefuroxim (43.8%) and fosfomycin (55.1%), on sqp fosfomycin (36.4%), ofloxacin (88.1%). for enterobacteriaceae amikacin (100%), gentamicin (100%), ciprofloxacin (100%) performed best on rp; worst nalidixinacid (32.5%), piperacillin (82.5%). concordance on sqp amikacin (100%), cefotaxim (100%), gentamicin (100%), nitrofurantoin (72.5%), cotrimoxazol (90%). very major errors were seen in b/1% of all test performed. interpretation: osiris is a rapid and reliable system for susceptibility testing with round and square agarplates and has an excellent expert system. altindis m a , aktepe oc a , kocagoz t b . a kocatepe university school of medicine, microbiology, afyon, turkey , b diomed inc. tr, istanbul, turkey dio-bacit, in a two section plate, that contains 5% sheep blood agar on one side and sheep blood agar with bacitracin (2 mg/ml) was compared for its efficiency in identification of group a beta hemolytic streptococcus (gabs) with other two different growth plates, one containing 5% sheep blood agar with bacitracin (b) and the other containing b-sxt. we used latex-agglutination for this comparision. throat specimens obtained from 490 cases were inoculated to dio-bacit plates, first to one side with 5% sheep blood agar and to the other side with b. after an overnight inoculation at 37 8c, colonies with beta hemolysis an 5% sheep blood agar but no growth with b, were inoculated to 5% sheep blood agar again and antibiogram identification discs containing 0.04 u b and 1.25 and 23.75 mg sxt (oxoid, uk) were placed onto the plate and incubated overnight at 37 8c. after that, colonies with beta hemolysis were defined as b-sensitive while colonies resistant to sxt were defined to be gabs. all colonies are serologically classified by latex-agglutination (oxoid, uk). seventy-one (14.5%) inoculations revealed growth of gabs at dio-bacit plates. after inoculating these colonies to 5% sheep blood agar, 72 of them were found to be sensitive to b, while 67 were found to be sensitive to b but resistant to sxt and 58 of them were defined as gabs by latex test. when compared with latex-agglutination test, we found dio-bacit method's sensitivity and spesificity to be 92 and 96.9%, respectively. method: agar diffusion technique as recommended by din 58940 was used to determine izs (read using aura and manually) for 178 staphylococci and 22 enterobacteriaceae . variations in automated measured zone sizes of 9/3 mm to the manual readings were considered to be within acceptable range. results: six thousand and fifty-two zone sizes were determined for staphylococci and 748 for enterobacteriaceae . mha displayed tendency to smaller zone sizes in automated readings than isa, as well in staphylococci and enterobacteriaceae . on the other side automated readings presented on isa more precise results than mha. overall less major discrepancies ( b/3 mm) were found on isa. izs were generally smaller on mha. the tables below show differences in manually and automated measured zone sizes on different media and species. we discovered seven more cases of resistance in the case of metronidazole. we did not have such experience with clarithromycin. conclusion: our results show that e -test is comparable to ad for clarithromycin, but for metronidazole our findings confirm nccls recommendantion. classical ad is time consuming for every day use in the laboratory. the use of screening agar plate with 8 mg/ml of metronidazole to detect possible resistance could be the solution. rokosz a a , sawicka-grzelak a a , meszaros j b , luczak m a . a department of medical microbiology, the university medical school, warsaw, poland , b department of bacteriology, state institute of hygiene, warsaw, poland purpose: to identify esbl-positive strains and to compare two methods applied for the detection of extended-spectrum beta-lactamases (esbls). methods: two hundred and sixty strains of gram-negative rods were cultured from clinical specimens from hospitalized patients. identification of strains was performed in the automatic atb system (biomerieux, france). these strains were identified as esbl-positive on the basis of the double-disc synergy test (ddst according to jarlier et al., 1988) results. all strains were also determined using a novel method of esbl detection (dd, diagnostic disc) according to appleton (1999) . two discs were applied in this test: cpd (cefpodoxime) and cd 01 (cefpodoxime/clavulanic acid) (oxoid, england). results: consistent results of two methods (ddst and dd) were obtained in the case of 166 from among 260 of examined strains (60.4%). consistent results concerned 161 out of 222 strains of enteric rods (72.5%) and only five from among 38 other strains (mostly nonfermenting rods). conclusions: the novel method of esbl-producers detection (dd) is more objective and easier for interpretation than the double-disc synergy test (ddst). diagnostic disc test should be used as the basic one or to confirm the results of ddst in difficult cases. assessment of e -test for determining penicillin resistance in pneumococci pm172 sener b, yeniþehirli g, ercis s, hasçelik g. department of clinical microbiology, hacettepe university medical faculty, ankara, turkey there is a greater need for susceptibility testing methods that distinguish between susceptible and resistant pneumococci. an alternative method could be the e -test, which is compared with the reference agar dilution method in this study. penicillin susceptibility of a total of 149 pneumococci was determined by e -test and agar dilution methods. streptococcus pneumoniae atcc 49619 and enterococcus faecalis atcc 29212 were used as controls. the results were given in the effect of anoxic conditions on the minimum inhibitory concentration of metronidazole in helicobacter pylori pm173 de la obra sanz p, lomas e, roman jl, alarcon t, lopez-brea m. the objective of this study was to determine the effect of incubation under anoxic conditions on the metronidazole resistance of helicobacter pylori . methods: a total of 35 clinical isolates were used in this study. mics were determined by an agar dilution method using mueller-hinton agar plus 7% lysed horse blood. three plates series contained twofold dilutions of metronidazole from 256 to 0.008 mg/l were prepared. the first one was incubated under microaerophilic conditions (oxoid) for 3 days; second and third series were incubated anaerobically (anaerobic system, oxoid) for 8 and 18 h, respectively, and were then transferred to the microaerophilic enviroment up to complete 3 days of incubation. results: with microaerophilic incubation, 12 of 35 strains were resistant (mic50 and mic90 were 1 and 32, respectively). with 8 h anaerobic preincubation, four of 35 strains were resistant (mic50 and mic90 were 0.5 and 4, respectively). with 18 h anaerobic preincubation, 1 of 35 strains was resistant (mic50 and mic90 were 0.25 and 1, respectively). conclusions: anaerobic preincubations causes an increase in sensitivity to metronidazole, the extent of which was dependent on the length of the anaerobic period. methods: the susceptibility to antibiotics was performed by microdilution method according to nccls guidelines. the production of b-lactamase was tested by nitrocefin sticks (oxoid). results: the mics50/mics90 (mg/ml) appeared, respectively: ampicillin 1/4, amoxicillin/clavulanic 0.06/0.12, cefaclor 1/1, ceftriazone 0.06/0.5, erythromycin 0.12/0.25, azithromycin 5/0.03/0.06, clarithromycin 0.12/0.12, ciprofloxacin 0.03/0.06, imipenem 5/0.015/0.06, tetracycline 5/0.25/5/0.25, trimethoprim/sulfamethoxazole 0.25/0.25. b-lactamase was detected in 98.5% of the strains. conclusions: (1) m. catarrhalis isolates were uniformly susceptible to all tested antimicrobials except ampicillin. (2) the production of blactamase was responsible for ampicillin resistance (98.5%). (3) m. catarrhalis strains had almost the same behavior 'in vitro' to the tested microlides (erythromycin, azithromycin, clarythromycin). rokosz a, sawicka-grzelak a, luczak m. department of medical microbiology, the university medical school, warsaw, poland purpose: to isolate, identify and determine the drug-susceptibility of fungal strains cultured from fecal samples routinely submitted for detection of clostridium difficile and its toxins in cases of antibioticassociated diarrhea (aad). methods: one hundred fecal samples from hospitalized patients were examined (may á/october 2001). c. difficile toxins a/b were detected directly in stools with c. difficile tox a/b ii test (techlab † , usa). fecal specimens were inoculated on ccca and candida id (biomerieux, france) media. c. difficile and fungi were identified with standard microbiological procedures. susceptibility of fungal strains to anti-fungal agents was determined (atb fungus, biomerieux, france). results: c. difficile toxins were detected in 38 and c. difficile strains were isolated from 23 of examined specimens. sixty-two fungal strains of 8 genera were cultured from 50 stool samples (24 c. albicans isolates). massive fungal growths were observed on primary plates in all cases. fifty-five fungal strains were susceptible to nystatin, 53-to 5fluorocytosine, 52-to amphotericin b, 45-to ketoconazole, 43-to miconazole and 39-to econazole. conclusions: in some cases of antibiotic-associated diarrhea fungal strains are responsible for symptoms of this disease. certain persons having aad should be treated with anti-fungal agents. results: a total of 291 isolates (33.2%) were penicillin nonsusceptible (intermediate and resistant) and 308 (35.1%) were erythromycin non-susceptible. non-susceptibility to both antibiotics was found in 183 (20.9%). conclusions: if penicillin administration eliminates all penicillin susceptible strains, the prevalence of penicillin non-susceptible strains will increase as well as the erythromycin non-susceptible ones. this means that the proportion of erythromycin non-susceptible strains should increase from 35.1 to 62.9%. at the same time, if erythromycin eliminate all susceptible strains to this antibiotic, the prevalence of penicillin non-susceptible strains would increase from the initial 33.2 to 59.4%. these data can explain the co-selection results observed in different surveillance studies. antimicrobial susceptibility and capsular types/groups of streptococcus pneumoniae isolates causing pneumococcal diseases in bulgaria pm178 setchanova l a , gergova r a , ioneva m b , sredkova v c . a department of microbiology, medical university, sofia, bulgaria , b department of microbiology, ii city hospital-sofia, sofia, bulgaria , c department of microbiology, faculty of medicine, pleven, bulgaria a prospective study of pneumococcal infections was performed in cooperation with five clinical microbiology laboratories in bulgaria. mics values to 12 antimicrobials and serotype/serogroup distribution were determined for 242 strains of streptococcus pneumoniae . pneumococci were isolated from patients with systemic or respiratory infections. the incidence of penicillin g-intermediate and penicillin gresistant isolate was 27.3 and 10.3%, respectively. the rates of resistance to other antimicrobials were: cefotaxime/ceftriaxone */ 6.2%; erythromycin */21.5%; clindamycin */7.8%; tetracycline */ 24%; chloramphenicol */26.4%; trimethoprim/sulfamothoxazole */ 38%; ciprofloxacin */10.7%; rifampin */3.7%. the s. pneumoniae isolates belonged to 19 capsular types/groups. the most common serotypes/serogroups in bulgaria are 1, 3, 5, 6, 9, 14, 19 we aimed to determine the pneumococcal antibiotic resistance rates and the serotypes of those resistant isolates in our hospital. the mic values of 212 isolates (year 1996 á/2001) were determined by agar dilution method. serotyping was performed by using 12 pooled antisera of the pneumo-test. the results were as follows (n 0/212). objectives: to asses the antimicrobial susceptibility of clinical isolates of pseudomonas aeruginosa obtained from 1997 to 2000 and to monitor trends in antimicrobial resistance. methods: mics were determined by microdilution testing according to nccls. the antibiotics tested were: ceftazidime (caz), aztreonam (atm), imipenem (imp), gentamicin (cn), tobramycin (tb), amikacin (ak) and ciprofloxacin (cip). results: a total of 1293 isolates were included. urine was the most common site of isolation for outpatients isolates (61.2%) while for hospitalized patients respiratory samples were the most frequent (40.2%). susceptibility to antibiotics was: 90% caz, 87.5% atm, 93% imp, 76.8% cn, 95.5% tb, 96.7% ak and 81.9% cip. comparison of susceptibility data through 1997 á/2000 showed that the increase in the resistance rate was significative for caz (4.6 vs 12.9%), atm (9.9 vs 18%), cn (18 vs 26%), tb (1.85 vs 6.9%), ak (1.6 vs 5.7%) and cip (15.17 vs 21%). significant differences were found under the following circumstances: isolates from intensive care units and from inpatients were significatively more resistant to caz, atm and imp. isolates from respiratory samples were more resistant to caz and atm and isolates from urine samples were more resistant to cip. conclusions: although the antimicrobial susceptibility level has been decreasing p. aeruginosa isolates still show good susceptibility percentages for all antibiotics tested. antimicrobial susceptibility testing of clinical isolates of bordetella pertussis : report on 27 isolates from rouen, france pm181 lemee l a , nouvellon m a , caron f b , lemeland jf a . a chu rouen, bacteriologie, rouen, france , b chu rouen, maladies infectieuses et tropicales, rouen, france reports of an increased clinical incidence of pertussis and the development of resistance by bordetella pertussis to erythromycin prompted the collection and antimicrobial susceptibility testing of recent clinical isolates from patients, who were hospitalized in rouen between 1991 and 1999. mics of nine antimicrobial agents (erythromycin, josamycin, spiramycin, roxithromycin, ketolide hmr 3647, cotrimoxazole, ciprofloxacin, rifampicin and amoxicillin) were measured by agar dilution method on mueller-hinton agar containing 5% horse blood. mbcs of erythromycin and rifampicin were also determined against four isolates of b. pertussis . all isolates were fully susceptible to the nine antimicrobial agents tested. mics 90 (mcg/ml) were 0.03 for erythromycin, ketolide hmr 3647 and ciprofloxacin, 0.06 for josamycin, 0.25 for spiramycin, roxithromycin and rifampicin, 0.5/2.5 for cotrimoxazole, and 1 for amoxicillin. mbcs (mcg/ml) were 0.125 á/0.5 for eyrthromycin and 0.5 á/1 for rifampicin. in conclusion, our isolates of b. pertussis remain extremely susceptible to all antimicrobial agents tested, especially macrolides. no resistance was detected. finally, if erythromycin remains the molecule of choice, other macrolides (c14 and c16) also confirm their good in-vitro activity. in addition, the good in-vitro potency of rifampicin, together with its great diffusion within the respiratory tract, suggests that rifampicin has potential clinical efficacy in pertussis too. the emergence of streptococcus pneumoniae (sp) with diminished susceptibility to penicillin g (psdp) suggests the use of other antibiotics such as newer fluoroquinolones (fq). the resistance phenotypes of 252 consecutive pneumococcal strains isolated from patients of four hospitals (observatoire régional des pneumocoques du nord-pas de calais) were studied: 39 strains were susceptible to penicillin g and 213 were psdp. reference strains provided from the centre national de réfeacute;rence des pneumocoques were added to the study. the activity of pefloxacin, ciprofloxacin, norfloxacin, sparfloxacin, levofloxacin and moxifloxacin was studied. reserpine was used to detect the efflux phenotype. methods used were performed according to the recommendations of the comité de l'antibiogramme de la société française de microbiologie. for each strain, the resistance phenotype to fq was deduced by comparison of mics or diameters obtained with those obtained with the reference strains of known phenotypes. fq resistance phenotypes were not correlated to blactam agent susceptibilities. wild type phenotype was observed among 76.9 and 77.5% of the susceptible and psdp strains, respectively. a 'wild efflux' mechanism, deduced by addition of reserpine to norfloxacin, represented the predominant phenotype. it was detected among sp susceptible to penicillin g (23.1%) as well as among psdp (17.4%). the aim of this study was to determine macrolide resistance phenotypes of sp isolated in three french departments (alpes maritimes, doubs, nord) from nasopharyngeal aspirates of children aged 3 months to 3 years attending a dcc. a random sample of children attending randomly selected dccs was obtained during three periods (spring, autumn and winter 1999) in each department (10 494 children attending dccs and 2565 children sampled). analysis of macrolide susceptibility of sp strains was performed using the ca-sfm method. out of 1262 strains, 64.7% had decreased susceptibility to penicillin (spdp) and 77.5% were resistant to erythromycin. the triple disk diffusion method (erythromycin (e), clindamycin (cl) and spiramycin) was used to determine resistance phenotypes. macrolide resistance is a well known phenomenon in france and is confirmed by our study. these results show that the constitutive phenotype is predominant as in other parts of europe and the frequency of efflux mechanism is lower than that observed in the usa and canada. developing antibiotic resistance surveillance of helicobacter pylori in england and wales pm184 elviss nc, owen rj. central public health laboratory, laboratory of enteric pathogens, london, uk purpose: helicobacter pylori antibiotic resistance is a key contributing factor in â/10% of infected patients failing drug treatment. our aim was to survey rates of primary in-vitro resistance at different locations, and links to disease severity. antral gastric biopsies/cultures were received from phls in chelmsford, mid-essex (1115 isolates 1995 á/2001); london (103 isolates 1999 á/2000); and bangor, north wales (165 isolates1999 á/2001). susceptibilities to metronidazole (mtz), clarithromycin (cla), tetracycline (tet) and amoxicillin (amx) were tested by disc diffusion and also by e -test for cla and mtz. results: overall resistance rates (1383 isolates) were 38% for mtz and 8% for cla. all were susceptible to amx and tet. dual resistance rate was 5%. breakdown by location showed some marked differences. mtz resistance was highest in london (63%) compared to 37% in chelmsford and 20% in bangor. by contrast cla rates were 11% for london, and about 5% for bangor and chelmsford. in london, the majority of mtz resistant isolates were from non-uk borne individuals (75% non-uk vs 25% uk). comparison of duodenal ulcer-associated isolates with those from non-ulcer patients indicated similar rates of mtz resistance (28%). conclusion: resistance rates may vary significantly between locations depending on the local population with non-uk birth being a key risk factor for primary resistance with a mtz resistant strain. local resistance rates should be taken into account in test and treat strategies. potrykus j, benetkiewicz m, wegrzyn g. department of molecular biology, university of gdansk, gdansk, poland purpose of the study : because of their ability to extrude a wide range of compounds, multidrug efflux pumps have recently become an important issue in combating bacterial infections. acrab-tolc is the major efflux system of escherichia coli . we investigated the effect of acra on plasmid-borne and intrinsic chloramphenicol, tetracycline and ampicillin resistance. results and conclusions: recently, we reported a chloramphenicol sensitivity of e. coli mutant expressing cat , the chloramphenicol resistance gene. the strain was shown to bear a nonsense mutation in the acra gene. our studies indicate that this mutation is, at least in part, responsible for the observed chloramphenicol sensitivity phenotype. the mutation seems also to influence the strain's susceptibility to ampicillin and teta (c )-mediated (plasmid-borne) tetracycline resistance. although the teta(c) protein retained its biological function, there was a considerable growth impairment of the mutant strain when cultured in tetracycline containing medium. deletion of the acrab locus prevented any growth in the presence of tetracycline. upon the addition of ampicillin, the mutant underwent lysis more rapidly than the control strain. such was also observed in acrab deletion derivatives of other e. coli strains. we are trying to elucidate the role of the acra gene product in the phenomena described above. existence of efflux pumps in wild type isolates of drug-resistance bacteria pm186 raja ray rr. medical microbiology and parasitology, calcutta university, kolkata, india efflux pumps possessed by the bacterial cells of different kinds of bacteria had presented as a newer mode of drug resistance in many organisms. the capacity of bacterial cells to cause outward flow of noxious agents was known, however, for a considerable time with respect to tetracycline. recently, interest in the efflux pump system has brought to light some previously ill-understood mechanisms of drug resistance, involving noxious agents, toxins or poisons. we have found high level of resistance in pseudomonads towards cetrimide and other germicides for which no definite chromosomal/plasmid-mediated genes/mechanisms could be identified. likewise, occurrence of nonantibiotic sensitive vibrios, staphylococci and pseudomonads in the background of their high level of resistance to most of the common antibiotics suggest a mechanism of interference with the efflux pump, which accounts for such sensitivity in such cases. involvement of multiple resistance of marine isolates of v. parahaemolyticus to numerous clinically used antibiotics to which they have never been exposed also suggests a possible role of efflux pumps in determining such resistance */that these can simultaneously develop against multiple marine toxins/poisons and other noxious agents. interaction between oxacillin and glycopeptides in a teicoplanin-resistant mutant of staphylococcus epidermidis with reduced susceptibility to vancomycin pm187 greco aa, ben hassen a. laboratory service of national bone-marrow transplant center, tunis, tunisia we selected a laboratory-generated mutant of staphylococcus epidermidis capable of growing in the presence of 256 mg/l of teicoplanin (353b tm256), from a methicillin-resistant (mic!/256 mg/l), teicoplanin-sensitive (mic 4 mg/l) and vancomycin-sensitive (mic 2 mg/l) clinical isolate of s. epidermidis (353b so). in a previous work, we studied the different phenotypic characteristics acquired by the teicoplanin-resistant mutant 353b tm256 (20th interdisciplinary meeting on anti-infectious chemotherapy, december 2000, poster sessions, 82/p1). in this work, we examined the interaction between oxacillin and glycopeptides against this teicoplanin-resistant mutant of s. epidermidis with reduced susceptibility to vancomycin. to study the combined antibiotic activity of oxacillin and glycopeptides, we used different methods: a modified disk diffusion test, the e -test, time-kill assays and population analysis profiles. the synergistic activity of glycopeptides in combination with oxacillin against the teicoplaninresistant mutant 353b tm256 was demonstrated with a bactericidal effect. no synergy was seen against the parental strain 353b so. moreover, the synergy between glycopeptides and oxacillin occurred with suppression of the subpopulation with the highest level of glycopeptides resistance. we concluded that combination of glycopeptides and oxacillin may be a possible alternative in the treatment of infections caused by methicillin-resistant, teicoplanin-resistant s. epidermidis . compositional changes in microcosm biofilms induced by application of minocycline: a preliminary study pm188 the aim of the study was to observe the effect of application of minocycline upon microcosm dental plaques. the plaques were cultivated in a constant departmenth film fermentor (cdff), which produces biofilms under conditions mimicking those present in vivo. the composition of the biofilms was determined by viable counting on selective and non-selective media. the proportion of antibiotic resistant genera within the biofilm was determined by viable counts utilising media containing minocycline (16 mg/ml). before commencing antibiotic pulsing, the biofilms had a total viable anaerobic count of 1.26)/10 10 cfu per biofilm, with negligible (6 cfu/biofilm) minocycline-resistant bacteria. however, 24 h after introduction of the antibiotic, the total count had been reduced to 1.02)/10 9 cfu/biofilm whilst the number of minocycline-resistant bacteria had risen to 1.23)/10 6 cfu/biofilm. at the final sampling time point (192 h) the total viable anaerobic count was 5.82)/10 8 cfu/biofilm whilst the number of minocycline-resistant bacteria was 1.12)/10 7 cfu/biofilm. hence, there is a very low basal level of inherent resistance to minocycline within microcosm dental plaques, but this increases considerably once the biofilms are exposed to minocycline. mechanism of resistance to aminoglycosides (amg) e. coli isolated from children with community-acquired urinary tract infections (cau-tis) pm189 methods: during the 2000 á/01 years nine centers took part in the study. the mics of antimicrobials were determined by the agar dilution method as described in the nccls guidelines. results: a total of 710 consecutive urine isolates from 692 children aged 1 month to 18 years with cauti were collected. the most frequently isolated species from children with cauti was e. coli (52.3%), followed by klebsiella spp. (8.0%) and proteus spp. (7.6%). results of the in vitro susceptibility testing of e. coli to amg are shown in table below. resistance of the strains was conditioned on production of amg-modifying enzymes. there has been found following phenotypes among resistance strains: gentamicin á/ tobramycin á/netilmicin (77.8% */aac(3)-v and 13.9% */aac(3)-iv enzymes) and gentamicin á/tobramycin (8.3%, due to ant(2ƒ) enzyme). conclusion: amikacin is most active amg against e. coli . resistance to gentamicin and netilmicin was mainly determined by production of aac(3)-v enzyme. effect of b-lactamase inhibitors (b-l-i) on the evolution of resistance (r) to b-lactams (b-l) in gram the b-lactams antimicrobials still are the most frequently used. among the bacteria responsible of high resistance to b-lactams are gramnegative rods; its most frequent mechanism is the production of b-lactamase. the use of b-l-i has reversed partially this mechanism of resistance. we expect changes in the frequencies of r using b-lƒci after more than 10 years. since 1988, the venezuelan group of bacterial resistance, with 29 health institution in the country; analyse and publish data on bacterial resistance of isolates from patients with bacterial infection coming from hospitals. it was used diffusion disk, according nccls. the software program whonet (world health organization net) was used. we follow the trends of r of gram-negative rods to b-l alone and with b-l-i during the decade 1988 á/1998. statistical analyses were made by evaluating the differences among percentages of resistance between the two series (p 5/0.05). results and discussion: the difference in r between b-l and b-l/b-l-i are: (1) piperacillin, piperacillin/tazobactam: between 10 and 30% of r for most isolated, except for escherichia coli (45%) and serratia spp. (60%); (2) ampicilin, ampicilin/sulbactam: between 10 and 30%; (3) cefoperazone, cefoperazone/sulbactam: between 5 and 25%. how is expected gramnegative rods resistance to b-lactams with a b-l-i is lower than the b-lactam alone; furthermore the difference between both series, grows higher with time. these results are relevant and they were not expected, since b-l-i have been shown to be b-lactamase inductors. trends in the resistance (r) to b-lactams and others antimicrobials in p. aeruginosa in venezuelan medical centres. nosocomial (nos) and communitarian resistance pm191 in order to approach the infection produced by resistant bacteria, it is convenient to consider the hospital and the community as two separate ecosystems. the hospital ecosystem has special relevance in the infection and r of gramnegative aerobic bacilli. today, they are the main responsible of nos infection, with special reference to pseudomonas aeruginosa . infection by resistant bacteria is a world wide problem, specially related to nos. since 1988, the venezuelan group of bacterial resistance, with 29 health institution in the country; identify, analyse and publish data on bacterial r to antimicrobials: b-lactams, quinolones and aminoglicosides of isolates from patients with bacterial infection coming from hospitals and the community. it was used diffusion disk, according nccls. the software program whonet (world health organiza-tion net) was used. statistical significance (p 5/0.05) was determined by application of the x 2 technique. we show significant differences in the r of p. aeruginosa nos and communitarian (highest differences: piperacilina 40/7%, tobra 40/4%). we also established significant differences between the r arising in public hospitals and private hospitals (highest differences: ceftazidime 50/5%, amika 25/ 5%). we show the tendency in decreasing of frequency of r since 1998; this is more evident in private hospitals (b-lactam and aminoglicosides). new antimicrobials and new mechanism of action, and in the future the new technology will solve today's problem. however, the most important tools we have today are prevention and antimicrobials, and we must make them suitable. susceptibility to antibiotics of enterobacter cloacae and citrobacter freundii from drinking water pm192 quintera sm, sousa jc, peixe l. department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal the increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the number of antibiotic resistant bacteria present in aquatic environment. our objective was to determine the antimicrobial susceptibility, with focus on b-lactam resistance, among enterobacteriaceae strains isolated from raw drinking water samples. several isolates (n0/107) of enterobacter cloacae and citrobacter freundii obtained from drinking waters were screened for antibiotic susceptibility patterns, using the agar diffusion technique, according to nccls's procedures. only 55% of e. cloacae strains, as well as 37% of c. freundii strains show resistance to amoxicillin and amoxicillin/ clavulanic acid. a reduced incidence of resistance to several others antibiotics was also observed. the obtained results suggest that strains isolated from raw drinking water have greater susceptibility to antimicrobial agents than pathogenic strains from hospital or outpatients infections. the 'natural' antimicrobial resistance phenotypes, usually described for c. freundii and e. cloacae , only seem to apply to strains isolated from human infections. notwithstanding the high susceptibility of the tested isolates to b-lactams, the role of environmental bacteria as a reservoir of resistance genes justify its periodical monitoring as a valid index for resistance spreading. a snapshot of the soil. using bacterial communities for tracing the evolution of metal-resistance pm193 quintera sm a , sousa jc a , peixe l a , monteiro nm b . a department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal , b department of zoology and anthropology, faculty of sciences, university of oporto, oporto, portugal it is well known that pathogenic bacteria, specially those resistant to antimicrobial agents and heavy metals poses public health risks of great concern, and its detection, namely in soils is generally related to pollution. in this study, the heavy metal resistance patterns of the microflora isolated from polluted (dump area) and unpolluted soil environments were examined. the plate growth covering percentage in the soil samples was determined using mueller-hinton plates supplemented with different heavy metal (al3'/, cd2'/, cu2'/, pb2'/, hg2'/ and zn2'/) concentrations. parallelly, using icp-aes, it was possible to ascertain the real heavy metal concentration for each soil sample. we found that the percentage of plate growth covering from the used samples was closely linked to the level of chemical pollution measured for each location. moreover, using anova, we found significant differences between locations. the dump site showed the highest tolerance to all the tested metals (newman á/keuls test). this pattern of results was consistent when using the data from the icp-aes. furthermore, it was possible to observe that pseudomonas spp., with a relatively high mic for the studied metals, might become a relevant model for both public health issues and eco-toxicological studies. biochemical characteristics of environmental isolates of listeria monocytogenes pm194 moshtaghi h a , garg sr b , mandokhot uv b . a shahrekord university, food hygiene, shahrekord, islamic republic of iran , b haryana agricultural university, food hygiene, hisar, india purpose: the investigations were carried out to study the biochemical reactions of listeria monocytogenes isolated from different sources in the environment. results: a total of 12 isolates of l. monocytogenes were obtained from 230 samples of agricultural soil, faecal matter of animals and sewage. all the isolates were gram-positive, small rods, catalase positive, oxidase negative, motile with tumbling motility in hanging drop at 22 á/25 8c, aerobic, facultative anaerobic, fermentative and produced acid from glucose. all the isolates of l. monocytogenes were beta haemolytic and positive for camp reaction with staphylococcus aureus . all the isolates were negative for phenyl alanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonate utilization and beta galactosidase tests. these were also negative for acid production from arabinose, d-xylose, mannitol, soluble starch and sucrose but acid was produced in rhamnose, salicin, and trehalose. hydrogen sulfide production was recorded in tripticase soy broth with lead acetate paper strips but negative with triple sugar iron agar. all the isolates were found to hydrolyse aesculin. out of 12 isolates of l. monocytogenes only two produced acid from lactose. in serotyping all the isolates were serotype 4b. conclusion: we can conclude that l. monocytogenes serotype 4b at least in fermentation of lactose shows different reactions. methods: the samples were pre-enriched in bhi broth with and without vancomycin (6 mg/l) and then plated onto m-enterococcus agar with and without antibiotics: vancomycin (6 mg/l), gentamicin (125 mg/l), kanamycin (500 mg/l), and streptomycin (1000 mg/l). representative colonies of each morphology were isolated and identified as enterococcus sp as previous described. pcr was used to identify e. faecium and e. faecalis and to characterise vancomycin resistant genotype. api20strep was also used in the identification. susceptibility testing to 11 antibiotics was performed by an agar dilution method (nccls). results: three hundred and fifty-three enterococci were isolated from 92 of a total of 99 faecal samples (93%, n 0/92/99). the majority of enterococci were identified as e. faecium , e. faecalis and enterococcus sp. resistance to almost all antibiotics studied was observed: vancomycin */3.0%; teicoplanin */3.0; ampicillin */21.2%; tetracyclin */81.8; erythromycin */86.9%; ciprofloxacin */78.8%; chloramphenicol */54.5%; gentamicin */17.2%; streptomycin */ 78.8%; kanamycin */77.7%; linezolid */0%. the vancomycin resistant enterococci presented a vana genotype. conclusion: resistance to several common antibiotics used in therapy was observed among enterococci isolated from healthy human from community. many of these isolates presented multi-resistance. of concern is the presence of vana genotype among these populations that may constitute a reservoir of vancomycin resistant genes. antimicrobial resistance in tetracycline-resistant oral bacteria pm196 mercury release from dental amalgam may select for mercuryresistant oral bacteria. mercury resistance is often associated with multiple antibiotic resistances. the aims of this study were to determine whether tetracycline-resistant oral bacteria from children with and without amalgam fillings were also resistant to: (a) mercury; and (b) multiple antibiotics. tetracycline-resistant organisms were isolated on iso-sensitest/blood agar containing tetracycline (8 mg/ml). the mic of hgcl 2 and several antibiotics were determined using agar dilution (bsac). one hundred and three organisms were isolated from patients without amalgam. ninety-one were streptococcus species, seven neisseria species, three veillonella dispar and two rothia species. fifty-seven percent exhibited resistance to at least one antibiotic, 12% were mercury-resistant, 42% were penicillin-resistant, 11% were ampicillin-resistant and 23% erythromycin-resistant. fiftytwo organisms were isolated from patients with amalgam. forty-five were streptococcus species, five neisseria species, one v. dispar and one staphylococcus aureus . sixty-three percent exhibited resistance to at least one antibiotic, 23% were mercury-resistant, 38% penicillinresistant, 11% were ampicillin-resistant and 31% showed erythromycin-resistance. statistically, the results showed that in tetracyclineresistant organisms, the presence of dental amalgam did not affect the level of resistance to mercury or to the antibiotics tested. conway-wallace hl a , mullany p a , bedi r b , wilson m a . a eastman dental institute, university college london, microbiology, london, uk , b eastman dental institute, university college london, transcultural oral health, london, uk the purpose of this study: to determine the prevalence of antibioticresistant oral bacteria in children who had not received antibiotics during the 3 months prior to sampling. plaque samples were obtained from 16 children aged 4 á/6 years and plated onto media containing: penicillin, ampicillin, tetracycline, erythromycin and vancomycin. resistant isolates were enumerated, sub-cultured and frozen for subsequent identification. the process was repeated 6 and 12 months later. the results obtained: bacteria resistant to each of the antibiotics were present in all of the children at each sampling time (except in the case of ampicillin and penicillin at 0 months). the proportion of antibiotic-resistant bacteria in the oral microflora ranged from ]/13.7 (erythromycin) to 5/0.5% (ampicillin). the proportions of bacteria resistant to a particular antibiotic remained reasonably constant over the 12-month sampling period. in only two cases (penicillin and ampicillin) was there a statistically significant change in the proportions of resistant bacteria at different time periods. the conclusion reached: the results of the study have revealed that bacteria resistant to a wide range of antibiotics may be isolated from children who have not been administered these agents during the 3 months prior to sampling. furthermore, in many cases the proportion of bacteria resistant to a particular antibiotic remains constant over a 12-month period. antimicrobial use in the intensive care unit: results of a pharmacoepidemiological study in italy pm198 periti p. e.i.f.t. srl, firenze, italy a retrospective survey of antimicrobial chemotherapy use in 560 intensive care units in italy was carried out in 1999 using a computerized questionnaire under the auspices of the journal of chemotherapy. of the icus contacted, 43.4% replied, being mainly general or post-surgical and pediatric units having a mean of 10 beds, nine doctors and 20 nurses. the antimicrobial agents used in these wards were almost always polychemotherapy with prevalent use of beta-lactams, aminoglycosides and glycopeptides or as empirical treatment during the first 72 h after hospital admission. the continual use of medium-high dose combinational antimicrobial chemotherapy was justified by microbiological testing, which revealed that more than one-third of bacterial pathogens were resistant. approximately, 60% of gram-positive bacteria were methicillin-resistant, whereas about 13% of gram-negative strains were resistant to at least one of the tested antibiotics. forty percent of the responding icus furnished microbiological testing data, of which three quarters indicated the incidence of chemoresistance of the isolated strains. fungal infections were less frequent than bacterial, the most commonly isolated agent being candida spp. in conclusion, the sample of icus examined showed adequate and reasonable use of antimicrobial agents, with heavy reliance on medium-high dose combination therapy due to the elevated incidence of resistant isolates found. plasma concentrations (p), urinary excretion (u) and bactericidal activity of gatifloxacin (gat) 400 mg versus ciprofloxacin (cip) 500 mg in 12 healthy volunteers after a single oral dose pm199 boy d a , kinzig-schippers m b , sö rgel f b , well naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, ibmp, nürnberg-heroldsberg, germany twelve volunteers received a single oral dose of 400 mg gat versus 500 mg cip to assess p up to 36 h, u (by hplc), and urinary bactericidal titers (ubt) in eight intervals up to 120 h. the mean pmax of gat/cip was 3.35/2.12 mg. the ucum (mean) for gat/cip was 81.0/43.2%. the ubts, i.e. the highest twofold dilution of urine still bactericidal, were determined for nine uropathogens and one reference strain */mics (mg/ml) (microdilution) for gat/cip: escherichia coli atcc 25922 (0.008/0.008); e. coli (0.06/0.06); klebsiella pneumoniae (0.03/0.016); proteus mirabilis (0.25/0.06); pseudomonas aeruginosa (1/ 0.125); s. saprophyticus (0.125/0.25); two strains of s. aureus (0.06/ 0.125); two strains of e. faecalis (0.5/1 and 8/32). the median ubts measured within the first 6 h for gatifloxacin were between 1:16 and 1:1024 for the five gram-negative strains (incl. p. aeruginosa ) and between 1:8 and 1:1024 for the five gram-positive strains. the median ubts for ciprofloxacin were between 1:64 and 1:1024 for the gramnegative strains (incl. p. aeruginosa ) and between 1:1.5 and 1:768 for the five gram-positive strains. for the ubts up to 12 h, gat was significantly superior to ciprofloxacin in all gram-positive strains, not different in the two e. coli strains, and inferior in the klebsiella , proteus and pseudomonas strains. for the ubts at 12 á/24 h, gat was generally superior to cip, but showed no difference in the proteus and pseudomonas strains. gat showed overall comparable urinary bactericidal activity as cip. this is in agreement with a clinical study performed previously. malaria is one of the most prevalent endemic infectious disease affecting humans. in bichat hospital 111 cases of malaria acute illness were reported during 2001. among them, 96 patients were hospitalised and intravenously treated by quinine. this retrospective study consisted of comparing the therapeutic drug monitoring (tdm) of quinine distinguishing, respectively 36 and 60 patients cured in infectious medical department (imd) and intensive care unit (icu) where a standardised quinine regimen was established (62 and 47% malaria attacks, respectively). in icu, the treatment consisted of an infused loading dose 16 mg/kg/4 h of quinine diluted in 5% glucose followed by 24 mg/kg/day. plasma quinine maximal concentrations were assessed after selective liquid á/liquid extraction and spectrofluorometry detection. statistical analysis was performed using t -test. results showed that patients had comparable weight (71.49/19.7 and 70.89/17.9 kg) but quinine doses and plasma concentrations were significantly different in icu and imd, respectively (20.29/6.5 versus 22.79/3.7 mg/kg/day, pb/ 0.001 and 11.49/3.3 versus 10.39/3.2 mg/l, p b/ 0.01). in icu and imd, respectively: 57 and 49% were in the therapeutic range (10 á/15 mg/l) with 29 and 43% below the requested therapeutic concentration (10 mg/l) and 13 and 8% above the limit of toxicity (15 mg/l) conveying the importance of tdm in intravenous quinine treatment to avoid infra-therapeutic or toxic concentrations. simultaneous central nervous system distribution using microdialysis and pharmacokinetic á/pharmacodynamic modelling of the electroencephalogram effect of norfloxacin in rats pm201 chenel m, marchand s, dupuis a, bouquet s, couet w. university of pharmacy, pharmacology, poitiers, france purpose: to investigate the epileptogenic activity of norfloxacin by a pharmacokinetic á/pharmacodynamic (pk á/pd) modelling approach and to assess the contribution of distributional processes across the blood á/brain barrier (bbb) to the delayed effect. methods: rats (n0/10) received an iv bolus dose of norfloxacin (150 mg/kg). convulsant effect was quantified by electroencephalogram (eeg) recording during 9 h post-dose. arterial blood samples were collected for drug assays in plasma. unbound norfloxacin concentrations were monitored in brain extracellular fluid (ecf) using microdialysis with in vivo calibration of the probes by retrodialysis with ciprofloxacin. results: the eeg effect reached its maximum between 70 and 190 min post-dose. a pk/pd effect compartment model was successfully fitted to these data. the relationship between effect and concentration at the effect site was best described by a spline function. norfloxacin concentrations in brain ecf were relatively low compared to plasma levels (ecf/plasma areas under curve (auc) ratio equal to 9.79/ 2.8%), but central distribution was rapid. therefore, the effect versus brain ecf concentrations curves still exhibited a marked hysterisis. conclusion: the delay observed between plasma concentrations and norfloxacin convulsant effect cannot be explained by a slow distribution of norfloxacin across the bbb. pagoulatou a a , kanellakopoulou k b , vafiadou m a , kostakopoulos th c , kastriotis i b , giamarellou h c . a department of anesthesia, sismanoglio general hospital, greece , b 4th department of internal medicine, athens medical school, athens, greece , c department of urology, athens medical school, athens, greece csf kinetics of van and fu were studied in 57 patients who underwent short urological surgery under spinal anesthesia. patients were excluded if they were already receiving an antibiotic or were suffering from renal and hepatic dysfunction. van was administered at 1 g over 1 h infusion. serum and csf samples were collected post-dose and the mean serum levels were as follows: 30 min á/1 h: 21.1 mg/ml (five patients), 1 á/2 h: 10.1 mg/ml (five patients), 2 á/4 h: 8.5 mg/ml (six patients), 4 á/6 h: 6.7 mg/ml (six patients) and 6 á/8 h: 5.15 mg/ml (seven patients). fu was administered at 500 mg dose over 1 h infusion. serum and csf samples were taken post-dose and the mean serum concentrations were found as follows: 0 á/30 min: 43.08 mg/ml (six patients), 30 min á/1 h: 41.7 mg/ml (six patients), 1 á/2 h: 33.8 mg/ml (five patients), 2 á/4 h: 27.8 mg/ml (six patients), 4 á/6 h: 25.6 mg/ml (five patients). in csf, both van and fu were undetectable. it is concluded that in the absence of meningeal inflammation van and fu do not penetrate (with the applied microbiological assay) the csf barrier. comparison of the pharmacology of intravenous and orally given moxifloxacin in an in-vitro model pm203 wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: the intravenous form (iv) of 400 mg moxifloxacin (mox), one of the newer fluoroquinolones, has been recently approved by the fda. during the iv treatment higher peak serum concentrations are achieved in comparison to the oral administration (po) of the same dose. the antibacterial activity of fluoroquinolones is concentration dependent. we therefore simulated human pharmacokinetics of single po and iv dosages of 400 mg mox in an in-vitro model using six different gram-negative and -positive pathogens to elucidate the different effect of these two dosing schedules. results: the comparison of the pharmacological parameter auc/ mic shows an increase ( table 1 ) that could predict an enhanced antibacterial effect. however, the analysis of the killing curves with the following parameters, ka.max (maximal killing activity) and aac (area above the killing curve between 0 and 24 h), reveals no major difference between the po and iv dosage. conclusion: the serum concentration after oral administration is already sufficiently high to show the optimal bactericidal effect of mox that can only be slightly increased by higher peak concentrations and higher auc/mic ratios. thus the concentration dependence is not linear but ends already at concentrations achievable by oral dosing and documents that auc/mic calculations cannot easily be translated into dosing schedules. background : bacteria growing in vivo multiply much more slowly than in vitro. whether the bactericidal activity of quinolones may be affected by an increase in generation time (g) was studied in batch cultures. methods: by limiting the nutrient supply, generation times were lengthened from approximately 0.45 to 1.5 h up to 3.9 h. alternatively, the quinolones were added to the bacterial cultures during the lag-, exponential-and stationary phase. recent clinical isolates of escherichia coli were exposed to multiples of the mics of ciprofloxacin or norfloxacin. the 'killing rates' were calculated in analogy to the growth rate. results: the bactericidal activity of the quinolones tested against e. coli was minimally influenced by the reduced generation time. ciprofloxacin concentrations of ]/2)/mic eliminated the test strains within 5/2 h from the test system if added during the lag or exponential growth phase; four times higher concentrations were needed to reduce cfus by 99% within 2 h, if added during the stationary phase. norfloxacin was significantly less active. conclusion: in contrast to norfloxacin, the bactericidal activity of ciprofloxacin is minimally affected by the generation time or growth phase of the bacteria. wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: moxifloxacin (mox) is one of the newer fluoroquinolones, now available for parenteral application. the pharmacology of an intravenous once-daily dose (od) of 400 mg mox was determined with five gram-negative and -positive pathogens (streptococcus pyogenes , streptococcus pneumoniae , moraxella catarrhalis , escherichia coli , and klebsiella pneumoniae ). a twice-daily dose (bid) of 400 mg mox was simulated with the gram-positive species in order to increase the bactericidal effect. results: to determine the efficacy, killing curves were analyzed, and following parameters were calculated: ka.max: maximal killing activity [log cfu]; ka. conclusion: an intravenous once-daily dose of mox is active against all tested pathogens. the gram-negative species are rapidly killed (ka.1 h similar to ka.max). there is no pronounced initial effect on the two gram-positive species but a general slow reduction in the viable cell count (ka.max is reached after 24 h). the efficacy of mox (measured as aac and ka.max) on s. pyogenes and s. pneumoniae is to some extent increased after the second dose. however, the analysis of the killing curves reveals no major difference between od and bid. even the od nearly gives the maximal bacterical activity of mox against gram-positive pathogens. objectives: to evaluate the dose proportionality of amoxicillin and to compare the respective pk/pd parameters of two dosage regimens. methods: the dose proportionality of amoxicillin was evaluated using linear regression of mean auc 0-inf and c max data of 13 different bioequivalence studies (n0/477 volunteers) performed with formulations containing various amounts of amoxicillin alone or in the combination with clavulanic acid. the volunteers received a single oral dose in the range of 250 á/1000 mg. amoxicillin plasma concentrations were determined by hplc/uv or lc/ms/ms methods. time above mic (tmic) expressed in% of dosing interval was calculated with three target mic values (0.5, 1.0 and 2.0 mg/l) for 500 mg 8hourly and 1 g 12-hourly dosage regimens. results: the absorption of amoxicillin (auc 0-inf ) showed a linear dependence with a correlation coefficient of 0.975. the correlation coefficient of the linear regression for the cmax dependence on the actual dose was 0.909. the respective tmic for both dosage regimens were very similar, with largely overlapping confidence intervals, supporting a pd breakpoint of 2 mg/l for the 1 g 12-hourly regimen (tmic ]/2 mg/l: 42.8%, 95% ci 38.6, 46.9%). conclusion: this analysis shows the dose proportionality of amoxicillin over the dosage range of 250 á/1000 mg and supports the pharmacodynamic rationale for a 1 g bid dosage regimen. piperacillin/tazobactam concentration profile after high dose administration pattern in nosocomial pneumonias due to mecanical ventilation pm207 pedeboscq s a , gruson d b , bassoua v a , hilbert g b , pometan jp a . a st. andré hospital, pharmacy, bordeaux, france , b pellegrin hospital, reanimation, bordeaux, france the piperacillin (p)/tazobactam (t) antibacterial spectrum covers the largest part of bacteria responsible for pneumonias due to mechanical ventilation. but, due to important bacterial inoculum and pharmacokinetic parameter modifications in intensive care patients, high doses of beta-lactamines seem to be necessary to obtain antibiotic concentrations above suspected bacteria's mic (minimal inhibitory concentration) . this led us to compare, in patients with pneumonia due to mechanical ventilation, two intermittent administration patterns: 4 g three times a day (usual pattern) versus 4 g four times a day (high dose pattern). this study is carried out in collaboration with intensive care unit, bacteriological department and pharmacy where antibiotic concentrations are determined. twenty-three takings of blood are executed within a 48 h period, in addition to two bronchial secretion samples. concerning p seric concentrations, the high dose pattern seems to be more adapted because of relatively high residual concentrations ( !/20 mg/ml). three hours after each injection, t seric concentrations are lower than the 4 mg/ml activity threshold. first and second day residual bronchial concentrations of p seem to be sufficient although t concentrations are below activity threshold. these results are to be correlated with the mic determined by the bacteriological department, and only this correlation will make us able to conclude the better efficacy of the high dose pattern in intensive care patients. anti-inflammatory drugs interference in absorption and tissue penetration of amoxycillin pm208 del fiol fs a , menon sz b , caramez th b , celotto tf b , lopes ras b . a university of sorocaba, pharmacology, sorocaba, brazil , b school of pharmacy, university of sorocaba, sorocaba, brazil antibiotics and anti-inflammatories are frequently associated in clinical practice. there is some concern about the quantity of antibiotic that reaches the infection sites, which may be reduced in the presence of an anti-inflammatory drug. the purpose of the present study was to analyse how steroids (dexamethasone (dexa)) and aines (celecoxib (cele)) influence on the penetration of amoxicillin to inflamed tissues. thirty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs to form granulomatous tissue. one week later the animals were divided into three groups. one group received only amox (40 mg/kg), another received amox (40 mg/kg) plus cele (6.0 mg/kg) and the last received amox (40 mg/kg) plus dexa (0.3 mg/kg). one hour later the animals were sacrificed and the concentration of amoxicillin in the serum and tissue investigated. there was no difference among the groups in the quantity absorbed (amox0/18.879/2.05 mg/ml; amox'/cele0/18.209/1.89 mg/ml and amox'/dexa0/18.139/2.28 mg/ml). there was a reduction in the tissue concentration of amoxicillin (p b/ 0.01 tukey-kramer) for the group that received the drug with dexamethasone. for the other groups, there was no difference in the tissue concentration of amoxicillin. the results indicated that in inflamed tissue, a significant reduction of antibiotic penetration was induced by sinultaneous dexamethasone therapy. prediction of the optimal amoxicillin dose regimen based on coupling of pharmacokinetic data and bactericidal activity pm209 background: given its short half-life, amoxicillin (amx) should be administered at least three times a day to patients with acute exacerbations of chronic bronchitis, in order to achieve serum concentrations well above the mic of the responsible pathogen. however, several authors have recommended twice-daily administration of a higher dose for a shorter period. we assessed the relationship between amx sputum concentrations and antibacterial activity following two treatment schedules in healthy volunteers. subjects and methods: twelve healthy volunteers were randomized to receive amx for 4 days at a dose of either 1 g bd or 500 mg bd. serum and sputum were collected every day, 3 h after the morning administration, and again 2 days after the last dose. amx concentrations were determined by hplc with fluorometric detection. sputum killing activity was determined against haemophilus influenzae , streptococcus pneumoniae and moraxella catarrhalis . results: mean serum concentrations measured 3 h after the morning administration were 1.5 (500 mg bd) and 1.95 mg/l (1 g bd), and were above the mics of the three microrganisms. in contrast, sputum concentrations were always below 0.5 mg/l. in terms of sputum killing activity, 1 g bd was more effective than 500 mg bd against s. pneumoniae and m. catarrhalis , whereas no sputum samples were active on h. influenzae . conclusion: the optimal amoxicillin treatment schedule cannot be established on the basis of serum pharmacokinetics only. galmiche h, louchahi k, tod m, drugeon h, giroud jp, rouveix b. service de pharmacologie clinique, hopital cochin, paris, crepit 93, hopital avicenne, bobigny, service de microbiologie, hopital laennec, nantes, france background: cysteine-based mucolytics are commonly used in combination with antibiotics to treat patients with acute exacerbations of chronic bronchitis (aecb). they are also used to allow in vitro mic determination in sputum specimens. we conducted an in vitro and ex-vivo compatibility study designed to detect a possible interaction between mucolytics and antibiotics. methods: serial samples of bronchial secretions were collected from aecb patients and from healthy volunteers who received 1 g of amoxicillin twice a day for 4 days. two mucolytics were used to fluidify sputum specimens: 2,3-dihydroxy-1,4-dithiolbutan (digest-eur † ) and acetylcysteine (10% solution). amoxicillin was assayed using a chromatographic system with fluorometric detection. each sample was also tested in a microbiological assay. results: amoxicillin could not be detected in the presence of the mucolytic agents. conclusions: this mucolytic á/amoxicillin interaction may be explained by amoxicillin fixation to fluidified mucoproteins, and should be taken into account when assessing antibiotic efficacy in vivo. del fiol fs a , ferro c b , albuquerques et b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil physicians frequently recommend that macrolides should be administered with milk to decrease the discomfort they cause. thus the objective of this study was to verify the interference of milk in the absorption and distribution of erythromycin (eryt); clarithromycin (clar); roxithromycin (roxi) and azithromycin (azit). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs eryt, clar, roxi and azit with and without milk (3.5 ml/kg [ca'/'/]0/1.1 mg/ml). the animals were sacrificed and the serum and tissue concentration of the drugs was investigated. there was no reduction (pb/ 0.05 tukey-kramer) in the serum and tissue concentration in the presence of milk for azit and clar. there was a 27% reduction for roxi in the serum concentration in the presence of milk (11.84 9/1.35 and 8.639/1.34 mg/ml), but no alteration in the tissue concentration. there was a 33% reduction for eryt (p b/ 0.05), in the serum concentration in the presence of milk (10.209/ 1.06 and 6.839/0.88 mg/ml) and a 40% reduction in the tissue concentration. the milk decreased the effectiveness of treatments with erythromycin and roxithromycin and the bioavailabilities of this macrolides were affected by co-administration with milk. del fiol fs a , souza gp b , duzzi mr b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil the degree to which tetracyclines are absorbed differs greatly. this absorption is impaired by the concurrent ingestion of divalent and trivalent cations. thus the objective of this study was to investigate the interference of milk in the absorption and distribution of tetracycline (tetr), oxytetracycline (oxyt), minocycline (mino) and doxycycline (doxy). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their back for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs: tetr; oxyt; mino; and doxy with and without milk (3.5 ml/kg [ca'/'/]0/1.1 mg/ml). the animals were sacrificed and the drug concentrations in the serum and tissue were determined. there was no reduction (pb/ 0.05 tukey-kramer) in the serum and tissue concentrations in the presence of milk for mino. there was a 25% reduction (p b/ 0.05) for doxy in the serum concentration in the presence of milk (11.249/0.61 and 8.409/0.41 mg/ml) and 32% in the tissue concentration. for oxyt, there was a reduction of 34% (pb/ 0.05) in the serum concentration in the presence of milk (14.579/1.51 and 9.549/1.51 mg/ml) and 16% in the tissue concentration. the tetr results show a 35.4% reduction (p b/ 0.05) in the serum concentration in the presence of milk (13.469/3.39 and 8.699/3.03 mg/ml) and 40% in the tissue concentration. milk decreased tetracycline bioavailability and effectiveness. isotopic studies with oxine labelled platelet. platelet kinetics in thrombocytopenic malaria patients pm213 introduction : thrombocytopenia is a common feature in human malaria (1). excessive splenic platelet pooling has been suggested to play a role in uncomplicated cases of malaria, but a moderately shortened platelet life span during the period with decreasing parasitaemia seems the most plausible cause of the frequently observed thrombocytopenia (2, 3). consumption coagulopathy, eventually manifested as disseminated intravascular coagulation, has been described in malaria (4). in uncomplicated malaria, however disseminated intravascular coagulation is rarely found (3). results: in malaria patients the sequestration was not different to normal. platelet half-life was reduced in patients with p. falciparum malaria to 10 á/17 h (normal â/8 days). in one patient with p. vivax malaria platelet half live was 59.5 h. conclusion: no significant differences in the sequestration of platelets when compared to healthy individuals could be detected by 111 in-labelled platelet scintigraphy. especially, no enhanced splenic sequestration, as previously expected, was the cause of the thrombocytopenia. therefore, other mechanisms than sequestration are responsible for the dramatically reduced life span of the platelets during acute malaria. zaharanka ag, rozhdestvensky da. vitebsk state medical university, vitebsk, belarus aim: the studying of chromatingeterogenic test (ct) results in sperm of subjects taking doxycycline (d) and some macrolides (erythromycine (e), jozamycine (j), and azytromycine (a)) in moderate therapeutic doses. methods: forty healthy volunteers (20 á/23 years) were studied. daily dose of d was 0.2; e was administered in dose 0.25 four times per day 10 days; j */0.5 before meals twice daily 10 days; a */0.25 before meals once daily 5 days. ct for evaluation of dna condition in human spermatozoids was performed before treatment (twice), on the 5th and 10th days of treatment, as well as after 1 and 2 months after treatment course completing. results: ct data analysis revealed that the mean amount of defective spermato-zoids before treatment was 18.5'/3.5%. by the 5th day of d treatment the index of de-natured dna was 63.4'/6.5% (pb/ 0.001), by the 10th day */80.4'/7.2% (pb/ 0.001). one and 2 months after the d treatment course the amount of generative cells with denatured dna was 54.5'/4.6 and 42.5'/3.8%, respectively (p b/ 0.001 in both case). under e treatment the amount of defective spermatozoids changed as 38.2'/4.2 (5th day), 42.4'/2.7 (10th day), 35.6'/2.5 (after 1 month), and 30.5'/4.2% (after 2 months) (pb/ 0.05 in any case). under a using the ct results at the same control points were 39.5'/4.7, 46.5'/3.2, 40.2'/3.5, 25.6'/3.2% (pb/ 0.05 in any case); and under j treatment */18.2'/2.5, 20.3'/2.5, 17.2'/3.2, 15.2'/ 4.2%, respectively (p !/0.05 in any case). conclusions: the data obtained permit to conclude that d demonstrates the high level of toxicity to male generative cells. this effect preserves during 2 months after the course of d treatment. objective: to study the effect of aggressive isolation and decontamination measures to control an outbreak of multi-resistant acinetobacter baumanii (mr-ab) in an icu. the outbreak: the index case was transferred from a mediterranean hospital, directly into an open-plan 10-bedded icu, with severe injuries to his head and thorax. he died shortly after admission. sputum, bronchoalveolar lavage fluid, blood cultures and a chest drain swab grew mr-ab, resistant to ampicillin, co-amoxiclav, aztreonam, amikacin, ceftazidime, cefotaxime, cefuroxime, ciprofloxacin, gentamicin, meropenem, piptazobactam, tobramycin and sensitive only to colistin. within 10 days, mr-ab was isolated from two further icu patients. all isolates demonstrated identical antimicrobial susceptibility profiles. the icu was closed to admissions and thoroughly cleaned. all patients were isolated and their contacts screened. the icu was reopened, however, mr-ab was isolated from a fourth patient. this patient was isolated, the icu closed, for a second time, thoroughly cleaned, and all contacts isolated until discharge. all subsequent patients screened were negative for mr-ab conclusion: this illustrates the importance of aggressive isolation measures and thorough supervised cleaning in control of an outbreak, and the need to screen patients for resistant bacteria before admission to the intensive care unit in a general hospital. extended spectrum beta-lactamase-positive bacteria isolated in neonatal intensive care unit pm216 sandorcinova z a , siegfried l a , kmetova m a , viragova s b . a institute of medical microbiology, faculty of medicine, university of p.j. safarik, kosice, slovakia , b hospital, kosice-saca, neonatal intensive care unit, kosice, slovakia extended-spectrum beta-lactamases hydrolyse all penicilins, cephalosporins, including third-generation cephalosporins and aztreonam. esbl are predominantly produced by klebsiella spp. but may be presented in other enterobacteriaceae, too. the aim of present study was to investigate the occurrence of esbl-producing bacteria isolated from patients hospitalized at the neonatal intensive care units (icu). fifty escherichia coli and 35 klebsiella spp. were isolated from rectum of patients hospitalized at the neonatal icu. the mics of antimicrobial agents were determined by the standard agar plate dilution method according to the nccls guidelines. for screening of esbl production we investigated strains showing reduced susceptibility (mic equal and/or more than 2 ml/l) to at least one of third generation cephalosporins. esbl production was detected by double disk synergy test (ddst), e -test for esbl, and pcr employing specific primers for the presence of blashv and blatem genes. by using ddst and e -test, among e. coli isolates, an expression of esbl was detected in the three by the former method, while among klebsiella spp. isolates, a production of esbl was found in the two by the latter method. in esbl-positive e. coli strains the presence of blatem genes fragments was detected while in esbl-positive klebsiella spp. genes encoding for shv-type beta-lactamases were found. isolation of staphylococci from wound swabs and their susceptibility to antibiotics pm217 markov mij, shopovski e, despotovski v, angelevski a, nikolovski b. military hospital, microbiology, skopje, the former yugoslav republic, macedonia purpose: to determine percent of staphylococci from wound swabs and to establish their susceptibility to antibiotics. material and method: the wound swabs have been evaluated with standards microbiological techniques. bacteria have been identified with strips from the 'atb expression' system. the susceptibility testing has been performed with strips with dilution technique, read by the same system. results: during the last 5 years (01 01 1997 á/25 02 2002), a total of 1978 wound swabs have been evaluated in the military hospital in skopje. positive bacterial finding have been determined in 1345 (68%) swabs with 1575 isolated bacterial species, from which 587 (37.3) were staphylococci: staphylococcus aureus 460 (29.2%) isolations (173 methycillin resistant s. aureus ); s. epidermidis 79 (5.0%); s. haemolyticus 21 (1.3%); s. hominis 11 (0.7%); s. chromogenes and s. lugdunensis nine (0.6%); s. intermedius , s. lentus , s. sciuri and s. warneri all with seven (0.4%) isolations. the susceptibility of s. aureus was to: penicillin 2%, ampicillin 15%, amoksicillin/clavulonic acid 67%, ceftazidime 66%, gentamicin 29%, tetracycline's 28%, erythromycin 61%, lincomycin 44%, ciprofloksacin 95%, cotrimoxazole 82%, vancomycin 100%, fusidic acid 96%, cefixime 57%, and azitromicin 78%. conclusion: in our study most frequently isolated bacteria from the wound swabs were staphylococci, especially s. aureus . susceptibility, except for the penicillin (2%), was high to other antibiotics. the study went on from january 1999 to december 2000. at maribor teaching hospital, 2288 staphylococcus aureus isolates were collected. in 1999, 114 (10%) and in 2000, 73 (6.4%) were mrsa. mrsa were recovered from routine clinical material and from surveillance swabs (nose, throat, skin). for isolation, conventional culture media were used and for surveillance swabs mrsa-screening plate (manitol salt agar with 6% oxacillin) and trypticase soy broth with 6.5% nacl were added. s. aureus was identified by catalase, dna-se and tube-coagulase test. antibiotic susceptibility was determined by the disk-diffusion method according to ncci guidelines. all 187 mrsa isolates were tested for sensitivity to the following antimicrobials: gentamicin, netilmicin, ciprofloxacin, erythromycin, chloramphenicol, tetracycline, cotrimoxazol, vancomycin and clindamycin. it was found that all mrsa isolates were sensitive to vancomycin and partially or totally resistant to the rest. there were no important differences between the years 1999 and 2000. our mrsa isolates were completely (100%) susceptible to vancomycin, but resistant to the other antimicrobials in use to some extent. although the monitoring of mrsa susceptibility to antimicrobials once a year did not show any important change in antimicrobial resistance, the periodical monitoring of mrsa susceptibility to antimicrobials and revaluation of current treatment regimens of mrsa infections is necessary. staphylococcus aureus strains with reduced susceptibility to vancomycin among clinical isolates in university hospital in warsaw pm219 the visa and especially h-visa are very difficult to be found in the routine laboratory. in our investigations we examined 1011 of s. aureus strains isolated and stored in our laboratory for several last years (1997 á/2001) . most strains were isolated in 2000, and some in 2001. for all staphylococcal strains mrsa as well as mssa the mics of vancomycin were performed by the standard dilution method. among strains isolated in the last year three strains were recognized as visa (mic values were 8 mg/l). the frequency of visa was 0.3%. in the aim of founding the h-visa strains the population analysis was used. for this analysis all strains growing on the concentration 4 mg/l of vancomycin from the inoculum 10 6 were chosen. it was 54 strains, but only 32 of them were recognized as h-visa. the frequency of h-visa among investigated strains was about 3%. most but not all of the h-visa and all visa strains were methicillin resistant. in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci isolated from patients of surgical intensive care unit pm220 kucukates e, karayel n, kansiz e. institute of cardiology, university of istanbul, istanbul, turkey objectives: oxacillin-resistant staphylococci have emerged as a major infection control problem in our hospital. the aim of this study was to evaluate the in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci. material and methods: this study was performed between january 2000 and december 2001, at university of istanbul, institute of cardiology. the antimicrobial susceptibilities of 179 staphylococci isolates for vancomycin, teicoplanin and oxacillin have been investigated by e -test according to nccls guidelines. results: fifty-five (30.73%) of 179 clinical isolates were staphylococcus aureus . one hundred and twenty-four (69.27%) of 179 clinical isolates were coagulase negative staphylococci (cns). none of 179 staphylococci isolates were resistant to vancomycin. but three of cns isolates were intermediate and six of cns isolates were resistant to teicoplanin. twenty-eight (50.9%) of 55 s. aureus were resistant to oxacillin. ninety (72.60%) of 124 cns isolates were also resistant to methicillin. conclusions: nosocomial staphylococcal infections, especially in intensive care units increase day by day. staphylococcal infections are a major problem in many hospitals. according to our experiences the rate of oxacillin resistant staphylococci isolates in our hospital has also increased. methicillin-resistant staphylococcus aureus (mrsa) is a clinically significant pathogen because mrsa is resistant to many kinds of antibiotics and causes nosocomial infections around the world. the antiseptics are used for prevention of infections by mrsa. antisepticresistant mrsa strains have been isolated from clinical specimens. antiseptic resistance genes confer resistance to many kinds of drugs structurally and the resistance mechanism is the energy-dependent drug efflux system. in addition, the fluoroquinolone (fq)-resistance gene, nora , confers also resistance to many kinds of antiseptics. we studied the relation of the susceptibility to antiseptics and fqs of mrsa strains isolated in japan. a total of 420 strains of mrsa were isolated from 14 hospitals in japan from 1998 to 1999. acriflavin (af), acrinol, benzethonium chloride, benzalkonium chloride and chlorhexidine digluconate were used as the antiseptics. norfloxacin and sparfloxacin were also used in this experiment. the mic was determined by agar double-dilution method as recommended by the nccls. about 65% of mrsa showed resistance to af (mic: !/64 ug/ml). no strain was resistant to a specific antiseptic. fq-susceptible strains were susceptible to all antiseptics. this study showed that antiseptic-resistant mrsa are widely spread at hospitals in japan. the drug of choice in treatment of serious infections caused by mrsa was still vancomycin, however sometimes failures were observed, especially in monotherapy. some conflicting are present in literature about an effect of combined action of vancomycin and betalactams. in the present work, the common effect of vancomycin and methicillin against chosen staphylococcus aureus strains was examined. the investigated strains were characterized as visa, h-visa and clones obtained from h-visa in population analysis. two methods were performed: e -tests with methicillin and vancomycin placed on the media supplemented with the second antibiotic and the chessboard micro-analysis with increasing concentrations of both antibiotics. the fic indexes were calculated for different combinations of concentrations. on the basis of the fic indexes it was shown that the simultaneous action of vancomycin and methicillin was synergistic in all examined strains visa and h-visa, but only in appropriate concentrations. in different combinations the observed effect was addition or indifference. antagonism was never observed. the synergistic effect was not observed in the case of standard s. aureus strain sensitive to methicillin. supplementation of media with 4% of nacl substantially decreased the observed effect. incidence of antibiotic resistance in staphylococcus aureus strains in hungary with special reference to mrsa pm224 ghidán á , maró di c, csukás z, kamotsay k, szabó d, ostorházi e, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary between january 1997 and december 2000, a total of 3109 staphylococcus aureus strains isolated from patients admitted to the clinics of the semmelweis university were examined for antibiotic sensitivity with the disc diffusion test. resistance to individual antimicrobials were as follows: penicillin 84%, oxacillin 25%, erythromycin 31%, ciprofloxacin 15%, amikacin 12%, netilmicin 8%, tobramycin 10%, gentamicin 20%, clindamycin 22%, mupirocin 4%, tetracyclines 34%, chloramphenicol 9%, teicoplanin 2% and vancomycin 0%. all mrsa were b-lactamase producer. they showed coresistance to erythromycin (53%), ciproflxacin (36%), amikacin (29%), netilmicin (23%) and mupirocin (10%). multiple resistant mrsa strains to mupirocin'/tetracyclines'/chloramphenicol amounted to 0.9%. triple resistance to oxacillin'/ciprofloxacin'/netilmicin was 14%. the detection of meca gene by pcr in randomly chosen mrsa qualified with 1 mg oxacillin disc resulted in only 15% meca positivity indicating that the traditional disc diffusion test overestimates the frequency of mrsa strains particularly in such an environment where the usage of penicillins and cephalosporins is so liberal as in hungary. consequently, the selective pressure for blactam-resistance and b-lactamase induction exists everywhere. this conclusion is coherent with the relatively low frequency of multiple resistant mrsa strains and urge the need of a routinely available genetic method to apply for mrsa detection. objectives: the main objectives of this study were to monitor antibiotic resistance, identify new/emerging resistance mechanisms at an early stage, prevent their dissemination, early detection and prevent the outbreaks. methods: our laboratory used antibiotic disc sensitivity testing methodology (nccls 1993) . zone sizes were measured objectively using a biomic automated radius zone reader. results: throughout 2 years (january 1999 till december 2000) we have surveyed 3190 organisms collected from outpatient departments (1145, 35.8%), radio-oncology department (992, 31.1%), medical department (765, 23.9%), obg department (217, 6.8%), surgical-oncology non icu department (207, 6.5%), icu department (175, 5.5%). from 1543 (100%) strains of enterobacteriaceae 68 (4.4%) were resistant to 4th generation fluoroquinolone and 44 (2.9%) strains were esbl positive. from 416 (100%) strains of staphylococcus aureus were only five (1.2%) strains resistant to methicilin (mrsa). we collected 370 (100%) strains of enterococci, whereabout only two (0.5%) were resistant to glycopeptides (vre). from 235 (100%) strains of pesudomonas aeruginosa , 97 (41.2%) were resistant to aminoglycosides. conclusions: national restrictive antibiotic policy hand in hand with local hospital antibiotic policy and regular rotation of antibiotics used in prevention and treatment on all departments is leading in our case in positive situation in antibiotic resistance in comparing with other slovakian and european centers. antimicrobial resistance of nosocomial strains of staphylococcus aureus pm226 dekhnich av, stratchounski ls, edelstain ia, narezkina ad. institute of antimicrobial chemotherapy, smolensk, russian federation purpose: to determine the antimicrobial resistance of staphylococcus aureus causing nosocomial infections in smolensk regional hospital. results: a total of 140 s. aureus strains isolated during 2000 á/2001 were studied. antimicrobials tested included oxacillin (oxa), erythromycin (ery), clindamycin (cli), gentamicin (gen), vancomycin (van), linezolid (lnz), tetracycline (tet), chloramphenicol (chl), rifampicin (rif), fusidic acid (fus), trimethoprim/sulfamethoxazole (ts), ciprofloxacin (cip), mupirocin (mup), quinupristin/dalfopristin (qd). susceptibility testing and its interpretation were performed by agar dilution according to nccls guidelines where applicable. results are presented in the conclusions: the most active antimicrobials were vancomycin, linezolid, quinupristin/dalfopristin, fusidic acid, mupirocin, followed by co-trimoxazole, rifampicin. beta-lactams, macrolides, lincosamydes, tetracyclines and chloramphenicol should not be used for the treatment of nosocomial s. aureus infections. we investigated all staphylococcal infections within 2 years among 246 neonates hospitalized for infection in the neonatal icu in a tertiary neonatal referral center. univariate analysis, to assess risk factors for neonates infected with staphylococcus aureus (121) vs. without s. aureus (125) was performed. from the total number of 246 cases, in 121 cases s. aureus was isolated from various samples; in 16 cases from blood cultures, in 36 cases from urine, in 18 cases from eye swabs and in 29 cases from gastric content (no significant differences in comparison with control group). colonization with s. aureus , was a predictor of infection: nasal swabs, throat swabs, ear swabs, skin swabs and umbilical swabs were significantly more commonly observed in neonates infected with s. aureus , than with other infections. etiological analysis showed that co-pathogens escherichia coli and viridans streptococci were significantly more frequently associated with neonatal infection caused by s. aureus , in comparison to other organisms. according to localization of infection site, conjunctivitis and thrush stomatitis was the commonest s. aureus neonatal infections. outcome was similar to other infections and without any significant differences between both groups. mortality was similar to other infections, probably because: initial therapy in our centre contains an antistaphylococcaly active agent (cefuroxime or cefotaxime plus aminoglycosides). morozova ot, semina na. laboratory of hospital infections, central research institute of epidemiology, moscow, russian federation purpose is to study the role of enterococcus spp. in the aetiology of nosocomial infections among the patients of the childrens clinical hospital and susceptibility of these strains to antibiotics. methods: the strains of enterococci were isolated from 66 patients with hospital infections in 2000 á/2001. results: the aetiological structure of enterococcus -infections showed the predominance of skin and soft tissue infections (39.4%), urinary tract infections (33.3%), bloodstream infection (10.6%), pneumoniae (4.5%), infection of central nervous system, gastrointestinal tract, eye, surgical wound infections were of rare incidence (3 á/ 1.5%). various nosological forms of infections were caused more often by e. faecalis than e. faecium (72.7, 27.3%). the antibiotic resistance to ampicillin and other beta-lactams occured in 100% of e. faecium isolates, but all strains of e. faecalis were susceptible to these drugs. high-level gentamicin resistance demonstrated e. faecalis isolates */ 29.0%, e. faecium */100%; and high-level streptomycin resistance showed e. faecalis */67.7%, e. faecium */88.8%. all the e. faecalis were active against fluoroquinolones, but e. faecium were resistant in 22.0%. there were no vancomycin resistant enterococci. conclusion: e. faecalis predominated in the aetiological structure of nosocomial infections due to enterococcus spp. antibiotic resistance patterns for two species of enterococci were different, all the strans were susceptible to vancomycin. evaluation of antimicrobial resistance of enterococcus spp. experience of 6 years (1996 á/2001) pm229 lopez-barba j, jesus de la calle i, solino-ocañ a i, rodríguez-iglesias m, perez-ramos s. microbiology laboratory, puerto real university hospital, cádiz, spain objective: determination of quantitative changes in antimicrobial resistance of enterococcus isolated from clinically significant not urinary samples of patients remitted to the laboratory of microbiology during a 6 years period (1996 á/2001) . material: the period of the study was comprised between 1996 and 2001. the samples has been processed for the isolation of enterococcus following conventional methods. were isolated 3424 enterococci strains. the identification and susceptibility to antibiotics have been performed in automated system microscan(c) dade-behring(c) through panels combo cgp. the data were processed by the statistical system statgraphics plus 4.1. results: of the 3424 enterococcus , have been identified 3298 e. faecalis and 126 e. faecium . the resistance is shown in table 1 . conclusions: the resistance to va and tei of e. faecalis remains through last 4 years (2 á/3%). the high resistance to erythromycin and tetracilin ( !/70%) and the resistance (30 á/31%) to quinolones, antibiotics all of them used in community-acquired infections justify the susceptibility testing to the clinical strains isolated of this group of microorganism. in e. faecium the antimicrobial resistances was high and increasingly to imipenem, meropenem, erythromycin and quinolones. characteristics of strains e. faecium colonizing the neutropenic patients pm230 abbassi ms, achour w, gréco a, ben hassen a. laboratory of bone marrow transplant center, tunis, tunisia digestive colonization by enterococcus faecium in the neutropenic patients under gut decontamination is important. seventeen multiresistant strains of e. faecium isolated from stools of seven neutropenic patients were the target of an epidemiological analysis through the determination of the mics of amoxicillin, gentamicin, vancomycin, the transferability gentamicin resistance to the recipient strain e. faecalis jh2-2 by filter-mating assay, analysis of plasmid profiles of e. faecium -strain and of transconjugants and the amplification by pcr of the gene aac(6?)-aph(2??) coding for the bifunctional enzyme by using primer m13771 who gives a fragment of 174 kb. among the seventeen strains, eleven had the same antibiotype a1, 12 had a gentamicin mic!/4096 mg/l. the mic90 of the amoxicillin was of 128 mg/l. all the strains were sensitive to vancomycin. ten strains harbored a plasmid of 70 kb transfered at a frequency of 6.10 á/5, also found in gentamicin-resistant transconjugants. however, strains belong to nine distinguished plasmids profiles. all high-level gentamicin resistant-strains had a positive pcr amplification of the aac (6?)-aph (2ƒ) gene. the features of the studied strains establish their endogen origin, specific for every patient, sharing only high-level resistance to gentamicin. gut decontamination treatment with gentamicin enhance either the spread and the preservation of easy-transferable plasmid carrying genetic transposable element. frequency and antibiotic resistance of bacteria isolated from patients suffering infectious complications following the implantation of prosthetic devices pm231 kristó f k, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary for patients with indwelling joint prosthesis, early recognition and prompt therapy for infection in any location may be critical to reduce the risk of seeding the joint implant heamatogenously. a year period (2001) a total of 2440 swabs of aspiration from patients with infectious complications following the implantation of prosthetic devices were cultured. cultivation and identification of the strains were performed by conventional methods and by vitek system (biomȇrieux) and susceptibility testing by disc diffusion. potentional pathogens were recovered in 324 cases (13.27%). gram positive cocci, in particular staphylococcus spp. proved to be the most commonly isolated bacteria. coagulase-negative staphylococcus (cns) was isolated more frequently (48%), followed by s. aureus (28%), enterococcus faecalis and e. faecium (11%), gram-negatives (3%), anaerob isolates (7.5%). resistance to individual antimicrobials of s. aureus and cns were as follows: methicillin 8 and 47%, clindamycin 4.5 and 21.7%, fluoroquinolones 0 and 18%. mupirocin resistant strains of s. aureus were not found, while 7.7% were among the cns strains. our results could be essential for the rational selection of treatment at our orthopedic wards. results: in the analysed period the percentage of isolated enterococcus sp. strains among all non-repetitive clinical isolates in 1999, 2000 and 2001 was 3.9, 3.1 and 51%, respectively. cultured strains were identified as e. faecalis, e. faecium, e. gallinarum and e. avium . the most prevalent was e. faecium strains, isolated with a frequency of 75, 62 and 75%, respectively. vancomycin-resistant strains were all identified as e. faecium and in 2001 they comprised 17% of all isolates of this species. conclusions: (1) the frequency of isolation of enterococcus sp. in blood cultures of haematological patients remained relatively stable in 1999 á/2001. (2) the predominant enterococcal species isolated from these patients was e. faecium . (3) in 2001 we recorded for the first time an emergence of vancomycin-resistant e. faecium , which comprised 17% of all isolates of this species. kalai s, ben hassen a, achour w, greco a. laboratory of microbiology, national bone marrow transplantation center, tunis, tunisia from april 1998 to june 2000, 47 non-repeated strains of pseudomonas aeruginosa were isolated from 42 immunocompromised patients. thirty-six percent of strains were isolated from abscess, 27% from blood culture and 11% from urine. susceptibility to antibiotic was studied by the routine disk diffusion method (ca-sfm). mics were determined using agar dilution to 11 antibiotics (5b-lactams, four aminosides and two fluoroquinolones). serotyping of the different trains was performed using antisera to the international antigenic typing systems serotypes. the study showed 80% of resistance to c cochin port royal hospital, service de gynecologie-obstetrique site st vincent-de-paul, paris, france , d cochin port royal hospital, cclin, paris, france aims: to determinate risk markers of an outbreak of postpartum endometritis due to group a streptococcus . design: a case-control study using data collected with a structured form. setting: the cases of postpartum endometritis were diagnosed in the department of obstetric of the paris hospital network during 5 days (december 1999 á/january 2000). the group of controls consisted of women delivered in the same department during the same period. participants: cases (n0/3) and controls (n0/59). findings: cases had smoked more often during pregnancy (67 vs. 16%; p 0/0.089) and received more often immunosuppressive treatment than controls (33 vs. 0%; p 0/0.060). instrumental delivery has been needed more often for cases than controls (67 vs. 13%; p 0/0.062). cases had been hospitalized after delivery in a ward z of the department more often than controls (100 vs. 23%; p 0/0.017). they had been examined after delivery more often by a midwife x (100 vs. 18%; p0/0.009) and a nurse y has provided care to cases and not to controls (33 vs. 0%; p 0/0.051). conclusion: smoking, receiving immunosuppressive treatment during pregnancy, and instrumental delivery were significantly associated with postpartum endometritis (pb/ 10%). a midwife and a nurse might be involved in the transmission of the infection. petoukhova i a , sokolova e a , dmitrieva n a , nummaev b b . a laboratory microbiology, cancer research center of russia, moscow, russian federation , b department of oncogynecology, cancer research centre, moscow, russian federation the aim of the study was to assess efficacy of perioperative ap. total 241 pts were included. two hundred and one pts with cervical cancer (cc) undergone extensive hysterectomy, 22 pts with cancer of vulva (cv)-extensive vulvoectomy, 18 pts with ovarian cancer (oc) á/ extensive/combined operations. fifty-one pts (group 1) received ap with amoxicillin/clavulanate (am/cl) 1.8 g iv 30 min prior to operation, then 1.2 g iv thrice per day for 3 á/5 days. fifty pts (group 2) received cefotaxime (ctx) 2 g iv 30 min prior to operation, then 1 g four times per day for 3 á/5 days'/metronidazole (mtz) 500 mg two times per day for the same period. one hundred and forty pts were retrospective control (they received ii á/iii generation cephalosporins or linkosamides only after operation). the rate of swi in pts with cc, cv and oc was 13.5, 73, 29%, respectively; dwi */16.2, 0 and 21%, respectively; uti */72, 80, 42.8%, respectively. the ap with am/cl was more effective compared to ctx'/mtz (swi */6 vs 18%, respectively, pb/ 0.05, dwi */9.8 vs 14%, respectively, p b/ 0.05). the rate of postoperative uti was equivalent in two groups (62 vs 74%, p 0/n.s.). thus, ap with am/cl is preferrable option in extensive operations in og pts. fifty-eight patients were randomized into two groups. group 1, a treatment consisting of 31 patients and group 2, consisting of 27 patients as a control. the patients were at the age of 679/1.2 and had had different surgical interventions with general anaesthesia from 1 to 3 h and accompanying copd (89%). artificial pulmonary ventilation was used in 45 cases. in the early post-surgery period the patients of group 1 were administered inhalation therapy, including ipratopium of bromide in the combination with fenoterol (atrovent, berodual) and ambrocsol (lazolvan) through a nebulizer. the inhalation therapy was not administered to the patients of group 2. under the influence of the inhalation therapy pulmonary ventilation and respiratory metabolism was restored faster in all the resuscitation patients (in group 1 */by the end of the first 24 h, in group 2 */on the 3rd á/4th day). the percent rate of pef1 was 76.7'/4.1 and 56.4'/4.5, respectively. artificial pulmonary ventilation ended in 9.2 and 7.3 h, respectively. the time of the patients' stay in the resuscitation department was 3.2 days in group 1 and 4.6 days in group 2. by the end of the 1st week pneumonia developed in one patient from group 1 and in eight patients in group 2. aerosol therapy application accelerates medication delivery to the respiratory tracts, increases the local activity and effects good prophylaxis for surgical hospital pneumonia. variation in ethiology of early and late onset ventilator associated pneumonia pm250 nikolopoulos j, daganou m, michailidou m, karabela e, kavada k, retsou s, antoniadou a, rasidakis a. department of respiratory abstracts s100 failure and icu, sotiria general and chest disease hospital, athens, greece purpose: to compare the distribution of causative microorganisms, their susceptibility to antibiotics and outcome of 'early' and 'late' vap in a greek icu. methods and results: retrospective study of mechanical ventilated (mv) patients (pts) with early and late vaps during a 6-month period. diagnosis of vap was made by clinical, radiographic criteria and quantitative cultures of bronchial secretions. vap was diagnosed in 17 pts (26%) out of 67 consecutive admissions in icu. all pts before the development of vap received antibiotics. three episodes of vap (17.6%) were developed before the 7th day of mv (early vap) and were caused: (1) by multi-resistant acinetobacter; and (2) by antibiotic-susceptible pseudomonas aeroginosa . in this group one pt died from septic shock related to vap and two pts survived. fourteen pts (82.4%) developed vap after the 7th day of mv (late vap). five cases were caused by multi-resistant p. aeroginosa , two cases by mrsa, two cases by multi-resistant acinetobacter, two cases by susceptible to antibiotics klebsiella pneumoniae , and three were polymicrobial and caused by multi-resistant microorganisms (mrsa and gnb). four pts died (28.6%) from septic shock related to vap, five pts (36%) died because of another cause and five pts (36%) survived. conclusions: early and late onset episodes of vap were caused by 'potentially drug-resistant bacteria'. p. aeroginosa as a cause of early vap was susceptible. mortality attributed to early and late vap was similar. antibiotic prophylaxis in oncological and major reconstructive orthopaedic surgery pm251 de biase p, ciampalini l, astone a, capanna r. azienda ospedaliera careggi, oncological and reconstructive centre, aoc, florence, italy during last year patients scheduled for oncological surgery or major reconstructive procedures were randomised to either vancomycin or teicoplanin prophylaxis. prophylaxis was performed with either vancomycin 1 g i.v. twice daily or teicoplanin once daily 400 i.v. two hundred patients were included. four patients did not agree the study protocol and were excluded. we treated 98 patients with teicoplanin and 98 patients with vancomycin. out of the 196 patients 117 were operated for oncological disease, while the remaining 79 underwent major orthopaedic procedures. we experienced 10 cases of red man syndrome, and five cases of moderate hypotension. five patients had postoperative complications: two deep venous thrombosis, one pulmonary embolism, two postoperative haematoma. in five patients we observed a wound dehiscence; two of these patients showed clinical sign of ssi and microbiological examinations were positive for mrsa. one patient recovered from infection with medical therapy, while the other patient showed a local tumour recurrence and was amputated at the thigh. at last surgery infection was still present clinical and at microbiological examination. in conclusion we had an infection rate of 1.02% which is comparable to the infection rate of a 'clean' surgery in patients with normal risk of infection. teicoplanin showed lower toxicity, has a longer half-life and has a simpler way of infusion and it is our current choice in high risk surgery. injuries with contaminated sharp articles in health care workers in general hospital celje, slovenia pm252 lesnicar g, sibanc b. department of infectious diseases, medical center celje, celje, slovenia in a prospective study carried out from january 1997 till june 2001, we registered 133 subcutaneous injuries with sharp objects, mostly in nurses and cleaning service workers. in 19% of cases the incident occurred outside the hospital, in persons who were not medical workers. in 69 cases the injury causing object was a needle that had been used in known patients, 14 of which were hepatitis b positive. fifty-five (48.2%) of the injured health workers had been previously vaccinated against hepatitis b; the protective antibodies to hepatitis b in the blood were found in 35/114 (30.7%) health workers only, while the tests for antibodies to hepatitis c and hiv were negative in all cases. following the incident, the majority of the injured persons, i.e. 102, were vaccinated against hepatitis b, while 46 persons (34.6%) also received passive prophylaxis with human immunoglobulin against hepatitis b. none of the injured persons have developed the disease or showed evidence of sero-conversion. in the year 2000 the general as well as specific preventive measures practised in our hospital became more rigorous. thus, approximately 80% of our health workers at risk have already been vaccinated against hepatitis b. to improve measures preventing dissemination of multidrug-resistant bacteria (mrb), a cross-sectional survey (2000) was conducted to analyse healthcare workers' (hcws) isolation precaution knowledge for mrb infection at 21 investigation and outpatient departments excluding four declaring not to be involved in care to mrb patients (emergency, obstetrics, nuclear medicine, and bacteriology). two hundred and eight hcws answered (67% of the paramedical staff, 28% of the physicians). thirty-three percent of them reported they do not know frequently or always the patient mrb status. they (82%) wish mrb status to be mentioned on the test form or on the advice request letter. mrb patient visit or test was appropriately timed in 62% answers. gowns (61%) or masks (58%) use were not systematically reported. other hcws (74%) reported better isolation precaution knowledge than physicians (58%) and nurses (81%) than investigation assistants (58%). physicians declared lower compliance with use of gowns, gloves or draw-sheets than other hcws. they had also lower education in isolation precautions and were less interested in education programs. this study suggests the necessity to improve mbr infection information. physicians and investigation assistants seem to be insufficiently aware of hospital infection control. therefore, education strategies targeted at physicians and investigation assistants working at outpatient and investigation departments should be developed. outbreak of clostridium difficile -associated diarrhoea in infectious disease department: risk factors and hygiene measures assessment pm254 henoun loukili n, martin m, remy v, hansmann y, christmann d. hôpitaux universitaires de strasbourg, maladies infectieuses et tropicales, strasbourg, france (shock)'/4* (bedridden status)'/4* (age !/65 years)'/3* (previous antibiotic treatment) and points (women)0/6* (shock)'/4* (bedridden status)'/4* (age !/65 years)'/3* (immunosuppression). the vast majority of patients (89 and 87% of males and females, respectively) could be classified in the subgroups with lower scores (six points or less) which had a very limited risk of death (0 á/2.5 and 0 á/2.3% for men and women, respectively), whereas for patients in the highest score subgroup (11 points or more), the risk was 100% for men and 91% for women. conclusion: risk stratification of patients with ap is possible from simple clinical variables available on admission. procalcitonin (pct) and c-reactive protein (crp) for differentiation of systemic inflammatory response syndrome (sirs), sepsis and severe sepsis pm259 lupse m a , ursu l b , slavcovici a a , carstina d a . a clinical department, teaching hospital of infectious diseases, cluj-napoca, romania , b laboratory department, teaching hospital of infectious diseases, cluj-napoca, romania objectives: to evaluate the value of pct in the differentiation of patients with sirs, sepsis, severe sepsis and bacteremia in comparison to crp. design: prospective study including patients who meet criteria for sirs, sepsis or severe sepsis (consensus conference of the accp/ sccm) admitted over 18-month period. patients and method: a total of 67 patients were included: eight with sirs, 35 with sepsis and 24 with severe sepsis. sixteen from 59 patients had bacteremia. pct and crp were evaluated in the first 24 h after admission: pct by brahms pct-q test and crp by turbidimetric assay. the sensitivity, specificity, predictive value of different cutoff points for crp and pct were determined. results: with a cut off point of 0.5 ng/ml for pct and 0.6 mg/dl for crp sensitivity and specificity for sepsis were 73%, respectively 75% (ppv 95.6, npv 27.3) and 96%, respectively 17% (ppv 87.2, npv 75). a cutoff point of 2 ng/ml for pct accurately predict severe sepsis (sensitivity 80%, specificity 100%, ppv 100). a pct level of at least 10 ng/ml was a good predictor for bacteremia (sensitivity 86%, specificity 90%, ppv 73.7 and npv 95). conclusion: pct is a good discriminating marker to characterize the level of inflammation caused by infection and can predict bacteremia . giamarellou h a , aoun a b , klastersky j b , anagnostopoulos n c , galani l c , grecka p c , panaretou e c , papageorgiou e c , repoussis p c , syrseloudis pct has been considered as a useful diagnostic marker in neutropenic patients with bacteremia and/or severe sepsis (giamarellos-bourboulis ej et al. clin infect dis 2001; 32:1718) . in an attempt to define its value in the diagnosis of localized infections in neutropenic hosts, daily determinations of pct and of c-reactive protein (crp) were performed before and after the onset of fever in 50 subjects 27 male and 22 female aged 48.69/18.7 years with various haematologic malignancies (aml 26, nhl 17, mds 5, all 2) developing neutropenia ( b/500 pmns/mm 3 ) after chemotherapy. thirty-three patients were presented with fever of unknown origin (fuo) and 17 with localized bacterial infections (lbi; pneumonia 7, acute pyelonephritis 4, soft tissue infections 3, acute pharyngitis 3). pct was determined by an immunoluminometric assay and crp by nephelometry. it is concluded that febrile neutropenia followed by a localized bacterial infections is accompanied by significantly higher levels of pct than in case of fuo (47.99/3.64 vs 0.519/0.16 ng/ml). similar differences are not observed with crp, which lacks the appropriate specificity. our study included 35 patients who were categorized as having proven (9), probable (11), or possible (5) systemic fungosis according to eortc criteria; 10 showed no sign of infection, and were used as controls. blood samples were received on the 1st, 3rd, and 5th day from the onset of signs of a fungal infection, and then twice a week. pct levels were determined by an immunochemioluminent assay, and candida and aspergillus antigen levels by elisa. in only five patients pct indicated early signs of infection, albeit at barely detectable limits. six patients, however, showed significantly increasing titres preceding time of death. positive antigens titres were observed only in 10 patients who had proven or probable systemic fungosis. only half of the control group had negative antigen titres; a high rate of false negatives was also observed. both pct and antigens titres increased in parallel in 4/6 patients with unfavorable outcome. pct and antigens titres cannot reliably indicate early diagnosis of systemic fungal infections although may be used as a prognostic tool of severity. lactulose, a factor that decreases endotoxaemia, in obstructive jaundice? pm262 koutelidakis im a , papaziogas v a , makris i a , giamarellos-bourboulis ej b , giamarellou h b , papaziogas t a . a thessaloniki med school, 2nd surgical clinic, thessaloniki, greece , b 4th department internal medicine, athens medical school, athens, greece bacterial translocation is a process implicated in the pathogenesis of spontaneous peritonitis. in order to evaluate the impact of lactulose administration on systemic endotoxaemia, obstructive jaundice was induced in 11 rabbits by common bile duct ligation. animals were divided into two groups, group a of five rabbits not receiving lactulose and group b of six rabbits, which received 1.5 ml/kg of lactulose orally by an oral catheter. blood was collected daily, before and after operation for a total duration of four days. samples were applied for culture and for determination of endotoxins (lps) by the lal qcl-1000 assay. concentrations of lps (mean9/sd) of group a were 1.159/0.74, 2.249/0.74, 1.609/0.81 and 2.349/0.70 eu/ml on the 1st, 2nd, 3rd and 4th day, respectively. respective concentrations of lps (mean9/sd) of group b were 0.849/0.28, 0.659/0.12, 0.819/0.20 and 0.609/0.42. all blood cultures were sterile in both groups. differences activity of linezolid against nosocomial strains of staphylococcus aureus in russia: results of multicentre study pm221 were included in the study. antimicrobial susceptibility testing was performed by agar dilution method in accordance with the nccls recommendations. all tested strains including 295 mrsa strains (33.6% of all strains) were found to be susceptible to linezolid with the mic ranged from 0.5 to 4 mg/l. both mic 50 and mic 90 were 2 mg/l. conclusions: linezolid had excellent in vitro activity that was not affected by resistance to other classes of antimicrobials susceptibility to antiseptics of mrsa isolated in japan during 1998 á 60% to piperacillin, 55% to ceftazidime, 71% to cefepime, 13% to imipenem, 51% to amikacin and 55% to ciprofloxacin. fiftythree percent of p. aeruginosa strains were multiresistant (25 strains) and were isolated in 12 patients. wild phenotype to b-lactams was observed in 30% of strains. the most frequent b-lactams resistance phenotypes were: cephalosporinase over production (34%) and penicillinase (11%). imipenem, ceftazidime and piperacillin-tazobactam were the most active b-lactams (mic 50 of 4.16 and 32 mg/l, respectively) these results showed high rates of antibiotic resistance and predominance of o11 serotype in multiresistant strains compared to the o12 serotype in europe. infectious complications sustained by stenotrophomonas (xanthomonas ) maltophilia in hiv at present, very limited informations are available about s. maltophilia infections in the setting of hiv disease. patients and methods: a retrospective survey of clinical and microbiological records of 1374 hiv-infected patients referring to out tertiary care centre between 1991 and 2000 was performed, in order to identify all episodes of s. maltophilia infections, and analyze its epidemiological, clinical, and microbiological variables. results: sixty-one episodes of s. maltophilia infection were observed in 59 patients: sepsis/bacteraemia in 48 cases (78.7%), lower airways infection in five, urinary tract infection in four, pharyngitis in two, lymphadenitis and liver abscess in one case each. forty-seven out of 61 episodes of s. maltophilia infections (77%) occurred as nosocomial disease, generally in association with advanced immunodeficiency, neutropenia, instrumentation, and prior antimicrobial therapy. bacterial isolates showed an elevated resistance profile against many betalactam compounds, aztreonam, imipenem, and aminoglycosides. conclusion: s. maltophilia represents an emerging opportunistic pathogen in hiv-infected patients extended spectrum beta-lactamases producing germs in intensive care units pm235 university of medicine and pharmacy 'victor babes ', microbiology, timisoara, romania injury and complications lasted 22 months, demanded for 11 surgical procedures and total cost was 1 comparison of different methods for detection of extended spectrum beta lactamases (esbls) and their genetic relatedness among enterobacteriaceae clinical isolates in a research medical institute pm242 methods: one hundred out of 178 isolates that were screened positive for esbls were tested with double disk synergy test (ddst), three dimensional test (tdt), e -test-esbl and vitek-esbl test. pulsed field gel electrophoresis (pfge) analysis was applied to 13 esbls; five klebsiella pneumoniae and eight escherichia coli . results: revealed the prevalence of esbls in 25.8% of clinical isolates. the sensitivities of the ddst, tdt, e -test and vitek were 100, 73, 86.9 and 47.8%, respectively. in the ddst, aztreonam was the most sensitive indicator (93.4%). pfge demonstrated that 80% of k. pneumoniae were derived from a single clone whereas 62.5% of e. coli isolates were derived from two different clones. non-clonal origin was demonstrated in 20% of k. pneumoniae and 37.5% of e. coli . conclusion: there is an increased prevalence of esbls. the ddst is the most sensitive, practical and cost effective diagnostic method reliable for routine use in our laboratory. both clonal spread and plasmid dissemination contributed to the concurrent nosocomial outbreaks caused by esbl-producing k severe nosocomial infections due to stenotrophomonas maltophilia pm243 the teaching hospital of infectious diseases total 241 og pts after extensive hysterectomy (201 pts), extensive vulvoectomy (22 pts) and extensive/combined operations for ovarian cancer (18 pts) were analysed. ic developed in 74 pts. one hundred and sixtyseven pts had no ic. twenty-eight of 50 rf analysed were independent rf of ic. most important included: age !/50 years (p0/0.0002), grade 2 á/3 obesity (p 0/0.0002), diabetes mellitus (p 0/ 0.0002), diagnosis of cervical cancer (p 0/0.0001), history of pre-cancer of vulva postpartum endometritis due to group a streptococcus : a case-control study pm247 unite operationnelle d 'hygiene all isolates were sensitive to vancomycin. among gram-negative bacteria klebsiella spp. was isolated in 21.6% of cases, acinetobacter baumanii in 13.5%, enterobacter spp. in 8.1%, providencia spp. in 2.7%. of the klebsiella spp. isolates 25% were resistant to amikacin, 85% to cephalosporins, 87% to piperacillin/ tazobactam. all were sensitive to imipenem. of the a. baumanii isolates 100% were resistant to amikacin, aztreonam, cefoperazone, cefotaxime, ceftriaxone, piperacillin; 60% to ampicillin-sulbactam, 80% to ceftazidime, and 40% sensitivity to imipenem antimicrobial activity of selected pharmacopoeial antiseptics analysed according to european standards pm270 european committee for standardisation approved several european standards (en), describing test methods establishing, whether an antiseptic has or does not have a bactericidal or fungicidal activity under the laboratory conditions defined by en. the aim of the study was to investigate, if some chemical compounds in concentrations recommended by polish pharmacopoeia for skin disinfection, comply european standards requirements. methods: basic bactericidal (en 1040) and fungicidal (en 1275) activity were investigated as well as bactericidal activity of products for hygienic and surgical handrub and hand wash used in human medicine (pren 12054). all methods and used neutralizers were validated. standard strains: staphylococcus aureus , pseudomonas aeruginosa , escherichia coli , e. hirae , candida albicans and a. niger were used, when en standards were evaluated. results: ethanol, izopropanol and n -propanol caused viable microbial count reduction required by ens in pharmacopoeial concentrations the purpose of the study: we collected 226 bacteriological samples from adult and neonate patients who were admitted in intensive care units (icu) . the aim was to observe the colonization status with microbes that may have a nosocomial potential and to establish circulating phenotypes in icus. the results obtained from a total of 226 samples 61 strains of gram negative bacteria (enterobacteriaceae family) were isolated. fourteen strains showed extended spectrum beta-lactamases (esbl) phenotype (eight strains of klebsiella pneumoniae , three of escherichia coli , two of klebsiella ornithynolitica , one of klebsiella oxytoca ). we used both disc diffusion test (extended antibiotic susceptibility test and synergy test to visualize 'champagne stopper' pattern) and mini api † system.the conclusion reached: we put in evidence a massive colonization with germs that may have a nosocomial potential especially microbes that produce esbl (22.9% from all enterobacteriaceae isolated) which implies a rational policy in prescribing antibiotics in hospitals from western part of romania.carbapenem activity against nosocomial gram-negative rods pm236sawicka-grzelak a a , rokosz a a , meszaros j b , luczak m a . a department of medical microbiology, university medical school, warsaw, poland , b department of general and transplantation surgery, university medical school, warsaw, poland purpose: to determine a susceptibility of nosocomial gramnegative rods to carbapenems.methods: two hundred strains of gram-negative rods were cultured from clinical specimens from hospitalized patients (july á/november 2001). identification of strains was performed in the automatic atb system (biomerieux, france). susceptibility of strains to carbapenems: imipenem and meropenem was determined with disc diffusion method according to nccls recommendations. esbl-producing strains were detected with double-disc synergy test (ddst according to jarlier et al., 1988) or a novel method of esbl detection (dd, diagnostic disc) according to appleton (1999) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd 01) (oxoid, england) .results: one hundred and ten strains of enteric rods and 90 strains of non-fermenting rods were cultured. twenty eight (14%) esblpositive strains were detected. carbapenems were active against 96% of enteric rods. the percentage of non-fermenting rods susceptible to imipenem was 70 and to meropenem */60.conclusions: carbapenems: imipenem and meropenem demonstrated high activity against clinical strains of enteric rods. however, the antibiotics were less active against nosocomial strains of nonfermenting rods.inhaled antibiotics against multiresistant bacteria in bronchial secretions of icu patients: a preliminary report pm237horianopoulou m a , kanellopoulou m b , paraskevopoulos i a , valakis k a , kyriakidis a a , lambropoulos s a . a intensive care unit, sismanoglio general hospital, athens, greece , b department of microbiology, sismanoglio general hospital, athens, greece purpose: the aim of this study was to assess the effectiveness of aerosolized ampicillin/sulbactam, ceftazidime and colistin, in icu patients with multiresistant acinetobacter baumannii or pseudomonas aeruginosa colonization of the respiratory tract.methods: fifty-three intubated, mechanically ventilated patients participated in the study. multiresistant a. baumannii , sensitive only to ampicillin/sulbactam, or p. aeruginosa , sensitive to ceftazidime or colistin, were isolated from the bronchial secretions (10 5 á/10 8 cfu/ ml). all 53 patients were subsequently treated with intravenous ampicillin/sulbactam, ceftazidime or colistin, whereas 27 of them were also given the same antibiotic in aerosolized form.results: a decrease in the number of colonies by 10 3 á/10 6 cfu/ml was observed, following 2 á/4 days of combined treatment with both intravenous and inhaled antibiotic. none of the 27 patients developed vap. in the 26 patients who only received the antibiotic intravenously, the decrease ranged from zero to 10 4 cfu/ml, after 7 days of treatment. two of 26 patients developed vap.conclusions: our results suggest that the administration of aerosolized antibiotics represents an effective means of preventing ventilator-associated pneumonia caused by a. baumanni and p. aeruginosa . introduction: pseudomonas aeruginosa is an important nosocomial pathogen. resistance to certain beta-lactam antimicrobial agents among p. aeruginosa is increasing. despite the development of new antibiotics multiresistant strains of p. aeruginosa represent an important therapeutic problem. the aim of this study was to investigate the activity of imipenem, amikacin, piperacillin, ciprofloxacin, ceftazidime, against clinical isolates of p. aeruginosa . methods: a total of 54 isolates by tracheal aspiration from hospitalized patients, admitted to intensive care units were identified as p. aeruginosa using an algorithm that included: gram stain, pigment, oxidaze ('/0,) and gram negative identifications microscan walkaway-96 (dade behring) were used according to the manufactures instructions. minium inhibitory concentrations were determined using walkaway, interpretation based on ncclsm 100-s9, january '99.results: the respiratory tract was the single site of isolation for this study. the best activity was showed by imipenem 25%, followed by amikacin 22%, piperacillin, pip/tazobactam, ciprofloxacin had the same sensitivity 11%.conclusion: a high level resistance to antibiotics was observed to p. aeruginosa isolated from tracheal aspiration. carbapenems seem to be the most active against p. aeruginosa in this study. materials and methods: clinical samples were collected from patients admitted to this hospital. only one isolate per patient was included. antimicrobial susceptibility testing was performed as recommended by the nccls. all bacterial isolates were tested by dd and ad to provide a comparison of both test results. very major error was considered when the strains were resistant (r) by ad and susceptible (s) by dd and major error when s by ad and r by dd. categories of s and r were stablised using the breakpoints suggested by mensura (2000) . colistin r strains was typed by rep-pcr.results: among the 35 strains included in this study 30 (85.7%) were s to colistin and five (14.2%) were colstin r by ad, of this five colistin r strains four (11.4%) were s to colistin by dd and one was r by both methods. all r isolates were similar by rep-pcr.conclusions: most of the ad colistin resistant strains were s when tested by dd indicating that this method is not useful to determine the resistance to colistin. rep-pcr patterns show that the spread of a colistin r clone seems to be involved. methods: gnb were isolated from per-operative biopsies (pob) and/ or from articular punction (ap). patients (pts) received cfp, 2 g bid'/ ofl, 200 mg tid or cip, 400 mg bid intravenously for 28 days, followed by a prolonged oral fq monotherapy. cure was defined as: resolution of all clinical signs of infection, normalization of the biological inflammatory profile at the end of treatment (eot) and absence of infection at the same site during the post-treatment followup period (ptfu).results: all of the 20 studied patients [mean age 0/48 years] had hospital acquired bji. seventeen/20 had an infected orthopedic device (prosthetic joints0/6, other orthopedic prosthetic devices0/11). culture of 17 pob and 5 ap yielded to pseudomonas sp. (11), enterobacter cloacae (6), others (4). vancomycin was added for six pts co-infected by gnb-mrsa. nineteen/20 pts underwent a surgical intervention (debridement0/11, removal-replacement0/7, amputation0/1). after ptfu period of 17 months (range 3 á/29), the overall success rate was 12/17 (88.2%) without serious adverse events.conclusion: cfp á/fq combination was safe and efficient in the treatment of hypercase gnb, bji.treatment of posttraumatic mrsa osteomyelitis of the femur with longterm cotrimoxazole */a case report pm241 the authors report a case of posttraumatic osteomyelitis of the femur caused by methicillin-resistant staphylococcus aureus (mrsa), following the shot injury. relapses of the infection occured in 3 months interval and were treated by revision, debridement, lavage and vancomycin. because of laboratory signs of renal insufficiency vancomycin became contraindicated for treatment of the third relapse of infection and the different approach was employed: classic open treatment of bone infection sec. orr was combined with a long-term administration of high-dose cotrimoxazole. the patient was given cotrimoxazole 9600 mg daily divided in four doses (120 mg/kg/24 h) for 2 months, then for gastrointestinal complaints with lowered dose of 5760 g daily for next 6 months. the wound completely healed. during 18 months after the final surgery there was no relaps of infection, but the atrophic pseudoarthrosis of the femur resulted. the patient can walk with a rigid orthesis and two crutches. the whole treatment of the objective: to present a variety of severe nosocomial infections due to stenotrophomonas maltophilia in patients hospitalized in tertiary medical units from cluj.results: during the last year nine strains of s. maltophilia obtained from patients with severe infections and hospitalized in different wards were isolated. all but one were considered nosocomial infections: four cases of pneumonia, one urinary tract infection, three cases of surgical wound infections and one case of endocarditis under surgical treatment. the cases of pneumonia were either primary occurring in a granulocytopenic patient with leukemia or secondary in patients that underwent surgical treatment. in the case of endocarditis the ethiology was established after surgery from the damaged valve in a negative hemoculture patient with a poor outcome under medical treatment. in all cases of surgical wound infection bacteremia occurred diagnosed on clinical basis in the presence of severe sepsis or hematogenous dissemination in the lung. the urinary tract infection occurred in a patient after urinary surgery and having a catheter in place. the immediate evolution was favorable in all cases but treatment was difficult due to the highly resistant strains and to underling diseases.conclusions: s. maltophilia should be considered in nosocomial severe infections and prophylaxis by interrupting environmental transmission has to be promoted. sawicka-grzelak a, rokosz a, luczak m. department of medical microbiology, university medical school, warsaw, poland purpose: to identify and determine the drug-susceptibility of esblpositive strains isolated from urine samples.methods: seven hundred and twelve strains of gram-negative rods were cultured from 4000 urine samples from hospitalized patients during 5 months (july á/november 2001). identification and susceptibility were performed in the automatic atb system (biomerieux, france) using id 32 e, id 32 gn and atb ur 5 strips. esbl-activity was detected with double-disc synergy test (ddst according to jarlier et al., 1988) or using a novel method of esbl detection (dd, diagnostic disc) according to appleton (1999) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd 01) (oxoid, england).results: five hundred and ninety-five strains (83.6%) belonging to enterobacteriaceae family, 115 strains (16.1%) of non-fermenting rods and two strains (0.3%) of other gram-negative rods were isolated. eighty-two esbl-producing strains (11.5% of all strains) were detected. fifty-nine esbl-positive strains were susceptible to nitrofurantoin, 58-to norfloxacin and ciprofloxacin and 54-to fosfomycin.conclusions: esbl-positive strains were detected most frequently among enteric rods (80 strains). nitrofurantoin and quinolones were the most active in vitro antibacterial agents against examined esblpositive uropathogens. results: the mechanisms of resistance were evaluated phenotypically using 12 different aminoglycosides. a total of 257 aminoglycoside resistant gram-negative strains were studied. one hundred fifty-eight strains were collected in 1995 á/1996, 52 in 1998 and 47 in 2000. the resistant profiles were determined: enterobacteriaceae */152; pseudomonas aeruginosa */62; acinetobacter spp. */43. the most frequently phenotypes were gt (gentamicin, tobramycin) */48% and gtnet (gentamicin, tobramycin, netilmicin) */35%. the gt phenotype due to production ant(2ƒ)-i enzyme, the gtnet á/aac(3)-v (87 strains), aac(3)-iv */one strain and aac(2ƒ)-i */one strain. the resistance to amikacin in 7% strains was due to production aac(6?)-i (3%) and aph(3?)-vi (4%). the most of examined strains were simultaneously resistant to kanamycin and neomycin caused by production of aph(3?)-i (69%). only seven strains were resistant to all aminoglycosides due to impermeability of outer membrane. no substantial differences were observed between years.conclusions: the main mechanism of aminoglycoside resistance is fermentative modification. the high rate to gentamicin and tobramycin was due to production of ant(2ƒ)-i and aac(3)-v. amikacin and isepamicin were the most active aminoglycosides against gramnegative nosocomial isolates.the incidence of clostridium difficile associated diarrhoea (cdad) increased in our department from january to june 2001.objective: to confirm the out break of cdad, to identify the risk factors and assess the effectiveness of the measures implemented for controlling this outbreak.methods: cdc definitions were used to identify the cases. the scope of the outbreak was defined. cdad incidences during the outbreak period and during the same period in 2000 were compared. risk factors (reduced mobility, antibiotic treatments . . .) were studied for patients whom length of hospital stay (lhs) was more than 6 days. contact precautions and environmental cleaning with clona implemented were assessed.results: seventeen episodes of cdad were identified. sex ratio: 1.41, mean age0/58.5, mean lhs0/36 days, mean delay for cdad occurring0/21 days. one hundred and fifty-two patients involved in the study of risk factors. relative risk (r.r.) evaluated were: blactams (r.r.0/7.62, ic95%: 1.03 á/56.56), reduced mobility (r.r.0/ 7.61, ic95%: 1.76 á/32.85). incidence of cdad was less than two cases per 1000 days of hospitalisation after june 2001.conclusion: we confirmed the outbreak of cdad in our department b-lactams and reduced mobility were identified as risk factors for cdad. measures implemented to control the outbreak were effectiveness.positive heart transport fluid cultures associated with severe infections in heart transplant recipients pm255 at the mount sinai hospital in new york city 256 heart transplants were performed between 1986 and june 2000. cultures were routinely performed on all heart transplant transport fluids. culture data was available for 204 of these patients. in total 24/204 (11.8%) were positive for bacteria, fungi or both. the organisms isolated included coagulase negative staphylococci (13), pseudomonas aeruginosa (2), staphylococcus aureus (1), acinetobacter baumanii (1), serratia mercescens (1), enterobacter cloacae (1), escherichia coli (1), proteus mirabilus (1), enterococcus faecalis (1), viridans streptococci (1), and fungi (aspergillus fumigatus (1), penicillium species (1), and rhodotorula rubra (1). two heart transplant recipients had two organisms isolated from the transport fluid. isolation of resistant gram-negative bacilli in the transport fluid was associated with significant infection in 3/5 patients (60%) with the same organism. the observed infections were pneumonia secondary to e. cloacae , sternal wound infection secondary to p. aeruginosa , and bacteremia secondary to p. aeruginosa . it appears prudent to provide prophylaxis against resistant gram negative bacilli to prevent infections. zacharof ak, flevaris c, petrogianopoulos c, karachalios g, vroulis j, chartzoulakis g, drakogiorgos g, loizidou a, svoukas g. 2nd department of internal medicine, hellenic red cross hospital, athens, greece objective: we studied the trends of nosocomial bloodstream infection and calculated the population-attributable risk for death among hospitalized patients. methods: we perform a 15-year retrospective study for all patients (n 0/79 160), admitted to our department between 1986 and 2000.results: between 1986 and 2000, a total of 1577 patients developed 1763 episodes of nosocomial bloodstream infection. the crude infection rates increased linearly from 7.2 to 25.4 per 1000 discharges (1.11 á/1.93 episodes per 1000 patient-days) during the 15-year study period. increases in the infection rates were due to gram-positive cocci, yeasts and essentially explained by infections caused by coagulasenegative staphylococci, staphylococcus aureus , enterococci, and candida species, respectively. although the crude mortality in patients with nosocomial bloodstream infections decreased from 45% in 1986 to 28% in 2000, the in-hospital population-attributable mortality among infected patients increased from 5.5 deaths per 1000 discharges in 1986 to 8.2 per 1000 discharges in 2000. the etiologic fraction or the proportion of deaths in patients with bloodstream infection to all deaths occurring in the hospital increased from 15.4% in 1986 to 26.4% in 2000.conclusions: the incidence and the population-attributable risk for death among patients experiencing nosocomial bloodstream infections increased progressively during the last 15 years in our department.ventilator-associated pneumonia before and after intensive care unit temporary closure pm257 results: we compared the incidence, causative organisms and mortality of vap in two different time periods. period 1 june to december 1999. period 2 june to december 2000. between those two periods the icu remained closed for 4 months because of reconstruction works.period 1: sixty-seven consecutive patients (pts) were studied with bronchial secretions cultures at least 2 days after mechanical ventilation (mv) initiation. the vap was diagnosed by clinical, radiological and microbiological criteria in 17 pts (26%). causative organisms included: pseudomonas aeruginosa 8, acinetobacter 5, staphylococcus aureus 4, klebsiella pneum. 3, enterobacter 1. in three cases vap was proved polymicrobial. fourteen (14) episodes of vap (82%) were developed after 7 days mv (late vap) and were attributed to multiresistant microorganisms. mortality of vap was 29%.period 2: among 66 consecutive pts, vap was diagnosed in 21 (32%). causative organisms included: acinetobacter 10, p. aeruginosa 8, s. aureus 4, k. pneumoniae 3, escherichia coli 1. five (5) cases were polymicrobial and 14 cases were 'late vap' (67%). causative microorganisms had similar patterns of sensitivity to antibiotics (compared to period one). mortality of vap was 38%.conclusion: temporary icu closure had no significant influence on the incidence, distribution of causative organisms, their sensitivities to antibiotics and mortality of the vap. efstathiou sp a , pefanis av a , tsioulos di a , tsiakou ag a , zacharos id a , kanavaki s b , mountokalakis td a . a third university department of medicine, sotiria general hospital, athens, greece , b microbiology laboratory, sotiria general hospital, athens, greecepurpose: the aim of this study was to derive a scoring system for the prediction of outcome in adult patients with acute pyelonephritis (ap) severe enough to need hospitalization. therefore, the charts of 225 patients (102 men, median age 67 years) were reviewed.results: logistic regression analysis identified in both sexes four independent correlates of in-hospital mortality, the coefficients of which divided by 0.5 and rounded to the nearest interval, resulted in the following integer-based scoring system: points (men)0/6* between concentrations of lps of the two groups were statistically significant on the 2nd and the 4th day (p b/ 0.05). it is concluded that the administration of lactulose may decrease systemic endotoxaemia in the field of obstructive jaundice.nosocomial infections: a prevalence study in the island of crete pm263 doukakis s a , tzimis l b , perogambrakis g a , kalloniatou m a , christodoulakis n a , evaggelopoulos a a , koutsoumba d a , kastanakis s a . a first medical department, 'saint george ' general hospital, chania, greece , b pharmacy department, 'saint george ' general hospital, chania, greeceprevalence surveillance is a rapid and inexpensive mode to estimate the problem of hospital-acquired infections (hais). to study the problem of nosocomial infections in our hospital, a prevalence study was made from our team in 1999. the study included 265 patients (the total number of hospitalized patients at the time of the study). from these patients 129 were males (49.0%) and 136 females (51.0%). one hundred and ninety-one patients (72.0%) belonged in the groups of age between 50 and 89 years. fifty-seven patients had a urine catheter (22.3%). one hundred and fifty-five/265 patients (58%) received antibiotics and from these 99 patients received one antibiotic and the remaining 56 patients two or more. a nosocomial infection was found in 13 patients and consequently the prevalence of hais was 5.0%. among these, urinary tract infections were six (46.1%), lower respiratory tract infections were three (23.0%), surgical site infections were three (23.0%), and bloodstream infections was one (7.7%). the incidence of multiresistant bacteria was primarily enterococcus spp and secondary, pseudomonas aeruginosa , enterobacter spp, klebsiella pneumoniae , escherirchia coli , staphylococcus aureus , enterobacter cloacae . unfortunately prophylactic chemotherapy of long duration was found despite the suggestions of the infection control committee. regarding age the highest incidence of hais occurred in the third age group. bagirova ns, dmitrieva n. laboratory of microbiology, cancer research center of russia, moscow, russian federation objectives: to determine the pathogens and susceptibility to antimicrobials.methods: blood samples were collected from 430 adult pts (1997 á/ 2001). the bacteraemic episodes were classified according to the definitions of the cdc. laboratory detection of bacteraemia and fungaemia was performed according to cumitech 1b (blood cultures iii, 1997). susceptibility testing was performed by disk diffusion method (nccls).results: the total number of blood samples */1371, 249-positive (18.2% episodes of significant bacteraemias). bsi was confirmed microbiologically in 137 of 430 febrile pts (31.9%). the most frequent pathogens were gram('/) cocci (62.7%) (p b/ 0.0001), gram((/) bacilli */22.7%, fungi */11.4%. coagulase-negative staphylococci (cns) represented 38.9%, staphylococcus aureus 12.4%, streptococcus spp. 4.9%, enterococcus spp. 5.9%, enterobacteriaceae 15.1%, pseudomonas aeruginosa 3.2%, other non-fermenting */5.9%, yeast 8.1%, mould 3.2%, anaerobes 2.7%. one hundred percent cns were resistant to penicillin, 68.4% to oxacillin, 36.6% to clindamycin, 39.5% to cefazolin, 80.3% to ceftazidime, 48.7% to ciprofloxacin, 51.3% to gentamicin, and no isolate was resistant to vancomycin. the predominant pathogens in all types of hm were gram('/) cocci (mainly cns). all gram('/) microorganisms were sensitive to vancomycin. katashinsky o a , opriatova t b , tchuev p c . a state clinical hospital, anaesteziology, odessa, ukraine , b state clinical hospital, bacteriology, odessa, ukraine , c medical university, anaesteziology, odessa, ukrainethis investigation was carried out in odessa state clinical hospital during 2000 á/2001. of the patients with post-operation complications, 60% of 518 gram-negative cultures were sensitive to ceftazidime and 72% to amikacin. sixty-nine percent of 143 gram-positive cultures were resistant to penicillin g, but they were sensitive to vancomycin and nitrofurantoin in 100 and 92% of cases, respectively. sixty percent of 366 isolates of pseudomonas aeruginosa were sensitive to ceftazidime and 71% to amikacin. rates of resistance to carbenicillin and gentamicin were 60 and 66%, respectively. one hundred and fortythree isolates of escherichia coli were studied and 65% of them were resistant to ampicillin 67% to cephalothin and 70% to tetracycline. of the isolates tested against ciprofloxacin, all were sensitive. one hundred and eight isolates of staphylococcus aureus were studied and they were resistant only to penicillin g (69%). staphylococcus aureus was sensitive to erythromycin (65%), tetracycline (65%), oxacillin (83%) and vancomycin (100%). all 34 isolates enterococcus faecalis were sensitive to nitrofurantoin and 70% to ciprofloxacin. the majority of s. aureus and e. faecalis isolates were susceptible to most other antibiotics, but the majority of e. coli isolates were resistant to the studied antibiotics expect ciprofloxacin.campylobacter foetus bacteraemia in an immunocompromised patient: a case report pm266monno r a , ierardi e b , rendina m b , ceci g a , luzzi i c , de vito d a , rizzo g a , francavilla a b . a department of internal medicine and public health, university of bari, bari, italy , b department of emergency and organ transplantation, university of bari, bari, italy , c istituto superiore di sanità, rome, italy a 33-year-old woman was admitted for recurrent fever. the patient underwent a liver transplantation and splenectomy in 1985. she had followed immunosuppressive therapy until 1995 when tacrolimus was added for chronic rejection. in 1999 non-hodgkin lymphoma was diagnosed and chemotherapy was started. six months later because of the presence of two metastatic encephalic foci affecting the optic chiasm, a new chemotherapy course was started with the regression of lesions. in january 2000 she was treated with steroid recycle and cyclosporine á/azathioprine á/prednisone reintroduction. fever occurred after 2 months and cytomegalovirus (cmv) infection was diagnosed. treatment with ganciclovir was started with clinical remission. in november 2000 cmv infection recurred and blood cultures were positive for a bacterium that was identified as campylobacter fetus . the patient was successfully treated with intravenous ciprofloxacin. bacteremia frequently occurs in cancer patients. bacteremia due to c. fetus are rare, occurring mainly in immunocompromised patients. c. fetus expresses a proteinaceous surface layer that confers serum resistance. in our patients steroid and immunosuppression may have contributed to the development of lymphoma. all of these factors and chemotherapy have contributed to cmv infection and all have made the patient susceptible to bacteremia with this infrequently found bacterium. the clinical microbiologist should be aware of this infection in immunocompromised host. minenko sv, dmitrieva nv, sokolova en, ptushkin vv. bone marrow transplantation department, n.n. blokhin cancer research center, moscow, russian federation c'/a is the standard regimen as empirical therapy for febrile neutropenia (fn). activity of c against g'/ bacteria and g(/ bacteria, producing chromosomally-mediated b-lactamases (e.g. ampc) is suboptimal. cep is active against a broad range of g'/ and g(/, including ampc producing bacteria. the purpose of the study was to compare the efficacy of two regimens in the treatment of fn.methods: patients with fn received either cep (2 g/8 h) or c (2 g/8 h) plus amikacin (15 mg/kg/day) or netilmicin (5 mg/kg/day). data were collected prospectively.results: a total of 35 pts with 41 episodes (35/41) of fn were included. fifteen/19 in cep group and 20/22 in c'/a group. the median duration of neutropenia grade 4, distribution of age, sex and underlying disease were comparable in both arms. mdi was in 37 and 24%, cdi in 21 and 18% fuo in 57 and 63% in fep and c'/a groups correspondingly. response to the initial empirical regimen according to ihs criteria was in 58% of cep and 27.3% of c'/a groups (p 0/ 0.05). modification of therapy with a change of cep or c to carbapenem took place in 31 and 40% of cep and c'/a groups (p0/0.5). no patient in either treatment group died due to the presenting infection. tolerability of cep was good and no laboratory abnormalities took place. transient elevation of serum creatinin level was observed in two patients c'/a group.conclusions: cep monotherapy is as effective as c'/a combination in the treatment of patients with fever and granulocytopenia purpose of the study: retrospectively, we analysed 60 patients with intraabdominal infection for aetiology, risk factors, and outcome from 11 hospitals in slovak republic within 1 year (march 00 á/march 01). results: in this group significantly more frequent were older patients ( !/65 years) with cancer (65 vs. 16%, p b/ 0.0007). acinetobacter spp. (50 vs. 7%, p b/ 0.0001) and enterobacteriaceae (83 vs. 39%, pb/ 0.04) were predictive for monoinfection. pre-treated patients with other antibiotics had inferior prognosis and more risk factors: permanent urinary catheter (95 vs. 67%, pb/ 0.05), ventilation or intubation (91 vs. 17%, pb/ 0.0001) and polymicrobial infection (86 vs. 33%, pb/ 0.0002). the risk factors with poor prognosis were enterobacteriaceae (30 vs. 14%, pb/ 0.04), diabetes mellitus as underlying disease (18 vs. 54%, p b/ 0.2) and uraemia (4 vs. 40%, p b/ 0.002). surprisingly, negative prognostic factor was also non-effective previous antibiotic therapy. failed patients died significantly more frequently patients due to underlying disease. cefoperazone/sulbactam was shown to be useful, effective and well tolerated also in one group of patients with 75% efficacy of treatment, and it belongs to a group of antibiotics suitable for treatment of nosocomial infections. key: cord-350571-6tapkjb6 authors: nan title: 45th escp-nsf international symposium on clinical pharmacy: clinical pharmacy tackling inequalities and access to health care. oslo, norway, 5–7 october 2016 date: 2017-01-10 journal: int j clin pharm doi: 10.1007/s11096-016-0404-4 sha: doc_id: 350571 cord_uid: 6tapkjb6 nan pharmacy, sint maartenskliniek, ubbergen, 2 pharmacy, radboud university medical centre, nijmegen, 3 clinical pharmacy and toxicology, maastricht university medical centre, maastricht, netherlands please specify your abstract type: research abstract background and objective: according to literature adherence to statins ranges from 32 to 71%. medication adherence is affected by both practical barriers and patient's beliefs about medication. however, physicians also have their beliefs about medication. several studies have shown that these beliefs also impact the decision of patients to agree with a particular treatment or not. as current published interventions on medication adherence (which focus predominantly on patients) are not or just partly effective, physicians' beliefs might be a promising target for interventions to improve adherence. however, there is currently no information available on physician's beliefs about statins and whether these beliefs affect patient's beliefs and adherence. therefore, the objective of this study is to examine whether physicians' beliefs about statins influence the beliefs and adherence of patients using a statin. setting and method: this cross-sectional study was conducted in gp practices and community pharmacies, between september 3, 2014 and march 20, 2015. physicians' and patients' beliefs about statins were assessed with the beliefs about medicine questionnaire (bmq) specific. patients' adherence on statins was assessed with both the mars-5 and the morisky-8 questionnaires. please specify your abstract type: research abstract background and objective: nhs highland and nhs western isles are the most remote and rural health boards in the united kingdom, with high numbers of dispensing medical practices. a pilot is underway in dispensing practices with clinical pharmacists undertaking targeted medication reviews. a previous quantitative service evaluation demonstrated its value, with pharmaceutical care issues identified in almost all patients, the vast majority of which (86.7%) were managed by the pharmacist without any need for general practitioner (gp) referral. the objective was to undertake a qualitative exploration of the service. setting and method: all patients and staff involved in the service were invited to participate. a semi-structured interview schedule was developed and piloted. telephone interviews were conducted with all consenting staff and a purposive sample of consenting patients recruited to the point of data saturation. interviews were audiorecorded, transcribed verbatim and analysed thematically. nhs ethics and research and development approvals were obtained. were the most confident with doacs (range from 73.4 to 75.7%) please specify your abstract type: research abstract background and objective: patients are at risk of drug-related problems (drps) at transition points during hospitalization. the community pharmacist (cp) is often the first healthcare professional patients visit after discharge. cps lack sufficient information about the patient and so they may be unable to identify problems in medications, which may lead to dispensing the wrong drugs or dosage, and/or giving wrong information. we aim to assess the impact of a complex intervention comprising of medication reconciliation performed at discharge by a hospital pharmacist (hp) with communication between the hp and cp on drps during the 7 days following discharge. setting and method: cluster randomized crossover trial involving medical and surgery care units (each unit corresponding to a cluster) in french hospitals during two consecutive 14-day periods, randomly assigned as 'experimental'(e) or 'control' c (usual care) periods. during the experimental period, the hp performed a medication reconciliation that was communicated to the patient's cp. main outcome measures: the primary outcome was a composite outcome of any kind of drp (prescription/dispensation, gap or patient) during the 7 days following discharge assessed at day seven post-discharge by phone from patient and cp. the secondary outcomes were 1/unplanned hospitalizations assessed by phone contact at day 35 after discharge and 2/the iatrogenic potential exposure scale from 0 to 3 for each patient established by a clinical team. analysis was conducted in intention to treat. results: 22 hospitals corresponding to 48 clusters enrolled 1092 patients (536 e group v/s 548 c group). no difference was observed on age, sex, autonomy, and number of drugs in home medication at admission and discharge. at day 7; 236 (45.6%) patients in e group had at least one drp v/s 280 (52.6%) in c group (or 0.77; ic 95% [0.61; 0.98] p = 0.034). intervention was especially efficient for patient discharged from surgery unit (or 0.64 ic 95% [0.43; 0.94]) and aged less than 75 years (or 0.71 ic 95% [0.53; 0.94] . although intervention decreased patient exposure to drp with high iatrogenic potential (from 8.7 to 5.2% p \ 0.0006), un-planned hospitalizations at day 35 weren't different between groups (5.8 vs. 4.5% p = 0.50). conclusion: medication reconciliation associated to communication between hospital and community pharmacists is efficient to decrease patient exposure to drp but not sufficient to decrease un-planed hospitalization. hp-pc003: clinical pharmacists bridging health care levels by medication reviews in primary care katherine wendelbo *,1 , kristine lundereng 2 1 namsos hospital pharmacy, central norway hospital pharmacy trust, namsos, 2 levanger hospital pharmacy, central norway hospital pharmacy trust, levanger, norway please specify your abstract type: descriptive abstract (for projects) background and objective: nord-trøndelag county is sparsely populated and many inhabitants live far from the hospital. additionally, only half (12 of 23) of the municipalities have a local pharmacy. traditionally, namsos and levanger hospital pharmacies have performed quality audits of the implementation of drug administration procedures in primary care units. since 2013, a service where clinical pharmacists participate in multidisciplinary medication reviews in 19 municipalities throughout the county has been established. the objective of this poster is to describe the practical approach and design of the service. design: a descriptive report of an implemented clinical pharmacy service in primary care where clinical pharmacists, as part of multidisciplinary teams, perform medication reviews. results: medication reviews are performed on patients admitted to nursing homes and patients in home care, receiving help with handling of their drugs. primary care nurses prioritise patients (by selecting frail elderly with multiple co-morbidities and polypharmacy), usually five patients in each meeting. prior to the review, nurses collect medical information using a checklist including; diagnosis, drug-related symptoms, standard laboratory tests and an updated medication list. the clinical pharmacist receives de-identified medical information by postal mail or e-mail before the meeting. based on this information the pharmacist identifies possible drugrelated problems (drps) and provides recommendations on how to solve them. this is performed in a structured approach according to the integrated medicine management (imm) model. subsequently, the pharmacist visits the municipality and discusses the medication reviews in a multidisciplinary team meeting with nurses and physicians. in addition, the pharmacist gives lectures in a medication related topic (e.g. treatment of insomnia and anxiety, oral anticoagulants and cognitive side effects). following the meeting, the pharmacist reports the drps and suggested interventions to the multidisciplinary team, for further follow-up. during 2015, totally 220 medication reviews were performed in 19 municipalities. in the same period, 25 lectures were given by the clinical pharmacists. conclusion: this clinical pharmacy service enables multidisciplinary medication reviews even in municipalities with limited health professionals and resources. as a part of multidisciplinary teams, the clinical pharmacists contribute with medical competence. camille castel 1 , arnaud de la blanchardière 2 , vincent cattoir 3 , guillaume saint-lorant *,1 1 pharmacy, 2 infectious and tropical diseases, 3 microbiology, chu caen, caen, france please specify your abstract type: research abstract background and objective: antimicrobial stewardship have clearly demonstrated their efficiency towards a more adequate use of antibiotics. since 2014, the use of daptomycin, a ''critical last resort antibiotic'' has intensified in our hospital, occasionally outside the scope of its approved indications. this situation has led to the implementation of an antimicrobial stewardship and the drafting of local guidelines. the aim of this study is to analyse the evolution and pertinence of daptomycin prescriptions, after distribution of these guidelines within our institution. setting and method: a monocentric prospective study was conducted between july 2015 and november 2015 in a 1500-bed university hospital. each daptomycin prescription recorded by pharmacy department was analysed by an infectious diseases specialist in the presence of the prescriber and considering local guidelines and the patient's clinical conditions. main outcome measures: the indicators chosen to determine prescription pertinence were: treatment indication, prescribed dose and other antibiotics associated with the daptomycin prescription. results: 20 daptomycin prescriptions were analysed. observed indications were: sepsis (35%), infective endocarditis (25%), bone and joint infections (25%) and vascular prosthetic infections (10%). identified pathogens were: mrsa (35%), methicillin-resistant coagulase-negative staphylococci (25%), methicillin-sensitive staphylococcus aureus (10%), enterococci (10%) and methicillinsensitive coagulase-negative staphylococci (5%). daptomycin was prescribed as first-line treatment in 50% of cases. the mean dose was 7 mg/kg/day [3-10 mg/kg/day] for a mean duration of 17 days [2; 55 days] . local guidelines were followed in 20% of cases. daptomycin use was relevant for 70% of prescriptions. the irrelevant prescriptions triggered the modification or stoppage of antibiotic therapy in 50% of cases, respectively, generating an 18% decrease in consumption and an economy of over €6700 for our institution. conclusion: this study shows the efficiency of antimicrobial stewardship in adequately using antibiotics, limitating ecological impacts, improving patient care and decreasing healthcare costs. it also shows that guidelines alone are insufficient to ensure a proper use of antibiotics. without a close prescription follow-up, constant reminders and sustainable evaluations, guidelines only affect a few prescribers. within the context of an ''antimicrobial crisis'', further development of guidelines and antimicrobial stewardship is essential to fight increasing bacterial resistances and requires a close collaboration between all healthcare professionals including pharmacists. interviews were transcribed verbatim and data were analysed using systematic text condensation. results: three major themes were identified: benefits, unrealised potential and criteria and barriers for success. (1) benefits described by physicians included increased patient safety, increased awareness on drugs, and an ease of workload. drug interaction management was emphasized as one of the clinical pharmacists' most important work tasks, as well as being a resource for collaborating healthcare professions and to the patient himself. (2) the clinical pharmacists expressed that they had an unrealised potential and could contribute to a greater extent in the multidisciplinary team than they did already. they mentioned education towards physicians and nurses, contribution in treatment decision-making and patient counselling as examples for possible extended work tasks. (3) as criteria to succeed as a clinical pharmacist, physicians highlighted the importance of oral communication and physical presence on the wards. as barriers for integration in the team, the clinical pharmacists identified the physicians' lack of knowledge about the clinical pharmacists' skills as well as unclear expectations regarding their responsibilities. conclusion: physicians agreed that the clinical pharmacist represent a valuable contribution to the multidisciplinary team, where patient safety and drug interaction management are highlighted as main benefits. clinical pharmacists should to a greater extent educate healthcare professions in drug related topics and provide patient counselling. continuous effort on making the clinical pharmacist a natural part of the multidisciplinary team is crucial for the development of clinical pharmacy. by gathering perceptions from the collaborating professions as well as educating them on what clinical pharmacists can provide, we can develop a multidisciplinary team that enhances patient safety. hp-pc006: assessment of dual antiplatelet therapy following acute coronary syndrome using grace and crusade sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: mortality and morbidity benefits of dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) have been unequivocally demonstrated in a large body of evidence. with the availability of more potent antiplatelet agents, balancing ischemic and bleeding risks to prevent adverse outcomes is an on-going challenge, in particular, recognising that patients with high bleeding risk were excluded from clinical trials. grace and crusade scores stratify risk of mortality and in-hospital major bleeding post acs respectively. these tools should be used to support antiplatelet choice in light of newer more potent agents that equally pose a greater risk of bleeding. design: grace and crusade scores were calculated for patients presenting with acs. clopidogrel was recommended for patients with a high or very high crusade score (greater bleeding risk). ticagrelor was recommended for patients presenting with st-elevation myocardial infarction (stemi) or those with nsteacs with a grace score of intermediate or above (greater ischemic risk) and a crusade score of moderate or less (low bleeding risk). in either case, treatment was at the discretion of the clinician and patients received concomitant aspirin. a registry was collated of risk scores, diagnosis and choice of antiplatelet therapy. results: 1030 patients were included in the registry, of which 587 (57%) presented with stemi and 443 (43%) presented with nsteacs. 558 of 752 (74%) patients with a greater ischemic risk received ticagrelor as part of their dapt regime. advanced age, concomitant anticoagulation and those awaiting surgery were the most common reasons for patients with a greater ischemic risk to receive clopidogrel. 145 (14%) had a high or very high crusade score. of these, 130 (90%) received clopidogrel as part of their dapt regime. conclusion: risk stratification was streamlined using the data collection tool and useful to support choice of dapt. european society of cardiology (esc) guidance recommends use of established risk scores for prognosis and bleeding; however evidence to correlate to choice of dapt is lacking. outcome data is currently being reviewed and will provide further evidence to correlate choice of dapt to grace and crusade scores. please specify your abstract type: descriptive abstract (for projects) background and objective: in europe, approximately 25% of the patients with the human immunodeficiency virus (hiv) infection are co-infected with the hepatitis c virus (hcv). treatment recommendations in hiv/hcv co-infected patients are identical to those in patients with hcv mono-infection. however, potential drug-drug interactions (ddis) between antiretroviral agents and new direct-antiviral agents (daas) imply the need of a careful selection of the hcv treatment regimen. the aim of the present study was to evaluate the need of a change in the antiretroviral therapy (art) due to potential ddis in patients with hiv/hcv co-infection who started treatment for hcv with new daas. we also assessed the effectiveness of hcv treatment 12 weeks after hcv treatment completion. design: we retrospectively registered clinical data about hcv and hiv management: hcv genotype, fibrosis metavir score, initial hcv viral load, hcv treatment and previous art regimen. we recorded the changes in art prior to starting hcv treatment and the reason of this switch (ddi, simplification or duplication of the therapy). results: between february 2015 and january 2016, 50 hiv/hcv coinfected patients started hcv treatment with a daas regimen. of them, 39 had advanced liver disease (fibrosis score: f3/f4) and 27 were infected with hcv genotype 1. prior to starting hcv treatment, 29 patients needed a switch in art regimen due to potential ddis with daas. simeprevir and the co-formulation ombitasvir/paritaprevir/ritonavir were the daas most frequently implicated in ddi with protease inhibitors or non-nucleoside reverse transcriptase inhibitors: 19/29 and 5/29, respectively. also, we observed some changes of art due to other causes. five switches occurred to adequate the regimen (discontinuation of ritonavir in candidates to take the co-formulation ombitasvir/paritaprevir/ ritonavir or art improvement to decrease pill burden). as for hcv treatment effectiveness, 42/50 (84%) patients achieved sustained viral response 12 weeks after therapy completion. conclusion: a large proportion of patients with hiv/hcv co-infection who initiate treatment with daas for hcv need to switch art due to potential interactions that may impact on effectiveness and safety of both treatments. additionally, some changes in art treatment are made to facilitate therapeutic adherence. these results highlight the need of a multidisciplinary approach in which interactions between art and hcv treatments should be carefully assessed. please specify your abstract type: descriptive abstract (for projects) background and objective: the potential impact of polymedication, iatrogenic events and medication error is a serious concern in hospitalized patients. clinical pharmacists can limit these risks by identify high risk. the aim of this study are to identify in six medical units high risk patients by using three predictive scores of rehospitalisation (8 ps) 1 , early mortality (charlson) 2 and drug related problems (drp) 3 . design: 49 clinical and therapeutic variables in 216 patients were collected through medical records and prescriptions by clinical pharmacists. scores were calculated during 2 months in six units (internal medicine, n = 30; nephrology, n = 35; geriatrics, n = 35; rheumatology, n = 45; cardiology, n = 54 and endocrinology, n = 29). the data were analysed by mann and whitney test for the continuous variables and chi square test for the qualitative variables. the coefficient of correlation between the three scores were calculated by a pearson test for normal distribution and by a spearman test for non normal distribution. patients were considered at a high risk for re-hospitalization (8ps [ 2) , early mortality (charlson [ 5) and iatrogenic events (drp c 8) . results: in the general population, the average age was 67.4 ± 18.1 years old and the sex ratio was 0.94. the average treatment used was 7.8 ± 4. charlson scores were higher in geriatric unit (6.3 ± 1.8) follow by medical interne unit (5.7 ± 2.7). the 8ps and drp scores were higher in nephrology unit respectively 2.5 ± 0.9 and 9.1 ± 2.6 follow by internal medecine unit 2.2 ± 1.3 and 7.6 ± 2.7. on contrary the rheumatology unit presented the lower level for the three scores. 51 patients were considered at high risk for three scores, 33% (n = 17) in nephrology unit (almost 49% of unit), 20% (n = 10) in geriatric unit, 18% (n = 9) in internal medicine unit, 18% (n = 9) in cardiology unit, 6% (n = 3) in endocrinology unit and 6% (n = 3) in rheumatology unit. conclusion: knowledge of the variables associated with these predictor scores could help clinical pharmacists to prioritise various medicine units and target those at risk. we identified especially three units at risk: nephrology, geriatric and internal medicine. thanks to these results, clinical pharmacists can rapidly and efficiently target patients who present iatrogenic and/or re-hospitalization risks. design: a retrospective observational analysis was conducted in our hospital, based on medical records of patients presenting atrial fibrillation (af) and treated by doacs from january 2011 to may 2016. to identify patients hospitalized due to severe bleeding, we analysed prothrombin complex concentrates (pccs) and activated pccs prescriptions, as well as pharmacovigilance declarations. results: 1328 patients were treated with doacs: 763 with rivaroxaban (57.5%), 286 with dabigatran (21.5%) and 279 with apixaban (21%). fifty-nine (4.4%) patients experienced at least one bleeding leading to hospitalization: 35 with rivaroxaban (4.6%), 16 with dagibatran (5.6%) and 8 with apixaban (2.9%). thirty-eight severe bleeding were identified (2.9%): 24 occurred with rivaroxaban (3.1%), 10 with dabigatran (3.5%) and 4 with apixaban (1.4%). they included 10 intracranial bleeding (26%) and 21 gastro-intestinal bleeding (55%). seven haemorrhages resulted in hypovolemic shock (dabigatran:4, rivaroxaban:2, apixaban:1) and 2 of them were fatal (dabigatran:2). rates of bleeding (p = 0.86, v 2 test) and of severe bleeding (p = 0.85, v 2 test) were not statistically different for the three molecules. in case of major haemorrhage, the recommended factor concentrate in our protocols differs between the anticoagulant. with dabigatran, the antidote idarucizumab (5 g, intravenously) should be administered, without waiting for plasma concentration results. with rivaroxaban, apixaban or unknown doacs, pcc (30-50 units/kg) is indicated. in case of pcc failure, activated pcc (30-50 units/kg) is suggested. pcc, activated pcc or idarucizumab (2) were used in 15/38 patients (40%). in rivaroxaban and apixaban-related haemorrhages, 10 patients received activated pcc: two had a 20 ui/kg dose and one had a 60 ui/kg dose. regarding dabigatran-related bleeding, one patient received pcc instead of idarucizumab. compliance with local recommendations was 92% (35, p [ 0.003, v 2 test) 0.9 pharmacovigilance reports were issued. conclusion: management of doacs-associated severe bleeding in our hospital respects local protocols. it should also be pointed out that patients with life threatening bleeding may benefit from pcc. however, the risk of thrombosis associated with pcc must be weighed against the risk of haemorrhage. since specific antidotes are emerging, like idarucizumab or andexanet alpha, new guidelines for doacs-related haemorrhage are expected. please specify your abstract type: descriptive abstract (for projects) background and objective: this project is part of a prospective quasi experimental proof-of-concept investigation of a clinical pharmacist intervention to reduce drug-related problems among people admitted to a ward in a rural hospital in northern sweden. the aim of this particular study is to explore doctors' and nurses' expectations of having a ward-based pharmacist providing clinical pharmacy services in a rural hospital. design: eighteen face to face semi-structured interviews were conducted with a purposive sample of doctors and nurses working on the ward were the clinical pharmacy service was going to be implemented. semi-structured interviews were digitally recorded, transcribed and analysed using thematic analysis. results: the majority of participants had limited experience or a vague idea of what pharmacists are able to do in a ward. most participants described traditional roles such as inventory, drug distribution and dispensing. most respondents were unaware of the pharmacists' knowledge, skills and competences. for some it was unclear how having a clinical pharmacist in the ward was going to impact on their workload this was particularly important for the nurses. some doctors (mainly experienced) were concerned that having a pharmacist may mean losing or not gaining competence on drugs. for others it was unclear how the pharmacists' will work with patients or what clinical skills they have. however most participants were positive about the implementation of the new service. conclusion: this study provided a rare opportunity to explore the doctors' and nurses expectations of the role of clinical pharmacists before a clinical pharmacy service was implemented. the results showed that the participants' expectations of the clinical pharmacist role were unclear. to successfully implement clinical pharmacy services in a clinical pharmacy ''naïve'' setting; roles, clinical competence and responsibilities should be clearly described. furthermore, it is important to focus on inter professional collaborations between doctors, nurses and pharmacists. practical for the local hospital setting. seven out of 9 experts agreed with pharmacist prescribing for the conditions identified. pharmacists (n = 31) were more willing to prescribe antihypertensive and antidiabetic medication (87.1%) when compared to oral anticoagulants (64.5%). these values are higher than those obtained by vella in 2014. the majority of pharmacists (87.1%) recommended that pharmacists should take up further studies to a master or doctorate level degree in a clinical aspect in order to be authorised to prescribe. conclusion: the developed framework for pharmacist prescribing and the guidelines developed for pharmacist prescribing of oral anticoagulants and pharmacotherapy of hypertension and diabetes mellitus were shown to be reliable and were accepted by pharmacists and physicians. please specify your abstract type: research abstract background and objective: valproic acid (vpa) and its derivates and mycophenolate mofetil (mmf) and mycophenolic acid used during pregnancy increase risk of congenital malformation and cognitive impairment. thus, the french national agency for medicines and health products safety (ansm) decided to establish new conditions of prescription and dispensation of drugs containing vpa (may 2015) and mmf (april 2016). a signed care agreement and a co-prescription of contraceptives are now mandatory in the drug dispensation for reproductive-age adolescent girls and adult women. this study will describe the impact of these new guidelines on our practice. our objective is to compare the vpa and mmf media coverage and the impact on the prescriptions. setting and method: we compared the mass communication between vpa and mmf on social media, webpages and journal article (public and professional journal) on google and googletrends in the first months around these new rules. we combined different keywords such as ''accord de soins'' and the drug name. in the same time, we collected and analysed vpa and mmf prescribing and dispensing data and compared it to the 2015 data for the first 6 months. main outcome measures: results: just before the vpa rule, the vpa was presented in the general press as the new health scandal after benfluorex mediator°w ith 100 google searches in march 2016 compared to 10 searches usually per month. simple research combining keywords reveal always more than twice more webpages concerning vpa than mmf. at the same time, a patients association (renaloo for renal failure) wrote to the ansm to contest the new rule with the double contraception and without any consultation of patients association. in our daily practice we also faced some physician reluctant to sign this prescription agreement with patient (too many agreements already asked, decision of ansm without any consultation of learned societies). the care agreements are kept in the patient records, a statement ''care agreement signed'' is reported in the electronic prescription of vpa and mmf. the overall consumption of mmf and vpa increase for respectively the first 2 and 3 months after rule implementation (from +122 to +326%) except for the micropakin 500 mg. the 6 months mmf data will be presented for the final communication. conclusion: the media pressure and the new regulation have an impact on prescription trends. these new prescription and dispensing rules concerned two different contexts: pathology, media coverage, possible drug alternatives. we were faced to some difficulty in implementing the new guidelines, which reveals a certain reluctance of the prescribers or the patients represented by associations. tdmp001: vancomycin trough serum concentrations are frequently subtherapeutic in a population of critically ill patients: a prospective observational study please specify your abstract type: descriptive abstract (for projects) background and objective: to design and characterise a framework of international pharmacy standards for pharmaceutical care application on oral anticoagulation for prevention of atrial fibrillation (af) related strokes. design: literature review (including existing international guidelines and quality measures) was conducted to characterise the standards and design an international framework for pharmaceutical practice application on oral anticoagulation for prevention of af-related strokes. expert opinions were sought through a delphi method to reach consensus on the framework domains and standards. results: the framework consisted of twelve overarching standards, which were defined and grouped into four domains as follows; ([personal care package]:-communication with patients, support decision making process, education and counselling, adherence. [medicines optimisation]:-clinical review and therapy optimisation, initiation and control, maintenance, supply and transfer between care settings. [workforce]:-workforce planning, training and development, analysing information; and [governance]:-assurance of service provision) specific to oral anticoagulation in prevention of af-related stroke. each standard was also categorised within dimensions and supporting statements to describe what a quality pharmacy service should deliver. a total of forty-five dimensions and twelve statements were incorporated into the framework. conclusion: a clearly defined framework of international standards was developed as a clinical tool and quality assurance to optimise the delivery of care for oral anticoagulation in prevention of af-related strokes. it will support pharmacists and their teams to develop their professional practice, improve services, and deliver safe and high quality patient care across all pharmacy settings. poster discussion forum i: community pharmacy and public health cp-pc004: nurses' and pharmacists' learning experiences from participating in inter professional medication reviews in primary health care: a qualitative study hege t. bell *,1 , anne gerd granås 2 , ragnhild omli 3 , ingela enmarker 4 , aslak steinsbekk 5 1 nord university/ntnu, trondheim, 2 hioa, oslo, 3 nord university, namsos, norway, 4 department of nursing, østersund, sweden, 5 ntnu, trondheim, norway please specify your abstract type: research abstract background and objective: traditionally, drug prescription and follow up have been the sole responsibility of physicians. however, interprofessional medication reviews (imrs) have been developed to prevent drug discrepancies and patient harm. what participating nurses and pharmacists learn from each other during imr is poorly studied. the aim of this study was to investigate nurses' and pharmacists' perceived learning experience after participating in imrs in primary health care for up to 2 years. setting and method: a qualitative study with semi-structured focus group interviews and telephone interviews with nurses and pharmacists with experience from imrs in nursing homes and home based services. the data was analysed thematically by using systematic text condensation. main outcome measures: a qualitative method is useful when looking at objects from the perspective of how they are experienced. results: sixteen nurses and four pharmacists were interviewed. the nurses' perception of the pharmacist changed from being a controller of drug management routines towards being a source of pharmacotherapy knowledge and a discussant partner of appropriate drug therapy in the elderly. the pharmacists became more aware of the nurses' crucial role of providing clinical information about the patient to enable individual advice. increasingly the nurses learned to link the patient's symptoms of effect and side effect to the drugs prescribed. with time both professions jointly spoke of an increased awareness of the benefit of working as a team and the perception of contributing to better and more individual care. conclusion: imrs in primary health care meet some challenges especially concerning how to ensure participation of all three professions and how to get thorough information about the patient. possible solutions might be to use shared communication tools like internet based communication programs and to introduce the patient as a participant at the imrs. please specify your abstract type: research abstract background and objective: international good pharmacy practice guidelines describe how pharmacists should counsel the patients about their medicines, offer additional services where needed, and intervene at drug related problems. daily practice often differs from theory. this study aimed at illustrating the whole process of prescribed medicines dispensing in daily community pharmacy practice. part b of the project focuses on pharmacists' opinions. setting and method: community pharmacies in basel, switzerland, were invited in random order for study participation. one master student in pharmacy performed non-participant observations during 1 day at each included community pharmacy. at dispensing of prescribed medicines, patient data, content of counselling, communication style, and provision of further services (e.g. follow-up offer) were documented on a checklist with predefined themes. interventions were documented systematically. a semi-structured interview on the pharmacists' opinions about the counselling, triggers, facilitators and barriers, and the documentation of interventions was conducted at each community pharmacy. main outcome measures: counselling content at prescription dispensing by numbers and by pharmacists' opinions; barriers, facilitators, and triggers for counselling at prescription dispensing. results: in march and april 2016, 18 of 49 invited community pharmacies participated in the study. out of 561 documented observation periods, 556 encounters were analysed (first prescription: 269/refill prescription: 287). counselling was provided to 367 (66.0%) clients with an average of 2.9 (±3.1) themes per encounter. a total of 148 clients refused counselling. themes most counselled at first and refill prescription dispensing were: drug intake (476/80), dosage (191/50) , and administration (163/38). for the pharmacists (n = 18), most important themes to be discussed at first prescription dispensing were indication (11), administration (9), and anamnesis (8); for refill prescription dispensing they were adherence (9), therapy benefits (9), and adverse effects (7). the majority of pharmacists (13) felt that it was their obligation to ask questions about patients' health during the dispensing of prescription medicines and named trigger (e.g. patient knowledge gap, patient motivation, interactions), but one-third reported difficulties with it. barriers were refusal by patients (13), communication problems (language, 7), lack of medical data (3) , and lack of time (3) . conclusion: a discrepancy in counselling content by observation compared to pharmacists' opinions was revealed. this might indicate that pharmacists are aware but hindered by barriers to practice according to good pharmacy practice guidelines. please specify your abstract type: research abstract background and objective: the 2020 workforce vision in scotland envisages 'making more and better use of technology …to increase access to services and improve efficiency' across the healthcare interface. services offered by community pharmacy remain limited by lack of shared access to patients' clinical information. in scotland, every patient has a unique identifier, their chi (community health index), which facilitates identification of/searching for patient records. the aim of this research was to explore the experiences of community pharmacists granted clinical portal access to patients' records. setting and method: from april 2015, community pharmacists across nhs tayside (n = 21) who had completed technical and information governance training were invited to maintain a portal log of their experiences of using the clinical portal to access patient records. each was asked to record when/why they considered accessing a patient's record and whether their information needs were met. portal logs were subject to independent summative content analysis by two researchers. this study gained ethical approval from robert gordon university. main outcome measures: not applicable. results: clinical portal logs were received from most participating pharmacists (n = 18/21). two were unavailable (moved to hospital setting; maternity leave). a third had not had occasion to access the clinical portal which he speculated was due to not working at weekends but also raised concerns about gaining patient consent. frequency of seven identified themes provided a partial indication of balance of reasons for usage. firstly (#1), to confirm a patient's prescription (n = 48), secondly (#2), for additional information (n = 46). less frequently (#3), portal access was to check repeat medications (n = 23). other reasons for access were (#4) to check hospital discharge (n = 21) followed by (#5) check on multi-compartment appliance aid status (n = 14) or (#6) check the emergency care summary (n = 11). there were also instances (#7) when portal access was not found to be helpful (n = 16) so traditional offline routes were followed. conclusion: preliminary findings indicate mainly positive experiences with no technical issues raised and community pharmacists' information needs largely met. although limited to a small number of pharmacists in only one health board, findings support scottish policy aims, including 'prescription for excellence.' further work is underway around patients' perspectives of community pharmacist access. please specify your abstract type: research abstract background and objective: multicompartment compliance aids (mcas) such as the multidrug punch cards pharmis ò are used to support patients in the daily management of their medication. solid oral medicines are unpacked from their original packaging and repacked in mcas although controversy exists about the stability of repackaged medicines. different countries published contradictory lists of medicines not recommended for repackaging. we aimed to define and apply criteria able to assess visual alteration of medicines repackaged in mcas. setting and method: eight criteria describing physical alteration of tablets/capsules were retrieved from the who international pharmacopoeia 2015: chipping, swelling, capping, rough surface, cracking, crushing under pressure, mottling, and discoloration. absence of one criteria gives 1 point. a maximum score of 8 points can be obtained. twenty-two critical medicines and three half tablets were repackaged in the multidrug punch cards pharmis ò and stored at accelerated conditions (40°c/75% rh) for 4 weeks. original blisters of medicines were stored at room temperature as control. each tablet/capsule was visually inspected after 7, 14, 21 and 28 days. main outcome measures: score according to eight criteria. results: after 4 weeks, 17 tablets/capsules including 2 of the half tablets showed no visual alteration and were identical to controls. a reduced score (3-7 points) was given to seven repackaged medicines and one half tablet within 4 weeks: madopar ò (3), pravastatin sandoz ò (4), carvedilol mepha ò (6), plavix ò (6), pantoprazol nycomed ò (7), adalat ò cr (7). swelling and rough surface were the most frequent. chipping and capping were not observed. conclusion: our eight visual criteria are able to detect physical alteration of repackaged solid oral medicines under stress conditions. in absence of reliable data we suggest to apply this simple quality control for repackaged medicines in pharmacy practice. because chemical stability testing is not feasible in practice, pragmatic solutions are sought. further studies are needed for storage at room temperature. cp-pc008: drug-related problems and symptom burden in nursing home residents kerstin bitter *,1 , ulrich jaehde 1 , christina pehe 2 , gabriela heuer 3 , manfred krü ger 3 1 clinical pharmacy, university of bonn, bonn, 2 aok rheinland/ hamburg, 3 pharmacists' association north rhine, düsseldorf, germany please specify your abstract type: research abstract background and objective: drug-related problems (drp) are common in the elderly due to polymedication. community pharmacies supplying drugs to nursing homes may play an important role in detecting and solving drp in nursing home residents. this project aims to evaluate whether community pharmacists can enhance the medication safety of nursing home residents by solving drp by means of a simple medication review (mr). furthermore, the applicability of a new tool to detect symptoms as potential adverse drug reactions in elderly patients should be tested. setting and method: nursing home residents at the minimum age of 65 years insured by aok rheinland/hamburg and regularly taking at least five drugs per day were invited to participate. pharmacists performed a mr based solely on the patients' medication data, including dosage regimens of the nursing home and self-medication data. the detected and solved drp were counted. additionally, a simple questionnaire (sympel) was distributed to the patients periodically in order to assess their symptom burden. main outcome measures: frequency and type of the patients' drp as well as their symptom burden before and after the mr. results: after testing the feasibility of this intervention in a pilot study, 54 patients were included in the main study so far. in average, the pharmacists identified two drp per patient and reported to the responsible general practitioner (gp) . as in the pilot study, most frequent drp documented by pharmacists were drugdrug-interactions (34%). 29% of the pharmacists' recommendations were accepted by the gp. 46% of the patients took at least one drug considered as potentially inadequate in the elderly. out of six different symptoms, patients reported dizziness and bruises most frequently. conclusion: pharmacists can detect many drp in nursing home residents by means of a simple mr. however, the full potential of this service to solve drp can only be exploited if the cooperation with the gps is improved. please specify your abstract type: research abstract background and objective: medicines for rare diseases (rd) are costly and have limited efficacy evidence but they represent around 20% of all innovative medicines. therefore, countries are facing challenges in providing patient access to them. the purpose of the study was to assess the patient access for slovenia and compare it with 23 other european countries in the last decade. setting and method: the medicines for rd that obtained marketing approval between 2005 and 2014 via centralised procedure were included in the study based on the orphanet list from january 2016. using the quarterly ims health database, sales data were analysed for slovenia and 21 other european union countries, norway and switzerland. patient access was assessed for each country and comparisons between the countries were made using the three main outcome measures. main outcome measures: the number of medicines for rd available; time to first continuous use after marketing approval; total pharmaceutical expenditure for medicines for rd in euros. results: altogether, 125 medicines for rd were approved between 2005 and 2014. complete sales data were available for 120 medicines which were included in the comparison. for germany and the united kingdom the continuous use of 102 (85%) and 94 (78%) was observed, respectively. the following italy, france, denmark and sweden use 60-70% of all the medicines. in slovenia, 66 (55%) medicines for rd were introduced. germany and the united kingdom times to first continuous use were the shortest (median time 1-3 months after marketing approval). median times below 12 months were observed for norway, sweden, austria, the netherlands, france and switzerland. other countries were slower in enabling first continuous use (median time from 12 to 34 months). germany, france, switzerland had the largest pharmaceutical expenditure per inhabitant in 2014 (29.2, 25.0 and 24.0 euros/inhabitant) while slovenia amounted to 18.5 euros/inhabitant. in slovenia more than a half of the medicines for rd approved in europe are used which ranks it in the middle of all the countries in comparison. comparing to the important european pharmaceutical markets like germany, united kingdom, france and italy, slovenia's median time to first continuous use is longer and pharmaceutical expenditure per inhabitant for these medicines is lower. pec002: mapping of the dlqi scores to eq-5d utility values using ordinal logistic regression and monte carlo simulations: is it plausible? please specify your abstract type: research abstract background and objective: converting dermatology life quality index (dlqi) scores to generic measure data would allow utility calculations and enable cost-effective analysis. this would meet the needs of health technology assessment agencies (htas) such as nice, who preferentially use the general health measure eq-5d. the dlqi is a specialty-specific measure unlike the eq-5d, a generic measure from which utility values can be derived. often several measures are implemented in studies with increased cost and patient burden. ordinal logistic regression (olr) was used to develop a model to convert dlqi scores to eq-5d based utility values for use in economic appraisal of medicines. setting and method: data from 4010 patients were randomly divided into estimation and validation sets to fit and test the model. a series of ordinal logistic regressions were fitted in spss v22 for the five eq-5d dimensions based on age, sex and all 10 individual items of the dlqi as predictors. the model produced three estimated probabilities per subject per eq-5d domain. using these estimated probabilities, a series of 10 monte carlo (mc) simulations were run for each subject resulting in predicted domain responses. from these, utility values were calculated and compared to actual patient values. main outcome measures: conversion of dlqi scores to eq-5d domain data results: there are conceptual overlaps between items of the dlqi and eq-5d. the validation data set (which was not included in the creation of the model), demonstrated that the models were highly predictive compared to actual responses, except for minor differences for the pain/discomfort domain. for example, for the eq-5d ' mobility' domain, 1389 patients answered 'no' (predicted 1392 and 440 patients answered 'some or extreme' (predicted 437) . we examined the model's latent variables, which also demonstrated high predictability at individual level. for example for the 'usual activities' domain the mean latent variable scores were -2.49, -1.62 and -0.86 for those responding 'no', 'some' and 'extreme' respectively, showing a clear increase in the scores with response. after excluding subjects with missing variable data there were 1769 patients in the estimation set and 1773 in the validation set. the model was shown to be highly predictive and repeated simulations demonstrated a stable model. the average predicted utility value for the entire validation set ranged from 0.742 to 0.753 across the 10 mc simulations compared to the actual average utility value of 0.754. conclusion: using olr, we have developed a method of mapping the disease-specific dlqi onto the eq-5d: utility values may then be derived for population data sets, and possibly for individuals. the olr technique could be used to convert data from other disease-specific quality-of-life measures to generic utility data for incorporation into cost-effective analyses, greatly enhancing the potential value of such information. tomi laptoš *,1 , tanja kersnik levart 2 1 hospital pharmacy, 2 the division of paediatrics, department of nephrology, university medical centre ljubljana, ljubljana, slovenia please specify your abstract type: descriptive abstract (for projects) background and objective: having to take medication during time in school may present certain discomfort for some children. suboptimal dosing regimens (i.e. dosing more frequent than determined by drug's trough:peak ratio) can be prevented by a clinical pharmacist's overview and therapy optimization. the aim of the study was to review and evaluate dosage regiments in paediatric patients on antihypertensive therapy. dosage regiments are defined as schedule of doses of a therapeutic agent per unit of time and the amount of a medicine to be given at specific time. design: electronic health records (ehr) review, evaluation of actual dosage regiments against regiments recommended in smpcs and clinical database (uptodate ò ), a consecutive case series study. results: ehrs of 254 patients, admitted to or discharged from the department of nephrology of the division of paediatrics, umcl in 2015 with suspected icd-10 diagnoses from i10 to i15 were reviewed. 107 patients were excluded (diagnosis not confirmed or lifestyle-change disease management only). the remaining 147 patients (average age 15.7 years (range 3-20)) received daily on average 1.59 medications (range 1-4) in 2.06 individual doses (range [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . most frequently used drugs were perindopril (n = 57), ramipril (n = 47), amlodipine (n = 34), bisoprolol (n = 32) and doxazosin (n = 13). 10 patients received ex tempore oral suspensions. dosage regimen was not optimized in 17% (n = 25) of the patients, among those 4% (n = 6) receiving one medication only and 13% (n = 19) receiving more than one medication where at least one was not optimized. furthermore, 9% (n = 13) patients on stabledosage therapy (no dosage change of either medication in last 6 months with satisfactory clinical outcomes) were eligible for fixeddose combination medication. with optimized dosage regimens patients would receive daily on average 1.51 medication (range 1-4) (-5%) in 1.76 individual doses (range 1-9) (-15%). the difference was statistically significant (95% ci, p \ 0.05) in both cases. conclusion: our data show that the majority of the paediatric patients on antihypertensive therapy (83%) received their medication in optimal dosage regimens. however, with an estimated every fifth patient not being on optimal dosage regimen, a multidisciplinary approach is crucial to assure that the individual patient achieves the best clinical, humanistic and economic therapy outcomes. ph005: polypharmacy management programmes in the elderly: a case study in greece dimitra gennimata *,1 , christos kampolis 1 , aggelos vontetsianos 1 , jennifer mcintosh 2 , alpana mair 3 , on behalf of simpathy consortium please specify your abstract type: descriptive abstract (for projects) background and objective: polypharmacy management and medication adherence in the elderly are significant public health issues throughout the european union (eu). simpathy (stimulating innovation management of polypharmacy and adherence in the elderly) is a consortium of 10 organizations representing eight european countries, aiming at stimulating innovation around management of appropriate polypharmacy and adherence, ultimately providing tools for eu policy makers to develop and/or improve, implement and evaluate programs addressing these issues. design: a mixed-methods case study was carried out in greece, to identify policies on the management of polypharmacy and adherence issues in the elderly. a desk review of the polypharmacy and adherence policies at the government, regional and institutional level has been completed. key informant interviews were conducted with policymakers and health professionals responsible for developing and implementing strategies. focus groups consisting of policymakers, clinicians and patients validated the research findings. results: although e-prescription implementation is widespread (&98% coverage nationwide) and disease-specific guidelines have been developed, polypharmacy management is only associated with direct economic indicators. no formal policies or programmes are identified. significant contributions are coming from different health professional organizations that have chosen to provide expanded services to their patients, aiming at optimising drug therapy and thus polypharmacy management and medication adherence in the elderly. community pharmacists offer pharmaceutical care to patients, which includes management of prescribed medication, otc remedies, vitamins and supplements and food-drug interactions. hospital pharmacists in some state (public) hospitals review medication for inpatients and out-patients, communicate with prescribers and confirm the ''benefit-no harm'' principle in the prescribed medication (e.g. incompatibilities, side effects, 7-rights of medication). medical doctors, mostly general practitioners and some specialized ones, usually in primary healthcare settings, keep health records of their patients and have an overview of all administered medication. however, key barriers still remain the lack of coordination of institutions and authorities and overlap of their responsibilities, healthcare workforce and infrastructure shortages and several cultural issues. conclusion: all initiatives to medication management and medicines optimisation are provided without directive from national policies or guidelines. therefore, these activities rely on the goodwill of the health professionals to address pharmacotherapy and therefore polypharmacy management but they are not necessarily representative of what is happening nationwide. a national policy to implement the management of polypharmacy nationwide could mobilise the willingness of health professionals and ensure consistency of care. development and implementation of this policy should build on the grassroots efforts currently underway in this area. ph006: clinical profile and treatment discontinuation in a tuberculosis control state programme in brazil: preliminary results from sinan database simone s. bezerra 1 , mara guerreiro *,2,3 , nathany pessoa 4 , maria paula athayde 5 , rodrigo auad 6 , joão josé gomes 7 , josé lamartine soares sobrinho 1 1 post graduation program in therapeutic innovation, federal university of pernambuco, recife, pernambuco, brazil, 2 centro de investigação interdisciplinar (ciiem), instituto superior de ciências da saúde egas moniz, monte de caparica, 3 please specify your abstract type: research abstract background and objective: challenges remain in tuberculosis (tb) control. discontinuing treatment can leave patients infectious and contributes to the emergence of resistance. this study aimed to describe the clinical profile and cure and discontinuation rates of tb patients enrolled in the pernambuco tuberculosis control programme (pect). setting and method: the study was conducted in three sites in recife, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were extracted from the notifiable diseases information system (sinan) for all pect outpatients, from 01/2012 to 12/2014 (n = 440). analysis was performed with the aid of action for excel; there is on-going analysis to further explore differences across sites. ethical approval was granted. main outcome measures: clinical form of the disease, hiv testing, new cases, cure and treatment discontinuation. results: sociodemographic data were available for sites a and b only. most patients were male (70%, n = 291), with age raging from 20 to 49 years old (60.5%, n = 252); most had a low education level (46%, n = 192) and low socioeconomic status (92%, n = 382). the most common clinical presentation was pulmonary tb. most cases were new (78.4%, n = 345); recurrence and enrolment after discontinuation were respectively 5.9 and 10.9% (n = 26 and n = 48). with respect to hiv, 37.27% of patients were seronegative (n = 164); about a third (35%; n = 155) had not performed hiv test. rates for cure were respectively 59.1% (n = 260), 28.95% (n = 128) and 16.6% (n = 73) in the sites a, b and c. correspondent rates for treatment discontinuation were 21.2%(n = 93), 24.9% (n = 110), 4.3% (n = 19), respectively. tb-related mortality ranged from 0 in site c to 5.4% in site b. conclusion: missed hiv tests may represent undetected tuberculosis/ hiv coinfection. site b presented the highest rates of intrapulmonary plus mixed tb forms, discontinuation of treatment and tb-related mortality; additionally, it had the lowest rate of enrolment after treatment discontinuation. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings may help managers allocating resources and assist clinical pharmacists in planning their interventions. findings also suggest the need of more intensive interventions in tb patients, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: pharmacists working in primary health clinics have various roles. pharmaceutical care is one of them. how to provide this service varies across countries and settings. the most optimal way to provide pharmaceutical care is important to define when developing clinical pharmacy services in a new setting such as primary care practices. general practitioners are key stakeholders in this endeavour. the aim of this study was to find the most optimal approach to providing pharmacist-led pharmaceutical care in primary health care clinics in iceland in collaboration with general practitioners. setting and method: action research provided the framework for this research. data was collected from pharmaceutical care interventions with patients, field observations, field notes, and interviews with general practitioners over the period of the study. the study ran from september 2012 to june 2015. three separate semi-structured in-depth interviews were conducted with five general practitioners from one primary health care clinic in iceland at different time points throughout the study. pharmacist-led pharmaceutical care was provided to patients (n = 125) before and between general practitioners' interviews. the study settings was a primary health care clinic in reykjavik area and the patients' homes. main outcome measures: how to provide pharmaceutical care in collaboration with general practitioners in the icelandic health care environment. results: direct contact between pharmacists and general practitioners over short distances are essential to providing optimal pharmaceutical care services. pharmacist's access to medical records is necessary even though face-to-face communication between pharmacist and patients are most effective in providing pharmaceutical care. pharmacist-led clinical service was deemed most needed in dose dispensing polypharmacy patients. patients require more information about drugs prescribed to them coupled with an accurate drug list with greater detail. conclusion: the most efficient collaboration when pharmacist and general practitioner is obtained when they work side by side at the primary health care clinic. when new services are developed it is vital to identify different requirements of the primary health care clinics to optimize the running of a clinical pharmacist service. ph008: accompaniment of patients treated with oral chemotherapy: a survey on patients' experience laure napoly *,1 , pascal paubel 2,3 , sylvie burnel 1 1 oncorif -regional cancer network, 2 health law and health economics deparment, 3 health law institute, inserm, umr s 1145, paris descartes university, sorbonne paris cité, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: antineoplastic agents taken orally are more and more used in cancer care. these medicines confer autonomy to patients. although, they may cause adverse effects that can lead to treatment adherence issue, unjustified hospitalizations, or premature treatment interruption. proper accompaniment of patient can prevent these issues. in order to define how to organize this accompaniment, we conducted a survey on patients' experience. the objective is to describe patients care pathway and identify their needs. design: a descriptive, qualitative, prospective, survey was conducted using self-administered questionnaires, in hospitals of ile-de-france region. inclusion criteria were: having being treated with oral neoplastic agents during minimum 2 months, age [18, solid tumor, no concomitant iv chemotherapy. collected data were: patients' sociodemographic and clinical profile, their insight about different steps of care pathway (information, treatment delivery, follow up), their behaviours and interactions with healthcare professionals, and their overall opinion. results: 44 patients were recruited in six hospitals. their sociodemographic and clinical characteristics were variable. 72% were treated with targeted therapy, 12% with cytotoxic agent, and 16% with endocrine therapy. patients showed high satisfaction for given information at the beginning of the treatment. principal source of information identified was the oncologist (100%). while the delivery of treatment, 40% of patients beneficiated of advices from the pharmacist. accompaniment of patients during treatment seemed unequal, with: a frequency of visits with the oncologist ranging from less than 1-3 months; 36% of patients not knowing any telephone number they can call in case of worries or questions about their condition or treatment; 39% not remembering any particular accompaniment treatment (visits or calls from nurses or doctors for example). for adverse effects management, three principals actors were identified: oncologist (95%), general practitioner (52%) and pharmacist (31%). regarding the use of oral chemotherapy, patient are satisfied with: it's comfort (93%); being more actively involved in their cancer care (90%); and state not having any anxiety taking chemotherapy home (95%) . asked openly about their concerned and needs three major concepts were identified: high concern about adverse effects, positive feedback about maintaining contact with health professionals between cures and difficulties of communication between health professionals. conclusion: there is a high satisfaction regarding oral chemotherapy and health professionals. the oncologist has a primordial place in patient pathway, whereas implication of pharmacist and general practitioner stays variable. adverse effects are a major concern that needs proactive accompaniment. the variability of our results suggests that accompaniment must be flexible and adapted to the needs of each patients. the key to flexibility could be good coordination and communication between healthcare professionals. ph009: general beliefs about medicines among independent elderly adults in sweden: data from an rct lina hellström *,1,2 , victoria throfast 2 1 the pharmaceutical department, kalmar county council, 2 ehealth institute, linnaeus university, kalmar, sweden please specify your abstract type: research abstract background and objective: there is a need to improve prescription and use of medications by the elderly. the objective of a recent rct was to investigate the effects of e-learning about medicines among elderly adults. a positive impact on the primary outcome measure, knowledge about medicines, is reported elsewhere. a secondary outcome measure, general beliefs about medicines, is reported below. setting and method: the study was a randomized controlled trial in elderly people (aged c65 years). participants were recruited from patient associations and pensionerś associations. participants were randomized to either an intervention group that participated in the e-learning, i.e. internet modules with video and audio, or a control group that did not take part in the e-learning. post-intervention data was collected using paper-based questionnaires, completed within two weeks after agreeing to participate in the study. the general beliefs about medicines questionnaire (bmqgeneral), comprising the subscales necessity, harm and overuse, was used to elicit beliefs. higher scores indicate stronger endorsement of scale constructs (range [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . main outcome measures: bmq-general subscale scores. results: a total of 195 elderly people were included in the study, 96 in the intervention and 99 in the control group. the mean age in the total population was 74.5 years and 53% were women. eleven percent did not use any prescribed drugs while 27% used more than five prescribed drugs. the patients' scores were very similar in the two groups for all three bmq subscales. the median ''overuse'' score was 12 in the intervention group versus 13 in the control group, the median ''harm'' score was 10 (iqr 8-12) in both groups and the median ''necessity'' score was 18 in both groups. in the total population the most commonly expressed negative beliefs referred to overuse of drugs. 62.4% of respondents agreed with the statement ''if doctors had more time they would prescribe fewer medicines'', 43.4% stated ''doctors prescribe too many medicines'', and 37.9% stated ''doctors place too much trust in medicines''. a majority of the respondents agreed with the four items on the ''necessity'' scale. for example ''medicines help people to live a better life (96.9% agreed)''. conclusion: the studied e-learning intervention was not shown to have any impact on general beliefs about medicines. beliefs about medicines have been associated with a number of background variables which might explain why increased knowledge about medicines alone cannot change such beliefs. in general, respondents in the study had highly positive beliefs about the necessity of medicines. nevertheless, the results indicate that overuse of medicines is regarded as a problem. ph010: maf-plus: pharmacists' contributions to provision of financial assistance for medications ian wee * , charlene ong, niron naganathar 1 changi general hospital, singapore, singapore please specify your abstract type: descriptive abstract (for projects) background and objective: the medication assistance fund plus (maf-plus) is a government scheme introduced in singapore in 2011 to provide financial assistance to needy patients who meet pre-set criteria based on means testing. unlike previous schemes, maf-plus provides broader discretion to institutions when providing financial assistance. in our institution, pharmacists reviewed patients' case and medication histories, and filed recommendations to a multidisciplinary committee tasked with approving deserving maf-plus applications. the pharmacists' contributions to the committee, and the outcomes of the applications, are presented in this study. design: all maf-plus applications received between 1st october 2011 and 31st december 2015 were reviewed. pharmacists' comments for each application, where provided, were noted, as well as the range of medicines applied for, review approval rates, and cumulative percentage of available funds utilised. the effect of a recent widening of the scheme's scope to include notable high-cost items was also evaluated. results: between 2011 and 2015, 2001 maf-plus applications were reviewed, of which 1737 (86.8%) were approved. of the 264 rejected applications, 145 (54.9%) were channelled to alternative financial assistance schemes on the recommendation of the pharmacists. the medicines most commonly applied for were intended for the treatment of cardiac (24.3%), respiratory (14.7%), and psychiatric (13.5%) conditions. pharmacists' recommendations also led to a gradual expansion of our institution's list of pre-approved medicines-from 5 in 2011-2012 to 47 by end-2015. from 2014 onwards, pharmacists previewed increasing numbers of applications for high-cost medicines, particularly those for treatment of retroviral disease, hepatitis c, and rare diseases. cumulative utilisation of maf-plus funds (inclusive of annual replenishment) rose from 3.3% in 2011-2012 to 38.8% by end-2015, representing an average year-on-year growth of 151.1%. conclusion: using a process of judicious previewing of maf-plus applications, and recommendations to the maf-plus committee, pharmacists contributed to a high percentage of patients receiving financial assistance for medications. despite a steep growth in the number of applications received between 2011 and 2015, this approach helped to prevent over-extension in fund utilisation. pharmacists will likely be increasingly relied upon due to an anticipated rise in the number of applications for high-cost medicines. please specify your abstract type: research abstract background and objective: adolescents often treat themselves and take medications without parental supervision. lack of experience and knowledge of medicines in this age group frequently leads to inappropriate use of medicines and adverse drug reactions. data about the use of medicines among slovak adolescents and their knowledge of medicines have not been studied yet. setting and method: for our study we used the questionnaire method. the questionnaire contained 23 multidimensional items with closed-ended and open-ended questions, which focused on the characteristics of the adolescentś health status, use of medicines, also in relation to parents and adolescentś knowledge and perception of medicineś risk. we distributed 930 validated questionnaires for adolescents aged from 12 to 18 at secondary schools in all regions of slovakia. response rate was 70.6%. 657 questionnaires were finally analysed. the differences in the distribution of categorical variables between groups were evaluated using the chi square test. sas 9.4. was used as statistical software. main outcome measures: to determine adolescentś knowledge of medicines in terms of efficacy, self-medication, safety of therapy and analyse which medicines are the most frequently used by adolescents. to compare adolescents with chronic disease and healthy ones from the perception of pharmacotherapy point of view. results: in the analysed group 40.8% (n = 268) of adolescents are treated for chronic disease. mostly they suffer from allergy (25.0%, n = 67) and skin diseases (6.8%, n = 18). adolescents with chronic disease use regularly prescription medicines (36.9%, n = 99, p \ 0.001) and over the counter medicines (11.6%, n = 31, p \ 0.001). this group of adolescents better accept the pharmacotherapy with parental supervision (33.2%, n = 89, p = 0.0047 vs. healthy adolescents) and they believe in effectiveness of prescription medicines (71.3%, n = 191, p = 0.0226 vs. healthy adolescents). most frequently prescribed medicines were azithromycin, levocetirizine, ofloxacin and over the counter medicines were ibuprofen, paracetamol, ascorbic acid. we found out in all group of adolescents that 81.3% (n = 534) prefer self-medication without check-ups, 73.5% (n = 483) used drugs in the last 6 months without a prescription, 51.8% (n = 340) take over the counter medications independently without the supervision of parents, 14.9% (n = 154) buy medicines themselves in the pharmacy, 44.6% (n = 293) do not take medications as recommended, 56.6% (n = 372) believe that they have enough knowledge of medicines which they take, 51.4% (n = 338) resp. 49.6%, (n = 326) believe that prescription medicines resp. over the counter medicines are safe. conclusion: questionnaire analysis pointed out that slovak adolescents have not enough knowledge of medicines. the study provides new information in the field of risk perception and adolescentś knowledge of medicines in the slovak republic and highlights the areas that need to be studied in the future in terms of adolescentś education. federal university of pernambuco, 6 state technical school prof. agamenon magalhães, recife, pernambuco, brazil please specify your abstract type: research abstract background and objective: the effectiveness of tuberculosis (tb) control programmes depends critically on patients completing appropriate treatment. this study aimed to outline the cure and discontinuation rates of patients enrolled in the pernambuco tuberculosis control program (pect), based on dispensing data. setting and method: the study was carried out in three sites in recife public health system, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were collected between 07-11/2014, through reports from the stock management software for public pharmacies (horus) for pect outpatients. reports corresponded to a total of 948 patients (232, 348 and 368 in sites a, b and c, respectively). horus defines ''cure'' as medicines collection for three, six or nine consecutive months without interruption, depending on the treatment scheme; discontinuation is defined as non-sequential collection of medicines or treatment interruption for two consecutive months or more. patients were assigned an ''undetermined'' status if treatment was ongoing. data were inputted onto an excel spreadsheet and checked for accuracy. quisquared test, fisher's exact test and bootstrap analysis were performed with r statistical computing. ethical approval was granted. main outcome measures: cure and discontinuation rates for pect outpatients. results: demographic data are not available for the sample. rates for cure were respectively 35.9% (83), 23.6% (82) and 31% (114) in the sites a, b and c, while rates for treatment discontinuation were 3.4% (8), 27.8% (97) and 9% (33), respectively. discontinuation rates were significantly different among the sites a, b and c (p \ 0.05). bootstrap analysis showed that overall the proportion of patients with an ''undetermined'' status in each site did not significantly change these differences. conclusion: only site a had an acceptable discontinuation rate, in light of the world health organization recommendations. this deserves attention as default treatment leaves patients infectious for longer, increases the risk of poor outcomes and fosters resistance to antibiotics. pharmacists could use dispensing data to signal tb patients at-risk of discontinuation, and subsequently tailor interventions addressing its causes. site b had the greater number of patients which discontinued treatment. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings suggest the need of more intensive interventions in patients co-infected with tb and hiv, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: many efforts are done to organise good quality and safe pharmaceutical care. in general, the involvement of a hospital pharmacist or hospital pharmacy personnel in the process of medication reconciliation results in a reduction of the number of medication discrepancies. however, in case of emergency admissions this topic is still insufficiently studied. the introduction of good medication reconciliation on the emergency department (er) requires firm logistical and organisational efforts. we investigated the effects of a drug reconciliation intervention by pharmacy personnel during emergency admissions in order to identify discrepancies between medication lists taken by er physicians and by pharmacy personnel. setting and method: this observational, comparative, non-randomised intervention study was performed in 2011. we calculated that a population size of 65 patients was sufficient to perform reliable measurements. inclusion criteria: all patients presented at the er and admitted to a hospital ward \24 after presentation with usage of one or more prescription drugs. exclusion criteria: age \18 years, residency outside the region delftland, inability to undergo an oral interview, absence of a medication list of the public pharmacy (ozislist), decease of the patient during er-stay and patients undergoing surgical procedures. discrepancies between both medication lists taken by an er physician or pharmacy technician were classified in four categories of increasing severity (1 = no discrepancy to 4 = clinical relevant discrepancy) using the index of the national coordinating council for medication error reporting and prevention (www.nccmerp.org). discrepancies were categorised by a panel consisting of a pharmacy technician, a (senior) hospital pharmacist and a 6th year pharmacy student. statistical analysis was carried out with a statistical software package (spss 18) using the mann-whitney u test and chi squares test. main outcome measures: during the intervention measurement we analysed the reconciliated medication by comparing the er's physician's list with the list of the pharmacy technician after a medication verification interview. the number of discrepancies were measured and judged by the panel. discrepancies were given a category 1, 2, 3 or 4 as defined. results: during the intervention measurement 768 patients were admitted to the er. sixty-five (65) patients (8.5%) met the in-and exclusion criteria. the number of medication discrepancies decreased significantly after intervention of the pharmacy technician by 52%, from 161 to 77 discrepancies. the average number of discrepancies per patient after intervention decreased by 68.0%, from average 2.5 to 0.8 discrepancies per patient. conclusion: medication verification by pharmacy personnel in the er reduces the number of medication discrepancies by half. medication lists generated with a standard interview by pharmacy technicians in combination with an ozis-list on admission of patients at the er is more complete and accurate than the current method. hp-pc015: discharged patients: a problem for community pharmacists? information transfer, as well as the role, needs, and objectives of pharmacists when they care for recently discharged patients. setting and method: a focus group was conducted with a sample of six community pharmacists from personal contacts to represent different characteristics. the focus group consisted of different questions and the recording was transcribed, fragmented and categorised. based on these results, a nationwide online-survey was created with the following questions: a) responder's characteristics, b) number and origin of prescriptions, c) role fulfilment of the joint-who/fip-guideline on good pharmacy practice, rated with a 5-point likertscale, d) 30 information items derived from the focus group discussion grouped into four categories and evaluated for their availability and for their usefulness by likert-scales, e) goals for discharge optimisation, f) additional comments. the questionnaire was piloted and translated forward and backward to french and italian by native speakers. it was sent to all managers of pharmacies belonging to the swiss pharmacist's association in summer 2015 (n = 1348). main outcome measures: conclusions from focus group discussion and responses to questions a-f from the nationwide questionnaire. results: the focus group participants (47.3 ± 13.7 years, 50% female, 50% employees) emphasised the importance of an expanded information transfer, especially for medication changes, unclear prescriptions, and information about a patient's medication acquisition. they were concerned about their extensive workload of discharge prescriptions, and mentioned treatment continuity as one of their goals. the questionnaire was answered by 194 pharmacists (response rate 14.4%, 49.7 ± 10.8 years, 50.5% female). there were 56.7% of responders who reported to fulfil their role (to manage a patient's therapy, function b) not satisfyingly. unavailable but essential information were allergies and the specification of off-label use prescription. unavailable although desired information were the reasons for therapy changes, indications, appointments, contact information, or compounding formulations. concerning design and transfer, information should be written in a structured way but no clear preference for a transfer method was found. goals of community pharmacists were: improved treatment continuity, patient safety, and pharmaceutical care. conclusion: swiss community pharmacists rarely receive sufficient information on discharge prescriptions. appropriate pharmaceutical care is therefore impeded. the knowledge and application of the findings enable directed optimisation of discharge. hp-pc016: patients attitude for using antipsychotic medication in the norwegian early intervention in psychosis, tips 2 study rafal yeisen *,1 , stein opjordsmoen 1,2,3 , inge joa 1,4 , jan olav johannessen 1,4 , jone bjørnestad 1 on behalf of centre for clinical research in psychosis, psychiatric division, stavanger university hospital, stavanger, norway background and objective: poor drug adherence in patients with psychosis leads to relapse, re-hospitalization, poor outcome and increased consumption of health services. pharmacoclinical studies have demonstrated that the treatment response decreases with each relapse. it is estimated that 50% of patients suffering from chronic illness are not taking medication as prescribed after 6 months. the purpose of this study is to investigate which experiential factors that potentially might affect adherence with medication in adults with psychotic disorders. setting and method: in a descriptive qualitative sub-study in the ongoing norwegian early intervention in psychosis, tips 2 study, where twenty-first episode patients (7 male, 13 female) participated in semi-structured interviews 2 years after inclusion. they were still using or had used antipsychotics during the last 2 years. data were analysed using interpretative phenomenological analysis. main outcome measures: adherence to antipsychotics. results: the data suggested four main themes, reflecting the patients' subjective experiences and their impact on the desire to adhere to antipsychotics: (1) admission experience as a psychotic's patient; (2) information from healthcare staff; (3) limited involvement in decision-making; (4) attitude to antipsychotics. conclusion: a number of factors had a positive influence on adherence to antipsychotics. pleasant admission/stay experiences, feeling that antipsychotics had therapeutic effects, mild or no side effects, and believing that antipsychotics are necessary and useful, were typical statements. please specify your abstract type: research abstract background and objective: the hospital-to-home transition is a vulnerable stage in a patient's care. patients can experience problems with medication supply, which possibly lead to therapy interruptions. the objectives of this study were to investigate medication supply after discharge, and patients' and physicians' opinions about the current discharge process and possible optimisations. setting and method: a telephone interview was conducted with 100 discharged patients from the surgical and internal medical wards from the cantonal hospital in baden (switzerland). inclusion criteria were: patients c50 years old, discharged home with a discharge prescription. patients were called between the 2nd and 6th day after discharge and a piloted, structured interview was performed, consisting of questions on experiences and optimisations. afterwards, semi-structured interviews were conducted with five physicians from the study hospital. results from patient interviews and the general discharge process were discussed. main outcome measures: proportion of filled prescription, frequency and type of supply problems including therapy interruptions. opinions of physicians and patients on current discharge process and possible optimisations. results: discharged patients were 65.6 ± 17.4 years old, 39% female, 53% from internal medicine, and 97% regularly visit the same pharmacy. of the 100 interviewed patients, 23 have not filled their prescriptions yet and 77 had their prescription filled when they were called. of these, 78% of them visited the pharmacy on the day of discharge, but it took up to the 6th day until all of them received their medication. supply problems were encountered by 14 patients (18%), mainly because of the medication not being in stock in the community pharmacy. only four patients experienced therapy interruptions, which took up to the 3rd day post-discharge. patients discharged from internal medical wards had more supply problems compared to surgical wards (relative risk = 5.56, p = 0.007). patients experiencing supply problems had statistically significant more medicines on a daily basis (8.0 ± 4.32 vs. 4.9 ± 3.04, p = 0.010). physicians were surprised about the late prescription filling and worried about the disease outcomes. however, interruptions were interpreted as unfrequent. when asked if, in future, hospitals should transfer prescription to the community pharmacy prior to discharge, 71% of patients refused and physicians were undecided, mainly because of a questionable benefit. but both groups indicated that giving some bridging supply would be welcome. conclusion: this study showed that patients discharged from a swiss hospital encounter supply problems, but therapy interruptions are seldom. giving some bridging supply was preferred over an early information transfer by patients and physicians. interventions should consider these opinions and focus on internal medicine patients with high number of medication. please specify your abstract type: research abstract background and objective: adherence to secondary prevention evidence-based medical (ebm) therapies for patients with st-segment elevation myocardial infarction (stemi) is essential to reduce long-term rates of major adverse cardiovascular events. current guidelines recommend the long-term use of low-dose aspirin, highintensity statins, angiotensin-converting enzyme inhibitors (acei)/ angiotensin receptor blockers (arb) and beta-blockers (bb), in addition to p2y 12 inhibitors for 1 year. we aimed to assess the adherence to secondary prevention ebm therapies from discharge to one-year follow-up among patients with stemi undergoing primary percutaneous coronary intervention (pci) in contemporary practice. setting and method: observational single-centre study including consecutive patients with stemi undergoing primary pci in a tertiary hospital in switzerland over a one-year period. secondary prevention ebm therapies were assessed at discharge and at one-year follow-up. main outcome measures: prescription of key secondary prevention ebm therapies (aspirin, p2y 12 inhibitors, statins, acei/arb and bb) from discharge to one-year follow-up after stemi. bb was recommended only for patients with heart failure or left ventricular ejection fraction (lvef) \40%. results: a total of 179 patients were included. ebm drug prescription at discharge was 99.4% for aspirin (n = 178), 97.8% for p2y 12 receptor inhibitor (n = 175), 97.2% for statin (n = 174), 93.9% for acei/arb (n = 168) and 87.5% for bb (n = 28, among 32 patients with lvef \ 40%). ticagrelor (84.6%) was the major p2y 12 inhibitor prescribed. overall, 25 ebm drugs were missing at discharge, with 13 of these missing drugs having no justification for no-prescription (contraindications, allergy or intolerance). at one-year follow-up (median 13.4 months, n = 156), aspirin, statins and acei/ arb prescription rates were 92.9% (n = 145), 92.3% (n = 144) and 82.1% (n = 128) respectively. 19 out of 23 patients (82.6%) with lvef \ 40% received a bb. among patients treated with ticagrelor at discharge, 31 (23.5%) were receiving ticagrelor at follow-up, whereas 21 (15.9%) were switched to another p2y 12 inhibitor. among patients who discontinued ticagrelor (n = 80, 60.6%), duration of dual antiplatelet therapy was 12 months for 80% (n = 64) and discontinued prematurely (\1 year) for 15% (n = 12) patients. reasons for ticagrelor early discontinuation or switch were not specified. conclusion: in a real-world cohort of patients with stemi undergoing primary pci, prescription of recommended secondary prevention medications at discharge is excellent. adherence to ebm therapies at 1 year remains high with more than 80% of patients receiving all ebm drugs. early discontinuation of dual antiplatelet therapy was observed in 15% of patients, whereas ticagrelor was switched for another p2y 12 inhibitor in 15.9% of patients. these observations highlight key opportunities to improve longitudinal use of secondary prevention therapies after stemi in routine clinical practice. although side effects are less common than traditional chemotherapies, certain ones such as pain, fatigue, nausea and vomiting can still be bothersome. in oncology outpatient clinics, side effects are monitored by oncology nurses; however due to high patient turnover and limited numbers of nurses, the assessment of side effects might not be performed adequately. therefore, aim of this study was to determine side effects of immunotherapy and targeted therapy and to compare the severity assessment of side effects by clinical pharmacist and nurses. setting and method: the study was conducted in the hacettepe university oncology hospital outpatient clinic. the patients who have been taking ipilimumab, nivolumab, pembrolizumab, bevacizumab, panitimumab or cetuximab during october 2015-march 2016 were included. the assessment of side effects were undertaken by a clinical pharmacist and nurses separately on each visit using the common terminology criteria for adverse events version-2 toxicity assessment scale. an independent clinical pharmacist compared the side effects' assessments by pharmacist and nurses for analysis. ethical approval was obtained from hacettepe university ethics committee. main outcome measures: to compare the severity of side effects of targeted drug therapies which were assessed by a clinical pharmacist and nurses. results: during the study period 204 visits of 43 patients were evaluated. a total of 5508 side effects assessments were recorded. among those assessments 909(16.5%) was assessed in different ranking by nurses and pharmacist. the differences in the number of assessments were mainly seen in criteria related to pain (n = 34; 51), sensory loss (n = 24; 59), fatigue (n = 114; 26), stress (n = 16; 28), insomnia (n = 18; 28) which was performed by nurses and pharmacist respectively. other side effects detected only by clinical pharmacist were oedema, cough, gastrointestinal complaints (heartburn, cramp) and sensitivity of odour which require close monitoring and in-depth counselling by clinical pharmacist. conclusion: this study explores the differences in assessment of side effects by pharmacist and nurses in targeted therapies. routine assessment of side effects between chemotherapy cycles might yield to misinterpretation or inadequate assessment due to workload of outpatient clinic. therefore, inter-professional interactions in outpatient clinics might close the communicational gaps and improve patient care. hp-pc021: implementation of clinical pharmacy in the acute psychiatric wards: improving quality of medical treatment across health care sectors amila zekovic *,1 , signe kristensen 1 , lisbeth lund pedersen 2 1 clinical pharmaceutical services, capital regional pharmacy, 2 head of clinic, mental health services, copenhagen, denmark please specify your abstract type: descriptive abstract (for projects) background and objective: a study from 2011 shows that people with a mental disorder had a two-to threefold mortality compared with the general population in denmark. life style diseases are the major reason for the excess mortality, partly due to undertreatment of physical disease and well known side effects from medicines such as obesity, diabetes, and heart disease. in may 2015, a clinical pharmacy service (cps) was implemented in all acute psychiatric wards (apw) in the capital region as a part of a three-year project funded by the danish health authorities. the objective is to illustrate how the implementation of clinical pharmacy in the apw in copenhagen increases the focus on drug related problems, rational pharmacotherapy and side effects, increasing the quality of medical treatment and patient safety across health care sectors. design: data was collected at the apw in copenhagen which consists of three wards and has a total capacity of 39 beds. inclusion criteria were patients to which two or more of the following apply: • c65 years of age • c6 drugs • high risk drugs (clozapine, sertindole and opioids) • combination of antipsychotics and benzodiazepines • diagnosed with liver/kidney disease the secondary inclusion criteria were all patients receiving c6 drugs as a single criterion. to obtain a valid medication history and secure medication reconciliation, the pharmacist interviewed included patients. the patients were also asked about side effects, compliance, and perceived effects of treatment. a medication review was conducted based on the patient interview, screening for interactions in an interaction database, and consideration of biomedical data in order to evaluate if treatments should be adjusted, initiated or discontinued. the pharmacist's input was discussed with the doctor, as inputs are more likely to be considered if they are communicated orally. finally, all inputs were documented in the patient's journal as a pharmacist note. the model for improvement was used as a tool for implementing the cps and is being used continuously for improving the service. results: between may 26th 2015 and june 24th 2016, 2285 patients were screened at admission to the apw, of which 30.7% met the inclusion criteria (702 patients). in this period the pharmacist conducted 475 notes, indicating that 67.7% of the included patients were seen by the pharmacist. in april 2016, 30 patients were in average admitted with 8.3 drugs and 1.5 inconsistencies between the hospital's medication orders and the medications that the patient had been taking. regarding patients who are discharged to community care shortly after admission, the pharmacist note is sent to their general practitioner for follow up. conclusion: overall, the implementation of a cps in the apw has been successful. medication reconciliation ensures that the patient is provided with correct medicine at admission, transfer or discharge. by performing a thorough medication review based on a consultation with the patient, the service contributes to an increase in quality of medical treatment. please specify your abstract type: descriptive abstract (for projects) background and objective: the importance of the role of a clinical pharmacist resident in the operating room during 6 months, in a private hospital belonging to a group devoted to healthcare for over 70 years. the hospital is recognized as a reference centre of excellence of hospital care in portugal. it has 145 inpatient beds, two surgical blocks with 9 rooms and 12 beds in the intensive care unit. the aim of the clinical pharmacist in the operating room is to ensure compliance with good clinical practice, safety and pharmacotherapeutic effectiveness, as well as optimization of drug costs. design: 1. logistics restructuring of pharmaceutical services and the need of the physical presence of the pharmacist in the operating room. 2. furthermore, the workstation of the pharmacist is moved to the operating room and the in-depth study of all medicines used in the operating room. 3. in compliance with the joint commission, definition, optimization and adjustment of drug stocks to the needs of the service itself. in close collaboration with the nursing staff, consumer kits were created for registration of drugs by type of surgery in order to facilitate registration and ensure billing efficiency. control of the analgesic drug's dispensation circuit in hospitalized surgical patients that stay less than 24 h in the hospital. ensure compliance with the project through which the health regulatory authority evaluates several hospitals in the country, creating a national ranking among hospital specialties. 4. clinical phase: creation of prescription protocols by type of surgical intervention based on national clinical guidelines. validate prescriptions in the intra-surgical block in compliance with antibiotic prophylaxis, antiemetic and thromboembolic, checking deviations in therapy according to good practice. identify pharmacologic hypersensitivities of patients by consulting the clinical process and anaesthesiology records. provide information on drugs, drug efficacy monitoring and adverse drug reactions in risk management platform. check off label use of drugs. results: of a total of 772 interventions, 360 relate to revenue optimization and 412 relate to clinical interventions. there was an increase of approximately 25% in billing. on what regards to clinical interventions, the majority of them showed deviations from good clinical practice. the physical presence of a clinical pharmacist in the surgical block is essential as the prescription and administration of drugs is carried out simultaneously, allowing immediate therapy validation, in order to increase the safety and efficacy. the pharmacist has the ability to interact with the multidisciplinary team, as well as monitoring the patient's clinical process, the pharmacotherapeutic profile and drug allergies, allowing the detection of any adverse drug reaction on-time. all these interventions are possible in the pre, intra and postoperative phases. results: counselling (av.(±sd) duration: 9 ± 4 min) was performed in 773 patients (57.9% female; av.(±sd) age: 51.7 (±21) years; av.(±sd) medicines at discharge: 5.4 (±3.9)). in 30% of patients mrps were intercepted. the five most common mrps (%) were: need for organisational support (30.5, e.g. proper prescriptions' writing), therapy-related discussions (14.6), untreated indications (12.8), errors in documentation (11.1), and medicines without an indication (9.7). 437 patients (56.5%) classified for study inclusion, of whom 157 (35.9%) consented to be followed-up and 113 (72%) provided data. roughly 80% of patients report having received information about medicines at discharge, of which three-fourths remember being informed by the pharmacist. more then every second patient (54.7%) reported having received valuable new information. changes in chronic-use medicines occurred in 40.6, 42.4, and 34.5% of patients at 1-, 3-, and 6-month, respectively. at 6-month, in 27.4% of patients chronic-use medicines were newly prescribed, in 23.9% discontinued. medical specialists initiated these changes in 72.8% of patients. one out of five patients couldn't recall the reasons for changes in medication. nearly 30% of patients showed moderate to little medication adherence at 6-month. it did not significantly change during the follow-up period. conclusion: clinical pharmacists' counselling prevents mrps at the transition from hospital to home. follow-up data show that changes occur in one out of three patients. medication adherence remains stable, but generally needs to be improved. please specify your abstract type: research abstract background and objective: until 2013, prescription analysis was based in our hospital pharmacy. clinical pharmacy has been deployed in care units since 2014. many clinical pharmacy services were developed: medication reconciliation, patient's therapeutic education and counselling, and prescription analysis unit based. the purpose of this study is to assess the impact of the clinical pharmacist as a direct patient-care team member on prescription analysis. setting and method: we collected pharmaceutical interventions (pis) of the first 6 months of 2013 and at the same period of the year 2015 in the neurology unit when the pharmacist was unit based. we studied and compared type of pis (medication, drug related problem-drp), rate of pis acceptance and clinical impact. focus was made on high alert risk medications and potentially inappropriate medications. 4.5% in 2015 versus none in 2013. when prescription analysis was based in the pharmacy unit, 31% of drp detected by the pharmacist had a potential clinical impact versus 59% when the pharmacist performed prescription analysis in the care unit (p \ 0.05). three drp detected in 2015 had serious potential harm. results: ward-based prescription analysis allowed detecting five more times drp with a significant more important clinical impact than pharmacy unit based prescription analysis. the clinical pharmacist as a direct patient-care team member is more efficient in detecting serious potential harm. indeed, the pharmacist has a greater knowledge of the patient's clinical condition. nevertheless the global rate of acceptance of pis was greater when the prescription analysis was based in the pharmacy unit even if the difference is not significant. but prescription analysis is more complex when performed in the care unit, taking account adherence of the patient, and potentially inappropriate medications resulting in much higher risk-taking by the ward-based pharmacist. conclusion: this study showed that unit based prescription analysis is the best way to detect drug related problem. it must be competed by medication reconciliation and medication review to improve medication safety process. hp-pc027: qt-prolongation in an acute psychiatric setting: fact or fiction? eva jacxsens *,1 , hans van den ameele 2 , jü rgen de fruyt 2 , yves vandekerckhove 3 , frank vancoillie 1 , veerle grootaert 1 1 pharmacy, 2 psychiatry, 3 cardiology, az sint-jan brugge-oostende av, bruges, belgium please specify your abstract type: research abstract background and objective: several psychotropic drugs can induce qt-prolongation, which is a well-known risk factor for developing torsade de pointes (tdp) and sudden death. the clinical relevance of this side effect of psychotropic medication remains unclear, especially in patients hospitalized in an acute hospital. to interpret the clinical importance of psychotropic drug induced qt-prolongation, we investigated the prevalence of these electrocardiographic changes. setting and method: a prospective study was conducted on four psychiatric wards in a general hospital: two acute, short-term psychiatric units (asp1 and asp2), one addiction service unit (asu) and one geriatric-psychiatric ward (gpw). all adult patients admitted between october 1st 2015 and march 15th 2016 on a psychiatric ward were eligible for inclusion. at admission, an ecg (ecg0) was performed and creatinine and potassium levels were measured. a second ecg (ecg1) was performed at least 7 days after the start of a psychotropic drug associated with a risk of qt-prolongation. qtcprolongation was defined as 470 ms for males and 480 ms for females. clinically relevant qtc-prolongation was defined as c500 ms. statistical analysis (r software) was done as appropriate. main outcome measures: prevalence of psychotropic drug induced qtc-changes and correlating factors. results: 268 patients (mean age 55 years, 59%female) were enrolled in the analysis. in 85 patients, an ecg1 was performed. qtc 0+1 were prolonged in 2.3%(5/220) of females and 3.7%(5/136) of males. no clinical relevant prolongation (c500 ms) was registered. higher qtc intervals were measured in the geriatric population. 28.5%(36/126) of all measured qtc were situated between [450 c qtc 0+1 b500 ms] in gpw versus 9.4%(22/233) in the other units. significant difference in qtc-changes was associated with sex (p = 0.02246). there was no correlation assessed between qtc-prolongation and age, number of psychotropic drugs or a specific single psychotropic drug (p [ 0.05). conclusion: in this study qtc-prolongation due to psychotropic drugs is less common than previously described. ecg monitoring may be unnecessary in the follow up of patients without risk factors and could reduce hospital and community costs. however, considering the potential harm associated with tdp, qt-prolongation should be avoided. we recommend recording an ecg before the start of a qt-prolonging psychotropic drug in risk patients: patients with a chronic alcohol or drug addiction, a cardiac history, on concomitant therapy with at least two qt-prolonging psychotropic drugs, or geriatric patients ([65 years). hp-pc028: implementation of medication reconciliation aase m. raddum *,1 , anne-lise sagen major 2 1 sykehusapotekene i midt-norge, sjukehusapoteket i å lesund, avd. volda sjukehus, volda, 2 sykehusapotekene i midt-norge, sjukehusapoteket i å lesund, å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: a correct and accurate medication list should accompany patients at transitions in care from one setting to another, including admission to hospital. complete information on drug use is a prerequisite for all hospital treatment, whereas incomplete information represents a potential patient safety risk. medication reconciliation is defined by the world health organization (who) as ''…the formal process in which health care professionals partner with patients to ensure accurate and complete medication information transfer at interfaces of care.'' the objective of this study was to investigate the quality of the medication history obtained for admitted patients. furthermore, measures to improve the quality of medication histories, i.e. implementation of medication reconciliation, were initiated. design: the study included patients admitted to the internal medicine ward. a comprehensive medication history was determined by performing a standardized patient interview and/or by using relevant sources of information. the primary endpoint was discrepancies between the medication history obtained on admission and the one determined prospectively by a clinical pharmacist. the clinical relevance of the discrepancies was not determined, but sorted according to six major categories, such as: medication not in chart, but patient reports using (omission) and medication in chart, but patient reports not using (commission). further on, in order to minimize the risk of discrepancies, it was focused on implementation of medication reconciliation. a campaign was initiated, where a clinical pharmacist held information meetings regarding the medication reconciliation procedure. for the next 9 weeks, the degree of medication reconciliation was recorded. to spur the degree of medication reconciliation, each ward's weekly numbers were published and the ward with the highest degree of medication reconciliation won a prize. results: among the 74 patients included, a total of 120 discrepancies were revealed. in summary, 50 patients had at least one discrepancy in their medication history, resulting in discrepancies in the medication lists of 68% of the included patients. at the start of the study, the level of medication reconciliation varied among the wards (23-54%), while at the end of the study the levels were increased (75-92%). conclusion: all the included wards improved their level of medication reconciliation during the study period. however, these new combinations have potential drug-drug and herb-drug interactions which can affect the safety and effectiveness of the treatment. in our clinical practice, the clinical pharmacist provides patient education about direct acting antiviral drugs (daa) based-regimens, promotes medication adherence and manages potential interactions with hcv treatment. the aim of the present study was to determine the prevalence of use of herbal products in the patients on hcv treatment, and to describe the potential hepatotoxicity of the herbal products and their interactions with hcv treatment. design: we included all adult patients on daa treatment for hcv who were dispensed drugs from 01/09/2014 to 31/12/2015. we retrospectively recorded demographic data (age and gender), clinical data related to hcv infection (hcv genotype, fibrosis stage, daa regimen and treatment outcomes) and type of herbal products consumed. we then assessed the presence of herb-drug interactions and the potential hepatotoxicity of herbal products. results: we obtained data from 359 patients on daa-based treatment for hcv. the prevalence of consumption of herbal products prior to starting the treatment was 20.61% (74/359). the most consumed herbal products were (prevalence [ 10% among herbal products users): milk thistle, green tea, chamomile, valerian, pennyroyal, boldo and artichoke. we detected four herbal products with potential hepatotoxic effects according to the literature: milk thistle, green tea, pennyroyal and aloe vera. the prevalence of consumption of these hepatotoxic plants among herbal products consumers were, respectively: 21.62, 17.57, 13.51 and 5.41%. we detected herb-drug interactions or potential for hepatotoxicity in 47 out of 74 patients who consumed herbal products. the management of these potential interactions consisted of stopping the herbal product before starting the hcv treatment. conclusion: the consumption of herbal products in our hcv patients was frequent. the management of potential interactions was conservative, recommending to stop herbal products. clinical pharmacists have an important role in the counselling, detection and management of potential herb-drug interactions and herbal products-related hepatotoxicity. poster discussion forum iii: hospital pharmacy and pharmaceutical care 2 please specify your abstract type: research abstract background and objective: anaemia is a common comorbidity of chronic kidney disease. intravenous (iv) iron is used when oral iron formulation became insufficient or to reduce the use of erythropoiesis-stimulating agents (esas) in haemodialysis (hd) patients. the lack of generic group for iv iron sucrose (is) preparations leads to a controversial issue about their clinical effectiveness. in this study, we evaluated the effectiveness of original is compared to is similar (iss) in hd patients. setting and method: a retrospective monocentric observational cohort study was conducted from 01/09/2014 to 31/08/2015, in a stable hd population to compare is and iss. the follow-up periods lasted 24 weeks and were separated by a one-month wash-out period. original is and iss were administered respectively during the first (p1) and the second (p2) periods. the comparisons were performed using the paired student's t test or the paired wilcoxon test for continuous data and the fisher's exact test for categorical data. main outcome measures: the main endpoint was the difference in haemoglobin (hb) levels between p1 and p2 per patient. anaemia parameters (serum iron, serum ferritin, transferrin saturation ratio), the number of transfused patients, the doses of iv is and the doses of erythropoiesis stimulating agents (esas) were compared before and after the switch from is to iss, as secondary endpoints. results: a total of 105 patients were included. there was no significant difference in mean hb value between p1 and p2 (6.78 ± 0.63 mmol/l versus 6.79 ± 0.59 mmol/l p = 0.97). anaemia parameters were significantly different between p1 and p2 (mean serum ferritin, serum transferrin and transferrin saturation ratio) with p \ 0.0001, except to the mean serum iron. the mean monthly dose of iv iron per patient and the mean dose of esas were respectively in p1 and in p2: 248.31 ± 159.18 mg versus 259.74 ± 158.92 mg (p = 0.39) and 0.46 ± 0.50ui/kg/week versus 0.59 ± 0.60ui/kg/ week (p = 0.005). transfusions occurred less frequently in p1 than in p2 (p = 0.02). conclusion: this study showed that iss was as effective as original is regarding hb levels. however anaemia parameters appeared to be in favour of is; the mean dose of esas seemed to be higher after switching from is to iss. these outcomes should be further explored using prospective comparative clinical studies. please specify your abstract type: research abstract background and objective: the pharmacy residents are sometimes up to deliver chemotherapy when they are on night or week-end duty at the hospital. a dispensation's error (delivery of metoject ò (methotrexate) for intrathecal (it) injection whereas it doesn't have the indication for this use), led us to test the pharmacy residents' knowledges about the it access in order to underscore the points to be improved. the final aim of this work is to secure the pharmaceutical care of the patient 24 h a day, 7 days a week. setting and method: an online and anonymous survey of 14 questions was sent to the 35 residents of our area. it was composed of three parts: specific general information, questions about the chemotherapy specifically (indication, maximum dose and volumes, molecules used), illustrated questions about real situation for the dispensation on duty. the answers were collected over a two weeks period. main outcome measures: we studied the rate of good answers in global and by respondent. results: twenty-five residents answered the survey, among them 60% never achieved any internship in a centralized unit of reconstitution of chemotherapy (urc). all the levels of internship are represented: 1st year (n = 4), 2nd year (n = 10), 3rd year (n = 7) and 4th year (n = 4). only 32% know where a medicine is injected intrathecally on the spinal column, 64% know on which level of the meninges. three residents think that a nurse can inject intrathecally. they also had to select the molecules which can be injected by this access: 20% answered vincristine, 8% vinblastine, 4% bortezomib; despite these three molecules are mortals if they are injected intrathecally. the majority know the indication of the it chemotherapy: prevention and treatment of cancers' meningeal localizations. sixty percent do not know that several molecules can be injected for the same patient in the same time. the maximum dose of methotrexate is known for half of the respondents, but only 28% for the cytarabine'one. only 3 residents out of 25 know that 10 ml is the maximum volume allowed to be injected for an adult. six residents would have delivered metoject ò 15 mg in pre syringe filled if a doctor had asked for an it during a night. lastly, there are only two people who know that aracytine ò (cytarabine) 100 mg must be reconstituted with sodium chloride for it use and not with the provided solvent containing benzylic alcohol. the score of the residents having already done an internship in an urc is 8.4/14 compared with 6.3/14 for those who never did. the respective scores per year of internship are 6.2 (1st year), 6.4 (2nd year), 7.4 (3rd year) and 7.9 (4th year). conclusion: results and answers have been presented in a meeting and sent to the residents. we initially note many gaps in knowledge. the residents who already worked in an urc and the elders got better results. all the residents could be on duty at the hospital and all must be formed. a second session will be organized in a month to evaluate the formation's impact. it also has been presented to the assistants during an interactive lesson. this formation is essential to guarantee the dispensation of the adequate product and a secured medical care of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: patient adherence to prescribed medications is crucial for reaching metabolic control goal. to better understand the impact of polypharmacy on medication adherence, we undertook a detailed survey of medication use among patients with endocrinologic diseases. the aim of this study was to determine medication adherence in a cohort of patients with endocrinologic diseases and to test the hypothesis that adherence decreases with increased number of medicines prescribed. design: we conducted structured interviews to determine self-reported adherence of patient on a scale of 0 (high) to 4 (low observance) (srap-4) and a measurement using morisky medication adherence scales . demographic and medication information were collected from medical record. for statistical analysis, mann-whiney u-test for continuous variables, with chi square for categorical variables and kendall test for correlation were used. results: our cohort included 149 patients, 52% were women and 76% were diabetic (65% suffering from type 2 diabetes). the mean age was 56 ± 14 years, the average number of medication was 7.3 ± 4.1. 53 (36%) patients were not able to estimate their adherence. patients reported srap-4 scale with an average of 1.1 ± 0.9, this estimation was significantly higher than mmas-4 with an average of 0.9 ± 0.9 (p \ 0.05). the proportion of adherence level were identical between srap-4 and mmas-4 with respectively 51 and 60% of high, 45 and 35% of medium and 4 and 5% of low adherence. a significand correlation between srap-4 and mmas-4 scales (r 2 = 0.58, p \ 0.0001) was found. however no correlation between adherence scale and number of treatment (r 2 = 0.0001 for mmas-4 and 0.01 for srap-4 scale) nor number of daily doses (r 2 = 0.0011 for mmas-4 and 0.016 for srap-4 scale). on the 1080 medications, 11% presented difficulties with observance. cardiovascular (34.5%), diabetes (25.9%) and psychiatric (13.8%) treatment are the three most involved drug classes in nonadherence. conclusion: in this cohort, patients reported high medication adherence. we highlighted a correlation between srap-4 and mmas-4 scales. surprisingly, we didn't find correlation between adherence scales and number of treatment or dose by day. the next step of this work will be the identification of risk factor of nonadherence using logistic regression analysis. hp-pc033: the office of access to healthcare: how to optimize secured access to treatments? claire chatron, adeline flatres, claudine hecquard, guillaume saint-lorant * , alexandra muzard pharmacy, chu caen, caen, france please specify your abstract type: research abstract background and objective: office of access to healthcare (oah) is an organization which offers a medical and social coverage to people who can't access to care and to medication because of the absence of social welfare, living conditions, or financial difficulties. medications are free dispensed thanks to retrocession activity in hospitals pharmacies. the aim of this study is to analyse this activity and to improve communication with patients and access to treatments by an adapted pharmaceutical interview. setting and method: this study includes all dispensations of year 2015. in order to get medications in our hospital, a social worker and the patient come at the hospital's pharmacy. one people of retrocession team (four assistants, two externs, two residents and two pharmacists) dispenses necessary drugs to the patient according to hospital drug formulary and operating protocol. a switch or a special order can be purposed if the drug is not available. then, we give the patient a medication management plan (mmp) to explain him how to take his treatment at home. retrocession team filled a quiz about this activity and ways to improve it. main outcome measures: the main topics included in the quiz and evaluated were: dispensation organization, english talking, feeling during the interview and evaluation of the mmp. results: three hundreds and ten patients were admitted in oah 2015. these patients come mainly from the eastern europe and do not speak french in most of cases. social workers, who can help for communication, are not always present because these patients can come during on-call duty. quiz results showed that weak points occurred during the interview: explanation of the mmp, languages barriers, mention '' if needed '' not understood by the patient. explanation of the order for a particular drug was difficult to operate too. mmp were only drafted in french which was not convenient for foreign people. however, modalities of dispensation were well understood by the retrocession team. following quiz results, mmp was translated into english by the retrocession team. mentions '' if needed '', '' number of maximum tablet a day: … '', ''your medication is in order, thank you for coming to look for this treatment back to the hospital on … '', '' … is the same as …, prescribed by your doctor on your medication list '' have been added and translated. results of the study and new mmp will be presented to the pharmaceutical team and to social workers in staff. an index card for ''communication in english with a patient'' has also been drafted. it contains sentences meadow drafted in english. conclusion: access quality health care service is important to achieve health equity and to increase the quality of everyone's life. these documents improve communication with patients and by the way their understanding about their treatment. the use and the impact of these documents on well understanding will be soon evaluated with social workers and patients. hp-pc034: improve the medication in an associated to general hospital nursing home luisa alonso * , marta vidal iglesias, lucia gómez carrasco, guillermo goda, laura garcia, laura marin, ana hernandez, alvaro moreno please specify your abstract type: descriptive abstract (for projects) background and objective: in order to improve the medication reconciliation and to implement training programs for the medical team in an associated to general hospital nursing (asnh) home we measured the discrepancies between pharmacy registered treatments (prt) and medical prescriptions (mp), and we analysed potentially inappropriate prescriptions according to ''american geriatrics society 2015 beers criteria'' and ''stopp-start 2014 criteria. design: retrospective observational study that included 143 patients admitted in the asnh. the ''consensus document on terminology and classification in medication reconciliation'' was considered for discrepancy classification. data collected: discrepancies between mp and prt. in 84 patients from the original group of 143, we reviewed potentially severe drug interactions, potentially inappropriate mp and drug classes to avoid in older adults and medications to be used with caution in older adults (according to stopp-start2014 and beers 2015) . all data were registered, measured and analysed in excel ò . results: 143 patients and a total of 1487 mp were reviewed. 367 discrepancies (24.7%) were found between the medical order and the prt, those discrepancies included errors of omission in prt (11.2%), absence of discontinuation of medication (8.3%), incorrect dosage (5.2%). potentially moderate to severe interactions: the most frequent drug groups were proton pump inhibitors (ppis) (12.9%), benzodiazepines (bzds) (9.7%), oral hypoglycemiants (9.7%), other groups with frequency over 5%, oral antihistaminic, statines, low molecular weight heparine (lmwh), laxatives, calcium salts and iron salts. 176 stopp criteria were identified that affected to 440 mp and the distribution was as follows: laxative combinations (40.5%), long term ppis (15.5%), cns depressants combinations (11.4%), long half life bzds combinations (10.9%), aspirin incorrect drug strength (9.1%) and other groups with lower frequencies, nsaids and prokinetics. start criteria: 12 being all of them by omission of the drug at the time of admission. beers 2015 criteria: 90 prescriptions in the ''avoid prescription in adults'' group of which corresponded largely to concomitantly cns depressants and long term use of ppis in no risk patients. conclusion: the difficult working conditions, the excessive workload and the high staff turnover, where doctors have a patient ratio over 100/1, make difficult to update treatments according to patient daily needs. a clear communication problem between the hospital pharmacy and the asnh prescribers exists due to lack of infrastructures, and it has been demonstrated with the high percentage of discrepancy, that implies an important logistic problem (not a safety problem) since the nurse team works directly with the original medical orders. the analysis of prescriptions showed the need for updating the medical knowledge. the high volume of stop and beers criteria and lack of doctors time made impossible the individual acting upon each patient, so short summaries of continuous training related to most frequent problems have been designed. please specify your abstract type: descriptive abstract (for projects) background and objective: our french university hospital is one of the most active centre for liver transplant (100 transplants annually). various professionals are involved in the graft patient care and education. much information and education sessions are exempted before and after the transplant. the objective of this work was to realize a short movie for patients (1) to get them ready for transplant (2) to give the key messages to support their transplant (3) to make family understanding the process and to promote the life behaviour changes. design: three members of the pharmaceutical team with nurses-led care coordination and a surgeon wrote the scenario. we requested two directors for 3 days of shooting. we defined the key points for the patient and places to film, and fixed the duration (7-12 min). the scenario was validated by the chief of the liver transplant unit and nurses-led care coordination. after the 3 days of film shooting, we selected sequences. results: the movie was a succession of six parts. (1) the movie has been burned onto cds, put on flash-drives and will be uploaded on the internet. because of the international origin of our patients, the video will be subtitled at least in english. the video will be broadcast to hospitals which do not transplant patients and refer them to our hospital. since the medical team was involved in a collaborative project, the making of the video has permitted to strengthen the cohesion. indeed, this work would not succeed if everybody did not express himself. patients understood the interest to testify about their lived experience with the liver transplant, because they wished to have such information when they were waiting for the graft. conclusion: this movie is very useful for patients and families who are looking for information before and after liver transplant. it is a tool to get them into condition patients. this video presents the advantage of being personalized (local and caregivers that the patient will encounter are filmed). furthermore, it maintains a dynamic involvement of the pharmacy (already well established with clinical pharmacy, patient education and medication reconciliation) in the liver transplant unit. the making of the film has been an opportunity to bind the members of the team together, by valuing the work of everyone. the film could be screened if this abstract is selected for an oral communication. please specify your abstract type: descriptive abstract (for projects) background and objective: numerous procedures on medication management at oslo university hospital aim to minimize the risk of medication-related errors. error reports and observations show great variation in the use of these procedures, primarily due to difficulties in their implementation and maintenance. our aim was to assess the effect of a novel teaching strategy, the impala project, on doctors and nurses compliance with the medication management procedures. design: the project was carried out at 12 general medicine wards at oslo university hospital for a period of 6 weeks at each ward. assessment of medication-related error reports yielded the following areas of focus: (i) correct medication prescription, (ii) specification of doses for medications given on an ''as required''-basis, (iii) double control of medication dosing, (iv) correct and documented generic substitution. weekly presentations by pharmacist(s), lasting for a maximum of 15 min, were given to doctors and nurses as part of daily ward routines. this was repeated over 4 weeks. data on medicationmanagement procedure compliance were recorded before the start of the intervention, during and after each intervention period. the results were presented and made available to both leaders and employees throughout the project period both as an incentive to improvement and as a motivation factor for continued effort. results: there was a marked increase in medication-management procedure compliance among the nurses, especially after the second week of intervention. the most marked increase was shown for double control. increase in medication-management procedure compliance was also present among the doctors, but was less prominent. the data presented gave an extra motivational kick according to the participants. the leaders and the employees stated that the impala strategy was easy to follow and gave results without much organizational effort. conclusion: fifteen minutes presentations given by a pharmacist(s) as part of daily ward routines, combined with presentation of results demonstrated considerable improvement in medication-management procedure compliance. please specify your abstract type: research abstract background and objective: high unexpected serum vancomycin concentrations (svcs) were observed in patients without impaired renal function during the therapeutic drug monitoring (tdm) in our pharmacokinetic service. the aim of this study was to analyse the evolution of the svcs and its relationship with the markers of renal function. setting and method: retrospective study conducted at a university hospital with a follow-up period of 3 months. only adult patients having at least two tdm were selected. trough svcs were measured by cmia (architect i-1000 analyser, abbott ò ) and fitted to a two-compartment model by using bayesian analysis (pks ò , abbott). clinical and demographic data and daily dose, as well as timings of vancomycin administration and of blood sample collection were accurately recorded. spss ò , version 22.0 was used to compare data from both tdm by student t-test (parametric data) and wilcoxon (nonparametric data). main outcome measures: concentration-to-dose ratio (cdr: trough concentration *1000/daily dose); glomerular filtration rates (gfr) estimated by cockroft-gault formula; measured and predicted svcs levels. results: 30 adult patients were included (females: 20%); median age 68 [35-93] years).the first and the second tdm were carried out after 1.5 [0.5-4 .0] and 3 [1.5-6.5 ] days from the beginning of the treatment, respectively. in the first tdm, no difference was found between the measured concentrations (11.93 (5.07) lg/ml) and those predicted (12.22 (5.58) mg/l. however, predictions were less accurate in the second tdm and predicted concentrations were significantly higher svcs (15.96 (4.32) mg/l vs. 12.88 (3.70) mg/ml, p \ 0.05). the median cdr in the second tdm was significantly higher than that calculated in the first one (6.2 [2.2-13 .6] l -1 vs. 9.6 [3.3-20.2] l-1; p \ 0.05), indicating a lower clearance and a drug accumulation. however, no statistically significant differences in the glomerular filtration rates were found (114 [30-230] ml/min vs. 107 ml/min) in the first and second tdm, respectively. conclusion: although the markers of renal function did not change during the treatment, a decrease in vancomycin clearance was observed. the pharmacokinetic model does not accurately predict evolution of the svcs over the treatment. the introduction of covariates such as the length of treatment or the cumulative dose in the pharmacokinetic model could improve its predictive performance. please specify your abstract type: descriptive abstract (for projects) background and objective: genetic polymorphism or major physiological changes have to be considered in patient therapeutic management. clinical pharmacists have a role to evaluate and optimize the appropriateness and effectiveness of patient's medications. we report here the impact of the clinical pharmacist and his collaboration with the clinical pharmacologist in the therapeutic management of a patient suffered from anorexia nervosa, a psychiatric disorder leading to body composition change that may influence drug pharmacokinetics and efficacy. design: case report. results: the patient was a 35-year old woman hospitalized for chronic pulmonary aspergillosis previously treated by voriconazole, posaconazole and itraconazole. her medical history included anorexia nervosa since 1997 with a body mass index of 9.8 kg m -2 , pulmonary tuberculosis in 2011 with relapse in 2013, and chronic pulmonary aspergillosis since 2014. at admission, a treatment by oral voriconazole at 100 mg/12 h was introduced. the trough concentration of voriconazole at steady state was .1 mg/l (therapeutic range 1-4 mg/l) despite taking drug on empty stomach. although the voriconazole dosage increased in 200 mg/12 h, the trough concentration did not increase significantly (0.2 mg/l). we hypothesized anorexia led to a significant mucosal atrophy and accordingly, a significant decrease in intestinal absorption surface which is a major determinant of the level of drug absorbed. thus, a switch from oral to intravenous route was performed (voriconazole 200 mg/12 h). according to subtherapeutic voriconazole concentrations (trough concentration: 0.2 mg/l) despite the use of intravenous route, we decided to perform genotyping to look for mutations of cytochromes p450 3a4*22, 2c19*2 and 2c19*17, particularly implicated in voriconazole metabolism. the presence of an ultrarapid metabolizer genotype (17* allelic variant of the 2c19 isoenzyme) in our patient should lead to increase drug dosage from 50 to 75%. finally, the patient was treated by intravenous voriconazole at 7 mg/kg/12 h (i.e., increase by 75%). the maximum concentration performed 24 h after iv route initiation was at 2.8 mg/l, suggesting a better efficacy. conclusion: this case report highlights the potential complexity of therapeutic management in some patients given anatomical and functional changes or genetic polymorphism, which can affect drug efficacy. clinical pharmacists in collaboration with clinical pharmacologists have to be able to help physicians in this type of situations. please specify your abstract type: research abstract background and objective: posaconazole (pcz) is widely used for invasive fungal infections as prophylactic, pre-emptive or curative therapy in lung transplantation. recently, a new formulation of pcz has been available in enteric-coated tablets. this new formulation improves pcz bioavailability, as compared to the oral suspension, which leads to increase pcz plasma trough concentrations (c min ) in haematological patients. no data related to pcz exposure and its effects on tacrolimus (tac), an immunosuppressant with narrow therapeutic index widely used, exists in lung transplantation. we aimed to assess the consequences of the treatment by pcz entericcoated tablets on pcz and tac exposure in lung transplant patients. setting and method: a single-centre retrospective study was conducted among lung transplant patients receiving tac and either enteric-coated pcz or both galenic forms. main outcome measures: pcz and tac exposure were estimated by the measurement of c min . to overcome the influence of dose (d), c min were adjusted on dose (c min /d) for both pcz and tac. a spearman test (nonparametric distribution) was performed to assess the correlation between pcz c min /d and tac c min /d. results: eighteen lung transplant patients (median age [q1; q3] = 48. 5 [34.7; 57.4] years; 50% female) were included between june 2015 and march 2016. eight patients received only pcz entericcoated tablets. pcz enteric-coated tablets were associated to an increase in pcz c min /d as compared to oral suspension (0.008 ± 0.006 l -1 vs. 0.001 ± 0.001 l -1 , p \ 0.0001). overall, pcz therapy initiation led to an increase in tac c min /d (0.002 ± 0.001 l -1 before initiation vs. 0.008 ± 0.007 l -1 after initiation, p = 0.02). tac c min /d was significantly higher with pcz enteric-coated tablets, as compared to pcz oral suspension (0.004 ± 0.004 l -1 vs. 0.009 ± 0.006 l -1 , p \ 0.0001). a weak correlation was observed between pcz c min /d and tac c min /d, independently to pcz galenic form (r = 0.37, p = 0.0001 with pcz enteric-coated tablets and r = 0.33, p = 0.02 with pcz oral suspension). conclusion: this pilot study in lung transplantation confirms the better bioavailability of pcz enteric-coated tablets as compared to oral suspension. our results show a more important increase in tac exposure with pcz enteric-coated tablets compared to pcz oral suspension, suggesting a concentration-dependent cyp450 3a4 inhibitor effect of pcz. these findings are of interest in clinical practice to monitor transplant patients treated by the new formulation of pcz. further analyses, including the consideration of confounders, will be conducted. please specify your abstract type: descriptive abstract (for projects) background and objective: within 8 months, two patients receiving apixaban developed agranulocytosis. based on temporal and clinical plausibility as well as published literature, the objective was to determine the causal relationship between agranulocytosis and apixaban. design: description of two agranulocytosis cases reported in our hospital. results: first case is an 89 years old male, admitted to the neurology unit (d0) for ischemic stroke. at admission, blood count showed no abnormalities. four days after admission, treatment administered consisted in: dextrose 5% infusion iv, sodic heparin iv, acetaminophen, atorvastatin, metoprolol. neutrophils count was normal (5.6 g/l). heparin was stopped at d9 and replaced with apixaban according to following dose regimen 2.5 mg twice a day. at d15, patient presented with hyperleukocytosis (neutrophils count 15 g/l) and high crp (109 mg/l). thus, a cytobacteriological urine test was performed. at d17, patient presented with hypothermia followed by hyperthermia related to acute sepsis. blood count showed agranulocytosis (neutrophils count 0.45 g/l). broad spectrum antibiotherapy was started (ceftriaxone and gentamycin). despite treatment, death of patient occurred at d17. the suspected cause of death was septic shock added to severe febrile neutropenia. following haemocultures confirmed sepsis (e. coli) possibly originating from urinary tract infection. second patient is a 76 years old male, admitted to the cardiology unit (d0) for bronchopneumopathy associated with tachycardia and atrial fibrillation. a treatment with heparin was immediately started in association with patient usual treatment (bisoprolol, valsartan, rosuvastatin, hydrochlorothiazide and manidipine). in addition, broad spectrum iv antibiotherapy was started with ceftriaxone and spiramycine followed at d7 by an oral treatment with cefixime and spiramycine until d12. heparin was replaced by apixaban at d1 (2.5 mg twice daily). antihypertensive treatment was adapted throughout patient's stay. patient presented neutropenia at d15 (neutrophils count 0.8 g/l), followed by agranulocytosis at d19 (neutrophils count 0.42 g/l) when it was decided to replace treatment with apixaban by fluindione. the following day, neutrophils count was about 0.28 g/l and patient received filgrastim. a myelogram showed a possible peripheral neutropenia. in the absence of other confounding factors (hiv, hbv, hcv, cmv), an iatrogenic agranulocytosis related to apixaban was suspected. conclusion: causal association with heparin is unlikely as neutropenia is not an adverse drug reaction known included in the smpc of this drug having a well-established safety profile. since the two patients were taking their usual treatment for a significant period of time, a causal relationship is deemed unlikely. temporal and clinical plausibility seem to indicate a possible relationship between agranulocytosis and apixaban. as this medicine has been recently approved, this might explain why no case has been reported in the literature and the absence of agranulocytosis as an adverse drug reaction of apixaban. please specify your abstract type: research abstract background and objective: taste is tightly connected to children's acceptability of medicines. two ways to overcome lack of acceptability are to administer solid formulations which are easier to taste mask and change to better tasting medicines. dicloxacillin is an antibiotic known for its unpalatability, and taste studies suggest that this might jeopardize its adherence. the aim of this study was to explore if prescription data can be used to estimate acceptability of antibiotics among children on a population level using dicloxacillin as an example drug. the research questions were: when comparing dicloxacillin with other antibiotics commonly used in children, (1) is there a difference in the age of conversion from liquid to solid formulation and (2) is there a difference in re-prescription rates on day 1 and 2 after the initial prescription? setting and method: we included all initial prescriptions of oral dicloxacillin, phenoxymethylpenicillin, amoxicillin and erythromycin for children 0-12 years registered in the norwegian prescription database (norpd) 2005-2007 due to dicloxacillin mixture being discontinued from the norwegian market in 2007. the age of conversion was defined as the age where half of the children were prescribed liquids and the other half prescribed solid formulations. re-prescription rates were defined as re-prescriptions of a different antibiotic or formulation on day 1 and 2 after the initial prescription, divided by the total number of prescriptions. main outcome measures: age of conversion and re-prescription rates of dicloxacillin compared with other common antibiotics. results: the age of conversion for dicloxacillin was 4.5 years, compared to 7 years for other common antibiotics. the average represcription rate for dicloxacillin was 8.2% for children 0-6 years and 1.8% % for children 7-12 years. the highest re-prescription rate of 13.3% was found in 2-year olds. corresponding numbers were 2.1, 1.8 and 2.3% for common antibiotics. conclusion: the lower age of conversion from liquid to solid formulation and higher re-prescription rate of dicloxacillin mixture compared to common antibiotics indicates that prescription data can be used to identify antibiotics with low acceptability for children 0-12 years. further studies are needed to investigate if this also holds true for other antibiotics. please specify your abstract type: research abstract background and objective: attention deficit/hyperactivity disorder (adhd) or hyperkinetic disorders (hkf) is among the most common mental disorders in children, and may persist through adolescence into adulthood. pharmacotherapy used for treating the disorders also has potential for misuse/abuse. the aim was to describe the prevalence and magnitude of use of stimulant drugs and atomoxetine, and compare consumption in the nordic countries. setting and method: a descriptive pharmacoepidemiological study from the * 26 million inhabitants of the five nordic countries in the period 2004-2014. data were collected from national prescription registers, public drug reports and by correspondence with public health institutions. population data were obtained from official statistical databases or by correspondence with public health institutions. main outcome measures: trend over time, comparison between countries, type of pharmaceutical, gender, age, comparability of data. results: the annual consumption has been increasing from 2004 to 2014, both in volume and prevalence of use. denmark had the largest increase in volume, from 0.6 to 8.2 ddd/1000 inhabitants/day. sweden had the highest increase in prevalence of use over the period, from 1.6 to 8.6 users/1000 inhabitants. iceland had the largest consumption of adhd medications in 2014, 21.9 ddd/1000 inhabitants/day. prevalence data was not available for iceland but sweden was highest in prevalence of use among the other countries in 2014: 8.6 users/1000 inhabitants. males aged 10-14 years had the largest volume and prevalence of use in 2014, but females' consumption had been increasing faster both in terms of numbers of users (* 1.5 9 faster) and in volume (*29 faster) than men's consumption. conclusion: variation in consumption is considerable and cannot be explained by diagnostic and prescription guidelines, as these are similar in the five countries. consumption has been increasing fast in the period in all the countries, and faster for women than for men, although men still consume larger volumes than women, and are more frequent users. please specify your abstract type: research abstract background and objective: in 2009 and 2013, regulatory bodies in usa (fda) and europe (ema), issued warnings on use of metoclopramide due to an increased risk for serious adverse drug reactions (adr), especially neurological adrs. ema recommended that metoclopramide only should be prescribed for up to 5 days while fda concluded that treatment longer than 12 weeks should be avoided. metoclopramide is commonly used to treat nausea and vomiting in pregnancy (nvp) and deficient breast milk production (10 days course). ema did not make any recommendations concerning use during pregnancy and lactation. the objective of this study is to assess the disproportionality of reporting of adr from metoclopramide, with special emphasis on neurological adrs and women in reproductive age. setting and method: data from whos global adr database vigibase ò for the time period november 1967 to may 2016 was used. the measure of disproportionality of reporting calculated was the proportional reporting ratio (prr), and 95% confidence intervals (ci). analyses were performed according to gender and age. time-toonset of adr was calculated. main outcome measures: proportional reporting ratio (prr) results: vigibase contains over 13 million adr reports. metoclopramide is a suspected/interacting drug in 47 407 of the reports, most common (72%) are neurological adrs. the majority (84%) of the metoclopramide adrs occurred within the first 3 days of use. a total of 65% of the reports was received the last 5 years (2011) (2012) (2013) (2014) (2015) . the reporting of neurological adrs was higher for metoclopramide than other medications in vigibase. women in reproductive age (18-44 years) reported higher proportion of neurological adrs (prr = 3.0, 95% ci 2.9-3.0, n = 4533) than women 45 + years (prr = 2.5, 95% ci 2.4-2.5, n = 3057) but a similar proportion as men 18-44 years (prr = 3.1, 95% ci 3.0-3.2, n = 1823). conclusion: there is a 2.5 to three fold higher proportion of all reports regarding neurological adrs for metoclopramide than for other drugs. patients initiating treatment with metoclopramide should be informed about risks of adrs and that most adrs occur within 3 days, and instructed to contact health care personnel and stop treatment if adrs occur. please specify your abstract type: descriptive abstract (for projects) background and objective: self-induced drug intoxications (sidi) are one of the most frequent reasons of hospitalization in emergency service (1%) with around 4-5/1000 inhabitants and represent around 6% of admissions in intensive care unit (icu). it is the most frequently used method of suicide attempts (sa) and the leading cause of hospitalization for young people under 30. the main objective of our study was to analyse, stratify and pharmaceutically map the different sidi identified in our icu. design: this is a prospective study over 20 months, including all icu patients following sidi from june 2014 to january 2016. we have collected psychiatric history and previous sa by sidi, usual treatment, state of consciousness, incriminating drugs, drug classes stratified according to the clinical severity score igsii, evolution, transfer in a specialized centre and average cost of stay. results: ninety-two cases were reported, representing 6% of icu admissions. the average hospital stay was 3 days for an average cost of 3396.5€. this amount is low compared with the average cost of all stays gone through the icu for the period (14,957€). ninety percent of patients had a psychiatric history and 48% a previous sa. the usual treatment was involved in 77% of sidi. half of the patients arrived conscious with an average of severity score igsii of 35/163, 88 being the highest found for a patient who had swallowed simultaneously pregabalin and nitrazepam. clinical severity of these patients is less than that found on average for all patients in the icu in this period (59/163). eighty-seven percent had a favorable evolution. only one death was observed after ingestion of propranolol. fifty-six and a half percent of patients were then hospitalized in a specialized centre. the great family of psychotropic is the most frequent with benzodiazepines 70%, neuroleptics 36%, antidepressants 31.5% and antiepileptic 17.5%. the main drugs involved are oxazepam 16%, alprazolam 15%, cyamemazine 14%, bromazepam 13% and quetiapine 8%. antihypertensives then arrive and represent 14% of sidi. the stratification of severity scores does not appear to show significant differences between drug classes, nor between mono or polydrug ingestions. conclusion: sa by drug ingestion are very common and are often linked to risky behaviours. for these epidemiological and economic findings, it is necessary to continue and develop prevention strategies avoiding the appearance of intoxication (primary), limiting the consequences (secondary), and reducing the risk of recurrence (tertiary). please specify your abstract type: research abstract background and objective: interpretation of quality of life scores to render them meaningful to aid clinical decision-making is an ongoing challenge. interventions often result in statistically significant quality of life (qol) improvement, but may not reach the threshold of clinical importance. the minimal clinically important difference (mcid) is the minimal score change of relevance clinically. the aim of this systematic review was to assess the impact on quality of life of topical, systemic and biologic treatments for psoriasis in randomised controlled trials (rcts). setting and method: prisma guidelines were followed. all available articles describing rcts of therapies for psoriasis that included qol measurements published up to november 2014 were identified. six databases were examined with 388 search terms. abstracts of articles were reviewed independently by two assessors: a third adjudicator resolved any opinion differences. risk of bias was assessed using the jadad scale. main outcome measures: reporting of the use of qol endpoints and impact of interventions in psoriasis. results: of 3597 screened article abstracts, 329 articles were selected for detailed review: 100 trials met the eligibility criteria, describing research on a total of 33,215 patients. reports of psoriasis interventions that fulfilled inclusion criteria have gradually increased over time : 1998-2004 = 12, 2005-2009 = 33, and 2010-2014 = 55 (1) to evaluate the relationship between the use of different therapeutic agents and the severity of osa, and (2) to determine the effects of commonly used medications on continuous positive airway pressure (cpap). setting and method: patient medical records (n = 2688) of 183 patients, that underwent sleep studies between the years 2009 and 2013 were collected over an eight-month period from the sleep laboratory department at mater dei hospital using a random sampling technique. data collected included body mass index, gender, age, epworth sleepiness score (ess), drug history, apnoea hypopnoea index (ahi) and cpap therapy prescription. likelihood ratio chi square test, paired samples t-test and multinomial logistic regression were the statistical tests used for data analysis. main outcome measures: assessment of the drug history in response to osa control using the ess and ahi scores. results: one hundred and seventy (92.9%) patients of the 183 patients (131 males, 52 females) were diagnosed with osa. forty-five (24.6%), 43 (23.5%) and 82 (44.8%) patients suffered from mild, moderate and severe osa respectively. patients had a mean age of 54 years. angiotensin ii receptor antagonists (arbs) (p-value = 0.022), sulphonylureas (p-value = 0.050), insulin therapy (p-value = 0.040) and non-benzodiazepine sedating agents (p-value = 0.037) were found to be associated with the presence of osa. a decline in the use of the arbs (p-value = 0.000), angiotensin converting enzyme inhibitors (p-value = 0.000) and non-benzodiazepine hypnotics (pvalue = 0.001) was observed over the study year period. reduction in the cpap therapy benefit was detected with the use of histamine (h 1 ) antagonists (p-value = 0.015), b-adrenergic blocking agents (pvalue = 0.001) and antiplatelets (p-value = 0.003). conclusion: it is confirmed that hypertension and diabetes mellitus type ii are the main co-morbidities associated with the presence of osa. reduction in the use of certain therapeutic agents is observed secondary to cpap therapy use. patients using specific drugs have been identified as being at risk of a reduced cpap therapy benefit. please specify your abstract type: research abstract background and objective: people are using increasingly more common of social networks such as facebook, twitter and youtube for different purposes. many people are using these networks with the aim of getting information and knowledge sharing. there are many groups that pharmacist is a member in social networks at turkey. the largest of these groups has 14,000 members. pharmacists are shared common problems, information and experiences in these groups. but the accuracy of the information shared on social networks are not always conclusive. the study aim to evaluate the impact of social network information sharing in the knowledge and attitude of pharmacists. setting and method: clinical pharmacy group has been created to share information on facebook. 2400 pharmacist joined this clinical pharmacy group. the group was fed by information which include new drugs, fda alerts, adverse event and case report and also drug related problems during the 6 months. pharmacists were assigned in two major groups, group a active pharmacist who becomes a member of our clinical pharmacy group, share and discuss information through the network and group b who is not a member. a knowledge measurement survey (ams) was given to both of them. main outcome measures: acknowledge measurement survey (ams) was developed and the difference in the score was used to evaluate the difference between the two study groups. results: 142 pharmacists participated in the study, 34.50% of the participants were a member of our facebook group and 65.49% of participants were not. 4.9% of the participants have doctoral degree or student, 15.4% have master degree or student, 32% have bachelor degree from 4 year-pharmacy faculty, 47.1% have bachelor degree from 5 year-pharmacy faculty. the education level distribution between the two groups was not statistically significant. while 63.64% of the ams questions were answered correctly in the member group only 44.09% were answered correctly in the non-member group. conclusion: the study emphasizes the importance of social network in providing the accurate and fastest information for the daily use of the pharmacists, there is a significant difference in knowledge between the pharmacist who join, share and discuss information on the social network and the one who do not join. cp-ce004: impacts of a community pharmacy practice experiences on student professionalism yunn-fang ho 1,2 , hung-wei lin *,1 , fang-ju lin 1,2 , sheng-ping chang 2 , yen-ming huang 1 1 graduate institute of clinical pharmacy, 2 school of pharmacy, college of medicine, national taiwan university, taipei, taiwan, r.o.c please specify your abstract type: research abstract background and objective: professionalism is valued globally and pharmacy schools are expected to nurture competent practitioners to better serve the public with humanity attitudes and behaviours. the study aims: (1) to understand possible differences in professionalism between pharmacy students and potential community pharmacist preceptors, and (2) to evaluate student changes in professionalism upon completing the community pharmacy practice experiences (cppe) at the end of the third (p3) year. setting and method: a modified chisholm's pharmacy professionalism instrument (18-item, 10-point likert scale) was administered to p3 students, pre-cppe and hopefully post-cppe in september, and community pharmacist practitioners who participated in a two-day preceptor training workshop. participants also provided their significance ratings toward ten traits, namely altruism, accountability, excellence, duty, honor and integrity, respect for others, communication, ethics, humanism, and teamwork. main outcome measures: differences or changes in chisholm professionalism scores. results: thirty-two students and fifty pharmacists participated in the survey. honor and integrity (8.9 ± 1.5) and communication (9.2 ± 1.1) were recognized by students (31.3%) and pharmacists (23.5%), respectively, as the most significant trait. humanism was rated the lowest in both groups (students, 8.2 ± 1.9; pharmacists, 8.1 ± 1.9). the 18-item professionalism scores ranged from 6.6 ± 1.6 (''i do not expect anything in return when i help someone.'') to 9.3 ± 0.9 (''i am respectful to individuals who have different backgrounds than mine.'') in the student group; whereas 8.1 ± 1.9 (''i do not expect anything in return when i help someone.'') to 9.5 ± 1.1 (''it is wrong to cheat to achieve higher rewards (i.e., grades, money).'') in the pharmacist group. in general, pharmacists' professionalism scores were higher and, in certain items, statistically significant differences were achieved. conclusion: professionalism might grow with professional competency and practice experiences as demonstrated by potential pharmacist preceptors. upon completion of cppe, students could probably exhibit gains in professionalism. more investigations are still underway. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, a significant consumption of benzodiazepines (bzd) is observed in prisons. they are widely used during incarceration to treat or prevent anxiety and insomnia. furthermore, it is known that, an important traffic exists with these drugs because of the releasing properties of bzd in case of misuse. based on these observations, the pharmacist has set up a plan to improve the use of bzd in prison. the purpose of the study was to evaluate the impact of these measures after 1 year of implementation. design: in january 2015, we shared with physicians in a meeting to explain our plan for a better use of bzd and to set up new rules of prescription in prison: • regularly reducing the dose to limit drug tolerance • promoting the use of long half-life molecules which allow reducing addiction and misuse • advising sedatives anti-histaminics to treat insomnia • providing information to patients about addictives risks of bzd on the tv channel please specify your abstract type: descriptive abstract (for projects) background and objective: some drug combinations (described in thesaurus of national agency of drug) are contraindicated because they appear to increase the risk of torsade de pointes. the aim of this work is to standardize our pharmacists' intervention and to propose guidelines for doctors and pharmacists, depending on the situation and drugs, to limit these combinations and to reduce this risk at our hospital centre (1105 beds). design: a prospective survey was realized over a period of 5 months to identify the drug combinations prescribed in medical prescription software, from the national drug agency thesaurus, that might be inducing torsade de pointes. a multidisciplinary staff was then constituted composed of a cardiologist, a geriatrician, a paediatrician, an anaesthesiologist, a psychiatrist and pharmacists to identify the different situations and to establish guidelines. results: from the survey 18 drug combinations were found to be contraindicated due to increased risk of inducing torsade de pointes on a list 415 interventions realized by pharmacists. the work group identified three drugs with a therapeutic alternative: hydroxyzine, domperidone, escitalopram, the other drugs can't be switched because they are vital or have no alternative. the work group decided to maintain hydroxyzine but only on premedication and child anxiety, to eject domperidone from our therapeutic index and substitute it with metoclopramide or metopimazine, to not initialize escitalopram but to keep it if the patient has no have others risk factors associated or no contraindication. if the patient has a contraindication with a risk factor the doctor could prescribe other ssri. in addition, pharmacists alert doctors about the risk of torsade de pointes on medical prescription software if some contraindications are identified. conclusion: the contraindications identified must not be underestimated. this work allows identification of torsadogenic drugs commonly prescribed and provides guidance for doctors and pharmacists regarding drug combinations. the collective decision will be disseminated to sensitize all the doctors in the establishment. some treatments could not be substituted despite the contraindication; these must be retained but with clinical monitoring. conclusion: a substantial proportion of medication waste in the community pharmacy could have been prevented. unused medicines in the community pharmacy are generally of low economic value, making it unlikely that the costs that pharmacies will make with the redispensing of unused medicines will be covered. therefore, other actions to decrease medication waste in the community pharmacy, such as preventing that too much medicines are dispensed, should be considered. please specify your abstract type: research abstract background and objective: flaws in usage technique for inhalationmedicines is common, as much as half of the users may need some correction measures, to get the active substances down to the lungs and provide the intended effect. inadequate compliance, especially for regular-use preventive medications, is common. good guidance in pharmacies enhances correct use of medicines. the new norwegian pharmaceuticals policy (legemiddelmeldingen) from 2015 opened up for paid cognitive services, leading to the first such service being implemented in march 2016. the service can contribute to a more correct use of the medicines and, as a consequence, lead to better control of the symptoms for patients with asthma or copd. our objective was to map the variation in pharmacies' handling of an inquiry regarding lack of effect of an inhalation-medicine. the study was done prior to the implementation of the standardized service ''inhalation-guidance'' in norwegian pharmacies. setting and method: simulated patient (mystery shopper) visits in 100 pharmacies in oslo, akershus and buskerud in november/december 2015. the mystery shopper expressed just having started to use an inhaler because of her asthma, but not experiencing effect. structured data collection sheets were used to register the handling immediately after the visit. main outcome measures: scoring of the quality and contents of the information based on the products' patient information leaflets. results: the issue of inhalation-technique was mentioned in 74 of the pharmacies, whereof 18 asked the ''patient'' to show their inhalationtechnique, in order to correct and advice and 32 used an inhaler or demo-inhaler as an aid in the guidance. going through the instructions or watching a video-demonstration with the simulated patient also occurred, or referring the patient to read the instructions and/or watch the video-demonstration on his own. half of the pharmacies discussed the difference between use for preventive treatment of asthma and inhaler that is being used for treatment of attacks. sixty-five pharmacies gave no information about the importance of regular use of the preventive treatment. conclusion: there was considerable variation in how the pharmacies guided, which indicates a potential for improvement. the new guidance-service, implemented in norwegian pharmacies in march 2016, will contribute to better guidance. please specify your abstract type: research abstract background and objective: in portugal, tobacco addiction was responsible for over 12,000 deaths in 2013 (11% of the total deaths). the community pharmacist's contribution to control this public health problem is insufficiently documented. the aim of this study is to assess the contribution of the community pharmacist for smoking cessation. setting and method: a retrospective and longitudinal study of a convenience sample of patients integrating quit tobacco consultations, as part of a pharmaceutical care programme implemented by an outsourced pharmacist was performed at several community pharmacies. the smokers, aged 18 or over, were invited to join the programme. patients signed an informed consent and were submitted to a comprehensive approach by face-to-face consultations and telephone contacts. richmond and fagerström tests were used to evaluate motivation and nicotine dependence, respectively. the therapeutic plan (pharmacotherapy and behavioural counselling) was personalised to each smoker. the quit rates were evaluated by patient selfreport and confirmed by carbon monoxide measurements. the continuous variables are expressed as mean ± standard error of the mean. main outcome measures: quit rates at 1, 3, 6 and 12 months. results: between january 2009 and june 2016, 87 smokers joined the programme, 22 dropouts (25.3%). the remaining 65 smokers, 40 (61.5%) were male, with mean age of 48.4 ± 1.91 years. on average, each smoker consumed 21.0 ± 1.53 cigarettes per day. the mean age of initial tobacco use was 15.8 ± 0.51 years with 31.4 ± 1.98 years of consumption. about 70% reported moderate or high motivation and 60% medium or high dependence. a total of 315 consultations were held and, on average, each patient received 7.3 ± 0.82 interventions. all smokers received non-pharmacological interventions (e.g. motivational approach) and 61 (93.8%) also accepted pharmacological interventions, usually nicotine replacement products. the quit day was achieved by 50 patients (57.5%). a month after quit date, 41 patients were abstinent (41.7%). the number reduced to 32 after 3 months (36.8%), to 27 after 6 months (31.0%) and to 19 after 1 year (21.8%). these data upgrade and are consistent with our previously published results (2014). the smoking cessation consultation in the scope of a pharmaceutical care programme in community pharmacy seems to effectively contribute to the reduction of tobacco addiction in portugal. cp-pc013: patient counselling at dispensing of oral anticancer drugs in european countries from the pharmacists' perspective andreja eberl * , on behalf of epic working group pharmacy, institute of oncology ljubljana, ljubljana, slovenia please specify your abstract type: research abstract background and objective: the number of oral anticancer drugs (oads) available on the market grows constantly. consequently the number of patients, which have to manage the complex treatment with oads at home is increasing. the pharmacists present an important member of healthcare team, since they are dispensing oads to the patients, which need a high quality information at that crucial moment. therefore, our aim was to evaluate pharmacists perceived confidence and needs for specific continuing education in connection to oads dispensing in european countries. setting and method: we used an electronic mailing approach and a standardized online survey to ask practicing pharmacists in european countries about their experience with dispensing of oads. main outcome measures: frequency of patient counselling and fields of counselling, assessment of knowledge and skills. results: the frequency of patient counselling varied widely in participating countries between ''never'' and ''more than 80%'' at initial fill of an oad. at following refills the frequency of counselling was generally even lower. counselling mostly encompassed directions of use, the proper use of antiemetics and side effects. however many pharmacists stated, that they do not feel comfortable counselling patients of oads (25%) and even more acknowledged that they were uncomfortable with managing patients' side effects (20%). on the other hand only 45% of pharmacists believed, that they have received adequate knowledge of oads through undergraduate program, continuing education (ce) events and professional practice. many of pharmacists (70%) have not attended any of ce events related to oncology in last 2 years. pharmacists' responses differed little between the countries. conclusion: the proportion of pharmacists who regularly counsel their patients on oads is insufficient in view of importance of the patients' needs to manage their therapy at home. however the pharmacists seems to be aware of their knowledge deficits and educational needs. the field of oads needs better coverage in under-and postgraduate education. the number of ces has to be increased in order to improve the knowledge and skills in the areas of oads counselling. please specify your abstract type: research abstract background and objective: treatment guidelines for diabetes recommend that patients are well-informed about their disease, treatments and treatment goals, e.g. glycosylated haemoglobin (hba1c). the objective was to describe diabetes patients' self-monitoring of blood glucose (smbg) and potential need of guidance. please specify your abstract type: descriptive abstract (for projects) background and objective: in 2015, the international pharmaceutical federation collected data of remuneration models for community and hospital pharmacy and identified large variations between remuneration models and highlighted that the focus is largely on products and not on cognitive services. the aim of the study is to map the remuneration models of different pharmacist-led cognitive services in primary care across europe, with a special interest on medication reviews and to update a prior survey by bulajeva (bulajeva a et al. medication review practices in european countries. res social adm pharm 2014;10:731-40.). the definition of terms is pivotal for such a european survey to avoid results based on pseudoconceptions. hereafter we present the development of the survey and we will present first results from pilot tests. design: pharmacist-led cognitive services were selected based on a previous study by our group and by searching the literature, official government websites, the pcne wiki and arising links. the definitions of the terms of these services were based on searches in the mesh browser, medline and google scholar. additionally, a search in grey literature and in the internet was conducted to find appropriate foundation for the formulation of the definitions. the questionnaire will consist, of a first part about the remuneration of the pharmacist-led cognitive services. the focus is on country-specific differences in remuneration and the different levels of supply across europe. the second part of the survey is about the different types of medication review services with a focus on e.g. the implementation level, addressed issues, eligibility criteria. this survey will have a cross-sectional study design with an online questionnaire specific for invited participants across europe. to achieve the best quality of answers we will send this survey to at least two researchers with references in pharmacy practice, in each european country (purposive sample). the answers from each country will be checked for discrepancies and these potential discrepancies will be solved by a discussion with the responders. results: by the end of the pre-pilot phase, 22 different pharmacist-led cognitive services were identified and the correlating definitions of the terms were developed. conclusion: at the time of submission the pre-pilot phase has been finished and the pilot will start july 2016. please specify your abstract type: research abstract background and objective: medication adherence is one of the key aspects in assuring optimal health outcomes in majority of chronic diseases. the aim of the study was to evaluate copd patients' medication adherence in slovenia and its association with health outcomes. setting and method: patients were recruited by community pharmacists at the time of dispensing medication for copd. medication adherence was evaluated by using morisky medication adherence scale (mmas-8). patients who scored b6 points, 6.25-7.75 points and 8 points were regarded to have poor, moderate and good adherence, respectively. quality of life was evaluated by saint george's respiratory questionnaire (sgrq) and the impact of disease by copd assessment test (cat). the study was conducted in september 2014 and february 2015. the association between potential predictors and copd impact or quality of life was estimated using multiple linear regression in ibm spss statistics version 23. main outcome measures: medication adherence rate (mmas-8), quality of life (sgrq total score) and impact of disease (cat score). results: of 65 patients, majority were men (68%) with mean age 70 years. in average, patients were prescribed 2.5 medicines for copd and 3.8 medicines for other diseases. good, moderate and low adherence to copd medication regimens was found in 53.4, 32.8 and 13.8% of patients, respectively. mean cat scores and sgrq scores were 17.3 (range 3-34) and 41.3 (range 2-79), respectively. thirtyeight percent of patients experienced an exacerbation in the past year. linear regression showed no statistically significant association between medication adherence and quality of life or copd impact on patient. factors that statistically significantly predicted patients' quality of life were exacerbation in the past year, education level and number of concomitant medicines for other diseases. the latter was found to be the only factor associated with copd impact. conclusion: the study showed half of the copd patients to be optimally adherent to their treatment and only a small proportion of patients not taking their medicines regularly. due to the nature of the disease medication adherence does not seem to play the most important role in assuring optimal health outcomes in copd patients. please specify your abstract type: descriptive abstract (for projects) background and objective: intermediate care units (imcu) are designed to serve patients in need of more advanced medical care than the ordinary nursing home units can provide. the aim of this study was to see; (1) how medication information follows patients in and out of icmu and nursing home short-termcare units (stcu) (2) the type and amount of drug related problems (drp), focusing inappropriate drugs, and (3) if there are differences between the icmu and stcu in drug use and drps. design: patients c65 years old admitted and submitted at the imcu or stcu in the study period (10 weeks) were included. transfer of medication information were evaluated and given a score. the clinical pharmacist provided medication reconciliation upon admission, medication review and monitoring, and presented identified drps and a suggestion for solving the problem, to the multidisciplinary team. inappropriate drugs, identified by screening tools (stopp/norgep), and systematic medication reviews, were recorded. results: 14 patients from imcu and five from stcu were included. a hospital discharge summary including medical history followed mostly all patients. the score of the medication history was 7.5 points out of 16. by submission from either imcu or stcu, the score was 8.6. systematic drug review identified 3.9 drp in the imcu and 2.0 in the stcu. imcu patients used 9.5 drugs, stcu patients 10. in the icu, 14% of the identified drps was inappropriate drugs, none in the stcu. the clinical pharmacist in the multidisciplinary team presented 92% of the identified drps. the doctors agreed in 80% of the suggestions for solution, and started immediate changes in 43%. conclusion: a hospital discharge summary followed the patients, but the medical history part needs improvement. although few patients, the results suggest that imcu patients had more complicated medication and more inappropriate drugs than stcu patients did. clinical pharmacist in a multidisciplinary team provides useful contribution to identify, solve and prevent clinical relevant drps, including inappropriate drugs. please specify your abstract type: research abstract background and objective: lack of clinical effects of medication review on health-related quality of life of older people may be due to insufficient focus on health-related complaints. goal attainment scales (gas) are an instrument to formulate specific health-related goals. the objective of this early process-evaluation of the dreamer-study (drug use reconsidered in the elderly using goal attainment scales during medication review) is to investigate if pharmacists are able to formulate gas during a medication review of older people with polypharmacy. setting and method: older patients aged 70 years or older using 7 or more medicines are included in this study. half of the patients were randomized into the intervention group, where they received a medication review. during the patient interview, the pharmacist formulated gas in concordance with the patient. recommendations were made to reach these goals in collaboration with the gp. main outcome measures: number of performed medication reviews, total number of formulated gas and the three most frequent types of gas. results: until now 453 patients have been included in the drea-mer study (60% of the target). half of them (220) were randomized into the intervention group. by now 179 (81%) of these patients have received a patient interview. 143 goal attainment scales were formulated yet. the number of gas ranged from 0 to 3 per patient. the four most frequent gas were: polypharmacy-reducing the number of medicines (28), reducing pain (23), increasing mobility (16), reducing fatigue (15). conclusion: gas seem to be a feasible approach during medication review that increased focus on patient's needs and health-related complaints. cp-pc019: oral transmucosal fentanyl citrate: a regional survey of dispensing practices in community pharmacy please specify your abstract type: descriptive abstract (for projects) background and objective: oral transmucosal fentanyl citrate (otfc) is an opioid analgesic indicated for management of breakthrough cancer pain in patients with malignancies who are already receiving and who are tolerant to opioid therapy for their underlying persistent cancer pain. otfc are usually use off-label prescription, especially in noncancer patients or patients without opioid maintenance treatment. this practice can expose to iatrogenic risks, lack of efficacy, abuse and addiction. the observatory of drugs, medical devices and therapeutic innovation of upper normandy, conducted a study to assess the knowledge of pharmacists on these medications and assess dispensing practices (pharmaceutical analysis and advice to patients). design: between june and september 2015, two quizzes were sent to the 1344 pharmacists and 512 pharmacies in upper normandy: one included questions of knowledge and general practice, the other assess dispensing practices of otfc prescriptions received at the counter, regarding indication, dosage and associated opioid medication. results: of the 93 pharmacists who participate in the survey, 21% know the all of the 7 oftc specialties, 46% of them confuse transdermal and transmucosal fentanyl specialties. indication, dosage, titration methods and the main interest of oftc are known by 52, 43, 18 and 71% of them. only 30% have dispensed oftc more than 10 times over the past 12 months, 28% never have. they already have dispensed oftc in noncancer patients (45%) or without opioid maintenance treatment (36%). they consider not know enough about these drugs to be able to provide the necessary advice to patient (57%) and would like specific training on oftc (89%). of the 21 analyzed prescriptions, only 24% are consistent with the marketing authorization: otfc medicines are prescribing in noncancer patient (52%) and/or dosage is higher than four units per day (24%) and/or there is no prescribed opioid maintenance treatment (24%). only two prescriptions have been discussed with the prescriber, and all were approved and dispensed. conclusion: otfc specialties are occasionally dispensed and often misunderstanding by pharmacists. a good knowledge of otfc is necessary to achieve the pharmaceutical analysis and provided appropriate advice to patients, in order to guarantee the good use of these medicines. support tools for dispensation, recalling indication, . the most frequent interventions were drug substitution (n = 132), dose adjustment (n = 57), and clarification of information (n = 48). common services were reconstitution of suspension (n = 7), provision in advance for continuing supply (n = 26), and follow up offers (n = 3). conclusion: the observation of the dispensing process in community pharmacies revealed a broad range of tasks performed by the pharmacy and identified several variables likely to influence the counselling. in addition, pharmacy activities could be pictured by the documentation of pharmaceutical interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a multidisciplinary process to correct medication errors resulting from miscommunicated information at transitions of care. development of this activity is essential but it is hindered by the time required for its implementation. we must carefully choose which services can develop this activity. as it was recently introduced in cardiac surgery unit, this study aims to evaluate impact of this process to hospital admission (severity of potential harm of medication error intercepted) and to determine the relevance of this activity in this unit. design: prospective study conducted from january 2016 to april 2016. the data is recorded in an excel table, filled after each mr. there are five items: patient's age, best possible medication histories (bpmh), implementation period of the mr, inadvertent discrepancies (ids) and clinical impact. to assess the severity of ids, a scoring method was used (doerper et al. 2015) with the cooperation of surgeon and pharmacist. results: eighty-two patients (mean age 68 ± 8 years old) were included in the study, which represents 52% of the patients hospitalized in this service. the mean number of drugs per patient was 6 ± 3. the bpmh were obtained within 24 h to 72 h of admission to hospital. a total of 34 ids were detected, with a mean of 0.41 ids per patient. the most frequent type of ids was omission (52%, n = 16), error of dose (39%, n = 12). the three most common classes involved in ids were hypolipaemic drug (n = 9), antidiabetic drugs (n = 6) and the drugs for acid related disorders (n = 4). the mean of ids per patient (0.41) as well as the percentage of patients affected by a ids (32%) are less important in cardiac surgery than those observed in other services of the institution and in the literature. about clinical impact, 31% of patients presented with ids considered as minor, 44% significant and 25% major. among the major ids, none was evaluated as critical or catastrophic. in our study, this process remains retroactive. conclusion: one of challenge experienced when implementing mr process in hospitals is demonstrating its clinical impact. in order to address this concern, we found that the little ids with a serious clinical impact in this unit. mr is an interesting process to detect drug errors. to optimize our study we will improve our organization in order to be closer to the patient and to strengthen the doctor-pharmacist collaboration. please specify your abstract type: research abstract background and objective: special packaging like multidose drug dispensing (mdd) may optimize medication use in patients with a decreased ability to manage their own medication. however, it remains unclear how a 'decreased ability to manage medication' is defined. the objective of this study is to assess potential medication problems that contribute to a decreased ability to manage medication in patients starting with mdd compared to patients who use manually-dispensed drugs. setting and method: patients starting with mdd (cases) and patients using manually-dispensed drugs (controls) were interviewed in 45 community pharmacies. questions to assess potential medication problems covered three domains; medication adherence (12), practical management issues (12) and medication knowledge (2) . every potential medication problem was scored with one point. cognition was assessed with the mini-cog and frailty with the groningen frailty index (gfi). main outcome measures: mean scores of potential medication problems on the domains medication adherence, practical management issues and medication knowledge. results: 188 patients starting with mdd and 230 patients using manually-dispensed drugs were interviewed. patients starting with mdd scored more potential medication problems on all domains: adherence 5.5 versus 1.7, practical management issues 3.7 versus 2.1, medication knowledge 1.1 versus 0.3. on the three domains together, patients starting with mdd scored 10.2 [9.6-10.9] potential medication problems compared to 4.1 [3.7-4.6 ] for patients with manuallydispensed drugs. forty-two percent of the patients starting with mdd might be cognitive impaired and 63% was classified as frail compared to 20 and 27% respectively of the patients using manually-dispensed drugs. conclusion: patients starting with mdd reported significantly more potential problems on three domains that may contribute to a decreased ability to manage their medication. cp-pc024: fifteen key questions to assess patient knowledge on new oral anticoagulants corina metaxas * , valerie wentzky, sonja luginbü hl, kurt e. hersberger, isabelle arnet please specify your abstract type: research abstract background and objective: knowledge on new oral anticoagulants (noacs) is crucial for their safe and effective use. validated tools that assess patient knowledge exist for vitamin k antagonists, but not for noacs. we aimed to identify which questions are relevant for patient knowledge on noacs. setting and method: based on a systematic literature search, 45 questions were compiled for the assessment of noacs knowledge. key questions were selected through three rounds of ranking by an expert panel (four physicians, four pharmacists, four nurses). round 1 (online survey; importance): the 45 questions grouped into the nine educational topics of wofford,adapted for noac (disease, mode of action, risk-benefit, adherence, accessing healthcare professionals, diet/life-style, lab-monitoring, medication interactions, self-care) were to be rated as important/not important and educational topics were to be ranked according to decreasing importance. round 2 (online survey; relevance): the questions were to be ranked according to decreasing relevance. round 3 (focus group): number of questions was reduced by voting. main outcome measures: ranking of educational topics and questions (1 = most important/relevant) in march/april 2016. results: experts ranked adherence (2.2 ± 0.9) as the most important topic, followed by risk-benefit (3.0 ± 1.6), disease (3.7 ± 2.7), accessing healthcare professionals (4.1 ± 2.0), self-care (5.3 ± 2.6), lab-monitoring (5.6 ± 1.6), medication interactions (6.5 ± 1.8), diet/life-style (7.3 ± 0.9) and mode of action (7.8 ± 1.5). one question was judged as unimportant by all experts. out of the remaining 44 questions, 10 (22.7%) were selected as relevant for basic knowledge, 7 (15.9%) were combined into four questions and one new question was generated. a total of 15 key questions remained after the focus group discussion. conclusion: a multiprofessional expert panel was able to select key questions retrieved from literature and ensured content validity. the selected questions will be compiled into a tool to assess patient knowledge on noacs. background and objective: medicines use review (mur) was defined by the slovene chamber of pharmacies in december 2014 and an education program was set to assure pharmacists competencies. in june 2015 the first pharmacists were certified and implemented the service in the community pharmacies. additionally, an online database was established to collect mur reports and provide feedback on pharmacists' performance. the aim of the study was to evaluate identified drug related problems (drp) as well as pharmacists' interventions from mur documentation. setting and method: a preliminary retrospective analysis of documentation for mur services provided in the first year after implementation was performed. drps were classified using a slovenian drp classification system, which is based on the pcne classification v 6.2 [1] . data were analysed with descriptive statistics measures. main outcome measures: number and type of identified drp and pharmacists' intervention. results: a preliminary analysis was performed on 129 mur cases, performed by 11 certified pharmacists. in total 258 drps were identified: 116 (44.96%) manifested and 142 (55.04%) potential. patient had on average two drps, however 11 patients had none. main risk factor for potential drps was inappropriate use of medicines. adverse drug events (ades) presented 50.86% of manifested drps; the main risk factor was again inappropriate use. in two cases ades happened due to an allergic reaction. 29 different medicines were the cause of ades; mainly statins resulting in muscles pain and sleeplessness. another frequently manifested drp was insufficient effectiveness of treatment. drug interactions were risk factors in 12 cases of manifested drps, mainly in connection with antidepressants: serotonin syndrome due to escitalopram, bleedings in concurrent use of escitalopram and ginkgo, sleepiness, etc. pharmacist intervened independently in 74.4% of cases; 57 times recommendations were given to physicians. however, in 92.6% of cases the outcome of intervention is unknown. the preliminary results of the first mur cases points to a high number of identified manifested drps. however, the knowledge of intervention outcomes is lacking and therefore more attention has to be put on establishing adequate follow up on this issue. official definition represented harmonisation of several similar activities that have already been performed in slovenian pharmacies and also provided an educational program to assure pharmacists competencies. in may 2015 the first 18 pharmacists were certified and implemented the service in the community pharmacies. therefore, the aim of the research was to get an insight into the implementation of mur in slovenia from the perspective of the first community pharmacists that provide the service in practice. setting and method: a focus group with seven community pharmacists, that provide mur in practice, was run in february 2016. guided discussion included three main themes: the development and assurance of competencies, experience with the provision of service in practice and the future of the service. the discussion was voice recorded and analysed with the nvivo 11. written consent from included participants was obtained. main outcome measures: views, challenges and opportunities for the medicines use review service in slovenia. results: in total 364 themes were identified and organized in three main categories: competencies for quality provision of mur, mur's recognisability and organizational aspects of mur provision. participants emphasized broad knowledge in pharmacotherapy is pharmacists' key competence and advantage in performing mur when compared with other healthcare professions. recognisability of mur among other health care professions as well as participants' work environments is low. hence a comprehensive approach in marketing of the service is needed. positive patient's feedbacks were reported, however persuading patients to attend mur presented a challenge. another barrier was the time to perform mur, which could be overcome by suitable work organization and special time intended for mur. conclusion: participants of the focus panel had positive experience with the development of competencies and implementation of the service in the practice. several challenges were presented connected with the recognition of the service by patients, physicians and health care payer. they strongly believe that continuing professional development forms the base for quality of the service in the future. cp-pc027: evaluation of rational antibiotic dispensing in the community pharmacy setting: a simulated patient study betul okuyan * , mehmet ali savan, fikret vehbi izzettin, mesut sancar please specify your abstract type: research abstract background and objective: in the present study, it is aimed to evaluate rationale antibiotic dispensing without prescription in the community pharmacy setting; this will be done by using a simulated patient methods. setting and method: this study was conducted in malatya, located in the east part of turkey. the simulated patient visited the community pharmacies to meet the pharmacist, posing as the husband of a patient with acute uncomplicated rhinosinusitis. the simulated patient was trained regarding the standard information to be provided by the researchers and informed about the privacy of all information that would be gathered during the present study. the sample size was sixty-seven pharmacies, with a confidence interval of 95% and error of margin of 10%. the study was conducted over a total of 70 pharmacies. all the pharmacies were listed alphabetically and were randomly selected and allocated random numbers by a computerbased program. main outcome measures: after each community pharmacy was visited, the simulated patient filled the check list which had been drawn up for the purpose of the present study. due to ethical concerns, no audio or video records were used during the study. any suggested medications were not purchased from the community pharmacy. results: of the total community pharmacies that were visited 55.7% of them had female pharmacists and 44.3% were run by male pharmacists. the mean number of questions asked by pharmacists to the simulated patient was 3.17 ± 1.65. only eleven pharmacists did not suggest any medication for the simulated patient. however, thirty-two (45.7%) pharmacists recommended various medication regimens, including antibiotics. of them, 67.1% referred the simulated patient to a physician. conclusion: in conclusion, it was observed that dispensing antibiotics without prescription was still high, pharmacists did not take comprehensive medical or medication history from patients, and pharmacists provided insufficient medication information to the patient regarding suggested medications at community pharmacy setting. to avoid irrational antibiotic dispensing, it is essential to educate both health care providers and the general population. although dispensing antibiotics without prescription is illegal in some countries, it is necessary to actualize new regulations to avoid antibiotic dispensing without prescription. please specify your abstract type: research abstract background and objective: the medication adherence is an important part of active (as well as passive) attitude of a patient to the disease treatment. it represents the level of keeping the treating procedure as well as the recommendations of doctors, pharmacists and other healthcare professionals. this study deals with the adherence in patients with hypertension. the hypertensive patients are a substantial part of patients, daily visiting the community pharmacy to pick their prescriptions. these patients represent group of patients with typical asymptomatic disease. this means that they do not take the medicines or use them according to their own will. the result of their non-adherence could lead to later complications. the aim of the study was to evaluate the level of adherence and its relation to the clinical outcome-the blood pressure in hypertensive patients. setting and method: the methodology was based on a single anonymous questionnaire survey combined with the blood pressure measuring in a community pharmacy in slovakia. the modified morisky 4-item medication adherence tool was used in this study. main outcome measures: the results of medication adherence were evaluated as follows: 3-4 points = full adherence, 2 points = partial adherence and 0-1 = non-adherence. each participant should use at least one antihypertensive agent and fulfil the anonymous questionnaire in the community pharmacy. the pharmacist measured the blood pressure in each participant twice, within the interval of 10 min and used the average value in data sheet. results: the research included 150 hypertensive patients (55.33% females and 44.67% males). the results showed that almost 46% of the respondents were non-adherent to the prescribed pharmacotherapy (57.98% of those were males and 42.02% were females). the group of partially adherent patients consisted of 35.33% of the respondents (66.04% of those were female). only 18.67% respondents were fully adherent according to modified morisky score (67.86% of those were women). fully adherent patients reached an average blood pressure 117.393/76.464 mmhg; partially adherent hypertensive patients recorded an average blood pressure 124.162/80.623 mmhg; and in the non-adherent patients has been observed the average blood pressure 154.116/95.319 mmhg. the results showed an alarming situation, and confirm the published data. non-adherent patients could not goal the good clinical outcomes. this leads to adding of another medications, raising the risk of interactions and adverse drug reactions, complications of undertreated disease, and finally, to pharmacotherapy costs increasing. please specify your abstract type: research abstract background and objective: in psychology, depression is a mental state characterized by feelings of sadness, dejection, inner tension and indecision. in psychiatry, the depression is defined as a severe mental affective disorder which paralyzes clarity of thought, psychomotorics, sleep cycle and raises pessimistic and depressing emotions often lead to pathological changes of personality. during treatment of depression is often needed psychotherapy and pharmacotherapy as well. using of antidepressants requires the sufficient level of medication adherence in patients. non-adherence to antidepressant medication significantly contributes to the undertreatment of depression in primary care populations. the aim of this study was to evaluate the level of medication adherence to antidepressants to better understand the socio-behavioural factors associated with non-adherence. setting and method: the anonymous, face-to-face questionnaire survey was set in the community pharmacy in slovakia. questionnaire obtained questions on socio-behavioural factors and adherence tool-modified 8-item morisky score (mmmas-8). main outcome measures: respondents were 150 patients (39 males, 111 females) using at least one antidepressant. the results were evaluated as follows: 8 points = full adherence, 6-7 points = partial adherence and 0-5 = non-adherence. results were evaluated in relation to socio-behavioural factors. results: average level of the medication adherence in our group was 5113, which means the line between partial and full adherence. the results showed non-significant higher medication adherence level in males (5179) compared to females (5090). the highest level of medication adherence (5367) has been shown in patients 45-64 years old, the lowest average adherence level (non-adherence) was observed in patients up to 24 years old (4656). patient living in the city were more adherent to their medication (5127) compared to patients living in countryside (5083). the highest level of the partial medication adherence has been shown in secondary educated patients (5344). partial adherence level was higher in patients with monthly income over 1000 € (5750) compared to non-adherent patients with monthly income up to 300€ (4878). in patients using no other medications, only antidepressant, we have observed the highest partial adherence (5219). conclusion: our survey showed the partial antidepressant medication adherence levels in our study group. poor adherence results in low stabilization of clinical state in patient, in using more types of therapy and in increasing costs. there might be very important role of the community pharmacists and other health care professionals to improve the medication adherence and persistence through counselling and education patients on importance and need of antidepressant medication. (1) and medication regimen complexity was assessed by using the medication regimen complexity index (mrci) (2) . five and more medication usage has been defined as polypharmacy. results: a hundred and two elderly subjects (74.5 ± 6.0; 65 male) were included in this study. of them, 89.2% had two and more chronic diseases. the most common chronic diseases determined in study population were cardiovascular diseases (especially hypertension), diabetes and hyperlipidaemia. the polypharmacy has been defined in 77.5% of them. the mean of mrci per elderly patient was 12.5 ± 7.0. one or more pims use was observed in seventy-four elderly subjects (72.5%). of all elderly subjects, 59.8% were dispensed one and more medicines with a potential for drug-disease/ syndrome interaction. pims use was more frequently determined in patients with polypharmacy (78.5 vs. 52.2%, p \ 0.05). the total score of mrci was significantly increased with elevated number of pims (r = 0.304, p \ 0.01). conclusion: this study highlights a significant association between utilization of pims and both polypharmacy and higher total score of mrci in elderly patients. pharmacists could be evaluated utilization of pims in especially elderly patients with used five or more medications and/or higher total score of mrci. please specify your abstract type: research abstract background and objective: nursing home patients with multimorbidity often use multiple drugs simultaneously, which makes these patients more susceptible to adverse drug events. several studies have pointed to a need to increase the quality of prescribing to this population. to achieve this there is a need for reliable information about patients' diagnosis, and what is recorded as the drug's indication in different electronical and handwritten health records. the aim of this study was to examine the registered diagnoses, and indications for drug use in nursing home patients. we also wanted to study the extent to which diagnoses are untreated with drugs, as well as the extent to which drugs have a registered indication for use and a suitable recorded diagnosis. setting and method: data was collected for 70 long-term patients, on average 83 years old, and 66% females from four nursing homes in tromsø municipality, norway. we retrieved information about patients' diagnoses and indication for drug use from the electronic health record and written drug charts. two pharmacists conducted the linkage between the reported diagnoses and drug use. main outcome measures: percentage of untreated diagnoses and the percentage of drugs with a registered indication for use. results: as considered by the pharmacists, 70% of the registered diagnoses was untreated with drugs. dementia, gout and osteoporosis were the most commonly untreated diagnoses with, 97, 60 and 57%, respectively. in comparison, the indication for use listed on the patients' drug charts was reported for 82% of the drugs. the drugs with the highest percentage of recorded indications were acetylcysteine (n = 15), oxycodone (n = 14) and zopiclone (n = 11), where 100, 93 and 90% had a listed indication, respectively. conclusion: a high percentage of nursing home patients' diagnoses seem to be untreated. however, most drugs that patients received were listed with indication for use in the drug charts. to increase quality of drug prescribing, one should put emphasis on improving the recorded information in electronical health records. cp-pc033: personal changes in drug regimen: dangerous for health system? inga urtane, raivis pastars, dace bandere please specify your abstract type: research abstract background and objective: patient compliance is a key factor for a successful treatment and lack of it is the main reason for predicting treatment failure. in multiple researches patient adherence is determined to be as low as 50%. therefore it is important to identify the reasons of patients not following their drug regimen. objective. to analyse the patient comprehension of their drug regimen depending on the duration of hypertension and received treatment. setting and method: during the period from december 2015 to march 2016 a quantitative survey was conducted to include respondents who have been diagnosed with arterial hypertension and whose regimen includes at least one fixed dose combination drug. main outcome measures: in an anonymous survey data was collected about their demographic information, co-morbidity, other prescribed medication, intake regime, the average blood pressure during treatment, and patient's assessment of the prescribed therapy. collected data was analysed with spss. results: the study included 103 participants, most of whom (64.1%) were women. participants average age was 62.5 ± 12.5 years and the median arterial hypertension duration was 8 (5; 16) years. the study participants, who sometimes consciously adjusted dosing regimen, observed arterial hypertension for a longer period of time compared to the group, which follows the prescribed regimen according to their doctor's recommendation, respectively, 10 (6; 28) vs. 8 (4; 15); p = 0.110. group of respondents (n = 16) receiving c3 prescription drugs, more often deliberately adjusted treatment regimen compared to respondents (n = 8) treated with b2 prescription drugs, respectively 26.7 versus 18.6%; p = 0.310. respondents who deliberately adjusted drug were more often not satisfied with the number of longterm daily use of tablets (n = 13) compared to the group (n = 11), which had to intake fewer tablets every day, respectively, 54.2 versus 45.8%; p = 0.005. conclusion: arterial hypertension duration was associated with more frequent conscious adjustment of therapy without consulting a doctor. more individual prescriptions (c3) and an increase in the number of tablets per day at the same time also increases the risk of patients deliberately changing their dosing regimen. long-term drug users should receive additional attention during pharmaceutical care process to their respective treatment schedule in order to promote proper use of medication. please specify your abstract type: research abstract background and objective: diabetes is a health issue and real burden for 1 in 6 belgians. better adherence to the treatment could potentially reduce complications, decrease morbidity and mortality, and have a beneficial economic impact due to fewer consultations and hospitalizations. setting and method: a one-year program was started in 370 belgian pharmacies to accompany diabetes patients taking dpp-4 inhibitors and encourage them to be compliant with their treatment. this study concerns 270 of these pharmacies, all part of the same cooperative group. all pharmacists received prior training in motivational techniques and reviewed the bases of diabetes therapy with an e-learning program. materials developed for the patients included brochures on diabetes and its treatment, nutritional advice, physical exercise, foot care and tips and tricks for diabetics. main outcome measures: the impact on pharmacological adherence was measured using mmpr and pdc. two control groups were included: a historical control group and a group of patients that were not included in the project. non-pharmacological adherence was assessed using questionnaires. results: in the subgroup of 270 pharmacies, 495 patients were included in the program. by the end of april 2016, only 44 of them had completed the program; 78 patients came only once to the pharmacy. they either stopped their treatment after one prescription, or were occasional clients. adherence rates were found to be high in all groups (5.7-9.1% of patients with mmpr b 80%). only for the pdc, a statistically significant difference was measured between the intervention and control group (135.37 vs. 129.22%; p = 0.015). no other statistically significant impact was measured (neither pharmacological, nor non-pharmacological). conclusion: adherence was very high in all groups. the underlying reasons still need to be investigated (choice of adherence measure, healthy user effect, etc.). however, both patients and pharmacists were very pleased with this type of program. this new role of the pharmacist will definitely be more developed in the future. please specify your abstract type: research abstract background and objective: oral anticoagulants (oac) have a beneficial effect on the long term survival of patients with atrial fibrillation and venous thromboembolism. however oacs have also side effects such as bleeding, especially when used inappropriately. pharmaceutical care interventions aim to optimize medicines use and improve patient health outcomes. the literature lacks a review on the impact of pharmaceutical care interventions in patients using oac. therefore, we systematically assessed the impact of pharmaceutical care interventions on the effective and safe use of oac compared to usual care. setting and method: a systematic review was performed in pubmed and embase with synonyms/detailed specifications of the terms oral int j clin pharm (2017) . it was motivate for the need to sort the instruments for urm, including professional participation, and on the basis of the clinical management unit, and reduce variability in decisions. the p&t or ''multidisciplinary commission rational use of medicines'' is constituted by 13 people: one hospital medical director (president), head of pharmacy (secretary), and three directors of healthcare centre, three directors of department of specialities, one epidemiology, one hospital pharmacy, one primary care pharmacy and one paediatric. because some of these members are far between them, and normally dose not have too much time, we create an online platform to work, discuss and download all the necessary documents. setting and method: we used the facilities of the andalusian agency for healthcare quality (www.acsa.junta-andalucia.es), and as a base the law of the administrative decision. we have organized a session to discuss methodology with the participation of all members. main outcome measures: number of meeting and number of internal discussion emails. drug or protocol decisions. design of the platform. results: the design platform consists of five tabs: (1) has the member information, position, telephone and address, (2) email forum, following a subject line, (3) a place for meeting requests and then hang up the meeting minutes. (4) a tool allows you to upload documents to the portal (5) a search engine. two sessions are schedules and total of 28 mails. we have 4 of 13 members who have never participated online. at this moment we have adopted two decisions. conclusion: it is an online experience of one andalusian p&t committees, the low turnout makes go slower than expected, therefore physical meetings are necessary in this moment. we are working how to get more participation and involve in the project the committee members. please specify your abstract type: research abstract background and objective: liver cirrhosis can have a major impact on drug metabolism, requiring evaluation of drug safety and dosage in individual patients. currently, there are no guidelines on safe prescribing for medications in patients with liver cirrhosis, and these patients have many questions about safety and side effects of medication. the objective of this study is to explore the patient's needs on information about medication. setting and method: qualitative, semi-structured interviews were performed in 28 patients with a (history of) liver cirrhosis. the patients were approached through an item in the newsletter of de dutch association of liver patients. topics in the interview guide were preferences about information about medication, side effects, safety, drug dosage, and how patients preferred to receive this information. interviews were audiotaped and transcribed verbatim. interviews were analysed using thematic content analysis. main outcome measures: the experiences and needs of patients with liver cirrhosis concerning information about medication. results: patients indicated they had received sufficient information about the indication, possible drug-drug interactions and the duration of treatment. they preferred (more) information about how medications work, what adverse drug reactions could be expected and practical aspects concerning intake of medication. informational needs were related to questions 'how to act': patients with more informational needs took a more active role in responsibility for their own medication management. patients needed information to know what to do, e.g. in case of adverse drug reactions or when a dosage was forgotten. the doctor and internet were the preferred sources of information: doctors because of the personal contact and internet because of the accessibility. facilitating factors were 'taking time' in healthcare provider-patient contact and 'everyday language' for texts on the internet and in package leaflets. a combination of verbal information by the healthcare professional and written information was preferred. conclusion: patients with liver cirrhosis need information about medication to take an active role in their drug management. comission for medicines and medical devices, chu de toulouse, toulouse, france please specify your abstract type: descriptive abstract (for projects) background and objective: due to its common use, insulin is often considered as a harmless medication by lots of health professionals while an overdose can lead to dramatic consequences and death. between january 2014 and june 2016, in our university hospital, 6% (7 out of 121) of the declared adverse drug events have involved insulin: 2 were caused by prescription errors and 5 by administration errors. all were discovered after the medicines have been administered but thankfully none had serious consequences. the british national health service (nhs) and the french medication safety national agency (ansm) made a list of ''never events'': avoidable events which should never happen and misadministration of insulin is among them. the objective was to increase patient safety in the hospital by setting different actions to promote and improve the appropriate use of insulin and warn health professionals about the real dangers of this medicine. design: different actors participated in the implementation of these actions: the commission for medicines and medical devices (which is composed by doctors and pharmacists) directed a group made by physicians and clinical pharmacists from the department of cardiological and metabolic diseases'. results: in addition of the usual analysis of any adverse event linked to medication declared in the hospital, several actions were set up: • a didactic document summarizing all the ''sensitive'' steps during the prescription, stocking, dispensation and administration of insulin has made the front page of the hospital's intranet and was also diffused throughout the establishment. • a chart resuming all the different insulins commercialized in france has also been diffused. it contains their types, durations and onsets of action, conditions of storage and pictures of their packaging. • the 49 computerized protocols involving insulin are going to be reviewed in order to lower their numbers and harmonize their content. • a revision of the list of insulins available at the hospital is in progress to reduce their number and avoid any confusion between the different products. • an evaluation of insulin's computerized prescription practices will be made via a data request. : this topic about insulin shows a greater willingness to secure the medication circuit in the hospital. other action plans such as this one will be set up involving other medications among the never-events list. meanwhile, the commission for medicines and medical devices pursues its actions of promoting the appropriate use of medication. please specify your abstract type: descriptive abstract (for projects) background and objective: one of the hospital pharmacist tasks is to suggest substitutions to ensure conformity of medical prescription with the hospital formulary. indeed, when an eye drop isn't available at the hospital, there is a specific supply circuit which has to remain exceptional: it's ordered directly to the pharmaceutical wholesaler. in this context, ophthalmologists and clinical pharmacists created a table proposing therapeutic equivalencies with eye drops available at the hospital. after approval by the commission for medicines and medical devices, this tool has been diffused within the establishment via the intranet website since the beginning of 2013 to the medical and paramedical staff. the purpose of the study is to evaluate professional practices concerning the use of the eye drops' equivalence chart. design: in the study, we compared eye drops' orders made to the pharmaceutical wholesaler before and after the table's diffusion, thus between january 2012 and december 2015. for each order, we used the table available at this time to determine if equivalencies could have been proposed or not. if so, we identified the hospital ward and the pharmaceutical specialty. market changes have also been considered. results: we noticed a decreased frequency of eye-drops ordered despite available equivalencies: 52% in 2012 (before the table's diffusion), 25% in 2013 (after its diffusion), 33% in 2014 and 33% in 2015. prisons units are responsible of 95% of these orders: they have the lowest rates of substitutions. their most ordered pharmaceutical specialties are ophthalmic glaucoma agents: 46% ganfort ò (bimatoprost 0.3 mg/ml/timolol 0.5%), 20% xalacom ò (latanoprost + timolol 0.5%), 23% azopt ò (brinzolamide) for which the authorized substitutions are for the first two specialties: monoprost ò (latanoprost) + ophtim ò (timolol 0.5%) and for the third: dorzolamide ò . conclusion: equivalence table diffusion throughout the hospital has facilitated and improved the prescription and substitution of eyedrops. orders of pharmaceutical specialties despite authorized equivalencies available have declined by half. probably for practical reasons regarding long-term treatments, prison units make less substitutions but an awareness campaign will be carried out to reduce these rates. please specify your abstract type: research abstract background and objective: the patient's education and information is a mean to reduce medicine misuse and it can be performed with support of a leaflet or informative material about medicines. in brazil, there is a lack in regulation about this type of informative material to compounded medicines. the aim of this study was to evaluate the quality and effectiveness of leaflets developed to compounded medicines' users through knowledge's level and medicine treatment adherence. setting and method: analytical and quantitative study; 3 month prospective study through interviews, at time zero (t0) and after 30 days (t1) in a university pharmacy in goiás, brasil; fisher's exact test to measure effectiveness; ethics committee number 008/12. main outcome measures: categorization into high adherence and low adherence by morisky test; categorization into sufficient, regular or insufficient knowledge about medicine prescription; perceptions and suggestions about delivered leaflet in medicine dispensing process. results: of 52 patients (82.7% female, mean = 48.7 years), 93.5% considered as relevant the leaflet's content, as well 88.5% of them kept it and 67.4% of them read it. suggestions of 16.1% included a desire in increase font size, more emphasis on drug interactions and images. there was a predominance of regular knowledge in both analysed times (48.4% e 38.7%), however there was a decrease in high adherence to medicine treatment (19.2-7.7%). among patients who read the leaflet, no statistically significant association was found on these two variables at t0 and t1 (p = 0.24 and p = 0.84, respectively). knowledge about ''administration schedules'' showed a significant improvement after intervention (p = 0.02). 61.3% of patients considered that there was no need to obtain more information. conclusion: this study demonstrates the evaluated leaflets had relevance to patients and demonstrate clinical relevance. however was not observed statistically significance. this highlights the need of using different ways to measure the effectiveness of an informative material to promote rational use of medicines and depth studies and stimulation of greater attention from the health professionals to the topic. di006: chlormethine gel: effectiveness and tolerance to treat mycosis fungoides françois dugre *,1 , anne lefebure 1 , sonia martelli 1 , marion pin 1 , eve maubec 2 , philippe arnaud 1 1 pharmacy, 2 dermatology, bichat-claude bernard hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: to determine the effectiveness and tolerance of chlormethine gel in treating mycosis fungoides. design: mycosis fungoides is the most common form of cutaneous t-cell lymphoma (mf-ctcl). early stages (ia and ib) can be controlled by skin-directed therapies such as chlormethine and carmustine. these drugs which are solutions for injection are usually used for skin application. chlormethine or mechlorethamine gel is an alkylating agent representing an alternative for previously treated patients diagnosed with mf-ctcl, in case of therapeutic failure and intolerance, or in case of chlormethine and carmustine solutions supply disruption. a retrospective observational analysis was conducted based on medical records of all patients treated by chlormethine gel in our hospital from the first of july to the first of september 2015. the following data were collected with an excel table: body surface area or bsa affected by disease, location of the lesions, therapeutic management, effectiveness and treatment tolerance. results: fourteen patients (7 women, 7 men, mean age 55 [min 33; max 84]) were treated with chlormethine gel in our hospital. twelve (86%) were treated three times per week, 2 (14%) once a day. before treatment by chlormethine gel, 4 (29%) patients were treated by dermocorticoids, 4 (29%) by dermocorticoids and phototherapy, and 1 (7%) by bexarotene, all of them stopped their treatment on account of inefficacity. one (7%) patient was treated by carmustine and dermocorticoid, and 3 (21%) by only carmustin, all of them stopped it because of supply disruption. one (7%) patient received it in first line therapy. ten (71%) patients showed a response (partial or complete), one (7%) experienced a stabilization of his disease. before treatment with topical chlormethine, seven patients (50%) had an involved bsa [ 10% and four of them (57.1%) experienced adverse effects. seven patients (50%) had an involved bsa \10% and three of them had (42.9%) side effects. a total of seven patients (50%) presented at least one adverse effect. five patients (36%) stopped the treatment on account of adverse effects; two of them (14%) interrupted it temporarily. reported side effects were: irritant dermatitis and erosive toxicity (5), rash (2) and telangiectasia (2) . conclusion: our results indicate that chlormethine gel can be effective to treat mycosis fungoides. however, it involves side effects that seems to be more frequent than those observed with chlormethine solution (used for skin application). indeed, the french national authority for health reports 28% of adverse effects for chlormethine solution versus 50% in our study for chlormethine gel. moreover, telangiectasia was never documented with chlormethine. this significant number of side effects of chlormethine gel can be explained by the gel formulation which induces patients to apply more product, especially in patients with plaques affecting more than 10% of the bsa. it is important to explain to patients to apply a thin film of chlormethine gel to involved skin areas and allow the skin to dry completely. sophie dumas *,1 , capucine devaux 1 , nathalie le guyader 2 1 diaconesses croix saint-simon hospital, 2 diaconesses croix saint-simon hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: aprepitant, a neurokinin-1 receptor antagonist, prevents nausea and vomiting due to high and moderate emetogenic chemotherapy in combination with other antiemetic agents. it induces cytochrome p450 (cyp) 2c9 and moderately inhibits cyp3a4. drug-drug interaction could occur with intravenous anticancer or antiemetic drugs metabolised by these isoenzymes. it may lead to adverse effects or loss in efficacy. regarding recent international antiemetic guidelines, emergence of new intravenous chemotherapy and lack of bibliographic data, a report on aprepitant interactions is performed in oncology. the aim of this study is to review pharmacokinetic interactions with aprepitant in order to prevent potential toxic effects of intravenous anticancer or antiemetic agents and provide the best patient care. design: anticancer and antiemetic agents metabolised by cyp3a4 and 2c9 were identified. pharmacokinetic literature review was performed using medline ò database and laboratory data. clinical assessment and non-aprepitant pharmacokinetic studies were excluded. a table was established to summarize data. results: ten intravenous anticancer agents used in oncology are identified as cyp3a4 substrates. pharmacokinetic assessments are achieved for docetaxel, cyclophosphamide, vinorelbine, irinotecan and trabectedin. studies dealing with the five other drugs are strictly clinical assessments. among the different pharmacokinetic studies, only trabectedin showed relevant interaction with aprepitant. in this association, aprepitant dose needs to be adjusted. cyp2c9 catalyses the cyclophosphamide activation pathway with minor contribution. however, it would have few repercussions on cyclophosphamide pharmacokinetic. corticosteroids and 5 hydroxytryptamine type 3 (5ht-3) receptor antagonists are also metabolised by cyp3a4. aprepitant significantly increases corticosteroid plasma concentrations. in this case, corticosteroid dose adjustment should be applied. furthermore, no interaction has been found with 5ht-3 receptor antagonist. conclusion: regardless of the emetogenic level of anticancer agents, all drugs have been studied because of theirs potential combinations. two relevant pharmacokinetic interactions have been demonstrated leading to dose adjustment recommendation. corticosteroids doses, in association with aprepitant, should be reduced one-fourth for intravenous form and one half for oral form. aprepitant first dose should be decreased to 80 mg when it is co-administrated with trabectedin. these two results lead us to re-evaluate our prescription practices. please specify your abstract type: research abstract background and objective: nsaids are associated with serious adverse reactions which in turn are responsible for significant risks of morbidity and mortality. the aims of this project is to identify risks involved in nsaid administration including over-usage and significant drug interactions, and to analyse occurrence of side-effects. the trends of nsaid prescribing by physicians and pharmacists are also determined. setting and method: a pharmacy from each electoral district was chosen by stratified sampling. a sample population (n = 100) was obtained from 13 pharmacies in malta. data was collected through the completion of questionnaires carried out by the patients. the trends of nsaid prescribing were determined by another questionnaire directed to 49 pharmacists and physicians that was available online. main outcome measures: use of nsaids by patients and prescribing trends. results: back pain (n = 19), muscular pain (n = 13), headache (n = 12) and arthritic pain (n = 10) accounted for the most frequent use of nsaids. diclofenac accounted for the most commonly administered nsaid, taken by 58 of the patients, of which 48 use the 50 mg dose. chronic disorders of symptoms experienced by the patients included hypertension (n = 24), heartburn (n = 22), dyspepsia (n = 21), asthma (n = 4) and a history of helicobacter pylori infection (n = 2). other disorders suffered by single individuals include epilepsy, crohn's disease and renal dysfunction. more than half of the respondents (n = 55) admitted to self-prescribing regardless the fact that the majority of nsaids are prescription-only medications. epigastric pain (64.6%), stomach ulcers or gi bleeding (30.6%) and elevated blood pressure (29.8%) were the most common sideeffects that pharmacists and physicians come across. nsaids were frequently found to be co-administered with antihypertensives (68.1%) and ssris (37%) regardless of their significant risks of interacting with nsaids. 75.5% of the pharmacists and doctors believe that nsaids are being over-used and 95.9% state that closer monitoring of nsaid adverse effects is necessary. conclusion: the risk involved with nsaid administration due to over-usage and drug interactions is identified, and healthcare professionals are aware of this risk. pierre leduc, antoine lanneluc, christophe gellis * , sylvie poux, dominique plats, regine larnaudie corrèze, ch brive la gaillarde, brive la gaillarde, france please specify your abstract type: research abstract background and objective: proton pump inhibitors (ppi) are widely prescribed in hospital while their long-term use may be responsible of many potentially serious long-term side effects (hypomagnesemia, neutropenia, gastric cancer) and drug interactions (ppi are inhibitors of cyp2c19). the objective of this study was to assess the appropriateness of ppi prescriptions in a geriatric department in order to optimize their conditions of prescriptions. setting and method: this prospective study involved patients hospitalized between january 2016 and april 2016 in a geriatric department. the accordance of the prescriptions with the marketing authorization indications and the french guidelines 1 was analysed. data collection was done using a table excel. main outcome measures: collected information were related to patients (age, sex) and ppi prescriptions (active substance, administration route, dosage, duration of therapy, therapy indication and reassessment of ppi therapy). results: ninety-one patients were included: sr: 0.3, mean age: 87.3 years [78; 102]. ppi therapy prevalence over the period was 38%. the ppi were prescribed in the geriatric department in 33 patients (mostly esomeprazole) whereas 58 patients had ppi therapy (mostly esomeprazole) at the admission, for more than 2 years in 42 patients. oral route was the most frequent one (n = 86). 89 ppi were administered once a day and only three ppi were administered in the morning. 40% of ppi prescriptions were considered unjustified; the indications were prevention of haemorrhage with antiplatelet therapy (n = 19), prevention of haemorrhage with corticoid (n = 4), prevention of haemorrhage with anti-vitamin k (n = 3), dyspeptic disorders (n = 2), gastralgy (n = 4) and others reasons (n = 4). 60% of ppi prescriptions were considered relevant. the reassessment of ipp therapy (n = 25) lead to prescribe another dosage (n = 9), to stop therapy (n = 11) or no change (n = 5). conclusion: the study showed that the majority of ipp prescriptions were not in accordance with french guidelines. limiting the prescription to the indications, reassessing the therapy or respecting the therapy duration should reduce the risk of long term side effects and the economic burden of ppi in a long term use. please specify your abstract type: descriptive abstract (for projects) background and objective: to evaluate the effectiveness and safety of the use of high dose of tigecycline (200 mg followed by 100 mg every 12 h) a tertiary care hospital. design: retrospective observational study. period: january to december 2015. inclusion criteria: episodes use of tigecycline (200 mg followed by 100 mg every 12 h. exclusion criteria: time less than 4 days treatment. data source: corporate program stories electronic health. results: we identified 24 episodes in 23 patients (15 men, mean age: 71 years (23-88)). treatment was directed to multidrug-resistant organism infection in 11 cases (seven klebsiella pneumoniae oxa-48, two enterobacter cloacae, two enterococcus faecium and one methicillin resistant staphylococcus aureus. in one episode they coincided e. cloacae and e. faecium). in 14 cases had severe sepsis or septic shock (seven abdominal focus, six respiratory focus and one unknown focus). the median number of days of treatment was 12 (4-119). tigecycline was administered as monotherapy in three cases, bitherapy 13 and triple combination therapy in 13. the antibiotics were associated were: beta-lactam (11), aminoglycosides (10), quinolones (3) colistin (three, two inhaled cases), cotrimoxazole (1) and vancomycin (1) . in 13 episodes produced clinical and/or microbiological resolution and 7 antibiotics are rotated by progression picture or lack of improvement, death occurred in three cases and 1 was suspended on suspicion of hepatotoxicity. among the seven episodes of klebsiella pneumoniae oxa-48 infection there were four pneumonias, three with favourable evolution and one patient died, two bacteraemia, both with resolution clinical and microbiological, and one urinary tract infection resolved. among the 14 episodes in severe/ septic shock were five cures, six cases of antibiotic rotation progression or lack of improvement and three deaths while patients receiving therapy tigecycline. 2 patients showed an adverse effect possibly related to therapy tigecycline: 1 diarrhoea after 15 days of treatment and 1case of liver toxicity after 4 days of tigecycline and piperacillin-tazobactam which led to their withdrawal. int j clin pharm (2017) 39:208-341 253 conclusion: tigecycline has been used in double dose defined in data sheet especially in situations of severe sepsis or septic shock and infection multiresistant microorganisms. the effectiveness is conditioned by the clinical situation patient, being worse in severe/septic shock sepsis. tigecycline high dose was well tolerated and there was only a case of stopping the medication for suspected damage hepatic. di012: wikipedia and medicines: who edits medicine articles on the english wikipedia? kristian husvik skancke 1 , kristian svendsen *,2 1 department of history, uit -the arctic university of norway, 2 hospital pharmacy of tromsø, tromsø, norway please specify your abstract type: research abstract background and objective: the medical profession and pharmacists are divided on the usability of wikipedia for looking up health information. nevertheless wikipedia is widely used, more than half of us physicians and 94 percent of all medical students use wikipedia as a source of health-related information. there is a potential for incorrect and biased information being added by the pharmaceutical industry. the aim of this project was to examine who edits wikipedia articles on medicines and to investigate whether the pharmaceutical industry edits these articles. setting and method: two different groups of articles has been examined; the top ten bestselling medicines (substances) in the world in 2014 and the ten most recently approved medicines on the european market (until december 2014). the top ten medicines were selected from a consultancy report by evaluatepharma/ep vantage. the ten most recently approved medicines (new substances) were found on the european medicines agency webpage. we queried the english wikipedia on 11 january 2015 and information from the edit history and the editors' user information were extracted. unregistered editors were checked using a whois service. for the new medicines all editors were checked, while for the bestselling medicines large edits and initial edits was checked. main outcome measures: edits suspected of being made by the pharmaceutical industry. results: ten bestselling medicines: there are many users editing these articles and/or watching them, limiting the risk of misinformation from the industry. there was no indication that the pharmaceutical industry had edited any of the articles. ten most recent medicines: no article existed for dasabuvir. for the nine other substances there were relatively few editors and watchers. in four out of the nine articles we found evidence of edits from the pharmaceutical industry. these edits, were done by registered editors with very few edits except for the medicine in question and they had made large additions to the articles sometimes even before the medicine was marketed. conclusion: the pharmaceutical industry seems to edit articles about medicines on english wikipedia however we found no evidence of harmful edits and bestselling medicines have many editors monitoring the quality of articles. please specify your abstract type: research abstract background and objective: the pharmaceutical professional service of the monitored dosage systems (mds) tries to improve the adherence of the patients to the treatment. the aim was to analyse the relevance of the repackaging of the most sold medicines in our country being used by patients included in the mds professional service and to determine the information discrepancies according to the source used by the pharmacist. setting and method: cross-sectional descriptive study. community pharmacy and healthcare institutions. all the patients included in the pharmaceutical professional service of mds on june 1, 2016. data source: patients' records in the professional service, database of medicines ranked by sales in units in our country to december 2015 (400 medicines), information sources on medicines: (1) vademecum of medicines and (2) the centre of drug information of our agency of medicines. main outcome measures: number of institutionalized and ambulatory patients included in the professional service of the mds and demographic characteristics, sum of different repackaged medicines belonging to the studied patients, analysis of the repackaged medicines of major use, number of discrepancies on the repackaging of the medicines according to the information source. results: 88 patients were included in the professional service of the mds. 67 of them were institutionalized (average age: 39.9 years, 65.7% men, 76.1% polymedicated defined as using c4 prescribed chronic medicines) and the remaining 21 were ambulatory (average age: 76.8 years, 57.1% women, 23.7% polymedicated). 130 different medicines prescribed in the institutionalized patients were taken into account, 29 of them included in the sales ranking in our country. according to the first source, 18 of 29 medicines were eligible for repackaging, 6 medicines could be repackaged according to the laboratory manufacturer and the 5 remaining ones could not be repackaged. according to the second source, 21 of 29 medicines could be repackaged, and the 8 remaining ones could not. 128 different medicines prescribed in the ambulatory patients were taken into account, 37 of them included in the sales ranking in our country. according to the first source, 27 of 37 medicines could be repackaged, 8 medicines would depend on the laboratory manufacturer and the 2 remaining ones could not be repackaged. according to the second source, 27 of 37 medicines could be repackaged, and the 10 remaining ones must remain in the original package. discrepancies were observed in the information for 8 (27.6%) and 14 (37.8%) medicines in institutionalized and ambulatory patients, respectively, based on the sources used. conclusion: a considerable number of discrepancies in the information on the relevance of the repackaging of medications in the mds were found between two analysed sources. these findings have already improved the quality of this professional service. it would be necessary to alert the pharmacist of the existence of the above mentioned discrepancies to be able to prevent errors from occurring at the time of repackaging the medicines in the mds and, thus, increasing patient safety. please specify your abstract type: research abstract background and objective: despite the global advances of pharmacy practice and subsequently pharmacy education, students experience insufficient opportunities to practice the activities, tasks and processes essential to deliver pharmaceutical care. objective: to describe the development, implementation, and assessment of a clinical pharmacy practice (cpp) experience course in internal medicine, cardiovascular, respiratory clinics and drug information centre that is newly integrated into pharmacy curriculum at a university in north cyprus. setting and method: a 8 weeks structured pharmacy practice experience was designed for fifth year students. student competence was assessed using formative osces and summative written exams before and after the course, and mapped in eight main cpp competences. the course utilized a wide variety of learning and practical activities including rounds participation, morning case reports, interdisciplinary activities, carrying interventions, role-play, direct patient care, formal case presentations, journal clubs and answering drug queries. competencies tested and strengthened include: taking medication history, response to the symptoms, pharmacotherapy knowledge application, comprehensive patient assessment, data interpretation using evidence-based approach, public health counselling, drug related problems management, patient counselling and communication skills. student perceptions and experience was assessed using semi-structured group interview and a questionnaire. main outcome measures: student scores in osce; student's perceptions. results: student reported that the course met pre-set objectives with substantial learning in different areas of cpp. students scored best in communication skills (83.4 ± 1.74%), public health promotion (76.6 ± 2.32%) and patient counselling (68.0 ± 2.74%) than in resolution of drps (49.0 ± 4.33%) and pharmacotherapy application (49.0 ± 3.37%), while they significantly enhanced in di manipulation (88.1 ± 2.6%) compared to baseline assessment (33.1 ± 2.41%)(p = 0.0038). conclusion: the course provided a rich experiential learning environment rather than just theoretical knowledge of clinical pharmacy. students well perceived the course structure assessment and knowledge attained. this could be implemented in other faculties of pharmacy through turkey. please specify your abstract type: descriptive abstract (for projects) background and objective: clinical pharmacy and clinical pharmacology have many similar aspects. both areas present professionals who have groundings in drug therapy principles and who aim to optimize the efficacy and safety of therapies for patient's benefits. however, there are clear distinctions. clinical pharmacologists are in general doctors with an additional education in clinical pharmacology. many of these are prescribers of drugs in practice but are in usual connected to academic parts responsible for education and research. they belong to a well-recognized but small sub-specialty of medicine. in contrast, clinical pharmacists are part of a much greater group of professionals working in most hospitals in developed countries. while the former one is restricted and subordinate to distributing the drugs requested by the medical prescribers, the role of the pharmacist has increasingly developed to encircle monitoring outcomes of medicine treatment and report management, patient safety and budgetary responsibilities. pharmacists are currently capable to take on prescribing responsibilities in developed countries and have been actively involved in collaboration in practice of prescribing with doctors. they also take on a great part in education related to rational prescribing that was once thought the area of the clinical pharmacologist. given the difference in size of the two areas there is understandably increasing confusion in the minds of managers in health services as to the continuing role and identity of clinical pharmacology. this may illuminate, in part, the diminishing in numbers and visibility of clinical pharmacologists in certain countries. in fact, some might see the continuous development of clinical pharmacy as a direct danger to the viability and future existence of the specialty of clinical pharmacology. however, clinical pharmacy and clinical pharmacology working synergistically would serve for the well-being of the public. design: . results: . conclusion: . maxime apparuit *,1 , lea boissinot 1 , ngauv melodie 1 , stephanie charles weber 2 , isabelle lopez 1 , françois chast 1 1 pharmacy, hopital cochin, 2 pharmacy, hopital hotel-dieu, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: hereditary angioedema (hae) is a rare disease characterized by episodic attacks of swelling which can be life-threatening. treatment for hae involves prophylaxis and management of acute attacks. the objective of this study was to evaluate patients' knowledge of their disease and their treatment. design: a questionnaire about the disease and drug treatment has been implemented. it was distributed to patients through either a pharmacist during patients stay at the hospital, or the french association des malades souffrant d'angioedèmes (amsao). answers were collected by electronic or conventional mail. results: 39 patients completed the questionnaire. the average patients age is 25.8 ± 27.2 years. all of those interviewed could name their disease. for 23% of patients, the crisis happened unpredictably but in most cases a triggering factor was described, such as stress (21%), fatigue (18%) or an emotional shock (17%). oedema were located mainly in extremities (31%), abdomen (19%), ent sphere (12%) or face (11%). 17 patients (43%) reported having more than 12 crisis each year (eligible to prophylaxis), among them, 5 patients (13%) said they had no preventive treatment. all patients knew the difference between prophylactic and curative treatment of crisis. among the 38 patients receiving treatment for crisis, 16 were able to define which treatment to be used depending on the intensity and location of the crisis. the majority of patients used icatibant during a crisis, but the most frequently cited prophylaxis treatments were tranexamic acid (38%) and danazol (35%). for injectable drugs to treat acute episodes, icatibant (subcutaneous) and c1 esterase inhibitor (intravenous) were self-administrated respectively in 79 and 8% of patients. conclusion: this study showed that patients generally knew their disease and its treatment. however, they are insufficiently informed on drugs to be used according to the clinical situation and especially intravenous self-administration. therefore, it seems necessary to increase pharmacist involvement in patient's information about therapeutic strategy and drugs routes of administration. this for a major objective: an optimal self-care in a skilled patient. please specify your abstract type: descriptive abstract (for projects) background and objective: hospital pharmaceutical educations (hpe) on patients with oral anticoagulant (oa) can improve their overall management by providing skills on proper use. an ambulatory monitoring is necessary to ensure good compliance and understanding of the treatment. our study aimed at the establishment of hpe for patients with oa, the establishment of a hospital-city link in burgundy, and an evaluation of the expectations of ambulatory health professionals (ahp). design: the development of hpe has been performed in our centre for patients with oa and assessed between may and september 2015. in order to ensure continuity in their support, patients then received a binding document to the attending physician, pharmacist and nursing home stating the treatment and acquired skills. a satisfaction survey, with anonymous electronic questionnaire circulated by the representative boards evaluating the expectations of ahp, took place in order to improve and make the programme more attractive. results: two hundred and ninety-one patients could benefit from hpe and 252 came out with an oa. one hundred and forty-three answers were collected: 38 officinal pharmacists and 105 nurses. ninety-seven percent of ahp have judged relevant the following stated security goals: the name of the drug, its use, its risks and to be able to inform all ahp. ''associated pathologies and treatments,'' ''the last coagulation test'' and ''potential factors for non-adherence'' seem necessary for the binding document. more than 90% of participants found that this action will facilitate the establishment of pharmaceutical anticoagulant educations in cities, the dialogue around the oa with the doctor, patient's compliance and will secure the treatment. conclusion: hpe certainly help patients. its implementation for patients with oa in our hospital has generated a real interest. the addition of an ambulatory link allows continuing at best their support. the questionnaire has also allowed us to know the opinions of ahp involved and some improvements to the binding document may have been done. participants were asked to associate the task to the profession by determining whether each profession had the main responsibility for undertaking the task, a supportive responsibility, or whether they should not be involved at all. data was analysed using spss ò, version 22. the chi squared test was used to assess any significant association between categorical variables. main outcome measures: perception of the oncology pharmacist's role by healthcare professionals. results: from a total of 84 completed questionnaires, it was found that for tasks listed as ''patient education and counselling'', 18% were considered as the pharmacists' main responsibility, whereas 42% were believed to be supportive roles. main tasks included educating the patient regarding which medication to avoid during their treatment. for tasks listed as ''drug related problems'', 20 and 45% of tasks were found to include pharmacists as having main and supportive roles respectively. supportive tasks included dose calculation of anti-tumour therapy required per patient. in the ''authorisation of medication'' category pharmacists' main roles carried a total of 13% and supportive that of 50% of the total number of tasks. this included ordering anti-tumour medication. further analysis of data revealed that years of experience did not have a significant association with results obtained (p-value = 0.074); however physicians, pharmacists and allied healthcare professionals were found to involve the pharmacist most extensively (pvalue = 0.000). conclusion: tasks associated with the pharmacist were representative of the current role they possess within the oncology setting; however this association was limited to professionals having a close working relationship with pharmacists. this may be due to the lack of an established multidisciplinary team approach within this scenario thus limiting the perception of the oncology pharmacist's contribution. an implemented multidisciplinary team may improve communication between the professionals involved and optimises patient care. the aim of the study is to analyse from a qualitative and quantitative point of view the pharmacy resident's activity in pneumology service. setting and method: the study included all the daily prescriptions of three units of pneumology from january to april 2016. pi and data were extracted from the software pharma ò and collected in a summary excel ò table: nature of potential errors, nature of the proposals offered by residents, way of transmitting pi, and rate of pis' acceptance. main outcome measures: potential errors are collected by following the validated and standardized criterions of french society of clinical pharmacy. results: over 4 months, 7968 lines of prescriptions from 412 patients aged 67 years old (median [28-85]) were evaluated. sex ratio (m/f) was 1.46. one hundred and two medication problems have been found: overdose (21.6%), contraindication (ic) (20.7%), under dosage (7.8%), wrong rhythm of administration (7.8%), forgotten treatment (7.8%), dose unit error (5.9%), antibiotic indication missing (5.9%), drug not listed in the hospital formulary (4.9%), potassemie unchecked (4.9%), dose unadapted to renal function (4.9%) or to inr (2.9%), treatment not indicated (1.9%), wrong administration route (0.98%), antibiotic unreevaluated (0.98%), redundancy (0.98%). the proposals made to the doctors were: stopping treatment (25.5%), posology adaptation (19.6%), substitution (19.6%), dose unit modification (7.8%), adding information about the indication (6.7%), treatment renewal (6.7%), administration modalities changing (4.9%), biological monitoring (3.9%), therapeutical monitoring (2.9%), antibiotic treatment reevaluation (2.94%). all pi were made by informatical way. all medicinal classes were found in this study. hydroxyzine, cyamemazine and escitalopram were often found in contraindication errors. they are involved in cardiac disorders with qt extension. pis' acceptance rate was 82%. conclusion: this study shows the importance of pharmaceutical analysis on the quality of access to healthcare. the statement of pi allows us to identify the most frequent errors, warn and prevent doctors from these potentials errors by proposing solutions. the rate of acceptance is high which means that doctors agree with our proposals. pharmacists' implication in clinical pharmacy activities and their participation to medical rounds will improve this activity and by the way optimization of the management of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: ppis consumption is largely practiced in europe, because of their excellent tolerance in short time, and their misuse with regard to indications, dosage and treatment duration (in 2007, france was the 2nd,ppi consumer in eu). the result is drug iatrogenic disease and unjustified expenses in health insurance. objectives: assess the ppis consumption and appreciate conformity according to the latest recommendations for relevant prescriptions of ppis. design: prospective study via an audit (model created internally), every hospitalized patients with a ppis prescription, in two hospitals, on a given day. data collected through the patient's medical record. prescriptions conformity defined, by taking account of indication level (1: approved by the ma (marketing authorization), 2: non-valid but certified by international publications or learned society, 3: nonvalid without scientific proofs, 4: non-indicated), dosage and treatment duration. analysed situations with no conformity (inappropriate dosage despite conform indication, treatment duration unjustified and ppis prescribed in wrong indications (3 and 4) . results: 172 patients have ppis prescriptions (78 male, 25 £ 99 years] among 325 patients (53%). 45% ppis prescriptions began during the hospitalization. 53 (30%) of the ppis prescriptions are in accordance with the experiment (indication + dosage +treatment duration), as well in community than in hospitals. details: indication level 1 (92.5%), indication level 2-gi bleedings-(7.5%). 119 of the ppis prescription aren't in accordance. details: treatment duration (6%), dosage (3%), indication level 3-prevention of iatrogenic bleeding risk without nsaids prescription-(75%), indication level 4 (16%). regarding level 3 indications, ppis are always taken with anticoagulant and/or platelet aggregation inhibitors and/or corticoid. conclusion: the part of ppis prescriptions in this study is high. the majority of non-conformity is caused by ppis prescribed with an indication level 3. the improvement program will involve feeding back ppis' good use, to educate physicians (junior and senior) about the relevant ppis prescription and give advice in complex situations (indication 3 and 4). in collaboration with prescribers, shutdown protocol of ppis, prescribed in long term, could be implemented in order to avoid the acid rebound effect after brutal treatment discontinuation. hp-ce014: impact of a self-management program on inflammatory bowel disease patient in a university hospital caroline egon * , xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: inflammatory bowel disease (ibd) is a group of chronic inflammatory diseases that affects the colon and the small intestine. crohn's disease and ulcerative colitis are the principal types of ibd and involve severe diarrhoea, pain, fatigue and weight loss. ibd affects young adult with an increasing annual incidence (2.5 million concerned people in europe). patients with ibd are affected by somatic or psychosocial problems and patient education may contribute to their well-being. since september 2010, individual educational sessions have been set up and since september 2015, collective educational sessions. these sessions have been developed to improve patient's understanding of treatment options and medical adherence. the aim of this study was to demonstrate that a therapeutic education program (tep) could have a significant effect on ibd patient's skills with regards to their disease. design: after individual education sessions with a nurse, a group education session was introduced for outpatients with ibd. the collective session include approximately six to ten patients and is organized in a half day workshops (about disease and treatment) conducted by a multidisciplinary team. the workshops were performed by an education nurse, two hospital gastroenterologists, two hospital pharmacists and a community pharmacist. these sessions were wrapped up by a short satisfaction and knowledge questionnaires. results: in total, 141 ibd outpatients participated to the educational program, 112 patients with crohn's disease and 29 patients with ulcerative colitis (52.5% male; median age: 39). for the individual educational sessions, two competence questionnaires were performed about anti-tumour necrosis factor alpha (tnfa) therapy: one about general knowledge, another one about self-administration subcutaneous injection. 43 patients completed these questionnaires. for the collective educational session, the competence questionnaire developed consisting of six questions covering few items: disease, symptoms, treatment and complications. 14 patients completed this questionnaire. after the questionnaire, each participant received a summary document about drugs, side effects, therapeutic and medical advice. conclusion: the patient education program contributes to the improvement of self-management skills when it comes to ibd. pharmacists joining medical specialists and nurses provided pharmaceutical care with a positive impact on compliance, which is a determining factor for the success of the treatment and the quality of life in patients living with an ibd. this program will be continued and a new program for teenagers is to be established as well. hp-ce015: desensitization study of paclitaxel and carboplatin drug in the ovary tumor protocol in cuf descobertas hospital miguel â . freitas * , daniela brites, ana bota pharmacy, hospital cuf descobertas, lisbon, portugal please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacy should be an integral part of the multidisciplinary team and implement strategies that meet the patient's needs. pharmacy, in oncological area, is in constant renewal. josé de mello saúde uses paclitaxel/carboplatin protocol as first line in ovarian tumor. although the antineoplastic agents are essential for the treatment of cancer, they can also cause hypersensitivity reactions, which may carry serious consequences. both immunoallergologist and oncologist create a desensitization protocol, which allows the reintroduction of the drug with greater security. the desensitization protocols involves the gradual administration of small quantities of the drug, resulting in a refractory period of the white blood cells (mastocytes) and a lower production of cytokines until the dose has been totally administrated. objective:to evaluate the efficacy of methods used to prevent and treat hypersensitivity reactions of carboplatin and paclitaxel, in order to carry on the treatment. design: a retrospective review of the patient files was performed in the day hospital between 2014 and 2016. we included only patients with moderate to severe immediate hypersensitivity reactions (b24 h) receiving carboplatin and paclitaxel. the desensitization protocol brigham and women's hospital was applied using three solutions with increasing concentrations (dilution 1: 100, 1:10 and 1:1) in twelve successive steps for about 6 h. results: in the period 2014-2016 were desensitized five patients with platinum group drugs, carboplatin (n = 4) and paclitaxel (n = 2) and the total elapsed six desensitization. almost all patients reached the scheduled daily dose, except a patient, which suspended the desensitization program for disease progression. conclusion: the desensitization protocol allowed the successful reintroduction of antineoplastic drugs in patients with a history of hypersensitivity reactions, in order to treat the disease. please specify your abstract type: research abstract background and objective: in the context of harmonization of clinical pharmacy activities within our region, a common medication reconciliation project was developed between two general hospitals. the objectives of this study were to initiate, a common medication reconciliation activity in the two hospitals, to analyse the results, and to communicate to all professionals in the area. setting and method: a working group composed of pharmacists of each hospital was formed to develop analytical documents. a 3-month prospective study was conducted in two general hospitals: in the first one, in an emergency department, and the other one, in a medicine department. patients included in the study were either elderly and/or had polypharmacy and/or were hospitalized for iatrogenic reason. at int j clin pharm (2017) 39:208-341 259 the point of admission and discharge for each patient, the pharmacist has completed a conciliation record, and has detected potential discrepancy. unintentional discrepancies were reviewed and corrected by doctors. at the discharge, medication changes were sent to general practitioner and community pharmacies. a satisfaction survey about this process was sent to 39 healthcare professionals (gp, pharmacist and nurses). a medication reconciliation's workshop was organized for a hundred healthcare professionals in the area. main outcome measures: at the point of admission, the conciliation record included the list of patient's home medication, admission medical orders, and the types of discrepancies. at the discharge, drugs prescribed were compared to admission medical orders. the satisfaction survey included seven questions to assess the process. results: during the study period, 35 patients were included corresponding to 351 prescription lines. reconciliation process required about 47 min per patient. we identified at admission 33 unintentional discrepancies. the most common unintentional discrepancy was the omission of medication (61%). 25% concerned alimentary tract and metabolism group. at the discharge, no discrepancies were found; the process required 35 min per patient.41% of healthcare professionals answered to our satisfaction survey to date. 100% are satisfied and believe that the process of medication reconciliation secures the patient medicinal treatment.56 healthcare professionals were present at the medication reconciliation's workshop, indicating an interest in the process. conclusion: in this experience of medication reconciliation, due to unintentional discrepancies observed, we had better implement this activity in the two general hospitals. a pharmacist devoted to this activity will be hire in each hospital. this relevant practice is well accepted by clinician. thus, we will improve communication with gp and community pharmacies. please specify your abstract type: descriptive abstract (for projects) background and objective: the sickle cell disease (scd) is a genetic, chronic disease, paroxystic in its unpredictable and polymorphic acute events. this most frequent genetic illness in the world is a major public health concern in french overseas territories. haute autorité de santé (has) recommendations for the care of scd advocate the development of therapeutic patient education (tpe). in martinique (french west indies), we consider the population of patients with scd in 1500 among which 1000 are followed in the adults sickle cell centre (ascc). one of the actions carried out by the ascc of our hospital is the tpe. the objective is to set up an original (because specific in the scd) tpe method, which enables the patient to live better with his disease on a daily basis, by teaching him and his family to recognize prematurely certain complications. design: we analysed needs from the outcomes of a national french survey has which one participated martinique and retained the following themes: the red blood cell, the genetic transmission, the main symptoms, the role of the water, the medicinal treatments and the questions of everyday life. we chose the innovative educational tools called the ''malles des savoirs ò **'', a set of unusual experiments, accessories and models, which, by using a method of active pedagogy ''omca*: observer, manipuler, comprendre, agir ***'', value the learner by offering to him to manipulate and to experiment by himself. results: in 2016, 24 healthcare professionals (doctors, pharmacists, nurses) and a president of patients with scd association followed one week of formation in the omca* method for the animation of six workshops for 10-15 teenagers and adults. every ''malle des savoirs ò **'' contains the necessary material for the animation and a guide of the organizer, including, for every tackled issue, a generic introduction, a presentation of the themes, the index cards of educational animations proposing the activities and one time of synthesis grouping the approaches concepts. the interactive manipulation allows the appropriation of the discoveries become then long-lasting experiences. a final evaluation allows to spot the problems met by learners to understand, to analyse the difficulties and to proceed to the useful adaptations during the next activity. conclusion: this tool, playful and perfectly adapted to the scd, engages, accompanies and helps patients in the construction of their own knowledges to return them actors of their disease. in 2017, we shall estimate the impact of the development of this specific tpe programme of the patient with scd. please specify your abstract type: research abstract background and objective: to investigate the frequencies and clinical relevance of unintentional medication discrepancies, between preadmission medication lists and discharge medication lists, at discharge from hospital. a discrepancy is considered unintentional if there is no documentation explaining the intent of the medication change or if it is unintentional according to the prescribing physician. setting and method: systematic literature review. main outcome measures: frequency of unintentional medication discrepancies per patient and per medication; frequency of clinically relevant medication discrepancies. results: of the patients included 14-88% experienced at least one unintentional medication discrepancy. of the medications used by the patients, 3-50% were involved in unintentional medication discrepancies. of unintentional medication discrepancies found in five studies, 15-58% were clinically relevant. conclusion: the review documented a high frequency of medication discrepancies, of which many were clinically relevant. ensuring sufficient communication of correct and complete medication information in transitions of care is a process which should be better implemented, to enhance patient safety. please specify your abstract type: research abstract background and objective: to investigate the frequency of medication changes not documented in the discharge letter, at discharge from hospital, for both regular, as needed and over-the-counter medications, supplements and herbal remedies (otc). secondary, differences between variables and patients with undocumented medication changes were investigated. setting and method: the patients included were all part of the intervention groups from an intervention study, conducted by one of the authors (tg), from april 2013 to december 2014. the best possible discharge medication list was compared against the medication list in the discharge letter and any discrepancy between the two lists was noted, taking into account the text in the discharge summary. main outcome measures: the proportion of patients affected by at least one undocumented medication change at discharge and proportion of medications with undocumented changes. the proportion of patients was compared using a test according to gender, age, number of preadmission/discharge medications and length of hospital stay. results: two hundred patients were included in the study. the proportion of patients experiencing at least one undocumented medication change for the three subgroups: regular medications; as needed; otc, were 78, 65 and 55% respectively. the proportion of medications involved in undocumented changes for the three subgroups were 34, 71 and 77% respectively. the proportion of patients experiencing undocumented medication changes was significantly higher in patients with more than five regular medications at admission, (p \ 0.001) and at discharge (p \ 0.001). in both regular and as needed medications, the proportion of patients experiencing undocumented medication changes was higher in patients hospitalized longer than 2 days (p \ 0.001 and p: \ 0.05 respectively). for otc, the rate of patients experiencing undocumented medication changes, was higher in females (p: \ 0.05). conclusion: a high proportion of patients are affected by at least one undocumented medication change and many medications are involved in undocumented changes. correct and complete medication information at admission and discharge may resolve many of these errors, ensuring patent safety at transitions of care. hp-ce020: participation in courses at learning and mastery centre and the impact on patients' beliefs about medicines merethe nilsen *,1 , erik oie 2 , kirsten k viktil 1 1 diakonhjemmet hospital pharmacy, 2 department of internal medicine, diakonhjemmet hospital, oslo, norway please specify your abstract type: descriptive abstract (for projects) background and objective: patients with chronic diseases are referred to learning and mastery centre (lmc) where the main objective is to support patients to cope with chronic diseases. education about the disease(s) (by a physician) and the medication treatment (by a clinical pharmacist) are important elements of these courses. little is known about how the participation at lmc influences the patients' beliefs about medicines. design: patients c18 years participating at a 2 days course at lmc regarding acute coronary disease or atrial fibrillation were included in the period september 2014-december 2015. the patients filled out 'beliefs about medicines questionnaire'(bmq) before and immediately after the course, and also 3 months after the course to evaluate their concern (bmq-concern) and necessity (bmq-necessity) of their cardiovascular medications. the bmq scores were dichotomized at scale midpoint (scale 1-5) to evaluate high and low concern and necessity, and these scores were combined to calculate the 'ambivalence'and 'acceptance', 'sceptical', and 'indifferent'rate to medications, and also the mean scores of the bmq were calculated. results: fifty patients were included, mean age 65 years, 14% were women, using a mean of 3.5 cardiovascular drugs taken regularly. fifty-eight percent of the patients had high concern prior to the course, whereas 37 and 60% had high concern immediately after and 3 months after the course, respectively. ninety-nine percent of the patients assessed their medication as highly necessary before the course, 100% immediately after, and 97% 3 months after the course. the mean score for bmq-necessity was 3.82 (sd 0.64) prior to course and 3.88 (0.56) and 3.78 (0.69) immediately after and 3 months after the course, respectively. the corresponding scores for bmq-concern were 2.58 (0.78), 2.39 (0.72), and 2.57 (0.77), respectively. the proportions of patients classified to be 'accepting'were 40, 63, and 43% at the three time points, respectively, and the corresponding numbers for patients classified as 'ambivalent'were 56, 38, and 60%, respectively. conclusion: the lmc course had an immediate positive influence on the patients' concern about their medicines and on 'acceptance'. however, the effect seems not to persist over time. a closer follow-up could be discussed. please specify your abstract type: research abstract background and objective: the narrative-based medicine was intended primarily for health care professionals, and the use of narratives can be applied in any settings to better understand the meaning of own profession, to rediscover/strengthen the motivation to work as a team. the italian society of hospital pharmacist (sifo) promotes a qualitative study aimed at getting the real picture of pharmacist's role within the national health system (nhs), the interaction with other health professionals and patients through the narratives of under specialization pharmacists (ui) and pharmacists already working in the nhs (hp). these data can be further investigated to increase the perceived value/role of the pharmacist. setting and method: sifo hps and uis joining the national pharmacy school specialization network were invited to participate. all pharmacists participating to the study were given a semi-structure interview. the methodology was developed within the conceptual framework of the grounded theory (gt) a research methodology that arises in the context of qualitative research. gt is a systematic methodology involving the construction of theory grounded in data systematically gathered and analysed. main outcome measures: analysis of narratives. narratives were analysed according to the classifications of kleinman, frank and launer and robinson together with transitional analysis (ta). results: a total number of 31 narratives were collected (16 ups and 15 hps). narratives from both group of participants show the need of strengthening the professional identity already in the early years of the pharmacy curriculum and more effectively during the years of specialization as well as the need of being educated to deliver patientcentred care as members of an interdisciplinary team. conclusion: this is the first step of a study that also includes patient's contribution to the definition of pharmacist's professional identity. hp-ce023: impact of pharmaceutical counselling on cancer patients' information desire and treatment satisfaction stephanie wuyts *,1 , jacques de grève 2 , veerle foulon 3 , hilde collier 1 , pieter-jan cortoos 1 1 pharmacy, 2 medical oncology, university hospital brussels, brussels, 3 faculty of pharmaceutical sciences, catholic university of leuven, leuven, belgium please specify your abstract type: research abstract background and objective: appropriately educating onco-/haematological patients is a prerequisite to improve patient empowerment, satisfaction and outcomes. objective: to quantify patients' information need and satisfaction on cancer drug therapy and how this can be improved by clinical pharmacist's counselling. additionally, the pharmacist's impact on therapy quality and costs is assessed. setting and method: setting: prospective, randomised study in the ambulatory (26 beds) and in-hospital onco-/haematology unit (34 beds) in a tertiary hospital. inclusion criteria: adult patients on intravenous or oral cancer therapy, with informed consent. methods: all patients were asked to complete standardised surveys (extent of information desired, eid; patient satisfaction with cancer treatment education, ps-cate and cancer satisfaction of treatment questionnaire, ctsq) on three occasions (at the start of a new therapy, during the second cycle and after 3 months). patients in the intervention group received additional counselling by a clinical pharmacist including medication reconciliation and review. control patients received standard of care (information on drug therapy was provided by the onco-/haematologist, followed by limited administration instructions by nursing staff). main outcome measures: patient information desire and satisfaction on cancer treatment results: 83 patients were included over a period of 6 months (control (n = 43); intervention (n = 40)). no significant differences were found between contact moments or patient groups for eid, ps-cate and ctsq-scores. however, scores for ps-cate on medication side effects were positively correlated with contact moment (r s = 0.198; p = 0.022). multiple linear regression analysis showed a similar trend (b = 0.207; p = 0.101). patients receiving first-line therapy (b = 0.270; p \ 0.001) and ambulatory patients (b = 0.027; p = 0.018) were more satisfied on treatment education. the clinical pharmacist documented more drugs than were recorded in the patient file (8 vs. 4.9 drugs/patient; p \ 0.001). on average, each patient required two pharmacist's interventions per occasion. intervention acceptance rate on drug related problems was high (72%). during the study, interventions shifted from therapy adjustments towards advice on supportive measures (1st contact: 12%; 3rd contact: 41%). improved medication stock control on the ward led to a savings of €36,890. conclusion: the clinical pharmacist can play an important role on the onco-/haematological ward, leading to improved drug reconciliation, patient counselling and cost savings. hospitalised patients and patients receiving salvage therapy appear to have higher educational needs, making them possibly overlooked target groups. finally, pharmaceutical counselling should be repeated and primarily focused on side-effect management to have a meaningful impact on patient satisfaction. please specify your abstract type: research abstract background and objective: europe is ahead of the usa and canada on approval, regulatory and marketing aspects of biosimilars. however, there is still uncertainty about interchangeability and substitution of biosimilars. the aim of the study is to assess pharmacists' perceptions about biosimilar interchangeability. setting and method: a cross-sectional study was carried out in june-july 2016. hospital pharmacists from quebec and france were invited to respond to an online survey of nine questions (surveymonkey ò , palo alto, ca, usa). the survey focuses on pharmacist's exposition to biosimilars (general knowledge, dispensing) and their perceptions about biosimilar interchangeability. a 5-item likert scale was used to answer to 15 statements based on key issues about biosimilar interchangeability. main outcome measures: levels of agreement on biosimilar interchangeability key issues. results: a total of 229 pharmacists responded (62% in quebec vs. 38% in france). the global response rate is: 27% (23% quebec vs. 34% france) (n = 229/880). 64% attended at least to one conference on biosimilars (57 vs. 74%). 36% had already dispensed biosimilars (7 vs. 81%). more than 95% of the pharmacists knew that: biosimilars can cause immunogenicity, clinical studies are requested for their approval, automatic substitution is not permitted. 43% considered that post-marketing surveillance for biosimilars should be reinforced. pharmacists considered that biosimilars are cheaper than the reference product (89 vs. 75%). there was no difference between the level of agreement of french and quebec pharmacists for the 15 statements. pharmacists agree that a list of biosimilar and interchangeable biologic products is necessary (85 vs. 77%), using the international nonproprietary name to prescribe a biological product can create confusion between the reference product and its biosimilar (60 vs. 55%), pharmacists should check if patients already experienced an immunogenic reaction before dispensing a biological product (82 vs. 70%). pharmacists disagree that a biosimilar can be used for all the indications of the reference product (53 vs. 46%). conclusion: perceptions of quebec and french hospital pharmacists about biosimilar interchangeability issues are very similar. this study highlights the need to deal with the lack of clarity of national guidances. clinical studies on biosimilar interchangeability must be conducted in the future to help pharmacists and physicians to take clear-headed decisions. please specify your abstract type: research abstract background and objective: analgesics are essential drugs in hospitals and especially in emergency units. medical and nurse staffs are used to the narcotic status of opioids. for some drugs, a regulatory change to narcotic status can discourage their use. for others, it could limit their access particularly in developing countries; that's why who did not recommend ketamine to be placed under international control (http://www.who.int/medicines/access/controlled-substances/ recommends_against_ick/en/). yet, the french drug agency has recently considered to register drugs containing ketamine as narcotics. the aim of this study was to assess the impact of this possible regulatory change on the pharmaceutical and medical practices in some paediatric french hospitals. setting and method: the survey was conducted in january-february 2016 in four parisian paediatric hospitals: four pharmacies, paediatric neurology and anaesthesia departments, intensive care units and pain management services. main outcome measures: pharmacists, clinicians, health managers and nurses were interviewed, using a standardized questionnaire with closed and opened questions, on the drug circuit including ordering, storage, distribution, prescription, administration and destruction. results: all the 20 health professionals (five pharmacists, ten clinicians, five nurses) indicate that the change to narcotic status would not preclude the use of an analgesic drug. they consider that the pharmaceutical aspects (dispensation, storage and transport, etc.) are not limiting, provided that clinical usefulness is demonstrated: short action onset allowing rapid efficacy, short duration of action allowing the replacement by another drugs if needed, and moderate clinical monitoring. change to narcotic status was rather seen as advantageous since allowing better traceability, use and prescription. half of the pharmacies (n = 2/4) had a computerized register of narcotics and 80% of care units (n = 4/5) had a drug staffing in addition to nominative prescriptions, which was used in all care services. the drugs were kept into secured rooms. none of the emergency units (n = 0/5) had a computerized secured cabinet. conclusion: according to this survey, narcotic status is not a limiting factor for a drug use in paediatric hospitals, when its clinical usefulness is clearly demonstrated. to promote its use, it is important to inform medical and nurse staffs and include it into care protocols. beyond the nominative prescription, implementation staffing is a key step. please specify your abstract type: research abstract background and objective: port-a-cath is an implanted venous access device most commonly used for frequent or continuous chemotherapy administration. however, the procedure and its subsequent maintenance are not free of complications and requires additional intervention by the clinical pharmacist who can provide further patient care to make a positive impact on. to assess the effective provision of appropriate patient counselling offered by a clinical pharmacist on reducing port-a-cath relatedcomplications in cancer patients. setting and method: a controlled prospective observational study carried out on 110 patients newly diagnosed with cancer eligible for chemotherapy administration at the oncology unit. assessment of port-a-cath related-complications were assessed at regular schedule of chemotherapeutic protocols administration. main outcome measures: to assess, reduce and solve port-a-cath related-complications. results: the most significant port-a-cath related complications were skin rash 55.5% (p \ 0.05) with occurrence in males (n = 40) and females (n = 21), skin erythema 5.5% with equal occurrence in both genders, followed by skin discharge 1.8% with also equal occurrence in both genders. a high occurrence of skin rash 88.2% occurred among diabetic cancer patients. a significant improvement in port-a-cath related complications after the provision of patient counselling by the clinical pharmacist was observed as skin rash (3.6%), skin discharge (0.9%), and skin erythema (0.9%). conclusion: results of this study pointed out the essential role of clinical pharmacist in argumenting patient care and improving port-a-cath related-complications in cancer patients. please specify your abstract type: research abstract background and objective: polytherapy, frequently used in the elderly, is associated to an increased risk of potential drug-drug interactions (pddis) and adverse drug reactions (adrs). literature demonstrated that medication reconciliation and medication review performed by hospital pharmacists are correlated to drug related problems (drps). aim: to define a structured and feasible model where hospital pharmacists support clinicians identifying drps and promote the safe use of medicines. setting and method: prospective, feasibility study conducted in four internal medicine wards of a hospital in northern italy. inpatients (c65 years old, treated with c5 drugs) were consecutively included; the recognition/reconciliation process was performed by pharmacists in order to identify changes between prescription profile at home and during the admission (active principles, dose, administration route). these changes were classified as intentional documented discrepancies (id), not documented (ind), not intentional (ni). prescriptions during the first 24-hours of hospitalisation were analysed to retrieve drps (ddis, inappropriate medications for elderly, off-label, over/ under dosage, duplications, adrs) then discussed with clinicians. based on literature, referring almost 1 drp in 70% of patients, a sample size of 50 patients should allow an estimate of drp rate over 50% (need of intervention) with a 80% power and a confidence interval of 95% (software stata version 12.0). main outcome measures: rate and type of: discrepancies, drps at admission and discharge, pharmacists consultations accepted by clinicians. results: ad interim results are presented. between october/2015-february/2016, 30 inpatients (16 male, 85.4 mean age) were included. overall, patients were admitted with 257 drugs used at home and 240 prescribed during the first 24-hours; pharmacists retrieved 99 discrepancies (45%id, 52%ind, 3%ni) and 118 drps, of which 57% ddis, 1% off-label, 3% overdoses, 3% duplications, 30% inappropriate drugs, 6% not notified adrs. the 70% of drps was known to clinicians and 52% considered clinically relevant for the patients. please specify your abstract type: research abstract background and objective: hypertension is a major risk factor for cardiovascular morbidity and mortality worldwide, for which management is based on two principal, complementary approacheslifestyle modification and lifelong treatment with antihypertensive medication. adherence to hypertension therapy is a major public health challenge, despite the availability of multiple classes of antihypertensive agents. factors contributing to non-adherence are multifactorial and include intolerances to drugs at standard doses that result in therapy discontinuation. medication intolerance (mi-htn) refers to patients who experience adverse drug reactions (adrs) to at least one antihypertensive medication, without a known immunological mechanism and the need to discontinue them. we sought to determine factors associated with mi-htn and to identify patients' beliefs and concerns about their antihypertensive treatment and medication in general. setting and method: a cross sectional survey consisting of selfreported questionnaires including beliefs about medicines questionnaire (bmq), perceived sensitivity to medication (psm) and quality of life was undertaken in an unselected patients attending a hypertension centre of excellence out-patient clinic based in london. main outcome measures: to determine factors associated with mi-htn and the impact of health beliefs and self-reported perceived sensitivity to medications on mi-htn and bp control. chi squared tests for comparisons between cases/controls and multiple logistic regression analysis were used for statistical analysis. results: 102 participants were included, of which 46 (45%) participants had mi-htn. two-thirds were female (p = 0.002) with a mean age of 70 ± 10 years (p 0.001), of whom 67.4% had uncontrolled hypertension (p = 0.063). calcium channel blockers were the most commonly reported intolerance by drug class followed by diuretics. being female and age [60 were statistically associated with a greater likelihood of reporting medicines intolerance (p \ 0.05). patients who believed that medicines are harmful were [5-times more likely to report mi-htn (p = 0.009) and 4-times more likely to have uncontrolled bp ([140/90 mmhg) (p = 0.024). patients with high self-perceived sensitivity to medication was 4-times more prone to mi-htn (p = 0.007). conclusion: our findings suggests the need for greater focus on behavioural change interventions to both improve patients' perception of the necessity to persist with lifelong antihypertensive medication and allay concerns regarding harmful effects of drugs may help with long term control of hypertension. please specify your abstract type: research abstract background and objective: today, the number of medical problems in heart transplant recipients has increased due to aging and complications common to immunosuppressive drugs. the co-existence or emergence of other disease states such as renal dysfunction, infection, diabetes, obesity, hypertension, hyperlipidaemia, malignancies, and osteoporosis necessitates the use of other medications. the use of these medications in combination with immunosuppressive agents increases the risk of drug-drug interactions. the aim of this study is to identify the frequency and significance of drug-drug interactions for the patients who received cardiac transplantation. setting and method: this retrospective study was conducted at a cardiovascular specialty hospital. all patients who received cardiac transplantation from the same surgery team between 2009 and 2014 (6 years) were included in the study. all data were collected from the medical records of the patients. only the most recent prescription before discharge was analysed for the presence and significance of drug-drug interactions. drug-drug interactions were checked using micromedex(r) interaction checker. main outcome measures: main outcome measures were the frequency and significance of drug-drug interactions. results: a total of 58 patients met the inclusion criteria and 58 prescriptions were analysed. each prescription contained an average of 11 drugs. a total of 529 drug-drug interactions were identified: 51.8% was classified as moderate; 43.4% as major and 3.7% as contraindicated. almost half of all interactions (n = 271) included immunosuppressive agents (59.4% was classified as moderate; 35.4% as major and 4.8% as contraindicated). conclusion: cardiac transplant recipients were found to have a high number of drug-drug interactions. in order to advise on these interactions which increase with poly-pharmacy, drugs with narrow therapeutic index or drugs that require intensive monitoring, it is recommended to include a transplantation pharmacist in the transplantation team. please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of patient education provided by the pharmacist on gylcemic control, medication knowledge level and medication adherence of patients with type 2 diabetes. patients who were diagnosed with type 2 diabetes for at least one-year time and were receiving at least one antidiabetic medication, attending to the outpatient diabetes clinic for the control visit were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. setting and method: the setting is a diabetes outpatient clinic of a state hospital. the medication knowledge levels, medication adherence scores, fasting blood glucose levels, hba1c levels and blood pressure of the patients were measured before pharmacist's education. after provision of standard information and individualized patient education all these parameters were measured again after 3 monthstime and the impact of the education was assessed. main outcome measures: main outcome measures are change in the clinical parameters (hba1c; fasting blood glucose; blood pressure), as well as improvements in medication knowledge and adherence levels. results: the study was conducted on 54 patients who met the inclusion criteria; none of the patients were lost to follow-up. majority (83%) of the patients was female and the mean age was 53.7 years. pharmacist intervention resulted in positive outcomes at all clinical parameters. systolic blood pressure decreased by 6 mmhg, while diastolic blood pressure decreased by 1.76 mmhg (p \ 0.05). hba1c level decreased by 0.39% (from 6.94 to 6.55%; p \ 0.05) and fasting blood glucose level by 7.1 mg/dl (p [ 0.05). on the other hand, the number of patients reaching the blood pressure goal increased from 36 to 46; and those reaching to hba1c goal increased from 23 to 32 (p \ 0.05 for all). similarly, the medication knowledge level [usual range 0-8] increased from 4.43 to 5.82 (p \ 0.001); and the medication adherence score [usual range 0-4] increased from 3.4 to 3.09 (p \ 0.001). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control and management of co-morbid conditions of type 2 diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of chronic diseases. hp-pc043: impact of a pharmaceutical care program on glycemic control, medication knowledge and medication adherence levels of type 2 diabetic patients residing at a nursing home nimet saglam *,1 , sule apikoglu-rabus 1 , betul okuyan 1 , fikret v. izzettin 1 , nuran yildirim 2 1 clinical pharmacy department, marmara university faculty of pharmacy, 2 darulaceze nursing home, istanbul, turkey please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of pharmaceutical care provided by the pharmacist on glycaemic control, medication knowledge level and medication adherence of patients with type 2 diabetes residing at a nursing home. setting and method: this prospective cohort study was conducted in a state nursing home (darülacaze nursing home) in istanbul, turkey on 39 patients who completed the whole study. all the patients received pharmaceutical care provided by the pharmacist. this pharmaceutical care program was held for 3 months. it consisted of an initial visit, followed by 5 ''care and control'' visits and a final control visit; each visit was held at two-week time intervals. at the initial visit, demographic and general clinical data were collected and medication knowledge and medication adherence levels of the patients were also assessed. pharmaceutical care needs were identified for each patient and recommendations addressing these issues were structured. education regarding the medications of the patients was provided in both verbal and written forms using the standard patient education leaflets prepared by the pharmacist. at each visit pharmaceutical care needs are assessed and pharmaceutical care is tailored accordingly. main outcome measures: main outcome measures are change in the clinical parameters (hba1c; fasting blood glucose), as well as improvements in medication knowledge and adherence levels. results: majority (74%) of the patients was male and the mean age was 68.8 years. pharmacist intervention resulted in positive outcomes regarding hba1c levels. hba1c level decreased by 0.35% (from 7.02 to 6.67%; p \ 0.05) and fasting blood glucose level by 11 mg/dl (p [ 0.05). similarly, the medication knowledge level [usual range 0-8] increased from 2.67 to 5.44 (p \ 0.001); and the medication adherence score [usual range 0-4] increased from 2.9 to 3.28 (p \ 0.01). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control of type 2 diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of type 2 diabetes at the nursing home setting. please specify your abstract type: research abstract background and objective: haemoglobin variability is related to mortality and morbidity in haemodialysis, renal transplantation and pre-dialysis patients. some demographic, haematological and pharmacological variables may affect hb variability. but there are some controversies about the influences of different erythropoiesis stimulating agents (esa).the objective of this study is to determine the influence of different esa on haemoglobin variability in pre-dialysis patients. setting and method: we conducted a prospective observational study with chronic kidney disease patients recruited from outpatients of nephrology department of a tertiary university hospital (from january 2011 to june 2012). exclusion criteria were: stage i and ii, not treated with esa, haemodialysis, peritoneal dialysis, renal transplantation, thalassemia, and deficit of glucose-6-phosphate dehydrogenase . main outcome measures: patients included were treated with esa in maintenance phase (stable 6 months prior).hb variability was calculated by standard deviation (sd) and residual standard deviation (residual sd) of hb levels. statistical analysis was performed with spss 15.0 (spss inc, chicago). observation period was 18 months and data were recorded from the clinical records. (2) 5.7%, sofosbuvir/daclatasvir/ribavirin (2) 5.7%, sofosbuvir/simeprevir (4) 11.4%, sofosbuvir/ledipasvir (6) 17.1%, sofosbuvir/ledipasvir/ribavirin (3) 8.5%, dasabuvir/ombitasvir/paritaprevir/ritonavir (2) 5.7%, dasabuvir/ombitasvir/pari taprevir/ritonavir/ribavirin (14) 40% ombitasvir/paritaprevir/ritonavir/ ribavirin (1) 2.8%, sofosbuvir/ribavirin (1) 2.8%. viral load at week 4 was \15 iu/ml in 32 patients and at the end of treatment 33. conclusion: the results of rapid viral response at end of treatment were similar to those obtained in studies published to date. due to its recent access to these treatments it is necessary to continue monitoring these patients to assess virologic sustained response at 24 weeks after end of treatment. please specify your abstract type: research abstract background and objective: fragile patients are considered those vulnerable patients with a certain degree of complexity in their care (polypharmacy, multi-pathological, palliative and/or residents in social and healthcare institutions). to ensure their continuity of care and safety in the use of drugs we applied a medication reconciliation process at admission, transition of care and/or hospital discharge. objective: to analyse the results of the medication reconciliation process of a fragile patient. setting and method: we developed a list of current medication with the following sources of information: medical history, clinical databases and information provided by the patient (interview). clinical case: 95-year-old woman admitted through emergency department due to severe dyspnoea. no known drug allergy. background: heart failure, chronic hypertension, hypercholesterolemia, hyperthyroidism, hyperuricemia, gouty arthritis, chronic kidney disease and cognitive impairment by alzheimer disease. exploration and complementary tests: echocardiogram and analytical control. clinical judgment: acute decompensated heart failure. acute myocardial infarction. prerenal acute kidney injury. main outcome measures: medication reconciliation made at admission with the detection of discrepancies and deprescribing criteria at hospital discharge. results: fragile patient (high-risk) with 13 medicines as home treatment. patient was hemodynamically stable during the hospital stay. 9 discrepancies were detected between the prescribed medication and the home treatment. discrepancies justified (7): five by omission of medication (two new clinical situation, two therapeutic exchanges to adapt to the pharmacotherapy guide and one wrong drug) and two beginning of medication. discrepancies unjustified (2): by omission of medication. to discharge: one antiplatelet therapy was. after the comprehensive review, we made the following recommendations of deprescription: suspend one non-steroidal anti-inflammatory drug-nsaid (by risk of bleeding in association with concomitant antiplatelet and antidepressant therapy) and one benzodiazepine (central nervous system-cns side effects); modify treatment: reduce doses of diuretics (blood pressure lowering effect). pharmacotherapeutic recommendations were accepted. conclusion: detection of discrepancies in the medication reconciliation and deprescription process are effective and safe strategies that allow optimization of pharmacotherapy in fragile patients. the use of drugs such as nsaids (gastrolesive effect), the combination of drugs with cns side effects and hypotensive action (associated with falls) in elderly patients constitute situations of risk that should be reviewed in fragile patients, as an essential part of the clinical evaluation. please specify your abstract type: descriptive abstract (for projects) background and objective: there are no positions for clinical pharmacists at the hospital, so we are dependent on projects to be able to show how pharmacists can contribute in the clinical team. our aim in this project was to introduce pharmaceutical knowledge by implementing medication reconciliation and medication review in different hospital wards. we wanted to show that many patients have discrepancies in their medication lists during hospital stay and that some of the drugs or doses given can cause drug related problems for the patient. our final goal was to get the physicians to be more aware of these issues when treating their patients. design: the method used was based on the two first parts of the integrated medicines management. the pharmacist conducted a standardized drug interview with patients who prior to admission were responsible for their own drugs. for patients who could not be interviewed or were not responsible for administering their own drugs, a current medication list from relevant care level was obtained. the medication lists obtained were compared to the documentation in the patient's drug chart and discrepancies communicated to the physician. during the hospital stay, a medication review and monitoring was also conducted by the pharmacist. results were presented to the patients physician and discussed. results: a total of 129 patients were included and of these 63% had c1 discrepancy identified by the process. the most frequent type of discrepancy was the use of a drug that was not registered on admission (omission discrepancies). other discrepancies were wrong dose, dosage or formulation and registration of a drug the patient didn't use. drug-related problems were discovered in 61% of the patients and the most frequent were use of anticholinergic drugs in elderly, interactions, lack of treatment and monitoring and too high doses regarding kidney function. many of the detected drug related problems results in change in medication, other times the physician addresses the problem to the gp. the physicians were surprised of the high numbers of discrepancies in medication lists and drug related problems discovered. almost all the physicians considered that the pharmacist could be an important part of the treatment team and they wanted the participation of the pharmacist to be permanent. conclusion: the project led to increased awareness of the importance of medication reconciliation and medication review and showed the importance of pharmaceutical knowledge in the treatment team. unfortunately this was not sufficient to create positions for pharmacists in our hospital. new projects will focus on pharmacists teaching interns to improve the reconciliation at admission. please specify your abstract type: descriptive abstract (for projects) background and objective: we aimed to assess the quality of fluoroquinolones (fq) prescriptions at the toulouse university hospital emergency department as part of significant increase in consumption. design: retrospective mono-centric study of fq prescriptions written to adult patients managed at the emergency department (february 29th, 2016 -march 6th, 2016 . a pair consisting of a biologist pharmacist and a clinical pharmacist has analysed them using tools provided by the centre de coordination de lutte contre les infections nosocomiales (cclin). various criteria (pertinence of prescription, choice of antibiotic, dosage, duration of treatment, method of administration…) were faced with the guidelines issued by the société de pathologie infectieuse de langue française (spilf). results: about 1229 files were examined, 17 contained fq prescriptions for systemic use. the most frequently prescribed antibiotic was ofloxacin (59%) and the most frequent indications were urinary tract infections (47%). among the 17 prescriptions of fq, the establishment of fq was justified in 71% of cases and the antibiotic chosen was always the most suitable. nonconformities of dosage and/ or treatment time were found in a quarter of cases. overall, 47% did comply with guidelines. the prescriptions, due to the particularity of emergency were still permormed probabilistic. however, a reassessment of them was scheduled for two-third of outpatients. conclusion: this study highlights the conformity of less than half of the prescriptions. this demonstrates that there are still actions to ensure the accuracy of fq prescriptions. and it is in this sense that this audit should be registered under the impetus of the committee on anti-infectives. it will raise awareness among doctors in the proper use of this family of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the number of persons suffering from end-stage renal disease (esrd) is growing worldwide, mainly due to the aging of the population. esrd incidence has been increasing by 3-7% per year for 10 years. it is estimated that worldwide, more than 1.5 million patients with established renal failure are being treated with haemodialysis (hd). water for haemodialysis must meet the physicochemical and bacteriological compliance standards defined by the european pharmacopoeia. as a medicine, this water is placed under the responsibility of hospital pharmacists. addressed to hospital pharmacists, this methodology guide will enable them not only to validate controls of haemodialysis water as well as drug prescriptions for dialysis patients, but also to familiarize themselves with the best currently existing dialysis techniques and medical devices. we have tried to simplify and synthesize existing circulars and guidelines so as to render them more readily understandable for the pharmacist in charge of a haemodialysis service, and thereby help to ensure optimally safe treatment of haemodialysis patients. design: the themes developed in this guide are: • a review of the different existing dialysis techniques, • a review of the different sampling points for controls of hd water, • a review of the physicochemical and bacteriological standards of these controls according to the latest recommendations of the european pharmacopeia, and of appropriate conduct for exceeding established thresholds, • a review of the main international recommendations with regard to clinical signs of chronic kidney disease: anaemia, mineral and bone disorders (ckd-mbd), high blood pressure. • a review of the various medical devices used in haemodialysis and haemodiafiltration. results: the recommendations of good practices summarized in this guide are integrated perfectly adapted to the concept of quality assurance and its role in the accreditation process. they are focused on improving patient safety by harmonizing pharmaceutical haemodialysis practices in different dialysis centres. conclusion: these types of recommendations may be transposable to other pharmaceutical fields and/or be used as a training tool for pharmacy students or young pharmacy school graduates. the format of this guide makes it convenient, easy to use every day. it will be revised regularly to ensure the sustainability of quality plans. please specify your abstract type: descriptive abstract (for projects) background and objective: combination antiretroviral therapy (cart) has strongly improved disease control in hiv-infected patients. however, aging and comorbidities are becoming a major problem in this group of patients. most hiv-infected patients are treated with five or more medications, and harms by polypharmacy increase proportionally with number of medications. possible risks include: poor medication adherence and consequently inefficient care, increased risk of drug interactions and adverse events, with prolonged hospitalization. the problem is worsened when patients are of nonnative language and so their comprehension and adherence to drug therapy can be very poor, compromising efficacy. the hivig study is designed to evaluate the impact of the interventions promoted by the clinical pharmacist in the optimization and comprehension to personal drug therapy, favouring compliance, in a cohort of patients, hiv infected with comorbidities like cancer; the cohort includes a high number of non-native italian language individuals. design: hivig is a randomised, parallel groups clinical trial. in april 2016 the study protocol was approved by the local ethical committee, aviano. the project is scheduled to start in autumn 2016. main objective: evaluation of the impact of a series of tools-''drug therapy setting interventions'' (dtsis) applied by the clinical pharmacist on a cohort pf hiv-infected patients with comorbidities, afferent for care at cro aviano. the treatment arm will be submitted to dtsis. dtsis interventions (treatment group) consist in: motivational interview, sharing and delivery of printed, explanatory material in the patient's native language, reconciliation of patients medications at hospital admission and at discharge; identification of potential risks due to drug-drug interactions; monitoring of compliance to drug therapy, and finally detection of adverse drug reactions (adrs) occurring in the course of care. the control group will undergo only to scheduled standard medical visits at cro. results: we expect to recruit a total 350 patients for a 24-months period of follow-up. statistical analysis will be performed by intention-to-treat and by protocol. at cro aviano, the italian cooperative group on aids and tumors (gicat) has studied malignancies in hiv-positive patients since 1986 and has a leading role for studies conducted in italy (vaccher, 2014) conclusion: previous collected data from the previous trial performed at cro aviano (target-vig), showed a positive impact in the optimization of individual drug therapy and in the reporting of adrs. hiv has an enormous impact on life of infected patients and represents a priority issue for the entire community. we consider the method of dtsi, combined with a close monitoring of patients by means of telephonic motivational interviews, the best added value performed by the profile of the clinical pharmacist in optimizing drug therapy and personal awareness about medicines. please specify your abstract type: research abstract background and objective: the world health organization reports that ''one in four people in the world will be affected by mental or neurological disorders at some point in their lives. around 450 million people currently suffer from such conditions, placing mental disorders among the leading causes of ill-health and disability worldwide». to review the evidences published about the roles and the impact of pharmacists in psychiatry. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, mental illness and psychiatry from january 1st 1990 until june 14th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in psychiatry. results: a total of 62 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (7), medication reconciliation (26), patient care needs assessment (25), drug therapy assessment (74), patient follow-up (61), interdisciplinary work (26), knowledge transfer (104), competencies maintenance (2). the impact of pharmacists interventions was studied using a total of 369 indicators from which 146 (40%) had outcome measures. of these 146 outcome indicators, 68 (47%) were positive, 77 neutral and 1 negative (knowledge transfer strategy). positive impacts of pharmaceutical interventions were identified in the following areas: morbidity (24), patient adherence (11), patients or clinicians satisfaction (7), side effects management (2), medication errors prevention (2), mortality (2) please specify your abstract type: research abstract background and objective: the world health organization reports that 8.2 million people die each year from cancer, an estimated 13% of all deaths worldwide and that there is a 70% increase in new cases of cancer expected over the next two decades. to review the evidences published about the roles and the impact of pharmacists in cancer. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, neoplasms from january 1st 1990 until june 20th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions as well as descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in cancer. results: a total of 42 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (6), patient care needs assessment (76), drug therapy assessment (84), drug compounding/dispensing (1), patient follow-up (93), interdisciplinary work (15), knowledge transfer (39). the impact of pharmacists interventions was studied using a total of 274 indicators from which 112 (41%) had outcome measures. of these 112 outcomes indicators, 98 (88%) were positive, 13 (12%) neutral and 1 (1%) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity (59), patient adherence (2), patients or clinicians' satisfaction (3), side effects management (8), medication errors prevention (7), mortality (0), costs (2) setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, myocardial infarction, acute coronary syndrome from january 1st 1990 until june 14th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in myocardial infarction. results: a total of 35 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (11), medication reconciliation (39), patient care needs assessment (2), drug therapy assessment (102), drug compounding/dispensing (8), patient follow-up (51), interdisciplinary work (60), knowledge transfer (52). the impact of pharmacists interventions was studied using a total of 177 indicators from which 107 (61%) had outcome int j clin pharm (2017) 39:208-341 269 measures. of these 107 outcome indicators, 49 (46%) were positive, 56 (52%) neutral and 2 (2%) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity (8), patient adherence (10), side effects management (1), mortality (5) and others (21). conclusion: the role and the impact of pharmacists have been studied in myocardial infarction and 46% of outcome indicators used in these studies show a positive impact of pharmaceutical interventions. pharmacists should pay attention to these evidences to improve their practice, contribute to prevention or insure treatment of patients with potential or found myocardial infarction. hp-pc055: impact of pharmaceutical care in vaccination: a review of literature please specify your abstract type: research abstract background and objective: the world health organization reports that ''immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine and a proven tool for controlling and eliminating lifethreatening infectious diseases and is estimated to avert between 2 and 3 million deaths each year. it is one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations». to review the evidences published about the roles and the impact of pharmacists in vaccination. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, vaccination and immunization from january 1st 1990 until july 5th 2016. manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in vaccination. results: a total of 43 articles were included. described pharmaceutical interventions included patient-pharmacist relationship (37), medication reconciliation (19), patient care needs assessment (37), drug therapy assessment (26), patient follow-up (39), interdisciplinary work (11), knowledge transfer (47), competencies maintenance (1). the impact of pharmacists interventions was studied using a total of 212 indicators from which 81 (38%) had outcome measures. of these 81 outcome indicators, 65 (80%) were positive, 15 (19%) neutral and 1 negative. positive impacts of pharmaceutical interventions were identified in the following areas: cost (3), errors (2), morbidity (21), patient adherence (14), patients or clinicians satisfaction (3) please specify your abstract type: descriptive abstract (for projects) background and objective: evaluating the appropriateness and effectiveness of the patient's medications by analysing prescriptions is pharmacist side work. bedside drug administration and computerised drug administration traceability (cdat) in nursing care plan (ncp) are nurse's one. however, in order to check adherence, efficiency and tolerance of a drug, pharmacist has to ensure that the patient takes the medication appropriately. therefor ncp could be a useful tool. the aim of this study is to evaluate the effectiveness of cdat, and if not, define causes of divergences with real life situation. design: • comparison between unused drugs remained in individual patients' seven daily pill dispensers (considered as not taken) which come back from the evaluated service to the pharmacy, and their cdat status completed by nurses (taken, not taken or no status) • two recorded data, each collecting a three-week period, separated by a period of discussion with nurses: first results presentation, analysis of divergences by taking into account their feedbacks, and actions to raise their awareness about the importance of cdat. • pill dispensers' cdat is correct only if all returned drugs' status in ncp is ''not taken''. results: during the first period (n = 112 pill dispensers), 29.5% of pill dispensers had an incorrect cdat status. on average, 1.4 drug per pill dispenser didn't have an appropriate status in ncp (taken or no status). major causes of divergences were the lack of time and insufficient human resources, the fact that they often are interrupted in the middle of this task, a software which isn't ''user-friendly'' and a deficit of information about the issue. corrective actions were implemented, prior to the second recorded data period, targeting human factor of divergences (oral and written reminders about cdat with didactic memorandum on computers). after awareness actions, results (n = 110) were 23.6 and 1.5 respectively. conclusion: efforts about cdat have been done but not enough to observe a significantly improvement in short terms. ncp's level of reliability is not optimal yet and still dependent on nurses' practices. this study allowed us to strengthen the relationship between clinical service and pharmacy, and opens the way for further works particularly through corrective actions targeting material and organizational causes of divergences. please specify your abstract type: descriptive abstract (for projects) background and objective: in 2015, our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. because broad spectrum antibiotics have to be followed with the attention of resistance prevention, we focused our analysis on these antibiotics in our hospital (carbapenems, piperacillin/tazobactam and amoxicillin/clavulanic acid). design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j01, j02, j04a, p01ab, a07, j05ah, p01b from the atc classification) prescribed at 8 a.m. on the day of the survey, were involved. from those who were treated by carbapenems, pip/taz or amx ac, following data were collected: age, gender, weight, doses, indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. results: the survey was carried out from april to june 2015 in 51 wards. among the 1435 patients included, 369 patients (25.7%) were treated with antimicrobial agents.114 patients (30.9%) were treated with broad spectrum antibiotics: 53 (14.1%) with amx-ac, 47 with pip-tz, 6 with imipenem and 8 with meropenem. the mean age of patients was 60.5 ± 17.7 and the weight was 67.9 ± 13.9 kg. their prescriptions were concentrated in three types of wards: 24 (21.1%) in icu, 47 (41.2%) in medicine, 43 (37.7%) in surgery. moreover, we observe that bsa were used to treat 33 (28.9%) community acquired infections, 54 (47.4%) nosocomial infections, 6 (5.2%) used as medical prophylaxis, or surgical prophylaxis (n = 19, 16.7%). in relation to the type of treatment: 66 were empirical treatment (including 25 prophylaxes) and 48 were targeted treatments (3 bacteraemia, 5 joint and bones infections, 1 cardiovascular system infection, 12 urinary tract infections, 10 lower respiratory tract infections, 10 skin and soft tissues infections and 7 others infections). finally, 23 extended spectrum beta-lactamase (esbl) producing enterobacteriaceae and 1 third generation cephalosporin resistant enterobacteriaceae non-esbl producing were targeted by bsa regimen. conclusion: in this survey, use of bsa is globally compliant to french guidelines and we identified no improper prescription: multidrug resistant bacteria infections, several diseases and empirical treatments with limited duration of regimen. this shows that control of the proper use of antibiotics especially those with a broad spectrum is efficient in our hospital and has to be continued. this has been made possible due to a multidisciplinary approach including physicians, bacteriologists and pharmacists. please specify your abstract type: descriptive abstract (for projects) background and objective: in 2015, our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. from these results, we observed that sulfamethoxazole/trimethoprim (tmp/smx) was largely prescribed in our hospital. we focused then our analysis on these results with the attention of check of its proper use. design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j01, j02, j04a, p01ab, a07, j05ah, p01b from the atc classification) prescribed at 8 a.m. on the day of the survey, were involved. from those who were treated by tmp/smx, following data were collected: age, gender, weight, doses, and indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. please specify your abstract type: research abstract background and objective: in france, benzodiazepine (bzd) is frequently prescribed in elderly people (ep). long-term efficacy is often questioned, and treatment has to be regularly re-examined, especially in ep. in our geriatric day-hospital for assessment of frailty, a multidisciplinary team evaluates the patients and gives them preventative measures against the loss of autonomy. medication evaluation is part of these measures. the aim of our study was to evaluate the impact of a standardized intervention on the optimization of bzd treatment. setting and method: after a short interview and the delivery of an information booklet about bzd, patients were proposed an optimization of their bzd treatment (dosage reduction, occasional medication, switch to a short half-life bzd, or total discontinuation). patients were followed up monthly by a phone-interview over a 6-months period. main outcome measures: the main outcome measure was the prevalence of bzd optimized treatments after a 6 months follow-up. results: 18 patients were included. among them, 50% have been taking a bzd for more than 10 years, and 29% were prescribed a long half-life bzd, which can be qualified as inappropriate in ep. 50% of the subjects were frail and 44% pre-frail according to the fried criteria. at the end of the study, 33% of the patients had their bzd treatments optimized, including 17% of total discontinuation. conclusion: in frail or pre-frail elderly population, a standardized intervention can be useful to improve bzd treatment. an extension to this intervention would be the creation of an organisation tasked with routinely monitoring the patients withdrawal over a 6 month period. hba1c and weight were significantly reduced by 1.36 ± 1.79%, p \ 0.000 and 3.21 ± 3.52 kg, p \ 0.000, respectively; systolic bp (12.91 ± 10.57 mmhg, p \ 0.000), diastolic bp (6.39 ± 7.34 mmhg p \ 0.000) and triglycerides (45.58 ± 115.65 mg/dl, p .011).genital-and urinary tract infections were reported by 6.7% patients. any diabetic ketoacidosis case was reported. conclusion: sglt-2 inhibitors added to other oral antidiabetic drugs or insulin in patients with uncontrolled t2dm significantly improved glycaemic control, reduced weight, blood pressure and triglycerides, and was generally well tolerated. in conclusion, sglt-2 inhibitors, appears to be an important addition to the therapeutic options for the management of type 2 diabetes, particularly when used as add-on therapy. (1). treatment safety takes part of the decision to undergo bariatric surgery. during multidisciplinary team meetings, the clinical pharmacist must rely on guidelines to limit drug-induced iatrogenesis. this review aims at assessing influence of bariatric surgery on the clinical impact and pk of cardiotropic drugs so as to document pharmacists' notifications. setting and method: literature review on medline-1946 to may 2016-with terms: cardiovascular drugs and bariatric surgery or malabsorption syndrome. related articles were reviewed. main outcome measures: pharmacokinetic or pharmacodynamic data and clinical impact of cardiotropic drugs. results: a total of 924 titles, and abstracts when necessary, were screened for eligibility. after reviewing process, 15 studies were included: nine concerning digoxin, five beta-blockers (bb) and one amiodarone. published studies varied in methodology: five case report, seven case control and three cohort studies. studies reported variations of digoxin plasmatic concentrations among 22 patients versus 66, suggesting liquid oral form are preferred. no clinical event was notified. more the bb is liposoluble (propranolol), the higher the toxicity is, such as heart rate and blood pressure decreasing, with potential fatal outcomes. a case of amiodarone-induced hyperthyroidism is described after bariatric procedure showing an increase plasma concentration adjusted to weight. conclusion: while the impact on narrow therapeutic range drugs is documented, others cardiotropic drugs may cause serious patient injury justifying their monitoring. therefore, risk must be identified for all patients undergoing bariatric surgery to setting up closely therapeutic monitoring. further studies are still expected to lead to recommendations about posology and treatment withdrawal to improve patient safety. please specify your abstract type: research abstract background and objective: the issue of non-compliance to prescribed medical treatment has been reported to be a crucial problem in psychiatric outpatients. the aims of this study were to assess the extent of non-compliance in a cohort of psychiatric outpatients in malta and to investigate the applicability of using a 7-day multi-dose pill box in terms of practicality, ease of use and impact on compliance within this patient group. setting and method: the study was conducted at mount carmel hospital, a psychiatric hospital in malta. twenty outpatients were recruited by convenience sampling. the study was divided into two phases. during phase 1, patient compliance was assessed using the medication adherence rating scale (mars) survey and patients were administered part a of a questionnaire entitled 'assessment of the 7-day multi dose pill box'. this questionnaire evaluated the patients' opinion regarding the 7-day multi dose pill box before and after its use. in phase 2, the chosen patients were given a demonstration on how to use the 7-day multi dose pill box and the device was given to them to use at home for one week. after one week, part b of the questionnaire was completed and compliance was re-assessed using mars . main outcome measures: evaluation of adherence before and after use of the compliance aid device. results: of the 20 patients recruited, 9 were male and 11 were female. the mean age was 46 years (range 33-70) and the mean number of daily medications 6 (range 3-16). upon initial scoring using mars, 15 patients were adherent and 5 patients were nonadherent. a higher adherence was observed in patients taking 5 or more medications daily. ten patients accepted to move on to phase 2 of the study and took the device home to use for one week. out of these 10 patients, 4 felt that the way they take their medication improved following use of the device and 7 out of 10 patients would consider buying the device since they found it practical and easy to use. statistical analysis of mars score before and after use of the device showed no significant improvement in compliance (p [ 0.05). there was no significant association between level of adherence and type of psychiatric condition (p [ 0.05). furthermore, results did not indicate increased adherence in patients who have a carer in-charge of their medication administration or in patients using a compliance aid device (p [ 0.05). conclusion: the use of a compliance aid device in psychiatric patients is challenging due to difficulty in establishing patient communication and motivation. the pharmacist is in a position to identify patients who would benefit from the compliance aid device. adrien borowik * , anne fratta, fabien hernandez pharmacy, ap-hp, armand trousseau paediatric hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: enoxaparin, a low molecular weight heparin, is the most prescribed anticoagulation treatment in paediatric indications. however, the marketing authorization mentions that due to the lack of data, the use of enoxaparin is not recommended to children nor anyone weighing less than 40 kg. thus, expert recommendations described specific dosage for the paediatric use: we aimed to compare these with our hospital practices. (3), sofosbuvir/ledipasvir (20), ombitasvir/ paritaprevir/ritonavir (4), ombitasvir/paritaprevir/ritonavir + dasabuvir (12), simeprevir + ifn (1) . twenty-six patients (44%) were treated for 24 weeks (12 week pay-back policy). twenty pharmacists' interventions were carried out with an acceptance rate of 80%. the interventions included treatment adjustments due to drug interactions (4), inappropriate treatment according to genotype (2), duration of treatment (5) and switch to a more cost-effective therapy (9). seven pharmacists' interventions concerning treatment switch were applied (78%) resulting in a cost saving of €102,102.7. all assessable patients (28) have a negative serum hcv rna 12 weeks after the end of treatment (svr = 100%) while 1 patient died during follow-up (due to the disease). conclusion: the hospital pharmacist, as an active member of the multidisciplinary team, has an essential role in guaranteeing optimal care for hcv patients at the best cost. monitoring has also shown to be fundamental to evaluate the real world effectiveness of these drugs approved with surrogate endpoints. hp-pc068: are hospital pharmaceutical staff educated on the criticality of thermosensitive drugs? camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: in the use of thermosensitive drugs, the safety of patient care involves compliance with allowed temperatures. having the right information at time of care is essential. the aim of this study is to assess, within a french university hospital, pharmaceutical staff knowledge on the criticality of thermosensitive drugs and to educate them accordingly, including associated patient risks. setting and method: an assessment of knowledge using a questionnaire was led in january 2016 among pharmaceutical staff in a 1500-bed hospital (11 pharmacists, 14 pharmacy residents, 28 pharmacy technicians). evaluation criteria were: storage temperature of refrigerated drugs and frozen drugs, thermosensitive drug retention period after removal from the refrigerator, highest risk situation for a thermosensitive drug (t [ 8°c or t \ 2°c) and action to be taken during a temperature excursion. main outcome measures: to determine shortcomings in the management of thermosensitive drugs in order to adapt appropriate tools. results: 43 completed questionnaires were collected. collected questionnaires included 12% from pharmacists (n = 5), 23% from pharmacy residents (n = 10) and 65% from pharmacy technicians (n = 28). regulatory variations in storage temperatures of refrigerated and frozen drugs are known in respectively 79 and 32% of cases. 3% of pharmaceutical staff are aware of thermosensitive drug retention periods after removal from the refrigerator and 51% of the highest risk situation for a thermosensitive drug (t \ 2°c). the measures to adopt during a temperature excursion are understood in 84% of cases. conclusion: this study highlights the lack of knowledge on the management and criticality of thermosensitive drugs and the lack of information available to pharmaceutical staff. dissemination of data and questionnaire reponses have been beneficial for the pharmacy department and have reduced inequalities in available information among pharmaceutical staff. subsequent to the study, thermosensitive drug management procedures have been revised. the deployment of this questionnaire is continuing via the university hospital intranet in order to train all health professionals in good patient care. please specify your abstract type: research abstract background and objective: temocillin is a beta-lactam antibiotic exclusively active against gram-negative pathogens. its use can avoid that of broad spectrum antibiotics, such as carbapenems, for the treatment of infections due to extended-spectrum beta-lactamase producing enterobacteriaceae. however, the absence of recommendations by learned societies on temocillin use could lead to misuse and the emergence of resistance. the aim of this study is to identify the role of temocillin in a french university hospital arsenal in order to limit ecological risks. setting and method: a retrospective study was conducted in a 1500-bed university hospital. all adult patients having received at least 2 days of treatment between june 2015 and april 2016 were included. data collected for the study were: age, sex, treatment indication (type of infection, identified pathogen, dosage and treatment duration), previous antibiotics and therapeutic outcomes. main outcome measures: the indicators chosen were: treatment indication, prescribed dose and treatment duration. results: two patients were included. in july 2015, temocillin was used in a 47 year old female as first-line treatment of intraperitoneal haematoma infection due to multiresistant klebsiella pneumoniae. prescribed at a dose of 2 g twice daily by an infectious diseases specialist, treatment was continued at the same dose for up 3 weeks with therapeutic success. in august 2015, temocillin was used in a 59 year old male for the treatment of bacteraemia due to multiresistant enterobacter aerogenes. previously treated by imipenem/cilastatin, temocillin was prescribed as second-line treatment at a dose of 2 g twice daily by an infectious diseases specialist. treatment was continued at the same dose for up 6 weeks with therapeutic success. conclusion: the dissemination of antibiotic resistance among gramnegative enterobacteriaceae continues to be an increasing threat for healthcare worldwide. within this context, temocillin could be an interesting alternative. determining the role of temocillin in a therapeutic arsenal is essential. our hospital considers temocillin as a ''critical antibiotic'' although its use is not exclusively limited to the new drug application. therefore, temocillin prescriptions are monitored permanently by infectious diseases specialists, microbiologists and pharmacists in order to improve the good use of this antibiotic and to optimise patient safety. please specify your abstract type: research abstract background and objective: drinkable solutions are more susceptible to deterioration and can lead to a potential risk for patient care. having the right information at time of care is essential. the aim of this study is to assess nursing staff knowledge in a french university hospital on the management of drinkable solutions to elaborate tools to help health professionals and to enhance equality of information in order to optimise patient care. setting and method: an assessment of practice using a questionnaire was conducted in may 2016 among a share of the nursing staff in a int j clin pharm (2017) results: 133 completed questionnaires were collected. 20% of nursing staff replied that the period-after-opening is the same for all of drinkable solutions. this period is estimated at 1 month in 21% of cases, 2 weeks in 10% of cases and 7 days in 8% of cases. 58% of nursing staff do not know how to store drinkable solutions after opening. the date of opening or the date of expiry after opening are specified on the medicine bottle in respectively 77 and 3% of cases. only 11% of nursing staff have tools pertaining to the management of drinkable solutions. these observations led the pharmacy to create and distribute appropriate tools. storage methods for the drinkable solutions available in our hospital were collected directly from pharmaceutical laboratories. this information has been made available to nursing staff via drug control software (pharma ò , computer engineering, paris). conclusion: this study highlights the lack of knowledge on the management of drinkable solutions and the lack of information available to nursing staff. in our hospital, the dissemination of appropriate data reduced inequalities in available information between care units. data will soon be integrated within the drug prescription software (mc kesson usv2 ò , crossway, san francisco) in order to homogeneously train all health professionals in good patient care. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a process which allows prevention of iatrogenic injuries during patient's hospitalisation and transfers. since 2013, a clinical pharmacist has been integrated into the orthopaedic surgery care. he has performed mr at patients' admission. the aim of this study was to evaluate the impact of medication reconciliation performed by a clinical pharmacist. design: a prospective monocentric study was conducted on patients admitted in an orthopaedic surgery care (elective or unplanned surgery), during 3 months. the clinical pharmacist established the best possible medication history (bpmh) from at least three sources of information (including patient interview when possible). then, it was compared to the admission medication order (amo) (from anaesthetists when elective or orthopaedists when unplanned). unintended medication discrepancies (umd) detected were discussed with prescribers in order to be corrected. epidemiological data, number and type of umd, therapeutic classes involved and the percentage of corrected umd were collected and their potential clinical impact was assessed. results: in this study, 325 patients were included during 3 months. elective surgeries were concerned in 72% of the cases. at least one umd was identified in 158 patients (49%) (median age: 77.1 years old; male/female ratio: 0.65). of these, 133 (84%) were older than 65 years old. finally, 406 umd were detected, being 2.6 by patient. main therapeutic classes concerned cardiovascular system (31%), nervous system (20%) and digestive system (18%). of the 406 umd detected by mr, there were 55% of omissions, 27% of inappropriate dosing and 13% of renewal prescriptions stopped by the patient. finally, 87% of umd were corrected. of these 406 umd, 2% were major errors (i.e. causing potential harm), 39% were significant errors (i.e. monitoring or intervention potentially required to preclude harm) and 59% were minor errors (i.e. without potential harm to the patient). conclusion: medication reconciliation process performed by a clinical pharmacist allows detection and correction of umd on half of patients in surgery care particularly on elderly patients. the high proportion of umd can be explained by the multiplicity of actors involved in medication management. health information technology could help to focus mr on patients at high-risk of adverse drug events. please specify your abstract type: research abstract background and objective: darunavir plus ritonavir (drv/r) have shown optimized results in simplification strategies (monotherapy (mt) or dual therapy (dt)) for selected hiv + in randomized clinical trials and real life experience. recent introduction of one pill drv plus cobicistat co-formulation (drv/c) may be particularly suited for both mt/dt allowing once daily administration optimizing dosage and adherence. the objective of our study is to evaluate efficacy and security of drv/c in mt and dt. setting and method: all hiv + adults with antiretroviral change to drv/c in mt/dt at a reference hospital in the northwest of spain were included in this retrospective study. a statistical analysis was performed using the spss v.19.software. main outcome measures: epidemiological, clinical, antiretroviral regimen, serum creatinine, lipids and inmunovirological data (rna-hiv and lymphocytes cd4) were compared previous and after change to drv/c. results: 71 hiv treatment-experienced patients have received drv/c in dt (27) or mt (44). 76.1% were men with a mean age of 48 years. main risk factors were: 45.1% heterosexual, 28.2% msm, 18.3% injection drug users, 2.8% mother-to-child transmission, 1.4% transfusion in haemophiliac patient and 4.2% unknown. cdc category distribution was 64.8% a, 4.2% b, 28.2% c and 2.8% unknown. overall mean nadir cd4 counts were 196.7 ± 115.1cells/mcl. mean time since drv/c prescription to discontinuation or until analysis was 182.5 days [range 61-296]. 86.4% drv/c mt were prescribed to patients with prior drv/r mt in order to simplify treatment and the mean time of the duration of these prior therapies were 3.1 years. in case of dt, 66.7% were prescribed on patients with prior drv/ r + 3tc with a mean duration of 1.3 years. serum creatinine increases (1.03 vs. 1.08; p \ 0.001) and cd4 decrease (714.1 vs. 663.3; p = 0.031) when patients move to drv/c. no significant change in the other analytical parameters and all patients maintained undetectable. 2 patients discontinue drv/c due to intolerance and inability to swallow in each case. conclusion: this preliminary study concludes that drv/c in mt or dt is efficacy (no viral rebound) and safety. although an increase in creatinine was observed, it would not be considered clinically significant. of note, lymphocytes decreased significantly and it will be important closely monitored to check that maintain effectiveness during the follow up. hp-pc073: developing clinical pharmacy in emergency department setting up a medication reconciliation process marion collignon *,1 , antoine gantier 1 , florent lapacherie 1 , hélène dewaele 1 , laura foucault 1 , anne-laure raso 1 , emmanuel cirot 1 , said laribi 2 , xavier pourrat 1 1 pharmacy, 2 emergency department, chru tours, tours, france please specify your abstract type: descriptive abstract (for projects) background and objective: in emergency department (ed), if a drug related problem (drp) happens at the patient admission, the risk is the error remains until discharge. one part of drp may be avoided with using medication reconciliation (mr). the objective of this study was to evaluate the feasibility of setting up a medication history (mh) of patients in ed in an acceptable lap of time before they were transferred in another unit or discharged. design: a 6 months prospective study was conducted in ed in a university hospital in france. two junior pharmacists coached by a senior pharmacist, after a 2 months training for mr, were in charge of the data and mh collection. for all patients, we collected age, mh according to number of sources, discrepancies identified, adherence to treatment (according to the social security questionnaire), type of sources. mh were established according to community pharmacies, patients, previous electronic patient files, prescription sheets, patient's family, packs of pills and to the general practitioner (gp). for patients from long term care facilities (ltcf), the mh was established only by communication with the ltcf. then, the current prescription was compared with the home medication regimen. mh and discrepancies (omitting medication, incorrect dose, ambiguous name) were recorded in the electronic patient files to be available during hospitalization. because ed does not have a pharmaceutical review of prescriptions, only major discrepancies were transmitted to physicians. results: we collected 1426 mh (187 from ltcf), with a sex ratio of 1.0 and a medium age of 74.5 years old. it represented an average of 9.5 mh per day or 19 min per mh. among patients who did not come from ltcf, sources used by pharmacy students were patient's community pharmacy (967, 78% of cases), patient (890, 72%), previous electronic patient file (769, 62%), prescription sheets (634, 51%), call to gp (80, 6.5%), gp mail (78, 6.3%), patient's family (47, 3.8%), packs of pills (29, 2.3%), community nurse (26, 2.1%). finally, 683 patients (48%) had been hospitalized, others were discharged. we analysed mh for 1099 patients: at least one drp occurred for 599 patients (55%). among 386 patients, 150 (39%) had an immediate pharmaceutical intervention because of the risk due to discrepancy. among 203 patients who did not come from ltcf and who could communicate, 162 were good adherent to treatment (80%). conclusion: this study highlights the great interest of the mh by pharmacists at ed, which avoids many drp. the presence of pharmacists in ed contributes to maintain a safe environment for medication and to assist prescribers in the continuity of treatment between home and hospital. spending 20 min by mh, we identify one drp every 11 min. nevertheless, it could be benefit to develop this activity because of the satisfaction of the emergency physicians. currently, mr is the first step to develop clinical pharmacy in the ed. please specify your abstract type: research abstract background and objective: emerging evidence in the literature suggests a high prevalence of suboptimal vitamin d (vitd) and an association between lower serum levels and higher mortality in cancer. the objective of this study was to quantify vitd deficiency in patients after surgery for head and neck cancer, and to determine the effect of one cholecalciferol intramuscular dose. setting and method: intervention study with a follow-up period of 5 months (november 2015-february 2016) performed on patients followed by the nutrition support unit after surgery for head and neck cancer. demographic and physiopatological data, including admission diagnosis, age, gender, calcium, magnesium and phosphate were collected. nutrition screening by conut index was carried out. a single intramuscular dose of 200.000 ui cholecalciferol (vitamine d3 bon ò ) was administered to vitd-deficient patients and serum 25-hidroxy-vitamin d (s25ohd) records after the administration, including primary carés records after discharge, were evaluated (reference range 30-70 ng/ml). main outcome measures: s25ohd (\20 ng/ml: deficiency; 20-31 ng/ml: insufficiency; c32 ng/ml: sufficiency). results: data from 25 patients with a mean (sd) age of 63.8 (14.8) years were collected (males: 92%). the admission diagnosis was laryngeal squamosis cell carcinoma (n = 14), glottis carcinoma (n = 6) and nasopharynx, tongue and skull base cancer (n = 5). at baseline, 1, 17 and 7 patients were considered have high, medium and low risk of malnutrition, respectively. the mean (sd) serum 25ohd was 8.46 (5.68) ng/ml (deficiency: 24 patients; insufficiency: 1 patient). despite the role of vitd in mineral balance, calcium, magnesium and phosphate mean (sd) serum levels were between the normal range 9.15 (0.36) mg/dl, 2.08 (0.22) mg/dl, and 2.49 (0.89) mg/dl, respectively. 17 s25ohd records were available 1 week after the administration (mean (sd) = 21.46 (13.79) ng/ml). 7 and 4 patients still showed deficiency and insufficiency, respectively. primary care's records from 3 patients were available after discharge (30.2, 36.5 and 52.5 ng/ml). conclusion: poor nutritional status and high prevalence of suboptimal vitd in patients with head and neck cancer were found. a single dose of intramuscular cholecalciferol slowly raises s25ohd. follow-up after discharge is essential to evaluate the achievement of the therapeutic objective. setting and method: this is a descriptive retrospective study. it took place in a teaching hospital. antifungal broad spectrum therapies (liposomal amphotericin b, caspofungin, micafungin, posaconazole, voriconazole) used between 1st january 2013 and 31st december 2015 were included. main outcome measures: indications, type of combination and patients specifications were analysed. results: only 19 patients (1.9% over all patients receiving antifungal therapy; n = 19/977) received an antifungal combination therapy during the study period. majority of patients presented risk factors: 31% of patients had an organ transplant (n = 6), 53% suffered from malignant blood disorders (four acute myeloid leukaemia, two chronic lymphoid leukemia, one non-hodgkin's lymphoma, one hodgkin's lymphoma and two refractory anaemia with excessive blast), 11% suffered from solid cancer (one lung cancer and one breast cancer) and 5% suffered from chronic obstructive bronchopneumopathy (n = 1). antifungal combination therapy was used against invasive aspergillosis in 68% of cases (n = 13) among which complications such as brain and cardiac impairment were found in 32% of patients (n = 6). the six remaining patients (32%) were co-infected with candidiasis for three patients and mucomycosis for three patients. voriconazole was logically the most used in combination, and just one patient received oral form. it was in majority prescribed with caspofungin (38%, n = 8) and intravenous liposomal amphotericin b (33%; n = 7). combination including liposomal amphotericin b and caspofungin (n = 3, 14%) or posaconazole with liposomal amphotericin b (n = 1) were found in our study. five patients deceased during the hospitalization of the fungal infection (26%) which shows the gravity of these cases. majority of patients ([50%) was treated less than 10 days with these combinations. conclusion: this retrospective study shows that patients who received antifungal combination therapy were mostly immunocompromised, co-infected or experienced a severe infection with severity factors. the antifungal combination was in majority initiated because monotherapy failed to cure the patient. all prescriptions were discussed with a mycologist who tried to shorter the combination treatment duration. this multidisciplinary approach is a major key in the process of these type of treatments. please specify your abstract type: research abstract background and objective: because of its broad spectrum and the risk of resistance mutation, delivery of posaconazole is nominative and controlled by hospital pharmacists. the aim of this work was to describe the use and pharmaceutical follow-up of posaconazole tablets over a 7-months period. setting and method: this is a descriptive retrospective study over a 7-months period from november 2015 to may 2016 in a teaching hospital. all patients who received posaconazole tablets were included. main outcome measures: indications and dosage were reported. results: 23 patients were included in the study. posaconazole tablets were used for: fungal invasive infection prophylaxis in case of stem cell transplantation (52%; n = 12), fungal invasive infection prophylaxis if a chemotherapy was started to treat a chronic myeloid leukaemia or a myelodysplasic syndrome (26%; n = 6); treatment of invasive aspergillosis (13%; n = 3); mycetoma (5%; n = 1); zygomycosis or mucormycosis while patient had renal impairment (5%; n = 1). all of these indications were approved for posaconazole (marketing authorization and local guidelines). only 10 patients (43%) received a loading dose (300 milligrams twice a day) as recommended in approval authorization. posaconazole blood levels were monitored by pharmacologists: 70% of patients (n = 16) did not need dosage modulation which shows that variability is not so important. but three patients did not have any assay to monitor posaconazole blood concentration. 1 patient received a loading dose and was switched to intravenous voriconazole after icu transfer. 3 patients needed increase and/or reduction dose to obtain optimal posaconazole blood levels. conclusion: this study describes the use and the follow-up of posaconazole tablets during the first months after its approval in europe. all indications are approved for posaconazole but this analysis shows that pharmacist have to remind the necessity of a loading dose. dosage can be adjusted according to assays results. please specify your abstract type: research abstract background and objective: due to the acute, hectic environment in a fast-paced work-flow emergency department (ed) it is a challenge to verify the correct and updated medication list for the admitted patients. when performing medication reconciliation (mr) in this environment, these challenge has to be taken into account and prioritizing patients for mr could be necessary. the objective of this study was to identify risk factors correlated to clinical relevant medication discrepancies (crmds) among patients admitted to ed, and based on these revealed risk factors, develop a model for prioritizing patients for mr in the fast-paced work-flow at the ed. setting and method: 276 patients continuously included at the ed, diakonhjemmet hospital (dh), oslo, norway. trained pharmacists and emergency nurse conducted mr. patient specific factors and revealed crmds, between hospital admission records and information about prehospital medication use, were recorded. binary linear regression was used to identify risk factors correlated to crmds. the prioritizing model was built using statics and clinical experiences. main outcome measures: what risk factors is correlated to crmds and how precisely do the prioritizing model classify the patients as high-and low-risk patients. results: 62% of the patients had c1 crmd. the following were identified as risk factors correlated to crmd and were suitable for inclusion in the prioritizing model; gender (woman), age (c60), c1 admission to hospital last 12 months, admission causes; surgical, malfunction, cancer. the model correctly classified 76.1% of the patients with crmds as high risk. further, 23.9% of the patients with crmds were classified by the model as low-risk patients (false negatives). the model classified 27.1% of the patients who did not have a crmd as high-risk patients (false positives). conclusion: the prioritizing model developed can be helpful in identifying what patients are at increased risk of having crmds in the fast-paced work-flow at the ed. identifying these patients will result in using the resources available in the ed in the most efficient manner and utilizing the full potential of the mr method. as a consequence of this, patient safety would be increased. hp-pc078: intravenous potassium chloride: quick audit of prescribers knowledge and recommendations regarding safe practice and proper usage asmaa damou * , vincent zaugg, martine postaire please specify your abstract type: descriptive abstract (for projects) background and objective: our hospital has established methods that try to ensure the safe use of high alert medications. intravenous potassium chloride (kcl) was the subject of preventive measures: separation of different dosages (kcl 7.46% vials reserved for paediatric services and kcl 10% vials reserved for adult services); creation of an advice record for doctors and nurses; specific labelling of storage areas; double-check the prescription and administration. the objective of this study was to evaluate the knowledge of the safe use of intravenous kcl by prescribers. design: multiple-choice questions were developed for prescribing recommendations established by our hospital with the collaboration of the doctor who is chairman of the central committee of vigilance and risk associated with care (cvris). a link to the online survey was sent by email to 85 physicians practicing in 14 departments (eight paediatric services and six adult services). the results were extracted and interpreted in excel ò . results: 57% of physicians responded to the survey (17 medicine residents, 32 hospital doctors). in paediatric services, 93% of doctors know that only the kcl 7.46% should be used. 87% know the unit of prescription to be used (mmol/kg or meq/kg), and 90% know that the maximum recommended infusion rate is 0.5 mmol/kg/hour (or 1 mmol/kg/h in recovery unit). in adult services, the recommended maximum rate of infusion (1 g/h) is known to all prescribers, but only 56% know that the concentration of kcl must be less than 4 g/l. 74% of paediatric doctors say that their kcl prescriptions are checked by a second doctor, but the answers in the same service area are sometimes contradictory. in adult services, only 6% of physicians say that the prescriptions are double-checked. the information brochure available on the intranet of the hospital is known by 16% of prescribers. the response rate of physicians to the survey was satisfactory. therefore, the recommendations are rather well known by prescribers, except the value of the maximum concentration of infusion for adults. the results of this audit were returned to the doctors, accompanied by a reminder stating the need to double-check the prescription and the existence of advice records on the website of the hospital. conclusion: this audit is an approach to increase the safety of the use of high alert medications. it will be completed a second time, by an evaluation of prescriptions collected and the storage conditions of potassium chloride in the care units. please specify your abstract type: research abstract background and objective: data listed behind each unit dose of a primary packaging of a pharmaceutical product are essential for a safe identification for the patient. however, the last medical services of the lausanne university hospital where nurses remove the solid form drugs (sfd) from their blisters when they prepare in advance the week container were in the vaud's prisons. the aims of the study were: (194), quetiapine (173) and ibuprofen (124) and 978 were psychotropics (39.8%). part 2. the four data identified as essential: brand name, dosage [mg], batch number, expiration date. the sfd unit doses were classified as green when the blister included four data, yellow with two or three and red with less than two. of the 273 sfd in cupboards, 90 were green (33%), 57 yellow (21%) and 126 red (46%); an infovigilance was sent to each manufacturers. part 3. potential barriers identified: trays' sizes and space in drug's cupboards; preparation time to cut versus to remove the blisters; risks of self/hetero-aggression with pre-cut blisters; drugs packaged in bulk; multidose liquid medications. using containers larger than is usual was rarely necessary; space in cupboards was sufficient. the preparation time gradually decreased during the study. ingestion or aggression with pre-cut blisters was considered as limited, based on literature and experiences of two others prisons (geneva; lyon). for bulk sfd and multidose liquid drugs: proposals to the pharmacy to store some alternatives blistered sfd; blistering expensive bulked drugs; availability of the entire package delivered to inmates. the pilot phase was initiated in may 2015. conclusion: a majority of inmates takes a drug treatment. half of sfd unit dose is identifiable (trade name and dosage) but an effort from manufacturers would better secure the drug supply chain. the study of the barriers helped to further implement the pilot phase. since early 2016, none of the five prisons medical wards are removing the blisters and no incident was reported. please specify your abstract type: research abstract background and objective: nefopam is a widely used antalgic in hospital. its use is contraindicated in the epileptic patient as it results in lowering the epileptogen threshold and is likely to trigger epileptic seizures. the clinical pharmacist should systematically warn the prescriber against this contraindication when analysing prescriptions. following the onset in our establishment of an epileptic condition in a patient treated with nefopam, who had not been subject to any pharmaceutical intervention (pi), we set about analysing the validation practices regarding this contraindication and possibly implementing actions designed to improve those practices. setting and method: retrospective collection over a period of 17 months of prescriptions for patients hospitalized in 210 hospital beds with clinical pharmacy service (associating med-reconciliation, checking prescription according to medical file and participation to medical rounds): orthopaedic surgery, hepatic-gastro-enterology, general surgery, liver transplant and chest surgery. records of patients with nefopam prescription associated to medication belonging to the therapeutic class of antiepileptics were consulted with a view to finding cases of epilepsy. the pharmaceutical alerts were extracted from the pharmaceutical software. main outcome measures: number of epileptic patients treated with nefopam, number of pharmaceutical interventions issued when prescribing nefopam in epileptic patients. the study focused on 11,252 patients. 3980 (35.4%) of them were prescribed nefopam, and 143 (1.3%) of them were prescribed nefopam associated to medication belonging to the therapeutic class of antiepileptics. after analysis of the patients' records has shown that 55 of them were really epileptic. only 29 pi's were effected (52.3% of problematic prescriptions), and 25 (86%) of them had an immediate prescription change. 77.8% (14/18) of the patients have a pi in medicine services compared to 40.5 (15/37) in surgery services (p \ 0.01). the results of this study show that 47% of the contraindications related to the use of nefopam in epileptic patients are not reported to the prescriber. these results will be presented to our pharmacists so they can take them into account. subsequently a new study will be conducted to measure the relevance and efficiency of this program. hélène dewaele * , anne-laure raso, emmanuel cirot, marion collignon, laura foucault, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) has been demonstrated to reduce drug-related problems in inpatients. in our university hospital, mr has been performed for 200 beds for 10 years at the same time as prescriptions review. the aim of this study was to assess the impact of mr on pharmaceutical interventions (pi) during prescriptions review. design: a 6-month prospective study in orthopaedic surgery, hepato-gastro-enterology, general surgery, liver transplant and chest surgery was conducted. during medication review all pis were collected and those related to mr (rpi) were identified. thereafter for each patient we collected age, type of hospitalization unit (med or surgery) and for pis the drug associated and its acceptance by the medical team. results: during the study 4756 patients had a daily prescription review. 1576 patients (33%) had at least one drug-related problem. 928 lines of prescriptions were mentioned to have at least one rpi. rpi represent 33% of drug-related problems. 697 (75%) discrepancies were corrected by prescribers. the age of the patient was significantly different between patients with rpi (mean age: 70 years old) and with pi (mean age: 65 years old; p \ 0.05). the type of unit did impact the percentage of prescriptions with drug-related problems (medicine: 46.8; surgery: 66.8; p \ 0.01), the rate of corrected pi (medicine:75%, surgery: 61%, p \ 0.01), but did not impact the rate of corrected rpi (p = 0.49). in surgery units the rate of corrected rpi (967/1571) is significantly higher than corrected pi (601/804; p \ 0.001). medicines belonging to the four classes of: digestive and metabolism system, blood and blood flow, cardiovascular system, neurological system represent more than 75% of all the medication concerned by a resolved pi or rpi. the proportion of medicines from the digestive and metabolism class is the only class among those four that is not significantly different between resolved pi and rpi. conclusion: mr highlights a large number of discrepancies in inpatients. a modification of prescriptions due to mr occurs in 10% of the patients. in surgery units, these rpi are more frequently taken into account than drug-related problem warned by pis. indentifying patients for whom mr has the bigger impact could help us to reinforce our actions. please specify your abstract type: research abstract background and objective: diabetes is very frequently causing cardiovascular complications, thus impairing various systems and organs. therapy for these multiple conditions has to be revised and improved constantly. the aim of this closed retrospective study lead in bucharest emergency clinical hospital was the assessment of some of the diabetes mellitus (dm) complications and the related medication. setting and method: data was collected from cardiology, neurology, gastroenterology, internal medicine wards from bucharest emergency clinical hospital. only patients diagnosed with type 2 dm were included in the study. there were analysed 105 records from patients aged 36-89 of whom 65 were men, following the presence, signalling and monitoring of diabetic nephropathy and arteriopathy. main outcome measures: we investigated the relationship between diagnosis and/or biochemical signs of kidney disease (serum urea, serum creatinine levels), diagnosis of arteriopathy, and the drug therapy administered in the respective cases. we also assessed the sex and age distribution of the patients diagnosed with diabetes mellitus and facing at least one of its complications. results: kidney disease, as a dm complication, was present in 30% of cases, patients aged 55-89, of whom 57% were men. 53 patients received diuretic treatment, 5 of them being given hydrochlorothiazide, contraindicated in dm because of its hyperglycaemia-inducing effect. of the 105 patients, 36 had high serum urea levels ([50 mg/dl), 39 had high levels of serum creatinine ([1.2 mg/dl), and 26 presented risen levels for both, but only 16 were also diagnosed with kidney disease. 9 patients with kidney disease were given furosemide, known for altering the renal function. circulatory failure was found in 10% of the patients, aged 61-80 and 6% of subjects, aged 62-79, had both diabetic complications. conclusion: the present study emphasizes the role of the clinical pharmacist in adapting the medication of the diabetic patient, an inappropriate pharmacotherapy worsening dm complications. this is essential especially for elders, where polypathology and polymedication lead to a significant increase of dm complications risk. hp-pc084: epileptic seizure after treatment with thiocolchicoside: discussion about a case report valérie dobremez *,1 , adeline martin-dupray 2 , jacqueline berlioz 1 , pierric giraud 2 1 pharmacy, 2 neurology, centre hospitalier annecy-genevois, metz-tessy, france please specify your abstract type: descriptive abstract (for projects) background and objective: thiocolchicoside is a semisynthetic derivate of naturally occurring colchicoside, which is largely used in humans as a centrally acting muscle relaxant. this compound also has anti-inflammatory and analgesic effects. the objective of this work is to report a recent case of serious adverse effect of thiocolchicoside occurring in context post traumatic brain damage without sequalae. design: a 28-year-old woman suffered from headaches and neck pain since 5 days, she was treated with thiocolchicoside. she took 8 mg in the evening and 8 mg the next morning. five generalized tonic-clonic seizures, without recovery of normal consciousness between seizures, have occurred suddenly 15-30 min after the second administration. the patient was admitted to intensive care unit in order to control the epileptic seizures. a status epilepticus was diagnosed requiring intravenous drugs with clonazepam, phenobarbital and propofol. the patient was controlled and transferred in neurologic unit in order to complete paraclinical investigations. its main antecedent was a severe head injury at the age of 7 years following a public road accident. the brain scan revealed an old frontal hypodensity. rest of etiological assessment was negative (lumbar puncture, no infectious disease), numbers were normal. the definitive diagnosis was a status epilepticus on post-traumatic sequelae, sensitized by taking a proconvulsant drug. a treatment with levitiracetam was initiated at 750 mg twice a day. outcome was favourable with no recurrence 10 months later, a recommendation was requested to pharmacovigilance. results: the muscle relaxant activity of thiocolchicoside results of an agonist action on glycinergic receptors located primarily in the brain stem and spinal cord. however, thiocolchicoside also acts as an antagonist of the gaba-a receptor (mainly located in the cerebral cortex), this pharmacological action can cause a proconvulsant effect. epilepsy is a very rare adverse effect, only few cases have been reported in literature. the epileptogenic activity of thiocolchicoside occur mainly in patients with a history of epilepsy, acute brain injury or possible blood-brain barrier disruption. the chronology is consistent with the responsibility of the drug as a promoting factor. pharmacovigilance retains after analysing drug causality. conclusion: the case history indicates that thiocolchicoside has a powerful epileptogenic activity. thiocolchicoside can precipitate seizures in predisposed patients, and that its use should be avoided in patients with brain diseases (and therefore lower seizure thresholds) or blood-brain barrier disruption. pharmacists could warn physicians and should verify the absence of notable history before dispensing thiocolchicoside. hp-pc085: acute exacerbation generalized myasthenia after red yeast rice use: a case report valérie dobremez *,1 , amélie serra 2 , déborah grosset-janin 2 , jacqueline berlioz 1 , aymeric dopter 3 , jean-henri ruel 2 1 pharmacy, 2 neurology, centre hospitalier annecy-genevois, metz-tessy, 3 nutrivigilance, french agency for food, environmental and occupational health and safety, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: many drugs can induce acute exacerbations or reveal myasthenia gravis. self-medication or complementary and alternatives medicines expose patients. the objective of this work is to report a recent case of acute exacerbation of myasthenia gravis because of a dietary supplement use. design: intermittent vertical diplopia and ptosis of the left eye settled in a 69-year-old man. its main antecedent is hypertension treated with perindopril. the neurovascular origin was ruled out. the electromyogram (emg) found a significant decrement (11%) of a postsynaptic block in the tongue and right orbicularis muscle. acetylcholine receptor-antibodies were positive. myasthenia gravis was diagnosed (osserman score 90/100) and the patient was treated with pyridostigmine. the identification of carotid atheroma required a treatment with a statin that the patient refused. he preferred a cholesterol lowering dietary supplement, containing red yeast rice. six days later, he was hospitalized for an acute decompensation of myasthenia with bilateral ptosis, oculomotor paresis, drooping head, int j clin pharm (2017) 39:208-341 281 chewing trouble and dysphagia (osserman score 42/100). the patient is treated with high-dose intravenous immunoglobulins then corticosteroids. the dietary supplement is stopped. an opinion was requested to the clinical pharmacist of neurology. the osserman score gradually increases to 78/100. results: red yeast rice contains a range of compounds known as monacolins, of which monacolin k-renamed lovastatin, which was found to be an inhibitor of cholesterol synthesis and the progenitor of the statin family. a literature review has highlighted the responsibility of statins in acute exacerbations or reveal myasthenia gravis occurrences. in this case, the chronology is consistent with the responsibility of red yeast rice. the case was reported to the french system of nutrivigilance, which retained after analysing a probable intrinsic imputability score. conclusion: dietary supplement with red rice yeast are not recommended in case of myasthenia gravis. this is the first case of acute decompensation of myasthenia recorded with red yeast rice in the french system of nutrivigilance. multidisciplinary collaboration (neurologists, clinical pharmacist) has optimized the patient management. fanny durand * , camille lambert, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: development of computerized prescription highlights the need to harmonize pharmaceutical analysis practices. the aim of this study is to analyse the antibiotics prescriptions in the treatment of urinary tract infections, to develop a pharmaceutical validation tool. design: a prospective observational study was conducted for one week, in 20 care units. pharmacists, interns, and pharmacy students were trained on spilf (french society of infectious pathology) recommendations, on pharmacist's role in the management of urinary tract infections, and on the data collection. all patients with antibiotic prescription for urinary tract infection were included. some data were collected: reason for hospitalization, clinical signs, results of susceptibility testing, risk factors for complications (organic or functional abnormality of urinary tract, male, pregnancy, elderly, severe immunodeficiency, severe renal impairment) and signs of severity (severe sepsis, septic shock, interventional surgical drainage). then, the treatments prescribed to the patient, probabilistic on the one hand and documented on the other hand, were compared to spilf recommendations. finally, during a multidisciplinary meeting (pharmacist, expert in infectious diseases), we selected the relevant pharmacist interventions. results: twenty-three patients were included (14 women, 9 men), 42% had a urinary catheter. 52.7% of prescriptions were concordant with spilf recommendations: probabilistic and documented treatment, and duration. among the non-conforming prescriptions, nine pharmacist interventions have been formulated: four prescriptions did not specify the duration of treatment, one antibiotic was prescribed on an insufficient period, two cases of severe acute pyelonephritis without prescription of aminoglycoside, one prescription was not reassessed according to results of susceptibility testing, one pregnant woman with urinary colonization without clinical signs, was treated before obtaining results of susceptibility testing. three cases of poor management are identified: two cases which treatment began only after results of susceptibility testing (a urinary tract infection linked to care, an acute pyelonephritis with complication risk), and a cystitis treated with nitrofurantoin while the germ was resistant. conclusion: a synthetic tool was created. there are three elements for helping pharmaceutical analysis: the questions to ask oneself facing a prescription of antibiotic for urinary tract infection, a flowchart to identify the recommendation adapted to the case, and finally a summary table showing spilf recommendations. this tool will be distributed and evaluated. hp-pc088: off-label use of rituximab in refractory antisynthetase syndrome (as) through a long-time experience in a neuromuscular diesases center lise durand * , carole metz, patrick tilleul, helga junot pharmacy, gh pitié salpêtrière, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: as is an idiopathic autoimmune inflammatory myopathy, characterized by presence of antisynthetase antibodies: anti-jo1, anti-pl7, anti-pl12. patients are usually first treated by corticosteroids (cs) or immunomodulating drugs. rituximab (rtx) has become another option for refractory as, supported by few uncontrolled studies 1 . because of its off-label use, our hospital pharmacy has implemented a controlled drug delivery. this work assesses a 2-years follow-up of patients treated by rtx and the resulted drug costs. design: patients registered in our database since december 2014 who received c1 injections of rtx to treat as, were analysed to describe their eligibility criteria, conditions of management and the clinical and biological effects of the treatment (creatine kinase (cpk) used as biomarker). patient files were consulted to collect all individual data and pharmaceutical software was used to review deliveries. drug costs were also reckoned based on prices from french health insurance. results: for 18 months, 14 patients (median age (min-max): 52 (20-76), 64% women) have been treated with rtx for refractory as, the majority with anti-jo1 antibodies (8). all patients suffer from muscular and lung affections, particularly interstitial pneumonia. many are also living with arthropathies (10) or cutaneous disorders (9). cardiac involvement is seldom (4 symptomatic patients). the mean age of diagnostic is 7.3 years and the mean treatment period is 2.6 years. the common treatment is 1 g at day1 (d1) and d15, then 1 g all 6 months. before rtx treatment, seven patients received c5 other drugs such as cs (93%), azathioprine (71%), methotrexate or mycophenolate mofetil (57%). prednisone and azathioprine are also prescribed with rtx respectively for 79 and 23%. treatment is associated with cures of intravenous immunoglobulins for four patients. to date, median number of administrations per patient is 4 (1-8), d1 and d15 included. all patients have presented positive effects on both clinical and biological markers, mainly during the first 6 months after treatment induction. wilcoxon tests show a significant difference in cpk level between d1 and m6, also between d1 and the last known result. today, three complete remissions are specified in patient file; only one hepatitis b virus reactivation is reported. since 2014, budget impact due to drug cost amounts to 119 000€. conclusion: whereas the use of rtx is controverted for treatment of all types of myopathy, as could have one of the best response 1 . our cohort shows real clinical results and positive effect on usual biomarker. our experience demonstrates the safe and successful use of repeated administrations in refractory as. however, there is a need for further controlled studies to assess the efficacy/safety of rtx and to define its place in the strategy in view of its cost-effectiveness ratio. the pharmaceutical controlled drug delivery has to be continued to supervise, support and document its proper off-label use. please specify your abstract type: descriptive abstract (for projects) background and objective: as a part of the national patient safety program, the northern norway regional health authority are implementing new procedures for medication reconciliation (mr) in hospitals in the region. the procedure defines that mr is the doctor's responsibility and describes how it should be performed. the aim of this study was to investigate whether the implementation of the procedure reduces medication discrepancies (mds) in the charts at bodø hospital. and 53.9% (7/13) of the patients died before discharge. parenteral nutrition was administered an average of 21.5 days (95% ci 2.5-40.5), of which 7.7 (95% ci 3.0-12.4) were with ipn. previous spn had been administered in 84.6% (11/13) of the patients. before beginning ipn, the average triglycerides level was 608.1 mg/ dl (95% ci 388.1-828.0) but at the end of the ipn it was 324.9 mg/ dl (95% ci 245.4-404.5), which lead to a mean reduction of 283.2 mg/dl (95% ci 45.3-520.0; p = 0.02). regarding to the total amount of lipids provided with parenteral nutrition, with ipn there was a mean reduction of 30.4 g (95% ci 12.4-48.5; p = 0.002) comparing to those administered with spn. conclusion: usage of ipn in critically ill patients with htg permits to adjust parenteral nutrition formulations to meet specific nutrition needs, enables to reduce the total amount of lipids administered and, therefore, it allows to significantly decrease triglycerides levels. jennifer a. esteban gonzález * , elisabet nogué pujadas, angels andreu crespo, xavier bonafont pujol, nuria romero pascual please specify your abstract type: descriptive abstract (for projects) background and objective: the incidents involving patient misidentification (pm), or wrong patient medical errors (wpme), are medication errors (me), near-miss or close-call situations which can pose a considerable threat to patient health. pm may be under-reported due to the unawareness of the error or the difficulty of identifying them. the aim of this study is to describe the incidence and categories of wpme in a university hospital. design: observational, retrospective analysis of the voluntary reported wpme in the pharmacy database since march 2010 until june 2016. these were classified in prescription, transcription, dispensing, administration and drug system errors. in addition, the national coordinating council for medication error reporting and prevention (nccmerp) taxonomy was used for classifying me according to the severity of the outcome. results: of 1767 me registered, 50 of them were wpme (2.8%). 40.0% of them were due to prescription errors, which consist on wrong labelled medical orders, intermingled patient prescriptions or patient misidentification in computerized physician order entry (cpoe). the administration errors supposed a 30.0% of the total amount of wpme and dispensing errors were 18.0%. 6% of wpme were transcription errors, which occurred previously to the implementation of cpoe, and the remaining 6.0% were system errors after cpoe. the wpme reported took place in the hospitalization wards (44.0%), pharmacy (20.0%), outpatient services (16.0%), intensive care unit (16.0%) and day-care hospital unit (4.0%). 88.0% occurred at working days and 12.0% at the weekends. wpme were notified by pharmacists (62.0%), nurses (34.0%) and physicians (4.0%). referring to the classification according to nccmerp, 48.0% of wpme didn't reach the patient (category b) whereas 40.0% reached the patient but didn't cause harm (category c) and 8.0% required patient monitoring (category d). the remaining wpme (4.0%) caused harm to patients and required medical intervention (category e). finally, in int j clin pharm (2017) 39:208-341 283 more than half of wpme (62.0%), reporters suggested measures to prevent these errors. conclusion: wpme represents near 3% of total me reported in our hospital. given that more than 50% reached the patient, safety measures must be implemented to reduce the risk of hazardous events. additionally, further encouragement in notification is necessary in order to improve patient safety. results: two men diagnosed with rrms aggressive evolution were included in the study. age: 23 and 31. both of them without any treatment by the time they started being treated with alemtuzumab (previously one of the patients had been treated with fingolimod, suspended by inefficiency). the protocol design for the elaboration and control of alemtuzumab in the pharmacy service ensures greater safety and represents a saving strategy. in addition, the development of the protocol in the electronic prescription system (silicon ò ) facilitates the prescription, proper administration and standardization of treatment among patients. the protocol includes daily alemtuzumab infusion for 5 days and other necessary medications including premedication (metylprednisolone, omeprazole, paracetamol and metoclopramide) and anti-infective prophylaxis (aciclovir). developed adverse effects during infusion were skin erythema, pruritus and fever. it was not necessary to stop the alemtuzumab infusion in any patient. during treatment, one patient developed a severe lymphopenia and upper respiratory tract infection (influenza a). conclusion: the role of the pharmacist is critical at various stages, from the preparation and the administration guidelines, to detection, monitoring and reporting of adverse effects. alemtuzumab is presented as an alternative for those patients who do not respond to standard therapies or who have rapidly evolving severe rrms. because of its mechanism of action it is important to closely monitor patients, with particular emphasis on prophylaxis of possible infections. hp-pc093: descriptive analysis of patients receiving oral anticoagulation following acute coronary syndromes sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: triple therapy with concomitant anticoagulant and dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) increases bleeding risk by 50% compared to patients on dapt. bleeding post acs increases mortality and reinfarction risk; balancing ischemic and bleeding risks is particularly challenging in this population. european society of cardiology (esc) produced a consensus document, providing guidance for patients presenting with acs requiring concomitant anticoagulation; however optimal duration of triple therapy and safety and efficacy of novel oral anticoagulants (noacs) and more potent antiplatelet agents requires further evidence. design: a registry was collated of patients presenting with acs requiring concomitant anticoagulation. baseline characteristics, bleeding and ischemic risk scores, periprocedural treatment and antiplatelet/anticoagulant choice and duration was recorded and analysed for trends in prescribing. results: 71 patients have been included in the registry between oct 2015 and june 2016, of which 40 (56%) were naïve to anticoagulation prior to admission, 24 (34%) were taking warfarin and 7 (10%) were on noacs. atrial fibrillation (af) accounted for 51 (73%) cases, (average chadsvasc score of 5, hasbled score of 2), and 15 (21%) were for lv thrombus. of those naïve to anticoagulation, 25 (63%) were initiated on warfarin and 15 (37%) on a noac (last 10 patients all received noacs). of those on a noac for af, 17 (81%) were dose reduced on triple therapy; apixaban being the most commonly prescribed (59% apixaban, 35% rivaroxaban, 6% dabigatran). background and objective: solid oral formulations are more convenient than liquids to manufacture, store and administer for most adults. given this superiority, one would think that children were prompted to use solid formulations when available in an eligible dose. there are indications, however, that the conversion from liquid to solid formulation in children is influenced by characteristics of the liquid medication, rather than the child's ability to swallow solid medications. the aim of this study was therefore to explore if the proportion of oral liquid formulations differed between antibiotics commonly used for upper respiratory tract infections (urti) in hospitalized children. setting and method: we collected the sales data for 2015 for the children's department of the five university hospitals in norway. the three most common oral antibiotics used for urti in children were included: penicillin v, amoxicillin and erythromycin. the proportion of oral liquids was calculated by dividing the number of defined daily doses (ddd) of liquids by the total oral ddds for each substance. main outcome measures: the proportion of ddds of oral liquid antibiotics. results: a total of 2575 ddds of common oral urti antibiotics were sold in 2015, distributed as 30% erythromycin 31% amoxicillin and 39% penicillin v. amoxicillin had the highest proportion of liquid with 97%, followed closely by erythromycin at 94%. in contrast, only 70% of the ddds sold of oral penicillin v were liquids. conclusion: higher proportions of liquid amoxicillin and erythromycin compared to penicillin v were sold to children's departments in hospitals. there are several limitations regarding the quality of sales data, as we lack information of the administered doses as well as the child's age, gender, infection and specific needs. infections in hospitals often require initial intravenous treatment, and oral switch will often be based on the initial treatment. despite these limitations, the results fit well with earlier findings which indicate that children prefer liquid amoxicillin and erythromycin to penicillin v. hp-pc095: proactive medication reconciliation: a preliminary study to identify barriers before its implementation in surgery departments laura foucault * , marion collignon, hélène dewaele, anne laure raso, emmanuel cirot, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: it's well known that medication reconciliation (mr) decreases drug-related problems at patient admission (pa). in surgery departments, for planned hospitalizations, mr is performed 24-48 h after the pa (pourrat x and al, 2013). during this period, some chronic treatments are unintentionally not prescribed to patients. the aim of proactive mr (pmr) is to anticipate the pa by collecting their medication history before their hospitalization. the objective of this study was to identify the barriers preventing pmr implementation in our hospital. design: one week prospective study in digestive and orthopaedic surgery units in a 600 beds' university hospital. the main outcome is to identify which barriers prevent the collection of mr before pa including the evaluation of time required to collect the relevant information, reconcile any discrepancies after the pa and identify the right sources from which to perform the mr. results: eighteen patients with a median age of 59 years old (14-84) were contacted by phone one week before their scheduled surgery. these calls were conducted by pharmacy residents mainly between 6 and 8 p.m. (a more practical time for patients and at the end of pharmacist's routine tasks). an average of 1.7 (1-3) calls per patient were conducted. one patient was unreachable by phone. the average duration of the calls was 7 min (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) . twelve community pharmacy (cp) were contacted. in all cases, cp have accepted to share information about the patient's prescriptions by phone and sending it by fax during the day. five pharmacists were not contacted because patients had no chronic treatment and consequently no regular cp. on 53 lines of prescriptions, 12 discrepancies between the patient's information and prescriptions were identified and 7 between prescriptions and the anaesthesia records. drug history was reported in the patient's records by pharmacy students on the day of pa in order to be used immediately by prescribers. surgery was cancelled for one patient. conclusion: the first step of an mr is made by a hospital anaesthetist some weeks before hospitalization but we have demonstrated that this step is not able to avert all potential errors. our study highlights that the time necessary to perform an mpr appears to be shorter than for an mr. in fact, it's sometimes difficult to properly interview patients during hospitalization (patient in operating room, drug-induced drowsiness). additionally, a key hurdle is to obtain any necessary modification of the prescriptions by surgeons. pmr can be expected to produce time saving efficiencies given that at pa, prescribers will have their full medication history. this study also allowed us to highlight the good cooperation between patients, cp and the hospital. it is worth noting that efforts were made to accommodate the schedules of a majority of working patients. however, as we would expect pharmacy student to perform the pmr, they will most likely attempt to contact patients during standard working hours which may impact the number of patients they are able to reach. laura foucault * , hélène dewaele, marion collignon, emmanuel cirot, anne laure raso, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: the french legislation has clearly defined and integrated the therapeutic education of patient (tep) for healthcare professionals. the pharmacist is invited to get involved in tep as a caregiver around the patient. in our study, we are investigating how the pharmacist's role is viewed by patients with chronic diseases that are included in a tep program. design: prospective study on 17 patients included in a tep program (chronic inflammatory bowel diseases, rheumatoid arthritis, ankylosing spondylitis) between september 2015 and april 2016. in july 2016, the participants of group sessions (gs) conducted with health professionals, including a pharmacist, were interviewed on the phone. the principal outcome of the interviews was to evaluate how their view of the involved health professional's roles evolved before and after gs; to evaluate if they would consider being followed by their pharmacist for individual sessions (is) in a community pharmacy (cp); and if the information supplied by the pharmacist during gs was understandable. to health care. however, discussions between patients appear to be essential to facilitate their acceptance of a chronic condition. some patients also questioned the cp's skills and knowledge when it comes to their particular disease. nevertheless, 88.2% of patients have found that the vocabulary and documents used by pharmacist during gs was adapted and that the information supplied was very useful. conclusion: this study highlights that although the pharmacist is the drug's specialist, a majority of patients will more likely ask their physician about medication. their participation to the gs hasn't changed their habits even if the pharmacist intervention was relevant and understandable. the fact that the pharmacists took into account the level of health literacy of each participant was an appreciated aspect. cp should be more proactive in their relationship with the patients in order to highlight their skills and the assistance they can provide in a chronic disease. however, it's important to take in consideration that in some cases, patients have lived with their disease since childhood. the role of is is likely to be much more limited than in other situations given their key need is to interact with patients afflicted with the same condition. hp-pc097: use and safety of trastuzumab emtansin in her2 + metastatic breast cancer in a tertiary hospital c. chaguaceda galisteo * , alba manzaneque gordon, héctor josé del río torres, natália creus baró please specify your abstract type: descriptive abstract (for projects) background and objective: novel anti her2 drugs have changed the management of her2 + metastatic breast cancer patients. the aim of this study is to describe the use of trastuzumab emtansin (tdm-1) in clinical practice in a tertiary hospital and to evaluate its safety profile. design: we performed a retrospective study of patients who started tdm-1 between january 2013 and december 2015. we recorded demographic data, clinical and treatment variables, number of doses received, reasons for discontinuation, progression-free survival (pfs) and adverse effects (aes). data were obtained from the chemotherapy prescription program and medical records. aes were classified according to the common terminology criteria for adverse events version 4.0 of the national cancer institute. results: eleven female patients with a median age of 55.6 years [40.7-80.1] and an ecog 1 (9/11) were included. tdm-1 was prescribed as a third or further line treatment in 8/11 patients and as firstline in one patient who develop disease recurrence within 6 months of completing adjuvant therapy. median number of tdm-1 cycles was 8.5 . all treatments discontinuations were due to disease progression (6/11). pfs was 6.0 [1.9-20.7 months] (patients that received less than three cycles were excluded (n = 2)). most frequent aes were plaquetopenia, neutropenia and transaminitis but only grade 3 in three patients (two transaminitis and one neutropenia). conclusion: the lower pfs obtained comparing to the pivotal study (6.0 vs. 9.6 months) could be explained by the later use of tdm-1 in clinical practice (8/11 patients received tdm-1 as third or further line while 61% in the pivotal study were first or second line). tdm-1 safety profile was according to the summary product characteristics. few data are currently available regarding the use of tdm-1 in clinical practice. further data are required to position this drug in clinical practice. please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacist for their specialized training in the area of medicines, possess a greater responsibility in the detection and reporting of adverse drug reactions (adrs), as well as other problems related to treatment, which may be subject to monitoring and reporting to the regulatory authorities and the respective laboratories. thus, the pharmaceutical services of the cuf infante santo hospital has implemented a pharmacovigilance program, with two main objectives: 1. optimization of the detection and reporting of problems related to therapy; 2. implementation of corrective and/or risk minimization measures. the pharmacovigilance program is based on the following methodology: 1. detection of adrs/problems related to therapy/medical device: the detection can be performed by the pharmacist or other health professional that guides the process to the pharmaceutical services. 2. information processing by the pharmaceutical services and realization of spontaneous reporting: the notification is performed both for the portuguese regulator (infarmed) as to the appropriate laboratory (if applicable). after evaluation by both entity, the conclusions are communicated to the pharmaceutical services, which has the responsibility to share it with all the other hospital services. 3. report of the event in the internal risk management platform: when applicable, the pharmaceutical services internally report the adverse event to the hospital's risk management department, leading to an internal evaluation of the current process. 4. completion of the process and implementation of corrective measures: when the regulatory authority and/or the laboratory sends the report/technical advice about the notification, the pharmaceutical service in partnership with the risk management team perform a reassessment of the whole process. if needed, corrective and/or monitoring measures are implemented. 5. monitoring of implemented measures: after the implementation of corrective and/or monitoring measures there is a period of evaluation. results: the implementation of this program for the period of 1 year, has led to a total of fourteen spontaneous reports. from all of these notifications, seven were related to quality defect of medicines, four were of adr, one was due to suspected lack of therapeutic efficacy, and lastly, one of the notifications was medication error derived. conclusion: the obtained results, over a 1 year period, by the pharmacovigilance program were satisfactory but the aim of the pharmaceutical services is to consolidate and optimize the same program with a view to achieving better results. the pharmaceutical services will continue to take responsibility for the pharmacovigilance circuit management in this hospital, by promoting a proactive approach to monitoring the safety, quality and efficacy of medicines, which possess the primary objective to patient safety assurance. please specify your abstract type: descriptive abstract (for projects) background and objective: for prematures, parenteral nutrition (pn) is essential for medical care but is complex (specific needs, daily change of intakes…). now, the software logipren ò , developed by the french society of neonatology, allows the prescription of pn as well as all the childish therapeutics. it is also in link with our production robot (baxa pomp) for individual pn bags. our objective was to integrate this software while optimizing our pharmaceutical validation process. design: the software implementation was lead by a physician/ pharmacist collaboration with several preliminary steps: • identification of pharmaceutical validation settings (pertinence of individual pn vs. industrial bags, parenteral approach, elements…). before the life-sized use of logipren ò , a base test has been experimented to identify possible difficulties and to realize some correctives actions of the software or our process. results: logipren ò leads us to a change in our pharmaceutical validation process, by introducing new elements: • the pharmaceutical validation of pn bags is done in collaboration with the physician, during the prescription step. • all the therapeutics are known, which allow the pharmacist to take into consideration all the intakes (micro-nutrients, vitamins…). • remove the transcription step of pn bags in our production software (abacus ò ) thanks to an interface with our production robot. • less production problems because of the coverage of those pharmaceutical aspects during the prescription. since 2 months, this reorganization helped us to propose 22 pharmaceutical notices for 234 prescriptions: • omissions (remove lipids, levocarnyl ò , micro-nutrients, electrolytes, remove industrial bags…) • modification (reduce proteins according to urea level, micronutrients and electrolytes posology, duration of lipids infusion…) conclusion: the implementation of logipren ò enabled us to reorganize of the pharmaceutical validation process with a consolidation of the role of the pharmacist during the prescription step, in the paediatric ward. it had a beneficial aspect by the reduction of the validation and production time, a decreased risk of error (suppression of job interrupts and better communication) and an improved production by the end of transcription step to abacus ò . furthermore, during our experimentation, we could bring to the software editor new ways to improve it and make it more efficient. 57% (52.9% in 2015) , difficulties in swallowing/psycho-behavioural distress in 60. 71% (41.2% in 2015) , and rejection of oral drug in 10. 71% (5.9% in 2015) . physicians and nurses indicate the reason in the medical record in 77.78% of case versus 50% last year. this year, 287 drug were crushed versus 134 drugs in 2015: 22% concerned nervous system group (vs. 25% in 2015), 18% concerned cardiovascular system group (vs. 19% in 2015) , and 15% concerned alimentary tract and metabolism group (vs. 13% in 2015) . nurses use guideline in 50% of cases versus 2.9% last year. as the previous year, in 100% of cases, washing hands before preparation and after administration are met. last year, none of them was wearing mask and gloves during this operation while this year, 17% was wearing mask and gloves. finally, in the two assessment, for each patient, drugs are systematically crushed together and then mixed with the patient's meal. conclusion: this study shows that crushing drugs is still problematic in our units. however, best practices were observed, such as the indication of the reason of crushing in the medical record, or the consultation of guideline. a new training for nurses will be conducted to create awareness about risks of crushing drug. please specify your abstract type: research abstract background and objective: in invasive candidemia, three echinocandins are indicated: caspofungin, mycafungin and anidulafungin. the aim of this work is to establish which echinocandin to prescribe in a french university hospital, given the scarcity of available clinical data in the literature regarding obese patients. setting and method: in a french uhc with 1500 beds, a multidisciplinary working group composed of a microbiologist, an infectious disease specialist and a pharmacist has been set up to analyse the various therapeutic options. main outcome measures: analysis of the literature, pharmacoeconomic study. results: four medications have been identified as possible therapeutic options. their adverse effects are similar and their administration rhythm is the same. according to recommendations by the esmid (2012) and the idsa (2016), the level of evidence for these three echinocandins in initial treatment of candidemia is equivalent. concerning obese patients, no weight limit is mentioned, int j clin pharm (2017) 39:208-341 287 despite recommended dosage adjustment. caspofungin must be prescribed at a dose of 70 mg/day for patients weighing over 80 kg. micafungin must be administered at a dose of 100 mg/day regardless of patient weight. in the case of persistence of cultures or if clinical condition does not improve, the dose may be increased to 200 mg/day. anidulafungin, which is not referenced in our establishment, must be prescribed at the same dose regardless of patient weight. from an economic point of view, in our hospital, micafungin at a dose 100 mg/day remains the least costly therapy. however, if its posology is doubled as indicated, caspofungin then becomes the most economic therapy. amphothericin b, an optional treatment, is never the most economically advantageous therapy. conclusion: as a result of this study, the chosen prescribed therapy for obese patients is caspofungin at a dose of 70 mg/day. this work has improved access to healthcare for obese patients. pharmacokinetics and survival data must be collected on the basis of various patient weights in order to predict clinical efficacy. kristin f. heier * , liv czynski please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to develop a system to prioritize patients for medication reconciliation by pharmacists in the emergency department. it also proved a useful setting for evaluating how other health care professionals perceived the role of the pharmacist performing medical reconciliations within the emergency department. design: the study was located in the østfold municipal hospital, located in kalnes, norway. pharmacists used a prioritization model to identify ''high-risk patients'' having clinically relevant prehospital medication discrepancies between hospital admission records and the information obtained via medication histories, general physician referrals and nursing homes. pharmacists registered patient information such as age, gender and drug-related problems (drps). seventeen physicians and thirty nurses in the emergency department answered structured questionnaires anonymously. main outcome measures: • number of patients with medication reconciliation performed by a pharmacist. • number of drug-related problems denoted in the electronical journal and presented to the physician. • the overall experience physicians and the nurses had with pharmacists when located in the emergency department. results: pharmacists performed medication reconciliation for 262 patients, identifying 443 drps and 178 potential drps in total. fourteen of the physicians had read the journal notes from pharmacist and found them helpful (n = 4, 29%) or greatly beneficial (n = 10, 71%). most physicians (n = 14, 82%) and nurses (n = 19, 63%) reported a good cooperation with the pharmacist in the care of the patients. some of the physicians (n = 4, 29%) and most nurses (n = 21, 70%) wanted more information about the pharmacists work in the emergency department. the majority of ed staff (100% of physicians and 63% of nurses) found pharmacist as a good academic resource in the emergency department. conclusion: the physicians reported an improvement regarding the quality in the medication reconciliation made by pharmacists in the emergency department and both physicians and nurses expressed a need that pharmacists work in the emergency department on a more permanent basis. more information in general and especially better communication with nurses regarding the care of the patients are important actions need to optimise collaboration with pharmacist in the emergency department. results: a total of 105 patients were included in the study, median age was 53 years and 63% were males. they used in average four drugs regularly (range 1-15). almost three-quarters (70%) of the patients reported high or moderate adherence to all their regularly used drugs (mmas-8 c 6 (max 8)). of the 39 patients using oral spasmolytics, 74% reported high or moderate adherence to these drugs. the majority (97% of the patients) had high perceptions of necessity to their treatment (bmq [ 2.5 (max 5)), and 54% had a high level of concern (bmq [ 2.5 (max 5)). logistic regression analysis showed that there was no association between adherence and pain, nor between adherence and spasticity. younger age was found to be associated with higher risk of nonadherence. conclusion: even though overall adherence was high, the patients were more concerned to take their medicines compared to other patients with other chronic conditions. further studies are required for understanding adherence and attitudes toward medication in this population, and to help the patients feel safe about their medication regime. please specify your abstract type: descriptive abstract (for projects) background and objective: errors in medication lists often emerge in transition between health care levels, and there is need for strategies to communicate medication information. therefor we aimed to describe reasons why medication discrepancies (md) occurs in the transfer of patients between hospital and primary care service. design: in conjunction to a study based on use of structured medication report at transition from hospital to primary care service, we observed different reasons to why mds occurs. our observations and experiences linked to communication between health care levels is outlined. results: we observed that many md's disclosed at discharge could most likely be attributed to lack of medicines reconciliation at admission to hospital. for instance, several medicines were prescribed in primary care service prior to admission, but not at admission to the hospital. in addition, at admission, some medicines were listed as prescribed medications although not found in the medication lists in primary care service. we also observed that newly started and discontinued medicines were documented in the hospital discharge letter, but not implemented in primary care service. according to health care personnel in primary care service, insufficient communication about the patients' medications at discharge from hospital, led to corrections in the medication lists based on their previous knowledge about the patients. in addition, justified medication changes at discharge from hospital were not always implemented in primary care service due to professional disagreement. some stated that lack of trust was one reason for not always taking changes into account, often based on earlier experience. conclusion: these observations indicated that mds occurred both with and without intent when patients from primary care service were admitted to hospital and returned back due to poor communication. medication errors during hospitalisation and unproven intentional changes may be the consequences. due to this, it is important to improve the communication and confidence between professionals in the hospital and primary care service in order to reduce the number of mds and to enhance patient safety. please specify your abstract type: descriptive abstract (for projects) background and objective: intravenous human immunoglobulins (iv igs), plasma protein products, may cause in patient to a range of adverse side effects (headache, skin rash, kidney failure, thromboembolic event). in the framework of securing medicinal care, an assessment of professional practices has been conducted within our university hospital. the overall goal of this study is to evaluate the process of intravenous administration of human immunoglobulins done by the nurse staff. design: this prospective study has been carried out in three departments of neurology. an observation grid was established on the basis of guidelines on good practices. all in all, 53 criterions have been examined resuming: prerequisites before administration, patient setup, iv igs administration, monitoring, traceability of drug delivery and management of adverse side effects. results: during the course of this investigation, 51 administrations were observed. only 26% of nurses deliver information about the treatment to their patients before administration and 46% question patients about previous hypersensitivity reaction. the presence of spontaneous diuresis is verified in 14% of cases. emergency cart is not reachable in 33% of all cases. 78% of nurses ask patients to decline their identity. the use-by date on the bottles is checked in 51% of cases. at the time of preparation of perfusion, labelling does not mention either patient's name (48%) or date and hour of perfusion (93%). int j clin pharm (2017) 39:208-341 289 during perfusion, only 11% of nurses follow diuresis and 70% watch rate of administration. hydration is not always kept 20 min after the end of perfusion (80%). patient monitoring varies between 5 min and 1 h after perfusion's end. in 14% of cases, diuresis is monitored after the end of administration. 85% of nurses explain to patients side effects that may occur remotely. finally, administration traceability is was conform in 100% of all cases and in the event of adverse side effects, statement was made in 96% of cases. conclusion: best compliance scores have been achieved in myology department where patients are fewer than in the two others departments (6 vs. 20 and 25). a presentation of those results will be given in theses three departments in order to improve patient management and securitization of iv igs administration. this audit will be carried out soon in other departments. please specify your abstract type: descriptive abstract (for projects) background and objective: a new human polyvalent immunoglobulins dose (40 g) for intravenous administration is available on our establishment since 2014. in order to secure the administration, this new dosage was initially reserved for the healthcares using administration pumps (being four health-care). the aim of this survey was to evaluate the satisfaction of the nursing staff already user of the new 40 g dose and to estimate the motivation of the nonuser nursing staff by the audit date. design: this satisfaction survey was carried out with the most igiv consumer services (being internal medicine, neurology, cardiology and haematology). the questionnaire was structured in two sections: the first section regarding igiv in general, the second section concerning the new 40 g dose. the survey included multiple choice questions or questions with answers based on a four levels evaluation scale (not satisfied, mildly satisfied, satisfied, and very satisfied). results: the audit was realized on eight health-care, involving 41 nurses. among the 41 interviewed, 17 (42%) have already used the 40 g dosage. in 80% of cases, users were very satisfied and 20% were satisfied. the most positive points noted were: gain of time provided (89.5% of satisfaction), less manipulation needed (99.9% of satisfaction), and reducing of infectious risk (94.7%). moreover, the influence of the injection technique on users' satisfaction was further reported. indeed, according to nurses interviewed, the use of an injection pump is safer and improves the job comfort of nursing staff, unlike the injection by gravity (used in 14% of cases), which seems to slow down the use of this new dosage. in two cases, a positive opinion given by patient was also reported. finally, negatives points noted were related to administration instruments (use of pump or not) and to less flexibility in daily dose regulation. among the 58% not-user of this new dose, the 89% showed a strong interest for the product apart from services making the igiv administration by gravity. conclusion: in light of these results, the use of 40 g dose will be spread to other services. the general diffusion of this dosage will provide a gain of time also at the pharmacy, during the unitary delivery and the computer-based administration of every units. a second survey will be soon effected within patients involved in the switch 20 g/40 g. the capital region pharmacy, 2 clinic of neurology, rigshospitalet, blegdamsvej, copenhagen, denmark please specify your abstract type: research abstract background and objective: the clinic of neurology, rigshospitalet, copenhagen, denmark experience continuous medicine-related patient safety incidents (psi) related to newly admitted patients and patient transfers between wards. in order to prevent drug related problems (drp), the pharmacists increased their focus on these patients and provided systematic medication reconciliation. thus, the objective of this pilot study was to investigate if the intervention would help identify drug discrepancies (dd) and prevent drp. four wards were included in this study; two neurological, one neuro-anaesthetic (icu), and one neurosurgical ward(s). three wards use electronic medication module (epm), whereas the icu uses critical information system (cis). furthermore, all patients' prescriptions are registered on shared medication record (smr), which provides an overview of prescribed medicine. prescriptions cannot be transferred from smr and epm to cis and vice versa. we suspected that psi resulted from these system incompatibilities. setting and method: patients admitted or transferred from may 2nd 2016 to june 3rd 2016 were included. medication reconciliations using smr, epm and cis were conducted by a pharmacist on weekdays. dd were presented to a physician orally and documented. only dd accepted by physicians led to drug prescribing change. main outcome measures: number of identified dd. results: the study included 186 patients, of which 147 (79%) were newly-admitted. 39 patients (21%) were transferred between wards. of the transferred patients, 37 (95%) were transferred from the icu to other wards and 2 (5%) were transferred from other wards to the icu. of the newly-admitted patients, 44 (30%) were admitted to the icu and 103 (70%) were admitted to other wards. the pharmacists identified 16 dd; 3 dd (19%) in the transferred and 13 dd (81%) in the newly-admitted patients. in the transferred, 3 dd were all related to the icu. in the newly-admitted, 11 dd (85%) was related to the icu and 2 dd (15%) to other wards. of the 16 dds, 11 (69%) were accepted by the physician. an example of a severe dd identified was an omission of prednisolone to a patient admitted to the icu. conclusion: most dd were identified in patients admitted to or transferred from icu, which uses the incompatible system cis. pharmacist systematic medication reconciliation helps identify these dd and prevent drp. please specify your abstract type: research abstract background and objective: antibiotic related drug interactions are more likely in intensive care unit patients due to common polypharmacy and antibiotic usage. the aim of this study is to determine the antibiotic related drug interactions with three different online databases (micromedex-paid, medscape-free and drugs.com-free) and to evaluate these interaction information by clinical pharmacist. setting and method: a retrospective, descriptive study was set up in hacettepe university hospital's intensive care units, between november 10 and december 31, 2015. 62 patients who use at least one antibiotic were involved in this study. all drugs were assessed by each three databases and only antibiotic drug interactions were evaluated. clinical significance of identified drug interactions were evaluated by clinical pharmacist. main outcome measures: clinical pharmacist's assessment in significance of drug interactions indicated by three online databases. please specify your abstract type: research abstract background and objective: an implementation of clinical pharmacy practice by postgraduate students in intensive care units is a new way of learning in postgraduate education which creates opportunities in multidisciplinary collaboration in clinical pharmacy research, and also has influence on clinicians' routine patient care process. this system in educational program was ongoing in the department of clinical pharmacy since 2014. as a part of this educational program, drug related problems in intensive care units were described and analysed, an influence of clinical pharmacy postgraduate students on patient treatment process was sought. setting and method: a prospective, cross-sectional study was performed between the march-june 2016 in hacettepe university hospitals, department of internal diseases intensive care units which consists of 17 beds. three postgraduate pharmacy students from the department of clinical pharmacy, faculty of pharmacy conducted medication reconciliation in order to identify any problems in patients' medical orders. drug related problems (drps) were identified by the students and recommendations for management were approved by a supervisor of clinical pharmacy department before they were directed to physicians for approval. the students were not authorized to undertake any action in patient care process, therefore all required interventions for drp were undertaken by physicians and the acceptance ratio of the interventions were recorded. the pharmaceutical care network europe foundation classification system (v.6.2) was used to asses drps. main outcome measures: determination of drps by pharmacists and evaluation of their interventions' acceptance by physicians in intensive care units. results: during the study period, 106 patients were admitted to the intensive care units. each patient's medication orders were evaluated and 80 interventions were recommended by postgraduate students. the number of interventions per patient was 0.75. the acceptability rate of interventions by physicians was 96.3%. in addition, physicians were provided drug information on seven different occasions. recommendations regarding drug therapy were mainly related with treatment effectiveness and adverse reactions. the common causes of drps were requiring dose adjustment due to pharmacokinetic problems (42.5%), no therapeutic drug monitoring (18.8%), inappropriate timing of administration and/or dosing intervals (11.3%), requiring dose adjustment due to deterioration/improvement of diseases (6.3%), inappropriate drug selection (5%) and new indication for drug treatment presented (5%). the most common drugs responsible for drps were ranitidin, levothyroxine, allopurinol, pantoprazol, piperacillintazobactam and vancomycin. the study showed that the most common drps was dose-related, therefore close monitorisation of the intensive care unit patients by students in clinical pharmacy postgraduate program can help physicians in terms of detecting, preventing and minimizing drps in order to improve patients' health outcomes. please specify your abstract type: research abstract background and objective: antibiotic stewardship is the process of salvaging important antibiotic agents from becoming ineffective due to bacterial resistance. this is important because throughout the world antibiotics continue to be one of the most important classes of therapeutic agents due to their vital role in saving patient lives. key goals of antimicrobial stewardship are to improve clinical outcomes, prevent antibiotic resistance, promote patient safety, and reduce health care cost. pharmacist are in the frontlines because they perform antibiotic stewardship activities, such as selecting the most optimal antibiotic agent, adjusting drug-dosage, and stopping use of unnecessary antibiotics. as a result of the continuous rise in antibiotic resistance and decline in development of new antibiotics, antibiotic stewardship programs are proving to be indispensable in a health care settings. setting and method: 100 adult and paediatric inpatients receiving antibiotic therapy in the hospital medipol university has been evaluated. patients were selected randomly in the hospital system. patients were evaluated for antibiotic susceptibility results and compliance with antibiotic management guidelines. main outcome measures: to evaluate the antibiotic therapy in patients with culture results and to determine according the treatment guidelines. results: it was observed 10 different pathogens in blood culture results of 51 inpatients out of 100 patients who were treated with antibiotics in hospital. antibiotic susceptibility results for acinetobacter spp, staphylococcus spp, enterococcus spp, pseudomonas aeruginosa, klebsiella spp, e. coli spp, streptococcus spp, corynebacterium spp, streptococcus pneumonia and enterobacter spp are evaluated in the study. klebsiella spp was the most isolated pathogen at total of 85 culture results. most frequently resistance were int j clin pharm (2017) results: a total of 289 (28%) questionnaires were completed. of these, approximately 80% were answered by hospital nurses, the remaining mainly by physicians (18%) and 2% ''other''. on the question ''what is your general perception of the benefit of the clinical pharmacy service; for collaborating health professionals? for the patient?'' the total benefit was ranked 5.45 and 5.54 respectively (scale from 0 (''no benefit'') to 6 (''beneficial to a very large extent''). the open questions: ''what disadvantages/advantages have you experienced by the introduction of clinical pharmacist into multidisciplinary teams?'' received 153/185 comments respectively. physical obstacles regarding office space, interference with the decision making process, more time consuming processes and the issue of relying too much upon the advices given was reported as possible disadvantages. 120 respondents answered ''none'' to this question. the comments regarding advantages dealt mainly with general increased patient safety and quality assurance. in addition, advantages as work-load relieve, time saved, collegial support, practical help, and learning interchange between professions, were highlighted. conclusion: health-professionals assessed the clinical pharmacy service as highly beneficial. the advantages outlined were higher patient safety and quality regarding medication, in addition to collegial support, practical help and learning interchange. please specify your abstract type: descriptive abstract (for projects) background and objective: in june 2015, the french health authority, the «has», published an index resuming the recommendations of benzodiazépines (bzd) prescriptions and proposing an approach to stop using it. indeed, it has been established that there is a too high and too long consumption of bzd in france. a study of prescriptions' prevalence has been done in our hospital centre. the aim of this study was to know our situation regarding the use of bzd in order to set up some improvements and take part in their proper use. design: a prospective study has been done on a 5 months period in different services: geriatric, post-op and rehabilitation facilities, endocrinology, internal medicine, pneumology and cardiology services. the data were raised on a given day in each services and recovered thanks to the prescriptions software but also through interviews with the patients and their doctors. it was examined whether there was a bzd prescription (hypnotic or anxiolytic), whether the duration was superior or not to the duration of the amm and whether the prescription was done in our hospital centre. if the prescription was already part of the patient treatment, we looked if it was possible for the patient to stop using it, according to the has criteria. on their discharge, the letters and bzd prescriptions were also analysed and some patients' general practitioner were contacted after their discharge. results: 185 patients (median age 83 years old) were included from november 2015 to march 2016. 59.5% (110/185) of the patients had at least one bzd prescription the day we collected the data. we found only one bzd in 81 prescriptions (73.6%) and among them 77.7% (63/81) were anxiolytic bzd. among those prescriptions, 62.7% (69/ 110) already existed before the hospitalization and 37.3% (41/110) were given during the hospitalization (24 were prescribed automatically). 59.4% (60/110) of the prescriptions did not respect the legal duration of the amm (9 pieces of data were not found). 12.2% (5/41) already exceeded this duration limit. among the patients who already had a bzd treatment before going to hospital, 24.6% (17 out of 69) could consider stopping their use of bzd. by the end of this study, 143 patients were discharged from hospital, among them 48.3% (69/ 143) with a prescription of bzd. 55.2% (16/29) of the prescriptions established during the hospitalization had been renewed when the patient came out of the hospital, we managed to contact ten general practitioners (approximately 53.4 days after their discharge), nine patients carried on their bzd treatment, among them one patient had reduced his consumption. conclusion: this study is an example of the high proportion of bzd prescriptions in france which the majority doesn't respect the legal length of the amm. the prescriptions of bzd in the hospital are generally systematically renewed by the general practitioners. the patients must be informed about the risks of using those molecules. in order to ameliorate this practice in the hospital, a proper use leaflet, reminding the prescriptions of bzd, has been created and distributed in each services to make people aware. main causes of admission were infections (34%) (respiratory disease (23%) and other (11%)), hepatic disease (20%) and neoplasias complications (13.5%). 11 patients died during their admission; 5 due to hepatic disorder, 3 due to neoplasia, and 3 due to infections. conclusion: last diagnosis of hiv or no art treatment are causes of admission. immunovirological situation is related with their adherence but isńt with admissions. coinfection with hcv or hbv or others infections are risk factors for admission. center for psychopharmacology, diakonhjemmet hospital, oslo, norway please specify your abstract type: research abstract background and objective: complex medical history and treatment can potentially cause problems. the objective of this study was to investigate the prevalence of drug-related problems (drps) and medication discrepancies in internal medical patients with complex treatment at hospital admission. further, to investigate to which extent drps were identified as a result of medication reconciliation, and to which extent drps could be associated to the hospitalization. setting and method: patients with at least four regular medicines from two different therapeutic groups were consecutively included at admission to an internal medicine ward at a university hospital in norway in the period 01.09.14-15.02.15. pharmacists used the integrated medicines management (imm) model for medication reconciliation and medication reviews at admission. a medication discrepancy was defined as any discrepancy between the recorded medication list at admission and the patient's actual use of medications, as revealed by medication reconciliation. the patients' actual use of medications, medical journal and laboratory results, were used to perform a medication review at admission time and identify drps. the proportion of drps revealed due to medication reconciliation was calculated. moreover, the project group retrospectively assessed possible drp-induced hospitalizations based on clinical history, cause(s) of admission and identified drps. main outcome measures: the main outcome was the median number of drps per patient at admission. the proportion of drps revealed due to medication reconciliation, the proportion of patients with drps possibly associated to the hospitalization, and the median number of medication discrepancies, were included as secondary outcomes. results: 120 patients were included, 50.0% women. median patient age was 79 (range 27-96) and most of the patients were home-living before admittance (89.2%). in total 1359 drps were identified at admission, with a median number of 11 (range 2-27) per patient. 99 drps (7.3%) were identified due to medication reconciliation. for 25 patients (20.8%) a causal relationship between the hospitalization and the drps was assessed as ''possible''. medication discrepancies were revealed in 113 of the 120 included patients (94.2%), with a median number of 4 (range 0-14) per patient. conclusion: internal medical patients with complex drug regime are frequently exposed to drps and medication discrepancies at hospitalization. medication reconciliation could be essential to identify drps, which is likely a common cause of hospitalization in the studied patient population. hp-pc117: assessment of oral anticoagulant prescriptions and pharmaceutical analysis at the hospital by regional audit damien fuss *,1 , clélia monchablon 1 , anaïs breteau 1 , marie lefebvre-caussin 1 , rémi varin 2 , jean doucet 1 , mikael daouphars 3 , doreya monzat 1 1 omedit normandie -chu rouen, 2 chu rouen, 3 please specify your abstract type: descriptive abstract (for projects) background and objective: oral anticoagulants (oa) are the most common drug class associated with preventable adverse drug events in hospitalized patients that require optimizing the pharmaceutical analysis (pa) process. in this context, a regional audit was conducted on pa of prescriptions oral of oa. the aim of this study is to provide an overview of the treatment by oa in the hospital by evaluating the consistency of the oa prescriptions compared with national and european guidelines and evaluate the pharmaceutical interventions. design: this study is based on the collection of pa data (demographics, indication, posology, drug interactions, monitoring) as well as the collection of pharmaceutical interventions and discordance int j clin pharm (2017) 39:208-341 293 between guidelines recommendations and clinical practice. the inclusion criteria were any patient treated with oa (vitamin k antagonists (vka), non-vitamin k antagonist oral anticoagulants (noacs)). included patients were followed minimum 2 months. the primary outcomes include description of baseline characteristics of patients, the number of inappropriate prescriptions compared to the different clinical recommendations, the number of pharmaceutical interventions, the number of adverse drug reactions (adrs) related to oa use and the assessment of patient monitoring. results: during the 6-months study period, 588 patients were included in six health institutions. the average age was 78 years (70% of patients over 75 years old) and 59% of the patients were women. 32% of patients had renal impairment. 73% of patients were treated with vka, and 27% with noacs. it was the first prescription of oa for 27% of patients (56% with vka; 44% with noacs). the most common indication was the non-valvular atrial fibrillation (60%). in this indication, 99% of patients had cha2ds2-vasc score c2, and nearly 30% had a high risk of bleeding (has-bled score c3). 8 drug interactions were observed, and 35 adrs occurred related to oa. 42% of patients with an adrs had a has-bled score c3. 12.5% of prescriptions were considered inappropriate, including 45% noacs (no monitoring renal function in 13% of patients over 75 years initiating treatment, inappropriate posology in 14%, and 3% of contraindications). the rate of pharmaceutical interventions was 4%. nearly 60% of the prescriptions were already adapted when the pharmacist was starting analysis. conclusion: prescribers are sensitized of the risks on the oa prescriptions, which explained the delay upon pa and low rate of pharmaceutical interventions. however, the high number of inappropriate prescriptions shows the necessity to improve the pa process on these drugs, particularly by actions on therapy initiation and patient monitoring, especially for noacs. for this class, the impossibility of assess the level of anticoagulation by laboratory monitoring requires appropriate initiation and monitoring, especially an assessment of baseline renal function. please specify your abstract type: descriptive abstract (for projects) background and objective: the development of bacterial resistance these last 10 years is a public health major problem in the world and needs to implement actions. in france, the national drug safety agency has defined a list of ''critical antibiotics''. this list includes antibiotics particularly generator of bacterial resistance (amoxicillinclavulanate, cephalosporine, fluroquinolone) and antibiotics called ''last resort'' (antibiotics against gram-positive cocci, car-bapenem…). at our regional level, an evaluation of prescription of these critical antibiotics was proposed to all medical centers. the aim was to evaluate the quality of prescription of these critical antibiotics. design: the regional working group (pharmacists, infectious diseases physicians and biologists) had developed a collection grid including data on patients, antibiotics and four criteria: adequate molecule, compliance with medical prescriptions, duration of antibiotic therapy and reassessment at 72 h. this is a prospective study proposed to all health institutions (public and private), which had to be completed on a given day in all care units and had to be conducted by a team of multi-professional evaluators. the study included a quantitative part (number of patients hospitalized in the audited units, number of patients receiving antibiotics and number of critical antibiotic prescriptions) and a qualitative part (adequate to the four criteria). results: response rate was of 84%. the study investigated on 7026 patients hospitalized in the audited units, including 1391 patients (20%) receiving antibiotics. among the 1391 patients, 89% were hospitalized in medical, surgery or obstetrics units we recorded 865 prescriptions of ''critical antibiotics particularly generator of bacterial resistance'' (53% amoxicillin-clavulanate, 30% ceftriaxone, 14% fluoroquinolone and 3% other third-generation cephalosporine) and 42 prescriptions of antibiotics called ''last resort'' (74% carbapenem). the average age of the population was 69.9 years (±20 years). sex ratio was 1.1. 91% corresponded to curative use and 9% to prophylactic use. the expertise of infections diseases physician was requested in only 13% of the cases. the antibiotics were prescribed in majority to treat bronchopulmonary infections (38%), urinary tract infections (16%) and intraabdominal infections (16%). ninety-two percent of the prescriptions had a proper indication. 66% of the prescriptions complied to the guidelines. the duration of antibiotic therapy was adequate in 82% of the cases. only 45% of the prescriptions were correct according to these three criteria. forty-four percent of the prescriptions were reassessed and adapted by the physician. conclusion: this study is original because of its regional dimension and antibiotic analysis. the number of analysed prescriptions was significant with an overall proper prescription in adequate with the guidelines. however, actions must be implemented on duration and reassessment and adjustment of treatment. these results were presented to the participating hospitals. these three points will be reevaluated during a new regional audit. the criterion «no more 2 psychotropic drugs» has been met in 88.95% of assessment. otherwise, 2 or more psychotropic drugs are prescribed in 88.89% of assessment from the point of admission. the criterion «no more a benzodiazepine drug» has been met in 91.72% of assessment. otherwise, more than one benzodiazepine drug is prescribed in 59.26% of assessment from the point of admission. no contra-indication is detected in 93.25% otherwise, a contra-indication between two drugs causing torsade de pointes is detected from the point of admission in this department. no more 1 anticholinergic drug is prescribed in 84.05% of assessment. according to the french criteria, one or more inappropriate drug is prescribe in 46.93% of assessment. the most common inappropriate drug group prescribed was alimentary tract and metabolism drug (60.85%) (the hospital at home team needs these class of drug) followed by nervous system (25.95%) (prescribed at the point of admission) and by cardiovascular drugs (12.34%) (prescribed at the point of admission). finally, the criteria «no more one non-steroidal anti-inflammatory drug» and «no illogical association» have been met in all cases. conclusion: this analysis shows that most of criteria for «assessment of prescription among elderly in a «hospital at home» department have been met. when one has not been met, either the hospital at home team needs the drug prescribed, or this drug have been yet prescribed from the point of admission in this department. this study could be used for the next certification. hp-pc120: access to health care: case of autologous serum eye drops batiste martel, fabien lindenberg, camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: autologous serum eye drops (ase), prepared from patient's serum, are indicated in the treatment of severe dry eye syndrome and defective epithelial healing. its in-hospital preparation within a controlled-atmosphere zone unable it to be dispensed by non-equipped hospital pharmacies. the aim of this study was to implement security measures to allow transport towards distant hospital pharmacies and all patients even those residing far from a regional university hospital (uh). setting and method: this study was conducted in a 1495-bed french university hospital. patient blood samples were taken within the university hospital every 12 weeks. serum was then biologically controlled (negative tests for hiv, hbv, hcv, tpha, vdrl). preparation was conducted 3 days after blood sampling. sterile preparations were then stored at a temperature of -18°c. studies showed that eye drops were stable 14 days after being thawed. transport of eye drops to distant hospital pharmacies requires to be conducted under controlled temperature i.e. below 8°c, to ensure the stability of eye drops. these pharmacies are located close to patient's homes. the entire process was examined by a pharmacy team in order to study and secure each step, transport in particular. main outcome measures: validation of each step of the autologous serum eye drop dispensing process, from sampling to receipt by different hospital pharmacies, transport in particular. results: 4 patients benefitted from the preparation. all patients resided more than 100 kilometres from a uh. a follow-up form was completed to qualify dispatching and to trace each step during transport. a temperature sensor was placed inside the box. the receiving agent was required to stop and control the sensor. a double retrospective control was performed by a pharmaceutical team via the recording of temperature sensors. a second follow-up form was drafted in order to track dispensation reviews, ongoing dispensation and future planning. a patient information booklet was distributed to hospital pharmacies to inform patients about good practice concerning eye drops. conclusion: technological necessities concerning autologous serum eye drop preparation and transport limit access to health care. in this study, the role of the pharmacist consisted in reducing inequalities among patients residing at a distance from the only regional uh. the role of the pharmacist is to ensure absolute quality of preparation between the uh and the patient. hp-pc121: computerized medication reconciliation: overview of pharmaceutical software used and support for development of integrated modules julie mocquard, anaïs berthe, elise rochais * , nicolas prévost, jean-claude maupetit, on behalf of centre de ressources régional en conciliation médicamenteuse omedit pays de la loire, nantes, france please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation aims to improve continuity of care for patients. in 2015, a national survey identified barriers for implementation of this activity in france, among which computerized systems were judged unsuitable for hospital practices. in the absence of appropriate hospital information systems (his), medication reconciliation remains a time consuming process implying manual transcriptions, potentially leading to a lack of traceability and medication errors. the objective of the study was to assess the current his used in a french region including the integration of medication reconciliation into the software and to define courses of action to assist this integration. design: an online survey conducted in may 2016 was addressed to head pharmacists of the 134 health facilities in the region, giving a total of 100 head pharmacists concerned. it included questions on the software used by the health facility, the development of medication reconciliation and its traceability, formulation of operational requirements to the editors of software and availability of a module integrating medication reconciliation provided by the software. results: seventy-eight pharmacists (78%) participated in the study, with all types of health facilities represented: public hospitals, clinics, home health agencies, haemodialysis structures and after care and rehabilitation facilities. thirty different software were identified in the region. 27 (35%) pharmacists planned to develop medication reconciliation in their health facility and 20 (26%) were already carrying out this activity. within these 26%, medication reconciliation was conducted on paper only for 9 (45%) of them, while 11 (55%) were using a computerized system (patient file, pharmaceutical software, other) for traceability. the most widely used software in the region contains a module enable for computerized medication reconciliation, and three other editors are currently developing one. no development is scheduled for another three editors nonetheless commonly used in the region. 15 (19%) pharmacists had contact with the editor of the software, and 10 had given thought to the preparation of requirement specifications to the editor to develop an integrated module of medication reconciliation. conclusion: despite the interest attributed to medication reconciliation and despite the need of a fully integrated module of medication reconciliation to his, only a few health facilities of the region possess an appropriate computerized system to develop this activity. this study underlines the approaches already made by pharmacists to editors in order to integrate medication reconciliation to the his. subsequently, retrieving these approaches and writing specifications common to all health facilities is scheduled, in order to assist them in providing a strong incentive for the editors to integrate medication reconciliation to existing his. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is an interactive and multiprofessional process that ensures the continuity of care by integrating the ongoing treatment to the new hospital prescription. this helps securing the patient's care pathway particularly at transition points. the objective is to initiate the mr process in our medical institution with a pilot study in the department of internal medicineemergency downstream to validate a methodology and adapted tools. design: the mr takes place in three steps performed by a pharmacy student: (1) realization of best possible medication histories (bpmh), combining at least three sources of information and using sources' collection form. this research begins with a patient interview done in pairs with a medical student using an interview guide. (2) comparison bpmh with the initial hospital prescription in the department (after passing through the emergency department) on the treatment reconciliation form. a status is assigned to each line of drugs and then the differences are identified (stopped, changed or added). these two steps are validated by a pharmacist. (3) discussion and characterization of observed differences (intentional/unintentional and documented/undocumented) with the senior physician. results: twenty-six mr were performed over 3 weeks in 2015. the mr is performed within 2 days after admission. on average, 2.3 information sources per patient were used for the bpmh: mainly drug prescription (dispensed in pharmacies community); analysis of emergency medical records and patient interview. for the 26 patients included, 268 drugs were listed. 148 discrepancies were observed and 62 were studied (status stopped or changed only): one documented intentional discrepancy, 43 undocumented intentional discrepancies and 18 unintentional discrepancies (ud). these uds affected 12 patients (1-3 medication errors per patient) and corresponded to a non-prescribed drug in 90% of the cases. vitamins, antihistamines, anti-reflux and proton-pump inhibitors were involved in 59% of cases; cardiovascular drugs in 17% and antiinfectious in 12%. through this pilot study, the methodology was validated: (a) need to have a minimum of three sources to achieve a relevant bpmh and to confirm each information with two sources; (b) need for a dedicated time with trained staffs; (c) development of tools to improve the traceability of information obtained from each source and traceability of medication reconciliation activity. conclusion: the mr establishment in the internal medicine department was helpful in identifying 18 medication errors that have been corrected. it is proposed to archive the treatment reconciliation form in the patient file to contribute to the traceability of information on treatment. this study strengthens the deployment of this method and mr tools to other services of the hospital. alma mulac * please specify your abstract type: research abstract background and objective: clinical pharmacists have an important role in improving healthcare services. there is lack of knowledge of clinical pharmacists' experiences in interprofessional collaboration. our objective was to explore the challenges and barriers experienced by clinical pharmacists in interdisciplinary teams in norway and incorporation of expanded pharmacist roles in hospital settings. setting and method: this qualitative study was conducted using semi-structured interviews. a total of 13 clinical pharmacists from four (government) hospitals were included in the study. the interviews were audio recorded using a digital recorder. the recordings were transcribed verbatim. main outcome measures: challenges and barriers clinical pharmacists experience in interdisciplinary teams in hospital setting. results: the main findings are that the pharmacists' role is little known to other health care professionals, particularly at hospitals with short tradition for clinical pharmacy services. clinical pharmacists have great motivation from being able to influence drug treatment for patients. from the perspective of the participating pharmacists they succeed in interdisciplinary cooperation when their professional knowledge solves the patients' drug-related problems. communicating recommendations to physicians with professional credibility has great importance for the intervention to be implemented. using the theoretical framework of communicating tensions, we argue that the pharmacists in our study use indirect communication to prevent physicians defensiveness to recommendations. lack of education in interprofessional cooperation and communication is apparent in this study. the participants also stated that there should be some form of quality assurance or education requirements before one can work as a clinical pharmacist. conclusion: training in communication for graduates and interprofessional collaboration during the undergraduate pharmacy education, can possibly help pharmacists with integration in interdisciplinary teams. increased attention to teamwork from the hospital leadership is essential for the implementation of interprofessional collaboration in a larger context. please specify your abstract type: descriptive abstract (for projects) background and objective: antifungal therapy in the icu, particularly therapy targeting resistant aspergillosis, mucormycosis and systemic candida, is often of lifesaving importance. posaconazole and voriconazole are the antifungal agents of choice. our aim was to compile a tool that can be used at the icu to address aspergillosis, mucormycosis and systemic candida in an optimal manner. design: female patient, age 50 + , liver transplant, crp [ 300 mg/ l, creatinine [ 150 lmol/l. abdominal x-ray imaging revealed four large abscesses and laboratory analyses confirmed mucormycosis. posaconazole intravenous (300 mg one times daily) and liposomal amphotericin b (1 mg/kg/day) were initiated. the inflammatory markers remained unchanged 5 days following initiation of therapy with no change in size or number of abscesses and the patient developed sepsis. amphotericin b dose was increased to 3 mg/ kg/day. after 1 week the inflammatory parameters and size of abscesses began to fall. the dosage form of posaconazole was switched from intravenous to mixture. the dose remained the same and within 24 h the crp rose to 600 mg/l. results: pharmacist intervention revealed a missing loading dose of intravenous posaconazole as well as incorrect dosage of the per oral form due to bioavailability variation. posaconazole mixture dose was increased to 400 mg two times daily. through serum concentration analysis of posaconazole was suggested prior to the dose increase. the serum concentration was 0.6 mg/l (range [1.0-1.25) . through serum concentration 4 days later was 1.2 mg/l. both crp and abscess size were on the decline. a dosage and tdm pocket card for posaconazole therapy of mucormycosis, aspergillosis and candida was compiled. conclusion: optimal systemic fungal infection therapy is essential, especially in the critically ill. of special importance is tdm and correct dose adjustment when dosage-form changes occur. please specify your abstract type: research abstract background and objective: potentially inappropriate prescriptions and omission of prescription, respectively ip and op, are common issues in the pharmacotherapy, especially in vulnerable population, such as elderly and children. there are many available tools detecting ip and op for geriatrics, however, similar tools are less common in paediatrics. therefore, a first target tool for paediatric population: popi «paediatrics: omission of prescriptions and inappropriate prescriptions» was created and was validated by delphi method in 2014. we aim to evaluate inter-rater reliability between health care professionals, who apply popi. our study also assessed their satisfaction and the accessibility of this tool. setting and method: twenty cases with or without ip or op were selected. these cases were identified in a previous retrospective ip-op prevalence study on 15.973 patients. these patients were admitted to the emergency department of a university mother and child hospital, between october 2014 and march 2015. one doctor and one pharmacist, who participated in the creation of popi tool, identified ip and op in 20 cases and composed ''standard answers''. these cases were then reviewed independently by eleven clinicians (including generalists, paediatricians, pharmacists, residents, general practitioners), who did not experience this tool before. inter-evaluator agreement was calculated by using the agreement kappa test. the satisfaction of users was also evaluated. main outcome measures: inter-evaluator agreement, the median time of use and the satisfaction of users. results: a high level of agreement of ip and op detection was recorded (ip: k median = 0.80; op: k median = 0.71). the easy use of popi was approved by 91% evaluators. the median time of use was 2 min 45 s per case (quartiles : 2.4-3.4) . as a result, there were 82% of clinicians satisfied with the provided popi and they would like to apply this tool in their daily practice. conclusion: popi demonstrated a good interrater reliability and is easy to use. this strong validation by many specialists prove popi is a reliable tool. it can be applied daily at work in paediatric section by doctors and pharmacists. other multicentre and prospective study should be conducted to evaluate economical and clinical impacts of popi. please specify your abstract type: descriptive abstract (for projects) background and objective: drug dosing during cvvh is challenging due to changes in pharmacokinetic parameters brought about by the patients' deterioration in health and factors associated with the physical process of filtration. this is of particular significance in the icu. in addition, there is the issue of the patients' diuresis or lack of such. this will affect the total clearance (cl total ) of the drug. the dose of antibiotics must therefore be calculated individually taking into account all of the above as well as changes of filtration parameters. our aim was to illustrate how such dosage calculations can be undertaken. design: a 50-year-old male patient, weight 75 kg, diagnosed with stenotrophomonas maltophilia infection. the trimethoprim/sulfamethoxazole dose was 7.5 mg trimethoprim/kg/day every 8 h as specified for anuric patients on cvvh. patient was initially anuric for 3 days after which diuresis was started. the dose was recalculated. results: creatinine clearance (crcl) related to cvvh during the anuric period was calculated accounting for ultrafiltration rate, sieving coefficient, blood-flow, haematocrit concentration and pre-dilution. the value was 22 ml/min. following diuresis on day 4, remaining kidney function was assessed by measuring urine and serum creatinine. the value for crcl renal (18 ml/min) was added to the extracorporeal clearance, and gave a total clearance of40 ml/min. this warranted dose adjustment of trimethoprim/sulfamethoxazole since this drug requires normal dosage at crcl [ 30 ml/min. conclusion: during cvvh, the presence or absence of diuresis must be taken into consideration when dosing antibiotics. in anuric patients, the cvvh-machine set up constitutes crcl total , but in patients with diuresis, the remaining crcl renal should be added. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of the study is; to evaluate patients' home (prescribed and non-prescribed) and hospital medication during hospital admission by computing medication regimen complexity index and investigating possible drug-drug interactions. design: patients (aged 18 and older) who applied internal service during 6 months (2 days/a week) were included to the study. patients' medical profile were obtained from patients' file. their home medication and hospital medication were calculated with medication regimen complexity index (1) and checked drug interactions with micromedex drug interaction program. results: a total of 151 from 360 of patients who applied to the internal service (male 46.4%, female 53.6; the mean age of patients was 69.01 ± 16.28.) were included to this study during 6 months. of them, 75.5% had low education level (\8 education years), 53.6% had 2 and more chronic diseases of them, 45% hospitalized last 6 months before this hospital admission. the most prevalent diagnoses documented at admission were kidney disease (19.8%), cardiovascular disease (15.3%) and cancer (13.2%). the mean of patients' home medication number was 5.64 ± 3.50 and the mean of their mrci scores was 16.02 ± 10.89. 45% in patients hospitalized in the last 6 months. at least one possible drug-drug interactions were found in 66.6% of patients at home medication and in 78.8% of patients at hospital medication, respectively. the mean number of possible drug-drug interactions at patients' home medications was 2.88 ± 3.62, while the mean number of possible drug-drug interactions at patients' hospital medications was 4.07 ± 4.06. of them, 53.6% had polypharmacy at home medication. the frequency of possible drug-drug interactions and the score of medication complexity index was found high among patients' hospital medications when compared with their home medications. conclusion: the potential role of pharmacist including medication reconciliation and medication review could improve rationale drug use during hospital admission. coronavirus. experts' local committee has approved to use oral ribavirin for the treatment of these respiratory viral infections. we aimed to assess the effectiveness and safety of oral ribavirin as main treatment in respiratory viral infections. setting and method: from may 2013 to october 2015, we performed a retrospective monocentric study including patients who received oral ribavirin for non-hcv infections. main outcome measures: viremia negativation was used to determine the response rate to oral ribavirin. specific toxicities (anaemia, cytopenia, liver dysfunction) and renal function were assessed biologically. results: thirty-five immunocompromised patients (f/m: 3/4, age: 57) were included. underlying conditions were lung transplant (n = 32), heart transplant (n = 1), pulmonary fibrosis (n = 1) and acute myeloblastic leukaemia (n = 1). the median duration between transplantation and infection was 1.8 years (0.1-10.8). nine patients were exclusively infected by rsv, 19 by hpiv (2 hpiv-1; 2 hpiv-2; 10 hpiv-3; 4 hpiv-4; 1 non-identified hpiv), 4 by hmpv and 1 by coronavirus. there were six co-infections: rsv/ hpiv-1, rsv/coronavirus, hpiv-2/hpiv-4 and hpiv-3 or 4/coronavirus (3 patients). all the patients were admitted in pulmonary division, except for the patient with heart transplant who was in cardiac intensive care unit. the administered dose was 400 mg tid or 200 mg tid if there was renal insufficiency (9 patients). the median duration of the treatment was 8 days . four patients prematurely discontinued the treatment due to severe toxicity or therapeutic change; three didn't respond to the treatment (no data for the last one). four patients were re-treated despite having a virological response to the first cure. one patient treated for a hpiv-3/coronavirus coinfection had an hpiv-3 relapse 64 days after ribavirin discontinuation. concerning the three other patients, they received a second cure to treat a new infection (coronavirus, hpiv-4 and hmpv, in opposition to hpiv-3 twice and hpiv-2 respectively). virological response rate was 82% (7/11 for rsv, 22/25 for hpiv, 4/5 for coronavirus and 4/4 for hmpv). two non-negative viremia patients (rsv and hpiv-4/coronavirus) received intravenous ribavirin after oral ribavirin therapy. no patient died from viral infection. twelve patients presented specific toxicity: one hepatic cytolysis and cholestasis, eight haemoglobin decrease, two pancytopenia and one mucositis. conclusion: despite the poor number of patient, our study shows that oral ribavirin seems to be efficient to treat hpiv, hmpv and coronavirus in immunocompromised adults. we observed known side effects that could generally be managed. oral ribavirin may thus represent a therapeutic strategy in several respiratory viral infections. please specify your abstract type: descriptive abstract (for projects) background and objective: reconciliation of medicine lists is important to ensure correct medical treatment of patients both in hospital and other healthcare levels. while reconciliation upon admission is part of the normal routine at surgical ward b, molde hospital, there has been less focus on reconciliation at discharge. as such, this study aimed to ensure reconciliation and correct transfer of medical information at discharge. design: medicine lists of all patients discharged from surgical ward b, molde hospital between week 39 and 51 in 2015 (n = 240) were investigated. the forms were gathered and counted based on the tasks signed for to ensure completed reconciliation and sufficient information given to the patient. the count was performed every 2-3 weeks, and the forms in each count was pooled together as one point of measure. the quality of medicine lists in discharge lists was evaluated based on the norwegian patient safety program criteria. medicine lists in discharge lists from week 39 to 51 (n = 102) were pooled together and compared to medicine lists in weeks 36-39 (n = 46). results: the results of reconciliation was divided into the subsections of surgical ward b, and represent the number of completed tasks as signed for in the reconciliation form. the surgical subsection showed a significant increase in patients with pre-checked medicine lists and reconciled medicine lists over the measured time period. similar results were not found in the orthopaedic subsection. as for the quality of medicine lists in the discharge lists, significant improvement was seen in all set criteria, with the exception of ''source'' in the surgical subsection. in the orthopaedic subsection however, no significant improvement was seen in any of the criteria other than ''indication for use''. conclusion: the implementation of reconciling medicine lists at discharge was successful. however, both subsections need to work further to ensure continuation and improvement of the process. furthermore we found varying results in the writing of medicine lists depending on subsection. still, regardless of the individual results of the two subsections there is big room for improvement to ensure that sufficient medical information is included in the discharge papers. please specify your abstract type: descriptive abstract (for projects) background and objective: from july 2015 clinical pharmacists began conducting medication histories and reviews (pharmacist notes) at the emergency surgical ward (esw), north zealand hospital (nzh). inclusion criteria are acute patients using c5 drugs or c1 risk drug (antidiabetics, anticoagulants, antipsychotics, benzodiazepines, opioids and digoxin). the aim of the service is to identify drugrelated problems and secure correct medication reconciliation between the medicine the patient is admitted with and the medicine in the electronic medication system (ems) in the hospital. the service ensures that the patients' medication follows across healthcare sectors. the objective is to determine if the discrepancies between the medicine the patient is admitted with and the medicine in ems (documented in the pharmacist notes) are used by the physicians. in addition to determine if the pharmacist interventions increase the physicians' acceptance rate of the discrepancies. design: data were collected at the esw at nzh (capacity of 27 beds). data consist of pharmacist notes conducted from august 2015 to may 2016. pharmacist notes were compared to the patient record and ems to identify if the pharmacist notes were considered by the physicians. in order to increase physicians' acceptance rate of the discrepancies suggested in the pharmacist notes, interventions were made according to the model for improvement. throughout the period, the focus was on oral delivery of the pharmacist notes. in december 2015 the pharmacist optimized the clinical relevance of the discrepancies, by creating and testing a list of products (including vitamins, herbal drugs, glucosamine etc.) which the pharmacist should not intervene on. in december 2015 the pharmacist also started to follow up on the pharmacist notes not considered by a physician the previous day to ensure that the physician considered the discrepancies. results: there were identified 599 discrepancies between the patients' actual medication at admission and ems at the hospital in 306 patient records (1.96 discrepancies per patient). in total 424 discrepancies were accepted by the physicians (1.39 discrepancies per patient). the physicians' acceptance rate was based on the acceptance of one or more of the discrepancies in the pharmacist note. baseline data were collected from august to november 2015, where 54 out of 85 pharmacist notes were accepted by the physicians resulting in an acceptance rate of 63.5%. from december 2015 to may 2016 the interventions made by the pharmacist contributed to an increase in acceptance rate to 81.8% (108 out of 132 notes accepted). if the pharmacist notes were not delivered orally to the treating physician the acceptance rate was 9% (8 out of 89 notes accepted). conclusion: the pharmacist interventions contributed to an increase in the physicians' acceptance rate of discrepancies from 56.5 to 81.7%. a result indicating that the pharmacist notes contributes to an increase the quality of the medication process across sectors. hp-pc134: how the centralization of medicines manufacturing enable to generalize the pharmaceutical validation? samantha oses * , soizic vandierdonck, vincent servant, dominique breilh please specify your abstract type: research abstract background and objective: the centralization of the reconstitution of injectable anti-infective drugs enhance to decrease costs and several risks. this minimization of the risks operates at several levels such as i) reduction of the staff exposure and external contamination of preparations during the reconstitution phase (with controlled atmosphere areas, isolators, etc.), ii) improvement in the quality of the management of infective diseases thanks to a pharmaceutical validation systematically performed after the prescription and before the reconstitution phase. the main objective of the study was to describe and quantify pharmaceutical validation on injectable anti-infective drugs prescriptions restored in a pharmaceutical reconstitution unit. setting and method: an observational descriptive study was carried out on each prescription with at least one injectable anti-infective drug that has to be reconstituted before administration. the process was as follows: 1-prescription by the physician on an electronic prescription software, 2-pharmaceutical validation and if necessary pharmaceutical intervention (pi) made by phone call, 3-reconstitution at the pharmacy, 4-administration to the patient. the pharmaceutical validation methodology followed the french society of clinical pharmacy (sfpc) guidelines ''prescriptions screening and analyses level'' published in the good practices of clinical pharmacy and one resident and one pharmacist were devoted to the activity every day. main outcome measures: the pharmaceutical validation was quantified by the number of pi by patient, which were categorized according to the sfpc guidelines. results: during 4 months, a total of 222 pi were collected. they concerned 93 patients with an average of 2.4 pi per patient. among them, 11.8% (11) concerned paediatric population. antibiotics were involved in 53.2% (118) then followed by 36.9% (82) cases (59.5% (69) biological assessment issues, 37.1% (43) absence of therapeutic drug monitoring (tdm) and 3.4% (4) the drug hasn't been adapted to the weight), dosage adjustments in 25.2% (56), information missing concerning the treatment indication in 15.8% (35) and miscellaneous pi in 6.8% (15) such as wrong clinical service on the prescription, etc. approval rate of physicians was 79.3% (176). conclusion: this study has shown that even if prescriptions were secured by electronic prescription software, the pharmaceutical validation remains essential. in that case, the centralization of the reconstitution of injectable anti-infective drugs enabled to generalize this activity on all prescriptions of the hospital. however, the pharmaceutical validation was focused only on anti-infective drugs, that was not fully efficient and must be extended to the whole prescription. it is a priority to develop a comprehensive and exhaustive validation on every medical prescription; however, this activity is highly time consuming and needs larger and more trained staff. hp-pc135: the start/stopp criteria as a helping tool to the pharmacists' medication review in the acute admissions unit of the regional hospital in horsens hans pedersen * please specify your abstract type: research abstract background and objective: polypharmacy occurs often increasing the need for patients' medications to be reviewed. the start/ stopp criteria help detects potentially inappropriate prescriptions in older people. in this study we aimed to measure and categorize the different start/stopp criteria found in medication reviews in the acute admissions unit of horsens and the acceptance rate. setting and method: patients admitted to the acute admissions unit were selected based on their age and the number of prescriptions in a period of 4 months. patients 65 years or more which received six or more drugs were included in the study. only patients who later were transferred to another medicine ward were included in the study. the pharmaceutical medicine review was performed by a clinical pharmacist using minimum two different sources; the electronic medical record and medication-lists. the guideline of pharmaceutical medicine review in the hospital pharmacy central denmark region was used as the standard-guideline. in addition, thestart/stopp criteria version 2 was used. main outcome measures: the number of start/stopp criteria found in medication reviews. the different start/stopp criteria were scored equally with one point each. results: 33 patients, 17 males and 16 females, out of 55, were included. the mean age was 79 years and the patients received in average 14 prescribed drugs. at admission the average number of stopp criteria were 1.1 ± 0.9 and 0.4 ± 0.6 for the start criteria. in average, 27% of the purposed stopp criteria were accepted by the physicians. the most frequently accepted stopp criteria were in the category of drugs that predictably increases the risk of falls in older people. the benzodiazepines where the most common drugs to be discontinued. in the start category, 25% of the suggested start criteria were accepted, which included: calcium and vitamin d3 supplement, beta-2 agonist and bisphosphonate. conclusion: the present study demonstrates that it was possible to integrate the start/stopp criteria as a helping tool in the medication reviews in the acute admissions unit of horsens. the start/stopp criteria were found within the different categories, however only a minor part of the registered start/stopp criteria were accepted by the physician. please specify your abstract type: research abstract background and objective: the objective of this work is to assess prescribing practices of somatostatin analogues in a surgery department, and to analyse the conformity of switching from immediateacting octreotide to the long-acting release (lar) form, in accordance to laboratories' guidelines. setting and method: retrospective observational study. a focus was realized on patients admitted in a digestive surgery unit between january 1 and december 31, 2015. the patients' medical records were reviewed for clinical features, diagnosis workup and treatment strategies. main outcome measures: medical records for patients with diagnosis of gastro-entero-pancreatic or endocrine tumors who had received injections of lar octreotide during hospitalization were reviewed and the economic impact of prescriptions errors has been evaluated. results: of the evaluated 234 patients, 73 (31%) were hospitalized in surgery digestive unit; mean age at first administration of octreotide was 66 years and 58% were male. the male and female ratio was 1.35:1. reasons for hospitalization were: digestive system neoplasms (75%), fistula (7%), intestinal obstructions (4%) and other pathologies (14%). of the 73 patients treated with octreotide, 41 (56%) received a lar form. only four patients received doses in accordance with guidelines: one at 20 mg/month lar form and three at 30 mg/month lar form, after having respectively been treated by intravenous octreotide at 300 and 600 mcg/day during 7-10 days. medical prescriptions of the 37 remaining patients did not comply: all patients received 30 mg/month after an intravenous treatment of 300 mcg/day, instead of 20 mg/month. from a financial perspective, these misuses have led to an additional cost of 6659.7 euros for the hospital, excluding tax (30 mg: 1389.29€/unit and 20 mg: 1212€/unit). conclusion: despite the publication of octreotide release form proper use recommendation in our hospital, 90% of patients of digestive unit are not right treated. a new guideline will be written added by doses of long-acting release and economic data. this work will be transmitted to specialists by clinical pharmacists. hp-pc137: pharmaceutical process for intrathecal analgesia in clinical oncology practice vivien pigeon * , guillaume binson, claire grignon, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: in some cases, patients with cancer pain remain painful despite the use of high dose of intravenous opioids and intrathecal analgesia becomes the ultima recourse to manage acute pain. until 2015, intrathecal syringes were prepared by nurses in the unit care which involve a risk for patients. therefore, the aim of this work is to describe the set-up of the prescription and preparation process with the potential benefits for the safety. design: multidisciplinary concertation took place between pharmacists, physicians and surgical teams and several points were discussed to secure the process: • identification of patients with high level of infection risk; • identification of critical points of the pharmaceutical process; • validation of quality control and drug stability studies regarding drug compounding involving morphine, ropivacaine, baclofen and clonidine, alone or in admixture. results: multidisciplinary concertation lead us to define the most important points to set up the pharmaceutical process for intrathecal analgesia: • chosen patients are cancer patients; • implementation of a prescription software to secure the prescription step; • production of syringes by the pharmacy department implying several criteria: • preparation in controlled atmosphere area; • training of pharmacy technicians; • implementation of quality control and drug stability studies at 4°c in syringe over 48 h and at 37°c in pumps over 1 month; • microbiological control and bacterial endotoxin level. the implementation of a pharmaceutical process for intrathecal analgesia gave us the opportunity to reorganize the care of cancer patients tolerant to high dose of opioids. in this process, the pharmacy department plays a major role leading to decrease the risk of infections and errors of dosing. ingrid plessala *,1 , xavier deviot 1 , thomas sidibe 1 , zohra mostefaï 2 , michèle minvielle 2 , marta wyrtwal 2 , roselyne gervais 1 1 pharmacy, 2 geriatrics, saint-denis hospital centre, saint-denis, france please specify your abstract type: descriptive abstract (for projects) background and objective: proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. design: prospective study in three geriatric wards. the study included ppis treated patients from these three geriatric wards. dose, indication of the ppi, age, gender and duration of treatment have been recorded for each patient. the relevance of each ppi treatment has been reassessed by a geriatrician, a pharmacist and a junior pharmacist, regarding the indication and the patient's clinical condition. following this re-evaluation, three situations arose: • to maintain ppi at the same dose (30 mg or 15 mg) • to maintain ppi but half dose (from 30 mg to 15 mg) • to stop ppi corrective actions have been recorded in patients' files to allow their traceability. results: 109 patients were included in the study. 61% of ppis prescriptions were off-label, 24% had no indication mentioned in patient's file and 11% were conform to the marketing authorization. 98% of patients have been on ppis medication longer than 2 months, which is the recommended treatment' duration in france, 55% longer than a year and 29% longer than 4 years. in collaboration with the geriatricians, ppi prescriptions were maintained for 52% of patients. we reduced the dose in 14% of cases. finally, we decided to stop a third of the ppis prescriptions. conclusion: ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since 2009 may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in 2017. ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since 2009 may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in 2017. hp-pc139: oral anticoagulants and heparin for children: standardized protocols for prescription, dispensation and administration alexandra liauzu 1 , marie-françoise hurtaud-roux 2 , ronan bonnefoy 3 , caroline farnoux 4 , philippe sachs 5 , theresa kwon 6 , olivier bourdon 7 , sophie ajzenfisz 8 , sonia prot-labarthe *,7 1 pharmacy, 2 hématologie clinique, ap-hp hôpital robert-debré, 3 cardiologie, 4 néonatologie, ap-hp hôpital robert debré, 5 réanimation pédiatrique, ap-hp hôpital robert-debré, 6 néphrologie, 7 pharmacy, ap-hp hôpital robert debré, 8 coordonnateur de la gestion des risques associés aux soins/ responsable du système de management de la qualité de la prise en charge médicamenteuse, ap-hp hôpital robert-debré, paris, france please specify your abstract type: research abstract background and objective: high-alert medications (ham) are medications that are associated with a high risk of serious harm if used improperly. we already identified paediatric ham used in our institution to identify safety measures for their use. anticoagulants and heparin were part of these high-alert medications. we aim to write standard protocol of use for low weight heparin and oral anticoagulant used in our mother-child teaching hospital. our secondary objectives were to decrease medication errors, anti-xa and inr unexplained variability and to help nurses to administer the drugs (standard dilution, oral solution available) setting and method: we carried out a literature search on pubmed ò , on websites of several learned or professional societies and agencies. the results of the literature search were compiled on written protocol and presented to our institute drug safety-steering committee composed of four doctors, two head nurses, two pharmacists, and one risk manager. main outcome measures: not applicable. results: the protocols concerned enoxaparin, tinzaparin, warfarin but we chose to also include protamine. the most difficult issue was to have standardized dilution and protocol for all ages and weight: from premature to adolescents and all units of care (from cardiology to intensive care unit, nephrology and neonatology). we took into account the administration errors we had in our hospital and the preexisting protocol to avoid any drastic error-prone change. the final version of these protocols will be presented on the final communication with web link to upload them. conclusion: for now we did note evaluate the impact of these protocols but a before/after analysis of error reports and users evaluation will be done. however, these protocols can help all health professionals working in paediatric units for benchmarking. hp-pc140: does a hospital formulary system impact timely medication administration and quality of inpatient care? anne-valérie putallaz *,1 , vera jordan-von gunten 1 , pierre-auguste petignat 2 , pierre turini 3 , johnny beney 1 1 division of pharmacy, institut central des hôpitaux, 2 division of internal medicine, 3 medical coordinator for quality of care and patient safety, hôpital du valais, sion, switzerland please specify your abstract type: research abstract background and objective: the prevalence of drug omissions is often underestimated but their impact can be clinically relevant. we hypothesized that delays in the administration of non-formulary/nonstored drugs could impair the quality of care. the aims of this study were: 1°to determine the time between the prescription and the administration of the first prescribed dose and, if applicable, to calculate how many doses were omitted. 2°to analyse the clinical relevance of the identified delays. setting and method: three months retrospective study of electronic records of patients hospitalized on the internal medicine wards of a network of hospitals supplied by a centralized pharmacy. this pharmacy is located in one of the sites; other sites are 15-45 km apart. main outcome measures: 1. for the main hospital site and the three distant sites: • median time between the prescription and the administration of the first prescribed dose • mean number of omitted doses for formulary and non-formulary/ non-stored drugs. 2. categorization of patient's harm caused by the delays of timecritical drugs, according to the ncc-merp taxonomy of medication errors. results: 16'954 prescriptions were analysed. calculated delays for non-stored/non-formulary drugs were longer than for formulary drugs. however, the median time to administration is less than 1 h for both formulary and non-stored/non-formulary drugs; and more than 95% of formulary drugs and around 90% of non-stored/non-formulary drugs were administered within 24 h following their prescription. there was no significant difference in the mean number of omitted doses or in the delays between the site where the centralized pharmacy is located and the other sites, except for one of them. a delay representing 1.5 or more omitted doses was found for 332 (1.96%) prescriptions. among them, only 17 were considered potentially clinically relevant. none of them caused severe harm to the patients involved. conclusion: in our setting, non-stored/non-formulary drugs take more time to be delivered than formulary drugs, but more than 95% of formulary drugs and around 90% of non-stored/non-formulary drugs are administered within 24 h following their prescription. none of the 17 patients who experienced delays underwent severe harm. our study showed that delays also occur for formulary drugs but no systematic cause of omission was identified; further studies should focus on all dose omissions during hospitalization. penelope randuineau *,1 , roger jeremy 1 , lauriane cornuault 1 , anne lecoeur 1 , franck lemercier 1 , isabelle javerliat 2 , thomas tritz 1 1 service de pharmacie à usage intérieur, 2 service de chirurgie vasculaire, hôpital ambroise paré, boulogne-billancourt, france please specify your abstract type: descriptive abstract (for projects) background and objective: a french national survey of inpatient adverse events reveals that nearly half of adverse drug events (ade) are preventable. medication errors behind these ade occur mainly during the transition steps of care pathway. in this context, medication reconciliation process has been implemented in our vascular surgery department. the objective of this study is to identify unintentional discrepancies (uid) and assess their potential clinical impact design: a pharmacy resident or a pharmacy student reconciliated patients: aged older than 65 or with at least five chronic treatments at admission or suffering from chronic diseases. patients were considered reconcilable if at least two reliable sources on usual patient's treatment were available. these many sources of data (patient interview, prescription or interview of general practitioner, reference dispensary, drug box …) were compared to the admission prescription during the first 48 h of hospitalization to detect and correct uid. based on gravity scale promoted by the french high authority of health, two pharmacists (a resident and a senior) and a vascular surgeon reviewed every uid in order to define their potential clinical impact. the uids were considered minor if it leads to no consequence for the patient, clinically significant if it leads to essential monitoring, major if it could cause temporary clinical consequences, and critical if it could result in permanent clinical consequences or the involvement of the prognosis. results: between february 15th and may 31st 2016, a total of 102 patients have been reconciled. 10 patients were excluded due to a lack of reliable sources. mean age was 73.9 years old (±11.4) and sex ratio m/f was 0.7. 85% of the reconciliated patients' admissions were scheduled. the mean number of medication was 10.8 (±2.9). 38 patients (41%) had at least one uid and the mean uids per patient was 2.0 (±1.6). the most common types of uids were omission (73%), incorrect dose (15%) and incorrect administration frequency (11%). more than 60% of these uid presented a potential clinical impact: an adverse effect (high blood pressure, hyperglycaemia) was observed for nine patients and lead to therapeutic optimization and monitoring; 37 uid were considered to have potential clinically significant impact (49%), 11 a potential major impact (15%) and 1 a potential critical impact. conclusion: these results appear consistent with those reported in literature. vascular surgeons have appreciated the approach and would like systematic medication reconciliation before surgery. as a major part of admissions were scheduled, we would like to establish the reconciliation before the patient's hospitalization every time it's possible. this new organization should facilitate the care pathway before surgery and decrease preventable postoperative adverse events. hp-pc142: delirium in elderly patients: successful use of melatonin gaëlle jouin 1 , aurélie reiter-schatz 1 , pierre bentzinger 2 , fatem-zohra laalou 2 , bénédicte gourieux *,1 1 pharmacy-sterilization, 2 orthopedic's intensive care unit, university hospital of strasbourg, strasbourg, france please specify your abstract type: descriptive abstract (for projects) background and objective: postoperative delirium happens to about one-third of elderly patients and is a major cause of morbidity and mortality. it is reported that haloperidol, an antipsychotic, has been the agent of choice for managing delirium. however, it induces cerebrovascular adverse effects and greater mortality. the hyperactive type of delirium is known to be associated with a low melatonin level and the loss of a normal melatonin secretion rhythm. the postoperative administration of melatonin to elderly could decrease the symptoms of delirium. the purpose of this study was to evaluate melatonin effectiveness in a cohort of patients suffering from postoperative delirium. design: a retrospective study of melatonin prescriptions has been conducted over a 12 months period. medical background, type of surgery, symptoms of delirium, use of antipsychotics and benzodiazepines have been studied in all patients who received melatonin in an orthopaedic surgery unit. length of hospital stay, time between delirium and melatonin administration and the effect of melatonin had been evaluated. results: a total of 14 patients were included: average age was 77.6 years (64-87), sex ratio m/f = 1. twelve patients (86%) were hospitalized because of an infection (prosthesis or osteoarticular). in 64% of cases (n = 9), the prescription of melatonin was started when the patients were hospitalized in our intensive care unit. nine patients (64%) were under chronic treatments like benzodiazepines or antipsychotics. the average length of hospital stay was 76 days (11-186). melatonin was started on an average of 14 days after surgery , and administered at the dose of 2 mg xr in the evening, during an average of 35 days (7-113). cognitive impairments requiring a prescription of melatonin were: confusion (86%, n = 12), agitation (64%, n = 9), daytime sleepiness (64%, n = 9), temporal-spatial disorientation (57%, n = 8), nocturnal awakening (14%, n = 2), hallucination (14%, n = 2), difficult falling asleep (7%, n = 1). the average time to recover from confusion was 8 days, agitation 4 days, daytime sleepiness 10 days, temporal-spatial disorientation 7 days, nocturnal awakening 16 days, hallucination 5 days and falling asleep 24 days. melatonin treatment helped stopping benzodiazepines treatment in six patients (66%). conclusion: after administration of melatonin, delirium symptoms were improved for all patients and benzodiazepines treatment stopped for six patients. earlier prescription of melatonin could regulate sleep-wake cycle and reduce the duration and incidence of delirium. please specify your abstract type: research abstract background and objective: denosumab (xgeva ò ), a fully human monoclonal antibody targeting rankl, which inhibits bone resorption, is indicated to prevent skeletal complications in patients with solid tumors and bone metastases. about 10% of patients develop hypocalcaemia, a common adverse event that may induce spasms, muscle cramps, paraesthesia, prolonged qt interval, tetany, convulsions… we report the management of ionic supplementation and physicochemical incompatibilities in a case of hypocalcaemia due to denosumab. setting and method: the clinical case was analysed with the pharmacovigilance regional center. main outcome measures: a 61 year old patient, with nodal and bone metastasis in prostate cancer, was treated with denosumab (stopped with the last injection 2 months before, on the 29th of march). he went to emergency on the 30th of may with asthenia, anorexia, nausea, diarrhoea, qt prolongation. biological results showed hypocalcaemia (corrected calcaemic = 1.94 mmol/l) and hypophosphatemia (phosphorus \ 0.21 mmol/l). concomitant calcium and phosphorus intravenous supplementation started with loading doses (10 g of calcium and 1.8 g of phosphorus) and then a week of following daily intakes: phosphorus (1 g iv and 1.2 g oral); calcium (1 g iv and 4.6 g oral). however, low-serum corrected calcium and phosphorus levels persist at 1.89 mmol/l and 0.24 mmol/l. results: incompatibility between phosphorus and calcium by formation of soluble or not-soluble complexes is described in literature. in our case, calcium and phosphorus were mixed in a same infusion. after a week of supplementation, calcium infusion is continued with increased dose (4 g/day) and phosphorus infusion is stopped. phosphorus oral supplementation remains stable (1.2 g per day); calcium oral supplementation is increased (9.3 g per day). 2 h between intakes is applied to avoid digestive complexation. 48 h later, corrected calcium levels are normalized at 2.19 mmol/l and phosphorus levels are still low. therefore, as hypocalcaemia due to denosumab induced a secondary hyperparathyroidism and thus hypophosphatemia; phosphorus levels are expected to increase subsequently. conclusion: this case report shows that recurrent hypocalcaemia with denosumab is possible few months after administration. supplementation with large amount of calcium is needed and administration methods may impact the effectiveness of supplementation. indeed, it seems that the incompatibility between phosphorus and calcium did not allow an effective supplementation. gunnhild langdal *,1,2 , ida rudberg 1 , lone holst 2 , anne-lise sagen major 1,3 1 central norway hospital pharmacy trust, å lesund, 2 centre for pharmacy, university of bergen, bergen, 3 møre og romsdal health trust, å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: drug interactions (dis) can cause side effects and lack of therapeutic effect. the objective of this study was to describe the prevalence of dis at the medical department of å lesund hospital, and to investigate how dis were managed by clinical pharmacists and physicians. design: at the medical department, å lesund hospital, clinical pharmacists serve seven out of ten wards, from which patients were included during a five weeks period. the clinical pharmacists selected patients for screening for potential dis (www.interaksjoner.no) as int j clin pharm (2017) 39:208-341 303 usual (= pharmacist group). detected dis were classified according to a predetermined classification system, and it was registered whether the physician implemented suggested changes in prescription. for patients not selected by clinical pharmacists (= non-pharmacist group), a pharmacy student performed the search for dis. results: in total 373 patients were admitted. on average, each patient had 1.6 dis, and 56.6% of the admitted patients had at least one di. the prevalence of dis was significantly higher among the 194 patients in the pharmacist group compared to the 179 patients in the non-pharmacist group (median@@@ 1 vs. 0, respectively, p \ 0.001). the groups differed significantly regarding number of drugs used, age, duration of hospital stay and number of warfarin users. 10.5% of the dis detected in the pharmacist group were discussed with the physician. the remaining 89.5% were considered not necessary to discuss for various reasons e.g. because they were considered not clinically relevant (30%) or already adjusted for in clinical practice (20%). for 24 dis the clinical pharmacist suggested a change in prescription, and 20 of these suggestions (83%) were implemented by the physician. conclusion: just over half of the patients were selected by the clinical pharmacist for screening of dis, and the pharmacist seemingly made a reasonable priority of patients with many drugs, old age, a long hospital stay and users of warfarin. only 1 of 10 dis was discussed with physicians. this indicated that pharmacists do a considerable work in assessing the relevance of dis before discussing with the physicians. it also seemed that changes in prescription suggested by the clinical pharmacist were reasonable. hp-pc145: securing the paediatric use of oral chemotherapy: a proactive risk assessment samia mouffak *,1 , linda an 1 , anne fratta 1 , anne auvrignon 2,3 , nadia marquis 3 , karine morand 1 1 pharmacy, 2 risk management committee, 3 hematology, armand trousseau hospital -aphp, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: oral chemotherapy is an important part of the therapeutic strategy in childhood cancer or haematological malignancy. it also represents an emerging risk area in oncology practice. several medications errors involving oral chemotherapy were reported in children of our onco-haematology department, fortunately without clinical consequences. nevertheless, the potential severity of such errors led us to implement a failure analysis of the paediatric oncology care pathway in order to identify and prevent potential risks, and secure the paediatric use of oral chemotherapy. design: we conducted a failure modes, effects and criticality analysis (fmeca) which is a proactive risk assessment approach. first, process maps were detailed for each step of the oncology care pathway. it was performed by a multi-disciplinary group composed of 2 physicians, 1 coordinating nurse, 2 hospital pharmacists and 1 pharmacy resident. then, for each step of the medication-use process, the team identified the failure modes, their main causes and effects. finally, participants rated the expected severity, frequency and detectability for each failure mode, assigning a score on a five-point scale. a risk priority number (rpn) was then calculated by multiplying those three indexes. the risks getting a high rpn were categorized as critical risks and have been the object of safety improvements. results: 69 failure modes were identified, including 15 critical risk failure modes. 9 critical failures were related to hospital discharge prescriptions and 6 were about the dispensation of oral chemotherapy by pharmacy assistants. most failures were due to prescriptions heterogeneity, lack of clinical information reported on prescriptions, and lack of training of pharmacy assistants in reading oral chemotherapy prescriptions and in mistake detection. two improvement strategies were implemented. first, physicians' awareness led to the harmonisation of practices and to the standardisation of discharge prescriptions. then, to enhance pharmacy assistants' abilities, an educational program on oral chemotherapy dispensation was planned. conclusion: the implementation of a fmeca has highlighted the most critical risks of oral chemotherapy medication-use process. the awareness of all caregivers and the targeted changes in our practices allowed us to improve the safety of the paediatric oncology care pathway. please specify your abstract type: descriptive abstract (for projects) background and objective: the purpose of this study was to investigate if medication reconciliation and medication review, by using the integrated medicines management (imm) model, were suitable to assure the quality of patients' medical treatment at a gastrointestinal surgical ward. furthermore, to analyse frequency, type, handling and clinical relevance of medication discrepancies (mds) and other medication related problems (mrps). design: patients, above 18 years of age, from two departments at a gastrointestinal surgical ward at a norwegian university hospital were included consecutively. medication reconciliation was performed at admission by a clinical pharmacist. the resulting medication histories were compared with the medications documented in the medical records. mds were detected and categorized. thereafter the clinical pharmacist identified mrps by reviewing the medical records systematically and categorized the revealed mrps. mds and mrps were presented for the physician with proposed solutions. the physician's actions to manage the mrps were registered. later a multidisciplinary team assessed the clinical significance of mds and mrps in a subset of patients. results: a total of 48 patients were included. overall, 144 mds and 153 mrps were identified. at least one md was revealed in 90% of the included patients, whereas at least one mrp was identified in 88% of the patients. the most frequent type of mds was omission, whereas mrps most often were related to medications that were considered unnecessary. totally, 76% of the mds and mrps were discussed with the treating physician. the physicians followed the pharmacist's input in 85% of the discussed md-cases and 62% of the mrp-issues. longterm consequences of mds and mrps were considered more serious than short-term consequences for the patients. conclusion: medication reconciliation and medication review revealed, solved and prevented a large number of mds and mrps in this study. the results emphasize that pharmacist involvement, by using the multidisciplinary imm-model, contributed to more correct medical records and furthermore to quality assurance of the patients' medical treatment at a gastrointestinal ward. hp-pc147: prevention and management of drug interactions in oncology day-hospital: results from a 6 months study involving drug assessment and pharmaceutical report to oncologist pauline-saraï zeller *,1 , chloé hugard 2 , céline mongaret 1,3 , juliette vella-boucaud 2 , antonin maréchal 1 , olivier bouché 2 , dominique hettler 1 , florian slimano 1,3 1 pharmacy, 2 oncology day hospital, university hospital reims, 3 clinical pharmacy, faculty of pharmacy, reims, france please specify your abstract type: research abstract background and objective: quality during transitions of care is a major concern in drug safety for patients. traditional hospitalization allows to reconciliation medication but there is not possible for dayhospitalization (patient's hospitalization short time and no outpatient medication prescribe by oncologists). however, lack of communication between health professionals may expose patients to drug-drug interactions (ddi). while ddi between oral antineoplastic and other drugs are well known, there is a lack of knowledge about ddi between parenteral antineoplastic (ak) and other drugs. in this pilot study, we aim to investigate prevalence and characteristics of ddi between ak and other drugs in real life and to propose a pharmaceutical report model to enhance patient's drugs safety. setting and method: during 6 months, all new oncologic patients (thoracic and digestive) receiving chemotherapy in day-hospital have been recruited by clinical pharmacist. first it was conducted a patient-clinical pharmacist interview and carried out the best possible medication history (bpmh) by contacting at least three different sources of drug information. then, the bpmh has been confronted with oncologic treatment (including concomitant medications such as like antiemetic) with support at least with two different database of ddi analysis. finally pharmaceutical recommendations in order to manage potential relevant ddi were reviewed with oncologists then reported and inserted in personal health record (phr). main outcome measures: prevalence and description of potential clinically relevant ddi in an ambulatory oncology population. results: from november, 2015 to april, 2016 n = 90 oncologic patients were included with following characteristics: mean age of 63.2, sex ratio 2:1, majority of oncologic thoracic localization (56%). number of oncologic concomitant medications per patient was 3.97 ± 0.94 (mean ± standard deviation). patients present an average of 2.2 ± 1.93 comorbidities (excluding cancer) and 5.97 ± 3.76 linked medications per patient. pharmaceutical analysis revealed 33 potential clinically relevant ddi (0.37 ± 0.97 per patient): 72% of them concern antiemetics (ondansetron and aprepitant): pharmaceutical interventions were formulated (including recommendations to adapt chronic treatment) and 39% of them involved biological monitoring (for renal function, inr, potassemie or magnesemie). conclusion: our pilot study confirms high prevalence of ddi between oncologic and non-oncologic drugs. clinical pharmacy services with bpmh performing and pharmaceutical recommendation appears to be useful to enhance patient' drug safety in oncology dayhospital. we currently are deploying our study in order to convey a pharmaceutical letter to general practitioner and community pharmacist. hp-pc148: loading dose of anti-infectives: elaboration of a tool helping pharmaceutical analysis julie soyer *,1 , cécile sanchez 1 , guillaume beraud 2 , nicolas venisse 3 , pauline lazaro 1 , antoine dupuis 1 1 pharmacy, 2 infectiology, 3 pharmacokinetics, university of poitiers, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: the recent data on vancomycin and ceftazidime confirm that continuous infusion is the best way of administration of these antibiotics. moreover a loading dose before the administration is required for the antibiotics to prevent from the infratherapeutic period at the start of infusion and limiting the risk of resistance emergence. long half-life antibiotics and antifungals also require a loading dose to be effective. the aim of this study is to analyse the prescriptions of anti-infective requiring a loading dose in order to develop a tool to help pharmaceutical analysis. design: a prospective observational study was carried out during 15 days in 16 units. initially, pharmacists, residents and students were trained (role of the loading dose, drugs concerned). then, all patients with anti-infective requiring loading dose were included. some data were collected: weight patient, creatinine clearance, loading dose or not, dose, administration mode, monitoring of steady state concentrations (vancomycin and ceftazidime) and dose adjustment. the results were analysed and compared to bibliographic research before discussion during a multi-disciplinary meeting (pharmacists, infection control specialist and pharmacokinetic specialist). finally, a list of relevant pharmacist interventions was selected. results: out of the 393 patients, 44 were enrolled for prescription of anti-infective requiring loading dose. twenty-six prescriptions including vancomycin, 8 ceftazidime, the others fluconazole, caspofungine, voriconazole and posaconazole. concerning vancomycin, the loading dose was prescribed in 85% of case, monitoring of steady state concentrations was performed in 90% of case and dose adjustment after first dosage was required in 56% of case. selected pharmacist interventions were: • to favour continuous infusion (excepted paediatric) • to keep loading dose at full dose even in patient with renal failure • to monitor steady state concentrations after the first 24 h in patient with renal failure or obesity • to adapt dosage when the target concentration is not reached concerning ceftazidime, the interventions were: • to recommend continuous infusion: 6 g/24 h after loading dose of 25 mg/kg • to monitor steady state concentrations in patient with renal failure a total of 27 interventions (dosage, adaption of posology at the monitoring, patients with renal failure, obese, paediatric patient, administration…) were identified by the group of experts. conclusion: this study allowed creating a recap data sheet for students and hospital pharmacists. the selected interventions will allow the harmonization of practices. these recommendations have been validated by the commission of the anti-infective. finally, this study shows that the pharmacist has a key role in the management of antiinfective requiring loading dose. hp-pc149: assessment of potentially inappropriate medications in orthogeriatric patients using the rasp list the detection of inappropriate prescribing. the objective of this study was to investigate if the rasp list (rationalization of home medication by an adjusted stopp list in older patients), an explicit screening method adapted to the belgian context, can be used to reduce the number of potentially inappropriate medications (pims) in orthogeriatric patients. setting and method: single-centre, interventional study conducted at the orthogeriatric department of the uz brussel, a 721-bed university hospital. the rasp list was first applied by a last year pharmacy student to the admission medication of orthogeriatric patients hospitalised in october 2015. after potential adaptations to the medication by a liaising geriatrician, the rasp list was additionally applied by the same pharmacy student to the discharge medication of these patients. main outcome measures: detection and reduction of the number of pims. results: in total, 59 orthogeriatric patients, from whom an informed consent was obtained, participated in this study. on admission, a total of 136 pims were detected in this population. at discharge, the number of pims decreased to 101. the median number of pims per patient decreased from 2 (on admission) to 1 (at discharge). this difference was statistically significant (p \ 0.001; wilcoxon signed rank test). drugs of atc class n (nervous system) were responsible for the highest number of pims. conclusion: pims can be detected and reduced in the hospital using the rasp list. a structured and collaborative medication review between (student) pharmacists and physicians appears a good approach to reduce the number of potentially inadequate drugs. nevertheless, more research is necessary to substantiate this further as well as to assess the clinical impact of the findings. hp-pc150: impact of implementing ward based dispensaries across a hospital site on both service delivery and patient care michelle sullivan, paul wright, christopher watson, malcolm smith, sotiris antoniou * please specify your abstract type: descriptive abstract (for projects) background and objective: waiting for medication at discharge is often quoted as a key factor for delaying patients leaving hospital. feedback from service users (patients and healthcare professionals) was for a more patient facing pharmacy service. this led to a phased installation of remote dispensaries on wards within the hospital to supply medicines. this new and innovative service enabled the supply function to be fully co-ordinated on the ward. this model was initially implemented on 8 wards, which coupled with one-stop dispensing meant 91% of discharges require nothing to be supplied at the point of validation, 100% of discharge prescriptions meeting key performance indicator of being dispensed and ready within 1 h with average turnaround time of 18 min for a discharge prescription and a reduction in missed doses-3.1% in september 2015 to 2.1% in march 2016. this success prompted further installation of remote dispensaries in all clinical areas on site. design: implementation included; purchasing hardware, pharmacy labellers, locating appropriate computer terminals and stock cupboards. the main pharmacy labelling and stock control system was fully integrated at ward level, enabling the automatic reordering of replacement stock. identification of items and quantities to stock for remote dispensaries was also needed prior to role out. there was a need to scope staffing requirements including the redeploying of roles from a main inpatient pharmacy to patient facing areas. results: over 6000 items are supplied at ward level each month via satellite pharmacies for all wards, equating to more than 80% of the total dispensing workload for the site allowing for pharmacy staff to be redistributed from dispensary to the ward. this offered the benefit of being more patient facing and supporting other initiatives such as patient counselling and medicines reconciliation. the project has impacted the pharmacists as it has enabled them to focus on clinical aspects of service delivery, including attendance of ward rounds as well as supporting a ward team approach with the pharmacy technician. results of missed dose audit from june 2016 shows across the site 41% (7) wards scored below the national 1.4% target and (29%) 5 wards had no unintentional missed doses. conclusion: ward based dispensing has led to pharmacists and pharmacy technicians being 100% ward based. as a constant presence on the ward, the team offer consistency within the pharmacy service for patients, nursing and medical staff. impact of pre-discharge planning has been beneficial to nurses, patients and work flow of the pharmacy teams. ward based dispensing has improved supply at discharge as well as promoting a more patient facing pharmacy service that has seen the pharmacy team instilled as integral to service delivery at ward level. kutay demirkan * , nursel surmelioglu, aygin bayraktar-ekincioglu clinical pharmacy, hacettepe university, ankara, turkey please specify your abstract type: research abstract background and objective: hacettepe university hospitals clinical pharmacy unit was established in april 2014. this unit runs its services by clinical pharmacy postgraduate students under the supervision of two qualified clinical pharmacists as part-time and oncall basis, in adults, paediatrics and oncology hospitals. the aim of this study was to identify drug related problems and describe its management strategies in inpatient and outpatient settings by pharmacists in clinical pharmacy postgraduate education program. setting and method: during a total of 9 months study period (period i: february-july 2015, and period ii: november-february 2016), clinical pharmacy postgraduate students followed patients for 2-3 times in a week in different services in hospitals (internal medicine, internal medicine intensive care, infectious diseases, neurology intensive care, paediatric bone marrow transplant/haematology unit, paediatric intensive care, geriatrics and nutrition units) and drug related problems were identified and pharmacists' recommendations were listed. main outcome measures: determination and evaluation of drug related problems by pharmacist in hospital. results: a total of 114 recommendations was provided for 93 patients. those recommendations were classified as alteration or discontinuation of drug treatment (33.3%), dose adjustment (31.6%), change in drug administration time (14.9%), inadequate treatment (7.9%), healthcare staff training/consulting (6.1%), patient education (5.3%) and error/deficit in therapeutic drug monitoring (1.7%). a majority of recommendations (n = 38) were related with alteration or discontinuation of drug treatment provided mainly in departments of internal medicine (n = 9, geriatrics (n = 7), neurology intensive care (n = 5) and infectious diseases service (n = 5). the following main reason for pharmacist's recommendation was related with dose adjustment (n = 36) which were provided in departments of internal intensive care (n = 16), infectious diseases service (n = 6), neurology (n = 5) and internal medicine (n = 5). conclusion: clinical pharmacy practices are being carried out effectively in many services, particularly in internal medicine services, internal medicine intensive care unit and infectious diseases services. a collaborative and bed-side education in postgraduate programs in clinical pharmacy help to increase the knowledge and skills of students in real life circumstances and also maintain safe and effective drug therapy by an involvement of clinical pharmacists in hospital services. hp-pc152: development of a tool to help pharmaceutical analysis in patients with hepatic failure barbara troussier *,1 , eric gautier 1 , astrid bacle 1 , florian charier 2 , christine silvain 3 , pauline lazaro 1 1 pharmacy, 2 gastroenterology, 3 hepatology and gastroenterology, university hospital of poitiers, france, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: hepatic impairment can cause significant changes in the pharmacokinetics of many medicines. however hospital pharmacists can be helpless in performing pharmaceutical analysis behind the lack of precise guidelines. we need a strategy to first detect accurately patients with hepatic impairment, then lead us in dose adjustments. the objectives of this project were to develop a helping tool for hospital pharmacists in the pharmaceutical analysis of patients with hepatic failure's prescription and to select relevant pharmacist interventions. design: we first planned an investigation of patients with hepatic failure's management, with multidisciplinary experts groups. the study was conducted during one week in post-surgical, gastro-enterology, endocrinology, cardiology, pulmonology, geriatric departments and reanimation care units. a flowchart based on hepatic's biomarkers helped us including patients. criteria used to assess hepatic impairment could be: a stage c child-pugh score, prothrombin score inferior to 70%, bilirubin superior to 50 micrograms per millilitres of blood without haemolysis, aspartate and alanin aminotransferases superior to three times the high normal value, and presence of a vitamin k antagonist interfering with those results. after a review of each included patient's prescription, we checked the major pharmacokinetic elimination pathway of each prescribed molecule (biliary or renal) and if hepatic biotransformation was expected. we also checked if the molecule could cause hepatic side effects. results: out of 474 patients, 15 patients were included for liver failure (3.2%) and 4 for a cholestasis (0.8%) mainly in reanimation care units (17.5%) and gastro-enterology (10%). among the 232 lines of prescribed medicines, the main pharmacological classes encountered were cardiology, (5%) pain (5%), psychiatry (4%), haemostasis (2%) and antibiotics (1%). at the end of the investigation, the expert group decided on the relevant pharmacist interventions. these were based on dose adjustment of anti-infectious, psychotropic drugs, painkillers, oral anti-diabetics, anti-coagulants and corticosteroids. alternatives are proposed for each class. conclusion: to conduct a better pharmaceutical analysis, 3 steps are necessary. first, any liver failure or cirrhosis must be detected thanks to the patient's biological results and medical record. then the patient's prescription can be analysed in order to highlight drugs that need a dose adjustment in a context of hepatic impairment. finally, the physicist and the pharmacist discuss about dose adjustments or alternatives if presence of contraindication with the drugs prescribed. soon the designed tool will be available to all pharmacists to harmonize clinical pharmacy practices. please specify your abstract type: research abstract background and objective: data regarding adherence rates to oral chemotherapy in lymphoma patients is limited. the aim was to assess pharmacist intervention on adherence to oral chemotherapy in patients suffering from hodgkin's (hl) and non-hodgkin's lymphoma (nhl). setting and method: following ethics approval, 5 hl and 41 nhl patients attending chemotherapy sessions at the medical investigations and treatment (mitu) at mater dei hospital accepted to participate. a questionnaire was compiled to evaluate adherence to oral chemotherapy and to assess pharmacist intervention. the questionnaire was divided into 3 sections (a-c). the same questionnaire was used for both the first interview (t = 0) and after 6 weeks (t = 1). an additional section (d) was incorporated at t = 1 to evaluate pharmacist intervention. section a consisted of questions regarding patient management of lymphoma. section b incorporated the morisky 8-item medication adherence scale (mmas-8) 1 to evaluate adherence to oral chemotherapy. a total mmas-8 score of zero indicates high adherence, a score between 1 and 2 indicates medium adherence and a score between 3 and 8 indicates low adherence. section c consisted of additional questions regarding medication adherence. between t = 0 and t = 1, pharmacist intervention involved providing each patient with an information leaflet which was developed in this study, an individualised treatment chart and verbal advice. ibm ò spss version 22 and the wilcoxon signed-rank test were used to assess changes in medication adherence between t = 0 and t = 1. main outcome measures: evaluation of pharmacist intervention on adherence to oral chemotherapy in patients suffering from hl and nhl. results: out of the 5 patients with hl at t = 0, 3 'never' missed a dose, 1 missed a dose 'once in a while' and 1 'sometimes' missed a dose. for the 41 patients with nhl at t = 0, 38 'never' missed a dose, 2 missed a dose 'once in a while' and 1 'sometimes' missed a dose. the reason for missing a dose was forgetfulness. all 41 nhl and 5 hl patients indicated the haematologist as their source of information about the management of lymphoma. of the 41 nhl patients, 3 scored low adherence and 38 scored medium adherence at t = 0 and after 6 weeks (t = 1) all 41 nhl patients who participated scored medium adherence. of the 5 hl patients, 2 scored low adherence and 3 scored medium adherence in the first interview (t = 0) and after 6 weeks (t = 1) all 5 hl patients who participated scored medium adherence. there was a statistically significant increase (p \ 0.05) in the number of patients who scored medium medication adherence between t = 0 and t = 1 for both nhl and hl patients. conclusion: this study shows how pharmacist intervention and extended professional services could be implemented in the clinical setting to impact on the management of hl and nhl patients. please specify your abstract type: descriptive abstract (for projects) background and objective: in may 2015, an activity of medication reconciliation was implemented in the gastroenterology service to carry on the optimization of the medication care of patients due to the recent computerization of their prescriptions. design: this project, worked in collaboration with the gastroenterology service has been introduced in two medical committees. this activity gathers pharmacy students, the pharmacist, senior and junior doctors. reconciled patients are selected according to several criteria (advanced age, poly pathological, poly-medicated and those for whom a drug background is difficult to retrieve for the medical team). a minimum of 3 information sources is used for the collection of the drug background. all information are synthetized on a paper, validated by the pharmacist and discussed again with the prescriber. results: on a 10-month period, 61 patients were reconciled with on average age of 72. the reconciliation is executed on average 2.4 days after the entry in the service. 96.7% of reconciliations are retroactive. the main sources of information used for the collection of the drug background are: in 85.2% of the cases an oral interview with the patient and/or the family; in 82% of the cases the prescriptions, the hometown pharmacist (78.7%) and a medical letter (67.2%). 6.7 drugs are on average on the hospital prescription, and 47.5% (29/61) of the patients are concerned with at least one non intentional divergence (nid). on average there are 1.2 nid/patient and 3.9 intentional divergences (id)/patient. the main types of nid are omissions (44.7%), drug dose errors (19.7%) and errors in administration frequency (11.8%). after the detection of nid, the proposed modifications to the prescribers are accepted in more than 50% of the cases (31/61). the average time of a reconciliation is 49 min. exchanges on the id and nid are made with the junior doctors in 85.2% of the cases. conclusion: some nid are occurring for 47.5% of the reconciled patients. it is therefore necessary to extend this new activity to reconciliation in other services in order to increase the interception of eventual medication mistakes and allow their correction. please specify your abstract type: research abstract background and objective: diuretic therapy is routinely used in the management of congestive heart failure (chf).,compliance with clinical practice guidelines is reported to result in improved outcomes for patients with chf such as reduced exacerbations. the aim was to assess the effect of pharmacist intervention on adherence to diuretic treatment in a hospital and community pharmacy scenario. setting and method: the study was undertaken at karin grech hospital (kgh), a geriatric and rehabilitation hospital, and in one community pharmacy. inclusion criteria for patients recruited from kgh were age over 60 years, suffering from chf and on bumetanide therapy. the validated 8-item morisky medication adherence scale (mmas-8) 1 was administered to patients on admission (t = 0), repeated after two weeks hospital stay (t = 1) and again one-month post-discharge (t = 2). a total mmas-8 score of zero indicates high adherence, a score between 1 and 2 indicates medium adherence and a score between 3 and 8 indicates low adherence. in the community setting patients on diuretic therapy were chosen by convenience sampling. the same adherence scale was administered prior to pharmacist intervention (t = 0) and one-month after pharmacist intervention (t = 1). pharmacist intervention in the community setting involved dissemination of an informative leaflet regarding chf and diuretic therapy developed for the purpose of this study. main outcome measures: impact of pharmacist intervention on adherence to diuretic therapy in chf patients. results: a total of 37 patients were recruited from the hospital setting, of whom 19 were female and 18 were male with a mean age of 80 years (range 67-97 years). on admission (t = 0), 16 patients scored high adherence, 11 scored medium adherence and 10 scored low adherence to bumetanide therapy. following 2 weeks at the hospital (t = 1), the number of patients scoring high adherence increased from 16 to 31 and the number of patients scoring low adherence decreased from 10 to 1. one-month post-discharge (t = 2), patients scoring high adherence decreased from 31 to 19 and patients scoring low adherence increased from 1 to 3 (p \ 0.05). a total of 38 patients were recruited from the community pharmacy, of whom 21 were female and 17 were male, with a mean age of 79 years (range 68-97 years). after pharmacist intervention (t = 1), the number of patients scoring high adherence increased from 21 to 23, while the patients scoring low adherence decreased from 9 to 5 (p [ 0.05). conclusion: pharmacist intervention in the hospital setting improved adherence to bumetanide therapy. in the community pharmacy setting, there was a slight improvement in the compliance. pharmacist monitoring and patient support is important post-discharge to ensure patient compliance to therapy. conclusion: surveillance of aeds may be followed by combination of data from adverse drug reaction databases and drug utilisation data from prescription databases. focus on reporting adverse reactions is important for pharmacists and clinicians, especially for newly approved drugs. awareness of increased exposure of aeds to new groups of patients followed by data regarding safety aspects is important and contributes to improved pharmacovigilance. please specify your abstract type: research abstract background and objective: the medication review of polymedicated patients is a priority shared among all healthcare professionals. a multidisciplinary approach of these patients is necessary to achieve the best results for their treatment (1) . the objective was to analyse the rate of acceptance of the recommendations made by the primary care pharmacist (pcp) to the general practitioner (gp) regarding the treatment of polymedicated patients. setting and method: setting: a primary health care centre (27,521 population). method: a review of the medical records of polymedicated patients (c5 chronic drugs for c6 months). the patients' data were collected from january to june 2015 from their clinical records. statistical descriptive analysis of data was performed. main outcome measures: drug related problems (drp) for each patient: interactions, contraindications, inadequate dosages, nonindicated drugs, omission of a necessary drug, duplications, medication with low therapeutic effect, and inappropriate medication for patients c75 years old. treatment alternatives proposed to gp's by pcp were also measured. results: 34 patients were included in the study (average age: 68.6 ± 11.7, 74% women). out of the 34 patients, 88 interventions were laid out to reduce the risks of drp's and to improve the efficiency of treatments. 97% of patients presented some drp or some intervention to improve the efficiency of their treatment, this mean an average 2.5 interventions for patient. the prevalence of intervention proposals were: non-indicated drugs (28%), interventions for improve the efficiency of treatments (20%), interactions (16%), inappropriate medication for patients c75 years old (10%), contraindicated drugs (9%), duplications (6%), medication with low therapeutic effect (5%), inadequate dosages (5%) and omission of a necessary drug (1%). 97% of these intervention proposals were accepted by the gp: 38% of the accepted proposals were carried out and from the remaining 62, 43.6% led to a prescription from a specialist physician. in 51% of the cases, the patient did not accept the changes. 5.4% were not carried out due to other issues. the main drug related problem was the prescription of non-indicated drugs and the most involved drug was omeprazole. conclusion: acceptance by gp's to changes proposed by primary care pharmacists was high. a significant number of changes was not accomplished due to the negative response by some patients and led prescriptions from a specialist physician. the gp greatly values the multidisciplinary aid in approaching the complexity of polymedicated patients. background and objective: case-reports provided evidence that influenza infections, particularly severe episodes, may exert neuronal damage in the cns and thereby increase the risk of depression. it was the aim of this study to analyse the association between influenza infections and the risk of developing incident depression. setting and method: we conducted a case-control analysis using the large uk-based primary care database clinical practice research datalink (cprd). this database contains anonymous longitudinal data from primary care. at present, it contains over 100 million person-years of data from some 10million active patients. the study encompassed 103,307 patients below the age of 80 years with an incident major depression diagnosis between 2000 and 2013, and we matched each case to one control patient on age, sex, general practice, number of medical encounters, and years of history in the cprd prior to the index date. main outcome measures: major depression diagnosis was identified by read-codes based on icd-10 codes (f32), with a minimum of three prescriptions for antidepressant drugs recorded after the diagnosis. we calculated relative risk estimates of developing depression in association with previous influenza infections, stratified by the number, timing and severity of such events, and we adjusted for a variety of comorbidities, smoking status, alcohol intake, body mass index, use of oral corticosteroids, and benzodiazepines. results: patients with a previous influenza infection had an increased risk of developing depression (or 1.30, 95% ci 1.25-1.34) compared to patients with no history of influenza infections. a recent influenza infection recorded within 30-180 days prior to the index date yielded an adjusted or of 1.57 (95% ci 1.36-1.81), and an increasing number of previous influenza infections was associated with increasing odds ratios (c3 recorded influenza infections, adjusted or 1.48, 95% ci 1.22-1.81). we did not see any differences in the relative depression risk associated with influenza with regard to a previous influenza vaccination. conclusion: this study suggests that influenza infections are associated with a moderately increased risk of developing depression. please specify your abstract type: research abstract background and objective: warfarin is known for its interactions with many drugs. elderly patients are particularly sensitive to warfarin interactions. to evaluate the incidence of potential drug interactions when prescribing new drugs to elderly patients on warfarin, a prospective observational study was conducted. setting and method: patients on warfarin older than 65 years were included and monitored for 6 months in 4 community pharmacies in croatia. data regarding new prescribed drugs was obtained from pharmacy records at the moment of dispensing or by patient selfreporting. the potential interacting drugs were identified using the lexicomp ò lexi-interact online software. only the clinically significant (levels c, d, x of clinical significance as classified by lexicomp ò lexi-interact online) interactions were included in this analysis. main outcome measures: number of new proscribed drugs, level of interaction with warfarin, mechanism of interactions. results: we included 157 elderly patients with an average age of 73 years. in the follow-up period, new drugs were prescribed to 54 patients (34.4%). there were 79 prescriptions of new drugs and 57 (72.2%) of those were drugs with a clinically significant interaction with warfarin. there were 39 prescriptions of drugs with level c of interaction (68.4%), and 18 (31.6%) with level d. there were no drug interactions of level x. in the group with level c the most prescribed drugs were antibiotics with 26 prescriptions: amoxicillin/clavulanate 28%, clindamycin 8%, ciprofloxacin 8%, norfloxacin 8%, azithromycin 5%, cefuroxime 5%, clarithromycin 3%, doxycycline 3%. the remaining 13 prescriptions included tramadol with paracetamol 18%, rosuvastatin 5%, simvastatin 3%, fluvastatin 3%, levothyroxine 3% and torasemide 3%. the dominant mechanism of the potential interactions was pharmacokinetic. in the group with level d the most prescribed drugs were nonsteroidal anti-inflammatory drugs with 12 prescriptions-diclofenac 35%, ibuprofen 23%, indomethacin 12%. among other drugs, 6 prescriptions were antibiotic sulfamethoxazole with trimethoprim 12%, fenofibrate 6%, miconazole 6%, and fluconazole 6%. the dominant mechanism of the potential interactions was pharmacodynamic. conclusion: pharmacists should actively monitor prescribing of new drugs to elderly patients on warfarin in order to reduce the risk of clinically significant drug interactions. please specify your abstract type: research abstract background and objective: explicit criteria of potentially inappropriate medications in the elderly (pims) have been published in the usa, canada, australia and many eu countries. there is a lack of studies describing prevalence of pim use in central and eastern europe. the aim of the eu cost action 1402 initiative wg1b (2015 wg1b ( -2018 is to evaluate the registration rates and use of pims in central and eastern europe compared to other eu countries participating in this initiative. this abstract describes preliminary findings on different registration rates of pims in different eu countries. setting and method: researchers/members of the eu cost action 1402 initiative from the czech republic, serbia, hungary, spain, turkey and portugal were asked to fill in evaluation tables for the list of 484 pims in the period 01-06/2016. items available in these evaluation tables related to: registration of individual pims on the pharmaceutical market, registered doses, drug forms, availability of pims on prescription or as otc drugs, prescription limits and the most frequently used brand names. data were evaluated using comparative descriptive statistics. main outcome measures: overall prevalence of registered pims in different countries, cross-country differences in availability of individual pims. results: of 484 pims 81.8% were registered in at least 1 participating country. for the czech republic (45.2%), turkey (48.6%), spain (52.1%) and hungary (54.1%) overall prevalence rates of registered pims were found to be similar. however, these prevalence rates substantially differed in serbia (low prevalence-33.9%) and portugal (high prevalence-68.5%). substantial differences were found also in the lists of individual pims registered in different countries. these lists were similar in spain and portugal compared to the czech republic, hungary, serbia and to turkey. conclusion: although overall prevalence rates of registered pims were similar in the majority of evaluated countries (except serbia and portugal), availability of individual pims was substantially different. our pilot results confirmed that there are substantial geographical/ regional differences in europe in the lists of pims available (in spain and portugal compared to central and eastern europe and compared to turkey). please specify your abstract type: research abstract background and objective: inappropriate prescribing is a common circumstance found in polymedicated patients. screening tools for identifying potentially inappropriate prescription (pip) and pharmacist interventions for evaluating them have been developed to decrease this (1) . the aim of this study was to evaluate the effectiveness of a pharmacist provided intervention to reduce pips in polymedicated patients. setting and method: the design was a quasi-experimental study focusing on a single group before and after intervention. the study took place from july to december of 2015 at three primary care centres (52,992 population). polymedicated patients were those using c10 chronic drugs for c6 months. main outcome measures: reduction in the rate of pip per polymedicated patient (number of pips found divided by the total number of polymedicated patients) before and after intervention, and the influence of the following variables: type of pip (inappropriate medication for patients c75 years old, medication with low therapeutic effect, duplication of benzodiazepines (bzd) or angiotensinconverting enzyme (ace) inhibitors, combination of anticoagulant and antiplatelet, combination of non-steroidal anti-inflammatory drug (nsaid) with a diuretic and ace inhibitor, nsaid in cardiovascular disease, chronic antipsychotic in dementia, chronic bzd, or chronic nsaid), gender and age of patients with at least one pip, and the main prescribed drugs involved in the pips based on atc classification system of world health organization. results: there were 1093 and 959 polymedicated patients before and after intervention, respectively. 71.36% (n = 780, before) and 68.30% (n = 655, after) of the total patients had at least one pip. the number of pips was reduced from 1373 to 1108, while the rate of pip per polymedicated patient decreased from 1.26 to 1.15, achieving the limit established by the regional health authority. 50.90% (before) and 48.70% (after) of patients had more than one pip at the same time, up to 5 pips per patient. before and after intervention, more than half of patients with at least one pip were c75 years old, and approximately 9 out of 10 were c65 years old. also before and after intervention, 8 out of 10 patients with chronic nsaid and with bzd duplication were women. 6 out of 10 patients with combination of anticoagulant and antiplatelet were men. the main pips before and after intervention were, respectively: chronic prescription of bzd (39.77 vs. 37.99% of the total pip), medications with low therapeutic effect (19.81 vs. 21.30%) and inappropriate medication for patients c75 years old (16.82 vs. 17.42%). the main atc group involved in the total of pips was drugs for the nervous system, and the five most prescribed drugs were all bzd (lorazepam being the first). conclusion: pharmacist provided intervention was able to reduce pip in polymedicated patients. gender, age and atc classification of drugs involved were factors in the pips. please specify your abstract type: research abstract background and objective: up to 10% of women are exposed to selective serotonin reuptake inhibitors (ssris) during pregnancy. information on their effect on birthweight and gestational age remains conflicting. the aim of this sibling-controlled prospective cohort study is to address shared genetic and family-level confounding to investigate the effects of prenatal ssri exposure and maternal depression on birthweight and gestational age. setting and method: we used the norwegian mother and child cohort study (moba) and the medical birth registry of norway (mbrn). our study population consisted of 27 756 siblings; 194 were prenatally exposed to ssris and 27 500 were unexposed to any antidepressant medication. random and fixed effects analysis with propensity score adjustment was used to evaluate the effects on birthweight and gestational age. main outcome measures: birth weight. gestational age. results: ssri exposure during two or more trimesters was associated with a decrease in birthweight of 205 g [95% confidence interval (ci) 372 to38] and a decrease in gestational length of 4.9 days (95% ci9.1 to1.4). neither maternal ssri use in one trimester, lifetime history of major depression nor depressive symptoms during pregnancy were associated with these pregnancy outcomes. conclusion: prenatal exposure to ssris during two or more trimesters may decrease birthweight and gestational length. our results indicate that neither maternal depression nor shared genetics and family environment fully explain this association. please specify your abstract type: research abstract background and objective: the drugs burden index (dbi) is a tool to evaluate the burden of medications with anticholinergic and sedative effects and this exposure has been associated with poorer physical and cognitive function in older people. objectives were; to determine the cumulative burden of anticholinergic and sedative medicines in older adults with intellectual disability (id) using the dbi, to examine the relationship between dbi score with demographics and comorbidity. setting and method: data from wave 2 of the intellectual disability supplement to the irish longitudinal study on ageing (ids-tilda), a nationally representative study of ageing people with id in ireland. dbi scores were calculated for all participants with available medication data (n = 677). bivariate associations between dbi and demographic and clinical characteristics were examined with a significance level of 0.05 main outcome measures: dbi scores of participants categorised into low (0), medium (0-1) and high (c1). dbi score categories were related to demographics, cognitive effects and to a modified functional comorbidity index (fci), which is associated with physical function in older adults. results: of 677 participants, 95.1% (644) had dbi exposure; 51.3% were exposed to any anticholinergic medication, 32.1% to any sedative medication; mean number of dbi medications 2.91 (±1.68), mean dbi score: 1.30 (±1.24). 145 (21.4%) participants had dbi score 0, 165 (24.4%) 0-1, and 367 (54.2%) c1. antiepileptics accounted for the greatest contribution to cumulative score (27.6%), antipsychotics (25%) and antidepressants (14%). there was no significant association between higher dbi score and sleep difficulties (p = 0.135). there was a significant age gradient associated with higher dbi score (p = 0.016) and significant association between higher scores and increased comorbidity scores; mean fci of 3.28 in those with dbi c 1, 2.73 in dbi 0-1 and 2.35 in those with dbi 0. conclusion: cumulative exposure to sedative and anticholinergic medicines was high in older adults with id. higher dbi scores were associated with higher comorbidity and associated poorer physical function. optimising use of medications with anticholinergic and sedative effects through medicines review by pharmacists as part of multidisciplinary teams using a tool such as the drug burden index may reduce functional decline and improve quality of life among older adults with id. please specify your abstract type: research abstract background and objective: poor adherence to pharmacotherapy may have considerable consequences for the patients' health and for healthcare costs to society. there was observed that diabetes patients have higher risk of later health complications development. it is necessary to be adherent to non-and pharmacological recommendations as well, to improve the clinical outcomes and decrease the cardiovascular risk (cvr). the aim of this study was to evaluate the medication adherence and cvr in group of patients with diabetes, and to find an association between them. setting and method: the methods were based on a questionnaire survey using a modified 8-item morisky score and score charts (2012). medication adherence and cvr were evaluated in the whole group (n = 107, 51 males and 56 females, range 22-86 years) as well as in subgroups according to age, gender, (no-/ex-) smoking, level of education, residence, number of used medicines, exercises, compliance to the diabetic diet, and total cholesterol levels. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava -ruzinov. all participants signed an informed consent. main outcome measures: the results of medication adherence were evaluated as follows: 8 points = full adherence, 6-7 points = partial adherence and 0-5 = non-adherence. the cvr (estimating 10-year cardiovascular attack risk) was evaluated according to score charts using data from questionnaire and medical records-gender, age, smoking, total cholesterol levels and blood pressure. the results showed a partial medication adherence in the study group in average (6.84 ± 0.28). the average value of cvr in the study group was 3.7%. the highest average medication adherence has been observed in males b65 years (7.03), with elementary education (7.0), in ex-smokers (7.1), in patients with regular physical activity-at least 3 times a week (7.29), in patients non-adherent to the diabetic diet (7.05), in patients using 2 medications (7.11), and in patients with satisfactory (4.5-5.0 mmol/l) total cholesterol levels (6.97). the lowest cvr has been observed in females b65 years (1.8%), in no-smokers (3.2%), with elementary education (3.0%), in patients with irregular physical activity (2.9%), in patients adherent to the diabetic diet (3.14) , in patients using 4 medications (2.9%) and in patients with satisfactory (4.5-5.0 mmol/l) total cholesterol levels (2.97) . on the other hand, the highest cvr has been observed in males [65 years (7.3%), smokers (5.9%), secondary educated patients (3.8%), without any physical activity (4.7%), in patients partially adherent to diabetic diet (4.7%), using 6 medications (4.1%) and, surprisingly, in patients with satisfactory (\4.5 mmol/l) total cholesterol levels (4.76%). conclusion: our survey has showed that medication adherence in our study group has been decreased and cvr has been increased. cvr and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, cvr and their relationships are specific in every patient. please specify your abstract type: research abstract background and objective: studies show that quality of life (qol) of patients with diabetes mellitus can influence medication adherence, satisfactorily improving clinical outcomes and reducing the morbidity and mortality rates and disease progression. this applies even upside down-medication adherence could significant contribute to improving patient qol. the aim of this study was to evaluate the medication adherence in group of patients with diabetes, to evaluate their qol and find a correlation between them. setting and method: the methodology was based on a questionnaire survey using a modified 8-item morisky score and questionnaire eq-5d-5l, including visual analogue scale (vas). medication adherence and qol were evaluated in the whole group (n = 107) as well as in subgroups according to age, gender, level of education, monthly income, number of used medicines and type antidiabetic treatment. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava-ruzinov. main outcome measures: the results of medication adherence were evaluated as follows: 8 points = full adherence, 6-7 points = partial adherence and 0-5 = non-adherence. the qol in 5 levels of 5 dimensions results were evaluated as follows: the lowest qol in every dimension = 1 point, the highest = 5 points. the highest vas evaluation has been 100 points and every patient should mark number on the scale 0-100 to indicate his/her health on current day. results: the results showed a partial medication adherence in the whole group in average (6.84 ± 0.28). the average value of the qol in the study group was 21.21 and vas 69.29. the highest medication adherence has been observed in males (7.04 ± 1.29), patients \40 years old (7.0 ± 1.05), with primary education (7.07 ± 1.04), with monthly income over 600€ (7.38 ± 0.99) and in patients using 2 medications (7.11 ± 1.6). the highest qol and vas (qol; vas) has been observed in males (22.0; 74.24), patients \40 years old (23.22; 77.22), university educated (23.11; 75.55) , with monthly income over 600€ (22.75; 75.63) . qol has been highest in patients using 3 medications (23.92), vas has been highest in patients using 1 medication (83.33). we have observed the highest level of medication adherence in patients treated with combined therapy-with oral antidiabetic agents and insulin (7.06), the lowest in patients treated with only insulin therapy (6.95). highest qol was recorded in patients treated with oral antidiabetic agents (22.04), and the lowest qol in patients with insulin therapy (19.97). the highest vas has been observed in patients using only oral antidiabetic agents (72.45), the lowest in patients using combined therapy (62.06). conclusion: survey has showed that medication adherence and qol in our study group has been decreased. qol and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, qol and their relationships are specific in every patient. the role of health care professionals should be in education and counselling with patients to improve qol and medication adherence as well. please specify your abstract type: research abstract background and objective: to assess the appropriateness of antibiotic prescriptions used for urinary tract infections (uti) in the elderly. setting and method: we included patients aged 70 years and older, hospitalized in the geriatric department and for whom a urine culture was performed between march and may 2016. a prescription was qualified as inappropriate: when the antibiotic prescribed was not the narrowest compared to the culture result, or when there was a contra-indication, or when the treatment duration was shorter or longer than recommended. prescriptions were consistent with the guidelines when they were identical to those adopted by the french society for infectious diseases in december 2015. main outcome measures: appropriateness of antibiotic prescription (type and duration) results: 47 elderly patients were included (women: 74.5% (n = 35), mean age: 85.9 years). 68% of antibiotic choices were appropriate and 64% of treatment durations were consistent to the guidelines. urinary clinical signs were mentioned in the medical files for 29.8% of the cases (n = 14). 28 patients received an empirical antibiotherapy (59.6%). 70.2% (n = 33) of urine cultures were positive with bacteria, escherichia coli being the most prevalent (n = 18). the urine culture results led to a change in antibiotics for 66.7% of the cases. for cystitis, 53.8% of the antibiotics chosen were appropriate (n = 7). the main reasons of non-conformity were the lack of deescalation (to amoxicillin or pivmecillinam), and the prescription of ciprofloxacin when the bacteria was in vitro resistant to other fluoroquinolones. the average duration of effective antibiotherapy for cystitis was 9.3 days (appropriateness: 53.8% (n = 7)). for pyelonephritis, 88.9% of the antibiotics chosen were appropriate (n = 8). the average duration of effective antibiotic treatment was 10.1 days (appropriateness: 77.8% (n = 7)). 40.4% of the patients had a transurethral catheterization (n = 19). another infection was diagnosed for 48.9% of the patients (n = 23). conclusion: according to these results, it appears important to reemphasize to the prescribers the guidelines around the uti diagnosis and treatment in order to improve the prescriptions appropriateness in elderly patients. it is particularly necessary to promote the de-escalation of antibiotherapy (with pivmecillinam for example which has recently become available in our hospital) and to insist about the recommended durations of treatment. please specify your abstract type: research abstract background and objective: to measure the use of potentially inappropriate medications (pim) in the general elderly population several criteria lists exist, e.g., beers criteria. last year, a set of explicit criteria for assessing pharmacologically inappropriate medication use in nursing homes was developed; the norwegian general practice-nursing home criteria (norgep-nh). the aim of this study was to investigate the prevalence of pims in nursing home patients using this new assessment tool. furthermore, we studied possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. setting and method: cross-sectional study comprising 103 nursing home patients from two geographical different regions in norway; tromsø city (n = 70) and lofoten islands (n = 33). data was collected from november 2015 to january 2016. pims were identified by norgep-nh. we used logistic and poisson regression to examine possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. main outcome measures: number of pims per patient, and odds ratios (or) and marginal effects for associations. results: nursing home patient used a mean (sd) of 10.9 (4.3) drugs; 7.2 (3.6) regularly and 3.7 (1.9) as needed. at least 69% of patients used one pim. concomitant use of three or more psychotropic drugs was the criterion most commonly identified (33%), followed by the use of antidepressant (26%) and hypnotics (23%). an increasing number of regularly used drugs increased the odds of having pims (or: 1.74), as well as it lead to 0.18 more pims per extra drug used. on average, patients c80 years had 0.46 fewer pims than patients \80 years. no statistical significant associations were seen between having pims and gender, nor geographical area and the use of as-needed medication. yet, statistical significant differences were identified in some criteria. conclusion: this is the first study that explicit uses norgep-nh. our results confirm that nursing home patients often use potentially inappropriate medications. this is an area where further work is necessary, not to measure the prevalence of pim, but to develop interventions in order to prevent pims from being used. pe020: use of pharmacy dispensing data to measure adherence and identify nonadherence with oral hypoglycaemic agents please specify your abstract type: research abstract background and objective: a framework for calculation of adherence for oral hypoglycaemic agents (ohas) based on data from health-insurance claims is available. pharmacy dispensing data aid identification of nonadherent patients in pharmacy practices. however, use of these data for calculation of oha adherence requires additional methodological categories. we examined the impact of different methodological choices on estimation of oha adherence using pharmacy dispensing data. setting and method: a framework for adherence calculation for pharmacy dispensing data was developed from health-insurance claims. a basic scenario was developed from 16 methodological categories. consequences of choices for different parameters within these categories on the scores of the three adherence measures were calculated from dispensing data. main outcome measures: for oha use between july 2013 and july 2014, three adherence measures were calculated: (1) average medication availability (ama); (2) mean rate of adherent patients with an ama c80% (mrap80); (3) please specify your abstract type: research abstract background and objective: ulcerative colitis (uc) is a chronic inflammatory disease usually affecting young adults and impacting on patient's quality of life. although many biological agents (bas) have been approved for the treatment of moderate-to-severe uc in patients who have responded inadequately to conventional therapy, the selection of bas is controversial due to the lack of head-to-head trials. indirect economic comparisons of these costly drugs are available from national healthcare perspectives that are not the italian ones. therefore, the objective is to evaluate cost-utility of bas for the treatment of refractory moderate-to-severe uc both in italy and in the lombardy region. setting and method: a markov model (considering 3 transition states: remission, clinical response, relapse) was constructed using the software r 3.3.1 markovchain-package to evaluate incremental cost-utility ratios (icur) of adalimumab, infliximab, infliximab biosimilar, golimumab and vedolizumab treatments of patients over a ten-year time horizon from the perspective of the italian (n) and lombardy region (r) healthcare system. clinical parameters were derived from clinical trials. costs (which have been actualised-1.5%) were obtained from the national database and regional public tender. utility was expressed as qaly (quality adjusted life years). main outcome measures: icur. results: costs per treatment were different from a n and r perspective (adalimumab -55%; infliximab -16.7%; infliximab biosimilar -29.6%; golimumab -9.6%; vedolizumab -10%). direct healthcare costs (treatment cost, visits, lab tests, hospital admissions) were calculated over 10 years of treatment per patient: adalimumab (n: €114,226.70, r: €68,314.12, -40.2%), infliximab (n: €130,594.90, r: €103,081.00, -21%), infliximab biosimilar (n: €110,437.80, r: €78,852.03, -28.6%), golimumab (n: €118,602.10, r: €96,922.20, -18.3%), vedolizumab (n: €113,851.80, r: €102,932.20, -9 .6%) with associated qaly respectively of 6.68, 6.66, 6.66, 6.70, 7.02. from a n perspective, infliximab biosimilar was dominating compared to all other treatments. the icur of vedolizumab/infliximab biosimilar was €9483.33 for 10 years (willingness to pay (wtp) €948.33/qaly). from a r perspective, adalimumab was dominating compared to all other treatments. the icur of vedolizumab/adalimumab was €101,817.88 for 10 years (wtp €10,181.78/qaly). conclusion: national and regional cua produced different results. as regional price discounts can occur, local analyses are needed to estimate the economic impact of therapies to ensure optimal choice. please specify your abstract type: research abstract background and objective: automated dispensing systems (ads) have been implemented to reduce overall medication errors related to picking, preparation and administration of drugs. costs of drug storage between ads and classic dispensing system (cds) had not been yet performed in france. our objective was to assess economic impact of ads compared to cds. setting and method: retrospective quasi experimental study was conducted in 2 university hospitals in 2015, one with ads (800 beds, 43 ads) and one with cds (600 beds, 31 cds (17) for ads and 53 (15) for cds (p \ 0.001). mean number of costly drug per system was 3 for ads and 1 for cds. the global stock value in the wards was 205,915€ in ads and 54,908€ in cds representing respectively 14.5 and 6.1% of total pharmacy stock value. conclusion: our data demonstrate that despite the same storage capacity, ads allow the storage of more expensive drugs such as innovative drugs fully reimbursed up to national reimbursement prices, due to the lower risk of pilferage. this preliminary study was focused mainly on stock value. subsequently, another study is conducted to evaluate cost of these two drug storage systems, satisfaction of pharmaceutical technicians and nurses and time allowed for systems reloading. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, pharmacists are not entitled to substitute an original biological drug with its biosimilar, due to specific issues of efficiency, safety, and patient monitoring. our hospital referenced a biosimilar of infliximab on 6 january 2015. according to the french medication safety national agency's recommendations, it has been decided that naïve patients would be treated with biosimilars, and changes between specialties would be proscribed. the objective is to compare prescribing practices between infliximab and its biosimilar, 1 year after its introduction. design: a database tracking patients treated with infliximab was set up. data comparing prescribing practices of biosimilar and reference treatment were analysed between june 2015 and may 2016. regional and national infliximab consumption between january 2015 and february 2016 were used to compare the practices of our hospital with other hospitals. the past and future savings were estimated from repayments data of the regional health agency. results: infliximab was administered to 633 patients, of which 201 (32%) were naive. 111 patients were treated with biosimilar (i.e. 17.5% of all patients), of which 97 were naive. in the end, nearly 48% of naive patients actually received the biosimilar and 2.5% of patients treated with infliximab switched specialties during treatment. in 80% of cases, biosimilar prescriptions were consistent with the recommendations (vs. 94% for infliximab). in 79% of cases the off-label prescriptions of the biosimilar were explained in the patient record (vs. 75% for infliximab). in february 2016, the share of biosimilars was 14% in france, 12% at regional level and 15% locally. in 1 year, infliximab and its biosimilar's consumption in our hospital have increased by 12% in quantity and only 2% in expenditure (+€ 6 m expenditure). negotiating a lower purchase price and costs has enabled the hospital to save € 137,629 (vs. € 182,961 during the previous year). because of the decline of refund rates, the gains would have been zero without using the biosimilar but € 377,172 if it had been prescribed to every naive patient. conclusion: current data from the literature on security and effectiveness of infliximab biosimilars are very reassuring and the french medication safety national agency doesn't exclude the possibility of changing specialties during treatment. in our hospital, there is room to improve the efficiency of treatment with infliximab. feedback on prescribing practices will be given to prescribers and a campaign to widespread prescriptions of biosimilars will be made. the arrival of biosimilars on the market is a real economic opportunity for hospitals, which are increasingly financially constrained in particular by the arrival of therapeutic innovations which are more and more expensive. setting and method: the study used health claims data on prescription ppis from 1st january 2011 to 31st july 2014 obtained from the health insurance institute of slovenia. to assess medicine use and costs before and after trp implementation data were aggregated into four periods: jan-dec 2011, pre-baseline period; jan-dec 2012, baseline period; jan-sept 2013, transition period between announcement and introduction of trp; oct 2013 to jul 2014, period after trp enforcement. main outcome measures: medicine costs; defined daily doses (ddds) dispensed per 1000 inhabitants per day; market share; herfindahl-hirschaman index (hhi); number of active substance switches; number of exceptions when medicine is fully reimbursed since physicians may choose option ''not to switch medicine'' when adverse consequences are predicted. results: average monthly cost of ppis declined from € 1,350,289 in pre-baseline period to € 800,125 in period after trp introduction although the consumption increased from 52.4 to 55.2 ddds/1000 inhabitants/day. cost of ppis decreased the most in baseline period (26%), however trp induced 9.5% cost reduction compared to the transition period. the reference pantoprazole was market leader already in the transition period, but its use increased significantly after trp introduction and represented 51% of total ppis consumption. manufacturers' market shares were constant before trp, whereas trp caused decrease of the largest market share for 5%. still, this resulted in the minor market concentration change; hhi was on average 0.351 before and 0.307 after trp introduction. further, at least one active substance switch was detected in approx. 15 and 21% of patients before and after trp introduction, respectively. similarly, the proportion of exceptions when medicine was fully reimbursed increased from 6.7% in transition period to 23.7% in period after trp introduction. conclusion: enforcement of trp for ppi contributed to approx. € 1 m annual cost savings. from the payer's perspective the new policy was proven to be effective in reducing pharmaceutical expenditure; however trp also affected physician prescribing pattern and use of ppis. pec007: blood coagulation factor: improvements of the supply chain samantha oses * , serri traore, sonia caroline sorli, lea damery, philippe cestac, sylvie pomies, julien tourel please specify your abstract type: descriptive abstract (for projects) background and objective: most of the antihemophilic factor (ahf) must be held by a teaching hospital to face serious bleeding events. to ensure better availability, offsite-stocks at critical points are required (emergency unit, intensive care unit, etc.). however, this management system increases the risk of economic loss and alteration of the quality due to expired products. in this context, we carried out an optimization of the supply and management system of the ahf. to identify critical points of the supply and management system and to implement improvement solutions. design: a multidisciplinary working group belonging to a regional management centre of haemophilia was set up. two lines of improvement were discussed: i) optimization of stocks ii) optimization of the supply system. results: the optimization of stocks has led to the modification of the threshold of the lowest stock (ls) for 19 ahf out of 38. in 70% of cases, this stock modification has exceeded 15%. the overall cost of ls has been reduced by 15.0% (83,000 €) for the general stock at the central hospital pharmacy (hp) and by 8.5% for offsite-stocks (20,000 €). the ahf mainly involved in this reduction was fvii 5 mg (27,000 €), then followed by the strengths of 2 mg and 1 mg (13,000 € for each). in order to improve the ahf management, several propositions have been implemented: (1) developing an online, easily accessible and monthly updated spreadsheet that displayed several accurate data such as the shortest expiry date and the storage location. this operative tool is shared between all pharmacists involved in ahf management in order to facilitate a stock rotation and decrease economic losses, (2) regular reminders to physicians and health care staff concerning the guidelines for inventory management and the importance of checking the drug expiry date, (3) presentation of the financial results and raising awareness on ahf costs to the medical consultant[ppip1] and (4) optimizing stock distribution based on consumption on the different hospital sites for better patient care management (pcm). conclusion: this optimization of stocks and improvement of the supply chain have led to a direct cost saving of 83,000 €. however, a more accurate assessment has to be performed to quantify the direct and indirect impact on pcm and cost saving. this work has been done in a context of a sharing operative network at a regional level. the aim of such project is to share, to optimize and to improve practices, knowledge, human and medical health resources at a widespread level to enhance the security and quality of health services and to promote cost and time saving. please specify your abstract type: descriptive abstract (for projects) background and objective: the overall pharmaceuticals consumption in hospitals is rising, which has led to an increasing expenditure, challenging health care professionals and threatening patients safety. clinical trials in hospitals have increased over the past few years and currently play an important role, giving access to new investigational medicinal products and also avoiding costs with standard treatments. the objective of this study is to evaluate the savings of centro hospitalar do porto, a central university hospital with 800 beds and currently 80 clinical trials, with patients included in clinical trials between january 2013 and may 2016. design: retrospective observational study over 41 months. all the clinical trials ongoing between january 2013 and may 2016 were analysed and the data was collected based on: pathology and doses established; number of treatments per patient and the medium prices of standard treatments that patients would be receiving if they were not in the clinical trial. results: there were 112 clinical trials ongoing between january 2013 and may 2016, but only 30 were selected to be included in this study. the total number of patients included was 652. the clinical trials selected for this study were conducted in 6 medical specialties: 4 in dermatology, 6 in immunology clinical unit, 11 in hemato oncology, 1 in gastroenterology, 5 in ophtalmology and 3 in neurology. during these 41 months, with all ongoing clinical trials, centro hospitalar do porto was able to save, in medical products, more than 2 million euros. conclusion: during the period of time established, 82 of the clinical trials ongoing, were not selected due to: not including patients or not having an alternative treatment. hospitals and patients can benefit from clinical trials not only financially but also by preserving resources and medication. on centro hospitalar do porto, the pharmacists specialized in clinical trials, as members of the study team, are more and more required to perform specific tasks, their contribution has been increasing over the years and also have become more aware of all the advantages from participating in clinical trials. these savings can be used to provide a better assistance and contribute, in general, to a higher quality health care. please specify your abstract type: descriptive abstract (for projects) background and objective: several studies show a misuse of opioid maintenance treatment (omt) in detention. in fact, buprenorphine (bup) when it's misused, could present the same effects as heroine. in order to reduce misuses, the pharmacist decided to switch all the patients under bup to buprenorphine/naloxone (bup/nlx). bup/ nlx prevents patients from misusing by a withdrawal syndrome when it's issued by another route of administration than sublingual route. in france, bup/nlx is more expensive than bup which may explain why this therapeutic strategy is not often observed. the purpose of this study is to evaluate the extra cost after switching patients from bup to bup/nlx in order to decide if this choice could be maintained. design: to identify our population, we used the administration reports drugs written by nurses. please specify your abstract type: research abstract background and objective: haemophilia b is an x linked genetic disorder characterized by spontaneous or prolonged haemorrhages due to factor ix (fix) deficiency 1 . within the next few years, new treatments are willing to hit the market. among them are recombinant extended half-life products that will reduce by half the number of injections and will potentially improve the patient quality of life. the aim of the study is to describe the development of haemophilia treatments market between 2011 and 2014 and to forecast the potential impact of these new therapies on the haemophilia market. setting and method: national and french hospitals of paris (aphp) consumption data of 4 fix between 2011 and 2014 have been studied. new therapies in development or soon to be marketed have been identified. potential benefits and interest in the therapeutic care of these new products were discussed with haemophilia's medical experts. main outcome measures: quantity (ui) and value (euros) of fix aphp and national consumption. results: in 2014, 1 recombinant (rfix) and 3 plasma-derived factors (pfix) were on the french market. the ap-hp's purchases of these 4 factors represent almost 15 million ui and 10 million euros, which comprise 24% of national fix expenditures. in france and aphp, ambulatory care is a major part of the use of these treatments with nearly 90% of the fix purchases in 2014. french rfix consumptions are higher than pfix consumptions (64% against 36%). in the ap-hp hospitals, rfix even account for 89% of consumptions against 11% for pfix. both national and ap-hp rfix purchases have steadily increased between 2011 and 2014. the added competition arising from new treatments may lead to more competitive market procedures in hospitals and may reduce costs of haemophilia treatments. according to haemophilia doctor, long-acting (la) fix would offer obvious benefits like fewer infusions and presumably fewer bleeds. these treatments will mainly be used in a prophylactic wayin ambulatory care-than in a curative way (such as surgical use). conclusion: the therapeutic extent of these new treatments is still hard to define. the choice of treatment must remain consensual between physicians and patients. please specify your abstract type: descriptive abstract (for projects) background and objective: good practice about medicines imposes to health institutions a close monitoring of prescriptions, especially off-label prescriptions. patient care should take into account clinical profile, respect of guidelines and health expense control. we report here a case highlighting the significant role of the clinical pharmacist in care units to ensure medication good use in a castleman syndrome, a rare disease due to human herpesvirus 8 (hhv-8) and associated with human immunodeficiency virus (hiv) infection. design: case report. results: our patient, a 49 years old man (creatinine clearance rate (crcl): 95 ml/min), was diagnosed with hiv infection in february 2016 (cd4 at 160ui/l), leading to introduce a therapy by emtricitabine-tenofovir, darunavir, and ritonavir. the evolution was hampered by repeated episodes of acute renal failure (arf; crcl: 21 ml/min) and pancytopenia (hemoglobinemia at 8.6 g/dl, leucopoenia at 3.3g/l, and thrombopenia at 55g/l). because of hhv8 blood pcr at 30 000copies/ml, transient crises with pancytopenia, arf, and hiv infection, a diagnostic of kaposi sarcoma herpesvirus (kics), an atypical castleman syndrome, was retained. given the lake of data in literature for this rare disease, a multidisciplinary team (medical specialists and clinical pharmacists) was gathered to choose an appropriate therapeutic strategy. treatment regimen consisted of: day 1, intravenous etoposide at 250 mg; day 4, rituximab at 375 mg/ m 2 ; following one week later by rituximab 1 day and oral etoposide at 250 mg the day after. good communication between medical specialists and pharmacists enables the patient to get an optimal and personal treatment. relaying the information by clinical pharmacists in care units to pharmacists in charge of good practice facilitate the reimbursement. conclusion: clinical pharmacists in care unit help to optimize therapeutic strategies according to their experiences and scientific works. cooperation with physicians is improved, as well as prescriptions follow-up of off-label drugs, and health patients fully respected. quality and relevance of prescriptions are strengthened, with a better control of economic expenses. please specify your abstract type: research abstract background and objective: the maltese government launched the hpv vaccination scheme in 2013 and the national healthcare system (nhs) has since provided the cervarix ò vaccine free of charge to girls aged 12. the aim of this study was to assess the cost of the administration of hpv vaccines in the healthcare system of malta. this study was based on the scheme provided by the nhs. the number of girls born per year was used to estimate the annual cost for vaccinating 12 year old girls, based on the wholesale price and tender price respectively. the estimated yearly cost using the wholesale price was approximately €547,000 while the average estimated cost based on the tender price was approximately €157,000. this signifies that cost savings based on the tender price compared to wholesale costs were of approximately €390,000. the cost for the cohort who completed the three dose schedule using the tender price on average was of €171,000 per year. this result proved to be more than the anticipated cost. a reason for this could be that the number of girls aged 12 increased possibly due to an influx of immigrants. including boys in the vaccination scheme would increase costs by an average of €165,000 per year. conclusion: this study shows that procuring branded vaccines using the tendering process reduces expenditure for the government and the tax payer. wholesale prices were found to be more expensive than tender prices. this proves that the tendering system in malta is a potent system with many advantages for the tax paying public. the impact of the tendering process must therefore, be safeguarded. please specify your abstract type: research abstract background and objective: with the old age, presence of comorbidities, and overcrowding in mass gatherings such as the annual hajj pilgrimage in saudi arabia, there is a high risk of spreading infectious diseases among pilgrims and then within their country of origin. knowledge and application of hygiene principles in such an environment is therefore important to reduce the transmission of infectious diseases. up to date, there have been no studies to evaluate pilgrims' knowledge, attitude and practices toward mers-cov during the annual hajj pilgrimage in order to see whether there is a need for these aspects to be improved. setting and method: a cross-sectional survey study was conducted with a convenience sample of 257 participants. participants were pilgrims, aged over 18, and able to speak arabic or english. a selfadministered structured questionnaire was distributed during hajj season in mecca. descriptive and multiple linear regression analysis were used in data analysis. main outcome measures: assessing pilgrims' knowledge, attitude and practices regarding mers-cov. results: two hundred and fifty-seven participants completed the study, 80% of whom were female, and the median (iqr) age was 35 (24.5-43.5) years. pilgrims had moderately correct knowledge and accurate attitudes towards mers-cov with median scores of 5 (iqr 4-7) and 6 (iqr: 5-7) respectively. they were less educated about management (80%), hallmark symptoms (77%), high-risk individuals (45%) and source of coronavirus (38%). almost 40% of participants showed a negative attitude towards the use of protective measures such as avoiding food prepared under unsanitary conditions and contact with live animals. some participants (30%) were unable to comply with hygiene practices, particularly washing hands with soap and water or disinfectant after sneezing/coughing and wearing a face mask in crowded areas. educational level and employment status were significantly associated with knowledge whereas gender and age were significantly associated with attitude and practices respectively (p \ 0.05). the correlation between knowledge, attitude and practices was significant (correlation coefficient: 0.207; p \ 0.05). better knowledge was found to be a predictor for positive practice. conclusion: these findings aided in the assessment of the adequacy of current pilgrims' educational measures. they will also provide insight when designing future interventions to promote specific messages to improve knowledge, change attitude and improve practice regarding mers-cov. please specify your abstract type: research abstract background and objective: the prevalence of type 2 diabetes significantly increased in the paediatric population, which is affected by obesity worldwide. today, type 2 diabetes accounts for 45% of all cases of new-onset diabetes in adolescents. preventive health care particularly taking place at community pharmacies may involve risk assessment for the children and the adolescents, early referral for seeking relevant medical care and patient education on healthy lifestyle choices. the aim of the study is to conduct a type 2 diabetes risk assessment program for the kids b18 years of age of whose parents visited the community pharmacies involved in the study and also to identify the behavioural parameters that might be associated with this risk. setting and method: the study was conducted in 4 community pharmacies. all patients with kids aged b18 years who visited the study pharmacies during one-week period were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. all data were provided by the parents. demographic data, height and weight of the kid, as well as data regarding the behavioural features (eating habits, exercising, time spent in front of a screen, etc.) of both the children and the parents were collected using standardized forms. type 2 diabetes risk test consisted of 8 questions and identified subjects at risk. the parent of the kid who was identified to have risk for type 2 diabetes was referred to a physician for further examination. also, information regarding type 2 diabetes and the importance of preventive measures such as converting to a healthy life-style was provided. main outcome measures: main outcome measures were the percentage of kids identified to be at risk of developing type 2 diabetes and the behavioural parameters associated with type 2 diabetes risk. results: the study involved 212 subjects. of the subjects 26% were identified to be at risk of type 2 diabetes. more girls than the boys had the risk (36 vs. 9.3%). those with type 2 diabetes risk were older, taller, heavier and had higher body mass index. they were spending more time in front of a screen (tv, pc, tablet, smart phone); 22.6% were spending more than 6 h a day. although the kids' eating habits were similar for those with and without risk, the parents' of the kids with risk ate out more frequently, consumed rice, pasta and pastry more frequently. both the kids with risk and their parents exercised more regularly and frequently. conclusion: this study shows that pharmacist have a vital role in identifying children and adolescents at risk for type 2 diabetes; thus at early management of this condition. identifying and addressing the behavioural parameters associated with the risk will be helpful in lifestyle modification interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: analyse and promote the reporting of adverse drug events (ade), to improve the quality and safety of care to be able to control the risks. design: a software is available on the intranet website of the institution, to enable health professionals to report ade. the drug and medical devices commission (comedims) of the hospital, centralizes these statements and always makes a multidisciplinary and overall analysis of the event, using a collection sheet which is based on the pdca model (plan, do, check, act). it proposes the nursing and medical teams axes of improvement. results: in 2015, only 48 ade were reported and analysed by the comedims, including 20 from the paediatric centre (44%), particularly sensitized to this issue. health professionals are divided as follows: healthcare executives (60%), nurses (23%), pharmacists (11%), residential students (2%), doctors (2%) and others (2%). the main impacted steps of the drug circuit are: administration (62%), prescription (27%) and the use or implementation of a sterile medical device (4%). identified causes include related following factors: operational tasks and procedures (37%), health professionals (31%), work environment (13%), organization and management (7%), drugs or associated medical devices (5%). the number of ade reports, taking into account the size of the institution, remains very low. in january 2016, the comedims decided to broadcast a communication campaign to promote ade reporting, on the hospital website via the intranet. three months after the release, this document was viewed 1059 times, and the number of reports increased by 229% compared to the same period in 2015. conclusion: in front of the low number of returns of adverse drug events, and relying on the charter of non-punishment, the come-dims wants to increase health professionals' awareness. in our hospital, where e-learning about drug-related iatrogenesis is already available, the communication campaign with poster and analysis of adverse events seems to be a useful complementary tool to enhance awareness of medication safety concerns. please specify your abstract type: research abstract background and objective: the migration of modern social networks to the internet has facilitated the transition of traditional pharmacy networks online. the ubiquitous nature of social media (some) combined with merging of personal and professional personas have led to organisations publishing guidance on online behaviour and responsible use of social media. the research to date on the use of social media as a support for professional practice in general is limited. as the pharmacy profession evolves to embrace the technologies which underpin core services and mainstream online daily social activities, it is important that research tracks and evaluates its use and impact within the profession. the objective of this research was to explore and describe how and why pharmacists interact with hosted networks on social media. setting and method: two one-hour online hosted micro-blogging twitter chats were held in december 2015 via the #weph network. topic guides were developed around 'exploring the use of twitter and wepharmacists' in line with the wenetwork guidelines (#wecommunities), informed by existing literature, discussion with the #weph moderator after review by an expert panel. all research was carried out in accordance with university governance processes and association of internet researchers guidelines. themes were inducted from analysing the textual content of the chats using the topic guide as a framework. the research was approved by the school of pharmacy and life sciences ethics committee. main outcome measures: tweets per chat results: each of the chats had over 2 million impressions with participants representing international pharmacy practice. themes of e-professionalism and online privacy emerged as concerns; however, the benefits included using social media for education, networking, support mechanisms and career development. tweets highlighted personal experiences of 'trolling' (angry, offensive behaviour) and the effect on user interaction with social media. twitter was also recognised as a career development tool and, in particular, collaborative outcomes around mentorship networking early career pharmacists with more experienced colleagues. conclusion: results support the responsible use of social media as a force for inclusion, breaking down geographical barriers in support of pharmacy practice. further research is underway including a systematic review of guidance on the use of social media by registered healthcare professionals. please specify your abstract type: research abstract background and objective: it is estimated that half of the 350,000 persons with diabetes in norway have not been diagnosed. with early treatment, life expectancy can be increased and the incidence of longterm complications and health costs reduced. community pharmacies may be able to help uncover undiagnosed diabetes, but being diagnosed with diabetes can lead to strong emotional reactions, and how the diagnosis is given may influence the experience. the aim of this study was to explore how norwegian people living with type 2 diabetes (t2d) experienced being diagnosed, and what led up to the diagnosis. in addition, their attitudes towards a planned community pharmacy service to identify undiagnosed t2d was investigated. setting and method: three focus group interviews with people with t2d were conducted using a semi-structured interview guide. eleven participants were recruited through a course about type 2 diabetes. the interviews were audio-taped and transcribed in modified verbatim form and analysed in accordance with malteruds principles of systematic text condensation. the study was approved by the norwegian data protection authority, and did not require approval from the regional committee for medical and health research ethics. main outcome measures: how people with t2d describe their experiences of being diagnosed with t2d, how the disease was revealed and reactions towards using community pharmacies to perform risk assessment for t2d. results: none of the participants were diagnosed due to their own suspicion of having diabetes. some saw their doctor because of unspecific symptoms such as fatigue and thirst, and were thereafter diagnosed with t2d. others were diagnosed through a routine checkup. negative reactions like shock, discontent and denial were commonly used to describe the experience of being diagnosed with t2d, but some participants also expressed a more relaxed attitude, especially if they were familiar with the disease through family members. participants expressed a strong wish for more and better information following the diagnosis. ''it's a jungle out there'' was used to describe how difficult they felt it was to find trustworthy and understandable information. they described change of lifestyle, side effects from drug use, and stigma as challenges following the diagnosis. while in general the participants were positive to using community pharmacies to uncover undiagnosed diabetes as this could help reduce the number of people who were undiagnosed, some were sceptical. they questioned whether the pharmacy staff had the necessary competence of the for this type of service, and saw it as the doctor's responsibility. conclusion: more information and support when people are diagnosed with diabetes may lead to that the experience being diagnosed will be more adaptable and that the challenges living with diabetes are reduced. community pharmacies are important healthcare providers, and risk assessment of t2d at the pharmacy can be valuable. however, the pharmacies may also be helpful to reduce the information gap. please specify your abstract type: research abstract background and objective: chemotherapy-induced nausea and vomiting (cinv) is a disruptive and unpleasant side effect in chemotherapy patients and is associated with decline in patients' quality of life and decrement in the adherence to effective chemotherapy regimens. setting and method: 100 chemotherapy naive patients were included in this study. consistency with guidelines were assessed according to mascc/esmo 2014. flie questionnaire was administered to patients before chemotherapy, and 5 days after receiving chemotherapy to assess the difference in the quality of life due to chemotherapy administration. main outcome measures: patients were categorized into two groups as consistent with guidelines group (acute (gcga) and delayed (gcgd)) and inconsistent with guidelines group (acute (giga) and delayed (gigd)). flie score differences between the two groups were assessed. results: the median flie score for patients prior to chemotherapy was 126 and a dramatic decline was noticed post chemotherapy (flie score 108; p \ 0.001). the post-chemotherapy score were for nausea and for vomiting (49.5, 63 respectively). although the flie score differed significantly between gcgd and gigd (p \ 0.01), these differences were not significant in gcga and giga. conclusion: the significant drop in flie scores in the study (126 pre-to 108 post-chemotherapy) reflected substantial declination in patients' quality of life. the lower postchemotherapy flie score of nausea emphasized the negative impact of nausea, and to a lesser extent vomiting on the patients ability to complete normal daily activities such as enjoying meals and maintaining social activities. although there were no significant differences in flie scores between giga and gcga groups for acute cinv prevention, significant differences were noted between gigd and gcgd (p \ 0.001). the flie score was lower for gigd patients. this result implied guideline inconsistency associated with high incidence of nausea which negatively affect patient quality of life. as for the degree of compliance with gp, the results are expressed as percentage of compliance compared to the ideal of 100%. prescription criterion was fulfilled to 100%: all requirements of pntb were performed using standardized procedure. in what concerns validation, 94% of pntb prescriptions were validated by a pharmacist. the invalidated prescriptions were made outside opening hours of the pharmacy service, which is open monday to friday from 08:00 to 20:00 and on weekends and holidays from 08:00 to 15:00. 100% of the dispensations were individualized and not pntb stocks were found in hospital wards. as for preparation, 36% were supplemented with micronutrients. pntb of kabiven peripheral administration 1920 ml are not supplemented in our centre. of the remaining 325 prescriptions central administration, 81% were supplemented. in all cases, the addition of micronutrients was performed in laminar flow hood in pharmacy service and the corresponding galenic validation was performed. finally, in the process of administration, 94% of pntb identified with a complete label: name of the patient, medical record number, type of pntb, qualitative and quantitative composition, date of administration and infusion rate. conclusion: use practices of pntb of our centre are far from those recommended by the sefh standards. this initial evaluation will serve for improvement measures that increase the quality of prescribing and safe use of pntb, in order to minimize errors that can occur with the use of this therapeutic modality. please specify your abstract type: research abstract background and objective: methadone maintenance treatment was developed in malta in 1987 and is provided to patients by sedqa, the national agency against drug and alcohol abuse. methadone is the most frequently prescribed opioid in opioid substitution treatment and is dispensed through a centralised service through the substance misuse outpatients unit. in 2013, 1078 patients were in opioid substitution treatment, 976 of who were on methadone. in 2005, the government introduced a take-home methadone program. the prescribing, purchasing and dispensing of methadone are regulated by subsidiary legislation 101.06. the objectives were to determine whether community pharmacists in malta would be willing to dispense and supervise the consumption of methadone and to investigate the involvement of community pharmacies in the development of a regionalised methadone dispensing service. setting and method: the study was set in community pharmacies. a cross-sectional study, through the use of a questionnaire, was performed to quantitatively analyse whether pharmacists in malta would be willing to dispense methadone. the questionnaire consisted of 19 questions divided into 3 sections, with each section assessing a particular aspect of community pharmacists' attitudes towards methadone dispensing. community pharmacies were then chosen via a systematic sampling procedure. a hard copy of the questionnaire, addressed to the managing pharmacist, along with a cover letter, instructions on how the questionnaire was to be returned, and a prepaid self-addressed envelope was distributed via postage to 103 community pharmacies. an online format of the questionnaire was also circulated to 311 community pharmacists through the pharmacy council. data was analysed using spss version 21. main outcome measures: community pharmacist's attitudes towards methadone dispensing. results: a total of 109 responses were obtained and a response rate of 35.04% was achieved. eighteen percent of the pharmacists (n = 109) who responded to the questionnaire worked in a community pharmacy located in the north of malta, 24% in the centre, 17% in the south, 8% in the southeast and 4% in gozo. thirty-two percent of community pharmacists were willing to dispense methadone to drug misusers. the number of community pharmacists who are willing to dispense methadone increased to 41% if they were provided with appropriate education and support. twenty-nine percent of community pharmacists were prepared to handle the duty of supervising the consumption of methadone while 86% had never learnt about methadone and its clinical application within opioid substitution treatment. conclusion: community pharmacists should be provided with education and training regarding methadone substitution treatment before embarking on a new regionalised methadone dispensing service within community pharmacies. this would allow more community pharmacists to become involved in a new dispensing methadone service. pt009: evaluation of regorafenib in patients with colorectal cancer please specify your abstract type: research abstract background and objective: the colorectal cancer is the second more frequent cancer in europe and the third in the world. regorafenib is only approved in adult patients with metastatic colorectal cancer who are previously been treated with available therapies or are not considered suitable candidates to these treatments. regorafenib is an oral anti-tumor drug that blocks the kinases involved in the tumor angiogenesis (vegfr1, -2, -3, tie2), the oncogenesis (kit, ret, raf-1, braf, brafv600e) and the tumor microenvironment (pdgfr, fgfr).in this study, we are reviewed the reports of the patients with colorectal cancer who are been treated with regorafenib in our hospital and analysed the information in order to evaluate the efficacy and safety of regorafenib. setting and method: descriptive and observational study about the use of regorafenib from april 2015 to the present day. the variables studied, obtained from the software applications archinet and diraya, were: sex, age, pathology, location of metastasis, posology and adverse effects of regorafenib, tumor markers (cea y ca 19.9) before and after the treatment with this drug and the mutational state of kras. main outcome measures: the tumor markers cea and ca 19.9 only decreased in the 22.22% of the patients after the regorafenib treatment. results: regorafenib was taken by 9 patients (78%men).the average age of these patients was 64.78 ± 8.48 years old. the patients took regorafenib to treat: metastatic and non-intervened gastrointestinal stromal tumors (gist) e-iv that progressed with the previous treatment of imatinib and sunitinib (11.11% patients), intervened colon adenocarcinoma e-iv (33.33% patients), sigma adenocarcinoma e-iv (33.33% patients) and unresectable and non-intervened rectal adenocarcinoma e-iv (22.22% patients).all patients presented metastasis in different locations on the body: liver (55.55% patients), diaphragm (11.11% patients), intestine (11.11% patients) and lung (44.44% patients).the 77% of the patients started the treatment with 160 mg of regorafenib, administrated once a day for 3 weeks followed by one week without this drug; while the 22.22% of the patients started the treatment with 120 mg. however, the 33.33% had to decrease the initial dose and the 55.56% of the total patients had to get off the treatment because of the development of side effects. the most frequent adverse effects were: hypertension associated with headache, hyperbilirubinemia, elevation of ast and alt, intense asthenia. the 66.67% of the patients presents native kras. the native kras was presented in the 100% of the patients treated with regorafenib who had an appropriate development of the illness (decrease of cea and ca 19.9) conclusion: the decrease of cea in the 22.22% of the patients and the high development of side effects reveal that regorafenib has low effectiveness and security in the control of the progression of colorectal cancer. in addition, it is supposed that this drug has better results in native kras patients. however, more studies are necessaries in order to demonstrate the effectiveness of regorafenib in this pathology. pt010: evaluation of nintedanib in patients with non-small-cell lung carcinoma (nsclc) please specify your abstract type: research abstract background and objective: the nsclc means a high rate of mortality in developed countries. patients diagnosed with nsclc who debut with advanced or metastatic disease have a median survival of 13 months. one of the innovative drugs approved to improve survival in nsclc is nintedanib: an inhibitor of multiple tyrosine kinases, which can be found in some receptors on the surface of cells involves in the growth and spread of cancer cells (''pdgfr'', ''fgfr'' and ''vegfr''). nintedanib is not yet marketed in spain. hospital pharmacists are responsible for applying this treatment as ''expanded drug'', only after the elaboration of an exhaustive report. in this study, we have reviewed all the reports and classified the information in order to present our clinical practice. the objective of this study is to evaluate the effectiveness and safety of nintedanib in patients with nsclc treated in a tertiary hospital. setting and method: descriptive observational study of the use of nintedanib from november 2014 to september 2015. sex, age, body mass index (bmi), pathology, smoking habits, line of treatment, posology and adverse reactions of the treatment with nintedanib and tumor markers (cea an ca 19.9) before and later the treatment with nintedanib were collected from medical history through archinet informatic application. main outcome measures: the tumor marker cea decreased in 57% of the patients and ca 19.9 no decreased in any patient after nintedanib treatment. results: nintedanib was used in 7 patients (57% men and 43% smoker).the average age of these patients was 58 years old. the average bmi was 28 kg/m 2 (18-50).all patients received nintedanib together with docetaxel for metastatic nsclc with adenocarcinoma histology and with non-mutated egfr and alk in third line treatments. posology: all patients started the treatment with nintedanib 200 mg/12 h from day 2 to day 21 every 3 weeks; but 2 patients had to reduce the initial dose to 300 mg/24 h (1 patient) and 150 mg/12 h (1 patient) because of some adverse reactions. the side effects were: asthenia, diarrhoea, alteration of transaminases, muscle pain and cramps, weight loss and mucositis. conclusion: the decrease of cea in 57% of the patients reveals that nintedanib is effective in controlling nsclc progression which involves an increase of the survival and the quality of life of these patients. however, more studies are required to demonstrate the efficacy of nintedanib in this illness. please specify your abstract type: research abstract background and objective: patients with sore throat symptoms often seek fast, meaningful relief when presenting to their local pharmacy. flurbiprofen is a non-steroidal anti-inflammatory drug, which has been developed as a spray and lozenge to provide targeted relief for the main underlying process responsible for the symptoms of sore throats, inflammation. to study the relief provided by flurbiprofen 8.75 mg delivered as a spray or lozenge, we conducted a multicentre, randomised, double-blind, double-dummy, parallel group, activecontrolled, single-dose, non-inferiority study. setting and method: adult patients with acute sore throat were randomly assigned to take one dose of either flurbiprofen 8.75 mg spray plus a placebo lozenge, or flurbiprofen 8.75 mg lozenge plus placebo spray at 16 sites across russia. main outcome measures: patients rated sore throat relief using the sore throat relief rating scale (strrs; a 7-point scale, 0 = no relief, 1 = slight relief, 2 = mild relief, 3 = moderate relief, 4 = considerable relief, 5 = almost complete relief, 6 = complete relief) at timed intervals throughout 2 h starting from 1 min post completion of first dosing (1 min after administration of the spray, and 1 min after the lozenge had fully dissolved). adverse events (aes) were recorded over 2 h post-dose. results: 417 patients were assessed (n = 205 for spray, n = 212 for lozenge). [90% of patients in either treatment group experienced some relief (a score of [1 on the strrs) at 1 min post-dose, which increased to 98% of patients by 2 h. 55-60% of patients reported 'at least moderate relief', which is a well-recognised measure of a clinically meaningful effect at 1 min post-dose, which increased to 74-78% of patients by 2 h. over the 2 h post-dose, a total of 17 drugrelated aes were reported by 13 patients across both treatments and no severe adverse events were reported. conclusion: flurbiprofen 8.75 mg delivered as a lozenge or spray provides fast, clinically meaningful relief from sore throat. pt012: analising antiangiogenics prescription in an ophtalmology service after a protocol implementation silvia cornejo-uixeda * , ivan de la vega-zamorano, celia aparicio-rubio, olga carrascosa-piquer, manuel prieto-castello, agustin sanchez-alcaraz pharmacy, hospital universitario de la ribera, alzira, spain please specify your abstract type: descriptive abstract (for projects) background and objective: after some years using antiangiogenics in our hospital, we observed a large variety of use. considering the high cost of these treatments, we proposed ophthalmology service to develop a protocol of use, attending efficiency criteria. in this paper, we analyse the protocol implementation repercussion. design: a protocol of use was designed with the main of unify criteria and to use the most efficient treatment depending on the specific situation on each patient. once it was implemented, we compared two periods, the period after the implementation (january-may2016) and the period before of it (january-may2015). the protocol designed is the following: the cost for each injection and patient was the following: aflibercept 207€, bevacizumab 10€, ranibizumab 857€. results: in the 2015 period, 303 patients were treated with antiangiogenics.181(60%) with aflibercept, 110(36%) with bevacizumab and 12(4%) with ranibizumab. in the 2016 period, 297 patients were treated, 147(49%) with aflibercept, 134(46%) with bevacizumab and 16(5%) with ranibizumab. the consumption of aflibercept decreased a 19%, bevacizumab consumption increased 22% an ranibizumab increased a 33%.we also observed, some patients had more than one diagnostic at the same time. once the protocol was implemented, the percentage of use was the following: 38% 1. please specify your abstract type: research abstract background and objective: drug prescribing is the most common medical intervention in the elderly. however, elderly patients are more sensitive to the drug's effects due to pharmacokinetic and pharmacodynamic changes associated with aging. chronic diseases and co-morbidities often require the use of a large number of medications. therefore, when prescribing drugs for the elderly, the choice of suitable drugs, dosage and duration of treatment should be carefully considered as well as clinically significant drug interactions. inappropriate prescribing is often associated with an increased risk of adverse drug reactions, increased morbidity and mortality, and health care costs. the aim of this study was to determine the incidence of potentially inappropriate medications (pim) prescriptions in the elderly (c65 years) using the original protocol developed by mimica matanovic and vlahovic-palcevski. setting and method: we enrolled 240 patients hospitalized in clinic of internal medicine. data about patients' medications was collected during patient interview taken by the pharmacists on hospital admission. pharmacotherapy was analysed using the original protocol developed by mimica matanovic and vlahovic-palcevski in order to detect pims. main outcome measures: number and type of potentially inappropriate medications, potential clinically significant interactions. results: the average age of patients was 74 years (range 65-92), and the average number of drugs per respondent was 6.7 (range 1-15). a total of 109 patients (45.4%) were taking at least one pim. the most common pim were long-acting benzodiazepines, central antihypertensive moxonidine and non-steroidal anti-inflammatory drugs (nsaids) in patients with hypertension. in the study population, 110 patients (45.8%) have taken at least one combination of drugs that could result in a clinically significant interaction. the most common combinations included application of nsaids and antihypertensive drugs or diuretics, concomitant use of multiple medications with effects on the central nervous system and drug combinations that can cause hyperkalaemia. conclusion: this study revealed the high prevalence of inappropriate prescribing. clinical application of this protocol could be an effective method for improving and optimizing drug prescription with the aim to reduce the number of side effects and the morbidity and mortality associated with the drug use in the elderly. please specify your abstract type: research abstract background and objective: to reduce adverse effects of conventional amphotericin b formulation (deoxycholate or d-amb) it can be infused in intralipid ò (a fat parenteral nutrition), or lipid-based formulations can be used (i.e. amphotericin b lipid complex (ablc), amphotericin b colloidal dispersion (adcd) and liposomal amphotericin b (l-amb)). studies evaluating safety profiles present conflicting results. the aim of our study was to gather evidence on nephrotoxicity rates of d-amb versus lipid-based formulations in immunosuppressed patients susceptible to invasive fungal infection. setting and method: a systematic review, including randomized controlled trials (rcts) that compared the use of d-amb and amphotericin b lipid-based was performed. a search was conducted in pubmed, scopus, web of science and scielo. results were synthetized and meta-analysis was performed using software review manager 5.3. main outcome measures: nephrotoxicity rates. results: eighteen rcts were identified (n = 2525 participants). the result from the meta-analysis favours the treatment with the lipidbased amphotericin b formulations (or: 0.32 (0.25, 0.41) and presents a low heterogeneity (i 2 = 18%). about 22% of patients from lipid-based treatment group presented an increase in serum creatinine of one to two times, which corresponds to stage one or two of acute renal failure (arf). and 2% presented an increase of tree times in serum creatinine achieving a stage three in arf (severe) which will require dialysis. while in group treated with conventional formulation int j clin pharm (2017) all of these 23 patients, except one whose treatment adherence was inadequate, were cirrhotic (15/23), liver transplanted (5/23) and/ or presented hepatocellular carcinoma (3/23). 6/23 patients were coinfected with hiv. 8/23 patients (35%) were genotype 3. the total genotype 3 patients treated with daas (svr12/relapsed) were 79, which means that 10.1% (8/79) of all genotype 3 patients has had a relapse. 10/23 patients (43%) were treated with ledispavir/sofosbuvir (2.4% of a total of 411 patients (svr12/relapsed) treated with this option). 39% of patients who suffered a relapse were treated with daas sofosbuvir, simeprevir, daclatasvir, previously to the introduction of the newest antivirals (dasabuvir + ombitasvir/ paritaprevir/ritonavir, ledispavir/sofosbuvir), which represents 6.3% of the total of 142 patients treated with the older option. conclusion: relapses rate was 3.1%, slightly lower than reported in other studies. according to the references, these results show that genotype 3 is the one presenting more relapses. all the patients presented a deteriorated performance status, except for one whose treatment adherence was inadequate. patients treated before april 2015, when the newest daas where introduced, showed more relapses. more studies have to be developed in the near future since other daas will appear, the treatment options will be amplified and the number of relapses is expected to decrease. please specify your abstract type: research abstract background and objective: the inappropriate use of antibiotics remains a major issue since it causes bacterial resistance, longer hospital stay and increased mortality. antibiotic prescriptions must be monitored: the clinical pharmacist has a key role in ensuring patient safety and quality of pharmaceutical care. therefore, an antimicrobial stewardship program has been implemented as part of a national project of the italian society of hospital pharmacy (sifo). the objective is to describe the results obtained at the hospital. setting and method: a multidisciplinary antimicrobial management team has been implemented including clinical pharmacists, microbiologists and infectious disease specialists. the pharmacist examines drug charts on a daily basis in the department of medicine and supports clinicians to improve the appropriate use of antibiotics. data from 2 time-points were extracted from medical records and collected in an excel database: t0 (november 2015-january 2016) and t1 (february 2016-april 2016). main outcome measures: type of infection, antibiotic consumption data, type of isolated pathogens, patient allergies, clostridium difficile infection assessment and adverse drug reactions (adr). results: 465 records were analysed (t0-t1), 277 of which contained at least one antibiotic prescription. the most frequent infections were urinary tract (27%), respiratory (20%) and gastro-intestinal (14%). antibiotic therapy was started in 15.9% of cases due to aspecific increase of c-reactive protein (crp). ddds were calculated for each treatment and were grouped by type of infection and setting (empiric vs targeted): ceftriaxone, meropenem and metronidazole were the most widely used antibiotics for empiric therapy. at t1, an increase in the use of piperacillin-tazobactam instead of meropenem was observed. the ddd of ceftriaxone for targeted therapies decreased significantly, while an increase was observed for carbapenems, levofloxacin, glycopeptides and, in case of mdr bacteria, tigecycline. three allergies to antibiotics were reported in medical history. there were 20 clostridium difficile infections (5 relapses), confirmed by antibiogram. a total of 22 adrs were identified: 3 of these were related to antibiotics. conclusion: antimicrobial stewardship is a fundamental step to optimise antibiotic management, ensure patient safety and improve quality of care. the results obtained so far demonstrate the added value of a multidisciplinary team in controlling bacteria resistance and in the improving the use of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to analyse effectiveness and safety of pirfenidone, an anti-inflammatory and antifibrotic agent used for treatment of idiopathic pulmonary fibrosis. design: a retrospective, descriptive, observational study including all patients treated with pirfenidone at the hospital between march 2015 and june 2016 (15 month) was carried out. to identify patients and collect data the outpatient medication dispensation software farhos ò and the electronic medical record software hcis ò were used. statistical analysis was carried out using microsoft excel ò . demographic (age and sex), clinical (forced vital capacity (fvc), diffusing co capacity (dlco) and six-minute walk test (wt6 m)) and therapeutic (dosage and adverse reactions) variables were collected. results: throughout the study period, a total of 22 patients (16 males) started treatment with pirfenidone, with a median age of 74.5 years (46-81). during this period 5 patients were excluded for lack of monitoring. the median fvc, dlco, wt6 m values prior to pirfenidone therapy, were 59% (50 [ 81%), 38.5% (17 [ 65%) and 357 m (200-620 m) respectively. all patients met the inclusion criteria of capacity trial according to fvc and wt6 m; however 8 of them didn't meet the dlco criteria (at least 35%).'' all patients were monitored every 3 months. the median in fvc percentage change at the end of the study was -1% (-13% to +9%). 8 patients (50%) showed an improvement on fvc during treatment with a median change of 7%. in the other eight patients fvc value decreased with a median of -6%. only one patient would be candidate to discontinue treatment due to a lack of efficacy, according to discontinuation criteria established at the hospital (absolute decrease of c10% in fvc during first year of treatment). dlco percentage was measured in 14 patients, with a median change of 2% (-17% to +10%). dlco decreased in 6 patients. wt6 m was monitored in 12 patients, with a median change of -44.5 m (-222 m to +35 m). adverse effects related to pirfenidone were gastrointestinal disorders (9/17), increase of hepatic ggt (5/17), and dermatologic toxicity (2/17). six patients (35%) required a dose reduction because of gastrointestinal adverse effects. five patients (29%) discontinued treatment with pirfenidone due to hepatotoxicity (2), gastrointestinal (1) and dermatologic effects (1). one patient died. conclusion: half of the patients improved fvc during the period of the study. the other half, showed a decrease in fvc value which was similar to the median obtained in capacity trial. gastrointestinal disorders were the most frequent adverse effects and cause of discontinuing treatment. treatment monitoring is important to achieve therapeutic benefit and control the adverse effects. the national centre for epilepsy, oslo university hospital, oslo, norway please specify your abstract type: research abstract background and objective: systematic medication reviews in interdisciplinary teams can help to identify potential and actual drugrelated problems (drp). the centre for development of institutional and home care services in oslo, norway, conducted medication reviews for polypharmacy patients with mental disabilities in 2015-2016, based on a lack of knowledge about drug-related problems in this patient group. the objective was to examine prescribing patterns, frequencies and types of drp in patients with mental disabilities. setting and method: the forms for medication reviews were developed by the national patient safety campaign in norway. the nurse/social educator recruited eligible patients, observed them, and ordered test if needed. the clinical pharmacist (jwa) reviewed the medications to identify drps. the interdisciplinary case conference took place at the different general practitioners' offices being responsible for the individual patients. the general practitioner, the nurse/social educator and the pharmacist were present, and in some cases, also patients took part. main outcome measures: an independent researcher (aqm) collected and analysed the data based on the drp-forms containing information on the prescribed medicines, strength, dose, indication, a description of drp and suggested interventions to resolve them. results: overall, 40 patients with mental disabilities, aged 34-77 years, consented to have a medication review. they used on int j clin pharm (2017) 39:208-341 327 average 12 medicines (range 5-23). the team identified 191 drp in 39 of the 40 patients (average 4.9, range 0-13). overall, 79% of all drp were resolved. for one-third of the medicines, an action was taken to improve the prescribing. the most commonly medicines were analgesics (62%), antiepileptics (58%) and anxiolytics (52%). the most frequent drps were unnecessary drug choice (24%), side effects (11%) and too low dose (11%). drps were most common in antipsychotics (10%), antidepressants (9%) and anxiolytics (7%). conclusion: patients with intellectual disabilities take more medicines and have many drps compared to other patient groups. they are also more prone to taking combinations of cns-active medicines and therefore more at risk of side effects and drug interactions. pt020: protocol feasibility and patient findings when using a dry extract of zingiber officinale roscoe (ginger extract gr10) during pregnancy please specify your abstract type: research abstract background and objective: there is limited information about the use of dry extracts of ginger root. the objectives of this study are (1) to evaluate the feasibility of a pilot study with a food supplement among pregnant women (2) to learn what the patient findings are when using the dry extract of ginger during pregnancy. this abstract deals with the intermediate evaluation of a study conceived to investigate the safety of the ginger extract gr10 during pregnancy. setting and method: a prospective, interventional and real life pilot study with pregnant women between 4 and 14 weeks of gestation and having symptoms of nausea and vomiting or digestive complaints. the included patients can use the ginger extract gr10 for digestive comfort during pregnancy when needed. during the use, the score of digestive discomfort is noted and the researcher reports adverse events. main outcome measures: (1) number of included patients as an indicator of feasibility: including a number of 50 patients was taken as a target (2) analysis (qualitative and quantitative) of the patient diaries, more particularly patient behaviour, wellbeing and impressions. results: within twelve weeks, 51 patients were included with an average age of 29.9 years and a median age of 29 (19-42) years. 45 patients used gr10: 3 patients were dissatisfied, 15 patients had a neutral opinion and 19 patients were satisfied to very satisfied. one miscarriage occurred at a gestational age of almost 17 weeks (only 2 tablets of gr10 were used, with no relevant medical history in preceding pregnancies). two patients were hospitalized, of which 1 with hyperemesis gravidarum. one patient complained about heartburn and one patient experienced a bad taste and heartburn. three patients have indicated that they experienced more nausea after taking the tablets. 29 patients experienced no adverse events. the remaining 8 patients were not yet evaluated. of the 51 included patients, six patients decided not to use the product: 3 because their gastrointestinal complaints were not serious enough, 1 because problems of swallowing (using ginger gums instead). one patient was afraid for the negative consequences for her unborn child. the last of the nonusers indicated that she had no confidence in the product. conclusion: conducting a pilot study with the ginger extract gr10 in case of pregnancy is feasible. the majority of the evaluated patients were satisfied. signing the consent form does not guarantee the intake of the product. pregnant women remain very cautious in the use of unknown products during their pregnancy, even though it concerns a food supplement and not a drug. the severity of symptoms does not give a good indication whether or not and how often the product will be used. please specify your abstract type: descriptive abstract (for projects) background and objective: to analyse effectiveness and safety of ibrutinib, an oral inhibitor of bruton tyrosine kinase, in patients with mantle cell lymphoma (mcl) who have received at least one prior therapy. design: a descriptive observational study was carried out. all patients with relapsed or refractory mcl who started treatment with 560 mg of daily ibrutinib between september 2014 and june 2016 were included. patients were identified and followed through electronic medical record. demographic and baseline clinical characteristics of patients were collected: age, sex, ecog (eastern cooperative oncology group scale), number and type of prior regimens, simplified mipi status (mantle-cell lymphoma international prognostic index), and disease stage (relapsed or refractory). progression free survival (pfs) and response to treatment were recorded to evaluate effectiveness. adverse effects related to ibrutinib and possible interactions with concomitant medication were documented to measure safety. statistical analysis of the data was carried out using microsoft excel 2013 ò and spss ò 18. results: throughout the period of study a total of 5 patients (4 males and 1 female) with a mean age of 62.5 ± 8.2 years started treatment with ibrutinib. the median of previous treatments were 2 (1) (2) (3) (4) (5) including first-line treatment with high dose chemotherapy (100%), steam-cell transplantation (80%), rituximab (100%), bortezomib (60%) and lenalidomide (20%). the median ecog value prior to ibrutinib therapy was 0 (range 0-1). the mipi status was intermediate risk in 4 patients and high risk in 1, the disease stage was relapsed in 80% of the patients. partial response was reported in 3 patients. the mean pfs estimated at the end of the study period was 13 months (95% 4.4-21.5). adverse effects related to ibrutinib were: fatigue (10%), diarrhoea (10%) leucocytosis (30%) and infections (50%), including upper respiratory and urinary tract infections, sinusitis and pneumonia. one possible interaction between ibrutinib and everolimus was found in a liver transplant patient. close monitoring of everolimus plasmatic levels was recommended. conclusion: the mean pfs estimated in our study was similar to the median obtained in the pivotal phase ii trial. infections were the most frequent adverse effects. concomitant medication to ibrutinib should be checked, as ibrutibib is metabolised by cyp3a4 and interactions may be frequently present. 1 pharmacy, 2 hiv unit, germans trias i pujol hospital, badalona, spain please specify your abstract type: descriptive abstract (for projects) background and objective: dolutegravir (dtg) is one of the preferred options for initial antiretroviral therapy (art) due to its high efficacy, good tolerability and low potential for drug-drug interactions. nevertheless, an unexpectedly high rate of dtg discontinuation (up to 16%) due to adverse events in the clinical practice has been recently reported. therefore, we aimed at assessing the dtg discontinuation rate and reasons for discontinuation in our hospital. design: single-centre, retrospective study from september 2014 to june 2016 of 2709 patients cohort with art both naive and pretreated. patients who had started dtg-based art containing regimen were identified and the reasons for the discontinuations were analysed. data were collected using the primary care service program and the electronic prescription program. results: out of 2700 patients attended by pharmacy department in our hospital, 563 patients (494 males, mean age 47 years (range 16-84)) had started a dtg-based art. out of them, 61 patients were art naive and 502 art-experienced. at the moment of starting dtg, mean cd4 cells were 654cell/mm 3 (range 7-2147) and hiv-1 rna load in plasma was detectable in 69 patients. treatment discontinuation was reported in 52/563 patients (9.2%) with a median treatment time of 241 days (range 7-842). 8/52 patients (15.4%) were naïve and 44/52 patients (84.6%) pre-treated. most of the patients (404) were in single tablet regimens (str) containing dtg in combination with abacavir and lamivudine, whereas the rest were in combination with other antiretroviral drugs. the main reason for treatment discontinuation was toxicity in 38/52 patients (73.1%). the rest of the patients discontinued due to other motives (clinical trial inclusion (3/52), treated in another hospital (4/52), exitus (1/52) and others (6/52). reasons for the discontinuation were classified in different side effects: 17/38 (44.7%) related to central nervous system (cns) (insomnia, psychiatric disorders such as anxiety, nightmares and depression), 14/38 (36.8%) gastrointestinal effects, 4/38 (10.5%) headaches, 6/38 (15.8%) musculoskeletal effects, 4/38 (10.5%) fatigue, 1/38 (2.7%) allergy and 6/38 (15.8%) for other reasons. some patients reported various toxicities at once. conclusion: more than 6% of patients treated with dtg discontinued by toxicity reasons. it is important to note that half of these patients had cns adverse effects. please specify your abstract type: research abstract background and objective: hcv therapy has been revolutionised recently by the approval of antiviral agents direct-acting (daa) facilitating the treatment of patients coinfected with hiv/hcv. however, potential drug interactions and overlapping toxicities of both treatments represent the major challenges in adapting therapy. to analyse the prescription profile of direct acting antivirals (aad) in patients coinfected with hiv/hcv. setting and method: retrospective observational study from january 2015 to january 2016 in a specialty hospital. the data were collected from the hospital program of clinical stories, archinet ò , and the outpatient program farmatools ò . the results were analysed using the statistical program r-commander. main outcome measures: inclusion criteria: adult patients coinfected with hiv/hcv with undetectable viral load. the following variables were collected: age, gender, hcv genotype, degree of fibrosis, patient type (naïve or pre-treated), baseline cd4 count, cd4 levels end of treatment, sustained viral response (svr) and hcv treatment. results: 20 patients, of whom 16 were men, mean age 52 years were included. 9 patients received daclatasvir and sofosbuvir for hcv, 3 patients had genotype 1a and 1b respectively, 2 patients genotype 3 and 1 patient genotype 4. 8 patients had fibrosis f3 f4, 1. of the 9 patients 3 they had not received previous treatment (naïve) and 6 had failed to treatment. hiv treatment was modified in 8 patients, 6 patients achieved svr. the cv was undetectable to hiv treatment change for all patients. cd4 levels increased in all patients at the end of treatment for hcv with a median of 304 cells/ul and 398 at the beginning and end respectively. 2 patients received ombitasvir/paritaprevir/ritonavir and dasabuvir, who had a genotype 1a. these two patients had received previous treatment and had a f2 and f4 fibrosis. none of them was modified hiv treatment and only one got svr. cv remained undetectable and cd4 slightly increased after the treatment. 9 patients received ledipasvir and sofosbuvir, 6 patients had genotype 1a, 2 patients genotype 1b and 1 patient genotype 4. 4 patients had f4 fibrosis and 5 had f3. 9 patients had received previous treatment (naïve). the hiv treatment was modified only in one of the patients, 8 patients achieved svr. cv increase in 2 patients after the treatment while cd4 followed the trend of increasing. conclusion: the aad that caused fewer changes in the hiv treatment were ombitasvir/paritaprevir/ritonavir and dasabuvir followed by ledipasvir/sofosbuvir. sofosbuvir and daclatasvir present a greater number of interactions with hiv drugs so they behaved to a major change. more patients are needed to assess more accurately the aad leading to a minor modification. please specify your abstract type: research abstract background and objective: the simplification strategies reduce the amount of tablets and the toxicity in order to facilitate adherence in patients with virological suppression. the strategy more studied is monotherapy with a ritonavir-boosted protease inhibitors (pi/r). to analyse the effectiveness of monotherapy with pi/r in pre-treated patients infected with hiv. setting and method: retrospective observational study. selected hiv patients treated with pi/r monotherapy at any time of pharmacotherapeutic history to 30/12/2015, with at least one clinical and analytical control 6 months before the beginning. data were collected from the medical record archinet ò and outpatient farmatools ò program. variables included were age, sex, duration of monotherapy, virological failure, treatment failure, cd4% during monotherapy. main outcome measures: inclusion criteria: virological suppression for 1 year prior to the start of monotherapy, no previous ip virological failure, high cd4 count ([300 cell/ml) and a high level of drug adherence. the effectiveness is defined as the percentage of patients without virological failure (2 consecutive plasma viral load (vl) [200 copies/ml) and without treatment failure (any event causing retirement monotherapy). results: 141 patients with monotherapy, which represent 22% of patients with antiretroviral therapy (art) at our institution were identified. 29 were excluded (8 co-infected with hepatitis virus, 3 with insufficient data and 18 no had more than 6 months included), including 112 patients in the analysis, with a mean age of 45 years and 60% were men. the median of time monotherapy treatment was 1.75 years (639.5 days), 89(79.4%) patients received darunavir/r and 23 (20.53%) lopinavir/r. the effectiveness of monotherapy treatment during the follow up period was 100% with undetectable pvl at follow-up. the median of cd4% over the treatment time was 794 cell/ml (34%). conclusion: the effectiveness of treatment with ip/r monotherapy in our hospital obtained good results. according with our results treatment adherence plays a very important role. this is a current and valid strategy that brings benefits to the patient and to the healthcare system. please specify your abstract type: research abstract background and objective: the access to investigational drugs for patients who are not included in a clinical trial and without authorized therapeutic alternatives is known as compassionate use. the incorporation of the evidence-based medicine in the area of oncohaematology has implied that an important part of clinic therapy validated by evidence that could not be controlled from an administrative point of view. this is due to the continuous and progressive development of investigation and information on cancer treatment and the delay of the administration regulation. the use of drugs in this way is regulated by royal decree 1015/2009 (19/6). the objective of the study is to describe the use of cancer drugs through compassionate use in the last 5 years in a specialty hospital. setting and method: descriptive retrospective study on a specialty hospital. all the applications for a compassionate use drugs were analysed from january 2011 until october 2015. the data were obtained from medical records programme diraya ò and from an excel database of medicines in compassionate use of the pharmacy service. main outcome measures: the following variables were registered: • number of patient clinic history • authorized medicine • authorization date • applicant service results: we recorded 80 requests of cancer drugs in compassionate use during the 5 years of study. oncology was the service that recorded more authorizations with 95%, followed with gynaecology with 2.5% and finally endocrinology and haematology with 1.25%. 64 drugs of the 80 requests were approved (80%) and 16 unauthorized (20%) in the 5 years of study. the year in which more applications were received was 2013 (31.25%) and the least requests were received in 2012 (6.25%), being the year where all requests were authorized. in 2015 fewer applications were authorized, 75%. in the years 2011, 2013 and 2014 were authorized 88.3, 76 and 88.3%, respectively. a total of 34 different active drugs were received during the study, the most requested bevacizumab (24%) for grade iii oligoastrocytoma, ovarian cancer (monotherapy), metastatic gall bladder cancer and metastatic platinum-resistant ovarian cancer, everolimus (18%) for indications of neuroendocrine carcinoid tumour and metastatic breast cancer, nab-paclitaxel (18%) for invasive lobular carcinoma indications of high-grade and metastatic pancreatic cancer, ipilimumab (12%) for the indication of metastatic melanoma, and regorafenib for indications of colorectal cancer and metastatic gist i pre-treat with imatinib (12%). the solicitude of drugs through compassionate use needs effective commissions of pharmacy and therapeutics, along with the medical management to establish an agile and faster requesting circuit and the consequent use monitoring. please specify your abstract type: research abstract background and objective: to describe the standard procedure for the elaboration and control of a magistral formula (mf) to assess their effectiveness in two patients with cutaneous metastases of malignant melanoma refractory to other treatment. setting and method: medication for compassionate use was requested for two patients of 78 and 49 years with histopathologic diagnosis of cutaneous metastases of malignant melanoma in the left thigh and left heel in which the lack of response to first-line treatments made to be valued to start with adesleukina intralesional therapy. the first week was infiltrated 3 mu (1 ml) in 5 lesions less than 1 cm, 9mu (3 ml) in the larger lesions and repeating each week until complete remission of the lesions. in the 2nd patient we proceed in the same way but the second week was infiltrated 5mu (8 ml). the following week, infiltrated 9 mml, in 15 metastases and we turn to 2 weekly infiltrations. the response was assessed by clinical disappearance of the lesions treated. complete response (cr) is defined as a clinical disappearance of lesions and partial response (pr) greater than 50% reduction of the lesion diameter. main outcome measures: we performed a literature search (pubmed, trissel, spc) for all studies published to determine the standard procedure for preparing and monitoring the mf (processing, preservation, stability, dose and indication). results: the standard procedure of preparation and quality control was carried out following the rules established in rd 175/2001. it was made in a vertical laminar flow cabinet. the aldesleukin vial was reconstituted with 1.2 ml api (18 mu/ml) and then diluted with 4.8 ml of a solution of 0.1% albumin, 5% glucose as stabilizer, to avoid aggregate formation, preparing 1 ml syringes (3 mu/ml). it was obtained a homogeneous and clear solution without precipitate or opalescence appearance. stable 6 days in a refrigerator (2-8°c), protected from light. initially patients had approximately a total of 60 injuries. after 2 months of treatment it was obtained a cr of most lesions in the first patient and rp of the second patient injuries. treatment was well tolerated. the side effects presented were only a flu-like syndrome in the second patient. conclusion: intralesional administration aldeslukina has been effective in treating malignant melanoma skin metastases in our patients, allowing the extension of its use in patients with the same involvement refractory to other primary treatments. the results are similar to those of the publications consulted. please specify your abstract type: research abstract background and objective: chronic infection with hepatitis virus c (hcv) affects about 170 million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. the new direct acting antivirals against hcv have revolutionized the treatment of this disease. due to the high cost of these drugs it is necessary to assess their use in clinical practice. to evaluate the effectiveness of daclatasvir in combination with sofosbuvir in patients with hcv monoinfected in a specialty hospital. setting and method: retrospective observational study of patients who began treatment with the combination of daclatasvir and sofosbuvir from january 2015 to january 2016 in a specialty hospital. the data were collected from the hospital program of clinical stories archinet ò and the outpatient program farmatools ò . the results were analysed using the statistical program r-commander. main outcome measures: the sustained virologic response (svr) was considered the primary endpoint of the study. as secondary variables were analysed: sex, duration of treatment, naïve patients or pre-treated, degree of fibrosis, hcv genotype, concomitant use with ribavirin, viral load (vl) before treatment and medical service. results: there were included 28 patients of whom 23 were men. baseline characteristics were: 17 patients with genotype 3, 8 genotype 1b, 2 with genotype 1a and 1 genotype 4. the degree of fibrosis in the study was 15 patients with f4, f3 9 and 3 to f2. among the 17 patients infected with hcv genotype 3, 9 had not received prior treatment (naïve) and 8 had failed therapy. the duration of the treatment was 12 weeks to 16 patients and 24 weeks for 12 patients. only 7 patients receiving ribavirin of these 5 had genotype 3 and 2 genotype 1b. from ribavirin patients it was greater the number of patients in whom the treatment duration was 24 weeks (6 patients versus 1 with p-value = 0.008151). the digestive service attended to 17 patients while 11 patients were followed by infectious. the median cv was 3,067,940 iu/ml. svr was achieved in 81.2% of patients with hcv genotype 3 in 87.5% with genotype 1b and 100% with genotype 4 and 1a. after 12 weeks of treatment 61% of patients achieved svr and 39% after 24 weeks. only one patient died during treatment. the results are similar to those obtained in clinical trials. svr has not been influenced by hcv subtype, duration of treatment, degree of fibrosis, pre-treatment or by concomitant use of ribavirin. further studies are needed to evaluate the efficacy of this treatment. please specify your abstract type: research abstract background and objective: the safety and efficacy of medications can vary significantly between patients as a result of genetic variability. as genomic screening technologies become more widely available, pharmacists are ideally suited to utilize this tool to optimize medication management. the objective of this study is to evaluate the feasibility of implementing personalized medication services into community pharmacy practice and to assess the number of drug therapy problems identified as a result of pharmacogenomic screening. setting and method: the study was designed as open-label, nonrandomized, and observational. two community pharmacies in toronto, ontario offered pharmacogenomic screening as part of their professional services program. prior to initiation, participating pharmacists received structured, comprehensive training in pharmacogenetics. pharmacists then facilitated voluntary subject enrolment among patients who they believed would benefit from screening and met inclusion criteria. eligible patients received a simple buccal swab followed by dna analysis using pillcheck ò . pillcheck ò is a genotyping assay that translates genomic data and generates a personalized, evidence-based, report that provides insight into patients' inherited drug metabolic profile. upon receiving the report, pharmacists invited patients back to the clinic for interpretation of the results. clinically significant drug therapy problems were identified and recommendations for medication optimization were forwarded to the primary care physician. main outcome measures: number of clinically significant drug therapy problems identified by pharmacists as a result of pharmacogenomic testing. results: 100 patients were enrolled in the study. average age was 57.4 years and patients were taking a mean of 5.6 chronic medications. pharmacists cited the most common reasons for testing as ineffective therapy (44.6%), to address an adverse reaction (35.5%), and to guide initiation of therapy (11.8%). an average of 1.3 drug therapy problems were identified per patient. pharmacist recommendations included change in therapy (57.1%), dose adjustment (14.3%), discontinuation of a drug (7.1%), and increased monitoring (19.6%). generally, physician feedback was positive but did reveal an opportunity for a broader understanding of the technology. conclusion: these results highlight the readiness of community pharmacists to adopt pharmacogenetic screening into practice and their ability to leverage this novel technology to positively impact medication management. community pharmacists are ideally suited to both offer personalized medication services and interpret genomic results. please specify your abstract type: descriptive abstract (for projects) background and objective: visual impairment is a common geriatric syndrome and glaucoma/miotic eye drops treatment is a frequent therapeutic option. pharmacist's role in medication reconciliation is an effective process for reducing medication errors and supporting safe medication use. we observed that mentioned medication reconciliation was occasionally not performed during hospital stay and could be cause of delirium because of visual impairment. the aim of this study was to evaluate the influence of omission errors of eye drops treatment on incidence of acute confusional state. design: we conducted an observational, descriptive and retrospective study in an orthogeriatric unit with an average of 600 patients with hip fractures per year (95% surgically treated). data collection was performed from june 2015 to march 2016. reconciling medications at admission was performed by implementing the tools and resources of the canadian patient safety institute (cpsi). we extracted from our electronic database (filemaker pro ò ): • demographic patient data (age and gender). • name and posology of the glaucoma/miotic eye drops treatment. • medication reconciliation performed and identification of professional in charge (pharmacist, geriatrician or orthopaedic surgeon) registration during hospital stay. • protocolar management of delirium with tiapride occasional intramuscular administration performed if necessary was also registered to establish the incidence of acute confusional state. results: thirty-two patients (26 women and 6 men) were included, median age 86 year-old . in 21 patients, eye drops reconciliation treatment was performed by the pharmacist in 17 of the 21 patients, the geriatrician in 3 cases and the orthopaedic surgeon in 1. in 11 patients, the mentioned medication reconciliation was not performed (pharmacist absentism). considering the 21 patients on eye drops treatment during hospital stay, 4 (19.0%) of them suffer from acute confusional state. on the other hand, among the 11 patients without medication reconciliation, delirium was registered in 7 cases (63.6%). concerning ocular topic treatment, 2.4 ± 1.1 active principles per patient were observed, being the most frequent timolol (68.8%), brinzolamide (40.6%) and latanoprost (37.5%). conclusion: we consider of paramount importance the pharmacist evaluation availability at an orthogeriatric unit, minimizing the impact of acute confusional state during hospital stay by medication reconciliation. please specify your abstract type: descriptive abstract (for projects) background and objective: to report the therapeutic management of haemorrhagic rectocolitis onset in a lung-transplanted patient with mycophenolate-induced diarrhoea. design: case report. results: a 54-year-old-man lung transplant patient for alpha 1-antitrypsin deficiency in 2003 receiving mycophenolate mofetil, tacrolimus and corticosteroid developed chronic diarrhoea worsened by sigmoid and cecal necrosis in 2011, and treated successfully by sigmoidectomy. severe diarrhoea attributed to mycophenolate mofetil reappeared in april 2015, which motivated a switch to mycophenolate sodium. the absence of clinical improvement in june 2015 led to stop mycophenolate sodium and introduce azathioprine at 100 mg/day (absence of mutation for the thiopurine methyl transferase gene). one month later, the patient presented melena, diarrhoea, bloating, nausea, and knee pain, attributed to azathioprine. this latter was stopped and mycophenolate mofetil was rechallenged associated with symptomatic treatment (i.e., diosmectite and loperamide). in january 2016, a colonoscopy, performed in a context of profuse chronic diarrhoea with mucus during 3 months, highlighted haemorrhagic rectocolitis. therefore, the patient initiated sulfazalasine therapy with no clinical improvement, and then high doses of oral corticosteroids. because high-dose of oral corticosteroids was not recommended as a long-term treatment, mercaptopurine was proposed as a new therapeutic option. mercaptopurine has no indication as an immunosuppressive treatment in solid organ post-transplant supportive care. however, as the active metabolite of azathioprin, an immunosuppressive drug widely used in transplantation, mercaptopurine has immunosuppressive functions towards t-lymphocytes. after multiprofessional collaboration between gastroenterology, pneumology and pharmacy specialists, it was decided to stop mycophenolate mofetil and introduce mecaptopurine at 1.5 mg/kg/day, as immunosuppressant for haemorrhagic rectocolitis as well as lung transplantation. this unusual lung transplant immunosuppressive therapy, associated with tacrolimus, improved digestive disorders and patient's quality of life. currently, mercaptopurine is biologically and clinically well tolerated. the dosage of blood residual concentrations of purinethol metabolites (6-thioguanine and 6-methylmercaptopurine) is going to be performed. conclusion: immunosuppressive therapy in solid organ transplantation is a real challenge for patients who have comorbidity onset. despite a lack of data in the literature, a multidisciplinary collaboration based on comprehensive pharmacology skills is essential to choose the best therapeutic option in this type of patients. please specify your abstract type: descriptive abstract (for projects) background and objective: the use of complementary medicines (cm) in oncology is the subject of broad but still controversial interest. a large part of patients with cancer uses cm, including complementary drugs, during their treatment period. indeed, according to different studies, this proportion ranges from 18 to 83%. importantly, the risk of interaction between cm and anti-cancer drugs is not negligible; hence we need to identify these cm to ensure the security of our patients and the success of their treatment. design: to achieve this purpose, a monocentric retrospective analysis was conducted with collection of data by pharmacy students during medication reconciliation of hospitalized patients from january to june 2016. collected data are patients' characteristics, prevalence of cm use and potential cm-anticancer drug interactions. results: 161 patients were included in the study (91 men-70 women); median age was 65 [34-88 years]. a total of 24.2% (n = 39) were using a least one cm, most frequently homeopathy (62%, n = 24) or phytotherapy (36%, n = 14); some patients were using a combination of two cm (41%, n = 16). cm are mainly used by women in comparison to men (32.9% versus 17.6% and p = 0.025, chi square test). for phytotherapy, at least 36 different herbs were described by patients and among them the most frequently used were mistletoe (viscum album), propolis and fireweed (epilobium angustifolium). data analysis showed that 23% (n = 9) of patients were at risk of potential cm-anticancer drug interaction. moreover this risk was increased to 50% if we considered only patients taking phytotherapy. interactions included pharmacokinetic (15%, n = 3), such as altered hepatic metabolism, and pharmacodynamics ones (85%, n = 17). conclusion: in conclusion, our work clearly demonstrates that the use of cm by patients is associated with high risk of relevant drug interaction with their anti-cancer treatment. even if further investigations are necessary to clarify the clinical impact of these interactions, the use of cm must be considered during prescribing process. please specify your abstract type: research abstract background and objective: since their reimbursement, the direct oral anticoagulants (doacs) are increasingly used for stroke prevention in atrial fibrillation (af). the objective of this study was to identify the proportion of real life patients with af eligible for doac therapy, based on the inclusion and exclusion criteria used in the clinical studies and based on the officially approved indications as mentioned in the summary of product characteristics (smpc). setting and method: data for this retrospective cross-sectional study was extracted from the uz brussel stroke registry, containing anonymized data of 2205 patients with a suspected stroke. characteristics of patients with documented af were compared with the patient characteristics in clinical trials and the approved indications in the smpc. main outcome measures: proportion of real life patients with af eligible for doac therapy. results: data of 468 patients with af was analysed. based on the selection criteria of the clinical trials, significantly less patients were eligible for treatment with rivaroxaban compared to dabigatran etexilate (39.3% versus 47.6%; p = 0.010), but not compared to apixaban (45.5%; p = 0.055). based on the indications and contraindications in the smpc, significantly fewer patients were eligible for apixaban compared to dabigatran etexilate and rivaroxaban (62.0% for apixaban, 72.9% for dabigatran etexilate and 75.6% for rivaroxaban; p \ 0.001 and p \ 0.001, respectively). significantly more patients were eligible for doac therapy based on the indications and contraindications in the smpc compared to the inclusion and exclusion criteria of the clinical trials (72.9% versus 47.6%; p \ 0.001 for dabigatran; 75.6% versus 39.3%; p \ 0.001 for rivaroxaban and 62.0% versus 45.5%; p \ 0.001 for apixaban). conclusion: when taking into account the selection criteria from the pivotal clinical trials with doacs for stroke prevention in af, less than half of real life patients are eligible for therapy with one of the doacs. however, the indications mentioned in the smpcs of these drugs are less strict. please specify your abstract type: research abstract background and objective: idiopathic pulmonary fibrosis (ipf) is a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. the formation of scar tissue is called fibrosis. pirfenidone is an anti-fibrotic and anti-inflammatory agent, thus offers a new hope for treating progressive fibrotic diseases. int j clin pharm (2017) 39:208-341 333 our objective is to set a description of idiopathic pulmonary fibrosis patients treated with pirfenidone, as well as the adverse reactions observed. setting and method: descriptive study in which all patients have received pirfenidone. the data were obtained through the dispensing program of outpatient (farmatools) and review of medical records of the hospital database (archinet) and clinical station (diraya). main outcome measures: we have extracted from each patient baseline data, comorbidities, dose received, reported adverse reactions and data about haematology and biochemistry. results: we have a total amount of 5 patients treated with pirfenidone, all diagnosed with idiopathic pulmonary fibrosis, including 2 women and 3 men. the age of patients is between 66 and 81 years, with an average of 70.8 years. all patients are ex-smokers and one of them is also ex-alcoholic. concerning concomitant pathologies, 3 patients have diabetes mellitus, 2 have arterial hypertension, and one of them has ischemic heart disease. another has upper gastrointestinal bleeding prior, among others chronic pathologies. pirfenidone dose received was the usual dose in 4 of the 5 patients: days 1-7 267 mg every 8 h, days 8-15 534 mg every 8 h and a maintenance dose of 801 mg every 8 h. in one patient due to its low imc the dose received was smaller (1-7 days 267 mg every 12 h, days 8-15 267 mg every 8 h and maintenance dose of 534 mg every 8 h). in relation with the adverse effects, digestive discomfort were observed in 2 of the 5 patients, causing the interruption of the treatment in one of them (with prior gastrointestinal bleeding). in the other patient it was relieved by lowering the dose received. also, one patient has experienced photosensitivity. alterations in transaminase levels were observed in 2 patients but that didn't force to discontinue the treatment. no alterations were observed in the blood count. conclusion: treatment with pirfenidone is being generally well tolerated by patients. it has improved their life-quality and reached the objective data of a slowdown in disease progression. currently, the number of patients is no enough to give conclusive information in relation to the drug effectiveness. please specify your abstract type: research abstract background and objective: to describe the total amount of patients treated with a magistral formula of sodium cromoglycate 200 mg without excipients: indications, concomitant therapy and the response to therapy. setting and method: we run a descriptive study in which we included the totality of patients in treatment with a magistral formula of sodium cromoglycate 200 mg without excipients in a tertiary hospital. the data were obtained through paracelso (development of magistral formulas program), as well as with farmatools (dispensation program of outpatient) and the review of medical records from the hospital database (archinet), and diraya clinical station. main outcome measures: from each patient we extracted data relative to sex, age, diagnosis, time in treatment with the formula, dose received, response to therapy, concomitant antihistamines treatments and adverse effects. results: a total of 9 patients in treatment with a magistral formula of sodium cromoglycate 200 mg without excipients were reviewed: 2 women and 6 men with a mean age of 45.3 years old (range 38-59 years). regarding the indication of the prescription, 3 patients have been diagnosed of indolent systemic mastocytosis and the remaining 5 were diagnosed of mast cell activation syndrome. in all cases, the diagnosis was established by examination of the bone marrow in the mastocytosis studies institute of castilla la mancha (spain). on average, patients took the treatment 12.75 months, with a range between 3 months and 24 months. the dose received was 200 mg every 8 h in 7 patients, having to be increased to 400 mg 3 times daily in a case with poor response to the therapy. in the remaining patients, the treatment response has been optimal. in relation to the concomitant anti-allergic treatment received, 6 patients took fexofenadine daily during the study. no cases of adverse effects related to the therapy received have been reported. conclusion: both indolent systemic mastocytosis and mast cell activation syndrome are considered rare diseases, and we should indicate that in spain there are no commercial medicines available of sodium cromoglycate without excipients for its treatment. the treatment with this magistral formula of sodium cromoglycate 200 mg without excipients has been effective and well tolerated in all patients, improving the symptoms associated with their condition as well as their quality of life, and also, assuming a solution to the lack of marketing of the drug currently in spain. please specify your abstract type: research abstract background and objective: to analyse the prescription profile, safety and effectiveness of new therapies available for the treatment of hcv genotype 1b in a tertiary hospital. setting and method: we run a retrospective observational study in which we included a total amount of 59 patients infected with hcv genotype 1b treated with the new therapies against hcv from february 2015 to december 2015 in a tertiary hospital. the data were obtained through the outpatient dispensing program farmatools and the review of the medical records from the hospital database, archinet and prescription hepatitis c portal of the andalusian health service. main outcome measures: from each patient the following information was collected: sex, age, viral genotype (gen.), naive/nonnaive, hiv coinfection, presence of cirrhosis, degree of hepatic fibrosis measured by fibroscan, treatment prescribed and duration, adverse effects, sustained viral response (svr) and the service that made the prescription. results: a totality of 59 patients with hcv gen. 1b were reviewed which 64.4% of them were men with a mean age of 58.76 years (range 38-78 years). 21 of the patients were naive and only 2 of them were hiv co-infected, there were a 57.63% of cirrhotic patients. regarding the degree of hepatic fibrosis, 36 patients had grade f4, f3 grade 13 patients, 8 patients grade f2 and f1 grade 2 patients. the most commonly therapy prescribed was lepidasvir + sofosbuvir in 28 patients (14 without ribavirin and with ribavirin 14) using a treatment schedule of 12 weeks in 23 of them. the treatment was discontinued in one case because of the adverse effects, achieving svr in the remaining patients. the combo treatment with paritaprevir/ombitasvir/r + dasabuvir was prescribed in 16 times (10 without ribavirin and 6 with ribavirin) choosing only in one of them for a treatment period of 24 weeks. there were no treatment discontinuations and svr was achieved in all patients treated in this way. 8 patients received simeprevir + sofosbuvir for 12 weeks (3 without ribavirin and 5 with ribavirin), one patient of the left the treatment due to adverse effects. svr was found in the remaining patients who completed treatment. sofosbuvir + daclatasvir was prescribed to 5 patients, associating ribavirin in only one case. a treatment duration of 12 weeks was used in 3 patients and 24 weeks in the remaining two. one patient failed rvs without any incidences of adverse effects in any case. interferon + ribavirin sofosbuvir + was prescribed to 2 patients in 12-week regimen which was well tolerated achieving svr. digestivo service treated the 83% of the total amount of patients. conclusion: new therapies for hcv have been used in all the treated patients and the older drugs have been relegated. about the effectiveness, svr was achieved in 98.30% of patients. regarding the safety, only 2 patients have discontinued the treatment due to adverse effects representing less than 5% dropout rate of the therapy. please specify your abstract type: descriptive abstract (for projects) background and objective: thanks to pharmacogenetics we can identify and predict different responses to the same drug among different individuals. during these last years we have noted a big increase of dosing guidelines and advices about the use of several drugs due to the influence of different polymorphisms. the aim of this study is to describe and evaluate the use of pharmacogenetics in our hospital from april 2012, when we started our first research about pharmacogenetics, to the actual time, using these information in our daily clinical practice; and indeed quantify the number of different tests and the number of different clinical advices done because of pharmacogenetic information, by different healthcare specialty areas and drugs. design: we reviewed all the pharmacogenetic test requests in our hospital from april 2012 to april 2016, noting which health specialty and for which drug was asked the test. polymorphisms were genotyped using taqman ò genotyping assays technology by 2 independent laboratories to confirm the results. results: from april 2012 we were asked for 2208 pharmacogenetic tests from 7 different healthcare specialty areas: rheumatology (9.78%), infectious diseases (1.49%), oncology (3.53%), cardiology (71.51%), vascular surgery (5.11%), neurology (4.53%), ophthalmology (4.03%); this information was asked about 5 different drugs: clopidogrel (81.16%), trastuzumab (3.53%), ranibizumab (4.03%), azathioprine (2.99%) and tocilizumab (8.29%). from all the genotypes, 713 (32.29%) were done after using the drug (study phase) and 1495 (67.71%) were done previous to the use of the drug in daily clinical practice to make a ''clinical recommendation''; from these recommendations 1429 affected to the prescription of clopidogrel. conclusion: during the last 4 years we could implement the use of pharmacogenetics in the daily clinical practice in our hospital in 5 different healthcare areas affecting 2 drugs and we started research studies previous to its use on the clinical practice for other three different drugs. please specify your abstract type: descriptive abstract (for projects) background and objective: the drug burden index (dbi) is a tool used to quantify the anticholinergic and sedative burden of medication on an individual. it has been independently associated with poor physical and cognitive performance in community-dwelling older people. objectives were: to create an inventory of medications used in ireland with clinically significant anticholinergic and/or sedative activity and to decide upon the minimum daily dose (mdd) for each medication. design: medications with potential anticholinergic and/or sedative burden were identified by literature review and examination of the summary of product characteristics (smpc) for all medications registered in ireland. each medicine was classified as anticholinergic or sedative. drugs with both anticholinergic and sedative properties were classified as primarily anticholinergic. the mdd, a key component of the dbi score calculation, was selected by reference to the irish smpc. other options which were also considered for this value include the defined daily dose (ddd) of a medication, as available from the world health organisation (who), and the mdd as outlined in the british national formulary (bnf). mdds were decided upon regardless of indication as the lowest effective therapeutic dose as specified in the smpc for the medication. the final list of medicines and mdds to be included in the inventory was then defined by consensus of three pharmacists. results: in total, 383 medicines with potential anticholinergic and/or sedative activity were considered for inclusion. a final list of 258 medications was identified by consensus (117 anticholinergic, 141 sedative). of these, 128 (50%) were agents which act primarily on the nervous system. the three main therapeutic groups contributing to the inventory of dbi medications were antipsychotics (25 medications), antidepressants (21 medications) and antiepileptics (20 medications). conclusion: creation of an inventory of medications with anticholinergic and/or sedative properties, in combination with the individual mdds, was achieved. this is a useful resource for use in analysis of drug burden in an older population. it could help in both identifying patients who would benefit from medication review as well as analysing population medication data. please specify your abstract type: research abstract background and objective: vancomycin is an antibiotic widely used to treat infections such as bacteraemia, infective endocarditis, osteomyelitis, meningitis and pneumonia. nowadays, optimal trough concentration is stablished between 10 and 15 mg/l to avoid development of resistance or 15-20 mg/l to improve penetration in complicated infections. some articles 1 have been published explaining the methodology to calculate an expected trough level in steady state. our aim was to compare the trough serum value estimated by the mathematical method with a two-compartimental bayesian forecasting model. setting and method: observational retrospective study carried out in a tertiary hospital from january to december 2015. non obese adult patients with creatinine clearance (crcl) \ 120 ml/min and who have achieved steady state level were included. vancomycin serum values were measured using a chemiluminescence's immunoassay (cmia) and bayesian analysis was performed with abbottbase pksystem ò (pks ò ). the statistical analysis was made with medcalc software ò . bland-altman plot and passing-bablok regression were used to compare both methods. main outcome measures: sex, age, weight, dose, creatinine, and size were collected from clinical history. serum trough values (cminr) were collected from cmia. trough values were estimated using two methods: mathematical method (cminf) and bayesian calculations (cminb). results: 50 patients were included, with a mean age of 61 (±16.17) years. 52% were male and 48% female. they received a median dose per 24 h of 2000 (1000-3000) mg. the mean of cminr was 17.21 mg/l (95% ci 13.86-20.58), cminb 17.28 mg/l (95% ci 13.87-20.69), cminf 18.21 (95% ci 14. 2399-22.1856) . correlation coefficients (r) comparing both methods were significantly different: r between cminf and cminr was 0.75 (95% ci 0.5866-0.8467), while r between cminb and cminr was higher: 0.99 (95% 0.9900-0.9968). bland-alman plot analysis showed both methods cannot be used interchangeably. the regression equations estimated by passing-bablok regression were y = -3.873368 + 1.309775x and y = -0.110207 + 1.003266x. conclusion: bayesian method has demonstrated better correlation with real measures than mathematical method. most part of our patients could be underestimated or overestimated using mathematical methods which could cause toxicity or lack of efficacy, so this method is unsuitable for clinical use. bayesian estimation remains the best option for optimal dosing of vancomycin. please specify your abstract type: research abstract background and objective: combination therapy with digoxin and acenocoumarol is common in patients with atrial fibrillation (af). getting optimal concentrations of digoxin leads an appropriate response; taking into account its narrow therapeutic range and all the factors which can affect to its pharmacokinetics. interaction between them has been studied, even though its mechanism is not clear yet. patients who are taking both drugs need higher doses of digoxin; because they get lower concentrations by using the same dosage. the objective of this study was to analyse digoxin concentrations in patients treated with this combination compared to expected concentrations according to population parameters. setting and method: retrospective observational study from december 2015 to march 2016 performed by pharmacokinetic unit. patients included had chronic treatment with acenocoumarol and digoxin, which determination were realized in the steady state before the next dosage. patients with toxics concentrations of digoxin, or who were suspected nonadherence, were excluded. the plasma digoxin concentrations were determined through the autoanalyzer architect c-4000 ò (petinia). dosage adjustment was realized by the program abbot pharmacokinetics system (pks). a comparative between the real measured concentrations in patients and estimated concentrations were realized based on population parameters. finally, in order to get optimal concentrations, some dosage changes were proposed based on pharmacokinetic monitoring. data collected: population characteristics (gender, age, weight, and height), analytical data (potassium, urea, creatinine and clearance). main outcome measures: digoxin serum concentrations (optimal range 0.8-1 ng/ml). results: data from 73 patients, 65.8% women with a mean (sd) age of 77.84 (9.07) years were included in the study. at baseline, potassium, urea, creatinine and clearance mean (sd) was 4.43 (0.52) mmol/l; 55.99 (34.05) mg/dl; 0.96 (0.39) mg/dl; 48.76 (22.14) ml/min. 69.86% of the patients had lower concentrations than expected according to population parameters. finally, digoxin dosage was increased in 41.07% of patients, it was maintained in 47.96%, and it was decreased in 10.97%. conclusion: digoxin concentrations in patients with af in combination therapy of digoxin and acenocoumarol are lower than would be expected in most cases. it is important monitoring digoxinaemia to achieve optimal concentrations and a good clinical response. further studies are needed to determine the relevance of this interaction in clinical practice. please specify your abstract type: research abstract background and objective: tocilizumab (tcz) is a humanized monoclonal antibody inhibitor of il-6 receptor, indicated in combination with methotrexate in the treatment of rheumatoid arthritis (ra) in patients with inadequate response or intolerance to prior therapy. interleukin 6 is involved in the pathogenesis of rheumatoid arthritis via its broad effects on immune and inflammatory responses. previous studies have shown that c-allele at the -174g[c (rs1800795) polymorphism is related with a bad response to tocilizumab (according to eular criteria). the aim of our study was to explore the potential role of il-6 genetic polymorphisms as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients and check this association depending on the genotype. setting and method: the il-6 (g[c) (rs1800795) genetic variant was genotyped using predesigned taqman ò genotyping assays technology and analysed on a viia7 ò real-time pcr system. main outcome measures: clinical response was evaluated at 3, 6, 9 and 12 months according to the eular criteria. patients were classified as ''responders'' (good and moderate response according to eular criteria) and ''non-responders''. the statistical analysis was performed using spss v.20. results: we recruited 163 patients with ra treated with tocilizumab, these were aged 54.02 ± 11.11 (mean ± sd), 132 (81%) were women. the mean das28 at baseline was 5.66 ± 1.14. of these 163 patients, the il-6 g[c genetic polymorphism was significantly associated with ''responders'' at 3 months after the baseline (cc vs non-cc p = 0.039, or 0.270, 95% ci 0.072-1.005) but not at 6 (p = 0.666), 9 (p = 0.233) and 12 (p = 0.244) months. conclusion: the il-6 g[c may be useful as a genetic marker of tocilizumab efficacy at 3 months. other polymorphisms, clinical parameters and other pharmacological treatment during the follow-up may be checked about their influence on the response to tocilizumab. tdmp014: daptomycin pk/pd profile in neutropenic cancer patients with beta-lactam-resistant gram-positive infection nancy perrottet *,1 , frederic tissot 2 , laurent decosterd 3 , thierry buclin 3 , guy prod'hom 4 , christina orasch 2 , oscar marchetti 2 , farshid sadeghipour 1,5 , thierry calandra 2 , véronique erard 2 1 pharmacy service, 2 infectious diseases service, 3 laboratory and division of clinical pharmacology, service of biomedicine, 4 institute of microbiology, lausanne university hospital, lausanne, 5 school of pharmaceutical sciences, university of geneva, university of lausanne, geneva, switzerland please specify your abstract type: research abstract background and objective: the pharmacokinetics (pk) and pharmacodynamics (pd) of many antibiotics are modified in neutropenic patients and few data are available on daptomycin in this population. this prospective study aimed to assess the pk/pd profile of daptomycin in the treatment of neutropenic patients with beta-lactamresistant gram-positive cocci infections. setting and method: this substudy was performed in the context of a prospective pilot study on daptomycin versus vancomycin in adult hemato-oncological patients with febrile neutropenia and proven or suspected infection with methicillin-resistant staphylococci or betalactam-resistant enterococci. patients received daptomycin 6 mg/ kg/day (8 mg/kg/day for enterococci) for c7 days as a 2-min infusion. main outcome measures: pk analysis using a published non-linear mixed effect model with nonmem ò , followed by comparison of parameters with values published for healthy subjects. pd analysis based on auc/mic (area under the concentration-time curve/minimal inhibitory concentration). according to eucast, an auc/mic ratio [438 is required for bacteriostatic effect against staphylococci and [800 for a two-log reduction in bacterial count. for e. faecium, an auc/mic ratio of 0.94 has been suggested for bacteriostasis and 4.14 for a 1-log bacterial count reduction. results: model-derived mean auc observed in 13 patients was 539.3 ± 340.1 mg h/l, maximum concentration (cmax) 88 ± 28 mg/ l, minimal concentration (cmin) 7.2 ± 6.7 mg/l. clearance was 0.98 ± 0.36 l/h and volume of distribution at steady sate 11.3 ± 3.3 l, both values found higher than those reported in healthy subjects. all patients (7/7) with a staphylococcal infection achieved auc/mic values predictive of bacteriostatic effect on staphylococci, and 6 out of 7 values associated with two-log bacterial killing. of note, infection relapse occurred in the only patient with suboptimal daptomycin exposure (auc/mic of 580). the pd targets were also reached in the two patients with e. faecium infection. an asymptomatic elevation of creatine phosphokinase was reported in two patients (568 u/l and 218 u/l) with cmin of 25.7 and 13.7 mg/l, respectively. conclusion: daptomycin pk profile in 13 neutropenic cancer patients indicated higher total clearance and volume of distribution, along with lower total exposure, compared to healthy subjects. despite this, standard dosages allowed attainment of pd targets in 6/7 patients with a staphylococcal infection (two-log drop) and 2/2 with e. faecium infection (1-log drop) . please specify your abstract type: research abstract background and objective: individual clinical response to infliximab can be influenced by their pharmacokinetics and immunogenicity, so therapeutic monitoring of drug levels (tdm) can guide these biologic treatments. the objective was to analyse the suitability of serum infliximab trough levels (sitls) in patients with inflammatory bowel diseases (ibd) receiving dose schemes based only on clinical response. setting and method: prospective and descriptive study of patients with ibd treated with infliximab and under tdm. medical records were reviewed. dose schemes were established according to clinical guidelines (5 mg/kg every 8 weeks) and optimized based on an index of clinical response (mayo, pcr…). sitls (therapeutic range 3-9 mcg/ml) and anti-drug antibodies (ada) were measured in all of patients by elisa (promonitor ò ). ada presence was considered as a therapeutic failure indicator. informed voluntary consent was obtained from all patients. main outcome measures: sitls and ada. results: a total of 61 patients, with a median age of 48 years (range ), were included in the analysis. infliximab standard dose according to clinical guidelines were administered to 39 patients: 46.1% showed sitls under the therapeutic range (11.1% with ada). in eight patients with maintained good clinical response, dose decrease or interval elongation had been implemented: 25% of these patients showed sitls below the therapeutic range (100% with ada). it had been necessary to increase the dose or shorten the interval in 14 patients due to inadequate clinical response: 28.6% of these patients with sitls below the therapeutic range (50% with ada). conclusion: optimization based on clinical response of infliximab treatments in patients with ibd is not always an effective strategy, since it leads to a high percentage of patients with sitls below the therapeutic range and adas. tdm together with clinical response should guide the optimization of infliximab treatments. please specify your abstract type: research abstract background and objective: in addition to its anticonvulsive properties, valproate is also used as a mood stabiliser in bipolar disorder and as augmentation treatment of other psychiatric disorders. the unpredictable relationship between dose-plasma valproate concentrations and correlation between concentrations-efficacy suggest therapeutic drug monitoring (tdm) of plasma valproate concentrations might be useful. the aim of our study was to evaluate the rationale of a new protocol for measuring valproate concentrations and the incorporation of a clinical pharmacist in the process of valproate tdm service, compared to pre-existing standard measuring. setting and method: in the retrospective study we analysed the process of measuring plasma valproate concentrations at the department of psychiatry and at the unit for forensic psychiatry of a large teaching hospital in slovenia before the enrolment of a clinical pharmacist. for the prospective study we created a protocol for tdm of valproate in adults based on literature research. the protocol included reference range, sampling time, indications for sampling and schedule of other laboratory tests that have to be monitored during valproate therapy. main outcome measures: percentage of plasma valproate concentrations in reference range (c trough = 50-125 mg/l) before/after the enrolment of a clinical pharmacist, percentage of measured valproate c trough . results: in the retrospective study 30 randomly chosen patients with measured plasma valproate concentrations were included (56% male, age 49 ± 15 years, length of hospital stay 56 ± 40 days). plasma valproate concentrations were measured 5.8 ± 4.4 times per patient, 22% were in the reference range (other 78% subtherapeutic), 3% were drawn at c though , 15.5% were drawn for assessing compliance (nontrough). in the prospective study 19 patients were included (37% male, age 46 ± 16 years, length of hospital stay 36 ± 22 days). plasma valproate concentrations were measured 2.95 ± 1.5 times per patient, 43% were in the reference range (other subtherapeutic), 71% were drawn at c trough , 14.3% were drawn for assessing compliance. conclusion: the inclusion of a clinical pharmacist in valproate tdm service increased the number of valproate plasma concentrations in the reference range by almost 100% and increased the number of concentrations drawn at c trough , when indicated. including a clinical pharmacist in valproate tdm is beneficial and the new protocol is useful for optimising valproate therapy. concurrent and predictive validity of a self-reported measure of medication adherence the effect of pharmacist-led interventions in optimising prescribing in older adults in primary care: a systematic review aflibercept: 1. neovascular membranes with visual acuity higher than 0.1 2. one eye affection severe cardiovascular pathology (severe episodes in the last 6 months) non-responders to other anti-vegf bevacizumab: 1. diabetic macular edema macular edema secondary to vascular pathology setting and method: a longitudinal study was carried out in primary care centres. participants: patients aged c65, under treatment with 5 or more drugs and belonging to 7 primary care areas in 5 different towns. patients should have at least one of the following potential safety problems: (a) concomitant use of a non-steroidal anti-inflammatory drug (nsaid) with an antihypertensive drug, anticoagulant or antithrombotic drug; (b) use of two or more benzodiazepines. two clinical management units (cmu) were randomized per area to be included in the study. thirty patients per cmu were randomized to be enrolled and monitored during 30 months number of adverse effects (0.40; p \ 0.001) and number of clinical problems (0.19; p \ 0.001).with each year increase in age 026) and a significant rise in physician (0.08; p \ 0.001) and nurse (0.06; p \ 0.001) home visits. women compared to men resulted in a significant decrease 043) but a significant increase in visits to nurses (0.43; p \ 0.001), hospital admissions (0.39; p \ 0.053) and hospital visits (0.35; p \ 0.030). age, sex and npsp had no significant effect on falls, fractures or cardiovascular events. conclusion: the npsp in elderly patients contributes to an increase in the use of health services and comorbidity. effective interventions should be addressed to general practitioners to reduce inappropriate prescriptions bpa was found in the dialysate (39 ng/l) and ls (1033 ng/l) wherein the concentration of bpa decreases over time to reach 265 ng/l at the end of a session. finally, bpa was present in all tested dialysis at concentrations of up to 149.0 ng/dialyzer in the compartment mimicking the blood and to 174.0 ng/dialyzer in the dialysate despite prior rinsing with 2l of 0.9% nacl. conclusion: our study is the first one to show the risk of exposure to bpa and bpa-clx hdf-ol. while assessment of the impact of this exposure in a patient under treatment remains to be done, it is now possible to better master contamination by bpa and its four chlorinated derivatives through better practices (choice of medical devices) and improvement of the overall water treatment process san cecilio university hospital, 2 genomic unit san cecilio university hospital, 2 genomic unit, genyo, centre for genomics and oncological research the aim of this study is to compare the apparition of stroke, acs, cardio-vascular death and the need of surgery in patients after percutaneous transluminal angioplasty (pta) or stroke depending on the presence of cyp2c19*2*3 polymorphisms. setting and method: retrospective cohort study. we recruited patients treated with clopidogrel after a pta of the lower limb or stroke (without surgery) from 2010 to 2013 in our hospital. data collected: age, sex, cyp2c19*2 (rs4244285) and cyp2c19*3 (rs4986893) genotypes and the primary end-point: stroke, acs, cv death and surgery of the affected vessel during 12 months after discharge. polymorphisms were genotyped using taqman ò genotyping assays technology. main outcome measures: we recruited 58 patients with stroke (62.07% men; mean age 68.30) and 72 patients after pta (77.7% men; mean age 67.44) treated with clopidogrel after discharge 8%) suffered the primary end-point during 12 months after discharge; 1 of these patients had the cyp2c19*2 allele. among patients with pta of the lower limb: 25% of them had the cyp2c19*2 allele and no one a cyp2c19*3 allele; 25 (34.72%) of these 72 patients suffered the primary end-point during 12 months after the discharge and 11 of these had the *2 allele of the cyp2c19 isoenzyme *2 allele and treated with clopidogrel have a higher risk of the primary end-point than those patients not carrying it spain please specify your abstract type: research abstract background and objective: the engagement of fcgrs by tnf antagonists could affect to macrophage-mediated clearance of immune-complexes. the aim of our study was to evaluate the potential role of fcgr3a (a[c) (rs366911) single nucleotide polymorphism (snp) as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients. setting and method: the fcgr3a (a[c) (rs366911) snp was genotyped using predesigned taqman ò genotyping assays technology and analysed on a viia7 ò real-time pcr system. the statistical analysis was performed using spss v the mean age of the patients was 53.25 ± 12.42 years and 79% were women. the mean das28 at baseline was 5.71 ± 1.13. we found no statistically significant association between our end-point and the genetic polymorphisms studied tdmp011: therapeutic drug monitoring of infliximab biosimilar and anti-infliximab antibodies in inflammatory diseases patients with dermatological conditions and inflammatory bowel disease being treated with ifx-b (5 mg/kg/8 weeks after the induction dose) were included. the concentrations of ifx-b and ati-b were quantified by two sandwich-type elisa immunoassays (triturus ò analyser). main outcome measures: plasma levels of ifx-b and ati-b, clinical response and infusion reactions. the clinical response was assessed according to pathology of each patient (based on specific clinical variables for the pathology into the electronic history) pharmacokinetic results (% assessments): (a) 30.0% no ifx-b detection (c \ 0.035 mcg/ml) and positive ati-b (c [ 2 ua/ml) (3 assessments/1 patient). atis = 114, 282 y 309 ua/ml. no clinical response (nr) in 66.6% assessments. (b) 10.0% ifx-b and ati-b (c b 2 ua/ml) no detection (1 assessments/1 patient). nr 0%. (c) 60.0% ifx-b detection (c [ 0.035 mcg/ml) and negative ati-b (6 1 assessments/4 patients) weight: 63 (21-90) kg. twenty assessments, 2.5 (1-5) assessments per patient, 4 (4-8) ifx-b doses, 80% concomitant treatment (16/16-azathioprine, 8/16-corticosteroid) the incidence of ati-b was low. a correlation was observed between the presence of ati-b and loss of clinical response, as infliximab original. tdmp012: serum concentration of non-vitamin k antagonist oral anticoagulants (noacs) in older hip fracture patients ina linnerud 1 , mette i martinsen 2 estimation of t of noacs by t1/2 = ln2/kel [kel; elimination constant] using two s-concentration measurements. results: we included 167 patients (median age 84 years, 73.1% women). noac use was detected be serum analysis in 11 patients (6.6%; 100% coherent with mr), while 15 patients (9.0%) used warfarin. 7 of the 11 noac users (63.6%) had s-concentrations of noacs above the reference range at admission, and five patients (45.5%) had s-concentrations within the reference range before surgery. patients using noac had significantly longer median waitingtime for surgery than warfarin-users (50 vs 36 h, p = 0.004). blood transfusions were given to 36.4% of noac-users vs 21.4% of warfarin-users (p = 0.651). mean estimated t of noacs were 33, 16.5 and 14.5 h for dabigatran (n = 2), apixaban (n = 4) and rivaroxaban (n = 2), respectively. conclusion: mr is effective in detecting noac use in older hip fracture patients, but importantly s-concentrations are higher than expected in this population. this might reflect the significantly longer waiting-time for surgery this column is supplied with packing material made of totally porous spherical silica coated with a silicone polymer monolayer containing octadecyl (c18) groups. the mobile phase was composed of 0.5% na 2 po 4 h 2 o (ph 2.5), acetonitrile, and methanol (55:25:20, v/v/v), which was degassed in an ultrasonic bath prior to use. the flow rate was 1.0 ml/min at ambient temperature and sample detection was carried out at 250 nm. plasma samples were obtained from 11 patients with cml receiving nilotinib treatment. sampling was performed at the steady state. blood samples were collected by venipuncture 24 h after oral administration of nilotinib. plasma was separated by centrifugation at 1900 9 g for 15 min and stored at -40°c until analysis. plasma samples (100 ll) were then extracted as described above. the same samples were also sent to a commercial laboratory (bml, inc.) for assaying nilotinib concentration by liquid chromatography-tandem mass spectrometry (lc-ms/ms). in addition, we applied this method to tdm of cml patients receiving nilotinib at our hospital. main outcome measures: the calibration curve exhibited linearity over the nilotinib concentration range of 50-2500 ng/ml at 250 nm, with relative standard deviations (n = 5) of 7.1, 2.5, and 2.9% for 250, 1500, and 2500 ng/ml, respectively. the detection limit for nilotinib was 5 ng/ml due to three blank determinations (q = 3). in addition, we compared the results with those measured by lc-ms/ ms at bml, inc. (a commercial laboratory). as a result, a strong correlation was observed between the nilotinib concentrations measured by our hplc method and those obtained by lc/ms-ms (r 2 = 0.988, p \ 0.01). in addition, tdm of nilotinib was performed to six cml patients. there was the case which participated in dosage adjustment of nilotinib in hepatic dysfunction and poor glycaemic control. results: we have developed a simple ultraviolet detection method for the determination of nilotinib, which has high sensitivity and large dynamic range please specify your abstract type: research abstract key: cord-000083-3p81yr4n authors: nan title: poster exhibition date: 2009-01-31 journal: hepatol int doi: 10.1007/s12072-009-9123-4 sha: doc_id: 83 cord_uid: 3p81yr4n nan background: biliary atresia (ba) is one of the most common causes of neonatal cholestasis and the most frequent hepatic cause of death in early childhood. the incidence rate of ba is higher in asian countries, occurring in approximately 1 of 8,000 (asian countries ) to 1 of 18,000 (european countries) live births. early identification and prompt intervention is very important. to im prove the early diagnosis, we used proteomic technology to screen serum biomarker for ba. methods: two-dimensional electrophoresis (2-de) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (maldi-tof-ms) were employed to screen serum biomarkers specific to ba sera from idiopathic neonatal hepatitis. after pretreatment including albumin and immunoglobulin (igg ) depletion, sera were subjected to 2-de and there after image analysis. the differentially expressed protein spots were identified by maldi-tof-ms. result: from optimized 2-de gel images, thirty-four spots were differentially expressed and identified by maldi-tof-ms to be eight proteins. overall, kininogen 1 variant was under expressed and alpha-1-b-glycoprotein, leucine-rich alpha-2-glycoprotein 1, 'sp40,40', a1bg protein, vitamin d-binding protein/group specific component , apolipoproteina-, aqgv 3103 were over expressed in ba group com pared to idiopathic neonatal hepatitis. conclusion: 2-de based serum proteome analysis can be useful in detecting protein expression alteration and new discovered biomarkers might be an aid in the diagnosis of ba, though further validation is needed. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 ims, bhu, varanasi, india background: a variety of autoreactive antibodies are detected in patients with chronic liver disease. this prospective, nonrandomized study was undertaken to evaluate the nature & prevalence of various autoantibodies in patients with chronic liver disease of diverse etiologies. methods: study population included 53 patients (75% males), who met defined criteria for chronic liver disease. detailed clinical, laboratory and sonographic evaluation was done. sera were tested for asma, anti-lkm type1, ama, apa, ana, by standard methods. p<0.05 was considered significant. results: among various etiologies for chronic liver disease, hepatitis b was most common (28%), followed by alcohol (19%), autoimmune hepatitis in 15%, hepatitis c (6%) and miscellaneous (2%). 30% of patients were labeled as cryptogenic after detailed investigations. ana (>1/80) was positive in 100% of definite aih, 33% of hcv related cld but at titer of >1/40, 66.6% of hcv related cld & 60% of probable aih were found positive. asma (>1/40) was positive in 6% of hbv related cld, 10% of alcohol related cld, 33% of definite aih, 40% of probable aih, 33% of hcv related cld but asma in titer of >1/80 was positive only in 33% oh definite aih. apa was detected in 12.5% of cryptogenic cld, 13.3% of hbv related cld & 20% of alcohol & probable aih related cld each. ama was detected in 1% of cryptogenic, hbv, aih (definite) & hcv related cld each, and 2% of alcohol related cld & 100% of pbc. conclusions: apart from aih there is high prevalence of ana & sma in hcv related cld while other antibodies has low prevalence in non-aih related clds. this study also suggests that prevalence of various autoantibodies should be borne in mind while considering the diagnosis of cld especially of mixed etiology. conclusions: oa infusion did not lower ammonia levels or improve survival. results: the mortality (50.0%) of patients in lamivudine group with meld score from 30 to 40 was lower than that (86.1%) of control group ( 2 =23.319, p=0.000). univariate analysis showed that mortality was significantly related to age (p=0.005), meld score (p=0.009), treatment method (p=0.000), pretreatment hbv dna load (p=0.000), the decline of hbv dna load during therapy (p=0.006) and encephalopathy (p=0.007). in multivariate analysis, in patients with meld scores 30-40, treatment method (p=0.004), pretreatment hbv dna load (p=0.009), decline of hbv dna load during therapy (p=0.014) and encephalopathy (p=0.019) were independent predictors of mortality; for meld scores above 40, only meld score (p=0.015) was independent predictive. conclusions: lamivudine treatment significantly decreases the 3 month's mortality of patients with meld score 30-40, and a low viral load pre-treatment and quick decline of hbv dna load are good predictors for the survival of lamivudine treatment. background/aims: early identification of patients with fulminant hepatic failure (fhf) who need a liver transplantation is very important. to construct a prediction model for early diagnosis and prognosis of fhf, we studied dynamics of metabolic profiles using a d-galactosamine/lipopolysaccharide (galn/lps)-treated mouse model. methods: balb/c mice were used to construct fhf model and sacrificed for blood collection at 4, 5, and 6 hour after treatment, respectively. levels of plasma metabolites were quantified using gas chromatography/time-of-flight mass spectrometry and data were processed using partial least squares discriminant analysis (pls-da). results: distinct clustering differences were observed 5 and 6 h after treatment between survival and dead groups. at 5 h, plasma levels of some metabolites differed significantly between survival, dead and control groups. ketogenesis and the tca cycle were inhibited in both survival and dead groups, but in dead group, the urea cycle was also inhibited and glycolysis was elevated. pls-da indicated that principal component weighting was greatest for plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate. the y-predicted scatter plot in pls model assigned samples to survival or dead groups using an apriori cutoff of 0.10 with 100% sensitivity and specificity. similar results were observed in 11 fhf patients with different outcomes. pe012 association between polymorphisms in the interleukin-10 gene promoter and hepatitis b-related acute liver failure conclusions: the pls model based on metabonomics analysis can be used to predict outcomes well, and plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate may constitute a set of markers for early diagnosis and prognosis of fhf. il-10 promoter are associated with the susceptibility to hepatitis b-related alf in the chinese population. il-10 a-592c may be a regulatory polymorphism that affects gene regulation. hepatocyte cell death in aclf: mechanism and significance -an immunohistochemical study. p. sakhuja 1 , a. rastogi 2 , s. s hissar 1 , a. singh 1 , a. kumar 2 , r. gondal 1 , s.k. sarin 1 1 gb pant hospital, 2 institute of liver and biliary sciences, new delhi, india background: acute on chronic liver failure (aclf) is defined as acute hepatic insult complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. caspases play an essential role in apoptosis. cox-2 is an inducible immediate early gene responsible for the release of prostaglandins during inflammatory response. we studied the immunohistochemical expression of cox-2 and caspase-1 in liver tissue to assess their role in pathophysiology and in predicting outcome of aclf. method: a retrospective analysis of 50 liver biopsies with clinical diagnosis of aclf was undertaken. patients were divided into two groups a and d based on clinical outcome (alive/died respectively). immunohistochemical analysis for cox-2 and caspase was performed on 39 and 36 cases respectively and scored from 0-8 as per intensity and distribution. score 6-8 indicated high intensity with focal to diffuse distribution, and was considered significant. results: etiology of acute liver failure was viral or alcoholic. increased expression of caspase was observed in 10/21 cases in group d and none of the cases in group a(n=15) (p=0.001). increased expression of cox-2 was observed in 4/21 cases in group d and none of the cases in group a (n=18) (p=0.052). conclusion: increased immunoreactivity of caspase in liver biopsies of patients of aclf may indicate worse prognosis and its important role in the pathophysiology of aclf. immunostaining for caspase is useful for assessment of prognosis and possibility of anti-apoptotic and anti-fibrotic therapies in future. conclusion: hhgf expression vector (pcmv-hhgf) has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hhgf in vivo. j.h. kim 1 , k.w. kim 1 1 asan medical center, seoul, korea background: splenic artery embolization (sae) is performed to increase hepatic arterial flow or to decrease portal venous flow in recipients of liver transplantation (lt). thus, the purpose of this study was to estimate sae effect on the basis of changes in caliber of related vessels and splenic volume on pre-sae and serial post-sae ct scans in lt recipients. methods: between 2003 and 2007, among 73 lt recipients who underwent sae and serial follow-up ct, 43 with no compounding factor that may obscure sae effect were included in this study. they underwent ct before and after (1week, 1month, and 1year) sae. a radiologist retrospectively measured diameters of ca, cha, sa, sv and splenic volume on serial ct scans. their diameters and splenic volume on each ct were compared with those on the prior and pre-sae ct. the difference was compared using repeated-measures anova tests. results: cas decreased between 1week and 1month after sae (p<.05), but were stable before 1week and after 1 month. chas increased within 1week (p<.05) but decreased between 1week and 1month (p<.05) and remained stable after 1month. compared with pre-sae ct, chas were larger for 1month after sae. sas continuously decreased for 1year (p<.05). svs decreased for 1 month (p<.05) and remained stable after 1 month. compared with pre-sae ct, sas and svs were smaller from 1week after sae and on. splenic volume continuously decreased for 1year except a period between 1week and 1month. conclusion: the increase of hepatic arterial flow persists for 1month after sae, but returns to baseline thereafter. the decrease of portal flow may lasts for at least 1year after sae. poster exhibition -cholangioca and other liver neoplasm poster session, hall 5b background: now, rfa has becoming an important practice of hcc therapy. in this study, we evaluated whether rfa therapy for metastatic liver tumor has a beneficial effect on patients' survival. methods: forty six patients were treated by rfa for metastatic liver tumor from july 2001 through february 2008 in our hospital, of the 46, 33 patients were metastasis either form colon or stomach cancer. these 33 patients were analyzed in this investigation. cumulative survival rate from initial rfa therapy was calculated by kaplan-meier method. predictive factors for survival were identified using cox proportional hazard regression model. results: the mean age of the 33 patients were 64. 6 (range, 40-79) . the mean size of the tumor is 28mm (range,8-70mm) and the numbers of tumor foci are 2.7 nodules range,1 18. the survival rates of patients treated by rfa were 49.5% at 3 years and 31.8% at 5 years in colon cancer, 15.6% at 3 years and 15.6% at 5 years in gastric cancer. in this series of 33 patients, primary cancer: colon (p=0.002 odds ratio 0.132 95%ci 0.037-0.473), younger patients ( 64) (p=0.041 odds ratio 0.312 95%ci 0.102-0.955) and multiagent chemotherapy (p=0.012 odds ratio 0.223 95% ci 0.069-0.723) were significantly correlated with better survival. conclusion: the survival of patients treated by rfa for metastatic colon cancers had better survival than those of gastric cancers. in addition, good indication of rfa is for metastatic colon cancers, younger patients and has to be treated by multiagent chemotherapies. utility of contrast enhanced ultrasonography with sonazoid in radiofrequency ablation (rfa) for liver metastasis e. goto 1 , s. shiina 1 , r. tateishi 1 , r. masuzaki 1 , k. enooku 1 , t. sato 1 , j. imamura 1 , t. goto 1 , y. sugioka 2 , h. ikeda 1,2 , h. yoshida 1 , m. omata 1 1 department of gastroenterology, university of tokyo, 2 department of clinical laboratory, tokyo, japan background & aims: contrast enhanced ultrasonography (ceus) with sonazoid is effective for liver metastasis because enhance defect in kupffer imaging is well delineated. the aim of this study is to investigate the detection ability of ceus and the utility of sonazoid in rfa for metastasis liver tumors. material & methods: from january 2007 to december 2007, a total of 346 liver metastatic nodules in 87 patients (62 colon cancer, 13 breast cancer, 3 gastric cancer, 3 islet cell tumor, and 6 others) admitted to receive rfa were studied. the detection ability of liver metastasis was compared between ceus and conventional us using enhanced ct as reference standard. the mean numbers of treatment session of rfa were compared between patient treated with ceus assistance and historical controls matched for size and number of tumors. results: the detection rate was 78.6% with conventional us and 96.4% with ceus (p=0.0004). 83 nodules in 25 patients were not detected by conventional us and detected after injection of sonazoid. in addition, 12 nodules in 2 patients were detected not by ct but only by ceus. the mean number of session was 1.5±0.5 as compared to 2.0±1.0 in the historical controls (p<0.001). conclusions: ceus with sonazoid is useful for detection of liver metastasis. sonazoid is an excellent supportive agent in rfa of liver metastasis. background/aims: carcinogenesis of intrahepatic cholangiocarcinoma (icc)-associated liver fluke infection accumulated genetic and epigenetic alterations. cholangiocarcinoma cell line (kku-m213) is adenosquamous carcinoma which rare variants and not commonly found in icc. however, interactions of liver fluke-associated icc proceed to genetic alterations in adenosquamous carcinoma that have been not elucidated. objectives: to analyze the whole genome-wide genetic alterations in kku-m213 using microarray comparative genomic hybridization. methods: dna of kku-m213 and matched-sex reference were differentially labeled with fluorescence dries (cy3 and cy5) and mixed together with cot-1 dna. the mixture was hybridized on array with spotting 2,464 human bacterial artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13.1, 4q21.1, 4q21.2, 5p tel, and 5p15.3, whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3. conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13. 1, 4q21.1, 4q21.2, 5p tel, and 5p15.3 , whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3 . conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. acknowledgements: this work was supported by faculty of medicine, kku, thailand (grant no. i51117 background/aims: we studied the clinical efficacy of arterial chemoinfusion therapy through an implanted port system for patients with intrahepatic cholangiocarcinoma (icc). thirty patients with unresectable icc or intrahepatic recurrence of icc after surgery were studied. comparison was made between patients who received arterial chemoinfusion therapy through an implanted port system with adriacin and lecithin-added lipiodol emulsion in 5 patients and 5-fluorouracil (5-fu) in 7 patients. eighteen patients were treated without port system. results: disease was stable in 5 patients with adriacin and lecithin-added lipiodol emulsion and in 3 patients with 5-fu. disease was progressed in 4 patients with 5-fu. the mean survival period was 20.8 months in patients with adriacin and lecithin-added lipiodol emulsion, 9.3 months in patients with 5-fu, and 10.5 months in patients without port system (p=0.02, p=0.04). conclusion: arterial chemoinfusion therapy through an implanted port system is useful for patients with intrahepatic recurrence of icc after surgery. pe038 s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh. india background: a number of dietary factors have been involved in the pathogenesis of cholelithiasis. cholesterol overfeeding is the primary means of inducing supersaturated bile and cholesterol gallstones in animal models.aim of the study was to investigate the rate of epithelial cell death and proliferation in gallbladder during gallstones formation. methods: balb/c mice was divided into two groups control in this group animals were fed normal chow diet, high fat diet group in this group (2% cholesterol,0.5% sodium cholate, 5% butter fat and 15% coconut oil) mixed with chow diet was fed to the mice for 4 weeks. cell apoptosis and proliferation was assayed in gallbladder epithelial cells. histological analysis of gallbladder sections were done with hematoxylin and eosin staning. results: mice fed high fat diet had apoptotic as well as necrotic epithelial cells. rate of proliferation was enhanced after 24 and 48 hrs in mice fed high fat diet group as compared to the control group. the histopathological section of control gallbladder has normal morphology whereas gallbladder wall thickness was markedly increased; epithelial cells appeared more elongated in mice fed high fat diet. conclusion: results obtain show that high fat diet markedly induced biliary epithelial cell proliferation and biliary epithelial cell apoptosis. it has been determined that when there is an injurious stimulus that leads to apoptosis, it is later followed by reparative proliferation and when there is no injurious stimulus, apoptosis occurs late in the course as part of remodeling. background: obstructive jaundice can be caused by malignancy. the treatment can be drainage by biliary stenting. in advanced malignant jaundice, the stent placement is often difficult. objective: to evaluate the success rate of malignant obstructive jaundice evaluation of ercp and success rate of stent placement. methods: retrospective study based on data of ercp from october 2004 until july 2008. results: we evaluated 139 patients who has done ercp examination, 131 (94,2 %) patients have clinical diagnosis of obstructive jaundice. there were 73 (55,7%) male and 58 (44,3%) female, age range 20 -84 (median age was 51). there were no malignancy in 66 (50,4 %) patients; malignancy in 48 (36,6%) patients and 17 (13%) patients need further evaluation.. from 114 patients, 57 (50 %) patients attempted to have stent placement, 50 (43,9 %) patients do not and 7 (6,1 %) patients have no data. we done descriptive study on 57 patients attempted to have stent placement, 32 (56,1 %) patients succeed in stent placement whereas 25 (43,9 %) failed. malignancy was showed to be a factor of stent failure (malignancy: 23 fail and 10 success (30,3 %) vs non malignancy: 2 fail and 22 success (91,7%)). conclusion: ercp can identify the cause of obstructive jaundice in 87 % patients. the success rate of stent placement was 56,1 %. the success rate of biliary stenting in malignant obstructive jaundice was 30,3 % whereas in non-malignant cases was 91,7 %. papillary carcinoma was the most frequent cause of malignant obstructive jaundice. background: in hydatid disease of the liver cystobiliary fisula (cbf) constitutes an entity characterized by the occurrence of a life-threatening cholangitis with increased morbidity. aim: to study the different diagnostic and therapeutic aspects of cystobiliary fistula in hydatid disease of the liver. patients and methods: fourteen patients with complicated cysts were divided into 2 groups; group a: nine patients presented with cholangitis, and group b: five patients had history of jaundice. in all patients, the diagnosis of cbf was confirmed by erc (endoscopic retrograde cholangiography). preoperative endoscopic sphincterotomy (es) was done in group a with retrieval of hydatid daughter cysts. seven patients (subgroup a1) were subsequently submitted to surgery entailing endocystectomy in 5 and hepatic resection in two. the remaining 2 patients in group a (subgroup a2), were managed by endoscopic therapy only. patients of group b (n=5), were not submitted to preoperative es and were subsequently managed by hepatic resection in one patient and endocystectomy in four. results: there was no mortality in the studied group. postoperative bile leak occurred in four cases in group b. in contrast, none of the patients who were submitted to preoperative es (subgroup a1) had bile leak. all patients received albendazole treatment. conclusion: erc is important in confirming the diagnosis of cbf. also, therapeutic erc has a place in the treatment algorithm of cbf as it was found to be a safe and a reliable therapeutic alternative especially in high risk patients for surgery. v. singh 1 , g. singh 1 , g.r. verma 1 , v. gupta 1 , s. ghosh 1 , r. gupta 1 , r. kapoor 1 , n. sharma 1 , a. bhalla 1 , s.k. mahi 1 1 background: endoscopic palliation in malignant hilar biliary obstruction requires ercp. however, contrast injection leads to cholangitis. recently, contrast-free metal stenting with or without mrcp has shown encouraging results. however, mrcp and metal stents are costly. there have been no reports on the use of air cholangiography in these patients. methods: we prospectively studied the role of air cholangiogaphy assisted unilateral plastic stenting in these patients. results: ten patients with unresectable malignant hilar biliary obstruction were studied. air cholangiography detected type ii obstruction in 8 and type i in 2 patients which is similar to mrcp. all patients underwent unilateral plastic stenting. a successful endoscopic drainange was achieved in 100% patients. cholanngitis occurred in none and there was no 30-day mortality. no major complications were observed. conclusion: air cohlangiography assisted plastic stenting in these patients is a safe and effective method of palliation. however, it requires a larger study. introduction: a description of igg4-related sclerosing cholangitis (igg4-sc) without pancreatic lesion has recently been reported. in addition to imaging, diagnosis relies on findings of elevated serum igg4 and immunodetection of invading igg4-positive cells. here we report a case of igg4-sc with only slight common bile duct abnormalities and normal pancreatic findings. case study: a 65-year-old man suffering from cephalalgia, general malaise and muscle ache was admitted to our hospital. his blood examinations on admission revealed eosinophilia, mild anemia, liver dysfunction and an igg level of 2820 mg/dl (igg4 374 mg/dl). although ercp did not reveal typical stenosis or irregularities of the bile duct wall, visualization of peripheral bile ducts was slightly impaired. echography revealed thickening of the intrahepatic bile duct and gallbladder walls as well as adenopathy. due to a gradual increase in pleural effusion and a progression of anemia, oxygenation was begun on the seventh day of illness. based on the combination of eosinophilia, elevated serum igg4 levels, image findings and a negative result for helminth, igg4-sc was suspected. liver biopsy was performed on the ninth day of illness and steroid therapy was initiated, after which symptoms and laboratory findings improved. the igg4-positive plasmocytic infiltrate present around the portal region at the time of biopsy disappeared within eight months of treatment. summary: this case displayed two unusual features that are not generally observed with igg4-sc: complications due to hemolytic anemia, and destruction of the peripheral bile duct with little damage to the common bile duct. introduction: various systemic diseases have been reported to be associated with igg4. although steroids are effective in the treatment of igg4-related diseases, there are some reports on relapses with their treatment, and cases are often difficult to differentiate from malignant diseases. we encountered a case of autoimmune pancreatitis with sclerosing cholangitis (aip-sc), in whom ca19-9 was elevated with episodes of exacerbation and an elevated serum igg4 concentration. igg4 staining was also useful for the diagnosis. case study: an 81-year-old woman noticed tumors beneath the bilateral jaw and was found to have an elevated level of ca19-9 (304) seven years previously. her left submandibular gland was removed and diagnosed as sclerosing sialadenitis. four years previously, she was diagnosed as having diabetes mellitus complicated by a recurrence of ca19-9 (419) elevation and liver dysfunction. cholangiocarcinoma was suspected based on ercp, but was not confirmed by histologic findings of bile duct biopsy. elevated igg4 and other test results established the diagnosis of aip-sc, so steroid therapy was initiated, after which symptoms and laboratory findings improved. this recurrence of ca19-9 elevation (634) was diagnosed as a relapse of aip-sc based on an increased igg4 level and histologic findings. summary: some papers have reported that igg4-positive cells are found in liver tissue in this disease, but such cells were not detected in the liver specimens in our case. this might be because intra-liver sites may have differed in the degree of morbidity, and long-term steroid therapy might have suppressed inflammation in the liver tissue. s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh, india background: cholelithiasis, a gallstone disease is major cause of morbidity affecting millions of people throughout the world. aim of the present study was to investigate the predisposing factors that lead to the formation of gallstones. methods: the study was carried out on gallstones, bile and serum of patients. gallstones and bile were divided into three groups' cholesterol, pigmented and mixed gallstones. blood of the patients was divided into two groups with gallstones and without gallstones patients. trace elements and various biochemical estimations were carried out. clinical history of the gallstones patients was recorded from the hospital records. results: trace elements analysis in bile and gallstones showed that calcium is the main element in all the three types of stones. iron was the main element in mixed gallstones. in pigmented gallstones magnesium and zinc were the major trace elements. liver function tests and lipid peroxidation levels in sera were significantly increase whereas, antioxidant enzymes concentrations in sera were significantly decreased in patients with gallstones. clinical history of the gallstones revealed the cases had jaundice, diabetes mellitus and estrogen replacement therapy respectively. conclusion: results suggest that trace elements in gallstones and bile as well as clinical history of patients with chronic cholelithiasis could be the underlying factor in the pathogenesis of gallstones. the concentration of products derived from the free radicals reactions increases with degree of inflammation. such a condition increases risk of bile saturation which would further contribute to the progress of gallstones formation. background and aims: diseases of the biliary tree and gallbladder are being described with increasing frequency among patients with the acquired immunodeficiency syndrome (aids).therefore there is a need to do a research about the risk factors of gallbladder diseases in hiv/aids patients. so it can be useful to clinicians to predict the possibility of a patient having gallbladder disease and consider the options of further plans. the aim of this study was to find the prevalence and varieties of gallbladder diseases in hiv/aids patients. methods: a cross sectional study was performed in patients with hiv/aids who visited ciptomangunkusumo hospital, jakarta. the risk factors (route of transmision,cd4,arv,hepatitis) and clinical presentations were studied.ultrasonography examinations were performed to detect gallbladder annormalities. results: 68 patients with hiv/aids match the study criteria. there were gallbladder abnormalities in 22 (32.4%) subjects, which 19 (27.9%) had acalculous cholecystitis and 3 (4.4%) had cholecystitis with cholelithiasis. on bivariate analysis, there was a significant association between abdominal pain, jaundice and the use of arv to gallbladder abnormalities (p = 0.000; 0.000; 0.004; 0.012). however, there was no association between age, sex, transmision route of hiv, hepatitis and cd4 to gallbladder abnormalities. conclusion: hiv/aids patients are susceptible to opportunistic gallbladder infection. acalculous cholecystitis is the most frequently encountered gallbladder abnormalities of hiv/aids patients in this study. poster exhibition -hbv poster session, hall 5b long-term stopping therapy t.b. trung 1 , p.h. phiet 1 1 university medical center, hochiminh city, vietnam background: among the approved nucleos(t)ide analogues therapies for chronic hepatitis b, lamivudine was used widely, sometime inappropriate in practice due to high safe and low price but lamivudine is associated with the highest rate of drug resistance. objectives: the aim of the study was to determine the ymdd variants after long-term stopping treatment in lamivudine-resistant patients using more sensitive technique. methods: 16 blood samples from lamivudine resistant patients were collected after long-term stopping therapy. the ymdd variants are detected using technique pcr restriction fragment length polymorphism (pcr-rflp) at hcmc university medical center results: after stopping lamivudine treatment 25 months (6-72 months) ymdd mutants were detected in 14 (87,5%) of 16 patients. among them 13 (92.9%) had the most important m204v/i mutant, 1(7 1%) had accompanying l180m mutant. it means that once drug resistant mutants have been selected, they are archived for the long time even if treatment is stopped. many of patients have the features characterized for the patients in immune tolerance phase (young age, hbeag positive, normal alt). the treatment of this group is not strongly recommended due to low efficacy and high risk of drug resistance. conclusion: the most important m204v/i mutant was still detected with significant portion of the virus population after long-term stopping therapy in lamivudine resistant patients. the options of retreatment for this patients when necessary are limited due to cross-resistance. the management of chronic hepatitis b should be followed strickly the recommendations of specialized association to avoid this problem. background/aim: whether liver stiffness measurement (lsm) using transient elastography is reliable to assess liver fibrosis in the settings of severe acute exacerbation of chronic hepatitis b (chb) is uncertain. methods: we prospectively recruited consecutive patients with severe acute exacerbation of chb (alanine aminotransferase or alt >10x upper limit of normal). the relationship of alt levels and lsm were serially assessed and liver biopsy was performed after alt normalization. results: eleven patients (10 male, median age 43 years) were followed up for 25 weeks; 9 patients received anti-viral therapy. overall, lsm was positively correlated with alt levels (r=0.67, p<0.001). at initial presentation, the median serum alt and lsm was 1136 (581-2210) iu/l and 26.3 (11. 1-33. 3) kpa. a progressive reduction in lsm was observed during subsequent visits in parallel with the reduction of alt levels. even after the normalization of alt at week 12, lsm of 9 patients continued to drop at week 25. at the last visit, the median alt was 27 (11-52) iu/l and lsm was 7.7 (4.7-10.8) kpa. among the 5 patients who had liver biopsy performed at week 25, 4 patients had f2 fibrosis (lsm 5.7-8.1 kpa) and 1 patient had f3 fibrosis (lsm 8.6 kpa). conclusions: lsm using transient elastography may misdiagnose liver cirrhosis in patients suffering from severe acute exacerbation of chronic hepatitis b. lsm should be assessed after normalization of alt levels in order to accurately assess the degree of fibrosis. h.c. lai 1 , s.w. lai 1 , k.f. liao 1 , c.s. liu 1 , t. lin 1 , c.c. lin 1 1 china medical university hospital, taichung, taiwan background: in 2007, chronic liver disease was the seventh leading cause of death in taiwan. hepatitis b and hepatitis c are two major causes of chronic liver disease in taiwan. the purpose was to investigate the seroepidemiology of hepatitis b surface antigen (hbsag) and hepatitis c virus (hcv) antibody in taiwan. method: this was a hospital-based cross-sectional study. we analyzed viral hepatitis data from 2695 subjects who received health checkups at one medical center in taichung from 2003 to 2004. all subjects were divided into three age groups, including 20-39, 40-64 and 65. this study emphasized the prevalence of hbsag and hcv antibody by gender and age. the statistical analysis was performed by t test and 2 . result: there were 1526 men (56.6%) and 1169 women (43.4%). the mean age was 49.2 (standard deviation 12.2, range 20-84). the overall prevalence of hbsag was 14.7%, with statistically significant difference(ssd) between gender (17.4% for men vs 11.2% for women, p <0.001). the prevalence of hbsag was decreased with age in men, with ssd (p <0.001), and also decreased in women, without ssd (p =0.08). the overall prevalence of hcv antibody was 5.2%, without ssd between gender (4.8% for men vs 5.7% for women, p =0.272). the prevalence of hcv antibody was increased with age both in men and in women, with ssd (p <0.001). conclusion: we hope this study can provide the epidemiological data for further studies of hepatitis b and hepatitis c virus infection in taiwan. s.m. wu 1 , x. zhou 2 1 wuhan medical treatment center, 2 center for gene diagnosis, zhongnan hospital, wuhan university, china e-selectin is revealed to facilitate leukocyte adhension to the endothelium and migration into inflamed tissue in inflammatory diseases. chronic hepatitis b virus infection is regarded as a chronic inflammatory process. to examine the possible involvement of e-selectin in the etiology of chronic hbv infection, we analyzed two polymorphisms of e-selectin and determined the plasma souble e-selectin levels in patients with chronic hbv infection and controls. the frequency of c allele of the a561c polymorphism was significantly increased in patients with lc campared with controls. no significant positive association was observed between the g98t polymorphism and chronic hbv infection. but in patients with lc, divided according to the child-pugh classification, the frequency of t allele was of significant difference between child'class a and class b plus c. plasma levels of soluble e-selectin were significantly increased in patients with chronic hepatitis and liver cirrhosiscompared with controls. in the liver cirrhosis group, levels of se-selectin were significantly decreased from child' class a to class c. in each group, patients with c allele of the a561c polymorphism showed higher soluble e-selectin levels than those with a allele. this is the first report describing the association between e-selectin polymorphisms and hbv-related hepatic fibrosis. our data showed the a561c polymorphism of e-selectin gene is associated with disease progression in patients with hbv infection and controls the expression of plasma soluble levels, the g98t polymorphism may be related to fibrotic severity in patients with liver cirhosis. background: chronic hepatitis b (chb) patients with high serum hbv-dna and normal serum alanine aminotransferase (alt) levels might be considered for treatment if histopathological findings show fibrosis stage 2 or more. however, to our knowledge there is no recommendation with regard to the therapeutic agents for this group of patients. objective: this study was aimed to evaluate the efficacy of nucleoside analogues (entecavir or telbivudine) in treating chronic hepatitis b patients with high serum hbv-dna and normal serum alt levels. patients and method: this was an open-label study in chb patients with high level serum hbv-dna levels between january 2007 and october 2008. patients were included if they showed normal serum alanine aminotransferase (alt) level at two measurements within a 3-month interval and had fibrosis stage > 2 on liver biopsy specimens. patients were treated with entecavir 0.5 mg/day or telbivudine 600 mg/day. the primary endpoint was the reduction or undetectable of serum hbv-dna at 24 week and 48 week of treatment, while the secondary endpoint was hepatitis b e antigen (hbeag) seroconversion. results: during a 2-year period, 37 chb patients with high level serum hbv-dna with normal alt two times with 3 months interval underwent a liver biopsy. twenty-eight (75.7%) of 37 pts showed fibrosis stage 2 on histological findings (metavir score). twelve of these 28 patients received nucleoside analogues, 7 (58.3%) of them were men. patients' median age was 42 (range: 24-52) years. there were 5 patients with stage-2, 6 patients with stage-3 and 1 patient with stage-4 fibrosis. eleven (91.7%) patients had genotype b virus. at baseline, the mean serum alt level was 32 + 11.8 u/l and mean hbv-dna level was 2.48 x 10 6 iu/ml, ranging from 1.23 x 10 3 to 2.4 x 10 7 iu/ml. six patients received entecavir and the other six received telbivudine therapy. undetectable hbv-dna was achieved by 9 (75.0%) patients at week-24 and 2 (16.7%) patients at week-48 of treatment. one patient who had the highest hbv-dna level had viral load reduction to 1.6 x 10 4 iu/ml at week-48 of treatment. two out of 5 patients with positive hbeag achieved hbeag seroconversion at week-48 of treatment. conclusion: this preliminary study has shown that nucleoside analogues might be considered in the treatment for chronic hepatitis b patients with high serum hbv-dna and normal serum aminotransferases levels. j. chen 1 , x.j.. wu 1 , y. wang 1 , g.q. wang 1 1 department of infectious diseases, peking university first hospital, beijing, china background: the dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are new members of cd28/b7 family, as co-stimulatory molecules expressing on t cells and antigen present cells (apcs). their engaging can downregulate the t cells function, including proliferation, cytokines secretion and cytotoxicity. in periphery blood, pd-1 was upreguated on virus specific-t cells, leading to the impairment of t cells. blocking the pd-1/pd-l can improve the function of t cells. methods and patients: 21 patients with chronic hepatitis b (chb) were treated by pegylated ifn -2b (pegintron from schering-plough, once a week, 0.5 or 1 g/kg/weight). the periphery blood were taken at 0 weeks, 4 weeks, 8 weeks, and 12 weeks. periphery blood mononuclear cells (pbmc) were isolated from fresh heparinized blood by ficoll-hypaque (density: 1.077g/l) density gradient centrifugation. then the cells were incubated with apc-conjugated anti-pd-1 antibodies. the pd-1 expression on lymphocytes was detected by flow cytometry (fcm). results: the pd-1 expression on lymphocytes at 0 weeks was 14.47±5.8%, at 4 weeks was 9.68±3.75%, at 8 weeks was 6.95±2.39%, at 12 weeks was 6.08±1.31% (p<0.05). conclusion: treatment with ifn -2b can downregulate the pd-1 expression on lymphocytes and may partially restore the function of t cells. to investigate the effects of nucleoside analogs therapy in hepatitis b related acute-on-chronic liver failure, we treated 55 hbv related acute-on-chronic liver failure patients with entecavir. as control, the remaining 74 were not treated with nucleoside analogues. results show the survival rate of entecavir therapy group has no significantly difference with none-treated group (p>0.05). although entecavir greatly reduced hbv replication during different therapy times (p<0.001), the meld score and liver function (alt, albumin, bilirubin, prothrombin time) had no significant changes (p>0.05). further more, we analyzed the meld score and liver function in different hbv-dna level patients .no significantly difference was observed (p>0.05). there is no significant correlation between hbv-dna level and meld score in different therapy times (p>0.05).the hbv-dna level between patients with over 3 months and less than 3 months survival patients showed no significant difference either (p>0.05). however, meld score and some parameters of liver function (albumin, bilirubin, prothrombin time) showed significant difference (p<0.05). these results suggest hbv-dna loading may not be a direct factor to increased liver injury and suppression of hbv replication may not reduce the severity of liver failure in hbv related acute-on-chronic hepatitis. s. firdoos 1 , u. adeeb 1 , a. mehmood 1 , m. gill 1 1 islamabad specialists clinic, islamabd, pakistan background: before the availability of etv, it was common to use adv for treatment of chronic hepatitis b patients. primary nonresponse and suboptimal response is a common problem with adv treatment. methods: we wanted to study the outcomes of entacavir therapy in this subset of patients. study was conducted between april 2007 to april 2008. we enrolled 30 chb patients who had non response to 12-24 weeks of 10 mg adv therapy. non response and suboptimal response was defined as non dimunition of at least one log of hbvdna from baseline after 12 weeks of therapy and persistence of 3 log10 after 24 weeks of therapy respectively.they were switched to 1mg entacavir before breakfast daily for at least 12 months.they had serial alt cbc and hbvdna measured every 12 weeks. results: out of 30 patients 20 male and 10 were female. only 8 patients were hbeag(+).mean hbvdna level prior to adv exposure was 6.5 log copies/ml.mean duration of exposure to adv was 26 weeks.5 patients lost to f/u.we did intention to treat analysis. 15 out 30 (50%) patient has, undetectable level of hbvdna after 12 weeks of therapy labelled as group 1.6 out of 30 (20%) had hbvdna level reduced by a mean of 3 log 10 copies/ml labelled as group 2.on week 24 treatment analysis all 15 patients from group 1 was hbvdna undetectable, 2 additional patients from group 2 had undetectable hbvdna. conclusion: entacavir therapy results in rapid suppression of hbvdna levels in majority of patients with primary nonresponse or partial non response to adv therapy. background: except for serum alt level, baseline factors predictive of therapeutic response to lamivudine in patients with hbeag-positive chronic hepatitis b remain largely unknown. we thus studied the influence of pre-therapy viral factors on end-of-treatment responses to lamivudine therapy. methods: a total of 116 treatment-naïve hbeag carriers who had pre-therapy serum alt level> 5xuln and received lamivudine for 18 months reimbursed by the national health insurance were prospectively enrolled. hbeag seroclearance and combined hbeag seroclearance, alt normalization as well as undetectable hbv dna at the end of therapy were defined as primary and secondary endpoint, respectively. the pre-therapy viral factors including viral load, genotype, precore stop codon (pc)/ basal core promoter (bcp) status, and pre-s deletion were determined to correlate with therapeutic endpoints. results: the frequency of patients with detectable pc mutation (g1896a), bcp mutation (a1762t/g1764a), and pre-s deletion at baseline was 22.4%, 21.6%, and 12.1%, respectively. after completing 18-month lamivudine therapy, overall hbeag seroclearance rate was 56.0%. patients with hbeag seroclearance had a higher prevalence of baseline pc mutation than those without (30.8% vs, 11.8%, p= .015). by multivariate analysis, the odds ratio of patients with pc mutation to develop hbeag seroclearance was 3.33 (p= .024). in addition, the presence of pc mutation also correlated with the combined response. conclusions: for hbeag-positive chronic hepatitis b patients with serum alt> 5xuln, pc mutation could predict a higher hbeag seroclearance rate at the end of 18-month lamivudine therapy. the efficacy of adefovir dipivoxil against all patterns of lamivudine resistant hepatitis b d.j. kim 1 , y.d. park 1 , y.g. kwon 2 , h.g seo 1 1 daegu fatima hospital, 2 kunngpook national university hospital, daegu, korea background: our aim was to evaluate the efficacy of adefovir dipivoxil (adv) and determine patient-dependent or laboratoroy variables that are predictive of hbeag loss and ivr for hepatitis b patients resistant to lamiduvine. also we evaluated the activity of adv against all patterns of lamivudine-resistant hbv. method: 179 hbv-infected patients with lamivudine resitance received adv for 6 months. quantitative hbv dna, hbeag/anti hbeag, alt was checked every 3-6months. the hbv polymerase of 161 patients were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. result: there is no significant difference in all patterens of hbv mutation about hbv dna reduction at 24w, 48w, 72w. there is no significant difference in all patterens of hbv mutation about alt normalization at 24w, 48w, 72w. conclusion: adefovir dipivoxil demonstrated similar potent anti-hbv efficacy regardless of the different patterns of lamivudine-resistant hbv mutations. g. novelli 1 , m. rossi 1 , v. morabito 1 , f. pugliese 1 , p. berloco 1 1 la sapienza university, rome, italy background: hepatitis b (hbv)-related end-stage liver disease is one of the most common indication for liver transplantation (lt). a number of patients dying while on the waiting list or removed because of being too ill is progressively increasing. we valued the possibility to improve the model end-stage liver disease (meld) of patients awaiting liver transplantation using a albumin dialysis: molecular adsorbent recirculating system (mars). methods: we treated 34 patients (19 male and 15 female) with a mean age 49.5. inclusion criteria: serum bilirubine > 15mg/dl, meld 25, inr > 2.1, encephalopathy grade ii. all patients were treated with mars mean 9±2.5 hr cycles and mean 9 treatments (range 3-15). all patients received standard medical treatment in addition to mars dialysis. the patient survival was valued at six months. results: we obtained a significant change of cytokines levels as interlukine 6 (p< 0.02) and tumor necrosis factor alfa (p<0.01) in association with an improvement of kidney, hepatic and hemodynamic parameters. at the end of mars treatments we observed a significant reduction of meld score (p<0.003). the results of meld show a rebound effect between the end of treatment and the follow up at six months without returning at starting values (p<0.005). twenty patients lived and 14 dead for clinical complications. conclusion: the improved meld score with mars gave patients on lt waiting list more time of survival, thus allowing them more opportunity for liver transplantation. entecavir for treatment of lamivudine-refractory patients chronic hepatitis b h.t. dat 1 , p.t.t. thuy 1 1 medic medical centre, ho chi minh, vietnam lamivudine treatment is associated with frequent development of resistant hepatitis b virus. this incidence especially is higher in longer time of treatment and loss of treatment benefit. entercavir is a new antiviral agent shown its high efficacy even in cases of mutations with lamivudine resistance. in this study, we evaluate the efficacy, the safety of entercavir in treatment of lamivudine-refractory patients chronic hepatitis b. sixty chronic hepatitis b patients with evidence of lamivudine resistance were randomly divided into two groups in proportion of 3:1. group i (n=45) used entecavir 1mg/day, group ii (n=15) used lamivudine 100mg/day. treatment time was 48 weeks. histology, alt, hbvdna were evaluated in the end of the treatment. age, sex, alt, hbvdna, genotype, hbeag were analyzed to evaluate their influences to the treatment. the results have showed hbvdna<2000 copies/ml in entecavir group 37.78% vs. 0% lamivudine group (p<0.01). hbvdna negative in entecavir group was 17.77% and incidence of seroconversion of hbeag was 8.82%. alt was normal in entecavir group 77.77% vs. 26.66% in lamivudine group (p<0.001).histologic improvement in entecavir group was 37.77% vs.6.66% in lamivudine group (p<0.05). patients with hbeag negative, genotype b, low viral load were shown better results. entecavir was shown to be efficacious in treatment for chronic hepatitis b patients experienced with lamivudine resistance. entercavir is safe, with almost no side effects. factors such as hbeag negative, genotype b, low viral load seems to be better in response to treatment. recurrence or mutation of entecavir resistance should be studied further in future. j.m. kim 1 , s.k. hwang 1 , b.h. choe 1 1 department of pediatrics, kyungpook national university hospital, daegu, korea backgrounds: by analyzing the characteristics of children with chronic hepatitis b who have lost hbsag by long-term lamivudine treatment, the selection of target patients could be relevantly predictable in the treatment of chronic hepatitis b in children. methods: a total of 75 hbeag positive children (< 18 y-o) were recruited who have visited kyungpook national university hospital from mar. 30, 1999 to may 8, 2008 . they were treated with lamivudine for at least 6 months. hbeag seroconversion occurred during lamivudine treatment in 49 out of 75 children. they were divided into hbsag clearance and non-clearance group. parameters influencing treatment results were analyzed according to hbsag loss. result: thirteen out of the 49 (26.5%) patients with hbeag seroconversion were classified as hbsag clearance group, while 36 (73.5%) as non-clearance group after lamivudine treatment. twenty five of 49 patients with hbeag seroconversion were under 6 years old, in 10 (10/25, 40%) of whom hbsag loss occurred as well. twenty four of 49 patients were over 6 years old, in 3 (3/24, 12.5%) hbsag loss occurred, that showed significantly difference (p-value= 0.029, or: 4.667, ci: 1.094-19.902) compared to younger group. age was significantly lower in hbsag clearance group (5.1±4.3 years) than non-clearance group (8.2±5.0 years) (p=0.043), but no difference was observed in other parameters. anti-hbs appeared in 12 patients. conclusion: in the treatment of hbeag positive chronic hepatitis b with lamivudine, age was significantly lower in hbsag clearance group than non-clearance group. background: dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are members of cd28/b7 family, was reported to transfer inhibitory signal, leading to the dysfunction of t cell. background: hepatitis b viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. mutations of hbsag allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus in reduced to undetectable levels. methods: immunohistochemical analysis of tissue samples from 56 patients with chronic hepatitis b (chb), 12 acute hepatitis b (ahb) patients and 10 health controls was performed. results: pd-1 was positively expressed on lymphocytes infiltrating the portal area.pd-l1 expression was the same as pd-1,also expressed in interlobular.pd-l2 expressed on kupffer cells and dendritic cells.pd-1-,pd-l1-,and pd-l2-positive cells express index of chb patients were much more than that of health controls and ahb patients(p 0.05).between groups in chb,the expression rate increase with the disease progression (p 0.05). methods: 58 chronic hepatitis b patients with both positive for hbsag and hbsab were studied.serological markers of hbv were detected by elisa and microparticle enzyme immunoassay. hbv dna levels were determined by fluorescent quantitative pcr, s gene fragments were directly sequenced, liver function was analyzed by automatic biochemistry analyzer au400. correlation test was conducted to evaluate their dependablity. conclusion: overexpression of pd-1 and pd-l within liver might be involved in inhibiting the immune response and be a mechanism of chronicity in hbv infection. results: the level of hbsag and hbsab was 254.4±68.3 s/n and 39.4±38.1 miu, respectively. hbv dna was detectable in 46 patients. fifty-one mutations of s gene were detected in 38 patients, and the relating amino acid substitution was at the sites of 36, 39, 47, 63, 77, 89, 90, 115, 126, 129, 139 and 154. eight (15.7%) out of 51 mutations were located at the "a" determinant region in 14 patients, while no mutation was found at the sites of 124, 137 and 147. however, the mutation did not affect hbv replication. hbv dna was positive correlated with hbeag. conclusions: change in hbsag antigenicity due to s gene resulted in concurrent hbsag and hbsab. the existence of hbsab did not affect hbv replication. the damage of liver failure in those patients was slight. background: hbv infection is common in bangladesh. we often encounter young patients incidentally detected with hbeag negative chronic hepatitis b (chb) in our clinical practice. however the characteristics of these patients is yet to be studied in this country. the aim of this study was to study the characteristics of young bangladeshis incidentally detected with hbeag negative chb. methods: we did percutaneous liver biopsies of 36 chb patients aged between 8 to 20 years. they were all hbeag negative with persistently normal or raised serum alt values. we did pre-core mutation (pcm) study in 4 patients who were randomly selected. results: 56% patients had significant necro-inflammation (hai-ni >3), while significant fibrosis (hai-f >2) was seen in 17.6%. serum alt (cut off 42 u/l) was raised in 38.2%, while high hbv dna load (>10 5 copies/ml) was observed only in 26.5%. pcm was negative in all 4. conclusion: although chb patients between 10-20 years of age are supposed to be in immune clearance phase, which is characterized by low hbv dna and hbeag positivity, the study shows that hbeag negative chb is an entity that can also be seen in this age group and a significant percentage of such patients may have considerable hepatic involvement. this challenges our current concept about immune clearance state of hbv infection, although much larger study is needed to draw any specific conclusion. background: hbv infection is common in bangladesh, but characteristics of young patients incidentally detected with chronic hepatitis b is yet to be studied in this country. methods: we did percutaneous liver biopsies of 88 chb patients aged between 8 to 20 years. results: significant necro-inflammation (hai-ni >3) was seen in 79.6% patients with hbeag positive and 56% patients with hbeag negative chb, while significant fibrosis (hai-f >2) was seen in 20.3% and 17.6% patients in these two groups respectively. serum alt (cut off 42 u/l) was raised in 37% hbeag positive and 38.2% hbeag negative patients, while in these two groups 87% and 26.5% patients respectively had high hbv dna load (>10 5 copies/ml). conclusion: hbeag negative chb is an entity that can also be seen in young population. a significant percentage of both hbeag positive and negative patients may have considerable hepatic involvement. profile of hbeag +ve chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: inactive hbv carriers constitute the major reservoir of hbv. present management guidelines provide inadequate treatment modalities. they are recommended for regular check-up; treatment is only recommended when patients exhibit evidence of liver damage. this is due to lack of information about their extent of liver damage. aim of this study was to assess extent of liver damage in hbeag +ve patients, unaware of their infection. methods: in this retrospective study, records of 206 hbeag +ve chb patients from our pool of 561 chb patients were reviewed. they were tested for hbsag, hbeag, hbv dna, anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 78.2% (161/206) patients were males and 21.8% (45/206) females. they were between 8-45 years of age. alt was raised >2times unl in 17% (35/206). 92. 2% (190/206) patients had high hbv dna (>10 5 copies/ml), while low hbv dna (<10 5 copies/ml) was seen in 7. 8% (16/206) . in high hbv dna group, significant necro-inflemmation (hai-ni >7) was seen in 48. 9% (93/190) and significant fibrosis (hai-ni >3) in 24. 7% (47/190) . figures were 37.5% (6/16) and 31.3% (5/16) respectively in low viral load group. none tested positive for hcv infection. conclusion: study indicates that machinery should be developed to characterize undetected hbv carriers in developing countries by conducting multi-center clinical studies. we have shown that considerable number of patients, unaware of their hbv infection, suffer from progressive liver damage. the overall strategy of management of chronic hbv infection should also be revisited. high viral load does not necessarily represent significant liver damage in patients with chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: in general, it is assumed that patients with chronic hepatitis b virus (hbv) infection with high viral load exhibit increased liver damages. treatment guidelines also emphasize on reducing viral load. these observations were mainly accumulated from developed countries. >80% chronic hbv carriers live in the developing nations, but little is known about relationship between hbv viral load and extent of liver damage in these countries. in this study, we addressed this issue. methods: in this retrospective study we reviewed records of 306 chb patients from our pool of 561 patients. all had high hbv dna (>10 5 copies/ml). 62. 1% (190/306) were hbeag +ve and 37.9% (116/306) hbeag -ve. they were alsotested for anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 51.1% (97/190 ) hbeag +ve patients with high hbv dna had non-significant hepatic necro-inflammation (hai-ni <7); this figure was 53.4% (62/116) in hbeag -ve patients. non-significant hepatic fibrosis (hai-f <3) was observed in 75. 2% (143/190) and 69.8% (81/116) in hbeag +ve and -ve patients respectively. none tested positive for hcv. conclusion: correlation doew not exist between viral load and liver damage in chb in bangladesh. many with both hbeag +ve and -ve chb with high hbv dna do not have significant hepatic necro-inflammation and fibrosis. further study may be needed to find out influence of other factors on liver damages in chb in bangladesh. most of these patients have not been characterized and treatment modalities have not been defined for them. background/aims: expression of intrahepatic hepatitis b core antigen (hbcag) is related to the immunopathogenesis of hepatitis b virus (hbv) infection. the role of hbv genotype and basal core promoter (bcp) mutation in expression of hbcag was investigated. methods: seventy hbeag-positive chronic hepatitis patients (genotype b in 52 and c in 18; bcp t1762/a1764 mutation in 16) were enrolled. clinical, virologic and histologic features were compared with regard to localization and expression of intrahepatic hbcag. the effects of hbv genotype and bcp t1762/a1764 mutation on the expression of hbcag were further evaluated by in vitro assays. results: cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic hbcag were found in 38 (56.7%), 25 (37.3%) and 4 (6.0%), respectively. fifty-eight (80.6%) of these patients expressed a high level of hbcag. in multivariate analysis, cytoplasmic localization of hbcag correlated only with low serum viral load (p=0.045) and bcp mutation (p=0.04). high expression level of hbcag also correlated with high serum viral load (p=0.015) and bcp wild-type sequence (p=0.037). in vitro assays supported that hbv bcp mutant had lower subcellular expression of hbcag compared with bcp wild-type strain. conclusions: hbv bcp mutation and viral load but not genotype contributes to the expression of intrahepatic hbcag. hepatitis b virus (hbv) genotypes show distinct geographical distributions and virological and clinical differences. in some of genotypes, specific substitutions and mutations have been described in association with hepatitis b e (hbe) protein expression and viral replication. in this study, genetic characteristics of hbv genotype e (hbv/e) were investigated using clinical samples obtained from 12 hepatitis b e antigen (hbeag)-positive, and 11 anti-hbe-positive asymptomatic carriers (ascs) in west-africa. full-genome analysis of isolated hbv strains revealed strong association between precore (pc) mutation and hbeag to anti-hbe seroconversion. furthermore, using 53 partial genome sequences, correlation among hbeag/anti-hbe status, viral load and key mutations were analyzed. the data showed that pc mutation is associated with hbeag seroconversion and enhanced viral replication efficiency. comparison between hbv/e and hbv/d strains reveals these two genotypes to have an identical sequence in their core-promoter-upstream and basic core promoter (curs/bcp) regions. it has been known from the previous phylogenetic studies, that hbv/d and hbv/e cluster together in trees reconstructed on x and precore/core orfs. in addition, this study, demonstrates that in spite of the high sequence similarity of curs/bcp region, the seroconversion-related mutation patterns are different between hbv/e and hbv/d in asc. further studies are needed to clarify the clinical significance of the regulatory sequence similarity between hbv/e and hbv/d. necro-inflammation and fibrosis p. siddappa 1 , p. kar 1 , b. das 2 , r. gondal 1 , m. asim 1 1 maulana azad medical college, 2 icpo, new delhi, india background: chronic hepatitis b(chb) is an important cause of morbidity and mortality. methods: pilot study involving 30 patients of chb, were equally randomized to receive either adefovir or lamivudine for 6 months. quantification of serum and hepatic hbv dna levels by real time pcr and liver biopsy done at start and end of 6 months. results: after 6 months there was significant and comparable reduction in serum and hepatic hbv dna viral load and liver biopsy showed significant reductions in hai scores in both the groups. serum alt which was elevated to 2 or more times normalized in both the groups. in the adefovir group 2 patients became hbeag negative and 2 patients who were hbeag negative at the start of therapy remained so. in the lamivudine group one patient became hbeac negative and 2 patients who were negative at the start of therapy remained so. in the adefovir group 4 patients became hbv dna (qualitative test) and in the lamivudine group 2 patients became hbv dna negative. there was strong correlation between serum and hepatic hbv dna levels both before and after the completion of therapy. conclusion: both the drugs bring about biochemical, histological and serological improvement with significant reduction in viral load in serum liver after 6 months without complete clearance of virus. there was not enough evidence to show therapeutic advantage of one drug over the other. the serum and hepatic hbv dna levels correlate well with eachother before and after treatment. aim: assessing efficacy and safety of treatment of chronic hepatitis b in children with pegylated ifn. materials and methods: 13 children (9 boys and 4 girls) aged 11-17 years with chb treated with peg-ifn alfa-2a, 100 g/m 2 /week during 48 weeks, 5 hbeag-positive and 8 hbeag-negative children, 4 previously treated with recombinant interferon. no child had liver disease greater than grade 2, stage 2. serum hbv dna was quantified at baseline, tw 4, ("rvr") tw 24, tw 48 (etr) and w 72 (svr) with rt pcr method (roche taqman). alt activity, haematology and adverse events were monitored. results: after 4 weeks treatment median hbv dna level decreased from 7.8x10 3 iu/ml at baseline to 1.43x10 2 iu/ml (p<0.01). "rvr" -undetectable hbv dna at tw4 was observed in 6/13 children and associated with lower pretreatment alt levels <25 iu and pretreatment viral load <750 iu/ml. all children with "rvr" were hbeag-negative pretreatment. at tw 24 and tw 48 seven children including all with "rvr" had undetectable hbv dna. 5 children achieved svr (undetectable serum hbv dna in w 72), among them 3 with "rvr". in 2/6 children with "rvr" hbsag disappearance was observed since tw 48. leukopenia was reported in 7 children, thrombocytopenia in 3. no adverse events were observed following dose modifications. conclusions: 1. peg-ifnalfa-2a is a good therapeutic option for children with chb, in particular with hbeag-negative chb 2. low pretreatment viral load and "rvr" seem to be predictive factors of efficient therapy. control by investigating the sanitizing modes among appliances used in the public service places (psp) and hbsag among appliances and practitioners worked in those places. methods: 63 beauty parlors, barber shops and bathing centers selected by stratified randomization sampling, 682 workers were investigated in questionnaire. the hbsag in appliances of psp and employee was detected by ria. results: the rate of hbsag among appliances of psp was 2.13%. the rate of hbsag in large-, medium-and small-sized appliances was 0.63%, 2.67% and 3.70%. the rate of hbsag has different( 2=6.68 p 0.05). the rate of hbsag among appliances of beauty parlors, barbering shops and footbath inns was 2.97%, 0.61% and 3.42%. different appliances had different rate of hbsag, such as the rate of acne needle and the forceps was 5.13% and 4.17%. the positive of hbsag amongworkers in psp was 7.13%. the rate of hbsag among workers in large-, medium-and small-sized psp was 7.34%, 8.33% and 2.94%. the rate of hbsag among workers in beauty parlors, barbering shops, footbath inns and bathing centers was 9.01%, 6.37%, 4.35% and 7.29%. the hbsag rate among workers was different in different works, the rate was higher in tattoo workers (13.33%), pedicures workers (12.68%), massagists (8.03%). conclusions: it is important to enhance the sanitizing management in psp and improve workers kap) of hepb. and we should promote health education to enhance the knowledge of hepatitis b control and build up supervision consciousness. background: integration of hepatitis b virus (hbv) dna into host chromosomes is often found in chronic liver disease and hepatocellular carcinoma, which is likely an early event of hbv-related carcinogenesis. however, the molecular mechanism of integration remains unclear. here we describe a potential mechanism of hbv integration and identify that ku70 and ku80, the gatekeepers of non-homologous end-joining (nhej) repair pathway, can serve as targets for anti-hepatitis virus integration. methods: using i-sce endonuclease-based system, we induced a dna double-strand break (dsb) in human hepatoma cell line huh-7. the cells were then incubated with serum from patients with chronic hbv infection. pcr amplification and direct sequencing were used to detect the inserted sequence in the site of dsb. finally, we employed taqman-based real-time pcr assay to quantify the integrated hbv dna and evaluate the effects of shrna on hbv integration. result: when huh-7 were exposed to viral serum and incubated for several days, hbv dna was detected in integrated form at the exact site of dna damage. furthermore, small interference rna (sirna) targeted against gatekeeper genes for nhej can down-regulate nhej repair and even the frequency of hbv integration. conclusion: thus, this project provided us with the first direct evidence that dna double-strand breaks are potential targets for hbv integration. the study has also shown that shrnas targeted against gatekeeper genes for nhej can regulate the frequency of hbv integration. objective: to screen proteins of human pancreas cdna library interacting with hbsag protein. methods: the library was amplifed, purified and evaluated, and then the puried library plasmids were transformed into yeast strain y187. the reconstructed plasmid pgbkt7-hbsag was transformed into yeast strain ah109 and screened on the nutrient deficiency medium sd/-trp. the transformed ah109 mated with y187 containing the library plasmid. the diploid yeast cells were plated on nutrient deficiency medium sd/-trp/-leu/-his/-ade and sd/-trp/-leu/-his/-ade containing x--gal for selecting. the plasmids in diploid yeast cells were extracted and electrotransformed into e.coli dh5 . the plasmids in dh5 were extracted, sequenced and analyzed by bioinformatic methods. results: sixteen proteins interacting with hbsag were founded. conclusions: these results show that hbsag protein may be related with metabolism of glucose and lipid. comparison of the sensitivity and specificity of the elecsys ® hbsag ii assay with other available assays in china for detection of hbsag j.d. jia 1 , l. wei 2 , x.x. zhang 3 , y.l. mao 4 , l.l. wang 5 , z.l. gao 6 , j.l. hou 7 , j. zhang 8 , w. melchior 9 , w. van der helm 9, 10 1 beijing friendship hospital, beijing, china, 2 beijing people hospital, beijing, china, 3 ruijin hospital, shanghai, china, 4 beijing 302 hospital, beijing, china, 5 west china hospital, chengdu, china, guangzhou, china, 7 guangzhou nanfang hospital, guangzhou, china, 8 shanghai public health clinical centre, china, 9 roche diagnostics ltd, rotkreuz, switzerland, 10 conclusions: in this patient population the prevalence of hbsag positive and anti-hcv were much higher than reported in community studies. genotypes 1 and 6 accounted for most of hcv. these very high rates of viral hepatitis in a hospital setting challenge to healthcare providers in terms of patient management as well as caregiver's prevention. hepatitis b is one of the major diseases of mankind that kills about one million persons each year in the world. accoring to primary study about 3% of iranian population is chronic hbv carriers. among iranian cirrhotics, 70-84% has evidence of exposure to hbv and 51-56% is carriers. because increase demand of blood transfusion, high blood dependent patients and long term window period of hbv infection, any controlling hbv infection program in blood donors can enhance the blood safety and public health. pe078 in this descriptive study included all the blood donors that referred to dezful blood transfusion center during 2005-2008. all the blood donors screened for hbs ag by using enzyme immuno assay and repeatedly reactive (r.r) samples confirmed by hbc-ab or confirmatory (neutralization) tests. the data analyzed by using spss 11.5. we found that in the first year 1.052 % were repeatedly reactive and 1.045 % confirmed. the results for other years as the followed: 0.782 %(r.r) and 0.770 % confirmed and in the last 0.725 % (r.r) and 0.700 % confirmed. the repeated blood donors increase in this period (42.11 %, 50.15 % and 51.68 % respectively). aim: we aimed to evaluate the cost-effectiveness of telbivudine versus entecavir with reference to lamivudine by roadmap model. methods: decision analysis model was used to study the incremental cost-effectiveness ratios (icer), i.e. the additional cost (in usd) required to achieve undetectable hbv dna or hbeag seroconversion for a patient at 2 years in america and hong kong. entecavir was used as a continuous monotherapy. lamivudine and telbivudine would be shifted to entecavir if hbv dna was detectable at month 6 and continued otherwise with drug resistance treated by add-on adefovir. weighted event rates based on previous reports were estimated for analysis. according our study, although the prevalence was higher than other region in our province, the hbv prevalence showed good decrease after stablishment strategies such as of repeated blood donor recruitment , improvement the donor selection and other educational programs . good following up those strategies to enhance the blood safety recommended. results: telbivudine was generally cheaper than entecavir to achieve an incremental case of undetectable hbv dna from lamivudine at 2 years. entecavir was least effective and most costly for hbeag seroconversion. conclusions: telbivudine is a cost-effective alternative to entecavir particularly when its cost is low in hong kong. h. tang 1 , g.l. zhang 1 , y.x. li 1 , r.q. tian 1 , m. liu 1 , x. li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china micrornas (mirnas) are single-stranded noncoding rnas of 18 to 25 nucleotides that play critical roles in a wide spectrum of biological processes. we investigated whether the mirnas-silencing machinery influences hbv replication or antigen expression. on the basis of elisa and mtt, the effect of 328 mirnas on the hbsag expression and cell proliferation was examined. three micrornas efficiently inhibited hbsag expression without significant effect on the proliferation of hepg2 2.2.15 cells compared to lacz control. subsequently, bioinformatics analysis were used to predict targets for the three mirnas, and the prediction results were conformed by cdna microarray analysis. the target region in hbv genome and the 3'utr region of one cellular gene were identified by fluorescent reporter assay, semi-quantitative rt-pcr and western blot. the results demontrated that mirna may play an important role in replication and gene expression of hbv. hepatitis b virus (hbv) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. although considerable progress has been made, the pathogenesis of hbv infection is still elusive. there's an urgent need to elucidate the mechanisms of hbv-host interactions, to discover novel biomarkers for diagnosis and prognosis and to develop therapeutic targets for anti-hbv treatment. herein, we applied a two-dimensional gel electrophoresis and maldi-tof/ms based comparative proteomics approach to globally analyze the host response to hbv by using an inducible hbv-producing cell line hepad38. of the 23 differentially expressed proteins identified, glucose regulated protein 78 (grp78) was one of the most striking proteins elevated by hbv replication, which was confirmed by real-time pcr and western blotting. knockdown of grp78 expression by rna interference resulted in a significant increase of both intracellular and extracellular hbv virions in hbv-transfected hepg2 cells. reversely, grp78 overexpression led to hbv suppression. the expression levels of hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) were determined by enzyme linked immunosorbent assay (elisa). immunofluoresce further revealed a positive correlation between the expression levels of grp78 and hbsag in both hbv-transfected hepg2 cells and hbv-infected human liver tissues. altogether, these data demonstrate for the first time that grp78 is an endogenous anti-viral factor in hbv-transfected hepg2 cells and may serve as a potential prognostic indicator of viral status in anti-hbv therapies. background/aims: to evaluate the predictors of response to long-term treatment of adefovir dipivoxil (adv) in patients with emerging lamivudine (lam)-resistant hepatitis b e antigen (hbeag)-positive chronic hepatitis b (chb) patients. methods: one-hundred-thirty-four lam-resistant hbeag-positive chb patients were treated with adv for a median of 28.0 months (range, 18 -54 months), following lam therapy for a median of 25.5 months (range, 5 -66 months). 65 patients (48.5%) were switched from lam to adv monotherapy, 43 (32.1%) were switched to adv with 1 month of lam overlap therapy, and 26 (19.4%) were switched to adv with 3 months of lam overlap therapy. the influence of baseline parameters on treatment response to adv in patients with lam-resistant hbeag positive chb was analyzed. result: during the follow-up period, 28 (20.9%) of 134 patients achieved complete response, defined as normalization of alt level, negative hbv dna by a digene hybrid capture assay and achievement of hbeag loss. sixteen (11.9%) patients achieved hbeag seroconversion. twenty-eight (20.9%) patients developed adv-related mutations during adv treatment. in multivariate analysis, virological response at 3 months (or=9.70, 95% ci: 32.63-35.78, p=0.001), defined as serum hbv dna levels less than 5 log10 copies/ml or a reduction in serum hbv dna levels greater than 2 log10 copies/ml after 3 months of adv therapy, independently predicted complete response. conclusions: virological response at 3 months was the strongest predictor of adv response in lam-resistant hbeag-positive chb patients. background/aims: to explore the effects of hbv dna level hbv genotype/subgenotype on the pathogenesis of severe liver diseases in chongqing. methods hbv dna level was analyzed in patients with severe liver diseases in retrospect,and hbv genotype/subgenotype hbv dna level and hbeag were determined in patients with hepatocellular carcinoma (hcc,n=78 ), liver cirrhosis(lc, n=58),chronic hepatitis b(chb, n=20) and acute on chronic liver failure(aclf, n=42). results hbv level from high to low with chb were lc, aclf and hcc in turn(p 0.05). hbv genotype was mainly genotype b.the rate of genotype b and c were 51. 3% and 47.4 respectively in hcc patients, 60.3% and 39.7 in lc patients, 55% and 40 in chb patients, 55% and 40 in aclf patients. the percentage of genotype b/c in aclf patients was higher in compared with other groups. but the distribution of hbv genotype among groups was not statistically different(p 0.05).subgenotypes of genotype b were almost ba but one. subgenotypes of genotype c were mainly ce in chongqing area, and there was no statistical difference among the 4 groups (p 0.05). conclusion: hbv dna level seems not to be a determining factor at end point of severe liver disease. both genotype b and c of hbv can lead to severe liver diseases, and there are more mixed infections by different genotypes in aclf. the efficacy of switching to entecavir (etv) monotherapy in japanese lamivudine (lvd)-experienced patients. background: this study aims to determine the efficacy of switching to 0.5mg etv daily in chronic hepatitis b (chb) patients previously treated with lvd. method: retrospective analysis of chb patients (n=134) previously on 100mg lvd daily and switched to 0.5mg etv daily. results: lvd-experienced patients were divided into three groups based on hbv viral load at time of switching to etv (<2.6 log10 copies/ml; 2.6-5.0 log10 copies/ml and >5.0 log10 copies/ml). detection of lvd-resistant virus at the time of switching was higher in the group with hbv dna 2.6 log10 copies/ml (76% in both 2.6-5.0 and >5.0 log10 copies/ml groups versus 23% in <2.6log10 copies/ml group) and was higher in patients treated with lvd for 3 years (52% versus 24% for patients on <1 year of lvd). a year after switching to 0.5mg etv daily, hbv dna undetectable rates were 100% (42/42), 94% (17/18) and 43% (6/14) for <2.6, 2.6-5.0 and >5.0 log10 copies/ml groups, respectively. alt normalization occurred in more than 90% patients at the end of the first year of switching to etv for all three patient groups. only one patient in the 2.6-5.0 log10 copies/ml group, who had lvd-resistant mutants at the time of switching, developed etv resistance during follow-up. conclusion: switching from lvd to etv maintains or improves viral suppression and alt normalization, especially in patients with viral load <5.0 log10 copies/ml. background/aims: we investigated the association between on-treatment hbsag decline and sustained response in patients treated with pegasys±lamivudine. methods: hbsag levels were measured retrospectively pre-treatment and at weeks 12, 24, 48 and 72 using the abbott architect hbsag assay in sera from 542 patients (83% asian) treated with pegasys alone (180 g qw; n=271) or combined with lamivudine (100mg qd; n=271) alone for 48 weeks as part of a large multinational trial. response was measured 6 months post treatment. results: more patients treated with combination therapy had >1 log decline in hbsag from baseline to week 48 ( figure) . hbsag level <1500 iu/ml at week 12 was associated with higher rates of response to pegasys±lamivudine 6 months post treatment ( figure) . data comparing hbsag and hbv dna as on-treatment predictors of response will be presented. conclusion: on-treatment hbsag monitoring may be useful for predicting response in patients treated with pegasys. y. wakui 1 , j. inoue 1 , y. ueno 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university graduate school of medicine, sendai, japan background/aim: chronic hepatitis b patients are clinically treated with nucleot(s)ide analogues and ifn-. nucleot(s)ide analogues have problems including drug resistance in continuous treatment, and ifn-has disadvantages of limited effectiveness and side effects. therefore, novel antiviral drugs are still needed. in this study, the suppressive effect to the replication of hbv was examined in vitro by using bezafibrate and rosiglitazone, which are ligands of peroxisome proliferator activated receptor (ppar) and , respectively. methods: the cytotoxicity of bezafibrate and rosiglitazone to hepg2 cells was examined with mts assay, and the concentration of 50% cytotoxicity (cc50) was calculated. hepg2 cells were transiently transfected with the plasmid containing 1.3-fold hbv genome of a genotype b strain. after 24 hours of transfection, rosiglitazone and bezafibrate was added to the cells. using the medium at day 3 after the addition of drugs, hbv dna was quantified with real-time pcr. results: the cc50 of bezafibrate and rosiglitazone in hepg2 cells were 250 m and 150 m, respectively. the amount of hbv-dna in the medium was decreased when the density of bezafibrate was over 200 m, but the density demonstrated considerable cytotoxicity. in contrast, rosiglitazone of 5 m, which showed no cytotoxicit, decreased the amount of hbv dna. the 50% effective concentration (ec50) was calculated to be 9.8 m. conclusions: in this study, it was suggested that the replication of hbv was inhibited by rosiglitazone of the density without cytotoxicity. the mechanism is uncertain and being investigated now. q. zheng 1 1 center for liver diseases, the first affiliated hospital, fujian medical university, fuzhou, p. r. china background: the objective of this study was to evaluate the early virologic response for prediction of achievement of hbeag seroconversion and hepatitis b virus (hbv) dna negativity after two years of lamivudine treatment in chronic hepatitis b (chb) patients. methods: in this retrospective study, adult patients with chronic hepatitis b (255 hbeag-positive and 122 hbeag-negative) were treated with lamivudine (100 mg/day), and followed-up up to 72 months. response and resistance to the treatment were assessed during the treatments with lamivudine. results: it was found that gender, age, baseline levels of alt and hbv dna, serum hbv dna at week 24 (p =0.019 , or = 0.442) were closely related to the achievement of hbeag seroconversion, undetectable hbv dna level and emergence of drug resistance after 2 years of lamivudine treatment. hbeag positive patients with baseline serum hbv dna in 10 6 -10 8 copies/ml and serum hbv dna 10 3 copies/ml at week 24 showed high response rate of alt normalization rate (91.7%), undetectable hbv dna rate (84.5%), hbeag seroconversion rate (55.2%), as well as low drug resistance rate (25.4%) after 2 years of treatment. similarly, hbeag negative patients with serum hbv dna 10 3 copies/ml at week 24 could achieve high 2-year response rate of alt normalization rate (72.41%), undetectable hbv dna rate (82.3%), and low drug resistance rate (15.5%). conclusion: serum hbv dna 10 3 copies/ml at 24-week provide the best prediction of 2-year lamivudine treatment response. background/aims: unlike oral antivirals, a finite course of (peg)interferon can induce sustained post-treatment response in patients with chronic hepatitis b (chb), with increasing rates of hbsag clearance observed in patients who respond during post-treatment follow-up. hbsag clearance is considered to be the closest outcome to a cure, being associated with improved histological outcome, reduced incidence of hcc and increased survival. methods: in a randomised multinational study, patients (hbeag-negative) received 180µg pegasys+placebo (n=177); 180µg pegasys+100mg lamivudine (n=179); or 100mg lamivudine (n=181) for 48 weeks, and were assessed 6 months post-treatment. from this initial study, 230 of those who had received pegasys±lamivudine and 85 patients who had received lamivudine monotherapy participated in a long-term observational study to investigate post-treatment response. hbsag clearance at yearly post-treatment follow-up visits up to 5 years post-treatment was analysed. results: hbsag clearance in patients treated with pegasys±lamivudine increased post-treatment (3% at 1 year to 6%, 8%, 11% and 12.2% at years 2, 3, 4 and 5). at year 5, 28 pegasys-treated patients (12.2%) had cleared hbsag compared with 3 (3.5%) of lamivudine-treated patients (p=0.022). 16/28 pegasys-treated patients had anti-hbsag (hbsag seroconversion). detailed analysis of the 5-year follow-up data will be presented. conclusion: the ability of a finite course of pegasys to induce sustained response with increasing hbsag clearance rates in responders during post-treatment follow-up supports its use as first-line therapy in hbeag-negative patients with chb. background/aims: recent studies suggest that quantification of hbsag levels early during treatment can be used to predict post-treatment response to pegasys. elecsys ® hbsag ii (roche) is a sensitive assay for the detection of hbsag. this assay can be used for the quantification of hbsag levels using a simple dilution algorithm. we compared results obtained using the elecsys ® hbsag ii method with those of a commonly used quantification assay. methods: hbsag levels obtained using the elecsys ® hbsag ii assay were compared with those obtained using the abbott architect ® hbsag assay for a total of 40 samples from patients infected with hbv genotypes a (n=8), c (n=1), d (n=29) and f (n=2). samples were diluted 1:100 in diluent provided by the manufacturer. samples with hbsag levels >250 iu/ml were retested at a final dilution of 1:1000. samples with hbsag levels <0.05 iu/ml were retested undiluted. results: overall, hbsag levels measured with the two assays correlated well (r 2 =0.9718) over a wide range (3-3x10 5 iu/ml). discrepancies in hbsag levels >±20% were reported for a minority of the samples (n=15), mainly distributed evenly above and below the ideal line (n=11). in the four low titre (range 3-9x10 3 iu/ml) samples with greatest discrepancy elecsys ® underestimated values (in two cases by >50%). conclusion: the elecsys ® hbsag ii assay provides a simple and reliable means for determining hbsag levels. this simple assay format could be used to provide useful information during on-treatment monitoring of hbsag levels in patients with chronic hepatitis b undergoing therapy. conclusions: hbv/a has been increasing in chb patients in japan as the consequence of ahb, spreading in the younger generation through promiscuous sexual contacts, thrust by an inclination of hbv/a to induce chronic hepatitis. the spread of hbv/a infection in japan should be prevented by universal vaccination programs. introduction: chronic viral hepatitis is common in end-stage renal disease (esrd), from endemic hepatitis b (chb) and nosocomial hepatitis c (chc). reduced outcomes post-renal transplant were reported thus chb and chc cirrhosis became contraindications to listing. however, these predated effective anti-viral therapies. we reviewed outcomes of patients with chronic viral hepatitis following assessment for renal transplantation. methods: prospective database of esrd patients with viral hepatitis referred for renal transplantation was reviewed. results: 110 patients were assessed. 23 patients underwent kidney transplantation. two were cirrhotic and had liver/kidney transplantation; both died within 6 months. 21 were non-cirrhotics, of whom 20 are alive. 14/20 have functioning allografts; predictors were normal alt and low viral load. of the 87 non-transplanted, 31 had cirrhosis; 17/31 received anti-virals. mortality was 35% -7 liver-related (3 hepatoma,1 bacterial peritonitis,3 sepsis -5 inactive cirrhosis); 4 non-liver related (2 cerebral,1 haemorrhage,1 renal -3 inactive cirrhosis). 12/20 surviving cirrhotics received anti-virals. in non-transplanted non-cirrhotics, mortality was 18%; 80% of survivors had inactive disease. 23 chb patients received lamivudine; 11 adefovir (lamivudine resistance). 11 chc patients received ifn-based therapy. conclusion: excellent outcomes are achieved in esrd patients with chb/chc post-renal transplant, in absence of cirrhosis. normal alt/non-detectable viral load can predict graft function. however, cirrhosis is associated with high mortality on dialysis whereas non-cirrhotics with inactive disease do well. the role of kidney transplantation in cirrhotics with suppressed viral replication needs to be reassessed. the truncated hbc149 interferes with replication of hepatitis b virus j.c. han 1,2 , x.b. pan 1,2 , l. wei 1,2 , k. deng 1,2 1 institute of hepatology, 2 peking university people's hospital, china hepatitis b virus (hbv) capsids play an important role in production of progeny virus and other elements of the virus life cycle. misdirection of capsid assembly and formation of aberrant particles may be an effective approach to interfere with virus replication. hbv capsids can be assembled in vivo and vitro from the dimeric hbv core protein (hbcag). the interaction of single and dimeric hbcag with some truncated hbcag is verified in vitro. the truncated hbcag consists of the first 149 amino acids and lacks the c-terminal, 34-residue rna-binding domain. method: we transiently transfected hepg2.2.15 with pcdna3.1hbc149 by fugene6.after 48 and 96h, hbvdna, hbeag and hbsag in culture supernatant were detected and cell subjected to southern blot and immunofluorescence analysis. result: the level of hbsag and hbeag had gentle change, we found that hbvdna decreased at 96h after transfection( 10 3 10 4 copies/ml p<0.01) ,but replication intermediates obviously decreased from 48h. some positive signal of hbcag located around the nuclear and conglomerated in cytoplasm compared to the control. conclusion: the truncated hbc149 can inhibit replication of hepatitis b virus. misdirection of capsid assembly and formation of aberrant particles could be an important cause. y.p. li 1 , r.c. li 1 1 objective: to assess the long-term efficacy of recombinant yeast derived hepatitis b vaccine in infant s born to hbsag and hbeag carrier mother. methods: a total of 273 neonates born to hbsag, hbeag both positive mothers were vaccinated with 5 ,5 ,5 g doses of recombinant yeast derived hepatitis b vaccine by 0 ,1 , and 6 months schedule. they were all followed for 129 years after the primary vaccination. results: twelve infant s (6.63 %) become hbsag positively conversed in 9 year after primary vaccination ,and the positive rate of hbsag in 1-9 year was 0.72 %-6.8 % ,17.18 % of child in no/ lowly respond become hbsag positively. at the ninth year, the positive rates of anti-hbs were 60 % above. anti-hbs positive rates and immunity level were higher at 3-5 year old by repetition immunity than others. conclusion: the recombinant yeast derived hepatitis b vaccine have good immunogenecity and long-term protective efficacy to hbv interruption of perinatal transmission , a booster dose seems necessary in aged 3-4 years to the mother with hbsag and hbeag.it is high risk tobecome hbsag positively in the baby of norespones to hepatitis b vaccine. chb patient group-initiated programme to improve awareness, adherence and treatment outcomes in asia pacific n. leung 1 1 founding chairperson of asiahep background: worldwide, over 400 million people live with chronic hepatitis b (chb); 300 million in asia pacific. regional survey data from 1,500 patients in 10 countries showed a lack of knowledge and understanding of chb, its severity and impact on quality of life. this initiative aims to coordinate patient groups in the region and devise programmes to improve knowledge and healthcare outcomes. methods: the patient groups met in hong kong in may 2008 and identified common needs to: (1) improve educational resources; (2) raise awareness; (3) increase diagnostic yield; and (4) enhance treatment compliance through education about the need for sustained viral suppression to reduce long-term complications. results: a patient engagement programme was developed for people with newly diagnosed or known chb. the programme comprises: -detailed information about chb -a health-tracking tool for self-monitoring of blood tests and treatment progress -detailed information for carers/family -a patient-physician communications video (including role-play) -mobile phone text messages providing advice and compliance/appointment reminders conclusion: this programme was developed to address the needs of patients and clinicians. improved knowledge and long-term support, particularly for patients on antiviral medication, is expected to improve quality of life. the programme encourages clinicians and patients to develop enduring therapeutic partnerships to promote optimal outcomes. acknowledgement: the chb patient group meetings and the patient engagement programme are supported by an unrestricted educational grant from glaxosmithkline. serum hbv rna level reflects the potency of nucleos(t)ide analogue y.w. huang 1,2 , k. chayama 3,4 , m. tsuge 3,4 , s. takahashi 3,4 , t. hatakeyama 3,4 , m.y. lai 2,5 , h.l. you 1 , j.t. hu 1 , c.j. liu 2,5 , p.j. chen 2,5 , d.s. chen 2,5 , s.s. yang 1 , j.h. kao 5, 6 1 liver unit, cathay general hospital medical center, 2 background and aims: serum hbv rna is detectable in patients treated with lamivudine (lmv) or entecavir (etv) (hatakeyama, 2007 and huang, 2008) . the aim of this study was to determine the clinical significance of serum hbv rna levels in patients treated with nucleos(t)ide analogues of different potency. methods: serum hbv rna was serially determined in 20 patients treated with nucleos(t)ide analogues for 48 to 52 weeks (9 with adefovir (adv), 5 with lmv, and 6 with etv). serum hbv rna was quantified by reverse transcription of hbv nucleic acid extract with subsequent real-time pcr. results: hbv rna was detectable in 11 patients as follows: 2 of 9 in adv (22%), 3 of 5 in lmv (60%), and 6 of 6 in etv (100%) (p = 0.002). mean log serum hbvdna levels at baseline were 10.1 ± 0.6 for adv, 6.6 ± 2.0 for lmv, and 9.5 ± 0.9 for etv, which were comparable between less potent adv and most potent etv (p = 0.14). during antiviral therapy, peak log hbv rna level of patients with etv was significantly higher than that of those with adv or lmv (3.2 ± 0.9 vs. background: in the phase iii clinical trials, clevudine 30mg for 6 months showed potent antiviral activity along with a marked post-treatment antiviral effect. the objective of this study is to compare the anti-hbv activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis b (chb) patients in a randomised way. methods: the patients are received clevudine for 24 weeks and then combination of clevudine and vaccine for another 24 weeks or clevudine alone for 48 weeks. eligible patients were treatment-naïve hbeag(+) chb patients with hbv dna levels 500,000 copies/ml. the primary endpoint is the proportion of patients with hbeag loss. preliminary results are presented here. results: thirty-one patients have completed week 24 visits and from them, 15 patients (11 in clevudine alone and 4 in combination group) have completed week 36 visits. at week 24, 26% of patients had hbeag loss. at week 36, 46% in clevudine alone and 25% in combination group (3 months on combination after clevudine monotherapy) had hbeag loss. at week 24, 63% of patients had negative hbv dna by amplicor pcr (<300 copies/ml). at week 36, all of patients in both groups had negative hbv dna by pcr and 73% in clevudine alone and 80% in combination group had normal alt. conclusion: clevudine demonstrated good serologic response as well as significant viral suppression and alt normalization. with this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. background/aims: to determine the reasonable number of clones for hbv quasispecies analysis. methods: 16 chronic hepatitis b patients were enrolled with hbvdna levels range from 4~10 log copies/ml. hbvdna was extracted. hbv reverse transcriptase (rt) gene encompassing the overlapping surface s gene was amplified by polymerase chain reaction, then cloned and sequenced. ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. the characteristics of hbv quasispecies including shannon entropy and genetic distance were calculated. results: the shannon entropy and genetic distance of 10 clones group was higher than those of 20 and 30 clones group, either in rt gene or in s gene (p<0.01). while the shannon entropy and genetic distance of 20 clones group showed on difference with those of 30 clones group, neither in rt gene nor in s gene (p>0.05). the number of different quasispecies detected in 30 clones group was higher than that of 20 and 10 clones group (p<0.01). the shannon entropy and genetic distance in three different clones group had no correlation with hbv dna levels (p>0.05). conclusion: although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than 20. the information contained in 20 clones per sample could well represent the quasispecies characteristics. the clone number was not necessary modulated according to different hbv dna levels. background: recent studies reported that basal core promoter mutation (a1762t and g1764a) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and hcc. but the effect of the double mutations on the activity of enhancer ii/basal core promoter is still uncertain. objectives: to evaluate the influence of nt1762 a/t and nt1764 g/a mutations on hbv enhancer ii/basal core promoter activity. methods: the pcr fragments of hbv enhancer ii/basal core promoter (nt1601 to nt1815) from the serum-derived genotype b hbv dnas of one hbv carrier aged 66 and one hbv related hepatocellular carcinoma patient aged 26 were introduced into the pgl3-basic-vector from promega via restriction sites of xho i and hind iii. the nt1762 a to t and t to a, the nt1764 g to a and a to g mutations were carried out by genetailor site-directed mutagenesis system from invitrogen. the promoter activity was evaluated by comparing firefly luciferase measurement with renilla luciferase as the internal control using the dual-luciferase reporter assay system from promega. results: the luciferase reporter assay results indicated that the 1762 t to a combined with 1764 a to g mutations increase (p<0.001) while the 1762 a to t combined with 1764 g to a mutations decrease (p<0.05) the hbv enhancer ii/basal core promoter activity significantly. conclusions: associated with increased risk of hepatocellular carcinoma, a1762t and g1764a double mutations of hepatitis b virus reduce the enhancer ii/basal core promoter activity. background/aims: a substantial proportion of chronic hepatitis b (chb) patients with mildly elevated alanine aminotransferase (alt) have significant fibrosis. we evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. methods: one hundred five chb patients with alt less than two times the upper limit of normal underwent liver biopsy. multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. results: there were 27 patients in the low normal alt group, 21 in the high normal alt group, 37 in the low elevated alt group, and 20 in the high elevated alt group. fifty eight patients (55.2%) had significant fibrosis ( stage 3) and 35 (21.0%) had significant inflammation ( grade 3). the age, platelet count and grade of inflammation were factors associated with significant fibrosis. progressive liver disease was observed in 23 (27.4%) of the 84 followed-up patients. the stage of fibrosis, alt group and antiviral therapy were significant predictive factors for progressive liver disease. conclusion: liver biopsies should be recommended in patients over 35 years with mildly elevated alt levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. background: four nucleos(t)ide analogues (nas) are currently approved for the treatment of hbv infection in china. however, long-term benefits are limited by the emergence of drug-resistant viruses. methods: patients accepted the examination based on physician's instruction. hbv reverse transcriptase gene was amplified from serum via nested pcr and sequenced directly. results: well-recognized drug-resistant mutations were detected in 567 of 2,000 patients. in patients receiving na monotherapy, corresponding drug-resistant mutations were detected in 214/381 for lamivudine (lam), 35/333 receiving adefovir (adv), 0/57 for entecavir (etv), and 9/17 for telbivudine (l-dt). the mutations were detected in 272/615 patients receiving 31 kinds of sequential/combined usages of the 4 nas. m204i (32%), m204v+l180m v173l (32%), and m204i+l180m (21%) were identified as major mutant patterns of lam monotherapy. n236t a181 substitution was the dominant adv-resistant mutation. t184 substitution was the dominant etv-resistant mutation always accompanied with lam-resistant mutation. l-dt-resistant mutation was m204i l180m exclusively. adv-resistant mutation was frequently seen in lam-resistant patients receiving adv sequential therapy rather than those receiving adv add-on therapy. controversial lam/adv-resistant mutations including a181t, v214a, q215s and i233v were detected in some patients singly or with the well-recognized drug-resistant mutations. interestingly, the drug-resistant mutations were also observed in a few of patients naïve to nas. conclusions: the exploration of hbv drug-resistant mutation profile in large clinical samples furthers our understanding of hbv drug-resistant status in china with implications for administrating anti-hbv therapy more reasonably. toll-like receptor (tlr) 2, tlr4 and cd4+cd25+cd127low/-regulatory t cells correlate with hepatitis b virus infection y. zhang 1 , j.q. lian 1 , c.x. huang 1 , x. wei 1 , j.p. wang 1 , p.z. wang 1 , x.f. bai 1 1 center of infectious diseases, tangdu hospital, the 4th military medical university, xi'an, china background: tlrs play a crucial role in sensing and initiating innate antiviral response and tregs actively suppress immune response, contributing to viral persistence and chronic tissue damage. in this study, we determined tlr2 and 4 expression and treg frequency, as well as their function in the effect of hbv infection. methods: tlr2 and tlr4 expression on monocytes and circulating cd4 + cd25 + cd127 low/-tregs were determined by flow cytometry in 16 ahb, 42 chb, 22 asc and 20 nc. spearman correlation was performed to investigate associated variables on treg or tlrs. pbmcs were stimulated with hbeag or hbcag and the tlrs profile was examined. result: tlr2 expressions were up-regulated in chb and asc, while tlr4 were increasingly expressed in ahb and asc. treg frequency in chb was significantly higher than that in nc. in chb, the increased tlr2 negatively correlated with hbv dna loads and treg frequency negatively correlated with tlr4 expressions. tlr2 was up-regulated after hbeag stimulation in both nc and chb. conclusion: increased tregs may be associated with chb and there might be possible interactions between hbeag, tlr signaling and the innate immune response, which may partially explain the mechanism of hbv infection induced immuno-tolerance. (6.7±1.35) . hbv-dna was quantitatively determined by polymerase chain reaction (pcr) technique, and hbv genotype was determined by pcr microwave gene chip technique. antiviral efficacy was assessed using measuring the following scales: the alt normalization rate, hbv-dna negative conversion rate and the hbeag/anti-hbe seroconversion rate. results: among 55 serum specimen, hbv genotype distribution was 38 genotype c, 14 genotype b, and 3 genotype non-b or c respectively. in genotype b, alt normalization rate was 78.57%(11 cases), hbv-dna negative conversion rate was 35.71%(5 cases) and the hbeag/anti-hbe seroconversion rate was 14.28%(2 cases). in genotype c, alt normalization rate was 76.31% (29 cases), hbv-dna negative conversion rate was 47.37%(18 cases) and the hbeag/anti-hbe seroconversion rate was 18.42%(7 cases). the efficacy of adefovir dipiroxil showed no significant differences between genotype b and c in the treatment of chronic hepatitis b p>0.05 . conclusion: adefovir dipiroxil is an effective antiviral drug. hbv genotype is irrelevant to the antiviral efficacy of adefovir dipiroxil in treatment of patients with chronic hepatitis b. the effect of anti-hbv drugs on albumin and bilirubin levels, and platelet count h. yoshida 1 , h. taniguchi 1 , r. nagano 1 , k. sakitani 1 , e. seki 1 , t. serizawa 1 , y. ito 1 , h. mizuno 1 , y. mitsuno 1 , r. nakata 1 , m. omata 2 1 japanese red cross medical center, 2 university of tokyo, japan background/aim: we assessed the efficacy of anti-hbv drugs on the liver function. methods: patients with hbv-related disease followed at our center between 2005 and 2007 were enrolled. lamivudine (100mg), lamivudine (100mg) +adefovir (10mg), or entecavir (0.5mg) was administered to the patients with detectable hbv dna and elevated alt. liver function (alt, alb, and t.bil) and platelet count were observed. alt, alb, t.bil, and platelet count of treated group at pretreatment, year 1, and year 2 were compared with untreated group. results: eighty six patients with positive hbsag were enrolled between jan 2005 and dec 2007. seven patients (2 acute infection, 1 overlap infection with hcv, 7 lost of follow up) were excluded. in total 76 patients were followed up for a median follow up of 26 (range 2-39) months. of 76 patients, 32 received anti-viral treatment. twenty one patients were treated with lamivudine, 4 with lamivudine+adefovir, and 7 with entecavir. the mean of levels of pre-treatment-year1-year2 were alt:129-37-43 (u/l), alb:4.0-4.4-4.4 (g/dl), t.bil:1.0-0.8-0.7 (mg/dl), and plt:14.3-15.4-16.0 (x10 4 mcl) respectively. markers of untreated group (n=44) (at baseline-year1-year2) were alt:33-40-35 (u/l), , t. bil:0.8-0.8-0.9 (mg/dl), and plt:18.7-18.0-18.5 (x10 4 mcl) respectively. although all of four markers in treated group were significantly worse than untreated group at baseline, all of four markers did not showed significant difference from untreated group at year2. conclusion: treatment with anti-hbv drugs showed the efficacy not only transaminase levels, but also on albumin, bilirubin, and platelet count improvement-improvement of "hepatic reserve" which is valuable for prevention of cirrhosis. background: currently, hbeag-negative chronic hepatitis b(chb) is increasing. but there are still controversial on the treatment of hbeag-negative chb with alt 2×uln. we have investigated the clinical efficacy of nucleotide analogues(nas) in the treatment of hbeag-negative chb with alt 2×uln. methods: the data of patients who were treated by nas for more than 2 years and with alt 1 2×uln (n=87) , alt 1×uln(n=43) and alt 2×uln(n=88) were collected, and 12w and 24w virologic response, 48w and 96w complete response, virologic breakthrough and clinical resistance were analyzed. results: compared with the base line, hbv dna level in all three groups were significantly decreased (p 0.01), and there was no significant difference between alt 1 2×uln group and alt 2×uln group. the viral load was significant decreased in alt 1×uln group at 12w, 24w and 36w (p<0.05). virologic response at 12w and 24w complete response at 48w and 96w was 71.3%, 82.8% , 82.8% and 86.2% respectively in alt 1 2×uln group and was 51.2%, 60.5%, 65.1% and 74.4% respectively in alt 1×uln group. there was no significant difference between alt 1 2×uln group and alt 2×uln group. virologic response at 12w and 24w and complete response at 48w were significant decreased (p<0.05) in alt 1×uln group. there was no significant difference among the three groups in virologic breakthrough and clinical resistance. conclusion: hbv replication can be satisfactory inhibited by nas in hbeag-negative chb patients with alt 2×uln, which suggests that in these patients the indication of alt is different from hbeag-positive patients. quantitative hbeag assay as a predictive factor of hbeag seroconversion induced by peg-ifn -2a therapy to hbeag-positive chronic hepatitis b y.y. zhu 1 , j. dong 1 , y.t. chen 1 , j. chen 1 , j.j. jiang 1 1 liver diseases research center, the first affiliated hospital of fujian medical university, fuzhou, fujian, china rp, 350005 background: to find predictive factor for hbeag seroconversion in the treatment of hbeag-positive chronic hepatitis b (chb) by peg-ifn -2a. methods: hbeag-positive chb patients were given peg-ifn -2a treatment for 48 weeks. clinical data were collected every 3 months. receiver operator characteristic (roc) curve was employed to calculate positive predictive value (ppv), negative predictive value (npv), sensitivity and specificity. results: sixty-five patients completed peg-ifn -2a therapy. among them, 17 (26.15%) were found hbeag seroconversion and 19 (29.23%) were found hbeag loss at cessation of therapy. none of age, gender, alt level and hbv dna load at baseline had relationship with hbeag seroconversion. hbeag level of baseline was correlated to hbeag seroconversion, with p value as 0.003 (table 1) . according to roc curve, supposed auc as 0.705 and p value as 0.042, the ppv, npv, sensitivity and specificity of hbeag level as 61 at 12 week were 0.361, 0.862, 0.765 and 0.521, respectively. supposed auc as 0.828 and p value as 0.001, the ppv, npv, sensitivity and specificity of hbeag level as 15 at 24 week were 0.452, 0.912, 0.824 and 0.646, respectively. the hbeag level (s/co) and decreased degree (percentage) at 12 week and 24 week were significant related to hbeag seroconversion (table 2) . conclusion hbeag level at baseline and at 12th and 24th week and its decreased degree (percentage) during the treatment course could be used as predictive factor for hbeag seroconversion. background: it is well documented that perinatal transmission is the major cause of chronic hbv infection in china. the aim of this study was to evaluate the efficacy of interruption of hbv intrauterine infection with hepatitis b immunoglobulin (hbig) in pregnant women with hbeag positive. methods:: a prospective randomized controlled trial was adopted. each subject in the trial group (28 cases) was given 200 iu hbig intramuscularly every 4 weeks from 28-week of gestation, while each subject in the control group (24 cases) received placebo in the same way. the cord blood of newborns were collected for detecting hbsag, hbeag and hbv-dna. results: for newborns, hbeag positive rate in trial group was 21.4%(6/28).hbeag positive rate in control group was 79.2%(19/24). there was significant difference in hbeag positive rate of newborns between the two groups( p < 0.01, rr = 0.27). hbv-dna positive rate in trial group was 25%(7/28). hbv-dna positive rate in control group was 83.3%(20/24). there was significant difference in hbv-dna positive rate of newborns between the two groups( p < 0.01, rr = 0.30). hbv-dna load of 7 cases of newborns in trial group was lower than that of their mothers(t = 28,p = 0.02). there was no significant difference in hbv-dna load between women and their newborns after delivery in control group (t = 81.5,p > 0.1). conclusion: it is effective and safe to prevent hbv intrauterine infection with hbig from the 28(th) wk in pregnant women with hbeag positive. ), especially, the cirrhosis and hcc cases obviously more in both hbeag and anti-hbe patients are negative than hbeag-negative but anti-hbe positive patients (.36.7% vs 26.7%; 7.9%vs5.5%, p ) .the prevale nce of pre-core g1896a mutate have no significant difference regardless of hbv serum marker status or the state of illness. conclusion: recent 5 years the hbeag-nagative chronic hepatitis b patients are gradually increasing in yunnan province. while the hbeag disappear but no anti-hbe serum transfer, and the virus still active replication -it may be a crucial phase determined the diseases outcome, which should be pay more attention by physicians. the clinical significant of pre-core g1896a mutate remain unknow. efficacy of interferon for chronic hepatitis b patients with normal or paranormal alt z. liu 1 , j.z. guo 1 , y.j. lin 1 , y.j. zhang 1 , z.w. lang 1 1 beijing ditan hospital, beijing, china background: we reported interferon treatment for 4 cases with normal or paranormal alt but in which liver histologic exam showed g2-4 and/or s3-4. methods: 4 patients were male with an average age of 28 years.mean alt was 46.7 iu/l and hbv dna level was 3~5 log10copies/ml. two patients were hbeag positive; one patient was both negative for hbeag and anti-hbe and one patient was anti-hbe positive. liver biopsy showed g2~g4 and s3~s4 respectively. 3 patients were treated with ifn-alpha, liver biopsy was repeated after 1 year.only one patient had received combination therapy with ifn and adefovir after 10 months treated ifn monotherapy and liver biopsy was taken after 1.5 years. results: all patients got normal alt after 1 year treatment. hbv dna was undetectable in 3 patients. 2 patients with initial positive hbeag cleared. but 3 patients still were anti-hbeag negative.liver biopsy showed change fromg4 s3-4 to g2 s2-3 in 1 patient; fromg2 s3 to g3 s4 in 1 patient and no change in the other 2 patients. conclusions: though alt and hbv dna improved after 1 year treatment, histological improvement is not satisfying. 1 patient's improvement in liver histology may be due to seroconversion before treatment and adding adefovir after 10 months of interferon therapy. after 8 months of combination therapy we did liver biopsy again. the other 3 patients were hbeag negative, but hbeab were also negative, liver biopsy was taken 1 year later without combination of nucleoside analogs. evaluation of long term efficacy of hepatitis b vaccination r.c. li 1 , j. gong 1 , j.y. yang 1 1 objective: to evaluate the long term effectiveness of preventive hbv infection and to monitor the incidence of hepatitis b in children to see possible impact on the program of long an that was launched in 1986. methods: (1) set up a surveillance systemof hepatitis ,to evaluate the possible impact on incidence of hepatitis b. (2) to serologically evaluate the effect of the program, a stratified random sampling of 1000 subjects in 1987 birth cohorts was recruited for long term follow up at the age 1-20 years. (3) cross-sectional seroepidemiolgical survey was carried out in the county in 1985 before the program and 20 years later. hbsag , anti-hbs and anti-hbc were tested by ria. results: the average coverage of hepatitis b vaccine was 89. 1 %. at 20 years after vaccination, the seropositivity for hbsag in population of 1-20 years has decreased from 16. 0 % to 2. 3 %, the annual effectiveness was 89. 7 %. hbv accumulated infection rate was 3. 5 %, protective rate was 96. 2 %. the incidence of acute hepatitis b was 1. 60 per 100,000 in population aged 1 -20 years , it decreased by 91.3 % as compared with the incidence of 18.38 per 100,000 in same age group in 1985 -1987. conclusion: mass hepatitis b vaccination program in long an county has proved to be effective in control of hbv chronic infection and incidence of acute hepatitis b. background and aims: although the evolution of viral quasispecies may be related to the pathological condition of disease, little is known about this in hepatitis b virus (hbv), especially during hbeag seroconversion. methods: nucleotide sequences of hbv precore/core genes from 5 time points were analyzed in four cohorts of chronic hepatitis b, interferon-induced seroconverters (is, n=9), interferon non-responders (in, n=9), spontaneous seroconverters (ss, n=9) and non-seroconverters (sn, n=9), followed during 60 months on average. only patients with genotype c were used. viral diversity was then estimated after nucleotide genetic distance was assessed and phylogenetic trees were constructed. results: analysis of 1800 nucleotide sequences showed that the nucleotide genetic distance of seroconverters (is and ss; 9x10 -3 substitutions/site and 8.5x10 -3 subsititutions/site, respectively) was similar to that of non-seroconverters (in and sn; both 7x10 -3 substitutions/site) before seroconversion. compared to that of nonseroconverters (in and sn; substitutions/site and 7.5x10 -3 substitutions/site, respectively) the viral diversity of seroconverters (is and ss; 13x10 -3 substitutions/site and 12x10 -3 substitutions/site, respectively) was significantly higher after seroconversion (p<0.05) and it was higher after seroconversion in seroconverters compared with that berore seroconversion (p<0.05) while it almost didn't change in non-seroconverters irrespective seroconversion. phylogenetic trees also showed that complex trees appeared in secoconverters and relatively simple in nonseroconverters. conclusions: the distinctly higher viral diversity after seroconversion in hbeag seroconverters could be related to increased hbv-specific t-cell responses and escape mutant which arise from stronger selective pressure caused by host immune activity. adefovir dipivoxil 10mg (adv) resistance at 5 yrs in chinese hbeag+ve chronic hepatitis b (chb) j.l. hou 1 , y.z. wang 2 , x.q. zhou 3 , j.q. niu 4 , y.m. wang 5 , h. wang 6 , y.m. mao 7 , k.f. barker 8 1 nanfang hospital, guangzhou, prc, 2 jinan infectious disease hospital, jinan, prc, 3 ruijin hospital, shanghai, prc, 4 1st hospital of jilin university, changchun, prc, 5 xinan hospital, chongqing, prc, 6 people's hospital, beijing, prc, 7 renji hospital, shanghai, prc, 8 glaxosmithkine r&d, london, uk background: long term adv provides clinical and histological improvement in chb, but may lead to emergence of treatment associated resistant mutations. we report on adv resistance data from chinese hbeag positive subjects treated for 5 years. methods: 480 hbeag positive chb subjects were randomized in an initial 52 weeks controlled adv study (with a 12 weeks placebo period in half of patients) and then offered open label adv treatment for a further 208 weeks. a total of 474, 456, 443, 421 and 390 subjects completed the 1 st , 2 nd , 3 rd ,4 th and 5 th yr, respectively. at the end of each year samples were analysed from those subjects with protocol-defined hbv dna breakthrough for the rtn236t or rta181v adv mutations associated with resistance. sera from subjects with breakthrough were analysed at all subsequent yearly timepoints whenever possible. results: at the end of the 1 st yr, none of the 45 subjects with hbv dna breakthrough had either mutation. sera were available for analysis from 137, 199, 228 and 187 subjects with viral breakthrough at the end of the 2 nd , 3 rd , 4 th and 5 th yr, respectively, with new mutations identified in 6, 20, 24 and 20 subjects at the same timepoints. of the cumulative 70 subjects at the 5 th yr analysis 31 had rtn236t, 29 had rta181v, and 10 had both mutations. conclusion: treatment with adv in chinese hbeag positive chb subjects for up to 5yrs resulted in a cumulative rate of 14.6% (70/480) adv resistance-associated mutations with hbv dna breakthrough. background and aims: to evaluate the predictive significance of rapid virologic response (rvr) for achieving an end-of-treatment virologic response (er) or hbeag seroconvertion and the predicting indicator of nonresponse (nr). methods: 205 patients with chronic hepatitis b were treated with adv and prospectively observed to 48 weeks. we assessed the values of virus load reduction at weeks 4, 8, and 12 weeks to predict the er and hbeag seroconversion. the association between less reduction of viral load at 12 and 24 weeks and nonresponse was also analyzed. results: of 146 etv-treated patients enrolled in etv-901, 98 met criteria for inclusion into 5 year etv treatment analyses. the proportion of patients achieving efficacy endpoints through 5 years of etv therapy is presented the table. results: after 48 weeks of therapy, serum hbv dna levels decreased with a median 4.07±2.47 log10 copies/ml. twenty-three(11.2%) of patients had er. twenty-six (12.7%) patients achieved hbeag seroconversion. hbv dna <4 log 10 copies/ml at week 4 predict both er and hbeag seroconversion. hbv dna> 4log10 copies/ml at 4 weeks but decline to <4log10 copies/ml at 12 weeks or 24 weeks both can predict er and hbeag seroconversion. less than 2 log 10 hbv dna reductions at 24 weeks might predict nr. conclusions: the majority of patients experienced durable serum hbv dna suppression (94%) and alt normalization (80%) after 5 years etv therapy. conclusions: the virologic response within 4 weeks could be useful for prediction of er and hbeag seroconversion of adefovir therapy. failing to evr might not predict nr. objectives: to determine the accuracy of hbcigm in diagnosing ahb and the correlation between hbcigm and liver inflammation (alt), bilirubin & biosynthetic functions (albumin,pt). result: a total of 233 patients were included: in 40 patients, adv was added on lam (add-on therapy), and in 193 patients, lam was switched to adv (switch therapy). during 16.5 months of follow-up, 25 patients developed adv resistance (rta181v and/or rtn236t) and all had undergone switch therapy. the cumulative probability of adv resistance at the 24th month was 17.5%. although add-on therapy induced no adv resistance, it failed to show significant superiority over switch therapy (p=0.220). in multivariable analysis, female (odds ratio [or], 2.75; 95% confidence interval [ci], 1.22-6.17; p=0.014), liver cirrhosis (or, 2.39; 95% ci, 1.07-5.33; p=0.033), and age >45 yr (or, 3.31; 95% ci, 1.14-9.66; p=0.028) were independent risk factors of adv resistance. methods: a retrospective cross-sectional study involving patients with hbcigm positivity between june 2006-december 2007, satisfying the definition for ahb and chbf,and fulfilling the exclusion criteria was performed. hbcigm test were done by using microparticle enzyme immunoassay (meia) and results were expressed as an index value.hbcigm positivity was defined as index value of >1.00 results: 74 patients were positive for hbcigm and 60 fulfilled the criteria(33 ahb, 27 chbf).hbcigm was significantly higher in ahb compared with chbf(median 3.46 vs 1.70;p <000.5).the hbcigm arbitary index value of 2.76 was highly sensitive(97%) and specific(85%) in diagnosing ahb with high accuracy(auroc 0.964;95% ci:0.925-1.003;p<0.0005).among patients in both groups, there was a weak, but significant negative correlation between hbcigm and pt above control(r = -0.309,p =0.016).however, among patients with chbf,the negative correlation between hbcigm and pt above control was moderately strong(r = -0.557,p =0.003).there was also a weak, but significant positive correlation between hbcigm and albumin in with chbf(r = +0.434,p =0.024). conclusion: adv add-on therapy developed no adv resistance during the observation period. therefore, add-on therapy is recommended to lam-resistant chb patients with genotype c who have any risk factors for development of adv resistance: female, liver cirrhosis, and age >45 yr. hbcigm-hepatitis b core igm antibody ahb-acute hepatitis b,chbf-chronic hepatitis b flare alt-alanine transaminase,pt-prothrombin time pe117 detection of emerging drug resistance mutations associated with major approved hbv antivirals using a novel line probe assay (lipa). j. doutreloigne 1 , f. shapiro 1 , r. maertens 1 , e. van assche 1 , e. sablon 1 1 hepatitis diagnostics unit, innogenetics nv, belgium background: in study etv-022, etv demonstrated superior virologic, histologic and biochemical benefit compared to lamivudine (lvd). this study (etv-022/-901) presents efficacy and safety results for patients who received 5 years continuous etv treatment. background/aims: an increasing number of antiviral drugs are being used to treat chronic hepatitis b virus (hbv)-infected patients. however, induced viral escape mutants -some potentially cross-resistant -lead to viral non-responsiveness and treatment failure. effective treatment strategies must therefore take possible drug resistance (dr) into account with respect to monitoring and selection of alternative drugs. we evaluated the use of an updated inno-lipa hbv dr v2+v3 reverse hybridization assay versus sequence analysis to detect resistance mutations. methods: the study evaluates etv-treated nucleoside-naïve hbeag (+) patients who completed etv-022 and enrolled into etv-901 with a treatment gap 35 days. the proportion of patients with hbv dna <300copies/ml, alt normalization, hbeag loss or hbeag seroconversion was evaluated at week 240. background: etv resulted in improved liver histology compared to lvd at 1 year. histologic data for patients on etv for a median of 6 years is evaluated. methods: 273 clinical samples (from untreated hbv patients or treated with different antivirals; hbv genotypes a-h) were tested for mutations with the lipa assay and sequencing. for lipa, samples were extracted with the qiaamp® dna blood mini kit (qiagen), and then tested on the lipa strips. sequencing-derived reference data were subjected to phylogenetic analysis (kodon version 3.10 applied maths, neighbour joining, with kimura-2 parameter). sequential samples from 9 patients were evaluated as well. methods: etv-treated patients completing etv-022 or etv-027 received etv (1.0mg daily) in etv-901. primary endpoints included 2-point decrease in knodell necroinflammatory score, no worsening of knodell fibrosis score and improvement in ishak fibrosis score (ifs) ( 1-point decrease) vs. baseline. secondary endpoints included proportions with hbv dna<300 copies/ml, alt normalization, and ifs normalization in patients with advanced fibrosis/cirrhosis. results: quasi-perfect concordance (> 99.6%) was obtained between the two assays for the samples tested. no indeterminate results were observed. for one sample, lipa provided additional information (wild-type/mutant mix), whereas sequencing showed only wild type. for sequential samples, lipa was clearly able to detect emerging treatment-resistance mutations associated with viral breakthrough. results: etv treatment led to significant histological improvements and improved ifs in 96% (55/57) and 88% (50/57) of patients respectively. of 10 patients with baseline fibrosis/cirrhosis (ifs 4), all demonstrated 1-point improvement in ifs (median change of -3). conclusions: lipa accurately detects the complex quasispecies nature of hbv and can help unravel the dynamics of emerging hbv resistance during treatment with different antiviral drugs. like its predecessor, it is useful for the monitoring and early detection of drug resistance. conclusions: long-term etv therapy in nucleoside-naïve chb patients results in durable virologic suppression, continued histologic improvement and regression of fibrosis/cirrhosis. precore (pc, g1896a) and basal core promoter (bcp, a1762t and g1764a) mutations of hbv are important for predicting the risk of hepatocellular carcinoma (hcc). we developed a new mass spectrometry-based assay using restriction fragment mass polymorphism (rfmp) to detect a1896 and t1762/a1764 mutations, and applied it to analyze their clinical significance in type b liver diseases (n=336), including 35 hccs, 118 liver cirrhosis (lc), 157 chronic hepatitis b (chb), and 25 hbsag-positive with low level viremia (inactive hbsag carrer, ihc). we devided patients into 4 major groups according to the presence of wild (w) or mutant (m) genes in bcp/pc regions; w/w, w/m, m/w and m/m gene types. each proportion was 9.5%, 3.6%, 41.4% and 23.5%, respectively. mixed infection (x) was also found as minority; 4 w/x, 28 m/x, 15 x/w, 4 x/m and 13 x/x. disease distributions (hcc, lc, chb and ihc) in each group were as follows; [w/w (n=32)] 3.1%-6.3%-90.6%-0; [w/m (n=12)] 0-16.7%-58.3%-25%; [m/w (n=139)] 9.4%-42.5%-46%-2.2%; [m/m (n=79)] 11.4%-45.5%-20.1%-13.9%. these results suggest that, in korea where only genotype c has been identified, bcp dual mutation is predominant (>73.2%), while bcp wild alone is only 13.1%. especially, a1896 mutation alone without bcp mutation (w/m type) is uncommon, while bcp mutation alone without a1896 mutation (m/w type) is most common. it might be suggested that prognosis of wild type in bcp and pc region (w/w type) is much better than that of m/w or m/m types. background/aims: entecavir is a potent inhibitor of hbv dna polymerase, which has been shown to be safe and effective for the treatment of chronic hepatitis b (chb) patients. the aim of this study was to evaluate the virologic, biochemical, and serologic responses of entecavir through 1 year in chb patients. methods: from 2007 may to 2008 october, we reviewed 82 patients (mean age 46±12 years, male:female=57:25) who were diagnosed as chb patients (hbeag (+) 64). forty-seven patients (57.3%) had been treated with 0.5 mg of entecavir and 35 (42.7%) with 1 mg of entecavir, respectively. mean follow-up period was 25±17 weeks. hbv dna was quantified by bdna assay with a lower limit of detection of 141,500 copies/ml. results: median hbv dna levels before therapy was 7.64 log10 copies/ml and the median decreases from baseline in hbv dna were -4.19, -4.31, -5.09, -5.31, and -5.59 log10 copies/ml at 4 (n=39, p<0.001), 12 (n=62, p<0.001), 24 (n=42, p<0.001), 36 (n=22, p<0.001), and 52 (n=15, p =0.053) weeks of follow-up, respectively. at baseline, overall median alt was 106 iu/l and the proportions of patients with normal alt were 13%, 48%, 56%, 71%, 67%, and 69% at baseline (n=82), 4 (n=42), 12 (n=63), 24 (n=44), 36 (n=24), and 52 weeks (n=16) after entecavir therapy, respectively. thirteen cases (15.9%) of hbeag seroconversion were noted. background: hepatitis b virus (hbv) infection is a major risk factor for the progression of liver diseases. because its clinical course varies, it is difficult to detect the predictive factor for the prognosis of patients with hbv infection. the aim of the present study was to determine the risk factors for the occurrence of hcc. methods: a total of 620 patients who tested positive for hepatitis b surface antigen and were referred to chiba university hospital between february 1985 and march 2008 were included in the study, and their following characteristics were analyzed: age, gender, the status of hbeag, alt, hbv-dna level, and plt. result: hcc was detected in 30 cases during the follow-up period (5.4 ± 5.1 years). multivariate analysis revealed that age [compared with young patients: odds ratio (or) = 1.07, 95% confidence interval (ci) = 1.03-1.11] and plt level (compared with patients with low plt level: or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. in patients with age more than 35 years, the hbv-dna level (compared with <5.0 log copies/ml: or = 3.29, 95% ci = 1.40-11.5) and plt level (or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. conclusion: advanced age and low plt level were the risk factors for hcc occurrence in patients with hbv infection irrespective of the plt level at baseline. in patients with age more than 35 years, viral load was also a risk factor for hcc. results: before treated by lps, the total mapk p38 level of pbmcs have no significant difference among the healthy control group, different stage groups with hbv infection, however, after treated with lps, the phosphorylated mapk p38 (ptpy180/182) in healthy control group are significant elevate than hbv infected groups(120.0±52 vs 80.2±84.8, p<0.05). in the two groups which hbsag, hbv dna are positive, alanine aminotransferase elevate than normal and hbsag positive, but hbv dna lower under the detect limited level, after treated by lps the ptpy180/182 although lower than healthy control yet, but significant elevated than themselves before treated by lps(86.6±38.6 vs117.8±21.3, 63.3±24.7 vs 93.1±21.8; p<0.05).otherwise, in the group of both hbsag and hbv dna are positive, but alt is normal, before and after treated by lps, the level of ptpy180/182 have no significant difference. conclusion: mapk p38 is a important signal transduction pathway which involving in inflammation and immune response, especially, mapk p38 activated up-regulate the ifn-gamma mrna. according to the result shown, we propose a hypothesis, hbv infection and virus active replication inhibit the mapk p38 activated, consequent on host immunotolerance and hbv persistence, thus, mapk p38 may be as a potential therapeutic target to break immnotolerance and establish host anti-viral states. van der helm 10 1 siriraj hospital, bangkok, thailand, 2 ramathibodi hospital, bangkok, thailand, 3 phyathai hospital, bangkok, thailand, 4 singapore general hospital, singapore, 5 national university hospital, singapore, 6 changi general hospital, singapore, 7 cheil general hospital, korea, 8 hwasun jeonnam university hospital, korea, 9 st mary hospital, korea, 10 roche diagnostics ltd, rotkreuz, switzerland background/aims: hepatitis b virus (hbv) surface antigen (hbsag) is one of the most important markers for diagnosis of acute and hbv infection. high sensitivity of hbsag assays can reduce the diagnostic window during course of disease. in addition, the presence of hbv mutants may be affected by the performance of the hbsag kit. therefore, the technical performance of the elecsys ® hbsag ii assay was explored, using samples (including recombinant mutants), at multiple sites in three countries. methods: nine hbsag screening centers in thailand, korea and singapore compared the sensitivity of elecsys ® hbsag ii assay with that of their routine testing procedure -abbott architect ® (7 centers), abbott axsym ® (1 center) and bayer advia ® centaur hbsag assays (1 center) using preselected seroconversion panels (n=5), recombinant hbv mutant panels (n=13) and routine clinical practice samples (n=1,863). results: the sensitivity of elecsys ® in seroconversion samples was equivalent to the architect ® assay, but more sensitive than the axsym ® and advia ® centaur assays (26 vs 23 and 24 vs 18 positive bleeds, respectively). there was concordance between the elecsys ® and architect assay results with respect to potentially cross-reactive samples (99.74%). the elecsys ® and architect ® assays detected all recombinant mutant samples, whilst axsym ® and advia ® centaur failed to detect three and nine samples, respectively. conclusion: elecsys ® hbsag ii assay was not only highly sensitive and specific when compared with established hbsag screening assays, but also reliably detected hbsag mutants. therefore, this attractive assay is suitable for hbv diagnosis and assessing safety of blood products. background: recent studies have shown a higher rate of adefovir-resistant mutation in lamivudine-resistant chronic hepatitis b (chb) patients treated with switch-to therapy than those treated with add-on therapy. we compared the clinical efficacy of adefovir monothrapy and lamivudine-adefovir combination therapy in lamivudine-resistant chb. methods: a prospective cohort study was performed in 49 patients with lamivudine-adefovir combination therapy and 77 patients with adefovir monotherapy for lamivudine-resistant chb over 18 months. result: biochemical response was achieved in 41 patients (83.7%) treated with combination therapy and in 77 patients (92.8%) treated with monotherapy (p=0.101). virologic response was observed in 19 patients (38.8%) in combination therapy and in 26 patients (31.3%) in monotherapy (p=0.383) and treatment periods for virologic response was significantly shorter in patients with combination therapy than in monotherapy (8.9±4.8 months vs. 13.9±5.0 month, p=0.001). cumulative rate of virologic response was significant higher in patients with combination therapy than monotherapy (p=0.033). hbeag loss was found in 6 patients (16.2%) in combination therapy and 17 patients (20.5%) in monotherapy (p=0.485). biochemical breakthrough was found in 17 patients (20.5%) with monotherapy significantly more frequent than 3 patients (6.1%) with combination therapy (p=0.026). genotypic resistance to adefovir was developed in 1 patient (2.0%) in combination therapy and 9 patients (10.8%) in monotherapy conclusion: to achieve a complete virological response and reduce the risk of adefovir-resistant mutants in lamivudine-resistant chb patients, adefovir in combination with lamivudine is preferable. background/aims: adefovir dipivoxil (adv) effectively inhibits both wild-type and lamivudine (lam)-resistat chonic hepatitis b virus (chb) replication. the aims of this study were to determine the factorts associated with antiviral effect of adv in lam-resistant chb. methods: one hundred-eighteen lam-resistant chb patient (74.6% hbeag-positive) were treated with adv plus lam (n=96) or adv monotherapy (n=22) for a mean of 33.0 months. restriction-fragment mass polymorphism analysis was used for detection ymdd and adv mutants. results: fifty-eight patients (49.2%) achieved complete response(cr) defined as hbv-dna levels <2000 copies/ml and alt normalization. twenty-eight patients (23.7%) achieved initial vilologic response(ivr) defined as hbv-dna levels <10 4 copies/ml within the first 6 month of treatment. 47 (53.4%) of 88 hbeag-positive patients exhibited hbeag loss and 31% seroconverted to anti-hbe ab. five (4.23%) patients developed adv-related mutations. factors associated with ivr were pretreatment level of alt (p=0.00), ast (p=0.00), pretreatment hbv dna level (p=0.03), hbeag negativity (p=0.01) and hbeab positivity (p=0.00). factors associated with cr were ivr (p=0.00), hbeab positivity (p=0.01), pretreatment level of alt (p=0.01), ast (p=0.02) and y-glutamyl transferase (p=0.04). age, sex, presence of liver cirrhosis, pretreatment hbv dna level and the type of ymdd mutants were not related to an cr during adv treatment. conclusions: adv therapy achieved cr in more than 49% of lam-resistant chb. factors associated with cr were ivr, hbeab positive status, high base line alt, ast, ggt levels. q.j. sheng 1 , y. ding 1 , x.g. dou 1 1 department of infectious disease, shengjing hospital affiliated to china medical university, shenyang, 110004, china objectives: to study a kinetics of hepatitis b virus during 12-week and 24-week of treatment with enticavir (ent); to compare the detecting results of hbvdna levels from different detection reagents. methods: thirty-seven cases of chronic hbv infections were selected randomly, treated with daily dose of ent 0.5-1.0mg (0.5mg for nucleoside-naïve patients, 1.0mg for lamivuding-refractory patients). evaluation indexes: serum hbvdna, hbv serological markers, and liver function tests. hbvdna levels were measured by pcr assay, using both domestic reagents and roche cobas amplicor,. the lower limits of measure level of hbvdna were 1000 copies/ml and 300 copies/ml, respectively. results: mean baseline of hbvdna was 5.24log10copies/ml for detection using domestic reagents and 5.70log10copies/ml for that using roche cobas amplicor, (p>0.05). the ratios of cases with undetectable (<1000 copies/ml) hbvdna at week-12 and week-24 were 46.2% and 72.9%, respectively. the ratios of cases with undetectable (<300 copies/ml) hbvdna at week-24 were 54.5%. among the cases whose hbvdna were lower than 1000 copies/ml(using domestic reagents), the ratio of hbvdna lower than 300 copies/ml(using roche cobas amplicor) was 94.7%. conclusions: ent can suppress hbv dna rapidly no matter the patients with alt elevation or not. there is a concordance on hbvdna levels detection between domestic reagents and roche cobas amplicor. background the study on the effect of nucleoside analogue therapy on the quantity of hepatocellular cccdna and tdna and sera hbv dna, hbsag to probe reliable marks for evaluation of therapy endpoint. methods the quantity of hepatocellular cccdna and tdna and sera hbvdna were assayed by fq-pcr, and sera hbsag by elisa in 4 chb patients over 2 years nucleoside analogue therapy satisfied the china criteria of therapy endpoint (therapy group)and 12 chb patients without antiviral therapy and sera hbvdna<10 3 copies/ml(control group). results: the quantity of hepatocellular cccdna, tdna and sera hbvdna, hbsag in therapy group were lower than that of control group,but low level hepatocellular cccdna in therapy group could be detected. conclusion: long term nucleoside analogue therapy may consume hepatocellular cccdna with decreasing of hepatocellular tdna and sera hbvdna, hbsag; although the patients have satisfied the china criteria of therapy endpoint, low level of hepatocellular hbvcccdna were detected, cessation of therapy may cause relapse. peptides that lead nuclear entry of nucleocapsid of hepatitis b virus in hepg2.2.15 cells x.b. pan 1,2 , l. wei 1,2 , j.c. han 1,2 , k. deng 1,2 1 peking university hepatology institute, 2 peking university people's hospital background: the nuclear entry of nucleocapsid is a key step for the hbv life cycle and the formation of covalently closed circled dna (cccdna). it has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (nls). whereas hbcag was primarily distributed in cytoplasm and no marked cccdna was detected in hepg2.2.15 cells. methods: we designed peptides containing a cell-penetrating sequence (rrrrrrr) and a nucleocapsid binding sequence (gsllgrmkga) with/without a classic nuclear localization sequence (pkkkrkv) and these sequences were linked by a soft linker acp. hepg2.2.15 cells were treated with the peptides at levels of 0 m, 25 m and 50 m for 4 days. results: compared with that of control cells, the results showed hbv dna levels in culture medium decreased at least 2 log10 both in 50 m of peptide rrrrrrracpgsllgrmkga treatment group and rrrrrrracpgsllgrmkgaacppkkkrkv treatment group; whereas hbsag and hbeag increased at 1.34+0.21 folds and 2.15+0.37 folds respectively. the signal strength of cytoplasmic hbcag increased at about 2.5-fold in both groups. in rrrrrrracpgsllgrmkgaacppkkkrkv treatment group, nuclear hbcag increased about 3.2-fold and obvious cccdna signal was detected by southern blot. conclusion: our results implied that the nls of core protein likely does not expose to surface of nucleocapsid in hepg2.2.15 cells, the artificial peptide containning nls binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccdna. our study presents a tool for study on cccdna formation and nuclear entry of nucleocapsid. b. tang 1 , j. xia 1 , y.m. wang 2 , h.f. wang 1 1 liver failure treatment and research center, 302rd military hospital, 2 the dept. aims: to establish a reliable real-time fluorescence quantitative (rfq) pcr method to quantify hbv cccdna, basing on lightcycler system and taqman probe. methods: hbv genotypes a-g were aligned to obtain a conserved sequence, crossing rcdna gap, which was used to design cccdna primers and taqman-mgb probe. also another pair of primers for quantify hbv total dna (tdna) was designed, utilizing the same probe. to increasing specificity, we added plasmid-safe atp-dependent dnase (psad) digestion step just before cccdna pcr amplification. a standard curve from 9 standard plasmid samples, from 2.0×10 9 to 2.0×10 1 copies, was created to examine our system. 50 hbv cccdna samples with known-amount were quantified by creating standard curve from 5 standard samples. results: the standard curve had clear log-phase and excellent parallelism, which means nice and equal amplification efficiency in all reaction capillarys. the slope (regression coefficient) of standard curve was -3.141, mean square error was 0.0990 and regression coefficient was -1.0. all of these key indexes measured up. in 50 tests, we got right results if the starting templates of cccdna copies were between 10 2~1 0 9 copies. the range was superior to commercial hbv kits. by quantifying 16 samples containing different amounts of cccdna and rcdna, digested or undigested, psad digestion would eliminate 10 7 rcdna molecules, leaving 10 2 cccdna molecules untouched. the test specificity was maintained up to 1:100,000 ratio of cccdna:rcdna. conclusions: the rfq-pcr based on lightcycler system for hbv cccdna quantification is reliable, sensitive, with high specificity and low cost. background: to study the changes of toll-like receptor (tlr)3 on dendritic cells derived from peripheral blood mononuclear cells(modc) and its role in the pathogenesis of chronic hepatitis b(chb) and chronic severe hepatitis b(cshb). methods: the expressions of tlr3 on 10000 modc were stimulated by poly i:c, and then were determined by flow cytometry in 20 healthy controls, 28 patients with chb and 30 patients with cshb.the level of interferon ifn-was determined by elisa. the differences of expression of tlr3 on modc and serum ifn-among the three groups of study subjects were determined by student-t test.the correlation between tlr3 and ifn-were determined by linear correalation test. results: the values of mean fluorescence intensity(mfi) of tlr3 on modc of the healthy controls, patients with chb and cshb were 1593.00±349.65,1369.56±287.08,and 1203.96±192.40.the serum ifn-(pg/l) of respective groups was 172.66±37.96,107.98±31.15 and 72.06±29.58. there was a gradual decrease of these values from the group of healthy controls to the group of patients with chb and cshb .significant positive correlations between tlr3 and serum ifn-were found. conclusion: tlr3 may have a role in the pathogenesis of chb and cshb. background/aim: to evaluate the efficacy and safety of entecavir treatment in patients with hbeag-positive chronic hepatitis b who had not previously received a nucleoside analogue. methods: fifty-five patients received 48-week entecavir 0.5mg/d therapy. serum hbv dna load was measured with quantitative real-time-pcr. alanine aminotransferase (alt) activity, hbeag, anti-hbe-antibodies, hbv dna level in serum were evaluated at baseline, week 2, 12, 24 and 48 during therapy. evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. results: hbv dna levels declined sharply by around 3 log10 copies/ml during the first two weeks, with a highly significant reduction (p<0.0001) at week 2 and thereafter, as compared to those at baseline; 31%, 51% and 78% of the patients had undetectable serum hbv dna levels at week 12, 24 and 48 respectively. highly significantly decreasing serum alt (p<0.0001) occurred during the first 2 weeks of the study. at week 48, alt levels were normalized in 84% of the patients. hbeag seroconversion (hbeag negative, hbeab positive) was achieved in 7.3% and 14.5% of patients by 24 and 48week. at the end of 24 th and 48 th weeks, complete response (alt normalization and hbv dna and hbeag loss) was observed in 11% and 15%, respectively. there was no evidence of drug resistance or adverse effect in chb patients treated for up to 48 weeks. conclusion: entecavir treatment through 48 weeks was well tolerated and resulted in continued benefit for patients with hbeag-positive chronic hepatitis b. aim: to assess the associations of single nucleotide polymorphisms of the mxa gene promoter and sustained treatment response of chronic hepatitis b or c patients with interferon treatment by meta-analysis of individual dataset from all studies published till date. methods: to clarify the impact of mxa gene promoter polymorphisms on sustained treatment response of chronic hepatitis b or c patients with interferon treatment, we performed a meta-analysis of the published data from eight studies comparing the frequencies of mxa gene promoter polymorphisms at nt -88 g/g, -88 g/t, -88 t/t and nt -123 c/c, -123 c/a , -123 a/a alleles in individuals with interferon treatment. as we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. results: the sustained treatment response rate was higher in patients with the nt -88 g/t and nt -123 c/aalleles in the mxa promoter snp . the meta-analyses yielded summary estimatesodds ratio (or) were 2.07 [95%ci (1.58, 2.7), p <0.00001] and 1.9 [95%ci (1.32, 2.73), p =0.0006] of the nt -88 g/t and nt -123 c/a alleles, respectively. conclusion: mxa gene promoter polymorphisms at nt -88 g/t and nt -123 c/a may be useful as a marker to predict the sustained treatment response of chronic hepatitis b or c patients with interferon treatment, and further investigation regarding their real significance is warranted in a large series of patients. background: to determine whether hbv with the same characteristics causes dissimilar mutations in different hosts. methods: full-length hbv genome was amplified and linked with pmd t18 vector. positive clones were selected by double-restriction endonuclease digestion (ecori and hindiii) and pcr. twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (s)]. bioeditor, clustal x and mega software were used to perform phylogenetic and mutational analysis. potential immune epitopes were determined by the stabilized matrix method (smm), smm-align method and emini surface accessibility prediction. result: all of the 27 sequences were genotype c, the inner-divergence for the mother and son was 0%-0.8%.13 specific nucleotides differed from the other pubished genotype c isolates were co-exist in the mother and her son. aa 1-11 deletion in pres1 was the dominant mutation in the mother (14/18). the 1762t/1764a double mutation existed in all clones of the mother, 3 of them were also coupled with g1896a mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential hla class i epitopes and one b cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the p gene. conclusion: the son was infected hbv from his mother, and discrepant mutation occurred in the mother and her son during infection. background/aims: nucleos(t)ide analogues have been recognized as an effective treatment for chronic hepatitis b. this randomized, double-blind trial compared the efficacy and safety of telbivudine and lamivudine after 104-week therapy in patients with compensated chronic hepatitis b in taiwan. methods: we analyzed 114 taiwanese patients from globe trial receiving telbivudine 600mg (n=58) or lamivudine 100mg (n=56) once daily for 104 weeks. the primary efficacy endpoint was therapeutic response with serum hbv dna <5 log10 copies/ml and either hepatitis b e antigen (hbeag) loss or alanine aminotransferase (alt) normalization. results: the therapeutic response at week 104 was 74.8% in telbivudine group versus 50% in lamivudine group (p=0.005). more patients with telbivudine achieved nondetectable serum hbv dna (<300 copies/ml) (p=0.024) and alt normalization (p=0.021) at the end of treatment. the cumulative resistant rate was significantly lower in those with telbivudine treatment (p=0.0032). the rate of hbeag seroconversion was comparable in both groups (p=0.407). although a lower percentage of patients in lamivudine group (83.9%) reported adverse events than those in telbivudine group (89.7%), the difference was not significant. conclusions: telbivudine demonstrates a significantly greater efficacy and a lower resistant rate than lamivudine in treatment of chronic hepatitis b in taiwan. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. aim: to define the potential role of pd-1/pd-lpathway in different hbv infection status; we examined the expression of pd-1on cd8+ t cells in pbmc of patients with chb and aehb infection. methods: the pd-1 level on cd8+ t lymphocytes and the number of hbv specific cd8+ t lymphocytes in patients and healthy controls were analyzed by flow cytometry. pcr was used to measure the serum hbvdna levels. results: the level of pd-1 expression on cd8+ t cells in chb patients was higher than that in aehb patients and healthy individuals. compared to aehb patients, lower frequency of hbv-specific cd8+ t cells was detected in chb patients. there was an inverse correlation between the strength of hbv-specific cd8+ t-cell response and the level of pd-1 expression. besides, there was a significant positive correlation between hbv viral load and the percentage of pd-1 expression on cd8+ t cells in chb and aehb subjects. however, pd-1 expression was not associated with alt levels. conclusion: our results confirm previous reports that hbv specific cd8+ t-cell response in the peripheral blood is more intense in patients with aehb than in chb with persistent viral infection. moreover, there is a negative correlation between the level of pd-1 and the intensity of virus specific cd8+ t cell response. observed in 10.9% of patients with a mean age of 51.8±12.9. the ethnic composition was 53.1% chinese; 27.3% malay; 14% indigenous sabahans; 2.9% indigenous sarawakians; 1.8% indians and 0.8% others. chinese patients were on average, older (mean 45.6±14.5 years), indians patients had higher mean alanine transaminase and indigenous sarawakian patients had the highest rate of cirrhosis (p<0.0001). during the study period, 20.7% of patients were on treatment and they were significantly older than those who were not on treatment (mean age 44.6 ± 14.5 vs 40.7 ± 14.3). lamivudine was the first agent used in 86.9% of cases. conclusions: in malaysia, chb remains a public health issue and significantly afflicts males in the productive age groups and of chinese ethnicity. the observed differences among ethnic groups could point to different disease severity which needs to be addressed in the local treatment guideline and policy. background/aims: liver stiffness measurement (lsm) has been validated for predicting fibrosis stage in patients with chronic hepatitis c. however, studies on lsm for chronic hepatitis b (chb) are few, and the relationship between histologic findings and liver stiffness needs to be further elucidated. this study was conducted to assess the association of histologic activity on liver stiffness in addition to fibrosis in patients with chb methods: thirty three patients who had taken liver biopsy and lsm at korea university ansan hospital between march 2008 and october 2008 were enrolled. necroinflammatory activity and fibrosis stage were assessed by metavir system. activity, fibrosis, and the sum of both score were included for the correlation analysis with lsm results: among 33 patients, 29 (87.9%) were male, and median values were as follows: age, 42 (20~54); ast, 49 iu/l (19~627); alt, 62 iu/l (8~778); total bilirubin, 1.03 mg/dl (0.4~2.39); lsm, 10.1 kpa (4.2~43.5). fibrosis stages were f1 in 4 (12.1%), f2 in 9 (27.3%), f3 in 12 (36.4%), and f4 in 8 (24.2%) patients. spearman correlation coefficient with lsm were 0.457 (p=0.008) for activity, 0.694 (p<0.001) for fibrosis stage, and 0.724 (p<0.001) for the sum of activity and fibrosis. in linear regression analysis, only the sum of activity and fibrosis remained to be significant. conclusions: not only fibrosis but also activity was an important factor for determining lsm for chb. it would be more appropriate to consider both activity and fibrosis for interpretation of lsm in patients with chb background: hbeag seroconversion is a key goal of chb therapy. hbeag kinetics may predict hbeag seroconversion during treatment. we aim to develop a robust hbeag quantitative method as the value of hbeag quantitation is undefined and data is limited. methods: we evaluated two commercially available qualitative hbeag assays (abbott architect, siemens centaur) for their linear range and validated them against paul-ehrlich institute (pei) standards. hbeag levels were determined from samples of untreated and telbivudine-treated chb patients. results: as a pre-requisite for quantitative use, the linear range for the architect (0.5-43 peiu/ml) and centaur (0.05>5 peiu/ml) assays were defined. architect was selected for further investigation. hbeag levels of 44 untreated patients (mean hbv-dna 9.8 log10 copies/ml, mean alt 166.7 iu/ml) varied from 0.4 to 1073.5 peiu/ml (median 161.7 peiu/ml). in 23 patients (mean hbv-dna 9.9 log10 copies/ml, alt 166 iu/ml) treated with telbivudine for 12 weeks, baseline hbeag levels varied from 1.6 to 664 peiu/ml (median 150.7 peiu/ml). after 12 weeks of telbivudine treatment, median hbeag level was 4.4 peiu/ml, with 76% decline from baseline (median decline 95.1 peiu/ml, range 1.4-657.6 peiu/ml). individual hbeag decline from baseline varied but occurred in all patients and was not correlated to baseline or decline from baseline hbvdna. conclusion: hbeag quantitation is feasible and robust with architect hbeag assay. hbeag decline occurred in all telbivudine-treated patients, and was not correlated to hbvdna. whether the magnitude of hbeag decline is predictive of future hbeag seroconversion merits further investigation. experience from the combined globe (nv-02b-007/cldt600a2302) and 015 (nv-02b-015) study clinical safety database. c. avila 1 , r. laeufle 2 , w.b. bao 3 1 novartis pharma ag, fabrikstrasse 6, basel, switzerland, 2 novartis pharma ag, basel, switzerland, 3 novartis pharma, east hanover, us background: creatine phosphokinase (ck) is a commonly used marker of muscle damage and is elevated by many factors (e.g. exercise, injury, drugs). normal ck levels are affected by muscle mass and elevated levels are described during the natural course of chb. 22% of patients in the globe study had pretreatment grade 1-4 ck elevations. methods: we reviewed data from this combined study clinical safety database, and describe the experience of ck elevation and its relationship to adverse event reports of muscle related symptoms. results: the frequency of new onset of grade 3-4 ck elevations in telbivudine-treated patients (combined database itt population) was 1.8% (15/847), 6.3% (53/838), 3.2% (27/826) and 5.1% (40/786) from weeks 0-24, 24-52, 52-76 and 76-104 respectively. the frequency of grade 3-4 ck elevations for all patients from week 0-104 was 12.6% (107/847). the majority of grade 3-4 ck elevations were asymptomatic, rarely resulted in discontinuation or interruption, spontaneously declined within 1 or 2 visits and were not associated with more frequent muscle-related adverse events. cumulative data from this combined database showed no relationship of the degree of increased ck to acute or persistent muscle disease. conclusion: ck elevations are associated with hbv disease and were also common during the globe and 015 trials and were not predictive of the development of muscle related symptoms. onset of muscle-related symptoms should prompt clinical and treatment review, including concomitant medications. backgrounds: leptin plays a crucial role in the regulation of energy balance and body weight control by activating the long form of the leptin receptor (ob-rl). epidemiologic studies showed that obesity is one of the factors associated with hbv related hepatocellular carcinoma. methods: huh7 cells were transiently transfected with 1.3 copies of hbv-replicon plasmid. after 48h, cells were harvested and total rna of the cells were extracted and reverse-transcribed into cdna. long form and short form leptin receptor (ob-rl, ob-rs) mrna transcription levels were assayed by real-time pcr respectively. and mrna transcription levels and protein expression of ob-rl and ob-rs in hepg2.2.15 cells were also detected. results: after transfected by 1.3 copies hbv-replicon plasmid, the mrna transcription level of ob-rl was inhibited significantly (**p<0.01), but the mrna transcription level of ob-rs did not change, and the ob-rl protein expression was reduced. in hepg2.2.15 cells, the mrna transcription level of ob-rl was also significantly lower than the mrna transcription level of ob-rl in hepg2 cells, while the mrna transcription of ob-rs in hepg2.2.15 and hepg2 cells didn't show significant difference. besides, the protein expression level of ob-rl in hepg2.2.15 was also lower than it in hepg2 cells. conclusion: hbv replication down-regulated the expression of long form leptin receptor in cell cultures, which could in part explain the clinical observation of obesity in association with development of serious sequelae in hbv infections. the results of entevavir treatment in patients with chronic hepatitis b s. kose 1 , g. akkoclu 1 , m. turkeri 1 , a. gozaydin 1 1 the ministery of health tepecik training and research hospital, izmir purpose: we evaluated the short and long term effectiveness of entecavir. patients and methods: those patients had received diagnosis of chronic hepatitis b. their pretreatment transaminases, hbsag, anti-hbs, hbeag, anti-hbe, hbv-dna were checked and a liver biopsy and a resistance test for lamivudine (lam) and adefovir (adv) were performed. a total of 44 patients who were taking entecavir for at least 24 weeks were included in the study. findings: the biochemical and virologic response were observed in 88.8 % at 3 and 6 months and in 75 % at 12 months. in 9 hbeag positive patients who had received therapy previously, the biochemical response was observed in 88.8 % at 3 and 6 months and in 100 % at 12 months. the virologic response in 77.7 % at 3, in 88.8 % at 6, and 100 % at 12 months. posttreatment hbeag seroconversion did not develop. in 9 hbeag negative patients the biochemical and the virologic responses were observed in 88.8 % at 3 months and 100 % 6 and 12 months, respectively. in 17 hbeag negative patients had received therapy previously, the biochemical response was observed in 76.4 % at 3, in 100 % at 6 and 12 months. the virologic response in 94.1 % at 3, in 100 % at 6 and at 12 months. conclusion: in our study, a higher therapy-response rate was achieved, especially in hbeag negative patients. in hbeag positive patients biochemical and virologic response rates were high. background/summary: patients with liver disease are known to have a higher prevalence of glucose intolerance. preliminary studies suggest that viruses can be an additional risk factor for the development of diabetes mellitus. individuals with type ii diabetes have an increased prevalence of cirrhosis, and a proportion of patients with acute and chronic liver disease develop diabetes mellitus. there is now emerging epidemiological data to suggest that hepatitis c virus (hcv) infection may also contribute to the development of diabetes reported to be higher than expected compared with the general population. while these investigations suggest an epidemiological association between hcv infection and diabetes, large controlled studies are required to observe association between hbv infection and diabetes. the present study was designed to study the relative proportion of diabetes mellitus in patients suffering from hepatitis b virus (hbv) infection. background: in easl 2008, we reported significant liver disease among hbeag negative patients with serum hbv-dna level <4log10copies/ml (i.e. 2.0x3log10iu/ml) and alanine aminotransferase (alt) <55iu/l. this reflects fluctuating nature of these levels. the aim of this retrospective study is to demonstrate the frequency of fluctuation among this group of chb patients. methods: clinical records of hbeag negative treatment naïve chb patients with at least one serum hbv-dna <4log10copies/ml were reviewed. results: there were 194 (62.5% male, median age 48.0 years) chb patients with negative hbeag and hbv-dna <4log10 copies/ml (roche cobas amplicor pcr assay, lod<300copies/ml). 32 had serial hbv-dna measurements within 2 years; 7 of them (21.9%) had increase serum hbv-dna level by >1log10 copies/ml; 2 patients had associated serum alt elevation from normal (normal range <55 iu/l), 4 had persistent normal alt, and one had persistently raised alt. 5 (15.6%) had serum hbv-dna level decrease by >1log10 copies/ml. another 20 patients had hbv-dna levels fluctuating within 1log10 copies/ml. 159 hbeag negative patients with single hbv-dna measurement showing <4log10 copies/ml had serial serum alt measurements within 3 years. 35 (22.0%) patients had intermittent / persistently raised alt; while 124 (78.0%) patients had persistently normal alt. conclusions: hbeag negative chb is common among chinese. serial serum hbv-dna and alt measurements are necessary to detect fluctuating levels and progressive liver disease that may require antiviral therapy. background: to determine the best vaccination strategy, a model that reflects the country-specific infection profile is needed. methods: a model was built in order to obtain the age-specific infection frequency q(t) for neonates(n), infants(i), children(c), and adults(a). the infected group can either become hbsag(+) or anti-hbs(+)* based on f(t). q(t) can be found from p(t) = [q(t) x (1 -f(t)) x crs + q(t) x f(t) x (1 -cras)], where p(t) represents the proportion of the late anti-hbs(+)** group and crs/cras denote natural conversion of hbsag/anti-hbs. to test the model, cross-sectional serologic marker data in korea were used. because f(t), crs, and cras were known(f(n)=0.1, f(i)=0.5, f(c)=0.8, f(a)=0.95, crs=0.015, cras=0.02), in order to determine q(t), only p(t) values were needed, which were evaluated from logistic modeling using the glm() function of s-plus. results: the infection frequencies during neonate, infant, children, and adult periods in non-vaccinees were 15.4%, 33.3%, 16.6%, and 34.7%, respectively. each group's likelihood of infection compared to adults was then: neonates 170.2 times more likely, infants 33.5 times, and children 0.9 times, making a strong case for neonatal and infantile vaccination for the studied region. conclusions: the hbv infection model can be used for determining the most cost-effective strategy for hb vaccination in nations where longitudinal data are not available. and where longitudinal data are available, it can be used to determine the appropriate time of transition of vaccination strategy to maintain cost-effectiveness. the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b x.c. pan 1 , f. yang 1 , m. chen 1 1 objective: to investigate the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b. methods: 36 patients with hbeag positive chronic hepatitis b were recruited and receiving telbivudine treatment for 9 months. before and during 3 6 9 months of treatment , flow cytometry was used to detect the proportion of peripheral blood tregs; real-time pcr was used to detect the levels of hbv dna in surum, markers of hepatitis b virus infection were detected by elisa assay and levels of alanine aminotransferase in serum were measured. results: the proportion of peripheral blood tregs in patients with chb was significantly higher than that in healthy controls and decreased over 6 or 9 months of treatment to a level comparable to that of healthy controls. after 3 months of treatment, the rate of alt normalization in patients which the proportion of peripheral blood tregs was unreduced was significantly lower than that in patients which the proportion of peripheral blood tregs was reduced (p<0.01). 3, 6 or 9 months of telbivudine treatment resulted in negative hbeag in 4(11%) patients, 7(19%) patients or 9(25%) patients respectively. within 9 months of treatment, 7 (19%) patients seroconverted from hbeag to anti-hbe , in which the proportion of peripheral blood tregs had decreased to a level comparable to that of healthy controls over 3 or 6 months of treatment. conclusion: during antiviral treatment with subsequent reduction of the viral load or alt levels, the proportion of tregs decreased to a level similar to that of normal healthy controls. in addition, seroconversion from hbeag to anti-hbe was prone to be established in patients which the proportion of tregs decreased quickly at the early phase of antiviral treatment with telbivudine. background: in china a part of patients with alt <1.5×uln and hbv dna >10 5 copies/ml will advance into hepatic cirrhosis even hepatoma. so these patients should not only be monitored but also be treated. this study was made to determine the safety and efficacy of combining therapy of pegylated interferon alpha 2a (peg-ifn -2a) and entecavir in treating naive patients with alt <1.5×uln and hbv dna>10 5 copies/ml. methods: nine patients with hbsag positive over 6 months and alt<1.5×uln hbv dna >10 5 copies/ml were taken as research subjects. before treatment,liver biopsy was used to assess histological damage. patients were treated with peg-ifn -2a 180 g /week for 48 weeks, and in the first 12 weeks entecavir 0.5 mg/day was applied, then it was stopped. results: 1 liver biopsy showed that 7 patients had mild inflammation. 2 after 12 weeks' treatment , hbv dna level in all patients decreased to less than 10 4 copies/ml, and after 24 weeks' treatment(12 weeks after entecavir was stopped)hbv dna in all patients was less than 10 3 copies/ml. 3 normal alt was seen in all patients after 12 weeks' treatment and 24 weeks' treatment. 4 none of the patients had peripheral neuropathy with combining treatment. conclusions: 1. bulk of patients with alt <1.5×uln and hbv dna>10 5 copies/ml had mild inflammation and need treatment. 2 combing treatment of peg-ifn and entecavir was safe and effective to this group. 3 it proved that it was safe for patients to stop treatment with entecavir after short time use. background & aims: quantification of serum hbv dna levels is important to monitor viral replication in chronic hepatitis b (chb) patients. both abbott realtime hbv and roche cobas amplicor hbv monitor are updated fully automatic commercial assays for hbv dna quantification. the aim of this study is to compare the performance of these two assays on the hbv dna quantification in chb patients. methods: serial serum samples from 30 chb patients were collected at the baseline and at days 4, 7, 10 and 14 and weeks 3, 4, 8 and 12 after the commencement of therapy. genotype was determined by sequence alignment. abbott and roche assays were employed for hbv dna extraction and quantification according to the instructions of manufactories. results: hbv dna quantification results of abbott assay was significantly correlated with those of roche assay (r=0. 972, p<0.0001). for genotype c, the difference in hbv dna levels [median (range): 1.22 (-0.16-2.36) log units] measured by these two assays was significantly higher than that for genotype b [0.66 (-0.52-1.63) log units, p<0.0001]. moreover, the difference in serum hbv dna levels after 12 weeks antiviral treatment [1.18 (-0.21-1.76) log units] measured by these two assays was significantly higher than that in baseline serum hbv dna levels [0.68 (-0.12-1.24) log units, p<0.0001]. conclusion: the quantification results of abbott realtime hbv showed a good correlation with those of roche cobas amplicor hbv. but the performances of these two assays have significant difference in the quantifications of serum hbv dna levels in genotype c patients and in patients after 12 weeks antiviral therapy. background: guidelines suggest hepatitis b virus (hbv) vaccination to all hepatitis c virus (hcv) infected patients and healthcare workers. we attempted to find out hbv vaccination status in our hcv infected population, and healthcare workers. methods: prospective survey of 100 consecutive hcv infected patients and also doctors and paramedical staff in our hospital. results: major sources of viral infection in study patients (58 males; average age 40 years -range 18 to 65 yrs) were reused syringes (38 pts). twenty had a household member infected with hcv. twenty were co-infected with hbv. eighty five of hcv infected patients were not vaccinated.against hbv. twenty five of them (29%) had financial reasons and 45 patietns (52%) had lack of awareness. out of 30 doctors, 12 and 15 did not know about their hbv and hcv status respectively, but none was known to have either of these infections. four (13%) were not vaccinated against hbv. out of 29 paramedical staff, 1 was hcv positive, 11 each were unaware of their hbv and hcv status, and remaining were negative for these markers. thirteen of them (44%) were not vaccinated against hbv. conclusion: thirty eight percent hcv infected patients were infected by reuse of syringes. eighty five percent were not vaccinated against hbv, out of which 52% had no awareness about it, whereas 29% could not financially afford it. a significant number of paramedical staff and some doctors were also not vaccinated background: early prediction of efficacy could decrease unnecessary interferon exposure of patients with chronic hepatitis b. methods: a multi-center clinical study. patients were injected interferon alpha 2b 5 million iu subcutaneously every other day for 24 weeks and 24-week follow-up was followed. results: 53 patients (44 male) were enrolled, 27.5 ±9.1 years old. 24 hours after administration, hepatitis b virus (hbv) load decreased significantly (6.96±1.03log copies/ml, p<0.05) from baseline (8.00±0.93 log copies/ml). hbv load was 4.94±1.47 and 5.25±1.58 log copies/ml at week 24 and 48, respectively. at the two points upwards, complete response rate was 4.2%(2/48) and 7.9%(3/38), partial response rate was 35.4%(17/48) and 34.2%(13/38), respectively. at week 4 and 12, hbv dna levels of complete responder and partial responder were lower than those of non-responder (p<0.05) at week 24. at baseline, on hour 12, day 2, 3, week 2, 4 and 12, hbv dna levels of complete responder were lower than those of non-responder at week 48 (p<0.05). multiple linear regression showed that baseline hbv dna was the independent variables to predict the response at week 24 and 48. conclusions: interferon alpha 2b was effective in treating patients with hbeag positive chronic hepatitis b. it could decrease the hbv dna level rapidly. early hbv dna levels were predictive to response at the end of treatment and follow-up. baseline hbv dna level was the independent predictor of the response at the end of treatment and follow-up. aim: to investigate features of pd-1 expression on peripheral tcells and pd-l1 expression in liver in chronic hepatitis b (chb) patients in immune clearance phase. methods: pd-1 expression on total peripheral t cells were evaluated by using flow cytometry. immunostaining was performed according to the envision chemmate methods. the degree of pd-l1 expression was scored and assessed according to the percentage and staining intensity of positive cells. results: compared to health control, the percentage of total peripheral t cells expressed pd-1 was elevated in chb with repeatedly increasing alt level. no specific association between the percentage of pd-1 positive and the mean fluorescence intensity mfi of pd-1 expression on total t cells with serum viral load were found. but alt level was correlated with the mfi of pd-1 expression on total cd8+t cells significantly. pd-l1 is up-regulated on hepatocytes by viral infection, and high expressed in fibrosis section. conclusion: the mfi of pd-1 on cd8+t cells plays important role in regulating the immune-host interaction in chb in immune clearance phase. and pd-1 expression on t cells is correlated with high immune inflammatory refection. aim: to study the quantity, characteristic of hbv-specific t-cell and the extent of liver damage in chronic hepatitis b (chb) patients with different hbeag status. methods: 103 chb patients were enrolled and divided into two groups according to the hbeag status, and the liver damage index were analyzed. the frequency and foxp3 expression of cd4 + cd25 + regulatory t cells (treg) were measured, as well as the frequency and phenotypic molecules expression of hbv-pentamer+ t-cell. hbv specific t-cell responses including cellular proliferation and ifn-production, with or without anti-pd-l1 and/or anti-ctla-4 blocking, were also observed. results: the demographic characters, serum alt, ast levels, the frequency and foxp3 expression of cd4 + cd25 + treg were similar, while the serum hbv dna levels were higher in hbeag+ patients (p <0.05). the liver necroinflammation was comparatively more severe in hbeag-patients (p =0.056), but the median percentage of liver cirrhosis was much higher in hbeag+ patients (p <0.05). the difference of hbv-specific t-cell frequency was not significant between two groups, while the expression levels of pd-1 and ctla-4 on hbv-specific cd8 t cells were significantly higher in hbeag+ patients (p both <0.05). combined using of anti-pd-l1 and anti-ctla-4 mab significantly increased the cellular proliferation in either hbeag+ or hbeag-patients, but only markedly enhanced the ifnproduction in hbeag+ patients. conclusion: hbeag persistency could probably induce higher expression of pd-1 and ctla-4 on the hbv-specific t cells and result in t-cell impairment, high hbv dna load and high percentage of liver cirrhosis in hbeag+ chb patients. hepatocyte apoptosis in patients with chronic hepatitis b y. liu 1 , k. wang 1 1 background: to investigate the relationship between hepatocyte apoptosis and the level of inducible nitric oxide synthase (inos) in hepatic tissue in the patients with chronic hepatitis b chb . methods: we observed 37 cases with chb and 10 normal controls. transferase-mediated-utp-biotin nick-end labling ( tunel) technique was used to detect apoptosis cells and immunohistochemical staining were also performed to investigate the expression of inducible nitric oxide synthase (inos) in biopsy samples .the serum level of alt hbv-dna grading of necroinflammatory activity and staging of fibrosis were also assessed. results: hepatocytes in all chb liver tissues were positively stained by tunel in various degree. in contrast, control tissues did not show dna fragmentation. a significant correlation was seen between apoptosis index (ai) and necroinflammatory grading ((r=0.404, p=0.015) and serum inos level r=0.465, p=0.004 . it did not correlate with fibrosis stage and serum alanine aminotransferase level. conclusion: the oxidative stress.in patients with chb may reflected the apoptosis of hepatocyte. apoptosis involves in liver injury of chb,but with no significant correlation to serum level of alt. objectives: to investigate the genotype-dependent development of lamivudine resistance in hepatitis b virus (hbv). methods: 215 patients with chronic hepatitis b who had been treated with lamviudine for more than 1year, and become lamviudine resistance were analysed for the hbv genotypes and cumulative rate of rt region mutant with standard dna sequencing technology. results: among the 215 patients, 60 patients were infected with hbv genotype b (hbv/b)(27.9%), and 155 with genotype c (hbv/c)(72.1%). in the hbv/b patients, 16/60(26.7%) were of subtype ba, and 44/60(73.3%) were of of subtype bj. the cumulative type and ymdd mutation rates in patients with genotype c were showed as l180m+m204v (67/155,43.2%) > l180m+m204i (52/155,33.5%) > m204i (36/155, 23.3%), while in patients with genotype b as l180m+m204v 36/60(60.0%) > m204i(24/60, 40.0%), none of l180m+m204i. conclusions: our results indicated that in patients with lamivudine resistance, hbv genotype c (hbv/c) were higher than genotype b (hbv/b). in both genotypes the combined mutations (180+204 sites) were found more than the single 204 site, showed some significance for monitoring lamivudine resistance. background & aims: il-35, a novel identified inhibitory cytokine specifically produced by regulatory t cells (tregs), is an ebi3-il-12 heterodimer encoded by epstein-barr-virus-induced gene 3 (ebi3) and interleukin-12 alpha (il12 ). the aim of the study is to determine the expression levels of il-35 in peripheral blood mononuclear cells (pbmcs) of chronic hepatitis b (chb) patients in different phases. methods: a total of 36 treatment naïve chb patients, including 17 in immune-tolerant phase [group 1, alt: 21 (12-51) u/l, serum hbv dna: 2.48 x 10 9 (6.30 x 10 6 -1.20 x 10 10 ) copies/ml] and 19 in immune-clearance phase [group 2, alt: 221 (71-1530) u/l, serum hbv dna: 5.10 x 10 8 (1.62 x 10 6 -1.77 x 10 10 ) copies/ml] were enrolled in the study. the relative mrna expression levels of ebi3, il12 and foxp3 were determined by semi-quantitative pcr. results: the significant correlations were observed between the expression of ebi3 and il12 (r=0.661, p<0.001), ebi3 and foxp3 (r=0.388, p<0.05), il12 and foxp3 (r=0.431, p<0.01). the relative expression levels of ebi3 and il12 in pbmcs were significantly higher in group 2 when compared with those in group 1 (1.46 ± 0.23 vs 0.80 ± 0.10 and 1.26 ± 0.19 vs 0.65 ± 0.09, p<0.05, respectively). furthermore, the relative expression levels of ebi3 and il12 in group 2 were significantly correlated with alt levels (r=0.473, r=0.474, p< 0.05, respectively), but not with serum hbv dna levels. conclusions: the expression levels of il-35 in pbmcs were significantly higher in chb patients in immune-clearance phase than that in immune-tolerant phase. increased il-35 expression levels were associated with liver injury. background: there are a number of oral antivirals approved for chronic hepatitis b. lamivudine, the first oral nucleoside analog, is associated with increased rates of drug resistance with prolonged use--from 20% at one year to 50% at three years. therefore, an alternative or add-on treatment is necessary. adefovir, an oral nucleotide analog, is used either in combination with lamuvudine or as monotherapy in lamivudine-resistant chronic hepatitis b. we did a meta-analysis to compare the efficacy of adefovir in combination with lamivudine versus adefovir alone in the treatment of lamivudine-resistant chronic hepatitis b infection. methods: a comprehensive literature search was performed using the following databases: medline, cochrane, and embase. a total of 3 randomized controlled trials were retrieved and analyzed. outcomes measured were virologic response, biochemical response and resistance rates. results: meta-analysis on virologic response showed that combination treatment with adefovir and lamivudine is as effective as adefovir monotherapy (or 1.04, 95% ci 0.50-2.15, p=0.92). likewise, in terms of biochemical response, both regimens were equally effective (or 1.06, 95% ci 0.47-2.40, p=0.90). one study showed statistically significant increase in adefovir resistance rate in the monotherapy arm compared to combination arm (p= 0.0182) after the first year of therapy. conclusion: in patients with lamivudine-resistant chronic hepatitis b infection and compensated liver disease, adding adefovir to lamivudine is as effective as switching to adefovir alone in terms of virologic and biochemical response. r. safadi 1 , q. xie 2 , y.g. chen 3 , y.k. yin 4 , l. wei 5 , s.g. hwang 6 south korea, 7 bnai zion medical center, haifa, israel, 8 beijing friendship hospital, beijing, china, 9 novartis pharma ag, basel , switzerland background: ldt produces greater viral suppression than lam. we investigated whether patients receiving lam can benefit from switching to ldt. methods: hbeag positive and negative persistently viraemic patients (median hbv dna 5.0 (ldt), 5.3 (lam) log 10 copies/ml) and lam treated for 3-12 months, were randomized to either switch to ldt or continue lam. we report the benefit of ldt switch assessed by primary treatment failure (tf, <1 log hbv dna decline) and viral breakthrough (vb, >1 log above nadir). results: 17% (21/122) of the ldt switch and 15% (18/124) continuing lam patients had pre-existing m204 mutations at screening. tf was 5% (ldt) versus 21% (lam, p<0.05). in patients with >24 weeks prior lam treatment, tf was 10% (ldt) versus 41% (lam). 83% ldt tf (5/6) was associated with resistance at screening versus 56% lam tf. in ldt switch with <24 weeks prior lam, no ldt tf occurred versus 12% lam. in hbeag positive, tf occurred in 6% (ldt) versus 29% (lam). among hbeag positive with >24 weeks prior lam treatemtn, vb was 19% (ldt) versus 44% (lam, p<0.05). differences were not significant for hbeag positive with >24 weeks lam or for hbeag negative regardless of duration of prior lam treatment. conclusions: early switch to ldt is associated with better virological outcomes in these patients. persistent viraemia for >6 months on lam treatment is associated with a high risk of tf and vb. for these patients, genotypic analysis is recommended prior to screening. objective: the aim of this study is to evaluate the proper endpoint in the treatment of chronic hepatitis b with antivirals by investigating the viral rebound ratio after one year's nucleosides or (three months) sustained treatment with lamivudine, adefovir, entecavir, or interferon when viral response and seroconversion response have been finished . methods: eag positive chronic hepatitis b naïve patients with alanine aminotransferase (alt) more than 2 uln were assigned to receive 100 mg of lamivudine, 10mg of adeforvir, or 0.5mg of entecavir once daily, respectively. patients in the interferon group were administrated with 5,000,000 iu of 2a interferon on every other day, and the therapeutic duration lasted for another three months after eag-ab seroconversion appeared. hbv dna and eag-ab in the serum were tested during the off-treatment period of 12 months. results: thirty four patients in lamivudine group of 148 cases got eag-ab seroconversion after treatment with 20±5 months of average duration, and the viral rebound ratios in the off -treatment 6 an 12 months follow up period were 20.6 7/34 and 40.1 15/34 , respectively. in adeforvir group were 19 4/21 and 33. 7/21 . in enticavir group were 20 3/15 and 46.7 7/15 . in interferon group was 16.2 6/37 in the off-treatment 6 months follow up period. conclusions: we conclude that eag-ab seroconversion in the treatment of eag positive chronic hepatitis b patients is the goal but not an endpoint of therapy physicians should aim at. to gain everlasting effect, longer duration of treatment may be needed. background: universal hepatitis b(hb) vaccination of hbsag negative people (especially infants) is widely recommended and practised. objective: to assess whether there is robust evidence of protective efficacy to back such practice. methods: this cochrane review included randomised trials identified from six databases through detailed electronic searches. trials comparing hb vaccine versus placebo/another vaccine, in hbsag negative persons were included without any restrictions. the primary outcome was hb infection (developing hbsag or anti-hbc). robustness of evidence was assessed through comparison of available-case analysis versus intention-to-treat(itt) analysis using four different models: (i)assuming unfavourable event for all missing data, (ii)assuming favourable outcome for all missing data, (iii)best-case-scenario and (iv)worst-case-scenario results: twelve trials were eligible among 2964 citations; all were methodologically poor (high risk of bias). data from four trials could be included in meta-analysis. efficacy of vaccination varied with the type of data analysis. available-case analysis suggested efficacy in reducing risk of developing hbsag (rr=0.17;95%ci=0.09-0.31;n=1341) and anti-hbc (rr=0.42;95%ci=0.31-0.57;n=1235). itt analysis results varied depending on the model chosen (table) , but liberal approaches suggested high efficacy, whereas conservative approaches did not. the available evidence on efficacy of hb vaccination in hbsag negative people is not robust; there are serious limitations in quality and quantity. background: open-label rollover study (etv-060) assessed histologic improvement in chb patients on at least 3 years etv therapy. methods: 100% nucleoside-naïve patients and 98% lamivudine (lvd)-refractory patients from etv-053 and etv-052 studies, respectively, entered etv-060 study and received etv at 0.5/1mg for greater than 96 weeks. improvement in knodell necroinflammatory (ni) score and knodell fibrosis score at weeks 48 and 148 were studied. results: at week 148, 95% of nucleoside-na ve patients and 56% of lvd-refractory patients achieved hbv dna <400copies/ml. furthermore, 95% of nucleoside-na ve patients and 93% of lvd-refractory patients had normalized alt levels. mean platelet counts in both naïve and lvd-refractory patients were improved at weeks 48 and 148 compared with baseline. conclusions: naïve and lvd-refractory chb patients showed significant improvement in liver histology after 3 year etv therapy, and improved dna and serum alt levels. results: 84.7 percent of patients were younger than 30 years old, 15.3 percent were older than 30 in this study. 48.0% patients' mothers were hbsag positive. high levels of serum hbv dna were founded in all patients, >10 7 copies/ml were 78.6%. only 5 cases (5.1%) whose liver inflammation grade were g0, the rest patients were mild inflammation, in which g1 were 64 cases (65.3%), g2 were 29 (29.6%); there were 56 patients (57.1%) had no signifecant liver fibrosis, the rest 42 cases (42.9%) had different fibrosis, among those s1 were 23 cases (23.5% , s2 were 14 14.3% , s3 were 5 5.1% , none of patients had cirrhosis. the fibrosis stages of higher alt level were markedly severer than lower alt in patients with normal alt p < 0.01 . conclusions: most of patients with chronic hepatitis b virus in immune tolerant phase present mild inflammation in liver, part of them have already appeared fibrosis, so some patients determinated by clinics are actually not in immune tolerant phase. although alt testing are in the normal range, but the possibility of liver fibrosis is increased in patients with relative higher alt level, so liver pathology should be recommended to judge illness correctly. background/aims: hepatitis b virus infection (hbv) is a global health problem. in bangladesh, 5-7% of people are hbsag positive. this study was carried out to evaluate the efficacy and safety of peginterferon alfa-2a in chronic hepatitis b patients. methods: a total of 60 patients with chronic hepatitis b, 32 (53.3%) were hbeag positive (group a) while 28(46.7%) were hbeag negative (group b) were included in this study after meeting the following criteria: age 18 to 60 years, hbsag positive for more than 6 months, serum hbv-dna was >5 log(10) copies/ml and alt more than two times the upper normal limit. they were given peginterferon alfa-2a (180 microgram once weekly) for 24 weeks and followed for an additional 24 weeks. results: after 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hbvdna levels below 20,000 copies per milliliter was significantly higher in hbeag positive patients (59 percent and 43 percent, respectively) than among hbeag negative patients (45 percent and 33 percent). loss of hepatitis b surface antigen occurred in 3 patients in group a, as compared with 1 patients in the group b (p<0.01). adverse events including pyrexia, fatigue, myalgia, headache and haematologic abnormalities were similar in both groups. conclusions: patients with hbeag positive chronic hepatitis b had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a. background: the effect of hepatitis b vaccination on individuals with isolated anti-hbc in endemic areas is not clear. we investigated the prevalence of individuals positive for anti-hbc only and their antibody response after hepatitis b vaccination in a single healthcare center. methods: the study included 1,812 healthcare workers. after screening for hbsag and anti-hbs, the individuals negative for both hbsag and anti-hbs were examined for anti-hbc and were vaccinated with a recombinant hepatitis b vaccine at 0, 1, and 6 months. the serum anti-hbs level was measured after the vaccination. results: of the subjects, 334 (220 females) were negative for both hbsag and anti-hbs. forty (2.2%) subjects had isolated anti-hbc, including more males (60.0% vs. 30.6%) and older people (45.7±8.2 vs. 37.3±8.5 years), compared with individuals negative for all of the viral markers. the anti-hbs seroconversion rate and anamnestic response in the individuals with isolated anti-hbc after the first vaccine injection were 60% and 27.5%, respectively. in the 294 persons who were negative for all hepatitis b viral markers, the seroconversion rate after the first vaccination was 52.6%. the anti-hbs seroconversion rate did not differ between the isolated anti-hbc positive individuals and those negative for all hepatitis b markers (89.5% vs. 96.6%) after the full course of vaccination. conclusions: serum hbsag and anti-hbs tests are sufficient for screening before hepatitis b vaccination, especially in healthcare workers. objective: to understand the quantity and distribution of cd83 + mature dendritic cells in patients with hepatitis b virus in immune tolerant phase. methods: there were 30 immune tolerant phase patients with hepatitis b virus infection (fibrosis stages were s0), 10 immune clerance phase patients, 10 non-active status patients and 5 healthy controls involved in our research. the quantity and distribution of cd83 + mature dendritic cells in liver were determined by immunohistochemical staining. result: the liver inflammation grades were between g1-g2 in patients who in inmmune tolerant phase and non-active status, moreover, patients in immune clerance phase were between g2-g4. there were a small amount of cd83 + dendritic cells in healthy liver tissue, scattered in portal areas and hepatic lobules. the quantity and distribution of cd83 + dendritic cells in patients who in inmmune tolerant phase and non-active status were similar to the healthy, and the quantity were no difference among them p 0.05 .the number of cd83 + cells in patients of immune clerance phase was significant increased compared with other groups, there were differences among them p 0.05 , the cd83 + cells mainly distributed in portal areas infiltrated with inflammatory cells and hepatic lobules with inflammatory necrosis. conclusion: cd83 + mature dendritic cells are involved in liver immune response in patients of inmmune clerance phase, is likely to related to hepatitis b virus clearance. lack sufficient mature dendritic cells may be one of the mechanisms of immune tolerance. background: local hospitals provide obstetric services including antenatal care to women normally living in the mainland china, whose prevalence of hepatitis b carrier is unknown. objectives: compare prevalence of hbv carrier of pregnant women from the mainland china with local counterparts and discuss the implications of results. materials and methods: antenatal serological results were retrieved from corporate laboratory information system databases. pregnant women from the mainland china were identified by a specific set of temporary-allocated identity number during january 2007 -october 2008. results: 7491 pregnant local residents and 1397 pregnant women from the mainland china underwent antenatal serological tests for hepatitis b surface antigen. positive hepatitis b surface antigen results were more frequent in pregnant women from the mainland china (12.7%) than in local pregnant women (8.17%) (p<0.001). discussion: because infected pregnant women can transmit the hepatitis b virus to the infant at delivery, specific management could entail maternal medication, injection of hepatitis b immune globulin to the infant at birth and immunization later on. however, early repatriation to the mainland china, which is common, will make completion of immunization program difficult. these babies will be at a higher risk to be infected by hbv, particularly when breast-fed by hbv carriers. their return to hong kong later will dilute the effects of local immunization program. the volume of work derived from the provision of obstetric services to women from the mainland chinese is larger with regard to medication, counseling and immunization for babies born to hbv carriers. immunosuppressive or anticancer therapy k. hirano 1 , t. kodani 1 , s. sato 1 , y. narita 1 , t. kikuchi 1 , t. genda 1 , k. iijima 1 , k. ogawa 1 , t. ichida 1 1 background/aim: we compared the prevention of hbv reactivation in (hbsag)-positive patients with hbsag-negative patients who were positive for antibody to (anti-hbc) and/or (anti-hbs) undergoing immunosuppressive, anticancer or molecular target therapy. methods: from sep 2004 to nov 2008 hbsag-positive patients and 22 anti-hbc and/or anti-hbs-positive patients were enrolled in this study. we compared with 2 groups about background disease, age, blood examination, and nucleoside analogues. results: in hbsag-positive patients mean age were 56.6±10.2 years old, median ast levels were 30 (18-706) iu/l, and median alt levels were 36 (13-544) iu/l for 8 (47%) haematological disease and 9 (53%) collagenosis disease. in anti-hbc and/or anti-hbs-positive patients mean age were 71.3±9.6 years old, median ast levels were 20 (9-106) iu/l, median alt levels were 16.5 (5-247) iu/l for 17 (77%) haematological disease and 5 (23%) collagenosis disease. serum hbv-dna levels >5.0 log copies/ml were 8 (47%), 2.6~5.0 were 7 (41%), <2.6 were 2 (12%) in hbsag-positive patients, and serum hbv-dna levels <2.6 were all cases in anti-hbc and/or anti-hbs-positive patients. 14 (82%) of hbsag-positive patints received nucleoside analogues (7 lam and 7 etv), and 12 (55%) of anti-hbc and/or anti-hbs-positive patients received nucleoside analogues (8 lam and 4 etv). mean duration of treatment for 14.2 months in hbsag-positive patients, and for 4.1 months in anti-hbc and/or anti-hbs-positive patients, the resistance virus occurred to 3 (75%) of 4 hbsag-positive patients treated with lam for collagenosis disease more than two years. conclusion: when hb carriers of collagenaous disease undergoing immunosuppressive therapy required the nucleoside analogues more than two years, we recommended treatment to prevent hbv reactivation with etv. background: adefovir dipivoxil is used for the initial treatment of chronic hepatitis b or rescue treatment of lamivudine-resistant chronic hepatitis b, and exhibits excellent antiviral activity. however, the presence of resistance to adefovir dipivoxil was more frequently in lamivudine-resistant chronic hepatitis b patients than in lamivudine-naïve patients during adefovir dipivoxil monotherapy. but the rate of adefovir resistance related mutations is little known in lamivudine-resistant patients before adefovir dipivoxil treatment. the aim of this study was to investigate the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus in lamivudine-resistant patients not treated with adefovir dipivoxil. methods: the existence of adefovir resistance-related mutations was examined in 240 lamivudine-resistant chronic hepatitis b patients with breakthrough hepatitis and 100 antiviral-naïve chronic hepatitis b patients. both polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) and directly sequencing of pcr product were used to detect resistant viruses. results: rta181t mutants were detected in only two sera of lamivudine-resistant patients, while none in the antiviral-naïve chronic hepatitis b patients. there was no rtn236t detected in the two groups. conclusion: our results suggest that the rta181 mutant virus were present in a few lamivudine-resistant chronic hepatitis b patients before they have been treated with adefovir dipivoxil, but the rtn236t mutant was not detected in any of the two groups. the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus was low in such lamivudine-resistant patients before adefovir dipivoxil treatment. objective: in this study, we tried to detect and identify the special protein of hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. to find new opinion on the developing of chronic liver disease. methods: the sera of health adult, hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were respectively detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms). the arrays of every group were analysised by clustering analysis and to establish disease predictive model. then the sample was eluted with different ph tris, trypsinization on-chip, mass determination and peptide database comparison. results: according cm10 chip we find 18 protein with obviously deviation (p<0.05) among hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. clustering analysis for the data from seldi-tof-ms confirmed 42 differentially expressed proteins. then we developed disease predictive mathematic models ( decision tree model, dt model ) with average validity up to 73.9 . the 5805 da protein peak was identified to be chondroitin sulfate synthase 2 (chss2), which is a potential molecule involved in the pathologic process and a potential serum marker for the hbv related hepatic diseases as well. conclusions: our results suggest that seldi-tof-ms is a usefull technique for differential expressed proteins screening and analysis in hbv related chronic liver disease. chss2 may be useful during the developing of hbv related chronic liver disease. backgound: clevudine is a new nucleoside analogue with potent antiviral activity in chronic hepatitis b patients. however, the efficacy and safety of clevudine in cirrhotic patients are not well recognized. this study was conducted to evaluate the early virologic and biochemical response rate as well as safety of clevudine in cirrhotic patients with chronic hbv infection. methods: 46 patients with chronic hbv infection who visited korea university ansan hospital and guro hospital between may 2007 and may 2008 were included. 24 patients had chronic hepatitis b (group a) and 22 had liver cirrhosis (group b). early virologic response was defined as hbv dna less than 200 iu/ml at week 12. early biochemical response was defined to be normalization of alt (<45 iu/l) at week 12. result: pretreatment hbv dna levels were higher in group a compared with group b (8.06 log iu/ml vs 7.09 log iu/ml, p=0.06). pretreatment alt levels were not significantly different between the two groups (166 iu/l vs 139 iu/l, p=0.725). the rate of early virologic response was significantly higher in group b compared with groups a (72.7% vs 33%, p=0.01). the rate of early biochemical response were not significantly different in both groups (75% vs 72.7%, p=0.725). conclusion: clevudine is considered to be safe and effective in cirrhotic patients with chronic hbv infection as well as chronic hepatitis b patients. long term safety and efficacy need to be evaluated in the future. objective: the aim of this study was to evaluate the role of nucleos(t)ide analogues against hbv reactivation in immunosuppression. methods: non-active hbsag carriers suffering from cancer, autoimmune diseases and needing the treatment of immunosuppressants or cytotoxic chemotherapy were enrolled in the study. the outpatients or in-patients from april 2007 to july 2008 were enrolled. the nucleos(t)ide analogues were used in cancer patients 1-2 weeks before chemotherapy, and the duration lasted 6-12 months according to patients' compliance after completion of chemotherapy. patients with other diseases used nucleos(t)ide analogues in 1-3 months before using glucocorticoids or other immunosuppressive agents, and continued to use for 6-12 months after accomplishing the course of immunosuppressant treatment. the characheristics and clinical manifestations about hbv reactivation were investigated. results: of the thirty two patients in prospective group, twenty two patients suffered from cancer, eight patients suffered from idiopathic thrombocytopenic purpura, two patients suffered from chronic nephritis. the amount of hbv dna was detected in the first, third, sixth and 12th month after the use of nucleos(t)ide analogues. after chemotherapy or immunosuppressant treatment, only 9.4% (3 / 32) of them suffered from hbv reactivation, which presented with hbv dna positive and abnormal liver function. conclusion: non-active hbsag carriers would appear potential incidence of hbv reactivation during use of chemotherapy or immunosuppressant. nucleos(t)ide analogues could be used in early phase as prophylaxis for reactivation of hepatitis b in immunosuppression and to improve clinical prognosis. background: hbv therapies are evolving toward combination antivirals. this study evaluated the combination of clevudine (clv), a potent nucleoside analog, with tenofovir dipivoxil (tdf). methods: a phase i, single-arm, multi-dose study in 18 healthy adult volunteers to evaluate pharmacokinetic and safety interactions between clv and tdf. subjects received 21 days of clv 30mg followed by 7 days of clv 30mg +tdf 300mg. pk profiles were obtained on days 1, 21 and 28. clv auc and cmax were compared on days 21 and 28. day 28 tenofovir pk was compared to historical data. safety assessments were conducted throughout. results: 18 subjects were enrolled (13m/5f);17 completed the study. the mean (range) age was 37y (22-49) and body mass index (kg/m2) was 27.6 (22.9-31.9). 22 aes were reported by 6 subjects, with 10 aes reported during clv-only dosing and 12 aes reported during clv+tdf dosing. aes included nausea (2) and pharyngolaryngeal pain (2). the majority of the aes were mild. there were no clinically significant changes in ecgs or laboratory parameters. comparisons of clv auc and cmax on day 21 and 28 revealed no significant impact of tdf upon the plasma clv exposure (d28/d21 auc ratio=0.98, d28/d21cmax ratio=0.89). there is no significant effect of clv on tenofovir when comparing auc and cmax of tdf to historical values. conclusion: safety and pharmacokinetic results demonstrate that clv and tdf may be safely co-administered, supporting the further study of this drug combination for the treatment of chronic hbv infection. s. kuznecovs 1,2 , i. kuznecovs 1 , k. jegina 2 , g. kuznecova 1 1 background: dolichyl (dol), the main lipid intermediator of dolichyl phosphate cycle (dpc) has been reported to be elevated in urine of patients with multidrug resistance in cancer. drug resistance poses a major threat to nucleoside analogue-based therapies for chronic hbv infection. methods: with focus on a risk predictor for susceptibility to the development of hbv drug resistance the present study was carried out to estimate urinary levels of dol in chronic hbv infection. the samples obtained every week before and during the course of treatment from 42 patients with hbv. the occurrence of exacerbations of chronic hbv were registered for 2 years. dol in urine was assayed by hplc method. results: the normal amounts of dol in healthy persons urine (n=1500) are 6,0 + 10,0 mkg/mmol creatine. during the period of observation 36 (86%) of patients treated with nucleoside analogue-based therapies were diagnosed with exacerbations due to resistance of hepatitis b virus to antiviral drugs. from this group of hbv patients 35 (98%) have had elevated urinal dol excreation (45,8±5,2 g/ml vs . 8,2±1,9 g/ml, p<0.0001) in more than 3 months of observation. conclusion: there is a reason to suggest that elevated urinal dol detected in patients with exacerbations during hbv treatment may evidence of possible defect of host mechanism of drug resistance development to nucleoside analogue-based therapies. the interest drawn to the employment of dol as a predictor for exacerbation of chronic hbv is explained by the role of dpc in p-glycoprotein regulation in human hepatocytes. background: a significant proportion chb patients treated with adv have a suboptimal response, increasing the risk of disease progression and development of resistance. we report clinical results from patients who either failed or relapsed following adv therapy and were subsequently switched to etv. methods: study etv-079 was a randomized, open-label study comparing antiviral efficacy of etv (0.5mg/day) vs adv (10mg/day) in nucleoside-naïve hbeag-positive patients. after up to 96 weeks of treatment in etv-079, 24 patients treated with adv (13 suboptimal responders) rolled over into study etv-901 (1.0mg/day). hbv dna viral suppression and safety was evaluated during 48 weeks of etv treatment. results: at entry to etv-901, the median hbv dna was 5.72log10 copies/ml. median exposure to etv (1.0mg) in etv-901 was 46 weeks and 18 patients currently remain on study therapy. at week 24, the mean reduction in hbv dna was 4.5log10 copies/ml and 8/16 (50%) reached hbv dna levels <300 copies/ml. nine patients have achieved week 48 and all have achieved hbv dna <10 4 copies/ml and 8/9(89%) had hbv dna levels <300 copies/ml. no patients experienced virologic breakthrough on etv. the safety profile of etv in adv-treated patients remained consistent with the previously reported experience. conclusions: the majority of patients who were suboptimal responders or virologic rebounders following adv treatment in study etv-079, experienced rapid reductions in hbv dna levels when switched to etv. hbv dna levels continued to decline to undetectable levels with 48 weeks of etv treatment. y. li 1,2 , t. han 1 1 tianjin third central hospital, 2 aim:to quantify hepatitis b virus (hbv) total dna and covalently closed circular dna (cccdna) in liver biopsies and sera which from chronic hepatitis b(chb) liver cirrhosis of hepatitis b(lc) and hepatitis b relevance hepatocellular carcinoma(hcc) patients, and analysis hbv replication under the circumstances of different diseases. methods:total hbv dna and cccdna in serum and liver biopsy samples were measured in 21 chb 23 lc and 25hcc patients by the real-time pcr assay. results: the levels of total hbv dna in serum,intrahepatic total hbv dna, intrahepatic cccdna, as well as the proportion of intrahepatic cccdna in total hbv dna decreased progressively in chb,lc and hcc ,moreover chb had significantly higher levels of total hbv dna in serum and liver biopsy samples than lc (log [total serum hbv dna] p = 0.024;log [total intrahepatic hbv dna] p = 0.034); chb and lc had significantly higher levels of intrahepatic cccdna and the proportion of intrahepatic cccdna in total hbv dna than hcc(p <0.01); cccdna couldn't be detect in all patients'serum. in chb ,the levels of serum's total hbv dna,intrahepatic total hbv dna and cccdna in hbeag-positive group had significantly higher than the hbeag-negative group(p <0.01) ,but in lc only intrahepatic total hbv dna had statistical difference between hbeag-positive and negative group (p=0.026) , no statistical difference between hbeag-positive and negative group in hcc. conclusions: the replication activity of hepatitis b virus in chb,lc were higher than hcc, hbv reproduction reduced significantly in hcc. duplication of hbv in lc was lower than chb but had no statistical difference. the levels of hbv reproduce in hbeag-positive group was higher than hbeag-negative group of all three desease. background: clevudine is a pyrimidine analogue with potent and sustained antiviral activity against hbv in the 24 week therapy. the present study assessed the efficacy and viral resistance of 48 week clevudine therapy in patients with chronic hepatitis b. method: a total of 42 patients (26 hbeag positive and 16 hbeag negative) who were received clevudine 30 mg once daily for 48 weeks were included in this analysis. serum hbv dna was quantified by real time pcr assay. result: at week 48, median reductions of serum hbv dna from baseline were 4.98 log10 iu/ml (5.00 log10 iu/ml for hbeag positive and 4.95 log10 iu/ml for hbeag negative) and 76.2% of patients showed undetectable serum hbv dna (<60 iu/ml) (65.4% for hbeag positive and 93.8% for hbeag negative). the normalization of alt levels (<35 iu/l) was achieved in 81.0% (88.5% for hbeag positive and 68.8% for hbeag negative). 15.4% of hbeag positive patients showed hbeag loss or seroconversion. hbv dna negativity at week 48 was associated with hbeag negativity (p =0.037), hbv dna <2,000 iu/ml at weeks 4 and 24 (p =0.019 and 0.001, respectively). two hbeag positive patients showed viral breakthrough with m204i mutation during 48 week. conclusion: clevudine therapy in patients with chronic hepatitis b showed potent virologic responses at week 48, especially in those with hbeag negativity and complete early virologic response (hbv dna <2,000 iu/ml at weeks 4 and 24). but clevudine resistance can occur in hbeag positive patients. background: serum alanine aminotransferase (alt) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. current standards for "normal" alt level were defined by using populations that included persons with subclinical liver disease. there is no study regarding normal level of alt and its modulating factors in healthy thai people. objective: to definitions of normal ranges for serum alt level in thai people. design: prospective observational study setting: phramongkutklao hospital and army institute of pathology pramongkutklao medical center(a.i.p.), bangkok, thailand participants: 200 persons who were first-time blood donors from august through december 2007 were negative for anti-hepatitis c virus(hcv), negative hbsag(hbv), and had no contraindications to donation. measurements: univariate and multivariate analyses examined associations between clinical and laboratory factors and alt levels. normal ranges for alt were computed from the population at lowest risk for liver disease. results: serum alt activity was independently related to body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. normal ranges for serum alt level in thai people upper limits for men 26 u/l and for women 21.67 u/l. conclusion: in men serum alt is strongly associated with body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. the normal range of alt should be defined for male and female separately. background: the open-label rollover study etv-060 was conducted after etv phase ii clinical study etv-047 for nucleoside-na ve adult chb patients in japan. in this analysis, we report etv long-term efficacy and safety in patients who were switched from 24-week lvd treatment to etv therapy. methods: ninety-seven percent (33/34) of lvd-treated patients from etv-047 were rolled over into etv-060 treated with 0.5mg of etv. thirty patients completed 96 weeks of etv therapy and were evaluated for hbv dna level, alt normalization, hbeag seroconversion, resistance and safety. results: comparing to baseline before switching to etv, after 96 week of etv treatment, the proportion of patients achieving undetectable hbv dna (<400 copies/ml) increased from 21% to 90%. increases were also observed for alt normalization (81% to 90%) and hbeag seroconversion (10% to 19%). three patients had detectable hbv dna at week 96 after etv treatment and samples from two were tested for resistance. neither demonstrated substitutions associated with etv or lvd resistance. five patients had grade 3-4 laboratory abnormalities, including increased ast/alt and increased lipase levels. conclusions: switching patients from lvd therapy to etv resulted in increased proportions of patients achieving hbv dna suppression, alt normalization and hbeag seroconversion, with no evidence of etv resistance. etv was well tolerated during treatment. backgrounds/aims: hepatitis b virus (hbv) reactivation in patients undergoing chemotherapy hampers an adequate administration of cytotoxic agents and even causes an treatment failure. prophylaxis failure occasionally results from viral breakthrough or withdrawal flare. the aims of this study were to identify predictors of anti-viral prophylaxis failure and to determine the optimal strategy for anti-viral prophylaxis. methods: cancer patients with positive hbsag who underwent cytotoxic chemotherapy in a tertiary medical center from january 2005 to june 2008 were included. prophylactic lamivudine was started with initiation of chemotherapy, continued during the chemotherapy, and discontinued within 6 months after the completion of chemotherapy. all patients were followed up even after withdrawal of lamivudine. results: 115 patients were enrolled. twenty-nine patients (23.7%) had hematologic malignancies and eighty-six (76.3%) had solid tumors. median follow-up duration was 15.9 months and twenty-six patients (22.6%) experienced the prophylaxis failure: viral breakthrough (11 patients, 9.6%), withdrawal flare (15 patients, 13.0%). ymdd mutation developed in four patients. withdrawal flare occurred at a median 2.5 months after discontinuation of lamivudine. using log-rank test and cox multi-variate analysis, our results showed that the type of underlying malignancies (hr 2.46, 95% ci, 1.11-5.43; p=0.026) and baseline hbv dna titer (hr 3.91, 95% ci, 1.63-9.39; p=0.002) were significant independent risk factors for antiviral prophylaxis failure. conclusion: cancer patients with high viral load of hbv and hematologic malignancies may need more prolonged and potent anti-viral prophylaxis to avoid interruption or delay of chemotherapy. back ground: the usefulness of hepatitis b virus (hbv) dna and hbv core-related antigen (hbcrag) was evaluated for timing hepatitis flare after viral breakthrough or withdrawal of antiviral treatment in chronic hepatitis b. method: a total of 32 events of hbv reactivation due to withdrawal of lamivudine (lam) or emergence of mutants resistant to lam or adefovir dipivoxil (adv) virus were analyzed in 25 patients with chronic hepatitis b (20 men, median age 56 years [range: 30-66]). they were followed monthly for serum alt, hbv dna and hbcrag before, during and after the treatment. result: high alt flare (alt > 100 iu/ml) after viral breakthrough or withdrawal was related with baseline hbeag positivity (p=0.016), hbcrag level at hbv dna elevation (p=0.038) and duration from hbcrag elevation to salvage therapy (p=0.044). in multivariate analysis, hbcrag > 4.0 log u/ml (or 22.9, 95%c.i. 1.7-304.1, p=0.018) and salvage therapy after 8 weeks from hbcrag elevation (or 10.6, 95%c.i. 1.6-71.4, p=0 .015) were selected as related factor with high alt flare. after appearance of resistant-virus or withdrawal of lam or adv therapy, hbv dna re-elevated without increase of hbcrag, then hbcrag elevated with hbv dna. re-elevations of alt occurred in 27 of the 32 (84%) events. in 23 of the 27 (85%) events, alt re-elevated within 8 weeks from the start of hbcrag increase. conclusion: hbcrag was useful for timing the re-elevation of alt after hbv dna re-elevation induced by drug-resistant virus or withdrawal of lam or adv therapy. background and objectives: the aim of the study was to observe the efficacy of a patient's therapy for switching lamivudine + placebo to adefovir dipivoxil (adv), and modeling the viral dynamics. methods: the studied object was a chinese chb patient with lamivudine mutation. used the lvd + placebo for 12 weeks' therapy. then switched adv for another 95 weeks. after that stopping the treatment and following up for 24 weeks. based on our modified basic virus infection model, we introduce a personalized model consisting of four variables: x, y, v, e , representing uninfected cells, infected cells, free virus, and ctl cells, respectively. results: selected the model parameters, the simulation data of hbv dnas of our model are good in agreement with the clinical ones. observe that after 24 weeks' treatment cessation, the benefit (hbv dna < 250 copies/ml) for suppressing hbv replication can still be kept. numerical simulation show that if the patient's immune functions can be kept after therapy stops, it needs 9 years to replace all infected cells by normal ones. conclusion: for lvd mutation patients, lvd+ placebo to avd therapy scheme may help patients to suppressing hbv replication. further researches are promising. acknowledgments: this work is jointly supported by the nnsf of china background: g-a-1896 pre-core mutants (p-c-mt) cause hbeag-negative chronic hepatitis (chb) in genotype d infected mediterranean adults. we studied their emergence during chronic hbv infection in children. methods: eighty consecutive hbsag carriers (50/30 males/females, age 11y, range 0.2-17y) with vertical (66%) or intra-familial (16%) transmissions were followed-up for 12.5y (range 1-25 y). hbv genotype and hbeag status were determined at the admission, hbeag/anti-hbe every 2 years thereafter. during the follow-up, hbv-dna was measured in 185 sera (1-7 sera/patient) (cobas-amplicor, roche); p-c populations were characterized by direct sequencing (ds), by oligo-hybridization (oha) and allele-specific-pcr (as-pcr) with 30%, 10% and 0.1% sensitivities, respectively. results: seven children were genotype a and 73 d; 70 (87.5%) were hbeag-positive. fifty-five (78.6%) underwent hbeag/anti-hbe seroconversion (median age 13y, range 1.3-27y). baseline hbv-dna (cp/ml) was lower in seroconverters (7.9+1.9 vs 9,4+0,6, anova p=0.012). ds/oha p-c-mt were 4.2% at the admission and 45.8% after follow-up; as-pcr p-c-mt 33.3% and 50% respectively. after seroconversion 47 (85.5%) became inactive carriers, 6 (14.5%) lost hbsag (5 genotype d/p-c-wt); 8 p-c-mt had chb. hbv-dna (cp/ml) was lower in 31 p-c-wt than in 14 p-c-mt inactive carriers (3.42+1.05 vs 4.58+0.91; anova p=0.007). conclusions: in genotype-d infected children p-c-mt is selected progressively after hbeag/anti-hbe seroconversion to become predominant in hbeag-negative chb. early and efficacious immune control of hbv replication avoids p-c-mt selection and leads to hbsag loss. , a, , d, k, u, m, n , k1 ,k2 and k3. here k1, k2, and k3 are the rate of ctl production and dead, killing virus, respectively. results: the patients with hbv dna levels less than 1000 copies/ml were reported in 17.5% (7/40). a patient whose hbv dna levels were higher than 40000 copies/ml can keep treatment benefits even stopping the therapy for over ten weeks. the simulation data of our model are in agreement with the patient's hbv dna data. our simulation also shows that it needs to spend about 18 years for clearing all infected cells. conclusion: the simulation result implies that some chinese patients may need long term's therapy to clear all infected cells. patients' ctls assays are needed to confirm the effectiveness of the personalized modeling, and help doctors to decide whether stop the drug treatment even patients' hbv dnas are higher than undetectable levels. background/aim: recent reports have shown that programmed death 1(pd-1) expression is associated with t cell exhaustion and persistent viral infection. we studied longitudinally 28 chronic hepatitis b(chb) patients undergoing treatment with nucleos(t)ide analogues or pegylated interferon-(peg-ifn-) in 12-16 weeks to determine the relationship between pd-1 expression levels on t cells and early reduction of viral load induced by treatment. methods: our investigations were focused on three points: baseline (time point 1, t1), treatment weeks 4-6(time point 2, t2) and treatment weeks 12-16(time point 3, t3). pd-1 expression on total cd4 and cd8 t cells in chb patients during antiviral therapy was detected by flow cytometry. serum hepatitis b virus (hbv)-dna load was measured by real time polymerase chain reaction. results: between t1 and t2, pd-1 expression on total cd8 (p<0.01) and cd4 t cells (p<0.01) dropped concurrently with treatment-induced hbv-dna decline(p<0.01). between t2 and t3, however, only the hbv-dna levels reduced significantly (p<0.01). conclusion: early suppression of hbv replication induced by antiviral treatment results in a significant decrease in pd-1 expression on total cd8 and cd4 t cells in chb patients. c. zhao 1,2 , w. zhang 2,3 , x.c. tian 1 , c.y. fang 2,3 , h.j. lu 2,3 , p.y. yang 2,3 , y.m. wen 1,2 background/aims: hepatitis b virus (hbv) is still regarded as one of the major causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. the interactions between hepatitis b surface antigen (hbsag) and host cells still remain largely unknown and need to be explored in detail. methods: differential protein expression profiles of hepg2-s-g2( stably expressing hbsag cell line) and hepg2-neo-f4 ( control cell line) were compared using two dimensional gel based differential proteomic approach. cell proliferation assay and survival assay were used for further studies on the candidate protein. results: compared with the control down regulation of 44 proteins and up regulation of 38 proteins were found in hepg2-s-g2 cell. all these regulated proteins were identified by ms/ms and could be fell into several categories including metabolism-associated, immune-response-related, protein modification, signal transduction and others. among them, a group of proteins in putative pathways associated with apoptosis were found out and discussed, including glucose-regulated protein 78kd (grp78/bip), heterogeneous nuclear ribonucleoprotein (hnrnp), far upstream element-binding protein (fusebp), rho gdp dissociation inhibitor (gdi), cystatin b and some scaffold proteins. grp78, an important chaperone protein involved in multiple functions in host cells, was consistently decreased in hepg2-s-g2 and in huh7 cell transiently transfected with hbsag expression plasmid. decreased crp78 inducing by hbsag or blockage of rnai consistently led to the less resistance to staurosporine-induced cell death. conclusions: these results revealed a possible pathogenesis induced by hbsag via grp78. background/aim: to evaluate the efficacy of adefovir dipivoxil alone and in combination with lamivudine in treating patients with lamivudine-refractory hbeag-positive chronic hepatitis b. methods: eighty-five hbeag-positive patients who had received lamivudine treatment for various periods and had a lamivudine-resistant liver function abnormality, documented ymdd mutations and persistent viremia were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. the primary efficacy measure was virological response. the secondary efficacy measure was serological response (hbeag loss rate and hbeag seroconversion rate) and alt normalization rate. results: after 24 weeks of therapy, mean reduction of hbv-dna level, the percentage of patients with hbv-dna lower than 5 log10 copies/ml and the percentage of patients with hbv-dna level decrease of more than 2 log10 copies/ml in patients of adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups were significantly higher than those in patients of lamivudine group (2.58, 2.21 log copies/ml vs. 1.02 log copies/ml, 92.3%, 88.5% vs. 33.6%, 76.9%, 75.8% vs. 28.8%; p < 0.001, respectively). at the end of 52 weeks, mean reduction from baseline in serum hbv-dna level at was 0.08, 4.25, and 4.12 log10 copies/ml in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. alt normalization rates were significant hihger in adefovir dipivoxil/lamivudine and adefovir dipivoxil recipients than those in lamivudine recipients (62%, 55% vs. 8%, p < 0.001, respectively). a similar pattern was observed in hbeag loss among three groups. conclusions: adefovir dipivoxil is an effective treatment option for patients with lamivudine-refractory hbeag-positive chronic hepatitis b. aim: to find the prevalence of hbv virologic flare as defined by hbv dna viral load of > 2,000 iu/ml in inactive chronic hepatitis b (hbv dna less than 2,000 iu/ml), hbeag-negative patients who have not received any treatment and to identify if there are any predictors that can predict virologic flare. methods: we retrospectively analyzed medical records of the patients who have attended hepatitis clinic, siriraj hospital from january 1, 2002 to february 28, 2008 . the patients were eligible if they were naïve to any treatment and hbv dna less than 2000 iu/ml at entry. co-infection with hiv and/or hepatitis c virus were excluded. hbv dna measurement determine by roche amplicor ® (detection limit of 60 iu/ml). hbv virologic flare was defined as hbv dna more than 2000 iu/ml during follow up period. result: there were 84 patients with mean follow up time was 598 days with annual prevalence of hbv virologic flare of 12.8, 4.8, and 4.2% for the first, second and third year of follow up, respectively. initial hbv dna level was the only predictor that can predict reactivation. no patients with hbv dna at entry below detection limit developed flare and the patients with hbv dna above 740 iu/ml had 22 times higher chance to develop flare during follow up. conclusion: hbv dna flare is not uncommon in inactive chronic hepatitis b patients. most of the virologic flares occur in the first year. the most important predictor or virologic flare is higher hbv dna at beginning. background: multi-drug resistant hbv developed with multiple antiviral agents. there existed difficulty in dealing with multi-drug resistant hbv. methods: retrospective analysis of 23 consecutive patients who exhibited chronic hepatitis b associated with multiple drug-resistant mutations to lamivudine and adefovir during antiviral treatment. multiple drug-resistant mutations were detected in those patients by dna direct sequencing. result: before multiple drug-resistant hbv emerged, 20 patients accept sequential antiviral therapy, 3 patients accept na monotherapies. there were 16 cases of rta181t/v rtm204v/i mutation, 2 cases of m204v/i +n236t mutation, 4 cases of a181t/v+m204v/i +n236t mutation, 1 case of l180m+a181t/v mutation. 14 cases received rescue therapy of interfronand hbv dna level of 8 cases decreased; other 9 cases received combination treatment and hbv dna level of 4 cases decreased. conclusion: the main reason of multiple drug-resistant mutations was sequential antiviral therapy. another reason may be pre-exist drug-resistant mutation before nucleoside or nucleotide analogue treatment. de novo combination of antiviral agents should be recommended. combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant hbv. interfron may be one choice for hbv of multiple drug-resistant mutations. background: to furnish basis for an accurate evaluation of hbeag negative chronic hepatitis b (e chb), the present study studies the clinical features and hepatic pathology, and analyzes the relation between the data and the grade and stage of hepatic pathology in e chb. methods: a study is performed in 120 chinese e chb patients (106 men and 14 women; mean age sd, 34.3 9.4 years). the relationship between the clinical features and the grade and stage of hepatic pathology was analyzed by spearman's rank correlation test or kruskal-wallis test by applying stata 7.0 software. result: negative correlation is shown between the grade and leucocyte count methods: 80 patients with hbeag-positive compensated chb with hbv dna >6 log10 copies/ml, serum alt 2 x uln were divided two groups:one treated with telbivudine and the other treated with entecavir. results: baseline characteristics were well balanced between treatment groups. at 12wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 50 52.5 (p 0.05), the rates of hbeag negative were 10 0 respectively, the rates of hbeag seroconversion were 20 5 respectively; at 24wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 80 70 (p 0.05), the total rates of hbeag negative were 20 15 respectively, the total rates of hbeag seroconversion were 27.5 17.5 respectively(p 0.05).no adverse reactions were found in both groups conclusion: there was no significant difference in hbv dna undetectable rates between two nucleotide analogs in short-term (24 weeks).the telbivudine group has better effect in hbeag seroconversion rate than the entecavir group in early stage,but no statistical significance. j.w. song 1,2 , z. xin 1 , j.x. tang 1 , l. yao 1 , b. wu 1 1 sun yat-sen university, 2 zhuhai sinochips biosci. co.,ltd., china background: to develop a equipment free, and can be widely used in clinical practice biosensor-based microarray for hepatitis b virus pre-c/bcp mutation assay. methods: a thin film optical biosensor were applied for amplification the microarray signal in situ. and hbv sites 1762, 1764,1768,1770 and 1896 were selected as the targets and the microarray were be fabricated. the 5 mutated plasmids contained 1762, 1764,1768,1770 and 1896 sites and 30 hbv sera were be tested in our study and all the plasmids and sera pcr products were be assayed by really time pcr and sequencing. results: the biosensor based microarray signal can be easily record by digital camera or even by the naked eyes and the detection signal for positive discriminated from negative were sharply contrasted as whole yes or no and it looks be significantly superior to classical microarray technique; 2. the sensitivity of the detection limitation of sera hbv load is 2x10e3 copies/ml with 95% reproducibility. the concordance index of 50 times negative and mutated plasmids were 98%(kappa=0.932). 3. 30 sera samples of hbv>10e4 load and 20 sera of hbv negative tested by pcr fragment sequencing were showing very good agreement between sequencing with our biosensor based microarray and the concordance index kappa was 0.7619. conclusion: our biosensor-based microarray for pre-c/bcp mutation assay were a both sensitive and accurate method. and its advantages of equipment free, sharply contracted signal of positive vs negative and easily be perform in testing were make it be a promised assay for clinical application. objective: to investigate the frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b, we assayed the differences among hbsag-positive and healthy subjects by flow cytometry. the results might offer some experimental evidence to explain the high rates of hbv persistent infection in vertical transmission of hbv from hbv-infected mothers. methods: 12 newborns born from hbsag positive mothers were recruited , 10 healthy subjects being used as a control group. the cord blood and peripheral blood of mothers were collected respectively .frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b were analyzed by flow cytometric analysis. result: the number of cd4 + cd25 + regulatory t cells/pbmcs in the cord blood of newborns born from hbsag positive mothers 4.49 ±1.18 significantly exceeded that in normal controls 2.26 ±0.97 ,p 0.001 ;and newborns born from hbsag positive mothers presented a much higher fraction of cord blood cd4 + cd25 + /cd4 + 9.62 ±2.34 than those in normal controls 7.93 ±1.43 ,p p=0.0025 p 0.05 . conclusions: the results indicate that the proportion of cd4 + cd25 + regulatory t cells in hbsag positive mother cord blood was higher than those of healthy cord blood. objective: to study the clinical features of chronic severe hepatitis b with negative hepatitis b e-antigen (hbeag) and positive hepatitis b e-antigen (hbeag) methods: a total of 353 in-patients with chronic severe hepatitis b were recruited into the study and divided into two groups according to the hbeag status. the serological chemistry data, hepatitis b virus (hbv) dna quantification data were detected, and morbility of cirrhosis, its complications and prognosis were also studied. results: of the 353 in-patients, 236(66.8%) patients were hbeag-negative. 117(33.2%) patients were hbeag-positive. the ratio of hbeag-positive patients was significantly higher than that of hbeag-positive patients (p<0.05).the average age of hbeag-negative patients was older than that of hbeag-positive patients (p=0.048). the serum hbv dna level of hbeag-negative patients was significantly lower than that of hbeag-positive patients (5.49±2.02) vs(6.64±1.41) log copies/ml (p<0.01).the ratio of patients who had a serum hbv dna level less than 5log copies/ml in hbeag-negative patients was significantly higher than that in hbeag-positive patients (41.8% vs 11.9% ,p=0.000). there was no significant difference in serological chemistry data, morbility of cirrhosis and its complications on infections, ascites, hepatoencephalopathy, gastrointestinal hemorrhage, as well as prognosis of the patients between those two groups. conclusions: the study suggested that serological chemistry data, morbility of complications and prognosis of the disease of hbeag-negative patients mimics that of hbeag-positive patients. the hbeag-negative patients had a higher level of age, while a lower level of serum hbv dna. to reduce the incidence of liver failure, more frequent monitoring and earlier antiviral therapy prone to be reasonable for chronic hepatitis b patients with negative hepatitis b e-antigen. background: the emergence of lam-resistant virus greatly limits the efficacy of therapy and induces the liver injury. the aims of this study were to assess the related factors of lam-resistant mutation in hbeag positive chb patients. methods: thirty-five patients carrying lam-resistant with hbeag positive were enrolled in this study. all of them underwent percutaneous liver biopsy, histological findings and had detectable viral load. age, viral load, levels of alt, types of mutation and hbv genotype was monitored. result: the median year of mutation found was 23months. 85.71% were genotype c and 14.29% were genotype b. the mutation of l80i, l80v, g173l, l180m, m204v and m204i were detected. the emergence rates were 34.3%(12/35), 25.7%(9/35), 17.1%(6/35), 60%(21/35), 57.1%(20/35), 54.3%(19/35) respectively. the rate of patients with two or three mutation were much more than one or four mutation. 62.9% patients were found to have significant histological findings, even 5 had established cirrhosis. two had no histological finding. one had rtl80i and rtm204i. the other had rtl80v, rtl180m and rtm204v. the number of resistant mutation has no significant finding with histological finding, basic alt level and basic viral load. conclusions: the emergence rate of l180m, m204v and m204i were higher than that of l80i, l80v, g173l in hbeag positive chb patients with lam-resistance. most of them have two or three lam-resistant mutation regardless of histological finding severity, level of basic alt and viral load. we must select the efficacious method to treat the patients with lam-resistant. objective: to investigate the therapeutic efficacy of foscarnet sodium in the treatment of patients with severe chronic hepatitis b. methods: forty four patients were randomly divided into foscarnet sodium treatment and placebo groups.each group consisted of 22 patients, 22 patients in foscarnet sodium group were treated with foscarnet sodium twice daily 3.0g given by intravenous infusions ,in addition to general therapy for 28 days.the other 22 cases were treated without any form of antiviral therapy as control.all patients were followed up for 6 months.the hbv markers, quantification of hbv-dna, serological chemistry data were measured at baseline , during therapy period and the end of follow-up period . results: clinical symptoms were improved in two groups patients, meanwhile alanine aminotransferase (alt) and total serum bilirubin (tbil) decreased. compare alt and tbil at the end of trentment, there were no significant differences between the two groups (p>0.05). in foscarnet sodium treatment group, the level of serum hbv-dna descreased from (6.993±0.898) log copies/ml to( 4.033±1.286) log copies/ml (p<0.05), the rate of hbv-dna descrease of more than two log was 81.1% 18/22 . in the control group, the level of serum hbv-dna descreased from( 7.068±0.938) log copies/ml to (5.188±1.926 )log copies/ml, the rate of hbv-dna descrease of more than two log was 45.4% 10/22 .a comparison of serum hbv-dna showed significant differences between the two groups(p<0.05) conclusion: foscarnet sodium administered can inhibit hbv replication in treating severe chronic hepatitis b.it can rapid lower the level of serum hbv-dna obviously.but the relapse rate was 47.0% in foscarnet sodium treated at the end of follow up period objective: evaluation of efficacy and safety of five years trail of entecavir for chronic hepatitis bpatients failed with lamivudine therapy in the chongqing area. methods: thirty-two eligible patients were enrolled who had documented lvd failure.in the double-blind phase,patients were randomized(4:1)to etv1.0mg/d (n=28)and placebo (n=4) for 12 weeks.in the open-lable phase ,patients received etv 1.0mg/d for 240 weeks.hbv-dna level,liver function tests,hbv serology and safety assessments were conducted. results: the mean reduction in hbv dna levels at week 12 was 4 05 logl0 copies ml in etv group compared to 0.08logl0 copies ml in placebo group(p< 0 05). the mean of hbv dna levels after 240 weeks of etv treatment decreased to 2.58logl0 copies m1 the proportion of hbv dna<3log10copy/ml raised from 0 at baseline to 6.25% at week 8,to 15.6% at week 24,to 50% at week 96,and raised to 57.14% at week 240.there were two patients with hbsag seroconversion and four patients with hbeag seroconversion at the end of study. the mean of alt became normal at week 12 and remained normal throughout week 240.there was one patient who had a severe adverse event during the trail. conclusion: the findings from this study demonstrated the antiviral activity and safety of etv in adults with chb who have failed lvd pe198 showing delayed response on t cells as increased on day 7.the mrna expression of il-5 and il-2 showed no response to hbv vaccine but highly regulated in tt after day 7(p=0.00,0.001).myd88andtraf 6(p=0.042)upregulated in hbv vaccine group followed by of ifn-(0.012)no change of ifn found in tt conclusions: i) hbv vaccine stimulates innate response by day 3 which potentiates further cascade,peripheral dendritic cells plays significant role in generating immune flare follows myd88 pathway and releases ifn-.ii) whereas t cells marjory involved in tt showed delayed immune response.iii) identification of key factors at different time points may prove to be a novel model to study the initial events after vaccination. objective: to compare th1/th2 cytokines' dynamic change and its clinical significance in hepatitis b e antigen-positive patients treated with telbivudine. methods: twelve hepatitis b e antigen-positive patients treated with telbivudine.the blood sample was collected at baseline, week 4, week 8, week 12, week 24 and week 48 and stored at -70c; serum il-2, il-4, il-6, il-10, tnf-and ifn-were tested at each time point by cytometric bead array (cba), compare th1/th2 cytokines' dynamic change at different time point in each group and compare th1/th2 cytokines' dynamic change cross four different groups: complete response, partial response, non-response and break through . results:the level of th1 type cytokines in complete response group are obviously higher than the group of partial response non-response and breakthrough,but the level of th2 type cytokines are lower than the group of partial response, non-response and breakthrough. conclusions: th1/th2 cytokines is essential for the regulation of the immune function of the body. after treated with telbivudine, the level of th1-type cytokines in the complete response group increased significantly, while the level of th2 cytokines declined trend. a. soamni 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background: chronic infection with hepatitis b virus causes spectrum of manifestations ranging from asymptomatic carrier state (often inactive with low replication) to the development of cirrhosis-related complications.the characterization of asymptomatic state has not been done in this part of the country, which forms important objective of present study. methods: 61 incidentally detected asymptomatic hepatitis b surface antigen positive (idahs) subjects having hbsag positivity for >6 month presenting to our liver clinic were enrolled after appropriate consent. detailed clinical, laboratory and sonographic evaluation was done. they were divided into two groups according to presence or absence of e antigen. group a -hbeag + (n=48) group b -hbeag -(n=13) results: most of our patients (49%) were young adults (21-30 years) with male to female ratio of 3.6:1. approximately half of our patients were detected during routine medical checkup, followed by family screening of contacts. most of our patients were asymptomatic, and fatigue was most common symptom found in 16%. all demographic and biochemical parameters other than ast & alt were comparable in both groups. among hbeag negative 48 (79%) subjects, hbv dna level >10 5 copies/ml was found in 31%. subjects with positive hbeag as compared to non-replicative infection (antihbe positive and hbv dna negative) had more frequent elevation of transaminase levels (62% versus 31%, p<0.05). antihbe antibody was positive in all hbeag negative subjects. mean age of seroconverted (antihbe positive) individuals was a decade older than hbeag positive. conclusion: from our study we can suggest that ongoing liver disease is present in approximately one-thirds of incidentally detected asymptomatic hepatitis surface antigen positive subjects previously referred to as carrier state. hbsag testing should be mandatory in all routine medical checkup and family and sexual contacts of index case should be screened. background and objectives: this research was carried out to determine the prevalence of hbcab among the hbsag negative first-time blood donors who had referred to khorramabad and borujerd centers for blood donation. materials and methods: this study was established on a descriptive cross-sectional basis in which hbsag test (elisa) was primarily performed on all of the donors having referred to khorramabad and borujerd blood centers; then, out of all those referred 1000 subjects, who were first-time and hbsag negative, were selected for furthur investigation. the information concerning age, gender, job, blood transfusion, and hbv vaccine injection was included in the questionnaire of the study. hbcab (total & igm) and hbsab tests were performed on the selected donors. data were collected and finally the prevalence rate of hbcab was determined. results: the results of the study showed that out of 1000 hbsag-negative first-time blood donors, only 47 were hbcab+, from which 27 were hbcab (total)+, and 3 were hbcab (igm)+. 18 were both hbsab+ and hbcab+, and 53 were seropositive only for hbsab. conclusions: it was demonstrated that the first-time blood donors who are seronegative for hbsag marker will be easily identified through hbcab test if they are in the so-called core window period of the virus. meanwhile, this group of donors have been implicated as high-risk for transfusiontransmitted hbv infection. so, detecting this marker will remarkably reduce the chance of latent cases of hbv infection and help promote blood safety. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. method: 20 hbv transgenic mice were randomly divided into physiologic saline group and matrine injection group. another10 normal mice at the same species and age with hbv transgenic mice were regarded as the normal group. the mice in matrine injection group were administrated at dosage of 82.2 mgkg -1 d -1 by intraperitoneal for 30 days. the mice in physiologic saline control group and normal group were administrated normal saline with the same volume at same time. the contents of hbv dna in serum and liver were quantitated by pcr. and the spleens were separated for cultivating dendritic cells. the surface molecules of dendritic cells were tested by flow cytometry. ifn-mrna and tnf-mrna in liver were tested by rt-pcr. result: there was no significant difference of the serum hbv-dna level between physiologic saline and matrine injection groups. the content of serum hbv-dna after treatment showed a significant decrease in two groups. the content of serum hbv-dna in matrine injection dropped significantly as compared with that in the physiologic saline group. but there was no significant difference in the content of hbv-dna in liver between physiologic saline and matrine injection groups. the expression level of mhc-ii on dendritic and hepatic ifn-r mrna and tnf-a mrna showed a significant decrease in hbv transgenic mice than normal mice. in comparison with physiologic saline group the expression level of them in matrine injection group showed a significant increase. conclusion: matrine injection was effective on depressing hbv-dna in hbv transgenic mice. its antiviral action may be achieved through regulating mhc-ii on dcs surface and promoting the production of antiviral factor such as ifn-and tnf-. purpose: to stimulate non-specific immune response capacity as the main content of the study to explore the hbv-dna and non-specific immune responses in the relationship between the low response capability, methods: 190 cases of asymptomatic carriers, double-blind, randomized into mycobacterium fu 36, lamivudine and traditional chinese medicine for the treatment group, mycobacterium fu36 with traditional chinese and lamivudine with traditional chinese medicine were in the control group, a total of 12 weeks of treatment, follow-up six months after the termination of treatment. results: different treatment of hbv -dna effect of the existence of significant differences; p> 0.01, the performance of different types of asymptomatic carriers negative rate of hbv-dna there is a significant difference; p> 0.01, as well as the performance of the different types of asymptomatic carriers continued application a treatment plan presented hbv-dna rebound rate there is a significant difference; p> 0.01, conclusion: hbv-dna and non-specific immune responses in response to the lower capacity, anti-hbv therapy is not associated with non-specific immune response capacity or improve is the anti-hbv drugs alone can not solve the asymptomatic carriers in anti-hbv therapy where the cause of the problem, solve the asymptomatic carriers in the anti-hbv treatment although the need for anti-hbv drugs with non-specific immune activation synchronous drugs on the basis of the joint application , but the simultaneous combination of two drugs rather than as a result of hbeag and hbv-dna can hbeag-positive asymptomatic carriers receive hbv-dna negative effect of the results. background: adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine (lam)-resistant hepatitis b virus (hbv) replication and resistance to this drug is infrequent compared with lam. in this study, we tried to identify factors affecting the emergence of resistant mutants after adefovir monotherapy in lam-resistant chronic hepatitis b (chb) patients. methods: the subjects were 87 chb patients with lam-resistance who had received adefovir for more than 12 months (range 12-39 months). the initial viral response (ivr) was defined as hbv dna <4.0 log copies/ml. the adefovir resistant mutant was assayed at baseline and every 6 months during adefovir administration. results: ivr was observed in 38% of patients. the cumulative emergence rates of adefovir resistance were 2.6% at 6 months, 10.4% at 1 year, 14.6% at 2 years and 21% at 3 years. in univariate analysis, factors contributing to the emergence of adefovir resistant virus were baseline hbv dna > 6 log copies/ml (p=0.003) and ivr (p<0.0001). the presence of precore mutation and type of ymdd mutants were not related. in multivariate analysis, only ivr was an independent factors affecting the emergence of adefovir resistant virus (p<0.0001). conclusion: ivr is a useful predictor for emergence of adefovir resistant mutants after adefovir monotherapy in lam-resistant chb patients. for ivr-negative patients, the change of therapeutic options such as add-on lam or switch to other drugs should be considered because of the high incidence of the emergence of adefovir resistant mutants. background: elevated hbv dna is strongly associated with the risk of disease progression. this study investigated the early viral suppression effects of etv and lvd in nucleoside-naïve chinese patients with active hbeag (+) chb. methods: this open-label study was conducted in 5 major hospitals in china. at study entry all patients had hbv dna levels 10 7 copies/ml, elevated alt (1.3-10xuln) and compensated liver function. patients received either 0.5mg etv or 100mg lvd daily. hbv dna measurements were taken at baseline and at weeks 2, 4, 12 and 24 during treatment, using roche cobas amplicor assay (llod 300 copies/ml). results: a total of 97 patients were enrolled; 42/50 etv patients and 40/47 lvd patients completed 24 weeks of treatment. at baseline, mean hbv dna levels were 8.45 0.81 in etv group and 7.67 1.76 log 10 copies/ml in lvd group (p<0.05). the mean change in hbv dna from baseline (log10 copies/ml) was -2. 96±0 ngo's/funding-agencies representative at apasl2009-conference need to address-this-issue. we ngo-representatives from developing-nations need exposure to research treatments used by european/american experts. do we all failed in addressing socio-economic issues of cancer-sufferers? we need to address these socio-economic issues of affected population in resource-poor-nations. background: a garlic derivative s-allylcysteine (sac) has anti-cancer effect in human prostate and colon cancers. we aimed to investigate the effect of sac and combination of chemo-drug on tumorigenesis and metastasis of liver cancer. methods: the orthotopic liver tumor model using a metastatic liver cancer cell line mhcc97l labeled with luciferase gene was applied. sac was given at day 7 after tumor implantation at 1mg/g/day, or 1mg/g/day combined with low dose cisplatin for 5 weeks. tumor growth and metastasis were monitored by xenogen in vivo imaging system. hepatic stellate cell (hsc) activation and tumor-associated macrophage (tam) in the tumor tissue were detected by -sma and ed1/ed2 staining. tumor micro-vessel density (mvd) and apoptosis were also analyzed. in vitro functional tests including proliferation assay, cell cycle analysis and apoptosis analysis were performed. results: tumor growth was inhibited by sac combined with cisplatin treatment at different time points accompanied by lower incidence of lung metastasis compared with other groups. the observation of xenogen ivis was confirmed by histopathological examination. the hsc activation by -sma staining in the liver tumors was suppressed by sac and cisplatin treatment accompanied with less tam infiltration. consistent with in vivo study, in vitro functional study also demonstrated that sac not only induced cell cycle arrest, apoptosis, and inhibited tumor cell proliferation, but also sensitized the anti-cancer effect of cisplatin. conclusion: sac treatment inhibited liver tumor growth and metastasis by inhibiting tumor cell proliferation, inducing apoptosis and sensitization of chemotherapy. background: anti-angiogenic therapy would be a promising approach against hepatocellular carcinoma (hcc). although a sorafenib has survival benefits in patients at advances stages of hcc, there seem to be several serious concerns to employ this agent for chemoprevention against hcc. branched-chain amino acid (bcaa) reportedly inhibits the incidence of hcc in patients with insulin resistance (ir). however, the possible mechanism is still obscure. the aim of the current study was to examine the effect of bcaa on hepatocarcinogenesis under the condition of ir, especially in conjunction with angiogenesis. methods: the effect of bcaa on the development of liver enzyme-altered pre-neoplastic lesions and angiogenesis in the obese diabetic otsuka long-evans tokushima fatty rats was examined. we also performed an in-vitro study to elucidate the possible mechanisms involved. result: treatment with bcaa markedly inhibited the glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions along with suppression of neovascularization in the liver. the hepatic expression of the vascular endothelial growth factor (vegf), a potent angiogenic factor, was also attenuated. bcaa treatment significantly suppressed the glucose-and insulin-induced in-vitro angiogenesis in the presence of vegf. these results indicate that bcaa exerted a chemopreventive effect under the condition of ir via suppression of vegf-mediated angiogenesis. conclusion: since bcaa is widely used in the clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against ir-based hcc in the future. background: krüppel-like factor 8 (klf8) is a member of transcription factors. whether and how klf8 signaling pathways contribute to hepatocellular carcinoma (hcc) development and progression is unknown. this study investigated role of klf8 in hepatocellular carcinoma cell line hcclm3 proliferation, invasiveness and epithelial to mesenchymal transition (emt). methods: the expression of klf8 in different liver cell lines was detected by quantitative real-time pcr and immunocytochemistry. we used small interfering rna (sirna) to down-regulate klf8 expression in hcclm3. the change of proliferation and invasive ability of klf8 down-regulated hcclm3 was investigated by mtt reduction assay and trans-well invasive assay respectively. the change of proliferation, invasiveness and emt related gene in klf8 down-regulated hcclm3 was evaluated by quantitative real-time pcr. result: klf8 protein expressed predominantly in the nuclei of cancer cells and its expression is positively correlated with metastatic potential of these cell lines. hcclm3 has the highest klf8 level. decreased klf8 expression can notably inhibit the proliferation (p<0.001, n=6), mobility and invasiveness of hcclm3 (p<0.001, n=8). we found that the mrna level of n-cadherin, fibronectin and vimentin is much higher than that of e-cadherin in hcclm3. the expression of cyclin d1, focal adhesion kinase (fak) and fibroblast markers including n-cadherin and fibronectin was obviously suppressed in klf8 down-regulated hcclm3. conclusion: klf8 plays an important role in the process of hcclm3 proliferation, invasiveness and emt. background: insulin resistance (ir) has shown to play an important role in the progression of chronic liver diseases, including liver fibrosis development and hepatocellular carcinoma. the aim of this study was to elucidate the possible mechanisms of ir on the liver fibrosis development and hepatocarcinogenesis using obese diabetic otsuka long-evans tokushima fatty (oletf) rats. methods: to induce liver fibrosis, 1.0ml/kg of pig serum was injected twice a week for 4 weeks in the oletf and leto rats. in the hepatocarcinogenesis model, glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions were induced by a single injection of 200mg/kg of diethyl nitrosamine (den). we also performed in-vitro studies to examine the mechanistic insights. results: the liver fibrosis development and gst-p-positive pre-neoplastic lesions were both markedly accelerated in oletf. in the fibrosis experiment, -smooth muscle actin-positive activated hepatic stellate cells (hsc) also increased in oletf along with augmentation of the hepatic collagen content and transforming growth factor-b1. in the den model, the neovascularization was up-regulated in oletf almost in parallel with the pre-neoplastic lesions development and a potent angiogenic factor, the vascular endothelial growth factor. our in-vitro study showed that both glucose and insulin stimulated the proliferation of the activated hsc and augmented the neovascularization. conclusion: these results indicated that the ir status directly accelerated the liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated hsc proliferation and hepatic neovascularization, respectively, in the rat. n. wakui 1 , t. ikehara 1 , r. takayama 1 , m. takahashi 1 , k. shiozawa 1 , h. nagai 1 , m. watanabe 1 , k. ishii 1 , k. iida 1 , y. igarashi 1 , y. sumino 1 1 case: a 68 years old man diagnosed with chronic hepatitis c regularly visited our hospital. in april of 2005, ultrasonography revealed a tumor 20mm in diameter in s2 of the liver and another tumor 15mm in diameter in s6/7 of the liver. the patient was hospitalized for further examination. computer tomography (ct) revealed that the tumor localized in s6/7 presented a pattern of hypervascular hepatocellular carcinoma (hcc). for the tumor localized in s2, the following were revealed. 1) contrast-enhanced ultrasound findings: a tumor vessel passed from outside the tumor to the center of the tumor in the early vascular phase, then radiated in a wheel-like shape at the center of the tumor; parenchymal phase perfused imaging in the area produced a similar imaging obtained from the area surrounding the liver. 2) ct: no tumor was detected. 3) spio-mri (t2 weighted imaging): iso-low intensity images were obtained. although these imaging findings indicated fnh, the patient was hcv positive. in order to disprove the possibility of hcc, a biopsy was performed on the tumor at s2 in the liver. the resulting diagnosis was well-differentiated hcc. discussion: until now, a characteristic finding of fnh has been spoke-like vasculariation, which is considered diagnostically quite important. however, some recent cases of hcc have been reported to present fnh-like vascularization. from now on, when evaluating a tumor that presents spoke-like vascularization underlining chronic hepatitis, the possibility of hcc should be considered and a close examination may be needed. chronic infection with hcv is problem .clinical management of chronic hcv depend on extent liver fibrosis .liver biopsy gold stander an invasive procedure responsible for severe complications and sample variability interpretation. serum biomarkers for inflammation/fibrosis investigated to wave liver biopsy. diagnostic accuracy panel of non-invasive serum biomarkers for hepatic fibrosis (fibrosure , apri score, forn's score) versus liver biopsy. 20 hcv patients subjected for: apri, forn's , fibrosure scores pcr quantitative hcv-rnaliver functions .lipid profile cbc . ultrasound guided liver biopsy. forns score; auroc (0.917) with 95% ci(0.791-1.042) for(fof1) vs. (f2f3f4) while(0.688)with 95% ci(0.464-0.91)for(fof1f2) vs.(f3f4). cutoff(>6.9)sensitivity for significant fibrosis(f2f3f4)and extensive fibrosis (f 3f4)were (100%) and with low specificity ,with accuracy(40%) and (20%)respectively.-apri score; auroc(0.792)with 95% ci( 0.568 -1.015)comparing(f0f1) vs.(f2f3f4)while was(0.875)with 95% ci(0.703 -1.047)for( f0f1f2)vs.(f3f4).cutoff(<0.5) had low sensitivity and specificity(100%)with accuracy(60%)for significant fibrosis and(80%)for extensive fibrosis.-fibrosure(fibro-acti test); showed best auroc(1.00)in different fibrotic stages with 95 % ci (1.00-1.00).cutoff(>0.59) sensitivity(50%)for significant fibrosis and(100%)for extensive fibrosis while specificity(100%)in all fibrotic stages. the ppv (100%)for significant and extensive fibrosis .npv and accuracy(75%, 80%)respectively for significant fibroses,while it was (100%) for extensive fibrosis respectively.significant correlation between liver biopsy and fibro-test(p0.002)and acti-test(p0.000).significant correlation between liver biopsy hepatitis activity score and apri (p 0.047)and forns score (p0.000). conclusion: forns score wasn't considered since does not discriminate between significant and extensive fibrosis. low sensitivity of apri prohibtes detection of minmal fibrosis and allow undetermined results. fibrosure classified all cases of chronic hcv sufficient to wave liver biopsy pe215 introduction: hcc is the 6 th common cancer. global increase of hepatitis b and c infection, the incidence of hcc steadily increasing. egypt seroprevalence of hcv in nile delta 20 -35%. afp had limited sensitivity 60% and specificity 90% for small hcc. gpc-3 oncofetal protein over expressed in hcc. evaluating validity of glypican-3 as early detector of hcc.:10 healthy controls and 40 hcv positive patients:10 patients chronic hepatitis c virus infection.10 patients compensated cirrhosis [child-pugh class a and b].10 patients decompensated cirrhosis [child-pugh class c]. 10 patients hcc. liver functions: alt, ast, bilirubin(t), albumin, gt.tumor markers: afp and gpc-3.viral markers: hcv antibodies, hbs ag and hbc ab. the median value of gpc-3 in hcc, dc, cc significantly higher than chronic hepatitis and control groups. no significant correlation between afp and gpc-3. auroc of afp 0.85 & auroc of gpc-3 0.84. the diagnostic sensitivity of afp (20 ng/ml) 70% with ppv 53.8%. the specificity85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) 100% with ppv 27%. the specificity 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved specificity to 87.5%. conclusion:gpc-3 although a serological test for early detection of hcc, showed limited specificity, where detected in different stages of chronic liver disease,it is oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 improve prediction accuracy of hcc patients in those showing seronegativity to afp. results: patients with hcv infection (n=388) were significantly older (mean age, 69 years) than patients with dual virus (n=75, 65 years) and hbv infection (n=752; 60 years) (p< 0.0001). the male-to-female ratio for hbv, dual virus and hcv group was 5.2, 3.4 and 1.3, respectively (p< 0.0001). patients in the hbv group more often had higher total tumor volume (mean, 409 cm 3 ) than the dual virus group (244 cm 3 ) and hcv (168 cm 3 ) group (p< 0.0001). no significant differences of the severity of liver cirrhosis, performance status, cancer staging and tumor cell differentiation were noted among the three groups. patients in the hcv group had a significantly poor survival in comparison to the hbv group only in the subset of patients with small tumor volume (< 50 cm 3 ) in the cox proportional hazards model (relative risk: 1.44, p=0.041). conclusions: dual hbv and hcv virus infection does not accelerate the speed of hcc formation in patients with chronic hepatitis b, and appears to have a modified course of carcinogenesis pathway diverted away from the biological behavior of hbv and hcv infection. background: patients presenting with hcc is not infrequent in our clinical practice. the aetiology vary ranging from hbv, hcv, nash and alcohol. the aim of this study was to see the aetiology of hcc in bangladeshi patients. methods: in this retrospective study, records of 976 patients who attended our opd between july 2004 to august 2008 were reviewed. patients having hepatic sol and/or heterogeneous echotexture of liver on usg and/or ct scan were included. diagnosis of hcc was confirmed at usg guided fine needle aspiration cytology with or without elevated serum afp (>500 ng/ml). results: of the 976 patients, 75% (732/976) had hbv infection. hcv infection was diagnosed in 17% (165/976). nash was responsible for 5% (49/976) cases, alcohol in 1% (10/976), while in the rest 2% (20/976) cases no specific aetiology could be established. conclusion: the study shows that hbv is the commonest cause for hcc in bangladesh followed by hcv. background: the aim of this study was to determine whether the hepatitis b virus (hbv) dna viral load and antiviral therapy is associated with hepatocellular carcinoma (hcc) recurrence. methods: this retrospective study involved 93 patients who underwent hepatic resection or radiofrequency ablation for initial hcc curative treatment. the patients were divided into four groups. fifteen patients with low serum hbv dna levels ( 4 log10 copies/ml) at the time of initial hcc treatment received antiviral therapy (lamivudine, adefovir, dipivoxil, entecavir) before hcc appeared (pre antiviral therapy group; pre-tg). thirty-four had low serum hbv dna levels without antiviral therapy (low virus group; lvg). fourteen had high serum hbv dna levels and received antiviral therapy after hcc appeared (post antiviral therapy group; post-tg). thirty patients had high serum hbv dna levels without antiviral therapy (high virus group; hvg). results: the cumulative hcc recurrence rates at 3 years in the hvg, lvg, pre-tg, and post-tg groups were 68.9%, 42.2%, 44.3%, and 57.1%, respectively. there were significant differences in the hcc recurrence rates between the hvg and lvg groups (p = 0.016), and between the hvg and pre-tg groups (p = 0.013). the recurrence rate was lower, though not significantly, in the post-tg group than in the hvg group (p = 0.18). conclusions: not only hbv dna viral load but also antiviral therapy is associated with hcc recurrence. antiviral therapy before hcc appears is important for patients with high serum hbv dna levels to prevent hcc recurrence. background/aims: few reports have described methods for predicting prognosis in unresectable hepatocellular carcinoma (hcc) patients, especially those treated by repeated transcatheter arterial chemoembolization (tae). to determine risk factors for death and determine prognosis in patients treated with repeated-tae, we evaluated clinical data. methodology: we retrospectively analyzed clinical parameters of 224 unresectable hcc patients treated with repeated-tae from january 1997 to december 2007. tae was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. factors affecting survival were evaluated using multivariate analysis after univariate analysis. next, we combined the score for each significant factor into a single prognostic score, after which the results were compared with jis and clip score methods. results: multivariate analysis revealed that bilobular hcc, alpha-fetoprotein ( 400 ng/ml), tumor invasion of the portal vein, tumor size ( 10 cm), and albumin (<2.8 g/dl) were related to poor prognosis, using those 5 factors, we developed a new prognostic scoring system. the 50% survival period was 29.2 months for all subjects, while it was 54.6, 29.2, 15.0, 5.6, and 1.0 months for those with scores of 0, 1, 2, 3, and 4 or over, respectively (p<0.0001), using our new system. clip score was not useful to predict prognosis, while jis score was better. however, subjects with jis scores of 1 and 2 were difficult to differentiate. conclusion: our scoring system was easy to perform and the results showed that repeated-tae was effective for unresectable hcc with a score of 2 or less. local ablative therapies and intrahepatic pressure c. kawamoto 1 , a. yamauchi 1 , k. kaneko 1 , n. miyagi 1 , k. kani 1 , t. aoyama 1 , k. yakabi 1 1 saitama medical center, saitama medical university, japan background: some of the unexpected recurrence observed after radiofrequency ablation (rfa) might be caused by increased intratumoral pressure. the present study examined the relationship between local ablative therapies and intrahepatic pressure. methods: a. basic study: under general anesthesia, laparotomy was performed on 19 pigs. a leveen needle and a percutaneous ethanol injection (pei) needle were inserted into the liver and intrahepatic pressure was monitored using an invasive blood pressure monitor. ablation was performed as follows: 1. rfa. 1) single-step method: after fully deploying the electrode, the power was initially applied at 30 w, then increased in increments of 10 w/min until power roll-off. 2) multi-step method: the array was deployed in 8 steps. at each step, the power was fixed at 30 w until power roll-off. 2. pei. injection of ethanol (2 ml). b. clinical study: we examined the multi-step rfa and pei for hcc. under local anesthesia, intratumoral pressure was monitored. 1. rfa. 39 patients with a mean tumor size of 15.3±4.9 mm were studied. 2. pei. in 10 patients with a mean tumor size of 21.4±8.8 mm, 5 to 10 ml of ethanol was injected per session. results : a. basic study: the intrahepatic pressures were: single-step method, 154.5±30.9 mmhg; multi-step method, 24.1±18.2 mmhg; and pei, 12.0±8.5 mmhg. b. clinical study: intratumoral pressure was 39.5±27.9 mmhg for rfa and 12.2±9.0 mmhg for pei. conclusion: these results suggest that consideration of intrahepatic pressure is crucial in local ablative therapies. background: a late evening snack (les) is recommended for liver cirrhosis. however, no clinical study has evaluated the nutrition status and the effect of les in cirrhotic patients with hepatocellular carcinoma (hcc). we investigated the effect of les undergoing hepatic arterial infusion chemotherapy (haic) in patients with hcc. method: nineteen patients with hcc were enrolled. ten patients were les group, and nine were control group. in the les group, the patients received les supplementation with a branched-chain amino acid (bcaa)-enriched nutrient mixture. in the control group, the patients received ordinary food. there were no significant differences in relation to age, gender, etiology, child-pugh scores, tumor stage, clinical responses to haic between two groups. blood biochemical data, nutrition status using an indirect calorimeter were evaluated at before and at the end of chemotherapy. results: the non-protein respiratory quotient (nprq) and molar ratio of branched-chain amino acid to tyrosine (btr) were significantly improved in the les group but not in the control group. there were no significant differences in the area under the concentration curve for glucose between before and the end of chemotherapy in two groups. background & aims: hepatocellular carcinomas (hccs) often show hypoor mixed vascularity, and the prognosis of these relatively hypovascular hccs is not fully elucidated. cytokeratin (ck) expression profiles may also be useful prognostic indicators, and specifically ck19 may reflect metastastic potency in hccs. this study was to assess the prognostic implication of tumor vascularity and its relation to ck19 expression in hcc patients. methods: a total of 150 patients who underwent surgical resection for hcc were enrolled. tumor vascularity was evaluated according to arterial enhancement pattern on ct scans and ck19 expression was evaluated using tissue microarray methods. clinicopathologic data were analyzed using kaplan-meier and cox proportional hazard model. results: during follow-up period, 91 (60.7%) patients experienced tumor recurrence. forty-five patients (30%) had hypovascluar tumor at the time of diagnosis, and they showed significantly higher positivity for ck19 expression (p=0.001) and shorter disease-free survival (p=0.023) than patients with hypervascular hccs. in addition, recurred tumors in these patients showed more frequently hypovascular pattern than in patients with hypervascular hccs (p=0.001). hypovascularity at initial diagnosis and microvascualr invasion were independent poor prognostic factors predicting survival. following treatment of recurred hccs, hypovascular tumors showed poor response to transarterial chemoembolization (tace), which resulted in shorter overall survival than hypervascular tumors (p=0.057). conclusions: these results demonstrate that tumor hypovascularity in hccs is associated with positive ck19 expression, early tumor recurrence, poor tace response and poor survival. therefore, tumor vascularity may also be a prognostic indicator in hcc patients. background: hepatic stellate cells (hscs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma(hcc). in this study, we determine gene expression changes in two different models of hscs activation and investigate whether induction-activated hscs(ihscs) gene expression changes are different from culture-activated hscs(ahscs). methods: hscs were isolated by density centrifugation and exposed to conditioned medium from rat hcc cell lines c5f. twenty-seven thousands and one hundred gene expression between quiescent hscs(qhscs), ahscs and ihscs was analyzed by microarray and confirmed by real-time rt-pcr and western blot. results: sixteen hundreds and seventy-one probe sets were differentially expressed in ahscs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors. seven hundreds and eleven probe sets were differentially expressed in ihscs. induction-activated hscs showed specific gene expression patterns including raf1, rac2, adam17, wnt6, mmp-9 and tnf, suggesting that hcc cells can specifically induce hscs activation. induction-activated hscs might play a important role in invasion and metastasis of hcc. conclusions: induction-activated hscs gene expression patterns are different from ahscs. culture-activated hscs does not properly regulate gene expression in hscs, suggesting that ihscs may be considered the model for the study of hscs biology in hcc. background: hepatocellular carcinoma (hcc) is a hypervascular tumor, and angiogenesis is important for tumor growth. ephrin receptors are related with vascular system development and the polymorphism of ephb1 in the carcinogenesis of digestive tract has been reported. our aim was to examine the polymorphsims of ephb 1 with the occurrence of hepatocelluar carcinoma in korean population. methods: genomic dna was extracted from 182 patients with hepatocellular carcinoma (hcc), 266 healthy subjects. ephb 1 polymorphism was determined by polymerase-chain reaction-based assays, and the association with hcc was investigated. results: with regard to ephb 1 polymorphism, a/a genotype at rs11926992, t/t genotype at rs7644369, a/a genotype at rs6776570, t/t genotype at rs3821502 and g/g genotype at rs6766459 were significantly associated with hcc but these were not associated with clinical characteristics of hcc. conclusions: five out of seven polymorphisms on ephb1 gene were statistically associated with hcc, in the korean population. therefore, more studies of ephb1 gene polymorphisms including various risk factors should be performed to use as genetic markers of hcc occurrence. background: we aimed to compare the results of hepatectomy for hcc in patients older than 70 years old with those for younger patients. methods: clinicopathological data and outcomes for 155 elderly patients and 333 younger patients with hcc who underwent hepatectomy between 1992 and 2007 were retrospectively compared. results: although postoperative delirium was more common in the elderly group, there were no significant differences between the 2 groups with regard to operative morbidity, hospital death, disease-free survival, and overall survival. the overall recurrence rate was significantly higher in the elderly patients with alcohol abuse than in younger patients with alcohol abuse. multivariate analysis revealed that preoperative alcohol abuse was a prognostic factor for elderly patients. conclusions: elderly patients with preoperative alcohol abuse should be followed closely, even after r0 surgery, because alcohol abuse is strongly correlated with postoperative recurrence and worse survival. background: little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. methods: among 410 patients who underwent curative resection between 1992 and 2005, 180 patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group a), while 46 patients were only transfused with packed red cells (group b), 43 patients were only transfused with fresh frozen plasma (group c), and 141 patients had no transfusion (group d). results: group c had significantly fewer postoperative complications and a shorter hospital stay than group a. preoperative coagulation was significantly worse in group c. survival was significantly better in groups c and d than in group a. conclusions: perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma. background: this study investigated risk factors for postoperative liver failure after resection of hepatocellular carcinoma to detect markers that could identify candidates for hepatectomy. methods: perioperative risk factors for liver failure after hepatectomy were analyzed in 191 patients with hepatocellular carcinoma. results: liver failure occurred postoperatively in 16 patients, 3 of whom died. the hyaluronate/gsa-rmax ratio was a risk factor for postoperative liver failure by univariate analysis and was the only risk factor according to multivariate analysis. all 3 patients who died had a hyaluronic acid/gsa-rmax ratio 500 mg min/dl. conclusions: to reduce postoperative liver failure, preoperative planning should employ various measures of the hepatic functional reserve, including tests of both parenchymal and nonparenchymal liver function. the hyaluronate/gsa-rmax ratio can predict liver failure after hepatectomy, and a ratio greater than 500 mg min/dl is a relative contraindication to liver resection. the patient was a 73-year old japanese man with chronic hepatitis c(ch-c) who achieved a sustained virological response(svr) to interferon(ifn) therapy. as a result the liver functions were normalized and the histological findings of the liver also improved. however, 13 years after svr, mild liver dysfunction was noticed along with a marked increase of tumor markers. several modalities revealed huge liver tumors about 13 cm in greatest diameter in the left lobe invading the bile ducts and another tumor about 3 cm diameter in segment v. we performed liver biopsy and confirmed that this tumor was well-differentiated hepatocellular carcinoma (hcc). only mild fibrosis development could be observed in the adjacent non-cancerous lesions. we successfully treated these tumors with transcatheter arterial chemoembolization and stereotactic radiosurgery. recent studies revealed that the risk of developing hcc still exists even after svr. since most of hcc that develop in patients with svr are usually detected within 5 years, several investigators speculate that hcc is already present but too small to be detected at the time of completion of ifn therapy. this speculation is not the case in our patient, since svr was achieved 13 years ago and no hcv-rna could be detected when hcc appeared. therefore, another possible mechanism should be considered. an annual follow-up with strict surveillance program for hcc should be performed for more than 10 years after the completion of ifn therapy. background/aims: in order to investigate the role and importance of oxidative stress as to carcinogenecity of hepatocellular carcinoma (hcc) we analyze the expression of 8-hydroxydeoxyguanosine (8-ohdg) in the liver tissue of the hcc patients with and without hepatitis viral marker. methods: patients undergoing hepatic resection for the first hcc from 1995 to 2004 were enrolled into the study. only the cases that took no alcohol or small amount of alcohol were enrolled. 24 cases were negative for hepatitis b surface antigen (hbsag) and antibody to hepatitis c virus (hcvab) (nbnc group). 24 were positive for hbsag and negative for hcvab (b group). 21 were positive for hcvab and negative for hbsag and antibody to hepatitis b core antigen (c group). staining with hematoxylin and eosin (h&e) and berlin-blue, and immunohistochemical staining for 8-ohdg were performed using the non cancerous liver regions. the degree of 8-ohdg immunostaining was expressed as the labeling index, which means the percentage of positive hepatocytes per 1000 hepatocytes. results: the labeling index of 8-ohdg for nbnc group is 19.3(±55.3), significantly lower (p=0.014) than that for b group 49.7(±89.5), and also lower (p=0.016) than that for viral group (b group and c group)(35.4±71.8). the labeling index of 8-ohdg had no correlation with grading, staging, fatty and iron deposit among all cases. conclusions: there is possibility that oxidative stress might not associate with the carcinogenesis of hcc in some cases without hepatitis viral infection. background: no effective chemopreventive agent has been approved against hepatocellular carcinoma (hcc) yet. since neovascularization plays a pivotal role in hcc, an angiostatic agent is considered as one of the promising approaches. recently, it has reported that vitamin k2 (vk) and angiotensin-converting enzyme inhibitor (ace-i) exert anti-angiogenic activity. the aim of the current study was to elucidate the combination effect of the clinically used vk and ace-i on cumulative recurrence after curative treatment, especially in consideration of neovascularization. methods: vk (menatetrenone; 45 mg/day) and/or ace-i (perindopril; 4 mg/day) were administered for 36 to 48 months after the curative therapy for hcc. the cumulative recurrence and several indices were analyzed. results: a 48-month follow-up revealed that the combination treatment with vk and ace-i markedly inhibited the cumulative recurrence of hcc in association with suppression of the serum level of vascular endothelial growth factor (vegf); a central angiogenic factor. the serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with vegf. these beneficial effects were not observed with single treatment of vk or ace-i for 36 months. conclusions: the combination treatment of vk and ace-i may suppress the cumulative recurrence of hcc after the curative therapy, at least partly through suppression of the vegf-mediated neovascularization. aim: the aim of this study was to clarify the cilnicopathologic features and management of hepatocellular carcinoma (hcc) patients surviving more than 5 years after hepatectomy. materials & methods: retrospective study was carried out on 823 hcc patients who underwent curative hepatectomy between 1973 and 2002. clinicopathologic factors in 5-year survivors and patients who died within 5 years were compared. the prognostic factors affecting survival were examined among the 5-year survivors. results: there were 290 patients who survived for more than 5 years after initial hepatectomy, and 83 of those patients survived for more than 5 years after hcc recurrence. the overall 3-, 5-, 10-and 20-year survival rates were 57.4%, 40.9%, 17.2%, and 8.5% respectively. in multivariate analysis, absence of underlying cirrhosis, solitary tumor, alfa-fetoprotein less than 1000 ng/ml, and absence of microscopic vascular invasion were favorable independent factors associated with 5-year survival. negative hepatitis c virus antibody status was favorable independent factor associated with longer disease-free interval and survival after tumor recurrence. multimodal treatments such as repeat hepatectomy or percutaneous ablation led to improved survival after recurrence, compared with the survival after transarterial chemoembolization (p<.05). conclusions: the results suggest that patients without underlying cirrhosis who have a solitary hcc that does not demonstrate vascular invasion or high afp levels might survive for longer than 5 years after the initial hepatectomy. close follow-up and multimodal treatment could contribute to prolongation of survival in such patients, even if cancer recurrence occurs. the history of the use of carbon ion radiotherapy (cirt) for treating hepatocellular carcinoma (hcc) goes back to 1995, when clinical trials were initiated at the national institute of radiological sciences. we have already reported that cirt used for the treatment of hcc is safe and effective, and that it causes only minor liver damage. in a phase ii clinical trial, the local control and cumulative overall survival rates were 94 % and 35 % at 5 years, respectively. however, the patients with tumor adjacent to the gastrointestinal tract are thought to be ineligible for cirt because of the high risk of radiation injury of the digestive organs. in order to extend the indication of cirt, we have challenged the cirt for such patients under the use of spacers. a case was a 67-year-old female with 8cm tumor in segment 4. in radiological findings, the tumor revealed typical enhancement pattern for hcc, and was near the ec junction. she had been judged ineligible for hepatectomy because of the high retention rate of indocyanine green. she could undergo the 42.8 gye/2-fraction cirt after the placement of gore-tex soft tissue patch under the laparoscopic procedure. up to the present date, no adverse effect due to the spacer has been occurred, and an apparent anti-tumor effect has been observed. this method seems to have a promising efficacy for extension of the indication of cirt to the patients with tumors adjacent to the gastrointestinal tract. background: previously we reported that high ubiquitination was marker of human hepatocellular carcinoma. on the basis of these finding, we firstly analyzed the effect of bortezomib(proteasome inhibitor) on human hcc cell line. we also reported that hhm/dip1/gcip1 was early marker for human hepatocarcinogenesis. hhm was suggested to be a new tumor suppression gene, but the mechanism was not well confirmed. we analyzed change of hhm signal by bortemib. method and result: we used hcc cell line (huh7, hlf, hepg2) . the inhibitory effect of bortezomib was evaluated using mtt assay. 20nm bortezomib significantly inhibited proliferation of hcc cell line. the inhibitory effect by 20nm bortezomib was similar with 10 m cisplatin. on the other hand, bortezomib has no inhibit effect in isolated hepatocyte from rat. in this condition, we analyzed the expression of cyclin d1, phospho-rb and hhm in hcc cell line by western blot analysis. expression of cyclin d1, phospho-rb decreased, but hhm was increased with time. next we analyzed cell cycle by facs. bortezomib induced hcc cell line into cell cycle arrest in g2/m. the transcriptional activity of hhm was also activated by bortezomib administration using ptimer-promoter-hhm plasmid. conclusion: bortezomib has specific anti-proliferative effect on hepatocellular carcinoma. the induction of hhm by bortezomib might be related with cell cycle arrest. bortezomib will be a useful drug for hcc. neovascularization is required for carcinogenesis of non-alcoholic steatohepatitis: experimental and clinical study m. kitade 1 , h. yoshiji 1 , r. noguchi 1 , k. kaji 1 , t. namisaki 1 , y. aihara 1 , h. background/aim: non-alcoholic steatohepatitis (nash) may progress to liver cirrhosis, and finally hepatocellular carcinoma. recent study suggested that development of hepatic angiogenesis correlates the risk for hepatocarcinogenesis in liver cirrhosis patient. we therefore examined the role of angiogenesis in the hepatocarcinogenesis of nash in both experimental and human study. methods: as an experimental nash model, zucker (z) rats, which naturally develop leptin receptor mutations, and their lean littermate (l) rats were fed a choline-deficient, amino acid-defined (cdaa) diet. in human study, 11 patients with nash-related cirrhosis or pre-cirrhosis, regarded as high risk group of hepatocarcinogenesis, and 13 with simple fatty liver (fl) were enrolled and underwent clinico-pathological examinations. immunohistochemical analysis of 4-hydroxy-2-noneal (4-hne) and cd34 were employed for detection of reacrive oxidative stress (ros) and angiogenesis in the liver tissues, respectively. results: in experimental nash model, both groups showed marked steatohepatitis by feeding cdaa diet. in sharp contrast, the development of glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions and hcc could be observed only in the l-rats. the hepatic neovascularization was also significantly increased only in the l-rats. in human study, both nash and fl exerted a marked elevation of ros. in sharp contrast, significant development of hepatic neovascularization was observed only in nash, whereas almost no neovascularization could be observed in fl. conclusion: in conclusion, these results suggested that neovascularization might play a important role in hepatocarcinogenesis in nash. background: paternally expressed gene 10 (peg10), which was an imprinted gene with an active paternal allele but silent maternal allele, was highly expressed in a great majority of hepatocellular carcinoma(hcc). the aim of this study was to generate transgene mice expressing peg10 in the liver under the control of mouse albumin (alb) promoter and study the integration, transcription, expression of peg10 gene in the transgenic mice methods: the linearized 1716bp transgene fragments, which contained alb promoter and structural gene of peg10, were microinjected into fertilized eggs of mice. then manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. all the newborn mice were screened and identified by pcr detecting genomic dna in tail tissue. as the transgene was driven by the alb promoter, we examined its expression in the liver of transgenic mice by rt-pcr and western blotting. results: the transgene fragment was microinjected into the male pronucleus of 3741 fertilized oocytes. the injected eggs were implanted into oviducts of 94 pseudo-pregnant foster mothers, of which 22 mice became pregnant and give birth to 108 offspring. 65 of them died from unknown reason. among the 43 offspring, 3 were identified to carry peg10 cdna as demonstrated by pcr, and peg10 transgene could be expressed successfully in the liver of the established transgenic mice. the ratio of transgene integration were 6.97% (3/43) by pcr. conclusions: the peg10 transgenic mouse model should be valuable for studying the in viro function of this imprinted gene in hcc. background/aims: brivanib alaninate is the l-alanine ester prodrug of bms-540215, an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. it is being developed in treating hepatocellular carcinoma (hcc), a disease highly prevalent in asia-pacific region. this analysis investigated whether bms-540215 exposure was different between asian and non-asian subjects. methods: a population pharmacokinetic (ppk) model was developed with data collected in 125 subjects (78 non-asian, 47 asian) with advanced and metastatic solid tumors (including hcc) from 2 clinical studies. potential effects of the following covariates on model parameters were examined: age, gender, race, and baseline body weight. model-based simulation was performed to examine bms-540125 exposure in asian and non-asian patients following brivanib doses of 800 mg qd (phase iii dose). results: the ppk of bms-540215 was characterized by a 2-compartment model with first-order absorption and elimination. clearance was found to slightly increase with body weight (p<0.01). however, effects of age, gender and race on clearance were not statistically significant. the median of apparent clearance in asian was 9.5% lower than that of non-asians, which was adequately explained by 16% lower body weight in asians. there was substantial overlap in steady-state bms-540215 auc of asian and non-asian patients, simulated based on their observed body weight distributions in these patient groups. conclusions: bms-540215 pk can be adequately described by a linear 2-compartment model; exposures in asian and non-asian subjects are similar following brivanib doses of 800 mg qd. background/aims: hepatic resection is the standard treatment for hepatocellular carcinoma. in some patients with multiple hcc, one-block resection can not be feasible due to either the tumor location or the reserved liver function. in this study, we attempted to analyze the outcome of multiple-site resection or combined resection and rfa in patients with multiple hcc. the prognostic factors for postoperative survival were also investigated. methods: among 507 patients who received resection from january 1996 to august 2006, 58 patients had a radiologically detected multiple hcc. patients with multiple hcc were divided into: group a, patients treated with one-block resection (n=40) and group b, patients with multiple-site resection or combined resection and rfa (n=18). results: in group b, 6 received multiple-site resection and 12 underwent combined resection and rfa. the clinicopathological variables and postoperative complication rate were not significantly different between the two groups. the 5-year disease-free survival rates for group a and b were 24.1% and 18.3%, respectively (p=0.386). the overall survival rates were also not significantly different (36.9% vs. 39.4%, p=0.528). the multivariate analysis revealed that radiological tumor number 3, edmondsons-steiner grade (iii-iv) and indocyanine green retention rate at 15 minutes> 10% were adverse prognostic factors for overall survival. conclusions: active treatments including multiple-site resection and combined resection and rfa showed similar treatment outcomes compared with one-block resection in patients with multiple hcc. the prognosis after treatment was associated with tumor number, tumor grade and icg r15. background: nasopharyngeal carcinoma (npc) is endemic to southern china. mortalities are mostly associated with secondary metastases. novel treatments for npc metastases are thus urgently needed. we aim to test the efficacy of a physiologically stable gold compound, gold (iii) meso-tetraarylporphyrin 1a (gold-1a), in treating intrahepatic npc metastasis in athymic mice. methods: twenty million of c666-1 human npc cells were injected into the livers of athymic mice to induce primary tumors. gold-1a was administrated by intraperitoneal injection. survival times, tumor volumes and degrees of metastasis of the animals were evaluated. intratumoral microvessel density was determined by immunohistochemical staining for cd34. tube formation by ms1 mouse endothelial cells were conducted with an in vitro angiogenesis assay kit. gene expression level was determined by semi-quantitative reverse transcription-polymerase chain reaction. cell proliferation was performed by methylthiazolyldiphenyl-tetrazolium bromide assay. result: gold-1a prolonged the survival and inhibited intrahepatic and lung metastasis of the tumor-bearing animals. the compound induced tumor tissue necrosis and reduced tumor microvessel formation. in in vitro studies, gold-1a inhibited tube formation and proliferation of ms1 cells, and downregulated the expression of stanniocalcin 1 (stc1), which plays roles in angiogenesis. furthermore, our preliminary data showed that overexpression of stc1 in ms1 cells rescued cells from gold-1a-induced death. conclusion: gold-1a is a novel anticancer agent that prolongs survival of the npc metastases-bearing mice. it inhibits intrahepatic and lung metastasis in vivo and inhibits angiogenesis in vitro, in part via downregulation of stc1. tbx3 is a transcriptional repressor that is important for embryonic development. overexpression of tbx3 was found in a large variety of cancers, including breast cancer, ovary cancer, cervical cancer, lung cancer, bladder cancer and liver cancer. tbx3 promote carcinogenesis by bypass cellular senescence via suppression of p14 arf . our resent studies revealed that two key motifs composed of 7 + 3 residues are essential for its transcriptional repression. based on this finding, we designed a set of peptides to block its transcriptional repression activity and tested their antiviral effects. we found that tat-tagged peptides (taps) effectively transduced hepatoma hepg2 and bel7404 cells at almost 100% efficiency and inhibited cell growth in a dose dependent manner. further studies revealed that the tap treated cells underwent up-regulate apoptosis via suppression of p14 arf both at mrna and protein levels, demonstrating the potential of novel taps for anti-hcc treatment in the future. safety and long-term outcomes of radiofrequency ablation therapy in elderly and cirrhotic patients with hepatocellular carcinoma k. kakisaka 1 , h. kuroda 1 , k. kasai 1 , y. takikawa 1 , k. suzuki 1 1 iwate medical university background and purpose: a tendency of the aging in patients with hepatocellular carcinoma (hcc) is predominantly seen in japan. in fact, the mean age of patients with hcc in our institute in 1990 was 60.1 years old, while that in 1999 was 67.8 years old. it is not still remained whether the percutaneous radiofrequency ablation (rfa) therapy in elder patients with hcc is safety and equal in therapeutic usefulness compared to the non-elder patients with hcc. subjects and methods: two hundred six cirrhotic patients with hcc (376 tumor nodules) received rfa therapy curative intent since august, 2006 were enrolled. we divided all patients into two groups: over 65 years (elder group: n=135) and under 65 years (non-elder group: n=71), and compared the patient's characteristics, tumor factors and survival rate and causes of death in two groups. results: the characteristics of patients, tumor factors, cumulative survival rate and recurrence rate were not revealed in two groups. although in elder group two patients complicated aspiration pneumonia and respiratory depression due to sedation under rfa respectively, total occurrence rate of complications did not differ between two groups. conclusion: rfa therapy is safety and effective even in elder patients with hcc, although their care is necessary to prevent any complications which are often occurred during the rfa therapy. background and purpose: the aim of this study is to evaluate whether administration of the branched-chain amino acid (bcaa) enriched nutrient (namely, aminoleban en, ostuka pharmaceutical company, japan) might improve protein-energy malnutrition (pem) status and quality of life (qol) in cirrhotic patients with hcc receiving rfa therapy. subjects and methods: thirty-five cirrhotic patients with hcc who had received rfa therapy from october 2005 to october 2006 in our institute were randomized into two groups: diet with supplementation of aminoleban en (en group: 20 patients, 420 kcal/day) and diet only (control group; 15 patients). the total intakes of calories (30-35 kcal/kg) and protein (1.0-1.5 g/kg) were equal between tow groups. the primary end point was event-free survival rate (development of liver cancer, rupture of esophageal varices, or progression of hepatic failure) and second end points were serum albumin levels and the health-related qol by shortform-8 questionnaire (sf-8). results: total intakes of calories and protein were similar during the one year after rfa. no significant differences in event-free survival rate were seen between two groups. however, decreased serum albumin levels and one (general health perception) of domains in sf-8 were significantly improved in en group compared to the control group. conclusion: supplementation of bcaa-enriched nutrient may improve the impaired liver function and qol after rfa therapy. large scale prospective study should conduct to confirm these results near the future. backgrounds and aims to investigate the effects of selective cox-1 and cox-2 inhibitor on proliferation and apoptosis of hcc cell. methods hep3b and snu 387 cells were treated with ns-398 and sc-560. mtt assay, caspase 3/7 activity assay and tunel assay were performed. cox protein and mrna expression were measured by western blot and real time rt-pcr. results in hep3b cell line, cox-1, cox-2 (50, 100, 200 um) and combination (25+25, 50+50, 100+100 um) treatment after 24hr showed a significant dose dependent inhibitory effect on cell growth (p<0.05). cox-1, cox-2 (100 um) and combination (50+50, 100+100 um) treatment after 24hr significantly increased caspase 3/7 activity (p<0.05) and induced apoptosis (p <0.05). however, the combination treatment could not showed a additive effect to cox-1 or cox-2 inhibitor (p>0.05). in snu 387 cell line, cox-1 inhibitor and combination treatment showed a inhibitory effect on cell growth (p <0.05) similar to hep3b cell line but any of treatment could not induce apoptosis significantly (p >0.05). in cox protein and mrna expression, snu 387 cell line showed significant cox-1 predominency (p=0.037) but hep3b cell line showed cox-2 predominency (p=0.032). conclusions in hcc cells, no additive effect of the combination treatment of cox-1 and cox-2 inhibitors could be anticipated. the apoptosis inducing effect of cox inhibitor could be different between hcc cell lines. more studies for the mechanism of different response to cox inhibitor between cell lines is needed. background: the aim of this study was to determine the maximum tolerated dose and recommended dose of combination chemotherapy with mitoxantrone and uracil/tegafur (uft) (phase i part), and to clarify its efficacy (tumor response, overall survival, and progression free survival) and safety in patients with advanced hepatocellular carcinoma (hcc) at the recommended dose (phase ii part). methods: patients eligible for study had histologically confirmed, chemo-naïve advanced hcc, who were unsuitable for resection, local ablation therapy or transcatheter arterial chemoembolization. the therapy consisted of mitoxantrone dosages (6, 8 and 10 mg/m 2 /day) intravenously on day1 and oral administration of uft 300 mg/m 2 on day 1 through day 21. the treatment was repeated every four weeks if there was no evidence of tumor progression or unacceptable toxicity. results: a total of 25 patients were entered into the study. all had a good ecog performance status score of 0-1. in phase i part, dose limiting toxicities occurred in all three patients (two patients: grade 4 neutropenia, one patient: grade 3 creatinine elevation) given mitoxantrone at dosage of 10 mg/m 2 /day, and the recommended mitoxantrone dosage was 8 mg/m 2 /day. among 19 patients administered at the recommended dosage, one patient (5.3%) achieved a partial response, 8 patients (42.1%) had stable disease and 10 patients (52.6%) had progressive disease. one-year survival proportion, median survival and median progression free survival were 26.3%, 8.4 months and 2.5 months, respectively. the most common toxicities were grade 3-4 leucopenia (15.4%) and neutropenia(10.3%). conclusion: mitoxantrone 8 mg/m 2 with uft 300 mg/m 2 /day is recommended dose. this regimen is generally well tolerated, but appears to have little activity for advanced hcc. these findings do not support its use in practice, and further trials with this regimen in patients with advanced hcc are not recommended. the study assessed the benefits of 3-d reconstruction of spiral ct scans for the diagnosis of and surgical guidance to large liver tumors or tumors at the hepatic hilum. we retrospectively analyzed 33 cases of children with such tumors treated in past 5 years.the patients were examined by 3-d reconstruction using 64 slice spiral ct. in 28 cases, the volume of tissue removed exceeded 1/3 the entire volume of the liver. in 5 cases, the excised tissue represented less than 1/3 of the total liver volume, but the location of the tumor was adjacent to major hepatic vessels. pathological diagnoses included hepatoblastoma (n = 20), hepatocellular carcinoma (n = 4), mesenchymal hamartoma (n = 4), teratoma (n = 1) and adenoma (n = 4). all children had curative resections with tumor-free microscopic margins. 3-d ct imaging can provide high quality images and accurate location of the tumors. it could help the surgeon identify the tumor borders accurately and devise a safe surgical strategy. with its help the surgeon could identify vital hepatic blood vessels before operation, and can avoid massive hemorrhaging during operation. background: to investigate the association between c-509t polymorphism of transforming growth factor (tgf)-1 gene and hbv-related hepatocellular carcinoma (hcc). methods: patients with hbv infection (196 cases were hbv carriers, 379 cases were hcc) and 299 healthy volunteers were enrolled. the polymorphism of tgf-1 gene c-509t was identified by polymerase chain reaction-restriction fragment length polymorphism method. the concentrations of plasma tgf-1 were measured by enzyme linked immunosorbent assay (elisa). tgf-1 mrna expression was quantified by real-time pcr. a recombinant construct containing -509c>t variant as promoter and cat as reported gene was transfected into hepg2 cells. the reporter gene cat was detected with elisa. results: the ct genotype at position -509 of tgf-1 gene prevailed in all three groups, the frequency of genotype cc and allele c at -509 in hcc were significantly higher than those of the hbv carriers and controls. the plasma tgf-1 concentration among the three genotypes did not show any significant difference in three groups. however, both the tgf-1concentration and liver mrna levels were statistically higher in patients with cc genotype than in those with tt genotype in the hcc group. reporter gene cat was elevated when hepg2 were transfected with -509c-cat recombinant construct compared to that with -509t-cat one (p<0.05) conclusion: the presence of c allele at position -509 may play an important role in the development of hbv-related hcc through influencing tgf-1 expression both at mrna level and protein level. background: to assess diagnostic value of n-glycan markers in identifying hepatocelluar carcinoma (hcc) from liver fibrosis after hbv infection. methods: a total of 273 cases of hbv related liver fibrosis (n=128) and hcc (n=145) patients as well as matched healthy controls (n=120) were recruited. routine liver function and tumor markers were detected by automatic biochemistry or immunological analyzer. n-glycome of serum protein was profiled by dna sequencer-assisted fluorophore-assisted carbohydrate electrophoresis with a capillary electrophoresis-based abi3130 sequencer. results: the abuncance of a single agalacto biantennary glycan (ng1a2f, peak 4) was increased in liver fibrosis and decreased in hcc, while that of a branching triantennary glycan (na3fb, peak 9) was decreased in fibrosis and increased in hcc. the efficacy of the log ratio of above two n-glycan abundance [log (p9/4)] was similar to afp in differentiation hcc from fibrotic patients. with logistic regression analysis, the accuracy and sensitivity of the diagnostic model combining afp with n-glycan analysis(cscore b) were increased 7-10% compared to afp. log(p9/4) was even more powerful in monitoring the progresison of hcc with the specificity improved 16% and accuracy improved 8% compared to that of afp. besides, log(peak 9/4) was correlated well with other tumor markers and tnm stages. conclusions: the log ration of the abundance of a branching triantennary glycan (na3fb, peak 9) to a single agalacto biantennary glycan (ng1a2f, peak 4) and the model combining afp with n-glycome markers are promising in hcc diagnosis and progression monitoring. the low incidence of tumor seeding and post-procedure bleeding after radiofrequency ablation (rfa) of hepatic tumors has been attributed to the use of thermocoagulation of the tract, which results in necrosis, upon electrode withdrawal. however, different investigators use different techniques with no experimental evidence of the effectiveness of a particular technique. objective: we aimed to compare the necrotic zone produced using different electrode withdrawal techniques. methods: eighteen tract ablation zones were created in ex vivo porcine livers by withdrawing an internally-cooled rfa electrode (cool-tip radiofrequency system, valleylab) 1-2 mm/second using energy-dependent (20 vs.40 vs.60 vs.80 watts) and temperature-dependent (80 vs. 90 0 c) techniques. horizontal mathematical modeling suggests an impractical number of radiofrequency ablation (rfa) zones needed in order to ablate a medium-large hepatic tumor. however, overlapping rfa zones may increase the necrotic diameter disproportionately to that deduced from single ablation alone. objectives: to compare the necrotic diameter in single (group1), dual overlapping (group2) and dual non-overlapping (group3) ablation. methods: single (n=5) and dual (overlapping n=18; non-overlapping n=6) ablation zones were created in ex vivo porcine livers using cool-tip rfa electrodes. necrotic diameter was measured at the midpoint (maxd) of the single and the two distinct rfa zones of the dual ablation groups and compared with the necrotic diameter at the tip of the second ablation (maxd-o), corresponding to the point of overlap in group2. the rfa electrode was withdrawn 2.5 and 4.1 cm before re-ablating for group2 and group3, respectively. results: despite no difference in end-rfa temperature between 3 groups (group1=75.6+7.4cvs.group2=73.9+7.3cvs.group3=74.8+4.8c; p=0.873), maxd was significantly greater (p=0.011) in group2 (4.1+0.7cm) as compared to group1 (3.3+0.6cm) and group3 (3.3+0.4cm), with no difference between group1 and group3(p=1.00). further proof of synergism between two overlapping ablations is that the maxd-o in group2(4.3+0.6cm) was larger than maxd of group1 (p=0.042) and group3 (p=0.028), and was similar to maxd of group2 (p=1.00). conclusions: overlapping two rfa zones results in incremental increase in necrotic diameter compared to single and dual non-overlapping ablation. this may explain the discrepancy in the number of ablation zones needed between clinical and mathematical modeling studies. background: hepatocellular carcinoma (hcc) is the fourth most common cancer worldwide, main etiological factors being chronic infections with hepatitis b and c viruses. the present study was undertaken to evaluate the association of glutathione-s-transferase (gst) t1 and m1 null genotypes and microsomal epoxide hydrolas e(mephx) polymorphisms with hepatitis virus related hcc risk in indian population. subjects and methods: three groups of subjects were considered viz. control (n=169), chronic viral hepatitis (n=174) and hcc (n=63). pcr-rflp was used for this polymorphic study. genotype distributions between categories were compared using the 2 test; odds ratios (ors) and 95% ci were calculated to express the relative risk. results: presence of gstm1 null genotype significantly (p<0.05) decreased the risk for hcc development among chronic viral hepatitis subjects. however, gstt1 null genotype was associated with an increased risk for hcc by 2.23 and 1.42 times among control and hepatitis subjects respectively. in case of mephx, tyr113his and his113his genotypes significantly (p< 0.05) reduced the risk of hcc development in both viral hepatitis and control subjects. in case of mephx exon 4 genotypes, arg139arg imposed an approximate 2 fold risk for hcc development in the two groups. combination of heterozygous mutant genotypes at mephx exons 3 & 4 also imposed around 2 fold risk (non-significant) for hcc. conclusions: polymorphic forms of gst and mephx share an association with viral related hcc risk in indian population and should be further evaluated as the candidate genes to determine individual susceptibility for viral related hcc. background : the association between type 2 diabetes mellitus (dm2) and hepatocarcinoma (hcc) has been identified in the last ten years. methods: to clarify the temporal relationship between dm2 and hcc and the possible effects of antidiabetic therapy on hcc risk, we recruited 465 patients with hcc compared with 490 control subjects without liver diseases and 618 cirrhotic patients. results: prevalence of dm2 was 31.2% in hcc, 23.3% in cirrhotic and 12.7% in control group. in univariate and multivariate analysis, the odds ratio (or) for hcc in diabetic patients were respectively 3.12 (ci 2.2-4.4; p <0.001) and 2.2 (ci 1.2-4.4; p= 0.01). or in univariate analysis were higher in male than in female patients. in 84.9% of the patients dm2 pre-exists the diagnosis of hcc from a mean time of 141.5 months. moreover, the insulin treatment was more frequent in diabetic hcc patients than controls and we report an or for hcc of 2.99 (ci 1.34-6.65; p= 0.007) in patients treated with insulin or sulfonylureas, and an or of 0.33 (ci 0.1-0.7; p= 0.006) in patients treated with metformin. conclusion: our study confirms that male patients with type 2 diabetes mellitus have a significantly increased risk of hcc independently of other cofactor such as hbv, hcv and alcoholic abuse. dm2 is a pre-existing disease in most hcc patients and suggests that insulin and sulphonylurea treatments in dm2 are associated with an increased risk of hcc development, while metformin may have a protective effect. background & aims: over the last few years, techniques that allow systematic analysis of chromosome aberrations at a genome-wide level were applied to hcc. the purpose of this study is to apply gene loss expression profiling in the attempt to discover new related genomic regions not revealed by loh or cgh, and search the new tumor suppression genes for hcc. methods: primary hcc and corresponding non-tumor liver tissues were obtained from surgery. serologically, 13 cases were with hepatitis b virus infection and 12 cases were with hepatitis c virus infection. four non-viral infected tissues from four patients receiving surgical resection for hepatic adenoma or focal nodular hyperplasia.affymetrix genechip, u133a, was used to compare the loss and gained gene expression in liver needle biopsy samples (n=54). results: after adjusting by chromosome arm length, 16p, 17p, 19p, 19q and 22q showed higher gene loss-expression ratio (>= 10 loss / cm) in the comparison between normal samples and tumor samples; 1q, 2p and 4q showed higher gene loss-expression ratio in the comparison between tumor and non-tumor tissues. more than 50 genes showed different loss expression level in this study. for example, cd10 was loss expression in all non-tumor samples comparing to four normal samples. ficolin 3 and ficolin 2 were loss expression in hcc samples with hbv infection and with hcv infection, respectively. conclusion: our results revealed the potential tumor suppression genes and the genomic region they harbored. further study is needed to validate the observation. background/aims: hepatocellular carcinoma is common malignancy in human, accounting for 1 million deaths in the world annually. caspase 8, as an initiator caspase, is involved in the induction of apoptosis. survivin, a novel inhibitor of apoptosis is related to the ability to inhibit caspases and involved in critical steps of onset and progression of hcc with unfavorable prognosis. methods: to explore the possibility that the epigenetic alteration of caspase 8 and survivin genes is implicated in the development and progression of hcc, promoter methylation of two genes was analyzed in 73 cases of primary hcc by methylation specific pcr. the relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathologic parameters was also investigated. results: the methylation of caspase 8 (34.2%, 25/73) and survivin (32.9%, 24/73) demonstrated a negative correlation with immunohistochemical expression of capsase 8 (47.9%, 35/73) and survivin (43.8%, 32/73) (p=0.042 and p=0.001 respectively). methylation of caspase 8 and immunohistochemical expression of its gene product was significantly correlated with apoptosis (p=0.026 and p=0.032). survivin nuclear immunoreativity revealed significantly correlated with proliferative activity of tumor cells (p=0.001). by survivial analysis, the negative caspase 8 expression and positive survivin expression showed worse prognosis in hcc, that was statistically insignificant (p>0.05). conclusion: in conclusion, caspase 8 and survivin may contribute an important regulatory mechanism for tumor cell proliferation and apoptosis, and may be prognostic predictors in hcc. injection was recently reported to be effective against hcc with pvi, though the therapy is not always applicable for the patients with arterial abnormality. therefore we tried combination therapy of transcatheter arterial cisplatin embolization and radiation, and will report the effectiveness and toxicity of the therapy. methods: the combined therapy was conducted in 7 hcc patients with pvi. transcatheter arterial embolization with 1mg/kg cisplatin powder (ia call) was performed against intralobar lesions, followed by external radiation targeted for pvi (60gy in 2 gy fractions). the following variables were evaluated with the survival rate: gender, age, viral etiology, child's class, performance status, and location of pvi. results: one (16%) patient showed complete response and another two (33%) partial response. two (33%) showed no change, and one (16%) showed progress of disease. the survival rates at six months among overall patients were 85.7%. adverse events were limited to nausea and appetite loss. one of the patients with partial response underwent curative resection, and is still alive without any recurrence for 530 days. conclusions: the combination therapy of cisplatin embolization and radiation is safe, effective and also feasible to the patients with arterial abnormality. this therapy is suggested to be a useful alternative therapy for the patients with extensive pvi. recently, the injection port has been used for hepatic arterial infusion chemotherapy (hai) in japan. hai is usually used for the treatment of multifocal bilobar tumors of the liver or hccs combined with portal vein tumor thrombosis (pvtt), not amenable to tace. this study examined the efficacy and toxicity of repeated hepatic hai using lipiodol suspension mixed with cisplatin powder. methods: from april 2005 to september 2008, 20 patients with inoperable advanced hcc were enrolled in this study. all received cisplatin powder (50mg) and lipiodol (2ml) suspension, with an intervening 4 weeks interval. the drugs were delivered from an injection port. 13 patients had hcc with pvtt, and 7 had hcc without pvtt. 11 patients with liver function of child grade a, 7 of grade b, and 2 of grade c were enrolled. result: the mean number of hai given during the follow-up period was 4.1 times. we found complete response in 1 case, partial responses in 6, no change in 6, and progressive disease in 7. the overall response rate was 35.0%. the 1-year survival rate was 36.7% and the 2-year survival rate was 12.1%. although 3 patients had cisplatin-induced anaphylaxis, no severe adverse events (hepatic failure and renal failure) were observed. conclusion: chemo-lipiodolization using cisplatin powder delivered via an injection port provides some clinical benefits without severe adverse events in patients with far advanced hcc. background: recently, the antitumor efficacy of angiogenesis inhibitors is expected in the treatment to hepatocellular carcinoma. the gene expression relevant to the vascularization, which is a target of these inhibitors, has a difference according to each case and it is thought that it influences the therapeutic effect of them. however, there are still few reports of mrna expression of vascular endothelial growth factor (vegf) receptors in hcc. methods: the relative mrna level of vegf and its receptors (kdr and flt-1) was analyzed using quantitative rt-pcr in 51 patients with hcc. matched samples of hcc (t) and non-tumor liver tissue (nt) were obtained by fine needle (21gauge) biopsy. results: gene expression level of vegf and flt-1 was significantly higher in hcc than nt (vegf; p<0.001, flt-1; p<0.001). according to the clinicopathological findings, gene expression level of vegf and kdr in hcc was significantly high in hypervascular hcc compared to hypovascular hcc (vegf; p=0.002, kdr; p=0.042). additionally, flt-1 tended to be expressed higher in hypervascular hcc than hypovascular hcc (p=0.09). moreover, gene expression level of vegf, kdr and flt-1 tended to be higher in advanced-stage hcc than early-stage hcc. conclusion: not only vegf but kdr and flt-1 were highly expressed in hypervascular and advanced hcc. aims: fibrinogen-like protein 2/fibroleukin (fgl2) has been reported to play a vital role in the pathogenesis in mhv-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo-and xeno-graft rejection by mediating "immune coagulation". fgl2 functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. therefore, this study was designed to examine the role of fgl2 in tumor development. methods: tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (hcc) model on nude mice (established from high metastasis hcc cell line mhcc97lm6) were obtained. results: hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human hcc nude mice. hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, nk cells, and cd8 + t lymphocytes and vascular endothelial cells. hfgl2 mrna was localized in cells that expressed hfgl2 protein. fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. in vitro, il-2 and ifn-increased hfgl2 mrna by 10-100 folds and protein expression in both thp-1 and huvec cell lines. one-stage clotting assays demonstrated thp-1 and huvec cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. conclusion: the hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction. . the therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least 2 cycles, when responses to treatment were evaluated by eastern cooperative oncology group criteria. results: of 146 patients treated (male, 79%; median age, 64 years), 64% had child-pugh a, and 31% had b. 90% had either metastasis or vascular invasion. 71% had metastasis and 49% had vascular invasion. on the basis of independent assessment, three (2.1%) patients achieved a complete response, thirteen (8.9%) had a partial response, and 42 (28.8%) had stable disease. there was no grade 3/4 drug related toxicities. median overall survival was 6.9 months. conclusion: combination therapy of ifn +5-fu has modest efficacy in hcc. background: amt is a mixture of approved pharmaceuticals in low therapeutic doses (human insulin and chlorpheniramine) and herbal components (aqueous camomile extract). preclinical and phase i data in healthy volunteers showed a favourable safety profile for amt. this pilot study should examine efficacy and safety of amt in the patients with advanced hepatocellular carcinoma (hcc). methods: thirteen patients with advanced hcc (tnm stage iii-iv), who did not respond to existing therapy, were treated with i.m. amt at 0.066 ml/kg up to a maximum volume of 5 ml twice daily for 1-10 months. primary study objectives: clinical benefit response (cbr). secondary objectives: safety of amt, tumor response according to who-recist criteria, quality of life (qol) and iimmunomodulatory effects. the effects were evaluated by cytokine production of pbmcs before and after the treatment. results: there were no significant safety issues. four and 3 patients showed positive and stable responses for cbr, respectively. tumor response was 1 pr, 6 sd and 6 pd. even in the patients with pd, 2 and 1 patients showed positive and stable responses for cbr. qol data showed clear improvement. immune monitoring demonstrated effects of amt on the functional immune parameters in about half of patients. in the patients with pr, histological examination showed tumor necrosis and many lymphocytes including plasmacytes infiltrating in the tumor. conclusion: these results suggest that a promising rate of patients with advanced hcc respond clinically to the amt treatment without significant safety issue and amt has some immuno modulatory capacities. background/aims: dysplastic nodules are important due to premalignant potential. the aim of this study was to evaluate the electron microscopic findings of liver dysplastic nodule in patients with liver cirrhosis. methods: a total of 5 patients (mean age: 64±9 years old, male 4) with dysplastic nodules which suspected as malignant nodule (mean size 1.38±0.22 cm) was enrolled from 48 cases of liver cirrhosis undergone ultrasonography-guided biopsy from december 2006 to january 2008. the etiologies of liver cirrhosis were as follows; alcohol (1 patient), hepatitis b virus (2), and hepatitis c virus (2). results: hepatocytes showed rosette formation of regenerative hepatocyte or degeneration. the nucleus was round or oval shaped and the nucleus membrane was irregular. the nucleolus was prominent and clear, the mitochondria were crowded to one side in the cytoplasm with megamitochondria. glycogen granules and lipofuscin pigments were abundant. sinusoid formation was poorly developed and collagen fiber bundles were increased. the hepatocytes of rosette formation and bile ductules cell made of canal of hering, which was dilated and microvilli was decreased. the number of canal of hering was 7, which was composed of 1.7±0.8 with hepatocyte and 2.1±0.7 with bile ductule cell, respectively. there was no oval cell in the canal of hering, which was relatively well developed. schwann cells were clustered together in nerve plexus. therefore, these electron microscopic findings showed that dysplastic nodule was similar to early hepatocellular carcinoma. conclusions: this study showed that dysplastic nodule in liver cirrhosis is nearly identified to early hepatocellular carcinoma. dcp is an important risk factor for recurrence after radiofrequency ablation of single hepatocellular carcinoma -< 3cm in diameter r. kuromatsu 1 , a. takata 1 , n. fukushima 1 , s. sumie 1 , m. nakano 1 1 division of gastroenterology, department of medicine, kurume university school of medicine background and aims: the aim is to analyze the risk factors for local recurrence + intrahepatic metastasis after radiofrequency ablation (rfa) and hepatic resection (hr) for single hepatocellular carcinoma (hcc) < 3cm in diameter. methods: between 1999 and 2007, 219 patients with single nodule < 3cm in diameter and child-pugh grade a were treated by hr and rfa, and recurrence rate and survival rate using kaplan-meier method, and important risk factors for recurrence using cox's proportional-hazards regression model were analyzed. factors used for multivariate analyses were age, gender, viral marker, tumor diameter, afp, afp-l3, dcp, and platelet count. results: mean age was 66 years old, m/f ratio was 136/83, hr/rfa was 59/160, and mean observation period was 1194 days. five-year survival rates, and 2-year local recurrence-free + intrahepatic metastasis-free rates were not significant between hr group and rfa group (82/72%, 92/89%). in rfa group, the only independent risk factor for local recurrence-free + intrahepatic metastasis-free survival was dcp (p=0.0034). tumor diameter was not significant for recurrence. in hr group, there was no risk factor for recurrence. in pathological analyses of hr group, dcp had a tendency to associate with microvascular invasion (p=0.065). conclusions: rfa was effective for hcc < 3cm in diameter and dcp < 40 mau/ml. hepatic resection should be selected for single hcc with dcp > 40 mau/ml even though hcc <3cm in diameter. background: radiofrequency ablation (rfa) is now a common treatment for small hepatocellular carcinoma (hcc). however, critical complications after rfa such as rapid intrahepatic dissemination have been reported. in this study, we investigated the method how to estimate the malignant potential of small hcc by dynamic ct before rfa. methods and results: firstly, 61 hccs less than 3cm in diameter were analyzed. those tissues were classified into 4 groups as followed, small nodular type with indistinct margin (type e), simple nodular type (type 1), simple nodular type with extranodular growth (type 2), confluent multinodular type (type 3). in the type 2 and 3 groups, portal invasion over vp1 were observed more frequently than those in the type 1 and e groups. at the next step, these 61 hccs were classified into above-mentioned 4 types by two radiologists according to the shape of early stain or defect of delay phase of dynamic ct before operation. the accorded rate was 87% between those classifications. next, 45 patients, which had solitary hcc less than 3cm in diameter and treated with rfa, were classified into those 4 types by dynamic ct before rfa. the recurrence rate and prognosis of those patients were examined. in the type 2 and 3 groups, the recurrence rate was higher and significant worse prognosis was showed than those in type 1 and e group . conclusion: it was suggested that hcc with type 2 and 3 might process higher malignant potential and rfa should be carefully performed on those types of hcc. background/aims: hypoxia-inducible factor-1 (hif-1 ) is the central transcriptional factor in the cellular response related to various aspects of cancer biology, including proliferation, survival, angiogenesis, and extracellular matrix metabolism to hypoxia. il-8 became known to replace hif-1 functions in the other cancer cell lines. the aim of this study was to evaluate whether il-8 may induce angiogenic factors without hif-1 by inflammation signal of hypoxic condition. methods: hif-1 knockdown cell lines of hcc (huh7 and hepg2) were constructed by rna interference tools, and cultured under normoxia (21%o2, 24 hours) and hypoxia (1%o2, 24 hours) conditions. following transfection, the amounts of hif-1 , il-8, angiogenic factors and matrix metalloproteinase (mmp) were examined using rt-pcr and western blotting, respectively. results: the expression of hif-1 , angiogenic factors, mmp, il-8 was markedly enhanced in wild types that were cultured under hypoxia, and the hypoxic induction of angiogenic factors and mmp was partially blocked in hif-1 knockdown hcc cell lines. nf-b inhibitor suppressed angiogenic effects by blocking il-8 activity. conclusion: these data suggest that il-8 induced tumor angiogenic factors in hif-1 knockdown hcc cell lines. background there were some reports that liver caner related to the levele of sera hbvdna our research focused on the relationship between the quantity of hepatocellular hbv cccdna , tdna and liver cancer. methods the samples included the liver tissue of 24 chb patients (chb group) and the para-liver cancer tissue of 26 primary liver cancer patients (phc group) the quantity of hepatocellular hbvcccdna, tdna were assayed by fq-pcr in both groups. result: the quantity of hepatocellular hbv cccdna in chb group was 11.56±16.14 copys/cell higher than phc group(3.96±9.27 copys/cell), p=0.011; the quantity of hepatocellular hbv tdna in chb group was 57.38±91.64 copys/cell higher than that of phc group(35.07±93.03 copys/cell),p=0.13.; conclusion: the quantity of hepatocellular hbvcccdna, tdna can not be used as predictors of liver cancer for hepatitis b patients. hepatic cancer predominantly occurs in males. this is almost a commonsense to most of us. but the detailed mechanisms underlying such phenomenon are still not well-known. the average age of liver cancer patients are about 30-40 years old. so most female patients have udergone pregnancy at least one time. pregnancy is a very important event before or during the development of liver cancer in females. in this special period, not only sex hormones secrete in a strange manner, but also immune system functions in a special module which is very different from normal. so it is urgent to investigate the impact of process of pregancy on the development of hepatic cancer. in this study, 100 female sd rats are randomly divided into two groups: pregance group and controll group. rats in both groups are injected iv diethylnitrosomine(a chemical carcinogen). in pregnance group, rats are raised together with male rats in 3:1 ratio(3 female, 1 male) to make every rats undergo pregnancy. while in controll group, rats are coupled with spermaduct-ligated male rats. the size and amount of hepatic cancers in pregancy group are smaller and less than those in controll group. the survial rate is also significantly higer than that in controll group. we conclude that the process of pregnancy exerts an inhibitory role in the development of chemical induced hepatic cancer in rats. acknowledgement: this project was sponsed by the national natural science foundation of china. the number of the grant is 30600727: the use of alpha-fetoprotein measurement in detection of recurrent hepatocellular carcinoma after living donor liver transplantation n. yamashiki 1,2 , y. sugawara 1,3 , s. tamura 3 , r. tateishi 2 , h. yoshida 2 , j. kaneko 3 , y. matsui 3 , n. kokudo 1,3 , m. omata 2 1 organ transplantation service, 2 department of gastroenterology, university of tokyo, 3 department of surgery, university of tokyo background: the recurrent hepatocellular carcinoma (rhcc) after liver transplantation (lt) can occur in 10 to 20 % of transplant recipients despite with a careful patient selection. for the surveillance of rhcc, frequent measurement of alpha-fetoprotein (afp) and annual ct scan is commonly used. however, the usefulness of afp is not clear. we report the update of our experience using our surveillance protocol. methods: between 1996 and march 2008, 330 adult living donor lt were performed at the university of tokyo. among them, 100 recipients with hcc in their explanted liver were subjected to analysis. we used monthly measurement of afp and des-gamma carboxy prothrombin (dcp) with annual dynamic ct scan. results: 83 met milan criteria pre-operatively and 12 did not. 5 were incidental hcc. rhcc was experienced in 9 patients at 10 (2-24) months after lt. recurrence sites were graft (1), lung (2), bone (3), and multiple organs (3). rhcc was first suspected from elevation of tumor markers in 8; afp in 4, dcp in 2, and both afp and dcp in 2. rhcc was confirmed with ct scan (7) or mri (2) in 4 (0-6) months after the first sign of rhcc. when the cutoff level of afp>20ng/ml was used, the sensitivity and specificity for rhcc were 66% and 100%. six cases were treated surgically of which two achieving prolonged survival. conclusions: although the confirmation of the rhcc sites required multiple imaging studies, afp measurement was useful as for the first sign of rhcc. purpose: to evaluate the therapeutic effect of heated (60 c) lipiodol via hepatic artery administration in vx2 rabbit liver cancer model. materials and methods: thirty male new zealand white rabbits were randomly divided into 3 groups with 10 rabbits for each group. vx2 carcinoma cells were surgically implanted into the left liver lobe. the tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of tumors detected by ultrasonograph reaching 2 to 3 cm. under the anaesthesia, transcatheter hepatic arterial embolization was performed and doxorubicin-lipiodol (37ºc) (1 ml), lipiodol (60ºc) (1 ml) and control (physiological saline (37ºc) (1 ml)) were injected into hepatic artery of the 3 different groups. one week later, the volume of tumor was measured by ultrasonograph again. the serum of all rabbits was collected before injection and at 4 and 7 days after injection and the level of aspartate aminotransferase (ast) was checked. the survival period of 3 groups of rabbits after treatment was also recorded. during the last course of their disease, the rabbits were given some analgetics to relieve suffering. results: the tumors' growth rate in lipiodol (60ºc) background/aims: hepatocellular carcinoma (hcc) is one of the male-dominant cancers, and hepatitis c virus (hcv) is one of the causes of hcc. it was reported that androgen receptor (ar) is expressed in hcc and its surrounding tissues. androgen signaling and ar may be involved in hepatocarcinogenesis. in this study, we investigated whether hcv interacts with androgen signaling in human hepatocytes. methods: hcv protein expression vectors were co-transfected with ar-expression vectors and ar-responsive element-driven reporter vector into immortalized human hepatocytes (ihhs) and human hepatoma cell lines. kinase inhibitors were used to examine the activation of the akt, mapk, and jak/stat3 pathways. real-time pcr and western blotting were performed. cell culture grown hcv (hcvcc) were also used, and angiogenesis was evaluated by tubule formation assays in human coronary microvascular endothelial cells in the presence of 5 -androgen-1 -ol-3-one. results: hcv enhances ar-responsive gene expression in the presence of androgen. hcv core protein has the strongest effects and induced ar activation associated with jak/stat signaling. hcvcc enhances vegf mrna expression and angiogenesis. conclusions: hcv core protein is an enhancer in androgen signaling and can be expected to play an important role in hcv-related hepatocarcinogenesis. background: to evaluate the therapeutic outcomes and the toxicity of the combination of arsenic trioxide and the chinese traditional jianpiliqi (jplq) formula in the treatment of advanced hepatocellular carcinoma (hcc). methods: patients with advanced hcc, not suitable for resection but with normal major organ functions, were enrolled to receive a therapeutic regimen consisting of intravenous arsenic trioxide (6 mg / m 2 ) administration from days 1-14, and an oral administration of jplq formula twice daily from days 1-28. each cycle was composed of 28 days and treatment could expand up to 4 cycles before evidences of intolerable toxicity or disease progression. result: one patient had partial response, one had minor response, 15 showed stable disease and 15 (46.9%) had disease progression. total disease control rate was 53.1%, median survival time was 5.8 months (2-23.9 ms), and time to progression was 4.2 months (1-16.9 months). the incidences of grade 1-3 abdominal distention and nausea/vomiting were 65.6% and 37.5%, respectively. increases in ggt occurred in 4 patients (2 grade 2, 1 grade 3, and 1 grade 4) and increases in serum creatinine in 2 patients (1 grade 3 and 1 grade 4), respectively. conclusion: compared with the single arsenic trioxide treatment reported in past literature, treatment by arsenic trioxide combined with jplq showed modestly higher anti-tumor activity and tolerable toxicity in patients with advanced hcc; its manageable toxicity and increased tumor response rate may offer a better treatment regimen, and deserve further investigation. aim: to investigate the effect of osteopontin (opn) expressions down-regulated by rna interference (rnai) on the invasion and metastasis of human hepatocelluar carcinoma (hcc). methods: hcc cell line (hcc-lm3) was transfected with the chemically synthesized small interfering rna (sirna) in study arm and with non-specific sirna in control arm. real-time pcr and western blotting were used to quantify the mrna and opn protein levels. the malignant phenotypes including cellular growth rates, colony formation and matrigel invasion activities of the hcc cell line were analyzed. results: in study arm opn mrna expressions decreased 75% and opn protein decreased 80% compared to those of blank arm. the number of formed colonies and migrating numbers of the cells in vitro decreased significantly (64.6% and 59.6% respectively) in study arm compared to these of blank controls (p<0.05). the parameters in the control arm did not differ from those of the blank arm (p>0.05). conclusion: the specific sirna was able to reduce opn expressions at both the mrna and protein levels and significantly diminished the invasiveness of hcc cells. methods: the expressions of mif and vegf in hcc and adjacent tissues were detected from 4 patients. specific sirna targeting mif gene was synthesized, and transfected into the hcc cell lines (plc and hepg2) in study group and non-specific sirna was used in controls. the mrna and protein expressions of mif and vegf were examined by pcr and western blot. results: mif and vegf mrnas were overexpressed in the hcc tissues compared with adjacent tissues (rq=8.91±1.85 and 3.00±0.86, p 0.01). the mrna and protein expressions of mif and vegf of hcc cell lines significantly decreased in study group compared with controls (p 0.01). vegf mrna levels decreased 75.6%±2.6%; 79.8%±1.2% in plc, and 73.6%±4.6%; 80.7%±2.2% in hepg2 cells when disposed with sirna 50nm and 100nm. vegf protein levels also significantly reduced in study group p 0.01 . conclusions mif and vegf mrnas were overexpressed in the hcc tissues in vivo, and mif sirna was able to knock down the expressions of mif and vegf in hcc cell lines in vitro. y.y. li, y.c. zhang, y.j. zhou, y.m. wei aim: to identify tumor-associated genes by constructing transcription profiles of pure hepatocellular carcinoma (hcc) tissues and normal liver tissues with the combination of laser capture microdissection and microarray. methods: hcc cells and normal liver cells from resection samples of 4 patients were laser capture microdissected. micro-rna was isolated from them for linear amplification then crna was tested with whole genome microarray. differentially expressed genes were screened. results: the quality control of this technique was satisfactory with rna integrity number>7, a260/a280 ratio for crna measurement=2.0~2.1 and good pictures for microarray. compared with normal liver tissues, hcc had 1361 differentially expressed genes, with 607 being up-regulated and 754 being down-regulated genes respectively. among the top ten ranked up-and down-regulated genes (total 20), 5 genes were known as hcc differentially expressed genes, 11, known as other tumors expressed genes previously. four unknown tumor related genes (depdc1b, aspm, fcn2 and bbox1) were detected in this study. conclusion: the combination with laser capture microdissection and microarray was effective in screening the differentially expressed genes of hcc. background/objective: young patients present with large hcc on initial presentation are not uncommon. our aim is to study the computed tomography(ct) imaging of hcc and the clinical features of this special group of patients. methods: 521 hcc patients had ct imaging of liver peformed in a three year period, 385 patients had ct imaging peformed at the time of initial hcc diagnosis in our centre and were selected. they were divided into three age groups: young patients with age </= 40 year-old(group 1), age 41 to 60(group 2), age >60(group 3) to study imaging and clinical factors. univariate and multivariate analysis by cox regression model done to look for prognostic predictive factors. results: infiltrative tumour in ct scans, symptomatic presentation, child's and tm staging are prognostic factors in hcc. conclusion: young hcc patients have larger infiltrative tumour in initial ct scans and more being symptomatic. age is not an independent prognostic factor. aim: to investigate the expression change of nk cells receptor nkg2d from human peripheral blood in patients with primary carcinoma of liver and study the relationship between nkg2d expression and cytotoxicity of nk cells. background/aims: lens culinaris agglutinin-reactive alpha-fetoprotein (afp-l3) is a specific protein produced by hepatocellular carcinoma(hcc), which is more valuable than afp in the diagnosis of hcc. aptamers are oligonucleotide ligands binding to target molecules sensitively and specifically, which are screened from a great capacity of synthetically oligonucleotide library by systematic evolution of ligands by exponential enrichment (selex). our aims were to select the aptamers against afp-l3 from a self-designed ssdna library for potential application in diagnosis of hcc. methods: a random ssdna library and its corresponding primers were designed and synthesized. aptamers against afp-l3 were selected by selex. individual aptamers were separated by polymerase chain reaction-single strand conformation polymorphism (pcr-sscp) analysis and characterized. results: a ssdna library of 78 nucleotides with 40 random nucleotides in middle were designed and used for the selection. the binding rate of library against afp-l3 was increased from 5.1% to 48.2% after 13 round selection. seven aptamers (s1 to s7) were isolated, and their sequences in random region and secondary structures were different from each other. all aptamers could bind afp-l3 in a different extent, and the dissociation constants of s4 and s7 are 650nmol/l and 132nmol/l. conclusions: aptamers for afp-l3 are successfully screened out and could bind afp-l3 specifically. methods: flow cytometry was used to determine the number of nk cells and the expression of nk cells receptor nkg2d from human peripheral blood in patients with 20 case primary carcinoma of liver 23 case hepatitis b cirrhosis 20 case hepatitis b and 20 healthy cases and enzyme mark instrument was used to detect cytotoxicity of nk cells in all cases. results: killing rate of nk cell for k562 cell,nkg2d expression level of nk cells, and the number of nk cells in the patients with primary carcinoma of liver decreased significantly p<0.05 compared with those in the healthy subjects and hepatitis b group ,and decreased a little compared with those in the hepatitis b cirrhosis (p>0.05).the activity of nk cells showed a obvious positive-correlation with the number of nk cell and expression level of nk cell receptor nkg2d. conclusion: the cytotoxicity of nk and the nkg2d expression of nk cells decreased significantly from human peripheral blood in patients with primary carcinoma of liver .the activity of nk cells is closely related to the nkg2d expression level of nk cells. enhancing the nkg2d expression level of nk cell may provide a new idea for adoptive immunotherapy of primary carcinoma of liver. and alpha-fetoprotein afp in serum and tissues for primary hepatic cancer(phc). methods: sixty-six phc and 32 cirrhotic patients were enrolled. in phc patients,male /female was 48:18, age was 55.4 ± 9.91.of them, 16 patients were defined as stage a-a. in cirrhotic patients, male /female was24:8, age was 53.3 ± 5.81. serumgpc3 was detected using elisa. serum afp was detected using electrochemiluminescence. the hepatic expressions of gpc3 and afp were measured using immunohistochemistry in 21 phc and 6 cirrhotic patients. results: the cutoff value of afp diagnosis for phc was 400 g / l or more, afp positive in phc patients was 28.8% (19/66); the cutoff value of gpc3 diagnosis for phc was 300ng / l or more, gpc3 positive was in 47.0 % (31/66), p = 0.031. in a-a stage phc patients,the positive of gpc3 and afp was 62.5% (10/16), 0(0/16), respectively,p = 0.000. in serum afp negative or positive patients, the positive of gpc3 was 46.7% (14/30) , 47.2% (17/36), respectively,p = 0.964. the relationship between gpc3 with age, sex, child-pugh grade, hbv infection, tumor size and metastasis were not observed.the positive expression of gpc3 and afp in hepatocellular carcinoma tissue was 85.7% (18/21), 4.8% (1 / 21), respectively, p = 0.000. neither gpc3 ,nor afp in the paracarcinomatous and cirrhotic tissue, was expressed. conclusions: diagnosis of glypican-3 protein for primary hepatic cancer is superior to afp.gpc3 can be regared as a early marker to diagnosis phc. objective: to investigate the effects and the possible mechanism of curcumin on the proliferation and the invasion of human hepatocellular carcinoma in vitro and in vivo. methods: hcclm3-rfp cell lines were maintained in dmem medium supplemented with 10% fetal bovine serum. the fluorescent areas of hcclm3-rfp were photographed daily and repeated in 4 consecutive days after curcumin treatment for obtaining cell growth curves. the cell morphologic changes were also observed. cell invasion experiment was performed with boyden chamber array. the rfp-expressing human hcc xenograft model in nude mice was established to study the anti-tumor effects of curcumin. the ctc was detected by facs. the expression of cyclind1 and mmp-2 was detected by sybr green real-time pcr. results: after incubation with 20 m, 10 m and 5 m curcumin respectively for 24, 48 and 72 hours, the growth of hcclm3-rfp was significantly inhibited and some morphologic changes were observed. the mean tumor size in nude mice treated with curcumin since day 21were significantly less than those of the control group(p 0.01). the mean metastasis area of lung and the number of ctc in curcumin group on day 35 were remarkably less than in the control group(p 0.01). the mrna levels of cyclin d1 p 0.01 and mmp-2 p 0.05 in curcumin group on day 35 were significantly lower than in the control group. conclusion: curcumin can inhibit the proliferation and invasion of hcclm3 cell line not only in vitro but also in vivo mainly by down-regulating the expression of cyclin d1 and mmp-2 in mrna levels. phosphorylated erk is a potential predictor of sensitivity to therapy with sorafenib in hepatocellular carcinoma -evidence from in vitro study z. zhang 1 , y.h. wang 1 1 background: sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (hcc) and thus sets the new standard for the first-line treatment of advanced hcc. however, it remains unresolved to predict the drug sensitivity in treating hcc with sorafenib. pretreatment perk level has been shown to be associated with favorable response to such therapy in a phase 2 preclinical study, indicating that perk may be a potential biomarker for treatment of hcc with sorafenib. methods: the effects of sorafenib and 5-fluorouracil on cell proliferation were evaluated by cell viability assay in four types of hcc cell line (smmc-7721, mhcc97-l, mhcc97-h and hcclm6), with different metastatic potential and basal perk expression. levels of perk expression were determined by immunocytochemical analysis and quantification, along with western blot analysis. correlation analysis was carried out between the ic50 values of drugs and mean optical density values of perk. results: the basal perk levels increased stepwise in cell lines in accordance with their metastatic potential. sorafenib inhibited erk phosphorylation at a concentration between 5 and 20 m dose-dependently, while no changes were observed after 5-fu treatment. correlation analysis between the ic50 values and mod values of perk revealed that the effects of sorafenib were significantly correlated with basal perk levels (spearman r=-0.8671, p=0.0003). on the other hand, the resistance to 5-fu were significantly associated with basal perk expression in these hcc cell lines (spearman r=0.7846, p=0.0009). conclusions: in this vitro study, perk was confirmed to be a useful biomarker predictive of sensitivity in treating hcc with sorafenib. the raf/mek/erk pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in hcc. background: to investigate the dynamic expression of igf-ii and igfbp-3 and its alteration of bcl-2 in hcc. methods: hcc models were induced with 2-faa on male sd rats. morphological changes of livers were observed and the dynamic changes of liver or serum igf-ii, igfbp-3, and bcl-2 were quantitatively analyzed by elisa. the expression and distribution of liver igf-ii were observed by immunohistochemistry. result: hepatocytes from granule-like degeneration to a typical hyperplasia to hcc and the progressing increasing of the levels of hepatic igf-ii after rats induced by 2-faa. the levels of igf-ii in hepatoma and sera were significantly higher than any of other groups. the positive relationship of igf-ii was found between liver and sera (p<0.01). the igfbp-3 levels in hepatoma were significantly lower than that in other groups (p<0.01) and the progressing increasing of the levels of hepatic bcl-2 expression during the course. the levels of bcl-2 in hepatoma tissues were significantly higher than those in normal and degeneration ones. the immunohistochemistry evidences indicated the positive expression and hepatocyte distribution of bcl-2 in rat hepatoma. conclusion: hepatic igf-ii, igfbp-3 and bcl-2 may participate in hepatocyte canceration and accelerate the occurrence and development of hcc. the expression of igf-ii and igfbp-3 could be useful molecular markers for early diagnosis and prognosis of hcc. background: this study was done to assess the etiological role of hepatitis b virus (hbv), hepatitis c virus (hcv) and aflatoxin b1(afb1) in development of hepatocellular carcinoma (hcc) in bangladesh. it was also investigated whether alpha-feto protein (afp) and protein induced by vitamin k absence or antagonist ii (pivka-ii) has any diagnostic advantage over each other methods: fifty five histologically proven hcc patients were tested for serological markers of hepatitis b and hepatitis c, and afb1-dna adduct. during the diagnosis, they were also investigated for liver function tests, afp pivka-ii. results: out of fifty five hcc patients, 41(74.5%) were found positive for serological markers of hbv, 21(38%) for hcv and 15(27%) for both. eight cases (14.5%) were negative for the markers of hbv and hcv. however, none had afb1-dna adduct above normal range. both pivka-ii and afp is strong marker for hcc with satisfactory level of sensitivity and specificity; but pivka-ii is more sensitive (92.7%) and afp is more specific (96%). conclusions: hbv and hcv is the major etiological agent responsible for the development of hcc in bangladesh. background: to investigate the influences on the malignant transformation of hepatocytes through the intervention of nf-b activation pathway. method: hcc models were induced with 2-faa on sd rats, thalidomide was administered intragastrically and rats were sacrificed fortnightly interval to the twelfth week. morphological changes were observed by he staining. nf-b expressions were detected by ihc. the relationship between nf-b expression and pathological characteristics in hcc and non-hcc were analyzed. results: rat hepatocytes showed vacuole-like denaturations at the early stages, then dysplastic nodules appeared at middle stage, and finally progressed to tubercles of cancerous nest, all of which were highly differentiated hcc. thalidomine can repress the morphologic change of liver cells. there were only punctiform denaturations at the early and middle stage; nodosity hyperplasy and minority atypical hyperplasia were found at the finally stage. the ihc results demonstrated that nf-b level was significantly higher than those in normal ones, and the nf-b level of livers in hcc was higher than those in thalidomide group. an increasing tendency of nf-b was found from normal to hcc. nf-b in hcc were significantly higher than those in nc. the nf-b levels with thalidomide intervence raised first and decreased later. nf-b expressions in hcc were higher than that in their non-cancerous tissues. no positive relationship presented between nf-b expression and histological differentiation grade or the number of tumor, and size of tumor. conclusion: decrease nf-b expression can inhibit hcc development and nf-b is expected to be a new molecular target of hcc therapy. method: the cellular distributions of vegf expression in hcc tissues were investigated by immunohistochemistry. the levels of total rna and vegf were quantitatively detected in hcc, their paracancerous, and distal cancerous tissues, respectively. simultaneitily, serum vegf were analyzed in patients with chronic liver diseases for clinical values. results: the positive expression showed palm-yellow or palm-brown granules and distributed in hepatocyte plasma of hccs. the incidence of vegf was 63.9% in hcc tissues, 78.3% in non-encapsulated hccs, and 90.9% in hccs with extrahepatic metastasis, respectively. no significant difference was found between hepatic vegf and hcc diameter or differentiation degree. the specific concentration (pg/mg liver) of vegf expression was significantly higher (p<0.01) in hcc than their paracancerous or distal cancerous tissues, respectively. the circulating vegf was abnormally elevated in hcc. if the cut off values was more than 280 pg/ml, the incidence of serum vegf was 88.4% in hcc, 14.3% in chronic hepatitis, and 10% in liver cirrhosis, respectively. the combined vegf and afp can increase positive rate up to 94.2% for hcc. conclusion: the vegf overexpression is a useful marker for vascular invasion and metastasis of liver tumors. background: hepatocellular carcinoma (hcc) represents a major health problem world wide. it accounts for 90% of all primary liver cancers and is the fifth most common malignancy (1). objectives: evaluation of radiofrequency thermal ablation versus transarterial hepatic chemoembolization with the effect of viscum (fraxini 2) on tumour recurrence. methods: 60 patients with hcc were enrolled in the study. group 1 include 30 patients and were treated with radiofrequency thermal ablation (15 patients of them received viscum 2 by subcutaneous route for 2 years). group 11 included 30 patients with hcc and were treated by tace (15 patients of them received viscum 2 subcutaneously for 2 years). results: group 1 patients showed total ablation in 70% with persistant inactivity during 2 years follow up. group 11 did not show significant difference from group 1 as regards relapse rate nor the performance status. complications as nausea, vomiting, fever, jaundice, and elevation of transaminases were significantly more encountered with tace. viscum 2 did not significantly arrest tumour recurrence. conclusion: non surgical patients with hcc can achieve curative treatment with radiofrequency with minmal side effects. tace is a palliative treatment option for large hcc. a new technique had been attempted to increase the field of radiofrequency ablation of expandable electrode needles in the treatment of hepatic neoplasms much larger than the routinely covered size of 5-7 cm according to the needle size overcoming the technical difficulties usually met with in the overlapping balls technique due to the hyperechoic focus that develops at the needle tip making reinsertion difficult and inaccurate. in this technique, two or three needles were inserted from the start into the mass with accurate estimation of the exact field of ablation of each needle trying to cover the whole extent of the mass before application of radiofrequency waves. 40 patients were included in the study, all presented with hepatic neoplastic mass lesion that range in size between 7 and 10 cm in its maximum diameter. all had a pretreatment helical (triphasic) ct study for accurate delineation of the whole extent and vascularity of the mass. two needles were sufficient to cover the whole extent of the mass in 23 patients (57%) while in the remaining 17 patients (43%) three needles were necessary. the procedure was done under general anathesia and ultra sound guidance, patients tolerated procedure well with smooth recovery. no major complications. follow up spiral (triphasic) ct was done 2 weeks after ablation revealed percentage of tumour necrosis of 90% or more in 30 patients (75%), 70-90% in 6 patients (15%) while in the remaining four patients (10%) the percentage was 50-70% necrosis. in conclusion this technique should be considered in the treatment of hepatic masses larger than the usual field of the needle. results: the median value of gpc-3 in hcc, dc, cc was significantly higher than chronic hepatitis and control groups. no significant correlation found between afp and gpc-3. auroc of afp was 0.85 & auroc of gpc-3 was 0.84. the diagnostic sensitivity of afp (20 ng/ml) was 70% with ppv 53.8%. the specificity was 85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) was 100% with ppv 27%. the specificity was 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved the specificity to 87.5%. conclusion:gpc-3 although it is a serological test for early detection of hcc, it showed limited specificity, where it is detected in different stages of chronic liver disease, as it is an oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 may improve the prediction accuracy of hcc patients in those showing seronegativity to afp. outcome of inoperable hepatocellular carcinoma patients receiving transarterial chemoembolization: retrospective analysis in an asian regional hospital w.m. yip 1 , k.f. li 1 , k.k. li 1 , m.l. szeto 1 1 background: hepatocellular carcinoma (hcc) is a common cancer worldwide causing substantial mortality. although surgical resection is a form of curative treatment in hcc, only a minority of patients is suitable for this treatment and the postoperative recurrence remains high. transarterial chemoembolization (tace) is a treatment option for inoperable hcc and it was proven by randomized control trials that tace can prolong survival in selected patients. the aim of this study is to evaluate the survival and the prognostic factors in patients with advanced hcc treated by tace. methods: seventy four patients with inoperable hcc diagnosed from january 1998 to december 2003 were analyzed retrospectively in this study. only patients with unresectable hcc or who refused operation were included. patients with advanced cirrhosis, extrahepatic metastasis or previously treated hcc were excluded. multiple host, tumor and treatment variables were analyzed in order to evaluate the predictive factors of favorable response to treatment and better survival. results: the median survival of the study patients was 213.5 days. the cumulative survival rates at 1 year, 2 year and 3 year were 28.4%, 12.2% and 6.8% respectively. by multivariate analysis, superselective cannulation performed in tace (hazard ratio: 0.47, 95% ci: 0.23-0.95, p=0.034), embolization with gelfoam (hazard ratio: 0.30, 95% ci: 0.11-0.80, p=0.017), treatment interval more than 45days (hazard ratio: 0.33, 95% ci: 0.15-0.72, p=0.006), child-pugh grade b (hazard ratio: 5.62, 95% ci: 2.11-14.97, p=0.001), and pre-treatment serum fp level (hazard ratio: 2.93, 95% ci: 1.50-5.73, p=0.002) were independent predictors of survival. conclusions: survival of patients with inoperable hcc is still grave despite treatment. this study provided information in predicting the survival of patients with inoperable hepatocellular carcinoma treated by transarterial chemoembolization. result: age <65, total bilirubin (tb) <2.0mg/dl, albumin (alb) 3.5g/dl, prothrombin time (pt) 70%, platelet counts (plt) 10.0 10 4 /mm 3 , single nodule, and type of treatment (surgery or local ablation therapy) were linked to increased survival at univariate analysis of clip 0-1 hcc patients. of clip 2-6 hcc patients, tb <2.0mg/dl, alb 3.5g/dl, des-gamma-carboxy prothrombin (dcp) <100mau/ml, absence of vascular invasion, and type of treatment were correlated with survival. the following factors were related to survival by multivariate analysis: clip 0-1 hcc patients; age, alb, single nodule, and absence of vascular invasion, clip 2-6 hcc patients; age, tb, alb, alpha-fetoprotein (afp) <100ng/ml, dcp, absence of vascular invasion, and type of treatment. conclusion: age, albumin, vascular invasion were significant predictors of survival both clip 0-1 and clip 2-6 hcc patients. clip 0-1 hcc patients: single nodule; clip 2-6 hcc patients: lower levels of tumor markers and patients receiving promising treatment had a better chance of prolonged survival. the role of gross classification as the predictor of microvascular invasion in hepatocellular carcinoma. s. sumie 1 , r. kuromatsu 1 , k. okuda 1 , e. ando 1 , a. takata 1 , n. fukushima 1 , m. sata 1 1 background; the presence of microvascular invasion (mvi) as the risk factor in hepatocellular carcinoma (hcc) is controversial. the aim of this study was to determine the outcomes and predictive factors after hepatic resection for hcc with mvi. methods; one hundred and ten patients who underwent curative resection for hcc were included in this retrospective study. the risk factors of these patients for recurrence-free and disease-specific survival were investigated, and the clinicopathological factors predicting the presence of mvi were also evaluated. result; multivariate analysis showed that cirrhosis and mvi were identified as independent risk factors for recurrence-free survival. the 2-year recurrence-free survival rates for patients with and without mvi were 44.6% and 76.7%, respectively. multivariate analysis showed that the number of tumors, presence of mvi, and im were identified as independent predictors of disease-specific survival. the 5-year disease-specific survival rates for patients with and without mvi were 59.3% and 92.0%, respectively. by univariate analysis, mvi was significantly associated with greater tumor size, gross classification, histological grade, and intrahepatic micrometastasis (im). gross classification proved to be the only independent predictive factor for mvi by multiple logistic regression analysis. the gross classification could be evaluated by preoperative imaging diagnosis. conclusion; mvi is strongly associated with recurrence and survival in hcc patients after curative resection. furthermore, gross classification of hcc can be helpful in predicting the presence of mvi. background: hcc is a common cause of cancer morbidity and mortality. pxd101 is a novel, low molecular weight, histone deacetylase inhibitor. this phase i study aims to determine dose limiting toxicity (dlt) and maximum tolerated dose (mtd). methods: patient eligibilities include unresectable disease, ecog 2, adequate organ functions. pxd101 was given intravenously on day 1-5 every 3 weeks; dose levels were: 600 (level 1), 900 (level 2), 1200 (level 3) and 1400 mg/m 2 /day (level 4). dlts are defined as grade 4 hematological toxicity or grade 3/4 non-haematological toxicity during cycle 1 (according to nci ctc v3), or treatment delay >2 weeks. the mtd is defined as the dose below which > 2 of 3 or > 2 of 6 patients experiencing dlt. results: 18 patients were entered; level 1 (3), level 2 (3), level 3 (6) and level 4 (6). grade 3/4/5 toxicities in cycle 1 included: raised alt 1/0/0, diarrhea 1/0/0, abdominal distension 1/0/0, anaemia 1/0/0. a total of 56 cycles were administered; overall grade 3/4/5 toxicities: raised alt 1/0/0, bilirubinaemia 1/0/0; cardiac ischaemia 1/0/0; diarrhoea 1/0/0, abdominal distension 2/0/0, anaemia 2/0/0; variceal haemorrhage 1/0/0; hypercalcaemia 1/0/0; hyperkalaemia 0/1/0; hyponatraemia 1/1/0; infection 2/0/0; liver dysfunction 0/2/0; muscle weakness 1/0/0; abdominal pain 1/0/0; prolonged qtc 1/0/0; syncope 1/0/0; seizure 1/0/0. there were 3 sd and 15 pd. conclusion: at the maximum dose of 1400 mg/m 2 /day, mtd has not been reached. pxd101 is very well tolerated. sponsor: the division of cancer treatment and diagnosis, national cancer institute, usa. tumor thrombus (pvtt) is prone to be produced in the portal vein near the main tumor nodule for hepatocellular carcinoma (hcc) patients and its molecular mechanism is still unclear. in this study, we first established a hcc cell line named csqt-01 from resected tumor thrombus in portal vein in a patient with histopathologically proved to be a moderately differentiated hepatocellular carcinoma . this cell line was composed of polygonal shaped cells and its peaks of the chromosome number was 69 and 77. study on stem cell biology in this cell line suggests that cd133 cells represent about one fourth of the tumor cell population and cd133(+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. with this cell line model and resected tumor thrombi specimen, we also studied the different expression of proteins in primary tumor and tumor thrombus and found 20 proteins expressed differentially between primary tumor and the pvtt. from these proteins, annexinv, prx , cycb were selected for further analysis to find potential biomarkers of pvtt in hepatocarcinogenesis. for clinical study, we recommended a new tumor thrombus type system ( type i iv) according to anatomic features of portal vein and tumor thrombus of hcc developing modes, then evaluate this type system to predict prognosis of hcc patients. the retrospective data of 406 hcc patients with pvtt underwent resection shows that the 1y, 2y, 3y overall survival rates were 44.7 , 26.3 and 19.7 for type i, 29.9 , 20.6 and 12.4 for type ii, 25.0 , 12.5 and 3.6 for type iii, 27.3 , 0 and 0 for type iv, respectively, suggests tumor thrombus type system may be helpful to determine treatments and prognosis of hcc patients with pvtt. polyprenol could decrease the risk of hepatocarcinogenesis in hbv g. kuznecova 1,2 , s. kuznecovs 1,2 , i. kuznecovs 1,2 background: over-expression of p-glycoprotein (pgp) is associated with liver cancer development from hbv . glycoprotein synthesis in malignant tissues is limited by dolichyl phosphate (dolp). the aim of the present study was to investigate the effect of polyprenol (pp) which provides a dolp substitute in regulation of n-glycosylation on pgp over-expression in the development of liver cancer in hbv infection. methods human hepatocytes, infected with hbv and human hepatocarcinoma hep 3b cell line were used. pgp was assessed by an immunohistochemical technique. dolp fractions were analysed by hplc methods. results it is confirmed that plasmatic membrans of hepatocytes cells contain 7,9 -9,4% of pgp (the total protein amount) as a resistance marker. hbv infected cells differ from normal hepatocytes in pgp content by 4-5 times and hep 3b cells differ by 10-12 times the study showed 5-fold dolp decrease in hbv infected cells and 10-fold dolp decrease in hep3b cells. the investigations demonstrate that the situation can be changed by treatment with dolp and pp. the dolp concentration in hbv infected hepatocytes was returned to the normal level. it is established that dolp in the concentration 10 -6 m aid 6-8-fold reducing pgp in membranes of hbv infected cells. background: metastasis is one of the most complicated and major pathological processes responsible for poor prognosis of hepatocellular carcinoma. snail was recently highlighted as a critical transcriptional factor for tumor metastasis. method & result: real time rt/pcr and western blot analysis demonstrated that snail mrna and protein, respectively, were induced by 12-otetradecanoylphorbol-13-acetate (tpa) in hepatoma cell hepg2. blockade of gene expression of snail by antisense oligodeoxynucleotide and/or sirna technique can prevent not only the tpa-triggered emt/cell migration and growth inhibition of hepg2 but also tpa-induced down-regulation of e-cadherin and up-regulation of p15ink4b. moreover, the tpa-triggered promoter activation of p15ink4b was also prevented. on the other hand, two of the hepg2 clone overexpressing snail, namely s7 and s15, had a scattered fibroblastic morphology and acquired higher motility than parental hepg2. also, the proportion of g0/g1 phase of s7 and s15 was higher than that of parental hepg2, consistent with the longer doubling time of both cells. semiquantitative rt/pcr analysis demonstrated a greatly elevated gene expression of snail accompanied with decreased e-cadherin and increased p15ink4b in both snail-overexpressing cells. on the transcriptional level, p15ink4b promoter activity was 2.6-fold higher in s7 as compared with parental hepg2. furthermore, electrophoretic mobility of dna fragments encompassing proximal p15ink4b promoter can be retarded by incubation of nuclear extract of s7. conclusion: our results demonstrated that snail play diverse trans-regulatory roles in hepg2. notably, we suggested that snail may upregulate p15ink4b gene expression by directly activating its promoter. a. schmitt-graeff 1 , r. fischer 1 , m. grosse-perdekamp 1 , o. skalli 2 1 universityhospital freiburg , 2 louisiana state university health sciences center, shreveport background/aims: synemin is an intermediate filaments (if) protein which affects the motility of several cell types by modulating the dynamic properties of alpha-actinin and f-actin. we have previously shown that synemin is expressed in resident hepatic stellate cells (hsc) and myofibroblasts (mf) in hepatic inflammation and fibrosis. in the present study we evaluated systematically the expression of synemin in a large cohort of western european hepatocellular carcinoma (hcc). methods: single and double immunolabelin for alpha-smooth muscle actin (sma), vimentin, cd31, cd34, cd68, cea, cd10, cellular retinol-binding protein1 (crbp-1) and synemin were performed on 75 paraffin-embedded hccs and 10 controls. results: synemin-positive hscs/ mfs were a hallmark of non-neoplastic fibrotic liver tissue at the border to the neoplastic lesion but were absent from normal controls. tumour cell plates of the trabecular and pseudoglandular types of hcc were covered by scattered synemin-positive cells outlining sinusoidal structures. a subpopulation of these cells showed features of pericytes while others resembled endothelium. this pattern correlated with the degree of differentiation and was not observed in poorly differentiated hccs which generally contained rare intratumoral mfs. conclusion: the presence of synemin-positive hscs/mfs in the vicinity of hccs suggests a possible contribution of mesenchymal cells to the promotion of liver carcinogenesis. since synemin expression is linked to motility, a migration of this cell type into the tumour and a differentiation in vascular mural cells may be implicated in sinusoidal remodeling and the expansion of the neoplastic population. a.s. butt 1 , a. ahmed 1 , s. hamid 1 , w. jafri 1 , h. ali shah 1 1 aim: to estimate the prevalence of viral marker negative hcc and to compare the clinical, biochemical, histological, radiological characteristics and initial treatment response among patients with viral marker negative and viral marker positive hcc. methods: medical records of patients diagnosed to have hcc visiting aga khan university hospital, karachi during january 1998 to december 2007 were reviewed. patients were divided in to nbnc-hcc(those who have negative hbsag and anti-hcv antibody)and viral hcc(those who have positive hbsag and anti-hcv antibody)group. results: out of 433 patients 68(15.7%) had nbnc-hcc. over all mean age was 57.48± 10.9 years and 69.5% were males. the proportion of hcc detected under surveillance was significantly smaller in nbnc-hcc group(p0.02). there was no difference in distribution of age, gender, bmi, child score, bilirubin, serum albumin, prothrombin time and alfa feto protein in both groups. however, patients with viral-hcc were found to be more thrombocytopenic(52.67±86.7vs.226.15±153.9,p<0.001) and had hepatopulmonary syndrome. on liver biopsy greater proportion of moderate to poorly differentiated hcc was observed in nbnc group(19.2%vs.7.9%,p<0.001). hcc measuring 5cm in diameter(60.3%vs.41.9%, p 0.01), non -solitary hcc(p0.038) and portal thromboses(p0.01) were strongly demonstrated in nbnc-hcc group. involvement of right hepatic lobe and extra hepatic tumor spread was greater in nbnc-hcc group but that difference was not statistically significant. out of 178 patients who underwent for liver transplantation(0.5%),tace(36.7%),resection(1%),ethanol ablation(2%) and chemotherapy(2%), poorer responses were observed in nbnc-hcc group (p 0.04). conclusion: hcc secondary to nbnc-cirrhosis is not uncommon. patients with nbnc-hcc tended not to be under surveillance that leads to diagnoses at more advanced stage and poor prognosis. background: hepatocellular carcinoma is a common malignancy in asia and is related to the high prevalence of chronic viral hepatitis. we examined the clinical features, treatments and survival rates in asian americans with hcc. methods: retrospective cohort study of 278 hcc patients who presented to the ucla liver cancer center in los angeles, california, usa from september 2000 to december 2007. results: two hundred and seventeen of 278 (78%) hcc patients were male, 58% and 30% had hbv and hcv infection respectively, and 73% had cirrhosis. hcc patients detected by surveillance had smaller tumor sizes, more within the milan and ucsf criteria, lower hcc tokyo system scores and had improved 1,3,5 year overall patient and disease free survival rates compared to hcc patients who presented with symptoms (p<0.01 to p<0.0001). by multivariate analysis, independent predictors of patient survival were tumor volumes greater than 5cm (hr1.48, p=0.04), afp per unit log10 increase (hr 1.12, p=0.02), hcc tokyo score per unit increase (hr 1.3, p<0.0001), liver transplantation (hr 0.14, p<0.0001), hepatic resection (hr 0.18, p<0.0001), rfa (hr 0.17, p<0.0001), tace (hr 0.44, p=0.0006), and hepatitis b infection (hr 0.71, p=0.03). factors associated with disease free survival were age per year increase (hr 1.01, p=0.03), meld per unit increase (hr 0.97, p=0.0049), liver transplantation (hr 0.23, p<0.0001), and hepatic resection (hr 0.39, p<0.0001). conclusion: hbv and hcv infection accounts for the majority of hcc in asian americans. hcc detected by surveillance resulted in treatments which improved overall patient and disease free survival. treatment of small ( 2cm) hcc tumours can be achieved by surgical resection and complete eradication always correlates with good patient's outcome, with low local recurrence and high survival rates. indeed, surveillance program for the early detection of small hcc tumour is imperative to facilitate curative treatment, and hence better survival. discovery of new blood-based biomarkers is obligatory and vimentin is a distinct novel small hcc tumour marker herein identified using proteomics. experimental design: a total of 76 liver tissues were evaluated by 2-de analysis. differentially expressed proteins were unequivocally identified by maldi-tof/tof and validation of the best candidate from protein to gene levels. indirect elisa assay was developed to detect soluble vimentin from 152 serum samples. results: vimentin was significantly over-expressed in small hcc tumours compared to non-malignant controls and maintained expression in >2cm tumours using 2-de analysis. blind verification displayed over-expression of vimentin in both transcripts and proteins levels. soluble vimentin was significantly detected at high level in small hcc as well as in overt hcc tumours. receiver operating characteristic analysis showed vimentin exhibited 40.91% sensitivity and 87.50% specificity in detecting small hcc at a cutoff of 245ng/ml. combined diagnostic performance of soluble vimentin and serum afp increases the detection sensitivity and specificity to 50.53% and 98.15%, respectively. conclusion: in this context, over-expression of vimentin is associated with the favourable 2cm sub-class of hcc thus may potentially be used as an effective serum-based diagnostic marker for cancer surveillance in high-risk cirrhotic patients. purpose: our recent comparative oncogenomic analysis in mouse model has identified yap (yes associated protein) as a novel oncogene in hcc. however, its clinical significance is unknown. in this study, we aimed to investigate the clinical values of yap as an independent prognostic marker in hcc. experimental design: a total of 177 hcc cases with retrospective clinicopathologic and follow-up data were recruited in this study. both tumor and adjacent non-tumor tissues were examined for immunoreactivity of yap expression by immunohistochemistry. clinicopathologic features and yap expression were investigated with pearson 2 test. hcc-specific disease free survival and overall survival with yap expression were analyzed by kaplan-meier curves and log-rank test. cox regression was used to test the independence and magnitude of the effects. results: yap was found over-expressed in hcc (62.1%) with nuclear expression pattern. positive yap immunoreactivity was significantly correlated with worse tumor differentiation grade (p=0.021) and high serum alpha-fetoprotein (afp) level >400 ng/ml (p<0.001). kaplan-meier plot and cox regression showed that yap was an independent predictor for hcc-specific disease free survival (hazard ratio, 1.653; 95% ci, 1.084-2.522; p=0.02) and overall survival (hazard ratio, 2.226; 95% ci, 1.312-3.778; p=0.003). conclusions: yap expression in hcc is correlated with tumor differentiation and serum afp level. it served as an independent prognostic marker for hcc. background: integrative analysis of global protein and mrna expression patterns could help researchers to understand cancer cell physiology without the need of any prior hypothesis. methods: we used a 2d-page approach to profile and compared the global protein expression profiles of hepatitis b virus-related hcc tissues, adjacent non-tumor liver tissues, normal liver tissues and hcc cell lines. subsequently, we established the bioinformatic tools for integrative analysis of gene expression and protein expression data. we compared the dysregulated protein list and the dysregulated gene lists obtained by meta-analysis of microarray gene expression data from 6 research centers in different countries. results: we identified 66 proteins dysregulated in hcc. hierarchical clustering analysis revealed that there was a progressive change of protein expression patterns from normal liver, adjacent non-tumor liver tissues, hcc tissue, then to hcc cell lines. according to the biological functions, the differential proteins could be classified into various groups, including heat shock protein, chaperone, kinase substrate, cell signaling, apoptosis regulation, transcription regulation, free-radical scavenger and metabolic enzyme. ontology analysis of the genes with consistent dysregulations at both mrna and protein levels identified specific pathways down-regulated during the progression of hcc. the inhibition of those pathways provides new insights in the hepatcarcinogensis and treatment strategies. results: in pre-s/surface regions, hcc patients had higher frequencies of pre-s deletions, amino acid substitutions at codon 4, 7, and 81 in pre-s1 genes, at the start codon in pre-s2 genes, and at codon 68 in surface genes. but they had a lower frequency of amino acid substitution at codon 2 in pre-s2 genes than those without hcc. in bcp/precore regions, hcc patients had higher frequencies of c or g1753, a1762/t1764, t1846, and a1899 than those without hcc. multivariate analysis showed that pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. the hbv with a complex mutation pattern (pre-s deletion, t1762/a1764, and a1899) rather than a single mutation was associated with hcc. patients with combined mutations of t1762/a1764 and pre-s deletion, t1762/a1764 and a1899, pre-s deletions and a1899, and t1762/a1764, pre-s deletions and a1899 had a 7.81, 7.7, 7.0, and 16.88 fold increased risk of hcc, respectively, compared to patients with wild-type at both or three genomic regions. conclusions: pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. combination of these viral mutations appeared increasing hcc risks. high peritumoral expression of placental growth factor in hepatocellular carcinoma is a poor factor for survival after curative resection h.x. xu 1 , x.d. zhu 1 , p.y. zhuang 1 , w.zhang 1 , h.chuan sun 1 1 background/aims: angiogenesis plays a significant role in the metastasis and recurrence of hepatocellular carcinoma (hcc). placental growth factor (plgf), which is one member of the vascular endothelial growth factor family, may have prognostic values in patients after curative resection of hcc. methods: expression of plgf was assessed by immunohistochemistry in tissue microarray containing paired peritumoral liver tissue and tumor from 105 patients underwent hepatectomy for histologically proved hcc. prognostic values of plgf and clinicopathological factors were evaluated. result: plgf staining was mainly on the cytoplasm of tumor cells or hepatocytes. the mean integrated optical densities of peritumoral and intratumoral density of plgf were 0.018±0.018 and 0.006±0.007 respectively. peritumoral plgf density was significantly higher than that in tumor (p<0.001), and this result was also validated in another cohort of 37 patients by quantitative real-time reverse transcription-pcr (p=0.007). intratumoral density of plgf was not correlated with common clinicopathological factors (eg, tnm stage, tumor size, microvascular invasion, intra-hepatic metastasis) or overall survival (os) (p=0.425) and time to recurrence (ttr) (p=0.419). however, peritumoral density of plgf, which was correlated with tumor size (p=0.003) and intrahepatic metastasis (p=0.004), was a prognostic factor for both os (p=0.015) and ttr (p=0.018). in multivariate analysis, peritumoral expression of plgf was also an independent prognostic factor for os (p=0.015, rr: 2.500 95% ci: 1.191-5.253 ) and ttr (p=0.045, rr: 2.013 95% ci: 1.014-3.996). conclusion: peritumoral expression of plgf in hcc patients is an independent risk factor for survival and recurrence, and may be a target of anti-angiogenic therapy in preventing post-operative recurrence. purpose: to further research rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection for treatment of advanced hepatocelluar carcinoma (hcc). methods: 25 cases were treated with transhepatic artery chemoembolization (tace) + radiofrequency ablation (rfa) + introportal vein chemotherapy (pvc) + percutaneous ethanol injection (pei) (four combined group) and this method was compared with 23 cases that were performed tace + pei (two combined group) . the serum level of afp was measured respectively after 2 and 6 months, ct scan and color doppler ultrasound were measured after treatment for six months. results: the serum level of afp declined in two groups after 2 months. for treatment after six months, afp in four combined groups was rose lower than two combined group (x 2 =5.06, p<0.05). ct and doppler ultrasound examination, four combined groups was superior than two combined groups to the control in tumor shrinkage (x 2 =8.29, p<0.01) and blood supply x 2 =6.81, p<0.01), relapse and mortality are also less. conclusions: rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection is a safe, effective combined method and has less complication in treatment of advanced hcc. poster exhibition -hcv poster session, hall 5b on the average, hepatitis c virus infects 1.2% of the population worldwide. in egypt, the prevalence rates reach 20% in some areas. ability of the virus to persist in about 75-85% of infected individuals is related to the virus higher mutation rate. six major hcv genotypes have been identified. genotype 4 seems to be confined to the middle east and central africa. extra hepatic syndromes have been reported in up to 1/3 of hcv patients. we aim in this study to determine the relationship between viral genotypes and specific extra hepatic haematological disease in patients with chronic hepatitis c. the study group included 60 selected patients with chronic hepatitis c having various haematological problems. we studied hepatitis c virus genotypes using rt-pcr. we found among 60 patients , 57 genotype 4 (95%) and 3 patients genotype 1a (5%).28 patients (46.66%) were diagnosed as chronic hepatitis c with associated thrombocytopenia, 16 patients (26.66%) were diagnosed mixed essential cryoglobulinemia(mec), 13 patients (21.66%) were diagnosed non-hodgkin's lymphoma, and 3 patients (5%) were aplastic anemia. positive serum cryoglobulins level was found in 20 patients (33.3%).no significant correlation was found between the level of viraemia and specific haematologic disease, biochemical liver markers or liver enzymes (p>0. 05). we did not find correlation between hcv genotype and specific extrahepatic haemological disorder in hcv infected patients. several environmental, genetic and immunological factors may contribute in disease progression. results: in the targeted area 111 shops of barbers were successfully interviewed and total 715 questionnaires were filled by both groups. the mean age were found in both groups of barbers (n=186) and clients (n=529), 28.47 years. the both groups showed that there are no any drugs which can protect us from diseases. both of the groups were not vaccinated for hepatitis b diseases. regarding the care providers the barbers replied that they prefer registered medical practitioners and the clients generally prefer the hakeems. those who knew hepatitis as liver disease, were 73 (39.2%), out of 186 barbers only 68 ( 36.6%) were knowing about hepatitis-b&c, when we enquired about routes of hbv& hcv transmission only (37%) replied correct routes of transmission in both groups. about hbv vaccination (49.7%) were aware, only (14.8%) were vaccinated against hbv. 60% barbers claimed for disinfection of instruments before shaving (88.9%) claimed for use of new blades. in the sero-surveillance the hbv found was very low and hcv became epidemic (5.7% -14.4 %) respectively. conclusion: the both groups need awareness for transmission. the use of new blade for the clients reduces the burden of hbv and hcv. the study highlights the roles of male sex, older age, and genotype 1b in the progression from chc infection to hcc. patients with higher hcv viral load potentially tend to develop hcc; however, hcc occurrence could be prevented using antiviral treatment. these two points need to be clarified further by a larger study population with longer follow-up period. an approach have recently been described that retroviral vectors encoding t cell receptor (tcr) genes are used to redirect the specificity of normal peripheral blood lymphocyte (pbl)-derived t cells to recognize the tumor antigens. the therapy in which t cells have been genetically modified with tcr genes to recognize hcv would represent a novel approach for the treatment of hcv infections and hcv-related malignancies. we have previously shown that hcv+ liver transplant patients that have received hla disparate liver allografts have hcv reactive t cells of host origin in their peripheral blood that are restricted by the donor hla molecules. initial studies indicate that the tcrs expressed by hcv reactive t cell clones from these patients have relatively high affinity for their ligands. we have cloned and expressed two tcrs which mediate recognition of the 1406-1415 and 1073-1081 epitopes from the hcv ns3 protein. the results indicate that these tcr transduced t cells can recognize the wild type epitopes, as well naturally occurring mutant variants of these epitopes. most importantly, the tcr transduced t cells could also recognize hcv+ hepatocellular carcinoma cells. these data suggest this high affinity hcv-specific tcr might have potential new immunotherapeutic implications. background: factors associated with svr in patients without an rvr remains unclear. methods: 200 hcv-1 (100 for 24 and 48 weeks, separately) and 150 hcv-2 (100 for 24 weeks, 50 for 16 weeks) patients were randomized to peginterferon-alpha-2a and ribavirin for analysis. results: multivariate analysis showed that treatment duration and a complete evr were the strongest independent factors associated with an svr. a higher svr rate and a lower relapse rate were observed in the standard regimen group than in the abbreviated group in patients who had a cevr (table 1). the best levels of viral loads in predicting cevr at week 4 were < 10 4 iu/ml (table 2) . conclusion: it was crucial to achieve a cevr with adequate treatment duration in patients who failed to achieve an rvr. our aim was to evaluate the impact of some biochemical, histological and viral factors on both evr and svr in patients with genotype 1 chronic hepatitis c (chc) treated with peginterferon plus ribavirin. patients and methods: we evaluated retrospectively 188 naïve patients with chc treated with peginterferon plus ribavirin at standard weight-based doses for 48 weeks. biopsies were assessed for inflammatory activity and fibrosis. steatosis was categorized by the proportion of hepatocytes per low-power field with fatty changes: >5%, >5-33%, 34-66%, >66%. biopsies were also assessed for stainable iron using the brissot scoring system. all patients were evaluated for metabolic syndrome (ms) using the ncep-atp iii criteria. results: evr was achieved in 115/188 pts (61.17%) while svr occurred in 82/115 (71.3%). after adjusting for sex and age, independent factors that negatively interfered with both evr and svr were: fibrosis score, steatosis, iron score, homa-ir index and viral load. after excluding the patients with ms criteria (n=32), evr was observed in 102/156 (65.4%) and svr in 82/102 (80.4%). factors that independently influenced both evr and svr were: fibrosis score, steatosis, iron score and viral load. conclusion: fibrosis, steatosis and iron scores, as well as viral load are independent parameters that can affect both evr and svr in genotype 1 chc patients, regardless the presence of ms. if ms is present, high homa-ir index can also additionally impair viral response. issue/argument: asia has rising cases of hepatitisb/c. alcohol/food-habits cause high prevalence in rural/tribal areas. lack of monitoring/follow-up complicates management. vaccines emerge as hope. clinical-trials of vaccines debated-issue. design of hepatitis-vaccine-trials in developing-countries complex ethical-issue. we focus on controversies identified in international/regional/local cme/pharma programs as vulnerability of volunteers to exploitation by foreign/local research-groups/funding-agencies. critical task is protect interests of vaccine-subjects in face of substantial-risks. determine if hepatitis-vaccine-volunteers will have access to treatment during trial. access to vaccine-trial-outcome. interaction with seniors 19 th apasl-congress from developed-countries will give voice to such burning-issues. methodology: researchers/pharmaceuticals/govt-policy planners need to develop forum to solve these problems. ngo's can play pivotal role. obligation on part of researchers to create mechanisms to offset anticipated risks of participation in controversial, risky vaccine-development. conclusion: counselling/right to withdraw from trial be made basic guideline. apart from monetary aspects unsuspected adverse reactions/deaths be properly evaluated/monitored. researchers need to evolve policy-guidelines to overcome barriers as variation in interpretation of essential ethical ideas, legal-system-differences, educational/economic-status. need to develop common consensus between research-community/pharma sector to reduce suffering of hepatitis-affected patients community. recommendations: researchers/ngos should come together at 19 th apasl-congress platform to form workgroup to settle these issues. we shall raise our this burning issue & present hepatitis-prevention-advocacy plan of our ngo graphically to apasl-2009 participants. results: 53% patients expressed that alternative-medicines-rx most important factor to cope with hepatitis. higher scores of qol (anova p < 0.001) correlated with alternative-medicines-rx. our ngo-initiative suggests that over 70% patients will need well trained specialist for home-based-care unit. conclusions: life-span/qol of hepatitis-sufferers depends on appropriate-palliative-care. ngo-personals should be trained in palliative-care-services. our data is being used for palliative care advocacy. field of spiritual/psycho-social/community support is fertile ground for further investigations. such use of complimentary indian medicinal plant extracts needs further evaluation in a large group in multicentre trial. treatment with adacolumn in patients with hepatitis c related who have undergone kidney transplantation: preliminary study g. novelli 1 1 la sapienza university introduction: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. background: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: the treatment was found to be safe without hemodynamic or infective complications. considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. m. sharaf-eldin 1 , h. el batae 1 , n. abd el-ghaffar 2 , w. rasheed 2 1 tanta faculty of medicine , egypt., 2 national research centre, cairo, egypt aim: we aimed to characterize serum cytokine levels of interleukin-1 beta (il-1 ) and interleukin -6 (il-6) in hcv infected patients & in patients with hepatocellular carcinoma (hcc) in comparison to control group and their possible use as markers of disease progression. patients and methods: sixty patients were divided into three groups: group i: twenty hcv infected patients without cirrhotic changes. group ii: twenty hcv infected patients with liver cirrhosis (lc). group iii: twenty hcv infected patients with hcc and 20 healthy subjects as control group. all patients and control group were subjected to biochemical and serological tests, anti hcv, hcv (rt-pcr) and cytokines measurements of serum il-1 & serum il-6 levels. results: showed a high statistically significant elevated serum il-6 and il-1 levels in patients with chronic hcv infection in comparison to control group. highly statistically elevated levels of il-6 and il-1 in liver cirrhosis and higher levels were found in hcc group in comparison to control group. the levels of il-6 and il-1 increased significantly in hcv infected patients as the disease progress. conclusion: serum il-1 , and il-6 levels are elevated in patients with hepatitis c-related liver diseases, especially in lc and hcc patients. their levels reflect hepatic dysfunction better than liver inflammation parameters; accordingly, we may use serum il-1 and il-6 as markers for liver disease progression in hcv-infected patients instead of invasive techniques. atsushi tanaka 1 , naoko hanawa 1 , mitsuhiko aiso 1 , yoriyuki takamori 1 , hajime takikawa 1 1 teikyo university school of medicine background and aim: pegylated interferon (peg-ifn) therapy is not indicated for many cases with hcv-related cirrhosis due to various adverse effects. however, patients with hcv cirrhosis are at high risk for development of hepatocellular carcinoma (hcc). thus we have introduced low-dose peg-ifn treatment for patients for compensated hcv cirrhosis. patients and methods: selection criteria for low dose peg-ifn is 1) compensated hcv-related cirrhosis, and 2) either the elderly (>65) or presence of thrombocytopenia (<8.0x10 6 / l). we have treated patients who met these criteria with low-dose peg-ifn, consisting of either peg-2a 90 g/1 -2w or peg-2b 0.5 g/kg/w+ribavirin 200mg/d. [results] twenty patients with compensated hcv cirrhosis (all patients genotype 1b) have been treated with low-dose peg-ifn (peg-2a:9, peg -2b+rib:11 . the age, platelet counts (x10 6 / l), and alt (iu/l) of 20 patients at baseline were 64.1±8.3, 7.8±4.6, and 90.4±68.6 respectively. all patients were well tolerated. low-dose peg-ifn has been continued 59.6±43.8 weeks on average. although viral response was not detected, biological response (br), defined as maintenance of alt within normal range, was obtained in 12 patients (12/20=60%). of note, neither development of hcc nor decompensated cirrhosis was observed in these 12 br cases. by contrast, hcc and decompensation developed in 5 and 1 patients respectively among 8 patients who failed to achieve br. conclusion: low-dose peg-ifn treatment was safe and well tolerable, and could potentially prevent hcc or decompensation in patients with liver cirrhosis when br was obtained. aims: to study the efficacy of peginterferon and ribavirin in treating chronic hepatitis c (chc) with genotype 6a in hong kong chinese. methods: to assess sustained virological response (svr) (serum hcvrna< 500iu/ml) at 6-months follow-up. results: nine patients with genotype 6a chc (included from jan2003 to dec2007) received peginterferon and ribavirin. mean age: 50 (range 28-64). mean alt before treatment: 96 iu/l (range 29-173 iu/l). seven patients had liver biopsy performed, only one showed stage 3-4 fibrosis and others showed active hepatitis without advanced fibrosis. mean serum hcv-rna: 9.17x10 5 iu/ml(range 9.0x10 3 -3.3x10 6 iu/ml). six patients had received peginterferon alfa-2b (1.5mcg/kg/week), other 3 received peginterferon alfa-2a (135mcg/week). ribavirin dosage ranged from 600mg-1000mg/day depending on body weight and baseline haemoglobin. treatment durations were 48-52 weeks in 7 patients, 24-26 weeks in 2 patients as one showed rapid virologic response at week 4 and the other was intolerant to side effect of peginterferon. eight patients had early virologic response at week 12 and one had >2log drop of hcvrna. eight patients had end-of-treatment response. eight patients (88.9%) achieved svr at end of follow-up. two patients who received only 24-26 weeks of combination therapy also achieved svr. the one who failed to achieve svr was at older age of 60 and had advanced fibrosis. conclusions: the efficacy of pegylated interferon and ribavirin in treating chinese patients with chronic hepatitis c genotype 6a can achieve high sustained virologic response rate of 88.9%. t. bharati 1,2 , p. kar 2 , a. mohammad 2 , k. mariappan 1 , j. annamalai 1 , r. introduction: hcv is a recognized cause of hcc. information on hcv genotypes in hcc are scanty in india. methods: a total of 154 hcc cases from delhi, 96 hcc cases from madurai and 246 cases of chronic hepatitis without hcc were controls in the study. rt-pcr for hcv rna and genotyping were carried out in all the cases results: in group-i, hcv rna was positive in 26.58% hcc cases in which genotype 3 was found in 57.1% genotype 1 was observed in 26.2% hcc cases. whereas 16.6% cases remained nontypable. in group-ii, hcv rna was positive in 23.95% hcc cases, with genotype 3 in 30.4% cases, genotype 1 in 52.2% cases and genotype 4 in 4.34% cases. however, 13.04% cases remained nontypable. out of the 246 control cases, 187 were ch and 59 were cirrhosis. in ch group, hcv rna was positive in 22.45% cases in which, genotype 3 was detected in 71.4% cases whereas genotype 1 was observed in 21.4% cases . however 7.1% cases remained nontypable. in cirrhosis group, hcv rna was positive in 37.2%cases. genotype 3 was found in 59.1% cases. while genotype 1 was present in 31.8% cases and 9.1% cases remained nontypable. conclusion: genotype 3 in delhi and genotype 1 in madurai were predominant. in hcc cases. our study demonstrates that no particular hcv genotypes were associated with hcc and genotype did not appear to influence the development of hcv-associated hcc. background/aims: the standard treatment for chronic hepatitis c infected with hcv genotype-1 is a combination of pegylated interferon alfa and ribavirin for a 48weeks. it is unclear if 24weeks treatment is possible for patients showing a rapid virologic response (rvr) without compromising the sustained virologic response (svr) in korea. method: between june 2005 and july 2008, among patients chronically infected with the hcv genotype-1 (hcv-1) who were treated with pegylated interferon alfa subcutaneously once weekly plus ribavirin (weight-based), 20 consecutive paients who had low pretreatment viral load ( 1.5x10 6 copies/ml) and rvr were treated for 24weeks and then followed up for 24weeks. the hcv rna was quantitatively assessed pretreatment, at 12weeks of treatment and was qualitatively assessed at 4weeks of treatment, the end of treatment (24weeks), 24weeks after end of treatment. rvr was defined as undetectable hcv rna at the 4weeks. results: baseline characteristics of patients was as followed; age (30-65years:mean 45years), bmi (21-27kg/m²:mean 23.5kg/m²), hcv rna titer (0.3-1.4×10 6 copies/ml:mean 0.5×10 6 copies/ml), alt (5-751iu/l:median 77iu/l). among the 20 patients, all patients (100%) had sustained virologic response (svr). conclusions: hcv-1 infected patients with a low baseline hcv rna concentration ( 1.5×10 6 copies/ml) who had hcv rna negative at week 4 of treatment may be treatment for 24weeks without compromising sustained virlolgic response. however, an additional trial will be needed to optimize the treatment duration. background/aims: acute hepatitis c (ahc) has a high chronicity rate of up to 50~84% if it is not treated. although the good treatment response to pegylated interferon (peg-interferon) therapy has reported, there is not definite guideline to treat of ahc in korea yet. the aim of our study was to investigate the clinical course and treatment outcome of ahc in single center of korea. methods: we performed a retrospective analysis of 35 patients who were diagnosed with ahc during the period from may 1996 to december 2007. the diagnosis of ahc was based on seroconversion to anti-hcv antibody or the clinical and biochemical diagnostic criteria satisfactory to ahc and on the presence of hcv rna in first serum sample. the spontaneous resolution was defined as loss of hcv rna in serum for 6-month in untreated group, and in treatment group, the sustained virological response (svr) was defined as a index of treatment success. results : thirteen of thirty-five patients were treated, six of thirty-five were untreated and observed clinical course, and sixteen patients were not followed up after diagnosis. in treatment group, nine of thirteen (69%) acquired svr, and two of six (33%) showed spontaneous resolution in untreated group. ten of thirteen treatment patients used conventional interferon, and another three patients used peg-interferon. conclusion : compared with untreated group, there was higher svr rate in treatment group (33% vs. 69%). so early interferon treatment in acute hepatitis c should be considered. background: this study was conducted to identify predictors of thyroid dysfunction and to determine whether virologic factors or treatment response affect thyroid dysfunction development during peginterferon (pegifn) therapy in chronic hepatitis c patients. methods: sixty chronic hepatitis c patients treated with pegifn -2a or -2b in combination with ribavirin from 1st july 2004 to 30th july 2008 were included in this study. treatment responses were evaluated and thyroid functions were assessed every 4 weeks. results: seventeen patients (28.3%) experienced thyroid dysfunction during treatment, and that occurred more frequently in women and in patients with a lower body mass index (bmi). the proportion of patients with a high viral load (a serum hcv rna titer >750,000 iu/ml) was significantly higher in the thyroid dysfunction group rather than in the euthyroid group(88.2% vs. 48.8%, p=0.005). among patients with hcv genotype 1, the rate of sustained virologic response was lower, and relapse occurred more frequently in the thyroid dysfunction group than in the euthyroid function group during pegifn-based therapy(svr, p=0.024; relapse, p=0.017). the female gender and the high viral load were independent predictors of thyroid dysfunction in multivariable analyses (female, or 9.44, p=0.009; high hcv rna titer, or 8.40, p=0.030) . conclusion: the risk of thyroid dysfunction during pegifn therapy for chronic hepatitis c was found to be higher for women and for those with a low bmi and a high viral load. background: in peginterferon alpha2b (peg-ifn 2b) and ribavirin (rbv) combination therapy for 48weeks for patients with chronic hepatitis c, it is still difficult to predict which patients will achieve sustained viral response (svr) at the completion of this therapy. aim: to predict svr and non-svr (relapse) at the end of this combination therapy by determining changes of serum hyaluronic acid (ha) levels. methods: eighteen patients were enrolled and their serum ha levels were measured before therapy, and after the 1st, 2nd, 3rd, and last trimesters during therapy. results: eleven patients achieved svr and 7 became relapsers. all patients showed higher ha levels in the 1st trimester than the pretreatment levels. in the svr group, 3 of 11 (27.2%) patients in the 2nd, 5 of 10 (50.0%) in the 3rd, and 9 of 11 (81.8%) in the last trimester showed lower ha levels than the pretreatment levels. by contrast, in the relapser group, none in the 2nd, 1 of 6 (16.7%) in the 3rd, and 1 of 7 (14.3%) (p<0.05) in the last trimester showed lower ha levels than the pretreatment levels. this study revealed that as the 48-week therapy went on, ha levels were more likely to fall below the pretreatment levels by the last trimester in patients achieving svr. however, ha levels of relapsers tended to continuously be above the pretreatment levels. conclusion: determination of changes of serum ha levels during peg-ifn 2b and rbv therapy predicts svr and non-svr at the completion of this therapy. results: the most frequent lymphomas were with high malignancy (40%), intermediate (32.5%) and low degree (27.5%). cryopathy was negative (87.5%). the presence of viral markers was performed soon as possible after the nhl diagnosis, at the same time (52.5%) or during the first year of evolution (32.5%). the prevalence of the hcv infection was 15%, comparable to the one in the control patients group (22.68%), admitted in a gastroenterological clinic. on the other hand, this prevalence is significantly increased compared to the one in the general romanian population (4.9%). the patients with nhl and hcv infection belonged especially to the low and intermediate malignancy degrees; the survival was influenced by the malignancy degree and not by the presence of hcv infection. the prevalence of hbv infection in the tested patients was 2.5%, being lower than that of hcv infection (2.5% vs. 15%, p = 0.113) but comparable to the one in the general population (2.5% vs. 6.3%, p = 0.525). conclusions: the prevalence of hcv infection in the patients having nhl was 15%, comparable to the one in the control group, but significantly increased compared to the one in the general population, leaving open the issue of a causal relationship between hcv infection and nhl. iron hepatic overload and hepatitis c d. damian 1 , m. grigorescu 1 , m.d. grigorescu 1 , t. zaharie 1 1 third medical clinic, cluj napoca, romania aim: evaluation of the prevalence and the degree of iron loading and the relationships with the clinical, biological and morphological changes. method: 212 patients with chronic hepatitis c were included, to whom we tested the blood iron level. in order to evaluate the hepatic iron accumulation we performed the perls staining, using a qualitative analysis and a semiquantitative scoring system (deugnier). results: from a total of 212 patients, 16.5% presented increased blood iron level (p = 0.000). the evaluation of the liver iron loading was performed in 54 patients, some having normal blood iron level (n = 34) and others (n= 20) increased (p = 0.007). the stainable iron was observed in 27 patients. the iron loading was usually low, the deposits were observed mostly at the sinusoid cells and the hepatocyte and less in the portal spaces, usually as a pale staining or of small, nonmerging granules. the total iron score deugnier was low. the increased blood iron correlated with the alt and ggt levels, the necroinflamatory activity and fibrosis. no correlationships between stainable iron and increased blood iron. the presence of liver iron accumulation only correlated with the fibrosis degree. conclusions: of the 212 patients to whom we tested the blood iron, 16.5% had increased levels. the perls staining was positive in 50% of the patients. the iron loading was mainly low, with a more frequent distribution in the sinusoid cells and in the hepatocytes and correlated only with the stage of fibrosis. response patients whose hcv rna became negative at 12 -16 weeks t. ide 1 , t. arinaga 1 , k. ogata 1 , i. miyajima 1 , k. kuhara 1 , r. kuwahara 1 , m. background/aim: chronic hepatitis patients whose hcv rna became negative at 4 weeks of peg-interferon/ribavirin treatment achieved excellent svr(sustained viral response) rate of almost 90%. however, in patients whose rna became negative after 20 weeks, the svr rate is very low. since many patients became rna negative at 12-16 weeks, it is important to clarify the characteristics of the patients. material and method: among 234 patients, 41 became rna negative at 12-16 weeks and the therapy completed (total 48-60 weeks). the characteristics were analyzed by using sex, age, weight, bmi, alt, gtp, hemoglobin, platelet counts, ccr, hyaluronic acid, the mutation of hcv core region (aa70, 91) and interferon sensitivity determining region, adiponectine, home-ir, rna dynamics, dose and the treatment period. result: the svr rate was 75.6%(31/41). because all 10 patients of non svr were female, we compared these 10 patients and 15 female svr. the platelets counts were low in non svr (non svr 15.0 ± 2.8 (x10 4 /mm 3 ) vs svr 20.2 ± 4.8 (p<0.05)). the mean dose of ribavirin was lower (447 ± 126 mg/day) in non svr (p<0.05) than in svr (625 ± 127). conclusion: as for the characteristics of the patients whose hcv rna became negative at 12-16 weeks but became non svr, female, low platelet count and low dose of ribavirin were important factors. in the patients who received reduced ribavirin doses, the idea to increase the ribavirin dose and to maintain it are necessary. (ex, use 400mg and 600mg alternately) pe329 background: chronic hepatitis c virus (hcv) infection poses a challenge for a growing number of infected patients who exhibit disease complications, including cirrhosis, hepatocellular carcinoma, and liver failure in china. the combination treatment of peginterferon alpha (peg-ifn alpha) plus ribavirin (rbv) is recommended as a standard care for hcv infections, which can improves hepatic markers and eradicates the virus in about 50% of patients. however, a significant number of patients do not respond to therapy or relapse following treatment discontinuation. several viral, hepatic, and patient-related factors influence response to therapy. methods: in our clinical practice, a total of 77 interferon-naïve patients (61% male; median age 47 years) with chronic hepatitis c include 11 cirrhotic patients (no genotyping) received peg-ifn alpha-2a 180 mcg/week plus rbv 900-1200mg/day for 48 weeks and follow up 24 weeks. results: show that the patients have more rvr and evr rate (54% and 90.9% respectively). while the svr (undetectable hcv-rna 24 weeks after treatment completion) rate is only 51.5% in conclusion: comparing with the data of clinical trail, the rvr, evr and eotr were higher, while svr was the same in chinese patient with chronic hepatitis c patients received the combination therapy of peg-ifn plus rbv. the reason of high relapse was still unknow. although optimal duration of retreatment and benefits and safety of maintenance therapy have not been determined, an extended duration is likely needed, even for the patients who achieved evr. s. nakamoto 1 , f. imazeki 1 , k. background/aims: recently amino acid (aa) substitutions in hepatitis c virus (hcv) core region (double wild (dw); arginine at aa 70, leucine at aa 91) were reported to be associated with sustained virological response (svr) in a combination therapy of peginterferon and ribavirin. we evaluated the viral factors influencing treatment response. methods: nucleotide sequences of core region were determined directly in 104 patients with genotype 1 and high viral load ( 100 kiu/ml) treated with peginterferon-alpha 2b and ribavirin for 48 weeks. rapid virologcal response (rvr) was defined as more than 2 log decrease of hcv-rna during the first four weeks of therapy and early virological response (evr) as that during the first 12 weeks. svr was defined as negative hcv-rna 6 months after the end of treatment and non-virological response (nvr) as less than 2 log decrease of hcv-rna during the treatment. results: dw at aa 70 and 91 was shown in 17/44 (35%) patients with rvr and in 5/44 (11%) with non-rvr (p=0.003), in 25/72 (35%) with evr and in 1/28 (3.6%) with non-evr (p=0.001), in 16/37 (43%) with svr and in 6/44 (14%) with non-svr (p=0.003), and in 1/24 (4%) with nvr and in 25/79 (32%) with non-nvr (p= 0.005). in multiple logistic regression analysis, dw was significantly associated with rvr, evr, svr and nvr. conclusions: dw at aa 70 and 91 in hcv core region was closely associated with virological response in a combination therapy of peginterferon and ribavirin. medicine and hepatology, henry dunant hospital, athens, greece, 3 biometrics, ist gmbh, mannheim, germany, 4 background: among patients with chronic hcv treated with pegylated interferon and ribavirin, the highest sustained virologic response (svr) rates are achieved in patients with a rapid virological response (rvr). here we investigate how the time taken to become hcv rna undetectable influences the probability of relapse during untreated follow-up. methods: data from 569 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180µg/week plus ribavirin 1000/1200mg/day were included in the intent-to-treat analysis. response was classified as rvr, complete early virological response (cevr) slow responder and non-evr. results: there was a correlation between the time required to become hcv rna undetectable and the relapse rate after stopping treatment. patients with an rvr had the lowest relapse rate (4%); this increased among patients with slower responses. conclusion: there was an inverse correlation between the time taken to achieve a virologic response and the probability of relapse. background: rapid virologic response (rvr; hcv rna <50iu/ml) at week 4 of treatment with pegylated interferon plus ribavirin can be used to predict the probability of achieving an svr. patients with detectable hcv rna at week 4 have a lower probability of achieving an svr than those with an rvr; further subdivision of these patients may be useful in predicting outcomes. methods: we conducted a retrospective analysis including 569 genotype 1 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180 g/week and ribavirin 1000/1200 mg/day. patients were categorized as rvr and non-rvr. those without an rvr were further subdivided into detectable but unquantifiable, 3, 2, 1 or <1 log10 drop in hcv rna. the proportion of patients with undetectable hcv-rna at week 12 and achieving an svr was calculated within each category. results: rvr and non-rvr patients had an 88% and 43% rate of svr respectively. among non-rvr patients, rates of svr depended on the categorical response at week 4: detectable but unquantifiable hcv rna, 77%; 3 log10 drop in hcv rna, 61%; 2 log10 drop, 43%; 1 log10 drop, 27%; and <1 log10drop, 13%. independent of week 4 response, undetectable hcv rna at week 12 was also highly predictive of svr. conclusions: patients achieving an rvr have high rates of svr. among patients who do not achieve an rvr a more precise prediction of svr can be achieved by considering the extent of viral load reduction at week 4 and week 12. retrospective japanese validation study of fibrotest and actitest in patients with chronic hepatitis c. n. nagata 1 , t. mine 1 1 background: fibrotest (ft) and actitest (at) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis c virus. the aim of this study was to perform a validation study the discordances between ft and at(ft/at) and liver biopsy in patients with chronic hepatitis c in japan. methods: 117 serum samples of chronic hepatitis c patients sended at -80°c at the biochemistry department of pitié salpetrière hospital were analysed between july and august 2007. ft/at components were assessed on thawed sera for 110 patients. 96 from 110 patients had liver biopsy at the moment of serum analysis. liver biopsy fibrosis and activity scores were assessed by a pathologist in japan according to metavir scoring system. for each individual test-ft/at the following statistical analysis were performed result: ft observed auroc for the diagnosis of advanced fibrosis was 0.81 and after adjustment according to the prevalence of different stages of fibrosis the auroc was 0.89. this difference could be explained by the non-homogenous distribution of different stages of fibrosis (low prevalence of extremes stages of fibrosis -f0 and f4-and high prevalence of adjacent intermediate stages -f1 and f2). the observed auroc of ft for the diagnosis of precirrhosis and cirrhosis was 0.86 and the observed auroc of at for the diagnosis of moderate to severe activity was 0.74 . conclusion: these results are similar to those observed in all independent validations worldwide. background: accurate monitoring of hcv-rna level throughout anti-hcv therapy is key factor for predicting sustained virological response (svr). real-time detection polymerase chain reaction (rtd-pcr) based methods are sensitive, have wide dynamic range of quantification and carryover contamination caused by classical pcr. aim: to compare rtd-pcr based assays; cobas ampliprep/cobas taqman (cap/ctm) and recently developed abbott realtime hcv for hcv rna quantification and measurements differences by 2 assays in different genotypes. methods: in total, 253 serum samples were used including, 135, 39, 24, 15, and 40 with genotypes 1b, 2a, 2b, 3a and 4 respectively were tested quantatively for hcv-rna by cap/ctm and abott realtime. results: good correlation between two assays as overall (r=0.96) with correlation coefficient (r) in genotypes 1b, 2a, 2b, 3a ranged between 0.99 to 0.98 and least in genotype 4 (r=0.78). mean differences between cap/ctm and abott realtime was significnat in genotypes 1b and 4. significantly hcv-rna genotype 4 underestimation by cap/ctm (4.3+0.9 log iu/ml) than abbott realtime (4.8+0.9 log iu/ml; p=0.01). in genotype 1b, significantly higher hcv-rna measurement by cap/ctm (5.7+1.7log iu/ml) than abbott realtime (5.0+1.4log iu/ml, p=0.001). two hcv genotype 4 samples showed measurement differences (cap/ctm minus abbott realtime) of -3.75 and -1.68 log iu/ml. studying genotype 4 sequences within 5 utr , target for cap/ctm rt-pcr amplification, revealed nucleotide polymorphisms at positions a107, a165, t203, a205, and a243. conclusion: different measurement efficiency by commonly used cap/ctm in different genotypes compared to abbott realtime. 6 new york, new york, usa, 7 vertex pharmaceuticals, 8 cambridge, ma, usa, 9 duke clinical research institute , 10 duke university, durham, nc, usa background: prove1 is a placebo-controlled study of 250 subjects with genotype 1 chronic hepatitis c randomized to 48 weeks of peginterferon-alfa-2a 180 ug/week (p) plus ribavirin 1000-1200 mg/d (r) (pr48, n=75), or 3 regimens of 750 mg q8h telaprevir (tvr) with pr: tvr/pr for 12wks followed by pr for 0wks (t12/pr12, n=17), 12wks (t12/pr24, n=79) or 36wks (t12/pr48, n=79). the impact of african american race (aa) and bridging fibrosis on sustained virologic response (svr) was examined. methods: subjects with cirrhosis were excluded from study. fibrosis was categorized as mild/minimal, portal, or bridging from biopsy within 2 years. itt analysis was performed. results: overall, svr was achieved by 41% of subjects in the pr48 group, 35% in t12/pr12 group, 61% in t12/pr24 group, and 68% in t12/pr48 group. subgroup analyses indicated svr was improved with tvr/pr (tvr/pr arms pooled) vs pr48 alone in aa subjects (44% (8/18) vs 11% (1/9)), and in subjects with bridging fibrosis (69% (22/32) vs. 26% (5/19)). adverse events leading to discontinuations were more frequent in the tvr/pr groups (21% vs. 10%). rashes, gastrointestinal events and anemia were more common in the t/pr arms, and rashes were more frequently severe (7% vs 1%). conclusions: tvr-based treatment for 24 or 48 weeks was associated with an increase in svr rates compared to pr48. subgroups with impaired response to standard peg-ifn/rbv therapy appeared to benefit from the addition of telaprevir. adverse events leading to discontinuation were more frequent in tvr-based regimens. background and aims: induction of type i ifns is a core issue in antiviral responses and must be tightly controlled. the protein kinase tbk1 is critically involved in virus-triggered type i ifn signaling. in previous studies, an alternatively spliced isoform of tbk1, termed tbk1s, was identified to be induced in both human and mouse cells. bound to rig-1, it is able to disrupt the interaction between rig-i and visa. this study was designed to observe the expression of tbk1s in hcv-infected patients. methods: total rna was extracted from samples of peripheral blood mononuclear cells obtained from 11 hcv patients, 9 hcv patients treated with ifn-/ribavirin and 9 healthy controls, and subjected to real -time pcr using the primer-probe sets for human tbk1s, tbk1 and ifn-genes. results: the tbk1s expression was significantly elevated in hcv-infected patients, while treatment of hcv-infected patients with ifn-/ribavirin resulted in down-regulation of tbk1s to the normal level. conclusions: the study strongly supports the idea that expression of tbk1s is correlated with hcv infection, and indicates that tbk1s may play an important role in the regulation of hcv infection.this work was supported by nsfc(30571643, 30672380, 30700702 background: infringement of iron metabolism is one of fibrosis progressing factors during diffuse liver diseases. the interrelation between the syndrome of iron overload (sio) and svr achievement is studied during chronic hcv infection treatment. methods: 68 patients with chronic hcv infection (genotyping: 1-34; 1+3-5; 3-22; 2-6; 4-2) are investigated. sio criteria: iron increase-more than 37 mkmol/l, ferritin -more than 200 mkmol/l, percent of transferriny saturation with iron (%tf) -more than 50%. results: sio revealed in 10 patients (14.7%): 5 patients -1 genotype (2 assotiative with a diabetes) and 5 patients-genotype 3 (3 -in combination with liver steatosis and obesity). venipuncture series were done up to getting ferritin referential parameter values before therapy beginning. rvr: sio -5 patients, normal metabolism -48; evr: 6 and 54, svr: 4 and 54 relatively. 5 nonresponding patients (sio) had steatosis and diabetes, hereditary hemochromatosis (c282y/h63d) is verified in 1 case. increase of ferritini values and %tf during therapy and positive hla-a3 and hla-b7 is registered in nonresponding patients. conclusions: sio in hla-a3, hla-b7 and c282y/h63d positive patients is independent predictor of nonresponse during peginterferon alpha-2a (40 kd)" ribavirin treatment. a.p. srivastava 1 , g. dogra 2 , s. sachdeva 1 , n. nigam 1 , a. chakravarty 2 1 dr. rml hospital, new delhi (india)-110001, 2 maulana azad medical college & associated hospitals, new delhi, india background: hepatitis c virus (hcv) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. genotyping and assessment of viral load in hcv patients are vital for designing therapeutic strategies. we aimed to determine the pattern of hcv genotypes and its association with viral load and biochemical profile. methods: 71 hcv rna positive patients were included in the present study attending the medical-opd and wards of dr rml hospital, a tertiary care hospital in new delhi during 2006-2008. hcv genotyping was carried out by restriction fragment length polymorphism (buoro et al 1999) followed by the type specific primers from the core region (ohno et al 1997) . viral load estimation was carried out by taqman real time pcr system using previously described method (martell et al 2000) . result: 67.6% of cases were having genotype 3 (3a, 3b, 3f & 3i) followed by genotyping 1 (1a & 1b) in 26.8% and genotype 2 in 5.6%. there was no statistical significant difference seen in the biochemical profile between the three groups of genotypes. genotype one was associated with a significantly higher viral load as compared to the genotypes three and two. parentral mode of transmission was accounted for the 68% of all the infected cases. conclusion: hcv genotypes 3 and 1 accounted for 94% of our cases. the genotype 1 is associated with higher degree of disease severity as assessed by viral load. also two unusual subtypes 3i and 3f were identified from this geographical region. a.p. srivastava 1 , g. dogra 2 , s sachdeva 1 , n. nigam 1 background: the development and resolution of an inflammatory process is regulated by a complex interplay among cytokines that have pro and anti-inflammatory effects. regulatory mechanisms that control the production of cytokines include genetic polymorphism in particular promoter/leader region. polymorphisms may directly or indirectly affect the binding of transcriptional factors, consequently increasing or decreasing the production of mrna, thus regulating cytokine production. we aimed to determine the polymorphism of tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) genes in chronic hepatitis c patients. methods: 40 hcv rna positive patients were included in the present study conducted during 2006-2008. 25 healthy controls were also included. genomic dna was extracted by using q1a amp dna blood kit protocol according to manufacture's instruction and desired fragment was amplified by using the primer's of vidigal et al 2002. result: genotyping of -308-promoter variant of tnf-alpha was performed by pcr. polymorphism in the tnf-alpha (g/g, g/a and a/a allele) was different between hcv patients and healthy controls. il-10 variants (c/t, c/c) were more frequent among hcv patients as compared to healthy controls. conclusion: genetic polymorphism analysis on il-10 promoter have indicated that distribution pattern of il-10 polymorphism was significantly different between controls and hcv patients. furthermore, polymorphism in promoter region of tnf-alpha (-308) was found, though the difference was not significant. since this is a preliminary study, we believe that our findings may stimulate further research on larger number of patients. introduction: the assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. although liver biopsy is the gold standard for fibrosis assessment, it is invasive and may have some risks, this has led to the development of non-invasive biochemical markers of liver fibrosis. fibro-test which have five parameters used for the quantitative assessment of liver fibrosis. our aim is to validate the performance of fibro-test in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 50 patients with chronic hepatitis c genotype 4. all biopsies were scored using metavir system by two independent pathologists. fibro-test was done with (biopredictive, houilles, france) for the assessment of liver fibrosis. sensitivity, specificity, ppv and npv were measured for distinguishing between different degrees of severity of fibrosis. results: patients (45 male and 4 female) age ranged 21-56 years, liver biopsy showed 4% (f0), 40% (f1), 20 % (f2), 28% (f3), 8% (f4). the efficacy of fibrotest is 63.26%, sensitivity 83.3%, specificity 53%, positive predictive value 50% and negative predictive value 85%. conclusion: fibrofast has a low performance in assessment in fibrosis in chronic hepatitis c genotype 4. introduction: liver biopsy is the reference method for assessing liver fibrosis. however, it is invasive, costly and has some limitations. european liver fibrosis (elf) markers have shown to be accurate in assessing liver fibrosis in a range of chronic liver disorders. our aim is to test the performance of elf markers in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 199 patients with chronic hepatitis c genotype 4. all biopsies were staged for fibrosis using metavir system by two independent pathologist. elf markers were done by (diagnostic & operations, england) and fibrosis scores were derived using the published elf algorithm. the area under the curve (auc) for receiver operator characteristic curves was measured along with sensitivity and specificity, positive (ppv) and negative (npv) predictive values for distinguishing between different stages. results: patients (179 male and 20 female), age was ranged 25-51 years, liver biopsy showed 2% (f0), 27% (f1), 34% (f2), 26% (f3) and 11% (f4). elf markers had no correlation with fibrosis score where r = -0.003, p = 0.963, aucs: 0.469, specificity 87.9 %, sensitivity only 9.3 %, ppv: only 6.03 %, npv: 61.5 % and efficacy 58.2%. conclusion: the performance of elf marker is low and can not be used for assessment of fibrosis in chronic hepatitis c genotype 4. background: the prove2 trial is a randomized, placebo-controlled study that assessed the safety and efficacy of 750mg q8h telaprevir (tvr) combined with 180 g/week peg -ifn alfa-2a (p) ± 1000-1200mg/day ribavirin (r) in chronic hcv genotype 1-infected treatment-naïve patients without cirrhosis. methods: overall, 323 patients received tvr + pr for 12 weeks (t12/pr12; n=82), tvr + pr for 12 weeks then pr for 12 weeks (t12/pr24; n=81), tvr + p for 12 weeks (t12/p12; n=78), or to pr for 48 weeks (pr48; n=82). primary endpoint: sustained virologic response (svr, undetectable hcv-rna 24 weeks post-treatment). results: baseline characteristics were well balanced across groups. numerically higher svr rates were observed in patients receiving t12/pr24 (69%; p=0.004 for difference vs. pr48) than t12/pr12 (60%), t12/p12 (36%) or pr48 (46%). relapse rates were lower in the t12/pr24 group (14%) than the t12/pr12 (30%), t12/p12 (48%) and pr48 (22%) groups. the relapse rate in patients receiving t12/pr24 with 4-week and 12-week undetectable hcv-rna was 7% (3/45). the aes occurring more frequently with the t/pr regimen were pruritus, rash, asthenia, nausea and anemia. in the t/pr arms, 12 patients discontinued due to rash, 2 discontinued due to pruritus, and 2 patients due to anemia. conclusion: these results showed that a telaprevir-based regimen led to significantly higher svr rates than pr, and indicate that this regimen could shorten the overall treatment duration from 48 weeks to 24 weeks for most patients infected with hcv genotype 1. a.c. cardoso 1 , c. stern 1 , r. moucari 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: this study evaluated the effect of the response (svr) to therapy on fibrosis stage, as assessed by ls, in patients with advanced fibrosis (f3) or cirrhosis (f4). methods: hcv patients with f3 or f4 who received interferon-based treatment were studded. ls was assessed after treatment (median delay of 36 months, 2-206) in patients with or without svr. correlations between ls and clinical and treatment characteristics were analyzed. results: 114 patients were included: male gender (72%), mean age (54±9 years), diabetes (26%), mean bmi (26±6 kg/m2), genotype 1 (59%). 33% had svr. ls was performed 0-3, 3-6, >6 years following treatment. by linear regression, the median of the ls was independently associated with svr (p=0.005) and diabetes (p=0.008). svr patients had lower ls (8.4 kpa; range 3.3-45) than non svr patients (15.7 kpa; range 5.3-75) (p<0.001). among the svr patients the median ls was lower when the delay between ls and the end of treatment was longer (10.9,8.8,6. 3) (p=0.02). on the opposite, among the non-svr patients the median ls was not significantly different (p=0.57). the median of liver stiffness was higher in patients with diabetes (p=0.006). bmi and dyslipidemia did not influence the median of the ls. conclusion: in patients with advanced fibrosis or cirrhosis, ls was lower in patients with svr and decreased with time while it was higher and did not decrease in non-svr patients. ls could be important for assessment of fibrosis stage during the post-treatment follow-up. to study peripheral blood and intrahepatic natural killer (nk) cells in patients with chronic hepatitis c in relation to disease activity and severity of hepatic fibrosis. patients & methods: fifteen untreated patients with histologically-proven chronic hepatitis c, and 12 matched healthy subjects. the nk cells and natural killer t (nkt) cells were identified in fresh whole blood samples using two-color flow cytometric assay as cd3 -cd56 + and cd3 + cd56 + positive cells. immunohistochemical staining of liver biopsies taken from all patients was done using monoclonal antibody against cd56 for detection of nk cells and rabbit polyclonal antibody against smooth muscle actin (sma) for identification of activated hepatic stellate cells (hscs). results: patients with chronic hepatitis c showed significant decreases in the percentages of nk cells and nkt cells in peripheral blood. a negative correlation was found between serum hcv rna levels and the percentages of peripheral blood nk cells and the intensity of intrahepatic nk cells. the percentages of circulating nk cells and nkt cells and the intensity of intrahepatic nk cells were inversely correlated with the metavir fibrosis stage and the steatosis grade, and also with the intensity of intrahepatic activated hscs. conclusion: patients with chronic hepatitis c had significant deficiency in circulating nk and nkt cells as well as in intrahepatic nk cells. this may provide a possible mechanism for the suppression of innate immunity against hcv. background: hcv infection is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. the virus is classified in six genotypes and more 50 subtypes, which are related distinct with antiviral therapies reply. in brazilian amazon, epidemiologist's studies in blood donors had pointed high frequency of genotype 1 (74%) followed by genotypes 3 (25%) and 2 (1%). however, epidemiological research in populations of risk to the infection still is scarce. aim: to determine hcv genotypic frequency in 116 blood donors, 55 patients with blood transfusions multiples, 57 patients in hemodialysis and 52 drugs users in the state of pará, brazilian amazon. methods: using real time pcr and nucleotide sequencing followed phylogenetic analysis had been gotten viral diagnosis and genotyping. results: in blood donors, hcv distribution was constituted by genotypes 1 (93.1%) and 3 (6.9%). in multitransfunded patients occurs maximum prevalence of genotype 1 (100%), probably reflect of genotype 1 specific transmission of blood donors population. on the other hand, in hemodialysis patients had been detected genotypes 1 (85.7%), 2 (3.6%) and 3 (10.7%), result of a bigger diversity of transmission routes (transfusional, interfamilial, nosocomial, etc) . in drug users occurs the biggest frequency of genotype 3 (38.1%) with prevalence of genotype 1 (61.9%), suggesting that the sharing of abuse machinery is allowing strains diffusion of genotype 3. conclusions: the genotype 1 possesses the biggest frequency in different population. moreover, through hcv genotypic frequency if it detached the contribution of transmission distinct routes indicated by previous epidemiologists researches. virol. 2007 ). we established real-time polymerase chain reaction (pcr) assays for the easy detection of these hcv mutations. methods: plasmids p-core-w, including wild type hcv core coding region (70r and 91l), and p-core-m, including mutant type hcv core (70q/h and 91m), were constructed by cloning and pcr-based mutagenesis for control vector of wild type core and that of mutant core, respectively. using serially diluted forms of these vectors, sybr green-based real-time pcr detections with mutation-specific primers were performed. results: analysis of known scalar concentrations of references indicated that the detection limits of these methods were at least 10 copies, 10 copies, 1000 copies, and 10 copies of 70-wild, 70-mutant, 91-wild, and 91-mutant, respectively. each primer could clearly distinguish the difference between p-core-w and p-core-m at the same copy numbers. concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-w versus p-core-m could be distinguished. on the other hand, for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. conclusions: this method could represent a useful alternative for the detection of genotype 1b hcv core amino acid substitutions 70 and 91 and be reliably applied for rapid screening. efficacy and tolerability of hcv treatment in asian patients according to age and genotype at a tertiary centre in western australia n. saroj 1 , n. kontorinis 1 , t. lorenzo 1 , m. marion 1 , s.l. chen 1 , w. cheng 1,2 1 royal perth hospital, 2 centre for international health, curtin introduction: race and ethnicity can influence efficacy and tolerability to treatment in hcv. the higher response rate in asians is thought to be associated with better adherence and tolerability. objectives: (1) to evaluate the adherence according to age and genotype (2) to assess the effect of age on treatment efficacy (3) background: hepatitis c virus (hcv) infection is a major health problem. there is huge regional variation in its prevalence and genotypic distribution. voluntary blood donors are thought to have somewhat lesser prevalence than the rest of the community. reliable statistics are not available for the entire country, particularly for the rural areas. it is important to know local situation and rationalize use of limited resources. methods: retrospective study of the records of patients attending the free liver clinic (flc) of our hospital located in a rural area of pakistan, and those screened for hcv infection prior to voluntary blood donation. results: patients at flc (324 out of 1638 [20%; males 65%] were found to have higher chances of being reactive for hcv antibodies as compared to voluntary blood donors (121/804 [14%]; p = 0.004; or 1.39 -95% ci = 1.11 -1.75). out of a total of 1022 hcv reactive patients, 904 (88%) were found to be positive on hcv rna testing. out of a total of 166 typeable genotypes, 125 (75%; 95% ci = 68.7 -81.9, estimated odds = 3.05) were infected with a single genotype, and only 7 patients (4%) were infected with genotype 1, either alone (n=4) or in combination with 3a. conclusions: one out of every 5 people tested in our flc is seropositive for hcv, and 14% of "healthy" voluntary blood donors have the same results. genotype 1 is very rare in our region. s.a. batool 1 , s.z. abbas 1 1 department of gastroenterology, muhammad hospital, mirpurkhas, pakistan background: hepatitis c viraus (hcv) infection is common in our region. data is not available on success rates of conventional interferon (inf) based products here. we attempted to find out the dominant genotype, and to determine the success rate of conventional inf-based treatment in eradicating hcv. methods: retrospective case series study of hcv infected patients' records treated with 14 different brands of inf. results: 320/1858 (17%) of all patients tested were positive for hcv antibodies. hcv-rna was tested by pcr for 1022 patients, of which 904 (88%) turned out to be positive. genotype type 3 was the dominant genotype -found in 118/168 (70%) patients. 101 men and 57 women were treated with various brands of inf with the same manufacturer's brand of ribavirin. the overall etr achieved was 100/142 (70%) -58/85 (68%) men and 42/57 (74%) women. 41/62 (66%) of genotype 3 achieved etr. there was no significant difference in average ages for those who achieved good etr and those who did not (39 years each). the etr achieved by different brands ranged from 48% to 91%. svr was achieved by 17/30 patients. conclusions: 17% of all people tested positive for hcv antibodies, of which about 88% had evidence of active hcv infection. etr achieved by different brands averaged 70%. this was 74% in female sex, although age did not appear to be a factor in determining a favourable etr. patients & methods: a total of 439 consecutive diabetic patients of either sex were evaluated for hcv and hbv infection by using enzyme linked immunosorbant assay (eliza-3) along with serum alt levels. on the basis of this test, the patients were divided into two groups, sero +ve and sero -ve. different variables were: age, sex, bmi, area of residence (rural or urban), type and duration of dm, smoking, literacy and alt. results: males 50.3% and females 49.7%. age ranged from 18 to 95. majority were married (98.4%), from rural area (70.4%), had type-2 dm (99.8%), normal weight (39.2%), normal alt(60.1%) and non-smokers (78.6%). seroprevalence for hcv, hbv and both were 25.1%, 1.8% and 1.6%. two groups were made, sero +ve and sero -ve. raised alt (59.2%) was significant (p<0.05) factor while all others variables were insignificant (p>0.05). conclusion: hbv and hcv infections are more prevalent in dm with increased alt levels. while hcv infection is more common than hbv in patients with dm. hepatitis c virus (hcv) envelope proteins (e1 and e2) mediate the entry of virus into host cells by binding to its cellular receptors and resulting in the fusion of the viral membrane with host cell membrane. the expression and secretion of biologically active envelope proteins in vitro have proven to be a difficult task due to the high degree of glycosylation and the existence of hydrophobic domains within these sequences. in order to obtain glycosylated, correctly-folded hcv envelop proteins in large quantities, we optimized the dna sequences of hcv envelop proteins by substituting the encoded sequence with human preferable codons and expressed them in human embryonic kidney (hek) 293 cells. both proteins were detected intracellularly, with a small portion secreted into supernatant. in order to enhance secretion, truncated forms of envelop proteins including e2 tm, e2384-661, e2484-661 were also expressed. both full-length and truncated forms of envelop proteins were glycosylated and expressed at high level. in addition, we also expressed the codon-optimized hcv receptors cd81 and claudin-1 in 293 cells. by comparing the expression level of codon-optimized sequences and the sequences that were obtained from cdna library by pcr, we found that codon-optimization enhance protein expression significantly in 293 cells. these results not only lay solid foundation for further research concerning the mechanism of hcv entry, including the optimal ph and right protein conformation for fusion, cell types that permit viral entry; but also potentiate a useful cell model for testing antiviral agents. background: prolactin (prl) is an immunoregulatory hormone secreted from lymphocytes, however, prl induction in relation to hepatitis c virus (hcv) infection has not been elucidated. methods: serum prl levels were measured in both 232 subjects of our hcv cohort study and 31 male patients of the hospital, who were chronically infected with hcv. furthermore, serum prl levels were compared in 27 male patients before and after interferon therapy. we measured expression of prl mrna level in pbmcs in 12 male patients, and also investigated prl mrna of pbmcs collected from 5 healthy men that stimulated by hcv produced by huh7.5 cells in vitro. result: serum prl levels were significantly higher in the hcv-infected subjects than in the controls (p< 0.01). they were significantly higher in hcv-infected male subjects than in the controls (p< 0.001). serum prl levels were significantly higher in male patients than in the controls (p<0.01). serum prl levels decreased significantly after interferon therapy in patients with sustained virological response to therapy (p<0.05). the levels of prl mrna in pbmcs derived from hcv-infected patients were significantly higher in 12 male patients than in the controls (p<0.001). conclusion: the high levels of prl expression are associated with hcv infection in carriers. background and objectives: hepatitis c virus (hcv) is a major cause of chronic liver hepatitis, cirrhosis, and hepatocellular carcinoma.current clinic standard therapy is interferon alpha (ifn-) combination with ribavirin, but this treatment is associated with adverse effects and often fails to induce a sustained response. until recently, development of a hcv cell culture system (hcvcc) provides a suitable tissue culture system to study the complete hcv life cycle. in this study, we tested the effect of ifn omega (ifn-)-a member of type interferon on hcv compared with ifnbased on hcv 1b replicon and hcvcc. methods: we compared ifn-and ifn-2a effects on hcv rna replication and protein expression, as measured by ribozyme protection assay and western blot. we also compared the intracellular protein level of phosphorylated signal transducer and activator of transcription 1 (p-stat1) treated with different interferon type and concentration with western blot analysis. results: hcv rna and protein level were inversely related with ifnconcentration and compared with ifn-2a, at the same concentration, the hcv rna and protein levels treated with ifn-were lower than that treated with ifn-2a p 0.05 .also based on the hcv rna analysis, ec50 of ifn-was 10 folds lower than ifn-2a. ifns increased intracellular p-stat1 level at a dose dependent manner and compared the same concentration of ifn-and ifn-2a, p-stat1 protein level was higher in ifn-treated group p 0.05 . conclusions: these results demonstrate distinct antiviral effect of ifncompared with ifn-2a and this difference maybe partly caused by the stronger stimulation of ifn receptor . outstanding antiviral activity of ifnmay be useful for developing new hcv treatment strategies. background & aim: hepatitis b virus (hbv) infection with undetectable levels of hepatitis b surface antigen (hbsag) is called an occult infection, which although has been described among subjects with chronic hepatitis c liver disease in the western world, it's prevalence and clinical significance are still ambiguous in the indian subcontinent. materials and methods: we investigated hbv-dna pcr in serum samples of 260 hbsag negative subjects with chronic hcv-related liver disease, and 70 apparently healthy volunteers negative for hbsag and anti-hcv as control. results: serum samples found positive by at least two independent pcr assays were considered hbv dna positive. hbv-dna was detected among 19 hcv-related chronic liver disease (cld) patients (7.3%), which was higher (p = 0.2) as compared with the control volunteers (4.3%). it was more frequent (37.5%) in 24 anti-hbs negative/anti-hbc positive patients than in 180 anti-hbs/anti-hbc positive (5 %, p < 0.05). hcv rna by qualitative pcr was significantly (p <0.001) higher in occult hbv compare to non-occult. hcv genotype 1b was predominantly associated with occult hbv (73%), especially among subjects with hepatocellular carcinoma (hcc) (p<0.05) as compared to non-occult hbv cases. though not significant, frequency of occult hbv infection was higher than healthy controls and hcv 1b genotype was significantly associated in patients with hcc. conclusion: this study suggests that in all hbv-endemic areas, the possibility of occult hbv in patients with hcv should be considered and hbv-dna should be performed. j. zhao 1,2 , w.d. cai 2 , l. chen 2 , y.x. gan 2 , m.l. he 1 , x.r. wang 1 1 background: besides hiv and syphilis, hepatitis c virus (hcv) is also rapidly spread among men who have sex with men (msm). this study was designed to identify the prevalence of these 3 sexual transmitted diseases in msms in shenzhen, china. methods: a cross sectional study was conducted by using time location sampling method from april to july, 2008. 831 msm participants (including 454 male sex workers) were recruited and finished guided self-administered questionnaires (or interviews if they have difficulty in reading or understanding) in 37 venue-date-time randomly selected from 48 active venues. results: results were analyzed using spss. 736 blood samples were collected for hiv, syphilis and hcv test. participated msms were between the age of 18 to 51 years (25.5±5.9) with a majority of 20-29 years (73.7%). most of them finished junior high school education (74.9%). 84.2% had high level of knowledge on modes of transmission and prevention. likewise, 56.0% msms have ever sold sex to men, 38.3% of them were self identified as gay, 35.1% as bisexual. 78.3% msms had multiple male sexual partners and 50.3% msms always used condom. 48.6% of them had sex with women in the past 6 month, and the condom use rate decline to 35.1% during both male and female sex. hiv positive rate is of 6.7% and syphilis for 18.3%, hcv is only found in 3 cases (0.4%). conclusions: a greater number of the participants have both male and female sex partners. this survey shows that hcv infection rate is still low among msms in shenzhen, although the hiv and syphilis rate is high and continuing increased in the past few years. the change of insulin sensitivity in hepatitis c patients with normal insulin sensitivity s.g. park 1 , y.k. cho 1 , j.w. lee 1 , j.w. yun 1 , h.j. kim 1 , w.k. jeon 1 , b.i. background: hepatitis c virus (hcv) infection is associated with a high prevalence of diabetes mellitus (dm). insulin resistance (ir) is known to play a crucial role in the development of dm in chronic hepatitis c (chc) patients. we prospectively investigated the change of insulin sensitivity in chc patients during 5-year period, and analyzed factors significantly associated with ir. methods: subjects consisted of 62 non-cirrhotic chc patients with normal alanine aminotransferase (alt) and normal insulin sensitivity (chc group), and healthy control group of 172 subjects matched by age, sex, body mass index and life styles. we compared initial baseline insulin sensitivity, metabolic parameters and incidence rate of ir at the end of follow up period in both groups. the change of insulin sensitivity and metabolic parameters and development of ir was analyzed, and factors associated with development of ir were evaluated. results: ir developed in 22.5% of 62 chc patients and 5.2% of 172 normal individuals (p<0.001). hcv infection per se and genotype 1 were independent risk factors of ir. initial fasting glucose 90-100 mg/dl, fasting insulin 10 uiu/ml, homa-ir 2.3-2.7 were significantly associated with development of ir in chc group. conclusions: hcv infection is independent risk factor of ir. even if chc patients with normal insulin sensitivity, careful monitoring for ir is necessary. prevalence of viral hepatitis c in latvia i. tolmane 1,2 , b. rozentale 1,2 , j. keiss 1 , f. arsa 1 1 sa infectology center of latvia, 2 riga stradin's university background and aim: viral hepatitis c (vhc) because of its prevalence and clinical course has become one of the most actual infectious diseases in the world. to date chronic hepatitis c affects over 170 million individuals worldwide. chronic vhc is a leading cause of cirrhosis and hepatocellular carcinoma. the aim of this study was to investigate how many residents of latvia, that are over 18 years of age have been exposed to vhc (anti-hcv prevalence) and how many are infected at the moment (hcv-rna prevalence). until now such research has not been performed in latvia. methods: from the register of general practitioners there were randomly selected 26 gp's from different regions of latvia, 60 persons over 18 years of age were selected out of each gp register and tested for anti-hcv with screening test (elisa). in case of positive result antibodies were confirmed with western-blot reaction and person was tested for hcv-rna (pcr). results: in total 1591 person was invited by general practitioners for the test and 1442 persons responded (response rate 90.6%). confirming test (western-blot) was positive in 34 participants and out of which hcv rna test was positive in 25 patients. conclusions: there are 2.4% of people exposed to hepatitis c virus in latvia and 1.73% are infected with hepatitis c virus, respectively, 1734 infected persons per 100 thousand individuals. genetic variation in the ikk/nf-b pathway and the live fibrosis progression in chronic hepatitis c r. sho 1 , k. ishii 1 , r. ishii 2 , h. watanabe 2 , k. sugahara 2 , y. nishise 2 , k. okumoto 2 , t. saito 2 , s. kawata 2 , a. fukao 1 1 department of public health, 2 department of gastroenterology, yamagata university faculty of medicine background/aims: i b kinase/nf-b (ikk/nf-b) signaling pathway is thought to play critical roles in liver inflammation and fibrogenesis. we carried out a haplotype-based association study to examine the contribution of common genetic variations in the genes encoding nf b inhibitor kinase alpha and beta (ikbka and ikbkb; the major components of ikk/nf-b pathway) to the progression of live fibrosis in chronic hepatitis c. methods: based upon the common single nucleotide polymorphisms (snps; minor allele frequency(maf) 0.05) and linkage disequilibrium (ld) information derived from the hapmap, we selected 5 and 3 tag snps from ikbka, and ikbkb, respectively, for genotyping. by using melting curve analysis, snps were genotyped in 217 chronic hepatitis c patients, including 80 patients with hepatocellular carcinoma. association between common genetic variations in ikbka/ikbka and platelet count (plt) was tested by both genotype-and haplotype-based approaches. results: we succeeded in genotyping a total of 8 tag snps that efficiently capture common variation across the 32 kb-block of ikbka and the 25 kb-block of ikbkb. for each of genes tested, 5 haplotypes were found in population studied. all snps were in hardy-weinberg equilibrium, but no significant association was observed between any single tag snp or haplotype and decreased plt in patients analyzed. conclusions: our data suggest that it is unlikely that polymorphisms within the ikbka and ikbkb genes are involved in the progression of live fibrosis in chronic hepatitis c. further studies on genetic variations in other nf-b-related genes in chronic hepatitis c are needed. background: hepatitis c virus infection is a major burden after liver transplantation. the effective treatment for patients who underwent liver transplantation has not been well established. management of these patients is the most challenging task. cyclophilins are essential host factors for hcv replication. we report here the efficacy of divided administration of ifn plus cyclosporine a in the treatment of chronic hepatitis c patients who failed peg-ifn or ifn combined ribavirin. patients and method: we prospectively included 59 patients (median age, 63) with genotype 1b and, failures to combination ifn plus ribavirin or combination pegylated ifn plus ribavirin. the present treatments consisted of an induction therapy, an intensified therapy and a maintenance therapy. the induction therapy comprised intravenous 1 mu ifn every 4 hours for the first 3 days, 1.5 mu ifn every 6 hours for the next 4 days and 2 mu ifn every 8 hours for the following 3 weeks, totaling 168 mu of ifn . the intensified therapy was induction therapy shortened to 2 weeks. the maintenance therapy comprised of pegylated ifn 2b and ribavirin. csa was given 4 times daily during the induction and the intensified therapies. ribavirin was given twice daily during the maintenance therapy. results: the end treatment response and sustained virological response rate of the present study were 73 % (43/59) and 59% (35/59), respectively. the relapse rate was 19 %(8/43). non-responders was 16 % (3/19). all adverse effects were completely reversible. the treatment protocol was well tolerable. conclusion: we concluded that our protocol should be effective in failures to the previous combination therapies. host factor targeting treatment will become a promising treatment option. cyclophilin targeting treatment is a promising new anti-hcv treatment k. inoue 1 , t. watanabe 1 , s. yoshiba 1 1 background: hepatitis c virus (hcv) is the most common cause of chronic liver disease. however, the efficacy of currently available treatments is limited. we recently reported the effects of combined interferon-/cyclosporin a treatment. cyclophilins are associated with hcv replication and bind cyclosporin a. which cyclophilins are closely associated with hcv replication remains controversial. in this study, several cyclophilins were found to be essential host factors for hcv replication and hcv replication was rescued by overexpression of cyclophilin a in the presence of cyclosporin a. methods: we evaluated the effect of cyclosporin a and its analogues on the replication of hcv in vitro using several types of hcv replicon. the gene expression of representative cyclophilins and pin-1 was knocked down using small interfering rna2 (sirna) to identify cyclophilins associated with hcv replication. the specificity of the effect of sirna was confirmed by western blot analysis. the effect of overexpression of cyclophilins on hcv replication in the presence of cycloporin a was also studied. results: cyclosporin a and its analogues suppressed hcv replication in a dose dependent manner. cyclophilin f, cyclophilin lc1 and cyclophilin lc2 as host factors which are closely associated with hcv replication, in addition to the previously reported cyclophilin a. knockdown of chclophilin b showed little effect on hcv rna replication. cyclophiln-dependent hcv replication varied among the three hcv replicon cell-lines used. overexpression of cyclophilin a rescued hcv replication in the presence of cyclosporin a. conclusions: these findings suggest several cyclophilins are essential host factors for hcv rna replication. thus potent cyclophilin inhibitors have the potential to be anti-hcv drugs. background/aims: hepatitis c virus (hcv) genotypes 1-6 have a worldwide distribution. types 1a and 1b are predominant in northern europe and north america, and in southern and eastern europe and japan, respectively. type 3 is endemic in south asia and is variably distributed in different countries. genotype 4 in egypt, genotype 5 in central and south america and genotype 6 is common in china, japan and south east asia. in pakistan 3a is the commonest genotype, which is associated with the most favorable outcome regarding end treatment response and sustained virological response after 24 weeks of therapy. the aim of this study is to find out hcv genotypes in newly diagnosed chronic hepatitis c patients. methods: this observational study was conducted in chronic hepatitis c patients. all patients had raised alt levels for last 06 months, had positive polymerase chain reaction (pcr) for hcv rna by real time method and liver biopsy was done in all patients under national program for prevention and control of hepatitis during year 2006 -2007. genotyping was done on roche genotyping kit. data was analyzed by spss 13.0 results: out of 164 patients, 85.9% (n=141) were genotype 3a. 6.1% (n=10) were genotype 3b. 3.0% (n=5) were genotype 1a. n= 01 had genotype 1b. 4.2% (n= 7) had mixed genotype (3a,3b/1a,1b,3a,3b). conclusion: majority (85.9%) of chronic hepatitis c patients were genotype 3a which is associated with favorable outcome after 24 weeks of interferon and ribavirin therapy and only 3.0% had genotype 1a in this cohort. s.t. zhou 1 , y. zhao 1 , f.j. zhang 1 background/aims: as human immunodeficiency virus (hiv) infected children who are receiving antiretroviral therapy (art) are living longer in china, comorbidities of hepatitis b virus (hbv) and hepatitis c virus (hcv) coinfection should be carefully considered when making management decisions. however, the coinfection rate of either hbv or hcv is unknown in hiv-infected children in china. we evaluated the seroprevalence of hbv and hcv in the china national pediatric art cohort of hiv-infected patients. methods: patients were selected from hiv infected children medically eligible for art who were enrolled into the china national pediatric art cohort since 2004. interviews, medical assessment, serology for hbsag, anti-hcv antibody, transaminase levels, and hiv serostatus and cd4 counts at baseline of patients were obtained. results: 42 of 763 hiv-infected children were hbsag seropositive (5.50%; 95%ci: 3.88%-7.12%), and 69 of 662 children were anti-hcv antibody seropositive (10.42%; 95%ci: 8.09%-12.75%). only age was associated with hbv coinfection. multivariate analysis revealed that children infected with hiv through contaminated blood or transfusion of blood products were 6.35 times more likely to be anti-hcv antibody positive than those infected with hiv through other routes. and children from central china provinces, henan, anhui, shanxi, and hubei were 2.5 times more likely to be hcv seropositive. conclusion: the high seroprevalence of hbv and hcv coinfection in hiv-infected children attending china national pediatric art cohort calls for routine screening for hepatitis viral coinfection and modification of the management of hiv-infected children in china. background: bms-790052 is a first-in class and highly selective hepatitis c virus (hcv) ns5a inhibitor with picomolar in vitro potency against genotypes 1a and 1b. in a sad study with healthy subjects, bms-790052 was safe, well-tolerated, and had a pharmacokinetic profile suggestive of once-daily dosing. methods: the objectives of this randomized, double blind, placebo-controlled, sad study were to evaluate the safety, tolerability, antiviral effect and pharmacokinetics of bms-790052 in patients with genotype 1 chronic hepatitis c (chc). treatment naïve or experienced patients were randomized to receive 1, 10, or 100 mg of bms-790052 or placebo. results: all bms-790052 single doses were well tolerated and had a safety profile similar to that of placebo. following oral administration, bms-790052 was readily absorbed with dose proportional exposures over the studied dose range. the mean terminal half-life of bms-790052 was approximately 12 hours. mean decline in hcv rna 24 hours after a single 1, 10 and 100 mg dose of bms-790052 was 1.8 log10 (range 0.18 to 3.0 log10), 3.2 log10 (range 2.9 to 4.0 log10) and 3.3 log10 (range 2.7 to 3.6 log10), respectively. the 100 mg dose resulted in a mean decline of 3.6 log10 (range 3.0 to 4.1 log10) 48 hours after dosing, which was maintained at 144 hours. conclusions: single doses of up to 100 mg of bms-790052 were safe and well tolerated in patients chronically infected with hcv genotype 1. bms-790052 produced a robust decline in hcv rna and has a pharmacokinetic profile that potentially supports once-daily dosing. background: the global infection rate of hcv is approximately 3%, and nearly 3.2% in china. only 42%-46% of patients with genotype 1b can achieve sustained virological response (svr) after antivirus therapy, nearly half of them experienced treatment failure. the study aimed to determine hcv-1b sequence evolution in patients experienced treatment failure during and after therapy, and further analyze relations between the mutations and treatment outcome. methods: 19 patients with genotype 1b accepted antiviral treatment of ifn plus ribavirin for 48 weeks, and long-term follow-up after therapy. 2 patients experienced treatment failure were further analyzed (one for relapser, another for nonresponder). sera were reserved at baseline, 12w, 48w and 4-year after therapy. hcv-rna was extracted. hcv full-length orf was amplified by rt-nested-pcr and sequencing. result: 9 of the 19 patients achieved svr (47.4%). from sequence alignments of relapser at baseline and 48w, we find that p7, ns5a and ns4a have higher mutation rate both in nucleotide and amino acid level (7.41% and 6.35%, 4.08% and 5.63%, 4.32% and 5.56%, respectively). but there is no significant difference in the alignments of 48w and 4-year after therapy, the mutation rate is lower. mutation rates of the non-responder among baseline, 12w, 48w and 4-year after therapy are very low. conclusion: antivirus effect is correlated with specific hcv sequences in chronic hepatitis c, mutations in hcv non-structure protein p7, ns5a and ns4a have important impacts on treatment outcome in ifn-based therapy. background: the results of antiviral therapy for hepatitis c (hcv) have improved recently with the use of peg-interferon (peg-ifn)/ribavirin therapy. however, age of patients are concerned because of side effects and safety. as we known, a few studies have targeted therapy in elder with chronic hcv. aim: we reviewed the results of interferon based antiviral therapy in the elderly with chronic hcv at our institution. methods: patients were defined as elderly if they were 65 years and elder who received therapy for hcv. the prescribed treatment duration, end of treatment response were mention. the data recorded included laboratory tests, adverse events (ae), dose modification, and withdrawal rate of therapy. results: 304 of chronic hcv patients treated with peg-ifn/ribavirin between nov 2004 and feb 2008. 50 patients were older than 65 years old. the mean age of the elder patients was 70.3 ± 4.8 years old. 21 were male and 29 were female. histological studies showed 18 with cirrhosis. almost all patients had experienced ae/side effects. the most common abnormalities were anemia and neutropenia. therapy was discontinued in 22% (11/50). the rate of dose modification was 46% (18/39) patients who received 24 weeks therapy. transaminases were normalized in 66% (33/50) after 24 weeks treatment and sustained in 66% (29/44) one year later. conclusion: the elder patients are more at risk of developing ae while on treatment. most patients should be discontinued or decreased dosage of medication. however, the elder patients with chronic hcv can be treated successfully. background: in the general population the incidence of interstitial lung disease is estimated to be 0.03% and has also been reported with the use of interferons. the higher reporting rate of ip in japan has created interest and warrants further investigation. methods: using both data from 12 randomized clinical trials (ex-japan) and the roche world-wide safety database (advent), the frequency of ip was estimated in patients treated with peginterferon alfa-2a ± ribavirin. ip was defined as: interstitial lung disease, alveolitis, pulmonary fibrosis, pneumonitis and pulmonary toxicity. results: one case of ip was reported among the 6180 patients included in the clinical trials (0.02%). in the advent database considering the estimated 926,000 patients with cumulative exposure to peginterferon alfa-2a (42,600 in japan and 883,400 us/row) the 228 reported cases of ip represent a rate of 0.02% with a proportional reporting ratio (prr) of 1.7 (p<0.0001). of these cases, 140 were reported in japan (prr 2.9; p<0.0001), 34 in the usa (prr 0.7; p=0.06) and 54 row (prr 1.2; p=0.11) representing reporting rates of 0.3% in japan and 0.01% in the usa and row. japanese patients with reported ip were older (66 versus 50-55 years) and were more likely to have been treated with peginterferon alfa-2a monotherapy (81% versus 21-39%). furthermore, the yearly incidence rate has remained unchanged. conclusions: the apparently higher rate of ip reported in japan may result from differences in patient demography, diagnostic criteria and treatment patterns. the overall incidence of ip remains low. background: hepatitis c virus (hcv) infection carries a significant risk for development of insulin resistance (ir) and/or diabetes (dm). recently, retinol-binding protein 4 (rbp4) has been reported as a protein contributing to ir. this study aimed to assess the different expression of serum rbp4 between chronic hcv infection (chc) patients and non-chc controls. methods: serum rbp4 was measured in 105 treatment-naïve chc patients and its correlation with the homeostasis model assessment of insulin resistance index (homa-ir), liver histology, virology and metabolic factors was investigated. patients were stratified into different stages of glucose tolerance by oral glucose tolerance test. another 100 sex-and age-matched non-chc adults served as the controls. results: the mean rbp4 level of controls tended to be higher than that of chc patients (32.46 ± 20.92 vs 25.48 ± 13.13 g/ml, p=0.07). the mean rbp4 level of 34 igt control-group subjects was 43.7 ± 24.0 g/ml, which was significantly higher than that of 34 ngt (24.4 ± 13.0 g/ml, p<0.001) and 32 dm controls (29.0 ± 19.5 g/ml, p<0.01). in contrast, the mean rbp4 level (22.2 ± 10.3 g/ml) of 32 dm/chc patients was not significantly different from that of ngt/chc (24.9 ± 10.5 g/ml, n=28) and igt /chc (28.1 ± 15.8 g/ml, n=45) patients. amongst chc patients, there was a significant decreasing linear trend of rbp4 dependent of both histological grading and staging progression, whilst a significant increment of homa-ir was found. conclusion: serum rbp4 is dysregulated in chc patients. introduction: sustained viral response (svr) in hepatitis c treatment with interferon alfa and ribavirin is affected by adherence and compliance due to severe myalgia, fatigue-anxiety and disturbed sleep. pregabalin, an orally effective gabasergic drug is not metabolized via cytochrome p450 and is used in fibromyalgia and fatigue-anxiety syndromes without hepatic toxicity.this study evaluates the addition of pregablin to standard agents in achieving svr by reducing side events. methods: thirty patients with chronic hepatitis c {mean age -46 years, male: female -2:1,genotype(g)1(n=29), g6 (n=1), fibrotic score f2-3 (n=24) and f4 (n=6), mean bmi > 29 kg/m 2 , initial viral load > 800,000 iu/ml} were randomized to pregablin100mg (n=15) or duloxetine 20mg (n=15) both orally daily with interferon alfa 2a 180mcg sq once a week and ribavirin 1200mg daily for 48 weeks. myalgia anxiety scale, modified quality of life score -evaluated at entry and tri-monthly. all were tested for rapid viral response, early viral response and end treatment viral response and svr. results: at the end of 48 weeks, in the pregablin arm, 14(97.3%) completed the therapy without interruption, one stopped due to excessive somnolence. duloxetine arm -10(66.6%) completed with interruptions, 4(22.2%) withdrew from the trial due to side events, one left the country. 9(62.2%) achieved svr in pregablin arm and 5(33.3%) with duloxetine. conclusions: pregablin may be considered with ifn and rbv for better adherence and compliance in achieving svr in treatment of chronic hepatitis c. larger randomized studies are needed to confirm the findings. in this study we extended this treatment approach to on treatment nonresponders (defined as having detectable hcv-rna after at least 24 weeks of soc). methods: so far, 5 pts. hcv-rna pos. after 24 weeks of soc (3 male, 2 female, genotype 1:4; genotype 3a:1, 3 with cirrhosis) participated in this protocol; 4 were treatment naïve pts, 1 relapser to two previous therapies (24 and 48 weeks). 20 mg/kg/d sil was given for 14 days, soc was continued. hcv-rna was quantified by taqman (roche diagnostics, usa) at monthly intervals on standard treatment and weekly after starting sil. results: all patients received at least 24 weeks of soc, at week 24 3 had a log drop < 3, two patients had detectable but unquantifiable hcv-rna (< 15 iu/ml). after 14 days of sil all 5 had undetectable hcv-rna, in one hcv-rna increased to 100 iu/ml and recived after a second course of sil. .all 5 patients are still on soc and are hcv-rna negative. conclusion: sil iv. is an effective "rescue treatment" for on treatment nonresponders to full dose of peginterferon/ribavirin combination therapy. poster exhibition -imaging modalities poster session, hall 5b background: levovist-enhanced ultrasonography using subtractions makes it possible to depict the perfusion of hyperechogenic nodules. our institution performs sonazoid-enhanced ultrasonography using a toshiba aplio80 that is set to a ps low images, as generally recommended. the resulting images, however, are difficult to evaluate the kind of staining image that is obtained from a hyperechogenic nodule. these staining images were then compared to advanced dynamic flow (adf) images of a hyperechogenic nodule recorded using levovist-enhanced ultrasonography. methods: the subjects were five nodules who had undergone sonazoid-enhanced ultrasonography. two patients had experienced a recurrence of hcc after tace, while three patients had a hyperechogenic nodule of hcc that had never been treated. one patient with hcc after tace was imaged at a ps low. the second patient with hcc after tace and the three patients with hcc showing a high echoic nodule, were imaged using adf. results: in the patients with hcc after tace, the remaining tumor was difficult to observe in both the vascular phase and the kupffer phase taken at a ps low. in the other patients, however, images taken using adf clearly showed the residual tumor. also, with regard to the findings from the perfused images obtained from the three patients with hyperechogenic nodules of hcc, the hcc was more easily detectable in the adf images than in those taken at a ps low. conclusion: hyperechogenic perfused nodules are easier to identify in images taken using adf than in images taken using ps low. y. komorizono 1 , t. shibatou 1 , k. sako 1 1 nanpuh hospital background: this study aimed to evaluate the usefulness of sonazoid enhanced radiofrequency ablation under real-time virtual sonography (rvs) guidance in a series of patients with hepatocellular carcinoma (hcc). method: twenty-five patients with a solitary hcc tumor measuring < = 2.5 cm in greatest dimension were enrolled in this study. eight patients received an initial treatment, seven also received an additional treatment for local recurrent tumors, and the remaining ten had distant recurrent tumors. all patients were easy to scan by multiple detector ct (mdct), but not by conventional ultrasound ( conclusions: the combination of the rvs system with sonazoid-enhanced us appears to have a high potential for use on patients that are difficult-to-scan by us examinations for percutaneous radiofrequency ablation. background & aims: contrast enhanced ultrasonography (ceus) with sonazoid can be expected to be useful not only for detection of tumor but also for us guided ablation therapy because kupffer imaging lasts for long time. the aim of this study is to investigate the usefulness of sonazoid in rfa for hcc. material & methods: a total of 716 hcc nodules in 316 patients admitted to receive rfa were studied. the detection ability of hcc was compared between ceus and conventional us using dynamic ct as reference standard. the effectiveness in the treatment was assessed by comparing the mean numbers of treatment session of rfa in patient treated with ceus assistance and that in historical controls matched for tumor and background conditions. results: the detection rate was 83.5% in conventional us and 93.2% in ceus (p=0.04). sixty-nine nodules in 52 patients were not detected by conventional us and detected after injection of sonazoid. the mean increase in detected tumor number with contrast enhanced us were well correlated with serum albumin level (p=0.016). ceus was not superior to conventional us in patients with low albumin level. the mean number of session was 1.33±0.45 as compared to 1.49±0.76 in the historical controls (p=0.0019). conclusions: ceus with sonazoid is useful for detection of tumor in patients with well-conserved hepatic reservoir. the decrease in the mean number of sessions compared to historical controls suggested that sonazoid is an excellent supportive agent in rfa treatment of hcc. direct measurement of peri-operative change in portal blood flow and pressure is difficult in human. in the present study, computational simulation of pre-and post-operative portal blood flow and pressure was performed using computational flow dynamic (cfd) software in patients with primary liver cancers. methods: patients with fibrotic or non-fibrotic livers were analyzed. according to preoperative md-ct, mesh models of portal branches were constructed. cfd software (fluent 6.2, fluent inc.) was employed for flow simulation. on the fluent 6.2, changes in flow dynamics in the remnant portal branches were simulated by virtual cutting of an interested portal branch. the simulation was also performed 14 days after the operation using dicom data obtained at that time. results: relative increase in blood flow in each remnant portal branch was not uniform throughout the liver in each patient. the sudden increase in portal pressure just after the virtual cutting of interested portal branch was almost normalized by day 14 in non-fibrotic liver according to the flow simulation, while the increase in fibrotic liver did not return to the pre-operative values by day 14. these results suggest that responsive dilatation of remnant portal branches and subsequent regional regeneration could normalize the sudden increase in portal pressure after surgery in non-fibrotic livers, while the mechanism is impaired in fibrotic livers. discussion: computational flow dynamic simulation is useful to analyze the differences in the peri-operative portal flow dynamics and liver regeneration between non-fibrotic and fibrotic livers. aim: to determine if roi analysis can characterize washout in hepatocellular carcinoma (hcc) better than visual analysis. methods: surgically proven hccs from a single institution were studied. the patients' gender, age, date of scan, date of surgery were recorded. 94 patients with pre-operative triphasic (n=67) and quadriphasic ct scans (n=27) were included. a representative section containing the lesion was selected for each case. the hu change between the precontrast and arterial (huabsolute hypervascularity) and the hu change between the peak attenuation and late portovenous phases (huabsolute washout) were recorded. cases were deemed positive if the hu change was more than the standard deviation (11 hu). this was compared against visual analysis to determine if our method would increase sensitivity of ct for hcc. results: the mean patient age was 63.7 years (range 19 to 84 years); there were 77 males and 17 females. the mean duration between surgery and the scan was 39.5 days (range 1 to 348 days). peak enhancement was seen in the early portal venous phase in 76.6% cases. the mean huabsolute washout was 23.8 hu (range -5 -54). roi analysis detected 86/94 cases (91.5%). this was 12.8% more than visual assessment, which detected 74/94 cases. this was statistically significant (p=0.014). conclusion: visual assessment of lesion density is subjective. quantitative measurement of lesion attenuation changes between scan phases is a simple and objective method that is more sensitive than visual assessment in determining lesion washout. background: abdominal ultrasonogram(usg) is a common available diagnostic tool to screen and follow up for hepatocellular carcinoma(hcc). but it has been reported that the specificity of ultrasonogram is high but the sensitivity of it is insufficient. we investigated the characteristics of hccs that was missed in the usg but was detected in the ct. methods: total 122 patients who were diagnosed with hcc between december, 2003 and february, 2008 , were enrolled and analysed retrospectively. all patients were performed with a usg prior to a spiral ct. the period between usg and spiral ct was limited within 1 month. we investigated age, gender, cause(hbv, hcv, alcohol), the size of hcc(the length of long diameter), stage(modified uicc), child-pugh grade, cirrhosis, tumor number, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns), and the tumor location at segement 8 as the possible related factors. results: the mean period between usg and spiral ct was 3.04±5.70 days. the diagnostic accuracy rate to hcc was 84.4%(103/122). there was no interobserver variation. in analysis of associated factors, there was no statistical significance in age, gender, cause(hbv, hcv, alcohol), stage(modified uicc), child-pugh grade, cirrhosis, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns) (p > 0.05). there was statistical correlation in the tumor size less than 2 cm, the solitary tumor and location at segement 8. (p > 0.05). conclusion: tumor size less than 2 cm, solitary lesion and location at segment 8 are significant factors to miss hccs in usg diagnosis. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 background: histopathological examination is required in the evaluation of various liver diseases for both diagnosis and prognosis. earlier blinded percutaneous liver biopsy was done commonly but now there are various studies suggesting that sonographic guided percutaneous liver biopsy could be more precise and safer. our aim was to compare the safety and diagnostic utility of sonographic guided versus blind percutaneous liver biopsy. methods: it was a retrospective single center study done between june 2003 and may 2007. trucut liver biopsy needle was used in all patients. demographic, clinical and histological characteristics between the two groups were evaluated. insufficient biopsy was defined as a sample with less than 6 portal spaces. we reviewed the type of complications and if hospitalization was required, or any mortality related to the procedure. results: out of 256 liver biopsies done in this period after excluding 16 patients we included 240 patients, 144 in group a(60%, blind approach) and 96 in group b (40%, sonographic guided approach). mean age was 38±12.4 years and male: female ratio was 1.6:1. biopsy was sufficient in 76% in group a and 94% in group b (p < 0.05). minor complications occurred in 58% in group a and 49% in group b which was not significant. major complications occurred in 2.8 % in group a and 1.2% in group b which was statistically significant. mortality was 1.2% in group a and 0.4% in group b which was statistically significant. conclusion: our study suggest that sonographic guided percutaneous liver biopsy is superior in the diagnosis of liver diseases in all aspects when compared to blind approach as it is more safe, has more diagnostic utility with significantly less complications and mortality. poster exhibition -liver fibrosis poster session, hall 5b background/aims: hmg-coa reductase inhibitors have been shown to reduce hepatic stellate cell proliferation and collagen production and decrease oxidative stress and hepatic vascular tone in cirrhotic patients. therfore, the aim of the present study was to examine whether the lipid lowering agents atorvastatin (ato) or rosuvastatin (ros) would prevent experimentally-induced acute or chronic hepatic damage in rats. methods: liver cirrhosis was induced by thioacetamide (taa, 200 mg/kg, i.p.) twice a week, for 12 weeks. acute damage was induced by two consecutive taa injections (200 mg/kg in a 24 h interval). rats were treated concurrently with taa only or taa and either ato or ros daily by nasogastric gavage. another group was treated with taa+pentoxifyline (ptx), an agent with known antifibrotic effect through a different mechanism and served as positive control. results: presented in the conclusions: the lipid lowering agents used in our study had no effect on the development of acute or chronic hepatic damage in rats or on oxidative stress induced by taa. purpose: the development of hepatic fibrosis in patients with chronic liver disease increases the risk of liver cancer. the present study was conducted to determine whether an easily performed myocardial examination technique can be applied to the assessment of hepatic fibrosis. strain rate imaging is a new method based on tissue doppler imaging (tdi). the usefulness of strain rate imaging in assessing the degree of hepatic fibrosis was evaluated. this time, it mede comparative study with fibroscan in 11 cases. methods: strain rate imaging was performed using a diagnostic ultrasound system (aplio tm , toshiba medical systems corporation, tochigi, japan) in a total of 47 subjects:25 in the chronic hepatitis group, 12 in the cirrhosis group, and 10 in the normal control group. tdi-q, the tissue doppler analysis software installed in the aplio system, was used for analysis. measurement was performed five times from the epigastrium, with the roi size set to 10 mm and the derivative pitch to 3 mm. results: (i): both scores were largely reproducible among the different laboratories. however, compared to the histological findings, the error ratio was 77% for all results calculated by fibrotest and actitest. (ii): calculated scores varied among f2 (9%), f3 (31%), f3-f4 (6%), and f4 (54%) (fibrotest), as well as a1/a2 (48%), a2 (9%), a2-a3 (5%), and a3 (38%) (actitest). results: the mean strain value was 0.156 in the chronic hepatitis group, 0.055 in the cirrhosis group, and 0.26 in the normal control group.the correlation was not thought to be fibroscan. conclusion: the results of the present study suggest that this noninvasive method permits quantitative assessment of the degree of hepatic fibrosis to be performed easily and in a short time. it is expected that the accuracy of the strain rate imaging method in determining the degree of hepatic fibrosis will be improved when it is used in combination with histological examination. conclusion: despite reproducibility of fibro-and actitest results among the six laboratories, large scale investigation (n=64) displayed increasing variability of the results depending on interlaboratory differences that were still in a quality controlled, analytically acceptable range. furthermore, calculated scores coincided with histological findings only in less than 25% of all cases. thus, the diagnostic accuracy of these tests seems low, if histology is accepted as gold standard. background: current knowledge attributes connective tissue growth factor (ctgf/ccn2) a crucial role in enhancing tgf-actions during hepatic fibrogenesis. recently, we demonstrated that caffeine leads to an upregulation of ppar in hepatocytes, thus sensitizing these cells to the well known inhibitory effect of 15-deoxy-12,14 -prostaglandin j2 (15-d-pgj2) on ctgf expression. however, upregulation of the receptor alone is not sufficient per se, its physiological ligand 15-d-pgj2 is required for exerting its inhibitory effect on ctgf synthesis. aim and methods: this study compares serum concentrations of 15-d-pgj2 in caucasian patients with fibrotic liver diseases (n=289), caucasian controls (n=136) and caucasian non-liver disease sick (n=307), as well as of chinese patients with hepatocellular carcinoma (n= 43) and chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with ppar inducing (i.e. ctgf inhibitory) drugs such as caffeine. results: presented data show that caucasian patients with ongoing hepatic fibrogenesis (mean 6.2 ± 5.9 µg/l) display impressingly higher serum concentrations of 15-d-pgj2 than healthy probands (mean 2.3 ± 1.0) and caucasian patients with non-liver disease (mean 2.7 ± 1.4 µg/l). similar results are found in chinese patients with fully developed hcc (mean 1.3 ± 0.7 µg/l) compared to chinese healthy controls (mean 0.4 ± 0.2 µg/l). we identified the predictors of tumor recurrence using cox-regression model. introduction: non-invasive, i.e. serum-based multiparametric panels of biomarkers have been proposed for the diagnostic assessment of liver fibrosis. aims/methods: (i) haptoglobin, alt, ggt, alpha 2-macroglobulin, apolipoprotein a1 and bilirubin in sera of 4 patients with histological proven fibrosis (f1-f4, a1-a3) were determined in 6 different quality-controlled laboratories. interlaboratory variations of the calculated fibrotest score for staging and actitest score for grading (both biopredictive tm ), and their error ratios compared to biopsy results were calculated. (ii) the variability of obtained fibrotest/actitest scores depending on 64 differential combinations of the allowed analyt-specific maximum/minimum permissible values as determined by the external quality control of the german association of laboratory medicine was determined and the frequency distribution of the results calculated. results: a total of 51 patients (mean age, 54.3±9.8 years; male, 78.4%) were included. median follow-up duration was 14.2 months (range, 5.6-37.3) and 20 patients (39.2%) experienced local tumor recurrence during the observational period. multivariable analyses showed that low p2/ms level (relative risk, 0.98; 95% confidence interval [ci], 0.96-0.99; p=0.035) and serum alpha-fetoprotein level >100 ng/ml (relative risk, 5.41; 95% ci, 1.59-18.18; p=0.007) were independent risk factors for tumor recurrence. patients with p2/ms level <45.0 revealed 3.79-fold (95% ci, 1.05-13.76; p=0.042) increase in the risk of recurrence after adjustment for serum alpha-fetoprotein level, as compared to those with p2/ms level >45.0. however, tumor size, child-pugh score, and hepatitis b virus dna level failed to significantly affect the time-to-recurrence. conclusion: our study suggests that lower p2/ms value, which means more severe liver fibrosis, is an independent predictor for hcc recurrence after rfa. background/aims: despite of its high prevalence, osteoporosis is an underestimated complication of liver cirrhosis. the aims of this study is to prove the prevalence of osteoporosis and osteopenia in patients with liver cirrhosis and to identify the principal risk factors associated. methods: the prevalence of osteoporosis and osteopenia was studied in patients with alcoholic or viral liver cirrhosis who were admitted to the institute of gastroenterology and hepatology, cnuh between march 2008 and september 2008. osteoporosis and osteopenia was evaluated by measuring their bone density using dual energy x-ray absorptiometry (dexa) at lumbar spine and femoral head. the variables taken into consideration were: sex, body mass index (bmi), presence of cholestasis, severity and duration of liver disease. results: total 45 patients (male 32 and female 13, respectively) were estimated for association of liver disease and osteoporosis. of these, 39 patients were estimated for bone density of lumbar spine and neck of femur by dual x-ray absorptiometry (dexa). morning blood samples were taken for hormonal and biochemical analysis from all patients. among 39 patients, 25 patients (64%) were found to have osteopenia or osteoporosis. there was no statistically significant correlation between age, bmi, severity and duration of liver disease, pth, vitamin d, alp and igf-1. conclusion: there is high prevalence rate of osteopenia or osteoporosis in liver cirrhosis. although the causes of osteopathy are heterogeneous, the early diagnosis and treatment of osteopathy in patients with liver cirrhosis is important. background: to build and to evaluate mathematical models for predicting liver fibrosis progression by using conventional laboratory indicators in chronic hepatitis b. methods: liver biopsy and routine laboratory tests were performed in 391 patients with chronic hepatitis b. using multiple logistic regression to analyze evidently relevant indicators, then the predicting models were built and analyzed by roc curve. results: after spearman analysis, factors such as age, platelet count(plt), international rate(inr), total bilirubin(tbil), albumin(alb), aspartate aminotransferase (ast), gamma glutamyltranspeptidase (ggt), total bile acid(tba) and cholinesterase(che) were found to be correlated with liver fibrosis p 0.01 . three models (s 2, s 3, s=4, respectively) were built by plt, inr, alb, ggt and che, which were independent predictors after multiple logistic regression analysis.finally, fibrosis score (fs) was calculated to predict different liver fibrosis stages. roc curve analysis revealed that the auc of fs was 0.784 in model1 (s 2), 0.768 in model2 (s 3) and 0.806 in model3(s=4) fig1 .the cut-off fs in model1 was at 7.09 with 67.4% sensitive, 79.3% specificity and the accuracy was 71.1%. the cut-off fs in model2 was at 5.67 with 75.0% sensitive, 67.7% specificity and the accuracy was 72.9%. the cut-off fs in model3 was at 3.65 with 71.4% sensitive, 78.5% specificity and the accuracy was 73.7%. conclusions: the predicting models, built by using conventional laboratory indicators, have fairly well value for diagnosing hepatic fibrosis or hepatocirrhosis in chronic hepatitis b. background: to investigate the effect of liver cirrhosis on the development of atherosclerosis in the rabbits chronically fed with high fat diet. methods: normal male new zealand white rabbits were randomly divided into four groups: a control group, a high fat diet group, a carbon tetrachloride (ccl4) group and a complex group. pathologic changes in ascending aortas and livers were observed. the levels of serum alanine aminotransferase alt , lipid, c-reactive protein (crp) were also determined. results: significant hepatic steatosis, inflammation and fibrosis could be observed in the three treatment groups; while atherosclerosis and typical arteriosclerotic plaques in ascending aortas could only be observed in the two high fat diet groups. compared with the control group, serum alt and lipid levels in ccl4 group were increased significantly (p<0.05), but no difference of arterial intima-media thickness (imt) and i/m ratio between these two groups. the levels of serum alt, lipid, crp and imt in two high fat diet groups were significantly increased compared with the control group (p<0.05). the level of serum alt in the complex group was significant higer than that in the high fat diet group, but the i/m ratio was just opposite (all p<0.05), and there was no difference of imt between the two groups. conclusions rabbits treated with ccl4 can elevate serum lipid levels, but can not induce atherosclerosis. though the activity of liver inflammation was aggravated in the complex model group, it has no effect on atherosclerosis possibly partly because of malnutrition. higher values of liver stiffness in males with mild chronic hepatitis c c. stern 1 , a.c. cardoso 1 , r. moucari 1 , a.d. pumpo 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: liver stiffness (ls) measured by fibroscan (echosens) is a noninvasive method to assess liver fibrosis in patients with chronic liver diseases. we evaluated the impact of factors on ls results in mild chronic hepatitis c (chc). methods: chc patients with metavir fibrosis stage 1 at liver biopsy and a reliable ls exam were eligible. all patients had no prior antiviral treatment. the ls values were compared to clinical and biochemical data. results: 93 patients were included with the following characteristics: mean age 50 11, male gender (46%), mean bmi 23 2.7, median ls 5.8 kpa (3.2-21.8), diabetes (7%), genotype 1 (61%), metavir activity a1 (86%), a2 (10%), steatosis at biopsy 30% (92%), mean glucose 4.9 1, abnormal alt (78%), abnormal ggt (47%), homa (2 1.2). the ls values were associated wtih male gender (median 6.1 in males vs 5.2 in females) (p=0.04), bmi (p=0.03), alt (p=0.006), ggt (p=0.02) and glucose levels (p=0.04). no association was found between ls and activity stage (p=0.34) or steatosis (p=0.14). in the linear regression, the only factor independently associated with higher ls was gender (p=0.038). in men, higher ls was related to levels of alt (p=0.005), but not to necro-inflammation grade (p=0.4). in women, ls was not associated with alt levels, but with bmi (p=0.045) and ggt levels (p=0.035). conclusion: in patients with mild chc, liver stiffness values are higher in males. these results suggest that different cut-off for fibrosis stage 1 should be proposed according to gender. aims: to investigate the effects of shuanghu qinggan granule(sqg) on prevention and treatment of hepatic fibrosis induced by carbon tetrachloride in rats. methods: 60 sd rats were divided into 6 groups, normal control groupamodel groupb, sqg largec1, middlec2small dose groupsc3 and silymarin positive contrast groupd. the rats of bc1c2c3d were injected with carbon tetrachloride for 8 weeks. the rats of c1c2c3 were then administered with sqg for 8 weeks. the rats of d were then administered with silymarin for 8 weeks. results the liver structure of rats of b was severely damagedlarge amount of liver cells became obviously degeneratedand hepatic veins were clearly congested. the hepatic cells fatty degeneration and infiltration of inflammatory cells in rats of c1c2c3d reduced significantly. there was no fiber hyperplasia in liver tissues of rats of c1c2c3d. blood serum ha cp p levels in rats of b were significantly higher than those in ac1c2c3d. conclusion: sqg has remarkable therapeutic effects on rats with hepatic fibrosis induced by carbon tetrachloride, the higher the dosage of sqg was, the more effective the results would be. conclusions: none of sophisticated biomarkers had value in addition to readily available laboratory data for the prediction of significant fibrosis in hbeag positive patients. two markers out of 7 sophisticated biomarkers provide additional diagnostic information in hbeag negative patients. before new biomarkers are accepted, their superiority to routine laboratory data should be meticulously appraised. objective: to evaluate the efficiency and safety of "tinmax" hb-3 herbal compound (cpd) in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. methods: a double-blind randomized method was employed. 60 patients of hepatofibrosis or cirrhosis post hepatitis b were separated into study group ("tinmax" hb-3 group) and control group (natural vitamin group) by randomized method. the course was 52 weeks. patients visited once every 12 weeks and the last visit at 12 weeks after the cessation of treatment. part of patients had liver biopsy before and after treatment. before, during the course and at the end of therapy, clinical symptoms and physical signs were evaluated, hepatic function, and serum markers of hepatofibrosis (such as hyaluronate acid, laminin, serum type iii procollagen and collagen iv) were tested, and ultrasound evaluation was performed. results: 60 patients enrolled in the evaluation. 58 patients completed the evaluation according to the protocol. 20 patients had liver biopsy twice, 10 from the study group and 10 from the other one. at the end of therapy, the total effective rate of hepatofibrosis in histopathology is 74.13% in the study group, much higher than that of 21.95% in the control group (p<0.05). the total effective rate of serum markers of hepatofibrosis at the end of therapy in the study group was 70.10%, much higher than that of 30.24% in the control group (p<0.05). the total effective rate of non-invasion markers of hepatofibrosis at the end of therapy in the study group was 76.08%, much higher than that of 19.41% in the control group (p<0.05). the drugs of adverse event had not happened in both groups. conclusion: "tinmax" hb-3 herbal compound (cpd) is effective and safe in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. w.h. sha 1 , xiaohui zeng 1 , yuyuan li 1 1 gi department, first municipal hospital of guangzhou, guangzhou aim: to investigate the clinical value of serum indices for hepatic fibrosis in chronic liver diseases. methods: competitive radioimmunoassay was used to determine the serum level of collagen type ( c), laminin (ln) and hyaluronic acid(ha) in 193 patients with different severity degree of chronic liver diseases, and in 30 healthy subjects. results: the serum levels of c, ln, and ha in the patients with liver diseases increased to different extent, compared with those in the healthy subjects. of which the highest of c, ln, and ha were found in the patients with primary carcinoma of liver or hepatocirrhosis and the serum level of ha is highlight. the combination detection of serum c, ln, and ha is more valuable than single index. conclusion: joint detection of serum c , ln, and ha is of higher significance in clinical diagnosis and prognosis of hepatocirrhosis, and is also available for successive observation on the development of liver diseases. aims: to investigate the mechanism of fuzheng huayu decoction (fzhy) on hepatic stellate cells (hscs) activation relating to tgf-1 signal transduction pathway. methods: hscs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. at day 4 after isolation, cells were stimulated with 100pm tgf-1 for 24h, then incubated with 10% fzhy pharmacological serum or 10 m t r -i inhibitor (sb-431542) for 24h. protein expression of -sma, smad3 was assayed by immunofluorescent stain; total protein expression of -sma, t r -i, smad2/3 and nuclear expression of smad3 was analyzed by western blotting. results: fzhy pharmacological serum significantly decreased expression of -sma, t r -i, and inhibited smad3 nuclear expression and translocation in tgf-1 stimulated hscs. conclusions: fuzheng huayu decoction can prevent hscs activation through tgf-1 signaling transduction pathway in hscs, which may be the important molecular pharmacological mechanism of fuzheng huayu decoction action against liver fibrosis. background: fatty liver disease has become a health problem related to metabolic syndrome worldwide although its molecular pathogenesis has remained further studied and it is unclear whether advanced fibrosis induced by steatohepatitis will regress when diet is controlled. aim of this study is 1) to study the involvement of endoplasmic reticulum stress (er stress) in the occurrence of seatohepatitis and 2) to obtain the evidence of resolution of fibrosis by changing the diet. methods: non-alcoholic steatohepatitis with advanced fibrosis was produced in rats by giving methionine-choline-deficient diet (mcdd) for 10 weeks. methionine-choline-control diet (mccd) instead of mcdd was given for the last 2 weeks in an experimental group. fibrosis and inflammation was determined by several tissue stainings. gene expression related to fibrosis and inflammation was determined by immunoblotting and real-time pcr. expression of caspase-12, caspase-7, and glucose-regulated protein 78 was evaluated to clarify the presence of er stress aim against liver fibrosis relating to hypoxia and angiogenesis regulation. methods: the rats were divided into normal, model, sa-b and perin control group. rats in sa-b and perindopril group were administrated with sa-b and perindopril respectively. liver fibrosis was induced by ip dimethylnitrosamine (dmn) for 4w. fibrosis degree was observed by sirius red staining. col-i protein expression was analyzed by western blot; col-i , vcam-1, icam-1, hif-1 and vwf expression in liver tissue was checked by immunohistochemistry; gelatinase activities in liver tissue were detected by gelatin zymography and in situ flourescent zymography. result: compared to normal group, col-i, hif-1 , icam-1, results: 1) changing the diet from mcdd to mccd triggered the reduction in fat in hepatocytes, the decrease of inflammatory gene expression and oxidative stress, and the regression of fibrosis accompanied by the disappearance of activated stellate cells and macrophages. 2) immunohistochemistry, immunoblotting, and rt-pcr analysis all indicated the occurrence of er stress in steatohepatitis while it recovered immediately after changing the diet from mccd to mcdd. vwf protein expression and gelatinase activity in liver tissue were increased obviously in model group, while sa-b and perindopril treatment significantly decreased these protein expressions and gelatinase activity. conclusions: this simple experiment clearly shows that the changing diet from steatohepatitis-causing mcdd to mccd triggers the resolution of inflammatory and fibrotic reaction in the liver, suggesting that food intake is a very important factor for controlling the state of fat and pathology of the liver. er stress is involved in the process. background: liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver disease. under injury conditions, hepatic stellate cells (hscs) are activated to transdifferentiate into myofibroblasts, which are capable of secretion of many connective tissue elements, especially collagens i, iii, and iv. gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in asia. gypenosides are the major saponins derived from g. pentaphyllum. in previous study, gypenosides have hepatoprotective and anti-fibrotic activities in rat chronic liver injury induced by ccl4, and anti-proliferative effect in rat isolated hscs. methods: in cultured hscs model, we detected type1 procollagen protein and mrna by western blot and rt-pcr. result: we found that g. pentaphyllum inhibited type1 procollagen protein expression in 66% at 48 hours. furthermore, g. pentaphyllum also inhibited type1 procollagen 1 and 2 mrna expression in 39% and 11% respectively. in addition to transcriptional inhibition, we found that g. pentaphyllum also enhanced the degradation rate of type1 procollagen protein. base on the effect of enhancing protein degradation, we used some protease inhibitors like ca-074 me, z-fa-fmk, aebsf, tpck and tlck to identify the potential target of g. pentaphyllum. on the other hand, in the ubiquitin-proteasome system analysis, we quantified the change of some target proteins of proteasome in the presence or absence of g. pentaphyllum. conclusion: g. pentaphyllum reduced type1 procollagen protein by inhibiting transcription and enhancing protein degradation. aim: excessive oxidative stress in diabetic patients has been implicated in the pathology and complication of liver. the present study was designed to examine whether ginger has a direct hepatoprotective effect in diabetic cases. methods: wistar strain albino rats were selected for this study. the rats were divided into 4 groups: (i) control, (ii) ginger treated (200mg/kg b.w. orally, 30 days) (iii) diabetic (50 mg/kg b.w., i.p.) and (iv) diabetic + ginger treatment. the lipid metabolic profiles such as total cholesterol, triglycerides, phospholipids and lipid peroxidation as stress markers and histopathological studies were carried out to assess the damage in hepatic tissue. results: ginger treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals. diabetic rats have shown increased total cholesterol, triglycerides, phospholipids and lipid peroxidation content in hepatic tissue compared to control, indicate prevailing of oxidative stress and alterations in fatty acid metabolism in these rats. further, degenerative changes of hepatic cells in diabetic group are minimized to nearness in structure by administration of ginger as evinced by histopathological examination. conclusion: we summarize that the hypolipidemic and antioxidant compounds present in ginger may be useful in delaying the complicated effects of diabetes. this results also reveal that ginger possess hepatoprotective properties in diabetic cases. anti-fibrotic action m. naime 1 , s. ali 1 1 hamdard university rhizomes of valeriana jatamansi (family, valerianaceae) have long been used in indian subcontinent by the traditional healers for the treatment of various diseases. this study provides experimental evidence suggesting the therapeutic effect of the crude extract of rhizomes on rat liver fibrosis, and demonstrates its antiproliferative role. crude extract (50% ethanolic) at a dose level of 800 mg/kg body weight was administered to rats to study the effect on biochemical and other markers of liver fibrosis. administration of the extract for 9 weeks could bring down elevated the levels of biochemical markers of liver injury, and modulate several other biochemical responses. morphology and hisopathological examination cooroborated with the biochemical changes, and indicated partial reversal of fibrosis. dpph assay confirmed the antioxidant property of the extract, which is suggested to be due to -ionone, -sitosterol and other chemical constituents. further, treatment could restore depleted glutathione level, inhibit lipid peroxidation, and inhibited elevated xanthine oxidase activity in fibrosis. the study also reports anti-tumour promotion activity of the extract as evident by a significant decrease in [ 3 h]-thymidine incorporation by hepatic dna in extract treated rats. results suggest that v. jatamansi extract has curative effect and can partially reverse biochemical and histological changes associated with liver fibrosis. with chronic hepatitis c c. wongjitrat 1 , s. chainuvati 1 , a. manuyakorn 1 , s. aroonparkmongkol 2 , t. tanwandee 1 1 mahidol university, 2 background: leptin is a peptide hormone that mainly regulates food intake, energy expenditure and reproductive function. leptin also releases from activated hepatic stellate cells and may have a role in regulation of fibrogenesis and inflammation. in human chronically infected by hcv, the role of leptin-associated fibrosis of the liver is still unclear. there is no data in thai patients chronically infected by hcv regarding leptin level and its correlation with hepatic histology and fibrosis.the purpose of this study was to evaluate the relationship between leptin level and severity of liver fibrosis in thai patients chronically infected by hcv. methods: sixty-six patients (31 men, 35 women) with chronic hcv infection and liver biopsy was done within 3 months were enrolled. fasting blood samples were obtained and serum leptin levels were measured by elisa. bmi, blood sugar, liver function test, lipid profile, hcv rna viral load and hcv genotype were also measured and related to histological findings. results: mean serum leptin levels were significantly higher in women than in male. there was a significantly correlation between serum leptin and bmi (r = 0.469, p < 0.001). leptin levels were not associated with hepatic fibrosis (r = 0.166, p = 0.183) and necroinflammation (r = 0.203, p = 0.102). steatosis was significantly associated with severe necroinflammation (r = 0.261, p = 0.034), but not fibrosis (r = 0.22, p = 0.076). conclusions: these findings failed to demonstrate correlation of serum leptin and hepatic fibrosis in thai patients chronically infected with hcv. background and aim: liver cirrhosis is one of the leading causes of mortality in our country as well as in our region. even though deterioration of glucose metabolism and existence of insulin resistance in liver cirrhhosis has been well documented in many studies, it is still unclear how insulin resistance mechanism develops. the aim of the present study is to assess insulin resistance, cytokines and crp levels in patients with liver cirrhosis and control subjects. in additon, we aimed to investigate the relation of insulin resistance in liver cirrhosis with such parameters as age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp. material and method: a total of 79 patients with cirrhosis of different etilogy (49 male, 30 female) were included into the study. as controls, 50 (23 male and 27 female) subjects were taken. the two groups were compared with each other in terms of glucose, insulin, c-peptid, homa-ir, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. in the second part of our study, the liver cirrhosis group was divided into two subgroups: patients with homa-ir value >2.7 as insulin resistance positive, and those with homa-ir value >2.7 as insulin resistance negative. these two groups, i.e. , homa-ir positive and homa-ir negative, were compared in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. results: in liver cirrhosis group, glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp levels were determined to be significantly higher than controls. between patients with homa-ir positive and negative, however, statistically no significant difference was found in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels, but il-8 level was seen to be significantly low in patient homa-ir positive. conclusion: in patients with liver cirrhosis, the levels of glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp increase with respect to normal population. determination of increased homa-ir level in liver cirrhosis supports the view that insulin resistance develops in liver cirrhosis as reported in related studies. in the study, it was also determined that the mechanism of insulin resistance development occurs independent of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels. the determination of statistically lower level of il-8 in patients with homa-ir positive with respect to those with homa-ir negative does not indicate similarity with the studies carried out earlier. ) in patients (48.08%) than in controls(14%)in group i hla-b5 significantly increased in patients (60%)as compared to controls (14%) . in group ii hla -b5 significantly higher in patients (45.46%)than controls (14%) also hla-aw19 significantly higher (40.91%) in patients than controls (12.67%).in group iii hla-aw19 significantly increased in patients (46.67%) compared to controls.no significant association between hla antigens and cases with hbv or hcv infection. conclusion: the significantly high association of hla-aw19 and hla-b5 in patients with hepatic schistosomiasis as compared to normal controlstogether with the lack of any association with active intestinal schisto . antigens predispose to liver affection.individuals possessing hla-aw19 appear to be more prone to severeform of liver disease background: atp8b1 mutation is one of the factors that result in cholestasis and progress to chronic liver disease, but has never been reported in the mainland china before. the aim of this study was to elucidate the role of atp8b1 mutation in mainland chinese patients with progressive intrahepatic cholostasis and low ggt. methods: 24 children who presented with progressive intrahepatic cholostasis and low ggt were admitted in a tertiary pediatric hospital in eastern china. abcb11 gene was analyzed firstly to exclude bsep deficiency. afterwards, all the encoding exons and their flanking areas of atp8b1 gene were sequenced in the remaining 19 patients in whom only one or no mutations of abcb11 were found. results: 10 mutations of atp8b1 gene were found in 9 patients. i694n had been reported in taiwanese patients with pfic1, and the others were novel. p209t and ivs6+5t g were linkage and found in 4 of 9 patients, including 2 homozygote and 2 heterozygote. liver biopsy had been performed in 6 patients with atp8b1 mutations and 5 with abcb11 pe408 mutations. variety portal fibrosis was showed in 2 patients with atp8b1 mutations and 4 patients with abcb11 mutations. giant cell transformation was detected in one patient with atp8b1 mutations and 4 patients with abcb11 mutations. 1 laboratory of exercise biochemistry, taipei physical education college, jhongcheng rd., no. 101, sec.2, taipei city-11153, taiwan, roc background/aim: generation of reactive oxygen metabolites are depends on the consumption of oxygen and their cumulative effects may be different from lean to obese population. this study was designed to investigate the deleterious effects of oxidants on hepatic antioxidant defence system in lean and obese rats under hypoxic condition. methods: zucker rats lean (200±10gms) and obese (450±15gms) were divided into control and acute hypoxia groups. the acute hypoxia treatment was performed in a hypoxic chamber at 14% oxygen consumption. objectives: to compare the diagnostic value of morning urine copper to zinc (copper/zinc) ratio and 24 hour urinary copper excretion in wilson's disease (wd) children. results: in the results, acute hypoxia caused a significant (p<0.05) decrease in major antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px), glutathione reductase (gr) and glutathione (gsh) content in lean groups when compare to their controls. the decrease in the activities of all antioxidant enzymes was also noticed in obese group with hypoxia treatment. however, this decrease was not significant in case of cat, gsh-px activities and gsh content. the mda levels (lipid peroxidation marker) were higher in obese rats compare to lean rats. methods: morning urine and 24 hour urine were collected from 96 patients over three years age who were hospitalized in a tertiary pediatric liver service. each patient was re-evaluated according to wd scoring system, and was assigned to one of the three groups: wd, suspecting wd, and non-wd. 24, 6, and 64 cases were assigned to wd, suspecting wd, and non-wd respectively. urine copper and zinc concentration was determined simultaneously by using inductively coupled plasma mass spectrometry. conclusions: the higher hepatic mda values observed in obese rats indicate that accumulation of free radicals may be more in obese rats thus leads to promote the lipids oxidation. from this study it is concluded that decrease of antioxidant enzymes (except gr) with acute hypoxia treatment were more in lean group compared to with that of obese group. results: the morning urine copper/zinc ratio and 24hr urinary copper excretion correlated well (r=0.758, p < 0.001). the median of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, 24h copper excretion, and 24h zinc urinary excretion were 0.370, 0.394, 87.1 and 398.7 in wd group, and 0.051, 0.061, 24.2 and 358.9 in the non-wd group respectively. the differences of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, and 24h copper excretion were significant (z-value -6.502, -6.020 and -6.208 respectively, all p values < 0.000 chd1l is a recently discovered oncogene localized at 1q21, one of the most frequently amplified chromosomal regions in hcc. herein, by yeast-two hybrid assay, we demonstrate that the anti-apoptotic ability of chd1l is associated with its interaction with nur77, a critical member of a p53-independent apoptotic pathway. as the first cellular protein identified to bind nur77, chd1l inhibits the nucleus-to-mitochondria translocation of nur77, and subsequently hinders the release of cytochrome c and the initiation of apoptosis ( figure 1 ). further study found that c-terminal macro domain of chd1l is responsible for the interaction with nur77, and a chd1l mutant lacking residues 600-897 failed to interact with nur77 and prevent nur77-mediated apoptosis. we also find that chd1l confers cellular chemoresistance to drugs that induce apoptosis via the nur77-mediated pathway, which may lead to the identification of new therapeutic targets for hcc treatment. background/aim: accumulation of oxidative damage to proteins, lipids and mitochondria could increase with advancing of age. the current study was aimed to test the hypothesis that swimming exercise training could revert the age dependent oxidative damages in liver. methods: sprague-dawley rats of young (3 months) and old (12 months) were divided into four groups; young control (n=5), young exercise (n=5), old control (n=5) and old exercise (n=5). 90 minutes of swimming exercise was given to the exercise group for a period of two weeks. results: the estimated antioxidant enzyme activities including, superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px) and glutathione reductase (gr) were decreased with age and significantly (p<0.05) increased with exercise training. however, elevated protein carbonyls and mda levels were noticed in old animals, which indicate that old liver had greater accumulated oxidative damages. the significant drop in protein carbonyl content and increase in mitochondrial succinate dehydrogenase (sdh) activity was observed with response to swim training in old rats. conclusions: this data implied that swim exercise training could revert the oxidative damages in liver. this was also proven by enhanced antioxidant enzyme status with response to exercise training in old rats. to sum-up these results it is cleared that age induced detrimental effects to the liver might be reversed by regular swimming exercise training in old rats. results: peg10 was expressed in l02/peg10 (fig.1) . peg10 accelerated the growth of l02. after treatment with 400mm h2o2 for 24 h, the inhibitory rate of l02/peg10 cells was 32.5%; the chromosomal condensation and ladder-like dna fragmentation were not observed (fig.2) . methods: hepg2 or hepg2.2.15 were co-cultured with jurkat cells, with blocking test by adding anti-pd-1 antibody. the pd-1 expression was detected by flow cytometry (fcm); cytokines in culture supernatant in blocking groups and controls were measured by enzyme-labeled immunosorbent assay (elisa); cytotoxic test of t cells were measured by methyl thiazolyl tetrazolium (mtt). conclusions: over-expression of peg10 can significantly promot l02 proliferation and ameliorate apoptosis-inducing effects of h2o2 on l02. results: the pd-1 expression on jurkat cells was induced by hepatoma cells, the expression rate were 16.17±6.5% (by hepg2) and 17.43±6.8% (by hepg2 2.2.15), respectively. the cytokines il-2 level (202.9±53.0 pg/ml), inf-level (88.6±4.6 pg/ml) and il-10 level (63.7±13.4 pg/ml) in culture supernatant of blocking groups were significant higher than that of controls (il-2, 102.9±53 pg/ml, inf-, 39.3±4.2 pg/ml and il-10, 34.6±13.7 pg/ml, respectively. p < 0.05). the cytotoxic test (od value) was markedly higher in blocking group (0.29±0.06) than that of control group (19±0.09 p<0.05) . conclusion: the pd-1 expression on lymphocytes can be induced by hepatoma cells, and cytokines expression and cytotoxic test were recovered by blocking pd-1/pd-l1 interaction. background: hedgehog (hh) pathway is well known as a positive regulator for tissue construction( during development) and reconstruction (in adults). our aim to observe the expression change of hh pathway on rat hepatic regeneration . materials and methods: adult male sprague-dawley rats underwent approximately 70% partial hepatectomy (ph) or sham operation (so). liver specimens were collected at 2, 6, 12, 24, 36, 48, 72 , and 168h after ph or so. hedgehog expression was determined in mrna level by rt-qpcr as well as in protein levels via immunohistochemical staining and western-blotting. results: so treatment did not induce remarkable changes in hedgehog expression; however, the level of transcript for hedgehog was significantly upregulated after ph. we found sonic hedgehog(shh )and glioblastoma (gli1-3) mrna expression in the regenerating liver arrive at its peak at as early as 24 h and returned to its physiologyical level 168 h later. it is similar to the change of proteins (shh and gli1) .as seen from immunohistochemistry experiments; shh protein was expressed uniquely in regenerating hepatocytes. similarly, ph induced over expression for shh protein occurred from 12 h with a peak level at 36 h after surgery. but gli protein mainly located in nucleus and no significantly changes in the phrase of liver regeneration. conclusion: hedgehog pathway may play a role in the activation of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as ph. background: to investigate whether peg10, an imprinted gene with an active paternal but silent maternal allele, was involved in hydrogen peroxide (h2o2) induced cellular apoptosis. methods: peg10 gene was stable transfected into l02. cellular gene expression was determined by rt-pcr, western blot and immunocytochemistry. cell proliferation was analyzed by mtt. after treatment with different concentrations (50-400 mm) of h2o2, cell proliferation inhibition rate was measured by mtt. morphological changes of apoptotic cells were determined by hoechst33342 staining, dna fragmentation was observed by agarose gel electrophoresis. hua tang 1 , xiao-yan tang 1 , min liu 1 , xin li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china we determined how afp modulates the proliferation of hepatoma cells. a recombinant adenovirus expressing sirna against afp (adv-afpsirna) was created and found that it reduced expression of afp specifically in hepatoma cells, and markedly inhibited the proliferation of hepatoma cells in vitro. local treatment using adv-afpsirna caused significant repression of the growth of hepatoma derived hepg2 cells in xenograft in nude mice. knockdown of afp resulted in an obvious delay in the g1/s transition of cell cycle, but did not affect apoptosis in hepg2 cells, as analyzed by flow cytometry and tunel assay. also, differential expressions of some genes related to the cell cycle, including skp2, cyclin d1, csk and ebag9 were identified by microarray and rt-pcr in hepg2 cells and hepg2 cells with knocked down afp. these results suggest that endogenous afp is a critical determinant of the growth of hepatoma cells. hematopoietic stem cell (cd 34+) therapy can improve liver function in patients with cirrhosis. these cells can be mobilized into peripheral blood using granulocyte colony stimulating factor (gcsf). this study was undertaken to assess feasibility and safety and of gcsf induced cd 34+ cell mobilization and its impact on liver function in patients with cirrhosis. patients with liver cirrhosis (cryptogenic or alcoholic with 6m abstinence) with cpt > 7 and < 12, and splenic diameter < 17 cm were included. gcsf injection was given for 5 days (5mcg/kg/dose). baseline & day-6 cd 34+ counts in peripheral blood were done by flow cytometry. follow up was weekly for 4 weeks and then monthly. cpt was compared at baseline and 6 months. 9 patients (median age 51 y, range 33-64 y, 8 males; etiology: 6 alcohol, 3 cryptogenic; median cpt 10, range 8-12) were included. cd 34 + cell counts at baseline and day 6 were 2(0-3) and 15 (13-41) respectively (median, range). side effects were fever in 8, allergic reaction in 1 and increase in splenic size in 1 (excluded). in follow up, 3 patients died (1, 3 & 4m after therapy, 1 after olt), 1 lost to follow-up. 4 patients showed improvement in 10 -7) at 6-month follow-up. gcsf treatment is safe and yields adequate cd 34+ cells in peripheral blood. in short term it results in improvement in liver function in patients with cirrhosis pe415 molecular cloning and transcriptional analysis of kctd9 gene promoter b. pi 1 , j.s. wang 1 , m.f. han 1 , y.y. zhou 1 , x.j. liu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science and technology aim: our previous work has shown that high expression of kctd9, a potassium channel associated gene, correlated with the disease severity of patients with severe chronic hepatitis b(schb). to further understand the gene transcription and regulation, kctd9 promoter was cloned and gene transcription was studied. methods: a full length of isolated promoter and series of 5' truncated promoter of kctd9 gene was subcloned into the luciferase report vector pgl2-basic to form the promoter-report constructs. the kctd9 promoter-report construct upstream of the luciferase report gene was cotransfected with constructs expressing hbv x,c and s protein respectively or stimulated with cytokines (il-2, ifn and tnf ) in 293 t cells to investigate kctd9 gene regulation upon both viral factors and host cytokines. result: a 759bp kctd9 segment upstream of atg translation start site was evidenced to contain potential regulative domains. an important regulation site located between -268bp and -81bp upstream of atg translation start site. based upon the luciferase activity assay, il-2 was able to upregulate the transcription of kctd9 whereas there was no effect from neither hbv viral proteins nor ifn and tnf . conclusion: here we first successfully cloned the full length promoter of kctd9. il-2 significantly enhanced the transcription of kctd9, a gene which has been shown to be involved in t cell activation and disease severity of schb from our group. this work was supported by nsfc(30571643, 30672380, 30700702) 1 1 istanbul university, cerrahpasa medical school, 2 marmara university, 3 okmeydani teaching hospital, 4 background: the treatment in chronic hepatitis c virus (hcv) is not highly effective, and cost, duration, and side effects are challenging. predicting favorable factors of response to treatment would make it possible to give it only responsive patients. recent studies report more conclusive results about the role of apoptosis in inflammation and fibrosis seen in chronic viral hepatitis. hepatocyte damage in hcv is mediated by cytotoxic t-cells. apoptosis primarily developed by the interaction between fas antigen on hepatocyte and fas ligand on t-cell corresponds to a main mechanism for hepatocyte damage. methods: in this study, we aimed to detect any relationship between apoptotic markers (fas, fas ligand, fas-associated death domain, caspases 3,8, and 9, insitu apoptosis) in liver biopsy taken before the treatment and response to the treatment of interferon+ribavirin. additionally, any relationship between these parameters and the other ones predicting the response to therapy including alt level, viral load, genotype, and gender were studied. results: the study includes the patients in 4 centers managing chronic hcv infection. all parameters were studied in 180 patients. study results revealed that histological activity index is correlated with cd95 staining density, caspase 8 intensiveness, and portal and parenchymal fas ligand scores. fibrosis is also seen to be correlated with the same parameters. apoptotic parameters of the responsive cases were not significantly different from unresponsive ones. conclusion: apoptotic parameters studied in the liver tissue is associated with inflammation and fibrosis, however these parameters may not predict the response to the treatment. s. gao 1 , d. xi 1 , j.w. guo 1 , c.l. zhu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science objective: this study was designed to explore the opportunity of microrna interference technique in the inhibitory application of human fgl2, human fas and tnfr1 expression. methods: the eukaryotic expression plasmids of human fgl2, fas and tnfri genes were constructed and have successfully expressed hfgl2, hfas and htnfri protein. mirna expression plasmids of hfgl2, hfas and htnfri complimentary to the sequence responsible for hfgl2, hfas and htnfri respectively were also constructed, meanwhile an irrelevant mirna plasmid was used as control. by respective cotransfection of p-hfgl2mirna and pcdna3.1-hfgl2, p-hfasmirna and pcdna3.0-hfas, p-htnfri mirna and pcdna3.0-htnfri expression construct into 293t cells, the inhibition of hfgl2, hfas and htnfri expression were analyzed by quatitative real time pcr and western blot. results: the experiments showed the significantly inhibitory effect of p-hfgl2mirna on hfgl2, p-hfasmirna on hfas and p-htnfri mirna on htnfri expression at 48h post-transfection both at rna level and at protein level, as well in 293t cell lines the inhibitory efficiency reached as high as 89.3% for hfgl2, 87.5% for hfas and 80% for htnfri, respectively. conclusions: the study demonstrated the constructs of p-hfgl2mirna, p-hfasmirna and p-htnfri mirna successfully interfered their target genes expression in vitro, which provides the foundation for further investigation of these constructs' application in vivo and further more as a therapeutic strategy for a targeting intervention in the diseases which the gene fgl2, fas and tnfri contribute to. this work was supported by nsfc30571643, 30672380, 30700702; 2005cb522901, 2007cb512900 pe418 influence of the id2 on the anti-tumor activity of histone deacetylase inhibitor in hepatocellular carcinoma cells r. tsunedomi 1,2 , s. harada 1 , n. iizuka 1 , m. oka 1 1 dept. of digestive surgery and surgical oncol., yamaguchi univ. , 2 research fellow of the japan society for the promotion of science for young scientists background: our recent study revealed that levels of the inhibitor of dna binding/differentiation 2 (id2) were associated with the progression of hcv-related hepatocellular carcinoma (hcc) and can affect susceptibility of hcc cells to histone deacetylase (hdac) inhibitors. we here aimed to investigate how and whether id2 expression affected on the anti-tumor activity of sodium butyrate (nab), one of hdac inhibitors. methods: two hcc cell lines, hle and huh-7, were used for gene targeting experiments. the id2 over-expressing and knockdown cells were subjected to mts assay to evaluate the susceptibility to nab. time-course of the expressional change of bcl-2 and bcl-xl genes after nab administration was measured by real-time rt-pcr. result: upregulation and downregulation of id2 levels in hcc cells resulted in decreased and increased susceptibility to nab, respectively. we observed that after nab administration, the id2 expression was induced gently, the bcl-2 expression was greatly increased immediately, and the bcl-xl expression was decreased to less than half once and then recovered. these increase and recovery of the expression of anti-apoptotic genes were inhibited in the id2 knockdown cells. in the id2 overexpressing cells, the bcl-2 expression was more upregulated than mock-transfected cells. conclusion: in hcc cells, id2 influences the susceptibility to the hdac inhibitor by regulating the expression of anti-apoptotic genes caused by the hdac inhibitor stimulus. we suggest that id2 could serve as a fascinating marker predictive of response to hdac inhibitors. the role of zinc finger protein 146 in liver cancer m.m.y. waye 1,2 , t.l. yeung 1,2 , j.l. zhu 1,2 1 the chinese university of hong kong, 2 croucher laboratory for human genomics background/aims: the aim of this research project is the characterization of a krüppel zinc finger protein, zinc finger protein 146 (znf146) using hepatocellular carcinoma as a disease model. zinc finger protein 146 (znf146), also named as only zinc fingers protein (ozf), is a 33kda nuclear zinc finger protein consisting solely of ten c2h2 zinc finger motifs of the krüppel type. like most of krüppel proteins, it is assumed to be the transcription factor and involved in the regulation of gene expression. the znf146 gene is amplified in 15-25% of pancreatic carcinomas and overexpressed in more than half of the tumors including liver cancer. it is thus proposed that overexpression of the znf146 may contribute to the development or progression of hepatocellular carcinoma. methods: we used flow cytometry, microarray, green fluorescent recombinant protein, rt-pcr site-directed mutagenesis, and transfection to study the effect of expression of znf146. results: our results shown that znf146 was over-expressed in two human hcc cell lines hepg2 and hep3b. expression profiles of znf146 over-expressing shown that genes related to the p53 tumor suppressor activity or dna damage, repair response and control were deregulated upon overexpression of znf146. conclusions: znf146 is possibly involved in liver carcinogenesis by affecting dna repair and cell cycle control upon induced dna damage. background: in the present study, we reported the establishment of a real-time monitor system for directly observing the catalytic, kinetic characteristics of dnazyme 10-23 in vitro cleavage on the target rna molecules as well as for rapid, accurate, high-throughout evaluation of varied dnazymes on their counterpart rna molecules. methods: dnazyme named dz-hcv-9 specific to hepatitis c virus (hcv) orf aug were designed and synthesized. dz-hcv-mis-9 with mismatched substrate-recognition domains, dz-hcv-mut-9 with mutant catalytic domains, antisense oligonucleotide ason and nonsense oligonucleotide nson were synthesized respectively as controls. a chimeric oligonucleotide of 29nt containing both rna and dna bases was designed and synthesized as the substrate: 5' fam-gt agaccgugcaccaugagcacgaaucct-bhq 3', corresponding to the330-354 nt (underline) of hcv genome(gi: 329873) the reporter fam/bhq was incorporated at the 5' and 3' end, respectively. under simulated physiological conditions (37 ), kinetic characterization of rna-cleaving dnazyme was analyzed in a real-time way. factors that influencing dnazyme cleavage were analyzed. results: dz-hcv-9 specific to hcv orf aug could cleave target rna at a•u site, a continuous change of fluorescence intensity was monitored. while the control oligonucleotides couldn't cleave rna, there were no change of fluorescence intensity. factors that influencing dnazyme cleavage concluded different substrate-recognition domain, mg 2+ concentration and ph. conclusion: a real-time monitoring system for kinetic characterization of rna-cleaving dnazyme was successfully established in the first time. the study on the apoptosis of hepatoma cells synergeticly induced by plasmid-mediated anti-angiogenesis and immunopotentiation therapy p.y. li 1 , q. zhang 1 , y. chang 1 , j.s. lin 1 , d.a. tian 1 1 background: angiogenesis is improtant to hepatoma and decreasing of host immunity promotes the development of tumor. we want to study the effect and mechanism of apoptosis of mice implanted hepatoma cells induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. methods: mouse endostatin eukaryotic plasmid (pseces) and mouse il-12 (interleukin 12) eukaryotic plasmid (pmil-12) were extracted and purified from e. coli. h22 hepatoma cells were inoculated into the leg muscle of mice, which was divided into four groups and injected with pseces, pmil-12, pseces+pmil-12 or pcdna3.1 naked plasmid dna respectively into implantation sites repeatedly. tumor formation and its weight was evaluated. tumor microvessel density, tumor infiltrating lymphocytes and apoptosis of tumor cells were assayed by cd31 staining, he staining and tunel assay respectively. results: inoculated mice received pseces, pmil-12 injection formed tumor slowly with less microvessel density, more tumor infiltrating lymphocytes in the latter and more tumor apoptosis cells in both groups compared with pcdna3.1 injection. there were much more tumor apoptosis cells in pseces+pmil-12 group (19.9±5.5 per 400× microscope field p<0.05) than any other single plasmid injection group (400× microscope field: pseces 11.3±4.1, pmil-12 14.6±3.2, pcdna3.1 1.4±1.3). conclusion: tumor cells of implanted hepatoma in mice could be synergeticly induced to apoptosis by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocytes to infiltrate, by which mice implanted hepatoma was inhibited. (20, 40, 80, 160, 200 ng/ml) in a serum-free medium for 24 h. cell proliferation was measured by brdu incorporation analysis, untreated wb-f344 cells were taken as controls. after treatment with wnt3a (160ng/ml) for 24 h, subcellular localization and protein expression of -catenin in wb-f344 cells treated and untreated with wnt3a were examined by immunofluorescence staining and western-blot analysis. cyclind1 mrna expression was determined by semi-quantitative reverse-transcript polymerase chain reaction (rt-pcr). mrna levels of some phenotypic markers (afp, ck-19, alb) and two hepatic nuclear factors were measured by rt-pcr. expressions of ck-19 and afp protein were detected by western-blot analysis. results: wnt3a promoted proliferation of wb-f344 cells. stimulation of wb-f344 cells with recombinant wnt3a resulted in accumulation of the transcriptional activator -catenin, together with its translocation into the nuclei, and up-regulated typical wnt target gene cyclind1. after 3 d of wnt3a treatment in the absence of serum, wb-f344 cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as afp, ck-19 following activation of the canonical wnt signaling pathway. conclusion: the canonical wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells. the expression level of bid and other pro-and anti-apoptotic proteins were detected by immunoblotting. results: hbx/51 showed the most sensitive towards dox treatment, and truncated bid (tbid) was also only detected in this cell line. the level of bax was also increased in hbx/51 cells. conclusions: the carboxy-terminal of hbx may enhance the processing of bid into tbid, which may contribute to increased sensitivity of the cell towards the dox treatment. cell homeostasis were performed with concentrations of oxysterol (3x10 -5 -10 -4 m) faraway from the physiological and/or pathological one (0.2 and 2x10 -7 m). in our study, we asked the effects of oxysterols (7k and 5'6s) on hepatoma cell lines homeostasis. to this purpose we used concentrations similar to those described in physiological or pathological conditions. sub-physiological (10 -9 m) to pathological (10 -7 m) oxysterol (7k and 5'6s) concentrations were used to stimulate hepg2 cells. a surprising pro-proliferative effect of 5'6s at sub-physiological (10 -9 m) concentration was observed. this behaviour was confirmed by the synergic increase of erk1/2 levels. facs analysis revealed an early progression of cells in s phase at the lowest concentration of 5'6s, while all the remaining concentrations of the two studied oxysterols induced a weakly accumulation of cells in g2/m phase. apoptosis was absent at all concentration used, except for the highest one (10 -5 m). at this point we asked if cells didn't undergo apoptosis but acquired a senescent profile. effectively, both 7k and 5'6s, at all concentration used (except for 10 -9 m), induced cell senescence (revealed by sa-ß-gal staining and sirt1 and p21 over-expression). in conclusion the two oxysterols analyzed have different and in same case opposite effects on hepatocellular line. the main effect is surely the senescence induction, but it is important to highlight the proproliferative effects of 5'6 secosterol at low concentration. mortalin, a member of hsp70 family protein, has been shown to play an important role in hepatocellular carcinoma (hcc). it has been reported that mortalin is binding to the c-terminal of p53, which acts as a safety guard and is a commonly mutated gene in hcc. in this study mortalin was silenced by specific shrna in plc/prf/5, a hcc cell line constitutively expressing p53ser249, and normal liver cells miha, and we found that suppression of mortalin can selectively trigger the mitochondria mediated apoptosis pathway by p53 dependent way in plc cells. tunel staining positive cells were only found in the plc cells mortalin knockdown group, and apoptosis associated protein, such as p53, bax, bcl-xl, cleaved-caspase 3, have been screened by western blot after transfection. quantitative-pcr data also showed that p53 mrna level are upregulated about 2 folds in mortalin knockdown group compared with the control groups in liver tumor cells. two p53 inhibitors, pft-and pft-, which can reverse this apoptosis was applied to demonstrate p53 dependent way. in summary, knockdown mortalin can selectively kill liver cancer cells through reactive apoptosis by sensitizing mutant p53 in plc cells, but had no effect on normal cells. the clinical application of this study suggested that motalin specific shrna might be a potential anti-cancer drug for hcc. background: nafld can proceed to nash and are at risk of cirrhosis and hcc. aim was to study profile of bangladeshi nafld patients. methods: 52 patients with nafld were included. of them 59.6% were males and 40.4% females. patients were between 12-60 years of age. they presented with dull right upper abdominal ache and/or incidental detection of raised alt/ast and/or fatty liver on ultrasonography. all tested negative for hepatitis b and c. none had history of alcohol. all underwent per-cutaneous liver biopsy for histopathology. they were also tested for dm, dyslipidaemia, insulin resistance, hypothyroidism and hepatitis c. their bmi and bp were recorded. results: 88.5% had nash. 63.0% of them were males and rest 37.0% females. 11.5% had nafl. of them 50% each were males and females. majority had nash. 47.8% were obese and 41.3% had dyslipidaemia. 28.3% had hypertension, 28.3% insulin resistance and 13% were diabetics. 6.5% had hypothyroidism. none had hepatitis c. alt was raised in 72% and ast in 40%. although all patients with nash did not have elevated alt, it was raised in majority, contrary to ast, which was normal in most. conclusion: majority nash patients in bangladesh are obese. other leading causes of nash include dyslipidaemia, hypertension and insulin resistance. some nash also had diabetes and hypothyroidism. this study also reveals that elevated alt in patients with nafld is suggestive of fibrosis, although normal serum alt does not exclude nash. the study further suggests that alt is superior to ast in predicting nash. background: non-alcoholic fatty liver disease is prevalent in obese patients. liver biopsy remains the best diagnostic tool for confirmation. we tried to find out the correlations of laparoscopic parameters with histology and laboratory data. besides, we also evaluated the effectiveness of laparoscopy in liver disease diagnosis. methods: in the period of one year and five months,126 morbidly obese patients submitted to laparoscopic bariatric surgeries at our institutions were prospectively studied. results: laparoscopic parameters of significant correlations with histologic steatosis, inflammation and fibrosis were summarized in table 1 . besides, important parameters with relationships to laboratory data were summarized in table 2 department of internal medicine, seoul national university hospital gangnam healthcare center, seoul, south korea, 2 department of internal medicine and liver research institute, seoul national university college of medicine, seoul, south korea background/aims: hepatic fibrosis is associated with poor prognosis in non-alcoholic fatty liver disease (nafld). recently, many non-invasive fibrosis markers have been studied to overcome the limitations of liver biopsy. among them, bard score and guha's simple panel are easy to use in clinical practice. in this study, we evaluated the efficacy of bard score and guha's simple panel as a noninvasive fibrosis marker in korean nafld patients. methods: data from 79 patients with biopsy-proven nafld in seoul national university hospital from 2000 to 2007 were used. bard score and guha's simple panel were calculated by using clinical and biochemical data and were compared with the histological fibrosis stages. results: stage 0 fibrosis were found in 67 patients, stage 1 in 4, stage 2 in 2, stage 3 in 1 and stage 4 in 5. the relationship between fibrosis stage and bard score ( = 0.43, p < 0.001) was statistically significant. all patients with advanced fibrosis (stage 3-4) had bard score greater than 2. mean values from original guha's simple panel for no fibrosis were not different between the patients with and without fibrosis. however, after adjusting coefficients by logistic regression analysis, the differences in mean values became statistical significant (p < 0.001). conclusions: our data suggest that bard score may be effective for detecting high risk patients for advanced fibrosis, and modification of coefficients within the guha's simple panel may be needed to use as a fibrosis marker in asian nafld patients. s.k. mohan 1 , s. subramaniam 2 , s. subramaniam 3 1 assistant professor, department of biochemistry, saveetha medical college & hospital, saveetha university, t.n, india., 2 consultant, department of biochemistry, apollo hospitals, chennai, t.n, india. , 3 department of biochemistry, apollo first med hospitals, chennai, t.n, india. background: non-alcoholic fatty liver disease (nafld) covers a spectrum of liver diseases from simple fatty infiltration to progressive fibrosis. non-alcoholic steato hepatitis (nash) is a severe form of nafld and progresses to the end stage of liver disease. it is becoming the leading cause for referral to liver clinics in most areas. the prevalence of nafld in indian population is estimated around 7 -13%. the nafld has the potential to progress to hepatocellular carcinoma or liver failure, both events that ultimately lead to early death. aim: to evaluate the combination of inter cellular adhesion molecule -1 (icam -1), adiponectin and type-iv collagen, a new biomarker profile for nash in patients with nafld. methods: 76 patients with nafld and age & sex matched 68 normal healthy individuals as controls were selected for this study. levels of serum icam -1, adiponectin, type-iv collagen, lipid profile and liver function test parameters were estimated in patients and compared with controls. results: serum icam -1 & type -iv collagen levels were significantly increased in patients with nash among the nafld patients compared to controls. the serum adiponectin levels were significantly reduced in patients with nash among the nafld patients compared to controls. compared to liver function test parameters and lipid profile levels, nash profile has got positive negative predictive value among the nafld patients. conclusion: in patients with nafld, nash profile test -a simple, noninvasive and reliable to predict the presence or absence of nash. background/aim: oxidative stress and cytokines plays an important role in the pathogenesis of nonalcoholic fatty liver disease (nafld). aim of study was to assess lipid peroxidation, serum levels of transforming growth factor-( tgf-) and tumor necrosis factor-( tnf-) in patients with nafld and compare it with patients of chronic viral hepatitis (cvh) and healthy controls (hc). methods: lipid peroxidation was studied by estimating plasma malondialdehyde (mda) levels as per the methodology described by buege and aust and tgf-& tnf-levels were measured by elisa kits (ray biotech, usa, & diaclone, uk) in the stored sera in 10 biopsy proven patients with nafld (m: 4, f: 6, mean age: 41.7 ± 11.0 yrs), 15 patients with cvh ( m:14, f:1, mean age: 33.7 ± 11.4 yrs) and 5 hc (m:5, mean age: 25.2 ± 2.7 yrs). results: there was no difference in mean plasma mda levels amongst patients with nafld (17.28 ± 3.6 mol/l), cvh (15.29 ± 2.3 mol/l) and hc (16.79 ± 1.2 mol/l). serum tgf-levels between nafld (0.56 ± 0.41 ng/ml) and cvh (0.52 ± 0.25 ng/ml) patients and hc (0.58 ± 0.57 ng/ml) were also comparable. though patients with cvh (17.2 ± 27.0 pg/ml) and nafld (7.5 ± 6.3 pg/ml) had higher levels of tnf-than hc (5.5 ± 1.1 pg/ml), the difference was not significant statistically. conclusion: lipid peroxidation, tgf-and tnf-need to be studied in a larger number of patients with nafld. background/aim: burnt out nonalcoholic fatty liver disease (nafld) may be responsible for cirrhosis and hepatocellular carcinoma (hcc) in the absence of other causes. aim of this study was to evaluate the surrogate markers of nafld in patients with cryptogenic cirrhosis (cc) and cryptogenic hcc (chcc). methods: sixty five patients with cc and 31 patients with chcc were analyzed for the presence of abnormal body mass index (bmi) and type 2 diabetes mellitus (dm aim: to investigate the relation of phosphatidylethanolamine n-methyltransferase pemt gene g175a single nucleotide polymorphism (snp) with the susceptibility to nonalcoholic fatty liver disease nafld . methods the genotypes and allele frequencies of pemt exon 8 snp g175a were analyzed by using pcr-rflp in 51 nafld patients and 50 controls. results: the g to a variation of the pemt gene g175a snp was significantly higher in nafld group compared with controls. the frequencies of gg ga and aa genotypes were 58.8 39.2 and 2.0 in nafld and 78.0 22.0 and 0.0% in controls (p=0.038 . the a allele of the pemt gene was significantly more frequent in nafld group (21.6%) than that (1.0%) in controls p=0.042 .there were significant differences in serum levels of cholesterol, triglyceride, hdl-c and ldl-c between gg and ga/aa genotypes p <0.05 . n. assy 1,2 , g. lipez 3 , s. korem 3 , m. grozovski 3 1 sieff hospital, safed, israel, 2 2technion institute, faculty of medicine, haifa, israel, 3 ort braude college, karmiel, israel background: previous studies reported increase in serum protein c and decrease in serum paraoxonase levels in patients with non alcoholic fatty liver diseases (nafld). conclusion people with pemt gene g175a snp were more susceptible to develop nafld aim: 1) determine whether there is a relationship between nafld, protein c and paraoxonase levels in quiescent and in regenerating rats fatty liver 2) determine the effect of isa on hepatic "protein c" and paraoxonase mrna. pe439 methods: forty-eight sd rats were treated with fructose enriched diet (fed), or fed with metformin (2 mg/kg/d), fed with rosiglitazone (3 mg/kg/d), or the combination of both drugs for 5 wks. 30% phx was performed at wk 5. protein c, paraoxonase mrna expressions, lipids, mda were measured before and 24 hours after phx. results: hepatic "protein c" mrna was higher in rats with fatty liver than control rats (+105%, p<0.01) whereas hepatic paraoxonase mrna was lower in rats with fatty liver than control rats (-28%, p<0.005). hepatic protein c and paraoxonase mrna increased in rats with fatty liver in regeneration (+116%, p< 0.01, and +15%, p<0.01 respectively). the combination of metformin and rosiglitazone decreased hepatic protein c expression at 24 hours after phx by -170% (p<0.001) and increase paraoxonase mrna by +50% (p<0.01). serum paraoxonase correlates with serum protein c (r=-0.2), mda (r=0.4), background: non-alcoholic steatohepatitis (nash) is a type of non-alcoholic fatty liver disease (nafld), and may progress to hepatic fibrosis and cirrhosis. the pathogenesis of nash remains unclear. the aim of this study was to explore the arginase change in the progress of steatohepatitis in rats. methods: male sd rats weighing 70-80g were obtained. twenty animals were randomly divided into two groups. in the model group, five animals were fed with high lipid forage that includes 3% cholesterol and 20% lard for 6 weeks, five were fed for 12 weeks, while the control group ate normal foods. the animals were sacrificed after 6 weeks. the animals were sacrificed after 6 weeks and 12 weeks. liver and blood serum were collected while the serum levels of alt, ast, tg and tc were measured. the pathology of liver was observed by he staining. western blot was used to investigate the expression of arginase in control and model group. tg (r=-0.23). conclusion: hepatic "protein c" mrna levels are high at baseline, up regulated during liver regeneration and decrease after treatment with (isa) whereas hepatic paraoxonase mrna levels are low at baseline, up regulated during liver regeneration and increase after treatment with isa. results: vacuolization were observed extensively in hepatic cells in the model group after 6 weeks and 12 weeks of high-fat diet. it is demonstrated that rats fed with high-cholesterol food are indeed fatty liver models. western blot showed that the level of arginase ii increase in the liver of model group rats as compared to the control group. furthermore, the level of arginase was higher in liver samples obtained from model rats that were 12 weeks on a fat diet as compared to rats that were only 6 weeks on the same diets. conclusion: the level of arginase ii was altered in the progress of non-alcoholic steatohepatitis in rats suggesting that arginase ii is putative biomarkers and may represent new targets in the development of therapeutic strategies against fatty liver disease hepatic fibrosis and cirrhosis. methods: c57bl6/j mice were fed with mcd diet to induce hepatic fibrosis and rosiglitazone was given in treated group. effect of rosiglitazone was assessed by comparison of the severity of hepatic fibrosis in liver sections, expression of mmp-2/9, timp-1/2 mrna and protein detected by rt-pcr and western blot respectively. the ethanolic extract of fructus schisandrae chinensis decreased hepatic triglyceride level in mice fed with a high fat/cholesterol diet results at week 8, fibrosing nash models showed severe hepatic steatosis, infiltration of inflammation and fibrosis, which is associated with down-regulated mmp-2/9 mrna and protein, up-regulated timp-1/2 mrna and protein. rosiglitazone significantly reduced mcd-induced fibrosis by induced mmp-2/9 expression and reduced timp-1/2 expression by activating ppar . s.y. pan 1 , z.l. yu 2 1 beijing university of chinese medicine, 2 hong kong baptist university effects of the ethanolic extract of fructus schisandrae chinensis (etfsc) on serum and liver lipid contents were investigated in mice fed with normal diet or high fat/cholesterol diet for 8 or 15 days. single dose of etfsc (1 or 5 g/kg/day, i.g.) increased the serum triglyceride (tg) level (40 and 142%, respectively), but decreased hepatic total cholesterol (tc) level (15 and 16%, respectively) in normal mice. the hypertriglyceridemia produced by etfsc was suppressed by the co-administration of fenofibrate. the induction of hypercholesterolemia by high fat/cholesterol diet caused significant increases in serum and hepatic tc levels (up to 165%) and hepatic tg levels (up to 528%) in mice. etfsc treatment (1 or 5 g/kg/day for 7 days, i.g.) significantly decreased the mouse hepatic tg level (by 35%) and slightly increased the hepatic index (by 8%). whereas fenofibrate treatment (0.1 g/kg/day for 7 days, i.g.) significantly lowered the hepatic tg level (by 61%), it significantly elevated the hepatic index (by 77%) in hypercholesterolemic mice. the results indicate that etfsc treatment can invariably decrease hepatic tg in hypercholesterolemic mice, suggesting its potential use for fatty liver treatment. aim: to investigate the influence of multiple gene polymorphisms in the susceptibility of nafld. methods: the data of single nucleotide polymorphisms (snps) in 201 nafld patients who had at least one of the genetic variations at the sites of tnf--238, adiponectin -45 and leptin-2548 were analyzed. the genotypes were determined by using pcr-rflp. our previous studies showed that the variations of these sites increased the susceptibility of nafld. results: the prevalence of nafld in adiponectin variation alone group (n=45) was 35.6%; in tnf-alone group (n=33) 42.4%; in leptin alone group (n=54) 35.2% (p>0.05). in comparison with the above groups with single snp, the prevalence of the groups with two gene variations of tnf-plus adiponectin (57.9%, n=19) increased significantly (p<0.05). however the prevalence of other two groups i.e. adiponectin plus leptin (34.6%, n=26) and tnf-plus leptin (40.0%, n=15) did not differed significantly from those of groups with single snp (p>0.05). the prevalence in the group with three gene variations (55.6%) differed significantly from all (p<0.05) except that of tnf-plus adiponectin group (p>0.05). the metabolic features of the nafld patients in the 7 groups mentioned above were not different significantly (p>0.05). conclusion: nafld is a polygenic disease. multiple gene polymorphisms may, but not always, increase the susceptibility of nafld. chronic hepatitis b patients with nonalcoholic fatty liver disease r.d. zheng 1 , c.r. xu 1 , j. chen 1 , b.f. chen 1 1 southeast hospital background: to investigate clinical pathological characteristic in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). methods: we measured fasting blood glucose, insulin, triglyceride, cholesterol, alanine aminotransferase (alt), aspartate aminotransferase (ast) in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). and we detected hepatitis b virus marker, hbv-dna, counted body mass index, insulin resistance index and observed pathological characteristic. all these patients with diagnosis were confirmed by clinical and pathological evidence. result : the body mass index, homeostatic model assessment (homa) of insulin resistance, fasting blood glucose, insulin, triglycerides, cholesterol, were significantly higher in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld) than hbeag negative chronic hepatitis b patients. but the alanine aminotransferase (alt), aspartate aminotransferase (ast), hbv dna levels were significantly lower in hbeag negative chb patients with nafld than in hbeag negative chronic hepatitis b patients. histologic features in hbeag negative chronic hepatitis b(chb) patients with nonalcoholic fatty liver disease (nafld) are in zone 3 predominate macrovesicular steatosis and mild inflammatory infiltrate in portal region. conclusion: the hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease, whose hepatic steatosis changes are mainly caused by the metabolic factors. to carry out liver biopsy selectively for the patients with hbeag negative chronic hepatitis b having metabolic factors, which is helpful for early diagnosis in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). aims: to investigate the preventive effect of cordyceps sinensis and its possible mechanism on apoptosis of nafld. methods: rats were randomly divided into basic diet group (b group), pathologic group (nash group) and cordyceps sinensis group(cs group).the latter two groups were administered with high-fat diet to establish nafld animal models. cs group were treated with cs at the 9th week after high fat diet. rats were sacrificed at the end of the 18th week. biochemical examination were used to detect superoxide dismutase (sod) of liver tissue. hepatocyte apoptosis was assessed in each group using the tunel assay and immunohistochemistry for activated bax bcl-2 caspase-3 and nf-kb p65. results: (1) compated with the b group, severe hepatosteatosis, inflammative necrosis and local fibrigenisis were showed in liver of nfsh group. sod lever was significantly decreased (p<0.01) and tunel-positive cells were significantly increased (p<0.01). immuunohistochemistry test demonstrated active bax caspase-3was increased (p<0.01) while no apparent change was observed in bcl-2. (2) in cs group, only diffusive steatosis but not inflammation or fibrosis was found. sod lever was increased than that of nash group (p<0.05). tunel-positive cells and active bax caspase-3 were significantly decreased (p<0.05 p<0.01) that those of nash group. bcl-2 and nf-kb p65 were increased (p<0.01) than those of nash group. conclusions: hepatocyte apoptosis is a prominent feature of nafld. cordyceps sinensis may be useful as an antiapoptosis theraphy in this syndrome through increasing activity of sod, decreasing express of bax and increasing express of bcl-2 and nf-kb p65. background: non-alcoholic steatohepatitis (nash) is a leading cause of chronic liver disease. insulin-sensitizing , anti-inflammatory and anti-fibrotic effect of thiazolidinediones support their use in the treatment of nash. we aimed to evaluate the efficacy of thiazolidinediones in the treatment of nash. methods: we have identified randomised clinical trials, evaluating the efficacy of thiazolidinediones versus placebo in nash, through medline, embase, ami, cochrane central register of controlled trials. data were abstracted from each study and disagreements were resolved by consensus. dichotomous outcomes were reported as relative risk with 95% confidence interval based on fixed-effects model. results: we included three trials, two evaluating pioglitazone and another rosiglitazone. a total of 171 patients were involved in the analysis. thiazolidinediones was noted to improve liver function tests. it was effective in the reduction of steatosis among patients with nash (rr 0.67, 95% ci 0.52-0.87). it was found to be beneficial in improving ballooning necrosis (rr 0.79, 95% ci 0.66-0.95). it was also found to improve lobular inflammation (rr 0.72, background: it is well known that the weight reduction is effective for alt normalization in patients with non-alcoholic fatty liver disease (nafld). the necessary condition for alt normalization is still unclear. to clarify the necessary and sufficient condition for alt normalization, we investigated the effects of body fat decrease in nafld patients by body composition analyzer. methods: forty-six nafld patients (23 male, 23 female, mean age 49.8±12.9 years old) with abnormal alt levels were evaluated. the volume of skeletal muscle, body fat and bmr were examined by using the body composition analyzer (in body 720; biospace co. ltd., tokyo japan). all patients were received an individualized diet consultation by dietician every 4 weeks for 6 months. daily energy was bmr (basal metabolic rate) x1.2 kcal and protein was 1.0-1.2g per ideal body weight. result: twenty-eight of 46 patients (60.9%) were achieved normal alt level. in alt normalized group, the body weight and fat loss were 3.6±2.3 kg, 3.0±1.5kg (2.8±1.7 %body fat) respectively. on the other hand, in cases with alt remained abnormal level, the body weight and fat loss were 0.5±1.5kg, 0.4±1.6kg (0.5±1.6 %body fat). conclusion: our results demonstrate that the fat loss of 3 kilograms or more was necessary to normalize alt level in nafld patients. a. somani 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background : nafld is often clustered within families and the causes include both genetic and environmental factors. family studies done thus far have been limited by small sample size. to examine the familial patterns , we performed a prospective study to see (a) whether nafld is more common in first degree relatives (b) genetically determined risk factors associated for clustering. methods: first degree relatives of histologically confirmed nafld patients and spouses (controls) were included after excluding other causes of fatty liver. those having raised transaminases >3 months or sonographic examination consistent with fatty liver, had undergone liver biopsy for histological confirmation. they were divided into three groups. group i patients group ii first degree relatives group iii spouses results: nafld was more prevalent among first degree relatives then spouses (37% and 17%, p<0.01). anthropometric measurements, systolic and diastolic blood pressure, lipid profile and liver function tests were comparable in three groups. homa-r was similar in group i and ii (p=0.073), but was significantly different in group i and iii (p= 0.0001) and group ii and iii (p=0.0001) respectively. metabolic syndrome was present in >70% of patients and were comparable in three groups except for fasting glucose > 110, which was present in 76%, 77% and 57% of patients in group i, ii and iii respectively. majority (>50%) of our patients among groups i, ii and iii were having only steatosis while nash was present in 20%, 13% and 14% of patients. a. somani 1 , v. dixit 2 , a. jain 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha background: normal levels of alanine aminotransaminase (alt) have been demonstrated in nafld patients. alt levels are also modulated by age, gender, bmi, fasting glucose, and serum triglyceride levels. we performed a prospective study of patients with histologically confirmed nafld and having alt < 1.5 times and compared them with those having raised alt to determine (a) clinico-pathologic features of nafld patients with normal alt (b) to observe any differences between them. methods: patients with fatty liver on sonography had under gone biopsy for histological confirmation after excluding other causes of fatty liver. participants were divided into two groups (a) those having alt > 1.5 times normal (n=97) (b) those having normal alt (n=47) results: mean age was comparable with slight male predominance. there were significant differences in anthropometric measurements like bmi (p=0.0001) and whr (0.90±3.57 and 0.88±3.23, p=0.0071). mean bp, lipid profile, fasting glucose, insulin, and homa r were comparable. there were significant differences in both mean ast (50.2±5 and 37.4±3.21, p=0.0001) and alt (104±7.29 and 69.9±11.5, p=0.0001) levels. metabolic syndrome was present in >75% of patients and individual components were comparable except for increased waist circumference which was significantly more in those with raised alt (78.35% and 44.68%, p<0.001). majority of our patients were having only steatosis, while nash was present in (27.82% and 8.5%, p<0.05) of patients. conclusion: nafld can exist in patients with normal alt values. although more work is needed to determine who should be screened for nafld and how such individuals should be evaluated, this study is a step toward the identification and characterization of nafld patients with normal alt. we can suggest that patients having metabolic syndrome or insulin resistance, despite having normal alt, should be screened for nafld. also alt values should be adjusted for variables like bmi to appropriately screen nafld patients. background: scientific evidence has demonstrated that traditional chinese medical (tcm) approaches and products can be beneficial for managing non-alcoholic fatty disease (nafld), but few rigorous criteria of patterns of tcm therapy are available to guide practitioners in deciding the cam interventions. objectives: to evaluate criteria of patterns of tcm therapy for the management of nafld identified by biomedicine. methods: literature research, clinical epidemiological investigation and mathematical statistics were employed to make information collecting tables and to establish database. descriptive analysis, factor analysis, and cluster analysis were involved. results: (1) background/aim: serum uric acid level has been suggested to be associated with factors that contribute to the metabolic syndrome. the aim of this study was to investigate the association of serum uric acid level with nonalcoholic fatty liver disease (nafld). methods: a cross-sectional study was performed among the employees of zhenhai refining & chemical company ltd., ningbo, china. results: the study included 8925 subjects (6008 men) with a mean age of 43 years. the prevalence rate of nafld and hyperuricemia was 11.78% and 14.71%, respectively. nafld patients had significantly higher level of serum uric acid than controls (370.3 ± 86.6 vs. 321.1 ± 82.6 mol/l; p < 0.001). the prevalence rate of nafld was significantly higher in the subjects with hyperuricemia than those without hyperuricemia (24.75% vs. 9.54%; p < 0.001), and the prevalence rate increased along with serum uric acid levels (p value for trend < 0.001). multiple regression analysis showed that hyperuricemia was associated with increased risk for nafld (odds ratio [or]: 1.291, 95% confidence interval [ci]: 1.067 -1.564; p < 0.001). conclusion: serum uric acid level is significantly associated with nafld, and increased serum uric acid level is an independent risk factor for nafld. background: development of fatty liver is believed to be an early and reversible consequence of excessive alcohol consumption. however, the cellular and molecular events in the early development of alcoholic liver diseases (ald) and the contributory effects of a high fat diet are not fully understood. methods: this study was designed to quantify specific enzymatic and cytokinetic activity as well as the development of hepatic steatosis in a rat model of alchohol-induced liver injury without high fat diet. results: ethanol-fed rats exhibited high blood ethanol levels (0.85+0.31%) and significant increases in serum alt (67.7+ 13.4 unit/l), ast (136.3+ 60.2 unit/l), and alp (400+108.9 unit/l) when compared with control rats (p<0.01, respectively). histopathological examination found unevenly raised knodell scores (5.33+1.15 in the ethanol-fed livers vs. 0.33+0.58 in control), which were characterized by scattered hepatocyte ballooning, portal inflammation and collagen fiber deposition. however, typical steatosis lesions were absent. qpcr demonstrated up-regulation of genes in the ethanol-fed livers, including hepatocyte metabolism enzymes/receptor (adh1, p<0.05; cytochrome p450 2e1, cyp2e1, p<0.05; gsta2, p<0.01; ppar , p<0.05), and genes coding for pro-inflammatory cytokines (il-1 , p<0.01 vs. control livers; tnf-p<0.05; tgf -, p<0.05; rantes p<0.05), ecm components and proteinases (collagen-1, p<0.01; sma, p<0.01; mmp -9, p<0.05 and timp-1, p<0.05). conclusion: chronic administration of ethanol to rats without high fat diet productively induces alcohol hepatitis in the absence of fatty liver, suggesting that alcohol hepatitis may precede steatosis in the development of ald. the aim of the present study was to evaluate the changes of several cytokines associated with inflammatory liver disease and liver regeneration by molecular adsorbent recirculating system (mars) in 15 aclf patients versus 15 patients treated with medical standard therapy (smt) that presented alcoholic liver disease etiology and similar model end-stage liver disease (meld). methods: mars group: fifteen (10 male and 5 female) patients were treated with mars® (gambro). five patients were excluded by study.the number of mars applications was about 9, the length of applications was about 10h. smt group: fifteen patients (10 male and 5 female) were treated medical standard therapy such as prophylaxis against bacterial infections, albumin and fresh plamsa and judicious use of diuretics. three patients were excluded by the study. the patients were valued during 30 days from inclusion with a survival follow up a three months. results: mars group: we observed a significant changes in levels of il-6(p<0.01), il-1(p<0.002), il-8(p<0.04) and tnf-alfa (p<0.03) in association with improvement of hepatic growth factor (p<0.001). the patient's survival at three months was 50%. smt group: we observed only a significant changes in il-1(p<0.01) and tnf-alfa (p<0.2). the patient's survival at three months was 33%. conclusion: the mars liver support device has corrective effects on disturbed pathophysiology of aclf and may be used to enhance spontaneous recovery or as bridge to transplant. a study of protective effect of centella asiatica in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury n. haleagrahara 1 , s. chakravarthi 1 , p. kumar 1 1 international medical university, malaysia background: centella asiatica has been used for centuries as a medicinal herb for wound healing, memory enhancement, cancer, vitality, respiratory ailments, psoriasis and eczema, revitalizing connective tissue, burn and scar treatment, skin infections, arthritis, rheumatism, periodontal disease, varicose veins, hypertension, sedative, anti-stress, anti-anxiety, aphrodisiac, and as immune booster. results: ppc significantly reduced hepatocyte damage, hepatitis, and hepatic fibrosis, but did not affect steatosis. phosphorylation of apoptosis signal-regulating kinase 1, p38 mitogen-activated protein kinase, and protein kinase c, as well as activation of nuclear factor-kappa b, were markedly suppressed by ppc. these effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ros)-generating enzymes, including cytochrome p450 2e1, acyl-coa oxidase, and nadph oxidases, in addition to restoration of ethanol-induced increases in toll-like receptor 4 and cd14. ppc also decreased the pro-apoptotic proteins bax and truncated bid, thus inactivating mitochondrial permeability transition. furthermore, ppc suppressed overexpression of transforming growth factor-1 and hepatic stellate cell activation, which retarded hepatic fibrogenesis. conclusion: ppc exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ald as a result of inhibition of alcohol-induced ros production. background: dysctamnus dasycarpus has used for the promotion of health in south korea. but, there were rare a report concerning the hepatotoxicity. we report cilinical features of liver injury by dysctamnus dasycarpus. method: eighteen patients diagnosed as acute toxic hepatitis by dysctamnus dasycarpus in chungnam national university hospital between january 2005 and arpil 2008 was enrolled. toxic hepatitis was diagnosed by rucam score ( 4). the medical records were reviewed, retrospectively. result: eleven patients (61%) were female and the mean age was 53.5. most common symptom was jaundice. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4 g/dl, platelet 212×10 3 /l,alt 1375iu/l, total bilirubin 11.3 mg/dl, alkaline phosphatase 159 u/l, ggt 244u/l, prothrombin time(inr) 1.1. the mean hospitalization was 21.5days. peak laboratory findings were as follows: alt 1382iu/l, total bilirubin 15mg/dl. recovery time of each biochemical finding was as follows: alt 37days, total bilirubin 39.4 days. recovery rates of alt and total bilirubin were 27.8% and 38.9%, 89.9% and 89.9% at 4 weeks, 8 weeks, respectively. the main biochemical pattern of hepatotoxicity was hepatocellar (72.2%) type. prednisolone was prescribed in six patients. progressive anemia and thrombocytopenia were detected in one patient diagnosed as pure red cell aplasia. other one patient had prolonged jaundice (117 days). but, all patients had recovered without sequelae. conclusion: in south korea, liver injury by dysctamnus dasycarpus was more frequent in women. the main pattern of hepatotoxicity was hepatocelluar type. most patients had prolonged icteric phase and hospitalization. patients were recovered by supportive management after drug cessation or prednisolone therapy. in korea, traditional medicine that is based on the use of herbal medicine developed from a long time ago. however, clinical study of the herbal medicine is not conducted in a structured manner. we report three cases of toxic hepatitis caused by the intake of dictamnus albus. the first patient, a 44 year old woman was admitted due to nausea after ingestion of liquor containing dictamnus albus for 2 months. total bilirubin was 2.55 mg/dl ast/alt 774/1,424 iu/l on admission. liver biopsy observed hepatocyte necrosis and cholestasis. the elevated bilirubin and transaminase returned to normal 2 weeks later after cessation of dictamnus albus. the second patient, a 64 year old man was admitted due to jaundice after ingestion of boiling dictamnus albus for 3 months. total bilirubin was 11.37 mg/dl ast/alt 2,010/2,522 iu/l on admission. liver biopsy observed pericellular fibrosis and necrosis. the bilirubin decreased slowly compared to the transaminase and normalized 3 months later after cessation of dictamnus albus. the third patient, a 48 year old man was admitted due to jaundice after ingestion of liquor containing dictamnus ablus for 1 month. total bilirubin was 12.8 mg/dl ast/alt 1,097/1,869 iu/l the hepatocyte necrosis was observed by liver biopsy. the elevated bilirubin and transaminase levels normalized 1 month later after cessation of dictamnus albus. all patients had negative viral markers and non-specific ultrasonographic findings. the above mentioned three cases demonstrate that liver may have been damaged by dictamnus albus, which indicated clinical characteristics. background/aims: cmili poses a diagnostic challenge as no tests are available to confirm the causality. the aims of this study were 1) to evaluate clinical features and patterns of cmili and 2) to assess the likelihood of causality among patients with liver impairment and exposure to chinese medicine (cm) by a multidisciplinary approach. method: between 5/2005 and 8/2007, patients who had liver derangement and cm or proprietary cm exposure within six months managed in the united christian hospital were studied. clinical features and the cm were reviewed by a multidisciplinary team involving a hepatologist, a toxicologist and cm experts. literature search of relevant herbs in chinese and western journals were performed. cm samples or residue were sent to toxicology laboratory for analysis to look for any toxic constituents, adulterant or contaminant. the likelihood of causality was ranked by various experts independently and disagreements were settled by a consensus meeting. results: there were forty-six cases of suspected cmili, nineteen cases with alternative causes of liver diseases were excluded. twenty-seven cases of cmili proceeded to detailed analysis. median age of patients was 51 (21-76) with female predominance. the median duration of cm exposure to presentation was 20 (1-150) days. majority of them (82%) had hepatocellular liver injury pattern. one case of adulteration with nsaid and erroneous substitution of herb was identified respectively causality were classified as unlikely, possible, probable and highly probable in 3, 12, 8 and 3 patients respectively. conclusion: a multidisciplinary approach allows systemic evaluation of suspected cmili. mouse model i. nassar 1 , t. pasupati 1 , i. segarra 1 , j.p. judson 1 1 international medical university, kuala lumpur, malaysia background: imatinib, a selective tyrosine kinase inhibitor, exhibits drug interactions with other drugs that are metabolised via the cytochrome p450 pathway. acetaminophen, a widely used analgesic and anti-pyretic drug is also metabolised via p450 pathway. this study aimed to evaluate the nature of hepatotoxicity after co-administration of imatinib and acetaminophen in a preclinical mouse model. methods: four groups of male icr mice (30-35g) were used. the mice were administered either saline solution orally, imatinib 100 mg/kg orally (control), acetaminophen 700 mg/kg intraperitoneally (positive control) or co-administered imatinib 100 mg/kg and ip acetaminophen 700 mg/kg (study group). the mice (n=4 per group) were fasted overnight, dosed respectively and sacrificed at pre-determined time intervals of 15, 30 minutes, 1, 2, 4, 9 and 12 hours and liver samples obtained by dissection. h&e stained liver sections (3µm thick) were histopathologically analysed. results: the liver samples showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, with both imatinib and acetaminophen, administered separately. the damage increased gradually with time, peaked at 2 hours and then resolved completely by 6 hours. liver samples showed irreversible damage (cytolysis, karyolysis and karyorrhexis) when both drugs were administered concurrently, the damage increased with time and had not resolved after 12 hours duration. conclusion: co-administration of acetaminophen and imatinib increased the hepatoxicity caused by acetaminophen and imatinib to become irreversible. this may be due to the fact that both drugs are metabolised by the cytochrome p450 pathway in the liver. background: a higher risk of antituberculosis drug (att) induced hepatotoxicity has been reported in indian subcontinent compared to the western counterparts. slow acetylator genotype of n-acetyltransferase2 (nat2) and ci genotype of cytochrome p4502e1 (cyp2e1) gene are two known risk factors associated with this disease. cyp2e1 gene encodes a rifampicin inducible enzyme which increases hepatotoxicity. therefore slow acetylation of isoniazid and simultaneous use of rifampicin may augment the toxicity of isoniazid. objectives: to analyze the allelic distribution of nat2 and cyp2e1 gene in patients of pulmonary tuberculosis who developed att induced hepatitis materials and methods: the study included cases of pulmonary tuberculosis (190) and att induced hepatitis (95). polymorphism of nat2 and cyp2e1 gene was studied by pcr-rflp method in both these groups. results: occurrence of att hepatotoxicity was 17.89%. there was a higher prevalence of slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 in patients with hepatotoxicity compared to patients without hepatotoxicity (72.09% vs 44.2%, p value < 0.05). no association of cyp2e1 rsai polymorphism could be considered with att hepatotoxicity. however, drai c/d genotype of cyp2e1 appears as a risk factor for predicting the occurrence of antituberculosis drug induced hepatitis (or 5.06, p value < 0.05). conclusion: the study demonstrates that patients with slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 and heterozygous mutant c/d genotype of cyp2e1 gene are predisposed to develop antituberculosis drug induced hepatotoxicity. regular monitoring of clinical and biochemical profile may be considered in these patients when they receive antituberculosis treatment. background: drug-induced liver injury is the most common adverse drug reaction. we often use two kinds of diagnostic scales to evaluate suspected patients. however, we still can't diagnose accurately without the direct drugs history and the pathological evidence. methods: twenty-seven drug-induced liver injury cases with liver biopsies from 2001 to 2008 were reviewed retrospectively by maria and japanese scale. result: there were 11.1% of cases with increasing eosinophils. herbs (29.63%) were the most common suspected drug and unknown drugs intake history (14.81%) were described in these cases. the high possibility and possibility were 25.93%, 29.63% by maria scale and 85.19%, 3.7% by japanese scale, respectively (p=0.015). conclusions: japanese scale seems more sensitive than maria scale in these cases. however, there are still some definite cases ignored as low possibility due to absence of obvious drug using history. early treatment and suspected drugs prohibition interferes the outcomes of the two diagnosis systems and lead to a false result. it is still a clinical challenge without strong drug using history or pathological evidence of liver biopsies to diagnose the drug-induced liver injury quickly and accurately. background: previous study suggested that oxidative stress may be an important mediator of methamphetamine-mediated tissue injury. the study was to examine the mechanism of antioxidant activity and methamphetamine-mediated liver injury. materials and methods: the 25 days old male sprague-dawley rats were subcutaneous injected daily with methamphetamine (10 mg/kg body weight) for 15, 30, 60 and 120 days. control group received equal volumes of vehicle. the liver tissues were extracted to measure the activities of sod, catalase, glutathion reductase (gr), and glutathione peroxidase (gpx), and the level of glutathione. western blot were used to measure the expression of rho and phosphor-ezrin-radixin-moesin (p-erm). results: compared with vehicle group, treated with methamphetamine for 15 and 30 days, the activities of liver sod, gpx, and catalase were significantly decreased. in 60 and 120 days group, the activities of antioxidant enzymes of methamphetamine-treated liver was not different from that of vehicle group. the levels of glutathione production also had the same trend. the activities of gpx and catalase on vehicle group gradually reduced following the days of treatment. however, administration of methamphetamine resulted to a lower activity of catalase through the treated days. there was no difference on the activity of gr between vehicle and methamphetamine group. the expression of rho and p-erm were also increased by methamphetamine treated for 30 days. conclusion: these results suggested the methamphetamine lead to liver remodeling via decreased antioxidant activity. finally, the situation of mechanism needs taking in advantage discussion. background: to observe intervening effects of preventive and theraptical treatment of radix sophorae tonkinensis's polysaccharides(rstp) on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: kunming mice intoxicated with anit 50mg/kg orally and treated with rstp 50mg/kg for 7 days before anit exposure and for 2 days after anit exposure respectively, the general condition,mortality rate and serum alt activity are obeverated. result: it was found that by preventive treatment the general condition and mortality rate were improved, serum alt activity reduced.by therapeutic treatment,the general condition deteriorated,mortality rate and serum alt activity increased. conclusion: the preventive treatment of rstp reduce the liver damage due to increasing the anti-stress ability such as the antioxidant capacity,its therapeutic treatment increase the injuried liver damage due to increasing the non-specific immune response and aggregating the preexisting liver inflammation. background: the product's instruction pointed out that in some patients polyphenolic acids' salt from salvia miltiorrhiza(ppas-sm) may lead to a temporary increase in serum alt activity.so we observe effects of ppas-sm on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: 24 hours after intoxicated with anit 50mg/kg orally, kunming mice were treated with ppas-sm 50, 25,10mg/kg/days for 2 days orally, then serum alt activity was measured. result: all doses of ppas-sm led to rise of serum alt activity in mice, most obvious in group of high dose.but the general situation and mortality rate did not increase significantly. conclusion: ppas-sm lead to rise of serum alt activity in mice with damaged liver.the auther suggests as a double-edged sword,the antioxidant ppas-sm may have a prooxidative effect in some condition too. *this project was supported by grants from shanghai municipal education commission under high school high-tech characteristic development programme (no smec finance (2005) 81) pe467 a. somani 1 , a. jain 2 , v. dixit 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha introduction: hepatic encephalopathy, a complex neuropsychiatric syndrome secondary to acute liver failure, chronic parenchymal liver disease or portal-systemic shunting, may possibly develop through mediators of endotoxin and tumor necrosis factor-alpha (tnf-). several studies have shown that serum levels of (tnf-) are significantly elevated in patients with acute and chronic liver diseases, where these elevations are independent of the etiology of the underlying disease. it has been shown that plasma levels of tnf-correlate with the severity of hepatic encephalopathy (he) in fulminant hepatic failure. however, still there is very few published data regarding the relationship between serum levels of tnf-and the presence or severity of he in patients with chronic liver failure. methods: the aim of this study is to determine the relationship between serum levels of tnf-and clinical grades of he in patients with chronic liver failure. this prospective study included 149 consecutive male patients with alcoholic cirrhosis in various clinical grades of he (according to west haven criterion). detailed clinical, biochemical and sonographic examination was done in all patients. circulating levels of tnf-was measured using solid-phase elisa. results: the mean±sem values of serum tnf-at presentation in patients with mhe (n=37), grade 1 (n=17), grade 2 (n=41), grade 3 (n=44), and grade 4 (n=10) were 6.2±0.4, 9.5±0.6, 15±0.7, 26.3±1.7, and 46±5 .9 pg/ml, respectively. significant positive correlation was found between serum levels of tnf-and severity of he (correlation coefficient = 0.7). conclusion: from the present study we can suggest that there is significant relationship between tnf-and he in patients with alcoholic cirrhosis and it could be involved in its pathogenesis. background: acute hepatitis a (aha) is one of the most common infectious diseases and usually a self-limiting disease. although extrahepatic manifestations are not common, a few cases associated with acute renal failure (arf) have been reported. methods: we reviewed clinical features of aha patients complicated with arf (group a) and compared with non-complicated aha patients (group b). medical records of 191 patients with aha were reviewed between january 2003 and december 2007. we experienced 11 patients (5.8%) with arf associated aha. result: there were no differences between group a and group b in sex ratio and age. the peak value of alt (median: 6133 iu/l vs 1685 iu/l, p<0.001), alkaline phosphatase (median: 235 iu/l vs 201 iu/l, p=0.03), prothrombin time (inr, median 1.72 vs 1.09, p<0.001) was significantly higher in group a than b. nine patients (81.8%) recovered completely with hemodialysis (6 patients, 66.7%) and only conservative management (3 patients, 33.3%), while 1 patient underwent liver transplantation and 1 patient died due to fulminant hepatic failure. there were 4 patients who underwent kidney biopsy. two patients were diagnosed as acute tubular necrosis and 2 patient as acute interstitial nephritis and iga nephropathy. conclusion: aha patients with arf had higher alt and more prolonged prothrombin time. the prognoses were poorer than those without arf. however, arf patients with nonfulminant aha had a good prognosis with a proper treatment and should not be confused with hepatorenal syndrome. background/aims: to investigate the hev infection among different animals and people with special profession, and to analyse the genotype of hev isolated in this study. methods: serum and fecal samples were collected from various animals and people with special profession in the south suburbs of beijing. hev antigen and anti-hev antibody were detected by das-elisa. hev rna was extracted from fecal samples and amplified by rt-npcr. the nucleotide sequence homology and phylogenetics of hev strains isolated from swine were analysed. results: the anti-hev antibody positive rate of adult swine, cow, sheep and younger swine were 98.23% (222/226), 29.35% (54/184), 9.80% (20/204) and 60.73% (99/164), respectively. the hev antigen positive rates of adult swine, cow, sheep and younger swine were 2.65% (6/226), 4.35% (8/184), 1.45% (3/207) and 9.75% (16/164), respectively. the hev antigen and anti-hev antibody positive rate of professional group was 0.40% (1/247) and 42.51% (105/247) respectively. the hev rna positive rate of fecal samples from younger swine was 22.89 %(19/83). 16 of 19 samples were hev rna positive by pcr with primers of hev orf1 and orf2. the sequence analysis of the 16 samples showed that there were 2 groups designated as bj-1 (11/16) and bj-2 (5/16). the nucleotide homology of bj-1 and bj-2 was 99%. phylogenetic analysis of hev orf2 indicated that both of them belonged to genotype 4d. conclusion: phylogenetic analysis of hev orf2 indicated that hev isolated in the south suburbs of beijing belonged to genotype 4d. 6 bracops hospital brussels , 7 st. jan hospital, bruges , 8 chu brugmann, 9 chu sart tilman, liège , 10 gent university hospital , 11 zna middelheim, antwerp hepatitis delta virus is a subviral satellite requiring hepatitis b virus to propagate, usually leading to severe, chronic liver disease. as data on epidemiology and management practice of hdv infection in belgium are lacking, a retrospective and prospective, multi-centric questionnaire-based registry is performed in 2008. results of 32 patients are reported. background/ aims: hepatitis a is an acute infectious disease that is transmitted by fecal-oral root. because the incidence of hepatitis a has been increased in gwangju and chonnam province of korea recently, hepatitis a patients in chonnam national university hospital employees had been increased. so we investigated the seroprevalence of igg anti-hav in hospital empolyees less than 40 years old. methods: we analysed seroprevalence of anti-hav igg from 1,002 hospital employees (men: 190, women: 812) . serum alt and bilirubin at admission were 2,979 1,711 iu/l and 3.9 2.4 mg/dl, respectively. these levels were elevated up to 3,397 1,789 iu/l and 6.8 3.7 mg/dl, respectively. ana was positive in 81 patients (64.3%). age, duration from peak-alt day, duration from peak-bilirubin day, alt level, and peak-bilirubin level were not different between ana(-) patients and ana(+) patients. in the while, sex, duration from symptom-onset day, and bilirubin level, and peak-alt level were significantly different. in 51 (62%) of 81 patients with positive ana, ana was followed after 1 month and ana became negative in 38 patients (74.5%). among 13 patients with positive ana after 1 month, titer decreased from the baseline in 7 patients, showed no interval change in 4, and increased in 2. conclusions: positive ana result is not rare in patients with acute hepatitis a. it is considered that ana transiently appear during the course of acute hepatitis a and then, disappear with the improvement of acute hepatitis. (89), 2007(107). the clinical data such as sex, admission period, ast, alt, total bilirubin, prothrombin time, crp, alt normalization time did not show difference. just wbc and gtp were higher on 2008 group. the older age patients were more on 2008 group. the patients admitted mainly on april, may, june, july (84%) on 2008 while admitted even on past years. conclusion: acute hepatitis a ptients is increasing. it is occurring in older age people and mainly on specific period. the more concern to prevention should be needed. background: we analyzed the 5' non-translated region (5'ntr), non-structural proteins 2b and 2c of hepatitis a virus (hav) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all of our data and examine whether genomic differences in hav are responsible for the range of clinical severities. methods: our accumulated hav strains of 5'ntr (nt 200 and 500), entire 2b and 2c from 25 japanese patients with sporadic hepatitis a, consisting of 7 patients with fulminant hepatitis (fh), 5 with severe acute hepatitis (ahs), and 13 with self-limited acute hepatitis (ah), in whom the sequences of all 3 regions were available, were subjected to phylogenetic analysis. results: fh patients had fewer nucleotide substitutions in 5'ntr, had a tendency to have more amino acid (aa) substitutions in 2b, and had fewer aa substitutions in 2c, than ah patients. four fh and 2 ahs with higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis a and genomic variations in 5'ntr, 2b and 2c of hav. conclusions: our study suggests that genetic variations in some parts of hav might cooperatively influence replication of the virus, and thereby affect virulence. viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis a. aims: the incidence of acute viral hepatitis a in adults is increasing very much in south korea, 2008. the aim of this study was to the clinical features and course in daejoen and its surrounding area. methods: forty seven patients admitted as acute viral hepatitis a in chungnam national university hospital between january 2008 and june 2008 were enrolled. the medical records were reviewed, retrospectively. results: the mean age was 30.5. common occupations were company employee and studuents. most common symptom was jaundice. presumptive infection sources were raw fish or shellfish and raw meat. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4g/dl, platelet 212×10 3 /l, ast 2268iu/l, alt 2755iu/l, total bilirubin 5.2mg/dl, alkaline phosphatase 207u/l, ggt 379u/l, prothrombin time(inr) 1.2. hospitalization was 21.5days. peak laboratory findings were as follows: alt 3181iu/l, total bilirubin 8.8mg/dl. leukopenia (<4000/ul) and thrombocjtopenia (<10×10 5 /l) were ocurred in sixteen and six patients, respectively. recovery time of each biochemical finding was as follows: alt 20.2 days, total bilirubin 26 days. recovery rates of altand total bilirubin were 74.4% and 74%, 88.5% and 92% at 4 weeks, 8 weeks after diagnosis, respectively. prolonged jaundice (115days) was detected in one patient. all patients were recovered by supportive management. conclusions: in south korea, acute viral hepatitis a was more prevalent in young adults, recenlty. presumptive infectious sources were raw fish or 1 guangxi center for disease prevention and control shellfsh and raw meat. if it can not change the food style that many korean enjoy raw seafood, vaccination for adults must be considered to prevent it. objective: to assess the safety and immunogenicity of a new inactivated hepatitis a vaccine (vero cell). pe479 methods: 1507 subjects were selected in gongcheng city of guangxi zhuang autonomous region, and the clinical trail was carried out according to the random, double-blind and parallel principle from january to august, 2005. after vaccination by 0, 6 schedule, adverse events of the subjects were observed, the seroeonversion rate and geometric mean titer (gmt) were tested by the competitive inhibition elisa. results: after immunization, the systemic and local reaction rates of adults were 8.80% and2.67%, which was no significantly statistical difference compared with control group, 12.41%and 4.41%; while the rates of children were 10.60and 2.28%%, and no significant statistical difference compared with control group, 10.71%and2.86%. one month after first dose of vaccination, the seroconversion rates of children and adults were 88.2% and 93.8%, and one month after second dose of vaccination, the rates were all 100%, the gmts of children and adults were 16447miu/m1 and 8555miu/ml, which was significant statistical difference in children compared with control group, 1946 miu/ml and 5881 miu/ml, respectively. methods: igg anti-hav was measured in a total of 3905 subjects under the age of 50, who visited hanyang university seoul and guri hospitals between january 2001 and may 2008. results: fig. 1 shows the relatively low positive rates of the antibody in ages of 11 to 30 and the lowest rates of 9.9% and 9.2% in the age group of 16 to 20, following the ages of 21 to 25 with rates of 12 background: some viruses encode proteins that affect their cap-independent internal ribosomal entry site (ires)-mediated translation and their replication. it was recently reported that hepatitis a virus (hav) proteases interact with intracellular dsrna-induced retinoic acid-inducible gene (rig-i)-mediated signaling, but it remained unknown whether hav proteins have any effects on hav ires-independent translation. in this study, we investigated the effects of 7 hav non-structural proteins on their ires-mediated translation using a reporter assay. pe480 methods: the bicistronic reporter constructs, termed psv40-hm175-ires, psv40-a1-ires, psv40-a2-ires, psv40-f1-ires, and psv40-f2-ires, contain the sv40 promoter that controls the expression of a bicistronic message coding for renilla and firefly luciferases separated by hav ires, and are derived from strain hm175, acute convalescent hepatitis clones a1, a2, fulminant hepatitis clones f1, f2, respectively. human hepatoma cell lines were co-transfected with psv40-hav-ires and each hav protein-expression vector. luciferase activity was determined 48 h after transfection. were from other countries within asia, africa, middle east, and eastern europe. patients of a wide age range were affected by hepatitis delta (mean age 46.0, median 47.0, range 19-79). 18 (40%) of 45 were co-infected with hcv. hepatitis b virus (hbv) dna was detectable in 43 (78%) patients and negative in 8 (15%) patients. all hepatitis delta patients were extracted from a prior study conducted by this collaboration. there were 1,191 chronic hbv carriers. 18 (1.51%) were hbv/ hcv/hdv infected. 32 (58%) of 55 patients carried a diagnosis of cirrhosis compared to 262 (22%) of 1191 chronic hbv patients. 13 (72%) hcv co-infected patients had evidence of cirrhosis while 4 (22%) patients did not. conclusion: individuals with hbv/hdv co-infection have higher rates of cirrhosis. individuals with hbv/hcv/hdv infection have rates of cirrhosis significantly higher than individuals with either chronic hbv infection or hbv/hdv co-infection. testing for hdv should be performed in all patients, especially those with advanced liver disease or high risk behavior. clinical characteristics were compared between the patients with significant endoscopic findings (group a) and without such findings (group b). peak ast and alt level were higher in group a (p<0.01). there were no statistical differences in age, gender, comorbidity, and etiology of acute hepatitis between group a and group b conclusion: significant endoscopic findings were found in considerable proportion of patients with acute hepatitis. severity of acute liver injury was associated with significant upper gastrointestinal endoscopic findings. in patients with severe acute hepatitis who complain of upper gastrointestinal symptom, esophago-gastro-duoenoscopy should be performed. background: in japan, hepatitis e virus (hev) testing is not allowed as routine one. to study the role of hev testing, we checked 22 sera of the patients diagnosed as etiology-obscure acute liver injury. methods: we have seen 54 cases of acute liver injury from january 2004 through december 2007 in our hospital and 25 cases of them were etiology-obscure. in 25 cases, 22 were retrospectively tested for hev-igm, hev-iga and hev-rna (rt-pcr) by direct sequence method on stored sera taken at the time of presentation. result: two of 22 cases (9.1 ) were positive for both hev-igm and hev-iga and one case was positive for hev-rna. in 54 cases of acute liver injury, the cause of virus was 31 cases (57.4%) and unknown was 8 cases (14.8%). hev was occupied in 3.7% in all cases and 6.5% in the cases caused by virus. one of the two cases had been misdiagnosed as "drug induced hepatitis". hev of genotype 3 was detected in one case and its nucleotide sequences of hev showed quite a high degree of similarity to the reported one at closed city in the same year. conclusion: hev is not rare in japan and the hev testing can reverse the diagnosis of acute liver injury. hev testing sould be used as routine one for acute liver injury. association of progesterone receptor gene with hepatitis e disease severity in pregnancy p.d. bose 1 , b. das 2 , a. kumar 1 , p. kar 1 1 maulana azad medical college, 2 background/aims: incidence of fulminant hepatic failure (fhf) in hepatitis e is high in pregnancy particularly during 3 rd trimester when there is an altered status of hormone and immunity. progesterone receptor (pr) up regulation provides fetal protection via immunosuppression but lower immune status in pregnancy may add to the disease severity. till now, no data is available whether pr can play any role in hepatitis e disease severity during pregnancy. progins, a haplotype of pr consisting of 320-bp insertion in intron g together with point mutations in exons 4 and 5 is associated with increased stability and higher transcriptional activity. the aim of the study is to analyze pr mutation (progins) and m rna expression in hepatitis e virus infected pregnant women with avh and fhf. methods: a total of 68 avh and 32 fhf cases were studied. blood and placental tissue were collected from the medicine and gynecology wards of lnjp hospital, new delhi. cases were screened for acute viral markers by commercially available elisa kit. extraction of dna from blood and rna from placental tissue was done by qiagen kit. mutation in pr was detected by pcr-rflp. semiquantitative rt-pcr for pr expression was performed in placental tissue using beta-actin as internal control. results: pr mutation (progins) was significantly more in fhf compared to avh (28.1% vs 10.3%, p value<0.05). protein expression was found higher in progins carriers. conclusion: progesterone receptor mutation (progins) may have a role in the hepatitis e disease severity in pregnant women. results: the hepatitis e was predominantly sporadic, some patients superinfected with other viral hepatitis, especially hepatitis b. in the old patients, jaundice lasted longer and the length of stay was longer, the incidence of complication was higher than the young men. the incidence of complication in the superinfected group was higher than the simple infection. the transaminase in the simple infection group was obviously raise than superinfected with liver cirrohsis. methods: liver sample were paraform-glutaral fixed, paraffin-embedded, sectioned and immunohistochemical stained, and positive samples were selected for histological analysis and rt-pcr detection. result: positive rate of hev immunohistochemistry ranged from 90% to 100% (fig. 1) . hepatocyte degeneration, scattered singled karyopyknosis, lymphocytic infiltrate, hyperplasia of bile canaliculus at the portal area and fibrous connective tissue hyperplasia been observed during histological analysis (fig. 2) , and two genotype 4 hev which closely related to many strain isolated from patients with sporadic acute hepatitis been detected. conclusion: the patients infected with hepatitis e of young men were frequently. jaundice lasted long in the old patients, the incidence of complication was higher in the superinfected men and the old men. conclusion: additional public-health concerns might be placed on pork safety and the risk of hev infection via the consumption of undercooked pork products. poster session, hall 5b aim: esophageal varices (ev) recurs frequently after endoscopic variceal ligation (evl) or endoscopic injection sclerotherapy (eis). we retrospectively investigated risk factors for early recurrence of ev after endoscopic treatment. methods: we treated 110 patients with ev, who had no past history of ev, at ehime prefectural central hospital from october 2005 to june 2008. of those, 78 (71%) were observed for at least 2 months after treatment and enrolled. we divided them into rupture cases at initial endoscopic treatment [(bleeding group; n=25 (32%)], and cases with preventive evl or eis performed [preventive group; n=53 (68%)]. all received periodic upper endoscopy examinations to confirm recurrence or no recurrence of ev. results: recurrence of ev occurred in 18 of all subjects and the average period after treatment was 6.5±3.0 months. the recurrence rate was significantly higher in the bleeding group (11/25) as compared to the preventive group (7/53) (p=0.0026). there was a significant relationship between recurrence of ev and hepatic reserve function (child-pugh a+b, c; 11/64, 7/14 respectively; p=0.0083). in logistic multi-variant analysis, ev rupture at initial treatment and child-pugh c were risk factors for recurrence. in contrast, age, sex, hepatocellular carcinoma, portal tumor thrombosis, continuous alcohol consumption, therapeutic modality (evl or eis), number of treatment sessions, and operator experience did not have a significant relationship with recurrence. conclusion: in cases with ev rupture at initial treatment or child-pugh c, the risk for early recurrence must be considered and patients carefully observed in follow-up examinations. endoscopic cyanoacrylate injection: less oil for less ectopic embolism c.z. li 1 , l.f. cheng 1 , z.q. wang 1 , f.c. cai 1 , q.y. huang 1 , e.q. linghu 1 1 general hospital of chinese pla background and aim: endoscopic injection sclerotherapy with n-butyl-2cyanoacrylate (nbca, histoacryl) has been reported to be effective for hemostasis of bleeding gastric varices, but occasionally the gel flows to other organs and causes ectopic embolism. the present study aimed to determine whether less amount of iodized oil preload in nbca injection helps in decreasing ectopic embolism. methods: from january 1997 to april 2006, 2 different methods of endoscopic nbca injection, "sandwich method" and "modified sandwich method" (in which iodized oil preload was minimized), were applied on 635 gv cases, to evaluate if decrease of iodized oil preload resulted in less ectopic embolism. results: altogether 5 cases of ectopic embolism occurred in the whole group (0.8%), including 3 cases of splenic infarction, 1 case of transient paralysis and 1 case of minor infarction of the lung. the modified sandwich method showed some superiority over original method in decreasing ectopic embolism (0/276 vs. 5/359, p=0.049). less cough during procedure was also found with the modified method (3/276 vs. 18/359, p=0.006). conclusions: less amount of iodized oil preload in endoscopic nbca injection is beneficial to decrease ectopic embolism. background: portal hypertension is closely associated with serious complications of liver cirrhosis which contribute to bad prognosis. hepatocellular carcinoma (hcc) and low serum sodium (sna) are manifestations of end-stage liver disease (esld) and are associated with poor survival in decompensated cirrhosis patients. therefore, we aimed to determine the relationship between hepatic venous pressure gradient (hvpg) and the development of hcc or low sna in decompensated alcoholic cirrhosis patients. methods: child-pugh scores, meld scores, and hvpg at baseline, and the development of low sna (sna <130 meq/l) or hcc during follow-up were analyzed prospectively in 170 patients with decompensated alcoholic liver cirrhosis. the predictive values of different risk factors for the progression to the esld were investigated by multivariate analysis and the kaplan-meier method results: twenty-four patients developed hcc during the follow-up period. in the multivariate analysis, only baseline hvpg>15mmhg was an independent predictive factor for the development of hcc (relative risk (rr)=1.128, p<0.05). those with hvpg >15 mmhg showed a significantly shorter time for the development of hcc on kaplan-meier analysis. twenty patients developed low sna during follow-up. initial hvpg was also an independent predictive value for the development of low sna in the multivariate analysis (rr=1.169, p<0.05). those with hvpg>15 mmhg also showed significantly shorter times for the development of low sna on kaplan-meier analysis. conclusions: in decompensated alcoholic cirrhosis, hvpg may be a useful predictive factor for the development of hcc and low sna, both of which are characteristic of esld and poor prognosis. the effectiveness of the treatment of octreotide on chylous ascites after liver cirrhosis d.x. zhou 1,2 , h.p. hu 1,2 background: octreotide is a crucial drug used for treating patients with chylous ascites; however, there have been few reports related to octreotide that are being used in cirrhotic patients. thus, this thesis is designed to determine the effects of octreotide on patients with chylous ascites after liver cirrhosis. methods: eight patients were diagnosed with chylous ascites, on the basis of laboratory findings on ascites samples, between january 2003 and may 2008. octreotide was given to the six patients, while the remaining two were treated as a control. all patients had persistent peritonea drainage with the quantity and quality of the drainage fluid observed once every other day. all the necessary care was individually given to the patients during the therapy results: all patients properly received combined therapy including low fat and sodium diet, and diuretic and peritoneal drainage. the volume of the peritoneal drainage was reduced to zero in one of the six patients who received octreotide therapy, while the other five had the drainage volumes decreased from 2000 ml to 50 ml with a clear appearance and negative qualitative analysis of chyle for those two patients who did not receive octreotide therapy, the conditions of peritoneal drainage seldom changed both from the qualitative and quantitative aspects. conclusion: octreotide, along with combined therapy, can rapidly relieve portal hypertension and reduce fat absorption from intestinal mucosa. it appears to be an effective therapy available for the treatment of chylous ascites caused by liver cirrhosis. albumin <38g/l were the best predictors of large varices. a model using these predictors in a validation cohort study is planned. background-aim: cirrhosis is associated with raised acute phase proteins (app), irrespective of infection. it is, however, unclear whether their values differ significantly or whether a particular app might be more indicative of infection, and these questions were addressed in our study. methods: we measured serum crp, fibrinogen, ferritin, haptoglobin, 2-microglobulin, c3, c4, and c1 inhibitor in 88 consecutive, cirrhotic patients, on admission. all patients were investigated according to a standard protocol for infection. child-pugh scores (cps) were calculated. results of app were expressed as means sem and compared with the mann-whitney test. results: 19 (21,6%) patients, median age 60 years, (cps: a=0; b=7; c=12), were diagnosed with infection (spontaneous bacterial peritonitis=7; pneumonia=5; septic shock=4; extensive cellulitis=1; listeria monocytogenes meningitis=1; viral infection=1), while 69 (78%) patients, median age 59 years, (cps: a=25; b=28; c=16), showed no infection. although most app values were raised, there was no statistically significant difference between patients with or without infection, or among different cps groups, except for crp, which was significantly more raised in patients with infection (p<0.01). this difference remained even after cps a cases in the non-infection group were excluded from analysis. interpretation: a significantly raised crp in cirrhosis would seem to be independent of cps staging and should prompt a thorough work up to exclude infection. by contrast, the discriminating power of all other app in the face of possible infection is negligible. the predictive value of crp towards infection is under investigation prospectively. although bleeding from ectopic varices such as duodenal, jejunal, ileal, colonic, and rectal varices is less common, it can also cause life-threatening problem, which is often difficult to diagnose and treat successfully. here we present a novel endoscopic approach for hemorrhagic rectal varices using endoscopic injection sclerotherapy with ligation (eisl). patients and methods: in 2000-2008, we performed endoscopic treatment in 215 patients with portal hypertensive varices. among those, four cases of hemorrhagic rectal varices were treated with the combined evl and sclerosing technique. the etiology of portal hypertension included oen idiopathic portal hypertension and three hcv cirrhosis. all patients had a history of prior abdominal surgery or endoscopic treatment for gastro-esophageal varices. results: hemostasis was obtained easily by the evl initially. furthermore, to avoid recurrent bleeding, the patients underwent endoscopic varicerography injection sclerotherapy (evis) using 5% ethanolamine oleate with iopamidol and the feeding vein was sclerosed successfully with no major complication occurred during the entire course of the treatment. conclusions: it is important to recognize the possibility of ectopic varices as a cause of gastro-intestinal haemorrhage especially in patients with a history of variceal therapy or abdominal surgery. the eisl technique is useful to control the initial and recurrent bleeding from rectal varices. t. hirano 1 , t. okada 1 , j. yamanaka 1 , y. iimuro 1 , n. kuroda 1 , k. oh 1 , y. yoshida 1 , j. fujimoto 1 1 aim: interferon (ifn) therapy is a powerful treatment for hcv-related hepatitis and is known to decrease the incidence of progression of hepatocellular carcinoma (hcc). however, thrombocytopenia is a common side effect of ifn treatment, often leads to discontinuance without insufficient therapeutic effect. in this study, we investigated the efficacy and safety of laparoscopic splenectomy ( ) in reversing thrombocytopenia in patients with hepatitis c cirrhosis and portal hypertension. patients and methods: out of 41 patients who underwent ls in our department during aug 2003 and december 2007, 13 patients associated with portal hypertension. among these patients, three patients had hcc, and they were simultaneously underwent partial hepatectomy after splenectomy. platelet count, operative time, blood loss, complications and length of stay were calculated. results: thirteen patients underwent laparoscopic splenectomy; their mean age was 59 years (range 20 to 68 years). six patients were child's class a and seven patients were class b. mean operative time was 178 minutes (range 97 to 255 minutes). blood loss was little, and none required transfusion with packed red cells. a hand-assisted laparoscopic technique was used in four cases (30.8%). average length of stay was 12.7 days. there have been no major complications during follow-up. platelet counts improved from a preoperative mean of 62000/ul (44000 to 108000) to 273000/ul (126000 to 469000) postoperatively. six patients are ongoing ifn treatment without remarkable thrombocytopenia. conclusion: laparoscopic splenectomy is safe and in patients with portal hypertension and thrombocytopenia. it may allows these patients by reversing thrombocytopenia. background: hepatic encephalopathy (he) is a significant cause of mortality in advanced cirrhosis patients. l-acyl-carnitine has been suggested as an alternative treatment for patients with he patients. to assess the clinical efficacy of acetyl-l-carnitine in the treatment of hepatic encephalopathy in cirrhotic patient, especially in diminishing the recurrence and reduction serum ammonia level. methods: we performed a randomized placebo-controlled, cross-over study. we administered acetyl-l-carnitine to group 1 during 3 months first then placebo during later 3 months, and administering acetyl-l-carnitine to group 2 alternatively. results: between january 2008 and february 2008, thirty two selected cirrhotic patients were enrolled in this study. following randomization, the patients were divided into two groups (group 1=14, group 2=18). during administering acetyl-l-carnitine period, serum ammonia level was decreased significantly in both groups significantly (p=0.005, vs. p=0.001 respectively). however, during administering placebo period, serum ammonia level changes were not significant. in group 1, the first recurrence cases of hepatic encephalopathy were more than group 2(group 1=5, group 2=2), and the first recurrences were occurred during first 3 months in all groups. conclusion: our study demonstrates that acetyl-l-carnitine administration reduced serum ammonia level, but not definitely diminishing the recurrence of hepatic encephalopathy. sodium (na + ) and water retention are the most common abnormalities in cirrhotic patients and the magnitude varies from patients to patients. aim: to assess the relationship between the meld score and urinary excretion of na+ in non-azotemic cirrhotic patients. methods: fifty four cirrhotic patients with ascites and normal serum creatinine (<1.5 mg/ml) were admitted and placed on a low sodium diet (4g/day), while all diuretics were withdrawn for 3 days. the electrolytes (na + , k + , na + / k + ) were measured in a random urine and both the volume and na + concentration of urine collected for 8 h after administration of furosemide 80 mg i.v. were determined. results: table. conclusions: the meld score was significantly correlated with the degree of impairment of urinary na+ excretion. the ratio of na + /k + in a random urine specimen and furosemide-induced na+ excretion reflect the degree of impaired natriuresis in non-azotemic cirrhotic patients with ascites. background: portal hypertensive gastropathy (phg) is common finding in patients with liver cirrhosis and portal hypertension. despite portal hypertension remains the crucial trigger for the development of phg, the relationship between portal hypertension and phg has not been widely investigated. methods: fifty-three cirrhotic patients (48 males, mean age 51 years) who were performed hepatic vein catheterization between november 2006 and august 2008 were prospectively included in this study. the degree of phg was assessed according to the third baveno international consensus workshop, and classified three degrees as no, mild and severe. the hepatic venous pressure gradient (hvpg=whvp-fhvp) measurements were performed by triplicate in each case, and results were given as arithmetic means of the three determinations. result: hvpg values did not differ between the patients without phg (13.28±4.72 mmhg) and those with phg (14.91±3.84, p=0.187), nor between those with mild (15.31±3.69 mmhg) or severe phg (14.27±4.12mmhg, p=0.323). the degree of phg and hvpg did not differ regarding the etiology of the cirrhosis(p=1.0, p=0.085) nor regarding the child pugh classification(p=0.085, p=0.738). no correlations were found between the degree of phg and child pugh score, age, with or without ascites, albumin, bilirubin, creatinine, meld score and the degree of gastroesophageal varices. conclusions: our data show that the presence and the severity of phg does not correlate with the degree of hvpg, and that correlate with esophageal varices in patients with liver cirrhosis. introduction: phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the colonic wall due to fibrous degeneration of the submucosal layer and fibrotic sclerosis of the venous wall. there are a few reports those this entity might be related to portal hypertension with disturbed venous return from the colon and mesentery. case description: a 61-year old man with alcoholic liver cirrhosis presented with right lower abdominal pain/tenderness and bloody diarrhea. a colonoscopy revealed multiple circumferential ulcerations in the transverse colon and the scope could not get through the ascending colon due to luminal stenosis, showing histologic finding of ulcerative inflammation with inflammed granulation tissue. abdominal computed tomography demonstrated liver cirrhosis with splenomegaly, multiple portosystemic venous collaterals, diffuse vascular engorgement and the wall thickening of right proximal to mid ascending colon with increased density in the surrounding fatty tissue. a follow-up colonoscopy performed one month later showed still remained multiple ulcerations in the transverse colon and could not further advance to ascending colon. superior mesenteric angiography revealed no main branch occlusion but pooling at the venous phase on ascending colon. a right hemicolectomy was performed because of the colonic obstruction. gross findings on operation showed thickening of the cecum and ascending colon. microscopic examination showed fibrous thickening in the submucosa, abundant neurovascular bundles in the mesentery and several intravascular hyaline thrombi of the mesenteric vessels. here we report the first case of early stage of phlebosclerotic colitis in a cirrhotic patient in korea. spontaneous bacterial peritonitis (sbp) is one of the severe complications in advanced cirrhotic patients with a high mortality rate. although a more rapid diagnosis should lead to the better survival, it takes several days to detect the causal bacteria from ascitic fluid cultures. furthermore, despite the use of sensitive methods, ascitic fluid cultures were negative in more than 50% of patients with suggestive clinical manifestations of sbp. therefore, diagnosis of sbp is based on the polymorphonuclear leucocytes (pmn) cell count in the ascitic fluid. the hybrizep kit (fuso pharmaceutical industries, osaka, japan) detects the dna of bacteria that have been phagocytized in neutrophils and macrophages, using in-situ hybridization method within one day. here we present a case of the patient for whom the hybrizep kit was used to detect the causal pathogen of sbp. a 76-year-old man had been admitted for the treatment of ascites and esophageal varices. one week after the admission, he complained abdominal pain and fever. because the pmn cell count in ascites fulfilled the criteria of sbp (1141/mm 3 ), we started an empirical antibiotic therapy without waiting for a result of the culture, and his symptoms improved within a few days. on the following day of the onset, in situ hybridization showed the positive signals by the ek probe, which detected the genomeic dna of e.coli species. however, the ascitic fluid culture was negative. this case suggested that the hybrizep kit was useful for the rapid diagnosis of sbp with high sensitivity. background: it has not been known that the hemodynamic effect of a portal hypertension for splenomegaly or esophageal and gastric variceal formation. this study was performed to access the parameters of doppler ultrasonography associated with splenomegaly or varices in patients with cirrhosis. patients and methods: from may 2007 to may 2008, 144 cirrhotic patients were performed the doppler ultrasonography. 91 of these patients were accessed the severity of varices endoscopically. the three dimensional volume of spleen was measured from a length, width and thickness on sonography. results: the splenic volume (415.2ml vs 505.6ml, p=0.048) and blood flow of main portal vein (12.6cm/s vs 15.1cm/s, p=0.012) were statistically significant different in alcoholic (38/144) and non-alcoholic (105/144) cirrhosis groups. the splenic volume (600.1ml vs 384.4ml, p=0.001), damping index (0.52 vs 0.38, p=0.024), and blood flow of main portal vein(12.5cm/s vs 15.7cm/s, p=0.006) were statistically significant different in esophageal variceal groups (55/91) and non-esophageal variceal groups(36/91). the only splenic volume (669.4 ml vs 461.7 ml, p=0.004) were statistically significant different in gastric variceal groups (24/90) and non-gastric variceal groups (66/90). the hemodynamic parameters venous ammonia and cff at baseline and after one month of treatment with lactulose. mhe diagnosed by abnormal psychometry and/or p300erp.response defined by normalization of abnormal test parameters. results: mhe diagnosed in 35(58%) patients. of 35 patients 26 (74%) had both abnormal psychometry and p300erp whereas30 (86%) alone had abnormal psychometry, 31 (89%) had abnormal p300erp.cff was <39hz in 28(80%) patients. mhe recovered in 55% with treatment and cff >39hz was seen in 22(69%) of 32 patients. cff sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy before and after treatment is shown in table. conclusions: critical flicker frequency is a simple and accurate test without any age or literacy dependence for the diagnosis and recovery of patients with mhe. background/aims: endoscopic injection of n-butyl-2-cyanoacrylate (histoacryl) is an effective treatment of varix bleeding. but nontarget embolizations and septicemia are unwanted complications. we evaluate the risk factors for complications. methods: thirty-three patients with esophageal or gastric varix bleeding received endoscopic histoacryl therapies (54 procedures). baseline varix size, ctp score were checked. serum leukocyte, blood culture and body temperatures were repeated checked within one week after procedure. average volume of histoacryl per each session was 1.6 ml, and dilution volume ratio of histoacryl/lipiodol was 1/1 or 1/2. results: average of ctp score was 8.0 ± 1.7. three cases of septicemia were correlated with ctp score rather than session frequency or injection volume. two cases of systemic embolizations (pulmonary and splenic arterial embolism) were correlated with high lipiodol dilution ratio (1/2) and lipiodol volume rather than histoacryl volume or ctp score. conclusion: ctp score, lipiodol volume and dilution ratio of histoacryl/lipiodol were significant risk factors for complications. detection of circulating toll-like receptor 2 and 4 and cd4+cd25+ regulatory t cells in patients with hbv-related liver cirrhosis x.q. wang 1 , y. zhang 1 , x.f. bai 1 , j.q. lian 1 1 background : to detect circulating cd4 + cd25 + regulatory t cells and toll-like receptor(tlr)2 and tlr4 expression on the peripheral blood mononuclear cells (pbmcs) of patients with hbv-related liver cirrhosis (lc), and to explore the correlation between them. methods: pbmcs isolated from 14 lc patients , 21 chronic hepatitis b (chb) patients and 16 normal controls(nc) were stained with fluorescent labeling anti-tlr2-pe, anti--tlr4-apc, anti--cd14-fitc monoclonal antibodies and anti-cd4-percp anti-cd25-fitc anti-cd127-pe. samples were collected and detected of three-color immunofluo rescence by flow cytometry. results: the expression of tlr2 and tlr4 were significantly up-regulated in patients with lc than those in the controls.the expression of tlr2 was significantly increased in patients with lc than those in patients with chb, but there were no differences of tlr4 expression between lc and chb.treg/cd4 + t cells were significantly increased in patients with chb than those in patients with nc and lc, but there were no differences between lc and nc. there were no correlation between the expression of tlr2,tlr4 and treg in patients with lc . the expression of tlr2 and tlr4 on pbmcs in patients with lc were positive correlation.the expression tlr4 and hbv dna level were negative correlation in patients with lc. conclusion: the expression of tlr2 and tlr4 were up-regulated on pbmcs in patients with lc. it seems to be expression of tlr2 and tlr4 invovlved in the pathogenesis of lc. evaluation of 13c-phenylalanine breath test for the measurement of hepatocyte function in patients with chronic liver disease z.j. bao 1 , d.k. qiu 2,3 , x. ma 2,3 , g.s. zhang 1 , y.q. huang 1 , z.p. fan 2,3 , s.m. yin 1 1 huadong hospital, fudan university, 2 renji hospital, shanghai jiao tong university school of medicine, 3 background: the objective is to investigate whether the 13c-phenylalanine breath test(pbt) would be useful for the evaluation of hepatic function in patients with chronic hepatitis b, liver cirrhosis and minimal hepatic encephalopathy (mhe). methods: l[1-13c] phenylalanine was administered orally in a dose of 100 mg to 80 patients with liver cirrhosis, 20 with chronic hepatitis b and 20 healthy subjects. the pbt was measured at 8 different time points (0, 10, 20, 30, 45, 60, 90, 120 min) to obtain the values of delta over baseline, percentage 13co2 exhalation rate and cumulative excretion (cum). the relationships of the cumulative excretion with the 13c-%dose/h and blood biochemical parameters were investigated. results: the 13c dose h -1 at 20 and 30 min combined with the cumulative excretion at 60 and 120 min showed correlations with the chronic liver diseases, especially child-pugh score and mhe or not. and the data showed correlations with serum albumin hemoglobin platelet and child-pugh score. prothrombin time, total and direct bilirubin were significantly increased, while serum albumin, hemoglobin and platelet, the cumulative excretion at 60 and 120 min values decreased by degrees in healthy controls, child-pugh a, b, and c patients (p<0.01). similar results of pbt were in the patients with and without mhe, while only prothrombin time prolonged and total bilirubin increased (p<0.05). conclusions: the pbt can be used as a non-invasive assay to evaluate hepatic function in patients with liver cirrhosis and mhe. the %13c dose h-1 at 20 min, %13c dose h-1 at 30 min and cumulative excretion at 60 min may be the key value for determination at a single time-point. branched chain amino acids in improving survival and decreasing risk of liver failure among cirrhotic patients: a meta-analysis h. flores 1 , e.l. ang 1 , n. iv estanislao 1 1 philippine general hospital background: the state of a patient's nutritional status greatly affects disease outcome. among cirrhotic patients, approximately 60-90% are in a state of protein-energy malnutrition. hence, adequate nutritional support is essential to improve their general medical condition and long term prognosis. several studies have shown than branched chain amino acids (bcaa) may be of benefit for this purpose. it is the aim of this study to evaluate the effectiveness of diet plus bcaa compared to diet alone in improving survival and in decreasing liver failure among cirrhotic patients. methods: pubmed, cochrane, and embase search was done for articles which compared the clinical effects of bcaa supplementation versus diet alone among patients with liver cirrhosis. the following free-text terms and mesh words were used -"branched chain amino acids", "amino acids, branched chain", "bcaa", "liver cirrhosis", "cirrhosis", "randomized controlled trials'" and "meta-analysis". after critical appraisal of the included studies, a random effects model using odds ratio was used to synthesize the results (revman 4.2). results: 3 rcts were included for analysis with a total study population of 836. combination of the studies showed a significant decrease in the risk of liver failure (or 0.45, 95% ci 0.25-0.82, p=0.009) and a trend towards benefit in improving survival (or 0.57, 95% ci 0.27-1.17, p=0.12). conclusions: the overall trend appears to show benefit in the use of branched chain amino acids for patients with cirrhosis with respect to liver failure and survival. background: the proxisome prolifrator-activated receptor gamma (ppar ) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation, carcinogenesis and gastric ulcer. on the other hand, the frequency of gastrointestinal ulceration is higher in cirrhotic patients compared with the normal population. the present study was designed to investigate the effect of specific ppar ligand, pioglitazone, on the mucosal lesions induced by ethanol in cirrhotic rats and the possible involvement of nitric oxide in the pioglitazone effect. methods: cirrhosis was induced by surgical ligation of bile duct and sham-operated rats served as controls. both cirrhotic and sham rats were kept for 28 days after the operation. different groups of sham and cirrhotic animals received saline, or 5, 10 or 15 mg/kg pioglitazone, daily during last 5 days of the fourth week after the surgery. another 2 groups of bdl or 2 groups of sham rats received l-name, a non selective inhibitor of nitric oxide synthase, alone or along with 5 mg/kg pioglitazone for 5 days. on day 28, rats were killed 1 hour after ethanol administration and the area of gastric lesions was measured. results: the ethanol-induced gastric mucosal damage was significantly more sever in cirrhotic rats than sham-operated ones (p < 0.001). pretreatment with pioglitazone dose dependently attenuated gastric lesions induced by ethanol in both sham and cirrhotic rats, but this effect was more significant in cirrhotic ones. concurrent treatment of l-name and pioglitazone decreased the ulcer index in bdl rats more than the groups that received l-name or pioglitazone alone. conclusion: we conclude that chronic treatment with pioglitazone exerts a potent gastroprotective effect on the stomach ulcers of cirrhotic rats probably due to inhibition of nitric oxide synthase. inhibition of phosphodiesterase 5 -a novel therapeutic strategy for portal hypertension l. halverscheid 1 , p. deibert 2 , b. pannen 1 , r. schmidt 2 , m. roessle 2 , w. kreisel 2 1 university hospital duesseldorf, germany, 2 university hospital freiburg, germany introduction: the no-cyclic gmp system is a key factor in the regulation of splanchnic and hepatic blood flow and may be a target for medical treatment of portal hypertension. clinical data have shown that inhibitors of phosphodiesterase 5 (pde5) lower portal pressure in cirrhotics. methods: we monitored in rats the effects of the pde5 inhibitors vardenafil and sildenafil on systemic and hepatic hemodynamic parameters up to 60 minutes after the drug. the drugs were administered intravenously into the tail vein at 1 (group a), 10 (group b), and 100 g/kg body weight (group c). 0.9% nacl was the control. n = 7 for each group. results: the most prominent changes were observed in the vardenafil b group: mean arterial and portal venous pressure decreased (-9%, -8%), as well as portal venous, hepatic arterial, and systemic vascular resistance (-31%, -30%, -12%). portal venous and sinusoidal flow increased (+31%, +13%). in the vardenafil c and sildenafil b and c groups there was an increase of portal venous flow by 20-30%, an increase of sinusoidal flow by 12-30%, and a decrease of portal venous resistance by about 25%. there was a trend for reduction of portal venous pressure. conclusions: vardenafil and sildenafil influence portal hemodynamics in the rat. portal venous flow increases by 20-30%, portal venous resistance decreases by >25%. dependent on the dose, portal venous pressure decreases significantly. these data yield further evidence that pde5 inhibitors may be a novel therapeutic option for portal hypertension. groups of liver cirrhosis e. havrilyuk 1 1 lviv national medical university introduction: rupture of esophageal varicose resulting in posthemorrhagic anemia is a common life-threatening complication of liver cirrhosis. but it is not clear, why the other patients, having the same degree of sclerosis and histologic activity index, die from hepatocellular failure or other reasons. aims & methods: 3713 autopsy cases performed in lviv regional hospital in 2004-2007 were analyzed. screening of slides with liver tissue allow to select 580 cases (15,6%) with cirrhosis (complete and incomplete), which are examined in order to evaluate the frequency of lethal portal hypertension complications in the different etiologic groups of liver cirrhosis. results: according to the etiologic factor the following groups of liver cirrhosis were examined: alcoholic disease (49,8%), viral hepatitis (7,8%), nonalcoholic steatohepatitis (14,8%), secondary biliary cirrhosis (2,9%), cardiac sclerosis (0,9%), combined lesions (11,9%) and cryptogenic cirrhosis (11,9%). analysis shows that in 287 cases (49,5%) patients die from cirrhotic complications (hepatocellular failure, jaundice, portal hypertension) and only in 105 cases (18,3%) -from posthemorrhagic anemia caused by rupture of esophageal varicose. in the latter cases correlation between the etiologic types of cirrhosis is almost the same, as in the main group and only alcoholic lesions (60%) and biliary cirrhosis (6,7%) are more frequent. conclusion: analysis shows that development of lethal complications of portal hypertension can not be explained only by etiologic factor. probably additional stimuli are more important for morphogenetical variants of cirrhotic transformation. patients with viral cirrhosis k. mumtaz 1 , s. ahmed 1 , h. ali shah 1 , s. hamid 1 , w. jafri 1 background and aims: increased nitric oxide (no) production is incriminated in the pathogenesis of arterial vasodilation and hyperdynamic circulatory state in non cirrhotic models of portal hypertension (pht). we investigated the relative roles of constitutive nos (enos) and inducible nos (inos) isoforms in the development of rabbit models of endotoxemia induced portal hypertension (eipht) methods: eipht was induced by chronic injection of lipopolysaccharide via an indwelling cannula placed in the gastrosplenic vein of rabbit and maintained for 6 months. the concentration of no, expression of nos (enos and inos) mrna and protein was measured in eipht and sham operated control animals. results: rabbits with eipht compared with controls had raised portal pressure (in mmhg-14.34±1.78 vs 6.30±0.60;p<0.05;1mo; 14.91±0.56 vs 7.04±0.42; p<0.05, 3mo; 19.8±3.10, vs 10.2±4.80;p<0.05), arterial hypotension (in mmhg-65.40±3.2 vs 80.04±1.40, p<0.05, 1mo; 62.90±5.40 vs 76.05±2.60,p<0.05, 3mo; 65.85±2.50 vs 79.1±5.10, p<0.05,6mo), splenomegaly (in g-0.92±0.14 vs 0.60±0.13,1mo; 0.90±0.16 vs 0.62±0.04, 3mo; 0.97±0.12 vs 0.67±0.07, 6mo), normal liver functions and preserved hepatic architecture at 1,3 and 6 mo. serum levels of no2 as well as the no3 were significantly elevated in eipht rabbits as compared to the controls. the expression of enos, at the level of mrna, was significantly increased in eipht rabbits consistent with increased levels of expression of inos as compared to the controls. the enos but not inos protein expression was elevated in eipht than control rabbits. conclusion: vascular dysfunction in the splanchnic circulation during the development of endotoxemia induced portal hypertension is predominantly characterized by enos and partly by inos gene up-regulation. liver cirrhosis contributed to the immunocompromised status by shedding the membranous tnfrii t.n. lin 1 , c.h. chao 1 , i.s. sheen 1 , y.p. ho 1 , w.t. chen 1 , c.j. lin 1 , c.t. yeh 1 , c.y. lin 1 1 liver research unit, linkou medical center, chang gung memorial hospital, chang gung university, taoyuan, taiwan background & aim: patients with decompensated liver cirrhosis (dlc) were regarded as immunocompromised, reflected by high incidence of bacterial infection. paradoxically, the proinflammatory cytokine like tnfincreased significantly in patients with dlc even in the face of this immunocompromised status. on the other hand, regulatory t cell (treg cell) is believed to play an important role in inhibiting immune responses, including innate immune responses like blockade of tnf-effect through soluble tnfrii. here, we studied the role of treg cells and tnfrii in patients with decompensated liver cirrhosis. patients and methods: 33 healthy volunteers and 78 cirrhotic patients were enrolled. the percentage of treg cells were enumerated by flow and serum levels of il-10, tgf-and tnf-by elisa. results: the percentage of treg cells increased significantly in patients with dlc associated with increased serum levels of il-10 and tgf-. in addition, these treg cells were mainly memory type reflected as high cd45ro. furthermore, the tnfrii expression increased significantly on these treg cells of dlc. interestingly, these membranous tnfrii on treg cells could be shed-off. lastly, we found the serum soluble tnfrii concentration increased significantly in patients with dlc when compared with normal volunteers. conclusion: our results demonstrated memory treg cells with high tnfrii expression increased significantly in patients with decompensated liver cirrhosis that could possibly blocked the biological effect of tnf-by shedding membranous tnfrii and contributed to the immunocompromized status of dlc. background: portal pressure measured as hepatic venous pressure gradient (hvpg) correlates with severity of portal hypertension and the development of complications. hvpg measurement is invasive. recently, liver stiffness measurement has been shown to correlate with liver biopsy and helps predict outcome in chronic liver disease patients. this study was conducted with the aim to study the correlation between portal pressure as measured by hvpg and liver stiffness as measured by fibroscan among patients with portal hypertension due to various causes. methods: between august and september 2008, consecutive patients with portal hypertension were included and were subjected to hvpg measurement and fibroscan (echosens, france). results: of the 18 patients with portal hypertension, both hvpg and liver stiffness were measurable in 12[9 (75%) males; mean age 37.5(10.6) years]. the etiological distribution was hbv related cirrhosis in 3 patients, hcv cirrhosis in 3, cryptogenic cirrhosis in 2, alcoholic cirrhosis in 2, hbv and alcoholic cirrhosis in 1and primary extra-hepatic portal vein obstruction in1. the mean hvpg and liver stiffness of this group were 13.9 (5.1) mm hg and 26.6 (16.5) kpa respectively. there was a strong positive correlation between hvpg and liver stiffness [r = 0.708; p = 0.01]. conclusions: non-invasive measurement of liver stiffness correlates well with invasive measurement of portal pressure. liver stiffness measurement could be used as a prognostic indicator to predict the severity of portal hypertension. endpoints were rate of rebleeding and mortality till day 30 after inclusion and to see for any adverse events. results: the bleeding was stopped in all 15 patients (100%). rebleeding till day 30 was observed in 4 (26%) patients (2 each in group a and b). total 2 patients (13%) died (1 each in both groups) due to rebleeding. transfusion needs were higher in group a (4.2±2.8 versus 2.3±2.1, p<.05). serious adverse effects leading to treatment discontinuation were not seen in any patients in both groups. conclusion: prolonging terlipressin treatment did not confer any significant decrease of mortality or bleeding recurrence. however transfusion requirements were significantly decreased in patients receiving prolonged treatment. serious adverse effects leading to treatment discontinuation are rare. poster exhibition -miscellaneous poster session, hall 5b background: it is important to know the prevalence of atp7b gene mutations of different geographical areas to justify the local screening strategies for wilson disease (wd). materials: eleven unrelated lithuanian families, including 13 wd patients were tested. genomic dna was extracted from whole venous blood using a salt precipitation method. firstly, semi-nested pcr technique was used to detect the c.3207c>a (p.h1069q) mutation. patients not homozygous for c.3207c>a (p.h1069q) mutation were further analyzed. the 21 exons of the wd gene were amplified in a thermal cycler. direct sequencing of the amplified pcr products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer. results: total of 13 wd patients (mean age 26.4 years; range 17-40; male/female, 3/10) presented with hepatic disorders and 16 their first degree relatives were studied. some of wd patients in addition to hepatic symptoms have had extrahepatic disorders (haemolytic anaemia 3; fanconi syndrome 1; neurophsychiatric and behavioural disorder 2). twelve of 13 (92.3%) wd patients have had c.3207c>a (p.h1069q) mutation, 6 of them in both chromosomes, 5 were presented as compound heterozygotes with additional c.3472 -82delggtttaaccat, c.3402delc or c.3122g>a (p.r1041q) mutation. for one patient with liver cirrhosis and psychiatric disorder no mutations were found. out of 16 first degree wd relatives 11 (68.7%) were heterozygous for c.3207c>a (p.h1069q) mutation. conclusion: c.3207c>a (p.h1069q) missense mutation is characteristic for lithuanian wd patients. even 92.3% of wd patients with hepatic presentation of the disease are homozygous or compound heterozygote for this mutation. background: diabetic dyslipidemia is a crucial problem of diabetic patients with inadequate control. we investigated the relationship between glutamic-pyruvic transaminase (gpt) and high-density lipoprotein cholesterol (hdl-c) in diabetic patients. methods: with informed consents, we recruited outpatients with diabetes at a hospital in rural area in taiwan in [2004] [2005] [2006] [2007] . anthropometric measures, blood tests and urine screening were examined in diabetic patients. results: overall, there were 1241 diabetic patients aged 19-91 years enrolled in this study and 660 (53.2%) of them had low hdl-c. diabetic patients with the highest quintile of gpt had higher average of body mass index (p<0.0001), diastolic blood pressure (p = 0.003), but lower average of hdl-c (p<0.0001) compared with diabetic patients with the lowest quintile of gpt. the prevalence of obesity (45.7% vs. 24.8%, p<0.0001) and low hdl-c (64.6% vs. 48.3%, p<0.0001) were higher in diabetic patients with highest quintile of gpt than in diabetic patients with lowest quintile of gpt. in the multivariate logistic regression, diabetic patients with highest quintile of gpt had higher odds ratio (or) of low hdl-c compared with diabetic patients with lowest quintile of gpt (or = 1.88, 95% confidence interval [ci] = 1.21-2.92). the corresponding or of low hdl-c in patients aged 70 years and older was 3.30 (95% ci = 1.15-9.47). conclusion: high gpt is one of factors associated with low hdl-c in diabetic patients. the effect of desferrioxamine as supplement to cefotaxime in the treatment of spontaneous bacterial peritonitis na. seda 1 , m. el-hamamsy 2 , r. el-wakil 3 , m. al azizi 4 1 ain shams specialized hospital, 2 faculty of pharmacy,ain shams university, 3 faculty of medicine,ain shams university, 4 faculty of pharmacy ,ain shams university background: oxidative damage lead to cell damage, organ dysfunction and death in sepsis. desferrioxamine (dfx), an antioxidant iron chelators. the aim was to assess the efficacy of desferrioxamine supplemented to cefotaxime in the treatment of spontaneous bacterial peritonitis (sbp) in cirrhotic patients. methods: thirty patients divided into two groups: group i (n=15) with sbp and receiving cefotaxime (1g iv every12 hours) alone and group ii (n=15) with sbp receiving cefotaxime (1g iv every 12 hours) with desferrioxamine (500mg im twice daily).all patient were monitored for seven days, their vital organs were screened and their ascitic fluid was assessed completely including microbiological investigations. results: the concomitant administration of desferrioxamine with cefotaxime significantly at (p<0.001)and(p<0.01) improved the therapeutic outcome and the cure rate after 5 days of treatment as compared to patients using cefotaxime only. conclusions: desferrioxamine can improve the therapeutic outcome by preventing iron-induced organ damage and inhibiting bacterial growth. oligella ureolytica is a gram-negative, nonfermenting rod that is infrequently recovered from clinical specimens and is most commonly isolated from the urine of patients with chronic indwelling urinary catheters or other urinary drainage systems. bacteremia due to this organism is an extraordinary finding. we describe here a case of oligella ureolytica being detected in the blood of a patient with decompensated cirrhosis. a 51-year-old male man was admitted to hospital with 9-month duration of debility, poor appetite and abdominal distension. decompensated cirrhosis was diagnosed based on clinical findings such as hepatic face, ascites, edema of lower limbs and icteric sclera. laboratory results showed positive serum anti-hcv and high serum hcv rna level. the patient received a therapeutic regimen of pegylated interferon alpha 2a plus ribavirin after being admitted to hospital. during hospital stay, a fever of 2-day duration with shivering occurred to the patient. three blood cultures were drawn, which all grew oligella ureolytica in pure culture. the organism was identified by the viteck 2 compact (biomerieux, france). additional tests for identification resulted positive for nitrate reduction and urea hydrolysis, strongly positive for phenylalaninedeaminase activity and showed no growth at 42.7c. tests for nitrite reduction and motility resulted negative. the organism was resistant to amikacin, cefoperazone, levofloxacin, piperacillin/tazobactam, trimethoprimsulfamethoxazole, aztreonam, cefotaxime, piperacillin and was susceptible to gentamicin, imipenem, meropenem, and netilmicin. a 14-day of combined therapeutic regimen with cefminox and isepamicin was administered to the patient. within 2 days, the patient became afebrile. background: endoscopic ultrasound (eus) is often performed in patients with unexplained liver tests to assess the gallbladder, bile ducts and pancreas. an unremarkable eus exam and negative hepatology workup often leads to a liver biopsy. eus may provide histopathologic evaluation of the liver in these cases under direct, real-time visualization. aim: to assess the feasibility and efficacy of eus guided core biopsy of the liver in a porcine model. methods: female pigs were used and live procedures were performed under general anesthesia. a linear echoendoscope was used and the liver identified endosonographically. transgastric core biopsies of the liver were obtained with a 19 gauge quick-core ultrasound biopsy needle (wilson-cook) and sent for histopathologic evaluation. live animals were euthanized at the end of the procedure and necropsy performed. results: core biopsies of the liver biopsy were obtained in 4 animals (1 cadaver and 3 live anesthetized). a total of thirteen needle passes were made (mean 3.25; range 2 -4 needle passes per animal) and a visible core of tissue obtained. the maximum length of liver tissue obtained was 10 mm and considered adequate for assessment as more than one such specimen could be obtained. microscopic evaluation confirmed liver tissue. no complications were noted. necropsy did not show any evidence of bleeding, perforation or damage to surrounding structures. conclusion: eus-guided liver biopsy is feasible and can be performed at the time of routine echoendoscopic exam in select patients undergoing eus examination for abnormal liver tests. background: as the common indexes, alt, ast and plt play an important role in disease diagnosis, treatment and prognosis. many researchers suggested that there was inflammatory changes and fibrosis in chronic hepatitis b and c patients whose alt level was persistently normal. a large sample investigation showed that the serum level of alt in healthy persons is lower than the normal reference value. this study re-evaluated the normal serum level of alt, ast and plt. methods: 3815 people were enrolled in the study between sep. and oct. 2007. the platelet count and serum alt and ast levels were measured. frequencies, one-sample kolmogorov-smirnov test and nonparametric tests were used to analyze the difference between age groups, male and female, glucose groups, cholesterol groups and triglyceride groups. result: in the five groups, there is significant difference in alt and ast levels between male and female. in group 1, the alt and ast levels showed a significant difference between different age groups, between different glucose groups and triglycide groups. in the three groups the plt level is significantly different between male and female, and the serum level in male is higher than female. there is significant difference between different age groups conclusion: the serum levels of alt, ast and plt are all significantly different between male and femal. there is significant difference between different genders and age groups for plt. the serum level of plt is higher than the reference value. background/aims: myeloproliferative disorders (mpd) (like polycythemia vera, essential thrombocythemia and primary myelofibrosis) are responsible for 50% cases of hepatic venous thrombosis (hvt) and 35% of portal venous thrombosis (pvt) in western series. latent form of mpd lacks the characteristic blood picture and may be classified as idiopathic thrombotic disorder. a point mutation at val617phe of janus kinase 2 tyrosine kinase gene (jak2 v617f mutation) occurs in high proportion of the patients with mpd. this non-invasive test with high positive predictive value is now considered to be essential for diagnosis of various mpd. this test may be useful in diagnosing latent form of mpd in splanchnic venous thrombosis methods: 232 patients with confirmed pyogenic liver abscesses admitted from 1995 to 2007 in our institution were included. there were 145 men and 87 women ranging in age from 19 to 91 years. the medical records were reviewed for clinical, laboratory and radiographic characteristics. results: among 232 patients, 81 (34.9%) experienced at least one complication. there were 67 pulmonary (pleural effusion, pneumonia, empyema) complications, 16 septic shock, 12 acute renal failure, 2 abscess rupture, 2 pseudomembranous colitis, and 2 pericardial effusion. the predictive factors for its complications were: systemic inflammatory response syndrome (sirs, 2 factors), thrombocytopenia ( 80,000/ml), hypoalbuminemia ( 3.0g/dl), elevated ast or alt (>200 iu/l), hyperbilirubinemia ( > 2.0 mg/dl), k. pneumonia, air within abscess cavity (p<0.05). conclusions: the incidence of complications in the pyogenic liver abscess was 34.9%. the various predictive factors of complication should be monitored carefully. further large scaled study should be warranted. background/aims: hepatic iron deposition is a common feature in chronic hepatitis c (ch-c), however, whether it could enhance the progression of fibrosis or not is controversial. the aim of this study was to evaluate the status and significance of hepatic iron deposition in the korean patients with chronic hepatitis c. methods: untreated, 78 ch-c patients who underwent liver biopsy were included. the hepatic iron was assessed by scheuer's scoring system, and activity, fibrosis, and steatosis were scored by a pathologist in a blind manner to the clinical features. clinical and laboratory data including serum iron indices, virological, biochemical results were analyzed to search for significant factors associated with hepatic iron deposition. results: hepatic iron staining was positive in 26(33%). among 26 patients with hepatic iron deposition, serum levels of ferritin (p=0.005) and -fetoprotein (p=0.002), and body mass index(bmi) (p=0.039) were significantly elevated. there was no significant association between the degree of hepatic iron deposition and fibrosis stage (p=0.321), although elevated levels of serum hyaluronic acid (p=0.049), -glutamyl transpeptidase (p=0.028), and prothrombin time (p=0.012) were associated with advanced fibrosis. conclusions: hepatic iron deposition in asian-pacific ch-c patients seemed to be neither frequent nor related to hepatic fibrosis, but related to obesity. therefore, phlebotomy might not commonly applicable to this area. further studies on the pathogenic role of iron in ch-c in asian-pacific countries are warranted. a late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture, necrosis of hepatocytes and the formation of regenerative nodules contributes to cirrhosis. limitations like organ donors shortage, high cost, absence of proliferation in cultured hepatocytes, inherent risks of infection, rejection in xenogenic cells and other socio-economical complications emerges advanced regenerative human hepatic stem cells(hhpscs) transplantation. hhpscs are located in the ductal plates in fetal and canals of hering in adult livers [schmelzer et al.(2006) ]. hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [schmelzere etal(2006) ].hhpscs express cd326(epcam)marker. scjelzer etal demonstrated that during embryogenesis 90% of the epcam positive cells had hepatoblast phenotype. in animal study, on transplantation of freshly isolated hhpscs in scid mice results in mature liver tissue expressing human-specific proteins. recently, we(aleem etal 2008)have shown clinical improvement in study in patients with crigler-najjar syndrome, biliary atresia using hhpscs infusion. in the present study we transplanted hepatic progenitors to five subjects of end stage liver cirrhosis with meld score >30. hhpscs were sorted using macs with cd326 antibody microbeads and infused through hepatic artery via femoral artery catheterization, a safe procedure provided portal pressure to monitor cell infusion route in order to prevent vascular thrombosis. all the patients showed improvement clinical and functional biochemical parameters after first month of cell infusion. ascites was decreased and changed encephalopathy grade into normal level was observed. meld score system falling to normal level from >30 to <22 after infusion. 1 the aga khan university patients. the apri of 1.5 in combination with a cut-off ha of 300 ng/ml can best detect patients with moderate to severe fibrosis (stages 2-4). it has a ppv of 93.7%. also, for patients without moderate to severe fibrosis, the test is hardly ever positive (specificity of 98.9%). but the apri of 1.5 in combination with different ha as cut-off points is not possible to detect patients with no or mild fibrosis. objectives: terlipressin is used in esophageal variceal bleed (evb) along with endoscopic band ligation (ebl) for 3days (uc). due to its high cost, it was stopped <3days (sc) who could not afford & were stable after achieving hemostasis with ebl. we retrospectively assessed the efficacy of sc vs uc of terlipressin for control of evb and length of stay. conclusion: the apri of 1.5 in combination with ha 300 ng/ml as cut-off points to predict patients with moderate to severe fibrosis (stages 2-4) is an easy and accurate method. methods: patients with evb who had achieved hemostatsis with ebl from jan 2004-dec 2005 were included. all were managed on standard protocol on hospital variceal bleeding pathway. the course of terlipressin as sc or uc was based on patient's inability to afford the cost of hospitalization and terlipressin. the efficacy of terlipressin in the control of evb was defined based on baveno iii criteria. results: total of 117 patients were admitted during the study period. out of them, 66 received uc & 51 sc of terlipressin. the base line characteristics were comparable except younger age in sc. there were 2 re-bleed (3%) in uc and 1 (2%) in sc terlipressin group. the length of stay was shorter in sc group. (2.47±0.57 vs 6.15±2.92 days). conclusions: sc seems as effective as uc terlipressin in the control of evb after initial control of hemostatsis with ebl and may reduces the length of hospital stay. rcts are needed to assess this as all stable patients may not need to continue terlipressin for 72 hours. background/aims: to analyze the relationship between conventional laboratory results and death risk in patients with esophageal varices bleeding due to cirrhosis (cevb), and establish a simple model for timely predicting death rsik of the patients. outcome of patients with gastro-oesophageal bleeding in a tertiary center j. wat, w.h. li, m.t. cheung methods: the medical documents of cevb patients were reviewed retrospectively and the data were collected. univariate and multivariate logistic regressions were performed, in which the discharged results (survival or death) as dependent variable and the results of liver function, kidney function, serum electrolytes and blood cell analysis as independent variables. the multivariate regression equation was as the model for the prediction of patient outcome and its predictive performance was evaluated. objectives: to determine the rebleeding rate, mortality and long term survival in cirrhotic patients presented with acute gastro-oesophageal variceal bleeding. method: this is a retrospective review of adult patients who were admitted to our hospital with the diagnosis of acute gastro-oesophageal variceal bleeding for the first time regardless of their underlying causes for cirrhosis. the study period was from january 2000-october 2008. data were collected from our hospital computer system and records. results: in univariate regression, the significant positive variables for death outcome were dbil, akp, k, wbc and plt, and the significant negative variables were tp, ap, a/g, na, cland ca 2+ . the variables entered the multivariate regression are alt, tbil, dbil, gp, a/g, cr, na + , cl -, ca 2+ , wbc, hb, plt. the sensitivity, specificity and accuracy of the regression model for predicting death of cevb patients were 97.1%, 95.1% and 95.8%. results: a total of 172 patients were included in this study, with 122 male and 55 female. their mean age was 61.9. the initial failure rate in endoscopic haemostasis was 15.1%. the 5-day and 6-week mortality rates were 11.8% and 21.9 %, respectively. poor child's grading, multiple columns of oesophageal varices, high grade of varices, failed initial endoscopic haemostasis, presence of inoperable hcc, low platelet count on admission, and short duration from index bleed to rebleed were factors associating with increased risk of 6-week mortality (p < 0.05). mean duration from index bleed to first rebleed was 15.1 months. poor child's grading and presence of inoperable hcc were associated with both early or multiple rebleed (p <0.05). overall, 37.2% of our patients developed rebleed before their variceal eradication. 5-year survival in patients with child's a, b and c were 65%, 22%, and 10%, respectively (log rank test p 0.000). conclusions: the liver function, kidney function, serum electrolytes and blood cell analysis are generally independent factors for cevb patient death risk, especially dbil, a/g and ca 2+ . the established model shows a excellent predictive performance. an imbalance in plasma amino acids of advaced cirrhotic patients impairs the maturation of dendritic cells via mtor/s6k signaling pathway e. kakazu 1 , y. ueno 1 , y. kondo 1 , k. fukushima 1 , m. shina 1 , j. inoue 1 , k. tamai 1 , m. ninomiya 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university hospital conclusion: although endoscopic haemostasis is an effective treatment modality; rebleeding is still commonly seen among patients with poor child's grading and inoperable hcc. this will result in significant bleeding-related death and poor overall survival. further advancement in treatment strategies for this group of patients are required to improve their outcome and prognosis. background: we have demonstrated that extracellular branched-chain amino acids (bcaas), especially valine, regulate the maturation and function of monocyte-derived dendritic cells (j immunol. 179: 2007) . however, it is not clear whether an imbalance in plasma amino acids of advaced cirrhotic patients influence the function of dendritic cells (dcs). methods: we used human pbmcs and cd1c+dcs in this study. we made two mediums: a serum free culture medium consistent with the average concentration of the plasma amino acids from a healthy volunteer (n=100) was defined as the healthy control medium (hcm); whereas that from advanced cirrhotic patients (n=50) was defined as the advanced cirrhotic active hepatitis b replication is defined as hbeag + or hbv dna > fibrosis was scored according to the metavir system. alt levels were characterized as being normal, < 2 x normal, and > 2 x normal. conclusions: pe494 frequency of portal hypertensive gastropathy and its non-invasive predictors in patients with viral methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent for screening egd for varices in last 2 years was reviewed. phg was defined endoscopically by using mccormack classification. noninvasive markers such as spleen/platelets ratio, meld score and child score of all the patients who underwent for egd were recorded. results: out of 360 patients 226(62.77%) were males. out of 300(83.3%) patients who had phg, 136(45.3%), 93 (31%) and 71(23.7%) had mild, intermediate and severe phg respectively. higher proportion of esophageal varices (89.7%) was present among those who have phg (p<0.001).196(65.3%) with phg has child score of 7. meld score >15 and 15 were seen in 32.3% and 67.7% of patients with phg, respectively. platelet/spleen ratio was 916.08± 400 in patients with phg as compared to 1476 methods: retrospective analysis of cirrhotics undergoing surveillance endoscopy was undertaken assessing for oesophageal varices. clinical, biochemical and radiological indices were analysed. results: 81 cirrhotics underwent surveillance endoscopy during the study. childs pugh scoring (cps) to assess prognosis of liver disease showed cps a(58%), cps b(27%) and cps c(14%). 68% were male albumin (42g/l vs 35g/l significant factors on multivariate analysis were albumin (p=0.003) and platelet count (p=0.026) conclusion: in our cohort there were significant biechemical and radiological differences in differentiating patients with large varices (grade 2-4) on surveillance endoscopy aims and objectives: to study the frequency of ev in patients with cirrhosis due to viral etiology and its correlation with different non-invasive markers. methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent screening egd for varices in last 2 years was reviewed. ev were divided in two grades (small and large) as proposed in consensus development workshop. noninvasive markers such as spleen/platelets ratio, meld and child turcotte pugh (ctp) scores of all patients were recorded. results: out of 360 patients, 226 (62.77%) were males on multivariate analysis ctp score of 7 (or 2.06, p<0.01), meld score >15 (or1.63, p<0.05) and platelet/spleen ratio 900(or 2.35, p=0.005) were found as significant predictors of large ev. conclusion: the frequency of ev is high in viral cirrhosis patients on screening egd. meld score>15, ctp score 7 and spleen/platelets ratio 900 can be used as non-invasive predictors of large ev. pe519 treatment outcome and prognostic factors of spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with hepatitis b virus-related thirty-seven (28.5%) patients had sbp while 91 (71.5%) had cnna. except the higher proportion of renal failure at admission in patients with sbp than cnna (32.4% vs. 7.5%, p=0.001), no significant difference in the clinical and laboratory data related to liver and renal function was observed. overall mortality during hospitalization was higher in patient with sbp than that of cnna evl was repeated every 2-weeks till varicial eradication.bb dose was titrated to achieve a resting heart-rate of 55bpm or a maximum dose 320mg/d or when side-effects began to appear.primary end-points were rebleed and death.secondary end-points were complications as a result of evlor beta-blocker,variceal recurrence afterevl,and decrease in variceal grade inbb limb. results: 71 patients (median age 14[range2-68] yrs, males65%) were included (evl arm[n=37]and bb arm[n=34]. median grade of varices was iii(range ii to iv) nitric oxide synthase isoforms play distinct roles in the evolution of hyperdynamic state in endotoxemia induced portal hypertension m.r. rizvi 1 , m. shahid institute of genomics and integrative biology, mall road, delhi 110 007, 4 department of physiology a seconderc was performed after two or more years to assess the progression of the disease. results: 79 ehpvo patients(median age 20[range 5-62]yr, males 67%) were studied.history of present or previous jaundice was present in 24%,ascites 18% and pain 9%. on erc,92% had portal biliopathy.the type of bile duct involvement was categorized as: type 3 b(51%) and type 1(42%).the pattern of involvement included indentations(49%) and dilatation and strictures(36%).20%of the patients had bile duct stone and 9% had history of cholangitis.the mdian bilirubin was1.1(range0.3-15.9) mg/dl and median serum alkaline phosphatase 171(range 42-617) iu/l.all patients were treated endoscopically by endoscopic stone extraction, dilations with/without stenting. 24(30%) patients underwent second erc after a median interval of 21(range 1-111)months.the type of involvement progressed,75% patients developed type-3 involvement compared to54% at the baseline (p=ns).indentations progressed to develop strictures, from46% to71%.the frequency of new bile duct stones per year was 4%(p= ns). conclusions: portal biliopathy is very common in ehpvo, often remaining asymptomatic.however,it is slowly progressive leading to development of biliary strictures objective: to investigate the mechanisms of angiotonin ii (angii)-induced ca (2+)-independent pathways mediated by rho kinase in hepatic stellate cells (hscs) various vasoactive drugs that reduce portal pressure are used in treatment of esophageal variceal bleeding alongwith endoscopic treatment. terlipressin use decreases both, recurrent bleeding and mortality. it is given usually for 3-5 days, nevertheless there is very little data comparing different time periods. our aim was to compare the efficacy and safety of 5-days versus 10 days of terlipressin treatment in bleeding esophageal varices. methods: out of 15 patients who presented with variceal bleeding, 8 were randomized to receive terlipressin 2 mg 8 hrly, i.v. daily for first 5 days and placebo for next 5 days (group a) and 7 to receive terlipressin 2 mg 8 hrly, i.v. daily for 10 days (group b). both groups were both age and sex matched. (svt) {consisting of hvt and pvt}. there is no such data from india a comparative study of male vs background: mucosal lesions are frequently observed.gender differences are expected due to food habits, nature of job, mobility related to work, consumption of alcohol, tobacco etc. material and methods: procedure done in 1395 m & 19(3.4%) f& duo 48(5.73%) m & 73%)f.varices (48) eso varices 31 (4.3%) m &10(1.8%) f & gastric varices 1 (.11%) m & 1(.11%) f .growth (17)eso growth 5 (.59%) m &1(.17%) f & stomach growth 9(1.07%)m & 1(.17%) f & laryngeal 1(.11%)m. eso monoliasis (7) 3(.35%) m & 4(.71%) f.polyp (8) eso polyp 4(.47%) m & 1(.17%) f & gastric polyp 1(.11%)m & 1(.17%) f & moniliasis and ulcers are more common in female lee 1 1 department of internal medicine, gyeongsang national university school of medicine background/aims: although the pyogenic liver abscess is a common intraabdominal inflammatory disease, this complications are not rare. however, reports dealing with this complications are not good enough and results are often variable. the aim of this study was to identify the predictive factors of complication in the pyogenic liver abscess. pe538 effects of saikosaponinsd (ssd) on expression of c-myc and pcna in experimental hepatocarcinoma of all rats were killed in the 18th week, then general conditions of rats were recorded, the serum alt akp ggt afu was detected and pathological examination was made. the expression of pcna and c-myc were tested by immunohistochemistry. results: he staining showed that rats were induced to hepatocellular carcinoma,the results of liver function in the 18th week displayed that alt akp ggt and afu of all groups were increased than that of normal control group conclusion: ssd can inhibit development of hepatoma induced by den, possibly by down-regulating the expression of pcna and c-myc protein lethal endotoxic shock was induced by single endotoxin (e.coli) 6mg/kg injection into abdominal cavity. ppc in 5% gs was given via tail vein at 1ml/100g (232.5mg/kg) 24h and 6h before endotoxin. rats' behavior and 24h survival were recorded, venous blood taken for ast and alt, liver preserved for he staining and liver/body weight ratio l/b and liver wet/dry weight ratio (w/d) calculated. intercellular adhesion molecule-1 (icam-1) expression in liver tissue was observed with 2-step immunohistochemistry assay. results: rats of ns and ppc group demonstrated similar normal activity, histology and other characters (p>0.05), while lps group showed sag and less water-intak and severe inflammation in liver including inflammatory cell accumulation, parenchymal cells edema and tissue exudation. p+l group turned tired but could drink water the role of insulin resistance, adipokine and cytokine pro-inflammatory in non-alcoholic fatty liver disease n. ratnasari 1,2 , s. anam 2 , p. bayupurnama 1,2 , s. maduseno 1,2 , s. nurdjanah 1, 2 1 dr sardjito general hospital yogyakarta indonesia, 2 background: non-alcoholic fatty liver disease (nafld) is a benign disease during 5-10 years period, with 67%-59% survival. nafld can progress to fibrosis, cirrhotic and cancer of the liver. the etiopathogenesis of nafld is still unknown, however genetic and environment factors are predicted. objective: to know the role of insulin resistance, adipokine and cytokine pro-inflammatory on nafld. methods: the cross sectional study was performed on general check-up population at dr. sardjito general hospital yogyakarta, indonesia. the study was begun from january 2007 until november 2007 at internal medicine outpatient department. inclusion criteria: adult, alcohol consumtion 20g/day, metabolic syndrome patients, and healthy subjects. exclusion criteria: the diseases with increasing liver enzymes (hbv, hcv, ischemic hepatitis, congestive liver), a "bright liver" on ultrasound examination (malnutrition, rapid weight decreased, post gut surgery on obesity patients, and drug induced). based on liver ultrasound subjects were devided into steatosis group and non-steatosis group. data were analyzed by t-test and non-parametrical test. results: 101 subjects that were enrolled the study 61 steatosis (60.4%) and 40 non-steatosis (39.6%) and the subjects who completed cytokine and adipokine examination were 48 steatosis and 30 non-steatosis. there were significantly different on homa -ir and adiponectin level in steatosis group (homa-ir 3.367±4.901 vs. 1.430±1.889, p=0.019; adiponectin 4.049±1.929 vs. 7.034±3.777, p=0.000). there were not significantly different on tnf-, il-6, leptin and visfatin level (p>0.05). conlusions: there were significantly different on homa-ir and adiponectin level in nafld patients compared non-nafld patients. background: to optimize management of nonalcoholic fatty liver disease (nafld), a simple screening tool is necessary. in this study, we aimed to devise a simple index that reflects the presence of nafld in the korean population. methods: a cross-sectional study was conducted on 10,724 health check-up subjects at a healthcare center (5,362 cases with nafld versus age-and sex-matched controls). study subjects were randomly assigned to a derivation cohort or a validation cohort. an index reflecting the presence of nafld was derived in the derivation cohort and validated in the validation cohort. results: multivariable analysis indicated that body-mass index (bmi), serum alanine aminotransferase (alt) to serum aspartate aminotransferase (ast) ratio, sex, and the presence of diabetes mellitus were independent predictors of nafld. using these variables, a formula was derived using a linear regression model: nafld index (nafldi) = 8×alt/ast ratio +bmi (+3, if female; +2, if diabetes mellitus).nafldi had an area under receiver-operating curve of 0.812 (95% confidence interval, 0.801-0.824). at a value <31.0, nafldi ruled out nafld with a sensitivity of 89.0% and a negative likelihood ratio of 0.22, and at a value >36.0, nafldi detected nafld with a specificity of 91.2% and a positive likelihood ratio of 5.42. in the validation cohort, the predictive power of nafldi was maintained at similar levels.aim: since nash could progress to liver cirrhosis and hepatocellular carcinoma, it is important to correctly diagnose between nash and simple steatosis (ss). the aim of this study was to determine the prevalence of nash among nafld patients and to clarify differences in clinical features between nash and ss. subjects and methods: thirty-one patients with nafld showing abnormalities in serum transaminase (ast and /or alt >40 iu/l) were enrolled (sex: male 11, female 20; mean age: 49.5 yrs, mean body mass index: 29.3), after obtaining informed consent. differential diagnosis between nash and ss was performed histologically according to the matteoni classification and clinical features were compared. results: among the patients with nafld, 81% and 19% were diagnosed with ss and nash, respectively. no significant differences in the sex, mean age and bmi were seen between nash and ss groups. the levels of ast, alt, homeostasis model assessment-insulin resistance (homa-ir) and hyaluronic acid were significantly elevated in nash patients compared to ss patients. no significant differences in serum levels of adiponectin, as well as the rates of occurrence of diabetes, hypertension and hyperlipidemia were observed between the two groups. conclusion: the prevalence of nash in nafld patients was about 20%. nash patients showed higher levels of serum transaminase, homa-ir and hyaluronic acid, compared to ss patients. a large-scale biochemical study is required to accurately diagnose nash patients and confirm these results. conclusion: nafldi was a simple, efficient screening tool for nafld that could be utilized for selecting individuals for liver ultrasonography and for determining the need for lifestyle modifications.pe432 aim: this study was conducted to evaluate the hepatoprotective effects of the centella asiatica extract in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury in rats. methods: sprague dawley rats were treated with alcohol extract of centella asiatica orally in two doses (20 and 40 mg/kg/day) for 3 months along with intraperitoneal injection of mptp (1 ml/kg). biochemical parameters such as serum total protein, albumin and marker enzymes were estimated. histopathological studies of liver were also carried out to confirm the biochemical changes.results & discussion: mptp -induced hepatotoxic effects were evident by a significant (p < 0.05) increase in the serum marker enzymes and a decrease in the total serum protein and albumin. administration of extract of centella asiatica effectively inhibited these changes in a dose-dependent manner; maximum effect was with 40 mg/kg. histopathological examination of liver tissue corroborated well with the biochemical changes. hepatic steatosis, hydropic degeneration and necrosis were observed in mptp-treated group, while there was a significant reduction in these changes in the treatment group. conclusion: centella asiatica extract exhibited hepatoprotective action against mptp induced liver injury. further optimization of tuberculosis chemotherapy requires a comprehensive evaluation of the effects of antitubercular drugs on metabolic processes in organism.wistar albino male rats, body weight (b.w.) of 160-200 g, were divided into three groups: group i received pyrazinamide per os at a dose of 1000 mg/kg b.w./day, whereas group ii received a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; the control group was composed of intact animals. the contents of free amino acids were determined using an amino acid analyzer -881 (czech republic). the study of the effects of pyrazinamide administered in different doses on the liver contents of free amino acids showed the largest number of changes at a dose of 1000 mg/kg b.w./day. the content of free amino acids at the level of 16 amino acids and total sum of amino acids significantly differed from controls. part of these changes could be regarded as compensatory answer of organism to this drug action. further pyrazinamide dose increasing caused exhaustion of liver adaptive possibilities. the study of the influence of pyrazinamide on liver contents of free amino acids allows to fully estimate the effects of this substance on metabolic processes in this organ. moreover, the effect of pyrazinamide on the majority of free amino acids in the liver is dose-dependent. background: taiwan is an endemic area of hbv and hcv infection, chemotherapy for lymphoma patients who has been hbv infection, may induce serious clinical sequela due to reactivation of hbv. this study want to clarify the difference of the chemotherapy induced hepatitis between hbv and hcv carrier in lymphoma patients. methods: from july, 2003 to july 2006, 537 non-hepatocellular carcinoma patients were enrolled, 49 (12.5%) cases were lymphoma, in these lymphoma patients, 24(48.9%) cases have been hbv infected, and 22 (44.9%) cases have no hbv or hcv infected. 3 (0.6%) cases have been infected with hcv. all patients received chemotherapy with the regimen of chop or r-chop. liver function , viral markers, hbv dna, hcv rna were checked before and after chemotherapy. results: hepatitis happened in 16 (32.7%) lymphoma patients, 11 (45.8%) cases were in hbv infected patients, 3 (12%) cases were in non-hbv infected patients, 2 cases of hcv infected patients suffered from hepatitis. hbv infected hepatitis patients hbv dna elevated more than 2 log as before chemotherapy. non of the hcv infected patient has elevated of hcv rna after chemotherapy. the mortality rate in hbv infected patient is 33%. no mortality in hcv infected patients after chemotherapy. conclusions: 1.high rate of hbv reactivation and mortality in chemotherapeutic lymphoma patients who has been hbv infected. screening of hbv viral markers among candidates for cancer chemotherapy is mandatory, especially in lymphoma patients. large number and prospect study for chemotherapy induced hepatitis in hcv carrier are needed. background: excessive drinking leads to social, psychological, physical and other problems. this study investigated the epidemiology of ald and analyses the associated risk factors. methods: from 6,043 residents 3,815 blood samples were collected. alcohol consumption and the impact of alcohol on liver function, blood lipids, blood pressure and bmi and mcv have been evaluated. results: the drinking rate and average daily alcohol intake was 35.0% and 36.97±48.76g respectively. the total alcohol intake was 297.90±506.52kg and the average drinking age was 19.21±11.34 years. the average -gt, ast, alt, mcv, chol, tg, ldl-c, hdl-c and bp increased gradually with increase in alcohol intake. the population ald prevalence was 3.98%. the prevalence of ald among the drinking population and the alcoholic population was 11.76% and 44.17% respectively. conclusion: chol, -gt, ast, alt, and mcv were highly correlated with daily alcohol intake which closely related to the occurrence of ald. n. tanaka 1 , w. okiyama 1 , t. aoyama 1 background: alcoholic liver disease (ald) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. polyenephosphatidylcholine (ppc), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons. however, its precise mechanism remains uncertain. we examined the effects of ppc on ald using peroxisome proliferator-activated receptor (ppara)-null mice treated with an ethanol-containing diet, which showed pathological features similar to human ald. methods: male ppara-null mice were pair-fed a lieber-decarli control or 4% ethanol-containing diet with or without ppc at a clinically comparable dose (30 mg/kg/day) for 6 months. o. parkash 1 , a. almas 1 , s.h. ali shah 1 , w. jafri 1 , s. hamid 1 , j. akhtar 1 1 aga khan university hospital karachi 1.hdv-hbv co-infection presents mostly as moderate to advanced liver disease. background: liver injury due to dengue infection is not uncommon. acute liver injury is a severe complicating factor in dengue, predisposing to life-threatening hemorrhage, dic and encephalopathy. results: there were no significant differences of demographic features and laboratory parameters such as peak serum alt, total bilirubin and creatinine between 2 groups. however, peak ast was higher in superinfected group than control group (median: 2,000 iu/l vs 731 iu/l, p=0.035,). additionally, the peak serum albumin levels, prothrombin time and platelet counts were lower in superinfected group than control group (median: 3.0 mg/dl vs 3.3 mg/dl, p=0.02, 51.8 % vs 87.2 %, p=0.027 and 103 x 10 3 /mm 3 vs 165 x 10 3 /mm 3 , p<0.001, respectively). of superinfected group, 9 patients were followed over 6 months after resolution of aha. interestingly, serum hbv-dna levels decreased significantly over 3 months following resolution of aha, then rebounded subsequently (median: -1.89, -1.85, -0.38, 0.58 and 1.10 log10 copies/ml at 1, 3, 6, 12 and 24 months, respectively).methods: the overlapping fragments of hev isolate swgx40 were amplified with reverse-transcription nested polymerase chain reaction (rt-npcr) and the 5' and 3' ends of viral genome were amplified with rapid amplification of cdna ends (race). the pcr products were cloned and sequenced. the phylogenetic analysis of swgx40 was performed.result: the genome of swgx40 consisted of 7,233 nucleotides, excluding the poly (a) tail of 36 residues. the genome contained three open-reading frames (orfs), orf-1 encoding 1705 amino acids, orf-2 encoding 674 amino acids and orf-3 encoding 114 amino acids. the full-length genomic sequencing showed that swgx40 strain shared similarity with all known hev genotype 1, 2 and 3 isolates by 73.4% to 76.5%, and with an identity of 83.1% to 91.2% among genotype 4 hev isolates, and a high nucleotide identity as 94% with chinese guangxi human strain lz-105.conclusions: acute hav super-infection may suppress hbv-dna replication in chronic hbv carriers and chronic hepatitis b, although the suppressive effect did not seem to sustain longer than 3 months. conclusion: the swine hev strain swgx40 was phylogenetically close to the human hev strain lz-105, both from the same region in south china. therefore it was concluded that hev sub-genotype 4b might have existed in south china at least for 6 years and now it was prevalent both in local human and swine, which also strongly supported the zoonosis hypothesis of hepatitis e. associated with splenic volume were damping index (r=0.213, p=0.022) and blood flow of portal vein (r=-0.314, p=0.001). conclusions: the splenomegaly in portal hypertension was more frequent in non-alcoholic groups, and associated with a damping index and a blood flow of portal vein. the measurement of blood flow of portal vein, damping index, and splenic volume by a doppler sonography can be helpful to predict the varices.z.q. zhang 1 , j. cao 1 , w. lu 1 , l.g. shi 1 1 objective: to appraise the clinical efficacy of simple non-invasive models of ast-to-alt ratio (aar), ast-to-platelet ratio index (apri), spleen-to-platelet ratio index (spri), age-platelet index (api), age-spleen-to-platelet ratio index (aspri) for predicting hepatitis b associated cirrhosis. methods: 138 patients and 32 patients were diagnosed pathologically as non-cirrhosis and cirrhosis, respectively. the simple non-invasive models were calculated as described originally. spss 13.0 was used for statistical analyses.background: to reveal the microrna (mirna) expression profile of the hepatic fibrosis inducing cells, rat hepatic stellate cells (hscs), during in vitro activation. results: the areas under roc curve of aar, apri, spri, api, aspri for predicting the cirrhosis were 0.81, 0.62, 0.71, 0.73, 0.74, respectively which were larger than those under the diagnosis reference line (p 0.000, 0.033, 0.000, 0.000, 0.000, respectively).methods: the hscs were isolated from male sd rats by in situ perfusion and density-gradient centrifugation. the quiescent and activated hscs, which were harvested at day 2 and 14, respectively, were then subjected to immunocytochemical staining (desmin and -sma), oil red o staining and quantitative rt-pcr (desmin, -sma, albumin, cd31, cd68 and cytokeratin-19). after extraction and labeling, the hy3-labeled cellular rna samples and hy5-labeled reference pool rna samples were mixed pair-wise and hybridized to the lna mercury microarray. differentially expressed mirnas were filtered and randomly verified by stem-loop rt followed by quantitative pcr.conclusion: all of the simple non-invasive models of aar, apri, spri, api, aspri can be used for predicting hepatitis b associated cirrhosis; and aar has the most practical efficacy for predicting hepatitis b associated cirrhosis. results: both the purity and the total activation of hscs were validated. global analysis of the mirna expression profile based on quiescent and activated hscs demonstrated 21 differentially expressed mirnas. among these, 12 mirnas were up-regulated more than 2-fold in activated hscs as compared to that in quiescent hscs, while 9 mirnas were less than the threshold level (0.5-fold) during the hsc activation. furthermore, the expression of mir-16, 15b, 122, 138, 143 and 140 had been proved. background/aims: only limited patients with chronic hepatitis b virus (hbv) infection will develop liver cirrhosis, and no effective methods to precisely predict ones who will develop cirrhosis. we try to establish a model to predict the patients with the risk of cirrhosis development basing on a clinical epidemiological factor survey. z.q. zhang 1 , w.y. bao 1 , w. lu 1 , l.g. shi 1 methods: cirrhosis patients with hbv markers (case group) and asymptomatic hbsag carriers (control group) were recruited and inquired by researchers with a specific designed questionnaire including 98 items. a multivariate logistic regression analysis were conducted to establish a predictive model, in which two third patients selected randomly as model sample and another 1/3 patients as validating sample, key factors screened out as variables. the predictive performance of the model was evaluated. objective: to explore the practical significance of the peripheral blood corpuscle counts for prediction of hepatitis b associated cirrhosis. methods: 122 and 31 male patients with chronic hepatitis b were pathologically diagnosed as non-cirrhosis and cirrhosis. peripheral blood corpuscle counts were measured by coulter ac•t diff hematology analyzer. results: red blood cell (rbc), platelet (plt), neutrophil (n) counts in cirrhosis were significantly lower than those in non-cirrhosis; and lymphocyte, mid-cell counts were similar to those in non-cirrhosis. the areas under the roc curves of rbc, plt, n counts for prediction of cirrhosis were 0.24, 0.32, 0.34 respectively; according the optimal cut-off determined by the roc curves, the sensitivity, specificity, positive predictive value, negative predictive value, accuracy of rbc, plt, n counts for prediction of cirrhosis were 0.55-0. method: pbmcs of 8 active chb patients under pyg-interferon treatment were analyzed for their th17, treg and pdc by flow cytometry. they were determined by cd4/il-17 for th-17, cd4/cd25/foxp3 for treg and cd123/cd86/cd14-for pdc. pbmc were collected every 4 weekly during the treatment until end of therapy (week 48) and every 12 weekly until the end of follow-up (week 72). alt was quantified at every time of the pbmc collection. ifn-gamma release cells were analyzed by elispot to hbv-core and s ag.background: alpha fetoprotein (afp) is a well-recognized tumor marker for hcc; elevated level of afp is found in at least 70% of hcc. other liver diseases such as cirrhosis and chronic hepatitis are also related with an elevated level of afp. the regulation of afp gene expression has been relatively less studied although the gene has been suggested to play a role in hcc development. in this study, we tried to identify genetic variations in afp gene and analyze its effect on serum afp level and possible hcc progression.results: in parallel with decline in alt for the first 12 weeks, we found decline in both pdc (r=0.71, p=0.03) and elispot (r=0.65, p=0.03 for hbv-core ag; r=0.48, p=0.05 for hbv-s ag). although there is trend that th-17 decline with treg decline, but they are not statistically significant differences in the same period. there is no significant difference between the svp and non-svp patients.methods: direct dna sequencing was carried out to sequence afp promoter and 500 bp upstream and downstream of afp coding regions in dna samples isolated from 99 hcc subjects and 105 controls respectively. for each samples serum afp levels were determined using commercially available elisa kits. conclusions: under pegyintron treatment, pdc, ifn-gamma change in the same trend of alt during 12 weeks of treatment. it implied that pdc take regulatory effects on ifn-gamma releasing cells and be very tightly related to alt. there wasn't a significant difference in both treg and th-17, which implies that treg and th17 might be of important cells in keeping the stability of the immune system.results: a total of 30 snps were detected in the afp genomic region analyzed, including 29 known snps and one novel snp. among the identified snps, the c>g nucleotide change in the position -250 bp upstream of afp transcriptional start site showed a significant association with hcc (p < 0.05) and a decrease in afp gene expression level. conclusion: our preliminary results indicated a possible association between serum afp expression and -205g allele. the identified snp is located in afp promoter region with possible binding sites for known transcription factors, such as tfiid, coup, apf and nfiii. -30°tail-suspension (ts) rats were used as the model to simulate the physiological effects of weightlessness. thirty-two wistar male rats were randomly divided into 4 groups: control for 7d (7d con), 14d con, ts for 7 d (ts7d) and ts14d. histopathological changes of testicle of the rat were observed by he stain. localization and expression of ar and hsp70 in testicle of rat were observed comparatively by means of immunohistochemistry, and the density of ar and hsp70 immunoreactivety in four groups were compared. results: signal molecules mrna level are shown in the table 1 (** p<0.01, * p<0.05). tnfa, il1, il6 and il8 were higher in group 1, 2 and 4 than those in group 5. ifna was higher in group 5 than that in other groups. there are no significant difference in infc, il2, and il4. there was a positive correlation between tnf and myd88 in group 2, tnf and nfkb in group1 and 2. results: obvious pathological lesions presented in testicle of ts7d and ts14d rat. germinal epithelium irregularity and malformed spermatozoa were found in seminiferous tubules. degeneration and necrosis of germinal epithelium appeared in testicle of ts7d and ts14d rat. ar immunoreactive cell density in the ts7d and ts14d groups were significantly decreased compared with the in-phase normal control groups ( p < 0. 01). while hsp70 immunoreactive cell density in the ts7d and ts14d rats were significantly increased than those of control rats( p < 0. 01), and in testicular interstice or extracellular there were very strong ehsp70 immunoreactive positive staining signals. the results indicate that ground simulated weightlessness induced by 7d-14d tail-suspension in rats can lead to the serious injury , depressed expression of ar and enhanced expression of hsp70 in testicle. despite the absence of any serologic marker of hbv recurrence, however, it remains unknown whether there is occult reinfection in the liver graft. we aimed to detect and quantify the presence of intrahepatic hbv dna in the liver grafts of patients who remain seronegative for hbsag for more than 1 year after liver transplantation. materials and methods: liver biopsy and blood samples were obtained from 31 patients who had been receiving nucleoside analogue prophylaxis alone and remained persistently seronegative for hbsag for at least 1 year (median 44.5 months, range 13.6 to 126.4 months) after liver transplantation for chronic hepatitis b. quantitative polymerase chain reaction was performed to detect and quantify total and covalently-closed circular (ccc) hbv dna in the liver (lowest detection limit, 10 copies/ml), serum and pbmc. direct sequencing was used for hbv quasispecies screening. results: liver biopsy was performed and intrahepatic hbv dna as measured by quantitative real-time pcr was detectable in 26 of 31 recipients. donors anti-hbc status before liver transplant was significantly related to the presence of intrahepatic hbv dna in the recipient's study biopsy (p=0.038). donor intrahepatic hbv cccdna levels correlated with recipient post-liver transplant intrahepatic hbv cccdna levels (p=0.004). hepatitis b virus sequencing results and phylogenetic analysis revealed that hbv reinfection in two recipients were of donor origin, four recipients were of recipient origin and four recipients were of both donor and recipient origins. conclusions: our findings demonstrate the presence of occult hbv reinfection with persistence of hbv dna in liver allografts despite long term nucleoside analogue prophylaxis after liver transplantation, suggesting the need to continue indefinite antiviral therapy. the use of liver grafts from anti-hbc-positive donors might increase the risk of occult hbv reinfection. both donor and recipient hbv dna could contribute to occult hbv reinfection in liver transplant recipients. aim: to construct one noninvasive assessment model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with chronic hepatitis b(chb). methods: we have retrospective analyzed 137 consecutive patients with chronic hepatitis b who underwent percutaneous liver biopsy. we calculated sensitivity, specificity, positive predictive value(ppv), and negative predictive value(npv) of an apri 1.5 in combination with different hyaluronic acid(ha) cut-off points medium (acm). we stimulated pbmcs or dcs under hcm and acm, and evaluated the function. results: after adding the stimulants under hcm, the cd83 and cd86 expression of dcs from cirrhotic patients (lc) were lower than those from healthy contorols (hc). in both hc and lc, the cd83 and cd86 expression of dcs stimulated under acm was lower than that under hcm. the il-12 production in acm was lower than that in hcm. the expression of cd98, which is related to amino acid transport, was not different between hcm and acm. however, dcs cultured in acm expressed lower levels of phospho-p70 s6k than those cultured in hcm. finally, we ascertained that the ifn gamma production by pbmcs was significantly decreased under acm.conclusions: an imbalance in plasma amino acids of advanced cirrhotic patients suppresses the maturation of dcs via mtor/s6k signaling pathway. key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-321773-5fw9abzl authors: cheng, wenyu; chen, guohua; jia, huaijie; he, xiaobing; jing, zhizhong title: ddx5 rna helicases: emerging roles in viral infection date: 2018-04-09 journal: int j mol sci doi: 10.3390/ijms19041122 sha: doc_id: 321773 cord_uid: 5fw9abzl asp-glu-ala-asp (dead)-box polypeptide 5 (ddx5), also called p68, is a prototypical member of the large atp-dependent rna helicases family and is known to participate in all aspects of rna metabolism ranging from transcription to translation, rna decay, and mirna processing. the roles of ddx5 in cell cycle regulation, tumorigenesis, apoptosis, cancer development, adipogenesis, wnt-β-catenin signaling, and viral infection have been established. several rna viruses have been reported to hijack ddx5 to facilitate various steps of their replication cycles. furthermore, ddx5 can be bounded by the viral proteins of some viruses with unknown functions. interestingly, an antiviral function of ddx5 has been reported during hepatitis b virus and myxoma virus infection. thus, the precise roles of this apparently multifaceted protein remain largely obscure. here, we provide a rapid and critical overview of the structure and functions of ddx5 with a particular emphasis on its role during virus infection. dead-box polypeptide 5 (ddx5), also called p68, is a prototypical member of the large asp-glu-ala-asp (dead)-box atp-dependent rna helicases family, which was first identified in 1980 [1] . ddx5 has extensive amino acid sequence homology in various organisms from escherichia coli to humans [2] [3] [4] . the protein plays multifunctional roles involved in all aspects of rna metabolism, including translation, splicing, transcription regulation, ribosome biogenesis, mrna nuclear export, and micro rna (mirna) processing [5] [6] [7] [8] [9] [10] [11] . in addition, the functions in dna modification, cell cycle regulation, tumorigenesis, apoptosis, cancer development, adipogenesis, and nuclear translocation of the wnt-β-catenin protein through phosphorylation have been established [12] [13] [14] . in line with the function of the protein in transcription regulation and mrna splicing, ddx5 has been found to shuttle between the nucleus and the cytoplasm, albeit it predominately localizes in the cell nucleus [15] . given the crucial roles of ddx5 in rna biology, several rna viruses were found to interact with the protein to promote viral replication (table 1) , including severe acute respiratory syndrome (sars) coronavirus (cov) [16] , human immunodeficiency virus 1 (hiv-1) [17] , hepatitis c virus (hcv) [18] , japanese encephalitis virus (jev) [19] , porcine reproductive and respiratory syndrome virus (prrsv) [20] , and influenza virus [21] . additionally, it has been reported that ddx5 is inhibitory for viral replication for two dna viruses, hepatitis b virus (hbv) and myxoma virus (myxv) [22, 23] . in view of the importance of ddx5 in regulating virus infection, we here provide a brief and critical overview of the known functions of ddx5, with a particular emphasis on its role during the virus life cycle. rna or dna helicases are categorized based on the substrates they bind, and can be classified into three superfamilies and two families (named sf1 to sf5) based on the occurrences and characteristics of conserved motifs in their primary sequences [26, 27] . the dead-box helicase family is named for its conserved asp-glu-ala-asp motif and belongs to the sf2 group [26] [27] [28] , which shares nine conserved motifs shown to be involved in atpase and helicase activities [29] [30] [31] . ddx5 is a 68 kd dead-box helicase with 614 amino acids in its full length [2] . consistent with the members of the sf1 and sf2 helicases, ddx5 contains the motifs q, i, ia, ib, ii, iii, iv, v, and vi, which constitute the core region of its helicase function ( figure 1 ). the q motif is named for the conserved glutamine residue that is present in more than 99% of dead-box helicase sequences. the q motif has been found to be necessary for efficient binding of single-stranded rna (ssrna) as well as for the conformational changes that are driven by nucleotide binding and atp hydrolysis [31] . motif i has been found to be crucial for atpase and helicase activities through its interaction with motif ii and motif iii [26, 32] . motifs ia and ib participate in rna-binding and structural rearrangements that occur upon atp binding and hydrolysis [26, 33] . motifs ii and iii are necessary for atpase activity, and they interact with motifs i and vi to collectively form the atp binding pocket, which leads to the correct positioning of the residues necessary for hydrolysis [26, 30] . motif iv is believed to bind ssrna and functions as a functional connect ion between motifs iv and v [26] . motifs v and vi are both important for rna binding in association with motifs ia, ib, and iv [34] . ddx5 also contains a arg-gly-ser-arg-gly-gly (rgs-rgg) motif and an ile-gln (iq) motif, which locate in the c-terminal region. the rgs-rgg motif can function as an rna-binding site to interact with rna or ddx3 for modulating biological functions [35] . (q, i, ia, ib, ii, iii, iv, v, and vi) and the corresponding conserved amino acid sequences of motifs are presented. rgs-rgg: arg-gly-ser-arg-gly-gly. given the ubiquity of rna helicases in plants and animals and the highly conserved asp-glu-x-asp/his (dexd/h) atp-binding domain in all helicases, rna helicases are assumed to play essential roles in a broad array of biological processes, especially rna metabolism. growing evidence shows that ddx5 is implicated in the infection of several viruses. by exploiting the driving force of ddx5-mediated atp hydrolysis or assembling large ribonucleoprotein complexes, rna viruses can complete their life cycles. however, ddx5 exhibits an antiviral activity during hbv and myxv infection. severe acute respiratory syndrome coronavirus (sars-cov), the causative agent of sars, was responsible for a large outbreak associated with a 10% fatality rate in 2003 [36, 37] . this positive rna virus encodes a large replicase polyprotein made up of 16 nonstructural proteins (nsp1-16), among which nsp13 is thought to be essential for the virus life cycle [38, 39] . as the helicase of sars-cov, nsp13 characterizes the enzymatic activities that can unwind both rna and dna duplexes [40, 41] . although sars-cov carries its own rna helicases, host rna helicases may be hijacked by viruses to facilitate the replication of their viral genome; examples include hiv-1 and hcv [42] . using the sars-cov nsp13 gene as a probe for two-hybrid screening in yeast and mammalian cells, only the ddx5 gene was identified as interacting with the helicase [16] . further rna interference (rnai) assays confirmed that inhibition of ddx5 results in the suppression of viral replication [16] . through direct binding to the sars-cov helicase, ddx5 may act as a coactivator to enhance viral genome transcription and virus proliferation [16] . like ddx5, another dead-box rna helicase, ddx1, has been reported to be associated with sars-cov and coronavirus infectious bronchitis virus (ibv) nonstructural protein 14 (nsp14) [43] . ddx1 utilizes its c-terminal region, containing motifs v and vi, to interact with nsp14 via the latter's n-terminal portion. further manipulation of ddx1 expression by small interfering rna and overexpression confirmed that this interaction enhances coronavirus ibv replication [43, 44] . moreover, ddx1 has been identified as a member of the cellular interactomes for the ibv n protein, a nucleocapsid protein encoded by subgenomic mrnas of coronaviruses [44, 45] . recruitment of ddx1 to the phosphorylated-n-containing complex mediated by n phosphorylation can facilitate template readthrough and longer subgenomic mrna synthesis [44] . therefore, the main role of ddx1 and ddx5 when hijacked by coronavirus is to positively modulate viral genome transcription and virus proliferation. as the causative agent of acquired immune deficiency syndrome (aids), human immunodeficiency virus 1 (hiv-1) is a retrovirus of the lentivirus genus with an rna genome of 9 kb. nine polypeptides are encoded by the genome rna, containing three structural proteins env (envelope), gag (group-specific antigen), and pol (polymerase); four accessory proteins, nef, vif, vpu, and vpr; and the regulatory proteins, tat and rev [46, 47] . this "intelligent" pathogen utilizes many host cell factors for its replication. genome-wide screening technologies have been applied by many groups to clarify host factors that affect hiv-1 replication. more than 300 cellular factors have been identified that may be involved in this process [48] [49] [50] . among these factors, several members of the dead-box helicase family that act as co-factors to facilitate hiv-1 proliferation, such as rna helicase a (rha), ddx1, ddx3, ddx5, ddx10, ddx17, ddx21, ddx28, dhx36, ddx47, ddx52, and ddx56 [17, 49, [51] [52] [53] [54] [55] . research has revealed that host rna helicases rha and ddx3 act as cofactors of tat and enhance hiv-1 genes expression [56] [57] [58] . knockdown of ddx3 expression or depletion of ddx3 in cells effectively suppresses the translation of tat and rev as well as hiv-1 mrna transcription [57, 58] . ddx3 directly interacts with hiv-1 tat, which is partially targeted to cytoplasmic stress granules upon ddx3 overexpression or conditions of cell stress, suggesting a potential role of tat/ddx3 complex in translation. more findings have indicated that ddx3 is recruited to the trans-activation responsive (tar) hairpin by interaction with viral tat to facilitate hiv-1 mrna transcription and translation [57, 58] . recently, ddx5 was found to be a new co-factor of hiv-1 that interacted with hiv-1 rev through the rev response element (rre) axis, and thus affects rev function to enhance hiv-1 replication [17] . meta-analysis of host cell genes linked to hiv replication showed that ddx5 was involved in rev-associated complex, suggesting its specific links to hiv-1 replication [50] . ddx5 was confirmed in co-localization with rev in the nucleus, an interaction that is largely dependent on rna [17] . since the main function of rev is to bind with unspliced and partially spliced hiv-1 transcripts and shuttle them from the nucleus to the cytoplasm [59, 60] , ddx5 might be a co-factor to bind with rna and then affect splicing or export of rev/rre-dependent mrnas ( figure 2 ). findings made by lever's group showed that ddx5 and ddx17 exist as homo-or heterodimers to be used by hiv but function in different phases of the virus' lifecycle [55, 61] . ddx5 has a phenotype consistent with its involvement in viral transcriptional control, as mentioned above, while it is speculated that ddx17 acts at a later stage, after transcription [61] . similarly, ddx17 has also been shown to associate with hiv-1 rev, with helicase knockdown leading to reduced virus release from infected cells [53] . in addition to ddx3, ddx5, and ddx17, other rna helicases, including rha, ddx1, ddx21, and ddx56 also have been shown to associate with hiv-1 rev and to stimulate the rev function, suggesting distinct dead-box rna helicases could cross-talk to enhance the hiv-1 life cycle at multiple stages through modulating the hiv-1 rev function ( figure 2 ). compared with other etiological agents, the genome of viruses encodes fewer proteins. consequently, numerous host factors are harnessed by viruses to complete their life cycles, among which some are pro-viral host factors while others are anti-viral host factors. viral dna/rna replication may be the prime target for the development of efficient and safe novel vaccines or anti-viral therapeutics [62] [63] [64] . unlike sars-cov, hiv-1 does not encode its own rna helicase. taken together, host ddx5 could be a new potential molecular target for the development of anti-viral drugs. there are several studies that focus on specific inhibitors or drugs of the host dead-box helicase to inhibit virus replication or treat cancers [65] [66] [67] , but it remains to be determined whether small molecular inhibitors of the interaction between ddx5 and rev can be found. hepatitis c, a contagious liver disease, is caused by hcv, which belongs to a positive-stranded rna virus of the family flaviviridae. hcv infects several hundred million people worldwide and results in cirrhosis, steatosis, and hepatocellular carcinoma [68, 69] . at least 10 proteins are encoded by the positive single-stranded 9.6 kb rna genome of hcv, including three structural proteins (the core protein and envelope (e) proteins e1 and e2) and seven nonstructural (ns) proteins (p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b) [68, 69] . as the viral rna-dependent rna polymerase (rdrp), ns5b can transcribe the positive-and negative-strand rna genome of the virus; it has therefore become a common target for antiviral agents [70] . viral replication requires interaction between rna, viral, and host proteins [71] . ns5b has been shown to interact with a mass of host proteins through yeast two-hybrid screening, small interfering rna (sirna) screening, and transcriptome expression analysis, including t-plastin, eukaryotic initiation factor 4aii, peptidyl-prolyl isomerase pin1, and ddx5 [18, [72] [73] [74] [75] . using yeast-two-hybrid screening, ddx5 was identified as an interacting partner of hcv ns5b, one that affects hcv replication [18] . overexpression of ns5b causes the redistribution of endogenous ddx5 from the nucleus to the cytoplasm; deletion of the c terminal of ns5b abolishes this interaction, while the deletion of the n terminal of ns5b does not influence the distribution of ddx5 [18] . similarly, another study revealed that ddx5 is redirected from the nucleus to the cytoplasm in huh7 cells infected with cell culture-produced hcv (hcvcc) [76] . further experimentation has demonstrated that there are two independent ns5b-binding sites in ddx5: one located at the n-terminus and another at the c-terminus [77] . the first 51 residues of n-terminal fragment are variable, which could fold back to block one of the ns5b binding sites located between 61 and 305 residues in ddx5, suggesting the highly dynamic interaction between ddx5 and ns5b in infected cells [77] . previous studies have shown that knockdown of ddx5 by rnai in 293 cells reduced the synthesis of negative-strand hcv rna, suggesting that ddx5 may be beneficial to the synthesis of the hcv genome (hcv-s1 strain) ( table 2 ) [18] . similarly, hcv rna replication was decreased and the infectivity of hcv (hcv-jfh1 strain) was significantly suppressed in the ddx5 knockdown rsc cells [76] . in line with this observation, the knockdown of ddx5 in huh7.5 cells dramatically inhibits hcv replication as determined from the 5 -nontranslated region of the hcv genome [78] . however, contrary results have been shown in huh7.5 cells by knockdown of the ddx5 through rnai, in a study that revealed that the level of hcv rna in the ddx5 sirna treated cells was higher than in the cells treated with control sirna, suggesting that ddx5 has the ability to inhibit hcv rna replication [79] . further experiments using cell-free infectious virus particles to infect sirna treated cells showed, curiously, that the increased hcv rna in the ddx5 sirna treated cells did not cause increased production of cell-free infectious virus particles (table 2 ) [79] . this finding may indicate that ddx5 was involved in a regulatory role at a later stage of the hcv life cycle. ddx5 was also found to be interacted with the 3 -untranslated region (utr) of hcv, which acts as an enhancer of hcv ires (internal ribosome entry site) function in hepatic cell lines [80] . the knockdown of ddx5 results in reduction viral translation efficiency, but has no effects on hcv replicon, suggesting the essential roles of ddx5 in hcv ires translation [80] . so far, limited focus has been trained on the precise role of ddx5 in hcv replication. hence, further investigations and studies should aim to elucidate the precise role of ddx5 in hcv replication and determine if ddx5 can interact with other hcv proteins. japanese encephalitis virus (jev), a mosquito-borne neurotropic flavivirus, is one of the common causes for epidemic encephalitis in humans and animals. the resulting disease, je, affects large parts of asia and the pacific, where over 3 billion people at risk of exposure to the virus [81, 82] . jev possesses a single-strand positive-sense rna approximately 11 kb in length, encoding a single polyprotein composed of three structural proteins and seven non-structural (ns) proteins in the order 5 -c-prm-e-ns1-ns2a-ns2b-ns3-ns4a-ns4b-ns5-3 [83] . of these, the core (c), premembrane (prm), and envelope (e) proteins are components of extracellular mature virus particles [84] . the seven c-terminal non-structural proteins (ns1-ns5) are involved in multiple steps of viral life cycles such as rna replication, virus assembly, and innate immunity evasion [83] . recently, it has been shown that ddx5 can interact with jev core protein (c), ns3 and ns5, and can host ddx5 act as a positive regulator for jev replication [19] . both knockdown of ddx5 and overexpression of ddx5 mutants lacking helicase activity can decrease jev replication, suggesting that ddx5 and its helicase activity are required for jev replication [19] . ddx5 binds to the viral 3 -utr and colocalize with viral rna, which promotes virus rna replication but not in viral protein translation. as mentioned above, ddx5 has an interaction relationship with 3 -utr of hcv and plays a role in hcv ires translation. similarly, ddx5 might participate in the translation of jev ires. the interaction between ddx5 and the three viral proteins (c, ns3, and ns5) co-localized with viral rna in the cytoplasm during infection might facilitate the unwinding the intermediate rna duplexes [19] . the c protein of jev was found to localize in both the cytoplasm and the nucleus; the mature c protein, released from the endoplasmic reticulum (er) membrane, is believed to bind to the genomic rna [85] . kinetic study of viral rna and protein syntheses have shown that, following translation at the early step of infection, it was translocated into the nucleus, and then facilitated rna replication at the late phase of infection. ns3 functions as rna helicase, rna triphosphatase, and rna-stimulated nucleoside triphosphatase. ns5 works as rna-dependent rna polymerase and methyltransferase [86] . these two viral proteins (ns3 and ns5), together with ns2a, specifically bind the 3 -untranslated region and lead to the formation of replication complex (rc) [87] . host ddx5 is recruited by the viral proteins and binds to this rc, and then utilizes its helicase activity to regulate jev replication [19] . since viral rna replication is a quite dynamic process, ddx5 shuttles between nucleus and cytoplasm to increase the efficiency of rna separation [19] . in addition to ddx5, helicase ddx3 was also found to be involved in jev replication [88] . viral ns3 and ns5 may interact with ddx3, which binds to jev 5 -and 3 -untranslated regions to positively regulate viral rna translation. when using chemical compounds (cmp6 and cmp8) to inhibit the helicase activity of ddx3 in hiv and jev infected cells, viral rna replication was remarkably inhibited [65, 88] . however, no drugs or molecule inhibitors targeting ddx5 are available. therefore, ddx5 could be a novel target for the development of anti-viral drugs. besides hcv and jev, other members of the flaviviridae family were also found to hijack ddx5 during virus infection. the genus pestivirus, a group of small positive-stranded rna viruses, belongs to the family flavivirus, which cause economically important diseases of farm animals and includes bovine viral diarrhea virus (bvdv), classical swine fever virus (csfv), and border disease virus (bdv) [89] . the enveloped pestiviruses contain single-stranded rna genomes of approximately 12.3-12.5 kb that consists of a single open reading frame, encoding only 12 proteins. these are coand post-translationally processed from a single rna into four structural and eight nonstructural proteins in the order nh2-n pro -c-e rns -e1-e2-p7-ns2-ns3-ns4a-ns5b-cooh [90, 91] . n pro , named for the viral n-terminal protease, is a 168-amino-acid autoprotease, with cysteine protease activity in a glu22-his49-cys69 triad that acts to cleave itself from the nascent polypeptide [92] . early work using mass spectrometry showed that n pro binds to ddx5, and also to ddx3x and dhx9 [25] . the distribution of n pro in ribosomal and ribonucleoprotein particles, as well as its interactions with several rna helicases, suggest that n pro is involved with virus rna translation; ddx5 possibly contributes to this process. nevertheless, the function of ddx5 in modulation of viral replication remains to be determined. porcine reproductive and respiratory syndrome virus (prrsv), the causative agent of one of the most economically important global swine diseases, is classified in the genus arterivirus of the family arteriviridae [93] . prrsv contains a single-strand, positive-sense rna of approximately 15 kb in length, composed of at least 10 open reading frames (orfs), and a poly-a tail at the 3 -terminus [94] . orf1a and orf1b encode the replication-related polymerase proteins and are processed into at least 13 nonstructural proteins (nsps) by self-cleavage [95] . among the nsps encoded by the orf1b region of prrsv, nsp9 is considered to be involved in viral replication and genomic transcription [96] . this protein possesses a rna-dependent rna polymerase (rdrp) domain in its c-terminal portion that contributes to its polymerase activity and the virulence of prrsv [97] . using the yeast two-hybrid method, the host ddx5 was found to interact with the nsp9 of prrsv [20] . overexpression of ddx5 in marc-145 cells showed an enhancement effect on the replication of prrsv; meanwhile, silencing of ddx5 expression in marc-145 cells might significantly inhibit the replication of virus particles, suggesting that ddx5 might function as a cellular cofactor that positively regulates prrsv replication [20] . confocal immunofluorescence assay has revealed that ddx5 co-localizes with nsp9 in the cytoplasm with a perinuclear pattern in hek293 cells, marc-145 cells, and the pam cell line, suggesting that endogenous ddx5 might diffuse from nucleus to cytoplasm and help to unwind the viral rna through its helicase activity. this hypothesis was confirmed by co-immunoprecipitation (co-ip) assay, which showed that the dead and helicase domains (aa1-436) of ddx5 bind with the c-terminal portion of nsp9 [20] . interestingly, prrsv carries its own helicase, nsp10, with helicase activity; the recruitment of host ddx5 by nsp9 may function as a cofactor for rna unwinding, export, and/or translation of the prrsv during viral genomic replication and transcription [20, 96] . influenza a virus is a major public health problem, causing seasonal epidemics in humans and occasionally lethal global pandemics, as happened in 1918-1919 (h1n1), 1957 (h2n2), 1968 (h3n2), and 2009 (swine-origin h1n1) [98] . currently, the h5n1 subtype circulating in wild and domestic birds is capable of crossing the species barrier into humans and constitutes huge threats to both animals and public health [99] . as many as 500 million people a year are infected with influenza a virus worldwide, with more than 500,000 deaths [100] . characterized by its high potential for mutations and adaptations, influenza virus presents a significant challenge to disease control and the pharmaceutical industry [101] . it has been demonstrated that the trimeric viral polymerase (a complex consisting of the pb1, pb2, and pa subunits) and nucleoprotein (np) contribute to the pathogenicity of h5n1 viruses in humans and other species of mammals [102] . functional genomics and proteomics approaches have identified a number of human proteins that associate with viral polymerases, including ddx5 and ddx17 [21] . the required role of ddx5 and ddx17 in h5n1 virus replication has led to a significant reduction in the virus titer after knockdown of these proteins [21] . it has been observed that ddx5 colocalizes with viral nps in the nucleus during viral rna replication and transcription. likewise, ddx17 has been found to colocalize with h5n1 np. however, the predominant distribution of ddx17 in uninfected cells is in the nucleus, where ddx17 exhibits as punctated [21] , whereas, in h5n1 infected cells, ddx17 was shown to be relocated in the cytoplasm, a change that exhibits its essential roles in efficient h5n1 mrna and viral rna (vrna) synthesis in both human and avian cells. given the multiple functions of ddx5 and ddx17 in ribosomal pre-rrna (ribosomal rna) processing and rrna nuclear export [103] , we speculate that influenza viruses have evolved to hijack host ddx5 and/or ddx17 in order to facilitate their own proteins' nuclear export. epstein-barr virus (ebv), a type of oncogenic human herpesvirus, is ubiquitous, causing over 90% infection in the exposed human population [104] . infections cause no symptoms in young children but result in the development of infectious mononucleosis at adolescence or later [105] . as with other herpesvirus, ebv possesses a 170 kb double-stranded dna genome encoding different sets of viral proteins and micrornas during lytic and latent infection. epstein-barr virus nuclear antigen 2 (ebna2) is a latency type iii expressed protein that interacts with various types of host cell proteins to regulate cellular and viral gene transcription [106] . recent work using immunoprecipitation identified ddx5 as an interacting partner of ebna2 [24] . as both ebna2 and ddx5 contain rg-repeat elements and mono-methylated arginine (mma) residues [107] , it has been proposed that ddx5 probably serves as methylation substrate to facilitate gene expression activation by ebna2. however, the relevance of ebna2-ddx5 interaction and functions during virus infection still remains elusive, and more studies are needed. being a member of the leporipoxvirus genus, myxoma virus exhibits a specific rabbit-restricted host tropism but reveals a much broader cellular host range in cultured cells [108] . myxv has been shown to selectively infect a variety of human cancer cell lines derived from a diverse group of tissues, leading to study of myxv as a potential oncolytic virotherapeutic for various classes of human cancer [109] . recently, a sirna library screen targeting the 58 human dead-box rna helicases has shown that ddx5 knockdown enhances myxv replication. the knockdown of ddx5 in multiple human cell lines increased myxv replication and enhanced foci size and virus spread, suggesting its potential roles in antiviral responses or regulation of innate immune responses [22] . in addition, ddx5 has shown increased expression in human monocyte-derived dendritic cells infected with attenuated strains of vaccinia virus (vacv) [110] . however, the exact function of this molecule in vacv infection was not established in that study. unlike myxv, bunyavirus replication is unaffected by ddx5 silencing, so that the antiviral function of ddx5 remains undefined, as ddx5 depletion upregulated ddx17 expression, restricting bunyavirus infection in an interferon-independent manner [111] . additionally, ddx5 has a role in transcription of hbv minichromosome by a mechanism not yet determined [23] . hbv is also the causative agent of chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma (hcc) worldwide [112, 113] . although hbv is a dna virus, the replication of its dna genome depends on reverse transcription [112] . after entry, the hbv dna is transported into the nucleus and is converted into covalently closed circular (ccc) dna [114] , which can assume a minichromosome-like structure and acts as the template for transcription of four viral rnas [114] . recently, it was shown that ddx5, by regulating prc2 (polycomb repressive complex 2) stability and function, represses hbv minichromosome and the expression of specific host genes involved in hbv-mediated hepatocarcinogenesis [23] . ddx5 interacts with and stabilizes suppressor of zeste 12 homolog (suz12), which is an essential subunit of prc2 [23, 115] . as an expression of viral protein hbx in hbv-infected cells, ddx5 was downregulated and leads to the degradation of suz12, which results in the transcription viral cccdna-encoded genes [23] . moreover, ddx5 expression was found to be reduced in a group of hbv-associated hccs and this can be an important molecule in pathogenesis of poor prognosis hbv-mediated liver cancer [23] . in recent years, as the emerging roles of some rna helicases in host innate response have been established, studies have shown that viral nucleic acids are sensed by ddx1, dhx9, ddx21, dhx33, dhx36, ddx41, retinoic acid-inducible gene 1 (rig-i/ddx58), and melanoma differentiationassociated gene 5 (mda-5), leading to type i interferon production and an antiviral response [116] [117] [118] [119] [120] [121] . these helicases (rig-i and mda-5) are characterized by caspase activation and recruitment domains (card) at the n-terminal region, which interact with other card-containing proteins to transduce the signaling cascade [122] . alternatively, the functions in nucleic acids recognition of helicases-such as ddx1, dhx9, ddx21, dhx33, dhx36 and ddx41-which are lacking in card, have all been established in certain human plasmacytoid dendritic cells [116] [117] [118] [119] [120] . ddx5 lacks the card signaling domains, and no literature indicates the function of ddx5 in regulating the production of type i interferon. the issue of whether ddx5 contributes to antiviral responses or regulation of innate immune responses remains uncertain, and more studies are needed. as summarized in this review, the multifaceted protein ddx5 has been shown to be involved in rna metabolism and viral infection, especially for rna viruses. it is intriguing that ddx5 is the target of manipulation by different viruses. each rna virus seems to hijack host ddx5 in order to facilitate its own replication. with conserved atp binding motif and helicase motif, ddx5 utilizes the free energy to change of binding and hydrolyzing a nucleotide triphosphate to dissociate duplexes or displace bound proteins, which, on one hand, ddx5 may translocate along with viral dna/rna; on the other hand, ddx5 may unwind double-stranded regions to promote the expression of viral proteins [26] . however, ddx5 exhibits a role in antiviral responses during hbv and myxv infection. this might indicate that a requirement of ddx5 for viral replication is a more universal feature of rna viruses. the opposite roles of ddx5 between dna and rna infection likely reflect the different modes of the biosynthesis of rna and dna viruses. as mentioned, the using of ddx5 for viral replication could be a therapeutic target for development of antiviral drugs. even though emerging studies on ddx5 have supplemented our knowledge about the multiple functions of ddx5 in recent years, the overall picture still remains fragmentary. research is still needed to resolve seemingly contradictory data on the role(s) of ddx5 in response to different virus. as conserved motifs in the primary sequence, dexd/h helicases are highly conserved from viruses and bacteria to humans, and these proteins have overlapping functions. in addition, these proteins generally act as components of large multi-protein complexes, with each other or other factors, to regulate transcription. therefore, studies should also attempt to establish whether the effects of ddx5 on viral replication after knockdown are unique or whether other helicases supplement abolished function via small interfering rna. sv40 large t shares an antigenic determinant with a cellular protein of molecular weight 68,000 rna helicase activity associated with the human p68 protein identification of a putative rna helicase in e. coli 68 rna helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts a subfamily of rna-binding dead-box proteins acts as an estrogen receptor α coactivator through the n-terminal activation domain (af-1) with an rna coactivator rna helicase is an essential human splicing factor that acts at the u1 snrna-5 splice site duplex the p68 and p72 dead-box rna helicases interact with hdac1 and repress transcription in a promoter-specific manner atpase/helicase activities of p68 rna helicase are required for pre-mrna splicing but not for assembly of the spliceosome redundant role of dead-box proteins p68 (ddx5) and p72/p82 (ddx17) in ribosome biogenesis and cell proliferation the rna helicase ddx5/p68 is a key factor promoting c-fos expression at different levels from transcription to mrna export rna helicases ddx5 and ddx17 dynamically orchestrate transcription, mirna, and splicing programs in cell differentiation rna helicase ddx5 is a p53-independent target of arf that participates in ribosome biogenesis a chicken embryo protein related to the mammalian dead-box protein p68 is tightly associated with the highly purified protein-rna complex of 5-mec-dna glycosylase phosphorylations of dead-box p68 rna helicase are associated with cancer development and cell proliferation p68 rna helicase is a nucleocytoplasmic shuttling protein interaction between sars-cov helicase and a multifunctional cellular protein (ddx5) revealed by yeast and mammalian cell two-hybrid systems ddx5 facilitates hiv-1 replication as a cellular co-factor of rev cellular rna helicase p68 relocalization and interaction with the hepatitis c virus (hcv) ns5b protein and the potential role of p68 in hcv rna replication the dead-box rna helicase ddx5 acts as a positive regulator of japanese encephalitis virus replication by binding to viral 3 utr the dead-box rna helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp9 in vitro host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins identification of host dead-box rna helicases that regulate cellular tropism of oncolytic myxoma virus in human cancer cells rna helicase dead-box protein 5 regulates polycomb repressive complex 2/hox transcript antisense intergenic rna function in hepatitis b virus infection and hepatocarcinogenesis antibodies against the mono-methylated arginine-glycine repeat (mma-rg) of the epstein-barr virus nuclear antigen 2 (ebna2) identify potential cellular proteins targeted in viral transformation host factors that interact with the pestivirus n-terminal protease, n pro , are components of the ribonucleoprotein complex the dead-box protein family of rna helicases happy birthday: 25 years of dead-box proteins helicase structure and mechanism the newly identified q motif of dead-box helicases is involved in adenine recognition dead-box proteins: the driving forces behind rna metabolism the newly discovered q motif of dead-box rna helicases regulates rna-binding and helicase activity dexd/h box rna helicases: from generic motors to specific dissociation functions rna helicase dynamics in pre-mrna splicing comparative structural analysis of human dead-box rna helicases the dead-box rna helicase ddx3 interacts with ddx5, co-localizes with it in the cytoplasm during the g2/m phase of the cycle, and affects its shuttling during mrnp export crystal structure and functional analysis of the sars-coronavirus rna cap 2 -o-methyltransferase nsp10/nsp16 complex rna 3 -end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex mechanisms and enzymes involved in sars coronavirus genome expression mechanism of nucleic acid unwinding by sars-cov helicase cooperative translocation enhances the unwinding of duplex dna by sars coronavirus helicase nsp13 sars-cov orf1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets viral and cellular rna helicases as antiviral targets the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology cell biology of hiv-1 infection of macrophages making sense of multifunctional proteins: human immunodeficiency virus type 1 accessory and regulatory proteins and connections to transcription a whole-genome association study of major determinants for host control of hiv-1 identification of host proteins required for hiv infection through a functional genomic screen host cell factors in hiv replication: meta-analysis of genome-wide studies a dead-box protein facilitates hiv-1 replication as a cellular co-factor of rev role of rna helicases in hiv-1 replication distinct ddx dead-box rna helicases cooperate to modulate the hiv-1 rev function multiple functions of ddx3 rna helicase in gene regulation, tumorigenesis, and viral infection identification of rna helicases in human immunodeficiency virus 1 (hiv-1) replication-a targeted small interfering rna library screen using pseudotyped and wt hiv-1 a role of rna helicase a in cis-acting transactivation response element-mediated transcriptional regulation of human immunodeficiency virus type 1 human ddx3 interacts with the hiv-1 tat protein to facilitate viral mrna translation ddx3 rna helicase is required for hiv-1 tat function hiv rev assembly on the rev response element (rre): a structural perspective the hiv-1 rev response element (rre) adopts alternative conformations that promote different rates of virus replication the roles of dead-box helicases in the life cycle of hiv-1 poxvirus dna replication host factor-targeted hepatitis b virus therapies recent strategies and progress in identifying host factors involved in virus replication discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors ketorolac salt is a newly discovered ddx3 inhibitor to treat oral cancer targeting ddx3 with a small molecule inhibitor for lung cancer therapy virus-specific mechanisms of carcinogenesis in hepatitis c virus associated liver cancer the core of hepatitis c virus pathogenesis nucleoside inhibitors of hepatitis c virus ns5b polymerase: a systematic review recent advances in the molecular biology of hepatitis c virus interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis c virus rna-dependent rna polymerase peptidyl-prolyl isomerase pin1 is a cellular factor required for hepatitis c virus propagation hepatitis c virus non-structural 5b protein interacts with cyclin a2 and regulates viral propagation chemical proteomic identification of t-plastin as a novel host cell response factor in hcv infection pml tumor suppressor protein is required for hcv production the variable n-terminal region of ddx5 contains structural elements and auto-inhibits its interaction with ns5b of hepatitis c virus identification of cellular factors associated with the 3 -nontranslated region of the hepatitis c virus genome understanding the interaction of hepatitis c virus with host dead-box rna helicases berzal-herranz, a. the cis-acting replication element of the hepatitis c virus genome recruits host factors that influence viral replication and translation japanese encephalitis: the virus and vaccines. hum. vaccines immunother japanese encephalitis surveillance and immunization-asia and western pacific regions regulation of flavivirus rna synthesis and replication capsid protein c of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence japanese encephalitis virus core protein inhibits stress granule formation through an interaction with caprin-1 and facilitates viral propagation an rna cap (nucleoside-2 -o-)-methyltransferase in the flavivirus rna polymerase ns5: crystal structure and functional characterization architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments cellular ddx3 regulates japanese encephalitis virus replication by interacting with viral un-translated regions pestiviruses: how to outmaneuver your hosts the molecular biology of pestiviruses structures and functions of pestivirus glycoproteins: not simply surface matters autocatalytic activity and substrate specificity of the pestivirus n-terminal protease n pro prrsv structure, replication and recombination: origin of phenotype and genotype diversity overview: replication of porcine reproductive and respiratory syndrome virus the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins nsp9 and nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china inhibition of porcine reproductive and respiratory syndrome virus by specific sirna targeting nsp9 gene h5 influenza, a global update a systematic review of the comparative epidemiology of avian and human influenza a h5n1 and h7n9-lessons and unanswered questions functional genomics reveals linkers critical for influenza polymerase a membrane-based purification process for cell culture-derived influenza a virus mutations in the pa protein of avian h5n1 influenza viruses affect polymerase activity and mouse virulence the dead-box proteins ddx5 (p68) and ddx17 (p72): multi-tasking transcriptional regulators epstein-barr virus lymphoproliferative disease after solid organ transplantation epstein-barr virus hijacks dna damage response transducers to orchestrate its life cycle proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest myxoma virus and the leporipoxviruses: an evolutionary paradigm oncolytic myxoma virus: the path to clinic distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors mva and nyvac stem-loop recognition by ddx17 facilitates mirna processing and antiviral defense hbv cure: why, how, when? hepatitis b virus infection and alcohol consumption molecular biology of hepatitis b virus infection hepatitis b virus-associated hepatocellular carcinoma and hepatic cancer stem cells ddx21, and dhx36 helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells aspartate-glutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome the helicase ddx41 senses intracellular dna mediated by the adaptor sting in dendritic cells is a novel antiviral factor promoting rig-i-like receptor-mediated signaling rig-i and mda-5 detection of viral rna-dependent rna polymerase activity restricts positive-strand rna virus replication emerging roles in viral replication and the host innate response this study was supported by grants from the natural science foundation of china (31402199) key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control. key: cord-009567-osstpum6 authors: nan title: abstracts oral date: 2008-04-23 journal: am j transplant doi: 10.1111/j.1600-6143.2008.02254.x sha: doc_id: 9567 cord_uid: osstpum6 nan abstracts evl (target 3-8ng/ml), aza or mmf with standard (sd) or reduced (rd) csa. data at 6 months post-tx is presented here. results. the proportion of patients with cmv events, including serious adverse events and laboratory evidence of cmv infection, has remained low and broadly similar following introduction of cc administration of evl and use of concomitant rd-csa. cmv syndrome and cmv organ involvement occurred in <3% of recipients receiving evl in each of the studies. the highest rates of all cmv parameters occurred with mmf + sd-csa or aza + sd-csa. conclusion. the low incidence of cmv infection and cmv-related events observed in heart tx patients receiving fd-evl and sd-csa as de novo immunosuppression has been maintained in newer regimens of cc-evl with rd-csa. cmv syndrome and cmv organ involvement are rare with evl-based immunosuppression after cardiac tx. the low rate of cmv infections in evl-treated patients may contribute to improved long-term outcome and a lower incidence of cav; confirmatory data are awaited. fibronectin-α 4 β 1 interactions enhance p38 mapk phosphorylation and metalloproteinase-9 expression in cold liver ischemia/reperfusion injury. sergio duarte, 1 xiu-da shen, 1 takashi hamada, 1 constantino fondevila, 1 ronald busuttil, 1 ana j. coito. 1 1 the dumont ucla transplant center. expression of endothelial fibronectin (fn) is an early event in liver ischemia/reperfusion (i/r) injury. we have recently shown that cs1 peptide facilitated blockade of fn-α4β1 leukocyte interactions regulates metalloproteinase-9 (mmp-9) expression in steatotic orthotopic liver transplants (olt). this study tests the function of the cs1 peptide therapy upon mmp-9, and further dissects putative mechanisms, in an alternate model of cold ischemia/reperfusion (i/r) injury. methods and results: cs1 peptides were administrated through the portal vein of sprage-dawley (sd) rat livers before and after 24 h cold storage (500 µg/rat). sd recipients of olts received an additional dose of cs1 peptides 1h post-olt. cs1 therapy significantly increased the 14d olt survival rate (100% vs. 50%, n=8/gr, p<0.005). cs1 peptides reduced sgot levels (u/l) at 6h (1413±420 vs. 2866±864 p<0.008) and 24h (1350±142 vs. 4000±1358, p<0.006) post-olt. cs1 treated olts showed good preservation of lobular architecture, contrasting with severe necrosis and sinusoidal congestion in controls. moreover, cs1 treated livers were characterized by a profound decrease in t (31±3 vs. 64±8, p<0.0002), nk (19±2 vs. 41±3, p<0 .0003) and ed1 (21±7 vs. 57±16, p<0.008) cells as early as 6h after i/r. neutrophils, as indicated by mpo activity (0.48±0.02 vs. 3.18±0.94, p<0.02) were depressed by cs1 therapy. this correlated with decreased mrna expression of tnf-α (0.032±0.04 vs. 0.83±0.26, p<0.005) and cycloxygenase-2 (0.8±0.1 vs. 2.2±0. 6, p<0.05) . leukocyte transmigration is dependent upon adhesive and focal matrix degradation mechanisms. mmp-9, which is inducible and expressed by infiltrating leukocytes, was profoundly depressed in 6h cs-1 peptide treated olts, at both mrna (0.1±0.1 vs. 0.5±0.2, p<0.03) and protein (0.15±0.07 vs. 0.7±0.14, p<0.03) levels. moreover, mmp-9 activity evaluated by zymography was reduced by ∼3-fold in the cs-1 group. interestingly, phosphorylation of mitogen-activated protein (map) kinase p38 (0.05±0.01 vs 0.14±0.06, p<0.03) was selectively downregulated in the 6h cs-1 treated olts. in conclusion, this work supports a broad regulatory role for fn-α4β1 interactions on mmp-9 expression by leukocytes, likely mediated through activation of p38 mapkinase, and matrix pathological breakdown associated with leukocyte infiltration. this data provides the rationale for the development of novel therapeutic approaches in cold liver i/r injury. ischemia reperfusion injury is the most common cause of acute kidney injury in both native and allograft kidneys. the pathogenic mechanisms of renal ischemia reperfusion injury include changes at the level of the microvasculature as well as the tubules. within the microvasculature, leukocyte-endothelial interactions likely play a role and contribute to the well-established microvasculature dysfunction (e.g., endothelial permeability) and alterations in endothelial-leukocyte interaction occur during ischemic acute kidney injury. our previous studies have demonstrated that t cells modulate renal ischemia reperfusion injury in a murine model, we hypothesized that t cells could mediate changes in renal vascular permeability during ischemia reperfusion injury. we performed a 30 min bilateral renal ischemia followed by reperfusion in c57bl6 wild type mice and in t cell deficient (nu/nu) mice with or without t-cell adoptive transfer from their wild type littermates and evaluated rvp by evans blue dye extravasations (ebde). the time course studies of rvp showed marked increases in renal ebde within the early 3-6 hrs after ischemia. cd3 positive pan t-cells but neither cd4 nor cd8 t cells were found to infiltrate into post ischemic kidney within 6 hrs, and comparison was made with other leukocytes using immunohistochemistry technique. gene microarray analysis demonstrated that the gene for tnf-α (tnfaip1), a potent mediator of microvascular permeability as well as a t cell product, was increased in the kidney early after ischemia. tnf-α, ifn-γ and il-4 protein by an intracellular cytokine staining technique was found increased early in peripheral circulating t cells and later in renal t cells after renal ischemia. the rise in rvp was significantly attenuated in t cell deficient mice nu/nu at 6 hrs compared to the wild type littermates and this attenuated rvp in t cell deficient mice was restored after adoptive transfer of splenic t cells from wild type littermates into these nude mice. these data demonstrate that t cells traffic early into post ischemic kidney, produce tnf-α and other cytokines that can increase rvp, and directly participate in the increased rvp during acute ischemia reperfusion injury. t cell-endothelial interactions are a likely mechanisms underlying the pathophysiologic role of t cells in renal ischemia reperfusion injury . background: ischemia/reperfusion injury (iri) is a major cause of organ dysfunction after intestinal injury and transplantation. the interaction between the innate and adaptive immune systems has become a major area of focus in this field. our preliminary work showed that mice undergoing intestinal iri experienced worse survival and tissue injury in conjunction with increased infiltration of pmn and cd3+ cells as compared to sham mice. the purpose of this study was to investigate the role of the t cell in intestinal iri through the use of genetically deficient mice. methods: under anesthesia, male c57bl6 wild-type mice (wt) and cd4 knockout mice (cd4ko) underwent 100 min of warm intestinal iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at 4h and 24h. tissue was analyzed by histology, cd3 immunostaining, myeloperoxidase activity (mpo), and semi-quantitative pcr for several cytokines/chemokines. results: wt had significantly worse survival compared to cd4ko (30% vs. 100%, p=0.002), and worse histopathological injury mostly involving mucosal sloughing. wt had higher mpo activity than cd4ko (1.9±0.88 vs. 0.70±0.73 at 4h, p=0.059, and 1.5±0.05 vs. 0.22±0.065 at 24h, p=0.004) and increased cd3+ cell infiltration. there was also increased mrna production of cytokines/chemokines in wt vs. cd4ko at both timepoints; specifically, the data with respect to b-actin at 24h was: il-2 (0. conclusion: this study demonstrates for the first time in the intestine that cd4+ t cells are important mediators of iri. in the absence of cd4+ t cells, better outcomes were associated with less pmn infiltration implying a link between the innate and adaptive immune systems. an alteration in chemotactic signaling potentially effected by cd4+ t cells may play a role in this process. these results confirm the important function of cd4+ t cells in intestinal iri and justify further investigation into their complex role in this process. messenger rna assessment in urine sediments from renal transplant patients is rapidly evolving as a non-invasive diagnostic tool. t cells, macrophages, and often b cells are present in rejecting kidney grafts. we questioned whether urinary mrna levels of markers representing these cell types ((regulatory) t cells: cd3ε, foxp3, cd25, tgf-ß; macrophages: cd68, s100a9; b cells: cd20) are associated with rejection. materials in our institute 520 urine samples a year from over 100 renal transplant patients are being collected for mrna quantitation purposes. urine sediments are pelleted and stored in rna-preserving solution. we initially investigated the effect of incubation time of urine on mrna integrity. next, in a case-control study 17 urine samples taken at time of biopsy-proven rejection were compared to 17 samples taken during stable graft function. median time of sampling (55 days versus 43 days posttansplant) did not significantly differ between groups. rna (0.90 ± 1.18 µg) was extracted using rneasy spin columns. message of the markers mentioned above was quantified with q-pcr and normalized. results incubation of urine for 7h or longer at room temperature after acquisition resulted in a 75% decrease in 18s rrna signals (p<0.05). however, storage of urine for up to 24h at 4ºc did not result in decreased mrna expression. in the case-control study, all markers tested showed the highest expression levels in urine rejection samples. when corrected for multiple comparisons, only tgf-ß (26-fold, p=0.007) and foxp3 (21-fold, p=0.002) expression was significantly increased in rejection samples compared to non-rejection samples. tgf-ß levels highly correlated with foxp3 levels (r = 0.90, p<0.00001), suggesting a mechanistic relationship in vivo between the two molecules. conclusion rna integrity in urine samples stored at 4ºc is maintained for at least 24h. detection of increased tgf-ß and foxp3 message in urine from patients with a kidney transplant represents a means for indicating occurrence of graft rejection. this implies that mrna urinalysis renders a suitable molecular tool for non-invasive patient monitoring in clinical practice. the data furthermore suggest that rejection is associated with tgf-ß-mediated immune mechanisms in which regulatory t cells play a role. the significance of b cell and plasma cell infiltration in renal allografts remains controversial. we previously established that transcript sets associated with ifng effects or t cells reflect the inflammatory burden in renal allografts. in the present study, we identified b cell associated transcripts (bats) and immunoglobulin transcripts (igts), reflecting b cell and plasma cell infiltration, respectively. using microarrays, we analyzed bat and igt expression in relationship to histologic lesions, diagnosis, and renal function in 177 renal allograft biopsies. immunostaining confirmed that bat and igt expression was associated with b cells and plasma cells in the graft. expression of bats and igts was increased in biopsies with rejection (1.2 ± 0.2, 1.6 ± 0.9) compared to non-rejection (1.1 ± 0.2, 1.1 ± 0.5), but was not different between t cell (1.3 ± 0.3, 1.5 ± 1.1) and antibody mediated rejection (1.2 ± 0.2, 1.5 ± 0.9) and also occurred in some non-rejecting biopsies (recurrent gn). bat and igt scores correlated strongly with time post transplant (fig1), which was best modeled as a dichotomous relationship: biopsies ≤ 5 months did not express bats or igts above the level of control kidneys. other inflammatory markers were not time dependent. in biopsies ≥ 5 months, bat and igt expression correlated with interstitial inflammation, tubular atrophy, and interstitial fibrosis. in a multiple regression analysis, only time post transplant and interstitial inflammation were independently related to bat and igt scores. when correcting for time post transplant, bat and igt scores did not correlate with renal function at the time of biopsy or future function 6 months post biopsy. bats and particularly igts are a time dependent feature of injured and inflamed renal allografts. corrected for the effect of time, bats and igts do not correlate with outcomes, indicating that b cell and plasma cell infiltrates have no specific role for the mechanism of injury independent of the inflammatory burden. their accumulation in late allografts may indicate emergence of specialized lymphoid compartments in tissues with long standing low level inflammation. pearson correlation coefficient between pbts expression and renal function pbts egfr at %change in egfr from biopsy 6 months after biopsy baseline to biopsy biopsy to 6 months after qcats -0.17* -0.16* -0.19* -0.05 grits -0.23** -0.25** -0.20** -0.10 irit_d1 -0.02 -0.01 -0.09 0.13 irit_d3 -0.51** -0.29** -0.36** 0.16* irit_d5 -0.28** -0.22** -0.19* -0.02 kt2 0.27** 0.13 0.22** -0.02 * p<0.05, ** p<0.01 thus the pbts have prognostic value. surprisingly, the transcripts in the biopsy with greatest prognostic value for future gfr or recovery of gfr were not related to rejection (cytotoxic t cell or ifng associated) but to the degree of injury response. we propose that the common pathway linking various injuries (immune and non immune) to gfr is through the degree of injury response these events induce in the epithelium, particularly those in the irit_d3 set. early rejection from anamnestic response in offspring-to-mother or husband-to-wife kidney transplant. kwan tae park, 1 song chul kim, 1 duck jong han. 1 1 surgery, asan medical center, seoul, korea. accelerated rejection can be developed in the immediate post-transplant period resulting from anamnestic response due to the exposure to fetal hla antigen during the previous pregnancy in case of offspring-to-mother or husband-to-wife. however, accelerated rejections in these groups have been rarely reported. 81 cases of offspring-to-mother (offspring group) and 53 cases of husband-to-wife (spouse group), who has been sensitized to spouse via their children, underwent kidney transplants from january of 1997 to august of 2007 at our institution and retrospectively reviewed. control group was female kidney recipients transplanted at the same period from living related donors other than offspring and from living unrelated donors other than husband. acute rejection (ar) rate within 3 months after transplant were 19.7% (16/81) in offspring group and 18.8% (10/53) in spouse group. the ar rates were not different between the two groups, however they were significantly higher than control group in both groups, namely 10.2% (24/235) for offspring control and 13.5% (10/74) for spouse control. the mean onset of ar was 7.5 day (0-29) and it was significantly later than that of control groups (16.5 day, p<0.05) and 54% of ar were accelerated rejections within 7 days after transplant. proportion of acute humoral rejection was significantly higher in both groups than control group (37.5% vs 8.3% in offspring group, 60% vs 10% in spouse group). most of the ar was successfully reversed by steroid pulse and/ or plasmapheresis, ivig, rituximab. any risk factors for the ar such as number of pregnancy, preoperative cdc cross matching, pra, immunosuppressant and antibody induction couldn't be identified. serum creatinine level in ar patients were higher than ar free patients by postoperative 1 month, but last follow up creatinine level didn't show any statistical difference (1.43 mg/dl in ar vs 1.14mg/dl in ar free). 5 year graft/patient survival rate were not different between ar and ar free groups and study and control groups. risk of higher rejection rate accompanied with higher accelerated and humoral rejection was identified in female kidney recipients from offspring or spouse donor in comparison with control group. considering the anamnestic response of this cohort of female recipients, more prudent preoperative screening and careful immediate postoperative immune monitoring are required for the avoidance of rejection and impaired graft survival. kaplan-meier survival curves showed that ∆upc > 0.2 were associated with decreased patient and allograft survival. these data show for the first time that regardless of the histopathology, ∆upc > 0.2 one month after rejection is associated with poor patient/allograft outcomes. from histology to microarrays the histopathological hallmark of t cell mediated rejection (tcmr), is interstitial infiltration. assessing infiltration by histology is arbitrary, limited in reproducibility, and has never been assessed against independent standards. we recently reported that a set of cytotoxic t cell-associated transcripts (qcats) could quantitatively assess the t cell burden in tissue. objective of the present study was to re-examine the current diagnostic criteria in relationship to the qcats. in 129 renal allograft biopsies, we assessed how histology predicts the qcat burden. an independent diagnostic threshold for qcats was established in control samples. applying this qcat threshold revealed current diagnostic criteria for histology to be flawed; the threshold for interstitial infiltration is too high (100% specificity, 37% sensitivity), the types of infiltration to be considered (i.e. i-banff) are wrongly defined, and tubulitis is not increasing diagnostic accuracy. changing the criteria by lowering the histological threshold for cortical infiltration from 25% to 10%, taking into account all interstitial cellular infiltration (= i-total) and ignoring tubulitis increased sensitivity to 91% with a decreased specificity of 50%. but, this includes biopsies having interstitial infiltration without qcats, indicating that histology might not discriminate between 'active' and 'inactive' infiltration. both the refined histological and the qcat threshold had prognostic value in terms of future renal allograft function. we propose a refined histological scoring system that better predicts the active t cell burden and outcome than the current banff criteria for renal allograft rejection. there has been an increasing and well justified interest regarding the long term renal consequences of kidney donation. the collective evidence suggests, however, that kidney donors enjoy a normal life span and their lifetime risk of esrd may not be different from non-kidney donors. assessing kidney function utilizing serum creatinine is not without limitations. therefore, to better assess kidney function, 5 yeas ago, we began a large effort to measure gfr using the plasma disappearance of iohexol and first void urinary albumin/creatinine ratio (acr) in randomly selected kidney donors who donated at our institution. results: we have performed over 3600 donor uninephrectomies since the inception of our program in 1963. of these, 242 donors underwent iohexol gfr at our general clinical research center. microalbuminuria is defined as acr between 30-300mg/g and macroalbuminuria as acr>300mg/g. the mean age was 53.0±9.6 years, 11.9±8.8 years have elapsed since donation, hemoglobin was 13.7±1.23g/l, systolic blood pressure (sbp) was 121.7±14.7mmhg and diastolic blood pressure (dbp) was 73.2±9.1mmhg. 82.6% of donors had a gfr greater than 60ml/min/1.73m 2 and 17.4% had a gfr between 30-60ml/min/1.73m 2 . the detailed renal profile of these donors is shown in the table below. multivariate analysis that adjusted for age, gender, ethnicity, time from donation, sbp, dbp and body mass index (bmi) identified age at donation, female gender and bmi as independent predictors for gfr<60ml/min/1.73m 2 and time from donation and systolic blood pressure as independent predictors for micro and macroalbuminuria. conclusion: this is the largest effort describing measured gfr in previous kidney donors. it is reassuring to find out that the majorities have a preserved gfr and only a minority has albuminuria. the risk factors for reduced gfr and albuminuria are analogous to what has been described in the general population. cystatin c levels and long-term donor health markers cystatin-c <1 (n=65) psychosocial and physical health of lkds following donation is of utmost importance. unfortunately, this issue has been neglected to a large extent. we sought to examine the current practices regarding psychosocial follow-up of lkds after surgery across transplant (tx) centers. we conducted a 68-question online survey regarding this practice and issues related to lkd. the survey was e-mailed via listservs of 2 professional tx societies. several questions allowed for more than one response. characteristics of the 69 tx centers that participated in the survey are found in table 1 . only 38.3% actively follow donors regarding mental health, substance abuse, or quality of life issues after donation. the professionals most often involved are social workers (72%) and nurse coordinators (69%). the majority of follow-up is conducted via phone (83.3%); though appointments (69.4%) and questionnaires (11.1%) are also utilized. 65% initiate follow-up with donors 1-4 weeks post-operatively, whereas only 27% of programs maintain contact at both 3-6 months and 12 months. 83.3% offer post-operative psychosocial support; 62% only under certain circumstances. this support is provided by social workers (90%), psychologists (30%), and/or psychiatrists (28%). support is available indefinitely in 68% of programs. 30.5% assume the costs of post-operative medical and psychosocial care indefinitely; 50.8% for a specified period of time. 40.7% bill the recipient's insurance and 15.2% bill the donor's insurance. 83.1% of centers accept donors without health insurance and only 5.1% purchase insurance on behalf of donors to cover post-operative health care needs. the results of this survey clearly demonstrate that post-operative psychosocial follow-up of lkds is uncommon and that current practices are widely variable. without routine follow-up of donors, tx centers are less likely to capture post-operative psychosocial issues that may result from organ donation. standardized post-operative follow-up of lkds should become a mandatory part of their care, which will require increased support from health care policy makers. the role of hepatitis c and race in patient and graft survival in combined kidney and liver transplantation. dilip moonka, 1 ravi k. parasuraman, 2 kim a. brown, 1 alissa kapke, 3 dean y. kim. 4 1 gastroenterology, henry ford health systems, detroit, mi; 2 nephrology, henry ford health systems, detroit, mi; 3 biostatistics, henry ford health systems, detroit, mi; 4 transplant institute, henry ford health systems, detroit, mi. the influence of hepatitis c (hcv) and race are not well understood in combined kidney-liver transplant (klt). hcv has a negative impact on patient and graft survival in liver recipients whereas african-american (aa) race is a negative prognostic factor in kidney recipients. aim: to determine the influence of hcv and aa race on patient and graft survival in klt. methods: the unos public use database was used to identify 2296 patients undergoing klt who had known hcv ab status. 13% were aa and 68% were non-hispanic white (nhw). 39.5% were hcv ab positive. groups were assessed for patient and graft survival. results: there is a significant gradient in patient survival and both kidney and liver survival from nhw patients without hcv to aa patients with hcv (table) . the patient survival at five years drops from 72% to 54% along this gradient. there is also a 14% drop in liver and an 18% drop in kidney graft survival. on pairwise testing, the difference in patient survival at 5 yr between all patients without hcv and those hcv ab positive drops from 72% to 60% (p=0.0003). the difference in 5 yr survival between all nhw and aa patients (regardless of hcv status) drops from 68% to 57% (p=0.221). on multivariate analysis, hcv and the combination of hcv and aa race remains associated with diminished survival but race alone does not. conclusions: patients with hcv who undergo a klt transplant are at increased risk for poor overall survival and poor survival of kidney and liver grafts. this effect appears even more pronounced in aa patients. this knowledge is critical to individual centers in assessing risk and benefit from klt in these groups and these groups represent an opportunity for improved interventions. kidney: pediatrics background: pediatric renal transplant recipients have excellent short-term outcomes but long-term success is compromised by complications of chronic immunosuppressive medications and chronic allograft nephropathy. studies show that calcineurin inhibitors and steroids can be individually avoided in pediatric renal transplantation. building on that experience we designed this study to optimize short and long-term renal allograft function with minimal chronic immunosuppression using a steroid-free, calcineurininhibitor withdrawal protocol in low risk pediatric renal transplant recipients. methods: unsensitized pediatric recipients of a first living donor kidney transplant received 2 doses of campath-1h ® (0.3 mg/kg), 1 day pre-and post-transplant. subjects received tacrolimus and mmf immediately post-transplant until week 8-12 when they underwent protocol renal biopsy and were changed to sirolimus and mmf if rejection free. the planned 35 subjects have been enrolled; this report describes the clinical outcomes of the 23 with 1 year of follow up. results: the mean subject age is 12.9 yrs; 59.3% are female and 70.4% caucasian. at transplant, 16/23 were cmv seronegative and 16/23 were ebv seronegative. protocol therapy was discontinued in eight subjects due to: rejection (3), mouth ulcers (2), leukopenia (1) , unrelated (2). clinical acute rejection (ar) occurred in 4 subjects (17%) and 2 had subclinical ar; ar was cellular rejection in 5 subjects, and humoral in 1 at 4 days post-transplant who had an undetected positive crossmatch to class ii hla. there were two graft losses, one due to recurrent fsgs and one due to medication non-adherence. there were no cases of ptld and no deaths. leukopenia occurred in 11 subjects (47.8%). there were 9 infections of which 7 were urinary tract infections (34.7%) and 2 were pneumonia (8.7%). conclusions: minimization of immunosuppression using a steroid-free, calcineurinwithdrawal protocol in low risk pediatric renal transplant recipients appears to be well tolerated with acceptable rates of clinical ar and no serious infections 12 months after transplantation. male; 57% white). substantial increases in bmiz were observed within the first 6 mo.; no changes were seen from 6 to 48 mo. baseline bmiz category (<-2.0, -2.0 to +2.0, >+2.0) influenced the pattern of change. subjects with low bmiz (<-2.0) at baseline experienced the greatest increases in bmiz, but overweight was rare; increases tended to result in a bmiz in the healthy range. those with high bmiz (>+2.0) at baseline demonstrated no significant change in bmiz post-tx. younger age at tx (highest risk for those 2 to 5 y. at tx) more remote date of tx, and baseline bmi between the 25 th and 75 th percentiles were significant independent risk factors for unhealthy weight gain both at 12 mo. and persisting at 48 mo. post-tx. weight gains occur early after tx and tend to persist. counseling focused on prevention of weight gain should be a routine part of post-tx care, with the most intense efforts concentrated on the highest risk patients: young, healthy weight children. avoidance of early weight gains may have an important impact on bmi over the long term. referral. lindsey a. pote, 1 jennifer trofe, 1 erin h. wade, 1 jorge baluarte, 3 alden doyle, 2 simin goral, 2 karen warburton, 2 robert grossman, 2 jo ann palmer, 3 roy d. bloom. 2 1 pharmacy, hosp of the univ of penn; 2 nephrology, univ of penn; 3 transplant surgery, children's hospital of philadelphia. intro: few data exist on outcomes in pediatric kidney recipients who transition to an adult transplant program. purpose: to examine outcomes in pediatric kidney recipients who transition to an adult transplant program and to identify characteristics associated with non-adherence related graft loss. methods: retrospective, single center analysis of 41 pediatric kidney recipients who transitioned to an adult program. results: for the cohort overall, transition to the adult program occurred a mean of 82 ± 8.7 months following transplantation. mean serum creatinine at transition was 2.6±3.2 mg/dl and mean patient age 21.2 ± 0.3 years. 61% of patients received living donor kidneys. within 31±3.5 months of transition, 13 (31.5%) of patients experienced graft loss. causes of graft loss included admitted non-adherence (n=6), recurrent disease (n=3), chronic progressive graft dysfunction (n=3) and bk nephropathy (n=1). graft loss occurred a mean of 16±7 months post transition for non-adherent patients and 20.5± 3 months for adherent patients (p=ns). the characteristics of the cohort is shown in the table according to whether or not patients had documented non-adherence related graft loss. conclusions: 1) graft loss commonly occurs within 3 years following transition and is attributable to both patient non-adherence and late referral by the pediatric transplant program, 2) non-adherence related graft loss was more common in males, 3) factors unassociated with non-adherence include ethnicity, prior transplantation, age at transplant, & duration of dialysis, 4) the development of collaborative pediatric-toadult transition clinics may enhance adherence and lead to improved graft outcomes in this population. this 2-year, prospective randomized pilot study compares the effect of conversion to sirolimus (srl) vs. continued mycophenolate meofetil (mmf) in patients with chronic allograft nephropathy (can), on histological progression. we present 2-year data on safety and renal function. participants >1 year post-transplant with can (banff ≥ci1, ct1) on tacrolimus, mmf and prednisone were randomized to continue mmf or convert to srl (target 8-12 ng/ ml). tac dose was minimized (target 3-5 g/l), and renal biopsy performed at baseline, 1, 2 years. 3-month interval monitoring included adverse event (ae) reporting, egfr (schwartz) and immunosuppressant levels. 16/20 (mmf=10, srl=10) have completed 2-year follow-up. baseline gender, ethnicity, previous acute rejection episodes, can grade, proteinuria (upcr) were similar. srl had lower baseline egfr (79±17 vs. 109±49 ml/min/1.73m 2 , p=0.22). 285 aes were reported (mmf=117, srl=168), 43 serious ae (sae: mmf=25, srl=28). common saes were similar: dehydration & elevated creatinine (30), gastroenteritis (10) and rejection (ar). 2 episodes (1 patient) of ar occurred in mmf group and 5 (2 patients) in srl group, all attributable to non-adherence. there were no sustained change in electrolytes, hg and total wbc counts in the 1 st 2 year, except neutrophil counts were lower in the srl group (24 mo: mean 4.7 vs. 2.8, p<0.05). platelets were significantly lower at 3 months (srl) but not thereafter (24 month: mean 271±71 vs. 196±61 x10e9). cholesterol and tg increased early (p<0.05), but only cholesterol persisted after 2 years (mean 5.2 vs. 3.7 mmol/l, p<0.05). in srl group only, upcr increased over 2 years (∆upcr 94±87 mg/mmol, p<0.05). this was not associated with hypoalbuminemia at 2 years. egfr was lower in srl vs. mmf after 18 months (p<0.01), but the rate of change in egfr from baseline was similar between groups (-28±23 vs. -30±39 ml/min/1.73m 2 , p=ns). serious adverse events did not differ significantly between the srl and mmf groups. sustained srl treatment was associated with mild neutropenia, hypercholesterolemia and proteinuria after 2 years. egfr was reduced at baseline compared with mmf, and both groups had similar rates of decline in gfr over time. allograft recipients. maarten naesens, 1 oscar salvatierra, 1 li li, 1 minnie sarwal. 1 1 department of pediatrics, stanford university school of medicine, stanford, ca. background: in contrast to adult kidney recipients, little is known about the long-term evolution of tacrolimus pharmacokinetics in pediatric kidney transplant recipients. methods: one-hundred five pediatric recipients of a kidney allograft, all treated with a corticosteroid-free immunosuppressive protocol, were included. the evolution of tacrolimus doses and exposure was recorded at 3, 6, 9, 12, 18 and 24 months after transplantation, as well as all pre-dose trough levels (c 0 ; n=9376) obtained in the first 2 years after transplantation. results: dose-corrected tacrolimus exposure (c 0 /dose/kg) increased in the first 2 years after kidney transplantation in pediatric recipients (table 1, figure 1 ). this decrease in dose requirement by time was only significant in children older than 5 years at the time of transplantation ( figure 1 ). in addition, the younger patients had significantly higher dose requirements compared to older recipients, which translated in marked underexposure in 72-79% of patients <12 years of age in the first days after transplantation. conclusion: pediatric kidney transplant recipients exhibit maturation of tacrolimus pharmacokinetics with time after transplantation. this can not be explained by differences in corticosteroid use, as all patients were treated with a corticosteroid-free protocol. the higher dose requirements for younger recipients and the absence of tacrolimus maturation in the youngest recipients suggest that age-dependent changes in tacrolimus intestinal first-pass effect, metabolism or distribution play a role. whether age-specific tacrolimus dosing algorithms will improve outcome needs further study. determinants of dose-corrected pre-dose trough levels (c0/dose/kg) aih may present as acute hepatitis in 25% of patients (pts) and result in alf. early administration of corticosteroids may obviate the need for liver transplantation (lt), but features which identify aih in pts with alf have not been determined. aim: to identify clinical and histological features which distinguish alf due to aih. methods: 197/1033 pts in the alf study group registry had no evidence of viral, metabolic, vascular, and drug/toxic liver injury. all had alf defined by acute disease, encephalopathy, and coagulopathy. based upon admission clinical features, 52/197 had probable aih, and 145 were considered "indeterminate." liver biopsies (lbx) available from 56 pts were reviewed by a blinded expert hepatopathologist. clinicopathologic correlations were analyzed retrospectively. results: all lbx available from pts with suspected aih had classical histologic features of aih. moreover, 23/46 (50%) lbx from pts with indeterminate alf also had aih features: extensive necrosis (100%), fibrosis (84%), cirrhosis (22%), interface hepatitis (75%), and plasma cell-rich inflammation (69%). of all 33 lbx with aih features, centrilobular lesions associated with acute, severe aih (am j surg path 2004;28:471) were frequent: plasma cellrich central venulitis (97%), exclusive centrilobular necroinflammation (19%), and pericentral dilatation/congestion (46%). clinical characteristics and outcomes of the 75 pts with aih based upon laboratory and histologic findings differed significantly from pts whose alf remained indeterminate: aih pts were older (44 v 38 y), predominantly female (75 v 56%), and had longer jaundice-encephalopathy interval (20 v 12 d) , lower alt (660 v 1949 u/l), higher globulins (3.7 v 2.7 g/dl), lower creatinine (1.7 v 2.3 mg/ dl), and a higher prevalence of ana (68 v 24%) and asma (51 v 29%) (all p<.01). although spontaneous survival did not differ, more aih pts underwent lt (63 v 34%), and more survived 1 month after enrollment (75 v 59%; p<.0001). conclusions: using histologic criteria to classify pts with indeterminate alf, aih accounted for at least 7% of the alf study group registry, and represented 50% of indeterminate alf. lbx should be performed in all patients with alf of obscure etiology, in particular to identify centrilobular necroinflammatory lesions, which appear to be specific indicators of acute and severe aih. (u-01 58369 from niddk). (2002) (2003) (2004) (2005) (2006) . mikel gastaca, 1 miguel montejo, 1 lluis castells, 2 antonio rafecas, 3 antonio rimola, 4 ramon barcena, 5 federico pulido, 6 magdalena salcedo, 7 martin prieto, 8 manuel de la mata, 9 jose r. fernandez, 1 jose m. miro, 4 the spanish lt in hiv-infected patients working group. 1 hospital de cruces, bilbao, spain; 2 hospital vall d´hebron, barcelona, spain; barcelona, spain; univ. of barcelona, barcelona, spain; 5 hospital ramon y cajal, madrid, spain; 6 hospital 12 de octubre, madrid, spain; 7 hospital gregorio marañon, madrid, spain; 8 hospital la fe, valencia, spain; 9 hospital univ. reina sofia, cordoba, spain. background and aim: we report on the preliminary results of the prospective multicenter spanish study in hiv-1 infected patients who underwent olt. methods: the prospective multicenter spanish study fipse-olt-hiv 05-gesida 45-05 was initiated in january 2002. inclusion criteria follows the rules previously described in the spanish consensus document. hiv-infected patients transplanted between january 2002 and december 2006 were included in this study. results: 89 olt were consecutively performed in 85 hiv-1 infected patients in the period of the study. median (iqr) follow-up is 16 (8-29) months. median (iqr) age was 42 years (39-46), 74% of the recipients were male and former drug abuse was the most common hiv-1 risk factor (78%). 83% of the patients were transplanted due to hcv-related cirrhosis and 6% due to hbv cirrhosis. median (iqr) meld score was 14 (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . pre-olt median (iqr) cd4 cell count was 288 (165-486) cells/mm3 and 64%patients had undetectable plasma hiv viral load. immunosuppression was based on tacrolimus in 70% of the patients. haart was re-started in a median (iqr) time of 10 (5-21) days after olt and was based on efavirenz in 48% of the cases. acute rejection occurred in 38 patients (43%). twenty patients died (22.5%) mainly due to hcv recurrence (8%). antiviral therapy with peg-interferon plus ribavirin was initiated in 18 patients obtaining a sustained viral response in 6 of them (33%). patient survival (95% confidence intervals) at 1, 2, 3 and 4 years was 90% (81-95%), 74% (60-84%), 67% (52-79%) and 67% (52-79%), respectively. conclusion: for selected hiv-1 infected patients under haart therapy, olt is a safe and effective procedure at mid-term. as in the hiv-negative population, hcv recurrence is the mayor cause of concern and response to antiviral therapy is still disappointing. hepatitis c virus and objectives: describe the 12-year experience of a university hospital treating chronic hepatitis c relapse, histologically diagnosed after liver transplant. material & methods: from september 1991 through july 2006, all the patients subject to liver transplant with histological diagnostics of chronic hepatitis relapse were submitted to antiviral treatment. treatment was suspended if there was a severe adverse reaction to the drugs used, non-adherence, severe rejection or no response after at least 12 months. hcv positive serology patients, alt increase (1,5x nl) and hepatic biopsy with metavir rating showing structural 1 and/or periseptal or parenchymatous portal inflammatory activity /= 2 were included. from 1995 to 2003, all patients were treated with conventional interferon and ribavirine, regardless of the genotype. from october 2003 on, patients with genotype 1 were treated with ribavirine and pegylated interferon and genotype 3 were treated with conventional interferon and ribavirine. results: 47 patients were treated during this time period, thirty-seven male (78.7%). their average age was 48. 51.1% were genotype 1, 25.5% were genotype 3. average time between transplant and beginning of treatment was 30 months. average treatment was 16 (4-36) months. 74.5% received conventional interferon, 6.4%, pegylated alpha 2a and 19.1%, pegylated alpha 2b. 48.9% had one or more cellular rejection episodes before vhc relapse diagnostics and were treated with corticosteroids. sustained virological response (svr) occurred in 48.8%. three patients had a virological response at the end of treatment (evr) and have not completed six months post treatment. out of 21 patients with svr, 20.9% were genotype 3, 9.3% genotype 1 and 18.6% were not genotyped. considering genotypes, svr was detected in 16.7% with genotype 1, in 75% with genotype 3. srv was detected when we examined hepatic biopsy, in 85.7% of the f1/f2 patients (18 out of 21), 9.5% of the f3 patients (2), 4.8% of the f4 patients (1) . survival period of 5 years for svr patients was 94% (20 out of 21) and 68.2% for patient without svr (p = 0.03 -kaplan-meier). five-year survival was 79.2% for patients with genotype 1 and 100% for patients with genotype 3. conclusions: our results show it is possible to get good five-year survival rates for treated patients. however, handling adverse reactions and long term treatment still pose difficulties in pursuing better svr rates. the impact of alcoholic liver disease (ald) and background: although liver transplant survival benefit has been shown to be associated with meld score, disease-specific analyses have not been reported. we evaluated, using srtr data, the effect of ald and/or hcv infection on transplant waitlist and post-transplant mortality, and survival benefit of deceased donor liver transplants. methods: 38,889 patients age ≥18, who were listed between sept 2001 and dec 2006, and followed until dec 31, 2006, were classified into 4 cells according to hcv or ald status: hcv+, ald-: 12,322; hcv+, ald+: 4,220; hcv-, ald+: 6,581; hcv-, ald-: 15,776). cox regression was used to estimate waiting list mortality and post-transplant mortality separately. survival benefit, which encompasses both pre-and post-transplant events, was assessed using sequential stratification; an extension of cox regression which matches transplant recipients by meld and organ procurement organization to patients on active on the waitlist at the time of transplant. results: hcv significantly (p=0.0001) increased waitlist mortality, with a covariate-adjusted hazard ratio (hr) for hcv+ vs. hcv-of hr=1.19 (p=0.0001). the impact of hcv+ was greater among ald+ candidates (hr=1.36; p<0.0001), but was also significant among ald-candidates (hr=1.11; p=0.02). the contrast between ald+ and ald-waitlist mortality was only significant among hcv+ candidates (hr=1.14; p=0.006). post-transplant mortality was significantly higher among hcv+ vs. hcv-recipients (hr=1.26; p=.0009); there was no difference between ald+ vs. ald-recipients. survival benefit of liver transplantation was significantly lower among hcv+ compared to hcv-recipients with meld 9-29, but significantly higher for hcv+ recipients with meld scores of ≥30. a diagnosis of ald did not influence the survival benefit of transplantation at any meld score. conclusion: despite higher waitlist mortality, hcv+ recipients had significantly lower liver transplant survival benefit than hcv-recipients, within categories defined by meld score. in contrast, transplant survival benefit was not influenced by ald. transplanted for hbv-related cirrhosis. giuseppe tisone, 1 daniele di paolo, 1 ilaria lenci, 1 laura tariciotti, 1 andrea monaco, 1 manuele berlanda, 1 linda de luca, 1 giuseppe iaria, 1 alessandro anselmo, 1 irene bellini, 1 mario angelico. 1 1 hepatic surgery and transplantation, university of rome tor vergata, rome, italy. background and aim: post-transplant active immunization with hbsag vaccine is a potential prophylaxis strategy against hbv-recurrence after liver tranplantation due to hbv-related disease. previous studies showed conflicting results using standard vaccines, whereas the use of the new adjuvant 3-deacylated monophosphoryl-lipid-a (mpl) significantly increased patient's immunization rate. we investigated the efficacy of a long-term (12 months) accelerated (monthly doses) vaccination schedule using the mpl-adjuvanted vaccine administered with and without concomitant hbig. methods: 18 patients (m/f:13/5) transplanted for hbv-related cirrhosis 73±38 months earlier were recruited. all were hbsag and hbv dna negative in serum and cccdna negative in liver tissue; 5 (27.7%) were co-infected with hcv and 5 (27.7%) with hdv. study protocol consisted of 12 consecutive monthly intramuscular vaccine doses (hbsag 20mg plus mpl 50mg) given together with lamivudine (100 mg/daily). each of the initial 6 doses (first cycle) was administered within 7 days after 2000 iu hbig i.v. infusion, while the last 6 doses were given after complete hbig withdrawal (second cycle). hbsab titre was determined before each vaccine dose and during the follow-up. all patients were maintaiened on low-level immunosuppression. preliminary results: all patients completed first vaccination cycle; 14 (77.7%) patients received 12 adjuvanted vaccine doses (first and second cycles) and were monitored during 3 months follow-up after vaccination end. no side effects occurred, nor evidence of hbv recurrence. at the end of first cycle all patients achieved an anti-hbs titre >100 iu/l (mean 343±209 iu/l) and 3 (16.6%) a titre greater than 500 iu/l. at the end of follow-up 8/14 (57.1%) and 4/14 (28.5%) had an anti-hbs titre greater than 100 (mean 411±599 ui/l) and 500 iu/l, respectively. conclusions: nine months after hbig withdrawal more than half of the patients reached and maintained a protective anti-hbs titre (>100 iu/l). this intensive schedule using the mpl-adjuvanted vaccine, given in combination with hbig and lamivudine, seems to be more effective than previous hbv vaccination protocols, although a longer follow-up is needed to assess its final effectiveness. heterologous immunity via alloimmune responses to hepatitis c virus replication after liver transplantation. hideki ohdan, 1 masahiro ohira, 1 yuka tanaka, 1 kohei ishiyama, 1 nobuhiko hiraga, 2 michio imamura, 2 kazuaki chayama, 2 toshimasa asahara. 1 1 surgery, hiroshima university, hiroshima, japan; 2 internal medicine, hiroshima university, hiroshima, japan. the immunosuppressive environment in liver transplantation (lt) recipients infected with hcv is believed to be associated with the progression of hcv reinfection. in the present study, we simultaneously monitored hcv levels and alloimmune status in hcv-infected lt recipients. for evaluating the immune status of 33 hcv-infected lt recipients, we employed a mixed lymphocyte reaction (mlr) assay using a cfselabeling technique. the kinetics of the stimulation index of anti-donor reactive cd4 + t cells clearly mirrored that of the hcv rna titer, and a significant reverse correlation was observed between the 2 parameters (r 2 = 0.67). we did not observe a similar relationship between the stimulation index of an anti-third party cd4 + t cells and the hcv rna titer (r 2 = 0.03). one possible explanation for this phenomenon might be that cytokines outputted by t cells responding to allostimulation display the anti-hcv activity either directly or indirectly through the activation of bystander immunocytes. to investigate such possibilities, we performed a transwell culture assay comprising a mlr culture in the upper chamber and genomic hcv replicon cell culture in the lower chamber to mimic the anatomical features of the interaction between hcvinfected hepatocytes and alloreactive t cells that infiltrate the portal area in the liver. when one-way mlr was performed using one-halpoidentical combination in the upper chamber, hcv replication was significantly suppressed in the lower chamber containing hcv replicon cells. hcv replication was further suppressed when mlr was carried out with a complete allogeneic combination. this inhibiting effect was dependent on their ifn-γ secreting activity. in addition, a similar result was obtained when ifn-γ-secreting nk/nkt cells stimulated with il-2 were cultured in the upper chamber. in conclusion, there was a close relationship between the anti-donor and the anti-hcv immune status in the lt recipients infected with hcv. cytokines such as il-2 and ifn-γ may be produced in response to allostimulation, and even these cytokines do not cause graft rejection, display anti-hcv activity. the elucidation of such a possible heterologous immunity via alloimmune responses to hcv replication might lead to the establishment of a novel method to prevent the progression of hcv reinfection in hcv-infected lt recipients. hepatitis c e2 region diversity after liver transplantation: a report from the hepatitis c iii trial. juan f. gallegos-orozco, 1 hugo e. vargas, 1 georges netto, 2 gary l. davis, 3 , and 7.3% are morbidly obese . the goal of this study was to quantify the effect of bmi on access to transplantation (att), likelihood of receiving a transplantation (lrt), turndown rates (tr), and survival benefit (sb). methods: att was defined as either registering for the deceased donor waiting list or receiving a live donor transplant, and was analyzed based on the usrds registry using logistic regression. lrt was based on time spent in active status on the waiting list before receiving a transplant, and was analyzed based on the unos waiting list dataset using cox proportional hazards time-to-event analysis. tr was defined as the relative likelihood of being turned down for an organ offer by a provider other than the patient, and was analyzed based on the unos organ turndown dataset using negative binomial regression. sb was defined as survival after kidney transplantation versus survival on the deceased donor waiting list, and was analyzed based on the unos waiting list dataset using a time-dependent cox survival benefit model. all models were adjusted for known factors influencing those outcomes. lgbp lsg lsg mean follow-up (months) 15.4 (range 3-24) 9 (range 3-18) 12.5 (12, 13) patients > 9 months since operation (n) 6/7 4/6 2/2 mean % ewl at > 9 months 61 (range 41-75) 33 (range 24-40) 62 (50, 73) transplant candidate at > 3 months 7/7 5/6 2/2 underwent transplant 0/7 1/6 1/2 complications developed in two patients (both with cirrhosis) and there was no mortality. mean follow-up was 12.3 months, and mean ewl at 9 months or later was 61% (esrd), 33% (cirrhosis) and 61.5% (esld). obesity associated comorbidities were improved or resolved in all patients. serum albumin and other nutritional parameters 9 months or later after surgery were similar to preoperative levels in all 3 groups. at most recent follow-up, 14 out of 15 patients (82%) have reached our institution's body mass index (bmi) limit for transplantation and are awaiting transplant; one patient with esld has undergone a successful lung transplant and one patient with cirrhosis has undergone a successful liver transplant. factors that contribute to inequitable access to the transplantation network (unos/ optn) include socioeconomic status, geographic location and delayed referral. the goal of the meld allocation system was to assign priority to the sickest candidate and assure equitable access to liver transplantation (lt). the meld has been validated as a reliable marker for liver disease severity. at the time of referral to the transplant center or listing a high meld (hm) is an indirect measure of delayed referral. therefore, the aim of this study is to identify factors associated with a hm at the time of listing for lt. method: using the unos database, we identified all adult candidates listed for lt from 2002 to 2006. patients who received meld upgrade (i.e. hcc, fhf) and those listed for multi-organ transplant were excluded. the data collected included demographics, insurance payor, diagnosis, and meld score. meld score at time of listing was categorized as low (< 20) and high (>20) and insurance type as private, medicaid and government (excluding medicaid) . results: during the study period 15,323 candidates were added to the lt waiting list. of these, there were 10,302 (67.2%) males with a median age of 53 (range 18 -83) . caucasians were 11,511 (75.1%), hispanic (11.6%), african americans (8.9%) and the rest (4.4%). the underlying liver disease was hepatitis c (31.9%), alcohol (15.1%), alcohol/hepatitis c (7.2%), pbc/ psc (9.9%), cryptogenic (7.5%) and others (28.4%). age, gender and liver disease etiology were not associated with a hm at listing. a hm was associated with ethnicity: aa (49.5%), hispanic (44.2%), caucasian (36.6%)[p<0.001] and insurance type: medicaid (46.3%), government (38.8%) and private (36.5%)[p<0.001].there was no strong interaction between ethnicity/race and insurance in combination as predictors of hm i.e. both were independent. conclusion: (1) aa and lt candidates with medicaid insurance are more likely to have a hm score at initial listing (2) hispanics had the lowest rate of private insurance compared to caucasians with the highest rate. the above results suggest that type of insurance and ethnicity are independently associated with a hm (i.e. sicker patients) at listing. since a hm score is associated with increased mortality, implementation of strategies that result in timely and equitable access to the transplantation network regardless of the insurance type or ethnicity of the candidate(s) should be a priority. marked increase in the use of inactive status on the kidney transplant waiting list. kim nguyen, 1 valarie ashby, 1, 2 further wait time accrued only after active status was restored. on 11/5/03, the optn implemented a change in policy that provides for the accrual of waiting time during the entire interval that wl candidates are designated as s7. methods: we investigated the impact of this policy change on the patterns of s7 designation, using the srtr/optn database. initial and subsequent status on the wl were determined for 130,986 candidates placed on the ki tx wl from 11/5/00-11/4/06. the probability of becoming active (including receiving a living donor tx) on the wl was calculated for those who were s7 at listing before and after the policy change. results: table 1 shows trends in the use of s7 before and after the policy change. of the 1,396 candidates listed as s7 before the policy change, 76% became active within 6 months and 83% within 1 year of wl. for the 11,248 candidates listed as s7 after the policy change, the corresponding figures are 48% and 60% respectively. figure 1 demonstrates that, since the implementation of the new policy, the % of patients initially wl in s7 at most us tx centers has increased. conclusion: since the implementation of this policy, the number and % of pts wl as s7 at most ki tx centers, and the duration of s7 for those initially wl as s7 has increased dramatically. the implications of this practice on access to ki tx and survival warrants further investigation. can abstracts (11%) patients were retransplanted, and 13 (9%) patients had panel reactive antibody >30. the mean cumulative thymoglobulin dose administered was 5.7 mg/kg, and the primary maintenance immunosuppression started prior to discharge was a sirolimuscontaining regimen (59%). at the end of the follow-up period, 98 (65%) patients had functioning grafts and 52 (35%) patients experienced graft loss. of the 52 patients with graft loss, 15 (10%) patients experienced graft loss secondary to death. no pertinent differences were identified between groups. the graft survivals for african american recipients with african american or caucasian donors were 95% vs. 91% at 1 year and 83% vs. 72% at year 2. conclusions: our study results suggest that donor race does not adversely affect graft outcomes in african american cadaveric kidney recipients with modern immunosuppression. donor racial differences may not play a primary role in the inferior graft outcomes of african american cadaveric kidney recipients. cardiac evaluation before kidney transplantation: are we screening too often or not enough? krista l. lentine, 1 m. a. schnitzler, 1 j. j. snyder, 2 d. c. brennan, 3 p. hauptman, 1 p. r. salvalaggio, 1 b. kasiske. 4 evaluation for ischemic heart disease (ihd) is a common but non-standardized practice before kidney transplant. we retrospectively studied pre-transplant cardiac evaluation (ce) practices among a national sample of renal allograft recipients. methods: we examined usrds data for medicare beneficiaries transplanted in 1991-2004 with part a and b benefits from dialysis initiation through transplant. clinical traits defining "high" expected ihd risk were defined as diabetes, prior ihd, or >2 other coronary disease risk factors. pre-transplant ce were identified by billing claims for noninvasive stress tests and angiography. we quantified individuals with claims for coronary revascularization procedures between ce and transplant, and abstracted post-transplant acute myocardial infarction (ami) events from claims and death records. results: among 27,786 eligible patients, 46.3% (65.4% of high-risk and 20.4% of lower-risk) underwent ce before transplant. overall, 9.5% of patients who received ce also received pre-transplant revascularization, including only 0.3% of lower-risk patients studied by ce ( table a) . the adjusted odds of transplant without ce (higher or for no ce, table b ) increased sharply with younger age and shorter dialysis duration. increased likelihood of transplant without ce also correlated with black race, female sex, and certain geographic regions. post-transplant ami rates in patients transplanted without ce allow assessment of whether ce was appropriately deferred in those who indeed face low ihd-risk after transplant. among patients transplanted without ce, the 3-yr incidence of post-transplant ami was 3% and 10% in lower and high-clinical risk groups, respectively, but varied by clinical traits within these groups. in lower-risk patients transplanted without ce, blacks patients faced increased ami-risk compared to whites (adjusted hr 1.52, 95% ci 1.16-2.00). conclusions: observed ce practices demonstrate a low yield of pre-transplant revascularization but also raise concern for socio-demographic barriers to evaluation access. women who become pregnant in the first two years after kidney transplantation have a higher risk of graft loss. nadia zalunardo, 1 olwyn johnston, 1 caren l. rose, 1 john s. gill. 1 1 division of nephrology, university of british columbia, vancouver, bc, canada. existing information regarding the risks of pregnancy is derived from voluntary data sources. using medicare claims files from the usrds (1990 usrds ( -2003 , we examined pregnancies (defined by presence of an inpatient icd-9 billing code for pregnancy or pregnancy-related complication) in the first 3 years after kidney transplantation (ktx) among women aged 15-45 years whose primary insurance payor was medicare (n=16,195) . patients were followed until graft failure, death or december 31, 2003. there were 530 pregnancies identified in 483 women during the first 3 post-transplant years. women who became pregnant during the 1st or 2nd post-transplant year had a shorter time to graft loss than women who became pregnant during the 3rd year (figure). to minimize the survivor bias among women who became pregnant during the 3 rd posttransplant year, we determined the association between the timing of pregnancy and graft survival among the subset of women (n=455) who had graft survival of at least two years using a cox multivariate regression adjusted for age, race, cause of esrd, donor source, calendar year of transplant, dialysis vintage, maintenance immunosuppression, and gfr at 6 months after ktx. pregnancy in the 1 st year (hr 2.01, 95% ci 1.35 -2.99), and second year (hr 1.79, 95% ci 1.23 to 2.62) was associated with an increased risk of graft loss compared to pregnancy during the third post-transplant year. we concluded that women who are able to wait should be counseled to become pregnant after the second post transplant year. the aging donor and recipient populations have led to new challenges in simultaneous kidney-pancreas transplantation (skpt). the purpose of this study was to retrospectively review our single center experience in skpt with respect to extended (ex) donor (d) and recipient (r) criteria. methods: over a 65 month period, we performed 83 skpts with enteric drainage (79 portal venous drainage). ex ds were defined as age <10 (n=4), >45 (n=12, mean age 50.2 yrs), or donation after cardiac death (dcd, n=4). all dcd donors were managed with extracorporeal support. ex rs were defined as age >50 (n=20, mean age 55.8 yrs) or those with a pretransplant serum c-peptide level >2.0 ng/ml (n=7, mean 5.7 ng/ml). all rs received depleting antibody induction (57 ratg, 26 alemtuzumab) with tacrolimus, mmf, and tapered steroids (39 steroid-free). results: a total of 20 ds (24%) and 23 rs (28%) met the above ex criteria. median waiting time was 10 months, mean pancreas preservation was 17 hours, and median length of stay was 10 days. with a mean follow-up (f/u) of 28 months, patient (pt) (95% ex d vs 94% non-ex d), kidney (90% ex d vs 89% non-ex d) and pancreas graft survival (85% ex d vs 81% non-ex d) rates were similar between d groups (all p=ns). the incidences of delayed kidney graft function (5% in each group) and early pancreas graft loss due to thrombosis (5% ex d vs 8% non-ex d) were also comparable between d groups. with regard to r groups, pt (87% vs 97%, p=0.13) and kidney (87% vs 92%, p=ns) graft survival (gs) rates were slightly lower in the ex r group compared to the non-ex r group, respectively. however, death-censored kidney gs rates (100% ex r vs 95% non-ex r) were comparable between groups. uncensored pancreas gs rates (83% ex r vs 82% non-ex r) were similar. the incidences of acute rejection, surgical complications, infection, and other morbidity were comparable regardless of d or r group. at 1 year (or latest) follow-up, renal and pancreas functional parameters were similar between d groups. however, the ex r group demonstrated slightly compromised renal and pancreas allograft function and a greater need for oral hypoglycemic agents. conclusion: intermediate-term outcomes in skpt from selected ex ds or rs have comparable outcomes, although ex r criteria may represent a risk factor for pt survival and functional outcomes. conclusion: spk recipients with functioning pancreas grafts have significantly improved kidney and patient survival compared to ld ka and dd ka. however, early pancreas graft failure results in kidney and patient survival rates similar to ka recipients. spk provides optimal outcomes for patients with dm1, but long-term risk associated with early pancreas loss may be a consideration when selecting spk vs ka transplantation. the impact of long-term metabolic control on renal allograft and patient survival in type 1 diabetes. christian morath, 1 martin zeier, 1 bernd dohler, 2 jan schmidt, 3 peter p. nawroth, 4 gerhard opelz. 2 1 nephrology, university of heidelberg; 2 transplantation immunology, university of heidelberg; 3 transplantation surgery, university of heidelberg; 4 endocrinology, university of heidelberg. it is a matter of debate whether pancreas allografts independently contribute to renal transplant and patient survival in type 1 diabetics who received a simultaneous pancreas kidney transplant (spk). using the data of the collaborative transplant study (cts), we studied type 1 diabetic recipients of deceased donor kidneys (ddk), living donor kidneys (ldk), or spk performed during two time periods: 1984 to 1990 and 1991 to 2000 . we analyzed graft and patient survival rates for a maximum of 18 years. ddk recipients showed inferior graft and patient survival compared to ldk and spk recipients in both time periods. ldk recipients had a superior graft survival rate initially, but the survival rate of kidneys in spk recipients reached the level of ldk toward the end of the follow up period. the results of patient survival paralleled those of kidney graft survival: an early advantage of ldk as compared to spk faded away during follow up. multivariate analysis, in which pretransplant cardiovascular risk assessment was appropriately considered, showed that patient survival of spk recipients was superior to that of ldk recipients beyond the tenth year after transplantation (hazard ratio hr = 0.55, p = 0.005). this was reflected by a lower cumulative cardiovascular death rate in recipients of spk (37.0 %) compared to recipients of ddk (45.8 %) or ldk (49.3 %) . the early survival benefit of ldk compared to spk is lost during long-term follow up, probably related to improved glycemic control in spk recipients. proposal for a grading rejection schema in pancreas allograft biopsies from a multidisciplinary panel of pathologists, surgeons and nephrologists. 1.normal 2.indeterminate septal inflammation that appears active but the overall features do not fulfill the criteria for mild cell mediated acute rejection. active septal inflammation involving septal structures and/or focal acinar inflammation. -grade ii / moderate acute cell mediated rejection multifocal (but not confluent or diffuse) acinar inflammation with spotty acinar cell injury and drop-out and/or minimal intimal arteritis -grade iii / severe acute cell mediated rejection diffuse, (widespread, extensive) acinar inflammation with focal or diffuse multicellular /confluent acinar cell necrosis.and/or moderate or severe intimal arteritis and/or transmural inflammation -necrotizing arteritis chronic active cell-mediated rejection. chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) 4.antibody mediated rejection =c4d positivity + confirmed donor specific antibodies + graft dysfunction -hyperacute rejection -accelerated antibody mediated rejection severe, fulminant form of antibody mediated rejection with morphological similarities to hyperacute rejection but occurring later (within hours or days of transplantation). -acute antibody mediated rejection specify percentage of biopsy surface with interacinar capillaries positive for c4d. 5.chronic allograft rejection/graft sclerosis stage i (mild graft sclerosis) <30% of the core surface stage ii (moderate graft sclerosis) 30-60% of the core surface. stage iii (severe graft sclerosis) >60% of the core surface 6. other histological diagnosis e.g. cmv pancreatitis, ptld, etc. a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and positively affect patient care. rank test). similar results were obtained when death was included as a cause of graft loss (data not shown). conclusion: par is associated with worse long term kidney graft survival than kar. it is likely that patients that experience par within the first year have also experienced undiagnosed sub-clinical kar. this adds associative data to the argument that the likelihood of concordance between par and kar is high and therefore, the need for increased monitoring of kidney function after par is warranted. the most efficacious immunosuppressive (immuno) regimen for spkt is debated, and information on the best regimen to prevent amr is particularly scarce. we performed a retrospective comparative cohort study that included 136 spkt patients (pts) transplanted in 2002-2005, who received maintenance immuno with tac, mmf and steroids. two groups were compared: pts who received induction with alemtuzumab (alem) (n=97) and pts who were induced with basiliximab (basilmab) (n=39). donor and recipient characteristics were similar. kt acute cellular rejection (acr) was more frequent with basilmab (2-yr 12.8% vs 3.1%, p=0.04), but the incidence of biopsy-proven kt amr was similar (2-yr 18% with basilmab vs 13.8% with alem, p=ns). no differences in prevalence were detected considering early amr (<90th day) or late amr (>90th day) separately. multivariate analyses showed that the only significant risk factor for amr was female gender (adjusted hr 2.97, p=0.016). the biopsy-proven kt rejection was associated with clinical pt rejection in 13 out of the 21 amr cases (62%), without differences between the groups. post-rejection kt graft survival was similar in both groups (2-yr basilmab/alem 94.7/91.2%), but deathcensored kt survival was lower with alem (100/91.2%, p=0.05). the predominant cause of kt loss in pts induced with basilmab was death-with-function (n=3), while in pts induced with alem acute or chronic amr was the most common cause of kt attrition (n=5). pancreas survival was similar between both groups. more pt losses due to ar occurred in alem-treated group than in the basilmab group (4 vs 1) . nearly all early episodes resolved with treatment and were not associated with worse graft survival. conversely, late amr episodes carried a worse prognosis, with decreased 2-yr kt (61.5% vs 96.2%, p<0.0001) and pt graft survival (47.7 vs 92.3%, p<0.0001). late amr was associated with graft loss in multivariate cox models (kidney loss hr 3.34, p=0.05 and pancreas hr=3.22, p=0.049) . amr is common in spkt recipients. acr is better prevented with alem than with basilmab, but no relevant difference is found between alem and basilmab in amr. late (as opposed to early) amr episodes are associated with significant reduction in spkt survival rates despite treatment. preventive and/or different treatment strategies are required to address late amr in spkt. intraoperative fluorescence imaging (ifi) in pancreas transplantation (pt) to determine vascular patency and allograft perfusion. edmund q. sanchez, 1 srinath chinnakotla, 1 marlon f. levy, 1 robert m. goldstein, 1 goran b. klintmalm. 1 1 baylor regional transplant institute, dallas/ft. worth, tx. thrombotic complications of pt are well known. we report ifi using the spy device to assess immediate vascular patency and allograft perfusion. our controlled experience is presented here. methods: pts were imaged intraoperatively using the spy device under our irb approved study. indocyanine green solution (2.5 mg/ml) was injected into central venous catheters after completion of the vascular anastomoses of whole pancreaticoduodenal allografts. the pancreas transplants were performed in the retroperitoneal portal and enteric drained technique described by boggi, et al. imaging of the allograft vasculature, perfusion of the pancreas, and perfusion of the duodenal segments was performed and recorded intraoperatively. all video sequences were archived for later review. results: ifi on 7 pancreas transplants (6 simultaneous pancreas-kidney and 1 pancreas after kidney) was performed and video sequences were recorded. all pancreas allografts demonstrated intraoperative vascular patency and complete pancreatic and duodenal perfusion. there were no side effects seen. all pancreas transplants had immediate graft function. one patient was re-explored on postop day 0 due to persistent acidosis and hypotension. repeat ifi demonstrated vascular patency and perfusion, despite an ischemic external physical appearance. there were no vascular complications that required reoperation, nor were there any graft thromboses. characteristic slow venous outflow was seen in each case. conclusion: ifi with the spy device is a simple and effective method to determine immediate patency and perfusion of the whole pancreaticoduodenal allograft. its use is beneficial in re-exploration to rule out infarcted pancreas allografts. further development in quantification of vascular flow rates and allograft perfusion indices by this device is abstracts in progress. once these are established, this information will be studied in a controlled fashion, and will be compared with clinical outcomes. we have demonstrated safety and developed a protocol for using the spy device in ifi of pancreas transplants. tolerance/immune deviation i tolerance without immunosuppression: exploiting epigenetic regulation as a new approach to achieving donor-specific allograft tolerance. liqing wang, 1 ran tao, 1 joel a. friedlander, 1 wayne w. hancock. 1 1 pathology & immunology, children's hospital of philadelphia & upenn, philadelphia, pa. given toxicities of all current immunosuppressive agents, plus complications of malignancy, infection and chronic rejection, maintaining the search for new approaches to tolerance induction is essential. while weaning of immunosuppression is fraught with risks and costimulation blockade has not yielded the expected boon, use of a broad histone deacetylase inhibitor (hdaci), such as trichostatin a (tsa) or suberoylanilide hydroxamic acid (saha), promotes foxp3 acetylation of foxp3 and binding to target genes, leading to enhanced treg suppressive function, and 2 wks of therapy with hdaci and low-dose rapamycin can induce allograft tolerance. we now show that in addition to acetylation, modulation of a second major epigenetic mechanism, dna methylation, has salutary effects, such that combined use of an hdaci and a dna methyltransferase inhibitor (dnmti, e.g. 5-aza-2 deoxycytidine) has potent effects. microarray analysis of the effects of hdaci on tregs showed down-regulation of multiple genes associated with dna methylation, including dnmt, methyl-cpgbinding domain and associated proteins, leading us to test the effects of dnmti use on treg function. dnmti administration decreased foxp3 gene methylation, enhanced treg gene expression, and increased treg suppression in vitro, and led to a dose-dependent prolongation of cardiac allograft survival (balb/c->c57bl/6) in vivo (p<0.01). the combination of hdaci and lower doses of dnmti, administered for just 2 wks, led to permanent engraftment (>100 d, p<0.001), and the permanent acceptance of second donor allografts but acute rejection of third-party (cba) cardiac allografts indicated induction of donor-specific tolerance post-epigenetic therapy. analysis of long-surviving cardiac allografts showed a minor infiltrate consisting primarily of foxp3+ tregs and an absence of chronic rejection (transplant arteriosclerosis, myocardial fibrosis), whereas combined epigenetic therapy led to only minor prolongation of allograft survival in treg-depleted recipients. in summary, brief use of 2 clinically approved agents (an hdaci plus an dnmti), can synergistically enhance treg function and induce tregdependent donor-specific allograft tolerance without use of any immunosuppression. we conclude that insights gained through epigenetic targeting may provide completely new approaches to the taming and regulation of otherwise powerful immune responses post-transplantation. a novel epigenetic approach to generate mouse and human cd4 + cd25 + foxp3 + regulatory t cells. girdhari lal, 1 nan zhang, 1 william van der touw, 1 yaozhong ding, 1 jonathan s. bromberg. 1 1 gene and cell medicine, mount sinai school of medicine, new york city, ny. background: constitutive foxp3 expression is required for stable and suppressive cd4 + cd25 + regulatory t cells (treg) . we previously showed that demethylation of an upstream cpg island of the foxp3 promoter is characteristic of stable and suppressive treg. using dna methyltransferase inhibitors, we present a novel method to generate antigen-specific treg from mouse and human cd4 + cd25 -t cells. methods: naïve cd4 + cd25 -t cells and natural treg (ntreg) were purified from wild type or foxp3-gfp transgenic mice. human naïve cd4 + cd25 -t cells were purified from pbmc. t cells were cultured with irradiated antigen presenting cells in the presence of il-2, anti-cd3ε mab, tgfβ or the dna methyltransferase inhibitor 5-aza-2'-deoxycytidine (zdcyd), which demethylates the upstream promoter. foxp3 expression was determined by flow cytometry and qrt-pcr. results: naïve t cells cultured under stimulatory conditions with zdcyd express increased foxp3 mrna (30 fold) and intracellular foxp3 protein (35% of cells vs 2% without zdcyd). foxp3 expression is synergistically enhanced with tgfβ (76 ± 4.6% vs 38.8 ± 5.3% zdcyd alone or 44.6 ± 1.4% tgfβ alone), similar to ntreg (92.4 ± 2.5%). tgfβ in combination with other dna methyltransferase inhibitors (procainamide, hydralazine, rg108) also induces foxp3. zdcyd plus tgfβ induced treg stably express foxp3 and similar surface markers and cytokine mrna as ntreg. zdcyd plus tgfβ induced treg suppress proliferation of cd4 + cd25 -t cells, prevent cd4 + cd25 -cd45rb hi induced colitis in scid mice, and enhance islet allograft survival. zdcyd plus tgfβ induce foxp3 expression in antigen-specific wild type or t cell receptor transgenic cd4 + t cells stimulated with alloantigen or peptides, and in naïve cd8 + cd25 -t cells. in vivo administration of zdcyd preferentially preserves thymic cd4 + cd25 + foxp3 + treg generation. zdcyd alone or zdcyd plus tgfb induce strong foxp3 expression in human cd4 + cd25 -t cells compared to tgfβ alone. zdcyd plus tgfβ induced human treg suppress the proliferation of naïve cd4 + cd25 -t cells, whereas tgfβ-induced treg do not. conclusion: dna methyltransferase inhibitors induce stable and suppressive foxp3 + treg from peripheral cd4 + cd25 -t cells. these finding have important implications for understanding t cell development and differentiation, and provide a clinically applicable technique for manipulating epigenetic regulation for the generation of treg for tolerance. macrophages driven to a novel state of activation can promote tolerance. katharina kronenberg, 1 seiichiro inoue, 1 james a. hutchinson, 2 beate g. brem-exner, 2 gudrun e. koehl, 1 hans j. schlitt, 1 fred fandrich, 2 edward k. geissler. 1 1 surgery, university of regensburg, regensburg, germany; 2 surgery, university hospital schleswig holstein, kiel, germany. background: the use of immunomodulatory cells in organ transplantation (tx) is a promising tolerance-induction strategy. here we describe a novel macrophage population capable of enriching t regulatory cells (treg) and promoting tolerance. methods: balb/c bone marrow, spleen and blood mononuclear cells were cultured with m-csf for 5 days, with a 24 hr pulse of ifn-γ on day 4; the resultant adherent cells are referred to as ifnγ-induced monocyte-derived cells (ifnγ-mdc) . residual lymphocytes from these cultures were recultured with the adherent ifnγ-mdc for an additional 3 days. ifnγ-mdc were phenotyped by facs and immunomodulation of lymphocytes was determined by cell counting and facs analysis for tregs. mouse heterotopic heart tx was used for in vivo testing. results: ifnγ-mdc highly express cd11b/c, f4/80 and pd-l1. compared to classically-activated (m1) macrophages, ifnγ-mdc express less cd40/cd86, but higher cd11c. ifnγ-mdc profoundly delete activated, but not resting, lymphocytes (>60%), with cell contact (via transwell system) and caspases (via inhibitors) being essential. most interestingly, ifnγ-mdc highly enrich lymphocytes for cd4 + cd25 high foxp3 + treg (41±2%; n=27). the same, or higher, proportions of treg developed when ifnγ-mdc were produced from macs-purified cd11b + and cd4 + cells (1:1) . ifnγ-mdc culture supernatant showed increased il-10 (elisa) vs. monocyte control cultures (183±42 pg/ml vs. <30 pg/ml, respectively; n=6), suggesting il-10 as one potential mediator. ifnγ receptor signaling and cd40 interactions are required for treg enrichment, as confirmed using ifnγ receptor and cd40 knock-out mice (c57bl/6 mice). ifnγ-mdc derived from cd11b + cells and lymphocytes of ova-transgenic otii mice showed enrichment of treg by 10-fold with high-affinity ova peptide (323-339) vs. treg levels using a low-affinity ova peptide (ova 323-334), suggesting ag-specific treg cells can be enriched with ifnγ-mdc. finally, a single post-heart tx (d+1) i.v. injection of 5x10 6 donor (c3h)-derived ifnγ-mdc prolonged allograft survival in balb/c recipients from 8.5±0.8d in controls (n=5) to 21.4±8.3d (n=11; p=0.001). conclusions: ifnγ-mdc are a novel macrophage population capable of deleting activated t cells and highly enriching remaining lymphocytes for tregs. therefore, ifnγ-mdc could be useful in a clinical setting for promoting transplant tolerance. plasmacytoid dc therapeutic potential is independent of ido induction due to elevated dap12 expression. tina l. sumpter, 1 bridget l. colvin, 1 zhiliang wang, 1 andrew l. mellor, 3 angus w. thomson. 1,2 1 starzl transplantation institute, dept. of surgery, university of pittsburgh, pittsburgh, pa; 2 immunology, university of pittsburgh, pittsburgh, pa; 3 immunotherapy center, medical college of georgia, augusta, ga. optimizing the mechanisms of pdc tolerogenicity may facilitate their use in the development of cell-based tolerance induction strategies. pdc may undermine t cell function via inducible expression of ido, a tryptophan catabolizer that enhances t cell apoptosis. ido is negatively regulated by dap12. in some systems, loss of dap12 correlates with immunostimulatory activation of pdc, while in others, its absence correlates with enhancement of pdc tolerogenicity through induction of ido. in these studies, we characterized the ability of wt and ido-deficient pdc to attenuate murine heart allograft rejection in order to define the contribution of ido and its regulation by dap12 relative to the immunoregulatory potential of pdc. methods: pdc and control myeloid (m) dc were generated from bm cultures of wt or ido ko c57bl/6 (b6; h2 b ) mice. wt mdc, pdc or ido ko pdc were pulsed with alloag (balb/c; h2 d ), stimulated with cpg, and then injected i.v. into syngeneic (b6) recipients 7d before heart transplant. donor ag-specific activation of t cells was analyzed by mlr and elisa. dap12 expression was evaluated by rt-pcr and abrogated with sirna. results: administration of ido ko pdc 7d before transplant prolonged graft survival beyond that seen with control mdc, but not to the extent seen with wt pdc, suggesting involvement of ido. pdc cultures from ido ko mice exhibited a more mature phenotype than their wt counterparts with reduced expression of co-regulatory molecules (e.g., icosl). although ido ko pdc induced enhanced t cell alloactivation compared to wt pdc, use of the ido inhibitor 1-mt indicated that wt pdc do not produce functional ido. further analyses showed wt pdc exhibited high levels of dap12 mrna expression. silencing dap12 had no effect on the ability of wt pdc to activate allogeneic t cell proliferation, but significantly enhanced ifnγ secretion. addition of 1-mt to mlr with dap12-silenced pdc increased t cell proliferative responses to alloag, verifying ido induction in dap12-silenced pdc. conclusions: these data underscore the prophylactic potential of pdc for cell-based therapy that may be independent of ido, due, in part, to elevated dap12 expression. additionally, our data support a role for dap12 in both immunostimulation and immunoregulation by pdc. rapamycin preferentially blocks the expansion of potentially tolerogenic plasmacytoid dendritic cells in vivo. heth r. turnquist, 1 angus w. thomson. 1, 2 1 starzl transpl inst and dept of surgery; 2 dept of immunol, univ of pittsburgh sch of med, pittsburgh, pa. rapamycin (rapa), a 'tolerance-sparing' immunosuppressant with anti-proliferative properties, inhibits myeloid (m) dendritic cell (dc) differentiation/maturation in vitro. rapa decreases cd11c + dcs in the mouse spleen and suppresses the expansion of total cd11c + dc following administration of the dc growth factor, fms-like tyrosine kinase 3 ligand (flt3l). however, the influence of rapa on plasmacytoid (p) dc,the principal type-1 ifn producers in the body, known to regulate innate and adaptive immune responses, and reported to promote experimental transplant tolerance, has not been evaluated. methods: dc were propagated from bone marrow for 10 days (d) in flt3l, in the absence or presence of a clinically-relevant dose of rapa (10 ng/ml). in addition, dcs were mobilized in c57bl/10 mice by i.p. flt3l (10 mg/d for 10 d; d1-10). untreated and flt3l-treated groups also received rapa (0.5 mg/kg/d i.p.; d3-10). on d11, dc were isolated by density gradient centrifugation and/or cd11c + positive selection. mdc (cd11c + cd11b + ) and pdc (cd11c lo cd11b -b220 + ) were identified by flow cytometry. results: rapa suppressed pdc and mdc generation in vitro; rapa-dc-treated cultures had only 22% of the pdc and 40% of the mdc found in control conditions. in normal mice, both mdc (54% of control) and pdc (24.5%) numbers were reduced significantly by rapa administration. rapa, when given concurrently with flt3l, blunted the typical profound expansion of mdc. specifically, flt3l and rapa-treated mice displayed a 46±8x increase over control (steady state) numbers compared to a 145±39x increase in mice treated with flt3l alone. flt3l-induced expansion of pdc was to a much greater extent impacted by rapa, as absolute numbers of pdc increased only 6.5±2.2x over control numbers compared to 51±3x increase in absolute splenic pdc in flt3l-treated mice. conclusion: these data identify rapa as a selective suppressor of pdc generation, as described for corticosteroids. this has significant implications, given the use of rapa following organ transplantation, and the suggested importance of secondary lymphoid tissue pdc for promotion of transplant tolerance mediated by treg. due attention to these disparate effects of rapa on regulating cell populations will be necessary to optimize therapeutic regimens for safe promotion of tolerance. the liver is tolerated better than other transplanted organs. this may reflect the inherent tolerogenicity of liver dendritic cells (dc) and interactions with foxp3 + regulatory t cells (treg). recent studies have shown that cd103 expression on dc correlates with induction of foxp3 + treg, and can be enhanced in the presence of transforming growth factor-β (tgf-β) and retinoic acid (ra), both of which are produced in the liver. this study evaluates cd103 expression on liver plasmacytoid (p) and myeloid (m) dc and the potential of liver dc subsets to induce tregs. methods: pdc and mdc were magnetically isolated from livers and spleens of c57bl/10 mice and co-cultured with cfse-labeled allogeneic (balb/c) cd4 + cd25 -t cells. after 4 or 5d of co-culture, t cells were assessed for cfse dilution and intracellular foxp3 expression. rhtgf-β and either all trans or cis-ra were added to co-cultures. cd103 expression was evaluated by flow cytometry. results: cd103 expression was elevated on liver pdc and mdc compared to spleen pdc or mdc, with higher expression on liver mdc. pdc from the liver and the spleen were poor inducers of foxp3 in cd4 + cd25 -t cells compared to mdc. foxp3 induction was enhanced with tgf-β when splenic pdc were used as stimulators. however, when liver pdc were used as stimulators, tgf-β had no effect on foxp3 induction. ra (either all trans or cis) enhanced induction of foxp3 + cells in cd4 + cd25 -t cells in the presence of tgf-β when splenic pdc but not liver pdc were used as stimulators. tgf-β and tgf-β with ra also enhanced foxp3 induction in cd4 + cd25 -t cells when either spleen or liver mdc were used as stimulators, though splenic mdc were superior inducers of foxp3. conclusions: many studies have focused on naturally-occurring foxp3 + tregs in systemic tolerance. our data show that liver mdc and pdc are poor inducers of foxp3 in t cells, even in the presence of tgf-β and ra. these data suggest that the inherent tolerogenicity of liver dc subsets may be independent of treg induction or that liver dc interact with treg through alternative mechanisms. background: t cell-mediated immune rejection occurs in organ transplantation. in addition to mhc-tcr signaling, t cell activation requires costimulation from antigen presenting cells. b7 molecules (cd80/cd86) and cd40 are critical costimulatory molecules in t cell activation. insufficient or lack of costimulation results in inactivation or tolerance. we hypothesized that blocking the costimulation pathway using small interfering rna (sirna) expression vector can prolong allogeneic heart graft survival. method: vectors that express hairpin sirnas specifically targeting cd40 and cd80 were prepared. recipients (balb/c mice) were treated with cd40 and/or cd80 sirna vectors, 3 and 7 days prior to heart transplantation. control groups were injected with a blank vector and sham treatment (pbs). after sirna treatment, a fully mhcmismatched (balb/c to c57/bl6) heart transplantation was performed. result: allogeneic heart graft survival (>100days) was approximately 70% in the mice treated simultaneously with cd40 and cd80 sirna vectors. in contrast, allogenic hearts transplanted into recipients treated with blank vector and pbs stopped beating within 10 days. hearts transplanted into cd40 or cd80 sirna vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. real time pcr and flow cytometric analysis showed an upregulation of foxp3 expression in spleen lymphocytes and a concurrent downregulation of cd40 and cd80 expression in splenic dendritic cells of sirnatreated mice. an mlr, using splenic dendritic cells (dcs) isolated from tolerant recipients, showed a significantly lower t cell proliferation capacity in cd40-and cd80-sirna vector-treated mice, compared to control groups. tolerant dcs from cd40-and cd80-treated recipients promoted cd4+cd25+foxp3+ regulatory t cell differentiation. finally, tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema in mice treated with cd40 and cd80 sirna vectors. conclusion: this study demonstrates that the simultaneous silencing of cd40 and cd80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation. costimulation blockade (cob) of the cd28/b7 pathway has long been suggested as a means of attaining indefinite, well-tolerated, antigen-specific prophylaxis from allograft rejection. although effective in rodent models, cd28/b7 blockade is not alone sufficient to prevent rejection in more robust primate models. the relative presence of allocrossreactive memory t cells is one mechanism of cob resistant rejection. lfa3-ig is an approved agent for the treatment of psoriasis, shown clinically to deplete tem cells in psoriatic lesions. we hypothesize that lfa3-ig specifically targets cob resistance cells, including heterologous alloreactive t cell memory, while avoiding interference of peripheral mechanisms that foster cob-mediated allograft acceptance. rhesus monkeys underwent mhc mismatched renal allotransplantation. ctla4-ig was given 20 mg/ kg iv on days -1, 0,3,7,14,21,28 and 10 mg/kg iv on days 35,42,49,56. lfa3-ig, (0.3 mg/kg iv) was given on day -1,0,3,7 then weekly (8 wks). sirolimus was given orally (1mg/kg) days 0-90 and donor specific transfusion (7 ml/kg iv) on day -1. animals were followed by polychromatic flow-cytometry to quantify t cell subsets. lfa3-ig synergized with ctla4-ig successfully preventing renal allograft rejection in this model and leading to indefinite survival in 2/5 animals (table) . this enhanced survival was associated with a significant reduction in tem cells in animals treated with lfa3-ig compared to animals without lfa3-ig treatment (figure). lfa3-ig withdrawal led to tem re-population and rejection. this therapy regimen, all clinically available agents, promotes cob-mediated graft acceptance without the use of calcineurin inhibitors, steroids, or gross t cell depletion. the aim of this study was to investigate the immunological pathways that regulate t cell dependant allograft responses in the hope of finding novel immunomodulatory compounds. islet (and skin) allografts were transplanted into full mhc-mismatched wild type (wt) and baff-transgenic (baff-tg) recipient mice. allograft function/ rejection was monitored by blood glucose levels and/or confirmed by nephrectomy. histology, splenocyte populations & t cell functions were analyzed. the b cell activation factor from the tnf family (baff) is critical for b cell survival. t cells express baff receptors suggesting that baff may also play a role in t cell responses. contrary to expectations, we found that baff-tg mice accepted a full mhc-mismatched islet allograft for >100 days. in addition, baff-tg mice also showed delayed skin graft rejection. this was due to a t cell intrinsic change in allo-responsiveness as shown by a failure of purified baff-tg t cells to reject an allograft in adoptive transfer experiments. however, baff-tg t cells were not anergic per se as they proliferated normally to mitogens in vitro and to antigenic challenge in vivo. intriguingly, baff-tg mice harbored an increased frequency of t regulatory cells in the periphery as compared to wt mice. elimination of cd25 + t cells restored normal allograft rejection to baff-tg mice, demonstrating that the increased number of treg cells were responsible for their altered allo-immunity. in a second approach, baff-tg t cells depleted of cd4 + cd25 + t cells were adoptively transferred to transplanted rag recipients, and in this case the baff-tg cd4 + cd25 -t cells rejected their allograft. proliferation assays showed that excessive baff was not promoting treg expansion by driving proliferation in the periphery. adoptive transfer of gfp expressing syngeneic splenocytes did not exhibit enhanced survival in the presence of excessive baff. however, analysis of treg cells in the thymus of baff-tg mice showed an increased intrathymic frequency. together these results demonstrate that baff-tg mice harbor an expanded number of treg cells that prevent th1-type immune responses including allograft rejection. furthermore, we demonstrate that manipulating baff levels may provide a means to generate immunosuppression free dominant allograft tolerance. we have previously reported the outcome of pig-to-baboon thymokidney transplants from galt-ko miniature swine using an immunosuppressive regimen designed to facilitate the induction of tolerance. although the results were superior to results using hdaf thymokidneys, there was a high rate of early post-operative complications. we investigated whether the elimination of steroids and whole body irritation (wbi) from the treatment protocol would decrease the complication rate in the perioperative period. methods: 7 baboons received thymokidney transplants from galt-ko miniature swine. the immunosuppressive regimen was based on the previously published protocol, but eliminated steroids and substituted rituximab for wbi. it consisted of thymectomy day -21, splenectomy day 0, atg, rituximab, mmf and anti-cd154 mab. renal function was assessed by serum creatinine levels. evidence for baboon thymopoiesis in the pig thymus was assessed by facs and immunohistochemistry. results: one animal died due to a drug reaction at pod 18 with normal renal function and no evidence of infection. the remaining 6 baboons showed no signs of rejection although igm deposition was observed, likely due to preformed non-gal nab. there were no deaths due to rejection. although early infectious complications were not seen, late complications leading to mortality were still observed, including systemic cmv infection (pod 28), pleural effusions (pod 40, 57) , ards with pulmonary hemorrhage (pod 49, 81) and acute myocardial infarction (pod 83). two animals showed evidence for early thymopoiesis in the pig thymus by facs and immunohistochemistry. cd4+/cd8+ thymocytes were seen in the thymic grafts, indicating t cell development was supported by the pig thymus. the average survival of the steroid-free group was 51 days including the animal with the drug reaction and 56 days excluding it, compared to 34.6 days in the regimen that included steroids/wbi. conclusions: a steroid-free and wbi free regimen designed for the induction of tolerance across the pig-to-baboon xenogeneic barrier had fewer early post-operative complications than previous regimens. greater than 50-day average survival of recipients of life-supporting xenogeneic thymokidneys was observed without rejection. this regimen also appears to permit baboon thymopoiesis in the pig thymus. purpose: the effects of human decay accelerating factor (hdaf) addition to the galactosyl transferase knock-out (galt-ko) background in transgenic pig kidney xenotransplantation in baboons has not been previously studied. methods: baboon recipients of galt-ko (n=4) or galt-ko/hdaf (n=2) pig kidneys received flolan, heparin and/or aspirin. steroids (s), cobra venom factor (v), atg (a), mmf (m), and/or a low-dose cd154 blockade (c) regimen were given. results: pre-transplant (tx) anti-non-gal antibody (ab) titers were inconsistently associated with early galt-ko kidney xenograft failure. high d-dimer levels 4 hours post-tx were closely associated with early galt-ko graft loss but not when assessing levels of c3a, btg, or increased cd62p expression on circulating platelets. regimens lacking mmf and/or cd154 blockade elicited by day 7-14 large anti-donor non-gal ab responses . post-operative creatinine levels for the galt-ko/hdaf recipients were lower along with superior graft survival compared to galt-ko; d-dimer, anti-donor non-gal ab, c3a, btg, and f1+2 measurements are in progress for the galt-ko/hdaf recipients. galt-ko/hdaf v,a,m,s,c >10* tbp 239 1.9; 1.5 * graft survival at time of abstract submission as graft still functioning at time of abstract submission; tbp --to be processed ! --anuric; non-life supporting, but appeared viable, pink, and well-perfused until day 7 conclusion: early failure of vascularized galt-ko organs is closely associated with activation of coagulation pathways; whether this is triggered primarily by anti-non-gal ab or by other mechanisms is not addressed by our study. preliminary results using galt-ko/hdaf pig kidneys show promise in attenuating or possibly preventing these and/or other such mechanisms. the established efficacy of cd154-based costimulation blockade with mmf to prevent induced anti-non-gal antibody elaboration is confirmed by our preliminary findings. recovery of cardiac function after pig-to-primate orthotopic heart transplant. christopher g. a. mcgregor, 1 william r. davies, 1 keiji oi, 1 henry d. tazelaar, 1 randall c. walker, 1 krishnaswamy chandrasekaran, 1 guerard w. byrne. 1 1 mayo clinic, rochester, mn. xenotransplantation has been proposed as a method to alleviate the shortage of donor organs. we report survival of up to 8 weeks of three orthotopic transplants after pig-tobaboon cardiac xenotransplantation. pig-to-baboon orthotopic transplantation was performed using an adult baboon recipient and cd46 transgenic pig. the recipients were treated with an α-gal polymer to block the effects of anti-gal antibody and with atg induction and a tacrolimus and sirolimus based maintenance immunosuppression. heart function was continuously monitored by intramyocardial electrocardiography and every 3 -4 days by serial echocardiography. standard hematological, serum chemistry and cardiac troponin levels were monitored every 2 -3 days. the animals survived 34, 40, and 57 days in a healthy condition. mortality was a result of pneumonitis, respiratory failure and bowel infarction respectively. one recipient (survival 40 days) showed impaired perioperative left ventricular function with an ejection fraction of 25% on echo. over the next two weeks, the ejection fraction in this animal returned to normal (60%) and troponin levels normalized. this study shows the longest survival of orthotopic cardiac xenografts to date with recovery of impaired heart function after early ischemia reperfusion injury. this significant improvement in ventricular function suggests that the normal reparative processes appear to function across the xenotransplantation barrier, supporting the potential clinical potential of cardiac xenotransplantation. purpose: to determine whether the galactosyl transferase gene knock-out (galt-ko) protects pig lungs from hyperacute lung rejection (halr) by human blood. the effects of adding human decay accelerating factor (hdaf) to the galt-ko background will also be examined for the first time. methods: heparinized fresh human blood was used to perfuse galt-ko pig lungs (n=10). lung function was assessed by flow, pulmonary vascular resistance (pvr), oxygen transfer, and tracheal edema using pre-defined survival endpoints. historical wild-type (wt) pig lungs (n=15) provide context for analysis. additionally, two pig lungs expressing galt-ko/hdaf were recently examined, one of which received treatment with xigris. results: median lung survival in galt-ko lungs was 114 minutes (range 15min to >240min) vs. 10 minutes in wt lungs (range 4min to 36min, p= 0.01); 2/2 galt-ko/hdaf pig lungs survived >240 min. pvr at 5 minutes for galt-ko lungs was 62±35mmhg-min/l vs.wt lungs (308±178 mmhg-min/l at 5 minutes) vs. 23mmhgmin/l for each galt-ko/hdaf lung. complement activation (∆c3a) at 15 minutes was significantly lower with galt-ko (∆c3a 133±243 ng/ml) than wt lungs (vs. 2080±1332 ng/ml, p=0.039). galt-ko was also associated with reduced platelet activation: only 4±4% of circulating platelets express cd62p at 15 min. vs. 16±13% in the wt group (p=0.05); ∆βtg at 15 min [223±151 iu/ml (galt-ko) vs. 1196±1186 iu/ ml (wt)], and less thrombin formation (∆ f1+2) at 15 minutes (galt-ko: 5±7.9 nm vs. wt: 22±39 nm). ∆c3a, % of circulating platelets expressing cd62p, ∆βtg, and ∆ f1+2 measurements are in progress for galt-ko/hdaf. platelet sequestration was delayed as mean % of the initial platelets remaining in the galt-ko lung perfusate was 52±19% at 15min vs. 27±9% in the wt lung group (p=0.02). neutrophil sequestration was also diminished in galt-ko (58±15% residual) vs. wt lung (5±3% residual, p=0.004). conclusion: galt-ko pig lungs are significantly protected from several facets of halr: complement and coagulation cascade activation, neutrophil sequestration, and platelet activation are all partially attenuated by this modification alone. preliminary results suggest that galt-ko/hdaf lungs offer even further protection. disseminated intravascular coagulation is associated with tissue factor expression on recipient platelets and monocytes. chih che lin, 1, 3, 4 mohamed ezzelarab, 1 corin torres, 1 david ayares, 2 anthony dorling, 3 david k. c. cooper. 1 1 thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; 2 revivicor inc., blacksburg, va; 3 department of immunology, imperial college london, hammersmith hospital, london, united kingdom; 4 department of surgery, chang gung memorial hospital, kaohsiung, taiwan. purpose acute humoral xenograft rejection (ahxr), frequently associated with disseminated intravascular coagulation (dic), remains a challenge in pig-to-primate xenotransplantation (tx). a previous in vitro study showed that recipient platelets and monocytes were induced to express tissue factor (tf) after incubation with porcine endothelial cells, and we speculated this may contribute to thrombosis. the present study investigated whether circulating (extragraft) monocytes and platelets express tf and whether this relates to the development of dic after pig tx. methods baboons (n=7) received an aortic patch or kidney from either a wild-type (wt) or α1,3galactosyltransferase gene-knockout (gtko) pig, with or without immunosuppressive therapy. baboon monocytes and platelets were isolated from blood before and after tx. surface tf phenotype was determined by flow cytometry. functional tf activity was determined by clotting time assays after mixing monocytes and platelets with recalcified factor vii (fvii)-deficient plasma with or without added fvii. before tx, monocytes and platelets did not express tf or display tf-dependent procoagulant activity. after gtko aortic patch tx, the baboons were euthanized by day 35 without developing dic. however, by day 28, circulating platelets (but not monocytes) expressed tf and promoted tf-dependent clotting in recalcified plasma. in the absence of immunosuppression, wt kidneys survived <1 day before the onset of dic, at which time tf activity was detected on both platelets and monocytes. after gtko kidney tx in immunosuppressed baboons, platelets expressed tf as early as day 3. in contrast, monocytes began to express tf only at the onset of dic. this study links expression of tf on recipient monocytes and platelets with the development of dic. expression on platelets appeared to predict the subsequent development of dic, whereas that on monocytes was associated with the onset of dic. whilst our observations do not establish a causal relationship, they provide the basis for further study. international human xenotransplantation inventory. antonino sgroi, 1 leo buhler, 1 megan sykes, 2 luc noel. 3 1 surgical research unit, department of surgery, geneva university hospital, geneva, switzerland; 2 transplantation biology research center, massachusetts general hospital, boston, ma; 3 world health organization, geneva, switzerland. background: xenotransplantation carries inherent risks of infectious disease transmission to the recipient and even to society at large, and should only be carried out with tight regulation and oversight. a collaboration between the international xenotransplantation association, the university hospital geneva and the world health organization has established an international inventory (www.humanxenotransplant. org) aiming to collect basic data on all types of xenotransplantation practices on humans that are currently ongoing or have been recently performed. methods: we collected information using publications in scientific journals, presentations at international congresses, internet-based information, and declarations of ixa members. an electronic questionnaire is available on the website www. humanxenotransplant.org, which can be filled out and sent to the office in geneva. results: we identified a total of 16 recent or current human applications of xenotransplantation, eight were currently ongoing and one will start soon. the source animal was: pig (n=7), sheep (n=3), calf (n=1), rabbit (n=2), blue shark (n=1), hamster (n=1), and unknown (n=1). all trials transplanted xenogeneic cells, i.e. islets of langerhans (n= 6), hepatocytes (n=1), kidney cells (n=1), chromaffin cells (n=1), embryonic stem cells (n=2), fetal (n=4) and adult cells (n=1) of various organs. the treatments were performed in 10 different countries, 7 in europe, 2 in russia, 2 in asia, 2 mexico, 1 in usa and 1 in africa. six countries had no national regulation on xenotransplantation. conclusion: several clinical applications of cell xenotransplantation are ongoing around the world, often without any clear governmental regulation. this information should be used to inform national health authorities, health care staff and the public, with the objective of encouraging good practices, with internationally harmonized guidelines and regulation of xenotransplantation. cells from α1,3-galactosyltransferase gene-knockout pigs additionally transgenic for human membrane cofactor protein demonstrate resistance to human complement-mediated cytotoxicity. hidetaka hara, 1 cassandra long, 1 mohamed ezzelarab, 1 peter yeh, 1 carol phelps, 2 david ayares, 2 david k. c. cooper. 1 1 surgery, thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; 2 revivicor, inc., blacksburg, va. purpose: (1) to compare the antibody binding and complement-mediated cytotoxicity (cdc) of human sera to pig peripheral blood mononuclear cells (pbmc) and porcine aortic endothelial cells (paec) from (i) wild-type (wt), (ii) α1,3-galactosyltransferase gene-knockout (gtko), (iii) human membrane cofactor protein transgenic (mcp) and (iv) gtko/mcp pigs. (2) to investigate the effect on binding and cdc of human sera following activation of paec. methods: pooled human serum was tested by flow cytometry for binding of igm and igg (5% serum concentration) and cdc (25% serum concentration) to pbmc and paec from wt, gtko, mcp, and gtko/mcp pigs. paec from all 4 pig types were activated by ifn-γ, and again tested for antibody binding and cdc. results: there was higher binding of igm and igg to wt and mcp than to gtko and gtko/mcp pbmc and paec, but there were no differences in binding (i) between wt and mcp cells or (ii) between gtko and gtko/mcp cells. cdc of wt pbmc and paec was significantly greater than of gtko, mcp, and gtko/mcp pbmc (wt 73%; gtko 37%; mcp 30%; gtko/mcp 4%) and paec (wt 51%; gtko 18%; mcp 1%; gtko/mcp 0%) (both p<0.05). importantly, there was no lysis of gtko/ mcp paec. after activation of paec, although cdc was increased against wt and gtko paec, mcp and gtko/mcp paec demonstrated significant resistance to lysis (wt 73%; gtko 41%; mcp 5%; gtko/mcp 0%). conclusions: (1) cdc of all 3 types of genetically-engineered pbmc and paec was significantly lower than of wt pbmc and paec, with lysis of gtko/mcp paec being significantly lower than to gtko or mcp cells. (2) paec from both mcp and gtko/mcp showed resistance to lysis even after activation of the cells. (3) organs from gtko/mcp pigs should provide considerable protection against cdc. role for cd47-sirpα signaling in human t cell proliferation in response to stimulation with porcine antigen presenting cells. hiroyuki tahara, 1 hideki ohdan, 1 kentaro ide, 1 toshimasa asahara. 1 1 department of surgery, hiroshima university, hiroshima, japan. we have previously proven that genetic induction of human cd47 on porcine cells provides inhibitory signaling to signal regulatory protein(sirp) α on human macrophages; this provides a novel approach to preventing macrophage-mediated xenograft rejection. a recent report indicated that the similar cd47-sirp system negatively regulates the functions of both t cells and antigen presenting cells(apcs) in humans. we hypothesize that the interspecies incompatibility of cd47 may also act as an additional barrier of t cell-mediated xenograft rejection. we have analyzed the frequency and proliferative activity of human t cells responding to either porcine or allogenic apcs by using in vitro mixed lymphocyte reaction (mlr) assay with a cfse-labeling technique. irradiated stimulator porcine or human peripheral blood mononuclear cells(pbmcs) were cultured with cfse-labeled responder human pbmcs. by fcm analysis, the number of division precursors was extrapolated from the number of daughter cells of each division and from the mitotic events, and precursor frequencies in cd4 + and cd8 + t cell subsets. using these values, stimulation index(si) was calculated. the frequencies of alloreactive and xenoreactive cd4 + t cell precursors were almost identical, 4.5±0.8% and 4.4±0.9%, respectively (n=10-12, for each type of precursor). however, the si of xenoreactive cd4 + t cells was significantly higher than that of alloreactive cd4 + t cells, indicating a stronger reaction by a single xenoreactive cd4 + t cell. in the presence of human cd47-fc(containing the extracellular domain of human cd47 fused to the fc portion of human ig, 10µg/ml), the si of xenoreactive cd4 + t cells was significantly reduced to a level similar to that of alloreactive cd4 + t cells (n=12, p<0.05), although the frequencies of their precursors were absolutely uninfluenced. the frequencies of alloreactive and xenoreactive cd8 + t cell precursors were also identical, i.e. 3.5±0.3%, 3.9±0.9%, respectively (n=10-12). the si of both alloreactive and xenoreactive cd8 + t cells did not differ: however, that of xenoreactive cd8 + t cells was significantly suppressed in the presence of human cd47-fc (n=12, p<0.05). these results suggest that the t cell responses to porcine cells are stronger than those to allogeneic cells because of the interspecies incompatibility of cd47. moreover, genetic manipulation of porcine apcs to induce human cd47 expression might attenuate human t cell-mediated xenograft rejection. reactivation of human cytomegalovirus (hcmv) is a potential risk following the clinical application of pig-to-human xenotransplantation. since little is known about undesirable side-effects arising from hcmv infection of porcine organs, we investigated the capabilities of various hcmv strains to infect porcine endothelial cells (pec) and putative immunological consequences thereof. pec from different anatomical origins were incubated with the hcmv laboratory strains ad169 and tb40/e or a clinical isolate at a multiplicity of infection (moi) ranging from 0.1 to 100. viral replication kinetics, evolution of cytopathology, and lytic end points were analyzed. consequences of pec infection on human nk cell activation were evaluated using xenostimulation assay assessing nk cell ifng secretion and cytotoxicity. infection was evident and a maximum percentage of infected cells was reached at a moi of 10. hcmv replicated in all tested pec types, with a fraction of infected cells ranging from 1% to 50%. ad169 infection of 2a2 (microvascular ec) resulted in cytopathic effect (cpe) development by 3 dpi and in lysis of 70% of the cells at 7 dpi. contrary, no cpe was observed in ped (aortic ec) up to 15 dpi. infection of 2a2 with tb40/e was non-lytic and resulted in accumulation of both extra-and intracellular virus. virus titers reached a maximum at 5 dpi, peaking at levels 20-fold higher than residual input virus. we then determined whether infected pec supported a complete replication cycle and produced viral progeny. after 5 dpi pec supernatants and lysates revealed the production of significant amounts of virus. preliminary results showed that coculture of human nk cells and infected pec resulted in increased nk killing and ifng production. altogether, these findings provide evidence that pec are permissive and support the complete productive replication cycle of hcmv. however, cpe are cell-type and strain dependent. moreover, pec infection leads to modification of the xenogeneic cytotoxicity mediated by human nk cells. our findings allow a better estimation of the potential role of hcmv cross-species infection following xenotransplantation and may be crucial to guide future clinical trials. chronic rejection/injury: innate and adaptive immunity i testing for donor-specificity of antibodies post-transplantation increases the predictability of chronic rejection. mikki ozawa, 1 arundhati panigrahi, 2 paul i. terasaki, 3 narindar mehra. 2 1 one lambda, inc., canoga park, ca; new delhi, india; 3 terasaki foundation laboratory, los angeles, ca. purpose: post-transplant hla antibodies have been shown to be linked to chronic graft failure, yet some recipients do well in the presence of antibodies. we investigated whether testing for donor-specificity of antibodies improves the predictive value of antibodies in regards to chronic rejection. methods: in a prospective study of 400 renal transplant recipients, post-transplant sera were tested for hla and mica antibodies, and positive sera were tested by single antigen beads to determine whether the antibodies were donor-specific. one year after testing, biopsy and clinical data on the graft status were collected. patients who did not have follow-up or died with function were excluded from analysis. results: of the 350 patients with follow-up and abdr typing info, 102 (29%) were found to have antibodies. sixteen of those had donor-specific a, b, or dr antibodies (dsa), while 26 had non-donor specific ab's (ndsa). sixty patients had only dp, dq, cw, or mica antibodies, and were grouped into "donor-specificity unknown", as these typing were not available at the time of this report. table 1 summarizes the antibody groups and transplant outcome one year later. interestingly, the mean serum creatinine values at the time of antibody testing were very similar among the groups, regardless of the presence of antibody or the type (ranged 1.34 to 1.73mg/dl). one year later, however, a striking 63% of dsa patients had either returned to hemodialysis, were regrafted, had died, or had biopsy-proven chronic rejection, compared to only 23% in ndsa patients and 13% in those who were negative (dsa vs. neg, p=0.00009). mica antibodies also showed significant association with graft failure or can (p=0.03), and donor mica typing is underway. conclusion: in this prospective study of 350 renal patients, dsa detected posttransplantation were highly correlated with chronic rejection. inclusion of specificity analysis in post-transplant antibody monitoring would significantly improve the predictability of chronic rejection. donor cd4 t cells within solid organ transplants contribute to graft rejection. thet su win, 1 sylvia rehakova, 1 margaret negus, 1 kourosh saeb-parsy, 1 martin goddard, 2 eleanor bolton, 1 andrew bradley, 1 gavin pettigrew. 1 1 university of cambridge; 2 papworth hospital, united kingdom. this study examines the contribution of donor cd4 t cells within heart allografts to the development of graft vasculopathy, by providing help to recipient b cells for generating effector autoantibody responses. b6 mice were transplanted with mhc class ii-disparate bm12 hearts. the allo-and auto-antibody responses were quantified, and their contribution to allograft vasculopathy assessed by incorporating b cell deficient mice as recipients. the role of donor cd4 t cells in providing help for autoantibody was examined by removing cd4 t cells from donor hearts before transplantation by three approaches: treating with anti-cd4 mab, administering 1300gy lethal irradiation, and using bm12 donors that are genetically deficient in t cells. the contribution of donor cd4 t cells to autoanitbody development was further assessed by challenge with bm12 cd4 t cells two weeks prior to transplantation. bm12 heart grafts developed progressive vasculopathy and were rejected slowly (mst=95 days, n=17). the contribution of antibody-mediated effector mechanisms was confirmed by histopathological evidence (fibrinoid necrosis and vascular proliferation), by complement c4d staining, and by a reduction in the severity of vasculopathy and long-term graft survival in b cell deficient recipients. surprisingly, no alloantibody was detected, but recipients instead developed autoantibody. the autoantibody response was completely dependent upon the provision of help from donor cd4 t cells; it was abrogated by transplanting cd4 t cell deficient hearts. to further confirm an effector role for autoantibody in vasculopathy development, b6 mice were primed for humoral autoimmunity, but not for alloimmunity, by injecting with highly purified bm12 cd4 t cells prior to transplantation. heart grafts were rejected more rapidly (mst=29 days, n=4, p<0.0001), and demonstrated severe vasculopathy. finally, bm-chimeric recipients that lacked mhc class ii expression only on b cells did not develop autoantibody, suggesting that cognate interaction between the donor cd4 t cells and mhc class ii of recipient b cells is crucial for post-transplant humoral autoimmunity. our results demonstrate the novel finding that help for autoantibody production is provided by graft-versus-host cognate recognition of recipient b cell mhc class ii by donor cd4 t cells. this autoantibody contributes to the development of vasculopathy independently of alloantibody. allograft rejection. gang chen, 1 huiling wu, 1 jainlin yin, 1 josette m. eris, 1 steven j. chadban. 1 1 collaborative transplantation research group/renal medicine, university of sydney/rpah, sydney, nsw, australia. toll like receptors (tlrs) are innate immune receptors that play an important role in innate immunity but also provide a link between innate and adaptive immunity. myd88 is a key tlr signal adaptor. a recent clinical study reported that activation of innate immunity in heart-transplantation recipients through tlr4 contributes to the development of chronic rejection after cardiac transplantation. in this study, we aimed to determine whether tlr4-myd88 signaling is required for the development of chronic allograft damage using tlr4 -/and myd88 -/mice in a murine model of chronic cardiac allograft rejection. methods: cardiac transplants were performed: b6.c-h-2 bm12 (b6.h-2 bm12 ) hearts to wt, myd88 -/and tlr4 -/mice (all c57bl/6-h-2 b -single mhc class ii mismatch) as allografts (n = 6-8/group) and c57bl/6 to c57bl/6 isografts (n = 5). blood and tissue were harvested at day 49 after transplantation. cell infiltration, fibrosis and vasculopathy were assessed histologically (grade 0 -5). cd4 + foxp3 + cells in the blood and spleen were measured by flow cytometry analysis. results: all hearts remained pulsatile until sacrifice. histology of tlr4 -/and myd88 -/recipients showed protection from leukocyte infiltration (1.2±1.1&1.0±1.1vs 3.2±1.4, p < 0.01), fibrosis (0.75±1.0& 0.6±1.2 vs 2.6±2.0, p < 0.05) and vasculopathy (0.28±0.6 & 0.04±0.1 vs 1.9±2.2) at day 49 post transplantation compared to wt recipients. by facs, the ratio of cd4 + foxp3 + regulatory t (treg) cells to total cd4 + cells at day 49 post transplantation in spleen was significantly increased in tlr4 -/and myd88 -/recipients versus wt allograft and isograft mice (18.1±0.2 & 19±0.9% vs 12.9 ±1.1 & 11.8±0.2 %, p <0.01). conclusion: absence of tlr4 and myd88 signaling reduces chronic allograft damage in a murine model of chronic cardiac rejection. one mechanism of protection may be enhancement of regulatory t cell function. promotion of treg generation via the tlr4/ myd88 pathway may be one important consequence of cross-talk between innate and adaptive immunity. pathway: implication in transplant arteriosclerosis. thibaut quillard, 1 stéphanie coupel, 1 flora coulon, 1 juliette fitau, 1 maria-cristina cuturi, 1 elise chiffoleau, 1 béatrice charreau. 1 1 inserm u643, itert, nantes, france. endothelial cell (ec) activation, injury and apoptosis are the key events associated with transplant arteriosclerosis (ta) and chronic allograft rejection (cr). notch is a major signalling pathway controlling vascular function and injury. nonetheless, the involvement of notch, at endothelial level, in both ta initiation and progression remains unknown. using a fully mhc mismatched rat cardiac allograft model of cr, we found that ta at day100 correlates with a strong decrease in both expression and activity of the notch pathway in transplant as compared to tolerant and syngeneic controls. the present study investigates the contribution of notch activity to ec survival. in this purpose, a recombinant adenovirus, encoding the notch2 intracellular domain (nicd) and the reporter gfp cdnas was constructed and used to maintain notch activity in cultured primary human arterial ec. our results demonstrate that nicd protects ec from cell death induced by tnfα in the presence of chx or by anoïkis as measured by annexinv and dna content staining. nicd mediates cytoprotection through the regulation of key-apoptosis molecules. using an apoptosis-dedicated qpcr array, we found that 14 out of a panel of 88 apoptosis-related genes were significantly regulated. nicd upregulated bcl2, bcl2a1and bcl-xl (2.5-, 8.2-and 2.8-fold compared to noninfected cells, respectively). in addition, pro-apoptotic genes bim, drp1, bok and cd40 were markedly downregulated in transduced cells (6.7-, 11.4-, 3.9-and 14 .5-fold decrease, respectively). western blotting analysis confirmed the induction of protective molecules (bcl2 and bcl-xl) and the inhibition of pro-apoptotic proteins (bad, bak and bax). consistent with these data, nicd conducted phosphorylation of akt as well as a reduction of pten expression, suggesting that the cytoprotective activity of nicd may be mediated by recruitment of the pi3k survival pathway. to conclude, our findings indicate that ta correlates with a decreased notch signaling in transplant and that activation of the notch pathway in vascular ec prevents apoptosis by promoting protective gene expression and survival pathway. these data suggest that controlling notch activity may prevent ec dysfunction associated with ta and cr. donor age intensifies the early immune response. christian denecke, 1 xupeng ge, 1 irene kim, 1 daman bedi, 1 anne weiland, 1 anke jurisch, 1 steven mcguire, 1 johann pratschke, 2 stefan g. tullius. 1 1 div. of transplant surg, bwh, transplant surg res lab, boston; 2 dept. of surgery, charite berlin, germany. increasing numbers of organs from elderly donors are currently utilized for transplantation. advanced donor age may not only be associated with physiological impairments but also with a modified immune response of the recipient. we hypothesized a more potent early immune response following the transplantation of elderly donor organs and we analyzed the immune response in a mouse heart transplant model. young b6 mice received heart allografts from 3, 12 and 18mths old bm12 donors. the recipients immune response and intragraft changes were analyzed. elderly, non-manipulated hearts contained overall significantly elevated frequencies of cd4 + and cd8 + t-cells and dcs (cd11c + ) (18mths vs. 3mths: cd4 + : 2.77% vs. 1.35%, cd8 + : 3.90% vs 1.71%, cd11c + : 46.1% vs.11.8%, p<0.05). following engraftment of 18mths old heart grafts numbers of activated dc's (cd11c + i-a b+ and cd11c + cd40 + ) had significantly increased in recipient spleens (day 14:p<0.05). in parallel, frequencies of effector/memory phenotype t-cells (cd4 + cd44 high cd62l low and cd8 + cd44 high cd62l low ) were significantly elevated with increasing age (3 vs. 12 vs, 18mths: cd8 + cd44 high cd62l low :6.7% vs. 8.4% vs. 12.2%, respectively, p< 0.01). in addition, tregs (cd4 + cd25 + foxp3 + ) were also elevated (3 vs. 12 vs, 18mths: 2.9%vs.9.6%vs.11%,p<0.05) t-cell alloreactivity, as measured by ifnγ-production, increased with donor age (3mths vs. 12mths vs.18mths: 24.4±1.5 vs 74.4±21.9 vs. 73.2±7.8 ifnγ-producing spots/5x10 6 cells, p<0.05). mixed lymphocyte reaction (mlr) at day 14 revealed a gradual increase in splenocyte proliferation with advancing donor age (p=0.0002) indicating an enhanced immunogenicity of older organs. immunohistochemical staining confirmed augmented cd4 + and cd8 + t-cell infiltrates and an intense ki67 positivity of gics in 18mths old heart grafts, emphasizing an intensified immune response towards organs from old donors. in summary, old native heart transplants contain an overall higher number of passenger leukocytes contributing to an increased immunogenicity of these organs. after transplantation, a more potent dc and t-cell activation and intragraft t-cell infiltration was observed in elderly organs. nox and chronic kidney allograft interstitial fibrosis. shannon reese, 1 madhu adulla, 1 surmeet bedi, 1 jose torrealba, 1 deb hullett, 1 arjang djamali. 1 1 nephrology, uw madison, madison, wi. to determine the role of oxidative stress (os) in kidney allograft fibrosis, we are developing strategies that would decrease the generation of reactive oxygen species (ros) instead of using ros scavengers, the standard approach to antioxidant therapy so far. we therefore started to examine the expression of nadph oxidase (nox) subunits (nox2, nox4, p22 and p47phox) and their distribution in human and rat kidney allografts with chronic interstitial fibrosis (iftanos). using double-staining immunofluorescent studies in human allografts (n=16) we showed that nox2 was present in injured tubules costained with αsma ( figure 1.1) similarly, interstitial macrophages (cd68 + -nox2 + ) and myofibroblasts (αsma + -nox2 + ) but not cd3 + t cells or s100a4 + fibroblasts expressed high nox2 levels, suggesting that nox is involved in the pathogenesis of allograft fibrosis via epithelial-tomesenchymal transition (emt), macrophage and myofibroblasts activation. we then examined the coexpression of nox2, nox4 and p22phox in normal and transplant kidneys with iftanos and showed that these molecules were upregulated in the latter group and that nox2 and nox4 were associated with p22phox expression. these results were confirmed in the fisher to lewis rat kidney transplant model (figure 1 .2). immunoblot analyses at 3 weeks and 6 months showed that nox4 and nox2 levels were increased in the allogeneic (n=7) compared to syngeneic transplants (n=3). greater nox levels were composite tissue allotransplantation (cta) is a recently introduced option for limb replacement and reconstruction of tissue defects. as other allografts, a cta grafts can undergo immune-mediated rejection, and standardized criteria are required for characterizing and reporting severity and types of rejection. this manuscript documents the conclusions of a symposium on cta rejection held at the ninth banff conference on allograft pathology in la coruna, spain on june 26, 2007, and proposes a working classification scheme, the banff cta-07, for the categorization of cta rejection. this classification was derived from public international consensus discussions regarding all published scoring systems for cta rejection. given the current limited clinical experience in cta, a formal histological classification was established for acute skin rejection with the understanding that other types of rejection involving other tissues will be developed with periodic review of this emerging field. it was agreed that the defining features to diagnose acute skin rejection would include inflammatory cell infiltration with epidermal and/or adnexal structure involvement, epithelial apoptosis, dyskeratosis, and necrosis, and that severity of rejection will be graded under five categories. this classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for cta reporting. the role of skin biopsies in diagnosing clinical rejection in hand composite tissue allotransplantation (cta). christina l. kaufman, 1, 4 ruben n. gonzales, 1 kadiyala v. ravindra, 3, 4 brenda w. blair, 1 joesph f. buell, 3, 4 warren c. breidenbach. 1, 2, 4 1 christine m. kleinert institute, louisville, ky; 2 kleinert kutz and associates, louisville, ky; 3 jewish hospital transplant center, louisville, ky; 4 surgery, university of louisville, louisville, ky. aim: traditionally allograft biopsy has dictated treatment of allograft rejection. we hypothesize that histological grade of the biopsy may over diagnose rejection in cta hand cta is unique in that the organ is external and early signs of rejection can be viewed directly. skin is the first tissue in the cta graft to show rejection. methods: for this study, rejection by defined by requirement of treatment. these episodes were compared with histological grade, and hand appearance. we reviewed 127 skin biopsies taken during 9, 7 and 1 years of follow up. results: three observations surfaced. first, a rejection grading scale for cta is still evolving. a review of the literature showed at least four different criteria are in use. the criteria used to grade biopsies at our center changed over the 9 year follow up. bias towards calling higher grades of rejection, and more aggressive treatment occurred in the first two patients. a re-read of random biopsies taken from the first two patients showed a down-grade of rejection to grade ii or i in five cases. secondly, concomitant cmv infection in the last patient prevented the aggressive treatment of rejection. despite high grade histology, the swelling, rash and redness responded to topical immunosuppression. systemic treatment was not necessary. analysis indicated that swelling and/or rash, and the percentage of skin involvement seemed to be more informative markers of existing (or resolving) alloreactivity than was histology. the biopsies taken from the graft shown below showed a grade iii histology. thirdly, changes in hand function were not associated with changes in biopsy grade. conclusion: the histologic grade of skin biopsies from hand allografts appears to over estimate rejection. alterations in immunosuppression shoud be based on appearance of the allograft, and clinical course as much as on the histologic grade of the biopsy. expression of molecular mechanisms of lymphozyte trafficking correlates closely with skin rejection in human hand transplantation. theresa hautz, 1 bettina zelger, 2 gerald brandacher, 1 hans g. mueller, 3 andrew w. p. lee, 4 raimund margreiter, 1 stefan schneeberger. 1 1 dept. of general and transplant surgery, innsbruck medical university, austria; 2 dept. of pathology, innsbruck medical university, austria; 3 dept. of dermatology, innsbruck medical university, austria; 4 div. of plastic surgery, university of pittsburgh. introduction: to understand in greater depth the molecular mechanisms involved in skin rejection in hand transplantation, we investigated 8 key molecular markers of lymphocyte trafficking, cellular rejection and antibody mediated rejection in human hand transplantation. methods: a total of 130 skin biopsies taken from three bilateral hand transplants were assessed by h&e histology (grades as per previously a published classification 0-4b) as well as immunohistochemistry using antibodies for following markers: lymphocyte function-associated antigen (lfa)-1 = cd11a, intercellular adhesion molecule (icam)-1 = cd54, selectin e = cd62e, selectin p = cd62p, ve-cadherin = cd144, human leukocyte antigen (hla) ii (dp, dq, dr), psoriasin = s100a7 and c4d. levels of expression were assessed (0, +, ++, +++) and read in the light correlated with the rejection as well as time after transplantation. results: rrejection ranged between grade 0 and 4a with an average score of 0.9. in healthy skin, none of the markers investigated was consistently up-regulated. upon rejection, cd54, cd62e and cd62p staining in endothelial cells was significantly increased. expression of cd62e and cd62p correlated well with severity of rejection. the majority of infiltrating lymphocytes stained positive for cd11a. interestingly, also kerationcytes were highly positive for cd11a at the onset of rejection. cd144 was detected on endothelial cells, but its occurrence did not correlate with rejection. psoriasin expression was observed in keratinocytes in a basal and focal pattern and correlated well with rejection. for c4d, no consistent staining pattern was observed indicating that antibody mediated rejection did not play a role in these patients. conclusion: molecular markers involved in lymphocyte trafficking are up-regulated upon skin rejection after hand transplantation and represent promising target for prophylaxis and treatment of rejection in composite tissue allotransplantation. investigation of the immunomodulatory phenotype of infiltrating lymphozytes in skin rejection of human hand allografts. theresa hautz, 1 gerald brandacher, 1 bettina zelger, 2 hans g. mueller, 3 andrew w. p. lee, 4 raimund margreiter, 1 stefan schneeberger. 1 1 dept. of general and transplant surgery, innsbruck medical university, innsbruck, austria; 2 dept. of pathology, innsbruck medical university, austria; 3 dept. of dermatology, innsbruck medical university, austria; 4 div. of plastic surgery, university of pittsburgh. introduction: skin rejection has complicated the postoperative course in human hand transplantation. to better define the characteristics of the lymphozytic infiltrate human hand transplant biopsies have been investigated for expression of foxp3 and indoleamine 2,3-dioxygenase (ido), a key regulatory enzyme to induce t-lymphocyte unresponsiveness. methods: a total of 104 skin biopsies taken from three bilateral hand transplant recipients during the first 6 years after transplantation were assessed by h&e histology (graded as per previously published classification 0-4b) as well as immunohistochemistry for ido and foxp3. levels of expression were assessed (0, +, ++, +++) and interpreted in the light of clinical courses, time after transplantation, severity of rejection as well as markers for lymphozyte migration. results: overall, rejection ranged between grade 0 and 4a with an average score of 0.94. ido was found constitutively expressed in the endothelium independent of rejection. ido expression in the cellular infiltrate was significantly increased upon and correlated well with severity of rejection (rejection grade 1, 0.91+/-0.85: rejection grade 3, 2.30+/-0.82 p<0.001). foxp3 positive t-cells were mainly found in severe rejection (rejection grade 1, 0.14+/-0.35: rejection grade 3, 0.75+/-0.75 p=0.019). ido expression correlated well with foxp3 expression, although the overall staining intensity for foxp3 was lower. a strong tendency towards higher expression of ido as well as foxp3 towards later time-points after transplantation was observed (year 1 -foxp3 0.07+/-0.26, ido 0.75+ /-0.92 [n=68] , year 3 -foxp3 0.45+/-0.74, , year 5 -fox 0.25+/-0.45, ). expression of ido correlated closely with expression of e-selectin, p-selectin, icam1 and lfa-1. conclusion: characteristics of the cellular infiltrate indicate a strong tendency towards self limitation of the alloimmune response towards the skin with both time after transplantation as well as severity of rejection in human hand transplantation. further studies are warranted to clarify the clinical relevance of these findings. prospective analysis of the immunologic profile of a hand transplant recipient in the first year. kadiyala v. ravindra, 1 warren c. breidenbach, 2 joseph buell, 1 suzanne t. ildstad. 3 1 department of surgery, university of louisville, louisville, ky; 2 kleinert, kutz, and associates, louisville, ky; 3 institute for cellular therapeutics, university of louisville, louisville, ky. introduction: a major obstacle to the wider application of hand transplantation is the long term complications associated with immunosuppression. minimization of immunosuppression is an important goal in all transplant recipients. currently there are no accurate tools to evaluate the immunological responsiveness which might help tailor the level of immunosuppression for an individual patient. the response of recipient lymphocytes to pha, candida, and alloantigen may represent laboratory tool towards this end. it has been reported that these 3 responses are hierarchical with response to alloantigen being the first to be lost, followed by candida and finally pha. a 54 year old male received a proximal forearm transplant in november 2006. immunosuppression included induction with a single 30 mg dose of alemtuzumab and maintenance with tacrolimus and mycophenolate mofetil. the patient developed an episode of cytomegalovirus infection followed by acute rejection after reduction of his immunosuppression during the 3 rd post-operative month. these were successfully treated with ganciclovir and topical tacrolimus & steroids respectively. blood samples were drawn at selected time points, and subjected to phenotyping of lymphocyte subsets and immune monitoring for circulating peripheral blood regulatory t cells (t reg ) and proliferative responses to phytohemagglutinin (pha), candida, and alloantigen. results: alemtuzumab induction resulted in profound lymphopenia. at week 2 and 1 month, the response to pha was intact (stimulation index 110 and 24 respectively), but response to alloantigen and candida suppressed (si <3). a similar immunologic profile persisted up through 6 months. at 1 year, the pha and candida responses are robust (si 69 and 38 respectively), but alloresponses have not returned. current immunosuppression consists of tacrolimus (6-9 ng/ml) and mycophenolate mofetil (500 mg b.i.d.). there is no gross evidence of acute or chronic rejection. conclusions: induction with alemtuzumab alters the recovery of immune response: recovery to candida was delayed beyond 6 months and to alloantigens beyond a year. in light of this, further reduction of immunosuppression may be contemplated in future hand transplants without the risk of rejection. composite tissue allotransplantation has achieved significant clinical advances despite an adequate preclinical model to study technical and immunosuppressive strategies. we have developed a non-human primate model of facial composite tissue allografts (cta). unilateral lower hemi-facial cta (bone, muscle, skin) transplants were performed between mismatched cynomolgus monkeys. immunosuppression consisted of 28 days of continuous iv tacrolimus monotherapy followed by tapered daily im doses. six animals received prophylactic gancyclovir. all animals had serial transplant biopsies. ten transplants have been performed, with one loss secondary to line infection on day 27 without evidence of rejection. two ctas had evidence of chronic rejection (day 56, 99); with development of alloantibody after 30 days. five ctas had prolonged survival (day 60, 86, 108, 150, 177) , but developed ptlds resulting in experimental endpoints. all animals had clinically normal grafts, but 3 animals showed histological evidence of mild rejection not treated with any additional immunosuppressive therapy. ptld tumors were analyzed using short tandem repeats (str) to define donor or recipient origin. str analysis demonstrated donor origin of 4 ptld tumors and recipient origin in 1 animal. none of the ptld animals had clinical evidence of rejection of skin, bone, or muscle. ebv was not detected in the serum of 2 tested animals, and ganciclovir therapy had no effect on the development of tumors. tacrolimus levels of ptld animals were higher than animals with rejected grafts (45 vs. 34 ng/ml; p=0.02). two additional animals have healthy grafts (day 24+, 52+) without evidence of rejection or ptld, and have been converted to rapamycin after day 28. we have developed a preclinical model for facial cta transplantation that achieves prolonged graft survivals with tacrolimus monotherapy. the high incidence of ptld tumors of donor origin represents an outcome similar to bone marrow transplantation in contrast to its rarity in solid organ transplantation. our findings are a cautionary note regarding ctas that include vascularized bone elements. immunosuppressive protocol modifications have been made in an effort to decrease the incidence of these donor-derived ptlds. heterotopic heart transplantation: the united states experience. jama jahanyar, 1 tarek a. sibai, 1 matthias loebe, 1 michael m. koerner, 1 guillermo torre-amione, 1 george p. noon. 1 1 dept. of surgery, baylor college of medicine, houston, tx. heterotopic heart transplantation (hht) is utilized in patients (pts) who do not qualify for standard orthotopic heart transplantation (oht). specific indications include refractory pulmonary hypertension and a donor-recipient size mismatch. the objective of this study was to analyze the unos database and compare outcomes of hht to oht. the unos database with more than 58000 pts undergoing thoracic organ transplantation in the u.s. between 1987 and 2007 was reviewed (based on optn data as of may 1, 2007) . primary endpoint of this study was overall survival and subgroup survival [pts with transpulmonary gradient (tpg)>15, ischemic (icm) and dilated cardiomyopathy (dcm)]. secondary endpoint was assessment of pretransplant criteria. exclusion criteria were retransplantation and missing transplant dates. of 41379 who underwent oht and 178 who underwent hht, 32361 and 111 respectively, were enrolled in this study. [5] [6] [7] [8] [9] [10] survival after oht is superior to hht. this survival benefit however, disappears in pts with a tpg>15. overall the survival after hht is superior to the reported survival in pts who undergo lvad implantation as destination treatment (rematch-trial/1year survival of 52%). thus in selected pts, especially those with elevated tpgs, hht should be considered a viable option with overall good results. ventricular assist device as a bridge to heart transplantation in children: can we afford it? william t. mahle, glen ianucci, robert n. vincent, kirk r. kanter. sibley heart center cardiology, emory university school of medicine, atlanta, ga. ventricular assist devices (vads) allow children with severe heart failure to be bridged to successful heart transplantation (ht). vads are being used with increasing frequency in the pediatric population and newer devices allow even young infants to be supported. vad implantation and maintenance, however, is quite expensive and the cost-effectiveness of vad use in adults has been questioned. to date, an economic analysis of vad support in children has not been undertaken. methods: we used pediatric health information system (phis), an administrative database of the child health corporation of america (a consortium of children's hospitals in north america), to determine the costs related to vad use in children. data on subjects <18 yrs of age from 2002-2007 were reviewed. hospital charges were converted to costs based on hospital-specific cost-to-charge ratios. projected survival for subjects who were successfully bridged to ht was derived from published data. costutility was expressed as cost per quality-adjusted life years (qalys) saved, expressed in 2006 us dollars. all future costs and benefits were discounted at 3%. results: the median age at implantation from the phis database was 11.2 years, range 2 days to 17 yrs. the mean hospital cost per patient was $522,489. estimated survival to heart transplantation was 77% and estimated successful explantation without transplantation was 4%. the calculated cost-utility for vad as a bridge to transplantation was $123,232 /qaly saved. if one assumes that the children who survived to vad explantation would otherwise have a high risk of hospital death (65%) without vad support, then the calculated cost-utility would be $117,235/ qaly saved. even if abstracts survival to transplantation exceeds 90%, vad implantation does not achieve a favorable cost-utility ratio. vad support in pediatric heart only becomes cost-effective if recovery and explantation can be achieved in over 22% of subjects. conclusions: vad supports serves as an effective bridge to heart transplantation in children. however, the cost-utility of this strategy is above the generally accepted threshold for cost-effectiveness ($100,000/qaly). in a setting of limited healthcare resources vad as a bridge to transplantation may not be justified. purpose: heart transplantation (tx) in patients with hla sensitization presents challenges in organ allocation. virtual crossmatch (vxm), in which recipient hla antibodies, identified by labscreen pra beads, are compared to the prospective donor hla-type, could increase the use of allografts from distant donors (dd). accuracy of vxm and outcomes of this approach in heart tx are not known. methods and materials: to increase time-efficiency at the time of organ allocation, crossmatch testing is frequently initiated on pre-set trays which contain sera of multiple prospective sensitized recipients. we used results from these studies to determine expected accuracy of vxm. we assessed outcomes of allocation algorithm implemented in 2001 in sensitized patients. conventional prospective crossmatch was done when allografts were procured from local donors (ld), while vxm was utilized when allografts were offered from dd. there were 257 direct t-cell ahg crossmatch tests done with sera of 12 potential allograft recipients who had preformed hla-antibodies of known class i hla specificities. as shown in table 1, the positive and negative predictive values (ppv, npv) of vxm were 92% and 79%. table 2 shows outcomes in 30 sensitized patients who were eligible for vxm approach. 14 received allografts from ld with negative prospective crossmatch while 16 (53%) received allografts from dd with negative vxm. three dd patients had a positive retrospective crossmatch -npv of vxm was 81% in this cohort. vxm has high negative and positive predictive values, accurately predicting results of standard direct crossmatch in most patients. vxm allows use of allografts from dd and is likely to improve organ allocation in the disadvantaged group of sensitized patients. single center experience with new heart allocation system implemented by united network of organ sharing. biljana pavlovic-surjancev, 1 nilamkumar patel, 1 linda dusek, 1 james sinacore, 1 jennifer johnson, 1 cassie bessert, 1 alain heroux. 1 1 heart failure/heart transplant program, loyola university medical center, maywood, il. purpose: in july 2006, united network of organ sharing (unos) implemented a new allocation system for adult heart transplant (tx) candidates with the following sequence: local status 1a→local status 1b→zone a status 1a→zone a status 1b→local status 2. the purpose of this study is to evaluate the impact of new system on heart tx patients at a single center in region 7. methods: patients transplanted during 1 year (y) prior to new system (7-11-05 to 7-11-06, group 1, n=19) and 1 y following new system (7-12-06 to 7-12-07,group 2, n=26) were compared for unos status at the time of transplant, waiting time, ischemia time, length of the hospital stay (los) before and after transplant, donor age, procurement-team travel distance and cost. results: new system significantly decreased median waiting time, but increased median ischemia time without affecting short-term survival: 1 patient died in each group. number of transplants increased 37% in group 2 mostly due to increased number of status 1a patients supported by iabp without change in number of status 1b and 2 patients. median pre-transplant los increased 16-fold in group 2 (p=0.072), whereas mean pre-transplant los increased by 5 days. in group 2, thirteen patients had donor heart procured in zone a and thirteen patients had donor heart procured locally, whereas in group 1, all donor hearts were procured locally. median procurement-team travel distance increased 15-fold and median travel cost 10-fold in group 2 (p<0.05). clinical outcomes associated with simultaneous heart-kidney transplantation. tariq shah, 1, 3, 4, 5 suphamai bunnapradist, 2 jagbir gill, 2 steven k. takemoto. 1 the figure below indicates recipients of shk transplants (open symbols) had lower rates of rejection when compared to heart (square) or kidney (diamond) recipients. survival rates were initially higher for kidney recipients with rates for shk similar to heart recipients. the hazard ratio (hr) for graft loss was higher for shk recipients compared to kidney, but lower when compared to heart recipients. the lower hazard for shk in heart allografts might be attributed to risk associated with pretransplant dialysis (hr=4.09, 3.81-4.39, p<0.001) . approximately 10% (2,122) of cardiac transplant recipients initiated dialysis prior to transplantation. the differing rates of shk rejection observed in the heart and kidney analytic files might be attributed to susceptibility or treatment of heart and kidney allografts, rejection monitoring or reporting. conclusion: retrospective examination of data provided to the optn indicates simultaneous heart-kidney transplantation seems to be effective for cardiac transplant candidates who require dialysis. abstract# 123 objective: krp203, a novel structural analog of fty720, has been documented to display 5-fold greater selectivity of agonism for sphingosine-1-phosphate (s1p) type 1 (s1p 1 ) receptor compared with s1p 3 receptor. clinical trial has shown that fty720 produced dose-limiting toxicity-bradycardia, due to its effect on s1p 3 receptor. we have tested the effect of krp203 on the survival of islet allografts either alone or combined with local delivery of cd4 + cd25 + foxp3 + t regulatory (treg) cells. previous studies using knockout mice have documented a key role for the integrin cd103 in promoting allograft rejection. these data are consistent with a critical role for cd103 expressing cells in this process. however, a direct test of this hypothesis has proven problematic due to the lack of mabs that efficiently deplete cd103 + cells in wild type hosts. to circumvent this problem, we conjugated the non-depleting anti-cd103 mab, m290, to the toxin, saporin (sap), to produce an immunotoxin (m290-sap) that selectively depletes cd103-expressing cells in vivo. treatment of naive mice with m290-sap selectively depleted cd103 + cd8 + cells and dramatically reduced the overall frequency of cd8 + lymphocytes in diverse compartment including intestinal intraepithelial lymphcyte,spleen and mesenteric lymph node. m290-sap also depleted cd103 + dendritic cells (cd11c + ) and t regulatory cells (tregs, cd4 + cd25 + ) in the above compartments. in the thymus, m290-sap depleted cd103-expressing cells in both cd4 -cd8and cd4 -cd8 + subpopulations, both of which express cd103, leading to a dramatic reduction in the number of thymocytes(77.08 ± 5.74 ×10 6 vs. 2.71 ± 0.82 ×10 6 , p<0.0001, in control vs. treated respectively). we next assessed the effect of m290-sap in a fully allogeneic islet transplantation model (balb/c→c57bl/6). m290-sap produced long-term (lt) graft survival (>100d, n=9,vs. untreated median survival time 13d, n=8 ). unconjugated m290 or isotype control (igg-sap) did not significantly prolong islet allograft survival. graft histology showed little, if any, lymphocyte infiltration surrouding islet transplants in lt mice. in contrast, intense lymphocyte infiltration with disruption of islet morphology was observed in untreated mice. pretreatment of donor islets with m290-sap did not significantly prolong allograft survival indicating that the immunosuppressive effect of m290-sap resides at the level of host. interestingly, we found a 3-4 fold increase in both percentage and number of foxp3 + cd4 + cd25 + cells in the spleen and draining lymph node from lt mice, though it remains to be determined whether such cells account for graft acceptance in m290-sap treated recipients. in summary, these data document that depletion of cd103 expressing cells promotes long-term islet allograft survival. these findings point to a novel strategy for therapeutic intervention in islet allograft rejection. pancreatic objective: to engineer pancreatic islets in a rapid and efficient manner with a novel form of fasl protein chimeric with core streptavidin and test the efficacy of engineered islets for long-term survival in allogeneic hosts. methods: balb/c pancreatic islets were engineered first by cell surface modification with biotin followed by the display of a chimeric form of fasl protein that consists of extracellular domain of fasl fused c-terminus with core streptavidin (sa-fasl). sa-fasl-engineered islets were transplanted into streptozotocin diabetic c57bl/6 mice under transient cover of rapamycin. unmodified islets or those engineered with streptavidin protein (sa) served as controls. results: all the islets showed effective engineering with sa-fasl, which persisted on the surface of islets for weeks in vitro as assessed by confocal microscopy. all the islets (n=23) engineered with sa-fasl survived over the observation period of 100-400 days without detectable signs of rejection. in marked contrast, all the unmodified (n=9) and sa-engineered (n=14) islets underwent acute rejection within 40 days. the observed tolerance was localized to the engineered islets as unmodified second set of islets transplanted under contralateral kidney of long-term (>90 days) graft recipients were rejected in a normal tempo (mst=17 ± 9 days) without any effect on the survival of primary islets. conclusions: engineering pancreatic islets with exogenous immunomodulatory molecules, such as sa-fasl, in a rapid (∼ 2 hrs) and efficient (100% of targeted islets) manner represents a novel means of immunomodulation with considerable therapeutic potential for the treatment of type 1 diabetes. supported in parts by nih (r21 dk61333, r01 ai47864, r21 ai057903, r21 hl080108), jdrf (1-2001-328) the ability of embryonic stem (es) cells to form cells and tissues from all three germ layers can be exploited for the generation of cells that can be used to treat diseases. in particular, successful generation of hematopoietic cells from es cells could provide safer and less immunogenic cells than bone marrow cells, that require severe host preconditioning, when transplanted across mhc barriers. in the past, it has been difficult to derive hematopoietic cells from es cells. it has now become clear that this was due to the lack of self-renewal properties by these newly developed progenitor cells. here, we exploited the self-renewal properties of ectopically expressed hoxb4, a homeobox transcription factor, to generate hematopoietic progenitor cells (hpcs) that successfully induce high level mixed chimerism and long-term engraftment in recipient mice. hoxb4-transduced 129svj es cells (h2 b ) were allowed to form embryoid bodies. these were dismantled after 5 days and the cells treated with a cocktail of hematopoietic cytokines. by day 26, es cells had formed hpcs. these newly generated hpcs were cd45+, cd34+, cd117+ but poorly expressed mhc class i molecules and no class ii. the hpcs fully restored splenic architecture in rag2 -/-γ c -/immunodeficient mice, abstracts comparable to bone marrow. additionally, hpc-derived newly generated t cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus (lcmv) and specifically secreted il-2 and ifn-γ upon cd3 stimulation. further, hpc-derived antigen presenting cells (apcs) in chimeric mice efficiently presented viral antigen to wild type (wt) t cells. in syngeneic recipient mice, hpcs engrafted and formed more robust t and b cell populations. the majority of the hpc-derived cells were however, gr-1 + , suggesting a bias towards myeloid cells by the hoxb4. interestingly, these cells successfully engrafted in allogenic mrl (h2 k ) and balb/c (h2 d ) recipients without the need for immunosuyprresion. this ability to form mixed chimerism across mhc barriers is a consequence of their lack of mhc, cd80 and cd86 expression. our results demonstrate for the first time that leukocytes derived from es cells ectopically expressing hoxb4 are immunologically functional and escape immunological rejection when transplanted across mhc barriers allowing the induction of mixed chimerism. normalization the de is derived from the anterior segment of the primitive streak which corresponds to the early and mid-gastrula organizer during early embryo development, from which many of the major visceral organs, including the liver, pancreas, lung, thyroid and intestines are derived. es cells were cultured in a serum-free medium containing activin a and bfgf for 6-8 days, the differentiated cells developed into an epithelial monolayer yielding more than 70% of cxcr4 expressing cells. cxcr4 has been reported as a cell surface marker of the definitive endoderm. molecularly, the differentiated es cells express typical definitive endodermal genes in particular, foxa2, sox-17, gsc, and hnf4α. the cxcr4 + definitive endodermal cells were further purified using immunomagnetic bead separation to more than 99% purity in order to eliminate teratoma-forming cells. to study their engraftment and regenerative capacity, these newly differentiated cells were intravenously infused into a mouse with carbon tetrachloride-induced liver injury. harvested livers from these animals showed large de-derived cells positive for albumin suggesting that they were de novo generated hepatocytes. a second cell type was ck-19 expressing, suggesting that the engrafted cells also differentiated into cholangiocytes. therefore, we further transplanted these de cells into a factor viii null mouse and asked whether these cells could correct factor viii activity. plasma factor viii activity (coamatic assay) fully normalized to that of wild type mice and has remained stable over 60 days. these data suggest that es cell-derived de progenitor cells can restore factor viii activity in hemophilia a mouse model, presumably through protein production in de novo generated hepatocytes. more importantly, none of these animals developed teratomas. thus, es-cell derived cells can potentially be coaxed to form cellular transplants with curative capabilities. objective: we have established a novel approach, protex, to rapidly and efficiently engineer primary cells, tissues, or organs to display on their surface exogenous proteins of interest for immunomodulation. this approach involves generation of chimeric proteins with core streptavidin, biotinylation of cells, and the transient display of chimeric proteins on the cell surface. in this study, we displayed a chimeric form of fasl (sa-fasl) on the surface of bone marrow cells and tested the efficacy of these cells to establish mixed chimerism in allogeneic hosts under nonmyeloablative conditions. methods: balb/c bone marrow cells were engineered with sa-fasl and 30 million of these cells were transplanted into c57bl/6 mice subjected to various doses of total body irradiation 2 days earlier. a short course of rapamycin was used to enhance the tolerogenic effect of sa-fasl. bone marrow cell recipients were typed for multilineage chimerism at various times post-transplantation and tested for donor-specific tolerance using skin grafts. results: all the animals (n=8) treated with 300 cgy total body irradiation and transplanted with sa-fasl-engineered donor cells showed significant levels of chimerism (10-60%) on day 14 post-transplantation that showed a steady increase overtime and reached to 40-90% on day 60 post-transplantation. in marked contrast, none of the control animals (n=11) receiving bone marrow cells and a short course of rapamycin showed detectable chimerism. chimerism was multilineage and associated with donor specific tolerance since chimeric animals accepted donor, but rejected third party skin grafts. conclusions: engineering bone marrow cells in a rapid (∼2 hrs) and efficient (100% targeted cells) manner with exogenous proteins having immunoregulatory functions provides a new and effective means of immunomodulation to establish mixed allogeneic chimerism under nonmyeloablative conditions with significant potential in clinical bone marrow transplantation. funded in parts by nih (r21 dk61333, r01 ai47864, r21 ai057903, r21 hl080108), jdrf (1-2001-328) , ada , and aha fellowship 0725348b. chronic allograft dysfunction is still a major clinical problem in organ transplantation. morphologically it is characterized by changes suggestive of an alloantibody mediated mechanism such as glomerulopathy and vasculopathy or by non-specific changes such as interstitial fibrosis and tubular atrophy. alloantigen dependent as well as -independent factors contribute to the pathogenesis of these changes. in this study we analysed allospecific t cells from the peripheral blood of kidney transplant patients under different immunosuppressive protocols with or without chronic allograft dysfunction. 107 renal allograft recipients of our renal transplant clinic were screened at least six months after transplantation. all patients were on a calcineurin-based immunosuppressive protocol consisting of cyclosporine (csa)/mycophenolate mofetil (mmf)/steroid, tacrolimus (tac)/mmf/steroid, or csa/steroid. patients had to be mismatched for one or more of the five candidate hla-dr antigens for which synthetic peptides were available (dr1, dr2, dr3, dr4, and dr7). patients with biopsy proven chronic allograft nephropathy (can)with an elevated serum creatinine level of ≥1.6 mg/dl were compared with patients with stable allograft function (serum creatinine <1.6 mg/dl). t cell lines were generated from peripheral blood lymphocytes of renal transplant recipients against donor-derived hla-dr peptides presented by self apc. t cell lines generated from patients with can produced significantly more ifn-γ, while those generated from stable patients produced il-10 associated with a low proliferation index in response to the donor-derived mismatched hla-dr allopeptide in vitro. moreover, significantly more cd4+cd25+foxp3+ t cells were found in stable patients on tac and mmf as compared to patients on csa and mmf. interestingly, a higher gene expression of cd4, cd25, ctla-4, foxp3, and il-10 was observed in those patients. taken together a th1 (ifn-γ) alloimmune response is deleterious and promotes chronic graft damage, while a th2 (il-10) response seems to be associated with a lower incidence of chronic allograft nephropathy. an immunosuppression based on tac and mmf seems to favour cd4+cd25+fosp3+ t cells and to allow long-term engraftment with stable renal function. cyclosporin induces epithelial to mesenchymal transition in renal grafts. the expression of epithelial to mesenchymal transition (emt) markers is a reliable predictor of the progression towards interstitial fibrosis and tubular atrophy of the renal grafts. in vitro experiments suggest that calcineurin inhibitors (cni) can induce emt of tubular epithelial cells. although no evidence was ever provided in vivo, this suggests that emt could be involved in the pathogenesis of renal fibrosis induced by cni. we have previously reported the results of a prospective randomized trial comparing the elimination at month 3 of either cyclosporine (csa, n=54) or mycophenolate (mmf, n=54) from a triple drug regimen in 108 de novo renal transplant patients. all of them had 2 systematic graft biopsies at months 3 and 12 post engraftment. in the leftover material, we retrospectively detected in tubular cells and by immuno-histochemistry the expression of two validated markers of emt: the de novo expression of vimentin (vim) and the cytoplasmic translocation of b-catenin (cat). we were able to measure the emt score at both months 3 and 12 in a total of 68 patients (34 in each group). in the csa group, the vim and cat scores had progressed between 3 and 12 months from 1. calcineurin inhibitors (cni) are efficacious but nephrotoxic immunosuppressives. arteriolar hyalinosis is one of the characteristic histological correlates of this toxicity. yet, time course of this lesion, its reversibility, dose-dependency and the discrimination between drug-related effects, diabetic and hypertensive vasculopathy remain unclear. aim of this study was to evaluate the prevalence and time course of cni-related vascular changes after renal transplantation (tx) in protocol (pbx) as well as in indication biopsies (ibx) and to correlate this to the cni blood levels, blood pressure and diabetes. from 491 patients, a total number of 1239 pbx, taken at 6 weeks (n=380), 3 (n=420) and 6 months (n=439) after tx and 360 ibx were classified according to banff-criteria. assessement of cni toxicity included: isometric vacuolisation of tubules (isovac), intimal arteriolar hyalinosis (iah) and nodular arteriolar hyalinosis (nah) and vacuolisation of small vessel smooth muscle cells (vsm). 96% of patients received either cyclosporine or tacrolimus. in pbx, isovac was present in 15%, 17% and 15% of patients at 6 weeks, 3 and 6 months post-tx, respectively; iah in 11%, 9% and 10%; nah in 5%, 5% and 7%; vsm in 19%, 19% and 15%. in late ibx (>2 years post-tx) the prevalence of the analyzed parameters apart from isovac (9%) was markedly higher: 42% for iah, 22% for nah and 38% for vsm. in pbx, vsm, iah and nah were associated with each other but not significantly dependenct on blood pressure, rejection episodes or diabetes. through levels of cnis were not different between patients with and without vascular hyalinosis. in patients with vsm and iah in late ibx one third had these lesions already present in earlier biopsies. conclusion: the prevalence of presumed morphological signs for vascular cni-toxicity in pbx is low and constant and apparently, not associated with cni blood levels, hypertension or diabetes. in contrast, in ibx later than two years after transplantation, prevalence of vascular cni-toxicity signs is much higher. this emphasises that cni reduction protocols should be regarded within the first six months after transplantation, when vascular changes are still marginal. further elucidation of precursor lesions in pbx would help to find out patients at risk for cni-induced vascular changes. donor introduction: achieving donor specific tolerance has been the goal of the transplant community. success has been reported with donor stem cell transfusion in animal studies and prevention of chronic rejection in cardiac allograft recipients in the clinic. this report details the results of the initial phase of a study in humans. methods: a prospective phase i/ii fda approved pilot protocol was initiated to evaluate the effects of donor graft facilitating cell (fc)/stem cell infusion in kidney transplant recipients. conditioning was performed with 200 cgy of total body irradiation. bone marrow processed to remove gvhd-producing cells but retain cd8 + /tcr -fc and stem cells was infused 24 hours post-operatively. the dosage of stem cells was limited by the t cell dosage. the starting dose was 1 x 10 5 t cells. this was increased in steps of 2 x 10 5 per patient. as the study spans a 7-year period, the immunosuppression changed: 3 patients received cyclosporine (cya), mmf and prednisone; 3 were induced with basiliximab and maintained on cya(2)/fk(1), cellcept and prednisone; and 3 received alemtuzumab induction and maintenance with fk and mmf. all patients underwent tolerance testing and immunoprofiling studies. results: of the 9 patients, 6 received live donor kidney transplants. one graft was lost from arterial thrombosis on day 2. delayed graft function was seen in 3 patients. good long-term graft function was seen in the other 8 patients. acute rejection was noted only in 1 and infectious complications (cmv-1, histoplasma-1) in 2 patients. two patients died with functioning grafts -one at 4 years from lung cancer and another from complications from diabetes at 6 years. six patients are alive with functioning grafts at mean follow-up of 4.3 years with a mean creatinine of 1.3 mg/dl. no gvhd was detected in any of the patients. macrochimerism was not detected in any of the patients at any point. notably, in spite of the fact that durable engraftment was not yet achieved, none of the patients were sensitized as a result nor did they experience immunologic sequelae. conclusions: in our patients there were no untoward sequelae related to either the conditioning regimen or marrow infusion. the incidence of acute rejection even on longterm follow up has been low. we currently propose to reduce the immunosuppression further in these patients. a pilot study was performed to evaluate whether immune cell depletion with alemtuzumab would permit post-transplant weaning of maintenance immunosuppression in well-matched renal transplant recipients. patients received alemtuzumab 30 mg intravenously on the day of the transplant and the subsequent 2 days while sirolimus and tacrolimus were started on day 1. tacrolimus was discontinued at day 60 in all patients. extensive immune monitoring was performed at 1 year. at current follow-up (22 to 34 months), all patients are alive with a functioning graft (median mdrd gfr=46 ml/ min). one patient experienced clinical and biopsy-proven rejection at 9 months. all other patients remain on sirolimus monotherapy. four patients have been weaned to 1 mg of sirolimus daily as their sole immunosuppressive agent, with resulting blood levels of 3-4 ng/ml. these 4 patients have no evidence of donor-specific alloantibody, are unresponsive or hyporesponsive to donor cells by the cytokine kinetics test, and have a regulator phenotype to soluble donor antigens by trans-vivo dth. flow cytometry of peripheral blood demonstrated increased foxp3 expression in the cd3+cd4+ population (p=0.002). naive b cells (cd19/cd27neg) cells increased in 9 of 10 patients (p=0.02) and memory b cells increased in all 10 recipients (p=0.0005) when comparing pretransplant to 1 year timepoints. other than the patient with rejection, 12-month protocol biopsies did not show any evidence of rejection, although 2 of 9 showed focal c4d positivity and 1 diffuse positivity. these 3 patients also had evidence of alloantibody by luminex xmap testing. in conclusion, the cytokine kinetics test, alloantibody testing, and trans-vivo dth assay abstracts results correlated with clinical evolution of patients who successfully weaned both tacrolimus and sirolimus without rejection or alloantibody. the flow cytometry findings described occurred regardless of clinical evolution and may represent alterations of the immune system inherent in the treatment protocol independent of individual patient responses to the graft. however, the functional assays of cytokine kinetics assay and trans-vivo dth may be of potential use to correlate with the clinical immune status of the kidney transplant recipient. we have previously reported the short-term results of alemtuzumab (campath-1h) pre-conditioning with tacrolimus monotherapy and subsequent spaced weaning in living donor kidney transplantation (ldkt). we report here our 5 year experience. methods: we performed 411 consecutive unselected ldkt (donor kidneys were removed laparoscopically) from 12/11/2002 to 11/26/2007 using 30 mg (0.5mg/kg) alemtuzumab and tacrolimus monotherapy. at 6 months post-transplant and every 2 to 6 months interval, we used clinical data (including elisa antibody titers, cylex t-cell activation assay, and identification of donor specific antibodies) to wean tacrolimus when possible (bid-->qd-->qod-->tiw-->biw-->qwk). the recipients included 5 hiv+, 35 pediatric recipients, and 60 re-transplants. the mean follow up was 843.1+478.7 days. results: actuarial recipient survivals at 1-, 2-, 3-years were 98.4%, 95.6%, and 92.7%, respectively. graft survivals at 1-, 2-, 3-years were 97.6%, 90.4%, and 85.4%, respectively. the mean creatinine (mg/dl) at 1-, 2-, 3-years were 1.46+0.62, 1.56+1.08, and 1.54+0.89, respectively. the mean gfr (ml/min/1.73m 2 ) at 1-, 2-, 3-years were 73.0+28.5, 71.8+28.8, and 68.9+28.6, respectively. the cumulative incidence of acute cellular rejection (acr) at 6-, 12-, 18-, 24-, 30-, 36-, 42-, and >42 conclusions: in the current era low risk patients infrequently have ar, and have excellent short and long term graft survival without the use of depleting antibodies. given the increased costs of these drugs, the indications for using depleting antibodies in low risk ktrs of scd kidneys should be further clarified. kidney transplantation prolongs survival in hepatitis c virus-positive (hcv+) patients with end-stage renal disease (esrd). however, the effects of induction therapy and chronic immunosuppression are unknown on the course of hcv infection and potential for cirrhosis in renal transplantation (rtx) recipients. we have retrospectively assessed parameters of liver function, child-pugh (cp) and meld scores in hcv+ esrd patients who received induction therapy with t-cell depletion (group 1: thymoglobulin, n=30) or an il-2 inhibitor (group 2: basiliximab, n=46). pre-rtx liver biopsies were similar in group 1 and 2. patients were followed for a mean of 826 days (range 47 to 2679 days) following rtx and received tacrolimus, mycophenolate mofetil, and sometimes steroids post-transplant. overall graft survival was 84% in group 1 and 85% in group 2 (p > 0.05). data were analyzed pre-rtx, at 30 and 365 days and at time of last follow-up. serum ast, alt, platelets, inr, albumin and bilirubin did not change following rtx in either group. cp scores in group 1 were not significantly changed after rtx (5.5 ± 0.9 to 5.7 ± 0.9 at last follow-up, p=0.53). group 1 patients on steroid-free protocols (n=8) demonstrated declining cp scores from 6.0 ± 1.0 to 5.3 ± 0.5 at last follow-up that were not statistically significant (p=0.31). group 1 patients on steroids showed opposite trends of cp: 5.3 ± 0.6 pre-rtx vs 5.8 ± 1.1 at last follow-up that similarly did not reach statistical significance (p=0.18). group 2 cp scores declined from 5.8 ± 0.8 to 5.4 ± 0.9 at last follow-up (p=0.04). there was no difference between cp or meld scores at any point between the groups. as expected, meld scores improved significantly following rtx and remained low up until the final visit (p=0.001); this was attributed to the drop in serum creatinine post-rtx. neither the use of thymoglobulin or basiliximab resulted in acute hepatitis resurgence or the development of cirrhosis post-transplant. we have not identified any association between choice of induction agent or maintenance immunosuppression regimens, including steroid withdrawal, with impaired hepatic function or progression to liver cirrhosis in hcv+ rtx patients. t cell depletion was well-tolerated by hcv+ rtx patients and resulted in good graft outcomes. interestingly, cp scores declined after renal transplantation in the basiliximab induction group. kidney: complications i prevalence background: a few years ago we observed an expansion of blood gd t cells following cytomegalovirus (cmv) infection in kidney transplant recipient (ktr). we recently demonstrated that these cells share a strong reactivity against cmv infected cells and tumor epithelial cells in vitro. an implication of gd t cells in the immune surveillance against cancer has been demonstrated in mouse and strongly suggested in human. we tested here the hypothesis of a protective role of cmv-induced gd t cells against neoplasia in ktr through: 1/ a longitudinal case / control (ktr with cancer / ktr without cancer) study where gd t cell percentages were determined before and after cancer diagnostic (n=63), 2/ a retrospective follow-up of 131 ktr for 8.23 years looking for risk factors for malignancy. results:the median of gd t cell percentage in patients with malignancies was significantly lower when compared to control patients 18, 12 and 6 months before the diagnostic of the cancer (p<0.005). using a conditional logistic model, we determined that patients with a gd t cell percentage above 4 % were protected from cancer (p<0.008). a significant association between increase of the vd2 neg gd t cell subset and lower cancer occurrence was only retrieved in the ktr who experienced pre-or post-graft cmv infection. finally, using univariate and multivariable analysis, absence of pre-or post-graft cmv infection in ktr was associated with a risk of cancer 5.69 times more elevated (p=0.006). this study reveals an unexpected protective role of cmv against cancer in ktr most probably via the expansion of gd t cells cross-reactive against cmvinfected and tumor cells. background: viral infection (vi) is a morbidity factor in transplant recipients (tx pts). induction therapy (ind-rx) is a known risk factor for vi. although cam is thought to be a more potent ind-rx than zen, we have previously shown similar cmv infection rates in each. we have also shown that cmv-tc analyzed by cytokine flow cytometery (cfc) are consistently detectable in cmv sero(+), but not sero(-) individuals. cmv-tc(-) was associated with persistence of cmv infection in tx pts. here, we report on the effect of ind-rx on cmv-tc in kidney tx pts. methods: 58 pre-tx samples from 39 cmv-sero(+) pts and 62 post-tx samples from 44 pts were submitted for cmv tc-cfc. whole blood was incubated with a pooled overlapping peptide mixture consisting of 138 peptides from cmv pp65 and brefeldin a at 37 degrees for 6 hours and room temperature overnight. ifnγ+cd8+ cells were enumerated by cfc and results were expressed as ifnγ+cd8+ cell%. results >0.2% were considered as (+ infection associated graft loss during the entire study period is shown in figure1. infections contributing to renal allograft loss increased significantly from 1990 to 2006. this may be due to increase use of both induction agents and potent maintenance regimens. this is an important cause for poor long-term graft outcome despite decreasing rejection rates and a balance has to be maintained between prevention of rejection and avoidence of infection. serum creatinine (scr) at procurement was 101±51 µmol/l. the incidence of donor hypertension, diabetes, and death from cerebrovascular origin was 31%, 15%, and 55% respectively. multivariate analysis showed that the only clinical parameters associated with a low egfr were donor scr and donor hypertension. nyberg or pessione scores were not significantly associated with a low egfr. regarding d0 biopsies, univariate analysis showed that % of sclerotic glomeruli (sg, p=0.02), arteriolar hyalinosis (p=0.03), mean remuzzi score (p=0.03) and mean cadi score (p=0.04) were all significantly associated with a low egfr. a logistic regression showed that an integrated score including: i) donor scr (±150 µmol/l), ii) hypertension, and iii) sg (±10%) had the highest performance in predicting a low egfr at 1 yr compared to clinical or histological parameters alone. using this composite score, the adjusted or for the prediction of a low 1-yr egfr ranged from 1 if none of the 3 factors were present, to 7.1 (if sg >10% was associated with one of the 2 clinical factors), and to 27.5 (if the 3 factors were present, p=0.0003). conclusion: this study highlights that d0 biopsies are useful to predict graft outcome particularly in md population, and may perform better than clinical scores alone. in this population, a simple and routinely applicable integrated scoring strongly predicts a poor graft outcome, which may allow an optimized allocation of marginal donors. prospective kidney transplantation from small pediatric donors is increasingly being utilized as a means to optimize the organ supply, however the single most common specified reason for the discard of pediatric kidneys is vascular damage, such as shortening of the suprarenal aorta or injury to the renal artery orifices, which often precludes en bloc transplantation (ebk). at our center, damaged kidneys were salvaged by transplantation as singles (sk background: in an effort to maximize the number of recipients transplanted per donor, transplant centers in our donor service area (dsa) voted to preferentially allocate local and imported en-bloc pediatric donor renal allografts to centers willing to transplant two individuals with single allografts. after 1 year of implementing this policy into action, we report on our initial experience. methods: from july 2006 to june 2007 we reviewed our experience with 12 adult single allograft recipients of pediatric donors less than 60 months of age. there were no exclusions based on age or size with exclusion criteria consisting of donor age < 2 months and single allograft size < 5 cm. all but 1 recipient received rabbit anti-thymocyte globulin induction, tacrolimus, mycophenolate mofetil, and rapid steroid withdrawal. results from this cohort were compared to 86 consecutive recipients of adult single allografts from standard criteria donors with the same immunosuppression protocol used as historical controls. results: 12 pediatric single allografts with median donor age of 24 months (range 8-58) were transplanted into 12 adults with median age 46 years (range 24-67). showed that non-white race was associated with increased risk of death (p<0.01). this effect of race was attenuated when ltx center was taken into account [b] . finally, with the addition of meld in the model, the effect of race on waitlist mortality all but disappeared [c] . conclusions: on the surface, minority patients may appear to have higher mortality on ltx waitlist compared to caucasian counterparts. however, this association is predominantly a result of minority patients having a higher meld score, although ltx center-specific mortality may contribute. these data suggest that waitlist outcome may be improved by optimizing referral of minority patients. background: in cirrhotic patients awaiting liver transplantation (lt), low serum sodium (na) predicts short term pre-lt mortality, independently of meld. incorporation of na into meld has been recommended to improve prognostic accuracy (meld-na; gastroenterology 130:1652 gastroenterology 130: , 2006 ). however, short term interventions such as water restriction that improve na may have little effect on prognosis. hypothesis: the lowest level of serum sodium in the preceding 30 (na30), 90 (na90) or 180 days (na180) may be better than the current serum sodium (nac) for predicting pre-lt cirrhotic mortality. methods: we reviewed electronic records of 764 cirrhotic veterans referred for consideration of lt, 2/28/02-6/30/07. date of most recent na at referral was chosen as time zero for determining nac, na30, na90, and na180 and for assessing subsequent survival. findings: within 90 days, 94 patients died pre-lt (12%) and 27 underwent lt (3%). na at all time points was associated strongly (p<.001) with prelt death (censored at lt). areas under receiver operating characteristic curves (aurocs) for na30, na90 and na180 as predictors of 90d prelt mortality (mean±se) were .808±.026, .807±.026 and .783±.025, respectively, compared to .714±.032 for nac (all p<.05 vs. nac). on multivariable logistic regression analysis, meld and na90 were independent predictors of 90d prelt mortality, with best discrimination given by the following model: meld-na90 = meld + (135-na90)*1.04 with value of na90 capped at 135. aurocs for meld-na90, meld-na, and meld were .875±.027, .865±.025 and .838±.027, respectively. findings were similar when patients with hcc at referral (n=155) were excluded (aurocs .884±.025, .876±.026 and .840±.031, respectively), and when 90 day survival endpoint was changed from "death censored at lt" to "death or lt" (auroc's .890±.021, .882±.022, and .855±.026, respectively). conclusion: short term improvement in na may mask true prelt mortality risk. the lowest na in the preceding 90 days is a better prognostic indicator than current sodium. substitution of meld-na90 for meld would permit more accurate "sickest first" organ allocation, while at the same time allowing prelt correction of na without loss of priority. prospective validation of meld-na90, in comparison to meld-na and meld, is warranted. hyponatremia does not affect survival following liver transplantation. byung cheol yun, 1 w. ray kim, 1 y. s. lim, 1 joanne t. benson, 1 walter k. kremers, 1 terry m. therneau. 1 1 gastroenterology and hepatology, mayo clinic college of medicine, rochester, mn. background: hyponatremia is a common yet important complication of cirrhosis. serum sodium (na) has been found to be an important predictor of survival in patients with cirrhosis. models incorporating na have been proposed for liver allocation. concerns have been raised, however, that liver transplantation (ltx) in hyponatremic patients will adversely affect the outcome. in this work, we assessed the effect of pre-ltx na on the short term survival following ltx. methods: patient-level data on all waitlist registrants in the us for 2005 and 2006 were obtained from the organ procurement and transplantation network. demographic, clinical and laboratory data at the time of ltx and outcomes following ltx were extracted. the relationship between na pre-ltx and survival post-ltx was analyzed using multivariable regression analyses. results: there were 7411 primary transplants that met the inclusion criteria between 2005 and 2006. the median na in meq/l at the time of ltx was 136 (interquartile range, iqr: 132-139). there were 1255 patients who had a na ≤130 meq/l. the mean meld score was 22.5 (sd 9.2). median follow up was 279 (iqr: 155-371) days. the overall 30-and 90-day survival post-olt was 96% and 93%, respectively. in a multivariable logistic regression model, meld was associated with 1.04-fold increase in 90-day mortality (95 confidence interval: 1.03-1.05), while na did not have impact on survival (hr=0.99, 95% ci: 0.98-1.02). the figure represents the risk of 90-day mortality according to na after adjustment for meld, which clearly shows absence of mortality increase over a wide range of na. conclusions: hyponatremia at the time of ltx has no detrimental impact on short term patient survival following ltx. although these data do not address morbidity (e.g., central pontine myelinolysis), there is no evidence that incorporation of na in organ allocation will lead to diminished survival. objective. this study examined the relationship between meld at liver transplantation (lt) and post-lt quality of life (qol). methods. adult lt recipients (n = 247) at two centers completed the sf-36 and transplant symptom frequency questionnaire (tsfq) 1-year post-lt. high sf-36 scores indicate better qol; high tsfq scores indicate more symptomatology. clinical (lab) meld at lt, demographic characteristics, presence of ascites, encephalopathy, and variceal bleeding pre-lt, current employment status, presence of co-morbid medical conditions, and bmi were collected from medical records. results. primary lt indication was viral hepatitis (57%), cholestatic liver disease (17%), or hepatocellular disease (27%), and 63% had ascites, 51% encephalopathy, and 29% gastroesophageal bleeding. mean meld at lt was 20±9. there was almost no correlation between meld and sf-36 physical (r = 0.11) and mental (r = 0.001) functioning. statistically significant yet weak correlations were found between meld and physical functioning (r = -0.15) and role functioning -physical (r = -0.15). meld was not significantly correlated with any other sf-36 scales (r's -0.11 to -0.01). meld was not significantly correlated with any tsfq domains: affective distress (r = 0.08), neurocognitive symptoms (r = 0.05), gastrointestinal distress (r = -0.02), physical appearance changes (r = 0.09), appetite and weight changes (r = 0.09), and miscellaneous symptoms (r = 0.08). older age (ß = -0.28), female sex (ß = -0.26), viral hepatitis (ß = 0.67) or cholestatic disease (ß = 0.45), higher bmi (ß = -0.62), and >1 medical co-morbidity (ß = 0.57) were significant predictors of lower qol as measured by the sf-36 (adj r 2 = 0.12, f = 4.4, p < 0.001). older age (ß = 0.27), female sex (ß = 0.29), higher bmi (ß = 0.71), history of variceal bleeding (ß = 0.19), and >1 medical co-morbidity (ß = 0.17) were predictive of more symptoms on tsfq (adj r 2 = 0.10, f = 4.1, p < 0.001). meld was not predictive of qol. conclusions. higher disease severity, as measured by meld, at lt does not portend a worse qol outcome for patients 1-yr after transplantation. other pre-lt indicators of decompensation also do not predict post-lt qol. post-lt qol is affected more by other variables, including age, sex, bmi, and medical co-morbidities. introduction: racial disparities in access to cadaveric renal allografts have been well described for renal transplantation. however, little is known about differences in orthotopic liver transplantation (olt) rates for patients of minority racial groups following listing. the purpose of the current study was to determine if there is difference in rate of transplantation among racial groups and to examine the potential reasons for the disparity. methods: the united network for organ sharing (unos) database was obtained. data was extracted for adult olts greater than 18 years of age performed from 2/2002-12/2005. transplants for which recipient race or model for end-stage liver disease (meld) score at listing were unknown and patients active on the list were excluded. rates of transplantation as well as differences in reasons for de-listing (transplantation, death/deterioration, and improvement) were examined. in an effort to examine only patients with chronic liver disease, further analysis was performed excluding patients with acute fulminant liver failure and retransplants. results: the database contained complete meld and race information on 17,916 olts. seventy-four percent of patients were caucasian, 12% hispanic, 9% african-american, and 4% were asian. as seen in table 1 , laboratory meld score at removal differed between racial groups. examining pair-wise comparisons of the three minority groups to caucasians, only hispanics differed in reason for delisting (table 1) . subgroup analysis excluding acute hepatic failure patients and retransplants showed similar results with hispanic patients being more likely to die/deteriorate as compared to other racial groups (31% deaths vs. 24% deaths for caucasians), and being less likely to receive a transplant (69% of hispanics vs. 75% of caucasians, p<0.001). conclusion: hispanic patients, although listed with higher meld scores, are transplanted less often than caucasian patients and are more likely to die/deteriorate while awaiting olt. reasons for this discrepancy are unclear and merit further attention. background: since the implementation of the model for end-stage liver disease (meld) for liver allocation, an increasing number of candidates with renal insufficiency have undergone orthotopic liver transplantation (olt). since candidates with renal insufficiency have higher post-transplant morbidity and mortality, meld-based allocation may be shifting some waiting list mortality to the post-transplant period in these candidates. the objective of this study was to evaluate the survival benefit among candidates with renal insufficiency who underwent olt. methods: scientific registry of transplant recipients data for adult candidates age ≥18 initially listed for olt between 9/1/01 and 12/31/2006 (n=38,899) were analyzed. the effect of serum creatinine on the survival benefit (contrast between waiting list and post-transplant mortality) was assessed by sequential stratification, an extension of cox regression. each recipient was matched with candidates active on the waiting list in the same organ procurement organization with the same meld score. results: for meld scores 12-40, the survival benefit of olt significantly decreased as serum creatinine increased. among candidates transplanted at meld 12-14, the 23% with serum creatinine >1.1 mg/dl (23%) experienced no significant survival benefit ( figure) . candidates transplanted at meld ≥15 experienced significant olt benefit irrespective of serum creatinine level. conclusions: comparing two patients with meld ≥12, the patient with higher creatinine experiences significantly less survival benefit from liver transplantation. almost onequarter of patients transplanted at meld 12-14 experienced no survival benefit from olt based on 5 years of follow-up. therefore, more careful assessment of candidates is required in order to maximize the survival benefit gained by the wait-listed end-stage liver disease population as a whole. liver: living donors and parial grafts i assessment introduction: consideration of the risks and benefits of a procedure are critical in medical decision making. however, relatively little is known about risk tolerance amongst donors and transplant professionals in live-donor liver transplantation (ldlt). we conducted confidential semi-structured interviews in a convenience sample of donors, non-donors (individuals who had been assessed for donation but did not donate) and transplant team members. in addition to examining issues surrounding decision making for ldlt donation, we sought to assess the tolerance of participants, above which they would no longer contemplate donation, for a number of potential outcomes following ldlt. the outcomes that participants were asked to consider included their tolerance for risk of donor death, risk of serious donor complication, as well as risk of recipient death following transplantation. the interviews were conducted sequentially, data was coded quantitatively, and the study terminated once saturation was reached. (pre, weeks 1, 4, 12, 26 and 52 post-donation) . ambivalence detected by staff or described by donor was recorded. donor and recipient characteristics were examined and compared between ambivalent and non-ambivalent groups. results: staff identified and self identifed ambivalent donors were not equivalent. staff assessments indicated 20 ambivalent donors (16 male, 4 female). 18 donors self-identified as ambivalent (11 male, 7 female); 7 donors were on both lists (5 male, 2 female). the combinations of brother to brothers and sons to fathers were the most common pairs among ambivalent donors and more common than in total donor cohort. recipient diagnosis of alcohol or hepatitis c related liver disease was more common in ambivalent donors. ambivalent donors were more likely to be college educated and to express significant religious affiliations than the total rhl donor group. all but 1 ambivalent donor indicated that they would donate again on the 1 year qol survey. conclusions: ambivalence about rhl donation is present in approximately 20% of candidates who complete donation. staff-identified and self-identified groups showed only 20% overlap; however, both groups showed similar characteristics. brother-tobrother and son-to-father pairings and recipients with perceived self-induced liver failure were more common in both groups compared to total donor cohort. ambivalent donors had more education and stronger religious or spiritual identification than the entire cohort. only 1 donor indicated persistent doubt about donation. these results suggest that expressed or perceived donor candidate ambivalence may represent a process of careful consideration and should not be used sole basis for donor disqualification. the impact of donor age on recipient outcome for adult right-lobe living donor liver transplantation (rldlt) is unclear. aim: to analyze the effect of donor age on recipient outcome following rdldt. methods: since 2000 we have performed 226 rldlt (mean donor age 37 years, range 18-60 years), including 20 donors age 55 years or older. we analyzed the effects of donor age, as a continuous or categorical (< 54 vs > 55years) variable, on recipient outcome. recipient outcome measures included biochemical markers of hepatocytes injury (ast, alt) and graft function (inr, bilirubin), postoperative infections, bleeding, biliary complications, acute cellular rejection, as well as patient and graft survival. analyses were carried out stratified for higher recipient meld scores (< vs. >25), recipient age (< vs. > 60 years), and hepatitis c virus (hcv) infection (presence vs. absence). results: 5-year patient and graft survival after rldlt was 80% and 78%, respectively. donor age as a continuous variable was associated with increased ast (p=0.04) and alt (p= 0.022) release after transplantation, while no effect was observed on inr or bilirubin. rldlt using donors above 55 years of age resulted in an increased incidence of biliary strictures (20% vs. 6%, p= 0.028), postoperative cholangitis (25% vs. 9%, p= 0.023). no effect of donor age was found for the following recipient outcome measures: the number of bile ducts supplying the graft, type of biliary reconstruction required; rejection, hemorrhage, pulmonary or urinary tract infections, renal failure, or length of hospital stay. 5-year patient survival was identical for patients receiving grafts from donors below or above 55 years of age (81% vs 77%, p= 0.86). similarly, 5-year graft survival was comparable for young and old grafts (78% vs 77%, p= 0.71). recipient age (< vs > 60 years), recipient meld score (< vs >25), or hepatits c status of the recipient did not impact on the effect of age on patient or graft survival. conclusion: in this single center series of 226 rldlt, the use of selected older donors did not impair graft and patient survival, but was associated with an increased rate of biliary strictures. background: biliary stricture rate after living donor liver transplant (ldlt) in adults remains relatively high in comparison to the stricture rate after adult cadaveric liver transplant or ldlt in pediatric patients. the etiology or risk factors for biliary stricture development at present time are uncertain. purpose: to determine the risk factors for biliary stricture after right lobe (rl) ldlt. methods: from 5/99 to 12/07, 109 ldlt procedures were performed in 109 adult recipients. eleven patients were excluded from analysis due to <90 days follow up or need for retransplant. the following data was prospectively collected: 1. demographics, 2. acuity of illness, 3. number of bile ducts, 4. type of biliary reconstruction, 5. graft to recipient weight ratio, 6. hemodynamic parameters, 7. outcomes. these parameters were compared in patients with and without strictures. results: mean follow-up for 98 patients is 1642 days (range: 120-3052). 8 of 109 patients died during the follow-up range and 3 required whole liver re-transplants. 33 patients (34%) developed a biliary strictures during the follow-up period. comparison of risk factors in patients with and without strictures revealed the following results: mean meld >1 bile duct grwr* < 1. neither meld score, number of bile ducts or type of biliary reconstruction appear to be contributing factors to the development of bile duct stricture following rl ldlt. the biliary stricture rate was related to the volume of transplanted liver and post transplant graft recovery. therefore, the development of biliary strictures in some patients may represent yet another feature of small-for-size syndrome. background: ox40 and cd154 can be expressed by both foxp3+ tregs and activated t effector cells. however, the question as to how ox40 and cd154 function, individually or collectively, in regulating such functionally different t cell subsets in transplant models remains poorly understood. in some models, blocking cd154 costimulation is remarkably effective in prolonging graft survival, but targeting cd154 alone rarely creates tolerance. but the role of ox40 in regulating the cd154 blockade induced tolerance is completely unknown. in the present study we critically examined the role of ox40 in the activation of cd154 deficient t effector cells as well as in the regulatory function of foxp3+ tregs. we also examined the effect of ox40 on the induction of new foxp3+ tregs/th17 cells from activated cd154 deficient t effector cells. the impact of ox40 in the induction of allograft tolerance was examined using an islet transplant model. we found that cd154 deficient foxp3+ tregs constitutively expressed ox40 on the cell surface, but the cd154 deficient t effector cells did not. however, when the t effector cells were sorted and stimulated in vitro, ox40 expression could be abstracts readily induced on the t effector cells. to further examine how ox40 regulates such functionally different t cell subsets, we found that ox40 delivers potent costimulatory signals to t effector cells, which prevent the induction of new foxp3+ tregs from activated t effector cells but promote their differentiation to th1 cells but not th17 cells. surprisingly, ox40 costimulation to cd154 deficient foxp3+ tregs completely inhibited their regulatory functions. in an islet transplant model, we showed that cd154 deficient mice can reject the dba/2 islet allografts, but blocking ox40 costimulation readily induced donor specific tolerance (mst>150 days), and this tolerant status was critically dependent on the induction of foxp3+ tregs. in contrast, treatment of cd154 deficient recipients with a agonist anti-ox40 mab precipitate rapid islet allograft rejection, suggesting that ox40 costimulation is critically important in the induction of transplant tolerance. conclusions: our data suggest that ox40 is a costimulatory molecule to t effector cells but a powerful negative regulator for foxp3+ tregs. thus, a key role for ox40 in the induction of transplant tolerance is the control of t cell mediated regulation. background: foxp3 is a winged-helix family transcription factor that is the master regulator for the development and function of regulatory t cells (treg). we investigated the molecular mechanisms important for regulation of foxp3 expression, and defined the structure of the active foxp3 promoter in cd4 + t cell lineages. methods: purified cd4 + cd25 -foxp3 -gfp -t cells (naïve) and cd4 + cd25 + foxp3 + gfp + treg were cultured with antigen presenting cells in the presence of il-2, anti-cd3ε mab, tgfβ or the dna methyltransferase inhibitor 5-aza-2'-deoxycytidine (zdcyd). foxp3 promoter structure and activity were monitored with methylation-specific pcr, disulfite-sequencing, chromatin immunoprecipitation (chip) assays, electrophoretic mobility shift assay (emsa) and luciferase promoter assay. the foxp3 promoter has an upstream cpg island ∼5kb from the transcriptional start site. disulfite-sequencing and methylation-specific pcr analysis showed that this region is heavily methylated in naïve cd4 + t cells and tgfβ induced peripheral treg, but demethylated in thymic derived natural treg (ntreg). chip analysis showed that the methylated cpg island is bound specifically by the dna methyltransferases 1 and 3b. zdcyd causes demethylation of the cpg island, and in combination with tgfβ, synergistically induces foxp3 expression. chip assays for acetylated histone 3 and sp1, both markers of gene activation, showed that the cpg island is acetylated and bound by sp1 in ntreg and zdcyd plus tgfβ induced treg, but not in activated cd4 + t cells or tgfβ induced treg. emsa likewise shows the cpg island binds sp1. in contrast to the upstream promoter, the structure of the first intronic promoter differs markedly between ntreg and tgfβ induced treg, but is not affected by zdcyd. the upstream cpg island also possesses enhancer activity that is repressed by dna methyltransferases. zdcyd plus tgfβ induced treg have stable foxp3 expression and enhanced suppressive functions in vitro and in vivo. conclusion: these results demonstrate that ntreg and tgfβ induced treg are genetically distinguished from each other by the epigenetic structure of a unique upstream cpg island of the foxp3 promoter. the function of this region is regulated by dna methylation and histone acetylation. zdcyd demethylates the promoter, leading to enhanced and stable expression of foxp3 and suppressor activity, similar to ntreg. this has important implications for biology, and generating treg for tolerance. chemokine background: trafficking of lymphocytes through lymphatics to secondary lymphoid organs is crucial for immune responses. we previously showed that regulatory t cell (treg) function required trafficking from the inflammatory graft site to the local draining lymph node (dln). since the mechanisms that regulate migration through afferent lymphatics are poorly understood, we explored the role of chemokine receptors on treg for afferent lymphatic migration in an islet transplantation model. methods: islets were transplanted from balb/c mice into foxp3 gfp c57bl/6 mice. treg from wild type, ccr2 -/-, ccr4 -/-, ccr5 -/-, or ccr7 -/-c57bl/6 mice were isolated, labeled with red dye pkh26, and transferred intravenously, or locally into the islet allograft. treg migration to islet grafts and dln was determined by flow cytometry and immunohistochemistry. endogenous foxp3 gfp+ treg and transferred pkh26 labeled treg were sorted from the islet grafts and the dln, and chemokine receptor and sphingosine 1-phosphate receptor (s1p1) expression were determined by rt-pcr. islet allograft survival was determined by measurement of blood glucose. results: freshly isolated treg expressed s1p1 and the chemokine receptors ccr2, ccr4, ccr5, and ccr7. endogenous treg, and both intravenously and locally transferred treg, that were recovered from islet allografts and dln expressed similar levels of ccr2 and ccr5. ccr4 was expressed preferentially on islet migrating treg, while s1p1 and ccr7 were expressed preferentially in dln migrating treg. locally transferred treg migrated to the dln, but ccr7 -/-treg were not able to migrate to the dln. ccr2 -/and ccr5 -/-treg were impaired in their ability to migrate to the dln. this suggested that these three chemokine receptors all regulated treg entry into afferent lymphatics and migration from the graft to the dln. in contrast, ccr4 -/-treg migrated normally from the islet to the dln. importantly, ccr2 -/-, ccr5 -/and ccr7 -/-, but not ccr4 -/-treg, were impaired in their ability to prolong islet allograft survival when transferred locally in the islet allograft. conclusion: treg migrate from the inflammatory site of the allograft to draining secondary lymphoid tissue through afferent lymphatics. this process depends on ccr2, ccr5, and ccr7; and is crucial for full treg function in vivo. these results demonstrate a novel role for sequential migration from the graft to the dln in treg function and suppression. epigenetic regulation of gene expression provides a major, and especially beyond oncology, largely unexplored means to regulate host immune cell functions. our ongoing analysis of histone deacetylase (hdac) expression by foxp3+ naturally occurring murine regulatory t (treg) cells showed tcr-activated tregs had 5-6 fold more hdac6 mrna than corresponding resting treg or non-treg cells. in various cell types, hdac6 deacetylates alpha-tubulin, cortactin, and hsp90, abrogates formation of the aggresome, and blocks the unfolded protein response, though nothing is known regarding these pathways in tregs. we found that an hdac6-specific inhibitor, tubacin (but not the control compound, niltubacin), increased treg suppressive function in vitro (p<0.01), in association with increased expression of ctla, il-10, gitr, pd-1 and other treg-associated genes (p<0.05), and increased treg foxp3 protein (though not mrna) expression. tubacin enhanced the conversion of cd4+cd25-cells into cd4+ foxp3+ treg in vitro, and globally decreased cytokine production, with the exception of il-10 and il-17 mrna. comparable and dose-dependent effects were seen using the hsp90 inhibitor, geldanamycin, suggesting that the effects of hdac6 inhibition were mediated, at least in part, by blocking the chaperone effect of hsp90. use of tubacin in vivo significantly decreased the severity of colitis in two murine inflammatory bowel disease models (p<0.05), dextran sodium sulfate-induced colitis and the cd4+cd62lhigh adoptive transfer model of colitis, as assessed by standard clinical and histologic criteria. in addition, 14 days combined use of tubacin and a subtherapeutic dosage of rapamycin led to significantly prolonged cardiac allograft survival (balb/c->c57bl/6) compared to use of either agent alone (p<0.05). our data show that use of the first known small molecule inhibitor of one specific hdac has important therapeutic effects, including enhancing the production and suppressive function of tregs. while ongoing studies are directed towards unraveling the interactions of hdac6-dependent pathways and treg functions, the current data indicate the importance of understanding the functions of hdacs to the development of entirely new ways to regulate host immune responses. dendritic cells supply paracrine il-2 for treg cell functional activity. regulatory cd4+cd25+ t cells (tregs) are important for the maintenance of immune tolerance, and immunotherapy with tregs is being explored for organ and cell transplantation. treg development, expansion and function depend on il-2. because tregs do not make il-2, they must obtain il-2 from another cell. although cd4+ teffectors are a logical candidate, the identity of the paracrine source of il-2 for tregs is not substantiated. we explored whether dendritic cells (dcs) could serve as the paracrine source of il-2 for treg and rd6, a cd4+cd25+ regulatory hybridoma. using four dimensional live cell imaging we demonstrate that treg and rd6 cells establish tight contact with dcs, and cd25 is localized at these contacts. using the il-2 elispot and real-time rt-pcr we found that splenic dcs and the jawsii dc cell line constitutively make il-2. lps and cpg increases dc production of il-2. co-culture with jawsii dc cell line significantly upregulates cd25 expression on alloreactive do11.10 tregs and rd6 cells, but not on do11.10 cd4+ teffector cells. tregs and rd6 cells are functionally suppressive after activation by wild type but not il-2 knock-out allogeneic dcs, and anti-cd25 inhibits the function of treg and rd6 cells in a dose response fashion. in contrast, wild type and il-2 knock-out dcs are equally able to activate alloreactive cd4+cd25-cells. supplemental il-2 at high (1000 u/ml) but not low doses (100 u/ml) restores the function of alloreactive tregs and rd6 that were activated by il-2 ko dcs. these data indicate that treg cells acquire il-2 from dendritic cells for their gain of function and validate dendritic cells as a paracrine source of il-2 for treg. introduction: previously, it has been demonstrated that foxp3, a gene required for the development and function of regulatory t cells, was highly expressed in the graft during cardiac rejection, suggesting infiltration of regulatory t cells in the transplanted organ during an allogeneic response. in this study, we investigated whether graftinfiltrating t cells expanded from rejecting human cardiac allografts exhibit immune regulatory activities. methods: graft-infiltrating lymphocytes (gils) cultured from endomyocardial biopsies (emb; n=13) with histological signs of acute cellular rejection were expanded in the presence of donor-antigens in il-2/il-15-enriched medium for 2-3 weeks. flow cytometry was used to analyze the expression of cd3, cd4, cd8 and foxp3. to analyze the immune regulatory function, we performed mlrs with peripheral blood mononuclear cells (pbmc) of the patients and irradiated donor or third party spleen cells in the absence and presence of gils (ratio 5:1). results: of the cd3 + gils, 9% (median; range: 1-21%) stained positive for foxp3. this foxp3 expression was detected in both cd4 + and cd8 + t-cell population (median: 8% and 9%, respectively). functional analysis demonstrated that gils suppressed the antidonor proliferation of responder t cells (range % inhibition: 55-81%). interestingly, this suppression was predominantly achieved by cd8 + gils: depletion of cd8 + cells from the gils population diminished the inhibitory effect, whereas addition of solely cd8 + gils to the mlr abundantly suppressed the anti-donor response (range % inhibition: 62-77%). in contrast, gils did not inhibit the proliferation of t cells stimulated with third-party antigens. the figure below depicts a representative example. graft-infiltrating lymphocytes expanded from rejecting cardiac allograft exhibit donor-specific immune suppressive activities. these results suggest that during acute cellular rejection, graft-infiltrating lymphocytes not only consist of graft-destructing effector t cells, but may also comprise immune regulatory cells of the cd8 + phenotype. the context: it has been previously suggested that a liver allograft is immunoprotective and able to decrease the rate of rejection of a donor-specific allograft of another organ. it has been recently proposed that allografts other than the liver may also be immunoprotective. objective: the aim of this analysis was to examine one year rejection rate and the incidence of rejection free survival of all combined transplants in the collective us experience to gain insight to any possible protective effect of one organ for another. methods: the united network of organ sharing (unos) provided de-identified patientlevel data. analysis included all recipients transplanted between january 1, 1994 and october 6, 2005 who were 18 years or older (except intestinal transplants). rejection at one year was defined as treatment for one or more episodes of rejection. results: analysis included a total of 83,424 patients who received either one, or combined, simultaneous or sequential, organ transplants in all possible combinations. results are summarized in figure 1 (one-year organ allograft rejection rate). the collected data demonstrate that the rejection rate of donor-specific organ allografts which accompanied primary liver, kidney, and heart transplants was significantly lower in combined transplants as compared to that of the primary allograft transplanted alone. this was not true, however, for intestinal and pancreatic allografts where protection for the accompanying organ was not observed. we further demonstrate that transplantation of two organs of the same type (double kidneys or double lungs), i.e. increase in antigen load, also leads to decreased rates of rejection of the allografted organs. conclusions: in combined simultaneous transplants, the heart, liver, and kidney allografts appear themselves to be protected, and to protect the other organ from rejection. increased antigenic load of identical antigens in case of double lung and double kidney transplants appears to also offer immunologic protection against rejection, perhaps by different mechanisms. background: a2all (9-center adult-to-adult living donor liver transplantation cohort study) has identified risk factors for mortality after aaldlt, including center experience. the aim of this study was to determine if a2all findings are reflected in the national experience. methods: aaldlt at a2all (n=681) and non-a2all centers (n=1598) from 1/1/98 to 8/31/07 in the scientific registry of transplant recipients database were analyzed. cox regression models adjusted for recipient and donor characteristics were fitted to test associations with mortality risk after aaldlt, including center type (a2all vs. non-a2all) and case number (for each aaldlt at each center). results: aaldlt were performed at 9 a2all and 63 non-a2all centers. there was no significant difference in overall mortality risk between a2all and non-a2all centers. significant predictors of death (both groups combined) included donor age (hazard ratio (hr)=1.14 per 10 years, p=0.003), recipient age (hr=1.25 per 10 years, p<0.001), diagnosis of hcv (hr=1.22, p=0.04) or hcc (hr=1.99, p<0.001), and earlier center experience (aaldlt case number ≤15, hr=1.58, p<0.001). there was no significant effect of transplant year after adjusting for experience. cold ischemia time >4.5 hours was associated with higher mortality (hr=1.83, p=0.004); this effect was similar in a2all and non-a2all centers. there were no significant interactions between center type and any predictor except center experience ( figure) . compared to later experience, earlier center experience was associated with significantly higher mortality risk in both a2all (hr=2.24, p<0.0001) and non-a2all centers (hr=1.38, p<0.004). survival during early experience was significantly worse at a2all vs. non-a2all centers (hr=1.42, p=0.024), but survival in later experience was similar. conclusions: after the first 15 cases, aaldlt survival was similar at a2all and non-a2all centers, and similar significant mortality risk factors were identified, including center experience. these analyses support the generalization of findings from a2all centers to others performing aaldlt. abstract# 173 rejection with hemodynamic compromise (hc) and chronic allograft vasculopathy (cav) impact survival in pediatric heart transplantation (phtx). we showed that high pro-inflammatory / lower regulatory cytokine gene polymorphism (gp) profile increased the risk for acute rejection. in this analysis, we assessed the effect of genetic factors on hc and cav. methods: 406 phtx with clinical and gp data for cytokines (tnf-α a-308g; inf-γ t+874a; il-10 g-1082a, c-819t, c-592a ; il-4 c-590t; il-5 t-746c; il-6 g-174c), growth factors (tgfβ-1 t+869c, c+915g; vegf a-2578c, c-460t, g+405c), effector molecules (fas a-670g; fasl c-843t) and pharmacogenomics (abcb1 c3435t, g2677t/a) were analyzed regarding hc and cav. results: adjusting for recipient black race and age with cox regression models, we identified the following risk factors: il-10 high was associated with lower rates of hc. low th1 (inf-γ, tnf-α) with high th2 (il-4, il-5) cytokine gp profiles were protective for hc in combination with il-10 high. carriers of fas high experienced higher rates for hc and cav and high fas-fasl combination doubled the relative risk for cav. abcb1 3435cc/2677gg genotypes were also associated with lower rates of hc (table 1) . conclusion: in this large multi-center study gps with higher regulatory profiles and increased drug transport were associated with a lower incidence of hc. a genetic proapoptotic profile might contribute to the pathogenesis of cav. sponsorship: this work was supported by 5p50 hl 074 732-03 from the national heart lung and blood institute, national institutes of health. it has recently been reported that cd1d-restricted nkt cells that express invariant tcr (inkt cells) play an important role in the production of autoantibodies through the interaction with b-1 cells. this observation prompted us to investigate the possible role of inkt cells in the production of antibodies (abs) against transplant-related antigens, such as abo blood group carbohydrates and histocompatibility complex allopeptides, in a mouse model. we have previously demonstrated that b cells with receptors for blood group a carbohydrates were found exclusively in a cd11b + cd5 + b-1 subpopulation of mice, resembling humans with blood group o or b. immunization with human blood group a red blood cells (a-rbcs) elicited the extensive production of anti-a igm and igg. furthermore, the number of b-1 cells with receptors for a carbohydrates increased in the peritoneal cavity. in cd1d -/and vα14 -/-balb/c mice, which lack inkt cells, such elicited production of anti-a igm was not observed, even after immunization with human a-rbcs. however, class ii -/-balb/c mice, which lack cd4 + t cells but maintain normal levels of inkt cells, exhibited levels of anti-a igm production comparable to those in wild-type (wt) balb/c mice. moreover, anti-a igg production was absent in cd1d -/-balb/c mice even after the immunization, indicating that although inkt cells crucially contribute to anti-a igm production and igg class switching, helper t cells do not. notably, the proportion of b-1 cells in the livers of cd1d -/-balb/c mice was significantly reduced (2.66 ± 0.43%, n = 4) when compared to that in wt mice (4.76 ± 1.93%, n = 4). we next immunized cd1d -/and wt balb/c mice twice with 2 ×10 6 allogeneic b6 mouse thymocytes, and thereafter detected the anti-b6 (allopeptides) abs by flow cytometry. in the cd1d -/mice, anti-b6 igm production was comparable to that of wt mice, and igg class switching also occurred normally. these findings indicated that inkt cells play a pivotal role in the production of abs specific for blood group carbohydrate determinants that are believed to be t cell independent, but are not required in the production of abs for allopeptides that are believed to be t cell dependent. the depletion of inkt cells or the suppression of their function might constitute a novel approach for preventing antibody-mediated rejection in abo-incompatible transplantation, or in xenotransplantation, which involves similar carbohydrate antigens. background static cold storage (cs) is the most widely used organ preservation method for deceased donor kidney grafts. retrospective analyses have indicated that preservation by hypothermic machine perfusion (mp) may lead to improved outcome after renal transplantation. however, there is a lack of sufficiently powered prospective studies to test the presumed superiority of mp. in an international prospective randomized controlled trial we enrolled kidney pairs of 336 consecutive deceased donors and randomly assigned one organ to mp and the contralateral kidney to cs preservation. follow-up was directed at all 672 recipients of these grafts. the primary endpoint was delayed graft function (dgf). mp significantly reduced the risk of dgf (or 0.63; p=0.02) and more than halved the incidence of primary non-function after transplantation, when compared to cs (2.1 vs. 4.8%; p=0.04). furthermore, mp significantly reduced the risk of graft failure in the first 6 months post-transplant (hr 0.46; p=0.05). in recipients who developed dgf, 6-month graft survival was better if their transplanted kidney was machine perfused (87 vs. 76%; p=0.05). hypothermic machine perfusion reduces the risk of delayed graft function, primary non-function, and graft failure in deceased donor kidney transplantation when compared to static cold storage. furthermore, mp alleviates the deleterious effect of dgf on graft survival. we investigated the trafficking of cells after skin and heart transplantation in a dynamic fashion through the use of in vivo microscopy. antigen presenting cells were followed using mhc-cl-ii-gfp and cd11c-gfp transgenic mice. vascularized and non vascularized skin grafts as well as heart transplants were used in syngeneic as well as allogeneic settings. after syngeneic non-vascularized skin transplantation, we observed an early and massive cellular infiltration of host cells into the graft as early as 3 hours post-transplant with a gradual accumulation in the dermis. the accumulation of host-derived cells was accelerated after graft vascularization at day 4/5 post transplantation. this graft infiltration by recipient cells was more pronounced with vascularized skin grafts, and to a higher degree in heart transplants. recipient cells similarly infiltrated allogeneic grafts early on and in larger numbers than for syngeneic grafts by day 4/5 post-transplantation. when visualizing mhc-cl-ii-gfp recipient cells in a syngeneic skin transplant, recipient dcs invaded the graft early on and, by 3 weeks post transplant gradually replaced graft dcs in the dermis (dermal dcs) and the epidermis (langerhans cells). donor dcs could still be seen in the graft up to 8 days post transplant. however, virtually all donor langerhans cells were eventually replaced by recipient ones in a concentric fashion suggesting that the new langerhans cells originate from the recipient skin adjacent to the graft and not from centrally-derived precursor cells. the vascular endothelium of a syngeneic transplant was partially replaced by recipient vascular endothelial cells in a centripetal fashion with more recipient-derived vascular endothelium present at the periphery of the graft and more donor-derived endothelium remaining in the center of the graft. therefore, the graft can be seen as a "chimera" of cells from donor and recipient origin. the presence of recipient endothelial vascular cells and dcs within the graft may be important for maintaining the indirect response thought to be responsible for chronic rejection. objective: maturation resistance and tolerogenicity can be conferred on dendritic cells (dc), -crucial regulators of t cells, by exposure to rapamycin (rapa), a tolerance-sparing immunosuppressant. the mechanisms underlying this acquired unresponsiveness, typified by diminished responses to toll-like receptor (tlr) or cd40 ligation, have not been identified. thus, our objective was to elucidate a molecular basis for rapa-induced dc maturation resistance. methods: rapa administration was used to condition splenic dc in vivo and bone-marrow derived dc in vitro. dc maturation was monitored by assessment of co-stimulatory molecule expression, cytokine production, and t cell allostimulatory capacity. to identify negative regulators of maturation, microarray analysis and quantitative rt-pcr was completed, and findings confirmed via western and flow cytometric analyses. results: in vitro or in vivo exposure of myeloid dc to rapa elicited de novo production of il-1β by otherwise immature dc (cd86 lo ). interestingly, dc il-1β production, acting in an autocrine/paracrine fashion, promoted dc overexpression of the il-1 receptor(r) family member, st2l, and enhanced its surface expression. st2l is the receptor for il-33, an il-1 family member, and has also been implicated as a negative regulator of tlr signaling. consistent with this regulatory function, il-1β-induced st2l expression suppressed the responsiveness of rapa-conditioned dc to tlr or cd40 ligation. conclusion: rapa causes de novo production of il-1β by immature dc, upregulating st2l, and establishing a barrier to dc maturation following exposure to tlr or cd40 ligation. as such this work identifies a novel mechanism by which a clinically-important immunosuppressant impedes the capacity of dc to mature and consequently stimulate effector/adaptive t cell responses. these findings are particularly relevant to the potential use of rapa-conditioned dc as "negative" cellular vaccines to block alloag-specific responses, as exposure to endogenous and exogenous inflammatory stimuli can induce dc maturation and negate the tolerogenic properties of immature dc. exosomes are nanovesicles (50-100nm) released to the extracellular milieu by different cell types. exosomes secreted by dendritic cells (dcs) and other apcs express mhc ag, adhesion molecules and costimulatory molecules oriented on the membrane surface with their binding domains facing outwards. thus, exosomes released by graft-infiltrating leukocytes (gils) could function as "ag-presenting vesicles" or as vehicles to transfer alloag between recipient's apcs during elicitation of t-cell allo-immunity. aims: to test if (i) gils activate anti-donor t-cells in secondary lymphoid organs by releasing exosomes with alloag into systemic circulation; or (ii) gils that traffic to the spleen as passenger leukocytes use exosomes as a local mechanism to transfer alloag to recipient's dcs. methods: exosomes were isolated from supernatants of bm-derived [c57bl/6(b6), ia b ] dcs pulsed with the balb/c iea 52-68 allopeptide and purified by ultra-filtration and ultra-centrifugation on a 30%sucrose/d 2 o gradient. we used pkh67 + exosomes and cd45.1 congenic b6 mice for traffic studies, heart (heterotopic) and skin transplantation models (balb/c→b6, thy1.2 + ), and cfse-labeled 1h3.1 tcrtg cd4 t-cells (thy1.1 + ) specific for ia b (b6) loaded with iea 52-68 (balb/c). dcs were genetically engineered to release exosomes expressing green fluorescent protein (gfp). we have previously shown that blood-borne exosomes carrying balb/c alloag are reprocessed by different subsets of splenic dcs for presentation to indirect pathway 1h3.1 cd4 t-cells. here, we demonstrated that although gils of cardiac and skin allografts release exosomes ex vivo, they did not secrete enough concentrations of exosomes with alloag into circulation to stimulate donor-reactive t-cells in lymphoid organs. instead, our findings indicate that migrating dcs (generated in vitro or isolated from gils), once homed in the spleen, they transfer exosomes expressing gfp and carrying allopeptides to spleen-resident dcs of the recipient, identified by the congenic marker cd45.1. conclusion: exchange of exosomes between dcs in lymphoid organs might be a mechanism by which passenger leukocytes transfer alloag to recipient's apcs in secondary lymphoid organs. t cell activation is critical in initiating adaptive immunity, and pkcθ, a novel member of the pkc family, mediates non-redundant functions in the t cell receptor; however, its role in the mediation of allograft rejection remains unclear. this study is aimed at investigating whether alloimmune response can be alleviated by a deficiency of the pkcθ molecule, and whether transgenic expression of anti-apoptotic bclmethods. wild-type (wt) cardiac allografts were transplanted into pkcθ -/mice, with or without sub-therapeutic anti-cd154 mab. purified pkcθ -/or pkcθ -/-/ bcl-x l t cells were adoptively transferred into rag2 -/mice engrafted with cardiac allografts. lymphocyte proliferation assays were performed (cfse). nf-kb activation was assessed by bioluminescence imaging (bli) using luciferase transgenic mice under the control of a nf-kb promoter. results. the cardiac allografts were rejected in a delayed fashion in pkcθ -/mice with increased nf-kb activation; however, sub-therapeutic anti-cd154 mab (that normally delays rejection of cardiac allograft) induced long-term survival of cardiac allografts. the cardiac allografts were permanently accepted in rag2 -/mice with adoptive transfer of pkcθ -/-t cells, and the rejection can be elicited by transfer of pkcθ -/-/ bcl-x l t cells. in a lymphocyte proliferation assay, pkcθ -/-t cells displayed greatly reduced proliferation. in response to cd3 and cd28 stimulation, pkcθ -/-t cells underwent accelerated apoptosis and reduced th1, th17, and treg subsets compared to the wt t cells. bcl-x l restored the survival of the pkcθ -/-t cells. conclusions. the results suggest that pkcθ mediates the alloimmune response. bcl-x l transgene prevents pkcθ -/-t cell apoptosis and re-elicits allograft rejection. tolerogenic dendritic cells (dc) are immature, maturation-resistant(mr) or alternatively-activated dc that express mhc molecules and low levels or absent costimulatory signals. although mrdc administration has successfully prolonged allograft survival in murine models, the mechanism of action in vivo remains unknown. aim: to test in vivo if the down-regulation of the anti-donor response induced by donor-derived tolerogenic dc is due to: (i) direct interaction of the tolerogenic dc with donor-reactive t cells or (ii) by reprocessing of the tolerogenic dc into alloantigen (alloag) by recipient apc for interaction with indirect pathway t cells. methods: dc were generated in vitro by culturing balb/c bone marrow cells for 6-8 days in medium with gm-csf + il-4 supplemented with 10nm 1α,25-(0h) 2 vitamin d 3 (vd 3 ). we used a model of heterotopic vascularized allogeneic heart transplantation [balb/c into c57bl/6 (b6)] and cd4 t cells from 1h3.1 tcrtg mice that recognize b6 ia b loaded with the balb/c allopeptide ieα 52-68 (indirect pathway) . results: we demonstrated that vd 3 renders dc maturation resistant (vd 3 -mrdc) as vd 3 -mrdc fail to up-regulate co-stimulatory molecule expression, release il-12p70, or stimulate allo-responsive t cells after challenge with potent dc-maturation stimuli. adoptive transfer (i.v.) of balb/c vd 3 -mrdc (day -7) significantly prolonged survival of balb/c heart grafts in b6 mice in the absence of immunosuppressive therapy. interestingly, we found that in vivo, balb/c vd 3 -mrdc induced proliferation of indirect pathway 1h3.1 cd4 t cells in the spleens of b6 recipient mice, indicating that reprocessing of the balb/c dc by host (b6) apc does occur. proliferation of 1h3.1 cd4 t cells in response to balb/c vd 3 -mrdc resulted in defective activation (cd62l high , cd69 low ) of 1h3.1 t cells, leading to their peripheral deletion and outgrowth of cd4 + foxp3 + treg cells. reprocessing of balb/c vd 3 -mrdc was performed by recipient splenic cd11c high cd8α neg dc, and donor alloag continued to be presented through the indirect pathway for 3 days after donor dc administration. conclusion: these results suggest that dc-based therapies downregulate t cell allo-immunity and prolong allograft survival, at least in part, through reprocessing of the tolerogenic dc into alloag by recipient apc. early introduction: alloreactive memory t cells are present in all transplant recipients due to prior direct sensitization or heterologous immunity. these cells are known to circumvent tolerance induction and/or prevent indefinite graft survival in several models, but mechanistic details of their function are unknown. the goal of this study was to test the hypothesis that cd8 memory t cells initiate alloreocognition and express effector functions within hours of reperfusion. methods: syngeneic or a/j (h-2 a ) hearts were transplanted into wt c57bl/6 (h-2 b ), cd4-/-, cd8-/-, or rag1-/-recipients. rna and protein were prepared from total graft homogenates and analyzed by qrt-pcr and elisa. rag1-/-mice received 1x10 6 wt or ifng-/-2c cells and were used as recipients 10 weeks after reconstitution. donor-specific cd8 memory cells were purified from wt spleens 8 weeks after a/j skin grafting, and donor-specific effector cd4 cells were purified from spleens of cd90.1 mice 8 days after a/j heart transplantation. flow cytometry was used to quantify graft infiltrates. results: allografts contained elevated levels of ifng and cxcl9 mrna at 24, 48 and 72 hrs post-transplant vs. isografts. detectable cxcl9 protein was produced in allografts from wt and cd4-/-recipients but not in isografts or allografts from cd8-/-or rag1-/recipients. treatment with ctla4-ig and mr1 failed to reduce cxcl9 production. reconstitution of rag1-/-mice with ifng sufficient or deficient 2c tcr transgenic cd8 cells indicated that early allospecific cxcl9 production absolutely requires ifng made by recipient cd8 cells. although donor-specific ifng production was undetectable in splenocytes until day 4-5 post transplant, graft-infiltrating cd44 hi cd62l lo cd8 t cells were present as early as 24 hrs post-transplant. in adoptive transfer studies, effector-memory cd8 t cells reconstituted early allospecific cxcl9 production in cd8-/-mice. lastly, primed cd4 t cells adoptively transferred at day 2 post-transplant readily infiltrated allografts in control but not cd8 depleted recipients. conclusions: cd8 memory t cells infiltrate allografts rapidly post-transplant, produce ifng, and propogate an inflammatory environment which optimizes recruitment of primed effector t cells. successful neutralization of this early allorecognition pathway should provide valuable adjunctive therapy to improve graft function and survival. background: allogeneic t cell stimulation requires not only antigen-specific signals but also costimulatory signals, most importantly between cd80/86 on the antigen presenting cell (apc) and cd28 and ctla4 on the t cell. engagement of the t cell receptor without costimulation can lead to anergy and the induction of regulatory t cells (tregs). t cell activation is also controlled by expression of the tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (ido). depletion of this essential amino acid, and/or the production of tryptophan metabolites inhibits t cell proliferation. methods: a genetic approach to confer tolerogenic properties on murine dendritic cells (dcs) has been explored using lentiviral vectors, based on the equine infectious anaemia virus. firstly, an intracellular method that prevents costimulation has been developed: a fusion protein consisting of ctla4 and kdel [an endoplasmic reticulum (er) retention signal] is expressed in dcs. the ctla4-kdel binds to cd80/86 in the er and prevents expression of these proteins on the dc surface. a second approach uses an elevated expression of the ido enzyme by transduced dcs. results: ctla4-kdel-or ido-transduced dcs were unable to induce allogeneic t cell proliferation. however, using two-stage dc:t cell co-culture assays, it was shown that ctla4-kdel-, but not ido-transduced dcs, can induce donor-specific t cell anergy in vitro and in vivo. tolerance to both the direct and indirect pathways was shown using ctla4-kdel-transduced dcs. linked suppression was mediated by the generation of donor-specific tregs. ido-transduced dcs did not generate tregs. furthermore, it was shown separately that dcs expressing ido whilst lacking cd80/86 expression for potential ligation by ctla4 (although ctla4-cd80/86 ligation upregulates ido, it downregulates t cell activation) failed to generate or even sustain foxp3+ treg populations. the ability of the transduced dcs to induce tolerance to allografts was assessed in a complete mismatch and cbk→cba (indirect pathway) corneal graft model. these results support a clinical strategy to induce treg-mediated, donorspecific transplantation tolerance using ctla4-kdel-, rather than ido-expressing dcs. indirect cd4 t cells that recognise processed alloantigen on recipient apc can provide help to alloreactive cytotoxic cd8 t cells that recognise intact mhc i alloantigen on donor apc, but exactly how such 'un-linked' help is provided is not clear. the respective abilities of direct and indirect pathway cd4 t cells to provide help for cytotoxic cd8 alloimmunity were examined in a mouse model of heart graft rejection in which the recipients contain only monoclonal helper cd4 t cells, specific for self-restricted h-y antigen (female b6 mar/rag1 -/mice). mice were additionally reconstituted with 10 6 b6 cd8 t cells, and then challenged with female balb/c (no cd4 t cell help), or male balb/c (indirect pathway help), or male b6xbalb/c f1 hearts (direct pathway help) . un-reconstituted mar/rag1 -/mice lack effector b and cd8 t lymphocytes, and consequently all heart grafts survived indefinitely. in contrast, reconstituted mar/ rag1 -/mice rejected male f1 grafts rapidly (mst 8d), whereas female balb/c grafts survived indefinitely, confirming a cd4-dependent effector role for the transferred cd8 t cells. cd4 t cell help through the indirect pathway, although sufficient to elicit graft rejection, was less efficient than direct pathway help, because male balb/c grafts were rejected more slowly than the f1 grafts (mst 12d, p<0.001). we next considered whether indirect pathway cd4 t cells provide help through recognition of mhc ii complexes on the surface of alloreactive cd8 t cells, in analogous fashion to the cognate interaction between b and t lymphocytes. in support, reconstitution of mar/rag1 -/recipients with instead, mhc ii-deficient cd8 t cells, resulted in slower rejection of male balb/c hearts (mst 21d, p<0.01), whereas male f1 grafts, that still permit provision of linked help, were rejected at the same tempo. most tellingly, mar/rag1 -/mice that received simultaneously a female balb/c heart and male b6 apc (to activate mar cd4 t cells) rejected their grafts rapidly when reconstituted with male cd8 t cells (mst 9d). in contrast, grafts survived indefinitely when female cd8 t cells were transferred. flow cytometric analysis of mitogenstimulated cd8 t cells revealed surface mhc ii expression. indirect allorecognition can provide help for generating cytotoxic alloimmunity, but not as effectively as through the direct pathway. indirect pathway help is potentiated by linkage through recognition of cd8 mhc ii. purpose: tolerogenic properties of dendritic cells (dc) are supported and preserved by conditioning with the immunosuppressant rapamycin (rapa). the ability of rapaconditioned, recipient-derived dc pulsed with alloantigen (alloag) to suppress both direct and indirect alloag-specific t cells in the absence of immunosuppression has been demonstrated in a murine allograft model. dc can acquire intact mhc from cells or cell lysates. however, the ability of alloag-pulsed rapa-dc to immunomodulate directly-reactive alloag-specific t cells has not been formally demonstrated. methods: dc were generated from c57bl/6 (b6; h2 b ) bone marrow cells in gm-csf and il-4. rapa was added to indicated cultures (rapa-dc) beginning on day (d) 2. on d7, cd11c + bead-purified rapa-dc or non-treated control dc (ctr-dc) were incubated with balb/c (h2 d ) splenocyte lysates ("alloag pulsing"). following incubation, the dc were harvested and the level of donor and recipient mhc molecules on cd11c + cells determined by flow cytometry and immunofluorescent imaging. surface levels of cd86, b7-h1 (programmed death ligand-1; pd-l1), and fas-l were compared. pulsed-dc were also incubated with cd8 + t cells from rag -/-2c mice for 3d. 2c cd8 + cells express t cell receptors specific for h2-l d , a mhc class i molecule of balb/c. following incubation, 2c cell proliferation and apoptosis were both assessed. results: ctr-and rapa-dc presented detectable levels of directly-transferred mhc class i and ii on their surface after incubation with allogeneic balb/c cell lysate. donor mhc presented by "pulsed" recipient dc stimulated directly-reactive, alloag-specific 2c t cell proliferation. however, only rapa-dc induced apoptosis in the overwhelming majority of these cells responding via the direct pathway. induction of apoptosis correlated with an increased level of surface fas-l on rapa-dc and their comparatively low level of cd86 relative to pd-l1. conclusions: rapa-conditioned dc can present intact mhc molecules acquired from lysates of allogeneic splenocytes and concurrently induce apoptosis of directlyreactive alloag-specific cd8 + t cells. as such, we provide mechanistic insight into a mechanisms by which alloag-pulsed, recipient-derived rapa-dc may facilitate allograft tolerance. background: t regs actively regulate alloimmune responses and promote transplant tolerance. atg, a widely used induction therapy in organ transplantation, depletes peripheral t cells but may preferentially spare t regs . sirolimus is thought to expand natural t regs . b7 t cell costimulatory blockade inhibits effector t cell (t eff ) expansion and may promote regulation. we investigated the effect of combining mouse atg (matg), ctla4ig and sirolimus on stringent skin allograft survival, and studied the mechanisms by determining t reg /t eff balance in vivo using a unique model (abm-tcrtg-foxp3/gfp reporter mouse conclusion:this is the first report to establish that t cell depletion with matg combined with ctla4ig and sirolimus synergize to prolong stringent fully allogeneic skin allograft survival by promoting regulation and tipping the t reg /t eff balance by both preserving t regs and facilitating generation of new t regs by a conversion mechanism. these results provide the rationale for translating such a novel therapeutic combination to promote regulation and tolerance in primates and human organ transplantation. expansion of cynomolgus cd4+cd25+foxp3+ regulatory t cells using low dose anti-thymocyte globulin. to test low dose atg in vivo, 2 mg/kg (10% of depleting dose) was administered thrice (day 0,1 and 2) to a naïve monkey and to a monkey that was treated concurrently with sirolimus (trough 15-20 ng/ml) after heart transplantation. in the naive monkey, lowdose atg led to expansion of cd4+cd25+foxp3+ tregs in peripheral blood (baseline 0.57%, day 7=4.09%, day 12=2.7%) and in lymph nodes (baseline 2.47%, day 7=9.35%, day 12=7.08%) without causing t cell depletion. similarly, in the transplanted monkey peripheral blood tregs expanded from 1.4% at baseline to 4.64% on day 6. low dose atg is not only able to expand tregs ex vivo by proliferation of natural cd4+cd25+ cells, but can equally induce tregs in vivo without lymphodepletion. these findings provide the rationale for development of tolerance inducing strategies based on enhancing regulatory mechanisms in human transplant recipients. immunological background⁄aim: previously, we have shown that combination of human anti-cd40 mab, 4d11 and tactolimus exerts additive immunosuppressive effect and markedly prolongs renal allograft survival in cynomolgus monkeys. in this study, we further evaluated the immunological aspects among these transplant recipients. method: kidney transplantations were performed across mhc mismatched cynomolgus monkeys. transplant recipient was given either no-treatment, tacrolimus (1 mg⁄kg⁄day, po), 4d11 (5 mg⁄kg, iv) or tacrolimus+4d11 (n=3⁄group). peripheral lymphocyte population, mlr and serum anti-donor antibody levels and graft histology were assessed. results: mean graft survival for no-treatment, tacrolimus, 4d11 and tacrolimus+4d11 treatment groups was 6.0±1.0, 27.7±3.2, 120.3±90.6 and 213.7±28.0 days, respectively. peripheral cd20 + cells partially declined in both 4d11 alone and 4d11+ tacrolimus given animals at the early post-operation period, although the numbers recovered thereafter. cd4 + and cd8 + cells were unaffected. cd4 + effector memory population was reduced by addition of tacrolimus to 4d11 (fig. 1a ). mlr against donor and 3rd party antigens were suppressed in both 4d11 and tacrolimus+4d11 groups (fig. 1b) . addition of tacrolimus further reduced graft cd4 + , cd8 + and cd20 + cellular infiltration (fig. 1c ). anti-donor antibodies were detected in sera during the treatment course of 4d11; however, they did not develop under the tacrolimus+4d11 treatment. graft c4d deposition correlated with serum anti-donor antibody levels. the 4d11 inhibits both cellular and humoral responses against donor antigens. addition of tacrolimus strengthens these immunosuppressive effects of 4d11, leading to further prolongation of graft survival. objectives: allogeneic islet transplantation offers the potential for cure from diabetes. application of this therapy, however, is limited by immunologic mechanisms requiring medical therapy to prevent rejection of the islets. costimulatory blockade of the cd28/ cd80/cd86 and the cd40/cd154 pathways has shown promise in ameliorating the immune response to allow engraftment and function of islets. we have evaluated a new drug regimen consisting of induction therapy with 3a8, a murine anti-cd40 antibody, and basiliximab and maintenance treatment with ctla4ig and sirolimus in diabetic rhesus macaques which received allogeneic islets. methods: allogeneic rhesus macaque islets (14,100 ie/kg ± 1,802) were transplanted intraportally into diabetic rhesus macaques (n=4) under the following immunosuppressive regimen: short term administration of anti-il-2 receptor (basiliximab) and anti-cd40 (3a8), with maintenance immunosuppression using sirolimus for 134 days and abatacept (ctla4ig) for long term therapy. weekly peripheral blood flow cytometric and cmv viral load monitoring was performed. results: recipients treated with this immunosuppressive regimen had immediate return to normoglycemia following islet transplant. the graft survival in the first three animals was 141, 283 and 297 days. the fourth animal continues to exhibit good glycemic control at his current post-operative day 30. each of these animals had monthly intravenous glucose tolerance tests with monitoring of blood glucoses and c-peptides with further evidence of glycemic response and c-peptide generation. flow cytometry confirms cd40 blockade during the administration of 3a8 and return of cd40 after cessation of therapy. the treatment was well tolerated with minimal evidence of cmv reactivation and no evidence of thrombocytopenia or thromboembolism. conclusions: these preliminary results indicate that cd28/cd40 costimulatory-based immunosuppressive regimens can protect allogeneic islets from rejection. furthermore, 3a8 appears to adequately block cd40 to facilitate this engraftment and function as demonstrated by flow cytometry. iwami, 1,2 qi zhang, 1 osamu aramaki, 1 nozomu shirasugi, 1 katsuya nonomura, 2 masanori niimi. 1 1 surgery, teikyo university, tokyo, japan; 2 renal and genitourinary surgery, hokkaido university, sapporo, japan. many studies have shown immunosuppressive effects of dietary intake of fish oil containing eicosapentaenoic acid (epa) in various models such as autoimmune diseases and transplantation. however, its mechanisms remain uncertain. furthermore, there have been no studies examining the effect of purified epa. here we determined the ability of purified epa to inhibit alloimmune response in mouse cardiac transplantation model. methods: cba recipients (h-2 k ) were given single injection of purified epa intraperitoneally on the same day as transplantation of a heart from c57bl/10 donors (h-2 b ). mixed leukocyte reaction (mlr) assay and enzyme linked immunosorbent assay (elisa) were also performed to evaluate the effect of purified epa on cell proliferation and cytokine production. to determine the presence of regulatory cells, adoptive transfer study was conducted. results: untreated cba recipients rejected c57bl/10 cardiac allografts with median survival time (mst), 8 days. in contrast, cba recipients treated with purified epa (1.0g/kg) had significant prolongation of allograft survival (mst, >100 days). cba recipients treated with 0.1g/kg purified epa eventually rejected allografts (mst, 13 days). in mlr assay, treatment with 1.0 g/kg purified epa suppressed alloproliferation of splenocytes in the recipients. the treatment also inhibited production of il-2, il-12 and ifng by the splenocytes in the recipients. when splenocytes were harvested from the recipients treated with 1.0g/kg purified epa 50 days after cardiac allografting and were adoptively transferred into naïve secondary recipients, the adoptive transfer induced significant prolongation of cardiac allograft in nave secondary recipients (mst >30 days, compared to that in the recipients with adoptive transfer of naïve splenocytes, mst, 10 days). conclusions: purified epa induced significantly prolonged survival of fully mismatched cardiac allografts, and generated regulatory cells. background: chronic allograft nephropathy (can), the most common cause of late kidney allograft failure, is not effectively prevented by the current regimens. activation of extracellular signal-regulated kinases 1/2 (erk1/2) mediating intracellular signal transduction from various growth factor stimuli is required for tgf-β production, which plays a key role in the development of can. hence, the therapeutic potential of disruption of erk1/2 signaling to prevent can was examined in an experimental model. methods: kidney donors from c57bl/6j mice (h-2 b ) were transplanted to bilaterally nephrectomized balb/c recipient mice (h-2 d ). the recipients were treated with ci1040 (mek-erk1/2 inhibitor) or vehicle after 14 days post-transplantation for 28 days. can was evaluated with the banff 97 working classification. results: all six allografts receiving ci1040 treatment were survived, while two out of seven grafts were lost in vehicle-treated group. at the end of experiment, the function of grafts in ci1040 treated recipients had been maintained, indicated by lower levels of serum creatinine and bun (30±6 µm and 22±8 mm, n=6) as compared to those (94±39 µm and 56± 25 mm, n=5) in vehicle group (creatinine, p=0.0015; bun, p=0.0054). pathological evaluation indicated that ci1040 reduced can, reflected by a lower can score in ci1040-treated group (3.93, n=4 ) as compared to that (9.96, n=5) in vehicle controls (p=0.0234). further examinations showed that ci1040 treatment resulted in inhibition of phosphorylation of erk1/2 and reduction of tgf-β levels in grafts. in vitro ci1040 potently suppressed not only growth factors-stimulated erk1/2 activation and tgf-β biosynthesis in renal tubular epithelial cells, but also attenuated alloantigenstimulated t cell proliferation. conclusion: our data suggest that interference of erk1/2 signaling with pharmacological agent (i.e. ci1040) has therapeutic potential to prevent can in kidney transplantation. objective: this is the first study to investigate the role of a novel jak3 and sykinhibitor, r348, in the prevention of obliterative airway disease (oad), the major obstacle after lung transplantation. methods: trachea from brown-norway (bn) donors were heterotopically transplanted in the greater omentum of lewis (lew) rats. recipients were treated for 28 days with r348 (10, 20, 40, or 80 mg/kg), rapamycin (0.75 or 3 mg/kg), or left untreated. allografts were recovered and processed for histological evaluation determining degree of luminal obliteration, percentage of respiratory epithelial coverage, and mononuclear cell infiltration. donor reactive (igg) antibodies from the recipient's serum were determined using flow cytometry. results: r348 at 20, 40, and 80 mg/kg significantly inhibited luminal obliteration in a dose dependent manner (69±20%, 20±13%, 15±7%; p=0.003 vs. no medication). rapamycin in both concentrations significantly inhibited luminal obliteration (37±15%, 11±6%; p<0.001 vs. no medication) similarly to r348 at 40 and 80 mg/kg. r348 at 40 and 80 mg/kg significantly preserved respiratory epithelium compared to r348 at 10 and 20 mg/kg (49±35%, 76±27% vs. 0±0, 3±7%; p=0.004) and was superior to rapamycin in epithelial preservation (49±35%, 76±27% vs. 27±17%, 36±15%; p=0.01). all r348 and rapamycin-treated recipients expressed decreased numbers of peritracheal mononuclear cells in a dose dependent manner (p<0.0001). r348 20, 40 , and 80 mg/ kg treated recipients had significantly reduced igg levels versus untreated recipients (381±136, 371±61, 230±56 vs. 853±207; p<0.03). all r348 treated recipient thymus and spleen weights were significantly lower compared to the untreated group (p=0.001). bun, cr, and cholesterol levels were unaffected in r348 treated recipients. conclusion: r348 potentially exhibits its inhibitory effect by preserving the respiratory epithelium, rather than by rapamycin's mechanism of reduced smooth muscle cell (smc) proliferation. r348 occupies a beneficial pharmacokinetic profile, lacks nephrotoxic and atherogenic properties, and provides a favorable alternative to rapamycin in the treatment of chronic rejection in lung transplant recipients. genz-29155 is a novel, oral immune-modulatory agent identified in a high-throughput screen designed to find inhibitors of tnfα-induced apoptosis. the molecular target of the compound remains under investigation but is likely downstream of the tnfα cell surface receptor. in vitro studies have shown genz-29155 to be an effective inhibitor of the tnfα-triggered caspase cascade but not anti-cd3 or fas-mediated apoptosis, and thus may act by inducing allograft resistance to immune attack rather than suppressing the alloimmune response per se. it has been shown to synergize with sirolimus in murine heterotopic cardiac allotransplant models. in order to test this promising new agent in a more clinically relevant model of solid organ transplantation, we studied genz-29155 in a mismatched nhp (rhesus macaque) renal transplant model. genz-29155 (n=3) was administered (3mg/kg, iv, days 0-14) with sirolimus (1mg/kg, po, days 0-30). five control animals received only sirolimus and vehicle (1mg/kg, po, days 0-30). all animals were followed serially by polychromatic flow cytometry to determine the relative and absolute number of cd4+ and cd8+ t cell subsets. time to allograft rejection, the primary end point, was determined by a significant rise in serum creatinine and bun, as identified with biweekly monitoring. after diagnosis of rejection, allografts were removed for histological and transcriptional studies, along with splenocytes for immune function assays. in this pilot study, prolongation of rejection-free survival was significantly improved with genz-29155 and sirolimus combined vs. sirolimus alone (42.67 days vs. 17.20 days, respectively, p = 0.05). given these initial results, we have initiated a larger study (n=12) to optimize the dose and duration of genz-29155. five animals, transplanted within the past month remain alive and well in this study. further investigation of this agent will allow us to better understand the benefit of inhibiting tnfα-mediated apoptotic effects in both cellular alloimmune response and allograft injury in solid organ transplantation. targeting purpose: allospecific t memory cell responses are present in transplant recipients from exposure to cross-reacting antigens. we have previously reported that lfa-1 inhibition suppresses primary cd8-dependent rejection responses which are not controlled by any conventional immunosuppressive strategy. these studies were conducted to analyze the efficacy of this anti-lfa-1 ab for control of cd8-dependent responses in sensitized hosts. methods: fvb/n (h-2 q ) donor hepatocytes were transplanted into c57bl/6 (h-2 b ) or cd4 ko (h-2 b ) recipients. memory responses were analyzed by retransplantation with a second fvb/n allogeneic hepatocyte transplant. cohorts of mice were treated with anti-lfa-1 mab and observed for hepatocyte survival or magnitude of cd8 + t cell mediated allospecific cytolytic activity. results: the untreated secondary cd4 ko and c57bl/6 recipients rejected hepatocyte allografts with enhanced kinetics in comparison to the primary graft (mst= day 10 vs day 14, and mst= day 7 vs. day 10, respectively; p < 0.05). anti-lfa-1 mab treated cd4 ko recipients demonstrated delayed rejection (mst= day 35 vs day 10; p=0.001) compared to secondary rejection in untreated cd4 ko hosts. anti-lfa-1 mab treatment did not delay rejection in sensitized c57bl/6 recipients (mst= day 7) but did significantly reduce the in vivo allospecific cytotoxic effector function in c57bl/6 secondary recipients (20.6±4.1%; p=0.001) as compared to untreated controls (97±1.3%). the residual cytotoxicity observed in anti-lfa-1 mab treated c57bl/6 recipients is comparable to the in vivo cytotoxicity of cd8-depleted c57bl/6 secondary recipients (12.1±8.9%) and is likely mediated by alloantibody. in fact, the level of allospecific cytotoxicity in anti-lfa-1 mab treated sensitized c57bl/6 recipients correlated with the amount of alloantibody present in recipient serum. conclusion: in conclusion, treatment with anti-lfa-1 mab delayed (cd4-independent) cd8-dependent rejection in sensitized recipients but did not delay rejection in sensitized cd4-sufficient c57bl/6 recipients. despite the efficacy of treatment with anti-lfa-1 mab to significantly reduce the in vivo allospecific cytotoxic effector function in sensitized c57bl/6 mice this strategy did not delay rejection. this is likely due to alloantibody mediated rejection in sensitized c57bl/6 (but not cd4 ko recipients) which is not suppressed by treatment with anti-lfa-1 mab. abstract# 194 cytomegalovirus (cmv) represents a major cause of infectious complications after transplantation. recently, chronic infections with lcmv, hiv or hcv were shown to be associated with functionally anergic t-cells characterized by high expression of the programmed death (pd)-1 molecule. this study was carried out to characterize functional exhaustion of cmv-specific cd4 t-cells as determinant of impaired cmv-control and to elucidate whether the pd-1 pathway may be operative in active cmv-infection after renal transplantation. cmv specific cd4 t cells from 13 controls, 31 hemodialysis patients, and 51 renal transplant patients were quantified using flow cytometry and analysed for their expression of pd-1 and cytokines ifnγ and il2. cmv specific proliferation was analysed by cfda-se dilution. in viremic transplant-recipients, a significantly higher proportion of cmv-specific cd4 t-cells were pd-1 positive (median 40.9%) as compared to non-viremic transplant patients (8.8%), dialysis-patients (8.8%) or controls (3.1%, p<0.0001). in line with functional impairment, pd-1 positive t-cells produced significantly less ifnγ per single cell as compared to pd-1 negative t-cells (mean fluorescence intensity 129.4±52.6 versus 256.6±96.1, p<0.0001). moreover, unlike controls or non-viremic patients, the majority of cmv-specific t-cells from viremic patients showed a long-term loss of il-2 production. interestingly, functional anergy of pd-1 positive cmv-specific cd4 t-cells was reversible in that antibody-mediated blockade of pd-1 signaling with its ligands pd-l1/-l2 led to a 10fold increase in cmvspecific proliferation. in conclusion, expression of pd-1 defines a reversible defect of cmv-specific cd4 t-cells, and blocking pd-1 signaling may provide a potential target for enhancing the function of exhausted t-cells in chronic cmv-infection. differential background: some patients with cmv disease may be simultaneously infected with multiple viral strains. it is unknown if different strains clear differently with the commencement of antiviral therapy. we assessed response to antiviral therapy in patients with simultaneous co-infection with multiple strains of cmv. methods: pcr-based strain typing of cmv was performed using the glycoprotein b gene of cmv (gb1-4) in a cohort of organ transplant recipients with cmv disease. from this, 89 patients were identified that had simultaneous infection with ≥ 2 cmv strains. quantitative assessment of each of the strain types was performed at regular intervals after starting antiviral therapy. results: the different types of multi-strain infections were gb1+gb2 (11/89, 12%), gb1+gb3 (22/89,25%), gb1+ gb4 (7/89, 8%), gb2+gb3 (10/89, 11%), gb2+gb4 (6/89, 7%) and gb3+gb4 (8/89, 9%). 25/89 (28%) were simultaneously infected with 3 or 4 different genotypes. within individual patients, there was trend for gb1 cmv load (4.27log genomes) to be lower than the other genotypes (p=0.05-0.07) at the onset of disease. decay kinetics for all genotypes showed a bisphasic response with a 1 st phase decline of ∼0.7 days and a 2 nd phase of ∼3days. 1 st phase delines were fastest for gb1 (p=0.0001 vs gb2 and 4) while gb4 decline was slower than gb3 during the 1 st phase. 2 nd phase declines were similar between gb1 and 3 (3days and 2.9 days) but were slower for gb2 and 4 (3.45 days; p=0.01). there was a significant correlation between 1 st phase decline and log decline from baseline by day 21 (r=0.37; p=0.002). relative fitness calculations revealed complex fitness dynamics between genotypes although gb3 was always less fit than gb1, 2 and 4, and gb4 and 2 were always less fit than gb1. conclusion: in patients with cmv disease who have simultaneous coinfection with multiple strains, the 1 st and 2 nd phase declines in gb1 are significantly slower than either gb2 or 4 and have a lower log decline from baseline by day 21. these data indicate that either a significant fitness difference exists between cmv strains or that antiviral control of replication may be linked to cmv gb genotype and should aid our understanding of treatment success and failure. one introduction: parvovirus b19 (pvb19) is a single-stranded dna virus that was first reported to affect transplant (tx) recipients around 20 years ago. in the kidney tx setting pvb19 has been reported to cause anemia and proteinuria. reported incidence in a general kidney transplant cohort has been reported to be between 0%-12% and as high as 38% in an anemic kidney tx population. here we report our incidence of pvb19 infection over a 12 year span. patients and methods: all records of kidney tx recipients from 1996 until 2007 were reviewed for the presence of pvb19 infection. there were 834 kidney tx performed during this period. diagnosis of pvb19 infection was made either by detection of pvb19 via pcr in a blood/tissue sample or by detection of virus on renal tissue by immunostain. in patients found to have pvb19 infection; presence of anemia, proteinuria, concurrent infection and acute rejection rates were examined. response to treatment with ivig was also evaluated. results: incidence of infection was 2.4% as 20 patients were found to have evidence of infection. average time from tx to diagnosis of infection was 21.6 months (range 3 days-86 months). average creatinine at diagnosis was 2.32 mg/dl. anemia was present in 85% of patients with an average hematocrit of 30.8%. proteinuria was present in 45% of patients with evidence of pvb19 infection. co-infection was noted in 5 patients (4 cmv, 1ebv) and acute rejection was noted in 30% of individuals within 3 months of diagnosis. collapsing glomerulopathy (cg) was present in 5 patients and they all had subsequent graft loss at an average of 6 months after diagnosis. 4 of the 5 patients with cg had proteinuria along with anemia and were caucasian. 90% (18/20) of all patients with evidence of pvb19 infection received ivig and cleared their infection. one of the remaining 2 pts without ivig spontaneously cleared their virus. conclusion: although the incidence of pvb19 infection in our kidney tx cohort was very low, its presence portends an unfavorable outcome. the presence of cg associated with pvb19 is an especially devastating lesion with very poor outcomes. response to treatment with ivig and reduction of immunosuppression is variable. based on our data it seems reasonable to screen all tx patients with unexplained anemia and concurrent proteinuria as early detection of pvb19 may be crucial. background: prior to transplant, screening for latent tuberculosis (ltbi) by tuberculin skin test (tst) is recommended. the accuracy of tst in end stage renal disease however may be limited. the quantiferon®-tb gold assay (qft) detects interferon-δ produced by peripheral blood t-cells in response to tb specific antigens and may be more accurate for diagnosis of ltbi. methods: this prospective single center study compared the tst to qft for the diagnosis of ltbi in a cohort of adult patients listed or undergoing workup for renal transplantation. all patients had both tst and qft performed. additional data collected included demographics, tb risk history and chest x-ray results. based on demographic and radiographic findings, patients were classified as high or low risk for ltbi. a positive tst was defined as ≥5 mm and positive qft as ≥0.35 iu/ml. results: a total of 45 patients were enrolled. complete data was available for 38 subjects (5 did not return to have tst read). the mean age was 50.4 +/-12.2 years with 21 (55.3%) males and 17 (44.7%) females. the most common etiologies of renal diseases were diabetes (52.6%) and glomerulonephritis (26.3%). most subjects (35 of 38) were on renal replacement therapy (hemodialysis in 73.7% and peritoneal dialysis in 18.4%). twenty (52.6%) subjects had received bcg and 10 (26.3%) were born in or lived in a country in with tb prevalence rate > 20/100000 population. fifteen (39.5%) subjects were considered to be at high-risk for ltbi. overall 5 (13.2%) had a positive tst and 3 (7.9%) had a positive qft. the qft was indeterminate in 1 subject due to a low mitogen response. agreement between the tests was 92% (k=0.72, p<0.0001). in low-risk subjects (n=23) the tst was negative in all and the qft was negative in 22 and indeterminate in 1. in clinically high-risk subjects, 5 (33%) had a positive tst and 3 (20%) had a positive qft. the 2 subjects with discordant results, both from tb endemic countries, had both completed treatment for ltbi 4 years prior and remained tst positive, but were qft negative. in renal transplant candidates, the tst and qft are comparable for the diagnosis of ltbi. the qft has the advantage of being completed in a single visit and in our cohort indeterminate results were uncommon. optimal utilization of htlv i/ii positive organs -a nationwide survey. objective: we recently presented data from the unos database that demonstrated no significant difference in graft or patient survival between htlv i /ii (+) and (-) liver recipients. several organ procurement organizations (opo) including our own, do not offer htlv i /ii positive organs while many others find it difficult to place them. despite this, the number of htlv (+) organs is increasing with 31 utilized in 2006 alone. this prompted us to evaluate the practical difficulties in placing these organs so as to improve utilization of these "high risk" life saving organs. medthod: a telephone/email survey of all the 65 opos in usa was done over a 2 month period from october to november 2007. results: of the 65 opos, 42 responded. all screen patients for htlv i/ii with elisa. 38 centers confirm with repeat elisa, 27 confirm with western blot and 6 centers do not pursue further. 36 of the 42 centers offer the htlv i/ii positive organs. there were a total of 231 positive donors in the past 5 years of which organs from 109 donors (47.2%) were placed. 27 centers offer all the organs while 15 offer one or more organs selectively based on accepting centers. none have been able to place the pancreas. only liver and kidney were commonly accepted. several centers noted a high false positive rate. based on the unos regional analysis data, 43% of the organs are utilized in ny state alone. many opos did not know which particular centers accept these organs and consequently spend a lot of time and effort in order to place them. a majority wanted to have a list of transplant centers that accept these organs. conclusions: htlv i/ii organs are being underutilized. moreover, our prior analysis of unos data shows that these life saving organs are shared more nationally than loco-regionally which is associated with a poorer outcome. increased knowledge of successful htlv (+) donation and the centers that are willing to utilize these organs in the appropriate setting will help expand the donor pool and decrease mortality on the waiting list. increasing traditional two-drug chronic immunosuppression (is) used in organ transplantation (tx) is associated with development of ebv-driven complications because of impairment of anti-viral cd8 + t cell surveillance. since the long-term impact of alemtuzumab preconditioning combined with tacrolimus monotherapy on ebv immunity after tx has not been studied, here we aim to analyze the frequency and function of peripheral blood ebv-specific cd8 + t cells. thirteen ebv + stable kidney transplant (ktx) recipients and 12 ebv + healthy controls were recruited to this cross-sectional study. all patients received alemtuzumab preconditioning, followed by tacrolimus monotherapy. blood samples were collected at least 1 year post-tx to allow immune reconstitution. the ebv-specific cd8 + t cell phenotype and function were screened by flow cytometry and ifng elispot assay. hla-a201 restricted ebv-lytic (bmlf1) and latent (lmp2a) peptides were used to generate tetramer (tmr) probes, and for functional screening in elispot. circulating cd8 + t cells from ktx patients had recovered by 1 year, and were comparable to those of healthy controls (28.6%±12.9 vs 23% ±6.6, p=0.2). moreover, the memory distribution and the frequency of ebv-specific cd8 + t cells detected in patients and controls (bmlf1-specific: 0.56%±0.96 vs 0.88%±1.08, p=0.46, and lmp2specific: 0.05%±0.06 vs 0.24%±0.40 p=0.09) were similar. in contrast, the frequency of functional type-1 (ifn-g producing) ebv-specific cd8 + t cells was significantly lower in ktx patients than in healthy controls (bmlf1: 47±24 spots/10 5 cd8 t cells vs 164±134 p=0.06, and lmp2: 9±8 vs 64±54 p=0.03). accordingly, on average, only 8-12% of circulating ebv-specific cd8 + t cells from ktx patients produced ifn-g, while 20-30% of effector cells were functional in healthy controls. addition of il-2 (20iu/ml) during the elispot assay reversed the hypo-responsiveness of type-1 (ifn-g) ebv-specific cd8 + t in patients (range 3-8 fold increase), suggesting that these effector cells were anergic. these results support the notion that alemtuzumab-induced lymphocyte depletion followed by tacrolimus monotherapy renders ebv-specific cd8 + t cells anergic in vivo, a state that can be readily reversed by cytokines such as il-2, which are commonly released during immune activation. background: the epidemiology of the transmission of cytomegalovirus (cmv) from organ donors to recipients is not completely understood. we studied donor to recipient transmission patterns by analyzing viral genomic variants through the use of cmv glycoprotein b (gb) genotyping by real-time pcr. polymorphisms in gb ul55 allow discrimination of 4 distinct genomic variants (gb 1-4). methods: organ transplant recipient pairs or triplets were included in the study if: a) they had cmv infection, b) they received an organ from a cmv seropositive donor, and c) there was at least one other recipient from the same donor that also developed cmv infection. genotyping (gb 1-4) was performed by quantitative real-time pcr on stored blood samples. clinical charts were reviewed to evaluate the clinical characteristics and outcome of cmv infection. results: of the 78 cmv seropositive donors screened, 21 were multiple organ donors for which 2 or more of their recipients developed cmv infection. the total number of recipients from these 21 donors was 86 (median of 4 recipients per donor). of these recipients, 47 (55%) had cmv infection (16 recipient pairs and 5 recipient triplets). the prevalence of genotypes was gb1 (n=22; 47%), gb2 (n=11; 23%), gb3 (n=4; 9%), gb4 (n=0). mixed infection with two concurrent genotypes was present in 10 patients (21%). overall concordance between cmv gb genotype in recipient pairs was 62.5% (10/16). if both recipients were cmv seronegative (d+/r-) the gb concordance in recipients was 67% (2/3 pairs). gb concordance was 70% (7/10 pairs) if one of the recipients was seronegative and the other seropositive. concordance was 33% (1/3 pair) if both recipients were seropositive. concordance between genotypes was seen in 2/5 (40%) recipients triplets. in seropositive recipients with cmv viremia, the origin of the cmv strain was thought to be donor derived in 11/16 (69%) and of reactivation of the recipients own virus in 5/16 (31%) of the cases. no difference in clinical outcome or organ tropism was seen between genotypes. based on an analysis of strain concordance within recipients from common donors, transmission patterns of cmv can be assessed. in d+/r+ transplant patients, donor strain superinfection accounts for the approximately two-thirds of cmv infection. backgroud: although map kinases have been implicated in the pathophysiology of liver iri, their functional significance in the mechanism of tlr4 mediated pro-inflammatory immune regulation, remains to be elucidated. methods: map kinase activation in a murine model of liver warm iri (90 min. ischemia, 6 h reperfusion) was determined by western blots. chemical inhibitors of erk (u0126, 10 µm in vitro or 160 mg/kg in vivo), jnk (sp600125, 50 µm or 30 mg/kg), and p38 (sb203580, 10 µm, 100 mg/ kg) map kinases were utilized in vitro in primary bm-derived macrophage cultures stimulated with lps (10 ng/ml); or in vivo in liver pro-inflammatory immune responses induced by lps (1 µg/mouse, i.p.) or iri. results: erk and jnk, but not p38, map kinase activation were readily detected in liver iri. in primary macrophage cultures, lps induced pro-and anti-inflammatory genes, including tnf-α, il-1β, il-6, il-10, inos and cxcl10. erk inhibitor mainly suppressed il-1β and il-10 (80% and 90% resp), whereas jnk inhibitor suppressed the majority of genes. in lps-induced liver inflammation, erk inhibitor suppressed il-6, il-1β, inos and il-10 by >50%, but failed to affect tnf-α/cxcl10. jnk inhibitor, on the other hand, preferentially inhibited pro-inflammatory genes, but marginaly affected il-10 (<30%). and produced comparable suppression of pro-/anti-inflammatory genes (figure1). interestingly, tnf-α was the least responsive gene subjected to map kinase regulation. conclusion: erk and jnk map kinase activation: 1/ are required for tlr4 activationinduced pro-inflammatory gene induction; 2/ play critical role in the development of ir-mediated liver immune response/tissue injury. background: the jak/stat signaling is one of the major pathways for cytokine signal transduction. the signal transducer and activator of transcription 1 (stat1) is mainly activated by ifn-α/ß/ifn-γ. the activation of stat1 by ifn-γ has been implicated in hepatic inflammation. we have shown that activation of toll-like receptor (tlr) 4 complex initiates pro-inflammatory response leading to liver ischemia/reperfusion abstracts injury (iri). indeed, tlr4 signaling in vitro activates stat1, which in turn triggers production of type-1 ifn-dependent cxcl10 (ip-10). this study was designed to analyze the cross-talk between stat1 and the map kinase (erk) downstream of jak/ stat signaling pathways. methods & results: we used a mouse liver model of partial warm ischemia (90 min), followed by reperfusion (6 h) . first, we employed stat1 ko (n=17) and control wt (n=16) mice. the hepatocellular damage, as measured by salt levels (iu/l), was significantly decreased in 10/17 stat1 ko mice (p<0.005); the remaining 7/17 of stat1 ko showed salt levels comparable with wt. hence, we distinguished two groups of stat1 "protected" vs. "nonprotected" ko recipients. histology revealed minimal sinusoidal congestion without edema/vacuolization or necrosis in stat1 ko "protected" group. the induction of mrna coding for tnfα/ il-6 was higher in stat1 ko "nonprotected" livers. the expression of cxcl10, the product of stat1 activation downstream of tlr4 in type i ifn pathway, was profoundly and selectively depressed in livers from stat1 ko "protected" mice, as compared to iri susceptible livers. similarly, western blot-assisted phospho-erk expression was up regulated selectively in the stat1 ko "protected" group. in the second series, c57bl/6 mice were treated 1 h prior to liver ischemic insult with jak-2 inhibitor (tyrphostin ag490; 40 mg/kg, i.p.; n=5), or vehicle (n=5). the hepatocellular damage, as measured by salt levels (iu/l), and histology was significantly decreased in ag490 group, as compared with controls (mean = 12770 vs. 28770; p<0.05). the disruption of jak/stat signaling by inhibiting jak2 uniformly ameliorates the inflammatory immune response in liver iri. however, the blockage of stat1 alone is insufficient to reproducibly exert cytoprotection. as jak2 is upstream of stat1 as well as upstream of map kinase (erk), this study highlights the role of both signaling pathways in hepatic iri. purpose: tlr4 is required for maximal ischemic injury of the heart, liver, lung, and kidney. to better understand the mechanisms of tlr4 action, we investigated a murine model of ischemic kidney disease and examined endothelial tlr4 expression. methods: 1. animal ischemia reperfusion injury(iri): the right kidney of wildtype(wt) c57bl/10, or tlr4-deficient(ko) c57bl/10scn mice was removed. the left pedicle was clamped for 23 min, followed by 4 hr reperfusion. sham animals were controls. 2. bone-marrow chimera: four groups of bm chimeras were created: wt→wt; ko→wt; wt→ko; ko→ko (8 recipients/grp). 8 wks later, chimerism was confirmed by tail and blood genotyping, and mice subjected to renal iri. 3. tlr4 mrna detected by dig-labeled antisense; tlr4 on endothelium by anti-tlr4 and anti-cd31. 4. total genome mrna expression on ischemic vs. sham wt mice, ischemic vs. sham ko mice(4 mice/grp) determined using affymetrix mouse genome 430.2 genechips followed by genesifter analysis. quantitative real-time rt-pcr confirmed candidate genes. 5. ms1 endothelial cells were treated with h 2 o 2 (500um) for 30 min, cultured for 4 hr and then expression of tlr4 and endothelial genes determined. results: tlr4-deficient mice had less renal injury as assessed by pathology and function. radiation chimeras showed that radioresistant parenchymal cells and radiosensitive leukocytes were both required for maximal injury. immunohistology and in situ hybridization identified endothelia in the outer medulla as a major tlr4 expressing cell type at 4 hr post-reperfusion. genechip analysis revealed a panel of cytokine, chemokines, proinflammatory and cell-cycle related genes that were differentially expressed on iri versus sham kidneys. real-time rt-pcr confirmed that the following pro-inflammatory endothelial genes increased after ischemia in wildtype but not tlr4 ko mice: tlr4, pentraxin related gene(ptx3), and endothelial cell-specific molecule 1(esm1). real-time rt-pcr showed that in vitro h 2 o 2 treatment of endothelial cells induced expression of tlr4(6.6-fold), ptx3(4.6-fold), and esm1(69-fold). we found that endothelia in the outer medulla increase their expression of tlr4 after ischemia, and that the endothelial genes ptx3 and esm1 increase only in wt ischemic kidneys. we also found that h 2 o 2 , which mimics reactive oxygen species generated during iri, directly increases these same endothelial genes in vitro. nkg2d is an activating receptor expressed on nk cells and cd8 + t cells. ligands for nkg2d including rae-1, mult-1 and h60 in mouse, may be upregulated by tissues in response to stress. a study in mouse macrophages described upregulation of rae-1 in response to stimulation through tlr4 by lps. we have shown that tlr4 mediates kidney ischemia reperfusion injury (iri) and also observed rae-1 upregulation in iri kidneys. we now determined whether: 1) kidney iri could induce the expression of nkg2d ligands: 2) expression of nkg2d ligands is tlr4 dependent; 3). bone marrow (bm) derived cells or parenchymal kidney cells express nkg2d ligands. methods. kidney-ischemia was induced in tlr4 -/-, myd88 -/and wt mice for 22 min. blood and tissue were harvested at days 1, 3 and 5. primary cultures of mouse tubular cells (tecs) were also subjected to ischaemia (mineral oil overlay for 1 hr) or tlr4 activation (lps stimulation). bm chimeric mice were generated by transplanting bm into irradiated recipient mice before iri was induced. results. rae-1 mrna level in iri kidney was increased from day 1 to day 5, peaking at day 3 compared to sham operated kidney (14-48 fold increase, p < 0.001) measured by real time pcr. rae-1 protein was detected in renal tubular cells from ischemic kidney but not from shamoperated control by flow cytometry. mult-1 mrna expression was also increased from day 1 to day 5 (15-51 fold increase, p <0.005). both rae-1 and mult-1 mrna levels were reduced in tlr4 -/and myd88 -/-iri kidneys (2-8 fold reduction) versus wt controls (p < 0.01). tlr4 -/and myd88 -/primary cultured tecs submitted to iri in vitro also showed less rae1 and mult-1 mrna expression than wt controls (p < 0.01). lps stimulated rae-1 and mult-1 expression in wt but not in tlr4 -/-tecs. tlr4 -/mice bearing wt hemopoietic cells had significantly lower kidney rae-1 and mult-1 mrna expression after iri versus wt mice with tlr4 -/-bm (p < 0.05). conclusion. kidney iri causes rae-1 and mult-1 expression and kidney parenchymal cells are the dominant source. tecs can be stimulated to express rae-1 and mult-1 by ischemia or lps via the tlr4 pathway in vitro and deficiencies in this pathway provide protection against iri and rae-1 and mult-1 expression in vitro and in vivo. thus, kidney iri causes upregulation of nkg2d ligands by parenchymal kidney cells via tlr4. background: intestinal ischemia/reperfusion injury (iri) is a major clinical problem. although toll-like receptor 4 (tlr4) has been implicated as a potential link between the innate and adaptive immunity, little is known on its role in intestinal iri. our preliminary research in intestinal iri has shown that, compared to sham controls, wt mice had decreased survival, worse tissue injury/apoptosis, increased pmn infiltration, increased cd3+ cell infiltration, and increased production of tlr4, chemokines, and adhesion molecules. here we used tlr4ko mice to further investigate the role of tlr4 in intestinal iri and its effects on cytokine/chemokine programs and apoptotic signaling. methods: c57bl10 wt and tlr4ko mice underwent 100 min of total jejunoileal warm iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at 4h and 24h. tissue analysis included histopathology, cd3 immunostaining, myeloperoxidase (mpo) activity, rt-pcr for chemokines/ cytokines, and western blots for apoptotic and ho-1 protein expression. results: tlr4ko had superior survival compared to wt (100% vs. 33%, p<0.02). on histopathology tlr4ko had near normal-appearing villous architecture, while in contrast, wt showed mucosal erosions and villous congestion/hemorrhage. tlr4ko had reduced cd3+ cell infiltration as compared to wt (3.9±0.7 vs. 6.2±1.4 per hpf at 4h, p<0.001; and 4.8±1.1 vs. 7.0±2.3 per hpf at 24h, p<0.02). early mpo activity was also reduced in tlr4ko (0.11±0.06 vs. 0.88±0.5 u/g at 4h, p<0.005). rt-pcr analysis demonstrated decreased production of mrna for ip-10, mcp-1, rantes, and ifn-γ and increased production of il-13 in tlr4ko. there was decreased protein expression of caspase-3 and increased expression of bcl-2 and ho-1 in tlr4ko mice. conclusion: the genetic absence of tlr4 exerts protection against intestinal iri, demonstrating for the first time that tlr4 is required for intestinal iri. the absence of tlr4 signaling reduces iri through reduced neutrophil and t cell chemotaxis, and up-regulation of protective molecules. these results support data that tlr4 is a mechanistic link between the innate and adaptive immunity, implicating tlr4 as a potential therapeutic target for the prevention of intestinal iri. it is well known that liver steatosis increases hepatic vulnerability to ischemia/ reperfusion (i/r) injury as part of the transplantation process. endotoxin (lps) is thought to be a major contributing factor to the pathogensis of i/r. during portal occlusion, lps is translocated across the mesenteric tissue barrier into the portal circulation, and is delivered as a large bolus to the liver at the point of reperfusion. at this time, lps is mainly recognized by toll-like receptor 4 (tlr4). this leads to downstream signaling and the production of proinflammatory products that ultimately lead to cellular inflammation, necrosis, and apoptosis. it is well known that steatotic livers are highly sensitive to endotoxin as compared to their lean counterparts post-i/r, and we have previously seen that monoclonal antibody blockade of endotoxin dramatically improves animal survival after i/r. therefore, we propose the novel hypothesis that tlr4 signaling is a major contributor to cellular damage after steatotic hepatic i/r. to test this hypothesis, we subjected male 4-week-old c57bl/10j (control) or c57bl/10scn (tlr4 deficient, tlr4ko) mice to a high-fat diet (hfd) for four weeks. then, we subjected the animals to 35 minutes of total hepatic ischemia and 1 or 24 hours of reperfusion. there was a dramatic improvement in animal survival in the hfd tlr4ko animals versus control hfd animals at 24 hours (74% vs. 31% in control hfd animals, p<0.05). there was significantly more liver necrosis (as measured by a grading scale from 0-3) in the control hfd animals as compared to the tlr4ko hfd animals (1.4±0.3 in control vs. 0.4±0.2 in tlr4ko, p<0.05). in addition, we see significant increases in the message level of the proinflammatory cytokines il-6, il-12, and ifn-γ at one hour in the control hfd animals that is abrogated dramatically in the tlr4ko hfd animals. we do not see these dramatic changes in the control animals fed a normal diet. despite the significant increases in inflammation in the control hfd animals versus the tlr4ko hfd and normal diet control animals, we do not see changes in the tlr4 message level or endotoxin boluses, implying an increased sensitivity in the absence of an increased number of receptors. tlr4 is a critical molecule in the pathogenesis of steatotic liver ischemia/reperfusion, and represents a potential therapeutic target for expansion of the donor pool. the background: neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia and reperfusion injury (iri). particularly, neutrophil elastase (ne) accounts for a substantial portion of the neutrophil function. this study was designed to explore the role of, and mechanism by which ne exerts its function in a mouse model of liver warm iri. methods: partial warm ischemia was produced in the left and middle hepatic lobes of c57bl/6 mice for 90 min, followed by 6-24h of reperfusion. mice were treated with ne inhibitor (nei; 2mg/kg p.o.; gw311616a; n=6) or control (n=6) at 60 min prior to the ischemia insult. after 6h or 24h of reperfusion, sast/salt levels and intrahepatic neutrophil accumulation (myeloperoxidase [mpo] activity) were assessed. the pro-inflammatory cytokine (tnf-α, il-6), chemokine (cxcl-1, cxcl-2, ip-10) and toll-like receptor (tlr) 4 gene expression profiles were screened by rt-pcr. liver samples were collected for histological grading, and detection of neutrophil infiltration by the naphtol as-d chloroacetate esterase stains. results: nei treatment significantly reduced sast/salt levels, as compared with controls (17695±3413 vs 10990±2411; p<0.05 / 33650±3793 vs 13510±6809; p<0.01 at 6 h, and 11778±3113 / 14483±3972 vs 3565±957 / 4810±1063; p<0.01 at 24h). the expression of pro-inflammatory cytokines, and chemokines was significantly reduced in the nei treatment group (tnf-α in 6h; p<0.05, il-6 in 6h and 24h; p<0.01 and p<0.05, cxcl-1 in 24h; p<0.01, cxcl-2 in 6h and 24h; p<0.01 and p<0.05, ip-10 in 24h; p<0.01). the mpo activity (u/g) was also significantly reduced following nei treatment (14.1±7.7 vs 4.0±1.2; p<0.05). tlr4 expression was selectively diminished in nei pretreated livers (6h; p<0.01). histological examination of liver sections has revealed that unlike in controls, nei treatment markedly reduced edema, diminished centrilobular ballooning/sinusoidal congestion, ameliorated hepatocellular necrosis, and decreased local neutrophil infiltration. conclusion: the inhibition of ne ameliorated hepatocellular damage, reduced local inflammatory responses, and neutrophil activity/infiltration in a stringent mouse liver model of warm iri. interestingly, it also downregulated the innate tlr4 signaling. this study documents the previously unrecognized ne -tlr4 cross talk, and implies neutrophil elastase in the signal transduction pathway instrumental for liver iri. lymphocyte lymphocytes are involved in the early pathogenesis of ischemia-reperfusion injury (iri) in kidney; however, their role during healing is unknown. this has direct clinical consequence since lymphocyte-targeting agents are currently administered to prevent rejection during recovery from iri in renal transplants. c57bl/6 mice underwent unilateral clamping of renal pedicle for 45 min, followed by reperfusion, and were sacrificed at day 10. mice were treated with saline (c), methylprednisolone (pred) or mycophenolate mofetil (mmf) i.p. daily from day 2 until sacrifice (n=12/group). lymphocytes were isolated from the kidneys, counted and stained with monoclonal antibodies. kidney damage (% damaged tubules) and proliferation (ki67 assay) were assessed. flow cytometry analysis demonstrated increased numbers of tcrβ + cd4 + and tcrβ + cd8 + t and tcrβ -nk1.1 + nk, but not cd19 + b cells at day 10 in the ischemic (ir) kidneys compared to contralateral. regulatory t cells, tcrβ + cd4 + cd25 + foxp3 + , and t cell subsets tcrβ + cd8 + cd122 + and tcrβ + nk1.1 + also increased. moderate tubular damage in cortex, severe injury in outer medulla and increased proliferation in both compartments characterized the repair phase. pred improved histological damage in ir kidneys, while mmf worsened it. proliferative index correlated with histology in outer medulla. pred reduced the total counts and activation of tcrβ + cd4 + and tcrβ + cd8 + t cells in ir kidneys, and increased the percentage of tcrβ + cd8 + cd122 + among total tcrβ + cd8 + t cells. mmf reduced all lymphocyte subsets, decreased the percentage of tcrβ + cd4 + cd25 + foxp3 + among total tcrβ + cd4 + t cells, and lowered il-10 tissue levels. il-6 and platelet derived growth factor-bb protein levels were also decreased in ir kidneys from mmf-treated mice. in conclusion, specific trafficking and phenotypic changes of kidney-infiltrating lymphocytes occur during recovery from renal iri, and lymphocyte-targeting agents, pred and mmf, alter tubular cell structure, proliferation, and inflammatory response in the repair phase. background: ho-1 plays an important cytoprotective role in a variety of organ injury models. we have shown that ho-1 exhibits potent cytoprotective effects against liver i/r injury. this study explores the function and mechanism of ho-1 in liver i/r injury by using sirna that suppress ho-1 expression both in vitro and in vivo. methods: using a partial liver warm ischemia model, c57bl/6 wide-type (wt) mice (n=6/gr) were injected with ho-1 sirna/nonspecific control sirna (2 mg/kg, i.v. at day -1) or ad-ho-1/ad-β-gal (2.5x10 9 pfu, i.v. at day -2). sham control wt underwent the same procedures, but without vascular occlusion. mice were sacrificed at 6 h of reperfusion; liver tissue and blood samples were collected for future analysis. in in vitro studies, ypen-1 endothelium cells were transfected with ho-1 sirna (100 nm) or ad-ho-1/ ad-β-gal. results: ho-1 sirna treated mice showed significantly increased sgot levels (iu/l), as compared with nonspecific control sirna or ad-ho-1 (7593±2859 vs. 3104±1777 and 211.5±40, respectively; p<0.05). these correlated with histologic suzuki's grading of liver i/r injury, with ho-1 sirna showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis; nonspecific control sirna showed moderate edema, sinusoidal congestion/cytoplasmic vacuolization. in contrast, ad-ho-1 revealed only minimal sinusoidal congestion without edema or necrosis. ho-1 sirna significantly increased local neutrophil accumulation and caspase-3 activity, and increased the frequency of apoptotic cells (31.8±5.5 vs. 18.5±5.4 and 3.5±2.2, respectively; p<0.005), as compared with nonspecific control sirna or ad-ho-1. both ypen-1 endothelium cells and wt mice treated with ho-1 sirna revealed markedly increased caspase-3 activity and reduced ho-1 expression. in contrast, ad-ho-1 significantly decreased caspase-3 activity and increased ho-1 and anti-apoptotic bcl-2/bcl-xl expression. conclusion: this study provides evidence that ho-1 exerts cytoprotection against i/r injury by regulating liver apoptosis and inhibiting caspase-3 activation pathway. organ specific sirna is not only a powerful tool to study local gene function, but it may also provide novel therapeutic application in transplant recipients. islet cell transplantation has recently emerged as one the most promising therapeutic approaches for diabetic patients to improve glycometabolic control. one major problem of the procedure is the requirement of an immunosuppression protocol capable of counteract both auto and allo-immune response. recent data suggest that anti-thymoglobulin (atg) can halt efficiently the mounting of an alloresponse and the recurrence of autoimmunity in nod mice by expanding antigen specific t-regulatory cells. we retrospectively reviewed our casuistry type 1 diabetic kidney-transplanted patients who underwent islet transplantation using an immunosuppressive protocol based on atg or daclizumab (as induction treatment) plus cyclosporine and mmf as maintenance therapy. 45 patients underwent islet after kidney transplantation in our center. thirty-four patients received a time course of atg as induction (125 mg per day for 7-10 days), (number of islet infused=499,596±28,780), and 9 patients received daclizumab at induction (1 mg/kg every 2 weeks for 10 weeks); (number of islets=486,108±70,644). no major adverse events were recorded in our center; no malignancies or infections outbreaks were evident. patients in the atg induction group showed a better islet survival rate compared to daclizumab (p=0.01), according to c-peptide>1ng/ml. a sustained and prolonged c-peptide secretion was evident in the atg group; while in the daclizumab group only 1 patient was functioning at 1 year. interestingly, in the atg, which reached a longer follow-up, we cannot observe a loss of beta cell mass according to our metabolic test, suggesting the preservation of islet mass. in conclusion, atg can provide a good protection towards both allo and auto immune response. the next step will be to use atg in a calcineurin free protocol, allowing a better expansion of t-regs. introduction: despite consistent achievement of insulin-independence, recent data indicate that the long term success of islets transplants using the edmonton protocol is <10% at 5 years. the cause of the nearly universal late islet allograft failure remains unknown but hypotheses include: allo or autoimmune injury, marginal mass exhaustion, hepatic site related dysfunction, and immunosuppression toxicity. we examined the series of islet transplants performed at our institution and noted marked differences in the outcome and complications in ia and iak groups. our results may provide insight as to the cause of chronic islet loss. methods: thirty-one islet infusions were administered to ia (n=9) and iak (n=8) type-1 diabetics between 9/2001-11/2007. ia and iak had similar demographics and transplanted islet mass (13,838 vs 13,411 ieq/kg). ia received edmonton like immunosuppression with zenapax induction and cni/srl, whereas iak patients received zenapax and: cni/mmf/pred (6), cni/mmf (1), cni/srl (1)). results: insulin-independence was achieved in all but 2 patients who completed therapy (2 others withdrew). compared with ia, iak exhibited better glycemic control (6-month mean hba1c 6.5 vs 6.1, stimulated c-peptide 2.3 vs 6.0), and improved islet survival; all ia eventually failed and only 1/8 were insulin free for >2-years, whereas only 3/7 iak grafts have failed with 4 exhibiting continued robust function at >38,>38,>46, and >58 months with 3/4 fully insulin independent. in addition, all ia patients demonstrated immune sensitization post graft failure versus 0/8 iak. all 9 ia developed mouth ulcers versus only 1/8 iak. conclusions: ia and iak exhibit striking differences in outcome and complications. the absence of mouth ulcers and lack of sensitization in iak may relate to steroid use and continued immunosuppression for the kidney graft, respectively. the superior outcome of the iak cohort may be a result of differences between the two groups including the use of maintenance steroids, prior exposure to thymo or the chronically immunosuppressed state of the iak recipient. perhaps the most interesting correlation with outcome is the absence of the anti-proliferative agent sirolimus in the iak group. our results provide clues to the cause of chronic islet transplant failure and may lead to novel approaches to avoid it. islet background: although islet transplantation has become an option for treatment of type 1 diabetes, all currently used immunosuppressive protocols have significant renal and islet toxicity. we describe a novel immunosuppressive protocol using sirolimus and the anti-lfa 1 antibody efalizumab that permits prolonged islet allograft survival without the need for steroids or calcineurin inhibitors (ci). methods: between february and august 2007, 4 consecutive type 1 diabetic patients with hypoglycemic unawareness and normal renal function received allogeneic pancreatic islet transplants. induction immunosuppression consisted of 2 doses of thymoglobulin given on pre-transplant days -2 and -1, efalizumab (5mg/kg sq/week starting on d -1), and sirolimus. maintenance immunosuppression consisted of sirolimus and efalizumab. results: all patients achieved insulin independence after single islet infusions (mean ieq/kg=8,905). three of 4 remain insulin independent 4 or more months after transplant (table 1) . patient 1 resumed low dose insulin (approximately 15% of original dose) 6 weeks after transplant and is awaiting a second islet infusion. her blood glucose control is markedly improved and she has not experienced any hypoglycemic episodes. all patients show persistent c-peptide secretion and have stable renal function. side effects due to efalizumab were limited to transient irritation at the injection site. conclusions: thymoglobulin induction followed by sirolimus and efalizumab maintenance is well tolerated and allows prolonged islet allograft survival. this protocol is the first ci/steroid free islet regimen resulting in insulin independence with a single donor islet infusion. by eliminating ci, this protocol minimizes renal and islet toxicity and may thus improve long-term islet survival and function. long . clinical and metabolic profiles were assessed every 3 months for18 months. results: si-exn group was on this drug for median time of 171 days pre-si. both groups were similar except for duration from post-completion to graft dysfunction (648±34 vs 221±58 in si-exn and si-c group respectively, p<0.05) and duration of graft dysfunction before si (664±83 vs 326±93, p=0.03). si-c and si-exn groups received mean of 8713±2123 and 5613±485 ieq/kg, respectively (ns). only 3/5 of si-c patients achieved insulin independence for 303, 403 and >1670 days after si. all subjects in si-exn group achieved insulin independence for more than 443, 621, 641, 654 days. at 18 months insulin independence was 20% in si-c and 100% in si-exn group. comparing pre and post-si, si-exn group had significantly lower a1c at 3-18 months and lower auc glucagon at 6 and 15 months (p<0.05). auc c-peptide in si-exn group was significantly higher than si-c at 3 and 9 months. intravenous glucose tolerance test showed significantly increased acute insulin responses to glucose at 3-18 months in si-exn and at 3 and 6 months in si-c group. si-exn group had more acute c-peptide response to glucose than si-c at 3 and 6 month (p<0.05). acute exenatide administration during intravenous glucose tolerance test at 15 month in si-exn revealed significantly increased acute insulin and c-peptide response to glucose which indicates improved first phase insulin release. conclusion: supplemental islet infusions under exenatide lead to insulin independence, restore first phase insulin secretion and result in long term insulin independence. exenatide this prospective phase 1/2 trial aimed to demonstrate safety and reproducibility of allogeneic islet transplantation (tx) in type 1 diabetic (t1dm) patients and implement a strategy to achieve and maintain insulin-independence with minimal islets. ten c-peptide negative t1dm subjects with hypoglycemic unawareness received 1-3 intraportal allogeneic islet tx. four subjects (group 1) received the edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). the next 6 subjects (group 2) received etanercept, exenatide and the edmonton regimen. we followed all subjects for 15 months after the first tx. the primary efficacy end point was insulin independence. secondary endpoints were hba1c, fructosamine, ogtt, mixed meal test, glucagon stimulation test, ivgtt and hypoglycemia. to study the effect of exenatide, we compared frequently sampled ivgtt, c-peptide, proinsulin, amylin and glucagon with and without exenatide. two self-limiting bleeds occurred in 18 infusions. all subjects became insulin independent. group 1 received a mean total number of islets (ein) of 1,460,080±418,330 in 2 (n=2) or 3 (n=2) tx, whereas group 2 became insulin independent after 1 tx (537,495±190,968 ein, p=0.028). all group 1 subjects remained insulin free through the 15-month follow-up. two group 2 subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with severe gastroparesis and exenatide intolerance. hba1c reached normal range in both groups (6.5±0.6 at baseline to 5.6±0.5 after 2-3 tx in group 1 vs. 7.8±1.1 to 5.8±0.3 after 1 tx in group 2). baseline hba1c was significantly higher in group 2 than group 1 (p=0.046). pre-and post-tx hypo scores were 841.8±1217.9 and 0 in group 1 vs 812.8±941.7 and 33.5±50.0 in group 2. glucagon levels decreased significantly in all subjects. in group 2, the decrease in glucagon levels after challenge tests with and without exenatide was 18.7 fold more significant after exenatide in all subjects ( background istx has been investigated as treatment for type 1 dm. however, the hope this approach would result in long-term freedom from exogenous insulin has failed in practice. techniques for isolating islets have advanced and with availability of new is agents, strategies can now be developed specifically for istx that will provide greater immunologic protection w/o diabetogenic side effects. rejection/ vascularization still remain major limitations for success of istx. we hypothesize that bm is an accessible, immunologic privileged space with natural well-developed vasculature and may be a suitable site for istx. method wistar rats were used as donors/ recipients. dm was induced by iv-streptozocin. rats who had morning glc levels > 300 mg/dl on two separate occasions were used as recipients. ptx was performed as normal rodent standard. islets were isolated from pancreas by distending pancreatic duct with liberase. islets were separated on a discontinuous histopaque density gradient, further purified, counted, divided into aliquots transplanted into different sites (liver, bm). bw, glc and c-pep were measured in all groups before/after tx. background: in february 2007, the islet community was notified of a possible bovine product contamination in the collagenase enzyme (liberase hi) used for human islet isolations. to eliminate the potential hazard of bovine spongiform encephalopathy, we successfully adapted our human islet processing procedure to utilize a different gmp collagenase and neutral protease (nordmark/serva). here we describe what we consider the most important factors for achieving reproducible and clinically useable islet isolations. methods/results: a standard isolation protocol involving controlled enzymatic digestion followed by density gradient purification was used. seventeen donor pancreata were processed and ten were ultimately used for clinical transplantation. eight of these successful isolations were performed using the nordmark/serva enzymes (table 1) . the following factors were identified as being important for ensuring successful islet yields: 1) donor age and size: male donors 17-45 years old who were tall (>180cm) and heavy (>100kg) conclusions: incorporation of several important modifications into our existing islet isolation protocol has allowed us to routinely obtain high quality islet isolations using an alternative, bovine product-free gmp enzyme. (n=187)], and in 121 pts immuno was cni-free. tac-treated pts were younger, more sensitized and more frequently re-kt. dgf was more frequent in pts receiving cni-free immuno. as a group, these pts were older, more frequently received a kt from a donor after cardiac death and less frequently from a living donor. acute cellular rejection (acr) was diagnosed in 41 pts (6.8%), 13 occurred before day 90th (e-acr), and 28 after this date (l-acr). c4d-positive amr was diagnosed in 128 pts (21.2%), of which 58 were early (e-amr) and 70 were late episodes (l-amr rates of acute rejection (ar) and background: an increasing number of hs patients are being transplanted using desensitization protocols. these patients are considered high risk for ar, particularly antibody mediated rejection (amr). here we examined our ar rates and treatment outcomes for our hs kidney transplant (kt) recipients using our most current desensitization strategies. methods: between june 2004 (when rituximab was introduced to our desensitization and treatment protocols) to july 2007, 130 hs kt patients were transplanted using combinations of high-dose ivig, rituximab, and plasmapheresis (pp) for desensitization. we examined the overall ar, c4d-cell-mediated (cmr), and c4d+amr rates. amr episodes were treated with steroids, high-dose ivig, and rituximab. refractory and rapidly progressive amr was treated with pp. treatment outcomes and differences in ar rates between deceased (dd) and living donors (ld) was examined. recent reports suggest that treatment with the monoclonal anti-cd20 antibody, rituximab (rtx) may improve renal graft survival in antibody mediated rejection (amr); however optimal dosing for this indication is unknown. we examined the efficacy of a single low dose (500mg) of rtx for treatment of refractory amr in order to limit significant infective complications associated with conventional dosing regimens (375mg/m 2 ). rtx was used in seven consecutive patients who had refractory amr as judged by ongoing biopsy evidence of amr after 4 weeks of standard therapy consisting of pulse methylprednisolone and plasma exchange with low dose ivig (100mg/kg) (pe/ivig). all patients received tacrolimus and mycophenolate mofetil. amr was defined as 1) characteristic histology on biopsy (banff criteria), 2) graft dysfunction and 3) presence of a donor specific anti-hla antibody (dsab). b-cell counts (cd19) and serum creatinine (scr) were monitored. pe/ivig was ceased in all cases after rtx dosing. the average follow-up since rtx dosing is 16.8 months (range 5.3-28.9 mths). all patients still have functioning grafts (100% 12 month graft and patient survival), with current mean scr levels (156±17µmol/l) significantly lower than mean peak rejection levels (514±355µmol/l) p=0.05. cd19 counts fell to zero and remained <5x10 6 /l for >6 months in all patients. dsabs remain detectable by luminex flow beads in all patients despite stabilization of scr. two of 7 patients developed an infective complication post rtx dosing. one patient developed bacterial pneumonia requiring hospital admission whilst a second patient developed cmv viraemia and later bk nephropathy which has not led to significant graft dysfunction. hence, infectious complications are far less than those reported for multiple standard dose regimens in similar patient groups. large clinical trials are required to confirm the efficacy of rtx in treating amr as well as optimal dosing. standard dosing is based on oncology treatment regimens which are likely to be excessive for amr in patients already markedly immunosuppressed. our data suggests single low-dose rtx for refractory amr results in excellent patient and graft survival and leads to low rates of serious infective complications in the short-term. renal the demographic and clinical data were similar between +cxm and negative cxm groups. the rejection rate was significantly higher and the length of stay was significantly longer in +cxm group as shown in the table. the ra survival rates were 8%, 7% and 6% lower at 1, 3 and 5 years post transplant respectively among + cxm recipients. however, this did not reach significance mostly due to small sample size (p=0.11). the inferior ra outcome is seen during the initial few years after transplantation that disappears after 9 years. in clkt, pre-transplant +cxm can result in higher ra rejection rate and longer length of stay in hospital. la may not always confer immunological protection to ra in + cxm clkt and its true burden may be under recognized since cxm is not routinely performed in all clkt. study of antibody specificities or la volume to determine the effect of +cxm on ra outcome is essential. skpt in patients with positive cdc b-cell and/or flow cytometry crossmatch is associated with high amr rate despite low dose ivig and r-atg/alemtuzumab induction. pancreas graft survival is inferior in patients with positive crossmatch while kidney graft and patient survival are similar to that of negative crossmatch recipients. majority of amr can be reversed with treatment but further long-term follow-up is needed to determine the impact on graft survival. ten the first european phase iii trial with tacrolimus in the early 90´s clearly showed advantages for tacrolimus in terms of acute rejection (ar) vs the original cyclosporin formulation. however, no advantages were seen in survival rates. the present investigator-initiated, observational follow-up study collected data on patients included in the original cohort of 448 patients who participated in this large study and who were randomised to a triple immunosuppressive regimen consisting of tacrolimus, azathioprine and steroids (tac/aza/ster, n= 303) or cyclosporine, aza and steroids (csa/aza/ster, n=145). efficacy and safety parameters assessed at follow-up included: acute rejection; patient and graft survival; renal function, vital signs, basic lab results and immunosuppressive regimen for the patients 10 years after completion of the original study. results: currently, data are available from 75% of the patients. all assessments were conducted following the intent-to-treat principles. more patients in the tac than in the csa group remained on the randomised treatment. while kaplan-meier (k-m) estimates for patient survival at year 11 show comparable (72% tac vs 72%csa) results, k-m estimates for graft survival demonstrate an advantage for the tacrolimus cohort (51% tac vs 46% csa). graft half-life estimates (gjertson and terasaki method) yielded overall results of 14.2 years (tac) and 11.4 years (cs). in both treatment groups, ar was associated with inferior long-term results. for patients with ar, half-life estimates were 11.1 and 7.8 years for tac and csa, respectively, while in patients without ar half-lives were 19.2 (tac) and 15.8 (cs) years. mean serum creatinine after 10 years was significantly lower in the tac cohort vs the csa group (1.65mg/dl tac vs 2.01mg/ dl csa, p<0.05). mean glomerular filtration rates (mdrd4 estimate) were 48.7 ml/ min in tac and 41.9 ml/min in cs. after ten years of follow-up, the mean (c-0) levels were 7.6 mg/ml (tac) and 120 mg/ml (cs). conclusion: analysis of this long term follow-up of a large study confirms the benefits of a tacrolimus-based therapy. it also confirms the concept that freedom from early acute rejection is associated with superior long term results in renal fuction. therefore, long term renal allograft function is remarkably good in tac patients. cyclophosphamide a novel therapy for ivig/plex-resistant antibody-mediated rejection. (c)). moreover subgroup analyses within the different groups were made to identify potential differences. groups were analyzed for survival, graft rejection and graftvasculopathy (cad). kaplan-meier analysis was used and log-rank test was performed to detect differences. results: a total of 81 transplants (8.9%) were blood group compatible. the majority (n=32) were 0a transplants (40%) followed by 0b (n=19; 23%), aab (n=17; 21%), bab (n=7; 9%) and 0ab (n=6; 7%). overall survival comparison showed no significant difference in long-term survival (10-year) recent data has shown the utility of urinary biomarkers to predict delayed graft function early following renal transplantation, but their ability to predict longer-term allograft function is less clear. we evaluated whether urinary biomarkers measured in the early post-transplant period would predict allograft function at 6 and 12 months after renal transplantation. urinary biomarkers including n-acetyl-β-d-glucosaminidase (nag), kidney injury molecule-1 (kim-1) and matrix metalloproteinase-9 (mmp-9) were measured in 20 patients at hours 0, 6, 12, 24 and days 2 and 3 following renal transplantation. glomerular filtration rate (gfr) at 6 and 12 months were calculated using the mdrd equation. all 20 patients had functional allografts at 6 months and 2 patients lost allografts after 6 months. levels of individual biomarkers at different post-transplant time points were correlated with 6 and 12 month gfr using spearman's correlation (rho) and are shown in the urinary nag levels at post-transplant hours 0, 6, 24 and days 2 and 3 showed significant negative correlation with 6-month gfr. nag levels at hour 24 and days 2 and 3 maintained the significant negative correlation with 12-month gfr. urinary kim-1 levels at the 24 hour point showed significant negative correlation with 6-month gfr and kim-1 levels at hour 24 and day 2 displayed significant negative correlation with 12-month gfr. urinary mmp-9 levels at no time points had significant correlation with either 6-month or 12-month gfr. urinary biomarkers particularly nag shows promise as a tool in the early prediction of longer-term allograft function following renal transplantation. in kidney disease chemokines are involved in the recruitment of leukocytes causing kidney damage and progression to endstage renal failure. similar mechanisms are proposed for chronic allograft failure in renal transplant recipients. the chemokine attracted leukocytes produce proinflammatory and profibrotic cytokines contributing to fibroblast proliferation, matrix production and tubular atrophy. the role of chemokines in the acute post-ischemic tubular necrosis early after renal transplantation and their impact on long term allograft outcome has not been investigated yet. methods 30 patients (23m, 7f, mean age 49.9yrs) with dgf (with a median number of 6.4 dialysis sessions) developed biopsy proven acute tubular necrosis during the first three weeks after transplantation. the biopsies were studied by immunostaining for ki67, ccr1, ccr2, rantes, mcp-1, cd20 and cd68, respectively. the patients were followed for a mean time of 4.2yrs. none of them developed a rejection episode in the follow up time. after follow up the mean serum creatinine was 2.4mg/dl (0.8-6. the goal of a tx is for the recip to achieve long-term survival (surv.), with continued graft function, equivalent to the gen. population. we studied subsequent outcome in 2202, 10-yr kidney tx survivors (tx 1963-1997) ; 62% living donor (ld); 87% 1 st tx; 41% female; 28% type 1 diabetes; mean age at tx (±se), 33±15 yrs. actuarial 25-yr surv. is shown in table 1 ; ld patient, graft, and death-censored graft surv. is signif. ↑vs. deceased donor (p<.0001). mean creatinine (± se) at 10 yrs was 1.7±.8; 15 yrs, 1.7±.9; 20 yrs, 1.5±1; 25 yrs, 1.6±1. the 2 major causes of late graft loss (gl) were death with function (dwf) and chronic rejection (can). by multivariate analysis, risk factors for gl after 10 yrs were age ≥50 yrs at tx; type 1 diabetes, retx, hla mm ≥3; ≥1 acute rejection episode, and pretx cardiac, peripheral vascular, or liver disease (p<.05). the most common cause of dwf was cardiovascular disease (cvd); however, >20% deaths were due to malignancy. the use of kidneys from donors with a positive serology for hcv into hcv(+) recipients still remains controversial. in 1990, our units adopted this policy, subsequently modified in 1993, so these kidneys were limited to recipients with a positive rna hcv before transplantation. the aim of the present analysis was to review the long-term safety of our policy. since january 1990 to february 2007, 474 hcv(+) patients with a negative hbsag and not treated with interferon received a kidney transplant in our units. 313 patients were transplanted from an hcv(-) donor(group 1) and 161 from an hcv(+) donor(group 2). median follow-up time was 65(ir 24-117) months. remarkably, group 2 showed a significantly higher donor age (46.6±13.6 versus 41.1±18.8 years;p<0.0001) and recipient age (50.4±13.1 versus 44.9±14.6 years;p<0.0001), as well as a worse hla compatibiliy than group 1. immunesuppressive therapy did not significantly differ between the two groups. group 2 notably, only 5 patients died because of a liver disease (3 in group 1 versus 2 in group 2) and only 2 patients developed a hepatocarcinoma (both in group 1). in summary, no significant differences were observed in hcv(+) recipients according to hcv serology of the donor, in terms of death censored graf survival, patient survival and evolution of liver disease. therefore, our experience clearly demonstrates that the use of kidneys from hcv(+) donors into hcv(+) recipients is a safe strategy in the long-term and a wise way of using these kidneys, that otherwise would be lost at a moment of shortage. introduction: deceased donor kidneys are allocated to adult candidates in the u.s. primarily by waiting time and hla similarity. we investigated two alternative allocation systems, lyft-scd and lyft-dy, that prioritize offers of kidneys to adults based in part on lyft. methods: based on characteristics of each candidate and donor, lyft was calculated as the difference between expected median years of life for the candidate with that kidney donor transplant (tx) versus without a kidney. years on dialysis were weighted at 0.8 of years with a functioning graft. the relative risk of graft failure for each donor was calculated from donor factors using a cox model and the donor profile index (dpi), the percentile score of that risk among kidneys used in 2003 (100%=greatest risk of graft failure). using actual candidates and deceased donors and acceptance patterns from 2003, we modeled allocation with the kidney-pancreas simulated allocation model (kpsam). kpsam sequentially offers kidneys to candidates according to user-specified allocation rules, and models the probability for kidney placement and post-tx survival. the lyft-scd model allocated scd kidneys by lyft and ecd kidneys by dialysis years (dy). the lyft-dy model allocated organs according to the formula [.8lyft(1-dpi)]+[dy(.8dpi+.2)]+[4pra/100] to adult kidney tx candidates. results: the table compares a year's worth of kidney-alone recipient characteristics, lifespan after tx and additional lifespan (v. dialysis) due to tx among all kidney (including spk) recipients, and the donor/recipient age correlation (r) under current national kidney allocation, allocation using lyft for scd, and allocation using lyft-dy within each donation service area. conclusions: allocation systems incorporating lyft have the potential to increase the number of years of life attainable from tx and are being considered for the allocation of kidneys within the u.s. introduction : organ shortage for transplantation has led to the use of marginal kidneys, which has been associated with poorer but still acceptable results. in our center, the transplantation of two very marginal kidneys into a single recipient was used as a strategy to increase the number of organs available. the present study reports renal function and survival of dual-kidney transplants (dkt) performed at our institution. methods : from october 1999 to june 2007, 392 transplants were performed at our center, from which 63 were dkt. kidney selection for a dkt was based on refusal of the kidneys for single kidney transplantation (skt), donor's age ≥ 60 years and ≥15% of glomerulosclerosis on pre-implantation biopsy. the calculated creatinine clearance (crcl, ml/min), graft and recipient survival from dkt were compared to those of skt from ecd (based on unos criteria, n=66) and ideal donors (id, aged 10 to 39 y, n= 63) over a seven-year period. results : dkt donors were significantly older than the two other groups (dkt: 69 year old, ecd: 62, id: 24). dual transplants were offered to older recipients by design (60, 50, 44). delayed graft function was more prevalent for dkt and ecd than with id (27%, 29%, 11%). twelve, 36 and 84-month crcl were similar for dkt and ecd but lower than id (twelve months: 58, 59, 81; 36 months: 54, 60, 82; 84 months: 62, 51, 86, respectively). patient survival, actuarial graft survival and actuarial graft survival censored for death were similar between the 3 groups. conclusion : to our knowledge, this study reports short and long-term outcome of the largest cohort of dkt performed at a single institution. it shows that dual-kidney transplantation is a more complex intervention with more risks of surgical and post-op complications (data not shown). however, with adequate surgical expertise, dkt patients can expect long-term results comparable or even better than ecd. in our center, the use of dkt has increased the number of transplantations by 19% for the period of study. it allowed older patients to receive a graft within shorter delays. moreover, it permitted a more judicious allocation of a resource that is still limited. an results: very few differences resulting from donornet were seen. for regionally exported kidneys, cold ischemia time (cit) was 22.9h before donornet (bd) and 23.3h after donornet (ad); 6.4% of regionally exported kidneys had cit>36h bd, as compared with 9.0% ad. for nationally exported kidneys, cit was 27.3h bd and 26.8h ad; 17.9% of nationally exported kidneys had cit>36h bd, as compared with 16.1% ad. the same hucs of exported kidneys bd were also hucs ad. of the top 10 centers utilizing exported kidneys bd, only one was not within the top 10 centers utilizing exported kidneys ad. when we looked at cit for kidneys exported to hucs, again there was little difference before or after donornet, with a trend to possibly longer cit for hucs. for kidneys regionally exported to hucs, cold ischemia time (cit) was 23.1h before donornet (bd) and 23.4h after donornet (ad); 7% of regionally exported kidneys had cit>36h bd, as compared with 10.4% ad. for kidneys nationally exported to hucs, cit was 27.2h bd and 27.4h ad; 17.5% of nationally exported kidneys had cit>36h bd, as compared with 16.8% ad. conclusion: the first 6 months of national implementation of donornet were not successful in decreasing cit for regionally or nationally exported kidneys. furthermore, the centers to which a high proportion of these kidneys were exported did not change as a result of donornet, and for these centers allocation efficiency is no better and might even be worse than before. machine cold machine preservation has been shown to improve outcome following transplantation of deceased heart-beating donor kidneys. to determine whether cold machine preservation also improves outcome of kidneys donated after cardiac death (dcd) we undertook a multicentre randomized controlled trial in the uk comparing machine perfusion with simple cold storage. one kidney from each dcd donor was randomized to machine perfusion (organ recovery systems lifeport device with kps-1 solution), the other to perfusion with uw (viaspan) solution followed by simple cold storage. the primary outcome measure was the need for dialysis in the first 7 days (delayed graft function, dgf); secondary outcome measures included gfr at 1 week (mdrd technique); 3 and 12 month graft and patient survival. at the time of writing, one week outcome data are available for all patients. a sequential design was used with the data inspected after 60 transplants and then every 20. all recipients received basiliximab, mycophenolate sodium, tacrolimus and prednisolone. the significance of the initial results has been tested using paired t tests and mcnemar's exact tests. recruitment was stopped after 46 donors when the unadjusted sequential analysis concluded that there was no difference in the incidence of dgf. 91 of the 92 kidneys were transplanted; one was not used for anatomical reasons. one kidney suffered primary non function; it had been machine preserved. the results shown below represent intention to treat. introduction: split liver transplantation has been adopted as an alternative to expand the organ donor pool. while the adult/child split liver (a/csl) in which an extended right graft (erg) is transplanted into an adult and a left lateral segment (lls), transplanted into a child, has gained a wide acceptance within the transplantation community, adult/ adult split liver (a/asl) in which the liver is split into a full right (fr) and full left (fl) is still performed very rarely. we analyzed the experience at our center with split liver grafts over a period of 10 years. material and methods: between october 1997 and october 2007 we performed a total of 676 liver transplants in 625 recipients (370/328 children and 306/297 adults). among the 593 (310 children and 283 adults) recipients of a primary isolated liver transplant, 281 (247 children and 34 adult) were transplanted with a a/c or a/a sl. the recipients of a whole size graft (249 adults and 63 children) were used as a control. results: adults: 34 patients received a split liver graft (26 erg graft and 8 fl/fr grafts) and 249 a whole liver graft. overall the incidence of biliary complications using a split liver graft was 29% and with a whole liver graft 15% (p= 0,0506).for the recipients of a split liver graft 1 and 5 years patient and graft survival was 91%/88% and 88%/85%. recipients of a whole liver graft had a 1 and 5 years patient / graft survival of 85%/84% and 80%/78% respectively. children: 247 children received a split graft (230 lls, 12 erg and 5 fl) and 63 a whole size graft. biliary complications occurred in 42% of the recipients of a split liver graft and 5% of the recipients of a whole size graft (p < 0,0001). among the recipients of a split graft 1 and 5 years patient / graft survival was 91%/84% and 87%/80% respectively. the recipient of a whole size graft had a 1 and 5 years patient / graft survival of 84%/78% and 83%/74%. conclusion: incidence of biliary complications is significantly higher using a split graft. at a high volume center, use of split liver grafts reached comparable and even better results of a whole liver graft in terms of patient and graft survival. thus, the use of split grafts should be strongly encouraged to expand the donor pool. a groups of primary ldlt and ddlt recipients were identified by matching diagnosis, meld score, and recipient age. cost data, acquired from the hospital financial database and inflation adjusted and clinical outcomes were compared between the 2 groups. background: liver transplant recipients with (gw/rw) < 0.8% are thought to have a higher incidence of post-operative complications, including small for size syndrome (sfss), and overall inferior outcomes. we analyzed a cohort of partial liver graft recipients and compared those with gw/rw < 0.8% to those with gw/rw > 0.8%. results: between 1999 and 2007, 107 adult patients underwent partial graft liver transplant. seventy-six grafts were from live donors (ldlt) and the remaining 31 were from deceased donor split-liver transplants (slt). of these, 22 patients had gw/ rw < 0.8% (12 ldlt, 10 slt), and 85 had gw/rw > 0.8% (64 ldlt, 21 slt). median follow-up was 46.1 months and did not differ between the two groups. baseline demographics including donor and recipient age, and meld at the time of transplant also did not differ between groups (table) . three-month and 1-year graft survival for the two groups was comparable. three recipients with gw/rw < 0.8% developed sfss (13.6%); all three had inflow modification with splenic artery ligation (sal), and 1 of the 3 grafts was lost at one-year. eight recipients with gw/rw > 0.8% developed sfss (9.4%) (p = ns); six underwent sal and none had graft loss at one-year. there was a trend toward increased hepatic artery thrombosis with the smaller grafts that did not reach statistical significance (p = 0.10). the incidence of other surgical complications was similar between the two groups (table) . the diagnosis of rejection in post-transplant kidney disease depends largely on the assessment of biopsy pathology, which suffers from arbitrary scoring, subjectivity, and sampling variance. with microarray gene expression data from 186 biopsies for cause, we used predictive analysis of microarrays (pam) to build a rejection classifier. because of the imperfect gold standard, the classifier cannot and should not have extremely high predictive accuracy. the goal was to see if microarrays could detect a robust rejection signature despite this uncertainty, and to identify the top genes distinguishing rejecting from non-rejecting biopsies. by examining discrepancies between pathology diagnoses and pam predictions, and using clinical follow-up information, we combine the strengths of each method to produce a more accurate diagnostic system. biopsies with rejection plus clinical episodes (fig. 1 ) have higher positive predictive value than those lacking episodes. the majority of episodes occur in the top part of fig.1 , indicating that a valid rejection signature is being detected. of the exceptions, most had been treated with steroids before the biopsy, suppressing their gene expression patterns. samples called borderline rejection separated into two distinct high/low probability groups using pam, with all the cases classified as clinical rejection episodes occurring in the high probability group. of the 39 genes chosen by the classifier, 36 had previously been annotated in our studies of mouse kidney graft rejection. gzma, gzmb, and prf1 ranked 26th, 32nd, and 38th respectively. the top five genes were: cxcl11, gbp1, cxcl9, indo, and cxcl10, all previously annotated in rejecting mouse kidneys as interferon-γ induced genes. in summary, gene expression-based classifiers, combined with histopathology, promise more accurate diagnostic assessment of the state of kidney transplants at the time of biopsy. moreover our data driven classifier independently identifies the genes previously annotated in experimental studies. influence of donor background: renal-cell associated tlr4 activation was found to be important in mediating ischemia reperfusion injury to murine kidneys. we hypothesized that genetic variations within the tlr4 gene of the kidney donor affects the rate of delayed graft function (dgf). methods: we genotyped the functional tlr4 polymorphisms (snps) d299g (rs4986790) and t399i (rs4986791) in 268 kidney donors from 2 centers and correlated them with the occurrence of dgf. dgf was defined as the need of dialysis within 7 days post-transplant or less than 25% drop in creatinine within the first 24 hours after transplant. in a sample of 67 patients from one center, we analysed intragraft hmgb-1 (high mobility group box protein-1) gene expression in pre-and post-implantation biopsies. statistical analyses were performed using the spss statistical package. results: both tlr4 snps were in hardy-weinberg equilibrium, and were combined for further analysis. recipients of a tlr4 mutated kidney showed a significant lower rate of dgf (p=0.005), which was persistent after correction for known donor-derived risk factors (donor age, ecd vs. dcd-kidney, cold ischemia time, p=0.007, hr 4.42, cl 1.51-12.94). additionally, we confirmed the presence of a possible tlr4 ligand, hmgb-1, in pre-and post-implantation biopsies. we also confirmed the presence of a functional tlr4 receptor in human proximal tubular cells which expressed mcp-1, il1-β, il-6 and tnf-α after specific tlr4 agonist treatment. conclusion: human renal tubular epithelial cells express tlr4 and a functional tlr4 snp in donor kidney is associated with lower rate of delayed graft function. background: methylation of promoter cpg islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. we postulate that differences in methylation patterns observed in tissues ranging from normal to cirrhosis to hcc may lead to the discovery of direct precipitating events that contribute to tumorigenesis in hcv-hcc patients. methods: dna from 20 hcv-hcc tumors and corresponding non-tumor hcv cirrhotic tissues as well as 20 independent hcv cirrhotic tissues and 20 normal liver tissues were bisulfite treated and hybridized to the illumina goldengate methylation beadarray cancer panel i for interrogating cpg sites in the promoter regions of 808 distinct genes. for each cpg site, a summary statistic representing "percent methylated" was estimated. for each cpg site, a jonckheere-terpstra test was applied to identify whether there was a significant monotonic trend in percent methylated across the independent normal, cirrhotic, and hcc tissues. in addition, the paired hcc and non-tumor cirrhotic tissues were analyzed using a paired t-test. the q-value method for estimating gene-wise false discovery rates (fdr) was used to adjust for multiple comparisons; cpg sites with an fdr<0.05 were considered significant. to identify if serological responses to allogenic non-hla renal compartmentspecific antigens can be detected after renal transplantation (txp). methods: 36 paired pre-and post-transplant (mean time 25 months) serum samples from 18 pediatric kidney allograft recipients were used for identification of de novo non-hla antibody formation assessed using invitrogen protoarray (v3), measuring signal post-pre. probes from the protoarray and cdna microarray platforms were re-annotated to current ncbi entrez gene identifiers using ailun. 34 cdna arrays (gse:3931) from 7 normal kidney regions (inner and outer cortex, inner and outer medulla, papillary tips, renal pelvis and glomeruli; see table) were analyzed for compartment-specific genes by sam, and the highest-ranked genes in each compartment (ks two-sample test p < 0.001) and were ranked against each individual patient's numerical antibody response across the 5057 proteins on the protoarray. results: in 83% of patients (15/18), kidney-specific serological responses against the renal pelvis were the first to be detected, followed by responses against renal cortex. control gene expression data sets from other solid organs (gse: 1133; lung, heart, pancreas) did not demonstrate immune-response enrichment. the presence of these antibodies was not associated with donor specific hla class i or ii antibody levels. conclusion: we demonstrated de novo formation of circulating non-hla non-abo antibodies after txp, irrespective of hla antibodies, are detected against kidneyspecific protein targets. as these antibodies are not seen against other solid organs, the response is likely allogeneic. the renal pelvis and renal cortex have the highest immunogenic potential. we conclude that urinary cell mrna profiles that include levels of mrna encoding tubular proteins and mrna encoding proteins implicated in emt offers a noninvasive means ascertaining renal allograft status with respect to presence or absence of can. functional functional annotation of these genes identified cell cycle pathway and carboxylic acid metabolism pathway as potentially relevant for both datasets. altogether, these data suggest that as compared with recipients requiring on-going immunosuppression, operationally tolerant liver recipients exhibit traits of immunological quiescence and activation of natural killer related pathways. liver and kidney tolerant states appear to be fundamentally different biological processes, although some common pathways could be relevant to both settings. genetic introduction: both normal and fibrotic renal allografts develop a large number of persistent changes in pro-inflammatory gene transcripts early after transplantation (1) . the aim of the current study was to determine if this "transplant effect" is attenuated by immunosuppression, fibrosis or hla-match. methods: we studied 41 histologically normal implantation biopsies (t0) and 45 12m protocol biopsies (t12) from living donor kidney transplant recipients who had no identifiable post-transplant complication (no acute rejection, polyoma virus, etc). patients were stratified into 4 distinct clinicopathologic groups. the first three groups had normal histology at both t0 and t12 and included: 1) hla identical-tac treated (n=10); 2) non-hla identical-tac (n=19); 3) non-hla identical srl (tac-free, n=8). a fourth group was histologically normal at t0 but developed mild fibrosis by t12 (n=7). mrna expression from these biopsies was measured using the u133plus2.0 microarray and custom taqman low density arrays (tlda). data for each group was tested using a generalized linear model and changes common to each dataset identified (p<0.05). results: in addition to transcript changes specific for each clinicopathologic condition, a large number of transcripts (n=1152) were altered in all groups. 68% (n=778) had higher expression in all comparisons at t12 versus t0, including fibronectin and collagen iv. 32% (n=374) of transcripts were considered down-regulated, including interleukin 6 and 1β. gene ontology and signaling pathway analyses showed many of the transcripts to be involved in pro-inflammatory and immune response signaling pathways (ex. tlr, il-10/-2, etc). custom tldas were used to study several genes considered not detected by microarray. this included tgf-β1, cd-3e/-28/-74 and vegf, all of which were significantly up-regulated in multiple comparisons. conclusions: persistent changes in pro-inflammatory transcripts occur in all renal transplants. this "transplant effect" cannot be explained by calcineurin-inhibitors (occurs in tac-free immunosuppression), increased alloreactivity (similar in hla identical/ non-identical) or fibrosis. a likely cause is persistent changes from ir injury. the role of the "transplant effect" in long-term allograft survival is unclear, but it likely interacts with other forms of graft injury. background: a critical 26 gene-set for acute renal rejection (ar) diagnosis in peripheral blood has been previously identified by our group using cross-platform hybridization of 91 unique peripherla blood samples with matched biopsy diagnosis, with ar (n=39) and without ar (sta; n=52), with cross-hybridization across 3 human microarray platforms: 30k cdna lymphochip, 44k agilent and 54k affymetrix hu133plus. in this study we proposed to verify and validate this gene-set for ar diagnosis and prediction by peripheral blood pcr-based analysis. method: 57/91 peripheral blood samples from the microarray gene-set were selected for 384-well format multiplex abi-taqman qpcrv validation of ar diagnosis across the 26 gene-set. an independant set of 66 new, time and immunosuppression matched, peripheral blood samples (30sta, 36ar) were used as a test-set for blinded prediction of ar diagnosis using qpcr across the most informative 8 genes. 42 sequential samples from 14 ar patients (at ar, 1-3 months prior to, and after ar) were also examined by qpcr for these 8 genes for blinded ar prediction, prior to biopsy proven ar. prediction models were generated using logistic regression analysis and p<0.05 considered significant. result: in the validation set of 57 samples, 8/26 genes were highly significant for confirming peripheral blood-ar diagnosis (p<0.03). in the test group of 66 samples, ar was predicted with a very high level of sensitivity and specificity (ppv and npv> 90%). in the group of 14 ar patients with sequential samples pre-and post-ar, expression for these 8 genes were significantly elevated in the pre-ar samples (vs. sta, p<0.01) but were not different between pre-ar and ar values. conclusion: a highly specific and sensitive biomarker panel of 8 genes has been derived and tested by cross-platform microarray analysis. thsi gene-set has now been validated and further verified by multiplex pcr, for ar diagnosis and prediction, even 3 months prior to the rejection event. utility of this gene-set should be further validated in real-time by serial longitudinal peripheral blood testing, for more senstive and specific minimally invasive monitoring for graft rejection. tolerance/immune deviation ii background: our previous studies showed that regulatory t cells (treg) execute suppressive function in both the inflammatory site of the allograft and the draining lymph node (dln) to suppress allograft rejection. we explored how treg influenced the migration and function of antigen specific effector t cells and dendritic cells (dc) at these two sites in an islet transplant model. methods: treg were sorted from wild type or ccr7 -/-c57bl/6 mice, labeled with pkh26, and transferred directly to islet allografts (balb/c into c57bl/6). effector cd4 t cells (te) from alloantigen specific t cell receptor transgenic mice were labeled with csfe and transferred intravenously. treg and te migration to islet grafts and dln were analyzed with flow cytometry and immunohistochemistry. chemokine expression was determined by rt-pcr. cx3cr1 gfp mice, in which dc are internally labeled with gfp, served as islet donors, and donor dc migration was tracked with fluorescence microscopy. results: during priming and rejection, islet allografts expressed a panel of inflammatory chemokines shortly after transplantation that recruited te to the islets. te accumulated and proliferated in both the islet allograft and the dln. concomitantly, donor derived dc migrated from the islet to the dln as early as 1 day after transplantation. local transfer of treg into the islet allograft inhibited islet chemokine expression, inhibited donor dc migration in an il-10 and tgfb dependent fashion, and reduced te migration to and proliferation in the graft. the transferred treg also migrated from the islet allograft to the dln, and directly inhibited te accumulation and proliferation in the dln, and migration of te to the islet. ccr7 -/-treg, which could not migrate to the dln but were retained within the graft, and were far less effective than wild type treg in inhibiting te migration and proliferation into both the islets and dln. conclusions: treg migrate to the islet and suppress parenchymal cell chemokine expression, dc migration, and te responses in the islet allograft. treg also migrate to the dln to suppress te proliferation and migration. treg trafficking from the allograft to the dln is crucial for suppressive function in order to target the separate effector mechanisms. these results demonstrate novel and important functions for migration in treg induced suppression and graft survival. tregs cd4+foxp3+ regulatory t cells (tregs) play an important role in transplant tolerance, yet basic aspects of treg biology including the mechanisms involved in their induction remain unclear. α-cd45rb is a potent tolerogenic agent that induces a 2x increase in number of tregs in wt mice, even in the absence of allo-antigen. here we use foxp3red fluorescent protein reporter knock-in mice to study treg homeostasis and identify the mechanisms by which α-cd45rb induces tregs. cfse-stained highly sort-abstracts purified foxp3+ or foxp3-cells were adoptively transferred into fully replete naive wt congenic mice. whereas only 5-10% of transferred foxp3-cells undergo homeostatic proliferation (hp) over 10d, 50-70% of foxp3+ cells proliferate -a surprisingly high rate of hp. moreover, treatment with α-cd45rb markedly enhanced hp by transferred foxp3+ cells, resulting in a 10x increase in cell number. α-cd45rb induced de novo foxp3 expression in 13-25% of transferred foxp3-cells (many of which subsequently underwent hp). thus, α-cd45rb induces both conversion of foxp3-to foxp3+ cells and promotes hp in foxp3+ cells in the absence of exogenous antigen. we then addressed the signals controlling basal hp of tregs and both hp and conversion mediated by α-cd45rb. cfse-stained congenic cd4+ cells adoptively transferred into naive wt mice were untreated, or received csa, α-cd45rb, or both and foxp3+ and foxp3-cd4 cells were assessed on d10. calcineurin inhibition greatly reduced basal hp of transferred foxp3+ cells compared to untreated mice. moreover, csa completely abrogated increased treg hp induced by α-cd45rb, although conversion still occurred. next we assessed the role of tgfβ. we found that blocking tgfβ signaling with α-tgfβ shortened allograft survival, but had no effect on basal treg hp, or on treg hp or conversion by α-cd45rb. finally, although exogenous antigen is not required, basal and α-cd45rb-induced hp may still require recognition of self-ag bytregs. indeed, preliminary results reveal that when cfse-labeled cd4+ cells are transferred into mhcii ko mice, basal hp by tregs is reduced and not restored by α-cd45rb. thus, α-cd45rb alters normal controls regulating hp by treg. moreover, both basal and α-cd45rb-mediated hp requires intact calcineurin activity and tcr signaling, but not tgfβ. understanding the signals controlling hp in treg may reveal new therapeutic targets for tolerance induction. we report the first demonstration of a tolerance-inducing strategy based on posttransplant administration of allo-ag-specific treg (aastreg) and rapamycin (rapa), in a fully allogeneic, unmanipulated mouse heart transplant model. enrichment of naturally-occurring aastreg represented the first step to counterbalance the high frequency of alloreactive t cells. selection was achieved by co-incubation of freshly-isolated cd4 + cd25 + t cells with donor bone marrow-derived dendritic cells (dc). the source of stimulatory factors necessary to sustain treg proliferation was the supernatant of cd4 + cd25 -t cells co-cultured with allogeneic dc (mlrsup). use of mature (cd86 high ) dc favored extensive expansion of foxp3cells capable of il-17 production (t h 17). co-culture of treg with immature dc in the presence of mlrsup rendered a t cell population with regulatory phenotype: foxp3 + , gitr + , ctla-4 + , ccr7 + , cd62l -. these cells inhibited in vitro effector t cell proliferation at 1:20 and 1:40 treg:t cell ratios. the suppressive activity was ag-specific; no significant inhibition was evident when effector t cells were stimulated by third party dc. aastreg were then tested for their ability to induce transplant tolerance in a mouse heterotopic heart allograft model (balb/c to c57bl/10; unmanipulated). rapa was used: i) to inhibit effector t cell proliferation, while sparing treg activity and thus enhancing the in vivo treg:effector t cell ratio; ii) to promote resolution of the inflammatory state and preserve the susceptibility of conventional t cells to treg suppression. graft recipients received rapa (1mg/kg/d; d0-9) and 2x10 6 aastreg i.v. on d7. in comparison to the untreated group (median survival time, mst=11d; n=14), rapa alone extended mst to 30 d (n=7), with no long-term graft survival. under cover of rapa, aastreg exerted a profound tolerogenic effect: >80% recipients exhibited longterm graft survival (mst>150d; n=6). this effect was stronger than polyclonal treg administration: 40% long-term survivors (mst>50d; n=5). moreover, the tolerogenic effect was ag-specific, as aastreg selected against third party dc (c3h/hej) did not prolong graft survival in comparison to the rapa-only control group. these results indicate the feasibility and therapeutic potential of ag-specific tolerogenic cell therapy based on post-transplant administration of selected aastreg. direct intravenous immunoglobulins (ivig) is an effective treatment for t-cell mediated graft rejection and autoimmune diseases. ivig treatment is associated with rapid clinical improvements without the side effects of global immunosuppression. this prompted us to ask whether ivig might enhance directly the suppressive function cd4+cd25+foxp3+ regulatory t cells in vitro and in vivo. in vitro, mouse cba/ca (h2 k ) total cd4 + or cd4 + cd25responder cells were stimulated with c57bl/10 (h2 b ) splenocytes, and human cd4 + or cd4 + cd25cells were stimulated with allogeneic antigen presenting cells. in vivo, 1*10 5 total cd4 + or cd4 + cd25 -t cells of cba/ca mice were adoptively transferred into cba/rag1 -/mice, and one day later transplanted with a tail skingraft of c57bl/10 mice. ivig was administered i.v. on day 1,3,7,10 and 14. human serum albumin (hsa) was used as a control. binding of ivig and activation status of foxp3 + cd4 + t cells were determined. in vivo, only when total cd4+ t cells, but not cd25-t cells, were adoptively transferred, ivig protected against t-cell mediated rejection of the fully mismatched skingraft (mst: ivig >100 vs hsa 16 days, p<0.01). ivig binds to 16±2% of foxp3 + t cells, while binding to foxp3 -t cells was minimal. this binding was partially fcγ receptor mediated. furthermore, ivig treatment resulted in activation of cd25 + t cells as detected by increased expression of phosphorylated zap70/syk, which could be abrogated by specific tyrosine kinase inhibition. in vitro, ivig inhibited the alloproliferative response of murine cd4 + t cells by 75±25%, but after depletion of cd25 + t cells, this inhibition decreased to 17±12% (n=6, p<0.01). similar results were found in human mlr, where depletion of cd25+ t cells resulted in twofold reduction in ivig mediated suppression. significantly, incubation of human cd4 + cd25 + with ivig enhanced their ability to suppress allogeneic t-cell proliferation (ivig 63±8% vs hsa 37±10% of inhibition, n=5, p<0.05) . our results identify a novel pathway through which ivig treatment induces direct functional activation of both mouse and human regulatory t cells. immediate binding and activation of regulatory t cells is one of the mechanisms in the immunomodulatory repertoire of ivig, which allows rapid inhibition of allogeneic responses, and therefore can be a valuable tool after organ transplantation. generation foxp3 is a dna-binding protein that is necessary for regulatory t cell (treg) function, though recent data indicate that detection of foxp3 mrna or protein alone does not necessarily indicate whether the associated foxp3+ cell is functional or not. to that end, our analysis of foxp3 sequence showed the presence of an rxxr motif, conserved between mice and humans, that is located 12 amino acids (aa) from the carboxy terminus of foxp3 and is a potential recognition sequence for cleavage by proprotein convertase enzymes. we found several proprotein convertases are expressed by naturally occurring tregs, with further upregulation upon tcr activation. we generated an antibody against the 12 aa carboxyl peptide of foxp3, and by western blotting detected a peptide of about 1.3-kda, consistent with a 12-aa long carboxy-terminal foxp3 cleavage product. both cleaved and uncleaved forms of foxp3 were resolved by sds-page, and the cleaved form was found only in the dna-bound fraction. the requirement of proteolytic cleavage, at the intact tetrabasic rxxr motif (414rkkr417), for full treg function was shown by abolishing the motif through mutagenesis, followed by retroviral expression of mutants, and analysis of proteolytic cleavage by western blotting. assays of treg function by cells expressing either long-or short-foxp3 mutants showed short-foxp3 (missing the carboxy-terminal 12-aa) suppressed teff cell proliferation significantly more effectively than wt foxp3 or an engineered and cleavage-resistant mutant, termed long-foxp3 (p<0.01). moreover, adoptive transfer of cells expressing short-foxp3 prevented experimental colitis more effectively than wt-foxp3 (p<0.01), and both were more effective than cells expressing long-foxp3. animals that received cells expressing short-foxp3 gained weight and were protected from disease. thus, function of foxp3 is regulated at a post-translational level by proteolytic cleavage and the short form of foxp3 represents the active form. our data shows proteolytic cleavage of foxp3 is key to the generation of functional tregs, with enzymes(s) of the proprotein convertase family likely playing an important role in this process and providing an extra and hitherto unrecognized important level of regulation for foxp3. blocking since tgf-β is secreted in a latent form and active tgf-β is rapidly cleared from circulation. in order to make long-lasting active form of tgf-β, the mutant tgf-β was fused to a human igg fc component. the secreted human mutant tgf-β/fc (hmtgf β /fc) fusion protein stained with both anti-human tgf-β and anti-human igg antibodies, confirming the cytokine and isotype specificity of tgf-β moiety and fc domain. moreover, in vitro bioassay, hmtgf-β/fc inhibited il-4 dependent ht-2 cell proliferation in a dose dependent manner and had a circulating half-life of 36 hours in mice. in vitro, anti-cd3 and anti-cd28 triggered cd4+ or cd8+ t cell proliferation, measured by 3h-thymidine uptake, could be synergistically inhibited by tgf-β and rapamycin. at the same time, the two reagents when added together could synergistically promote the induction of cd4 + foxp3 + t cells from peripheral naïve cd4 + foxp3 -t cells. in a pancreas islet transplantation model in which the fully mhc mismatched dba/2 islets were transplanted into c57 bl/6 mice rendered diabetic by streptozotocin, mean survival time of islet was 19 days in control recipients (n=6). administration of muttgf-β/fc resulted in a delayed islet allograft rejection (mst; 38 days, n=5). treatment with rapamycin prolonged islet grafts survival in 63% of recipients. in contrast, combined treatment with tgf-β/fc and rapamycin by four doses produced indefinite islet allograft survival in 83% cases. we have produced the long-lasting active hmtgf-β/fc fusion protein. the tgf-β/fc is synergistic with rapamycin to convert naïve cd4+foxp3-t cells into cd4+foxp3+ tregs and promote long-term islet allograft engraftment. the the recognition of microbial motifs by the innate immune system leading to the stimulation of adaptive immune responses may explain the relationship between infections and susceptibility to graft rejection. a number of infectious agents have been reported to prevent the induction of allograft tolerance, however, none of these can reverse established tolerance, a situation that is highly relevant to clinical transpalntation. we here report that established allograft tolerance (induced with anti-cd154 + donorspecific transfusion) can be reversed in a cardiac transplantation mouse model following infection with listeria monocytogenes. this reversal of tolerance was dependent on the presence of both cd4+ and cd8+ cells, as well as of the tlr/il-1/il-8 adaptor molecule, myd88. we hypothesized that the reversal of tolerance requires that alloreactive conventional t cells (tconv) escape dominant regulation by pre-existing tregs. these alloreactive tconv can then proliferate resulting in an increased ratio of tconv:tregs favoring the reversal of established tolerance. indeed, we observed that tolerant grafts harbored low numbers of infiltrating cd4 + and cd8 + t cells (1-1.4 x 10 4 /heart), with 10-43% of the infiltrating cd4 + cells co-expressing foxp3 (n=10). following the reversal of tolerance and allograft rejection, a 7-10 fold increase in cd4 + foxp3and cd8 + foxp3 -tconv was observed in the graft, but no increase in the foxp3 + subset. in contrast, we observed no significant changes in the splenic t cell subsets, raising the possibility that the escape from tregs occurs locally within the graft. infection of tolerant il-6-or ifnar1-deficient recipients with listeria monocytogenes failed to reverse tolerance, consistent with a hypothesis that the initial escape of tconv from regulation may depend on il-6, while their proliferation may be type i ifn-dependent. in summary, the conditions for the reversal of tolerance is more stringent than the prevention of tolerance, and requires myd88-signalling and the presence of both cd4 + cells and cd8 + cells, as well as il-6 and type i ifn signaling. these studies point to the potential impact of bacterial infections on established tolerance, and to novel strategies to monitor and facilitate the maintenance of tolerance in the clinic. th17 our laboratory has identified kα1 tubulin, as an epithelial autoantigen to which immune response occurs following human ltx. further, we have shown a strong correlation between the presence of antibodies (abs) to kα1 tubulin and development of chronic rejection ie bronchiolitis obliterans syndrome (bos). goal of our study is to test the hypothesis that epithelial damage due to allo immunity results in remodeling exposes otherwise cryptic self antigens including kα 1 tubulin and collagen type v (coll v) leading to cellular immune reactivity and ab production against these auto-antigens which may play a role in the pathogenesis of bos. 10 patients who developed anti-hla ab and 10 patients who had no detectable anti-donor hla abs post-ltx by luminex assay were analyzed for development of abs to kα1 tubulin and coll v using elisa method developed in our laboratory. the elisa for kα1 tubulin used recombinant protein (1µg/ml) purified on affinity resin. the elisa for collagen v uses commercially available human collagen v (1µg/ml). elispot assay for ifnγ and il-10 were performed for 5 bos-and 5 bos+ patient pbls (at the time of bos diagnosis), after 3 stimulations with kα1 tubulin protein. the levels of anti-tubulin abs and anti-coll v abs were increased significantly in bos+ ltx recipients with anti-hla compared to those with no anti-hla, table 1 (p<0.05). . surprisingly, both cd4 and cd8 subsets induced long-term survival of secondary test grafts (>100 days). however, only the cd4 + t-reg prevented tvs (ni=19+5, 21+6% of vessels), as compared to cd8 + t-reg (ni=40+9 in 50+15% of vessels). the cytokine profile indicated a dominant il-10 response. allo-antibody analysis showed up-regulation il-4/il-12 dependent igg1 and igg2c. conclusion: allochimeric protein-therapy and csa treatment generate t-reg that prolong graft survival, but only cd4 + t-reg generated from allochimeric protein-therapy prevent tvs. this cd4 + t-reg may be responsible for long-term graft maintenance through its unique ability to control anti-inflammatory and alloantibody responses. immunodominant h60 background: minor histocompatibility ags (mihas) are self peptides, derived from proteolytic processing of normal cellular proteins. miha incompatibility can induce t cell response to dominant mihas and facilitates expansion of ctls and graft rejection. however, the contribution of ctls recognizing these mihas in solid organ transplantation has not been fully evaluated. methods: balb.b (h-2 b ) donor hearts were transplanted heterotopically to c57bl/6 (h-2 b ) recipients. in vivo alloreactive cd8 t cells were monitored with peptide/mhc multimers. α-lfa-1 mab was given to recipients to prevent rejection. various numbers of h60-specific cd8 t cells or cd8 t cells lacking h60 specificity were adoptively transferred into balb.b graft bearing b6.scid recipients. results: 50% of transplantation recipients developed acute rejection and showed markedly increased numbers of h60-specific cd8 t cells at day 8-12 in spleen. abundant cd8 infiltration was found and selective infiltration of h60-specific cd8 t cells in the graft was confirmed with flow cytometry and in situ tetramer staining. α-lfa-1 mab treatment prevented acute rejection (mst>100) and allogeneic t cell expansion. it also profoundly attenuated neointimal hyperplasia ( graft-bearing b6.scid mice. we found that the degree of acute rejection positively correlated with the number of h60-specific cd8 t cells transferred. however, transferred h60-specific cd8 t cells did not cause chronic rejection. interestingly, greater numbers of cd8 t cells with h4 immunodominance were found in spleen, blood and graft at 50 days after adoptive transfer of cd8 lacking h60 specificity compared to h60-specific cd8 t cell transfer. conclusion: h60 responses dominate other miha immune responses during acute rejection after balb.b to b6 cardiac allograft. we confirmed a role of immunodominant h60 specific cd8 t cells as pathological effector t cells in acute rejection but not in chronic rejection. maintaining a h60 response may be beneficial in allotolerance to suppress miha responses that could induce chronic rejection in long-term grafts. chronic lung allograft rejection, bronchiolitis obliterans syndrome (bos), affects up to 60% of transplant survivors 5 years post-ltx. human neutrophil peptides (hnp 1-3/ α defensins), have been identified in the lavage fluids from patients with chronic rejection by proteomics. goals of our study were to determine the role of anti-hla abs and defensins in bos and to define the interactions between α-1antitrypsin (aat), and defensins in regulating inflammation leading to epithelial cell proliferation and bos pathogenesis. bal and serum samples (post-ltx and pre bos) from 21 bos+, 15 bos-patients and 12 normals were analyzed by elisa for α defensins (hnp1-3), human β defensin2 (hbd2) and aat. small airway epithelial cells (saec) were treated with hnp1 or 2, with or without equimolar aat or with anti-hla abs and analyzed for levels of hbd2 production (elisa), cytokine and chemokine (luminex), and cell surface adhesion molecules (icam, vcam) by facs. bal and serum from bos+ patients had high levels of hnp1-3 and hbd2 compared to bos-recipients or normal sera (p<0.05) ( table 1 ). there was also a significant decrease in aat levels in bos+ compared to bos-or normal serum (p=0.01) ( table 1) . saec produced human β defensins following anti-hla ab stimulation or by hnp treatment (table 2) . there was increase in adhesion molecules (icam, vcam, 2 folds) cytokines {il-6, il-1r α (2 folds), il-1 β, il-13(20 folds)}, il-10(2 folds decrease), chemokines (il-8, mcp1, 2-4 folds increase) and growth factors (egf and vegf, 2.5 folds increase) in response to treatment with hnp1 or hnp2 compared to untreated saec, that was inhibited by aat. increased defensins and decreased aat levels were seen in lavage and serum of ltx recipients with bos. anti-hla abs further stimulate defensin production by saec. we conclude that chronic stimulation of epithelial cells both by defensins and anti-hla abs can lead to increased growth factor production contributing to the pathogenesis of bos. under tubular atrophy/interstitial fibrosis (ta/if) remains a major cause of late kidney allograft loss and epithelial mesenchymal transformation (emt) is now appreciated as a key feature in this process. we hypothesize that macrophages play a direct role in the development of ta/if by creating a profibrotic microenvironment and stimulating emt within the allograft. in human kidney allografts with ta/if (n=128), macrophage infiltration detected by immunostaining was significantly upregulated (mean score 2.8±0.5) compared to biopsies without corresponding pathological changes from recipients with stable function (n=51; 2.0±0.1; p<0.0001) and the extent of this staining also correlated to the magnitude of graft dysfunction (p<0.0001). rt-pcr in ta/if grafts also showed marked upregulation for emt markers αsma (3.0±1.1-fold; p<0.05), s100a4 (8.7±2.7-fold; p<0.05), and vimentin (4.2±2.3-fold; p<0.01), compared to stable function allografts. to explore the macrophage-emt relationship, we co-cultured freshly isolated human monocytes over a primary culture of human proximal tubular epithelial cells (ptecs) using culture inserts allowing media exchange but forbidding contact between the two cell populations. by rt-pcr, co-cultured epithelial cells showed significant downregulation of bmp7 (0.45±0.09-fold; p<0.001), a negative regulator of emt, and epithelial marker e-cadherin (0.001±0.001-fold; p<0.01), with marked upregulation of emt genes s100a4 (2.43±0.40-fold; p<0.001) and αsma (5.31±2.23-fold; p<0.05) compared to ptecs cultured in media alone. investigating the mechanism of monocyte driven emt, we evaluated the effects of am80, a synthetic retinoid that also inhibits il-6 and vegf signaling. am80 markedly reversed the transcriptional profile with reduction of emt markers s100a4 (0.34±0.10-fold; p<0.01), αsma (0.29±0.06-fold; p<0.05) and vimentin (0.45±0.13-fold; p<0.01), and a simultaneous increase in expression of bmp7 (151.7±53.9-fold; p<0.01), thus reversing the pro-emt milieu. these results indicate that human monocytes induce transcription of emt related genes in ptecs, even in the absence of physical contact. moreover, this phenomenon appears to be mediated in part by il-6, vegf, and other pathways mediated by the retinoic acid receptor. thus, in addition to their typical immune functions, macrophages support a milieu within allografts that promotes ta/ if in humans. further investigation into this novel pro-ta/if pathway could ameliorate chronic injury and improve graft survival. old donor kidneys are more likely to develop long-term graft failure, especially if they are exposed to stresses (e.g. rejection). this limited ability of old tissue to withstand stress may be due to its reduced capacity of replication and regeneration. telomere shortening determines lifespan and regenerative capacity. we showed that telomere shortening occurs in old human kidneys. late-generation terc ko mice with critically short telomeres have a reduced lifespan, show an accelerated aging phenotype and are an ideal model to resemble the situation in old human donors. this study addressed the question whether iri causes greater damage in kidneys from late-generation terc ko mice. we studied terc wildtype (wt), early-(g1) and late-generation (g4) terc ko mice 1 day (wt:n=7; g1:n=8; g4:n=8), 3 (wt:n=8; g1:n=8; g4:n=5), 7 (wt:n=6; g1:n=8; g4:n=8) and 30 days (wt:n=8; g1:n=9; g4:n=6) after iri. acute tubular necrosis was found mainly on days 1 and 3 and was significantly higher in terc ko g4 kidneys. tubular atrophy (ta) and intersitial fibrosis (if), reflecting chronic damage, were first detected at day 7 and increased in all mice with a significantly higher extent of ta/if in terc ko g4 kidneys at day 30 ( fig.) . the cell cycle inhibitor p21, a downstream mediator of senescence induced by telomere shortening, was significantly upregulated in all groups after iri. p21 levels were highest in terc ko g4 kidneys at day 30 ( fig.) . proliferative capacity, as measured by ki-67 immunostainings at day 30, was significantly lower in tubular, glomerular and interstitial cells of kidneys from terc ko g4 mice. we show that late-generation terc ko mice with critically short telomeres have a greater susceptibility towards acute injury and develop more chronic renal damage. this is likely due to the reduced capacity of these kidney to proliferate and thereby to regenerate. our data strongly suggest a pathogenetic role of telomere shortening, an important senescence mechanism, for the development of long-term graft failure. results: belatacept treatment had no effect on the number of peripheral blood treg cells and t cell suppression assays. the percentage of foxp3 cells was significantly elevated in rejecting kidney allografts in belatacept-treated patients compared to cnitreated patients. conclusions: following chronic belatacept therapy, the number and function of peripheral blood treg cells are maintained in kidney transplant patients. belatacept enhances the treg population in the allograft in patients with acute rejection. therefore, our data suggest that co-stimulation blockade with belatacept does not affect treg homeostasis. the increased number of treg cells in rejecting allografts in belatacepttreated patients may provide a novel mechanism whereby belatacept can mitigate the severity of acute rejection and improve graft survival. the steady-state auc of n-desmethyl-aeb071 was minor in comparison to aeb071 and similar between the patient groups: 200 ± 90 ng.h/ml in transplantation vs 393 ± 220 ng.h/ml in psoriasis (p=0.08). demographic covariates: in the first week posttransplant with patients receiving fixed-dose aeb071, intersubject variability for c0 was 78% and for auc was 49%. aucs were similar in men vs women (8350 ± 4163 vs 10784 ± 5369, p=0.26). age, which ranged from 18-64 years, did not influence auc based on regression analysis (r 2 = 0.005, p=0.65). there was a borderline-significant negative correlation between weight (range, 51-110 kg) and auc (p = 0.07); however, its clinical relevance was low in that it could explain <9% of the variability in auc (r 2 = 0.086). there was a significant positive correlation between aeb071 c0 and auc (r 2 = 0.724, p<0.001). conclusions: (1) in the first week posttransplant, patients achieved aeb071 blood levels anticipated for this regimen. (2) there was notable intersubject pharmacokinetic variability at this time but it was not attributable to standard demographic factors such as sex, age, or weight. (3) a good correlation was noted between c0 and auc suggesting that c0 might serve as a marker for total drug exposure. a we studied which is protocol would be best after rapid discontinuation of p. between 9/01 and 4/06, 440 1st and 2nd kidney tx recips were randomized: csa-mmf (n=151) vs. high tac-low srl (n=149) vs. low tac-hi srl (n=140) (for tac and srl levels, high = 8-12, low = 3-7). all received thymoglobulin (tmg) 5 doses, and p for 5 days; tmg was continued in dgf. min f/u = 1 yr; mean = 42±19 mos. there was no diff between groups in recip age, gender, ethnicity, prim dis, donor source, % retx, pra; or in donor age, gender, ethnicity. there was no signif diff. between groups (intention to treat) in actuarial patient, graft, death-censored (dc) graft, acute rejection (ar), or biopsy-proven chronic rejection rates (cr), serum cr level or calculated (mdrd) gfr or in studied side effects. cr(sd) 1.6(.7);1.7(.9);1.8(1.3); 1.9(2);1.6(.7) 1.5(.4);1.7(1);2(2.5); 1.7(1.1);1.6(.6) 1.5(.5);1.5(.7); 1.6(.6);1.6(.6);1.7(.7) mdrd gfr(sd) 63(24),58 (21),60(26), 61(26),62 (20) 64 (20),65(24),61(27), 56(23),63 (28) 65 (20) the most common cause of graft loss was death with function (csa 40%, high tac 35%, low tac 57%). the majority of recips in each group remained p-free (csa 72%, high tac 86%, low tac 74%) but a large % were not on the medications which they were randomized to (csa 16%, high tac 51%, low tac 42%). we found a signif ↑ of new onset diabetes (nodm) (p=.03) in the tac-srl groups: csa 2%, high tac 11%, low tac 5%. there was a trend towards more use of lipd lowering medications in the tac/srl groups. in summary, all 3 is protocols were effective; with min f/u 1 yr for each group, patient and graft survival and ar rates are similar. we found an increased nodm and possibly hyperlipidemia in the tac/srl groups. purpose: we present preliminary 2-year results from a worldwide trial comparing 2 srl regimens with tac+mmf. methods: renal transplant recipients (n=451) were randomly assigned to the following treatment regimens: group 1: srl (8-15 ng/ml, then 12-20 ng/ml after week 13) + tac (6-15 ng/ml) with elimination at 13 weeks (n=155); group 2: srl (10-15 ng/ ml through week 26, 8-15 ng/ml thereafter) + mmf (up to 2 gm/day) (n=155); or group 3: tac (8-15 ng/ml through week 26, 5-15 ng/ml thereafter) + mmf (up to 2 gm/day) (n=141). all patients received corticosteroids and daclizumab. in june 2006, group 2 was terminated (after all patients were accrued) because of increased acute rejection (ar) rates. results: demographic characteristics were similar between groups except for more females in group 3. patient and graft survival were also similar among groups (see table) . biopsy-confirmed ar (bcar) was significantly greater in group 2 compared with groups 1 and 3, p<0.001, and most occurred in the first 6 months posttransplant with preponderance during the first 3 months. subtherapeutic srl trough concentrations were reported in a large number of rejectors in group 2. most of the rejections in group 1 occurred within the first 3 months before tac was eliminated. all ars were mild to moderate; grade ii ars were proportionally greater in group 3. mean nankivell gfr was numerically higher in group 2. preliminary results at 2 years show excellent patient and graft survival and similar renal function among treatment groups, despite higher ar rates in group 2. early adequate exposure to sirolimus is mandatory to achieve desired (low bcar rates) results. the goal of rapid discontinuation of prednisone (rdp) after kidney transplantation is to minimize prednisone-related side effects without increasing acute rejection (ar) rates or decreasing long-term graft survival. to date, studies have shown that rdp is associated with decreased prednisone (p)-related sided effects, and randomized trials of rdp (vs. long-term p) have shown little or no ↑ in ar rates. however, concern remains that long-term graft survival will be worse with rdp protocols. we studied t½ (the time it takes for ½ of the grafts surviving at 1 year to subsequently fail) for 1st tx recipients treated with rpd (1999-2007) (n = 652) (antibody, cni, antimetabolite [mmf or srl] , and rdp) vs. 1st tx historical controls (1995) (1996) (1997) (1998) (1999) (2000) (n = 388) treated with a protocol of antibody, cni, antimetabolite [mmf] , and long-term p. table 1 shows characteristiscs of the 2 groups. pra; rdp were more likely to get a living unrelated donor transplant. t ½ is shown separately for living (ld) and deceased (dd) donor transplants in table 2 . there was no significant difference in t½ between groups. we conclude that, compared to historical controls, rapid discontinuation of prednisone can be done without any detrimental impact on long-term outcome. a prospective, randomized study to confirm this observation is necessary. successful 1a) . we enrolled 152 patients (table 1) with an average follow-up of 19.7 + 11.7 months. subjects 19 -65 years old included primary and re-transplants, with primary endpoints of renal function and can. follow-up averaged 16.6 + 9.5 months in 47 patients withdrawn from ci (26 from group 3, 21 from group 4). we found no increased rejection after ci discontinuation and that both ratg induction dosing regimen and ci discontinuation significantly impacted renal function and the development of can. we successfully discontinued both calcineurin inhibitors and steroids with either single or divided-dose ratg induction, and single-dose ratg induction independently associated with improved renal function and reduced can. study demographics single-dose ratg (6 mg/kg x 1) divided-dose ratg (1.5 mg/kg x 4) group 1 (n = 38) group 3 (n = 38) group 2 (n = 37) group 4 (n = 39) age 45.7 ± 11.9 45. in subset analysis, we also find an enhanced negative impact of srl when combined with steroids (str) in patients without dgf. conclusion: this data supports previous findings that srl may be associated with a sustained survival disadvantage apparent early post transplant, and that this effect appears exacerbated when combined with dgf or str. several potential explanations for this effect may include srl-associated prolonged early graft dysfunction, hyperlipidemia or proteinuria. however, patients initiating srl after discharge may not evidence this survival disadvantage, and this question was not addressed here.these findings may be of particular importance to centers employing combined srl and str therapy, as well as to define the best mode of support during recovery from dgf. prospective randomized studies would be necessary to evaluate these. table 1 shows cai in both groups from 1 to 5 years. in slr group the doses of cin were significantly lower from one through 5 years (p=0.05). cai due to interstitial fibrosis/ tubular atrophy was significantly lower in slr group at 5 years. our data shows that 5 year patient and graft survival, graft function, bpar and scar were comparable between mmf and slr groups despite lower prevelance of interstitial fibrosis/tubular atrophy in slr group. registry analyses suggest that tac/mpa immunosuppression is associated with superior kidney graft survival vs. tac/srl. large single-center experience may assist in clarifying these findings, by examining outcomes related to specific utilization practice. we retrospectively examined the outcomes of 529 consecutive first renal transplants (55% deceased donor, 45% living donor) at a single center, treated with tac/srl or tac/mpa. graft and patient survival, acute rejection rates, and 1yr egfr were analyzed by era of transplant (2000-2002 vs. 2003-2006) . changes in tac/srl utilization between eras included elimination of the srl loading dose and a reduction in tac target trough concentrations. summary. compared to tac, srl+cya was associated with a 55% decreased risk of skin ca that was statistically significant. although srl+cya was associated with a 32% decreased risk of de novo solid ca, it did not reach statistical significance. this reduced risk may not reflect the unique aspects of srl itself, but may be a reflection of practice pattern, patient selection or center effect. the backgrounds: a number of studies have observed increase of malignancies following renal transplantation. however, the incidence and the site of malignancies were quite different by the follow-up times, the era and the region. methods: we reviewed the records of 694 renal transplant recipients in our institute between 1970 and 2007 and recorded the incidence and types of de novo malignancies. they were divided into two groups by immunosuppressive era; azathioprine (aza) era (1970.4-1982.3: n=172) and calcineurin inhibitor (cni) era (1982.4-: n=522) . results: a total of 57 (27 in aza era and 30 in cni era) kidney recipients out of 694 developed 60 malignancies. the tumors included 10 gi-tract cancers, 9 liver cancers, 12 skin cancers, 4 tongue cancers, 6 breast cancers, 6 renal cell carcinomas, 2 thyroid cancers, 5 leukemia, 3 lymphoma, one lung cancer, one uterus cancer and one kaposi's sarcoma (ks). the average interval between transplantation and development of malignancy was 134±86 (8-340) months. mortality was high in liver cancer (89%) and leukemia (100%). cumulative incidence of malignancies of all 694 recipients in 5, 10, 20, 30 years were 2.3%, 4.5%, 10.7% and 14.3%, respectively. graft-loss censored cumulative incidence, which was calculated to see the incidence among graft survivors under continuing immunosuppression, of all recipients in 5, 10, 20, 30 years were 2.8%,6.3%, 16.3% and 26.2%. that of 5 ,10 and 20 years in cni era was 3.0%, 6.8% and 13.9%, while that in aza era was 1.8%, 4.9% and 19.5%, showing early higher incidence in cni era outstripped by aza era by 12 years. site of malignancy in cni era occurring within 3 years, which was never observed in aza era, was focused on liver, leukemia (including atl), ks and ptld. discussions:our results demonstrated that recent potent immunosuppressive regimen shortened the interval between transplantation and viral-related malignancies. however, long-term incidence of whole malignancies has been decreasing by minimizing chronic immunosuppression in our institute. the impact of transplant center practice on the association between pulsatile perfusion and delayed graft function. jagbir gill, 1 david gjertson, 1 suphamai bunnapradist, 1 michael cecka. 1 1 ucla, la, ca. the use of pulsatile perfusion (pp) is increasing in the us, but practice varies widely among transplant (tx) centers. we describe the variability of pp use and its impact on post transplant outcomes. methods: we identified all cadaveric kidney tx from 2000-2005 using optn/unos data. the cohort was stratified by tx center pp use as follows: low pp centers (0-10% pp use), med pp centers (10-30% pp use), and high pp centers (>30% pp use). donor characteristics and the incidence of dgf were compared between and within each strata. results: pp was used by 97 % of centers, however most centers used pp <10% of the time (70.7%). compared to low and med pp use centers, kidneys pumped in high pp centers were from donors that were younger, had a lower mean terminal serum creatinine, and had a lower incidence of cva and hypertension. the overall incidence of dgf was lowest in the high pp centers (13.3%), compared to the med (24.7%) and low pp (24.8%) centers. the rates of dgf within each strata (high, med, and low pp centers) for tx performed using pp versus cold storage (cs) are outlined in the table below . within each strata, the rate of dgf did not differ between tx performed with and without pp. in ecd transplants, pp was associated with lower rates of dgf across all center groups. however, the impact of pp on scd and dcd transplants was less significant and varied across centers by pp use. hla sensitized patients (20-100%) in our dsa are now transplanted at a rate of 40%, which is significantly higher (p = 0.02) than the 23% rate when ua weren't entered into unet. furthermore, of 9 kidneys imported into our dsa and allocated to the dsawide renal candidate list (since ua entry started), 63% (5/9) were transplanted into sensitized candidates (85% to 27%), each of whom had a negative flow or ahg t cell igg crossmatch. conclusion. the higher transplantation rate for hla sensitized patients in our dsa shows that virtual a, b, & c crossmatching yields a dsa-wide ranked list of sensitized candidates likely to have a negative final class i (t cell) crossmatch and be transplanted. the data also lend support to the notion that sharing kidneys across dsa boundaries for hla sensitized candidates, based on a negative virtual hla class i crossmatch, has merit. predicting introduction: predicting graft outcome after renal transplantation based on donor histological features has remained elusive and is subject to institutional variability. we propose a pre-transplant donor path scoring system that reliably predicts graft outcome regardless of recipient ® characteristics. methods: we retrospective analyzed 286 imported cadaveric renal transplants which were initially rejected by other centers due to donor parameters between 1/00-6/05. all kidneys were re-biopsied at our center prior to implantation. morphometric analysis performed consisted of measuring glomerular-size arterioles, interlobular, and arcuate/ interlobar arteries and wall to lumen ratio (wlr) was calculated; calculating % glomerulosclerosis (gs); the presence of arteriolar hyalinosis (ah), scar, periglomerular fibrosis (pfg), and acute tubular necrosis in the biopsies. the patients were followed for a mean of 33 months. multivariate cox analysis was done to evaluate the predictive value of these path variables to graft outcome.results: ah, gs>15%, wlr>0.5, pgf, and scar were found to independently predict graft outcome. the unos board of directors has approved the change from using panel reactive antibodies (pra) to calculated pra (cpra). the cpra is a formulated pra based upon the frequency of the specificities of hla antigens found in the donor pool and is expected to standardize the degree of patient sensitization. donor organs expressing unacceptable antigens will not be offered to a recipient with donor (hla) antigen specific antibodies (dsa). highly sensitive, single antigen bead and solid phase assays (flow pra and luminex) are used to identify these hla antibodies (abs). each transplant center can determine the criteria used for identifying an unacceptable antigen, for example, based upon dsa titer or the fluorescence intensity (fi) coming from the donor-specific single antigen bead. it is unclear whether abs identified by these techniques are clinically relevant for organ allocation. we retrospectively evaluated flow-pra, flow cytometry crossmatching (fcxm), hla ab specificities and titers of 300 pre-transplant (tx) sera from transplant recipients of deceased renal allograft donors transplanted following a negative cytotoxic-anti-human globulin crossmatch. the two year graft survival of 91% for the recipients (44/54, 81%) with low-titer (≤ 1:16) donor specific hla ab and a negative (-) fcxm was significantly better when compared to the 60% two year graft survival of the 19% (10/54) of recipients presenting with (+) dsa but a (+) fcxm (p< 0.001). recipients (55/110, 50%) with non-donor abstracts specific hla abs (high or low titer) and a (-) fcxm also experienced a better two year graft survival of 89% compared to the 74% for the other 50% of recipients with (+) fcxms (p < 0.001). these data suggest that in the presence of donor-specific or non-donor-specific hla abs you can not predict the crossmatch outcome without actually performing the crossmatch which will then influence donor organ allocation and graft survival outcome. in the face of low-titer dsa and a (-) fcxm recipients experienced excellent graft outcome when compared to recipients with (+) fcxms. therefore, donor organ allocation based on cpra (ab specificity and unacceptable ags) utilizing highly sensitive single antigen bead and solid phase luminex assays may disadvantage recipients (no donor crossmatch) who could otherwise be successfully transplanted. aim: to assess vegfr2 expression in hcc and the adjacent benign cirrhotic parenchyma and its correlation with tumor differentiation, vascular invasion, and tumor morphologic parameters. background: hcc is a highly vascular tumor in which angiogenesis is mediated in part by vegf. vegf is highly expressed in hcc and mediates its effects through multiple receptors including vegfr2. the tyrosine kinase inhibitor sorafenib inactivates the vegfr2 receptor and exhibits anti-tumor effects in hcc. clinical significance of vegfr2 expression with respect to tumor parameters has not been evaluated. patients and methods: immunohistochemical staining for vegfr2 was performed in hcc and corresponding adjacent cirrhotic liver from 79 patients undergoing liver transplant. 57 patients had hcc within mc. stains were scored by estimating the % of positive surface area in veins, arteries, and sinusoidal lining cells. data are presented as median [p25, p75]; wilcoxon signed rank and wilcoxon rank sum tests were used. results: vegfr2 levels in hcc were significantly correlated to levels in adjacent non-tumorous liver. higher levels of vegfr2 in non-tumorous liver were associated with higher levels in hcc from the same patient. vegfr2 levels were significantly higher in hcc compared to adjacent areas (p<0.05). vegfr2 levels in hcc were not significantly different between patients who fell within mc and those beyond mc. however, vegfr2 levels were significantly higher in the adjacent arteries of nontumorous liver (15.8 [0, 60] vs. 0 [0, 35]; p=0.03) in those patients with hcc beyond mc. subjects with moderate or poor differentiation had significantly higher levels of vegfr2 in sinusoids and veins of hcc and in the sinusoids of adjacent non-tumorous liver. there was no correlation with vascular invasion. conclusions: elevated vegfr2 in hcc correlates with elevated vegfr2 in adjacent cirrhosis, suggesting that high expressing hcc arise in a high vegfr2 expression, pro-angiogenic environment. moreover, higher vegfr2 expression in background cirrhosis correlates with advanced hcc (beyond mc), suggesting that anti-angiogenic agents may prevent tumor formation or progression in cirrhotic patients. this novel concept warrants further study. . we further attempted to find a subgroup of patients combining tumor size, number of nodules and various levels of afp which together would correlate strongly with pdiff tumors. however, the distribution was erratic and even in extreme outliers (>4 nodules, > 8 cm in maximum size, with or without high afp), where transplantation is currently not indicated according to any current expanded tumor inclusion criteria, the incidence of pdiff did not exceed 42%. conclusions: pdiff is most highly associated with tumors that exceed the milan criteria and the expanded criteria currently used for liver transplantation. thus, tumor biopsy would not appear to be of benefit except perhaps in those patients with a high afp level. however, if tumor criteria are further extended in the future, tumor biopsy may be justified in those with higher tumor burdens. < 2 cm) ), 2,511 (85.7%) at ls=t2 (1 tumor < 5cm or 3 tumors ≤ 2 cm), and 111 (3.8%) with ls>2. results: overall survival at 36 months was 76.5%, with significant differences seen for listing stage (ls 1= 82.2%, ls 2=75.9% ls>2=73.5%, p=0.0395) and by histologic stage as determined by pathology (p<.0001). survival for patients with histologic stage 4b hcc was only 29% at 36 months, versus 83% for stage 1. patients with tumors greater than 3cm both by listing stage and by pathology fared poorly compared to those with smaller tumors (p=0. 0.002). patients listed who received ablation treatment (at) preoperatively and who had their tumors "down-staged" had better results (p= 0.0061). we observed no difference in at types (tace vs rfa). those with micro or macrovascular invasion had lower survival rates, at 66.8% and 30.5%, respectively (p <.0001). afp >500 continues to be a significant predictor of lower post-transplant survival, with a survival rate of 57.9% at 36-months (p<.0001). conclusions: we conclude that listing and histologic tumor size, presence of micro/ macrovascular invasion, and high afp are associated with poorer lt results. at is emerging as potentially effective treatment for improving lt outcomes for hcc recipients. introduction: so far, milan criteria are used to select patients with hepatocellular carcinoma (hcc) for liver transplantation (lt). herein we compare prognostic markers in patients with pretreatment by transarterial chemoembolization (tace) to a second cohort transplanted without tace pretreatment. patients and methods: between september 1997 and october 2007, 134 patients with hcc underwent lt at our institution. eighty-two patients were pretreated by repeatedly performed tace whereas in 52 patients none or other forms of pretreatment had been used (non-tace group). tace was performed using lipiodol and mitomycin. every 6 weeks tace was repeated until transplantation. tumor response was assessed by ct scans (6-week intervals). results: sixty-seven percent of the patients transplanted after tace pretreatment exceeded the milan criteria compared to 37 % in non-tace patients. the proportion of recurrence-free patients was comparable in the tace and non-tace group (77.32 % and 78.45 %, respectively). in the univariate analysis grading, angioinvasion and progress-free tace were significant predictors for recurrence after lt in patients with tace pretreatment. progress-free tace was the only significant predictor of recurrence in the multivariate analysis. in the non-tace group t classification, number of nodules, grading, angioinvasion, milan criteria and underlying disease (hcv versus all other diseases) were significant. after tace pretreatment freedom from recurrence was 92.3 % in patients with stable disease or regress but only 38.4 % in patients with progress during tace. conclusions: tace pretreatment is capable of selecting a biological entity of tumors which differs significantly from untreated tumors. this statement is deduced from the remarkable differences of predictors for tumor recurrence in patients with and without tace pretreatment. moreover, tace patients can be separated into two groups: those who experienced tumor progress during repeatedly performed tace and those who did not. stable disease during the continued tace before transplantation resulted in remarkably low tumor recurrence. liver with a median follow-up of 30 months, there was no statistical difference in the 5 year overall survival in the m (76%) and m+ (69%) groups (p=0.40). the 5-year disease free survival was significantly higher in the m (80%) vs. m+ (61%) groups (p=0.02). when stratifying for ucsf criteria tumors, the 5-year disease free survival was milan (80%) vs. ucsf (72%) vs. beyond ucsf (54%) groups (p=0.01). univariate analysis demonstrated the following factors to be associated with disease recurrence: intermediate waiting time 2-4 months (p=0.01), preoperative tace (p=0.002) or resection (p=0.04), more than 3 tumors (p=0.03), and max tumor size over 5cm (p=0.03). multivariate analysis controlling for age, gender, and waiting time demonstrated that preoperative resection hr2.78 (95%ci 1.1-7.2), more than 3 tumors hr2.3 (95%ci 1.1-4.7) and max tumor size greater than 5cm hr2.68 (95%ci 1.2-5.9) were independently associated with disease recurrence. conclusions: the overall survival after liver transplantation is excellent in the m and m+ groups, far exceeding survival rates that can be obtained via any other modality. the current unos hcc algorithm should be reconsidered, to allow extra listing points for selected patients with hcc that exceed both the milan and ucsf criteria. postoperative use of intense insulin therapy in liver transplant recipients. lama m. hsaiky, 1 iman e. bajjoka, 1 dhaval patel, 1 marwan s. abouljoud. 1 1 transplant institute, henry ford hospital, detroit, mi. hyperglycemia and insulin resistance are common post liver transplant, even in patients with no history of diabetes. in the general surgical intensive care unit (sicu) population, the use of intensive control of blood glucose has recently been shown to reduce both morbidity and mortality. thus far, limited data exist in the liver transplant population. purpose: assess the impact of intensive insulin therapy to maintain blood glucose at or below 110 mg/dl immediately post liver transplantation in the surgical intensive care unit (sicu). methods: a retrospective evaluation of liver transplant recipients who received two different insulin protocols in the sicu was performed. prior to january 2003, patients were assigned to receive sliding scale insulin therapy (ssit) to maintain blood glucose (bg) less than 180 mg/dl. the intensive insulin therapy (iit) was implemented in august 2005 with a goal bg level between 80-110mg/dl. the following data was analyzed: bg ranges, need for mechanical ventilation, blood transfusions, infection rate in the sicu, rejection episodes and patient survival. results: a total of 97 liver transplant patients were evaluated; of which 47 patients were in the ssit group and the other 50 in the iit group. demographic characteristics were comparable between the two groups. in the iit, 24% of the bg readings were maintained at < 110 mg/dl versus 5% in the ssit. the incidence of hypoglycemia (bg <60mg/dl) was less than 1% in both groups. the need for mechanical ventilation was 3.0 days vs. 2.3 days and the overall number of blood transfusion was on an average of 5.0 vs. 2 units (p<0.05) in the ssit vs. iit group, respectively. iit also reduced overall sicu infection rate by 15% (p=0.01). the rate of acute cellular rejection at 3 months post transplant was less in the iit, 14% vs. 25% in the ssit group (p=0.02). moreover, mortality during hospital stay was reduced from 5% in the ssit to 2% in the iit group. conclusions: the use of intense insulin therapy immediately post liver transplantation has resulted in reducing infection rate and rejection episodes and a trend for reduced morbidity and mortality among post surgical liver transplant recipients without the adverse effects of hypoglycemia. medical epidemiology of patients surviving ten years after liver transplantation. kerri a. simo, 1 stephanie e. sereika, 1 david a. gerber. 1 1 abdominal transplantation division, department of general surgery, university of north carolina, chapel hill, nc. background: as the population of long term survivors of liver transplantation (olt) grows, their medical epidemiology has become increasingly important. the goals of this study were to define a collective profile of liver transplant recipients ≥ 10 years post olt and to compare their co-morbidities with those of the general population. in 2005, the national health survey reported that 22% of the us population had hypertension, 14.4% had diabetes/impaired fasting glucose, and 16.8% had chronic kidney disease. methods: a retrospective review of a prospectively collected database of 125 adult patients who underwent olt at a single transplant center from september 30, 1991 to october 1, 1997 was performed. inclusion criteria consisted of survival ≥10 years post olt with >1 year follow up. results: seventy-one patients met inclusion criteria. ninety percent of patients had ≥10 years follow up. the mean age at transplant was 43 (range 18-67). the mean calculated meld score was 22 (range 9-62, median=22). indications for olt were hcv(32%), alcohol(28%), cryptogenic(18%), autoimmune(10%), hbv(7%), psc(7%), pbc(7%), and other(10%). seven patients required retransplant during the first 10 years. an additional 38 patients underwent other operations: 10 arterial or biliary revisions, 13 hernia repairs and 19 non-transplant related (7 abdominal). during analysis, the following medical co-morbidities were found: 51 patients(72%) had hypertension (41 new onset), 20(28%) diabetes (10 new onset), 33(46%) renal insufficiency and 8 renal failure (28 new onset), 10(14%) cardiovascular disease (all new onset). nine patients (13%) were diagnosed with de novo cancer. medications for chronic health problems included 18 patients on diabetic medications, 46 on antihypertensives and 18 on lipid lowering agents. initial immunosuppression consisted of 100% on steroids, 61% on cyclosporine, 40% on tacrolimus, 24% on mycophenolate mofetil (mmf) and 23% on azathioprine. immunosuppression at 10 years consisted of 42% on cyclosporine, 41% on tacrolimus, 18% on steroids, 13% on mmf, 8% on sirolimus, 1% on mycophenolate sodium, 1% on azathioprine. no patients were on triple therapy, 29 were on dual therapy, and 42 were on monotherapy. summary: patients alive 10 years post olt have a significantly higher incidence of hypertension, diabetes, and renal disease than the general population. this study supports conscientious medical follow up to ensure continued meaningful survival. liver transplantation in the morbidly obese (bmi>40). c. quintini, 1 l. kauzman, 1 k. hashimoto, 1 p. ding, 1 t. doago uso', 1 n. sopko, 1 j. rosenblum, 1 f. aucejo, 1 c. winans, 1 d. kelly, 1 b. eghtesad, 1 d. vogt, 1 j. j. fung, 1 c. miller. 1 1 general surgery -liver transplant service, cleveland clinic oh. morbid obesity (mo) is a problem seen with increasing frequency among candidates for olt. mo is considered a contraindication for olt in some centers without clear evidence to support such a practice. our aim is to describe outcomes for olt in patients with a bmi>40kg/m 2 in a single center. methods. between 1/2004 and 4/2007, 364 olts were performed in 347 patients. 20/347 patients with a bmi>40 were compared to all other olt patients with a bmi< 40. we analyzed patient and graft survival, operative time, blood transfusion requirements, and post operative events (icu and overall length of stay los, surgical complications and infections). we also analyzed the post transplant weight records of our study group at 1-3 and 6 months. results. results are summarized in table 1. outcomes of olt in the morbidly obese are no worse than those of other patients undergoing olt. bmi is often artificially elevated in end-stage cirrhotic due to severe fluid retention. these patients exhibit rapid weight loss following transplantation that is likely due to extra-cellular fluid loss from the improved homeostatic milieu provided by the new liver and possible improvement in renal function. the presence of obstructive cad was not associated with increased peri-operative morbidity or mortality. these patients experienced similar patient and graft survival irrespective of the degree of cad. significant unmodified cad should not represent an absolute contraindication to liver transplantation. olt was also examined. we found that using a trj of 3.0 m/s as a cut-off created two age-matched groups with significantly different survival curves at one year (p = 0.006) with a relative risk of mortality of 3.98 for trj ≥3.0 m/s. this study underscores the importance of screening tte for the presence of pph in the evaluation of patients for olt, suggesting that clinically significant pph may be present at lower trj than typically prompt right heart catheterization. some of the limitations of the study are the variability of the time from pre-olt echo to transplant, and the assumption of rap = 5 without assessment of ivc size. we are using these data as a framework for further prospective trials to better assess the clinical applications of the findings. hyperlipidemia has been shown to predict faster chronic kidney disease (ckd) progression over the long term in lung allograft recipients. it is unknown whether disordered lipid metabolism may also aggravate the early loss of renal function often seen in this patient population in the immediate post-operative period. we studied 230 lung allograft recipients transplanted between january 1997 and december 2003. pertinent demographic and clinical variables were recorded at baseline and one month post-transplant, including creatinine levels and fasting lipid panels. logistic regression models were created to investigate an independent association between lipid levels and change in renal function by one month post-transplant. mean +/-sd baseline creatinine was 0.8 +/-0.2 mg/dl and low density lipoprotein (ldl) was 110 +/-35 mg/dl, the latter remaining unchanged at one month. in contrast, by one month post-transplant the mean creatinine level of survivors increased significantly to 1.2 +/-0.6 mg/dl (p < 0.001), with a overall mean increase of 52% above baseline. the highest quartile of patients that fared the worst experienced a rise in creatinine > 72% above baseline. on univariate analysis, there was a strong trend toward those with one month ldl values in the highest quartile (i.e., >140 mg/dl) having an increased odds of experiencing a rise in creatinine by one month > 72% above baseline (or 1.8, p=0.09). after controlling for age, gender, pre-transplant creatinine, bmi, and the presence of diabetes prior to transplant an ldl value in the highest quartile by one month post-transplant was the only variable independently predictive of a rise in creatinine > 72% above baseline at one month (or 2.5, p=0.05). in summary, hyperlipidemia occurring early post-lung transplant predicts faster loss of renal function soon after surgery. though speculative, given the known beneficial effects of lipid lowering agents such as statins on the rate of ckd progression, perhaps timely initiation of these medications after surgery may also attenuate the early decline in renal function often observed in this patient population. the risk for acute cellular rejection (acr) following lung transplantation (ltx) is still a problem despite heavy multidrug immunosuppression. induction therapy with potent t-cell depleting agents have facilitated the implementation of minimal post-transplant immunosuppression.the impact of this protocol on the activation of proinflammatory cytokines and effector molecules that affect the cellular rejection process is not well determined. in this study, we evaluated the relationship between upregulation of t-cell and macrophage-dependent inflammatory cytokines detected by molecular methods and the clinical status of ltx patients. we studied 28 ltx patients who received anti-lymphocytic induction therapy(thymoglobulin n=8 or campath-1h n=20) followed by maintenance immunosuppression with tacrolimus and low-dose steroids. we analyzed 137 bronchoalveolar lavage (bal) mrna samples (3-8 per ltx patient) by real-time pcr for gzmb, ifn-γ, il-15, mcp-1, rantes, tnf-α, and gapdh (control). the abi prism 7700 sds and 7500 fast real-time pcr systems were used and data were analyzed by the dct and 2-ddct methods. we determined the relationship of categorical outcomes (rejection-no rejection) with continuous variables (number of cycles) by anova. early acr that occurred within the first 60 days post-ltx was associated with an increase (>3-6 fold) of macrophage-specific mrna (mcp-1, rantes, tnf-α). 7/8 thymo-treated ltx patients experienced early acr while only 5/20 campath-1h-treated patients had early acr (p<0.005). in contrast, late acr that occurred greater than 60 days post-ltx was associated with a significant upregulation (4-64 fold) of t-cell dependent mrna (gzmb and ifn-γ) in addition to increases of rantes and mcp-1 mrna. overall, ltx patients who experienced early or late acr (n=20) exhibited higher mrna levels for the above mediators compared to stable patients (n=8). our data indicated that in early post-t-cell depletion, the acr phenotype was characterized mainly by macrophage activation. in contrast, greater than 3 months post-ltx with the recovery of cd8+ t-cells, the acr phenotype was associated with cytotoxic t-cell activation. furthermore, sensitive molecular methods may detect the activation of pro-inflammatory mediators within the allograft prior to the diagnosis of rejection by transbronchial biopsies and may impact optimal patient management. antibody-mediated rejection (amr) is defined by the presence of donor-specific alloantibodies, markers of complement activation and the clinical phenotype of organ dysfunction. compared to renal and cardiac allografts, very few amr cases are documented in lung transplantation (ltx). methods. we report here on seventeen ltx patients exhibiting: 1) donor-specific hla antibodies (dsa); 2) linear, continuous subendothelial c4d deposition in lung allograft; 3) lung allograft dysfunction. the presence and specificity of dsa were determined by elisa and/or luminex. c4d deposition was assessed by immunohistochemistry in transbronchial biopsy paraffin blocks. allograft dysfunction was considered when either biopsy-proven acute cellular rejection (acr, ≥ ishlt a2) or bronchiolitis obliterans (bos) were diagnosed. the average detection of dsa occurred in the first year post-ltx, on 283±218 postoperative day (pod), range 14 to 734 days. thirteen (76%) of amr patients were females. an anamnestic humoral response was encountered in 4 cases (one pregnancyrelated and three re-transplant patients), while de novo dsa were detected in 14 cases (82%). the percent-reactive antibody (pra) was lower in de novo cases (24%) when compared to memory response (71%, p<0.05). anti-class i dsa were found in 7 cases, anti-class ii in 5 cases, while 5 patients exhibited both class i and class ii dsa. specific vascular c4d deposition was detected in 16 (94%) patients. lung allograft dysfunction was considered in 14 (82%) cases, while three patients with dsa and specific c4d deposition fulfilled the criteria of sub-clinical amr. in patients where plasma-exchange/ ivig were applied, antibody titers dropped from 1:32 to 1:4 or 1:2 in two cases; in a third case, antibody titer remained high post-pheresis (1:512) and the graft failed. conclusions: both anti-class i and anti-class ii, low pra or high pra, pre-formed or de novo dsa can be detrimental for lung allograft. the presence of donor-specific alloantibodies, vascular c4d deposition and allograft dysfunction shows that amr criteria can also be met in ltx. rationale: recently, inhaled cyclosporine has been shown to reduce mortality and bos. previously, we demonstrated that apically-dosed cyclosporine poorly transmigrated differentiated human airway epithelial cells in vitro (using a transwell system). only ∼5% of the apically deposited cyclosporine passed through the epithelial layer whereas most of the drug accumulated within the epithelium. thus inhaled cyclosporine may not be capable of inactivating airway allogeneic t cells since it may not reach the airway wall in high concentration. in this study, we hypothesized that inhaled cyclosporine alters airway epithelial signaling cascades that may ultimately result in reduced inflammatory cell recruitment to the lung. methods: human tracheobronchial epithelial (htbe) cells from healthy donors were grown in ali media to confluence at an air-liquid interface in millicells. differentiated htbes were treated on their apical surface with cyclosporine concentrations of 1000 and 10,000 ng/ml or vehicle for 24hr to mimic the effects of inhaled cycolsporine. the basilar and apical compartments (n=8-12 for each) were then assayed for cytokine secretion (il8, il6, il1, il-12p40, tnf, eotaxin, mcp-1, gm-csf, rantes and egf) by luminex assays in pg/ml. results: 10,000ng /ml of cyclosporine markedly blunted the basilar secretion of il-6 (250 ±142 vs 71± 24, p= 0.002), rantes (22±8 vs 9±3, p=0.003), gm-csf (21±16 vs 3±3, p=0.008) and mcp-1 (181±112 vs 28±19, p=0.002). il8 and eotaxin were unaffected; tnfα, il1β and il12p40 were undetectable. at 1000ng/ml cyclosporine, cytokine secretion was decreased to a lesser extent. a similar pattern of diminished cytokine secretion was seen in the apical compartment of this system. last, apical, but not basal, egf secretion was augmented by cyclosporine (2231±817 vs 1114±614 pg/ml, p=0.02). conclusion: cyclosporine decreased the secretion of critical cytokines and chemokines from human airway epithelial cells. these mediators are known to enhance mononuclear and t cell recruitment in a large variety of animal models of disease as well as in clinical studies. inhaled cyclosporine may work by reducing cell recruitment. this hypothesis will require in vivo testing. funded by: cf foundation. background: cytomegalovirus (cmv), human herpes virus -6 and -7 (hhv-6 and -7) are β-herpesviruses that commonly reactivate and have been proposed to trigger acute rejection and chronic allograft injury. the role of these viruses in the development of bos after lung transplantation remains unclear. we assessed the contribution of β-herpesvirus infection in the allograft by a prospective molecular assessment of serial broncho-alveolar lavage (bal) samples in lung transplant recipients. methods: quantitative real-time pcr of bal samples were performed for cmv, hhv-6 and hhv-7 in a prospective cohort of lung transplant recipients. a time-dependent cox regression analysis was used to correlate the risk of bos and acute rejection in patients with and without β-herpesviruses infection. results: 93 patients were included in the study over a period of 3 years. a total of 581 samples from bal were obtained (median 6 per patient). 61/93 patients (66%) had at least one positive result for one of the β-herpesviruses: 48 patients (52%) for cmv, 19 patients (20%) for hhv-6, and 19 patients (20%) for hhv-7. median time to detection was 152 days (range 8-839) for cmv, 309 days (range 28-928) for hhv-6, and 286 days (range 17-928) for hhv-7. median peak viral load was 3,419 copies/ml (range 102-41,600,000) for cmv, 258 copies/ml (range 106-131,075) for hhv-6, and 665 copies/ml (range 103-66,100) for hhv-7. acute rejection (≥ grade 2) occurred in 46% and bos (≥ stage 2) in 19%. in the time dependent cox regression model, the relative risk of bos or acute rejection was not increased in patients with cmv, hhv-6, or hhv-7 reactivation. for example, the hazard ratio of cmv and bos was 1.04 (95% ci 0.62-1.73, p=0.9) and for cmv and acute rejection was 0.81 (95% ci 0.55-1.20, p=0.3). in many of the patients, β-herpesvirus reactivation occurred after the acute rejection episode likely reflecting augmented immunosuppression. abstract# 317 mannose binding lectin in this large cohort of lung transplant recipients, local reactivation of cmv, hhv-6 and hhv-7 in the allograft was very common. however, despite high viral loads in many patients, infection was not significantly associated with the development of acute rejection or bos. introduction: the role of chemokine receptors in regulating donor-specific responses to allografts is poorly understood. cd4 + cd25 + t cells regulate alloreactive cd4 t cell responses and acute rejection of single class ii mhc-disparate cardiac allografts in c57bl/6 mice. ccr5 is expressed by a small proportion of cd4 + cd25 + t cells but the requirement for these cells in regulating alloreactive t cell responses remains poorly understood. the goal of this study was to investigate the role of ccr5 + t regulatory cells in acute rejection of single class ii mhc-disparate cardiac allografts. methods: wild-type c57bl/6 (h-2 b ) and b6.ccr5 -/received heterotopically transplanted b6.h-2 bm12 mice heart grafts. the presence of cd4 + foxp3 + t cells in the recipient spleen and in heart allografts was determined by flow cytometry. foxp3 mrna expression in the heart grafts was analyzed by qrt-pcr. donor-specific cd4 + t cells producing ifn-g or il-4 in allograft recipient spleens were enumerated by elispot assay. cell sorted naïve wild-type cd4 + cd25 + t cells and cd4 + cd25 -t cells were adoptively transferred to wild-type and ccr5 -/mice before the cardiac transplant. results: in wild-type recipients >80% b6.h-2 bm12 cardiac grafts survived more than 100 days whereas ccr5 -/recipients rejected the allografts within 24 days (18 days mean survival) with intense cd4 + t cell infiltration in the graft. donor-reactive ifn-g and il-4 producing cd4 + t cell numbers were increased 2-fold in the spleens of ccr5 -/vs. wild-type recipients at day 7 post-transplant and in contrast to wild-type recipients these numbers were sustained for at least 7 more days. allograft infiltrating cd4 + cd25 + foxp3 + cells and intra-graft foxp3 mrna expression were clearly present in allografts from wild-type recipients and were virtually absent in allografts from ccr5 -/recipients. transfer of purified wild-type cd4 + cd25 + t cells to ccr5-deficient mice resulted in the long-term survival of 60% of b6.h-2 bm12 cardiac allografts. conclusion: ccr5 + regulatory t cells control the magnitude and function of the alloreactive t cell immune response to single class ii mhc-disparate cardiac allografts. profile that were distinct from those of cd4+cd25+ tregs, naïve cd4+cd25-t-cells, and activated cd4+ t-cells. furthermore, the cd4+ converted dn t-cells were highly potent in suppressing antigen specific alloimmune responses in vitro. in this study, we further characterized and test the functional potential of the converted dn t-cells in vivo. we showed that the converted dn t-cells retained a stable phenotype after re-stimulation in vitro and in vivo. il-2 was capable of breaking the anergic status and reserving the suppressive function of dn t-cells. in an immunocompetent mhc completely mismatched islet transplant model, the transfer of 13 x 10 6 dn t-cells (converted from cd4+cd25-t-cells of naïve c57bl/6 mice by co-culture with mature dba/2 dc plus ril-15 in mlr for 6 days) resulted in a statistically significant prolongation of alloantigen specific dba/2 strain, but not third party c3h strain, islet allograft survival in c57bl/6 recipients in comparison with that of untreated control group. as il-2 was capable of breaking the anergic status and reserving the suppressive function of dn t-cells, we added il-2/fc, a long-lasting form of il-2, and low dose rapamycin with dn t-cells in a mhc mismatched skin allograft model. the single transfer of 5 x 10 6 dn t-cells plus 28 days il-2/fc and low dose rapamycin treatment significantly prolong dba/2 skin allograft survival in c57bl/6 recipients in comparison with untreated group (mst 71 days vs. 11 days, p=0.0049) and il-2/fc plus rapamycin treated group mst (71 days vs. 34 days, p=0.0091). the results of using ex vivo cd4+ t-cells converted dn t-cells in skin and islet transplantation models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention of allograft rejection. jessamyn bagley, 1 jonathan g. godwin, 1 joren madsen, 2 john iacomini. 1 introduction: it has been suggested that natural killer (nk) cells are critical mediators that connect the innate and adaptive immune response. cytokine production by nk cells contributes to the polarization of immune responses to t helper 1, and nk cells express co-stimulatory molecules that may affect t cell proliferation. recent work has shown that nk cells are involved in the chronic rejection of parental cardiac grafts by f1 recipients. we hypothesized that given the role of nk cells in t cell activation and proliferation, nk cells may play a role in the development and function of t regulatory cells (treg) which control alloreactive responses. methods: nk, cd4+ t cells and cd4+cd25+ treg were purified from the spleens of c57bl/6j mice using macs bead separation followed by fluorescence activated cell sorting. naïve cd4+cd25-t cells from c57bl/6j mice (h-2b) were placed in culture with tgf-beta in the presence or absence of nk cells, and the development of treg was monitored by assessing foxp3 expression by intracellular cytokine staining and flow cytometry. in addition, the effect of nk cells on the function of treg was measured by elispot assay. results: following 4 days of culture with tgf-β and anti-cd3 antibody, cd4+cd25-t cells acquire foxp3 expression. the addition of activated nk cells to cultures with cd4+cd25-t cells and tgf-β prevented the acquisition of foxp3 expression by cd4 cells. tregs induced by stimulation of cd4+cd25-t cells with anti-cd3 in the presence of tgf-β are capable of suppressing the production of il-2, ifn-γ and il-4 by cd4 t cells in response to fully allogeneic balb/c stimulators. however, in the presence of activated nk cells, induced tregs fail to function, and production of il-2, ifn-γ and il-4 by cd4 t cells is restored. these experiments were repeated with natural cd4+cd25+ tregs isolated directly from mice without induction, and found that the ability of natural treg to suppress a cd4 t cell response to alloantigen was impaired in the presence of activated nk cells. conclusions: the presence of activated nk cells in culture can prevent the development of induced treg. furthermore, the presence of activated nk cells interferes with the function of mature treg in culture and allows a productive cytokine response by cd4 effector cells in response to alloantigen. results: both the syngeneic b6 cells and allogeneic dba/2 cells survived nicely in the rag-/-il-2rg-/-mice. however, the allogeneic dba/2 cells, but not the syngeneic b6 cells, were readily killed by the nk cells in the rag-/-mice. however, both rag-/-and rag-/-il-2rg-/-mice accepted the dba/2 skin allograft long term without any sign of rejection (>100 days). thus, nk cells by themselves, though cytolytic to dba/2 cells, fail to reject the dba/2 skin allografts. to test the hypothesis that the activation status of nk cells may dictate their alloreactive potential, we treated the rag-/-mice with il-5/il-15ra complex to maximally stimulate the nk cells in vivo. we found that il-15 is remarkably potent in stimulating nk cells in vivo; and nk cells stimulated by il-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells, as il-15 treated rag-/-, but not the rag-/-il-2rg-/-mice, readily rejected the dba/2 skin allografts (mst=18 days). nk cell-mediated graft rejection doesn't show features of memory responses. and suggests that the fate of the allografts may depend on the activation status of nk cells and the availability of nk stimulating cytokines. background: t-bet is a transcription factor that promotes th1 development. both t-bet and the cytokines ifng and il-4 have been implicated as negative regulators of th17. in contrast, il-6 promotes th17 development. il-17 production is associated with granulocytic pathologies in several disease states. hence, this study assessed the relationship between t-bet, ifng, il-4 and il-6 in th17 induction and granulocytic infiltration of cardiac allografts. (ifng-/-) mice were transplanted with balb/c cardiac allografts. recipients were left untreated, depleted of cd4+ or cd8+ cells, treated with anti-cd40l mab, and/or treated with neutralizing anti-il-4 or anti-il-6 mab. graft histology was assessed and primed donor-reactive th responses were quantified by elispot. intragraft expression of il-17 and the th17 transcription factor rorgt were quantified by real-time rt-pcr. results: wt and t-bet-/-mice rejected their allografts at a similar tempo but with distinct pathologies: allografts in t-bet-/-recipients were heavily infiltrated with granulocytes while graft infiltrating cells in wt recipients were primarily mononuclear. while th1 and th17 responses were readily detectable in t-bet-/-recipients, th1 dominated the response in wt mice and th17 were not detectable. depletion of cd4+ cells prolonged graft survival in wt, but not in t-bet-/-recipients suggesting that cd8+ cells mediated rejection independent of cd4+ help in t-bet-/-mice. cd8+ cells were the source of il-17 in t-bet-/-recipients. anti-cd40l therapy promoted long-term allograft survival in wt recipients, but not t-bet-/-mice unless cd8+ cells were depleted. additionally, anti-cd40l therapy inhibited th1 responses in both wt and t-bet-/-recipients, but not the cd8+ th17 response in t-bet-/-mice. eliminating ifng and il-4 failed to induce il-17 production, while neutralizing il-6 reduced the th17 response in t-bet-/-mice. conclusions: while cd4+ th17 have been described in detail, cd8+ th17 have received less attention. in t-bet-/-allograft recipients, cd8+ th17 emerge independent of cd4+ help. cd8+ th17 are resistant to anti-cd40l therapy and are associated with granulocyte infiltration of the graft. these data implicate t-bet, as opposed to ifng and il-4, as a negative regulator of the th17 response while il-6 is required for cd8+ th17 induction. nan zhang, 1 bernd schroppel, 1 girdari lal, 1 jordi c. ochando, 1 jonathan s. bromberg. 1 1 background: cd4 + cd25 + foxp3 + regulatory t cells (treg) are important in suppressing immunity to prolong allograft survival. treg migration and its effects on suppressive activity are poorly understood. we determined treg migration patterns and effector function in an islet allograft model. ccr2 -/-, ccr4 -/-, ccr5 -/-, ccr7 -/-, l-selectin (cd62l) -/-, and fucosyltransferase (fuct) iv-vii -/-c57bl/6 mice were used to generate treg. treg were transferred intravenously, or locally to islet allografts, following islet transplantation (balb/c into c57bl/6). transferred treg were labeled with red dye pkh26 and migration to islet grafts, draining lns (dln), peripheral lns and spleen were tracked with fluorescence microscopy and flow cytometry. islet allograft survival was determined by measurement of blood glucose. results: treg expressed p-selectin ligand, cd62l, and a panel of chemokine receptors similar to other t cell subsets. intravenously transferred wild type treg migrated to both islet grafts and dln, and prolonged allograft survival. cd62l -/or ccr7 -/-treg, which migrated to islets but not dlns, prolonged allograft survival as potently as wild type treg. fuct iv-vii -/-treg, which lack e-selectin and p-selectin ligands, migrated to dln, but not islets, and did not prolong graft survival. similarly, ccr2 -/-, ccr4 -/-, and ccr5 -/-treg migrated to dln, but not islets, and did not prolong graft survival. when locally transferred to the islet graft, treg also migrated from the allograft to the dln, and prolonged graft survival even longer than after intravenous transfer. locally transferred ccr2 -/-, ccr5 -/-, or ccr7 -/-treg were not able to migrate from the islet to the dln, and were impaired in their ability to prolong islet survival. conclusion: treg migration to allografts is essential for their suppressive function; migration to lymphoid tissues alone is not sufficient to prolong graft survival. treg migration from the islet allografts to the dlns, via afferent lymphatics, is also required for optimal suppressive function and graft survival. the sequential migration from the site of inflammation and then to dlns is necessary for treg to execute fully their suppressive program. these results demonstrate a novel and important aspect of migration in treg suppression and tolerance. abstracts failure is unknown. methods:40 patients with iothalamate gfr <40ml/min (n=34) or on dialysis (n=6) at the time of liver transplant evaluation had undergone a percutanous ct guided renal biopsy. prior to the biopsy an inr ≤1.5 and platelet count ≥50,000/ ml were achieved in the majority of cases. all patients were monitored overnight for complications. candidates were listed for slk if pathology showed ≥ 40% glomerulosclerosis (gs) or ≥30% interstitial fibrosis (if). results:13 patients were eligible for slk and 27 for lta.creatinine was higher in slk candidates but not clinically different. background -it is well known that delayed graft function (dgf) is costly for those who received a renal transplant. however, the true cost of dgf is unknown. methods -we estimated the cost of dgf for adult cadaveric renal recipients in the usrds 1995-2004 who had medicare as their primary payer. those included were restricted to single organ recipients as well as those who had no previous transplants. cost was defined as the accumulated average cost per day for everyone with a functioning graft on that day. we examined the total cost of dgf, cost associated with dialysis, and non-dialysis cost for all patients combined and separately by donor type; standard criteria donors (scd), expanded criteria donors (ecd), and non-heart beating donors (dcd) as well as time to graft failure or no graft failure. background: based on adverse outcomes during the first year post-transplant, the optn membership and professional standards committee (mpsc) peer-review process flags transplant programs for further review using one of two methods. programs performing at least 10 transplants during at 2.5 year cohort are flagged based on the comparison of observed to expected event counts (death or graft failure) and the corresponding p-value (<0.05). programs performing 9 or fewer transplants are flagged if they have any adverse events. this leads to different flagging rates for programs of different sizes. the p-value has low sensitivity to identify poorly performing programs with a "moderate" number of transplants (10 to 20) whereas flagging every event results in a high false positive rate for "small" centers with <10 transplants. during the july 2007 review, 27% of "small" centers and 7% of centers with 10+ transplants were flagged for review (all organs). the scientific registry of transplant recipients (srtr) has developed alternative approaches for flagging centers with more consistent flagging rates. methods: the new method would allow for different choices of sensitivity (rr) and specificity (p-value) for different purposes and would flag program if either {p-value < .05 (a different value could be chosen)} or {observed / expected > rr}. the resulting false negative rate is less than 50% for centers with the given rr. this approach avoids an arbitrary definition of small versus large programs, and has sensitivity (or power) > 50% to flag programs with the selected rr, regardless of size. results: the following discussion: this approach gives a more balanced distribution of flagging across programs of different sizes and has sensitivity >50% for transplant programs of all sizes. with the choice of rr=2.5, the overall number of centers flagged would be nearly unchanged from current methods. center performance ratings are of increasing importance to the transplant community with the introduction of the cms final rule. ongoing debate exists regarding how much center ratings are directly a reflection of quality of care or whether ratings can be substantially influenced by exogenous factors. the study examined data from the national srtr database from 2000-2007. centers' semi-annual graft and patient survival were calculated along with a comparison with expected outcomes adjusted for covariates used by the srtr. centers meeting three criteria for poor performance were categorized within each cohort. patient characteristics at each center were compared with performance evaluations. overall, half of transplant centers met criteria for low performance in at least one of the semi-annual intervals. several center factors investigated were not significantly associated with the likelihood to meet low performance criteria including the proportion of older, obese and privately insured patients. in contrast, centers with higher levels of ecd transplants (most ecds=70% vs least ecds=40%), african american recipients (most aas=75% vs least aas=35%) and patients with low albumin level (lowest albumin =60% vs highest albumin =32%) were more likely to meet low performance criteria. approximately half the centers that initially met criteria for low performance no longer met criteria when excluding ecd transplants and african american recipients from the performance assessment. conclusions given the extreme implications of performance ratings for transplant centers including possible loss of funding to centers with low performance, it is critical that we recognize potential weaknesses and biases of performance ratings. our results are important towards understanding factors related to performance ratings and raising questions as to whether risk adjustment techniques are adequate for fair transplant center performance evaluations. rather than only risk adjustment, stratified evaluations may be a partial solution to remove disincentives to performing higher risk transplants. it is also important to recognize factors not associated with low performance such that centers do not unnecessarily limit access to groups based on perceived deleterious impact on ratings. model. olaf boenisch, 1 takaya muramatsu, 1 francesca d'addio, 1 robert padera, 1 hideo yagita, 2 nader najafian. 1 1 transplantation research center, brigham and woman's hospital and children's hospital, boston, ma; 2 immunology, juntendo university of medicine, tokyo, japan. t cell immunoglobulin and mucin domain (tim)-3, a molecule expressed on terminally differentiated th1 cells, is an important regulator of th1 autoimmunity and in induction of transplantation tolerance. its functions in alloimmune responses during acute and chronic rejection are unknown. tim 3-23, a novel blocking antibody of tim-3, was administered to recipients in various donor-recipient strain combinations on days 0 (500ug), 2, 4, 6, 8 and 10 (250ug) after transplantation. the frequency of ifng-, il-4-, and granzyme b-producing splenocytes was measured by elispot. these data establish the regulatory functions of tim-3 in alloimmune responses in solid organ transplantation models. the inhibitory actions may be secondary to modulation of effector or regulatory t cells and appear to dominate in conditions of low levels of t cell activation, due to a restricted degree of allogeneic mismatch or absence of cd28 costimulation. the i-r injury in transplanted kidney is a major cause of dgf, an event associated with an increased risk of acute rejection. adaptative immunity was suggested to play a role in the pathogenesis of renal i-r injury, although the influence of the th1/th2 bias in this scenario is still debated. thus, the aim of the present study was to evaluate the features of t cell response during i-r injury at the peripheral and tissue level in renal graft recipients with dgf. the mrna levels of specific th1 (t-bet) and th2 (gata-3) transcription factors were evaluated in circulating lymphomonocyte of kidney transplant recipients with early graft function (egf) (n=10) and dgf (n=10), before (t0) and 24 hours after transplantation (t24) by real time pcr. infiltrating lymphocytes were characterized in graft biopsies of patients with dgf (n=40) and in a control group of patients with tubular damage by acute cni toxicity (n=10) by immunohistochemistry. in addition, we evaluated the th1/th2 bias at the renal level in a pig model of i-r injury. t-bet/gata-3 mrna ratio was similar in the 2 groups of patients at t0. at t24 the dgf group presented a significantly higher increase of t-bet/gata-3 ratio compared with the egf group (798±346 vs 288±147% of t0, p<0.001). moving to the tissue level, dgf patients presented a number of interstitial cd4 + (8.0±5.1 vs 2.6±2.1, p=0.04) and cd8 + (10.8±6.7 vs 4±2, p=0.02) t cells significantly higher compared to the control group, while no significant differences were observed in cd20 + cells number between the two groups. also at the tissue level the ratio between t-bet + and gata-3 + cells was significantly higher in the dgf compared with the control group (3.5±1.8 vs 1.1±0.9, respectively, p=0.005). to confirm that these changes were due to i-r, we investigated the presence of t-bet + and gata-3 + cells in a pig model of i-r injury. interestingly, the ratio was significantly increased after 24 hours of reperfusion (basal 2.5± 0.9 vs 24 hours 8.1±3.6; p=0.02). in conclusion, our results suggest that kidney transplant recipients with dgf present a bias toward a th1-driven immune response both at the peripheral and at the tissue level. this event, due to the i-r process, as suggested by the animal model, may represent a link between dgf and acute graft rejection. as expected a strong negative association between duration of dialysis and patient survival was seen in caucasian recipients. in contrast the relationship between patient survival and duration of dialysis was u shaped in minorities with the worst patient survivals seen among preemptive transplants and those patients with over 60 months of dialysis. the difference in outcomes between caucasians and minorities could be related to the biologic differences in the effect of dialysis on subsequent transplantation or to a differential selection bias introduced by duration of dialysis in the two populations. anti-carbohydrate natural antibodies. lorenzo benatuil, 1 jonathan g. godwin, 1 shamik gosh, 1 john iacomini. 1 1 transplantation research center, boston, ma. background. we constructed immunoglobulin gene knock-in mice lacking expression of the αgal epitope that carry rearranged vh and vl genes encoding antibodies specific for the carbohydrate antigen galα1-3galβ1-4glcnac-r (αgal). here, we describe two novel populations of b cells in the spleen of these m86vhvlgt 0 mice and their role in the production of αgal specific antibodies. methods. m86vhvlgt 0 mice were sacrificed and single cell suspensions from spleen, bone marrow, peripheral lymph nodes and peritoneal cavity were prepared and stained with different antibodies and with fluorescently labeled αgal-bsa for flow cytometric analysis. using multiparameter cell sorting, we purified marginal (mz) and follicular (fo) zone b cells, in addition to two novel splenic b cell populations. cells were adoptively transferred into b cell deficient µmt/gt 0 double knock-out mice. serum samples were collected, and production of αgal specific igm antibodies was assessed by elisa. to investigate these b cell tolerance mechanisms, we employed mice with naturally occurring antibodies (abs) against human blood group a carbohydrates in their sera and possessing b cells with receptors for blood group a determinants. b cells with receptors for a carbohydrates in mice belong to the cd5 + b-1 subset, with phenotypic properties similar to those of human b cells. when these cells were temporarily eliminated by injecting synthetic a carbohydrates, subsequent treatment with cyclosporin a or tacrolimus, which blocks b-1 cell differentiation, completely inhibited the reappearance of b cells with receptors for a carbohydrates in mice. it is probable that calcineurin inhibitors used for preventing t cell-mediated rejection simultaneously suppress b-1 cell differentiation. however, despite a very limited dose of calcineurin inhibitors, the b cell tolerance toward blood group a antigens was persistently maintained in the blood group a-to-o liver transplant recipient. b cell tolerance after abo-incompatible transplantation might be a consequence of presentation of blood group carbohydrate antigens by cells in the engrafted liver. immune fluorescence staining of the human liver reveals that blood group antigens are predominantly expressed on the liver sinusoidal endothelial cells (lsecs). we have previously proven that lsecs, which constitutively express fas-l and pdl-1 have the capacity to tolerize alloreactive t cells. taken together, we hypothesize that blood group antigen-reactive b cells are also tolerized through the interaction with the lsecs. in order to address this possibility, we used α-1,3galactosyltransferase-deficient mice. when the α-gal-expressing lsecs isolated from wild-type mice were adoptively transferred via the portal vein into the splenectomized congeneic α-gal-deficient mice, these mice lost the ability to produce anti-α-gal abs (n = 4). this finding suggests that the lsecs expressing blood group carbohydrates play a pivotal role in the tolerization of newly developed b cells specific for the corresponding carbohydrate antigens after abo-incompatible liver transplantation. success in kidney transplantation has resulted from control of t-cell-mediated acute rejection. however, little has been done to improve the fate of patients who possess pretransplant donor-specific antibody (dsa), and no proven therapies exist to specifically prevent dsa formation post-transplant. while methods to detect and characterize dsas are clearly useful, the b-cell subsets that produce dsas or more importantly sustain their production are poorly characterized. this study set out to investigate the fundamental mechanisms determining the formation of donor-specific memory b cells and plasma cells in novel mouse models of dsa formation. we have developed two complementary and novel systems to track the phenotypic and functional properties of polyclonal donor-specific b-cells. the first system involves allosensitization between normal c57bl/6 and balb/c mice. we used advanced flow cytometric methods to track donor-specific b cells with h-2k b or h-2k d tetramers. tetramers are comprised of four identical h-2 molecules bound to a fluorescently labeled steptavidin molecule that is able to bind the donor-specific b-cell antigen receptor (surface immunoglobulin). in our second system, we use donor mice that constitutively express full-length membranebound chicken ovalbumin (mova) protein in all tissues. analogously, ova specific-b cells can be analyzed flow cytometrically with the use fluorescently-labeled ovalbumin. both systems allow us to identify donor-specific memory b cells (7aad -cd4 -cd8 -ova/ tetramer + igd -b220 + cd138 -) and plasma cells (7aad -cd4 -cd8 -ova/tetramer + igd -fb220 lo cd138 + ) during skin graft rejection (day 14 post transplant). we were also able to track the development of a stable memory cell population in the bone marrow and spleen for > 90 days cells.for the ova system quantitative elisa and elisot measurements for serum dsa and dsa-secreting cells, respectively, correlated with the development of donor-specific memory b cells. using these assays we will be evaluate polyclonal donor-specific b memory subsets under multiple conditions of immunity and tolerance and for the first time, characterize their functional properties and migration patterns. these experiments will provide new information on the basic biology of the memory b-cell response to allografts in mice and facilitate the development of these methods in sensitized patients that may lead to critical therapeutic opportunities for dsa production. in the tnf-related b-lymphocyte survival factor, blys/baff, is critical for primary follicular (fo) and marginal zone (mz) b-cell survival. in vivo neutralization of blys/ baff, using a newly developed monoclonal antibody, depletes the fo and mz b-cell compartments. here, we hypothesized that targeting b-lymphocytes, via the blys/baff pathway, could promote humoral tolerance to islet allografts. cohorts of stz-diabetic c57bl/6 mice were transplanted with isolated islets from balb/c donors. treatment with anti-blys/baff alone (100mcg/mse x2 doses+15mcg/wk/mse) did not protect islet allografts from acute rejection (mst=14d; n=4) . on the other hand, a 14-day course of rapamycin (1mg/kg) prevented acute allograft rejection (mst=35d; n=9). when rapamycin was combined with the anti-blys regimen, islet allograft survival was markedly prolonged (mst>150d; n=4). importantly, islet allograft survival was coincident with the absence of detectable serum alloantibodies and blunted donor specific t-cell responses. following treatment with anti-blys, b-lymphocyte compartment reconstitution was detectable at 30 days following treatment and was characterized by stringent selection at the transitional→fo b-cell tolerance checkpoint. overall, these data indicate that the blys/baff pathway may be a logical target of immunomotherapy for the achievement of humoral transplantation tolerance. reza tavana background: highly sensitized patients' ability to be transplanted is severely compromised because of high level of antibodies against various hla antigens. interleukin-21 is a type i cytokine that signals through a receptor composed of the il-21r and the common cytokine receptor -chain ( c ). it is produced by t-cells and has been shown to be the contributing factor in terminal differentiation of memory b-cell to anti-body producing b-cell and plasma cells. objective: we evaluated the effect of il-21 co-stimulation on antibody production capacity of b-cells and also measured the expression of il-21r on b lymphocytes of highly sensitized patients compared to non-sensitized patients on the kidney transplant list. methods: patients with a pra level >20% (sensitized) were compared to non-sensitized patients from the transplant waiting list. after consent was obtained, peripheral blood was taken from patients before initiating hemodialysis. leukocytes were labelled with anti-cd19, anti-il-21 antibodies and paraffin fixed after rbc lysis. il-21r expression was measured by flow-cytometry over facscan machine on the same day. the expression of the il-21r is significantly higher on b-lymphocytes of highly sensitized patients (hsp) compared with non sensitized patients (nsp) (fig 1.p<0.05 ). in-vitro co-stimulation of isolated peripheral b-cell with il-21 and anti-cd40 results in higher igg1 production compared with anti-cd-40 or il-21 stimulation alone (fig 2) . conclusions: il-21 is an important cytokine in b-lymphocyte stimulation and increases igg1 production. il-21 receptor on b-lymphocytes is up-regulated in sensitized kidney transplant recipients. il-21 to il-21r interaction between t and b-lymphocytes may be an important pathway in antibody production in highly sensitized renal transplant recipients. we created mixed bone marrow chimeras in irradiated µmt (b cell-deficient) recipients by transplantation of syngeneic bone marrow from µmt, wildtype (wt) and mhcko (lack mhc i & ii expression on all cells) mice. µmt+mhcko chimeras lack expression of mhc i & ii on b cells but not other professional apcs such as dcs hence antigen presentation specifically by b cells is disrupted. allograft rejection and development of alloreactive memory t cells in µmt+mhcko chimeras was compared to µmt+wt chimeras that had intact antigen presentation by both b cells and apcs. skin allograft rejection was comparable between µmt+mhcko and µmt+ wt chimeras (mst = 17 and 16 days, respectively, p = 0.6, n = 5/grp). however, heart allograft rejection was significantly delayed in the µmt+mhcko chimeras compared to µmt+ wt chimeras (mst = 23 and 15 days, respectively, p=0.03, n = 5/ grp). development of alloreactive memory t cells was assessed in chimeras at 8 weeks after skin allograft rejection by quantitation of antigen-specific ifnγ producing t cells. alloreactive cd4 and cd8 memory t cells were significantly fewer in µmt+ mhcko chimeras (7-fold fewer cd4, p = 0.02, and 5-fold fewer cd8, p = 0.003, n = 5/grp) than in µmt+ wt chimeras. these results show that the disruption of antigen presentation by b cells significantly delays heart but not skin allograft rejection. development of alloreactive cd4 and cd8 memory t cells is significantly impaired in the absence of antigen presentation by b cells. conclusions: antigen presentation by b cells accelerates heart allograft rejection and leads to development of alloreactive memory t cells. these findings emphasize antibodyindependent functions of b cells in promoting alloimmune responses and highlight the need for b-cell targeted therapies to improve long-term allograft survival. purpose: to test whether depletion of cd20+ b cells at the time of engraftment alters the prevalence of anti-donor alloantibody (ab)or severity of cav in the context of therapeutic immunosuppression with (csa) or high dose cd154 inhibition. methods: forty-five mlr-mismatched heterotopic cardiac cynomolgus allograft recipients were treated with high dose anti-cd154 monotherapy (αcd154; n=14, 5 with atg induction) or αcd154 with additional anti-cd20 therapy (rituximab 20mg/ kg q wk for 4 weeks: αcd154 + αcd20; n=18, 11 with atg). thirteen other animals received therapeutic csa (target trough >500ng/ml), five of which received additional αcd20. graft survival was censored at 90 days. ab was deemed (+) if present (by flow cytometry) around time of explant. results: 12 animals died with beating grafts, mainly with atg-associated lung pathology or infectious etiologies and are excluded from this analysis. 3 animals that had sustained αcd154 levels <100µg/ml after protocol day 30 were considered "subtherapeutic" for this reagent and also excluded from further consideration. graft survival with αcd154 + αcd20 (median >90d) and proportion of grafts surviving to 90 days (6/6) was significantly increased relative to αcd154 alone (median 43d, p=0.007; 4/14 >90d). graft survival with csa + αcd20 (median >90d) and proportion of grafts surviving to 90 days (4/4) was increased relative to csa alone (median 66d, 3/6 >90d). with therapeutic αcd154 (trough level >100 µg/ml until graft explant), 12/13 (92%) developed ab vs. 1/6 with αcd154 + αcd20 (17%) (p=0.006). average cav score for the αcd154group was 2.44 ±0.44 vs. 0.7 ± 0.8 in the αcd154 + αcd20 group (p=0.006 using unpaired t test). preliminary cav scoring suggests that added αcd20 inhibited cav (scores ranging 0.0-0.1) relative to csa alone (median 2.1; range 1.5-2.4); ab analysis is in progress for these groups. conclusions: using αcd20 is associated with significant attenuation of cav when used with either "therapeutic" αcd154 or csa. our findings demonstrate for the first time that αcd20 reduces the severity of cav in conjunction with both conventional immunosuppression and costimulation pathway blockade. mechanisms include inhibition of ab production, and perhaps others that remain to be defined. subsaturating concentrations of anti-hla antibody ( sensitized recipients having donor specific anti-hla abs have been successfully transplanted following a conditioning regimen employing plasmapheresis with or without pooled human immunoglobulin. in vitro studies have shown that exposure of ecs to subsaturating concentrations (ssc) of anti-hla ab (priming) followed by subsequent exposure to high concentrations (hc) of anti-hla induces the expression of protective genes, bcl2 and ho-1 and confers protection to complement mediated lysis of ecs. however, the molecular events following priming with exposure to ssc of anti-hla ab still remains undefined. to determine the priming events and to define the kinetics of protective gene expression, we exposed human aortic ecs to ssc of anti-hla ab for 72 hrs and re-exposed them to saturating concentrations of anti-hla abs. ecs were collected at 24, 48, and 72 hrs after exposure to ssc as well as 24 hrs after exposure to hc. expression profile of signaling intermediates (mapk, wnt, nf-kb, hedgehog, pi3kinase, stress pathway, tnf family) in the ecs were analyzed by gene array. analysis of the bcl2 and ho-1 expression showed no significant increase in expression following exposure to ssc of w6/32 (priming) or control ab alone at any of the time points ( background/aims: microcirculation disturbance, endothelial injury and cytokine overproduction are implicated in the pathophysiology of hepatic ischemia reperfusion injury (iri). thrombomodulin (tm) is a membrane-bound endothelial thrombin receptor that accelerates thrombin-catalyzed protein c activation, inhibits thrombin-induced fibrin formation, and also regulates inflammation. in the present study, we investigated the effects of recombinant human soluble tm (rhstm) on hepatic iri in the rat. methods: wister hannover rat was used for preparing ischemia/reperfusion model. hepatic iri was induced by subjecting 70 % area of the rat liver to 90 minutes of ischemia followed by 6 h of reperfusion. the rats were randomly assigned to a group receiving an intravenous injection of rhstm (3 mg/kg body weight) and to a group treated with saline 60 minutes prior to the beginning of reperfusion. sinusoidal endothelial cells (secs) and kuppfer cells (kcs) were isolated using centrifugal elutriation. the plasma levels and the concentration in cultured cell supernatant of il-6 and tnfα were measured by specific enzyme immunoassays. results: the plasma alt, ast and hyaluronic acid levels were significantly decreased, and the histological damage of the liver was attenuated in rhstm-treated rats as compared to control rats. using laser doppler flow-meter we found that rhstm treatment improved hepatic microcirculation. the intrasinusoidal fibrin deposition, injury of secs and liver dysfunction during hepatic iri were weaker in rhstm-treated rats than in control rats. tm activity in secs was significantly recovered, and plasma il-6 and tnfα were significant decreased in rhstm-treated rats as compared to control rats. further, il-6 and tnfα production in isolated kcs was also significant decreased in rhstm-treated rats as compared to control rat . conclusion: the present results suggest that rhstm is useful for the prevention of secs damage and kcs activation induced by iri. our present study also suggests that disturbance of hepatic microcirculation is induced in part by intrasinusoidal microthrombus formation and by locally released inflammatory cytokines from kcs. protective effects of preservation solution including activated protein c in small-for-size liver transplantation in rats. background: small-for-size liver graft is a serious obstacle of partial orthotopic liver transplantation (olt). however, various therapeutic strategies including surgical innovations, pharmacological agents and gene therapies to protect small-for-size liver graft have not yet been developed. aims: activated protein c (apc) is known to have cell protective properties via its anti-inflammatory and anti-apoptotic activities. this study aimed to examine the cytoprotective effects of preservation solution containing apc on olt using smallfor-size rat liver graft (20% partial liver). methods: liver grafts were assigned to two groups: in the control group, the grafts were flushed and stored in histidine-tryptophan-ketoglutarare (htk) solution alone for 6 h; in the apc group, in htk solution containing apc for 6 h. results: the apc group significantly increased 7-day graft survival from 60% to 100%, decreased levels of transaminase, and improved histological features of hepatic iri compared to the control group. myeloperoxidase activity demonstrated that the apc group markedly suppressed the infiltrations of neutrophil. hepatic expressions of tumor necrosis factor-α and il-6 of the apc group were remarkably decreased. the apc group significantly reduced serum hyaluronic acid levels, indicating attenuated sinusoidal endothelial cell injury. moreover, the apc group markedly increased hepatic levels of nitric oxide caused by upregulated endothelial nitric oxide synthesis (nos) together with downregulated inducible nos, and decreased hepatic levels of endothelin-1. finally, hepatocellular apoptosis of the apc group was remarkably suppressed by downregulated hepatic caspase-8 and caspase-3 activities. conclusions: preservation solution containing apc inhibited pro-inflammatory cytokine synthesis, which leads to hepatocellular apoptosis and liver injury. one of the cytoprotective effects of the apc treatment was to upregulate hepatic enos, followed by increased expression of hepatic no, and to decrease expression of hepatic et-1, resulting in the prevention of microcirculatory disturbance. preservation solution containing apc is a potential novel and safe product for small-for-size liver transplantation to improve liver graft function and animal survival. deletion of cd39 on natural killer cells attenuates hepatic ischemia/ reperfusion injury. living donor liver transplantation (ldlt) has emerged as a solution to ease organ shortage in orthotopic liver transplantation. however, ldlt is often complicated by small-for-size liver graft that is highly susceptible to injury and shows decreased liver regeneration. suppression of liver regeneration in small-for-size grafts correlates with impaired priming as a result of limited nf-κb activation and decreased production of the priming cytokines tnf and il-6. we have shown that the hepatoprotective protein a20 promotes liver regeneration, partly through blockade of the cyclin dependent kinase inhibitor p21. however the impact of a20 expression in livers on il-6 production and/ or signaling were still unknown. in this study we demonstrate that secretion of il-6 (elisa) following treatment with lps or tnf was moderately lower in hepatocytes transduced with a recombinant a20 adenovirus (rad) as compared to non-transduced or rad.β-galactosidase transduced cells. this indicates that il-6 production in hepatocytes is not solely nf-κb dependent (not totally blocked by the nf-κb inhibitor a20). despite similar or lower il-6 levels, il-6 signaling as evaluated by phosphorylation of stat3 (western blot; wb) was enhanced in a20 expressing hepatocytes. this was confirmed in experiments showing that a20 increases stat-3 phosphorylation in response to exogenous human il-6. accordingly, hepatocyte proliferation was significantly higher in rad.a20 transduced hepatocytes as opposed to controls. the pro-proliferative function of a20 mapped to the 7zn domain. since the balance of il-6 signaling in hepatocytes is finely regulated through a negative feed-back loop provided by the il-6/stat-3 dependent induction of suppressor of cytokine signal-3 (socs3), we investigated whether a20 affects il-6 signaling by modulating socs3 expression. our results indicate that a20 indeed decreased il-6 mediated upregulation of socs3 (wb). this later result was confirmed in vivo. improved regeneration and survival in a20 treated livers correlated with a substantial decrease in socs3 levels before and 24 hours following extended (78%) liver resection in mice. these results suggest that a20 enhances priming of hepatocytes by il-6 likely through down-regulation of socs3. this added to the effect of a20 on p21 would further enhance its pro-proliferative function in hepatocytes to benefit survival and function of small-for-size liver grafts. background: we have previously demonstrated that silencing inflammatory, apoptosis and complement genes can prevent ischemia-reperfusion (i/r) injury occurring in heart transplantation. however, the method for efficiently delivering sirna into donor organ has not been established. this study was designed to develop a new method to induce gene silencing by coronal artery infusing with sirna solution for prevention i/r injury in heart transplantation. methods: multiple sirnas that specifically target tnfa, caspase 8, and c5a receptor (c5ar) genes were generated and selected. sirna protection of donor organs was evaluated in a rat heart transplantation model. heart grafts from lawis rats were infused with sirna solution via coronal artery and preserved in hkt solution at 4° c for 18 hrs, and subsequently transplanted into syngeneic lawis rats. cardiac functions were assessed by heart beating rate. gene expression at mrna level was determined by qpcr. the i/r injury was assessed by immunohistochemistry. results: after donor heart perfusion with sirna solution, sirna was found to enter the myocardial cells, indicated by the fluorescence emitted from the dye labeled sirna. the levels of tnfa, caspase 8 and c5ar genes were significantly up-regulated in the grafts after ex vivo preservation for 18 hrs. these up-regulated tnfa, caspase 8, and c5ar genes were significantly knocked down by sirna infusion. using a sirna infused organ as a donor, the graft survival was significantly prolonged in heart transplantation. while sirna solution-treated heart grafts retained strong heartbeat up to the end point of observation (>10 days), the control grafts lost function within 3 days. in addition, an improved cardiac function was observed in the graft preserved in sirna solution. the protection of graft by sirna solution is associated with prevention of i/r injury. sirna solution-treated organs exhibited almost normal histological structures as well as less neutrophil and lymphocyte infiltration, compared with control solution-treated organs. conclusions: this study developed a novel ex vivo sirna delivery system using coronal artery infusion, which can effectively silencing genes in donor hearts and prevent cardiac i/r injury. background: ischemia/reperfusion (i/r) insult is a prime factor leading to liver dysfunction. apoptosis plays key role in the early graft loss following orthotopic liver transplantation (olt). bcl-xl has been showed to exert an anti-apoptotic function both in vitro and in vivo. this study was designed to evaluate potential cytoprotective effects and mechanisms for bcl-xl in liver i/r injury by ad-bcl-xl gene transfer. methods: a mouse model of partial 90 min warm hepatic ischemia followed by 6 h of reperfusion was used. balb/c wide-type (wt) mice (n=6/gr) were injected with ad-bcl-xl or adβ-gal reporter gene (2.5x10 9 pfu, i.p. at day -2). sham control wt mice underwent the same procedure, but without vascular occlusion. mice were sacrificed after 6 h of reperfusion; liver tissue and blood samples were collected for future analysis. results: ad-bcl-xl treated mice showed significantly lower sgot levels (iu/l), as compared with ad-β-gal or wt controls (509±361 vs. 2819±706, and 2212±841, respectively; p<0.005). these correlated with histologic suzukis grading of hepatic i/r injury, with wt/ad-β-gal controls showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. in contrast, wt mice treated with ad-bcl-xl revealed minimal sinusoidal congestion without edema/vacuolization or necrosis. ad-bcl-xl gene transfer significantly reduced local neutrophil accumulation and apoptosis (3.8±2.9 of tunel+ cells in ad-bcl-xl vs. 21.5±5.3 and 23.5±6.5 of tunel+ cells in wt or ad-β-gal treated mice; p<0.01). unlike in controls, intragraft expression of mrna coding for tnf-α, e-selectin/icam-1, and ip-10/mcp-1 remained depressed in the ad-bcl-xl group. ad-bcl-xl gene transfer markedly depressed the activation of nf-κb, caspase-3, and increased ho-1, a20, and bcl-2/bcl-xl expression, as compared with wt/ad-β-gal controls. conclusion: this study demonstrates that inhibition of nf-κb activation contributes to the cytoprtective effects after bcl-xl gene transfer in hepatic i/r injury. the induction of anti-oxidant ho-1 and anti-apoptotic a20, bcl-2 by bcl-xl gene transfer exerts synergistic cytoprotective effect against antigen-independent hepatic inflammatory injury induced by i/r. hepatocyte background: primary graft non-function (pnf) affects survival and function of renal allografts. pnf, secondary to the ischemic and inflammatory injury in the peri-transplant period, leads to acute tubular necrosis and predisposes to acute rejection. defining new preconditioning regimens to reduce pnf are desirable. a20 is part of a negative antiinflammatory loop aimed at inhibiting nf-κb in renal proximal tubular epithelial cells (rptec). hepatocyte growth factor (hgf) is a pleiotropic growth factor upregulated in acute kidney injury and acute rejection likely to modulate inflammation and promote repair. in the present study we evaluated the effect of hgf on rptec and hypothesized that some of its protective functions may relate to the upregulation of a20. methods and results: treatment of rptec with hgf (50ng/ml) led to a 2.4±0.8 (n=4; p=0.04) fold increase in a20 mrna (real time-pcr), which translated into a significant 3.0±1.5 fold increase (n=5; p=0.04) in a20 protein by 6h, as shown by western blot (wb). two lines of evidence suggested that upregulation of a20 by hgf was nf-κbindependent. hgf did not degrade iκbα in rptec (wb) nor upregulated the nf-κb dependent molecule icam-1, as shown by flow cytometry analysis (facs). further, a20 was still upregulated in rptec expressing the nf-κb inhibitor iκbα, both at the mrna and protein levels. upregulation of a20 by hgf protected rptec from a subsequent inflammatory insult, here mimicked by the addition of tnf. pretreatment of rptec with hgf for 6 hours blunted tnf-induced (10u and 25u/ml) upregulation of icam-1, as analyzed by facs. conclusion: to our knowledge this is the first demonstration that a20 could be upregulated in rptec, in a non-nf-κb dependent manner. further studies are carried out to elucidate the transcription factors involved in hgf-induced upregulation of a20. from a clinical standpoint, these results highlight the unique ability of hgf to protect rptec from inflammation by inducing the anti-inflammatory protein a20, remarkably without triggering other pro-inflammatory signals. we propose that hgf-based therapies could serve in preconditioning regimens to prevent ischemia/reperfusion injury and reduce pnf and acute rejection in renal transplantation. background. liver ischemia reperfusion injury (iri) is one of the main causes of graft dysfunction and rejection in liver transplantation. it has been documented that iri is associated with inflammatory and complement pathway activation. this study was designed to investigate the efficacy of small interfering rna expression vector (shrna) targeting tnf-α and complement 3 (c3) genes in the protection of mouse liver iri. methods. shrna expression vectors were constructed for tnf-α and c3 genes. mice received shrna by hydrodynamic injection prior to iri, which consisted of interrupting blood supply to the left lateral and median lobes of the liver for 45 minutes followed by reperfusion. iri was evaluated using liver histopathology, as well as levels of serum alanine transferase (alt) and aspartate transaminase (ast). neutrophil accumulation was determined by a myeloperoxidase (mpo) assay. lipid peroxidation was assessed by malondialdehyde (mda) levels. realtime pcr was used to test gene silencing efficacy in vitro and in vivo. result. we demonstrated that iri is associated with an increase in tnf-α and c3 mrna levels in liver tissue 6 hours after reperfusion. shrna-treatment effectively down-regulated tnf-α and c3 expression in iri livers. in comparison with vehicle control, the serum levels of alt (9843.31 ±2610.66u/l vs 1960.00 ±1361.98 u/l) and ast (7188.25 ±3295.99u/l vs 1405.13 ± 774.65u/l), were significantly reduced in mice treated with tnf-α and c3 shrna. additionally, the neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by mpo and mda respectively, were improved after shrna treatment. tissue histopathology showed an overall reduction of injury area in shrna-treated mice. conclusion. this is the first demonstration that liver iri can be prevented through gene silencing of inflammatory genes and complement genes, showing potential for shrna-based clinical therapy. kidney -acute rejection: antibody-mediated rejection the (2) in the first three days after transplantation, a temporary decrease in dsa was observed in all amr cases, and all of them quickly rebounded thereafter; (3) c4d can be detected very early (can be seen on day 1, 89% in day 4 protocol biopsies, frequently in the absence graft dysfunction, and 100% in index biopsies); (4) the pathologic changes observed in sequential biopsies were c4d deposition followed by acute tubular injury, then interstitial inflammation and peritubular capillary margination seen in index biopsies; (5) pure amr occurred early (100% at day 3), usually evolving into mixed amr with accompanying cellular rejection (87% in index biopsies); (6) most recipients (87%) had initial graft function before developing amr; (7) background: antibody-mediated rejection (amr) has been recognized as a major problem in abo-incompatible (abo-i) renal transplantation (rtx). however, little is known about the long-term impact of amr in the abo-i renal transplant setting, especially after the introduction of a tacrolimus (fk)-based immunosuppressive regimen. the aim of this study was to assess the long-term impact of amr on the clinical and pathological outcomes in abo-i rtx. methods: fifty-eight patients who underwent abo-i rtx at our institution between march 1999 and december 2004 under an fk-based immunosuppressive regimen were enrolled in this study. protocol biopsies were performed regardless of renal function at one month and one year after rtx. fifty-six of the 58 patients received the biopsy at one month and 43 of 56 patients underwent biopsy at one year posttransplant. amr was diagnosed by morphological features based on the banff '05 update and other characteristic findings for amr previously reported, such as mesangiolysis, interstitial hemorrhage, and cortical infarction. we evaluated graft survival, incidence of chronic rejection characterized by transplant glomerulopathy (tgp) at one year posttransplant, and renal function using serum creatinine at three years posttransplant according to the incidence of amr at one month posttransplant. the overall graft survival rate at 3, 5, and 8 years after rtx was 93%, 87%, and 63%, respectively. the incidence of amr at one month was 34% (19/56). the graft survival rate of the patients with amr was significantly lower than that of the patients without amr (p<0.01, 3 years: 79% vs 100%, 8 years: 38% vs 94%). the incidence of tgp in the patients with amr was significantly higher than that of the patients without amr (p<0.001, 57% vs 7%). the serum creatinine concentration at three years after rtx was significantly higher in the patients with amr than in those without amr (p<0.001, 1.95 mg/dl vs 1.32 mg/dl). in this study, we revealed that amr in abo-i rtx is associated with not only graft loss but also the progression of chronic renal impairment, functionally as well as pathologically, even after the introduction of an fk-based immunosuppressive regimen. further studies are needed to establish a more effective immunosuppressive regimen, such as rituximab induction therapy; against amr in abo-i rtx. conclusions: de novo dsa in ar is an independent predictor of graft loss and its degree of influence is comparable to other established risk factors (aa race, dgf, increased baseline creatinine). additional studies are warranted to: 1) confirm the predictive ability of dsa and 2) determine whether reduction/eliminattion of dsa will allow improvements in graft survival. was performed in all the recipients before and six months after lrkt. graft biopsies were performed as well within and after six months of the transplantation (tx). all the data of 87 recipients were collected prospectively during the period of follow-up. humoral rejection rate, donor specificity, and time of appearance of the de novo abs were retrospectively studied. results among the 87 lrkt recipients, 47 (54%) showed negative/negative results, 15 (17%) showed positive/positive results, 12 (14%) showed positive/negative results, and 13 (15%) showed negative/positive results (de novo abs) in the pre-/post-transplant flow-pra analysis. among the 13 cases with de novo abs, 5 (38%) had donorspecific abs (dsa) and the remaining 8 (62%) had non-donor specific abs (ndsa) as determined by lab single antigen analysis. four of the five recipients (80%) with dsa showed evidence of both vascular and humoral rejection in the graft biopsies performed within 6 months of the transplantation, while one of the eight recipients (13%) with ndsa showed evidence of cellular rejection during the same period. a 5-year graft survival rate of the recipients with de novo abs was 69%, compared with 96%, 88% and 93% in other groups without de novo abs (p=0.009). conclusions lrkt recipients with developing de novo abs has much higher incidence of humoral rejection and worse prognosis, especially those with donor-specific de novo abs. cautious monitoring for the appearance of anti-hla antibodies should be adopted after transplantation, even in patients without anti-hla ab prior to the transplantation. despite significantly higher response than the 4 males w/o amr (p<0.03), the other 5 females did not experience amr. conclusions: 1) cfc is a novel assay to measure allo/ do cd3-cell responses, assess the degree of sensitization, and predict amr in hs, 2) allo/do cd3-cell numbers are elevated in many hs, but not nc, 3) hs w/ high(+) cfc are at increased risk for amr and may need additional pre-tx desensitization, 4) allo/do reactivity are higher in hs females, which may explain their higher rate of amr, 5) cfc cut off levels for amr prediction may be higher in females than males, 6) monitoring hs using the cfc pre-and post-desensitization may help determine the efficacy of desensitization and risk for amr. were treated with plasmapheresis, ivig and rituximab, and pts with l-amr received ivig and rituximab. the 2-year gs post-amr in pts with e-amr and focal c4d staining was 33% vs. 50% in pts with diffuse staining; while cases of l-amr with focal c4d deposition had a gs of 55% vs. 60% in cases with l-amr and diffuse staining. the number of cases with focal staining was low, and the numerically evident differences were not statistically significant (log-rank p=ns). notably, when losses due to death with a functional graft were censored, post-amr gs was significantly lower in pts with e-amr and focal staining than in their counterparts with diffuse c4d deposition (41% vs. 67%, log-rank p=0.04). in this retrospective single center study, focally positive c4d amr carries a worse prognosis than previously thought, and causes a significant reduction of gs. whether any degree of c4d staining in the context of kt dysfunction should be treated as amr remains a pending question. association time of biopsy was 29.1±43.0 mo after kt. however, 172 cases were biopsied in the 1st year posttransplant. the extent of c4d staining was graded as <10% (0), 10-50% (1), and ≥50% (2) of ptc, and the intensity was graded as none (0), light (1), and strong (2) staining. these findings demonstrate the significant discordance between detection of dsa and c4d, which is a relatively specific histological evidence of ab-mediated injury. this factor should be taken into account when clinical decisions for treatment of patients with either c4d or dsa positivity are made. the observed discrepancy could be partially due to the technique used for staining of the biopsy specimens, inability to detect anti-hla dsa with the available technology, or non-hla dsa. long term follow up data are needed to evaluate the impact of these markers on graft outcomes. introduction epithelial to mesenchymal transition (emt) is a potential mechanism of tissue fibrogenesis. in a previous study, we had reported that the early expression of emt markers was associated with the progression of renal grafts towards interstitial fibrosis and tubular atrophy (if/ta). here, we report the long-term follow-up of this cohort, paying a special attention to the evolution of graft function. patients and methods 83 patients engrafted with a kidney from a cadaveric (n=63) or a living (n=20) donor, and in whom sequential protocol biopsies had been performed at 3 and 12 months, were included. the phenotype of epithelial cells was studied at three months according to the expression of vimentin (an intermediate filament normally expressed by fibroblast-like cells) and to the cellular localization of β-catenin. grafts in which these two markers were abnormally expressed by more than 10% of tubular cells were considered as emt+ grafts. serum creatinine and creatinine clearance (estimated abstracts by gault and cockcroft index) were collected from 12 to 24 months post-transplant and compared according to the emt status of the graft. results multivariate analysis demonstrated that the early expression of emt markers was an independent risk factor of the progression of graft fibrosis between 3 and 12 months. more importantly, these early phenotypic changes were associated with a progressive and sustained deterioration of the graft function : emt+ patients had a statistically higher serum creatinine from twelve months after transplantation, and a significantly lower creatinine clearance from 18 months after transplantation (emt+ 49.4±4.5 ml/min vs emt-61.1±2.3 ml/min, p=0.01). the difference was persistent at 24 months. conclusion the expression of emt markers by tubular epithelial cells at an early time point post-transplant (three months) is highly suggestive of an ongoing fibrogenic process, and has repercussions on the long-term graft function. therefore, these epithelial phenotypic changes are relevant and promising biomarkers for an early detection of if/ta. we recently reported that 73% of transplant glomerulopathy (tg) has evidence of alloantibody-mediated injury in biopsies for cause. we found that 1/3 of tg is c4d+ab+ and 1/3 is c4d-ab+ suggesting that c4d staining is not sensitive enough to detect all biopsies with antibody-mediated injury. we aimed to develop a new laboratory test to detect biopsies with antibody-mediated rejection (abmr) which are missed by c4d. using affymetrix microarrays, we analyzed gene expression in 173 renal allograft biopsies for cause. we previously reported that both abmr and t cell-mediated rejection (tcmr) biopsies show increased expression of transcript sets associated with cytotoxic-t cells (cats) and gamma-interferon effects (grits) compared to biopsies without rejection (p<0.05). however, abmr biopsies were discriminated by a selective increased expression of 25 "endothelial cell-associated transcripts" (endats). these genes included established endothelial markers such as vwf, pecam1, sele, cd34, and cadherin 5, which are involved in endothelial-cell activation. hierarchical clustering of 82 biopsies with ab+ using endats identified a group of c4d-ab+ biopsies (n=20) clustered with c4d+ ab+ biopsies (n=18). thus 25% of biopsies with antibody (20 of 82) had increased endat-scores despite being negative for c4d. these c4d-ab+ biopsies with high endats, had higher scores for cats and grits, increased incidence/severity of tg, tubular atrophy/interstitial fibrosis, and worse future graft function (p<0.05), but similar incidence of tcmr or borderline lesions, in comparison to c4d-ab+ biopsies with no increase in endats. the c4d-ab+ cases with endothelial activation show extensive inflammation in the allograft, as measured by the gene sets, which is similar to c4d+ abmr. there are a significant number of cases with alloantibody and no c4d that show increased expression of endothelial genes. thus the transcriptomics detects deteriorating c4d-allografts with ongoing alloantibody mediated injury. we conclude that increased expression of endothelial genes provides a new feature of abmr, and can be used as a new diagnostic test to detect and treat c4d-abmr. probabilistic ( 001) . the bayesian network model was analyzed and, interestingly, cd86 was critically related to tg, suggesting a b-cell mediated process. tg was also predicted by upregulation of ccl2, ccl3, ccl5, cxcl9, il-8, il-10, and icam gene expression. ten percent of the samples were excluded randomly from the initial model, and subsequently used for cross validation. in the validation analysis, the model effectively predicted tg (auc of 0.92, 90% ppv) and sf (auc of 0.866, 83% ppv). this study provides a compelling and clinically relevant example of the combination of quantitative gene expression with probabilistic bayesian modeling to predict renal allograft pathology. potentially important molecular pathways associated with transplant glomerulopathy were also identified. the application of this integrated approach has broad implications in the field of transplant diagnostics and interpretation of large data sets. 1, 6, 12, 24, 36, 48 and 60 months respectively. the daily dose and blood levels of tac were significantly lower in tac/slr group compared to tac/mmf group. renal function is shown. despite lower prevelance of cai in tac/slr group long-term graft function and patient and graft survival are comparable between tac/mmf and tac/slr groups. objective: the aim of this study was to determine if ethinicity impacts graft outcomes in kidney transplant patients converted to sirolimus (srl) and either maintained on calcineurin inhibitors (ci) or mycophenolate (mmf) with steroids. methods: this was a retrospective analysis of all kidney transplants converted to srl and transplanted from 7/91 to 4/07. patients were divided into 4 groups: group 1: aas converted to srl + continued on ci; group 2: non-aas converted to srl + continued on ci; group 3: aas converted to srl + continued on mmf; group 4: non-aas converted to srl + continued on mmf. pediatrics and multiorgan transplants were excluded. results: a total of 257 patients were included (61% aa). demographics, baseline immunosuppression, and reason for srl conversion were similar between groups. table 1 displays characteristics and outcomes. patients converted to srl+ci regimens had higher rates of acute rejection before srl conversion (p<0.02), but equal rates after conversion. development of proteinuria was similar across groups. figure 1 displays the graft survival rates for each group. aa patients converted to srl+mmf tended to have poorer outcomes compared to aa patients converted to srl+ci. non-aa patients converted to srl+mmf tended to have better graft outcomes compared to non-aa patients coverted to srl+ci, although this did not reach statistical significance(p=0.186). conclusion: aas converted to srl may benefit from continued ci, while non-aas converted to srl appear to have better outcomes with mmf. further prospective studies are warranted to confirm these findings. aa srl+ci (n=113) there are no large registry studies evaluating the correlation of allograft failure for recipients of kidneys from the same deceased donor. we examined outcomes in such recipient pairs using data from the united states renal data system. methods: we studied the correlation of graft failure events within 19,461 pairs of same-donor recipients transplanted during 1995 through 2003. analyses were limited to patients with functioning grafts 3 months post-transplant (tx) and adjusted for known donor, recipient, and tx management factors. we estimated odds ratios to measure the increased risk for 1, 2, and 3-year graft failure and death-censored graft failure when the contralateral kidney had such an event. we also evaluated the effect of recipient pairs transplanted at the same center vs different centers. results: there is a strong correlation in outcomes for 2 recipients with the same donor (table) . the correlation was stronger within pairs transplanted at the same center than for those transplanted at different centers. differences in the correlation of graft failures within pairs transplanted at the same versus separate centers diminished over 2 and were absent by 3 years post-tx. results for death-censored allograft failure were similar. conclusion: unmeasured donor factors contribute significantly to the correlated graft failure outcomes in paired recipients of deceased donor kidneys and need further study. kidney tx outcomes in the first year may be affected by differences in management between transplant centers, more so than in subsequent years post-tx. 1 odds ratio of death-censored graft failure and graft failure in recipients of a donor pair, given that the outcome occurred in the recipient of the contralateral kidney, with both recipients being transplanted at the same (s) center or at different (d) centers. all odds ratios significant with p<0.001. the deterioration of kidney allograft function (dekaf) study is a nih-funded multicenter observational study of late allograft (ktx) loss. the study examines two cohorts: a "long-term cohort" (ltc) of prevalent ktx with scr < 2.0 mg/dl with deterioration of function (25% increase in scr or proteinuria) and a "prospective cohort" (pc) of incident ktx developing a persistent >25% increase in scr. we examined the pathologic features of the first renal biopsy (bx) obtained for new onset deterioration in each cohort. all bx were read and scored centrally using a modified banff schema. on average, bx were obtained at 6 (pc) and 75 (ltc) months post-tx. mean scr was similar, but more patients (pts) in ltc had proteinuria. moderate to severe interstitial fibrosis and tubular atrophy (ta) were more prevalent in ltc than pc (33 vs 13% ci score ≥2; 33 vs 9% for ta). the rate of vascular sclerosis >25% was similar (10.87% pc vs 15.46% ltc); however, hyaline arteriolar sclerosis >25% was more common in the ltc (30 vs 4.35%). interstitial inflammation (i) and tubulitis (t) scores were similar in both cohorts. however, more pts in ltc had peritubular capillary infiltrates (>5 cells) and evidence of tx glomerulopathy. while rates of interstitial inflammation and tubulitis sufficient to warrant diagnosis of acute rejection are similar in the prospective and long-term cohorts, long term cohort pts had more proteinuria, interstitial fibrosis, tubular atrophy, hyaline arteriolar sclerosis, and transplant glomerulopathy. analyses of histologic findings and renal outcome are ongoing. the term chronic allograft nephropathy (can) has been abolished by the last banff meeting report (am j transplant, 2007) and 2 categories have been introduced for chronic changes: chronic active t cell-mediated rejection and chronic active humoral rejection (cahr). aim of the study was to review all cases of can diagnosed in the last 4 years and to identify immunohistochemical markers of chronic rejection. a cohort of 79 cad pts with biopsy-proven can was analyzed. each case was reviewed and assigned into 3 groups according to banff 2005 criteria: chronic rejection (cr), chronic calcineurin toxicity (cnit) or chronic lesions not otherwise specified (nos). cd4+, cd8+, cd20+, cd68+ cells and c4d deposits were assessed by immunohistochemistry. twenty-eight pts were classified as cnit,34 as cr,19 of which were cahr, and 17 as nos. serum creatinine and 24h proteinuria at renal biopsy, extent of interstitial fibrosis and glomerulosclerosis were not significantly different among 3 groups (table1).the number of cd4 + cells was higher at ti level in cr compared to cnit (table1;*p=.05). cd8 + cells were higher at ti and g level in cr compared to cnit (table1;*p=.05). ti and g cd20 + cells were not different among the 3 groups (table1). the number of g cd68 + cells was increased in cr compared to cni and nos (table1;*p=.05). no significant difference in cd20, cd4, cd8, cd68 expression was found at ti and g level between c4d + and c4dcases of cr. cd68, cd8, cd4 but not cd20 expression at ti level correlated with ti fibrosis (r 2 =.105, .077, .156, respectively, p<.05) at the univariate analysis. only ti cd4 + cells independently correlated with fibrosis at multiple regression analysis. in conclusion, our data suggest that: morbid obesity limits access to kidney transplantation and predicts adverse transplant outcomes. there are limited data on the safety and efficacy of gastric bypass (gb) as a weight reduction therapy among transplant candidates and recipients. methods: we examined usrds registry data to identify medicare-insured kidney transplant candidates and recipients with billing claims for gb procedures. gb were categorized according to occurrence before listing, on the waitlist, or after transplant. we studied the clinical characteristics of gb-treated patients, and subsequent outcomes including progression from listing to transplant and 30-day mortality. usrds surveys bmi data at dialysis start, waitlist entry, transplant, and transplant anniversaries.we computed changes between most recently reported body mass index (bmi) values preceding and following gb, when available. results: we identified 72 transplant candidates treated with gb before listing, 29 who underwent gb on the waitlist, and 87 gb cases after transplant. patients treated with gb were most commonly female, white race, and without diabetic or hypertensive renal failure (table) . 30-day mortality after gb, calculable for listed and transplanted patients, was 3.4% and 3.4%, respectively. 1 transplant recipient experienced graft failure within 30 days of gb. of 29 patients treated with gb on the waitlist, 20 proceeded to transplant. post-gb weight loss was detected for 60% with gb pre-listing, 70% with gb on the waitlist, and 90.7% with gb after transplant. among patients listed for transplant in the same era and bmi >35 at first dialysis who were not treated with gb, 22% had lost weight between dialysis start and listing. conclusions: gb has been performed in small numbers of kidney transplant candidates and recipients, and is followed by weight-loss in the majority of cases. peri-operative mortality is comparable to reports in patients without kidney disease. gb warrants prospective study as a strategy for reducing complications of obesity in esrd. introduction: the present study investigated the incidence of posttransplant diabetes mellitus (ptdm) and calculated the risk of developing ptdm under a tacrolimus and mycophenolate mofetil (mmf)-based immunosuppression based on clinical characteristics, tacrolimus-pharmacokinetics, and genetic polymorphisms related to tacrolimus-pharmacokinetics, cytokines and diabetes mellitus. methods: seventy-one non-diabetic adult kidney recipients (male 37, female 34) were studied. patients with continuous high plasma glucose levels, over 6.5mg/dl of hemoglobin a1c, or requiring insulin and/or oral anti-diabetic agents for more than 3 months after transplantation at 1-year after transplantation were diagnosed as having ptdm. fifteen genomic polymorphisms were assessed. results: one year after transplantation, 21 recipients (29.6%) developed ptdm. positive risk factors were age (p=0.028) and body mass index (p=0.048). there were no significant differences in acute rejection rate, total steroid doses, tacrolimuspharmacokinetics or its related to genetic polymorphisms between the two groups. the frequencies of ptdm were significantly higher in patients with adiponectin t45g tt genotype than in those with the g allele (p=0.034), and in patients with glucocorticoid receptor (nr3c1) bcl i cc genotype than in those with the g allele (p=0.004). conclusions: the incidence of ptdm at 1-yr after transplantation was 29.3% in our cohort. elder or obese patients were risky for the development of ptdm. the presence of the adiponectin t45g tt or nr3c1 bcl i cc genotype may also be risk factors for ptdm, suggesting that insulin and glucocorticoid sensitivity-related genes are associated with the development of ptdm. analysis of these genotypes is a possible method of predicting a patient's risk for developing ptdm and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals. pharmacokinetics persistent hyperparathyroidism (hpt) with hypercalcemia is common after renal transplantation. studies have shown that treatment with cinacalcet corrects hypercalcemia and lowers pth levels in these patients. so far cinacalcet's steadystate pharmacokinetics and their correlation with pharmacodynamics (pk/pd) have only been studied in hemodialysis patients, but not in renal transplant recipients with persistent hpt. to gain further insight into cinacalcet's effects on calcium-phosphate homeostasis, we determined its steady-state pharmacokinetics and pharmacodynamic effects in these patients. in a prospective, single center, open label study we examined the effect of a 2-week treatment with 30 mg and subsequent 2-week treatment with 60 mg cinacalcet daily on calcium-phosphate homeostasis over 24 hours and determined the steady-state pharmacokinetics of cinacalcet in stable renal allograft recipients. the urinary calcium excretion was determined in timed urine samples. median auc 0-24 was 784.8 ng*h/ml and c max was 68.5 ng/ml for 60 mg cinacalcet which is higher, and oral clearance (cl/f) was 76.9 l/h which is lower in renal transplant recipients compared to previously published data of hemodialysis patients (50 mg cinacalcet auc 0-24 179, c max 17.2, cl/f 279). we also observed a non-proportional increase of auc 0-24 after doubling of the cinacalcet dose. the once daily administration of cinacalcet dose-dependently reduced ipth and serum calcium. cinacalcet and parathyroid hormone (pth) concentrations showed an inverse correlation and were fitted to a simple emax model (e max =80 % reduction vs. baseline, ec 50 =13 ng/ml). the 8-hour fractional urinary excretion of calcium was increased after 60 mg cinacalcet (baseline 0.85±0.17 %, 30 mg 1.53±0.35 %, 60 mg 1.92±0.37 %). renal function remained stable. cinacalcet's higher and non-proportional increase of auc 0-24 in transplant recipients compared to hemodialysis patients evokes the possibility of a pharmacokinetic interaction with concomitant cyclosporine treatment. cinacalcet effectively corrected the biochemical abnormalities of persistent hpt. the transient calciuria could potentially favor nephrocalcinosis and reduce bone mineral density, suggesting that higher doses of cinacalcet need to be used with caution in renal transplant recipients with severe persistent hyperparathyroidism. screening for proteinuria in the kidney transplant clinic. bryce a. kiberd, 1 romuald panek. 1 1 dalhousie university, halifax, ns, canada. proteinuria is a predictor of progression in kidney disease. it is not clear whether measuring albuminuria will have greater clinical utility over measurement by dipstick or total proteinuria in kidney transplant recipients. there has also been a trend away from using 24 hour collections to using spot urine albumin/creatinine (ac) and protein/creatinine (pc) ratios. we compare the prevalence of proteinuria estimated by dipstick, ac and pc in prevalent patients (>6 months post transplant) in the kidney transplant clinic. significant albuminuria defined as ≥30 mg/g was present in 52% (211/403). albuminuria was seen in 29% (70/245) with negative and 67% (34/51) with trace dipstick proteinuria. significant predictors of albuminuria in a mulitvariate logistic analysis were egfr (or 0.977 per ml/min/1.73m 2 , 95% ci 0.965-0.989 p=0.001), diastolic bp (or 1.028 per mmhg, 95% ci 1.008-1.048 p=0.007), and mmf use (or 0.50, 95% ci 0.32-0.77 p=0.002). macroalbuminuria (ac>299 mg/g) was seen in 17.4% (70/403) and significant predictors in a mulivariate logistic analysis were lower egfr and higher systolic bp. sirolimus use was associated with more macroalbuminuria and mmf use with less macroalbuminuria. in a subset of patients followed for >2 years prior gfr loss was considerably greater (p=0.021) in patients with albuminuria (-0.88 ml/min/1.73m 2 /year) compared to those without (-0.15 ml/min/1.73m 2 /year). however other measures of proteinuria were also significantly (p for trend) associated with prior gfr loss (ml/min/1.73m 2 /year) as shown in the <0.13 (n=185) 0.13-0.49 (n=109) >0.50 (n=80) ∆ egfr/year -0.33 -0.26 -1.40 0.02 * a pc cut point of 0.129 g/g had a sensitivity and specificity for albuminuria > 30 mg/g of 87% and 88% respectively (c=0.92, 95% ci 0.89-0.95), and a pc cut point of 0.49 g/g had a sensitivity and specificity for macroalbuminuria >300 mg/g of 100% and 94% respectively (c=0.98, 95% ci 0.97-0.99). ac may be more sensitive and therefore have more clinical utility than other measures of proteinuria for progression. however prospective follow up of renal function change and cv outcomes is required. serum creatinine is a crude marker of gfr in renal transplant recipients and changes in gfr are frequently not accompanied by commensurate changes in serum creatinine concentration. serum cystatin c and estimates of gfr (egfr) based on cystatin c have been shown to be more accurate than serum creatinine and creatinine-based egfr in renal transplant recipients. the purpose of this study was to determine whether the filler, lebricon and rule cystatin c-based egfr equations were better able to detect changes in true gfr than the mdrd and cockcroft gault creatinine-based egfr equations. we performed two measures of 99m tc-dtpa gfr, serum creatinine and serum cystatin c on each of 183 stable renal transplant recipients at least 6 months apart. we calculated and compared the percent annual change in the measured gfr and the estimated gfr using the various gfr estimation equations. we also determined the sensitivity, specificity, positive predictive value and negative predictive value of each prediction equation for the detection of decline in measured gfr. results are presented below: the cystatin c and creatinine-based egfr equations all demonstrated poor sensitivity and diagnostic performance to detect a decline in gfr. novel equations derived and validated in the transplant population are needed to accurately assess kidney function over time. background: hypercalcemia, hypophosphatemia and renal phosphate wasting are common after kidney transplantation and are related to persistent hyperparathyroidism and hyperphosphatoninism. animal data suggest that these alterations in mineral metabolism may contribute to nephrocalcinosis and progressive graft dysfunction. supporting clinical data are limited. aim: to test the hypothesis that nephrocalcinosis is highly prevalent in the early posttransplant period and is related to a disturbed mineral metabolism. methods: biomarkers of mineral metabolism (including albumin-corrected serum calcium [ca c ], serum phosphorus [p], biointact pth, calcidiol, calcitriol and alkaline phosphatase) and renal calcium and phosphorus excretion parameters were prospectively assessed in 201 renal transplant recipients (62% male, mean age 55 ± 14 yrs) at the time of their 3-month protocol biopsy. these protocol biopsies were screened for the presence of microcalcifications. intratubular, interstitial and/or cytoplasmatic microcalcifications were observed in 30.4% of biopsies. calcifications were more prevalent in recipients of a living related donor as compared to cadaveric donor. high serum ca c levels, high serum pth levels, a high urinary ca×p product and high fractional excretion of p and low serum p levels were significantly associated with renal microcalcifications (see figure below). microcalcifications were not related to the fractional excretion of ca, use of diuretics, immunosuppressive regimen, serum alkaline phosphatase level and history of delayed graft function. the extent of microcalcifications correlated significantly with the severity of mineral metabolism disturbances. conclusion: our data demonstrate that nephrocalcinosis is highly prevalent in the early posttransplant period and suggest that a disordered mineral metabolism is implicated in its pathogenesis. polymorphism in abcb1, the gene encoding for p-glycoprotein, predicts recovery of graft function early after kidney transplantation. the pharmacokinetics of cyclosporine (csa) is characterized by wide inter-individual variability. this might be particularly relevant in the early post-transplant period, due to the detrimental effects of the drug on the kidney. p-glycoprotein (p-gp), the product of the abcb1 gene, plays a key role in the distribution of csa at cellular level. single nucleotide polymorphisms (snps) of abcb1 might potentially influence the response of patients to csa. in particular, the snp in position 3435 of the exon 26, despite its silent nature, has been recently associated with altered specificity for its ligand, such as csa (kimchi-sarfaty et al, science 2007, 315:525) . whether p-gp pharmacogenetics would help to guide csa treatment early post-transplant remains ill defined. we sought to evaluate the effects of the snps in the exon 26 on the rate of recovery of graft function, as estimated gfr early postoperatively, in 150 kidney transplant patients given csa as part of their immunosuppressive regimen. the frequency of dgf (as need for post-operative dialysis) among the different abcb1 genotypes was also estimated. of the 150 kidney transplant recipients, 35% had the abcb1 wild type (c/c) genotype in exon 26, 40% were heterozygous (c/t) and 25% were homozygous (t/t) for the polymorphic variant in position 3435. gfr values were significantly lower in patients carrying one of the two mutant alleles than in the wild type ( figure) . the frequency of dgf was 15%, 28% and 30% in patients with the cc, ct and tt genotypes, respectively. these findings demonstrate that in patients carrying the ct or tt mutant alleles in exon 26 of the abcb1 gene and given csa, the recovery of graft function is less prompt, and the risk to develop dgf higher than in wild-type cc genotype. pre-transplant screening for abcb1 polymorphism would help to identify patients who may safely receive csa early post kidney transplantation. cigarette cigarette smoking has shown to reduce graft and patient survival in renal transplant patients. however, whether it could directly produce allograft disfunction has not been investigated. the aim of this study was to assess the smoking influence on renal graft function. we studied a cohort of 827 adult renal transplant patients, transplanted from jan/96 to dec/05 and followed until dec/06. smoking habits were recordered at the time of transplant (never, former, current smoker). during summer of 2007, a telephonical survey allowed us to obtain complete information about smoking habits in 642 patients (aged 47.6 ±13, 65% male): status (never, former, current), years of habit, years of quit, and number of cigarette smoked per day. number of "pack-years" was calculated. renal function was measure by inverse serum creatinine at 3 rd month and then annually. time to decline a thirty percent in inverse serum creatinine was registered. patients were divided in two groups: those who always smoked during all transplant period (smokers, n=94) and those who did not (n=546 renal insufficiency occurs frequently after extrarenal transplantation as a result of acute tubular necrosis at transplantation, high blood pressure, and cni toxicity. we performed 56 renal biopsies in 54 patients after heart (4), lung (20) , liver (22), bone marrow (9), and cornea (1) transplantation since 2000. the time from transplantation to biopsy was 49±53 months in general and was longest after liver (60±61 months) and shortest after bone marrow transplantation (37±43 months). the histologic changes were: tubular atrophy/interstitial fibrosis of ≥20% in 52% of biopsies (heart biopsies 75%, lung 70%, liver 46%, bone marrow 22%); acute tubular changes in 54% (heart 25%, lung 65%, liver 46%, bone marrow 56%); arteriolar hyalinosis in 41% (heart 75%, lung 65%, liver 23%, bone marrow 22%); arterionephrosclerosis in 41% (heart 50%, lung 50%, liver 36%, bone marrow 22%); glomerular sclerosis of ≥20% in 21% (heart 50%, lung 25%, liver 18%, bone marrow 22%); glomerulonephritis in 13% (heart 25%, liver 18%, bone marrow 22%; that means iga-nephropathy after heart and liver, immune complex nephritis twice after liver, mpgn after liver, and membranous glomerulonephritis and minimal changes after bone marrow transplantation); thrombotic microangiopathy in 7% (lung 10%, liver 5%, bone marrow 11%); finally one case of polyoma nephritis after lung transplantation. among 1702 heart and lung transplantations, 13 patients needed kidney transplantation (0.8%) after 96±40 months; and among 2221 liver transplantations, 11 needed kidney transplantation (0.5%) after 112±65 months (sign. later, p=001). conclusion: as expected, most histologic changes were those of cni toxicity and hypertension. surprising is the high number of glomerulonephritis under immunosuppression. thrombotic microangiopathy without the typical clinical signs were interpreted as cni-related toxicity and seems to occur more often after extrarenal than after renal transplantation. patients with heart and lung transplantation reach end-stage renal failure more often and earlier than patients with liver transplantation. the context: living kidney transplantation, a superior therapy to deceased donor kidney transplantation, is underutilized. states have enacted legislation and the federal government has launched initiatives to compensate living organ donors, but the effect of policy on improving living kidney donation rates in the united states is unknown. objective: to determine whether public policies are associated with changes in living kidney donation rates in the continental u.s. design, setting, and study subjects: series of cross-sectional analyses using records of state legislatures in 48 continental states and living kidney donation rates from the united network for organ sharing. main outcome measures: living kidney donation rate during each year from 1988-2006 and change in donation rates before and after legislation enactment in each state and launch of federal initiatives. results: from january 1990 through december 2005, 28 states enacted legislation for living donors (24 mandating paid leave, 8 tax deductions, 3 unpaid leave, 2 encouraging paid leave). few states (n=5) enacted legislation prior to 1999. there was a steady increase in the mean living kidney donation rate in the continental u.s during the study period (mean (standard deviation) annual increase in donations 1.5 (0.04) donations per 1,000,000 population). in analyses accounting for length of time state legislation had been enacted, the types of legislation enacted, and the incidence and prevalence of esrd in each state, there was a slightly (but not statistically significantly) greater average annual increase in donations after compared to before state legislation enactment (annual increase in donations per 1,000,000 population [95% confidence interval ( introduction: accurate and precise renal function assessment is essential in the evaluation of prospective kidney donors. while direct measurement of gfr is the "gold standard", it is not widely available. moreover, creatinine (scr)-based estimation equations are suboptimal to assess kidney function in this setting. ct scans are increasingly being used to study renovascular anatomy in donors and has replaced angiographic exams in many institutions. 3d imaging reconstruction allows for kidney volumes (kv) measurements which have been shown to highly correlate with measured gfr in this population. thus, the purpose of this study was to develop a model to estimate measured gfr that not only incorporates scr and demographic data but also kv as measured by 3d ct scans. methods: 244 individuals who underwent donor evaluation were identified. an automated segmentation algorithm was used to measure renal parenchymal volume from preoperative abdominal cts. patient demographics and scr values were obtained from the medical records. gfr (normalized for bsa) was measured by i 125 iothalamate renal clearances (igfr). an analysis of covariance model was created to correlate measured igfr with kv, patient age, sex, race, weight, height and scr. pearson's correlation coefficient was calculated for each variable. results: kv (p<0.001), age (p<0.001), scr (p<0.001) and weight (p<0.001) significantly correlated with igfr. sex (0.60), race (0.90) and height (0.76) were not statistically significant. the new fitted regression model is: kv-egfr (ml/min/1.73 m 2 ) = 70.77 -(0.444*age) + (0.366*weight) + (0.200*volume) -(37.317*scr). we then compared the performance of the kv-egfr model to the re-expressed mdrd equation using calibrated scr assay. the r 2 was 0.61 vs 0.50, respectively; signed median % difference was +1.8% vs -12.7%, respectively (% difference b/w estimated gfr and igfr); and accuracy within 30% (% of estimated gfr values that fall within 30% of igfr) of 96.3% vs 89.8%, respectively. finally, the kv-egfr model was closer to igfr (in absolute values) than the mdrd eq. in 177/244 (70.5%) cases vs 72/244 (29.5%) cases, respectively. conclusions: kidney volumes highly correlate with igfr and the proposed gfr estimation model outperforms the mdrd equation in potential living kidney donors. the kv-egfr model could be used to estimate donor gfr in lieu of i 125 iothalamate gfr which is less clinically available. for donor selection, current reports identify unsuspected renal pathology by time 0-biopsy. aim: to explore whether the findings at time 0-renal biopsy (bx) correlates with pre-donation clinical data including renal function. methods: kt databases from 2 institutions were reviewed. time 0-renal bx are routinely performed from the upper pole during back-table and evaluated by 2 nephropathologist for interstitial fibrosis (if), tubular atrophy (ta), arteriolar hyalinosis (ah), mesangial increase (mi), and glomerulosclerosis (gs). pre-donation data gathered from the donors were demography, body weight, bmi, systolic/diastolic bp, scr, proteinuria, and egfr by levey equation clinical data is summarized in the table. and gs showed no correlation. multivariate analysis failed to sustain the significant associations found on bivariate analysis, most likely due to a low event/parameter relation. conclusions: a significant correlation was observed between time 0-bx findings and clinical pre-donation parameters. whether these histological findings at the time of kidney donation represent a higher burden/risk for the remaining kidney ought to be evaluated during follow-up. in an era where living donation is increasing, we should advise a closer surveillance of these donors in order to modify risk factors that participate in kidney damage progression. predictors of poor early graft function following laparoscopic donor nephrectomy (ldn). matthew cooper, 1 abdolreza haririan, 1 stephen jacobs, 1 michael phelan, 1 benjamin philosophe, 1 stephen bartlett, 1 joseph nogueira. 1 1 dept of surgery, urology, and medicine, university of maryland, baltimore, md. ldn has become the standard of care in many transplant centers. poor early graft function remains an important complication. we conducted a retrospective study to evaluate the risk factors for slow or delayed graft function following ldn methods: donor and recipient records from the first 1000 ldn were reviewed (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) . results: slow graft function (sgf) was defined as cr>3.0mg/dl at pod5 and dgf as the need for dialysis within the first week following transplantation. donor variables examined included age, sex, race, bmi, egfr, and number of renal arteries. recipient variables included age, sex, race, bmi, prior tx, pre-tx dm, history of smoking and drug usage. additional variables evaluated included degree of relationship (lurt), hla mismatch, antilymphocyte induction, de novo cni usage, r v. l nephrectomy, wit, total or time, and performance of simultaneous deceased donor pancreas tx (splk). univariate analysis was performed with significance defined as p<0.05. significant variables were then included in the multivariate analysis. background: a kidney exchange program is the logistic solution for patients with positive cross match (x + ) or abo incompatible donors. a major problem in all kidney transplantation programs is the sensitized recipient. we analyzed the success rate for immunized recipients in the dutch kidney exchange program. methods: from january 2004 till december 2007 242 donor-recipient pairs were registered. there were 117 couples in the x + group while 125 pairs were abo incompatible. in the x + group the median pra was 46% (2-100%). to create new combinations a match program was run 16 times (every 3 months) with a median of 46 (16-66) participating couples. allocation criteria included bloodtype (first identical, then compatible), hla match probability within the actual exchange donor pool to ensure that highly sensitized recipients have the best chance to receive a kidney, and the waittime on dialysis. cross matches between new donor and recipient were performed centrally in our reference laboratory with cdc-tests. results: after 16 match runs, we found matching couples for 83/117 (71%) x + pairs, for 21/87 (24%) abo incompatible pairs with o recipients and for 29/38 (76%) abo incompatible pairs with non-o recipients. median pra of the 83 recipients in the x + group was 42% (2-100%). after 3 match runs chances for success became small. the overall success rate for abo incompatible and x + pairs in the dutch kidney exchange program after 4 years is 55%. however, the success rate for immunized patients in the x + group is significantly higher (71%) as our match program gives priority to those recipients with the smallest chance of finding a compatible donor in each match run. thus our kidney exchange program is especially suited for immunized patients. although paired kidney donation (pkd) program is an established method to overcome incompatibilities between kidney donor-recipient pairs (drp), significant proportion of the incompatible drp participating in such program could remain unmatched. domino-kidney transplantation (kt) in which altruistic living non-directed donor kidney (lndk) is offered to a pool of incompatible drp, and is used to initiate a chain of pkd transplants, could provide more opportunities of kidney transplantation to drp in pkd program. we introduce our experience of multicenter domino kt for the last 7 years sixteen hospitals participated in the domino-kidney transplantation between february, 2001 and july, 2007. 181 domino-kidney transplants were performed with 69 domino-kt chains initiated by altruistic lndk. 2-pair chains were 43, 3-pair chains 14, 4-pair chains 9, 5-pair chains 2, and 7-pair chains 1. the development of a multi-regional kidney paired donation program. using an optimization matching algorithm the new england program for kidney exchange (nepke) efficiently matches incompatible donor/recipient pairs. in 2006, the mid-atlantic paired exchange program (mapep) and other individual centers began sharing data with nepke. this is a report of the success of two regional programs working together to increase the probability of participants in both programs finding a compatible match. methods: incompatible pairs and non-directed donors (ndd) are referred to nepke through transplant centers. donor and recipient abo, hla and recipient hla antibody screening are entered into the computer database. utilizing the optimization program, searches for compatible matches are conducted every 30 to 45 days. the program identifies potential 2 and 3-way matches, ndd chains, and list exchange chains. following the determination of compatibility nepke notifies transplant centers involved of the potential match and centers accept or decline the offer. transplant centers notify their pairs and preliminary crossmatches are performed. a conference call is scheduled to coordinate simultaneous donor nephrectomies and recipient transplants. surgeons speak prior to incision to ensure simultaneous donation. results: from july 2005 to december 2007, 186 pairs and 10 ndds have entered nepke. during this time frame over one thousand possible matches were identified, with the majority of these matches involving the same pairs in multiple matches. after "optimizing" and eliminating multiple matches 13 two-way exchanges; 15 three-way exchanges; 7 three-way list chain exchanges; and 25 ndd chain matches were offered to transplant centers as possible matches. most common reason offers were declined include: positive crossmatch (21.6%); donor factors (20%), and recipient inactive or transplanted (20%). four offers occurred in mapep alone pairs, 47 in nepke, and 14 offers were cross-regional. three matches are pending for 9 additional transplants. one previous and one pending transplant are the result of cross-regional exchanges. five transplants performed and 6 pending involve ndd chains. one pending match involves a list exchange chain. conclusion: using a computerized optimization algorithm to match 2 and 3 way exchanges, ndd and list exchange chains has lead to a substantial increase in the number of kpd matches and transplants performed. cross-regional coordination is feasible and expands the number of transplants performed beyond the ability of individual exchange programs. as living donors become an increasingly important source of life-saving organs, there is growing concern about the lack of comprehensive research on donor outcomes. in addition to possible long term consequences, there is a risk that donors will experience complications during or following surgery. of the 13,000 living kidney donors in 2005-2006, none died during surgery, and 0.5% (n=68) needed blood transfusions during surgery. in the six weeks following donation, 3.9% (n=512) had at least one serious adverse event (sae): 1.7% (n=220) needed readmission following initial discharge, 0.6% (n=72) needed an interventional procedure, 0.5% (n=61) needed re-operation, 0.3% (n=41) had vascular complications, and 2.1% (n=274) had other complications. when all saes were considered in combination, the rate was 3.9%. because over 50% of ldr forms were submitted by transplant centers fewer than 6 weeks post-donation, all complication rates should be considered minimum estimates. one donor was reported to have died from donation-related causes within 6 weeks of donation. the number of living donor kidney transplants performed by a transplant center in 2005 -2006 ranged from 1 to 362 transplants. additionally, the risk of donor complications is not equal across transplant centers. for example, there was a significant correlation between the number of living donor kidney transplants performed at a transplant center and the percentage of that center's patients who were readmitted within 6 weeks of donation, with greater donor volume associated with a lower rate of readmission. living kidney donation is relatively safe, but prospective donors should be made aware that there is a non-trivial risk (3.9%) of short-term complications post-donation. as with many other major surgical procedures, complication rates are lower, on average, at institutions that perform a larger number of these procedures. we sought to determine if intensive screening improves detection of polyomaviral reactivation in asymptomatic patients and pre-emptive stepwise modification can improve outcome of polyomaviral nephropathy (pvn). methods:this is a prospective single center study. we randomly assigned de novo kt (cluster randomization) to intensive screening (is:n=648) and routine care (rc: n=260) for the detection of decoy cells. is was initiated at week-4 of kt and rc at the time of increase in serum creatinine. this was complemented with urine and blood nucleic acid testing. all patients had biopsies performed for detection of polomya nephritis (pvn). both groups were treated with pre-specified stepwise modification of it based on cell cytology and viremia (step 1: decrease dose of cellcept by 50%, step 2: decrease dose of tacrolimus by 50%, or switch to sirolimus therapy, step 3: discontinue cellcept). primary outcome included persistence of decoy cells/viremia following each step in modification of it every three months and secondary outcomes included acute rejection, graft function and graft loss. results: polyomaviral reactivation developed in 11.7% in is group and 40% had pvn without changes in serum creatinine. the estimated cumulative rate of primary outcome in the is versus rc groups, 3-months (51% vs. 79%) relative risk (rr), 0.47;95%ci,0.20-1.08;p=0.051); 6-months (20%vs.52%) (rr=0.67; 95% ci,0.38-1.20; p=0.009); and 12-months (2%vs.33%) (rr=0.88; 95% ci, 0.41-1.43; p=0.003). secondary outcomes: despite similar degrees of step-wise is modification, rate of acute rejection non-significant (p=0.25), but 25% of patients in rc loss the graft vs no graft loss in is group. patients who continue to remain on tacrolimus vs. those who were switched to sirolimus therapy had persistent viremia (or=10.25; 95%ci, 1.2-92.0; p=0.003). conclusion: is for poloymaviral reactivation allows early detection of pvn in the presence of stable graft function. stepwise modification in it resulted in early resolution of decoy cells and viremia in both groups, albeit slowly in rc group, and it did not prevent the graft loss in rc group. background: dna sequencing of the bk viral (bkv) genome non-coding control region (nccr) from individual patient isolates demonstrate divergent sequence and alterations in the arrangement of modularly conserved sequence blocks (p-q-r-s) ranging in size from 39 to 68 base pairs. aim: primary aim to molecularly clone and analyze patient-derived bk virus nccr sequence variants. our secondary aim is to determine if reporter gene constructs of patient-derived nccr variants differ in their promoter activity upon transfection into a mammalian cell line (vero) and a human primary tubular epithelial cell line. methods: bkv dna was amplified and sequenced from blood and urine samples of 18 renal transplant recipients. via sequence alignment, unique nccrs were determined. these sequences were pcr amplified and cloned into reporter plasmids containing the renilla luciferase gene. promoter activity was measured via luminometer 24 hours after transfection into vero cells and human tubular epithelial cells. results: variation in naturally occurring bkv nccr promoter regions exist as single basepair insertions and deletions, and insertions or deletions of partial sequence blocks (p-q-r-s). single basepair substitutions were most commonly seen (46% of analyzed samples). promoter activity within vero cells ranged from 188% to 39% as compared to nccr activity of an archetypal strain (wwb). low promoter activity (<50%) was seen in isolates with duplications of the p block and large deletions of the r block. conclusion: these sequence blocks are rich in regulatory elements and control the expression of both bkv structural and regulatory genes. variation in these cisacting eukaryotic transcriptional promoter binding sites corresponds to differential promoter activity in naturally occurring bkv isolates. current plans include repeating the promoter activity studies in human primary tubular epithelial cells. humoral and cellular immunity to polyomavirus bk large t and vp1 antigens after pediatric kidney transplantation. polyomavirus bk-associated nephropathy (bkvn) has emerged as a cause of graft failure after kidney transplantation (ktx). in a cohort of 37 pediatric renal recipients undergoing prospective three-monthly monitoring for bk by blood and urine q-pcr, we evaluated antibody response, measured by enzyme immunoassay using bk vlp, and cellular immune response, reported as frequency of ifnγ-secreting cells in a elispot assay after 9-day stimulation with bkv large t (lt) and vp1 peptides. we could not observe any influence of recipient pre-transplant bkv-specific igg or t-cell levels, which were generally low, on bkv infection after allografting. after transplantation, both specific igg levels, and frequency of bkv-specific t cells increased according to the degree of viral exposure. in detail, bkv-seropositive patients who never reactivate the virus (group1, n=10) did not show significant increase in igg levels (from a median od of 0.37 at month +1 to 0.39 at the end of follow-up), while patients with urinary shedding alone (group 2, n=14) or with viremia (group 3, n=13) increased from a median od of 0.3 to 1.2 (p=0.09), and 0.26 to 2.8 (p>0.0005). in the case of cellular immunity, vp1-specific t-cells increased in the three groups. conversely, lt-specific t-cells, which were high (median 71 sfu/10 5 cells) and remained unchanged throughout the follow-up period in group 1 patients, had a significant increase in recipients belonging to both group 2 and 3. interestingly, patients with urinary shedding who do not progress to viremia show a median 3-fold increase in lt-specific t-cell levels at peak viruria, compared to no increase observed in patients who develop viremia. the latter group mount a significant response to lt only after therapeutic reduction of immunosuppression. at peak viruria, viremic patients already show a 8-fold rise in specific igg compared to the 1.5 increase observed in group 2 recipients. our data suggest that inability to reach protective levels of bkv lt-directed t cells, rather than specific igg, predispose ktx recipients to bkv replication. introduction:the antibody response to human bkv virus (bkv) is incompletely characterized. antibody responses to the vp-1 protein have been detected in kidney transplant patients, but it is not known if these have virus neutralizing activity. methods:recombinant bk, jc, and sv40 virus like particles were used to produce a panel of 20 monoclonal antibodies. these antibodies were characterized for isotype and for ability to bind the respective antigens in elisa assays. to test neutralizing activity, bkv gardner strain (atcc# vr837) viral particles were incubated with the corresponding antibodies for 2 hours at 37 degrees c, and used to infect wi 38 cells. bkv infection was monitored by quantitative real time pcr using primers directed against the vp-1 gene. neutralizing activity was defined as greater than 95% inhibition of viral yield. results:the monoclonal antibodies were of the igg 2a or igg 2b class with the exception of one igg 1 and one igm antibody. all 12 anti-bkv monoclonal antibodies bound to bkv capsids in-vitro in elisa assays. this binding affinity was species specific, as only 1 antibody showed weak binding activity to jcv and sv40 capsids. neutralization of infectious bk virus was shown for 10/12 antibodies. denaturation of capsid proteins indicated that the monoclonal antibodies recognized primarily conformational epitopes, with only monoclonal antibody appearing to have a linear component. paucity of linear epitopes was further suggested by lack of reactivity of sera from bkv seropositive subjects in elisa assays based on genotype-specific short peptide sequences derived from the bkv vp-1 loop region. four monoclonal antibodies each generated from jcv capsids and sv40 capsids did not show any bkv neutralizing activity. conclusions: bkv vp-1 protein capsids contain species specific conformational epitopes which can elicit virus neutralizing and non-neutralizing antibody responses. measurement of these antibodies in renal transplant recipients may have diagnostic and prognostic applications. bkv specific monoclonal antibodies deserve further study as potential therapy of acute infections in the viremic phase. impact of immunosuppression reduction in bk viremic patients: 3 year follow-up. s. kuppachi, 1 a. guasch, 1 c. p. larsen, 1 k. e. kokko. 1 1 transplant center, emory university, atlanta, ga. background: development of bk nephropathy is a risk factor for allograft loss. bk viremia (bkv) precedes the development of bk nephropathy. it has been reported with 1-year follow-up that reduction of immunosuppression leads to control of bkv. here, we report our 3-year follow-up experience of bkv patients that were identified by a prospective screening protocol and managed by sequential reduction of immunosuppression. methods: all kidney or kidney-pancreas transplant recipients at emory university between 5/03-5/04 were screened prospectively for bkv by real time pcr during follow-up visits (1-6, 9, 12, 24, 36) . patients with a bk viral load of greater than 10,000 copies/ml blood received a kidney biopsy to screen for bk virus nephropathy by immunohistochemistry. bkv without nephropathy resulted in reduced immunosuppression by a 50% reduction in mycophenolate dose. bkv with nephropathy resulted in discontinuation of mycophenolate. all identified bk patients were monitored every 2-4 weeks until viral load was below 10,000 copies/ml. immunosuppression was further reduced if viral loads failed to decrease. results: 135 recipients were followed over a 36-month period. 24 patients had received simultaneous kidney and pancreas, 2 a liver and kidney and the rest kidney alone. 27 of 135 (20%) patients developed bkv within a year from time of transplantation. average time to diagnosis of bkv was 4.7 months. average dose of mycophenolate at 3 years was 1.3 g/d in the bkv negative population as compared to 0.56 g/d in the bkv positive population. average 12 hour trough blood level of tacrolimus at 3 years was 9.1 ng/ml in the bkv negative population as compared to 7.4 ng/ml in the bkv positive population. survival rates for both patient and organ were comparable at 3 years in bk viremic vs bk negative patients (100% vs 92%) and (88% vs 83%) respectively. while bk viremia is a historic risk factor for organ loss, prospective monitoring and reduction of immunosuppression is associated with comparable 3 year patient and organ survival to patients that never develop bk viremia. background: there are currently no bk virus (bkv) specific therapies available for clinical use. this study evaluates viral large t antigen as a potential target for drug development, since (a) this is a key molecule that participates in several different stages of viral replication, (b) has no homologous human protein, and (c) offers multiple functional domains for chemical binding, particularly the atp binding site, the dna binding site, the hexamerization surfaces, and the hinge region. methods: virtual screening and protein modeling techniques were applied to bkv large t antigen using a model developed from the known crystal structure of sv40 large t antigen. two different structural states of large t antigen (monomer and dimer structure) in three different states (with the nucleotide pocket empty, with bound adp and bound atp), were evaluated for a total of 6 large t antigen receptor conformations. results: a computational solvent mapping analysis of small molecular probes allowed identification of multiple functional sites, which represent potential drug binding pockets on the large t antigen molecule. it was possible to classify 1200 molecular conformations centered on the atp binding site, hexamerization surface and hinge region of the viral protein. we docked 1209 medium sized fragments (<100 da) to confirm the results obtained by computational solvent mapping, and further characterize chemical properties of the atp binding site. in another approach, known chemical structures of hsp90 and rho-kinase inhibitors were used to search compound databases and obtain a subset of 8294 compounds (mean size of 350 da) capable of docking large t antigen. cross-referencing the top solutions obtained by energy ranking, we were able to identify 50 compounds that bind large t antigen in all 6 conformational states. a subset of 5 compounds simultaneously binds the atp binding site, hexamerization surface and hinge region of bkv large t antigen. conclusions: virtual screening and three dimensional homology modeling technology has allowed us to identify compounds that can bind multiple sites on the large t antigen. these compounds are predicted to preferentially inhibit viral replication without the toxicity expected from simultaneous inhibition of host cell kinases. bk virus (bkv), a human polyomavirus, causes bkv nephritis, which often leads to graft loss after renal transplantation. currently, the only efficient therapy against bkv nephritis appears to be a reduction/change of immunosuppressive agents, and this may increase the inherent risk of rejection. since human renal proximal tubular epithelial cells (hrptec) represent a main natural target of bkv nephropathy, the analysis of bkv infection of hrptec is likely to provide necessary additional insight into bkv biology and contribute to the development of strategies for treatment of bkv nephritis. here we report the ability of 3-hydroxy-3-methyl-glutaryl coenzyme a (hmg-coa) reductase inhibitor pravastatin, which is routinely used to treat hypercholesterolemia, to repress bk virus entry pathways in hrptec and, correspondently, prevent bkv infection. the percentage of hrptec infected with bkv was assessed by immunofluorescent analysis in the absence and presence of pravastatin. both, the percentage of bkv infected cells and the intensity of bkv infection, assessed by western blotting using antibodies against large t antigen, were significantly decreased in hrptec treated with pravastatin. it is likely, that pravastatin's inhibitory effect is explained by depletion of caveolin-1, a critical element of caveolae. we demonstrate that bkv enters hrptec by caveolarmediated endocytosis and disruption of caveolin 1 mrna and protein inhibits bkv infection of hrptec. we provide evidence that pravastatin dramatically decreased caveolin-1 expression in hrptec and interfered with internalization of labeled bkv particles. our data suggest that pravastatin, acting via depletion of caveolin-1, prevented caveolar-dependent bkv internalization and repressed bkv infection of hrptec. our data represent the first report of inhibitory action of statins upon bkv infection. results: 74.6% of patients were caucasian, 12.2% hispanic, 8.9% african-american and 4.4% asian. overall 1-and 3-yr patient survivals were 85% and 77%. stratified by race, only african-american survivals differed from caucasian (3-year survivals of 71% vs. 77%; figure 1 ). compared to caucasians, african-american patients were younger (48yrs ±11.5 vs. 52yrs ±9.9), were more likely to be status 1 (10.3% vs. 5.1%), to have a serum creatinine ≥1.5mg/dl (37% vs. 31%), to receive an multi-organ transplant (8% vs. 5%), to have fulminant hepatic failure (12% vs. 7%), to have a higher meld score (22.9 ± 10.4 vs. 20.1 ±9.4) , to be in the icu (16% vs. 11%), and to be ventilated pre-transplant (8% vs. 5%); all p-values <0.001. after adjustment for each of these variables, race was still an independent predictor of mortality (p=0.01, hr: 1.22, ci: 1.048-1.425). multivariate analysis of the african-american group (n=1481) alone revealed that a bmi greater than 40 (p=0.001, hr: 2.5, ci: 1.49-4.17), a creatinine greater than 1.5 mg/dl (p=0.002, hr: 1.6, ci: 1.18-2.10), and icu admission pretransplant (p=0.018, hr: 1.5, ci: 1.07-2.17) are independent predictors of mortality in this subset of patients. conclusion: in the current meld era, race is still a predictor of worse outcomes, even after adjustment for multiple clinical variables. further work is necessary to elucidate why this disparity exists. the purpose of this study was to analyze the effects of successive pregnancies in female liver transplant recipients on newborn and maternal outcomes. data were collected from the national transplantation pregnancy registry via questionnaires, phone interviews and hospital records. analyses for linear trends (proportions and continuous variables) were done by chi square and least squares regression. there were 217 outcomes of 213 pregnancies, including twins. of the 125 liver recipients who had a first pregnancy, 61 had between one and four subsequent pregnancies. there were no significant differences in the variables analyzed as noted in the conclusions: successive pregnancies in liver transplant recipients are not associated with adverse fetal outcomes and/or increased maternal graft loss. female liver recipients with excellent allograft function without significant recurrent disease or chronic rejection who wish to have more than one pregnancy should not be discouraged to conceive. recorded and categorized in a blinded fashion. univariate, multivariate and survival analyses were performed. over a 4.5-year period (2002) (2003) (2004) (2005) (2006) (2007) , 148 olt recipients were randomized to receive a cca with biliary stent (n=76) or cca alone (n=72). patients with hepatic artery thrombosis (n=8; 5.7%) were excluded. there was no significant difference in demographic or graft-related variables. the mean age at transplant was 55 years, 82% were male, the mean meld was 21 and 23% had hepatitis c. the mean donor age was 46 years, 59% of donors were male, and the mean cold ischemia time was 8.7hrs. the only complication related to the biliary stent was one occlusion. the rate of overall bc in the stented patients was 24.7% vs. 32.8% in non-stented patients, (p=ns). however, stented patients had significantly less bc in the first 60-days post-olt (6.8% vs. 21.0%, p<0.02) and significantly less anastomotic leaks (2.4% vs. 9.6%, p<0.05). over the year following olt, stented patients also required less biliary therapeutic interventions (mean 1.8 vs. 0.9 interventions/patient, p<0.04) and fewer readmissions (mean 2.8 vs. 1.6 readmissions/patient, p<0.01). we also observed improved late graft survival (>6mo) in the stented group. intraoperative stenting of the cca at olt does not appear to reduce the long-term rate of bc but it decreases the incidence of biliary leaks and significantly improves many facets of early patient management. is background: long-term outcomes after retransplantation of the liver (re-olt) is inferior compared to primary olt. however, the survival benefit of re-olt based on model for end stage liver disease (meld) is not known. a single-center analysis of 421 adult patients who underwent re-olt between february 1984 to february 2007 was performed. survival benefits, at a given meld score, were calculated by comparing re-olt survival at 3 months to expected 3-month survival without retransplantation results: of 421 pts, 380 underwent re-olt, and 41 received 3 transplants. the figure shows re-olt survival benefit at any meld score with increased significance in patients with meld scores > 18. although meld scores 30-40 predicted the highest mortality after re-olt, they also demonstrated survival benefit. multivariate cox regression identified cold ischemia time >10 hrs (rr 1.8, p 0.001), meld 30-40 (rr 2.0, p 0.04), time from first olt (8 days -1 year, rr 1.8, p 0.02) and third transplant (rr 1.7, p 0.03) as independent predictors for mortality following re-olt. conclusions: re-olt should be considered in all patients even with low meld scores. although patients with high meld scores (30-40) exhibit poor survival outcomes, a survival benefit is achieved. survival benefit that considers both probability of death without re-olt and expected survival with re-olt, should be used for selection of retransplantation candidates. this study analyzed posttransplant complications, meld score, and donor ages and their effect on length of stay (los). methods: this irb approved retrospective review of our prospectively maintained database included 1427 liver transplant recipients transplanted between 1999-2006. los <14 days, 14-30d, and >30d were analyzed. primary analysis to look at los, meld, and donor age was performed using wilcoxon two-sample test. complication groups were analyzed using logistic regression and odds ratio estimates were determined. kaplan-meier for patient survival for the los groups was performed. univariate analysis: allograft dysfunction, vascular complication of liver allograft, intra-abdominal (other than liver), biliary, cardiac, pulmonary, neurologic, sepsis, renal, and endocrine were statistically significant (p<0.001) using fisher exact test. multivariate analysis: using logistic regression, significant complications were analyzed to determine the complications linked with los >14d and >30d. analysis of 1427 liver transplants demonstrated increasing los with increasing meld in each donor age group (50-60, 61-70) p<0.0001. for donor age >70, this relationship was not significant (p=0.2206). multivariate analysis using logistic regression determined odds ratio estimates for each complication resulting in los >14 days and >30 days. los >14 days and los >30 days were associated with different complications (as shown above). allograft dysfunction (including pnf) and renal complications were not significant factors in multivariate analysis. patient survival significantly decreased (p<0.001) with increased los >14 days. introduction: some patients with primary biliary cirrhosis (pbc) may require longterm corticosteroid (cs) therapy following liver transplantation (olt) due to recurrent inflammation in the graft. our center has attempted to minimize cs use in all of our olt recipients. we reviewed our experience in this cohort of patients to determine 1) patient outcome including recurrent disease and 2) long-term requirement for cs use in pbc patients. methods: from 1988 to 2006, 1,102 olts were performed in 1,032 adults at the university of colorado of which 70 patients (6.8 %) with pbc received 74 allografts. recurrence was defined by characteristic histologic changes on biopsy. bivariate and multivariate analyses were used to evaluate predictors of cs withdrawal. 13 potential predictors of cs discontinuation were considered: age, gender, bmi, race, presence of inflammatory bowel disease (ibd), type of graft (cadaver or living donor (ld), recurrence of aih, warm ischemia time, follow up time (time since transplant), and immunosuppressant (is). results: overall survival at 5 years was 85%. the 1, 5 and 10 year recurrence-free survival was 90, 72, and 54%, respectively. disease recurred in 18 patients (25.7%). of these 18 patients, none received a second transplant because of recurrent disease. cs was withdrawn in 73% of patients at time of review. independent predictors of cs discontinuation are age (>median) (p = 0.0029) and ld graft type (p=0.0018). conversely, cyclosporine (csa) (p=0.0012), female gender (p=0.0197), and bmi > 31 (p=0.0306) were negatively associated with cs withdraw. interestingly, cs withdrawal did not influence pbc recurrence. conclusions: 1) long-term outcomes in pbc patients are favorable and disease recurrence can be managed medically without abstracts re-transplantation. 2) using an aggressive cs minimization approach, almost 3/4 of the patients were cs-free at the time of last follow-up. 3) increasing age and ld grafts were associated with successful cs withdraw; while csa, female gender, and increasing bmi were associated with unsuccessful cs withdraw. incidence cholestatic disease (cd), either chronic rejection or recurrent primary sclerosing cholangitis (psc), post liver transplantation (lt) occurs in 5-35% of psc grafts. the study objectives were to evaluate the incidence and long-term outcome of cd. from 1985 to 2006, 141 grafts in 125 consecutive psc patients and 85 grafts in 79 concurrent alcoholic liver disease (ald) patients were compared. cd was diagnosed by biliary imaging and/or histology. median follow-up was 57 months. the groups were similar including meld score and cold ischemic time; however, roux-en-y biliary anastomosis was more common in the psc group (95% vs. 13%, p<0.01). the psc group had more cmv hepatitis (22% vs. 6%, p<0.01) and acute rejection (60% vs. 30%, p<0.01), and fewer biliary anastomotic strictures (7% vs. 16%, p<0.01). cd occurred in 38 psc grafts and 7 ald grafts (p<0.01). the incidence of cd was greater in the psc group (p=0.02, fig. 1 ). no significant risk factors for cd were identified. in the psc group the graft survival was lower in the cd group (p=0.04, fig. 2) ; however, patient survival at 10 and 15 years was not effected by cd: 73% and 67% vs 75% and 60% because the re-lt rate was greater in this group (26% vs. 6%, p<0.01). at 15 years, patient survival in the psc group was better than for ald (64% vs 22%, p<0.01). long-term outcome post lt for psc is good and better than for ald; however, cd continues to develop beyond the first 5 years with a prevalence of 43% at 15 years. graft loss in patients with cd is high, but with re-tx, patient survival is similar to non-cd patients. further studies to distinguish chronic rejection vs. recurrent psc may provide insight into the prevention of cd following lt for psc. discussion: our analysis showed that patients with aih have a worse long term survival compared to pbc and psc after ddlt. this may be explained by possibly more advanced disease at the time of presentation or more septic complications due to pretransplant salvage therapy with immunosuppressants. also,pbc had the worst relative outcome after ldlt in this group-possibly explained by the older age of the recipients. overall, ldlt offered better outcomes than ddlt in terms of survival in patients with aih,pbc and psc. this study highlights an important and previously unvisited aspect of transplantation for autoimmune and cholestatic liver diseases. inflammatory bowel disease course in patients transplanted for primary sclerosing cholangitis. ariana wallack, 1 joel s. levine, 2 lisa forman. 2 1 internal medicine, univ. of colorado hsc, aurora, co; 2 gastroenterology and hepatology, univ. of colorado hsc, aurora, co. the natural history of ibd following liver transplant (lt) for psc is unknown. prior studies have not shown factors consistently associated with disease activity, but were limited by small sample sizes. the aim of the study is to describe our experience with ibd post-lt in patients with psc and determine factors predictive of disease activity. a survey was mailed to 115 liver recipients transplanted for psc between 1988 and 2006 asking about medications, ibd activity and quality of life after lt. responses were linked to our lt database. results 88 (77%) recipients responded. 76% were male with a median of 82 (7-215) months since transplant. 93% were caucasian with a median transplant age of 46 (16-71) years. 16% developed recurrent psc post-lt. 74% had a diagnosis of ibd (63% uc). immunosuppression included tacrolimus in 84% and cyclosporine in 11%. 71% experienced at least one episode of acute rejection. 73% rated their quality of health 4-5 on a scale of 1-5. there was no significant difference in demographic variables between ibd and non-ibd cohorts. 66% of respondents had ibd pre-lt. of the 30 patients without pre-existing ibd, 7 developed ibd post-lt. of the de novo ibd cohort, 43% were male, median transplant age was 45 years (39-57), and 86% were caucasian. ibd developed in 71% at more than 3 years post-lt. there was no statistical difference between the de novo ibd cohort and those with pre-existing ibd except for ibd type (57% uc vs 88%, p=0.008). significantly more patients were not on any ibd medications post-lt compared to pre-lt (75% vs 91%, p=0.02). fewer post-lt patients were on aminosalicylates (63% vs 79%, p=0.03) and prednisone (14% vs 31%, p=0.03) compared to pre-lt. post-lt recipients developed fewer ibd flares requiring hospitalization (27% vs 45%, p=0.004). 38% reported improvement in ibd activity post-l. 23% and 33% reported no change and worsening activity, respectively. there was no significant difference between reported disease activity and immunosuppression, cmv status, rejection rate, recurrent psc, transplant age, gender or race. background: symptomatic cmv continues to be a significant problem. the costs associated with its development remain high without an optimal method for prevention. the aim of this study was to measure the pharmacoeconomic impact of implementing an abbreviated pre-emptive monitoring strategy versus valganciclovir (vgc) prophylaxis in a large teaching hospital. methods: costs for this analysis were based on a societal perspective, including drug, personnel, hospital, outpatient infusion and monitoring costs related to resources needed to perform pre-emptive monitoring and treatment of cmv related events. time per hr costs were nurse/data coordinator $45, physician $200, and pharmd $52. cmv pcr cost was $203. vgc cost per mg was calculated for prophylaxis, treatment and 30 days of consolidation as needed based on an awp ($0.09 per mg). estimated cost of cmv syndrome was based on cost of picc line placement, drug, personnel and supply costs based on 21 days of therapy and was estimated to be $11,885.50. inpatient admission cost per day were $3727. results: a total of 119 patients were included in this analysis. baseline and transplant demographics were well matched. table 1 displays the total direct and indirect costs accumulated for each group. cmv syndrome occurred in three patients for each group. there was no cmv disease in either group. 27% of patients in the pre-emptive group had dnaemia, but only 4 patients required oral anti-viral therapy. provider time and lab monitoring costs were significantly higher in the preemptive group, while direct medication cost was significantly higher in the prophylactic group. conclusions: frequency of disease severity and outcomes were equal in each group. although the overall costs between strategies is equivocal, allocation of resources to provide pre-emptive monitoring places the burden of disease prevention on the health care system versus the patient necessitating further abbreviation of these strategies. we propose a non-simultaneous form of kidney paired donation that starts with a living, non-directed donor (lnd). a paired donation matching algorithm was developed to allow for lnds to start potentially never-ending altruistic donor (nead) chains in addition to closed loops of 2-, 3-and 4-way exchanges. results: in july 2007, a lnd from michigan traveled 1500 miles to donate a kidney to a woman whom he had never met in arizona. the following week, the arizona recipient's husband donated one of his kidneys to a 32-year-old woman in ohio. the following month, the mother of this recipient traveled to a city 3 hours away to donate her kidney to a patient whose incompatible donor simultaneosly gave a kidney to the fourth patient in the chain. the incompatible donor for this fourth recipient is now slated to give her kidney to a patient in maryland. over the past 9 months, 60 transplant programs have partnered to perform 10 paired donation transplants. demonstrating the advantage of nead chains over classic paired donation, 8 of 10 transplants resulted from altruistic donor chains. conclusion: in order to fully realize the potential of the above approach, one must be willing to supplant two prevalent ideas: 1) that kidneys from altruistic donors should be given to the top candidate on the deceased donor waiting list, and 2) that paired exchanges must be done simultaneously. while there are certain pitfalls to using chains of donors as opposed to traditional "swaps" (i.e. the possibility that a donor could renege, or the accumulation of type ab donors who are not likely to be able to begin another chain), this proposed paradigm shift could result in a very significant increase in both the number and quality of paired donation kidney transplants. purpose: medical literature and national best practices correlate families' understanding of brain death with organ donation rates. analysis of hospital data demonstrated variability in family communication. although surgical residents frequently interact with family members of potential donors and play a critical role in the donation process, they receive no formal cst. we sought to determine if pre-training residents improved performances in cst involving explanation and notification of brain death to family members and aided efforts to achieve the national donation rate goal of 75%. methods: in collaboration with a regional organ procurement organization (opo), an educational model for end of life cst was developed. 17 surgical residents were divided into 2 groups. the first group (n=9) attended a 3-hour didactic session at the opo that included role-playing exercises explaining brain death to families. opo staff, trained to serve as family role-players and skills station coaches, debriefed residents after each simulation. the second group (n=8) received no specialized training. six weeks later both resident groups participated in formal videotaped family communication simulations. independent observers (hospital faculty and senior opo staff), blinded to training, evaluated residents' communication skills using a 12-parameter assessment tool. all residents reviewed their videotaped performances and evaluations, then repeated the simulations after six months. results: during the study period, the pre-trained resident group assessment scores increased by 25% (p=0.0001), while the untrained group increased by 39% (p<.0001). while evidence of improvement existed in both groups, pre-trained residents consistently scored higher when compared to untrained (80% vs. 71%, p=0.037). during this same time period, donation rates in the surgical intensive care unit (sicu) increased by 21%. conclusion: our educational model demonstrated effective training in communication skills during end of life discussions. although a direct relationship cannot be established, donation rates in the sicu increased after resident training. incorporation of this training program into resident education has the potential to improve donation rates and increase the number of organs available for transplantation. program. saverio mirarchi, 1 graeme n. forrest, 1 benjamin philosophe. 2 1 medicine, university of maryland, baltimore, md; 2 surgery, university of maryland, baltimore, md. objective: to improve the efficiency of care for solid organ transplant patients by having internists work in conjunction with transplant surgeons to manage transplant patients admitted to the hospital more than 30 days post organ transplant. this includes patients who have undergone kidney, pancreas, and liver transplants which our center transplants over 300 of these organs/year. in 2002, the organ transplant program was divided into a surgical transplant service (sts) and medical transplant hospitalist service (mths). the mths consists of four full time physicians, one part time physician, and one nurse practictioner with daily rounds from a transplant surgeon on a weekly rotating schedule. methods: we analyzed our data from the past five years since the inception of the mths at a large university medical center. the length of stay (los) for the mths and sts were compared to data from the previous combined program as well as data from the university health consortium (uhc). in addition, we looked at the cost of care to determine if there were any savings related to reduced los and adjusted for the combined salary of the mths. results: in the review period, the total admissions for both the mths and sts averaged 1450 admissions/year. over the past five years the mths showed a major decrease in the los index, defined as the ratio between the observed los and the expected los based on uhc data. this index dropped from 1.27 to 0.91 for patients on the mths. there was also a parallel drop in the sts los index from 1.53 to 1.05. this was associated with a significant cost savings for the hospital. on average, the program has realized an average savings of approximately $1,000,000 per year. when accounting for the combined salary for the mths group which is 625,000 dollars/year, the total savings over the 5 year period this amounts to $1.9 million. conclusion: the creation of a mths working within an organ transplant program at a large university center has been able to demonstrate a major decrease in los for transplant patients which has resulted in decreased costs and improved efficiency. this has also allowed more focused care for a complex medical population. we believe that our program can serve as a model for similar programs at other busy transplant sites. with increasing demand for kidney transplants(tx), more patients are opting to travel outside the us to obtain transplantation. we describe the characteristics and outcomes of 32 kidney tx recipients followed at our center who traveled abroad for a kidney tx between 1995 and 2007. methods: data were obtained via chart review. we compared demographics to all tx recipients at our center during the same period and compared post-tx outcomes to a cohort of patients transplanted at our center matched for age, race, tx year, dialysis time, prior tx, and donor type. median follow-up time was 487 days (range 31-3056). results: demographics are outlined in table. patients transplanted abroad were more likely to be asian and had shorter dialysis times. most patients were transplanted in china (44%) followed by iran (16%), the philippines (13%), and india (9%). living unrelated tx were most common. all patients were discharged on a calcineurin inhibitor, pred, and either mmf (90%), aza (7%), or rapamycin (3%). 7 patients received induction. only 4 patients received cmv prophylaxis. the median duration of hospitalization was 15 days. the median time post-tx to initial visit at our center was 35 days. 4 patients required urgent admission to hospital, 3 of whom lost their grafts. 17 patients ( graft survival at 1-year was 96% for both "tourists" and matched recipients at our center. median time to graft loss was 500 (31-2832) days. mean scr and rejection 1-year post tx was not significantly different between recipients transplanted abroad and at our center. conclusion: compared to patients transplanted locally, patients transplanted abroad had similar graft survival and renal function post tx, but had a high incidence of infectious complications. [background] the primary benefits anticipated following successful induction of allograft tolerance are avoidance of the complications of long-term immunosuppression and prevention of chronic rejection. we have previously reported successful induction of renal allograft tolerance in recipients of hla mismatched combined kidney and bone marrow transplantation (ckbmt). no evidence of chronic rejection has been observed in these recipients after immunosuppression-free periods of 1 to 4 years. in the current study, we have evaluated the longer-term economic impact of this approach. [method] the conditioning regimen for ckbmt included cyclophosphamide, thymic irradiation, anti-cd2 mab, and a calcineurin inhibitor, which was discontinued after 9-12 months. 18 stable renal transplant recipients receiving ongoing triple or double drug immunosuppressive therapy with compatible follow up times were compared with the four tolerant recipients. tolerant patients and stable patients on maintenance immunosuppression were also comparable with respect to their age, their original disease and donor-recipient histocompatibility. [results] the perioperative charges for ckbmt were approximately $90,000 higher than those for conventional living donor kidney transplantation. after 9-12 months, continuing medications in ckbmt recipients included only occasional over-thecounter analgesics. in contrast, the stable conventionally treated recipients were taking an average of 8 pills daily. these included treatments required for de novo diabetes (11%), hypertension (67%), hyper lipidemia (22%) and gastro-intestinal symptoms/ prophylaxis (78%) in addition to their maintenance immunosuppression. these needs resulted in annual maintenance charges of over $15,000/year/allograft recipient and do not include unmeasured costs related to issues such as quality of life or absences from employment. [conclusion] even with this admittedly expensive approach to tolerance induction, the overall medical costs for conventional kidney transplantation with ongoing immunosuppression and treatment for complications will exceed the cost of tolerance after approximately 5 years. increasing african american donation rates in a midwest metropolitan community. susan gunderson, 1 susan mau larson, 1 david m. radosevich, 2 clarence jones, 3 bill tendle, 3 tiffany scott. 1 1 lifesource, st. paul, mn; 2 transplant information services, university of minnesota, minneapolis, mn; 3 southside community health services, minneapolis, mn. purpose: african americans are underrepresented in deceased organ donation in this +2 million community. historically minimal educational outreach had occurred and this study was designed to understand the community's disposition toward donation and to increase support for donation. methods: television, newspaper, and radio advertising aired over a 24 month period beginning in 2004 using the nationally produced donate life-african american campaign as the primary intervention. other components included related faith-based and community outreach. the opo partnered with a minority focused community health clinic to survey african americans pre-and post-intervention. a mailed, selfadministered survey was sent to a sample drawn from organizational lists with a high likelihood of including african american households. for the evaluation, the sample was separated into 1) community members and 2) church members exposed to faithbased campaigns. results: african americans in this community have a sophisticated understanding of the importance of donation (average 12 of 15 knowledge questions scored correctly) in pre-intervention survey. in both community and church groups media exposure and donation knowledge increased after the media campaign (p<0.001 and p=0.004 respectively). similarly, donor designation rates for the entire population on the drivers licenses increased (33.0% versus 40.1%, p=0.119). among all african americans the propensity to donate was, however, unchanged following the campaign. propensity to donate increased (p=0.034) in the community sample whereas there was a reduced propensity to donation (p=0.092) in the church sample. during the study time period the opo also experienced significant increases in donation authorization rates among african-americans, increasing from 37% to 75%. summary: a media based campaign combined with grassroots outreach is an effective tool to increase knowledge and awareness. partnership between the opo and community and faith based leadership was a significant positive byproduct of the project and should be included in future outreach efforts. background: it has been hypothesized that the clinical benefits of anti-thymocyte globulin (atg) induction therapy do not completely result from immunodepletion, but may also result from induction of immunoregulatory t-cells (treg). in this prospective, controlled study we investigated the effect of atg-induction therapy on the frequency and phenotype of peripheral cd4 + foxp3 + cd127 -/low t-cells in kidney transplant patients. methods: after transplantation, 16 patients received atg-induction therapy (thymoglobulin ® ) and triple therapy consisting of tacrolimus, mmf and steroids. the control group (n=18) received triple therapy only. by flow cytometry, t-cells were analyzed for markers associated with immune regulation: cd25, foxp3 and cd127. within the foxp3 + t-cell population, the cd45ro (memory) and ccr7 (homing receptor) markers were characterized. results: pre-transplant levels of cd4 + foxp3 + cd127 -/low t-cells in all patients were 3-17% (median 6%) of cd4 + t-cells. one wk post atg induction therapy, no measurable numbers of treg were present. at 12 wks post atg-induction therapy, a higher proportion of the first detectable t-cells expressed foxp3 compared to the control-group (atg vs. control-group; 7 vs. 4%, median, respectively, p=0.03) and then returned to pre-transplant levels at 26 wks. this increased proportion of cd4 + foxp3 + cd127 -/ low t-cells resulted in a significantly higher foxp3 + /foxp3 neg ratio than in the controlgroup at 12 wks; 0.074 vs. 0.046, median, p=0.02) . at 26 wks, we found a decline in the proportion of naive foxp3 + treg (cd45ro neg ccr7 + pre-transplant vs. post-transplant; 21 vs. 9%, median, p=0.02) which was associated with a rise in the proportion of memory foxp3 + treg (cd45ro + pre-transplant vs. 26 wks post-transplant; 65 vs. 82%, p=0.02). moreover, the proportion of memory t-cells exceeded that in the control-group at 26 wks (atg vs. control-group; 82% vs. 67%, median, p=0.05) which was mainly due to an increase in the proportion of effector memory foxp3 + treg (cd45ro + ccr7 neg ). conclusion: after atg-induced immune cell depletion, a shift towards cd4 + foxp3 + cd127 -/low peripheral regulatory t-cells with the memory phenotype was measured in kidney transplant patients. this finding suggests that atg treatment triggers the generation of de novo peripheral regulatory t-cells by homeostatic proliferation. introduction in a prospective study, we investigated whether donor-specific regulatory cd4 + cd25 bright+ foxp3 + t cells develop in kidney transplant patients. methods we analyzed the percentage and function of peripheral regulatory cd4 + cd25 bright+ foxp3 + t cells of 51 patients before, 3, 6 and 12 months after kidney transplantation. the immune regulatory capacities of cd4 + cd25 bright+ foxp3 + t cells were assessed by their depletion from pbmc and reconstitution to cd25 neg/dim responder t cells at a 1:10 ratio in the mlr. in the first year after transplantation, peripheral cd4 + cd25 bright+ foxp3 + t cells decreased from 8,7% ± 3,0 pre-transplantation to 6,1% ± 1,8 at 12 months (p<0.001). while mlr reactivity to 3 rd party-ag (3 rd p) significantly improved (p<0.05), the reactivity against donor antigens remained low. functional analysis demonstrated potent donor-specific regulatory activities by cd4 + cd25 bright+ foxp3 + t cells after transplantation. depletion of cd4 + cd25 bright+ foxp3 + t cells from pbmc resulted into increased proliferation upon stimulation by donor antigens (p<0.01). upon reconstitution, the capacity of cd4 + cd25 bright+ foxp3 + t cells to control the proliferation of anti-donor reactive cd25 neg/dim t cells increased over time: from 58% (median) pre-transplant to 85% post-transplant at month 12 (p<0.01). moreover, the anti-donor regulatory activities by the cd4 + cd25 bright+ foxp3 + t cells were significantly more vigorous than those controlling 3 rd p-ag stimulated responder t cells (65%, p<0.05). the generation of potent donor-specific regulatory cd4 + cd25 bright+ foxp3 + t cells in the periphery of kidney transplant patients prevents the development of adequate alloreactivity. conclusions: these results indicated that anti-donor responses could be detected even in a significant proportion of "stable" long-term hla identical kidney transplant recipients. speculatively this may be the cause of late graft failures in this group of patients. expression klotho is a gene almost exclusively expressed in renal distal tubules and loss of expression is associated with accelerated aging. in various models of acute and chronic renal injury, and with normal aging, renal klotho expression has been found to decrease. increased donor age is associated with poorer long term allograft function. we hypothesized that klotho mrna expression in renal implant biopsies would correlate with donor kidney quality, as determined by donor chronologic age and peri-transplant renal injury. our recent unsupervised microarray analysis of 87 renal implant biopsies revealed a continuum of organ quality across all samples ranging from the best living donor (ld) kidneys at one end to the worst performing deceased donor (dd) kidneys at the other (am j transpl 2007, nov 16,epub). three predominant groups were identified of ld, dd1 kidneys with low risk (9.5%) of delayed graft function (dgf) and dd2 kidneys with high risk (35%) of dgf (p < 0.05). analysis of klotho gene expression among these groups also revealed a spectrum of expression from highest levels in ld to lowest in dd2 kidneys, especially those who developed dgf. differences were highly significant among ld vs dd kidneys (p < 0.001), although donor age was not different between these groups. klotho transcript levels were significantly different among kidneys that developed dgf compared to those with immediate graft function (igf) (p < 0.05). in conclusion, reduced klotho gene expression is associated with grafts at risk of poorer function and appears to be independent of donor age. decreases in klotho expression likely reflect other factors impacting the renal tissue, which may impact potential for repair and ultimately allograft function. anti renal allograft rejection episodes produce a stereotypical response in the allograft characterized by infiltration by cytotoxic t lymphocytes (ctl), potent ifng response by donor and recipient cells, and decreased transcripts associated with the epithelium. we previously identified pathogenesis based transcript sets (pbts) that reflect the disturbance in rejecting allografts (qcats -ctl, grits -ifng response, kts -decreased function, ajt 7:2712 , 2007 . we hypothesized that anti-rejection treatment would reverse the transcriptome changes of rejection more than histopathologic lesions. using microarray analysis we measured expression of these pbts in 18 antibody mediated rejection (abmr)(11 untreated, 7 treated) and 31 t-cell mediated rejection (tcmr) (25 untreated, 6 treated) biopsies, normalized to nephrectomy samples. histopathology scoring of the primary rejection lesions were analyzed; interstitial inflammation(i), tubulitis(t), intimal arteritis(v), and glomerulitis(g) (fig 1a) . of these, only i and t differentiated between abmr and treated abmr (p<0.05) yet none differentiated between tcmr and treated tcmr. pbts on the other hand differentiated treated from untreated for both abmr (p<0.04) and tcmr (p<0.005) with all 3 pbts (fig 1b/c) . the changes in transcript expression in treated cases were dramatic. particularly impressive was the consistent correction of the disturbances in pbt expression despite the heterogeneous treatment following abmr episodes. unlike abmr treatment, tcmr treatment always included steroids which may contribute to the more pronounced changes compared to abmr. the time of treatment before the biopsy, which also varied within the groups, did not affect the changes in pbt expression since values in treated cases approached those of nephrectomy samples. thus, histologic rejection lesions persist following anti-rejection treatment whereas transcript disturbances of rejection are greatly reduced compared to untreated cases. this study suggests that assessment of anti-rejection treatment is more sensitively monitored by transcript expression than histopathology. blood deciphering the mechanisms of tolerance and chronic immune-mediated rejection remains a major goal in transplantation. data in rodents suggests that toll-like-receptors (tlr), regulators of innate immune responses, play a role in determining graft outcome. however, few studies have focused on tlr in human kidney transplant recipients. we addressed this issue by analyzing the peripheral blood (n = 75) and graft biopsies (n = 26) of renal transplant patients and healthy volunteers. we analyzed, for the first time, the expression of tlr4 in pbmc from kidney recipients with contrasted situations: operational tolerance and chronic immune-mediated rejection (banff 2005), compared to patients with normal histology and stable graft function, non transplant patients with renal failure and healthy volunteers. we found that myd88 and tlr4 were significantly contrasted in the pbmc, and in particular in monocytes, of patients with chronic immune-mediated rejection vs. operational tolerance. chronic rejection patients had significantly increased tlr4 and myd88 compared to operationally tolerant patients, who resembled healthy volunteers and non transplant patients with renal failure. interestingly, analysis of tlr4 transcripts in graft biopsies from patients with normal histology or chronic immune-mediated rejection reflected the blood findings, with a significant increase of tlr4 in chronic immune-mediated rejection. thus, we provide data to support a link between tlr4 expression and long-term graft outcome. the role of tlr and their endogenous ligands in mediating allograft rejection or acceptance therefore warrants further investigation and could give rise to new strategies of therapeutic intervention. our results suggest that peripheral blood tlr4 shows potential as a biomarker of chronic immune-mediated rejection and that measuring blood tlr4 levels may help to identify patients experiencing chronic rejection who require a biopsy. moreover, our data suggest that absence of tlr signaling may be a feature of operational tolerance to kidney grafts. identification of immune identification of immunological tolerance is an important prerequisite in order to establish an individually-tailored approach to the post-transplant management of allograft recipients. it will also provide new insight into the mechanism underlying the balance between tolerance and rejection. here we present data from a multi-centre study aimed at identifying tolerance to renal allografts. we have collected samples from five selected groups of renal transplant recipients: drug-free tolerant patients that were functionally stable despite remaining immunosuppression-free for more than one year; functionally stable patients on minimal immunosuppression (<10 mg/ day prednisone); stable patients maintained with calcineurin inhibitors (cni); stable patients maintained on cni-free immunosuppression regimen; and patients showing signs of chronic rejection. a group of age and sex matched healthy volunteers was also included as control. several biomarkers and bioassays, were combined to provide an immunological 'fingerprint' of the tolerant state. immunophenotype showed a selective expansion of peripheral blood b and nk lymphocytes in drug-free tolerant patients. this group of patients was also characterized by the absence of anti-donor specific antibodies. the differential expression of several immune relevant genes and a high ratio of foxp3/ α-1,2-mannosidase expression in these patients was observed. tcr landscape analysis highlighted differences between the vβ repertoires of drug-free tolerant recipients and chronic rejection patients. additionally, direct pathway donor-specific hyporesponsiveness by ifnγ elispot and lack of indirect pathway anti-donor responses assessed by trans-vivo dth were detected in drug-free patients. the diagnostic capabilities of the combined results of several of the above mentioned biomarkers and bioassays are as follows: specificity 0.964, sensitivity of 0.933 and a positive predictive value of 82.4%. these biomarkers could be used to inform drug weaning protocols of kidney transplant recipients. bile acid aspiration stimulates lung allograft immunity. bile acids detected in the broncho alveolar lavage (bal) as a marker of aspiration has been associated in a dose dependent fashion to earlier development of bronchiolitis obliterans syndrome. we sought to study the relationship between bile acids and active immune molecules as detected in the bal. methods: bal collected prospectively from lung transplant recipients at routine surveillance bronchoscopies were assayed for bile acids. samples were then assayed by luminex for cytokines , and by elisa for pulmonary collectins (sp-a, sp-d). results were analyzed according to levels of bile acids as per roc testing for accuracy for bronchiolitis obliterans syndrome diagnosis (high levels ≥3.5 µmol/l). results: we prospectively examined 120 lung transplant recipients and a total of 271 bal samples were collected. in 114 no bile acids were detected, low levels were present in 110, and high levels were detected in 47 samples. samples with high bile acids had significantly greater innate (tnf-a, il-1b, il-12, il-10, il-6) and adaptive (ifn-g, il-2) cytokines as well as greater chemokines (mcp-1, il-8) compared to the other samples. in contrast pulmonary collectins sp-a and sp-d were significantly reduced in samples with high bile acids. the figures shows the median and interquartile range for each molecule according to bile acid levels. conclusion: bile acids detected in the bal as markers of aspiration stimulate the lung allograft immunity in a dose dependent fashion. in particular high levels of bile acids are associated with an impaired lung specific innate defense system provided by the pulmonary collectins and with a broncho-alveolar district "cytokine storm". tolerance/immune deviation iii the results of using ex vivo cd4+ t-cells converted dn t-cells in nod mouse models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the treatment of autoimmune type i diabetes. horng-ren yang, 1 gouping jiang, 1 john j. fung, 1 shiguang qian, 1 lina lu. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. liver transplant tolerance was recognized by spontaneous acceptance of liver allograft in many species. the underlying mechanism remains unclear. interestingly, although liver allografts are accepted, hepatocyte transplants in the same combination are promptly rejected, indicating a crucial role of liver tissue cells in immune suppression. we have demonstrated a profound t cell inhibitory activity of hepatic stellate cells (hpsc), which are known to participating in repairing and fibrosis during liver injury. addition of activated (a) hpsc, but not quiescent hpsc, significantly inhibited allo-dc induced-t cell proliferative responses (mlr) in a dose dependent manner, which was associated with enhanced t cell apoptosis (tunel). neutralization of b7-h1 by anti-b7-h1 mab significantly reduced the hpsc-induced t cell apoptosis and reversed the inhibition of t cell proliferation, suggesting a key role of b7-h1. to evaluate this in vivo, balb/c islets (300) were co-transplanted with 3 x 10 5 activated hpsc (b6) into stz-induced diabetic b6 recipients. co-transplant with hpsc effectively protects islet allografts from rejection. this was associated with reduction of graft infiltrating t cells and enhancement of apoptotic activity. co-transplant with hpsc from b7-h1 -/livers markedly lost their islet graft protective capacity, associated with less apoptosis of infiltrating cells. to determine the subsets of apoptotic t cells, t cells were isolated from spleen, draining lymph nodes, and grafts for phenotype and function analyses. on pod 8, a marked reduction of graft infiltrating cd4 + (30.7 %) and cd8 + t cells (31.3 %) was seen in hpsc co-transplant group, as compared to islets alone, which was further progressed thereafter. cd8 t cells dropped ∼7 folds on pod 140. cd4 + / cd8 + ratio was increased from 0.5 at the early pod to 2.0 in the long-term survival grafts. adoptive transfer of cfse-labeled des transgenic t cells was used to track the response and fate of antigen-specific cd8 + t cells. the results showed active division of des + cells within the allograft in both the islet only and hpsc co-transplantation groups. however, accumulation of these des + cells was significantly lesser in the hpsc co-transplantation group as compared to that in the islet only group (1.9×10 3 vs. 10×10 3 cells per graft). these findings suggest that hpsc induce antigen-specific cd8 + t cell death, and may not inhibit their activation. donor-specific memory t cells are potent mediators of allograft rejection due to their ability to proliferate and give rise to cytotoxic and inflammatory cytokine-secreting effectors within hours of stimulation. furthermore, memory t cells have been shown to be relatively resistant to the effects of many tolerance-induction protocols, including blockade of the cd28 and cd154 pathways. while seminal studies have shown that donor-reactive memory cells, generated through pre-sensitization with donor tissue or infection with pathogens with cross-reactive epitopes, contribute to costimulation blockade-resistant rejection of fully mhc disparate allografts, the role of memory t cells specific for minor antigens in costimulation blockade-resistant rejection has not been well studied. we addressed the ability of memory t cells specific for a single donorderived class i epitope to mediate this process. tcr transgenic t cells (ot-i) specific for siinfekl/kb were adoptively transferred into naive b6 recipients, which were then infected with ovalbumin-(siinfekl) expressing listeria monocytogenes (lm-ova). at memory, mice received a skin graft expressing ovalbumin (mova), and therefore the siinfekl epitope. results showed that the transferred ot-i t cells proliferated in response to lm-ova, but not in response to a control infection with wild-type listeria (lm). at day 9, ot-i t cells comprised ∼15% of the total cd8+ t cell compartment. during memory, the siinfekl-specific cells comprised ∼1-2% of the total cd8+ t cell compartment. engraftment of mova skin on lm-ova memory recipients resulted in rejection with accelerated kinetics relative to lm infected controls (mst=13d vs 20d). following treatment with ctla-4 ig and anti-cd154, 9/10 lm-ova infected recipients experienced costimulation blockade-resistant rejection, while lm-infected controls went onto long-term graft survival (p<0.001). lm-ova-infected recipients also resisted the engraftment of mova-expressing donor bone marrow following a tolerance induction protocol containing busulfan, ctla-4 ig, and anti-cd154. these results suggest that memory t cells specific for a single surrogate minor antigen are sufficient to induce costimulation blockade-resistant rejection, and support the feasibility of using this model to study the specific requirements for donor-reactive memory t cell activation and tolerance induction during transplantation. the during an immune response, cd4 helper t cells can be instructed by non-antigenspecific signals to differentiate into functionally distinct subsets with mutually exclusive patterns of cytokine production. we find that ikaros, a zinc finger transcription factor required for lymphocyte development, is crucial for the development of polarized t helper subsets. in the absence of ikaros dna binding activity, cd4 t cells induced to undergo th2 differentiation in vitro or in vivo produce high levels of il-4, but fail to silence expression of ifn-gamma and il-17, cytokines that contribute to inflammatory disease processes such as autoimmunity and organ transplant rejection. similarly, ikaros is required for repression of il-4 and il-17 expression by th1 cells, and inhibition of ifn-gamma and il-2 production by th17 cells. our results show that ikaros controls the expression of transcription factors such as gata-3, c-maf, stat-6, and runx3, and in polarized th2 cells, ikaros inhibits ifn-gamma gene expression through direct repression of the t-bet locus. these studies place ikaros, a dna binding protein previously recognized only as a regulator of lymphocyte development, as a master regulator of peripheral t cell differentiation and function. introduction as the fastest growing subpopulation seeking organ transplantation is >65 yrs of age, it will be imperative to discern how aging impacts the acquisition of transplantation tolerance. prior work has demonstrated that viral infections induce the development of alloreactive t cells, which impede the induction of transplantation tolerance. in this study, we tested the hypothesis that aging alters host defense against viruses leading to the development of cross-reactive t cells, which impair transplantation tolerance induction. we first examined how aging modifies the function of plasmacytoid dcs (pdcs), as ifnα production by pdcs is essential for control of viral infections. using both in vitro and in vivo murine systems, we found that aged pdcs produced lower levels of ifnα in response to tlr9 activation with cpg sequences or herpes simplex (hsv)-2 virus (elisa). aged mice (18-20 mths) failed to the clear this virus as effectively as young (2-4 mths) mice. this was associated with increased liver inflammation, the release of systemic th17-skewing cytokines, il-6 and il-21, and augmented splenic il-17 levels in aged mice (elisa). prior to transplantation, unmanipulated aged mice (b6 or cba background) produced more donor-specific il-17 effector-memory t cells as compared to unmanipulated young mice (elispot). furthermore, mlr assays demonstrated that aged memory cd4+ t cells from unmanipulated mice produced significantly more il-17 in response to donor antigen compared to young t cells (elisa). to determine if aged recipients manifest an altered response to therapies that prolong allograft survival, aged and young b6 mice received balb/c skin allografts and perioperative anti-cd45 and anti-cd154. we found that aged mice rejected their allografts significantly faster (median survival 20 days) than young mice (mst, 76 days, p <0.01). similar results were noted when we employed perioperative treatment with anti-cd154 + dst and when we altered the donor-recipient (b6 to cba) strain combination. pre-treating aged mice with an anti-il-17 mab improved the efficacy of the graft-prolonging therapy compared to aged mice that received control mab (p = 0.04). conclusion our results suggest that impaired control of viral infections with aging leads to the generation of alloreactive il-17 producing t cells that impair therapies that may induce transplantation tolerance. prevention of type 1 diabetes by thymus genetic modification with protective mhc class ii molecules. jesus r. paez-cortez, 1 michela donnarumma, 1 chaorui tian, 1 john iacomini. 1 1 transplantation research center, boston, ma. introduction. susceptibility to type 1 diabetes is determined by multiple genetic factors, among the strongest of which is the inheritance of at-risk genes that lead to disease development. here we examined whether diabetes can be prevented by providing protective mhc class ii genes through directly infecting the thymus of diabetes prone nod mice. methods. after direct exposition of the thymus, lentiviruses encoding control (phage-cmv-dsred-ires-zsgreen-w) or protective mhc class ii iaβ d (phage-cmv-iaβ d -ires-zsgreen-w) genes were injected in a single thymic lobe of 3 to 4 week old female euglycemic nod mice. gene expression was determined by microscopic examination at different time points after injection and blood glucose levels were monitored weekly to examine whether this approach prevents the development of diabetes. the presence of diabetogenic cd8 + t cells were detected by flow cytometry via tetramer staining of splenocytes. pancreatic islet integrity and insulin production was assessed by immunohistochemistry. results. viral gene expression was observed exclusively in thymic epithelial cells beginning at 5 days post-injection in both groups. nod mice injected with control lentivirus developed diabetes by 22 weeks post injection, similar to non-treated animals (18-20 weeks). in contrast, phage-cmv-iaβ d -ires-zsgreen-w injected animals remained normoglycemic at 12 months post-injection. diabetogenic cd8 + t cell population were not detected in splenocytes of iaβ d injected animals using mhc class i tetramers. islet integrity and insulin production was preserved in the treated group, in marked contrast to controls, which exhibited characteristic lymphocytic infiltration of islet cells and low insulin storage. conclusions. thymic genetic modification with protective a mhc class ii iaβ d molecule can be used to prevent diabetes in nod mice. central deletion of diabetogenic t cell populations may be involved in prevention of autoimmunity in these animals. role of invariant nkt cells in liver sinusoidal endothelial cell-induced immunosuppression of t cells with indirect allospecificity. masayuki shishida, hideki ohdan, yuka tanaka, masataka banshodani, yuka igarashi, toshimasa asahara. surgery, hiroshima university, hiroshima, japan. we have reported that liver sinusoidal endothelial cells (lsecs) endocytose portally injected allogeneic splenocytes and can negatively regulate t cells with indirect allospecificity via the fas/fasl pathway. as a result of in vitro transmigration across the lsecs from balb/c mice treated with a portal injection (pi) of b6 mhc class iideficient (c2d) splenocytes, the naive balb/c cd4 + t cells lost their responsiveness to the stimulus of balb/c splenic antigen presenting cells (apcs) that endocytosed the donor-type alloantigens. however, they maintained a normal response to the stimulus of balb/c apcs that endocytosed third-party c3h alloantigens. in the present study, we examined whether invariant nkt (inkt) cells influence the ability of lsecs to endocytose irradiated allogeneic cells. balb/c wild-type (wt) mice or balb/c cd1d-deficient (cd1d -/-) mice that lacked inkt cells were portally injected with 30×10 6 irradiated b6 c2d splenocytes labeled with pkh-26. only 3.56 ± 3.19% lsecs endocytosed the labeled splenocytes in the balb/c cd1d -/mice at 12 h after pi, whereas 16.82 ± 2.69% lsecs endocytosed the labeled splenocytes in the wt control mice (p < 0.001, n = 4 each). when balb/c wt mice intraperitoneally received 4 µg α-galcer, before pi of b6 c2d splenocytes, the expression of mhc class ii on lsecs and endocytic activity of lsecs were enhanced. thus, we found that the endocytic activity of lsecs was regulated by the inkt cells. intraportal adoptive transfer of lsecs isolated from balb/c wt mice, treated with a pi of b6 c2d splenocytes, into balb/c mice significantly prolonged the survival of subsequently transplanted heart allografts (n = 4) as compared to the adoptive transfer of lsecs isolated from balb/c cd1d -/mice, treated similarly, into the balb/c mice (n = 4). however, intraportal adoptive transfer of lsecs isolated from balb/c wt mice, which received 4 µg α-galcer intraperitoneally prior to pi of b6 c2d splenocytes, into balb/c mice did not result in a further prolonging of the effect (n = 4). these findings indicate that inkt cells are required for such lsec-induced immunosuppression of t cells with indirect allospecificity; however, α-galcer-induced activation of inkt cells does not promote such suppressive effects on these t cells. in conclusion, naive inkt cells play a pivotal role in the lsec-induced immunosuppression of t cells with indirect allospecificity. notwithstanding the considerable amounts of in-vitro data supporting the nonimmunogenicity and immunomodulatory effects of mesenchymal stem cells (msc), scanty and conflicting data are available on their in-vivo immunomodulatory capacities. in this study we formally investigated whether msc had immunomodulatory properties in solid organ transplantation, using a semi-allogeneic heterotopic heart transplant mouse model, and studied the underlying mechanism(s). msc, isolated from bone marrow by adherence, were depleted from cd45+cd11b+ cells before injection. bone marrow-induced hematopoietic mixed chimerism is the most robust mechanism for the induction of transplantation tolerance. however, immunogenicity of bone marrow cells requires harsh immunosuppressive regimens that can lead to severe sideeffects including death. here, we examined whether embryonic stem (es) cells can be successfully coaxed to form hematopoietic progenitor cells (hpc) which potentially could be less immunogenic than bone marrow cells. here, we transduced es cells with hoxb4, a hematopoietic transcription factor that confers self-renewal properties to hematopoietic cells and differentiated them into hematopoietic cells. transduced cells had a 100-1000fold greater proliferation capacity than controls. at the end of the differentiation procedure, most cultures were >80 % cd45 + . hpcs were purified using immunomagnetic bead separation. the separated cells were further characterized for leukocyte markers and showed a high percentage of cd34, cd117, cd31 and low class i, but no class ii expression. further, they poorly express co-stimulatory molecules such as cd80 and cd86. when transplanted in rag2 -/γ c _/_ mice, hpcs fully reconstituted bone marrow, forming multi-lineage hematopoietic cells. to now determine whether these cells engraft in allogenic recipients, the cells were transplanted in syngeneic and allogenic mrl mice. all transplanted animals became chimeric (n>30), reaching 20-30 % after 28 days. thereafter donor cells declined as a result of out-competition by resident bone marrow cells. this pattern was identical in both syngeneic and allogenic recipients. unexpectedly, allogenic chimeric mice became tolerant to donor-type cardiac allografts as monitored over 100 days. grafts showed no mononuclear cell infiltration or signs of chronic rejection. interestingly, the t cells in tolerant animals showed responses to mrl alloantigen similar to that of controls, suggesting that our protocol was likely non-deletional, but could involve regulatory t cells. indeed, when stained for cd25 + foxp3 + cells, the allografts showed a high percentage of these cells, but not in controls, confirming our hypothesis. thus, these data show for the first time the potential of es-derived hpcs to regulate engraftment of allografts, providing an alternative approach for the induction of transplantation tolerance. we had previously shown that a20 is part of the regulatory atheroprotective response of endothelial (ec) and smooth muscle (smc) cells to injury. a20 is a nf-κb dependent gene with potent anti-inflammatory effects in ec and smc, through blockade of nf-κb. a20 also serves an anti-proliferative function in smc and opposite anti-apoptotic or pro-apoptotic functions in ec and neointimal smc. based on these functions, a20 would be a good candidate to prevent transplant arteriosclerosis (ta) and chronic rejection in vascularized organ grafts. this is supported by a20 expression in ec and smc correlating with the absence of ta in rat kidney allografts and long-term functioning human kidney allografts. fully mismatched c57bl/6 (h2 b ) and balb/c (h2 d ), were used as donors and recipients of an aortic to carotid allograft. in this combination, ta lesions start at 4 weeks and become occlusive by 8 weeks when left without immunosuppression. a20 expression in the graft was achieved by recombinant adenoviral (rad) mediated gene transfer prior to retrieval. control mice were infused with saline or control rad beta-galactosidase. the grafts were harvested at 4 weeks and analyzed for ta lesions by measuring intima to media ratios (i/m) and for markers of inflammation and of the immune response by immunohistochemistry. a20 expressing vessels were significantly protected from intimal hyperplasia with i/m reaching 0.4± 0.09 as compared to saline (1.62±0.17) and beta-gal (1.86±0.06) treated vessels. this effect of a20 did not associate with a decrease in infiltrating cd3, cd4 or cd8 t cells in a20 vessels as compared to controls. a possible modification of the phenotype of these t cells (t-regs vs. effector t cells) is being explored. rather, protection from ta correlated with increased expression of endothelial and inducible nitric oxide synthases (nos) in ec and smc of a20 expressing vessels, suggesting that this effect was, at least in part, related to increased in situ production of nitric oxide. in conclusion, we present the first direct evidence that expression in the vessel wall of the anti-inflammatory and atheroprotective protein a20 prevents transplant arteriosclerosis through a mechanism implicating increased expression of nos. carbon monoxide inhalation reverses established chronic allograft nephropathy through the no pathway. g. faleo, a. nakao, j. kohmoto, r. sugimoto, k. tomiyama, a. ikeda, m. a. nalesnik, d. b. stolz, n. murase. thomas e starzl transplantation institute, university of pittsburgh, pittsburgh, pa. chronic allograft nephropathy (can) is the most common cause of graft loss; however an established therapeutic strategy in preventing/treating can is not yet available. we have previously shown that carbon monoxide (co) effectively inhibits can development. here, we examine the mechanisms of co in overturning can through vascular endothelial cell protection. methods: orthotopic kidney transplantation (ktx) was performed in lewis to binephrectomized bn rats under brief tacrolimus (0.5 mg/kg, d0-6, im). by d60 after ktx, bn developed can with decreased creatinine clearance (ccr, 0.58 ±0.1 ml/min), significant proteinuria (92.8 ±30.5 mg/24h), and increased banff scores for intimal arteritis, interstitial fibrosis, and tubular atrophy. recipients were then treated with inhaled co 20 ppm from d60 to d150. results: inhaled co effectively reversed the severity of can and markedly improved renal function at d90 (table) and recipient survival (>150d vs. 81d air control). co treatment resulted in reduced cytokine mrna levels (tnf-α, ifn-γ) and improved banff scores compared to untreated controls. in untreated allografts, cd31 expression on peritubular capillaries (ptc) was markedly diminished, while co-treated grafts showed normal cd31 expression, suggesting significant improvement in maintaining ptc integrity with co. interestingly, enos and inos protein expression was significantly upregulated in untreated grafts, while it was maintained at steady levels in co-treated grafts. immunohistochemistry revealed enos expression on vascular endothelial cells while inos on infiltrates. further, serum nitrate/nitrite levels were significantly higher in untreated than in co-treated recipients. elevated levels of mda, a marker for oxidative stress, in air control group were accordingly reduced in co-treated grafts. renal cortical blood flow data showed a better perfusion in co-treated group at 90d (69. chronic allograft vasculopathy (cav) is a component of chronic rejection and a major cause of graft loss. non-immunologic factors and indirect allorecognition participate in the pathogenesis of cav. new therapies with tolerogenic dendritic cells (dcs) are based on in situ-delivery of alloag to "quiescent" dcs of the recipient's lymphoid organs via apoptotic cells, vesicles or particles. we have shown that the ability of apoptotic cells to deliver alloag and an inhibitory signal to dcs down-regulates the indirect alloresponse and prolongs allograft survival in mice. aims: to test if targeting of recipient's dcs in situ with donor apoptotic cells ameliorates cav by down-regulating indirect pathway allo-immunity. methods: we performed functional aortic (abdominal) transplantation in mice [balb/c→c57bl/6 (b6)]. b6 mice were injected i.v. with 10 7 balb/c uvb-induced early apoptotic splenocytes (d-7). sixty days later, grafts were evaluated in sections with h&e, vangieson's (elastic fibers) and masson's (collagen) techniques. cfse-labeled 1h3.1 tcrtg cd4 t-cells specific for ia b (b6) loaded with ieα 52-68 (balb/c) were used to evaluate the indirect pathway t-cell response. results: pkh67 + balb/c apoptotic cells injected (i.v.) in b6 mice were captured by splenic cd8and cd8α + dcs, but not plasmacytoid dcs. splenic dcs with apoptotic cells remained quiescent in vivo (mhc-i/ii lo , cd80/86 lo , icosl + , pdl-1/2 + ) and were unable to up-regulate mhc-ii and cd86 upon culture with gm-csf. injection of donor apoptotic cells induced defective activation and deletion of indirect pathway 1h3.1 cd4 t-cells. therapy with donor apoptotic splenocytes reduced intimal thickness in aortic allografts (100±21 vs.196±28mm in controls; p<0.001 ) and proliferation of α-smooth muscle cells and collagen deposition. the effect of apoptotic cells was allospecific, superior than that of cells alive, and depended on the physical properties of apoptotic cells, since necrotic cells did not achieve the effect. treatment with donor apoptotic cells decreased significantly the indirect pathway t-cell response (assessed by elispot for ifn-γ) and reduced the level of circulating alloab. conclusion: in situ-targeting of recipient's dcs with (early) apoptotic cells carrying donor alloag is a novel approach to prevent cav by down-regulating the indirect pathway alloresponse. the antifibrotic agent, pirfenidone, has direct inhibitory effects on t cell activation, proliferation, and cytokine and chemokine production, leading to suppression of host alloresponses. gary a. visner, 1 fengzhi liu, 1 hanzhong liu, 1 liqing wang, 2 wayne w. hancock. 2 1 medicine, children's hospital boston, boston, ma; 2 pathology, children's hospital of philadelphia, philadelphia, pa. there is an urgent need to develop new therapies effective against the fibrotic and other complications of chronic allograft rejection. while pirfenidone (pfd) is an established anti-fibrotic agent, we previously showed that pfd treatment reduced acute rejection in a rat lung transplant model suggesting that it might have direct immune modulating properties. accordingly, in this study, we tested the effects of pfd on t cell responses. we first evaluated whether pfd alters t cell proliferation and cytokine release in response to t cell receptor (tcr) activation in vitro. since pfd can inhibit tgf-β by mononuclear cell fractions, we also examined whether pfd affects the suppressive effects of regulatory t cells (cd4+cd25+). the effects of pfd on alloantigen-induced t cell proliferation in vivo were then assessed by adoptive transfer of cfse-labeled t cells across a parent->f1 mhc mismatch, as well as by using a murine heterotopic cardiac allograft model (balb/c->c57bl/6). pfd was found to significantly inhibit tcr-stimulated cd4+ t proliferation in vitro (p<0.01), whereas cd8+ t cell proliferation was not significantly affected. while the beneficial effects of pfd were not associated with increased cd4+ t cell apoptosis, pfd use inhibited tcr-induced production of multiple cytokines and chemokines, including . interestingly, there was no change on tgf-β production by purified t cells, and pfd also had no effect on the suppressive properties of naturally occurring regulatory t cells. similar to the in vitro studies, pfd inhibited allo-antigen-induced t cell proliferation in vivo (parent->f1 model), and showed synergistic effects with low dose rapamycin in this model. lastly, though pfd alone did not affect the tempo of acute cardiac allograft rejection across a full mhc mismatch, use of pfd plus a subtherapeutic regimen of rapamycin significantly prolonged allograft survival (p<0.05), decreased mononuclear cell infiltration and prevented development to chronic rejection, including arteriosclerosis and myocardial fibrosis. we conclude that pfd may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses. we have previously reported studies in miniature swine showing that transplantation (tx) of prevascularized donor islets as part of composite islet-kidney (i-k) reversed diabetic hyperglycemia across fully allogeneic barriers, while free islets did not. in order to test the potential clinical applicability of this strategy, we have extended it to a fully allogeneic nonhuman primate model. methods: two diabetic baboons received composite iks and one diabetic baboon received free islets across fully allogeneic barriers. (1) i-k preparation in donors: two i-ks were prepared by isolating islets from 60% partial pancreatectomies and injecting them under the autologous renal capsule, allowing for vascularization before allogeneic tx. these i-ks were harvested at days 203 and 102 for allogeneic ik tx. (2) induction of insulin-dependent diabetes (iddm) and allogeneic i-k or free islet tx: all recipients received streptozotocin at 2000 mg/m 2 x (body surface area). iddm was induced successfully in two animals, while one animal required total pancreatectomy to induce iddm. after confirming iddm by fasting blood sugar (fbs) >300mg/dl for 3 consecutive days, either i-ks or free islets were transplanted (single donor to each recipient) with atg (day -3) followed by mmf and low-dose tacrolimus. free islets were injected into the liver through the ileocolic vein. islet function was assessed by fbs and renal function was assessed by serum creatinine. immunologic status was examined by cml/mlr assays. results: all three recipients had strong ctl/mlr responses to donors pretx, indicative of a fully allogeneic combination. fbs decreased immediately after i-k tx and no insulin therapy was required throughout the experimental period (days 225 and 279). bs levels averaged 82.2+/-15.6 mg/dl in the first baboon and 125.4+/-42.7 mg/dl in the second. normal creatinine levels (<1.0mg/dl) were maintained by these life-supporting ik grafts. in contrast, the recipient of allogeneic free islets had unstable bs levels and required insulin from day 60 (bs 400 at day 60) conclusions: life-supporting i-ks from single donors achieved glucose regulation without insulin therapy and maintained normal renal function. these results demonstrate the feasibility of composite i-k tx in a non-human primate allogeneic model, with possible clinical applicability for the cure of diabetic nephropathy. donor cell infusion without immunosuppression as a novel therapy for induction of donor-specific tolerance in islet cell transplantation. xunrong luo, 1 kathryn pothoven, 1 derrick mccarthy, 2 matthew degutes, 2 aaron martin, 2 xiaomin zhang, 3 guliang xia, 3 dixon kaufman, 3 stephen miller. 2 1 medicine, northwestern university; 2 microbiology and immunology, northwestern university; 3 surgery, northwestern university, chicago, il. background in autoimmune models, peptide-pulsed splenic antigen presenting cells that are chemically fixed with ethylcarbodiimide (ecdi) have been used as a powerful and safe method to induce antigen specific t cell tolerance. ecdi fixed donor cells for allo-antigen specific transplant tolerance has not been well studied. material and methods c57bl/6 mice were rendered diabetic by stz. kidney subcapsular allogeneic islet transplant was performed 10 days after diabetes stabilization (day 0). 1x10 8 ecditreated donor splenocytes were injected i.v. either once (day -7) or twice (day -7 and day +1). animals were analyzed for graft outcome. results control mice receiving islet graft alone rejected the graft between day 11 to day 18 (mst = 14 days, n=8). mice receiving one dose of ecdi-treated donor splenocytes (on day -7) showed similar graft survival as controls (mst = 15 days, n=5). in contrast, mice receiving 2 doses of ecdi-treated donor splenocytes (on day -7 and day +1) showed significant prolongation of graft survival with 72.7% functional grafts at day 60 (n=11), and some remained functional >100 days. this protection is donor-specific as mice receiving ecdi-treated sjl cells rejected balb/c islet grafts as controls (mst = 18 days, n=4). immunohistochemistry of protected grafts showed positive insulin staining within well-defined islet architecture. peri-islet infiltrates were composed of cd4+, cd8+, and cd11c+ cells, with occasional foxp3+ cells. anti-donor antibody production (igg1,g2a,b, g3) was completely abolished in long-term graft survivers. this tolerance was undisturbed by anti-cd25 antibody treatment during maintenance stage, but could not be established if treatment was given around the time of the first donor cell infusion. in addition, lack of pd-l1 also impaired tolerance induction evidenced by using pd-l1 -/as recpients. conclusion 1. multiple infusions of ecdi-treated donor splenocytes significantly prolonged allo-graft survival in the islet transplant model. 2. the protective effect is donor-specific and is dependent on regulatory t cells as well as the pd-l1 signaling pathway. therapy with ecdi-treated donor cells may emerge to be a novel and potent agent for induction of donor-specific transplant tolerance. mice. rebecca stokes, 1 k. cheng, 1 c. scott, 1 w. hawthorne, 2 p. o'connell, 2 j. e. gunton. 1 1 garvan institute, darlinghurst, nsw, australia; 2 nptu, westmead, nsw, australia. the aim was to investigate the effects of increasing hif-1α protein in human islets upon islet-transplant outcomes. hif-1α is a transcription factor which co-ordinates a program of cellular responses to stressors including hypoxia. in other cell-types hif-1α improves survival following hypoxic-challenge. hif-1α functions as a heterodimer with arnt which we have shown to be important for normal β-cell function (1) . islet transplantation subjects islets to hypoxia. it is thought up to 70% of islets die within 1 week of transplantation and this is at least partly due to hypoxia. the role of hif-1α in β-cell function is unknown. we hypothesized that increasing levels of the protective factor hif-1α in islets before transplantation would improve survival and engraftment and thus improve islet transplant outcomes. isolated human pancreatic islets from 9 separate donors were cultured overnight in control media, or media supplemented with desferrioxamine (dfo), a small molecular stimulator of hif-1α protein. islets were transplanted into diabetic scid mice. there were 3 transplant groups, with mice receiving: 1.supra-physiological-mass transplant of 2000 control-cultured ieq (islet equivalents) 2.minimal-mass-transplant of 600 control-cultured ieq, or 3.minimal-mass-transplant of 600 ieq cultured with dfo. for each human donor, at least 1 of each of the 3 transplant groups was performed to avoid the confounder of inter-donor variability. recipients of 2000 control ieq cured in 42% of cases. minimal-mass-transplantation was ineffective: 600 ieq cured 0% mice at 28-days. however, minimal-mass-transplant of dfo treated islets had 53% success (p<0.001 vs group 2 and p=ns vs 2000-control-ieq). blood glucose levels were markedly improved in the 600 dfo treated group compared to 600 ieq control group (p<0.0001) and equivalent to 2000 control ieq transplants (p=ns). this data demonstrates increasing hif-1α in human islets prior to transplantation markedly improves islet transplant outcomes. hif-1α and dfo may have a therapeutic role in human islet transplantation. long-term disappearance of neovascularization of transplanted islets. eba hathout, nathaniel chan, annie tan, john chrisler, john hough, naoaki sakata, john mace, ricardo peverini, richard chinnock, lawrence sowers, andre obenaus. loma linda university, loma linda, ca. we recently reported an in vivo time-line for neovascularization of transplanted islets using dynamic contrast enhanced (dce) magnetic resonance imaging (mri) over a 14-day period. however, vascularization of transplanted islets must be maintained for extended periods to provide long-term function. in this dataset, we investigated whether vascularization was maintained in transplanted feridex-labeled syngeneic murine subcapsular islets (400 ieq per kidney) using dce imaging on an 11.7t mr scanner and subsequent immunohistochemistry over 180 days. sub-capsular transplants could be visualized at post-transplant days 3 and 14 using t2 weighted imaging. however, the islets could not be seen on mri at post-transplant day 180. injection of the contrast agent gadolinium (gd)-dtpa for dce at 3, 14 and 28 days showed increased signal in the transplant area. at 180 days, there was no change in signal intensity after contrast injection during dce. immunohistochemistry confirmed mri and dce findings. these results suggest that islet neovascularization occurs early after transplantation but is likely not maintained for the 180-day duration of our experiments. this work was supported by nih/niddk grant # 1r01dk077541. a) t2 imaging at day 3 clearly identifies iron-labeled islets (arrows) in the subcapsular region. no iron-labeled islets are observed at day 180 (arrows). b) dce imaging for neovascularization of transplanted islets in the subcapsular region demonstrates a temporal decline in signal intensity. oleanolic acid, a natural triterpenoid, significantly improves islet survival and function following transplantation. n. angaswamy, 1 d. saini, 1 s. ramachandran, 1 n. benshoff, 1 w. liu, 1 n. desai, 1 w. chapman, 1 t. mohanakumar. 1, 2 1 surg, wusm; 2 path & immunol, washington univ sch med, st. louis, mo. oleanolic acid (oa), a triterpenoid in medicinal herbs, is an integral part of normal human diet. oa has anti-oxidant, anti-inflammatory properties (inhibits inos & cox2) & lowers plasma glucose levels. we hypothesis that these properties of oa will prevent early islet cell loss following transplantation & also benefit long term function of allograft. c57bl/6 mice, made diabetic by streptozotocin (200mg/kg) were transplanted with 500 balb/c islets (isolated by collagenase digestion) under kidney capsule. oa (0.5mg/day) was administered i.p. in 100 µl of pbs (with 6m dmso) or pbs-dmso as vehicle control daily from day -1 onwards. blood glucose was monitored daily. immunohistochemical analyses of grafts were performed for cd4 & cd8 markers. cellular immune responses to donor antigens & cytokines produced by cells and in sera were measured using elispot & luminex assays. effect of oa on function of transplant with suboptimal dose of islets (100-250) was also analyzed. optimal dose of islets (500) transplanted into diabetic bl/6 mice administered with oa significantly reduced time taken to reverse diabetes following transplantation (<2±1 vs 4±2 days, p=0.003). further, oa treatment reversed diabetes even with suboptimal dose (200) of islets while untreated animals did not achieve normoglycemia. as expected, control diabetic mice rejected on 6±2 days whereas, oa administration alone prolonged islet allograft survival to 23±3 days (p<0001). oa treatment resulted in >3 fold increase in serum kc, il-10 & vegf (p<0.0003) & 2 fold decrease in mcp-1, ip-10 & il-4 (p<0.005) in luminex assay. stimulation of splenocytes from oa treated mice with donor balb/c cells resulted in significantly reduced ifng (4.5 fold), il-4 (3.5), il-2 (2.3) & il-17 (4). in addition, proliferation in mlr was also reduced 2.5 fold. immunohistochemical analysis of grafts showed significant reduction in cellular infiltration in oa treated animals with reduction in both cd4 and cd8 t cells. daily administration of oa markedly improved islet engraftment & function with reversal of diabetes even when suboptimal dose of islet were transplanted. further, oa treatment allowed significant long term survival of allograft with no other immunosuppression. we demonstrate that prevention of inflammatory signaling cascades by oa resulted in marked reduction of cellular infiltration into graft allowing long term function of allograft. endoplasmic reticulum stress may be an important cause of cell loss after human islet isolation. soon hyang park, 1 michel tremblay, 2 steven paraskevas. 1 1 surgery, mcgill university health center, montreal, qc, canada; 2 mcgill cancer center, mcgill university, montreal, qc, canada. purpose: to evaluate the presence of endoplasmic reticulum (er) stress, induced by conditions to which islets are subjected during isolation (ischemia, nutrient deprivation, thermal stress and cytokine release) in human islets and to determine if this leads to the unfolded protein response (upr), which could alter cell survival. methods: human islets were purified from cadaveric pancreata by collagenase dissociation and continuous density gradient purification. islet preparations were cultured in serum-free medium and sampled at the end of isolation and daily thereafter. total mrna was purified and gene expression evaluated by rt-pcr. activity in upr signaling pathways was evaluated by immunoblot. apoptosis was measured by a caspase-3 activity assay. representative trends observed in >5 isolations are described. results: following isolation, a rapid increase in upr signaling was observed in the perk and ire-1 modules of the upr. these include the phosphorylation of perk target eif2? and splicing of mrna for the transcription factor xbp-1. these changes occurred concurrently with a rapid spike in jnk activity and a rise in expression of the upr target gene chop. after these signals peaked, caspase-3 activity increased with time (apoptotic cells), as did expression of er chaperone bip (surviving cells). conclusion: we consistently observed upr activation in human islets. er stress and the upr may be one important and unrecognized cause of apoptosis in this context. current investigations focus on upr modification and determination of a causal relationship with apoptotic cell death. immunosuppression and the risk of renal transplant failure due to recurrent glomerulonephritis. atul mulay, 1 carl van walraven, 1 greg knoll. 1 1 the ottawa hospital, ottawa, on, canada. glomerulonephritis (gn) is the most common cause of end-stage renal disease among those who undergo kidney transplantation. recurrent gn is a major cause of kidney transplant failure. immunosuppressive medication is used to treat gn in the native kidney prior to the development of end-stage renal disease but the impact of different immunosuppression on recurrent gn post-transplantation is unknown. we used the united states renal data system to determine the association of routine post-transplantation immunosuppressant use with time to renal allograft failure due to recurrent gn. immunosuppressants were treated as time-varying covariates. the study-cohort included patients with kidney failure due to gn who received first kidney transplant between 1990 and 2003. the study cohort included 41,272 patients with a median follow-up of 51 months. ten-year overall graft survival (including death as graft loss) and death-censored graft survival was 56.2% and 70.5% respectively. use of cyclosporine (hazard ratio 0.93; 95% ci 0.62-1.41), tacrolimus (hazard ratio 1.03; 95% ci 0.67-1.60), azathioprine (hazard ratio 0.88; 95% ci 0.68-1.13) or mycophenolate mofetil (hazard ratio 1.10; 95% ci 0.85-1.40) was not associated with risk of graft failure due to recurrent gn after adjusting for important covariates. there was no difference of recurrent gn causing graft failure between cyclosporine and tacrolimus (p=0.4) or between azathioprine and mycophenolate mofetil (p=0.1). however, change in any immunosuppressant during follow-up was independently associated with graft loss due to recurrence (hr 1.31, 95% ci 1.07-1.60, p=0.01).when we restricted the analysis to patients who had no change in immunosuppression during follow-up we again found no association between any of the immunosuppressive medications and the risk of graft loss due to recurrent gn. despite the increased use of tacrolimus, cyclosporine and mycophenolate mofetil to treat gn in native kidney disease, the use of these medications following kidney transplantation had no impact on the risk of graft loss due to recurrent gn. glomerulosclerosis. junichiro sageshima, 1 gaetano ciancio, 1 alessia fornoni, 1 linda chen, 1 carolyn abitbol, 1 jayanthi chandar, 1 warren kupin, 1 giselle guerra, 1 david roth, 1 sherry shariatmadar, 1 gaston zilleruelo, 1 george w. burke iii. 1 1 university of miami miller school of medicine, miami, fl. background: disease recurrence is a major obstacle of kidney transplant for focal segmental glomerulosclerosis (fsgs). anti-cd20 antibody (rituximab) has been used for nephrotic syndrome of native kidney. the significant reduction of proteinuria in transplant recipients with fsgs recurrence was also reported after rituximab use for posttransplant lymphoma. we hypothesized that rituximab induction could alter the posttransplant course of fsgs recipients, particularly in those patients with rapid progression to end-stage renal disease who are higher risk of recurrence. methods: we compared the outcome of transplants for primary fsgs treated with and without rituximab. from jan. 2000 to dec. 2003 received renal allografts along with our "standard" immunosuppressive protocol, consisting of tacrolimus, mycophenolate, corticosteroids, antithymocyte globulin and/or daclizubab. from jan. 2004 to dec. 2007 received rituximab in addition to the "standard" immunosuppression. posttransplant proteinuria was treated with plasmapheresis (pp) and maintenance angiotensin blockade (ab). results: there was no adverse event related to rituximab infusion. the overall incidence of posttransplant proteinuria was significantly lower in recipients with rituximab induction (p < 0.05). four recipients treated with "standard" immunosuppression developed massive proteinuria (u-protein/creat. > 10) immediately following transplantation; they responded poorly to pp and ab. four other recipients had moderate proteinuria. in contrast to this, of the 18 patients induced with rituximab, only 2 had massive proteinuria and 4 had mild to moderate proteinuria which responded well to pp and ab. with a median follow-up of 26 months, there was no significant difference of graft survival between 2 groups (2-year survival: 81% without rituximab vs. 84% with rituximab). a half of the graft loss was related to non-compliance. conclusion: while the mechanism of action is unclear, our observation indicates that rituximab induction may decrease the incidence and severity of recurrence of fsgs following kidney transplantation. a larger-scale study is desirable to confirm this observation. mesangial chimerism in recurrent iga nephropathy. geoffrey talmon, 1 dylan miller. 1 1 department of pathology and laboratory medicine, mayo clinic, rochester, mn. background iga nephropathy (in) is the most common primary glomerulonephritis and nearly 50% of patients who undergo a renal transplant for in recur. data support that bone abstracts marrow-derived cells are capable differentiating into various mesenchymal cells within the kidney. the extent to which this phenomenon versus proliferation of resident mesenchymal cells is involved in populating mesangium is not well understood. the mesangial injury and/or hypercellularity seen in in provides a robust in vivo model for determining if this phenomenon is prevalent in human kidneys. design follow-up biopsies from male patients receiving female renal allografts for in that showed recurrent disease were selected. fluorescent in-situ hybridization and immunofluorescent staining was performed on unstained slides from the paraffinembedded tissue for smooth muscle actin, x, and y chromosome centromeres. cells within nonsclerotic glomeruli with triple positivity (y+) were assumed to be mesangial cells derived from the recipient. results four cases of recurrent in with nonsclerotic glomeruli were obtained, each displaying at least minimal mesangial proliferation by light microscopy (one "minimal", two "mild", one "moderate"). mesangial cells with y chromosome centromeric material were observed in each case (100%). between 6 and 34 mesangial cells were present in each glomerulus (mean 16.45) with no to three y+ cells seen (mean 1.8). these accounted for between 8% and 16% of mesangial cells in individual glomeruli. the ratio of y+ to total mesangial cells in each case ranged from 1:7.8. to 1:11.3 (mean 1:9.35 ). the case exhibiting minimal mesangial hypercellularity had a ratio of 1:9.6, those with mild had ratios of 1:7.8 and 1.8.7, and that with moderate 1:11.3. conclusions recipient-derived mesangial cells make up a fraction of the population of glomerular cells in renal allografts affected by recurrent in. although the number of cases is small, the number of recipient-derived cells does not seem to be directly related to the degree of mesangial hypercellularity seen by light microscopy. the consistent presence of these "colonizing" cells in patients with recurrent in does, however, suggest that there may be a role for targeted therapy directed against circulating recipient cells. in the mid 1970's, patients developing esrd secondary to systemic lupus (sle) were deemed to be poor transplant candidates because of concern for early recurrent lupus nephritis (rln) leading to allograft loss. subsequently, rln was considered an unusual complication of kidney transplantation, occurring in <4% of allografts. however, over the last decade, several reports have shown the frequency of rln to range from 8-30%. we sought to determine the frequency of rln at our center and to identify any clinical variables associated with rln. between 6/1977 between 6/ -11/2005 allografts in 166 patients with esrd due to sle functioned for more than 90 days after engraftment. immunosuppression consisted of azathioprine (aza), or cyclosporine (csa) and aza, or mycophenolate (mmf) and csa, or tacrolimus and mmf depending on the date of transplant. all received steroids. proteinuria was defined as 2+ on dipstick or urine protein/creatinine ratio >0.5. medical charts were reviewed. we found pathologic evidence of rln in 18 (24%) of 75 patients who underwent biopsy due to allograft dysfunction or proteinuria, comprising 10% of all patients transplanted for sle. characteristics of these patients are shown below: randomized studies have shown little or no increase in ar in kidney tx recips on p-free is; but concern remains about long-term outcome. we present 8-yr f/u of a protocol incorporating rapid (<6 days) discontinuation of p, with now over 1000 patients transplanted using this protocol. between 1/1999 between 1/ and 10/2007 between 1/ , 1003 adult tx recips were treated with thymoglobulin (5 doses)(extended in dgf), p (5 days), a cni, and either mmf or srl. of these, 658 were ld (260 lurd); 345 dd. of the 1003, 41% were female;88% white; mean recipient age was 47± 14 years and mean donor age was 39.7± 13.2 years. diabetes was present in 34.3% of the recipients and 12.5% of the transplants were retransplants. the peak pra was >10% in 20% of recipients; 14% had tx pra >10%. table 1 shows actuarial survival rates. graft survival rates were significantly better in ld vs dd transplants (p=0.002) and and acute rejection rates lower (p=0.04). compared to national data from srtr, overall outcomes were not significantly different. with mean follow-up of 3.5 years, a total of 110 (11%) recipients have died -the most common cause being cerebrovascular accident (14%) followed by malignancy (9%). there were only 5 (4.5%) patient deaths due to cardiac causes. of 195 (19.4%) graft losses, 86 (44%) were from dwf (death with function); 43 (10%) from cr/can. renal function has been stable with mean serum cr of 1.7 mg/dl at 3 and 5 years posttransplant with cretinine clearance of 63 and 60 respectively. at 5 years posttx, compared to ppretransplant values, recipients showed a 9.6% increase in weight, a 3% decrease in serum cholesterol, and a 10.8% decrease in serum lipid values. 84% of the kidney recips remain p-free; the most common reason for restarting p was acute rejection (ar). conclusion: short-term data suggests kidney tx recips do well with rapid discontinuation of p. our intermediate-term data suggests that patient and graft survival rates remain good and renal function remains stable. ongoing long-term follow-up is necessary. background. since 4/02 our program has employed a steroid-free, rapamycin and neoral maintenance immunosuppression regimen for kidney transplant recipients. prior to that time recipients were treated with prednisone, mmf and neoral. we noted a significant reduction in acute cellular rejection (acr) after implementing this regimen. this retrospective analysis was performed to examine the impact, if any, on the incidence, character, and outcomes of early ahr. results. this study includes 1611 consecutive kidney recipients transplanted between 1/99 and 12/06. there were 717 recipients in the prednisone, mmf, and neoral era (grp 1) and 894 in the steroid-free, rapamycin and neoral era (grp 2). recipient age, gender, african-american race, ab and dr mismatch, and frequency of pra>10% was not statistically significantly different between the 2 groups. however, 43% of grp 1 pts vs 63% of grp 2 pts received a living donor kidney due to a more recent volume increase in this procedure (p<0.001). there were a total 722 kidney biopsies in 466 pts performed in the first 6 months post-transplant; 478 in 287 pts when excluding pre-perfusion biopsies. nineteen percent (54/287) of these showed >50% peritubular capillary (ptc) c4d deposition. comparison of grp 1 to grp 2 demonstrated the following: 1) the incidence of clinical acute rejection in the first 6 months was 12.7% (91/717) in grp 1 and 5.6% (50/894) in grp 2 (p<0.001), 2) the overall incidence of a c4d+ biopsy was similar in the 2 groups ( conclusions. despite a significant reduction in the incidence of acute rejection using our newer, steroid-free immunosuppression protocol, there has been no reduction in the incidence of early (0-6 months) ahr evidenced by c4d+ kidney biopsy. however, the percentage of ahr unassociated with acr has significantly increased. the poor graft survival in pts with early ahr has not improved with our newer immunosuppression regimen. conclusions: four risk factors for ar were identified in the rdp study population: retx, aa race, age 18-50 (vs. >50) and pra >50 (vs. <50). four risk factors for gl were identified: pre-tx t1 dm, ar, dgf and dd tx (when ar and dgf omitted). these risk factors for ar and gl are the same as we observed in prednisone-containing protocols. additionally, many of these factors are not modifiable. identification of high risk groups allows for individualization of is. increasing lds and utilization of is protocols to decrease or minimize dgf and ar are goals for improving graft outcome. effect with the advent of more potent immunosuppression hla matching has been deemphasized in the allocation of deceased donor kidneys due to the limited impact on acute rejection and graft survival. an unforeseen consequence of poorer matching could be an increase in sensitization of patients in need for a repeat transplant. our study examined candidates listed in the us from 1988-2007 from the srtr database that were re-listed following loss of a primary kidney transplant (n=19,827). the primary outcome of the analysis was change in pra from the listing prior to recipient's initial transplant to the subsequent listing. absolute change in peak and current pra levels were examined in general linear models as well as the proportion of patients with a rise in pra level in a logistic model. results hla(a,b,dr)-matching in the primary transplant was strongly associated with change in pra level (p<.001, figure 1 ). among recipients with 6-hla mm, over 50% had a rise in pra at re-listing as compared to 25% of 0-hla mm recipients. younger recipient and donor age, males, deceased donor transplants and african american recipients were also significantly associated with elevation in pra. in addition, the effect was apparent stratified by primary donor type. while there might be a limited impact of hla matching on graft survival, many patients might be negatively impacted from poor hla matching from their first transplant when needing a second transplant. as high pra is one of the strongest risk factors for not getting transplanted, this should be taken into account when evaluating the impact of hla matching in kidney transplantation. this might be particularly important in younger patients and in patients with a long life expectancy in general because of the high likelihood of needing a second transplant during their lifetime. the were about 25% less likely to die or lose an ecd kidney from a donor aged 50-59 and 70% more likely with kidneys from donors older than 69. a similar trend was seen with older recipients, but the risks seemed to be lower. logistic regression indicates recipients older than age 65 were 5 times more likely to have a donor older than age 70 than recipients younger than 50, hypertension and creatinine >1.5 were less likely in older donor kidneys. interestingly, the use of these kidneys relative to the total number of ecd kidneys has decreased since 2002 (odds ratio=0.63, 0.53-0.76, p<0.001). conclusion: an increase in the supply of kidneys might be achieved with increased utilization from deceased donors older than age 70. outcomes were similar to those from donors age 60-69 in recipients that were older than age 50. outcomes , but approximates non-dcd survival thereafter. dcd listing for retransplantation and graft failure progressed continuously over 180 days versus 20 days in non-dcd. when retransplanted, dcd recipients waited longer and received higher risk allografts (p=0.039) more often from another region. more dcd recipients remain waiting for retransplantation with fewer removed for death, clinical deterioration, or improvement. conclusions: dcd utilization is impeded by early outcomes and a temporally different failure pattern that limits access to retransplantation. allocation policy that recognizes these limitations and increases access to retransplantaton is necessary for expansion of this donor population. orthotopic liver transplantation with allografts from dcd donors. roberto c. lopez-solis, 1 background: in the current era of liver transplantation, organ shortage continues to be a significant problem. the use of extended criteria allografts from donation after cardiac death (dcd) donors to increase transplantation rates is widely practiced. this study is a review of one of the largest single center experiences utilizing dcd donors in the world with a follow-up of almost 15 years. methods: from 03/01/1993 to 10/31/2007, 3,431 liver transplants were performed at our institution, 146 (4.2%) of which were liver allografts from dcd donors. patient and donor demographics, recipient and graft survival, and the incidence of primary non function, hepatic artery thrombosis, retransplantation, and bile duct complications were analyzed for this subset of recipients. results: kaplan meier analysis showed a 1-and 5-year patient and graft survival of 81% and 71%, and 71% and 59%, respectively. the mean age of recipients was 52 ± 10 years with an average meld score of 18.7 ± 9.3 (range, 6 to 40), and there were 101 male patients (69%). donor mean age was 36 ± 16 years and cold ischemia time was 646 ± 173 minutes. one hundred and three patients (70.5%) are alive and 24 (16.4%) underwent retransplantation. the incidence of primary non-function was 11.6% (17 patients) and hepatic artery thrombosis was 4.1% (6 subjects the fda warns against using sirolimus (srl) in liver transplants, reporting increased hepatic artery thrombosis (hat), excess mortality and graft loss when srl is used as initial immunosuppression (is) with calcineurin inhibitors. we report the largest experience to date of patients with srl used as initial is, assessing hepatic artery complications and survival outcomes. materials and method all 1554 olt pts from 1998-2007 were reviewed. those using srl as initial is were identified, and the remaining olt pts from that time period were used as controls. ultrasound assessed graft vascular status and any issues were verified by angiogram. . there were no significant difference in demographics variables, lt indication or pre-lt meld score between the two gr. mean follow-up was 6.8±3.9 months. all the enrolled pts were treated with abstracts an initial dose of cs of 2mg/kg/day, to target 100ng/ml for the first 10 days. pts were randomized on day 10 into one of the two following gr on a 2:1 basis. ev gr: initial dose of ev was 2 mg/day, to reach blood level of 8 ng/ml. the dose was increased on day 30, when cs was discontinued, in order to reach an ev blood level between 10 and 12 ng/ml. cs gr: after the 10 th post-operative day the dose of cs was adjusted to a target level of 250 ng/ml until day 30, then to 200 ng/ml until the end of month 6. all pts received basiliximab induction on day 0 and 5 after lt. pts were weaned off prednisone by 5 weeks. pt survival at 6 and 12 months was similar in the ev and cs gr (95.2% and 86.6% vs 87.5% and 87.5% respectively; p= ns). causes of death were sepsis (1), hcv recurrence (1), pulmonary embolism (1) in the ev gr, and sepsis (1), rupture of splenic artery aneurysm (1) the overall incidence of infection episodes was comparable between two gr (5.9% ev gr vs 12.5% cs gr; p=ns). cholesterol but not triglycerides increased in the ev gr compared with the cs gr (p<.05); ev dose reduction decreased such parameters without the need for statin implementation. conclusion ev monotherapy in de novo lt showed similar patient survival and incidence of morbidity compared to a cs immunosuppressive protocol. the primary endpoint was achieved inasmuch as renal function was statistically better in the ev gr. background: sir is a potent immunosuppressive agent that inhibits t-cell activation and proliferation. in lt recipients, sir has primarily been used as a renal-sparing agent, but its toxicity and tolerability in this population has not been well defined. aims: to identify the adverse effects and predictors of discontinuation of sir in lt recipients. methods: records from 327 adult lt recipients transplanted between 1/2000 and 12/2006 were reviewed. reasons for starting and discontinuing sir were captured, as were all significant adverse effects and laboratory abnormalities. factors predicting sir discontinuation in univariate analysis were further analyzed by multivariable logistic regression (mlr). results: mean age of the study group was 50 ± 11 years, and 75% were male. underlying liver disease was hcv ± alcohol in 56%, 24% had hepatocellular carcinoma, and 14% received living donor grafts. calcineurin inhibitors (cni) were started post-operatively in 91% (85% tacrolimus/15% cyclosporine), with or without mycophenolate and prednisone. 179 patients (54%) started sir a median of 15 days (iqr: 4-138) post-lt primarily for renal insufficiency (76%) or cni neurotoxicity (7%). sir was overlapped with tacrolimus and cyclosporine in 65% and 16%, respectively. prior to starting sir, total cholesterol was 139 ± 69 mg/dl, ldl-cholesterol 90 ± 49, triglycerides 193 ± 126. peak lipids after sir were 263 ± 97, 138 ± 81, and 475 ± 456 mg/dl, respectively, despite lipid-lowering therapy. serum creatinine was 2.05 ± 1.04 and 1.08 ± 0.5 mg/dl before and after sir, respectively. before sir, 70% of patients had no proteinuria, but only 36% had no proteinuria after sir. high range proteinuria (>300mg/dl) was noted in 3% before and 16% after sir. finally, sir was discontinued in total of 81 (45%) patients, for indications of cytopenias (20%), hyperlipidemia (19%), mouth ulcers (11%), sepsis (7.5%), skin reactions (7.5%), nephrotic syndrome (6%), gi intolerance (4%), pneumonitis/boop (2.5%), myopathy (2.5%), and combinations of above (20%). mlr failed to identify any pretreatment predictors of discontinuation. conclusions: immunosuppression with sir improves azotemia at the expense of considerable hematologic, metabolic, dermatologic, renal, pulmonary and muscle toxicity. considering the high incidence of proteinuria after sir treatment, the use of sir as a less nephrotoxic agent must be re-considered. and to identify the most effective protocol. peripheral blood was obtained from 4 ebvseronegative and 4 ebv-seropositive pediatric heart (h) tx patients. lcl vs dc-based methods were compared as follows: (i) lcl (ii) lcl + il-12 (iii) type-1 polarized dc (treated with il1-b, tnf-a, il-6 and ifn-g) loaded with mhc class i-restricted ebv-peptide pool (dc/pep.) and (iv) dc/pep. + il-12. the ebv-specific cd8 + t cell phenotype and function were screened using flow cytometry, ifng elispot and cytotoxicity assays. the yields and the functional activities of in vitro co-cultures differed based on the induction method employed, and on the ebv status of the patients tested. for the ebv-seropositive pediatric htx patients, all four methods resulted in the successful expansion of functional type-1 ebv-specific cd8 + t cells, suggesting that memory cd8 + t cell are readily reactivated in vitro. for the ebv-seronegative pediatric tx patients however, only the lcl + il-12 approach resulted in significant augmentation of type-1 ebv-specific ctls that were competent to secrete ifn-g (400±50/10 5 cells) and to kill (300±170 lu/10 7 cells) ebv + targets. we found that il-27 secreted by lcl (and not by dc) was critical in triggering expression of il-12rb2 on naive cd8 + t cells, and rendering these cells responsive to il-12p70. further addition of exogenous il-12p70 (which is generally not produced by lcl) proved to be essential for effective type-1 priming. however, blocking il-27 during ebv-priming has abolished il-12rb2 expression and subsequent ifng production. these results demonstrate that the inducible expression of il-12rb2 on naïve cd8+ t cells was dependent on il-27, and support the critical early role of ebv infected b cells in the in vivo priming of naïve precursors into potent ebv type-1 cd8 + t cell in children. serial ebv load monitoring in pediatric heart transplant patients (phtx) has identified a group of asymptomatic children that exhibit persistently high ebv loads in peripheral blood (≥16,000 copies/ml on at least 50% samples over a period of at least 6 months). these patients have a high rate of progression to late ptld. our goal is to characterize the deficiency of ebv-specific cd8 + t-cell immunity that allows this state to occur and be maintained. twenty-one stable ebv + phtx patients were categorized as follows: group 1 (n=6) no detectable viral load; group 2 (n=12) low viral load (≤16,000 copies/ ml); group 3 (n=4) high viral load (≥16,000 copies/ml). twelve healthy subjects were recruited as controls. flow cytometric analysis with hla-a2 ebv-tetramer (tmr) probes in conjunction with mabs against memory/activation markers was performed on peripheral blood cd8 + t cells, and their ebv-specific ifn-γ production was measured by elispot. ebv-"latent" specific cd8 + t cells in g2 patients were mostly cd62l + / cd45ro + (central memory) and expressed heterogeneous levels of pd-1 and high cd127 (il-7 receptor α), the ebv-lytic-specific cd8 + t cells were more frequent, and biased toward cd62l -/cd45ro + (effector memory) and cd62l -/cd45ra + (stable effector memory), corresponding to terminally differentiated memory compartments. this cell population also expressed heterogeneous levels of pd-1 and down-regulated cd127. in contrast, both ebv-lytic and -latent specific tmr + cd8 + t cells from g3 patients were homogeneously cd62l + /cd45ro + (effector memory), cd38 + and cd127 -, suggestive of "recently activated" phenotype. interestingly, although patients in groups g2 and g3 had high frequencies of ebv-specific tmr + cd8 + t cells (g2 0.65%±1.5, g3 1.9%±3.5, p=0.3), only g2 patients exhibited a direct correlation between tmr + cd8 + t cells and ebv-specific ifn-γ production. these results demonstrate that different levels of chronic ebv-antigenic pressure trigger significant differences in the phenotypic and functional features of ebv-specific cd8 + t cells from phtx, suggesting that the immunologic characterization of high ebv load carrier state is a combined "activated" phenotype with "exhausted" function of ebv-specific cd8 + t cells. ebv-encoded lmp1 indirectly activates the jak/stat pathway through induction of ifnγ. abstracts evidence of ptld. 15 children were enrolled at 5 sites. mean age at transplant 7.1 years, mean time to ptld 43 months . organs transplanted were lung 6, heart 5, kidney 4. all ptld were of b cell origin and expressed cd20 and all were ebv positive. histology was: polymorphic 11, monomorphic 3, hodgkins-like 1. 7 patients received 4 doses, 7 patients 8 doses and 1 patient 7 doses. treatment was associated with minimal side effects in 12, mild-moderate infusion related reactions in 2 and moderate reaction in 1. no patient had treatment discontinued because of side effects. twelve patients (80%) showed complete response after 4-8 doses, 2 had progressive disease and one had stable disease. at 24 months, 10 (67%) were alive with one graft loss (kidney) and none with residual disease. at latest follow-up (mean 60.4 months, 42-82), 10 remain alive with 1 further graft loss (lung) and no ptld. the 5 deaths occurred between 2.5 and 13 months and were associated with progressive disease (2), chronic rejection (2), and complications of elective surgery (1) . conclusions: these findings support prior registry data and suggest that rituximab without chemotherapy is a successful second line treatment in approximately two-thirds of children with refractory ptld. cancer after organ transplantation in france. jean michel rebibou, 1 fabienne pessione, 1 francois aubin, 1 bernard loty. 1 1 agence de la biomedecine, france. cancer prevention appears as a major challenge in transplantation. estimating cancer frequency is a major step toward designing prevention policy. we report on patterns of cancer incidence among 47000 transplantations registered in the french data base. ). the risk of lung cancer is higher for recipients of a thoracic organ and the risk of kidney cancer appeared higher for kidney recipients. ten year cumulative incidence was 8.6% for all cancer and transplantation types, 1.7% for nhl. multivariate analysis demonstrated that cancer risk increased with recipient age (p<10 -3 ), 10 year cumulative incidence was 15% for recipients older than 60 year. it was higher in male (p<10 -3 ) and in thoracic organ recipients when compared with kidney recipients (p<10 -3 ). cancer incidence did not vary according to the transplantation period (1995 ( -1999 ( vs 2000 ( -2005 .95 p=0.34) and nhl risk was significantly lower during the period 2000-2005 (rr=0.8, p=0.02). this work does not report any increase in cancer incidence among transplant recipients while cancer incidence increased in the general population. the observed decrease of nhl risk is of particular interest. the both costimulatory and co-inhibitory signals are delivered by b7 ligands through the cd28 family of receptors on t lymphocytes determining the ultimate immune responses. although b7-h4, a recently discovered member of the b7 family, is known to negatively regulate t cell immunity in autoimmunity and cancer, its role in transplantation rejection and tolerance has not been established. to study its role in physiologic rejection processes, we first treated b6 wt recipients of balb/c hearts with a blocking mab against b7-h4 or isotype igg control and found no difference in graft survival (mst 7 days, n=8 vs. 7, n=10). however, b7-h4 blockade resulted in accelerated allograft rejection in cd28 deficient b6 recipients (mst 9.5 vs. 19, n=8, p=0.003) , indicating that b7-h4 signaling can mediate negative regulation in the absence of cd28 costimulation. we next studied b7-1/b7-2 double deficient (dko) b6 recipients of balb/c heart allografts, as these mice are truly independent of cd28/ctla-4:b7 signals. while cardiac allografts were accepted in control dko recipients (mst>100, n=5), blocking b7-h4 precipitated rejection (mst 45, n=5, p=0 .01) demonstrating non-redundant functions of these two negative pathways. based on these results, we next evaluated the role of b7-h4 in acquired transplantation tolerance by blocking cd28:b7 using ctla4-ig. b7-h4 blockade abrogated prolongation of allograft survival by ctla4-ig (250 µg on day 2) in the fully mhc-mismatched cardiac allograft model (mst 13 vs. 46.5, n=8, p=0.0002) . we conclude that the novel b7-h4 molecule can regulate alloimmune responses independent of an intact cd28/ctla-4:b7 costimulatory pathway. the interplay between positive (stimulatory) and negative (regulatory) costimulatory signals is an important determinant of the outcome of the alloimmune response and could be exploited to induce tolerance. specific in a model of mhc-mismatched kidney allograft in the rat, treatment with anti-cd28 antibodies induced a form of tolerance independent of treg cells but associated with a two-fold accumulation of mdsc in the blood. to further characterize these cells, we analyzed their phenotype and mechanism of action by flow cytometry, western blotting and suppression assays. mdsc expressed cd80 and cd86, nkrp-1, cd172a (sirpa), cd11a, cd11b, his48 and for a fraction of them cd4 but did not express mhc class ii molecules. mdsc dose-dependently suppressed the proliferation of t cells in mlr and after stimulation with anti-cd3 + anti-cd28 antibodies. although detected in blood, bone marrow, spleen and lymph nodes, mdsc were only suppressive in blood and bone marrow. this suppression was lost after physical separation from the responding t cells by semi-permeable membranes in transwell assays, as well as after addition of l-nmma, a selective inhibitor of inducible nitric oxide synthase (inos), suggesting a role for no in the suppression. western blot analyses revealed that inos was expressed only after contact between mdsc and activated cd4 + cd25effector t cells and to a much lesser extent after contact with activated cd4 + cd25 high t reg cells. mdsc affected the viability of stimulated cfse-labeled effector t cells by blocking their proliferation but not their activation. in contrast, mdsc did not block the response of t reg cells stimulated by anti-cd3 + anti-cd28 antibodies. this selective suppression of effector but not of regulatory t cells was confirmed by cytokine profile analyses. in vivo, the expression of inos was higher in the blood of tolerant recipients, as well as in the graft, as compared with isografted recipients. in addition, the injection in stable tolerant animals of aminoguanidine, which inhibits inos, induced graft rejection within 3 weeks. in conclusion, these results suggest that mdsc, accumulated in the blood of tolerant recipients of kidney allografts, release high levels of no after contact with activated effector t cells and specifically control their proliferative response. liver non-parenchymal components inhibit dendritic cell differentiation and maturation. ching-chun hsieh, 1 horng-ren yang, 1 guoping jiang, 1 john j. fung, 1 shigunag qian, 1 lina lu. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. the inherent tolerogenicity of liver allografts could be due to comparatively large numbers of potentially tolerogeneic antigen presenting cells, in particular dendritic cells (dc). it is not clear whether the unique antigen presenting function of liver dc is intrinsic, or is altered by the microenvironmental factors within the liver. in the present study, we investigated the effect of hepatic stallet cells (hpsc), a unique tissue cells in the liver which were actively expanded in the liver allografts, on generation and function of bone marrow derived dc (bm dc). we hypothesize that liver hpsc may modulate immune response via inhibition of liver dendritic cells (dc) which are known of bone marrow (bm) origin. in this study, dc were propagated from b6 bm cells with gm-csf for 5 days. irradiated hpsc isolated from b6 mouse liver were added at the beginning into the culture at hpsc : bmdc progenitor ratio of 1:20. the differentiation, maturation and function of propagated dc were determined by characterizing their surface molecule expression and functions of instructing t cell activation / differentiation. the results showed that addition of hpsc markedly blocked the differentiation of dc from bm precursors (most cells remained in cd11b + cd11cprecursors stages). the incidence of cd11c + cells was 7% vs. 41.6% in normal bm dc culture (without hpsc). the presence of hpsc also prevented maturation of cd11c + dc, as evidenced by low expression of cd40, cd80 and cd86, but high of b7-h1. the inhibitory effect appeared to be mediated by soluble factor (s) produced by hpsc, since addition of the hpsc culture supernatant or transwell culture provided comparable inhibitory activity. culture of allogeneic cd4 + t cells with hpsc-dc elicited poor proliferative response in a 3d mlr assay, with low il-2 and ifn-γ production. three color staining of t cells stimulated by hpsc-dc showed that cd25 + foxp3 + t cells were preferentially expanded, suggesting that hpsc-dc were capable of inducting treg. in contrast, bm dc induced vigorous cd4 + t cells proliferation, most of activated cd4 + t cells were cd25 + foxp3associated with high levels of ifnγ and il-2 in the culture, indicating induction of th1 cells. in conclusion, liver tissue cells, such as hpsc markedly inhibit dc differentiation and maturation, suggesting that the tolerogenic property of liver dc may not be intrinsic, but is altered by the microenvironmental factors in the liver. . allograft rejection was also significantly accelerated when bm12 hearts were transplanted into cd28ko recipient (mst 14 days). to investigate the mechanisms of these findings, lymphocytes harvested at day 10 post-tx from spleens and regional lymph nodes were assessed by flow cytometry for phenotypic differentiation of the activation status, regulatory t cell markers and effector cell generation. the ratio of effector to regulatory cells was 1.4: 3.8: 5.2 in the wt, cd28ko and b7dko recipients, respectively. cytokine production was evaluated by elispot using donor specific antigen stimulation of recipient splenocytes. the mean ifn-γ production was 173 spots per 500,000 splenocytes in wt, 206 in cd28ko and 702 in b7dko. this study for the first time demonstrates the paradoxical role of b7-cd28 co-stimulation in fully allogeneic and class ii mismatched grafts and proposes a possible mechanism through the re-alignment of the effector to regulatory t cell ratio in favor of a highly alloreactive immune response. the major concern regarding kidney donation has been whether the occurrence of hyperfiltration, on the background of increasing prevalence of hypertension with aging and the decline in glomerular filtration rate (gfr) noted in some people as they get older, may put donors at a higher risk for progressive kidney disease. we have previously reported on donors > 20 years after donation and found them to incur no excessive risk of hypertension or kidney disease. herein, we report on renal and non-renal outcomes on the world's largest experience of kidney donors to date (n=302) who donated more than 35 years ago. methods: kidney donors were asked to fill out a survey detailing their medical history since donation and obtain a physical exam and laboratory testing by their local physician. results are expressed as mean±standard deviation (sd the graph below depicts the cross-sectional distribution of last serum creatinine available 35 years or more after donation regardless whether the donor is presently alive or not though the majority is from currently alive donors. conclusion: in the longest follow-up of kidney donors to date, this data indicates that 4-5 decades of living with one kidney has no serious adverse renal effects and a prevalence of hypertension that is probably similar to the general population considering the age of these donors. centers ) . before and after adjustment for comorbidity, 19 (7.8%) and 18 (7.5%) centers, respectively, met all criteria for review, but only 16 met criteria for review both before and after comorbidity adjustment. we conclude that failure to adjust for pre-existing recipient comorbidity results in grossly inaccurate estimation of expected gf per ktx center, more often resulting in expected gf that is too low. using data that are not adjusted for comorbidity to judge the quality of tx programs could encourage denial of access to high-risk patients. introduction: the purpose of our study was to examine temporal trends in the regionalization patterns and center volume-outcomes relationship for lung transplantation in the united states over the past decade. a retrospective analysis of all adult single-organ lung transplants included in the scientific registry of transplant recipients for three consecutive time periods between 1997 and 2006 was performed. for each time period, lung transplant centers were divided into three groups based on each center's annual volume of the procedure (low-volume group = 1-17 procedures per year, medium-volume group = 18-30 procedures per year, high-volume group = greater than 30 procedures per year). oneyear observed-to-expected patient death ratios were then calculated and compared for each group in each time period. a temporal analysis of the percentage of transplants being performed relative to center volume was also performed. statistical comparisons were made using chi square testing. results: a total of 12,603 lung transplant procedures were included in the analysis. in period 1, there was not a significant difference in the one-year observed-to-expected patient death ratio of low-volume lung transplant centers when compared to high-volume centers (ratio 1.15 for low-volume centers vs. 0.79 for high-volume centers, p = 0.07). by period 3, however, a significant relationship between center volume and outcomes had emerged (ratio 1.30 for low-volume centers vs. 0.79 for high-volume centers, p = 0.006). over this same time period, the percentage of lung transplants within the united states that are performed at low-volume centers has decreased significantly (from 43.3% of all lung transplants in period 1 to 22.7% in period 3, p < 0.0001), while the percentage being performed at high-volume centers has increased significantly (from 20.5% of all lung transplants in period 1 to 51.3% in period 3, p<0.0001). conclusions: a significant relationship between center volume and patient outcomes has emerged for lung transplantation over the past decade. at the same time, the percentage of these procedures being performed at high-volume centers has increased. these findings suggest that regionalization patterns for a given procedure may be influenced by the presence or absence of a volume-outcomes relationship for that procedure. liver transplantation (lt) has emerged as one of the few curative treatment modalities for patients with hepatocellular carcinoma (hcc). however, the increase in the incidence of hcc recurrence due to immunosuppressants administered after lt is a serious issue. we have recently proposed a novel strategy of adjuvant immunotherapy for preventing the recurrence of hcc after lt: intravenous administration of il-2-stimulated natural killer (nk) cells extracted from donor liver graft to liver transplant recipients. since the immunosuppressive regimen currently used after lt reduces the adaptive immune components but well maintains the innate components of cellular immunity, the augmentation of nk cells response might be a promising immunotherapeutic approach. we confirmed that the il-2-stimulated donor liver nk cells exhibited a significantly high level of trail and showed vigorous cytotoxicity against an hcc cell line without cytotoxicity against normal cells. after obtaining approval from the ethical committee of our institute, we successfully applied this therapy to 13 cirrhotic patients with hcc from january 2006. the average number of nk cells that had been administered to lt recipients at 4 days after lt was 304.2 ± 225.5 × 10 6 cells/body. the lt recipients were categorized as follows: (1) based on the milan criteria, 8 recipients met the criteria while 5 did not, and (2) based on the tnm stage, 2 recipients were categorized as pathological tnm stage i; 6, stage ii; 4, stage iii; and 1, stage iv. in our institute, the 2-year recurrence-free survival rates of the lt recipients treated with and without this therapy were 100% and 71.3%, respectively. kinetic studies revealed that in the early postoperative period, the peripheral blood obtained from the treated lt recipients exhibited a significant improvement in cytotoxicity against hcc cell line as compared to the untreated lt recipients (p < 0.01). furthermore, flow cytometric analyses revealed that the frequency of trail + nk cells increased remarkably in the peripheral blood of the treated lt recipients (p < 0.05). in conclusion, the administration of il-2-stimulated donor liver nk cells contributes to the promotion of host anticancer activity and has the potential to regulate hcc recurrence after lt. abstract# 500 vegf, a well-established angiogenesis factor, is expressed within allografts at high levels in association with acute and chronic rejection. in previous studies, we have reported that vegf possesses potent proinflammatory properties in part via its ability to mediate leukocyte trafficking into allografts. recently, we discovered that vegf mediates cd4+ and cd8+ t cell migration via interaction with its receptor kdr (also called vegfr2). blockade of t cell kdr significantly inhibits transendothelial migration. these observations suggest that kdr may be a novel t cell receptor for allogeneic lymphocyte recruitment. here, we first examined the expression of kdr on peripheral human cd4+ and cd8+ t cells by facs analysis. we found that ≤ 1% of circulating t cells express kdr, and its expression was at low levels on individual unactivated t cells. further, we found that the expression of kdr on t cells increased markedly following activation with mitogen and following interactions with activated allogeneic endothelial cells. induced expression of kdr on cd4+ and cd8+ t cells was at a similar level as that observed on endothelial cells. therefore, kdr appears to be selectively expressed on t cells, that traffick into allografts. to test the pathophysiological significance of these observations, we analyzed the expression of kdr in a total of 19 cardiac, and 5 renal, human allograft biopsies. we correlated the expression of kdr with cd3+ t cell infiltrates; and by double immunofluorescence staining, we determined co-expression of kdr on individual cd3+ t cell infiltrates. in cardiac allografts we found that kdr was expressed throughout the endomyocardium, and was most notable on endothelial cells in all biopsies examined. by grid counting of 3-4 areas of each biopsy, we found that the mean number of cd3+ t cell infiltrates ranged from 4 to 79 cells/hpf (x600 mag.). by double staining, we noted that kdr was expressed on 29 ± 4 % (mean±sem) of these cd3+ t cell infiltrates. similarly, we found that kdr was co-expressed on cd3+ t cells within renal allografts. while infiltrates were more focal, again 30 ± 2 % (mean±sem) of graft infiltrating t cells expressed kdr. collectively, these observations for the first time identify kdr as a novel receptor on allogeneic t cells. we suggest that intragraft vegf may interact with t cell kdr to facilitate homing and recruitment of allospecific lymphocytes into allografts. interaction of infiltrating cd8 + t cells and tissue cells in tolerant liver allografts: using tcr transgene approach. guoping jiang, 1 qiwei zhang, 1 horng-ren yang, 1 kathleen brown, 1 john j. fung, 1 lina lu, 1 shiguang qian. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. the liver allografts are accepted without requirement of immunosuppression in mice. the underlying mechanisms are not completely understood. we hypothesized that it resulted from an abortive t cell response within the liver due to hyporesponsiveness or apoptosis. to test this, we examined the activation and fate of allo-ag specific cd8 + t cells following liver transplants (ltx) compared with heart transplants (htx) that were acutely rejected. following transplantation [b10 (h2 b )→c3h (h2 k )], cfse labeled cd8 + t cells (10x10 6 ) from des tcr tg mice (h2k b specific tcr) were adoptively transferred into recipients. animals were sacrificed two days following des t cell administration for analyses of t cells in the grafts or draining lymph nodes (d-ln). host cd45 + leukocytes were quickly infiltrated following ltx. among cd3 population ∼48% were cd4 + and ∼52% cd8 + . cd8 + t cells were further increased thereafter in grafts and d-ln, associated with high inf-g production. cd8 + t cells in the liver grafts rapidly reduced to 36% by pod 14, and to 12% by pod 40. however, the incidence of cd4 + t cells remained high. cfse dilution assay and elispot showed an active division of des + t cells in liver allografts either on pod 7 or 40. these ag-specific cd8 + t cells functioned well evidenced by ifn-g production in response to allo-ag. however, compared to htx, the accumulation of des + cells in grafts was significantly lower in ltx [9.6% (17x10 4 /heart), and 0.59% (6.14×10 4 /liver)] on pod 7, and further dropped to 0.14% (3.14×10 4 /liver) on pod 40. the expended cohorts of adoptively transferred cells followed by their elimination suggested elimination of ag-specific cd8 + cells. to examine the role of liver environment, graft cd45non-parenchymal cells (npc) were isolated tested fro regulatory effect on des + t cell response. liver allografts showed significantly expansion of cd45 -npc, which were donor mhc class i + (h2b + ), b7-h1 + , trail + and low for cd40 and cd86. these cells did not inhibit des + t cell proliferation in response to b10 spleen stimulation, but significantly enhanced their death, which was dependent on b7-h1/trail. this was confirmed by using cd45 -npc from b7h1 -/or trail -/mice. in conclusion, activated t cells in liver grafts may stimulate tissue cells to express inhibitory and death inducing molecules, resulting in t cell death and graft acceptance. foxp3 + graft-infiltrating lymphocytes (gil) have been detected in the rejecting allografts of transplant patients. foxp3 is a marker for regulatory t cells (t reg ). published reports suggest that human foxp3 can be upregulated following tcr stimulation without induction of regulatory function. to investigate whether foxp3 + gil during acute rejection are t reg , we analyzed the phenotype of gil harvested from acutely-rejected non-human primate kidney allografts. methods: renal allografts with histologically-confirmed acute rejection were harvested at the time of necropsy from macaques that had undergone experimental transplantation. following digestion of kidney fragments using collagenase, gil were isolated using lymphocyte-separation media and cryopreserved. axillary lymph nodes (ax ln) were also isolated either at the time of necropsy or by biopsy. for seb-stimulated lymphocytes, ax ln cells were stimulated for 5 days in the presence of 25 ng/ml seb for 5 -6 days. to perform intracellular interferon-gamma (ifng) analysis, cells were stimulated with pma and ionomycin in the presence of brefeldin a for 5 -6 hours at 37°c. samples were prepared for flow cytometry by first staining for extracellular antigens. cells were then fixed and permablized (kit from ebiosciences) prior to intracellular staining for foxp3, ki67 and ifng. results: the percentage of foxp3 + in cd3 + gil was similar to that found in ax ln ( table 1 ). the majority of these cells were cd4 + . while 80% of the cd3 + /cd4 + /foxp3 + population of both ax ln and gil were cd25 + , a significantly higher frequency of cd39 + and ki67 + cells were found in gil (p < 0.01; student's t-test). simlar levels of cd39 and ki67 expression were found in seb stimulated lymphocytes. unlike the seb-stimulated lymphocytes, few ifng -producing cells were demonstrated following pma/ionomycin stimulation of gil. conclusion: our current data indicates that the majority of foxp3 + gil from acutely rejecting renal allografts are recently activated cd4 t cells that lack effector function. this suggests that foxp3 t cells within rejecting allografts may indeed be t reg . findings in mouse models of transplantation often fail to translate well in humans. three variables may account for the discrepancy: (1) evolutionary divergence between mice and humans, (2) influence of infection history on alloimmunity, and (3) use of highly inbred strains of laboratory mice. here, we investigated whether the use of inbred mouse strains skews the rejection phenotypes and their response to treatment due to decreased genetic diversity and/or fixation of undesirable genetic loci, known as inbreeding depression. we examined heterotopic cardiac allograft survival in outbred and inbred mouse populations in the presence or absence of immunosuppression. in the absence of immunosuppression, heart transplantation within or between outbred stocks of mice (n = 35) resulted in three distinct rejection phenotypes that resemble accelerated (1 -4 days), acute (8 -25 days), and chronic rejection (> 75 days), respectively. in contrast, all fully allogeneic grafts transplanted between inbred mice (n = 12) were rejected acutely (7 -13 days) as were historical controls (n > 50). the accelerated phenotype, present in 29% of outbred to outbred transplantations, was characterized by extensive hemorrhagic necrosis of the heart with thrombosis, neutrophil margination and neutrophilic arteritis, and did not correlate with donor:recipient mhc ii disparity. immunosuppression with t cell costimulation blockade did not prevent accelerated rejection (incidence = 27% in the treated group, n = 26) but did convert the acute rejection phenotype into longterm allograft survival in all groups studied. the same accelerated phenotype was observed if transplantation was performed from outbred to inbred mice (incidence = 39%; n = 23) but could not be duplicated if inbred to outbred transplantation was performed (n = 20). finally, c3 depletion with cobra venom factor abrogated the accelerated rejection phenotype in outbred to outbred transplantations (n = 16), suggesting a role for complement in the pathogenesis of this phenotype. in summary, our data (1) indicate that the use of outbred mouse stocks may uncover clinically-relevant rejection phenotypes not observed in inbred mouse strains, and (2) underscore the importance of the donor background in determining the phenotype of rejection. outbred mouse stocks may provide a platform to uncover mhc-unlinked genetic loci that play an important role in the outcome of solid organ transplantation. th17 are limited in their ability to reject allografts. elderly recipients represent the most rapidly growing segment of patients on the waiting list. however, little is known about age-dependent alterations of the immune response in organ transplantation. we examined age dependent t-cell functions in a transgenic mouse transplant model. effector t-cell phenotype, -function, cytokine production and regulatory t-cell function were analyzed in 3 and 18mths old b6 mice. in an in vivo transplant model, bl/6 nude mice were reconstituted with 2x10 6 young or old transgenic alloantigen-specific cd4 + tcells and engrafted with bm12 skin grafts. t-cell phenotype and cytokine secretion were sequentially analyzed in all lymphatic compartments. splenocytes of naïve old b6 mice contained significantly higher frequencies of t-cells with an effector/memory phenotype (cd4 + cd44 high cd62l low and cd8 + cd44h igh cd62l low; p<0.005). in vitro proliferation and ifnγ-production were significantly reduced in aged mice indicating an impaired t-cell response with increasing age as assessed by mlr (p<0.005) and elispot (p<0.001). in parallel, regulatory functions remained age-independent as alloantigen-specific cd4 + cd25 + foxp3 + t-cells isolated from sensitized old mice demonstrated a dose-dependent well preserved suppressor function. next, we tested the age-dependent alloantigen -specific cd4 + t-cell function in a transgenic skin transplant model: age did not significantly impact rejection kinetics (young vs. old: 10.1 vs. 14.3days, n.s.) however, t-cell migration and activation were significantly different: fewer numbers of activated cd4 + cd25 + and effector/memory phenotype t-cells (cd4 + cd44 high cd62l low ) were found in recipient spleens (p<0.05) and draining lymph nodes (dln) (p<0.05) after transfer of old t-cells. chemokine receptor staining revealed less cxcr3 + and ccr7 + t-cells in dln following the transfer of old t-cells (total cell numbers x104: cxcr3 + : 10.9±4.2 vs.0.95 ±0.2, ccr7 + : 4.7±1.0 vs. 0.35±0.2, p<0.05). this was paralleled by reduced intragraft t-cell infiltration as observed by immunohistochemistry. in summary, native elderly mice showed an increased frequency of effector memory t-cells but an overall impaired t-cell response. regulatory -t-cell function remained preserved. in vivo allospecific cd4+ t-cell activation and migration was impaired in elderly transplant recipients. the background: the sensitized transplant recipients may undergo an "accelerated" form of rejection, which is mediated by t cell-dependent mechanisms. these patients often experience increased rate of early rejection episodes, which are difficult to control with currently used immunosuppressive agents. methods: in our model of cardiac graft rejection in sensitized recipients, b6 mice are first challenged with b/c skin, followed 40-60 days later by b/c heart transplant (htx). unlike in naive hosts, htx rejection and alloreactive cd8 activation in this model are cd154 blockade-resistant. we first performed systemic analysis at the intragraft transcriptional level by microarray to identify disparities in local immune responses in naive vs. sensitized hosts. aiming to improve the efficacy of costimulation blockade in the sensitization settings, we then determined the role of cd4 t cells in costimulation blockade-resistant alloimmune response by using cd4 depleting (gk1.5) vs. cd4 blocking (yts177) ab, in conjunction with cd154 blockade (mr1). results: htx harvested from groups of naïve (day 4-6), sensitized (day 2-4), control ig or mr1 ab treated mice (n=3/gr) were subjected to microarray analysis. mr1 treatment suppressed htx expression of proinflammatory genes (il-1β, il-6, tnf-α), and t cell-targeted chemokines (rantes, mig, cxcl10) early after htx in naïve, but not sensitized recipients. five groups of sensitized mice treated at the time of htx with: (1) 5). ctl activation was determined by facs phenotyping at day 10 and 30. the simultaneous blockade of cd154 costimulation and cd4 help, but not a single blockade with mr1 ab or anti-cd4 ab, was required to inhibit peripheral alloreactive cd8 activation in sensitized mice. additionally, cd8 activation in the absence of cd4 help showed defective cytotoxic molecule profile, with suppressed perforin but upregulated granzyme b expression at the graft site. conclusion: cd154 blockade-resistant cd8 activation is critically dependent on cd4 t cells. this study provides novel immunological basis to study the potential synergy between adjunctive cd4 and cd154 targeted therapies to control accelerated graft rejection in sensitized hosts. mediators. g. einecke, l. g. hidalgo, p. f. halloran. department of medicine, university of alberta, edmonton, canada. the hallmark of t cell mediated rejection (tcmr) are interstitial inflammation and tubulitis. the mechanisms of tubulitis and epithelial deterioration during tcmr are unknown. we previously showed that tubulitis in mouse allografts is independent of cytotoxic molecules (gzma/b, prf) and is preceded by molecular changes with loss of epithelial genes, reflecting epithelial dedifferentiation. human tcmr is associated with loss of the same epithelial genes and re-expression of embryonic pathways (wnt, notch). we hypothesized that tcmr is mediated through soluble factors released by effector t cells or macrophages in the interstitium, and that supernatants of effector t cells would simulate these changes in epithelial cultures. we established an in vitro model in which cultured primary human renal epithelial cells are incubated with supernatants from effector t cell/monocyte co-cultures. the transcript changes in this model, analyzed by microarrays, closely simulated those in human and mouse tcmr (fig1), with loss of epithelial transcripts, activation of wnt/ notch pathways, and increased expression of ifng-inducible and injury-related transcripts previously defined in our mouse model. some of these changes were reproduced by incubation of epithelial cells with ifng or tgfb. the in vitro model identified additional epithelial transcript changes not previously identified in vivo (not affected by ifng or ischemic injury, not expressed in t cells, macrophages, b cells, or nk cells). expression of these transcripts (n = 305) was highly altered in human tcmr compared to nonrejecting biopsies of 177 human renal allograft biopsies and distinguished tcmr from antibody-mediated rejection in a hierarchical cluster analysis. thus we have established an in vitro model that closely simulates the epithelial events during human tcmr and confirms that these changes are independent of direct contact with inflammatory cells, supporting the hypothesis that interstitial effector t cells mediate allograft deterioration by soluble mediators. together with previous mouse and human data these results provide the first in vitro model of the epithelial consequences of tcmr. objective. c4 split product deposition to hla antigen-coated microparticles ([c4d] flowpra) was previously shown to be a specific marker of c4d-positive antibodymediated rejection (amr). the objective of this study was to assess the predictive value of [c4d]flowpra reactivity in a cohort of non-biopsied patients with stable graft function during the first year. methods. a total of 133 kidney transplant recipients were enrolled (inclusion criteria: functioning graft at 12 months; prospective collection of sera taken before and at 1-3, 6, and 12 months after transplantation). included patients were serially screened for humoral panel reactivity applying [igg] and [c4d]flowpra screening. results. fifty-four of the included 133 recipients had stable graft function within the first year and were not subjected to diagnostic renal biopsy. in this particular patient group, detection of complement-fixing hla reactivity tended to be less frequent than in the 79 patients with biopsied graft dysfunction (≥10% [c4d]flowpra before transplantation: 9% vs. 18% of recipients, p=0.2; ≥10% [c4d]flowpra after transplantation: 11% vs 24%, p=0.06). in line with our previous results, within the group of biopsied patients, pre-and/or post-tx [c4d]flowpra reactivity was tightly associated with the immunohistochemical detection of peritubular capillary c4d deposition (p=0.005) reflecting ongoing amr. remarkably, in initially stable patients, detectable [c4d] flowpra reactivity was not associated with inferior long-term outcomes. within this patient group, recipients with and without (pre-and/or post-transplant) c4d-fixing anti-hla reactivity did not differ with respect to 4 yr allograft survival (p=0.4), 4 yr serum creatinine levels (1.5 vs. 1.5 mg/dl; p=0.7), and proteinuria at 4 yrs (0.2 vs. 0.18 g/24h; p=0.8). similar results were obtained for a comparison of [igg]flowpra positive vs. negative subjects. conclusion. our data suggest that a considerable number of patients with initially stable graft function may have excellent long-term graft function despite serologically detectable levels of (complement-fixing) alloreactivity. for these antibody-positive recipients, a potential role of graft accommodation can be speculated. the immunoproteasome subunit beta 10 as a novel peripheral blood and intragraft biomarker of chronic antibody mediated allograft rejection in clinical transplantation. joanna ashton-chess, 1 in an attempt to identify non-invasive biomarkers of specific histological scarring, we compared publicly available gene sets derived from microarray studies of human renal transplant biopsies published in the literature with our own microarray data derived from studies of rat heart allografts. in this way we identified an immunoproteasome subunit (proteasome subunit beta 10 -psmb10) as a potentially interesting candidate. psmb10 is one of three members of the immunoproteasome that are induced by abstracts interferon gamma. messenger rna profiling in renal transplant biopsies (n = 52) with normal histology, interstitial fibrosis and tubular atrophy, calcineurin inhibitor toxicity, transplant glomerulopathy or chronic antibody-mediated rejection (banff 2005) revealed psmb10 to be strongly and significantly increased in chronic antibody mediated rejection vs. the other three histological diagnoses. receiver operator characteristic (roc) curve analysis showed that psmb10 mrna could diagnose chronic antibody-mediated rejection with an auc of 0.99, a sensitivity of 1.0 and a specificity of 0.95. moreover, psmb10 mrna was significantly increased in the pbmc (n = 24) of patients with chronic antibody-mediated rejection compared to those with normal histology. roc analyses revealed an impressive auc of 1.0 with all patients being correctly classified. similar results were also observed in a rat allograft model where psmb10 was significantly increased at day 100 post transplantation in both the heart allograft and the pbmc of animals presenting chronic transplant vasculopathy vs. syngeneic grafts. moreover, inhibition of the proteasome by administration of velcade® at 0.1 mg/kg every other day for the first 20 days post transplantation significantly and dose-dependently prolonged allograft survival (mst 31.7 days in velcade-treated vs. 6.3 days in untreated animals).together our data point towards psmb10 as a blood and intragraft biomarker of chronic antibody-mediated rejection as well as a potential therapeutic target. furthermore, our results suggest that using a threshold of psmb10 in the blood could help in guiding the decision to biopsy in the clinic. baff monitoring after b-cell depletion therapy for acute renal transplant rejection. valeriya zarkhin, 1 snehal mohile, 1 li li, 1 jonathan martin, 1 minnie sarwal. 1 1 pediatrics, stanford university, stanford, ca. introduction: the objective of this study was to investigate the interaction between b-cell activation factor of the tnf family (baff) level and circulating b-cell repopulation in pediatric patients with acute kidney transplant rejection treated with the b-cell-depleting agent rituximab. methods: 10 pediatric patients (3-23 yrs) with biopsy proven b-cell positive ar were treated with steroids and rituximab (4 x 375 mg/m 2 /dose/week). all patients were followed up for 12 months. peripheral blood cd19 cells and donor specific antibodies (dsa) were monitored monthly. serum level of baff was measured by elisa at ar, 1, 3, 6, and 12 months post-ar treatment and correlated with clinical outcomes. results: complete depletion of circulating and intragraft b-cells was observed with rituximab, with improvement in ar grade in all patients. the median time of peripheral b-cells repopulation was 5 months (range 3-12 months, fig. 1a) . no correlation was found between pre-treatment peripheral b-cell number and the b-cell repopulation time (r=0.59, p=0.09). baff levels rose significantly with b-cell depletion with maximum values at 3 months post-treatment (7.5 fold increase, p=0.0001) and returned to pre-treatment levels, with b-cell recovery, at 12 months (fig. 1b) . serum baff levels correlated positively with b-cell depletion >6 months (r=0.91, p=0.004, fig1b). a lack of depletion of dsa i, but not dsa ii correlated with higher baff levels (r=0.99, p=0.007). the timing of b-cell repopulation and depletion of dsa i may be dependant on serum baff level. anti-baff treatment may be considered in addition to rituximab or standard immunosuppressive treatment protocols in patients with persistent and/or antibody mediated rejection. the background: the development of donor specific hla antibodies (dsa) post-transplant has been associated with graft failure. we have shown in a longitudinal study that increases in dsa may precede rejection by months. this retrospective analysis evaluates changes in maintenance immunosuppression (mi) and appearance of dsa in stable transplant recipients. methods: sera from stable renal transplant recipients were collected at 4-6 month intervals and tested for the presence of dsa. the types and doses of immunosuppression were correlated with the appearance of dsa. two hundred eighty stable renal transplant recipients who received either a deceased or a living donor kidney were monitored post-transplantation for the development of dsa. patients have been followed for 1 to 7 years and had a minimum of 4 serum samples analyzed. all recipients received anti-lymphocyte induction therapy. maintenance immunosuppression (mi) consisted of a calcineurin inhibitor, prednisolone, and mycophenolic acid. hla single antigen beads analyzed in the luminex instrument were used to establish donor specificity of the antibodies. a chart review was undertaken to determine the doses of the mi posttransplantation. all mi was managed by the transplant team and changed according to clinical indications without regard to dsa. results: of the 280 patients monitored 37 developed dsa post-transplantation with a functioning graft. dsa was against hla-class ii antigens in 26 of 37 (70%); class i antigens in 8 of 37 (22%); and against both class i and ii antigens in 3 of 37 (8%). dsa against class ii was against dq in all except one case. in the majority of the recipients the appearance of dsa was preceded with dose reduction of the mi, either calcineurin inhibitor or mycophenolic acid or both. conclusions: our data show that dsa developed predominantly against hla-class ii antigens and that the appearance of dsa was often preceded with reduction of one or more of the mi. this data shows the importance of monitoring dsa with mi decreases in a stable allograft recipient. antibody production and antigen presentation are directly inhibited by mycophenolate mofetil. anat r. tambur, 1 joe leventhal, 1 nancy d. herrera, 1 joshua miller. 1 1 division of organ transplantation, northwestern university, chicago, il. immunosuppressive medications are primarily designed to target t cell proliferation. mycophenolate mofetil (mmf) exerts its effect by inhibiting de-novo synthesis of guanine, a dna building block. we, and others, have previously shown that mpa (active metabolite of mmf) affects the differentiation of monocytes into dendritic cells (dc). we further demonstrated that cell-surface receptors associated with antigen up-take and antigen-processing and presentation (cd83 and cd205) are down regulated when cells are matured in the presence of mpa. this phenotype translated into a decreased uptake of alloantigens and reduced stimulation of t cells. we concluded that mmf inhibits also cell functions requiring mrna synthesis. we now present data regarding the role of mpa in maturation and function of b-lineage cells. pbmcs from 10 subjects were cultured in the presence of cpg, il-2, il-10 and cd40l for 5 days to induce memory b and plasma cell maturation in-vitro. cultures were performed in the presence or absence of mpa (50 ugr/ul) for the length of the incubation period. in-vitro stimulation of b cells increased the memory population (cd19+ cd27+) from 2.8 +/-1.1% to 7 +/-2%. similarly, plasma cells were increased from 4.4 +/-1.3% to 6.4 +/-1.8%. the addition of mpa to the culture inhibited stimulation for both memory and plasma cells (4.7 +/-3% p=0.01; and 4.7 +/-2.3% p=ns, respectively). we have further analyzed the effects of mpa on antibody secretion using an elisa (measuring soluble antibodies) as well as a b-cell elispot assay (assessing the number of b cells that produce antibodies). while an expected increase in od values was observed for stimulated samples compared with non-stimulated samples, a significant decrease was observed when stimulation occurred in the presence of mpa. nonstimulated cells: 0.249 +/-0.28; stimulated cells: 0.391+/-0.33; mpa treated stimulated cells: 0.279+/-0.43; p<0.0005). the number of antibody producing cells was also significantly lowered when cultures were done in the presence of mpa (a mean of 106 cells were counted for the stimulated cells compared with 1-2 cells for non-stimulated and mpa-treated-stimulated cells). to our knowledge this is the first time where in-vitro experimental data document the inhibitory effect of mpa on b lineage cells and antibody secretion, although clinically known for some time. these results confirm our previous observations regarding the effects of mmf on non-proliferating immune cells. a non-allogeneic stimulus triggers the production of de novo hla and mica antibodies. luis e. morales-buenrostro, 1,2 lluvia a. marino-vazquez, 1 anh nguyen, 3 paul i. terasaki, 2 josefina alberu. 1 1 nephrology and transplantation., instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, df, mexico; 2 terasaki foundation laboratory, los angeles, ca; 3 one lambda inc., canoga park, ca. background: in a previous study, we found that healthy people developed hla abs after immunization against hepatitis b virus. the aim of this prospective study was to establish if stimulation with influenza vaccine is capable of triggering the production of hla and mica abs. methods: we determined the presence of hla and mica abs (de novo and preformed abs) in 3 groups of patients vaccinated against influenza: a) 42 healthy adults, b) 40 esrd patients, and c) 25 tr. additionally, we followed 22 healthy unvaccinated people without exposure to sensitizing factor: d) control group. sera samples were collected at baseline (pre influenza shot), at 1 week, and monthly up 6 months after immunization. hla abs were assessed with labscreen single antigen beads for luminex. all samples of each patient were tested simultaneously. a luminescence value higher than 500 was considered positive only if it was 3 times the baseline value. we analyzed the data using chi square, one way anova test, and logistic regression. results: the table shows the types of abs in each group. interestingly, we found preformed abs across all four groups, including the control group (which is free of any known sensitizing factors). the proportion of de novo abs was higher in the group b and c. multivariate analysis shows that the only independent factor associated with development of de novo abs was the presence of preformed abs. we observed a nonspecific immunologic response triggered by external stimulus and was not necessarily associated to the vaccine in people previously sensitized. a introduction. decisions about the minimization and ultimate withdrawal of immunosuppression (is) would be facilitated by the identification of biomarkers associated with operational tolerance (ot). methods. as part of an itn/nih supported study tolerant kidney transplant recipients (off all is for > 1yr with stable function, n=22) were compared to recipients with stable function on is (sis, n=34), recipients with chronic allograft nephropathy (can, n=20), and healthy volunteers (hv, n=18). pbmc, whole blood total rna, and urine samples from each group were examined using flow cytometry, microarrays, and rt-pcr respectively. results. analysis of microarrays revealed significantly higher expression of b cell differentiation genes in tolerant recipients compared to the sis and can groups. consistent with this finding, tolerant recipients also displayed higher numbers of naïve b cells in peripheral blood and increased expression of cd20 in urine relative to the sis and can groups. no differences in treg or genes associated with regulatory cells were observed in tolerant recipients relative to other groups. these analyses failed to demonstrate significant differences between tolerant recipients and hvs although support vector machine learning methods suggested potential differences in a number of genes including nfat and calcineurin. finally, relative to tolerant patients, those with can showed decreased numbers of t and nk cells and expressed lower levels of genes associated with immune cell activation in peripheral blood. conclusions. differences in b cell numbers may be useful in identifying tolerant renal transplant recipients or those predisposed to developing tolerance and could potentially provide insights into the mechanisms of tolerance. erythrocyte development of antibodies (abs) to mismatched donor hla antigens has been associated with acute and chronic rejection. complement activation, and c4d deposition, has been correlated with humoral rejection of allografts. however, utility of c4d staining in ltx has been controversial. a recent study (arthritis rheum. 2004 nov; 50(11) :3596-604) shows a strong correlation between erythrocyte bound c4d (e-c4d) in diagnosis and monitoring of sle. goal of our study is to determine the utility of measuring e-c4d in the diagnosis of humoral rejection following human ltx. 26 ltx recipients were analyzed post-ltx for e-c4d using facs of rbcs incubated with anti-c4d (quidel) followed by fitc-goat anti-mouse.10 normals were also analyzed. the serum was analyzed for development of anti-hla abs by solid phase assays and for the presence of autoabs to kα1 tubulin and collagenv (elisa). biopsies from 11 patients were stained immunohistochemically for c3d deposition. summary of results are presented in table 1 . infection=2/10 ar=0/10 % e-c4d in normals-10.35%; mfi in normals-5.46; mfi=mean fluorescence intensity; ar=acute rejection; dsa=donor specific abs 16 out of 26 patients show significant increase in the % bound e-c4d (p<0.05) as compared to controls.11/16 had anti-hla and 13/16 had autoabs which were significantly different from those with low e-c4d (p= 0.005). staining of the biopsies showed c3d deposition in 6 recipients with increased e-c4d. all 4 patients with acute humoral rejection had elevated e-c4d. we conclude that there is a significant correlation between increase in % e-c4d in ltx recipients and development of abs to either hla antigens or auto-antigens during the post-ltx period. biopsies from patients with increased e-c4d showed deposition of c3d in the allografts. preliminary data suggest that measurement of e-c4d using a non-invasive method of flow cytometry may be of value in monitoring ltx patients for humoral rejection. innate immunity: chemokines, cytokines innate immunity is emerging as an important initiator and modulator of the adaptive immune response. in the setting of transplantation, ischemia-reperfusion injury and tissue trauma appear to potentiate the alloimmune response. one of the mechanisms through which the innate immune system modulates an adaptive immune response is dendritic cells (dc). in this study, we examined dc activation in a mouse model of skin transplantation by monitoring the expression of mhc ii and p40 chain of the proinflammatory cytokine il-12. the p40 expression was detected in live cells using the yet40 reporter mouse, in which a transgene for yellow fluorescent protein (yfp) was placed downstream of the endogenous il-12p40 gene, thus faithfully "reporting" p40 expression. skins from c57bl/6 or balb/c donors were grafted on the dorsal thorax of c57bl/6.yet40 mice. draining and non-draining lymph nodes (ln) were harvested at 24 hours and examined for yfp expression by fluorescent microscopy. a marked increase in the numbers of yfp-expressing dc was observed in draining ln in both syngeneic and allogeneic graft recipients. surprisingly, yfp-expressing dc also increased in nondraining ln when compared to non-transplanted controls. similarly, dc in draining and non-draining ln showed higher mhc ii expression than those in non-transplanted controls. upregulation of mhc ii was highest at 24 hours and decreased significantly by 48-72 hours. to assess whether dc activation in non-draining ln was functionally significant, we monitored the activation of adoptively transferred ovalbumin-specific ot-ii tcr transgenic t cells in response to footpad antigen challenge in mice with or without a syngeneic skin transplant on the contralateral upper thorax. otii t cells in transplanted animals proliferated approximately 5-to10-fold better and a higher percentage of the otii cells produced il-2 than those from non-transplanted animals. thus, local surgical trauma results in a widespread, time-limited, functional activation of dc that appears to act as a partial adjuvant for t cell responses. together, these data suggest that surgical trauma may incite a systemic barrier to transplantation via the activation of dc and therapeutic interventions that reduce the surgical trauma and dc activation may help to improve survival of transplanted grafts. tolerance of cardiac allografts: studies with cx3cr1-deficient mice. takaya murayama, 1 katsunori tanaka, 1 takuya ueno, 1 mollie jurewics, 1 guleria indira, 1 fiorina paolo, 1 paez jesus, 1 smith n. rex, 2 sayegh mohamed, 1 reza abdi. 1 1 transplantation research center, renal division, brigham and women's hospital, harvard medical school, boston, ma; 2 department of pathology, massachusetts general hospital, harvard medical school, boston, ma. although donor/tissue dendritic cells (ddc) have long been known to play a key role in mounting alloimmune responses, however, their generation, trafficking and role in tolerance have not rigorously examined. we have used b6.fvb-tg (itgax-dtr/ egfp) 57 mice which has a gfp linked to the cd11c promoter. using these mice as the donors of heart allograft transplantation provided us a unique model to study ddc posttransplantation. our trafficking data indicate there is a rapid migration of ddc into spleen (3 hours post transplantation) but not lymph nodes and that ddc were unexpectedly detected in the spleen of the recipients long after rejection of heart allografts suggesting ddc could escape from the immunosurveillance of host immune system. our data also show that ddc proliferate in the lymphoid tissue of the recipients and co-express class ii molecule of the recipients. we then show that cx3cr1 pathway regulates generation abstracts of heart tissue dc constitutively. as compared to wt hearts, cx3cr1 -/hearts contain lower number of dc and transplanting cx3cr1 -/donor hearts into wt balb/c mice led to significant prolongation of allograft survival without immunosuppression (mst of 8 vs. 17 days, respectively). increasing cx3cr1 -/heart dc by implanting donors with flt3-producing hybridoma cells has restored the time of rejection. unexpectedly, induction of long-term survival with anti-cd154 blockade (mr1) and ctla-4 ig (but not low dose rapamycine) was abrogated when cx3cr1 -/hearts were used as donors, with concomitant lesser tregs in the cx3cr1 -/heart allografts as compared to wt. furthermore, co-transplanting hearts from wt and cx3cr1 -/into the same recipient treated with mr1 resulted in significant prolongation of cx3cr1 -/heart allograft survival. depleting the ddc of heart donors prior to transplantation with diphtheria toxin also worsened markedly chronic rejection in the recipients at day 100 post-transplantation. our data indicate that, in contrast to the widely accepted dogma, the presence of donor dc in graft tissue is not only central to allograft rejection but also is necessary for the induction and maintenance of peripheral tolerance. local c5a interaction with c5ar on dcs modulates dc function, subsequently up-regulating allospecific t cell responses. qi peng, ke li, steven h. sacks, wuding zhou. mrc centre for transplantation, department of nephrology and transplantation, king's college london, guy's campus, london, united kingdom. the innate system of immunity plays an important role in ischemia-reperfusion injury and allograft rejection. the early stages of inflammatory processes are accompanied by complement activation. one biological consequence of this activation is the release of potent inflammatory anaphylatoxins, c3a and c5a, which have been reported to regulate a range of inflammatory responses. we previously reported that dcs express c3ar and c5ar, and c3a-c3ar interaction has a positive impact on murine bm dcs, in terms of activation phenotype and capacity for ag uptake and allostimulation. however, the role of c5a in modulating dc function remains unclear. the aim of this study is to investigate the role of local c5ar signalling in modulating murine bm dc function and subsequent regulation of the allospecific t cell response. we first evaluated if c5a-c5ar interaction could result from local expression of factors. our results showed that c5ar mrna was detected in wt dcs at different stage of dc culture by rt-pcr, and c5a was detected by elisa in the culture supernatants from different stages of dc culture. we next determined if c5a-c5ar interaction modulates dc function in allospecific t cell stimulation in vitro and in vivo. we found that bm dcs cultured from c5ar-/-mice or treated with c5ar antagonist (c5ara, w54011) exhibited a less activated phenotype (producing significantly less il-12 and more il-10, in response to lps stimulation); both c5ar-/-and antagonist-treated dcs (lps stimulated) showed reduced capacity to stimulate naïve alloreactive t cells, as measured by ifn-γ production and thymidine uptake. as regards interaction in vivo, following i.p. administration of the c5ara-treated dcs into allogeneic mice for 10 days, ex vivo mixed lymphocyte reaction showed that cd4+ t cells from those recipients have reduced thymidine uptake, but increased il-4 production compared to that with untreated dcs. conversely, dcs treated with c5ar agonist (c5a) exhibited a more activated phenotype (producing more il-12 and less il-10) and were more potent in allospecific t cell stimulation. our findings demonstrate that murine bm dcs can express c5ar and c5a can be generated locally; c5a-c5ar interaction up-regulates murine bmdc activation and their allostimulatory capacity. thus, targeting c5a-mediated signal may be able to prevent allograft injury. role of tnfα in early chemokine production and leukocyte infiltration into heart allografts. daisuke ishii, 1 austin d. schenck, 1 robert l. fairchild. 1 1 immunology, glickman urological and kidney institute., cleveland clinic, cleveland, oh. objectives: the acute phase cytokines il-6 and tnfα are produced early during inflammatory processes, including wound healing and ischemia/reperfusion. the goal of this study was to investigate the role of these cytokines in the induction of early chemokine production and leukocyte infiltration into heart allografts. methods: c57bl/6 (h-2b), balb/c (h-2d), and balb/c.il-6-/-mice received vascularized syngeneic or complete mhc mismatched, a/j (h-2a), cardiac grafts. grafts were retrieved at different time points and total rna and tissue protein were prepared and analyzed by quantitative rt/pcr and elisa to test expression levels of tnf-α, il-6, cxcl1/kc, cxcl2/mip-2, and ccl2/mcp-1 in the grafts. anti-tnfα mab (500 ug) was given at the time of transplantation with or without anti-cd154 mab (200 ug on days 1 and 2). infiltration of cd4+, cd8+ t cells, neutrophils and macrophages was assessed by flow cytometry and immunohistochemistry. donor-reactive t cell priming to ifn-g producing cells in the recipient spleen was measured by elispot. results: expression of tnfα and il-6 mrna reached an initial peak at 3 hrs post-transplant and a second peak at 9-12 hrs with equivalent levels in both iso-and allo-grafts. the neutrophil and macrophage chemoattractants cxcl1/kc, cxcl2/ mip-2 and ccl2/mcp-1 reached peak levels at 9 hrs post-transplant in both sets of grafts and then declined to background levels. il-6 deficiency in the recipient or the cardiac allograft did not prolong allograft survival. in untreated mice, heart allografts were rejected at 8.6 ± 0.6 days after transplantation. anti-tnfα mab decreased neutrophil and macrophage chemoattractant levels 50% at 9 hrs post-transplant and subsequent neutrophil, macrophage and cd8+ cell infiltration into the allografts as well as extended graft survival to 14.1 ± 0.8 days. anti-tnfα mab also decreased the number of donor-reactive ifn-g producing cd8 t cells almost 90% on day 7 post-transplant. whereas anti-cd154 mab prolonged survival to day 21, administration of anti-tnfα and anti-cd154 mab delayed rejection to day 32 and resulted in the long-term (> 80 days) survival of 40% of the heart allografts. conclusions; these data indicate that anti-tnfα antibodies can delay donorreactive cd8 t cell priming and leukocyte infiltration into heart allografts rejection. as a conjunctive therapy, tnfa antibodies can promote long-term survival of the allografts. introduction recent evidence indicates that inflammation impairs immune regulation, yet the mechanisms behind this effect are not clear, in particular in regards to transplantation tolerance. in this study, we investigated the role of inflammatory cytokines, il-6 and tnfα, in transplantation tolerance induction. we first examined the impact of il-6 + tnfα on in vitro t cell alloimmune responses. t cells that were stimulated by allogeneic apcs in conditioned media harvested from lps-activated dcs proliferated more than apc-stimulated t cells that were cultured in conditioned media derived from non-lps-activated dcs. the ability of cd4 and cd8 t cells to respond to allogeneic apcs in the lps-activated conditioned media was significantly impaired by the addition of either anti-il-6 or anti-tnfα mabs. the addition of both mabs further diminshed t cell proliferation, indicating that il-6 and tnfα synergize to augment in vitro t cell allostimulation. in support of these findings, we noted reduced t cell proliferation during the mlr when t cells were cultured in conditioned media derived from either il-6-/-or tnfα-/-lpsactivated dcs. furthermore, these diminished responses was restored by the addition of recombinant il-6 or tnfα, respectively. to examine the in vivo implications of these findings, we employed a murine skin allograft model, with recipients that were either b6 wild type, il-6-/-or tnfα-/-. these groups were transplanted with a balb/c skin graft and treated with (or without) perioperative costimulatory blockade (ctla4 ig and anti-cd154). in the absence of immune modulation, all groups rejected balb/c skin allografts at a similar tempo (<12 days). in the presence of costimulatory blockade, il-6-/-recipients (median survival time, mst = 22 days, p =0.05) rejected their allografts at a slower tempo compared to wt (mst = 16 days) or tnfα-/-recipients (mst = 18 days). however, this response in il-6-/-recipients was further delayed by administering a tnfα inhibiting mab (mst = 60 days), indicating that synergy between il-6 and tnfα occurs in vivo and prevents the ability of costimulatory blockade to delay the onset of allograft rejection. conclusions we conclude that synergy between il-6 and tnfα augments t cell alloimmune responses and impairs the effects of costimulatory blockade to delay allograft rejection. abstract# 529 background: calcineurin inhibitors (cni) are involved in the development of post transplant diabetes mellitus (ptdm). changes in insulin secretion and sensitivity are central mechanisms involved in the development of ptdm. in addition alterations in endothelial function seem to be involved. the present study investigated the effect of cni's on these factors. methods: in a predefined sub-study of a previously published randomized trial it was aimed to compare the effect of cni treatment (n=27) with complete cni-avoidance (n=27) on insulin secretion and sensitivity as well as endothelial function. an oral glucose tolerance test and endothelial function investigation with laser doppler flowmetry was performed in 44 patients, 10 weeks and 12 months following transplantation. results: insulin sensitivity differed already 10 weeks posttransplant and was significantly better after 12 months in patients never treated with cni drugs (p=0.043). endothelial function was significantly correlated with insulin sensitivity (n=27, r 2 =0.22, p=0.013) at 10 weeks posttransplant, but not after 12 months (p=0.54). insulin secretion tended to be higher in cni treated patients both at week 10 and month 12 (p=0.068). conclusions: findings in the present study indicate that long-term cni treatment reduce insulin sensitivity which was associated with impaired endothelial function. in response to this peripheral insulin resistance a tendency towards a compensatory increase in insulin secretion was seen. these effects combined may indicate a future risk for premature cardiovascular disease in cni treated renal transplant recipients, but this hypothesis needs further study. group(n) ktx ptx(kptx) ecd re-tx pra(>20%) race(aa) low immunologic risk alem (113) overall pt, ktx, and ptx survival are 96, 92, and 86% at 16 months median follow-up. actuarial survival rates, initial length of stay, delayed graft function, steroid free rates, major infection, and incidence of ptld (1 ratg pt) were similar for alem and ratg groups, but treated acute rejection (ar) occurred in 17(15%) alem pts compared to 29(27%) ratg pts (p=0.03) and biopsy proven rejection (bpar) in 13(12%) alem pts compared to 23(21%) ratg pts (p=0.04). only 1 alem bpar has occurred after 12 months. total daily mmf doses were similar for alem and ratg groups at 3 months (1567±457mg vs 1670±508mg). neupogen use was greater in the alem group, 26(23%), than in the ratg group (14(13%), p=0.03). excluding pak, chronic allograft nephropathy (can) was observed in 19(17%) alem pts and 27(25%) ratg pts. (p=0.14). conclusions: alem and ratg induction both provide excellent 1 and 2 yr pt, ktx, and ptx survival. alem is associated with lower acute rejection rates and perhaps less can, but requires increased neupogen administration to help maintain mmf dosing. thymoglobulin dosing intensity and density: effects on induction efficacy ruth-ann m. lee, 1 adele h. rike, 1 background: alemtuzumab (campath 1h) has been used as induction therapy for kidney transplant recipients with acute rejection rates reported by us and others of 7 to 20% at one year. the histologic type of rejection and the time frame for occurrence after treatment with alemtuzumab have not been well established. this study is a retrospective single center review of acute rejection episodes of kidney transplant recipients treated with alemtuzumab induction with respect to the kinetics and histologic patterns of acute allograft rejection. methods: from 11/01/03 to 10/31/06, 416 kidney transplants were done meeting the inclusion criteria for this review. all patients had negative t and b cell flow cytometric crossmatches and received induction therapy with alemtuzumab 30mg iv intra-operatively, methylprednisolone 500 -750 mg during the first 24 hours, and were then maintained on tacrolimus (target level 5-7) and mycophenolate mofetil without steroids. all episodes of biopsy-proven acute rejection (ar) were reviewed. patients in pre-transplant desensitization protocols or with documented non-adherence with medications were excluded from the analysis. results: a total of 44 of 416 patients (10.6%) experienced ar during the study period, with a mean follow up of 28 months (range 14-48 months). of the ar episodes, 19 (43%) occurred within the first 3 months post-transplant, 13 (30%) occurred between 3-6 months, 5 (11%) occurred between 6-9 months, 1 (2%) occurred between 9-12 months, and 6 (14%) occurred more than one year post-transplant. of the rejection episodes within the first 3 months post-transplant, 9/19 occurred within the first 30 days. histologic analysis showed that 9/19 rejection episodes (47%) within the first 3 months included an antibody mediated component (6/9 within the first 30 days.) in contrast, 3/25 rejection episodes (12%) which occurred greater than 3 months post-transplant were antibody mediated. of the 12 patients with antibody mediated rejection, only 3 patients had panel reactive antibody (pra) levels > 25% at the time of transplant. conclusion: this large experience with alemtuzumab induction therapy with a steroidfree maintenance protocol, demonstrates that the majority of rejection episodes occur within the first 6 months post-transplant, with the largest fraction in the first 3 months. a significant number of early rejection episodes are antibody mediated and occur in unsensitized recipients. in a randomized, international, multicenter study, comparing the use of thymoglobulin (tmg) and basiliximab (bas) in recipients at high risk for delayed graft function (dgf) or rejection, tmg was associated with less acute rejection (15.6 % vs 25.5%, p=0.02) and a lower triple endpoint (rejection, death or graft loss, 20.6% tmg vs 33.6% bas, p=0.02) but not a significantly lower quadruple endpoint including dgf. the purpose of this study was to compare the efficacy of tmg and bas for induction stratified by donor source: standard criteria donor (scd), extended criteria donor (ecd) or donor with hypertension (htn). methods: retrospective review of data collected in the original randomized trial. data-capture limitations necessitated defining ecd as donor age > 60 or donor age between 50 and 60 with both a donor history of htn and donor renal insufficiency (history of atn or creatinine above 2.5 mg/dl during the 24 hours prior to organ recovery/start of cold ischemia time). results: 75 recipients received ecd kidneys [tmg n=40 (28.4%), bas n=35 (25.6%), p=ns]. outcomes are presented below. there were no differences in the rates of dgf between the groups examined. conclusion: standard and non-htn donor recipients had a tremendous benefit of tmg compared to bas with less acute rejection and death. contrary to the perceived niche of tmg in ecd recipients, tmg has its most beneficial effect in scd recipients and recipients of donors without htn at risk for acute rejection or dgf. evaluation we have changed our immnosuppressive protocol in abo-incompatible kidney transplantations and attempted to determine whether the changes in agents have resulted in better outcomes. we used tacrolimus(fk), mycophenolate mofetil(mmf) and methylprednisolone(mp) in immunologically high risk patients between 2000 and 2007. moreover, we performed splenectomy at the time of the transplant surgery in 117 patients (group 1) with aboincompatibilities between 2000 ansd 2004, and administered rituximab as an alternative to splenectomy in 38 patients(group 2) with abo-incompatibilities between 2005 and 2007. in this study, we compared the graft survival rates as well as the incidence of acute rejection in these two treatment eras. the graft survival rate at one year was 94% in group 1 and 97% in group 2 (p=ns). the graft was lost in one case of the 38 cases of group 2 due to insufficient doses of the immunosuppressive drugs. the incidence rate of acute rejection was 15% (18/117) in group 1, and 11% (4/38) in group 2 (p<0.01). there were no significant differences in serum creatinine level one year after transplanatation between two groups(1.5±0.3 in group 1 vs.1.4±0.4mg/dl in group 2). no serious adverse events associated with rituximab or splenectomty were encountered in either groups. in abo-incomaptible kidney transplantation, rituximab under fk/mmf combination as an alternative to splenectomy seems to yield an excellent result in terms of the incidence rate of acute rejection. introduction: the choice of immunosuppression in the elderly kidney transplant recipient remains unclear. the objective of this study was to compare outcomes with different t cell-depleting induction agents in the elderly. method: all solitary kidney transplant recipients over the age of 60 years that received induction therapy with either alemtuzumab or thymoglobulin from 2002 to 2007 were included in this unos analysis. overall graft survival, the risk of graft loss, and the risk of rejection were compared using kaplan meier, cox proportional hazards, and logistic regression, respectively. results: patients receiving alemtuzumab had a significantly lower 3-year graft survival (70.8%) than patients receiving thymoglobulin (79.3%), p=0.02) (figure) after adjusting for other risk factors, alemtuzumab had a higher risk of graft loss compared to patients given purpose. the aim of this prospective randomized study was the comparison of efficacy and incidence of adverse events in two induction therapy regimens (atg versus basiliximab) in patients receiving a dual immunosuppression. methods. 120 recipients of first or second deceased donor kidney transplants were prospectively randomized to receive either atg (fresenius) or basiliximab (novartis) as induction therapy. dual immnosuppression consisted of tacrolimus (astellas) and methylprednisolone. cmv prophylaxis was not applied on a regular basis. statistical analysis was performed with fisher's exact or chi-squared test, anova or mann-whitney u test, kaplan meier curves and log-rank test. results. patient characteristics of populations treated with atg versus basiliximab were similar concerning average age (48 years), gender and dialysis time prior to transplantation (78 vs. 88 months). average donor age and cold ischemia were also comparable (43 vs. 41 years and 833 vs. 852 minutes). the actuarial 5-year patient survival for the atg subpopulation is 91,7 % in comparison to 85 % in the basiliximab group (n.s.). analyzing graft survival after 5 years, rates of 88,3 % in atg patients compared to 75 % in the basiliximab group can be observed (n.s.). the incidence of acute rejection episodes was similar in both groups (atg: n=20 vs. basiliximab: n=16). 18 (30%) patients in the atg group and 20 (33,3%) in the basiliximab group showed a delayed graft function. serum creatinine was not significantly different at 1 and 5 years (atg: 1,4±0,6 mg/dl and 1,4±0,8 mg/dl vs. basiliximab: 1,3±0,7 mg/dl and 1,5±0,9 mg/dl). patients in the atg group had a higher rate of cmv infections (n=13 vs. n=3; p=0,05), whereas patients treated with basiliximab had significantly more hematological complications like anaemia, leukopenia and thrombocytopenia. conclusion. comparing induction therapy with atg and basiliximab, our data shows similar patient and graft survival rates with slightly better results in the atg group. patients treated with atg had a higher rate of cmv infections but less hematological complications. predicting cardiovascular events (cvd) and the varied effects of immunosuppressive medication on cvd risk factors requires an understanding of how traditional and nontraditional risk factors impact cvd after kidney transplantation (ktx). single-center studies have generally lacked statistical power and generalizability. registry studies have lacked sufficient data on cvd risk factors. the port project is creating a multicenter international database of ktx recipients with the primary objective of developing risk prediction models for post-transplant cvd. as a preliminary data assessment, an analysis was done on 19,669 ktx from 1990-2007 from 11 transplant centers representing 5 european centers, 4 north american centers, and 2 centers from asia/oceania. all data were extracted from preexisting databases at each individual transplant center and processed into the consolidated port database. 1,131 major adverse cardiac events (mace) were identified, defined as non-fatal or fatal myocardial infarction, cardiac abstracts arrest, and sudden death. the mace-free survival curves by participating center are shown in the figure. in this preliminary analysis, the overall one-year cumulative incidence of mace was 2.2%, ranging from 0.2% to 4.4% across centers; and the five-year cumulative incidence was 5.8%, ranging from 0.6% to 13.5%. the prevalence of diabetes pre-transplant varied from 10% to 47% across centers. in cox proportional hazards models adjusted for age, gender, race, donor type, transplant center, and reported history of diabetes, hypertension (htn), and ami, patients with a reported history of ami had a 112% increased risk (79%-152%, p<0.0001) for mace. for the final analysis, the definition of mace will be expanded to include major revascularization events. the final port database will be used to develop and validate an equation to predict mace and other health outcomes of interest, after accounting for differential cvd risk factors internationally. abstracts to center. the most commonly used bp medications were beta-blockers, followed by ccbs, and both were used with the same frequency at 1 and 6 months. in contrast, the percentage of patients on an acei or arb more than doubled between 1 and 6 months, suggesting reluctance to use these agents early after tx (a practice not necessarily evidence-based); however, more than 70% of subjects did not receive acei/arb therapy even at 6 months. fewer than half of all patients received aspirin, including only 60% with dm and/or cvd. similarly, only half with dm and/or cvd received a statin at 1 and 6 months. these data indicate current management of ktx recipients fails to utilize optimal cvd risk reduction measures in a timely fashion, perhaps missing an opportunity to reduce long-term morbidity and mortality from cvd in this at-risk population. background: cardiovascular (cv) risk reduction has been a primary reason for pursuing early corticosteroid withdrawal (ecswd). to date, actual cardiovascular event data (cve) (rather than cv risk) has not been reported for ecswd. therefore, we analyzed and compared actual cv events (cve) and cv-related survival in ecswd (≤7 days) and chronic corticosteroid (ccs) pts. methods: cve and heart failure (hf) data were prospectively collected. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/ transient ischemic attack. hf events were defined as pulmonary edema or hf diagnosis. conclusions: rtx recipients receiving ecswd experienced: 1) fewer cve and 2) a trend toward overall better pt survival. these differences in cve and pt survival do not present until at least 3 yrs ptx. and therefore require long term followup to become evident. abstracts immunohistochemistry was performed for cd4 + and cd8 + cells. results were compared with those of the patients on maintenance is (gr-is n=29) and the liver tissue from normal subjects (gr-normal n=11). results: the follow-up time in gr-tol was longer than that in gr-is.(gr-tol and gr-is: 121m and 52m, p<0.01) in gr-tol, typical features of neither acute nor chronic rejection were observed following banff criteria. the extent of graft fibrosis in gr-tol, however, was greater, than those in gr-is and gr-normal (gr-tol, gr-is and gr-normal ; 1.6, 0.9 and 0 (ishak's modified staging )(gr-tol vs. gr-is, gr-is vs.gr-normal p<0.01). each number of cd4 + and cd8 + cells in graft infiltrates was increased in gr-tol, compared with that in gr-normal, but equivalent with that in gr-is (cd4 + gr-tol, gr-is and gr-normal ;14.6,10.6 and 5.3 cells/field, gr-tol vs.gr-normal p<0.05, gr-tol vs.gr-is ns / cd8 + gr-tol, gr-is and gr-normal; 27.6, 26.3 and 10.5 cells/field gr-tol vs.gr-normal p<0.01, gr-tol vs.gr-is ns). conclusions+discussion : in tolerant graft after pediatric living-donor ltx, neither acute nor chronic rejection was observed, but fibrosis developed. because of the similar extents of cd4 + and cd8 + cells infiltrates and different follow up time between tolerant and immunosuppressed patients, it remains questionable whether fibrosis in tolerant graft is antigen-dependent. serial protocol biopsy before and after starting weaning is will detect fibrosis early, and observing whether reintroduction of maintenance is reverses fibrosis in that case will answer this question. operational tolerance may not always guarantee intact graft morphology. development of "operational tolerance" after pediatric liver background : in the setting of our pediatric living-donor liver transplantation (ltx), 15% of all the patients (significantly higher proportion, compared with those of other transplant centers) achieved complete withdrawal of immunosuppression (is), which is reffered to as "operational tolerance". nonetheless, some patients encountered rejection while they were undergoing weaning from is. it is,therefore,essential to identify and characterize the differences that will enable patients in these two distinct populations to be distinguished reliably. methods: the study groups consisted of group tolerance(gr-tol) in which 88 patients are successfully weaned off from is, and group rejection (gr-rej) in which 22 patients experienced clinically evident rejection during or after weaning process. the correlation between the clinical outcome (success or failure of weaning is) and following parameters was assessed ; donor/recipient age, donor/recipient gender, abo compatibility, hla mismatch, graft size, early (<1month) rejection episode and initial immunosuppression.results: there was no difference between gr-tol and gr-rej with respect to donor/recipient age (gr-tol and gr-rej;32y and 32y ns/35m and 26m ns), or donor/recipient gender (gr-tol and gr-rej (female);60% and 50% ns/60% and 73% ns). abo compatibility did not differ between the two groups(gr-tol and gr-rej (i dentical:compatible:incompatible);63%:27%:10%and 55%:27%:18% ns).the presence of hla-b mismatch was more frequent in gr-tol than that in gr-rej (gr-tol and gr-rej;95% and 76% p<0.05 ), while the presence of hla-a or dr mismatch did not affect success or failure of weaning is(hla-a gr-tol and gr-rej;73% and 76% ns, hla-dr ;83% and 82% ns). graft size did not differ between the two groups(gbwr gr-tol and gr-rej;2.9% and 3.2% ns). the patients in gr-rej experienced early rejection more frequently than those in gr-tol(gr-tol and gr-rej;17% and 50% p<0.05). mean trough level of tacrolimus within 7 days after ltx was compatible between the two groups (gr-tol and gr-rej;11ng/ml and 11ng/ml ns). conclusions: development of operational tolerance after pediatric ltx was associated with the absence of early rejection and the presence of hla-b mismatch between donors and recipients. auxiliary partial orthotopic liver transplantation (apolt) in children with fulminant hepatic failure. patients with fulminant liver failure (fhf) who undergo auxiliary partial orthotopic liver transplantation (apolt) have a chance to come off immunosuprresion (isp) when the native liver regenerates. it may be most beneficial for children with fhf; however, the literature regarding its use in children has been limited. from the beginning of the pediatric liver transplant program at our institution, 31 patients underwent liver transplantation for fhf. of those, 7 received apolt and the remaining standard liver transplantation (olt). seven children (age 8months to 8 years) who received apot (apolt group) were compared to matched control group of 11 patients (olt group). since apolt was offered routinely at out instituting since 2005, 6 of apolt cases were done since 2005. in apolt group, either left lateral segment or left lobe graft was used. recipients left lobe was removed in all cases. in olt group, 7 received whole liver graft and 4 received partial liver graft. all native livers showed submassive to massive necrosis at the time of transplant in pathology. all children (100%) in apolt group are currently alive with a median follow up of 761 days (range 116-4139 days) where 8 (72%) patients are alive in olt group (median follow up 1724 days). six of 7 children in apolt group (87%) showed native liver regeneration. first four apolt recipients (57%) are currently off isp with fully regenerated native liver. two of those patients developed complete atrophy of the graft liver, one underwent graft removal due to sepsis caused by severe rejection. one remaining patient who is off isp is displaying progressive atrophy of the transplant liver. incidence of acute rejection was 57% (4/7) in apolt group vs 30% (3/10) in olt group. other postoperative complications included hepatic artery thrombosis (hat) (n=1), bile leak (n=1), bilary structure (n=1) and bowel obstruction (n=1) in apolt group, hat (n=1), bile leak (n=2), bowel perforation (n=2), chylothorax (n=1) and aplastic anemia (n=2). median posttransplant length of stay was 15 days in apolt and 20days in olt group. conclusions: apolt was safely performed in children with fhf. significant proportion of recipients displayed native liver regeneration and came off immunosuppression. natural killer cell dysfunction in pediatric acute liver failure. nada yazigi, 1 greg tiao, 1 alexandra filipovich, 2 john bucuvalas. 1 in pediatric patients, indeterminate acute liver failure (alf) accounts for ∼50% of all cases, and carries a particularly poor prognosis without transplantation. evidence exists to suggest that acute liver failure may reflect a disproportionate immune response to a common stimulus. nk cells comprise a central component of the innate immune system. we hypothesized that nk cell dysfunction (innate or secondary to an antigenic insult) plays a pathologic role in indeterminate alf. we reviewed peripheral nk cell function in a series of 15 consecutive children cared for at cincinnati children's hospital, who met criteria for indeterminate alf as defined by the pediatric alf study group. peripheral blood testing was carried for nk cell number, cytolytic function and perforin and granzyme activity as part of our clinical alf protocol. seven of fifteen patients had nk cell dysfunction. only the severity of cholestasis was statistically higher in the nk cell dysfunction group. there was no statistical difference between the 2 groups with respect to age, inr, or peripheral blood cell counts. 3 of seven patients with nk cell dysfunction died in contrast to none of eight in the normal nk cell group. of the 3 patients with nk cell dysfunction who eventually received a liver transplant, 2 had severe early recurrence of chronic hepatitis in the graft at a year follow up. those outcomes are in sharp contrast to the group with no nk cell dysfunction where 7 patients needed transplantation, but all had no complications both on short or long term follow up. we documented nk cell dysfunction at the time of alf diagnosis in 7/15 pediatric patients with indeterminate alf. this subgroup of patients was found to have higher: mortality, risk of infections, as well as recurrent disease in the graft. our findings suggest that nk cell dysfunction is involved in the pathogenesis of indeterminate alf. as such, it could therefore be a prime target for therapeutic intervention, with goals to rescue the patient from liver failure and /or to improve post-transplantation outcomes. background hrs is a reversible renal failure which occurs in pts with advanced liver disease and portal hypertension and is characterized by a marked decrease in gfr and rpf in absence of other identifiable causes. vasodilation theory is currently the most accepted hypothesis to explain the pathogenesis. in decompensated cirrhotics, probability of developing hrs is 8-20%/yr and increases to 40% at 5 yrs. ideal treatment is ltx. however, there is an urgent need for effective alternative tx to increase surv chances for pts until ltx can be performed. interventions that have shown some promise are vasoconstrictors in splanchnic circulation and tips. main objective was to compare efficacy of two different regimens (albumin/terlipressin resp hes/terlipressin both w/ wo midotrine) against tips whereas grf was considered as primary efficacy endpoint. pts/tx dx of hrs was based on criteria, as proposed by international ascites club. only pts with esld on the waiting list for ltx were eligible to be enrolled. pts were assigned to tx arms and randomized w/wo midotrine. volume/vasoconstrictor tx lasted for 10d, mitodrine was continued; follow up for 90d. results iia (albumin/terlipressin); iib (hes/terlipressin); iic (tips) discussion combination of volume expansion/vasoconstriction improved effectively gfr in pts with hrs. use of albumin shows no advantage compared to (cost effective) hes. although a marked improvement was observed during iv-treatment, renal fct deteriorated upon treatment withdrawal whereas pts with continued mitodrine showed superior long term outcome. we analyze our single institution experience to quantify the long-term incidence of renal failure based on month 3 gfr compared to subsequent determinations. methods: this is an irb approved retrospective review of the prospectively maintained database of lt recipients. exclusion: patients on renal replacement therapy (rrt) at time of transplant, combined liver kidney, fulminant hepatic failure, and <1-year follow-up. gfrs (i 125 iothalamate glofil method) were measured at initial evaluation (ie), month 3 (m3), year 1 (y1), 2 (y2), 5 (y5), 10 (y10), and 15 (y15). patients were grouped by gfr >80 (g1), 60-80 (g2), and <60 (g3). ie and m3 gfr were used as starting points for longitudinal analyses. paired data analysis for ie, m3, y5 and y10 was also performed. renal failure was defined as gfr <30, received kidney transplant, on dialysis, or on kidney transplant list. results: 592 liver transplant patients were reviewed between 1985 and 1999. paired glofil data was available for the y5 and y10 analysis in 114 patients. m3 gfr correlated more with long-term renal function (p<0.0024). g1 demonstrated largest reductions in gfr over time. g2 and g3, when corrected for patients that got kidney transplantation and rrt, demonstrated progressive reduction in gfr. g3, g2, and g1 were statistically significant (p<0.001 wilcoxon two-sample test). this study clearly demonstrates progressive decline in gfr continuing out to 15 years after liver transplantation. m3 gfr correlates better with long-term renal function compared to ie gfr (not truly reflective of renal function at time of lt). if m3 gfr <60, data showed a high rate of renal failure in our paired data analysis by y5 (p<0.0024). by correcting for patients with renal failure, the previously reported stability in gfr between y1 and y5 is not seen. analysis of grouping demonstrates that g1 patients at m3 have lower incidence of renal failure >10 years after lt. g2 and g3 patients will be at higher risk for renal failure each year after transplantation. introduction: calcineurin inhibitors have demonstrated efficacy in liver transplantation. however, they have a potential to impair renal function. delayed tacrolimus (tac) administration may reduce the risk of renal dysfunction. methods: a prospective study included liver transplant pts randomised to delayed introduction of tac (day 5) + daclizumab (dac) (group a) or to immediate tac administration (group b). in both groups tac t0 was 10-20 ng/ml until week 4 and 5-15 ng/ml thereafter. mmf was given at 2 g/d for 2 months, and corticosteroids (cs) at standard doses. pts with a serum creatinine (scr) > 180 µmol/l at 12 hours (h12) were excluded. the primary endpoint was the rate of pts with a mean scr > 130 mmol/l at month 6. month 24 results are presented. results: 207 pts were randomised. baseline characteristics were similar. at month 6, mean tac t0 was 9.6 (group a) and 11.2 ng/ml (group b median follow-up post-tx was 8 years (5-21). most frequent tx indications were alcoholic (34%) and hcv (10%) cirrhosis. amdrd glomerular filtration rate (gfr) was < 60 ml/min/1,73m 2 in 11% (bt), 46% (1m), 48% (1y) and 55% (5y) of the patients. changes in gfr were then compared according to the immunosuppressive protocol: -group "cni+mmf" = a calcineurin inhibitor (cni) + mycophenolate mofetil (mmf). -group "cni" = a cni without mmf. in this group, some patients received only cni and some cni + azathioprine. there was no difference between those 2 sub-groups, neither on rf nor on cni doses. all those patients were thus pooled. in both groups, gfr decreased from bt: -14% in "cni+mmf" vs -25% in "cni" at 1m (p=0.06), -14% vs -29% at 1y (p=0.03), and -12% vs -33% at 5y (p=0.02). although their mean gfr bt was lower (86 vs 97 ml/min/1.73m 2 , p=0.0002), the decrease in rf in "cni+mmf" patients was less severe. nearly 50% of the patients had renal insufficiency in the 5 years following liver tx. the reduction in the gfr is less pronounced in patients treated with mmf even if they were significantly more at risk bt. except at 1m, there was no difference in cni doses between the 2 groups, suggesting that the sustained lower decrease in rf observed in "cni+mmf" may not be only explained by a cni dose reduction. acute rejection is a complex biologic process involving multiple cell types, cytokines and chemokines/chemokine receptors. we hypothesized that an mrna panel that included genes implicated in the anti-allograft response would distinguish allografts undergoing acute rejection from normal allografts with a high degree of accuracy. we tested this hypothesis by measuring levels of urinary cell mrna and peripheral blood cell mrna for cell surface proteins cd3, cd20, cd25, cd103, and ctla4; chemokines/ chemokine receptors ip10, mig, cxcr3; cytotoxic attack molecules granzyme b(gb) and perforin, and immunoregulators foxp3, tgf-beta1 and il-10. gene specific primer pairs and probes were used in pre-amplification enhanced real time quantitative pcr assays to measure mrna and transcripts for 18s rrna. for each cell source, we used logistic regression to identify a linear function of up to 5 log-transformed measures that would distinguish biopsies of ar patients from those of stable transplant patients. our study demonstrates that molecular signatures developed using urinary cell levels of just 5 genes (signature 1, urinary cell levels of mrna for ctla4, foxp3, gb, cd3, and mig), or a combination of 3 urinary and blood cell levels (signature 3, urinary cell level of ctla4 mrna and peripheral blood cell levels of cd103 and ctla4) differentiate ar from stable biopsies with 100% sensitivity and 100% specificity. blood cell levels alone are also informative, but less so. we conclude that molecular signatures, developed from noninvasively ascertained mrna profiles of urinary cells/ peripheral blood cells, predict acute rejection with extraordinary accuracy. clinical trials to validate the predictive value of these signatures are worthy of pursuit. we have described the association of cd20+ b cell infiltrates in renal transplant (tx) biopsies (bxs) with acute cellular rejection (acr) and tx dysfunction (dysfx). we have also found metabolically active plasma cells (pcs) staining for s6 ribosomal protein (s6rp) within these txs. herein, we report the significance of cd138+ pcs in rejection (rj) and evaluate the impact of cd20, cd138, and s6rp on long term tx fx by calculated creatinine clearance (crcl). we studied 46 tx bxs from 32 pediatric (ped) patients (pts) who were bxed for suspicion of rj from nov 2001 to nov 2004. pts were given daclizumab and maintained on prednisone, mycophenolate mofetil, tacrolimus or cyclosporine. immunohistochemical staining and quantification for cd20, cd138, s6rp, and c4d were performed under 500x light microscopy. bxs were classified by modified banff 97 criteria. crcl was followed 2 yr post-bx. cd138+ pcs were associated with c4d-negative acr (p=0.0002) but not antibody mediated rj (amr, p=0.82). roc analysis confirmed >5 cd138+ cells/hpf strongly associated with acr, yielding 89% sensitivity, 83% specificity, correctly classifying 84% and comprising total roc area 0.92 (95% ci 0.82, 1). higher cd138 counts at bx correlated with worse tx fx (fig 1) . a univariate regression model showed that cd20, cd138, s6rp and time were associated with a decline in tx fx at bx. multivariate model showed that cd20, cd138, and time had the main effects on crcl decline, with s6rp dropping out. all patients regardless of rj status had a 16 ml/min/1.73 m 2 crcl decline exerted by time (p=0.004). pts with cd20 had an additional sustained 19 ml/min/1.73 m 2 crcl decline seen 2yrs post-bx (p=0.03). pts with cd138 also had 19 ml/min/1.73 m 2 crcl decline at bx (p=0.03), but there was an interaction between time and cd138 that negated a sustained effect (p=0.04). this study identifies a numerical threshold of >5 cd138 cells/hpf that is associated with acr and tx dysfx. infiltrating cd20 cells had the greatest, sustained effect on tx dysfx. we conclude that cells of the b lineage, particularly cd20, play a key, but undefined, role in acr. intragraft there is now evidence that foxp3+ cells are not indicators of tolerance, since foxp3 is also increased during acute rejection. however, it is unknown whether foxp3+ cells are present during chronic antibody mediated rejection. moreover, the relative balance of regulatory, effector and cytotoxic pathways in chronic vs. acute injury has yet to be explored. here we addressed this issue. intragraft regulatory, effector and cytotoxic transcriptional profiles were analysed within renal transplant biopsies (n = 43) classified (banff 2005) as displaying normal histology, chronic calcineurin inhibitor toxicity (cnitox), chronic antibody mediated rejection (camr) and acute cellular rejection (acr). granzyme b, tbet and foxp3 mrna were measured by quantitative pcr and foxp3 -positive cells were additionally quantified in graft biopsies by immunohistochemistry. distinguishing mrna profiles were analyzed in the peripheral blood (n = 26). our data show that foxp3 mrna is increased not only in acr (p<0.0001) but also in camr (p<0.05). expression of foxp3 mrna correlated tightly with the density of foxp3 protein-positive cells by immunohistochemistry (spearman r = 0.71; p < 0.0001); foxp3+ cells were found in aggregates and within tubules. moreover, graft cytotoxic, effector and regulatory pathways were all found to be active in chronic as well as acute graft injury. significant increases in granzymze b, tbet and foxp3 mrna were observed in camr, cni-tox and acr compared to normal histology (p<0.0001, p<0.001 or p<0.05). however, differences in the relative contribution of each pathway were evident, with significant accumulation of foxp3 mrna predominating in acr and granzyme b predominating in camr. thus, camr can be distinguished from both acr and cni-tox by an unfavorable intragraft granzyme b/foxp3 mrna ratio (p< 0.05). interestingly, this ratio was reversed in the blood, suggesting different migratory patterns for regulatory and cytotoxic cells between the blood and the graft.our data thus confirm that intragraft and peripheral blood foxp3 accumulation is also a feature of camr of kidney grafts. moreover, camr can be distinguished from other graft injury types based on its intragraft or blood cytoxicity/regulatory profile. survival of solid organ grafts depends on life long immunosuppression which results in increased rates of infection and malignancy. induction of tolerance to allograft would represent the optimal solution for controlling both chronic rejection and side effects of immunosuppression. we previously showed that operational tolerance after kidney transplantation could occur in some patient. here, the potential of high throughput microarray technology allowed us to study the peripheral blood gene expression profile associated to operational tolerance and chronic rejection in a cohort of human kidney graft recipients (n=26). microarrays were used to compare the gene expression profile of pbmc from patients with chronic rejection and drug-free operationally tolerant recipients. results have been treated using a classical statistical and a non-statistical analysis based on the identification of key leader genes associated respectively to chronic rejection and operational tolerance, either as those mostly changing their expression or having the strongest interconnections. 343 differentially expressed genes were identified between operational tolerant patients and patients with chronic rejection. abstracts defined as missing > 10% of prescribed doses on mam and mems, and >2 sd among consecutive blood serum levels. results: participants were 28 transplant patients (m = 14.11 +3.38 years old, 75% male, 71.4% caucasian). on the mam, 67.9% of the patients acknowledged some non-adherence but minimized how many doses they missed. using mems technology, 90% had some non-adherence and specifically, 23.8% of the participants missed doses and only 56% of their doses were taken within the allowable time frame. using > 2sd criteria for blood serum levels, 42% of the participants were considered non-adherent. non-adherence worsened with years since transplant. more missed (r = .59, p = .001) and late doses (r = .59, p = 0.04) on the mam and >2 sd among blood serum levels (r = .83, p = .001) was associated with higher incidence of acute rejections. adherence data was examined for patients with documented acute rejections (n = 12). sensitivity and specificity of each detection method was also examined. the mam and sd detection methods each identified non-adherence in 67% of the patients with acute rejections; mems did not identify any additional non-adherent patients. only 25% of the patients with acute rejections were identified consistently by all three adherence detection methods; all patients with acute rejections were identified by at least one method. discussion: non-adherence worsening with time since transplant and was associated with acute rejections. since no single method of detecting adherence identified all the patients with acute rejections, multi-method adherence assessments should be used to accurately capture patients who are non-adherent. non na is a leading cause of allograft loss and results from multiple factors. locus of control (loc) and beliefs regarding health have been associated with adherence in other populations. 51 randomly chosen ktr's were interviewed using a confidential questionnaire administered by an outside investigator that included questions regarding loc, health beliefs and self-efficacy. the population was 55% female, 92% black, 15% hispanic, 71% deceased donor kidney, 47% diabetic, 49% greater than high school education, 36% employed, 30% married or cohabiting, 63% income <20k per year, 65% insured by medicaid. mean age 47.8±13.5 yrs, time on dialysis 72.8±51 mos, months since transplant 23.9±14.3, total meds 7.3±3.0. by pearson correlation, non-adherence (na), defined as "having missed doses of immunosuppression over the preceding 3 months", was not correlated with race, gender, income, type of insurance, age, marital status, type of txp, mos on dialysis, time since transplant, or number of medications. na was correlated with higher education level (r=0.4, p=0.006), current employment (r=0.3, p=0.05), and knowledge of most recent creatinine value (r=0.35, p=0.015). na was associated with concerns regarding prednisone (long term effects, dependency) r=0.33, p=0.018, feelings of greater personal control over illness (r=0.36, p=0.011), and inversely correlated with powerful others loc (feeling that one's health is dependent on other people), r=-0.31, p=0.029 and belief in the necessity of medication for maintenance of transplant health, r=-0.3, p=0.031. we conclude, in our population of inner-city patients: 1. na is not associated with standard demographic factors including income, race and gender. 2. contrary to findings in other populations, na is associated with higher education and current employment. 3. na is associated with knowledge about creatinine value, concern regarding long-term effects of prednisone and disbelief in the importance of transplant medications. 4. na is associated with feelings of personal control and feeling that powerful others (e.g. health care providers) are not of high importance in the outcome of illness. 5. education programs designed to address na in this population should be targeted towards altering negative beliefs regarding medications and stress the importance of partnering with the transplant team for optimal long-term outcome. purpose: the present study aimed to prospectively examine the relationships among nonadherence, health-related quality of life (hrqol), and family factors in adolescent kidney, liver, and heart transplant recipients. method: 68 adolescent transplant recipients aged 11 to 20 years (m = 15.8, sd = 2.5; 44% female; 57% kidney, 25% liver, 18% heart) and their parents participated. at baseline and 18-month follow-up assessments, adolescents and their parents independently completed phone interviews assessing self-/proxy-reported medication adherence, hrqol, and family cohesion and conflict. medical record reviews were conducted to obtain current medications, immunosuppressant drug assays, and clinical outcomes in the past year (i.e., rejection episodes, hospitalizations, graft loss). results: at baseline, adolescents classified as nonadherent based on self-report and tacrolimus standard deviation (sd) reported significantly lower general health perceptions (f(3, 64) = 3.63, p < .05), self-esteem (f = 4.58, p < .01), mental health (f = 3.09, p < .05), and behavior hrqol (f = 2.75, p < .05) compared to adolescents classified as adherent. similarly, parents of adolescents classified as nonadherent reported significantly lower physical functioning (f = 3.18, p < .05), self-esteem (f = 5.85, p < .01), and behavior hrqol (f = 2.82, p < .05) for their adolescents. family conflict was correlated with adolescent report of behavior (r = -.49, p < .01), physical functioning (r = -.33, p < .01), self-esteem (r = -.42, p < .01), and mental health hrqol (r = -.34, p < .01). family conflict was correlated with parent report of behavior (r = -.46, p < .01) and physical functioning (r = .24, p < .05). improvement and deterioration in hrqol from baseline to 18-month follow-up is currently being examined. it is expected that increased family conflict and decreased medication adherence will be associated with deteriorations in hrqol. the interrelationships between medication adherence, family conflict, and hrqol domains such as self-esteem and mental health suggest that interventions targeting these domains may result in improvements in medication adherence behavior. the use of cam in general is associated with non-disclosure by patients to physicians. 51 randomly chosen ktrs were interviewed using a confidential questionnaire administered by an outside investigator, including questions on cam usage, whether it was doctor-recommended, and whether the patient disclosed use. cam was defined as ingestion of herbal or other preparations, use of mind-body techniques or manipulation of the body for healing by someone not an allopathic medical provider. use of vitamins and spirituality were excluded. the population was 55% female, 92% black, 15% hispanic, 71% deceased donor kidney, 47% diabetic, 49% > high school education, 36% employed, 30% married or cohabiting, 63% income <20k per year, 65% insured by medicaid. mean age 47.8±13.5 yrs, time on dialysis 72.8±51 mos, months since transplant 23.9±14.3, total meds 7.3±3.0. 45% of patients (n=23) used cam. by pearson r, cam use was correlated with na to immunosuppressants, p= 0.041, r= 0.4, blood sugar-lowering medications, p= 0.003, r= 0.57, and cholesterol lowering medications, p= 0.0001, r=0.84, worries about long-term effects of medicines, p=0.02, r= 0.324, belief that doctors place too much trust in medication, p=0.032, r=0.3, and that natural remedies are safer than medicines, p= 0.034, r=0.3. cam use was inversely related to belief that health depends on allopathic medicines, p=0.014, r= -0.341, medicines protect from worsening disease, p=0.048, r= -0.28, and that following doctors orders is the best way to stay healthy, p= 0.04, r= -0.29, and that having a kidney transplant makes them feel happy, p= 0.005, r= -0.39. we conclude, in our population of inner-city patients: 1. use of cam is correlated with medication non-adherence. 2. patients who use cam are more worried about long term effects of medication, believe that natural remedies are safer than medications and that doctors place too much trust in medication. 3. patients who use cam do not believe that their health depends on allopathic medication, that medicines protect from worsening disease, or that following a doctor's orders is the best way to maintain optimal health. 4. patients who use cam are less happy with their kidney transplant. 5. disussing cam use and motivation for use is important in the transplant clinic and may alert the provider to possible risk for non-adherence. by multivariate cox analysis the risk of tg related to the presence of hla-iiab death censored graft loss occurred in 4.6% of patients without tg and in 38.4% of patients with tg (p<0.0001) hla-iiab are associated with higher risk of tg and reduced graft survival. furthermore, the risk of tg and its prognosis relate to the level of hla-iiab quantitated in a solid phase assay term survival of cardiac allografts in wild-type mice by alloantigen (alloag)-specific foxp3 + cd4 + cd25 + natural regulatory t (nt reg ) cells. guliang xia, 1 jie he methods: fresh naive cd4 + cd25 + nt reg were isolated from congeneic b6.pl mice via automacs and enriched for alloag specificity by in vitro culture with either anti-cd3/ cd28-coated dynabeads (d0-11), then donor bone marrow-derived dendritic cells 8% (d+16) for dc/beads-expanded nt reg , while total fold of expansion of nt reg remained similar (24.8∼30.2 for beads/dc-or 5.2∼7.1 for dc/beads-expansion) regardless of the presence or absence of tgf-β. introducing ra (100 nm) into bead/dc-based, tgf-β/ il-2-conditioned culture resulted in marginal improvement with 28.9% (d+18) nt reg being foxp3 + . in mlr assays, nt reg expanded with tgf-β/il-2 exerted more potent suppression than cells conditioned with il-2 alone. in vivo, beads/dc-expanded, tgf-β/ il-2-conditioned nt reg synergized with transient host t cell-depletion (anti-thy1.2 mab i.p. 50 µg at d-3 & 25 µg on d+2) in c57bl/6 mice to suppress balb/c heart allograft rejection with 57.1% (n=7) and 100% (n=10) allografts surviving over 100 days when 4x10 7 or 8x10 7 cells/mouse were injected immediately post-transplant, respectively. anti-thy1.2 treatment alone led to only 18.8% long-term survival. infused nt reg survived long-term (5.8 % circulating t cells (8x10 7 cell dose) or 1.2 % (4x10 7 cell dose) at d+7 post-transplant) and expressed high level foxp3 (40∼60%) in vivo. long-term surviving allografts showed characteristics of 'acquired immune privilege' with cellular infiltrates that were foxp3 + , tgf-β + , il-10 + and indoleamine 2,3-dioxygenase (ido) + , although signs of mild to moderate chronic rejection were still evident conclusion: t-bet deficiency results in up-regulation of il-17 expression in addition to th2 associated cytokines resulting in acceleration of chronic rejection despite profound deficiency of ifn-γ. t-bet deficiency may contribute to the alloimmune responses independent of ifn-γ by in situ hybridization, tir8 mrna level was higher (p<0.05) in cortical tubuli, glomeruli, perivascular and peritubular areas of kidney grafts at 1, 3 and 6-7 d post-tx, than in naive kidneys. to assess how local expression of tir8 affects the outcome of kidney grafts, we transplanted tir8 -/-b6x129 kidneys into dba/2 mice. most (83%) recipients of tir8 -/-kidneys rejected their grafts with a median survival of 9.5 d (n=12, p<0.05 vs wt) and had more severe graft dysfunction (bun levels) at day 1, 3 and 6-7 days post-tx, than recipients of a wt allograft (p<0.05) opticept trial: efficacy and safety of monitored mmf in combination with cni in renal transplantation at 12 months. r trough-based dose adjustments were made in the mmf cc arms. antibody induction and/or corticosteroids were administered according to center practice. primary endpoints were the proportion of patients with treatment failure (biopsy-proven acute rejection [bpar], graft loss, death), and mean percent change in calculated glomerular filtration rate (gfr; nankivell equation) at 12 months. safety endpoints were incidences of adverse events (aes) and serious aes baseline characteristics did not differ among treatment groups with living donors accounting for approximately 50% of grafts. 82% received tacrolimus (tac) and 18% cyclosporine (cya): cni doses and levels were significantly lower in group a. mmf doses were greater in cya-treated subjects in all groups cya treated patients and in group a (p=0.08); stability of renal function over time was greatest in group a. despite higher mmf doses in group a (p<0.001) at most time points, significantly fewer mmf withdrawals occurred in group a vs. groups b and c. conclusions: a concentration-controlled mmf and reduced level cni regimen is not inferior to that of fixed-dose mmf and standard-dose cni as regards bpar and other end points. this regimen facilitated higher mmf dosing without an overall increase in adverse effects, and with a trend toward preservation of kidney function versus standard-dose cni regimens comparison at one year of interstitial fibrosis (if) by automatic quantification in renal transplant recipients with cyclosporine (csa) discontinuation and sirolimus (srl) introduction introduction: we previsouly reported the clinical results of a multicentric study showing that csa conversion to srl at week (w) 12 is associated with a significant improvement in renal function. using routine renal biopsy (rb) performed at w52 during this study routine rb was performed at w52. for each rb, a section was imaged using a colour video camera and analyzed by a program of colour segmentation which automatically extracts green colour areas characteristic of if. results were expressed as percentage of if and grade according to banff classification. results: male donor gender was associated with higher if (30±2% vs. 23±2%, p =0.03). if was numericaly higher in patients who had experienced acute rejection (33±4%, n = 18 vs. 26±1%, n=99, p=0.06) there was a positive correlation between renal function and the percentage of if on rb (p=0.004). despite significant improvement of renal function at w52 in the srl group intent to treat (n=117) mean if (%) grade i (%) grade ii (%) grade iii (%) sirolimus (n=58) conclusion: despite significant improvement in renal function after csa to srl conversion at 3 months, we found no difference of if on rb at w52. the observed improvement of renal function may be due to a hemodynamic effect. a longer delay may be necessary to observe histological improvement. the higher if score than the one previously reported by others may be explained by the use of expanded criteria donors abstract# 528 effects of cni or mmf withdrawal on carotid intima media thickness in renal transplant recipients methods: we included 119 stable renal transplant patients on cni-based immunosuppression, including steroids (10 mg/d) and mmf (2g/d), who were randomized to mmf-withdrawal (group a: csa-auc 3000 ng*h/ml) or cni-withdrawal (group b: auc-mpa 75 µg*h/ml). patients were treated for traditional risk factors according to stringent predefined targets. ambulatory bloodpressure (abpm), lipids, estimated creatinine clearance (mdrd) and imt were measured at baseline and after 12 months. results: groups were comparable with respect to demographic characteristics, immunological profile, renal function, systolic and diastolic bloodpressure and lipids. mean duration of follow-up was 17.4±5.5 months. only 1 patient (1.3%) in group b and 3 patients (3.8%) in group c experienced acute rejection despite adequate exposure (p=0.31). imt did not change final renal function outcomes from the spare-the-nephron (stn) trial: mycophenolate mofetil (mmf)/sirolimus (srl) maintenance therapy and cni withdrawal in renal transplant recipients purpose: to compare the effect on renal function of maintenance immunosuppression with mmf and srl to that of mmf and a cni in renal allograft recipients. methods: in a 2-year open-label, prospective, randomized, controlled, multicenter study, 305 subjects maintained on mmf and a cni were randomized 30-180 days posttransplantation to either mmf (1-1.5 g bid) plus srl (2-10 mg followed by ≥ 2 mg/ day results: outcomes of the first 123 subjects receiving mmf/srl and 126 receiving mmf/cni (tac, n=98; csa, n=28) completing 1 year of follow-up will be reported here. final outcomes of all 305 subjects will be presented at the congress. mean time from transplant to randomization in both groups was 117 days. groups were similar at baseline for all reported renal function endpoints after 12 months of therapy, maintenance immunosuppression with mmf/ srl after cni withdrawal appears to preserve renal function when compared with a mmf/cni-containing regimen improved outcomes after de novo renal transplantation: 2-year results from the symphony study. h. ekberg, 1 h. tedesco-silva frei, 10 y. vanrenterghem, 11 p. daloze, 12 p. halloran at 2 years, the rate of uncensored graft loss was lowest in patients receiving tacrolimus (8% vs 10-13% in other groups; kaplan-meier estimates). gfr at the end of the core study was slightly better in the follow-up itt patients (65-70ml/ min) than in the core study itt patients (57-65ml/min), suggesting inclusion of betterperforming patients in the follow-up. renal function was generally stable over year 2. a slight improvement in gfr in the sirolimus group (+1.6ml/min) was observed, whereas the tacrolimus group still had superior gfr (69 vs 65-67ml/min in other groups). conclusions: in follow-up patients, renal function was stable during the second year and gfr differences were less marked than at 1 year a prospective randomized study of alemtuzumab vs rabbit anti-thymocyte globulin induction in kidney and pancreas transplantation gautreaux, 1 s. iskandar, 4 p. adams, 2 r. stratta. 1 1 surgery; 2 medicine; 3 pharmacy alemtuzumab (alem) and rabbit anti-thymocyte globulin (ratg) are the most commonly used t-cell depleting induction agents in kidney (k) and pancreas (p) transplantation (tx) expanded criteria donors (ecd) were included. results: between 2/1/05 and 8/24/07 225 pts enrolled and 222 pts were transplanted. of 222 pts, 180(81%) had ktx alone, 38(17%) kptx, and 4(2%) paktx. of 180 ktx alone, 152(84%) were deceased donor, and 61(34%) were ecds. recipient age, race, re-tx abstract# 535 purpose: to determine the impact of alginduction on long-term outcomes post-renal tx. methods: between 01/85 and 01/86, 123 consecutive adult pts received a deceased donor renal tx at a single institution results: the incidence of acute rejection was lower in gr.2 (28% vs. 75%, p<0.0001). the incidence of cmv infection was 10% in gr.1 and 18% in gr.2 (p=ns). the overall incidence of cancer was 22 abstract# 538 single-dose induction with rabbit anti-thymocyte globulin (ratg) safely improves renal allograft function and reduces chronic allograft nephropathy 1 clifford miles, 1 gerald groggel, 1 lucile wrenshall. 1 1 divisions of transplantation and nephrology we conducted a prospective, randomized trial in renal transplant recipients comparing two dosing protocols [single dose (6mg/kg) vs. divided doses (1.5mg/kg for 4 doses)] of rabbit anti-thymocyte globulin (ratg; thymoglobulin®). we present herein the results of the first 142 patients throughout the first 6 months post-transplantation, recipients of kidneys from non-marginal deceased donors derived the greatest benefit in renal function (egfr) from the single-dose regimen (p = 0.006). the incidence of chronic allograft nephropathy (can) was also lower in the single-dose group, in both clinically-indicated and protocol biopsies combined (p = 0.045) and in 12-month protocol biopsies alone high risk (race, pra) 8 (11%) in multivariable regression, allograft failure strongly predicted increased risk of subsequent cve. among listed candidates, receipt of a transplant was associated with significant time adjusted for baseline factors, cve after transplant predicted increased risk of subsequent mortality: hr 5.15 (ci 4.53-5.85) after is microalbuminuria post-renal transplantation is related to inflammation and cardiovascular risk our objective was to define the relationship between microalbuminuria and these risk factors in stable rtr. methods: over one year, we identified 223 stable rtr who were at least 2 months post-transplant and provided 3 successive urine albumin-to-creatinine ratio (acr) measurements, excluding those with recent illness and overt proteinuria. microalbuminuria was defined as averaged acr ≥ 2.0 in men and 2.8 in women (cda 2003). framingham-based traditional as well as novel cardiovascular risk factors associated with microalbuminuria were determined by univariate (p < 0.20), followed by stepwise backwards elimination (p >0.05) multivariate logistic regression analysis microalbuminuria did not correlate with prior acute rejection, delayed graft function, or any specific antihypertensive or immunosuppressive agents. conclusions: post-transplant microalbuminuria is highly prevalent and is associated with elevated crp, elevated bp, and smoking. its relationship to these other factors suggests that it reflects an inflammatory state in otherwise stable patients and thus may indicate graft and patient health the first year after kidney transplantation (tx) is associated with increased mortality relative to dialysis. early post-tx deaths are often cardiovascular (cv) and frequently occur after the first week post-tx. ctnt is a sensitive and specific maker of myocardial injury. in this study we investigated whether ctnt relates to early post-tx survival. methods: 396 patients received kidney tx from 9/2004 to 12/2006, 74% from living donors. ctnt was measured during the pre-tx workup and periodically while on the tx waiting list. 261 patients (64%) had a dobutamine stress echo (dse) and 103 (26%) had a coronary angiogram. the combined end point of the study was death or major cardiac events. survival was censored for graft loss. results: mean age was 51+12, 59% males. pre-tx ctnt level was elevated (>0.01 ng/ ml) in 56% of patients other dse derived parameters did not relate significantly to survival. ctnt further stratified the risk associated with other variables. thus, among patients with ef<55%, 2 year survival was 97%, 88% and 60% (p=0.0003) in patients with ctnt <0.01, 0.01-0.03 and >0.03, respectively. similarly, these ctnt ranges stratified risk in patients with low albumin conclusion: an elevated pre-tx ctnt is a strong and independent predictor of reduced early post-tx survival. ctnt allows stratification of risk in patients who have other risk factors such as low ef, low serum albumin and dialysis>2 years. in all patients, independent of any other variables, a normal ctnt was an excellent predictor (97%) of survival abstract# 545 validation of framingham risk assessment by actual cardiovascular event data in renal transplant recipients alloway, 2 michael cardi, 3 gautham mogilishetty, 2 shazad safdar excellent outcome after liver transplantation in children with cystic fibrosis some studies have reported benefits of liver transplantation (lt) in cf patients, but large outcome studies are not available. we report the outcomes of a large cohort of cf patients undergoing lt. methods: pre and post-lt patient characteristics, post-lt morbidity and mortality, and patient and graft survival were 2702 patients age < 18yr) received a 1 st isolated lt. 62 cf patients were listed for 1 st lt, neither waitlist deaths nor the probability of death from time of listing was different from non-cf. 42 (1.6%) cf patients underwent lt with an average followup of 3yrs (0-10yrs) average peld: 5.4 (86.5% had a peld < 10), median age: 11.9 yrs (0.7 -17.5) graft survival in cf patients was 85.0%, 81.2%, and 81.2% at 1, 3, and 5 yrs compared to 85.6%, 80.7%, and 78.1%. rejection rates were not different (50.6% cf vs 56.4% non-cf @ 5 yrs with 50% of these patients requiring dialysis. standardized height and weight scores showed no improvement over 2 years followup in the cf patients (height z -1.4 at tx to -1.4 at 2 yrs., weight z -1.2 to -1.5), but tended to improve in the non-cf group in addition, death rates from time of listing are not increased compared to non-cf patients. these data support lt as a treatment for cf liver disease, but studies investigating the lack of growth improvement and increased renal complications in these patients may further improve outcomes. abstracts full cni group. conclusion: compared to full cni, low cni/mmf a) allows renal function to recover in patients with impaired renal function at the time of ltx and b) preserves long term renal function. cni sparing in combination with mmf may become cellular islet autoimmunity influences clinical outcome of islet cell transplantation methods: twenty-one t1d patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (atg) induction and tacrolimus plus mycophenolate mofetil (mmf) maintenance immunosuppression. immunity against auto-and alloantigens was measured before and during one year after transplantation. cellular auto-and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic t lymphocyte precursor assays, respectively. humoral reactivity was measured by auto-and alloantibodies. clinical outcome parameters remained blinded until their correlation with immunological parameters. results: all patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. multivariate analyses showed that presence of cellular autoimmunity before and after transplantation was associated with delayed insulinindependence (p=0.001 and p=0.01, respectively) and lower circulating c-peptide levels during the first year after transplantation (p=0.002 and p=0.02, respectively). 7/8 patients without pre-existent t-cell autoreactivity became insulin-independent, versus 0/4 patients reactive to both islet autoantigens gad and ia-2 before transplantation. autoantibody levels and cellular alloreactivity were not associated with outcome. conclusions: cellular islet-specific autoimmunity affects clinical outcome of islet cell transplantation under atg-tacrolimus-mmf immunosuppression bmp-7 is downregulated & tgfβ1 to bmp-7 ratio favors emt during acute rejection of human renal allografts allospecific cd154+ t-cells predict rejection risk and measure immunosuppressive effect after abdominal organ transplantation in 100 recipients methods: allospecific cd154+t-cells were measured in <24 hours with polychromatic flow cytometry to identify rejectors (who had experienced acute cellular rejection within 60 days post-transplantation) in single mixed leukocyte responses (mlr) from 100 cross-sectional recipients-88 children with liver or intestine allografts, and 12 adults with renal allografts. where possible, results were correlated with proliferative alloresponses measured by cfse-dye dilution (n=45), allograft biopsies (n=46), and expression of ctla4, a negative t-cell costimulator, which antagonizes cd154-mediated effects (n=52). results: in the first 33 children, logistic regression identified donor-specific, memory cd154+ t-cytotoxic cells (tc) as enhanced among rejectors, compared with non-rejectors (408±231 vs 90±36 per 10,000 cells, p=0.003), relatively drug-resistant (r with drug levels =-0.5, p=ns), with greatest sensitivity/specificity (>93%) for rejectors noninvasively developed molecular signatures accurately predict acute rejection of human renal allografts greater emotional well-being (sf-36) and felt that their transplant interfered significantly less with various aspects of their life (iirs). conclusions: findings highlight the potential utility of assessing attachment style in transplant populations cni sparing in de novo renal transplantation: 3-year results from the symphony study one background: single center non-randomized results with steroid avoidance have shown patient and graft benefits. methods: 130 unsensitized, primary kidney recipients, 0-21 yrs of age, were enrolled from 12 us transplant programs (2004)(2005)(2006), in a prospective 1:1 randomized multicenter study of steroid-free (sf) vs. steroid-based (sb) immunosuppression with matched demographics. 24.1 % of sf and 27.5% of sb were african americans and 15.5% of sf vs. 8.7% of sb had esrd from fsgs. sf patients received extended (6 mo) vs. standard (2 mo) daclizumab induction in the sb group. patients in both arms received tacrolimus and mmf maintenance. protocol biopsies were performed at 0, 6, 12 and 24 mo, and for renal dysfunction. primary end-points were differences for standardized height scores and biopsy proven acute rejection (bpar) at 1 year. results at 1 year: 60 sf and 70 sb patients were enrolled; 10 sf and 16 sb were 0-5 yrs of age. patient survival was 100% in both arms. graft survival was similar (96.7% in sf vs. 98.6% in sb). intent to treat median delta height sds scores from baseline for different age groups were: 0.58 for sf and 0.48 for sb in the 0-5 yr old (p=0.80); 0.34 for sf and 0.34 for sb in the 6-12 yr old (p=0.86 protection of liver ischemia reperfusion injury by silencing of tnf-α and complement 3 genes. roberto hernandez-alejandro, 1 xusheng zhang, 2 dong chen, 2 xiufen zheng, 2 hongtao sun, 2 weihua liu, 2 marianne beduhn, 2 aminah shunnar, 2 motohiko suzuki, 2 norihiko kubo, 2 bertha garcia, 2 anthony jevnikar, 1,2 living kidney donation is rapidly increasing worldwide to offer a partial (?) solution for the numerous esrd wait-listed pts. in spite of properly followed guideline criteria conclusion: dcd donors are a viable source of liver allografts for transplantation. patients who receive dcd livers have outcomes comparable to subjects who receive grafts from brain dead donors. use of dcd livers from donors over 60 years of age is accompanied by a higher incidence of retransplantation and biliary complications. background: hypothermic machine perfusion (hmp) is in its infancy in liver transplantation (ltx). potential benefits include diminished reperfusion injury and improved early function. methods: the study was designed as a phase 1 trial of liver hmp. exclusion criteria included: multiple organ recipients, meld>35, icu patients, and patients >65 years of age. donor livers >65 years, biopsy with >30% macrosteatosis and dcd were also ineligible for hmp. seventeen patients were enrolled transplanted with livers that underwent hmp for 4-7 hours using dual centrifugal perfusion with vasosol solution at 4-6°c. patient, operative and early outcome variables were recorded. we compared outcomes to 17 matched cold stored (cs) controls from the same era. results: all 17 hmp grafts functioned immediately by usual clinical criteria with intraoperative bile production. results are summarized in table 1 . synergy between il-6 and tnfα promotes t cell alloreactivty and impairs the graft-prolonging effects of costimulatory blockade. hua shen, 1 bethany m. tesar, 1 wendy e. walker, 1 daniel r. goldstein. 1 1 internal medicine, yale university, new haven, ct. a novel role of th17 cells in allograft rejection and vasculopathy. francesca d'addio, 1 jesus paez-cortez, 1 m. javeed ansari, 1 laurie glimcher, 2 john iacomini, 1 mohamed sayegh, 1 xueli yuan. 1 1 transplantation research center, renal division, brigham and women's hospital, boston, ma; 2 harvard school of public health, boston, ma. introduction: transcription factor t-bet plays a crucial role in th1/th2 development. here, we investigated the role of t-bet in th17 differentiation and function of th17 cytokines in allograft rejection using an mhc class ii mismatched model of cardiac allograft vasculopathy. methods/results: cardiac allografts from bm12 mice were transplanted into wild-type as well as t-bet and ifn-γ deficient c57bl/6 recipients. t-bet-/-mice showed significantly accelerated allograft rejection (mst=14.75±0.96 days). however, as previously reported, all ifn-γ-/-and majority of the c57bl/6 mice accepted grafts for greater than 60 days. upon in vitro stimulation of recipient splenocytes by irradiated donor cells, t-bet-/-and inf-γ-/-lymphocytes produced significantly less inf-γ and more th2 cytokines. interestingly, production of the proinflammatory cytokines il-17 and il-6 was significantly higher in t-bet-/-(337±94.4 and 339±10.5 pg/ml) than c57bl/6 (135.1±68.3, 75.3±25.9pg/ml, p=0.0399 and 0.0001 compared to t-bet-/-) and inf-γ-/-(95.8±29.8, 20844.5 pg/ml, p=0.0135 and 0.0093 compared to t-bet-/-) mice. in vivo administration of il-17 neutralizing antibody (mab421) significantly prolonged survival of bm12 hearts (mst>30 days, p<0.01 compared to the 15.3±2.6 days of the control igg group) in t-bet-/-mice. immunofluorescence staining of bm12 hearts harvested from t-bet-/-recipients indicated that both cd4 and cd8 infiltrating lymphocytes produced il-17. however, t-bet-cd4 double knockout mice did not reject bm12 heart grafts, nor did the grafts exhibit chronic vasculopathy. in contrast, t-bet-cd8 double knockout mice rejected (mst:18.9±3.2 days). splenocytes from t-bet-cd4 knockouts produced significant lower il-6 (12.9±3.8) and il-17 (7.9±4.6 pg/ml) than observed in t-bet knockouts (940±176.3 and 210.2±73.4 pg/ml) and t-bet-cd8 knockouts (1240.5±215.6 and 113.1±61.2 pg/ml respectively) recipients when re-stimulated with donor cells, while there was no significant difference in inf-γ production. induction in the elderly transplant recipient: an analysis of the optn/ unos database. suphamai bunnapradist, 1 steven takemoto, 2 jagbir gill, 1 tariq shah. 2 1 medicine-nephrology, ucla, la, ca; 2 medicine-nephrology, national institute of transplantation, la, ca.we examined the incidence and mortality implications of cerebrovascular events (cve) after kidney transplant. we also compared variations in risk on the transplant waitlist and after allograft failure. methods: we used registry data from the us renal data system to retrospectively investigate ischemic stroke (is), hemorrhagic stroke (hs) and transient ischemic attacks (tia) among 29,614 adults who received kidney transplants in 1995-2002 with medicare as primary payer. patients with prior indications of cve in the registry were excluded. we ascertained events from billing claims, and estimated incidence of first events by the product-limit method. at-risk time was censored at: loss of medicare, 3yr transplant anniversary, non-cve death or end of study (12/31/2002). cox regression was used to identify independent correlates of cve, and to examine cve events as time-dependent mortality predictors. we estimated cve incidence after graft failure among patients without cve diagnoses prior to graft loss (n=2,599), and amongthe association between hyperuricemia at six months after kidney transplantation and the development of new cardiovascular disease, many studies have previously reported safe withdrawal of prednisone (pw) late after kidney transplantation (ktx). to determine the best immunosuppression regimen during the pw, we performed a prospective trial with stable ktx patients randomized to either csa or 's' based regimen. methods: all patients received antibody induction therapy at the time of rtx and maintained on csa, p and cellcept®. patients excluded if they had >1 acute rejection, >1 gm/d proteinuria or serum creatinine >2.5 mg/dl. 161 patients were enrolled and data presented for 141 patients with >52 weeks follow-up (f/u) with mean f/u of 112.2±29.2 weeks. no differences observed in baseline characteristics in both groups. all patients then randomized to either csa (n=69) or 's' (n=72) and cellcept® converted to equivalent dose of ms. csa dosed by c2 level (2-hour) with goal level of 600 ng/ml. sirolimus target level was 8 ng/ml. results: 5 patients withdrew from study, 12 patients on s returned to csa regimen because of side effects. 5 patients in the csa group and 1 patient in 's' group had ar (3 of them due to drug non-compliance). death censored graft survival was 100%. mean csa drug level acheived was 664±188 ng/ml and 's' drug level was 8.2±1 ng/ml. no significant differences noted in hematological values or bp measurements. csa ( purpose: the clinical significance of c4d positiviity in patients with acute rejection is well defined but its significance in stable graft function is undetermined. this study was performed to evaluate the clinical outcome of protocol biopsy-proven c4d positive renal transplants with stable graft function in the early posttransplantation period. methods: 151 renal allograft biopsies were included. protocol biopsies (n=79) were performed from stable allografts on day 14 posttransplantation, and indication biopsies (n=72) were performed from dysfunctioning allografts. incidence of c4d positivity was compared between protocol and indication biopsies. clinical characteristics, biopsy findings, graft function, acute rejection episodes, and graft survival rates were compared between the c4d-positive and c4d-negative grafts in each group. results: c4d deposition in protocol biopsies was detected in 4 of 79 biopsies (5.1%), whereas 9.7% (7 of 72 biopsies) in indication biopsies. the histological findings of c4d-positive protocol biopsies were minimal inflammation of tubulointerstitium. on the other hand, those of c4d-positive indication biopsies were various including acute humoral rejection, acute cellular rejection, acute tubular necrosis and calcineurin inhibitor toxicity. in the protocol biopsy group, graft function during 1 year after biopsy, acute rejection rate, and cumulative graft survival did not differ between the c4d-positive and c4d-negative grafts. all c4d-positive allografts maintained stable graft function without any antirejection therapy. in the indication biopsy group, graft function during 1 year after biopsy and acute rejection rate did not differ between the c4d-positive and c4d-negative grafts. however the cumulative graft survival rate was worse in the c4d-positive grafts than the c4d-negative ones (p=0.008). conclusion: c4d positivity associated with allograft dysfunction indicates a poor graft outcome. however, c4d-positive allografts with stable graft function in the early posttransplantation period take an indolent course. are methods: this was a retrospective single centre study reviewing all the adult patients who had a kidney transplant biopsy between april 2003 and october 2006 at guy's hospital. results: 45 patients had diffuse (>50%) c4d staining out of 228 who had kidney transplant biopsies in this time and had been followed up within the centre. of these 45 patients, 20 also had dsa prior or at the time of biopsy. the fall in egfr in this group a year post biopsy was greater than those with diffuse staining for c4d but no dsa. the mean change in egfr from the day of biopsy at a year was -3.1ml/min/1.73m2 (+/-12.1) in those with dsa compared with +17.7 ml/min/1.73m2 (+/-23.2) for those with c4d but without dsa. the changes in egfr from the pre-biopsy baseline at one year showed a fall in egfr in both groups but this was greater in those with dsa (-21.5 compared with -9.6 ml/min/1.73m2 ).of 179 patients who never had diffuse or focal c4d staining on biopsy, only 81 had had dsa tested. of these only 8 (10%) had a positive dsa result. from these eight, four had features of rejection and four did not. one person in each of these groups is dialysis dependant and one person in the rejection group has egfr <15 ml/min/1.73m2. although small numbers, this outcome appears to be worse than that of c4d negative patients with no dsa but features of rejection who in fact showed an improvement in egfr from the day of biopsy by 14.4 m l/min/1.73m2 or an improvement from their pre-biopsy baseline of 1.3 m l/min/1.73m2 at one year. conclusion: dsa is of additional value in evaluating risk of graft failure. this appears to be of value in those with and without diffuse c4d staining on biopsy. utility of post-transplantation flow cytometry crossmatching in predicting graft outcomes. michelle willicombe, 1 graham shirling, 2 ray fernando, 2 henry stephens, 2 paul sweny, 1 peter j. dupont. 1 1 department of renal medicine, royal free hospital, london, united kingdom; 2 histocompatibility laboratories, anthony nolan trust, london, united kingdom. de novo development of donor-specific anti-hla antibodies after renal transplantation may be associated with increased rejection and decreased graft survival. flow-cytometry crossmatches (fcxm) have been suggested as method of screening for development of donor-specific hla antibodies post-transplantation, but interpretation of crossmatch results can be confounded by antibodies directed against antigens other than hla. we assessed the impact of developing a positive fcxm post-transplantation on clinical outcomes in a cohort of live donor renal allograft recipients. methods: 29 patients were studied. 23/29 (79%) received tacrolimus-based and 6/29 (21%) ciclosporin-based immunosuppression. median follow-up was 24 months. all patients had negative complement-dependent cytotoxic (cdc) t cell crossmatches pretransplantation. 4/8 (50%) in the group with a positive fcxm had an acute rejection episode in the first 12 months compared with 10/21 (41%) in the group with a negative fcxm (p=ns). graft function at 12 months was not different between the groups (positive fcxm -median creatinine 137mmol/l; negative fcxm -median creatinine 141mmol/l; p=ns). 2/8 grafts (25%) were lost within the first year in the positive fcxm group compared with 1/21 (5%) in the group with negative post-transplant fcxm (p=ns). the development of a positive fcxm post-transplantation alone is not predictive of adverse clinical outcomes. this may be explained by the poor correlation between a positive fcxm and the presence of antibody directed against mismatched donor hla antigens. surveillance for development of donor-specific anti-hla antibodies after transplantation may be best performed using high-resolution bead technologies rather than fcxm. use of the fcxm alone, without establishing antibody profiles, is of limited predictive value. based upon the amount of antibody (ab) measured by the titer of donor specific hla antibodies, dsa, or the fluorescence intensity (fi) of the donor specific single antigen bead. it is unclear whether abs indentified by sensitive single antigen bead and solid phase assays (flow pra and luminex) correlate with and are predictive of a clinically relevant end-point (a + fcxm). we evaluated the pra, dsa, bead specific ag fi and fcxm reactivity of pre-transplant (pre-tx) sera from 219 recipients of a deceased donor renal allograft to determine whether amount of ab (measured by fi) predicts a (+) fcxm. patients with a (+) dsa, a (+) fcxm and pre-tx class i pra ≥ 80% (n = 103, mean pra of 91 ± 6%) when compared to patients with pre-tx pra < 80% (n = 58, mean pra 53 ± 21%) had comparable mean fis (7,407 ± 4,305 vs 7,483 ± 5,380) , fi ranges (1,000 -20,243 vs 1,552 -17,153 ) and median fis (5,681 vs 5,670). the class ii comparisons were of the same pattern. surprisingly, patients with a (+) dsa, a (-) fcxm and pre-tx class i pra ≥ 80% (n = 22, mean 90 ± 6%) compared to patients with pre-tx pra < 80% (n = 36, mean pra 50 ± 18%) had comparable mean fis (6,758 ± 4,841 vs 6,412 ± 3,844) , fi ranges (1,656 -16,596 vs 2,162 -11,637) we have recently showed that pre-treatment of the donor with epo causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. 5-aminoisoquinolinone (5-aiq) a potent water soluble parp inhibitor has proven to reduce renal ischemia-reperfusion (i/r) injury. the aim of our study was to determine the effects in the graft and in the receptor of the pre-treatment of the donor with epo and treatment of the recipient with 5-aiq, in a porcine model of dcd kidney transplantation. material/methods:24 landrace pigs were killed by lethal injection; their kidneys were subjected to 30 min of warm ischemic time (wit) and then transplanted after 24 h of cold storage in celsior. in the pre-treated group, donors received a single dose of epo (1000 iu/kg) 30 min before cardiac arrest. in the treated group, recipients received a continuous dose of 5-aiq (5mg/kg/h) 10 minutes before reperfusion and maintained during 60 minutes. blood, urine and renal tissue samples were collected at the end of the experiment for biochemical, histological and immunohistochemistry (pars, inos and cox-2) evaluation. data analysis performed with graph pad prism statistical package; p<0.05 considered statistically significant. results:transplantation of kidneys from dcd resulted in: a significant rise of the levels of creatinine, n-acetil-b-d-glucosaminidase, glutathione-s-transferase, ast, ldh, alt, fractional excretion of na+, interleucin 1 and 6, malondialdehyde levels and myeloperoxidase activity (p<0.05); a significant reduction in urine flow and creatinine clearance, disturbances in the histological and imunohistochemistry pattern. administration of epo before ischemia and 5-aiq before reperfusion reduced significantly the biochemical (p<0.01), histological and imunohistochemical evidence of glomerular dysfunction and tubular injury. they also reduced systemic injury, inflammatory response and oxidative stress. conclusions:pre-treatment of the donor with epo and treatment of the recipient with 5-aiq causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. in the hmp group perfusate ast levels strongly correlated with recipient peak ast by linear regression (p<0.001). conclusions: hmp of liver grafts provides safe and reliable preservation in our pilot series. perfusate ast may allow pretransplant prediction of reperfusion injury. a larger randomized trial will be necessary to demonstrate the magnitude of benefits of hmp over cs in ltx.purpose: liver discard rates have increased in a large, urban organ procurement organization from 8 % to 24 %. the reason is likely the result of increased transplant surgeon willingness to consider organs close to the margin of clinical acceptability. however, the costs associated with recovering these organs are high if the liver is discarded. we endeavored to determine whether a model based on pre-recovery data could predict liver discard introduction: we report our 6 years experience with the use of campath-1h (c1h) in adult liver transplantation. from december 2001 until july 2007 we administered c1h induction with low dose maintenance tacrolimus immunosuppression to 166 adult recipients of a liver allograft. most common primary diseases were laennec (n=60), cryptogenic cirrhosis (n=31) and autoimmune: psc (n=17), pbc (n=16) and aih (n=14). the first dose of c1h was administered immediately before (n=74) or after (n=92) the transplant procedure. follow up was until september, 2007. results: five year patient and graft survival was 90% and 85% respectively. there were 14 deaths due to stroke (n=2), chronic rejection (n=2), failure to thrive/pneumonia (n=3), sepsis (n=2), hepatic artery thrombosis, hcc, prostate cancer, graft lymphoma and non-compliance (one each). seven patients were retransplanted, for primary non function (n=2), portal vein thrombosis (n=1), hepatic artery thrombosis (n=1), hepatitis b (n=1) and chronic rejection (n=2). thirty six patients had biopsy proven rejection episodes: mild (n=28), moderate (n=9) or severe (n=3). the average tacrolimus 12 hour trough levels were 6.6, 5.2 ng/ml and 2.78 ng/ml for the 1rst, 3 nd and 5th year post-transplantation, respectively. there was no significant difference in the outcome of the transplant so far, between patients that received c1h before or after the transplant procedure. immunosuppression-related complications included a). opportunistic infections: most common were herpes zoster (n=19), cmv (n=3), and herpes simplex (n=3), b). neoplasms: skin cancer (n=3), kaposi sarcoma (n=1), lymphoma (n=2) and c). nephrotoxicity: five patients received a kidney graft for diabetic nephropathy (n=2), nephrotic syndrome (n=1) and calcineurin nephrotoxicity (n=2). conclusion: the use of c1h induction with half the usual dose of tacrolimus is an effective regimen in adult liver transplantation. the timing of c1h administration does not seem to affect the clinical outcome so far. a. david mayer, 1 james m. neuberger. 1 1 the liver unit, queen elizabeth hospital, birmingham, united kingdom. introduction: in the prospective respect study, primary liver transplant patients were randomised to 1 of 3 groups: a) standard-dose tacrolimus (target trough level > 10 ng/ml) for the 1 st month; b) 2 g mycophenolate mofetil (mmf) iv until at least day 5, 2 g po thereafter + reduced-dose tacrolimus (target trough level ≤ 8 ng/ml); and c) mmf as in group b + reduced-dose tacrolimus introduced on day 5 (target trough level ≤ 8 ng/ml) + daclizumab on days 1 and 7. steroids were given in all groups according to local centre protocol. results at 1 year showed that 2 g mmf + delayed and reduced tacrolimus + daclizumab is associated with significantly less impairment of renal function compared with standard treatment. here we present the results of the per protocol (pp) population. methods: the pp population, which was defined prior to the sub-group analysis, consisted of patients from the full analysis set who had no inclusion/exclusion criteria violation, had at least one creatinine clearance (crcl) value beyond 6 months, were treated according to the protocol and did not receive any prohibited medication during the first 14 days and for less than 1 week at any time during the study. a composite endpoint comprising freedom from renal dysfunction (≥ 20% decrease from baseline in calculated crcl), acute rejection, graft loss or death was also investigated. results: the full analysis set included 181, 168 and 168 patients, whereas the pp population only included 69, 67 and 67 patients in groups a, b and c, respectively. the mean difference in calculated crcl from baseline to 1 year was significantly smaller in group c compared with group a (-10.7 ml/min vs -22.0 ml/min, p = 0.038), but was not significantly different between groups a and b (-20.50 ml/min). the incidences of death (n = 2, 0, 1) and graft loss (n = 1, 0, 0) were similar in all 3 groups. the incidence of the composite endpoint at 1 year was in both the full analysis set and the pp population significantly lower in group c compared with group a (pp population: 70% vs 93%, p < 0.0001), but was not significantly different between groups a and b (85%). the pp analysis confirms the results from the full analysis set that 2 g mmf + delayed and reduced tacrolimus + daclizumab is associated with less impairment of renal function compared with standard treatment with no negative effect on death and graft loss. aims: post transplant lymphoproliferative disorder (ptld) is a serious complication of solid organ transplantation that is closely associated with epstein barr virus (ebv) infection. ebv + ptld lymphomas express several latent viral genes including latent membrane protein 1 (lmp1), a proven oncogene that is essential for human b cell transformation. lmp1 is able to activate erk, jnk, p38, nfκb and pi3k. the aim of this study is to determine whether lmp1 isolated from ptld tumors differs in signaling ability from lmp1 derived from the b.95 strain of ebv, originally isolated from a patient with infectious mononucleosis. methods: lmp1 variants isolated from a panel of ebv + ptld-associated b cell lines were cloned and sequenced. inducible chimeric constructs containing the lmp1 c-terminus and ngfr transmembrane domain were created for each tumor variant and expressed in the burkitts b lymphoma cell line bl41. lmp1 signaling in bl41 clones was induced by crosslinking of ngfr. activation of p38, erk, akt and jnk was assayed by western blotting (wb) with phospho-specific antibodies. nfκb activation was assayed by wb for iκb and cfos induction was analyzed by wb and the transam cfos binding assay. results: all three tumor variants of lmp1, as well as the b.95 lmp1 isoform, were able to induce p38 activation within 30min of ngfr crosslinking while akt and jnk were activated within 10min. all variants showed similar ability to activate nfκb. however, tumor lmp1 variants induced prolonged erk activation (up to 3hrs) while the b.95 lmp1 variant induced a transient response. cfos is induced only during the sustained phase of erk activation. indeed, the tumor variants of lmp1, but not b.95 lmp1, were able to induce cfos protein. similarly, cfos binding to the ap1 consensus site was only observed in tumor lmp1-induced nuclear lysates. two mutations in the c-terminus-aa212 (s vs g) and aa366 (t vs s) -are conserved in the tumor variants lmp1 compared to b.95 lmp1. point mutation of either of these amino acids from the b.95 to tumor variant version allowed for sustained activation of erk and subsequent cfos induction and binding to the ap1 site. conclusion: tumor-derived lmp1 has enhanced ability to induce the cfos oncogene and this property can be localized to two amino acids in the c terminus. these findings suggest that these specific amino acid residues of lmp1 are important in determining whether ebv infection is benign or results in ptld. the absence of interferon regulatory factor-3 (irf-3) confers protection against the liver ischemia and reperfusion injury through an il-10 independent pathway. elizabeth r. benjamin, 1 xiu-da shen, 1 feng gao, 1 yuan zhai, 1 genhong cheng, 1 ronald w. busuttil, 1 jerzy w. kupiec-weglinski. 1 1 surgery, dumont-ucla transplant center, los angeles, ca. toll-like receptor 4 (tlr4) mediated liver reperfusion damage after warm ischemia requires signaling through the myd88-independent, irf3-dependent pathway with cxcl-10 (ip-10) playing a central role in the injury development. studies using cxcl-10 ko mice have shown that these mice are protected through an il-10 dependent mechanism. we chose to investigate irf3, upstream of cxcl-10, to further characterize its role in the injury progression, and to better understand the involvement of il-10 in this pathway. methods: we used irf3 ko mice and their wt counterparts in a model of partial hepatic warm ischemia with 1, 2, and 6h of tissue reperfusion (n=2 ko, wt at 1 and 6h; n=3 ko, wt at 2h). wt bone marrow derived macrophages were generated and stimulated with lps to determine the kinetics of il-10 production. tissue was analyzed for histology and mrna levels were measured by qpcr. results: kinetic studies showed peak il-10 production at 1 and 2h post-reperfusion (pr). on pathology, irf3 ko mouse livers were protected from ir injury both early pr, at 1 and 2h, and at 6hrs pr when compared to wt. consistent with these data, il-6 mrna induction was decreased in irf3 ko, as compared with wt at 1, 2, and 6h. although il-10 induction was maintained in the cxcl-10 ko mice, the irf3 ko mice showed decreased levels of il-10 at 1h pr. by 2h, il-10 levels were normalized to wt. conclusion: irf3 ko mice are protected from liver ir injury with evidence of this protection as early as 1h pr. although cxcl-10 ko mice are protected from ir injury with maintained il-10 expression, the absence of the upstream molecule, irf3, confers protection in an il-10 independent manner. these data suggest a novel mechanism of ir injury mediated by irf3 in the liver. background: bone marrow (bm) transplantation may induce donor-specific tolerance to prevent rejection of allogeneic solid organs while maintaining immunity against infections and tumors. currently allogeneic bm transplantation is limited by donor t cell mediated graft-versus-host disease (gvhd), as well as a variable requirement for recipient marrow ablation and high numbers of donor bm cells. furthermore, sustained macro-chimerism has not yet been easily or predictably achieved in partially ablated patients or large animals. while rejection of allografts is mediated primarily by recipient t cells, recent studies have demonstrated the capacity of nk cells to reject allogeneic bm and to prevent long-term mixed chimerism. thus, nk cells represent a barrier to long term bm engraftment even with t cell tolerance. we have previously identified a novel type of regulatory t (treg) cell with a "double negative" (dn) phenotype (tcrab + cd3 + cd4 -cd8 -). dn-treg cells can effectively suppress anti-donor t and b cell responses and prolong graft survival in allo-and xenotransplantation models. we therefore tested the capacity of dn-treg to alter nk cell function. methods: c57bl/6 bm cells were i.v. injected into sub-lethally-irradiated (6.5 gy) cb6f1 (h-2b/d) in a "parent to f1" model, or into allo-disparate balb/c mice. bm cells were co-transplanted with various numbers of c57bl/6 dn-treg cells or cd4 + or cd8 + t cells as controls. recipient spleen cells were collected 7 days after to detect donor progenitors in a colony-forming-unit (cfu) assay. mice then received cardiac (n=8) or skin transplants (n=8) to confirm tolerance. we found that donor-derived dn-treg cells suppress nk cell-mediated allogeneic bm graft rejection in both "parent-to-f1" and fully mhcmismatched bm transplantation models. adoptive transfer of dn-treg cells with donor bm cells promoted the establishment of stable mixed chimerism and donor specific tolerance to bm donor cardiac and skin grafts (mst>160 days), without inducing gvhd in sub-lethally irradiated mice. perforin deficient dn-treg cells were unable to efficiently inhibit nk cell function, and donor bm did not engraft. these results demonstrate a potential approach to control innate immune responses and promote allogeneic bm engraftment and donor specific tolerance through the use of dn-treg cells.framingham risk score (frs) predicts cardiovascular (cv) risk in the general population, but may underestimate cv risk in kidney transplant (txp) patients (pts). frs has not previously been validated by prospective cardiovascular event (cve) data collection in kidney txp pts. the purpose of this study was to validate frs with actual observed cve data in kidney txp pts. methods: cve data was collected at routine intervals in our kidney txp pts and entered in a cardiovascular risk database. frs was calculated from baseline to 7 yrs posttransplant (ptx) individual frs factors of age, sex, smoking, diabetes mellitus (dm), high-density lipoprotein (hdl), total cholesterol (tc), and blood pressure (bp) were evaluated for their ability to predict acutal cve occurring after kidney txp. pts with coronary artery disease (cad) were excluded from the frs analysis. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/transient ischemic attack. frs factors were evaluated by cox proportional hazards in univariate (uva) and multivariate (mva) models. rho kinase (rok) modulates calcium sensitivity of vascular smooth muscle cells and contributes to the regulation of peripheral vascular tone in man. in essential hypertension increased rok-activity contributes to the generation of vascular resistance. arterial hypertension is a common complication in renal transplant recipients. in this study we were interested in the role of rok for systemic hemodynamics in hypertensive renal transplant recipients (tx). we tested the specific inhibitor of rok fasudil. 10 tx and 10 matched control subjects (c) received either fasudil (1600 g/min) or placebo over a period of 30 minutes intravenously. peripheral blood pressure and heart rate were recorded every 5 min over a total of 120 minutes. measurements for pulse wave analysis (sphygmocor vt) were performed every 10 minutes during this period. statistics by anova for repeated measurements.compared to placebo fasudil significantly reduced peripheral mean arterial pressure p<0 .05; figure 1) and increased heart rate (+ 6.31.1 bpm, p<0.001) in tx but not in c. likewise, central systolic pressure(p=0.006; (figure 2), augmented pressure and augmentation index were decreased in tx only.we conclude that acute inhibition of rok by fasudil consistently and effectively lowers blood pressure in tx with a calcineurin inhibitor-based immunosuppression. interestingly, rok-inhibition also reduces central blood pressure and arterial stiffness in these patients. improvement of both these parameters has been linked to a reduction in cardiovascular morbidity and mortality in large trials. hence rok inhibition might prove beneficial for the treatment of hypertension in renal transplant recipients. use death with function causes half of late ktx failure, and cardiovascular disease (cvd) is the most common cause of death. chronic kidney disease (ckd) is a cvd risk equivalent, justifying aggressive risk reduction with blood pressure (bp) control, statins, aspirin, and use of angiotensin converting inhibitors (acei) and angiotensin receptor blockers (arb). dekaf is an nih-sponsored prospective observational study examining causes of ktx failure at 7 transplant centers in the us and canada, with current enrollment of over 1800 subjects. we examined the use of cardioprotective medications among patients transplanted after 10/1/05 with at least 6 mos follow-up, focusing on subgroups with preexisting diabetes (dm) and/or cvd. we conducted a retrospective cohort study to asses the prevalence and the predictors for the development of hyperuricemia at 6 months after kidney transplantation and the association between hyperuricemia and clinical outcomes including patient and graft survival, new cardiovascular events and chronic allograft nephropathy (can). adult patients who underwent kidney transplantation at mount sinai medical center between 1.1.2001-12.30.2004 were included. patients who died or lost the allograft within 6 months after transplantation were excluded from analysis. of the 307 patients with a functioning allograft at 6 months after transplantation, 163 patients (53%) had normal uric levels and 144 patients (47%) had hyperuricemia. after age, race, sex adjustment, receiving a cadaveric kidney, having an egfr<50 ml/min, and taking diuretics and cyclosporine were associated with a higher odds ratio of hyperuricemia. over a mean of 4.3 years of follow-up, 81 patients had one, or more, of the pooled outcomes; 40 had new cardiovascular events, 41 developed biopsy-proven can, 4 patients died, and 20 had graft failure. kaplan-meier survival curves demonstrated that the pooled outcomes of events occurred more frequently in hyperuricemic patients (figure, p < 0.001). due to association between low egfr and hyperuricemia, we analyzed the clinical outcomes in patients with low and normal egfr. while 44.7% of hyperuricemic patients with an egfr<50 ml/min had one of the pooled outcomes, it was 20.8% in patients with normal uric acid levels (p=0.038). among patients with an egfr ≥50 ml/min, 19.4% of normouricemic and 25% of hyperuricemic patients had one of the events. these results suggests an important association between hyperuricemia at 6 months after transplantation and the new cardiovascular events, biopsy-proven can, and graft loss in kidney transplant recipients with decreased allograft function. background: long-term survival after liver transplantation (lt) is now the rule rather than the exception. hence, assessment of outcomes for children after lt must consider not only the quantity, but also the quality, of life years survived and restored. aim: to examine key hrqol themes after pediatric lt raised by both recipients and their parent proxies, with evaluation by time (1-3 yrs, 3-5 yrs, 5-10 yrs, and >10 yrs) since lt. methods: semi-structured 1:1 item generation interviews were conducted in person with children (c) and parents (p) at time of ambulatory lt follow-up at 4 pediatric lt programs in canada and uk. all interviews were audio-taped, transcribed verbatim, and subjected to content analysis utilizing qsr nvivo 2.0 software for hrqol related theme generation. the participants interviewed were part of a larger research program aimed at developing a disease-specific instrument to assess hrqol for children after lt. results: data representing 91 (47% male) pediatric lt recipients was obtained from a total of 146 (62 c, 84 p) item generation interviews. median recipient age at lt was 2.4 (range, 0.05 to 17) yrs, for primary indications including biliary atresia (45%), fulminant liver failure (17.6%), metabolic liver disease (4,4%), malignancy (8.8%) and others (24.2%). median patient age at time of interview was 9.9 (range, 1.1 to 18.8) yrs. themes emerging at all time points post-lt included infection risks, limitations on physical activities, side effects from immunosuppression meds, educational supports, and ongoing bloodwork. themes identified within the medium (1-5 yrs) follow-up included worries about rejection episodes, need for future re-transplantation, school absenteeism, and altered sibling and family dynamics. the impact of living with a surgical scar was a more frequent theme with recipients >3 yrs from lt. as time from lt increased to >10 yrs, themes suggest a focus on normalization and health promoting behaviours, along with expressed desires to be like healthy peers. conclusions: unique hrqol themes emerged from item generation interviews not captured by currently available generic hrqol tools. hrqol themes identified after pediatric lt suggest the importance of considering time trajectories from lt, and a focus on elements of 'everyday life' apart from lt. the shortage of cadaveric donors has led many transplant centers to expand their criteria for accepting life-saving organs. utilization of donation after cardiac death (dcd) donors has been estimated to increase the number of cadaveric donors. we report our experience with a recently established dcd program at a pediatric hospital and the outcome with the transplanted grafts. methods: in 2005 a protocol for dcd was established at a free standing pediatric hospital. from 2005 to 2006 all patients undergoing withdrawal of care were evaluated for dcd. patients meeting criteria for dcd underwent withdrawal by the critical care team and organ retrieval was initiated if asystole was reached in less than 60 minutes. in addition, one dcd liver was imported and was included in the liver results. results: during the 2 year study period 7 patients (25% of total donors) underwent dcd resulting in 14 organs (11 kidneys and 3 livers) transplanted. the 7 cases had a mean donor age of 9 yrs (range 3-16), wit of 22 min (9-55), time sbp < 60 of 11 min (7-15), and time from asystole to aortic flush of 8 min (6) (7) (8) (9) (10) (11) (12) . four kidneys were transplanted locally with cit 5-14 hrs, no dgf, and one month creat 1.2-1.7. the remaining 7 kidneys were exported for transplant. four livers were transplanted locally with donor age 7 mth-16 yrs, wit 12-28 min, recipient age 14 mth-61 yrs, cit 6-15 hrs, ast peak 190-3630, ast day 7 33-44, inr peak 1.4-1.8, inr day 7 1.0-1.1, total bili one month 0.3-1.0, and graft survival 1.5-2.9 yrs. there were no vascular or biliary complications. conclusions: a protocol for dcd at a pediatric hospital increased the number of pediatric donors by 25%. liver and kidney grafts from pediatric dcd donors demonstrated excellent graft function and survival. liver retransplantation in children. a 21 year single centre experience. christophe bourdeaux, andrea brunati, magda janssen, jean-bernard otte, etienne sokal, raymond reding. pediatric liver transplant program, université catholique de louvain, saint-luc university clinics, brussels, belgium. when graft failure occurs in liver recipients, secondary transplantation represents the only chance of long-term survival. in such instance however, several surgical and immunological aspects should be carefully considered, with respect to their impact on final outcome.in the present study, the epidemiology and outcome of graft loss following primary pediatric liver transplantation (lt) were analysed, with the hypothesis that early retransplantation (relt) might be associated with lower immunologial risks when compared to late relt. between march 1984 and december 2005, 745 liver grafts were transplanted to 638 children at saint-luc university hospital, brussels. among them, a total of 90 children (14%) underwent 107 relt, and were categorized into two groups (early relt, n=58; late relt, n=32), according to the interval between both transplant procedures (< or > 30 days).ten-year patient survival rate was 85% in recipients with a single lt, versus 61% in recipients requiring relt (p=0.001). ten-year patient survival rates were 59% and 66% for early and late relt, respectively (p=0.423), the corresponding graft survival rates being 51% and 63% (p=0.231). along the successive eras, the rate of relt decreased from 17% to 10%, whereas progressive improvement of outcome post-relt was observed. no recurrence of chronic rejection (cr) was observed after relt for cr (0/19). two children developed a positive cross-match at relt (2/10, 20%), both retransplanted lately for cr secondary to immunosuppression withdrawal following a post-transplant lymphoproliferative disease.in summary, the current need for relt has been decreasing over years, with a parallel improvement of its outcome. the results presented could not evidence better results for early relt when compared to late relt. the latter did not seem to be associated with higher immunological risk, except for children with immunosuppression withdrawal following the first graft. the background: a serum conjugated bilirubin greater than 100 umol/l (cb100), in neonates who receive parenteral nutrition (pn), has been demonstrated to be a predictor of end-stage liver disease requiring transplantation. given the recent interest in the role of omega-6 lipids in the development of parenteral nutrition associated liver disease (pnald), we sought to examine in a multiple variable model the role of days of maximal lipid (>2.5 g/kg/day), in the development of this outcome. method: between 2003 and 2004, data were collected prospectively on all neonates undergoing an abdominal surgical procedure. univariate logistic regression models for the prediction of cb100 were developed with the following predictors: gestational age, percentile weight, percent predicted small bowel and colonic length, resection of the ileocecal valve, presence of a stoma, post-operative enteral tolerance, number of septic episodes, days of pn amino acid > 2.5g/kg/day, days of pn lipid > 2.5g/kg/day, and total days of pn. univariate predictors significant at the 0.2 level were entered into a backward stepwise multiple variable logistic regression. results: 152 infants received pn post-operatively, and 22 developed cb100. predictors that met criteria for consideration in the multiple variable model were: age (p=0.079), weight (p=0.059), small bowel length (p=0.001), presence of a stoma (p=0.163), proportion of enteral feeds post-operatively (p=0.049), days of pn amino acid > 2.5g/ kg/day (p=0.00), days of lipid > 2.5 g/kg/day (p=0.00), and total days of pn (p=0.00).the final multiple variable model which had a negative predictive value of 97.6% and positive predictive value of 52.6% is presented in the table below. our model suggests a key role of pn lipids and intercurrent septic events in the development of cb100 from pnald. these data may provide targets, such as careful line care, reduction in maximal lipid dose, or the use of alternate lipids such as omega-3 fatty acids, to prevent cb100 an identified marker for the need of subsequent liver transplantation in infants with pnald. terminal renal failure occurs in more than 10 % of liver transplant recipients after 10 years. we have previously shown that, beside renal toxicity of calcineurin inhibitors, renal lesions may be related to diabetes, arterial hypertension, accumulation of hydroxyethylstarch (elhoes), and the etiology of the liver disease. we made the hypothesis that these lesions may be already present at the time of liver transplantation (lt), a finding that could lead to adapt the perioperative management. this work investigated prospectively whether renal histopathological lesions were present before lt by performing systematically a renal biopsy by endovenous route in 60 candidates to lt with end-stage liver disease. these patients were 58 ± 10 years old, 46 males ; 10/60 had a diabetes, and 21 an arterial hypertension ; the liver disease was related to alcohol in 32 cases, hcv in 12 cases, hbv in 5 cases, and to a cholestatic disease in 7 cases. at the time of the pre-lt workup, the biochemical parameters were : child score10 ± 2, meld score 18 ± 4, prothrombin rate 50 ± 12, creatinin serum level 90 ± 6 umol/l, proteinuria 0.12 ± 0.04 g/24h. severe side effects related to the procedure were limited to 2 cases of macroscopic hematuria, lasting less than 24 hours. in 10 cases, the material obtained during the procedure did not allow the histological analysis. among the 50 samples available, 21 were considered as normal ; in 29 cases, lesions related to mesangial iga glomerulonephritis (14 cases), diabetic glomerulosclerosis (12 cases), elhoes accumulation (5 cases), thrombotic microangiopathy (1 case) were found, often associated ; in 5 cases, the lesions were severe and lead to combined kidney/liver transplantation in 2 cases. in conclusion, significant renal lesions are detectable in more than 50 % of the candidates to lt. interestingly, histological findings often combined lesions related to the liver disease and to an associated cause (diabetes, previous treatment by elhoes or interferon). results of histological analysis could help to decide either to perform a combined renal/liver transplantation, to adapt the immunosuppressive regimen, or to abandon the lt project. . because serum creatinine is one of the components of meld, liver candidates with renal insufficiency have been transplanted in increasing numbers, with some candidates receiving a kidney along with the liver transplant. we aimed to compare the liver graft outcomes for liver alone (lta) transplants with those from combined liver-kidney transplants (clkt). a propensity score analysis was used to reduce the impact of selection bias in the comparison of outcomes in the two groups. methods. demographics, clinical factors and outcomes on lta and clkt recipients from 3/1/02 to 12/31/06 (n=10,388) obtained from the optn database were used for the analysis. univariate post-transplant survival rates were estimated using kaplan-meier survival, and multivariable post-transplant outcomes were analyzed using a cox regression model with and without stratification by categories of the propensity score. the propensity score (probability of receiving a clkt) for each recipient was estimated using a logistic regression model. several donor and recipient factors were included in both the cox and logistic regression models. in this cohort, liver graft outcomes for clkt were significantly better than those for lta based on the multivariable analysis. the results were similar, although slightly less significant, when the model was adjusted for propensity. the superior outcomes of clkt may be due to unobserved differences between these groups of recipients, reflecting data not currently captured by the optn. effect of liver the tgfβ1 to bmp-7 ratio was higher in the ar group (median ratio: 1635) compared to recipients with stable graft function & normal biopsy (median ratio: 438, p=0.0002).our observations that bmp-7 is specifically down-regulated during an episode of ar and that the balance between tgfβ1 & bmp-7 is in favor of emt advance a mechanism for the deleterious impact of ar on the long-term outcome of human renal allografts. the non-statistical bioinformatic approach identified 68 leader genes which define the highest interaction genes derived from the 343 sam-gene list. an interaction map between the genes identified has been calculated. this network is formed around 2 majors clusters: a network of interleukins and a network of signal transduction which allow us the identification of key genes such as bank1, a negative modulator of cd40mediated akt activation, thereby preventing hyperactive b cell response in blood from patients with operational tolerance and il7r, a specific marker absent on potentially regulatory cd4 + cd25 +high t cells in blood from patients with chronic rejection. we have identified by a non-statistical analysis of the peripheral blood gene expression in human kidney recipients a cluster of genes which are strongly interconnected and which could be a starting point for further analysis of the molecular mechanisms of kidney graft operational tolerance and chronic rejection. fecal algorithm based on multiparameter mixed lymphocyte reaction assay for tailoring maintenance immunosuppressants after living donor liver transplantation. yuka tanaka, hideki ohdan, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan.background: no reliable immunological parameters exist for identifying liver allograft recipients in whom immunosuppressants can be safely withdrawn. for minimizing maintenance immunosuppressants, we established an algorithm determining anti-donor alloreactivity based on multiparameter mixed lymphocyte reaction (mlr) assay, wherein the number and phenotype of alloreactive precursorscan be quantified. we enrolled 68 adults undergoing living donor liver transplantation (lt). the initial immunosuppressive regimen comprised tacrolimus/cyclosporine and methylprednisolone, which were gradually tapered off by 6 months after lt. thereafter, therapeutic adjustments were determined by a policy of slow tapering off in the case of normal liver function. mlr assay was performed at 6 month intervals to monitor immune status. in this assay, cfse-labeled pbmcs from recipients were used as responders. irradiated donor and third-party pbmcs were used as stimulators. after coculture, the responder cells were stained with cd4 or cd8 mabs along with cd25 mab, followed by fcm analyses. the proliferation and cd25 expression of cd4 + and cd8 + t cell subsets in response to anti-donor and anti-third-party stimuli were analyzed; the immune status of lt patients was categorized as hypo-response, norm-response, or hyper-response for cd4 + t cells and as hyper-response for cd8 + t cells. of the 68 patients, 33 had normal liver function at >6 months after lt. we examined the fluctuation of immunosuppressants at 6 months after mlr in these patients. in patients whose immune status was categorized as hyper-response for cd4 + or cd8 + t cells (n=4), immunosuppressants had to be increased. in patients with norm-response immune status (n=7), immunosuppressant tapering was abandoned. immunosuppressant therapy was successfully tapered off in patients with hypo-response immune status (n=22). of 10 patients with hypo-response immune status at >3 years after lt, immunosuppressants were completely discontinued in 3. in these "operational tolerance" patients, the precursor frequency of anti-donor cd4 + t cells (mean=5.2±1.9%) was not reduced compared to that in non-tolerance patients, suggesting that donor-specific immune tolerance is maintained via inhibitory/ suppressive mechanisms rather than via clonal deletion. conclusion: multiparameter mlr assay can provide a clinically validated rule predicting the success of tailoring/weaning immunosuppression. psychological factors associated with non-adherence among adolescents before and after kidney transplant. nataliya zelikovsky. 1 1 dept. of pediatrics, div. of nephrology, the children's hospital of philadelphia, philadelphia, pa.purpose: little is known about psychosocial risk factors for poor adherence among pediatric transplant patients. identification of variables that impact illness management can guide targeted interventions to improve adherence. methods: a longitudinal study was conducted to determine whether quality of life (pedsql), family functioning (fad), and parent adjustment (pip) would predict adherence in adolescent transplant patients. psychological questionnaires were administered prior to and 12 months after the transplant. medical adherence measure (mam), a semi-structured interview was used to assess adherence. adherence was calculated as % missed and % late doses of those prescribed. results: 60 patients (m =14.32 years +2.18, 75% male, 63% caucasian) and their parents were evaluated at the time of listing for kidney transplant. the rate of non-adherence prior to transplant was high, with 90% of patients reporting some degree of nonadherence. of these patients, 37% missed and 28% took late > 10% of prescribed doses. on the quality of life measure, behavior issues were associated with missed (r=-.42, p=.01) and late doses (r=-.39, p<.05), and mental health issues were associated with late doses (r=-.38, p<.05). adolescent reports of problems in affective responsiveness among family members was associated with missed (r=.33, p=.04) and late (r=.51, p=.001) doses. missed doses were also associated with mother reports of difficulties with overall family functioning (r=.34, p<.05), communication (r=.41, p<.01) and role definitions (r=.33, p<.05) among family members. 49 of the families were re-evaluated one year after the kidney transplant. 65% had been on dialysis prior to transplant and 42% received living-related transplants. 58% of the patients reported some degree non-adherence post-transplant, and using more stringent criteria, 17% of patients reported missing and 27% reported taking late >10% of prescribed doses. worse quality of life such as limitations due to emotional problems (r=-.32, p<.05), behavioral problems (r=-.39, p<.01), and difficulties with family cohesion (r=-.32, p<.05) was related to worse adherence. discussion: adolescent quality of life in behavioral and emotional domains, and family functioning play a significant role in adherence both before and after transplant. programs to improve adherence among transplant patients should incorporate psychosocial supports and behavioral interventions to improve adjustment of patients and families. kidney transplantation leads to marked improvements in health, yet transplant (tx) recipients often have difficulty with sexual functioning, which can affect quality of life. specific sexual concerns of tx recipients remain under investigated. the purposes of this study were to 1) further establish the psychometric properties of the sexual concerns questionnaire (scq) including reliability and preliminary construct validity and 2) identify the sexual concerns of kidney tx recipients. the scq was answered by 390 kidney tx recipients who rated each item on a 0 (not at all) to 5 (extremely) scale. a cronbach's alpha correlation coefficient was calculated to determine the reliability of the scq. an alpha value of .83 was calculated for the questionnaire indicating it was reliable. exploratory factor analysis (efa) was performed to establish preliminary construct validity of the scq. as a result from the efa, 14 items were dropped and a 5 factor structure was accepted. examples of items and responses include question 1: "how difficult is it for your vagina to get or stay wet or moist?" (for women) and "how difficult is it for you to get or keep an erection?" (for men) and question 2 "how comfortable are you talking about sexual concerns with your doctors and nurses?"participants were also asked to indicate how important their sexuality was to them on a 0 (not at all) to 6 (extremely) rating scale. twenty-six percent of participants rated their sexuality as quite a bit important, 26% rated their sexuality as very important, and 20% rated their sexuality as extremely important. the findings provide evidence of a reliable questionnaire with evidence for preliminary construct validity. they also indicate that sexuality is an important issue for a majority of kidney tx recipients. a cross-sectional study of fatigue before and after liver transplantation. james r. rodrigue, 1 timothy antonellis, 1 p=0.78 # 1 (none) to 10 (extremely high); ♣ higher score = more fatigue, poorer sleep quality, or more mood disturbance; ¶ higher score = better qol one-third of pre-lt (32%) and post-lt (33%) patients reported severe fatigue. poor sleep quality was reported by 68% and 79% of pre-and post-lt patients, respectively. pre-lt fatigue was predicted by higher bmi (ß = 0.21) and meld (ß = 0.20), depression (ß = 0.35), and poor sleep quality (ß = 0.48), adj r 2 = 0.44, f = 10.72, p < 0.0001. post-lt fatigue was predicted by older age (ß = 0.19), tension-anxiety (ß = 0.27), anger-hostility (ß = 0.38), and poor sleep quality (ß = 0.18), adj r 2 = 0.38, f = 7.45, p < 0.0001. more fatigue was associated with lower sf-36 physical (r = -0.34) and mental (r = -0.44) qol. conclusions. fatigue and poor sleep quality are clinically significant problems for lt candidates and recipients. bmi, psychological functioning and sleep quality are modifiable variables that predict fatigue severity and should be targets of intervention when addressing fatigue symptoms. health literacy in kidney transplant recipients. elisa j. gordon, 1 michael s. wolf. 2 1 medicine, albany medical center, albany, ny; 2 medicine, northwestern university. background: in order to successfully manage the transplant long-term, kidney recipients must have a basic understanding of key transplant-related concepts and terms indicative of their condition and treatment to properly communicate with health care providers and manage their health. kidney recipients must also possess numeracy skills to enable proper medication-taking and to monitor serum creatinine levels, bodily temperature, etc. the objective of this study was to examine health literacy levels among kidney transplant recipients. methods: we surveyed 124 consecutive adult renal transplant recipients using the test of functional health literacy in adults (s-tofhla), and a modified version of the rapid estimate of adult literacy in medicine, called the "realm-transplant," which measured patients' knowledge of 69 kidney transplant-related terms that patients are expected to have familiarity with. open-ended and multiple choice questions assessed numeracy related to kidney survival. results: most kidney recipients (91%) had adequate health literacy (s-tofhla), but 81% were unfamiliar with at least 1 kidney transplant-related term (realm-t). patients who were less educated (p<0.0001), had lower income (p<0.002), and were single or without a partner (p=0.046) had significantly lower health literacy levels (s-tofhla). patients less familiar with transplant-related terms (realm-t) had less education (p<0.0001), lower income (p<0.0001), and were nonwhites (p=0.033). the five least familiar terms were: sensitization (50%), urethra (45%), trough level (41%), blood urea nitrogen (32%), and toxicity (31%). sixteen percent wanted more information about their transplant. numeracy levels varied: 21% knew the likelihood of 1-year survival; 29% knew that half of kidney recipients have problems with the transplant in the first 6 months; 86% knew the normal range of creatinine for kidney recipients; and 86% were aware of the risk of death within the first year of transplantation. conclusion: at this clinic, kidney transplant recipients generally had high levels of health literacy. however, most had difficulty recognizing frequently used transplantrelated terms, which could impede their understanding of health information and self-care management. greater efforts are needed to educate kidney recipients about transplant concepts, which may foster better self-care management, and ultimately transplant outcomes. abstracts by a single pathologist using the banff, cadi and cnit classifications. all indication biopsies with clinical acute rejection (ar; 23.3% for sf and 21.4% for sb) were excluded from this analysis. the histological and clinical parameters were assessed using multivariate generalized-estimating-equations statistical analysis. results: subclinical ar was present in 12.2% sf vs. 8.5% sb bx at 6 mo and 0% sf vs 5% sb bx at 12 mo (p=ns); borderline ar was seen in 2.4% sf vs. 8.5% sb bx at 6 mo and 10% sf vs. 5% sb bx at 12 mo (p=ns). despite the pristine condition of the kidneys at implantation, regardless of steroid exposure, there was a significant trend increase (p<0.0001) in chronic tubulo-interstitial damage; 35% of 6 mo bx and 53% of 12 mo bx demonstrated ifta; with moderate/severe changes (ifta grade 2-3) in 6.8% and 10% of 6 and 12 mo bx respectively. the prevalence of biopsies with ischemic glomerular changes (p<0.0001), tubular microcalcifications (p=0.009), vascular intimal thickening (p=0.0008) and the number of sclerosed glomeruli (p<0.0001) increased over the first year after transplantation, without any difference between the sb and sf group. a critical risk factor for ifta injury by multivariate analysis, independent of time after transplantation, was smaller recipient size. in this first ever serial histological analysis, embedded in a randomized multicenter pediatric study of steroid avoidance, we found significant progression of chronic graft injury in the first year post-transplantation in both study arms. small recipient size is the primary risk factor for tubulo-interstitial damage, likely related to vascular size discrepancies between recipient and the graft, resulting in chronic graft ischemia. in the 1-year symphony core study, a regimen with 2g mycophenolate mofetil (mmf) + low-dose tacrolimus (3-7 ng/ml) + daclizumab + steroids resulted in less acute rejections and better glomerular filtration rate (gfr) compared with 2g mmf + steroids and either standard-dose cyclosporine (csa), low-dose csa (50-100 ng/ml) + daclizumab or low-dose sirolimus (4-8 ng/ml) + daclizumab. methods: 960 patients participated in an optional follow-up of 2 years. gfr data from 79% of included patients (48% of the core itt population) were available at 3 years.here we present results in the itt population. results: at inclusion into follow-up 47%, 34% and 17% of patients received csa, tacrolimus or sirolimus, and at 3 years 37%, 31% and 14%, respectively. many follow-up patients had been switched to tacrolimus in the 1st year, including 24% of patients randomized to sirolimus. at 3 years 95% of patients were on mmf and 69% on steroids.over the 2nd and 3rd year all arms had a low rate of biopsy-proven acute rejection (bpar; 2-4%) and of graft loss (3-5%). low-dose tacrolimus remained clearly superior in terms of bpar (13% vs. 26%-38% in the other arms). uncensored 3-year graft survival was 89% with low-dose tacrolimus and low-dose csa, 87% with standarddose csa and 85% with low-dose sirolimus (p=0.19). patient survival was between 94% and 97% (p=0.52). in the four arms, the mean gfr change over the 2 nd and 3 rd year was between +1 and -3 ml/min and low-dose tacrolimus still had the highest gfr (69 vs 64-66 ml/min, p=0.15). observational follow-up results based on approximately half of the core symphony population indicate that during the 2nd and 3rd year renal function was stable, bpar and graft loss rates were low and many patients changed treatment regimen substantially. still, the itt arm with 2g mmf + low-dose tacrolimus + daclizumab + steroids was superior at 3 years with respect to renal function and graft loss but differences were less marked and statistically not significant. discovering histological evaluation of time zero donor kidney biopsies has not conclusively predicted graft outcome. we hypothesize that gene expression analysis could provide additional information to determine graft outcome in the first year of transplantation. to this end, we evaluated all 49 implantation biopsies obtained post reperfusion in 18 deceased donors (dd) and 31 living donors (ld) at our center. biopsies were evaluated and scored using banff criteria. low density real time pcr arrays were utilized to measure intragraft expression of 95 genes associated with programmed cell death, fibrosis, innate and adaptive immunity, and oxidative stress signaling. results of expression were defined as folds compared to a pool of 25 normal kidney biopsies. in dd, histological features of atn were more common (44%) than in ld grafts (6%; p<0.001), whereas arteriosclerosis was infrequent in both groups (6% and 15%, respectively), as well as the extent of glomerular sclerosis (0% and 2%). there was no association between these histological features and renal function at 1 year post transplant. not surprisingly, dd grafts displayed a pattern of gene expression remarkably different from ld, including an increased expression of complement protein c3 ( background: isa247 is a novel calcineurin inhibitor (cni), developed using a pharmacodynamic approach for use in autoimmune disease and solid organ transplantation. in moderate to severe plaque psoriasis, a canadian phase iii trial has demonstrated that isa247 is efficacious with minimal changes to renal function and a european trial is presently underway comparing isa247 to cyclosporine a (csa).in renal transplantation, a phase iia study comparing isa247 to csa in stable renal transplant recipients demonstrated isa247 to be efficacious and well tolerated. a phase iib study in de novo renal transplant patients comparing isa247 to tacrolimus is ongoing and final data will be available may 2008. we hypothesize that isa247 is non-inferior to tacrolimus in terms of efficacy.methods: this is a 6 month, randomized, multicenter, open-label, concentrationcontrolled study comparing three oral isa247 dosing groups (0.4, 0.6, or 0.8 mg/kg bid) to tacrolimus in 42 north american transplant centres. all cni's were titrated to target trough concentrations. inclusion criteria included males and (non-pregnant) females between the ages of 18-65 who were receiving a first deceased or living donor renal transplant. cold ischemia times were to be ≤ 24 hours, and peak panel reactive antibodies ≤ 30%. the primary efficacy parameter of the trial is non-inferiority (in at least one dose group) in biopsy proven acute rejection (bpar) at 6 months as compared to tacrolimus. secondary objectives include: renal function; pk/pd relationships; patient and graft survival; and proportion of patients with hypertension, hyperlipidemia or new onset diabetes mellitus (nodm).results: interim data, as previously presented at the 2007 atc, demonstrated that isa247 had rejection rates similar to tacrolimus (isa247 0.4 mg/kg bid 11%, isa247 0.6 mg/kg bid 8%, isa247 0.8 mg/kg bid 3%, tacrolimus 9%) and confirmed previous results indicating an improved safety profile. 334 patients have now been enrolled between january 2006 and june 2007, with an optional extension to 12 months added to the trial. a recent approval by both fda and health canada has allowed continued use of isa247 in these patients until commercialization. the six month final results will be available for presentation at atc 2008. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-010092-uftc8inx authors: nan title: abstract of 29th regional congress of the isbt date: 2019-06-07 journal: vox sang doi: 10.1111/vox.12792 sha: doc_id: 10092 cord_uid: uftc8inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner 0 s fate illustrates the brain drain beginning in the early 30 s escalating in 1939 with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early 80 s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in 1995. as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu28. levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of 3% r&d intensity was first met in 2014 and is 2018 the sixth highest among oecd countries and the second highest in the eu28. austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon 2020 and the preceding 7th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd19 targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd19 directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd19car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about 30% of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to 75% of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in 4% of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il-1rap is expressed by the leukemic but not by the normal cd34 + /cd38-hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il-1rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il-1rap mab (#a3c3 clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with 3rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp9 (inducible caspase 9) and a monitoring/selection cell surface marker δcd19. we demonstrated in-vitro and in an in-vivo xenograft murine model that il-1rap car t cells can be activated in the presence of il-1rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a 4-year period does not affect transduction efficiency of cml patient t-cells by il-1rap car vector and that autologous cart-cells are able to target il-1rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il-1rap expression on a tissue macroarray comprising 30 normal human tissues (3 donors) , with #a3c3, detected various il-1rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a3c3 flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il-1rap. as expected, monocyte subpopulation is targeted by autologous il-1rap cart cells (ratio e: t = 1:1), but at a lower level that il-1rap cml cell line. in-vivo investigation of specific toxicities of autologous il-1rap cart-cells against hsc and/or immune cells on a human-cd34 + cord blood cell engrafted/nog murine model, but also by an in-vitro cd34 + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd34 + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp9/ rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than 90% of cart-cells, after exposure to ap1903. in conclusion, based on cml model, we demonstrated that il-1rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from 20 to 30% for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about 20% when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of 5% for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of 2 h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within 15 min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at 4°c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an 1/1/1 ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the 6th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and 24 h organ failure. this trial will be recruiting 200 patients in 6 french trauma centers and is planned to be initiated second half of 2019. local/neighbours day: innovation in germany 1c-03-01 mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct1: eudract number: 2011-005441-13; ortho-ct2: eudract number: 2012-002010-39; maxillo1: eudract number: 2012-003139-50) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. 2018 introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using 50 ml stirred flasks. cells were differentiated into mks using tpo, scf and il-3 in apel2 medium for a period of 22 days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il-2rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an 8-fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of 18.7 ae 6.8 9 10 7 in microcarrier-assisted bioreactors in comparison to 4.9 ae 1.3 9 10 7 mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il-2rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than 12 years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september 30, 2016 . it has travelled billions of kilometers, just to study a small (4 km diameter), black boulder named 67p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd19 expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. 1d-04-03 cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb2) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from 450-1200 ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between 3-28 days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation (450/470 ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard 450 ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline 28 days after blood donation, but endurance performance was normalized already after 14 days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, 4 -5 weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" (1970) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby 2004) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease 2a-02-01 immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be 29-36% and 53-72%, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be 8.4-14%, 12.5-27.7%, 3.3-3.5%, respectively, and the alloimmunization rates were 17.6%, 14.3-30%, 7.1%, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of 2-6% is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from 7% to 76%, the highest figures being established when an abo/rh1-only matching policy is implemented. in a meta-analysis of 24 publications, the overall alloimmunization rates were around 20%. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a 61% increased risk of producing additional antibodies versus 30% in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to 70% of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; 1) which antibody specificities are clinically relevant 2) which antigens are most immunogenic 3) what is the availability of specific antigen typings in the donor population 4) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over 50 years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to 15-30 min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first 72 h of life, and late thrombocytopenia, occurring after the first 72 h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet-2/matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of 50 9 10 9 /l had a significantly higher rate of death or major bleeding within 28 days after randomization than those who received platelet transfusions at a platelet-count threshold of 25 9 10 9 /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from 1% to 8%, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> 60 years or < 3 years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within 12 h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: 1) using normal pediatric values published in the nelson textbook of pediatrics; and 2) using patients as their own controls and comparing pre-and post-transfusion values with either a 10% or 20% difference threshold. we monitored for taco up to 24 h post-transfusion. a total of 136 patients were included. taco incidence varied from 1.5% to 76%, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of 96 samples. from november 2017 to january 2018, a total of 10,141 blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas 6800 platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than 5.00e+04 iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was 7.05e+03 iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence 1:1,268) , whereas 17 hev rna positive donations were identified by id-nat (incidence 1:597); all id-nat only positive donations had vl < 25 iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately 50 % higher if id-nat was used. however, vl were below 25 iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last 25 years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b 2 ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost 20 years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p1 and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p 1 and p 2 phenotypes. the system (isbt no. 003) currently includes three different antigens, p1, p k and nor. the p1 antigen was discovered already in 1927 by landsteiner and levine while p k and nor were described in 1951 and 1982, respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p1/p k carbohydrate structures. anti-p1 is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p1 have been reported to react at 37°c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p 1 k /p 2 k . the rbc of the fetus as well as of the newborn express low amounts of p1, p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p1. recently it was clarified that the same a4galtencoded galactosyltransferase synthesizes both the p1, p k and nor antigens and in addition the p 1 and p 2 phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p 1 allele in the 5'-regulatory region of a4galt, which enhance transcription of the gene. it has been debated whether the p k and p1 antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p1 antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p1, has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata1 nor klf1 represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf1 gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd44) and p1, among others. since the first description of klf1 variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf1 table of blood group alleles. other than klf1, a mutated gata1 gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata1 allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in 1995, when the disruption of a gata motif in the ackr1 gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata1 binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata1 binding to a control element located 3.7 kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata1 binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p1 antigen has been known for a long time to be determined by the a4galt gene but the molecular basis underlying the common p1/p2 phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon 2a, which showed a very good correlation with p1 antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx1 tf in the expression of p1 antigen, by selective binding to a regulatory site present in p1 but not in p2 alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p1 and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. 2c-06-03 clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of 1) guiding targeted rh prophylaxis in non-immunized rhd negative women and 2) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in 6-7 european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of 99.9%, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified 837 studies, but only 10 was eligible for full text review and 5 met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. 2d-07-02 applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in 1976. during the 1994 genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from 1995, the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs 4, 5 and 6. today, rwanda has an ambitious vision to put all 12 million citizens within 30 minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in 2016, the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in 15 -45 minutes depending on a hospital's location. the average duration was between 2 -4 hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to 25 health facilities throughout the country and are set to reach 90% of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of 3.1 hours per delivery and a total of 10,115 hours of lost time on road pick up they could instead dedicate to patient care. by march 2019, over 11,000 deliveries have been made, with 30% of those being emergency deliveries. a total of more than 19,500 blood units have been delivered. in february 2018, zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to 100 km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. 2d-07-03 scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna-1b, fcgriiib and hna-2 have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna-1a, -1b, -1c, -1d, hna-2, hna-3a, -3b, hna-4a, -4b and hna-5a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna-3a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna-1a, -1b, hna-2 and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna-1, -2, -4, -5 and hla class i antibodies are clearly detectable in the gift while hna-3 antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna-3 with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna-3, can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna-2 in the gift because the molecular reason for the hna-2-null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd177*787a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. 2d-08-02 norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa-1, -2, -3, -5 and -15), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa-4, -6 to -11 are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd109 and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the 19th international platelet immunology workshop of isbt (2018) . the investigations also include measurement of the anti-hpa-1a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa-1 typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. 2d-08-03 molecular basis of hna-2 expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen 2 (hna-2) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd177 (also known as nb1). hna-2 is absent on the neutrophil surface of 3-5% of the healthy individuals that divided the population to hna-2 positive and hna-2 null individuals. exposure of hna-2 null individuals to hna-2 positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna-2 and consequently the production of iso-antibodies. the hna-2 iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd177 on a neutrophil surface of hna-2 positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna-2 positive and negative subsets. the cd177 gene contains 9 exons encoding a protein of 437 amino acids. the lack of hna-2 (in hna-2 null individuals) is associated with the presence of a missense mutation, cd177*c.787a>t in exon 7 of cd177 gene inducing a premature stop codon in codon 263. this mutation alone or in combination with cd177*c.997delg has been introduced as the main reason for the absence of cd177 in hna-2 null individuals. a pseudogene (cd177p1) highly homologous to exons 4-9 of cd177 gene is located downstream of the cd177 gene. conversion of exon 7 of cd177p1 into cd177 gene is responsible for the generation of cd177*c.787a>t missense mutation. in addition, the heterozygosity or homozygosity of cd177*c.787a>t is accounted for regulation of hna-2 negative and positive neutrophils subpopulations. genotyping has revealed the hna-2 null individuals, heterozygous for cd177*c.787a>t mutation without cd177*c.997delg, indicating the presence of a complementary mechanism regulating cd177 expression. newly in hna-2 null individuals and individuals with atypical cd177 expression a cd177*1291g>a polymorphism in combination with cd177*787a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd177 expression on the neutrophil surface. this presentation will summarize recent findings on cd177 expression and highlights the potential genotyping methods for genetic assessment cd177 expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, 100 transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding 1) blood product ordered, 2) whether the patient experienced a reaction, and 3) the start and end times of the transfusion. for each of these fields across all 100 sampled notes, the claritynlp tool reproduced these data points with 100 percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than 1 h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and 24-h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid-1980s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:-1,2,3,4, (5),6,7. genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel*02.03/ 02.06 (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 5'and 3'-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than 1000 human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked 30 years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" 3a-s02-03 background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns1 proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at 650 nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of 10 -10 2 tcid 50 /ml for zikv and denv (serotypes 1/2/3/4) after a detection phase of around 5 min. the first results obtained on 16 zikv (+) clinical samples previously tested by commercial real-time pcr (ct < 36, altona) showed an 88 % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than 10 min (sampling + detection) instead of 2 h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during 2016, with up to~2% of blood donors reactive for zikv rna in id-nat testing at the peak in june 2016. aims: perform a serosurvey for anti-zikv igg using six panels of 500 donor specimens each collected in march 2015, at the beginning, peak and end of the 2016 epidemic, and from march 2017 and april 2018. methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns1 antigen from bio-techne to characterize zikv seroprevalence in the 6 9 500 cross-sectional sample sets (anonymized with selected demographic information). results: 500 pr donor samples collected in april 2015 were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative (3 positive, 3 equivocal) despite the high dengue virus seroprevalence (>90%) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected 1-3 months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > 99%, and an area under the curve (auc) of 0.999, demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional 5 sample sets (2500 samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in 6,636 female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, 17b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo-3am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp9 (a selective trpc6 activator). results: sex hormone intake by menstrual status was higher in premenopausal women (25.3%) than in postmenopausal women (7.4%). female hormone intake was significantly (all p < 0.0001) associated with reduced storage hemolysis in all females (0.32 ae 0.16% versus 0.35 ae 0.29% in controls), enhanced susceptibility to oxidative hemolysis (37.9 ae 9.3% versus 35.8 ae 9.9% in controls), and reduced osmotic hemolysis in postmenopausal women (23.1 ae 10.2% versus 26.8 ae 12.0% in controls). in vitro, supraphysiological levels of progesterone (10 or 20 lmol/l), but not 17b-estradiol or testosterone, inhibited spontaneous or hyp9-induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after 30 day cold storage (0.95 ae 0.18% versus 1.85 ae 0.35%, progesterone 10 lmol/l versus solvent control (dimethyl sulfoxide, 0.1%), p < 0.0001). co-incubations (2.5 h, 37°c) of rbcs in the presence of progesterone and a trpc6 activator (hyp9, 25 lmol/l) suggested that progesterone protected against hyp9-induced hemolysis (1.45 ae 0.13% and 1.01 ae 0.09% versus 2.63 ae 0.19%; hyp9 + progesterone at 10 or 20 lmol/l versus hyp9 alone, p < 0.05 by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc6 channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. 3a-s05-01 international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid-1970s, the red cross was active in the national blood programs in approximately 95% of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly 50% of all worldwide donations take place (home to only 17% of the population). who data shows that the median annual blood donation rate in high-income countries is 3.21% of the population compared to 0.46 % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last 30 years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their 2018 global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by 25 % in lebanon; vnrbds by 71% annually in kirgizstan, and from practically zero to 3'588 in south sudan. the importance of rc societies was also underlined in the 2018 published global mapping of gap, which showed that 36 (19%) of them provide level a (full blood service), 75 (39%) are level b (systematic blood donor recruitment) and 47 (25%) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ 132420/d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in 500 pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: (1) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. (2) corresponding educational materials for students and teachers were developed and distributed. (3) blood donation clubs were established in 500 pilot schools. (4) trainings were conducted for 688 personnel of moh and trc on blood donation regarding their responsibilities. (5) cascade trainings were conducted for 3218 personnel of transfusion centers and 4399 school principals in 81 provinces. (6) information seminars were delivered to 251.475 students and 15.739 teachers and family members of students during school campaigns. (7) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). (8) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. (9) media spots were produced and broadcasted 3.851 times in 33 different tv and radio channels. (10) billboard posters and brochures were prepared and distributed to 81 provinces for raising public awareness. (11) advertisements about the project and the importance of vnrd were displayed 386 times on national and local newspapers, 1.117 times on online news, and broadcasted on 6 national tv channels. (12) during the campaigns, 28.310 units of whole blood were collected in pilot schools. (13) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. (14) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to 32.07 % and 35.99 % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from 73.66% to 82.54 in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite 70% of pakistan's population being under 29 years, only 10% of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p0 checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with 3-10 walk-in donors and an average of more than 20 telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june 2019. the statistics generated since january 2019 are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to 31 patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since 01.01.2013, whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from 01.01.2013 until 01.03.2019, an extended determination of rbc antigens was performed in 352 subjects presenting for blood donation. twenty-nine rare donors (8%) were identified and included in the rare donor file: 25 fy(a-b-), 1 lu (a-b-), 1 lu(b-), 1 fy(a-b-) and s-, 1 ccddee (r'r'). overall, these 29 donors provided 105 rbc units (range . to date, all donors are still active and 14 are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately 100. -chf, resulting in a total financial effort of around 35,200.-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: 1.) avoidance of transfusion transmittable infections; 2.) quality of the blood product with a strong focus on component therapy; 3.) prevention of severe transfusion reactions; 4.) avoidance of clerical errors; 3.) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. 1.) sufficient availability of blood and proper utilisation; 2.) avoidance of transfusion transmittable infections; 3.) avoidance of clerical errors; 4.) prevention of severe transfusion reactions; 5.) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at -20°c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from 51 samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with 2654 probes covering exons of 64 genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, 51 samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > 2509, with 94% of target at a quality of q30. optimal sample plexity for a standard run was determined to be 14 samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted 99.5% of snp based red cell genotypes. script predictions were 100% concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna2 genotypes defined by cd177 could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p1pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to 14 samples can be reliably sequenced in a single run. our script correctly predicts over 99% of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type 600,000 uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > 1:20,000 identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = 7,871) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was 99.9% for 95,022 comparisons; 29 of the 89 discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across 48 antigens for which serology was available, genotyping provided a 3.6-fold increase in the number of typing results available per donor (47.9 vs 13.2). furthermore, genotyping provided data on an additional 224 clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from 3,146 patient referrals with > 3 alloantibodies between 2014 and 2018, 772 unique alloantibody profiles were identified. we found that there was a 2.6-fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the 4,721 nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from 293 to 246, representing an additional 164 patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. 3c-s06-04 biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near 1500 wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including 2 transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by 4 dna samples with targeted sequencing on illumina miseq, and specific variants were verified by 40 dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, 1) m, n: insufficient sequencing reads, 2) c, c: identical rhce exon 2 with rhd exon 2 for c allele, 3) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, 13 systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a2, has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. 8:14750, 2017) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine 27 (suppl.2) :42, 2017). here we have used it to analyse and document bel-a2 blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a2 cell-line. methods: bel-a2 cells (day 196) were cultured in expansion medium and genomic dna (gdna) was isolated from 1 9 10 6 cells on day 5. for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for 41 genes encoding all 36 known blood group systems and 12 further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a2 genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c4a/c4b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a2 did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr1, cdan1 and tmx4, these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a2 genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, 2017) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a2 blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from 16 countries were enrolled (67% in north america, 17% in europe, 7% in oceania, 5% in asia, and 4% in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = 360) differed significantly on a regional basis (p = 0.04) and ranged from 12 (8-41) x10 9 cells/l in the middle east to 45 (20-66) x10 9 cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = 0.08), nor did the tpc prior to therapeutic transfusions (n = 189) differ on either a regional (p = 0.16) or national (p = 0.57) basis. for children supported by ecls (n = 90), there were no regional (p = 0.06) or national (p = 0.40) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = 0.02) and national (0.04) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [28 (16-81) x10 9 cells/l]. for children with an underlying oncologic diagnosis (n = 233), no differences were seen in the tpc for prophylactic transfusions (n = 175) on a regional (p = 0.19) or national (p = 0.20) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional (0.86) or national (p = 0.33) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < 0.001) and national basis (p < 0.001). the median (iqr) dose based on volume ranged from 8.4 (5.0-11.2) ml/kg in north america to 12.6 (9.8-16.0) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < 0.001) and national (p < 0.001) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, 2015) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ 10 9 10 11 /l and a platelet dose of 1.1 9 10 11 per square meter (sm) of body-surface area (bsa) in inpatient and 2.2 9 10 11 /sm in outpatient setting. aims: in january 2018 we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of 1.1 9 10 11 /sm and outpatients a dose per transfusion of 2.2 9 10 11 /sm. platelets were transfused when the count was ≤ 10 9 10 11 /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january 2018 to december 2018 a total of 9839 platelet pediatric aliquots were transfused: 5534 (56.2%) were obtained from apheresis platelet concentrates and 4305 (43.8%) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots (7505-76.3%) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, 6365 aliquots were transfused in inpatient setting: 1675 (17%) in the hematology unit, 2772 (28.2%) in the oncology unit and 1918 (19.5%) in hsct unit. a total of 1140 (11.5%) aliquots were transfused in outpatient setting: 840 (8.5%) to patients affected by hematological malignancies and 300 (3%) to patients with solid tumors. five major bleeding events (who grade ≥ 3) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade 3 bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = 3, 1.2%) developed intracranial bleeding (who grade 4). the platelet count at the time of the event was 22 9 10 9 /l, 25 9 10 9 /l and 8 9 10 9 /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade 4 bleeding has been observed in inpatients setting only (1.2% vs 1% of plado trial, sj slichter, nejm, 2010) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ 3) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell 3000 elite (best theratronics, canada) at a dose of 25 gray. all rbcs were used for transfusion for 14 days from the harvest day. 70 pediatric patients with various oncological and hematological diseases were randomized to 2 groups of 35 members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. 3-5 days after the transfusion, the direct antiglobulin test (dat) was performed and after 14-21 days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = 0.2), as well as the concentration of potassium (p = 0.44) and haptoglobin (p = 0.25) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = 1). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = 0.39). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for 14 days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama 2018) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over 5 years duration from 2012 to 2016. methods: using 5 years data (2012) (2013) (2014) (2015) (2016) perioperative transfusions decreased steadily per year from 6.4% in 2012 to 5.5% (14% cumulative decline) in 2016 for children of all ages (or 0.966; 95% ci 0.957-0.976; p trend < 0.001). the cumulative change in elective procedures was 9.2% versus 27.2% decrease in urgent/emergent procedures (p trend < 0.001). summary/conclusions: in this large prospective registry study of > 350,000 children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across 5 years from 2012 through 2016 with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk 3c-s08-01 transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of 2-20% of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ 100 iu/ml and being id-nat (procleix-ultrio plus tm [95% lod: 3 iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < 6 iu/ml (n = 44) or nat repeat reactive (rr) with vl < 6 iu/ml (n = 32). french samples initially tested nat nr/nrr with procleix-ultrio (lod 95%: 11 iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: 6 iu/ml]). hbv dna was purified from 5 to 12 ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in 79% of all samples with undetectable or vl < 6 iu/ml. hbv genotypes were a1 (33.3%), a2 (18.4%), a3 (3.3%), b (3.3%), c (1.7%), d (25%), and e (15%). all samples were anti-hbc positive and 73% of ultrio-negative samples tested positive with ultrio plus. unusual 1-2 nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = 16) or deletions (n = 4) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of 30 samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. 3c-s08-03 background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since 1998, hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of 15 and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i1000 analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas 8800 platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p22cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in 69 blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, 59/69 (85.5%) donors were anti-hbc positive, and 10 (14.5%) negative. 18 donors had both anti-hbc and anti-hbe reactivities. a group of 11 young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and 9 patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g600 automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is 2 logu/ml and the lower limit of quantification (loq) is > 3 logu/ml, due to nonlinearity results between 2 and 3 logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss 19.0.0. results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: 3.0 logu/ml, range 2.0-4.9), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in 34/69 obi donors (49.3%), with a mean value of 2.21 logu/ml (range 2.0-3.20). hbcrag could be measured only in 2 obi donors (3.0 and 3.2 logu/ml), being below the loq of the test in the majority of obi (32/34). considering the presence of anti-hbc, hbcrag was detected in 29/59 (49.1%) anti-hbc+ and in 5/10 (50%) anti-hbc-obi, with no significant difference in their mean levels (2.21 ae 0.32 vs 2.14 ae 0.26; p = 0.44). interestingly, the presence of anti-hbe (18/62) was independently associated with higher hbcrag levels (2.38 ae 0.42 vs 2.14 ae 0.22; p = 0.03). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. 3c-s08-05 hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss 20.0 statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < 0.05 was considered statistically significant. results: 1. proliferation characteristics of t cells. the proliferation of cd4 + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group (3.0%, 3.3% vs. 1.7%), with significant difference (3.0% vs. 1.7%, p = 0.016, 3.3% vs 1.7%, p < 0.001). 2. the frequency of specific ifn-c secreted t cells. the response intensity of the obi group (25 sfc/10 6 pbmcs) and chb group (25 sfc/10 6 pbmcs) was higher than that of the hc group (5 sfc/10 6 pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group (64.0%). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term (15-20 years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under 10 themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = 2.11; 95% ci: 1.03-4.31). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for 72,000 participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = 150,000), were calculated for all dbds participants. 12,903 female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of 10 depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile 1 contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = 1 from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between -0.42 and 0.50 (mean 0.01). a total of 12,523 female donors were classified as "not tired" and 380 (2.9%) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: 0.00; not tired mean prs: 0.01). an age-adjusted logistic regression model found this to be insignificant (or: 0.74, 95% ci: 0.33-1.66), p = 0.47). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile 6) had or = 1.02 (p = 0.93) of being tired when compared to those in quantile 1 (or set as 1). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv-1 subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv-1 infected blood donors. methods: from 2016-2018, 177 blood donations collected from 24 blood centers, covering almost the whole of china, were confirmed as hiv-1 positive by national centers for clinical laboratories using abbott realtime hiv-1 assay or cobas taq-man hiv-1 test, version 2.0. then hiv-1 gag (973 bp, hxb2: 1074-2044), pol genes (1454 bp, hxb2: 2068-3521) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv-1 subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv-1 subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among 177 donations, gag and pr-rt regions of 160 samples were sequenced successfully. the distribution of hiv-1 genotype was as follows: crf_07bc = 66 (38.8%), crf_01ae = 58 (36.3%), b = 9 (5.6%), crf_08bc = 2 (1.3%), crf55_01b = 4 (2.5%), crf59_01b = 1 (0.6%), crf65_cpx = 1 (0.6%), crf67_01b = 2 (1.3%), crf79_0107 = 1 (0.6 %), crf85_bc = 1 (0.6 %), urf_0107 = 6 (3.8%) and urf = 9 (5.6%). 26 of 160 hiv-1 isolates were identified to have drms. there were 4 (15.4%, 4/26) protease inhibitors (pi) accessory drms, 3 pi major drms and 20 (76.9%, 20/26) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf01_ae and crf07_bc (61.5%, 16/26). 3 of 4 pi accessory drms were q58e. the pi major drms included m46l, m46i and n88s. n88s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v179d/e is main nnrti drm (70.0%, 14/20). a combination of v179d and k103r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k103n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf07_bc (38.8%), crf01_ae (36.3%). besides, other rare crfs and several urf_0107 and urfs were also found in these hiv-1 isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in 16.3% donors in the study, which may result in resistance to pis and nnrtis, especially the hiv-1 variants with n88s mutation in pr gene and k103n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv-1 in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv-1 among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in 100% plasma were biotinylated at day 1 and day 7 of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, 50 ml of pcs was washed twice and incubated with 5 mg/l biotin, dissolved in phosphate buffered saline-pas-e (1:9), for 30 min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd62p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in 65% pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, 98.4% ae 0.9% of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd62p expression was increased in biotinylated platelets 48.4% iqr(41.7-56.2%) compared to the control samples 12.3% iqr(9.5-12.7%) on day 1 of storage. however, biotinylated platelets were not more activated compared to sham samples 50% iqr(41.7-56.2%). thus only the procedural steps led to increased cd62p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day 7, a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd62p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd62p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs4 insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs4, these insulators are small-sized (119-284 bp vs 1.2 kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a1, one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il-3-dependent 32d cells, which upon transduction with oncogenic vectors become il-3-independent, leading to transformation. 32d cells were transduced with sin-lvs: the b-globin-τνs9.3.55-, the insulated b-globin-a1-tns9.3.55and the oncogenic sffv-gfp-vector. transduced cells were expanded in 10% il-3 and transduction efficiency was determined by vector copy number (vcn). transduced 32d cells were seeded in methylcellulose with 10% or 0-1% il-3 to detect the il-3-independent and potentially transformed clones. the il-3-independent clones were further expanded in 10% il-3 and infused in partially myeloablated and il-3-treated c3h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a1 insulator did not negatively affect vector titers (τνs9.3.55, a1-tns9.3.55, sffv-gfp: 2.8, 1.8, 2.5x10^8 iu/ml, respectively). 32d cells were successfully transduced with all vectors (%vcn positive colonies: 40-100%) and expanded up to 400-fold. the a1-insulator decreased the number of il-3-independent colonies by 78-93% over the uninsulated vectors. the uninsulated vector-transduced, il-3-independent colonies, were greatly expanded in culture with 10% il-3 over the a1-transduced colonies (sffv, τνs9.3.55, a1-tns9.3.55: 48, 40, 10 fold change, respectively). il-3 independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il-3-independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a1 insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a1 may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including 46 missense variants reported in rhd exons 6 and 7, was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published 3d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c.1065c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c.1154g>t (gly385val; d-negative) disrupts totally normal splicing, while c.1154g>c (gly385ala; weak d) and c.1154g>a (gly385asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a 3d model suggests that gly385asp, but not gly385ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons 6 and 7 by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in 15/46 (32.6%) variants, which correlates well with the quepasa-like prediction (sensibility = 0.93, specificity = 0.94). additionally, while normal exon inclusion is affected by c.1012c>g (weak d type 70), the associated leu338val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in 2014, participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of 6.210 compensated and 68.205 non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either 25 eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after 24, 36, 48 and 72 months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups (51.9%), refreshments (50.0%) and free (testing) laboratory parameters (38.3%) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by 28.6% and 25.3%, respectively. the lowest acceptance was for release from work (17.8%), complementary items (13.9%) and additional cash reimbursement (11.8%). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received 25 eur as a monetary reimbursement, the proportion of regular donors after 48 months (19.9%) was significantly higher than among comparable non-compensated donors (9.2%). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice (1-5 prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand 1) how deferral reasons and donor experience jointly affect donor lapse, and 2) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between 2013 and 2015 (n = 343,564). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included 25% new donors, 41% novice donors, 30% experienced donors, and 4% reactivated donors. deferral reasons included travel, hb, medical short-term (<28 days duration), medical long-term (>28 days duration), and miscellaneous. next, we interviewed 31 temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, 2012) to identify key topics and underlying themes. results: of target donations, 11% were deferred, mostly for travel (40%), medical short-term (23%), and hb (19%). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < 0.75, p's<0.001). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: 8.4 mmol/l (135 g/l) for men and 7.8 mmol/l (125 g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at 8 weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us 35% -75% of deferrals are due to low hb, especially in women (editorial, transfusion, 2012) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from 2007 to 2016. we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of 1000 new entrant donors (500 males and 500 females). prior information from the clinical literature (boulton, vox sanguinis 2004) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of 58 days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of 100 to 419 days, while the estimated recovery time for females varies between 54 to 503 days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of 23% reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, 2019) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (<30 years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: 330 ml water drink, 500 ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within 400 days after the index donation and we analysed their return for a donation attempt within 421 days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the 8199 episode participants who had received an invitation, 6538 (79.7%) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or 1.2, 95% ci 1.06-1.4 and 1.3, 1.12-1.5 respectively). return was slightly lower in women (or 0.88, ) and lower in first-time donors (or 0.86, 0.77-0.96) than after a 2 nd -4 th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or 0.47, 95% ci 0.37-0.60 and or 0.53, 0.46-0.61 respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, 79.7% returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > 50 years contributed 41% of all donations made in 2017. however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's 45 and up study baseline data collected between 2006 and 2009 to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up 22,058 active whole blood donors for 200,403 eligible person-years (average age at recruitment 56.4 years, 56.3% female, average follow up 9.1 years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the 2 years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made 1066 (95% ci 1056-1075), 987 (981-993), 900 (891-909), and 710 (690-730) donations per 1000 person-years, respectively. iron-deficiency was identified in 8.9% of donors in the study (n = 1964, 95% ci 8.5-9.3) . sixty percent of those with iron deficiency (n = 1,175, 95% ci 57.6-62.0) visited their general practitioner (gp) within 60 days of the identification of irondeficiency, and 48.4% (95% ci 45.5-51.3) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = 0.004). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting 25% of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to 30% of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp01-02 the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: 1) optimising the patient's own blood, 2) minimising blood loss and 3) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. 3d-s11-01 department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c3bc/c3d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c5 to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd163 and cd91-mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase-1 (ho-1) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho-1 has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. 3d-s11-02 understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. 3d-s11-03 cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il-12p70-producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il-12 production. in diseases characterized by local th1 inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band 3. other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd47. a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by 90%. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. 3d-s12-02 treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, 60,000 children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately 20% patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is 88% in paediatric and 65% in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss6 inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since 1987, include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with 204 national thalassemia associations in 62 countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive 2004/33/ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive 2005/61/ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to 15 days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers 95% of total blood supply. data on ttis in 3,067 patients with thalassaemia and scd-thalassaemia in 2010-2017 are analysed. results: tti prevalence in thalassaemia syndromes was: hbv 1.8% (occult type 1.3%), hcv 54%, hiv 0.3%, htlv 0.8%, wnv 0.5% and hev 3%. most frequent adverse reactions in 2015-17 were allergic (incidence 1:854), non-haemolytic febrile reactions 1:2,631, "other" 1:5,883, alloimmunisation 1:6,667, taco 1:100,013, tad 1:50,006, tt-hev 1:100,013. hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in 2010-2017 show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs10421768, rs117345431, and rs142126068 with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of 102 samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs10421768, rs117345431, and rs142126068. statistical analysis was performed on ibm*spss* statistic 23 using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.-582a>g variant (p = 0.02). for rs117345431 statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = 0.058). all samples were homozygous for allele t of rs142126068. summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to 20 years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by 2030 might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to 12 months in july 2016. since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s1. deferral of msm during the 4 months preceding the donation; s2. deferral of msm who have had more than one sexual partner in the 4 months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july 2016-december 2017 which is the baseline rr with the current 12month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤180 days) since all hiv-1 antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s1, from msm blood donors with the current deferral policy (12 months) and for s2, from monogamous msm of the general population. results: from july 2016 to december 2017, 8/28 (29%) hiv-1 positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at 0.16 in 1 million donations [95% ci: 0.04-0.34], or 1 in 6,380,000 donations. for s1, the number of additional msm donors was estimated at 733 and the number of additional hiv positive donations at 0.09, resulting in an hiv rr of 0.16 in 1 million donations [95% ci: 0.04-0.35] or 1 in 6,300,000 donations. for s2, the number of additional msm donors was estimated at 3,103 and the number of additional hiv positive donations at 4.92, resulting in an hiv rr of 0.23 in 1 million donations [95% ci: 0.05-0.56] or 1 in 4,300,000 donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by 1.5, the risk would be 1 in 6,225,000 donations for s1, and 1 in 3,000,000 for s2. summary/conclusions: for both scenarios, the hiv rr remains very low. for s1 (4-month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s2 (no more than one sexual partner, 4 months), the risk is 1.5 higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s1, since the risk could be 2 times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. 3d-s13-03 background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering 8 of 9 provinces in south africa. controls were frequency matched at a 3:1 ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < 1.5. eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the 6 months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november 2014 to january 2018, we enrolled 323 incident hiv cases and 877 controls; 202 (62.5%) cases and 544 (62%) controls were ≤ 29 years old. there were significantly more female cases 230 (71.2%) than female controls 439 (50.1%) (p < 0.0001). significant hiv risk factors (all p < 0.0001) reported within the 6-months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from 2000 to 2016, among male blood donors (mbds) found hiv-1 positive at blood donation screening, 31% did not disclose any risk factor for hiv infection during post-donation interviews, while 38% reported having sex with men (msm), and 24% and 7 % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv-1 infection in mbds, we performed an hiv-1 genetic network analysis, including hiv-1 positive mbds and patients included in the french primary hiv infection anrs co6 primo cohort (pc). methods: 284 mbds, who donated blood between 2000 and 2016, and 1340 pcs, included between 1998 and 2014, were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: <6 months). a partial transmission network was computed based on tamura-nei 93 nucleotidic distance (threshold for hiv-1 s/t b = 1.5%; for non-b s/ t = 0.8%) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv-1 strains from 81 mbds and 126 pcs were linked into 54 clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger (30 vs. 36 year-old; p < 0.01) and were more likely to have a recent infection (48% vs. 34%; p = 0.03). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < 0.001) and to have the same risk factor for hiv infection (p < 0.001) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: 38% vs. 49%, hts: 24% vs. 21%, other: 7% vs. 6% and unknown: 31 vs. 24%. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to 18% (31/176) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv-1 associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in 2002. monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in 2013 and 2015. aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every 2-3 years. results: in the uk 2002 -2017, 254 htlv-infected donors were identified. prevalence among new donors was steady around 5/100 000 donations. prevalence among repeat donors peaked in 2002 (2.7/100 000 donations), with most in previously untested. from 2004 to 2016, prevalence of 0.07 per 100,000 donations (average of one positive/year) was recorded. in 2017, prevalence among new donors increased to 9.9/100,000 donations (17 positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women (182/254, 72%), uk-born (125/254;49%) and htlv-1 infections (228/254;90%). mean age was 43 years. almost all positive donations were from previously untested donors (240/254), with seroconversion within a year of previous donation confirmed for only 5 of the 14 previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of 114 htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over 800-person years follow-up, none had developed atll or ham. summary/conclusions: over 16 years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in 2017 nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the 60°and 70°n latitude. the length of the country is 1150 km and width 550 km. by surface area it is the fifth largest country in eu. the population of the country is 5.5 million resulting in the lowest population density in eu (15.8 inhabitants/km3). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as 10-20% of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since 1948. frc bs collects annually about 200 000 whole blood units of which 50 % are collected in 10 fixed sites and 50 % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by 8 am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the 1930s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in 2007 and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice (4 courses) -pbm: general (7 courses) -pbm: medical (6 courses) -pbm: acute care and surgical (4 courses) -pbm: obstetrics and maternity (3 courses) -pbm: neonates and paediatrics (7 courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from 1 july 2007 to 31 january 2019: -489,600 people registered as learners -1,072,299 courses were completed -these learners came from 182 countries, with 11,141 (2.3%) of them from outside of australia. analysis by profession shows that: -82.4% are nurses and/or midwives -11.2% are medical -6.4% are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = 2,885) from 1 april 2015 to 31 january 2018 shows that these courses have a positive impact, with 88.7% of respondents stating they gained additional knowledge, 65.2% able to make changes to clinical practice, and 88.2% reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with 93.5% gaining knowledge, 76.4% able to change their clinical practice and 91.9% believing this will improve patient outcomes. analysis of red cell usage in australia shows that since 2012 there has been a 21.1% reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in 2012 and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. 3d-s14-03 prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a 12-month period (january 2018-december 2018). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > 50,000/ll, ≥2 doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy1) were compared to subsequent years (pgy > 1). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $500 for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < 0.05 considered statistically significant. results: there were 1446 platelet doses requiring approval with 670 (46%) routed to the pgy1 group and 776 (54%) to the pgy > 1 group. there were 847 (59%) ordered doses that were in compliance with hospital transfusion policy and 599 (41%) that were not in compliance with hospital policy. of the 847 appropriately ordered doses, the pgy1 group declined release of 7 necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > 1 group. when paged by the blood bank, pgy1 physicians approved product release not in compliance with policy for 191/670 (29%) doses while pgy > 1 physicians approved not indicated products for 79/776 (10%) of doses (p < 0.01). products not indicated by hospital policy were held from release by pgy1 physicians for 113/670 (17%) doses and 216/776 (28%) doses by pgy > 1 physicians (p < 0.01). the ordered doses not in compliance with hospital policy had an estimated cost of $299,500. of this cost, there was a calculated $164,500 savings of products not released due to prospective auditing. there was an additional potential savings of $135,000 for products not indicated but released ($95,500 from the pgy1 and $39,500 from the pgy > 1 group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > 1 group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy1 group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy1 physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. 3d-s14-04 what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in 2015 we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in 2017 and 2018 were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of 188 adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty (10.6 %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected 39 (20.7 %) events and formal internal or external reviews detected one event. seven (3.7 %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of 16 wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of 22 events related to blood ordering, two of seven errors in testing, six of 17 events where incorrect blood had been transfused, and eight of 64 events related to donor selection. in 80 reports human error was listed as the cause of the event and 27 of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average 1.2 g/l (0.9%) higher than from the venous blood samples. the range of the difference was -21 -+22 g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in 1.4 % (12/845) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + 9 g/l. 92 % of the hemoglobin results from the finger prick were in the range ae10 g/l compared to the venous hemoglobin results. 63 % of the results from the finger prick were between ae5 g/l (the precision of the device) compared to venous hemoglobin results. in 13 cases the difference between finger prick and venous measurements was outside 2 standard deviations from the mean i.e. 2.5 % from the bottom (n = 9) or top (n = 4) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about 8% (men) to 81% (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of 56 days, a donor needs to absorb 4.5 mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of 3-4 mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every 5th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < 15 lg/ l, or ≥ 15 and ≤ 30 lg/l (for 12 and 6 months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from 2020 onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. 3d-s15-02 superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (<7.8 mmol/l [<12.57 g/dl] for women and < 8.4 mmol/l [<13.54 g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since 2010 with more than 110,000 participants. extensive genotyping has been performed on approximately 72,000 dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than 150,000 icelandic individuals (an independent discovery cohort), we constructed 9 different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of 2017 was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at 5, 10, 25, 40, 60, 75, 90 , and 95 th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was 6.9 donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the 40-60 prs percentile group, donors below the 5 th percentile had lower (-0.23 hb mmol/l (95% ci: -0.25; -0.21)) and donors above the 95 th percentile higher (+0.19 hb mmol/l (95% ci: 0.18; 0.21) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the 40-60 prs percentile stratum as reference, donors below the 5 th percentile and donors above the 95 th percentile had odds ratios of deferral of or = 2.58 (95% ci: 2.27; 2.93) and or = 0.46 (95% ci: 0.39; 0.54), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, 2012) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles (58 features, 3,162 donations from 1,025 repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < 20 ng/ml for women and < 30 ng/ml for men), or absent-iron donation (ferritin < 12 ng/ml for men and women). we trained multiple machine learning models on 2,543 of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of 620 donations, which were not used to train or select the model. we then used our model to generate risk estimates for these 620 test donors as a function of days since their last donation, which varied from 56 days to 250 days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes 250 days after the last donation was lower than the risk 56 days after the last donation for most donors (risk of hemoglobin deferral decreased for 84% of donors; risk for low-iron donation decreased for 61%; and risk for absent-iron donation decreased for 94%). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, 2011) and from the danish blood donor study (rigas, transfusion, 2014) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for 15-24 month follow-up of donation frequency and iron status. a brief checklist of 8 food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of 2425 enrolled donors, 1406 were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was 7. 2, 13.1, and 22 .0 mg of heme iron weekly. these values are equivalent to approximately 3, 6, and 9 servings of beef per week, or alternately twice as many servings of chicken or pork. across 2236 follow-up visits with iron outcomes assayed, almost 33% of donor visits were associated with intermediate iron depletion (serum ferritin < 26 ng/ml) and 8.5% with complete depletion of iron stores, representing serum ferritin < 12 ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than 2fold higher risk for complete iron depletion during all follow-up visits (rr 2.40, 95% ci 1.51, 3.81, compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately 250 mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included 2,552 donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = 292). hb levels were measured in edta whole blood samples using a hematology analyzer (xt-2000, sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci8200, abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, 2,260 (1,186 female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) (95% confidence interval (95% ci)) in men and women respectively: 0.147 (0.069 to 0.225) and 0.094 (0.013 to 0.175) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (-0.015 (-0.026 to -0.004) and -0.018 (-0.032 to -0.005) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: 0.073 (0.046 to 0.107) and 0.065 (0.031 to 0.100) lg/l for heme and -0.003 (-0.008 to 0.000) and -0.007 (-0.013 to -0.002) for non-heme). more mvpa was negatively associated with hb levels in men only (-0.004 (-0.008 to -0.001)), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd20 moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. 4a-s16-02 thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple 1 , r aslam 2 , e speck 2 , j rebetz 1 and r kapur 1 1 lund university, lund, sweden 2 st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last 10 years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately 30% of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp4, amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood 2010) . methods: platelet glycoprotein (gp) iiia (cd61) knockout (ko) mice were immunized with cd61 + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra (1, 10 and 20 ug/kg) had significantly higher platelet counts by 72 h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after 2 weeks and were fully rescued to control levels after 3 weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: 112 itp sera were investigated in this study. 35 (31%) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): 3.23, range: 1.76-13.61, p = 0.0001). in addition, 23 (21%) sera caused higher ecl binding to test plts (ecl-fi: 2.31, range: 1.54-5.7, p = 0.0001). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between 2014 and 2018. the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from 64 patients (52% women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was 63 years (range 22-89 years). the mean hg level at diagnosis was 68.60 g/l. dat results were positive mostly with igg+c3d (59%) or igg (31%). most patients had warm aiha (70%). other types of aiha diagnosed were mixed aiha (15%), cad (11%), pch (1.5%) and dat negative aiha (1.5%). in 6 cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as 1st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of 80% of patients were transfused at a mean hemoglobin level of 67.88 g/l. during this period we detected 136 dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november 2017 to march 2018. completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented 42% (58/137) of english acute trusts. median number of adults with aiha diagnosed annually was 4-6. in the preceding 5 years, 31% (18/58) recalled at least one patient who had died due to aiha. although 7% (4/57) undertook a bone marrow biopsy in all patients, 93% required additional features, mainly: neoplasia, age over 60 or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, 59% (34/58) would not organise confirmatory tests, either because it was considered unnecessary (29/34), or because clinicians were unsure how to access tests (5/34). when determining aiha subtype, 29% (17/58) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with 12 considering this unnecessary and 5 unsure how to access tests. in 4 clinical scenarios of patients with aiha and dat positive to c3d ae igg ae cold associated symptoms, up to 87% (47/54) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone 1 mg/kg given before judging the patient refractory and reducing the dose was 3.5 weeks (sd 1.70, range 1-19 weeks). second line treatment of choice was rituximab for 82% (45/55) of respondents and splenectomy for 5%. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for 88% (49/56) but single agent steroid for 9%. we also explored the potential for future audit and research. 64% (37/58) of respondents were able to identify patients with aiha who previously required transfusion. 96% (55/57) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c3d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although 44% (25/57) would continue prednisolone at 1 mg/kg beyond the recommended 21 days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. 4a-s17-02 low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < 8.0 g/ dl, 5 mmol/l) vs. high-trigger (hemoglobin < 9.7 g/dl, 6 mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed (1) severe adverse transfusion reaction, (2) acute myocardial infarction, (3) stroke, (4) new-onset renal replacement therapy, (5) vascular reoperation, and (6) amputation of the lower limb. results: the primary outcome, mean hemoglobin within 15 days of surgery, was significantly lower in the low-trigger group: 9.46 g/dl vs. 10.33 g/dl in the hightrigger group (mean difference 0.87 g/dl; p = 0.022, longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november 1, 2017, standard double-unit rbc transfusion was indicated with a hb threshold ≤ 8.1 g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to 7.3 g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ 8.1 g/dl and ≤ 9.7 g/dl applied. we evaluated rbc blood product utilization over a 6 month period starting december 1, 2016 (liberal protocol) and december 1, 2017 (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ 7 days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, 137 patients were admitted who in total received 164 therapy cycles, including 56 acute myeloid leukemia (aml) induction cycles and 69 autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in 303/402 (75.4%) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from 29/112 (29.0%) to 81/111(73.0%) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units 4.0 (interquartile range (iqr) 2.0-8.0) during the liberal versus 2.5 (iqr 0.0-9.0) during the restrictive period (p = 0.36)). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: 0.25 (iqr 0.11-0.33) versus 0.15 (iqr 0.00-0.32) units per day during the liberal versus restrictive period, respectively (p = 0.06). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from 2.0 (iqr 0.0-2.0) to 0.0 (iqr 0.0-1.5) units (p = 0.06), corresponding to a reduction from 0.13 (iqr 0.00-0.21) to 0.0 (iqr 0.0-0.13) (p = 0.01) units per neutropenic day. moreover, 14/38 (36.8%) patients during the liberal versus 21/31 (67.7%) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. 4a-s17-04 assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain 1 , n marwaha 2 and s sachdev 2 1 transfusion medicine, aiims, new delhi 2 transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from 350 ml and 450 ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of 900 prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group 100 collections were done in double 350 ml, triple 450 ml and quadruple 450 ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) 9 volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = 0.007). hemoglobin concentration of prbc ranged from 14.2-29.6 g/dl; mean hb was 21.02 ae 2.90 g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = 0.0001) and rtvd (p = 0.008). the hb content of prbc units prepared from 450 ml collection ranged from 35.19-87.36 g and from 350 ml collection ranged from 30.77-65.78 g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = 0.730, p = 0.0001).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from 155 ml to 370 ml (including both 350 and 450 ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. 4a-s17-05 nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a 639 bedded tertiary care oncology centre with approximately 18,000 to 20,000 rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of 4413 rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < 0.05. results: total 4413 rbc transfusion events for 2012 patients were analyzed. there were 1877 (43%) events in 628 patients of medical oncology and 2536 (57%) in 1384 patients of surgical oncology. maximum transfusions were received by patients in age group of 41 to 60 years (47%). total 83% of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < 0.05. inappropriateness was more 53%(396/735) and significant in daycare setup (p < 0.05). anemia was the most common indication of rbc transfusion observed in 90% of events (3971/4413). total 62% rbc transfusions were given as planned and 38% as urgent transfusions. most common adverse transfusion event observed was allergic reaction in 0.3% of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. 4a-s18-01 paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed 0.38% of donors were positive for babesia dna or antibodies (moritz, nejm, 2016) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â 6800/8800 systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the 4 babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october 2017 under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of 6 (mp6), plus 3 idt replicates with cobas â babesia. reactive index donations were also tested with 2 validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, 256,802 valid donations have been screened with cobas â babesia, and 15 (0.006%) were reactive. 13 of 15 (87%) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. 1 of 13 (8%) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and 1 (8%) was collected in a state where babesia is not considered endemic (iowa). 11 of 13 (85%) confirmed positive donations were collected in states with high endemicity. 8 of 13 (62%) confirmed babesia-positive donations were detected in late fall or winter. all 13 (100%) confirmed babesia-positive donations were reactive in mp6. serology results are available for 12 of 13 confirmed-positive donations: at index, 6 of 12 confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; 3 were positive for both igg and igm. 3 of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of 99.999% (256,787/256,789; 95% exact ci: 99.997%>100%). summary/conclusions: the cobas â babesia test successfully identified 13 babesiapositive donations, including 3 confirmed-positive donations with no igm or igg reactivity. 2 donations were collected in states considered low-or non-endemic for babesia. 8 confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a 2013 study of~14,000 donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november 2018, 50,752 blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of 14,758 tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the 50,752 donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of 14,758 samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october 1, all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only 1 tma-confirmed-positive donation of 50,752 tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. 4a-s18-04 background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september 2015 and july 2017, 520 blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the 520 blood donors (mean age 38.55 ae 11.14; range 18-65 years old), 38.1% were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence (55.1%) followed by the north (37.2%) and the south (25.3%). blood donors living in rural areas had a significantly higher seroprevalence (p = 0.001) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence (60.2%) found in the age group of 46-55 years old (multiple logistic regression [mlr]: or = 7.68; ci: 4.08-14.46; p < 0.001), and decreased with educational level (p < 0.001). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = 0.02). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = 2.72; ci: 1.27-5.86; p = 0.001). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. 4a-s18-05 who is syphilis testing excluding? c reynolds 1 , c pearson 2 , k davison 2 and s brailsford 1 1 nhsbt/phe epidemiology unit, nhs blood and transplant 2 nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the 1940s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from 12 to 3 months in november 2017 and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between 2009 and 2018. methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within 12 months for regular donors. results: between 2009 and 2018 there were 153 recent syphilis cases, 121 hiv and 32 acute hbv infections identified by donation screening. recent syphilis rates per 100,000 donations increased from 0.66 to 1.64 whereas hiv decreased from 1.04 to 0.19 with less than 5 positive donations in 2018. acute hbv rates rose slightly from 0.28 to 0.38 in 2018. males outweighed females accounting for 71.9%, 63.6% and 59.4% of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below 25 years old. of the male donors with recent syphilis, 58.2% reported sex between men and women (sbmw), 19.1% sex between men (sbm) and 22.7% did not report a risk. this contrasted with hiv where 45.5% of male donors reported sbm, just 2.6% not reporting a risk. overall 19, 32 and 3 males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in 2018, 26 donors with recent syphilis aged 23-65 years (median 36 years) were excluded from the donor pool, including 3 non-compliant to the sbm deferral. there were fewer than 5 hiv cases identified in 2018, all over 40 years old, all compliant, reporting sbmw. of the 6 hbv acute cases in 2018, 5 were male, all but one in the 45 and over age-group. summary/conclusions: over the 10 year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a 3 month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated 113 million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last 10 years. plasma donors are bled up to 104 times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in 2014, the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into 6 categories (16 subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in 2018, with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between 1/1/2017 to 9/30/2018. severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade 2 or higher were individually evaluated by a physician for grading accuracy. results: in 1,511,758 needle-in collections, 31,320 dae were graded for severity. the majority (16,143, 51.5%) were vasovagal reactions (vvr), followed by 8,570 apheresis-related (27.4%), 6,572 needle-related (21.0%) and 35 allergic (0.1%) events. the majority of dae were grade 1 accounting for 98.6% of all dae, followed by grade 2 (1.2%), and grade 3 (0.1%). there were 2 grade 4 and no grade 5 dae. among the vvr, 98.1%, 1.6%, 0.2% and 0.01% were grade 1, 2, 3, and 4 respectively. grade 3 vvrs included 14 concussions, 11 fractures, 1 dental injury, and 2 pre-faint and 8 fainting events requiring hospitalization for work-up. two grade 4 vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only 6 and 5 grade 2 reactions respectively, and no grade 3 or 4 events. needle-related dae included 98.3% grade 1, 1.6% grade 2, 0.1% grade 3, and no grade 4 events. of the six grade 3 needle-related dae, 4 were nerve irritations lasting > 6 months, and 2 were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since 2006. aims: we analysed the data collected in 2006-2016 in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from 2008 the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in 2015. reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per 1000 donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was 7, iqr 2-8). the total number of country years (cy) was 138, covering 155 million donations. the overall complication rate was 6.3/1000 donations and the median country rate was 3.2 complications/1000 donations (iqr 1.1-10.1). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall 4.6/ 1000 donations, median country rate 3.1/1000 donations (iqr 0.6-7.7). rare and apheresis-related types of complications such as generalized allergic reaction (0.10 per 100,000, 40 cy), and major blood vessel injury (category available since 2015; overall 0.12 per 100,000, 6 cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total 89 cy, 101.6 million wbd and 26.3 million aphereses, total 128 million donations). for these hvs the median rate of vasovagal reactions was 3.4/1000 wbd (iqr 1.0-9.1) and 1.5/1000 apheresis procedures (1.0-4.2) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was 0.39/1000 wbd (iqr 0.31-1.2) vs 4.2/1000 aphereses (0.69-5.6); rates of arm pain and/or nerve injury (not separated in 2006-2013) also tended to be higher: median 0.09/1000 with wbd, iqr 0.03-0.34, vs 0.39/1000 with apheresis, 0.05-0.57. summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. 52000 registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in 8000 control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each 4 th (women) or 8 th (men) donation, and with yearly testing of young adult blood donors below 25 years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that 20% of female and 9% of male applicants cannot be registered because of low hb (125 and 135 mg/l respectively). adding ferritin testing, a preferred cut-off level of 30 lg/ml (male reference level), would result in additionally 24% female and 1.3% male applicant donor loss. as this would threat the blood demand, cut-off was set to 15 lg/ml for women, above the female reference 10 lg/ml, with an acceptable 6% loss of female applicant donors. for registered blood donors, 4000 mg of extra iron tablets were offered at low ferritin (10-29 lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above 30 lg/ml. donors with ferritin below 10 lg/ml (in 0.6% applicant donors, 0.8% registered donors) or above 600 lg/ml (0.5% applicant donors, 0.1% registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from 3 to 5 months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from 50% to 70% approved hb at return. the team of nurses processing ferritin and blood count results (1½ nurse fulltime weekdays) reacted to approximately 40 donor results daily, representing 20% of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november 2017 a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at 30 and 15 ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors 46% (44% of 16,433 male donors, and 48% of 13,525 female donors) have ferritin levels below 30 ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of 43 ng/ml. in contrast, we found that only 27% of new female donors (n = 13,283) and 1.7% of new male donors (n = 6,334) have a ferritin levels below 30 ng/ml. the average ferritin level in new donors was 148 ng/ml for males and 60 ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between 1.5 and 2.0 was observed in female donors and between 3.2 and 4.4 in male donors. both ratios increased with donor age. at the end of december 2018 2884 donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by 13 ng/ml per year whereas average ferritin levels in male donors increased by 34 ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from 1.5 to 2.0 in female and from 3.2 to 4.4 in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~7000 different rare diseases and the genes for half have been identified. approximately 3.5 million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts 2.2 years. the main aims of the 100,000 genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in 2013 and dna samples from 100,000 nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the 100,000 genomes project comprising phenotype and genotype data from 13,037 individuals recruited at 83 hospitals using approved eligibility criteria for 15 rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over 165 million unique variants in the 10,258 genetically independent samples, with 47% of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in 2,000 unique diagnostic-grade genes for the 15 domains. over 1,000 reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from 0.5% to 55%, while the proportion of novel causal variants ranged between 25% and 73%. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying >30 novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the 100,000 genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped 7588 donors from england and the netherlands with this new array and observed a 99.92% concordance when comparing 92,387 blood centredetermined antigen typing results with genotype-determined ones. for the 48 red cell and hpa antigens that were available for 7,473 donors, the array typing provided a 3.6-fold increase in typing results per donor (13.2 vs 47.9) and 38 rare donors were identified. using the genotyping data we identified 2.6 times more compatible units among this cohort of donors when blood demand was modelled using referral data from 3,146 english patients with more than three red cell alloantibodies. in conclusion the 100,000 genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another 500,000 dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early 1990s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found 4 amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons 266 and 268 are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than 250 abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a1,3-gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. 1: our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); 2: identification of novel abo alleles by others; 3: more snp data from genome sequences and potential problems for abo genotyping; 4: findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; 5: a1,3-gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and 6: the potential causes of generation of abo polymorphism and of species variations of the gbgt1 gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. 4c-s20-01 a large deletion spanning xg xg and gyg2 gyg2 constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd99. the cd99 gene resides within pseudoautosomal region 1 on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first 3 exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly 11% of women and 34% of men are xg(aà). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs311103c variant disrupts a gata motif between xg and cd99. this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from 13 archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the 1000 genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg2. database searches indicated that the most likely deletion was the infrequent genomic structural variant esv2662319 reported in the 1000 genomes project. further analyses with a short (714 bp) and a long (3555 bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately 114 kb and corresponded well with esv2662319. this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the 13 cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific 714-bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr6b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the 1000 genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg2 genes accounts for the xg null phenotype underlying the majority (10 of 11) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c.143c>t, p.thr48met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c.230c>t (p.thr77met) causing partial exon skipping and designated gybp*03n.01 (gypb*ny) and c.270 + 5g>t, an intron change causing complete skipping of exon 5, designated gypb*03n.03 (gypb*p2). aims: samples from three individuals, a previously transfused african american sickle cell patient (p1), a blood donor of unknown ethnicity (p2), and an african american patient (p3) (lapadat r. 2014 aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p2 were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p1 and p2. p1 was also tested by gypb*s/s as-pcr, exon 5 pcr-rflp for c.270 + 5g>t and as-pcr for c.230c>t. p3 was tested for gypb*s/s and c.230 c>t and c.270 + 5g>t changes by a real-time pcr-fluorogenic 5' nuclease taqman chemistry. for all, gypb exons 1-6 were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c.143t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c.230c>t and c.270 + 5g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sàu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c.143c/t, c.230c/t and c.270 + 5g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c.59t/g and c.60a/g (p.20leu/trp), c.67a/t (p.23thr/ser), c.71a/g and c.72g/t (p.24glu/gly), c.143c/t (s/s), c.208g/t (p.70val/leu), c.230c/t (p.77thr/met), and c.270 + 5g/t. all samples were also c.251g/g (p.84ser) and heterozygous for several previously recognized silent changes in exon 2, c.87t/c, c.96t/c and c.102a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sàs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c.230t or c.270 + 5t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c.270 + 5t change is on a gypb*03n.02 [gyp*he(ny)] background. c.230c>t (rs79492560) and c.270 + 5g>t (rs139511876) have a frequency of 0.0053 respectively 0.032 in the african population (exac). although we identified 3 samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of 25 antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin-5a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of 15 exons located on chromosome 19q13.2. a number of rare lutheran phenotypes have been previously recorded in israel, including lu:-6,9 observed among iranian jews, lu:-20 in one thalassemia patient and one case of lu:-21. in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with 2-aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:-5, lu:-6, lu:-8, lu:-13, lu:-21, lu:-22, and lu:-23 cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: -1,2,8,12,13,-19,21 . bcam sequence analysis confirmed the patient to be lu*02, lu*18 and revealed a novel homozygous mutation c.1351a>c in exon 11, encoding p.lys451gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c.1351a>c (p.lys451gln) in exon 11 of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises 25 antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome 19. currently, isbt lists 20 high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: -1, 2, 3, 5, 6, 8, 13, 20, 21 . bcam sequencing revealed a novel homozygous mutation c.121g>a in exon 2, encoding p.val41met in the lu glycoprotein. the c.121g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of 3.98 9 10 -6 and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val41met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema7a exon-specific primers and sequenced. results: the proband was a 32-year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at 27 weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted 1 + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge2 was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge2 and anti-ch1 could be ruled out. the serum was nonreactive with two jmh:-1 and positive with two jmh:-3 samples. the patient was found to be jmh1 positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg4. overall, these results were consistent with a probable jmh variant and prompted us to perform sema7a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon 7, c.709g>a (p.asp237asn, rs140707085, maf < 0.01, sift score = 1); a common synonymous change in exon 12, c.1545a>g (p.gln515gln, rs741761, maf = 0.5); a rare non-synonymous change in exon 14, c.1865g>a (p.arg622his, rs140128092, maf < 0.01, sift score = 0.36). the analysis of surface accessibility of asp 237 and arg 622 using the 3d structure of sema7a (rcsb pdb-3nvq https://www.rcsb.org/structure/3nvq) showed that only arg 622 was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg4 antibody showing a similar pattern of reactivity, and with the same three changes in sema7a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg622his substitution in sema7a. we propose to provisionally assign the name jmh7 for this antigen. interestingly, our two unrelated jmh:-7 individuals were from north african ancestry. background: the abo system was discovered almost 120 years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca3(fuca2)gal and gala3(fuca2)gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, 1985) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca3(fuca2)galb3galnaca3(fuca2)galb4glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a3-specific gh110 family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, 40% of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px2 but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a 1 b individual with the p1 k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p 1 k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen 500-litre reference preparation and confirmatory preparation from 24 freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bà human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a 1 b p 1 k plasma contained anti-px2 and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px2 was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex-4hexnac-hex-4hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type 2 hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala3(fuca2)galb4glcnacb3galb4glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 1990's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p2y12 antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. 4c-s21-02 university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between 0.05 -15% of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day 100 (33%) compared with those who had normal elpi at baseline (7%) (p = 0.00034). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < 50 ml plasma). until, in 2011, we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in 2017, we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under 50 years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (>24 days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (<10 days storage) red cells from ever-pregnant donors (3-year cumulative incidence of death 15.4% versus 4.8%). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. 7.2% versus 4.7%). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy (6-9 months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s-303) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (>5.3 log reduction) and plasmodium falciparum (>7.5 log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase 1 clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase 1 clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase 1 clinical trial in africa, 20 clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade 2) within the first 24 hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within 59 (ae3) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment 24 hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase 3 clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase 1 clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's (2014) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every 4 weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from 2 months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions 1 and 2, donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition 3) or the revised email (condition 4) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every 4 weeks, and a prompt to forward-book appointments. results: the final sample (n = 6788) comprised 3859 women (57%) and 2929 men (43%) aged 18-70 (mean = 32). after two months 37.2% of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition 4 (revised email + phone call), or = 1.305, ci = 1.128-1.510, and bau. donors assigned to the two telephone conditions (condition 3 and 4) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c282y mutation), but also carriers of other hfe mutations (p.c282y/p.h63d or homozygous h63d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< 200 ng/ml in females, < 300 ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis (2rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ 18-60 years, total blood volume ≥ 5 l, bmi < 35 kg/m 2 , hb ≥ 140 g/l, elevated sf levels and no end organ damage due to iron overload. 2rbcaph (360 ml rbc) were scheduled every 14 days and wbph (450 ml) every 7 days until sf was < 100 ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up 8 weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: 30 subjects (5 females; mean age 47 years) were randomized to wbph (n = 16; 1 female) or 2rbcaph (n = 14; 4 females). hfe mutations were p.c282/ p.c282y in 17 subjects, p.c282y/p.h63d in 9, and p.h63d/p.h63d in 4. at baseline, mean hb was 149 g/l (sd 7.8) and median sf was 504 ng/ml (iqr 406-620 ng/ml). 222 procedures (wbph n = 146, 2rbcaph n = 76) were completed; 9 were interrupted (local hematoma, insufficient flow); 35 (16 wbph, 19 2rbcaph) were postponed because of low hb and 15 for non medical reasons. there were 2 drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < 130 g/l in males, <120 g/l in females) occurred after 15 visits in 8 wbph subjects and after 5 visits in 3 2rbcaph subjects. fatigue was reported after 37 phlebotomies and 31 aphereses. only 5 participants (17%) completed the study per protocol. 136 blood components (94 rbc concentrates and 42 plasma units) for transfusion were obtained. overall, a median of 7.5 wbph (iqr 6.2-9.8) was needed to reach sf < 100 ng/ml, corresponding to 1.8 times of 2rbcaph (median 4.0, iqr 3.0-5.8) (p = 0.0001). analyzing separately p.c282/p.c282y and p.c282y/p.h63d carriers, the relation wbph to 2rbcaph was 1.6 and 1.8, respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = 0.0001 and 0.007, respectively). summary/conclusions: 2rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately 200 ml red blood cells (rbc) and 200-240 mg iron are lost with wb donation. double unit rbc (2rbc) collections of 360 ml (ca. 40 ml less than the rbc amount of two wb donations) lead to a loss of about 400 mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every 6 months for 2rbc and once every 3 months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and 2rbc at our institution. methods: we included 294 wb and 151 2rbc donors (n = 445) who donated with the maximal allowed donation frequency over 48 months between 2008 and 2013, yielding 4,704 wb and 1,208 2rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were 135 g/l for wb and 140 g/l for 2rbc donation. with 2rbc apheresis 360 ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was 53 (wb) and 48 years (2rbc), respectively. at the first donation, mean hb was 153 g/l (sd 13) in wb and 159 g/l (sd 8) in 2rbc donors; mean sf was 44 (sd 52) and 73 lg/l (sd 56), respectively. on average, hb and sf were higher in 2rbc donors (5.1 g/l and 26 lg/l, respectively; p < 0.001). there were 137 subjects with sf < 30 lg/l in wb and 19 in 2rbc group, and 85 with sf < 50 lg/l (but > 30 lg/l) and 40, respectively. in 2rbc donors, between the first and the last donation, mean hb declined from 159 g/l to 157 g/l (p < 0.05) and mean sf from 73 lg/l to 66 lg/l (ns). in wb donors, mean hb dropped from 153 g/l to 152 g/l (p < 0.05) and sf from 44 lg/l to 35 lg/l (p < 0.001). similar results were found when adjusting for age and season. hb values dropped from baseline until the 11 th donation for wb donors and until the 4 th donation for 2rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the 4 th donation in both wb and 2rbc donors (37 lg/l and 60 lg/l) and increased thereafter in 2rbc donors. in wb donors, sf followed a parabolic trend that peaked at the 10 th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and 2rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15% or 30% mfg (gelafundin 4%, b. braun melsungen ag) or hes (hespan 6%/450/0.7, b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd11b, cd62l and cd66b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < 0.001 respectively), whereas only the higher concentrations (15% and 30%) of mfg showed lower relative migration distances (p < 0.001 respectively). track straightness was reduced with both sedimentation agents at 15% and 30% to the same extent (p < 0.001 respectively). hes resulted in lower cd11b expression (p = 0.028) and higher cd62l expression (p = 0.007) compared to the controls, whereas the differences for cd66b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, 2001) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to 12 plateletpheresis donations per year, with a minimum 4 weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between 01/01/2014 and 08/31/2018 from the french civilian blood donors' base and then selected a cohort of donors with at least 24 donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. 8 measures for each donor. results: the cohort includes 2,186 donors (384 women and 1,802 men). mean platelet counts fluctuate between 276.4 and 278.6 platelets/ml. analysis of variance does not show any statistically significant difference (f = 0.462), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the 24 donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than 20,000 described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the 1950s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb1, -dqb1), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb3, -drb4, -drb5, -dqa1, -dpa1). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa1a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa1a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa 1a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa-1a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n162a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd61 (c17) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa 1a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n162a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, 166 plasma samples with anti-hpa1a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, 2016) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n162a-fccriiia correlated with the level of fucosylation of the hpa1a antibodies, as measured by mass-spectrometry (r = à0.524; p < 0.0001). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n162a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa 1a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa1a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa-1a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa-1a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa-1a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa-1a screening program in poland. aims: our aim was to assess the frequency anti-hpa-1a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa-1a screening of 24 244 pregnant women in 8-40 gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa-1a negative/hpa-1b/1b women were tested for hla drb3*01:01 and for anti-hpa-1a antibodies by maipa (followed up at week 17-20, 28, 32, 38-40 and 6 weeks after delivery). if anti-hpa-1a were detected, quantitative maipa was performed. all hpa-1a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa-1a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: 554 hpa-1a negative women were identified (2.3%). anti-hpa-1a was antibodies were detected by maipa in 44 women (two delivered tweens). in addition, anti-hpa-1a antibodies were later detected by paklx in further 3 women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was 47 (8.5%). they delivered 49 babies; 35 were boys. three women were treated by ivig: two by 4 and 8 injections since 33 th and 26 th gw respectively. the anti-hpa-1a concentration in the 1 st one was 0.4; 0.1; 5.88 iu/ml in 17, 28, 32 gw respectively and in the 2 nd <0.05 iu/ml in all examined samples. the decision on treatment was based on the low plt count~50 g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the 3 rd treated woman entered the program in 35 gw (anti-hpa-1a concentration was high 22.64 iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in 5/49 newborns: in 3 with anti-hpa-1a detected in paklx only and in 2 with antibody concentration in maipa -1 st : 6.86/12.91/ 23.36 at 28/32/38 th gw respectively; 2 nd : 8.85/17.58 at 32/38 th gw respectively. ich was observed in all of them; plt count was < 50x10 9 in four, 56 9 10 9 / in one. summary/conclusions: 1/ the severe thrombocytopenia due to anti-hpa-1a alloimmunisation in our prospective study occurred in 2/10 000 pregnancies 2/ the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative 3/ the hpa-1a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in 2014 that the diagnosis of alloimmunization was carried in only 33% of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by 3 different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all 2017 and 2018 data were collected using the laboratory information management system. results: 544 patient files were analyzed. no incompatibility is demonstrated in hpa-1 to -9, -11 and -15 systems in 19.3% (n = 105). hpa-1 and / or 3 and / or 5 incompatibilities were found in 271 cases (49.8%), hpa-2 and / or 15 in 87 cases (16%). platelet alloimmunization was globally confirmed in only 10.6% of the cases. 58 platelet alloabs were identified regardless of clinical manifestations: 24 anti-hpa-1a (41.4%), 20 anti-hpa-5b (34.5%), 8 anti-cd109 (13.8%), 2 anti-hpa-5a and anti-hpa-15b (3.4% respectively) and 1 anti-hpa-1b and anti-cd36 (1.7% respectively). 40 alloabs were found in the context of neonatal thrombocytopenia, 6 in ich and 10 in fdiu, and 1 in a follow-up of pregnancy. even if no anti-hpa-3 alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = 55, n = 32 and n = 17 on 114 cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa-3 and hpa-5 incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa-3 and hpa-15 systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa-1a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the 2% hpa-1a negative women. at present, hpa-1a typing is mostly done by genotyping. for costeffective implementation of anti-hpa-1a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa-1a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa-1a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg1 anti-hpa-1a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, 20 ll of the uppermost plasma of 3 -6 days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < 0.500 in the hpa-1a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, 506 consecutive samples were tested. in phase ii, the hpa-1a elisa was performed in another 62,171 consecutive samples, with confirmatory q-pcr in 1,825. the two phases combined, samples from in total 1,585 hpa-1a negative and 823 hpa-1a positive pregnant women were genotyped. the assay reached a 100% sensitivity with a cut-off od between 0.075 and 0.200, leading to a specificity of 99.6%. summary/conclusions: a quick, low-cost and reliable assay for hpa-1a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa1a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of 1047 pregnant women in 2002 found that 30.4% of irish women were cmv seropositive in comparison to 56% from western europe and 92% eastern europe and 97% from africa. an internal study carried out by the irish blood transfusion service (ibts) in 2010 indicated the rate of cmv seropositivity in irish blood donors was 21.97%. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in 2018 the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing 48 confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = 54). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing 6127 donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be 100%. the seroconversion sensitivity reported 42 out of 54 samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be 1.12 iu/ml. the validation reported 65 discordant results from 6127 donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. 60 discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified 27 discordant results as positive, 12 as negative and 21 as indeterminate. 5 discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be 99.80%. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity (99.80%) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of 6127 donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: 1) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, 2) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, 2014) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: 1. to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa 2. to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: 900 negative new donor samples and 100 confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was 0.56%, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than 750.000 blood and plasma donations are tested, on average 3.000 samples per day. for more than 10 years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july 2018, serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from 6 months before and 5 months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june 2018 (prism) and august to december 2018 (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: 1) hbsag p 0.01 % (42/390.736) versus a 0.03 % (87/322.394); 2) hiv p 0.07 % (262/390.735) versus a 0.06 % (201/322.470); 3) anti-hcv p 0.11 % (427/390.737) versus a 0.03 % (109/322.476). the rate of anti-hbc reactive samples was not significantly different between prism (0.39 %) and alinity s (0.42%). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s (0.39 %) and prism assays (0.34 %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to 0.16 %, which was significantly lower than for the three prism assays (0.33%). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by 0.17 % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on 2,748 blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag 6.0, enzygnost anti-hcv 4.0, and enzygnost hiv integral 4 assays and on the pk7300 with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i1000 system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas 8800 system) were available. results: based on the results from testing 2,748 blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around 90% of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e 801 analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e 801 analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e 801 analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using 30 preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar (99.81-100.00% versus 99.79-99.98%; n ≥ 5195). in specificity analyses, there were 14 discrepant results for hiv testing, 27 for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay (83.33%; 95% ci 65. 28-94.36) was higher than the prism hiv o plus assay (76.67%; 95% ci 57.72-90.07), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv (85.19%; 95% ci 67.33-95.97), hbsag (70.00%; 95% ci 50.60-85.27), anti-hbc (100.00%; 95% ci 85.75-100.00), and syphilis (100.00%; 95% ci 88.43-100.00); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e801 (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june 2018, close to 10,000 aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = 10%) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of 7,389 plasma and 2,242 serum samples, 41 test results representing 37 individual donations were found rr on one or both instruments. two samples were confirmed positive (1 9 hbsag, 1 9 hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e801): hbs ag: 0.04/0.03 % in a total of 9590/9589 samples tested; hcv ab: 0.01/0.09 % in 9620/9585, p < 10%; hiv ag/ab: 0.03/0.09% in 9362/9329, p < 10%; syphilis ab: 0.1/0.07% in 2971/2987; hbc: 0/0% in 1531/1549. no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd34 + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type 1 (hiv-1), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd8 + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd8 + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered 50 years of soviet occupation. we held hands in a 600 km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june 1 st , 2004. the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in 1991, the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in 1992. a lot of advice came from finnish colleagues. in 1997, it was decided to move towards non-paid voluntary donations. the process took 6 years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than 15 years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in 2006-2015. it resulted in 51% of the donations being unpaid. the second program initiated in 2016 is still ongoing, aiming towards 100% non-remunerated donations by 2020. by the end of 2018, they had reached 99.2%. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: 13.8 ae 0.01 vs 15.5 ae 0.01 g/dl; p < 0.000). percentage of females with low hb < 12.5 g/dl were 7.1%, 4.9%, 5.2%, 6.7% and 4.2%, percentage of males with hb < 13.5 g/dl were 0.5%, 0.6%, 0.7%, 1.7% and 1.2% for the age groups 1-5 respectively. ferritin values were higher in males compared to females (median; 25 th -75 th %>tile: 51;31-79 vs 157;106-231 lg/l; p < 0.000) and in older age groups compared to younger age groups (median; range in age groups 1-5 in females: 40;3-702, 52;6-619, 60;6-480, 60;8-725, 98; 10-604 and in males: 99;8-661, 161;18-1080, 206;15-1090, 220;26-1430, 221;33-981 respectively) . percentage of females with ferritin ≤ 15 lg/l were 8.6%, 3.8%, 3.7%, 6.7% and 2.0%, while percentage of males with ferritin ≤ 15 lg/l were 0.2%, 0.0%, 0.1%, 0.0% and 0.0% for the age groups 1-5 respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: 7.2 ae 0.02 vs 6.6 ae 0.03; p < 0.000). percentage of females with wbc > 12x10 9 /l were 1.8%, 1.3%, 1.7%, 1.4% and 0.3%, while percentage of males with wbc > 12x10 9 /l were 1.4%, 0.9%, 0.5%, 1.2% and 0.9% for the age groups 1-5 respectively. none had wbc < 2x10 9 /l. platelet counts (plt) were higher in females compared to males (meanaesem: 257 ae 0.6 vs 222 ae 0.6; p < 0.000).percentage of females with plt < 150x10 9 /l were 1.0%, 1.5%, 2.2%, 2.4% and 1.0%, while percentage of males with plt < 150x10 9 /l were 2.6%, 3.6%, 2.7%, 2.4% and 1.6% for the age groups 1-5 respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with 75% younger than 30 years of age and the female/male ratio was 58/42. of the 16 583 first time donors with data on ferritin available, 14% had low ferritin (≤ 30 lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb (6%<12.5 g/dl) and low iron stores (23%≤30 lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period (2017-2018), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november 2017 was assessed by comparing results in two study years. results: a total of 491 pdi were recorded: 266 in 2017 (1/396 donations) and 225 in 2018 (1/452 donations) with the following distribution: nonsexual risk as tattoo and piercing (17.5%), surgical procedures (17.1%), travel history (16.1%), infections/ contact (15.3%), other medical reasons (13.4%), endoscopy/invasive diagnostic procedures (12.2%), malignancy (4.3%), autoimmune diseases (3.7%) and sexual risks (0.4%). majority (76.4%) were late pdi, revealed on the future donation(s): 58.7% on the first next donation, 28.9% on the second and 12.3% after more than 2 subsequent donations. the mean age of blood donors associated with pdi was 40 ae 12 years (median 39 years), while the mean age of all donors in 2017/2018 was 38 years (median 37 years). of all pdi, 81.1% were related to male donors (84% in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > 0.05). the median number of all donations preceding pdi was 6 for female donors and 19 for male donors. implementation of education leaflet for blood donors resulted in 15.4% reduction of pdi in 2018 compared with 2017 (p > 0.05). the effect is more pronounced (p < 0.05) when comparing second and first half of 2018 (-25.6%). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from 21.1% in 2017 to 26.7% in 2018, which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. 45 % of omani populations are àa/àa gene carriers, 44% àa/aa, and 11% of the population are aa/aa. around 10 % of omani nationals carry the gene for hbs, and 2 -3 % carry the gene for b-thalassaemia. recent statistics show that there are around 400 patients with thalassaemia major and 3000 with scd in oman. the other rbc abnormality that is common in oman is g6pd deficiency which is found in 28 % of males and 12 % of females. omanis are known to have the highest frequency of a thalassaemia and g6pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get 40 new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: 418 patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the 418 scd patients, 49% of the patients were male and 51% female, mean age was 17 years, in the range of 1-72 years. 38 % of the scd cases were positive for the alloantibodies, 72% were female and 28% were male, the age range was from 6-68 years. 78% of the positive were scd, 19% s trait and 3% were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. 51% of the patients were with single alloantibody, 30 % of them with two antibodies, 10 % with three antibodies, 7 % with four antibodies and 2 % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority 23%, followed by anti-d 13%, anti-k 17%, anti-c 14%, anti-c 8%, anti-jk a 5%, anti-jk b 5%, anti-fy a 5%, anti-e 3%, anti-s 3%, antis 1%, anti-kp a 0.7%, anti-fy b 0.3% and igm being 2%. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and 19% of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in 2018, a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ 1, 2-5, 6-9 and ≥10), previous pregnancy, number of transfused units (2, 3-5 and 6-10 and > 10), and years after last transfusion (<1, 1-2, 2-5, >5y) with presence of alloantibodies results: 226 patients (100 males and 126 females, median age 17 years, range 1.7-66) were included. the median number of transfusions was 3 (range 2-40). the median years after last transfusion was 2 (range 2 weeks-55.5 years). in 56 patients, anti-rbc antibodies were detected. in 14 of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least 34 patients (15.0%) alloimmunization was demonstrated or suspected; in 11 patients the alloantibodies were 'enzyme only'. besides, the 36 alloantibodies of known specificity (8 anti-d, 2 anti-d+c, 16 anti-e, 1 anti-c, 3 anti-e, 1 anti-k, 1 anti-s, 1 anti-le a , 1 anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients (3 had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated (2 dau-alleles and 1 diii type 4 or diva-2). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the 34 patients. immunobiology -red cell alloimmunity 65 fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion (12 patients had dark urine). adverse reactions were associated with the number of units received (or 1.71 (95% ci, 1.25-2.33; p = 0.001), but not with the presence of antibodies (p > 0.7) summary/conclusions: in at least 15% of multi-transfused patients with scd alloimmunization could be demonstrated, mainly (80%) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of 12 patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately 1% of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman /3 rd pregnancy, 1 st delivery, 2 abortions in 1 st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd1) by biorad and anti-d duo igm+igg, clone: th28 + ms26 by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons 5,7,10) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon 1 to 9 revealed presence of a nucleotide deletion in position c.147dela, which is specific for allele rhd*01el.04. this nucleotide change results in the amino acid change p.val50leufs*5 causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the 2000 level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd*01el.04 allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt 00023736 and rvo-vfn 64165. 5a-s29-05 ea scharberg 1 , s rothenberger 1 , a st€ urtzel 1 , n gillhuber 1 , s seyboth 1 , e richter 1 , g rink 2 and p bugert 2 1 institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden 2 institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di6) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc4a1*c.1643c>t (p.pro548leu; isbt allele name: di*02.06) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of 125,742 sequenced genomes (allele frequency: 0.000004). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di6 in 1,700 blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in 3,652 patients and in 964 blood donors was performed in the gel technique (biorad ahg id-cards) using a 3 cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di*02.06 allele in 1,700 blood donors revealed no single positive individual. within the first 2 weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell 84 patients with anti-rb a were found. it was 2.3% of 3,652 patients tested in 21 laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in 18 of 964 randomly tested blood donors (1.9%) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di*02.06 allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around 1% of patients and donors. 5a-s30-01 national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since 1935. real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in 2003 in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in 2002, who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in 2017, national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since 2016, reporters to shot have been asked to score(0-10) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between 2010-2017(inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between 2016-2017 was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between 2010-2017; majority (56/67, 83.6%) were red cell transfusions but aboi plasma (9/67) and platelet transfusions (2/67) were also seen. most errors occurred in the clinical area (45/67, 67%), and could have been detected at point of administration. in 21(31%) cases, the error could not have be detected at the point of administration with a primary laboratory error in 10/21(48%) incidents. reviewing data from hfit for cases in 2016-2017 (13 aboi cases), the total score for staff culpability was 100, compared to a total score of 99 for all the other three organisational and system factors. this disparity is most obvious for the 4 aboi red cell cases, all of which scored the maximum 10 for staff culpability, i.e. 40/40 compared to 8/120 as the combined total score given to the other factors. in the preceding years (2010 to 2015), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by 37 cases that included an outcome of the local case review and 14 (37.8%) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with 2/56(4%) that resulted in death, 14/ 56(25%) major morbidity and 40/56(71%) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between 2015-2018 to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = 27 were possibly related to treatment, n = 2 trars were probable, and n = 2 were definitely related to treatment; n = 37 trars were grade 1, n = 4 were grade 2, and none were grade 4. in recipients of conventional wb, there were n = 13 (1.12%) ars, n = 32 (2.75%) fnhtrs, n = 1 (0.09%) taco, n = 0 trali, and n = 16 (1.38%) unclassified transfusion reactions. of the confirmed trars, n = 55 were possibly related to treatment, n = 1 trar was probable, and n = 8 were definitely related to treatment; n = 54 trars were grade 1, n = 1 was grade 2 and n = 1 was grade 4. there were 21 mirasoltreated wb transfusions in pregnant women and 2 trars (9.5%), both grade 1 and probably related. there were 80 transfusions of mirasol-treated wb and 84 transfusions of conventional wb in patients < 18 years old resulting in n = 7 (8.33%) trars in recipients of mirasol-treated wb and n = 10 (11.90%) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of 2181 wb transfusions in routine use in ghana, there were 4.02% trars in recipients of mirasol-treated wb and 5.59% in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january 2008 to october 2018 at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified 310 patients who received fludarabine (n = 52, 17%), bendamustine (n = 29, 9%), cladribine (n = 17, 5%), dacarbazine for hl (n = 164, 53%) and alemtuzumab (n = 48, 15%). the median age of patients was 46 years (range 8-92) and 171 (55%) were male. median follow-up was 30 months (range 0-132). post-exposure, 42 patients (14%) received transfusions with 33% correctly receiving irradiated rbcs. the remaining 28, all from haematology/oncology, received a total of 192 unirradiated rbcs. in 8 patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of 14.5 months (range 0-75) from first rbc transfusion. after medication administration, 99 patients had g&h requests after a median of 3 months (range 0-129). only 23% of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only 20% asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march 2017. for audited patients, these were written from 38 days prior to 104 days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february 2018 to february 2019. ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after 15 minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version 23.0. results: a total of 500 transfusions forms were analyzed. over all compliance rate was 18%. out of 500, 115(23%) forms were available in source notes and of 115, 88 (76%) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = 0.000). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion(3%) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion(89%). a total of 02(0.4%) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was 2 hours and 30 minutes for red blood cells and for platelets it was 1 hour and 13 minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after 6 hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. 5a-s31-01 as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately 15 cases per 100,000 but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of 2-4 weeks (range, 5 days to 5 months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and 130 (4.6%) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of 8.7 9 10 3 , 8.0 9 10 3 and 7.2 9 10 3 , respectively. mean cci decreased in a linear fashion between day ≤ 2 and day 7 pcs (9.0 9 10 3 , 9.1 9 10 3 and 8.5 9 10 3 at ≤ 2 days; 6.7 9 10 3 , 5.8 9 10 3 and 4.9 9 10 3 at 7 days, respectively), although the number of pc transfused on day 7 to autohsct patients was small (n = 71). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late 1990s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from 4 bcs (35% plasma in additive solution ssp+) were spiked with virus suspension (10% v/v). pcs (n = 2, 375 ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) j/cm 2 )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid 50 ) by endpoint titration in microtitre plate assays on vero 76 cells (atcc â crl-1587 tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of 6.2 (bag no. 1) and 6.5 (bag no. 2) log 10 tcid 50 /ml was received in the pcs. at a uvc dose of 0.10 j/cm 2 and higher niv was inactivated down to the detection limit of the system (1.9 log 10 tcid 50 / ml), resulting in log 10 reduction factors of ≥4.3 (bag no. 1) and ≥4.6 (bag no. 2). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. 364 ae 20.6 9 10e11 platelets/unit, p < 0.001), whereas the platelet content of apheresis pc did not change (305 ae 9.9 vs. 300, ae16.1, p = 0.57). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. 1478 first-time donors has attended to the study in 18 regional blood centres in 29 cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = 14). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = 3.55, close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: 0.47, 0.28. 0.19, 0.17, and 0.5, respectively). donation anxiety was the negative direct predictor of intention (-0.05). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy (0.90 and 0.08). paraphernalia anxiety was the negative indirect predictor of intention (-0.03). descriptive norm did not show any significance. our model accounted for 75.3% of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from 16 countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp1 coordination, wp2 dissemination, and wp3 evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp4 inventory of donor selection & protection practices; -wp5 development of risk-based guidelines for donor selection and protection; -wp6 development of a standard donor health questionnaire (dhq); -wp7 training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september 2017 and will complete in spring 2020. wp4 has completed its work in october, wp5 will complete its work in june 2019, and wp6 and wp7 have recently commenced. results: with the use of the deliverables created by wp4, we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp5's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from 16 countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to 1) provide a comprehensive inventory of current systems for selection and protection of donors and donations, 2) critically review them and 3) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of 163 experts (40 blood; 40 plasma; 27 tissues; 47 stem cells; 9 art) and 39 (24%) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of 24 stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years 2014-2017, both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by 77%), arterial puncture (77%) and nerve damage (54%). vasovagal reactions were also frequently included (75%); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma (86%), citrate reactions (86%) and arm pains and nerve damage (both 57%, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl-03-01 modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg1/2) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a 2 c 2 ). we have previously identified bcl11a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas9 editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas9:sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + 58 bcl11a erythroid enhancer results in highly penetrant disruption of gata1 binding motif, reduction of bcl11a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl11a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl11a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m101 was developed in the medical device named hemo 2 life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on 60 patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo 2 life â and grafted on recipients. in 2018, a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo 2 life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m101 making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since 2012; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at 9-11 march 2018. aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from 25 countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations 4 different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: 1. should have university degree preferably in marketing and business administration field. 2. should have a certificate and/or professional experience in public relation 3. should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team 4. should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter 5. should be a permanent staff 6. should have basic salary and performance bonus might be given 7. is eligible to monitor and modify mobile team working period at blood drive 8. should participate the mobile blood drive which he/she has organized 9. should participate the group who will create promotional materials for national blood service 10. number at each blood establishment should be defined based on annual blood collection such as 5 staffs for 50,000 whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga 1 and j emmanuel 2 background: africa is a large continent with 55 independent states and a total population of 1,275,710,034 (february 2018) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the 2 who regional offices; eastern mediterranean regional office (emro) supporting 8 arabic speaking countries and the african regional office (afro) responsible for 47 sub-saharan countries. population distribution is approximately 40.6% urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise 3 level accreditation program. in 2016 emro held a consensus meeting developing a "strategic framework for blood safety and availability for 2016-2025" with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in 2001 all 54 member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least 15% of the national fiscal budget. in 2013 ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha63.12 on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's 2016 global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the 2016 report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of 4 staff on the morning (am) shift (from 8:00 to 17:30) and 5 staff on the afternoon (pm) shift (from 13:00 to 22:00) on weekdays and 3 staff on the am shift and 5 staff on the pm shift on weekends. bdt lab has a staff strength of 15 to be rostered for the 2 work shifts. each staff is on a five-day work week and has to work 11 pm shift and 9 am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving 5 staff on am shift and 4 staff on pm shift was conducted. the total number of pm shift per month was reduced from 124 to 112 using the re-defined process. the 10% reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having 2760 licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of 70,000 blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of 6 months. after overcoming all the challenges we minimised hard copies to 6 registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by 20%. waiting time for attendant decreased by 10%. traceability of all the units became 100%.supervision of the activities being carried out was 90% accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by 50% thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p-007 ct smit sibinga 1 , y abdella 2 and f konings 2 1 iqm consulting, zuidhorn, netherlands 2 who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in 2013 blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only 9/22 countries are put in force by governments [1960 (egypt) till 2017 (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in 2008-2017 including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in 21 regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from 170 in 2008 to 133 in 2017 in favor of mobile collections, which increased from 8,672 to 13,189, the former is still the number one location for donating blood. on average 47.36% of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar (583,908 in 2008 and 588,184 in 2017); over 99% of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from 237,658 in 2008 to 143,038 in 2017). the total number of donations increased from 1,076,655 in 2008 to 1,249,655 in 2017; most frequent were whole blood collections (from 1,016,411 in 2008 to 1,171,302 in 2017) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs (996,678-1,154,239 units per year), fresh frozen plasma -ffp (1,090,369-1,289,021 units) and platelet concentrates -pcs (81,692-129,143 units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs (52-57.6% in respective years irradiated, 75.18-92.07% leukocyte-depleted), than to rbcs (4.46-8.46% irradiated, 7.65-20.7% leukocyte-depleted). in 2010, the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in 2017 approximately 11.5% of pcs and 8% ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland 80% of hospitals depend on blood for the treatment of patients; over 1.5 mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. 1 st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the 20 year-period in two stages. goals at 1 st stage: 1. data digitalization; scanning of paper documents. 2. development of a uniform template for collecting digital data from various sources. 3. standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. 4. selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) , b) digital stored in two file types (.doc and .xls, for the years 2005-2010 and 2011-2017, respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: 1. 1344 pages of paper documents were scanned. 2. models developed for data from 3 different sources: a. paper-data were rewritten and ascribed to its model; outcome -3 tables, 272 columns, 10,400 rows. b. .doc and .xls. filesdata were ascribed to 2 other models; outcome -6 tables, 1656 columns, 788 rows. 3. the 3 models were merged into 1 analytical table to create a 588 mb database (comparable to approx. 784 min of music). 4. the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. 5. selection of data for analysis at 2 nd stage. summary/conclusions: the 1 st stage provided a set of selected data for analysis in the 2 nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far 8 workshops were organized and each year around 30 countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. 6. supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from %96 whole blood (at 1996) to 5%. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management 7. around 11,000 blood bankers attended national courses, 7,000 attended national congresses, 18,000 attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in 2016 an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages 4.3% of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages 122.8% (2018) based on a 5-day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated (2016/2017) to be at 3.2% of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october 2018, an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level 6. prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the 12 months prior to emr implementation was compared with 3 months post. ntr numbers reduced from 4220/month to 2019/month (52% reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of 27/mth to 6/mth. wbit numbers have increased slightly: before having median 1 (range 0-2), after with median 2 (range 0-3). although it was hoped that wbit incidence may reduce with the new emr, 4 of the post implementation 6 wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa 1 , k teo 2 , s tsai 1 , p heng 1 , r sagun 1 and m wong 1 1 laboratory medicine 2 khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a 761-bed general and acute care hospital, opened in 2010, serving more than 800,000 people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase 1: an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase 2: the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase 3: elimination of the witness step for blood collection. specimen collection and rejection data from 2016 to 2018 was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january 2016 and march 2017, before the implementation of the e-t&s phase 1, the average rejection rate for blood bank specimens was 0.16% and 1.14% for identification and clerical errors respectively. during phases 1 and 2 of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february 2018, with the implementation of the final phase of the e-t&s system the specimen rejection rate was 0.38% and 0.12% for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after 15 min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on 30 patients admitted to the outpatient clinic for 45 red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january 2017-december 2017) 76 patients, 40 females and 36 males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was 12.0 months (interquantile range-iqr 26). the median length of stay was 16 days (iqr 30). in total 400 blood components were transfused in which of 227 red blood cell concentrates, 114 apheresis platelet concentrates, 6 granulocyte concentrates, 51 fresh frozen plasmas, and 1 cryoprecipitate and 1 whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < 5%, 5-10%, 11-15% and > 15%. most of the patients (63.2%) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were 5478.76 euros (iqr = 11280.02) and 130.57 euros (iqr = 354.86), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: 0.674, p < 0.01). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: 0.247, p = 0.032) there was also a significant weak positive correlation between the age and transfusion associated cost (p = 0.048, r: 0.227). a significant difference was found between the patients with and without hematological malignancies (p < 0.01) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < 0.05). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately 55.7% of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january 2016 to december 2018 were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total 177,370 ordered prc unit, 166,910 (94.1%) were subjected to pretransfusion testing and 63.1% (105,260) of ordering prc unit which are pretransfusion testing were transfused. this means that 5.9% (10,460) of ordering prc unit were not subjected to pretransfusion test. this showed savings of 1,098,300,000 rupiah. c/t ratio was 1.6 which demonstrate a good ordering pattern. however, 16.4% (27,451) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of 2,882,355,000 rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of 19 questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, 2014 to february, 2015 . results: the overall mean percentage of 'correct' responses for 19 questions in the baseline study was 61.75%, whereas post sensitization it was 77.21%. the mean percentage increase in general awareness related questions was 21.49%, 13.95% for storage of blood/blood components related questions, 17.37% for pre-transfusion checks and bedside transfusion practices related questions, 21.33% for testing and blood component preparation related questions and 27.27% for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < 0.001). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in 2014 to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online 24/7, complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during 2018, tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from 3/5 international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been 2,376 paediatric and neonatal courses completed from 6 march 2018 to 28 february 2019 with 89.6% of learners being nurses and/or midwives. analysis of course evaluation data (n = 33) showed that these courses: -provide knowledge (96.9%) -improve patient safety and outcomes (84.9%) -result in change to clinical practice (69.9%) -are relevant to clinical practice (70.9%) -are easy to use (67.7%) -are readily accessible (58.1%). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was 100%. the 2 nd group participants had an average seniority of 9 years . more than half of them (64%) had seniority of less than 10 years. only 12% had more than 20 years of experience. the rate of correct answers for all items combined was 44.4% for students versus 42.5% for practicing nurses. the theoretical knowledge part was more mastered in the 1 st group than that of practicing nurses (44.8% vs 33.4% of correct answers). on the other hand, the control of the transfusion act was better in 2 nd group (44% vs 50.5%). the overall "dangerous" response rate was 47% for students and 41.7% for practicing nurses. false practical knowledge was more common in group 1 (59.5% vs. 41.5%). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive 2004/33/ec on blood donor selection criteria is 15 years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than 24 stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive 2004/33/ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in 2018, we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive 2004/33/ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified 64 risks considered to be significant, distributed between donors and recipients. for 35/64 (55%) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for 9/20 criteria concerning general health and medication, 12/22 for transfusion transmissible infections, 10/13 for high-risk behaviour and travel, and 4/9 for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive 2004/33/ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of 5 possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since 2007, showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after 10 years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all 27 states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since 2007, when the assessment covered 109 blood services, marpsh reached 1218 blood services in 2017 (57% of the blood services registered) what corresponded to almost 100% of the inspection cover in this year. over this period (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from 26% to 9%. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over 10 years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = 2), training coordinator (n = 1), medical doctor for donor selection (n = 2), hemovigilance officer (n = 1), laboratory technicians performing donor sampling, blood qualification and production (n = 7), biomedical scientists (n = 3), microbiologist (n = 1), clinical biologist (n = 1), nurse (n = 3). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is >8 h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: 19.3 million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab0, rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version 4.2; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than 8800 blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over 1000 blood donors from more than 20 non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of 600 migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of 1.6%. amongst 509 hla-typed potential stem cell donors, we found 28 (5.5%) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund 2014-2020 (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of 1 year starting from june 2017 to may 2018 at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ 16) that were admitted within 24 h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period 885 patients (83.5% males) were enrolled. mean iss scores was 21.89 . mean time to hospital admission after injury was 9:03 (iqr 3.38-13:48) hours. mean time to first rbc transfusion following admission was 2:09 (iqr 0:27-2:45) hours. approximately 49.3% (436) patients were in shock (sbp < 90 mm hg &/or pulse rate > 110/min). whereas, 160 (18.1%) patients were coagulopathic (pt ≥ 1.5 times of normal). during initial 24 h of admission, these patients were transfused with 2929 (69.7%) rbc, 1986 (51.8%) ffp, 2327 (42.9%) rdp and 384 (81.5%) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ 5 units/4 h) was given to 190 (21.4%) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of 9% per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on 78 patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in 2016. pc consumption was examined from january 2016 to december 2018. the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched (10-8/10) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of 78 patients aged from 3 to 74 years were analyzed; 48 (62%) were male and 30 (38%) female; 35 (45%) were cmv-negative and 43 (55%) were cmv-positive. out of a total of 78 transplants, 14 (17.9%) were haploidentical and 64 (82.1%) hla-matched. according to the stem cell source, bm was transplanted in 22 cases (28.2%), and pbsc in 56 cases (71.8%). two patients also received a cd34 + stem cell boost. our analysis showed that, with a mean follow-up of 652 days, the number of pc transfused to our patients treated by hsct ranged from 0 to 383 units, with an average of 37 and a median of 15, illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = 0.034) higher number of pc transfused for patients treated with a haploidentical (89) versus hla-matched (26) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused (2016: 20% (371/1813), 2017: 19% (322/1742)). moreover, approximately 8 products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october 2017 to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july 2014 and december 2016 to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ 25% of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ 25%: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took 2.5 min using the pneumatic tube system (maximum of 2 units per tube) and 4.5 min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december 2017. one year following implementation of the change in policy 940 rbc units were issued to the or (a 54% reduction); 38% (361) were transfused, compared to 19% in 2017. wastage rates decreased from 8 products a month to 1 per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, 34% of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from 1 may 2018. the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired 5-days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july 2018 set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of 704 daily blood bank statements which were submitted between may and december 2018 from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was 130.4% (sd +/-101.6), the available stock was 142.3%: (sd +/-118.8) and the demand versus supply was at 95.6% (sd +/-11.3).the overall bsms was 122.8%; (sd +/-77.2) for the study period. gweru and masvingo nearly supplied all the demanded blood with 99.2%, overall bsms of 118.1% and 99.7%, overall bsms of 144.3% respectively. bulawayo supplied 98.3% of the blood demanded with an overall bsms of 99.1%. mutare supplied 97.8% with a bsms of 180.1% and harare 85.6% and a bsms of 78.0%. there were monthly variations but the service could supply above 90% of the blood demand. in the month of may the service met 91.6% of the demand and a bsms of 87.8%. in november and december it supplied 92.6%, bsms of 100.8% and 92.8%, bsms 131.8% respectively. august also had a below average supply of 93%, bsms -98.2%. june, october and september recorded above the average values; 97.3%, bsms of 126.8% and 98.7%, with a bsms of 117.2% respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in 2008. subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in 2016 and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where 4 or more red cell units are transfused in <4 h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: 65 from 10 treating units including cardiothoracic surgery (18) hepatobiliary/gastrointestinal/colorectal surgery (24), vascular surgery (6), neurosurgery (4), orthopaedic surgery (3), endocrine (2) and "other" (encompassing general surgery, urology, general medicine and oncology -8). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. 18 of 65 reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at à40°c, and irradiation. biolog-id tags are passive hf (13.56 mhz) tags. they are compliant with is0 15693, iso 18000-3 and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, 2010). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt 128 format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with 450 ml water. centrifugation at 4,500 rpm for 10 min. 270 tags processed, 15 tags per kit affixed at different positions. -shock-freezing at à80°c: shock-freezer (angelantoni, sf40), 50 units processed, reading immediately after removal from shock freezer. water, 30 tags irradiated at 30 gy and 30 tags at 50 gy results: all biolog-id tags were encoded and read with a 100% success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at à40°c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an 18-year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh34). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd*01/rhd*03n.01 and rhce*cevs.01/ rhce*cevs.03. aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion 2013 53(2s):174a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier 1 donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier 2 donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier 3 donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: 102 tier 1, 357 tier 2 and 100 tier 3 donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier 2 unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from 7 to 1.9 g/dl. at that time, she was transfused the only compatible units available -2 of the rare -dphenotype and her hb increased to 3.8 g/dl and eventually to 7 g/dl. the tier 2 rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform (1) assuring the patient identification with barcode techniques; (2) designing a structured order entry; (3) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; (4) automatically reminding physicians the happening of reaction and suggesting relevant test; (5) building a complete traceability log system; and (6) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected (0% after implementation, p < 0.01), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency 30-min blood crossmatch (98.1% after implementation, p < 0.05). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply (0% after implementation, p < 0.01). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow (2004 moscow ( -2011 . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during 7 days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in 7 days after were compared. results: in 2004-2011, 5 terrible ta occurred in moscow: 141 people died and more than 629 were injured. with the explosion in subway in 02/2004 42 people died, 250 were injured. the number of d-rbcus increased by 10% on ta-day, and by 30% during next 7 days. dbdn in the 1st day after ta increased 6,7 times, and in the next 7 days -1,6 times. second explosion in subway in 08/2004 resulted in 10 died, 50 injured. the number of d-rbcus increased by 80% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 1,1 times, and in the next 7 days -2,2 times. in 2006 (explosion on market) resulted in 14 died, 61 injured. d-rbcus delivery increased by 20% on ta-day, and by 10% during 7 days. dbdn in the 1st day increased 2,0 times, but decreased to 0,6 times during the next week. with subway explosion in 2010 40 people died, 88 were injured. the number of d-rbcus increased by 10% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 6,5 times, and in the next 7 days -1,6 times. with the explosion in airport in 2011 35 people died, 180 were injured. rbcus delivery increased by 50% on ta-day, and by 40% during next 7 days. dbdn in the 1st day after ta increased 6,1 times, and in the next 7 days -2,0 times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for 7 days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of 70,000 blood units. 2000 patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. 1000 patients were taken as control and the other 1000 were taken as cases. 2482 blood units of healthy donors were chosen (2442 were males and 40 were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to 1000 patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of 6 months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in 5 cases (0.5%) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common (99.44%) followed by d (95.45%), c (89.65%), c (53.1%) and e (17.65%). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of 12.5 million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab0, rhesus and kell systems were analyzed retrospectively in 352362 blood donors for 14 years (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab0, rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih-1000" (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) 35.8%, 0 (i) blood group 34.3%, b (iii) 21.3%, ab (iv) 8.6% (n = 352362). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was 82.8% and 17.2%, respectively. donor's phenotype detection was routinely conducted from the 2013 year, therefore the number of donors was 152883. the most common phenotype among donors ccdee (32.61%), the second in frequency ccdee (19.33%), the third in frequency rhesus negative phenotype ccddee (15.42%) in the studied population. the ccdee and ccdee phenotypes were 14.04% and 12.17%, respectively. the most rare are ccdee (2.65%), ccdee (1.86%), ccddee (1.54%). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of 0.37% (n = 152883). cw antigen was tested in 104230 donors and was detected in 5.28%. cw is most commonly found in donors with ccddee phenotypes (2.36%), ccdee (2.03%) and ccdee (0.79%), with other variants of the data phenotype, the antigen was detected in 0.1% of the examined (n = 104230). antigen k was detected in 6.8% of donors, in 93.2% of this antigen is absent (n = 352362). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in 2014 the number of outdated units at the hospital side dropped considerably. results: since 2008 when the issued number of red blood cell units (rbc) was 5170 the amount of issued units rose to 5678 in 2011 and then dropped more or less steadily to 4850 in 2018. in this period the hospitals' programmes rose for 10% in all areas. number of donated units declined from 6330 in 2011 to 4820 in 2018. after reorganization in 2009 the number of outdated units fell from 3% of stocked units to 1.5%. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from 397 to 41 in 2018. for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens 4 times a day; at 10 a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high (99%). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of 78% blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve 100% voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january 2013 to january 2017 were collected from donor management software. the data includes socio demographic data. data has been process with spss version -17 results: during 4 years study period, total of 276,290 blood donation happened from both mobile blood collection and in-house blood collection. out of 276,290 collection, 48351 (17.5%) are from 18-24 age group; 106924 (38.7%) are from 25-31 age group 53324 (19.3%) are from 32-38 age group; 34536 (12.5%) are from group 39-45 age group; 23761 (8.6%) are from age group 46-52 and 9394 (3.4%) from age group above 53 respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal 18-38 years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in 2018 to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by 80 of the 81 blood banks authorized nationwide between january and december 2018. results: sihevi-ins© received information of 854,462 accepted donors, 33% of them with more than one donation in the same blood bank in a period of 12 months. a total of 72 abo or rhd discrepancies were identified in 69 people, who made donations in 20 blood banks (estimated risk: one discrepancy per 11,876 accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of 6,086 ae 3,785 units, representing 3.6% of the national collect). the remaining blood banks are distributors (average collection: 31,389 ae 25,704 units per year, representing 44% of the national collect). 75% of blood group typing discrepancies (n = 36) were related to the abo group. the most common discrepancy was between a typing group and ab typing group (67%) . in 25% of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = 10) or b type (n = 4). rhd typing discrepancies account for 25% (n = 18) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of 4417 edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, 1 anti-s, 1 anti-jka and 6 anti-k. in one case ih-1000 failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples (0.045%) false-positive result were observed both on ih-1000 system and neo iris and in two cases (0.09%)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih-1000 system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from 2017 to 2018 in taiwan. aims: the proficiency testing (pt) held semiannually from 2017 to 2018 were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into 4 groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of 11 kinds of routine work categories of 841 blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the 11 categories were as follows: 1. characteristics of institution 2. the equipment of blood bank 3. the kinds of tube in blood bank 4. the present kinds of blood bank tests 5. abo and rh type tests 6. the cross-match tests 7. the irregular antibody tests 8. hemovigilance system 9. other blood bank tests 10. massive transfusion protocol 11. quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of 3rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum 7 days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since 2001 year with 844 collections of stem cells (686 in patients and 159 collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt 128 standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, 2017 to dec, 2018) qc data from archi-tect i2000sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of 6532 data points (3523 data points in 2017 & 3009 data points in 2018) were obtained for 10 different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% (5.70-12.05) were within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2018. five out of six positive quality control levels cv% (5.72-15.80) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2017 except syphilis tp positive control (15.8%). all four negative quality control levels showed the sd values within 0.009-0.41 in 2017 and 0.009-0.41 in 2018 respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: 1. to assess the existing transfusion practices in the institute with specific quality indicators 2. to introduce corrective reforms to improve the existing practice 3. to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, 295 transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within 30 min of release from the blood bank (iii) close observation of transfusions for the first 15 min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within 30 min of issue. transfusion practices were once again monitored by following up 306 transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. 57.3% transfusions were verified in phase i while 73.6% were verified in phase ii (p < 0.001). 69.5% transfusions were started within half an hour of issue while in the second phase, it rose to 83.1% (p < 0.001). 47.8% transfusions were observed in the first 15 min in phase i and 88.6% were observed in the second phase (p < 0.001). in phase i, 54.6% transfusions were completed within right time while the same in phase ii was 73.9% (p < 0.001). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a1 & b) antibodies were selected. the titers of 126 samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, 12 of these samples were tested in the 2 automated systems for 5 times (120 datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured (3 expert technicians) in 2 different sample workload (1 and 10 samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, 2 different sample workload (1 and 10 samples testing) were assessed in manual method (3 expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ 1 titer: 73% ≤0.5 titer). the results proved that both instruments were equivalent in performing titration (differences ≤ 1 titer; 85% ≤0.5 titer). the precision results showed no difference between titers obtained through the 100% of the runs performed with the grifols analyzers (differences ≤ 1 titer: 75% ≤0.5 titer). the manual hands-on in automated system was reduced in a 15% compared to manual method for 1 sample. when the number of samples was increased (10 samples), the difference in hands-on in was even more reduced (65%). in addition, the walk-away was 46% higher in automated system compared to manual method. furthermore, when the number of samples was increased (10 samples), the walk-away difference was increased even more (78%). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least 15% the hands-on, increasing at least 46% the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by 30%>50% of users and "easy" and by 50-80% of users; 90%>100% of the users considered "sufficient" the design to ease the interaction; and 60%>80% of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), 40% of users considered tasks "very easy" or "easy"; 40% of users considered "sufficient" the design to ease the interaction; and 40% of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), 100% of users considered tasks considered "very easy" or "easy"; 90% of users never found any situation of not knowing how to proceed; and 100% of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno (1 and 2) and correspondent two mp-readers lyra (1 and 2) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a1, b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of 4 + 2 tubes of whole blood and 2 tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno 1/lyra 1 system. ward to ward. methods: a prospective observational pilot study was done for around 140 prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to 90 min, with highest time taken 795 min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be 10 min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to 70 min, maximum being 310 min (jan 2019), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p-059 hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: 204 healthy blood donors of greek origin (18-24 years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < 0.05. results: b-thal-het represented 9% of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch (11% p = 0.039, 26% p = 0.008 and 30% p = 0.006, respectively) and b) increased rbc count (20%, p = 0.042) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = 0.053) and serum total protein concentration slightly reduced (p = 0.054) in the same group. a trend for higher plasma antioxidant capacity (p = 0.052) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by 13%, p = 0.022) and hemolysis (by 41%, p = 0.001) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = 10 per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > 0.05). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of 23) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding 100,000 per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years 2010-2017 obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from 400 in 2010 to 716 in 2017 (+79%). in the group of male donors this trend remained rather stable with a slight decrease: from 181 in 2011 to 151 in 2017 (à16.6%). in the group of female donors the growth was more prominent: from 219 in 2010 to 565 in 2017 (+158%). in terms of the recorded confirmed hcv infections a downward trend can be observed: from 63 in 2010 to 16 in 2017 (à74.6%). in the group of male donors from 43 in 2019 to 12 in 2017 (à72%), in the group of female donors from 20 in 2010 to 4 in 2017 (à80%). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last 6 months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a 6 month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, 18.3% of donors who received a reminder message attempted to return compared to 12.8% of donors in the control group (p < 0.05). looking at each time point, donors who received the message 1 week before their deferral ended were 64% more likely to attempt to return compared to the control group (p < 0.05). the 1 week prior reminder message was particularly effective with males, with 30.3% attempting to return to donate, compared with 25.2% of females (p < 0.05). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide 100% voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of 1000 interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: 1. provide 100% voluntary donation for safe blood. 2. establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with 60 questions in total was divided into 6 sections out of which 14 questions on blood practices were answered by all 1000 interviewees. 416 people answered 9 questions on the blood transfusion service. 5 questions on blood knowledge were answered by 270 people. 5 questions on the knowledge of the blood transfusion were answered by 220 people, 17 questions on blood donation were answered by 420 people and 10 questions on the communication channels were answered by 500 people. results: out of 1000 interviewees, 19% have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, 40% did not donate, but expressed the readiness to donate in the future, 10% have donated voluntarily only once, 24% were family donors, 3% regular volunteer donors, and 4% have donated voluntarily several times and did not want to donate anymore. from those who have donated, 59% have donated for one of their relatives, 28% have donated for thalassemic children, 10% have donated to benefit free check-up and 3% have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, 57% have answered yes, 22% no and 21% were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around 60% of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with 3 adults of 10 overweight and 1 of 10 obese in 2017 with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < 25 kg/m 2 , overweight with a bmi ≥ 25 and < 30 kg/m 2 , and obesity with bmi ≥ 30 kg/m 2 . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least 3 donations in the last 2 years, and were eligible to donate. results: of the 2468 blood donors enrolled between july 2017 and january 2018, 1390 were regular repeat donors, 1102 did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and 288 accepted, of which 83 were deferred from blood donation and were excluded from the analysis. among the 205 included participants 68.3% (n = 140) were male, age ranged from 19-61 years with a mean age of 39.8 ae 11.1 sd and 31.7% (n = 65) were female age ranged from 20-62 years with a mean age of 37.7 ae 11.0 sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found 50.7% in men and 16.9% in women. our survey showed that 84.4% of the participants evaluated their health as "good", without gender difference (men, 86.4% vs women, 80.0%). besides, 14.6% reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged 18-25 attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics 22.0. categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < 0.05 was considered statistically significant. results: a total of 600 students were surveyed (300 squ, 300 non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, 73% of the squ and 25% of non-squ students reported the university as the main source for information (p < 0.001), while 56% of squ and 45% of non-squ students reported that the social media was the main source respectively (p = 0.048). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = 0.868). about 84% of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation (44.3% vs 29.7%, p < 0.001). squ students reported greater influence of peers (84% vs 60.7%, p < 0.001), personal knowledge (82% vs 74.7%, p = 0.029) and personal experience (79.3% vs 69%, p = 0.005) when compared to non-squ students. they also reported more feeling of commitment to the society (90.3% vs 78%, p < 0.001). squ students reported lower influence of parents (53% vs 65%, p = 0.005), lower rates of fear from needles (18% vs 32%, p < 0.001) and lower rates of fear from blood (16% vs 29%, p < 0.001). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about 15% of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately 85% of the expatriate population (and 71% of the emirate's total population) are asian, chiefly indian (51%) and pakistani (16%). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in 2019. the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of 1080 donors were surveyed and asked about the languages which they can read and understand and 1860 responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = 760; 41%), arabic (n = 272; 14.6%), hindi (n = 268; 14.4%) and malayalam (n = 233; 12.3%). the donors come from different countries, most common 591 (54.7%) donors are indian and 73 (6.8%) are from uae. it was found that 45% donors can read and understand only one language. majority 884 (81.9%) donors can read and understand either of the official languages arabic or english. however, 196 (18.1%) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, 1290 responses were obtained. the most common mode of communication were sms and telephone (85% together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as 18.1% donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type 2 diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a1c (hba1c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period 2015-2017 and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was 83.7%, plasmapheresis donors -13.2%, blood cell apheresis, including plateletapheresis, donors -3.1%. for the period 2015-2017, the percentage of repeated and regular whole blood and apheresis donors increased from 66.2% to 72.2%. the percentage of first-time donors ranged from 27.8% to 33.8%. the largest proportion of plasmapheresis donors was observed in the volga fd (22.0%). about 28.9% of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from 1.9% to 2.3%. the largest percentage of plateletapheresis donors was observed in the central fd (2.9%), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to 69.9% in 2017. actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between 20 to 23 years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january 2016 to december 2018. the data were retrieved from the official reports of each mobile blood donation. results: a total of 828 young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is 25.6%. the main causes of deferral are low haemoglobin (hb) level (20.8%) followed by low blood pressure (16.0%), upper respiratory tract infection (11.3%) and sleep less than 5 h (9.4%). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than 5 h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is 12.7% and in non-pregnant women is 30.2%. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was 454913. repeated blood donors was 44.8% of total population and had the highest number of referrals, followed by first and lapsed donors with 31.2% and 24% respectively. in terms of gender distribution, 6.2% were female and 93.8% were male. the highest rate of hemoglobin level less than 12.5 g/dl was found in first-time donors with 51.6% and the lowest prevalence was observed in lapsed donors, followed by repeated donors with 24.7%. 44.8% of the repeated blood donors have hemoglobin higher than 16.5. there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit 50,000 blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of 10% (500,000) of the finnish population. (11.58%), dental examination 10(3.32%) and medication history 6(1.98%). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were 5(1.65%) and 8(2.64%) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years (2016-2018). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold (120 g/l in women, 130 g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies (15.9% versus 3.4%) or in continental france (2.9% and 0.8%). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march 11 and march 28, 2019. results: this study ferridon has been approved by ethical research committee. nine thousand (9000) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, 150 donations should be included in the french west indies and 120 in reunion island. additionally, 1500 whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than 26 ng/ml and iron overload is defined by ferritin higher than 300 ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may 2019 to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around 10 million people, only 3.8% are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of 95% in 2018, instead of 80% in 2016, which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the mean interval between donations is shorter for former regular donors (4.9 months, p < 0.01) whereas donors with an interval of 9 to 12 months are more likely to be regular (aor 16; 95% ci 1.6-164). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least 60 9 10 9 cells per dose), and in kazakhstanin therapeutic doses (at least 200 9 10 9 cells per dose). in 2017, the estimated consumption of platelets in kazakhstan exceeded the russian indicator by 28.0%. 86% of platelets in kazakhstan and 69.9% in russia are harvested by the apheresis method. inactivation of pathogens is performed in 92% of platelets in kazakhstan and in 15.4% in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in 2017 exceeded the russian indicator by 141.4%. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in 10% of plasma in kazakhstan and in 3.6% in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. 0.7% of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p-091 finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: 1. no bacteria growth near the puncture spot (result 0 cfu) in ≥ 90% of the samples. 2. total amount of bacteria on average is at most 2 cfu/25 cm 2 . at most 10% of samples are allowed to have 2-10 cfu/25 cm 2 . methods: microbiological samples were taken with contact plates from 30 voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from 20 to above 500 cfu/25 cm 2 in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean (0 cfu/25 cm 2 ) in 93.3% of the samples and 86.7% of the samples had 0 or only 1 cfu/25 cm 2 . the average number of bacteria after disinfection was 0,6 cfu/25 cm 2 and the maximum number was 4 cfu/25 cm 2 . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the 2018 meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb201 + (hemocue ab) point-of-care (poc) device. donors with hb < 125 g/l for females or < 135 g/l for males or with a decrease > 20 g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = 305) and control (n = 331) groups, the proportion of male donors (27% and 26%) and the mean initial skin-prick hb (122 g/l and 123 g/l) were similar. significantly less donors were deferred from donation in the study group (40%) than in the control group (51%; chi-square test p = 0.004). the mean difference in venous hb with the poc device versus the hematology analyzer was à3 g/l (range à12 to + 6 g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only 1 g/l below the limit of donation eligibility (124 g/l for a female and 134 g/l for a male). interestingly, a further 33 donors (27% of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above 60 years of age. this age group has been reported to use up to 50% the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged 60 years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ 60 years from blood centres of four countries. the estimated proportion of blood donors aged ≥ 60 years old for each country was 0.81% in south korea (sk); 2.57% in hong kong (hk), <3.18% indonesia (indo) and 9% in japan (jap). the minimum hb criteria varied between each country and ranged from 11.5-12.5 g/dl for women and 12.5-13.0 g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe-2100d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are 6 medication questions on the donor history questionnaire (dhq), including any medication use in the last 3 days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in 2017 were included in the analysis. donors' answers to each of the 6 medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were 975,067 donation attempts with a completed dhq. overall, 34% of donors answered yes to medications in the last 3 days, 8% to vaccination, and less than 0.1% to others (38% any). slightly more were female (42 vs 36%) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from 24% of 17-19 year olds to 57% aged 60 + (p < 0.0001 for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel 7 and tomes software j schreier 1 , a davison 1 , j gambarte 2 , y l opez 2 , c calonge 2 and e herranz 2 1 terumo bct, lakewood, united states 2 centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than 4000 apheresis platelet collections were completed in 2018, of which almost 3000 were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel 7 was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel 7 devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel 7 with tomes compared to trima accel version 6. methods: this was a retrospective study analyzing 1372 apheresis procedures on trima accel version 6 during the control period from 2 january 2018 to 10 september 2018 compared to 541 apheresis procedures on trima accel 7 during the test period from 11 september 2018 to 28 december 2018. this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a 2-proportions test, whereas donor demographic data were analyzed using a 2-sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = 3.0 or 3.5 9 10 11 ) or double (target platelet yield = 6.0 9 10 11 ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = 5148 ml, test = 5124 ml, p = 0.545), hematocrit (control = 43.7%, test = 43.6%, p = 0.233), or platelet pre-count (control = 249 9 10 3 / ll, test = 253 9 10 3 /ll, p = 0.091). females represented 21% of donors in the control arm compared to 23% of donors in the test arm. platelet split rate (platelet products per procedure) increased from 1.15 with trima accel version 6 to 1.18 with trima accel 7; procedure time decreased from 55.7 min to 53.3 min for single collections and from 63.0 min to 60.9 min for double collections with trima accel 7 (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from 62.4% to 84.8% (p < 0.001) and the rate of completed apheresis procedures increased from 89.8% to 92.4% (p = 0.083) with trima accel 7. manual transcription of data during the procedure was discontinued with the implementation of trima accel 7 with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel 7 significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the 19 th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec 2017, distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of 36 respondents, 24 bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering 2,888,390 plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per 10,000 plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to 16% of the estimated total blood volume but maximally 750 ml, including anticoagulant. respondents had differing practices and scale of collection program 15 be were aligned or lower, and 9 be had higher collection volumes. altogether 14 reported the immediate vvr with loc rate, which mainly was lower than 10/10000 collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by 16 respondents. the retention rate banded in the range of 50%>80% (mean = 60%, min = 25%, interquartile range = 14%, max = 90%). the association between maximum allowed yearly plasma collection (25 l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide 0 s limits for collection volume of maximum of 750 ml per donation and 25 l per year per donor. methods: serum ferritin concentrations were established from sera stored at à30°c from repetitive platelet donors between 2014 and 2016, using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn 1800 â . sixteen samples were obtained from women (age: 35.3 ae 12.4 years, range: 18-61) and 35 samples from men (age: 36.2 ae 9.4 years, range 20-61), corresponding to 55.2% and 37.2% of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, 43.8% of women and 40% of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was à38.4 ae 52.2 ng/ml when the donations had an interval less than three months, vs 3.3 ae 13.0 ng/ml when the time between donations was higher (p = 0.046). in men the change in the ferritin levels was à38.5 ae 61.2 ng/ml with donation times less than three months vs © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 à3.3 ae 38.9 ng/ml with prolonged donation times (p = 0.042). in women, the change in platelet count was à2 ae 46.2 9 10 3 /ul, when the donations had an interval less than three months vs à22 ae 45.3 9 10 3 /ul when the time between donations was greater (p = 0.43). in men, the delta of the platelet count was à12 ae 37.0 9 10 3 /ul in donation times less than three months vs à13 ae 26.6 with higher donation times (p = 0.93). no correlation was found between the concentrations of serum ferritin and the platelet count (r = 0.05, q = 0.75 for males, and r = 0.34, q = 0.22 for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version 7 (ta7) by comparing its routine performance with that of the previous software version 6.06 (ta6). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta6 and ta7 sequentially. data were collected from december 2017 to april 2018 on ta6 and from june to july 2018 on ta7. simple and double doses of platelets (respectively 5.0 9 10 11 , 5.5 9 10 11 and 8.0 9 10 11 , 9.0 9 10 11 ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from 1.06 (ta6) to 1 (ta7). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety (590) collections with ta6 and 607 with ta7 were recorded, with 92% and 95% complete procedures respectively. mean duration of procedures was 70 min on ta6 against 72 min on ta7, p < 0.05. the mean alerts number per procedure on ta6 was 4.1 against 0.5 on ta7, p < 0.05 whereas the maximum alerts number per procedure was 57 and 12 respectively. on ta7, 76% procedures did not require operator's intervention against 40% on ta6,. with ta7 the inlet flowrate was automatically adjusted in 97.2% procedures. the inlet flowrate was increased in response to access pressure in 45.6% of procedures, for 3% of the procedures the inlet flowrate was decreased and for 48.6% of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta7 autoflow system. summary/conclusions: ta7 with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel 7 (ta7) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta7 leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta7 software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version 6 (ta6) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta6 from 4th january 2016 to 10th october 2017 were compared to 995 procedures, started on ta7 from 18th october 2017 to 31 st december 2018. procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta6 vs. ta7 respectively) were comparable and were characterized by: tbv -5207 vs. 5344 ml; platelet count pre-procedure -252 9 10³/ll vs. 252 9 10³/ll; hematocrit pre-procedure -44% vs. 44%. gender distribution was 11% female with ta6 vs. 2% with ta7. venous access pressure alerts were significantly improved by ta7 with an average of 0.2 alerts per procedure as compared to 2.0 with ta6, i.e. 92% decrease. this decrease went down to 91% if only male procedures were analyzed. the maximum number of pressure alerts went down by 84% from 90 alerts in one particular run in the ta6 cohort to 14 alerts in one ta7 procedure. procedure time for single platelet products was reduced from 51 to 49 min and for double platelet products from 59 to 54 min (ta6 and ta7 respectively). donor qualification possible was 52% of procedures yielding single products and 48% of procedures yielding double products with ta6. the percentage of procedures qualifying for doubles increased to 91% with ta7. in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from 1.48 to 1.91 from ta6 to ta7, respectively. in fact, the observed split rate rose modestly from 1.01 to 1.05, as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta7 decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta7 has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel 7 to trima accel version 6 august 11 2017 to october 17 2017 or trima accel 7 during the test period from november 28 2017 to january 9 2018. this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: 63 donors completed the survey during the control period whereas 86 donors completed the survey during the test period. the mean number of previous donations for the control period was 3.0 (min 1 max 6) and for the test period was 3.2 (min 0 max 7). there were no first time donors during the control period and 20 first time donors during the test period. 91% of donors rated their overall donation experience as good on trima accel 7 compared to 90% on trima accel version 6. zero (0) donor rated their experience on either trima accel device as poor. 100% of donors who responded to the question said they would donate on trima accel 7 again. summary/conclusions: no significant difference was observed in donor experience between trima accel version 6 and trima accel 7 as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in 2014, a higher number of the platelets was consumed compare to 2011 by 7.8%. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in 2018, 635 (8.6%) of the 7,371 patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is 14,292, which is 668 higher (by 4.9%) comparing to 2017. platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + 9000 (haemonetics), upp and trima accel (terumo bct) version 6.0 protocols. methods: the data for 4868 protocols of platelet donations performed in 2018 were analyzed: 2773 on trima accel and 2095 on msc + 9000. all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was 32 years old, height -175 cm, weight -76 kg, platelet count before donation -254 9 10 9 /l, hematocrit -42%. detailed data are presented in table 1 . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than 0.05 was considered as significant. results: the data obtained showed significant difference (p < 0.01) between average number of platelets collected on trima accel (6.3 ae 1.06 9 10 11 /l) and on mcs + 9000 (5.09 ae 0.78 9 10 11 /l). while cost of consumables are comparable, trima accel demonstrated 23.7% higher efficiency. procedure duration also was comparable and averaged within 94 min for both devices. detailed data are presented in table 2 . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during 2018 and reached 56, 7% in total (2017-19.4%). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from 7.64% up to 43.7%. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by 23.7% compared to mcs + 9000 while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from 2010 till 2019. all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ 3.0 9 10 11 equal to 12 random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was 150.000/ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was 273.000/ll, with range from 182.000/ll to 397.000/ll. male were 77% of the donors and females were 23%. the single procedure usually took 45-70 min. the median platelet count collected was 4.0 9 10 11 , range 2-6.5 9 10 11 . the median processed blood volume was 3215 ml and median used acd-a was 352 ml. mean total volume of collected product was 312 ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso4) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso4 solutions with specific weight (y) 1.053 can to detect value of hemoglobin above or same with 12.5 gr/dl. but, cuso4 solution specific weight 1.053 can not to detect and elimination value of hemoglobin above 17 gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso4) with a specific weight (y) 1062 to detect value of hemoglobin below 17gr/ dl and determine value of hemoglobin above 17 gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above 17gr/dl with the test of 35 samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso4 solution with specific weight 1.062 in to detect and elimination value of hemoglobin donors above 17 gr/dl. methods: used the method cyanmethemoglobin and cuso4 (y) 1.062 determination value of hemoglobin donor. test results were analyzed with spss software version 23 using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso4 solution with specific weight (y): 1.062 in detecting and eliminating hemoglobin value donors above 17gr/dl. from data processing using spss with the wilcoxon test p value 0.02. summary/conclusions: it was found that the cuso4 solution (y): 1062 can detect hemoglobins value above 17gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version 23 with the wilcoxon test p value < 0.05. it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, 8194 whole blood donors (20,864 whole-blood donations) were included in the zinc study, 63% being female donors. previous zpp showed a statistically significant association (p < 0.001) with hb levels in females, but the size of the association was quite small (regression coefficient, b = à0.17, 95% confidence interval à0.22 to à0.11). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was 66 years until the end of year 2013 and since beginning of 2014 donors between 66 and 70 years have been able to donate blood. blood donation after the age 66 is possible if the donor has donated within the last 24 months. the upper age limit was raised up based on adverse event data from frcbs donors up to 66 years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from 2013 with upper age limit of 71 years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than 66 years have more adverse events compared to other donors. the most common donor adverse event for donors over 66 years, haematoma, was registered 211 times (0.05%). in the other age groups haematoma was registered 4408 times (0.56%) and the difference between the oldest age group compared to all other donors was statistically not significant (chi2, p = 0.012). vvrs with loc were registered 21 times (0.05%), vvrs without loc 25 times (0.06), and the total number of all daes was 293 (0.65%) in the age group 66 years or older. the respective numbers in the other age group were: 1152, 0.15%; 11055, 1.41%; 17550 (2.23%). the number of vvrs with and without loc, and total number of all daes in the age group 66 years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi2, p < 0.0001). summary/conclusions: donors over the age of 66 years have less donor adverse events than other age groups. decision to raise the age limit from 66 to 71 seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in 2016-2018 years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last 3 years (2016-2018 inclusive) were reviewed to look for reports of dvt post donation results: a total of 135 saeds were reported in uk from approximately 5.8 million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for 2% of the saeds reported and rate of dvt of 1 in 1.9 million donations collected. -case 1: a regular male whole blood donor in his early 30s reported worsening arm pain 12 days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case 2: a female donor in her mid-40s gave her sixth whole blood donation without event. 11 days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case 3: a female donor in her 20s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february 2013 to march 2015. the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study 6,955 whole blood donors were included, during the period of 2 years, 105 (1.50%) adverse donor reactions were reported. majority 89 (84.76%) of adverse donor reactions were mild in nature such as, sweating; 27 (25.72%), light headedness; 19(18.09%), nausea and vomiting; 15(14.28), allergy and bruises; 11(10.47%), sore arm; 9(8.58%) and hematoma; 8(7.62%) while 16 (15.24%) were severe adverse reactions similarly, anaphylaxis; 11(10.49%), loss of consciousness; 3(2.85%) and convulsive syncope; 2(1.90%). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with 18-23 years old at higher risk compared to 24-55 years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th1/th2 cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, 2008; ellertsen & hetland, clin mol allergy, 2009). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for 7 weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm (82%), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v 1-induced basophil activation in whole blood determined by cd63 expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct03198455, clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v 1 and anti-t3, were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for 2 months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in 20/9/2014. the data presented in this abstract is till 28/2/ 2019. data is collected from total of 114475 blood donors (86.67% male donors and 13.33% female donors). repeat donors accounted for 59.34% against 40.66% of first time donors. of the total number of donor adverse events recorded, 2.73% (2633) reported for male donors and 6.97% (1063) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group 18-24 years (male 5.45% and female 13%). among age group 25-40 years, (male 2.4% and female 4.02%), whereas in age group 41-65 years, (male 1.52% and female 2.11%) data analysis of total 6226 reported and registered donor adverse events, are categorized as hyperventilation (864), sweating (1458), dizziness (pre-syncopal 3160), loss of consciousness (416), vomiting (108), convulsions (220), hematomas with re-bleed (255), nerve irritation (8) and off-site reactions (333). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january 1 st 2016 to december 31 st 2018 and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january 1 st 2016 to december 31 st 2018 was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into 8 categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < 0.05 was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, 193,228 donors were registered from january 1 st 2016 to december 31 st 2018 and 41,037 (21.24%) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from 24.07% (15302/63563) in 2016 to 20.79% (13180/63394)in 2017 and further to 18.74% (12419/66271) in 2018 (p < 0.05). the specific deferral rate due to low hb also significantly (p decreased during these three years (86/1000 in 2016, 68/1000 in 2017, 58/1000 in 2018), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june (6/1000) and then increasing with a peak in october (19/1000) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of 4/1000 in june and increasing to 10/1000 in october (p < 0.05). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels 2017 thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of 28 days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from 2015 to 2018 on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the 7 days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in 2015-2016 and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in 2015-2018, 16,400 donation attempts led to onsite deferral for wnv risk; 87% at whole blood donation attempts, 13% at new donor examinations. in total 19,624 offsite deferrals could not be traced directly to a donation, but based on the next donation more than 90% were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from 920 in august 2015 to 1711 in august 2018. this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred (92% versus 95%); for wnv deferred new donors the return rate is 75% (versus 87% for no deferral). thus wnv deferral resulted in approximately 400-500 extra lapsing donors during these 2 years. however wnv deferred donors, that are older (odds ratio (or) 1.03; 95% confidence interval (95% ci) 1.02-1.03), of male sex (or 1.16; 95% ci 1.03-1.31) and whole blood donors as opposed to new donors (or 2.01; 95% ci 1.70-2.38) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since 2012 with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given 14 days of one tablet of iron supplementation (100 mg elemental iron) since 2012. an electronic questionnaire was sent to blood donors in 2017 to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: 3212 donors (male to female was 1:1.9) replied to the questionnaire. of them, 594 received iron supplementation (male to female was 1:1.6). most of the respondents (94%) had one or more donations in the preceding 12 months. of the 594 donors received iron tablets, only 246 (41%) took all; 69 (12%) took more than 50% but not all; 130 (22%) took some but less than 50% and 149 (25%) did not take any. gastrointestinal upset was reported in 113 (25%) donors and constipation seen in 193 (43%) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than 50%) to those who did not (took less than 50%), significantly more donors in the former knew vitamin c could enhance iron absorption (p < 0.05). on the other hand, no difference was seen when they were asked if 1) iron can only be absorbed from meat; 2) tea and coffee consumed during meal can enhance iron absorption; 3) everyone can take iron supplementation on their own; and 4) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by 27% for whole blood donors and 22% for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with 36 whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor 10,000 study in 2015. we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may 2015 and december 2017. participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer 2018. we included the 1435 participants (596 men and 480 premenopausal and 359 postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = 31), good (n = 483), very good (n = 684) or excellent (n = 237) health tended to report improved (58%), similar (63%), similar (55%) or poorer (55%) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal 51%, post-menopausal women 38%), or = 1.37 95% ci 1.02-1.85). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in 2018. methods: the data of volunteers from january to december 2018 were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were 9081 blood donors in 2018, 69 (0.76%) of whom had adverse reactions and 9 causes were induced, among which mental stress was the most common factor that accounted for 78.26% (54 cases). there was no significant difference in the incidence of adverse reactions between men and female (p > 0.05). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < 0.01). when it comes to age, the incidence was different and the 18-25 age group was the highest (1.61%). among different blood group donations, there was no significant difference (p > 0.05). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least 8 weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to 24 times per year (if spaced more than 14 days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥4 whole blood donations or 16 plasmapheresis or plateletpheresis donations, and female donors with ≥3 whole blood donations or 12 component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past 2 years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ 2.07. the repeat donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of 53 male and 57 female repeat donors were recruited, and 30 each male and female ft-ra donors were recruited to the control group. after 4week iron supplementation, among male donors, the prevalence of: low hb level (<13.0 g/dl) decreased from 24.5% to 1.9%; low ferritin level (<15.0 ng/ml) decreased from 58.5% to 3.8%; high tibc level (>380 lg/dl) decreased from 66.0% to 37.5%; low transferrin saturation (<20.0%) decreased from 58.5% to 35.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 28.3% to 2.3%. among female donors, the percentage of: low hb level (<12.0 g/dl) decreased from 43.9% to 9.8%; low ferritin level (<15.0 ng/ml) decreased from 78.9% to 11.8%; high tibc level (>380 lg/dl) decreased from 68.4% to 24.5%; low transferrin saturation level (<20.0%) decreased from 70.2% to 22.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 94.7% to 44.4%. in total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. a total of 38 male (71.7%) and 36 female (70.8%) blood donors were administered iron supplementations for 28 days. 89 participants (85.6%) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, 4-week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than 3 mg/l has been independently associated with a 60% excess risk in incident of coronary heart disease (chd) as compared with levels less than 1 mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci4100 (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl 180, erba mannheim). data was analysed using stata version 15 (stata corp) statistical software. results: in total of 313 blood donors, 278(90.8%) were males and 28(9.2%) were females, the mean age was 32.3 ae 9.3 years. two hundred and thirty four (74.8%) were first time donors and 79(25.2%) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors (0.95 ae 3.7 vs 0.35 ae 1.7 mg/l, p = 0.06) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors (87.56 ae 23.9 vs 46.44 ae 31.4 ng/ml, p = 0.02). interestingly, levels of serum hs-crp were significantly higher in male than female population (0.52 ae 2.4 vs 0.17 ae 0.2 mg/l, p < 0.03) and smokers than non-smokers (1.4 ae 4.6 mg/l vs 0.4 ae 1.9 mg/l, p = 0.02). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes 250 mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = 13.5 g/dl for male and > or = 12.5 g/dl for female • inter donation interval = 56 days • 5 donations/year for male and 3/year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: 1056 first time and regular donors accepted to enroll in this study. only 38 female donors (3.6%) participated to this study. 11.3% of the participants were first time donor. 21% of male and 26% of female frequent donors are iron deficient 173 out of 1018 male blood donors were iron deficient (17%) with serum ferritin < 30 ng/ml. 98.8% were repeat donors. 7 out of 38 female donors were iron deficient (18.4%) with serum ferritin < 13 ng/ ml, all were repeat donors. 19.2% of repeat donors were iron deficient 3/180 of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a 12-month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years 2014-2018 in the group donors of aged 18-24. we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years 2014-2018 the total number of 1904 adverse reactions among the donors was recorded which is 0.39% of the total number of collected donations. 50% of the adverse reactions occurred in the group of donors aged 18-24. vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled 50.2% of all adverse reactions. in the group of donors ages 18-24 it totalled 27% of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled 29.8%, in the analysed group of donors 15.96%. vascular reactions (bruises) totalled 10.2% of all adverse reaction, in the analysed group 5.6%. the remaining adverse reactions totalled 9.8%. summary/conclusions: 1. vasovagal reactions (with and without fainting) were proved to be 2 most common adverse reactions in the group of donors aged 18-24 i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. 2. it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. 3. it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" (2 nd day) and "old" (42 nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd235a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd235a, cd44, cd47, cd55, cd59, and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0.5 lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units (201 ae 139 mvs per 1 ll), as compared to the microvesicles in the "fresh" (67 ae 53 mvs per 1 ll). at day 2, the microvesicles had elevated expression levels of cd47 and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd55 and cd59 molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of 42-day vesicles. the phagocytosis index was significantly higher (28.44%) for the microvesicles isolated from 42-day stored rbcs than for microvesicles from the 2background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of 5-7 days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to 7 days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in 30% plasma/70% ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into 3 units (n = 6 of each) on day 2 and stored either at room temperature (rt; 20-24°c with agitation) for 5 days, cold stored for 21 days (2-6°c no agitation) or cryopreserved (à80°c with the addition of 5-6% dimethyl sulfoxide) for 33-109 days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at à80°c. the mean size and concentration of microparticles were measured using nanosight ns300 nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < 0.05 was considered significant. results: at day 2, sheep pcs had a microparticle concentration of 3.77 9 10 10 ae0.84 9 10 10 microparticles/ml with a mean size of 156.6 ae16.7 nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration (4.39 9 10 10 ae0.81 9 10 10 microparticles/ ml; p = 0.0087) and the mean size (170.9ae 20.5 nm; p = 0.0008) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day 21 was comparable to room temperature pcs stored for 5 days (165.4 ae17.5 nm vs. 163.6 ae20.9 nm; p = 0.4081 and 4.11 9 10 10 ae 0.54 9 10 10 microparticles/ml vs. 3.82 9 10 10 ae0.71 9 10 10 microparticles/ml; p = 0.16 respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the 21 days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and 2,3-dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p50: the oxygen tension (po2) at which 50% of the hemoglobin is saturated with o2. an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po2. this relationship is dependent on temperature, ph, pco2 and 2,3-dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p50 is at a po2 of about 26 mm hg. not much is known about p50 of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. 2010;50:2386-92). methods: rbcs were prepared in sagm (n = 3) or pagggm (n = 3). pagggm is designed to better maintain both atp and 2,3-dpg during storage. rbcs were stored at 2-6°c and sampled on day 1, 35 and 56 for (internal) ph, atp, 2,3-dpg and p50. p50 was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment (2%) using n2 gas. p50 was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. 2,3-dpg content of sagm-rbcs decreased during storage and was below the detection limit after day 14. 2,3-dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day 7 on. at day 56, pagggm-rbcs still contained 2.3-dpg (2.2 lmol/g hb). p50 values decreased during storage from 26 mmhg at day 1 to 20 mmhg at day 56 for sagm-rbc and from 31 mmhg to 26 mmhg for pagggm-rbc. p50 values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p50 decreased in all rbcs. the p50 was higher for the pagggm-rbcs during the whole storage period. the higher p50 in pagggm-rbcs seems to correlate with the higher 2,3-dpg content in these cells. background: in belgium 100% of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of 3.0 9 10 11 per platelet concentrates (pc). therefore routine pools are produced with 6 buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from 6 to 5 bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with 5 bc and 250 ml platelets additive solution (pas) instead of 280 ml for 6 bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ 3.0 9 10 11 platelets with a ratio plasma/pas between 32 to 47% required for pi. after validation, deploy a dual pooling strategy (5 or 6 bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce 44 ml bc with 40% haematocrit (htc) and > 90% platelets recovery with average platelets content of 1 9 10 11 . 5 random bc are pooled with 250 ml or 6 bc are pooled with 280 ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl 80; horiba). results: during the study 45594 bc were processed into 8225 pools (3756 (45.7%) with 5 bc and 4469 (54.3%) with 6 bc). before tacsi separation, bc mixture with pas-e had volumes of 421 ae 9 ml (5 bc) and 491 ae 8 ml (6 bc) with respectively htc of 19 ae 1% and 20 ae 2%. the plasma/ pas ratio was 37 ae 1% in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was 3.8 9 10 11 ae 0.5 with 5 bc and 4.7 9 10 11 ae 0.5 with 6 bc (average ae standard deviation). pools below the limit of < 3.0 9 10 11 were 139/3756 (3.7%) with 5 bc and 1/4469 (0.02%) with 6 bc. the platelets concentrations (10 3 /ll) were 1395 ae 167 (5 bc) and 1491 ae 155 (6 bc). platelets recovery was 85% ae5 for 5 bc and 86% ae 6 for 6 bc. summary/conclusions: 45594 bc could theoretically produce 7599 pools of 6 bc or 9118 pools of 5 bc. this means a maximum potential gain of + 20% pc. in practice during shortage periods we switched from 6 to 5 bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods (626 pc, +8%). the disadvantage of pooling randomly 5 bc is that 139 pools contained less than 3.0 9 10 11 platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the 5 bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica 2018, pmid: 30115655). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and 4°c in the presence or in the absence of caspase-3 inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: 13 ae 1% vs. 5 ae 1% p = 0.018). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: 6.13 ae 1.89 vs. 18.53 ae 3.64, p = 0.038). accordingly, a decrease of the procaspase-3 level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase-3 inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: 74 ae 3% vs. 22 ae 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o 2 , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o 2 (so 2 )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within 24 h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for 3 h on an orbital shaker (50 rpm) at 22°cae2 and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at 4°c. a g-irradiation treatment (25 gy, gammacell 3000 elan, theratronics) was applied at day 2 or 14 and the rccs (expiry dates at day 16 or day 28, respectively) were stored until day 43. hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so 2 values were of 63.7%ae18.4 (n = 12) in control and of 20.8%ae9.8 (n = 12) in treated bags, and reached 90.8%ae9.1 and 6.6%ae5.9 at day 43, respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around 70 mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day 2 and the values stayed equivalent through end of storage (at day 16, hemolysis (control) = 0.37%ae0.24, hemolysis (treated) = 0.34%ae 0.15, p-value > 0.9999). when irradiated at day 14, hemolysis was lower (p-value = 0.033) in treated rccs at the end of storage (day 28, 0.67%ae0.16) compared to control (1.06%ae0.33). seven days post-irradiation, two-third of the control rccs were above the limit of 0.8% whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o 2 content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day 2 and was an advantage when irradiated at day 14. importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o 2 depleted rbc. in summary, the reduction of o 2 level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, 2017] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as 0.5 ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a 3d printed support. the accuracy of flow rate measurements between 0.5 ml/min and 1.5 ml/min was evaluated as well as the optimal measurement time. for 4 different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under 8% was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under 3% for a measurement time of 2s. the mean error is still under 5% for a measurement time of 0.5s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags 2, 3 and 4. hematocrit estimation for bag 1 diverges from reference for values above 6%. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under 8% without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood (450 ml) was drawn into compoflow blood bags containing cpd and sag-m from 10 healthy controls and 10 newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing 20 ml prp (range 270-320 9 10 9 platelets/l). platelet quality tests were performed on days 0, 1, 3, 5 and 7 of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd41, cd42b, and cd62p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd40l and scd62p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included 7 males and 3 females. the mean age was significantly lower in the control group, 35 years (22-55 years), than in the hh group, 55.7 years (29-77 years) (p = 0.004) while ferritin levels were significantly higher in hh patients (median 847.5, range 498-1889) than in controls (median 45.8 ng/ml, range 9.28-97 ng/ml) (p < 0.001). in the hh group, 5 each had the c282y/c282y and c282y/h63d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < 0.05) with the exception of glucose (higher in hh patients on all time points, p < 0.05). platelet aggregation and the expression of activation markers (cd62p and cd42b) on platelets and in the supernatant (scd62p and cd40l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd42b expression decreased and cd62p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = 16) were collected using a trima accel platform in 40% plasma/60% pas (ssp+). after resting for 1 h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (<6 h; t0), and the others remained within the shipper, without agitation. the second component was removed at 6 h post-collection (t6), and the third was removed at 23.5 h post-collection (rounded up to 24 h; t24). platelets were tested on day 1, 5 and 7 post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < 0.01 was considered significant. results: platelets held without agitation for 24 h consumed significantly more glucose than those removed at 6 h or immediately upon arrival (p < 0.0001), even on day 1 post-collection. this was accompanied by increased lactate production (p < 0.0001), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t24 platelets (p < 0.0001), and on average it was 0.4 ph units lower than in platelets held in the shipper for 6 h or less. however, the ph remained above 6.9 in all components. mean platelet volume was also reduced in t24 platelets (p < 0.0001), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd62p and microparticle generation were significantly higher in the t24 platelets throughout the storage period (all p < 0.0001), suggesting platelet activation. release of scd62p was also increased in t24 platelets (p = 0.0006), whereas extended storage in a shipper did not affect release of rantes (p = 0.4488). adp-induced activation of glycoprotein iib/iiia, measured by pac-1 binding, was decreased in t24 platelets (p < 0.0001), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = 0.0001) and adp (p = 0.003) were significantly lower in t24 platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for 24 h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to 5 days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after 5 days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, 9, 2018) . our new target is to extend this research in 2 extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from 5 to 7 days. methods: in this study, platelets were collected from 11 normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of 450 ae 10 ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc 1200). samples were drawn aseptically with a needless access coupler (cair-lgl) on days 1, 5, and 7. platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day 5 and day 7, maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days 5 and 7 of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of 7 days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at 4°c or room temperature (rt) over 10 days for 4 apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with 5% or 10% dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either 5% or 10% dmso. results: for pcs standard quality assurance tests did not show a major difference between 4°c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day 1-7, p < 0.001 and day 10, p < 0.02). for red cells, we found by rt-dc no impact of gamma irradiation with 30 gy over the entire storage period of 42 days assessing 3 different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation (0.052 for 5% dmso versus 0.034 for the control without dmso; p < 0.00004). however, this did not differ to high extent whether 5% or 10% dmso were used for cryopreservation (0.035 and 0.041, respectively; p < 0.02). hpsc viability was lower after cryopreservation using 10% dmso in comparison to using 5% dmso. overall, blood cell regeneration is comparable between 5% and 10% dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight (16 h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day 0 was separated into plasma, bc and red cell concentrates either at day 0 or at day 1. bcs were then stored until the pooling step at 20°c without agitation and pcs were prepared at day 1 by pooling 5 isogroup bcs. seven "16 h-pcs" were prepared from bcs stored for 16 h (whole blood separation at day 0) and six "90 min-pcs" from bcs stored for 90 min (whole blood separation at day 1). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd42) and of activated platelets (marked with cd62 the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: 32 dd-bc-pc were prepared with 8 bc and 280 ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a 600 ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within 1 h of preparation and after storage for 8 h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po 2 , pco 2 , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = 32) was on average 6.9 ae 0.3.10 11 in a volume of 393 ae 6 ml. the mean of plasma ratio was 42% [min: 39.3max: 43.9]. all pc contain <1.10 6 wbc [min: <lq; max: 0.38]. po 2 decreased from 166 ae 12 mmhg after separation (1 h) to 44 ae 18 mmhg after 8 h, ph (+22°c) and pco 2 was stable during the same time period. glucose decreased from 9.0 ae 0.3 mmol/l (1 h) to 8.7 ae 0.3 mmol/l (8 h) while lactate increased from 6.07 ae 0.46 mmol/l (1 h) to 6.57 ae 0.54 mmol/l (8 h). the vpm was stable with 7.6 ae 0.2 at 1 h and 8 h. ldh increased from 120.4 ae 8.6 u/l at 1 h and 125.8 ae 10.3 u/l at 8 h as p-selectin 45.7 ae 7.6 ng/ml at 1 h and 57.7 ae 7.0 ng/ ml. all units have maintained swirling. the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in 2018 the ibts collected 130,183 whole blood units and performed 9,654 platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september 2017, the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years 2016 to 2018 were analyzed. in 2016, the ibts prepared 5,609 orbisac platelet pools of which 5,477 were qc tested. in 2017 5,031 orbisac and 1,625 tacsi platelet pools were prepared and tested. in 2018 6,783 tacsi platelet pools were prepared of which 4671 were qc tested. qc data comprised pc volume, platelet content (10 9 /unit), residual leucocyte content (10 6 /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the 10,508 o-pcs tested in 2016 and 2017, mean pc volume was 292 ae 9 ml. initially the t-pcs mean volume was 273 ae 13 ml but more recently the volumes were increased by addition of a larger volume of additive solution to 303 ae 13 ml. platelet content (10 9 /unit) was significantly improved in t-pcs (384 ae 47) compared to o-pcs (356 ae 50) [p < 0.01] as well as residual leucocyte content (10 6 /unit) which was lower in t-pcs (0.05 ae 0.04) compared to o-pcs (0.1 ae 0.2) [p < 0.01]. ph at expiry was lower in t-pcs (7.32 ae 0.07) compared to 0-pcs (7.38 ae 0.06) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately 48 t-pcs per hour compared to a lower throughput of 20 o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a 10% increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga 1 , f puente 1 , a aranda 1 , j domingo 1 , l call en 1 and a bah 2 1 banco de sangre y tejidos de arag on, aragon, spain 2 terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes 44,000 whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november 2012 and routine use started in july 2013. in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november 2012 to improve transfusion safety. in november 2015, the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in 2012, 369 whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from 2013 to 2018, qc data of rccs (n = 3025), plasma (n = 660) and pcs (n = 660) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from 2015 to 2018 were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: 296 ae 21 ml vs 251 ae 19 ml; hb: 59.9 ae 6.2 g/ unit vs 49.2 ae 7.8 g/unit; hct: 59.5 ae 2.7% vs 56.1 ae 6.3%). control plasma units had higher volume (280 ae 17 ml) and lower residual platelet content (9 ae 7 10 9 /l) compared to reveos plasma units (volume: 249 ae 23 ml; residual platelet content 19 ae 13 10 9 /l). for pcs, volume was higher for units processed with reveos compared to control (350 ae 20 ml vs 310 ae 10), but no statistical difference was observed in platelet content (reveos: 361 ae 60 9 10 9 /unit; control: 386 ae 80.10 9 /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the 5 years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from 4% to 0% between 2013 and 2018 and platelet content in pcs increased from 370 9 10 9 /unit (first six months in routine) to 390 9 10 9 /unit. plasma volume increased from 249 ml during validation to 267 ml in 2018. survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from 10% in 2015 to 59.7% in 2018 with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations (450 ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm (100 ml) and then filtered by gravity in average 2-3 h after blood collection to obtain leukoreduced rbc. average temperature during filtration was 26°c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = 27) were performed. rbc characteristics post-filtration and separation were as follows: volume (266.8 ae 17.1 ml), hematocrit (57.8 ae 2.1%) hemoglobin (19.8 ae 1.1 g/dl) and hemoglobin per unit (53.0 ae 5.4 g); residual platelets (11.7 ae 2.2 x10³/ll); residual wbcs (0 à 0.004 9 10³/ll). the mean filtration time was 19 ae 3 min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between 2 and 8 h from collection using the 3c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day (42 nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate 4 whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in 2018. the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to 8 h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, 1005 therapeutic units of lpcs were produced after overnight hold: 38 pools of 4 ipus and 966 of 5 ipus were made using platelet pooling set (terumo bct), using 250 ml ssp+ solution (macopharma). results: 42 therapeutic units of lpc were analyzed. the mean volume of lpc was 387 ae 13 ml, platelet content was 3.67 ae 0.46 x10 11 , and white blood cells (wbc) count was 0.15 ae 0.13 x10 6 , 100% all lpcs had wbc below 1 9 10 6 . ph measurements were made on the fifth day of storage, the ph was determined in 15 units of lpc and was 7.2 ae 0.04. summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for 5 days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term (10 days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about 250 ml) were obtained from a healthy blood donors of type o blood group and divided into a 30 ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd62p, gpiba (cd42b), annexin v), degree of reactive oxygen species and sialidase activity on the 7th, 10th and 12th day, and phagocytosis using the hepg2 cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the 2nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day 12 and sialidase activity was maintained lower than that of control group on day 12. the degree of phagocytosis by the hepg2 cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for 2 h and 24 h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos 3c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of 2 h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within 8 h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately 4 to 6 ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool (4 units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately 19 units of whole blood using the reveos system and approximately 4 units of pooled platelet products. a total of 19 units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the 19 units, 10 units have been processed on the reveos device using the fresh procedure (within 8 h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a 22-min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn 2000 cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than 1 million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = 4) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax 20 (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including 40 units in 450 ml quadruple bags (450q) with/without integrated leucofilter and 18 units in 350 ml quadruple bags (350q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into 2 groups, mixed at 2 revolutions per minute (rpm) for 2 min (n = 30) and 30 min (n = 28), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both 450q and 350q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for 2 or 30 min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for 30 min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as 3, 5 or 10 min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year 2000, universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs2 for rcc in comparison with sepacell tm r-s11 and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over 40,000 whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from 5 (routine in italy) to 4. the 4-bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the 4-bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from 4-bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory (2.0 9 10 11 cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february 2018 to february 2019. study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version 23.0 results: a total of 2880 blood products were available at our institute including 960 each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was 3.5%. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. 102(10%). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with 02 common causes, including the expiry of unused products 60(59%) followed by broken bags 30(29%). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early 2000s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of 1 liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of 2010-2017. results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than 200 blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are 18 blood centers in the republicone for each of the 18 administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since 2012, the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in 100% of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group 0 rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis 30 units of pooled platelet concentrate derived from buffy coats and 30 units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl 80 analyser. 1. pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: 4.12 9 10 11 /unit ae 0.50 the number of platelets after the reconstitution: 3.40 9 10 11 /unit ae 0.50 the loss in the number of platelets: 17.5% 2. platelet concentrates derived from apheresis the number of platelets before the reconstitution: 3.68 9 10 11 /unit ae 0.55 the number of platelets after the reconstitution: 2.91 9 10 11 /unit ae 0.49 the loss in the number of platelets: 20.7% summary/conclusions: 1. reconstitution results in the loss of 17.5% of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. 2. the average number of platelets after the reconstitution is 3.4 9 10 11 /unit, which fulfils the quality requirements. 3. reconstitution results in the loss of 20.7% of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. 4. the average number of platelets after the reconstitution is 2.9 9 10 11 /unit, which slightly deviates from the quality requirements. 5. the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf4 was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf4 antibody on the column. hereafter, pf4 was purified with concentration tubes having 5 kda molecular weight cut-off and dialyzed via dialysis tubing with 3.5 kda cut-off. pf4 concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf4 specificity. molecular weight of obtained pf4 was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf4 had 30 kda molecular weight which is corresponding with a tetramer pf4. in addition, elisa showed that the pf4 qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf4. summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf4. nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf4 is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf4. unused human pcs and specific anti-pf4 antibody can considered as an alternative to separate the pf4 particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first 2 weeks of storage at 4°c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from 7 days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: 34 units of whole blood were leucodepleted within 24 h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for 7 days at 4 ae 2°c. on the 8 th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of 35 days at 4 ae 2°c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, 2.3 dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units (0.23 ae 0.2.10e6/u)) while maintaining 80% of the initial platelet content (85 ae 20.10e9/u). mean volume (464 ae 24 ml), hemoglobin content (54 ae 6 g/u) and hematocrit (36 ae 3%) was within conformance range. after 7 days of storage, 50% of the initial platelet content at collection is maintained and mean hemolysis is 0.27 ae 0.15%. rcc processing at day 8 was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume (266 ae 20 ml), hematocrit (56 ae 3%), hemolysis (0.18 ae 0.19%) were in conformance with mandatory standards at day 8. after 7 days of storage (i.e. day 14 after collection), rcc quality parameters were maintained with however an increase in hemolysis (0.34 ae 0.36%) which was above usual values routinely observed with rcc produced from day 1 processed units. 3 rcc out of 34 were above the maximum limit of 0.8% at day 14 after collection such increased hemolysis was more pronounced at day 28 after collection (0.70 ae 0.45%) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold (20% of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after 7 days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within 21 days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland 1,2 , f mahmood 3 , l nissen-meyer 1 and i nentwich 1 1 immunology and transfusion medicine, oslo university hospital 2 immunology, university of oslo, oslo 3 immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, 2005) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were 1) to assess sensitization profiles against an array of 55 allergens in 100 randomly selected blood donors and 2) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from 100 blood donors randomly selected on one donation day outside the pollen season. we used 300 ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for 55 analytes (euroline atopy screen, euroimmun, germany), which comprised 28 food allergen extracts and single allergens, 24 inhalant allergen extracts, 2 insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of 44 serum samples for ige against 55 allergens in less than 3 h. results are expressed as east (enzymoimmunoassay) classes: class 0 (no sensitization), 1-2 (weak sensitization), 3 (moderate sensitization), 4-5 (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class 5none detected. class 4, sesame seed: 1. class 3, apple 1; hazelnut 2. class 2, apple 1; almond 2; hazelnut 1; sesame seed 1; soybean 1; cow milk alpha-lactalbumin 3. class 1: 26 donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class 5, 4 and 3 none, class 2, wasp venom 7; honey bee 2. lower classes (0-1) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor (1%) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to 5 days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = 53) were collected, stored at 22°c with agitation, and tested at day 1, day 5 and 7 post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd61 + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a 2 (txa 2 ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin-1 (rca-1; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to 4-fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to 4-fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only 27% of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa 2 was also higher in the arachidonic acid responders (p = 0.027). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day 5 was weakly associated with donor age (p = 0.009, r 2 =0.2613) but not bmi (p = 0.9143, r 2 <0.001), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than 10%) are b-thal-het that meet the criteria for blood donation (hb > 13.5 g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from 10 healthy non-smoker donors (5 b-thal-het carriers and 5 controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca 2+ and proteasomal activities were determined. for statistical analysis, significance was accepted at p < 0.05. samples from the red cell units were collected aseptically, processed (dual centrifugation at 3,000 g for 15 min) and stored at à80°c. processed samples were thawed, and then analysed using the nanosight ns300 nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d2, red cell units contained an average of 8.1 9 10 9 ae 4.0 9 10 9 mvs/ ml. the mean size of these mvs was 112.8 ae 10.5 nm and the mode size was 79.9 ae 10.3 nm. the concentration of mvs increased gradually throughout storage (p = 0.0276), reaching a maximum at d42 of 32.4 9 10 9 ae 1.8 9 10 9 mvs/ml. both the mean (p < 0.0001) and mode (p < 0.0001) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d2 vs. d7: p < 0.0001 for both mean and mode). by d42, mean and mode size of mvs was 176.1 ae 4.8 nm and 171.8 ae 5.6 nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than 400 nm in red cell units. both the concentration and size of mvs present in red cell units increased during the 42 days of routine storage. the concentration of these mvs was approximately 100-fold higher than we had previously detected using flow cytometry (aung, pathology, 2017) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than 24 h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six 14 day old leukocyte depleted red blood cells (ld-rbc) and six 4 day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated (25 gy). a volume of 350 ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for 6.5 min at 2888 9 g at 4°c (hettich roto silenta 630 rs) before removing the supernatant using compomat g5 extractor. wash procedure was repeated twice using 450 ml sagm solution, and after removal of the last supernatant, 100 ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after 24 h, 7 days and 14 days storage at 4 ae 2°c. results: all washed ld-rbcs met european specification for haematocrit (0.50-0.70) and all but one for hb content (≥ 40 g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than 0.8%) 14 days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration 14 days after washing and irradiation did not exceed those levels found at the end of shelf life (day 35) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than 0.05 mg/dl (0.5 mg/l). our iga method 0 s lower limit for detection is 0.05 mg/l and all results were below this level. total albumin were well below finnish specification (<250 mg/unit). background: room temperature has been the standard storage condition for platelets since the 1970s, when it was shown that this improved in vivo survival compared to when stored at 4°c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at 4-9°c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from 5 buffy coats and 300 ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for 23 days at 4-9°c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d1-d16: 7.05-6.77 with platelet count 400 9 10 9 /u and glucose still at 2 mm at least until 16 days of storage. platelet activation maintained acceptable levels with cd62p expression < 30%, while ps exposure increased rapidly; >20% after 6 days of storage. aggregation tests showed functional platelets until 13 days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at 4-9°c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at 4-9°c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets 0 rhd negative were frozen in 5-6% dmso and stored at à80°c for 6 months. cpp were thawed at 37°c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was 24%. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount 194 9 10 9 /unit, concentration 0.73 9 10 9 /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value 3 in fp and on value 1 in cpp. the average plasma content in fp was 31% compare to 3.22% in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low (0-1:2). cpp had faster clot initiation (rotem clotting time (ct) in cpp 69.2 s, fp 81.8 s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp 39.8 mm, fp 53.2 mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of 24 ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, 250 ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept 1 h on bench in room temperature followed by 1 h on agitator at 22 ae 2°c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of 420 ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of 7 days. results: during validation of the method, 12 pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po2, pco2 and lactate on day 2, 5 and 7 of storage. the platelet count was 2.27 ae 15 9 10 11 per unit on day 2. the ph value was 6.9 on day 2, 7.1 on day 5, and 6.9 on day 7. the glucose concentration decreased from 5.8 to 3.5 and 1.6 mmol/l on day 2, 5 and 7, respectively. the mean po2 level was 25.3, 25.0 and 28.7 kpa while the mean pco2 was 3.8, 1.7 and 1.8 kpa and the lactate concentration was 5.9, 9.6 and 12.9 mmol/l on day 2, 5 and 7, respectively. since routine implementation of the method in april 2017, regular quality controls showed an average of platelet count of 2.63 ae 34 9 10 11 (n = 161) with a volume of 204 ae 7 ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of 7 ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from 11 published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested 4-10 ml sample volume per culture bottle. the 11 studies classified results based on aabb bulletin 04-07 definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta 3d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of 217,932 platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day 3, 4, or ≥ 6, and represented 8.9%, 44%, and 47% of the units tested, respectively. true positives were detected in 174 platelet units representing 0.08% of the total platelets tested. the majority were reported from platelets tested on day ≥ 6 with a total of 128. data showed the bta 3d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤27 h with the majority detected in ≤24 h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in 5 of the 11 studies for platelets tested at 3 days and ≥ 6 days with a total false positive rate of 0.27% (range of 0-1.1%). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, 1,136 culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at 3 to 8 days post collection provided evidence that the bta 3d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert 3d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day 3 and up to day 7 when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts (0.5-2 mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with 2 mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~2 nm, medium 30 nm and long~100 nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at 0.5-1.0 mg/ml hsa coated particles with 0.57 ae 0.36 and 0.54 ae 0.39 pg/platelet, respectively. however, the 1.0 mg/ml hsa coating resulted in~100-fold higher binding affinity to platelets than particles coated with 0.5 mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of 80, 220, and 360 pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day 3, by the increased expression of cd36. the percentage of cd36-positive cells among the population of large platelets did not change during storage. on the day 3, increased expression of cd42b and cd62p was detected, but only on large platelets. small and medium-sized platelets had increased cd62p expression only on day 5. the expression of cd42a on total platelets increased significantly on day 3, and stayed unchanged until day 5. the same pattern of cd42a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd41-positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the 5-day storage period. increased annexin and pf4 concentrations were detected on day 5. the concentration of b-tg increased on day 3 of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february 2009, the transfusion service in our hospital has managed 3 autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of 3 types of autologous blood components in a single institution over an approximately 9-year period. methods: between february 2009 and december 2017, we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of 9,193 patients (9% of whom received operations) received blood transfusion, of which 5,080 (55%) received autologous blood transfusion alone, 2,101 (23%) both autologous and allogeneic blood transfusions, and 2,012 (22%) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was 78%. pacs units were transfused to 5,830 patients (60%), pad units to 2,845 patients (30%), and anh units to 982 patients (10%), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was 98.6%. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were 12 and 0.1%, respectively, of which the most common was febrile non-hemolytic transfusion reaction at 9 (75%), followed by allergic reactions at 2 (17%), and hypotensive transfusion reaction at 1 (8%). the severity of adverse reactions to pad transfusion was grade 1 (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was 0.55%, that to frozen pad units was 0.05%, and that to autologous ffp units was 0.10%. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was 78%, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp215 (haemonetics corp.), is approximately 1%. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: 3-40%) for up to 24 h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for 48 h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in 100% plasma were washed on day one after blood collection using the automated closed-system cell processor acp215 (n = 6). wpc, which were rested 1 h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at 20-24°c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was 110.9 ae 1.9 (910 10 /l) and the volumes of the control and test units after the division were 120 ae 11 and 123 ae 12 (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than 6.85 during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco 2 , po 2 , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd62p expression were significantly higher in the test units than in the control units on days three (52.6% ae 6.0 vs 40.9 ae 5.7, p < 0.01); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd42b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a 2-month field trial over 10,000 data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < 0.0001) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ 0.3246). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > 90% of ld components have less than 1 9 10 6 rwbc/unit. for ld failures (n = 14) there was a good correlation (r 2 =0.9848) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around 6 and 8 rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < 0.0001) in rrbc contamination, with lower levels in apheresis platelets (median = 15 rbc/ll, n = 1820) compared to those produced from buffy coats (median = 783 rbc/ll, n = 77) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately 2-3 h for 60-90 samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was 60 to 220 nm (without oxidation). the heights (h) of dips were 4 to 10 nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only 10% to 15% of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from 8 donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn 2+ (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to 600 nm. the young's modulus e = 10 ae 5 kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn 2+ ) significantly increased the absolute value of the young's modulus of rbc membranes up 2-3 times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from 200 nm to 600 nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january 2016 to december 2018, in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january 1st 2016 to december 31st 2018. results: a total of 54,599 whole blood units and 3,166 apheresis platelets were collected during the study period. of the 66,289 blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of 2,891 (4.4%) blood components were discarded, of those 75.4% due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in 2016 was 5.6%, in 2017 was 3.8% and 0.2% in 2018. similar tendency was shown in the pooled platelets for 2 consecutive years with 4.8% (2016) and 3.8% (2017), but with an increase in 2018 (7.7%). the rate of apheresis platelets had a more variable behaviour from 2016 to 2018 with rates of 1.3%, 0.6%, and 0.9% respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from 1 buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, 31 apheresis pcs and 66 pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba1c from red cells. triglyceride levels > 2.5 mmol/l and hba1c levels > 42 mmol/mol were defined as high. results: twenty two percent (21/97) of the donors were marked as 'rapid acidifiers' and 71% (15/21) of these donors had health issues. 'rapid acidifiers' were of age 57, 31-69 (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type 2 diabetes, indicated by high cholesterol and/or triglycerides, high hba1c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' (29%) did not have high triglyceride or hba1c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, 2012). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than 10 visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, 2016). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis (1246.8 ae 90.1/ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from à600 mv to +1100 mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse 80i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + 500 mv and + 800 mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than 10%) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from 4 healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph 7.4. we used ultraviolet (uv) irradiation of blood or nano 2 as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico 2800, usa) to measure the absorption and scattering of light (0.5 nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo2,l c hbo2 l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno2 -,l c methbno2 -l+ e methbno,l c methbno l+k+s/k 4 l (1), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano 2 action (up to 90%). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r 2 = 0.98). incubation of rbcs with cytoflavin leads to reduction of methb to hbo 2 . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr5, the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr5 leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl2 and mgcl2) in ph 7.2 containing 2 mm edta and 4%(w/w) dextran 40 without additive amounts of triton x100 was the most optimized buffering system for lcr5 filter back flushing. total cell content was also determined as 6.28*10 8 granulocytes, 4.27*10 8 lymphocytes and 0.8*10 8 monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis (2019) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of 5.2 g ig (70% intravenous -privigenand 30% subcutaneous -hizentra) and 26 g albumin (alburex) per kg plasma fractionated, corresponding to 998.000 g ig and 4.992.000 g albumin; based on present demand, this represents 97% and 100% self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted 6.000 g fibrinogen (riastap) and 6.000.000 iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of 5.000.000 iu at and 3.000.000 iu pcc, capable of ensuring self-sufficiency for naip regions until 2020. summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in 2017 in italy 923,944 litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, 151 of the 176 italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of 3 parts: a) "surrogate" bags (check-bags) of 250 and 700 ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period 2016-2018, at the pievesestina be of emilia romagna region 210,871 plasma units were frozen so as to allow complete freezing within 60 0 to a temperature below à30°c, in 8,349 freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be 108 tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of 8,349 freezing sessions carried out at the pievesestina be, 27 (0.3%) were detected to fail to reach à30°c at the core of the check-bags within 60 0 . of the latter, in most cases (70%) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (<24 units), ii) labelling time (<8 0 ), iii) optimal storage t (à40°c vs. à30°c), iv) optimal time between two openings of storage sites (>30 0 ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january 2018 -december 2018) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check (6 months) and data with double check (6 months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of 6818 from the first six months a 38 mismatch was found which equals 5573 dpmo and 4.1 sigma metric. and in a sample size of 5854 from the second six months a 25 mismatch was found which equals 4277 dpmo and 4.2 sigma metric. out of the whole 12672 pooled units 63 were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a 4.1 sigma metrics obtained from the first 6 months, were shifted to a sigma metrics of 4.2 in the second 6 months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from 150 ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (à20°c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced 7 mc; 1 mc as the control, 3 mc were lyophilized with excipient and 3 mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at 30 days at room temperature (30-32°c) and blood bank refrigerator temperature (2-8°c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher (9.8 iu/ml) than without excipient (4.5 iu/ml) and the storage at blood bank refrigerator (2-8°c) is better than at room temperature (30-32°c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature (2-8°c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from 2016 to 2017. they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of 27 patients were included (12 patients as control group) and (15 patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph 2.5 after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was 6.99 d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of 4.2 million habitants. annually, it collects about 100,000 whole blood and 3,500 apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for 7 days at 22°c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of 4 bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in 33% plasma and 65% pas and mcd were subsequently evaluated also in 38% of plasma and 62% pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days 1, 5 and 7. bacteria sterility test was performed at day 7 for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = 10) and ad pcs (n = 8) stored in 33% plasma showed at day 7 an average ph ≥ 6.9; swirl ≥ 2.5 and yield = 3.3 9 10 11 . their untreated counterparts showed average values for ph ≥ 7:0, swirl ≥ 2.9 and yield 3.3-3.4 9 10 11 . mcd stored in 33% plasma (n = 6) that underwent prt showed at day 7 average values for ph = 6.7, swirl = 1.2 and yield = 3.05. control mcd showed average values for ph = 7.0, swirl = 2.8 and yield = 3.1 9 10 11 . mcd stored in 38% plasma (n = 8) that underwent prt showed average values for ph = 6.6., swirl = 1 and yield = 3.1. their untreated counterparts had average ph = 7.1, swirl = 2.9 and yield = 3.2. total protein content in pcs derived from wbd (n = 12), ad (n = 15) and mcd (n = 27) was 22 g/l, 20 g/l and 20 g/l, respectively. while the coefficient of variation of wbd and ad ranged from 3% to 4%, plasma products 129 respectively, the one of mcd reached 14%. all prt-pcs were negative for bacterial growth at day 7. summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day 7 and passed the requirements of the eu guidelines (19 th edition). quality of mcd units met eu criteria at day 5; swirl decreased significantly at day 7 which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~25% of the annual produced buffy coat platelet concentrates (bcp) (~40.000 bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~25% pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period (2017) . this cost was offset by substituting a semi-automated production method for bcp, which was used in 2017 to produce 28.7% of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by 89.2%. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in 2018 by 0.19%. the extension of max. storage time from 5 to 7 days for 24% of the bcp-units that were pr resulted in decreasing our overall outdating rates by 29% (versus 2017). this reduction in outdating rates reduced our opc in 2018 by 2.2%. in 2018 we gamma-irradiated 25.9% fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~25% pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day 14 of storage then stored for a further 14 days. rcs were also irradiated on day 5 of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into 4 arms on day 1 of storage, with 10 units in each arm. all units received an irradiation dose of 25.9-48.3 gy. two arms remained as standard volume rcc and were either cor x-irradiated on day 14 of storage. the other two arms were both split into 6 rcs on day 4 of storage before being irradiated on day 5 (early arm) or day 14 (late arm) of storage. for each replicate in these arms, 3 splits were c-irradiated and 3 splits x-irradiated. all arms were tested a day prior to irradiation and 1, 7 and 14 days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and 2,3 dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, 1 split was tested on each testing day post-irradiation. a 2-way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (>75% of units with < 0.8% haemolysis) and 100% of units had atp levels above the recommended minimum for acceptable post-transfusion survival (2.3 lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz 1 , c coello de portugal 2 , m morales 3 , f solano 4 , c perez parrillas 5 , a rodriguez hidalgo 5 , t diaz rueda 5 and m flores 5 1 direccion, regional transfusion center toledo-guadalajara 2 transfusion service, toledo hospital complex, toledo 3 transfusion service, general university hospital of guadalajara, guadalajara 4 transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera 5 regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves 3 general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of 943,000 inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since 2008, rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from 5 to 7 days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last 8 years divided into four periods ( results: pc were predominantly obtained from whole blood collections with 85% of bc platelets/15% of apheresis platelets. 77% of the available bc were used in production for period a and 88% for periods b, c and d. after wastes of approximately 1.9%, the distribution of pc was stable for the 4 periods studied. 7075 pc were distributed for period a, 7047 pc for b, 7467 pc for c and 7083 pc for d. the % of pi platelets with 7-day shelf life available in the 3 hospitals was limited to 13% during period a. it was then increased to 14.1%, 20.9% and 26% for periods b, c and d respectively. the percentage of wastes was stable at 0.2-0.3% but the discards due to expiry went down from 24.24% (period a) to stabilize at 10.9% in periods b and c and 10.3% in period d. in the 3 general hospitals the expiry went down from 20% to 5.89%(hct), 29.4% to 14.5% (ughg) and 33.36% to 17.68%(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with 7-day shelf life allowed reducing discards for expiry from 24.24% to 10.3% in the last period analyzed at rtc and the 3 major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in 100% donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day 5. the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh-800, beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of 2-3. the average volume per unit of the pre-inactivation was 288 ml (278-302 ml) and post inactivation was 269 ml (254-284 ml), with average volume loss during inactivation was 36 ml (24-43 ml), corresponding to 13% (8-15%). the average platelet yield per unit pre-inactivation was 3.7 9 10 11 (3.0-4.4 9 10 11 ) and post inactivation 3.2 9 10 11 (2.8-3.9 9 10 11 ) with an average platelet loss of 13% (3-20%) . the average rbc contamination per unit (0.01-0.02 9 10 9 rbc/ml). the culture tests were negative, the average ph at day 1 was 7.4 (7.1-7.6), average ph at day 4/5 was 7.3 (7. 2-7.4 ). the average residual amotosalen concentration post treatment was 0.30 lm (0.23-0.35 lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below 2 lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of 106 independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of 2 ml pre-inactivation and another sample post-inactivation (16 h after pi). the platelet viability of each sample was evaluated by demonstrating the cd62p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of 106 independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd62p marker, was 18%, while the percentage of functional platelets post inactivation was 19%, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a 95% confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, 2016" in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than 10%. for the intermediate precision, the coefficient of variation was less than 15%, but for the pcs after treatment, this result rose up to 21%. the r² value for the linearity was 97.7%. the detection limit corresponded to a result of 6 edel and the loq (equal to 10xsd) is 19 edel. concerning roc curves, the men apcs threshold was 58.5 edel compared to women apcs with 62.5 edel. the threshold for bcpc was 54 edel. all of these results had 100% of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, 404 apcs (182 women and 222 men) and 263 bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, 14.0% of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was 89 edel. our study showed a threshold with 100% specificity and 90% sensitivity at 58.5 edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at 62.5 edel compared to 66.5 edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold (54 edel) was lower than abonnenc threshold which was at 59.8 edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a 3-ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: 1) complete treatment, 2) plasmas with mb without illumination, 3) plasmas without mb with illumination and 4) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol 1 where n = 20, were realized during the viro-inactivation process, t1 corresponds to a dosage of plasmas before treatment, t2 the plasma after the mb dry tablet passage, t3 is the time after illumination and t4 corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than 50%, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series 1 and 3, the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series 2 was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t4 (238 ae 16 edel at t3 and 209 ae 17 edel at t4). however, in the absence of mb (series 3 and 4), the results at t1 and t4 were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis (2019) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ 2.00 g/l was 94% (>70% required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were 75% and 90%, respectively. residual platelets were < 25 9 10 9 /l, leucocytes < 1 9 10 4 /l and red blood cells < 4 9 10 9 /l. all units had a protein content > 50 g/l. residual amotosalen was below 2 lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c3a and c5a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at 37°c was consistently short (6-7 min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine 8-10 to 6-7 min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included 5 experiments. for each experiment 5 male-donor, abo-compatible whole-blood derived plasma units (≥ 270 ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the 5-unit pools were split into 2 equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into 3 (≥200 ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from 5 single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, 2018) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ 18 lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, 2017) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ 18 lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade (0 to 70 lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to 2 different fluorochromes: phycoerythrin (pe) or brilliant violet (bv421). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and 4 populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): 0 (mfi 0.4), 4 (gmp mfi 12; non gmp mfi 9.5), 18 (gmp mfi 44; non gmp mfi 42), 70 (gmp mfi 174; non gmp mfi 180). streptavidin-bv421 brighter than streptavidin-pe is a promising tool because it amplifies by 1.7 the signal of fluorescence and allows a good differentiation of the 4 populations of rbc treated with only 0, 1, 4 and 18 lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ 18 lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases 2 (timp2) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, 2016) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = 9) were centrifuged within 3 h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = 9) were used as adult counterpart. platelet concentration was adjusted to 7-10 9 10 11 / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at à30°c/37°c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of 50,000 au spot density was defined as cut-off. data were analyzed by graphpad prism 8 using two-way anova. results: semi-quantitative evaluation of 105 analytes per array revealed significant differences. in plasma samples and platelets 63 and 48 analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (>200,000 au spot density) compared to adult plasma (6/63 vs. 3/63). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor 15 (gdf 15), platelet derived growth factor aa (pdgf-aa) and serpin e1 (p < 0.0001). more highly prevalent proteins were detected in npl (10/48) compared to apl (1/48), and 20 proteins were significantly elevated including vascular cell adhesion molecule-1 (vcam-1), platelet factor 4 (pf4/cxcl4), epidermal growth factor and lipocalin-2 (p < 0.0001). in adult samples only 10 proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < 0.05 to p < 0.0001). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, 2018) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, 1999) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are 1) to describe a procedure to label injectable human platelets with 2 densities of biotin, 2) to evaluate the impact of biotinylation on platelet functions, 3) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with 1.2 and 10 lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap (60 lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid (1 mm), amplitude of aggregation is 81.7 ae 2.5% (bio 0); 82.6 ae 2.3% (bio 1.2 lg/ ml); 79.2 ae 1.6% (bio 10 lg/ml). using collagen (2.5 lg/ml), amplitude of aggregation is 65.6 ae 4.2% (bio 0); 61.4 ae 4.0% (bio 1.2 lg/ml) 40.8 ae 6.6% (bio 10 lg/ml). the 2 biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than 48 h. after 4 h, the mean fluorescence intensities are 0.13 ae 0.02 for unlabeled circulating mouse platelets, 1.01 ae 0.03 and 8.2 ae 0.15 for circulating human biopl covered respectively with 1.2 and 10 lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, 10 ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, 9 ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to 30%. auto seds were administered in the form of 20% eye drops. results: auto seds have been applied to 17 ophthalmological patients (10 men and 7 women), previously resistant to standard therapy. in total 44 treatments were performed (each lasted 20 days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were 71.3 and 19.6 respectively). also, objective progress was present in 83% of all patients (p < 0.001). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. 1 platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of 2,3-dpg while reducing storage lesions stemming from oxidative stress. 2, 3 on the other hand, effects of steady hypoxia (pco2~10-20 mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for 3-week storage at 1-6°c. methods: 11 units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into 70 ml cp2d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o2-free bag. 5 ml of wb were collected from each unit at day 1, weeks 1, 2, 3. plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < 0.05. results: h-plt counts declined to~60% by the o2-reduction process, while similar decline was observed after 1 week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac-1 showed large increase after 1 week, then remained steady (h << c). plt activation by trap or adp declined modestly over 3 weeks (~15%) while h-plt showed additional~10% reduction for all time points. collagen activation for c-plt increased after 1 week (74%) and gradually increased to 100% after 3 weeks (~20% reduction with h compared to c). plt-derived mv (cd61 and cd61/annexin v) increased~4-fold over storage time; day 0 mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd42a) in wb supernatant increased 17-fold after 3 weeks for c, while h suppressed increase to 7-fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over 3-week period when stored at 1-6°c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated 500 ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted 1:1 by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, 19 patients were enrolled, 4 of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): 1) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); 2) autologous serum eye drops (sed). the creation of both types of platelet gel started in 2014 but only in 2017 we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from 2018, is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. 1) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between 800-1200 10 3 /ll and negative blood cultures, required by the italian law; the units are frozen at à80°c and last 5-year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. 2) the ophthalmologist's patients, with dry eye syndrome, donate 120 ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution (30%) are divided in 2 boxes containing 30 single-dose vials each: they are stored at à80°c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc 10231 strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. 10% calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of 1 9 10 3 cfu/ml and 1 9 10 4 cfu/ml were inoculated on sabouraud dextrose agar at the 1 st , 2 nd , 4 th , 8 th and 16 th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after 18-24 h of incubation at 30°c. chemokines and kinocidins (platelet factor-4, interleukin-8 and thymosin-b4) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp-10 3 group showed that the antifungal activity was still going on at the 8 th hour. the difference in colony production between the two groups at 8 th hour was statistically significant (p < 0.05). it was observed that the antifungal activity continued at the 4 th hour, decreased at the 8 th hour in the group prp-10 4 group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp-10 3 group. although there was an increase in il-8 levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb4 values results from the antifungal activity on the advancing hours in the prp groups. whereas pf-4 did not act an antifungal activity on prp-10 3 and prp-10 4 . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that 4% of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions (883 ml of red cells) in the preceding 10-day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of 225 ml, the hemoglobin decreased from 8.6 to 5.8. anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant (68% reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as 7-10 units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over 27,000 responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over 3,500 samples from 75 blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered (0.06%); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for 3-5 days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for 20 h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected 0.5 ml of ipm (2 mg/kg) intravenously between 15 to 30 seconds into each 5 health mice, weighing 17-22 grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during 24 h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after 24 h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother 0 s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the 1980s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than 6 years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since 2 years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab0-identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: 31 patients were identified receiving allogeneic sed, 15 patients had been treated autologous previously. in total, allogeneic sed have been produced 54 times since june 2017. indications were ocular gvhd (n = 15.48%), neurotrophic keratopathy (n = 9.29%), mucous membrane pemphigoid (n = 2.7%), sj€ ogren syndrome (n = 1.3%) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = 4.13%). contraindications for autologous donation were underlying disease (n = 19.61%), poor venous access (n = 12.38%), low haemoglobin (n = 8.25%), low body weight (n = 8.25%), very young age (n = 7.22%), circulatory disturbances (n = 3.10%) and lack of response to auto-sed (n = 2.7%). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = 2.4%), high donor haemoglobin (n = 2.4%) and unspecific positive serological findings (anti-hbs n = 2.4%). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a 8 year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood (20 ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for 1 h at 37°c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into 0.3 ml segments (capsules)and the artificial tears applied to the left eye 8 9 daily. results: ulcer healing was reported after 4 weeks of therapy with artificial tears. the dosage was reduced to 4 9 daily. no recurrence of corneal ulceration was observed after subsequent 8 weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > 80% of ab+/rnadonations since 2016 testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ 1.5), longstanding (ab+/rna+, lag odn > 1.5) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit 0.02 lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during 2017, 1671 donors tested hiv-positive of whom 1315 had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was 9.3% (n = 122) with efavirenz most frequently detected (115), followed by lopinavir (5) and nevirapine (2) . potential elite controller cases had the highest proportion of detectable arv (68/80; 85%) (p < 0.0001) followed by longstanding (37/741; 4.9%) and recent (17/366; 4.6%) infections. none of 82 acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older (35 to 64 years) hiv-positive donors (49/305; 16.1%) were significantly more likely to test positive for arv than younger (15 to 34 years) donors (73/1010; 7.2%) (p < 0.0001). arv use was more frequent among first time (101/ 716; 14.1%) than in lapsed (13/277; 4.7%) or repeat (8/322; 2.5%) donors (p < 0.0001). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites (10.4% vs 5.0%; p = 0.0051). summary/conclusions: the 9.3% prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the 4.7% prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year 2018. methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of 11 yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than 160.000 samples annually from whole blood and apheresis donations, testing on average around 600 samples per day. for the last 5 years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of 2019. although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the 4 mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to 2.000 unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: 19/33 anti-hcv, 19/21 hiv ag/ab and 04/22 hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: 1) hbsag aly 100. 00% (1994 00% ( / 1994 00% ( ) vs arc 99.95% (1993 00% ( /1994 ; 2) hiv aly and arc 99.95% (1992/1993); 3) anti-hcv aly 99. 90% (1994 90% ( /1996 90% ( ) vs arc 99.75% (1991 90% ( /1996 . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of 2 years from january 2016 to december 2017. blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. (78) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of 0.09%, thereby total reactivity being 2.39%. 55% (22/40) of these were hcv reactive & 45% (18/40) for hbv. the nat yield was 1 in 1086 and the viral loads of nat reactives ranged from 1-9 x10 4 iu/ml for hcv & all the hbv yields had an extremely viral load of < 06 iu/ml. 12/40 nat reactive showed sero-conversion after 4-8 months with follow-up eclia screening, and 7 of these were hcv reactive and 4 hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. (1/190,298) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect 283 infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e801 equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv-1/2, hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv-1/2 or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than 900 plasma samples collected in 7 sub-saharan africa countries (2011) (2012) and the amazon region of brazil (2016) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv-1 (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b19, chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv-1 genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in 2014 during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i2000sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m2000 realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a (5.78 log iu/ml), b (5.90 log iu/ml), c (5.62 log iu/ml), crf01 (5.61 log iu/ml), crf02 (6.19 log iu/ml), and urf (6.33 log iu/ml), as well as single donor high viral titer hcv genotypes 1a (6.84 log iu/ml), 1b (6.73 log iu/ml), 3a (6.64 log iu/ml), 2 (6.10 log iu/ml), 2q (6.65 log iu/ ml), and 4t (6.07 log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately 1 ml of residual sample volume, was then tested using the 0.6 ml abbott realtime hiv assay (lod 40 copies/ml) or 0.5 ml abbott realtime hcv assay (lod 12 iu/ml) and an hcv ag immunoassay (lod 1.24 fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = 24 per platform per virus schema) run on alinity s, alinity i, and architect i2000sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< 0.20 s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< 0.20 s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i2000sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in 2015 per 2.083.914 donations, 54% % of confirmed positive hiv, 87% of hcv, and 93% of hbv cases has been reported among first blood donors. in the end of 2015 a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after 3 months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in 2015, about 0.156% of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in 2017 was 0.082%, which shows a significant decreasing the ttis prevalence among blood donation from 2015 to 2017. the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in 2017 compared with the 2015. prevalence of hiv among donations reduce from 0.0032% in 2015 to 0.0024% in 2017, for hcv and hbv the same results have been experienced, respectively from 0.040% and 0.113% in 2015 reduce to 0.026% and 0.053% in 2017. it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from 80.19% in 2015 to 86.97% in 2017. it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from 0.0156% in 2015 to .082% in 20117. abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 61 samples were tested (19 for hcvab, 24 for havab igm and 18 for havab igg) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hcvab ranged from 0 to 4.474%. 5 samples were tested for havab igm in a total of 55 essays and the %cv ranged from 0 to 11.475%. havab igg was tested in 3 samples during 33 essays and the %cv ranged from 0 to 4.861%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 85 samples were tested (10 for hbsag, 20 for hbsab, 18 for hbcab, 14 for hbeag and 23 for hbeab) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 30 essays we found the %cv hbsag ranged from 0-4.375%. 3 samples were tested for hbsab in a total of 33 essays and the % cv ranged from 0-5.196%. hbcab was tested in 3 samples during 32 essays and the %cv ranged from 0-2.811%. using 3 samples and a total of 28 essays we found the %cv hbeag ranged from 4.176-5.147%. 4 samples were tested for hbeab in a total of 29 essays and the %cv ranged from 0-4.444%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 38 samples were tested (12 for hivag/ab, 14 for syphilis and 12 for htlv i/ii) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hivag/ab ranged from 0 to 3.05%. 2 samples were tested for syphilis in a total of 22 essays and the %cv ranged from 0 to 1.481%. htlv i/ii was tested in 3 samples during 28 essays and the %cv ranged from 3.342 to 7.5%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were 1.27%, 0.20% and 0.50% respectively. consecutive positive results for hbv were 9.20% (63/685), for hcv were 6.0% (16/266) and nil for hiv. there was no sample carry over in this. out of 79 consecutive reactive donors 69 were donated for same patients and 32 were related with infected patient which were statistically significant (p < 0.0001). summary/conclusions: among all tti reactive donors 7.4% (79/1061) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study 32 reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < 0.0001). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june 2018. a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using 100 positive and 100 negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were 65% and 70% (hightop), 67% and 85% (rightsign), 62% and 73% (wondfo), 70% and 80% (accu-chek), 68% and 77% (fastep), 73% and 85% (abon), 77% and 83% (immumed), 80% and 90% (insta-answer), 67% and 81% (biocheck) and 72% and 83% (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were 69% and 80% (hightop), 76% and 83% (rightsign), 69% and 81% (wondfo), 78% and 79% (accu check), 68% and 68% (fastep), 63% and 73% (abon), 71% and 70% (immu-med), 79% and 68% (insta answer), 62% and 66% (biochek) and 69% and 78% (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be 100%, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , 1940 and 1945 , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. ,906 donors were routinely tested for hbv dna by using cobastaqscreen mpx-1 and mpx-2 (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: 348 hbsag-/dna+ donors (1:1,462) including 294 confirmed obi were identified (1:1,731 prevalence). among obi donors, 160 (54.5%) tested anti-hbc+/anti-hbs-, 108 (36.7%) were anti-hbc+/anti-hbs+, 25 (8.5%) were anti-hbc-/anti-hbs+, and 1 anti-hbc-/anti-hbs-primary obi (0.3%). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: 21 years [range: 18-57 years]) than those with anti-hbc+/anti-hbs+ (mean: 44 years [range: 19-60 years]) and anti-hbc+/anti-hbs-(median: 45 years [range: 21-55 years]) profiles (p < 0.0001). hbv vaccination was documented for 18 (72%) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: <10-155 iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = 1/20) and genotype c (n = 19/20). preliminary analysis of core protein (n = 16) and bcp (n = 10) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for 15/ 25 anti-hbc-/anti-hbs+ donors (2-6 samples/donor; range: 2-56 months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between 12 and 1,000 iu/l. no significant difference in hla-a, -b (except hla-b*46 more frequently detected in anti-hbc negative obi), and -drb1*. summary/conclusions: overall, the 8.5% prevalence of anti-hbs-only in hbv dna positive obi carriers (1:20,355 of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in 15/25 donors by the over 50 months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after 1986 is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since 2000. hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december 31, 2018, allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between 2012 and 2018. background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about 130,000 blood donors are submitted to serological screening tests (hbv, hcv, hiv-1/2, chagas disease, syphilis and htlv-1/2) and nat for hiv, hcv and hbv per year. approximately 2% of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was 0.65% in 2016. aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly 74,000 donations from may 2016 to december 2016. hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity 95% lod 300 iu/ml for hbv). reagent samples (n = 407) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity 10-20 ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno2pheno [hbv] 2.0. socio-demographic and epidemiological data were also analyzed. financial support: fapesp 2017/16658-6. results: among the 407 hbv reactive samples, 377 were reactive for anti-hbc only (92.6%), 10 for hbsag only (2.5%) and 20 were reactive for both markers and hbv dna (4.9%). routine testing and id-pcr-hbv identified 20 (0.027%) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from 1.08e+01 to 2.45e+09 cop/ml (median, 2.12e+08cop/ml). hbv sub-genotypes a1, a2, c1, d3 and f1 were found in 70%, 5%, 5%, 15%, and 5% of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in 30% (6/20), with the following main substitutions 100c (3x), 120r, 123n and 144g. the mean age of donors with active hbv infection was 40 years, mostly donors were males (80%), mixed (40%) or white (40%) and had concluded high school (45%). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a1 and mutations associated with escape were found in 30% of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period 15-30 november 2018, all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of 620 edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in 24 (3.87%) samples, among which 7 (19%) were also anti-hbe(+), while anti-hbs was found > 200 m iu/ml in 15 (62.5%), between 100 and 200 miu/ml in 3 (12.5%), and < 100 miu/ml in 6 (25%). among anti-hbcore positive donors, 4/24 were foreigners (16.6%) and 20 were greeks, while foreigners consisted 6,29% (39/620) of donors examined. so, anti-hbcore was found positive in 10,25% of foreigners (4/39, all from countries with high prevalence for hepatitis b infection) and in 3,44% of greeks (20/581). in total, 285 (45.96%) samples had anti-hbs > 10 miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors (45.96%) were immunized, 261 by vaccination and 24 (3.87%) by natural infection. the incidence of natural infection was significantly higher in foreigners (10.25% versus 3,44%). if not all anti-hbcore+ donors, 25% with anti-hbs < 100 iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < 100 or < 200, or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately 2 million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in 20 samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than 20 km to each other. pcr was positive in 15 samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and 11 of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the 1a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with 1b subtype originated from the same village, and their difference was only 2 nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to 2 or 3 primary human sources. during 2011 and 2014, a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from 1993. however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march 2014 to december 2017, there were 2083 blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from 15 chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, 1178 of 2083 donors in the study who could be classified into two categories for hcv status: 201(17.1%) true positive and 974 (82.9%) false positive. a total of 905 of 2083 donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors (82.9%) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at 6.6% and 0.9% respectively. between january and december 2016, in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming 8.1%. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: 235 hcv seropositive blood samples from 3 bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that 45/50 seropositive blood (90%) was rna pcr negative. in december 2018, 156/249 (63%) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between 1.00-1.99 (1.00 is the cut-off indicating a positive sample). data from the re-testing of 235 seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december 2018. preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november 2017, the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of 3 months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a 3-month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for 2010-2018 (2018: preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within 12-months, and/or microbiological and clinical evidence of recent infection. for donors positive in 2018, compliance with the 3-month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from 2010 to 2018 among new donors, annual hiv prevalence decreased significantly by an average of 11.3% each year (p = 0.02) to 1.49/100,000 donations in 2018; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of 17.3% each year (p = 0.03) to 0.23/100,000-person years (pyrs) in 2018 (based on 2 seroconverters). there was no significant trend in hbv incidence over the study period, however between 2017 and 2018 incidence increased from 0.35/100,000 pyrs to 0.84/ 100,000/pyrs (based on 3 and 7 seroconverters respectively). with the information available to date, none of the 2018 seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in 2018 although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the 1980s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a 5-year deferral was implemented in 2013, reduced to 12-months in 2016; a 3-month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a 3-month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a 12-month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a 3-month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current 12-month deferral, calculated as hiv positive donors with a previous negative within 12 months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a 3-month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the 2.5th and 97.5th percentiles over the simulation (95% ci). results: incidence, per 100,000 donations, was estimated to be 0.15, 0.13 and 0.27 for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the 3month deferral "most likely" scenario, hiv residual risk was predicted to be 1 in 32.6 million donations (95% ci: 1 in 217,692 million to 1 in 8.3 million). for the "optimistic" scenario, hiv residual risk was estimated to be 1 in 34.3 million donations (95% ci: 1 in 257,692 million to 1 in 9.1 million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be 1 in 16.0 million donations (95% ci: 1 in 34,091 million to 1 in 5.7 million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every 30 years for the "most likely" scenario, every 32 years for the "optimistic" scenario and every 15 years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a 3-month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above 25%). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september 2018, a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated 22 times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang 9 urumqi blood center, urumqi, china 10 rti international, rockville 11 national heart, lung, and blood institute, bethesda 12 stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in 2013-2016 participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv-1/2 on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately 129 days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the 2013-2016 estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least 2 donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of 1,276,544 whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between 2013 and 2016, including 648,607 donations from first-time donors and 627, 937 donations from repeat donors. hiv prevalence among first-time donors was 68.04 per 100,000 donors (95% ci, 61.68-74.40). hiv ir was estimated to be 37.93 per 100,000 person-years (95% ci, 30.62-46.97) among first-time donors and 20.55 per 100,000 person-years (95% ci, 16.95-24.91) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than 25 years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over 10 years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during 2009 to 2018 at thai national blood centre. results: a total of 6,408,024 voluntary blood donations over 10 years, the repeated reactive results on hiv serological screening were 5,978 (0.093%) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for 6 months and tested by using the different three principles of hiv serological testing. a total of 5,978 hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was 1,130 (35.7%) cases, 742 (23.5%) cases and 1,292 (40.8%) cases respectively. out of 1,130 hiv positive results, we found that 1,105 (97.8%) cases were positive with all hiv serological testing for the first-time follow-up and 25 (2.2%) cases were tested and become to positive results after follow-up more than one time. in 742 cases of inconclusive results, 539 (72.6%) cases were reactive only 1 or 2 testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, 203 (27.4%) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, 1,292 negative results cases showed 762 (59.0%) cases were negative results after follow-up over 6 months while 530 (41.0%) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over 10 years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, 241 donors were found (80% men); of these people 256 donations were obtained (97% whole blood). 166 donors (69%) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average 174 ae 83 days to be reported in sivigila. 25 donors (10.4%) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. 20 donors (60% men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to 6 months they were reactive by sivigila (93% of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for 28 recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around 10-100 cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = 4-12 per species) was inoculated with approximately 10 cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for 22 to 24 h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with 3 random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to 3.5 9 10 3 cfu/ml in bcs and up to 0.9 9 10 3 cfu/ml in the corresponding pcs right after preparation. in total, 33 out of 48 pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., 6/6 pcs tested positive after spiking with streptococcus pyogenes, while only 1/8 pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male (69 yo and 65 yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day 4 platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after 18 h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit 1: a. baumanii tntc and s. saprophyticus with 8.5 9 10 4 cfu/ml unit 2: a. baumanii 6.6 9 10 7 cfu/ml and s. saprophyticus 2.2 9 10 6 cfu/ml blood cultures collected from both patients became positive within 8 h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a-105 and yersinia enterocolitica a-176) were distributed from the paul-ehrlich-institut to 17 laboratories in 10 countries for enumeration, identification and growth measurement in a 7-day interval for a total of 42 days after low-count spiking of rbc bags (10-25 colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a-105 and y. enterocolitica a-176 achieved >10 6 cfu/ml at day 14 and 10 9 cfu/ml at day 21. growth of l. monocytogenes was lower reaching a maximum concentration of >10 6 cfu/ml at day 35. in 9 out of 17 laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert 3d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber (10 or 16 ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc 29523), streptococcus agalactiae (atcc 13813), escherichia coli (atcc 25922), clostridium perfringens (atcc 13124). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert 3d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range 4 9 100-2.8 9 103 cfu/ml) and tested on bact/alert with a 1 ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two 10 ml ssk, which were held for a period of 6 h at 20-25°c. the process was repeated with a 24-h period. once elapsed, 8 ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of 7 days (36ae0.5°c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of 0.6-log10 after 6 h (2.0 9 102 to 8.9 9 102 cfu/ml) and 2.0-log10 after 24 h (3.1 9 101 cfu/ml to 3.4 9 103 cfu/ml). s. agalactiae increased by 0.7-log10 after 6 h (2.1 9 102 cfu/ml to 1.1 9 103 cfu/ml) and 0.09-log10 after 24 h (1.0 9 102 cfu/ml to 1.2 9 102 cfu/ml). c perfringens increased by 0.6-log10 after 6 h (1.3 9 102 cfu/ml to 5.0 9 102 cfu/ml) and 2.7-log10 after 24 h (4.0 9 100 cfu/ml and 2.2 9 103 cfu/ml). for e. coli, the concentration after 6 h was reduced by 0.28-log10 (2.8 9 103 cfu/ml and 1.5 9 103 cfu/ml), however this was possibly a result of inherent counting errors. at 24 h, an increase in growth by 2.7-log10 (1.5 9 102 to 7.9 9 104 cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to 24 h does not have a negative effect on bacterial viability and detection using the bact/alert 3d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in 100% plasma or amicus tm for 65% intersol pas/35% plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken 7 h after successful and complete transfusion of an apheresis intercept-treated pc in 65% pas/35% plasma, from a patient with a suspected septic reaction that occurred 2 h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a 7 day shelf-life. methods: a double apheresis pc collected in 65% pas/35% plasma was split into three equal components, yielding approximately 250 ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~6 log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at 3, 5 and 7 days of storage. the samples were analyzed by plating on lb plates (100 ll-10 ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were 5.9-6.0 cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after 3, 5 and 7 days of storage, corresponding to > 5.9 log 10 inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after 7 days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between 0,3%>17,5%, up to 5000 times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december 2018, a total of 2411 blood products were sampled, of which 979 in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), 662 in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and 770 in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, 16 ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, 4 ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at 35°c for 7 days. cultures were checked daily for signs of growth. in addition, 2 ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated 48 h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of 1.6% (39/2411) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, 2.7% (26/979) of blood products were contaminated, in hpkll 1.5% (10/662) and in hslk 0.4% (3/770) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = 0.00047). there was no significant difference between the other sites (p = 0.051 and p = 0.12). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. (46.7%) and bacillus spp. (33.3%). the remaining 20% of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than 10³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at 10³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than 2000 blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate (1.6%) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (<10³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the 18 to 65-year-old general population registered at the nphc. methods: altogether, 474,017 18 to 65-year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between 2015 and 2017. reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ 1:8 and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the 18 to 65-year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk 1,2 across 2015-2017. statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < 0.05. results: anti-tp reactivity was confirmed in 167 blood donors. aeid was proved in 85 cases with 36 ft and 49 rt donors. in that period, 1696 syc were notified. both in aeid and syc, the age group of 25-34 years with approximately 35% and 36% of individuals was the dominant. the proportion of men was 74% and 77% (p = 0.5) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher (0.020%; 0.023%; 0.032%, p < 0.0001) than that of rt donors (0.007%; 0.009%; 0.009%) and the proportion of syc (0.008%; 0.009%; 0.010%) in the general population. pp of aeid showed a roughly homogenous distribution in the 7 regions (0.011%-0.025%), however, pp of syc had a significant (0.058%; p < 0.0001) central hungary dominance in relation to the other regions (0.008%-0.016%). comparing the pp of aeid to syc, central hungary indicated a significant difference (0.025% vs. 0.058%, p < 0.0001), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised 4135 donations, including 112 bm (2.70%), 3787 pbsc (91.60%) and 236 cb (5.70%) donations processed in our laboratory in the years 1996-2016. bm was collected in operating theatre-conditions, pbsc with cell separators -cs-3000 (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and 35°c) using a variety of culture media. pbsc and bm were tested using 2 samples with trypcase-soy broth (tsb-t) and 1 with schaedler + vit k3 (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/10/anaerobic/f (becton-dickinson). results: in the 1996-2016 period 42 contaminated products were found: 25 pbsc (0.66% of all tested pbsc units) and 17 cb (7.20% of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at 0,1%. in 2010, three (3) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis (61.36%). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around 12 million new cases of syphilis in the world, more than 90% occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between 2015 and 2017 we observed in our institution a significant increase of 24% in positivity of syphilis among blood donors from 0.62% in 2015 to 0.73% in 2016 and 0.77% in 2017 (p < 0.0000001). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in 2017 was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp 2017/23028-9. results: among 123,851 samples screened in 2017, 958 (0.77%) presented a positive result for cmia -syphilis. of these, 626 (65.4%) were included in the study. a total of 106 samples (17%) showed vdrl+/igm+; 96 (15%) vdrl+/igmand 28 (4.5%) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in 28 (4.5%) samples that presented positive results for elisa igm and vdrl negative with 21 (3.35%) positive results, 1 (0.15%) undetermined and 6 (0.95%) negative. none of the 626 samples showed the presence of treponema dna by real-time pcr. the prevalence was 0.77%, the incidence was 0.1% in the year 2017, and the incidence in relation to the total positivity was 20.4%. both, prevalence and incidence were higher in men, white, not married, aging 18-29 years and high school educational level. we observed a 6% a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of 1.5% htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in 2017 (0.77%) with an incidence of 20.4% of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found 22 (3.5%) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to 5 days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to 7 days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; 22°c) or refrigerated (cs; 4°c). aims: the aims of this study were to 1) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and 2) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to 60 rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg4 + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first 3-4 days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day 4. bacterial numbers remained unchanged in refrigerated aliquots through day 5. rt storage resulted in significantly (p < 0.05) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < 0.05) delay in bacterial growth at day 1. while growth of e. coli and p. aeruginosa recovered by day 2, growth of a. baumannii was significantly (p < 0.05) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < 0.05) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day 3, indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a 20-ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle (10 ml each) within 48 h after collection. the bottles were then placed in the bact/ alert system and incubated for at least 7 days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a 2-ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a 2-ml and a 20-ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september 2008 and march 2018, after maintaining in liquid nitrogen for 1 to 17 years, 205 pbpc products collected from 45 patients, which was preserved in a volume between 35 and 60 ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these 205 final products were thawed and cultured with both the 20-ml and the 2-ml aliquot. one of these pbpc products had the positive culture result with the 20-ml retested samples. nevertheless, the same pbpc product had the negative result with the 2-ml post-thaw pbpc sample and the 20-ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the 2-ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) (10 surfaces, 1 air), hôpital provincial g en eral de r ef erence (hpgrk) (24 surfaces, 2 air) and the national blood transfusion centre (cnts) (20 surfaces, 2 air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates (23.7 cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd30, kikkoman), expressed as relative light units (rlu) per 100 cm². air was sampled by active sampling (500 liter; spinair, iul) on cled and macconkey medium. in parallel, particles > 0.5 lm and > 5 lm were counted using a particle counter (14,15 liter; metone 227a). culture media were incubated for 48 h at 35°c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was 27 (6-60) cfu/rodac, 69 (6-103) cfu/rodac, 82 (3-132) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes (43 and 60 cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator (132 cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per 100 cm² was 2.906 (778-26.497) rlu at hpkll, ) rlu at cnts and 35.235 (1.754-348 .052) at hpgrk. atp results and total viable count were not correlated (n = 49, p = 0.52). median (range) bacterial count in the air was 258 (210-375) cfu/500l for all sites together. there was no correlation found between total bacterial count and particles > 0.5 lm or > 5 lm (r = 0.7 and r = 0.6 respectively; p < 0.05; n = 5) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january 1st 2020. however, we already decided to voluntarily test all our blood products since january 2015. aims: in this study, we present our results of a 100% screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january 2015 to december 2018, a total of 386,307 allogenic blood donations from 69,956 individual german blood donors were screened in a minipool format of 96 samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from 4.8 ml plasma using the chemagen msm-i extractor (viral 5k, perkin elmer chemagen gmbh). the 95% lod of the assay was determined to 4.66 iu/ml (447 iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for 3 months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf1. results: in total, 274 hev rna positive donors were identified. of these, 274 hev rna-positive donors, 216 were nat-only positive donations (78.83%, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer (1.09%), 30 donors showed reactive igm and igg titers (10.95%), 25 donors already had isolated igg titers (9.12%). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely 62 donors showed elevated alt levels (22.63%), mostly within a double increase within the reference range (16.06%), only 6.57% of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > 1,000 iu/ml compared to the group with viral loads between 100 and 1000 iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype 3 in all cases. the month-dependent incidence ranges from 1:719 to 1:3,781 blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february 2016, and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within 1 to 2 weeks. however, in whole blood, zikv rna might present positive up to day 101 after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of 200 ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at 37°c for 3 days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at 37°c for 3 days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to 10 9 copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < 0.05) in type o rbcs and lower than those in plasma (p < 0.05) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting 20 residents from 2000 to 2014. during 14 august to 4 september 2018, another outbreak of 29 confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district (19 cases) and the other in cheung chau, an outlying island (10 cases). aims: we assessed the risk of dengue transmission from blood donors during the 2018 outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, 2013) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of 15-to 64-year-old patients in the 2018 outbreak and residents of the same age range in hong kong and wts district as at mid-2018 respectively (16-65 years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during 12 august to 8 september 2018 after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, 2009 ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in 778,000 (266,000-1,822,000) territory-wide for the 28-day study period but increased to one in 147,000 (50,000-345,000) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was 1 in 279,000 (118,000-752,000) territory-wide and 1 in 52,000 (21,000-188,000) in wts during the same period. the eufrat also predicted a territory-wide issue of 0.08 unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at 1 in 778,000 during the 2018 outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by 4 folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july 2017 to december 2017 at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version 21.0 (ibm). results: in our study population there were 445 (98.9%) males and 5 (1.1%) females. the mean age of recruited blood donors was 28.38 (sd ae 7.34), with a range of 18-55. younger donors were more common with a frequency of 18-27 year olds of 249 (55.3%). we found an overall hev igg prevalence of 17.5% and an hev igm prevalence of 9.1%. there were 10 (2.2%) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was 33.8 (sd ae 41.0) with a range of 4-554 iu/l. the serum alt levels were elevated (>45 iu/l) in 73 (16.3%) blood donors. there was significant correlation (p=<0.001) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are 20 million hev infections, 3 million acute hev cases and 56 thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively 4.0% in new zealand, 13.5% in britain, 20.6% in denmark, 16% in the united states and 27.0% in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from 5552 blood donors were collected from april 2017 to april 2008 at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were 20.05% (1113/5552), 0.76% (42/5552) and 0.04% (2/5552), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = 1.089, p < 0.001; igm or = 1.055, p < 0.001). donors who were zhuang minority (32.69%, 7.69%) showed higher anti-hev than those who were han ethnicity (19.89%, 0.70%), and the difference was statistically significant (igg or = 2.052, p = 0.023; igm or = 12.029, p < 0.001). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population (60-100% seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested 20 h after infection, washed and used for spiking of plasma. plasma units (n = 2, 290 ml) were spiked with infected cell suspension (10% v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b2 illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses (0 after addition of mb, 30, 60, 90 and 120 [standard] j/cm 2 ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of 4.2 (bag no. 1) and 4.5 (bag no. 2) log 10 tcid 50 /ml was achieved in the plasma units. in hold samples, a mcmv titer of 3.8 (bag no. 1 and bag no. 2) log 10 tcid 50 /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ 2.9 log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log 10 reduction factors ranged from 4.2 (bacillus cereus) to 6.1 (serratia liquefaciens) for the tested bacteria, and from 3.1 (emcv) to ≥ 4.9 (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below 0.8% until day 35 of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a-1,2-fucosyltransferase (fuct2), encoded by fut2 gene, determines the secretor status of an individual. about 80% of caucasian population have a functional copy of fut2 (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: 126 samples of semen, 68 from infertile men and 58 from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who 2010 criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners (76.9%) were significantly higher than those from fertile couples (21.6%) (p < 0.03) the results obtained by pcr in sperm cells correlated 100% with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut1 gene (h | h), mandatory linked with an active fut2 gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric231-pe and a 1:1 mixture of bric231-pe/bric231, ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from 29 blood donors with different abo phenotypes (o (5), a 1 (5), a 2 (5), b (5), a 1 b (5), a 2 b (4)) and 2 patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a 1:1 antibody mixture (bric231-pe/bric231) covering approx. half of the h-binding sites by unconjugated bric231. in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric231-pe. rbcs coated with bric231-pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric231-pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen 1,2 , x xu 1,2 , x hong 1,2 , k ma 1,2 , j he 1,2 , j chen 1,2 and f zhu 1,2 1 blood centre of zhejiang province 2 zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: 109 samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of 109 samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province (2018rc029). results: 69 samples in 80 blood donors showed increased expression of h antigen, of which 47 were identified as abo subgroups. there were 22 enhanced reactions in 29 hemopathic patients. however, 19 were finally confirmed as normal abo genotypes. no statistical significance (86.3% vs 75.9%, p > 0.05) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is 50 and 3 respectively in blood donors and hemopathic patients. extremely significant statistical differences (68.1% vs 13.6%, p < 0.005) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november 2018 to january 2019, 55 cases (0.6%) of abo discrepancies among 9,550 abo blood type tests performed using the 15-min reaction mode of ih-500 in chonbuk national university hospital blood bank were identified. we compared the test results of 15-min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a1 and abo genotyping. results: in the immediate reaction using different red cell reagents, 45 cases (81.8%) of discrepancies disappeared and 10 cases (18.2%) remained discrepancies. all abo discrepancies observed in the 15-min reaction were due to serum side causes, and one case (1.8%) was due to both of serum and red cells side cause. nonspecific response (28 cases, 50.9%), cold antibody (20 cases, 36.4%), rouleaux formation (3 cases, 5.5%), cis-ab (3 cases, 5.5%), and abo subtype (1 case, 1.8%) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the 15-min reaction. summary/conclusions: ih-500, an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the 2 years data from jan 2017 to dec 2018. the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel 2016 and spss (version 22). results: during study period a total of 1970 patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were 20.91%, 37.51%, 32.74%, 8.83% for a, b, o and ab respectively. the common neurological tumours found in our study were glioma (33.55%) followed by pituitary adenoma (20.05%), meningioma (18.58%), schwannoma (8.98%), cavernoma (2.54%), neuroma (2.23%) and space occupying lesions (14.06%). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in 47.73% o group patients as compared to other blood groups which was found statistically significant (p < 0.05). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen (38.2%) is more common as compared to o blood group(33.4%). background: rhd and rhce represent homologous genes in head-to-head position on chromosome 1 (chr1, p36.11). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band 3 and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, 2016). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, 1955) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad-3/brad-5/fog-1 (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric-69pe (ibgrl). results: a total of 146 samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr(21), r1r(20), r1r1(23), r2r(18), r0r(15), r1r2(27), r2r2 (22)). variant expression of rhd by different rhce phenotypes using brad-3-pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples (3) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad-5-pe and fog-1-pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c.48g>c, c.201a>g, c.203a>g of exon 1, exon 2 resp. and intron 2) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c.676c>g, exon 5) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon 5 is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh26 antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:-26, and the antibody specificity anti-rh26. most polyclonal anti-c contain anti-c and anti-rh26. previous studies have shown 2 of 10 monoclonal anti-c reagents are actually anti-rh26. these reagents will not detect the c antigen where the red cells are rh:-26. aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r 1 r) was inconsistent with the current donation phenotype c-(r 1 r 1 ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a 2 + reaction by tube with bio-rad seraclone â (2) [clone ms35] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk7300 using beckman coulter anti-c [clone 951] blood grouping reagent and tested positive (4) reaction on the immucor neo using immuclone â (1) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd*01/*01n.01 and rhce*01.15/*02 with a predicted phenotype of c+, c+ w , d+, e-, e+, rh:55 (locr+), rh:-26. a review of the donor's historical records indicated the donor tested as c+ on the pk7200, which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms33]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:-26 variant associated with the rhce*01.15 allele. reagents containing clones ms-33 and ms35 correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk7300 and associated anti-c [clone 951] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:-26 phenotype, which is in contrast to the previous report by faas et al, transfusion, 1997 where it was demonstrated that clone 951 reacted with c+ rh:-26 bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since 2017 at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of 6 monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in 32 samples: a) by using the partial d kit, 23 samples were typed: four samples were characterized as "partial d" (3 dfr, 1diii) and 19 as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type 1" three samples, "weak d type 4.0 or 4.3" one sample). b) the nine (9) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized 2 samples as "weak d type 1", one sample as "weak d type 3" and another as "weak d type 11". results are pending for 5 samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in 68,75% of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over 200 rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ 50 years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type 1, 2 or 3 are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type 1, 2 and 3. aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from 2013/01/01 to 2018/10/01 we analysed 119.845 samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs232, bs226) (biorad, dreieich, germany), swing maestro [lmh 59/20 (ldm3) + 175-2 and th-28 + rum-1 + ldm1] and ih-1000 [lmh 59/20 (ldm3) + 175-2] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from 119.845 samples 300 (0,25%) were swd. 71/300 (24%) were referred to rhd genotyping. 55/71 (77%) samples were weak d type 1, 2 or 3, while 16/71 (23%) were weak d type 14 and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types 1, 2 and 3. clearly negative serologic reactions were given in 27/29 samples with bs 226 and bs 232, in 30/33 samples with lmh 59/20 (ldm3) + 175-2 and in 18/39 samples with th-28 + rum-1 + ldm1. summary/conclusions: the prevalence of swd in this study is rather low (0,25%). after rhd genotyping 77% of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of 3243 randomly selected saudi donors, who donated blood between january and june 2018, were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k1 phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of 3243 saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in 93.7%, while k antigen was positive in 11.1%. among other studied rh antigens, e was the most common (98.1%) followed by c (78.2%), c (70.3%) and e(26.9%). dce/dce (34.2%) and dce/dce (5.3%) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent (11.9%) with almost 5 times higher frequency compared that reported in caucasians (2.0%). the rare phenotype dce/dce was found in 3 donors (0.09%), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a 58-year-old male patient with lung cancer without previous transfusions was admitted (06/2009) for surgery. his hemoglobin was 13.2 g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july 2017, the patient was admitted for colon cancer surgery with a hemoglobin of 11,0 g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge*01.-03. an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of 0.35% and the antibody was considered not to be clinically relevant. the mi was interpreted as following: 0-3% not significant; 3-5% inconclusive; >5% clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received 2 units of ge:-2,-3 prbcs without any transfusion reaction. one and a half year later (11/2018), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was 87 g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july 2017 indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following 16 days the patient received a total of 5 units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december 12 th and 16 th (12 days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of 0.8% und 1% respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from 1 st april, 2013 to 30 th september, 2015 (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a1 lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen (3-cell) and identification (11-cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected 104 (0.072%) abo discrepancies out of the total 144279 donor samples tested during the study period. out of these, 135043 (93.6%) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody (33/104; 31.73%), followed by weak anti-a antibody and weak subgroups of a (24/104 each; 23.07% each) and weak subgroups of b (5/104; 4.8%). the remaining 17.3% (18/104) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was 0.02% ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within 3 days after receiving a patient's sample. however, in some cases, retests could be performed after more than 3 days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih-500 and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih-500 (bio-rad, 1785 cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, 1 week after storage at 4°c and 1 month after storage at à20°c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih-500 and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, 15; anti-le a , 4; anti-di a , 4; anti-c+e, 3; anti-m, 3; anti-d, 2; anti-c, 1; anti-k, 1; anti-jk a , 1: anti-xg a , 1; unidentified antibody, 13; autoantibody, 2 cases. with regard to the changes in reactivity owing to storage, 26 (52%) samples (anti-e+c, 12; anti-m, 3; anti-di a , 3; anti-d, 2; anti-c+e, 2; anti-le a , 1; anti-c, 1: autoantibody, 1; unidentified antibody, 1) showed identical reactivities after 1 week and 1 month storage by both ih-500 and tube methods. however, 19 (38%) samples, comprising 12 unidentified antibodies, 3 anti-le a , 1 anti-c+e, 1 anti-e, 1 anti-e+c, and 1 autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after 1 month storage, while one sample with anti-xg a showed decreased reactivity only after 1 month storage. higher reactivities were observed in all samples detected using the ih-500 analyzer than manual tube methods (p < 0.005, wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih-500 and tube methods were the same in all storage states; however, reactivities were higher in ih-500 than in the tube method. twenty-six (52%) of 50 samples showed identical reactivities after 1 week refrigeration and 1 month freezing. nineteen (38%) samples showed decreased reactivities after storage; however, 12 (12/19, 63%) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than 3 days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel 1 , d huber-marcantonio 1 , n rufer 1 , g canellini 1 and c niederhauser 2 1 unit of transfusion medicine, interregional blood transfusion src, lausanne 2 laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of 3'753 dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (<14 days). results: a positive dat was found for igg and c3d in 421 out of 3'753 (11.2%) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, 244 (57%) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only 10.7% (26/244) of the cases. from the 161 patients transfused within the last 14 days and having a positive dat, 60 (14%) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms (244 versus 60 samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last 14-days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd38 monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin #16-02). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd38 antibodies. vox sanguinis (2019) 114 (suppl. 1), 5-240 methods: edta-anticoagulated whole blood samples coming from 30 patients in different stages of treatment with daratumumab and 5 with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd38 antibodies treatment. in these cases, 6 of 7 negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed 100% agreement with genotype id core xt results. focusing in the data obtained during the treatment, 8 negative antigens were type as positive in gel technology (12% of the tests). mdmulticard agreed with genotype in 100% of the analyzed antigens. as complementary data, 13 of 66 patient-treated samples had dat or ac positive and 59 showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd38-directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes 25 ll and 50 ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and 20 donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card (50 ll at 0.8%). further, sample dilutions were dispensed into the card (25 ll or 50 ll). subsequently, cards were incubated 15 min, 37°c (coombs technique) and 15 min, 18-25°c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = 80 titrations, titer ranged 0-256) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ 0.5 titer in the 98% of the cases). differences between dg gel coombs and neutral (saline technique) (n = 80 titrations, titer ranged 2-512) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at 37°c and igg antibodies. differences were > 1 titer in 35% of the comparisons and ≤ 1 titer in the rest of the cases (65%). comparing sample volumes of 25 ll and 50 ll in all cards (n = 160 titrations), higher titers were observed using 50 ll, as expected. differences were 1 titer in the 51% of the comparisons, <1 titer in 44% and > 1 titer in the 5% of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged 0-18 years, diagnosed with aiha, between april 2017-december 2018 (21 months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: 10 patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from 2.5 to 9 grams/dl. the initial presentation was severe anemia in 7 children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in 3. the trigger for haemolysis was infection in 4 children. rheumatological evaluation was performed for 5 children out of whom 2 were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for 4 and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in 9 out of 10 aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of 9 dat positive cases had igg & c3d positivity on monoclonal dat testing whereas rest 7 had only igg coating the red cells. dat titration was more than 1:300 in 4 patients; where only 1 of these 4 patients had both igg1 and igg3 coating and rest 3 had only igg1. alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a 4-6 weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in 2 and rituximab was used in 3. out of these children 5 children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a 64-year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and 5-fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day 1), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day 5), two units of rbcs (day 6) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day 24 were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at 1 and 5 mg/ml. results: dat was positive (anti-igg 2 + , anti-c3d 2 + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer 2) and enzymetreated (titer 8) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at 5 mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p-331 singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd38 used in the treatment of multiple myeloma and has been known to bind to cd38 on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october 2016 to december 2018 was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of 55 patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in 23 of the 55 patients while the use daratumumab was mentioned in the serology request form of the other 32 patients. the median tat for red cell antibody screen and identification is 212 min (range: 47-877) if information on daratumumab was provided prior to start of testing and 301 min (range: 133-1417) if information was not provided prior to testing. the median tat for routine testing is 198 min (range: 15-2245). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value 0.72634). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value 0.00694). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd38 monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd38 is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd38 on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd38 interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd38 tertiary structure, avoiding the binding and testing interference by the anti-cd38 daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since 2016, 32 patients were studied, from which 16 were transfused with 108 blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd38 and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd38 is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july 2014 and dec 2018 (54 months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of 22888 patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in 145 patients and autoantibody was detected in 53 patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha (58%) and 20% of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group (45% vs 34%, p < 0.05). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in 21 where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood (1.2% vs. 5%, p < 0.05). during the period of study we obtained 10 cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk7300, 250,599 donation samples were tested and 4,895 (1.95%) were found absc positive. antibodies to red cells were identified in 250 donations out of 4,895 (5.11%) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was 0.10%. the top 5 most frequent antibody specificities were: nonspecific cold antibodies (28.8%), anti-e (26.8%), anti-mi a (19.6%), anti-m (16.8%) and anti-le a (4.0%). a total of 225,124 donations were screened for atypical antibodies by ih-1000 and 2,275 (1.01%) were screened positive. among these, anti-red cell antibodies were identified in 1,239 samples (54.46%), which was significantly higher than those identified in pk7300 screened positive samples (p < 0.00001). the prevalence rate for atypical antibody as screened positive by ih-1000 and with confirmed red cell specificities was 0.55%, which was also significantly higher (p < 0.00001). the top 5 most frequent antibody specificities were: anti-mi a (55.3%), anti-m (21.1%), anti-le a (10.2%), anti-e (6.5%) and non-specific cold antibodies (3.6%). anti-fy b was detected in 7 cases, which would be missed detection by enzyme treated reagent cells on pk7300 system. summary/conclusions: the performance of the ih-1000 system using a 3-cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk7300 in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of 0.55% in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus 2019) reported a rbcalloimmunization prevalence of 11.4%, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a 18 years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from 1990 to 2018 thalassaemia patients, with a minimum follow up of 1 year and transfused with more than > 10 rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall 57 patients (38 females, 19 males) were included in our retrospective analysis: 54 patients were affected by thalassaemia major and 3 by thalassaemia intermedia. rbc alloimmunization prevalence was 12.3% (7 patients): 3 patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected (1 anti-h, 1 anti-cw, 4 anti-e, 1 kpa, 1 anti-jka, 1 anti-jkb, 1 anti-m and 1 anti-lua). in 2 out of 3 alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining 4 patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the 7 patients positive for alloantibodies, 6 were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: 12.3% vs 11.4%; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since 1998. recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april 2013. that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in 3642 patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in 759 patients, acute leukemia in 600, multiple myeloma in 390, chronic lymphatic leukemia in 319, chronic myeloid leukemia in 218, aplastic anemia in 147, hemophilia in 105, myelodysplastic syndrome in 73, and other hematological diseases in 1031. the frequency of detection of antibodies to antigen c (0.25% vs 0.06%) and to antigen e (0.46% vs 0.12%) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly (0.10% vs 0.06%, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since 1998, therefore the immunization index for these antigens remained unchanged and amounted to 0.10% vs 0.12% for anti-c antibodies; 0.36% vs 0.36%for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included 466 first time patients of hematology clinic in 2017-2018. typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in 84 (18.0%) patients. the total number of complex cases was 96. the double population of red blood cell was most often determined in antigens of the rhesus system (10.9% of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in 31 cases (60.8% of patients with the chimera for rhesus and kell antigens), cin 23 (45.1%), sin 15 (29.4%), e -5 (9.8%), cw -8 (15.7%), k -5 (9.8%). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in 0.6% of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients (1.1%) was due to a decrease in the production of antibodies -4 cases and the appearance of extra agglutinins -1 case. autoantibodies were detected in 3.9% of all patients, including 0.6% of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in 2.8% of patients, including specific anti-din 4 (0.86%), anti-dcin 2 (0.43%), anti-kin 1 (0.21%); antibodies of unidentified specificityin 2 (0.43%), polyspecificin 4 (0.86%). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of 3,49,497 crossmatches were performed over period of last four & half years, out of which 867 units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c3d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of 3,49,497 crossmatches, only 867 units were found to be incompatible (0.14%). the major cause for incompatibility found in patients was aiha-(32.87%). other causes of incompatibility were infections (27.44%), multiple transfusions (17.41%), trauma (11.23%), evan's syndrome (4.15%), rh negative mother (3.57%), sca (2.99%) & incompatibility due to dat positive packed red cells (0.34%).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb-1) was admitted to our hospital in january 2017 for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu4 / atg protocol (5 days of 250 mg iv. fludarabine, 4 days of busulfan 792 mg iv, 2 days of 300 mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of 2016, was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december 2016. results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and 0.2 m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a 75-year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in 2004, with unknown transfusion history. her obstetric history was g6p4a2. the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a 0 rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from 9.0 g/dl to 12.0 g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (>99% prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of 1.2%, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no 0 rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an 18-years-old female patient with scd presented to our hospital with hemoglobin level of 4 g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial 11 cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r1r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to 2.8 g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to 8 g/dl in 2 weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r1r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than 1% of most populations are known as low incidence antigens and those present on more than 90% are known has high incidence antigens. the mns blood group system consists of 49 antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are 35 low incidence and 10 high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns49) antigen. anti-jenu was first described in a thai patient with thalassemia where only 2 compatible units were found following screening of 3600 units. the jenu negative phenotype is a rare phenotype with an estimated frequency of 0.06%. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of 4 + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd47 is also expressed on red blood cells (rbcs). recently, many drugs targeting cd47 have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd47 targeted high affinity sirpa fusion protein alx148. methods: a 35-year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx148 clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past 8 months. she had no previous transfusion history during the past two years. after two infusions of alx148, two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and 37°c albumin phase, and 2 + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and 37°c albumin phase, but 2 + at ahg phase. antibody screening (2 cells) and identification (11 cells) all showed 3 + at ahg phase using gel cards. direct antiglobulin test was 4 + for anti-igg and 3 + for anti-c3d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to 7.8 g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between 11.1-12.0 g/dl but it decreased again to 9.2 g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx148, and antibody screening and dat decreased to 0-1+ reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to 13.6 g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd47 is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms15/seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons 4-11 was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk*01(28a,226a,303a,588g)/jk*02. to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk*01(28a,226a,303a,588g)/ jk*01 and jk*02/jk*02, respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk*01(28a,226a,303a,588g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a 60 y/o man who presented to the ed (hospital day 0, hd0) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (<3 days). methods: specimen #1 (s1) was sent on hd0 for type & abs (t&s) and crossmatch (xm) of 2 rbcs. abs and immediate spin xm were negative; there was no patient history. by hd9, he had 4 negative t&s specimens (hd0: s1; hd2: s2&3; hd6: s4) and had been transfused 4 rbcs (hd2: 2; hd5: 2) via electronic xm (exm). at 1730 hr on hd9, 2 rbcs were requested and could have been issued via exm since s4 was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s5 was requested and received at 1801 hr. results: s5 showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from 10.2 (14.0-17.5 g/dl)/30.1 (41.5-50.4%) on hd6 to 6.9/ 20.6 on hd9. during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd9, which he tolerated well with an increase of hemoglobin from 6.9 g/dl to 8.6 g/ dl. he did well post transfusion with stable h/h between 8.1/24.2.0 to 8.5/25.3. he was discharged on hd19. repeat abs on s4 was negative. of the 4 rbcs transfused before s5, one was e+ and four jk(a+). the family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. hospital #1 (h1) reported admissions 20 years ago (2 rbcs transfused) and 4 years ago; all abs were negative. h2 admission was 5 years ago with positive abs and inconclusive workup. h3 admission 4 years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every 3 days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a 75 year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong (4+). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr1), a high frequency antigen expressed by the abcg2 gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and 16 exons and adjacent intronic sequence of the abcg2 gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive (2+) in iat test, while the self-control was negative. the antiserums reacted strongly (4+ in iat test in gel card) with the papain-treated cells, but kept the same reaction strength (2+) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was 2. in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg2 gene in the patient and her brother revealed one homozygous nonsense mutation in exon 4 (c.376c>t, p.gln126x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals (98% or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, 76 years old, o rhd+, previously transfused with 4 red blood cells concentrates in 2006, was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected (2+ in all the 3 screening cells). in order to identify the specificity of the antibody, a panel of 11 cells was tested; the result was considered inconclusive, due to positive reactions (2+) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel (2+ with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg1 and igg3) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from 80865 blood samples, four donors with jk (a-b-) were selected, at a frequency of 0.0049%. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs5-1g>a combining with heterozygote of 359g>a (gly120glu) and heterozygote of 896g>a (gly299glu) combining with heterozygote of 956c>t(thr319met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs5-1g>a, 896g>a and 956c>t were common mutations in the before reports, while 359g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk3. background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of 10 blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: 5,213 blood donors from age 19 to 54 were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of 10 blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce 0.3%, rhce*ce 65.0%, rhce*ce 28.8%, rhce*ce 5.9% -kel*k_kpb_jsb allele 100% -jk*a allele 49.1%, jk*b allele 50.8%, jk*b_null allele 0.1% -fy*a allele 92.8%, fy*b allele 7.2% -gypa*m allele 51.1%, gypa*n allele 48.9% -gypb*s allele 5.1%, gypb*s allele 94.8%, gypb*mur allele 0.1% -di*a allele 4.7%, di*b allele 95.3% -do*a allele 10.1%, do*b allele 89.9% -co*a allele 100% -yt*a allele 100% -lu*b allele 100% summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding 16 blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu (8/14), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c.697c>g, and rhce*c.733c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c.667g>t were tested by pcr-ssp. a total of 1111 probands including 800 individuals from syria, 147 from iran, 123 from the arabian peninsula and 41 from northern african countries were included. results: 2% of the donors were homozygous for the fy null (fy*-67t>c, fy*02n.01) mutation, 16.2% carried the heterozygous mutation. 0.4% of the syrian probands were heterozygous for the do*350c>t mutation (both, do*jo1 and do*jo2; jo(a+/ a-)) that is virtually unknown in caucasian donors. 0.8% of the syrian donors heterozygously carried the kel*02.06 allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel*02.06 carriers were identified. 1.8% of the syrian and 1.5% of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c.251c>g, intron 5 + 5 g mutation, inducing the s-u+ w phenotype. 3.6% of all and 29.3% of northern african donors were heterozygous for the rhce*c.733c>g substitution, 0.3% of the syrian donors carried rhce*c.697c>g (heterozygously) and 0.004% of all donors were heterozygous for rhce*667g>t. heterozygosity for vel deficiency (vel*-01) was detected in 21 individuals (2%; 16 of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp1*c.601delg (co*01n.06) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals (2 blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: 1/gel cards diaclon id abo/d (anti-a: clone a5, anti-b: clone g1/2, anti-a,b: clone es131, es15+ birma 1+ es4; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m297/628 = la-2; bio-rad); 2/tube techniques with: anti-a (birma 1; a-11h5, a1 s.pa1m095, c.9113d10), anti-b (lb-2, b-6f9, c.9621a8). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of +7.21-kb site of abo gene to cover sequences ranging from the end of intron 1 to 3 0 utr of the abo gene. additionally sequence of exon 1 of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals (2 blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma 1 (1+ to 2+), a-11h5 (1+ to 2+) and c.9113d10 (weak+ to 1+); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a1 antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of 1061delc typical for abo*a2 alleles). in all these individuals abo sequencing of 7.21-kb fragment confirmed the heterozygous genotype with 7 polymorphisms characteristic for abo*b.01 allele (297a>g; 526c>g; 657c>t; 703g>a; 796c>a; 803g>c; 930g>a) and detected a novel abo*a allele sequence with duplication-based insertion of 21 bp after 564 position (abo*a c.dup543_563; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon 188 (p.dup_182_188qdvsmrr), with synonymous change of the repeated codon 182 (cgc>cgg) and 188 (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup543_563 allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a1.01 allele and the same abo*a c.dup543_563 allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in 1990 of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons 1-7 (including 50 base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a1 in plasma. abo genotyping gave the genotype abo*a1.01/o2.01 usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately 85%) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately 15% of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a1.01-like allele except for a novel mutation located in intron 5, c.239+4g. the o 2 allele had an additional snp, c.689g>a, consistent with the abo*o2.03 allele variant summary/conclusions: a previously unreported variant, c.239+4a>g, likely effecting the 5 0 -donor splice site of intron 5 was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. 374+4a>g mutation is located in the 5 0 -end of intron 6 and is predicted to cause partial skipping of exon 6. strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately 2% positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past 3 years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a2, b, o1 and o2 alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon 6, intron 6 and exon 7 were amplified using a forward primer targeting the 261g nucleotide (to exclude o1 alleles amplification) in combination with either b, a2 or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of 16 samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in 8 out of 12 samples with suspected a subgroup alleles, the c.804insg insertion was detected corresponding to the abo*ael.01 allele. the abo*aw31.02-05 variant, a hybrid a1-o1v allele, was found in 2 cases. in 1 case we found the c.722g>c change, previously reported associated with weak a antigen expression. finally, a novel c.961c>g change was detected in an a2 allele. b(a) or cis-ab suspected alleles: the abo*b(a)04 variant carrying the c.640a>g change was found in 1 of 3 samples with bo1 genotype but a weak antigen expression. in the remaining 2 cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b01.02 allele carrying the nucleotidic change c.926a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon 7 of the abo gene, in 14 out of 16 samples, including 2 novel alleles. chimerism was suspected in 2 cases of a antigen expression in samples with b1o1 genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but 8% of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a 20-year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih-500; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a 3 b 3 phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a102/b101/o01. however, in buccal swabs, the patient showed a102/o01 and his brother showed b101/o01. other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d8s1179, d21s11, d7s820, csf1po, th01, d13s317, d16s539, d19s433, d18s51, d5s818, and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case #1 is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.29-5t>g in heterozygosity on an otherwise common a1 allele, and in trans an abo*b.01 allele. case #2 is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c.29-5t>g in heterozygosity on an a background, and in trans an abo*o.01.01 allele. given that this variant is located near the intron 1 splice acceptor site, abo*29-5g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case #3 is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c.467c>t (pro156leu) and c.709t>a (tyr237asn), both in heterozygosity on an otherwise common a1 allele, with an abo*o.01.01 allele in trans. thus, the data establish an association of allele abo*467t,709a with an aweak-like phenotype. case #4 is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c.479c>g (pro160arg) in heterozygosity on an a2 background, and in trans an abo*o.01 allele. an interpretation of the data is that variant c.479c>g weakens the activity of the a2 transferase, with allele abo*a2(479g) encoding the aweak-like phenotype detected by serology. case #5 is a 9 year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.803g>c (gly268ala) in heterozygosity on an a2 background, and in trans an abo*o.01.02 allele. the serology and molecular results suggest that allele abo*a2(803c) encodes a cisab weak phenotype. case #6 is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c.739g>a (glu247lys) and c.871g>a (asp291asn), both in heterozygosity, in trans, and on a1 backgrounds. variant c.871g>a by itself constitutes allele abo*a3.01. the phenotype encoded by abo*739a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case #1 is a 35 year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a1/abo*o1. sequencing detected variant c.107insg (val36gly>fs56ter) in heterozygosity on an otherwise common a1 allele, and in trans an abo*o.01.01 allele. it is unclear how the early truncation of the a1 transferase encoded by allele abo*107insg still allows for some residual enzyme activity, as suggested by the reverse a type. case #2 is a recently-transfused 72 year-old black patient with an unresolved abo type. sequencing detected variant c.297a>g (silent) in homozygosity and variant c.1049c>t (ala350val) in heterozygosity, both on an o1 background, with an abo*b.01 allele in trans. although variants c.297a>g and c.1049c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o1(297g,1049t) allele. case #3 is a 40 year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon 7 detected variant c.436c>t (arg146cys) in heterozygosity on an abo*b allele background, and in trans an abo*o.01.01 allele. the phenotype encoded by allele abo*b(436t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case #4 is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a 2+ mixed field (mf) in tube. reverse type on a1 cells 1+ in gel, 0/1+ in tube. sequencing of genomic dna and cloned pcr products covering exons 6-7 detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*o.01.09 allele. case #5 is the newborn baby of case #4, with a forward type a 1+ mf in gel, a 3+ mf in tube. sequencing of the baby's dna detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*b.01 allele. from these results it is inferred that the phenotype encoded by allele abo*784c is a3-like. case #6 is an 18 year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons 5-6 and 6-7 detected variant c.979c>t (gln327ter) in heterozygosity, and in trans an abo*o.01.02 allele. the truncation of the a1 transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo*979t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. variants reported to date in the intron 1 enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut1 could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v5.1 170221 of red cell immunogenetics and blood group terminology working group of the isbt, 55 fut1 alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut1 alleles for the 19 chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut1 was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut1 sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut1 homozygous mutations were found in the 12 individuals and fut1 heterozygous changes were existed in 7 individuals after bidirectionally sequencing. .05%, 7.89%, 5.26%, 5.26%, 5.26%, 2.63%, 2.63%, 2.63% respectively in the 19 individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut1*01n.06(c.551_552delag), fut1*01w.09 (c.658c>t) and fut1*01w.01 (c.293c>t) were abolished in vitro assay, while fut1 mrna levels of them had no effect compared with wild type. summary/conclusions: the fut1 mutations in the para-bombay individuals were various. the most common fut1 allele in the chinese individuals with para-bombay phenotype was fut1*01n.06(c.551_552delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a1 and a2. later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a1, and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons 6 and 7 of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron 1 enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron 1 enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o1v/a 1 . dna sequence analysis showed result abo*a01 (28+5859a), abo*o.01.02. the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a01(28+5859a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d175-2, immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately 85% of o d+c+ cells. the patient's genotype was identified as abo*o.01/*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a1/*o1 genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a1 homozygote result. sanger sequencing of abo exons 6 and 7 also gave anomalous reactions: no abo*a allele was detected. homozygosity for c.261delg was observed as well as heterozygosity for c768c/a. this result therefore suggests the patient's genotype is abo*o.01/*o.01.26. next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr 3 0 -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing 4 copies of the enhancer region were present. presence of a single copy of the 43-bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, 9-30% of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd*01el.01 (rhd*1227a) is most prevalent (>99%) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from 2017 to 2018, the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th-28/ms-26, igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of 129 serologically apparent d-chinese patients (female, n = 128; male, n = 1) with alloanti-d were identified. different titers of alloanti-d from 1:2 to 1:4096 (≤1:16, n = 60; >1:16, n = 69) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = 11; anti-d mixed anti-e, n = 5). serological rhd typing confirmed the serologically apparent d-phenotype. rhd*01n.01/01n.01 (homozygous rhd gene deletion) genotype was identified in majority of them (123/129, 95.3%) by rhd zygosity analysis, while rhd*01n.03/ 01n.01 genotype (n = 5) and rhd*01n.05/01n.01 genotype (n = 1) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average 25% frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, 2005; legler, trans. med., 2001; richard, transfusion, 2007) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all 10 rhd exons were sequenced. the sequencing data revealed the mutation c.148+2delt in the splice donor site of exon 1. to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns4) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c.148+2delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing 9 monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor 1 were predicted to be rh:-1,-2,-3,4,5. however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position 1151 which is the second last nucleotide of exon 8 and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon 8 was identified. the mutation c.1151c>g leads to the amino acid substitution t384r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., 2011) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd*1151g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor 2. further serological determination of the rhd antigen with 9 different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c.526g>c in exon 4. this mutation causes a p.a176p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c.1151c>g and c.526g>c harbouring an amino acid substitution within a transmembrane segment. the c.1151c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c.1151c>g suggests that the t384r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c.526g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id1 is a d-negative donor self-declared as african descent. sample #id2 is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id1 was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id1 were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id2 was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional (3d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id1, a single nucleotide missense change, i.e. c.1151c>g in exon 8, was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position 384 (p.thr384arg) of the rhd protein. analysis in the 3d model clearly suggests a dramatic impact of the p.thr384arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id1. in sample #id2, the single c.325a>g transition was found in exon 2 leading to a threonine-to-alanine substitution at amino acid position 109 (p.thr109ala). amino acid 109 is located in rhd protein extracellular loop 2, and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id2. summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type 4 and diva cluster. in africans, the 16 most frequent were typically associated with alleles of the weak d type 4 (including dol and rhdpsi), diva and dau clusters with f223v occurring in > 10% of alleles; in addition the key mutations of weak d type 1 and dii and two inactivating mutations (c.1056_1057inst and c.1060delg) not reported in rhb were among the first 20 polymorphisms. in east asians, rhd(1227g>a) at 0.8% was most frequent, followed by dfv, weak d type 33, dbo-3, key mutations of diva and weak d type 4 cluster as well as rhce-like substitutions and the mutations of weak d type 25, type 15, type 66, rhd(a85v), dvl-1, weak d 119 and rhd(n135s). weak d type 151 and rhd(t148r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type 45 and type 33 in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r 0 haplotype and 1 probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r 1 r 1 , r 2 r 2 , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v3.1 cycle sequencing kit. sequence data was aligned to rhd_ng_007494.1. rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on 201 rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using 2 different anti-d igm clones (clone 1, dvi+: ldm1+esd1m; clone 2, dvi-: rum-1, th28) and 2 different anti-d igg clones (clone 1: ms26; clone 2: d415 1e4). all donors with d-negative results (n = 201, divided into 153 subjects with rhc+, 45 with rhe+ and 3 with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further 101 donors, studied prospectively. in 92.5% of donors (n = 186), the molecular analyses showed the complete deletion of the rhd locus, while in 15 cases (7.5%) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these 15 donors revealed the presence of 9 negative and 4 weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd*01n.82 (2 cases), rhd*01n.83 (1), rhd*01n.80 (1), rhd*01n.61 (1) , rhd*01n.05 (3), rhd*01el.08 (1), rhd*01el.18 (1), rhd*01el.17 (1), rhd*01w.54 (1) . moreover we found a donor with a lack of signal encompassing exons 1-5 of the rhd sequence (bioarray rhd beadchip), while 2 additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about 1 in 50 cases (4 subjects out of 201), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of 43960 serologically typed donors, 85.5% were d+, 14.0% d-and 0.5% weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in 22 blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: 62% weak d type 1, 28% weak d type 3, one donor was typed as weak d type 14 and another one as weak d type 11. these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from 2+ to 4+ with iat, except for the weak d type 11 with the score of <1+ which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was 19%. one of the weak d type 1 donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type 14, as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a 14 year-old young woman with parameters of hemolytic anemia: low hemoglobin level (10 g/dl), reticulocytosis (14%), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron 4 and the 3 0 untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c.920c>t mutation in rhag exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c.920c>t mutation is responsible for the p.ser307phe amino acid substitution predicted to be in the 10 th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c.920c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of 15692 individual blood donors within a 15 month period, 1688 (10.76%) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from 112 out of 151 (8.95%) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron 4, exon 7 and exon 10 by a multiplex pcr-ssp method. the reaction was conducted in a final volume of 20 ll with primers that were applied as described by f. wagner et al. (bmc genetics, 2001 ) except antisense primer for exon 10 and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a 4% agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of 112 d-individuals analyzed, 101 were ddccee, 8 ddccee, 2 ddccee and one had a ddccee phenotype. molecular analysis showed that 104 (92.86%) were negative for all four rhd dna regions. among the other 8 samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron 4, exon 7 and exon 10, three for rhd promoter and exon 10, and two for exon 10 alone. further genotyping revealed five hybrid rhd-ce-d alleles [3 rhd-ce(2-9)-d and 2 rhd-ce(3-7)-d], one allele represented the del(m295i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than 100 weak d types have been described to date. transfusion recipients with weak d type 1, 2, or 3 are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is 5 anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band 3, as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, 4 haplotypes containing the mcc b and sl2 polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr1 polymorphisms and haplotypes, especially those containing mcc b and sl2 snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr1 polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do*01 and do*02 backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a 42 year old woman from the northeast brazil with a history of 4 pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the 3 exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology 2 diagnostic immunohematology 3 experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises 360 blood group antigens. most antigens (322) belong to one of the 36 blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of 38 antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in 1967.~91% of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in 96% of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a 3-cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b4galnt2 gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad 3-cells panel, containing donor 626521 with high expression of sd a . additionally, 200 pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b4galtnt2 exon 1-11. results: sequencing of b4galtnt2 revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in 6 controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon 10, c.1396t>c (enst00000300404.2, rs7224888) changes a cysteine to arginine at position 466 of the protein. the second mutation in exon 11 c.1590a>g (rs16946912) does not change an amino acid. both snps have a maf of 0.11 and therefore we expect that 2.1% of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b4galtnt2 gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon 10 causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~4% sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b4galnt2 and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by 5ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl8 was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = 0.016). similar result was also observed in rbc chemokine scavenging of ccl2 (p = 0.038). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = 0.021). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp1pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a 66-year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp1pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was 12.9 g/dl. 800 ml of blood was withdrawn through a radial arterial catheter in two 400 ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of 6% hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged 48 h later with a hemoglobin level of 12.3 g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp1pk and sequencing of the a4galt gene, which were conducted according to the protocols by koda et al.(transfusion. 2002) . the proband and her brother were homozygous for c.1029dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[140a], gypb*s_null(ivs5 + 5t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c.140c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about 0.06% and in thais with 0.04%. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom*12 allele. the lack of the serf antigen, serf(à) on red cells is caused by a single nucleotide polymorphism, c.647c>t in exon 5 of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(à) and a serf(à) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom*12 allele among blood donors. aims: this study aimed to identify the crom*12 allele among thai blood donors leading to predicted serf(+) and serf(à) phenotypes. methods: dna samples obtained from 1,512 central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom*12(+) and crom*12(à) among 1,512 central thais were 0.992 (3,001/3,024) and 0.008 (23/3,024), respectively. the homozygous of crom*12(à/à) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen 100 samples together with 23 heterozygous crom*12(+/à) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom*12(+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than 25 years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o1, o2, a2 and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, 31 from patients and 25 from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd1227g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene 1227 g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd 1227g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd 1227g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to 5 0 ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd 1227g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon 9 sequencing was performed to determine rhd 1227g>a genotype. results: a total of 84 samples were genotyped for rhd 1227g>a by pcr with t mshift primers. 32 samples were typed as 1227a+/g-, 22 samples were typed as 1227a-/g+, 6 samples were typed as 1227a+/g+ and 24 samples were typed as 1227a-/g-. two samples typed as 1227a+/g+ by pcr-ssp but 1227a+/g-by pcr with t m -shift primers were confirmed as 1227a+/g-by rhd exon 9 sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd 1227g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the 115 dna samples, 75 (65.2%) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon 8, which is functionally not relevant as reported before, was found in 28 samples, including 11 and 17 samples carrying respectively rhd-ce(8)-d and rhce-d(8)-ce (two homozygous each). except two samples that require additional studies (1.7%), rhd zygosity was resolved successfully: 60 (n = 2 rhd gene copies; 52.2%), 49 (1; 42.6%) and 4 (0; 3.5%). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples (4/115, 3.5%) carrying rhd*dau5, rhd*dv.2, a rhd*diiia-like allele, and a novel rhd*d-ce(7:g329h-y330s-n331i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd*03n.01 and rhd*diiic, were also found in trans with a normal rhd*01 allele. in rhce, c/c genotype could be resolved. the rhce*ce16 (rhce*ce (48c)-d(9)-ce) allele, which is commonly cis-associated with rhd*ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = 19) vs. non-bleeding (n = 15) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd63), p-selectin (cd62p), activated gpiib/iiia (pac-1) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd42b). normal healthy reference levels were available. results: the platelet count in bleeding (72 9 10 9 /l) and non-bleeding (68 9 10 9 /l) patients was comparable (p = 0,66). bleeding patients had a higher bat score compared to non-bleeding patients (10 vs. 2, p < 0,01). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) (86,16% and 4,96mfi) were higher, compared to bleeding patients (64,08% and 2,44mfi, both p < 0,05), and the proportion of ps+cps correlated negatively with bat score (r 2 =0,26, p < 0,01). cd63 + cp was higher in non-bleeding (97,25% and 10,54mfi) compared to both bleeding patients (94,88% and 6,89mfi) and significantly higher than the reference level (88,44% and 5,36mfi, both p < 0,05). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist (134,12 mfi unstimulated vs 32,38 mfi reference, p < 0,01). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa-1, hpa-2, hpa-3, hpa-5 and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa-1a, anti-hpa-2b, anti-hpa-3a, anti-hpa-5a samples were used to validate the specificities of the luminex assay. the anti-hpa-1a, anti-hpa-3a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to 1/1024). results: 44 samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa-1a, hpa-2b, hpa-3a, or hpa-5a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa-1a, and anti-hpa-3a were 1:512 and 1:64, respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of 44 samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of 358 individuals with normal abo groups (101 group a, 100 group b and 101 group ab individuals, and 56 group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg1 anti-b antibody (9431pe bgrl1). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v5.01. the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than 0.05 were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about 66.22% of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: 40 cases were found with antibody positive. among them, 6 cases (15%) were only anti-hla-i positive, 4 cases (10%) were only anti-hpa positive, 30 cases (75%) were both anti-hla-i and anti-hpa positive. 8 cases were found without anti-hla-i or anti-hpa. among the 34 cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were 73.5%, 61.2%, 50%, 44.1%, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > 0.05). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of 303 blood donors and 302 hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v 2 test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr3b*01 (hna-1a), fcgr3b*02 (hna-1bd), and fcgr3b*03 (hna-1bc) alleles were 0.384, 0.584 and 0.032; for the slc44a2*1 (hna-3a) and slc44a2*2 (hna-3b) alleles, 0.804 and 0.196; for the itgam*1 (hna-4a) and itgam*2(hna-4b) alleles, 0.898 and 0.102; for the itgal*1 (hna-5a) and itgal*2 (hna-5b) alleles, 0.708 and 0.292, respectively. in hematological patients, the gene frequencies for hna-1a/1bd/bc, -3a/3b, -4a/4b, and -5a/5b were 0.376/0.588/0.036, 0.795/0.205, 0.887/0.113, and 0.699/0.301, respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna -1, -3-5 for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = 605). the predicted risk of hna-1, -3, -4, -5 incompatibilities in this cohort were 34.9%, 26.9%, 19%, and 32.9%, respectively. the possible risk of hna-1a, -1bd, and -1bc alloimmunization were 0.233, 0.143, and 0.064, respectively; of hna-3a and -3b alloimmunization, 0.037 and 0.231; of hna-4a and -4b alloimmunization, 0.01 and 0.163; of hna-5a and -5b alloimmunization, 0.080 and 0.250, respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons (5, 7 or 10) of the rhd gene. methods: 2004 samples were tested from 01/12/2017 to 01/12/2018 using free dna fetal kit â rhd. 44 samples (2,2%) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce(4-7)-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. 65% of the identified variants are rhd negative alleles including alleles leading to partial rh2 antigen expression. unexpected alleles are found such as weak d type 1, 2, 5 or 11. these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on 20 cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed 16 of 20 (80%) pregnant women were rhd*weak d type 1 and not at risk for anti-d. rhd*weak d type 3 were typed in 3 cases (15%) and 1 case was rhd*weak partial 4.0 and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that 95% of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may 2018, uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of 3 ml. all material used passed pre-acceptance serological testing; samples were dispatched to 298 participants in 17 countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of 1.1 ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the 36 participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed 2 + reactions vs. two reagents by tube, cf. 4 + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of 3.7 ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with 4/17 anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all 16 respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa-1a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from 133 plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = 29), s/s (n = 13), hpa-1a (n = 49), hpa-3 (n = 8), hpa-5 (n = 14), hpa-15 (n = 20) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of 100-16,000 reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from 0,01% to 26,4% (one case was for advanced pregnancy -38 week of gestation). in 19/133 tested samples false positive results were detected at the level of 1 or 2 unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa-1a, -3a/b, -5a/b, -15a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh1) and anti-c (rh4) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih-500 system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih-500 system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from 29 samples containing anti-d and 20 containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih-500 system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < 0.0001, r = 0.79 and p < 0.0001, r = 0.82 respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < 0.0001) but gel scores have a better correlation coefficient than tube scores (r = 0.78 versus 0.55). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were 75 and 35, corresponding respectively to 5 ui/ml (250 uchp/ml) of anti-d and 7.5 ui/ml (500 uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih-500 system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from 250 pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january 2008 to december 2016. we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ 1:8 for anti kell antibodies and ≥ 1:32 for other specificities. results: out of 250 women, 143 (57.2%) displayed clinically significant antibodies, 92 (36.8%) clinically insignificant antibodies and 15 (6%) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d (16.8%) also in combination with other rh antibodies (8.8%), while anti-k accounted for 10%, anti-e for 10% and antibodies against high-incidence antigens for 2.4%. anti-m and anti-le a antibodies were also found (16.8% and 8% respectively) but they were not clinically significant. among 143 women with clinically relevant antibodies, 37 showed a critic antibody title and they underwent gynecological and obstetric monitoring. 21 fetuses resulted affected by hdfn, displaying anti-d in 16 cases and anti-kell in 5. 11 fetuses with severe hdfn (anti-d in 7 and anti-kell in 4) required iuts, 2 were treated with et, 8 received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from 0,4% to 2,7% worldwide. there are over 400 erythrocyte surface antigens, of which more than 43 have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in 2016 and 2017. methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during 2 years, from january 2016 to december 2017. in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, 5982 pregnant women were attended in hospital de braga. the laboratory registered 100 positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was 1,7%. anti-d was the most common antibody found (58,5%). anti-d prophylaxis given during pregnancy was responsible for 51 of 62 cases and maternal antibody titer levels did not exceed 8 among these cases. the prevalence of non-rhd immunization was 33%. anti-e (9,4%) was the most frequent alloantibody other than anti-d followed by anti-m (5,7%) and anti-c (4,7%). multiple maternal antibodies were found in 5 pregnant women. four women had 2 types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had 3 types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in 45% of them and phototherapy was given in 14%. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was 1,7%. the erythrocyte antibody screening showed that anti-d was the most common antibody found (58,5%) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was 33%. the other most frequent alloantibodies were anti-e (9,4%), anti-m (5,7%) and anti-c (4,7%). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may 2018, the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih-500 system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard (01/572) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the 2-months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of 2 have lower values. the highest differences (more than 2 to 3 dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel1) and anti-m (mns1) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih-500 system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a 1-day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was 13 mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes (15.2%), ldh (1162 iu/l) and g6pd (25.3 u/ghb); dat (+/-), iat (-), anemia (hb 8.7 g/dl, hct 28%), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs (3 + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with 1:1024 and 1:2048. the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to 14.6, bilirubin level was within normal range, she was discharged. another 5-days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was 15 mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes (5.2%), dat (+/à), iat (à), hb 12. background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in:1,-2 phenotype (in(a+b-)) are observed with a frequency of < 0.1% in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon 2 of cd44. results: in a 27-year-old pregnant (para 1) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of 1:2 was detected by iat (negative with papain-treated cells) at gestational week (gw) 32 and 36. the mma, performed in duplicate on samples taken at these dates, showed a mi of 0.5%/4.8% and 7.7%/6.3% respectively. the mi was interpreted as follows: 0-3% not relevant; 3-5% inconclusive; >5% clinical significant. the patient's parents were typed heterozygous, in:1,2 whereas her husband was homozygous, in:-1,2. due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw 41 without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw 32, was inconclusive whereas the second mma, performed in gw 36, indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included 20 immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period 2013 and 2018. results: the "critical titre" in our study was 8, meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only 2 newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than 4. moderate hemolytic disease of fetus and newborn were caused between the titre values 8-32. the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, 16 weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons 4, 5 and 10. rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all 3 exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at 3 nucleotide positions (c.1154, c.1193, c.1227) located in rhd exon 9 suggesting exon 9 was either deleted or rhce-replaced. paternal and cord gdna sequencing detected 4 out of 6 snps (c.186g>t, c.410c>t, c.455a>c, c.602c>g) associated with diiia phenotype plus 2 additional snps (c.604g>a, c.733g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from 5.3 iu/ml (16 weeks gestation) to 166 iu/ml (33 weeks gestation). the fetus required 3 intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon 9 deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd109 is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of 170 kda. in addition to being expressed on human plts, cd109 is expressed on activated t-cells, endothelial cells, cd34 + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa-15a and -15b are 0.505 and 0.495, respectively. based on these data, the risk of alloimmunization against hpa-15 alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa-15 alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa 15b alloantibodies by maipa and icfa. methods: a 24-year-old mother, gravida 2/para 0. the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy 33 weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy 24 weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to 4.7 9 10 10 /l, wbc count dropped to 2.96 9 10 10 /l, including neutrophil 37%, lymphocyte 16%, mononuclear 43%, eosinophil 2%, basophil 2%, red blood cells were normal, hb was 111 g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa-1a/a, -2a/a, -3a/a, -4a/a. -5a/a, 6a/a, 7a/a, 15a/b, naka (+) and the maternal was hpa-1a/a, -2a/b, -3a/a, -4a/a. -5a/a, 6a/a, 7a/ a, 15a/a, naka (+). the paternal genotype was hpa-1a/a, 2a/b, 3a/a, 4a/a, 5a/a, 6a/a, 7a/a, 15a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa-15aa and -15bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa-15a/a (donors 1), hpa-15a/b (donors 2) and hpa-15b/b (donors 3) donors by maipa. the mother's serum showed no reactivity against 15a/a plts, weak positive reactivity against 15a/b plts (od values 0.28), but strong reactivity against 15b/b plts (od values 0.38).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa-15genotyped donors (anti-hpa-15b average value 9.8). summary/conclusions: in this study, we found anti-hpa-15b in a case of fnait (patient hpa-15aa, blood group o) using the maipa technique. we were able to detect the presence of hpa-15b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in 1:10000 live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = 40). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, 2014 ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at 4 weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. 19%) are deceased due to organ rejection, and 8/36 patients (22%) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of 8-methoxypsoralen (8-mop) and irradiation with uv-a light. however, the addition of 8-mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with 8-mop+uv-a compared to uv-c treatment without additional 8-mop. methods: we used an in vitro 72 h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with 0,2 lg/ ml 8-mop plus 2 j/cm 2 uv-a treated cells and 2 j/cm 2 (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and 7-aad flow cytometry standings. results: the apoptosis analysis of cd3 cd4 t-helper cells, cd3 cd8 cytotoxic tcells, cd19 b-cells, cd14 monocytes, cd3 neg cd56 nk-cells and cd3 cd56 nkt cells revealed no statistical differences in almost all of these cell types after treatment with 8-mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after 72 h were similar in both treatment groups. summary/conclusions: the addition of 8-mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the 8-mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of 8-mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with 8-mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with 16 bedded micu and received tpe therapy between 1 june, 2016 and 31 december 2018. all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln9000 apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone 269 tpe procedures. among them, thirty were female patients (43%). the median age 45 (13-75) years. guillain-barre syndrome (gbs) was the most common indication (72%) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were 48 (18%) episodes of patient related complications during the tpe treatments. in 8 (3%) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts 13 levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of 9 years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in 46 patients with diagnosis of maha (27-ahus, 16 -ttp, 1 each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was 19.94 ae 19.58 years with m:f as 1.5:1. number of procedures per patient varied from 1 to 27. post pe recovery was observed within 10-14 days with statistically significant increase in mean platelet count from 40.05 ae 5.9 to 82.11 ae 12.10 9 10 9 /l (p = 0.000) and significant decline in mean lactate dehydrogenase level from 48.91 ae 34.73 to 10.98 ae 6.49 lkat/l (p = 0.000). there was also significant decline in mean percentage of schistocytes in peripheral smear from 5.44 ae 3.96% to 0.56 ae 0.89% (p = 0.000). the mean serum urea changed from 48.88 ae 24.56 to 24.50 ae 17.78 mmol/l and creatinine from 266.11 ae 142.59 to 173.09 ae 127.34 lmol/l (p = 0.000 and 0.001 respectively) with significant increase in urine output from 0.71 ae 0.53 to 1.06 ae 0.33 ml/kg/h (p = 0.000). adverse events were observed in 10 patients (21%), allergic reaction to replacement fluid (n = 6) being the commonest followed by hypotension (n = 2), rigors and chills (n = 2). overall survival rate at 6 months was 89%. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a 17 year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since 2-3 days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, 6 cycles of tpe were performed on daily basis. after 6 cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [1] for motor performance, patient was assessed on a disability scale (0 = healthy; 1 = minor symptoms; 2 = able to walk 5 meters without support; 3 = able to walk 5 meters with support; 4 = confined to bed or wheelchair; 5 = requiring assisted ventilation; 6 = dead).patient's motor performance was increased to scale 1(upper limb) and 2(lower limb) from scale 5, deep tendon reflexes were normal. visual function began to improve 1 week after the treatment. visual acuity was 6/6 after 4 weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of 10% has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july 2014 to july 2018. a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > 10% but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than 10%, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were 10.6%, 11% and 31%. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused 46 days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between 9-16 days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from 01/01/2017 to 31/12/2018 in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of 6 patients met the inclusion criteria described. however, 1 patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded 88 exchange procedures (42 pmet and 46 aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why 2 patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = 2), stroke (n = 1) and recurrent vaso-occlusive crisis with multiorgan failure (n = 2). for both procedures, target values were to obtain a pre-exchange hbs level ≤ 30% for stroke and cerebral vasculopathy and ≤ 50-60% for other indications. the median hbs level before pmet was 42,6% (31,3-59,1) and 38,4% (18, 9) before aet. we documented a higher hbs level prior to the next procedure in 11,4% of patients (n = 10). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: 1356,1 vs. aet: 1314,7 ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: 360 vs. aet: 60 min and pmet: 21 vs. aet: 24 weeks, respectively). annual rbc requirements per procedure were superior (median 2 vs. 4 units) and the overall costs related with aet were 2,2 times higher -18.180,93€ and 8.174,79€ aet and pmet, respectively (estimated cost per session aet: 790,48€ and pmet: 389,28€). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo2) of 87% on air. the patient did not show improvement in spo2 level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo2 of 97% on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july 2016 to august 2017 were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these 46 (43%) were done on the mcs plus (haemonetics corporation) and rest 61 (57%) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = 0.000). the systolic (p = 0.010) and diastolic (0.001) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < 0.001), the age of the patient (p < 0.001) and the pre-procedure ph (p = 0.002). procedurerelated complications occurred during 30 procedures of which 13 complications (12.15%) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of 16 years (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . tpe procedures were done on two different apheretic devices (cs 3000 plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of 356 tpe (range 1-22/patient with mean of 6.2 procedures) were performed for 55 pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total 44 patients, neuromyelitis optica (category ii) in 4 patients, rapid proliferative glomerulonephritis (category i), c3 glomerulopathy in 3 patients each and one patient of infective hemophagocytosis. the average age of patient population was 7.8 yrs (1.2-13 years) . the male:female ratio was 3:1 with an average weight of 25.5 kgs. adverse events were observed during 20 (5.61%) procedures. most commonly observed adverse events were allergic reaction to replacement fluid (1.4%) followed by hypotension (1.1%), line occlusion (0.8%), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each (0.28%).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of 160 registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = 80)they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ 50 g); and group ii (n = 80)they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion 60, ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected 1 h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = 0.000) in group i (mean ae standard deviation: 67.86 ae 8.07 g; range: 50.80-92.13 g) than group ii (60.92 ae 8.29 g; range: 40.86-86.76 g). the mean hb increment in group i patients (3.26 ae 0.83 g/dl) was significantly higher (p = 0.04) than the group ii patients (3.00 ae 0.76 g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = 0.000 in groups i and ii), age (p = 0.001 for group i; p = 0.032 for group ii), body surface area (bsa) (p = 0.002 for group i; p = 0.000 for group ii) and blood volume (p = 0.006 for group i; p = 0.000 in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = 0.000 in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of 50 g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under 50 years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between 20/03/2004 and 01/09/2015 was collected. for the current study, male transfusion recipients under 50 years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (<10 days storage) or 'old' (>24 up to 36 days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was 0.47 day shorter when comparing the 221 patients who died, to 1,623 patients who survived (ci: à1.41 to 0.48). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was 0.64 day longer when comparing the 27 patients who died, to 101 patients who survived (ci: à2.53 to 3.80). in the single-transfusion cohort, 1,280 patients received a fresh rbc transfusion from a male donor, 52 of whom died; 138 patients received a fresh transfusion from an ever-pregnant female donor, 9 of whom died. 193 patients received an old transfusion from a male donor, 7 of whom died; 16 patients received an old transfusion from an ever-pregnant female, 2 of whom died. the 3-years cumulative incidence of death among young male recipients was 4.7% (confidence interval (ci): 3.6% to 6.2%) after a fresh transfusion from a male donor and 4.8% (ci: 2.2% to 10.4%) after a fresh transfusion from an ever-pregnant female donor. the 3-years cumulative incidence of death was 7.2% (ci: 3.8% to 13.6%) after an old transfusion from a male donor and 15.4% (ci: 4.1% to 48.8%) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at 3 years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from 50 to 80%. in our previous investigation, we found that premedication rate was 92.5% in the outpatients in 2017, which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, 2017 was retrospectively surveyed. evidence-based clinical practice was initiated since january, 2018. clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, 2018. the incidences of atrs and premedication rates in 2017 and 2018 were compared using chi-square test. a p value less than 0.05 was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, 2017 to september, 2018, a total of 5,018 blood units were transfused in the outpatients with 2,453 transfusion events. of these, 25 cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was 92.5% in 2017, and was significantly decreased to 77.9% in 2018 (p < 0.001). it was reported that the incidences of atrs in 2017 and 2018 were 0.48% and 0.52% per unit, respectively. there was no remarkable difference between the incidence of atrs in 2017 and 2018 (p = 0.642). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of 19 granulocyte transfusions in 15 hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) 5-10 lg/kg and tablet dexamethasone 8 mg, 10-12 h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within 30 days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of 1 9 10 10 per unit was fulfilled in 90% of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < 500/ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least 48 h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within 24 h) increased significantly (median value: 350/ll) as compared to baseline levels (median value: 40/ll) (p < 0.05). infection related mortality was observed in only 20% (3 out of 15) of patients. patients became afebrile within 2-4 days and culture negative within 3-6 days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: 1.5-3.0 9 10 8 cells/kg and high dose: >3.0 9 10 8 cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in 2016, we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for 2017. results are compared to the pilot audit and summarized below. methods: we collected data on 3 performance indicators from 7 acute public care hospitals for 4 weeks each in march and august 2017 (the pilot audit covered 2 weeks in 2016). the performance indicators were: 1). percentage compliance to documentation of red blood cell transfusion indications 2). percentage of patients screened for pre-operative anaemia, 14 to 45 days before surgery 3). peri-operative transfusion rates (3 days before to 3 days after surgery) for 6 commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator 3) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in 2012. results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator 1), 2 hospitals had a compliance of 57-61%, the remaining 5 had a compliance of 90-100%. all 7 hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from 75% in 2016 to 100% in a hospital that had newly mandated electronic ordering. for indicator 2), results ranged from 30% to 92%. 2 hospitals made notable improvements when compared to 2016, achieving 83% and 88% respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least 2 weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator 3), mean number of transfused units for each surgery ranged from 1.5 to 2.8 units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at 46% and lowest for tkr at 7%. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator 3) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november 2017 and march 2018. the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha 3 months after an allogeneic stem cell transplant, who has received multiple transfusions in the last 2 weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently 48 g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of 104 g/l and returns to clinic at a 2-week interval with symptoms of fatigue. he is actively haemolysing and commenced on 1 mg/kg prednisolone. results: there was a 42% (58/137) response rate by trusts. faced with a 4-6 h delay for allo-adsorption studies, 68% (38/56) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, 7% (4/56) would transfuse with o rh d negative red cells and 25% (14/56) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. 2017 british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the 2017 serious hazards of transfusion (shot) report, the most serious and fatal of 95 cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of 38 g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < 60 g/l after an hb fall of at least 40 g/l in the previous 2 weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits (2008) (2009) (2010) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between 14 th and 27 th may 2018 excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines (2008) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to 2 or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: 105 episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of 241 rbc units to 103 patients and the discard of 2 rbcs (due to incorrect transport). of the 105 episodes, 78 episodes (74%) involved an eventual switch to group-specific rbcs (range of emergency units, 1-13 units). the main requester was the emergency department (53%). the most common clinical indication for transfusion was acute gastrointestinal bleeding (49%). of the 105 episodes, 28 episodes (27%) did not meet the guidelines for emergency use because > 2 units of emergency uncrossmatched o rhd-neg rbcs were issued. 2 episodes (2%) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. 30 episodes (29%) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > 1 h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding (6%), receipt of an unsuitable pretransfusion sample requiring sample recollection (5%), delay in pre-transfusion sample processing (4%), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding (10%). only one patient was investigated for potential transfusion-related adverse outcome (1%) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, 105 episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with 241 rbcs issued and 2 rbcs discarded. a significant proportion of episodes (29%) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within 1 h before transfusion, 1 h after and 14-24 h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap-6) was assessed by flow cytometry by measuring p-selectin and pac-1 expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of 22 patients. the mean platelet count before transfusion was 8 9 10 9 /l (range 2-34 9 10 9 /l). 1 h cci was 11 9 10 9 /l and 14-24 h cci was 6 9 10 9 /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at 1 h after and 14-24 h after transfusion compared to before transfusion (p < 0.05). pac-1 expression after stimulation with adp was significantly higher at 14-24 h after transfusion (p < 0.001), but not at 1 h after transfusion. in rotem, clot amplitude at 10 and 20 min (a10 and a20) as well as maximum clot firmness (mcf) improved after transfusion (p < 0.05). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s =0.53 and 0.45 respectively, p < 0.001). absolute platelet count was also significantly correlated with mcf (r s =0.61, p < 0.001), where 84% of patients with a platelet count of more than 20 9 10 9 /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than 20 9 10 9 /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule-1 [soluble vcam-1]) in patients with scd in their non-crisis, "steady state." methods: patients, at least 10 years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: 15, hbsb thalassemia: 10) were enrolled (females: 15 [60%]), with a median age of 23 years (iqr: 11). following 6 months of hu, median values for wbc count (9.02 9 10 9 /l vs. 7.22 9 10 9 /l, p = 0.007) and d-dimer (1243.8 ng/ml vs. 830.2 ng/ml, p = 0.028) were significantly lower than baseline values, while the mean corpuscular volume (76.0 fl vs. 88.0 fl, p = 0.001) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin (9.1 g/ dl vs. 9.6 g/dl, p = 0.72), platelet count (250 10 9 /l vs. 196.5 10 9 /l, p = 0.71), lactate dehydrogenase (744 u/l vs. 567.5 u/l, p = 0.23) or soluble vcam-1 (567.8 ng/ ml vs. 526.4 ng/ml, p = 0.33) following 6 months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included 478 confirmed breast cancer patients planned for elective breast surgeries from january 2012 to december 2017. patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor 2 (her -2) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤40 years] and older group (>40 years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < 0.05 was considered statistically significant. results: of the total 478 patients most underwent wide local excision of the breast and modified radical mastectomy. a total of 16 patients received 71 units of blood and blood components in all categories of surgeries. only 103 were younger women (≤40 years) with mean age of 31 years. non-transfused patients were significantly more than transfused ones (p < 0.05). frequency of blood transfusion was more in young patients (4.9%). seven (22.6%) of the total 31 stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy (8.8%). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in 2009 and fully revised in 2016 by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a 700-bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in 2016. it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ 7 g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ 65 years, age ≤ 6 months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep 2016 to feb 2018 were analyzed. results: the number of transfusion-indications analyzed was 16138 for rbcs, 11158 for plts and 6024 for ffps. the most common indications for transfusion were chronic disease for rbcs (7977/16138, 49.4%), bleeding prevention for plts (5726/11158, 51.3%) and 'other' for ffp (2180/6024, 36.2%). 'hb ≤ 7 g/dl' was the most frequent sub-indication of chronic disease (3570/7977, 44.8%), and hematologic disease was the most frequent sub-indication of bleeding prevention (3432/ 5726, 59.3%). many clinicians entered transfusion indication as 'other': rbcs (2866/ 16138, 17.5%), plts (856/11158, 7.7%) and ffp (2180/6024, 36.2%). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; 82.9% of rbcs and 54% of plts. of the indications entered as 'other' in ffp, 80.3% were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep 2016. transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged 1-18 years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and 1 h post-transfusion, the expression of cd62p on platelet was determined by flow cytometry method. results: there were 102 subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd62p for nonleukodepleted and leukodepleted groups were 26.2% and 27.7%, and the mean increase of post-transfusion platelet cd62p for non-leukodepleted was 10.1% and the mean decrease of leukodepleted groups was 3.3%. it was shown the increase of post-transfusion platelet cd62p for non-leukodepleted group, and it was significantly (p < 0.05) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd62p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of 158 patients hospitalized in the period january -may 2018 in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < 12 g/dl and males < 13 g/dl), from the 158 patients included in the study, 61 (39%) were anaemic. from 117 males in the study, 40 (34%) of them were anaemic based on hb lab values, whereas from 41 women in the study anaemic were found to be 21 (51%) of them. from the 61 anaemic patients in the study, 35 (57.4%) of them with mild anaemia, 23 (37.7%) with moderate anaemia and 3 (4.9%) with severe anaemia. in the total of anaemic female 38.1% are under 65, while 61.9% are over/or 65 years old. in the total of anaemic males, 35% are under 65, while 65% are over/or 65 years old. it is noticed that most of them are with normochromic normocytic 67.2%, normocytic hypochromic anaemia 16.4%, hyperchromic microcytic anaemia 8.2%, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively 1.6% and microcytic normochromic anaemia 4.9%. the average value of preoperative hb decreased from 12.8 g/dl before surgery to 10.3 g/dl after surgery, so there is a decrease of approximately 2.5 g/dl of hb value. in our 158 patients, 48% (76) were transfused and the remaining 52% (82) were not transfused. from 76 transfused patients 41 (54%) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, 2003 to december, 2017, the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january 2008 to december 2009, the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of 988 patients were performed with unexpected antibody identification tests. of 1896 patients who underwent more than 2 screening tests, 25 (1.3%) were positive. 1871 were consistently unexpected antibody negative. the patients with solid tumors (n = 375, 56.3%) and those with hematologic diseases (n = 112, 16.8%) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = 0.0002). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group (65/988, 6.6%) than in the negative group (77/1871, 4.1%) (p = 0.006). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group (28/1871, 1.5%) than in the positive group (5/988, 0.5%) (p = 0.019). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in 2018 there were three female patients in the clinics of the centre for observation, age between 29 and 51 years (me = 39) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)-2, acute myeloid leukemia (aml)-1. individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac3/4 were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd41a. the density of fixed paig, pac was © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose 0,4 g/kg per day, for 3 days. one patient with aml received ivig-iggam therapy in the standard dose 5,0 ml/kg/day for 3. results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa-1 mfi-paigg reduced from 926 to 65; while the patient with aml: paiga reduced from 5506 to 187, and paigm from 1971 to 302, pac3 from 691 to 31, pac4 from 404 to 103. the patient with aa-2 over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the 3 patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells (60% vs 78%) and fresh frozen plasma (36% vs 84% p<o.oo1). more patients in the study group received platelets (72% vs 60%) and cryoprecipitate (50% vs 36%). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a 33-years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a 33-year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of 3 years at dubai thalassemia center. she was admitted to the hospital with 5 days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of 7.8 g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was 8.5 g/dl with a positive dat with both igg+4 and c3+4, patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin 5.6 g/dl, mcv 85.0, platelets count 216000, crp 3.6, retic count 2.18, blood culture no growth, total bilirubin 1.5, malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg 57.1 and igm 21.3, she was started on azithromycin for 5 days and received 2 units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to 9.3 g/dl. she did not show any improvement in her condition over the following 2 weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for 10 days. screening for collagen disease were all negative apart from high esr of 130 mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was 11.8 g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to 85 mm/hr and hemoglobin 10.2 g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of 1.2 g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first 60 min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january 1, 2011 to january 1, 2013 in 61 dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within 60 min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within 120 and within 180 min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of 1216 women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in 780 women (64.1%). in this cohort, seven maternal deaths (0.6%), 62 hysterectomies (5.1%) and 159 arterial embolizations (13.1%) were observed. plasma transfusion within 60 min in 114 women was matched on time-dependent propensity scores with 114 women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, 21.2% versus 19.9%, adjusted odds ratio 1.09 (95% confidence interval 0.57-2.08). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within 60 min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included 60 patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november 2017-july 2018. patients were previously taken asa at a dose of 100 mg per day, but therapy was discontinued 5 days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi 790-1410 au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (<790 au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately 30% of carrier females have factor ix clotting activity lower than 40% and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: 45 year old woman, with no history of abnormal haemorrhage, healthy, up to 2004 when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of 20% fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in 2008, after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since 2009. in 2012, she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with 2000 iu recombinant fix (rfix) daily, for four days, and up to 48 h after discharge maintaining basal levels of fix of 60% and with no major complications reported. in 2013, the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of 4000 iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of 3000 iu rfix are administered from day 1 (d1) and up to d7 maintaining fix levels around 60-80%. the patient is operated at d8 under the cover of 2000 iu of rfix, twice per day, and up to d14 after surgery reaching fix levels of around 80-110%. at d15 the dose was diminished to 2000 iu of rfix daily and at d17 she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february 2018 to january 2019. the study was approved from institutional review board. pack red cells (prc) transfusion in case of < 7 g/dl hemoglobin (hb) with cardiac history and < 7 g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < 50 9 10 9 /l, sepsis with bleeding and < 100 9 10 9 /l, patients on active chemotherapy without bleeding but platelet count < 10 9 10 9 /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version 23.0 was used for descriptive and inferential statistics. results: a total of 250 transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data 194(78%). out of 250, 134 (54%), 100(40%) and 16(6%) prcs, platelets and ffp were transfused respectively. out of 100 platelets, 10(10%) were platelet apheresis. 80/134 (60%) prcs and 22/90 (24%) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than 1 unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = 0.121). thirty six (45%) of transfusions were done with more than 1 units of prcs in non genuine group. the mean hb level of patients received 1 unit and more than 1 units prcs was statistically same (p = 0.868) and it was found out that there was same increment in hb in both the groups post transfusion. (p = 0.867). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = 4) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period (1 h) sheep were bled~40% of their estimated blood volume (estimated at 65 ml/kg) to a mean arterial blood pressure (map) of 30-40 mmhg. sheep were then resuscitated with either plasmalyte â (up to 20 ml/kg/ hr; n = 3) or transfused with sheep red cells (n = 1) to achieve a map > 65 mmhg. sheep were euthanised 4 h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of 931.7 ae 135.3 ml blood which combined with iatrogenic blood loss (~300 ml) corresponded to an average 40.2 ae 2.4% blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = 30-40 mmhg, lactate > 4 mm, svo 2 < 60%). across all four sheep the nadir map averaged 40.5 ae 13.1 mmhg, lactate peaked at 3.9 ae 1 mmol/ l, and nadir svo 2 was 41.3 ae 17.9%. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve 100% incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p-515 bilirubin were recorded within the 28-day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a 15-min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c3d. the rh group and non-rh group had 11 and 10 cases performed manually, and results were 2+ or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in 31/44 (70%) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in 2017, out of 7,276 notified atrs, 113 (1.5%) and 6 (0.1%) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january 1 st 2010 to june 30 th 2018. each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, 9,544 atr were reported, of which 29 included unconsciousness and/or convulsions (0.3%). of these 29 patients, 13 were females (44.8%) and 16 males (55.2%). unconsciousness alone was frequently observed (21 reports, 72.4%). convulsions were notified in 8 reports (27.6%) and were associated with unconsciousness in 2 of them. the diagnosis of seizure, with no other clinical signs, was established in 2 cases (6.9%). unconsciousness and/or convulsions were present in 8 allergic reactions (27.6%), 4 cases of transfusion-associated circulating overload (13.8%), 3 cases of suspected transfusion-transmitted bacterial infections and 2 hypertensive reactions. in allergic atrs, unconsciousness was notified in 7 cases and unconsciousness associated with convulsions in one. twelve atrs were severe (41.4%), 10 were life-threatening (34.5%) and in 4 cases, they resulted in the death of the recipient (13.8%). of the 8 allergic atrs, 4 were severe and 4 life-threatening. red blood cell concentrate was involved in 15 atrs (51.7%) and platelet concentrate in 9 (31.1%), including 5 cases with apheresis platelet concentrate and 4 cases with pooled platelet concentrate. fresh frozen plasma was involved in 5 atrs (17.2%). nevertheless, the imputability of the blood product was excluded or unlikely in 11 atrs (37.9%). in the 3 suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in 7 and 9 atrs respectively, but was certain in only 2 atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr (26 ars, 89.7%). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr 3.0, 95% ci 1.5-5.7, p = 0.001). this effect was most pronounced in the td-rs subgroup (hr 6.0, 95% ci 2.2-16.2, p < 0.001). similarly, elevated lpi levels were associated with inferior pfs (hr 3.4, 95% ci 1.9-6.3, p < 0.001) for the whole study population and the td-rs subgroup (hr 8.2, 95% ci 3.4-21.0, p < 0.001). in total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 5 patients desferrioxamine). lpi levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = 4 of each) was cultured with rpmi media (37°c, 5% co 2 ) alone or with the addition of lps (1-100 lg/ml; derived from escherichia coli 055: b5). the inflammatory response was assessed after 2 h (h), 6 h, 12 h, 24 h, 36 h and 48 h. supernatant was harvested at each time point and stored at à80°c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il-1b, il-6, il-8 and il-10). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il-1b and il-10 but not il-6 or il-8. il-1b production was significantly increased following stimulation of 100 lg/ml lps for 6 h (p = 0.043) and declined following 36 h incubation. release of il-10 was significant 12 h post-lps stimulation with 100 lg/ml (p = 0.030) and reached a maximum at 24 h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps (1 lg/ml), although the incubation times differed. il-1b was significantly increased following 2 h incubation (p = 0.002), with increasing levels observed up to 24 h post-lps stimulation. il-8 production was evident from 6 h and reached significance at 24 h post-lps stimulation (p = 0.009). il-10 was significantly increased following stimulation of 5 lg/ml lps for 6 hr (p = 0.043) with lower concentrations of lps resulting in il-10 production at 24 h (p = 0.008). release of il-6 was significant after 6 h of 50 lg/ml lps stimulation (p = 0.046), with lower concentrations of lps resulting in il-6 production at 24 h (p = 0.015). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. 1. rhdig inappropriately administered (unnecessary exposure) (n = 17, 40%) administered to: -rhd positive woman (n = 9) -rhd negative mother with rhd negative neonate (n = 5) -woman with immune anti d (n = 1) -administered in error (instead of other ig) (n = 2) 2rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = 17, 40%) -omitted (n = 14) -delayed (n = 1) -inadequate dose (n = 2) 3administration without correct patient identification (n = 6, 14%) 4storage & handling (n = 3, 7%) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo 2 agencia gallega de sangre, organos y tejidos, galicia 3 banco de sangre y tejidos de cantabria, cantabria 4 banco de sangre de la rioja, la rioja 5 banco de sangre y tejidos de navarra, navarra 6 banco de sangre y tejidos de arag on, aragon 7 fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon 8 fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain 9 terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in 17 autonomous regions, the hemovigilance system is structured in three levels: (1) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to (2) the regional hemovigilance coordinator, who communicates all the region's data to the (3) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in 2008. the mirasol prt system for platelets and plasma was introduced in 2010 and is currently being used in 10 of the 17 spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from 2012 to 2017 as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between 2012, when 4,196 mirasol treated blood products were issued to hospitals and 2017 with 56,229 mirasol products issued. due to low number of transfusions of mirasol-treated blood components in 2012 and 2013, notification rates began to be analyzed in 2014, showing ae rates of 0.2%, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around 0.23). rate of ae after transfusion of m-p is fluctuant between 0.06 and 0.17. this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around 80%) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > 2 reactions in 2016 and 2017 of respectively 0.18 and 0.07 no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period (2012) (2013) (2014) (2015) (2016) (2017) . at the national level, nine cases of bacterial transfusion transmission (with g&i > 2) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > 2 is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error (75%) with the lowest frequency in equipment failure (10%), compared to 21% and 29%, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes (25%) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in 2007-2017 were 80 in 8,385,448 blood units (1/104,818) issued for transfusion. these resulted in 27 serious reactions (34%) (1 fatal, 11 life-threatening) . another 21 (26%) were related with ibct that did not cause a reaction. near misses (component not transfused) were 24 (40%) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of 24 months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: 42,403 blood components were issued during the study period, while transfusion feedback forms for 37,816 components (89.1%) were received in the transfusion medicine department. delay in starting the transfusion (more than 30 min after issue) was observed in 2965 transfusion events (6.9%). the component transfusion time exceeded the permissible limits for 930 component (2.1%).the overall total permissible time for completion of components exceeded permissible limit in 2217 (5.2%) of transfusion events. the pediatric ward (23.9%), icu and ot complex (25.4%) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the 2965 delayed transfusions after issue, 2120 (71.5%) were during the routine hours i.e. between 7 am to 7 pm and 845 (28.5%) were in the non routine hours i.e. between 7 pm to 7 am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for 2013-2016 in comparison to previous years of surveillance, [2006] [2007] [2008] [2009] [2010] [2011] [2012] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for 2013-2016 89 sets of annual aggregated data from 25 countries covering 85,521,393 bcs issued were analyzed. all ars totalled 76,907 and infectious ars amounted to 285 (0.4%). the overall incidence of the infectious ars was 0.33/ 100,000 units of bcs issued. bacterial infections were the most frequent (188, 66%), next viral (84, 29.5%) and then parasitic (13, 4.5%). serious were 46% and there were 11 fatalities (3.9%, incidence 0.013/100,000). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the 84 viral sttis included 17 hbv (20%), 10 hcv (12%) and 2 hiv (2.5%). the 55 recorded as "other" (65.5%) including 24 cases of hev, one case of parvovirus b19, one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were 15 (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for 2006-2012 shows a consistent overall incidence in total sttis (0.33 vs 0.4/100,000). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in 2013-2016, p < 0.001) and an almost doubled rate of parasitic infections (p < 0.001). compared to the earlier period, there were many fewer hbv infections (17 vs 160) and many more hev. a similar reduction in the rate of hcv and hiv was observed in 2013-2016 in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january 2015-december 2018), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was 72 months (interquantile range-iqr 108). the median for the numbers of individual transfusions in children in a year was 12 (iqr 17). the median for the numbers of blood components individually transfused to patients was 18 (iqr 24). patients, anaphylactic transfusion reactions in 4 patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of 2.7 per 100000 units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of 11 and in 100000 units and 26 in 100000 units, respectively. while the incidence of transfusion reactions in children was found 0.62% in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of 2.7 per 100000 units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria 6 hospital general universitario, ciudad real, spain 7 hospital nostra senior de meritxell, andorra, andorra 8 banco sangre y tejidos, santander 9 banc de sang i teixits, barcelona, spain 10 fundaci on hematol ogica colombia, bogot a, colombia 11 centro regional de transfusi on de almer ıa, almeria 12 complejo hospitalario de navarra, pamplona 13 fundaci on banc de sang i teixits illes balears, palma de mallorca 14 hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in 2018 fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to 3 patients. two patients of 55 years of age diagnosed of acute myeloblastic leukemia (case 1 and 2) and chronic lymphatic leukemia (case 3). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors 1 month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all 3 cases, the story was judged as sufficient for analysis. the majority of reviewers (93%) diagnosed a chain of errors. there was agreement of 95% with respect to the initial process affected. the initial error was communication (42%), monitoring (45%) and compliance (62%), in cases 1, 2, and 3. 2-3 human errors were detected per case (average: 2.2, 2.7 and 2.4 errors respectively) and 2-3 latent errors per case (average: 1.8, 2.7 and 2.1, respectively). the latent errors most punctuated were: failures in the quality of the protocols (24%), in the transfer of important knowledge (22%), in the available technology (21%) and in the information to the patient (10%). all the participants contributed feasible measures of improvement according to root causes: 1) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, 2) train staff in knowledge important for safety, 3) communicate with computer application providers to improve the effectiveness and visibility of the alerts and 4) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from 2007 to 2017, reports of taco increased from 6 to 92; deaths from 1 to 7, major morbidity from 3 to 20. known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, <3 years, >60 years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last 3 years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case 1: a patient in their 50s weighing 67 kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb 37 g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of 100 g/l. -case 2: a patient in their 50s presented with a 4-week history of weakness and dizziness and had felt unwell for 6 months. the hb was 34 g/l, ferritin 26 lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of 51 g/l. a third unit was transfused over 125 min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case 3: a patient in their 90s with severe megaloblastic anaemia, hb 41 g/l and peripheral oedema developed taco after transfusion of 3 units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b12 deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b12 deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since 2015. results: total of 121,076 different blood components were applied in the period between 2015-2018. department for quality assurance and control has received 4 serious adverse reactions, 1 serious adverse event, 157 reports of transfusion reactions, of which 11 (7%) were inadequately filled, in the same period. from the above, 54 (34.39%) were transfusion reactions to erythrocyte blood components which were applied, 86 (54.77%) to platelet components and 17 (10.82%) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions (88 or 56.05%), followed by allergic reactions (50 or 31.84%). two transfusion reactions (1.27%) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was 0.004% (5 of 121,076) and 0.12% were reported transfusion reactions (157 of 121,076). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > 70 years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of 93 patients were analyzed (48 women, 45 men), with a median age of 86 years (70-97). in total, 189 ch were transfused. 70 patients received 2 ch, 3 patients 3 ch, 10 patients received 1 ch. 10 patients were transfused at two different times. the median hb prior to transfusion was 7.6 g/dl. in the patients who received 2 ch was 7.5 g/dl, those who received 3 ch: 6 g/dl and those who received 1 ch: 7.75 g/dl. the infusion time could be estimated in 84% of the patients. in those who received 2 ch was 223.62 min; 249.33 min in those who received 3 ch and 109.75 min in those who received 1 ch. 13 patients (14% of the total) suffered some type of adverse effect related to the transfusion. in 9 patients there was an increase in posterior ta, in 2 an increase in hr, in 1 an episode of hypotension and in another one episode of acute respiratory failure. 70% of those who had an adverse effect were older than 85 years. patients with aht after transfusion, 75% received 2 ch and the remainder 1 ch. among their background, 66% had a history of ischemic heart disease. 33% also had a positive balance. the average previous bp was 137/65 mmhg and the subsequent one was 182/72 mmhg. 55% of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. 2 ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > 85 years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse 2ch than 1ch. -we have appreciated that in those patients receiving 3ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february 2018 to december 2018. the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version 23.0. results: a total of 108 ida patients were analyzed in which 74(69%) were females and 34(31%) were males. the most common symptom in females and males was fatigue followed by body aches in females 12(16%) and pallor in males 10(29%). menorrhagia was present in 24(44%) of females of reproductive age. surgical history was present in 12(16%) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was 8.0 ae 2.2, 60.5 ae 9.6, and 20 ae 8.3 while in males it was 7.8 ae 2.3, 63 ae 14.3 and 18.4 ae 6.1 respectively. sixty two (84%) females were advised oral and i/v iron and 12 (16%) received transfusion. however, in males 8(24%) received transfusion and 26 (76%) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of 3 months follow up in males and females was same as that the transfusion (p > 0.05). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo (100 ng/ml). after 12, 14 and 18 days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd41a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day 0), cultured but unselected samples (day 7), and cultured, selected samples (day 12, 14 and 18) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of 65140 genes were detected, of which 16612 showed up-regulation and 48528 down-regulation. among these genes, 8 differentially expressed genes (degs) (fold change ≥ 10; false discovery rate < 0.05) were selected were further validated with rq-pcr, including gabre, cdhr1, wasf3, pkhd1l1, thbs1, pf4v1, lrrc32 and lgals12. the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf4v1 mrna expression level was highest at day 14, lgals12 was highest at day 18. summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: 7-8 g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between 2011-2018. patients with hb level measurement within 90 days pre-surgery were included. receiving > 1 blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, 2591 patients underwent hip or knee surgery; 231 were excluded from the analysis due to receiving > 1 blood unit. hb measurement within 90 days pre-surgery was available for 1202 patients. hip or knee surgery was performed in 588 (49%) and 614 (51%) patients, respectively. women comprised 60% (n = 356) of patients who underwent hip surgery. in the hip-surgery group, 14.5% of patients required bt, with this need being slightly higher among women (31.5% vs. 26.2%; p-value=ns). only 50 (8%) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it (11.93 g/dl versus 12.8 g/dl for women and 12.3 g/dl vs. 13.8 g/dl for men; p-value < 0.001). hospitalization was longer in transfused patients compared to non-transfused ones (mean 7.52 vs. 6.91 days, p-value = 0.018) and in patients with a low hb level (female < 12, male < 13.5) than in those with a high hb level, irrespective of receiving bt (p-values < 0.00045). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < 0.05). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for 1 blood unit was 0.43 in the hb 11 g/dl group and 0.15 in hb 13 g/ dl group (35%>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd36, known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd36 deficiency was cd36 not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd36 deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd36 is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd34 + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd36 monoclonal antibody on cd34 + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd36 monoclonal antibody on proliferation and differentiation of human cd34 + hematopoietic stem (progenitor) cells in vitro. methods: choose 3 healthy full-term maternal women without various obstetric complications, take cord blood 20 ml. after density gradient centrifugation of ficoll cell separation solution, cd34 + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. mtt was used to examine the effect of anti-cd36 monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd34 + hematopoietic stem (progenitor) cells. the effect of anti-cd36 monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after 10-14 days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd34 + were sorted by flow cytometry, and about 0.44% of cd34 + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd36 monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value (0.9 ae 0.15) of anti-cd36 monoclonal antibody group (2 mg/ml) was decreased than normal group (1.05 ae 0.12) (p < 0.05), and the od value (0.81 ae 0.11) was significantly decreased at the cd36 monoclonal antibody concentration of 32 mg/ml (p < 0.01). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > 0.05). in the annexin v flow detection, the apoptotic rate of anti-cd36 monoclonal antibody group (2 mg/ml) was statistically increased than the normal group (p < 0.05). anti-cd36 monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g1 phase cells increased, and g1/s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was 169 ae 12, the number of cfu-e/bfu-e clones in the control group was 172 ae 12, and the number of cfu-e/bfu-e clones in the anti-cd36 monoclonal antibody culture group was 85 ae 6. the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd36 monoclonal antibody culture group was significantly lower than that of the other groups (p < 0.05). summary/conclusions: anti-cd36 monoclonal antibody can reduce the proliferation of human cd34 + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. 11 (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of 159 autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd 34+ cells concentration in blood higher than 20/ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. 11 or cobe spectra v. 6, v. 7, terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in 56 transplanted patients were assessed. results: standard collections were performed in 52 patients. the yield of cd 34+ cells was high, and no significant differences were found between the numbers of cd 34+ cells prepared from spectra optia 8,6 (1,3-41) 9 10 6 and cobe spectra 10,9 (1, 6 ) 9 10 6 /kg b. w. (a = 0,05; pval 0,619). the dependence of cd 34+ cell yield on the precollection concentration of cd 34+ cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = 0,95, and cobe spectra r = 0,93. lvl collections were performed in 107 of patients, and there were no significant differences between the numbers of cd 34+ cells prepared by cmnc spectra optia 10,9 (2-61,2)9 10 6 and cobe spectra 9,3 (2,4-86) 9 10 6 /kg b.w. (a = 0,05; pval 0,35). the relations between the precollection cd 34+ cells concentration in blood and the numbers of cd 34+ cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = 0,93, and cobe spectra r = 0,78, respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia (45%) than in cobe spectra (57%). a group of 12 patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra 11 days, while in platelets from cmnc 14 days, and from cobe spectra 12 days, respectively. the number of 44 patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with 11 and 13 days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd 34+ cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd34 + cells/kg. this number depends on the pre-apheresis cd34 + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in 1 day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd34 number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in 1 day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in 1 day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd34 + cells collected/(pre-apheresis cd34 + number*processed volume)*100%. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd34 + number, patient weight and the requested number of cd34 + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in 1 day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = 64% (n = 348). a ce of 40% was chosen as the cut-off for the cd34 calculation tool. using the cd34 calculation tool: allogeneic donors (n = 61): mean ce = 61%, mean blood volume processed = 3.3 9 tbv, mean time: 253 min, 37 donors were finished in 1 day collection (61%) autologous patients (n = 205): mean ce = 65%, mean blood volume processed = 2.4 9 tbv, mean time = 218 min, 173 patients were finished in 1 day (83%). the calculation failed in only 1 case (0.4%). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd34 calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from 2001 till 2019. all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was 2 9 10 6 /kg cd34 + cells and 2 9 10 8 /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c53000, cobe spectra and spectra optia using conventional-volume apheresis processing the 2-2.5 total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after 4 to 5 days of g-csf subcutaneous administration at a dose of 10 lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were 159 apheresis procedures performed in 106 healthy sibling donors. there were 75 males and 31 females, aged 20-55. one to two apheresis procedures were required to collect adequate graft. the single procedure usually took 3-4 h and the volume of collected stem cells was 50-220 ml. the needed number of mnc and cd34 + cells was successfully collected by 1.5 apheresis. there were 29 abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia -57 patients (53.7%), acute lymphoblastic leukemia -14 patients (13.2%), chronic myeloid leukemia -9 patients (8.5%), myeloproliferative disorders -8 patients (7.5%), myelofibrosis -5 patients (4.7%), severe aplastic anemia -5 patient (4.7%), non-hodgkin lymphoma -3 patient (2.8%), multiple myeloma -3 patient (2.8%), chronic lymphoblastic leukemia -1 patients (0.9), hodgkin disease -1 patient (0.9%). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from 400 ml whole blood collected from 32 healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of 35 days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw 125,000, 98% of hydrolysis) using electrospinning technology (nanospider tm 1ws500u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of 700-900 x10 6 plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from 10% pva solution using water: ethanol (8:2) solvent system. materials with proteins were electrospun from solution containing 10% of pva and 10% of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: 340 ae 120 nm for pristine pva fibers and 350 ae 148 nm for pva with incorporated proteins (pva/pl). pva/pl layers contain 5-10 mg of protein per gram of pva. 60% of the proteins are released during the first day (burst release) followed by a gradual release of up to 2 weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv18-01-00332. background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) 1, 2 and 3 which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp-4, comprises less than 1% of the total defensins in neutrophils and has a distinct sequence from hnp1-3. the other two, human defensin 5 and 6, are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp 1-3) are 3.4 kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at 22°c by leukoflex lst-1 filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph 7.4, without mgcl2 and cacl2, containing 5 mm edta and 2.5% sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs 1x at a concentration of 1 9 10 7 cells/ml. for degranulation, cells were stimulated with 100 nm of formylmethionyl-leucyl-phenylalanine (fmlp) for 5 min followed by stimulation with 10 lm of cytochalasin b for 5 min. supernatant was collected by centrifugation at 200 9 g for 6 min. supernatant was incubated with mouse monoclonal antibody to hnp 1-3 and purification of hnp 1-3 was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the 3.4 kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst-1 filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing 5-aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct4, sox2 and nanog were performed after 24 and 72 h and 1 week results: the results indicated that after 24 h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a with extract expressed oct4 with weak intensity after 24 h. no expression of oct4, sox2 and nanog were observed in other groups at the same time. after 72 h, oct4, sox2 and nanog were over expressed in the cells that were treated with 5-aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct4 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct4 and sox2 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase (5-aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than 150 nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon 1, 5, 10 and 1227a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of 1227 g>a and rhd exon 1, 5, 10. samples with rhd gene deletion homozygous/heterozygous, 1227 g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd1-rhce(2-9)-rhd10 homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were 1 in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon 1 (87°c) to internal control (77°c) were 2.33 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.09-2.21 in alleles of rhd gene deletion/normal rhd, 2.53-2.66 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 10 (81°c) to internal control (77°c) were 3.35 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 4.29-4.39 in alleles of rhd gene deletion/normal rhd, 4.67-6.10 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 5 (81°c) to internal control (77°c) were 3.64 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.49 in alleles of rhd gene deletion/rhd1-rhce(2-9)-rhd10 3.47-3.59 in alleles of rhd gene deletion/normal rhd, 3.97-4.77 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types 1, 2 and 3 not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total 583 dna samples were tested in 87 pools. fifteen (15) pools (181 samples) gave rhd deletion genotype and seventy two (72) pools (402 samples) resulted to the presence of rhd gene. the 72 positive pools were also analyzed individually. the genotype results obtained were: rhd exon 1 no amplification (1), rhd exon 6 and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, 100% accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: 1 sample rhd*93-94inst (del), 1 sample rhd*ivs3+1g (del), 9 samples rhd*weak d type 11 (partial d), 1 sample rhd*weak d type 5 (weak d), 1 sample rhd*weak d type 61 (weak d) and not described: 1 sample rhd*del 1-5 (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to 20 pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type 1, 2, and 3 which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~12-kilobase duplication event, including exon 3, was predominantly identified in 58.3% d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the 70 rhd variants included in this study, 49 samples (70%) showed presence of indian specific allele i.e. exon 3 duplication. seventeen rhd variants samples showed presence of both exon 5 and 10. qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r 1 r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. -128 non-invasive fetal kel1 genotypes from allo-immunized anti-kel1 women were done (34 positive confirmed fetuses, 6 undetermined, 16 positive non-confirmed, 22 negative non-confirmed and 50 negative confirmed). -190 non-invasive fetal rhc genotypes from allo-immunized anti-rh4 women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh3 women were done (66 positive foetuses, 1 undetermined for 21,5% of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła 1 , m krzemienowska 2 , m pelc-kłopotowska 2 , m jurkowska 3 , m debska 4 , m uhrynowska 2 and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since 2016 but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type 1, 2 and 3. aims: summary of a 3-year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of 366 pregnant women determined with standard serology as rhd-negative (in 8-38 week of gestation) was examined for the presence of exons 5 and 7 of rhd and ccr5 by realtime pcr using lc480ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in 123 cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr5 indicated a maternal d variant (d ct ccr5-rhd >2); the genetic follow-up of six of them identified: rhd*01w.2 in 4 cases, rhd*01w.1 and rhd*15. in 235 cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining 67% of mothers. in 1.9% cases with maternal d variant rhd nipt was not possible. however, in 5/6 such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type 1 and 2 in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh23 (d w ), as these are almost exclusive to african populations. rh23 is encoded by several types of rhd*dv as well as by dau-5. anti-rh23 is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida 4, para 1, causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at 37°c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh23 in addition to anti-fy 5 , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd*10.05 which encodes rh23. the newborn's phenotype was a r0r k-, fy(a-b+) and most likely rh23-and jk (a+b+), considering both maternal and paternal (a r0r, k-k+, fy(a-b+), jk(a+b-), rh23-) predicted phenotypes. the neonatal serum contained maternal anti-a1, anti-rh23 and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy 5 during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh23, derived from several rh23 + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from 22% in the patients younger than 60 years to 58% in the patients older than 80 years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with 59%. only 48,5% achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene 17%-33% patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above 200 mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a 28y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at 30y, a sister with similar profile age 26y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin 75 mg od, ticagrelor 90 mg bd, rosuvastatin 40 mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux5a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column 5a20 (evaflux, kawasumi, japan) as the second step. a total of 1.5x plasma volume(4470 ml) was processed. the patient was given continuous calcium infusion. the flow rate of 50 ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by 60%. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from 2010. hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: 101, 95, 111 in the last 3 years. delayed transfusion in relation to mh was reported for 54 cases 2016-2018 contributing to death in 6 patients 7%) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in 31/71 (43.7%): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, 2 units); 14% of women were transfused with fresh frozen plasma (median, 2 units) and 2% with platelets. mean pre-and post-transfusion hemoglobin levels were 6.7 g/dl and 9.0 g/dl, respectively, representing an increment of 1.0 g/dl per rbc unit transfused (1.09 g/dl in soweto and 0.83 g/dl in durban). indications for transfusion included obstetric hemorrhage (31%), chronic anemia (29%), surgery or anesthesia (13%), other (9%) and not specified (17%). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ 26 weeks (odds ratio = 7.07, 95% confidence interval 3.52-14.24). surgical blood loss was a common indication in trimester 1 (21%) that declined to 7% then 1% in trimesters 2 and 3. summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period 2016-2018. results: there were evaluated 1,281 688 of blood products administrations in 117,414 patients in the cr during defined three years period. announced 1,456 (0,11%) transfusion reactions including 50 severe transfusion reactions (20 9 adjudged with grade 3). the most frequent types of severe transfusion reactions: anaphylaxis 20, trali 7x, taco 5x, hcv 4x, hbc 2x, bacterial infection 1x, delayed hemolysis 1x. transfusion reactions incidence according to administered bp: red blood cells products: 882,017 administrations, 883 transfusion reactions (fnhtr 386x, allergy 227x, circulatory overload 66x, anaphylaxis 6x, trali 4x, hbv 2x, hcv 1x) platelets: 88,438 thrombocyte administrations, including 178 transfusion reactions (allergy 129x, fnhtr 30x, anaphylaxis 5x, circulatory overload 8x, delayed immune hemolysis 3x, acute circulatory overload 8x. granulocytes: 203 administrations, 2 transfusion reactions plasma: 293,801 administrations, 359 reactions reported (allergy 278, fnhr 25, circulatory overload 14, anaphylaxis 17x, trali 4x, hbv 3x, ards 1x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: 1. plasma for clinical uses from male blood donors, 2. prestorage of leucocyte reduced blood products, 3. production of platelets in additive solutions, 4. implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of 5 they were mainly associated with deviation in processing (22.4%) and attributed to equipment failure and materials (71%) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < .001) annual rate of total aes by 28% (95% confidence interval 26-30) ) 10% fibrinogen-depleted phpl or (3) 10% fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene 2.1 st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd73+/90+/105+ and cd14-/19-/34-/45-/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) 5%) were male and 31(36.5%) female. mean age of abo matched and mismatched groups were (6.99 ae 4.6) years. most common indications for transplant included beta thalassemia major 53(60.2%), aplastic anemia in 25(28.4) and pure red cell aplasia 2(2.35%). source of stem cell was bone marrow in 45 and peripheral blood 40 patients abo matched while abo mismatched group comprised of 28(32.9%) patients with further subdivision into major (n = 13), minor (n = 13) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = 401) and (n = 1837) comparably(n = 102) and (n = 480) in mismatched group. primary and secondary graft failure in matched group was 8.77% and 12.28% patients while in mismatched group graft failure was observed in 4(14.28%) patients respectively. positive dct in abo matched group in 1(1.75%) patient, whereas 2(7.14%) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia 1 , c sabia 1 , f cavalca 1 , g nicoletti 2 and c napoli 1 in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr4 and dr16 in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested 5 sera pre-treated with fbs from non-sensitized patients that showed the dr4/dr16 pattern. in particular, 5 ll of fbs was added to 95 ll of patient serum; incubated for 30 min at 37°c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from 5 patients with documented dsa including dr4/dr16 and 5 patients without hla antibodies. results: dr4/dr16 non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr4 and dr16 dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre 2 transfusion medicine, national blood transfusion center 3 transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from 17,992 healthy donors from which group a (36,8%), group o (41,7%), b (16,2%) and group ab (5,3%) resulted. the study was conducted in 3727 patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period 2011-2017 results: among the 3727 patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a (39.7%), followed by 0 (39.5%), b (15.2%) and ab (5.4%). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a (42%), in gastric cancer a (43%), colorectal cancer a (39, 6%), breast cancer a (41%), cervical cancer a (43%) and ovarian cancer a (42%) versus a (36.8%) in the control group. a high incidence of blood b is seen in multiple myeloma b (22%) and cervical cancer b (20%) versus b (16.2%) in the control group. blood group ab has a high incidence in malignant lymphoma ab (10%) versus (5.3%) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types 1, 2 and 3, are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a 12-year-old patient typed as weak d type 3, receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma (2+ by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type 3. rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type 3. mma showed the anti-d was clinically significant (>5%). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type 3 allele. weak d type 3 patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band 3/diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: 1.1. predictprotein (https://predictprotein.org); 1.2. spider2 (http://sparks-lab.org/server/spider2/); 1.3. dsc (discrimination of protein secondary structure class): using an in-house implementation; 1.4. jpred4 (http://www.compbio.dundee.ac.uk/jpred4/); 1.5. raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: 2.1. robetta (http://robetta.bakerlab.org/submit. jsp); 2.2. falcon (http://protein.ict.ac.cn/treethreader/); 2.3. itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: 3.1. verify3d (http://servicesn.mbi.ucla.edu/verify3d/); 3.2. prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: 4.1. gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); 4.2. gramm (http://va kser.compbio.ku.edu/main/resources_gramm1.03.php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = 10) carrying fetuses with known genotype was loaded into 3% agarose gel casette (3% df marker q3, sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from 45 min to 2 h 30 min of electrophoresis. results for real-time pcr detection of fetal rhd, kel*1 and ccr5 (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr5) was significantly lower in dna samples tested after gel selection (from 8.4 to 25.9geq/pcr) versus the level obtained from non-processed plasma dna (from 130 to 133geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from 1,5 to 6.6geq/pcr versus 10.8-11.2geq/pcr for non-processed plasma dna. results for kel*1 detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel*1-negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel*1 positive amplification was observed. however, kel*1 detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel*1-negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel*1 positive genotype was obtained. the total dna level in samples from k-negative women was from 18.3 to 18.7geq ccr5/pcr after gel selection versus from 52 to 746geq ccr5/ pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh1 quantification assay using ih-500 (bio-rad â ): promising results for monitoring rh:-1 pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh1 immunoglobulins since 1970 complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh1 microtitration. it is a column agglutination technology using red blood cells rh: 1, -2, -3,4,5 (r0r) . it permits to quantify low levels of anti-rh1 in comparison to a range of an anti-rh1 standard. performed since 1999 at the cnrhp and automated on evo clinical base tecan in 2008 (dilutions and distribution), anti-rh1 microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih-500 system from bio-rad â . methods: on ih-500, the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r0r, anti-rh1 standard). the performances of the method were evaluated using three internal quality control (icq) (2 cnrhp home-made at 2 and 12 ng/ml and 1 bio-rad â at 12 ng/ml) on papainized r0r (plc) and native r0r (nlc). a comparison of results from patient sera ranging from 1.5 to 48 ng/ml was done between ih-500 and evo clinical base tecan. results: the results of the 3 qci are similar between the different reagents used. there is no significant difference between the 2 types of red blood cells except for the limit of detection: 1.5 ng/ml in plc -6 ng/ml in nlc. for the 3 qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between 0 and 15%, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on 50 samples in plc and 44 samples in nlc was satisfactory (deming plc: r2 = 0.80 y = 0.89x + 0.78 -nlc: r2 = 0.86 y = 1.08x-0.25). summary/conclusions: the anti-rh1 microtitration on the ih-500 offers similar performances to the method conducted at the cnrhp. the ih-500 allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh1. this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab0i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about 1.5-2% of cases haemolytic disease demands transfusion support. aims: analysis hdfn from 2011 to 2018. methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group 0 rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (<5 days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit 70 + 5%, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit 40% -60% and irradiated. the ec used in post-natal transfusions is usually divided into rates of 70 ml, hematocrit 55 ae 5%. results: in last 7 years, we calculated 59 neonates with hdfn (28 males and 31 females): 31 with rhdi hdfn, 19 with ab0i hdfn and 9 with hdfn due to incompatibility for other red blood cell antigens. we have produced 81 iut: 48 for our hospitalized patients and 33 for patients located in other hospitals. 25 of these 48 patients, who received iut, were immunized: 19 showed anti-d antibody and 6 antibodies different from anti-a and anti-b. 7, of the 31 infants with rhdi hdfn, were transfused in utero. 4 neonates on 59 (6.8%) have performed et: 1 with ab0i hdfn and 3 with rhdi hdfn; the latter had also been transfused in utero. 27 neonates on 59 were transfused after birth: 18 with rhdi hdfn, 3 with ab0i hdfn and 6 with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous (52.7% of the total) and are those who have requested the highest blood supply both in the antenatal period (39.6%) that postnatal (9.6% performs et, 66.6% performs postnatal transfusions). neonates with aboi hdfn are 32.3%: nobody has received iut, only one has been subjected to et, and 11% has transfused after birth. patients with hdfn due to other antigens are 15%, have undergone iut 12.5% and were transfused after birth 22.2%. background: according to british guidelines on neonatal transfusion, since 2012 we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ 30 weeks and/or a birthweight lower than 1500 g, until 4 months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab0 direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab0/rh confirmation. blood transfusions were performed with 0 negative kell negative (0 cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than 5 days. ec were subdivided in 70 ml aliquots with a hematocrit of 55 ae 5%. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with 72 h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october 2012 to the end of 2018, 694 premature newborns received 2328 ec transfusions within their first 4 months of life. in 51% of cases (n = 1186), transfusion requirement was comprised within the 'small volume transfusions'. another 44% of cases (n = 1035), requiring further ec administration, was requested of a blood sample for t&s determination and 5% (n = 107) for a cross-match test. in 79.4% of newborns (n = 551), being transfused within the " small volume transfusions", blood requirement of 1401 ec was fulfilled by the initial blood test (1102 blood samples). 13.3% of newborns (n = 92) received more than 5 transfusions (6-21; median = 8) accounting for 820 ec released and for this group 381 blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, 381 blood tests were performed to sustain 643 infants transfused with 2221 units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of 75% of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first 4 months of life. background: glucose-6-phosphate dehydrogenase deficiency (g6pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g6pd 202 genotype is the most common g6pd genotype in sub saharan africa (ssa). some studies have linked blood from g6pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g6pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g6pdd 202 genotype among donors in two regions in uganda. it also described the effect of g6pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: 3,255 blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g6pd 202, haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g6pd 202 and co-inheritance with a-thalassaemia (n = 2,546) and haemoglobin s (n = 2,642) on the haematological quality of blood packs. a subset of 142 donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g6pdd. results: based on g6pd 202 genotyping, 10.3% (n = 274) of the blood samples used in the trial were deficient for g6pd enzyme while 5.3% (n = 142) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = 0.010) and whole blood (p = 0.009) donations of heterozygous g6pd 202 genotype. co-inheritance of g6pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was 83.3% sensitive and 0.8% specific for detecting donor blood packs with g6pdd. summary/conclusions: the prevalence of g6pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g6pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in 39 hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after 21 days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < 1000) and cdc-xm became negative. cdcxm labeled positive at ≥ 10%. t-cell fcmx was considered positive above 42 mfi and b-cell fcmx was considered positive above 120 mfi. lmxm was considered positive above 500 mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in 8 patients (20.5%). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the 39 cases initially screened 35 showed dsa positivity in sab. desensitization was done in those 35 cases only. in our study, sab was positive for class ii alone in 13(37%) while in remaining 22 (63%) cases it was positive for both class 1 and class ii. the number of pre transplant tpe procedures required was 6.7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) . the mean number of post-transplant tpe sessions required was 0.7 (range, 1-6). during pretransplant and post transplant tpe procedures, five (14.3%) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean-8.5, median-7 days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx 17 months with the longest duration of follow up of 36 months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < 22%, we custom prime the tubing set with 5% albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient #1 is a 56-year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every 4 weeks, starting in 2012. the patient has completed 84 procedures with 3-4 units of washed red cells transfused to achieve a target hct goal of 45 to 57%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #2 was a 25-year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent 9 prx procedures with 2-3 units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient #3 is a 33-year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone 38 prx procedures with 3-5 red cell units transfused to achieve a hct goal of 30%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #4 is a 48-year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with 4-5 red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the 2016 american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since 2011. aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days 1, 2, 3, 5, 7, 9 along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos 7 aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of 63 variables was defined as possible confounders by a panel of experts. after discussion, 9 global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and 7 cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: 1018 patients were extracted from databases and further analyzed. the mean age of this group was 65,9 years (sd +/-14,1 years) and 67.3% of them were male. the mean duration of surgery was 256 min (sd +/-88,3 min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < 0.001). in 2011, the mean use was 2,34 units per operation (sd +/-2,277), which changed to 1,38 units (sd +/-2,158) in 2014. three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over 4 years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < 0.001). in the specific group of patients undergoing cardiac surgery with cpb (n = 640), the use of rbc was also significantly reduced (p < 0.001). in 2011, the mean use was 3,06 units per operation (sd +/-0,448) and this changed to 1,94 units (sd +/-0,167) in 2014. after correction for the 3 cpb variables that notably changed over the 4 years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over 4 years still remained statistically significant (p < 0.001). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between 2011 and 2014. this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from 14 adult pid and 13 adult sid patients from medical records and pathology reports, for their last 12 months of ivig and their first 12 months of scig. the starting and maintenance dose was 0.4 g/kg/month for ivig, transitioning immediately to 0.1 g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of 62.9 years versus 43.4 years in pid patients (p = 0.007). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = 0.041). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. 55% of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig (9.3 g/l) compared to ivig (8.4 g/l), and fewer infections on scig than ivig (mean annual infection rate of 1.64 vs 2.14 respectively). sid patients had higher mean serum igg trough levels on scig (8.4 g/l) than ivig (7.1 g/l) (p = 0.009) but experienced more infections while on scig versus ivig (mean annual infection rate of 2.15 vs 1.62 respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd62p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd62p expression. aims: to analyze the increase of platelet cd62p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante 2', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june 2014 to august 2018. the medical helicopter (ec 145t2) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and 35 days after collection, were performed. the statistical analysis used spss 23.0 version (significance p < 0,05). results: 138 prbc samples were evaluated, 48,5% (67) from case group and 51,5% (71) from control group. analyses were tested day 1 and day 35 after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered 35 prbc transfusion to 28 patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a 35-day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in 2010 the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of 11 deaths and 83 instances of harm due to delay over a 4-year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of 18 adult patients over a period of 7 months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all 18 cases. results: the abg analysis of all 18 patients showed decrease in the ph, increase in pco 2 , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the 18 cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of 18 cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in 4 cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in 2 cases. platelets were transfused in 5 patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of 40 adult surgical oncology patients who received massive transfusions and was conducted for a period of 7 months. every case of a massive transfusion was studied under the following headings (1) patient's details (2) patients base-line laboratory tests (3) resuscitation with transfusion (4) intra-operative laboratory tests (5) thromboelastography (teg) (6) post-operative complications (7) duration of stay in the hospital (8) 30 day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by 2 nd or 3 rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of 13 (32.5%) patients had ssi, 14 (35%) had surgical complications and 22 (55%) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, 38 (95%) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of 725 outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december 2017. laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted 15 ae 17.52%, but 49.72% of patients had a ttr less than 60%. patients were at high risk of thrombosis in 6.15% of time (inr < 1.5) and high risk of bleeding in 2.2% of time (inr > 4.5). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated 1.9 million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo 2 ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between 2005 and 2015 in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = 505) with two similarly exposed non-alloimmunized control recipients (n = 1010) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > 30 ml/min/1.73 m 2 , 'moderate renal failure' i.e. gfr ≥ 10-30 ml/min/1.73 m 2 during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < 10 ml/min/1.73 m 2 and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among 441 (87.3%) cases versus 838 (83.0%) controls; moderate renal failure among 24 (4.8%) cases versus 54 (5.3%) controls; and severe renal failure among 40 (7.9%) cases versus 108 (11.7%) controls. among the latter, 30 cases and 97 controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr 0.82, 95% ci 0.67-1.01 and adjusted rr 0.81, 95% ci 0.58-1.11, respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr 0.48, 95% ci 0.39-0.59) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir2dl1, kir2dl2/kir2dl3, kir3dl1, kir3dl2 and hla-a, -b, -c, -drb1, -dqb1 genotyping. 58 pairs of hla 10/10 identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c1/c1 homozygote group (48 cases), c1/c2 heterozygote group (8 cases) and c2/c2 homozygote group (2 cases). according to the expression of 3dl1, 53 cases were 3dl1 positive and 5 cases were 3dl1 negative. there were 5 cases of bw4/bw4, 24 cases of bw4/bw6 and 24 cases of bw6/bw6 in the 3dl1 positive samples. according to the expression of a3/a11, they were divided into three groups: a3/a11 negative group (28 cases), a3/a11 heterozygous group (28 cases) and a11/ a11 homozygote (2 cases). according to kir genotyping, kir haploidentical group (38 cases) and kir haploid mismatched group (20 cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of 58 cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v5.01. results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir3dl1 was positive. it was conducive to neutrophil and platelet remodeling when bw4/bw6 and a3/a11 was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to 5-7 days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = 3), prepared in 30% plasma/70% ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for 7 days (d) and sampled on d2, d5 and d7. cryopreserved sheep pcs (n = 3), prepared by the addition of 5-6% dimethyl sulfoxide, were stored at à80°c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), anti-inflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), 12-hete and 15-hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < 0.05 using paired t-test. results: in rt stored sheep pc supernatant il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete were detected at d2, d5 and d7. storage duration significantly increased accumulation of ip-10 at d5 (253.4 ae 266.7 pg/ml compared to 569.7 ae 272.7 pg/ml, p = 0.008) and further increased at d7, and il-8 at d7 (3477 ae 937.4 pg/ml compared to 5092 ae 521.1 pg/ml, p = 0.0460). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete than rt stored d5 pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il-8 with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than 10 cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = 5), prepared according to standard human procedures with minor modifications, were stored (2-6°c, 42 days (d) ) and sampled at d2 and d42. supernatant was prepared by double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), antiinflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), 12-hydroxyeicosatetraenoic acid (hete) and 15-hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < 0.05). results are mean ae standard deviation. results: at day 2, aa (75,142 ae 47,205 pg/ml), 12-hete (849.1 ae 235.6 pg/ml), 15-hete (87.6 ae 32.7 pg/ml) and il-1b (144.7 ae 198.9 pg/ml) were detectable in sheep prbcs supernatant. at day 42, storage duration significantly increased concentrations of aa (136,254 ae 60,433 pg/ml, p = 0.0425) and 15-hete (380.9 ae 116.3 pg/ml, p = 0.0022) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as 1 in 2,500 transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within 28 days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for 50 years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from 16% to 0.2%, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately 0.01 deaths per 1000. blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since 2007. since january 2015, rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = 93) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january 2015-december 2018, 45 reports were received; 43 reports were validated, with 2 reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in 2017 and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, 2019, depending on their iron parameters as having either: -idaserum ferritin < 30 mg/l -chronic inflammation with idaserum ferritin 30-100 mg/l with transferrin % of < 20%/crp > 5 mg/l -anaemia of chronic inflammationserum ferritin > 100 with transferrin % of < 20%/crp > 5 mg/l patients were considered eligible for iv iron if the following criteria were met:1. an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short 2. the anaemia pro-forma was completed 3. hb was ≤ 120 g/l 4. they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, 20 days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included 80 patients. 48 patients were classified as having ida and 32 patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of 96 g/l (55-120), a mean mcv of 77.4 fl and a mean serum ferritin of 16 lg/l. those with chronic inflammation with ida had a mean hb of 106 g/l (77-120), a mean mcv of 87.9 and a mean serum ferritin of 57 lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was 119 g/l (100-149) with an average increment of 23 g/l and in the group with chronic inflammation with ida the average post iv iron hb was 117 g/l (85-129) with an average increment of 11 g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb 20 days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january 2012-august 2018, which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were 154 products dispensed to 53 patients. the recipients were male patients diagnosed with prostate cancer with a mean age of 74 years. there were 3 doses (a complete course) administered to 50/53 (94%) recipients and a partial course (1-2 doses) administered to 3/50 (6%) recipients, for a total of 154 products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were 5 adverse events described in provider notes; 2 of them necessitating transfer to the emergency department, and 1 requiring hospital admission. of the 154 infusions, 6 infusions were documented in a chemotherapy note rather than a transfusion note (4%), and 59 (38%) were documented as both a transfusion and a chemotherapy administration. there were 6 additional deviations from the blood product administration policy: 2 cases where the consent check was not performed, 1 case where the product was infused with ringer's lactate rather than normal saline, and 3 cases where the 2-person 3-way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref 10310) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till 2017, at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ 20,000/ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = 31) and cmnc (n = 88) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in 134 consecutive cmnc procedures, including 88 autologous and 46 allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd34 + count ≥20/ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency-2 (ce-2) was defined as the total cd34 + amount in the collection bag divided by the amount of cd34 + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time (333 ae 59 and 289 ae 73 min, respectively; p = 0.001), the total blood volume processed (3.4 ae 0.8 and 2.4 ae 0.8, respectively; p = 0.001) and the final volume in the collection bag (337 ae 77 and 291 ae 84 ml; p = 0.001). the mean ce-2 in autologous versus allogeneic donors was 49 ae 24 and 66 ae 22, respectively (p = 0.003). using cmnc, the collection was effective in 94% of allogeneic and 63% of autologous donors. in autologous donors, a significantly lower collection bag volume (341 ae 84 and 396 ae 132, respectively; p < 0.01) and increased total wbc in the collection bag (263 ae 119 versus 149 ae 36, respectively; p < 0.000) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd34 + count following g-csf therapy; 7 of them achieved a cd34 count ≥20 and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd34 + count ≥ 20/ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd34 + cell counts are obtained with both methods. . tbv processed ranged from 1-4.8 tbv with mean of 2.6, average was 2.65 tbv for females and 2.74 tbv for males mean pre-apheresis cd34 + count was 92.89 cells/ll (range 33.14-152.64). mean postapheresis cd34 + count was 1402.3 cells/ll (559.02-2245.5). mean cd34 + cells x10 6 / kg recipients body weight was 7.5 (range: 2.77-12.33). our target yield was ≥3 9 10 6 cd34 + cells/kg body weight of the recipient and in only 2/34 (6%) cases, the yield was <3. 18/34 (52.9%) procedures were lvl and 16/34 (47.1%) were svl. summary/conclusions: most of our pbsc were done for haematological indications (85.3%) and the target dose was 3 9 10 6 cells/ll in single leukapheresis. in 32 cases (94%), target yield was achieved, only 2 cases had <3 but >2 yield. in our study donors <5 years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd34 + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd34 yield as high wbc count did not convert into high cd34 yield or vice versa. high prepheresis cd34 + count gave higher postpheresis cd34 + count. large volume leukapheresis (lvl), >3 tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd34 + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd34 + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid 1980s and early 1990s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our 18 year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine 11 hla loci (hla-a, -b, -c, drb1/3/ 4/5, dqb1, dpb1, dpa1, dqa1) in potential bone marrow donors from poland. the research included 18,500 potential bone marrow donors registered between 2017 and 2018. a novelty of this paper was that the amplification of all 11 hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the 11 hla loci (hla-a, -b, -c, drb1/3/4/5, dqb1, dpb1, dpa1, dqa1) of potential bone marrow donors was made by using the alltype tm ngs 11-loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of 18,500 donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c*12: 143, c*12: 30, c*05: 37, c*07: 151, drb1*11: 28, c*14: 04, b*51: 22, c*15: 13, dqb1*03: 12, drb1*14: 87, drb1*11:69. summary/conclusions: 1. new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. 2. the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. 3. the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn5 dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version 1.2.0(one lambda inc.)with the default setting. 94 cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: 94 samples were successfully tested with two methods and the coincidence rate between two sequencing methods was 100%. with the next-generation sequencing method, the probability of ambiguous results among 94 samples in our study is 1.06%(1/94) for hla-a, 5.31% (5/94) for hla-b and 0% (0/94)for hla-c. however, the probability of ambiguous results with the sanger sequencing method is 96.8% for hla-a, 95.7% for hla-b, 100% for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques.